JOHANNES GUTENBERG UNIVERSITÄT MAINZ Dissertation Small molecules targeting apoptosis and parthanatos in sensitive and drug-resistant tumor cells Min Zhou Mainz, August 2023 Small molecules targeting apoptosis and parthanatos in sensitive and drug-resistant tumor cells Kumulative Dissertation zur Erlangung des Grades “Doktor der Naturwissenschaften” im Promotionsfach Pharmazie am Fachbereich Chemie, Pharmazie und Geowissenschaften der Johannes Gutenberg-Universität in Mainz vorgelegt von Min Zhou geboren am 01.29.1994 in China Mainz, August 2023 Betreuer: Uni.-Prof. Dr. Thomas Efferth Gutachter Der Arbeit: Uni.-Prof. Dr. Thomas Efferth Uni.-Prof. Dr. Kristina Friedland Datum der mündlichen Prüfung: 04.10.2023 Prüfungskommission: Uni.-Prof. Dr. Peter Langguth (Vorsitzende) Uni.-Prof. Dr. Thomas Efferth Uni.-Prof. Dr. Kristina Friedland Frau Dr. Xiaohua Lu (Protokoll) D77 (Dissertation Universität Mainz) List of publications Publications as first author M. Zhou, J.C. Boulos, S. Klauck, T. Efferth, The novel cardiac glycosides ZINC253504760 induces parthanatos-type cell death and G2/M arrest via downregulating MEK1/2 phosphorylation in CCRF-CEM leukemia cells. Cell Biology and Toxicology (2023) 1-27. M. Zhou, J.C. Boulos, H.A. Rudbari, T. Schirmeister, N. Micale, T. Efferth, Two palladium (II) complexes derived from halogen-substituted Schiff bases and 2-picolylamine induce parthanatos-type cell death in sensitive and multi-drug resistant CCRF-CEM leukemia cells. European Journal of Pharmacology 956 (2023) 175980. M. Zhou, J.C. Boulos, E. Omer, S. Klauck, T. Efferth, Modes of action of a novel c-MYC inhibiting 1,2,4-oxadiazol derivative in leukemia and breast cancer cells. Molecules 28(15) (2023) 5658. M. Zhou, A. Varol, T. Efferth, Multi-omics approaches to improve malaria therapy, Pharmacological Research. 167 (2021) 105570. Publications as co-author M. Elbadawi, J.C. Boulos, M. Dawood, M. Zhou, W. Gul, M.A. ElSohly, S.M. Klauck, T. Efferth, The Novel Artemisinin Dimer Isoniazide ELI-XXIII-98-2 Induces c-MYC Inhibition, DNA Damage, and Autophagy in Leukemia Cells, Pharmaceutics 15(4) (2023) 1107. H.A. Rudbari, N. Kordestani, J.V. Cuevas-Vicario, M. Zhou, T. Efferth, I. Correia, T. Schirmeister, F. Barthels, M. Enamullah, A.R. Fernandes, Investigation of the influence of chirality and halogen atoms on the anticancer activity of enantiopure palladium (II) complexes derived from chiral amino-alcohol Schiff bases and 2-picolylamine, New Journal of Chemistry 46(14) (2022) 6470-6483. C. Di Chio, M. Zhou, T. Efferth, T. Schirmeister, M. Zappalà, R. Ettari, Synthesis and Cytotoxicity of Diarylpentanoids against Sensitive CCRF-CEM and Multidrug-Resistant CEM/ADR5000 Leukemia Cells, Chemistry & Biodiversity 19(2) (2022) e202100451. Conference papers 1. 6th Cancer World Congress, Lisbon, Portugal, Sep 28-30, 2022. Presentation: Cytotoxicity of ZINC253504760 in CCRF-CEM leukemia cells through inhibition of MEK1/2 phosphorylation, G2/M cell cycle arrest, and the induction of parthanatos- type cell death”. (Best Flash Oral Presentation of Young Researcher) 2. 7th Cancer World Congress, Palermo, Italy, May 29-31, 2023. Presentation: The mode of action of a 1,2,4-oxadiazole derivative as c-MYC inhibitor in leukemia and breast cancer cells. Contribution to articles included in the thesis 1. Title: The cardiac glycoside ZINC253504760 induces parthanatos-type cell death and G2/M arrest via downregulation of MEK1/2 phosphorylation in leukemia cells. Contribution: 1) Conceptualization and methodology. 2) Conduction of experiments including growth inhibition assay, RNA extraction, qRT-PCR, cell cycle, apoptosis, fluorescence microscopy of the microtubule cytoskeleton, protein extraction and western blot, mitochondrial membrane potential, immunofluorescence microscopy of AIF translocation, single cell gel electrophoresis (comet assay), molecular docking, ROS detection, microscale thermophoresis. 3) Data curation, visualization, and data presentation of all Figures 1-7. 4) Writing the original manuscript. 2. Title: Two palladium (II) complexes derived from halogen-substituted Schiff bases and 2-picolylamine induce parthanatos-type cell death in sensitive and multi-drug resistant CCRF-CEM leukemia cells. Contribution: 1) Conceptualization and methodology. 2) Conduction of experiments including isolation and cytotoxicity of human peripheral mononuclear cells, apoptosis, mitochondrial membrane potential, protein extraction and western blotting, immunofluorescence microscopy of AIF translocation, immunofluorescence microscopy of AIF and mitochondria staining, single cell gel electrophoresis (comet assay), cytotoxicity in the combination with PARP inhibitor. 3) Data curation, visualization, and data presentation of all Figures 1-10. 4) Writing the original manuscript. 3. Title: Modes of action of a novel c-MYC inhibiting 1,2,4-oxadiazole derivative in leukemia and breast cancer cells. Contribution: 1) Conceptualization and methodology. 2) Conduction of experiments including growth inhibition assay, molecular docking, microscale thermophoresis, RNA extraction, qRT-PCR, cell cycle, apoptosis, single cell gel electrophoresis (comet assay), protein extraction and western blotting, doxorubicin uptake assay. 3) Data curation, visualization, and data presentation of all Figures 1-11, except Figure 2G. 4) Writing the original manuscript. Erklärung Hiermit erkläre ich an Eides statt, dass ich diese Arbeit selbständig verfasst und keine anderen als die angegebenen Quellen und Hilfsmittel verwendet habe. _____________________ _____________________ Ort, Datum Min Zhou I Acknowledgment Time is the best gift for me. Five years ago, when I decided to study the anticancer activity of medical plants, I applied to Prof. Dr Thomas Efferth. The following life in Germany, the food, the weather, the environment, the people, everything was new but exciting to me. Now it is my graduation. My sincere and hearty appreciations go firstly to my supervisor. Prof. Dr. Thomas Efferth, who gave me this cherished opportunity to be a Ph.D candidate, work in an excellent international group, and experienced a rich and wonderful life in Mainz, Germany. It has been a great privilege and joy to study under his guidance and supervision, which brought me into the field of studying pharmaceutical biology about cancer. He constantly guided me in the correct direction with his wisdom and expertise, helped me move into new steps, and we shared progress and challenges regularly. I vividly remember the first time I verified our hypothesis (the treated cells induced parthanatos), how beautiful the sunset was when I reported to him, and how much happy we were! It is my honor to benefit from his personality, diligence, and encouragement, which I will treasure my whole life. My gratitude to Prof. Efferth knows no bounds. I would like to thank Prof. Dr. Peter Langguth, Prof. Dr. Kristina Friedland, and Prof. Dr. Thomas Efferth for becoming my Ph. D defense committee members, reviewing my thesis, and taking part in my defense. Thanks to the Chinese Scholar Council (CSC) for the financial support of my Ph. D life in Germany. This scholarship allows me to focus on study without distractions, and it encourages me to improve my overall research skills. Thanks to the teachers of the Generalkonsulat der Volksrepublik China in Frankfurt a. Main for their service to us Chinese students. Many greetings and helps deliver our country's care. Sincere gratitude should also go to Prof. Dr. Tanja Schirmeister, Dr. Sabine M. Klauck, Prof. Nicola Micale, and Prof. Hadi Amiri Rudbari for their collaborations in my projects. Their detailed feedback and corrections of manuscripts have been supporting me to solve problems and improve my articles in a scientific way. I am thankful to the scientists in IMB for their technical support in flow cytometry and microscopy, their suggestions and clearing up confusion help me gain better lab skills. II I would like to express my warm gratitude to all group members of the Department of Pharmaceutical Biology. Thanks to Dr. Mona Dawood who taught me cell culture and resazurin assay, this was my first lesson beginning in the cell culture room. Thanks to Mohamed Elbadawi who showed me IPA analysis and qRT-PCR, and we discussed potential pathways and targets, as well as many urgent occasions I need help, he was here. Thanks to Dr. Xiaohua Lu for her help in the lab and for my life. Thanks to Joelle C. Boulos for her showing me many important experiments, the efforts we spent in the lab were all about our exciting explorations, and we were happy for each other’s nice progress. My cordial thanks to the support and encouragement from Nasim Shahhamzehei, Ejlal Omer, Roxana Damiescu, Chunmei Jin, Matteo Rosellini, Assia Drif, Dr. Rümeysa Yücer, and so forth. There are also many thanks that will not be forgotten to our previous group members Dr. Onat Kadioglu, Dr. Mohamed Elfatih Saeed, Dr. Sara Abdelfatah, Dr. Nuha Mahmoud, and Dr. Nadire Özenver, they were generous in helping me in the early stage of my PhD. I also want to thank our secretary, Ms. Elena Zigutkin and Ms. Christine Bösler-Uzman for their patience and willingness for helping me at the university administration. Thanks to my Chinese friends who had been in Germany, Dr. Chunlun Hong, Dr. Ge Yan, Dr. Yuanping Hai, Dr. Yuhuan Yuan, and Xizhi Huang. I would love to extremely express my appreciation to my beloved family, my father, my mother, my twin sister, and my brother-in-law. I thank you from the bottom of my heart for staying healthy, happy, and positive. For four years, I was unable to return home due to the pandemic, missed too much happiness, and felt too much guilty that I could not be with you. But you always give me the greatest hope, inner strength, and faith. I believe that we can finally be reunited. I would thank my husband Feng Jiang, who accompanies me to Germany and also obtained his doctoral degree in the summer of this year. Thanks for his love. His enthusiasm for life makes me realize how wonderful it is and makes me determined to be the best I can be. We enjoyed life in Germany, we also traveled to 10 countries across Europe, Africa, and North America, and we preserved plenty of precious memories. Last but not least, I would like to thank time, for witnessing my growth, and making me brave, strong, and committed to my ideals. Min Zhou 周敏 Mainz, in July 2023 III Abstract Resisting cell death is one of the hallmarks of carcinogenesis, especially when it is relevant to tumor proliferation pathways and oncogene overexpression. Deregulation of cell death mechanisms (e.g., anti-apoptotic modalities) during cancer therapy also contributes to tumors that display multidrug resistance (MDR), which is a challenging obstacle for successful treatments. The aim of this thesis is two parts: 1) which novel cell death mechanisms that bypass apoptosis can be induced by three synthetic compounds (cardenolide derivative, palladium (II) complexes), 2) how does a natural product derivative of 1,2,4-oxadiazole inhibit the oncogene c-MYC and promotes apoptosis. These investigations in both drug-sensitive and -resistant cancer cells would be a promising way to overcome tumor resistance to apoptosis. The compound ZINC253504760 showed potent cytotoxicity to different drug-sensitive and multidrug-resistant cell lines, which showed the most lethal effect in CCRF-CEM cells. Transcriptome-wide mRNA expression profiling and pathway analysis pointed out a canonical pathway involved in G2/M phase cell cycle arrest, which was predicted to be linked with MEK1/2 and ERK in the network analysis. Afterward, G2/M phase arrest was measured by flow cytometry in a time- and concentration-dependent manner, which was supported by the microtubule-destabilizing observation using fluorescence microscopy. Interestingly, apoptosis was not the predominant mode of cell death observed by flow cytometry, nor was it autophagy. Using western blotting, ZINC253504760 induced parthanatos accompanied by p-histone H2A.X, PARP, and PAR accumulation, leading to the translocation of AIF from the cytoplasm to the nucleus. The dissipation of the mitochondrial membrane potential, AIF translocation, and DNA damage were further confirmed by flow cytometry, immunofluorescence microscopy, and alkaline single cell electrophoresis. Moreover, ZINC253504760 inhibited the phosphorylation of MEK1/2, which further affected the activation of ERK. Molecular docking also showed ZINC253504760 as an ATP competitive kinase inhibitor bound to the phosphorylation sites of MEK1 and MEK2. Their binding was confirmed in microscale thermophoresis (MST). Therefore, ZINC253504760 induced parthanatos as a major mode of cell death and downregulated MEK1/2 phosphorylation. Palladium (II) complexes J4 and J6 induced parthanatos-type cell death in CCRF-CEM and its multidrug-resistant CEM/ADR5000 cells. The biomarker p-histone H2A.X, PARP, and PAR were clearly hyperactivated by J4 and J6, followed by AIF translocated into the nucleus, mitochondrial membrane potential dysfunction, and large-scale DNA fragmentation. Furthermore, J4 and J6 specifically suppressed leukemia cells, but not healthy leukocytes. IV Therefore, J4 and J6 triggered parthanatos for cell death, which offers the prospect of more effective treatment of malignancies with drug resistance that are hampered by the inability to cause cell death. The 1,2,4-oxadiazoles derivative ZINC15675948 showed profound cytotoxicity towards CCRF-CEM and MDA-MB-231-pcDNA3 cells, while it was cross-resistant in P-glycoprotein- overexpressing CEM/ADR5000 cells and BCRP-overexpressing MDA-MB-BCRP cells. MST and molecular docking revealed a strong binding of ZINC15675948 to c-MYC with an interaction close to the c-MYC/MAX interface. C-MYC reporter assay and western blotting showed a downregulation of c-MYC by ZINC15675948 in a concentration-dependent manner. Furthermore, ZINC15675948 induced apoptosis and DNA damage in leukemia and breast cancer cell lines. Autophagy induction was only observed in CCRF-CEM cells. ZINC15675948 also caused G2/M phase or S phase arrest in CCRF-CEM cells or MDA-MB-231-pcDNA3 cells, accompanied by the downregulation of CDK1 or p-CDK2 in western blotting. Additionally, the microarray profiling of MDA-MB-231-pcDNA3 cells revealed an involvement of ubiquitination toward c-MYC, indicated by the upregulation of a novel ubiquitin ligase (ELL2) in the absence of c-MYC expression. Therefore, ZINC15675948 promoted apoptosis in c- MYC-driven cancers by targeting c-MYC. V Zusammenfassung Die Resistenz gegen den Zelltod ist eines der Kennzeichen der Karzinogenese, insbesondere im Zusammenhang mit der Tumorproliferation und der Überexpression von Onkogenen. Die Deregulierung von Zelltod-Mechanismen (z.B. anti-apoptotische Modalitäten) während der Krebstherapie trägt auch dazu bei, dass Tumore eine Multidrug-Resistenz (MDR) aufweisen, was eine Herausforderung für eine erfolgreiche Behandlung darstellt. Das Ziel dieser Arbeit besteht aus zwei Teilen: 1) ob und welche neuartigen Zelltodmechanismen, welche die Apoptose umgehen, durch drei synthetische Verbindungen (Cardenolid-Derivat, Palladium(II)- Komplexe) induziert werden können, 2) wie ein Derivat des Naturproduktes 1,2,4-Oxadiazol das Onkogen c-MYC hemmt und die Apoptose fördert. Diese Untersuchungen sowohl an medikamentenempfindlichen als auch -resistenten Krebszellen wären ein vielversprechender Weg zur Überwindung der Tumorresistenz gegen Apoptose. ZINC253504760 zeigte eine starke Zytotoxizität bei verschiedenen medikamentenempfindlichen und -resistenten Zelllinien, wobei die stärkste Wirkung bei CCRF-CEM-Zellen zu beobachten war. Die transkriptomweite mRNA- Expressionsprofilierung und die Analyse der Signalwege wiesen auf einen kanonischen Signalweg hin, der an der Zellzyklus-Arretierung in der G2/M-Phase beteiligt ist und mit MEK1/2 und ERK in der Netzwerk-Analyse verbunden war. Anschließend wurde der G2/M- Phasenstillstand mittels Durchflusszytometrie zeit- und konzentrationsabhängig gemessen, was durch die Beobachtung der Mikrotubuli-Destabilisierung mittels Fluoreszenzmikroskopie unterstützt wurde. Interessanterweise war Apoptose nicht die vorherrschende Form des Zelltods, die mittels Durchflusszytometrie beobachtet wurde, ebenso wenig wie Autophagie. Im Western Blot induzierte ZINC253504760 Parthanatos, begleitet von einer schnellen Akkumulation von p-Histon H2A.X, PARP und PAR, was zu einer Verlagerung von AIF aus dem Zytoplasma in den Zellkern führte. Der Abbau des mitochondrialen Membranpotenzials, die AIF- Translokation und die DNA-Schäden wurden durch Durchflusszytometrie, Immunfluoreszenzmikroskopie und alkalische Einzelzellenelektrophorese bestätigt. Darüber hinaus hemmte ZINC253504760 die Phosphorylierung von MEK1/2, was wiederum die Aktivierung von ERK beeinträchtigte. Molekulares Docking zeigte außerdem, dass ZINC253504760 als ATP-kompetitiver Kinaseinhibitor an die Phosphorylierungsstellen von MEK1 und MEK2 bindet, was durch mikroskalige Thermophorese (MST) bestätigt wurde. Daher induzierte ZINC253504760 Pathanatos als Hauptmodus des Zelltods und regulierte die MEK1/2-Phosphorylierung herunter. VI Die Palladium(II)-Komplexe J4 und J6 lösten in CCRF-CEM- und multiresistenten CEM/ADR5000-Zellen einen parthanatischen Zelltod aus. Der Biomarker p-Histon H2A.X, PARP und PAR wurden durch J4 und J6 eindeutig hyperaktiviert, gefolgt von einer Verlagerung von AIF in den Zellkern, einer Störung des mitochondrialen Membranpotenzials und einer großflächigen DNA-Fragmentierung. Darüber hinaus unterdrücken J4 und J6 spezifisch Leukämiezellen, jedoch nicht gesunde Leukozyten. Daher lösten J4 und J6 Parthanatos für den Zelltod aus, was die Aussicht auf eine wirksamere Behandlung bösartiger Erkrankungen mit Arzneimittelresistenz bietet, die durch die Unfähigkeit, den Zelltod herbeizuführen, behindert wird. Das 1,2,4-Oxadiazol-Derivat ZINC15675948 zeigte eine ausgeprägte Zytotoxizität in CCRF- CEM- und MDA-MB-231-pcDNA3-Zellen, während es in P-Glykoprotein- überexprimierenden CEM/ADR5000-Zellen und BCRP-überexprimierenden MDA-MB- BCRP-Zellen kreuzresistent war. MST und molekulares Docking zeigten eine starke Bindung von ZINC15675948 an c-MYC mit einer Interaktion nahe der c-MYC/MAX-Schnittstelle. C- MYC-Reporter-Assay und Western Blotting zeigten eine konzentrationsabhängige Downregulation von c-MYC durch ZINC15675948. Darüber hinaus induzierte ZINC15675948 Apoptose und DNA-Schäden in Leukämie- und Brustkrebszelllinien. Eine Induktion der Autophagie wurde nur in CCRF-CEM-Zellen beobachtet. ZINC15675948 verursachte auch einen G2/M- oder S-Phasen-Arrest in CCRF-CEM-Zellen oder MDA-MB-231-pcDNA3- Zellen, begleitet von einer Herunterregulation von CDK1 oder p-CDK2 im Western Blotting. Darüber hinaus zeigte das Microarray-Profiling von MDA-MB-231-pcDNA3-Zellen eine Beteiligung der Ubiquitinierung von c-MYC, was durch die Hochregulierung einer neuartigen Ubiquitin-Ligase (ELL2) in Abwesenheit der c-MYC-Expression angezeigt wurde. Daher förderte ZINC15675948 die Apoptose bei c-MYC-gesteuerten Krebsarten, indem es auf c- MYC abzielte. VII Table of contents Acknowledgment ........................................................................................................................ I Abstract .................................................................................................................................... III Zusammenfassung ..................................................................................................................... V Table of contents ..................................................................................................................... VII 1 Introduction ............................................................................................................................. 1 1.1 Cancer .......................................................................................................................... 1 1.2 Cancer treatment .......................................................................................................... 3 1.2.1 Classical chemotherapy ............................................................................................. 4 1.2.2 Targeted therapy ........................................................................................................ 6 1.3 Mechanisms of drug resistance ........................................................................................ 9 1.3.1 Multidrug resistance by ABC transporters ................................................................ 9 1.3.2 Apoptosis resistance ................................................................................................ 11 1.4 Targeting of cell death pathways for cancer therapy ...................................................... 13 1.4.1 Apoptosis ................................................................................................................. 14 1.4.2 Parthanatos .............................................................................................................. 17 1.4.3 Autophagy and other modes of cell death ............................................................... 18 1.5 The relevance of MEK1/2 for apoptosis in cancer therapy ............................................ 20 1.5.1 The RAS-RAF-MEK-ERK (MAPK) cascade in cancer ......................................... 20 1.5.2 MEK1/2 as targets for cancer treatment .................................................................. 22 1.6 The relevance of c-MYC for apoptosis in cancer therapy .............................................. 24 1.6.1 the c-MYC oncogene in cancer ............................................................................... 24 1.6.2 c-MYC as a target for cancer treatment .................................................................. 27 1.7 Parthanatos-inducing compounds in cancer cells ........................................................... 28 2 Objective of the thesis ........................................................................................................... 32 3 Results and discussion ........................................................................................................... 33 VIII 3.1 Cardiac glycoside (ZINC253504760), a novel MEK1/2 inhibitor inducing a state-of-art parthanatic cell death and G2/M arrest in leukemia cells .................................................... 33 3.2 Palladium(II) complexes induced parthanatos-type cell death in CCRF-CEM leukemia and its multidrug-resistant cells ............................................................................................ 34 3.3 1,2,4-oxadiazole derivative (ZINC15675948) as a novel c-MYC inhibitor by inducing DNA damage, cell cycle arrest, and apoptosis in leukemia and breast cancer cells ............ 35 4 Conclusion ............................................................................................................................. 37 5 Reference ............................................................................................................................... 38 6. Appendices (published articles) ........................................................................................... 47 Curriculum Vitae of Min Zhou ................................................................................................. 48 1 1 Introduction 1.1 Cancer Cancer is a disease caused by cells dividing uncontrollably and spreading into surrounding tissues. In about two-thirds of the world’s countries, cancer is the first or second leading cause of mortality before the age of 70 (Figure 1). According to the World Health Organization (WHO), the number of new cases globally was estimated to be 19.3 million, with 10 million cancer death in 2020 (1). Approximately one-half of all cases and 58.3% of cancer death occur in Asia, which reside 60% of the world’s population, followed by Europe and America. Female breast cancer (11.7 %), lung cancer (11.4 %), and colon cancer (10.0 %) are the top diagnosed cancer types. Lung cancer (18%) accounts for the highest cancer mortality (1). Some cancers are commonly diagnosed in children and adolescents, including leukemia, brain tumor, and lymphoma. Figure 1. National ranking of cancer as a leading cause of death for people under 70 in 2019. (1) Cancer has more than 100 different types, they can be broadly divided into six groups from a histological perspective: carcinoma, sarcoma, leukemia, lymphoma, myeloma, and mixed types (2). Carcinoma is a form of cancer that develops from epithelial cells, which are the cells that constitute the tissue that covers the surface of organs, glands, and other structures in the body. Carcinoma accounts for 80-90% of all cancer cases (3). Sarcoma refers to cancer of supporting 2 and connective tissues, such as bones, muscles, tendons, cartilage, and fat (4). Leukemias (“blood cancers”) are cancers developing in the bone marrow that are often associated with rapidly multiplying immature white blood cells. As a result, the production and function of normal blood cells, including white blood cells, red blood cells, and platelets are hampered. The patient is frequently at risk for infection and fatigue (5). Lymphomas originate in the glands or nodes of the lymphatic system, which is primarily responsible for cleaning bodily fluids and producing white blood cells that fight infections (6). Multiple myeloma is characterized by aberrant clonal plasma cells that possess the potential to expand out of control in the bone marrow. It causes anemia, hypercalcemia, acute kidney injury, and destructive osseous bone lesions (7). The mixed type may be within one or from different categories, such as carcinosarcoma and adenoid cystic carcinoma. Tumor initiation and progression are a process that normal cells transform into a metastatic cancer cell. Two decades ago, the landmark paper has been highlighted six enabling features (“hallmarks”) concerning the acquired functional capabilities that allow cancer cells to uncontrollably proliferate and survive: sustaining proliferative signaling, resisting cell death, inducing angiogenesis, enabling replicative immortality, evading growth suppressors, and activating invasion and metastasis (8). Since then, it has been evident that tumor is also contributed by reprogramming cellular energy metabolism and avoiding immune destruction. Meanwhile, there are probably two enabling characteristics that facilitate their acquisition for tumorigenesis: genomic instability and tumor-promoting inflammation (9). Furthermore, the latest study proposed four possibly emerging hallmarks and enabling characteristics, involving unlocking phenotypic plasticity, non-mutational epigenetic reprogramming, polymorphic microbiomes, and senescent cells, which could be incorporated into the hallmarks of cancer after further validation (10). The canonical and prospective new additions to the ‘‘Hallmarks of cancer’’ are shown in Figure 2. These aberrations have been recognized as essential principle in the treatment of cancer. 3 Figure 2. Hallmarks of cancer with new additions. Canonical and prospective new additions to the ‘‘Hallmarks of cancer’’. (10) 1.2 Cancer treatment The treatment of cancer depends on the cancer types and stages, the patient’s health status, and individual preferences. Currently, the main strategies of cancer treatment include surgery, radiation therapy, chemotherapy, immunotherapy, targeted therapy, and hormone therapy. Cancer surgery involves the removal of the tumor and surrounding tissues. It is the earliest oncological discipline, with descriptions of the removal of breast tumors dating back to ancient Egypt (1600 BC). The surgery for local tumor excision progresses commonly in breast, gastric, colorectal, esophageal, and neurological cancers (11). Surgery also provides prevention of tumorigenesis, including screening-associated surgery for premalignant conditions, and prophylactic surgery in high-risk people with heritable genetic disorders (12). Radiation is a physical agent and aims to target aberrant cancer cells with the maximum radiation dose while minimizing exposure to healthy cells (13). The radiation used is referred to as ionizing radiation. It deposits energy to damage the DNA of the cells and thus block further cell division and lead to cell death (14). Apoptosis and mitotic cell death account for the major types of cell death induced by radiation therapy to exhibit therapeutic effects (13). Immunotherapy provides passive or active immunity to target tumors by reactivating and harnessing the immune system (15). The passive immunotherapy applies effector molecules to direct attack tumor cells, such 4 as antibodies and genetically engineered T cells (chimeric antigen receptor [CAR]-T). The active immunotherapy is to enhance immune system activation through the modulation of endogenous regulation (16). Hormonal therapy is the administration of exogenous hormones that manipulate the endocrine system in patients with hormone-dependent cancer. Hormones are chemical messengers released by certain endocrine system organs to act on gene expression and cell proliferation (17). A high level of specific hormone therefore raises the probability of neoplastic transformation. Tumors caused by hormonal dysregulation include prostate, endometrial, ovarian and breast cancer, and uterine sarcomas (18). The following paragraphs will focus on classical chemotherapy and targeted chemotherapy. 1.2.1 Classical chemotherapy In the early 1900s, the use of chemicals to treat disease was defined as “chemotherapy” by the German chemist Paul Ehrlich. During World War II, an accidental leakage of sulfur mustard from a bombed ship led to the discovery that both bone marrow and lymph nodes had significantly declined in those personnel exposed to the mustard gas. Then, the management of lymphomas by nitrogen mustard observed remarkable regression of lymphoid tumors, which brought great initiation to the development of chemotherapy for cancer (19-21). Currently, chemotherapy with cytotoxic anticancer agents is the mainstay of therapy in cancer treatment. Most chemotherapy drugs act by killing or inhibiting the growth of cancer cells through an interaction with DNA or preventing chromosomal replication, this further prevents cell division and leads to programmed cell death (apoptosis) (22). However, due to the poor selectivity for malignant cells over normal tissue, cytotoxic chemotherapy agents cause a series of severe side effects, such as gastrointestinal toxicity, ototoxicity, hepatotoxicity, and nephrotoxicity (23). Chemotherapy drugs are classified into different types: 1) DNA-binding substance: DNA is the most extensive target of anti-cancer compounds. When exogenous chemicals that bind to DNA alter the structure of nucleic acid, which in turn affects DNA-interacting enzymes or transcription factors, disturbing cell cycle and affecting DNA replication, cell division, and cell proliferation (24). The types of DNA-binding substances are as below: a) Topoisomerase inhibitors: DNA topoisomerases (TOPO) are crucial enzymes that catalyze the modification of DNA topology for DNA replication. Topoisomerase activity is particularly elevated in rapidly dividing cells, such as cancer cells. There are two types of topoisomerases: 5 type I, which breaks single-stranded DNA, and type II, which cuts double-stranded DNA. Various anticancer drugs act as TOPO poison inhibitors, trapping covalent complexes of human TOPOs, leading to DNA damage and cell death (25). The DNA topoisomerase I inhibitors (e.g., camptothecin, topotecan, and irinotecan): alternating the DNA topology by breaking the phosphodiester bonds between nucleotides in DNA strands is the fundamental mechanism for this class of anticancer drugs. Campothecin was the first compound demonstrated to target topoisomerase I. It is a natural product that was isolated from the bark and stem of Camptotheca acuminate (‘tree of joy’). Clinical-approved topotecan and irinotecan are analogs of camptothecin. DNA topoisomerase I inhibitors are commonly applied in lung cancers, however, they exhibit notable dose-limiting toxicity, and cancer cells develop drug resistance (26). The DNA topoisomerase II inhibitors include anthracyclines (e.g., doxorubicin, daunorubicin, epirubicin, and idarubicin) and epipodophyllotoxins (e.g., etoposide, etopophos, and teniposide). Cutting both DNA stands is the main mechanism by which DNA topoisomerase II inhibitors disrupt DNA topology. Especially, anthracyclines involve in binding to DNA by inserting a polycyclic aromatic moiety between base pairs, at the same time extending and unwinding the helix. DNA topoisomerase II inhibitors are effective against leukemia, lymphomas, sarcomas, and breast carcinoma. However, they were hampered by the development of drug resistance and cardiotoxicity. Treatment with DNA topoisomerase II inhibitors can cause secondary malignancies such as acute myeloid leukemia (27, 28). b) Alkylating agents (e.g., chlorambucil, melphalan, cyclophosphamide, and ifosfamide): form covalent bond structures with DNA and cause single-strand or double-strand DNA breaks. These drugs are extensively used in leukemias, lymphomas, and solid tumors. Most alkylating agents cause gastrointestinal side effects and dose-limiting bone marrow toxicity (29). c) Platinum (e.g., cisplatin, carboplatin, and oxaliplatin) The mechanism of action of cisplatin is mediated by its binding with DNA to generate DNA lesions and block DNA synthesis, which suppresses RNA transcription and arrests the cell cycle, therefore inducing the activation of apoptosis (30). Cisplatin has been widely applied as adjuvant therapy in the treatment of a spectrum of cancers, such as ovarian, cervix, bladder, small and non-small cell lung cancers, Hodgkin’s and non-Hodgkin’s lymphomas, and melanoma (31). The major toxicities of cisplatin include general cell-damaging effects, nephrotoxicity, hepatotoxicity, neurotoxicity, and hearing loss (32). Despite emerging newer platinum-based complexes, only carboplatin and oxaliplatin received approval for clinical practice. 6 2) Microtubule inhibitors: this group is referred to as mitotic poisons, because they interfere with microtubule dynamics, leading to chromosomal dysregulation, aberrant spindle formation, and ultimately cell dead due to mitotic failure. Microtubule inhibitors are categorized into microtubule-destabilizing agents (Vinca alkaloids, e.g., vinblastine and vincristine) and microtubule-stabilizing agents (Taxanes, e.g., paclitaxel and docetaxel). However, they are associated with cardiotoxicity (33). 3) Antimetabolites: The early discovery by Sidney Farber found that aminopterin helped acute lymphoblastic leukemia relief in children and promoted the development of the drug class of antimetabolites (34). Antimetabolites are small molecules structurally similar to nucleotide metabolites, they interfere with critical enzymes that are used for DNA synthesis, thereby causing DNA damage, and induction of apoptosis. Notable examples include folate antagonists (e.g., methotrexate), purine antagonists (e.g., thioguanine, 6-TG), pyrimidine antagonists (e.g., fluorouracil, 5-FU), and other nucleoside analogs (e.g., gemcitabine and cytarabine) (35). 4) Antihormones: some breast cancers are known as hormone-sensitive (or hormone-dependent) breast cancers, are driven by estrogen and progesterone. Various antihormones therapies have been approved by FDA, such as tamoxifen treated in estrogen-positive early-stage breast cancer of premenopausal women, and aromatase inhibitors (e.g., letrozole) used in postmenopausal women. Hot flashes and night sweats are common sides effects (36). 1.2.2 Targeted therapy Targeted therapy refers to blocking specific biological transduction pathways or cancer proteins that are involved in tumor growth, progression, and metastasis. The molecular targets (e.g., receptors, growth factors, kinase cascades, cell cycle proteins, or modulators of apoptosis) present in healthy tissue but are mutated or overexpressed in cancer (37). The aim of targeted therapy is to inhibit one of the capabilities in hallmarks of cancer, impair uncontrolled tumor growth and progression, lead to the death of cancer cells, and avoid undesirable side effects (9). The outstanding clinical success of targeted therapy is witnessed by the Food and Drug Administration (FDA), which has approved a growing number of drugs in the oncology area in recent two decades. The types of targeted therapy can be classified into 1) small molecules; 2) monoclonal antibodies; 3) cancer vaccines; 4) gene therapy. 1) Small molecules 7 Small molecules are substances with low molecular weight (less than 800 Dalton) that can access cells to interfere with signaling pathways and act on targets inside the cells. They have ‘‘ib’’ as the suffix in the name representing the molecule has inhibitory properties (38). Encouraged by the approval of the first small molecule tyrosine kinase inhibitor (TKI) in 2001, imatinib, involved in the pathogenesis of chronic myeloid leukemia (CML), small molecules for targeted therapy are moving quickly from bench to bedside for both solid tumors and hematological malignancies (39, 40) (Figure 3). Currently, there are 68 FDA-approved therapeutic agents that target protein kinase, including receptor tyrosine kinase inhibitors (e.g., the epidermal growth factor receptor, EGFR; vascular endothelial growth factor, VEGF; platelet-derived growth factor, PDGF), serine/threonine kinase inhibitors (e.g., RAF-MEK- ERK; PI3K/AKT/mTOR; cyclin-dependent kinases, CDK;) (41). Furthermore, epigenetic inhibitors (e.g., histone deacetylase, HDACs), non-receptor tyrosine kinase inhibitors (e.g., Bcr- abl1; BTK; JAK), Poly (ADP-ribose) polymerase (PARP) inhibitors have been obtained approval (39). There is a substantial pipeline of valuable targets in the development, such as c- MYC. Two attractive targets MEK1/2 and c-MYC for cancer therapy are shown in details in section 1.5.2 and 1.6.2. Figure 3. Timetable for approval of small molecule targeted anti-cancer drugs. (39) 8 2) Monoclonal antibodies B-lymphocytes become active when a foreign substance enters the body, and antibodies take place in the recognition of epitope regions on the foreign substance (antigen). Monoclonal antibodies (mAbs) are produced in response to a single epitope (42). The use of mAbs for cancer therapy has achieved considerable success in recent years. mAbs specifically target extracellular proteins because they are generally too large to fit inside the cells. After attaching the cells, mAbs can exert their effects either directly or indirectly. The direct mechanism refers to the binding of mAbs to an antigen, cell receptor, or membrane-bound protein, allowing them to exert effects on the specific target to cause cell death (43). While the indirect mechanism refers to after stimulation via the binding of mAbs to cancer-specific antigens, the immune system can respond and further attack cancerous cells. The indirect mechanism is widely applied in immunotherapy (44). Furthermore, immunotherapeutic mAbs depend on whether or not they carry drugs or radioactive substances, can be categorized into self-acting, non- conjugated mAbs (e.g., transtuzumab and alemtuzumab), and conjugated mAbs (e.g., gemtuyumab otogamicin). Conjugated mAbs have been shown to improve drug pharmacokinetic profiles by decreasing volume of distribution and prolonging the distribution and elimination phases (45). Therefore, the application of mAbs is a promising strategy and also lies at the heart of personalized medicine. 3) Cancer vaccines Cancer vaccines are designed to specifically recognize aberrantly expressed tumor-associated antigens (TAAs), tumor-specific antigens (TSAs), peptides, and antigenic epitopes through CD4+ T cells and CD8+ T cells. This further stimulates cytotoxic T lymphocytes and helps T cell-mediated immune response against TAAs and TSAs (46, 47). Common therapeutic cancer vaccines include hepatitis C virus (HCV), viral proteins (e.g., human papillomavirus (HPV)), and oncofetal antigens (e.g., carcinoembryonic antigen (CEA)). Several cancer vaccines targeting oncoproteins (e.g., HER2) are in clinical trials. However, given the intricacy of cancer immunology and the optimal vaccine design, clinical translation of cancer vaccine has been challenging (37, 48). 4) Gene therapy Gene therapy is a treatment of genetic disease by transferring genetic material into the cells of the patients. It acts through one of three mechanisms: (i) blocking the expression of a target gene, (ii) enabling expression of the transferred gene, (iii) modifying a target gene (49). Gene 9 therapy drugs were biological medicines that are provided as nucleic acids, lipid complexes, viruses, or genetically engineered micro-organisms. There are two dozen of gene therapies officially licensed so far as clinical used drugs (50). 1.3 Mechanisms of drug resistance In response to therapies, cancer cells modify their dependence on an original particularly hallmark capability and increase dependent on another, this represents drug resistance. Innate or acquired resistance remains a major impediment to effective chemotherapy of cancer (9). Multidrug resistance (MDR) is characterized by drug resistance that develops not only to a single chemotherapeutic drug, but also occurs in a broad range of drugs with different chemical structures and various modes of action (51). Chemotherapy failure has been attributed to a number of reasons. Known mechanisms with clinical relevance represent alternations in intracellular drug concentration (drug efflux/influx pump), inhibition of cell death (apoptosis suppression), enhance DNA repair and gene amplification, change in drug metabolism, and epigenetic alternations (52, 53). This thesis will focus on drug efflux and deregulation of cell death, which are discussed in detail below. 1.3.1 Multidrug resistance by ABC transporters One of the major causes of MDR is the enhanced overexpression of transmembrane efflux pumps such as ATP-binding cassette (ABC) transporters. ABC transporters are a family of membrane proteins that mediate the ATP-driven transport process. They are found in all domains of life, including bacteria, archaea, fungi, plants, as well as humans (54). Forty-nine human ABC transporters have been found so far and divided into 7 subfamilies (55). The most of molecular architecture of ABC transporters is similar. They consist of a pair of conserved cytoplasmic domains refer as nucleotide-binding domains (NBDs). Substrates can pass through the membrane’s lipid bilayer, either be imported into or exported out of the cytoplasm due to the NBD’s hydrolysis of ATP, which drives the conformational changes in the associated transmembrane domains (TMDs). Some ABC transporters, termed half-transporters, are composed of only one TMD and NBD (56). ABC transporters are involved in fundamental cellular functions, especially regulating levels of biochemical substances to maintain cellular homeostasis and protect issues from xenobiotics (57). 10 Overexpression of ABC transporters plays an important role in MDR-cells and confers resistance to anticancer drugs. P-glycoprotein (P-gp, gene symbol ABCB1), multidrug resistance-associated protein 1 (MRP1, gene symbol ABCC1), and breast cancer resistance protein (BCRP, gene symbol ABCG2) are primary determinants of drug resistance in cancer cells. These drug transporters engage in active efflux of drugs and drug conjugates (58) . An example of a substrate is transported by P-gp is shown in Figure 4. Figure 4. Model of substrate transport by P-gp. (A) From the outside of the cell to the inside leaflet, the substrate (magenta) partitions into the bilayer and enters the drug-binding site (cyan spheres). (B) ATP (yellow) binds to the NBDs and results in a large conformational change, bringing the substrate and drug-binding site to the extracellular space. (59) P-gp was the first member of ABC transporter identified in cancer. P-glycoprotein-expressing organs (e.g., apical membrane of hepatocytes, kidney proximal tubules, endothelial cells of the blood-brain barrier) are crucial for absorption, distribution, metabolization, and excretion (ADME) pharmacological drugs, which also prevent the accumulation of harmful, carcinogenic compounds from tumorigenesis (55). However, a limited phase I trial reported that enrolled patients with sarcoma pulmonary metastases showed a 3-15-fold rapid increase in ABCB1 gene expression after around 1 h of doxorubicin treatment (60). In addition, in vitro study has indicated P-gp overexpressing CEM/ADR5000 cells were cross-resistant (6.9 ⁓ 1036-fold) to a number of unrelated anticancer drugs, such as anthracyclines (e.g., doxorubicin idarubicin, epirubicin), Vinca alkaloids (e.g., vincristine, vinblastine), taxanes (e.g., docetaxel, paclitaxel), epipodophyllotoxins (e.g., etoposide) (61). A great deal of effort has been expended to identify 11 potent and selective P-gp inhibitors to overcome multidrug resistance. The first generation of P-gp inhibitors such as verapamil and deverapamil were not specifically for inhibiting P-gp while with a low affinity. The second generation contained more specific inhibitors with less side effect, such as biricodar and valspodar. The third generation is composed of more potent and selective inhibitors, such as tariquidar and elacridar. The fourth generation obtained with multiple strategies, such as natural products with their derivates, peptides and lipids (62). The role of BCRP in drug disposition is highly resembles P-gp in tissue expression and distribution. BCRP substrates include broadly anticancer agents such as camptothecin derivates, methotrexate, and flavopiridol. Notably, some TKIs including imatinib, gefitinib, and nilotinib are BCRP substrates (63). Many BCRP inhibitors with various chemical structures have been found. The first part is P-gp inhibitors, which are also excellent BCRP inhibitors. The other outstanding BCRP inhibitors include tamoxifen, novobiocin, tryprostatin, and dietary flavonoids such as chrysin (64). 1.3.2 Apoptosis resistance An underlying hallmark of cancers is resisting cell death. Dysregulated apoptotic signaling enables cancer cells to escape the fate of death, resulting in uncontrolled proliferation, tumor survival, therapeutic resistance, and cancer recurrence (65). To date, apoptosis is still the most common form of cell death induced by radio- and chemotherapy in cancer cells. However, multiple molecular mechanisms can be altered leading to the reduction of apoptosis or acquisition of apoptosis resistance (Figure 5), including 1) disrupted balance between proapoptotic and anti-apoptotic proteins; 2) impaired 53 function; 3) overexpression of IAP proteins; 4) reduced caspase function; 5) impaired death receptor signaling (66). 12 Figure 5. Mechanisms contribute to deregulation of apoptosis. (67) Table 1. Bcl-2 members classification based on function and the Bcl-2 homology (BH) domains (68). Function Bcl-2 homology (BH) domain Protein Anti-apoptotic proteins Contain four BH domains Bcl-2, Bcl-XL, Bcl-w, Mcl-1, A1/Bfl-1 and Bcl-B/Bcl2L10 Pro-apoptotic proteins Restrict to the BH3 domain Bid, Bim, Puma, Noxa, Bad, Bmf, Bik and Hrk Pro-apoptotic proteins Contain four BH domains Bax, Bak, and Bok/Mtd First of all, the disruption of balance between proapoptotic and antiapoptotic proteins is a powerful mechanism of tumor growth and drug resistance. Bcl-2 family proteins consist of proapoptotic and antiapoptotic proteins that play a vital role in the regulation of apoptosis. The three groups of Bcl-2 family members are shown in Table 1. This unbalance can be one or more anti-apoptotic proteins may be overexpressed while one or more pro-apoptotic proteins may be downregulated, or both may be present (67). Secondly, the p53 protein is a well-known tumor suppressor protein encoded by the tumor suppressor gene TP53. The most conserved function of p53 is the induction of apoptosis in 13 response to cellular stress. A set of pro-apoptotic genes (e.g., Bax, Bid, and PUMA) and anti- apoptotic gene Bcl-XL of Bcl-2 family are p53 targets (69-71). However, mutated p53 leads to abnormal activity of p53 and inefficient apoptosis induction (66). Furthermore, dysregulated expression of a group of the inhibitor of apoptosis proteins (IAPs) that are negative regulators of caspase function has been correlated to many cancers. IAPs can bind and inhibit caspase 3, -7, or -9 by promoting their degradation or keeping caspases away from their substrates (72). X-linked IAP (XIAP) is the most widely characterized IAPs and is regarded as the most potent endogenous caspase inhibitor (73). Finally, as section 1.4.1 will show, caspases and death receptor signaling are important plays in initiating and executing apoptosis. Therefore, it is reasonable to believe that reduced expression of caspase and impaired death receptor signaling cannot activate apoptosis. 1.4 Targeting of cell death pathways for cancer therapy Cell death is an essential physiological process for human health to maintain homeostasis and response to stress (74). The homeostatic balance between survival and death is critical. Aberrant (excessive or deficient) cell death underpins multiple pathologies, including cancer, neurodegeneration, autoimmunity, and injury (75). Tumor cells gain the ability to avoid cell death pathway, which functions as a protective mechanism in normal cells to remove damaged cells. As a consequence, a population of death-resistant cells with accumulating genetic and epigenetic errors contributes to tumorigenesis (76). Thus, cell death has risen in importance as targets for the development of cancer therapies. Historically, cell death based on morphological alternations is classified into three different forms: (1) type I cell death/apoptosis, (2) type II cell death/autophagy, and (3) type III cell death/necrosis (75). As various types of cell death sprung up, the Nomenclature Committee on Cell Death in 2018 proposed an updated classification of more than 10 types of cell death with an emphasis on the molecular mechanisms involved in the initiation, execution, and propagation (77). Cell death can be categorized into accidental cell death (ACD) and regulated cell death (RCD). ACD is an uncontrolled process of cell death, in which cells are exposed to server physical (e.g., high temperature or pressure), chemical (e.g., extreme variations in pH), or mechanical damages. RCD involves genetically regulated cell death that maintains the stability of the internal environment in an orderly and autonomous manner. When RCD occurs as a physiological program without any external environment disturbance, it is referred to as 14 programmed cell death (PCD). PCD can be further classified into apoptotic cell death and non- apoptotic cell death. The classification of cell death is shown in Figure 6. Figure 6. Cell death classification. (78) 1.4.1 Apoptosis Apoptosis is the most widely studied form of cell death. It is a highly regulated and conserved process with multicellular organisms. The term "apoptosis" was proposed by Kerr and colleagues in 1972 to describe morphological features detected as cells were eliminated during embryonic development (79). From a morphological perspective, apoptotic cells show characteristics including membrane blebbing, cell shrinkage, loss of cytoplasmic organelle positional organization, membrane exposure of phosphatidylserine (PS) on the extracellular side, DNA condensation and fragmentation, and formation of apoptotic bodies (78, 80). They are mechanically induced by the activation of a set of cysteine-aspartic proteases known as caspase. To date, two types of caspases have been defined, the initiator caspase and the executioner caspase. Initiator caspases (caspase-2, -8, -9, and -10) are responsible for initiating caspase activation cascades. Effector caspases (caspase-3, -6, and -7) are in charge of the actual breakdown of the cell by cleaving cellular substrates (81). Furthermore, apoptosis is regulated by the components of the Bcl-2 protein family (see above 1.3.2). There are two major apoptosis pathways: the intrinsic or mitochondria pathway and the extrinsic or death receptor pathway (82). The intrinsic and extrinsic pathways are shown in Figure 7. 15 Figure 7. Apoptosis signaling pathways. (82) 1) Intrinsic apoptosis pathway: This pathway is initiated by the cell when it detects damage via numerous intracellular sensors. When cytotoxic stimuli activate proapoptotic molecules (BAX and BAK) to change the mitochondrial outer membrane permeabilization (MOMP), it caused subsequent release of cytochrome c from mitochondria to cytoplasm and binds to APAF-1, further triggering apoptosome to activate the formation of caspase 9, and subsequently a cascade of effector caspases (74). 2) Extrinsic apoptosis pathway Death receptors are members of the tumor necrosis factor (TNF) receptor gene superfamily. They are characterized by a cytoplasmic region known as the “death domain’’ (FADD), which plays a vital part in transmitting the death signal from the cell’s surface to intracellular signaling pathways (82). The best-studied death receptors are CD95 (APO-1/Fas), TNF receptor 1 (TNFR1), TNF-related apoptosis-inducing ligand-receptor 1 (TRAIL-R1), and TRAIL-R2. The corresponding death receptor ligands include CD95L (FasL), TNF, and TRAIL (83). Upon stimulation and ligation, the signals are transmitted via FADD to activate caspase 8. In ‘type I’ cells (primarily dependent on the extrinsic pathway), activated caspase 8 leads to sufficient activation of downstream effector caspases. While in ‘type II’ cells (primarily dependent on the intrinsic pathway), activator protein Bid enhances the apoptotic signal via the mitochondria, which eventually demands mitochondrial processes for cell execution (84). 16 Understanding of molecular mechanisms of apoptotic pathways and their deregulations has already provided new avenues for cancer treatment. Therapeutic approaches directly targeting the apoptosis intrinsic pathway (e.g., Bcl-2, Bcl-XL, Mcl-1 and IAP inhibitors) and extrinsic pathway (e.g., targeting death receptor agonists and targeting p53) are under development, which have brought small molecules into clinical research (85) (Figure 8). On the other hand, most of the approaches indirectly induce apoptosis, including targeting oncogenic signaling pathways (e.g., MEK inhibitors) and blocking the deregulated oncogenic downstream effectors (e.g., MYC, CDKs). However, targeting apoptotic pathways is particularly complex due to the abundance of therapeutic targets, each with particular resistance mechanism (85). Instead, much more remains to be explored about other modes of cell death, as shown in section 1.4.2 and 1.4.3. Figure 8. Therapeutic approaches targeting apoptosis pathways in cancer cells. (85) 17 1.4.2 Parthanatos Parthanatos known as a PARP-1-dependent cell death, is officially recognized as a non- apoptotic cell death. The word ‘‘parthanatos’’ was coined from Thanatos, the personification of death in Greek mythology to illustrate cell death carried on by PAR polymer, which is a product of PARP-1 activation (86). Parthanatos take part in many crucial pathogenic processes, such as Parkinson’s disease, Alzheimer’s disease, ischemia-reperfusion injury after myocardial infarction or brain ischemia. Increasing evidence recently suggests that pathanatos is involved in the regulation of cancer cells (87, 88). Parthanatos is distinctive with classical cell death pathways concerning of its biochemical and morphological characteristics. The biochemical features of parthanatos include rapid activation of PARP-1, synthesis and accumulation of PAR polymer, mitochondria depolarization, nuclear AIF translocation, and perhaps caspase activation at late stage (89). Caspase activation does not engage in parthanatos since caspase inhibitors are unable to protect cells from parthanatic cell death (90). Therefore, parthanatos is a caspase-independent type of cell death. The morphological characteristics of parthanatos include membrane breakdown, shrunken and condensation of the nucleus (87). Unlike apoptosis, parthanatos does not result in the production of apoptotic bodies or small-scale DNA fragmentation (90). Even though parthanatic cells exhibit loss of membrane integrity comparable to necrosis, but it does not cause cell swelling (91). The key steps of parthanatos are illustrated below in Figure 9. Figure 9. The pathway of parthanatos. (92) 18 PARP-1 is a fundamental member of the PARP family localized in nuclei. PARP-1 contains four major domains as shown in Figure 10. They are 1) two zinc fingers are in charge of detecting DNA breaks, 2) nuclear localization of signal (NLS) containing a caspase-3 cleavage site, 3) an automodification domain with a BRCT motif for protein-protein interactions, 4) a catalytic site has NAD+-fold (93). Hence, PARP-1 function ranges from supporting survival (e.g., DNA repair, genomic stability, and transcription) to inducing cell death (94). PARP-1 can be activated by posttranslational modifications such as phosphorylation, acetylation, or ADP ribosylation. In response to DNA damage, PARP-1 binds to PAR to various receptor proteins or on PARP-1 itself (automodification) by using NAD+. Mild genomic stress induces PARP-1 activation to repair DNA damage, whereas massive DNA damage generates excessive ADP ribosylation, PARP is overactivated, producing excess PAR (87, 95). PAR is a death signal that translocates to cytosol and induces AIF nuclear translocation (96). AIF is a flavoprotein involved in maintaining mitochondria structure and effecting cell death when it moves to the nucleus. AIF translocates from mitochondria to the nucleus has been recognized as a key factor to mediate parthanatos, where it causes cell death by chromatin condensation and significant DNA fragmentation (50 kb) (91). In addition, cytochrome c is also a mitochondria protein as introduced above, which is efficient in stimulation of apoptosis. Studies have reported that AIF release occurs ahead of that of cytochrome c upon PARP-1 activation (97). The parthanatos- inducing compounds in cancer are shown in section 1.7. Figure 10. The major domains of PARP-1. (86) 1.4.3 Autophagy and other modes of cell death Autophagy ("self-eating") is a nonselective, intracellular catabolic degradation process. The Nobel Prize in Medicine in 2016 was awarded to Dr. Yoshinori Ohsumi for the breakthrough in the discovery of autophagy mechanisms (98). There are three types of autophagy: macroautophagy, microautophagy, and chaperone-mediated autophagy, and "autophagy" 19 typically refers to macroautophagy (99). Autophagy involves cytoplasmic constituents being sequestered in double-membrane autophagosomes. These structures are then fused with lysosomes, which deliver their cargoes for degradation and recycling (100). This process is regulated by around 20 autophagy-related (ATG) proteins. Autophagy has been viewed as a survival mechanism, such as starvation adaption, organelle clearance, removal of microorganisms (101). However, autophagy has double roles in cancer. In the widespread hypothesis, autophagy performs an anticarcinogenic role in primary cells through protection from metabolic stress, further clearing the protein aggregates. While in established tumors, autophagy might provide survival benefits for tumor cells, which further contributes to acquired resistance to chemotherapy (102). Even whether to develop autophagy inducer or inhibitor is debated, recent evidence supported that inhibiting autophagy could be an effective cancer treatment (103). Pyroptosis is an inflammatory form of PCD triggered by caspase-1/4/5/11 which is activated certain inflammasomes (e.g., NLRP3), leading to the cleaving of gasdermin D (GSDMD) and activation of inactivated cytokines like IL-18 and IL-1β (104). Morphologically, the nucleus of pyroptotic cells is still intact despite chromatin condensation and DNA fragmentation. The formation of plasma membrane pores allows water influx and generates cell swelling and lysis (105). Pyroptosis has been shown to affect the proliferation, invasion, and metastasis of tumor (106). Ferroptosis is an iron- and ROS-dependent form of non-apoptotic cell death that was first described in 2012 (107). Morphological characteristics of ferroptosis involved intact cell membrane, decreased or absent mitochondrial crests, fractured mitochondrial outer membrane, and normal nuclear size and chromatin (108). Ferroptosis is initiated by the suppression of cystine glutamate antiporter (system Xc-), resulting in a reduction of glutathione (GSH) biosynthesis and phospholipid peroxidase glutathione peroxidase 4 (GPX4) inactivation. Generally, GPX4 converts harmful lipid hydroperoxides (L-OOH) to non-toxic lipid alcohols (L-OH). Inactivation of GPX4 ultimately leads to excessive lipid peroxidation, which interacted with free iron through the Fenton reaction, further forming lipid ROS and leading to cell death (109). Recently a novel FDA-approved drug (e.g., sulfasalazine) shows the induction to ferroptosis, implying ferroptosis has a tumor suppression function and a unique potential for reversing drug resistance (110-112). More recently, excess concentration of copper (Cu) causes a mitochondrial-induced cell death referred to as cuproptosis. It involves direct binding of Cu to lipoylated components of 20 tricarboxylic acid (TAC) cycle, resulting in aggregation of lipoylated protein, proteotoxic stress, and subsequent cell death (113). Interestingly, cells in a more respiratory condition may have higher levels of lipoylated enzyme expression, which lead to increased aggregation formation. The finding of cuproptosis suggests inhibiting mitochondrial respiration may be a strategy for cancer cells (114). Therefore, it is expected that additional regulated forms of nonapoptotic cell death will be discovered, which will increase the likelihood that cancer cells will die in different mechanisms, and overcome the challenges of drug resistance. 1.5 The relevance of MEK1/2 for apoptosis in cancer therapy 1.5.1 The RAS-RAF-MEK-ERK (MAPK) cascade in cancer Mitogen-activated protein kinase (MAPK) pathways relay, amplify and integrate intracellular signals from a number of cell surface receptors to response cell proliferation, development, differentiation, and apoptosis. In mammalian cells, there are four well-known MAPK cascades: the classical extracellular signal-regulated kinase (ERK) cascade, the c-Jun N-terminal kinase cascade, the p38MAPK cascade, and the ERK5 cascade. Each MAPK cascade consists of a MAPK kinase kinase (MAP3K), a MAPK kinase (MAP2K), and a MAPK (115, 116). Among the MAPK pathways, the RAS-RAF-MEK-ERK axis is the best charact characterized (Figure 11). Mechanically, signal transduction along this pathway is initiated by the binding of different ligands to growth factor receptors such as EGFR. Upon the upstream stimulation, inactivated Ras-GDP converts to activated Ras-GTP and subsequently recruits Raf to the cell membrane for dimerization and activation. Activated Raf phosphorylates and activates the downstream dual-specificity protein kinase MEK. Two Raf-MEK dimers constitute a transient tetramer, to promote MEK activation by Raf. Then, activated MEK dually phosphorylates and activates ERK (117, 118). Hundreds of proteins involved in diverse cellular response have been identified as ERK substrates and ERK-interacted partners. For example, ERK controls G1 to S phase cell cycle checkpoint for preventing the cell from abnormal replication (119). ERK also regulates several pro-survival Bcl-2 proteins such as Bcl-2, Bcl-XL, and Mcl-1 to achieve cell survival (120). 21 Figure 11. The RAS-RAF-MEK-ERK signaling pathway. (121) Deregulation of the Ras-Raf-MEK-ERK pathway has been established a thoroughly important role in human cancers. Expressing a constitutively activated mutant form of Ras (KRAS, NRAS, HRAS) and Raf (ARAF, BRAF, CRAF) have been associated with different types of cancers (122). For example, KRAS is the most frequently mutated form of RAS, which is mutated in more than 20% of all cancers, including pancreatic ductal adenocarcinoma (PDAC), non-small cell lung cancer (NCSLC), and colorectal cancer (CRC) (123). BRAF is also commonly mutated in 7%-10% of all cancer such as melanoma and ovarian cancer. Therefore, the efforts to target oncogenic RAS and RAF, have been extensively studied as promising targets for therapeutic development. However, drug resistance inevitably has been developed in patients within 6-7 months of initiating treatment, for example, in advanced melanoma treated with BRAF inhibitor (124, 125). The resistance is typically mediated through rapid recovery of the ERK pathway with several different mechanisms (126). Activation of ERK pathway has well-documented anti-apoptotic effects, which are related to the inactivation of pro-apoptotic proteins and the induction of anti-apoptotic proteins belonging to the Bcl-2 family (127, 128). For example, in transformed human mammary epithelial cells, Bcl-XL overexpression is vital for RAS-induced cancer cells and the maintenance of cancer- 22 initiating cells phenotype (129). On the contrary, suppression of inducible oncogenic HRASV12G expression causes melanoma to regress while triggering an extensive rise in apoptotic cell death (130). While applying RAS downstream MEK inhibitor has been shown promising anticancer potential. For example, the combination of Bcl-2/Bcl-XL inhibitor, navitoclax, and MEK inhibitor, G-963, resulted in dramatic enhancement of apoptosis in lung and pancreatic tumors harboring KRAS mutation (131). Hence, for maximum ERK pathway inhibition and minimizing resistance, MEK inhibitors are of growing interest as an alternative strategy to block ERK pathway, especially those cancers brought on RAS or RAF dysfunction. 1.5.2 MEK1/2 as targets for cancer treatment Compared with hot-spot RAS and RAF mutations, the occurrence of MEK mutations is sporadic (around 1% of human cancers) (132). Both MEK and ERK have two isoforms, known as MEK1/2 and ERK1/2. MEK1 and MEK2 share 80% homology, and are encoded by MAP2K1 and MAP2K2 genes, respectively. The molecular weight of MEK1 and MEK2 are 44 and 45 kDa, and they exhibit similar functions to activate ERK1 and ERK2. While MEK1 and MEK2 are the only known upstream regulators of ERK1 and ERK2, which serves as ‘‘ERK1 and ERK2 gatekeeper’’ kinases (133). Inhibiting MEK can inhibit ERK activation and its downstream process, resulting in inhibition of cell proliferation and survival. MEK1 and MEK2 consist of a small N-terminal lobe, which contains an ERK1 and ERK2 docking site, and a big C-terminal lobe, which contains the docking site for upstream activating MAP3Ks (Figure 12). At the interface between these lobes, conserved regions are important in ATP binding and hydrolysis, substrate binding, and phosphate transfer. MEK1 is activated by phosphorylation at Ser218 and Ser222, and MEK2 is activated by phosphorylation at Ser222 and Ser226 (117). Moreover, two potential phosphorylation sites are unique to MEK1. In addition to RAS, MEK1 integrates signals from integrins and is phosphorylated at Ser298 (134). MEK1 is also subjected to negative feedback from activated ERK, which is phosphorylated at Thr292. This phosphorylation induces inhibition of the pathway, decreases MEK1 activation, and further conferred to MEK2 via heterodimerization for accelerating MEK2 dephosphorylation as well (135). 23 Figure 12. Key functional domains of the human MEK1 and MEK2 proteins are shown in a linear format. (133) The first synthetic inhibitor of MEK1 and MEK2, PD098059, was reported in 1995 (136). Two other potent inhibitors U0126 (137) and Ro-2210 (138) were subsequently identified from cell- based assays. However, none of them moved to the clinical stage because of their pharmaceutical limitations such as low potency and physical properties. There are currently four FDA-approved clinical utility of MEK inhibitors, trametinib, cobimetinib, binimetinib, and selumetinib. They are all classified as ATP non-competitive, type III allosteric protein kinase inhibitors, which means they bind to a site that is adjacent to, but not on ATP-binding sites on MEK (139). Given resistance occurs to BRAF inhibitors alone, the combinations of MEK inhibitors with first-generations of BRAF inhibitors (e.g., trametinib plus dabrafenib, cobimetinib plus vemurafenib) have been demonstrated enhanced apoptosis than the respective monotherapies in metastatic melanoma, which have revolutionized treatment for many patients and approved by FDA (126). In recent years, a number of novel MEK1/2 inhibitors such as refametinib or pimasertib as monotherapy, as well as the combination of AZD6244 and Akt inhibitor MK-2206 have progressed into clinical trials (140, 141). The novel MEK1/2 inhibitors may take advantage of their role at the central part of the ERK pathway to inform future drug development, or to find potential combinations with other targeted agents or conventional cytotoxic drugs. 24 1.6 The relevance of c-MYC for apoptosis in cancer therapy 1.6.1 the c-MYC oncogene in cancer The MYC (known as c-MYC) oncogene is a ‘‘global’’ transcription factor. It controls the expression of the genes that are essential to a vast range of biological events, including proliferation, differentiation, programmed cell death, and immune regulation (142). C-MYC was the first gene to be discovered in myc family due to its homology to the oncogene v-myc of the avian myelocytomatosis virus (143). Two additional paralogs were found later: MYCN (N- myc) was first identified in human neuroblastomas (144), and MYCL (L-myc) was originally observed in human small cell lung cancer (145). Subsequently, both MYCN and MYCL were identified in many tissues and tumor types. MYCN and MYCL share function with c-MYC despite having a more restricted tissue expression pattern than c-MYC (146). All three MYC proteins frequently deregulate and contribute to human cancers. Figure 13. Crystal structure of the a MYC/MAX heterodimer binds to a canonical E-box (PDB code: 1NKP). C-MYC encompasses a C-terminal domain (CTD), an N-terminal transactivation domain (NAD), and a central region (142). The C-terminal domain contains a basic region helix-loop- helix leucine zipper (bHLHLZ) motif and promotes dimerize with its obligatory partner, the MYC-associated protein X (MAX) protein. The MYC/MAX heterodimers bind to the canonical E-box sequence 5’-CACGTG-3’ in promoters and enhance MYC-regulated genes (147) (Figure 13). The N-terminal domain contains conserved transcriptional regulation elements called ‘‘MYC boxes’’ (MBI and II). The control of c-MYC stability and activity in response to 25 cell growth signals involves two important phosphorylation sites, threonine 58 (T58) and serine 62 (62), which are located in MBI (148). The central region comprises a nuclear localization signal (NLS), and other conserved MYC boxes (MBIII and MB IV) (149). In addition, c-MYC mRNA is innately unstable, with a short half-life of 30 min. The stability and degradation of c-MYC protein are also subjected to other various of post-translational modifications, including ubiquitination, SUMOylation, methylation, acetylation, and glycosylation (150, 151). c-MYC expression is tightly controlled in healthy adult tissue. However, approximately 70% of all human malignancies exhibit deregulated c-MYC activity, which is frequently associated with a poor clinical outcome, an elevated risk of recurrence, aggressive biological behavior, and an advanced stage of disease (148). Generally, c-MYC overexpression does not result from gene point mutations. Aberrant c-MYC expression is involved in gene amplification, chromosomal translocation, enhanced cell signaling, retroviral promoter insertion, altered protein degradation and mutation (152). Of these mechanisms, gene amplification is the most widely mechanism of c-MYC deregulation in solid tumors. The Cancer Genome Atlas Program (TCGA) in 2018 showed c-MYC gene amplification contributes to ovarian cancer (33.2%), breast cancer (15.0%), pancreatic cancer (12.6%) and lung cancer (8.4%) (153). Depending on biochemical features of tumors, c-MYC gene amplification is more prevalent in certain cancer types. For example, in the comparison with ER+ (estrogen receptor α positive) or HER2+ (human epidermal growth factor receptor 2 positive) breast cancers, c-MYC is markedly elevated in triple-negative breast cancer (TNBC) (154). While the translocation of c-MYC gene into the T cell receptor locus or immunoglobulin is of the key events in the development of hematological malignancies such as acute lymphoblastic leukemia (ALL) and Burkitt’s lymphoma (155). Upstream oncogenic signaling from EGFR, ALK, Wnt, NOTCH, TGF, Hedgehog and Hippo pathways also drive irregular c-MYC expression in many cancer cells (156). c-MYC activation alone is generally insufficient to cause non-malignant cells to undergo neoplastic transformation. The tumorigenesis function of oncogenic c-MYC is limited by numerous physiological mechanisms including cell cycle arrest, apoptosis, and/or cellular senescence (157). C-MYC has an intrinsic function involved in cell death, which induces apoptosis requiring its DNA binding functions and dimerization with MAX. C-MYC does not act as a death effector but instead acts to sensitize cells to a variety of apoptotic stimuli such as hypoxia, genotoxic stress, and death receptor signaling. Plenty of evidence supports the view that this apoptotic sensitization substantially restraints the oncogenic potential of c-MYC (158). The prosurvival Bcl-2 pathway and the proapoptotic p53 pathway are primarily interrelated in cancer cells, which respond to c-MYC activation by proliferation or by inducing apoptosis (159, 26 160) (Figure 14). For example, Bax is one of the transcriptional targets of c-MYC and the main regulator of c-MYC-dependent apoptosis. Expression of c-MYC induces Bax upregulation and further releases cytochrome c from mitochondria. However, Bak is prevented from activating in response to c-MYC by the overexpression of Bcl-XL (161). C-MYC is also known as stimuli participates in the extrinsic apoptosis pathway to sensitize cells to Fas-induced and TNF-α induced cell death. As introduced above, caspase 8 is a crucial initiator caspase downstream of FADD. The expression of caspase 8 can be enhanced directly or indirectly by c-MYC even with neutralizing antibodies against Fas ligand and TNF-α, therefore increasing the susceptibility of apoptosis (162). Furthermore, stable p53 in normal cells can interact with pro-apoptotic genes such as Puma, Noxa, Apaf-1, and Bax, and suppress anti-apoptotic genes including Bcl-2, Bcl- XL, and Mcl-1. While ectopic MYC expression upregulates a tumor suppressor ARF, which further inhibits MDM2-mediated degradation of p53. Therefore c-MYC expression indirectly promotes p53 expression and triggers apoptosis (163). However, the oncogenic potential of c- MYC is released when continuous abnormal c-MYC expression occurs correlated with loss of stress response checkpoints such as p53 and Bcl-XL, or activation of mitogenic signals such as RAS. In these circumstances, oncogenic c-MYC binds to canonical E-boxes at promoters, amplifying the output of current gene expression, simultaneously, also invasive non-canonical, lower-affinity E-boxes at promoters in a concentration-dependent manner, causing previously silent genes to be ectopically deregulated. Through this mechanism, c-MYC activated multiple genes to initiate and maintain all the ‘‘hallmarks’’ of cancer (153, 164, 165). 27 Figure 14. The relevance of c-MYC with apoptosis in the normal cells and c-MYC contributes to cancer. (A) c-MYC and apoptosis pathway. C-MYC expression can sensitize cells to a variety of proapoptotic stimuli (163). (B) Oncogenic level of c-MYC regulates all hallmarks of cancer (164). 1.6.2 c-MYC as a target for cancer treatment c-MYC is a well-established therapeutic target. Experiments have shown that turning off c- MYC expression can regress tumor development. However, c-MYC has been considered to be 28 ‘‘undruggable’’. Due to the large size of the c-MYC/MAX bHLHZ interface, and c-MYC lacks catalytic activity, unlike the other oncoprotein (e.g., BRAF, EGFR, HEK2) with enzymatic pocket, many drugs are difficult to bind c-MYC with high affinity. This reality makes it extremely challenging in developing effective c-MYC inhibitors (166). Therapeutically, c- MYC has not yet been successfully targeted. Despite these challenges, direct inhibition of c- MYC and indirect inhibition of c-MYC are the major strategies and have been evaluated by a series of potential c-MYC inhibitors. Direct inhibition is inhibiting c-MYC/MAX dimerization and E-box binding. Small molecules and peptides, as well as applying RNA interferences to downregulate c-MYC translation, have effectively blocked c-MYC activity (167). 10058-F4, 10075-G4, and 10075-A4 are the early identified compounds disrupting c-MYC/MAX interaction (168). The bHLHLZ of MYC represented the most studied structure for drug design of MYC inhibitors so far, aiming to bind to c-MYC/MAX dimer and prevent it from binding to DNA (169). Recently, OmoMYC as a 90 amino acid MYC mini-mutant attracted great attention. OmoMYC consists of the bHLHLZ domain and competes with c-MYC for binding to DNA by excluding c-MYC/MAX heterodimers and preventing the transcription of target genes. Among the different types of OmoMYC, OMO-1, and OMO-103 have progressed into clinical trials (170). In addition, given the short-life of c-MYC and its stability is regulated by ubiquitination, the field of proteolysis- targeting chimeras (PROTACs) is expanding. A promising result has shown pan-BETi PROTAC ARC-771 decreased c-MYC expression and caused xenograft tumor regression in prostate cancer mouse models (171). Indirect inhibition is concerning c-MYC-related synthetic lethal interactions, which means the genes are the important mediators of c-MYC in the c-MYC-driven tumor cells (157). Diverse potential targets have been identified, including cyclin-dependent kinases (CDKs) related to cell cycle, ATR and CDHK1 related to DNA repair (172, 173). Moreover, one advantage of this group of novel synthetic lethal targets may already holding a clinical approved inhibitor that could be promptly utilized in c-MYC driven malignancies. 1.7 Parthanatos-inducing compounds in cancer cells Mechanisms for bypassing the apoptosis signaling pathways have received considerable attention to cause the death of cancer cells. Parthanatos, as described above, is a recently discovered cell death mechanism related to cancer. The first discovery of parthanatos induced 29 by chemicals was in fibroblasts, exposed to N-methyl-N’-nitro-N-nitrosoguanidine (MNNG), a DNA-alkylating agent. This groundbreaking study also confirmed parthanatos is a caspase- dependent mode of cell death (90). Several evidence reported small molecules inducing parthanatos have shown promise as anticancer strategies. They are summarized in Table 2. Interestingly, two known anticancer agents were found to be parthanatos-inducing compounds. For example, saurosporine was demonstrated to trigger AIF release and downstream DNA fragmentation in non-small-cell carcinomas (NSLCL2) cells, which are resistant to induce apoptosis by conventional anticancer treatment (174). Saurosporine is a microbial alkaloid isolated in 1977, followed by the approval for clinical use as a broadly selective and potent protein kinase inhibitor (175). Atiprimod is a kinase inhibitor against multiple myelomas. It was found to lead to cell death in an AIF-mediated pathway, but caspase-independent. However, some investigations were not accounted for here. For example, flavopiridol, a pan CDK inhibitor to kill cholangiocarcinoma cells, showed that the cell death mechanism was caspase- dependent, which raises the potential that might be parthanatos but lacks sufficient evidence. More verifications of biochemical relevant events are still required (176). Natural products have also induced parthanatos, such as cynaropicrin, isolated from edible plant (Cynara scolymus) (177), and deoxypodophyllotoxin, isolated from traditional plant (Anthriscus sylvestris) (178). These studies not only indicated parthanatos was triggered by the active compounds, but also affected deregulated proliferation pathways or transcription factors that are related to tumor progression. Therefore, parthanatos deserves to be investigated by established anticancer agents or natural products as a potential treatment for cancer drug resistance. 30 Table 2. Cancer therapeutic agents that inducing parthanatos verified in vitro and/or in vivo. Compound (resource) Chemical structure Parthanatos-related biochemical events Study model Reference Cynaropicrin γH2AX upregulation, PARP and PAR Multiple myeloma, (177) (natural product) hyperactivation, AIF translocation, AMO1; T-ALL tumor Mitochondria membrane potential xenograft zebrefish dysfunction, PARP-dependent cell death Staurosporine Mitochondria membrane potential Non-small-cell lung (174) (natural product) dysfunction, AIF release, nuclear carcinomas, U1810 fragmentation Deoxypodophyllotoxin Upregulation of PARP and PAR, nuclear Glioma, C6, SHG-44 (178) (natural product) translocation of AIF 31 YM155 γH2AX expression, PARP and PAR Esophageal (179) (synthetic compound) overexpression, AIF translocation squamous-cell carcinoma, KYSE410 Atiprimod AIF release, caspase-independent cell Mantle cell (180) (synthetic compound) death lymphoma, MCL; CB-17SCID mice 32 2 Objective of the thesis Although enormous advances in cancer biology and rapid growth in the designs of new chemotherapy agents, cancer cell resistance against the anticancer agent is still a challenging process that leads to major treatment failures. The cytotoxicity of antineoplastic drugs depends mainly on their ability to induce cell death, especially apoptosis. However, cancer cells are constantly evolving and adapting, allowing them the capacity to evade cell death, and the activation of oncogene, which confers the cells a selective growth advantage. Hence, the first part of this thesis focuses on a state-of-art cell death mechanism, parthanatos, induced by new synthetic derivatives, and investigates their underlying molecular modes of action. The other part of this thesis aimed to study a natural product derivative as a potential c-MYC inhibitor. ZINC253504760 is a synthetic cardenolide that is structurally similar to cardiac glycoside (CG) such as digitoxin. In recent years, CG exhibited potential anticancer effects with many potential mechanisms and has been attracted interest in cancer drug discovery. This study aimed to 1) investigate the cytotoxicity of ZINC253504760 towards different drug-sensitive and -resistant cancer cells, 2) explore the major mode of cell death, 3) verify the potential affected target and pathway in vitro and in silico informed by microarray-based mRNA profiling. J4 and J6 are two synthetic and characterized palladium (II) complexes, and were designed to be novel metal-based small molecules to overcome drug resistance. Despite displaying excellent cytotoxicity in drug-sensitive leukemia cells, J4 and J6 have not been shown to significantly induce apoptotic cell death. The aim of this study was continuously to find out the predominant mode of cell death induced by J4 and J6, as well as to verify whether the cell death mechanism efficiently works in multi-drug resistant cells. ZINC15675948 is a natural product derivative of 1,2,4-oxadiazole. The compounds, 10074-G5 and 10075-A4 possess oxadiazole scaffold, are well-known c-MYC inhibitors. The aim of this study was 1) explore whether c-MYC is the potential target of ZINC15675948, 2) investigate the downstream cellular functions when c-MYC was affected. 33 3 Results and discussion 3.1 Cardiac glycoside (ZINC253504760), a novel MEK1/2 inhibitor inducing a state-of-art parthanatic cell death and G2/M arrest in leukemia cells Cardiac glycosides have been identified as leading compounds in cancer treatment recently. The Raf-MEK-ERK signaling cascade is a well-characterized MAPK pathway that is aberrantly activated in human cancers. MEK currently being the only activator of ERK, making it an attractive target. In the present study, a cardiac glycoside compound ZINC253504760 displayed potent cytotoxicity to five pairs of drug-sensitive and -resistant cell lines except for MDA-MB- BCRP. CCRF-CEM cells were the most sensitive to ZINC253504760 with an IC50 value of 0.022 ± 0.002 µM, which were selected as the study model for further investigation. Transcriptome-wide mRNA expression profiling and pathway analysis pointed out the canonical pathway involved in “G2/M cell cycle arrest”, which was predicted to be connected with downregulations of MEK1/2 and ERK. Afterward, G2/M arrest was remarkably observed by flow cytometry in a time- and concentration-dependent manner. The results were confirmed by fluorescence microscopy that ZINC253504760 affected microtubule polymerization. A series of genes affected by ZINC253504760 in G2/M arrest was verified by the upregulation of HIPK2, PPM1D, CDK1, Wee1, CKS1, CKS2, and the downregulation of p53 and CDK7 using qRT-PCR. Interestingly, apoptosis was not the major mode of cell death observed on flow cytometry, nor was it autophagy. While ZINC253504760 resulted in mitochondrial membrane potential collapse. Western blotting showed caspase 3 expression but not its cleaved form. These results indicated the mode of cell death was caused by mitochondria dysfunction and were caspase 3-independent. Therefore, we further focused on a novel caspase-independent cell death mechanism parthanatos. The key features of ZINC253504760-induced parthanatos were confirmed by PARP activation, PAR accumulation, and AIF translocation by western blotting. AIF translocation from cytoplasm to nucleus was captured using immunofluorescence microscopy. Sing cell gel electrophoresis (comet assay) supported ZINC253504760 caused parthanatos and eventually the cells die from large-scale DNA damage. Moreover, ZINC253504760 inhibited phosphorylation of MEK1/2 upon treatment for 24 h, which further affected the activation of ERK. Molecular docking also showed ZINC253504760 as an ATP 34 competitive kinase inhibitor bound to the phosphorylation sites on MEK1 (SER218) and MEK2 (SER222). Their binding was confirmed in microscale thermophoresis. Further reading: Appendix I The cardiac glycoside ZINC253504760 induces parthanatos-type cell death and G2/M arrest via downregulation of MEK1/2 phosphorylation in leukemia cells Min Zhou, Joelle C. Boulos, Sabine M. Klauck, Thomas Efferth 3.2 Palladium(II) complexes induced parthanatos-type cell death in CCRF- CEM leukemia and its multidrug-resistant cells Due to severe-sides effects and the development of drug resistance with platinum complexes, palladium complexes as innovative anticancer drugs are emerging. The aim of this study was to reveal the major mode of cell death of two synthetic palladium (II) complexes with halogen- substitute Schiff bases and 2-picolylamine (pic), one with double chlorine-substitution, another with double iodine-substitution. Their profound cytotoxicity and G2/M arrest in CCRF-CEM leukemia cells had been investigated in previous study (IC50 value of J4: 1.78 µM, IC50 value of J6: 2.39 µM), but none of them significantly induce apoptotic cell death (181). In the present study, surprisingly, PARP-dependent parthanatic cell death was primarily induced by the two palladium(II) complexes, evidenced by PARP, PAR, and p-histone H2A.X overactivation by western blotting, mitochondrial membrane potential sharply dysfunction, AIF translocation from cytoplasm to nucleus by immunofluorescence microscopy, and DNA fragmentation in a concentration-dependent manner using alkaline single cell gel electrophoresis. The induction of parthanatos was also observed in P-gp overexpressing CEM/ADR5000 cells. PARP inhibitor PJ34 indicated cell viability was maintained in combination with PJ34 plus J4 or J6. Furthermore, J4 and J6 were unable to induce other cell death mechanisms such as autophagy, apoptosis, or necrosis, verified by western blotting and flow cytometry. Both J4 and J6 showed less cytotoxicity on human peripheral blood mononuclear cells (PBMCs) than leukemia cells. 35 Further reading: Appendix II Two palladium (II) complexes derived from halogen-substituted Schiff bases and 2- picoplylamine induce parthanatos-type cell death in sensitive and multi-drug resistant CCRF-CEM leukemia cells Min Zhou, Joelle C. Boulos, Ejlal A. Omer, Hadi Amiri Rudbari, Tanja Schirmeister, Nicola Micale, Thomas Efferth 3.3 1,2,4-oxadiazole derivative (ZINC15675948) as a novel c-MYC inhibitor by inducing DNA damage, cell cycle arrest, and apoptosis in leukemia and breast cancer cells The MYC oncogene contributes many hallmarks of cancer in various highly aggressive human cancers, such as acute leukemia and triple-negative breast cancer. 1,2,4-oxadiazole is one of the well-known oxadiazoles attracting increasing interest for their anticancer activities. In this study, (6S)-N-(4-methylphenyl)-6-(3-naphthalen-2-yl-1,2,4-oxadiazol-5-yl)-3,4,6,7- tetrahydroimidazo[4,5-c]pyridine-5-carboxamide, code name ZINC15675948, exhibited profound cytotoxicity in CCRF-CEM leukemia and MDA-MB-231-pcDNA3 breast cancer cells with IC50 values at 0.008 ± 0.001 µM and 0.08 ± 0.004 µM, respectively. Particularly, ZINC15675948 displayed a strong binding to c-MYC/MAX interaction site in molecular docking (LEB: −9.91 kcal/mol, pKi: 0.055 µM), compared with and microscale thermophoresis (Kd: 1.18 ± 0.1 µM). C-MYC reporter assay, western blotting, and qRT-PCR showed downregulation of c-MYC by ZINC15675948 in a concentration-dependent manner. For mechanistic studies of ZINC15675948, microarray hybridization and signaling pathway analyses predicted several cellular functions were commonly affected by ZINC15675948 in both CCRF-CEM cells and MDA-MB-231-pcDNA3 cells compared with their control samples, including “cellular growth and proliferation”, “DNA replication, recombination and repair”, “cell cycle”, as well as “cell death and survival”. The “cell cycle”, and “cell death and survival” networks of ZINC15675948 treated CCRF-CEM cells indeed revealed downregulation of c- MYC gene, implying they were downstream of c-MYC inhibition. However, the microarray profiling of MDA-MB-231-pcDNA3 cells revealed an involvement of ubiquitination toward c- MYC, indicated by the upregulation of a novel ubiquitin ligase (ELL2) in the absence of c-MYC 36 expression. DNA damage was significantly induced by ZINC15675948 indicated by “comet tail” in single cell gel electrophoresis. ZINC15675948 arrested CCRF-CEM cells in G2/M phase and MDA-MB-231-pcDNA3 cells in S phase. Furthermore, ZINC15675948 induced apoptosis in both of the cell lines. Autophagy induction was only observed in CCRF-CEM cells. We propose ZINC15675948 as a novel c-MYC inhibitor for leukemia and breast cancer treatment. Further reading: Appendix III Modes of action of a novel c-MYC inhibiting 1,2,4-oxadiazole derivative in leukemia and breast cancer cells Min Zhou, Joelle C. Boulos, Ejlal A. Omer, Sabine M. Klauck, Thomas Efferth 37 4 Conclusion Programmed cell death especially apoptosis is an important barrier to prevent cancer development. Cancer cells evolve multiple strategies to evade apoptosis. Therefore, resisting cell death is not only a hallmark to initiate tumors, but also a challenge for overcoming multi- drug resistance. This thesis reported three promising compounds that induce the novel mode of cell death termed parthanatos rather than apoptosis in leukemia cells to better attack drug resistance, and one potential compound that promotes apoptotic cell death in leukemia and breast cancer cells by inhibiting oncogene c-MYC. The synthetic derivative of cardiac glycoside compounds (CGs) (ZINC253504760) might be a potential therapeutic agent for blocking the RAS-RAF-MEK-ERK signaling pathway in CCRF- CEM leukemia cells. ZINC253504760 inhibited MEK1/2 phosphorylation, which further affected downstream ERK. G2/M phase arrest was significantly induced in a time- and concentration-dependent manner. Importantly, ZINC253504760 triggered parthanatos-type cell death. A series of in vitro investigations confirmed that this compound induced PARP and PAR hyperactivation, and nuclear AIF translocation, mitochondrial membrane potential collapse. This study will promote novel modes of cell death as an alternative strategy for cancer treatment. Two synthetic palladium (II) complexes (J4 and J6) containing double chlorine-/iodine substituted Schiff base and 2-picolylamine showed induction of parthanatos as their primary cell death in CCRF-CEM leukemia cells and its subline P-gp overexpressing cells. J4 and J6 overactivated p-histone H2A.X, PARP, and PAR, followed by AIF translocation from mitochondria to the nucleus, leading to large DNA fragmentation and eventually parthanatic cell death. The investigation of cell death mechanism of J4 and J6 will motivate further designs on the development of palladium-based complexes as cancer therapy, especially to kill cancer cells in other cell death modes that insufficiently induce apoptosis. The 1,2,4-oxadiazole derivative (ZINC15675948) may provide a novel treatment for c-MYC- driven CCRF-CEM and MDA-MB-231-pcDNA cells. ZINC15675948 bound to c-MYC/MAX interface and further affected c-MYC activity. As downstream of c-MYC inhibition, ZINC15675948 induced remarkable DNA damage, cell cycle arrest, and apoptosis in both cell lines. In addition, ZINC15675948 also induced autophagy in leukemia cells. This study will help to continue targeting apoptosis, as c-MYC oncogene is one of the master regulators for gene expression of apoptosis. 38 5 Reference 1. Sung H, et al. (2021) Global Cancer Statistics 2020: GLOBOCAN Estimates of Incidence and Mortality Worldwide for 36 Cancers in 185 Countries. CA: a cancer journal for clinicians 71(3):209-249. 2. Carbone A (2020) Cancer Classification at the Crossroads. Cancers 12(4). 3. Murali R, Soslow RA, & Weigelt B (2014) Classification of endometrial carcinoma: more than two types. The Lancet. Oncology 15(7):e268-278. 4. HaDuong JH, Martin AA, Skapek SX, & Mascarenhas L (2015) Sarcomas. Pediatric clinics of North America 62(1):179-200. 5. Minciacchi VR, Kumar R, & Krause DS (2021) Chronic Myeloid Leukemia: A Model Disease of the Past, Present and Future. Cells 10(1). 6. Jiang M, Bennani NN, & Feldman AL (2017) Lymphoma classification update: T-cell lymphomas, Hodgkin lymphomas, and histiocytic/dendritic cell neoplasms. Expert review of hematology 10(3):239-249. 7. Cowan AJ, et al. (2022) Diagnosis and Management of Multiple Myeloma: A Review. Jama 327(5):464-477. 8. Hanahan D & Weinberg RA (2000) The hallmarks of cancer. Cell 100(1):57-70. 9. Hanahan D & Weinberg RA (2011) Hallmarks of cancer: the next generation. Cell 144(5):646-674. 10. Hanahan D (2022) Hallmarks of Cancer: New Dimensions. Cancer discovery 12(1):31- 46. 11. Wyld L, Audisio RA, & Poston GJ (2015) The evolution of cancer surgery and future perspectives. Nature reviews. Clinical oncology 12(2):115-124. 12. Lippman SM & Hawk ET (2009) Cancer prevention: from 1727 to milestones of the past 100 years. Cancer research 69(13):5269-5284. 13. Baskar R, Lee KA, Yeo R, & Yeoh KW (2012) Cancer and radiation therapy: current advances and future directions. International journal of medical sciences 9(3):193-199. 14. Bernier J, Hall EJ, & Giaccia A (2004) Radiation oncology: a century of achievements. Nature Reviews Cancer 4(9):737-747. 15. Baxevanis CN, Perez SA, & Papamichail M (2009) Cancer immunotherapy. Critical reviews in clinical laboratory sciences 46(4):167-189. 16. Sanmamed MF & Chen L (2018) A Paradigm Shift in Cancer Immunotherapy: From Enhancement to Normalization. Cell 175(2):313-326. 17. Fairchild A, et al. (2015) Hormonal therapy in oncology: a primer for the radiologist. AJR. American journal of roentgenology 204(6):W620-630. 18. Henderson BE & Feigelson HS (2000) Hormonal carcinogenesis. Carcinogenesis 21(3):427-433. 19. DeVita VT, Jr. & Chu E (2008) A history of cancer chemotherapy. Cancer research 68(21):8643-8653. 20. Krumbhaar EB & Krumbhaar HD (1919) The Blood and Bone Marrow in Yelloe Cross Gas (Mustard Gas) Poisoning: Changes produced in the Bone Marrow of Fatal Cases. The Journal of medical research 40(3):497-508.493. 21. DeVita VT, Jr. (1978) The evolution of therapeutic research in cancer. The New England journal of medicine 298(16):907-910. 22. Lind MJ (2011) Principles of cytotoxic chemotherapy. Medicine 39(12):711-716. 23. Oun R, Moussa YE, & Wheate NJ (2018) The side effects of platinum-based chemotherapy drugs: a review for chemists. Dalton transactions (Cambridge, England : 2003) 47(19):6645-6653. 39 24. Portugal J (2018) Challenging transcription by DNA-binding antitumor drugs. Biochemical pharmacology 155:336-345. 25. Delgado JL, Hsieh CM, Chan NL, & Hiasa H (2018) Topoisomerases as anticancer targets. The Biochemical journal 475(2):373-398. 26. Hartmann JT & Lipp HP (2006) Camptothecin and podophyllotoxin derivatives: inhibitors of topoisomerase I and II - mechanisms of action, pharmacokinetics and toxicity profile. Drug safety 29(3):209-230. 27. Minotti G, Menna P, Salvatorelli E, Cairo G, & Gianni L (2004) Anthracyclines: molecular advances and pharmacologic developments in antitumor activity and cardiotoxicity. Pharmacological reviews 56(2):185-229. 28. Jang JY, Kim D, & Kim ND (2023) Recent Developments in Combination Chemotherapy for Colorectal and Breast Cancers with Topoisomerase Inhibitors. International Journal of Molecular Sciences 24(9):8457. 29. Godzieba M & Ciesielski S (2020) Natural DNA Intercalators as Promising Therapeutics for Cancer and Infectious Diseases. Current cancer drug targets 20(1):19- 32. 30. Siddik ZH (2003) Cisplatin: mode of cytotoxic action and molecular basis of resistance. Oncogene 22(47):7265-7279. 31. Florea AM & Büsselberg D (2011) Cisplatin as an anti-tumor drug: cellular mechanisms of activity, drug resistance and induced side effects. Cancers 3(1):1351-1371. 32. Ghosh S (2019) Cisplatin: The first metal based anticancer drug. Bioorganic chemistry 88:102925. 33. Joshi AM, et al. (2021) Microtubule Inhibitors and Cardiotoxicity. Current Oncology Reports 23(3):30. 34. Farber S & Diamond LK (1948) Temporary remissions in acute leukemia in children produced by folic acid antagonist, 4-aminopteroyl-glutamic acid. The New England journal of medicine 238(23):787-793. 35. Luengo A, Gui DY, & Vander Heiden MG (2017) Targeting Metabolism for Cancer Therapy. Cell chemical biology 24(9):1161-1180. 36. Plummer C, et al. (2019) Treatment specific toxicities: Hormones, antihormones, radiation therapy. Seminars in oncology 46(6):414-420. 37. Lee YT, Tan YJ, & Oon CE (2018) Molecular targeted therapy: Treating cancer with specificity. Eur J Pharmacol 834:188-196. 38. Joo WD, Visintin I, & Mor G (2013) Targeted cancer therapy--are the days of systemic chemotherapy numbered? Maturitas 76(4):308-314. 39. Zhong L, et al. (2021) Small molecules in targeted cancer therapy: advances, challenges, and future perspectives. Signal transduction and targeted therapy 6(1):201. 40. Chabner BA & Roberts TG (2005) Chemotherapy and the war on cancer. Nature Reviews Cancer 5(1):65-72. 41. Roskoski Jr R (2022) Properties of FDA-approved small molecule protein kinase inhibitors: A 2022 update. Pharmacological Research 175:106037. 42. Kimiz-Gebologlu I, Gulce-Iz S, & Biray-Avci C (2018) Monoclonal antibodies in cancer immunotherapy. Molecular biology reports 45(6):2935-2940. 43. van de Donk NW, et al. (2016) Clinical efficacy and management of monoclonal antibodies targeting CD38 and SLAMF7 in multiple myeloma. Blood 127(6):681-695. 44. Weiner LM, Surana R, & Wang S (2010) Monoclonal antibodies: versatile platforms for cancer immunotherapy. Nature Reviews Immunology 10(5):317-327. 45. Dosio F, Brusa P, & Cattel L (2011) Immunotoxins and anticancer drug conjugate assemblies: the role of the linkage between components. Toxins 3(7):848-883. 46. Morse MA, Gwin WR, 3rd, & Mitchell DA (2021) Vaccine Therapies for Cancer: Then and Now. Targeted oncology 16(2):121-152. 40 47. Lin MJ, et al. (2022) Cancer vaccines: the next immunotherapy frontier. Nature Cancer 3(8):911-926. 48. Burke EE, Kodumudi K, Ramamoorthi G, & Czerniecki BJ (2019) Vaccine Therapies for Breast Cancer. Surgical oncology clinics of North America 28(3):353-367. 49. Tang R & Xu Z (2020) Gene therapy: a double-edged sword with great powers. Molecular and Cellular Biochemistry 474(1):73-81. 50. Ma CC, Wang ZL, Xu T, He ZY, & Wei YQ (2020) The approved gene therapy drugs worldwide: from 1998 to 2019. Biotechnology advances 40:107502. 51. Gottesman MM & Pastan I (1993) Biochemistry of multidrug resistance mediated by the multidrug transporter. Annual review of biochemistry 62:385-427. 52. Mansoori B, Mohammadi A, Davudian S, Shirjang S, & Baradaran B (2017) The Different Mechanisms of Cancer Drug Resistance: A Brief Review. Advanced pharmaceutical bulletin 7(3):339-348. 53. Assaraf YG, et al. (2019) The multi-factorial nature of clinical multidrug resistance in cancer. Drug resistance updates : reviews and commentaries in antimicrobial and anticancer chemotherapy 46:100645. 54. Efferth T (2001) The human ATP-binding cassette transporter genes: from the bench to the bedside. Current molecular medicine 1(1):45-65. 55. Efferth T & Volm M (2017) Multiple resistance to carcinogens and xenobiotics: P- glycoproteins as universal detoxifiers. Archives of toxicology 91(7):2515-2538. 56. Locher KP (2016) Mechanistic diversity in ATP-binding cassette (ABC) transporters. Nat Struct Mol Biol 23(6):487-493. 57. Nobili S, et al. (2020) Role of ATP-binding cassette transporters in cancer initiation and progression. Seminars in cancer biology 60:72-95. 58. Sharom FJ (2008) ABC multidrug transporters: structure, function and role in chemoresistance. Pharmacogenomics 9(1):105-127. 59. Aller SG, et al. (2009) Structure of P-glycoprotein reveals a molecular basis for poly- specific drug binding. Science (New York, N.Y.) 323(5922):1718-1722. 60. Abolhoda A, et al. (1999) Rapid activation of MDR1 gene expression in human metastatic sarcoma after in vivo exposure to doxorubicin. Clinical cancer research : an official journal of the American Association for Cancer Research 5(11):3352-3356. 61. Efferth T, et al. (2008) Prediction of broad spectrum resistance of tumors towards anticancer drugs. Clinical cancer research : an official journal of the American Association for Cancer Research 14(8):2405-2412. 62. Palmeira A, Sousa E, Vasconcelos MH, & Pinto MM (2012) Three decades of P-gp inhibitors: skimming through several generations and scaffolds. Current medicinal chemistry 19(13):1946-2025. 63. DeGorter MK, Xia CQ, Yang JJ, & Kim RB (2012) Drug transporters in drug efficacy and toxicity. Annual review of pharmacology and toxicology 52:249-273. 64. Mao Q & Unadkat JD (2015) Role of the breast cancer resistance protein (BCRP/ABCG2) in drug transport--an update. Aaps j 17(1):65-82. 65. Mohammad RM, et al. (2015) Broad targeting of resistance to apoptosis in cancer. Seminars in cancer biology 35 Suppl(0):S78-s103. 66. Pistritto G, Trisciuoglio D, Ceci C, Garufi A, & D'Orazi G (2016) Apoptosis as anticancer mechanism: function and dysfunction of its modulators and targeted therapeutic strategies. Aging 8(4):603-619. 67. Wong RS (2011) Apoptosis in cancer: from pathogenesis to treatment. Journal of experimental & clinical cancer research : CR 30(1):87. 68. Youle RJ & Strasser A (2008) The BCL-2 protein family: opposing activities that mediate cell death. Nature Reviews Molecular Cell Biology 9(1):47-59. 41 69. Yu J, Wang Z, Kinzler KW, Vogelstein B, & Zhang L (2003) PUMA mediates the apoptotic response to p53 in colorectal cancer cells. Proceedings of the National Academy of Sciences of the United States of America 100(4):1931-1936. 70. Thornborrow EC, Patel S, Mastropietro AE, Schwartzfarb EM, & Manfredi JJ (2002) A conserved intronic response element mediates direct p53-dependent transcriptional activation of both the human and murine bax genes. Oncogene 21(7):990-999. 71. Song G, Wang W, & Hu T (2011) p53 facilitates BH3-only BID nuclear export to induce apoptosis in the irrepairable DNA damage response. Medical hypotheses 77(5):850-852. 72. Schimmer AD (2004) Inhibitor of apoptosis proteins: translating basic knowledge into clinical practice. Cancer research 64(20):7183-7190. 73. Tu H & Costa M (2020) XIAP's Profile in Human Cancer. Biomolecules 10(11). 74. Strasser A & Vaux DL (2020) Cell Death in the Origin and Treatment of Cancer. Molecular cell 78(6):1045-1054. 75. Galluzzi L, et al. (2007) Cell death modalities: classification and pathophysiological implications. Cell death and differentiation 14(7):1237-1243. 76. Cerella C, Teiten MH, Radogna F, Dicato M, & Diederich M (2014) From nature to bedside: pro-survival and cell death mechanisms as therapeutic targets in cancer treatment. Biotechnology advances 32(6):1111-1122. 77. Galluzzi L, et al. (2018) Molecular mechanisms of cell death: recommendations of the Nomenclature Committee on Cell Death 2018. Cell Death & Differentiation 25(3):486- 541. 78. Yan G, Elbadawi M, & Efferth T (2020) Multiple cell death modalities and their key features (Review). World Acad Sci J 2(2):39-48. 79. Kerr JF, Wyllie AH, & Currie AR (1972) Apoptosis: a basic biological phenomenon with wide-ranging implications in tissue kinetics. British journal of cancer 26(4):239- 257. 80. Xu X, Lai Y, & Hua ZC (2019) Apoptosis and apoptotic body: disease message and therapeutic target potentials. Bioscience reports 39(1). 81. McIlwain DR, Berger T, & Mak TW (2013) Caspase functions in cell death and disease. Cold Spring Harbor perspectives in biology 5(4):a008656. 82. Fulda S & Debatin KM (2006) Extrinsic versus intrinsic apoptosis pathways in anticancer chemotherapy. Oncogene 25(34):4798-4811. 83. Walczak H (2013) Death receptor-ligand systems in cancer, cell death, and inflammation. Cold Spring Harbor perspectives in biology 5(5):a008698. 84. Ozören N & El-Deiry WS (2002) Defining characteristics of Types I and II apoptotic cells in response to TRAIL. Neoplasia (New York, N.Y.) 4(6):551-557. 85. Carneiro BA & El-Deiry WS (2020) Targeting apoptosis in cancer therapy. Nature Reviews Clinical Oncology 17(7):395-417. 86. David KK, Andrabi SA, Dawson TM, & Dawson VL (2009) Parthanatos, a messenger of death. Frontiers in bioscience (Landmark edition) 14(3):1116-1128. 87. Andrabi SA, Dawson TM, & Dawson VL (2008) Mitochondrial and nuclear cross talk in cell death: parthanatos. Annals of the New York Academy of Sciences 1147:233-241. 88. Zhou Y, et al. (2021) Parthanatos and its associated components: Promising therapeutic targets for cancer. Pharmacol Res 163:105299. 89. Fatokun AA, Dawson VL, & Dawson TM (2014) Parthanatos: mitochondrial-linked mechanisms and therapeutic opportunities. British journal of pharmacology 171(8):2000-2016. 90. Yu SW, et al. (2002) Mediation of poly(ADP-ribose) polymerase-1-dependent cell death by apoptosis-inducing factor. Science (New York, N.Y.) 297(5579):259-263. 91. Wang Y, Dawson VL, & Dawson TM (2009) Poly(ADP-ribose) signals to mitochondrial AIF: A key event in parthanatos. Experimental Neurology 218(2):193-202. 42 92. Koehler RC, Dawson VL, & Dawson TM (2021) Targeting Parthanatos in Ischemic Stroke. Frontiers in neurology 12:662034. 93. Amé JC, Spenlehauer C, & de Murcia G (2004) The PARP superfamily. BioEssays : news and reviews in molecular, cellular and developmental biology 26(8):882-893. 94. Pascal JM (2018) The comings and goings of PARP-1 in response to DNA damage. DNA repair 71:177-182. 95. Bürkle A & Virág L (2013) Poly(ADP-ribose): PARadigms and PARadoxes. Molecular aspects of medicine 34(6):1046-1065. 96. Andrabi SA, et al. (2006) Poly(ADP-ribose) (PAR) polymer is a death signal. Proceedings of the National Academy of Sciences of the United States of America 103(48):18308-18313. 97. Daugas E, et al. (2000) Mitochondrio-nuclear translocation of AIF in apoptosis and necrosis. FASEB journal : official publication of the Federation of American Societies for Experimental Biology 14(5):729-739. 98. Levine B & Klionsky DJ (2017) Autophagy wins the 2016 Nobel Prize in Physiology or Medicine: Breakthroughs in baker's yeast fuel advances in biomedical research. Proceedings of the National Academy of Sciences of the United States of America 114(2):201-205. 99. Mizushima N & Klionsky DJ (2007) Protein turnover via autophagy: implications for metabolism. Annual review of nutrition 27:19-40. 100. Mizushima N (2007) Autophagy: process and function. Genes & development 21(22):2861-2873. 101. Mizushima N (2005) The pleiotropic role of autophagy: from protein metabolism to bactericide. Cell death and differentiation 12 Suppl 2:1535-1541. 102. Choi AM, Ryter SW, & Levine B (2013) Autophagy in human health and disease. The New England journal of medicine 368(7):651-662. 103. Onorati AV, Dyczynski M, Ojha R, & Amaravadi RK (2018) Targeting autophagy in cancer. Cancer 124(16):3307-3318. 104. Fang Y, et al. (2020) Pyroptosis: A new frontier in cancer. Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie 121:109595. 105. Wang H, et al. (2022) The emerging role of pyroptosis in pediatric cancers: from mechanism to therapy. Journal of hematology & oncology 15(1):140. 106. Tong X, et al. (2022) Targeting cell death pathways for cancer therapy: recent developments in necroptosis, pyroptosis, ferroptosis, and cuproptosis research. Journal of hematology & oncology 15(1):174. 107. Dixon SJ, et al. (2012) Ferroptosis: an iron-dependent form of nonapoptotic cell death. Cell 149(5):1060-1072. 108. Xie Y, et al. (2016) Ferroptosis: process and function. Cell death and differentiation 23(3):369-379. 109. Stockwell BR, et al. (2017) Ferroptosis: A Regulated Cell Death Nexus Linking Metabolism, Redox Biology, and Disease. Cell 171(2):273-285. 110. Liu J, et al. (2022) Iron plays a role in sulfasalazine-induced ferroptosis with autophagic flux blockage in K7M2 osteosarcoma cells. Metallomics : integrated biometal science 14(5). 111. Zhang C, Liu X, Jin S, Chen Y, & Guo R (2022) Ferroptosis in cancer therapy: a novel approach to reversing drug resistance. Mol Cancer 21(1):47. 112. Zhao L, et al. (2022) Ferroptosis in cancer and cancer immunotherapy. Cancer communications (London, England) 42(2):88-116. 113. Tsvetkov P, et al. (2022) Copper induces cell death by targeting lipoylated TCA cycle proteins. Science (New York, N.Y.) 375(6586):1254-1261. 43 114. Kahlson MA & Dixon SJ (2022) Copper-induced cell death. Science (New York, N.Y.) 375(6586):1231-1232. 115. Montagut C & Settleman J (2009) Targeting the RAF-MEK-ERK pathway in cancer therapy. Cancer letters 283(2):125-134. 116. Zhang W & Liu HT (2002) MAPK signal pathways in the regulation of cell proliferation in mammalian cells. Cell research 12(1):9-18. 117. Barbosa R, Acevedo LA, & Marmorstein R (2021) The MEK/ERK Network as a Therapeutic Target in Human Cancer. Molecular cancer research : MCR 19(3):361-374. 118. Roberts PJ & Der CJ (2007) Targeting the Raf-MEK-ERK mitogen-activated protein kinase cascade for the treatment of cancer. Oncogene 26(22):3291-3310. 119. Voisin L, Saba-El-Leil MK, Julien C, Frémin C, & Meloche S (2010) Genetic demonstration of a redundant role of extracellular signal-regulated kinase 1 (ERK1) and ERK2 mitogen-activated protein kinases in promoting fibroblast proliferation. Molecular and cellular biology 30(12):2918-2932. 120. Balmanno K & Cook SJ (2009) Tumour cell survival signalling by the ERK1/2 pathway. Cell Death & Differentiation 16(3):368-377. 121. Guo YJ, et al. (2020) ERK/MAPK signalling pathway and tumorigenesis. Experimental and therapeutic medicine 19(3):1997-2007. 122. Samatar AA & Poulikakos PI (2014) Targeting RAS-ERK signalling in cancer: promises and challenges. Nature reviews. Drug discovery 13(12):928-942. 123. Haigis KM (2017) KRAS Alleles: The Devil Is in the Detail. Trends in cancer 3(10):686-697. 124. Proietti I, et al. (2020) Mechanisms of Acquired BRAF Inhibitor Resistance in Melanoma: A Systematic Review. Cancers 12(10). 125. Sosman JA, et al. (2012) Survival in BRAF V600-mutant advanced melanoma treated with vemurafenib. The New England journal of medicine 366(8):707-714. 126. Subbiah V, Baik C, & Kirkwood JM (2020) Clinical Development of BRAF plus MEK Inhibitor Combinations. Trends in cancer 6(9):797-810. 127. Pylayeva-Gupta Y, Grabocka E, & Bar-Sagi D (2011) RAS oncogenes: weaving a tumorigenic web. Nature reviews. Cancer 11(11):761-774. 128. Raimondi V, et al. (2022) A personalized molecular approach in multiple myeloma: the possible use of RAF/RAS/MEK/ERK and BCL-2 inhibitors. Exploration of targeted anti-tumor therapy 3(4):463-479. 129. Carné Trécesson S, et al. (2017) BCL-X(L) directly modulates RAS signalling to favour cancer cell stemness. Nature communications 8(1):1123. 130. Chin L, et al. (1999) Essential role for oncogenic Ras in tumour maintenance. Nature 400(6743):468-472. 131. Tan N, et al. (2013) Bcl-2/Bcl-xL inhibition increases the efficacy of MEK inhibition alone and in combination with PI3 kinase inhibition in lung and pancreatic tumor models. Molecular cancer therapeutics 12(6):853-864. 132. Ullah R, Yin Q, Snell AH, & Wan L (2022) RAF-MEK-ERK pathway in cancer evolution and treatment. Seminars in cancer biology 85:123-154. 133. Caunt CJ, Sale MJ, Smith PD, & Cook SJ (2015) MEK1 and MEK2 inhibitors and cancer therapy: the long and winding road. Nature Reviews Cancer 15(10):577-592. 134. Eblen ST, et al. (2004) Mitogen-activated protein kinase feedback phosphorylation regulates MEK1 complex formation and activation during cellular adhesion. Molecular and cellular biology 24(6):2308-2317. 135. Kocieniewski P & Lipniacki T (2013) MEK1 and MEK2 differentially control the duration and amplitude of the ERK cascade response. Physical biology 10(3):035006. 44 136. Alessi DR, Cuenda A, Cohen P, Dudley DT, & Saltiel AR (1995) PD 098059 Is a Specific Inhibitor of the Activation of Mitogen-activated Protein Kinase Kinase in Vitro and in Vivo(*). Journal of Biological Chemistry 270(46):27489-27494. 137. Duncia JV, et al. (1998) MEK inhibitors: the chemistry and biological activity of U0126, its analogs, and cyclization products. Bioorganic & medicinal chemistry letters 8(20):2839-2844. 138. Williams DH, et al. (1998) Ro 09-2210 exhibits potent anti-proliferative effects on activated T cells by selectively blocking MKK activity. Biochemistry 37(26):9579-9585. 139. Pan Y & Mader MM (2022) Principles of Kinase Allosteric Inhibition and Pocket Validation. Journal of Medicinal Chemistry 65(7):5288-5299. 140. Singh VJ, Sharma B, & Chawla PA (2021) Recent developments in mitogen activated protein kinase inhibitors as potential anticancer agents. Bioorganic chemistry 114:105161. 141. Do K, et al. (2015) Biomarker-driven phase 2 study of MK-2206 and selumetinib (AZD6244, ARRY-142886) in patients with colorectal cancer. Investigational new drugs 33(3):720-728. 142. Dang CV (2012) MYC on the path to cancer. Cell 149(1):22-35. 143. Vennstrom B, Sheiness D, Zabielski J, & Bishop JM (1982) Isolation and characterization of c-myc, a cellular homolog of the oncogene (v-myc) of avian myelocytomatosis virus strain 29. Journal of virology 42(3):773-779. 144. Stanton LW, Schwab M, & Bishop JM (1986) Nucleotide sequence of the human N- myc gene. Proceedings of the National Academy of Sciences of the United States of America 83(6):1772-1776. 145. Nau MM, et al. (1985) L-myc, a new myc-related gene amplified and expressed in human small cell lung cancer. Nature 318(6041):69-73. 146. Nesbit CE, Tersak JM, & Prochownik EV (1999) MYC oncogenes and human neoplastic disease. Oncogene 18(19):3004-3016. 147. Amati B, et al. (1993) Oncogenic activity of the c-Myc protein requires dimerization with Max. Cell 72(2):233-245. 148. Meyer N & Penn LZ (2008) Reflecting on 25 years with MYC. Nature Reviews Cancer 8(12):976-990. 149. Conacci-Sorrell M, McFerrin L, & Eisenman RN (2014) An overview of MYC and its interactome. Cold Spring Harbor perspectives in medicine 4(1):a014357. 150. Farrell AS & Sears RC (2014) MYC degradation. Cold Spring Harbor perspectives in medicine 4(3). 151. Chen Y, Sun X-X, Sears RC, & Dai M-S (2019) Writing and erasing MYC ubiquitination and SUMOylation. Genes & Diseases 6(4):359-371. 152. Duffy MJ, O'Grady S, Tang M, & Crown J (2021) MYC as a target for cancer treatment. Cancer treatment reviews 94:102154. 153. Lourenco C, et al. (2021) MYC protein interactors in gene transcription and cancer. Nature Reviews Cancer 21(9):579-591. 154. Fallah Y, Brundage J, Allegakoen P, & Shajahan-Haq AN (2017) MYC-Driven Pathways in Breast Cancer Subtypes. Biomolecules 7(3). 155. Boxer LM & Dang CV (2001) Translocations involving c-myc and c-myc function. Oncogene 20(40):5595-5610. 156. Gabay M, Li Y, & Felsher DW (2014) MYC activation is a hallmark of cancer initiation and maintenance. Cold Spring Harbor perspectives in medicine 4(6). 157. Dhanasekaran R, et al. (2022) The MYC oncogene — the grand orchestrator of cancer growth and immune evasion. Nature Reviews Clinical Oncology 19(1):23-36. 158. Prendergast GC (1999) Mechanisms of apoptosis by c-Myc. Oncogene 18(19):2967- 2987. 45 159. Prendergast GC (1999) Mechanisms of apoptosis by c-Myc. Oncogene 18(19):2967- 2987. 160. McMahon SB (2014) MYC and the control of apoptosis. Cold Spring Harbor perspectives in medicine 4(7):a014407. 161. Juin P, et al. (2002) c-Myc functionally cooperates with Bax to induce apoptosis. Molecular and cellular biology 22(17):6158-6169. 162. Järvinen K, Hotti A, Santos L, Nummela P, & Hölttä E (2011) Caspase-8, c-FLIP, and caspase-9 in c-Myc-induced apoptosis of fibroblasts. Experimental Cell Research 317(18):2602-2615. 163. Ahmadi SE, Rahimi S, Zarandi B, Chegeni R, & Safa M (2021) MYC: a multipurpose oncogene with prognostic and therapeutic implications in blood malignancies. Journal of hematology & oncology 14(1):121. 164. Llombart V & Mansour MR (2022) Therapeutic targeting of "undruggable" MYC. EBioMedicine 75:103756. 165. Vita M & Henriksson M (2006) The Myc oncoprotein as a therapeutic target for human cancer. Seminars in cancer biology 16(4):318-330. 166. Beaulieu ME & Soucek L (2019) Finding MYCure. Molecular & cellular oncology 6(5):e1618178. 167. Madden SK, de Araujo AD, Gerhardt M, Fairlie DP, & Mason JM (2021) Taking the Myc out of cancer: toward therapeutic strategies to directly inhibit c-Myc. Mol Cancer 20(1):3. 168. Yin X, Giap C, Lazo JS, & Prochownik EV (2003) Low molecular weight inhibitors of Myc-Max interaction and function. Oncogene 22(40):6151-6159. 169. Sammak S, et al. (2019) Crystal Structures and Nuclear Magnetic Resonance Studies of the Apo Form of the c-MYC:MAX bHLHZip Complex Reveal a Helical Basic Region in the Absence of DNA. Biochemistry 58(29):3144-3154. 170. Massó-Vallés D & Soucek L (2020) Blocking Myc to Treat Cancer: Reflecting on Two Decades of Omomyc. Cells 9(4). 171. Raina K, et al. (2016) PROTAC-induced BET protein degradation as a therapy for castration-resistant prostate cancer. Proceedings of the National Academy of Sciences 113(26):7124-7129. 172. Hydbring P & Larsson LG (2010) Cdk2: a key regulator of the senescence control function of Myc. Aging 2(4):244-250. 173. Krüger K, et al. (2018) Multiple DNA damage-dependent and DNA damage- independent stress responses define the outcome of ATR/Chk1 targeting in medulloblastoma cells. Cancer letters 430:34-46. 174. Gallego MA, et al. (2004) Apoptosis-inducing factor determines the chemoresistance of non-small-cell lung carcinomas. Oncogene 23(37):6282-6291. 175. Gani OA & Engh RA (2010) Protein kinase inhibition of clinically important staurosporine analogues. Natural product reports 27(4):489-498. 176. Saisomboon S, et al. (2019) Antitumor effects of flavopiridol, a cyclin-dependent kinase inhibitor, on human cholangiocarcinoma in vitro and in an in vivo xenograft model. Heliyon 5(5):e01675. 177. Boulos JC, et al. (2023) Cynaropicrin disrupts tubulin and c-Myc-related signaling and induces parthanatos-type cell death in multiple myeloma. Acta Pharmacologica Sinica. 178. Ma D, et al. (2016) Deoxypodophyllotoxin triggers parthanatos in glioma cells via induction of excessive ROS. Cancer letters 371(2):194-204. 179. Zhao N, et al. (2015) YM155, a survivin suppressant, triggers PARP-dependent cell death (parthanatos) and inhibits esophageal squamous-cell carcinoma xenografts in mice. Oncotarget 6(21):18445-18459. 46 180. Wang M, et al. (2007) Atiprimod inhibits the growth of mantle cell lymphoma in vitro and in vivo and induces apoptosis via activating the mitochondrial pathways. Blood 109(12):5455-5462. 47 6. Appendices (published articles) Cell Biol Toxicol https://doi.org/10.1007/s10565-023-09813-w RESEARCH The cardiac glycoside ZINC253504760 induces parthanatos‑type cell death and G2/M arrest via downregulation of MEK1/2 phosphorylation in leukemia cells Min Zhou · Joelle C. Boulos · Sabine M. Klauck · Thomas Efferth Received: 23 February 2023 / Accepted: 23 May 2023 © The Author(s) 2023 Abstract Overcoming multidrug resistance (MDR) CCRF-CEM cells, while CDK1 was linked with the represents a major obstacle in cancer chemotherapy. downregulation of MEK and ERK. With flow cytom- Cardiac glycosides (CGs) are efficient in the treat- etry, ZINC253504760 induced G2/M phase arrest. ment of heart failure and recently emerged in a new Interestingly, ZINC253504760 induced a novel state- role in the treatment of cancer. ZINC253504760, a of-the-art mode of cell death (parthanatos) through synthetic cardenolide that is structurally similar to PARP and PAR overexpression as shown by western well-known GCs, digitoxin and digoxin, has not been blotting, apoptosis-inducing factor (AIF) translocation investigated yet. This study aims to investigate the by immunofluorescence, DNA damage by comet assay, cytotoxicity of ZINC253504760 on MDR cell lines and mitochondrial membrane potential collapse by and its molecular mode of action for cancer treat- flow cytometry. These results were ROS-independent. ment. Four drug-resistant cell lines (P-glycoprotein-, Furthermore, ZINC253504760 is an ATP-competitive ABCB5-, and EGFR-overexpressing cells, and TP53- MEK inhibitor evidenced by its interaction with the knockout cells) did not show cross-resistance to MEK phosphorylation site as shown by molecular ZINC253504760 except BCRP-overexpressing cells. docking in silico and binding to recombinant MEK Transcriptomic profiling indicated that cell death and by microscale thermophoresis in vitro. To the best of survival as well as cell cycle (G2/M damage) were the our knowledge, this is the first time to describe a card- top cellular functions affected by ZINC253504760 in enolide that induces parthanatos in leukemia cells, which may help to improve efforts to overcome drug Supplementary Information The online version resistance in cancer. contains supplementary material available at https://d oi. org/1 0.1 007/ s10565- 023- 09813-w. Keywords Cardiac glycosides · Leukemia · MEK M. Zhou · J. C. Boulos · T. Efferth ( )  inhibitors · Parthanatos · Synthetic derivative · * Department of Pharmaceutical Biology, Institute Transcriptomics of Pharmaceutical and Biomedical Sciences, Johannes Gutenberg University-Mainz, Staudinger Weg 5, Abbreviations 55128 Mainz, Germany AIF apoptosis-inducing factor e-mail: efferth@uni-mainz.de ATM ataxia-telangiectasia mutated S. M. Klauck  ATR ataxia-telangiectasia mutated and Division of Cancer Genome Research, German Cancer Rad3-related Research Center (DKFZ), German Cancer Consortium BCRP breast cancer resistance protein (DKTK), National Center for Tumor Disease (NCT), 69120 Heidelberg, Germany BSA bovine serum albumin Vol.: (0123456789) 1 Cell Biol Toxicol CDK1 cyclin-dependent kinase 1 and cardiac pump activity (Newman et al. 2008; Pras- CGs cardiac glycosides sas and Diamandis 2008). Chemically, CGs consist of DAPI 4’6-diamidino-2-phenylindole a steroid core, with a sugar portion at position 3 and an DSB double-strand break unsaturated lactone ring at position 17 (Fig. 1a). The ERK extracellular signal-regulated kinase two CGs classes are cardenolides and bufadienolides, IPA Ingenuity Pathway Analysis both of which have unsaturated five-or six-membered JC-1 5 ,5’6,6’-trtrachloro-1,1’3,3’-tetraethylbe- rings, respectively (El-Seedi et al. 2019a). nyimidazolylcarbocyanine iodide Since the 1980s, Stenkvist et al. noted that breast Kd dissociation constant cancer cells from women who received Digitalis ther- MAPK mitogen-activated protein kinase apy showed a lower risk of recurrence compared with MDR multidrug resistance untreated patients, suggesting CGs may have strong MEK m itogen-activated protein kinase kinase anticancer effects (Stenkvist et al. 1979, 1982). During MEKi MEK inhibitors the past three decades, the interest in developing CG MMP mitochondrial membrane potential as an anticancer drug has been steadily growing. The MNNG N-methyl-N’nitro-N-nitrosoguanidine initial anticancer mechanism of CGs is binding to Na+/ MST microscale thermophoresis K+-ATPase and altering signal transduction pathways NAD nicotinamide adenine dinucleotide to affect the growth of human malignant tumor cells, in PAR poly(ADP-ribose) particular glioblastoma, melanoma, and non-small cell PARP1 p oly(ADP-ribose) polymerase 1 lung cancer where Na+/K+-ATPase is vastly expressed PBS phosphate-buffer saline (Silva et al. 2021). This makes CGs promising chemo- PI propidium iodide therapeutic candidates for anticancer treatment (Ayogu PS phosphatidylserine and Odoh 2020). Subsequently, other modes of action qRT-PCR quantitative reverse transcription PCR of CGs have been elucidated, including activation of ROS reactive oxygen species ERK1/2, increased expression of cell cycle inhibitor p21 (Yuan et  al. 2019), inhibition of Akt and PI3K pathway, and inhibition of transcription factors such as Introduction NF-κB (Prassas and Diamandis 2008). Different types of cancer cells, such as leukemia (Zeino et al. 2015a, Cancer is one of the leading causes of death world- b; Saeed et  al. 2016), breast (Li et  al. 2021), mela- wide. Although chemotherapy made great progress as noma (Smolarczyk et al. 2018), lung (Hu et al. 2021), a major therapeutic strategy in oncology, the develop- pancreatic (Ha et al. 2021), liver (Reddy et al. 2019), ment of multidrug resistance (MDR) still limits drug and kidney carcinoma (Nolte et  al. 2017) induced efficiency and the successful treatment of patients. cell death upon treatment with CGs. A series of syn- Cardiac glycosides (CGs) are a class of steroid-like thetic CGs analogs raised interest because some of naturally derived products (Prassas and Diamandis them showed improved anticancer properties through 2008). CG-containing herbs have been used in folk structural modification (Ren et al. 2020; Ainembabazi medicine by native people of the ancient Egyptian cul- et  al. 2022; Wang et  al. 2017). Their synthetic route, ture, and Arab physicians treated heart and malignant structure-activity relationship (SAR), and the screen- diseases (Gurel et al. 2017). In the year 1785, extracts ing of anticancer activity are still in progress, and their from Digitalis purpurea containing CGs were first potential modes of action are not fully elucidated yet described for medical use in the Western world by Wil- and need further investigation. liam Withering (Bessen 1986). Nowadays, more than The mitogen-activated protein kinase (MAPK) cas- 100 CGs have been identified in plants and animals cades are key signaling pathways in transmitting signals (Mijatovic et  al. 2007). Digitalis-based drugs such as from cell surface receptors into the inside of the cell digitoxin and digoxin are still in clinical use as oral to regulate numerous cellular activities, including cell medications for treating heart failure and atrial arrhyth- proliferation, survival, and differentiation (Roberts and mias. In the case of myocardial fibrosis, CGs bind to Der 2007). The Ras-Raf-MEK-ERK signaling is the and inhibit N a+/K+-ATPase, allowing calcium to ele- most extensively studied pathway, which is aberrantly vate contraction and improve myocardial contraction activated in nearly one-third of all human cancers (Kun Vol:. (1234567890) 1 3 Cell Biol Toxicol et al. 2021). MEK is playing a central role in transmit- All these steps trigger parthanatos, which is distinct from ting signals from Raf to ERK, making it an attractive apoptosis and necrosis. Parthanatos has been involved drug target. MEK has two isoforms, MEK1 and MEK2 in retinal disease, ischemia-reperfusion injury, and neu- (Barbosa et al. 2021). The activation segments of pro- rodegenerative diseases including Alzheimer’s disease tein kinase commonly contain phosphorylation sites. and Parkinson’s disease (Wang and Ge 2020). Apply- The activation of MEK1 by Raf requires the phospho- ing PARP inhibitors to suppress parthanatos opens new rylation of two serine residues, S218 and S222 (Zheng treatment possibilities in these diseases. Several cancer and Guan 1994). The corresponding residues on cells such as glioma cells have a greater level of PARP MEK2 are S222 and S226. While both serine residues and are negatively correlated with patient survival rates are necessary for activation, the dephosphorylation (Galia et  al. 2012). Instead, parthanatos through hyper- of either one would fully inactivate MEK (Roskoski activation of PARP has been induced by chemotherapies 2012). MEK mutations are rare in cancer. (Ullah et al. in esophageal cancer and glioma cells (Zhao et al. 2015; 2022). However, constitutive MEK activation has been Ma et al. 2016), suggesting parthanatos as a promising observed in 50 tumor cell lines (Hoshino et al. 1999), strategy to kill cancer cells. resulting in cell transformation and, eventually, tumori- We recently screened a series of phytochemicals genesis (Cowley et al. 1994; Mansour et al. 1994). Cur- against MDR cancer cells, digitoxin and digoxin were rently, there are four FDA-approved MEK inhibitors ranked at the top with promising growth-inhibitory (MEKi), i.e., trametinib, cobimetinib, binimetinib, and potential, suggesting CG compounds have the crucial selumetinib (Frémin and Meloche 2010; Singh et  al. benefit to anticancer effect (Khalid et  al. 2022). The 2021). Tumor cells driven by mutated Raf are sensitive aim of this study was to explore the anticancer activ- to MEKi in vitro and in vivo. Significantly, the com- ity of a modified CG compound ZINC253504760 that binations of Raf and MEK inhibitors (e.g., dabrafenib could be a potential anticancer agent. We investigated and trametinib, vemurafenib and cobimetinib) were par- the cytotoxicity of ZINC253504760 toward MDR can- ticularly successful in B RAFV600E mutant melanoma cer cells and studied the molecular mode of action. and showed better efficiency than using a Raf inhibitor ZINC253504760 revealed the highest sensitivity in alone (Flaherty et  al. 2012). More importantly, MEK CCRF-CEM leukemic cells. After microarray-based is the only known activator of ERK up to date, which mRNA profiling, we applied flow cytometry to inves- can inhibit ERK activation and downstream molecules, tigate cell cycle, apoptosis and mitochondrial mem- thereby inhibiting cell proliferation and survival (Caunt brane potential. Molecular docking, western blotting et  al. 2015). This makes MEK a prime target sup- and immunofluorescence were used to confirm the pressing cell signaling. Therefore, targeting MEK is a potential targets and the major mode of cell death. rational solution to efficiently silence the MAPK signal- ing pathway in cancers. Parthanatos is a programmed cell death mode that Material and methods depends on poly(ADP-ribose) polymerase 1 (PARP1) hyperactivation independent of caspase activity. Mechan- Compound ically, PARP is rapidly activated by DNA damage, such as ultraviolet light, reactive oxygen species (ROS), or Compound ZINC253504760 (IPUAC name: alkylating agents (e.g., N-methyl-N’nitro-N-nitrosoguan- 3-[(3S,5S,8S,9R,10S,12R,13S,14S,17S)-3-[(2S,4R,5R,6R)- idine (MNNG)). PARP produces excess poly(ADP- 5-[(2R,4R,5R,6R)-5-[(2S,4S,5S,6R)-4,5-dihydroxy-6- ribose) (PAR), which migrates from the nucleus to the -methyloxan-2-yl]oxy-4-hydroxy-6-methyloxan-2-yl] cytoplasm. PAR binds to mitochondrial membrane pro- oxy-4-hydroxy-6-methyloxan-2-yl]oxy-12,14-dihy- teins, causing apoptosis-inducing factor (AIF) to translo- droxy-10,13-dimethyl-1,2,3,4,5,6,7,8,9,11,12,15,16,17- cate from mitochondria to the nucleus, which eventually tetradecahydrocyclopenta[a]henanthrene-17-yl]-2H- leads to large-scale fragmentation and chromatin conden- furan-5-one) was purchased from SPECS (Zoetermeer, sation. Simultaneously, PARP overactivation causes nic- Netherlands) (#SPECS AP-163/40806811). The chemical otinamide adenine dinucleotide (NAD+) and ATP dele- structure is shown in Fig. 1A. The stock solution (20 mM) tion, which further leads to mitochondrial depolarization. was prepared in DMSO and stored at -20 ℃. Vol.: (0123456789) 1 3 Cell Biol Toxicol Vol:. (1234567890) 1 3 Cell Biol Toxicol ◂Fig. 1 Dose-response curves of ZINC253504760 towards Following 72 h of incubation, 20 μL 0.01% resazurin drug-sensitive and drug-resistant cell lines as determined by (Promega, Mannheim, Germany) was added and incu- the resazurin assay. a Steroid nucleus, the core structure of car- bated at 37 °C for 4 h. The fluorescence signal was diac glycoside, and the chemical structure of ZINC253504760. b Growth inhibition of ZINC253504760 towards CCRF-CEM measured with the Infinite M200 Pro-plate reader and ABCB1/P-glycoprotein expressing CEM/ADR5000 leuke- (Tecan, Crailsheim, Germany). The experiment was mia cell lines. c Growth inhibition of MDA-MB-231-pcDNA3 repeated in triplicates independently and with six rep- and their transduced MDA-MB-231-BCRP clone 23 breast licates of each concentration. The growth inhibitory cancer cell lines. d Growth inhibition of HEK293 and their transduced HEK293/ABCB5 human embryonic kidney cancer effect of treatment was presented as the percentage of cell lines. e Growth inhibition of HCT116 (p53+/+) and p53-/- cell viability, plotted as a dose-response curve. The knockout HCT116 ( p53-/-) colon cancer cell lines. f Growth fifty percent inhibition concentration ( IC50) value was inhibition of U87MG wild-type and their transfected U87MG. calculated from the dose-response curve using Micro- ΔEGFR glioblastoma cell lines. The results are mean values ± SD of three independent experiments soft Excel 2021. The figures were generated using Prism 8 GraphPad Software (GraphPad Software Inc., San Diego, CA, USA). Cell culture RNA extraction The source and culture of cell lines used in this study were reported before (Saeed et  al. 2019; Doyle et  al. The procedure of RNA extraction has been previously 1998; Rudbari et al. 2022; Dawood et al. 2020b). Two reported (Mahmoud et al. 2018). One million CCRF- leukemia cell lines, drug-sensitive CCRF-CEM and CEM cells were treated in duplicates with 1-fold I C50 multidrug-resistant CEM/ADR5000 were cultured in of ZINC253504760 or DMSO (solvent control) and RPMI 1640 medium. Doxorubicin (5,000 ng/mL) has incubated for 24 h, followed by total RNA extraction been added to CEM/ADR5000 cells every 14 days to with InviTrap®Spin Universal RNA Mini Kit (Invitek maintain the resistance. Molecular, Berlin, Germany). The lysis solution was Human HEK293 embryonic kidney cells and prepared as 350 µL Lysis Solution TR containing 1% HEK293/ABCB5, breast cancer cells MDA-MB-231- β-mercaptoethanol for each sample. The RNA concen- pcDNA3 and MDA-MB-231-BCRP clone 23, wild- trations were quantified by a Nanodrop spectrophotom- type colon cancer cells HCT116 (p53+/+) and HCT116 eter (Nanodrop Technologies, Wilmington, DE, USA). (p53-/-), glioblastoma multiform cells U87MG and U87MG.∆EGFR was maintained in DMEM medium Microarray gene expression profiling and Ingenuity (Invitrogen, Darmstadt, Germany). Both media were sup- Pathway Analysis (IPA) plied with 10% fetal bovine serum (FBS) and 1% peni- cillin-streptomycin (Invitrogen, Darmstadt, Germany). The duplicate extracted total RNA was further used Cells were cultured at 37 °C in a humidified environment in microarray hybridizations, which were carried out with 5% CO2. In addition, 400 μg/mL geneticin was con- using Affymetrix GeneChips ® with human ClariomTM tinuously added to resistant cells HCT116 (p53-/-) and S assays (Affymetrix, Santa Clara, CA, USA) at the U87MG.∆EGFR, and 800 ng/mL geneticin for MDA- Genomics and Proteomics Core Facility at the German MB-231-BCRP clone 23 every two weeks. Cells in the Cancer Research Center (DKFZ, Heidelberg). logarithmic growth phase were used in the experiments. The differential gene expressions between the con- trol and ZINC253504760-treated groups were ana- Growth inhibition assay lyzed by applying Chipster software as we previously reported (Hegazy et  al. 2021; Kadioglu et  al. 2021). The growth inhibitory activity of ZINC253504760 Robust Multi-array Average (RMA) method was used was determined with the resazurin reduction assay to normalize the data. Genes were filtered using the (Saeed et  al. 2018). A 100 μL medium contain- percentage to exclude genes with a standard deviation ing suspension cells (1×104 cells/well) or adherent of 0.5 from the gene mean. Then, the missing value cells (5 × 103 cells/well) was seeded in a 96-well were removed. The subsequent assessment of signifi- plate. Cells were treated with a series of concentra- cantly deregulated genes used empirical Bayes t-test (p tions of ZINC253504760 diluted in 100 μL medium. < 0.05) (accessed in July 2021). Vol.: (0123456789) 1 3 Cell Biol Toxicol The significantly deregulated genes were analyzed based on forward and side scatter properties (FSC-A/ with the Ingenuity Pathway Analysis (IPA) software SSC-A), the single cells were gated (FL2-A/FL2-H) in a (Qiagen, Redwood City, CA, USA) (accessed in August linear manner to remove doublets or debris. 2021). The core analysis tool was used to generate the Annexin V/PI staining was applied to detect and canonical pathway, cellular functions, and networks that quantify apoptotic and necrotic cells (Reutelingsperger were affected by ZINC253504760 treatment. and van Heerde 1997; Vermes et al. 1995). The proto- col has been recently reported by us (Saeed et al. 2022; qRT-PCR Rudbari et al. 2022). The same amount of CCRF-CEM cells was treated with ZINC253504760 at different The primers design and protocol were previously reported concentrations, DMSO (negative control), or vincris- (Mahmoud et  al. 2018). One microgram of extracted tine (positive control, 5 μM) for 24, 48 or 72 h. Cells RNA was converted into cDNA with the Luna S criptTM were stained with Annexin V and PI (BioVersion/Bio- RT SuperMix Kit (New England Biolabs, Frankfurt, cat, Heidelberg, Germany) in the dark, and further ana- Germany). The cDNA sample, both extracted from lyzed using a BD AccuriTM C6 Flow Cytometer. All untreated cells (DMSO, 24 h) and treated cells ( IC50, experiments were performed three times. 24 h), were used for the verifications in qRT-PCR. Four genes, including two top-upregulated genes (CD82 and Fluorescence microscopy of the microtubule H2AC18) and two top-downregulated genes (HSP90AA1 cytoskeleton and HSP90AB1), were chosen to validate the microar- ray results. Eight genes (HIPK2, PPM1D, CDK1, Wee1, Briefly, human U2OS osteosarcoma cells were seeded CKS1, CKS2, TP53, and CDK7) involved in cell cycle in μ-Slide 8 Well (30,000 cells/well) (ibidi, Gräfelfing, regulation were selected to verify the mechanism. Primer Germany) and kept overnight to allow cells to adhere to sequences are presented in Supplementary Table S1. The the plate. Then cells were treated with ZINC253504760 real-time quantitative polymerase chain reaction (qRT- (IC50, 2 × I C50, and 4 × I C50) or DMSO as a negative PCR) was carried out with the CFX384TM Touch Real- control. After 24 h, cells were washed with PBS, fixed Time PCR Detection System (Bio-Rad, Munich, Ger- with 4% paraformaldehyde, and stained with 1 µg/ many). The expressions were normalized using GAPDH mL of 4’6-diamidino-2-phenylindole (DAPI, Sigma- as an internal control gene. The 2ΔΔCt method was applied Aldrich, Darmstadt, Germany) in the dark. Subse- to calculate the fold change of each gene between treated quently, the slides were immersed in Mounting Medium cells and untreated cells (Livak and Schmittgen 2001). (ibidi, Gräfelfing, Germany). Imaging was carried out using an AF7000 widefield fluorescence microscope Cell cycle analysis and detection of apoptosis (Leica Microsystems, Wetzlar, Germany). Images were analyzed with Image J software (National Institute of The cell cycle analysis was previously described using Health, Bethesda, MD, USA). The methods have been propidium iodide (PI) staining (Abdelfatah et  al. 2020; described by us (Boulos et al. 2021). Rudbari et  al. 2022). CCRF-CEM cells (1 × 1 06 cells/ well) were treated with ZINC253504760 at 0.5 × IC50, Western blotting I C50, 2 × IC50 and 4 × IC50, DMSO (solvent control) or doxorubicin (positive control), respectively. After corre- CCRF-CEM cells were treated with ZINC253504760 for sponded incubation (24 h, 48 h or 72 h), cells were fixed the indicated times, cells were washed twice with PBS. with ethanol and stored at -20 ℃ for 24 h. Then, samples Total proteins were extracted with M-PER Mammalian were centrifuged (4000 rpm, 10 min) and re-suspended Protein Extraction Reagent (Thermo Fisher Scientific, in 500 μL cold PBS containing 20 µg/mL RNase (Roche Darmstadt, Germany). Nuclear and cytoplasmic proteins Diagnostics, Mannheim, Germany), followed by staining were extracted using NE-PER Nuclear and Cytoplasmic with 50 μg/mL PI (Sigma-Aldrich, Darmstadt, Germany). Extraction Reagents kit (Thermo Fisher Scientific). CER The measurement was performed using a BD A ccuriTM I, CER II, and NER reagents were added as the volume C6 Flow Cytometer (Becton-Dickinson, Heidelberg, ratio at 200:11:100 μL following the manufacturer’s Germany). The results were analyzed using FlowJo soft- instructions. Both lysis buffer contained 1% Halt Protease ware (Celeza, Olten, Switzerland). Cells were firstly gated Inhibitor Cocktail and phosphatase inhibitor (Thermo Vol:. (1234567890) 1 3 Cell Biol Toxicol Fisher Scientific). Protein concentrations were quantified stained with 10 µL diluted JC-1 per well (1 µL JC-1 by a Nanodrop spectrophotometer. in stock: 9 µL medium) and incubated at 37 ℃ for 15 Equal amounts of protein extracts (30 µg) were separated min in the dark. Subsequently, cells were washed with by 10% SDS-PAGE and blotted onto a polyvinylidene fluo- 200 µL Cell-based assay buffer (Biomol, Hamburg, ride membrane ( ROTIPVDF®). The membrane was blocked Germany) and centrifuged at 400 × g for 5 min twice. in TBST buffer containing 5% bovine serum albumin (BSA) Finally, cells were suspended with 100 µL Cell-based for 1 h. Afterward, the membranes were incubated with spe- assay buffer and detected using a BD LSR Fortessa cific primary antibodies (anti-p44/42 MAPK (Erk1/2) rab- SORP equipment. JC-1 is the most specific fluores- bit antibody (1:1000, Cell Signaling Technology, Leiden, cence probe for measuring changes in mitochondrial The Netherlands), anti-phospho-p44/42 MAPK (Erk1/2) membrane potential. J- aggregates or monomers are (Thr202/Tyr204) rabbit antibody (1:1000, Cell Signaling the two detectable forms with emissions of JC-1 and Technology), anti-MEK1/2 rabbit antibody (1:1000, Cell can be detected by flow cytometry (Smiley et  al. Signaling Technology), anti-phospho-MEK1/2 (Ser217/221) 1991). In healthy cells with higher membrane poten- rabbit antibody (1:1000, Cell Signaling Technology), anti- tial, J aggregates emit red fluorescence at 520-570 AIF rabbit antibody (1:1000, Cell Signaling Technology), nm and were collected using a 586/15 bandpass fil- anti-Lamin B1 monoclonal antibody (1:10000, Proteintech, ter. While the fluorescence properties of the probe are Planegg-Martinsried, Germany), anti-PAR mouse anti- altered according to the aggregation. In lower mem- body (1:1000, Merck, Darmstadt, Germany), anti-caspase brane potential, JC-1 is predominantly a monomer 3/p17/p19 polyclonal antibody (1:1000, Proteintech), anti- (dead cells) that emits green fluorescence at 488 nm PARP rabbit antibody (1:1000, Cell Signaling Technology), and was collected using a 530/30 bandpass filter. All anti-phospho-histone H2A.X (Ser139) antibody (1:1000, experiments were performed in triplicates. The FSC Cell Signaling Technology), anti-GAPDH rabbit antibody files were analyzed by the FlowJo software (Celeza). (1:1000, Cell Signaling Technology), anti-β-actin rabbit antibody (1:1000, Cell Signaling Technology),  anti-p62, Immunofluorescence microscopy of AIF SQSTM1 polyclonal antibody (1:1000, Proteintech), or anti- translocation Beclin 1 polyclonal antibody (1:1000, Proteintech)) over- night at 4 ℃. Finally, the membrane was incubated in anti- Three concentrations of ZINC253504760 (IC50, 2 mouse IgG or anti-rabbit IgG, HRP-linked antibody (1:2000, × I C50, or 4 × IC50) or DMSO alone were treated in Cell Signaling Technology) for 1 h at room temperature. CCRF-CEM cells for 12 h. Cells were harvested and Horseradish peroxidase (HRP) substrate ( LuminateTM Clas- washed once with washing buffer (1% FBS in PBS) sico, Merck Millipore, Schwalbach, Germany) was used to and kept on ice. Then, 10,000 cells of each sample detect the immunoreactive band. The protein was visualized were cytospinned on cover slides (Thermo Fisher by an Alpha Innotech FluorChem Q system (Biozym, Old- Scientific, Dreieich, Germany). Subsequently, cells endorf, Germany). The bands were quantified using Image were fixed with 4% paraformaldehyde, then permeabi- J software (National Institutes of Health). Relative protein lized with 1% Triton X-100 in PBS. Afterward, sam- expression was normalized to GAPDH or β-actin. ples were kept in the blocking buffer containing 10% FBS and 1% BSA for 1 h. The primary antibody AIF Analysis of mitochondrial membrane potential (1:400, Cell Signaling Technology) was diluted in PBS (MMP) and applied to the slides overnight in a humidified chamber at 4 ℃. After rinsing three times with wash- To analyze the mitochondrial membrane potential, ing buffer, the secondary antibody (1:700) was applied 5,5’6,6’-trtrachloro-1,1’3,3’-tetraethylbenyimida- to the samples for 1 h in the dark at room temperature. zolylcarbocyanine iodide (JC-1; Biomol, Hamburg, Then, 1 µg/mL DAPI was added to the samples for 5 Germany) staining was used as previously reported min to stain cell nuclei. At last, cells were rinsed five (Özenver et al. 2018). Briefly, aliquots of 104 CCRF- times with washing buffer and immersed in the Mount- CEM cells were seeded in a 96-well plate and treated ing Medium  (ibidi). Images were taken by a Leica with 0.5-, 1-, 2- and 4-fold IC50 of ZINC253504760, AF7000 widefield fluorescence microscope (Leica or DMSO as a negative control, or vinblastine (1 μM) Microsystems) and analyzed using ImageJ software as a positive control, respectively, for 24 h. Cells were (National Institutes of Health). Vol.: (0123456789) 1 3 Cell Biol Toxicol Single cell gel electrophoresis (comet assay) trametinib to MEK1 and MEK2 was carried out using AutoDock 4.2.6 (The Scripps Research Institute, CA, The comet assay is one of the common methods for evalu- USA) (Saeed et  al. 2018). AutoDockTools 1.5.6 was ating DNA damage. Under electrophoresis, damaged DNA used for converting the proteins and ligands to PDBQT or denatured cleaved DNA fragments migrate from the format. The grid box was set to cover the whole pro- intact cells, creating a ‘‘comet tail’’ under the microscope. tein. Visual Molecular Dynamics (VMD) software was The OxiselectTM Comet Assay Kit (3-Well Slides) (Cell used to create the visualization of interactions. Biolabs/Biocat, Heidelberg, Germany) was applied to per- form the comet assay as recently described by us (Elbad- awi et al. 2023). Briefly, CCRF-CEM cells were plated in ROS detection a 6-well plate (106 cells per well). Cells were treated with ZINC253504760 at 0.088 µM, 0.15 µM, and DMSO (neg- Briefly, CCRF-CEM cells (2 × 106 cells/well) were ative control), respectively, for 3 h. H2O2 (50 µM) as posi- seeded and treated with ZINC253504760 at different tive control was added to the cells for 1 h. Collected cells indicated concentrations or DMSO (negative control) were centrifuged at 3,000 × g for 10 min. A ratio of 1:6 for 12 h, and 24 h, respectively. Cells were harvested and was used to mix cells ( 105 cells/ml) suspended in cold PBS washed with PBS, then resuspended in 1 mL PBS and with melting agarose at 37 ℃. After samples were spread incubated with 10 µM 2’7-dichlorodihydrofluorescein on comet slides and dried, the pre-chilled lysis buffer and diacetate ( H2DCFH-DA) (Sigma-Aldrich, Germany) at pre-chilled alkaline electrophoresis solution buffer were 37 ℃ for 30 min. 10 µL H 2O2 at the stock concentra- applied to the slides in the dark. Slides were then placed tion (positive control) (Sigma-Aldrich, Germany) was horizontally in alkaline electrophoresis solution buffer in treated in cells during the incubation at 15 min. The the electrophoresis chamber. Twenty Volts of voltage were measurement was performed on a BD AccuriTM C6 delivered to the chamber for 20 min. After that, the slides Flow Cytometer (Becton-Dickinson). Histograms were were washed by pre-chilled distilled water twice, followed analyzed using FlowJo software (Celeza). All the exper- by cold 70% ethanol. Vista Green DNA dye was diluted at iments were repeated three times independently. The a ratio of 1:10,000 in TE buffer and added in the slides (100 protocol was described by us (Wu and Efferth 2015). µL/well). DNA damage was observed by EVOS digital inverted microscope (Life technologies GmbH, Darmstadt, Microscale thermophoresis Germany). Fifty comets in each treatment were randomly selected and analyzed by OpenComet in Image J soft- The ligand-protein interactions between ZINC253504760 ware (National Institutes of Health). Tail DNA% was meas- and MEK1 or MEK2 were performed by microscale ured as a parameter for DNA damage (Gyori et al. 2014). thermophoresis (MST) as described (Dawood et  al. 2020a). The recombinant human MEK1 protein (Abcam, Berlin, Germany) and the MEK2 protein (Sino Biologi- Molecular docking cal, Beijing, China) were labeled using the M onolithTM NT.115 Protein Labeling Kit BLUE-NHS (NanoTemper The PDB files of MEK1 and MEK2 were down- Technologies, Munich, Germany). Subsequently, the loaded from the RCSB Protein Data Bank (PDB MEK1 protein (1530 nmol/L) and the MEK2 protein codes: 1S9J and 1S9I, respectively) (Ohren et  al. (990.1 nmol/L) were titrated against 16 different concen- 2004). ZINC253504760 and trametinib in SDF for- trations of ZINC253504760 in assay buffer. The fluores- mat were downloaded from the ZINC 15 database cence signal measurement was performed using a Mono- (ZINC253504760) and PubChem, respectively, and lith NT.115 instrument (NanoTemper Technologies) and were converted to PDB files. In addition, consider- the samples were loaded to the standard glass capillaries. ing GCs in human metabolism are normally hydro- The results were shown with 60% LED power and 10% lyzed to their deglycosylated congeners by removing MST power for MEK1, 40% LED power and 10% MST sugar moiety (Jortani and Valdes 1997), therefore, the power for MEK2. The curves of ZINC253504760 bind- deglycosylated form of ZINC253504760 was also per- ing to both proteins were generated using MO. affinity formed by molecular docking. The in silico binding analysis software (NanoTemper Technologies), and the of ZINC253504760 and its deglycosylated form, and dissociation constant (Kd) was calculated. Vol:. (1234567890) 1 3 Cell Biol Toxicol Results Table 1 IC50 values of ZINC253504760 in different drug-sen- sitive and drug-resistant cell lines Cytotoxicity assay Cell lines IC50 (μM) Resistance ratio ZINC253504760 revealed cytotoxicity against a panel CCRF-CEM 0.022 ± 0.002 0.95 of drug-sensitive and -resistant cell lines with IC50 val- CEM/ADR5000 0.021 ± 0.001 ues in the range from 0.022 ± 0.002 µM (CCRF-CEM) MDA-MB-231-pcDNA3 18.76 ± 3.50 > 5.33 to 25.95 ± 0.26 µM (HEK293) (Fig.  1 and Table  1). MDA-MB-231-BCRP clone 23 > 100 Except for MDA-MB-231-BCRP clone 23 that was HEK293 25.95 ± 0.26 0.99 cross-resistant, the other four drug-resistant cell lines HEK293/ABCB5 25.57 ± 1.52 did not show cross-resistance to ZINC253504760. HCT116 (p53+/+) 0.22 ± 0.03 0.81 The resistant ratios were 0.95 (CEM/ADR5000), HCT116 (p53-/-) 0.18 ± 0.03 0.99 (HEK293/ABCB5), 0.81 (HCT116 p53-/-), and U87MG 0.055 ± 0.004 0.98 0.98 (U87.MGΔEGFR), respectively. Evidently, U87MG.ΔEGFR 0.054 ± 0.01 ZINC253504760 induced cytotoxicity on both leuke- mia cells and glioblastoma cells in the nanomolar range. Among the panel of cell lines tested, ZINC253504760 exerted the most lethal effect with the lowest I C50 value upregulated genes CD82 and H2AC18 in the microar- in CCRF-CEM cells, we used CCRF-CEM as a model to ray data were upregulated in qRT-PCR. HSP90AA1 explore the molecular modes of action of ZINC253504760. and HSP90AB1, the top two downregulated genes in the microarray data also showed downregulation in Microarray hybridization and pathway analysis qRT-PCR. The correlation coefficient (R) between mRNA expression values measured by microarray and Gene expression analysis was performed to investi- qRT-PCR was 0.95 (Pearson correlation test), which gate the potential mode of action of ZINC253504760. indicated a high correlation between the two methods Therefore, total RNA was isolated to conduct transcrip- (Fig. 2e). tome-wide microarray assays after CCRF-CEM cells The IPA-based network analysis revealed that cas- were exposed to ZINC253504760 at the I C50 value pase 3 expression was increased (Fig. 3a). Fig. 3b shows (0.022 µM) or DMSO as negative control for 24 h. The the increased expression of the cell cycle biomarkers genes that were differentially expressed were further including CDK1, Wee1, Cyclin A, and Cdc25c, while subjected to IPA for signaling pathway analysis. MAP2K1/2 (MEK1/2) and ERK1/2 were downregu- IPA predicted that the “mitotic role of polo-like lated. Therefore, we conducted cell cycle and apoptosis kinase” and “cell cycle: G2/M DNA damage” could experiments, and explored the cell death mechanisms be potential molecular mechanisms among the panel of ZINC253504760. We chose MAP2K1/2 (MEK1/2) of canonical pathways (Fig.  2a). “Cancer and hemato- to further investigate whether these two proteins are tar- logical disease” appeared as important diseases affected gets of ZINC253504760. Moreover, Fig.  3c shows the by ZINC253504760 (Fig.  2b). Notably, the top cellu- network of top downregulated genes HSP90AA1 and lar function in Fig.  2c indicated that ZINC253504760 HSP90AB1 associated with the decreased expression of may affect “cell death and survival”, “DNA replication, c-Src. recombination, and repair”, “cell cycle”, as well as “cel- lular growth and proliferation”. Moreover, there were a Cell cycle analysis total of 916 genes that were deregulated between treated and untreated cells according to the expression fold To study cell cycle arrest, CCRF-CEM cells were change values. Among them, 368 genes were downregu- treated with ZINC253504760 and incubated for three lated, while 548 genes were upregulated (Supplemen- different timepoints. Fig. 4a shows that after 24 h treat- tary Tables S2 and S3). The technical verification was ment, the G2/M phase peak was elevated at all concen- quantified with four selected genes (CD82, H2AC18, trations tested. After 48 and 72 h treatment, the arrest of HSP90AA1, and HSP90AB1) with real-time RT-PCR cells in the G2/M phase was even more evident, and the to validate the microarray results (Fig.  2d). The top increased percentages of the G2/M cell fraction were Vol.: (0123456789) 1 3 Cell Biol Toxicol Fig. 2 Microarray gene expression profiling and Ingenu- by the red boxes. d Technical verification of four selected ity Pathway Analysis (IPA) in CCRF-CEM cells treated with genes by qRT-PCR analysis in CCRF-CEM cells treated with ZINC253504760. a Top canonical, b disease, and c cellular I C50 of ZINC253504760  for 24 h. e Person correlation coef- functional pathways affected by ZINC253504760 were marked ficient of microarray and qRT-PCR data statistically significant compared to untreated cells (p < To further confirm this result, 8 genes of the G2/M 0.05). Therefore, the flow cytometric analyses verified arrest pathway that were deregulated in the microar- the IPA prediction of G2/M cell cycle arrest. ray analysis were selected for biological verification Vol:. (1234567890) 1 3 Cell Biol Toxicol Fig. 3 Prediction of functional networks by transcriptome- the cell cycle linked with the downregulation of MEK1/2 and wide microarray and IPA analysis of ZINC253504760-treated ERK, and c The red circles highlight the downregulation of Src CCRF-CEM cells. a The red circles highlight the upregulation linked with the downregulation of HSP90AA1 and HSP90AB1 of caspase 3, b The red circles highlight the genes related to using qRT-PCR. As shown in Fig.  4b, HIPK2, Influence on microtubules PPM1D, CDK1, Wee1, CKS1, and CKS2 were upreg- ulated, whereas TP53 and CDK7 were downregulated The G2/M arrest caused by ZINC253504760 raises the compared with housekeeping gene GAPDH. These question of how this compound impacts on the micro- results were consistent between qRT-PCR and micro- tubules. Tubulin Alpha 1b (TUBA1B) is a microtubule array data. protein (Liu et  al. 2017). A characteristic microtubule Vol.: (0123456789) 1 3 Cell Biol Toxicol Vol:. (1234567890) 1 3 Cell Biol Toxicol ◂Fig. 4 Cell cycle analysis and influence on microtubules. of 4.18%-11.1%. At 72 h, the fractions of late apoptotic a Flow cytometric cell cycle analysis in CCRF-CEM cells cells were 7.98%-13.1%. Vincristine induced late apop- treated with different concentrations of ZINC253504760, or DMSO (control), or doxorubicin (positive control) for 24, 48, totic cell death with 14.3% after 24 h treatment, subse- or 72 h. b The cell cycle genes (red dotted boxes) predicted quently increased to 74.2% after 72 h. It can be clearly from transcriptome-wide microarray and IPA treated with seen that the fraction of late apoptotic cells treated with ZINC253504760 after 24 h for biological verification by qRT- ZINC253504760 was not significant at all times and PCR. The results are represented as mean values ± SD of three independent experiments. c Immunofluorescence analysis of concentrations tested. More than 80% of cells were U2OS cells treated with ZINC253504760, or DMSO (con- non-apoptotic. trol), and stained for DAPI (blue) and α-tubulin-GFP (green) Next, we investigated whether autophagy was after 24 h. The graph shows the mean fluorescence intensity of related to cell death, and the expression level of p62 U2OS cells expressing α-tubulin-GFP tubulin. Statistics analy- sis was done by paired student’s t-test, * p < 0.05, if compared and Beclin was measured by western blotting. Fig. 5b with untreated cells shows that p62 and Beclin were both downregulated. Especially after 48 h, the decline of both biomarkers was more significant. pattern was visible in the human U2OS cell line that Therefore, these results did not provide evidence endogenously expresses the fluorescent fusion pro- that apoptosis or autophagy were the predominant tein (GFP-TUBA1B, Green Fluorescent Protein (GFP) modes of cell death, and other cell death modes may gene attached to the genomic TUBA1B gene). There- contribute to the cytotoxicity observed in the resa- fore, U2OS cells were treated with different concentra- zurin assays with ZINC253504760. tions of ZINC253504760. Fig.  4c illustrates the effect of ZINC253504760 observed on the cellular microtu- bule network. In untreated U2OS cells, the microtubules Assessment of mitochondrial membrane potential extended continuously in the cytoplasm and polymerized to form an extensive intracellular network in addition to Mitochondria are essential for ATP generation for life the nucleus. By contrast, ZINC253504760 significantly and are the key players of cell death under cell stress disrupted microtubule distribution and reduced the mass conditions. Even if the major form of cell death regu- of the microtubule network. Specifically, the reduced lated by mitochondria is apoptosis, other types of cell microtubules aggregated around the nucleus, but the death also have been implicated with mitochondria aggregation diminished with increasing concentrations (Bock and Tait 2020). Therefore, we investigated the of the treatment, finally resulting in dense microtubules MMP to understand whether it is affected. CCRF- being less present in the cell periphery. The thickness of CEM cells were treated with different concentrations microtubules weakened compared to untreated cells. It is of ZINC253504760 or vinblastine (positive control, the same effects as vincristine (postitive control) as we 1 µM) and measured by a flow cytometry after 24 h. reported (Khalid et  al. 2022). Hence, these results fur- As shown in Fig.  5c, the percentages of dead cells, ther strengthened the observation that ZINC253504760 which shift from red fluorescence (unaltered potential) induced G2/M phase arrest in the cell cycle. to green fluorescence (defective potential), were in a range of 31.1%-57.0%. All treatments showed signifi- Apoptosis and autophagy detection cant percentages of dead cells compared with untreated cells (p < 0.05). Especially, the 4 × IC50 treated sam- As cell death and survival appeared in IPA as the first ple showed a comparable effect (57.0%) to vinblastine cellular function, we aimed to investigate the mode of (53.9%). Hence, we conclude that ZINC253504760 cell death induced by ZINC253504760. Apoptosis by caused mitochondrial dysfunction leading to cell death. annexin V-PI staining on the flow cytometer was first investigated. As shown in Fig. 5a, the percentage of late Western blot analysis of parthanatos apoptotic cells showed a slight increase in a time- and concentration-dependent manner after treating CCRF- To investigate other forms of cell death, we first CEM cells with ZINC253504760 (0.5 × IC50, IC50, examined caspase 3 since the functional network in 2 × I C50, and 4 × I C50) for 24 h, 48 h, or 72 h, respec- IPA software showed its upregulation (Fig.  3a). As tively. At 48 h, the late apoptotic cells were in the range depicted in Fig.  6a, caspase 3 expressions appeared Vol.: (0123456789) 1 3 Cell Biol Toxicol Vol:. (1234567890) 1 3 Cell Biol Toxicol ◂Fig. 5 Assessment of apoptosis, autophagy and mitochon- of control CCRF-CEM cells. However, if cells were drial membrane potential. a Flow cytometric analysis in treated with ZINC253504760 with IC , 2 × I C , CCRF-CEM cells treated with different concentrations of 50 50 ZINC253504760, or DMSO (control) or vincristine (positive or 4 × IC50 for 12 h, AIF accumulated in the nucleus, control) for 24, 48 or 72 h. The graph shows the mean frac- which was consistent with the western blotting results. tion of CCRF-CEM cells. The data represent as mean ± SD These AIF translocation findings further confirmed that of three independent experiments. b Western blot analysis of ZINC253504760 induced a parthanatos-type cell death. the proteins involved in autophagy in CCRF-CEM cells treated with different concentrations of ZINC253504760 for 24 or 48 h. Data represent relative expression intensity to GAPDH. Single cell gel electrophoresis (comet assay) Statistics analysis was done by paired student’s t-test, * p ≤ 0.05, ** p ≤ 0.001, if compared to DMSO untreated cells. AIF enters the nucleus, triggers cell death by binding Data represent as mean values ± SEM of three independent experiments. c Representative images of JC-1 fluorescence to DNA and leads to DNA fragmentation. For this rea- with flow cytometry of mitochondrial membrane potential son, we examined DNA damage at the level of individ- in CCRF-CEM cells treated with different concentrations of ual cells by the alkaline comet assay. ZINC253504760 ZINC253504760 or DMSO (control), or vinblastine (positive induced comet tails in CCRF-CEM cells upon treat- control) for 24 h. Statistical results of the dead cells defined as MMP collapse after 24 h treatment. Statistics analysis ment with different concentrations for 3 h (Fig.  6d). was done by paired student’s t-test, * p ≤ 0.05, if compared The median value (percentage of tail DNA) from 50 to DMSO untreated cells. The statistical analysis shows mean randomly selected cells was 5.2% with a range from values ± SD of three independent experiments 2.4% to 97.5% at a concentration of 0.088 µM. Only a few cells were damaged. Upon treatment with 0.15 but not its cleaved form, which means the mode of µM, the median value reached 39.4% with a range cell death was caspase 3-independent. Therefore, we from 3.9% to 96.2%, evidencing that DNA damage and further focused on parthanatos as a novel caspase- the number of damaged cells increased in a concentra- independent mode of programmed cell death. The tion-dependent manner. This finding further supports expressions of biomarkers in the parthanatos path- the hypothesis that ZINC253504760 caused parthana- way were examined. As shown in Fig. 6a, the expres- tos and implies that eventually the cells die from large- sion level of PARP (116 kDa) significantly increased scale DNA damage. upon increasing ZINC253504760 concentrations. PAR also showed an increased expression. Then, we Assessment of oxidative stress measured the nuclear and cytoplasmic AIF localiza- tion (Fig. 6b). Notably, the AIF expression decreased ROS are well recognized as DNA damage mediators. in the cytoplasm while increasing in the nucleus com- Since ZINC253504760 resulted in a pathanatos, G2/M pared with the control group. Moreover, the level phase arrest, collapse of mitochondrial membrane of p-histone H2A.X increased in a dose-dependent potential and a minor fraction of apoptosis, we further manner. In addition, ZINC253504760 also led to investigated whether they were induced via ROS gen- increased PARP cleavage (89 kDa), which appeared eration. Compared with H 2O2 that showed a high fold to be inconsistent with the absence of cleaved-cas- change of ROS production, ZINC253504760-treated pase 3 expressions, we give discussions below. Taken samples did not show ROS generation at 12 h or 24 h together, these results suggest that ZINC253504760 (Supplementary Fig. S4). Therefore, ZINC253504760 caused parthanatos as predominant form of cell death. did not induce ROS-dependent mitochondrial apopto- sis pathway, and the induction of pathanatos and G2/M Immunofluorescence microscopy of phase arrest were not ROS-dependent. apoptosis-inducing factor translocation Western blotting of MEK AIF is released from mitochondria and rapidly translo- cated to the nucleus, where it induces large-scale DNA Since MEK1/2 and ERK were predicted to be downreg- fragmentation (50 kb) and chromatin condensation. ulated and linked with G2/M cell cycle arrest in CCRF- This translocation can be captured by confocal immu- CEM cells using IPA-based evaluation of the microarray nofluorescence microscopy. Fig.  6c indicates that AIF data, we further evaluated their expression and phospho- was almost all extensively localized at the cytoplasm rylation status by western blotting. Upon treatment for Vol.: (0123456789) 1 3 Cell Biol Toxicol Vol:. (1234567890) 1 3 Cell Biol Toxicol ◂Fig. 6 Induction of parthanatos as major mode of cell death i.e., LYS97, LEU115, ASP208, PHE209, and SER212. by ZINC253504760. a Western blot analysis in total pro- Remarkably, ZINC253504760 bound to SER218, tein of parthanatos-related biomarkers (caspase 3, p-Histone H2A.X, PARP, and PAR) treated with different concentra- which is one of the important phosphorylation sites on tions of ZINC253504760 for 24 h in CCRF-CEM cells. b The MEK1. Regarding MEK2 (chain B), LYS101, ASP212, expression of AIF in the nucleus and the cytoplasm. Statis- and GLY214 interacted with both ZINC253504760 tics analysis was done by paired student’s t-test, * p ≤ 0.05, and trametinib. Similarly, ZINC253504760 bound to ** p ≤ 0.001, if compared to DMSO untreated cells. The bars represent the mean values ± SEM of three independ- another important phosphorylation site on MEK2, ent experiments. c Detection of AIF translocation from the SER222. Therefore, we conclude that ZINC253504760 cytoplasm to the nucleus detected by immunofluorescence is an ATP-competitive inhibitor and hereby affects microscopy. CCRF-CEM cells treated with different concen- MEK1 and MEK2 phosphorylation. Furthermore, trations of ZINC253504760 or DMSO (control) for 12 h and stained with antibody AIF to visualize AIF protein. Nuclear the binding affinity of the deglycosylated form of AIF (green) translocated into the nucleus (blue) was obvious. d ZINC253504760 to MEK1 was -6.26 ± 0.05 kcal/ Detection of DNA damage by alkaline comet assay in CCRF- mol, and to MEK2 was -6.57 ± 0.005 kcal/mol (Sup- CEM cells. Cells incubated in different concentrations with plementary Table  S5), which were closed to the gly- ZINC253504760 for 3 h and 50 µM of H2O2 (positive control) for 1 h. The parameter tail DNA% was measured from 50 ran- cosylated form of ZINC253504760. Even though the domly selected cells shown in the violin plot. interacting amino acids of deglycosylated form did not dock to phosphorylated sites on MEK1 or MEK2, they were at similar sites compared with trametinib. 24 h, ZINC253504760 significantly downregulated the phosphorylation level of p-MEK1/2, while neither sig- Detection of MEK1/2-compound binding by nificant effect on MEK 1/2 nor downregulation of ERK microscale thermophoresis protein expression was observed upon treatment, indicat- ing that ZINC253504760 inhibited MEK1/2 phospho- The in vitro interaction of ZINC253504760 with rylation (Fig. 7a). After 48 h treatment, ZINC253504760 MEK1/2 was confirmed by MST (Fig. 7c). The meas- further downregulated ERK and p-ERK through a more ured concentration-dependent fluorescence signals significant downregulation of p-MEK1/2, indicating indicated an interaction between the fluorescently that ZINC253504760 inhibited ERK protein expression labeled MEK1 and MEK2 protein with the compound. through inhibiting p-MEK1/2. However, there were still ZINC253504760 bound to MEK1 with a Kd value of 0.5 no noticeable changes observed on MEK1/2. The signifi- µM and to MEK2 with a Kd value of 2.2 µM. cant decrease of MEK1/2 expression with 4×IC50 at 48 h may result from cell death. Therefore, these data sug- gested that ZINC253504760 inhibited the phosphoryla- Discussion tion of MEK1/2. Natural products have been regarded as a ‘‘treasure Molecular docking box’’, which greatly contributed to pharmacological research and drug development (Newman and Cragg To further investigate the possible interaction of 2020). Cardiac glycosides (CGs) are one of the lead- ZINC253504760 with MEK1 and MEK2, molecular ing naturally derived classes of anticancer drug can- in silico docking studies were performed. Trametinib didates (Kumar and Jaitak 2019). To further develop was used as a known allosteric inhibitor of MEK1/2 cardenolides, the aim of the present study was to to compare the binding affinities with those of explore the molecular mode of action of a cardenolide, ZINC253504760 (Roskoski 2017). ZINC253504760 ZINC253504760. This is a synthetic CG compound showed binding affinity (lowest binding energy) to with structural similarity to digitoxin and digoxin. Dig- MEK1 (-8.15 ± 0.3 kcal/mol) and MEK2 (-7.85 ± itoxin and digoxin have been discussed as new antican- 0.4 kcal/mol), which were approximately the same as cer agents (El-Seedi et al. 2019b; Menger et al. 2013), trametinib (Supplementary Table  S5). As shown in indicating the likelihood that ZINC253504760 might Fig.  7b, five amino acid residues of MEK1 appeared also show anticancer activity. to interact with both ZINC253504760 and trametinib, Vol.: (0123456789) 1 3 Cell Biol Toxicol Vol:. (1234567890) 1 3 Cell Biol Toxicol ◂Fig. 7 Inhibition of MEK1/2 phosphorylation. a Western blot- (necrosis). Meanwhile, novel cell death modes have ting analysis of the effect of ZINC253504760 on MAPK sign- been attracting attention for intervention in disease aling in CCRF-CEM cells. GAPDH was used as the loading control. Digitalized graphs of affected protein levels are shown mechanisms. PARP is activated by DNA strand nicks below. The bars represent mean values ± SEM of three inde- and breaks and takes part in different pathways of pendent experiments. Statistics analysis was calculated by DNA damage repair. Different from mild DNA damage paired student’s t-test, * p ≤ 0.05, ** p ≤ 0.001. b Visualiza- that stimulates PARP to repair damage, parthanatos is tion of docking results. MEK1 (PDB code:1s9j, ice-blue) and MEK2 (PBD code:1s9i, purple) are presented in a new cartoon uniquely induced by severe DNA damage. It is impor- format. Ligands are presented in bond format with different tant to point out that PARP activation is required for AIF colors. ZINC253504760 (red) and trametinib (green). c Bind- translocation. AIF failed to translocate into the nucleus ing of ZINC253504760 with MEK1 and MEK2 as determined in PARP-knockout fibroblasts after MNNG treatment by microscale thermophoresis. M onolithTM NT analysis soft- ware was used to determine the fitted K on MEK1 and MEK2, (Yu et al. 2002). Furthermore, PAR has been identified d and to plot the ZINC253504760 fit curve as an AIF-releasing factor. AIF activity was abrogated by PAR glycohydrolase, an enzyme that plays an impor- tant role in the degradation of PAR (Yu et  al. 2006). We demonstrated that this compound was not involved Our western blot analysis revealed elevated full length- in the major mechanism of resistance. It is worth to point PARP (116 kDa) and increased PAR expression in a out that cross-resistance to ZINC253504760 was only concentration-dependent manner by ZINC253504760, observed in BCRP-overexpressing cells, multidrug resist- following decreased cytoplasmic but increased nuclear ance (except BCRP), making it attractive for further AIF expression. AIF translocation represents a key investigations. Previously, we examined the cytotoxicity event in response to PARP-mediated cell death (Susin of a library of 66 CGs that inhibited the efflux function of et  al. 1999). Immunofluorescence microscopy showed P-glycoprotein and overcame MDR (Zeino et al. 2015b). that AIF indeed accumulated in the nucleus of treated As a next step, we selected drug-sensitive CCRF-CEM cells. Currently, the mechanism of AIF release can leukemia cells as model to unravel the modes of action, be explained in two ways: (1) PAR translocates to the since the resazurin assay revealed that ZINC253504760 cytoplasm, interacts with mitochondria, and binds to showed the lowest nanomolar concentration against AIF (Wang et  al. 2011). (2) PAR formation consumes CCRF-CEM cells. In contrast, the viabilities in embry- the N AD+ stores, which leads to a disruption of the onic kidney cells (HEK293 and HEK293/ABCB5) and MMP and AIF release (Andrabi et al. 2006). Mitochon- breast cancer cells (MDA-MB-231-pcDNA3) were still dria act as death centers under cellular stress condi- more than 20% treated with 100 µM of ZINC253504760, tions to release apoptogenic factors including AIF and which clearly means these cells lines did not exhibit sig- cytochrome c (Andrabi et al. 2008). In our study, flow nificant lethality to ZINC253504760. While in glioblas- cytometric analyses showed that ZINC253504760- toma multiform cells (U87MG and U87MGEGFR), the treated cells lost their MMP in a concentration-depend- viabilities already decreased to 80% at 0.003 µM, which ent manner. Therefore, our experiments support the view means ZINC253504760 may begin to kill cells in lower that AIF translocated to the nucleus because of the mas- concentrations, while its IC50 value was the second only sive PAR accumulation and the loss of the MMP. The to leukemia cells. Leukemia remains a malignancy in phosphorylation of the histone variant H2A.X represents children and adults due to the frequent relapses after treat- an immediate response to DNA double-strand breaks ment (Siegel et  al. 2022). A variety of studies reported (DSB) and has been widely applied for the detection of that CGs including digitoxin, bufalin, ouabain, and per- DNA damage (Rahmanian et al. 2021). Our data showed voside, which showed already profound cytotoxicity in the p-histone H2A.X levels rose instantly upon treatment leukemia cells (Masuda et al. 1995; Jing et al. 1994; Feng with increasing drug concentrations. To further validate et al. 2016; Zeino et al. 2015a). Importantly, several CGs this result, DNA damage was also investigated by the indicated that the execution of apoptosis in leukemia cells alkaline comet assay, which indeed revealed increased and did not affect normal blood cells, suggesting that percentages of tail DNA induced by ZINC253504760. CGs may possess at least some specific tumor-specificity Taken together, from the key features of parthanatos, (Ayogu and Odoh 2020). we demonstrated that ZINC253504760 resulted in rapid Cell death was initially categorized into three types: PARP activation, PAR accumulation, mitochondrial type I (apoptosis), type II (autophagy) and type III depolarization, AIF translocation, and large-scale DNA Vol.: (0123456789) 1 3 Cell Biol Toxicol fragmentation. These data are consistent with the other (stage I), parthanatos is the predominant mode of cell reports where parthanatos was induced in cancer cells death, when AIF translocates into the nucleus. In stage (Zhao et  al. 2015; Ma et  al. 2016). Parthanatos is the II (after 24 h or more), caspase 3 was gradually acti- major mode of cell death of ZINC253504760 in CCRF- vated, while a small fraction of apoptosis was induced CEM leukemia cells. It contributes to the growth inhi- in dying cells. Furthermore, ZINC253504760 did not bition of ZINC253504760 in the resazurin assay and induce ROS-dependent parthanatos or mitochondrial explains the appearance of the cell death and survival apoptosis in CCRF-CEM cells, this finding is differ- pathway predicted by the microarray-based Ingenuity ent with parthanatos induced in glioma cells (Ma et al. Pathway Analysis. 2016). Finally, autophagy biomarkers including Beclin Even if parthanatos is different from other modes of and p62 were decreased in a time- and concentration- cell death regarding their morphological and molecular dependent manner, suggesting that ZINC253504760 pathways, they interact with each other and exert cross- did not induce autophagy in CCRF-CEM cells. Thus, talks. Firstly, parthanatos may interact with apoptosis. ZINC253504760-induced parthanatos did not correlate Early studies have been demonstrated that PARP acti- to autophagy. Our finding is consistent with parthanatos vation and AIF activity can be maintained in the pres- induced in esophageal cancer (Zhao et al. 2015). ence of wide-ranging caspase inhibitor Z-VAD-fmk in Using flow cytometry, we observed that MNNG treated wild-type fibroblasts, suggesting that ZINC253504760 induced G2/M phase arrest, and this pathanatos is a caspase-independent cell death mode result was consistent with the microarray-based pathway (Susin et al. 1999; Yu et al. 2002), while recent emerg- prediction. We also further verified a number of genes ing evidence indicates that caspases also can be acti- involved in the cell cycle using qRT-PCR. G2/M phase vated in parthanatos (Yu et al. 2002). In our study, the arrest is one of the main cellular responses to DNA dam- PARP fragment (89 kDa) and caspase 3 participated in age that prevents the transmission of damaged DNA to ZINC253504760-induced cell death. PARP is a switch undergo mitosis without repair of DNA lesions. It is worth point that directs cell death toward either apoptosis or to mention that ATM (ataxia-telangiectasia mutated) and parthanatos. Parthanatos starts in the overactivation of ATR (ataxia-telangiectasia mutated and Rad3-related) PARP and results in the deletion of energy stores, while kinase are the required sensors to recognize DNA damage apoptosis is initiated by cleaved PARP with abundant and to phosphorylate their target proteins, such as p53 and energy (Zhou et al. 2021). Hence, the switch of PARP H2AX (Sancar et al. 2004). In the present study, HIPK2, might be cross-dynamic. Meanwhile, we observed the a damage-activated checkpoint kinase that is activated by expression of cleaved PARP without cleaved caspase 3. ATM was upregulated after treatment (Hofmann et  al. In fact, it has been reported that PARP cleavage was not 2013), indicating that ZINC253504760 induced DNA defective in the absence of caspase 3 (Slee et al. 2001). damage. PPM1D, a p53-induced protein after chemi- Our results supported this study. Since we did not find cal stimulation, deactivates p53 by dephosphorylating significant late apoptosis of ZINC253504760 treatment, ATM/ATR following DNA damage. Therefore, PPM1D we propose here, ZINC253504760 induced parthana- has been categorized as an oncogene, since it suppresses tos as the major mode of cell death and with a minor the DNA repair function (Lu et  al. 2004, 2005). This role of apoptosis in leukemia cells. Furthermore, AIF may explain our results that PPM1D was upregulated may interact with caspase. As mentioned above, both while p53 was downregulated. CDK7 along with cyclin AIF and cytochrome c are released from mitochondria. H comprises the CDK-activating kinase (CAK), which Cytochrome c redistributes into the cytosol and triggers is necessary to activate CDKs by providing the T-loop the activation of caspase 3 or caspase 9. Kinetic stud- phosphorylation (Sava et al. 2020). In our experiment, the ies revealed the order of action that the mitochondrial downregulation of CDK7 by ZINC253504760 during G2 release of AIF occurred earlier than that of cytochrome phase actually disrupts cyclin B1-CDK1 assembling and c (Daugas et  al. 2000b). This means that AIF release blocks cells to enter mitosis (Larochelle et al. 2007). How- initiates stage I of chromatin condensation (caspase- ever, a connection between CDK7 and p53 was not pre- independent), and in stage II cell death relies on the dicted by our microarray data. CDK7 and p53 have been activation of caspase by cytochrome c (caspase-depend- raised questions because they both share functional simi- ent) (Daugas et al. 2000a). Along this line, we assume larities regarding cell cycle regulation, transcription, and that upon treatment with ZINC253504760 for 24 h DNA repair. P53 can be phosphorylated by CDK7-Cyclin Vol:. (1234567890) 1 3 Cell Biol Toxicol H in a p36MAT1-dependent manner both in vitro and in polymerization in U2OS cells expressing an α-tubulin- vivo (Ko et al. 1997). Hence, it is understandable in our GFP construct. In brief, our data obtained from flow study that CDK7 was downregulated if p53 was also cytometry, qRT-PCR, and fluorescence microscopy downregulated. Moreover, in our study, CDK1 was upreg- of the microtubule cytoskeleton supported the view ulated both in microarray and qRT-PCR experiments, that ZINC253504760 induced G2/M phase arrest and which seems to be conflicting with G2/M phase arrest at blocked the cellular entry into mitosis. first sight. In fact, even though the cyclin B1-CDK1 com- A wide of human tumors are under the control plex is inactive in the G2/M phase, it still can be activated of MAPK pathway for growth and survival (Sebolt- at the start of prophase, and cyclin B1-CDK1 activity Leopold and Herrera 2004). Since the downregulation reaches its maximum shortly after the nuclear envelope of MEK1/2 and ERK was associated with G2/M cell breakdown (Gavet and Pines 2010). This period maintains cycle arrest biomarkers as predicted by IPA, we con- the cells in their mitotic state. Therefore, we assume that firmed that ZINC253504760 indeed resulted in G2/M our experiments at 24 h captured this mitotic moment of phase arrest. Therefore, we also investigated the hypoth- CDK1 upregulation. On the other hand, cyclinB1-CDK1 esis that ZINC253504760 can downregulate MEK1/2 was inactivated after cyclin B1 degradation in arrested in vitro, supposing that MEK1/2 might be a target of cells (Porter and Donoghue 2003). The change from the ZINC253504760 in CCRF-CEM cells. Indeed, our active to the inactive cyclinB1-CDK1 complex has been study revealed that p-MEK1/2 was downregulated in a described as hysteresis (Pomerening et al. 2003). Regard- concentration- and time-dependent manner, which fur- ing our results, this may be another reason that CDK7 was ther affected p-ERK and ERK. While ERK was still already downregulated while CDK1 was still upregulated upregulated after 24 h, MEK1/2 dephosphorylation was and active. CKS1 and CKS2 are proposed to physically still ongoing and ERK was downregulated after 48 h. link with cyclinB1-CDK1 for further phosphorylation to Next, we carried out molecular docking to understand their substrates (Ellederova et al. 2019). The upregulation the mode of binding of ZINC253504760 to MEK1 and of CKS1 and CKS2 was consistent with the upregulation MEK2. As expected, ZINC253504760 bound to the of CDK1. Furthermore, Wee1 is a kinase which nega- phosphorylation sites, SER218 on MEK1 and SER222 tively regulates CDK1 by catalyzing the phosphorylation on MEK2. In human MEK1, the substitution of either on Thr14 and Tyr15 and which inhibits CDK1 activity SER218 or SER222 abrogates the MEK1 activation, (Du et al. 2020). Our data showed that Wee1 was upregu- implying that both serines are required for phosphoryla- lated by ZINC253504760, while CDK1 remained upreg- tion (Zheng and Guan 1994). Our results supported this ulated under hysteresis and inactivated cyclin B1-CDK1. finding. Therefore, we conclude that ZINC253504760 Furthermore, the ATR-CHEK1-CDC25 pathway was contributed to MEK1/2 inactivation, which further led predicted by IPA to be affected by ZINC253504760. This to downstream ERK downregulation and inhibition of is a classical pathway activated following DNA dam- cell proliferation. In addition, the lowest binding energy age and arrest in the G2 phase (Calonge and O’Connell of deglycosylated form of ZINC253504760 to MEK1/2 2008). Due to our limited treatment time (24 h) and con- was similar to that of glycosylated form. Regarding centration (IC50), this pathway was only predicted by IPA the fact that the bioactivity of GCs decreases with the software without expression of fold change, but our vali- loss of sugar, the monitor of bioactive metabolites of dation by qRT-PCR of the eight genes is sufficient to sup- GCs needs to be studied in the future, and if necessary, port cell cycle G2/M arrest induced by ZINC253504760. concomitant administration such as metabolic enzyme Microtubules have multiple functions in cellu- inhibitors or other drugs (e.g., quinidine increased lar processes, especially regarding the formation of absorption of digoxin) might be options to increase CGs mitotic spindles during cell cycle, making them vital bioavailability (Pedersen et al. 1983; Jortani and Valdes therapeutic targets in cancer treatment (Dumontet and 1997). Moreover, the ATP-binding pocket on kinases Jordan 2010). The microtubule-targeting antimitotic has been classified into many types (Pan and Mader drugs can be divided into microtubule-destabilizing 2022). MEK1/2 is one of the most thoroughly investi- agents (e.g., Vinca alkaloids and colchicine), and gated kinases for type III allosteric inhibitors. The four microtubule-stabilizing agents (e.g., taxanes). Both FDA-approved MEK1/2 are all ATP-noncompetitive drug classes block mitosis (Wang et  al. 2016). We kinase inhibitors (Roskoski 2017; Lu et al. 2020). Our found that ZINC253504760 interfered with tubulin results showed trametinib as a known allosteric inhibitor Vol.: (0123456789) 1 3 Cell Biol Toxicol bound adjacent to the ATP binding site which supported detail. In the light of developing CG compounds for previous studies and proved that our molecular dock- the treatment of malignant diseases, studies based on ing approach was correct. Finally, the molecular inter- pharmacokinetic properties, toxicological mechanisms actions of ZINC253504760 with MEK1/2 have been and further structural modifications should be con- confirmed by MST, and the fit curves proved once again ducted to achieve reduced toxicity of CG compounds. that ZINC253504760 could bind to MEK1 and MEK2. Personalized medicine by investigating gene polymor- Taken together, our in vitro and in silico results indicated phisms is also a strategy to delineate applicable popu- that ZINC253504760 bound to MEK1/2 and inhibited lations with increased efficacy and less toxicity (Zhai MEK1/2 phosphorylation. et al. 2022). Interestingly, numerous studies revealed the char- acteristic molecular mechanisms of CGs in cancer cells. First and foremost, apoptotic cell death has been Conclusion described in most cases (e.g., UNBS1450) (Juncker et al. 2011). Immunogenetic cell death was also induced Taken together, in this study we described the mecha- by CGs (e.g., oleandrin) (Li et al. 2021; Menger et al. nism of a new synthetic cardenolide compound, 2012). In the present investigation, ZINC253504760 ZINC253504760. This compound displayed cytotoxicity predominantly induced parthanatic cell death rather against different multidrug-resistant cell lines with overex- than apoptosis. Our finding opened a new door for pression of ABC transporters (P-glycoprotein, ABCB5), further studies on the cell death mode of CGs and overexpression of the activated oncogene ∆EGFR, or a pointed to the potential of ZINC253504760 to treat knock-out of the tumor suppressor TP53. Starting from anti-apoptosis- and drug-resistant cancers. Moreo- microarray-based mRNA expression profiling, we demon- ver, ZINC253504760 arrested CCRF-CEM leukemia strated that ZINC253504760 induced parthanatos-type cell cells in the G2/M phase of the cell cycle. This result death and G2/M phase arrest in CCRF-CEM cells, both is comparable to reports from many other CGs, such resulting from DNA damage. ZINC253504760 induced as proscillaridin A-treated glioblastoma cells (Denico- the overexpression of PARP and PAR and nuclear AIF laï et al. 2014), ouabain-treated melanoma cells (Wang translocation, and disrupted the mitochondrial membrane et  al. 2021), and lanatoside C-treated breast, lung, or potential, all of which are steps triggering parthanatos. Fur- liver cancer cells (Reddy et  al. 2019). Based on this thermore, ZINC253504760 blocked the MAPK pathway fact, the induction of parthanatos and G2/M phase by inhibiting MEK1/2 phosphorylation in a time- and con- arrest in cell cycle are the best evidence to support that centration-dependent manner. ZINC253504760 bound to ZINC253504760 leads to DNA damage. In addition, MEK1 and MEK2 at their phosphorylated sites, as demon- Src is a non-receptor protein tyrosine kinase, the acti- strated by microscale thermophoresis and molecular dock- vation of the Src-EGFR-MAPK pathway is one of the ing. To the best of our knowledge, parthanatos was shown accepted potential mechanisms of the anticancer effects for the first time to be induced by a cardenolide com- of CGs (Prassas and Diamandis 2008; Kometiani et al. pound. These results provide a basis for further exploring 2005). Our functional network identified from the tran- ZINC253504760 as an alternative strategy to treat cancer scriptomic analysis (Fig.  3c) showed that c-Src pro- cells that have the ability to escape apoptosis and are drug- tein, as well as the top affected genes Hsp90AA1 and resistant. Further assessments of ZINC253504760 are war- Hsp90 AB1 were downregulated after ZINC253504760 ranted regarding its toxicity. treatment. Luo et al have demonstrated that c-Src was weakly affected by the chaperone Hsp90 (Luo et  al. Acknowledgement We gratefully acknowledge the Microar- 2017). Therefore, our study revealed the molecular ray Unit of the Genomics and Proteomics Core Facility, German mode of action of ZINC253504760 resulting from Cancer Research Center (DKFZ), for providing excellent Expres-sion Profiling service. We thank Microscope Core Facility and DNA damage, parthanatic cell death and G2/M arrest. Flow Cytometry Core Facility at the Institute of Molecular Biol- Last but not least, CG compounds have a nar- ogy (IMB, Mainz, Germany) for their kind training and technical row therapeutic window and show different tox- support for microscopy and flow cytometry related experiments. icities. In the future, the selectivity and toxicity of We are grateful for the PhD stipend of the Chinese Scholarship Council to M.Z., and the stipend of the Sibylle Kalkhof-Rose- ZINC253504760 should be investigated in more Foundation to J.C.B. Vol:. (1234567890) 1 3 Cell Biol Toxicol Author contributions Min Zhou: Data curation, Methodol- Invest New Drugs. 2020;38(1):1–9. https://d oi. org/1 0. ogy, Investigation, Conceptualization, Writing – original draft, 1007/ s10637- 019-0 0752-0. Writing – review & editing. Joelle C. Boulos: Conceptualization, Ainembabazi D, Geng X, Gavande NS, Turchi JJ, Zhang Y. Syn- Methodology, Investigation. Sabine M. Klauck: Conceptualiza- thesis and Biological Evaluation of Cardiac Glycosides for tion, Methodology, Investigation, Software, Data curation, Writ- Cancer Therapy by Targeting the DNA Damage Response. ing – review & editing. Thomas Efferth: Project administration, ChemMedChem, 2022:e202200415. https:// doi. org/ 10. Supervision, Conceptualization, Writing – review & editing. 1002/c mdc.2 0220 0415. Andrabi SA, Dawson TM, Dawson VL. Mitochondrial and nuclear Funding Open Access funding enabled and organized by cross talk in cell death: parthanatos. Ann N Y Acad Sci. Projekt DEAL. 2008;1147:233–41. https:// doi.o rg/ 10. 1196/a nnals. 1427. 014. Andrabi SA, Kim NS, Yu SW, Wang H, Koh DW, Sasaki M, Data availability The authors declare that the data supporting et  al. Poly(ADP-ribose) (PAR) polymer is a death sig- the findings of this study are available within the paper. All other nal. Proc Natl Acad Sci U S A. 2006;103(48):18308–13. data are available from the corresponding author upon reason- https:// doi. org/ 10. 1073/p nas. 06065 26103. able request. Ayogu JI, Odoh AS. Prospects and Therapeutic Applications of Car-diac Glycosides in Cancer Remediation. ACS Comb Sci. 2020;22(11):543–53. https:// doi.o rg/ 10. 1021/ acsco mbsci. 0c0008 2. Code availability Not applicable. Barbosa R, Acevedo LA, Marmorstein R. The MEK/ERK Net- work as a Therapeutic Target in Human Cancer. Mol Can- Declarations cer Res. 2021;19(3):361–74. https:// doi. org/ 10. 1158/ 1541- 7786. Mcr- 20- 0687. Competing interests The authors declare no competing interests. Bessen HA. Therapeutic and toxic effects of digitalis: William Withering, 1785. J Emerg Med. 1986;4(3):243–8. https:// Conflict of interest The authors declare there is no conflict doi.o rg/ 10. 1016/ 0736- 4679(86)9 0048-X. of interest. Bock FJ, Tait SWG. Mitochondria as multifaceted regulators of cell death. Nat Rev Mol Cell Biol. 2020;21(2):85–100. Ethnical approval Not applicable. https://d oi. org/ 10. 1038/ s41580- 019- 0173-8. Boulos JC, Saeed MEM, Chatterjee M, Bülbül Y, Crudo F, Marko D, et al. Repurposing of the ALK Inhibitor Crizo- Consent for publication All authors have agreed to publish tinib for Acute Leukemia and Multiple Myeloma Cells. this manuscript. Pharmaceuticals (Basel). 2021;14(11). https:// doi.o rg/1 0. 3390/ ph141 11126. Consent to participate Not applicable. Calonge TM, O’Connell MJ. Turning off the G2 DNA dam- age checkpoint. DNA Repair (Amst). 2008;7(2):136–40. https:// doi. org/ 10. 1016/j.d narep. 2007. 07. 017. Human and animal rights This article does not contain any Caunt CJ, Sale MJ, Smith PD, Cook SJ. MEK1 and MEK2 studies with human or animal subjects. inhibitors and cancer therapy: the long and winding road. Nature Reviews Cancer. 2015;15(10):577–92. https://d oi. Open Access This article is licensed under a Creative Com- org/1 0. 1038/ nrc400 0. mons Attribution 4.0 International License, which permits Cowley S, Paterson H, Kemp P, Marshall CJ. Activation of MAP kinase use, sharing, adaptation, distribution and reproduction in any kinase is necessary and sufficient for PC12 differentiation and medium or format, as long as you give appropriate credit to the for transformation of NIH 3T3 cells. Cell. 1994;77(6):841–52. original author(s) and the source, provide a link to the Crea- https:// doi.o rg/ 10.1 016/0 092-8 674(94)9 0133-3. tive Commons licence, and indicate if changes were made. The Daugas E, Nochy D, Ravagnan L, Loeffler M, Susin SA, images or other third party material in this article are included Zamzami N, et  al. Apoptosis-inducing factor (AIF): a in the article’s Creative Commons licence, unless indicated ubiquitous mitochondrial oxidoreductase involved in otherwise in a credit line to the material. If material is not apoptosis. FEBS Lett. 2000;476(3):118–23. https:// doi. included in the article’s Creative Commons licence and your org/ 10. 1016/ s0014-5 793(00)0 1731-2. intended use is not permitted by statutory regulation or exceeds Daugas E, Susin SA, Zamzami N, Ferri KF, Irinopoulou T, Laro- the permitted use, you will need to obtain permission directly chette N, et al. Mitochondrio-nuclear translocation of AIF from the copyright holder. To view a copy of this licence, visit in apoptosis and necrosis. Faseb j. 2000;14(5):729–39. http:// creat iveco mmons.o rg/l icen ses/b y/4. 0/. Dawood M, Fleischer E, Klinger A, Bringmann G, Shan L, Efferth T. Inhibition of cell migration and induction of apoptosis by a novel class II histone deacetylase inhibitor, MCC2344. Pharmacol Res. 2020a;160:105076. https:// References doi.o rg/ 10. 1016/j.p hrs. 2020. 105076. Dawood M, Hegazy MF, Elbadawi M, Fleischer E, Klinger A, Abdelfatah S, Fleischer E, Klinger A, Wong VKW, Efferth T. Bringmann G, et al. Vitamin K(3) chloro derivative (VKT- Identification of inhibitors of the polo-box domain of polo- 2) inhibits HDAC6, activates autophagy and apoptosis, and like kinase 1 from natural and semisynthetic compounds. inhibits aggresome formation in hepatocellular carcinoma Vol.: (0123456789) 1 3 Cell Biol Toxicol cells. Biochem Pharmacol. 2020b;180:114176. https:// doi. image analysis. Redox Biol. 2014;2:457–65. https://d oi. org/ 10. 1016/j.b cp.2 020. 114176. org/1 0. 1016/j. redox.2 013.1 2.0 20. Denicolaï E, Baeza-Kallee N, Tchoghandjian A, Carré M, Colin Ha DP, Tsai YL, Lee AS. Suppression of ER-stress induction C, Jiglaire CJ, et al. Proscillaridin A is cytotoxic for glio- of GRP78 as an anti-neoplastic mechanism of the car- blastoma cell lines and controls tumor xenograft growth diac glycoside Lanatoside C in pancreatic cancer: Lana- in vivo. Oncotarget. 2014;5(21):10934–48. https:// doi. org/ toside C suppresses GRP78 stress induction. Neoplasia. 10. 18632/ oncot arget. 2541. 2021;23(12):1213–26. https://d oi. org/ 10.1 016/j.n eo. Doyle LA, Yang W, Abruzzo LV, Krogmann T, Gao Y, Rishi AK, et al. 2021.1 0. 004. A multidrug resistance transporter from human MCF-7 breast Hegazy MF, Dawood M, Mahmoud N, Elbadawi M, Sugimoto Y, cancer cells. Proc Natl Acad Sci U S A. 1998;95(26):15665–70. Klauck SM, et  al. 2α-Hydroxyalantolactone from Pulicaria https:// doi. org/ 10. 1073/ pnas. 95. 26. 15665. undulata: activity against multidrug-resistant tumor cells and Du X, Li J, Luo X, Li R, Li F, Zhang Y, et al Structure-activ- modes of action. Phytomedicine. 2021;81:153409. https:// doi. ity relationships of Wee1 inhibitors: A review. Eur J org/1 0.1 016/j. phymed. 2020.1 53409. Med Chem. 2020;203:112524. https:// doi. org/ 10. 1016/j. Hofmann TG, Glas C, Bitomsky N. HIPK2: A tumour sup- ejmech. 2020. 112524. pressor that controls DNA damage-induced cell fate and Dumontet C, Jordan MA. Microtubule-binding agents: a dynamic cytokinesis. Bioessays. 2013;35(1):55–64. https:// doi. org/ field of cancer therapeutics. Nat Rev Drug Discov. 10. 1002/b ies. 201200 060. 2010;9(10):790–803. https:// doi. org/ 10. 1038/ nrd32 53. Hoshino R, Chatani Y, Yamori T, Tsuruo T, Oka H, Yoshida El-Seedi HR, Khalifa SAM, Taher EA, Farag MA, Saeed A, Gamal O, et  al. Constitutive activation of the 41-/43-kDa mito- M, et al. Cardenolides: Insights from chemical structure and gen-activated protein kinase signaling pathway in human pharmacological utility. Pharmacol Res. 2019;141:123–75. tumors. Oncogene. 1999;18(3):813–22. https:// doi. org/ 10. https:// doi. org/ 10. 1016/j. phrs. 2018. 12. 015. 1038/ sj.o nc. 120236 7. El-Seedi HR, Khalifa SAM, Taher EA, Farag MA, Saeed A, Gamal Hu Q-Y, Zhang X-K, Wang J-N, Chen H-X, He L-P, Tang J-S, M, et al. Cardenolides: Insights from chemical structure and et al. Malayoside, a cardenolide glycoside extracted from pharmacological utility. Pharmacol Res. 2019;141:123–75. Antiaris toxicaria Lesch, induces apoptosis in human non- https:// doi. org/ 10. 1016/j. phrs.2 018. 12.0 15. small lung cancer cells via MAPK-Nur77 signaling path- Elbadawi M, Boulos JC, Dawood M, Zhou M, Gul W, ElSohly MA, way. Biochem Pharmacol. 2021;190:114622. https:// doi. et al. The Novel Artemisinin Dimer Isoniazide ELI-XXIII-98-2 org/1 0. 1016/j. bcp.2 021.1 14622. Induces c-MYC Inhibition, DNA Damage, and Autophagy in Jing Y, Ohizumi H, Kawazoe N, Hashimoto S, Masuda Y, Leukemia Cells. Pharmaceutics. 2023;15(4):1107. https://w ww. Nakajo S, et  al. Selective inhibitory effect of bufalin on mdpi. com/ 1999- 4923/1 5/4/1 107. growth of human tumor cells in  vitro: association with Ellederova Z, Rincon Sd, Koncicka M, Susor A, Kubelka M, the induction of apoptosis in leukemia HL-60 cells. Jpn J Sun D, et al. CKS1 Germ Line Exclusion Is Essential for Cancer Res. 1994;85(6):645–51. https://d oi.o rg/ 10. 1111/j. the Transition from Meiosis to Early Embryonic Develop- 1349-7 006.1 994. tb024 08.x. ment. Mol Cell Biol. 2019;39(13):e00590-18. https:// doi. Jortani SA, Valdes R Jr. Digoxin and its related endogenous fac- org/1 0. 1128/ MCB.0 0590- 18. tors. Crit Rev Clin Lab Sci. 1997;34(3):225–74. https:// Feng Q, Leong WS, Liu L, Chan W-I. Peruvoside, a Cardiac doi.o rg/1 0. 3109/ 10408 369708 9980 94. Glycoside, Induces Primitive Myeloid Leukemia Cell Juncker T, Cerella C, Teiten MH, Morceau F, Schumacher M, Ghelfi Death. Molecules. 2016;21(4):534. https:// www. mdpi. J, et al. UNBS1450, a steroid cardiac glycoside inducing apop- com/ 1420- 3049/ 21/4/ 534. totic cell death in human leukemia cells. Biochem Pharmacol. Flaherty KT, Infante JR, Daud A, Gonzalez R, Kefford RF, Sosman J, 2011;81(1):13–23. https:// doi.o rg/1 0. 1016/j. bcp.2 010.0 8. 025. et al. Combined BRAF and MEK inhibition in melanoma with Kadioglu O, Klauck SM, Fleischer E, Shan L, Efferth T. Selec- BRAF V600 mutations. N Engl J Med. 2012;367(18):1694–703. tion of safe artemisinin derivatives using a machine learn- https:// doi. org/ 10.1 056/ NEJMoa 1210 093. ing-based cardiotoxicity platform and in vitro and in vivo Frémin C, Meloche S. From basic research to clinical develop- validation. Arch Toxicol. 2021;95(7):2485–95. https://d oi. ment of MEK1/2 inhibitors for cancer therapy. J Hematol org/ 10. 1007/ s00204-0 21- 03058-4. Oncol. 2010;3:8. https:// doi. org/ 10. 1186/ 1756- 8722-3-8. Khalid SA, Dawood M, Boulos JC, Wasfi M, Drif A, Bah- Galia A, Calogero AE, Condorelli R, Fraggetta F, La Corte A, ramimehr F, et  al. Identification of Gedunin from a Phy- Ridolfo F, et  al. PARP-1 protein expression in glioblas- tochemical Depository as a Novel Multidrug Resistance- toma multiforme. Eur J Histochem. 2012;56(1):e9. https:// Bypassing Tubulin Inhibitor of Cancer Cells. Molecules, doi. org/ 10.4 081/e jh. 2012.e 9. 2022;27(18). https://d oi. org/ 10. 3390/ molec ules27 18585 8. Gavet O, Pines J. Progressive activation of CyclinB1-Cdk1 coor- Ko LJ, Shieh SY, Chen X, Jayaraman L, Tamai K, Taya Y, et al. dinates entry to mitosis. Dev Cell. 2010;18(4):533–43. p53 is phosphorylated by CDK7-cyclin H in a p36MAT1- https://d oi.o rg/1 0. 1016/j. devcel.2 010.0 2. 013. dependent manner. Mol Cell Biol. 1997;17(12):7220–9. Gurel E, Karvar S, Yucesan B, Eker I, Sameeullah M. An Over- https:// doi. org/1 0. 1128/ mcb. 17. 12. 7220. view of Cardenolides in Digitalis - More Than a Cardiot- Kometiani P, Liu L, Askari A. Digitalis-induced signaling by Na+/ onic Compound. Curr Pharm Des. 2017;23(34):5104–14. K+-ATPase in human breast cancer cells. Mol Pharmacol. https://d oi. org/ 10.2 174/ 13816 12823 666170 8251 25426. 2005;67(3):929–36. https:// doi. org/ 10. 1124/m ol.1 04. 007302. Gyori BM, Venkatachalam G, Thiagarajan PS, Hsu D, Clem- Kumar A, Jaitak V. Natural products as multidrug resist- ent MV. OpenComet: an automated tool for comet assay ance modulators in cancer. Eur J Med Chem. Vol:. (1234567890) 1 3 Cell Biol Toxicol 2019;176:268–91. https://d oi. org/ 10. 1016/j. ejmech. Menger L, Vacchelli E, Adjemian S, Martins I, Ma Y, Shen S, et al. 2019. 05.0 27. Cardiac glycosides exert anticancer effects by inducing immu- Kun E, Tsang YTM, Ng CW, Gershenson DM, Wong KK. MEK nogenic cell death. Sci Transl Med. 2012;4(143):143ra99. inhibitor resistance mechanisms and recent developments https://d oi. org/ 10. 1126/s citr anslm ed.3 0038 07. in combination trials. Cancer Treat Rev. 2021;92:102137. Menger L, Vacchelli E, Kepp O, Eggermont A, Tartour E, Zit- https://d oi. org/1 0. 1016/j. ctrv. 2020. 102137. vogel L, et al. Trial watch: Cardiac glycosides and cancer Larochelle S, Merrick KA, Terret ME, Wohlbold L, Barboza NM, therapy. Oncoimmunology. 2013;2(2):e23082. https://d oi. Zhang C, et al. Requirements for Cdk7 in the assembly of org/ 10. 4161/o nci. 23082. Cdk1/cyclin B and activation of Cdk2 revealed by chemi- Mijatovic T, Van Quaquebeke E, Delest B, Debeir O, Darro F, cal genetics in human cells. Mol Cell. 2007;25(6):839–50. Kiss R. Cardiotonic steroids on the road to anti-cancer https:// doi. org/1 0. 1016/j.m olcel. 2007. 02. 003. therapy. Biochim Biophys Acta. 2007;1776(1):32–57. Li X, Zheng J, Chen S, Meng F-D, Ning J, Sun S-L. Oleandrin, https:// doi.o rg/ 10.1 016/j.b bcan. 2007. 06.0 02. a cardiac glycoside, induces immunogenic cell death via Newman DJ, Cragg GM. Natural Products as Sources of New the PERK/elF2α/ATF4/CHOP pathway in breast cancer. Drugs over the Nearly Four Decades from 01/1981 to Cell Death Dis. 2021;12(4):314. https://d oi.o rg/ 10. 1038/ 09/2019. J Nat Prod. 2020;83(3):770–803. https:// doi. org/ s41419- 021- 03605-y. 10. 1021/ acs. jnatp rod.9 b012 85. Liu YB, Sun YY, Zhang JL, Zhu YS, Dai YP, D J, et al. Up- Newman RA, Yang P, Pawlus AD, Block KI. Cardiac glyco- regulation of TUBA1B promotes astrocyte proliferation sides as novel cancer therapeutic agents. Mol Interv. after spinal cord injury in adult rats. Int J Exp Pathol. 2008;8(1):36–49. https:// doi. org/ 10.1 124/m i.8. 1.8. 2017;10(2):1094–103 Nolte E, Wach S, Silva IT, Lukat S, Ekici AB, Munkert J, et al. A Livak KJ, Schmittgen TD. Analysis of Relative Gene Expres- new semisynthetic cardenolide analog 3β-[2-(1-amantadine)- sion Data Using Real-Time Quantitative PCR and the 2− 1-on-ethylamine]-digitoxigenin (AMANTADIG) affects ΔΔCT Method. Methods. 2001;25(4):402–8. https:// doi. G2/M cell cycle arrest and miRNA expression profiles and org/1 0.1 006/ meth. 2001. 1262. enhances proapoptotic survivin-2B expression in renal cell Lu X, Bocangel D, Nannenga B, Yamaguchi H, Appella E, carcinoma cell lines. Oncotarget. 2017;8(7):11676–91. https:// Donehower LA. The p53-induced oncogenic phosphatase doi. org/ 10.1 8632/ oncot arget.1 4644. PPM1D interacts with uracil DNA glycosylase and sup- Ohren JF, Chen H, Pavlovsky A, Whitehead C, Zhang E, presses base excision repair. Mol Cell. 2004;15(4):621–34. Kuffa P, et al. Structures of human MAP kinase kinase 1 https:// doi. org/ 10. 1016/j.m olcel. 2004. 08.0 07. (MEK1) and MEK2 describe novel noncompetitive kinase Lu X, Nannenga B, Donehower LA. PPM1D dephosphoryl- inhibition. Nat Struct Mol Biol. 2004;11(12):1192–7. ates Chk1 and p53 and abrogates cell cycle checkpoints. https:// doi.o rg/ 10. 1038/ nsmb85 9. Genes Dev. 2005;19(10):1162–74. https:// doi. org/ 10. Özenver N, Saeed M, Demirezer L, Efferth T. Aloe-emodin as drug 1101/ gad. 129130 5. candidate for cancer therapy. Oncotarget. 2018;9(25):17770– Lu X, Smaill JB, Ding K. New Promise and Opportunities for 96. https:// doi.o rg/ 10. 18632/o ncot arget. 24880. Allosteric Kinase Inhibitors. Angew Chem Int Ed Engl. Pan Y, Mader MM. Principles of Kinase Allosteric Inhibition 2020;59(33):13764–76. https:// doi. org/ 10.1 002/ anie. and Pocket Validation. J Med Chem. 2022;65(7):5288–99. 201914 525. https:// doi.o rg/ 10. 1021/ acs. jmedc hem. 2c000 73. Luo Q, Boczek EE, Wang Q, Buchner J, Kaila VRI. Hsp90 Pedersen KE, Christiansen BD, Klitgaard NA, Nielsen-Kudsk F. dependence of a kinase is determined by its conforma- Effect of quinidine on digoxin bioavailability. Eur J Clin Phar- tional landscape. Sci Rep. 2017;7(1):43996. https:// doi. macol. 1983;24(1):41–7. https:// doi. org/ 10.1 007/ bf006 13925. org/ 10.1 038/ srep4 3996. Pomerening JR, Sontag ED, Ferrell JE. Building a cell cycle oscil- Ma D, Lu B, Feng C, Wang C, Wang Y, Luo T, et al. Deoxypodo- lator: hysteresis and bistability in the activation of Cdc2. Nat phyllotoxin triggers parthanatos in glioma cells via induction Cell Biol. 2003;5(4):346–51. https:// doi. org/ 10. 1038/ ncb954. of excessive ROS. Cancer Lett. 2016;371(2):194–204. https:// Porter LA, Donoghue DJ. Cyclin B1 and CDK1: nuclear locali- doi.o rg/1 0.1 016/j. canlet.2 015. 11.0 44. zation and upstream regulators. Prog Cell Cycle Res. Mahmoud N, Saeed MEM, Sugimoto Y, Klauck SM, Greten 2003;5:335–47. HJ, Efferth T. Cytotoxicity of nimbolide towards multid- Prassas I, Diamandis EP. Novel therapeutic applications of car- rug-resistant tumor cells and hypersensitivity via cellular diac glycosides. Nat Rev Drug Discov. 2008;7(11):926– metabolic modulation. Oncotarget. 2018;9(87):35762–79. 35. https:// doi.o rg/ 10. 1038/ nrd26 82. https://d oi. org/ 10. 18632/ oncot arget.2 6299. Rahmanian N, Shokrzadeh M, Eskandani M. Recent advances in Mansour SJ, Matten WT, Hermann AS, Candia JM, Rong S, γH2AX biomarker-based genotoxicity assays: A marker of DNA Fukasawa K, et  al. Transformation of mammalian cells damage and repair. DNA Repair (Amst). 2021;108:103243. by constitutively active MAP kinase kinase. Science. https:// doi.o rg/ 10.1 016/j. dnarep. 2021. 103243. 1994;265(5174):966–70. https://d oi.o rg/ 10.1 126/ scienc e. Reddy D, Kumavath R, Ghosh P, Barh D. Lanatoside C 805285 7. Induces G2/M Cell Cycle Arrest and Suppresses Cancer Masuda Y, Kawazoe N, Nakajo S, Yoshida T, Kuroiwa Y, Cell Growth by Attenuating MAPK, Wnt, JAK-STAT, Nakaya K. Bufalin induces apoptosis and influences the and PI3K/AKT/mTOR Signaling Pathways. Biomole- expression of apoptosis-related genes in human leukemia cules. 2019;9(12). https://d oi.o rg/ 10. 3390/b iom9 120792. cells. Leuk Res. 1995;19(8):549–56. https://d oi.o rg/1 0. Ren Y, Ribas HT, Heath K, Wu S, Ren J, Shriwas P, 1016/ 0145-2 126(95)0 0031-i. et  al. Na(+)/K(+)-ATPase-Targeted Cytotoxicity of Vol.: (0123456789) 1 3 Cell Biol Toxicol (+)-Digoxin and Several Semisynthetic Derivatives. J anticancer agents. Bioorg Chem. 2021;114:105161. Nat Prod. 2020;83(3):638–48. https://d oi. org/ 10. 1021/ https:// doi.o rg/ 10.1 016/j.b ioorg.2 021.1 05161. acs. jnatp rod.9 b010 60. Slee EA, Adrain C, Martin SJ. Executioner Caspase-3, -6, and -7 Reutelingsperger CPM, van Heerde WL. Annexin V, the regu- Perform Distinct, Non-redundant Roles during the Demoli- lator of phosphatidylserine-catalyzed inflammation and tion Phase of Apoptosis*. J Biol Chem. 2001;276(10):7320–6. coagulation during apoptosis. Cell Mol Life Sci CMLS. https:// doi. org/1 0.1 074/j bc. M0083 63200. 1997;53(6):527–32. https://d oi. org/ 10.1 007/ s00018 00500 67. Smiley ST, Reers M, Mottola-Hartshorn C, Lin M, Chen A, Smith Roberts PJ, Der CJ. Targeting the Raf-MEK-ERK mitogen-activated TW, et al. Intracellular heterogeneity in mitochondrial mem- protein kinase cascade for the treatment of cancer. Oncogene. brane potentials revealed by a J-aggregate-forming lipophilic 2007;26(22):3291–310. https:// doi. org/ 10. 1038/ sj. onc. 12104 22. cation JC-1. Proc Natl Acad Sci U S A. 1991;88(9):3671–5. Roskoski R Jr. MEK1/2 dual-specificity protein kinases: struc- https://d oi. org/1 0. 1073/p nas.8 8.9. 3671. ture and regulation. Biochem Biophys Res Commun. Smolarczyk R, Cichoń T, Pilny E, Jarosz-Biej M, Poczkaj A, 2012;417(1):5–10. https:// doi. org/ 10. 1016/j. bbrc.2 011.1 1. 145. Kułach N, et  al. Combination of anti-vascular agent - Roskoski R Jr. Allosteric MEK1/2 inhibitors including cobimetanib and DMXAA and HIF-1α inhibitor - digoxin inhibits the trametinib in the treatment of cutaneous melanomas. Pharmacol growth of melanoma tumors. Sci Rep. 2018;8(1):7355. Res. 2017;117:20–31. https:// doi.o rg/ 10. 1016/j. phrs. 2016.1 2.0 09. https:// doi. org/ 10. 1038/ s41598- 018-2 5688-y. Rudbari HA, Kordestani N, Cuevas-Vicario JV, Zhou M, Efferth T, Stenkvist B, Bengtsson E, Dahlqvist B, Eriksson O, Jarkrans T, Correia I, et al. Investigation of the influence of chirality and hal- Nordin B. Cardiac glycosides and breast cancer, revisited. ogen atoms on the anticancer activity of enantiopure palladium N Engl J Med. 1982;306(8):484. (II) complexes derived from chiral amino-alcohol Schiff bases Stenkvist B, Bengtsson E, Eriksson O, Holmquist J, Nordin B, and 2-picolylamine. New J Chem. 2022;46(14):6470–83. Westman-Naeser S. Cardiac glycosides and breast can- Saeed MEM, Boulos JC, Mücklich SB, Leich E, Chatterjee cer. Lancet. 1979;1(8115):563. https:// doi. org/ 10. 1016/ M, Klauck SM, et al. Disruption of Lipid Raft Microdo- s0140- 6736(79) 90996-6. mains, Regulation of CD38, TP53, and MYC Signal- Susin SA, Lorenzo HK, Zamzami N, Marzo I, Snow BE, Broth- ing, and Induction of Apoptosis by Lomitapide in Mul- ers GM, et al. Molecular characterization of mitochondrial tiple Myeloma Cells. Cancer Genomics Proteomics. apoptosis-inducing factor. Nature. 1999;397(6718):441–6. 2022;19(5):540–55. https:// doi. org/ 10. 21873/ cgp. 20339. https://d oi. org/1 0. 1038/ 17135. Saeed MEM, Mahmoud N, Sugimoto Y, Efferth T, Abdel-Aziz Ullah R, Yin Q, Snell AH, Wan L. RAF-MEK-ERK pathway H. Molecular Determinants of Sensitivity or Resistance in cancer evolution and treatment. Semin Cancer Biol. of Cancer Cells Toward Sanguinarine. Front Pharmacol. 2022;85:123–54. https://d oi.o rg/1 0. 1016/j. semca ncer. 2021. 2018;9:136. https:// doi. org/ 10.3 389/ fphar.2 018.0 0136. 05. 010. Saeed MEM, Meyer M, Hussein A, Efferth T. Cytotoxicity of South- Vermes I, Haanen C, Steffens-Nakken H, Reutelingsperger C. African medicinal plants towards sensitive and multidrug- A novel assay for apoptosis. Flow cytometric detection resistant cancer cells. J Ethnopharmacol. 2016;186:209–23. of phosphatidylserine expression on early apoptotic cells https:// doi. org/ 10.1 016/j. jep. 2016. 04. 005. using fluorescein labelled Annexin V. J Immunol Meth- Saeed MEM, Rahama M, Kuete V, Dawood M, Elbadawi M, ods. 1995;184(1):39-51. https://d oi. org/ 10. 1016/ 0022- Sugimoto Y, et  al. Collateral sensitivity of drug-resistant 1759(95) 00072-i. ABCB5- and mutation-activated EGFR overexpress- Wang DD, Li XS, Bao YZ, Liu J, Zhang XK, Yao XS, et al. Syn- ing cells towards resveratrol due to modulation of SIRT1 thesis of MeON-neoglycosides of digoxigenin with 6-deoxy- expression. Phytomedicine. 2019;59:152890. https:// doi. and 2,6-dideoxy-d-glucose derivatives and their anticancer org/1 0. 1016/j. phymed.2 019. 152890. activity. Bioorg Med Chem Lett. 2017;27(15):3359–64. Sancar A, Lindsey-Boltz LA, Unsal-Kaçmaz K, Linn S. Molecular https:// doi. org/1 0. 1016/j. bmcl.2 017. 06. 008. mechanisms of mammalian DNA repair and the DNA dam- Wang L, Cai W, Han B, Zhang J, Yu B, Chen M. Ouabain age checkpoints. Annu Rev Biochem. 2004;73:39–85. https:// Exhibited Strong Anticancer Effects in Melanoma Cells doi. org/ 10. 1146/ annur ev.b ioch em. 73. 011303. 073723. via Induction of Apoptosis, G2/M Phase Arrest, and Sava GP, Fan H, Coombes RC, Buluwela L, Ali S. CDK7 inhibitors Migration Inhibition. Onco Targets Ther. 2021;14:1261– as anticancer drugs. Cancer Metastasis Rev. 2020;39(3):805– 73. https://d oi. org/1 0.2 147/o tt.S 2835 48. 23. https://d oi. org/1 0. 1007/ s10555-0 20- 09885-8. Wang X, Ge P. Parthanatos in the pathogenesis of nervous sys- Sebolt-Leopold JS, Herrera R. Targeting the mitogen-activated tem diseases. Neuroscience. 2020;449:241–50. https://d oi. protein kinase cascade to treat cancer. Nat Rev Cancer. org/ 10.1 016/j. neuro scienc e.2 020. 09.0 49. 2004;4(12):937–47. https://d oi.o rg/ 10. 1038/ nrc15 03. Wang X, Tanaka M, Krstin S, Peixoto HS, Wink M. The Inter- Siegel RL, Miller KD, Fuchs HE, Jemal A. Cancer statistics, ference of Selected Cytotoxic Alkaloids with the Cytoskel- 2022. CA Cancer J Clin. 2022;72(1):7–33. https:// doi. eton: An Insight into Their Modes of Action. Molecules. org/ 10.3 322/ caac. 21708. 2016;21(7). https:// doi.o rg/ 10. 3390/ molecu les21 07090 6. Silva CID, Gonçalves-de-Albuquerque CF, Moraes BPT, Garcia Wang Y, Kim NS, Haince J-F, Kang HC, David KK, Andrabi SA, DG, Burth P. Na/K-ATPase: Their role in cell adhesion et al. Poly(ADP-Ribose) (PAR) Binding to Apoptosis-Induc- and migration in cancer. Biochimie. 2021;185:1–8. https:// ing Factor Is Critical for PAR Polymerase-1–Dependent Cell doi. org/ 10. 1016/j. biochi. 2021.0 3. 002. Death (Parthanatos). Sci Signal. 2011;4(167):ra20-ra20. Singh VJ, Sharma B, Chawla PA. Recent developments in https://d oi. org/1 0. 1126/ scisi gnal. 200090 2. mitogen activated protein kinase inhibitors as potential Vol:. (1234567890) 1 3 Cell Biol Toxicol Wu C-F, Efferth T. Miltirone Induces G2/M Cell Cycle Arrest cardiotonic steroids. Biochem Pharmacol. 2015;93(1):11– and Apoptosis in CCRF-CEM Acute Lymphoblastic Leu- 24. https:// doi.o rg/ 10. 1016/j. bcp. 2014. 10. 009. kemia Cells. J Nat Prod. 2015;78(6):1339–47. https:// doi. Zhai J, Dong X, Yan F, Guo H, Yang J. Oleandrin: A Systematic org/ 10. 1021/a cs. jnatpr od. 5b001 58. Review of its Natural Sources, Structural Properties, Detection Yu SW, Andrabi SA, Wang H, Kim NS, Poirier GG, Dawson TM, Methods, Pharmacokinetics and Toxicology. Front Pharmacol. et al. Apoptosis-inducing factor mediates poly(ADP-ribose) 2022;13:822726. https:// doi. org/ 10.3 389/f phar. 2022. 822726. (PAR) polymer-induced cell death. Proc Natl Acad Sci U Zhao N, Mao Y, Han G, Ju Q, Zhou L, Liu F, et  al. YM155, a S A. 2006;103(48):18314–9. https:// doi.o rg/1 0.1 073/ pnas. survivin suppressant, triggers PARP-dependent cell death 06065 28103. (parthanatos) and inhibits esophageal squamous-cell carci- Yu SW, Wang H, Poitras MF, Coombs C, Bowers WJ, Federoff noma xenografts in mice. Oncotarget. 2015;6(21):18445–59. HJ, et  al. Mediation of poly(ADP-ribose) polymerase- https:// doi. org/ 10. 18632/ oncot arget. 4315. 1-dependent cell death by apoptosis-inducing factor. Sci- Zheng CF, Guan KL. Activation of MEK family kinases requires ence. 2002;297(5579):259–63. https://d oi. org/ 10.1 126/ phosphorylation of two conserved Ser/Thr residues. Embo scien ce.1 0722 21. J. 1994;13(5):1123–31. https:// doi.o rg/1 0.1 002/j.1 460- Yuan B, Shimada R, Xu K, Han L, Si N, Zhao H, et al. Multiple 2075. 1994. tb063 61.x. cytotoxic effects of gamabufotalin against human glioblas- Zhou Y, Liu L, Tao S, Yao Y, Wang Y, Wei Q, et al. Parthanatos toma cell line U-87. Chem Biol Interact. 2019;314:108849. and its associated components: Promising therapeutic tar- https://d oi.o rg/ 10.1 016/j.c bi.2 019. 108849. gets for cancer. Pharmacol Res. 2021;163:105299. https:// Zeino M, Brenk R, Gruber L, Zehl M, Urban E, Kopp B, et al. Cyto- doi. org/ 10.1 016/j. phrs. 2020.1 05299. toxicity of cardiotonic steroids in sensitive and multidrug- resistant leukemia cells and the link with Na(+)/K(+)-ATPase. Publisher’s note Springer Nature remains neutral with regard J Steroid Biochem Mol Biol. 2015;150:97–111. https://d oi. org/ to jurisdictional claims in published maps and institutional 10.1 016/j.j sbmb.2 015. 03.0 08. affiliations. Zeino M, Paulsen MS, Zehl M, Urban E, Kopp B, Efferth T. Iden- tification of new P-glycoprotein inhibitors derived from Vol.: (0123456789) 1 3 European Journal of Pharmacology 956 (2023) 175980 Contents lists available at ScienceDirect European Journal of Pharmacology journal homepage: www.elsevier.com/locate/ejphar Two palladium (II) complexes derived from halogen-substituted Schiff bases and 2-picolylamine induce parthanatos-type cell death in sensitive and multi-drug resistant CCRF-CEM leukemia cells Min Zhou a, Joelle C. Boulos a, Ejlal A. Omer a, Hadi Amiri Rudbari b, Tanja Schirmeister c, Nicola Micale d, Thomas Efferth a,* a Department of Pharmaceutical Biology, Institute of Pharmaceutical and Biomedical Sciences, Johannes Gutenberg University-Mainz, Staudinger Weg 5, 55128, Mainz, Germany b Department of Chemistry, University of Isfahan, Isfahan, 81746-73441, Iran c Department of Medicinal Chemistry, Institute of Pharmaceutical and Biomedical Sciences, Johannes Gutenberg University-Mainz, Staudinger Weg 5, 55128, Mainz, Germany d Department of Chemical, Biological, Pharmaceutical and Environmental Sciences, University of Messina, Viale Ferdinando Stagno D’Alcontres 31, 1-98166, Messina, Italy A R T I C L E I N F O A B S T R A C T Keywords: The use of cisplatin and its derivatives in cancer treatment triggered the interest in metal-containing complexes Cell death as potential novel anticancer agents. Palladium (II)-based complexes have been synthesized in recent years with Drug design promising antitumor activity. Previously, we described the synthesis and cytotoxicity of palladium (II) complexes Leukemia containing halogen-substituted Schiff bases and 2-picolylamine. Here, we selected two palladium (II) complexes Metallodrugs Palladium-based complex with double chlorine-substitution or double iodine-substitution that displayed the best cytotoxicity in drug- Parthanatos sensitive CCRF-CEM and multidrug-resistant CEM/ADR5000 leukemia cells for further biological investiga- tion. Surprisingly, these compounds did not significantly induce apoptotic cell death. This study aims to reveal the major mode of cell death of these two palladium (II) complexes. We performed annexin V-FITC/PI staining and flow cytometric mitochondrial membrane potential measurement followed by western blotting, immuno- fluorescence microscopy, and alkaline single cell electrophoresis (comet assay). J4 and J6 still induced neither apoptosis nor necrosis in both leukemia cell lines. They also insufficiently induced autophagy as evidenced by Beclin and p62 detection in western blotting. Interestingly, J4 and J6 induced a novel mode of cell death (parthanatos) as mainly demonstrated in CCRF-CEM cells by hyper-activation of poly(ADP-ribose) polymerase 1 (PARP) and poly(ADP-ribose) (PAR) using western blotting, flow cytometric measurement of mitochondrial membrane potential collapse, nuclear translocation of apoptosis-inducing factor (AIF) by immunofluorescence microscopy, and DNA damage by alkaline single cell electrophoresis (comet assay). AIF translocation was also observed in CEM/ADR5000 cells. Thus, parthanatos was the predominant mode of cell death induced by J4 and J6, which explains the high cytotoxicity in CCRF-CEM and CEM/ADR5000 cells. J4 and J6 may be interesting drug candidates and deserve further investigations to overcome resistance of tumors against apoptosis. This study will promote the design of further novel palladium (II)-based complexes as chemotherapeutic agents. Leukemia represents a group of malignancies with genetic errors that occur in normal cell regulatory processes and causes uncontrolled cell 1. Introduction proliferation of hematopoietic stem cells in the bone marrow (Davis et al., 2014). The incidence rate of leukemia has been increasing by 1% Cancer is still a major public health problem worldwide. According per year from 2009 to 2018. In 2022, an estimated 60,650 new leukemia to the American Cancer Society, there were an estimated 18.1 million cases were reported in the USA, and an estimated 24,000 deaths from people diagnosed with cancer globally in 2020, and the number is ex- this disease (Siegel et al., 2022). Importantly, leukemia is the most pected to reach 28 million by 2040 (Sung et al., 2021). * Corresponding author. E-mail address: efferth@uni-mainz.de (T. Efferth). https://doi.org/10.1016/j.ejphar.2023.175980 Received 26 May 2023; Received in revised form 29 July 2023; Accepted 8 August 2023 Available online 9 August 2023 0014-2999/© 2023 Elsevier B.V. All rights reserved. M. Zhou et al. E u r o p e a n J o u r n a l o f P h a r m a c o l o g y 956 (2023) 175980 water-solubility making them more attractive (Coskun et al., 2013; Abbreviations Kapdi and Fairlamb, 2014). A plethora of Pd (II) complexes was syn- thesized with remarkable progress in the antitumor activity in vitro and AIF Apoptosis-inducing factor in vivo as well as in the understanding of the underlying Bcl-2 B-cell CLL/Lymphoma 2 structure-activity relationships (SAR) (Alam and Huq, 2016). Many of DMSO Dimethyl sulfoxide these Pd(II) complexes displayed fewer side effects and exhibited JC-1 5,5′,6,6′-tetrachloro-1,1′,3,3′- powerful cytotoxicity against various tumor cell lines, some of which tetraethylbenzimidazolylcarbocyanine iodide were even greater than cisplatin and its platinum analogues (El-Morsy LC/MS Liquid chromatography-mass spectrometry et al., 2014; Ilić et al., 2014; Kovala-Demertzi et al., 2007). For example, NAD Nicotine amide adenine dinucleotide a series of comprehensive studies have evaluated Pd(II) dinuclear PAR Poly(ADP-ribose) complexes with spermine (Pd2Spm) and showed promising anti-invasive PARP Poly(ADP-ribose) polymerase 1 as well as anti-proliferative activities in human breast cancer cells. PBMCs Human peripheral mononuclear cells Furthermore, in vivo pharmacokinetic studies revealed that the overall PBS Phosphate-buffered saline tissue accumulation of palladium was lower than platinum, suggesting P-gp P-glycoprotein that Pd(II)-based agents may have less adverse effects (Batista de Car- PI Propidium iodide valho et al., 2016; Vojtek et al., 2021). Remarkably, a recent develop- ment is the approval of TOOKAD® Soluble (Padeliporfin, WST11), a palladium-based photosensitizer to treat low-risk prostate cancer with vascular targeted photodynamic therapy (VTP) in Israel, Mexico, and frequent type of cancer in children. Leukemia is mainly classified into European countries (McFarland et al., 2020). Impressively, clinical four subtypes based on the cell types and the rate of growth: acute phase III results showed that 24 months after VTP, 49% of men in the lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic treatment had negative prostate biopsy compared with only 14% of the myeloid leukemia (CML), and chronic lymphocytic leukemia (CLL). The men on active surveillance (Azzouzi et al., 2017). Palladium has, thus, most frequently identified risk factors of leukemia are genetic syn- raised increasing interest in the development of pharmaceuticals. dromes (e.g., Down syndrome and neurofibromatosis), family history, Furthermore, multiple modes of action of Pd (II) complexes have been radiation (e.g., occupational and therapeutic exposure), chemical identified, including inhibition of cancer cell metabolism, angiogenesis contamination (e.g., benzene and household pesticide exposure), and modulation, as well as mitochondria dysfunctions and apoptosis (Car- lifestyle factors such as smoking (Bispo et al., 2020). In previous de- neiro et al., 2020; Qin et al., 2018; Reigosa-Chamorro et al., 2021; cades, evolving therapeutic strategies in leukemia have been developed, Scattolin et al., 2020). such as radiotherapy, bone marrow or stem cell transplantation, In our previous study, we synthesized and characterized 8 new pure immunotherapy, and CAR-T cells (Döhner et al., 2021). Chemotherapy chiral Pd (II) complexes (four enantiopure pairs) with halogen- is the most common treatment modality, typical chemotherapeutic substituted Schiff bases and 2-picolylamine (pic) (Fig. 1A). The anti- agents include doxorubicin, daunorubicin, cytosine arabinoside, eto- proliferative activity of these Pd (II) complexes was evaluated on two poside, and vincristine. human leukemia cell lines, the drug-sensitive cell line CCRF-CEM cell Metal ions are essential cellular components and play critical roles in line and its multidrug-resistant CEM/ADR5000 sub-line (Rudbari et al., living systems (Frezza et al., 2010). They are frequently found in enzyme 2022). Afterward, the two most active compounds (Fig. 1B), one with catalytic domain and involve many biochemical processes, such as double chlorine-substitution (J4) and another one with double carrying oxygen, electron transfer, catalysis as well as regulating iodine-substitution (J6) were subject to detect cell cycle and apoptosis metabolism. Historically, metal-containing compounds have been (annexin V-FITC/propidium iodide (PI) staining) in flow cytometry in traditionally used in remedies or medicines to treat a variety of disorders CCRF-CEM cells. Our results revealed that all Pd (II) complexes showed (Orvig and Abrams, 1999). Cisplatin, cis-[Pt(NH3)2Cl2] was the first outstanding growth inhibition activity with IC50 value in the micromolar platinum-based chemotherapeutic anticancer agent approved by the range in leukemia cell lines. The active compounds J4 and J6 both Food and Drug Administration (FDA) in 1978, which was a landmark arrested the cell cycle at G2/M phase in CCRF-CEM cells without and opened a new era for platinum (II)- and other metal-based com- inducing apoptosis or necrosis. With the limited evidence at that time, pounds as potential anticancer candidates (Desoize and Madoulet, 2002; we only assumed that they may act as non-apoptotic cell death. Since Ndagi et al., 2017). Cisplatin has been widely applied as adjuvant our designed Pd (II) complexes in the combination of Schiff bases and an therapy in the treatment of a large spectrum of cancers. However, its auxiliary ligand (pic) exhibited optimal stability, solubility, and note- clinical use is hampered due to the notable dose-dependent toxicity and worthy IC50. We assumed that they may be promising compounds for drug resistance (Florea and Büsselberg, 2011; Ghosh, 2019). Therefore, anticancer agents and, therefore, worth further investigation. the research interests in medical inorganic chemistry have been Tumor cells that are unable to induce apoptosis and autophagy are extended to other metal ions. also resistant to chemotherapy. Apoptosis is currently the most common Palladium is frequently applied in medicine, e.g., for dental appli- mechanism for tumor cells to die in response to chemotherapy. How- cations (Kielhorn et al., 2002), for 103Pd radioactive plaque radio- ever, tumor cells evolved many de-regulating apoptosis signaling routes, therapy of intraocular melanoma (Finger et al., 2002), and for especially activation of anti-apoptotic systems to allow development and nanoparticles (Miller et al., 2017; Shibuya et al., 2019). Since palladium progression (Mohammad et al., 2015). For example, B-cell CLL/Lym- (II) is a d8 system compound close to platinum (II), the remarkable phoma 2 (Bcl-2) and its relative Bcl-2 family proteins function as con- structural similarities between Pd (II) and Pt (II) have triggered the trolling outer mitochondrial membrane integrity and apoptosis. They development of Pd (II) complexes with the aim to obtain more effective are classified into proapoptotic Bcl-2 proteins (Bax and Bak) and anti- and less toxic compounds (Lazarević et al., 2017). With the aid of co- apoptotic Bcl-2 proteins (A1, Bcl-2, Bcl-XL, and Bcl-w) (Chipuk et al., ordination chemistry, properties such as solubility, reactivity, toxicity, 2010). Tumor cells achieve anti-apoptosis through increasing expression or activity can be controlled by joining different types of ligands of antiapoptotic Bcl-2 proteins and downregulation of proapoptotic (Abu-Surrah and Abdalla, 2008). In the past two decades, the interest in Bcl-2 proteins, which is one of the hallmarks of cancer (Hanahan and Pd (II) complexes as potential anticancer agents has considerably Weinberg, 2011). Indeed, overexpression of Bcl-2 and Bcl-xL induced increased (Ferraro et al., 2022; Scattolin et al., 2021). The aquation and paclitaxel resistance in AML HL-60 cells (Huang et al., 1997). It is worth ligand-exchange rate of Pd (II) complexes are 105 times faster than that noting that the Beclin autophagy protein was initially isolated as a of Pt (II) complexes, which means that Pd (II) complexes have a better Bcl-2-interacting protein (Liang et al., 1998). Increased apoptosis 2 M. Zhou et al. E u r o p e a n J o u r n a l o f P h a r m a c o l o g y 956 (2023) 175980 Fig. 1. Synthesis of Pd (II) complexes. (A) Synthetic route of enantiopure Pd(pic) Schiff base complexes. (B) J4 and J6. Based on reference (Rudbari et al., 2022). resistance via Bcl-2 family members also inhibits autophagy by binding 2021). to Beclin and, therefore, protects cells from autophagic cell death (Sinha The aim of this study is to continue our previous work to get a deeper and Levine, 2008). Even though the role of autophagy in cancer is insight into the mechanism of cell death of the two Pd (II) complexes J4 controversial, Bcl-2 homologs to some extent participate in oncogenesis and J6 in leukemia cells. To understand the induction of cell death by inhibiting apoptosis and autophagy. Other mechanisms contribute to response to multidrug-resistant cells, we extended the investigation to- evasion of apoptosis including reduced caspase expression, mutations in ward both drug-sensitive CCRF-CEM and multidrug-resistant CEM/ p53, increased expression of inhibitor of apoptosis proteins, and ADR5000 cells. We firstly performed annexin V-FITC/PI staining with impaired receptor signaling pathways (Wong, 2011). Therefore, anti- these two compounds using flow cytometry to verify the absence of cancer compounds that kill cancer cells by different modes of cell death apoptosis and necrosis. Then, we investigated autophagy and other than apoptosis or autophagy may provide exquisite opportunities to mechanisms by means of western blotting and immunofluorescence improve the tumor cell killing rates. microscopy. This study on different modes of cell death provides an In recent years, multiple novel modes of cell death have been iden- explanation for the strong cytotoxicity of our Pd (II) complexes toward tified and provided new clues to tackle human diseases, especially the leukemia cells. The induction of parthanatos in otherwise apoptosis- and problem of drug resistance in cancer. Parthanatos is a non-apoptotic autophagy-resistant tumor cells represents a novel therapy concept and form of cell death and has been recently identified in cancer cells in eventually stimulates the development of novel metal-based drugs in response to different small molecules (Boulos et al., 2023; Ma et al., cancer chemotherapy. 2016; Zhao et al., 2015; Zhou et al., 2023). Mechanically, parthanatos does not require caspases for activation. It leads to an enhanced syn- 2. Materials and methods thesis of poly (ADP-ribose) (PAR) by poly(ADP-ribose) polymerase 1 (PARP1), causing nuclear translocation of apoptosis-inducing factor 2.1. Compounds (AIF), DNA fragmentation, chromatin condensation, and eventually cell death (Andrabi et al., 2006b; Fatokun et al., 2014). Targeting partha- The palladium (II)-based complexes were synthesized and charac- natos may offer alternative avenues for cancer management (Zhou et al., terized as previously reported. The purity of J4 and J6 accessed by liquid 3 M. Zhou et al. E u r o p e a n J o u r n a l o f P h a r m a c o l o g y 956 (2023) 175980 chromatography-mass spectrometry (LC/MS) was around 90% in San Diego, CA, USA). different assay media at both timepoints (0 h and 24 h), indicating that the compounds were stable (supplementary file of this reference (Rud- 2.4. Apoptosis bari et al., 2022) https://www.rsc.org/suppdata/d2/nj/d2nj00321j/ d2nj00321j1.pdf). The synthetic route and their structures are shown Apoptosis was detected with an Annexin V-FITC apoptosis kit (Bio in Fig. 1. Stock solution of palladium (II)-based complexes (20 mM) were Version/Biocat, Heidelberg, Germany) using flow cytometry. CCRF- prepared in dimethyl sulfoxide (DMSO) and stored at − 20 ◦C. The CEM and CEM/ADR5000 cells (1 × 106/well) were seeded into a 6- cytotoxicity of palladium (II)-based complexes J4 or J6 as well as well plate and treated with J4 or J6 at a concentration of 4 × IC50 for doxorubicin as positive control in sensitive CCRF-CEM and 48 h. DMSO or cisplatin (5 μM) were used as negative and positive multidrug-resistant CEM/ADR5000 leukemia cells were previously re- controls. Cells were collected to remove medium and centrifuged with 1 ported (Rudbari et al., 2022) and presented in Table 1. mL cold PBS or 1 ml 1 × binding buffer (Bio Version), respectively. Afterward, cells were resuspended in 1 × binding buffer, then each 2.2. Cell culture sample was stained with 2.5 μL annexin V/FITC and incubated at 4 ◦C for 15 min in the dark. Subsequently, cells were again stained with 10 μL The CCRF-CEM cell line is a human acute lymphoblastic leukemia T propidium iodide (PI). The measurements were performed using a BD lymphocyte line. It was originally isolated from the peripheral blood of a Accuri™ C6 Flow Cytometry (BD Biosciences, Germany) with 20,000 three-year- and eleven-month-old female ALL patient (Foley et al., events in each sample. All experiments were repeated three times. The 1965). Our CCRF-CEM cells were kindly provided by Prof. Axel Sauer- results were analyzed with FlowJo (Celeza, Switzerland). The protocol brey (Department of Pediatrics, University of Jena, Germany). has been described by us (Dawood et al., 2020). CEM/ADR5000 cells were treated with doxorubicin (5000 ng/mL) every other week to sustain P-glycoprotein overexpression. The molecular 2.5. Analysis of mitochondrial membrane potential (MMP) profile of CEM/ADR5000 cells has been described (Efferth et al., 2003; Kadioglu et al., 2016; Kimmig et al., 1990). The cells were grown in The effects of J4 and J6 on the mitochondrial membrane potential RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS) (MMP) were analyzed by 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzi- and 1% penicillin (1000 U mL− 1)/streptomycin (100 μg mL− 1) (Life midazolylcarbocyanine iodide (JC-1; Cayman Chemical, Ann Arbor, Technologies, Darmstadt, Germany). Cells were incubated at 37 ◦C, 90% Michigan, USA) staining as described before (Lu et al., 2020). JC-1 dye is humidity, and a 5% CO atmosphere. Cells were passaged every three widely used to monitor mitochondrial integrity. Based on the usage of 2 days. All the experiments were performed during the cell growth phase. two lasers, one is to excite green fluorescence of JC-1 monomers at 488 nm (dead cells) and the other one is to excite red fluorescence of JC-1 aggregates at 520–570 nm (healthy cells). The red fluorescence disap- 2.3. Isolation and cytotoxicity of human peripheral mononuclear cells pears if the cells lose their mitochondrial membrane potential (De Biasi (PBMCs) et al., 2015). Briefly, CCRF-CEM cells (1 × 104 cells/well) were seeded in a 96-well flat-bottom plate in a volume of 200 μL and treated with DMSO Human peripheral mononuclear cells (PBMCs) were isolated from as negative control, vinblastine (2 μM, positive control), IC50 or 2 × IC50 three healthy donors using Histopaque® as previously described of J4 or J6 for 24 h. After treatment, cells were incubated at 37 ◦C in (Dawood et al., 2020). Briefly, 3 mL fresh whole blood was layered dark for 15 min with JC-1 diluted solution (1:10 in culture medium, 10 carefully over 3 mL of Histopaque® and centrifuged at 400×g for 30 min μL/well). Then, cells were centrifuged with 200 μL cell-based assay at room temperature. Subsequently, the interface containing PBMCs was buffer (Cayman Chemical) at 400×g for 5 min twice. Cells were transferred into a clean tube and mixed gently with 10 mL re-suspended in 100 μL cell-based assay buffer and measured in a BD LSR phosphate-buffered saline (PBS, Invitrogen). Cells were centrifuged at Fortessa SORP equipment. For each sample, 104 cells were counted. The 250×g for 10 min several times, then resuspended in Panserin 413 green JC-1 signal (488 nm excitation) was detected using a 530/30 nm medium (PAN-Biotech, Aidenbach, Germany) supplemented with 2% bandpass filter. The red JC-1 (520–570 nm) signal was measured using a phytohemagglutinin M (PHA-M, Life Technologies, Darmstadt, Ger- 586/15 bandpass filter. All experiments were performed at least in many). Afterward, the growth inhibition effect was measured using triplicate. Data were analyzed using FlowJo software (Celeza, Olten resazurin reduction assay. PBMCs cells (104/well) were seeded into Switzerland). 96-well plates and treated with different concentrations of J4 or J6 ranging from 0.01 to 30 μM for 72 h. Then 20 μL 0.01% resazurin 2.6. Protein extraction and western blotting (Promega, Mannheim, Germany) were added and incubated for 4 h at 37 ◦C. The Infinite M200 Pro-plate reader (Tecan, Crailsheim, Germany) CCRF-CEM and CEM/ADR5000 cells (5 × 106 cells/flask) were was applied to measure the fluorescence intensity. The figure was treated with J4 or J6 at different concentrations (0.5 × IC50, IC50, and 2 generated using Prism 8 GraphPad Software (Graphpad Software Inc., × IC50) for 24 h and then washed with PBS. The total protein extraction was performed with M-PER Mammalian Protein Extraction Reagent Table 1 (Thermo Fisher Scientific, Darmstadt, Germany) (Mahmoud et al., Antiproliferative activity (IC50 values) of compounds J4, J6, and doxorubicin 2022). Cells were suspended in 100 μL extraction reagent containing 1% (positive control) towards the drug-sensitive CCRF-CEM leukemia cell line and Halt Protease Inhibitor Cocktail and phosphatase inhibitor (Thermo its multidrug-resistant sub-line CEM/ADR5000. The data have been previously Fisher Scientific). The lysates were shaken for 30 min at 4 ◦C in the dark published (Rudbari et al., 2022) and are shown here for illustration only. and centrifuged for 15 min at 14,000 rpm. The concentrations of protein Compound CCRF-CEM (IC50 CEM/ADR5000 (IC50 Degree of resistancea were determined using a NanoDrop 1000 spectrophotometer (Thermo μM) μM) Scientific). A quantity of 30 μg protein was separated by 10% SDS-PAGE Mean SD Mean SD gel electrophoresis followed by transfer to polyvinylidene difluoride J4 1.78 0.20 7.24 4.06 4.06 (PVDF) membrane. The membrane was blocked using 5% BSA for 1 h at J6 2.39 0.90 4.87 2.04 2.04 room temperature. The membrane was incubated with diluted primary Doxorubicin 0.01 – 26.56 2.31 2656 antibodies overnight at 4 ◦C against PARP rabbit antibody (1:1000, Cell a The degree of resistance was determined by dividing the IC50 value against Signaling, PAR mouse antibody (1:1000, Merk), phospho-histone H2A.X multidrug-resistant CEMADR5000 cells by the IC50 value against drug-sensitive (Ser139) antibody (1:1000, Cell Signaling), caspase 3/p17/p19 poly- CCRF-CEM cells. clonal antibody (1:1000, Proteintech), AIF rabbit antibody (1:700, Cell 4 M. Zhou et al. E u r o p e a n J o u r n a l o f P h a r m a c o l o g y 956 (2023) 175980 Signaling), anti-p62, SQSTM1 polyclonal antibody (1:1000, Pro- 50,000 cells were counted and cytospinned onto slides, followed by the teintech), Beclin 1 polyclonal antibody (1:1000, Proteintech), permeabilization and blocking process as described above. The primary anti-Lamin B1 monoclonal antibody (1:10,000, Proteintech), GAPDH antibody AIF, secondary antibody, and DAPI were incubated with cells rabbit antibody (1:1000, Cell Signaling), β-actin rabbit antibody at the indicated time. Images were taken using Stellaris 8 Falcon (1:1000, Cell Signaling). Finally, the membrane was incubated with confocal microscope (Leica Microsystems) controlled by LASX software secondary anti-rabbit IgG HRP-linked antibody or anti-mouse IgG (version 4.5.0). The microscope was equipped with a 100x/NA1.4 oil HRP-linked antibody (1:2000, Cell Signaling) for 1 h at room tempera- objective (HC PL APO CS2), a white light laser (WWL), laser diodes (405 ture. The membrane was treated with Luminata™ Classico Western HRP nm) and hybrid detectors. Fluorophores were detected with spectral substrate (Merk Millipore Darmstadt, Germany), and then the bands detection for DAPI (ex. 405 nm, em. 430–484 nm), Alexa Fluor 488 (ex. were visualized using an Alpha Innotech FluorChem Q System (Biozym, 488 nm WWL, em. 494–650 nm), MitoTraker® Deep Red (ex. 641 nm Oldendorf, Germany). The protein expression was quantified using WWL, em. 650–750 nm). ImageJ software (National Institute of Health, United States). The NE-PER Nuclear and Cytoplasmic Extraction Reagents kit 2.9. Single cell gel electrophoresis (comet assay) (Thermo Scientific) was used to extract nuclear and cytoplasmic protein (Zhou et al., 2023). CER I, CER II and NER reagents were added in the The comet assay is a sensitive and rapid method for detecting DNA following volume ratios: 200:11:100 μL, along with 1% Halt Protease damage. The extent of DNA damage present in the cells directly affects Inhibitor Cocktail and phosphatase inhibitor. Then, the lysates were DNA migration (Kumaravel et al., 2009). The OxiSelect™ Comet Assay vortexed according to the manufacturer’s instructions. Finally, the Kit (Cell Biolabs/Biocat, Heidelberg, Germany) was used to detect DNA cytoplasmic protein or nuclear protein was centrifuged at 16,000×g for damage according to the manufacturer’s instructions (Özenver et al., 5 or 10 min. The nuclear and cytoplasmic proteins were measured as 2018). Briefly, CCRF-CEM cells (1 × 106 cells/well) were treated with described above. 0.5- or 1-fold IC50 of J4 or J6, or DMSO (negative control) for 24 h. Cells were harvested, centrifuged at 3000 rpm for 10 min, and resuspended 2.7. Immunofluorescence microscopy of AIF translocation with 1 mL cold PBS (4 ◦C). After counting cells, 1 × 105 cells/mL were combined with milting agarose at a ratio of 1:6 and then applied to CCRF-CEM and CEM/ADR5000 cells (1 × 106 cells/well) were OxiSelect™ Comet Assay slides. The slides were kept at 4 ◦C in the dark treated with two different concentrations of J4 or J6 (0.5 × IC50 or IC50) for 30 min to be solidified. Next, slides were treated with pre-chilled or DMSO (negative control) for 24 h. The cells were harvested, washed lysis buffer (NaCl 14.6 g, EDTA solution 20 mL, 10 × lysis solution, with washing buffer (1% FBS in PBS), and resuspended with 1 mL pH 10.0, fulfill to 100 mL with distilled water, stored at 4 ◦C) for 1 h and washing buffer. Cell suspension (200 μL) containing 10,000 cells was alkaline electrophoresis solution buffer (NaOH 12 g, EDTA solution 2 transferred onto the slides (Super®frost plus slides, VWR International mL, fulfill to 100 mL with distilled water, stored at 4 ◦C) for 40 min in GmbH, Darmstadt, Germany) by centrifugation at 1000 rpm for 5 min. the dark. Subsequently, alkaline electrophoresis solution buffer was The slides were fixed with 3.7% paraformaldehyde (Sigma-Aldrich, added to the chamber, electrophoresis was performed with horizontally Darmstadt, Germany) for 15 min at room temperature, then rinsed for 3 placed slides with a voltage of 20 V for 20 min. The slides were washed × 5 min with washing buffer. Subsequently, the cells were per- with pre-chilled distilled water for 2 × 5 min, and then fixed with 70% meabilized with 0.5% Triton X-100 (AppliChem, Darmstadt, Germany) ethanol for another 5 min. Diluted Vista Green DNA dye (1: 10,000 in TE in PBS for 5 min. After rinsing 3 × 5 min with washing buffer, cells were buffer (121.14 mg, EDTA 200 μL, pH 7.5, fulfill to 100 mL distilled blocked with blocking buffer (1% BSA + 1% FBS in PBS) for 1 h. Then, water)) was applied on the slides (100 μL/well) in the dark. The slides the cells were rinsed again with washing buffer for 3 × 5 min and were photographed with an EVOS digital inverted microscope (Life incubated with the primary antibody against AIF (1:400, Cell Signaling) Technologies GmbH, Darmstadt, Germany). At least 50 cells were in a humidified chamber at 4 ◦C overnight. The next day, antibodies randomly selected and analyzed with OpenComet (Image J). The tail were removed, and the slides were rinsed for 3 × 5 min with washing DNA percentage was used to present DNA damage (Gyori et al., 2014). buffer. The anti-rabbit IgG secondary antibody Alexa Fluor® 488-conju- gate (1:700, Cell Signaling) was added and incubated for 2 h at room 2.10. Confirmation of parthanatos using PARP inhibitor temperature in the dark. The slides were rinsed 3 × 5 min with washing buffer, and nuclear straining was done using 1 μg mL− 1 4′6-diamidino-2- Since PARP overexpression is an important biomarker of parthana- phenylindole (DAPI) diluted in PBS for 5 min. Finally, cells were rinsed tos, this cell death mechanism was verified using PARP inhibitor evi- with washing buffer for 5 × 5 min and mounted (ibidi, Gräfelfing, denced by cell viability. The protocol was recently reported by us Germany) with coverslips (24 × 32 mm, VWR international). AIF was (Boulos et al., 2023). CCRF-CEM and CEM/ADR5000 cells (104/well) visualized by an AF7000 widefield fluorescence microscope (Leica were seeded in 100 μL medium in 96-well plates. Cells were treated with Microsystems, Wetzlar, Germany) controlled by the LAS-X software 100 μL medium containing J4 or J6 of a concentration at 0.5 × IC50 and (version x, Leica Microsystems). The microscope was equipped with a IC50, which are 0.89 μM and 1.78 μM of J4 in CCRF-CEM cells, 3.62 μM 63 × (oil) objective, LED light source (SOLA, lumencor), and a sCMOS and 7.24 μM of J4 in ADR/CEM5000 cells, 1.195 μM and 2.39 μM of J6 camera (Flash 4.0, Hamamatsu). Fluorophores were detected with band- in CCRF-CEM cells, and 2.435 μM and 4.87 μM of J6 in CEM/ADR5000 pass filters for DAPI (ex. BP 360/40, FT400, em. BP 470/40) and cells. These treatments were additionally also combined with the PARP Alexa488 (ex BP 480/40, FT505, em. BP 527/30). The obtained images inhibitor PJ34 (10 μM) (Sigma-Aldrich, Darmstadt, Germany). The were further analyzed with Image J software (Mahmoud et al., 2022). plates were incubated at 37 ◦C for 24 h as the detected timepoint of parthanatos. The cell viability was measured using resazurin reduction 2.8. Immunofluorescence microscopy of AIF and mitochondria staining assay as described above. The experiments were repeated three times with four replicates in each plate. CCRF-CEM and CEM/ADR5000 cells (106 cells/well) were seeded into a 6-well plate. After 24 h incubation, cells were harvested and 3. Results washed with washing buffer (800 rpm, 5 min), followed by incubation for 15 min at 37 ◦C with 100 nM MitoTraker® Deep Red (Invitrogen) as 3.1. Cytotoxicity on human peripheral mononuclear cells (PBMCs) manufacturer’s instruction. Cells were centrifuged and then fixed with 3.7% paraformaldehyde (Sigma-Aldrich) for 15 min at room tempera- To study the cytotoxicity of J4 and J6 towards normal cells, human ture. The washing buffer was used to wash cells twice. Subsequently, peripheral mononuclear cells (PBMCs) were isolated from healthy 5 M. Zhou et al. E u r o p e a n J o u r n a l o f P h a r m a c o l o g y 956 (2023) 175980 donors. As shown in Fig. 2, the cell viability did not decrease upon a induced by J4 and J6, we used CCRF-CEM cells as a study model. The series of increasing concentrations of J4 or J6, which means that PBMCs effects in CEM/ADR5000 cells were then confirmed in the key steps. were not affected by J4 and J6. Taken together with the previously re- ported antiproliferative activity on drug-sensitive CCRF-CEM and 3.3. Mitochondrial membrane potential (MMP) -resistant CEM/ADR5000 cells (see Table 1), J4 and J6 specifically inhibited leukemia cells but not healthy leukocytes. Even though apoptosis was not observed with J4 or J6, some types of cell death are regulated by mitochondria. Hence, we further detected the 3.2. Detection of apoptosis, necrosis, and autophagy mitochondrial membrane potential of J4 or J6, to understand if it could be positively affected in CCRF-CEM cells. Cells were treated at IC50 or 2- Our previous investigation revealed that J4 and J6 exerted cyto- fold IC50 with J4 or J6 for 24 h. Subsequently, the cells were stained with toxicity in CCRF-CEM cells without inducing apoptotic cell death at JC-1 and analyzed by flow cytometry. Fig. 5 shows that J4, J6, as well as different concentrations and timepoints (Rudbari et al., 2022). As a first vinblastine altered the MMP to different extents, as indicated by the step to illustrate the mode of cell death of J4 and J6, the detection of sharp shift of red fluorescence of JC-1 to green fluorescence. The per- apoptosis by annexin V-FITC/PI staining was carried out to verify our centages of reduced MMP with J4 were up to 94.8%–95.6%, and with J6 previous results. After treatment with J4 or J6 at 4 × IC50 for 48 h were 90.47%–95.7%, which was more significant than that of vinblas- (Fig. 3), 86.9% of cells treated with J4 were non-apoptotic in CCRF-CEM tine displaying a 54.9% alternation. Therefore, these findings suggested cells, J6 also showed 81.03% of non-apoptotic cells. Fractions of 95.9% that both J4 and J6 can cause mitochondrial dysfunction, and we as- or 88.9% of CEM/ADR5000 cells remained non-apoptotic upon treat- sume that they may induce other forms of programmed cell death ment with J4 or J6. However, over 90% of living cells appeared upon regulated by mitochondria. DMSO treatment in both cell lines. Cisplatin as a positive control induced 42.9% late apoptosis in CCRF-CEM cells and 24.7% early 3.4. Western blotting apoptosis in CEM/ADR5000 cells. J4 or J6 induced only minor fractions of apoptosis. This validation experiment was consistent with our pre- To study the predominant mode of cell death induced by J4 and J6, vious investigation (Rudbari et al., 2022) and proved that J4 or J6 did the protein expression level of poly (ADP-ribose) (PAR) polymerase-1 not significantly kill CCRF-CEM and CEM/ADR5000 cells by apoptosis (PARP-1) and caspase 3 were firstly measured by western blotting or necrosis. analysis in CCRF-CEM cells (Fig. 6A). Significantly increased levels of Afterward, to investigate whether J4 and J6 induce autophagy as the both full-length PARP (116 kDa) and cleaved PARP (89 kDa) were mode of cell death, we measured the expression level of p62 and Beclin observed upon increasing J4 or J6 concentrations. Caspase 3 and after treatment for 24 h. Fig. 4 shows that in CCRF-CEM cells, J4 grad- cleaved caspase 3 were expressed but the differences were not signifi- ually increased p62 expression and decreased Beclin expression, while cant in comparison to untreated cells, implying that the mode of cell J6 downregulated p62 and upregulated Beclin. It seems that J6 may death was caspase-independent. Afterward, we concentrated on another induce autophagy, but the expression of these biomarkers was not sig- potential mode of cell death that does not rely on caspase: parthanatos. nificant for both compounds. In CEM/ADR5000 cells treated with J4 or As assumed, poly (ADP-ribose) (PAR) and p-histone H2A.X showed an J6, the expression of p62 and Beclin showed slight variations but obviously increased expression. Then, we detected the expression levels without significant differences compared to control. Therefore, auto- of apoptosis-inducing factor (AIF) in the nucleus and cytoplasm, phagy was not the major mode of cell death of J4 and J6. These two respectively (Fig. 6B–C). Notably, AIF was upregulated in the nucleus in compounds presumably kill CCRF-CEM and CEM/ADR5000 cells by a concentration-dependent manner, particularly J6 treated with 2 × IC50 other cell death mechanisms. To evaluate the primary mode of cell death was very significant (p = 0.007). Cytoplasmic AIF showed a slight decrease with J4 and steady expression with J6. These results suggest that J4 and J6 predominantly induced parthanatos as the major mech- anism of cell death. 3.5. Nuclear localization of AIF Translocation of AIF, which occurs downstream of PAR over- expression, is a critical step of parthanatos. Using immunofluorescent imagining, the localization of AIF was monitored upon 24 h treatment with J4 or J6 to verify whether AIF can translocate to the nucleus. The experiments were carried out in both CCRF-CEM and CEM/ADR5000 cells. Fig. 7 indicates that J4 and J6 treated independently with 0.5- or 1- fold IC50 showed a nuclear fraction of AIF besides that at the cytoplasm in both leukemia cell lines, while AIF was on the edge of the nucleus and only localized at the cytoplasm in the untreated cells. Therefore, these findings of CCRF-CEM cells were consistent with the results from western blotting, confirming that both J4 and J6 caused the trans- location of AIF from the cytoplasm into the nucleus, further leading to parthanatic cell death. Similarly, the observed translocation of AIF in CEM/ADR5000 cells indicated parthanatos as the principal mode of cell death induced by J4 or J6. 3.6. AIF and mitochondria staining Fig. 2. Cytotoxicity of compounds J4 and J6 toward human peripheral Non-stimulated AIF resides in mitochondria (Yu et al., 2002). We mononuclear cells (PBMCs). The PBMCs were isolated from three healthy do- found that AIF was translocated from the cytosol into the nucleus of nors and the measurements were carried out after incubation with a series of CCRF-CEM and CEM/ADR5000 cells by treatment with J4 and J6. As concentrations of J4 or J6 for 72 h. shown in Fig. 8 and Supplementary Video 1, the triple 6 M. Zhou et al. E u r o p e a n J o u r n a l o f P h a r m a c o l o g y 956 (2023) 175980 Fig. 3. Assessment of apoptosis. CCRF-CEM leukemia cells and their multidrug-resistant CEM/ADR5000 subline treated with palladium (II)-based compounds J4 or J6, DMSO (negative control), and cisplatin (positive control) after 48 h. Samples were stained by annexin V-FITC/PI and detected on flow cytometry. The 4 × IC50 was 7.12 μM for J4 and 9.56 μM for J6 in CCRF-CEM cells. In CEM/ADR5000 cells, 4 × IC50 was 29.0 μM for J4, and 19.5 μM for J6. Q1 and Q2: necrotic cells or late apoptotic cells exhibit annexin V+/PI+; Q3: early apoptotic cells exhibit annexin V+/PI-; Q4: non-apoptotic cells exhibit annexin V-/ PI-. The graph presents the mean fraction of CCRF- CEM cells. The data represent as mean values ± SD of three independent experiments. The statistical significance was calculated by the Student’s t-test, *p < 0.05, **p < 0.01 compared with DMSO. immunofluorescence staining of untreated cells shows that AIF was further applied for the confirmation of parthanatos. As shown in Fig. 10, present with mitochondria and outside of the nucleus. This confocal cells treated with J4 or J6 alone resulted in lower percentages of visible microscopical experiment illustrated the migration of AIF localization. cells, while J4 or J6 in combination with PJ34 clearly prevented cell death. The cell viability of J4 treated in CEM/ADR5000 cells at a con- 3.7. Single cell gel electrophoresis (comet assay) centration of IC50 combined with PJ34 (p = 0.01), as well as J6 treated at a concentration of IC50 combined with PJ34 in both CCRF-CEM (p = As a result of nuclear AIF translocation, DNA fragmentation and 0.008), and CEM/ADR5000 (p = 0.008) cells are particularly significant chromatin condensation were observed. Since the protein expression compared with only J4 or J6. Therefore, PJ34 maintained cell survival levels of p-histone H2AX in CCRF-CEM cells treated with J4 and J6 were in the presence of J4 or J6, supporting that indeed parthanatos was the shown at 24 h, we also performed comet assay and detected DNA primary mode of cell death. damage at the same timepoint. As shown in Fig. 9, the cells with J4 or J6 treatment at a concentration of IC50 showed slight tails-like patterns. 4. Discussion Furthermore, the mean value (percentage of tail DNA) of J4 at 2 × IC50 was increased to 9.3%, and J6 at 2 × IC50 also significantly showed a Cells die through a variety of mechanisms that can be categorized mean value of 41.3%. The DNA damage extent of H O was less than J6 into non-programmed cell death as unexpected cell injury and pro-2 2 with a mean value of 26%. No effects were observed in untreated cells grammed cell death. Programmed cell death can be further classified (DMSO). Therefore, J4 and J6 induced DNA damage in a concentration- into apoptotic cell death and non-apoptotic cell death (e.g., autophagy, dependent manner followed by AIF translocation, which further induced entosis, pyroptosis, mitoptosis, ferroptosis, and necroptosis) (Yan et al., cell death. 2020). Previously, we synthesized a set of Pd (II) complexes, and the best two of them (J4 and J6) revealed excellent cytotoxicity in CCRF-CEM leukemia cells resulting in G2/M cell cycle arrest but without inducing 3.8. Confirmation of parthanatos using PARP inhibitor apoptotic cell death (Rudbari et al., 2022). In continuation of this work on Pd (II) complexes, the goal of this study was to pinpoint the principal Parthanatos is a PARP-dependent cell death form (Yu et al., 2006). In mode of cell death induced by these two palladium-based complexes in a series of experiments, we identified key events of parthanatos induced drug-sensitive CCRF-CEM and its multidrug-resistant CEM/ADR5000 by J4 and J6 in leukemia cells. The known PARP inhibitor PJ34 was 7 M. Zhou et al. E u r o p e a n J o u r n a l o f P h a r m a c o l o g y 956 (2023) 175980 Fig. 4. Western blot analysis of biomarkers related to autophagy. CCRF-CEM and CEM/ADR5000 leukemia cells treated with palladium (II)-based compounds J4 or J6 after 24 h. (A) p62 and Beclin were detected by western blotting in CCRF-CEM cells. (B) p62 and Beclin in CEM/ADR5000 cells. Graphs show quanti- fication of protein expression levels with J4 and J6. The expression of p62 and Beclin was normalized to GAPDH or β-actin. The histograms present the mean value ± SD of three independent experiments. Sta- tistics analysis was done by paired Student’s t-test. subline. Our current finding illustrated that parthanatos was the major staining (Ari et al., 2014; Coskun et al., 2013). As a second independent mode of cell death as evidenced by rapid accumulation of PARP and method, we further determined the expression level of caspase 3, PAR, dissipation of mitochondrial membrane potential, nuclear AIF cleaved caspase 3, and cleaved PARP by western blotting in CCRF-CEM translocation, and DNA fragmentation. Additionally, the cytotoxicity on cells. Fig. 5A indicates that J4 or J6 induced the expression of both PBMCs cells revealed that both J4 and J6 were less toxic toward PBMCs. caspase 3 and cleaved caspase 3, while almost equal expression levels We previously reported that J4 and J6 were more specific on eliminating appeared as negative control. Cleaved caspase 3 could be related to the leukemia cells (Rudbari et al., 2022). This difference in activity towards small apoptotic fraction observed in flow cytometry, indicating that J4 leukemia cells and normal leukocytes is consistent with the recently or J6 induced a minor cell fraction with apoptotic cell death but it was described safety profile that palladium nanoparticles showed mitigated not the major mode of cell death. Along this line, PARP-1 as one of the toxicity compared to formulations with conventional chemotherapeutic downstream substrates of caspases can be cleaved as an 89 kDa fragment drugs (Miller et al., 2017). during apoptotic cell death (Soldani and Scovassi, 2002). As expected, Initially, cell death was divided into three types: apoptosis (Type I), we observed expression levels of cleaved PARP which were in accor- autophagy (Type II), and necrosis (Type III) (Green and Llambi, 2015). dance with cleaved caspase 3. In our study, cleaved PARP by both J4 and Apoptosis is characterized as a series of well-organized distinct steps and J6 reached significant expression levels in a concentration-dependent biochemical modifications including cell shrinkage, phosphatidylserine manner. The expression of cleaved PARP and cleaved caspase 3 was (PS) exposure, DNA condensation and fragmentation, as well as mito- also found in human breast cancer cell lines treated with other Pd (II) chondrial membrane potential alternation. The initiation of apoptosis is complexes (Ari et al., 2013). PARP-1 can be also cleaved even in the dependent on the activation of caspases (Elmore, 2007). In our study, we absence of caspase 3 (Jänicke et al., 1998). However, it is important to analyzed apoptosis with two methods. First, using annexin V-FITC/PI note that the expression of caspase 3 was not significant, implying that staining, CCRF-CEM and CEM/ADR5000 cells treated with J4 or J6 at a the major mode of cell death may not rely on caspases. Among the concentration of 4 × IC50 for 48 h still did not show significant apoptotic various forms of cell death, one caspase-independent mode has been cells or necrotic cells were induced, which is in accord with our previ- addressed as parthanatos (Kang et al., 2004; Yu et al., 2002). Therefore, ously reported results (Rudbari et al., 2022). Annexin V and PI uptake is we conclude that J4 and J6 induced apoptosis only in a minor cell one of the most widely used assays to measure apoptosis and necrosis. PS fraction, and we further investigated if CCRF-CEM and CEM/ADR5000 located at the outer layer of the plasma membrane are bound to annexin cells would be killed through parthanatos. V, and DNA fragments that are released from apoptotic nuclei are PARP-1 is the best-known nuclear enzyme of the PARP superfamily. stained by PI (Crowley et al., 2016; Riccardi and Nicoletti, 2006). In It is a 116 kDa protein that serves as a DNA damage sensor. In the contrast to other studies, several Pd (II) complexes recently have been presence of moderate DNA damage, PARP uses nicotine amide adenine described as inducers of apoptosis as proved by annexin V-FITC/PI dinucleotide (NAD+) as a substrate to catalyze the addition of 8 M. Zhou et al. E u r o p e a n J o u r n a l o f P h a r m a c o l o g y 956 (2023) 175980 Fig. 5. Assessment of mitochondrial membrane potential. Representative images of JC-1 fluorescence with flow cytometry of mitochondrial membrane potential in CCRF-CEM cells treated with palladium (II)-based compounds J4 or J6, DMSO (negative control) or vinblastine (positive control) for 24 h. The IC50 was 1. 78 μM for J4 and 2.39 μM for J6. 2 × IC50 was 3.56 μM for J4 and 4.78 μM for J6. Death of cells were defined as MMP collapse after 24 h treatment. The graph presents percentages of CCRF-CEM cells. The statistical analysis was done by paired Student’s t-test, **p ≤ 0.01, if compared to DMSO untreated cells. Mean values ± SD were derived from three independent experiments. monoADP-ribose or PAR to various acceptor proteins or to PARP itself. Interestingly, our mitochondrial depolarization experiments showed a This early event fosters the recruitment of DNA repair proteins and sharp dissipation of the mitochondrial membrane potential by J4 and J6. nucleases to damaged sites and promotes DNA damage repair (Wang At the same time, we verified that the cell viability was increased if J4 or et al., 2019). Whereas in toxin-exposed cells with considerable DNA J6 was combined with the PARP inhibitor PJ34, indicating that the damage, PARP becomes hyper-activated, leading to PAR production, mode of cell death was PARP-independent. Following PARP and PAR which translocates to the cytosol, from where it affects the translocation overexpression as well as mitochondrial damage, AIF is released from of AIF to the nucleus and results in large-scale DNA fragmentation and the mitochondrial outer membrane layer and translocated through the chromatin condensation. These steps trigger the PARP-dependent and cytosol into the nucleus. AIF is a mitochondrial effector regulating cell caspase-independent parthanatos (Andrabi et al., 2008; Fatokun et al., death and survival. Reducing AIF abundance or blocking AIF release 2014). It acts through unique biochemical features which differ from protects cells against parthanatos (Wang et al., 2009). Therefore, AIF is a that of apoptosis. Parthanatos has been associated with ischemic commitment protein in this form of cell death. PARP, PAR, and AIF reperfusion injury after brain ischemia or myocardial infarction (Eli- regulate parthanatos in a tightly coordinated manner. Genetic deletion asson et al., 1997; Harraz et al., 2008; Zhang et al., 2019), neurode- of PARP caused a failure of AIF to translocate into the nucleus (Yu et al., generative disease (Kam et al., 2018), and retinal disease (Greenwald 2002). Furthermore, AIF is a PAR-binding protein (Wang et al., 2011), and Pierce, 2019). and AIF-release activity is abolished if cells are treated with PAR gly- Our western blotting analyses in CCRF-CEM cells showed that PARP cohydrolase, which can degrade PAR. This demonstrates that PAR is a was rapidly overactivated upon J4 or J6 treatment, which stimulates releasing signal of AIF (Andrabi et al., 2006a; Yu et al., 2006). Hence, mass PAR production and indicates the initiation of parthanatos. The PARP activation and PAR formation are fundamental for AIF trans- overactivation of PARP depletes NAD+, which is also required for the location into the nucleus. Our results of nuclear AIF by western blotting synthesis of PAR. In turn, increased PARP and PAR cause mitochondrial in CCRF-CEM cells indeed showed that AIF was released from mito- dysfunction. Mitochondrial depolarization could be reversed by PARP chondria and translocated to the nucleus. Immunofluorescence micro- inhibitor or supplementation with NAD+ (Jang et al., 2017). scopy demonstrated as well that AIF accumulated in the nucleus, which 9 M. Zhou et al. E u r o p e a n J o u r n a l o f P h a r m a c o l o g y 956 (2023) 175980 Fig. 6. Western blot analysis of parthanatos-related proteins. CCRF-CEM cells were treated with palla- dium (II)-based compounds J4 or J6 treated for 24 h. (A) The protein expression of full-length PARP, cleaved PARP, p-histone H2AX, PAR, caspase 3, and cleaved caspase 3 in total protein. (B) The protein expression of AIF in nuclear protein. (C) The protein expression of AIF in cytoplasmic protein. (D), (E), (F), and (G) Quantification of protein expression levels. Data represent relative expression intensity to GAPDH or β-actin. Statistical significance used paired Stu- dent’s t-test in comparison to control (DMSO), *p ≤ 0.05, **p ≤ 0.01. The data represent as mean values ± SD of three independent experiments. Fig. 7. Detection of AIF translocation from the cytoplasm into the nucleus by immunofluorescence microscopy. Cells were treated with different concentrations of palladium (II)-based compounds J4 or J6, or DMSO (control) for 24 h. (A) CCRF-CEM cells (Scale bar: 50 μm) and (B) CEM/ADR5000 cells (Scale bar: 80 μm). Samples stained with AIF primary antibody to visualize AIF protein. Images were merged with DAPI (blue) as cell nucleus to indicate AIF (green) translocation. particularly supported that J4 and J6 induced parthanatos also in (Fatokun et al., 2014). The phosphorylation of histone variant H2AX is CEM/ADR5000 cells. Moreover, AIF induces large-scale DNA fragmen- an early cellular response to DNA double-strand breaks induction and is tation after translocation, but apoptosis only causes small-scale DNA a sensitive biomarker and commonly used to monitor DNA damage fragmentation, which means that AIF induces more critical DNA damage initiation (Mah et al., 2010). Our results on p-histone H2A.X expression 10 M. Zhou et al. E u r o p e a n J o u r n a l o f P h a r m a c o l o g y 956 (2023) 175980 Fig. 8. The mitochondrial localization of AIF in (A) CCRF-CEM cells and (B) CEM/ADR5000 cells. Cells were stained with AIF (green), MitoTracker® Deep Red (magenta) to show mitochondria, and DAPI (blue) to show the nucleus. See also Supplementary Video 1. in CCRF-CEM cells showed a continuous upregulation upon the treat- cellular proteins and organisms and deliver the infusion with the lyso- ments, especially with J6, and the comet assay revealed the formation of some (Levy et al., 2017). This lysosomal degradation pathway is a highly J4- and J6-induced DNA tails in a concentration-dependent manner. In controlled process that is regulated by multiple signaling events. It in- addition, parthanatos as caspase-independent cell death cannot be volves a set of about 16–20 core conserved autophagy-related genes rescued by pan-caspase inhibitors. However, the activation of caspase (ATGs), which initiate the formation of the autophagosome (Levine and was found in some studies after AIF release (Yu et al., 2002). Our Kroemer, 2019). Human Beclin, an ortholog of the Atg6/vacuolar pro- western blotting also showed caspase 3 expression. In parallel to AIF, tein sorting (Vps)-30 protein in yeast, is important for the localization of mitochondria contain different apoptogenic factors including cyto- autophagic proteins to the phagophore, which interacts with the class III chrome C, pro-caspase 2, 3, and 9 to engage in the degradation of phosphatidylinositol 3-kinase (PI3K) complex and several cofactors (He apoptosis. AIF can initiate the release of cytochrome c, which further and Levine, 2010). By means of gene-transfer techniques, autophagic activates caspases. The activation of caspase after the executioner phase bodies have been identified in beclin-1-transformed yeast that were of AIF may assist in cell disintegration (Susin et al., 1999). Therefore, we hardly seen in non-transformed yeast, confirming that Beclin induces conclude that parthanatos is the most relevant mode of cell death autophagy (Liang et al., 1999). Our data revealed that J4 decreased the induced by the two Pd (II) complexes, J4 and J6. expression of Beclin 1, while J6 increased it in CCRF-CEM cells. The The crosstalk between apoptosis and autophagy is intricate. If cells effects of Beclin in CEM/ADR5000 cells were minor (Fig. 3). On the are under stress, autophagy can activate a cytoprotective switch by other hand, sequestosome 1 (SQSTM 1/p62) is an autophagy receptor inactivating apoptosis to help cancer cells to escape cell death (Gump and a selective substrate for autophagy. P62 interacts with light chain 3 and Thorburn, 2011). Hence, we also focused on autophagy to under- (LC3) for attachment to the autophagosomes, and thereby delivers stand whether autophagy contributes to death or even protects cells polyubiquitin cargos to the lysosome system for degradation (Islam from apoptosis by J4 and J6. Autophagy is a programmed self-digesting et al., 2018). A widely accepted notion is that the activation of auto- mechanism, which is characterized by the formation of phagy suppresses the expression of p62, as p62 degrades together with double-membrane vesicles (autophagosomes) that engulfs impaired the target cargos (Liu et al., 2016). Our results showed that p62 11 M. Zhou et al. E u r o p e a n J o u r n a l o f P h a r m a c o l o g y 956 (2023) 175980 Fig. 9. Detection of DNA damage by the alkaline comet assay. Representative images of CCRF-CEM cells treated with palladium (II)-based compounds J4, J6, or DMSO (control) for 24 h, or H2O2 (positive control) for 1 h. Scale bar, 60 μm. The tail DNA percentage was measured by Image J from 50 randomly represented as mean ± SEM shown in the bar diagram. Statistical significance (*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.0001) was compared to DMSO. expression in CCRF-CEM cells was upregulated with J4 and down- as the main cell death pathway. On the other hand, DNA represents the regulated with J6. The expression level of p62 in CEM/ADR5000 cells principal target of Pd (II) complexes (Ferraro et al., 2022). It has been was, however, almost equal to untreated samples (Fig. 3). Taken reported that DNA binding by Pd (II) complexes causes genetic damage together, J6 may induce autophagy only in CCRF-CEM cells. A crosstalk (Bjelogrlić et al., 2019). Our single cell electrophoresis observations and between autophagy and parthanatos modulated by PARP was reported upregulated p-histone H2AX expression both supported that J4 and J6 in rat stria marginal cells (Jiang et al., 2018). However, upon the caused DNA damage, which subsequently induced parthanatos. elevated treated concentration in western blotting, there was no statis- Another aspect we focused on was the relevance of the multidrug tical significance observed for these two autophagy biomarkers. There- resistance phenotype for our palladium (II) complexes. Drug resistance fore, we concluded that autophagy could not be the major mode of cell is a crucial factor leading to the failure of chemotherapy with fatal death induced by J4 and J6 in CCRF-CEM cells. consequences for patients. Apart from resistance to apoptosis, another Taking a broad look at the field of metal-based agents as chemo- major reason is multidrug resistance (MDR), where tumor cells confer therapeutics, they have been designed with a “multitargeted” approach resistance to a quantity of functionally and structurally unrelated com- to maximize anticancer activity and to overcome the problem of drug pounds via the ATP-binding cassette (ABC) transporters. P-glycoprotein resistance. The utilization of a certain central metal ion can have a (P-gp) is a well-known drug efflux pump, whose overexpression leads to significant impact on biological activity since different metals show lower efficacy of chemotherapeutics (Li et al., 2016). We previously distinct physicochemical characteristics (Lucaciu et al., 2022). In an reported that P-gp-overexpressing CEM/ADR5000 cells were moder- endeavor to replace platinum, palladium, gold, and ruthenium are the ately cross-resistant to J4 and J6 (2- to 4-fold) (Rudbari et al., 2022), most studied metals with potential anticancer activity (Ferraro et al., while they are highly cross-resistant to many clinically established 2022). However, the challenge of designing and synthesizing Pd (II) anticancer drugs (100- to 1000-fold) (Efferth et al., 2008). In the present complexes is their lower kinetic stability, which affects to address their study, we observed that CEM/ADR5000 cells are also able to induce drug targets (Prince et al., 2017). The stability of our Pd (II) complexes parthanathos upon J4 or J6 treatment. We conclude that at lower con- containing halogen-substituted Schiff bases and pic have been previ- centrations, P-gp is expelling these two compounds, while at higher ously confirmed by LC/MS analysis (Rudbari et al., 2022). Moreover, we concentrations the efflux capacity of P-gp might be exhausted leading to also confirmed that there is no cytotoxicity from the original material parthanathos. Parthanatos may be an alternative strategy to overcome palladium salt (PdCl2), and the cytotoxic activity of the Schiff base drug resistance if other mechanisms of cell death such as apoptosis or ligand is inadequate compared with the corresponding Pd (II) complex. autophagy fail. Therefore, our drug design strategy with excellent anticancer activity and stability is worthy to be further studied. Generally, the induction of 5. Conclusion apoptotic cell death pathways has been a main goal to achieve cyto- toxicity in designing non-platinum compounds (Ferraro et al., 2022). In conclusion, we demonstrated that two Pd (II) complexes con- Similarly, numerous investigations indeed have shown that Pd (II)-based taining double chlorine-/iodine- substituted Schiff base and 2-picolyl- complexes can cause cell death by apoptosis (Ari et al., 2014; Coskun amine (J4 and J6) induced parthanatos as the major mode of cell et al., 2013; Keswani et al., 2014). However, dysfunctional apoptosis death accompanied with a minor contribution of apoptotic cell death in allows tumor cells to escape from this mode of programmed cell death drug-sensitive CCRF-CEM and multidrug-resistant CEM/ADR5000 leu- for uncontrolled proliferation. To the best of our knowledge, our current kemia cells. Both compounds displayed non-toxic effects on human pe- study is the first to report that Pd (II) complexes can induce parthanatos ripheral mononuclear cells. They induced overactivation of PARP and 12 M. Zhou et al. E u r o p e a n J o u r n a l o f P h a r m a c o l o g y 956 (2023) 175980 Fig. 10. Cell viability of PARP inhibitor PJ34 in the presence of different concentrations (0.5 × IC50, and IC50) of J4 or J6. (A) CCRF-CEM and (B) ADR/ CEM5000 cells treated with J4 or in combination with PJ34. (C) CCRF-CEM and (D) ADR/CEM5000 cells treated with J6 or in combination with PJ34. PJ34 was 10 μM. Bar diagrams were shown as mean value ± SD from three repetitions). Statistical signif- icance used paired Student’s t-test (*p ≤ 0.05, **p ≤ 0.01), if compared with J4 or J6 treatment alone. PAR, nuclear AIF translocation, dissipation of mitochondrial membrane Investigation, Validation, Writing – review & editing. Ejlal A. Omer: potential, and DNA damage. These effects led to cells dying from par- Data collection, Investigation, Validation, Writing – review & editing. thanatos. Along with our previous biological investigations (Rudbari Hadi Amiri Rudbari: Resources, Visualization, Writing – review & et al., 2022), J4 and J6 arrest the cell cycle in the G2/M phase and editing. Tanja Schirmeister: Resources, Writing – review & editing. inhibited the proteolytic activity of the proteasome in CCRF-CEM cells. Nicola Micale: Resources, Writing – review & editing. Thomas Efferth: These results present the novel Pd (II) complexes J4 and J6 as promising Study conception and design, Project administration, Supervision, anticancer candidates. The induction of parthanatos by J4 and J6 im- Writing – review & editing. plies the possibility of better attacking drug-resistant cancers suffering from the inability to induce other modes of cell death. Our results may Declaration of competing interest encourage further studies on palladium-based drug design, and palladium-based complexes may offer great potential for cancer therapy. The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence CRediT authorship contribution statement the work reported in this paper. Min Zhou: Study conception and design, Data collection, Investi- Data availability gation, Validation, Software, Formal analysis, Data curation, Visuali- zation, Project administration, Writing – original draft, preparation, Data will be made available on request. Writing – review & editing. Joelle C. Boulos: Data collection, 13 M. Zhou et al. E u r o p e a n J o u r n a l o f P h a r m a c o l o g y 956 (2023) 175980 Acknowledgements De Biasi, S., Gibellini, L., Cossarizza, A., 2015. Uncompensated polychromatic analysis of mitochondrial membrane potential using JC-1 and multilaser excitation. Curr Protoc Cytom 72 (7), 32.31-37.32.11. We thank the IMB Microscopy and Histology Core Facility (Mainz, Desoize, B., Madoulet, C., 2002. Particular aspects of platinum compounds used at Germany) for their helpful training and technical support for present in cancer treatment. Crit. Rev. Oncol. Hematol. 42, 317–325. microscopy-related experiments with the AF7000 widefield fluores- Döhner, H., Wei, A.H., Löwenberg, B., 2021. Towards precision medicine for AML. Nat. Rev. Clin. Oncol. 18, 577–590. cence microscope (founded by DFG grant 212049334) and Stellaris 8 Efferth, T., Konkimalla, V.B., Wang, Y.F., Sauerbrey, A., Meinhardt, S., Zintl, F., Falcon confocal microscope (founded by the DFG grant 497669232). We Mattern, J., Volm, M., 2008. Prediction of broad spectrum resistance of tumors also thank the IMB Flow Cytometry Core Facility (Mainz, Germany) for towards anticancer drugs. Clin. Cancer Res. 14, 2405–2412. the analysis of mitochondrial membrane potential on BD LSRFortessa Efferth, T., Sauerbrey, A., Olbrich, A., Gebhart, E., Rauch, P., Weber, H.O., Hengstler, J. G., Halatsch, M.E., Volm, M., Tew, K.D., Ross, D.D., Funk, J.O., 2003. Molecular SORP. We are grateful for a Ph.D. stipend from the Chinese Scholarship modes of action of artesunate in tumor cell lines. Mol. Pharmacol. 64, 382–394. Council to M.Z., the stipend from the Sibylle Kalkhof-Rose-Foundation El-Morsy, F.A., Jean-Claude, B.J., Butler, I.S., El-Sayed, S.A., Mostafa, S.I., 2014. to J.C.B., and the stipend from the German Academic Exchange Ser- Synthesis, characterization and anticancer activity of new zinc(II), molybdate(II), palladium(II), silver(I), rhodium(III), ruthenium(II) and platinum(II) complexes of vice (DAAD) to E.A.O. 5,6-diamino-4-hydroxy-2-mercaptopyrimidine. Inorg. Chim. Acta. 423, 144–155. Eliasson, M.J., Sampei, K., Mandir, A.S., Hurn, P.D., Traystman, R.J., Bao, J., Pieper, A., Appendix A. Supplementary data Wang, Z.Q., Dawson, T.M., Snyder, S.H., Dawson, V.L., 1997. Poly(ADP-ribose) polymerase gene disruption renders mice resistant to cerebral ischemia. Nat. Med. 3, 1089–1095. Supplementary data related to this article can be found at https Elmore, S., 2007. Apoptosis: a review of programmed cell death. Toxicol. Pathol. 35, ://doi.org/10.1016/j.ejphar.2023.175980. 495–516. Fatokun, A.A., Dawson, V.L., Dawson, T.M., 2014. Parthanatos: mitochondrial-linked mechanisms and therapeutic opportunities. Br. J. Pharmacol. 171, 2000–2016. References Ferraro, M.G., Piccolo, M., Misso, G., Santamaria, R., Irace, C., 2022. Bioactivity and development of small non-platinum metal-based chemotherapeutics. Pharmaceutics Abu-Surrah, A.S., Abdalla, M.Y., 2008. Palladium-based chemotherapeutic agents: routes 14, 954. toward complexes with good antitumor activity. Cancer Ther. 6, 1–10. Finger, P.T., Berson, A., Ng, T., Szechter, A., 2002. Palladium-103 plaque radiotherapy Alam, M.N., Huq, F., 2016. Comprehensive review on tumour active palladium for choroidal melanoma: an 11-year study. Int. J. Radiat. Oncol. Biol. Phys. 54, compounds and structure–activity relationships. Coord. Chem. Rev. 316, 36–67. 1438–1445. Andrabi, S.A., Dawson, T.M., Dawson, V.L., 2008. Mitochondrial and nuclear cross talk Florea, A.M., Büsselberg, D., 2011. Cisplatin as an anti-tumor drug: cellular mechanisms in cell death: parthanatos. Ann. N. Y. Acad. Sci. 1147, 233–241. of activity, drug resistance and induced side effects. Cancers 3, 1351–1371. Andrabi, S.A., Kim, N.S., Yu, S.-W., Wang, H., Koh, D.W., Sasaki, M., Klaus, J.A., Foley, G.E., Lazarus, H., Farber, S., Uzman, B.G., Boone, B.A., McCarthy, R.E., 1965. Otsuka, T., Zhang, Z., Koehler, R.C., Hurn, P.D., Poirier, G.G., Dawson, V.L., Continuous culture of human lymphoblasts from peripheral blood of a child with Dawson, T.M., 2006a. Poly(ADP-ribose) (PAR) polymer is a death signal. Proc. Natl. acute leukemia. Cancer 18, 522–529. Acad. Sci. U.S.A. 103, 18308–18313. Frezza, M., Hindo, S., Chen, D., Davenport, A., Schmitt, S., Tomco, D., Dou, Q.P., 2010. Andrabi, S.A., Kim, N.S., Yu, S.W., Wang, H., Koh, D.W., Sasaki, M., Klaus, J.A., Novel metals and metal complexes as platforms for cancer therapy. Curr. Otsuka, T., Zhang, Z., Koehler, R.C., Hurn, P.D., Poirier, G.G., Dawson, V.L., Pharmaceut. Des. 16, 1813–1825. Dawson, T.M., 2006b. Poly(ADP-ribose) (PAR) polymer is a death signal. Proc. Natl. Ghosh, S., 2019. Cisplatin: the first metal based anticancer drug. Bioorg. Chem. 88, Acad. Sci. U.S.A. 103, 18308 102925. –18313. Ari, F., Cevatemre, B., Armutak, E.I., Aztopal, N., Yilmaz, V.T., Ulukaya, E., 2014. Green, D.R., Llambi, F., 2015. Cell death signaling. Cold Spring Harbor Perspect. Biol. 7, Apoptosis-inducing effect of a palladium(II) saccharinate complex of terpyridine on a006080. human breast cancer cells in vitro and in vivo. Bioorg. Med. Chem. 22, 4948–4954. Greenwald, S.H., Pierce, E.A., 2019. Parthanatos as a cell death pathway underlying Ari, F., Ulukaya, E., Sarimahmut, M., Yilmaz, V.T., 2013. Palladium(II) saccharinate retinal disease. Adv. Exp. Med. Biol. 1185, 323–327. complexes with bis(2-pyridylmethyl)amine induce cell death by apoptosis in human Gump, J.M., Thorburn, A., 2011. Autophagy and apoptosis: what is the connection? breast cancer cells in vitro. Bioorg. Med. Chem. 21, 3016–3021. Trends Cell Biol. 21, 387–392. Azzouzi, A.R., Vincendeau, S., Barret, E., Cicco, A., Kleinclauss, F., van der Poel, H.G., Gyori, B.M., Venkatachalam, G., Thiagarajan, P.S., Hsu, D., Clement, M.V., 2014. Stief, C.G., Rassweiler, J., Salomon, G., Solsona, E., Alcaraz, A., Tammela, T.T., OpenComet: an automated tool for comet assay image analysis. Redox Biol. 2, Rosario, D.J., Gomez-Veiga, F., Ahlgren, G., Benzaghou, F., Gaillac, B., Amzal, B., 457–465. Debruyne, F.M., Fromont, G., Gratzke, C., Emberton, M., 2017. Padeliporfin Hanahan, D., Weinberg, R.A., 2011. Hallmarks of cancer: the next generation. Cell 144, vascular-targeted photodynamic therapy versus active surveillance in men with low- 646–674. risk prostate cancer (CLIN1001 PCM301): an open-label, phase 3, randomised Harraz, M.M., Dawson, T.M., Dawson, V.L., 2008. Advances in neuronal cell death 2007. controlled trial. Lancet Oncol. 18, 181–191. Stroke 39, 286–288. Batista de Carvalho, A.L., Medeiros, P.S., Costa, F.M., Ribeiro, V.P., Sousa, J.B., Diniz, C., He, C., Levine, B., 2010. The Beclin 1 interactome. Curr. Opin. Cell Biol. 22, 140–149. Marques, M.P., 2016. Anti-invasive and anti-proliferative synergism between Huang, Y., Ibrado, A.M., Reed, J.C., Bullock, G., Ray, S., Tang, C., Bhalla, K., 1997. Co- docetaxel and a polynuclear Pd-spermine agent. PLoS One 11, e0167218. expression of several molecular mechanisms of multidrug resistance and their Bispo, J.A.B., Pinheiro, P.S., Kobetz, E.K., 2020. Epidemiology and etiology of leukemia significance for paclitaxel cytotoxicity in human AML HL-60 cells. Leukemia 11, and lymphoma. Cold Spring Harb. Perspect. Med. 10, a034819. 253–257. Bjelogrlić, S.K., Todorović, T.R., Kojić, M., Senćanski, M., Nikolić, M., Vǐsnjevac, A., Ilić, D.R., Jevtić, V.V., Radić, G.P., Arsikin, K., Ristić, B., Harhaji-Trajković, L., Araškov, J., Miljković, M., Muller, C.D., Filipović, N.R., 2019. Pd(II) complexes with Vuković, N., Sukdolak, S., Klisurić, O., Trajković, V., Trifunović, S.R., 2014. N-heteroaromatic hydrazone ligands: anticancer activity, in silico and experimental Synthesis, characterization and cytotoxicity of a new palladium(II) complex with a target identification. J. Inorg. Biochem. 199, 110758. coumarine-derived ligand. Eur. J. Med. Chem. 74, 502–508. Boulos, J.C., Omer, E.A., Rigano, D., Formisano, C., Chatterjee, M., Leich, E., Klauck, S. Islam, M.A., Sooro, M.A., Zhang, P., 2018. Autophagic regulation of p62 is critical for M., Shan, L.-t., Efferth, T., 2023. Cynaropicrin disrupts tubulin and c-Myc-related cancer therapy. Int. J. Mol. Sci. 19, 1405. signaling and induces parthanatos-type cell death in multiple myeloma. Acta Jang, K.H., Do, Y.J., Son, D., Son, E., Choi, J.S., Kim, E., 2017. AIF-independent Pharmacol. Sin. https://doi.org/10.1038/s41401-023-01117-3. Online ahead of parthanatos in the pathogenesis of dry age-related macular degeneration. Cell Death print. Dis. 8, e2526. Carneiro, T.J., Martins, A.S., Marques, M.P.M., Gil, A.M., 2020. Metabolic aspects of Jänicke, R.U., Ng, P., Sprengart, M.L., Porter, A.G., 1998. Caspase-3 is required for alpha- palladium(II) potential anti-cancer drugs. Front. Oncol. 10, 590970. fodrin cleavage but dispensable for cleavage of other death substrates in apoptosis. Chipuk, J.E., Moldoveanu, T., Llambi, F., Parsons, M.J., Green, D.R., 2010. The BCL-2 J. Biol. Chem. 273, 15540–15545. family reunion. Mol. Cell 37, 299–310. Jiang, H.Y., Yang, Y., Zhang, Y.Y., Xie, Z., Zhao, X.Y., Sun, Y., Kong, W.J., 2018. The dual Coskun, M.D., Ari, F., Oral, A.Y., Sarimahmut, M., Kutlu, H.M., Yilmaz, V.T., Ulukaya, E., role of poly(ADP-ribose) polymerase-1 in modulating parthanatos and autophagy 2013. Promising anti-growth effects of palladium(II) saccharinate complex of under oxidative stress in rat cochlear marginal cells of the stria vascularis. Redox terpyridine by inducing apoptosis on transformed fibroblasts in vitro. Bioorg. Med. Biol. 14, 361–370. Chem. 21, 4698–4705. Kadioglu, O., Cao, J., Kosyakova, N., Mrasek, K., Liehr, T., Efferth, T., 2016. Genomic and Crowley, L.C., Marfell, B.J., Scott, A.P., Waterhouse, N.J., 2016. Quantitation of transcriptomic profiling of resistant CEM/ADR-5000 and sensitive CCRF-CEM apoptosis and necrosis by annexin V binding, propidium iodide uptake, and flow leukaemia cells for unravelling the full complexity of multi-factorial multidrug cytometry. Cold Spring Harb. Protoc. 2016 https://doi.org/10.1101/pdb. resistance. Sci. Rep. 6, 36754. prot087288. Kam, T.I., Mao, X., Park, H., Chou, S.C., Karuppagounder, S.S., Umanah, G.E., Yun, S.P., Davis, A.S., Viera, A.J., Mead, M.D., 2014. Leukemia: an overview for primary care. Am. Brahmachari, S., Panicker, N., Chen, R., Andrabi, S.A., Qi, C., Poirier, G.G., Fam. Physician 89, 731 738. Pletnikova, O., Troncoso, J.C., Bekris, L.M., Leverenz, J.B., Pantelyat, A., Ko, H.S., – Dawood, M., Elbadawi, M., Böckers, M., Bringmann, G., Efferth, T., 2020. Molecular Rosenthal, L.S., Dawson, T.M., Dawson, V.L., 2018. Poly(ADP-ribose) drives docking-based virtual drug screening revealing an oxofluorenyl benzamide and a pathologic α-synuclein neurodegeneration in Parkinson’s disease. Science 362, bromonaphthalene sulfonamido hydroxybenzoic acid as HDAC6 inhibitors with eaat8407. cytotoxicity against leukemia cells. Biomed. Pharmacother. 129, 110454. 14 M. Zhou et al. E u r o p e a n J o u r n a l o f P h a r m a c o l o g y 956 (2023) 175980 Kang, Y.-H., Yi, M.-J., Kim, M.-J., Park, M.-T., Bae, S., Kang, C.-M., Cho, C.-K., Park, I.-C., Prince, S., Mapolie, S., Blanckenberg, A., 2017. Palladium-based anti-cancer Park, M.-J., Rhee, C.H., Hong, S.-I., Chung, H.Y., Lee, Y.-S., Lee, S.-J., 2004. Caspase- therapeutics. In: Schwab, M. (Ed.), Encyclopedia of Cancer. Springer Berlin independent cell death by arsenic trioxide in human cervical cancer cells: reactive Heidelberg, Berlin, Heidelberg, pp. 3371–3378. oxygen species-mediated poly(ADP-ribose) polymerase-1 activation signals Qin, Q.-P., Zou, B.-Q., Tan, M.-X., Luo, D.-M., Wang, Z.-F., Wang, S.-L., Liu, Y.-C., 2018. apoptosis-inducing factor release from mitochondria. Cancer Res. 64, 8960–8967. High in vitro anticancer activity of a dinuclear palladium(II) complex with a 2- Kapdi, A.R., Fairlamb, I.J., 2014. Anti-cancer palladium complexes: a focus on PdX2L2, phenylpyridine ligand. Inorg. Chem. Commun. 96, 106–110. palladacycles and related complexes. Chem. Soc. Rev. 43, 4751–4777. Reigosa-Chamorro, F., Raposo, L.R., Munín-Cruz, P., Pereira, M.T., Roma-Rodrigues, C., Keswani, T., Chowdhury, S., Mukherjee, S., Bhattacharyya, A., 2014. Palladium(II) Baptista, P.V., Fernandes, A.R., Vila, J.M., 2021. In vitro and in vivo effect of complex induces apoptosis through ROS-mediated mitochondrial pathway in human palladacycles: targeting A2780 ovarian carcinoma cells and modulation of lung adenocarcinoma cell line (A549). Curr. Sci. 107, 1711–1719. angiogenesis. Inorg. Chem. 60, 3939–3951. Kielhorn, J., Melber, C., Keller, D., Mangelsdorf, I., 2002. Palladium–a review of Riccardi, C., Nicoletti, I., 2006. Analysis of apoptosis by propidium iodide staining and exposure and effects to human health. Int. J. Hyg Environ. Health 205, 417–432. flow cytometry. Nat. Protoc. 1, 1458–1461. Kimmig, A., Gekeler, V., Neumann, M., Frese, G., Handgretinger, R., Kardos, G., Rudbari, H.A., Kordestani, N., Cuevas-Vicario, J.V., Zhou, M., Efferth, T., Correia, I., Diddens, H., Niethammer, D., 1990. Susceptibility of multidrug-resistant human Schirmeister, T., Barthels, F., Enamullah, M., Fernandes, A.R., 2022. Investigation of leukemia cell lines to human interleukin 2-activated killer cells. Cancer Res. 50, the influence of chirality and halogen atoms on the anticancer activity of 6793–6799. enantiopure palladium (II) complexes derived from chiral amino-alcohol Schiff bases Kovala-Demertzi, D., Boccarelli, A., Demertzis, M.A., Coluccia, M., 2007. In vitro and 2-picolylamine. New J. Chem. 46, 6470–6483. antitumor activity of 2-acetyl pyridine 4n-ethyl thiosemicarbazone and its platinum Scattolin, T., Bortolamiol, E., Visentin, F., Palazzolo, S., Caligiuri, I., Perin, T., (II) and palladium(II) complexes. Chemotherapy 53, 148–152. Canzonieri, V., Demitri, N., Rizzolio, F., Togni, A., 2020. Palladium(II)-η(3)-allyl Kumaravel, T.S., Vilhar, B., Faux, S.P., Jha, A.N., 2009. Comet Assay measurements: a complexes bearing N-trifluoromethyl N-heterocyclic carbenes: a new generation of perspective. Cell Biol. Toxicol. 25, 53–64. anticancer agents that restrain the growth of high-grade serous ovarian cancer Lazarević, T., Rilak, A., Bugarčić Ž, D., 2017. Platinum, palladium, gold and ruthenium tumoroids. Chemistry 26, 11868–11876. complexes as anticancer agents: current clinical uses, cytotoxicity studies and future Scattolin, T., Voloshkin, V.A., Visentin, F., Nolan, S.P., 2021. A critical review of perspectives. Eur. J. Med. Chem. 142, 8–31. palladium organometallic anticancer agents. Cell Rep. Phys. Sci. 2, 100446. Levine, B., Kroemer, G., 2019. Biological functions of autophagy genes: a disease Shibuya, S., Watanabe, K., Tsuji, G., Ichihashi, M., Shimizu, T., 2019. Platinum and perspective. Cell 176, 11–42. palladium nanoparticle-containing mixture, PAPLAL, does not induce palladium Levy, J.M.M., Towers, C.G., Thorburn, A., 2017. Targeting autophagy in cancer. Nat. allergy. Exp. Dermatol. 28, 1025–1028. Rev. Cancer 17, 528–542. Siegel, R.L., Miller, K.D., Fuchs, H.E., Jemal, A., 2022. Cancer Statistics, 2022. CA Cancer Li, W., Zhang, H., Assaraf, Y.G., Zhao, K., Xu, X., Xie, J., Yang, D.H., Chen, Z.S., 2016. J. Clin. 72, 7–33. Overcoming ABC transporter-mediated multidrug resistance: molecular mechanisms Sinha, S., Levine, B., 2008. The autophagy effector Beclin 1: a novel BH3-only protein. and novel therapeutic drug strategies. Drug Resist. Updates 27, 14–29. Oncogene 27 (Suppl. 1), S137–S148. Liang, X.H., Jackson, S., Seaman, M., Brown, K., Kempkes, B., Hibshoosh, H., Levine, B., Soldani, C., Scovassi, A.I., 2002. Poly(ADP-ribose) polymerase-1 cleavage during 1999. Induction of autophagy and inhibition of tumorigenesis by beclin 1. Nature apoptosis: an update. Apoptosis 7, 321–328. 402, 672–676. Sung, H., Ferlay, J., Siegel, R.L., Laversanne, M., Soerjomataram, I., Jemal, A., Bray, F., Liang, X.H., Kleeman, L.K., Jiang, H.H., Gordon, G., Goldman, J.E., Berry, G., 2021. Global Cancer Statistics 2020: GLOBOCAN estimates of incidence and Herman, B., Levine, B., 1998. Protection against fatal Sindbis virus encephalitis by mortality worldwide for 36 cancers in 185 countries. CA A Cancer J. Clin. 71, beclin, a novel Bcl-2-interacting protein. J. Virol. 72, 8586–8596. 209–249. Liu, W.J., Ye, L., Huang, W.F., Guo, L.J., Xu, Z.G., Wu, H.L., Yang, C., Liu, H.F., 2016. p62 Susin, S.A., Lorenzo, H.K., Zamzami, N., Marzo, I., Snow, B.E., Brothers, G.M., links the autophagy pathway and the ubiqutin–proteasome system upon Mangion, J., Jacotot, E., Costantini, P., Loeffler, M., Larochette, N., Goodlett, D.R., ubiquitinated protein degradation. Cell. Mol. Biol. Lett. 21, 29. Aebersold, R., Siderovski, D.P., Penninger, J.M., Kroemer, G., 1999. Molecular Lu, X., Saeed, M.E.M., Hegazy, M.F., Kampf, C.J., Efferth, T., 2020. Chemopreventive characterization of mitochondrial apoptosis-inducing factor. Nature 397, 441–446. property of Sencha tea extracts towards sensitive and multidrug-resistant leukemia Vojtek, M., Gonçalves-Monteiro, S., Pinto, E., Kalivodová, S., Almeida, A., Marques, M.P. and multiple myeloma cells. Biomolecules 10, 1000. M., Batista de Carvalho, A.L.M., Martins, C.B., Mota-Filipe, H., Ferreira, I., Diniz, C., Lucaciu, R.L., Hangan, A.C., Sevastre, B., Oprean, L.S., 2022. Metallo-drugs in cancer 2021. Preclinical pharmacokinetics and biodistribution of anticancer dinuclear therapy: past, present and future. Molecules 27, 6485. palladium(II)-spermine complex (Pd(2)Spm) in mice. Pharmaceuticals 14, 173. Ma, D., Lu, B., Feng, C., Wang, C., Wang, Y., Luo, T., Feng, J., Jia, H., Chi, G., Luo, Y., Wang, Y., Dawson, V.L., Dawson, T.M., 2009. Poly(ADP-ribose) signals to mitochondrial Ge, P., 2016. Deoxypodophyllotoxin triggers parthanatos in glioma cells via AIF: a key event in parthanatos. Exp. Neurol. 218, 193–202. induction of excessive ROS. Cancer Lett. 371, 194–204. Wang, Y., Kim, N.S., Haince, J.-F., Kang, H.C., David, K.K., Andrabi, S.A., Poirier, G.G., Mah, L.J., El-Osta, A., Karagiannis, T.C., 2010. γH2AX: a sensitive molecular marker of Dawson, V.L., Dawson, T.M., 2011. Poly(ADP-Ribose) (PAR) binding to apoptosis- DNA damage and repair. Leukemia 24, 679–686. inducing factor is critical for PAR polymerase-1–dependent cell death (parthanatos). Mahmoud, N., Hegazy, M.-E.F., Wadie, W., Elbadawi, M., Fleischer, E., Klinger, A., Sci. Signal. 4 ra20-ra20. Bringmann, G., Khayyal, M.T., Efferth, T., 2022. Naphthoquinone derivatives as P- Wang, Y., Luo, W., Wang, Y., 2019. PARP-1 and its associated nucleases in DNA damage glycoprotein inducers in inflammatory bowel disease: 2D monolayers, 3D spheroids, response. DNA Repair 81, 102651. and in vivo models. Pharmacol. Res. 179, 106233. Wong, R.S.Y., 2011. Apoptosis in cancer: from pathogenesis to treatment. J. Exp. Clin. McFarland, S.A., Mandel, A., Dumoulin-White, R., Gasser, G., 2020. Metal-based Cancer Res. 30, 87. photosensitizers for photodynamic therapy: the future of multimodal oncology? Yan, G., Elbadawi, M., Efferth, T., 2020. Multiple cell death modalities and their key Curr. Opin. Chem. Biol. 56, 23–27. features (Review). World Acad Sci J 2, 39–48. Miller, M.A., Askevold, B., Mikula, H., Kohler, R.H., Pirovich, D., Weissleder, R., 2017. Yu, S.W., Andrabi, S.A., Wang, H., Kim, N.S., Poirier, G.G., Dawson, T.M., Dawson, V.L., Nano-palladium is a cellular catalyst for in vivo chemistry. Nat. Commun. 8, 15906. 2006. Apoptosis-inducing factor mediates poly(ADP-ribose) (PAR) polymer-induced Mohammad, R.M., Muqbil, I., Lowe, L., Yedjou, C., Hsu, H.Y., Lin, L.T., Siegelin, M.D., cell death. Proc. Natl. Acad. Sci. U.S.A. 103, 18314–18319. Fimognari, C., Kumar, N.B., Dou, Q.P., Yang, H., Samadi, A.K., Russo, G.L., Yu, S.W., Wang, H., Poitras, M.F., Coombs, C., Bowers, W.J., Federoff, H.J., Poirier, G.G., Spagnuolo, C., Ray, S.K., Chakrabarti, M., Morre, J.D., Coley, H.M., Honoki, K., Dawson, T.M., Dawson, V.L., 2002. Mediation of poly(ADP-ribose) polymerase-1- Fujii, H., Georgakilas, A.G., Amedei, A., Niccolai, E., Amin, A., Ashraf, S.S., dependent cell death by apoptosis-inducing factor. Science 297, 259–263. Helferich, W.G., Yang, X., Boosani, C.S., Guha, G., Bhakta, D., Ciriolo, M.R., Zhang, J., Liu, D., Zhang, M., Zhang, Y., 2019. Programmed necrosis in cardiomyocytes: Aquilano, K., Chen, S., Mohammed, S.I., Keith, W.N., Bilsland, A., Halicka, D., mitochondria, death receptors and beyond. Br. J. Pharmacol. 176, 4319–4339. Nowsheen, S., Azmi, A.S., 2015. Broad targeting of resistance to apoptosis in cancer. Zhao, N., Mao, Y., Han, G., Ju, Q., Zhou, L., Liu, F., Xu, Y., Zhao, X., 2015. YM155, a Semin. Cancer Biol. 35 (Suppl. l), S78–s103. survivin suppressant, triggers PARP-dependent cell death (parthanatos) and inhibits Ndagi, U., Mhlongo, N., Soliman, M.E., 2017. Metal complexes in cancer therapy - an esophageal squamous-cell carcinoma xenografts in mice. Oncotarget 6, update from drug design perspective. Drug Des. Dev. Ther. 11, 599–616. 18445–18459. Orvig, C., Abrams, M.J., 1999. Medicinal inorganic chemistry: introduction. Chem. Rev. Zhou, M., Boulos, J.C., Klauck, S.M., Efferth, T., 2023. The cardiac glycoside 99, 2201–2204. ZINC253504760 induces parthanatos-type cell death and G2/M arrest via Özenver, N., Saeed, M., Demirezer, L., Efferth, T., 2018. Aloe-emodin as drug candidate downregulation of MEK1/2 phosphorylation in leukemia cells. Cell Biol. Toxicol. for cancer therapy. Oncotarget 9, 17770–17796. Zhou, Y., Liu, L., Tao, S., Yao, Y., Wang, Y., Wei, Q., Shao, A., Deng, Y., 2021. Parthanatos and its associated components: promising therapeutic targets for cancer. Pharmacol. Res. 163, 105299. 15 molecules Article Modes of Action of a Novel c-MYC Inhibiting 1,2,4-Oxadiazole Derivative in Leukemia and Breast Cancer Cells Min Zhou 1, Joelle C. Boulos 1, Ejlal A. Omer 1, Sabine M. Klauck 2 and Thomas Efferth 1,* 1 Department of Pharmaceutical Biology, Institute of Pharmaceutical and Biomedical Sciences, Johannes Gutenberg University-Mainz, Staudinger Weg 5, 55128 Mainz, Germany 2 Division of Cancer Genome Research, German Cancer Research Center (DKFZ), German Cancer Consortium (DKTK), National Center for Tumor Disease (NCT), Im Neuenheimer Feld 460, 69120 Heidelberg, Germany * Correspondence: efferth@uni-mainz.de; Tel.: +49-6131-392-5751; Fax: +49-6131-392-3752 Abstract: The c-MYC oncogene regulates multiple cellular activities and is a potent driver of many highly aggressive human cancers, such as leukemia and triple-negative breast cancer. The oxadiazole class of compounds has gained increasing interest for its anticancer activities. The aim of this study was to investigate the molecular modes of action of a 1,2,4-oxadiazole derivative (ZINC15675948) as a c-MYC inhibitor. ZINC15675948 displayed profound cytotoxicity at the nanomolar range in CCRF-CEM leukemia and MDA-MB-231-pcDNA3 breast cancer cells. Multidrug-resistant sublines thereof (i.e., CEM/ADR5000 and MDA-MB-231-BCRP) were moderately cross-resistant to this com- pound (<10-fold). Molecular docking and microscale thermophoresis revealed a strong binding of ZINC15675948 to c-MYC by interacting close to the c-MYC/MAX interface. A c-MYC reporter assay demonstrated that ZINC15675948 inhibited c-MYC activity. Western blotting and qRT-PCR showed that c-MYC expression was downregulated by ZINC15675948. Applying microarray hy- bridization and signaling pathway analyses, ZINC15675948 affected signaling routes downstream of c-MYC in both leukemia and breast cancer cells as demonstrated by the induction of DNA dam- age using single cell gel electrophoresis (alkaline comet assay) and induction of apoptosis using Citation: Zhou, M.; Boulos, J.C.; flow cytometry. ZINC15675948 also caused G2/M phase and S phase arrest in CCRF-CEM cells Omer, E.A.; Klauck, S.M.; Efferth, T. and MDA-MB-231-pcDNA3 cells, respectively, accompanied by the downregulation of CDK1 and Modes of Action of a Novel c-MYC p-CDK2 expression using western blotting. Autophagy induction was observed in CCRF-CEM cells Inhibiting 1,2,4-Oxadiazole but not MDA-MB-231-pcDNA3 cells. Furthermore, microarray-based mRNA expression profiling Derivative in Leukemia and Breast Molecules 2023 28 indicated that ZINC15675948 may target c-MYC-regulated ubiquitination, since the novel ubiquitinCancer Cells. , , 5658. https://doi.org/10.3390/ ligase (ELL2) was upregulated in the absence of c-MYC expression. We propose that ZINC15675948 is molecules28155658 a promising natural product-derived compound targeting c-MYC in c-MYC-driven cancers through DNA damage, cell cycle arrest, and apoptosis. Academic Editors: M. Mizerska- Kowalska, Wojciech Płaziński, Sylwia Keywords: 1,2,4-oxadiazole; c-MYC inhibitor; leukemia; natural product derivative; oncogenes; Sowa and Roman Paduch triple-negative breast cancer Received: 16 June 2023 Revised: 19 July 2023 Accepted: 24 July 2023 Published: 26 July 2023 1. Introduction Cancer is mainly caused by genomic alterations that result from the loss of tumor suppressor genes and the activation of oncogenes [1]. The MYC gene is one of the oncogenes Copyright: © 2023 by the authors. that has been described as a master regulator of gene expression in multiple biological Licensee MDPI, Basel, Switzerland. processes [2,3]. This gene encodes a family of basic helix-loop-helix zipper (bHLHZip) This article is an open access article proteins consisting of c-MYC, N-MYC, and L-MYC [4]. MYC acts as a transcription factor distributed under the terms and that binds to its obligatory partner, MYC-associated factor X (MAX). The MYC-MAX conditions of the Creative Commons heterodimer activates a large number of genes by binding to E box sequences (5′-CACGTG- Attribution (CC BY) license (https:// 3′) within gene promoters and enhancers [5]. creativecommons.org/licenses/by/ MYC-regulated transcription is tightly controlled in non-transformed cells. How- 4.0/). ever, MYC is estimated to be overexpressed up to 70% in various human cancer types [6]. Molecules 2023, 28, 5658. https://doi.org/10.3390/molecules28155658 https://www.mdpi.com/journal/molecules Molecules 2023, 28, 5658 2 of 28 MYC contributes to several hallmarks of cancer, including the escape from programmed cell death, promoting sustainable proliferation, genome instability, escape from immuno- surveillance, and change of cellular metabolism [7,8]. The major mechanisms of MYC deregulation are gene alternation and the activation of upstream MYC-related signaling pathways (e.g., NOTCH, WNT, and EGFR) [9]. Moreover, MYC is an unstable protein with a short half-life [10], and its stability is also regulated by post-translational modifica- tions [11]. For example, MYC gene amplification largely drives breast carcinogenesis [12], and the chromosomal translocation of the MYC gene contributes to the development of T-cell acute leukemia [13]. In mice with MYC-induced hematological cancers, an inactiva- tion of the MYC transgene led to tumor regression [14]. To date, there is no approved MYC inhibitor, despite several promising MYC inhibitors being investigated in preclinical or clinical studies [8]. Therefore, there is an ongoing quest for novel effective MYC inhibitors for an improvement of cancer therapy with targeted drugs. Natural products are a promising resource for drug discovery [15]. Oxadiazole rep- resents a five-membered heterocyclic scaffold with one oxygen and two nitrogen atoms. Oxadiazole-based compounds are a rapidly growing field in drug development. They are fundamental pharmacophores due to their stability in aqueous medium and are commonly employed as bioisosteric substitutes [16,17]. Based on the position of oxygen and nitrogen in the ring, oxadiazoles are classified into four regioisomeric structures [18]. In addition, 1,2,4-Oxadiazoles received considerable attention as witnessed by increasing numbers of published studies [19]. The first natural products from the class of 1,2,4-oxadiazoles were phidianidine A and B that were isolated from marine mollusk Phidiana militaris by Alder and Hancock [20]. Interestingly, phidianidine A was identified as an antifoulant agent which is a chemical defense of slow-moving marine organisms to deter predators, other colonizers, or competitors [21]. In the past two decades, a great number of 1,2,4- oxadiazole derivates were synthesized and studied for their pharmacological properties, which exhibit numerous bioactivities, including anti-cancer [22,23], anti-inflammatory [24], anti-bacterial [25], anti-viral [26], anti-malarial [27], anti-diabetic [28], and anti-Alzheimer activities [29]. Zibotentan represents an example of an oxadiazole that reached a clinical phase III trial for the treatment of hormone-resistant prostate carcinoma [30]. Studies re- vealed that 1,2,4-oxadiazoles derivatives exhibited more powerful anticancer activities than established drugs (e.g., doxorubicin and etoposide) by inhibiting cell proliferation which make them appealing as prospective therapeutic candidates [16,18]. However, the modes of action of 1,2,4-oxadiazoles derivatives in cancer have not been fully understood yet. Breast cancer is the most common malignancy in females worldwide. Triple-negative breast cancer (TNBC) is characterized by the absence of estrogen (ER)/progesterone (PR) expression and human epidermal growth factor receptor-2 (HER2) amplification [31]. MYC is remarkably elevated in TNBC compared with other breast cancer subtypes. TNBC is highly invasive leading to a poor five-year survival rate and distant recurrence rates [12,32]. On the other hand, acute lymphoblastic leukemia (ALL) is an aggressive hematological cancer frequently appearing in children and adolescents. A multitude of evidence supports the important role of MYC in the initiation and progression of ALL [33]. The aim of this study was to investigate the 1,2,4-oxadiazoles derivative ZINC15675948 as a novel c-MYC inhibitor. We determined the anticancer activity of ZINC15675948 against drug-sensitive and -resistant leukemia (CCRF-CEM and CEM/ADR5000) and triple-negative breast cancer (MDA-MB-231-pcDNA3 and MDA-MB-BCRP) cell lines. The effects of ZINC15675948 were studied by using western blot, qRT-PCR, molecular docking, microscale thermophoresis, and c-MYC reporter assay. By means of microarray hybridiza- tion and Ingenuity Pathway Analysis, we explored the underlying modes of action of ZINC15675948 regarding cell cycle, apoptosis, autophagy, and DNA damage. In addition, the interactions of ZINC15675948 with the multidrug-resistance-mediating ATP-binding cassette transporters’ P-glycoprotein and breast cancer resistance protein were investigated using molecular docking. Molecules 2023, 28, x FOR PEER REVIEW 3 of 30 resistance-mediating ATP-binding casse e transporters’ P-glycoprotein and breast cancer resistance protein were investigated using molecular docking. 2. Results 2.1. Growth Inhibition Assay Molecules 2023, 28, 5658 3 of 28 ZINC15675948 exhibited profound cytotoxicity toward both leukemia and breast cancer cell lines using resazurin reduction assays. Doxorubicin was used as a positive con- tro2l. R[e3s4u].l tsCCRF-CEM and MDA-MB-231-pcDNA3 cells were extremely sensitive to ZI2N.1C. G15ro6w7t5h9I4n8h iwbititiohn IACs5s0a vyalues of 0.008 ± 0.001 µM, and 0.08 ± 0.004 µM, respectively. It is interZeIsNtiCn1g5 6th75a9t 4C8EeMxh/iAbiDteRd5p0r0o0f ocuenllds cayst owtoexlli caitsy MtoDwAar-dMbBo-tBhClReuPk ceemllisa danisdplbaryeeadst cross- recsaisntcaenrccee ltlolwinaersdu sZinINg Cre1s5a6z7u5ri9n48re d(duecgtiroenesa sosaf yrse.siDstoaxnocreu:b 8ic.i3n7 wanasdu 9s.e0d, aressappecotsiivtievley). The docsoen-trreoslp[o34n]s.eC cCuRrvFe-Cs EaMre asnhdowMnD Ain- MFiBg-u23r1e- p1cCD,DN,A a3ncde ltlhs ew IeCre50e vxatrleumese lyansden dsietgivreeetos of re- sisZtIaNnCce1 5a6r7e5 p94r8esweinthteIdC 5in0 vTaalbueles 1o.f 0.008 ± 0.001 µM, and 0.08 ± 0.004 µM, respectively. It is inAtesr mesutilntgidtrhuagt -CrEesMis/taAnDt Rce5l0l0 l0incesll swaesrwe cerlloass -MreDsiAst-aMnBt -tBoC ZRIPNcCe1ll5s6d7i5sp9l4a8y,e fdurctrhoessr- inves- tigraestiisotnasn cuestionwga ZrdINZCIN1C56175657954984 8w(edreeg rpeeersfoofrmreesidst aoncley: i8n.3 7CaCnRdF9-.C0,ErMes paencdtiv MelyD).AT-hMeB-231- pcdDoNseA-re3s pceolnlss.e c urves are shown in Figure 1C,D, and the IC50 values and degrees of resis- tance are presented in Table 1. FigFiugruer e1.1 T. heT hcehecmheimcailc asltrsutcrutuctruer eanadn ddodsoes-er-ersepspoonnssee cucurrvvee oof fZZININCC1155667755994488 ddeetteerrmmiinneedd bbyy using thue sinregsathzeurriens azruerdinucrteidounc tiaonssaayss. ay(.A()A )11,2,2,4,4--OOxxaaddiiaazzoollee nunculceluesu. s.( B()BC) heCmhiceaml isctarul ctsutrreucotfure of ZIZNICN1C516576579549488. .(C()C G) rGorwowtht hinihnihbiibtiitoinon ofo Zf IZNINCC1515667755994488 totowwaarrdd lleeuukkeemmiiaa CCCCRRFF--CCEEMM aanndd PP--glyco- prgoltyecionp-orovteerienx-opvreersesxipnrge sCsiEnMg C/AEDMR/5A0D00R 5c0e0ll0 lcienlelsl.i n(eDs.) G(Dr)oGwrtohw itnhhiinbhitiibointio onf oZfIZNICN1C516576579549848 toward tritpolwe-anrdegtaritpivlee- nbergeaatisvt ecbarnecaestr cManDceAr -MMDBA-2-M31B-p-2c3D1-NpcAD3N aAn3da nBdCBRCPR oPvoevreerxepxpreressssiningg MMDDAA--MMBB--BCRP ceBllC liRnPesc.e lTl hlien edsa. tTah we deareta pwloereepdl oatste md eaasnm ±e aSnD± frSoDmfr tohmreteh rienedienpdeenpednednetn etxepxpereirmimeennttss wwiitthh eeaacchh of the 6 poaf rthaelle6lp mareaallseul rmemeaesunrtesm. ents. Table 1. Cytotoxicity of ZINC15675948 toward drug-sensitive and -resistant cancer cell lines measured by resazurin reduction assay. CEM/ADR5000 and MDA-MB-BCRP were the two cell lines displaying multidrug-resistant phenotypes by overexpressing P-glycoprotein and BCRP, respectively. Cell Lines IC50 (µM) Degree of Resistance CCRF-CEM 0.008 ± 0.001 8.37 CEM/ADR5000 0.071 ± 0.002 MDA-MB-231-pcDNA3 0.08 ± 0.004 9 MDA-MB-BCRP 0.72 ± 0.07 Molecules 2023, 28, 5658 4 of 28 As multidrug-resistant cell lines were cross-resistant to ZINC15675948, further inves- tigations using ZINC15675948 were performed only in CCRF-CEM and MDA-MB-231- pcDNA3 cells. 2.2. Molecular Docking The oncogene MYC is deregulated in various human cancers and drives several cancer- related hallmarks. Despite its unquestionable contribution to cancer development, MYC has been regarded as undruggable, and there are only a few MYC inhibitors so far [35,36]. The aim of this study was targeting c-MYC by ZINC15675948, as demonstrated below with different in vitro verifications and pathway analysis of microarray data. To investigate the possible interaction and binding affinity of ZINC15675948 to c-MYC, molecular docking was performed using AutoDock 4.2.6. As shown in Figure 2A, the binding site of ZINC15675948 bound to c-MYC was almost the same as for the known MYC-inhibiting control drugs. ZINC15675948 was particularly near to 10058-F4 and 10074-A4, and Arg925 and Gln927 were two common amino acid residues shown in the interactions, and Gln 927 displayed hydrogen-bonding (Figure 2B–D). Table 2 illustrates that ZINC15675948 displayed a strong binding to c-MYC, revealed by a lowest binding energy (LBE) value of −9.91 kcal/mol and a predicted inhibition constant (pKi) of 0.05 µM. In comparison, the binding affinities of known c-MYC inhibitors were weak (LBE value of 10058-F4: −4.92 kcal/mol; LBE value of 10074-A4: −6.42 ± 0.01 kcal/mol; LBE value of 10074-G5: −6.96 ± 0.01 kcal/mol). Table 2. Molecular docking results of ZINC15675948 and known inhibitors 10058-F4, 10074-A4, and 10075-G5 (positive control) to c-MYC. Compound Lowest Binding pKi ( M) Amino Acids InteractionsEnergy (kcal/mol) µ (Residues in H-Bond Bolded) ZINC15675948 −9.91 0.055 Arg924, Asp926, Gln927,Tyr949, Ile950, Val953 10058-F4 −4.92 247.03 ± 1.5 Arg925, Gln927, Pro929,Leu931, Glu932 10074-A4 −6.42 ± 0.01 19.53 ± 0.30 Arg925, Asp926, Gln927 10074-G5 −6.96 ± 0.01 7.93 ± 0.13 Pro929, Pro930, Lys945, Ala948 To better understand the cross-resistance of CCRF-CEM and MDA-MB-231-pcDNA3 cells toward ZINC15675948, molecular docking of ZINC15675948 towards P-gp and BCRP was carried out. Table 3 revealed that ZINC15675948 showed high binding affinities to P-gp and BCRP (LBE values: −10.55 ± 0.24 kcal/mol and −11.49 kcal/mol, respectively). The binding site of ZINC15675948 to P-gp was similar to doxorubicin (Figure 3A). They shared the same amino acid with Trp232 and Gln990 on P-gp (Figure 3B,C). ZINC15675948, likewise doxorubicin, bound to the substrate-binding site on P-gp. However, the binding site of ZINC15675948 was similar to both doxorubicin and Ko143 (Figure 3E–H). We further performed a doxorubicin uptake assay (Section 2.11) to pinpoint whether ZINC15675948 is an inhibitor or substrate of P-gp and BCRP. 2.3. Microscale Thermophoresis To confirm the in silico binding of ZINC15675948 to c-MYC with an in vitro assay, we applied microscale thermophoresis (MST). As shown in Figure 2F, the measurement of the concentration-dependent fluorescence signals revealed an interaction between the fluorescently labeled c-MYC protein and ZINC15675948. ZINC15675948 bound to c-MYC with a Kd of 1.08 ± 0.1 µM. MoleMcuolleescu2l0es2 32,02283,, 2586,5 x8 FOR PEER REVIEW 5 of 350 of 28 FiFgiugruere2 .2.I nInhhibibititiioonn ooff cc--MYC by ZIINCC115566775599484.8 (.A(A) I)nI nsilsiicloic mo omleocluelcaurl darocdkoinckgi onfg ZoIfNZCI1N5C67155964785 948 anadndth threreek knnoowwnni innhhiibiittors 100F4-58,, 1100007744-A-A44, a, nandd 101007047-4G-5G t5o tco-Mc-YMCY. (CB.)( IBn)teIrnatcetriancgt ianmginamo ainciodsa cids ofoZf ZININCC1155667755994488( (rreedd)),, ((CC)) 1100005588--FF44 ((bbluluee),) ,(D(D) )101007047-4A-A4 (4p(upruprlep)l,e ()E,)( E10)017040-7G44- G(v4io(lveito) lientt)erinactetirnagc ting wwithithc -cM-MYYCCa ass vviissuuaalliizzeedd bbyy DDisicsocovevreyr yStSutduido.i o(F. ) (BFi)nBdiinngd iknignektiicnse otifc Zs IoNfCZ1I5N67C5195468 7b5o9u4n8db toou cn-d to MYC obtained by microscale thermophoresis. (G) Inhibition of c-MYC activity by ZINC15675948 as c-MYC obtained by microscale thermophoresis. (G) Inhibition of c-MYC activity by ZINC15675948 as determined by a c-MYC reporter assay. Statistical significance (* p ≤ 0.05) was compared to DMSO (negative control). All experiments were performed three times independently. Molecules 2023, 28, x FOR PEER REVIEW 6 of 30 Molecules 2023, 28, 5658 6 of 28 determined by a c-MYC reporter assay. Statistical significance (* p ≤ 0.05) was compared to DMSO (negative control). All experiments were performed three times independently. Figure 3. Molecular docking of ZINC15675948 to the ABC transporters (A) P-gp and (E) BCRP. The proteins were presented in a new carton format. The ligands were displayed using a dynamic bond format with different colors: ZINC15675948 (green), doxorubicin (blue), verapamil (yellow), and Ko143 (cyan). The binding sites were visualized by P-gp residues that interact with (B) ZINC15675948, (C) doxorubicin, and (D) verapamil, as well as BCRP residues that interact with (F) ZINC15675948, (G) doxorubicin and (H) Ko143, are shown in a QuickSurf format. Molecules 2023, 28, 5658 7 of 28 Table 3. In silico molecular docking of ZINC15675948 and control drugs (doxorubicin, verapamil, and Ko143) to P-gp and BCRP. Protein Compound Lowest Binding pKi ( M) Amino Acids InteractionsEnergy (kcal/mol) µ (Residues in H-Bond Bolded) P-gp ZINC15675948 −10.55 ± 0.24 0.07 ± 0.01 Trp232, Phe239, Ala295, Asn296, Ile299, Phe770,Gln773, Gly774, Met876, Gln990, Phe994 Doxorubicin −6.42 ± 0.05 147.35 ± 72.62 Ala229, Trp232, Phe303, Ile306, Phe343, Gln725,Ala871, Gly872, Phe983, Met986, Ala987, Gln990 Verapamil −7.61 ± 0.31 3.0 ± 1.46 Ser228, Ala229, Trp232, Ile306, Phe336, Phe343,Gln725, Phe728, Tyr953, Phe983, Met986, Gln990 A: Leu405, Phe431, Phe432, Thr435, Asn436, BCRP ZINC15675948 −11.49 ± 0.01 0.007 ± 0.004 Phe439, Thr542, Met549, Leu555 B: Phe432, Thr435, Phe439 Doxorubicin −7.00 ± 0.49 122.79 ± 10.96 A: Phe431, Phe432, The435, Asn436, Phe439, Ser440B: The542, Ile 543, Val546, Met549, Ile555 A: Leu405, Phe431, Phe432, Thr435, Asn436, Ko143 −10.24 ± 0.19 0.03 ± 0.01 Phe439, Met549, Leu555 B: Phe431, Phe32, Thr435, Phe439, Val546, Met549 2.4. c-MYC Reporter Assay To determine whether the c-MYC activity could be diminished by ZINC15675948 binding, we performed c-MYC reporter assays in HEK293 cells with a transfected c-MYC- luciferase reporter construct. Notably, the c-MYC activity was suppressed by ZINC15675948 in a concentration-dependent manner. A significant inhibition was observed at a concen- tration of 320 nM (Figure 2G). Surprisingly, 10058-F4 as a positive control only showed a slight inhibition. Therefore, ZINC15675948 indeed inhibited c-MYC activity as consistently also demonstrated with molecular docking, microscale thermophoresis, and as illustrated below by western blotting and qRT-PCR. 2.5. Gene Expression Profile of Cell Lines Using Microarray Analyses The gene expression measured using mRNA microarray hybridization was filtered by Chipster software (version 3.16.3). A total of 329 and 314 genes were significantly deregulated in CCRF-CEM cells and MDA-MB-231-pcDNA3 cells, respectively, compared with their untreated samples (Supplementary Tables S1 and S2). The deregulated genes were further analyzed by Ingenuity Pathway Analysis (IPA) to predict canonical pathways, networks, and cellular functions and diseases affected by ZIN15675948. Here, we did not observe any potentially impacted canonical pathways. Interestingly, several affected cellular functions were commonly revealed in the two cell lines, including “cell death and survival”, “cell cycle”, “cellular growth and proliferation”, as well as “DNA replication, recombination and repair” (Figure 4). “Cancer” and “hematological disease” were affected correspondingly by ZINC15675948 in CCRF-CEM cells. MDA-MB-231-pcDNA3 cells also showed “cancer” as an important affected disease. Therefore, we further investigated the roles of the cell cycle, apoptosis, autophagy, and DNA damage to verify these microarray- based results by independent other methods. We further accessed the networks under different cellular functions to unravel the genes that were involved. The IPA-based comparison of untreated and ZINC15675948- treated CCRF-CEM cells indeed also revealed a downregulation of the c-MYC gene as illustrated in Figure 5A,B. These networks are related to cell cycle and cell death, implying that they were downstream and affected by the c-MYC gene. However, the c-MYC gene did not appear in the gene expression profiles of MDA-MB-231-pcDNA3 cells. The cell death network revealed that MCL-1 and BAD were downregulated accompanied by an upregulation of SQSTM1 (p62) (Figure 5C). Furthermore, Figure 5D shows that the ELL2 gene was upregulated, which is involved in the proteasomal degradation of c-MYC [37,38]. Molecules 2023, 28, 5658 8 of 28 Molecules 2023, 28, x FOR PEER REVIEW 9 of 30 The tumor suppressor TP53 was upregulated, which may be a consequence of c-MYC downregulation as we will discuss below. FiguFrieg u4.r eG4e.nGee enxeperxepsrseisosnio pnrporfiolfiinlingg aass ddeetteerrmmiinneedd bbyyI nInggenenuuityityP aPtahtwhawyaAyn Aalnyaslisys(IiPs A(I)PoAf )C oCfR CFC- RF- CEMC aEnMd aMndDMAD-MA-BM-2B3-123-p1-cpDcDNNAA33 cceelllsls uuppoonn ttrreeaattmmeennt tw witihthth tehIeC IC5c0o cnocnencetrnattr50 ioantioofnZ oIfN ZCI1N56C7155964875948 for 2f4o rh2. 4Tho.pT coepllcuellalurl faur nfucnticotniosn (sr(erded bbooxxeess)) aanndd ddiisseeaasseess( g(grereenenb obxoexse) sa)f faeffcteecdtebdy ZbyIN ZCI1N5C67155964785i9n48 in (A) C(AC)RCFC-CRFE-MCE aMndan (dB)(B M) MDADA-M-MBB-2-23311--ppccDDNNAA33 cceellllss. .C CCCRRFF−−CCEEMM. Molecules 2023, 28, x FOR PEER REVIEW 10 of 30 M olecules 2023, 28, 5658 9 of 28 FFiigguurree 55.. MMoolleeccuullaarr nneettwwoorrkk ggeenneerraatteedd uussiningg IPIPAA ssooftfwtwaarere (c(oconntetnent tvveresrisoinon: 5: 15916936831831)3 f)rofrmom mmRNRNA A mmiiccrrooaarrrraayy hhyybbrrididizizaattioionna affffecetceteddb ybyZ IZNINC1C51657657954984i8n iCnC CRCFR-CFE-CMEMan danMdD MAD-MAB-M-23B1-2-p3c1D-pNcDAN3 Ace3ll s. cells. (A) Cell cycle network in CCRF-CEM cells. The red circle highlights that c-MYC was down- (rAeg) uClealtlecdy acnledn reetlwatoerdk tion cCeCll RcFyc-CleE rMegcuellaltsi.oTnh. e(Br)e dCeclilr cdleeahtihg hnleitgwhotsrkth iant Cc-CMRYFC-CwEaMs dceolwlsn. rTehgeu rlaetde d acnirdcler ehlaigthedligthotsc ethllact ycc-MleYrCeg wualast iaolsno. d(oBw) nCreelgludleaatetdh annedtw inovroklvinedC iCn RceFl-lC dEeMathc. e(lCls). CTelhl ecyrcelde nciertc-le hwigohrkli ignh MtsDthAa-tMc-BM-2Y3C1-pwcaDsNaAlso3 cdeollws.n Trheeg ureladt ceidrcalensd hiingvholilgvhetd thinatc SeQllSdTeMat1h .(p(6C2)) Cwealsl ucypcrleegnuelattwedo,r k iwnhMileD MAC-ML-B1- a2n3d1- BpAc DN wAe3rec deollws.nrTehgeulraetdedc. i(rDcl)e Tshhei grehdli gcihrct ltehs ahtigShQliSgThMt t1ha(tp T62P)53w aansdu EpLrLe2g uwlearte d, wuphrileegMulaCtLe-d1 iann MdDBAD-MwBe-2re31d-powcDnNreAg3u lcaetlelsd.. (D) The red circles highlight that TP53 and ELL2 were upregulated in MDA-MB-231-pcDNA3 cells. 2.6. Quantitative Reverse Transcription PCR (qRT-PCR) The technical verifications of the results obtained from microarray hybridization were conducted with the top deregulated genes. Two upregulated and downregulated genes Molecules 2023, 28, x FOR PEER REVIEW 11 of 30 2.6. Quantitative Reverse Transcription PCR (qRT-PCR) Molecules 2023, 28, 5658 The technical verifications of the results obtained from microarray hybridiz1a0tioofn28 were conducted with the top deregulated genes. Two upregulated and downregulated genes (RAD21, HMGCS1, PGK1, and ATP5MF) in CCRF-CEM cells, and two upregulated a(nRdA dDo2w1n, HreMguGlaCteSd1, gPeGneKs1 (,HaSnPdDA1T, PC5ITMEFD)2i,n HC4CR3,F a-nCdE MDHcFeRlls) ,ina nMdDtwA-oMuBp-r2e3g1u-plactDedNaAn3d cdelolsw wnerereg usulabtjeedctegde ntoes p(eHrfSoPrmD 1q,RCTI-TPECDR2 (,FHig4uCre3 ,6aAn).d TDheH PFeRa)rsionnM coDrrAe-laMtiBo-n2 3co1e-pfficcDieNnAts3 wcerlles wcaelcreulsautebdje cbtedtwteoepne rthfoer mdeqteRrTm-PinCeRd (fFoilgdu rceh6aAng).eT ohfe mPeicarosoarnracoyr rheylabtriiodnizcaoteioffinc aienndts qwReTr-ePCcaRl cdualtaat.e Adsb sehtwoweenn int hFeigduertee 6rmB,iCn,e tdhef or lvdalcuhea nwgaes 0o.f9m8 iinc rCoCarRraFy-ChEyMbr cidelilzsa atniodn 0a.9n7d inq RMT-DPAC-RMdBa-t2a3.1A-pscsDhoNwAn3 icneFllisg, ucroen6fiBrm,Ci,ntgh ear hvigalhu edewgarsee0 .o9f8 cionnCcoCrRdFa-nCcEeM bectewlleseann tdhe0s.9e7 twinoM dDiffAe-rMenBt -m23e1t-hpocdDsN. FAu3rtcheelrlsm, cooren,fi trhme ienxgparehsisgiohnd leegvrele ooff cc-oMnYcoCr dwaansc edebteetrwmeiennedth beyse qtRwTo-PdCifRfe r(Fenigtumree t6hDo)d. sT.rFeautrmtheenrtm oof rZe,INthCe1e5x6p7r5e9s4s8io (nICle50v) esligonf icfi-cManYtClyw daoswdnertegrmulianted cb-y MqRYTC- (PpC ≤R 0.(0F5ig) uinr ebo6tDh) l.eTurkeeamtmiae nant do fbZreIaNsCt c1a5n6c7e5r9 c4e8ll( lIiCn5e0s), sinigdnicifiatcianngt ltyhadt oZwINnrCe1g5u6l7a5te9d48c - inMhYibCit(epd≤ c-0M.0Y5C) i ant btohteh gleenuek eemxpiareasnsdiobnr leeavset lc. ancer cell lines, indicating that ZINC15675948 inhibited c-MYC at the gene expression level. FFigiguurere 66. .TTeechchnniciacal laanndd bbioiolologgiciacla lvvereirfiificactaitoionns sbyb yqRqRTT-P-PCCRR ananaalylysesse sinin CCCCRRFF-C-CEEMM anandd MMDDAA-M-MBB- - 223311-p-pccDDNNAA33 cceellllss uuppoonn ttrreeaattmeenntt wiitthht htheeI CIC5050c coonncceenntrtaratitoionno foZf IZNINC1C51657657954984f8o rfo2r4 2h4. (hA. )(ATe) cThencihca- l nvicearilfi vceartiifiocnastiofntsh oeft tohpe ftoupr fdouere dgeurleagteudlagtende gseineCs CinR CFC-CREFM-CcEeMlls caenllds aMndD AM-DMAB-M23B1-2p3c1D-NpcAD3NcAel3ls , creellssp, ercetsivpelcyt.ivLeinlye.a Lr irneegarre srseigornessasinodnsP eaanrdso Pnecaorsrroenla ctoiornrecloateifofinc iceonetsffiocfiemnitcsr oaf rmraiycraonadrrqaRyT a-nPdC RqRdTat-a PoCbRta dinaetda ionb(tBai)nCedC RinF -(CBE) MCCcRelFls-CanEdM( Cce)lMls DanAd-M (CB)- 2M31D-pAc-DMNBA-2331ce-pllcsD. (NDA) D3 ocwellnsr. e(gDu)l aDtioown norfecg-MulYa-C tieoxnp roefs sci-oMnYiCn CexCpRreFs-sCioEnM ina nCdCMRFD-ACE-MMB -a2n3d1 -MpcDDAN-AM3Bc-2e3ll1s-pucpDoNn Atr3e actemllesn ut pwointh trZeIaNtmCe1n5t6 7w5i9t4h8 . ZSItNatCis1t5ic6a7l5s9i4g8n.i Sfitcaatniscteic(a*l psi≤gn0ifi.0c5a)nwcea s(*c po m≤ p0.a0r5e)d wtoasc coonmtrpolar(DedM toSO co).nTtrhoel r(eDsMulStsOa)r. eTrheep rreessuenlttse adraes represented as± mean values ± SD of three independent experiments. mean values SD of three independent experiments. 22.7.7. .SSiningglel eCCelell lGGele lEElelcetcrtoropphhooreressisis (A(Alklkaalilninee CCoommeet tAAssssaayy) ) AAss ““DDNNAA rreepplilcicaatitoionn, ,rereccoommbbininaatitoionn aanndd rereppaairi”r” aapppeeaareredd inin oouur rIPIPAA aannaalylyssisis aass ““cceelllluullaarr ffuunnccttiioonnss”” aaffffeecctteedd bbyy ZZIINCC1155667755994488, ,wwee ppeerrfoforrmmeedd aalklkaalilninee ccoommeet taasssaayyss toto ddeetetecct tDDNNAA dadmamagaeg aet athtet hleevelel voef lsionfgslein cgelels.c Relelps.reRseenptraetsiveen tiamtiavgeesi maraeg sehsoawren isnh oFwignurien 7F. igCuorme p7.arCedom wpaitrhe dnwonit-hdanmona-gdeadm caognetdroclo ncterlolsl c(eDllMs (SDOM), SOth)e,rteh ewreaws aasna ninicnrceraesaes einin ZZININCC1155667755994488-i-nindduucceedd ccoommeet ttatailisls inin bboothth CCCRRFF-C-CEEMM aanndd MMDDAA-M-MBB-2-23311-p-pcDcDNNAA33 ceclellsl,s , suggesting that DNA was indeed damaged. H2O2 as a positive control also led to clearly visible comet tails. The analysis of tails of each 50 cells revealed that ZINC15675948 induced DNA damage in both cell lines in a concentration-dependent manner. Molecules 2023, 28, x FOR PEER REVIEW 12 of 30 suggesting that DNA was indeed damaged. H2O2 as a positive control also led to clearly Molecules 2023, 28, 5658 visible comet tails. The analysis of tails of each 50 cells revealed that1 1ZoIfN28C15675948 in- duced DNA damage in both cell lines in a concentration-dependent manner. Figure 7. AnalysiFsigouf rDeN 7.A Adnaamlyasgise obf yDsNinAg ldeacmelalggee lbeyl esicntrgolep hceolrle gseels e(laelcktarolipnheocroemseest (aaslksaayli)nien cdoumceedt assay) induced by ZINC15675948. Representative comet images captured in (A) CCRF-CEM and (B) MDA-MB-231- by ZINC15675948p.cRDeNpAre3s ecneltlast itvreeactoedm wetitimh dagiffeesrceanpt tcuornedceinntr(aAti)oCnCs RfoFr- 2C4E hM. Hand (2O2 Ban)dM DDMAS-MO Bse-2r3v1e-d as positive or pcDNA3 cells trenaetgeadtiwveit chodntirffoelrse. nSctacloen bcaern, t5r0a tµiomn. sTfhoer g2r4aphh. Hsh2oOw2eadn tdailD DMNSAO pseerrcveendtaagse pporesisteinvteed as mean val- or negative contruoelss .±S ScEalMe bfraor,m5 050µ cmo.mTehtse. gSrtaatpishtischaol wsigendifitacialnDceN (A***p pe r≤c e0n.0t0a1g)e wparse sceonmtepdaraesdm toe aDnMSO. values ± SEM from 50 comets. Statistical significance (*** p ≤ 0.001) was compared to DMSO. 2.8. Cell Cycle Arrest 2.8. Cell Cycle Arrest The IPA analysis of the microarray data also revealed that cell cycle progression was The IPA adniasltyusribseodf btyh Ze ImNCic1ro56a7rr5a9y48d iant baoathls oCCreRvFe-aClEedMt ahnadt cMeDll Acy-McleB-p2r3o1g-precDssNioAn3 cells. There- was disturbed fboyreZ, IwNeC i1n5v6e7s5ti9g4a8teidn tbhoet hceCll CcRycFl-eC dEiMstriabnudtioMnD bAy -flMoBw- 2c3y1t-opmcDetNryA. F3igceulrles .8 shows that Therefore, we inZvIeNstCig1a5t6e7d59th4e8 cseigllnciyficclaendtliyst rinibcuretiaosnedb ythfleo fwraccytitoonm oeft rCyC. FRiFg-uCrEeM8 s cheolwls sinth tahte G2/M phase ZINC15675948asfitgenri fitrceaantmtlyenint cforera 7s2e dh,t hwehfircahc twioans oinf Ca CraRnFg-eC EofM 28c.e2l5ls–3in3.t3h%e Gat2 d/iMffeprehnats ceoncentrations after treatmentcfoomr p72arhe,dw whiitchh DwMasSOin (a15r.a0n%g)e. Wofh2e8r.e2a5s– 3th3e.3 S% pahtadsief fferraecntitocno onfc eMnDtrAat-iMonBs-231-pcDNA3 compared withinDcMreaSsOed(1 a5f.t0e%r 2).4W h.h Aetr ethase thhieghSepsth caosnecfernac×tr taiotinono f(2M ×D IAC-MB-231-p50), S phase caDrrNesAt 3reached 28.1% increased after(p2 4< 0h..01A) ctotmhepahriegdh wesitthc oDnMceSnOtr aast imonoc(k2 conItCro5l0,) w, Shipchh awsaes acrlroesset tore tahceh pedositive control, 28.1% (p < 0.01c)icsopmlatpinar (e3d6.w2%it)h inD MMDSOA-aMs Bm-2o3c1k-pcocDntNroAl,3w cehlilcsh. Hweanscec,l oZsIeNtCo1t5h6e7p59o4s8it iavrerested the leu- control, cisplatikne(m36ia.2 c%el)lsi ninM thDeA G-M2/MB- 2p3h1a-spec aDnNdA th3ec berllesa.sHt ceannccee,rZ cIeNllCs i1n5 6th7e5 9S4 p8haarsree sotfe dthe cell cycle. the leukemia cells in the G2/M phase and the breast cancer cells in the S phase of the cell cycle. Molecules 2023, 28, x FOR PEER REVIEW 13 of 30 Molecules 2023, 28, 5658 12 of 28 FigFuirgeu r8e. C8.eClle cllyccylcel eananaalylyssisis wiitth ZIINC115566775599484.8(.A (A) H) Histiosgtoragmrasmofs coefl lcceylcl lceydcilset rdibisuttriiobnuitnioCnC iRnF C- CRF- CECME Mcelclesl luspuopno ntrteraeatmtmeenntt wiitth diiffffeerreennttc oconncecnetnratrtaiotniosnosf oZfI NZCIN15C61759647859fo4r87 f2ohr .7V2i nhc. rVisitninceriasntidne and DMDSMOS Owewre reusuesded aass ppoossiittiive and nneeggaatitviveec ocnotnrotrlso.ls(.B ()BH) iHstoisgtroagmrsamofsc oelfl cyelcll ecydcislter idbuisttiroinbuintion in MDMAD-MA-BM-2B3-213-p1-cpDcDNNAA3 3cceelllsls upon ttrreeaattmmeennt tw withithZ IZNICN1C5617556974589f4o8r f2o4rh 2. 4C his.p Claitsinplantidn DaMndS ODMSO wewree uresuedse adsa ps opsoistiitvivee aanndd negattiivee ccoonntrtorolsl.sT. hTehree rsuesltuslwtse wreerreep respernetseednatsedm eaasn m±eaSnD ±fr oSmD tfhroreme three indienpdenpdenednet nmt meaesausurermemeennttss.. SSttaattiistiicall ssiiggnnifiificacanncecew wasaasn anlyazleydzeuds inugsiSntgu dSetnutd’sent-t’ess tt,-*teps1000) can also be observed in MDR cells [66,67]. P-gp overexpressing CEM/ADR5000 cells were 8.37 times more cross-resistant to ZINC15675948, while BCRP overexpressing cells were nine times more resistant to ZINC15675948 compared to their sensitive cell lines, indicating that ZINC15675948 was moderately cross-resistant to P-g and BCRP. Using molecular docking, we observed that ZINC15675948 bound at the substrate binding site on P-gp, but its binding site on BCRP is ambiguous concerning the substrate or inhibitor pockets. In the further doxorubicin uptake assay, our data confirmed that ZINC15675948 failed to block the efflux. Therefore, there was no intracellular accumulation of doxorubicin, which can be taken as a further hint that ZINC15675948 is a substrate of P-gp and BCRP. This finding is consistent with our recent report [68]. Future structure-activity relationship (SAR) investigations of derivatives of ZINC15675948 can be carried out to search for more therapeutically promising P-gp and BCRP inhibitors. 4. Materials and Methods 4.1. Compounds The compound ZINC15675948 (IUPAC name: (6S)-N-(4-methylphenyl)-6-(3-naphthalen-2- yl-1,2,4-oxadiazol-5-yl)-3,4,6,7-tetrahydroimidazo [4,5-c]pyridine-5-carboxamide) (C26H22N6O2) was purchased from Glentham Life Sciences (Corsham, United Kingdom) (Ref: 44011662 GLS05772). The structure of the 1,2,4-oxadiazole nucleus and ZINC15675948 is shown (Figure 1A,B). The stock solution was prepared with dimethyl sulfoxide (DMSO) at a concentration of 20 mM and stored at −20 ◦C until use. Since DMSO was the solvent of the compound, DMSO was used as a negative control in all experiments. DMSO was added at the same volume as the highest concentration of ZINC15675948 in the cells, which was less than 1% of the total medium. 4.2. Cell Culture The cell lines investigated in this study were reported previously [69,70]. The drug- sensitive CCRF-CEM and multidrug-resistant CEM/ADR5000 leukemia cell lines were cul- tivated in complete RPMI 1640 medium (Invitrogen, Darmstadt, Germany). The two breast cancer cell lines transduced with the control vector (MDA-MB-231-pcDNA3) or with a cDNA for the breast cancer resistance protein BCRP (MDA-MB-231-BCRP clone 23) were maintained in DMEM medium (Invitrogen, Darmstadt, Germany). Both media were sup- plemented with 10% fetal bovine serum (FBS) (Invitrogen) and 1% penicillin (100 µg/mL)- streptomycin (100 µg/mL) (Invitrogen). Cells were cultured at 37 ◦C in a humidified air incubator (90%) containing 5% CO2. In addition, CEM/ADR5000 cells were continuously treated with 5000 ng/mL doxorubicin every two weeks to maintain the overexpression of P-glycoprotein. The gene expression profiles of CEM/ADR5000 cells were previously described in [71–73]. MDA-MB-231-BCRP clone 23 cells were treated with 800 ng/mL geneticin (Sigma-Aldrich, Darmstadt, Germany) every two weeks. Molecules 2023, 28, 5658 19 of 28 4.3. Growth Inhibition Assay The resazurin reduction assay was used to access the effect of growth inhibition of ZINC15675948 [74]. The non-fluorescent dye resazurin is metabolically converted to the fluorescent dye resorufin by living cells [75]. Briefly, the CCRF-CEM and CEM/ADR5000 suspension cells were seeded into 96-well plates (1 × 104 cells/well) and then directly treated with 10 concentrations of ZINC15675948 in a range of 0.3–100 µM, respectively, in a total volume of 200 µL. MDA-MB-231-pcDNA3 and MDA-MB-231-BCRP clone 23 cells were seeded into 96 well plates (5× 103 cells/well) overnight and treated in the same series of concentrations as in leukemia cells with ZINC15675948 on the following day. After 72 h incubation, 20 µL of 0.01% resazurin (Promega, Germany) were added to each well and incubated for 4 h at 37 ◦C. The resazurin fluorescence was detected using an Infinite M2000 ProTM plate reader (Tecan, Crailsheim, Germany) at Ex/Em = 550 nm/590 nm wavelength. Relative cell viability was calculated in comparison to the DMSO-treated control. The final concentration of DMSO was 0.5%. The growth inhibition was accessed according to the effectiveness in inhibiting cell proliferation by half and was expressed as half-maximal inhibitory concentration (IC50) values. All IC50 values were expressed as mean ± standard deviation (SD). This experiment was repeated three times independently with six wells for each concentration. The figures were analyzed using GraphPad Prism Software (version 9.0.2) (GraphPad Software Inc., San Diego, CA, USA). 4.4. Molecular Docking In silico molecular docking was performed using the AutoDock 4.2.6 software (The Scripps Research Institute, CA, USA). The protocol was recently described by us [69]. The SDF format of ZINC15675948 was downloaded from ZINC 15 database (https://zinc15. docking.org/ accessed on 20 October 2019). ZINC15675948 was considered to perform molecular docking to P-glycoprotein (P-gp, MDR1, ABCB1) and the breast cancer resistance protein (BCRP, ABCG2), since growth inhibition of ZINC15675948 was determined in P-gp- or BCRP-overexpressing multidrug-resistant cell lines. The 3D structures of c-MYC (PDB code: 1NKP), P-gp (PDB code: 6QEX) and BCRP (PDB code: 6FFC) were downloaded from the RCSB Protein Data Bank (http://www.rcsb.org/, accessed on 20 October 2019) as PDB files. The known c-MYC inhibitors 10058-F4, 10074-A4 and 10074-G5 were used as positive control drugs to compare their affinity and binding mode with ZINC15675948 [76]. C-MYC was covered with grid box for the defined docking mode. The center of the grid box was set at x = 67.083, y = 64.481, and z = 42.943 with a spacing of 0.397 and with a number of grid points of 68 in x, 64 in y, and 72 in z). Doxorubicin as a known substrate of P-gp and BCRP was used as a control drug. Verapamil and Ko143 are inhibitors of P-gp or BCRP, respectively. Both were also used as control drugs. The grid boxes of P-gp and BCRP were placed around the drug-binding site [74]. Hydrogens were added to each protein structure, missing atoms were checked. The Lamarckian GA (4.2) was applied as an algorithm for 250 runs and 25,000,000 energy evaluations for each cycle. Docking was performed three times independently and the predicted inhibition constants were obtained from the docking log files (dlg). VMD (Visual Molecular Dynamics) software (version 1.9.3) (University of Illinois at Urbana Champaign, Champaign, IL, USA) and Discovery Studio Visualizer software (version v.21.1.0.20298) (Dassault Systems Biovia Corp, San Diego, CA, USA) were used as visualization tools to generate figures and gain a deeper understanding of the binding modes that were calculated from docking. 4.5. Microscale Thermophoresis Microscale thermophoresis (MST) was performed with human recombinant c-MYC protein and ZINC15675948. The protein was purchased from Abcam (Cambridge, UK) with a concentration of 0.5 mg/mL (ab169901). C-MYC was labeled with Monolith Protein Labeling Kit RED-NHS 2nd Generation (Nano Temper Technologies GmbH, Munich, Germany) according to the manufacturer’s instructions. The final concentration of c- MYC after labeling was 595 nM. ZINC15675948 was diluted from 400 µM to a series Molecules 2023, 28, 5658 20 of 28 of concentrations in assay buffer (50 mM Tris buffer (pH 7.4) containing 10 mM MgCl2, 150 mM NaCl, and 0.05% Tween-20). The labeled protein and diluted compounds were mixed (1:1). After 30 min incubation in the dark at room temperature, the fluorescence signal was measured on a Monolith NT.115 instrument (Nano Temper Technologies) with Monolith NT.115 standard capillaries. The MST with ZINC15675948 was performed with 70% LED power and 10% MST power. Fitting curved and dissociation constant (Kd) values were calculated with MO. Affinity Analysis software (version 2.2.4) (Nano Temper Technologies). The measurements were repeated three times independently. 4.6. c-MYC Reporter Assay A signal MYC reporter assay (Qiagen, Germantown, MD, USA) was used to determine the impact of ZINC15675948 on c-MYC activity as we described recently [58]. Briefly, a c- MYC-luciferase reporter construct was transfected into human embryonic kidney HEK293 cells and incubated according to the manufacturer’s instructions. Subsequently, cells were treated with two concentrations (4 × IC50 in CCRF-CEM or MDA-MB-231-pcDNA3) of ZINC15675948 (32 nM and 320 nM) and DMSO (negative control) or of the known c-MYC inhibitor 10058-F4 (positive control) for 48 h. A Dual-glo® Luciferase Reporter Assay System (Promega, Madison, WI, USA) was applied for the measurement of c-MYC promoter activity. Renilla and firefly luciferase luminescence were measured using an Infinite M2000 ProTM plate reader (Tecan, Crailsheim, Germany). 4.7. Gene Expression Profiles Total mRNA was isolated using the InviTrap®Spin Universal RNA Mini Kit (Invitek Molecular, Berlin, Germany). CCRF-CEM (1 × 106 cells/well) and MDA-MB-231-pcDNA3 cells (5 × 105 cells/well) were treated with DMSO as a solvent control and ZINC15675948 for 24 h in duplicate. ZINC15675948 was applied with a concentration according to the IC50 value (CCRF-CEM cells: 0.008 µM, MDA-MB-231-pcDNA3 cells: 0.08 µM). The RNA concentrations were determined using NanoDrop1000 (PEQLAB, Erlangen, Germany). Afterwards, quality control of total RNA, probe labeling, hybridization, scanning and data analysis of the samples were performed at the Genomics and Proteomics Core Facility of the German Cancer Research Center (DKFZ, Heidelberg, Germany). Affymetrix GeneChips® with human ClariomTM S assays (Affymetrix, Santa Clara, CA, USA) were applied for microarray hybridizations as previously described in detail [34]. 4.8. Pathway Analysis of Microarray Data The Chipster software (http://chipster.csc.fi/, accessed on 20 October 2019) (The Finnish IT Center for Science CSC, Espoo, Finland) was used to filter a set of differentially expressed genes acquired from microarray hybridization [77]. The Empirical Bayes t-test (p < 0.05) was applied to access the deregulated genes between DMSO and ZINC15675948- treated groups (accessed in July 2021). The filtered genes were analyzed with the Ingenuity Pathway Analysis (IPA) software (Qiagen, Redwood City, CA, USA) (content version 51963813) by the core analysis tool to determine the cellular functions and networks affected by drug treatment (accessed in August 2021). 4.9. Quantitative Real-Time Reverse Transcription PCR The same total RNA samples (DMSO control and IC50) used for the microarray analyses were also used for qRT-PCR experiments [78]. One microgram RNA was con- verted to cDNA using the LunaScript® RT SuperMix Kit cDNA Synthesis Kit (New England Bio Labs, Darmstadt, Germany) according to the manufacturer’s instructions. All PCR primers were designed using the NCBI Primer-BLAST (https://www.ncbi.nlm. nih.gov/tools/primer-blast/) website and purchased from Eurofins genomics (Ebers- berg, Germany) (https://eurofinsgenomics.eu/en/dna-rna-oligonucleotides/optimised- application-oligos/pcr-primers/) (accessed in September 2021). The primer sequences are shown in Table 4. The GAPDH gene served as an internal control. The reaction mix- Molecules 2023, 28, 5658 21 of 28 ture contained 4 µL master mix (5 × Hot Start Taq EveGreen® qPCR Mix (no Rox) (Axon Labortechnik, Kaiserslautern, Germany), 1 µL forward or reversed primer (250 nM final concentration), 13 µL nuclease-free water (Thermo Fisher), and 1 µL cDNA converted from 300 ng RNA. The qRT-PCR was performed on CFX384 Touch Real-Time PCR Detection System (Bio-Rad, Munich, Germany) using the 384-well plate. The initial denaturation of qRT-PCR was at 95 ◦C for 10 min followed by 40 cycles including strand separation at 95 ◦C for 15 s, annealing at 57.5 ◦C for 40 s and extension at 72 ◦C for 1 min. CFX Manager Software (version 3.1) was used to generate the Cq values. The fold-change of gene expres- sion was calculated using comparative 2−∆∆CT method as reported in the literature [79]. Specifically, for the DMSO control sample, the data of the target gene are presented as fold change in gene expression normalized to GAPDH gene. For the sample treat with the IC50 of the compound, the evaluation of 2−∆∆CT after normalization to GAPDH gene reveals the fold change of the target gene in gene expression relative to the untreated control. Table 4. The sequence (5′→3′) of qRT-PCR primers. Gene Symbol Forward Primer Reverse Primer RAD21 GAGTCAGCTATGCCTCCACC TGGAGGTTCTTCTGGGGGAA HMGCS1 CTTTCGTGGCTCACTCCCTT GTTTCCTCCTTCGGGCACA PGK1 TGTGTGGAATGGTCCTGTGG TGGCTTTCACCACCTCATCC ATP5MF CGGACACCAGGACTCCAAAA GGACTGAAGTCCCGCATCAA CITED2 GGCGAAGCTGGGGAATAACA AATCAGCCCTCCTCATCCTG HSPD1 GCCGCCCCGCAGAAAT AAGCCCGAGTGAGATGAGGA H4C3 CAGGGCATTACAAAACCGGC GTGCTCCGTATAGGTGACGG DHFR GCCACCGCTCAGGAATGAAT AGGTTGTGGTCATTCTCTGGAA c-MYC ACACTAACATCCCACGCTCTG CTCGCTAAGGCTGGGGAAAG GAPDH ATGAATGGGCAGCCGTTAGG AGCATCACCCGGAGGAGAAA 4.10. Single Cell Gel Electrophoresis (Alkaline Comet Assay) DNA damage was detected by alkaline comet assay using the OxiSelect™ Comet Assay Kit (Cell Biolabs/Biocat, Heidelberg, Germany) as described in [78]. The alkaline comet assay is a sensitive method for monitoring the migration of DNA fragments from nuclei under alkaline conditions. It detects DNA single- and double-strand breaks at a cellular level [50]. Briefly, CCRF-CEM cells (1 × 106 cells/well) were seeded into a 6-well plate and treated with different concentrations of ZINC15675948 (IC50, 2 × IC50 and 4 × IC50) and DMSO as a negative control for 24 h. MDA-MB-231-pcDNA3 cells (5 × 105 cells/well) were first seeded for 24 h to allow adhesion and then incubated with the same treatments for 24 h. Both cell lines were treated with H2O2 (50 µM) as a positive control for 30 min [80]. Cells were harvested and centrifuged at 3000× g for 10 min and were suspended in 1 mL cold PBS. Next, 1 × 105 cells were counted and mixed with agarose at 37 ◦C at a ratio of 1:6 and then spread on a comet slide. The following steps were conducted in the dark. Slides were left at 4 ◦C for 30 min to solidify and then immersed in pre-chilled lysis solution (NaCl 14.6 g, EDTA solution 20 mL, 10× lysis solution, pH 10.0, fulfill to 100 mL with distilled water, stored at 4 ◦C) for 1 h at 4 ◦C. Afterwards, slides were immersed in pre-chilled alkaline electrophoresis solution buffer (NaOH 12 g, EDTA solution 2 mL, fulfill to 100 mL with distilled water, stored at 4 ◦C) for 40 min. Next, electrophoresis was performed for 20 min at 20 V with alkaline electrophoresis solution buffer. Subsequently, slides were transferred into pre-chilled distilled water for 2 × 5 min for washing, followed by immersion in 70% ethanol for 5 min. After slides were dry, Vista Green DNA dye was diluted in TE buffer (Tris 121.14 mg, EDTA 200 µL, pH 7.5, fulfill to 100 mL distilled water) at a ratio of 1:1000. Then 100 µL diluted Vista Green DNA dye was added to each sample. DNA damage was captured by EVOS digital inverted microscope (Life Technologies GmbH, Darmstadt, Germany). At least 50 comets of each treatment were analyzed by Imaged J software (version 1.53q) using a plugin OpenComet (National Molecules 2023, 28, 5658 22 of 28 University of Singapore, Singapore). The tail DNA percentage was used as a parameter of DNA damage [81,82]. 4.11. Cell Cycle Arrest CCRF-CEM cells (1 × 106 cells/well) were seeded into a 6-well-plate and treated with ZINC15675948 at concentrations of IC50, 2 × IC50 or 4 × IC50, DMSO (negative control), or vincristine (positive control, 0.3 µM) (University Hospital Pharmacy, Mainz, Germany) for 72 h. MDA-MB-231-pcDNA3 cells (3 × 105 cells/well) were seeded and on the second day treated with ZINC15675948 at concentrations of 0.5 × IC50, IC50 or 2 × IC50, and DMSO (negative control) and cisplatin (positive control, 0.5 µM) (University Hospital Pharmacy) for 24 h. The cells were harvested and centrifuged with cold PBS (4 ◦C) twice (1500 rpm for 5 min). Ice-cold ethanol (80%) was used for fixation. Samples were kept at −20 ◦C at least for 24 h. Afterwards, cells were spun down from the ethanol by centrifugation at 4000 rpm for 10 min and washed twice with cold PBS. Before measurement, the cells were resuspended with 500 µL cold PBS containing 20 g/mL RNase (Roche Diagnostics, Mannheim, Germany) and incubated at room temperature for 30 min, followed by staining with 50 µg/mL propidium iodide (PI) (Sigma-Aldrich). After 15 min incubation in the dark at 4 ◦C, CCRF-CEM cells were analyzed on a BD AccuriTM C6 Flow Cytometer (Becton-Dickinson, Heidelberg, Germany). MDA-MB-231-pcDNA3 cells were analyzed on a BD LSRFortessa SORP (Becton Dickinson, Heidelberg, Germany). The cells were gated firstly using FSC-A/SSC-A gate in linear scale, 104 cells were recorded. Then doublets were removed using FL2-A/FL2-H gate also in linear scale. The DNA histogram was generated using FL2-A/histogram properties. All the experiments were repeated three times independently. The cell cycle distributions were analyzed by the FlowJo software (version 10.8.1) (Celeza, Olten, Switzerland) [34]. 4.12. Detection of Apoptosis in Suspension Cells Annexin V-FITC apoptosis kit (Bio Version/Biocat, Heidelberg, Germany) was applied to detect apoptosis in suspension cells [83]. CCRF-CEM cells (1 × 106 cells/well) were seeded in a 6-well plate, then treated with different concentrations (IC50, 2 × IC50 or 4 × IC50) of ZINC15675948, DMSO (negative control), or vincristine (positive control, 5 µM), and incubated for 24, 48, or 72 h. The cells were harvested, washed with cold PBS and 1 × binding buffer (Bio Version), respectively. Afterwards, the cells were stained with 52.5 µL annexin V master mix (2.5 µL annexin V, 50 µL 1 × binding buffer) (Bio Version), and incubated at 4 ◦C in the dark for 15 min. Then cells were stained with 440 µL PI master mix (10 µL PI, 430 µL 1 × binding buffer) (Bio Version). The detection was performed on a BD AccuriTM C6 Flow Cytometer (Becton-Dickinson) and 2 × 104 cells were recorded for each sample. Four different cell populations were obtained from flow cytometer, including living cells: annexin (−)/PI (−), early apoptosis: annexin (+)/PI (−), late apoptosis: annexin (+)/PI (+), and necrosis: annexin (−)/PI (+). The data were analyzed using FlowJo software (version 10.8.1) (Celeza). The experiments were repeated in triplicate. 4.13. Detection of Apoptosis in Adherent Cells MDA-MB-231-pcDNA3 adherent cells generally need trypsinization for cell-harvesting, which may result in false positive results by applying the Annexin V-FITC apoptosis kit. Therefore, the Violet Ratiometric Membrane Asymmetry Prob/Dead Cell Apoptosis Kit (Thermo Fisher Scientific, Darmstadt, Germany) was alternatively utilized to detect apoptosis in adherent cells [84]. Furthermore, 4’-N,N-diethylamino-6-(N,N,N dodecyl- methylamino-sulfopropyl)-methyl-3-hydroxyflavone (F2N12S) is a novel fluorescent probe to monitor the lipid composition on the plasma membrane in the early stage of apop- tosis. SYTOX® AADvancedTM can pass through the cell membrane only in necrosis or late apoptosis. Briefly, 1 × 105 MDA-MB-231-pcDNA3 cells were seeded in a 6-well plate overnight and then treated with different concentrations (0.5 × IC50, IC50, 2 × IC50 or Molecules 2023, 28, 5658 23 of 28 4 × IC50) of ZINC1565948, DMSO (negative control), or vincristine (positive control, 1 µM). After the incubation for 48 h, cells were detached using 500 µL Accutase (Thermo Fisher Scientific, Darmstadt, Germany) at room temperature and then washed with 1 mL cold Hank’s balanced salt solution (HBSS, 4 ◦C) twice. Cells were suspended with 1 mL HBSS. Subsequently, 1 µL F2N12S solution was added to each sample at a final concentration of 200 nM. Another 1 µL SYTOX® AADvancedTM dead cell stain solution was added at a final concentration of 1 µM. Samples were incubated at room temperature for 5 min and then analyzed using a BD LSRFortessa SORP (Becton Dickinson). The F2N12S was excited at 405 nm, and the emissions were collected with orange fluorescence (585/15 bandpass filter) and green fluorescence (530/30 bandpass filter). A ratio of the orange fluorescence channel to the green fluorescence channel was set as a derive parameter. The SYTOX® AADvanced TM dead cell stain was excited at 488 nm, and the emission was collected with a 670/30 bandpass filter. The detections were repeated three times independently. The data were analyzed by the FlowJo software (version 10.8.1) (Celeza). 4.14. Protein Analyses by SDS-PAGE and Immunoblotting CCRF-CEM cells (6,000,000 cells/flask) and MDA-MB-231-pcDNA3 cells (500,000 cells/well) were treated with varying concentrations of ZINC15675948 (IC50, 2 × IC50, or 4 × IC50) or DMSO as a negative control. After 24 h incubation, the cells were harvested and washed with PBS. Then 100 µL of ice-cold M-PER Mammalian Protein Extrac- tion Reagent (Thermo Fisher Scientific, Darmstadt, Germany) containing 1% Halt Protease Inhibitor Cocktail and phosphatase inhibitor (Thermo Fisher Scientific) were added to each sample. The cell lysis solutions were shaken for 30 min at 4 ◦C and centrifuged at 14,000× g for 15 min at 4 ◦C. The supernatants were harvested, and the protein concentrations were measured with NanoDrop1000 (PEQLAB, Erlangen, Germany). Each protein sample of 30 mg was electrophoresed on 10% SDS-polyacrylamide gels and transferred to polyvinylidene difluoride (Ruti®-PVDF) membranes (Millipore Corporation, Billerica, MA, USA) at 250 mA for 90 min. The membranes were washed with Tris-buffered saline containing 0.1% Tween-20 (TBST) for 5 min and then blocked in 5% bovine serum albumin in TBST for 1 h at room temperature. Afterward, the membranes were washed with TBST for 3× 5 min and incubated with diluted primary antibody at 4 ◦C overnight as follows: c-MYC antibody (1:1000, Cell Signaling Technology, Franfurt a. M., Germany), p62, SQSTM1 polyclonal antibody (1:1000, Proteintech, Planegg-Martinsried, Germany), Beclin 1 polyclonal antibody (1:1000, Proteintech), CDK1 (1:1000, Cell Signaling Technology), Phospho-CDK2 (Thr160) (1:1000, Cell Signaling Technology), GAPDH (1:1000, Cell Signaling Technology), or β-actin (1:1000, Cell Signaling Technology). After washing with TBST for 3 × 5 min, the membranes were incubated with diluted secondary antibody anti-rabbit IgG, HRP-linked antibody (1:2000, Cell Signaling Technology) for 1 h at room temperature. Finally, Horseradish peroxidase (HRP) substrate (LuminateTM Classico, Merck Millipore, Schwalbach, Germany) was added to membranes in the dark. The Alpha Innotech FluorChem Q system (Biozym, Oldendorf, Germany) was used for band detection. The quantification was carried out using Image J software (version 1.53q) (National Institute of Health, Bethesda, MD, USA). All the experiments were repeated at least three times independently. 4.15. Doxorubicin Uptake Assay Doxorubicin is a substrate of P-gp. P-gp-overexpressing CEM/ADR5000 cells were seeded in 12-well plates (104 cells/well). Doxorubicin was obtained from the University Hospital Pharmacy (Mainz, Germany) and used at a concentration of 10 µM in all samples. In parallel, different concentrations of ZINC15675948 (IC50, 2 × IC50 or 4 × IC50) were combined with doxorubicin treatment. Verapamil (20 µM; Sigma-Aldrich) was applied as a positive control for P-gp inhibition. Unstained CEM/ADR5000 cells were used as a negative control. In comparison, non-P-gp-expressing CCRF-CEM cells were used as a positive control for doxorubicin uptake. After 3 h incubation at 37 ◦C, cells were centrifuged Molecules 2023, 28, 5658 24 of 28 to discard the old medium and resuspended in 1 mL PBS. The doxorubicin fluorescence assay was carried out on a BD AccuriTM C6 Flow Cytometer (Becton-Dickinson) by blue laser at excitation 488 nm and emission 530 nm. In each sample, 3000 cells were counted. Dead cells and debris were removed by gating the cells in forward vs. side scatter, and forward-area vs. forward-height scatter, respectively. All experiments were performed in triplicate. The protocol has been described by us in [74]. MDA-MB-231-pcDNA3 and MDA-MB-BCRP (5000 cells/well) were seeded into 12- well plates. Doxorubicin treatment was combined with ZINC15675948 (IC50, 2 × IC50 or 4 × IC50). Ko143 (200 nM; Sigma-Aldrich) was used as a positive control for BCRP inhibition. The negative control is unstaining cells. Non-BCRP-overexpressing MDA-MB- 231-pcDNA cells were applied as a positive control of doxorubicin uptake. After 24-h incubation at 37 ◦C, the samples were washed and measured as described above. 5. Conclusions In conclusion, ZINC15675948 displayed remarkable cytotoxicity in CCRF-CEM and MDA-MB-231-pcDNA3 cells. ZINC15675948 bound to the c-MYC/MAX interface and in- hibited c-MYC activity and expression. The inhibition of c-MYC in MDA-MB-231-pcDNA3 cells involved ubiquitination. By means of microarray-based mRNA expression profiling, we verified that ZINC15675948 induced DNA damage and apoptosis in both cell lines as downstream effects of c-MYC inhibition. ZINC15675948 caused G2/M phase cell cycle ar- rest in CCRF-CEM cells and S phase arrest in MDA-MB-231-pcDNA3 cells. ZINC15675948 also induced autophagy in CCRF-CEM cells. Furthermore, ZINC15675948 was a substrate of two ABC transporters, P-gp and BCRP. These results illustrate that ZINC15675948 is a promising inhibitor of c-MYC worth further development as a novel anticancer drug. Supplementary Materials: The following supporting information can be downloaded at: https://www. mdpi.com/article/10.3390/molecules28155658/s1, Table S1: Deregulated gene expression upon treat- ment of CCRF-CEM leukemia cells with the IC50 concentration of ZINC15675948 for 24 h; Table S2: Deregulated gene expression upon treatment of MDA-MB-231-pcDNA3 breast cancer cells with the IC50 concentration of ZINC15675948 for 24 h. Author Contributions: Conceptualization, M.Z., S.M.K. and T.E.; Data curation, M.Z., J.C.B. and S.M.K.; Formal analysis, M.Z.; Investigation, M.Z., J.C.B., E.A.O. and S.M.K.; Methodology, M.Z., J.C.B., E.A.O. and S.M.K.; Project administration, T.E.; Software, M.Z., J.C.B., E.A.O. and S.M.K.; Supervision, T.E.; Validation, M.Z., J.C.B. and E.A.O.; Visualization, M.Z., J.C.B. and E.A.O.; Writing— original draft, M.Z.; Writing—review & editing, M.Z., S.M.K. and T.E. All authors have read and agreed to the published version of the manuscript. Funding: This research received no external funding. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: The authors declare that the data supporting the findings of this study are available within the paper. Acknowledgments: We are appreciative of the expression profiling service provided by the Microar- ray Unit of the Genomics and Proteomics Core Facility, German Cancer Research Center (DKFZ). We thank the Flow Cytometry Core Facility at the Institute of Molecular Biology (IMB, Mainz, Germany) for their helpful training and technical support for the usage of BD LSRFortessa SORP, which was applied for the detection of cell cycle and apoptosis in adherent cells. We are grateful for a stipend of the Chinese Scholarship Council to M.Z., the stipend of the Sibylle Kalkhof-Rose-Foundation to J.C.B., and the stipend of the German Academic Exchange Service (DAAD) to E.A.O. Conflicts of Interest: The authors declare no conflict of interest. Sample Availability: Samples of the compound ZINC15675948 are available upon reasonable request. Molecules 2023, 28, 5658 25 of 28 Abbreviations ABC, ATP-binding cassette transporter; ALL, acute lymphoblastic leukemia; BCRP, breast cancer resistance protein; CDK, cyclin-dependent kinases; DMSO, dimethyl sulfoxide; ELL, eleven-nineteen lysine-rich leukemia gene, elongation factor for RNA polymerase II; FBS, fetal bovine serum; HBSS, Hank’s balanced salt solution; IC50, half-maximal inhibitory concentration; IPA, Ingenuity Pathway Analysis; Kd, dissociation constant; LBE, lowest binding energy; MAX, MYC-associated factor X; MDR, multidrug resistance; MST, microscale thermophoresis; PBS, phosphate buffered saline; P-gp, P-glycoprotein; TNBC, triple-negative breast cancer. References 1. Bishop, J.M. Molecular themes in oncogenesis. Cell 1991, 64, 235–248. [CrossRef] [PubMed] 2. Dang, C.V. MYC on the path to cancer. Cell 2012, 149, 22–35. [CrossRef] [PubMed] 3. Lourenco, C.; Resetca, D.; Redel, C.; Lin, P.; MacDonald, A.S.; Ciaccio, R.; Kenney, T.M.G.; Wei, Y.; Andrews, D.W.; Sunnerha- gen, M.; et al. MYC protein interactors in gene transcription and cancer. Nat. Rev. Cancer 2021, 21, 579–591. [CrossRef] [PubMed] 4. Albihn, A.; Johnsen, J.I.; Henriksson, M.A. MYC in oncogenesis and as a target for cancer therapies. Adv. Cancer Res. 2010, 107, 163–224. [CrossRef] 5. Sammak, S.; Hamdani, N.; Gorrec, F.; Allen, M.D.; Freund, S.M.V.; Bycroft, M.; Zinzalla, G. Crystal structures and nuclear magnetic resonance studies of the apo form of the c-MYC: MAX bHLHZip complex reveal a helical basic region in the absence of DNA. Biochemistry 2019, 58, 3144–3154. [CrossRef] 6. Duffy, M.J.; O’Grady, S.; Tang, M.; Crown, J. MYC as a target for cancer treatment. Cancer Treat. Rev. 2021, 94, 102154. [CrossRef] 7. Gabay, M.; Li, Y.; Felsher, D.W. MYC activation is a hallmark of cancer initiation and maintenance. Cold Spring Harb. Perspect. Med. 2014, 4, a014241. [CrossRef] 8. Llombart, V.; Mansour, M.R. Therapeutic targeting of “undruggable” MYC. eBioMedicine 2022, 75, 103756. [CrossRef] 9. Dhanasekaran, R.; Deutzmann, A.; Mahauad-Fernandez, W.D.; Hansen, A.S.; Gouw, A.M.; Felsher, D.W. The MYC oncogene—The grand orchestrator of cancer growth and immune evasion. Nat. Rev. Clin. Oncol. 2022, 19, 23–36. [CrossRef] 10. Sears, R.C. The life cycle of C-myc: From synthesis to degradation. Cell Cycle 2004, 3, 1133–1137. [CrossRef] 11. Dingar, D.; Tu, W.B.; Resetca, D.; Lourenco, C.; Tamachi, A.; De Melo, J.; Houlahan, K.E.; Kalkat, M.; Chan, P.K.; Boutros, P.C.; et al. MYC dephosphorylation by the PP1/PNUTS phosphatase complex regulates chromatin binding and protein stability. Nat. Commun. 2018, 9, 3502. [CrossRef] [PubMed] 12. Fallah, Y.; Brundage, J.; Allegakoen, P.; Shajahan-Haq, A.N. MYC-driven pathways in breast cancer subtypes. Biomolecules 2017, 7, 53. [CrossRef] [PubMed] 13. Boxer, L.M.; Dang, C.V. Translocations involving c-myc and c-myc function. Oncogene 2001, 20, 5595–5610. [CrossRef] [PubMed] 14. Felsher, D.W.; Bishop, J.M. Reversible tumorigenesis by MYC in hematopoietic lineages. Mol. Cell 1999, 4, 199–207. [CrossRef] [PubMed] 15. Atanasov, A.G.; Zotchev, S.B.; Dirsch, V.M.; Orhan, I.E.; Banach, M.; Rollinger, J.M.; Barreca, D.; Weckwerth, W.; Bauer, R.; Bayer, E.A.; et al. Natural products in drug discovery: Advances and opportunities. Nat. Rev. Drug Discov. 2021, 20, 200–216. [CrossRef] 16. Hassan, A.; Khan, A.H.; Saleem, F.; Ahmad, H.; Khan, K.M. A patent review of pharmaceutical and therapeutic applications of oxadiazole derivatives for the treatment of chronic diseases (2013–2021). Expert Opin. Ther. Pat. 2022, 32, 969–1001. [CrossRef] 17. Camci, M.; Karali, N. Bioisosterism: 1,2,4-oxadiazole rings. ChemMedChem 2023, 18, e202200638. [CrossRef] 18. Atmaram, U.A.; Roopan, S.M. Biological activity of oxadiazole and thiadiazole derivatives. Appl. Microbiol. Biotechnol. 2022, 106, 3489–3505. [CrossRef] 19. Dhameliya, T.M.; Chudasma, S.J.; Patel, T.M.; Dave, B.P. A review on synthetic account of 1,2,4-oxadiazoles as anti-infective agents. Mol. Divers. 2022, 26, 2967–2980. [CrossRef] 20. Carbone, M.; Li, Y.; Irace, C.; Mollo, E.; Castelluccio, F.; Di Pascale, A.; Cimino, G.; Santamaria, R.; Guo, Y.-W.; Gavagnin, M. Structure and cytotoxicity of phidianidines A and B: First finding of 1,2,4-oxadiazole system in a marine natural product. Org. Lett. 2011, 13, 2516–2519. [CrossRef] [PubMed] 21. Labriere, C.; Elumalai, V.; Staffansson, J.; Cervin, G.; Le Norcy, T.; Denardou, H.; Réhel, K.; Moodie, L.W.K.; Hellio, C.; Pavia, H.; et al. Phidianidine A and synthetic analogues as naturally inspired marine antifoulants. J. Nat. Prod. 2020, 83, 3413–3423. [CrossRef] 22. Shamsi, F.; Hasan, P.; Queen, A.; Hussain, A.; Khan, P.; Zeya, B.; King, H.M.; Rana, S.; Garrison, J.; Alajmi, M.F.; et al. Synthesis and SAR studies of novel 1,2,4-oxadiazole-sulfonamide based compounds as potential anticancer agents for colorectal cancer therapy. Bioorg. Chem. 2020, 98, 103754. [CrossRef] 23. Caneschi, W.; Enes, K.B.; Carvalho de Mendonça, C.; de Souza Fernandes, F.; Miguel, F.B.; da Silva Martins, J.; Le Hyaric, M.; Pinho, R.R.; Duarte, L.M.; Leal de Oliveira, M.A.; et al. Synthesis and anticancer evaluation of new lipophilic 1,2,4 and 1,3,4-oxadiazoles. Eur. J. Med. Chem. 2019, 165, 18–30. [CrossRef] 24. Mohamed, M.F.A.; Marzouk, A.A.; Nafady, A.; El-Gamal, D.A.; Allam, R.M.; Abuo-Rahma, G.E.-D.A.; El Subbagh, H.I.; Moustafa, A.H. Design, synthesis and molecular modeling of novel aryl carboximidamides and 3-aryl-1,2,4-oxadiazoles derived from indomethacin as potent anti-inflammatory iNOS/PGE2 inhibitors. Bioorg. Chem. 2020, 105, 104439. [CrossRef] Molecules 2023, 28, 5658 26 of 28 25. Il’in, M.V.; Sysoeva, A.A.; Bolotin, D.S.; Novikov, A.S.; Suslonov, V.V.; Rogacheva, E.V.; Kraeva, L.A.; Kukushkin, V.Y. Aminon- itrones as highly reactive bifunctional synthons. An expedient one-pot route to 5-amino-1,2,4-triazoles and 5-amino-1,2,4- oxadiazoles—Potential antimicrobials targeting multi-drug resistant bacteria. New J. Chem. 2019, 43, 17358–17366. [CrossRef] 26. Kim, J.; Shin, J.S.; Ahn, S.; Han, S.B.; Jung, Y.-S. 3-Aryl-1,2,4-oxadiazole derivatives active against human rhinovirus. ACS Med. Chem. Lett. 2018, 9, 667–672. [CrossRef] 27. Dos Santos Filho, J.M.; de Queiroz, E.S.D.M.A.; Macedo, T.S.; Teixeira, H.M.P.; Moreira, D.R.M.; Challal, S.; Wolfender, J.L.; Queiroz, E.F.; Soares, M.B.P. Conjugation of N-acylhydrazone and 1,2,4-oxadiazole leads to the identification of active antimalarial agents. Bioorg. Med. Chem. 2016, 24, 5693–5701. [CrossRef] 28. Mohammad, B.D.; Baig, M.S.; Bhandari, N.; Siddiqui, F.A.; Khan, S.L.; Ahmad, Z.; Khan, F.S.; Tagde, P.; Jeandet, P. Heterocyclic compounds as dipeptidyl peptidase-IV inhibitors with special emphasis on oxadiazoles as potent anti-diabetic agents. Molecules 2022, 27, 6001. [CrossRef] [PubMed] 29. Wang, M.; Liu, T.; Chen, S.; Wu, M.; Han, J.; Li, Z. Design and synthesis of 3-(4-pyridyl)-5-(4-sulfamido-phenyl)-1,2,4-oxadiazole derivatives as novel GSK-3β inhibitors and evaluation of their potential as multifunctional anti-Alzheimer agents. Eur. J. Med. Chem. 2021, 209, 112874. [CrossRef] [PubMed] 30. Nelson, J.B.; Fizazi, K.; Miller, K.; Higano, C.; Moul, J.W.; Akaza, H.; Morris, T.; McIntosh, S.; Pemberton, K.; Gleave, M. Phase 3, randomized, placebo-controlled study of zibotentan (ZD4054) in patients with castration-resistant prostate cancer metastatic to bone. Cancer 2012, 118, 5709–5718. [CrossRef] [PubMed] 31. Engebraaten, O.; Vollan, H.K.M.; Børresen-Dale, A.L. Triple-negative breast cancer and the need for new therapeutic targets. Am. J. Pathol. 2013, 183, 1064–1074. [CrossRef] [PubMed] 32. Yin, L.; Duan, J.J.; Bian, X.W.; Yu, S.C. Triple-negative breast cancer molecular subtyping and treatment progress. Breast Cancer Res. 2020, 22, 61. [CrossRef] [PubMed] 33. Li, Q.; Pan, S.; Xie, T.; Liu, H. MYC in T-cell acute lymphoblastic leukemia: Functional implications and targeted strategies. Blood Sci. 2021, 3, 65–70. [CrossRef] [PubMed] 34. Hegazy, M.F.; Dawood, M.; Mahmoud, N.; Elbadawi, M.; Sugimoto, Y.; Klauck, S.M.; Mohamed, N.; Efferth, T. 2α- Hydroxyalantolactone from Pulicaria undulata: Activity against multidrug-resistant tumor cells and modes of action. Phytomedicine 2021, 81, 153409. [CrossRef] 35. Beaulieu, M.E.; Soucek, L. Finding MYCure. Mol. Cell. Oncol. 2019, 6, e1618178. [CrossRef] 36. Whitfield, J.R.; Soucek, L. The long journey to bring a Myc inhibitor to the clinic. J. Cell Biol. 2021, 220, e202103090. [CrossRef] 37. Ghobrial, A.; Flick, N.; Daly, R.; Hoffman, M.; Milcarek, C. ELL2 influences transcription elongation, splicing, Ig secretion and growth. J. Mucosal Immunol. Res. 2019, 3, 112. 38. Chen, Y.; Zhou, C.; Ji, W.; Mei, Z.; Hu, B.; Zhang, W.; Zhang, D.; Wang, J.; Liu, X.; Ouyang, G.; et al. ELL targets c-Myc for proteasomal degradation and suppresses tumour growth. Nat. Commun. 2016, 7, 11057. [CrossRef] 39. Newman, D.J.; Cragg, G.M. Natural products as sources of new drugs over the nearly four decades from 01/1981 to 09/2019. J. Nat. Prod. 2020, 83, 770–803. [CrossRef] 40. Yap, J.L.; Wang, H.; Hu, A.; Chauhan, J.; Jung, K.Y.; Gharavi, R.B.; Prochownik, E.V.; Fletcher, S. Pharmacophore identification of c-Myc inhibitor 10074-G5. Bioorg. Med. Chem. Lett. 2013, 23, 370–374. [CrossRef] 41. Hammoudeh, D.I.; Follis, A.V.; Prochownik, E.V.; Metallo, S.J. Multiple independent binding sites for small-molecule inhibitors on the oncoprotein c-Myc. J. Am. Chem. Soc. 2009, 131, 7390–7401. [CrossRef] 42. Massó-Vallés, D.; Soucek, L. Blocking Myc to treat cancer: Reflecting on two decades of omomyc. Cells 2020, 9, 883. [CrossRef] [PubMed] 43. Han, H.; Jain, A.D.; Truica, M.I.; Izquierdo-Ferrer, J.; Anker, J.F.; Lysy, B.; Sagar, V.; Luan, Y.; Chalmers, Z.R.; Unno, K.; et al. Small-molecule MYC inhibitors suppress tumor growth and enhance immunotherapy. Cancer Cell 2019, 36, 483–497.e15. [CrossRef] [PubMed] 44. Boike, L.; Cioffi, A.G.; Majewski, F.C.; Co, J.; Henning, N.J.; Jones, M.D.; Liu, G.; McKenna, J.M.; Tallarico, J.A.; Schirle, M.; et al. Discovery of a functional covalent ligand targeting an intrinsically disordered cysteine within MYC. Cell Chem. Biol. 2021, 28, 4–13.e17. [CrossRef] [PubMed] 45. Panda, D.; Saha, P.; Das, T.; Dash, J. Target guided synthesis using DNA nano-templates for selectively assembling a G-quadruplex binding c-MYC inhibitor. Nat. Commun. 2017, 8, 16103. [CrossRef] 46. Michel, J.; Cuchillo, R. The impact of small molecule binding on the energy landscape of the intrinsically disordered protein C-myc. PLoS ONE 2012, 7, e41070. [CrossRef] 47. Follis, A.V.; Hammoudeh, D.I.; Wang, H.; Prochownik, E.V.; Metallo, S.J. Structural rationale for the coupled binding and unfolding of the c-Myc oncoprotein by small molecules. Chem. Biol. 2008, 15, 1149–1155. [CrossRef] 48. Farrell, A.S.; Sears, R.C. MYC degradation. Cold Spring Harb. Perspect. Med. 2014, 4, a014365. [CrossRef] 49. Chen, Y.; Sun, X.-X.; Sears, R.C.; Dai, M.-S. Writing and erasing MYC ubiquitination and SUMOylation. Genes Dis. 2019, 6, 359–371. [CrossRef] 50. Lu, Y.; Liu, Y.; Yang, C. Evaluating In Vitro DNA Damage Using Comet Assay. J. Vis. Exp. 2017, 128, e56450. [CrossRef] 51. Goga, A.; Yang, D.; Tward, A.D.; Morgan, D.O.; Bishop, J.M. Inhibition of CDK1 as a potential therapy for tumors over-expressing MYC. Nat. Med. 2007, 13, 820–827. [CrossRef] 52. Gu, Y.; Rosenblatt, J.; Morgan, D.O. Cell cycle regulation of CDK2 activity by phosphorylation of Thr160 and Tyr15. EMBO J. 1992, 11, 3995–4005. [CrossRef] Molecules 2023, 28, 5658 27 of 28 53. Kurbegovic, A.; Trudel, M. The master regulators Myc and p53 cellular signaling and functions in polycystic kidney disease. Cell. Signal. 2020, 71, 109594. [CrossRef] [PubMed] 54. Kelly, G.L.; Grabow, S.; Glaser, S.P.; Fitzsimmons, L.; Aubrey, B.J.; Okamoto, T.; Valente, L.J.; Robati, M.; Tai, L.; Fairlie, W.D.; et al. Targeting of MCL-1 kills MYC-driven mouse and human lymphomas even when they bear mutations in p53. Genes Dev. 2014, 28, 58–70. [CrossRef] [PubMed] 55. Li, X.; He, S.; Ma, B. Autophagy and autophagy-related proteins in cancer. Mol. Cancer 2020, 19, 12. [CrossRef] 56. Kang, R.; Zeh, H.J.; Lotze, M.T.; Tang, D. The Beclin 1 network regulates autophagy and apoptosis. Cell Death Differ. 2011, 18, 571–580. [CrossRef] 57. Liu, W.J.; Ye, L.; Huang, W.F.; Guo, L.J.; Xu, Z.G.; Wu, H.L.; Yang, C.; Liu, H.F. p62 links the autophagy pathway and the ubiqutin–proteasome system upon ubiquitinated protein degradation. Cell Mol. Biol. Lett. 2016, 21, 29. [CrossRef] [PubMed] 58. Elbadawi, M.; Boulos, J.C.; Dawood, M.; Zhou, M.; Gul, W.; ElSohly, M.A.; Klauck, S.M.; Efferth, T. The novel artemisinin dimer isoniazide ELI-XXIII-98-2 induces c-MYC inhibition, DNA damage, and autophagy in leukemia cells. Pharmaceutics 2023, 15, 1107. [CrossRef] 59. Li, W.; Zhang, H.; Assaraf, Y.G.; Zhao, K.; Xu, X.; Xie, J.; Yang, D.H.; Chen, Z.S. Overcoming ABC transporter-mediated multidrug resistance: Molecular mechanisms and novel therapeutic drug strategies. Drug Resist. Updates 2016, 27, 14–29. [CrossRef] 60. Robey, R.W.; Pluchino, K.M.; Hall, M.D.; Fojo, A.T.; Bates, S.E.; Gottesman, M.M. Revisiting the role of ABC transporters in multidrug-resistant cancer. Nat. Rev. Cancer 2018, 18, 452–464. [CrossRef] 61. Tiwari, A.K.; Sodani, K.; Dai, C.L.; Ashby, C.R., Jr.; Chen, Z.S. Revisiting the ABCs of multidrug resistance in cancer chemotherapy. Curr. Pharm. Biotechnol. 2011, 12, 570–594. [CrossRef] 62. Doyle, L.; Ross, D.D. Multidrug resistance mediated by the breast cancer resistance protein BCRP (ABCG2). Oncogene 2003, 22, 7340–7358. [CrossRef] 63. Silva, R.; Vilas-Boas, V.; Carmo, H.; Dinis-Oliveira, R.J.; Carvalho, F.; de Lourdes Bastos, M.; Remião, F. Modulation of P- glycoprotein efflux pump: Induction and activation as a therapeutic strategy. Pharmacol. Ther. 2015, 149, 1–123. [CrossRef] 64. Szakács, G.; Paterson, J.K.; Ludwig, J.A.; Booth-Genthe, C.; Gottesman, M.M. Targeting multidrug resistance in cancer. Nat. Rev. Drug Discov. 2006, 5, 219–234. [CrossRef] 65. Modi, A.; Roy, D.; Sharma, S.; Vishnoi, J.R.; Pareek, P.; Elhence, P.; Sharma, P.; Purohit, P. ABC transporters in breast cancer: Their roles in multidrug resistance and beyond. J. Drug Target. 2022, 30, 927–947. [CrossRef] [PubMed] 66. Hall, M.D.; Handley, M.D.; Gottesman, M.M. Is resistance useless? Multidrug resistance and collateral sensitivity. Trends Pharmacol. Sci. 2009, 30, 546–556. [CrossRef] [PubMed] 67. Efferth, T.; Konkimalla, V.B.; Wang, Y.F.; Sauerbrey, A.; Meinhardt, S.; Zintl, F.; Mattern, J.; Volm, M. Prediction of broad spectrum resistance of tumors towards anticancer drugs. Clin. Cancer Res. 2008, 14, 2405–2412. [CrossRef] 68. Saeed, M.E.M.; Boulos, J.C.; Elhaboub, G.; Rigano, D.; Saab, A.; Loizzo, M.R.; Hassan, L.E.A.; Sugimoto, Y.; Piacente, S.; Tundis, R.; et al. Cytotoxicity of cucurbitacin E from Citrullus colocynthis against multidrug-resistant cancer cells. Phytomedicine 2019, 62, 152945. [CrossRef] [PubMed] 69. Saeed, M.E.M.; Mahmoud, N.; Sugimoto, Y.; Efferth, T.; Abdel-Aziz, H. Molecular determinants of sensitivity or resistance of cancer cells toward sanguinarine. Front. Pharmacol. 2018, 9, 136. [CrossRef] 70. Doyle, L.A.; Yang, W.; Abruzzo, L.V.; Krogmann, T.; Gao, Y.; Rishi, A.K.; Ross, D.D. A multidrug resistance transporter from human MCF-7 breast cancer cells. Proc. Natl. Acad. Sci. USA 1998, 95, 15665–15670. [CrossRef] 71. Kadioglu, O.; Cao, J.; Kosyakova, N.; Mrasek, K.; Liehr, T.; Efferth, T. Genomic and transcriptomic profiling of resistant CEM/ADR- 5000 and sensitive CCRF-CEM leukaemia cells for unravelling the full complexity of multi-factorial multidrug resistance. Sci. Rep. 2016, 6, 36754. [CrossRef] 72. Efferth, T.; Sauerbrey, A.; Olbrich, A.; Gebhart, E.; Rauch, P.; Weber, H.O.; Hengstler, J.G.; Halatsch, M.E.; Volm, M.; Tew, K.D.; et al. Molecular modes of action of artesunate in tumor cell lines. Mol. Pharmacol. 2003, 64, 382–394. [CrossRef] 73. Kimmig, A.; Gekeler, V.; Neumann, M.; Frese, G.; Handgretinger, R.; Kardos, G.; Diddens, H.; Niethammer, D. Susceptibility of multidrug-resistant human leukemia cell lines to human interleukin 2-activated killer cells. Cancer Res. 1990, 50, 6793–6799. 74. Abdelfatah, S.; Böckers, M.; Asensio, M.; Kadioglu, O.; Klinger, A.; Fleischer, E.; Efferth, T. Isopetasin and S-isopetasin as novel P-glycoprotein inhibitors against multidrug-resistant cancer cells. Phytomedicine 2021, 86, 153196. [CrossRef] 75. O’Brien, J.; Wilson, I.; Orton, T.; Pognan, F. Investigation of the Alamar Blue (resazurin) fluorescent dye for the assessment of mammalian cell cytotoxicity. Eur. J. Biochem. 2000, 267, 5421–5426. [CrossRef] 76. Yin, X.; Giap, C.; Lazo, J.S.; Prochownik, E.V. Low molecular weight inhibitors of Myc-Max interaction and function. Oncogene 2003, 22, 6151–6159. [CrossRef] 77. Kallio, M.A.; Tuimala, J.T.; Hupponen, T.; Klemelä, P.; Gentile, M.; Scheinin, I.; Koski, M.; Käki, J.; Korpelainen, E.I. Chipster: User-friendly analysis software for microarray and other high-throughput data. BMC Genom. 2011, 12, 507. [CrossRef] [PubMed] 78. Zhou, M.; Boulos, J.C.; Klauck, S.M.; Efferth, T. The cardiac glycoside ZINC253504760 induces parthanatos-type cell death and G2/M arrest via downregulation of MEK1/2 phosphorylation in leukemia cells. Cell Biol. Toxicol. 2023, 1–27. [CrossRef] [PubMed] 79. Livak, K.J.; Schmittgen, T.D. Analysis of relative gene expression data using real-time quantitative PCR and the 2−∆∆CT method. Methods 2001, 25, 402–408. [CrossRef] [PubMed] 80. Benhusein, G.M.; Mutch, E.; Aburawi, S.; Williams, F.M. Genotoxic effect induced by hydrogen peroxide in human hepatoma cells using comet assay. Libyan J. Med. 2010, 5, 4637. [CrossRef] Molecules 2023, 28, 5658 28 of 28 81. Gyori, B.M.; Venkatachalam, G.; Thiagarajan, P.S.; Hsu, D.; Clement, M.V. OpenComet: An automated tool for comet assay image analysis. Redox Biol. 2014, 2, 457–465. [CrossRef] [PubMed] 82. Collins, A.R. The comet assay for DNA damage and repair. Mol. Biotechnol. 2004, 26, 249–261. [CrossRef] [PubMed] 83. Crowley, L.C.; Marfell, B.J.; Scott, A.P.; Waterhouse, N.J. Quantitation of apoptosis and necrosis by annexin V binding, propidium iodide uptake, and flow cytometry. Cold Spring Harb. Protoc. 2016, 2016, 953–957. [CrossRef] [PubMed] 84. Shynkar, V.V.; Klymchenko, A.S.; Kunzelmann, C.; Duportail, G.; Muller, C.D.; Demchenko, A.P.; Freyssinet, J.-M.; Mely, Y. Fluo- rescent biomembrane probe for ratiometric detection of apoptosis. J. Am. Chem. Soc. 2007, 129, 2187–2193. [CrossRef] [PubMed] Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content. 48 Curriculum Vitae of Min Zhou Date of birth: 29 January 1994; Place of birth: Kunming, China; E-mail: minzhou1@uni-mainz.de; Phone: +49(0)15257225218 Address: Staudinger Weg 5, 55128 Mainz, Germany Education • Johannes Gutenberg-Universität Mainz, Mainz, Germany Ph. D candidate for Pharmaceutical Biology, October 2019-present, expected defense: October 2023 Supervisor: Prof. Dr. Thomas Efferth • Kunming Institute of Botany, University Chinese Academy of Sciences (UCAS), Kunming and Beijing, China Master, Ethnobotany, 2016-2019 Supervisor: Prof. Xuefei Yang • Yunnan Agricultural University, Kunming, China Bachelor, Land Resource Management, 2012-2016 Research experience • Ph.D. candidate: Prof. Dr. Thomas Efferth’s group, Johannes Gutenberg-Universität Mainz 1) Molecular mode of action and parthanatic cell death of a novel cardiac glycoside compound targets MEK1/2 in leukemia cells. 2) Molecular mode of action of a 1,2,4-oxadiazole derivate targets c-MYC in leukemia and triple- negative breast cancer cells. 3) Parthanatic cell death of synthesized palladium (II) -based compounds to overcome anti-apoptosis and anti-autophagy in leukemia cells. • Master student: Prof. Xuefei Yang’s group, Kunming Institute of Botany, Chinese Academy of Sciences 1) Ethnobotany research, nutritional and functional evaluation on edible flowers in Yunnan, China. 2) Traditional medicine in Myanmar: isolation of potential anti-diabetic compounds from medical plants 3) Traditional Tibetan herbal medicine and Ethnobotany: verification of plant extracts against anti-cold stress in Caenorhabditis elegans. Awards, Honors & Activities • Special Award of Best Poster Presentation, 7th Cancer World Congress, Palermo, Italy, 2023. • Special Award of Best Flash Oral Presentation, 6th Cancer World Congress, Lisbon, Portugal, 2022. • Outstanding Performance Award, 5th Youth Forum, Kunming Institute of Botany, Kunming, China, 49 2019. • Honours Student, University of Chinese Academy of Sciences, Beijing, China, 2017. • Merit Award, 11th Student Academic and Scientific Works Competition, Yunnan Agricultural University, Kunming, China, 2015. • Outstanding Volunteers, 1st China-South Asia Exposition, Kunming, China, 2015. Personal Skills Laboratory skills Software and analysis Language • Cell culture (leukemia, breast, • Ingenuity Pathway Analysis • Chinese colon, kidney, glioblastoma, • Chipster (native) osteosarcoma cancer cells) • Autodock • English • Cell growth inhibition assay • Pyrx Virtual Screening • German • Isolation of peripheral blood • Prism-GraphPad (Level: A2.2) mononuclear cells • Origin • Western blotting • Image J • PCR • R programming • qRT-PCR • Swiss target prediction • Single cell electrophoresis • String protein-protein • Wound healing assay interaction network • Immunofluorescence microscopy • Visual Molecular Dynamics • Flow cytometry (cell cycle, • FlowJo apoptosis, ROS, mitochondrial • ArcGIS membrane potential) • Motif Enrichment Analysis • Luciferase assay • mRNA and DNA extraction • Bioinformatical evaluation of microarray hybridization • Mycoplasma detection Scholarships • Doctoral Scholarship, Chinese Scholarship Council, 48 months, October 2019-September 2023 • Exchange Student Scholarship, Kagoshima University (Japan), 2 weeks, 2015. • The First Prize Scholarship, Yunnan Agricultural University, 12 months, 2013. Teaching experience • Pharmacological Biology III, Johannes Gutenberg-Universität Mainz, 2020-present 1) Responsible for Quality control: Identification of Panax ginseng by PCR; 2) Quantitative analytics of caraway oil. 50 Conference 1) 7th Cancer World Congress, Palermo, Italy, May 28th - 30th, 2023. (Awarded with Best Poster Presentation of Young Researcher) 2) 6th Cancer World Congress, Lisbon, Portugal, September 28th - 30th, 2022. (Awarded with Best Flash Oral Presentation of Young Researcher) 3) 9th National Symposium on Ethnobotany and 8th Asia-Pacific Forum on Ethnobotany, Huhhot, China, July 16-18 2016. Book chapters and contributions • Common Research Methods in Ethnobotany. Chapter: Relative importance values. Zhejiang Education Publishing House, 2018 (Writing). • Typical Vegetables in Myanmar, Press of University of Science and Technology of China, 2018. (Collection of plant information and photographs).