Authors:	Zhou, Min
Advisor:	Efferth, Thomas
Title:	Small molecules targeting apoptosis and parthanatos in sensitive and drug-resistant tumor cells
Online publication date:	17-Oct-2023
Year of first publication:	2023
Language:	english
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. 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.
DDC:	610 Medizin 610 Medical sciences
Institution:	Johannes Gutenberg-Universität Mainz
Department:	FB 09 Chemie, Pharmazie u. Geowissensch.
Place:	Mainz
ROR:	https://ror.org/023b0x485
DOI:	http://doi.org/10.25358/openscience-9585
URN:	urn:nbn:de:hebis:77-openscience-e04eb976-a087-4ad6-898e-ffdb2e0539488
Version:	Original work
Publication type:	Dissertation
License:	CC BY
Information on rights of use:	https://creativecommons.org/licenses/by/4.0/
Extent:	Getrennte Zählungen ; Illustrationen, Diagramme
Appears in collections:	JGU-Publikationen