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ARTICLE OPEN
Cynaropicrin disrupts tubulin and c-Myc-related signaling and
induces parthanatos-type cell death in multiple myeloma
Joelle C. Boulos1, Ejlal A. Omer1, Daniela Rigano2, Carmen Formisano2, Manik Chatterjee3, Ellen Leich4,5, Sabine M. Klauck6,
Le-tian Shan7 and Thomas Efferth1✉
The majority of blood malignancies is incurable and has unforeseeable remitting-relapsing paths in response to different
treatments. Cynaropicrin, a natural sesquiterpene lactone from the edible parts of the artichoke plant, has gained increased
attention as a chemotherapeutic agent. In this study, we investigated the effects of cynaropicrin against multiple myeloma (MM)
cells in vitro and assessed its in vivo effectiveness in a xenograft tumor zebrafish model. We showed that cynaropicrin exerted
potent cytotoxicity against a panel of nine MM cell lines and two leukemia cell lines with AMO1 being the most sensitive cell line
(IC50= 1.8 ± 0.3 µM). Cynaropicrin (0.8, 1.9, 3.6 µM) dose-dependently reduced c-Myc expression and transcriptional activity in
AMO1 cells that was associated with significant downregulation of STAT3, AKT, and ERK1/2. Cell cycle analysis showed that
cynaropicrin treatment arrested AMO1 cells in the G2M phase along with an increase in the sub-G0G1 phase after 24 h. With
prolonged treatment times, cells accumulated more in the sub-G0G1 phase, implying cell death. Using confocal microscopy, we
revealed that cynaropicrin disrupted the microtubule network in U2OS cells stably expressing α-tubulin-GFP. Furthermore, we
revealed that cynaropicrin promoted DNA damage in AMO1 cells leading to PAR polymer production by PARP1 hyperactivation,
resulting in AIF translocation from the mitochondria to the nucleus and subsequently to a novel form of cell death, parthanatos.
Finally, we demonstrated that cynaropicrin (5, 10 µM) significantly reduced tumor growth in a T-cell acute lymphoblastic leukemia
(T-ALL) xenograft zebrafish model. Taken together, these results demonstrate that cynaropicrin causes potent inhibition of
hematopoietic tumor cells in vitro and in vivo.
Keywords: hematological malignancies; multiple myeloma; cynaropicrin; c-Myc; microtubules; parthanatos; network
pharmacology; xenograft tumor zebrafish model
Acta Pharmacologica Sinica (2023) 0:1–17; https://doi.org/10.1038/s41401-023-01117-3
INTRODUCTION maturation in the thymus, leading to differentiation arrest and
Hematological malignancies are a group of cancerous tumors in abnormal spread of immature progenitors [5]. Although T-ALL
which immunological or defective hematological cells fail to survival rates have significantly increased over the past 50 years,
differentiate and perpetually proliferate, impairing the function of relapsed and refractory patients are still very difficult to treat, and
biological organisms [1]. They primarily fall into one of three the prognosis for those who cannot withstand intense treatment
categories: lymphoma, multiple myeloma (MM), and leukemia [2]. is still bleak [6].
Hematological malignancies are among the most fatal diseases, MM is a hematological plasma cell malignancy that comprises
posing a major threat to human health and life due to their high around 10% of all blood cancers [7]. The median survival rate for
mortality rate. In fact, hematological malignancies are unique in stage III MM patients is about 29 months, whereas stage I and
that they cannot be surgically extirpated as solid tumors, and their stage II patients survive almost 62 and 44 months, respectively [8].
clinical first-line therapies mostly consist of chemotherapy, Given the increase in MM incidence as well as the mortality rate,
radiation, and hematopoietic stem cell transplantation [3]. Even several anti-cancer therapies have been established, e.g., targeted
though standard first-line medicines have some effect, the general drug therapies (immunomodulatory drugs—IMiDs, proteasome
efficacy is unsatisfactory because of relapses and refractory inhibitors—PIs, monoclonal antibodies—mAbs-, etc.), combina-
conditions brought on by the emergence of primary and tion therapies based on lenalidomide, bortezomib, and others,
secondary drug resistance [4]. radiotherapy, stem cell transplantation, corticosteroids, and
T-cell acute lymphoblastic leukemia (T-ALL) emanates from bisphosphonate treatment [9]. Even though these therapies
genetic abnormalities that assemble in the course of T-cell protracted the anticipated life span of MM patients, many
1Department of Pharmaceutical Biology, Institute of Pharmaceutical and Biomedical Sciences, Johannes Gutenberg University, Staudinger Weg 5, 55128 Mainz, Germany;
2Department of Pharmacy, University of Naples “Federico II”, Naples, Italy; 3University Hospital Würzburg, Translational Oncology, Comprehensive Cancer Center Mainfranken,
Würzburg, Germany; 4Julius Maximilian University, Institute of Pathology, Würzburg, Germany; 5Comprehensive Cancer Center Mainfranken, Translational Oncology, University
Hospital of Würzburg, Würzburg, Germany; 6Division of Cancer Genome Research, German Cancer Research Center (DKFZ), German Cancer Consortium (DKTK), National Center
for Tumor Diseases (NCT), Heidelberg, Germany and 7The First Affiliated Hospital, Zhejiang Chinese Medical University, Hangzhou 310053, China
Correspondence: Thomas Efferth (efferth@uni-mainz.de)
Received: 15 February 2023 Accepted: 28 May 2023
© The Author(s) 2023
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concerns have been raised regarding the drawbacks resulting Human HEK293 embryonic kidney cells were kindly provided by
from the prolonged application of these treatments. Prof. Dr. Christina Friedland (Johannes Gutenberg University,
As scientists became aware of the imminent side effects of the Germany) and bone osteosarcoma U2OS human cells stably
current hematologic therapies, they searched for natural products expressing α-tubulin-GFP protein were kindly provided by Dr.
(NPs) as a possible replacement. Plant-based diets, focusing on the Joachim Hehl (Light microscope center, ETH Zurich). HEK293 and
consumption of not only vegetables and fruits, but also whole U2OS cells were cultured in DMEM (Life Technologies) supple-
grains, are inversely associated with the incidence of malignancies, mented with 10% FBS (Life Technologies) and 1% penicillin
as they are rich in phytochemicals [10]. Over the past decades, the (1000 U/mL)/streptomycin (100 μg/mL) (Life Technologies). Cells
application of phytochemicals as adjuvant anti-neoplastic agents were kept in a 5% CO2 incubator at 37 °C.
has been a rising trend [11], because NPs display the following Fresh blood samples were collected from four healthy donors at
main characteristics: (i) “metabolite-likeness”, (ii) active transport the Department of Hematology, Oncology, and Pneumology
metabolites, and (iii) high bioavailability [12–14]. Hence, studies on (University Medical Center of the Johannes Gutenberg University,
NPs skyrocketed in the past three decades [15]. Numerous Mainz, Germany) and flowed in plastic Monovette EDTA tubes.
phytochemicals have been shown to exhibit cell cycle arrest, Histopaque® (Sigma-Aldrich, Taufkirchen, Germany) was used to
apoptosis, anti-angiogenesis, and miRNA modulation in MM. isolate human peripheral blood mononuclear cells (PBMCs). In
Additionally, agaricus, curcumin, and neovastat have been applied brief, 3 mL of fresh blood were thoroughly layered on top of
in clinical trials for the treatment of MM [16–18]. Altogether, these Histopaque® and centrifuged for 30 min at 400 × g and 4 °C.
evidences shed light on the potency of NPs and their bioactive Subsequently, the buffy coat consisting of PBMCs was separated,
compounds in MM treatment [9]. Sesquiterpene lactones (SQL) washed with PBS, and subjected to centrifugation at 250 × g
constitute one of the major and most studied class of plant- thrice, for 10 min each. The cell pellet was suspended in Panserin
derived phytochemicals [19], as they have potent anti-inflamma- 413 growth media (PAN-Biotech, Aidenbach, Germany) supple-
tory, anti-tumor, antibiotic, phyto-toxic, insect-feeding deterrent, mented with 2% phytohemagglutinin M (PHA-M, Life
and schistosomicidal potential bioactivities [20]. Technologies).
Cynaropicrin, a SQLs of the guaianolide type, has a γ-
butyrolactone ring, a crucial pharmacophore that is implicated Cell viability assay
in several biological activities (Fig. 1). Cynaropicrin was first The resazurin reduction assay was applied to determine the
isolated from the edible artichoke plant (Cynara scolymus L.) [21], sensitivity of leukemia, MM cell lines, and PBMCs to cynaropicrin.
and it is currently regarded as a chemotaxonomic marker of the Brie 4fly, 10 cells/well were seeded in a flat bottom 96-well plate.
artichoke plant [22]. The bitter taste of artichokes is associated Cells were immediately treated with different cynaropicrin
with elevated SQL amounts, particularly of cynaropicrin, which concentrations for 72 h.
causes around 80% of the typical bitter taste of artichoke plants AMO1 cells (104 cells/well) were also seeded in a flat bottom 96-
[23]. Besides, cynaropicrin has been isolated from numerous well plate. Cells were immediately treated with 1.8, 3.6, and 7.2 µM
species of the genus Centaurea and Saussurea [24–28], and it has of cynaropicrin as well as 1.8, 3.6, and 7.2 µM of cynaropicrin in
several biological and pharmacological properties such as anti- combination with the PARP inhibitor PJ34 (10 µM) (528150,
inflammatory, anti-cancer, anti-parasitic, anti-protozoal, anti- Sigma–Aldrich, Darmstadt, Germany), or the caspase inhibitor z-
hyperlipidemic, anti-hepatitis C viral, anti-photoaging, anti-spas- vad-fmk (50 µM) (627610, Sigma–Aldrich, Darmstadt, Germany), or
modic, anti-feedant activities, and is also an inhibitor of NF-κB and the autophagy inhibitor Bafilomycin A1 (50 nM) (J61835.MCR,
activator of bitter sensory receptors [29]. Several studies reported Thermo Fisher Scientific, Dreieich, Germany), or the autophagy
the effect of cynaropicrin against various cancer types. Cynar- inducer Rapamycin (50 nM) (J62473.MF, Thermo Fisher Scientific,
opicrin induced apoptosis and weak G2/M arrest in a human Dreieich, Germany) for 48 h. The 96-well plates were then
gastric adenocarcinoma cell line [30]. Cynaropicrin was also incubated at 37 °C and 5% CO2 for 4 h with resazurin. The
investigated for melanoma and thyroid cancer [31, 32], triple- reduction of resazurin by living cells generated a fluorescence,
negative breast cancer [33], cervical cancer [34], colorectal cancer which was detected with an Infinite M2000 Pro plate reader
[35], and lymphoma and leukemia, especially multidrug-resistant (Tecan, Crailsheim, Germany) at an excitation wavelength of λ/
cell lines [24, 36]. nm= 544 and an emission wavelength of λ/nm= 590. Later on,
However, no studies have yet been performed to examine the GraphPad Prism 5 software (GraphPad Software, San Diego, CA)
response of MM to cynaropicrin in vitro or to evaluate the in vivo was used to plot the cell viability against the concentration of
antiproliferative activity of cynaropicrin through a leukemia model. cynaropicrin and to determine the IC50 values from three
Therefore, the aim of this project was to investigate the effect of independent experiments with six replicates each [37].
cynaropicrin on MM cancer cells in vitro and to assess the in vivo
effectiveness of cynaropicrin using a T-ALL zebrafish model. Gene expression profiles
AMO1 cells were treated with 1.8 µM of cynaropicrin or with
DMSO. After 24 h treatment, RNA extraction was performed using
MATERIALS AND METHODS the RNeasy Kit from Qiagen (Hilden, Germany). As previously
Cell lines described, gene expression profiling was obtained by microarray
The drug-sensitive CCRF-CEM T-ALL and their counterpart hybridization of duplicate samples using Affymetrix Clariom S
multidrug-resistant P-glycoprotein-overexpressing CEM/ADR5000 human chips (Affymetrix, Santa Clara, CA, USA) at the Genomics
leukemia cells were provided by Dr. Axel Sauerbrey (Children’s and Proteomics Core Facility of the German Cancer Research
Hospital, University of Jena, Germany). MM cell lines (AMO1, JJN3, Center (DKFZ, Heidelberg) [38].
KMS12BM, KMS11, L363, MolP8, NCI-H929, OPM2) were kindly
supplied by Dr. Manik Chatterjee and Dr. Ellen Leich (University of Real-time quantitative polymerase chain reaction (RT-PCR)
Würzburg, Germany). RPMI8226 cells were obtained from the Luna Script™ RT SuperMix Kit (E3010) (New England Biolabs
American Type Cell Culture Collection (ATCC® CCL-155™, USA). All GmbH, Frankfurt, Germany) was used to convert 1 μg of the
MM cells were mycoplasma free. MM and T-ALL cells were extracted RNA into cDNA following the manufacturer’s instruc-
cultured in RPMI-1640 (Life Technologies, Darmstadt, Germany) tion. 5 × Hot Start Taq EvaGreen® qPCR Mix (Axon-Labortechnik,
supplemented with 10% FBS (Life Technologies) and 1% penicillin Kaiserslautern, Germany) was used to carry out RT-qPCR
(1000 U/mL)/streptomycin (100 μg/mL) (Life Technologies). Cells following manufacturer’s instruction. The Primer-BLAST tool
were kept in a 5% CO2 incubator at 37 °C. was applied to design the primers (Table 1), which were
Acta Pharmacologica Sinica (2023) 0:1 – 17
Induction of parthanatos by cynaropicrin
JC Boulos et al.
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double-checked for rightness with the Oligo Analyze Tool from Apoptosis examination
Eurofins Genomics Germany GmbH (Ebersberg, Germany) and Apoptosis was examined using a Ratiometric Membrane Asym-
purchased from Eurofins Genomics. GAPDH was chosen as a metry Probe/Dead Cell Apoptosis Kit (Thermo Fisher Scientific,
reference gene. RT-qPCR was performed using CFX384™ Real- Darmstadt, Germany). A violet excitable dye named 4′-N, N-
Time PCR Detection System (Bio-Rad Laboratories GmbH, diethylamino-6-(N,N,N dodecyl-methylamino-sulfopropyl)-methyl-
Feldkirchen, Germany). Each sample was measured three times, 3-hydroxyflavone (F2N12S) is used to detect changes in mem-
each time in duplicates. The fold change FC was determined brane asymmetry through apoptosis [41]. Briefly, AMO1 cells were
based on the (Ct) of the gene of interest, represented as (gene), seeded in a 6-well plate (1 × 106 cells/well) and treated with
and the reference gene represented as (ref) obtained from the different concentrations of cynaropicrin (0.5, 0.9, 1.8, and 3.6 µM)
control and the sample [39]. or with DMSO, used as a negative control, for 48 h at 37 °C/5 %
¼  CO2. Cells were then harvested and washed with HBSS (HanksΔCt CtðgeneÞ CtðrefÞ Balanced Salt Solution) twice. Consecutively, cells were incubated
with F2N12S at a final concentration of 200 nM and SYTOX™
ΔΔCt ¼ ΔCtðsampleÞ  ΔCtðcontrolÞ AADvanced™ dead cell stain at a final concentration of 1 µM for
5 min, in the dark, at room temperature. Cells were analyzed with
FC ¼ log ð2ΔΔCtÞ a BD LSRFortessa SORP (Becton Dickinson, Heidelberg, Germany)2 flow cytometer. F2N12S was excited by a violet laser (λ/nm= 405)
and the emission light was assembled with a 585/15 bandpass
filter representing the orange fluorescence channel and 530/30
Cell cycle analysis bandpass filter representing the green fluorescence channel (The
The cell cycle perturbations of AMO1 cells were studied using ratio parameter (585/530) was set up in the software). SYTOX™
Propidium iodide (PI) 24, 48, and 72 h post-treatment with AADvanced™ dead cell stain was excited by a blue laser (λ/
different cynaropicrin concentrations (0.5, 0.9, 1.8, and 3.6 µM) or nm= 488) and the emission light was assembled with a 670/30
media alone. Cells were then harvested and fixed with 80% bandpass filter as suggested by the manufacturer [42]. Cells were
ethanol at −20 °C. After each specific incubation period, cells were then gated using the F- (forward) and S- (side) scatters. Singlets
incubated in PI staining solution (Thermo Fisher Scientific, were selected by gating according to the A- (area) and W- (width)
Dreieich, Germany) at 4 °C. Fifteen minutes later, PI staining was scatters. From each well, 104 events from the first gate (FSC/SSC)
measured using an Accuri C6 flow cytometer (Becton- were recorded. Data were then analyzed with FlowJo
Dickinson, Heidelberg, Germany). Total DNA content was detected V10.6.2 software (Becton Dickinson).
on FL2-A [38].
Mitochondrial membrane potential (MMP)
Fluorescence confocal microscopy of the microtubule MMP was measured using the JC-1 mitochondrial membrane
organization potential assay kit (Cayman Chemical, Ann Arbor, Michigan, United
Human osteosarcoma U2OS cancer cells stably transfected with an States). Briefly, 105 AMO1 cells/well were seeded in a flat bottom
α-tubulin-GFP construct were cultured in a sterile µ-Slide 8 Well 96-well plate. Cells were treated with different concentrations of
(ibidi, Gräfelfing, Germany). Aliquots of 30,000 cells/well were left cynaropicrin (0.5, 0.9, 1.8, 3.6, or 7.2 µM) or DMSO (negative
overnight in an incubator at 37 °C/5 % CO2 to attach, then cells control), or 1 µM of bortezomib (positive control) for 48 h at 37 °C/
were treated with 1.8 or 3.6 µM of cynaropicrin or DMSO (negative 5 % CO2. Cells were then incubated with 10 µL of JC-1 solution for
control) or vincristine (1 µM), a polymerization inhibitor, or 15min at 37 °C/5 % CO2. Afterwards, cells were centrifuged at
paclitaxel (1 µM), a depolymerization inhibitor, for 24 h. Later, 277 × g for 5 min and washed twice with 200 µL assay buffer.
cells were rinsed with PBS and fixed for 15 min with 4% Finally, cells were re-suspended in 200 µL assay buffer and JC-1
paraformaldehyde. After fixation, cells were washed three times staining was measured using a BD LSR Fortessa SORP flow
with PBS and the nucleus of each cell was stained for 5 min with cytometer. J-aggregates (living cells) were excited by a yellow-
1 µg/mL 4′,6-diamidino-2-phenylindole (DAPI) (Sigma–Aldrich, green laser (λ/nm= 561) and the emission light was assembled
Darmstadt, Germany) at room temperature. Afterward, cells were with a 586/15 bandpass filter. JC-1 monomers (dead cells) were
rinsed three times with PBS to get rid of excessive DAPI and ibidi excited by a blue laser (λ/nm = 488) and the emission light was
mounting medium (ibidi, Gräfelfing, Germany) was added to each assembled with a 530/30 bandpass filter [43]. If JC-1 accumulates
slide. Fluorescence images were obtained using an AF7000 in healthy mitochondria, it exhibits red fluorescence, however
widefield fluorescence microscope (Leica Microsystems, Wetzlar, when it accumulates in damaged mitochondria, it emits green
Germany). The blue laser (λ/nm= 470) was used to excite both fluorescence. Cells were then gated using the F- (forward) and S-
GFP and DAPI. The emission wavelength of GFP is λ/nm= 525, (side) scatters. Singlets were selected by gating according to the
however the emission wavelength of DAPI is λ/nm= 447. A- (area) and H- (height) scatters. Data were then analyzed with
Fluorescence images were analyzed with Fiji ImageJ software FlowJo V10.6.2 software (Becton Dickinson).
(National Institutes of Health, Bethesda, MD, USA) [40].
AMO1 cells were seeded in a six-well plate (500,000 cells/well) Western blot analyses
and treated with different concentrations of cynaropicrin (1.8 or AMO1 cells were seeded in a six-well plate (1 × 106 cells/well) and
3.6 µM) or with DMSO for 24 h at 37 °C/5 % CO2. Cells were then treated with different concentrations of cynaropicrin (0.9, 1.8, or
harvested, washed with HBSS, and cytospinned over slides 3.6 µM) or with DMSO, used as a negative control, for 48 h at
(Thermo Fisher Scientific, Dreieich, Germany) for 5 min at 37 °C/5% CO2. Cells were then harvested and washed with PBS
123 × g. Afterwards, cells were stained with Tubulin Tracker™ twice. To extract total proteins, cells were incubated in a 500 rpm
Deep Red (Thermo fisher Scientific, Dreieich, Germany) for 30 min shaker with M-PER® Mammalian Protein Extraction Reagent
at 37 °C and 5% CO2. Cells were then washed with HBSS thrice for supplemented with 1% Halt™ Protease Inhibitor Cocktail (Thermo
5min each. Cells nuclei were stained with Hoechst 33342 Nuclear Scientific, Frankfurt, Germany) for 30 min at 4 °C. Later, the cells
Stain (H3570, Thermo Fisher Scientific, Darmstadt, Germany) for were centrifuged at 277 × g for 15 min at 4 °C and the supernatant
30min at room temperature. After 30 min, cells were washed with containing proteins was collected. NE-PER Nuclear and Cytoplas-
HBSS thrice for 5 min each and polymerized tubulin in AMO1 cells mic Extraction reagent (Thermo Fisher Scientific, Dreieich,
was imaged at 40 × magnification using an AF7000 widefield Germany) was used to extract nuclear and cytoplasmic proteins
fluorescence microscope. following the manufacturer’s instructions. Protein concentration
Acta Pharmacologica Sinica (2023) 0:1 – 17
Induction of parthanatos by cynaropicrin
JC Boulos et al.
4
Darmstadt, Germany) for 5 min and slides were washed with
Table 1. Design of the primer’s sequences (5′→3′) for RT-PCR. PBS thrice. Finally, cells were mounted with Fluoromount-G®
Genes Forward primer Reverse primer (SouthernBiotech, Birmingham, AL, USA) and an AF7000 widefield
fluorescence microscope (Leica Microsystems, Wetzlar, Germany)
STAT3 TCT GTG TGA CAC CAA CGA GGA CTC AAA CTG CCC TCC was used for visualization. Alexa Fluor® 488 was excited at λ/
CC TG nm= 470 and emitted light at λ/nm= 525. DAPI was excited at λ/
MAP2K2 TTG TGA ACGAGCCACCTCC TGA GCA TCT TCA GGT CCG C nm= 470 and emitted light at λ/nm = 447 [46].
AKT1 GCG GCA GGA CCG AGC CGC CTG CTC CCG TCT TC
Cignal Myc reporter assay
c-MYC CTT CTC TCC GTC CTC GGA GAA GGT GAT CCA GAC TCT
TTC T GAC CTT c-Myc activity was determined using the cignal Myc reporter assay
kit (CCS-012L) from Qiagen (Germantown, MD, USA). Myc reporter
GAPDH GCTCTC TGC TCC TCC TGT TC GAC TCC GAC CTT CAC CTT CC assay is made to keep track of the Myc signaling pathway’s activity
in cells. A transfection-ready expression vector for c-Myc and a
was determined with a NanoDrop 1000 spectrophotometer reporter vector for luciferase are included in the kit. As a first step,
(Thermo Scientific, Frankfurt, Germany). Subsequently, 30 µg of HEK293 cells were transfected with a c-Myc-luciferase reporter
the lysate was loaded to each lane of a 10% SDS-PAGE gel. construct and cultured according to the manufacturer’s sugges-
Following the separation step, the proteins were transferred on a tions. HEK293 cells were used as hematopoietic cells are difficult
polyvinylidene difluoride membrane. The membrane was blocked to transfect. Cells were then treated with different concentrations
with a blocking buffer consisting of 5% BSA in TBST for 1 h at of cynaropicrin or DMSO (negative control), or the known Myc
room temperature. After the blocking step, the membranes were inhibitor (10058-F4) (positive control) for 48 h. The activity of the
incubated overnight at 4 °C with primary antibodies (1:1000) c-Myc promoter was quantified using the Dual-glo
® Luciferase
against c-Myc (#9402), Akt (pan) (C67E7) (#4691), phospho-Akt Reporter Assay System (E2920, Promega, Madison, WI, USA). An
(Ser473) (D9E) XP® (#4060), p44/42 MAPK (Erk1/2) (137F5) (#4695), Infinite M2000 Pro™ plate reader (Tecan, Germany) was used to
Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (D13.14.4E) XP® measure the luminescence of firefly and renilla luciferases [47].
(#4370), Stat3 (D3Z2G) (#12640), Phospho-Histone H2A.X (Ser139)
(20E3) (#9718), PARP (#9542), AIF (D39D2) XP® (#5318), caspase 7 cMyc activity ¼
(D2Q3L) (#12827), FoxO1 (C29H4) (#2880), GAPDH (D16H11) XP® ðfirefly luciferase luminescence=renilla luciferase luminescenceÞ
(#5174), β-Actin (13E5) (#4970) which were purchased from Cell
Signaling Technology (Frankfurt a. M., Germany), P62 (18420-1- Relative luciferase ¼
AP), Lamin B1 (66095-1-Ig), Beclin 1 (11306-1-AP), and Caspase 3/ 100 ´ ðfirefly luciferase luminescence=renilla luciferase luminescenceÞ
p17/p19 (19677-1-AP) which were purchased from Proteintech
(Planegg-Martinsried, Germany) and finally Anti-PAR (AM80-
100UG) which was purchased from Merck (Darmstadt, Germany). Normalized cMyc activity ¼
Membranes were then washed thrice with TBST for 10min each. relative luciferaseðsampleÞ=relative luciferaseðDMSOÞ
Following the washing step, the membranes were incubated with
either anti-rabbit IgG secondary antibody coupled to horseradish
peroxidase (HRP-linked; 1:1000) (#7074S, Cell Signaling Technol-
ogy, Frankfurt a. M., Germany) or anti-mouse IgG secondary Xenograft zebrafish model
antibody coupled to horseradish peroxidase (HRP-linked; 1:1000) Adult wild-type AB strain zebrafish was ordered from the China
(#7076S, Cell Signaling Technology, Frankfurt a. M., Germany) for Zebrafish Resource Center, Institute of Hydrobiology, China
1 h [44]. Finally, membranes were incubated with Luminata Academy of Science (Wuhan, China) and officially authorized by™
Classico Western HRP substrate (Merck Millipore, Darmstadt, the Association for Assessment and Accreditation of Laboratory
Germany) for 3 min and imaged using an Alpha Innotech Animal Care International (SYXK 2012-0171). After 48 h of
FluorChem Q system (Biozym, Oldendorf, Germany). Protein fertilization, natural pair-mating gave rise to zebrafish larvae
expression was calculated using ImageJ software [45]. which were kept in an aquaculture facility with an alternate
photoperiod of 14 h day/10 h night. Zebrafish larvae were fed with
one portion of fry flakes and two portions of live brine shrimps
Fluorescence microscopy of apoptosis inducing factor (AIF) per day.
AMO1 cells were treated with different concentrations of To initiate a T-ALL tumor xenograft zebrafish model, CCRF-CEM
cynaropicrin (0.9, 1.8, and 3.6 µM) and DMSO (negative control) cells were stained with red fluorescence CM-Dil (1:1000). After 48 h
for 48 h at 37 °C/5% CO2. After 48 h treatment, cells were of fertilization, 200 cells/fish were microinjected in the zebrafish
harvested and washed with PBS. Cells were then cytospinned on larvae yolk sac. After 24 h of tumor growth, a fluorescent
microscope slides (Thermo Fisher Scientific, Dreieich, Germany) at microscope (AZ100, Nikon, Tokyo, Japan) was used to verify the
a speed of 123 × g for 5 min. Subsequently, 4% paraformaldehyde model. The CCRF-CEM cell-bearing zebrafish were then treated
was added on top of the cells for 15min at room temperature to with different concentrations of cynaropicrin, and cis-platinum,
fix them. After fixation, cells were washed thrice with PBS, used as positive control, respectively, for 24 h (n= 5 zebrafishes/
permeabilized with 1% Triton X-100 in PBS for 10 min at room group). The fluorescence intensity (Fi) of CCRF-CEM cell mass in
temperature, and again washed thrice with PBS. Afterwards, cells every zebrafish was determined and the inhibitory rate was
were blocked with blocking buffer (1% BSA+ 10% FBS in PBS) for
® calculated as follows [48]:1 h. After blocking, the primary antibody AIF (D39D2) XP (#5318)
(Cell Signaling Technology, Frankfurt a. M., Germany) was added inhibitory rateð%Þ ¼
to the microscope slides which were incubated at 4 °C in a ½1ðFi of treated group=Fi of negative control groupÞ ´ 100%
humidified chamber overnight. The slides were washed thrice
with PBS. After the washing step, slides were incubated with
antirabbit secondary antibody-Alexa Fluor® 488 Conjugate (Cell
Signaling Technology) at room temperature in a dark humidified Statistical analysis
chamber. After 2 h, slides were rinsed three times with PBS. Cell The results were presented as mean ± S.D. Statistics were
nuclei were stained with 1 µg/mL of DAPI (Sigma–Aldrich, calculated using the unpaired two-tailed Student’s t-test
Acta Pharmacologica Sinica (2023) 0:1 – 17
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JC Boulos et al.
5
(Microsoft Excel, 2019). Significant results were those with a P- the expression of AKT, STAT3, c-MYC and MAP2K2 (Fig. 2d). To
value less than 0.05. further validate this assumption, we detected the gene expression
of STAT3, AKT, c-MYC, and MAP2K2 in L363, NCI-H929, and OPM2.
Our results were in line with that obtained in AMO1, and indeed
RESULTS STAT3, AKT, c-MYC, and MAP2K2 gene expression was down-
Cynaropicrin reduced MM and leukemia cells viability regulated in L363, OPM2, NCIH929, and AMO1 upon treatment
The cytotoxic effect of cynaropicrin was analyzed using the with cynaropicrin.
resazurin reduction assay in MM, drug-sensitive and multidrug-
resistant P-glycoprotein-overexpressing leukemia cells, as well as Cynaropicrin repressed c-Myc upstream regulators as detected by
PBMCs. A significant obstacle in cancer treatment is the Western blotting
emergence of multidrug resistance (MDR) to chemotherapy. We IPA revealed that c-Myc upstream regulators were downregulated.
have previously identified lead compounds in the early phases of These findings stimulated us to further evaluate the influence of
drug discovery that are not substrates of the ATP-binding cassette cynaropicrin on signaling proteins that are upstream of c-Myc.
(ABC) transporters [49]. We have also demonstrated that natural Especially, ERK signaling, JAK2-STAT3, and PI3K-AKT which are
products may provide attractive lead molecules for the generation strong mediators of MM cell proliferation, survival, anti-apoptosis,
of collateral sensitive anticancer compounds [50]. In this context, and resistance [53]. Our data showed that cynaropicrin treatment
we decided to study the cytotoxicity of cynaropicrin against CEM/ downregulated STAT3 in a dose-dependent manner. Furthermore,
ADR5000 cells. cynaropicrin significantly reduced the level of total AKT as well as
Cynaropicrin exhibited a strong cytotoxicity in all tested MM its activated form p-AKT. However, cynaropicrin significantly
cells after 72 h of treatment, with AMO1 being the most sensitive downregulated p-ERK1/2 without affecting total ERK (Fig. 3a).
cell line (IC50= 1.8 ± 0.3 µM) and KMS12BM being the least Our findings indicated that cynaropicrin downregulated the
sensitive (IC50= 3.2 ± 0.2 µM) (Fig. 1a, Table 2). Moreover, the protein level of c-Myc upstream regulators.
degree of responsiveness of CCRF-CEM (IC50= 2.9 ± 0.0 µM) and
CEM/ADR5000 (IC50= 2.6 ± 0.2 µM) to cynaropicrin was compar- Cynaropicrin inhibited c-Myc expression and transcriptional
able. The IC50 values of AMO1, JJN3, OPM2, and L363 obtained activity as detected by Western blotting and Myc reporter assay
with a narrow concentration range (three-fold apart from 100 µM mRNA expression profiles revealed that c-Myc-mediated apoptosis
to 0.005 µM) (Fig. 1b, Table 3) were comparable with the IC50 signaling was targeted by cynaropicrin and divulged a network
values obtained with a wide concentration range (ten-fold apart where c-Myc was downregulated. It is well-known that MM rely
from 100 µM to 10−6 µM) (Fig. 1a, Table 2) with a correlation strongly on c-Myc for growth and survival [54]. Therefore, it was
coefficient (R2) equal to 0.89, indicating that the IC50 values crucial to determine whether cynaropicrin prohibited c-Myc
obtained with the wide concentration range were accurate. expression and transcriptional activity. c-Myc expression was
The concentration of cynaropicrin needed to inhibit 50% of significantly downregulated in a dose-dependent way upon
normal leukocytes differed from one donor to another and it treatment with cynaropicrin (Fig. 3b).
ranged between 22.0 ± 4.5 µM and 39.3 ± 1.4 µM. The concentra- In an attempt to discover if cynaropicrin inhibitory effect was
tions of cynaropicrin needed to inhibit half of the normal not only restricted to c-Myc expression but could also include the
leukocytes were higher than the concentration needed to inhibit inhibition of c-Myc transcriptional activity, human embryonic
50% of KMS12BM, the least sensitive cell line. kidney 293 (HEK293) cells were transfected with a c-Myc reporter
luciferase construct. These cells were treated with different
Transcriptome-based expression profiles of cynaropicrin-treated cynaropicrin concentrations and with the known c-Myc inhibitor
AMO1 cells and Quantitative real time reverse transcription PCR 10058-F4 [55] (F3680, Sigma–Aldrich, Darmstadt, Germany),
(qRT-PCR) respectively. Indeed, cynaropicrin inhibited c-Myc transcriptional
AMO1 cells were treated with the IC50 concentration of activity in a dose-dependent manner. Additionally, low concen-
cynaropicrin and subjected to mRNA expression profiling. trations of cynaropicrin inhibited c-Myc activity more efficiently
Ingenuity Pathway Analyses software (IPA) (Qiagen, Hilden, than the known inhibitor 10058-F4, indicating that cynaropicrin
Germany) was used to analyze differentially expressed genes has a c-Myc inhibitory effect (Fig. 3c). To further validate that
compared to untreated samples. IPA reported several diseases and cynaropicrin inhibited c-Myc expression and transcriptional
considerable cellular functions that might be altered by cynar- activity, we studied the expression of c-Myc in HEK293 cells. As
opicrin, e.g., cell death and survival, cellular development, cellular expected, c-Myc expression significantly decreased upon cynar-
growth and proliferation, cancer, cell cycle, and most importantly opicrin treatment (Fig. 3b).
hematological disease (Fig. 2a). Additionally, IPA estimated many
canonical pathways that might be affected by cynaropicrin. Cell cycle analyses of cynaropicrin-treated AMO1 cells
Among these pathways, c-Myc-mediated apoptosis signaling Afterwards, the perturbations of the cell cycle caused by
drew our attention (Fig. 2b). Upstream regulator analyses showed cynaropicrin were explored, given that the cell cycle was one of
that STAT3, a transcription factor overexpressed in MM [51], was the cellular processes that appeared to be susceptible to change
significantly deregulated by cynaropicrin (P= 1.96 × 10−3) (Fig. 2a). The cell cycle status of AMO1 cells was evaluated after 24,
(Table 4). 48, and 72 h incubation with various cynaropicrin concentrations
Furthermore, IPA suggested a network where both STAT3 and c- (0.5, 0.9, 1.8, and 3.6 µM). After 24 h treatment, the fraction of
MYC, a downstream proto-oncogene target of STAT3, were AMO1 cells in the G2M phase and the sub G0G1 phase increased
downregulated. Besides these two genes, MAPKs (MAP2K2, proportionally with increased cynaropicrin concentration. After
MAP4K1, MAP2K1/2), that play a crucial role in cell survival, 48 h and 72 h treatment, the portion of cells in the sub G0G1 phase
proliferation, and death [52] were also downregulated by noticeably increased with increased cynaropicrin concentration,
cynaropicrin (Fig. 2c). In addition to that, PI3K and TUBA appeared implying that cynaropicrin induced cell death in AMO1 cells
as connecting genes within this IPA pathway disturbed by (Fig. 4).
cynaropicrin, rendering them eligible for further specific analyses.
Several genes (AKT, STAT3, c-MYC, and MAP2K2) were subjected Cynaropicrin distorted the microtubule network
to qRT-PCR, and their expression was normalized with the Microtubules are crucial for chromosome separation during
reference gene, GAPDH. Results were in line with the microarray mitosis, and it is likely that the inhibition of microtubule
analyses. In fact, treatment of AMO1 with cynaropicrin decreased polymerization triggers mitotic arrest [56]. Taking into
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Fig. 1 Chemical structure of the lipophilic compound cynaropicrin and the cytotoxic effect of cynaropicrin in nine different human
multiple myeloma (MM) cell lines (AMO1, KMS11, JJN3, MolP8, L363, NCI-H929, RPMI8226, OPM2, and KMS12BM), in human AML cells
(CCRF/CEM, CEM/ADR5000), and PBMCs. Each concentration is obtained by calculating the mean value ± SD of three experiments performed
at different time points with six replicates in each experiment. a Using a wide concentration range ten-fold apart from 10−7 to 102, b Using a
narrower concentration range three-fold apart from 10−2.35 to 102.
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of cytotoxic drugs in cancer cells. The ratiometric probe F2N12S
Table 2. IC50 values of cynaropicrin in MM, drug-sensitive and and the DNA-binding SYTOX™ AADvanced™ dye were used to
multidrug-resistant P-glycoprotein-overexpressing leukemia cells, and measure cell membrane asymmetry (apoptotic cells) and cell
normal leukocytes. permeability (dead cells), respectively. F2N12S and SYTOX™
Cell type Cell line IC (µM) AADvanced™ combined together discriminate between live,50
apoptotic, and dead cells. Our results revealed that only 7.5% of
Multiple myeloma AMO1 1.8 ± 0.3 control cells experienced apoptosis and the percentage of
JJN3 2.1 ± 0.7 apoptotic cells was the same even upon exposure to different
L363 2.5 ± 0.3 cynaropicrin concentrations (Fig. 6a), implying that cynaropicrin
did not induce apoptosis in AMO1 cells. To strengthen our
MOLP8 2.5 ± 0.3
findings, we further investigated apoptosis by detecting the
OPM2 2.6 ± 0.6 apoptotic markers (caspase 3 and caspase 7) by Western blotting.
NCI-H929 2.7 ± 0.2 Neither caspase 3 nor caspase 7 were cleaved by cynaropicrin
RPMI8226 2.7 ± 0.4 treatment (Fig. 6b), suggesting that cynaropicrin induced a
KMS11 2.8 ± 0.3 caspase independent cell death. Subsequently, we investigated
the programmed non-apoptotic cell death autophagy by evaluat-
KMS12BM 3.2 ± 0.2 ing the autophagy specific marker (Beclin 1 and P62) by Western
Leukemia CCRF-CEM 2.9 ± 0.0 blotting (Fig. 6c). Cynaropicrin did not affect the expression of
CEM/ADR5000 2.6 ± 0.2 neither Beclin 1 nor P62, indicating that autophagy is not induced
Normal leukocytes PBMCs- Donor 1 39.3 ± 1.4 by cynaropicrin. Moreover, the pharmacological inhibition of
caspase by z-vad-fmk, the inhibition of autophagy by bafilomycin
A1, as well as the induction of autophagy by rapamycin did not
significantly alter the sensitivity of AMO1 to cynaropicrin (Fig. 6d,
Table 3. Validation of IC50 values using a narrower concentration e). So far, our data revealed that neither apoptosis nor autophagy
range in four MM cell lines: AMO1, JJN3, OPM2, and L363 and in was implicated in cynaropicrin-induced cell death, therefore
PBMCs collected from three different donors. further investigations must be performed to decipher the type
Cell type Cell line IC (µM) of cell death induced by cynaropicrin.50
Multiple myeloma AMO1 1.8 ± 0.2 Cynaropicrin induced parthanatos as a novel cell death mode in
JJN3 1.9 ± 0.4 AMO1 cells
OPM2 2.3 ± 0.5 It is generally known that mitochondria release death factors
whenever a cell is under stress [57]. Therefore, to evaluate if the
L363 2.6 ± 0.6 mitochondria underwent critical changes upon cynaropicrin
Normal leukocytes PBMCs- Donor 2 28.4 ± 3.8 treatment, we investigated the changes of the inner transmem-
PBMCs- Donor 3 24.9 ± 7.8 brane potential Δψm in cynaropicrin-treated AMO1 cells using JC-
PBMCs- Donor 4 22.0 ± 4.5 1 probe. Cynaropicrin treatment led to a significant dose-
dependent loss of the mitochondrial membrane potential.
Bortezomib used as a positive control absolutely induced
consideration that cynaropicrin induced G2M arrest after 24 h, we mitochondrial membrane depolarization (Fig. 7a).
investigated whether cynaropicrin inhibited the tubulin network. Our findings so far implicated that cynaropicrin-induced cell
U2OS cells stably expressing the α-tubulin-GFP fusion protein death was mitochondrial-linked, though caspase-independent.
were exposed to different concentrations of cynaropicrin (1.8 and Because apoptosis-inducing factor (AIF) triggers cell death in a
3.6 µM) for 24 h in an effort to examine the drug’s impact on the caspase-independent way and because it is one of the most
microtubule network. The tubulin network in control cells was eminent parthanatic biomarkers [58], we next investigated
properly polymerized. This was evident by the substantial amount parthanatos as a potential novel mode of cell death induced by
of tubulin that was dispersed throughout the cytoplasm and that cynaropicrin. One of the hallmarks of parthanatos is the over-
resulted in a robust intracellular network. On the other hand, activation of poly (ADP-ribose) polymerase 1 (PARP1) [58].
cynaropicrin treatment and vincristine treatment both led to a Therefore, we determined by Western blotting the level of PAR
disorganized tubulin network. In contrast to paclitaxel-treated polymer, which is produced if PARP-1 is activated [59]. Our data
cells where the microtubule network seemed stiff, cynaropicrin revealed that PAR polymer expression clearly increased in AMO1
and vincristine reduced the expansion of microtubules at the cells after being exposed to different cynaropicrin concentrations
edges and increased the bulk of tubulin around the nucleus. In (Fig. 7c). Next, we examined cynaropicrin-induced changes in
addition, if compared to untreated cells, cynaropicrin-treated cells PARP1 protein level. Western blotting results revealed different
had thinner microtubules at their extremities (Fig. 5a). Moreover, PARP1 isoforms. The full-length PARP1 (116 kDa) indicated PARP1
similar results were obtained in AMO1 cells exposed to 1.8 and activity, while the 89 kDa band denoted PARP1 cleavage [60].
3.6 µM of cynaropicrin. Cynaropicrin treatment distorted the Although a portion of PARP1 was cleaved following cynaropicrin
tubulin network of AMO1 cells if compared to control cells treatment, the expression of PARP1 increased if AMO1 cells were
(Fig. 5b). These findings collectively showed that cynaropicrin, treated with 0.9 and 1.8 µM of cynaropicrin, however, if treated
such as vincristine, inhibited the polymerization of the micro- with 3.6 µM of cynaropicrin the expression of PARP1 as well as c.
tubule network. PARP1 dropped (Fig. 7c).
Considering that DNA damage is a crucial initiator of
Cynaropicrin did not induce apoptosis or autophagy in AMO1 cells parthanatos and that PARP-1 is triggered in response to various
Cell cycle analyses revealed that the portion of cells in the sub kinds of DNA damage [61], we then tested the protein level of
G0G1 phase noticeably increased with increased cynaropicrin phospho-Histone H2AX (Ser139) known as γH2AX and as a
concentration, indicating that cynaropicrin induced cell death in marker of DNA double strand breaks. Western blot results
AMO1 cells. In an attempt to deliberate the mode of cell death showed that γH2AX obviously increased when AMO1 cells were
induced by cynaropicrin, we examined the programmed cell treated with 0.9 and 1.8 µM of cynaropicrin, however, if treated
death, apoptosis, as it is one of the probable mechanisms of action with 3.6 µM of cynaropicrin γH2AX expression diminished
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Fig. 2 Ingenuity Pathway Analysis (IPA) using Affymetrix Clariom S human chips normalized microarray-based gene expression data.
a Illustration of the top “biological functions” and “diseases”. b Canonical pathways. A cut-off threshold (–log (P value)) of 1.3 is considered as a
default parameter. Only significant biological functions and diseases as well as canonical pathways with P < 0.05 are illustrated. c Prediction of
a functional network by IPA of cynaropicrin-treated AMO1 cells. The red circles emphasize the downregulation of c-MYC, STAT3, and MAPKs. In
addition, PI3K and TUBA appeared to be strongly involved within this pathway disturbed by cynaropicrin. Relative changes in gene expression
profiles are displayed by a color-coding system: red represents upregulated genes and green depicts downregulated genes. d The expression
levels of AKT, STAT3, c-MYC, and MAP2K2 upon treatment of AMO1, L363, NCI-H929, and OPM2 with the corresponding IC50 value of
cynaropicrin were determined by qRT-PCR. The X-axis represents the previously mentioned genes; however, the Y-axis shows the fold change.
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most sensitive cell line. Hence, we examined the mechanisms of
Table 4. Upstream regulators impaired by cynaropicrin. action of cynaropicrin in AMO1 cells. CEM/ADR5000 cells over-
Top Upstream Regulators P value expressing the ABC-transporter P-glycoprotein are known to efflux
chemotherapeutic drugs out of the cell, leading to chemotherapy
STAT3 1.96 × 10−3 failure [64]. Our data showed that CEM/ADR5000 cells exhibited
MED1 1.37 × 10−2 comparable sensitivity to cynaropicrin as their sensitive sublines.
MKNK1 1.84 × 10−2 Additionally, the resistance ratio was only 0.9, which is consider-
−2 ably lower than that of doxorubicin, a standard chemotherapeuticSLC2A4 1.84 × 10 drug with a degree of cross-resistance significantly higher than
ATF4 1.84 × 10−2 1000 [65]. These findings implied that P-glycoprotein does not
intervene in resistance to cynaropicrin. The doses of cynaropicrin
needed to inhibit the growth of normal leukocytes by 50%
(Fig. 7c), indicating that cynaropicrin prompted DNA damage. (22.0 ± 4.5 µM ≤ IC50 ≤ 39.3 ± 1.4 µM) were much greater than in all
Subcellular protein fractionation revealed that cynaropicrin examined leukemia and MM cell lines. This implies that the doses
treatment led to a dose-dependent substantial decrease in of cynaropicrin required to inhibit the proliferation of leukemia
cytoplasmic AIF, which was accompanied by a significant and MM cells could be attained without damaging healthy cells.
increase in nuclear AIF (Fig. 7e). Confocal microscopy further Microarray gene expression profiles were applied to evaluate
validated that AIF translocated from the cytoplasm into the the effects of cynaropicrin on AMO1 transcriptional activity.
nucleus following cynaropicrin treatment (Fig. 7d). The pharma- Various differentially expressed genes were identified in cynar-
cological inhibition of PARP by PJ34 restored the cell viability of opicrin treated cells. IPA elucidated a network where c-MYC,
AMO1 but not to 100% due to the cytotoxic activity of PJ34 STAT3, MAP2K1/2, MAP2K2, and MAP4K1 were downregulated.
(Fig. 7b). All in all, these findings demonstrated that cynaropicrin Furthermore, the PI3K complex and TUBA were targeted by
triggered DNA damage, leading to the hyperactivation of PARP1 cynaropicrin. Remarkably, several diseases and canonical path-
and the accumulation of PAR polymer in the cytoplasm, which in ways were affected by cynaropicrin, especially hematological
turn caused mitochondrial depolarization and AIF nuclear diseases and c-Myc-mediated apoptosis signaling. Upstream target
translocation. analyses proposed STAT3 as potential upstream target of
cynaropicrin. STAT3 is a transcription factor known to be basically
Anticancer activity of cynaropicrin in vivo activated in several human cancer cells as well as hematological
The antitumor activity of cynaropicrin has been investigated tumors. Another significant signal transduction pathway in B-cell
in vivo using golden hamsters or murine models [29]. In our study, neoplasms involved AKT and PI3K. In fact, aberrant activation of
a CCRF-CEM xenograft tumor model was developed in larvae PI3K pathway prevailed in hematological diseases. Previous
zebrafish and fluorescence intensity was assessed 24 h after studies demonstrated the critical roles of STAT3 and PI3K in
treatment to determine the inhibition rate of the tumor. The survival, growth, and chemotherapy resistance of plasma-cells and
positive control cis-platinum effectively repressed the fluores- B-cells neoplasms [66]. Yet, MAPK-pathway was also among the
cence intensity of CCRF-CEM tumor if compared to the untreated prominent upregulated signaling pathways in MM [67].
control group (Fig. 8). Similarly, cynaropicrin inhibited CCRF-CEM C-Myc is a transcription factor known to play a crucial role in
tumor growth with an inhibitory rate of 33.7% at low concentra- several cellular processes as transcription, translation, prolifera-
tion (5 µM), and this inhibitory rate increased in a dose-dependent tion, apoptosis, and metabolism [68]. It was firstly discovered as
manner to reach 41.01% at higher concentration (10 µM). Our the homolog of an avian retrovirus and as being upregulated in
findings revealed that cynaropicrin exhibited an anti-tumor Burkitt’s Lymphoma [69–71]. C-Myc controls the progression of
activity in a larvae zebrafish model. the cell cycle, therefore the inhibition of c-Myc led to cell cycle
arrest and impairment of cell cycle progression in human myeloid
and lymphoid cells [72]. C-Myc deregulation was associated with
DISCUSSION MM, therefore any therapeutic strategies targeting it would be
Despite recent improvements in medical science that quadrupled valuable in treating B-cells malignancy. However, therapies
the survival rate of MM patients over the past 40 years, this against c-Myc were and, by some means, are still challenging
disease is practically uncurable due to the outgrowth of resistant for the absence of a nuclear localization, a defined 3-D structure,
subclones, upon receiving the first-line treatment [62]. Therefore, and an enzymatic pocket. Despite these drawbacks, many studies
researchers are nowadays looking for alternative therapies to showed the potential impact of c-Myc inhibition. C-Myc is a
overcome resistance. Herein, natural products provided a variety downstream target of the MAPK, STAT3, and PI3K pathways.
of compounds that were applied in pharmacology and medicine Therefore, it is foreseen as the master integrator and regulator of
for their promising impact on the reduction and the suppression signaling networks associated with cancer [67]. Based on
of tumor development [63]. Cynaropicrin, the major ingredient of microarray analyses performed here, we assumed that cynar-
the edible parts of the artichoke plant, was applied as a safe diet opicrin exposure inhibited STAT3/AKT/ERK signaling and c-Myc,
for ages. Currently, the noteworthy anticancer activity of engendering the cynaropicrin anti-proliferative activities in MM.
cynaropicrin captivated the attention of several researchers based This assumption was validated on the RNA level through RT-qPCR,
on two important characteristics: its tiny size and water solubility on the protein level through immunoblotting, and by performing
which increase its membrane permeability, rendering therapeutic a c-Myc reporter assay. RT-qPCR revealed that c-MYC was
injections easy to formulate [29]. Cynaropicrin possessed a strong downregulated upon treatment with cynaropicrin. Moreover,
anti-proliferative activity against leukocyte cancer cells [36], and cynaropicrin remarkably inhibited c-Myc expression and exerted
chronic myeloid leukemia cells [63]. Yet so far, no studies have a strong inhibition of c-Myc DNA binding activity. Interestingly,
been conducted on MM. Hence, the aim of the present work was this inhibitory effect was much more significant than that of the
to investigate the cytotoxicity and the mode of action of known c-Myc inhibitor 10058-F4. Furthermore, the concentra-
cynaropicrin in MM in vitro and in vivo. tions of cynaropicrin needed to inhibit c-Myc were extremely
Therefore, we examined a panel of 9 MM cell lines in addition to lower than that of 10058-F4. To the best of our knowledge, we are
the multidrug-resistant CEM/ADR5000 T-ALL cells and their the first to report that cynaropicrin devastated MM cells by
sensitive counterpart, CCRF-CEM T-ALL cells. All cell lines were inhibiting c-Myc, implying a novel mode of action of cynaropicrin
impeccably sensitive towards cynaropicrin, with AMO1 being the as an anti-tumor drug.
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Fig. 3 Cynaropicrin affected p-AKT, AKT, p-ERK1/2, ERK1/2, and c-Myc expression as well as c-Myc transcriptional activity. a Effect of
cynaropicrin at different concentrations on the protein expression levels of p-AKT, AKT, p-ERK1/2, ERK1/2, and STAT3 in AMO1 cells as detected
by Western blotting. b Effect of cynaropicrin on the protein expression levels of c-Myc in AMO1 and HEK293 cells as detected by Western
blotting. The bar diagrams were obtained by calculating the mean value ± SD of three experiments performed at different time points. c Effect
of cynaropicrin and the known c-Myc inhibitor 10058-F4 on c-Myc transcriptional activity as percentage of the normalized HEK293 cells
transiently transfected with c-Myc luciferase reporter construct. The bar diagram was obtained by calculating the mean value ± SD of two
independent experiments with three replicates each. *P < 0.05, **P < 0.01, ***P < 0.001 if compared to control sample.
With an eye toward investigating how cynaropicrin inhibited cynaropicrin exerted significant inhibitory effects on STAT3, AKT,
c-Myc expression as well as activity, we checked the effect of and ERK pathways. The fact that the basal level of AKT was
cynaropicrin on upstream regulators of c-Myc. A previous study downregulated might imply that cynaropicrin affects its synthesis
revealed that cynaropicrin decreased various cellular malignant rate at any point of transcription and translation. STAT3, AKT, and
characteristics in melanoma through the suppression of ERK1/2 ERK pathways play a major role in controlling the stability, the
and NF-κB activity [31]. Butturini et al. also described that accumulation, as well as the transcriptional activity of c-Myc [75].
cynaropicrin regulated STAT3 function [73]. Moreover, cynaropi- Consequently, the inhibition of STAT3, PI3K, and MAPK pathways
crin exhibited anti-proliferative and apoptotic activities in lung might downregulate c-Myc. Our data revealed that reduced c-Myc
cancer cells through the inactivation of the EGFR/AKT signaling expression upon treatment with cynaropicrin was strongly
pathway [74]. Our data were in line with those findings as associated with the inhibition of STAT3, AKT, and ERK1/2. These
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Fig. 4 Cell cycle disturbance of AMO1 cells by cynaropicrin. DNA histograms of AMO1 cells subjected to several cynaropicrin concentrations
for 24 h (a), 48 h (b), and 72 h (c). These histograms are generated by flow cytometry using the blue laser (λ/nm= 488) for excitation and by
measuring the emission at λ/ nm =530. Bar diagrams representing the distribution of cynaropicrin-treated AMO1 cells in the different cell
cycle phases after 24 h (d), 48 h (e), and 72 h (f). The percentage of cells in each phase is obtained by calculating the mean value ± SD of three
experiments performed at different time points. *P < 0.05, **P < 0.01 if compared to the control sample.
Fig. 5 Disorganization of the microtubule distribution upon treatment with cynaropicrin. a U2OS cells stably express GFP-α-tubulin
protein. Micrographs of U2OS cells fixed with 4% paraformaldehyde were photographed 24 h post-treatment with DMSO, 1.8 µM and 3.6 µM
of cynaropicrin, 1 µM vincristine, or 1 µM paclitaxel. Cells nuclei were stained with DAPI (blue). The peripheral microtubule masses are
represented by the white arrows. Images were taken with an AF7000 widefield fluorescence microscope at 40 × magnification (scale bars =
10 µm). b The living AMO1 cells were stained with Tubulin Tracker™ Deep Red. Micrographs of AMO1 cells were photographed 24 h post-
treatment with DMSO, 1.8 µM and 3.6 µM of cynaropicrin. Cells nuclei were stained with Hoechst 33342 Nuclear Stain (blue). Images were
taken with an AF7000 widefield fluorescence microscope at 40 × magnification (scale bars = 7 µm).
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Fig. 6 Evaluation of apoptosis and autophagy in AMO1 cells treated with different cynaropicrin concentrations (0.9, 1.8, and 3.6 µM) or
DMSO for 48 h. a Cynaropicrin did not induce apoptosis in AMO1 cells. Apoptotic cells were measured using the Violet Ratiometric
Membrane Asymmetry Probe/Dead Cell Apoptotic kit. Q1 depicts dead cells, Q3 illustrates living cells, and Q4 displays apoptotic cells. The
numbers under Q1, Q3, and Q4 represent the percentage of cells. b Cynaropicrin prevented the cleavage of the executioner caspases (caspase
3 and caspase 7) as detected by Western blot. c Cynaropicrin did not affect the protein expression levels of the autophagy marker Beclin 1 and
P62 as detected by Western blot. The bar diagram representing the protein expression level of Beclin 1 and P62 was obtained by calculating
the mean value ± SD of three experiments performed at different time points. d Caspase inhibition by z-vad-fmk did not significantly alter the
sensitivity of AMO1 to cynaropicrin. e The inhibition of autophagy by bafilomycin A1, as well as the induction of autophagy by rapamycin did
not significantly alter the sensitivity of AMO1 to cynaropicrin.
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Fig. 7 Investigation of parthanatos as a novel cell death in AMO1 cells treated with different cynaropicrin concentrations (0.9, 1.8, and
3.6 µM) or DMSO for 48 h. a Analysis of the mitochondrial membrane potential. The bar diagram represents the level of mitochondrial
membrane potential (ΔΨm) which was discerned by JC-1-staining and measured by calculating the ratio of J-aggregates to J-monomers
fluorescence intensity. b PARP inhibition by PJ34 increased AMO1 cell viability in presence of cynaropicrin. c Effect of cynaropicrin on the
expression levels of several proteins involved in the parthanatic cell death as detected by Western blot. d Confocal microscopy images of
apoptosis inducing factor (AIF) immunofluorescence showing the translocation of AIF from the cytoplasm to the nucleus in AMO1 cells
treated with cynaropicrin for 48 h. The arrows highlight the release of AIF into the cytoplasm. Images were taken at 50 × magnification (scale
bars = 20 µm). e Effect of cynaropicrin on the expression level of AIF in the cytoplasm and the nucleus as detected by Western blot. Bar
diagrams were obtained by calculating the mean value ± SD of three experiments performed at different time points. *P < 0.05, **P < 0.01,
***P < 0.001 if compared to control sample.
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Fig. 8 Acute cytotoxicity examination (fluorescence intensity and inhibition rate) of cynaropicrin in CCRF-CEM xenograft tumor zebrafish
model (n= 5 zebrafish). The red fluorescence highlights CCRF-CEM tumor mass. Images were taken at 60× magnification (scale bars =
100 µm). *P < 0.05, **P < 0.01, ***P < 0.001 if compared to the model.
findings further validated the key role of those pathways in c-Myc organization was aberrant in both cell lines. Similarly to vincristine,
regulation. cynaropicrin reduced the density of microtubules at the edges
We also investigated the expression of c-Myc in HEK293 cells to and increased the mass of tubulin around the nucleus. Moreover,
confirm that cynaropicrin reduced c-Myc transcriptional activity. cynaropicrin-treated cells had thinner microtubules at their
As predicted, a considerable downregulation of c-Myc was extremities, if compared to untreated cells. Mastimoto et al.
observed. recently identified tubulin as a binding protein of cynaropicrin.
Afterward, cell cycle arrest was examined by analyzing the However, we demonstrated in this study for the first time that
distribution of cynaropicrin-treated AMO1 cells through the sub- cynaropicrin disassembled the microtubule network, and this
G0G1, G0G1, S, and G2M phases, using flow cytometry, after 24 h, could be an explanation of its cytotoxicity [79]. Transfected U2OS
48 h, and 72 h incubation. Cells were assembled in the G2M phase human cells were used as a model to detect the effect of
24 h after cynaropicrin treatment. Thus, mediators of the G2M cynaropicrin on tubulin network. Adherent cells are known to
phase may be affected by cynaropicrin. Therefore, we concen- have a well polymerized tubulin network, however, MM are small
trated on examining cynaropicrin’s effect on the microtubule suspension cells with a very tiny tubulin network, rendering it hard
cytoskeleton. Our results were in line with previous studies to visualize and making U2OS cells a much better model to
demonstrating that cynaropicrin promoted G2M arrest in lung observe effects on microtubules.
epithelial A549 cancer cells, in human anaplastic thyroid cancer, Sesquiterpene lactones are known to be potent inducers of
and in breast MDA-MB-231 cancer cells [32, 33, 76]. The cytotoxicity by initiating apoptosis [36]. Therefore, we investigated
inappropriate entry of cells in the mitotic process is known as whether cynaropicrin-induced cytotoxicity in AMO1 cells is related
mitotic catastrophe, a process that recognizes mitotic failure and to apoptosis. Our results indicated that cynaropicrin did not
reacts by sending a cell into an irrevocable antiproliferative death induce apoptosis as evident by the absence of changes in cell
[77]. This could assist in understanding the increase in the sub- membrane asymmetry and cell permeability in cynaropicrin trea-
G0G1 phase with increased cynaropicrin concentration after 24 h, ted AMO1 cells compared to untreated cells after 48 h incubation,
48 h, and 72 h treatment. Microtubules are extremely active the absence of activated caspase 3 and caspase 7 that are known
cytoskeletal elements that are crucial for a variety of cellular as apoptotic markers, and the unaffected sensitivity of AMO1 to
processes, including cell division, vesicle transport, and intracel- cynaropicrin upon caspase inhibition. These results suggested that
lular structure. Microtubule targeting agents suppress the dynamic cynaropicrin caused cell death in a caspase-independent manner.
of the spindle microtubules, which causes mitosis to slow down or We next addressed whether cynaropicrin-induced cytotoxicity is
stop. This block in the G2/M phase results in cell death [78]. linked to autophagy. The fact that the expression of Beclin1 and
Thereafter, we studied the impact of cynaropicrin on the P62, two autophagy markers, did not change after treatment and
microtubule cytoskeleton through confocal microscopy of U2OS the combination of cynaropicrin with either bafilomycin A1 or
cells stably expressing α-tubulin-GFP and by visualizing the rapamycin did not significantly alter the sensitivity of AMO1 to
stained tubulin network of AMO1 cells. Indeed, the microtubule cynaropicrin refuted this assumption as well. Unexpectedly, our
Acta Pharmacologica Sinica (2023) 0:1 – 17
Induction of parthanatos by cynaropicrin
JC Boulos et al.
15
findings were contradictory to previously published research that Several researchers confirmed the antitumor efficacy of cynar-
suggested that cynaropicrin-induced cytotoxicity is associated to opicrin in vivo using different animal models [35, 88]. Recently, the
either apoptosis or autophagy [76, 80, 81]. In an attempt to zebrafish model turned out to be a suitable in vivo model for drug
decipher the mode of cell death induced by cynaropicrin in more discovery as well as toxicity assessment [67, 89]. In the present
detail, we studied the effect of cynaropicrin on the mitochondrial study, we thus proved that cynaropicrin reduced tumor growth
membrane potential as mitochondria are the cellular powerhouse in vivo using a CCRF-CEM xenograft tumor zebrafish model.
and important regulators of cell death [82]. Our results indicated In summary, the insights acquired in this investigation suggest
that cynaropicrin treatment indeed resulted in a significant loss of that cynaropicrin is a natural product that effectively reduces
the mitochondrial membrane potential. Thus far, our findings tumor growth in zebrafish via the novel parthanatic cell death.
suggested that cynaropicrin-induced cell death was non-apopto- Cynaropicrin also causes potential cytotoxicity in vitro by
tic, mitochondrial-linked, though caspase independent. Given that inhibiting c-Myc, and subsequently STAT3, AKT, and ERK1/2, and
AIF is a potential caspase-independent cell death effector and by suppressing the tubulin network. Our findings revealed that
since it is one of the most prominent parthanatic biomarkers [58], the potential therapeutic value of cynaropicrin is the outgrowth of
we further focused on parthanatos as a potential novel cell death parthanatos, a novel mode of cell death.
modality induced by cynaropicrin. During parthanatos, excessive
DNA damage induces extensive PAR polymer production by
PARP1 hyperactivation. The translocation of PAR into the ACKNOWLEDGEMENTS
cytoplasm alters the activity and the location of cytoplasmic We gratefully acknowledge the stipend of the Sibylle Kalkhof-Rose-Foundation to
proteins, resulting in the translocation of AIF from the mitochon- JCB. Furthermore, we thank the Microarray Unit of the Genomics and Proteomics
dria to the nucleus and subsequently to cell death [58, 59]. DNA Core Facility, German Cancer Research Center (DKFZ), for providing excellent
damage is a crucial initiator of parthanatos. Therefore, we tested expression profiling services. This research was funded by a private donation of Marc
the level of marker protein γH2AX by Western blotting. Our results Strobel, Frankfurt, Germany, and by the Deutsche Krebshilfe (process number:
70112693 to EL)
showed that γH2AX clearly increased if AMO1 cells were treated
with cynaropicrin, indicating that cynaropicrin initiated DNA
damage. As DNA damage is a cognitive factor that induces AUTHOR CONTRIBUTIONS
PARP-1 activation, we consequently assumed that cynaropicrin- JCB: designed research, performed research, analyzed data, and wrote the paper;
induced DNA damage was implicated in the parthanatic cell EAO: performed research; DR: performed research; CF: performed research; MC:
death. In order to attract DNA repair machinery to DNA defects, contributed new reagents or analytic tools; EL: contributed new reagents or analytic
the nuclear protein PARP1, activated by adhering to DNA lesions, tools; SMK: performed research; LTS: performed research; TE: designed research. All
catalyzes the poly(ADP-ribosylation) (PARylation) of nuclear authors have read and agreed to the published version of the manuscript.
acceptor proteins, along with PARP1 itself [59]. PARP1 activation
was detected by the increase in its expression upon treatment
with cynaropicrin. However, at high concentration (3.6 µM) the FUNDING
expression of PARP1 as well as cleaved PARP1 dropped off. This Open Access funding enabled and organized by Projekt DEAL.
drop is accompanied by an increase in PAR polymer. The decrease
in PARP1 expression could be explained by the fact that PARP
enzymes regulate the function of AIF by adding ADP-ribose ADDITIONAL INFORMATION
polymers (PAR) to it while utilizing NAD+ as a substrate [83, 84]. Supplementary information The online version contains supplementary material
The binding of PAR to AIF was essential for AIF translocation from available at https://doi.org/10.1038/s41401-023-01117-3.
the mitochondria to the nucleus, and PAR binding was crucial for Competing interests: The authors declare no competing interests.
AIF to promote parthanatos [85]. Surprisingly, a partial cleavage of
PARP1 was observed, although caspase 3 was not activated. It has
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