Methoxyphenyl chalcone sensitizes aggressive epithelial cancer to cisplatin through apoptosis induction and cancer stem cell eradication

Article abstract of DOI:10.1177/1010428317691689

Current standard chemotherapy for late stage ovarian cancer is found unsuccessful due to relapse after completing the regimens. After completing platinum-based chemotherapy, 70% of patients develop relapse and resistance. Recent evidence proves ovarian ca

Full text of DOI:10.1177/1010428317691689

691689TUB0010.1177/1010428317691689Tumor BiologySu et al.
research-article2017
Original Article
Tumor Biology
May 2017: 1–12
Methoxyphenyl chalcone sensitizes
aggressive epithelial cancer to cisplatin
through apoptosis induction and cancer
stem cell eradication
© The Author(s) 2017
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httpDs:O//dIo:i1.o0r.g1/1107.171/1770/110041024823813716769911668899
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Yu-kai Su1,2*, Wen-Chien Huang3,4,5*, Wei-Hwa Lee6,
Oluwaseun Adebayo Bamodu7,8, Muhammad Ary Zucha7,8,
Indwiani Astuti9, Heri Suwito10, Chi-Tai Yeh7,8 and
Chien-Min Lin1,2
Abstract
Current standard chemotherapy for late stage ovarian cancer is found unsuccessful due to relapse after completing
the regimens. After completing platinum-based chemotherapy, 70% of patients develop relapse and resistance. Recent
evidence proves ovarian cancer stem cells as the source of resistance. Therefore, treatment strategy to target both
cancer stem cells and normal stem cells is essential. In this study, we developed a novel chalcone derivative as novel
drug candidate for ovarian cancer treatment. We found that methoxyphenyl chalcone was effective to eliminate ovarian
cancer cells when given either as monotherapy or in combination with cisplatin. We found that cell viability of ovarian
cancer cells was decreased through apoptosis induction. Dephosphorylation of Bcl2-associated agonist of cell death
protein was increased after methoxyphenyl chalcone treatment that led to activation of caspases. Interestingly, this
drug also worked as a G2/M checkpoint modulator with alternative ways of DNA damage signal–evoking potential that
might work to increase response after cisplatin treatment. In addition, methoxyphenyl chalcone was able to suppress
autophagic flux and stemness regulator in ovarian spheroids that decreased their survival. Therefore, combination of
methoxyphenyl chalcone and cisplatin showed synergistic effects. Taken together, we believe that our novel compound
is a promising novel therapeutic agent for effective clinical treatment of ovarian cancer.
Keywords
Ovarian cancer stem cells, chemo resistance, phytochemical, methoxyphenyl chalcone, apoptosis, tumorspheres,
autophagy
Date received: 14 October 2016; accepted: 23 December 2016
9Department of Chemistry, Faculty of Science and Technology,
Airlangga University, Surabaya, Indonesia
1Department of Neurology, School of Medicine, College of Medicine,
Taipei Medical University, Taipei, Taiwan
2Division of Neurosurgery, Department of Surgery, Shuang Ho
Hospital, Taipei Medical University, Taipei, Taiwan
3Department of Medicine, Mackay Medical College, Taipei, Taiwan
4Department of Thoracic Surgery, Mackay Memorial Hospital, Taipei,
Taiwan
5Mackay Junior College of Medicine, Nursing, and Management, Taipei,
Taiwan
6Department of Pathology, Shuang Ho Hospital, Taipei Medical
University, Taipei, Taiwan
7Division of Hematology and Oncology, Cancer Center, Shuang Ho
Hospital, Taipei Medical University, Taipei, Taiwan
8Department of Medical Research and Education, Shuang Ho Hospital,
Taipei Medical University, Taipei, Taiwan
10Department of Pharmacology and Clinical Pharmacy, Universitas
Gadjah Mada, Yogyakarta, Indonesia
*These authors contributed equally to this work.
Corresponding authors:
Chien-Min Lin, Division of Neurosurgery, Department of Surgery,
Shuang Ho Hospital, Taipei Medical University, Taipei 23561, Taiwan.
Email: m513092004@s.tmu.edu.tw
Chi-Tai Yeh, Department of Medical Research and Education, Shuang
Ho Hospital, Taipei Medical University, Taipei 23561, Taiwan.
Email: ctyeh@s.tmu.edu.tw
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use, reproduction and distribution of the work without further permission provided the original work is attributed as specified on the SAGE and
Open Access pages (https://us.sagepub.com/en-us/nam/open-access-at-sage).

2
Tumor Biology
Phytochemical chalcone is a bioflavonoid that can be
found in vegetables and fruits. Chalcone-based derivatives
have been yearly investigated to treat several diseases,
including inflammatory diseases and cancer. Cytotoxicity
of chalcone derivative was reported against several cancer
types, including leukemia, lung, breast, colon, and ovary.9
Chalcone is able to upregulate tumor suppressors through
inhibition of deubiquitination enzymes.10 In addition, cell
cycle checkpoint regulators are also downregulated by
chalcone to inhibit cell cycle progression.11 Here, we
developed a novel chalcone-based derivative, (E)-1-4(4-
aminophenyl)-3-(2,4-methoxyphenyl)prop-2-en-1-one or
methoxyphenyl chalcone (MTC) as drug candidate in
ovarian cancer. In this study, we explored the cytotoxic
activity of the compound on ovarian cancer cells and try to
overcome resistance in the cisplatin-resistant cells. We
found that ovarian cancer cell lines that were originated
from clear cell carcinoma and serous adenocarcinoma type
were responsive to MTC in a concentration-dependent
manner. MTC was able to suppress cell cycle progression
in G2/M checkpoint and induce cellular death. The process
might be mediated by dephosphorylation of BAD. More
importantly, MTC was able to suppress autophagy-related
protein 5 (Atg-5) that is important for autophagosome
elongation. Inhibition of autophagy in ovarian spheroids
successfully decreased their survival. Therefore, applica-
tion of MTC had beneficial effects in combination with
cisplatin. Taken together, we believe that our novel com-
pound is a promising novel drug candidate to treat ovarian
cancer, as well as to be an adjunct drug to improve treat-
ment outcome in cisplatin treatment.
Introduction
Ovarian cancer is usually diagnosed in late stage and is
one of the most significant cause of death from gyneco-
logic malignancy in the world. From the latest global can-
cer statistics, it is estimated that 225,000 female were
affected with 140,200 mortality rate annually.1 The cur-
rently available chemotherapy following cytoreductive
surgery gives valuable response in few months, but soon
within the first year of completing the regimen of plati-
num/Taxol, about 75% of patients were relapse cases.2
Therefore, development of a novel drug candidate for late
stage ovarian cancer is demanding. In addition, a better
treatment strategy to improve responsiveness against plat-
inum-based therapy is also very important.
Mode of action of platinum-based therapy is mediated
by interaction with DNAto form DNAadducts. Intrastrand
DNA crosslink causes activation of several pathways that
lead to DNA synthesis inhibition, cell cycle attenuation,
and cellular death. In molecular basis, DNA adduct acti-
vates DNA damage signals, including activation of p53,
p73, mitogen-activated protein kinase (MAPK), and
ATR.3 However, activation of those signaling can be
attenuated by several events that cause cisplatin resist-
ance. In late stage ovarian cancer, patients initially
respond well to platinum-based therapy up to 70%; how-
ever, they soon gain resistance that causes 5-year survival
rate below 20%.2 Therefore, alternative ways to evoke
DNA damage signal or any independent signals that pro-
mote cellular death are needed to improve responsiveness
against cisplatin. From a genome-wide study, a number of
genes were activated along with phosphorylation of Bcl2-
associated agonist of cell death (BAD) protein that may
correlate with acquired resistance of ovarian cancer
against platinum. As a result, BAD phosphorylation pre-
vents cisplatin-induced apoptosis. Patients with higher
level of phosphorylated-BAD (p-BAD) had inferior over-
all survival rate than patients with lower p-BAD.4 Thus,
dephosphorylation or inhibition of BAD could resolve
cisplatin resistance in ovarian cancer.5 In addition, recent
evidence supports the role of cancer stem cells (CSCs) as
the tumor initiator and also the source of relapse after
chemotherapy treatment in various tumors.6,7 CSCs have
higher autophagic flux that maintains the cells for high
metabolic demands and also serves as pro-survival mech-
anism. In bladder CSCs, inhibition of high autophagy flux
through small interfering RNA (siRNA) and pharmaco-
logic treatment successfully decreased the cell survival
and improved the response to chemotherapeutic drugs.8 In
ovarian cancer, ovarian CSCs are enriched in ovarian
spheroids that are commonly found in ascites of late stage
patients. Ovarian spheroids play putative roles in progres-
sion, metastasis, and drug resistance.6 Therefore,
autophagy in ovarian CSCs may also be another promis-
ing target of therapy.
Materials and methods
Compound synthesis
The mixture of 4′-aminoacetophenon (6mmol) and
2,4-dimethoxybenzaldehyde (6mmol) was dissolved in
30mL ethanol, and then 6mL of NaOH 40% solution was
added dropwise, while the temperature was kept under
10°C. The reaction mixture was stirred under this condi-
tion for 1h. Stirring was continued at room temperature for
4h. Thereafter, the reaction mixture was poured into ice-
water. The precipitated solid was filtered off and recrystal-
lized on aqueous ethanol. Purity of the compound was
clarified with melting point analysis, and the molecular
structure was confirmed with infrared (IR), nuclear mag-
netic resonance (NMR), and mass spectrometry (Bruker
Daltonik GmbH, Germany).
Cell culture
Human ovarian cancer cell lines (ES-2 and Hey-A8) were
received from American Type Culture Collection (ATCC,
USA). Cells were cultured in McCoy’s 5Amedium (Gibco,

Su et al.
3
USA) supplemented with 1% antibiotic/antimycotic
(#15140; Gibco) and 10% fetal bovine serum (FBS;
Sigma-Aldrich, USA). Cells were maintained in a standard
incubator (Shel Lab; Sheldon Manufacturing, USA) at
37°C and 5% CO2.
Immunofluorescence staining
For adherent cells, cells were seeded on 24-well plates
with a coverslip on the bottom of each well. After treat-
ment, cells were washed twice with PBS, fixed with 4%
formaldehyde, and probed with primary antibody (cleaved-
poly(ADP-ribose) polymerase (PARP), p21/Clip1, or
c-Myc) at 4°C for overnight. Fluorophore-conjugated anti-
body (Alexa Fluor; Life Technologies, USA) was used to
track the in situ interaction of protein–primary antibody.
Double-stranded DNA staining (4′,6-diamidino-2-phe-
nylindole (DAPI); Invitrogen, USA) was used as nuclear
staining. The fluorescence signal was captured under con-
focal microscopy (Nikon, Japan).
Spheroids formation
Ovarian spheroids were developed from ES-2 cells fol-
lowing the previously described protocol.12 Parental cells,
as much as 105, were seeded on non-attached flask with
stem cell medium (Nutristem-XF; Biological Industries,
Israel) that contains suitable growth factors for CSC
enrichment at 37°C in 5% CO2 incubator for 2days when
the spheroids were clearly visible under microscope.
Non–stem cells died due to starvation while CSCs kept
alive and compacted to form spheroids. In order to get
higher purity of CSCs, at least three passages were done
prior to studies.
Apoptosis analysis
Annexin V–propidium iodide (PI) double staining was
performed with flow cytometry to quantify the percentage
of apoptotic cells in each sample. Cells (106 cells for each
sample) were treated with Annexin V and PI at room tem-
perature for 15min in the dark. The samples were immedi-
ately analyzed using flow cytometry (LSRFortessa; BD
Biosciences, USA).
Cell viability assay
Cell viability for adherent cells against drugs was quanti-
fied with sulforhodamine B (SRB; Sigma-Aldrich). Cells
were seeded on 96-well plates (at a density of 3500 cells/
well) in 100µL of McCoy’s 5A medium and left to attach
for 24h. Cells were treated with cisplatin (Abiplatin injec-
tion; Pharmachemie BV, The Netherlands) or MTC. Cells
were washed with phosphate-buffered saline (PBS) and
then fixed with 10% cold trichloroacetic acid for 1h prior
to incubation with SRB for 1h at room temperature.
Attached SRB dye on the viable cells was dissolved in
weak base (20mM Tris base) and read under an absorb-
ance of 595nm.
GFP-LC3 transfection
GFP-LC3 was transfected to ES-2 cells to study the
autophagosome formation and function in ovarian cancer,
especially in response to MTC treatment. Two millions of
ES-2 cells were mixed with 10µM GFP-LC3, and trans-
fection was performed with the electroporation technique
following the manufacturer’s protocol (Invitrogen).
Cell cycle analysis
Cell cycle was analyzed with the properties of PI that is
able to analyze cellular DNA content. By this method,
three major cellular cycle phases can be detected, includ-
ing G1, S, and G2/M. Samples (105 cells per sample) were
pretreated with PI for 30min prior to analysis with flow
cytometry (LSRFortessa; BD Biosciences).
Western blot
Cells were washed with PBS twice and then lysed with ice-
cold lysis buffer (radioimmunoprecipitation assay (RIPA)
buffer; R0278; Sigma-Aldrich, USA). The protein lysate
was collected after centrifugation to remove cellular debris
on the pellet. The protein concentration then was quantified
using bicinchoninic acid (BCA) assay kit (Pierce, Thermo
Scientific, USA). The equal amount of protein lysate from
each sample was run on 10% sodium dodecyl sulfate poly-
acrylamide gel electrophoresis (SDS-PAGE), followed by
protein transfer to polyvinylidene difluoride (PVDF) mem-
brane. Primary antibodies originated from mouse or rabbit
were used to probe the protein level from each well (the list
of antibodies was presented in Supplemental Table 1). After
overnight probing, the interaction of protein–primary anti-
body was identified using horseradish peroxidase (HRP)-
linked secondary antibody. The signal was detected using
UVP imaging system (UVP, USA).
Trans-well migration assay
ES-2 cells were resuspended in serum-free McCoy’s 5A
medium and placed in trans-well chamber with or without
MTC administration. After 18h, migrated cells were fixed
and then stained with Giemsa.
Combination analysis
Combination of cisplatin and MTC was analyzed using
Chou-Talalay algorithm of multi-drug combination.
CompuSyn software (ComboSyn Inc., USA) was used

4
Tumor Biology
Figure 1. Chemical structure and molecular weight of chalcone derivative MTC. (a) The molecular mass and formula of
chalcone derivative was assessed with mass spectrometry. Calculated molecular mass was 284.1287 with formula of C17H18NO3.
(b) Molecular structure of (E)-1-4(4-aminophenyl)-3-(2,4-methoxyphenyl)prop-2-en-1-one (MTC) was presented.
following the guideline of two drug combination analysis.
Combination index (CI) was used to determine whether
both drugs have synergistic, additive, or antagonistic effects.
high-grade clear cell carcinoma. This subtype was men-
tioned as one of the poor prognosis predictors because the
patients have poorer outcome and high resistance to estab-
lished platinum-based chemotherapy.13 In this study, we
also used the most prevalent subtype, Hey-A8, that origi-
nated from late stage serous cystadenocarcinoma. Serous
type adenocarcinoma is the most common type of ovarian
cancer14; thus, cytotoxic activity against this cell type gives
opportunity to be translated into a majority of patients.
Statistical analysis
Statistical analysis was performed using GraphPad Prism
statistical tools (GraphPad Software, Inc., USA). In the
descriptive analysis, measures of central tendency (mean
and median) and dispersion (standard deviation) were
determined. Level of confidence was set at 95%.
Apoptosis induction by MTC is mediated by
activation of DNA damage signals through
p-BAD dephosphorylation
Results
Photomicrographs were taken after 48h of treatment with
MTC (Figure 3(a)). Then, cells were fixed with 4% formal-
dehyde and probed with antibody against cleaved-PARP
for immunofluorescent staining. Since PARP is the major
substrate of caspase, cleaved-PARP accumulation could
indicate higher activity of caspase. In order to quantify the
proportion of apoptotic cells in each sample, Annexin V–PI
double staining was performed with flow cytometry.
Anticoagulant proteinAnnexin V could bind exposed phos-
phatidylserine that is exposed on the surface of cell mem-
brane due to asymmetry in the early phase of apoptosis.
Thereby, fluorescein isothiocyanate (FITC)-conjugated
Annexin V is able to detect the early phase of apoptosis.
Only cells with co-staining with PI indicate chromatin con-
densation, the late stage process of apoptosis. The percent-
age of cells with Annexin V and PI staining was compared
with control with flow cytometry. We found that applica-
tion of MTC could promote cellular death via apoptosis
MTC has cytotoxic effects on ovarian cancer
cells
As a result of the synthesis process, (E)-1-4(4-amino-
phenyl)-3-(2,4-methoxyphenyl)prop-2-en-1-one or MTC
that is a chalcone-based derivative was generated. MTC has
calculated molecular weight of 284.1287 following our con-
firmatory findings by mass spectrometry (Figure 1(a)).
Molecular structure of MTC was represented in Figure 1(b).
For the detection of cytotoxic activity in ovarian cancer
cells, SRB assay was used to detect the viable cells. Ovarian
cancer cells were treated with MTC in various concentra-
tions for 24 and 48h. Viable cells were then quantified with
SRB assay. Figure 2 clearly show the concentration-depend-
ent effects of MTC on ovarian cancer cells. We found that
MTC has potential therapeutic effects to eliminate ovarian
cancer cells. Two ovarian cancer cell lines from different
subtypes were used in this study. ES-2 was originated from

Su et al.
5
Figure 2. MTC has cytotoxic effects on various subtypes of ovarian cancer. Cytotoxicity of MTC was assessed in two ovarian
cancer cell lines: ES-2 and Hey-A8. Cells were treated with MTC with concentrations ranging from 6.25 to 100µM for 24 or 48h.
Viability of survived cells after treatment was quantified with sulforhodamine B assay.
mechanism. The proportion of apoptotic cells increased
along with higher concentration given (Figure 3(b)).
Several proteins that are important in apoptosis regulation
were clarified with western blotting (Figure 3(c)). From the
western blotting, the apoptosis pathway was activated after
MTC treatment. We checked protein level of p-BAD,
cleaved caspase-9, and cleaved caspase-7. We found that
caspase was activated to promote apoptosis. This caspase
activation was consistent with BAD dephosphorylation.
contrast with the fluorescent in situ imaging, we also found
that expression of c-Myc was downregulated.
Autophagy suppression by MTC decreased
survival of ovarian spheroids through Sox-2
downregulation
Autophagy becomes important for cancer cells to meet the
high metabolic demand associated with rapid proliferation
or as a survival mechanism to protect their life under stress.
Autophagy-related stress tolerance was found to promote
cell survival by maintaining energy production that can
lead to tumor growth and chemoresistance.16 We initially
transfectedGFP-LC3intoES-2cellstostudytheautophago-
some formation during the autophagy process. Then,
autophagy-related proteins were clarified with western
blotting following MTC treatment. Suppression of
autophagosome formation by MTC was seen from photo-
micrographs with fluorescent microscopy. Autophagy is
highly modulated by autophagy-related genes, and Atg-5,
in particular, plays a major role in autophagy regulation
through promoting autophagosome elongation.17 We found
that after MTC treatment, Atg-5 was downregulated that
caused inhibition of mature autophagosome, as we can see
from inhibition of lipidated LC3. As a result, caspase-7 was
activated (Figure 5(a)). Therefore, because of Atg-5 sup-
pression, autophagy inhibition was seen under a fluorescent
microscope (Figure 5(b)). We then developed ovarian sphe-
roids from ES-2/GFP-LC3 cells following the previously
described protocol.12 Spheroid formation ability is one of
the CSC characters via self-renewal property, so it can also
be used to enrich CSCs.18 After 24h of treatment with 25
and 50µM MTC, ovarian spheroids were disrupted and the
viability was significantly decreased (Figure 5(c) and (d)).
From the three-dimensional immunofluorescence of ovar-
ian spheroids, inhibition of LC3 was consistent with Sox-2
suppression (Figure 5(e)). Sox-2 is one of the key
MTC inhibits cell cycle progression
in G2/M phase
Cellular status in cell cycle was also studied following
MTC treatment. After 48h of treatment, ES-2 cells were
analyzed for their cell cycle status with flow cytometry
and their cell lysates were assessed for cyclin expression
with western blotting. Immunofluorescent staining to
identify p21WAFI/CIP1 and c-Myc was also performed. From
western blotting, cyclin B1 was suppressed by MTC treat-
ment (Figure 4(a)). We found that compared with controls,
cells that were treated with MTC exhibited decreased cell
cycle progression. Proportion of cells in G2/M phase was
increased significantly (G2/M arrest) after treatment
(Figure 4(b)). G2/M arrest is also known as DNA damage
checkpoint that delays or arrests cell cycle progression in
response to DNA damage. The promotion of G2/M arrest
was caused by inhibition of cyclin B1 activity after MTC
treatment. The mechanism of cyclin B1 inhibition may be
initiated by the cyclin-dependent kinase (CDK) inhibitor
p21 (also known as p21WAFI/CIP1) upregulation. p21WAFI/CIP1
is a notable CDK inhibitor that acts as both sensor and
effector of certain anti-proliferative stimulus, including
DNA damage signals.15 p21WAFI/CIP1 could inhibit cyclin
B1/cdc2 activity that acts as key regulator in G2/M phase
checkpoint. Therefore, we also observed the upregulation
of p21WAFI/CIP1 after MTC treatment (Figure 4(c)). In

6
Tumor Biology
Figure 3. MTC induces apoptosis in ovarian cancer cells. (a) Photomicrographs of ES-2 cells treated with MTC for 24h showed
the concentration-dependent effect on cytotoxicity. Immunofluorescent staining to identify cleaved-PARP was performed to
represent caspase activity. Cleaved-PARP as the main substrate of caspase-3 was found abundant after MTC treatment, indicating
ongoing apoptosis. (b) After treatment with MTC for 24h, quantitative proportions of early and late apoptotic phases of ES-2 cells
were clarified with Annexin V/PI double staining. (c) Western blotting was performed to study the regulation of apoptosis after
MTC treatment for 24h. Status of BAD phosphorylation and activation of caspases were clarified. Apoptosis-inducing effect of MTC
was found consistent with dephosphorylation of p-Bad. As a result, caspase activation was present to allow apoptosis.
regulators for ovarian CSCs regulation.6 In addition to
MTC inhibitory effect on Sox-2, in a confirmatory assay,
using western blot, the ovarian spheroids when treated with
different concentrations of MTC exhibited a dose-depend-
ent significant downregulation of the pluripotent transcrip-
tion factors, Oct4, KLF4, and c-Myc (Figure 5(f)).

Su et al.
7
Figure 4. MTC inhibits cell cycle progression. (a) After treatment with MTC at different concentrations for 24h, expression level
of cyclin B1 in ES-2 cells was analyzed with western blotting. (b) Flow cytometry was performed to study the status of ES-2 cells in
cell cycle. Cells that were treated with 25µM of MTC for 24h were compared with controls. (c) From immunochemistry staining,
p21 as tumor suppressor was activated in ES-2 cells treated with MTC (25µM) for 24h. In contrast, c-Myc was found suppressed
after MTC treatment.
Therefore, elimination of ovarian spheroids by MTC treat-
ment might be mediated by autophagy inhibition.
trans-well migration assay. We found that N-cadherin and
c-Met were suppressed by MTC administration (Figure
6(a)). MTC treatment could successfully suppress migration
ability in vitro (Figure 6(b)).
MTC suppresses the migration ability of ovarian
cancer cells in vitro
MTC enhances the anti-cancer effect of
cisplatin
There is a significant positive correlation between metastatic
status and prognosis among ovarian cancer patients, as the
formation of metastases in ascites is a typical features of late
stage ovarian cancer. Therefore, inhibition of metastases is
very important to improve clinical outcome. N-cadherin and
c-Met are essential for metastasis and progression in various
tumors.19,20 We sought to study the suppression of N-cadherin
and c-Met in correlation with the migration ability of ovarian
cancer cells after MTC treatment. N-cadherin and c-Met
expression profiles were evaluated with western blotting,
while the migration ability of ES-2 cells was analyzed with
We co-administered MTC with cisplatin using various
concentration regimens (Figure 7(a)). After 24h of treat-
ment, we found that combination of MTC and cisplatin
had synergistic cytotoxicity on ES-2 cells (Figure 7(b)).
This observed synergistic effect was corroborated by data
obtained from the Chou-Talalay drug combination algo-
rithm using the CompuSyn software. Synergism is defined
with combination index (CI)<1, additive effect is achieved
when CI=1, and antagonism is inferred when CI>1.

8
Tumor Biology
Figure 5. MTC has cytotoxic effects on ovarian spheroids through autophagy inhibition and suppression of Sox-2. (a) Expression
levels of Atg-5, LC3, and cleaved caspase-7 in ES-2 cells treated with MTC (25µM) for 24h were assessed by western blotting.
β-actin was used as internal control in control and treated samples. (b) After 24h of treatment, ES-2 cells that have been
transfected with GFP-LC3 were fixed and assessed under a fluorescent microscope. Nuclear staining was done with administration
of DAPI. Red arrow indicates mature autophagosome, while white arrow represents autophagosome puncta. (c) Ovarian spheroid
was developed from ES-2/GFP-LC3 with stem cell medium. Then, cytotoxic effect of MTC to ovarian spheroids was seen under
a light microscope. (d) Quantification of viable cells from spheroids was analyzed with Alamar blue assay (mean SD). (e) Three-
dimensional immunofluorescent staining was performed to see the in situ expression of Sox-2, in colocalization with LC3. DAPI
was used to stain nucleus. **p<0.05. (f) Ovarian spheroids when treated with different concentrations of MTC exhibited a dose-
dependent significant downregulation of the pluripotent transcription factors, Oct4, KLF4, and c-Myc.
Synergism was seen as dots inside the triangle. This dem-
onstrated synergism is indicative of the potential role of
MTC as an adjunct to cisplatin.
Clear cell carcinoma subtype only represents 3.7%–12.1% of
all epithelial ovarian cancer cases.21 However, clear cell car-
cinoma patients in advanced stage have relatively poor prog-
nosis with lower 3-year and 5-year survival rates compared
with the non–clear cell carcinoma subtype.13,22 Late stage
clear cell carcinoma patients also have higher tendency of
platinum insensitivity.23 In this study, we used ES-2 cell line
which originated from late stage clear cell carcinoma patient
Discussion
Ovarian cancer has a high mortality rate with considerable
metastasis ability and resistance to established chemotherapy.

Su et al.
9
Figure 6. MTC inhibits ovarian cancer cell migration ability through N-cadherin and c-Met suppression. (a) In order to study
suppression of key regulators for invasiveness and metastases by MTC treatment, western blotting was performed with antibody
to probe N-cadherin and c-Met. (b) With lower concentration, starting from 6.25µM for 8h, ES-2 cells migration was assessed in
trans-well chambers. Images were captured after cellular staining with Giemsa.
Figure 7. Combination analysis of MTC and cisplatin shows synergistic effect. (a) ES-2 cells were treated with monotherapy of
cisplatin or combination of MTC and cisplatin. Cell viability was then assessed with SRB assay. (b) Combinational effects of cisplatin
and MTC were analyzed with CompuSyn software. Combination index (CI) was presented in a triangle diagram, where synergistic
effects were represented as dots inside the triangle. We found that combination of MTC and cisplatin had synergistic effect with
CI<1.

10
Tumor Biology
and found that the cell line was responsive to MTC (Figure
2). This finding is suggestive of a potential curative role of
MTC in the currently therapy-elusive late stage clear cell
ovarian cancer patients. In addition, another cell line, Hey-
A8, from late stage serous ovarian carcinoma. Serous ovarian
adenocarcinoma is the most common subtype of ovarian can-
cer. Thus, the potency of MTC is not only limited to clear cell
carcinoma but may also be beneficial for a majority of ovar-
ian cancer patients.
(Figure 4(b)). Single agent therapy with MTC enhanced the
activity of CDK inhibitor p21WAFI/CIP1. Inhibition of cell
cycle progression limits proliferation of cancer cells, how-
ever, the cell cycle arrest at G2/M phase also allows cells to
repair the damage. PARP-1 is the main player in DNArepair
machinery. In our study, despite the G2/M phase arrest-
related DNA repair, we observed that PARP-1 is cleaved
following MTC treatment. Thus, indicating a dysfunctional
DNA damage repair mechanism upon exposure to MTC. In
addition, we observed the upregulation of p21WAFI/CIP1 with
associated c-Myc suppression after MTC treatment (Figure
4(c)). c-Myc is pivotal in cancer survival and progression. In
breast and ovarian cancers, c-Myc overexpression is associ-
ated with poor prognosis through its induction of multi-drug
resistance (MDR) genes.27 In breast cancer, c-Myc signaling
can be deregulated by endogenous p21WAFI/CIP1 upregulation
which contributes to tumor suppression.28 In addition, inhi-
bition of c-Myc may also inhibit cell cycle progression29 and
decrease epithelial–mesenchymal transition (EMT) features
and properties of CSCs in breast cancer.28 In order to study
the metastasis inhibition effect of MTC treatment, we
treated ES-2 cells with a lower concentration of MTC and
found that lower concentration of MTC treatment could
limit metastases through inhibition of N-cadherin and c-Met
pathway (Figure 6(a)).
Impaired regulation, activation, assembly, or function for
autophagy has been increasingly studied, especially in cor-
relation with chemotherapeutic response. Autophagy plays
an important role in drug resistance of CSCs. A high
autophagic flux is associated with CSCs and may protect
them from stress and DNA damage.8 Moreover, inhibition of
autophagosome formation by gene silencing or co-treatment
with autophagosome inhibitor successfully induced cellular
death of pancreatic CSCs.30 To study autophagy regulation
by MTC in ovarian CSCs, we developed ovarian spheroids
from a clear cell carcinoma cell line. Ovarian CSCs are com-
monly found in ascites fluid of late stage ovarian cancer
patients. The spheroids are enriched with CSCs that are
responsible for resistance to chemotherapeutic drugs, metas-
tasis, and tumor relapse.6 We showed that MTC treatment
could decrease autophagosome elongation byAtg-5 suppres-
sion in ES-2/GFP-LC3 cells. MTC decreased autophagy in
ovarian spheroids developed from ES-2/GFP-LC3.
Suppression of autophagy was followed by Sox-2 suppres-
sion, altering CSC maintenance, as a result, ovarian sphe-
roids viability was decreased after MTC treatment.
Collectively, these data demonstrate that MTC inhibits can-
cer growth by inducing apoptosis through dephosphorylation
of BAD, limiting cell cycle progression by upregulation of
CDK inhibitor, blocking metastasis through inhibition of
N-cadherin, and importantly decreasing ovarian spheroids
viability through autophagy inhibition. Co-administration of
cisplatin and MTC increased tumor cytotoxicity in a syner-
gistic manner. A schematic summary of MTC mechanism of
action is shown in Figure 8.
While the late stage clear cell carcinoma is associated
with poor prognosis due to enhanced resistance to platinum-
based therapy, interestingly, early stage clear cell ovarian
carcinoma is highly responsive to platinum-containing
chemotherapy.23 Therefore, ES-2 is an ideal model to explore
and develop a therapeutic strategy against acquired resist-
ance in ovarian cancer. Several mechanisms involved in
platinum resistance in clear cell carcinoma have been pro-
posed, including drug efflux mechanism, drug detoxifica-
tion, and DNA repair mechanism. In addition, several
mechanisms of cell survival have been reported to correlate
with platinum resistance in clear cell carcinoma, such as
phosphorylation of BAD,4 and HER2/neu activity and/or
expression.24 We demonstrated that clear cell carcinoma
cells were apoptotic following MTC treatment, and that the
observed induction of apoptosis might be mediated by
dephosphorylation of BAD protein. The inhibition of BAD
phosphorylation was related to caspases activation that ini-
tiates apoptosis pathway (Figure 3). BAD is a proapoptotic
protein that can inhibit the Bcl-2 anti-apoptotic protein,
facilitate the activity of other proapoptotic proteins and
activate cytochrome-c release to induce apoptosis. This
proapoptotic property of BAD is regulated by its phospho-
rylation. Only unphosphorylated BAD could interact with
anti-apoptotic Bcl-2 family members, while p-BAD is
sequestered in the cytosol and remains inactive by binding to
14-3-3 proteins.25 In ovarian cancer patients, p-BAD has
been reported to be positively correlated with a lower overall
survival rate cisplatin resistance.4 By contrast, inhibition of
BAD phosphorylation reverses the cisplatin resistance to
improve treatment outcome.5 In our present study, we found
that BAD dephosphorylation level was consistent with cas-
pase-9 and caspase-7 activation to modulate cellular death.
PARP-1 plays a critical role in double-strand break (DSB)
repair, including the DSB repair process after cisplatin treat-
ment that leads to chemoresistance. Inhibition of PARP-1 was
found to be essential to sensitize cancer cells to cisplatin.26
However, PARP-1 is one of the essential proteins that are
digested fast and early by caspases -7 and -9. We found that
activation of caspases might cause PARP-1 cleavage as evi-
denced by increased cleaved-PARP expression after treat-
ment with MTC (Figure 3(a)). This observed cleavage of
PARP-1 by our novel drug, MTC, is indicative of its putative
role as a potentiator of cisplatin.
Apoptosis induction by MTC seems to be mediated by
DNA damage signals as cells were arrested in G2/M phase

Su et al.
11
(MOST-103-2113-M-324-001-MY2;MOST-103-2811-M-324-001)
and W.H.L. (MOST-105-2320-B-038-054). This study was also sup-
ported by grants from Taipei Medical University (105TMU-SHH-15)
to W.H.L. and grants from Taipei Medical University–National
Taiwan University of Science and Technology Joint Research
Program (TMU-NTUST-103-03) to C.T.Y.
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