Cisplatin in combination with emetine and patulin showed dose and sequence dependent synergism against ovarian cancer

Journal Pre-proof Cisplatin In Combination With Emetine And Patulin Showed Dose And Sequence Dependent Synergism Against Ovarian Cancer Md Nur Alam, Jun Qing Yu, Philip Beale, Fazlul Huq

PII:

S2213-7130(19)30017-3

DOI:

https://doi.org/10.1016/j.synres.2019.100060

Reference:

SYNRES 100060

To appear in:

Synergy

Received Date:

11 July 2019

Revised Date:

22 August 2019

Accepted Date:

19 November 2019

Please cite this article as: Alam MN, Yu JQ, Beale P, Huq F, Cisplatin In Combination With Emetine And Patulin Showed Dose And Sequence Dependent Synergism Against Ovarian Cancer, Synergy (2019), doi: https://doi.org/10.1016/j.synres.2019.100060

This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier.

Cisplatin In Combination With Emetine And Patulin Showed Dose And Sequence Dependent Synergism Against Ovarian Cancer

Md Nur Alam1, 3 , Jun Qing Yu1, Philip Beale2, and Fazlul Huq1

1

ro of

Discipline of Biomedical Science, Sydney Medical School, University of Sydney, Cumberland Campus C42, 75 East Street, Lidcombe, NSW 1825, Australia. 2Sydney Cancer Centre, Concord Hospital, Sydney, NSW 2139, Australia. 3Department of Pharmacy, Faculty of Biological Sciences, Jahangirnagar University, Savar, Dhaka 1342, Bangladesh

ur

na

lP

re

-p

Graphical Abstract

Jo

Abstract

Combating multiple drug resistant ovarian cancer is a major challenge of the day. Combined chemotherapy of platinum drugs with phytochemicals (e.g. paclitaxel) introduced new horizon among oncologists in overcoming drug resistance. The objective of the present study is to investigate the activity of cisplatin in combination with emetine and patulin within ovarian cancer models. Antitumour activity of the cisplatin, emetine and patulin as a single

drug was determined against human ovarian cancer cell lines (A2780 and A2780CisR) was determined using MTT reduction assay. Combination indices obtained from Chou-Talalay method were used to express combined drug actions. Proteomics was also carried out to identify the proteins which were responsible for the synergistic effects of the drug combinations. Cisplatin in combination with emetine produced synergism against ovarian cancer models depending on dose and sequence of drug administration. Patulin also demonstrated synergism when combined cisplatin at different doses and sequence of

ro of

administrations. Eight proteins (e.g. VIME, ENPL, GRP78, CARL, NACA, COF1, PPIA and RSSA) were considered for combined drug actions of cisplatin plus emetine. Synergistic

activity from combination cisplatin with patulin could be attributed to the downregulation of

-p

VIME, COF1 and CH10 as well as upregulation of CISY. Combination of cisplatin with emetine and cisplatin with patulin have the potential to overcome cisplatin resistance in

1. Introduction

lP

re

ovarian cancer, and could be warranted for further evaluation using suitable animal model.

na

Ovarian cancer is called the silent killer in women due to its insidious and subtle nature. It is the fourth leading cause of death among women in developed countries [1]. It can be said that

ur

ovarian cancer is a series of molecularly and aetiologically distinct diseases that merely share the same anatomical location [2]. In most of the cases, ovarian cancer is diagnosed at late

Jo

stages when it spreads into surrounding thus accounting for low survival rate. Although patients respond well with platinum based chemotherapy, recurrence is common within 18 months of treatment. Moreover, dose dependent side effects of platinum drugs also limit their use as a single therapy. Combination of carboplatin and paclitaxel has gained popularity among clinicians due to lower adverse effects and improved efficacy [3]. It has been postulated that combined chemotherapy can provide multiple advantages over monotherapy

such including improved therapeutic efficacy in a synergistic manner by targeting various signalling pathways, overcoming drug resistance by modulating genetic barrier and delaying tumour cell mutations [4, 5]. Natural compounds obtained from plants and other sources have great potential for use against various diseases including cancer. A number of such compounds and their derivatives including paclitaxel, docetaxel, vinblastine and vincristine have also been used in clinic to treat cancer. Moreover several natural compounds are currently at various stages of clinical

ro of

development for treatment against various cancers [6]. Combination of phytochemicals with existing chemotherapeutic drugs might provide a good opportunity to exploit the anticancer potential of both the compounds. Such synergistic combinations would result into cost-

-p

effective treatment of cancer without increasing the side effects. We have reported synergism from combination of platinum drugs (cisplatin, oxaliplatin, designed compounds) with

re

phytochemicals (curcumin, EGCG, resveratrol, thymoquinone, quercetin, capsaicin,

lP

colchicine and paclitaxel) against ovarian cancer models [7-10]. In this study emetine and patulin in combination with cisplatin have been selected for investigation in ovarian cancer cell lines for combined drug effects.

na

Emetine is an alkaloid usually found in Ipecacuanaha species which is an active component of Ipecac syrup and historically used for the treatment of dysentery. Anticancer potential of

ur

emetine was identified in early 1970s and the drug entered into clinical trials as a single agent

Jo

against different tumours, however withdrawn due to muscle weakness and cardiotoxicity [11, 12]. Recently, attention has been focused on emetine again to exploit its high antitumour activity in combination with other chemotherapeutics against leukaemia, bladder and ovarian cancer [13-15]. Patulin is an α, β-unsaturated γ-lactone produced by several filamentous fungi of the genera of Penicillium, Aspergillus, and Byssochlamys which is usually found in rotten

apple products. Although earlier studies reported carcinogenic activity of patulin [16, 17], recent discovery of its antitumour activity has opened the new direction of research [18-20]. In the present study, activity of cisplatin in combination with emetine and patulin has been investigated against A2780 parent ovarian cancer cell line and cisplatin resistant A2780cisR cell line as a function of concentration and sequence of administration. Proteomic study was also conducted to identify the proteins associated with the combined drug effect.

2. Materials and methods

ro of

2.1 Materials

Cisplatin was prepared according to modified Dhara method [21]. Emetine and patulin

was obtained from Toronto research chemicals, Canada. Cisplatin was initially dissolved

-p

in DMF (Sigma-Aldrich Pty Ltd, NSW, Australia) followed by the addition of mQ water (at a ratio 1:4) to give a 2.5 mM stock solution. Emetine was dissolved in mQ water

re

whereas patulin in ethanol to give 10 mM and 5 mM stock solutions respectively. The

lP

drug solutions were serially diluted from the stock solutions with freshly prepared RPMI1640 medium (Thermo Trace Pty Ltd Melbourne, Australia) to produce a range of

na

concentrations from 0.16 to 20 µM for cisplatin, 0.008 to 1 µM for emetine and 0.08 to 10 µM for patulin. 2.2 Cell culture

ur

Human ovarian cancer cell lines A2780 and A2780cisR were seeded in 25 cm2 cell culture

Jo

flasks in an incubator at 37˚C in a humidified atmosphere consisting of 5% CO2 in air. The ovarian cancer cell lines were obtained as gifts from Ms. Mei Zhang, Royal Prince Alfred Hospital, Sydney, Australia. The cells were maintained in logarithmic growth phase in a complete medium consisting of RPMI-1640, 10% heat-inactivated foetal calf serum, 20 mM Hepes, 0.11% bicarbonate, and 2 mM glutamine at physiological pH.

2.3 MTT [3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide] reduction assay Cytotoxicity of the compounds against cancer cells either alone or in combination were determined by MTT reduction assay [22]. In short, 4000 to 5000 cells per well in RPMI-1640 medium were seeded into flat-bottomed 96-well culture plate and allowed to attach overnight. When drugs were added alone, four different concentrations of each drug were prepared from the stock solutions and 100 µl of drugs were added to equal volumes of cell culture in

ro of

triplicate wells added to triplicate wells which were left in the incubator (37°C, 5% carbon dioxide in air, pH 7.4) for 72 h. During combination studies, cells were treated with

increasing concentrations of compounds at constant ratios of their IC50 values using the

-p

sequences: 0/0, 0/4 and 4/0, where 0/0 indicated bolus administration, 0/4 indicated that cisplatin was added first followed by the emetine/patulin 4 h later and 4/0 indicated the

re

converse. The concentration ranges were: cisplatin: 0.18-4.48 µM and 1.85-46.16 µM;

lP

emetine: 0.01-0.21 µM and 0.01-0.17 µM; patulin: 0.27-6.84 µM and 0.41-10.28 µM for A2780 and A2780cisR cell lines respectively. After 72 h of incubation of the drug treated cells and control in 5% CO2 incubator, the medium was removed and 50 µl of the MTT solution

na

were added to each well of 96-well plate and incubated for 4 h. After completion of incubation period, 150 µl of DMSO were added to each well. The viable cells remained

ur

attached at the bottom stained with MTT as purple formazan product. The mean absorbance

Jo

at 595 nm for each compound or drug treatments whether alone or in combination was expressed as a percentage of the untreated control well absorbance. The effect of combined drug treatments was studied using a median effect analysis whereby a combination index (CI) was calculated from pooled data from 3 to 5 individual experiments each comprising at least three data points for each drug alone and for the drug combinations. The combination index

(CI) for two compounds or drugs was calculated from the following calculation based on Chou and Talalay median effect [23, 24] equation:

where D1 and D2 in the numerator stand for the concentrations of compounds 1 and 2 in combination to achieve x% inhibition whereas D1x and D2x in the denominator represent

Dx can be readily calculated from the following equation: Dx=Dm[fa/(1–fa)]1/m

ro of

concentrations of compounds 1 and 2 to achieve x% inhibition when present alone.

-p

In the equation, Dx denotes the dose of drug, Dm is the median effect dose, fa is the fraction of cells affected (killed) by the dose, fu is the fraction of cells remaining unaffected so that

re

fu=1−fa, and m is the exponent defining the shape of the dose–effect curve. CI values of <1, =1 and >1 indicate respectively synergism, additiveness and antagonism in

lP

combined drug action. The CI, Dm and r values were obtained automatically using Calcusyn software (V2) (Biosoft, UK). The Dm sometimes reflect the values of IC50 value. The linear

na

correlation coefficient, r (where r=1 indicates perfect fit), of the median effect plot should be reasonably good; for the cell culture system, r should be greater than 0.95 (r>0.95).

ur

2.4 Proteomic study

Jo

Proteomic studies were carried out to identify the proteins that were responsible for combined drug actions in A2780 and A2780cisR cell lines. The cells were cultured in 50 cm2 petri dishes to produce the concentration of 106 cells/dish. A2780 cells were treated with solutions of cisplatin and emetine as 0/4 sequence of administration at IC50 concentration. A2780cisR cells were treated with solutions of cisplatin with emetine, cisplatin with patulin as 4/0 sequence of administration at IC50 concentrations. Control cells were treated will the medium only.

Following 24 h of incubation period after drug treatment, cell pellets were collected through washing with PBS and centrifugation. The pellets were lysed using cell lysis buffer, 1st dimensional electrophoresis was done using non-linear ReadyStrip™ IPG Strip in Protean i12 IEF (Isoelectric focusing) cell unit. 2nd dimensional gel electrophoresis (SDS-PAGE) was conducted by using 4–20 % SDS Criterion™ TGX™ pre-cast gels in a Criterion Dodeca™ cell separation unit (BIO-RAD, Australia) at constant 200 V for 100 min in a TrisglycineHCl buffer system. The detailed method has been described in our previous article [25]. The

ro of

gel images were taken by ChemiDoc™ MP Imaging system (BIO-RAD, Australia) and spots were analysed by using Melanie version 7.0 software (GeneBio, Switzeland). A 1.5 fold

change in the expression of a protein across the matched groups was used as the indicative

-p

point for significant expression. Analysis of variance (ANOVA), a statistical tool used to detect differences between experimental group means, was performed using a target

re

significance level of 0.05.

lP

2.5 Mass spectral characterization of differentially expressed proteins Bio-Safe Coomassie Stain was used for staining the spots before excision from 2-D gels. Destaining of the spots was done by using 120 μl of (50 % acetonitrile/50 mM NH4HCO3)

na

solution and digestion of spots was performed by trypsin. The obtained peptides were extracted with 0.1 % trifluoroacetic acid (TFA) then extracted and concentrated by C18 zip-

ur

tips (Millipore, μ-C18, P10 size) on Xcise (Proteome Systems). Matrix assisted laser

Jo

desorption ionisation mass spectrometry (MALDI-MS) was performed with 4800 plus MALDI TOF/TOF Analyser (AB Sciex). Detailed procedure has been described in our earlier article [25]. The data on peptides masses were analysed using database search program Mascot (Matrix Science Ltd, London, UK). The peak lists were searched against Homo sapiens entries in the SwissProt database.

3. Results 3.1 Anticancer activity of single drugs: The results of the antitumour activity of tested compounds (cisplatin, emetine and patulin) against ovarian cancer cell lines obtained from MTT reduction assay is shown in Table 1. The resistance factor (RF) is defined as the ratio of IC50 value of the compound in the resistant cell line to that in the parent cell line. It is evident from Table 1 that, emetine demonstrated highest anticancer activity among the tested compounds against all tested cell lines with IC50 values at nano-molar levels. Although the

ro of

cytotoxicity of patulin and cisplatin against A2780 parent cell line is similar; the RF values of patulin are much lower than cisplatin indicating greater potency of patulin against resistant ovarian cancer cell lines.

-p

3.2 Anticancer activity of the drugs in combination

Combined drug effect as a function of sequence of administration and added concentrations

re

from selected combinations was determined using combination indices (CI). Table 2 gives

lP

dose−effect parameters in terms of median-effect dose, shape (sigmoidicity), conformity (linear correlation coefficient), represented as Dm, m and r respectively. ED50, ED75 and ED90

na

represents the combined drug concentration required for 50%, 75% and 90% cell kill, respectively. Graphs obtained for dose effect curves, Fa-CI plots and median-effect plots

Jo

ur

from Calcusyn software is presented in Figure 1 and Figure 2.

ro of -p re lP na ur Jo Figure 1: Dose effect curves, Fa-CI plots and median-effect plots obtained from combinations of cisplatin with emetine and patulin within A2780 cell line using Calcusyn

ro of -p re lP na ur Jo Figure 2: Dose effect curves, Fa-CI plots and median-effect plots obtained from combinations of cisplatin with emetine and patulin within A2780CisR cell line using Calcusyn

It can be seen from Table 2 that, cisplatin in combination with emetine exhibited synergism at all sequences of administration (bolus, 0/4 and 4/0) and added concentrations (ED50, ED75 and ED90) against A2780 ovarian cancer cell line. Greater synergism was observed at lower concentrations with the 0/4 sequence of administration of cisplatin with emetine against A2780 cell line. On the contrary, against A2780cisR cell line, the same combination of cisplatin with emetine showed synergism with 0/4 and 4/0 sequences of administration. However, bolus combination of cisplatin with emetine produced antagonism at lower added

ro of

concentrations (ED50 and ED75) and additiveness at ED90 against A2780cisR cell line. Cisplatin in combination with patulin demonstrated mild synergistic to additive effects

against A2780 cell line for all sequences of administration (bolus, 0/4 and 4/0) and added

-p

concentrations (ED50, ED75 and ED90). But against A2780cisR cell line, the same combination of cisplatin with patulin showed synergism for bolus and 4/0 sequences of administration at

re

all added concentrations. However, antagonism was predominant with 0/4 sequence of

lP

administration of cisplatin with patulin against A2780CisR cell line. 3.3 Proteomics

The study was conducted to get mechanistic information concerning the changes in

na

expression of proteins observed in ovarian cancer cell lines after drug treatments. Cisplatin in combination with emetine using 0/4 sequence of administration was used for proteomics in

ur

A2780 cell line. In case of A780cisR cell line, cisplatin in combination with emetine using 4/0

Jo

sequence of administration and cisplatin in combination with patulin using 4/0 sequence of administration was employed in this study. The above mentioned combinations were chosen for proteomics because the selected combinations demonstrated significant synergism in combination study in the corresponding cell line. Also in this study, the expression of the proteins in cisplatin resistant cell line were compared to those observed in parent A2780 cell line before and after treatment to identify the proteins responsible for drug resistance.

In the gels obtained from A2780 untreated cell line, a total of 308 spots were detected by the software, whereas A2780cisR reference gels produced 107 spots. Among the spots observed in A2780 reference gels, only 7 spots showed significant changes in expression after treatment with combination of cisplatin with emetine using 0/4 sequence of administration. 77 spots demonstrated insignificant changes in expression (fold factor below 1.5) and remaining 224 spots were absent in treated gels. Figure 3 and Figure 4 shows the image of reference A2780 gel and treatment gel. Table 3 describes the protein which exhibited significant changes in

Jo

ur

na

lP

re

-p

ro of

expression in A2780 cell line after combined drug treatment.

ro of -p re lP na ur Jo Figure 3: Reference gel A2780 showing the selected spots

ro of -p re lP na ur Jo Figure 4: Cisplatin with emetine (0/4) treated A2780 gel

*Mascot score is insignificant, information not provided Whereas, among 107 spots observed in A2780cisR reference gels, only 2 spots demonstrated significant changes in expression after treatment with combination of cisplatin with emetine using 4/0 sequence of administratiob. In contrast, after treatment with combination of cisplatin with patulin using 4/0 sequence of administration, significant changes in expression were observed for 4 proteins in A2780cisR reference gels. Figure 5 and Figure 6 shows the

ro of

image of reference A2780cisR gel and treatment gels. Table 4 describes the protein which exhibited significant changes in expression in A2780cisR cell line after combined drug

Jo

ur

na

lP

re

-p

treatments.

ro of -p re lP na ur Jo

Figure 5: Reference gel A2780CisR showing the selected spots

ro of -p re lP na ur Jo Figure 6: Cisplatin with emetine (Up); Cisplatin with patulin treated A2780CisR gel (Down)

4. Discussion Among the investigated compounds, emetine was found to be the most active against all cancer cell lines tested in this study. The IC50 values of emetine were found to be at nanomolar range; lowest against ovarian A2780cisR cell line with 18 nM and highest in parent A2780 cell line with 23 nM. Anticancer potential of emetine was discovered in late 1910s and entered into Phase-I and Phase-II clinical trial in 1970s [26]. However, it was withdrawn due to dose dependent muscle weakness and cardiotoxicity [27]. Since the adverse effects are

ro of

observed mainly at high doses, emetine has been considered for combination study with

cisplatin where required amount of each drug would be very little and considered to give

activity with minimum side effects. Patulin has not been studied much towards its anticancer

-p

potential, but this study demonstrates that patulin is a prospective compound having greater

antitumour activity than cisplatin against A2780cisR and A2780ZDO473R cell lines. The result of

re

this study is supported by the earlier studies against HEK kidney cancer cell line (IC50 value

lP

2.5 µM) and CHO-K1 ovarian cancer cell line (IC50 value 2.8 µM) [28, 29]. Another in vivo model study reported that intraperitoneal administration of patulin caused significant

na

reduction of tumour in B16F10 cell-implanted mice [20]. The authors also mentioned that after 20 days no potential toxicity was observed among patulin administered mice. However

ur

several long term studies concluded that patulin can lead to immunotoxicity, genotoxicity, mutagenicity and neurotoxicity [30]. International Agency for Research on Cancer includes

Jo

patulin in ‘Class- 3 agents’ which are not carcinogenic towards human. That is why patulin has been selected here for combination study. From combination study it has been observed that cisplatin in combination with emetine displayed synergism at all concentrations and for all added sequences in A2780 cell line (Table 2). Among three sequences of administration, 0/4 sequence exhibited the most synergistic effect with the highest at ED50 level. Gradual decrease in synergism was observed

with the increase in added concentrations of drugs with 0/4 sequence but the converse was true for 0/0 and 4/0 sequences in A2780 cell line. Whereas in case of A2780cisR cell line, cisplatin in combination with emetine showed synergistic effect when administered using the sequences 0/4 and 4/0. Bolus administration of cisplatin with emetine displayed antagonism in A2780cisR cell line. Interestingly, a clear trend was evident with the increase in added concentrations of drugs producing greater synergistic action for all sequences of administration (Table 2). Sun et.al also found that co-administration cisplatin and emetine

ro of

increased the apoptotic cell death after 72 hours in SKOV-3 ovarian cancer cell line [31]. The authors suggested that synergism was linked with activation of caspase-3, caspase-7 and caspase-8. Sensitization of cancer cells towards cisplatin action was correlated with the

-p

downregulation of Bcl-xL by emetine. Another study concluded that the inclusion of low

dose of emetine as part of multi-modal therapy for bladder cancer could benefit patients by

re

enhancing the anti-tumour activity of standard of care chemotherapy by reducing dose of

lP

cisplatin and increasing the efficacy of carboplatin.

4/0 combination of cisplatin with patulin showed synergism at all added concentrations in A2780 cell line whereas 0/0 sequence displayed synergism only at ED90 level. But the same

na

combination demonstrated additiveness at lower concentrations when administered as a bolus and also for all 0/4 sequence of administration against A2780 cell line (Table 2). In contrast,

ur

bolus and 4/0 sequences of administration of cisplatin with patulin produced synergism at all

Jo

added concentrations against A2780cisR cell line. But 0/4 sequence of administration proved to be antagonistic to additive in A2780cisR cell line (Table 2). All told, it can be said that 4/0 combination of cisplatin with patulin was better than other sequences of administration pertaining to synergistic outcome in ovarian cancer and also in overcoming cisplatin resistance.

Proteomics has provided information on underlying mechanism for the combined drug action. Treatment of cisplatin with emetine (0/4) in A2780 cell line caused significant changes in expression of 7 proteins, among which six (vimentin, endoplasmin, 78 kDa glucose-regulated protein, calreticulin, polyubiquitin-B, nascent polypeptide-associated complex subunit alpha) were downregulated and one (peptidyl-prolyl cis-trans isomerase A) was upregulated. Change in folds of the respective proteins after treatment of cisplatin emetine is shown in Table 3.

ro of

Vimentin (VIME) is a cytoskeleton protein belonging to type III intermediate filaments and is found mostly in mesenchymal cells. VIME plays a key role in epithelial to mesenchymal

transition (EMT) where epithelial cells convert into mesenchymal phenotype and acquire the

-p

ability to move easily [32]. Previous studies also suggested the elevated expression of VIME

re

in prostate, ovarian, lung and gastric cancers [32]. In the present study, VIME was found to be significantly downregulated due to treatment with combination of cisplatin and emetine

lP

using 0/4 sequence of administration in A2780 cell line. The protein was also downregulated following treatment with combination of cisplatin with patulin using 4/0 sequence of

na

administration in A2780CisR cell line.

Endoplasmin (ENPL) is a well-known master immune chaperone involved in folding of

ur

several innate immune receptors e.g. toll like receptors, integrins, platelet glycoprotein, GARP and LRP6 [33]. Other than the association with autoimmune and inflammatory

Jo

diseases, ENPL has also been linked with carcinogenesis and metastasis [34]. A number of studies using various tumour models have shown that ENPL is upregulated in cancer cells compared to normal cells. Elevated expression of ENPL has been reported in oesophageal cancer, gastric cancer, pancreatic cancer, prostate cancer and lung cancer [35]. In the present study, the protein was significantly downregulated in A2780 cell line following treatment with combination of cisplatin with emetine administered using 0/4 sequence.

78 kDa glucose-regulated protein (GRP78) accelerates folding and assembly of nascent proteins; prevents their misfolding and intermediate aggregation; eliminates misfolded proteins via proteasome degradation [36]. Strong evidence regarding the upregulation of GRP78 in neoplasia exists as in breast cancer, hepatic cancer, lung cancer, and prostate cancer [37]. Lee has described the mechanisms through which GRP78 in involved in tumour progression, cancer cell resistance and chemotherapy resistance [38]. In the present study, GRP78 was found to be significantly downregulated in ovarian A2780 cancer cell line after

ro of

treatment with combination of cisplatin and emetine using 0/4 sequence. Calreticulin (CALR) is a 46 kDa protein primarily located in endoplasmic reticulum that performs critical functions inside and outside of endoplasmic reticulum. In addition to

-p

maintaining calcium homeostasis, CALR serves in regulation of protein folding, cell

re

adhesion, immune response, gene transcription and RNA stability. Recent studies also show that CALR is involved in tumour initiation and development. Upregulation of CALR has

lP

been observed in ovarian, colon, oral, breast, bladder and prostate cancers [39]. Treatment of A2780 cell line with combination of cisplatin with emetine using 0/4 sequence of

na

administration caused significant downregulation of CALR. Nascent polypeptide associated complex (NAC) is predominantly located in cytoplasm,

ur

which is involved in trafficking growing nascent polypeptides to suitable co-factors via its communication with the nascent chains on the ribosome. NAC is a heterodimer consisting of

Jo

two subunits, alpha and beta. Alpha subunit of NAC known as NACA has transcriptional activating activity and binds to DNA, ribosomal RNA and transfer RNA. NACA displays transcriptional activity by co-activating c-Jun. NACA has been found to be overexpressed in different tumour cells including hepatic carcinoma through activation of ERK pathway [40]. Treatment of A2780 cell line with synergistic combination of cisplatin with emetine using 0/4 sequence of administration caused significant downregulation of the protein.

Peptidyl-prolyl cis-trans isomerase A (PPIA) is the most abundant in human, contributing 0.1% to 0.6% of total cytosolic proteins. The protein has been reported to play a role in cell migration, proliferation and differentiation [41]. Upregulation of PPIA has been observed in different types of cancers including non-small cell lung carcinoma, hepatocellular carcinoma, pancreatic adenocarcinoma, colorectal cancer, endometrial cancer, gastric cancer, oesophageal cancer, glioblastoma and metastatic melanoma [42]. In the present study, treatment of A2780 cell line with combination of cisplatin with emetine using 0/4 sequence

ro of

of administration caused significant upregulation of PPIA compared to the level found in untreated A2780 cell line.

10 kDa heat shock protein, mitochondrial (CH10 or Hsp10) is a co-chaperone which acts in a

-p

coordinated fashion with Hsp60 during protein folding. Due to the elevated expression of CH10 in several tumour models, the protein is now being considered to be involved in

re

carcinogenesis. Evidence of upregulation of CH10 has been described in uterine, colorectal,

lP

prostate, bladder, bowel and ovarian cancer [43, 44]. However, in bronchial tumour the expression of CH10 was found to be downregulated [45]. In the present study, Hsp10 was significantly downregulated in A2780cisR cell line following treatment with combination of

na

cisplatin with patulin administered using 4/0 sequence.

ur

Cofilin belongs to a family of closely related low molecular weight proteins named as actin depolymerizing family (ADF). Among three members of cofilin family (ADF, Cofilin-1 and

Jo

Cofilin-2), COF1 or Cofilin-1 is the most abundant consisting about 95% of all expressed cofilin [46]. Overexpression of COF1 has been observed in a variety of cancers including: glioblastoma, lung, pancreas, breast, oral, renal and ovary [47]. In the present study, treatments with combinations of cisplatin with both emetine and with patulin administered using 4/0 sequence caused significant downregulation of COF1. However, treatment with

combination of cisplatin with patulin resulted in higher downregulation of COF1 than that resulting from combination of cisplatin with emetine. Citrate synthase (CISY) plays a key role in the central metabolic pathway of tricarboxylic acid cycle in all eukaryotes by catalysing the condensation reaction between acetyl-CoA and oxaloacetate to form citrate. CISY is synthesized in cytosol, but primary located in mitochondria. CISY has been reported to regulate cell proliferation via STAT-3 pathway [48]. Overexpression of the protein was observed in pancreatic and ovarian cancers [49].

cisplatin with patulin using 4/0 sequence of administration.

ro of

Upregulation of CISY was observed in A2780 cells after treatment with combination of

40 S ribosomal subunit A (RSSA) is a 67 kDa protein which is abundantly located in

-p

cytoplasm, but is also found in nucleus as a component of nuclear structures. Overexpression of RSSA has been considered as prognostic marker in variety of cancers including breast

re

carcinoma, small cell lung carcinoma, pancreatic adenocarcinoma, ovarian carcinoma and

lP

gastric carcinoma [50, 51]. Moreover, the protein has been reported to mediate resistance towards chemotherapy e.g. against vincristine in gastric cancer [52]. After treatment with combination of cisplatin with emetine using 4/0 sequence of administration, the protein was

na

upregulated in A2780cisR cell line.

ur

5. Conclusion

Combination of cisplatin with emetine and cisplatin of patulin demonstrated synergism

Jo

against in vitro ovarian cancer models depending on dose and sequence of drug administration. Combined treatments of cisplatin with phytochemicals (emetine/patulin) could be a better means of treating ovarian cancer patients with minimal side effects due to reduced dose of combined drugs. Synergism obtained from the combination of cisplatin with emetine might be due to downregulation of VIME, ENPL, GRP78, CARL, NACA and COF1 as well as upregulation of PPIA and RSSA. Synergistic activity from combination cisplatin

with patulin could be attributed to the downregulation of VIME, COF1 and CH10 as well as upregulation of CISY.

Acknowledgements Md Nur Alam is grateful to Australian Government for granting Endeavour Postgraduate Scholarship.

Competing Interests

ro of

The authors have declared that no competing interest exists.

CONFLICT OF INTEREST

Jo

ur

na

lP

re

-p

I am Dr. Md Nur Alam, states in favor of all authors of the submitted manuscript that, no conflict of interests exist.

References

Jo

ur

na

lP

re

-p

ro of

[1] G.C. Jayson, E.C. Kohn, H.C. Kitchener, J.A. Ledermann, The Lancet, 384 (2014) 1376-1388. [2] S. Vaughan, J.I. Coward, R.C. Bast Jr, A. Berchuck, J.S. Berek, J.D. Brenton, G. Coukos, C.C. Crum, R. Drapkin, D. Etemadmoghadam, M. Friedlander, H. Gabra, S.B. Kaye, C.J. Lord, E. Lengyel, D.A. Levine, I.A. McNeish, U. Menon, G.B. Mills, K.P. Nephew, A.M. Oza, A.K. Sood, E.A. Stronach, H. Walczak, D.D. Bowtell, F.R. Balkwill, Nature Reviews. Cancer, 11 (2011) 719-725. [3] W. Shen, X. Chen, J. Luan, D. Wang, L. Yu, J. Ding, ACS applied materials & interfaces, 9 (2017) 40031-40046. [4] J. Lehar, A.S. Krueger, W. Avery, A.M. Heilbut, L.M. Johansen, E.R. Price, R.J. Rickles, G.F. Short, 3rd, J.E. Staunton, X. Jin, M.S. Lee, G.R. Zimmermann, A.A. Borisy, Nature biotechnology, 27 (2009) 659-666. [5] C. He, Z. Tang, H. Tian, X. Chen, Advanced drug delivery reviews, 98 (2016) 64-76. [6] G.B. Maru, R.R. Hudlikar, G. Kumar, K. Gandhi, M.B. Mahimkar, World Journal of Biological Chemistry, 7 (2016) 88-99. [7] M.U. Nessa, P. Beale, C. Chan, J.Q. Yu, F. Huq, Anticancer research, 31 (2011) 3789-3797. [8] N.M. Yunos, P. Beale, J.Q. Yu, F. Huq, Anticancer Research, 31 (2011) 1131-1140. [9] M.U. Nessa, P. Beale, C. Chan, J.Q. Yu, F. Huq, Anticancer research, 32 (2012) 53-59. [10] L. Arzuman, P. Beale, J.Q. Yu, F. Huq, Anticancer research, 36 (2016) 2809-2818. [11] F. Panettiere, C.A. Coltman, Jr., Cancer, 27 (1971) 835-841. [12] R.C. Kane, M.H. Cohen, L.E. Broder, M.I. Bull, P.J. Creaven, B.E. Fossieck Jr, CANCER CHEMOTHER.REP., 59 (1975) 1171-1172. [13] M. Moller, K. Herzer, T. Wenger, I. Herr, M. Wink, Oncology reports, 18 (2007) 737-744. [14] Q. Sun, S. Yogosawa, Y. Iizumi, T. Sakai, Y. Sowa, International journal of oncology, 46 (2015) 389-394. [15] V.J. Davidson, D. Patel, R. Flanigan, G.N. Gupta, K.E. Foreman, 2017, (2017). [16] P.J. Becci, F.G. Hess, W.D. Johnson, M.A. Gallo, J.G. Babish, R.E. Dailey, R.A. Parent, Journal of Applied Toxicology, 1 (1981) 256-261. [17] D. Choudhary, G. Sahay, J. Singh, Journal of food science and technology, 29 (1992) 264-265. [18] M. Boussabbeh, I. Ben Salem, A. Prola, A. Guilbert, H. Bacha, S. Abid-Essefi, C. Lemaire, Toxicological Sciences, 144 (2015) 328-337. [19] M. Abastabar, A. Akbari, J. Akhtari, M.T. Hedayati, T. Shokohi, H. Mehrad-Majd, H. Ghalehnoei, S. Ghasemi, Current medical mycology, 3 (2017) 25. [20] M. Boussabbeh, I.B. Salem, K. Rjiba-Touati, C. Bouyahya, F. Neffati, M.F. Najjar, H. Bacha, S. Abid-Essefi, Tumor Biology, 37 (2016) 6285-6295. [21] S. Dhara, Indian J Chem, 8 (1970) 193-194. [22] T. Mosmann, Journal of immunological methods, 65 (1983) 55-63. [23] T.-C. Chou, P. Talalay, Advances in enzyme regulation, 22 (1984) 27-55. [24] T.-C. Chou, Pharmacological reviews, 58 (2006) 621-681. [25] Z. Al-Eisawi, P. Beale, C. Chan, J.Q. Yu, N. Proschogo, M. Molloy, F. Huq, BMC cancer, 16 (2016) 688. [26] E. S Akinboye, O. Bakare, The Open Natural Products Journal, 4 (2011). [27] K.E. Foreman, J.N. Jesse, P.C. Kuo, G.N. Gupta, The Journal of urology, 191 (2014) 502-509. [28] Y. Pillay, A. Phulukdaree, S. Nagiah, A. Chuturgoon, Toxicon, 99 (2015) 1-5. [29] N. Zouaoui, B. Mallebrera, H. Berrada, S. Abid-Essefi, H. Bacha, M.-J. Ruiz, Food and Chemical Toxicology, 89 (2016) 92-103. [30] F. Sarubbi, G. Formisano, G. Auriemma, A. Arrichiello, R. Palomba, Food Control, 59 (2016) 420-423. [31] Q. Sun, S. Yogosawa, Y. Iizumi, T. Sakai, Y. Sowa, International journal of oncology, 46 (2015) 389-394. [32] A. Satelli, S. Li, Cellular and molecular life sciences, 68 (2011) 3033-3046.

Jo

ur

na

lP

re

-p

ro of

[33] B.X. Wu, F. Hong, Y. Zhang, E. Ansa-Addo, Z. Li, Advances in cancer research, 129 (2016) 165190. [34] W.-T. Chen, D. Ha, G. Kanel, A.S. Lee, Neoplasia, 16 (2014) 617-626. [35] B.X. Wu, F. Hong, Y. Zhang, E. Ansa-Addo, Z. Li, GRP94/gp96 in cancer: biology, structure, immunology, and drug development, in: Advances in cancer research, Elsevier, 2016, pp. 165-190. [36] K.-W. Park, G. Eun Kim, R. Morales, F. Moda, I. Moreno-Gonzalez, L. Concha-Marambio, A.S. Lee, C. Hetz, C. Soto, Scientific Reports, 7 (2017) 44723. [37] M.M. Lizardo, J.J. Morrow, T.E. Miller, E.S. Hong, L. Ren, A. Mendoza, C.H. Halsey, P.C. Scacheri, L.J. Helman, C. Khanna, Neoplasia, 18 (2016) 699-710. [38] A.S. Lee, Cancer research, 67 (2007) 3496-3499. [39] M. Zamanian, L.A.Q. Hamadneh, A. Veerakumarasivam, S.A. Rahman, S. Shohaimi, R. Rosli, Cancer cell international, 16 (2016) 56. [40] M.-J. Lee, G.R. Yu, H. Lee, Y.A. Kim, J. Zhang, D.G. Kim. [41] H. Yang, M. Li, H. Chai, S. Yan, P. Lin, A.B. Lumsden, Q. Yao, C. Chen, Journal of Surgical Research, 123 (2005) 312-319. [42] H. Fujioka, A. Sakai, S. Tanaka, K. Kimura, A. Miyamoto, M. Iwamoto, K. Uchiyama, Oncology letters, 13 (2017) 289-295. [43] S. Akyol, C. Gercel-Taylor, L.C. Reynolds, D.D. Taylor, Gynecologic oncology, 101 (2006) 481486. [44] J. Wu, T. Liu, Z. Rios, Q. Mei, X. Lin, S. Cao, Trends in pharmacological sciences, 38 (2017) 226256. [45] F. Cappello, A. Di Stefano, S. David, F. Rappa, R. Anzalone, G. La Rocca, S.E. D'Anna, F. Magno, C.F. Donner, B. Balbi, Cancer, 107 (2006) 2417-2424. [46] W. Wang, R. Eddy, J. Condeelis, Nature reviews. Cancer, 7 (2007) 429. [47] S. Mousavi, R. Safaralizadeh, M. Hosseinpour-Feizi, A. Azimzadeh-Isfanjani, S. Hashemzadeh, Journal of Gastrointestinal Oncology, 9 (2018) 791. [48] S. MacPherson, M. Horkoff, C. Gravel, T. Hoffmann, J. Zuber, J.J. Lum, Cell Reports, 19 910918. [49] L. Chen, T. Liu, J. Zhou, Y. Wang, X. Wang, W. Di, S. Zhang, PloS one, 9 (2014) e115708. [50] L. Liu, X. Ning, L. Sun, Y. Shi, S. Han, C. Guo, Y. Chen, S. Sun, F. Yin, K. Wu, Molecular and cellular biochemistry, 303 (2007) 151-160. [51] S. Ménard, E. Tagliabue, M.I. Colnaghi, Breast cancer research and treatment, 52 (1998) 137145. [52] L. Sun, L. Liu, X. Liu, Y. Wang, M. Li, L. Yao, J. Yang, G. Ji, C. Guo, Y. Pan, Oncology reports, 32 (2014) 105-114.

Table 1: Cytotoxicity (IC50 values in μM) of the single drugs against ovarian cancer cell lines and RF values A2780cisR

RF

A2780ZDO473R

RF

Cisplatin

1.12 ± 0.13

11.54 ± 0.98

10.30

10.03 ± 1.02

8.95

Emetine

0.023 ± 0.0009

0.018 ± 0.0009

0.78

0.022 ± 0.001

0.95

Patulin

1.47 ± 0.12

1.28 ± 0.08

0.87

1.44 ± 0.10

0.97

Jo

ur

na

lP

re

-p

ro of

Compound A2780

Table 2: CI values (at ED50, ED75, ED90) and dose-effect parameters (median effect dose Dm, the exponent defining the shape of the dose effect curve m, correlation coefficient r) applying to combinations of cisplatin with emetine and patulin in the A2780 and A2780cisR cell line Cell line

Drug or drug combination

Sequence Molar (h) Ratio

CI values at ED50 ED75 ED90 Dm

r

N/A

N/A

N/A

1.88 1.40 0.91

Emetine

N/A

N/A

N/A

0.05 2.01 0.84

Cisplatin+Emetine 0/0

0.96

0.84

0.75

0.68 2.20 1.00

0.53

0.70

0.94

0.38 1.20 0.97

Cisplatin+Emetine 4/0

0.86

0.76

0.67

0.61 2.22 0.99

Cisplatin

N/A

N/A

N/A

1.88 1.40 0.91

Patulin

N/A

N/A

N/A

0.47 1.18 0.92

1.06

0.93

0.82

0.28 1.40 0.98

0/0

Cisplatin+Patulin

0/4

Cisplatin+Patulin

4/0

1:1.53

-p

Cisplatin+Patulin

1:0.04

ro of

Cisplatin

Cisplatin+Emetine 0/4

1.09

1.03

0.97

0.29 1.29 1.00

0.93

0.88

0.83

0.25 1.29 0.99

N/A

N/A

N/A

7.07 1.30 0.99

N/A

N/A

N/A

0.03 1.56 0.86

Cisplatin+Emetine 0/0

1.89

1.41

1.06

7.19 2.25 0.97

Cisplatin+Emetine 0/4

1:0.004 0.91

0.84

0.79

3.47 1.57 1.00

0.86

0.79

0.72

3.28 1.60 0.99

N/A

N/A

N/A

7.07 1.30 0.99

N/A

N/A

N/A

0.89 1.26 0.98

0.63

0.75

0.90

1.62 1.06 1.00

2.14

1.45

0.99

5.47 2.31 0.97

0.88

0.84

0.81

2.24 1.34 0.98

na

Emetine

lP

Cisplatin

re

A2780

m

Cisplatin+Emetine 4/0

ur

A2780CisR Cisplatin

0/0

Cisplatin+Patulin

0/4

Cisplatin+Patulin

4/0

Patulin

Jo

Cisplatin+Patulin

1:0.22

Table 3: Protein spots which underwent significant changes in expression after combined treatment of cisplatin with emetine using 0/4 sequence of administration in A2780 cell line and their identification (name, mass, Da/pI, mascot score, matched peptides, percent of sequence coverage). Spot No

Change in Expression

Fold change

Protein name

Mass (Da)/pI

Mascot No of score matched peptides

Sequence coverage

739

6

45

21

(%)

Downregulated

2.0

Vimentin

53619 / 5.06

18

Downregulated

3.05

Endoplasmin

92411/4 647 .76

36

45

Downregulated

1.71

78 kDa glucoseregulated protein

72288/5 820 .07

28

27

89

Downregulated

1.65

Calreticulin

48112/4 112 .29

8

17

118

Downregulated

3.91

PolyubiquitinB

25746

‫٭‬

‫٭‬

196

Downregulated

2.57

Nascent polypeptideassociated complex subunit alpha

23370/4 110 .52

12

32

235

Upregulated

Peptidyl-prolyl cis-trans isomerase A

18001/7 483 .68

21

68

-p

re

lP

na 5.26

ur Jo

ro of

12

40

Table 4: Protein spots that underwent significant changes in expression after combined treatment in A2780CisR cell line and their identification (name, mass, Da/pI, mascot score, matched peptides, percent of sequence coverage). Spot No

Treatment

Change in Expression

Fold change

Protein name Mass (Da)/pI

Mascot score

No of matched peptides

Sequence coverage (%)

Cisplatin + Emetine

Upregulated

3.04

40S ribosomal protein SA

32833 / 4.79

130

10

17

Cn69

Cisplatin + Emetine

Downregulated

1.86

Cofilin-1

18491/ 8.22

144

10

22

Cn41

Cisplatin + Patulin

Upregulated

2.27

Citrate synthase, mitochondrial

51680 / 8.45

97

14

16

Cn48

Cisplatin + Patulin

Downregulated

1.63

Vimentin

53619 / 5.06

285

25

30

Cn65

Cisplatin + Patulin

Downregulated

3.40

10 kDa heat 10925 / shock protein, 8.89 mitochondrial

78

9

33

Cn69

Cisplatin + Patulin

Downregulated

11.04

Cofilin-1

144

10

22

-p

re

lP

na ur Jo

ro of

Cn9

18491/ 8.22