OL Oncology Letters 1792-1074 1792-1082 D.A. Spandidos 10.3892/ol.2018.7743 OL-0-0-7743 Articles Cardamonin enhances the anti-proliferative effect of cisplatin on ovarian cancer NiuPeiguang ShiDaohua ZhangShusheng ZhuYanting ZhouJintuo Department of Pharmacy, Fujian Provincial Maternity and Children Hospital, Affiliated Hospital of Fujian Medical University, Fuzhou, Fujian 350001, P.R. China Correspondence to: Professor Daohua Shi, Department of Pharmacy, Fujian Provincial Maternity and Children Hospital, Affiliated Hospital of Fujian Medical University, 18 Daoshan Road, Fuzhou, Fujian 350001, P.R. China, E-mail: shidh@yeah.net 03 2018 08 01 2018 15 3 3991 3997 15082016 09092017 Copyright © 2018, Spandidos Publications 2018

The mammalian target of rapamycin (mTOR) is well-known as a promising therapeutic target in various cancer cells. mTOR activation decreases the sensitivity of ovarian cancer to cisplatin. Cardamonin inhibits the proliferation of various cancer cells by mTOR suppression. The present study examined whether cardamonin combined with cisplatin is efficacious for the anti-proliferation of ovarian cancer cells. The anti-proliferative effect was determined by MTT and cell cycle assays. Activation of the mTOR signal pathway and the expression of anti-apoptotic proteins were evaluated by western blot analysis. Cardamonin significantly enhanced the effects of cisplatin on cell proliferation and cell cycle progression. The expression of B cell lymphoma-2, X-linked inhibitor of apoptosis protein and Survivin was significantly decreased following combination treatment. Furthermore, the activation of mTOR and its downstream 70 kDa ribosomal protein S6 kinase was inhibited by cardamonin. These results demonstrated that the combinatorial effects of cardamonin and cisplatin on anti-proliferation were enhanced by suppressing the expression of anti-apoptotic proteins and activation of mTOR in ovarian cancer cells.

 cardamonin cisplatin mammalian target of rapamycin apoptosis ovarian cancer Introduction

Ovarian cancer is a major cause of gynecological malignancy-associated mortality worldwide. The majority of patients are diagnosed with epithelial ovarian cancer at the advanced stage and the 5-year survival rate is 20–40% (1). Platinum-based adjuvant chemotherapy following cytoreductive surgery is commonly used for treatment of ovarian cancer, which is initially effective in a high percentage of cases (2). However, an increasing number of patients eventually relapse due to the decreased sensitivity to cisplatin (3,4). Therefore, novel therapeutic strategies are required to increase the sensitivity of cancer cells to cisplatin.

Chemotherapeutic drug-induced apoptosis is usually disturbed by the anti-apoptotic proteins, including B cell lymphoma-2 (Bcl-2), X-linked inhibitor of apoptosis protein (XIAP) and Survivin (5). Bcl-2 regulates programmed cell death in various cancer cell lines. It exerts a survival function in response to a wide range of apoptotic stimuli and inhibits the release of mitochondrial cytochrome c (6). XIAP is well known as a direct inhibitor of apoptosis, which binds to caspase-3, −7 and −9 and prevents their activities (7). Survivin interferes with apoptosis by inhibiting the activity of caspases or via interaction with other regulators. It has been demonstrated that Survivin binds to XIAP and inhibits the activation of caspase-9 (8). The overexpression of these proteins is involved in the insensitivity of epithelial ovarian cancer cells to cisplatin (9–11).

Mammalian target of rapamycin (mTOR) activation is essential in the development of cisplatin insensitivity in ovarian cancer cells. It was reported that the phosphorylation level of mTOR was higher in cisplatin-insensitive clear-cell ovarian carcinoma cells compared with in cisplatin-sensitive cells (12,13). mTOR also performs a key role in the modulation of cisplatin-induced apoptotic cell death. Cisplatin activates the mTOR survival pathway in epithelial and clear-cell carcinoma ovarian cancer cells, which may counteract cisplatin-induced apoptosis and in turn lead to cisplatin resistance (14,15). The mTOR inhibitors, rapamycin and its analogs, including everolimus, efficiently enhance the therapeutic efficacy of platinum in clear-cell ovarian carcinoma cells (16,17). Therefore, mTOR is a promising therapeutic target for the treatment of ovarian cancer when combined with cisplatin.

Cardamonin is a natural chalcone compound, which increases cell apoptosis in nasopharyngeal carcinoma, prostate cancer and triple negative breast cancer cells and induces autophagy in colorectal carcinoma cells (18–21). Cardamonin inhibits a number of signaling pathways, including c-Jun N-terminal kinase, nuclear factor-κB, and signal transducer and activator of transcription 3 (21,22). Combined with doxorubicin, cardamonin sufficiently prevents the enrichment of drug-resistant triple negative breast cancer stem cells and retards tumor growth (23). It may also increase the anti-proliferative effect of cisplatin in cervical carcinoma, hepatocellular carcinoma, prostate cancer and colon cancer cells (24). However, the underlying mechanism has not been previously studied. Our previous studies demonstrated that cardamonin inhibits the proliferation, angiogenesis and metastasis of non-small cell lung carcinoma cells, epithelial ovarian cancer cells and Lewis lung carcinoma cells by inhibition of mTOR activation (25–27). In the current study, the therapeutic potency of cardamonin combined with cisplatin in ovarian cancer was evaluated and the underlying mechanism was investigated.

 Materials and methods <sec> <title>Chemical reagents

Solutions and supplements for cell culture were purchased from Gibco (Thermo Fisher Scientific, Inc., Waltham, MA, USA). Cardamonin, cisplatin, dimethyl sulfoxide (DMSO) and MTT were purchased from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany). Antibodies against mTOR (cat. no. 2972; dilution, 1:1,000), phospho-mTOR (Ser 2448; cat. no. 2971; dilution, 1:1,000), 70 kDa ribosomal protein S6 kinase (p70S6K; cat. no. 9202; dilution, 1:1,000), phospho-p70S6K (Thr 389; cat. no. 9205; dilution, 1:1,000), Bcl-2 (cat. no. 2872; dilution, 1:1,000), Survivin (cat. no. 2803; dilution, 1:1,000), XIAP (cat. no. 2042; dilution, 1:1,000) and actin (cat. no. 4967; dilution, 1:1,000), and the secondary antibody (anti-rabbit IgG, horseradish peroxidase-linked antibody; cat. no. 7074; dilution, 1:2,000) were purchased from Cell Signaling Technologies, Inc. (Danvers, MA, USA). The cell cycle assay kit was from BD Biosciences (San Jose, CA, USA).

 Cell culture

Epithelial ovarian cancer SKOV3 and A2780 cells were obtained from Wuhan Boster Bio-Engineering Ltd. Co. (Wuhan, China), and cultured, at 37°C in a saturated humidity incubator with an atmosphere containing 5% CO2, in McCoy's 5A and high glucose Dulbecco's modified Eagle's medium (DMEM; Gibco; Thermo Fisher Scientific, Inc.), respectively. The media was supplemented with 10% fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc.), penicillin (100 U/ml) and streptomycin (100 µg/ml) (HyClone; GE Healthcare Life Sciences, Little Chalfont, UK).

 Cell viability assay

MTT assay was used to analyze the effect of cardamonin on cell viability as previously described (28). Cells were cultured overnight at 37°C in a saturated humidity incubator with an atmosphere containing 5% CO2 in 96-well plates (5×103 cells/well). Cell viability was assessed following the addition of cardamonin and cisplatin at the indicated concentrations for 48 h in medium containing 10% FBS. Surviving cells were assessed using a microplate reader (Bio-Rad Laboratories, Inc., Hercules, CA, USA) for the determination of the absorbance at 570 nm of the DMSO-dissolved formazan product following the addition of MTT (5 mg/ml) for 4 h. Five independent experiments were performed.

 Clonogenic survival assay

The clonogenic survival assay was performed as previously described (28). Cells (1×103/well) were seeded on 6-well tissue culture plates in medium containing 10% FBS. The cells were cultured overnight at 37°C in a saturated humidity incubator with an atmosphere containing 5% CO2, and then treated with cisplatin in the presence or absence of cardamonin for 48 h, followed by two washes in McCoy's 5A and DMEM, respectively. The cells were then cultured in medium containing 10% FBS and allowed to proliferate for 7 days. Cells were fixed with ethanol (75%) at room temperature for 15 min and stained by 1% crystal violet at room temperature for 60 min. Colonies (>30 cells/colony) were counted with a Leica DMIL LED microscope (magnification, ×200; Leica Microsystems GmbH, Wetzlar, Germany) for each group to generate survival rate in triplicate wells. Five independent experiments were performed.

 Cell cycle assay

Cell cycle distribution was determined by flow cytometry based on DNA content measurement of nuclei, stained with propidium iodide (PI). Following treatment with cisplatin in the presence or absence of cardamonin for 48 h, cells were the washed with cold PBS and harvested by centrifugation at 400 × g at room temperature for 5 min. Cells were then fixed in cold ethanol (70%) overnight at 4°C. Next, the cells were stained with 50 µg/ml PI and 10 µg/ml RNase A (BD Biosciences, San Jose, CA, USA) in the dark at room temperature for 30 min in the dark. Cell cycle distribution was analyzed by flow cytometry (BD Biosciences). Results were calculated by the Mod Fit LT software version 2.0 (BD Biosciences). Five independent experiments were performed.

 Western blot analysis

Treated with indicated drugs for 48 h, cells were washed with ice-cold PBS twice and lysed in radioimmunoprecipitation assay lysis buffer (20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM Na2 EDTA, 1 mM EGTA, 1% NP-40, 1% sodium deoxycholate, 2.5 mM sodium pyrophosphate, 1 mM beta-glycerophosphate, 1 mM Na3VO4 and 1 µg/ml leupeptin) with the addition of 1 mM phenylmethylsulfonyl fluoride for 20 min at 4°C. Lysates were centrifuged at 15,000 × g at 4°C for 20 min, and protein concentrations of the supernatants were determined using a bicinchoninic acid protein assay reagent. Proteins (40 µg) were separated by 6–12% SDS-PAGE and transferred to polyvinylidene difluoride membranes. The membranes were blocked with 5% bovine serum albumin (Cell Signaling Technologies, Inc.) in 1X TBS-Tween 20 for 1 h at room temperature and then incubated overnight at 4°C with the following primary antibodies: mTOR (cat. no. 2972; dilution, 1:1,000), Ser 2448 (cat. no. 2971; dilution, 1:1,000), p70S6K (cat. no. 9202; dilution, 1:1,000), Thr 389 (cat. no. 9205; dilution, 1:1,000), Bcl-2 (cat. no. 2872; dilution, 1:1,000), Survivin (cat. no. 2803; dilution, 1:1,000), XIAP (cat. no. 2042; dilution, 1:1,000) and actin (cat. no. 4967; dilution, 1:1,000) all purchased from Cell Signaling Technologies, Inc. Immunoblots were visualized with horseradish peroxidase-conjugated goat anti-rabbit immunoglobulin (Cell Signaling Technologies, Inc.; cat. no. 7074; dilution, 1:2,000) at room temperature for 1 h using enhanced chemiluminescence (GE Healthcare Life Sciences) and exposure to X-ray film to produce bands within the linear range. The protein purity was evaluated by densitometry analysis using the Quantity One software version 4.6.2 (Bio-Rad Laboratories, Inc.). Three independent experiments were performed.

 Statistical analysis

All data are expressed as the mean ± standard deviation. Statistical analysis was performed with SPSS version 16.0 software (SPSS, Inc., Chicago, IL, USA). Data were analyzed by one-way analysis of variance and Tukey-Kramer multiple comparison test. P<0.05 was considered to indicate a statistically significant difference.

 Results <sec> <title>Anti-proliferation efficacy of cisplatin is enhanced by cardamonin

Our previous study demonstrated that 20 µM cardamonin effectively inhibited the activation of mTOR in SKOV3 cells (unpublished data). In the present study, whether 20 µM cardamonin enhanced the efficacy of cisplatin was determined. The anti-proliferation effect of cisplatin (1, 2, 4 and 8 µg/ml) was enhanced by cardamonin in a dose-dependent manner in SKOV3 and A2780 cells (Fig. 1A). Cisplatin at a concentration of 2 µg/ml was selected for the following experiments according to its nephrotoxicity in clinical use. Furthermore, the effect of cardamonin combined with cisplatin (2 µg/ml) on anti-proliferation was more potent in SKOV3 cells compared with that in A2780 cells (cell viability, 34.8±3.14 vs. 43.1±3.82%; P<0.05). In addition, the clone formation was significantly inhibited by cardamonin combined with cisplatin in SKOV3 and A2780 cells (Fig. 1B).

 Cell cycle is arrested by cardamonin and cisplatin at the G2/M phase

It was then determined whether cardamonin was able to enhance cisplatin-induced cell cycle arrest. As presented in Fig. 2, cardamonin and cisplatin induced the accumulation of cells in the S, and G2/M phase. As expected, cardamonin significantly increased the cisplatin-induced accumulation of cells in the G2/M phase, and the accumulation of cells in G0/G1 phases was significantly reduced (Table I).

 Anti-apoptotic proteins are decreased by cardamonin and cisplatin co-administration

Cisplatin-induced apoptosis may be disturbed by the anti-apoptotic proteins. The effect of cardamonin on anti-apoptotic proteins was examined. Cisplatin and cardamonin decreased the protein expression of XIAP and Survivin. Notably, it was demonstrated that combination of the two agents significantly reduced the protein expression of Bcl-2, XIAP and Survivin in SKOV3 cells (Fig. 3).

 Activation of mTOR and p70S6K is attenuated by cardamonin

Elevated phosphorylation of mTOR and p70S6K was detected in SKOV3 cells. To examine the effect of mTOR pathway inhibition by cardamonin on ovarian cancer cells, the effect of cardamonin and cisplatin on the phosphorylation of mTOR and p70S6K was examined. No significant effects were observed on the phosphorylation of mTOR and its downstream target (p70S6K) following cisplatin treatment; whereas the activation of mTOR signaling was significantly inhibited by cardamonin, or by the combination of cardamonin and cisplatin (Fig. 4).

 Discussion

Platinum-based chemotherapy is the most commonly used therapeutic approach for ovarian cancer following initial cytoreductive surgery. Although ovarian cancer is highly responsive to cisplatin, there is a substantial risk of emergence of drug resistance, which results in a poorer clinical outcome (29,30). Thus, improving the antitumor efficiency of cisplatin is an essential way for patients with ovarian cancer. A number of flavonoid compounds, including baicalein, apigenin and fisetin may enhance the cytotoxicity of cisplatin in head and neck squamous cell carcinoma, embryonal carcinoma and cervical carcinoma cells (31–33). Epithelial ovarian cancer accounts for almost 80% of all ovarian cancer cases and is the leading cause of cancer-associated mortality among women (34). Therefore, it was investigated whether cardamonin was able to potentiate the anticancer effect of cisplatin in epithelial ovarian cancer cells, including SKOV3 and A2780 cell lines. The results demonstrated that cardamonin significantly enhanced the anti-proliferative effect of cisplatin in SKOV3 and A2780 cells. The combinatorial effect on anti-proliferation using low concentrations of cisplatin was similar to that of cisplatin in the high concentration, which indicated that cardamonin may decrease the dose of cisplatin required to inhibit the proliferation of ovarian cancer cells, which may be beneficial in gaining clinical efficacy with less adverse effects.

Numerous chemotherapeutic agents, including cisplatin, paclitaxel and fluorouracil, disrupt the cell cycle, which ultimately inhibits cell proliferation (35,36). Cell cycle arrest is a major cellular response to DNA damage. Cisplatin was able to induce G2/M arrest in ovarian cancer cells (37,38). A previous study demonstrated that cardamonin inhibits cell growth and induces cell cycle arrest at the G2/M phase in colon cancer SW480 cells by suppressing the expression of cyclin D1, and c-myc (39). The results of the present study revealed that cardamonin increased the accumulation of cells in the S and G2/M phase, and the combination of cardamonin and cisplatin significantly increased the cisplatin-induced G2/M arrest, which indicated that the potential anti-proliferative effect of cardamonin was associated with cell cycle arrest.

The sensitivity of cancer cells to chemotherapeutic drug-induced apoptosis depends on the balance between pro-apoptotic and anti-apoptotic signals, and the inhibition of anti-apoptotic signals has been proposed as a promising strategy to enhance the efficacy of conventional chemotherapeutic agents (40). Several studies have reported that cardamonin is able to inhibit the expression of Bcl-2, Survivin and induce apoptosis (22,41). Furthermore, the mTOR inhibitor CCI-779 decreases the expression of anti-apoptotic proteins and restores the sensitivity of cisplatin-resistant small cell lung cancer cells to cisplatin (42). Therefore, additional study is required to clarify whether cardamonin potentiates the anti-proliferative effect of cisplatin through the anti-apoptotic proteins. The results of the present study revealed that cardamonin significantly enhanced the efficacy of cisplatin by inhibiting the anti-apoptotic signals (Bcl-2, XIAP and Survivin).

mTOR activation is associated with tumor malignancy and poor prognosis in numerous types of human cancer. In addition, mTOR is frequently hyper-activated in cisplatin-insensitive ovarian cancer cells (43). In the present study, it was demonstrated that the activation of mTOR and p70S6K was significantly inhibited by cardamonin, and cardamonin combined with cisplatin. This indicated that cardamonin may prevent the occurrence of cisplatin resistance and reduce the incidence of poor prognosis of patients with ovarian cancer induced by mTOR activation. mTOR inhibition enhances the sensitivity of cisplatin in numerous types of human cancers, including esophageal squamous cell carcinoma, lung cancer and ovarian cancer (42,44,45). RAD001, the analog of rapamycin, in combination with cisplatin was demonstrated to induce a distinct increase in the number of apoptotic cells by downregulating the pro-survival molecules, Bcl-2, Survivin and Cyclin D1 in hepatocellular carcinoma (46), which was consistent with the present results. Therefore, mTOR inhibition by cardamonin provides a strategy for increasing the efficacy of platinum-based treatment for ovarian cancer.

The clinical application of cisplatin is reduced by its dose-dependent toxicity to normal organs, including the liver and kidney. Cardamonin significantly enhanced the anti-proliferative effect of low concentration of cisplatin in ovarian cancer SKOV3 and A2780 cells, which may be useful in reducing the toxicity of cisplatin on normal tissues. In addition, compared with rapamycin, cardamonin has a number of advantages in vivo, it attenuated the oxidative stress and inflammation to protect against cisplatin-induced nephrotoxicity in rats (24).

In conclusion, these results indicated that the combination of cardamonin and cisplatin is more effective in anti-proliferation compared with the agent used alone in ovarian cancer cells. Cardamonin combined with cisplatin is a promising therapeutic strategy, particularly for patients with mTOR hyperactivation, which is beneficial for increasing the sensitivity to cisplatin in ovarian cancer.

 Acknowledgements

The present study was supported by the Youth Scientific Foundation of Fujian Provincial Health and Family Planning Commission (grant no. 2013-1-11) and the Natural Science Foundation of Fujian Province (grant nos. 2015J01368 and 2016J01492), China.

 References SiegelRNaishadhamDJemalACancer statistics, 2013CA Cancer J Clin631130201310.3322/caac.2116623335087 QianXQinJPanSLiXPanYMaSMaintenance therapy in ovarian cancer with targeted agents improves PFS and OS: A systematic review and meta-analysisPLoS One10e0139026201510.1371/journal.pone.013902626402447 ChenJSolomidesCParekhHSimpkinsFSimpkinsHCisplatin resistance in human cervical, ovarian and lung cancer cellsCancer Chemother Pharmacol7512171227201510.1007/s00280-015-2739-225894720 WangJWuGSRole of autophagy in cisplatin resistance in ovarian cancer cellsJ Biol Chem2891716317173201410.1074/jbc.M114.55828824794870 MakinGDiveCApoptosis and cancer chemotherapyTrends Cell Biol11S22S26200110.1016/S0962-8924(01)82111-511684438 ShaltoukiAFreerMMeiYWeymanCMIncreased expression of the pro-apoptotic Bcl2 family member PUMA is required for mitochondrial release of cytochrome C and the apoptosis associated with skeletal myoblast differentiationApoptosis1221432154200710.1007/s10495-007-0135-z17879164 ScottFLDenaultJBRiedlSJShinHRenatusMSalvesenGSXIAP inhibits caspase-3 and −7 using two binding sites: Evolutionarily conserved mechanism of IAPsEMBO J24645655200510.1038/sj.emboj.760054415650747 ZumbrägelFKMachtensDACurthULüderCGReuboldTFEschenburgSSurvivin does not influence the anti-apoptotic action of XIAP on caspase-9Biochem Biophys Res Commun482530535201710.1016/j.bbrc.2016.11.09427865841 YangQWangHChenSLanXXiaoHShiHMaYFiber-optic-based micro-probe using hexagonal 1-in-6 fiber configuration for intracellular single-cell pH measurementAnal Chem8771717179201510.1021/acs.analchem.5b0104026118725 ZhangZXieZSunGYangPLiJYangHXiaoSLiuYQiuHQinLReversing drug resistance of cisplatin by hsp90 inhibitors in human ovarian cancer cellsInt J Clin Exp Med866876701201526221207 ZhaoWJDengBYWangXMMiaoYWangJNXIAP associated factor 1 (XAF1) represses expression of X-linked inhibitor of apoptosis protein (XIAP) and regulates invasion, cell cycle, apoptosis, and cisplatin sensitivity of ovarian carcinoma cellsAsian Pac J Cancer Prev1624532458201510.7314/APJCP.2015.16.6.245325824780 MabuchiSKawaseCAltomareDAMorishigeKSawadaKHayashiMTsujimotoMYamotoMKlein-SzantoAJSchilderRJmTOR is a promising therapeutic target both in cisplatin-sensitive and cisplatin-resistant clear cell carcinoma of the ovaryClin Cancer Res1554045413200910.1158/1078-0432.CCR-09-036519690197 XuSFuGBTaoZOuYangJKongFJiangBHWanXChenKMiR-497 decreases cisplatin resistance in ovarian cancer cells by targeting mTOR/P70S6K1Oncotarget62645726471201510.18632/oncotarget.476226238185 FengXLiLJiangHJiangKJinYZhengJDihydroartemisinin potentiates the anticancer effect of cisplatin via mTOR inhibition in cisplatin-resistant ovarian cancer cells: Involvement of apoptosis and autophagyBiochem Biophys Res Commun444376381201410.1016/j.bbrc.2014.01.05324462866 PengDJWangJZhouJYWuGSRole of the Akt/mTOR survival pathway in cisplatin resistance in ovarian cancer cellsBiochem Biophys Res Commun394600605201010.1016/j.bbrc.2010.03.02920214883 WuHZhaoYMuXWuHChenLLiuWMuYLiuJWeiXA silica-polymer composite nano system for tumor-targeted imaging and p53 gene therapy of lung cancerJ Biomater Sci Polym Ed26384400201510.1080/09205063.2015.101203525624096 MabuchiSHisamatsuTKawaseCHayashiMSawadaKMimuraKTakahashiKTakahashiTKurachiHKimuraTThe activity of trabectedin as a single agent or in combination with everolimus for clear cell carcinoma of the ovaryClin Cancer Res1744624473201110.1158/1078-0432.CCR-10-298721622721 LiYQinYYangCZhangHLiYWuBHuangJZhouXHuangBYangKWuGCardamonin induces ROS-mediated G2/M phase arrest and apoptosis through inhibition of NF-κB pathway in nasopharyngeal carcinomaCell Death Dis8e3024201710.1038/cddis.2017.40729048425 ZhangJSikkaSSiveenKSLeeJHUmJYKumarAPChinnathambiAAlharbiSABasappaRangappaKSCardamonin represses proliferation, invasion, and causes apoptosis through the modulation of signal transducer and activator of transcription 3 pathway in prostate cancerApoptosis22158168201710.1007/s10495-016-1313-727900636 ShrivastavaSJeengarMKThummuriDKovalAKatanaevVLMarepallySNaiduVGMCardamonin, a chalcone, inhibits human triple negative breast cancer cell invasiveness by downregulation of Wnt/β-catenin signaling cascades and reversal of epithelial-mesenchymal transitionBiofactors43152169201710.1002/biof.131527580587 KimYJKangKSChoiKCKoHCardamonin induces autophagy and an antiproliferative effect through JNK activation in human colorectal carcinoma HCT116 cellsBioorg Med Chem Lett2525592564201510.1016/j.bmcl.2015.04.05425959811 WuNLiuJZhaoXYanZJiangBWangLCaoSShiDLinXCardamonin induces apoptosis by suppressing STAT3 signaling pathway in glioblastoma stem cellsTumour Biol3696679676201510.1007/s13277-015-3673-y26150336 JiaDTanYLiuHOoiSLiLWrightKBennettSAddisonCLWangLCardamonin reduces chemotherapy-enriched breast cancer stem-like cells in vitro and in vivoOncotarget7771785201610.18632/oncotarget.581926506421 El-NagaRNPre-treatment with cardamonin protects against cisplatin-induced nephrotoxicity in rats: Impact on NOX-1, inflammation and apoptosisToxicol Appl Pharmacol2748795201410.1016/j.taap.2013.10.03124211271 TangYFangQShiDNiuPChenYDengJmTOR inhibition of cardamonin on antiproliferation of A549 cells is involved in a FKBP12 independent fashionLife Sci994451201410.1016/j.lfs.2014.01.06624508654 NiuPGZhangYXShiDHLiuYChenYYDengJCardamonin inhibits metastasis of lewis lung carcinoma cells by decreasing mTOR activityPLoS One10e0127778201510.1371/journal.pone.012777825996501 XueZGNiuPGShiDHLiuYDengJChenYYCardamonin inhibits angiogenesis by mTOR downregulation in SKOV3 cellsPlanta Med827075201626393941 MabuchiSAltomareDACheungMZhangLPoulikakosPIHensleyHHSchilderRJOzolsRFTestaJRRAD001 inhibits human ovarian cancer cell proliferation, enhances cisplatin-induced apoptosis, and prolongs survival in an ovarian cancer modelClin Cancer Res1342614270200710.1158/1078-0432.CCR-06-277017634556 González-MartínASánchez-LorenzoLBratosRMárquezRChivaLFirst-line and maintenance therapy for ovarian cancer: Current status and future directionsDrugs74879889201410.1007/s40265-014-0221-924848752 MouldTAn overview of current diagnosis and treatment in ovarian cancerInt J Gynecol Cancer22Suppl 1S2S4201210.1097/IGC.0b013e318251c8e322543914 ChanLPChouTHDingHYChenPRChiangFYKuoPLLiangCHApigenin induces apoptosis via tumor necrosis factor receptor- and Bcl-2-mediated pathway and enhances susceptibility of head and neck squamous cell carcinoma to 5-fluorouracil and cisplatinBiochim Biophys Acta182010811091201210.1016/j.bbagen.2012.04.01322554915 TripathiRSamadderTGuptaSSuroliaAShahaCAnticancer activity of a combination of cisplatin and fisetin in embryonal carcinoma cells and xenograft tumorsMol Cancer Ther10255268201110.1158/1535-7163.MCT-10-060621216935 WangYWangQZhangSZhangYTaoLBaicalein increases the cytotoxicity of cisplatin by enhancing gap junction intercellular communicationMol Med Rep10515521201410.3892/mmr.2014.215724736991 SankaranarayananRFerlayJWorldwide burden of gynaecological cancer: The size of the problemBest Pract Res Clin Obstet Gynaecol20207225200610.1016/j.bpobgyn.2005.10.00716359925 EliasSTBorgesGARêgoDFE SilvaLFAvelinoSDE Matos NetoJNSimeoniLAGuerraENCombined paclitaxel, cisplatin and fluorouracil therapy enhances ionizing radiation effects, inhibits migration and induces G0/G1 cell cycle arrest and apoptosis in oral carcinoma cell linesOncol Lett1017211727201510.3892/ol.2015.345826622739 LiYLiWDengWGanYWuKSunJSynergistic anti-proliferative and pro-apoptotic activities of 5F and cisplatin in human non-small cell lung cancer NCI-H23 cellsOncol Lett1453475353201729098029 ChenXGongLOuRZhengZChenJXieFHuangXQiuJZhangWJiangQSequential combination therapy of ovarian cancer with cisplatin and γ-secretase inhibitor MK-0752Gynecol Oncol140537544201610.1016/j.ygyno.2015.12.01126704638 Taylor-HardingBOrsulicSKarlanBYLiAJFluvastatin and cisplatin demonstrate synergistic cytotoxicity in epithelial ovarian cancer cellsGynecol Oncol119549556201010.1016/j.ygyno.2010.08.01720837358 ParkSGwakJHanSJOhSCardamonin suppresses the proliferation of colon cancer cells by promoting β-catenin degradationBiol Pharm Bull3610401044201310.1248/bpb.b13-0015823538439 WangHZhangZWeiXDaiRSmall-molecule inhibitor of Bcl-2 (TW-37) suppresses growth and enhances cisplatin-induced apoptosis in ovarian cancer cellsJ Ovarian Res83201510.1186/s13048-015-0130-x25823945 YadavVRPrasadSAggarwalBBCardamonin sensitizes tumour cells to TRAIL through ROS- and CHOP-mediated up-regulation of death receptors and down-regulation of survival proteinsBr J Pharmacol165741753201210.1111/j.1476-5381.2011.01603.x21797841 WangpaichitrMWuCYouMKuoMTFeunLLampidisTSavarajNInhibition of mTOR restores cisplatin sensitivity through down-regulation of growth and anti-apoptotic proteinsEur J Pharmacol591124127200810.1016/j.ejphar.2008.06.02818585380 AideNKinrossKCullinaneCRoseltPWaldeckKNeelsODorowDMcArthurGHicksRJ18F-FLT PET as a surrogate marker of drug efficacy during mTOR inhibition by everolimus in a preclinical cisplatin-resistant ovarian tumor modelJ Nucl Med5115591564201010.2967/jnumed.109.07328820847160 HouGYangSZhouYWangCZhaoWLuZTargeted inhibition of mTOR signaling improves sensitivity of esophageal squamous cell carcinoma cells to cisplatinJ Immunol Res2014845763201410.1155/2014/84576324818169 CaiYTanXLiuJShenYWuDRenMHuangPYuDInhibition of PI3K/Akt/mTOR signaling pathway enhances the sensitivity of the SKOV3/DDP ovarian cancer cell line to cisplatin in vitroChin J Cancer Res26564572201425400422 TamKHYangZFLauCKLamCTPangRWPoonRTInhibition of mTOR enhances chemosensitivity in hepatocellular carcinomaCancer Lett273201209200910.1016/j.canlet.2008.08.01818824293

Cardamonin enhanced the anti-proliferative effect of cisplatin in SKOV3 and A2780 cells. (A) SKOV3 and A2780 cells were treated with 1, 2, 4, 8 µg/ml of cisplatin in the presence or absence of 20 µM of cardamonin for 48 h, and an MTT assay was performed (n=5). Data are presented as the mean ± SD. *P<0.05 vs. control; #P<0.01 vs. the corresponding group treated with cisplatin alone in A2780 cells; $P<0.01 vs. the corresponding group treated with cisplatin alone in SKOV3 cells. (B) SKOV3 and A2780 cells (1×103) were treated with cardamonin (20 µM), cisplatin (2 µg/ml) or the combination of the two agents for 48 h, and then cells were processed for clonogenic survival assay (n=5). Data are presented as the mean ± SD. *P<0.05 vs. control; #P<0.01 vs. cisplatin; $P<0.01 vs. cardamonin. SD, standard deviation; DMSO, dimethyl sulfoxide.

Cardamonin and cisplatin induced cell cycle arrest in G2/M phase in SKOV3 cells. SKOV3 cells were treated with cardamonin (20 µM), cisplatin (2 µg/ml) or the combination of the two agents for 48 h, and then cells were processed for cell cycle analysis (n=5). DMSO, dimethyl sulfoxide.

Cardamonin combined with cisplatin decreased the expression of anti-apoptotic proteins. SKOV3 cells were treated with cardamonin (20 µM), cisplatin (2 µg/ml) or a combination of the two agents for 48 h. The expression of Bcl-2, XIAP and Survivin was determined by western blot analysis. Actin was used as the loading control (n=3). (A) Representative western blotting protein bands for Bcl-2, XIAP, Survivin and actin. Quantification of (B) Bcl-2, (C) XIAP and (D) Survivin protein expression levels normalized to actin, and compared with control, cisplatin or cardamonin. Data are presented as the mean ± standard deviation. *P<0.05 vs. control; #P<0.01 vs. cisplatin; $P<0.01 vs. cardamonin. DMSO, dimethyl sulfoxide; Bcl-2, B cell lymphoma-2; XIAP, X-linked inhibitor of apoptosis protein.

Cardamonin inhibited the activation of mTOR and p70S6K. SKOV3 cells were treated with cardamonin (20 µM), cisplatin (2 µg/ml) or the combination of both agents for 48 h. The expression of mTOR, phospho-mTOR (Ser2448), p70S6K and phospho-p70S6K (Thr389) was determined by western blot analysis. Actin was used as the loading control (n=3). (A) Representative western blotting protein bands for mTOR, p-mTOR, p70S6K, p-p70S6K and actin. Quantification of (B) p-mTOR and (C) p-p70S6K protein expression. The relative density ratio of protein was normalized to actin and compared with control, cisplatin or cardamonin. Data are presented as the mean ± standard deviation. *P<0.01 vs. control; #P<0.01 vs. cisplatin; $P<0.01 vs. cardamonin. DMSO, dimethyl sulfoxide; mTOR, mammalian target of rapamycin; p, phosphorylated; p70S6K, 70 kDa ribosomal protein S6 kinase.

Effect of cardamonin combined with cisplatin on cell cycle distribution.

Cell cycle, %
Drug n G0/G1 S G2/M
Control 5 69.96±3.25 18.50±2.52 11.54±3.82
DMSO 5 70.38±2.87 19.01±4.28 10.61±1.60
Cisplatin 5 55.99±1.58a 22.67±1.29a 21.34±0.68a
Cardamonin 5 41.05±2.14a 28.54±2.74a 30.42±4.27a
Cisplatin + cardamonin 5 35.33±2.86a, b 31.48±3.54a, b 33.19±1.16a, b

Effect of cardamonin combined with cisplatin on cell cycle distribution. SKOV3 cells were treated with cardamonin (20 µM), cisplatin (2 µg/ml) or the combination of the two agents for 48 h, and then cells were processed for cell cycle analysis (n=5). The ratios of cell cycle distribution were calculated.

P<0.01 vs. control

P<0.01 vs. cisplatin. DMSO, dimethyl sulfoxide.