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Use of Resveratrol to improve the effectiveness of cisplatin and doxorubicin: Study in human gynecologic cancer cell lines and in rodent heart

Published:April 24, 2006DOI:https://doi.org/10.1016/j.ajog.2005.11.030

      Objective

      The purpose of this study was to investigate whether resveratrol adds to the growth inhibitory effects of cisplatin and doxorubicin on ovarian and uterine cancer cells and to evaluate whether resveratrol diminishes the cardiac toxicity of doxorubicin in rodent heart.

      Study design

      Human ovarian (OVCAR-3) and uterine (Ishikawa) cancer cells in culture were treated with cisplatin and doxorubicin, respectively, with and without resveratrol; and cell growth and viability were evaluated. Neonatal rat ventricular myocytes received doxorubicin in the presence and absence of resveratrol, and cell viability was evaluated. Mice received doxorubicin ± resveratrol, and electrocardiograms were evaluated. Data were analyzed with analysis of variance and Scheffe's test.

      Results

      Resveratrol combined with cisplatin or with doxorubicin demonstrated an additive growth-inhibitory anticancer effect with a left shift of the cisplatin and doxorubicin dose/response curves. Resveratrol increased the viability of neonatal rat ventricular myocytes that were treated with doxorubicin and reduced doxorubicin-induced bradycardia and QTc interval prolongation in mice.

      Conclusion

      Resveratrol adds to the growth inhibitory/anticancer activity of cisplatin and doxorubicin in vitro and protects against doxorubicin-induced cardiac toxicity both in vitro and in mice.

      Key words

      Cisplatin is the cornerstone of chemotherapy in advanced epithelial ovarian cancer; both cisplatin and doxorubicin are first-line agents for advanced endometrioid endometrial adenocarcinoma, which is the most common form of uterine cancer. Unfortunately, the clinical usefulness of cisplatin has been limited by the frequent development of drug resistance,
      • Vasey P.A.
      Resistance to chemotherapy in advanced ovarian cancer: mechanisms and current strategies.
      while a dose-dependent cardiac toxicity has limited the usefulness of doxorubicin.
      • Shan K.
      • Lincoff A.M.
      • Young J.B.
      Anthracycline-induced cardiotoxicity.
      This cardiac toxicity is thought mainly to be due to oxidative stress and the generation of free radicals by doxorubicin
      • Rajagopalan S.
      • Politi P.M.
      • Sinha B.K.
      • Myers C.E.
      Adriamycin-induced free radical formation in the perfused rat heart: implications for cardiotoxicity.
      and is noted by changes in electrocardiogram results and other measures of heart function.
      Resveratrol is a naturally occurring compound that is found in grapes and has been shown to have oncostatic activity, in large part through mediation of apoptosis,
      • Huang C.
      • Ma W.Y.
      • Goranson A.
      • Dong Z.
      Resveratrol suppresses cell transformation and induces apoptosis through a p53-dependent pathway.
      • Lin H.Y.
      • Shih A.
      • Davis F.B.
      • Tang H.Y.
      • Martino L.J.
      • Bennett J.A.
      • et al.
      Resveratrol induced serine phosphorylation of p53 causes apoptosis in a mutant p53 prostate cancer cell line.
      and cardioprotective properties, partly because of its antioxidant effects.
      • Cao Z.
      • Li Y.
      Potent induction of cellular antioxidants and phase 2 enzymes by resveratrol in cardiomyocytes: protection against oxidative and electrophilic injury.
      • Aggarwal B.B.
      • Bhardwaj A.
      • Aggarwal R.S.
      • Seeram N.P.
      • Shishodia S.
      • Takada Y.
      Role of resveratrol in prevention and therapy of cancer: preclinical and clinical studies.
      Combining resveratrol with each of cisplatin and doxorubicin is a novel strategy that has the potential for improving the antineoplastic activity of cisplatin and doxorubicin, while protecting against a dose-limiting cardiac toxicity of doxorubicin. This preliminary study was designed to test these hypotheses.

      Material and methods

      Resveratrol (Sigma Chemical Company, St. Louis, MO) was dissolved in 95% ethanol to 10−2 mol/L and diluted in water and then with culture medium. Cisplatin and doxorubicin were diluted in saline solution and then with culture medium. OVCAR-3 cells were grown in Rosewell Park Memorial Institution 1640 medium that was supplemented with 10 μg/mL insulin and 20% fetal bovine serum. Ishikawa (human uterine cancer) cells were grown in Dulbecco's Modification of Eagle's Medium (DMEM) supplemented with 10 ng/mL insulin and 5% fetal calf serum. Neonatal rat ventricular myocytes (NRVM) were freshly isolated and grown in DMEM that was supplemented with 10% fetal bovine serum.
      • Pham C.G.
      • Harpf A.E.
      • Keller R.S.
      • Vu H.T.
      • Shai S.Y.
      • Loftus J.C.
      • et al.
      Striated muscle-specific beta(1D)-integrin and FAK are involved in cardiac myocyte hypertrophic response pathway.
      For experiments, cancer cells were seeded at 2 × 105 cells/well in 24-well plates. Twenty-four hours after being seeded, resveratrol or vehicle were added; 48 hours after seeding, either vehicle (cisplatin or doxorubicin) was added. Media were changed every 2 to 3 days, with resveratrol added 1 hour before cisplatin or doxorubicin. On day 8, viable cells were enumerated with a hemacytometer.
      To evaluate the effect of resveratrol on doxorubicin-induced cardiac toxicity, NRVM were seeded on fibronectin coated 6-well plates (2.88 × 105 cells/well); the next day, doxorubicin (10 nmol/L to 100 μmol/L) with or without resveratrol (1 μmol/L) was added. Forty-eight hours later, cell viability (methylthiazol-tetra zolium [MTT] assay) was determined, and dose/response curves were generated for determination of IC50. In vivo, severe combined immunodeficient mice (5 per group) were treated with doxorubicin (2 mg/kg, intravenously, every other day × 5 days) after receiving resveratrol (3 mg/kg, intraperitoneally) that was administered 2 hours before and 1 hour after doxorubicin. Controls received vehicle and each drug alone. The electrocardiogram was recorded at baseline and on day 11. In vitro and in vivo amounts of cisplatin, doxorubicin, and resveratrol were based on previously published concentrations and doses that were shown to be biologically and therapeutically active in experimental systems.
      • Aggarwal B.B.
      • Bhardwaj A.
      • Aggarwal R.S.
      • Seeram N.P.
      • Shishodia S.
      • Takada Y.
      Role of resveratrol in prevention and therapy of cancer: preclinical and clinical studies.
      Standards for the care and use of such animals at our institution were followed.

      Statistical analysis

      Mean viable cell number in each group was determined and converted to percent inhibition relative to the control group. IC50s and IC25s were calculated with Prism software (GraphPad, Inc, San Diego, CA). Cell and animal groups were compared with the use of analysis of variance and Scheffe's test.

      Results

      Both resveratrol alone and cisplatin alone inhibited growth of OVCAR-3 cells (IC50: 24 μmol/L and 0.12 μmol/L, respectively). Similarly, both resveratrol alone and doxorubicin alone inhibited growth of Ishikawa cells (IC50: 15 μmol/L and 0.005 μmol/L, respectively). When resveratrol was combined with either cisplatin or doxorubicin, additive activity was found for each cancer cell line (Table). Similarly, in the presence of the IC25 of resveratrol, a left shift in the cisplatin or doxorubicin dose response curve was found for each of the gynecologic cancer cell lines (Figure 1).
      TableThe effect of IC-25 of cisplatin (0.065 μmol/L) and resveratrol (13.2 μmol/L) on OVCAR-3 cells and the effect of IC-25 of doxorubicin (0.0025 μmol/L) and resveratrol (10 μmol/L) on Ishikawa cells
      VariableOVCAR cellsIshikawa cells
      Vehicle00
      Resveratrol2626
      Cisplatin28
      Doxorubicin34
      Resveratrol + cisplatin54
      Resveratrol + doxorubicin60
      Values are mean percent growth inhibition ± SEM calculated as described in Figure 1. Growth inhibition that was obtained with resveratrol + cisplatin and resveratrol + doxorubicin is significantly greater than that obtained with either drug alone (P < .05, Scheffe's test).
      Figure thumbnail gr1
      Figure 1Effect of sequential combination of resveratrol plus cisplatin on growth of OVCAR-3 cells (A) and resveratrol plus doxorubicin on growth of Ishikawa cells (B). Cells were seeded in 24-well plates, and 24 hours later the IC25 of resveratrol was added 1 hour before various concentrations of cisplatin or doxorubicin. The medium was changed every 3 days with a fresh addition of drugs or vehicle. Cells were counted 8 days after seeding. Six replicate wells were seeded per drug concentration. Mean cell number in a treated group was compared with the control group and mean percent inhibition ± SEM was calculated.
      Against NRVM, resveratrol caused a right shift of the doxorubicin curves (data not shown). As shown in Figure 2, the amount of doxorubicin that was needed to kill NRVM was increased in the presence of resveratrol. Moreover, in mice, doxorubicin-induced bradycardia and QTc interval prolongation were ameliorated significantly after pretreatment with resveratrol (33% ± 6% to 19% ± 2% decrease in heart rate and 26% ± 4% to 6% ± 3% prolongation in QTc interval, respectively; P < .01; values are means ± SEM). No abnormal electrocardiogram changes were seen in the control or resveratrol alone groups. Future studies are planned to evaluate left ventricular function by use of echocardiography and isolated heart preparations.
      Figure thumbnail gr2
      Figure 2Effect of resveratrol on doxorubicin-induced cell death in NRVM. NRVM were treated with doxorubicin (0-10 μmol/L) in the presence (n = 3) or absence (n = 5) of resveratrol (1 μmol/L), and the viability of the cells was determined by MTT. IC50 of doxorubicin was calculated (A) from the viability curves (the asterisk denotes P < .05, Student t test). B, Percent viability of NRVM in the presence of doxorubicin (10 μmol/L; P = .06) with and without resveratrol (1 μmol/L). Values are expressed as mean ± SEM.

      Comment

      The proapoptotic effects of resveratrol have been found to play a major role in its anticancer effects,
      • Huang C.
      • Ma W.Y.
      • Goranson A.
      • Dong Z.
      Resveratrol suppresses cell transformation and induces apoptosis through a p53-dependent pathway.
      which has been substantiated in our laboratory.
      • Lin H.Y.
      • Shih A.
      • Davis F.B.
      • Tang H.Y.
      • Martino L.J.
      • Bennett J.A.
      • et al.
      Resveratrol induced serine phosphorylation of p53 causes apoptosis in a mutant p53 prostate cancer cell line.
      In the current study, we hypothesized that the proapoptotic effects of resveratrol should work cooperatively with the cytotoxic effects of chemotherapy. Resveratrol was added before cisplatin or doxorubicin so that activation of apoptosis-related signal transduction pathways could take place before the cells were exposed to the chemotherapeutic agent. The micromolar levels of resveratrol that were used in our study have been shown previously to be proapoptotic and antioxidant. The doses of resveratrol that were used in our study were also reported previously to achieve 1 μmol/L levels of resveratrol in the blood.
      • Aggarwal B.B.
      • Bhardwaj A.
      • Aggarwal R.S.
      • Seeram N.P.
      • Shishodia S.
      • Takada Y.
      Role of resveratrol in prevention and therapy of cancer: preclinical and clinical studies.
      However, the importance of dose and sequential treatment must be followed up with more comprehensive kinetics and scheduling experiments that correlate drug levels with biochemical markers of apoptosis and changes in cell growth and function. Nevertheless, the results of the study reported herein do support the aforementioned hypothesis that resveratrol works cooperatively with cytotoxic chemotherapy.
      Cardiac toxicity is an important dose-limiting factor of doxorubicin. The efficacy of this drug could be improved if this toxicity could be minimized. Unfortunately, antidotes that are designed to address this toxicity have not had much success clinically. Moreover, there is evidence to suggest that, even the most widely used agent in that setting, dexrazoxane, may interfere with the DNA-damaging effect of doxorubicin against cancer cells.
      • Sehested M.
      • Jensen P.B.
      • Sorensen B.S.
      • Holm B.
      • Friche E.
      • Demant E.J.
      Antagonistic effect of the cardioprotector (+)-1,2-bis(3,5-dioxopiperazinyl-1-yl)propane (ICRF-187) on DNA breaks and cytotoxicity induced by the topoisomerase II directed drugs daunorubicin and etoposide (VP-16).
      The value of resveratrol compared with these other agents culminates in the fact that it has both cardioprotective and antitumor activities. It is also well-tolerated by the host.
      • Aggarwal B.B.
      • Bhardwaj A.
      • Aggarwal R.S.
      • Seeram N.P.
      • Shishodia S.
      • Takada Y.
      Role of resveratrol in prevention and therapy of cancer: preclinical and clinical studies.
      • Juan M.E.
      • Vinardell M.P.
      • Planas J.M.
      The daily oral administration of high doses of trans-resveratrol to rats for 28 days is not harmful.
      The findings of our study suggest a protective role for resveratrol against doxorubicin cardiac toxicity. This response was quite different from the response found with cancer cells, where cell toxicity from chemotherapy was enhanced in the presence of resveratrol. These discrepant effects of resveratrol may be attributed to the proliferative nature and oxidative state of the responding cell. The nonproliferative nature of the cardiac tissue suggests that it would be spared from the cell-cycle inhibition and proapoptotic effects of resveratrol and mainly benefit from its antioxidant properties. Our data support this simultaneous cardioprotective and tumor-damaging properties of resveratrol and suggest that other tumor cell types and host tissues should be investigated with regard to the cooperative role of resveratrol with cytotoxic chemotherapy.

      Acknowledgments

      The authors would like to thank Patrick Timmins, III, MD, for his critical review of the manuscript and Amy Houghton for her work with Ishikawa cells.

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