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Resveratrol Induces Growth Inhibition, S-Phase Arrest, Apoptosis, and Changes in Biomarker Expression in Several Human Cancer Cell Lines1

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Resveratrol Induces Growth Inhibition, S-Phase Arrest, Apoptosis, and Changes in Biomarker Expression in Several Human Cancer Cell Lines1 Vol. 8, 893–903, March 2002 Clinical Cancer Research 893 Resveratrol Induces Growth Inhibition, S-phase Arrest, Apoptosis, and Changes in Biomarker Expression in Several Human Cancer Cell Lines1 Andrew K. Joe, Hui Liu, Masumi Suzui, Cyclin D1, cyclin B1, ␤-catenin, and apoptotic index could Muhammet E. Vural, Danhua Xiao, and be useful biomarkers to evaluate treatment efficacy. I. Bernard Weinstein2 INTRODUCTION Herbert Irving Comprehensive Cancer Center [A. K. J., H. L., M. S., Ј M. E. V., D. X., I. B. W.] and Department of Medicine [A. K. J., The polyphenolic compound resveratrol (3,5,4 -trihy- I. B. W.], College of Physicians and Surgeons of Columbia droxy-trans-stilbene) is a naturally occurring phytochemical and University, New York, New York 10032 can be found in approximately 72 plant species, including food products like grapes, peanuts, and various herbs (1). Its exact physiological function is not known, but it may have roles in ABSTRACT protecting plants against fungal infections and in conferring Purpose: We examined the effects of the phytochemical disease resistance. Red wine (1.5–3 mg/liter) and grapes (50– resveratrol in six human cancer cell lines (MCF7, SW480, 100 ␮g/g grape skins) are probably its main sources in Western HCE7, Seg-1, Bic-1, and HL60). diets. One of its richest sources is the herb Polygonum cuspi- Experimental Design and Results: Resveratrol induced datum, which has been used in Asian folk medicine. Previous marked growth inhibition in five of these cell lines, with IC50 investigations have demonstrated its antioxidant and anti- ␮ values of approximately 70–150 M. However, only partial inflammatory activities, its ability to induce phase II drug- growth inhibition was seen in Bic-1 cells. After treatment with metabolizing enzymes, and its ability to inhibit cyclooxygenase ␮ 300 M resveratrol for 24 h, most of the cell lines were arrested activity and transcription; thus, it has activity in regulating in the S phase of the cell cycle. In addition, induction of multiple cellular events associated with carcinogenesis (for re- apoptosis was demonstrated by the appearance of a sub-G1 view, see Ref. 1). It may also have in vivo activity in modulating peak and confirmed using an annexin V-based assay. Cyclin B1 indices of platelet activity and lipid metabolism, which could expression levels were decreased in all cell lines after 48 h of explain the epidemiological evidence that red wine may de- ␤ treatment. In SW480 cells, cyclin A, cyclin B1, and -catenin crease coronary heart disease mortality (for a review of its expression levels were decreased within 24 h. There was a potential benefits in atherosclerotic heart disease, please refer to decrease in cyclin D1 expression after only2hoftreatment, Ref. 2). and this persisted for up to 48 h. This decrease was partially Resveratrol has been shown to have growth-inhibitory ac- blocked by concurrent treatment with the proteasome inhibi- tivity in several human cancer cell lines and in animal models of tor calpain inhibitor I. Using a luciferase-based reporter assay, carcinogenesis. In HL60 promyelocytic leukemia cells, treat- resveratrol did not inhibit cyclin D1 promoter activity in ment with resveratrol led to growth inhibition, induction of SW480 cells. Furthermore, using a reverse transcription-PCR- apoptosis, S-G -phase cell cycle arrest, and myelomonocytic ␮ 2 based assay, only a higher dose of resveratrol (300 M) ap- differentiation (3, 4). Resveratrol also displayed antiprolifera- peared to decrease cyclin D1 mRNA. Seg-1 cells expressed tive activity in JB6 mouse epidermal, CaCo-2 colorectal, and basal levels of cyclooxygenase-2 (cox-2), which was further A431 epidermoid carcinoma cell lines (5–7). Its effects in breast induced by resveratrol. Neither basal levels nor induction of cancer cell lines are more complicated. Whereas some investi- cox-2 was detectable in the remaining cell lines. Thus, cox-2 gators have demonstrated antiproliferative effects in the MCF7, does not appear to be a critical target of this compound. MDA-MB-231, KPL-1, MKL-F, and T47D cell lines (3, 8, 9), Conclusions: These studies provide support for the use others have demonstrated growth enhancement in T47D and of resveratrol in chemoprevention and cancer therapy trials. MCF7 cells (10, 11). The latter effect appears to be due to the potential estrogenic effects of resveratrol (10–12). Resveratrol inhibited tumor formation in several animal models of carcino- genesis, including mouse 7,12-dimethylbenz(a)anthracene/12- Received 8/3/01; revised 12/19/01; accepted 12/21/01. O-tetradecanoylphorbol-13-acetate-induced skin cancers (1), The costs of publication of this article were defrayed in part by the azoxymethane-induced colon cancers (13), and transplanted Yo- payment of page charges. This article must therefore be hereby marked shida rat ascites hepatomas (14). In the mouse skin carcinogen- advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. esis model, resveratrol inhibited the three major steps of carci- 1 Supported by an award (to A. K. J.) from PureWorld Botanicals and an nogenesis, initiation, promotion, and progression (1). However, American Association for Cancer Research-Cancer Research Founda- the precise mechanisms by which resveratrol exerts these anti- tion of America Fellowship in Prevention Research award (to A. K. J.). tumor effects are not known. 2 To whom requests for reprints should be addressed, at Herbert Irving Comprehensive Cancer Center, 701 West 168th Street, HHSC-1509, Limited epidemiological and clinical evidence suggest that New York, NY 10032. Phone: (212) 305-6921; Fax: (212) 305-6889; resveratrol is well tolerated during human consumption and that E-mail: [email protected]. it may offer benefits with respect to atherosclerotic heart dis- Downloaded from clincancerres.aacrjournals.org on September 26, 2021. © 2002 American Association for Cancer Research. 894 Anticancer Effects of Resveratrol ease. In a small study of 24 healthy male volunteers, trial plated in flat-bottomed, 96-well microtiter plates (4 ϫ 103 participants tolerated the consumption of resveratrol-enriched cells/6.4-mm-diameter well). After 12–24 h, cells were treated beverages, but the effects of this compound on lipid metabolism with DMSO (0.1–0.3%) or increasing doses of resveratrol. and platelet activity were unimpressive (15, 16). Although res- After 48 h of treatment, cells were treated with 10 ␮lofMTT veratrol is available commercially as a dietary supplement, there reagent for4hat37°C and then treated with 100 ␮l of solubi- are no published controlled clinical studies demonstrating either lization solution at 37°C overnight. The quantity of formazan its efficacy or safety in the treatment or prevention of cancer or product was measured using a spectrophotometric microtiter coronary artery disease. plate reader (Dynatech Laboratories, Alexandria, VA) at 570 nm In the present study, we used a spectrum of six human wavelength. Results were expressed as a percentage of growth, cancer cell lines to further examine the range of antitumor with 100% representing control cells treated with DMSO alone. activity of resveratrol. To obtain insights into its mechanism of All experiments were performed in duplicate. action, we examined the effects of resveratrol on cell prolifer- Apoptosis Assays. The percentage of cells actively un- ation, cell cycle distribution, apoptosis, and on the levels of dergoing apoptosis was determined using annexin V-PE-based expression of several cell cycle control proteins. Our results immunofluorescence, as described previously (19). Briefly, cells provide support for the use of resveratrol in clinical chemopre- were plated in 10-cm culture dishes at concentrations deter- vention and chemotherapy trials. In addition, we have identified mined to yield 60–70% confluence within 24 h. Cells were then potential surrogate biomarkers, which may serve as intermediate treated with either DMSO (0.1–0.3%) or resveratrol (300 ␮M). clinical end points in these trials. After 48 h of treatment, both adherent and floating cells were harvested and then double-labeled with annexin V-PE and MATERIALS AND METHODS 7-aminoactinomycin (PharMingen, San Diego, CA), as de- Compounds and Antibodies. Resveratrol was gener- scribed by the manufacturer. Cells were analyzed using a FAC- ously supplied by PureWorld Botanicals (South Hackensack, Scan instrument equipped with FACStation running Cell Quest NJ) and isolated from the Chinese herb huzhang (P. cuspida- software (Becton Dickinson, San Jose, CA). All experiments tum). The compound was supplied in powder form (trans iso- were performed in duplicate and yielded similar results. mer), dissolved (stock solution, 100 mM) in DMSO (Sigma Cell Cycle Distribution Analysis. PI staining was used Chemical Co., St. Louis, MO), and added directly to cell culture to analyze DNA content. Cells were plated in 10-cm culture medium at a final concentration of 0.1–0.3% DMSO. Primary dishes at concentrations determined to yield 60–70% conflu- antibodies were obtained from the following companies: (a) ence within 24 h. Cells were then treated with either DMSO ␮ cyclins A, B1, and D1, Upstate Biotechnology (Lake Placid, (0.1–0.3%) or resveratrol (300 M). After a 24-h treatment, both NY); (b) ␤-catenin, Transduction Laboratories (Lexington, adherent and floating cells were harvested, and the cells were KY); (c) cox-2, Oxford Biomed (Oxford, MI); and (d) actin, labeled with PI using previously described methods (20). Sigma Chemical Co. LLnL3 and PI were obtained from Sigma Briefly, cells were resuspended in PBS, fixed with 70% ethanol, Chemical Co. labeled with PI (0.05 mg/ml), incubated at room temperature in ␮ Cell Lines and Cell Culture. Seg-1 and Bic-1, esopha- the dark for 30 min, and filtered through 41- m spectra/mesh geal adenocarcinoma cell lines established from patients with nylon filters (Spectrum, Rancho Dominguez, CA). DNA content Barrett’s esophagus, were developed and generously provided was then analyzed using a FACScan instrument equipped with by Dr.
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