Cancer Prevention Research Resveratrol Prevents Estrogen-DNA Adduct Formation and Neoplastic Transformation in MCF-10F Cells

Exposure to estrogens is a risk factor for breast cancer. Specific estrogen metabolites may initiate breast cancer and other cancers. Genotoxicity may be caused by cytochrome P450 (CYP)–mediated oxidation of catechol estrogens to quinones that react with DNA to form depurinating estrogen-DNA adducts. CYP1B1 favors quinone formation by catalyzing estrogen 4-hydroxylation, whereas NAD(P)H quinone oxidoreductase 1 (NQO1) catalyzes the protective reduction of quinones to catechols. 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) induces CYP1B1 expression through the aryl hydrocarbon receptor (AhR). Resveratrol has anticancer effects in diverse in vitro and in vivo systems and is an AhR antagonist that decreases CYP expression but induces NQO1 expression. The chemopreventive effect of resveratrol on breast cancer initiation was investigated in MCF-10F cells. Its effects on estrogen metabolism and formation of estrogen-DNA adducts were analyzed in culture medium by high-performance liquid chromatography, whereas its effects on CYP1B1 and NQO1 were determined by immunoblotting and immunostaining. The antitransformation effects of resveratrol were also examined. TCDD induced expression of CYP1B1 and its redistribution in the nucleus and cytoplasm. Concomitant treatment with resveratrol dose-dependently suppressed TCDD-induced expression of CYP1B1, mainly in the cytoplasm. Resveratrol doseand time-dependently induced expression of NQO1. NQO1 is mainly in the perinuclear membrane of control cells, but resveratrol induced NQO1 and its intracellular redistribution, which involves nuclear translocation of nuclear factor erythroid 2–related factor 2. Resveratrol decreased estrogen metabolism and blocked formation of DNA adducts in cells treated with TCDD and/or estradiol. Resveratrol also suppressed TCDD and/or estradiol-induced cell transformation. Thus, resveratrol can prevent breast cancer initiation by blocking multiple sites in the estrogen genotoxicity pathway. Carcinogenesis is a complex multistep process. Prolonged exposure to estrogens is a major known risk factor for breast cancer and other estrogen-mediated cancers (1). Besides the estrogen receptor (ER) pathway (2), reactive estrogen metabolites may also be involved in mutagenesis and breast cancer initiation via an estrogen genotoxicity pathway (3). The genotoxicity occurs via cytochrome P450 (CYP)–mediated oxidation of estrogens to quinones that react with DNA to form predominantly depurinating DNA adducts. This event may lead to the critical mutations that initiate cancer (3, 4). Two of the estrogen-metabolizing enzymes in this pathway, CYP1B1 and NAD(P)H quinone oxidoreductase 1 (NQO1), are implicated in the etiology of estrogen-mediated tumors by regulating the formation or clearance of genotoxic metabolites (5, 6). CYP1B1 is highly expressed in estrogen-related tissues, such as mammary gland, uterus, and ovary (7), suggesting that CYP1B1 is important in the local control of estrogen metabolism. CYP1B1 is expressed in normal tissues (8, 9) and its overexpression has been implicated in premalignant progression (10). CYP1B1 predominantly catalyzes the 4-hydroxylation of estrone (E1) and estradiol (E2) into 4-OHE1(E2) (refs. 11, 12), favoring quinone formation, whereas the phase II protective enzyme NQO1 catalyzes the reduction of quinones back to CEs (13), which can be methylated by catechol-O-methyltransferase to form 4-OCH3E1(E2). NQO1 is abundantly present in many human tissues (14), and genetic polymorphisms may cause variation in its activity (15, 16). Authors' Affiliations: Eppley Institute for Research in Cancer and Allied Diseases and Departments of Obstetrics and Gynecology and Environmental, Agricultural and Occupational Health, University of Nebraska Medical Center, Omaha, Nebraska Received 02/27/2008; accepted 03/10/2008. Grant support: F. Lu was supported by a fellowship from the University of Nebraska Environmental Toxicology Graduate Program. This study was supported by USPHS grant P01 CA49210 from the National Cancer Institute and Department of Defense grant DAMD17-03-1-0299 from the U.S. Army Breast Cancer Research Program. Core support at the Eppley Institute was provided by grant P30 CA36727 from the National Cancer Institute. Note: M. Zahid and C. Wang contributed equally to this work. Requests for reprints: Eleanor G. Rogan, Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, 986805 Nebraska Medical Center, Omaha, NE 68198-6805. Phone: 4025594095; Fax: 4025598068; E-mail: [email protected]. ©2008 American Association for Cancer Research. doi:10.1158/1940-6207.CAPR-08-0037 135 Cancer Prev Res 2008;1(2) July 2008 www.aacrjournals.org Cancer Research. on June 28, 2017. © 2008 American Association for cancerpreventionresearch.aacrjournals.org Downloaded from Low NQO1 activity and increased formation of depurinating adducts resulting from unbalanced estrogen metabolism can be critical factors leading to the initiation of breast cancer (17, 18). Therefore, the efficiency of CYP1B1 metabolism of estrogens and the enzymes, including NQO1, that regulate the clearance of quinones are important in the determination of cancer risk. A potential mechanism for the initiation of breast cancer involves altered expression of estrogenmetabolizing enzymes (18). The CYP1 family is inducible by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) through highaffinity binding to the aryl hydrocarbon receptor (AhR). The activated AhR translocates into the nucleus, then interacts with the dioxin response element Ah gene battery, responsible for regulating expression of CYP1B1 (19, 20). High CYP1B1 and low NQO1 activity seem to be associated with increased risk of developing breast cancer (21). Resveratrol (3,5,4′-trihydroxystilbene), a natural antioxidant present in grapes and many other plants (22), has anticarcinogenic effects in diverse in vitro and in vivo systems (23, 24). TCDD induces CYP1B1 expression through the AhR in breast epithelial cells (25, 26). Resveratrol acts as an AhR antagonist that decreases expression of the CYP isoforms catalyzing estrogen metabolism (27). However, it induces NQO1 activity in cultured cells (22) that are capable of metabolically detoxifying carcinogens (28). Induction of NQO1 may be regulated by the antioxidant response element (ARE)/nuclear factor erythroid 2–related factor 2 (Nrf2) pathway (29). Cytosolic transcription factor Nrf2 translocates into the nucleus, where it binds to the ARE to activate transcription of NQO1 mRNA (30, 31). In addition, resveratrol as an antioxidant may reduce semiquinones back to CEs, in turn preventing the formation of depurinating estrogen-DNA adducts (32, 33). Most anticancer studies emphasize resveratrol inhibiting cell proliferation and inducing apoptosis. Neither its preventive role in breast cancer initiation nor its mechanisms of action have been thoroughly characterized. TCDD-induced CYP1B1, which enhances estrogen metabolism and formation of DNA adducts, seems to play a major role in malignant transformation of human breast epithelial cells. Low catechol-O-methyltransferase activity and increased formation of estrogen-DNA adducts mediated through the CEs may be contributory factors in the development of breast cancer (34, 35). We hypothesize that resveratrol may prevent estrogen genotoxicity and neoplastic transformation via regulating estrogen-metabolizing enzyme expression and blocking CE and DNA adduct formation (Fig. 1). In this article, we used the human breast epithelial cell line MCF-10F (ER negative and AhR positive), a well-developed cell culture model for studying carcinogenesis through non-ER receptor– mediated pathways (36, 37), to further investigate the chemopreventive effects of resveratrol on breast cancer initiation. The profile of E2 metabolites and depurinating DNA adducts in resveratrol-treated cells pretreated with TCDD and treated with E2 was analyzed, as well as the effects of resveratrol on the expression pattern of CYP1B1 and NQO1 and the signal transductional mechanism of NQO1 induction. The antitransformation effects of resveratrol were determined by an anchorage-independent growth assay in agar methocel. This study helped us to obtain a deeper understanding of the roles of estrogen-metabolizing enzymes in the genotoxic mechanism of estrogen-initiated cancer. Materials and Methods Chemicals and cell culture MCF-10F and MCF-7 cells were obtained from the American Type Culture Collection and cultured in DMEM and Ham's F12 medium as described previously (34). Cell viability was determined by the 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. TCDD (>99% pure) was purchased from AccuStandard, Inc. Standards of E2 metabolites and depurinating DNA adducts were prepared as described previously (34). Resveratrol and other chemicals were obtained from Sigma.