Isoflavones are compounds found in plants that act like estrogens. The importance of these compounds relates to their action on the estrogen receptor, which may influence cancer risk. One of the most commonly consumed plant products is soy, which contains isoflavones. The two isoflavones in soy that have gained the most research attention are genistein and daidzein. Genistein (5,7,4′-trihydroxyisoflavone) binds to the active site of the estrogen receptor (1
) and increases luciferase in cells with an estrogen response element–luciferase reporter gene construct that coexpresses ERα and ERβ (2
). Genistein and other soy isoflavones inhibit ER negative (−) breast cancer cell growth, although some studies have indicated that they may increase ER positive(+) breast cancer cell growth and may interfere with the antitumor activity of tamoxifen (4
Treatment with dietary levels of genistein during puberty leads to a lower incidence of mammary tumors in rats challenged with carcinogens as adults (5
), whereas genistein administered during adult life but not during puberty had no effect on mammary tumors (6
). Asian women who consumed tofu during adolescence but not in adult life still had a lowered incidence of breast cancer compared with those who never consumed tofu or only consumed tofu as an adult (7
). The effect of early exposure to isoflavones on future breast cancer risk may be due to epigenetic changes such as alterations in DNA methylation.
Cytosine residues methylated by DNA-cytosine methyltransferase-1 (DNMT1) are associated with loss of transcription of the target gene (8
). DNMT1 activity is elevated in malignant cells, and this is associated with increased cell proliferation, tumorigenesis, and tumor progression (8
). Genistein (2–20 μmol/l) was found to inhibit DNMT, reverse DNA hypermethylation and reactivate RARβ, p16, and O6
-methylguanine methyltransferase (MGMT) in esophageal squamous carcinoma and prostate and mammary cancer cells in vitro (9
). Genistein also inhibited cell growth at these concentrations. In theory, prevention or reversal of hypermethylation-induced inactivation of key tumor suppressor or receptor genes by DNMT inhibitors such as genistein and daidzein could be an effective approach for cancer prevention, but little is known about the methylation effects of these compounds in humans.
Asian populations that have low rates of breast cancer consume 20 to 80 mg/day of genistein, whereas dietary intake of genistein in the United States is 1 to 3 mg/day (7
). In a case-control study, a significant reduction in breast cancer risk was found in both premenopausal and postmenopausal women who consumed supplemental isoflavones (11
). Circulating ovarian hormone levels were decreased (estradiol, 25%; progesterone, 45%) in premenopausal women after they received 36 oz of soymilk (113–207 mg/day isoflavones) through one menstrual cycle (12
). Six-month ingestion of 37 mg/day genistein led to increased plasma estradiol levels, increased nipple aspirate fluid (NAF) volume, and epithelial hyperplasia in NAF in a subset of premenopausal women (13
). In general, clinical studies of isoflavones have observed lesser effects in postmenopausal compared to premenopausal women (14
Genistein and daidzein belong to a larger class of compounds called polyphenols, which have documented effects on DNA methylation (15
). Genistein, and to a lesser extent daidzein, reversed DNA hypermethylation and reactivated RARβ, p16, and MGMT in mammary cancer cells in vitro (6
), whereas two animal studies have observed increased methylation effects with treatment (16
). Thus, the demethylation effects of genistein and daidzein on tumor suppressor genes in vitro appears to be at odds with the methylating effects of genistein in the two animal studies. Furthermore, no studies to date have examined the effect of dietary isoflavones on gene methylation in humans. In theory, prevention or reversal of hypermethylation-induced inactivation of key tumor suppressor genes by genistein and daidzein could be an effective approach for cancer prevention.
We examined the response of genes frequently methylated in breast cancer to orally administered genistein and daidzein in the breast tissue of healthy premenopausal women using mammary ductoscopy (MD). Our attempts to use methylation arrays were unsuccessful; however we observed treatment related methylation effects using quantitative methylation specific PCR (qMS-PCR) in our assessment of 5 genes (p16, RASSF1A, RARβ2, ER, and CCND2) known to be methylated in breast cancer (18
). Estrogenic effects of isoflavones were determined by measuring circulating levels of the estrogenic marker complement (C)3 (21
) and evaluating the cytologic profile of breast ductal epithelial cells. We correlated the estrogenic and methylation changes with isoflavone dose and with circulating levels of genistein.