Reversal of hypermethylation and reactivation of p16, RARβ, and MGMT genes by genistein and other isoflavones from soy

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We have previously demonstrated the reactivation of some methylation-silenced genes in cancer cells by treatment with (-)-epigallocatechin-3-gallate, the major polyphenol from green tea (Fang et al, Cancer Res. 63:7563, 2003). In the present work, using both methylation specific PCR and quantitative real-time PCR, we demonstrated that genistein, a major isoflavone present in soy bean, could reverse DNA hypermethylation and reactivate methylation-silenced genes in the KYSE 510 esophageal squamous cell carcinoma cell line. Retinoic acid receptor beta (RARβ), p16^INK4a, and O⁶-methylguanine methyltransferase (MGMT) were activated with concentrations of genistein in the 2-20 μM range. Genistein also inhibited cell growth at these concentrations. Reversal of DNA hypermethylation and reactivation of RARβ by genistein were also observed in esophageal cancer KYSE 150 cells and prostate cancer LNCaP and PC3 cells. In vitro, genistein showed an inhibitory effect on both DNMT activity in nuclear extracts from the KYSE 510 cells and on purified recombinant DNMT1. Kinetic studies indicate that genistein inhibits DNMT activity in both a substrate- and methyl donor dependent manner and that its effect on substrate binding differs from that of (-)-epigallocatechin-3-gallate. Two other closely related isoflavones from soy products, biochanin A and daidzein, were less potent inhibitors of DNMT in vitro than genistein and were less efficient in reactivation of the RARβ gene and inhibition of cancer cell growth. These results suggest that genistein and related soy isoflavones can reactivate methylation-silenced genes at least partially through a direct inhibition on the activity of DNMT1 and this activity may contribute to the cancer preventive activity of dietary isoflavones. (This work was supported by NIH grant CA105331 and CA88961)