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Tea Polyphenol (-)-Epigallocatechin-3-Gallate Inhibits DNA Methyltransferase and Reactivates Methylation-Silenced Genes in Cancer Cell Lines

¹ Department of Chemical Biology, Ernest Mario School of Pharmacy, Rutgers, The State University of New Jersey,
² Department of Pharmacology, Robert Wood Johnson Medical School, University of Medicine and Dentistry of New Jersey, Piscataway, New Jersey

Hypermethylation of CpG islands in the promoter regions is an important mechanism to silence the expression of many important genes in cancer. The hypermethylation status is passed to the daughter cells through the methylation of the newly synthesized DNA strand by 5-cytosine DNA methyltransferase (DNMT). We report herein that (-)-epigallocatechin-3-gallate (EGCG), the major polyphenol from green tea, can inhibit DNMT activity and reactivate methylation-silenced genes in cancer cells. With nuclear extracts as the enzyme source and polydeoxyinosine-deoxycytosine as the substrate, EGCG dose-dependently inhibited DNMT activity, showing competitive inhibition with a K_i of 6.89 µM. Studies with structural analogues of EGCG suggest the importance of D and B ring structures in the inhibitory activity. Molecular modeling studies also support this conclusion, and suggest that EGCG can form hydrogen bonds with Pro¹²²³, Glu¹²⁶⁵, Cys¹²²⁵, Ser¹²²⁹, and Arg¹³⁰⁹ in the catalytic pocket of DNMT. Treatment of human esophageal cancer KYSE 510 cells with 5–50 µM of EGCG for 12–144 h caused a concentration- and time-dependent reversal of hypermethylation of p16^INK4a, retinoic acid receptor ß (RARß), O⁶-methylguanine methyltransferase (MGMT), and human mutL homologue 1 (hMLH1) genes as determined by the appearance of the unmethylation-specific bands in PCR. This was accompanied by the expression of mRNA of these genes as determined by reverse transcription-PCR. The re-expression of RARß and hMLH1 proteins by EGCG was demonstrated by Western blot. Reactivation of some methylation-silenced genes by EGCG was also demonstrated in human colon cancer HT-29 cells, esophageal cancer KYSE 150 cells, and prostate cancer PC3 cells. The results demonstrate for the first time the inhibition of DNA methylation by a commonly consumed dietary constituent and suggest the potential use of EGCG for the prevention or reversal of related gene-silencing in the prevention of carcinogenesis.

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