Tea polyphenol (-)-epigallocatechin-3-gallate inhibits DNA methyltransferase and reactivates methylation-silenced genes in cancer cell lines.
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 microM. 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 Pro(1223), Glu(1265), Cys(1225), Ser(1229), and Arg(1309) in the catalytic pocket of DNMT. Treatment of human esophageal cancer KYSE 510 cells with 5-50 microM of EGCG for 12-144 h caused a concentration- and time-dependent reversal of hypermethylation of p16(INK4a), retinoic acid receptor beta (RARbeta), O(6)-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 RARbeta 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.
Fang
Ming Zhu
MZ
Department of Chemical Biology, Ernest Mario School of Pharmacy, Rutgers, The State University of New Jersey, 164 Frelinghuysen Road, Piscataway, NJ 08854-8020, USA.
Wang
Yimin
Y
Ai
Ni
N
Hou
Zhe
Z
Sun
Yi
Y
Lu
Hong
H
Welsh
William
W
Yang
Chung S
CS
D048868:Adaptor Proteins, Signal Transducing; D016588:Anticarcinogenic Agents / Q000494:pharmacology; D000970:Antineoplastic Agents / Q000494:pharmacology; D002294:Carcinoma, Squamous Cell / Q000201:enzymology / Q000235:genetics*; D002352:Carrier Proteins; D002392:Catechin / Q000031:analogs & derivatives* / Q000494:pharmacology*; D002455:Cell Division / Q000187:drug effects; D045744:Cell Line, Tumor; D004248:DNA (Cytosine-5-)-Methyltransferases / Q000037:antagonists & inhibitors*; D019175:DNA Methylation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D004938:Esophageal Neoplasms / Q000201:enzymology / Q000235:genetics*; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects* / Q000502:physiology; D020868:Gene Silencing / Q000187:drug effects* / Q000502:physiology; D019942:Genes, p16 / Q000187:drug effects / Q000502:physiology; D006801:Humans; D008958:Models, Molecular; D000070957:MutL Protein Homolog 1; D009363:Neoplasm Proteins / Q000235:genetics; D009687:Nuclear Proteins; D019853:O(6)-Methylguanine-DNA Methyltransferase / Q000235:genetics; D018168:Receptors, Retinoic Acid / Q000235:genetics
D048868:Adaptor Proteins, Signal Transducing; D016588:Anticarcinogenic Agents; D000970:Antineoplastic Agents; D002352:Carrier Proteins; D004791:Enzyme Inhibitors; C501486:MLH1 protein, human; D009363:Neoplasm Proteins; D009687:Nuclear Proteins; D018168:Receptors, Retinoic Acid; C082925:retinoic acid receptor beta; D002392:Catechin; C045651:epigallocatechin gallate; D004248:DNA (Cytosine-5-)-Methyltransferases; D019853:O(6)-Methylguanine-DNA Methyltransferase; D000070957:MutL Protein Homolog 1