Green tea inhibits methylation of the RXRalpha gene and selectively targets initial stages of intestinal carcinogenesis in the AOM-MIN mouse model.

摘要

We have improved a protocol where we treated the well-known MIN mouse with azoxymethane (AOM), a colon selective carcinogen. Our protocol significantly and selectively induced a 4-fold increase in the number of colon tumors ( P0.005). This refined model was then utilized to investigate the possible mechanisms of inhibition of colorectal carcinogenesis by green tea. Mice received water or a 0.6% (w/v) solution of green tea as the only source of beverage. Green tea treatment commenced after 8 weeks of age and lasted for either 4 or 8 weeks. Green tea significantly inhibited the formation of new adenomas (P0.05), but not preexisting tumors. Western blotting analysis showed green tea decreased the total levels of beta-catenin and its downstream target cyclin D1. Immunohistochemical analysis showed that green tea selectively inhibited the formation of new adenomas overexpressing beta-catenin and cyclin D1 (P0.05). In contrast, green tea failed to inhibit the expression of COX-2, a later event in colon carcinogenesis. To explain the decrease in beta-catenin levels, we studied the effects of green tea on the expression of Retinoic X Receptor alpha (RXRalpha), which has been previously reported to induce cytoplasmic degradation of beta-catenin in vitro. Our results are the first to show that RXRalpha is selectively down-regulated in MIN intestinal tumors. On the contrary, retinoic receptors such as Retinoic Acid Receptor alpha (RARalpha), RARbeta, RXRbeta and RXRgamma were all expressed in MIN adenomas. Furthermore, our results clearly show that RXRalpha downregulation is an early event in colorectal carcinogenesis. Green tea significantly inhibited the formation of new adenomas in which RXRalpha is down-regulated (P0.05). RT-PCR analysis showed a 2.1 fold greater loss of RXRalpha mRNA levels in the mice that received water compared to those that received tea. Bisulfite treatment of genomic DNA followed by pyrosequencing of 24 CpG sites in the promoter region of RXRalpha gene showed a 4-fold increase in CpG methylation in the absence of tea treatment. The current study supports previous reports of inhibition of DNA methyltransferase in vitro by EGCG and suggests a possible mechanism of action of green tea through which it inhibits early carcinogenesis.