Hypomethylation of LIMD1 and P16 by downregulation of DNMT1 results in restriction of liver carcinogenesis by amarogentin treatment.

Amarogentin (active component of Chirata) was found to prevent CCl4/NDEA-induced liver carcinogenesis at mild dysplastic stage through modulation of cell cycle, apoptosis, self-renewal pathways. The cell cycle regulatory genes LIMD1, P16 and RBSP3 were found to be upregulated in restricted liver lesions. To understand the mechanism of upregulation during restriction of cacinogenesis, the effect of amarogentin on epigenetic modification was evaluated in this study. It was also validated in vitro. Hypermethylation of LIMD1 and P16 was seen in mouse hepatocellular carcinoma (30th week carcinogen control mice); however, hypomethylation of these genes was seen in amarogentin-treated liver. In the case of RBSP3, no such change was seen. DNMT1 expression (mRNA/protein) was significantly increased in later stages of carcinogenesis, whereas its expression was comparable to normal liver in the case of amarogentin treatment. No significant change in expression (mRNA/protein) of HDAC1/2 was observed irrespective of treatment. Amarogentin treatment upregulated the expression (mRNA/protein) of LIMD1, P16 and RBSP3 in the HepG2 cell line. Here also treated cells showed LIMD1 and P16 hypomethylation with DNMT1 downregulation. Increased expression of LIMD1, P16 and RBSP3 after treating cells with demethylating agent 5-aza-2-deoxycytidine indicated epigenetic modulation by amarogentin treatment.