5-Aza-2’-deoxycytidine Induces Apoptosis In Human Hepatocellular Carcinoma Cells Huh7through Enhancing The Methionine Adenosyltransferase1A Gene Expression And Inducing S-Adenosylmethionine Production

Backgroud:Methionine adenosyltransferase (MAT) is the only enzyme that can catalyze the biosynthesis of S-adenosylmethionine, which is the principal biological methyl donor in the cell. SAMe can regulate hepatocyte growth and apoptosis. Exogenous SAMe inhibits the growth of hepatoma cells, prevents development of HCC. In mammals, two genes (MAT1A and MAT2A), encode for two homologous MAT catalytic subunits, MAT1A is expressed mostly in the liver, while MAT2A predominates in the fetal liver and is progressively replaced by MAT1A during development. Thus MAT1A can be considered a marker of differentiated liver phenotype. Our previous studies showed that hypermethylation of the MAT1A promoter may be one of events in the development of HCC. Low expression of MAT1A is likely involved in the progression of the tumor and was found to be an independent factor for poor prognosis of patients with HCC. Therefore, it is a reasonable strategy to reverse MAT1A gene epigenetic change by using drugs releasing epigenetic repression. The demethylating reagent5-aza-2’-deoxycitidine (5-Aza-CdR) inhibits DNA methyltransferases (DNMTs) and reverses DNA methylation. It has been found that5-Aza-CdR can inhibit cancer cell growth.5-Aza-CdR can inhibit hepatocellular carcinoma cells growth by inhibiting the telomerase activity and inducing p16expression. However, the mechanisms underlying its anticancer activity and other biological effects are not fully understood. In this study, we investigated the molecular mechanism underlying the effects of5-aza-2’-deoxycytidine on MAT1A:MAT2A expression and SAMe production, meanwhile, we explored the effect of5-Aza-CdR on apoptosis pathway in the Huh7cell line.Methods:Effects of5-Aza-CdR on Huh-7cells:3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide colorimetric assay (MTT) and cells apoptosis analysis with flow cytometry were applied to observe whether5-Aza-CdR could effectively induce apoptosis of Huh7cells and inhibit cells growth. Western blotting analysis was applied to observe expression of Bcl-2, Bax and cleaved-caspase-3protein. Study of the mechanism: 1. RNA extraction and quantitative real-time reverse-transcription polymerase chain reaction (qRT-PCR) was applied to observe the expression of MAT1A/MAT2A mRNA.2. Western blotting analysis was applied to observe the expression of MAT1A/MAT2A protein.3. Quantification of the levels of SAMe by RP-HPLC4. Methylation-specific polymerase chain reaction was applied to analyse the methylation status of MAT1A/MAT2A promoterResults:1.5-Aza-CdR inhibited cells growth and induced cells apoptosis by Bcl-2, Bax and caspase-3pathway in Huh7cells.2.5-Aza-CdR inhibited MAT2A expression and activated MAT1A expression in Huh7cells.3.5-Aza-CdR induced S-Adenosylmethionine production in Huh7cells.4.5-Aza-CdR increased demethylation of MAT1A promoter and had no obvious effect on MAT2A promoter methylation in Huh7cells.Conclusion:Our results demonstrated5-aza-2’-deoxycytidine can reactivate MAT1A and inhibit MAT2A expression, induce S-Adenosylmethionine production of human hepatoma Huh7cells through reversing the hypermethylation of Mat1A promoter, and it induced apoptosis through down-regulation of Bcl-2, up-regulation of Bax and cleaved-caspase-3in Huh7cells.