Influence of triptolide on P53/P73 gene methylation and inhibition effect against proliferation of breast carcinoma MCF-7 cells

Abstract

Objective To study the effect of triptolide(TP) on the proliferation of breast carcinoma cell line MCF-7 and its association with P53/P73 gene expression and methylation.Methods MCF-7 cells were treated with different concentrations of TP(10 ng/ml,20 ng/ml,and 40 ng/ml),and the proliferation of MCF-7 cells was measured by MTT method.The expressions of methyltransferase DNMT1,DNMT3a and DNMT3b mRNA were measured by RT-PCR in MCF-7 cells,P53/P73 gene methylation was analyzed by methylation specific PCR,and the protein expression of p53/P73 in MCF-7 cells was examined by Western blotting assay.Results TP inhibited the proliferation of MCF-7 cells in a dose-dependent manner(P0.05,P0.01),with the inhibitory rate being(70.1±3.52)% at 40 ng/ml TP,and the IC50 of TP was 20 ng/ml.TP significantly inhibited DNMT1,DNMT3a,and DNMT3b mRNA expression in MCF-7 cells(P0.05,P0.01),and it also significantly inhibited methylation of P53 promoter region.TP increased P53 gene expression at 20 ng/ml and the increase was significant at 40 ng/ml(P0.01).TP reversed the hypermethylation of P73 gene in MCF-7 cells;it also significantly increased P73 mRNA expression at 10 ng/ml(P0.05),and the increase was in a dose-dependent manner.Western blotting analysis showed that TP(20 ng/ml) increased the protein expression of P53 and P73 in MCF-7 cells.Conclusion TP can promote the expression of P53 and P73 genes through inhibiting methyltransferase-dependent gene methylation,and further inhibit the proliferation of MCF-7 cells.