Influence of triptolide on P53/P73 gene methylation and inhibition effect against proliferation of breast carcinoma MCF-7 cells / 第二军医大学学报

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 (P< 0. 05, P<0. 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 (P<0. 05, P<0. 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 (P<0. 01). TP reversed the hypermethylation of P73 gene in MCF-7 cells; it also significantly increased P73 mRNA expression at 10 ng/ml (P<0. 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.