Effects of 5-Aza-CdR on the Proliferation of Human Breast Cancer Cell Line MCF-7 and on the Expression of Apaf-1 Gene

Hypermethylation in the promoter region of tumor suppressor genes is a common mechanism of gene silencing,which tends to occur in cancer.The effects of 5-Aza-2'-deoxycytidine (5-Aza-CdR),a specific DNA methyltransferase inhibitor,on the cell proliferation of human breast cancer cell line MCF-7 and on the expression of Apaf-1 gene were investigated.Human MCF-7 cells were incubated with increasing concentrations of 5-Aza-CdR for 12 to 120 h.The growth inhibition rates of MCF-7 cells were detected by MTT assay.Changes of cell cycle distribution and apoptotic rates of MCF-7 cells were determined by flow cytometry.The expressions of DNA methyltransferase 3b mRNA and Apaf-1 mRNA were measured by reverse transcription polymerase chain reaction (RT-PCR).Meanwhile,the expression of Apaf-1 protein was detected by Western blotting.The results showed that 5-Aza-CdR significantly inhibited the growth of MCF-7 cells and the growth inhibition rate of MCF-7 cells was significantly enhanced with the concentration of 5-Aza-CdR and the action time.Flow cytometry indicated that 5-Aza-CdR could significantly induce G1/S cell cycle arrest and increase the apoptosis rate of MCF-7 cells.The mRNA and protein expressions of Apaf-1 were up-regulated in MCF-7 cells treated with 5-Aza-CdR,which was accompanied by down-regulation of DNA methyltransferase 3b mRNA.It is concluded that 5-Aza-CdR might retard the growth of tumor cells and promote the apoptosis of MCF-7 breast cancer cells by inhibiting the expression of DNA methyltransferase 3b and re-activating the Apaf-1 gene expression.