Effects of 5-Aza-CdR on the proliferation of human breast cancer cell line MCF-7 and on the expression of Apaf-1 gene

Abstract

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 G(1)/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.

Role of Methylation in the Regulation of Apoptosis Genes APAF1, DAPK1, and BCL2 in Breast Cancer

“…The data on APAF1 methylation in BC are presented in just one report of an in vitro study [12]. We have detected a high incidence of APAF1 gene hypermethylation (32%) in specimens of primary BC tumors, significantly higher than in specimens of normal tissue (0%).…”

“…The data on hypermethylation of DAPK1 and APAF1 in some tumors have been presented, while studies of the contribution of methylation to regulation of these genes in BC are rare [9,12]. The relationship between suppression of APAF1 expression and methylation is demonstrated only in vitro on MCF-7 cells [12], and there are no reports about APAF1 methylation in clinical specimens of BC. A manifest increase of BCL2 expression in metastatic ovarian cancer is demonstrated [2].…”

Promoter Methylation and mRNA Expression of APAF-1 Gene in Breast Cancer

“…Xiong et al (32) examined the effects of 5-Aza-CdR on cell proliferation of human BC cell line MCF-7 and expression of APAF-1 gene. They observed that 5-Aza-CdR significantly suppressed the growth of MCF-7 cells and increased mRNA and protein expression of APAF-1 in MCF-7 cells; also, down-regulation of DNA methyltransferase 3b mRNA was observed.…”

Low concentrations of 5-aza-2'-deoxycytidine induce breast cancer stem cell differentiation by triggering tumor suppressor gene expression

“…Previous studies have investigated the ability of DAC to induce expression of tumor suppressor genes in a variety of cell lines. Tumor suppressor genes that are reported to increase their expression in response to DAC exposure include Rb , 38 p53 , 39 p21 , 40 p27 , p15 , 41 , 42 p16 , 29 , 43 , 44 Apaf-1 , 45 PTEN , BRCA1 , and BRCA2 . 46 , 47 In this study, the effect of DAC exposure on p15 , p16 , p53 , PTEN , BRCA1 , and BRCA2 expression was similar to previous reports.…”

“…Treating MCF-7 cells with 0.5, 1, 2, and 5 µM DAC resulted in a 7%, 15%, 37%, and 45% decrease in proliferation, respectively. 45 Dai et al 64 used 102.4 µM DAC to decrease cell growth by 58.6%.…”