Effects of methylation of p53-bax mitochondrial apoptosis pathway regulated by 5-aza-2-deoxycyti-dine on the growth of cholangiocarcinoma cells

Objective To study the effects of 5-aza-2-deoxycytidine (DAC or 5-aza-dc) , on the growth of cholangiocarcinoma cells (QBC939) and the mechansim. Methods Methyl thiazolyl tetrazolium (MTT) method was used to measure the growth of QBC939 cells treated with DAC at different concentrations (0.1, 0.5, 1.0, 5.0, 25. 0, 100.0μmol/L) and different time points (24, 48, 72 h). The morphology was observed by electron microscopic technique (EM). Cell growth cycle and apoptosis were examined by flow cytometry. Promoter hypermethylation of DAPK, pl4 and ASC genes were detected by methylation-specific polymerase chain reaction (PCR) before and after treatment with DAC. Results DAC inhibited QBC939 cells in concentration-and time-dependent manners, and its half inhibition ratio (IC50) was 5μmol/L at 72 h. After treatment with DAC, apoptosis was observed through scanning and transmission electronic microscope. Flow cytometric analysis demonstrated that the apoptosis rate of QBC939 cells was inhanced greatly from 4.31% to 43.04%. The tumor suppressor genes, pl4 and DAPK, in QBC939 cells were methylated before treatment with DAC, and demethylated after treatment with DAC. Conclusion DAC inhibits the growth of QBC939 cells because of demethylation. DAC affects methylation of p53-bax mitchondrial apoptosis pathway in cholangiocarcinoma cells and recovers cell apoptosis.\r\n\r\nKey words: \r\nCholangiocarcinoma;  5-aza-2-deoxycytidine;  Methylation;  Apoptosis