X-F Liu1, H Jiang, C-S Zhang, S-P Yu, Z-Q Wang, H-L Su. 1. Department of Hepatobiliary Surgery, Affiliated Yantai Yuhuangding Hospital, Qingdao University Medical College, Yantai, China. liu634@263.net
Abstract
OBJECTIVE: To study the mechanism of 5-aza-2-deoxycytidine (DAC; a methylation inhibitor) on growth of the human cholangiocarcinoma QBC939 cell line. METHODS: A colourimetric assay was used to detect growth of QBC939 cells treated with DAC (0.1-100 μmol/l) over 24 h, 48 h and 72 h. Cell morphology was observed by transmission electron microscopy (TEM). The cell cycle and apoptosis were analysed by flow cytometry. Hypermethylation of the promoters of the p53-BAX mitochondrial apoptosis genes cyclin-dependent kinase inhibitor 2A (CDKN2A), death-associated protein kinase 1 (DAPK1) and PYD and CARD domain containing (PYCARD) was detected by methylation-specific polymerase chain reaction, with and without DAC treatment. RESULTS: DAC inhibited QBC939 cell growth with a half maximal inhibitory concentration of 5 μmol/l at 72 h. After DAC treatment, apoptosis was observed by TEM. Flow cytometric analysis of propidium iodide-positive cells demonstrated increased apoptosis of DAC-treated QBC939 cells (43.04%) compared with untreated cells (4.31%). DAC treatment resulted in demethylation of the gene promoters of CDKN2A and DAPK1 in QBC939 cells. CONCLUSIONS: DAC induces apoptosis of QBC939 cells by reactivation of hypermethylated p53-BAX mitchondrial apoptosis genes in cholangiocarcinoma cells.
OBJECTIVE: To study the mechanism of 5-aza-2-deoxycytidine (DAC; a methylation inhibitor) on growth of the humancholangiocarcinoma QBC939 cell line. METHODS: A colourimetric assay was used to detect growth of QBC939 cells treated with DAC (0.1-100 μmol/l) over 24 h, 48 h and 72 h. Cell morphology was observed by transmission electron microscopy (TEM). The cell cycle and apoptosis were analysed by flow cytometry. Hypermethylation of the promoters of the p53-BAX mitochondrial apoptosis genes cyclin-dependent kinase inhibitor 2A (CDKN2A), death-associated protein kinase 1 (DAPK1) and PYD and CARD domain containing (PYCARD) was detected by methylation-specific polymerase chain reaction, with and without DAC treatment. RESULTS:DAC inhibited QBC939 cell growth with a half maximal inhibitory concentration of 5 μmol/l at 72 h. After DAC treatment, apoptosis was observed by TEM. Flow cytometric analysis of propidium iodide-positive cells demonstrated increased apoptosis of DAC-treated QBC939 cells (43.04%) compared with untreated cells (4.31%). DAC treatment resulted in demethylation of the gene promoters of CDKN2A and DAPK1 in QBC939 cells. CONCLUSIONS:DAC induces apoptosis of QBC939 cells by reactivation of hypermethylated p53-BAX mitchondrial apoptosis genes in cholangiocarcinoma cells.