AIM:To investigate the effects of ESC-3 isolated from crocodile bile on the growth and apoptosis induction of human cholangiocarcinoma cells.METHODS:ESC-3 was isolated from crocodile bile by Sephadex LH-20 and RP-18 r...AIM:To investigate the effects of ESC-3 isolated from crocodile bile on the growth and apoptosis induction of human cholangiocarcinoma cells.METHODS:ESC-3 was isolated from crocodile bile by Sephadex LH-20 and RP-18 reversed-phase column.3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay was conducted to determine the effects of ESC-3 on the proliferation of human cholangiocarcinoma cell lines (QBC939,Sk-ChA-1 and MZ-ChA-1).Giemsa staining,Hoechst 33258 and acridine orange/ethidium bromide staining showed the morphological changes of Mz-ChA-1 cells exposed to ESC-3 at different concentrations.Flow cytometry with regular propidium iodide (PI) staining was performed to analyze the cell cycle distribution of Mz-ChA-1 cells and to assess apoptosis by annexin v-fluorescein isothiocyanate (VFITC)/PI staining.Rh123 staining was used to detect the alteration of mitochondrial membrane potential (ΔΨm).The protein levels of Bax,Bcl-2,Cdk2,cytochrome c and caspase-3 were further confirmed by Western blotting.RESULTS:ESC-3 significantly inhibited the growth of three human cholangiocarcinoma cell lines and arrested Mz-ChA-1 cell cycle at G0/G1 phase.Mz-ChA-1 cells showed typical apoptotic morphological changes after treated with ESC-3 (10 μg/mL) for 48 h.Cell death assay indicated that Mz-ChA-1 cells underwent apoptosis in a dose-dependent manner induced by ESC-3.In addition,ESC-3 treatment could downregulate the protein level of Bcl-2 and upregulate the Bax,leading to the increase in the ratio of Bax to Bcl-2 in Mz-ChA-1 cells.Meanwhile,cytochrome c was released from the mitochondria into the cytosol,which subsequently initiated the activation of caspase-3.All these events were associated with the collapse of the mitochondrial membrane potential.CONCLUSION:ESC-3,the active ingredient of crocodile bile,induced apoptosis in Mz-ChA-1 cells through the mitochondria-dependent pathway and may be a potential chemotherapeutic drug for the treatment of cholangiocarcinoma.展开更多
基金Supported by The National Natural Science Foundation of China,No.81072014the National Foundation for Fostering Talents in Basic Sciences,No.J1030626the Thailand Sriracha Tiger Zoo Co.Ltd.,Sriracha,Thailand
文摘AIM:To investigate the effects of ESC-3 isolated from crocodile bile on the growth and apoptosis induction of human cholangiocarcinoma cells.METHODS:ESC-3 was isolated from crocodile bile by Sephadex LH-20 and RP-18 reversed-phase column.3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay was conducted to determine the effects of ESC-3 on the proliferation of human cholangiocarcinoma cell lines (QBC939,Sk-ChA-1 and MZ-ChA-1).Giemsa staining,Hoechst 33258 and acridine orange/ethidium bromide staining showed the morphological changes of Mz-ChA-1 cells exposed to ESC-3 at different concentrations.Flow cytometry with regular propidium iodide (PI) staining was performed to analyze the cell cycle distribution of Mz-ChA-1 cells and to assess apoptosis by annexin v-fluorescein isothiocyanate (VFITC)/PI staining.Rh123 staining was used to detect the alteration of mitochondrial membrane potential (ΔΨm).The protein levels of Bax,Bcl-2,Cdk2,cytochrome c and caspase-3 were further confirmed by Western blotting.RESULTS:ESC-3 significantly inhibited the growth of three human cholangiocarcinoma cell lines and arrested Mz-ChA-1 cell cycle at G0/G1 phase.Mz-ChA-1 cells showed typical apoptotic morphological changes after treated with ESC-3 (10 μg/mL) for 48 h.Cell death assay indicated that Mz-ChA-1 cells underwent apoptosis in a dose-dependent manner induced by ESC-3.In addition,ESC-3 treatment could downregulate the protein level of Bcl-2 and upregulate the Bax,leading to the increase in the ratio of Bax to Bcl-2 in Mz-ChA-1 cells.Meanwhile,cytochrome c was released from the mitochondria into the cytosol,which subsequently initiated the activation of caspase-3.All these events were associated with the collapse of the mitochondrial membrane potential.CONCLUSION:ESC-3,the active ingredient of crocodile bile,induced apoptosis in Mz-ChA-1 cells through the mitochondria-dependent pathway and may be a potential chemotherapeutic drug for the treatment of cholangiocarcinoma.
