Brief Article
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World J Gastroenterol. Feb 21, 2012; 18(7): 704-711
Published online Feb 21, 2012. doi: 10.3748/wjg.v18.i7.704
Apoptosis of human cholangiocarcinoma cells induced by ESC-3 from Crocodylus siamensis bile
Wei Song, Dong-Yan Shen, Jin-He Kang, Shan-Shan Li, Hui-Wang Zhan, Yan Shi, You-Xiong Xiong, Ge Liang, Qing-Xi Chen
Wei Song, Dong-Yan Shen, Jin-He Kang, Shan-Shan Li, Hui-Wang Zhan, Yan Shi, Ge Liang, Qing-Xi Chen, State Key Laboratory of Cellular Stress Biology, School of Life Sciences, Xiamen University, Xiamen 361005, Fujian Province, China
You-Xiong Xiong, Thailand Sriracha Tiger Zoo Co. Ltd., Sriracha, Thailand
Author contributions: Song W and Shen DY contributed equally to this work; Song W, Shen DY, Chen QX designed the research; Song W, Kang JH, Li SS, Zhan HW performed the research; Shi Y, Xiong YX and Liang G analyzed the data; Song W, Shen DY and Kang JH wrote the paper.
Supported by The National Natural Science Foundation of China, No. 81072014; the National Foundation for Fostering Talents in Basic Sciences, No. J1030626; the Thailand Sriracha Tiger Zoo Co. Ltd., Sriracha, Thailand
Correspondence to: Qing-Xi Chen, Professor, State Key Laboratory of Cellular Stress Biology, School of Life Sciences, Xiamen University, Xiamen 361005, Fujian Province, China. chenqx@xmu.edu.cn
Telephone: +86-592-2185487 Fax: +86-592-2185487
Received: May 6, 2011
Revised: July 10, 2011
Accepted: July 17, 2011
Published online: February 21, 2012
Abstract

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 (V-FITC)/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.

Keywords: Crocodylus siamensis bile; Cholangiocarcinoma; Antiproliferation; Apoptosis; Mitochondria