title: Apoptosis of human cholangiocarcinoma cells induced by ESC-3 from Crocodylus siamensis bile.
abstract: OBJECTIVE
  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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: State Key Laboratory of Cellular Stress Biology, School of Life Sciences, Xiamen University, Xiamen 361005, Fujian Province, China.
authors.forename: Wei
authors.initials: W
authors.lastname: Song
authors.forename: Dong-Yan
authors.initials: DY
authors.lastname: Shen
authors.forename: Jin-He
authors.initials: JH
authors.lastname: Kang
authors.forename: Shan-Shan
authors.initials: SS
authors.lastname: Li
authors.forename: Hui-Wang
authors.initials: HW
authors.lastname: Zhan
authors.forename: Yan
authors.initials: Y
authors.lastname: Shi
authors.forename: You-Xiong
authors.initials: YX
authors.lastname: Xiong
authors.forename: Ge
authors.initials: G
authors.lastname: Liang
authors.forename: Qing-Xi
authors.initials: QX
authors.lastname: Chen
mesh_terms: D000489:Alligators and Crocodiles; D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology* / Q000627:therapeutic use; D017209:Apoptosis / Q000187:drug effects*; D001646:Bile / Q000737:chemistry*; D018281:Cholangiocarcinoma / Q000188:drug therapy / Q000473:pathology*; D004354:Drug Screening Assays, Antitumor; D006801:Humans; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism; D014020:Tissue Extracts / Q000494:pharmacology* / Q000627:therapeutic use
chemical_list: D000970:Antineoplastic Agents; D014020:Tissue Extracts