Apoptosis of human cholangiocarcinoma cells induced by ESC-3 from Crocodylus siamensis bile

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

Figure 1

Isolation of ESC-3 from the gallbladder of Crocodylus siamensis. A: Using Sephadex LH-20; B: Using RP-18 reversed-phase columns; C: QBC939, Sk-ChA-1 and MZ-ChA-1 were treated continuously with different concentrations of ESC-3 for 48 h. Cell growth inhibition was analyzed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay; D: Growth inhibitory effects of ESC-3 on Mz-ChA-1 cells. Exponentially growing Mz-ChA-1 cells were treated with different concentrations of ESC-3 for different periods of time. Cell growth inhibition was analyzed by the MTT assay.

Figure 2

Morphological changes in the Mz-ChA-1 cells after exposure to different concentrations (0 μg/mL and 10 μg/mL) of ESC-3 for 48 h. A: Morphological changes visualized under an ordinary inverted phase-contrast microscope with Giemsa staining (magnification 400 ×); B: Morphological changes visualized under fluorescence microscope with AO/EB staining (magnification 200 ×); C: Morphological changes visualized under fluorescence microscope with Hoechst 33258 staining (magnification 400 ×). The arrows indicate the cells undergoing apoptosis. AO/EB: Acridine orange/ethidium bromide.

Figure 3

Effects of ESC-3 on cell cycle distribution and apoptosis. A: Cell cycle analysis of Mz-ChA-1 cells using flow cytometry with propidium iodide (PI) staining and the DNA histograms; B: Assessment of apoptosis using flow cytometry with annexin v-fluorescein isothiocyanate (V-FITC)/PI staining and the dot-plot graph of Mz-ChA-1 cells.

Figure 4

Apoptosis induced by ESC-3 through the mitochondria-dependent pathway. A: Effect of ESC-3 on the ΔΨm of cholangiocarcinoma cells. The increase in Rh123 hypofluorescence indicates a reduction in ΔΨm, which is shown with arrows; B: Expression of cytochrome C, caspase-3, CDK2, Bax, and Bcl-2 in Mz-ChA-1 cells treated with 10 μg/mL ESC-3 for different periods of time; C: Effect of ESC-3 on the activation of caspase-3 activity in Mz-ChA-1 cells. The cells were treated with 0 μg/mL, 5 μg/mL, 7.5 μg/mL and 12.5 μg/mL ESC-3, and caspase-3 activity was analyzed after 24 h, 48 h or 72 h.