Published online Sep 7, 2005. doi: 10.3748/wjg.v11.i33.5109
Revised: February 15, 2005
Accepted: February 18, 2005
Published online: September 7, 2005
AIM: To explore the effect of six bile salts, including glycoc-holate (GC), glycochenodeoxycholate (GCDC), glycodeoxy-cholate (GDC), taurocholate (TC), taurochenodeoxycholate (TCDC), taurodeoxycholate (TDC), and two bile acids including cholic acid (CA) and deoxycholic acid (DCA) on esophageal cancer Eca109 cell line.
METHODS: Eca109 cells were exposed to six bile salts, two bile acids and the mixed bile salts at different concentrations for 24-72 h. 3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide (MTT) assay was used to detect the cell proliferation. Apoptotic morphology was observed by phase-contrast video microscopy and deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay. Sub-G1 DNA fragmentations and early apoptosis cells were assayed by flow cytometry (FCM) with propidium iodide (PI) staining and annexin V-FITC conjugated with PI staining. Apoptosis DNA ladders on agarose were observed. Activation of caspase-3 was assayed by FCM with FITC-conjugated monoclonal rabbit anti-active caspase-3 antibody and expressions of Bcl-2 and Bax proteins were examined immunocytochemically in 500 μmol/L-TC-induced apoptosis cells.
RESULTS: Five bile salts except for GC, and two bile acids and the mixed bile salts could initiate growth inhibition of Eca109 cells in a dose- and time-dependent manner. TUNEL, FCM, and DNA ladder assays all demonstrated apoptosis induced by bile salts and bile acids at 500 μmol/L, except for GC. Early apoptosis cell percentages in Eca109 cells treated with GCDC, GDC, TC, TCDC, TDC, CA at 500 μmol/L for 12 h, DCA at 500 μmol/L for 6 h, and mixed bile salts at 1 000 μmol/L for 12 h were 7.5%, 8.7%, 14.8%, 8.9%, 7.8%, 9.3%, 22.6% and 12.5%, respectively, all were significantly higher than that in control (1.9%). About 22% of the cell population treated with TC at 500 μmol/L for 24 h had detectable active caspase-3, and were higher than that in the control (1%). Immunocytochemical assay suggested that TC down-regulated Bcl-2 protein level and up-regulated Bax protein level.
CONCLUSION: GCDC, GDC, TC, TCDC, TDC, CA and DCA, except for GC, can inhibit growth and induce apoptosis of esophageal cancer Eca109 cells. Activation of caspase-3, decreased Bcl-2 protein and increased Bax protein are involved in TC-induced apoptosis of Eca109 cells.