Arsenic Trioxide Inhibits The Growth Of Human Gallbladder Carcinoma Cell And Induces The Cell Apoptosis

Part I: Arsenic trioxide induces gallbladder carcinoma cell apoptosis via downregulation of Bcl-2Background and Purpose: Gallbladder carcinoma, lacking of specific clinical manifestation, usually diagnosed at advanced stages of the diseases and fewer can be resected in operation. Chemotherapy has not shown significant activity in gallbladder carcinoma, so new agent should be applied to its treatment. Arsenic trioxide, verified as a breakthrough in the management of acute promyelocytic leukemia, has been applied to the research of a variety of solid tumors. This study is to investigate the biological effect of As₂O₃ on human gallbladder carcinoma cell GBC cells and and identify the contribution of Bcl-2 in the As₂O₃-induced apoptosis. Methods: Hoechest33258 staining, DNA gel electrophoresis and flowcytometric were used to determine apoptosis. Western blot was performed to analysis apoptotic proteins expression. GBC cells were transiently transfected with constructed Bcl-2 plasmid and its apoptosis was detected again. Results: The treatment of As₂O₃ in gallbladder carcinoma cells could induce apoptosis in a dose-dependent manner and downregulate the expression of anti-apoptotic protein Bcl-2. Moreover, Bcl-2 overexpression could protect gallbladder carcinoma cells from As₂O₃-induced apoptosis. Conclusions: As₂O₃ induces gallbladder carcinoma cell apoptosis via down regulation of Bcl-2 Part II: Arsenic Trioxide-mediated Growth Inhibition in Gallbladder carcinoma Cells via down-regulation of Cyclin D1 transcription mediated by Sp1 transcription factorBackground and Purpose: The internal characteristic in the development and progression of malignant tumor is non-limited growth, which is closely linked with the abnormal regulation of cell cycle. The family of cyclin D have played a major role in the regulation of cell cycle and the overexpression of cyclin D1 has participated the development and progression of gallbladder carcinoma. In additional, can stimulate the activity of varieties of the cell-growth-related gene. This study is to investigate the inhibiting effect of arsenic trioxide(As₂O₃) on human gallbladder carcinoma (GBC) cells as well as the whats and the hows of cyclin D1 and Sp1 transcription factor during the effect of As₂O₃. Methods: GBC cells were cultured with different concentrations of As₂O₃, the proliferative activity of cells was detected by MTT methods, the cell cycle status were detected by flow cytometry as well as Western blot and RT-PCR were performed to analyse the protein and mRNA expression of Cyclin D1,D2,D3,CDK4 and CDK6.GBC cells were transient transfected with overexpression of Cyclin D1 derived from the constructed plasmid as well as the As₂O₃-induced alteration of cell cycle was detected again. GBC cells were transient transfected with Cyclin D1 promoter construct pGL3 and then treated by different dose of As₂O₃. The Sp1 protein expression was also measured and its plasmid was constructed and transiently transfected into GBC cells as well as the luciferase activity of Cyclin D1 promoter was measured pre- and post the transfection. Results: The treatment of As₂O₃ in gallbladder carcinoma cells could inhibit the growth of cell in a time- and dose-dependent manner, make cells arrested in G₁ and downregulate the expression of Cyclin D1. Cyclin D1 overexpression played the negative role of As₂O₃-induced alteration of cell cycle. The activity of Cyclin D1 promoter was downregulated by As₂O₃ in a dose-dependent manner and decreased about 70 percent when treated with 4μmol/L As₂O₃. Moreover, the Sp1 was downregulated by As₂O₃ in a dose-dependent manner and the exogenous Sp1 transcription factor played the negative role to the effect of As₂O₃. Conclusion: As₂O₃ can significantly inhibit the growth of human gallbladder carcinoma cell via down-regulating the expression of Cyclin D1 mediated by Sp1 transcription factor.