Effect of lobaplatin on apoptosis of human cholangiocarcinoma cell line RBE and expression of B cell lymphoma/leukemia-2 associated X protein, B cell lymphoma/leukemia-2, Caspase-3/Caspase-8 and Cytochrome C

Abstract

Objective The object of this study was to determine the anti-tumor effect of lobaplatin in cholangiocarcinoma (CCA) cell line RBE and to investigate the mechanisms by determines the transcription and expression levels of B cell lymphoma/leukemia-2 associated X protein (bax),B cell lymphoma/leukemia-2 (bcl-2),Actived-Caspase-3,Caspase-8 and Cytochrome C (Cyt-C).Methods The RBE cells were cultured in vitro,followed by various concentration of lobaplatin for several times.The inhibition rates of cells were determined by methyl thiazol tetrazolium (MTT) assay and cell cycle changes were detected by flow cytometric analysis.Then,Annexin V/propidium iodide (PI) double-staining assay was employed to measure the apoptosis of cells.The change in mRNA and protein levels of bax,bcl-2,activedCaspase-3,Caspase-8 and Cyt-C were determined by Western blotting and real-time quantitative polymerase chain reaction (Real-time PCR).Results Lobaplatin was shown to be more Inhibition of cell proliferation in RBE cells,with the drug concentration and the role of time increasing.The proportion of G0/G1 and G2/M increased (P ＜0.05).In Annexin V/PI assay,the percentages of apoptosis cells were higher in lobaplatin groups with statistically significant (P ＜ 0.05).Both the Western blotting and Real-time PCR assays shown bax,Actived-Caspase-3,Caspase-8 and Intracytoplasmic Cyt-C were up-regulated at both the mRNA and protein levels,while bcl-2 was decreased at mRNA level.Conclusion Lobaplatin inhibits proliferation of RBE cell and induces apoptosis by up-regulating the bax,decreasing the bcl-2,in transcription and expression levels,releasing Cyt-C,and activating Caspase-3,Caspase-8. Key words: Cholangiocarcinoma; Lobaplatin ; Apoptosis B cell lymphoma/leukemia-2 associated X protein; Caspase; Cytochrome C