黄芩素诱导人肝癌细胞株SMMC-7721凋亡作用的研究

摘要

目的:研究12-脂氧合酶(12-lipoxygenase,12-LOX)在人肝癌细胞株SMMC-7721中的表达,以及选择性12-LOX抑制剂黄芩素对人肝癌细胞SMMC-7721生长、凋亡的影响.方法:采用免疫细胞化学染色及RT-PCR检测12-LOX的蛋白质和mRNA在SMMC-7721中的表达.应用MTT、流式细胞术及TUNEL检测细胞生长及凋亡.蛋白印迹法检测分析Caspase-3,Bcl-2,Bax,phospho-ERK1/2,ERK1/2和β-actin蛋白质的表达.结果:免疫细胞化学技术显示12-LOX蛋白表达于SMMC-7721细胞质.RT-PCR检测到黄芩素呈浓度-时间依赖性地抑制12-LOX mRNA表达.采用黄芩素小同浓度或不同时间段干预细胞SMMC-7721,MTT法检测细胞增殖呈剂量、时问依赖性的下降.原位末端转移酶标记技术及流式细胞仪分析证实12-LOX抑制剂诱导细胞凋亡.蛋白印迹法检测显示,黄芩素干预后,Bcl-2蛋白表达明显下降和Bax蛋白轻微上升,细胞凋亡蛋白酶-3活性水平与黄芩素浓度正相关,黄芩素对细胞凋亡相关蛋白的影响被12 (S)-HETE逆转,12(5)-HETE可能激活ERK1/2磷酸化.结论:人肝癌细胞株SMMC-7721存在12-LOX的表达,12-LOX抑制剂黄芩素可抑制细胞生长和诱导细胞凋亡.%Objective:To examine the expression of 12 - lipoxygenase (12 - LOX) in human hepatoma cell line SMMC -7721, investigate the effect of its selective inhibitor baicalein on cell growth and apoptosis. Methods: Im-munocytochemical staining and RT - PCR were used to detect the expressions of 12 - lox protein and mRNA in SMMC -7721cel)s. Cell growth and apoptosis were detemined by MTT assay,flow cytometry and TUNEL assay . Western blot analysis was used to detect the expressions of Caspase-3, Bcl-2, Bax, phospho- ERK1/2 , ERK1/2 and β -actin protein. Results: The expression of 12 - LOX protein was detected in SMMC -7721 cells cytoplasm. The mRNA expression of 12 - LOX was detected with RT - PCR which showed that baicalein inhibited the expression of mRNA in a concentration - and time - dependent manner. The SMMC -7721 cells were treated with various concentrations of baicalein or were exposed to baicalein at 20 μmol/L for different time periods, MTT assayed that the cell growth decreased in a dose - time dependent manner. TUNEL assay and flow cytometry were used to confirm that induction of cell apoptosis by 12 - LOX inhibition. After treatment with baicalein, a marked downregulation of Bcl -2 protein and a slight upregulation of Bax protein were observed. The levels of active Caspase - 3 were increased gradually in a concentration - dependent manner after exposure to baicalein. The effects of baicalein on the above apoptosis - related proteins were reversed by the exogenous 12 (S) - HETE. 12 (S) - HKTE may activate phosphorylation of ERK1/2. Conclusion; The expression of 12 - LOX protein was detected in SMMC -7721 cells cytoplasm. Inhibition of 12 - LOX caused inhibition of cell growth and induction of apoptosis in HCC cells.