黄芩素诱导人肝癌细胞株SMMC-7721凋亡作用的研究

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.

Effect of baicalein on apoptosis of human hepatoma cell line SMMC- 7721

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: Immunocytochemical staining and RT-PCR were used to detect the expressions of 12-lox protein and mRNA in SMMC-7721cells.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)-HETE 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.