5-溴甲基-2(5H)呋喃酮诱导人肝癌HepG-2细胞株凋亡的作用机制

Studies on the apoptotic mechanism of the human hepatoma cell line HepG-2 induced by 5-bromomethyl-2(5H)furanone

[目的]研究5-溴甲基-2(5H)呋喃酮诱导人肝癌HepG-2细胞株凋亡的作用机制.[方法]应用细胞体外传代培养方法培养人肝癌HepG-2细胞;利用四唑盐比色(MTT)法检测5-溴甲基-2(5H)呋喃酮对人肝癌HepG-2细胞株的抗增殖及细胞毒作用;采用流式细胞术检测癌细胞周期分布及药物对细胞凋亡的影响;采用免疫细胞化学技术检测5-溴甲基-2(5H)呋喃酮作用前后细胞凋亡相关基因Caspase-3,Caspase-8,Caspase-9,Survivin,Livin,NF-κB,Bcl-2,Bax,Fas蛋白的表达情况,并探讨其诱导人肝癌HepG-2细胞凋亡的作用机制.[结果]MTT检测结果显示,5-溴甲基-2(5H)呋喃酮对人肝癌HepG-2细胞的抑制作用表现出质量浓度和时间依赖性,IC50值为5 mg/L,48 h.流式细胞仪检测结果显示,5-溴甲基-2(5H)呋喃酮5 mg/L作用于人肝癌HepG-2细胞24,48,72 h后可见典型的亚二倍体峰,且G1期细胞数量增多,48 h时对照组G0/G1期细胞占40.30%,5-溴甲基-2(5H)呋喃酮组占46.21%,提示细胞阻滞在G0/G1期;对照组细胞凋亡率为0.02%,5-溴甲基-2(5H)呋喃酮组为19.74%,两组比较差异具有统计学意义(P<0.05).免疫细胞化学检测结果显示,与对照组比较,5-溴甲基-2(5H)呋喃酮组Survivin,Livin,NF-κB,Bcl-2蛋白的阳性表达率明显减少(P<0.05),Caspase-3,Caspase-8,Caspase-9,Fas及Bax蛋白的阳性表达率明显增加(P<0.05).[结论]5-溴甲基-2(5H)呋喃酮对人肝癌HepG-2细胞株具有明显的抑制增殖作用,且呈量-效和时-效关系,并将细胞阻滞于G0/G1期.5-溴甲基-2(5H)呋喃酮对人肝癌HepG-2细胞株有明显的诱导凋亡作用,作用机制可能是通过死亡受体途径和线粒体途径共同介导的.

OBJECTIVE To research the apoptotic mechanism of the human hepatoma cell line HepG-2induced by 5-bromomethyl-2（5H）furanone.METHODS Cell subculture in vitro method was used to culture the human hepatoma cell line HepG-2.MTT assay was used to detected the antiproliferation and cytotoxic effects of 5-bromomethyl-2（5H）furanone on HepG-2cell line.Flow cytometry was used to measure cell cycle distribution of cancer cells and the influences of drugs on cell apoptosis.Immunocytochemistry was used todetect the expressions of apoptosis-related genes Caspase-3,Caspase-8,Caspase-9,Survivin,Livin,NF-κB,Bcl-2,Bax and Fas before and after the use of 5-bromomethyl-2（5H）furanone,and to explorethe apoptotic mechanism of the human hepatoma cell line HepG-2induced by it.RESULTS MTT assay showed that the inhibition effects of 5-bromomethyl-2（5H）furanone on HepG-2 cell line was in a dose and time-dependent manner.The IC_（50） value was 5mg/L.48 hours flow cytometry results showed that a typical sub-diploid peak was observed at 24,48,72 hafter the treatment of 5-bromomethyl-2（5H）furanone,and the amount of cells in G_1 phase was increased.Cell count of G_0/G_1 phase in the control group accounted for 40.30%,and that in the 5-bromomethyl-2（5H）furanone group accounted for 46.21%,which hinted that the cells were blocked in G_0/G_1 phase.The apoptosis rates were 0.02% and 19.74%in the control and 5-bromomethyl-2（5H）furanone group respectively,there was statistical significance between the two groups（P〈0.05）.Immunocytochemistry showed that as compared with the control group the expressions of Survivin,Livin,NF-κB and Bcl-2in experiment group were significantly decreased（P〈0.05）,while the expressions of Caspase-3, Caspase-8, Caspase-9, Fas and Bax were markedly increased（P 〈0.05）.CONCLUSION5-bromomethyl-2（5H）furanone obviously inhibited the growth of human hepatoma cell line HepG-2in a dose and time-dependent manner,and arrested the cells in G_0/G_1 phase.It could induce the apoptosis of human hepatoma cell line HepG-2,and the mechanisms might be mediated by the death receptor pathway and the mitochondrial pathway.