桂皮酸诱导Bel-7402人肝癌细胞凋亡的作用及机制

Effect and Mechanism of Human Hepatocarcinoma Cell Bel-7402 Apoptosis Induced by Cinnamic Acid

【目的】通过检测不同浓度的桂皮酸对人肝癌Bel-7402细胞生存率及凋亡的影响,明确中药桂皮酸诱导该肿瘤细胞凋亡的作用和机制。【方法】采用MTT法检测0、1、3、5和10 mmol/L桂皮酸对正常肝细胞CL48及人肝癌细胞Bel-7402生存率的影响;采用PI/AnnexinⅤ双染法检测0、1、3、5和10 mmol/L桂皮酸对Bel-7402细胞凋亡作用的影响;应用Western-blot技术检测不同浓度桂皮酸对Bel-7402细胞中Bax、cytochrome c、caspase 8、caspase 9及caspase 3的作用。【结果】MTT结果显示桂皮酸能剂量依赖性地抑制Bel-7402细胞的生长,而对正常肝细胞的生存率无影响,P<0.05。PI/AnnexinⅤ双染法,流式细胞仪检测显示空白对照组细胞凋亡率为(5.43±0.57)%,浓度为1、3、5和10mmol/L的桂皮酸处理组细胞凋亡率分别为(7.37±0.74)%、(13.73±1.5)%、(25.67±2.15)%和(52.23±4.18)%。蛋白质印迹法结果显示,桂皮酸能促进Bel-7402细胞caspase 8、caspase 9及caspase 3的切割;促进Bax蛋白转位至线粒体及cytochrome c的释放。【结论】桂皮酸具有抑制肝癌Bel-7402细胞生长及促进其凋亡的作用,可能是通过促进Bax蛋白转位至线粒体,从而刺激cytochrome c释放至胞浆,激活caspase蛋白导致肿瘤细胞凋亡。

[Objective] To study the effects on viability and apoptosis of human hepatocarcinoma cell Bel-7402 and the molecular mechanism by cinnamic acid （CINN） treatment. [Methods] MTI＂ assay was performed to detect the viability of CIA8 cells and Bel- 7402 cells by 0, 1, 3, 5, and 10 mmol/L of CINN. The apoptosis of Bel-7402 cells induced by O, 1,3,5, and 10 mmol/L of CINN were detected through flow cytometry. Western blot assay were used to determine Bax, cytochrome c, caspase 8, caspase 9, and caspase 3 by CINN at different concentration. [ Results ] MTT assay showed that CINN dose-dependently inhibited growth of Bel-7402 cells, but had no effect on the viability of CIA8 cells （P 〈 0.05）. Annexin V and propidium iodide （PI） staining showed that the apoptosis rates of Bel-7402 cells induced by 0, 1, 3, 5, and 10 mmol/L of CINN were （5.43 ± 0.57）%, （7.37 ± 0.74）%, （13.73 ± 1.5）%, （25.67± 2.15）%, and （52.23 ± 4.18）%, respectively. Western-blot analysis showed that CINN prompted eytoehrome c release and activated easpase 8, caspase 9, and caspase 3. CINN had no effect on the total amounts of Bax protein and induced Bax translocating to mitochondria. [Conclusion] The reason that CINN induce the apoptosis of Bel-7402 cells might be through promotion translocation of Bax to mitochondria, cytochrome e release and then caspase proteins activation.