细胞色素C在氟尿嘧啶诱导肝癌细胞凋亡中的作用

The role of cytochrome C in the apoptosis of hepatoma cell induced by flurouracil

目的检测细胞色素C在氟尿嘧啶诱导肝癌细胞凋亡过程中的作用,分析细胞色素C在线粒体和胞浆中的分布及其与caspase-9活化的关系。方法用1×10-2mol/L的5-FU处理HepG2细胞,分别作用2,4,8,16,24 h,用荧光检测试剂盒检测HepG2细胞凋亡过程中细胞色素C分布变化;Western-blot检测caspase-9的活化和细胞色素C在胞浆及线粒体中的分布。结果氟尿嘧啶处理肝癌细胞4 h后,caspase-9活性开始升高,于16 h后达到高峰,与对照组比较差异有显著性(P<0.01)。Western-blot分析发现caspase-9被蛋白酶水解切断后形成10kD片段;细胞色素C在胞浆中的分布从4h后逐渐增加,16 h最明显,而其在线粒体中的分布却正相反;细胞色素C的分布改变和caspase-9的活化基本同步。结论在氟尿嘧啶诱导肝癌细胞凋亡过程中caspase-9被活化,并伴有细胞色素C自线粒体中释放到胞浆,提示细胞色素C可能在Caspase-9的活化和肝癌细胞凋亡中起重要作用。

Objective To investigate the role of cytochrome C in the apoptosis of hepatoma cell induced by flurouracil , and to analyze the distribution of cytochrome C in cytosol and the relationship between the cytochrome C distribution and Caspase-9 activation. Methods The human hepatoma HepG2 cells were treated with flurouracil at 1 × 10^-2 mol/L and 2,4,8,16,24h respectively. The chang of cytochrome C in HepG2 apoptosis was detected by using Fluorescent Assay Kit; and proteolytic cleavage of caspase-9 and distribution of cytochrome C in cytosol or in mitochondria was analyzed by Western blot. Results Four hours after cells exposure to flurouracil, the caspase-9 activity in HepG2 cells increased gradually and reached the peak at 16 h, compared with the control groups, the difference was significant （ P 〈 0.01 ）. In Western blot analysis, caspase-9 was found to be cleaved into 10 kD fragment, and distribution of cytochrome C in cytosol became more and more obvious from 4h on and reached a peak at 16h, but the distribution of cytochrome C in mitochondria was opposite. The distribution change of cytochrome C and activity alteration of Caspase-9 were synchronous. Conclusions Caspase-9 was activated in the apoptosis of HepG2 ceils induced by flurouracil, accompanied by release of cytochrome C from mitochondria to cytosol. This data implies that cytochrome C plays an important role in the activation of Caspase-9 and apoptosis of hepatoma cell induced by flurouracil.