羟基喜树碱诱导肝癌细胞凋亡前后线粒体疏水蛋白质组的定量分析

Quantitative Analysis of Mitochondrial Hydrophobic Proteome in HCPT-Induced Apoptosis of Hepatoma Cells

抗癌剂羟基喜树碱可以通过线粒体途径诱导肝癌细胞凋亡.应用定量蛋白质组学技术分析羟基喜树碱诱导肝癌细胞凋亡前后的线粒体疏水蛋白质差异表达,探讨癌细胞凋亡机制及羟基喜树碱的抗癌机理.分离提取羟基喜树碱诱导肝癌细胞凋亡前后的线粒体,并采用顺序抽提法提取疏水蛋白质;用含稳定同位素亲和标签的c-ICAT试剂标记蛋白,利用基于多维色谱-线性离子阱/静电场轨道阱质谱联用技术的鸟枪(shotgun)法策略分析鉴定了在肝癌细胞凋亡前后的线粒体中表达量差异有显著统计学意义(P<0·05)的疏水蛋白144种,其中,12种蛋白的表达量在凋亡细胞中下调,而表达量在羟基喜树碱诱导细胞凋亡后上调10倍以上的蛋白43种.这些蛋白主要与细胞分裂增殖、分化凋亡、能量代谢、核酸代谢以及信号转导相关.该研究结果为在亚细胞定量蛋白质组水平上深入探讨羟基喜树碱的作用机理提供了新的实验依据,亦为研究肿瘤细胞凋亡机制提供了新的思路.

Hydroxycamptothecin is an anticancer agent that is known to induce apoptosis in hepatoma cells through the mitochondrial pathway, To extend the study of the molecular mechanism involved in hydroxycamptothecin-induced apoptosis, we analyzed the differentially expressed mitochondrial hydrophobic proteins in hydroxycamptothecin-treated cells via a quantitative proteomics approach. The mitochondria extracts from hydroxycamptothecin treated hepatoma cells were analyzed by western blot; and the mitochondrial hydrophobic proteins were labeled with cleavable isotope-coded affinity tag（c-ICAT）reagent. A total of 144 proteins were significantly （P 〈 0.05） altered in hydroxycamptothecin-treated cells; they were subsequently identified and quantified based on a shotgun proteomic method using a multiple dimensional liquid chromatography-linear ion trap/orbitrap mass spectrometer. 12 proteins were down-regulated after hydroxycamptothecin treatment, whereas 43 proteins were up-regulated for more than 10-folds. The functions of these proteins include cell proliferation, differentiation, apoptosis, energy metabolism, nucleic acid synthesis and signal transductions. The results may help further investigation of the pharmacological impacts in hydroxycamptothecin treated cells.