MG132抑制肝癌细胞Bel-7404生长的机制研究

A Study on The Mechanism of The Inhibition of Bel-7404 Hepatocarcinoma Cell Growth by MG132

MG132(Z-Leu-leu-leu-CHO)是一种蛋白酶体抑制剂,它可逆地抑制蛋白酶体活性,从而抑制泛素-蛋白酶体通路所介导的蛋白质降解,诱导细胞凋亡.实验研究MG132抑制肝癌细胞Bel-7404生长的机制.采用不同浓度梯度和时间梯度,通过荧光显微镜、透射电子显微镜、Hoechst33342染色、MTT检测、AnnexinⅤ/PI流式细胞术、Westernblot分别检测MG132对Bel-7404细胞的形态学变化、细胞内质网压力变化、自噬泡形成、凋亡小体形成、细胞存活率、细胞凋亡水平、凋亡及自噬信号途径中相关蛋白质表达的影响.结果显示,MG132能明显抑制Bel-7404细胞生长.通过增加内质网压力激活Caspase-12,也可通过线粒体途径调节Bcl-2/Bax水平,促进细胞色素c的释放,两者皆可激活下游效应Caspase-3,剪切PARP,诱导细胞凋亡.同时,MG132可诱导Beclin1和LC3B的上调,促使Bel-7404细胞发生自噬,可在透射电镜下观察到自噬泡形成.上述结果表明,MG132作用Bel-7404细胞涉及两类细胞程序性死亡途径:细胞凋亡和自噬.

MG132（Z-Leu-leu-leu-CHO） is an inhibitor of proteasome, and it can reversibly inhibit the activation of proteasome, thereby inhibiting the degradation of protein which involved in ubiquitin-proteasome pathway （UPP）, and inducing apoptosis at last. Study demonstrated that MG132 was capable of inhibition the proliferation of Bel-7404 hepatocarcinoma cell. After treated with different-concentration of MG132 at different-time, the change of morphological change and endoplasmic reticulum stress, formation of autophagic vacuoles and apoptotic bodies, cells viability, cell apoptosis, the protein expression of both apoptosis and autophagy signaling pathway related genes formation of in Bel-7404 cells were assessed by fluorescence microscope, Hoechst33342 staining, MTT assay, AnnexinⅤ/PI flow cytometry, Western blotting and transmission electron microscopy analysis. The results suggested that MG132 can inhibit Bel-7404 cells growth remarkably . It was able to activate Caspase-12 through endoplasmic reticulum stress pathway, can also influence the level of Bcl-2/Bax, and consequently induced releasing of cytochrome c through mitochondrial pathway. Both of the two different signaling pathways can activate Caspase-3 and PARP. Furthermore, MG132 increased the expression of Beclin1 and LC3B. Autophagic vacuoles were also detected by transmission electron microscopy analysis. It was confirmed that MG132 inhibited the growth of Bel-7404 cells not only via apoptosis pathway, but also related with autophagy pathway.