蛋白酶体抑制剂MG-132通过JNK信号通路对人脑胶质瘤细胞SHG-44内质网应激相关机制的探讨

Mechanism of proteasome inhibitor MG-132 on endoplasmic reticulum stress in human glioma SHG-44 cells by JNK signaling pathway

目的探讨MG-132通过内质网应激途径在诱导人脑胶质瘤细胞SHG-44凋亡中的作用。选择内质网应激凋亡信号转导通路JNK,使用JNK特异阻断剂SP600125,观察MG-132通过JNK信号通路诱导胶质瘤细胞凋亡过程中凋亡指标的变化。方法传代法培养人脑胶质瘤细胞。选择浓度为5、10、15、50μmol/L的MG-132,分别作用于实验组,用噻唑噻(MTT)法筛选半数有效浓度后进行实验。实验分为5组:正常对照组、二硫苏糖醇(DTT)组、MG-132组、SP600125组、MG-132+SP600125组。试剂干预终止后,应用流式细胞仪和DNALadder评估肿瘤细胞凋亡,以聚合酶链反应(PCR)和蛋白印迹法检测内质网应激指标GRP78、JNK。结果5μmol/L为筛选的最佳抑制浓度,SHG-44细胞活力下降达39%(P<0.05)。MG-132干预后,与对照组比较,DNA电泳呈凋亡特征(梯状条带),细胞凋亡率显著增加(P<0.05),GRP78m RNA表达明显增加(P<0.05)。但给予DTT后,与MG-132组表现一致,差异无统计学意义(P>0.05)。SP600125+MG-132较DTT和MG-132组有所减低,但不如单独SP600125组降低明显(P<0.05)。蛋白印迹法结果显示,与对照组比较,MG-132组和DTT组内质网应激指标GRP78、JNK表达明显增加(P<0.05),SP600125+MG-132组表达有所减低,但不如单独给予SP600125组明显(P<0.05)。结论内质网应激是MG-132诱导胶质瘤细胞凋亡的主要途径之一,JNK通路抑制剂SP600125可以部分阻断内质网应激,即JNK信号通路为MG-132发挥凋亡作用的途径之一。

Objective To investigate the effect of MG-132 in inducing apoptosis of human glioma SHG-44 cells by endoplasmic reticulum stress. The JNK pathway was transduced by endoplasmic reticulum stress（ERS） signaling, and the JNK specific inhibitor SP600125 was used. The changes of apoptosis indicators during the process of MG-132 in inducing apoptosis of glioma cells by JNK signaling pathway were determined. Methods The passage culture of human glioma cells was performed. The concentrations of 5,10,15,and 50 μmol/L MG-132 were included and used in the study group. After screening the median effective concentration by MTT method,the experiment was carried out. The study was divided into 5 groups：the normal control group,DTT group,MG-132 group,SP600125 group, and MG-132＋SP600125 group. At the end of the reagent intervention,the apoptosis of tumor cells was evaluated by flow cytometry and DNA Ladder. The expression of the ERS indicators GRP78 and JNK were measured by PCR and Western-blotting. Results The optimal screened inhibitory concentration was 5μmol/L, and the viability of SHG-44 cells decreased by 39%（P〈0.05）. Compared with the control group, the DNA electrophoresis showed apoptotic characteristic（ladder-like band）,the apoptosis rate significantly increased（P〈0.05）, and the GRP78 m RNA expression significantly increased in the MG-132 group（P〈0.05）. The performance of the DTT group was consistent with the MG-132 group,and there were no statistically significant difference between them（P 〉0.05）. The performance in the SP600125＋MG-132 group was reduced compared with that in the DTT and MG-132 groups,but was not significantly lower than that in the SP600125 group（P〈0.05）. The findings of Western-blot showed that compared with the control group, the expressions of ERS indicators GRP78 and JNK in the MG-132 and DTT groups significantly increased（P〈0.05）,and the expression in the SP600125＋MG-132 group decreased,but was significantly lower than that in the SP600125 group（P〈0.05）. Conclusion ERS is one of the main pathways of MG-132 induced apoptosis in glioma cells. JNK pathway inhibitor SP600125 can partially block ERS. In other words, JNK signaling pathway is one of the ways in which MG-132 plays an apoptotic role.