【摘要】 目的:探讨蛋白酶体抑制剂MG-132逆转人结肠癌细胞获得性TRAIL耐药的作用及其可能的机制。方法:在MG-132和TRAIL蛋白联合处理获得性TRAIL耐药的人结肠癌细胞DLD1-TRAIL/R后,MTT法检测细胞的存活率,流式细胞术检测细胞凋亡率,Westernblotting检测细胞中各种凋亡相关蛋白的表达和JNK激酶的磷酸化水平。结果:MG-132联合TRAIL蛋白处理DLD1-TRAIL/R细胞后,其细胞存活率明显下降(P<0.01),而细胞凋亡率则明显增加(P<0.01)。Westernblotting检测显示,联合处理后DLD1-TRAIL/R细胞中各种凋亡信号分子包括caspase-8、caspase-9、caspase-3、Bid和PARP蛋白均明显活化,线粒体中细胞色素C和Smac蛋白大量释放;进一步的Westernblotting检测显示,死亡受体DR5和凋亡诱导蛋白Bik的表达水平明显增高,而其他凋亡信号分子包括DR4、Bax、Bak、Bcl-XL、XIAP和Survivin等则无明显改变;检测结果还显示,MG-132能诱导JNK激酶发生磷酸化,使用JNK激酶抑制剂SP600125能够阻断MG-132诱导的DR5表达,但不影响Bik的表达,并且不能减弱MG-132和TRAIL蛋白联合处理对DLD1-TRAIL/R细胞的致凋亡效应(P<0.05)。结论:蛋白酶体抑制剂MG-132能逆转人结肠癌细胞DLD1-TRAIL/R的获得性TRAIL耐药,其机制可能与Bik蛋白上调后启动线粒体凋亡途径有关,与JNK通路激活无关。更多还原

【Abstract】 Objective:To evaluate the role of proteasome inhibitor MG-132 in reversing the acquired TRAIL resistance of human colon cancer cell line DLD1-TRAIL/R and the related mechanisms.Methods:Colon cancer cell line DLD1-TRAIL/R was treated with MG-132 combined with TRAIL protein.The viability of DLD1-TRAIL/R cells was determined by MTT assay;the apoptotic rate was detected by flow cytometry,and the expression of apoptosis-related proteins was examined by Western blotting analysis.Results:The viability of DLD1-TRAIL/R cells was dramatically decreased after combined treatment with MG-132 and TRAIL protein(P<0.01) and the apoptotic rate was significantly increased(P<0.01).Western blotting analysis showed that MG-132 dramatically enhanced the cleavage of apoptotic molecules,including caspases-8,9,3,Bid,and PARP in DLD1-TRAIL/R cells after combined treatment and increased the release of cytochrome C and Smac from mitochondria.Further study demonstrated that MG-132 up-regulated DR5 and Bik proteins,but had no detectable effects on DR4,Bax,Bak,Bcl-XL,XIAP or survivin.Moreover,we found MG-132 induced phosphorylation of kinase JNK,and the inhibitor of JNK(SP600125) blocked MG-132-induced expression of DR5,but not the expression of Bik.Furthermore,SP600125 did not attenuate the apoptosis of DLD1-TRAIL/R cells induced by MG132 in the presence of TRAIL protein(P<0.05).Conclusion:Proteasome inhibitor MG-132 can reverse the acquired drug resistance to TRAIL and induce up-regulation of DR5 and Bik protein in DLD1-TRAIL/R cells.The underlying mechanism may involve the initiation of mitochondrion-related apoptosis caused by Bik protein expression,not by activation of JNK pathway.更多还原