蛋白酶体抑制剂MG-132逆转人结肠癌细胞获得性TRAIL耐药  

Proteasome inhibitor MG-132 reverses acquired resistance to TRAIL in human colon cancer cells

作　　者：胡静姿 朱洪波[2] 何超[2] 劳伟峰[2] 黄学锋[2] 

机构地区：[1]南京军区杭州空军疗养院妇科,浙江杭州310013 [2]浙江大学医学院附属邵逸夫医院肛肠外科,浙江杭州310016

出　　处：《中国肿瘤生物治疗杂志》2009年第2期140-144,共5页Chinese Journal of Cancer Biotherapy

基　　金：国家自然科学基金资助项目(No.30700970);浙江省自然科学基金资助项目(No.Y205093)~~

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。Objective： To evaluate the role of proteasome inhibitor MG-132 in reversing the acquired TRAIL resistance of human colon cancer cell line DLDI-TRAIL/R and the related mechanisms. Methods： Colon cancer cell line DLD1-TRAIIZR 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 treat- ment with MG-132 and TRAIL protein（P 〈0.01 ） and the apoptotic rate was significantly increased （P 〈0. O1 ）. 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 DRS, 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 pro- tein （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 mitoehondrion-related apoptosis caused by Bik protein expression, not by activation of JNK pathway.

关 键 词：蛋白酶体抑制剂 MG-132 结肠肿瘤细胞 TRAIL 耐药 Bik 

分 类 号：R735.35[医药卫生—肿瘤] R730.54[医药卫生—临床医学]