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，Western blotting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）。Western blotting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Western blotting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 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.

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）