蛋白酶体抑制剂硼替佐米逆转结肠癌获得性TRAIL耐药的研究  

Overcoming Acquired TRAIL Resistance in Human Colon Cancer by Proteasome Inhibitor Bortezomib

作　　者：胡静姿 朱洪波[2] 何超[2] 陈琳琳[2] 黄学锋[2] 

机构地区：[1]南京军区杭州窄勤疗养院,杭州310013 [2]浙江大学医学院附属邵逸夫医院肛肠外科,浙江省生物治疗重点实验室,杭州310016

出　　处：《中国药学杂志》2010年第1期19-22,共4页Chinese Pharmaceutical Journal

基　　金：国家自然科学基金资助课题(30700970);浙江省自然科学基金资助课题(Y205093)

摘　　要：目的探讨蛋白酶体抑制剂硼替佐米在逆转人结肠癌细胞获得性肿瘤坏死因子相关凋亡诱导配体(TRAIL)耐药中的作用及机制。方法在硼替佐米和TRAIL蛋白联合处理DLD1-TRAIL/R细胞后,通过噻唑蓝(MTT)法检测细胞的存活率,并通过免疫印迹法检测各种凋亡相关蛋白的表达情况。通过siRNA转染明确目的基因在硼替佐米逆转结肠癌细胞获得性TRAIL耐药中的作用。结果硼替佐米联合TRAIL蛋白处理DLD1-TRAIL/R细胞后,其细胞存活率明显下降,而细胞凋亡率则明显增加(P<0.01)。Western免疫印迹结果显示,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,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免疫印迹法结果显示,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其他Bcl-2家族成员以及XIAP和Survivin等则无明显改变。通过转染siRNA阻断Bik蛋白表达后,硼替佐米介导的TRAIL增敏作用被部分抑制(P<0.05)。结论蛋白酶体抑制剂硼替佐米可以逆转人结肠癌细胞DLD1对TRAIL的获得性耐药,其机制可能与Bik蛋白蓄积有关。OBJECTIVE To evaluate the effect of proteasome inhibitor of bortezomib on acquired TRAIL resistance in human colon cancer cell line DLD1-TRAIL/R.METHODS Cell viability of DLD1-TRAIL/R was determined by MTT assay after combined treatment of bortezomib and TRAIL protein.Western blot analysis was used for detecting the expression of various apoptotic proteins.Then,the effect of selected gene targeted by bortezomib on acquired TRAIL resistance was determined by siRNA transfection.RESULTS Cell viability of DLD1-TRAIL/R cells was decreased dramatically after combination treatment of bortezomib and TRAIL protein,whereas apoptotic ratio of combined group was increased significantly（P0.01）.Western blot analysis showed that combination treatment of bortezomib and TRAIL protein dramatically enhanced the cleavage of molecular markers of TRAIL-induced apoptotic signaling,including caspases-8,9,3,Bid and PARP.Moreover,western blot analysis also showed that the combination treatment increased the release of cytochrome C and Smac from mitochondria.Further study demonstrated that bortezomib upregulated Bik protein.However,no effects on Bax,Bak,Bcl-2,Bcl-XL,XIAP or survivin were observed.Finally,treatment with Bik siRNA significantly attenuated bortezomib-mediated TRAIL sensitization（P0.05）.CONCLUSION Proteasome inhibitor bortezomib can overcome acquired TRAIL resistance in DLD1-TRAIL/R cells,which might be mediated by proteasome inhibitor-induced accumulation of Bik.

关 键 词：蛋白酶体抑制剂 硼替佐米 肿瘤坏死因子相关凋亡诱导配体 耐药 Bik 

分 类 号：R965[医药卫生—药理学]