细胞外基质蛋白介导膀胱癌化疗耐药的研究  

Cell adhesion to extracellular matrix induces mitomycin C resistance in bladder cancer cells

作　　者：潘春武[1] 王伟明[1] 刘建河[1] 康健[1] 刘海龙[1] 齐隽[1] 沈周俊[2] 

机构地区：[1]上海交通大学医学院附属新华医院泌尿外科,200092 [2]上海交通大学医学院附属瑞金医院泌尿外科

出　　处：《现代泌尿生殖肿瘤杂志》2015年第3期165-170,共6页Journal of Contemporary Urologic and Reproductive Oncology

基　　金：国家自然科学基金项目(81101931);上海高校青年教师培养计划(ZZjdyx12083);上海市卫生局青年科研项目(2010Y073)

摘　　要：目的研究细胞外基质中的一种主要蛋白:纤维连接蛋白(FN)介导的人膀胱癌细胞(T24)对化疗药物丝裂霉素(MMC)的耐药现象,并阐明其发生的信号转导机制。方法 T24细胞与FN黏附后予以MMC处理及磷脂酰肌醇3-激酶(PI3-K)特异性抑制剂LY294002处理,分别检测其细胞凋亡及细胞周期情况。分别采用比色法、免疫荧光、蛋白印迹法及流式细胞术等技术检测Caspase-8、Caspase-9、凋亡诱导因子(AIF)3种凋亡通路的激活情况。蛋白印迹法检测细胞周期G1/S期转换的关键分子糖原合成激酶3β(GSK-3β)、Cyclin D1的表达水平。同时利用蛋白印迹法检测PI3-K/Akt信号通路的激活情况。结果 FN与T24细胞黏附后显著抑制了MMC诱发的细胞凋亡,且这种作用是通过抑制Caspase-9及AIF两种凋亡通路而非Caspase-8凋亡通路实现的。同时,FN通过失活GSK-3β而稳定了Cyclin D1的表达水平,从而拮抗了MMC诱导的T24细胞G1/S期停滞。FN介导的对T24细胞的保护作用是PI3-K/Akt依赖的,可以被LY294002阻断。结论 FN介导PI3-K/Akt信号通路激活促进了T24细胞对MMC的耐药。Objective To investigate that cell adhesion to fibronectin （FN）, a major protein of extracellular matrix, induces drug resistance in human bladder cancer cells, and to study fibronectin mediated survival signaling pathway in bladder cancer chemotherapy resistance in vitro. Methods T24 cells （human bladder cancer cell lines） were precoated with fibronectin, and treated with Mito- mycin C （MMC） and the specific PI3-K inhibitor LY294002, and the apoptosis and cell cycles were analyzed respectively. The activity of Caspase-8, -9 and apoptosis-inducing factor （AIF） apoptosis pathways were assessed using colorimetric assay, immunofluorescence, western blot and flow cytometry. The expression of glycogen synthase kinase-3β （GSK-3β） and Cyclin D1, as the key regulator of G1/S phase transition, were determined by western blot. The expression of PI3-K, Akt, phospho-Akt and β1-integrin were also examined by western blot. Results Apoptosis induced by MMC was significantly resisted by FN adhesion in T24 cells, and this effect was through inhibition of Caspase-9 and AIF apoptosis pathways but not Caspase-8 pathway. FN antagonized MMC-induced G1/S- phase arrest by inactivating GSK-3β to stabilize Cyclin D1 expression in T24 ceils. Furthermore, FN-medi- ated protection of T24 cells was dependent on activity of PI3-K/Akt signaling pathway, and the protection could be abolished by the PI3-K inhibitor LY294002. Conclusions FN-mediated PI3-K/Akt activation protects T24 cells from MMC-induced cell death through inhibition of both Caspase-9 and AIF medi- ated apoptosis and GSK-3βl/Cyclin D1 involved G1/S-phase arrest.

关 键 词：纤维连接蛋白 膀胱癌 化疗 耐药性 

分 类 号：R737.14[医药卫生—肿瘤]