凋亡抑制蛋白XIAP和促凋亡因子Smac在胰腺癌化疗抵抗中的作用

Effects of X-linked inhibitor of apoptosis protein and pro-apoptotic protein Smac on apoptosis regulation in response to chemotherapies in pancreatic carcinoma cells

目的探讨X-相关凋亡抑制蛋白(XIAP)和促凋亡因子Smac在胰腺癌细胞化疗抵抗中的作用,以及转染胞浆表达型Smac基因靶向下调XIAP对化疗药物诱导的胰腺癌细胞凋亡的影响。方法应用流式细胞术检测顺铂、5-FU介导的Panc-1、BXPC-3的凋亡率及胞浆染色分析细胞XIAP表达变化,Western blot分析XIAP、Smac、Caspase-3表达水平;构建pEGFP-N1/Smac真核表达载体并转染胰腺癌Panc-1细胞,流式细胞术检测转染Smac基因前后Panc-1细胞的凋亡敏感性。结果与BXPC-3细胞相比,Panc-1对顺铂或5-FU介导的凋亡具有较强抵抗性,Western blot分析显示Panc-1细胞高表达XIAP,在化疗药物作用下化疗敏感细胞BXPC-3胞浆内XIAP水平下降明显多于Panc-1细胞,而且凋亡的BXPC-3细胞释放入胞浆内的成熟Smac蛋白水平明显高于Panc-1细胞。转染胞浆表达型Smac基因至化疗抵抗Panc-1细胞,可明显下调其XIAP表达水平,促进效应Caspase-3分子活化,显著提高顺铂、5-FU诱导的细胞凋亡率。结论胰腺癌细胞XIAP的表达水平下调与其化疗敏感性有关,XIAP是克服化疗抵抗的重要靶分子,而上调Smac活性蛋白的胞浆表达作为一种有效调节信号,通过拮抗XIAP的凋亡抑制作用协同化疗药物促进胰腺癌细胞凋亡。

Objective To investigate the role of X-linked inhibitor of apoptosis protein（XIAP） and second mitochondria-derived activator of caspases（Smac） in apoptosis regulation as a response to chemotherapy of human pancreatic carcinoma cells （Panc-1 and BXPC-3）, and to examine whether ectopic overexpression of Smac down-regulates XIAP and sensitizes these cells to anticancer drug induced apoptosis. Methods The proportions of apoptosis of Panc-1 and BXPC-3 cells and XIAP expression in permeabilized cells induced by cisplatin and 5-fluorouracil（5-FU） were analyzed by flow cytometry. The expressions of XIAP, Smac and Caspase-3 were determined by Western blot. A recombinant plasmid vector pEGFP-N1/Smac was constructed and transfected into Panc-1 cells. The apoptosis sensitivity of Panc-1 cells before and after Smac gene transfection was evaluated by flow cytometry. Results Panc-1 was resistant to cisplatin or 5-FU induced apoptosis as compared to BXPC-3. Western blot analysis showed a high expression of XIAP in Panc-1. Down-regulation of XIAP expression in permeabilized cells was greater in chemo-sensitive BXPC-3 than Panc-1 during cisplatin or 5-FU-induced apoptosis. Moreover, mature Smac released from mitochondria to cytosol was much more in apoptotic BXPC-3 than Panc-1 cells. Ectopic overexpression of Smac significantly down-regulated levels of XIAP and promoted the activity of Caspase-3, increased cisplatin or 5-FU-induced apoptosis of chemo-resistant Panc-1 cells. Condusion The sensitivity of apoptosis induced by chemotherapeutic agents correlated with selective down-regulation of XIAP in pancreatic carcinoma. XIAP could be a potential target for overcoming chemo-resistance. The up-regulation of Smac/DIABLO may serve as a potential effective medifying signal to sensitize resistant pancreatic cancer cells to anticancer drug-induced apoptosis.