衣霉素通过上调TRAIL-R2增加胃癌细胞对TRAIL的敏感性

Tunicamycin enhances the sensitivity to TRAIL in gastric cancer cells by up-regulation of TRAIL-R2

目的:研究衣霉素(tunicamycin,TM)上调胃癌细胞对肿瘤坏死因子相关凋亡诱导配体(TNF-related apoptosis-inducing ligand,TRAIL)诱导凋亡敏感性的作用机制。方法:采用碘化丙啶染色的FCM法检测TM(1μmol/L)与TRAIL(100μg/L)单独或联合作用3、6、16、24和36h时对SGC-7901细胞凋亡率的影响;FCM法检测TM处理前后细胞表面TRAIL受体1(TRAIL receptor1,TRAIL-R1)、-R2、-R3和-R4的表达情况;实时荧光定量-PCR法检测TRAIL-R2mRNA的表达情况;蛋白质印迹法检测葡萄糖调节蛋白78(glucose-regulated protein 78kDa,GRP78)和CCAAT/增强子结合蛋白同源蛋白(CCAAT/enhancer-binding protein homologous protein,CHOP)的表达水平;RT-PCR检测X盒结合蛋白(X-box binding protein1,XBP1)mRNA的剪接情况。结果:TM单独作用引起的SGC-7901细胞的凋亡率低,TM和TRAIL联合作用能有效提高SGC-7901细胞的凋亡率;TM能明显上调SGC-7901细胞表面TRAIL-R2的表达水平,而对TRAIL-R1、-R3和-R4却无明显影响;TRAIL-R2mRNA的表达水平随TM作用时间延长而相应升高;GRP78蛋白的上调和XBP-1mRNA的剪接活化证实了TM可诱导未折叠蛋白反应(unfolded protein response,UPR)的发生;CHOP蛋白的表达水平也在TM作用后上调。结论:TM通过诱导UPR上调TRAIL-R2的表达,从而增加胃癌细胞对TRAIL的敏感性,CHOP介导了TRAIL-R2的上调作用。

Objective： To investigate the promoting effect of TM （tunicamycin） on apoptosis of gastric cancer cells induced by TRAIL [TNF （tumor necrosis factor）-related apoptosis-inducing ligand], and to explore its possible mechanism. Methods： The effect of treatment with TM （1 μmol/L）/TRAIL （100 μg/L） alone or in combination for 3, 6, 16, 24 and 36 h on the apoptotic rate of SGC-7901 cells was detected by FCM （flow cytometry） using propidium iodide DNA staining. The cell surface expression levels of different types of TRAIL receptors including TRAIL-R1, TRAIL-R2, TRAIL-R3 and TRAIL-R4 before and after TM treatment were detected by FCM, and the expression level of TRAIL-R2 mRNA was detected by RFQ-PCR （real-time fluoregenic quantitative-PCR）. regulated protein） and CHOP （CCAAT/enhancer-b detected by Western blotting. The splicing of XBP1 RT-PCR. Results： Treatment with TM alone induced The expression levels of GRP78 （78-kDa glucose- nding protein homologous protein） proteins were （X-box binding protein 1） mRNA was detected by minimal level of apoptosis of SGC-7901 cells. The apoptosis rate of SGC-7901 cells was increased significantly after treatment with TM in combination with TRAIL. TM could markedly up-regulate cell surface expression level of TRAIL-R2 on cell surface. In contrast, TM did not induce any changes in the expressions of TRAIL-R1, TRAIL-R3 and TRAIL-R4.The expression level of TRAIL-R2 mRNA of SGC-7901 cells induced byTM treatment was up-regulated in a time-independent manner. The results of up-regulation of GRP78 and the splicing ofXBP1 mRNA demonstrated the activation of UPR（unfolded protein response） induced byTM. Treatment with TM also resulted in a remarkable increase in the expression of CHOP protein. Conclusion： TM enhances TRAIL- induced apoptosis in gastric cancer cells by up-regulation of TRAIL-R2 expression via UPR. CHOP may be responsible for this effect involved in up-regulation of TRAIL-R2.