Caspase-4活化参与TRAIL诱导的胃癌细胞凋亡

Activation of caspase-4 was involved in TRAIL-induced apoptosis of gastric cancer cells

目的研究caspase-4在肿瘤坏死因子相关凋亡诱导配体(tumor necrosis factor-related apoptosis-inducing ligand,TRAIL)诱导胃癌细胞凋亡中的作用。方法采用碘化丙啶染色,流式细胞术检测TRAIL和caspase-4抑制剂(z-LEVDfmk)单独或联合作用对胃癌细胞凋亡的影响;化学合成3条针对caspase-4基因的小干扰RNA(small interfering,siRNA),用脂质体法转染胃癌细胞,采用Western blot验证沉默效果,流式细胞术观察转染后对TRAIL诱导胃癌细胞凋亡的影响;Western blot检测TRAIL作用后葡萄糖调节蛋白78(78-ku glucose-regulated protein,GRP78)的表达情况,以内质网应激经典诱导剂衣霉素(tunicamycin,TM)为参照;Western blot检测TRAIL作用后caspase-3和caspase-4蛋白的表达情况。结果 z-LEVD-fmk能明显地抑制TRAIL诱导的胃癌细胞凋亡(P<0.05);与阴性对照相比,转染caspase-4 siRNA后caspase-4蛋白表达水平明显下降,且转染后细胞凋亡率降低(P<0.05);和TM相比,TRAIL能诱导较低程度的内质网应激反应;在TRAIL处理的早期caspase-4和caspase-3即活化。结论 Caspase-4的活化参与了TRAIL诱导的胃癌细胞凋亡,这可能与TRAIL诱导的内质网应激有关。

Aim To investigate the potential involve-ment of caspase-4 in TRAIL （ tumor necrosis factor-re-lated apoptosis-inducing ligand ）-induced apoptosis in gastric cancer cells. Methods The effect of treatment with TRAIL /z-LEVD-fmk alone or in combination for 24 h on the apoptotic rate of gastric cancer cells wasdetected by FCM （ flow cytometry） using propidium io-dide DNA staining. Chemically synthesized three siR-NAs targeting caspase-4 gene were transfected into gas-tric cancer cells by Lipofectamine 2000 Reagent. The efficacy of gene silencing was confirmed by Western blot analysis . The apoptotic rates of gastric cancer cellsto TRAIL before and after transfection with caspase-4 siRNA were observed by FCM. The expression level of GRP78 （78-ku glucose-regulated protein） protein was examined by Western blot, the classic endoplasmic re-ticulum stress inducer tunicamycin （ TM） was used as a control. The expression levels of caspase-4 and caspase-3 after TRAIL treatment were also measured by Western blot. Results z-LEVD-fmk decreased TRAIL-induced apoptosis of gastric cancer cells signifi-cantly（P〈 0. 05）. As compared with negative control, the expression level of caspase-4 protein was reduced after transfection, and the apoptotic rate was also de-creased （ P 〈 0. 05 ） . While TM induced marked up-regulation of GRP78 , treatment with TRAIL resulted in, albeit to a lesser extent, increases in GRP78, in-dicative of induction of ER stress by TRAIL. Activa-tion of caspase-4 and caspase-3 occurred early after TRAIL treatment. Conclusion Activation of caspase-4 contributes to TRAIL-induced apoptosis and is asso-ciated with induction of ER stress by TRAIL in gastric cancer cells.