XAF1增强TRAIL诱导人结肠癌细胞株HCT116凋亡的实验研究

背景:肿瘤坏死因子(TNF)相关凋亡诱导配体(TRAIL)可特异性诱导肿瘤细胞凋亡。X染色体连锁凋亡抑制蛋白(XIAP)相关因子1(XAF1)是新型抑癌基因,在肿瘤细胞中呈低表达。目的:研究XAF1联合TRAIL诱导人结肠癌细胞株HCT116凋亡的作用。方法:构建重组腺病毒AdS/F35-XAF1、对照病毒Ad5/F35-Null,分别以相同感染复数(MOI)感染人结肠癌细胞株HCT116,并设立空白对照组,感染4 h后加入重组人TRAIL(rhTRAIL)。以RT-PCR法检测XAF1 mRNA表达;以蛋白质印迹法检测XAF1、PARP、caspase-3、-8、-9蛋白表达;以Annexin V-FITC/PI双染法检测细胞凋亡率;以MTT法检测细胞增殖抑制率。结果:Ad5/F35-XAF1组和TRAIL+Ad5/F35-XAF1组XAF1 mRNA和蛋白表达显著高于其余各组。TRAIL+Ad5/F35-XAF1组PARP、caspase-3、-8、-9蛋白可见明显凋亡裂解带,其余各组未见明显裂解带。AdS/F35-XAF1组、TRAIL组、TRAIL+Ad5/F35-Null组、TRAIL+Ad5/F35-XAF1组细胞凋亡率较空白对照组和Ad5/F35-Null组显著增加(P<0.05),以TRAIL+Ad5/F35-XAF1组增加最为显著。TRAIL+Ad5/F35-XAF1组HCT116细胞增殖抑制率显著高于TRAIL组和TRAIL+Ad5/F35-Null组(P<0.05),并呈浓度和时间依赖性。结论:XAF1可增强TRAIL诱导的HCT116细胞凋亡和抑制增殖的作用。

XAF1 Enhances TRAIL Induced Apoptosis in Human Colon Carcinoma Cell Line HCTll6

Tumor necrosis factor （TNF）-related apoptosis-inducing ligand （TRAIL） is a specific inducer of apoptosis in cancer cells. X-chromosome linked inhibitor of apoptosis protein （XIAP） -associated factor 1 （ XAF1 ） is a novel tumor suppressor gene and is low expressed in cancer cells. Aims ： To investigate the effect of XAF1 combined with TRAIL on inducing apoptosis in human colon carcinoma cell line HCTll6. Methods： Recombinant virus Ad5/F35-XAF1 and control virus Ad5/F35-Null were constructed. HCT116 cells were transfected with the constructed recombinant virus AdS/F35-XAF1 and AdS/F35-Null, respectively, at the same multiplicity of infection （MOI） for 4 hours, blank group was served as control, then co-cultured with recombinant human TRAIL （rhTRAIL）. The expression of XAF1 mRNA was determined by RT-PCR. The expressions of XAF1, PARP and easpase-3, -8, -9 protein were determined by Western blotting. The cell apoptosis rate was determined by Annexin V-FITC/PI double staining, and cell proliferation inhibition rate was measured by MTI assay. Results： Expressions of XAF1 mRNA and protein in AdS/F35-XAF1 group and TRAIL ＋ AdS/F35-XAF1 group were significantly higher than those in the other groups. Cleavage of apoptosis-associated proteins PARP and caspase-3, -8, -9 were seen in TRAIL ＋ AdS/F35-XAF1 group but not in other groups. The apoptosis rate of HCT116 cells in AdS/F35-XAF1 group, TRAIL group, TRAIL ＋ AdS/F35-Null group and TRAIL ＋ AdS/F35-XAF1 group were significantly higher than those in blank control group and AdS/F35-Null group （P 〈 0.05 ）, and was highest in TRAIL ＋ AdS/F35-XAF1 group. Compared with TRAIL group and TRAIL ＋ AdS/F35-Null group, the cell proliferation inhibition rate of HCTll6 cells was significantly increased in TRAIL ＋ AdS/F35-XAF1 group in a concentration and time dependent manner （ P 〈 0.05 ）. Conclusions： XAF1 can promote the TRAIL inducing apoptosis and inhibiting proliferation in HCT116 cells.