甲胎蛋白对人肝癌Bel 7402细胞caspase信号传递及耐受TRAIL的影响

Inhibition of alpha-fetoprotein on caspase mediated signal transduction and its effect on TRAIL resistance of human hepatoma Bel 7402 cells

目的研究甲胎蛋白(AFP)对人肝癌Bel 7402细胞内caspase信息通路信号传递的影响,以及对肿瘤坏死因子相关凋亡诱导配体(TRAIL)诱导肝癌细胞凋亡的对抗作用。方法激光共聚焦显微镜观察AFP与caspase-3、caspase-8在Bel 7402细胞内的共定位;免疫共沉淀(Co-IP)技术研究AFP与caspase-3、caspase-8的相互作用;RNA干扰(RNAi)技术封阻AFP表达,并用Western blotting检测RNAi干扰肝癌Bel 7402细胞内AFP表达的效果;用蛋白酶活性比色法检测AFP对肝癌细胞caspase-3、caspase-8活性的影响;MTT法分析干扰AFP表达30 h后再给予TRAIL(2 nmol/L)处理24 h,Bel 7402细胞的生长情况,并用荧光显微镜观察经TRAIL处理后肝癌细胞的凋亡状况。结果共聚焦显微镜观察发现AFP、caspase-3、caspase-8主要表达于Bel 7402细胞的细胞质,发现AFP能与caspase-3共定位于细胞质,但与caspase-8在细胞质的共定位可能性很小;Co-IP技术研究发现AFP能与caspase-3结合,但不能与caspase-8结合;2 nmol/L剂量的TRAIL单独处理Bel 7402细胞并不能显著改变caspase-3活性,但能提升caspase-8活性,而用全反式维甲酸(ATRA)40μmol/L加TRAIL(2 nmol/L)处理时,则能提高Bel 7402细胞caspase-3活性;干扰AFP表达后,单独用TRAIL(2 nmol/L)也能激活caspase-3,而干扰AFP表达前后对caspase-8活性没有显著性改变;MTT分析发现,2 nmol/L剂量的TRAIL对Bel 7402细胞的生长并没有显著性抑制作用,但是干扰AFP表达后TRAIL(2 nmol/L)能明显抑制Bel 7402细胞生长(抑制率为48.4%);荧光显微镜观察表明,干扰AFP表达后TRAIL(2 nmol/L)能诱导Bel 7402细胞凋亡。结论AFP选择性抑制caspase-3活性是阻断人肝癌Bel7402细胞内凋亡信息传递的重要机制,也是AFP抵抗TRAIL诱导肝癌细胞凋亡的关键环节。

Objective To explore the effects of alpha-fetoprotein （AFP） on the transduction of apoptotic signal in hepetocellular cancinoma cells （HCC） and the role of AFP in counteracting the apoptosis induced by tumor necrosis factor-related apoptosis-inducing ligand （TRAIL） of HCC. Methods Laser confocal microscopy was applied to observe the co-localization of AFP and caspase-3 or caspase-8. Co-immunoprecipitation （Co-IP） was utilized to analyze the interaction of AFP with intracellular apoptotic signal molecules, caspase-3 or caspase-8. Short small RNA interfering （siRNA） technique was used to knockdown the expression of AFP in Bel 7402 cells. AFPs effect on the activity of caspase-3 or caspase-8 was detected by protease activity colorimetric methods. Bel 7402 cells were administered with TRAIL （2 nmol/L） for 24 h after knockdowning the expression of AFP by siRNA and the growth of the cells was analyzed by MTT or apoptosis of the cells was observed by fluorescence microscopy. Results AFP could interact with caspase-3 in cytoplasm, but could not bind with caspase-8. Treated with all trans retinoic acid （ATRA） （40 μmol/L） plus TRAIL（2 nmol/L） also enhanced the activity of caspase-3 or caspase-8, whereas treated with TRAIL（2 nmol/L） alone for 24 h, the activity of caspase-8 was enhanced but the activity of caspase-3 has not any change. AFP was able to inhibit caspase-3 activity but it had little affect on the activity of caspase-8 in vitro. When RNAi knockdowned the expression of AFP followed by treatment with TRAIL （2 nmol/L）, the activity of caspase-3 was increased, but the activity of caspase-8 remained the same as knockdown before. MTT detection showed that the growth of Bel 7402 cells was inhibited, the ratio was 48.4%, and apoptosis in nuclei of Bel 7402 cells was found to be induced when observed by fluorescence microscopy. Conclusion AFP can block the transduction of apoptotic signal by selectively inhibiting the activity of caspase-3, and this is also the pivotal event that AFP counteracts the apoptosis induced by TRAIL of hepatoma cells.