摘要
目的研究p53凋亡刺激蛋白2(ASPP2)p53依赖以及非依赖的自噬抑制作用,在提高奥沙利铂(OXA)促细胞凋亡中的作用。方法 HCT116(p53-/-)细胞分为雷帕霉素(rapamycin)联合ASPP2组、ASPP2组、p53组、ASPP2联合p53组、3-甲基嘌呤(3-MA)组、对照组(OXA或单独饥饿处理)6组。凋亡检测时添加单独空病毒(GFP-Ad)组、rapamycin组作为对照。各组细胞转染绿色荧光蛋白微管相关蛋白1轻链3(GFP-LC3)质粒,在荧光显微镜下观察并计数LC3阳性细胞数;Western blot法检测各组自噬相关分子表达水平;annexinⅤ-FITC/PI双染色结合流式细胞术检测细胞凋亡水平。结果 3-MA处理组与ASPP2处理组具有相同的自噬抑制作用,且能够促进OXA或饥诱导的细胞凋亡,但促凋亡作用低于ASPP2组;rapamycin联合ASPP2处理能够有效抵消ASPP2的自噬抑制作用,但仍具有促进OXA或饥饿诱导的细胞凋亡,且促凋亡作用同样低于ASPP2组。结论OXA增强结肠癌细胞自噬,而ASPP2过表达能够抑制OXA的自噬诱导作用;ASPP2能够通过P53依赖以及P53非依赖途径促进细胞凋亡;ASPP2通过p53依赖以及p53非依赖途径所引起的促细胞凋亡并非完全通过抑制自噬来实现。
Objective To investigate the role of apoptosis stimulating p53 binding protein 2 (ASPP2)-induced p53-dependent and p53-independent autophagy inhibition in apoptosis-promoting function of oxaliplatin (OXA). Methods According to different treatments, HCTll6( P53^-/-) cells were divided into 6 groups: rapamycin combined with ASPP2 group, ASPP2 group, p53 group, ASPP2 combined with p53 group, OXA combined with 3-methyladenine (3-MA) group, control group (OXA treatment or starvation without OXA treatment). When the level of apoptosis was detected, green fluorescent protein-advirus (GFP-Ad) group and rapamycin group were supplemented as controls. Cells were transfected with GFP-microtubule- associated protein t light chain 3 (GFP-LC3) plasmid, and LC3-expressing cells were calculated under a fluorescent microscope. Expressions of autophagy-related molecules were detected by Western blotting. Cells were subjected to annexin V-FITC/PI staining and apoptosis was assessed by flow cytometry. Results The 3-MA group showed the same inhibitory ability on autophagy with the ASPP2 group, and both of them were able to promote OXA or starvation-induced apoptosis, but the cell apoptosis rate in the 3-MA group was lower than that of the ASPP2 group. Rapamycin combined with ASPP2 still promoted OXA or starvation-induced apoptosis, and the apoptosis rate was also lower than that of the ASPP2 group. However, rapamycin counteracted effectively the inhibitory effect of ASPP2 on autophagy. Condusion OXA can induce autophagy of colorectal cancer cells, while ASPP2 over-expression can suppress the OXA-induced autophagy. ASPP2 can promote apoptosis through p53-dependent and p53-independent pathways. The function of ASPP2 promoting cell apoptosis through p53-dependent and p53-independent pathways is not entirely achieved by inhibiting cell autophagy.
出处
《细胞与分子免疫学杂志》
CAS
CSCD
北大核心
2015年第7期898-904,共7页
Chinese Journal of Cellular and Molecular Immunology
基金
山东省医学科学院院级科技计划(2014-24)
国家科技支撑计划课题(2012BAI15B00)
国家自然科学基金(81272266)