p53凋亡刺激蛋白2对饥饿诱导的大肠癌HCT116 p53+/+细胞凋亡、周期和自噬的影响

目的 探讨p53凋亡刺激蛋白2(ASPP2)对饥饿诱导的大肠癌HCT116 p53+/+(p53野生型)细胞凋亡、周期和自噬的影响.方法 实验分6组:①对照组；②绿色荧光蛋白腺病毒(rAd-GFP)感染组；③ASPP2腺病毒(rAd-ASPP2)感染组；④饥饿处理组；⑤rAd-GFP+饥饿组；⑥rAd-ASPP2+饥饿组.利用rAd-ASPP2感染使细胞过表达ASPP2基因.无血清培养基培养24h诱导凋亡、自噬和细胞周期改变.钙黄绿素(Calcein)/碘化丙啶(PI)吸收试验观察各组细胞调亡水平.细胞转染红色荧光蛋白标记的CFP-Lc3自噬质粒,荧光显微镜下观察各组细胞自噬水平.流式细胞术观察细胞周期改变.组间比较采用单因素方差分析进行统计学分析.结果 ASPP2过表达显著促进了饥饿诱导的细胞凋亡、自噬及G2-M期阻滞,各组细胞的凋亡率为:rAd-GFP+饥饿组10.00％±1.42％,rAd-ASPP2+饥饿组18.44％ ±2.06％(q=9.548,P=0.000)；各组细胞的自噬发生率为:rAd-GFP+饥饿组35.00％±5.34％,rAd-ASPP2+饥饿组57.61％ ±6.06％(q=7.657,P=0.000).但无饥饿诱导时ASPP2过表达使G0-G1、G2-M期都发生阻滞.结论 ASPP2过表达促进饥饿诱导的大肠癌HCT116 p53+/+细胞凋亡和自噬,显著改变细胞周期进程.

Roles of ASPP2 in the apoptosis, cell cycle and autophagy of starvation-induced HCT116 p53 +/+ cell line

Objective To investigate the role of apoptosis stimulating protein 2 of p53（ASPP2）in the apoptosis, cell cycle and autophagy of starvation-induced colorectal cancer HCT116 p53^+/+ (p53 wild-type) cell line. Methods Six groups were included: ⑴ control group; ⑵ green fluorescent protein adenovirus (rAd-GFP) infection group; ⑶ ASPP2 adenovirus (rAd-ASPP2) infection group; ⑷ starvation group; ⑸GFP+ starvation group; ⑹ASPP2+ starvation group. HCT116 cells were infected with ASPP2 adenovirus (rAd-ASPP2), resulting ASPP2 gene over-expression. The apoptosis, autophagy and cell cycle changes were induced by culturing with serum-free medium for 24 h. Apoptosis was evaluated by Calcein／PI uptaking test, and autophagy was observed by counting the red fluorescent protein autophagy plasmid CFP-Lc3 which was transfected into cytoplasm. Cell cycle was detected by flow cytometry. Statistical analysis was performed by one-way analysis of variance (ANOVA). Results Over-expressed ASPP2 was found to significantly promote starvation-induced HCT116 apoptosis（cell apoptosis rate: GFP+ starvation group : 10.00%±1.42%；ASPP2+ starvation group: 18.44%±2.06% P=0.000） and autophagy（cell autophagy rate: GFP+ starvation group : 35.00%±5.34%；ASPP2+ starvation group : 57.61%±6.06% P=0.000）, and accelerate HCT116 G₂/M arrest under starvation. But over-expressed ASPP2 could result in both G₀/G₁ and G₂/M arrest without starvation. Conclusion These results suggest that ASPP2 can promote HCT116 p53^+/+ cell apoptosis and autophagy, and affect the cell cycle.