p53凋亡刺激蛋白2对饥饿诱导的大肠癌HCT116p53^＋/＋细胞凋亡、周期和自噬的影响

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

目的探讨p53凋亡刺激蛋白2（ASPP2）对饥饿诱导的大肠癌HCT116p53^＋/＋ （p63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％（g=9．548，P=0．ooo）；各组细胞的自噬发生率为：rAd-GFP＋饥饿组35．00％4-5．34％，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HCT116p53^＋/＋ 细胞凋亡和自噬，显著改变细胞周期进程。

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： （1） control group; （2） green fluorescent protein adenovirus （rAd-GFP） infection group; （3）ASPP2 adenovirus （rAd-ASPP2） infection group; （4）starvation group; （5）rAd-GFP ＋ starva- tion group; （6） rAd-ASPP2 ＋ 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 and autophagy. The cell apoptosis rate in rAd-GFP ＋ starvation group was 10.00% ±42%, and 18.44% ±2.06% in rAd-ASPP2 ＋ starvation group（q =9. 548, P =0. 000）. The cell autophagy rate in rAd- GFP＋ starvation group and rAd-ASPP2 ＋ starvation group was 35.00% ± 5.34% and 57.61%± 6. 06% respectively（ q = 7. 657, P = 0.000）. Over-expressed ASPP2 accelerated HCT116 G2/M arrest under starvation, but resulted in both G0/G1 and G2/M arrest without starvation. Conclusion These results suggest that ASPP2can promote starvation-induced HCT116 p53 ＋/＋ cells apoptosis and autophagy, and affect the cell cycle.