ASPP2过表达可增强DRAM介导的自噬对HepG2细胞的促调亡作用

ASPP2 Enhances the Effect of DRAM Induced Autophagy on Promoting Apoptosis in HepG2 Cells

p53凋亡刺激蛋白2(apoptosis stimulating protein 2 of p53,ASPP2)能特异性地与p53蛋白结合并增强其促凋亡功能,进而发挥抗肿瘤作用.最近文献提示,自噬对肿瘤发生、发展及肿瘤细胞对抗肿瘤药物的反应都具有重要作用.在本研究中,甲基磺酸(MMS)处理HepG2细胞24 h后,用calcein AM/PI和M30染色检测细胞凋亡,可引起早期(M30免疫组化阳性)和晚期细胞凋亡(PI染色阳性).给HepG2细胞转染GFP-LC3质粒后,发现MMS处理24 h可引起自噬的发生.ASPP2腺病毒(rAd-ASPP2)感染HepG2细胞引起ASPP2过表达后,再用MMS处理24 h,能引起更明显的早期、晚期细胞凋亡和自噬.荧光定量PCR检测发现,rAd-ASPP2诱导了更高的BCL-2相关X蛋白基因(BAX)和p53蛋白的目的基因p53诱导的自噬调节蛋白(p53-induced modulator of autophagy,DRAM)的表达.但仅用rAd-ASPP2处理HepG2细胞不能引起自噬和凋亡.利用2条DRAM特异性的siRNA下调DRAM的表达,发现rAd-ASPP2引起的自噬被完全抑制,早期和晚期凋亡均部分被抑制,同时BAX的mRNA水平也明显下降.以上结果说明,ASPP2可通过上调BAX和DRAM基因的转录而促进MMS引起的HepG2细胞凋亡;另外,DRAM介导的自噬是ASPP2促进MMS引起的肿瘤细胞凋亡的机制之一.该研究可为肝癌的基因治疗提供新的思路.

Apoptosis stimulating protein 2 of p53（ASPP2） specifically binds to p53 and enhance the pro-apoptotic function of p53,which plays a role in inhibiting tumor development.Present studies have suggested that autophagy may be important in the regulation of cancer development and progression and in determining the response of tumor cells to anticancer therapy.In this study,methyl methanesulfonate（MMS） was employed to stimulate HepG2 cells for 24 hours,calcein AM/PI staining and M30 immunoreactivity were used to assess apoptosis,we identified that MMS induces early（M30 positive） and late apoptosis（PI staining positive）.HepG2 cells were primarily transfected with GFP-LC3 plasmid and then treated by MMS for 24 hours,we identified that MMS treatment induced LC3 puncta development,indicating that autophagy was induced.Moreover,rAd-ASPP2 pretreatment induced ASPP2 overproduction leading to more early and late apoptosis and autophagy in HepG2 cells in response to MMS treatment.However,only rAd-ASPP2 preteatment could not induce apoptosis and autophagy.Using real time PCR,we identified that ASPP2 overproduction induced more Bcl-2-associated X protein（BAX） and DRAM expression.Using two specific siRNAs to inhibit DRAM expression,we identified that autophagy was completely blocked,early and apoptosis were partly blocked,and the mRNA level of BAX was significantly decreased.Thus,these results suggest that ASPP2 overproduction can promote MMS-induced apoptosis by up-regulating the level of BAX and DRAM transcription.In addition,DRAM mediated autophagy is one of mechanisms by which ASPP2 overproduction promotes MMS induced apoptosis.Our results could supply new ideas in the field of gene therapy for liver cancer.