ASPP2以p53非依赖形式促进自噬引起Hep3B细胞凋亡

ASPP2 Promotes p53-independent Autophagy-induced Apoptosis in Hep3B 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的功能,进而发挥抗肿瘤作用.本室前期研究发现,ASPP2可以通过p53-DRAM-自噬途径诱导细胞凋亡.在本研究中,利用ASPP2腺病毒感染Hep3B细胞(p53缺陷型肝癌细胞系)并用甲基磺酸(MMS)处理后;Calcein AM/PI和M30染色检测细胞凋亡;GFP-LC3质粒转染细胞后检测自噬;荧光定量PCR和免疫印迹检测自噬基因表达.结果表明,ASPP2在p53缺陷的Hep3B细胞内可诱导发生凋亡;在MMS存在和缺失条件下,Adr-ASPP2均引起自噬体水平升高及自噬基因的表达增加,且MMS协同Adr-ASPP2能使自噬水平增加;进一步用VPS34 siRNA和DRAM siRNA抑制自噬发现,细胞凋亡水平下降,说明由Adr-ASPP2诱发经损伤相关自噬调节蛋白(DRAM)介导的自噬参与了肝癌细胞系凋亡的发生.综上结果表明,ASPP2可以通过非p53依赖的DRAM介导自噬,并促进肝癌细胞凋亡.该研究可为肝癌的基因治疗提供新的思路.

ASPP2 （apoptosis stimulating protein 2 of p53） binds to p53 specifically and enhance the pro-apoptotic function of p53, which plays a role in inhibiting tumor development. Adr-ASPP2 induces apoptosis through activating p53-DRAM-autophagy pathway. In this study, methyl methanesulfonate （MMS） was emplyed to stimulate Hep3B cells for 24 hours, then four different methods were used to detect autophage. Results： Adr-ASPP2 treatment induced autophagosome formation and expression of autophagy genes with and without MMS treatment. Moreover, ASPP2 combined with MMS induced higher autophagy level and higher level of gene expression. Both VPS34 and DRAM （damage-regulatedautophagy modulator） siRNAs inhibited autophagy and blocked more ASPP2-or ASSP2 combined with MMS-induced apoptosis, indicating that ASPP2 induced DRAM-mediated autophagy involves in apoptotic death in Hep3B cells. The results showed that ASPP2 induces p53-independent DRAM expression- mediated autophagy, which promotes hepatocarcinoma death via apoptosis. Our results supply new idea in the field of gene therapy for liver cancer.