ASPP2Enhances L-OHP-induced Colorectal Cancer Cell Apoptosis In A P53-independent Manner By Inhibiting Protective Cell Autophagy

ObjectiveColorectal cancer is a serious threat to the health of people. p53apoptosis stimulatingprotein2(apoptosis stimulating protein of p53-2, ASPP2) is the mostly complete familymember of ASPP—a recently discovered apoptosis family of p53. As an importantsuppressor gene to regulate apoptosis of p53, ASPP2is able to specifically enhance theapoptotic pathway mediated by p53. While in75%of sporadic colorectal cancer, there existmutations or inactivation of p53. This paper aims to reveal the role of ASPP2inoxaliplatin-induced apoptosis on colorectal cancer in the state of p53inactivation, and thedepth of which roles autophagy plays.MethodsThe first part of the experiment: the cells over-expressing ASPP2gene by using infectionof ASPP2(rAd-ASPP2).Cells cutured with oxaliplatin-containing100uM (L-OHP) highglucose medium (DMEM) for12hours to observe apoptosis and autophagy levels. Calcein/PI absorption test and annexin V-PI test were used to observe apoptosis levels of each group.Cells were transfected with a red fluorescent protein CFP-Lc3to observe cell autophagyplasmids in each group autophagic under fluorescence microscopy; Western-blot assay wasused to observe autophagy-related proteins LC3-Ⅰ and LC3-Ⅱ expression levels. Cellscutured with oxaliplatin-containing100uM (L-OHP) high glucose medium (DMEM) for4 hours, then extracted RNA, RT-PCR was used to detect expression levels of apoptosis andautophagy-related gene.The second part of the experiment: extracted tissue RNA of samples of colorectal cancerand adjacent normal tissue, then reversed transcription to cDNA, using ASPP2primers foramplification, detection ASPP2expression levels of colorectal carcinoma specimens and theadjacent. Embedding fixed colorectal cancer and adjacent tissue specimens frozen sectionsafter, then fluorescence immunoassay was used to detect the expression of colorectal cancertissue specimens ASPP2and apoptotic gene caspase3and the correlation between them.The third part of the experiment: rAd-ASPP2adenovirus infected marker GFP-LC3mice,then oxaliplatin was injected, intestinal tissues were extracted from mice to observe theexpression level of ASPP2and the correlation with autophagy.ResultsThe first part: The over-expressions of ASPP2significantly enhanced the level ofnon-p53-dependent apoptosis, rAd-ASPP2group (21.13±1.03) and rAd-GFP group (7.17±0.61), P <0.01. Meanwhile ASPP2overexpression significantly inhibited non-p53-dependent,autophagy of cells, rAd-ASPP2group (19.57±2.76), rAd-GFP group (40.17±3.86), P<0.01.The secend part: The expression of ASPP2in colorectal cancer tissue gene (2.11) islower than the adjacent normal tissues (3.54) P <0.05, and immunofluorescence of ASPP2was positively correlated with the level of caspase3expression.The third part: The autophagy level of ASPP2haploid-deficient mice was significantly higherthan in normal mice after oxaliplatin treatment and autophagy levels of the ASPP2over-expression mice were significantly lower than the control group mice.Conclusion1. Compared with adjacent non-tumor tissue, ASPP2mRNA expression of colon cancertissues was significantly reduced.2. ASPP2can promote L-OHP-induced non-p53mediated apoptosis by inhibitingautophagy.3. ΔASPP2had no effect on cell autophagy, but can antagonize the inhibition of ASPP2and p53to autophagy. 4. ΔASPP2can antagonize ASPP2mediated p53-dependent and p53-independentapoptosis of cells, and p53-mediated apoptosis.