The Effect Of ASPP2 And Isomer ΔASPP2 On Autophagy And Apoptosis In Human Colon Cancer Cells

Objective Colon cancer is one of the most common malignant tumor of digestive tract, more than half of patients with colon cancer have the mutation or deletion of P53 gene. Apoptosis stimulating protein 2 of P53(ASPP2) plays an important role in selectively inducing cell apoptosis by P53 genes, it can induce cells apoptosis through P53 dependent pathway, thereby inhibiting the formation and development of tumor. ΔASPP2 is a new, shortened isoform of ASPP2 through the 5 ’RACE technology, containing 880 amino acids. Preliminary studies indicate that the ΔASPP2 not only can inhibit cell apoptosis, but also can promote autophagy. Meanwhile, we also found that the expression of ΔASPP2 was significantly increased in colon cancer specimens. In this study, we aimed to discuss the effect of ASPP2 and its isomers ΔASPP2 on apoptosis and autophagy induced by oxaliplatin in human colon cancer cell line HCT116-/-(P53 deletion) cells, and deeply discussed the effects of ASPP2 and ΔASPP2 in P53 apoptosis pathway and related mechanism.Methods Frozen sections of colorectal carcinoma tissues and paracancerous tissues were made to observe the expression of ASPP2 and P53 by immunofluorescence after immobilized. RNA of colorectal carcinoma tissues and paracancerous tissues was extracted, c DNA was got by reverse transcription. The primers of ASPP2,ΔASPP2 and P53 were used for amplification. The expressions of ASPP2 m RNA, ΔASPP2 m RNA, P53 m RNA in colorectal carcinoma tissues and paracancerous tissues were detected by q RT-PCR. HCT116-/-cells were transfected with ASPP2, ΔASPP2, P53 and GFP-LC3 plasmids, then treated withoxaliplatin(L-OHP). Cell autophagy was observed by immunofluorescence. Apoptosis was observed by M30, Calcein AM/PI and flow cytometry. The rate of cell survival was detected by MTT. HCT116-/- cells were transfected with r Ad-ASPP2, r Ad-ΔASPP2 and r Ad-P53, then treated with L-OHP. Cell proliferation ability was detected by flat cell colony formation assays. Detected the effect of ΔASPP2 on the combination of ASPP2 and P53 by Co-Immunoprecipitation(CO-IP).The expressions of autophagy and apoptosis related proteins were detected by Western blotting.Results Compared with adjacent normal tissues, the expression of ASPP2 m RNA is significantly lower in colon tumor tissues(tumor 2.27±1.66 VS para-tumor 3.63±1.73,P <0.05). ΔASPP2 m RNA expression is significantly higher in colon tumor tissues than in corresponding adjacent non-tumor tissues(tumor 0.54±0.26 VS para-tumor 0.40±0.23,P <0.05).The expression of P53 m RNA has no discrepancy in colon tumor tissues and adjacent normal tissues(tumor 5.31±1.14 VS para-tumor 5.55±1.07,P >0.05).ΔASPP2 can antagonist the combination of ASPP2 and P53,and inhibit the pro-apoptotic function of them.ΔASPP2 can increase the rate of cell survival and improve cell proliferation ability. Compared with the P53 sole treated group(autophagy 5.43%±0.21%, apoptosis 33.67%±0.76%), autophagy(14.77%±0.25%,P <0.05) was increased and apoptosis(21.33%±0.94%,P <0.05) was decreased in the group of ΔASPP2+P53. Compared with the ASPP2+P53 treated group(autophagy 3.37%±0.15%, apoptosis 50.61%±1.25%), autophagy(12.33%±0.26%,P <0.05) was increased and apoptosis(35.18%±1.12%,P <0.05) was decreased in the group of P53+ASPP2+ΔASPP2.Conclusion 1.Compared with adjacent normal tissues, the expression of ASPP2 m RNA is significantly lower in colon tumor tissues. ΔASPP2 m RNA expression is significantly higher in colon tumor tissues. 2.ΔASPP2 can antagonist the combination of ASPP2 and P53 and effect the functions of them. 3. ΔASPP2 can antagonist the function of inhibit autophagy of ASPP2 and P53. 4.ΔASPP2 can inhibit the pro-apoptotic function of ASPP2 and P53. 5.ΔASP P2 can suppresses cell apoptosis by promoting cell autophagy.