Effects Of ASPP2 And HIV Protease Inhibitors On Autophagy And Apoptosis In Colorectal Cancer Cells

Background Colorectal cancer(CRC),one of the malignant tumors threatening human health,has a high incidence and mortality.Mutation or inactivation of tumor suppressor gene P53 is an important cause of colorectal cancer.ASPP2(Apoptosis stimulating protein 2 of p53)can bind to P53 and selectively enhance the apoptotic activity of P53.HIV protease inhibitors(HIV PIs)are widely used to anti-human immunodeficiency virus,which effectively improves the survival of HIV infected people.Recently,PIs were found to have an activity of anti-tumor.So PIs are now being considered as a agent to be used for anti-tumor chemotherapy.PIs have been shown to be effective in suppressing the growth of lung cancer,breast cancer and hepatocellular carcinoma(HCC),and have been shown to be effective in the treatment of breast cancer,non-small cell lung cancer,hepatocellular carcinoma and Kaposi’s sarcoma.The effect of HIV protease inhibitors on colorectal cancer has been rarely reported,and it is known that oxaliplatin is a recognized chemotherapeutic agent for colorectal cancer and there are several articles reported that oxaliplatin has an effect on autophagy of colorectal cancer cells.Objective In this study,we study the effect of HIV protease inhibitor and oxaliplatin on the apoptosis and autophagy level of colorectal cancer cells and the overexpression or low expression of colorectal cancer cell line ASPP2.On the effect of protease inhibitor on cell autophagy and apoptosis,and to analyze its effect on chemosensitivity of tumor cells.Methods The first part of the experiment : 1.To obtain the stable transfected cell line of HCT116+/+-sh P53/HCT116+/+-sh ASPP2/HCT116+/+-sh Ctrl,we use the Lentiviral vector to knockdown its sh RNA.2.MTT assay was used to detect the growth of HCT116 + / +(p53 wild type)cells treated by three different HIV protease inhibitors(ritonavir,lopinavir and darunavir)at different time points(12 hours,24 hours and 36 hours)and to detect the growth inhibition of HCT16+/+-sh Ctrl / HCT116+/+-sh P53 cells by them at 24 hours.The second part of the experiment:1.HCT116+/+ cells were treated by the IC50 of DRV/OXA at 24 hours,then Calcein-AM/PI staining and western blot(WB)were used to analyze cell apoptosis.2.The cell line was cultured and divided into 6 groups:1)Ad-GFP single infected group;2)Ad-ASPP2 single infected group;3)sh ASPP2 transfected group;4)Ad-GFP+DRV group;5)Ad-ASPP2+DRV group;6)DRV+ sh ASPP2 transfected group;HCT116 cells were cultured to density 60%,infected with indicated adenovirus for 12 hours,then treated with DRV for 24 hours.Then Calcein-AM/PI staining and western blot(WB)were used to analyze cell apoptosis and autophagy.3.In order to detect the effect of DRV on cell cycle and the effect of different levels of ASPP2 on cell cycle,this study was divided into 6 groups :1)Ctrl group,Ad-GFP group,Ad-ASPP2 group,Ad-ASPP2 + DRV group,Ad-GFP + DRV group and DRV group.Results In the first part: 1.The knockdown of P53 / ASPP2 with its sh RNA(sh P53/sh ASPP2)were able to be stably transfected.The results were confirmed by WB.2.The HIV protease inhibitor darunavir could induce HCT116+/+-sh Ctrl,cell apoptosis,and the rate of apoptosis increased along with the increase of DRV concentration.3.n this experiment,MTT assay was used to detect the effects of RTV,LPV and DRV on the growth inhibition of HCT116+/+-sh Ctrl,cells at three different time points at 12 h / 24 h / 36 h.At three different time points,The obtained curve is relatively more stable,so select the best time for 24 h.The effect of three kinds of protease inhibitors on the growth inhibition of HCT116+/+-sh Ctrl,and HCT116+ / +-sh P53 cells was detected.The IC50 of R for HCT116+/+-sh Ctrl,was about 25μg / ml,the lopinavir was about 25μg / ml,and the darunavir was about 40μg / ml.But The IC50 of RTV for HCT116+ / +-sh P53 was about 20μg / ml,the lopinavir was about 18μg / ml,and the darunavir was about 30μg / ml.We found that HCT116+ / +-sh P53 was more sensitive to HIV PIs compared with HCT116 + / +-sh Ctrl.In the second part: 1.Compared with oxaliplatin,protease inhibitor darunavir also induced cell autophagy and apoptosis;2.Calcein-AM / PI staining was used to detect the apoptosis of each group,the apoptotic rate(4.85 ± 0.53)%,(4.57 ± 0.75)%,(1.95 ± 0.44)%,(39.5 ± 4.29)%,(47.7 ± 6.20)%,(32.68 ± 2.49)%,GFP group and ASPP2 group(P <0.01).There was significant difference between GFP + DRV group and ASPP2 + DRV group(P = 0.036 <0.05).Compared with sh ASPP2 + DRV group(P <0.01)The expression of autophagy and apoptotic protein was detected by WB.We found that ASPP2 overexpression could enhance the sensitivity of HCT116 cells to darunavir;low expression of ASPP2 could promote autophagy of HCT116+/+-sh Ctrl cells and decrease the sensitivity of HCT116+ / + cells to darunavir.3.Detection of cell cycle(48.7 ± 2.06)%,(45.00 ± 5.64)%,(40.25 ± 3.96)%,(18.55 ± 1.38)%,(26.70 ± 1.07)% and(28.56 ± 2.53)%.There was no significant difference between the normal culture group and the GFP group(P = 0.289> 0.05)and the roisenavir group and the oxaliplatin group(P = 0.482,> 0.05).There was no significant difference between the two groups Between the two were P <0.05,the difference was statistically significant.Conclusions 1.The suppression of p53 expression using its sh RNA increased the chemotherapeutic sensitivity of HCT116+/+-sh Ctrl cells to HIV protease inhibitors.2.Compared with oxaliplatin,the effect of protease inhibitors induce cell autophagy and apoptosis is not as good as the former.3.ASPP2 overexpression enhanced the level of autophagy in HCT116+/+-sh Ctrl cells and increased its sensitivity to PIs.4.HIV protease inhibitor darunavir can inhibit cell proliferation by causing G0 / G1 arrest,and ASPP2 enhances its inhibitory effect on cell proliferation.