Effects Of Hypoxia And Autophagy Induced By Hypoxia On Colon Carcinoma HCT116Cells

Objective: To investigate the effects of hypoxia on the proliferation, apoptosis andautophagy of colon carcinoma HCT116cells, and study the effects of autophagy onHCT116cells induced by hypoxia.Method:①We established the colon carcinoma HCT116cells culture system with5%CO2and95%N2, simulating the circumstance of hypoxia. colon carcinoma HCT116cells were cultured in the circumstance of hypoxia for12h,24h,48h as the experimentalgroup(cells were cultured for6h,12h,24h,48h as the experimental group in the detectionof protein of HIF-2α), colon carcinoma HCT116cells were cultured in the normoxiccondition for48h(cells were cultured in the normoxic condition for12h,24h,48h in MTT).The MTT assay was used to determine the inhibition effects on cell proliferation. Cellcycle ratio was measured by flow cytometry (FCM). The protein of HIF-2α and LC3wasdetected through western blot.②We selected3-MA as the autophagy inhibitor, andinhibited the autophagy of colon carcinoma HCT116cells which cultured in thecircumstance of hypoxia for24h. We detected the specific protein of autouphagy throughwestern blot, to confirm the efficiency of autophagy inhibitor of3-MA. We observed theshape changes of the cells by inverted microscope. Mitochondrial membrane potential wasmeasured using the fluorescent probe JC-1. PI-AnnexinV staining with flow cytometrywere took out to quantization the cell apoptosis.Results:①A fter thecolon carcinoma HCT116cells cultured under hypoxic system,the inhibition ratio of12h、24h、48h were23.53%±2.93%、38.47%±1.39%、50.94%±1.17%; FCM indicated hypoxia induced arrest of colon carcinoma HCT116cellsin G1phase. The number of G1phase cells increase gradually(from54.65%to74.80%),and that of S phase cells decrease gradually(from39.65%to12.46%) under hypoxiccondition. hypoxia induced rapidly the accumulation of HIF-2α protein in colon carcinomaHCT116cells. With the prolonged hypoxia the HIF-2α protein increase. The ratio of LC3-II/LC3-I increase from0.263±1.67%to0.79±1.47%.②After the colon carcinomaHCT116cells cultured with hypoxic system and3-MA for24h, the ratio of LC3-II/LC3-Iwere less than other groups; the percents of the cells with green fluorescence were largerthan other groups when the colon carcinoma HCT116cells were cultured with hypoxicsystem and3-MA for24h. PI-AnnexinV staining with flow cytometry were took out toquantization the cell apoptosis, and the result indicated that colon carcinoma HCT116cellscultured with hypoxic system and3-MA had a increased apoptosis compared other groups.Conclusion：①A fter thecolon carcinoma HCT116cells cultured under hypoxicsystem, the colon carcinoma HCT116cells were inhibited; the level of autophagy wasup-regulated.②Autophagy plays a role in colon carcinoma cells during oxygen stress, andinhibition of autophagy could enhance the apoptosis of colon carcinoma HCT116cells.