Effects Of Hypoxia And CO₂ On Autophagy In Gastric Cancer Cells Induced By Starvation

Purpose:To study the effect of autophagy induced by laparoscopic CO₂pneumoperitoneum in starved gastric cancer cells on whether induce apoptosis or promote survival.Secondly,to investigate the effect of autophagy on proliferation and invasiveness of gastric cancer cells.To study whether it is related to the metastasis of gastric cancer.METHODS:Gastric cancer cells SGC7901 were sealed in a sealed box connected with CO₂ gas or anoxic gas to simulate hypoxia and pneumoperitoneum.Flow cytometry was used to detect apoptosis.Electron microscopy was used to observe autophagosomes and count the number of autophagosomes.Western blotting was used to detect the expression of autophagy-related proteins LC3II/I and P62.CCK8 was used to detect the inhibition of cell proliferation in each group.Transwell tumor invasion assay was used to detect the effect of autophagy on the invasion ability of gastric cancer cells.RESULTS:Before inhibition of autophagy,the apoptosis rates of the control group,hypoxic starvation group and CO₂ starvation group were 5.14±2.20,10.54±1.76,15.61±1.26%,respectively.Compared with the control group,the apoptotic rate of the latter were significantly increased（P<0.05）.There was no significant difference in the apoptotic rate between the three groups after autophagy.Under electron microscopy,we found that autophagy occurred in all of the groups,and the hypoxic starvation group occur more autophagy.In the control group and hypoxia-starvation group,the cells were well-formed,but the cells in the CO₂starvation group were seriously damaged.Compared with the control group,the levels of autophagy-related protein P62 in CO₂ starvation group and hypoxia starvation group were significantly lower（P<0.05）.The expression of LC3II/I increased significantly（P<0.05）.After inhibition of autophagy,the increase of P62was observed in CO₂ starvation group and hypoxia starvation group compared with the control group（P<0.05）,but there was no significant difference between the latter two groups;CCK8 results showed that at the time of 48h,before autophagy inhibition,Compared with the control group,the inhibition rate of proliferation in the CO₂ starvation group did not change significantly,and the proliferation inhibition rate in the hypoxic starvation group was significant（P<0.05）.After autophagy inhibition,the CO₂ starvation group and the hypoxia starvation group both are increased compared with the control group（P<0.05）.However,with the accumulation of time,there was no significant difference in the proliferation of the three groups.In the Transwll experiment,we found that compared with the control group,the hypoxic starvation group was significantly increased（P<0.05）,while the CO₂ starvation group was significantly lower（P<0.05）,there was no significant difference between the three groups after inhibition of autophagy.In addition,the number of cells invading the CO₂ starvation group was significantly lower than that in the hypoxic starvation group（OD570 was 0.22±0.003 and 0.146±0.002,respectively,P<0.05）.Conclusion:Autophagy can be induced by starvation,hypoxia and CO₂pneumoperitoneum,and autophagy is the strongest induced by CO₂,followed by hypoxia.Proper autophagy can promote the proliferation of gastric cancer cells.CO₂-induced hyper-autophagy can induce autophagic death of gastric cancer cells,increase the apoptotic rate,and reduce the invasiveness of gastric cancer cells.