Bcl-2 Inhibitor ABT737 Promotes The Apoptosis Induced By Oxidative Stress Through Glycometabolism Reprogramming In Ovarian Cancer Cells

Inducing oxidative stress in tumor cells is one of the main anti-tumor mechanisms of some chemotherapeutic drugs.Reactive oxygen species?ROS?play an important role in maintaining the redox balance of cells and can induce cell apoptosis.People expect to increase oxidative stress through various methods to achieve maximum anti-tumor effect.In addition,it has been found that even with sufficient oxygen,tumor cells mainly undergo glucose metabolism through glycolysis.This is conducive to the rapid proliferation of tumor cells,People call this reprogramming of glucose metabolism the"Warburg effect."Mitochondria is the convergence point of redox balance,regulation of apoptosis,cell glycometabolism and signal transduction.Previous experimental studies have shown that the mitochondrial anti-apoptotic protein Bcl-2 may be involved in antioxidation.Recent studies have shown that Bcl-2 family proteins are involved not only in apoptosis but also in the regulation of cellular metabolism.Therefore,it is suggested that we explore the relationship between anti-apoptotic Bcl-2 protein and cellular glucose metabolism and oxidative stress,which may provide new clues for elucidating the anti-tumor mechanism of inducing oxidative stress.In this study,ovarian cancer SKOV3 cells as the research object,to investigate the effect of Bcl-2 inhibitor ABT737 on the regulation of glucose metabolism in tumor cells and the mechanism of integrated oxidative stress and glucose metabolism reprogramming to regulate apoptosis,for the purpose of targeting oxidative stress to reproduce the tumor to provide new ideas.Methods:1)MTT assay was used to detect the effect of H₂O_2,ABT737,H₂O₂ combined with ABT737 on the survival rate of SKOV3 cells.2)Flow cytometry was used to detect the effect of H₂O_2,ABT737,H₂O₂ combined with ABT737 on the apoptosis of SKOV3 cells,Western Blot was used to detect the effect of H₂O_2,ABT737,H₂O₂ combined with ABT737 on the expression of apoptotic protein.3)Using H202 replication oxidative stress model,the ROS levels,mitochondrial membrane potential and mitochondrial ROS levels were measured by flow cytometry and fluorescence microscopy.4)The levels of ROS were measured by flow cytometry,and the activity of SOD was detected by SOD kit after adding H₂O_2,ABT737,H₂O₂ combined with ABT737.5)The mitochondrial membrane potential and mitochondrial ROS levels were measured by flow cytometry after after adding H₂O_2,ABT737,H₂O₂ combined with ABT737.6)Western blot was used to detect the effects of H₂O_2,ABT737,H₂O₂ combined with ABT737 on the expression of Sirt3,HIF-1?and some key metabolites of glucose metabolism.7)Glucose uptake and lactate production kit were used to detect the glucose uptake and lactic acid production of adding H₂O_2,ABT737,H₂O₂ combined with ABT737.Results:1)Both H₂O₂ and ABT737 were able to inhibit the survival of SKOV3 cells,and this effect showed dose-dependent,the inhibitory effect of the combination on the survival rate of SKOV3 cells was more obvious.2)Both H₂O₂ and ABT737 can increase the rate of apoptosis,the increase of the combination was more obvious.Both H₂O₂ and ABT737 could affect the expression of apoptosis-related proteins,and the expression of Bax and caspase3 increased,which indicated that ABT737 could increase the sensitivity of H₂O₂-induced apoptosis through some matrices.3)The SKOV3 cells were treated with two concentrations of H₂O₂ at 100?M and 300?M,the elevated levels of ROS cells increased,mitochondrial membrane potential decreased,mitochondrial ROS level,and the results showed a dose-dependent,indicating H₂O₂ replication oxidative stress model successfully,and can through the mechanism of oxidative damage to kill tumor cells.4)Adding H₂O_2,ABT737,H₂O₂ combined with ABT737 can increase the level of cellular ROS,decrease the activity of SOD,and the effect of combination is more obvious,indicating that both H₂O₂ and ABT737 can cause oxidative stress.5)Adding H₂O_2,ABT737,H₂O₂ combined with ABT737 can decrease the mitochondrial membrane potential and increase the mitochondrial ROS level,combination membrane potential decreased obviously,but compared with the single H₂O₂,mitochondrial ROS almost no rise,indicating that H₂O₂ and ABT737 can damage the mitochondria.6)In the H₂O₂ group,the expression of Sirt3 decreased,the expression of HIF-1 increased,and the ratio of P-PDH/PDH decreased;in the ABT737 and the combination group,the expression of Sirt3 was increased,the expression of HIF-1 was decreased,the expression of Glut1,HK2,PKM2 and LDHA decreased,and the ratio of P-PDH/PDH decreased,indicating that ABT737 could regulate the reprogramming of glucose metabolism in tumor cells.7)The glucose uptake and lactate production of H₂O₂ group are increased slightly,but without statistical significance;ABT737,the glucose uptake and lactate production of ABT737 and the combination group are decreased,indicating that ABT737 can inhibit tumor cell glycolysis.Conclusions:1)Bcl-2 inhibitor ABT737 can regulate the reprogramming of glucose metabolism in ovarian cancer SKOV3 cells,inhibit glycolysis,reduce glucose uptake and lactate production,increase oxidative phosphorylation,and induce apoptosis.2)Bcl-2 inhibitor ABT737 can induce oxidative stress in SKOV3 cells and enhance the oxidative stress-induced apoptosis of ovarian cancer SKOV3 cells by regulating the reprogramming of glucose metabolism.In summary,we speculate that ABT737,a Bcl-2 inhibitor targeted to mitochondria,may increase the oxidative stress-induced apoptosis of cancer cells through the glycometabolism pathway,and may be the experimental basis for the treatment of tumors in combination with other drugs.