NOX4 Promotes Glutamine Metabolism And Proliferation In NSCLC Through ROS/PI3K/Akt/c-Myc Pathway

Object:NOX4 has been shown to be abnormally expressed in non-small cell lung cancer?NSCLC?and can promote NSCLC progression.However,the regulation that NOX4 promotes tumor proliferation and how NSCLC cells produce oxidative stress resistance remains unclear.NOXs are associated with tumor reprogramming and glutamine metabolism,as well as GSH content are highly dependent on NOXs activity.Therefore,this study will investigate whether NOX4 regulates glutamine metabolism in NSCLC;whether NOX4-mediated glutamine metabolism in NSCLC promotes its malignant proliferation.Whether glutamine-dependent GSH mediates redox adaptation in NOX4-overexpressed NSCLC cell.Finally,we aimed to explore the mechanisms of NOX4 regulation of glutamine metabolism in NSCLC.Methods:1.Western blottingAt the cellular level,Western blotting was used to detect ASCT2,GLS1,GS and c-Myc,after interfering with NOX4-overexpressed or siNOX4 in A549 and H460 cell lines.In addition,after treated with DPI or LY294002 in A549 cells over-expressing NOX4,the expression of c-Myc was observed.2.Real-time RT-PCR?RT-qPCR?Real-time quantitative gene amplification fluorescence detection system?RT-qPCR?was used to detect mRNA expression of glutamine metabolism-associated enzymes and c-Myc in NOX4-overexpressed A549and H460 cells.In addition,after treated with DPI or LY294002 in A549cells,the expression level of c-Myc mRNA was observed.3.MTTThe toxicity of different concentrations of glutamine on A549 and H460 cells was detected by MTT assay after A549 and H460 were inhibited by NOX4.4.Clonogenic survival assayThe long-term proliferation ability of A549 and H460 after BSO treatment was detected by the method of cell colony formation assay.5.Glutamine and Glutamate assay kitThe Glutamine consumption and Glutamate production was measured by Glutamine and Glutamate assay kit in NOX4-overexpressed or NOX4-depleted A549 and H460 cells.6.Amplex Red^?peroxide assay kitAfter treated with PEG-catalase,the H₂O₂ production was measured in NOX4-overexpressed A549 cells.7.Xenograft studiesAt the animal level,A549 cells or NOX4-overexpressed A549 cells were injected subcutaneously into nude mice and injected intraperitoneally with BSO.The size of the tumors was observed and measured every other day.After 36 days,nude mice were sacrificed to obtain tumor tissue and tumor tissue was weighed8.TUNEL assayA549 cells or NOX4-overexpressed A549 cells were subcutaneously injected into nude mice and intraperitoneally injected with BSO.After 36days,the nude mice were sacrificed and the tumors were removed.The apoptosis of A549 cells was detected by TUNEL assay.Result:1.This study examined the breakdown and uptake of glutamine and the expression of key metabolic enzymes in NSCLC cell lines by interfering with NOX4.It was found that inhibition of NOX4 significantly reduced the uptake and breakdown of glutamine,and the expression of key enzymes also decreased.At the same time,the opposite result was obtained in NSCLC cell lines over-expressing NOX4.The results explain NOX4 regulates glutamine metabolism in NSCLC.2.This study examined GSH and NADPH in NSCLC cell lines that interfered with or overexpressed NOX4 and found that NOX4 can modulate glutamine-dependent GSH,enhance the production of NADPH;and by blocking the synthesis of GSH,it can prevent NSCLC cells apoptosis,detected by H₂O₂,BSO-treated NOX4 over-expressed NSCLC cells significantly increased H₂O₂.Above instructions.It reveals that NOX4 promotes glutamine-dependent GSH and NADPH in NSCLC3.This study examined the proliferation of NOX4-overexpressed NSCLC cells in different concentrations of glutamine and we found that high concentrations of glutamine can promote NSCLC cell proliferation and its proliferation in NOX4-overexpressed NSCLC cells was more significant.By measuring the proliferation of NOX4-overexpressed NSCLC cells treated with BSO,it was found that NOX4 overexpression can promote NSCLC cell proliferation,but its proliferation effect is blocked in NSCLC cells NOX4-overexpressed.The results suggest that NOX4 promotes proliferation of NSCLC is dependent on glutamine,glutamine metabolism can make cells obtain oxidative stress resistance,so that tumor cells have a growth advantage.4.This study examined the expression of c-Myc in NSCLC cell lines that interfered with or overexpressed NOX4 and found that NOX4 can upregulate c-Myc protein expression.Interfering with c-Myc inhibited glutamine metabolism and no additional changes were observed in glutamine metabolism caused by NOX4 interference in NOX4 and c-Myc double-interfering cells.Following the use of PI3K/Akt pathway inhibitors or ROS scavengers,the NOX4-regulated c-Myc effect was eliminated.Therefore,in NSCLC,NOX4 can increase glutamine metabolism through ROS/PI3K/Akt/c-Myc,thereby affecting the malignant proliferation of NSCLC.Conclusion:NOX4 can promote NSCLC growth by synthesizing glutamine-derived energy and biomacromolecules,and against NOX4-mediated oxidative stress through maintaining the redox homeostasis of tumor cells via increasing glutamine-derived GSH,thereby promoting NSCLC malignant proliferation.Therefore,NOX4 can be an effective target for the treatment of NSCLC,and it provides a feasible treatment strategy for ROS treatment of NSCLC.