Nrf2 Mediates Redox Adaptation In NOX4-Overexpressed Non-Small Cell Lung Cancer Cells

Object:The redox adaptation in cancer cells can promote the function of endogenous antioxidant system and activate the cell survival signaling,however,the mechanism of redox adaptation in tumor cells is very complicated.Our previous studies had confirmed that NOX4-mediated oxidative stress confered anti-apoptotic activity on non-small cell lung cancer?NSCLC?cells.However,the mechanism of the resistance of tumor cells to NOX4-mediated oxidative stress remains unclear.Therefore,this study sought to determine whether Nrf2 mediates redox adaptation in NOX4-overexpressed NSCLC cells and explored the potential significance of Nrf2-mediated redox adaptation in NOX4-overexpressed NSCLC cells,in order to provide experimental basis for clinical development of anti-NSCLC strategy based on redox adaptation.Methods:To determine Nrf2 expression in NSCLC,we first performed immunohistochemistry assay in 122 human NSCLC samples and their corresponding adjacent.A549 and H358 cells were transfected with siRNA against NOX4 or pCMV-NOX4 cDNA plasmid to overexpress NOX4.The mRNA expression,the protein expression and the activity of Nrf2 were detected by RT-qPCR,Western blotting and ARE-luciferase reporter assay,respectively.Intracellular levels of total GSH,reduced GSH and H₂O₂ in A549 cells and NOX4-overexpressed A549 cells were detected after treatment with brusatol.The proliferation and apoptosis of A549 cells and NOX4-overexpressed A549 cells were detected by MTT assay,colony formation assay,flow cytometry and caspase-glo3/7 activity assay after treatment with brusatol,respectively.In vivo,the tumor volume and the survival time of each group of nude mice were recorded through the tumor formation test.The survival curve was made and the effect of Nrf2-mediated redox adaptation on the tumor was analyzed.Results:The data showed that Nrf2 was highly expressed in NSCLC samples.The expression of Nrf2 in NSCLC cell lines?A549,H460,H358,H441 and HCC827?and normal lung epithelial BEAS2B cells was also examined.Consistent with results from clinical specimens,the in vitro results showed that Nrf2 was expressed in all the NSCLC cell lines,and theexpression level was markedly higher in NSCLC cell lines than that in BEAS2B cells.Western blotting,RT-qPCR,and ARE reporter genes assays showed that NOX4 in A549 and H358 cells upregulated the expression of Nrf2 at the protein level,but had little effects on the expression of Nrf2 at the mRNA level.These results of GSH,GSSG,and H₂O₂ assay showed that in NOX4 over-expressed A549 cells,inhibition of Nrf2 caused a decrease in intracellular GSH content and a significant increase in H₂O₂ production.Both in vivo and in vitro experiments showed that inhibition of Nrf2 suppressed the growth of NOX4overexpressed-A549 cells by oxidative stress and caused apoptosis of tumor cells.Conclusion:The redox adaptation in NOX4-overexpressed NSCLC can be mediated by Nrf2.Combined targeting NOX4 and Nrf2 may be a promising strategy against malignant progression of NSCLC.