NOX4 Promotes Non-Small Cell Lung Cancer Cell Proliferation Through Regulation Of PI3K/Akt Signaling

Objectives: 1. To study the effect of NOX4 on the NSCLC cell proliferation; 2. To explore the mechanism underlying that NOX4 promotes NSCLC cell proliferation.Methods: 1. Immunohistochemistry To study the expression of NOX4 and p-Akt in 152 samples of NSCLC tissue specimen and adjacent normal lung tissue by immunohistochemistry staining for the correlation between NOX4 and clinical stages. 2. MTT assay Stable cell lines expressing the NOX4 or sh NOX4 or Akt were generated by transfection of p CMV-NOX4 or p RS-sh NOX4 or pc DNA3.1-Akt into A549 and H460 cells. The cells were seeded in 96-wells respectively then detected the cell proliferation viability. 3. Cell colony formation assay The A549 and H460 stable expressing NOX4, sh NOX4 and Akt were seeded in 6-wells 14 days then detected the NSCLC colony formation viability. 4. Xenograft studies 1×106 A549 cells and H460 cells with NOX4 overexpression and sh NOX4 were subcutaneously inoculated into the nude mice. The tumor volume was measured and recorded every other day. 5. Western blotting For analysis of NOX4 in NSCLC cell lines and detected the expression of p-Akt and Akt in NOX4 oversxpression NSCLC cells.Results: 1. Our study showed that NOX4 was upregulated in NSCLC clinical sample and it was correlatedly with clinical disease stage and prognosis(P<0.001). Compared with adjacent normal lung tissue, NOX4 protein levels were higher in NSCLC carcinoma tissue(P<0.05).2. NOX4 overexpression in NSCLC cell lines enhanced cell proliferation in vitro, and produced larger tumors in nude mice than control cells. On the contrary, NOX4 depletion inhibited NSCLC cell proliferation and tumor growth.3. Inhibition of PI3K/Akt pathway could sufficiently block the cellular proliferation of NOX4 overexpression in NSCLC cells both in vitro and in vivo. The enhancement effect of NOX4 on NSCLC cell growth would be blocked with treatment of PI3K/Akt pathway inhibitor LY294002 and Wartmannin. Compared with controls, NOX4 significantly enhanced levels of phosphorylated Akt in NSCLC cells, while NOX4 depletion caused a reduction in PI3K/Akt activity(P<0.05). In addition, the studies in 152 specimens showed that NOX4 levels were correlated with the expression of p-Akt(P<0.05). Moreover, treatment of NOX4 overexpression NSCLC cells with LY294002 could produced smaller tumors in nude mice than control cells.4. Reciprocal activation between PI3K/Akt pathway and NOX4 was important to proliferation of NSCLC cells. Inhibition of Akt led to a significantly decrease in NOX4 expression in A549 and H460 cells. While the Akt overexpression could substantially stimulate NOX4 expression in NSCLC cells. Moreover, both of NOX4 and Akt overexpression could significantly enhance NSCLC cell proliferation by comparison of NOX4 overexpression cells(P<0.05).Conclusions: 1. NOX4 is significantly upregulated in human NSCLC pathological specimen. 2. Overexpression of NOX4 enhances NSCLC cell proliferation. 3. The activation of PI3K/Akt pathway is involved in NOX4-mediated proliferation of NSCLC cells. 4. Reciprocal activation between NOX4 and PI3K/Akt pathway is critical for proliferation of NSCLC cells.