Abstract
Abstract NADPH oxidase (NOX) family enzymes (NOX1-5 and DUOX1-2) generate reactive oxygen species (ROS) and modulate cellular pathways that regulate proliferation. While high doses of ROS are clearly toxic, the effects of low doses of NOX-mediated ROS as signaling molecules are less clear. Recent work has proposed that NOX5 plays a role in cell proliferation in Hairy cell leukemia, Barrett's esophageal adenocarcinoma and in prostate cancer cells, yet relatively little is known of the signaling pathways mediated by NOX5. In this study we explored the functional role of NOX5 in cancer cells obtained from the NCI-60 cell line panel. Initial screening of the NCI-60 cancer cell line panel for expression of the Nox family members by real time PCR revealed several melanoma cell lines that express Nox5. Of the NOX5 expressing NCI-60 human cancer cell lines, subsequent studies were carried out in human UACC-257 melanoma cancer cells. Two molecular approaches were undertaken to determine the functional role of NOX5 in UACC-257 cells: 1) generating NOX5 over-expressing stable clones and 2) transiently silencing endogenous NOX5 and the stably over-expressed NOX5. Redox-sensitive assays for both intra- and extracellular ROS production confirmed that NOX5 is functional and generates superoxide in both UACC-257 cells and stable Nox5-over-expressing clones. NOX5 can be activated both by the protein kinase C activating phorbol ester PMA and by the Ca2+ ionophore ionomycin. Furthermore, this enzymatic activity is calcium- and flavin dehydrogenase-dependent; both the calcium chelator BAPTA-AM and the flavoprotein inhibitor DPI (200nM) significantly decreased NOX5-mediated ROS production. Silencing NOX5 over-expression significantly inhibited both endogenous and PMA-stimulated ROS production, suggesting that the ROS production is NOX5-mediated. Furthermore, knockdown of both endogenous and over-expressed NOX5 in UACC-257 cells resulted in increased p27Kip1 expression. Conversely, over-expression of NOX5 in UACC-257 cells resulted in decreased p27Kip1 expression both at the mRNA and protein levels. As the AKT signaling pathways are known to modulate p27Kip1 levels, we observed that knockdown of both endogenous and over-expressed NOX5 in UACC-257 cells resulted in decreased Akt and GSK-3β phosphorylation. Studies to characterize additional mechanisms of translational and transcriptional regulation of p27Kip1 by NOX5 are ongoing. In summary, our findings suggest that NOX5 expression could contribute to cell proliferation in human cancers in part due to the generation of high local concentrations of extracellular ROS that regulate the Akt/GSK-3β/p27Kip1 signaling network. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2060. doi:1538-7445.AM2012-2060
Published Version
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