NADPH Oxidase 1 Overexpression Enhances Invasion via Matrix Metalloproteinase-2 and Epithelial-Mesenchymal Transition in Melanoma Cells

NADPH oxidase 1 (Nox1) is a member of the NADPH oxidase family that has not been well characterized in the melanocytic cell lineage. Here we demonstrated that Nox1 and Nox4 were detected in melanocytic lineage, with only Nox1 detected in normal human melanocytes and Nox4 in a subset of metastatic melanoma cell lines. The protein level and enzymatic activity of Nox1 was elevated in all melanoma cells as compared with normal melanocytes. Overexpression of GFP-Nox1 protein in Wm3211 primary melanoma cells increased invasion rate by 4- to 6-fold as measured by Matrigel invasion assay, whereas knocking down or inhibiting Nox1 decreased invasion by approximately 40-60% in Wm3211 and SK-Mel-28 cells. Matrix metalloproteinase-2 (MMP-2) was increased by Nox1 overexpression at the mRNA, protein, and activity levels, and decreased by Nox1 knockdown. MMP-2 promoter activity was also regulated by Nox1 knockdown. In addition, stable clones overexpressing Nox1 exhibited an epithelial-mesenchymal transition (EMT) as examined by cell morphology and EMT markers; knockdown or inhibiting Nox1 led to a reversal of EMT. Supplementing MMP-2 to culture media did not induce EMT, suggesting that EMT induction by Nox1 was not through MMP-2 upregulation. In summary, Nox1 was overexpressed in all melanoma cell lines examined, and enhanced cell invasion by MMP-2 upregulation and EMT induction.