Poster presentation, P148

Official XXIst International Pigment Cell Conference website - 21-24 Sept 2011, Bordeaux - France | updated: September 04 2011

Expression of NADPH Oxidase 1 in Melanoma Cells and its effect on Invasion via induction of Matrix Metalloproteinase-2

INTRODUCTION: NADPH oxidase (EC 1.6.3. -) is a family of enzymes that catalyze transfer of an electron from NAD(P)H to molecule oxygen to generate superoxide or hydrogen peroxide. It consists of 7 members, represented by their catalytic subunits: Nox1, Nox2 (gp91phox), Nox3, Nox4, Nox5, Duox1 and Duox2. Among these members Nox4 was previously demonstrated to play a role in melanoma invasion down-stream of Akt pathway. Expression of Nox1 was not reported in melanoma cells. METHODS: RT-PCR and Western blot analysis was used to analyze expression of Nox1, Noxo1 and p22phox in normal melanocytes and melanoma cells; over-expression, knockdown or inhibition of Nox1 was achieved in radial growth phase Wm3211 melanoma cells, cell invasion was assayed using Matri-gel coated transwells. Expression of MMP-2 was analyzed by qRT-PCR and western blot in these cells. Promoter reporter plasmid of human MMP-2 is being constructed; promoter activity will be analyzed in cells clones with varied Nox1 expression levels. RESULTS: Nox1 was expressed in normal melanocytes and all melanoma cell lines examined, as in contrast to Nox4 which was not expressed in normal melanocytes but only expressed in a subset of metastatic melanoma cell lines. Nox1 subunits Noxo1 and p22phox were also expressed in normal melanocytes and all melanoma cell lines. Over-expression of GFP-fused Nox1 protein in Wm3211 cells increased cell invasion rate by 4 to 6 fold, while knocking down Nox1 decreased invasion rate by 2-3 fold. Inhibiting Nox1 by Diphenyliodonium (DPI) also inhibited invasion rate. We further found that secreted MMP-2 increased in cells over-expressing Nox1, and decreased in cells with Nox1 knockdown. Quantitative RT-PCR analysis demonstrated the regulation of MMP-2 may occur at transcription level as mRNA of MMP-2 increased about 10-11 fold in cell clones over-expressing Nox1. CONCLUSION: Our data shows that Nox1 is over-expressed in all melanoma cell lines examined; it contributes to enhanced cell invasion by activating MMP-2 at transcriptional level in melanoma cells.

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