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NADPH Oxidase 4 Contributes to Transformation Phenotype of Melanoma Cells by Regulating G₂-M Cell Cycle Progression

Departments of ¹ Dermatology and ² Molecular Biology and Biochemistry, Shinshu University Graduate School of Medicine, Matsumoto, Nagano, Japan

Requests for reprints: Tohru Kamata, Department of Molecular Biology and Biochemistry, Shinshu University Graduate School of Medicine, 3-1-1 Asahi, Matsumoto, Nagano 390-8621 Japan. Phone: 81-263-37-2601; Fax: 81-263-37-2604; E-mail: kamatat{at}shinshu-u.ac.jp.

Key Words: Nox4 • melanoma • reactive oxygen species • cell cycle • cdk1

Generation of reactive oxygen species (ROS) has been implicated in carcinogenic development of melanoma, but the underlying molecular mechanism has not been fully elucidated. We studied the expression and function of the superoxide-generating NADPH oxidase (Nox)4 in human melanoma cells. Nox4 was up-regulated in 13 of 20 melanoma cell lines tested. Silencing of Nox4 expression in melanoma MM-BP cells by small interfering RNAs decreased ROS production and thereby inhibited anchorage-independent cell growth and tumorigenecity in nude mice. Consistently, a general Nox inhibitor, diphenylene iodonium, and antioxidants vitamine E and pyrrolidine dithiocarbamate blocked cell proliferation of MM-BP cells. Flow cytometric analysis indicated that Nox4 small interfering RNAs and diphenylene iodonium induced G₂-M cell cycle arrest, which was also observed with another melanoma cell line, 928mel. This was accompanied by induction of the Tyr-15 phosphorylated, inactive form of cyclin-dependent kinase 1 (a hallmark of G₂-M checkpoint) and hyperphosphorylation of cdc25c leading to its increased binding to 14-3-3 proteins. Ectopic expression of catalase, a scavenger of ROS, also caused accumulation of cells in G₂-M phase. Immunohistochemistry revealed that expression of Nox4 was detected in 31.0% of 13 melanoma patients samples, suggesting the association of Nox4 expression with some steps of melanoma development. The findings suggest that Nox4-generated ROS are required for transformation phenotype of melanoma cells and contribute to melanoma growth through regulation of G₂-M cell cycle progression. [Cancer Res 2009;69(6):2647–54]