NADPH oxidase 4 contributes to transformation phenotype of melanoma cells by regulating G2-M cell cycle progression.
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 G(2)-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 G(2)-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 G(2)-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 G(2)-M cell cycle progression.
Yamaura
Maki
M
Department of Dermatology, Shinshu University Graduate School of Medicine, Matsumoto, Nagano, Japan.
Mitsushita
Junji
J
Furuta
Shuichi
S
Kiniwa
Yukiko
Y
Ashida
Atsuko
A
Goto
Yasuhumi
Y
Shang
Wei H
WH
Kubodera
Makoto
M
Kato
Masayoshi
M
Takata
Minoru
M
Saida
Toshiaki
T
Kamata
Tohru
T
D000595:Amino Acid Sequence; D000975:Antioxidants / Q000494:pharmacology; D016203:CDC2 Protein Kinase / Q000378:metabolism; D002455:Cell Division / Q000502:physiology; D048708:Cell Growth Processes / Q000502:physiology; D045744:Cell Line, Tumor; D016195:G2 Phase / Q000502:physiology; D006801:Humans; D008545:Melanoma / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D008969:Molecular Sequence Data; D000074663:NADPH Oxidase 4; D019255:NADPH Oxidases / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics; D017382:Reactive Oxygen Species / Q000378:metabolism; D014162:Transfection; D015854:Up-Regulation; D020687:cdc25 Phosphatases / Q000378:metabolism
D000975:Antioxidants; D034741:RNA, Small Interfering; D017382:Reactive Oxygen Species; D000074663:NADPH Oxidase 4; D019255:NADPH Oxidases; C498910:NOX4 protein, human; D016203:CDC2 Protein Kinase; C517009:CDC25C protein, human; D020687:cdc25 Phosphatases