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

Abstract

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.

p22phox-dependent NADPH oxidase activity is required for megakaryocytic differentiation

“…Moreover, in the presence of DPI, intracellular ROS levels were significantly lower in K562 and HEL cells (Figure 3b). In addition, supplementary analyses showed that DPI treatment was also hindering differentiation: (1) the compound prevented the increase in cell size ( Figure 3c) and acquisition of the megakaryocytic morphological features ( Figure 3d); (2) the increase in the megakaryocytic markers (CD41 and CD61) and the decrease in the erythrocytic marker GpA were partially inhibited by DPI (Figure 3e 21 This would imply that ROS production might also be involved in cell-cycle progression, thus being crucial for endomitosis. In this sense, while this paper was in preparation, a report appeared describing the potential role of NADPH oxidases in megakaryocyte polyploidisation, 22 in agreement with our results (Figure 3f).…”

Mechanisms of Superoxide Signaling in Epigenetic Processes: Relation to Aging and Cancer

“…This is typical for prostate cells [17][18][19][20][21][22][23][24][25], pancreatic cancer cells [26][27][28][29][30][31][32][33], melanoma cells [34][35], breast cancer cells [36][37][38][39], and other cancer cells [40][41][42][43][44][45][46][47][48][49][50]. It is important that the elevated ROS formation in cancer cells can have both surviving and hindering effects.…”

Reactive Oxygen Generated by NADPH Oxidase 1 (Nox1) Contributes to Cell Invasion by Regulating Matrix Metalloprotease-9 Production and Cell Migration

“…Overproduction of intracellular ROS has been considered as a risk factor in cancer development. In this context it should be noted that aberrant activation of the Nox activity benefits transformation phenotypes of a subset of cancer cells (2); that is, Nox1 in Rastransformed cells (3), Nox4 in pancreatic cancer (4) and melanoma cells (5), and Nox5 in esophageal adenocarcinoma cells (6). With regard to Nox1, Ras oncogene up-regulated the Nox1 expression by activating GATA-6 through mitogen-activated extracellular signal-regulated kinase (MEK)-extracellular signal-regulated kinase (ERK)-dependent phosphorylation (7).…”