Abstract
NADPH oxidase 4 (NOX4) is a redox active, membrane-associated protein that contributes to genomic instability, redox signaling, and radiation sensitivity in human cancers based on its capacity to generate H2O2 constitutively. Most studies of NOX4 in malignancy have focused on the evaluation of a small number of tumor cell lines and not on human tumor specimens themselves; furthermore, these studies have often employed immunological tools that have not been well characterized. To determine the prevalence of NOX4 expression across a broad range of solid tumors, we developed a novel monoclonal antibody that recognizes a specific extracellular region of the human NOX4 protein, and that does not cross-react with any of the other six members of the NOX gene family. Evaluation of 20 sets of epithelial tumors revealed, for the first time, high levels of NOX4 expression in carcinomas of the head and neck (15/19 patients), esophagus (12/18 patients), bladder (10/19 patients), ovary (6/17 patients), and prostate (7/19 patients), as well as malignant melanoma (7/15 patients) when these tumors were compared to histologically-uninvolved specimens from the same organs. Detection of NOX4 protein upregulation by low levels of TGF-β1 demonstrated the sensitivity of this new probe; and immunofluorescence experiments found that high levels of endogenous NOX4 expression in ovarian cancer cells were only demonstrable associated with perinuclear membranes. These studies suggest that NOX4 expression is upregulated, compared to normal tissues, in a well-defined, and specific group of human carcinomas, and that its expression is localized on intracellular membranes in a fashion that could modulate oxidative DNA damage.
Highlights
Cellular redox balance relies on a dynamic interplay between endogenous [mitochondria, cytochrome P-450, nitric oxide synthase (NOS) and NADPH oxidase (NOX) enzymes] and exogenous production of reactive oxygen species (ROS) and a broad array of intrinsic cellular antioxidant pathways [1,2,3]
While the NADPH oxidase 4 (NOX4) amino acid sequence does not display endoplasmic reticulum (ER) retention or endosomal translocation signals [62], previous studies have reported a lack of NOX4 enzyme at the plasma membrane surface in overexpression systems established in HEK293, COS-7, or HeLa parental cell lines [29,30,33,62]
NOX4 is among many ROS producing enzymes under study as potential sources of ROS producing oxidative DNA damage [87,88]
Summary
Cellular redox balance relies on a dynamic interplay between endogenous [mitochondria, cytochrome P-450, nitric oxide synthase (NOS) and NADPH oxidase (NOX) enzymes] and exogenous (environmental agents, pharmaceuticals, and industrial chemicals) production of reactive oxygen species (ROS) and a broad array of intrinsic cellular antioxidant pathways [1,2,3]. Understanding the mechanisms by which ROS imbalance results in epithelial dysfunction and promotes tumor growth and progression could focus development of new redox-based strategies for therapeutic intervention. The NOX enzymatic family members (NOX1-5, DUOX1-2), through isoform specific superoxide or hydrogen peroxide production, have been associated with tissue remodeling, resistance to apoptosis, tumor cell proliferation and metastasis, and enhanced angiogenesis. Tumor promotion by NOX isoforms occurs, in part, by inflammation- and hypoxia-mediated upregulation of oxidative DNA damage and tissue injury [4,5,6,7,8]. The relationship between NOX-related oxidant production and cancer is both NOX-isoform and tumor context specific. DUOX2 expression, unlike that of the DUOX1 homolog, is associated with progression of pancreatic cancer; the pro-inflammatory cytokine
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