The mechanism by which reactive oxygen species (ROS) are produced by tumour cells remained incompletely understood until the discovery over the last 15 years of the family of NADPH oxidases (NOXs 1-5 and dual oxidases DUOX1/2) which are structural homologues of gp91phox, the major membrane-bound component of the respiratory burst oxidase of leucocytes. Knowledge of the roles of the NOX isoforms in cancer is rapidly expanding. Recent evidence suggests that both NOX1 and DUOX2 species produce ROS in the gastrointestinal tract as a result of chronic inflammatory stress; cytokine induction (by interferon-γ, tumour necrosis factor α, and interleukins IL-4 and IL-13) of NOX1 and DUOX2 may contribute to the development of colorectal and pancreatic carcinomas in patients with inflammatory bowel disease and chronic pancreatitis, respectively. NOX4 expression is increased in pre-malignant fibrotic states which may lead to carcinomas of the lung and liver. NOX5 is highly expressed in malignant melanomas, prostate cancer and Barrett's oesophagus-associated adenocarcinomas, and in the last it is related to chronic gastro-oesophageal reflux and inflammation. Over-expression of functional NOX proteins in many tissues helps to explain tissue injury and DNA damage from ROS that accompany pre-malignant conditions, as well as elucidating the potential mechanisms of NOX-related damage that contribute to both the initiation and the progression of a wide range of solid and haematopoietic malignancies.

The family of NADPH oxidase (NOX) genes produces reactive oxygen species (ROS) pivotal for both cell signalling and host defense. To investigate whether NOX and NOX accessory gene expression might be a factor common to specific human tumour types, this study measured the expression levels of NOX genes 1-5, dual oxidase 1 and 2, as well as those of NOX accessory genes NoxO1, NoxA1, p47 phox , p67 phox and p22 phox in human cancer cell lines and in tumour and adjacent normal tissue pairs by quantitative, real-time RT-PCR. The results demonstrate tumour-specific patterns of NOX gene expression that will inform further studies of the role of NOX activity in tumour cell invasion, growth factor response and proliferative potential.

Significance: Reactive oxygen species (ROS) promote genomic instability, altered signal transduction, and an environment that can sustain tumor formation and growth. The NOX family of NADPH oxidases, membranebound epithelial superoxide and hydrogen peroxide producers, plays a critical role in the maintenance of immune function, cell growth, and apoptosis. The impact of NOX enzymes in carcinogenesis is currently being defined and may directly link chronic inflammation and NOX ROS-mediated tumor formation. Recent Advances: Increased interest in the function of NOX enzymes in tumor biology has spurred a surge of investigative effort to understand the variability of NOX expression levels in tumors and the effect of NOX activity on tumor cell proliferation. These initial efforts have demonstrated a wide variance in NOX distribution and expression levels across numerous cancers as well as in common tumor cell lines, suggesting that much remains to be discovered about the unique role of NOX-related ROS production within each system. Progression from in vitro cell line studies toward in vivo tumor tissue screening and xenograft models has begun to provide evidence supporting the importance of NOX expression in carcinogenesis. Critical Issues: A lack of universally available, isoform-specific antibodies and animal tumor models of inducible knockout or over-expression of NOX isoforms has hindered progress toward the completion of in vivo studies. Future Directions: In vivo validation experiments and the use of large, existing gene expression data sets should help define the best model systems for studying the NOX homologues in the context of cancer. Antioxid. Redox Signal. 20, 2873Signal. 20, -2889

We investigated the mechanism of action of LBH589, a novel broad-spectrum HDAC inhibitor belonging to the hydroxamate class, in Philadelphia chromosome-negative (Ph ؊ ) acute lymphoblastic leukemia (ALL) . IntroductionHistone deacetylases (HDACs) are responsible for deacetylating histones and nonhistone proteins, thus regulating gene transcription, protein function, and stability. 1,2 Aberrant recruitment of HDACs has been shown to have an important role in leukemogenesis, and alterations in the expression and/or activity of HDACs have been also observed in solid tumors. [3][4][5] HDAC inhibitors have been widely studied, clinical trials with several of these have started, and HDAC inhibitor-responsive tumors have been observed. [6][7][8][9] Clinical benefits of HDAC inhibitors may result from re-expressing and/or reactivating tumor suppressor genes and/or proteins, thereby inducing apoptosis, cell-cycle arrest, and differentiation. [6][7][8][9] In most cases of acute lymphoblastic leukemia (ALL), chromosomal abnormalities are found such as the gain or loss of whole chromosomes, but more often, translocations, deletions, or inversions occur. [10][11][12] Epigenetic alterations, including aberrant DNA methylation of promoter-associated CpG islands resulting in gene silencing, have been frequently observed in ALL both at presentation and at relapse. 13,14 There is a need for new agents with efficacy in Ph Ϫ ALL, especially for patients who relapse where the treatment options are limited. [15][16][17][18] Treatment of ALL cells with an HDAC inhibitor offers an appealing strategy, potentially promoting, in the resistant lymphoblasts, reactivation of genes responding to genomic instability and therefore elimination of the affected cells.In this study, we evaluated the antitumor efficacy and mechanism of action of LBH589, an HDAC inhibitor belonging to the hydroxamate class, in Philadelphia chromosome-negative (Ph Ϫ ) ALL cells. Two Ph Ϫ ALL cell lines (T-cell MOLT-4 and pre-B-cell Reh) were treated with LBH589. Our results show that LBH589, at clinically safe concentrations (approximately 10-50 nM), induces potent cell growth inhibition, cell-cycle arrest, and apoptosis in a time-and dose-dependent manner in both cell lines. We further show that the antitumor activities of LBH589 correlate with induction of histone (H3K9 and H4K8) hyperacetylation, activation of p21 and p27, and suppression of c-Myc in the 2 cell lines treated. Using polymerase chain reaction (PCR) arrays, we determined the effects of LBH589 on the expression of DNA damage and apoptosis genes. LBH589 treatment induced significant increases in mRNA levels of a number of genes with proapoptosis, growth arrest, and DNA repair functions. Some of these genes have been previously reported to be epigenetically down-regulated in human malignancies such as GADD45G. 19 Furthermore, we demonstrate that LBH589 response correlates with induction of histone H2A.X phosphorylation. Importantly, LBH589 also induces apoptosis and elevated GADD45G expression in culture...

Pancreatitis is associated with release of pro-inflammatory cytokines and reactive oxygen species and plays an important role in the development of pancreatic cancer. We recently demonstrated that dual oxidase 2 (Duox2), an NADPH oxidase essential for ROS-related, gastrointestinal host defense, is regulated by IFN-γ-mediated Stat1 binding to the Duox2 promoter in pancreatic tumor lines. Because lipopolysaccharide (LPS) enhances the development and invasiveness of pancreatic cancer in vivo following Toll-like receptor 4 (TLR4)-related activation of NF-κB, we examined whether LPS, alone or combined with IFN-γ, regulated Duox2. We found that upregulation of TLR4 by IFN-γ in BxPC-3 and CFPAC-1 pancreatic cancer cells was augmented by LPS, resulting in activation of NF-κB, accumulation of NF-κB (p65) in the nucleus, and increased binding of p65 to the Duox2 promoter. TLR4 silencing with siRNAs, and two independent NF-κB inhibitors, attenuated LPS- and IFN-γ–mediated Duox2 upregulation in BxPC-3 cells. Induction of Duox2 expression by IFN-γ and LPS may result from IFN-γ-related activation of Stat1, acting in concert with NF-κB-related upregulation of Duox2. Sustained extracellular accumulation of H2O2 generated by exposure to both LPS and IFN-γ was responsible for an ≈ 50% decrease in BxPC-3 cell proliferation associated with a G1 cell cycle block, apoptosis, and DNA damage. We also demonstrated up-regulation of Duox expression in vivo, in pancreatic cancer xenografts and in patients with chronic pancreatitis. These results suggest that inflammatory cytokines can interact to produce a Duox-dependent pro-oxidant milieu that could increase the pathologic potential of pancreatic inflammation and pancreatic cancer cells.

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Reactive oxygen species (ROS) play a critical role in cell signaling and proliferation. NADPH oxidase 1 (NOX1), a membrane-bound flavin dehydrogenase that generates O2˙̄, is highly expressed in colon cancer. To investigate the role that NOX1 plays in colon cancer growth, we used shRNA to decrease NOX1 expression stably in HT-29 human colon cancer cells. The 80–90% decrease in NOX1 expression achieved by RNAi produced a significant decline in ROS production and a G1/S block that translated into a 2–3-fold increase in tumor cell doubling time without increased apoptosis. The block at the G1/S checkpoint was associated with a significant decrease in cyclin D1 expression and profound inhibition of mitogen-activated protein kinase (MAPK) signaling. Decreased steady-state MAPK phosphorylation occurred concomitant with a significant increase in protein phosphatase activity for two colon cancer cell lines in which NOX1 expression was knocked down by RNAi. Diminished NOX1 expression also contributed to decreased growth, blood vessel density, and VEGF and hypoxia-inducible factor 1α (HIF-1α) expression in HT-29 xenografts initiated from NOX1 knockdown cells. Microarray analysis, supplemented by real-time PCR and Western blotting, revealed that the expression of critical regulators of cell proliferation and angiogenesis, including c-MYC, c-MYB, and VEGF, were down-regulated in association with a decline in hypoxic HIF-1α protein expression downstream of silenced NOX1 in both colon cancer cell lines and xenografts. These studies suggest a role for NOX1 in maintaining the proliferative phenotype of some colon cancers and the potential of NOX1 as a therapeutic target in this disease.

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.