Exogenous DNA enhances DUOX2 expression and function in human pancreatic cancer cells by activating the cGAS-STING signaling pathway.

Pro-inflammatory cytokines upregulate the expression of the H2O2-producing NADPH oxidase dual oxidase 2 (DUOX2)2 which, when elevated, adversely affects survival from pancreatic ductal adenocarcinoma (PDAC). Because the cGAS-STING pathway is known to initiate pro-inflammatory cytokine expression following uptake of exogenous DNA, we examined whether activation of cGAS-STING could play a role in the generation of reactive oxygen species by PDAC cells. Here, we found that a variety of exogenous DNA species markedly increased the production of cGAMP, the phosphorylation of TBK1 and IRF3, and the translocation of phosphorylated IRF3 into the nucleus, leading to a significant, IRF3-dependent enhancement of DUOX2 expression, and a significant flux of H2O2 in PDAC cells. However, unlike the canonical cGAS-STING pathway, DNA-related DUOX2 upregulation was not mediated by NF-κB. Although exogenous IFN-ß significantly increased Stat1/2-associated DUOX2 expression, intracellular IFN-ß signaling that followed cGAMP or DNA exposure did not itself increase DUOX2 levels. Finally, DUOX2 upregulation subsequent to cGAS-STING activation was accompanied by the enhanced, normoxic expression of HIF-1α and VEGF-A as well as DNA double strand cleavage, suggesting that cGAS-STING signaling may support the development of an oxidative, pro-angiogenic microenvironment that could contribute to the inflammation-related genetic instability of pancreatic cancer.

Effects of Iodonium Analogs on Nadph Oxidase 1 in Human Colon Cancer Cells.

Recent studies suggest that of the molecules postulated to function as inhibitors of the NADPH oxidase family of enzymes iodonium analogs known to broadly interfere with flavin dehydrogenase function demonstrate mechanistic validity as NADPH oxidase poisons. In recent work, we have produced a series of novel iodonium compounds as putative inhibitors of these oxidases. To evaluate the potential utility of two novel molecules with favorable chemical properties, NSC 740104 and NSC 751140, we compared effects of these compounds to the two standard inhibitors of this class, diphenyleneiodonium and di-2-thienyliodonium, with respect to antiproliferative, cell cycle, and gene expression effects in human colon cancer cells that require the function of NADPH oxidase 1. Both new agents blocked NADPH oxidase-related reactive oxygen production, inhibited tumor cell proliferation, produced a G1/S block in cell cycle progression, and inhibited NADPH oxidase 1 expression at the mRNA and protein levels at low nM concentrations in a fashion similar to or better than the parent molecules. These studies suggest that NSC 740104 and NSC 751140 should be developed further as mechanistic tools to better understand the role of NADPH oxidase inhibition as an approach to the development of novel therapeutic agents for colon cancer.

NADPH oxidase 1 is highly expressed in human large and small bowel cancers.

To facilitate functional investigation of the role of NADPH oxidase 1 (NOX1) and associated reactive oxygen species in cancer cell signaling, we report herein the development and characterization of a novel mouse monoclonal antibody that specifically recognizes the C-terminal region of the NOX1 protein. The antibody was validated in stable NOX1 overexpression and knockout systems, and demonstrates wide applicability for Western blot analysis, confocal microscopy, flow cytometry, and immunohistochemistry. We employed our NOX1 antibody to characterize NOX1 expression in a panel of 30 human colorectal cancer cell lines, and correlated protein expression with NOX1 mRNA expression and superoxide production in a subset of these cells. Although a significant correlation between oncogenic RAS status and NOX1 mRNA levels could not be demonstrated in colon cancer cell lines, RAS mutational status did correlate with NOX1 expression in human colon cancer surgical specimens. Immunohistochemical analysis of a comprehensive set of tissue microarrays comprising over 1,200 formalin-fixed, paraffin-embedded tissue cores from human epithelial tumors and inflammatory disease confirmed that NOX1 is overexpressed in human colon and small intestinal adenocarcinomas, as well as adenomatous polyps, compared to adjacent, uninvolved intestinal mucosae. In contradistinction to prior studies, we did not find evidence of NOX1 overexpression at the protein level in tumors versus histologically normal tissues in prostate, lung, ovarian, or breast carcinomas. This study constitutes the most comprehensive histopathological characterization of NOX1 to date in cellular models of colon cancer and in normal and malignant human tissues using a thoroughly evaluated monoclonal antibody. It also further establishes NOX1 as a clinically relevant therapeutic target in colorectal and small intestinal cancer.

IL-4 and IL-17A Cooperatively Promote Hydrogen Peroxide Production, Oxidative DNA Damage, and Upregulation of Dual Oxidase 2 in Human Colon and Pancreatic Cancer Cells.

Dual oxidase 2 (DUOX2) generates H2O2 that plays a critical role in both host defense and chronic inflammation. Previously, we demonstrated that the proinflammatory mediators IFN-γ and LPS enhance expression of DUOX2 and its maturation factor DUOXA2 through STAT1- and NF-κB‒mediated signaling in human pancreatic cancer cells. Using a panel of colon and pancreatic cancer cell lines, we now report the induction of DUOX2/DUOXA2 mRNA and protein expression by the TH2 cytokine IL-4. IL-4 activated STAT6 signaling that, when silenced, significantly decreased induction of DUOX2. Furthermore, the TH17 cytokine IL-17A combined synergistically with IL-4 to increase DUOX2 expression in both colon and pancreatic cancer cells mediated, at least in part, by signaling through NF-κB. The upregulation of DUOX2 was associated with a significant increase in the production of extracellular H2O2 and DNA damage-as indicated by the accumulation of 8-oxo-dG and γH2AX-which was suppressed by the NADPH oxidase inhibitor diphenylene iodonium and a DUOX2-specific small interfering RNA. The clinical relevance of these experiments is suggested by immunohistochemical, microarray, and quantitative RT-PCR studies of human colon and pancreatic tumors demonstrating significantly higher DUOX2, IL-4R, and IL-17RA expression in tumors than in adjacent normal tissues; in pancreatic adenocarcinoma, increased DUOX2 expression is adversely associated with overall patient survival. These data suggest a functional association between DUOX2-mediated H2O2 production and induced DNA damage in gastrointestinal malignancies.

NADPH oxidase 5 (NOX5)-induced reactive oxygen signaling modulates normoxic HIF-1α and p27Kip1 expression in malignant melanoma and other human tumors.

NADPH oxidase 5 (NOX5) generated reactive oxygen species (ROS) have been implicated in signaling cascades that regulate cancer cell proliferation. To evaluate and validate NOX5 expression in human tumors, we screened a broad range of tissue microarrays (TMAs), and report substantial overexpression of NOX5 in malignant melanoma and cancers of the prostate, breast, and ovary. In human UACC-257 melanoma cells that possesses high levels of functional endogenous NOX5, overexpression of NOX5 resulted in enhanced cell growth, increased numbers of BrdU positive cells, and increased γ-H2AX levels. Additionally, NOX5-overexpressing (stable and inducible) UACC-257 cells demonstrated increased normoxic HIF-1α expression and decreased p27Kip1 expression. Similarly, increased normoxic HIF-1α expression and decreased p27Kip1 expression were observed in stable NOX5-overexpressing clones of KARPAS 299 human lymphoma cells and in the human prostate cancer cell line, PC-3. Conversely, knockdown of endogenous NOX5 in UACC-257 cells resulted in decreased cell growth, decreased HIF-1α expression, and increased p27Kip1 expression. Likewise, in an additional human melanoma cell line, WM852, and in PC-3 cells, transient knockdown of endogenous NOX5 resulted in increased p27Kip1 and decreased HIF-1α expression. Knockdown of endogenous NOX5 in UACC-257 cells resulted in decreased Akt and GSK3ß phosphorylation, signaling pathways known to modulate p27Kip1 levels. In summary, our findings suggest that NOX5 expression in human UACC-257 melanoma cells could contribute to cell proliferation due, in part, to the generation of high local concentrations of extracellular ROS that modulate multiple pathways that regulate HIF-1α and networks that signal through Akt/GSK3ß/p27Kip1 .

Characterization of potent and selective iodonium-class inhibitors of NADPH oxidases.

The NADPH oxidases (NOXs) play a recognized role in the development and progression of inflammation-associated disorders, as well as cancer. To date, several NOX inhibitors have been developed, through either high throughput screening or targeted disruption of NOX interaction partners, although only a few have reached clinical trials. To improve the efficacy and bioavailability of the iodonium class NOX inhibitor diphenylene iodonium (DPI), we synthesized 36 analogs of DPI, focusing on improved solubility and functionalization. The inhibitory activity of the analogs was interrogated through cell viability and clonogenic studies with a colon cancer cell line (HT-29) that depends on NOX for its proliferative potential. Lack of altered cellular respiration at relevant iodonium analog concentrations was also demonstrated. Additionally, inhibition of ROS generation was evaluated with a luminescence assay for superoxide, or by Amplex Red® assay for H2O2 production, in cell models expressing specific NOX isoforms. DPI and four analogs (NSCs 740104, 751140, 734428, 737392) strongly inhibited HT-29 cell growth and ROS production with nanomolar potency in a concentration-dependent manner. NSC 737392 and 734428, which both feature nitro functional groups at the meta position, had >10-fold higher activity against ROS production by cells that overexpress dual oxidase 2 (DUOX2) than the other compounds examined (IC50≈200-400nM). Based on these results, we synthesized and tested NSC 780521 with optimized potency against DUOX2. Iodonium analogs with anticancer activity, including the first generation of targeted agents with improved specificity against DUOX2, may provide a novel therapeutic approach to NOX-driven tumors.

Decoding NADPH oxidase 4 expression in human tumors.

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-ß1 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.

Interleukin-4 and interleukin-13 increase NADPH oxidase 1-related proliferation of human colon cancer cells.

Human colon cancers express higher levels of NADPH oxidase 1 [NOX1] than adjacent normal epithelium. It has been suggested that reactive oxygen species [ROS] derived from NOX1 contribute to DNA damage and neoplastic transformation in the colon, particularly during chronic inflammatory stress. However, the mechanism(s) underlying increased NOX1 expression in malignant tumors or chronic inflammatory states involving the intestine are poorly characterized. We examined the effects of two pro-inflammatory cytokines, IL-4 and IL-13, on the regulation of NOX1. NOX1 expression was increased 4- to 5-fold in a time- and concentration-dependent manner by both cytokines in human colon cancer cell lines when a functional Type II IL-4 receptor was present. Increased NOX1 transcription following IL-4/IL-13 exposure was mediated by JAK1/STAT6 signaling, was associated with a ROS-related inhibition of protein tyrosine phosphatase activity, and was dependent upon activation and specific binding of GATA3 to the NOX1 promoter. NOX1-mediated ROS production increased cell cycle progression through S-phase leading to a significant increase in cellular proliferation. Evaluation of twenty pairs of surgically-resected colon cancers and their associated uninvolved adjacent colonic epithelium demonstrated a significant increase in the active form of NOX1, NOX1-L, in tumors compared to normal tissues, and a significant correlation between the expression levels of NOX1 and the Type II IL-4 receptor in tumor and the uninvolved colon. These studies imply that NOX1 expression, mediated by IL-4/IL-13, could contribute to an oxidant milieu capable of supporting the initiation or progression of colonic cancer, suggesting a role for NOX1 as a therapeutic target.

NADPH oxidase 1 supports proliferation of colon cancer cells by modulating reactive oxygen species-dependent signal transduction.

Reactive oxygen species (ROS) play a critical role in cell signaling and proliferation. NADPH oxidase 1 (NOX1), a membrane-bound flavin dehydrogenase that generates O2̇̄, 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.

Dual oxidase 2 and pancreatic adenocarcinoma: IFN-γ-mediated dual oxidase 2 overexpression results in H2O2-induced, ERK-associated up-regulation of HIF-1α and VEGF-A.

Several NADPH oxidase family members, including dual oxidase 2 [DUOX2], are expressed in human tumors, particularly gastrointestinal cancers associated with long-standing chronic inflammation. We found previously that exposure of pancreatic ductal adenocarcinoma cells to the pro-inflammatory cytokine IFN-γ increased DUOX2 expression (but not other NADPH oxidases) leading to long-lived H2O2 production. To elucidate the pathophysiology of DUOX2-mediated H2O2 formation in the pancreas further, we demonstrate here that IFN-γ-treated BxPC-3 and CFPAC-1 pancreatic cancer cells (known to increase DUOX2 expression) produce significant levels of intracellular oxidants and extracellular H2O2 which correlate with concomitant up-regulation of VEGF-A and HIF-1α transcription. These changes are not observed in the PANC-1 line that does not increase DUOX2 expression following IFN-γ treatment. DUOX2 knockdown with short interfering RNA significantly decreased IFN-γ-induced VEGF-A or HIF-1α up-regulation, as did treatment of pancreatic cancer cells with the NADPH oxidase inhibitor diphenylene iodonium, the multifunctional reduced thiol N-acetylcysteine, and the polyethylene glycol-modified form of the hydrogen peroxide detoxifying enzyme catalase. Increased DUOX2-related VEGF-A expression appears to result from reactive oxygen-mediated activation of ERK signaling that is responsible for AP-1-related transcriptional effects on the VEGF-A promoter. To clarify the relevance of these observations in vivo, we demonstrate that many human pre-malignant pancreatic intraepithelial neoplasms and frank pancreatic cancers express substantial levels of DUOX protein compared to histologically normal pancreatic tissues, and that expression of both DUOX2 and VEGF-A mRNAs is significantly increased in surgically-resected pancreatic cancers compared to the adjacent normal pancreas.

NADPH oxidases and cancer.

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.

NADPH oxidases: a perspective on reactive oxygen species production in tumor biology.

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, membrane-bound 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.

Characterization of NADPH oxidase 5 expression in human tumors and tumor cell lines with a novel mouse monoclonal antibody.

Reactive oxygen species generated by NADPH oxidase 5 (Nox5) have been implicated in physiological and pathophysiological signaling pathways, including cancer development and progression. However, because immunological tools are lacking, knowledge of the role of Nox5 in tumor biology has been limited; the expression of Nox5 protein across tumors and normal tissues is essentially unknown. Here, we report the characterization and use of a mouse monoclonal antibody against a recombinant Nox5 protein (bp 600-746) for expression profiling of Nox5 in human tumors by tissue microarray analysis. Using our novel antibody, we also report the detection of endogenous Nox5 protein in human UACC-257 melanoma cells. Immunofluorescence, confocal microscopy, and immunohistochemical techniques were employed to demonstrate Nox5 localization throughout UACC-257 cells, with perinuclear enhancement. Tissue microarray analysis revealed, for the first time, substantial Nox5 overexpression in several human cancers, including those of prostate, breast, colon, lung, brain, and ovary, as well as in malignant melanoma and non-Hodgkin lymphoma; expression in most nonmalignant tissues was negative to weak. This validated mouse monoclonal antibody will promote further exploration of the functional significance of Nox5 in human pathophysiology, including tumor cell growth and proliferation.

Functional activity and tumor-specific expression of dual oxidase 2 in pancreatic cancer cells and human malignancies characterized with a novel monoclonal antibody.

Dual oxidase 2 (Duox2), one of the seven members of the NADPH oxidase gene family, plays a critical role in generating H2O2 for thyroid hormone biosynthesis and as an integral part of the host defense system of the respiratory epithelium and the gastrointestinal tract. Recent evidence suggests that the regulation of Duox2 expression is under the control of pro-inflammatory cytokines and that Duox2-induced reactive oxygen species (ROS) contribute to the inflammation-related tissue injury that occurs in two pre-malignant, inflammatory conditions: chronic pancreatitis and inflammatory bowel disease. Because no reliable Duox antibodies are commercially available, we report the development of a murine monoclonal antibody (MAb) to Duox2 (clone Duox S-12) and its use for the characterization of Duox2 expression in human tumors, tumor cell lines and normal tissues. Duox S-12 specifically detected both endogenously- and ectopically-expressed Duox2 protein by immunoblotting, immunofluorescence microscopy and immunohistochemistry (where both membranous and cytoplasmic staining were present). Duox2 expression detected by Duox S-12 was functionally coupled to the generation of H(2)O(2) in pancreatic cancer cells that expressed Duox2 and its cognate maturation factor DuoxA2. Although Duox S-12 recognizes ectopically expressed Duox1 protein because of the extensive amino acid homology between Duox1 and Duox2, the lack of substantial Duox1 mRNA expression in human tumors (except thyroid cancer) allowed us to evaluate Duox2 expression across a wide range of normal and malignant tissues by immuno-histochemistry. Duox2 was expressed at elevated levels in many human cancers, most notably tumors of the prostate, lung, colon and breast while brain tumors and lymphomas demonstrated the lowest frequency of expression. The Duox-specific monoclonal antibody described here provides a promising tool for the further examination of the role of Duox-dependent reactive oxygen production in inflammation-related carcinogenesis, where alterations in oxidant tone play a critical role in cell growth and proliferation.

Effects of iodonium-class flavin dehydrogenase inhibitors on growth, reactive oxygen production, cell cycle progression, NADPH oxidase 1 levels, and gene expression in human colon cancer cells and xenografts.

Iodonium-class flavoprotein dehydrogenase inhibitors have been demonstrated to possess antiproliferative potential and to inhibit reactive oxygen production in human tumor cells, although the mechanism(s) that explains the relationship between altered cell growth and the generation of reactive oxygen species (ROS) remains an area of active investigation. Because of the ability of these compounds to inhibit the activity of flavoprotein-containing epithelial NADPH oxidases, we chose to examine the effects of several iodonium-class flavoprotein inhibitors on human colon cancer cell lines that express high, functional levels of a single such oxidase (NADPH oxidase 1, or Nox1). We found that diphenyleneiodonium (DPI), di-2-thienyliodonium (DTI), and iodonium diphenyl inhibited the growth of Caco2, HT-29, and LS-174T colon cancer cells at concentrations (10-250nM for DPI, 0.5-2.5µM for DTI, and 155nM to 10µM for iodonium diphenyl) substantially lower than needed for DU145 human prostate cancer cells, which do not possess functional NADPH oxidase activity. Drug treatment was associated with decreased H2O2 production and diminished intracellular ROS levels, lasting up to 24h, after short-term (1-h) exposure to the iodonium analogs. Decreased tumor cell proliferation was caused, in part, by a profound block in cell cycle progression at the G1/S interface in both LS-174T and HT-29 cells exposed to either DPI or DTI; and the G1 block was produced, for LS-174T cells, by upregulation of p27 and a drug concentration-related decrease in the expression of cyclins D1, A, and E that was partially prevented by exogenous H2O2. Not only did DPI and DTI decrease intracellular ROS, they both also significantly decreased the mRNA expression levels of Nox1, potentially contributing to the prolonged reduction in tumor cell reactive oxygen levels. We also found that DPI and DTI significantly decreased the growth of both HT-29 and LS-174T human tumor xenografts, at dose levels that produced peak plasma concentrations similar to those utilized for our in vitro experiments. These findings suggest that iodonium analogs have therapeutic potential for NADPH oxidase-containing human colon cancers in vivo and that at least part of their antineoplastic mechanism of action may be related to targeting Nox1.

Activation of TLR4 is required for the synergistic induction of dual oxidase 2 and dual oxidase A2 by IFN-γ and lipopolysaccharide in human pancreatic cancer cell lines.

Pancreatitis is associated with release of proinflammatory cytokines and reactive oxygen species and plays an important role in the development of pancreatic cancer. We recently demonstrated that dual oxidase (Duox)2, an NADPH oxidase essential for reactive oxygen species-related, gastrointestinal host defense, is regulated by IFN-γ-mediated Stat1 binding to the Duox2 promoter in pancreatic tumor lines. Because LPS enhances the development and invasiveness of pancreatic cancer in vivo following 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 small interfering RNAs, as well as 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 H(2)O(2) generated by exposure to both LPS and IFN-γ was responsible for an ∼50% decrease in BxPC-3 cell proliferation associated with a G(1) cell cycle block, apoptosis, and DNA damage. We also demonstrated upregulation 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.

Antiproliferative mechanisms of action of the flavin dehydrogenase inhibitors diphenylene iodonium and di-2-thienyliodonium based on molecular profiling of the NCI-60 human tumor cell panel.

Flavoprotein-dependent reactive oxygen species (ROS) play a critical role in cytokine-mediated signal transduction in normal tissues and tumor cells. The flavoenzyme inhibitors diphenylene iodonium (DPI) and di-2-thienyliodonium (DTI) have been used to inhibit membrane-bound, flavoprotein-containing NADPH oxidases, including epithelial and leukocyte NADPH oxidases (Nox1-5 and Duox 1 and 2). Recent evidence suggests that DPI can decrease tumor cell proliferation; however, the molecular mechanisms involved remain poorly defined. To explore the mechanisms underlying DPI- and DTI-related tumor cell growth delay, we examined growth inhibition patterns produced by both agents in the NCI-60 tumor panel, and determined expression levels of Nox gene family members across these cell lines. Possible molecular targets were predicted using the COMPARE program. DPI was more potent than DTI (GI(50): 10nM versus 10µM); DPI and DTI exposure produced unique patterns of growth inhibition when evaluated against the small molecule anticancer database of the National Cancer Institute. Growth inhibition profiling of DPI revealed a modest positive correlation with Nox1 levels; novel mechanisms of DPI and DTI action, including alterations in Stat, Erk1/2, and Akt pathways, were inferred by correlation with NCI-60 Affymetrix(®) array data. Exposure of HT-29 colon cancer cells, which express Nox1, to DPI and DTI confirmed their inhibitory effects on steady state ROS levels, and demonstrated decreased Stat, Erk1/2, and Akt signaling mediated by IL-4, IL-6, IL-13, and IL-22, possibly due to a concomitant increase in tumor cell phosphatase activity. These findings suggest that DPI and DTI may act therapeutically by altering ROS-related signal transduction.

Up-regulation and sustained activation of Stat1 are essential for interferon-gamma (IFN-gamma)-induced dual oxidase 2 (Duox2) and dual oxidase A2 (DuoxA2) expression in human pancreatic cancer cell lines.

Dual oxidase 2 is a member of the NADPH oxidase (Nox) gene family that plays a critical role in the biosynthesis of thyroid hormone as well as in the inflammatory response of the upper airway mucosa and in wound healing, presumably through its ability to generate reactive oxygen species, including H2O2. The recently discovered overexpression of Duox2 in gastrointestinal malignancies, as well as our limited understanding of the regulation of Duox2 expression, led us to examine the effect of cytokines and growth factors on Duox2 in human tumor cells. We found that exposure of human pancreatic cancer cells to IFN-γ (but not other agents) produced a profound up-regulation of the expression of Duox2, and its cognate maturation factor DuoxA2, but not other members of the Nox family. Furthermore, increased Duox2/DuoxA2 expression was closely associated with a significant increase in the production of both intracellular reactive oxygen species and extracellular H2O2. Examination of IFN-γ-mediated signaling events demonstrated that in addition to the canonical Jak-Stat1 pathway, IFN-γ activated the p38-MAPK pathway in pancreatic cancer cells, and both played an important role in the induction of Duox2 by IFN-γ. Duox2 up-regulation following IFN-γ exposure is also directly associated with the binding of Stat1 to elements of the Duox2 promoter. Our findings suggest that the pro-inflammatory cytokine IFN-γ initiates a Duox2-mediated reactive oxygen cascade in human pancreatic cancer cells; reactive oxygen species production in this setting could contribute to the pathophysiologic characteristics of these tumors.

Quantitative high-throughput screening identifies inhibitors of anthrax-induced cell death.

Here, we report the results of a quantitative high-throughput screen (qHTS) measuring the endocytosis and translocation of a beta-lactamase-fused-lethal factor and the identification of small molecules capable of obstructing the process of anthrax toxin internalization. Several small molecules protect RAW264.7 macrophages and CHO cells from anthrax lethal toxin and protected cells from an LF-Pseudomonas exotoxin fusion protein and diphtheria toxin. Further efforts demonstrated that these compounds impaired the PA heptamer pre-pore to pore conversion in cells expressing the CMG2 receptor, but not the related TEM8 receptor, indicating that these compounds likely interfere with toxin internalization.