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.

Chronic Exposure to HIV-Derived Protein Tat Impairs Endothelial Function via Indirect Alteration in Fat Mass and Nox1-Mediated Mechanisms in Mice

People living with human immunodeficiency virus (HIV) (PLWH) have increased risk for atherosclerosis-related cardiovascular disease (CVD), the main cause of death in this population. Notwithstanding, the mechanisms of HIV-associated vascular pathogenesis are not fully elucidated. Therefore, we sought to determine whether HIV-regulatory protein Tat mediates HIV-induced endothelial dysfunction via NADPH oxidase 1 (Nox1)-dependent mechanisms. Body weight, fat mass, leptin levels, expression of reactive oxygen species (ROS)-producing enzymes and vascular function were assessed in C57BL/6 male mice treated with Tat for 3 days and 4 weeks. Aortic rings and human endothelial cells were also treated with Tat for 2–24 h in ex vivo and in vitro settings. Chronic (4 weeks) but not acute (3 days and 2–24 h) treatment with Tat decreased body weight, fat mass, and leptin levels and increased the expression of Nox1 and its coactivator NADPH oxidase Activator 1 (NoxA1). This was associated with impaired endothelium-dependent vasorelaxation. Importantly, specific inhibition of Nox1 with GKT771 and chronic leptin infusion restored endothelial function in Tat-treated mice. These data rule out direct effects of HIV-Tat on endothelial function and imply the contribution of reductions in adipose mass and leptin production which likely explain upregulated expression of Nox1 and NoxA1. The Nox1 and leptin system may provide potential targets to improve vascular function in HIV infection-associated CVD.

Abstract P280: CCL5 Causes Vascular Injury Via CCR5 And Nadph Oxidase 1- Derived Ros

Chemokine (C-Cmotif) ligand 5 (CCL5) and its receptor CCR5 belong to the family of chemokines and are expressed and active in the vasculature. NADPH oxidases (Noxs) are the major source of reactive oxygen species (ROS) in vascular cells, but whether the activation of these oxidases is CCL5/CCR5 sensitive and whether such interaction participates in the genesis of vascular disease is not fully known. We investigated whether CCL5/CCR5 leads to vascular injury by activating Nox1. Carotid ligation model (CL, for 2-weeks) was used to induce pathological vascular growth in 10-weeks old (C57BL6/J) mice. Rat aortic smooth muscle cells (RASMC) were treated with recombinant CCL5 (100ng/mL) to study the molecular mechanisms. CL induces neointima formation, which was associated with increase in IL1β, TNFα, CCR3, CCR5 (3-fold increase), CCL5, and Nox1 gene expression. No difference was observed for Nox2 and 4. Treatment with CCR5 blocker (maraviroc, 25mg/Kg/day i.p) partially inhibited CL-induced vascular injury (media/intima ratio, CL: 1.2 ± 0.2 vs CL + maraviroc: 0.7 ± 0.2) and Nox1 expression (Fold changes: CL: 2.1 ± 0.4 vs CL + maraviroc: 1.2 ± 0.4). In RASMC, CCL5 induced Nox1 expression, which was blunted by pre-treating cells with maraviroc (10uM). Also, it increases p47phox content in membrane fraction (index of Nox activation), and elevated ROS production, analyzed by L012. CCL5 also induced cell migration, measured by transwell assay (number of cells per spot, control: 21.3 ± 3.1 vs CCL5: 31.1 ± 2.4), proliferation, analyzed by Edu+ cells (% of cells per spot, control: 10.6 ± 4.3 vs CCL5: 22.8 ± 5.1), and inflammation (studied by IL1β and TNFα levels). Lastly, CCL5 elevated NF-κB translocation into the nucleus, indicating NF-κB activation. Strikingly, inhibition of Nox1 (GKT771, 10uM), blocked CCL5-induced vascular migration, proliferation, and inflammation, as well as NF-κB activation. We propose that CCL5 activates Nox1 in the vasculature leading to local injury characterized by vascular inflammation and cellular migration and proliferation, perhaps by activating NF-κB signaling. Herein, we place CCR5 signaling as possible therapeutic target to reduce the cardiovascular risk in inflammatory diseases associated with dysregulation of CCL5 and/or CCR5

Astaxanthin Inhibits Interleukin-6 Expression in Cerulein/Resistin-Stimulated Pancreatic Acinar Cells

Acute pancreatitis is a common clinical condition with increasing the proinflammatory mediators, including interleukin-6 (IL-6). Obesity is a negative prognostic factor in acute pancreatitis. Obese patients with acute pancreatitis have a higher systemic inflammatory response rate. Levels of serum resistin, an adipocytokine secreted by fat tissues, increase with obesity. Cerulein, a cholecystokinin analog, induces calcium (Ca2+) overload, oxidative stress, and IL-6 expression in pancreatic acinar cells, which are hallmarks of acute pancreatitis. A recent study showed that resistin aggravates the expression of inflammatory cytokines in cerulein-stimulated pancreatic acinar cells. We aimed to investigate whether resistin amplifies cerulein-induced IL-6 expression and whether astaxanthin (ASX), an antioxidant carotenoid with anti-inflammatory properties, inhibits ceruelin/resistin-induced IL-6 expression in pancreatic acinar AR42J cells. We found that resistin enhanced intracellular Ca2+ levels, NADPH oxidase activity, intracellular reactive oxygen species (ROS) production, NF-κB activity, and IL-6 expression in cerulein-stimulated AR42J cells, which were inhibited by ASX in a dose-dependent manner. The calcium chelator BAPTA-AM inhibited cerulein/resistin-induced NADPH oxidase activation and ROS production. Antioxidant N-acetyl cysteine (NAC) and ML171, a specific NADPH oxidase 1 inhibitor, suppressed cerulein/resistin-induced ROS production, NF-κB activation, and IL-6 expression. In conclusion, ASX inhibits IL-6 expression, by reducing Ca2+ overload, NADPH oxidase-mediated ROS production, and NF-κB activity in cerulein/resistin-stimulated pancreatic acinar cells. Consumption of ASX-rich foods could be beneficial for preventing or delaying the incidence of obesity-associated acute pancreatitis.

Protein Disulphide Isomerase and NADPH Oxidase 1 Cooperate to Control Platelet Function and Are Associated with Cardiometabolic Disease Risk Factors

Background: Protein disulphide isomerase (PDI) and NADPH oxidase 1 (Nox-1) regulate platelet function and reactive oxygen species (ROS) generation, suggesting potentially interdependent roles. Increased platelet reactivity and ROS production have been correlated with cardiometabolic disease risk factors. Objectives: To establish whether PDI and Nox-1 cooperate to control platelet function. Methods: Immunofluorescence microscopy was utilised to determine expression and localisation of PDI and Nox-1. Platelet aggregation, fibrinogen binding, P-selectin exposure, spreading and calcium mobilization were measured as markers of platelet function. A cross-sectional population study (n = 136) was conducted to assess the relationship between platelet PDI and Nox-1 levels and cardiometabolic risk factors. Results: PDI and Nox-1 co-localized upon activation induced by the collagen receptor GPVI. Co-inhibition of PDI and Nox-1 led to additive inhibition of GPVI-mediated platelet aggregation, activation and calcium flux. This was confirmed in murine Nox-1−/− platelets treated with PDI inhibitor bepristat, without affecting bleeding. PDI and Nox-1 together contributed to GPVI signalling that involved the phosphorylation of p38 MAPK, p47phox, PKC and Akt. Platelet PDI and Nox-1 levels were upregulated in obesity, with platelet Nox-1 also elevated in hypertensive individuals. Conclusions: We show that PDI and Nox-1 cooperate to control platelet function and are associated with cardiometabolic risk factors.