Enhanced Reactive Oxygen Species Production Research Articles

C-Type Natriuretic Peptide Induces Anti-contractile Effect Dependent on Nitric Oxide, Oxidative Stress, and NPR-B Activation in Sepsis.

Aims: To evaluate the role of nitric oxide, reactive oxygen species (ROS), and natriuretic peptide receptor-B activation in C-type natriuretic peptide-anti-contractile effect on Phenylephrine-induced contraction in aorta isolated from septic rats.Methods and Results: Cecal ligation and puncture (CLP) surgery was used to induce sepsis in male rats. Vascular reactivity was conducted in rat aorta and resistance mesenteric artery (RMA). Measurement of survival rate, mean arterial pressure (MAP), plasma nitric oxide, specific protein expression, and localization were evaluated. Septic rats had a survival rate about 37% at 4 h after the surgery, and these rats presented hypotension compared to control-operated (Sham) rats. Phenylephrine-induced contraction was decreased in sepsis. C-type natriuretic peptide (CNP) induced anti-contractile effect in aortas. Plasma nitric oxide was increased in sepsis. Nitric oxide-synthase but not natriuretic peptide receptor-B expression was increased in septic rat aortas. C-type natriuretic peptide-anti-contractile effect was dependent on nitric oxide-synthase, ROS, and natriuretic peptide receptor-B activation. Natriuretic peptide receptor-C, protein kinase-Cα mRNA, and basal nicotinamide adenine dinucleotide phosphate (NADPH)-dependent ROS production were lower in septic rats. Phenylephrine and CNP enhanced ROS production. However, stimulated ROS production was low in sepsis.Conclusion: CNP induced anti-contractile effect on Phenylephrine contraction in aortas from Sham and septic rats that was dependent on nitric oxide-synthase, ROS, and natriuretic peptide receptor-B activation.

Highly effective antibacterial activity and synergistic effect of Ag-MgO nanocomposite against Escherichia coli

Ag-MgO nanocomposite, synthesized by loading small-sized Ag nanoparticles on the surface of MgO nanoparticles, was firstly used as an effective antibacterial agent against Escherichia coli. The microstructure, morphology and composition of the nanocomposite were examined by X-ray diffraction (XRD), transmission electron microscopy (TEM), scanning electron microscopy (SEM) equipped with an energy-dispersive X-ray (EDX) analyzer, X-ray photoelectron spectroscopy (XPS), Brunauer–Emmett–Teller (BET) and flame atomic emission spectroscopy (FAES) measurements. An interfacial interaction between Ag and MgO was confirmed by XRD and XPS analyses, which could effectively inhibit the release of silver ions (Ag+) from the nanocomposite. Antibacterial tests showed that the as-prepared Ag-MgO nanocomposite exhibited stronger antibacterial activity against Escherichia coli, compared to pure MgO and equivalent Ag nanoparticles alone, indicating a synergistic effect between Ag and MgO. Based on the results of reactive oxygen species (ROS) detection, the enhanced antibacterial activity of Ag-MgO nanocomposite was attributed to the promotion of ROS production, because Ag particles on the surface of MgO are in favor of electron transfer, which could enhance ROS production via one-electron reduction of oxygen. Taking into consideration easy preparation, low cost, low release rate of Ag+, and high antibacterial activity of Ag-MgO nanocomposite, it is a very promising candidate material for bacterial disinfection.

Superoxide dismutase 2 overexpression alleviates maternal diabetes-induced neural tube defects, restores mitochondrial function and suppresses cellular stress in diabetic embryopathy

Pregestational diabetes disrupts neurulation leading to neural tube defects (NTDs). Oxidative stress resulting from reactive oxygen species (ROS) plays a central role in the induction of NTD formation in diabetic pregnancies. We aimed to determine whether mitochondrial dysfunction increases ROS production leading to oxidative stress and diabetic embryopathy. Overexpression of the mitochondrion-specific antioxidant enzyme superoxide dismutase 2 (SOD2) in a transgenic (Tg) mouse model significantly reduced maternal diabetes-induced NTDs. SOD2 overexpression abrogated maternal diabetes-induced mitochondrial dysfunction by inhibiting mitochondrial translocation of the pro-apoptotic Bcl-2 family members, reducing the number of defective mitochondria in neuroepithelial cells, and decreasing mitochondrial membrane potential. Furthermore, SOD2 overexpression blocked maternal diabetes-increased ROS production by diminishing dihydroethidium staining signals in the developing neuroepithelium, and reducing the levels of nitrotyrosine-modified proteins and lipid hydroperoxide level in neurulation stage embryos. SOD2 overexpression also abolished maternal diabetes-induced endoplasmic reticulum stress. Finally, caspase-dependent neuroepithelial cell apoptosis enhanced by oxidative stress was significantly reduced by SOD2 overexpression. Thus, our findings support the hypothesis that mitochondrial dysfunction in the developing neuroepithelium enhances ROS production, which leads to oxidative stress and endoplasmic reticulum (ER) stress. SOD2 overexpression blocks maternal diabetes-induced oxidative stress and ER stress, and reduces the incidence of NTDs in embryos exposed to maternal diabetes.

Enhancement of anti-tumor effects of 5-fluorouracil on hepatocellular carcinoma by low-intensity ultrasound.

BackgroundHepatocellular carcinoma (HCC) accounts for 75 % of liver cancers and is the second most lethal cancer, associated with its multiple etiologies, poor prognosis and resistance to chemotherapy drugs. Chemotherapy treatment on HCC suffers low efficacy of drug uptake and can produce a range of side effects. Here we report an investigation on the effect of a combined treatment on human hepatocellular carcinoma BEL-7402 cells using low-intensity ultrasound (US) and 5-fluorouracil (5-FU).MethodsThe uptake of 5-FU was measured by the high-performance liquid chromatography (HPLC). DNA damage was detected by the comet assay. MTT assay was used to examine cell viability. Intracellular reactive oxygen species (ROS) and mitochondrial membrane potential (Δψm) were respectively detected by the fluorescent probes DCFH-DA or JC-1. Endogenous apoptosis-associated proteins were analyzed by the western blot and immunohistochemistry. Histopathological changes were evaluated by the hematoxylin and eosin (H&E) staining. Cell apoptosis was evaluated by the TUNEL and flow cytometry assays. Cell proliferation was measured using the immunohistochemical staining of PCNA.ResultsOur results showed that low-intensity US (1.1 MHz, 1.0 W/cm2, 10 % duty cycle) significantly enhanced the uptake of 5-FU, 5-FU-mediated DNA damage and reactive oxygen species (ROS) generation. The increased ROS production up-regulated the p53 protein level, which led to the up-regulation of Bax and down-regulation of Bcl-2. The enhancement of ROS generation and the activation of the apoptosis-associated proteins further triggered the collapse of mitochondrial membrane potential, released cytochrome c from mitochondria into cytosol and activated the mitochondria-caspase pathway, and cell apoptosis. Such enhanced effects could be partially blocked by the ROS scavenger N-acetylcysteine (NAC). Overall, low-intensity US combined with 5-FU led to an effective inhibition of tumor growth and prolonged overall survival of BEL-7402 HCC-bearing nude mice by more than 15 % compared with 5-FU treatment alone.ConclusionsOur results showed that low-intensity ultrasound combined with 5-FU produced much enhanced synergistic anti-tumor effects via enhanced ROS production in treating HCC.Electronic supplementary materialThe online version of this article (doi:10.1186/s13046-016-0349-4) contains supplementary material, which is available to authorized users.

Metformin Modulation of Dichloroacetate‐Induced Oxidative Stress and its Impact on Mitochondrial Integrity in VM‐M3 Glioblastoma Cells

There is accumulating evidence that altered mitochondrial function in cancer contributes to a metabolic phenotype that confers a survival advantage towards cancer cells. A hallmark of this phenotype is increased reactive oxygen species (ROS) production often coupled with upregulated antioxidant defense. This redox balance is an intriguing therapeutic target as many traditional chemotherapeutics elicit cytotoxicity through enhanced ROS production. Dichloroacetate (DCA), an inhibitor of pyruvate dehydrogenase kinase, promotes pyruvate flux into the mitochondria and oxidative metabolism consequently enhancing ROS production. As an adjuvant, DCA is shown to enhance cytotoxicity and reverse resistance to a number of chemotherapeutic agents through induction of oxidative stress. Dual targeting of glycolytic and mitochondrial metabolism in cancer may elicit a greater therapeutic response than either alone. Metformin, a first line therapy for type II diabetes mellitus, inhibits complex I of the electron transport chain (ETC), disrupts oxidative phosphorylation and subsequently increases glycolytic metabolism. As such, we investigated the impact of metformin on DCA cytotoxicity in VM‐M3 glioblastoma cells. We propose that metformin insult to the ETC will augment DCA‐induced ROS production and increase oxidative stress, thus enhancing cytotoxicity. We evaluated mitochondrial membrane potential (DYm) and superoxide production through confocal fluorescence microscopy as well as cytochrome c localization through immunofluorescence. We demonstrate that DCA and metformin independently promote dissipation of DYm (p<0.01) and that metformin treatment reduces superoxide production compared to untreated VM‐M3 cells (p<0.01). DCA increases superoxide production in a concentration dependent manner and co‐treatment with metformin reverses its reduction of ROS production (p<0.05). Moreover, the combination exhibits enhanced cytotoxicity towards VM‐M3 cells (p<0.05). These findings support continued investigation of DCA and metformin in combination as a potential anti‐cancer therapy.

MP90-03 REACTIVE OXYGEN SPECIES INDUCTION BY CABAZITAXEL VIA INHIBITION OF THE SESTRIN FAMILY IN CASTRATION-RESISTANT PROSTATE CANCER

You have accessJournal of UrologyProstate Cancer: Basic Research & Pathophysiology IV1 Apr 2016MP90-03 REACTIVE OXYGEN SPECIES INDUCTION BY CABAZITAXEL VIA INHIBITION OF THE SESTRIN FAMILY IN CASTRATION-RESISTANT PROSTATE CANCER Takeo Kosaka, Hiroshi Hongo, Yasumasa Miyazaki, Eiji Kikuchi, Akira Miyajima, and Mototsugu Oya Takeo KosakaTakeo Kosaka More articles by this author , Hiroshi HongoHiroshi Hongo More articles by this author , Yasumasa MiyazakiYasumasa Miyazaki More articles by this author , Eiji KikuchiEiji Kikuchi More articles by this author , Akira MiyajimaAkira Miyajima More articles by this author , and Mototsugu OyaMototsugu Oya More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2016.02.2547AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Cabazitaxel (CBZ) has become the most effective cytotoxic agent to demonstrate an improvement in survival in men with docetaxel-refractory CRPC. Accumulating evidence has suggested reactive oxygen species (ROS) production induced by taxanes in cancer cells may influence taxane-induced cell death or drug resistance. However, ROS production by CBZ or its mechanistic action remains to be characterized in CRPC. In this study, we investigated PCa cell sensitivity to docetaxel (DTX) or CBZ by measurement of ROS production. METHODS Three cell lines were used: LNCaP, a human PCa cell line that exhibits androgen-dependent proliferation; C4-2, a human PCa cell line that exhibits androgen-independent proliferation; and C4-2AT6, a CRPC cell line derived from C4-2. These cells were treated with increasing concentrations of docetaxel or CBZ in vitro. Gene expression profiles of CBZ- or DTX-treated cells were analyzed using a whole human genome microarray. FACS analysis was performed using a fluorogenic marker of ROS. Furthermore, we investigated the changes in expression of antioxidant enzymes induced by DTX and CBZ. RESULTS Cabazitaxel showed significantly higher cytotoxicity than docetaxel in human CRPC cells. FACS analysis revealed that treatment with cabazitaxel significantly increased ROS generation than docetaxel in C4-2AT6 cells, accompanied by higher expression of phospho-p38. Microarray analysis revealed significant changes in ROS regulation-related genes by CBZ. qPCR analysis revealed CBZ induced C4-2AT6 cell death, accompanied by significantly decreased expression of Sestrin-3 (SESN3). To investigate whether CBZ-mediated cell death was caused by induction of ROS generation, C4-2AT6 cells were incubated with CBZ in the presence of the antioxidant N-acetylcysteine (NAC), which was observed to reduce cabazitaxel-induced cytotoxicity. Microarray analysis revealed dynamic changes in ROS regulation-related genes by CBZ, and qPCR analysis demonstrated CBZ reduced mRNA expression of SESN3. We observed that ROS reduction by SESN3 was significantly inhibited by cabazitaxel in a dose-dependent manner, but not by docetaxel. When C4-2AT6 cells were treated with cabazitaxel in the presence of siRNAs against SESN3, significant cytotoxicity was observed, accompanied by enhanced ROS production. CONCLUSIONS Our results indicated that the higher sensitivity of human CRPC to CBZ, compared with DTX, involves ROS production via inhibition of SESN3 expression, an antioxidant enzyme. © 2016FiguresReferencesRelatedDetails Volume 195Issue 4SApril 2016Page: e1145 Advertisement Copyright & Permissions© 2016MetricsAuthor Information Takeo Kosaka More articles by this author Hiroshi Hongo More articles by this author Yasumasa Miyazaki More articles by this author Eiji Kikuchi More articles by this author Akira Miyajima More articles by this author Mototsugu Oya More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Lactobacillus rhamnosus GG conditioned media modulates acute reactive oxygen species and nitric oxide in J774 murine macrophages

Phagocytes such as macrophages are capable of detecting and killing pathogenic bacteria by producing reactive oxygen and nitrogen species. Formation of free radicals in macrophages may be regulated by probiotics or by factors released by probiotics but yet to be identified. Thus, studies were carried out to determine whether cell-free conditioned medium obtained from cultures of Lactobacillus rhamnosus GG (LGG-CM) regulate production of reactive oxygen species (ROS) and/or nitric oxide (NO) in macrophages. J774 macrophages in culture were loaded with either H2DCFDA for monitoring ROS or with DAFFM-DA for NO detection. Free radical production was measured on a fluorescence microplate reader and changes were analysed by Cumulative sum (CuSum) calculations. Low concentration of LGG-CM (10% LGG-CM) or LPS did not cause any significant change in basal levels of ROS or NO production. In contrast, high concentration of LGG-CM (75% and 100%) significantly enhanced ROS generation but also significantly reduced NO level. These findings are novel and suggest for the first time that probiotics may release factors in culture which enhance ROS production and may additionally reduce deleterious effects associated with excessive nitrogen species by suppressing NO level. These events may account, in part, for the beneficial bactericidal and anti-inflammatory actions ascribed to probiotics and may be of clinical relevance.

Impaired mitochondrial function due to familial Alzheimer's disease-causing presenilins mutants via Ca2+ disruptions

Mutants in presenilins (PS1 or PS2) is the major cause of familial Alzheimer's disease (FAD). FAD causing PS mutants affect intracellular Ca2+ homeostasis by enhancing the gating of inositol trisphosphate (IP3) receptor (IP3R) Ca2+ release channel on the endoplasmic reticulum, leading to exaggerated Ca2+ release into the cytoplasm. Using experimental IP3R-mediated Ca2+ release data, in conjunction with a computational model of cell bioenergetics, we explore how the differences in mitochondrial Ca2+ uptake in control cells and cells expressing FAD-causing PS mutants affect key variables such as ATP, reactive oxygen species (ROS), NADH, and mitochondrial Ca2+. We find that as a result of exaggerated cytosolic Ca2+ in FAD-causing mutant PS-expressing cells, the rate of oxygen consumption increases dramatically and overcomes the Ca2+ dependent enzymes that stimulate NADH production. This leads to decreased rates in proton pumping due to diminished membrane potential along with less ATP and enhanced ROS production. These results show that through Ca2+ signaling disruption, mutant PS leads to mitochondrial dysfunction and potentially to cell death.

Quercetin attenuates neuronal death against aluminum-induced neurodegeneration in the rat hippocampus

Aluminum is a light weight and toxic metal present ubiquitously on earth, which has gained considerable attention due to its neurotoxic effects. It also has been linked ecologically and epidemiologically to several neurological disorders, including Alzheimer’s disease (AD), Parkinson’s disease (PD), Guamanian–Parkinsonian complex and Amyotrophic lateral sclerosis (ALS). The mechanism of aluminum neurotoxicity is poorly understood, but it is well documented that aluminum generates reactive oxygen species (ROS). Enhanced ROS production leads to disruption of cellular antioxidant defense systems and release of cytochrome c (cyt-c) from mitochondria to cytosol resulting in apoptotic cell death. Quercetin (a natural flavonoid) protects it from oxidative damage and has been shown to decrease mitochondrial damage in various animal models of oxidative stress. We hypothesized that if oxidative damage to mitochondria does play a significant role in aluminum-induced neurodegeneration, and then quercetin should ameliorate neuronal apoptosis. Administration of quercetin (10mg/kg body wt/day) reduced aluminum (10mg/kg body wt/day)-induced oxidative stress (decreased ROS production, increased mitochondrial superoxide dismutase (MnSOD) activity). In addition, quercetin also prevents aluminum-induced translocation of cyt-c, and up-regulates Bcl-2, down-regulates Bax, p53, caspase-3 activation and reduces DNA fragmentation. Quercetin also obstructs aluminum-induced neurodegenerative changes in aluminum-treated rats as seen by Hematoxylin and Eosin (H&E) staining. Further electron microscopic studies revealed that quercetin attenuates aluminum-induced mitochondrial swelling, loss of cristae and chromatin condensation. These results indicate that treatment with quercetin may represent a therapeutic strategy to attenuate the neuronal death against aluminum-induced neurodegeneration.

Impaired Mitochondrial Function due to Familial Alzheimers Disease-Causing Presenilins Mutants via Calcium Disruptions

Mutants in presenilins (PS1 or PS2) is the major cause of familial Alzheimer's disease (FAD). FAD causing PS mutants affect intracellular Ca(2+) homeostasis by enhancing the gating of inositol trisphosphate (IP3) receptor (IP3R) Ca(2+) release channel on the endoplasmic reticulum, leading to exaggerated Ca(2+) release into the cytoplasm. Using experimental IP3R-mediated Ca(2+) release data, in conjunction with a computational model of cell bioenergetics, we explore how the differences in mitochondrial Ca(2+) uptake in control cells and cells expressing FAD-causing PS mutants affect key variables such as ATP, reactive oxygen species (ROS), NADH, and mitochondrial Ca(2+). We find that as a result of exaggerated cytosolic Ca(2+) in FAD-causing mutant PS-expressing cells, the rate of oxygen consumption increases dramatically and overcomes the Ca(2+) dependent enzymes that stimulate NADH production. This leads to decreased rates in proton pumping due to diminished membrane potential along with less ATP and enhanced ROS production. These results show that through Ca(2+) signaling disruption, mutant PS leads to mitochondrial dysfunction and potentially to cell death.

Inhaled Methane Limits the Mitochondrial Electron Transport Chain Dysfunction during Experimental Liver Ischemia-Reperfusion Injury.

BackgroundMethanogenesis can indicate the fermentation activity of the gastrointestinal anaerobic flora. Methane also has a demonstrated anti-inflammatory potential. We hypothesized that enriched methane inhalation can influence the respiratory activity of the liver mitochondria after an ischemia-reperfusion (IR) challenge.MethodsThe activity of oxidative phosphorylation system complexes was determined after in vitro methane treatment of intact liver mitochondria. Anesthetized Sprague-Dawley rats subjected to standardized 60-min warm hepatic ischemia inhaled normoxic air (n = 6) or normoxic air containing 2.2% methane, from 50 min of ischemia and throughout the 60-min reperfusion period (n = 6). Measurement data were compared with those on sham-operated animals (n = 6 each). Liver biopsy samples were subjected to high-resolution respirometry; whole-blood superoxide and hydrogen peroxide production was measured; hepatocyte apoptosis was detected with TUNEL staining and in vivo fluorescence laser scanning microscopy.ResultsSignificantly decreased complex II-linked basal respiration was found in the normoxic IR group at 55 min of ischemia and a lower respiratory capacity (~60%) and after 5 min of reperfusion. Methane inhalation preserved the maximal respiratory capacity at 55 min of ischemia and significantly improved the basal respiration during the first 30 min of reperfusion. The IR-induced cytochrome c activity, reactive oxygen species (ROS) production and hepatocyte apoptosis were also significantly reduced.ConclusionsThe normoxic IR injury was accompanied by significant functional damage of the inner mitochondrial membrane, increased cytochrome c activity, enhanced ROS production and apoptosis. An elevated methane intake confers significant protection against mitochondrial dysfunction and reduces the oxidative damage of the hepatocytes.

Abstract A160: Imprime PGG binds to neutrophils through complement, Fc, and dectin-1 receptors, priming these cells for enhanced ROS production and tumor cell cytotoxicity

Abstract Imprime PGG (Imprime), a soluble yeast 1,3/1,6 β-glucan, is being developed as a novel cancer immunotherapy in conjunction with anti-tumor antibodies in several cancers. In clinical studies, including randomized Phase 2 clinical trials in the 1st-line treatment of stage IV non-small cell lung cancer with bevacizumab, Imprime treatment has shown promising efficacy in both objective tumor response rates and survival. In numerous syngeneic and xenogeneic mouse tumor models, Imprime treatment in combination with an anti-tumor antibody reduced tumor growth and prolonged survival beyond that observed with antibody alone. Mechanistic studies have now demonstrated that, with endogenous anti-β-glucan antibodies (ABA), Imprime forms an immune complex. This Imprime-ABA complex then activates the classical complement pathway and is subsequently opsonized by iC3b. This immune complex then binds to and primes innate immune cells, including macrophages, monocytes and neutrophils, triggering a coordinated immune attack against antibody-targeted cancer cells. In this study, we sought to explore more fully the functional consequences of Imprime-ABA immune complex binding to, and priming of, neutrophils. Neutrophils are the first line of defense against fungal infections. Neutrophils detect fungal infections through recognition of β-glucans on the surface of yeast and fungi. These β-glucans serve as fungal pathogen associated molecular patterns (PAMPs) that are efficiently and effectively recognized by receptors on innate immune cells, initiating a coordinated immune response to infection. Here we demonstrate that Imprime, in individuals with high ABA levels, is recognized as a PAMP, directly interacting with neutrophils via multiple receptors, priming these cells and ultimately leading to enhanced effector responses including reactive oxygen species (ROS) generation and tumor cell killing. Our data now show that Imprime elicits enhanced neutrophil survival as measured by cell viability dye exclusion using flow cytometry. Imprime treatment also enhances the responsiveness of neutrophils to Fc receptor activation. As measured by luminol-based read-outs, ROS production in response to anti-Fc receptor antibody-coated beads is profoundly enhanced by Imprime treatment. Moreover, Imprime treated neutrophils show a remarkable surge in ROS production compared to vehicle–treated neutrophils when exposed to B cell lymphoma cells decorated with anti-CD20 monoclonal antibodies (i.e. rituximab). These Imprime-treated neutrophils also exhibit a robust increase in cytotoxicity against these B cell lymphoma cells coated with rituximab using in vitro Calcein AM dye release assays. We next sought to delineate which receptors might be most critical for Imprime binding and priming of neutrophils. As demonstrated by antibody blocking studies, the Imprime-ABA complex can bind to both complement receptors (notably CR3) and Fc receptors. Imprime can also block binding of a dectin-1 agonizing antibody to dectin-1 and can bind directly to HL-60 and HEK cells lines engineered to overexpress dectin-1 by retroviral transduction. Collectively, these data show for the first time that Imprime directly primes neutrophils to recognize and kill antibody-decorated tumor cells and interacts with the Fc, complement and dectin-1 receptors on the surface of neutrophils. Citation Format: Steven M. Leonardo, Ross B. Fulton, Lindsay R. Wurst, Keith B. Gorden, Adria Bykowski Jonas, Xiaohong Qui, Anissa SH Chan, Jeremy R. Graff. Imprime PGG binds to neutrophils through complement, Fc, and dectin-1 receptors, priming these cells for enhanced ROS production and tumor cell cytotoxicity. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr A160.

Anti-Inflammatory Effects of Hyperbaric Oxygenation during DSS-Induced Colitis in BALB/c Mice Include Changes in Gene Expression of HIF-1α, Proinflammatory Cytokines, and Antioxidative Enzymes.

Reactive oxygen species (ROS) and nitrogen species have an indispensable role in regulating cell signalling pathways, including transcriptional control via hypoxia inducible factor-1α (HIF-1α). Hyperbaric oxygenation treatment (HBO2) increases tissue oxygen content and leads to enhanced ROS production. In the present study DSS-induced colitis has been employed in BALB/c mice as an experimental model of gut mucosa inflammation to investigate the effects of HBO2 on HIF-1α, antioxidative enzyme, and proinflammatory cytokine genes during the colonic inflammation. Here we report that HBO2 significantly reduces severity of DSS-induced colitis, as evidenced by the clinical features, histological assessment, impaired immune cell expansion and mobilization, and reversal of IL-1β, IL-2, and IL-6 gene expression. Gene expression and antioxidative enzyme activity were changed by the HBO2 and the inflammatory microenvironment in the gut mucosa. Strong correlation of HIF-1α mRNA level to GPx1, SOD1, and IL-6 mRNA expression suggests involvement of HIF-1α in transcriptional regulation of these genes during colonic inflammation and HBO2. This is further confirmed by a strong correlation of HIF-1α with known target genes VEGF and PGK1. Results demonstrate that HBO2 has an anti-inflammatory effect in DSS-induced colitis in mice, and this effect is at least partly dependent on expression of HIF-1α and antioxidative genes.

Arabidopsis thaliana constitutively active ROP11 interacts with the NADPH oxidase respiratory burst oxidase homologue F to regulate reactive oxygen species production in root hairs.

Reactive oxygen species (ROS) play a key signalling role in cells. Plant NADPH oxidases, also known as respiratory burst oxidase homologues (Rbohs), are well characterised ROS-generating systems. In this study, we found that the constitutively active small guanosine triphosphatase (GTPase) ROP11 (CA-ROP11) interacted with RbohF by using a yeast two-hybrid analysis, a pull-down assay and an in vivo bimolecular fluorescence complementation assay. The mutation of amino acid L336 or L337 in RbohF abolished its interaction with CA-ROP11. Coexpression of CA-ROP11 and wild-type RbohF in Nicotiana benthamiana Domin enhanced ROS production compared with coexpression of CA-ROP11 and mutant RbohF or of dominant negative ROP11 and wild-type RbohF. Moreover, CA-ROP11 overexpression resulted in ROS accumulation and a swollen root hair phenotype in Arabidopsis thaliana (L.) Heynh. The deletion of RbohF partially reduced the increase in ROS in Arabidopsis plants overexpressing CA-ROP11. These results suggest that Arabidopsis ROP11 modulates ROS production by interacting with RbohF in root hairs.

Stres oksydacyjny indukowany hiperglikemią w cukrzycy ciążowej (GDM)

An imbalance between production of reactive oxygen species (ROS) and their clearance by antioxidant defence mechanisms results in the development of oxidative stress. Biological consequences of this state involve oxidative damage of key cellular components such as nucleic acids, lipids, or proteins and, in turn, impairment of cell and tissue function. Evidence from clinical and experimental studies supports the notion that oxidative stress is one of pathologic factors associated with gestational diabetes mellitus (GDM), metabolic disorder defined as any degree of glucose intolerance with onset or first recognition during pregnancy. It has been established that high blood glucose concentrations in diabetic pregnancy induce oxidative stress by several mechanisms, including an enhanced ROS production in mitochondria, the polyol pathway and the hexosamine pathway, as well as protein kinase C (PKC) activation and an advanced glycation end-products (AGEs) formation, and changes in biomarkers of free radical-induced damage and antioxidant defences have been detected in maternal diabetes. Moreover, hyperglycaemia-induced oxidative stress is related with some congenital anomalies in diabetic pregnancy. The current article provides an overview how oxidative stress is related to GDM, with special emphasis on the involvement of the hyperglycaemia-induced mechanisms in ROS overproduction, followed by discussion of indicators of oxidative stress. In addition, the relationship between oxidative stress and congenital malformations in diabetic pregnancy is described.

Targeting F2r/Beta-Catenin/JNK Signaling for Leukemia Stem Cell Eradication

G-protein coupled receptors (GPCRs) are the most successful drug targets with 36% of currently marketed drugs targeting human GPCRs. F2r, a GPCR that is overexpressed in various human cancers (Ribeiro et al., Oncol Rep 2009), is a positive regulator of the β-catenin pathway and an inhibitor of the JNK pathway in mammalian cells (Sun et al., Nat Cell Biol 2001). The expression of F2r is significantly elevated in aggressive leukemias including blast phase of CML and AML (Veiga et al., Blood Cells Mol Dis 2011). While these observations implicate an involvement of F2r in human leukemia, its function in AML remains unknown.Our preliminary data showed that shRNA-mediated knockdown of F2r markedly decreased active β-catenin in MLL-AF9 mediated pre-leukemia stem cells (pre-LSCs) and leukemia stem cells (LSCs), confirming that F2r is a positive regulator of β-catenin. F2r deficiency decreased LSC colony formation (p=0.0002) in a serial replating assay, indicating a reduction in self-renewal capacity. Furthermore, LSCs exhibited a significant enhancement in apoptotic activity in response to F2r deficiency, displaying a 12-fold increase in apoptosis. Our microarray expression analysis revealed that F2r inhibition significantly reduced the expression of several genes responsible for maintenance of mitochondrial integrity and energy metabolism (mtND4L (p=0.0013), mtND2 (p<0.0001) and mtCytB (p<0.0001)). To investigate this further we used a mitochondria-specific fluorogenic probe to measure reactive oxygen species (ROS) production. A significant increase in ROS production (p=0.0003) indicated that F2r inhibition destabilizes the mitochondrial membrane. This was accompanied by a marked increase, as observed by Western blot analysis, in the proapoptotic proteins Bcl-2-interacting mediator of cell death (Bim) and thioredoxin-interacting protein (Txnip) which permeabilize the mitochondrial membrane releasing cytochrome c and inducing apoptosis. Through oxidative phosphorylation, mitochondria play an essential role in the supply of metabolic energy (ATP) to the cell. F2r deficient LSCs had a significantly reduced rate of oxygen consumption measured using a phosphorescent oxygen probe (p=0.0066) and a significantly lower concentration of basal ATP (p=0.012) compared to control LSCs. F2r inhibition, therefore, induces substantial oxidative stress which triggers the intrinsic apoptotic pathway.To assess the therapeutic value of F2r inhibition, we used the selective non-peptide F2r inhibitor SCH79797. Alamar blue-based cell viability assays showed that SCH79797 was potent against LSCs and had no cytotoxic effects on lineage-negative normal mouse bone marrow cells. F2r inhibitor treatment resulted in a 2-fold reduction in colony forming ability, 3.8-fold enhanced ROS production and inhibited β-catenin activity. BrdU labeling revealed a significant reduction in in vivo short-term proliferation of LSCs that were pre-treated with SCH79797 for 48 hours in culture, transplanted into recipient mice and collected from bone marrow 8 days post-transplantation (p=0.0008). Additional in vivo studies using a mouse model of MLL AML are currently ongoing to further evaluate the therapeutic potential of F2r inhibition. Collectively, our findings suggest that F2r inhibition selectively targets LSC self-renewal, identifying a therapeutic window to eliminate LSCs while preserving normal blood cells.Previous studies suggest that F2r knockdown not only suppresses β-catenin but also activates JNK signaling. Consistently, our Western blot analysis revealed activation of JNK in response to inhibition of F2r. Sustained JNK activation has been reported in many types of AML cells and promotes survival signals during leukemia development (Hess et al., Nat Genet 2002). This suggests that JNK and F2r inhibition could be used in combination to impair LSC self-renewal, with a concurrent increase in cell death. In support of this hypothesis, we have showed that co-inhibition of F2r and JNK induced a potent anti-LSC effect, significantly increasing cell death and ROS production compared to single treatment. The efficacy of this co-treatment is currently being evaluated in primary human AML patient samples in addition to our in vivo mouse model system. Altogether our data suggest a novel LSC-eliminating treatment strategy targeting F2r/β-catenin/JNK signaling for aggressive AML. DisclosuresNo relevant conflicts of interest to declare.

Abstract A98: Imprime PGG, a soluble yeast β glucan, primes innate immune effector cells to recognize and eradicate tumor cells

Abstract Imprime PGG (Imprime), a soluble yeast 1,3/1,6 β-glucan, is being developed as a novel cancer immunotherapy in conjunction with anti-tumor antibodies in several cancers. In clinical studies, including randomized Phase 2 clinical trials in the 1st-line treatment of stage IV non-small cell lung cancer with bevacizumab, Imprime treatment has shown promising efficacy in both objective tumor response rates and survival. Mechanistic studies in humans have now demonstrated that Imprime, a fungal pathogen associated molecular pattern (PAMP), forms an immune complex with endogenous anti-β-glucan antibodies (ABA), which is then opsonized by complement. This immune complex binds complement receptor 3 (CR3) and FcγRIIA on innate immune cells (macrophages, monocytes and neutrophils). In numerous syngeneic and xenogeneic mouse tumor models, Imprime treatment in combination with an anti-tumor antibody reduced tumor growth and prolonged survival beyond that observed with antibody alone. We sought to understand better the anti-tumor effect elicited by Imprime binding to these innate immune effector cells by exploring both phenotypic and functional changes driven by Imprime binding to neutrophils, monocytes and macrophages. Neutrophils were isolated from human whole blood treated with Imprime or vehicle. Imprime treatment elicited an activated phenotype as demonstrated by downregulation of CD88 (C5aR) and CD62L (L-Selectin), along with the upregulation of CR1 and CR3. These surface changes coincided with functional changes as well. Neutrophils from Imprime-treated whole blood showed a profound increase in the generation of reactive oxygen species (ROS) when stimulated with phorbol myristate acetate (PMA). Similarly, Imprime- treated neutrophils exhibited further enhanced ROS production when co-cultured specifically with B cell lymphoma cells (Raji) that had been coated with the anti-CD20 antibody rituximab. ROS were not generated by vehicle-treated neutrophils nor when Imprime-treated neutrophils were co-cultured with Raji B cells that had not been coated with antibody. These data support the notion that that Imprime specifically “primes” neutrophils enhancing the recognition of, and responsiveness to, antibody-coated tumor cells. Monocytes showed similar “priming” effects post Imprime treatment, including downmodulation of CD88, upregulation of CR1 and CR3, and enhanced ROS production in response to PMA co-treatment. Differentiation of monocytes to macrophages, post Imprime treatment of human whole blood, enabled enhanced antibody-dependent cellular phagocytosis (ADCP) of multiple B cell lymphoma cell lines decorated with the anti-CD20 antibodies rituximab, ofatumumab and obinatuzumab. Collectively, these data highlight the impact of Imprime on key cell lineages of the innate immune system, priming neutrophils, monocytes and macrophages to better recognize and exert cytotoxic effector functions in response to antibody- coated tumor cells. Citation Format: Steven M. Leonardo, Adria Jonas, Nadine Ottoson, Xiaohong Qiu, Anissa Chan, Jeremy Graff, Nandita Bose, Keith Gorden. Imprime PGG, a soluble yeast β glucan, primes innate immune effector cells to recognize and eradicate tumor cells. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr A98.

Reperfusion injury and reactive oxygen species: The evolution of a concept

Reperfusion injury, the paradoxical tissue response that is manifested by blood flow-deprived and oxygen-starved organs following the restoration of blood flow and tissue oxygenation, has been a focus of basic and clinical research for over 4-decades. While a variety of molecular mechanisms have been proposed to explain this phenomenon, excess production of reactive oxygen species (ROS) continues to receive much attention as a critical factor in the genesis of reperfusion injury. As a consequence, considerable effort has been devoted to identifying the dominant cellular and enzymatic sources of excess ROS production following ischemia-reperfusion (I/R). Of the potential ROS sources described to date, xanthine oxidase, NADPH oxidase (Nox), mitochondria, and uncoupled nitric oxide synthase have gained a status as the most likely contributors to reperfusion-induced oxidative stress and represent priority targets for therapeutic intervention against reperfusion-induced organ dysfunction and tissue damage. Although all four enzymatic sources are present in most tissues and are likely to play some role in reperfusion injury, priority and emphasis has been given to specific ROS sources that are enriched in certain tissues, such as xanthine oxidase in the gastrointestinal tract and mitochondria in the metabolically active heart and brain. The possibility that multiple ROS sources contribute to reperfusion injury in most tissues is supported by evidence demonstrating that redox-signaling enables ROS produced by one enzymatic source (e.g., Nox) to activate and enhance ROS production by a second source (e.g., mitochondria). This review provides a synopsis of the evidence implicating ROS in reperfusion injury, the clinical implications of this phenomenon, and summarizes current understanding of the four most frequently invoked enzymatic sources of ROS production in post-ischemic tissue.

Antimicrobial activity of iron oxide nanoparticle upon modulation of nanoparticle-bacteria interface.

Investigating the interaction patterns at nano-bio interface is a key challenge for safe use of nanoparticles (NPs) to any biological system. The study intends to explore the role of interaction pattern at the iron oxide nanoparticle (IONP)-bacteria interface affecting antimicrobial propensity of IONP. To this end, IONP with magnetite like atomic arrangement and negative surface potential (n-IONP) was synthesized by co-precipitation method. Positively charged chitosan molecule coating was used to reverse the surface potential of n-IONP, i.e. positive surface potential IONP (p-IONP). The comparative data from fourier transform infrared spectroscope, XRD, and zeta potential analyzer indicated the successful coating of IONP surface with chitosan molecule. Additionally, the nanocrystals obtained were found to have spherical size with 10–20 nm diameter. The BacLight fluorescence assay, bacterial growth kinetic and colony forming unit studies indicated that n-IONP (<50 μM) has insignificant antimicrobial activity against Bacillus subtilis and Escherichia coli. However, coating with chitosan molecule resulted significant increase in antimicrobial propensity of IONP. Additionally, the assay to study reactive oxygen species (ROS) indicated relatively higher ROS production upon p-IONP treatment of the bacteria. The data, altogether, indicated that the chitosan coating of IONP result in interface that enhances ROS production, hence the antimicrobial activity.

Glycated human serum albumin induces NF-κB activation and endothelial nitric oxide synthase uncoupling in human umbilical vein endothelial cells

AimsNon-enzymatic glycated proteins could mediate diabetes vascular complications, but the molecular mechanisms are unknown. Our objective was to find new targets involved in the glycated human serum albumin (gHSA)-enhanced extracellular reactive oxygen species (ROS) production in human endothelial cells. Methods & resultsSome nuclear factors and phosphorylation cascades were analysed. gHSA activated nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), which up-regulated NOX4 and P22PHOX and enhanced ROS production. Pharmacological inhibition of NF-κB reversed gHSA-enhanced NOX4 expression and decreased gHSA-induced ROS production in extra- and intracellular spaces. The inhibition of activator protein-1 (AP-1) induced a rise in NOX4 and P22PHOX subunit expression and a down-regulation of endothelial nitric oxide synthase (eNOS). AP-1 inhibition also enhanced extracellular ROS production in the presence of serum albumin, but not with gHSA. These results were explained by the eNOS uncoupling induced by gHSA, also demonstrated in this study. Phosphatidylinositol 3-kinase or mitogen-activated protein kinase kinase 1/2 did not show to be involved in gHSA-induced ROS production. ConclusionsAll together, the results suggested that gHSA-enhanced ROS production in endothelium is mediated by: 1) NF-κB activation and subsequence up-regulation of NADPH oxidase, 2) eNOS uncoupling. AP-1, although is not directly affected by gHSA, is another target for regulating NADPH oxidase and eNOS expression in endothelial cells.