4th International Conference on Oxidative/Nitrosative Stress and Disease

Agenda

*Presentation times are subject to change.

Day 1: October 28, 2009

SESSION I: Inflammation, Lung Cancer and Fibroproliferative Diseases: Roles of Reactive Oxygen/Nitrogen Species

Reactive Oxygen/Nitrogen Species in Lung Cancer

Neelam Azad, PhD¹, Anand Iyer, PhD¹, Val Vallyathan, PhD², Yon Rojanasakul, PhD^3
1Department of Pharmaceutical Sciences, School of Pharmacy, Hampton University, Hampton, Virginia. ²Pathology and Physiology Research Branch, National Institute for Occupational Safety and Health, Morgantown, West Virginia. ³Department of Pharmaceutical and Pharmacological Sciences, School of Pharmacy, West Virginia University, Morgantown, WV

Lung cancer is the leading cause of cancer mortality worldwide. Reactive oxygen/nitrogen species (ROS/RNS) are known to be the key players in lung inflammation and cancer as increased ROS/RNS production leads to genetic mutations, predisposing individuals to cancer. Individuals are constantly exposed to increased levels of ROS/RNS generated from various carcinogens. Hexavalent chromium [Cr(VI)] compounds are redox cycling environmental carcinogens strongly associated with the incidence of lung cancer. Generation of reactive species and dysregulation of apoptosis have been implicated in Cr(VI)-induced lung carcinogenesis; however, the underlying mechanisms are largely unknown. In our study, we observed that ROS, specifically superoxide anion (·O2‾), mediated Cr(VI)-induced apoptosis of human lung epithelial cells. Cr(VI)-induced apoptosis mainly through the mitochondrial death pathway via caspase-9 activation, which was negatively regulated by the mitochondrial anti-apoptotic protein Bcl-2. Specifically, ·O2‾ induced apoptosis by downregulating and degrading Bcl-2 protein through the ubiquitin-proteasomal pathway. Consequently, overexpression of Bcl-2 blocked Cr(VI)-induced apoptosis, whereas Cr(VI)-induced nitric oxide (NO) production was involved in the stabilization of Bcl-2. This mechanism involved NO-mediated S-nitrosylation of Bcl-2, which prevented its ubiquitination and subsequent proteasomal degradation. Therefore, NO was found to exert an anti-apoptotic effect in response to Cr(VI) exposure. The mechanism of Cr(VI)-induced apoptosis resistance was verified in an in vitro model that facilitated mechanistic studies of Cr(VI)-induced carcinogenesis. Long-term exposure to Cr(VI) led to the malignant transformation of non-tumorigenic human lung epithelial cells. Cr(VI)-transformed cells exhibited loss of contact inhibition, colony formation, and increased rates of cell invasion, migration and proliferation as compared to passage-matched control cells. Cr(VI)-transformed cells showed decreased apoptosis and ROS production. The mechanism by which these cells evaded apoptosis involved NO-mediated S-nitrosylation and stabilization of Bcl-2 protein. Taken together, this study establishes an important in vitro model that facilitates mechanistic studies of Cr(VI)-induced carcinogenesis, and elucidates a novel mechanism of apoptosis-resistant and malignant transformation of non-tumorigenic lung cells in response to a human carcinogen. The study also reveals a novel mechanism linking ·O₂‾ and NO with Bcl-2 stability providing a new dimension to reactive species-mediated Bcl-2 stability, apoptotic cell death and cancer development.

Role of Reactive Oxidant Species in Environmental Lung Disease

William N. Rom MD, MPH, Division of Pulmonary and Critical Care Medicine, NYU School of Medicine, New York, NY

ROS contribute to inflammation and injury of the lower respiratory tract after inhalation of particulates that lead to fibrosis and/or cancer. The technique of bronchoalveolar lavage in humans has recovered alveolar macrophages (AMs) in dust diseases that release increased amounts of ROS. Peptide growth factors (PDGF, IGF-I) from AMs and matrix metalloproteinases contribute to the inflammation. Other cells are important: eosinophils cause acute eosinophilic pneumonia due to WTC dust; eosinophils from TPE release very high ROS leading to fibrosis, and neutrophils releasing elastase cause focal emphysema in CWP. Key to the fibrosing process is Transforming Growth Factor-β from myofibroblasts. Myofibroblasts develop from progenitor cells in the basal layers of the airway epithelium and migrate into the interstitium releasing excess amounts of collagen. Progenitor cells activate embryonic signaling pathways through Hedgehog that can be tagged with Gli1-lacZ to identify key players in the ROS-inflammation-fibrosis process.

Cell Signaling and Inflammatory Pathways in Asbestos-Related Diseases

Arti Shukla, PhD, Jedd M. Hillegass, PhD, Maximilian B. MacPherson, BSc, Stacie L. Beuschel, BSc, Sherrill A. Lathrop, BSc, Nicholas H. Heintz, PhD, Brooke T. Mossman, PhD, Department of Pathology, University of Vermont College of Medicine, Burlington, VT

Exposure to asbestos fibers is associated with the development of pulmonary fibrosis, lung cancers and mesotheliomas. Several years ago we questioned the conclusion that asbestos was primarily a DNA- damaging or genotoxic carcinogen, suggesting instead that the initial interaction of asbestos fibers at the plasma membrane triggered multiple signaling cascades that are linked to transcription factors (AP-1, NF-κB, CREB, etc.) governing abnormal cell proliferation and inflammation. Many of these signaling pathways are initiated by elaboration of oxidants both extracellularly and intracellularly. Recently we have shown that asbestos fibers stimulate NADPH oxidases in human macrophages, a key cell in early responses to asbestos, that leads to activation of the inflammasome (Dostert et al, Science 320:674-677). We addressed the hypothesis that NADPH oxidases were over-expressed after exposures to asbestos and in human mesothelioma cells, leading to activation of extracellular-signal regulated kinases (ERKs 1 and 2) critical to the development of asbestos injury, inflammation and mesotheliomas. In RNA interference experiments, human mesothelioma cells (kindly provided by Dr. Harvey I. Pass, NYU) were stably transfected with either shERK1 or shERK2 before characterization in vitro and injection into a mouse xenograft model. We show that ERK2 is causally linked to both asbestos toxicity and the development of mesotheliomas. Microarray experiments on shERK1, shERK2 and shControl mesothelioma lines reveal unique cell death and oxidant/antioxidant genes regulated by ERKs. These studies link ERK mechanistically to the development of inflammation and mesothelioma. (This work is supported by P01 grants from NCI and NHLBI and a training grant from the NIEHS.)

The Role of iNOS-Mediated DNA Damage in Inflammation-Related Carcinogenesis

Shosuke Kawanishi, PhD¹, Yusuke Hiraku, MD, PhD², Takamichi Ichinose, PhD³, Mariko Murata, MD, PhD^2
1Suzuka University of Medical Science, Suzuka, Mie, Japan, ²Mie University Graduate School of Medicine, Mie, Japan, ³Oita University of Nursing and Health Sciences, Oita, Japan

Chronic infection and inflammation considerably contribute to environmental carcinogenesis. Chronic inflammation has been estimated to account for approximately 25 % of human cancers. The International Agency for Research on Cancer (IARC) has estimated that approximately 18 % of cancer cases worldwide is attributable to infectious diseases. Moreover, not only infectious diseases but also various physical, chemical and immunological factors participate in inflammation-related carcinogenesis. Under inflammatory condition, reactive oxygen and nitrogen species are generated from inflammatory and epithelial cells and may play an important role in inflammation-related carcinogenesis by causing DNA damage. Nitric oxide (NO) is generated through the expression of inducible nitric oxide synthase (iNOS) and interacts with superoxide (O2^•-) to form highly reactive peroxynitrite (ONOO-), which reacts with guanine to produce 8-nitroguanine, a mutagenic DNA lesion.

We performed immunohistochemical analysis to examine 8-nitroguanine formation in various clinical specimens and animal models of cancer-prone infectious diseases induced by the liver fluke Opisthorchis viverrini, Helicobacter pylori, hepatitis C virus, human papillomavirus (HPV) and Epstein-Barr virus (EBV). We demonstrated that 8-nitroguanine formation was observed at the sites of carcinogenesis and increased during tumor development. 8-Nitroguanine formation in cervical biopsy specimens of HPV-induced cervical intraepithelial neoplasia was increased with its grade. 8-Nitroguanine formation in cancer cells of patients with EBV-mediated nasopharyngeal carcinoma was significantly stronger than that in biopsy specimens of nasopharyngitis patients. We have also reported that strong 8-nitroguanine formation was closely associated with a poor prognosis in patients with soft tissue tumor.

Asbestos fibers are potent carcinogens causing lung cancer and malignant mesothelioma in humans. We found that 8-nitroguanine was formed particularly in the nucleus of bronchial epithelial cells in the lung tissues of mice intratracheally administered asbestos fibers. Interestingly, quantitative image analysis revealed that crocidolite induced 8-nitrogunaine formation to a significantly greater extent than chrysotile in consistency with their carcinogenic potentials. iNOS expression was also observed in the bronchial epithelial cells and its immunoreactivity was correlated with that of 8-nitrogunaine. On the basis of these findings, we have proposed that 8-nitroguanine could be a potential biomarker to evaluate the risk of inflammation-related carcinogenesis and the prognosis of cancer patients.

Regulation of Apoptosis through Cysteine Oxidation: Implications for Fibrotic Lung Disease

Yvonne MW Janssen-Heininger, PhD¹, Vikas Anathy, PhD¹, Scott Aesif, PhD¹, Jos van der Velden, PhD¹, Amy S. Guala¹, Jessica N. Reiss¹, Elle Roberson¹, Ralph C. Budd², Niki L. Reynaert, PhD³
Department of Pathology¹ and Medicine² University of Vermont, Burlington VT, and Department of Respiratory Medicine³ Maastricht University Medical Center, Maastricht, the Netherlands

Tissue fibrosis is believed to be a manifestation of dysregulated repair following injury, in association with impaired re-epithelialization, and aberrant myofibroblast activation and proliferation. Numerous pathways have been linked to the pathogenesis of fibrotic lung disease, including the death receptor, Fas which contributes to apoptosis of lung epithelial cells. A redox imbalance also has been implicated in disease pathogenesis, although mechanistic details whereby oxidative changes intersect with pro-fibrotic signaling pathways remain elusive. Oxidation of cysteines in proteins, which encompass S-nitrosylation, S-glutathionylation (PSSG), among others are known to act as regulatory events that affect protein structure and function. The overall goal of the research that will be discussed was to determine whether cysteine oxidation events, notably protein S-glutathionylation (PSSG) are important in FasL-induced apoptosis. We recently demonstrated that Fas Ligand (FasL)-induced death of lung epithelial cells is accelerated following S-glutathionylation of the death receptor Fas (Fas-SSG) itself at cysteine 294. Fas-SSG occurs following caspase-8 induced degradation of glutaredoxin, 1, which under physiological conditions acts to decompose PSSG. Fas-SSG promotes its recruitment into lipid rafts, oligomerization, and Death Inducing Signaling Complex formation, leading to feed forward amplification of caspase 8 activation and apoptosis. Ablation of glutaredoxin 1, via SiRNA or gene ablation augments FasL-induced Fas-SSG and sensitizes epithelial cells to apoptosis, while conversely overexpression of glutaredoxin-1 attenuates Fas-SSG, and protects against apoptosis. Using glutaredoxin-1 mediated cysteine derivatization in situ, I will demonstrate evidence that protein S-glutathionylation is increased in two independent mouse models of fibrosis, and in patients with idiopathic pulmonary fibrosis. Lastly, the impact of modulation of glutaredoxin-1 in mouse models will be presented in order to demonstrate the functional significance of protein-S-glutathionylation in fibrogenesis. Funded by NIH R01 HL079331 and R03 HL095404.

Inhibition of Inflammation and Carcinogenesis in the Lung and Colon by Tocopherols

Chung S. Yang, PhD, Guang Xun Li, PhD, Gang Lu, PhD, and Jihyeung Ju, PhD, Department of Chemical Biology, Ernest Mario School of Pharmacy, Rutgers, The State University of New Jersey, Piscataway, NJ

Tocopherols, which exist in α, β, γ, and δ forms, are antioxidative nutrients known as vitamin E. Although α-tocopherol (α-T) is the major form of vitamin E found in the blood and tissues, γ- and δ-T have been suggested to have stronger anti-inflammatory activities. A robust cancer preventive activity of these tocopherols has not been demonstrated previously. In the present study, using a tocopherol mixture that is rich in γ-T (γ-TmT, which contains 57% γ-T), we demonstrated the inhibition of inflammation as well as of cancer formation and growth in the lung and colon in animal models. When given in the diet at 0.3%, γ-TmT inhibited lung tumorigenesis in the A/J mice, induced by the tobacco carcinogen NNK or NNK plus benzo[a]pyrene (a ubiquitous environmental carcinogen). Dietary 0.3% γ-TmT also inhibited the growth of human lung cancer H1299 cells in xenograft tumors in NCr-nu/nu mice. In both experimental systems, γ-TmT decreased the levels of 8-hydroxydeoxyguanosine (a product of DNA oxidation), γ-H2AX (an indicator of DNA repair caused by DNA double-strand breakage), and nitrotyrosine in tumors. The plasma levels of prostaglandin E2 and leukotriene B4 were also decreased by γ-TmT treatment in mice. More evident anti-inflammatory activity was demonstrated in mice treated with azoxymethane (AOM) and dextran sulfate sodium (DSS), as indicated by the inflammatory index and biochemical markers. The anti-inflammatory activity of pure γ-T and δ-T was also demonstrated in this model. Inhibition of colon carcinogenesis (reduction of adenocarcinoma and adenoma formation by ~80%) was observed when 0.3% γ-TmT diet was initiated either before or after AOM/DSS treatment. These results demonstrate the anti-oxidative, anti-inflammatory, and anti-carcinogenic activities of a mixture of γ-T-rich tocopherols (supported by NIH grant CA122474 and CA120915).

SESSION II: Reactive Oxygen and Reactive Nitrogen Species: Signaling and Detection

Signaling by Reactive Oxygen Species and Aldehydes

Henry Jay Forman, PhD, School of Natural Sciences, University of California, Merced, Merced, CA

Signaling by H₂O₂, α,β-unsaturated aldehydes, such as 4-hydroxy-2-nonenal (HNE) and related chemical species is thought to differ from signaling by other second messengers because the oxidants and other electrophiles can readily undergo non-enzymatic reactions and are therefore classified as “reactive.” Although there are some significant differences in the chemistry of these reactive species from classic second messengers, such as cyclic AMP, there are significant similarities between signaling by H₂O₂ and classic second messengers in terms of the kinetics and regulation of their production and degradation, and effect on signaling pathways including reversibility of action. HNE can form reversible adducts with proteins but its involvement in signaling appears to be less similar to that of the classic second messengers and more like other post-translational signaling, such as farnesylation or ubiquitination.The chemistry of cysteine provides a common factor that underlies signaling by H₂O₂ and HNE. Nonetheless, as H₂O₂ and HNE are rapidly metabolized in vivo and have very different physical properties, spatial considerations are also important in their actions. Therefore, the locations of sources of H₂O₂ and α,β-unsaturated aldehydes, the NADPH oxidases, mitochondria, membrane lipids, and redox cycling toxicants, as well as their targets are key factors. The activation of the JNK pathway by HNE and endogenously generated H₂O₂ illustrates these principles.

S-nitrosylation and Denitrosylation of Proteins in Health and Disease

Jonathan S. Stamler, MD, Duke University, Durham, NC

S-nitrosylation and denitrosylation, the covalent attachment and removal of NO groups from the thiol side chain of cysteine, have emerged as important mechanisms for dynamic, posttranslational regulation of most or all classes of protein. S-nitrosylation thereby conveys a large part of the ubiquitous influence of NO on cellular signal transduction, and provides a prototypic example of redox-based physiological regulation. Accumulating evidence suggests that alterations in S-nitrosylation-regulated signaling contribute to human disease.

Nitric Oxide - A Molecular "Switch" in Redox Signaling

Bruce A. Freeman, PhD, Department of Pharmacology and Chemical Biology, University of Pittsburgh, PA

Over the last 50 years, the posttranslational modification (PTM) of proteins has emerged as a central mechanism for cells to regulate metabolism, growth, differentiation, cell-cell interactions, and immune responses. By influencing protein structure and function, PTM leads to a multiplication of proteome diversity. Redox-dependent PTMs, mediated by environmental and endogenously-generated reactive species, exert both cell signaling and toxicological actions in organisms. PTMs induced by the electrophilic byproducts of redox reactions most frequently occur at protein thiols, with other nucleophilic amino acids serving as less favorable targets. Advances in mass spectrometry and affinity chemistry strategies have improved the detection of electrophile-induced protein modifications both in vitro and in vivo, revealing a high degree of amino acid and protein selectivity of electrophilic PTM. Some PTMs that are induced by electrophilic products of redox reactions appear to modulate physiological signaling pathways that have evolved to act as sensors of oxidative conditions. The identification of these biological targets of electrophiles has motivated further study of the functional impact of various PTM reactions on specific signaling pathways and how this might impact organisms. We also now appreciate that nitric oxide profoundly stimulates the post-translational modification of proteins via the formation of multiple reactive byproducts. This presentation will describe the formation, signaling actions and potential therapeutic uses of electrophilic fatty acid derivatives stemming from these reactions.

NO-Modification of Biomolecules in the Lung Lining: Inflammatory Implications

Andrew Gow, PhD, Rutgers University, Piscataway, NJ

The biological chemistry of Nitric Oxide (NO) is complex in that there are multiple reactive targets and its reactivity is flux rate dependent. However, this very complexity undoubtedly lies at the heart of how NO is capable of controlling such a wide range of biological processes. Nowhere is this more apparent than in the lung where NO controls a wide range of functions ranging from bronchial and vascular tone, to innate immunity and development; and there are multiple reactive targets. These reactive targets allow for the generation of novel NO-modified biomolecules such as S-nitrosylated Surfactant Protein D and nitrated lipids. We have investigated the capability of NO-modified biomolecules to activate cellular signaling pathways in both epithelial and monocytic cells. Our results indicate that these compounds are capable of eliciting a variety of responses and thus may provide a mechanism for the multifarious nature of NO. Specifically, we have begun to investigate how such NO-modifications are involved in a NOS-mediated lung injury model, intratracheal bleomycin administration. Our observations within this model indicate demonstrate the importance of NO-modified biomolecules in mediating lung injury and repair.

Pathophysiological Functions of Reactive Nitrogen Species in Vascular Disorders

Harry Ischiropoulos, PhD, Departments of Pediatrics and Pharmacology, The Children's Hospital of Philadelphia Research Institute and The University of Pennsylvania Philadelphia, PA

Over the last decade basic and translational medicine discoveries have implicated alterations in nitric oxide (NO) metabolism in atherothrombotic diseases. Specifically, we have employed mass-spectrometry-based proteomic approaches to identify site specific NO-mediated oxidative modifications to protein targets of pathobiological relevance to atherothrombotic vascular diseases. Published and new data will be presented relating to the functional consequences of fibrinogen modifications by NO-derived oxidants in animal models of atherosclerosis and in humans. Protein tyrosine nitration also results in the induction of immune responses in humans and animal models. Recent studies have indicated the presence of circulating immunoglobulins that specifically recognize the modified nitrated tyrosine residues in proteins. Preliminary data have indicated that the levels of these specific circulating immunoglobulins are strong predictors of angiographic evidence of coronary artery disease, as well as its acute adverse complications including incident risks for experiencing myocardial infarction, stroke or death. The data suggest that nitrated fibrinogen and immunoglobulins to nitrated proteins may represent two novel prognostic parameters for identifying individuals at increased risk for subsequent adverse cardiac outcomes.

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