Chemical basis of inflammation-induced carcinogenesis

Chronic inflammation induced by biological, chemical, and physical factors has been associated with increased risk of human cancer at various sites. Inflammation activates a variety of inflammatory cells, which induce and activate several oxidant-generating enzymes such as NADPH oxidase, inducible nitric oxide synthase, myeloperoxidase, and eosinophil peroxidase. These enzymes produce high concentrations of diverse free radicals and oxidants including superoxide anion, nitric oxide, nitroxyl, nitrogen dioxide, hydrogen peroxide, hypochlorous acid, and hypobromous acid, which react with each other to generate other more potent reactive oxygen and nitrogen species such as peroxynitrite. These species can damage DNA, RNA, lipids, and proteins by nitration, oxidation, chlorination, and bromination reactions, leading to increased mutations and altered functions of enzymes and proteins (e.g., activation of oncogene products and/or inhibition of tumor-suppressor proteins) and thus contributing to the multistage carcinogenesis process. Appropriate treatment of inflammation should be explored further for chemoprevention of human cancers.     

Suppression of inducible nitric oxide synthase and cyclooxygenase-2 by cell-permeable superoxide dismutase in lipopolysaccharide-stimulated BV-2 microglial cells

Oxidative stress plays a pivotal role in uncontrolled neuro-inflammation leading to many neurological diseases including Alzheimer’s. One of the major antioxidant enzymes known to prevent deleterious effects due to oxidative stress is Cu,Zn-superoxide dismutase (SOD). In this study, we examined the regulatory function of SOD on the LPS-induced signaling pathways leading to NF-kappaB activation, expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2), in BV-2 cells using cell-permeable SOD. Treatment of BV-2 cells with cell-permeable SOD led to a decrease in LPS-induced reactive oxygen species (ROS) generation and significantly inhibited protein and mRNA levels of iNOS and COX-2 upregulated by LPS. Production of NO and PGE2 in LPS stimulated BV-2 cells was significantly abrogated by pretreatment with a cell-permeable SOD fusion protein. Furthermore, cell-permeable SOD inhibited LPS-induced NF-kappaB DNA-binding activity and activation of MAP kinases including ERK, JNK, and p38 in BV-2 cells. These data indicate that SOD has a regulatory function for LPS-induced NF-kappaB activation leading to expression of iNOS and COX-2 in BV-2 cells and suggest that cell-permeable SOD is a feasible therapeutic agent for regulation of ROS-related neurological diseases.     

诱导型一氧化氮合酶抑制剂研发现状

一氧化氮(NO)对炎症性疾病的治疗作用近来引起了广泛的重视。诱导型一氧化合成酶(iNOS)被发现广泛地参与趋炎因子表达和反应性氧化产物(ROS)/反应性氮化产物(RNS)的产生,从而进一步证明了一氧化氮在炎症病理发生发展中的关键作用。由于传统的抗炎药物环氧合酶-2(COX-2)抑制剂被报导有较多副作用,新型抑制炎症药物的研究开发势在必行。本文分别介绍了化学来源、生物来源、植物来原性iNOS抑制剂阻的开发、研究现状,阐述了其在断炎症信息传递通道中的作用。表明了iNOS抑制剂防止炎症损害的相关机理,提出iNOs不仅能在初始阶段影响炎症的发生,也对抑制和终结炎症有作用。最后进一步介绍了用中草药研发iNOs抑制剂的可能性,展望了于中药在该领域内的巨大前景。     

Hemin inhibits cyclooxygenase-2 expression through nuclear factor-kappa B activation and ornithine decarboxylase expression in 12-O-tetradecanoylphorbol-13-acetate-treated mouse skin

Inflammation induced by various stimuli has been found to be associated with increased risk for most types of human cancer. Inflammation facilitates the initiation of normal cells, as well as the growth of initiated cells and their progression to malignancy through production of proinflammatory cytokines and diverse reactive oxygen/nitrogen species. These also activate the signaling molecules that are involved in inflammation and carcinogenesis. Our previous studies have demonstrated that hemin inhibited 7,12-dimethylbenz[a]anthracene (DMBA)-induced bacterial mutagenesis and oxidative DNA damage, reduced the level of DNA-DMBA adduct and 12-O-tetradecanoylphorobl-13-acetate (TPA)-induced tumor formation in DMBA-initiated ICR mouse skin, and inhibited myeloperoxidase and ornithine decarboxylase (ODC) activity and H(2)O(2) formation in TPA-treated mouse skin. In the present study, to further elucidate the molecular mechanisms underlying the chemopreventive activity of hemin, its effect on the expression of ODC and cyclooxygenase (COX)-2, and the activation of nuclear factor-kappa B (NF-kappaB) and mitogen-activated protein kinases (MAPKs) regulating these proteins were explored in mouse skin with TPA-induced inflammation. Topically applied hemin inhibited ear edema and epidermal thickness in mice treated with TPA. Pretreatment with hemin reduced the expression of ODC and COX-2, and also reduced NF-kappaB activation in TPA-stimulated mouse skin. In addition, hemin suppressed the TPA-induced activation of extracellular signal-regulated protein kinase (ERK) and p38 MAPK in a dose-dependent manner. Taken together, hemin inhibited TPA-induced COX-2 expression by altering NF-kappaB signaling pathway via ERK and p38 MAPK, as well as TPA-induced ODC expression in mouse skin. Thereby, hemin may be an attractive candidate for a chemopreventive agent.     

Measurement and meaning of markers of reactive species of oxygen, nitrogen and sulfur in healthy human subjects and patients with inflammatory joint disease

Reactive species of oxygen, nitrogen and sulfur play cell signalling roles in human health, e.g. recent studies have shown that increased dietary nitrate, which is a source of RNS (reactive nitrogen species), lowers resting blood pressure and the oxygen cost of exercise. In such studies, plasma nitrite and nitrate are readily determined by chemiluminescence. At sites of inflammation, such as the joints of RA (rheumatoid arthritis) patients, the generation of ROS (reactive oxygen species) and RNS overwhelms antioxidant defences and one consequence is oxidative/nitrative damage to proteins. For example, in the inflamed joint, increased RNS-mediated protein damage has been detected in the form of a biomarker, 3-nitrotyrosine, by immunohistochemistry, Western blotting, ELISAs and MS. In addition to NO?, another cell-signalling gas produced in the inflamed joint is H2S (hydrogen sulfide), an RSS (reactive sulfur species). This gas is generated by inflammatory induction of H2S-synthesizing enzymes. Using zinc-trap spectrophotometry, we detected high (micromolar) concentrations of H2S in RA synovial fluid and levels correlated with clinical scores of inflammation and disease activity. What might be the consequences of the inflammatory generation of reactive species? Effects on inflammatory cell-signalling pathways certainly appear to be crucial, but in the current review we highlight the concept that ROS/RNS-mediated protein damage creates neoepitopes, resulting in autoantibody formation against proteins, e.g. type-II collagen and the complement component, C1q. These autoantibodies have been detected in inflammatory autoimmune diseases.     

Prolonged poly(ADP-ribose) polymerase-1 activity regulates JP-8-induced sustained cytokine expression in alveolar macrophages

Environmental pollutants inducing oxidative stress stimulate chronic inflammatory responses in the lung leading to pulmonary tissue dysfunction. In response to oxidative stress, alveolar macrophages produce both reactive oxygen species and reactive nitrogen species, which induce the expression of a wide variety of immune-response genes. We found that a prolonged exposure of alveolar macrophages to a nonlethal dose (8 microg/ml) of JP-8, the kerosene-based hydrocarbon jet fuel, induced the persistent expression of IL-1, iNOS, and COX-2, as well as cell adhesion molecules (ICAM-1 and VCAM-1). Because poly(ADP-ribose) polymerase (PARP-1), a coactivator of NF-kappaB, regulates inflammatory responses and associated disorders in the airways, we determined whether JP-8 induces the poly(ADP-ribosyl)ation automodification of PARP-1 in alveolar macrophages. We observed that PARP-1 is activated in a time-dependent manner, which was temporally coincident with the prolonged activation of NF-kappaB and with the augmented expression of the proinflammatory factors described above. The 4 microg/ml dilution of JP-8 also increased the activity of PARP-1 as well as the expression of iNOS and COX-2, indicating that lower doses of JP-8 also affect the regulation of proinflammatory factors in pulmonary macrophages. Together, these results demonstrate that an extensive induction of PARP-1 might coordinate the persistent expression of proinflammatory mediators in alveolar macrophages activated by aromatic hydrocarbons that can result in lung injury from occupational exposure.     

iNOS signaling interacts with COX-2 pathway in colonic fibroblasts

COX-2 and iNOS are two major inflammatory mediators implicated in colorectal inflammation and cancer. Previously, the role of colorectal fibroblasts involved in regulation of COX-2 and iNOS expression was largely ignored. In addition, the combined interaction of COX-2 and iNOS signalings and their significance in the progression of colorectal inflammation and cancer within the fibroblasts have received little investigation. To address those issues, we investigated the role of colonic fibroblasts in the regulation of COX-2 and iNOS gene expression, and explored possible mechanisms of interaction between COX-2 and iNOS signalings using a colonic CCD-18Co fibroblast line and LPS, a potential stimulator of COX-2 and iNOS. Our results clearly demonstrated that LPS activated COX-2 gene expression and enhanced PGE(2) production, stimulated iNOS gene expression and promoted NO production in the fibroblasts. Interestingly, activation of COX-2 signaling by LPS was not involved in activation of iNOS signaling, while activation of iNOS signaling by LPS contributed in part to activation of COX-2 signaling. Further analysis indicated that PKC plays a major role in the activation and interaction of COX-2 and iNOS signalings induced by LPS in the fibroblasts.     

Nitric oxide in gastrointestinal epithelial cell carcinogenesis: linking inflammation to oncogenesis

Chronic inflammation of gastrointestinal tissues is a well-recognized risk factor for the development of epithelial cell-derived malignancies. Although the inflammatory mediators linking chronic inflammation to carcinogenesis are numerous, current information suggests that nitric oxide (NO) contributes to carcinogenesis during chronic inflammation. Inducible nitric oxide synthase (iNOS), expressed by both macrophages and epithelial cells during inflammation, generates the bioreactive molecule NO. In addition to causing DNA lesions, NO can directly interact with proteins by nitrosylation and nitosation reactions. The consequences of protein damage by NO appear to be procarcinogenic. For example, NO inhibits DNA repair enzymes such as human 8-oxodeoxyguanosine DNA glycosylase 1 and blocks apoptosis via nitrosylation of caspases. These cellular events permit DNA damage to accumulate, which is required for the numerous mutations necessary for development of invasive cancer. NO also promotes cancer progression by functioning as an angiogenesis factor. Strategies to inhibit NO generation during chronic inflammation or to scavenge reactive nitrogen species may prove useful in decreasing the risk of cancer development in chronic inflammatory gastrointestinal diseases.     

Oxidative and nitrative DNA damage in animals and patients with inflammatory diseases in relation to inflammation-related carcinogenesis

Infection and chronic inflammation are proposed to contribute to carcinogenesis through inflammation-related mechanisms. Infection with hepatitis C virus, Helicobacter pylori and the liver fluke, Opisthorchis viverrini (OV), are important risk factors for hepatocellular carcinoma (HCC), gastric cancer and cholangiocarcinoma, respectively. Inflammatory bowel diseases (IBDs) and oral diseases, such as oral lichen planus (OLP) and leukoplakia, are associated with colon carcinogenesis and oral squamous cell carcinoma (OSCC), respectively. We performed a double immunofluorescence labeling study and found that nitrative and oxidative DNA lesion products, 8-nitroguanine and 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG), were formed and inducible nitric oxide synthase (iNOS) was expressed in epithelial cells and inflammatory cells at the site of carcinogenesis in humans and animal models. Antibacterial, antiviral and antiparasitic drugs dramatically diminished the formation of these DNA lesion markers and iNOS expression. These results suggest that oxidative and nitrative DNA damage occurs at the sites of carcinogenesis, regardless of etiology. Therefore, it is considered that excessive amounts of reactive nitrogen species produced via iNOS during chronic inflammation may play a key role in carcinogenesis by causing DNA damage. On the basis of our results, we propose that 8-nitroguanine is a promising biomarker to evaluate the potential risk of inflammation-mediated carcinogenesis.     

Nitrative DNA damage in inflammation and its possible role in carcinogenesis.

Chronic inflammation has long been recognized as a risk factor for human cancer at various sites. Examples include Helicobacter pylori-induced gastritis for gastric cancer, inflammatory bowel disease (ulcerative colitis and Crohn's disease) for colorectal cancer and chronic viral hepatitis for liver cancer. Here we review the role in carcinogenesis of nitrative damage to nucleic acids, DNA and RNA, which occurs during inflammation through the generation of reactive nitrogen species, such as peroxynitrite, nitroxyl, and nitrogen dioxide. Enhanced formation of 8-nitroguanine, representative of nitrative damage to nucleobases, has been detected in various inflammatory conditions. The biochemical nature of DNA damage mediated by reactive nitrogen species is discussed in relation to its possible involvement in mutations, genetic instability, and cell death. Better understanding of the mechanisms and role of such nitrative damage in chronic inflammation-associated human cancer is a necessary basis to develop new strategies for cancer prevention by modulating the process of inflammation.     

Differential pattern of inflammatory molecule regulation in intestinal epithelial cells stimulated with IL-1

To better predict the consequences of blocking signal transduction pathways as a means of controlling intestinal inflammation, we are characterizing the pathways up-regulated by IL-1 in intestinal epithelial cells (IEC). IL-1beta induced increased mRNA levels of MIP-2, MCP-1, RANTES, inducible NO synthase (iNOS), and cyclooxygenase-2 (COX-2) in the IEC-18 cell line. IL-1beta activated NF-kappaB but not ERK or p38. Infecting cells with adenovirus expressing a mutated gene for IkappaBalpha (IkappaBAA) blocked IL-1-induced mRNA increases in MIP-2, MCP-1, and iNOS but not COX-2 or RANTES. Expression of IkappaBAA attenuated the IL-1-induced increase in COX-2 protein. Unexpectedly, RANTES mRNA increased, and protein was secreted by cells expressing IkappaBAA in the absence of IL-1. Adenovirus-expressing IkappaBAA, blocking protein synthesis, and IL-1beta all resulted in activation of JNK. The JNK inhibitor SP600125 prevented the RANTES increases by all three stimuli. A human enterocyte line was similarly examined, and both NF-kappaB and JNK regulate IL-1-induced RANTES secretion. We conclude that in IEC-18, IL-1beta-induced increases in mRNA for MIP-2, MCP-1, and iNOS are NF-kappaB-dependent, whereas regulation of RANTES mRNA is independent of NF-kappaB but is positively regulated by JNK. IL-1beta-induced mRNA increases in COX-2 mRNA are both NF-kappaB- and MAPK-independent but the translation of COX-2 protein is NF-kappaB-dependent. This pattern of signaling due to a single stimulus exposed the complexities of regulating inflammatory genes in IEC.     

Role of pro-oxidants and antioxidants in the anti-inflammatory and apoptotic effects of curcumin (diferuloylmethane)

Extensive research within the past half-century has indicated that curcumin (diferuloylmethane), a yellow pigment in curry powder, exhibits antioxidant, anti-inflammatory, and proapoptotic activities. We investigated whether the anti-inflammatory and proapoptotic activities assigned to curcumin are mediated through its prooxidant/antioxidant mechanism. We found that TNF-mediated NF-kappaB activation was inhibited by curcumin; and glutathione reversed the inhibition. Similarly, suppression of TNF-induced AKT activation by curcumin was also abrogated by glutathione. The reducing agent also counteracted the inhibitory effects of curcumin on TNF-induced NF-kappaB-regulated antiapoptotic (Bcl-2, Bcl-xL, IAP1), proliferative (cyclin D1), and proinflammatory (COX-2, iNOS, and MMP-9) gene products. The suppression of TNF-induced AP-1 activation by curcumin was also reversed by glutathione. Also, the direct proapoptotic effects of curcumin were inhibited by glutathione and potentiated by depletion of intracellular glutathione by buthionine sulfoximine. Moreover, curcumin induced the production of reactive oxygen species and modulated intracellular GSH levels. Quenchers of hydroxyl radicals, however, were ineffective in inhibiting curcumin-mediated NF-kappaB suppression. Further, N-acetylcysteine partially reversed the effect of curcumin. Based on these results we conclude that curcumin mediates its apoptotic and anti-inflammatory activities through modulation of the redox status of the cell.     

Differential regulation of cyclooxygenase-2 and inducible nitric oxide synthase by 4-hydroxynonenal in human osteoarthritic chondrocytes through ATF-2/CREB-1 transactivation and concomitant inhibition of NF-kappaB signaling cascade

4-hydroxynonenal (HNE), a lipid peroxidation end product, is produced abundantly in osteoarthritic (OA) articular tissues and was recently identified as a potent catabolic factor in OA cartilage. In this study, we provide additional evidence that HNE acts as an inflammatory mediator by elucidating the signaling cascades targeted in OA chondrocytes leading to cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) gene expression. HNE induced COX-2 protein and mRNA levels with accompanying increases in prostaglandin E2 (PGE(2)) production. In contrast, HNE had no effect on basal iNOS expression or nitric oxide (NO) release. However, HNE strongly inhibited IL-1beta-induced iNOS or NO production. Transient transfection experiments revealed that the ATF/CRE site (-58/-53) is essential for HNE-induced COX-2 promoter activation and indeed HNE induced ATF-2 and CREB-1 phosphorylation as well as ATF/CRE binding activity. Overexpression of p38 MAPK enhanced the HNE-induced ATF/CRE luciferase reporter plasmid activation, COX-2 synthesis and promoter activity. HNE abrogated IL-1beta-induced iNOS expression and promoter activity mainly through NF-kappaB site (-5,817/-5,808) possibly via suppression of IKKalpha-induced IkappaBalpha phosphorylation and NF-kappaB/p65 nuclear translocation. Upon examination of upstream signaling components, we found that IKKalpha was inactivated through HNE/IKKalpha adduct formation. Taken together, these findings illustrate the central role played by HNE in the regulation of COX-2 and iNOS in OA. The aldehyde induced selectively COX-2 expression via ATF/CRE activation and inhibited iNOS via IKKalpha inactivation.