Role of cyclooxygenase-2 and inducible nitric oxide synthase in pancreatic cancer

BACKGROUND AND AIM: Recently, it has been recognized that both cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) produce important endogenous factors of human tumor progression. However, the clinicopathological and biological significance of the expression of COX-2 and iNOS in pancreatic cancer remains unclear. The objective of this study is to find the possible roles and clinical significance of COX-2 and iNOS expression in pancreatic cancer. METHODS: Seventy-two pancreatic adenocarcinoma tissue specimens were obtained through surgical resection. We investigated the immunohistochemical expression of COX-2 and iNOS in respect to variable clinicopathological characteristics, proliferation activity (by Ki-67 expression), apoptosis (by terminal deoxyribonucleotidyl transferase-mediated dUTP nick-end labeling stain), and microvessel density (by CD34 expression; angiogenesis). RESULTS: Immunohistochemical investigations demonstrated immunolabeling of tumor cells with the primary antibodies, bovine anti-iNOS and anti-COX-2 antibodies. The COX-2 and iNOS positive rates were 41.7 and 66.7%, respectively. There was significant correlation between positive COX-2 and positive iNOS expression (P = 0.043). The proliferation index (Ki-67 labeling index) was higher in COX-2 positive specimens compared to COX-2 negative specimen (P = 0.015). The apoptotic index of positive iNOS expressions was significantly higher than negative expressions (P < 0.001). The expression of COX-2 and iNOS proteins did not correlate with age, sex, serum bilirubin, CA-19-9, location, size, American Joint Committee on Cancer stage, differentiation, distant metastasis, patient survival, or microvessel density. CONCLUSIONS: Although the pattern of positive expression was similar in both enzymes, the effect on tumor progression differed; iNOS expression may play a role in apoptosis of tumor cell, while COX-2 expression may contribute to tumor proliferation. However, COX-2 and iNOS expression is not related to prognosis in patients with pancreatic cancer.     

Interaction between inducible nitric oxide synthase and cyclooxygenase-2 after cerebral ischemia

Focal cerebral ischemia is associated with expression of both inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2), enzymes whose reaction products contribute to the evolution of ischemic brain injury. We tested the hypothesis that, after cerebral ischemia, nitric oxide (NO) produced by iNOS enhances COX-2 activity, thereby increasing the toxic potential of this enzyme. Cerebral ischemia was produced by middle cerebral artery occlusion in rats or mice. Twenty-four hours after ischemia in rats, iNOS-immunoreactive neutrophils were observed in close proximity (<20 μm) to COX-2-positive cells at the periphery of the infarct. In the olfactory bulb, only COX-2 positive cells were observed. Cerebral ischemia increased the concentration of the COX-2 reaction product prostaglandin E₂ (PGE₂) in the ischemic area and in the ipsilateral olfactory bulb. The iNOS inhibitor aminoguanidine reduced PGE₂ concentration in the infarct, where both iNOS and COX-2 were expressed, but not in the olfactory bulb, where only COX-2 was expressed. Postischemic PGE₂ accumulation was reduced significantly in iNOS null mice compared with wild-type controls (C57BL/6 or SV129). The data provide evidence that NO produced by iNOS influences COX-2 activity after focal cerebral ischemia. Pro-inflammatory prostanoids and reactive oxygen species produced by COX-2 may be a previously unrecognized factor by which NO contributes to ischemic brain injury. The pathogenic effect of the interaction between NO, or a derived specie, and COX-2 is likely to play a role also in other brain diseases associated with inflammation.     

Immunocytochemical evidence for inducible nitric oxide synthase and cyclooxygenase-2 expression with nitrotyrosine formation in human hibernating myocardium

Background: Myocardial hibernation may result from repetitive episodes of transient ischaemia leading to prolonged dysfunction. Inducible nitric oxide synthase (iNOS) expression has been demonstrated in animals following brief, non-lethal ischaemia-reperfusion injury. We therefore, hypothesised that in human hibernating myocardium: 1) iNOS would be present; 2) the reaction of nitric oxide and superoxide would form the strong oxidant peroxynitrite; 3) that this process would be accompanied by the expression of cyclooxygenase-2 (Cox-2) which interacts with NOS and whose products could further affect myocardial function. Method and results: In sixteen patients with coronary artery disease (CAD), left ventricular biopsies were obtained from chronically dysfunctional segments subtended by a stenotic artery (> 75 %) and shown to be viable by ¹⁸F-fluorodeoxyglucose positron emission tomography. Comparison was made with myocardial biopsies (n = 8) from normally contracting myocardium in patients undergoing coronary surgery, from unused transplant donors and at post-mortem. Regional wall motion score improved in all patients 6 months post-revascularisation (from 2.7 ± 0.7 to 1.5 ± 0.5; p < 0.001), confirming hibernation. Immunocytochemistry localized reactivity to iNOS, Cox-2 and nitrotyrosine (a marker of peroxynitrite formation) to cardiomyocytes from hibernating segments. No difference in reactivity to endothelial NOS was seen between hibernating and control cardiomyocytes. Conclusion: Cox-2 and iNOS are co-expressed in hibernating myocardium with nitrotyrosine suggesting nitric oxide production and peroxynitrite formation. We propose that this is secondary to ischaemia-reperfusion and that the products of these enzymes may have consequences for myocardial contractile function and survival. Received: 11 February 2002, Returned for revision: 14 February 2002, Revision received: 4 March 2002, Accepted: 11 March 2002     

Heterogeneous expression of cyclooxygenase-2 and inducible nitric oxide synthase within colorectal tumors: Correlation with tumor angiogenesis

BackgroundRecent studies have shown that the cyclooxygenase (COX) and the inducible nitric oxide synthase (iNOS) pathways are involved in the development of tumor angiogenesis in human cancers.AimsTo investigate whether a different pattern of COX-2 and iNOS expression/activity exists within different areas of colorectal tumors and to analyze the relationship between these two enzymes and tumor angiogenesis.MethodsMicrovessel density (MVD) and COX-2, iNOS, vascular endothelial growth factor (VEGF) and VEGF receptor-2 (VEGFR-2) protein expression were evaluated at both the invasive front (IF) and the tumor center (TC) in 46 human colorectal cancer specimens. We also investigated the concentration of PGE₂ and NO at the same sites.ResultsCOX-2 and iNOS protein expression and activity were significantly higher within the IF than the TC of the tumor specimens. Similarly, MVD and VEGF/VEGFR-2 expression significantly increased from the TC to the IF. Only COX-2 expression was significantly correlated with MVD and VEGF/VEGFR-2 expression at both the TC and the IF.ConclusionOur study shows a heterogeneous expression of COX-2 and iNOS in colorectal cancer. The up-regulation of COX-2 at the IF parallels an increase in vessel density and VEGF/VEGFR-2 expression, thus supporting the hypothesis that the tumor periphery is the most aggressive portion of a colorectal tumor.     

Up-regulation of inducible nitric oxide synthase expression in cancer-prone p53 knockout mice

High concentrations of nitric oxide (NO) cause DNA damage and apoptosis in many cell types. Thus, regulation of NO synthase (NOS) activity is essential for minimizing effects of cytotoxic and genotoxic nitrogen oxide species. We have shown previously that NO-induced p53 protein accumulation down-regulates basal and cytokine-modulated inducible NOS (NOS2) expression in human cells in vitro. To further characterize the feedback loop between NOS2 and p53, we have investigated NO production, i.e., urinary nitrate plus nitrite excretion, and NOS2 expression in homozygous p53 knockout (KO) mice. We report here that untreated p53 KO mice excreted 70% more nitrite plus nitrate than mice with wild-type (wt) p53. NOS2 protein expression was constitutively detected in the spleen of untreated p53 KO mice, whereas it was undetectable in the spleen of wt p53 controls. Upon treatment with heat-inactivated Corynebacterium parvum, urinary nitrite plus nitrate excretion of p53 KO mice exceeded that of wt controls by approximately 200%. C. parvum treatment also induced p53 accumulation in the liver. Splenectomy reduced the NO output of C. parvum-treated p53 KO mice but not of wt p53 controls. Although NO production and NOS2 protein expression were increased similarly in KO and wt p53 mice 10 days after injection of C. parvum, NOS2 expression returned to baseline levels only in wt p53 controls while remaining up-regulated in p53 KO mice. These genetic and functional data indicate that p53 is an important transrepressor of NOS2 expression in vivo and attenuates excessive NO production in a regulatory negative feedback loop.     

Nitric oxide-induced p53 accumulation and regulation of inducible nitric oxide synthase expression by wild-type p53.

The tumor suppressor gene product p53 plays an important role in the cellular response to DNA damage from exogenous chemical and physical mutagens. Therefore, we hypothesized that p53 performs a similar role in response to putative endogenous mutagens, such as nitric oxide (NO). We report here that exposure of human cells to NO generated from an NO donor or from overexpression of inducible nitric oxide synthase (NOS2) results in p53 protein accumulation. In addition, expression of wild-type (WT) p53 in a variety of human tumor cell lines, as well as murine fibroblasts, results in down-regulation of NOS2 expression through inhibition of the NOS2 promoter. These data are consistent with the hypothesis of a negative feedback loop in which endogenous NO-induced DNA damage results in WT p53 accumulation and provides a novel mechanism by which p53 safeguards against DNA damage through p53-mediated transrepression of NOS2 gene expression, thus reducing the potential for NO-induced DNA damage.     

诱导型一氧化氮合酶、环氧合酶-2与乳腺癌淋巴管生成的关系及其临床意义

目的探讨诱导型一氧化氮合酶(iNOS)和环氧合酶-2(COX-2)在乳腺癌间质淋巴管生成中的作用机制及其临床意义。方法收集乳腺浸润性导管癌组织90例和乳腺纤维腺瘤组织30例,应用寡核苷酸探针原位杂交法检测组织中iNOS和COX-2的mRNA表达,另以Elivision免疫组化法检测淋巴管内皮细胞透明质酸受体1(LYVE-1)和血管内皮生长因子-C(VEGF-C)蛋白的表达情况,分析iNOS和COX-2与LYVE-1标记的淋巴管密度(LVD)以及VEGF-C的关系。结果在90例乳腺癌中iNOS、COX-2的mRNA阳性率分别为66.7%、68.9%,LVD为(8.03±2.26)个/HPF,VEGF-C表达阳性率为47.8%,与良性对照组比较均有显著差异(P<0.01)。iNOS mRNA表达与乳腺癌肿瘤大小、分期、LVD和VEGF-C表达呈正相关(P<0.01);COX-2 mRNA表达与肿瘤分级、淋巴结转移、分期和LVD呈正相关(P<0.05),与VEGF-C表达无显著相关(P>0.05);iNOS和COX-2的mRNA阳性表达呈正相关(P<0.05)。结论 iNOS和COX-2具有协同促进乳腺癌淋巴管生成及侵袭转移的作用,有望在乳腺癌的临床预后判断及靶向治疗中发挥作用。     

Helicobacter pylori stimulates inducible nitric oxide synthase expression and activity in a murine macrophage cell line

BACKGROUND & AIMS: Helicobacter pylori uniquely colonizes the human stomach and produces gastric mucosal inflammation. High-output nitric oxide production by inducible nitric oxide synthase (iNOS) is associated with immune activation and tissue injury. Because mononuclear cells comprise a major part of the cellular inflammatory response to H. pylori infection, the ability of H. pylori to induce iNOS in macrophages was assessed. METHODS: H. pylori preparations were added to RAW 264.7 murine macrophages, and iNOS expression was assessed by Northern blot analysis, enzyme activity assay, and NO2- release. RESULTS: Both whole H. pylori and French press lysates induced concentration-dependent NO2- production, with peak levels 20-fold above control. These findings were paralleled by marked increases in iNOS messenger RNA and enzyme activity levels. iNOS expression was synergistically increased with interferon gamma, indicating that the H. pylori effect can be amplified by other macrophage-activating factors. Studies of lipopolysaccharide (LPS) content and polymyxin B inhibition of LPS suggested that the H. pylori effect was attributable to both LPS- dependent and -independent mechanisms. CONCLUSIONS: iNOS expression in macrophages is activated by highly stable H. pylori products and may play an important role in the pathogenesis of H. pylori-associated gastric mucosal disease. (Gastroenterology 1996 Dec;111(6):1524-33)     

Induction of inducible nitric oxide synthase (iNOS) expression by oxLDL inhibits macrophage derived foam cell migration

Objective

Deletion of inducible nitric oxide synthase (iNOS) in apolipoprotein E knockout mice was shown to mitigate the extent of arteriosclerosis. Oxidized low density lipoprotein (oxLDL) inhibits macrophage migration and traps foam cells, possibly through a mechanism involving oxidative stress. Here, we addressed whether a reduction of iNOS-mediated oxidative stress remobilizes macrophage-derived foam cells and may reverse plaque formation.

Methods

Migration of RAW264.7 cells and bone marrow cells was quantified using a modified Boyden chamber. iNOS expression, phalloidin staining, focal adhesion kinase phosphorylation, lipid peroxides, nitric oxide (NO) and reactive oxygen species (ROS) production were assessed.

Results

oxLDL treatment significantly reduced cell migration compared to unstimulated cells (p < 0.05). This migratory arrest was reversed by co-incubation with a pharmacologic iNOS inhibitor 1400W (p < 0.05) and iNOS-siRNA (p > 0.05). Furthermore, apoE/iNOS double knockout macrophages do not show migratory arrest in response to oxLDL uptake, compared to apoE knockout controls (p > 0.05). We documented significantly increased iNOS expression following oxLDL treatment and downregulation using 1400W and small inhibitory RNA (siRNA). iNOS inhibition was associated with a reduction in NO and peroxynitrite (ONOO–)– and increased superoxide generation. Trolox treatment of RAW264.7 cells restored migration indicating that peroxynitrite mediated lipid peroxide formation is involved in the signaling pathway mediating cell arrest..

Conclusions

Here, we provide pharmacologic and genetic evidence that oxLDL induced iNOS expression inhibits macrophage-derived foam cell migration. Therefore, reduction of peroxynitrite and possibly lipid hydroperoxide levels in plaques represents a valuable therapeutic approach to reverse migratory arrest of macrophage-derived foam cells and to impair plaque formation.     

Expression of inducible nitric oxide synthase and cyclooxygenase-2 in pancreatic adenocarcinoma： Correlation with microvessel density

AIM: Cyclooxygenases (COX) are key enzymes for conversion of arachidonic acid to prostaglandins. Nitric oxide synthase (NOS) is the enzyme responsible for formation of nitric oxide. Both have constitutive and inducible isoforms. The inducible isoforms (iNOS and COX-2) are of great interest as regulators of tumor angiogenesis, tumorigenesis and inflammatory processes. This study was to clarify their role in pancreatic adenocarcinomas. METHODS: We investigated the immunohistochemical iNOS and COX-2 expression in 40 pancreatic ductal adenocarcinomas of different grade and stage. The results were compared with microvessel density and clinicopathological data. RESULTS: Twenty-one (52.5%) of the cases showed iNOS expression, 15 (37.5%) of the cases were positive for COX-2. The immunoreaction was heterogeneously distributed within the tumors. Staining intensity was different between the tumors. No correlation between iNOS and COX-2 expression was seen. There was no relationship with microvessel density. However, iNOS positive tumors developed more often distant metastases and the more malignant tumors showed a higher COX-2 expression. There was no correlation with other clinicopathological data. CONCLUSION: Approximately half of the cases expressed iNOS and COX-2. These two enzymes do not seem to be the key step in angiogenesis or carcinogenesis of pancreatic adenocarcinomas. Due to a low prevalence of COX-2 expression, chemoprevention of pancreatic carcinomas by COX-2 inhibitors can only achieve a limited success.     

Gene therapy with inducible nitric oxide synthase protects against myocardial infarction via a cyclooxygenase-2-dependent mechanism

Although the inducible isoform of NO synthase (iNOS) mediates late preconditioning (PC), it is unknown whether iNOS gene transfer can replicate the cardioprotective effects of late PC, and the role of this protein in myocardial ischemia is controversial. Thus, the cDNA for human iNOS was cloned behind the Rous sarcoma virus (RSV) promoter to create adenovirus (Ad) 5/iNOS lacking E1, E2a, and E3 regions. Intramyocardial injection of Ad5/iNOS in mice increased local iNOS protein expression and activity and markedly reduced infarct size. The infarct-sparing effects of Ad5/iNOS were at least as powerful as those of ischemic PC. The increased iNOS expression was associated with increased cyclooxygenase-2 (COX-2) protein expression and prostanoid levels. Pretreatment with the COX-2-selective inhibitor NS-398 completely abrogated the infarct-sparing actions of Ad5/iNOS, demonstrating that COX-2 is an obligatory downstream effector of iNOS-dependent cardioprotection. We conclude that gene transfer of iNOS (an enzyme commonly thought to be detrimental) affords powerful cardioprotection the magnitude of which is equivalent to that of late PC. This is the first report that upregulation of iNOS, in itself, is sufficient to reduce infarct size. The results provide proof-of-principle for gene therapy against ischemia/reperfusion injury, which increases local myocardial NO synthase levels without the need for continuous intravenous infusion of NO donors and without altering systemic hemodynamics. The data also reveal the existence of a close coupling between iNOS and COX-2, whereby induction of the former enzyme leads to secondary induction of the latter, which in turn mediates the cytoprotective effects of iNOS. We propose that iNOS and COX-2 form a stress-responsive functional module that mitigates ischemia/reperfusion injury.