Cyclooxygenase Enzyme Activity Research Articles

Cyclo-oxygenase inhibition in colorectal adenomas and cancer.

Increasing evidence indicates that Non-steroidal anti-inflammatory drugs (NSAIDs), compounds that inhibit the enzymatic activity of cyclooxygenase (COX), can reduce the number and size of adenomas in patients with familial adenomatous polyposis as well as the incidence of colorectal cancer. The COX enzyme family consists of the classic COX-1 and a second enzyme, COX-2, which is induced by various stimuli, such as mitogens and cytokines. While it is well proven that COX-2 overexpression is a central event in colorectal carcinogenesis, that prostaglandins (PGs) can contribute to tumorigenesis, and that COX-2 selective inhibitors are active chemopreventive agents, the molecular mechanisms by which NSAIDs exert their chemopreventive effect is not fully understood. However, significant advances have been made in understanding the interference of NSAIDs with the pathways that control cell growth and survival even independently from their COX-inhibiting properties, making their use attractive both alone and in combination with standard therapies in the treatment of advanced colorectal cancer. In addition, the recently recognized anti-angiogenic and radiosensitizer properties of COX-2 inhibitors support, further suggest their use in the adjuvant setting.

Increased prostaglandin E 2 concentrations and cyclooxygenase-2 expression in asthmatic subjects with sputum eosinophilia

Background Prostaglandin E 2 (PGE 2) is known to be produced within human airways, but it is not clear whether in airway diseases it can play a deleterious or a beneficial role. Recently it has been reported that PGE 2 can enhance eosinophil survival in vitro. Objective To evaluate whether the concentrations of PGE 2 in asthmatic airways correlate with the number of eosinophils and can be responsible for eosinophil-enhanced survival and to identify the cyclooxygenase isoform contributing to the synthesis of PGE 2 by cells present in asthmatic airways. Methods Reversed-phase high-performance liquid chromatography and/or specific radioimmunoassay was used to measure PGE 2 concentrations in induced sputum supernatants from 14 control and 30 asthmatic subjects. Correlations between concentrations of PGE 2 and the number of eosinophils in induced sputum were evaluated. Expression of cyclooxygenase-2 (COX-2) in induced sputum cells was determined by immunocytochemistry, and the effect of COX-2 inhibition on PGE 2 production was evaluated with the use of radiolabeled arachidonic acid. The effects on eosinophil apoptosis by PGE 2 or induced sputum supernatants were studied by using peripheral blood eosinophils obtained by negative immunomagnetic selection. Results PGE 2 concentrations resulted in elevated samples from asthmatic subjects and directly correlated with the percentage of eosinophils and the concentrations of eosinophilic cationic protein. Immunostaining for COX-2 showed enhanced expression in macrophages of asthmatic subjects when compared with control subjects, and the use of a specific COX-2 inhibitor provided evidence that PGE 2 synthesis was the result of COX-2 enzymatic activity in asthma-induced sputum cells. Supernatant from induced sputum of asthmatic subjects with high eosinophil counts caused a decreased apoptosis of peripheral blood eosinophils when compared with control subjects, and immunoprecipitation of PGE 2 significantly reverted this phenomenon, suggesting that PGE 2 was present in biologically relevant concentrations in induced sputum. Conclusions The results obtained suggest that COX-2 expression in alveolar macrophages from asthmatic subjects may contribute to enhanced eosinophil survival through an increased PGE 2 production. (J Allergy Clin Immunol 2003;112:709-16.)

Neuronal overexpression of cyclooxygenase-2 increases cerebral infarction.

Increases in COX-2 enzymatic activity and prostaglandin production have been associated with neuronal injury in both acute and age-related degenerative neurological diseases. In this study, we tested the effects of increased COX-2 activity in a model of transient focal ischemia using a transgenic mouse model in which human COX-2 is constitutively expressed selectively in neurons of the striatum, cerebral cortex, and hippocampus. These COX-2 transgenic mice harbor elevated levels of PGE(2) that are 10-fold higher than nontransgenic levels. A significant increase in infarct volume was observed after middle cerebral artery occlusion with 4 days of reperfusion in COX-2 transgenic mice as compared with nontransgenic littermates. Pretreatment of nontransgenic mice with the selective COX-2 inhibitor SC58236 resulted in a significant reduction of infarct volume in nontransgenic mice, consistent with previous pharmacological studies. However, transgenic COX-2 mice treated with SC58236 did not show a significant reduction. This suggests that chronic increases in COX-2 expression and enzymatic activity, which can occur in aging and in pathological states characterized by oxidative stress and chronic inflammatory processes, can lead to downstream cellular changes that have a negative impact on neuronal survival in cerebrovascular disease.

Synergistic inhibitory effect of ascorbic acid and acetylsalicylic acid on prostaglandin E2 release in primary rat microglia.

Ascorbic acid (vitamin C) has been suggested to protect cerebral tissue in a variety of pathophysiological situations such as head trauma, ischemia or Alzheimer's disease. Most of these protective actions have been attributed to the antioxidative capacity of ascorbic acid. Besides the presence of elevated levels of oxygen radicals, prostaglandins produced by neurones and microglial cells seem to play an important role in prolonged tissue damage. We investigated whether ascorbic acid alone inhibits prostaglandin E2 (PGE2) synthesis and may augment the inhibitory effect of acetylsalicylic acid on prostaglandin synthesis. Ascorbic acid dose-dependently inhibited PGE2 synthesis in lipopolysaccharide-treated primary rat microglial cells (IC50 = 3.70 micro m). In combination with acetylsalicylic acid (IC50 = 1.85 micro m), ascorbic acid augmented the inhibitory effect of acetylsalicylic acid on PGE2 synthesis (IC50 = 0.25 micro m in combination with 100 micro m ascorbic acid). Ascorbic acid alone or in combination with acetylsalicylic acid did not inhibit cyclooxygenase-2 (COX-2) protein synthesis but inhibited COX-2 enzyme activity. Our results show that ascorbic acid and acetylsalicylic acid act synergistically in inhibiting PGE2 synthesis, which may help to explain a possible protective effect of ascorbic acid in various brain diseases.

Ceramide-induced and Age-associated Increase in Macrophage COX-2 Expression Is Mediated through Up-regulation of NF-κB Activity

We have shown that the age-associated increase in lipopolysaccharide (LPS)-stimulated macrophages (M phi) prostaglandin E(2) (PGE(2)) production is because of ceramide-induced up-regulation of cyclooxygenase (COX)-2 transcription that leads to increased COX-2 expression and enzyme activity. To determine the mechanism of the age-related and ceramide-dependent increase in COX-2 transcription, we investigated the role of various transcription factors involved in COX-2 gene expression. The results showed that LPS-initiated activations of both consensus and COX-2-specific NF-kappa B, but not AP-1 and CREB, were significantly higher in M phi from old mice than those from young mice. We further showed that the higher NF-kappa B activation in old M phi was because of greater I kappa B degradation in the cytoplasm and p65 translocation to the nucleus. An I kappa B phosphorylation inhibitor, Bay 11-7082, inhibited NF-kappa B activation, as well as PGE(2) production, COX activity, COX-2 protein, and mRNA expression in both young and old M phi. Similar results were obtained by blocking NF-kappa B binding activity using a NF-kappa B decoy. Furthermore, NF-kappa B inhibition resulted in significantly greater reduction in PGE(2) production and COX activity in old compared with young M phi. Addition of ceramide to the young M phi, in the presence or absence of LPS, increased NF-kappa B activation in parallel with PGE(2) production. Bay 11-7082 or NF-kappa B decoy prevented this ceramide-induced increase in NF-kappa B binding activity and PGE(2) production. These findings strongly suggest that the age-associated and ceramide-induced increase in COX-2 transcription is mediated through higher NF-kappa B activation, which is, in turn, because of a greater I kappa B degradation in old M phi.

Constituents of Hypericum laricifolium and their cyclooxygenase (COX) enzyme activities.

Investigation of the aerial parts of the medicinal plant Hypericum laricifolium led to the isolation of two new natural products, hentriacontanyl caffeate (1a), nonacosanyl caffeate (1b). In addition, stigmasterol, beta-sitosterol, 3-epi-betulinic acid (2), caffeic acid (3), ferulic acid, docosanol, p-hydroxybenzoic acid, 3,4-dimethoxy benzoic acid, quercetin (4), quercetin-3-O-galactoside (5), quercetin-3-O-rutinoside (6), quercetin-3-O-rhamnoside (7), quercetin-3-O-glucuronide (8) and shikimic acid were also isolated. The structures were determined by 1D- and 2D-NMR, mass spectrometry, and chemical transformations. The anti-inflammatory effects of the isolated compounds were discussed briefly.

Prevention of short-term ultraviolet B radiation-mediated damages by resveratrol in SKH-1 hairless mice

Nonmelanoma skin cancer is the most common cancer among humans and solar UV radiation, particularly its UVB component (290–320 nm), is its major cause. One way to reduce the occurrence of the cancer is via the use of substances (often antioxidants) termed “ photochemopreventive agents”. Resveratrol ( trans-3,4′,5-trihydroxystilbene), a phytoalexin found in grapes, nuts, fruits, and red wine, is a potent antioxidant with strong anti-inflammatory and antiproliferative properties. This study was designed to examine whether resveratrol possesses the potential to ameliorate the damages caused by short-term UVB exposure to mouse skin. Single topical application of resveratrol (25 μmol/0.2 ml acetone per mouse) to SKH-1 hairless mice was found to result in significant inhibition of UVB (180 mJ/cm 2)-mediated increase in bifold skin thickness and skin edema. The resveratrol treatment to mouse skin was also found to result in significant inhibition of UVB-mediated induction of cyclooxygenase and ornithine decarboxylase (ODC) enzyme activities and protein expression of ODC, which are well-established markers for tumor promotion. We also observed that resveratrol inhibits UVB-mediated increased level of lipid peroxidation, a marker of oxidative stress. Taken together, our results suggest that resveratrol may afford substantial protection against the damages caused by UVB exposure, and these protective effects may be mediated via its antioxidant properties.

Celecoxib, a selective cyclooxygenase-2 inhibitor, inhibits retinal vascular endothelial growth factor expression and vascular leakage in a streptozotocin-induced diabetic rat model

Overexpression of vascular endothelial growth factor (VEGF) is implicated in the development of vascular leakage and retinal neovascularization in diabetic subjects. The objective of this study was to determine whether celecoxib, a selective cyclooxygenase-2 enzyme inhibitor, reaches ocular tissues following oral administration and inhibits the retinal VEGF expression and vascular leakage in a streptozotocin-induced diabetic rat model. After administering a single intraperitoneal injection of streptozotocin (60 mg/kg) to Sprague–Dawley rats and ensuring the induction of diabetes at the end of 24 h, celecoxib was administered b.i.d. by oral gavage (50 mg/kg). On day 8, the animals were sacrificed and the retinal VEGF and cyclooxygenase-2 mRNA levels, ocular tissue celecoxib concentrations, and the vitreous/plasma protein ratio were determined. In diabetic rats, the retinal VEGF mRNA expression was 2.3-fold compared to controls, with a corresponding increase in cyclooxygenase-2 mRNA expression. Celecoxib treatment inhibited VEGF mRNA expression without any significant reduction in cyclooxygenase-2 mRNA. Furthermore, the retinal vascular leakage estimated as vitreous to plasma protein ratio increased in diabetic animals from 0.35±0.1 to 1.1±0.1 and celecoxib treatment significantly decreased this ratio to 0.4±0.1. Celecoxib levels were 24.8±6.6, 1.9±1, 1.7±0.8, and 6.9±0.9 ng/mg in the retina, vitreous, lens, and cornea, respectively. The plasma celecoxib levels were 85±24 ng/ml. Thus, celecoxib reaches the retina after oral administration and reduces diabetes-induced retinal VEGF mRNA expression and vascular leakage by inhibiting the activity of cyclooxygenase-2 enzyme.

Capsaicin exhibits anti-inflammatory property by inhibiting IkB-a degradation in LPS-stimulated peritoneal macrophages

Capsaicin, a major ingredient of hot pepper, was considered to exhibit an anti-inflammatory property. In order to clarify the signalling mechanism underlying the anti-inflammatory action of capsaicin, we investigated the effect of capsaicin on the production of inflammatory molecules in lipopolysaccharide (LPS)-stimulated murine peritoneal macrophages. The level of PGE2 was measured by EIA. The expression levels of COX-2, iNOS, IkB-a, and vanilloid receptor-1 (VR-1) were determined at the protein and mRNA levels. Significant inhibition of the production of LPS-induced PGE2 by capsaicin was observed in a dose-dependent manner. Capsaicin did not affect the COX-2 expression at either the protein or mRNA level, but inhibited the enzyme activity of COX-2 and the expression of the iNOS protein. Capsaicin completely blocked LPS-induced disappearance of IkB-a and therefore inactivated NF-kB. The inhibitory action of capsaicin on PGE2 production was not abolished by capsazepine, a specific antagonist to VR-1. A high expression level of the VR-1 like protein (VRL-1) was observed in peritoneal macrophages, while the expression of VR-1 was not detected. These findings suggest that the anti-inflammatory action of capsaicin may occur through a novel mechanism, not by a VR-1 receptor-mediated one. Both capsaicin and capsazepine may be a promising drug candidates for ameliorating inflammatory diseases and cancer.

Cyclooxygenase inhibitory and antioxidant compounds from the mycelia of the edible mushroom Grifola frondosa.

The bioassay-guided isolation and purification of the hexane extract of the cultured mycelia of Grifola frondosa led to the characterization of a fatty acid fraction and three compounds, ergosterol (1), ergostra-4,6,8(14),22-tetraen-3-one (2), and 1-oleoyl-2-linoleoyl-3-palmitoylglycerol (3). The composition of fatty acid fraction was confirmed as palmitic, oleic, and linoleic acids by GC-MS and by comparison with the retention values of authentic samples. The structures of compounds 1-3 were established by spectroscopic methods. The fatty acid fraction and compounds 1-3 showed cyclooxygenase (COX) enzyme inhibitory and antioxidant activities. The inhibition of COX-1 enzyme by the fatty acid fraction and compounds 1-3 at 250 microg/mL were 98, 37, 55, and 67%, respectively. Similarly, COX-2 enzyme activity was reduced by fatty acid fraction and compounds 1-3 at 250 microg/mL by 99, 37, 70, and 4%, respectively. The inhibitions of liposome peroxidation by the fatty acid fraction and compounds 1 and 2 at 100 microg/mL were 79, 48, and 42%, respectively. This is the first report of compounds 2 and 3 from the cultured mycelia of G. frondosa. The COX inhibitory activities of compounds 1-3 are reported here for the first time.

Diesel Exhaust Particle Extracts and Associated Polycyclic Aromatic Hydrocarbons Inhibit Cox-2-dependent Prostaglandin Synthesis in Murine Macrophages and Fibroblasts

Diesel exhaust particles (DEP) and their organic constituents modulate the immune system and exacerbate allergic airway inflammation. We investigated the role of DEP extract and associated polycyclic aromatic hydrocarbons (PAHs) on prostaglandin synthesis in endotoxin-activated murine macrophages and in mitogen-stimulated fibroblasts. In both macrophages and fibroblasts, DEP extract, phenanthrene, anthracene, phenanthrenequinone, and beta-napthoflavone inhibit prostaglandin production from endogenous arachidonic acid in response to ligand stimulation. However, DEP extract and PAHs do not block ligand induction of cyclooxygenase-2 (COX-2) protein, either in mitogen-stimulated fibroblasts or endotoxin-treated macrophages. Release of total arachidonic acid and total lipid products is not reduced by DEP or PAHs following ligand stimulation of macrophages or fibroblasts. DEP extract and the PAHs inhibit the activity of purified COX-2 enzyme in vitro but do not inhibit COX-1 activity. Thus, DEP and PAHs do not affect ligand-induced COX-2 gene expression, phospholipase activation, or arachidonic acid release in macrophages and fibroblasts but exert their inhibitory effect on prostaglandin production by preferentially blocking COX-2 enzyme activity.

Inhibition of prostaglandin E 2 production by taiwanin C isolated from the root of Acanthopanax chiisanensis and the mechanism of action

Five lignans, l-sesamin, savinin, helioxanthin, taiwanin C, and cis-dibenzylbutyrolactone, were isolated from the root of Acanthopanax chiisanensis (Araliaceae), a Korean medicinal plant, and their inhibitory effects on the production of prostaglandin (PG) E 2 stimulated by 12- O-tetradecanoylphorbol 13-acetate (TPA) in rat peritoneal macrophages were examined. Among the five lignans, taiwanin C was the most potent ( ic 50=0.12 μM), followed by helioxanthin, cis-dibenzylbutyrolactone, and savinin. l-Sesamin had no effect. Taiwanin C showed no inhibitory effect on the TPA-induced release of radioactivity from [ 3 H] arachidonic acid-labeled macrophages, nor did it inhibit the expression of cyclooxygenase (COX)-2 protein induced by TPA. However, the activities of isolated COX-1 and COX-2 were inhibited by taiwanin C ( ic 50=1.06 and 9.31 μM, respectively), reflecting the inhibition of both COX-1- and COX-2-dependent PGE 2 production in the cell culture system. These findings suggest that the mechanism of action of taiwanin C in the inhibition of PGE 2 production is the direct inhibition of COX enzymatic activity.

Byakangelicol, isolated from Angelica dahurica, inhibits both the activity and induction of cyclooxygenase-2 in human pulmonary epithelial cells.

We examined the inhibitory mechanism of byakangelicol, isolated from Angelica dahurica, on interleukin-1beta (IL-1beta)-induced cyclooxygenase-2 (COX-2) expression and prostaglandin E2 (PGE2) release in human pulmonary epithelial cell line (A549). Byakangelicol (10-50 microM) concentration-dependently attenuated IL-1beta-induced COX-2 expression and PGE2 release. The selective COX-2 inhibitor, NS-398 (0.01-1 microM), and byakangelicol (10-50 microM) both concentration-dependently inhibited the activity of the COX-2 enzyme. Byakangelicol, at a concentration up to 200 microM, did not affect the activity and expression of COX-1 enzyme. IL-1beta-induced p44/42 mitogen-activated protein kinase (MAPK) activation was inhibited by the MAPK/extracellular signal-regulated protein kinase (MEK) inhibitor, PD 98059 (30 microM), while byakangelicol (50 microM) had no effect. Treatment of cells with byakangelicol (50 microM) or pyrrolidine dithiocarbamate (PDTC; 50 microM) partially inhibited IL-1beta-induced degradation of IkappaB-alpha in the cytosol, translocation of p65 NF-kappaB from the cytosol to the nucleus and the NF-kappaB-specific DNA-protein complex formation. Taken together, we have demonstrated that byakangelicol inhibits IL-1beta-induced PGE2 release in A549 cells; this inhibition may be mediated by suppression of COX-2 expression and the activity of COX-2 enzyme. The inhibitory mechanism of byakangelicol on IL-1beta-induced COX-2 expression may be, at least in part, through suppression of NF-kappaB activity. Therefore, byakangelicol may have therapeutic potential as an anti-inflammatory drug on airway inflammation.

Interleukin-1beta elevates cyclooxygenase-2 protein level and enzyme activity via increasing its mRNA stability in human endometrial stromal cells: an effect mediated by extracellularly regulated kinases 1 and 2.

We investigated the regulation of PG production in human endometrial stromal cells (ESC) by IL-1beta. We found that cyclooxygenase-2 (COX-2) mRNA and protein levels and PGE(2) production in ESC were significantly increased by IL-1beta. COX-2 mRNA, protein, and PGE(2) levels in IL-1beta-treated ESC were decreased by a PKA inhibitor, a nuclear factor (NF-kappaB) inhibitor, and an ERK1/2 inhibitor, but not by a p38 MAPK inhibitor or a PKC inhibitor, suggesting the possible involvement of PKA, NF-kappaB, and/or the ERK1/2 signaling pathway(s) in IL-1beta-mediated COX-2 gene induction in ESC. We then transiently transfected deletion mutants of the COX-2 promoter fused to the luciferase reporter gene and variants of -360/+56 bp promoter construct carrying different site-directed mutations of selected cis-acting elements. We determined that a NF-kappaB site (-222/-213 bp), a nuclear factor for IL-6 expression site (NF-IL6, -132/-124 bp), and a cAMP response element (-59/-52 bp) were essential for the baseline COX-2 gene promoter regulation. The addition of IL-1beta, however, did not affect the activity of these COX-2 promoter constructs. To investigate the potential effects of IL-1beta on COX-2 mRNA stability, ESC were treated with actinomycin D, a general transcription inhibitor, in the absence or presence of IL-1beta. We found that 1) IL-1beta significantly increased COX-2 mRNA stability; 2) continuous transcription was not required to sustain the IL-1beta-induced COX-2 mRNA levels; and 3) COX-2 mRNA was highly unstable in the absence of IL-1beta. Additionally, we found that the ERK1/2 signaling pathway was essential for stabilizing COX-2 mRNA. We conclude that levels of COX-2 mRNA, protein, and enzyme activity in ESC are controlled by various signaling pathways, including PKA, ERK1/2, and NF-kappaB. Moreover, posttranscriptional mRNA stability is an important mechanism for IL-1beta-induced elevation of COX-2 expression in ESC.

An Analysis of Responses to Levosimendan in the Pulmonary Vascular Bed of the Cat

Calcium-sensitizing drugs, such as levosimendan, are a novel class of drug therapy for heart failure. We investigated the hypothesis that levosimendan is a pulmonary vasodepressor mediated through inhibition of phosphodiesterase, adenosine triphosphate (ATP)-dependent potassium channels, or both. We investigated responses to the calcium sensitizer levosimendan in the pulmonary vascular bed of the cat under conditions of controlled pulmonary blood flow and constant left atrial pressure when lobar arterial pressure was increased to a high steady level with the thromboxane A(2) analog U-46619. Under increased-tone conditions, levosimendan caused dose-related decreases in lobar arterial pressure without altering systemic arterial and left atrial pressure. Responses to levosimendan were significantly attenuated, although not completely, after the administration of U-37883A, a vascular selective nonsulfonylurea ATP-sensitive K(+)-channel-blocking drug. Responses to levosimendan were not significantly different after the administration of the nitric oxide synthase inhibitor L-N(5)-(1-iminoethyl)-ornithine or the cyclooxygenase inhibitor sodium meclofenamate or when lung ventilation was interrupted. These data show that levosimendan has significant vasodilator activity in the pulmonary vascular bed of the cat. They also suggest that pulmonary vasodilator responses to levosimendan are partially dependent on activation of ATP-sensitive K(+) channels and independent of the synthesis of nitric oxide, activation of cyclooxygenase enzyme, or changes in bronchomotor tone in the pulmonary vascular bed of the cat. Calcium-sensitizing drugs, such as levosimendan, are a novel class of drug therapy for heart-failure treatment. The lung circulation affects both right- and left-sided heart failure. Levosimendan decreased lobar arterial pressure via a partial K(+)(ATP) (potassium channel sensitive to intracellular adenosine triphosphate levels)-dependent mechanism. These data suggest that, in addition to calcium-sensitizing activity, levosimendan decreases pulmonary resistance, which may also aid in the treatment of heart failure.

An Analysis of Responses to Levosimendan in the Pulmonary Vascular Bed of the Cat

Calcium-sensitizing drugs, such as levosimendan, are a novel class of drug therapy for heart failure. We investigated the hypothesis that levosimendan is a pulmonary vasodepressor mediated through inhibition of phosphodiesterase, adenosine triphosphate (ATP)-dependent potassium channels, or both. We investigated responses to the calcium sensitizer levosimendan in the pulmonary vascular bed of the cat under conditions of controlled pulmonary blood flow and constant left atrial pressure when lobar arterial pressure was increased to a high steady level with the thromboxane A2 analog U-46619. Under increased-tone conditions, levosimendan caused dose-related decreases in lobar arterial pressure without altering systemic arterial and left atrial pressure. Responses to levosimendan were significantly attenuated, although not completely, after the administration of U-37883A, a vascular selective nonsulfonylurea ATP-sensitive K+-channel-blocking drug. Responses to levosimendan were not significantly different after the administration of the nitric oxide synthase inhibitor l-N5-(1-iminoethyl)-ornithine or the cyclooxygenase inhibitor sodium meclofenamate or when lung ventilation was interrupted. These data show that levosimendan has significant vasodilator activity in the pulmonary vascular bed of the cat. They also suggest that pulmonary vasodilator responses to levosimendan are partially dependent on activation of ATP-sensitive K+ channels and independent of the synthesis of nitric oxide, activation of cyclooxygenase enzyme, or changes in bronchomotor tone in the pulmonary vascular bed of the cat.

Influence of Cigarette Smoking on Prostaglandin Synthesis and Cyclooxygenase-2 Gene Expression in Human Urinary Bladder Cancer

The present study examines the effect of tobacco smoking on the expression of cyclooxygenase (COX)-2 gene, COX enzymatic activity and prostaglandin (PG) synthesis in urothelial mucosal tissues from patients with bladder cancer and from normal individuals. The detection frequency of COX-2 mRNA was 2-fold higher in bladder cancer patients compared to controls and it was accompanied by a significantly increased COX enzymatic activity and PGE2 synthesis (p<0.05). Smokers, in both control and patients groups, had higher COX-2 expression, COX activity, and PGE2 synthesis compared to the nonsmokers (p<0.05). The number of cigarettes smoked in the cases, but not controls, correlated well with COX enzymatic activity (r=0.42, p=0.016). The observed over-expression of COX-2 gene in human urinary bladder and the concomitant increases in PG synthesis may explain, at least in part, the mechanism by which cigarette smoking influences the development of urothelial neoplasia.

Anti-inflammatory and anti-allergic activities of hydroxylamine and related compounds.

The anti-inflammatory activities of several novel oximes and O-acyl oximes that we synthesized have been reported based on carrageenan-induced rat foot-pad swelling assay and histamine-induced rat vascular permeability assay. A cyclooxygenase (COX)-1 inhibitory effect has also been reported for 4'-piperidinoacetophenone and 4'-morpholinoacetophenone oximes and their O-acyl derivatives. To further search for more effective non-steroidal anti-inflammatory or anti-allergic drugs, 1-hydroxylamino-1-(4'-piperidinophenyl) ethane (P-HA) and 1-hydroxylamino-1-(4'-morpholinophenyl) ethane (M-HA) were synthesized from the corresponding oximes with sodium cyanoborohydride, and N,O-diacetyl hydroxylamines (P-HA-Ac and M-HA-Ac) were prepared from these hydroxylamines using acetyl chloride. These hydroxylamines and N,O-diacetyl hydroxylamines clearly exhibited inhibitory effects on mouse carrageenan-induced foot-pad swelling induced by oral administration (150, 37.5 mg/kg). An oral dose of P-HA-Ac (150 mg/kg) significantly inhibited the mouse anaphylactic reaction to ovalbumin measured by the abdominal wall (AW) method. Percutaneous administration of P-HA and M-HA significantly inhibited 2,4-dinitrofluorobenzene (DNFB)-induced contact hypersensitivity reaction (type IV) in mice at a dose of 0.5 and 0.1 mg/ear, respectively. All tested hydroxylamines and N,O-diacetyl hydroxylamines clearly inhibited both COX-1 and COX-2 enzyme activities with IC(50) values of 1.9-28.7 and 1.6-2.9 micro M against COX-1 and COX-2, respectively. Hydroxylamines (P-HA and M-HA) also showed a 5-lipoxygenase inhibitory effect.

Regulation of cyclooxygenase by the heme-heme oxygenase system in microvessel endothelial cells.

Heme oxygenase (HO) is a microsomal enzyme that oxidatively cleaves heme to form biliverdin, with the release of iron and carbon monoxide (CO). HO not only controls the availability of heme for the synthesis of heme proteins but also is responsible for the generation of CO, which binds to the heme moiety of heme proteins thus affecting their enzymatic activity. Cyclooxygenase (COX) is a heme protein that catalyzes the conversion of arachidonic acid to prostaglandin H(2), the precursor of prostanoids that participate in the regulation of vascular function. The goal of the present study was to determine whether the heme-HO system regulates COX enzyme expression and activity in vascular endothelial cells. Endothelial cells stably transfected with the human HO-1 gene exhibited a severalfold increase in human HO-1 mRNA levels, which was accompanied by an increase in HO activity and a marked decrease in prostaglandin (PG) E(2) and 6-keto-PGF(1alpha) levels. Exposure of cells to CoCl(2), an inducer of HO-1 gene expression, resulted in increases in HO-1 protein levels and HO activity. The increase in HO activity was associated with a subsequent decrease in COX activity, which returned to normal levels following normalization of HO activity. The addition of heme resulted in an increase in COX activity with an increase in PGE(2) and 6-keto-PGF(1alpha) levels. The degree of HO-1 expression and, consequently, the level of cellular heme, were directly related to COX activity. These results demonstrate that the heme-HO system can function as a cellular regulator of the expression of vascular COX, thus influencing the generation of prostanoids, PGE(2) and PGI(2), known to play a role in vascular homeostasis.

Role of human heme oxygenase-1 in attenuating TNF-alpha-mediated inflammation injury in endothelial cells.

Heme oxygenase (HO) is the rate-limiting enzyme in the formation of bilirubin, an antioxidant, and carbon monoxide (CO), a cell cycle modulator and a vasodilator. Cyclooxygenase (COX) is a hemeprotein that catalyzes the conversion of arachidonic acid (AA) to various prostanoids, which play an important role in the regulation of vascular endothelial function in normal and disease states. The influence of suppression or overexpression of HO isoforms on COX expression and synthesis of prostanoids is of considerable physiological importance. Consequently, the goal of the present study was to determine whether the heme-HO system regulates COX enzyme expression and activity in vascular endothelial cells in the absence and presence of TNF-alpha (100 ng/ml). Endothelial cells stably transfected with the retrovirus containing the human HO-1 gene exhibited a several-fold increase in HO-1 protein levels, which was accompanied by an increase in HO activity and a marked decrease in PGE(2) and 6-keto PGF(1alpha) levels. We also assessed the effect of retrovirus-mediated HO-1 gene transfer in the sense and antisense orientation on HO-1 expression and cell cycle progression in human endothelial cells. The levels of CO and HO activity were increased in cells transduced with the HO-1 sense and were greatly suppressed in cells transduced with HO-1 antisense as compared to control sham-transduced cells (P < 0.05). The percentage of the G(1)-phase in cells transduced with HO-1 significantly increased (41.4% +/- 9.1) compared with control endothelial cells (34.8% +/- 4.9). We measured COX activity by determining the levels of PGI(2) and PGE(2). The levels of PGI(2) decreased in cells transduced with HO-1 sense and increased in cells transduced with HO-1 in antisense orientation. The expression of p27 was also studied and showed a marked decrease in cells transduced with HO-1 sense and a marked increase in the HO-1 antisense transduced cells. Cell cycle analysis of endothelial cell DNA distributions indicated that the TNF-alpha-induced decrease in the proportion of G(1)-phase cells and increase in apoptotic cells in control cultures could be abrogated by transfection with HO-1 in the sense orientation. Tin mesoporphyrin (SnMP) reversed the protective effect of HO-1. These results demonstrate that overexpressing HO-1 mitigated the TNF-alpha-mediated changes in cell cycle progression and apoptosis, perhaps by a decrease in the levels of COX activity.