Constitutive COX-1 Research Articles

2,3-Diarylxanthones as Potential Inhibitors of Arachidonic Acid Metabolic Pathways.

In response to an inflammatory stimulus, arachidonic acid (AA), the main polyunsaturated fatty acid present in the phospholipid layer of cell membranes, is released and metabolized to a series of eicosanoids. These bioactive lipid mediators of inflammation arise physiologically through the action of the enzymes 5-lipoxygenase (5-LOX) and cyclooxygenases (constitutive COX-1 and inducible COX-2). It is believed that dual inhibition of 5-LOX and COXs may have a higher beneficial impact in the treatment of inflammatory disorders rather than the inhibition of each enzyme. With this demand for new dual-acting anti-inflammatory agents, a range of 2,3-diarylxanthones were tested through their ability to interact in the AA metabolism. In vitro anti-inflammatory activity was evaluated through the inhibition of 5-LOX-catalyzed leukotriene B4 (LTB4) formation in human neutrophils and inhibition of COX-1- and COX-2-catalyzed prostaglandin E2 (PGE2) formation in human whole blood. The results showed that some of the studied arylxanthones were able to prevent LTB4 production in human neutrophils, in a concentration-dependent manner. The xanthone with a 2-catechol was the most active one (IC50 ∼ 9μM). The more effective arylxanthones in preventing COX-1-catalyzed PGE2 production presented IC50 values from 1 to 7μM, exhibiting a structural feature with at least one non-substituted aryl group. All the studied arylxanthones were ineffective to prevent the formation of PGE2 catalyzed by COX-2, up to the maximum concentration of 100μM. The ability of the tested 2,3-diarylxanthones to interact with both 5-LOX and COX-1 pathways constitutes an important step in the research of novel dual-acting anti-inflammatory drugs.

Celecoxib administration reduced mortality, mesenteric hypoperfusion, aortic dysfunction and multiple organ injury in septic rats.

The cyclooxygenase (COX)-2 overexpression is associated with vascular injury and multiple organ failure in sepsis. However, constitutive COX-1 and basal COX-2 expressions have physiological effects. We aimed to investigate the effects of partial and selective COX-2 inhibition without affecting constitutive COX-1 and basal COX-2 activities by celecoxib on mesenteric artery blood flow (MABF), vascular reactivity, oxidative and inflammatory injuries, and survival in septic rats accomplished by cecal ligation and puncture (CLP). Wistar rats were allocated into Sham, CLP, Sham+celecoxib, CLP+celecoxib subgroups. 2h after Sham and CLP operations, celecoxib (0.5mg/kg) or vehicle (saline; 1mL/kg) was administered orally to rats. 18h after drug administrations, MABF and responses of isolated aortic rings to phenylephrine were measured. Tissue samples were obtained for biochemical and histopathological examinations. Furthermore, survival rate was monitored throughout 96h. Celecoxib ameliorated mesenteric hypoperfusion and partially improved aortic dysfunction induced by CLP. Survival rate was%0 at 49th h in CLP group, but in CLP+celecoxib group it was 42.8% at the end of 96h. Serum AST, ALT, LDH, BUN, Cr and inflammatory cytokine (tumor necrosis factor-alpha, interleukin-1 beta and interleukin-6) levels were increased in CLP group that were prevented by celecoxib. The decreases in liver and spleen glutathione levels and the increases in liver, lung, spleen and kidney malondialdehyde levels in CLP group were blocked by celecoxib. The histopathological protective effects of celecoxib on organ injury due to CLP were also observed. Celecoxib has protective effects on sepsis due to its preservative effects on mesenteric perfusion, aortic function and its anti-inflammatory and antioxidative effects.

Spontaneous Glutamatergic Synaptic Activity Regulates Constitutive COX-2 Expression in Neurons OPPOSING ROLES FOR THE TRANSCRIPTION FACTORS CREB (cAMP RESPONSE ELEMENT BINDING) PROTEIN AND Sp1 (STIMULATORY PROTEIN-1)

Burgeoning evidence supports a role for cyclooxygenase metabolites in regulating membrane excitability in various forms of synaptic plasticity. Two cyclooxygenases, COX-1 and COX-2, catalyze the initial step in the metabolism of arachidonic acid to prostaglandins. COX-2 is generally considered inducible, but in glutamatergic neurons in some brain regions, including the cerebral cortex, it is constitutively expressed. However, the transcriptional mechanisms by which this occurs have not been elucidated. Here, we used quantitative PCR and also analyzed reporter gene expression in a mouse line carrying a construct consisting of a portion of the proximal promoter region of the mouse COX-2 gene upstream of luciferase cDNA to characterize COX-2 basal transcriptional regulation in cortical neurons. Extracts from the whole brain and from the cerebral cortex, hippocampus, and olfactory bulbs exhibited high luciferase activity. Moreover, constitutive COX-2 expression and luciferase activity were detected in cortical neurons, but not in cortical astrocytes, cultured from wild-type and transgenic mice, respectively. Constitutive COX-2 expression depended on spontaneous but not evoked excitatory synaptic activity and was shown to be N-methyl-d-aspartate receptor-dependent. Constitutive promoter activity was reduced in neurons transfected with a dominant-negative cAMP response element binding protein (CREB) and was eliminated by mutating the CRE-binding site on the COX-2 promoter. However, mutation of the stimulatory protein-1 (Sp1)-binding site resulted in an N-methyl-d-aspartate receptor-dependent enhancement of COX-2 promoter activity. Basal binding of the transcription factors CREB and Sp1 to the native neuronal COX-2 promoter was confirmed. In toto, our data suggest that spontaneous glutamatergic synaptic activity regulates constitutive neuronal COX-2 expression via Sp1 and CREB protein-dependent transcriptional mechanisms.

Level of Arachidonic Acid and State of Peroxidation Processes in Patients with Aspirin-Intolerant Polypous Rhinosinusitis

The main peculiarity of aspirin-intolerant polypous rhinosinusitis pathogenesis is the presence of “genetic block” of constitutive cyclooxygenase being the key enzyme of the arachidonic acid metabolism. It justifies the necessity of studying its metabolic peculiarities.The objective of the research was to determine the level of arachidonic acid as well as the state of lipid and protein peroxidation processes in patients with aspirin-intolerant polypous rhinosinusitis.Materials and methods. The levels of arachidonic acid, malondialdehyde and oxidative modification of serum proteins were studied in 20 patients with aspirin-intolerant polypous rhinosinusitis and 7 healthy individuals.Results. Significantly elevated levels of arachidonic levels were observed. The search for alternative metabolic pathways stimulated lipid and protein peroxidation processes and led to the increase in the levels of malondialdehyde and oxidative modification of serum proteins. The peculiarities of biochemical changes indicated pro-inflammatory orientation of lipid metabolism.Conclusions. The obtained data confirmed the hypothesis of “genetic block” of the arachidonic acid metabolism as the main pathogenetic component of aspirin-intolerant polypous rhinosinusitis and allowed us to clearly interpret biochemical picture of the disease.

MFN2 provides antitumor efficacy in pancreatic cancer cells

Both the insulin-like growth factor-I receptor (IGF-IR) and cyclooxygenase-2 (COX-2) are frequently overexpressed in pancreatic cancer. We hypothesized that IGF-IR is directly involved in induction of COX-2 and sought to investigate signaling pathways mediating this effect. Pancreatic cancer cells (L3.6pl) were stably transfected with a dominant-negative receptor (IGF-IR DN) construct or empty vector (pcDNA). Cells were stimulated with IGF-I to determine activated signaling intermediates and induction of COX-2. Signaling pathways mediating COX-2 induction were identified using signaling inhibitors. IGF-I up-regulated COX-2 selectively via the MAPK/(Erk-1/2) pathway. In addition, IGF-IR DN cells showed a marked decrease in constitutive COX-2 and a blunted response to IGF-I. Similarly, treatment with an anti-IGF-IR antibody effectively inhibited IGF-IR and MAPK/Erk activation and decreased COX-2 in parental cells. In conclusion, activation of IGF-IR mediates COX-2 expression in human pancreatic cancer cells.

Cyclooxygenase-2 expression in hepatocytes attenuates non-alcoholic steatohepatitis and liver fibrosis in mice

Cyclooxygenase-2 (COX-2) is involved in different liver diseases but little is known about the significance of COX-2 in the development and progression of non-alcoholic steatohepatitis (NASH). This study was designed to elucidate the role of COX-2 expression in hepatocytes in the pathogenesis of steatohepatitis and hepatic fibrosis. In the present work, hepatocyte-specific COX-2 transgenic mice (hCOX-2-Tg) and their wild-type (Wt) littermates were either fed methionine-and-choline deficient (MCD) diet to establish an experimental non-alcoholic steatohepatitis (NASH) model or injected with carbon tetrachloride (CCl4) to induce liver fibrosis. In our animal model, hCOX-2-Tg mice fed MCD diet showed lower grades of steatosis, ballooning and inflammation than Wt mice, in part by reduced recruitment and infiltration of hepatic macrophages, with a corresponding decrease in serum levels of pro-inflammatory cytokines. Furthermore, hCOX-2-Tg mice showed a significant attenuation of the MCD diet-induced increase in oxidative stress and hepatic apoptosis observed in Wt mice. Even more, hCOX-2-Tg mice treated with CCl4 had significantly lower stages of fibrosis and less hepatic content of collagen, hydroxyproline and pro-fibrogenic markers than Wt controls. Collectively, our data indicates that constitutive hepatocyte COX-2 expression ameliorates NASH and liver fibrosis development in mice by reducing inflammation, oxidative stress and apoptosis and by modulating activation of hepatic stellate cells, respectively, suggesting a possible protective role for COX-2 induction in NASH/NAFLD progression.

Systematic study of constitutive cyclooxygenase-2 expression: Role of NF-κB and NFAT transcriptional pathways

Cyclooxygenase-2 (COX-2) is an inducible enzyme that drives inflammation and is the therapeutic target for widely used nonsteroidal antiinflammatory drugs (NSAIDs). However, COX-2 is also constitutively expressed, in the absence of overt inflammation, with a specific tissue distribution that includes the kidney, gastrointestinal tract, brain, and thymus. Constitutive COX-2 expression is therapeutically important because NSAIDs cause cardiovascular and renal side effects in otherwise healthy individuals. These side effects are now of major concern globally. However, the pathways driving constitutive COX-2 expression remain poorly understood. Here we show that in the kidney and other sites, constitutive COX-2 expression is a sterile response, independent of commensal microorganisms and not associated with activity of the inflammatory transcription factor NF-κB. Instead, COX-2 expression in the kidney but not other regions colocalized with nuclear factor of activated T cells (NFAT) transcription factor activity and was sensitive to inhibition of calcineurin-dependent NFAT activation. However, calcineurin/NFAT regulation did not contribute to constitutive expression elsewhere or to inflammatory COX-2 induction at any site. These data address the mechanisms driving constitutive COX-2 and suggest that by targeting transcription it may be possible to develop antiinflammatory therapies that spare the constitutive expression necessary for normal homeostatic functions, including those important to the cardiovascular-renal system.

Effect of Cyclooxygenase(COX)-1 and COX-2 inhibition on furosemide-induced renal responses and isoform immunolocalization in the healthy cat kidney

BackgroundThe role of cyclooxygenase(COX)-1 and COX-2 in the saluretic and renin-angiotensin responses to loop diuretics in the cat is unknown. We propose in vivo characterisation of isoform roles in a furosemide model by administering non-steroidal anti-inflammatory drugs (NSAIDs) with differing selectivity profiles: robenacoxib (COX-2 selective) and ketoprofen (COX-1 selective).ResultsIn this four period crossover study, we compared the effect of four treatments: placebo, robenacoxib once or twice daily and ketoprofen once daily concomitantly with furosemide in seven healthy cats. For each period, urine and blood samples were collected at baseline and within 48 h of treatment starting. Plasma renin activity (PRA), plasma and urinary aldosterone concentrations, glomerular filtration rate (GFR) and 24 h urinary volumes, electrolytes and eicosanoids (PGE2, 6-keto-PGF1α, TxB2), renal injury biomarker excretions [N-acetyl-beta-D-glucosaminidase (NAG) and Gamma-Glutamyltransferase] were measured. Urine volume (24 h) and urinary sodium, chloride and calcium excretions increased from baseline with all treatments. Plasma creatinine increased with all treatments except placebo, whereas GFR was significantly decreased from baseline only with ketoprofen. PRA increased significantly with placebo and once daily robenacoxib and the increase was significantly higher with placebo compared to ketoprofen (10.5 ± 4.4 vs 4.9 ± 5.0 ng ml−1 h−1). Urinary aldosterone excretion increased with all treatments but this increase was inhibited by 75 % with ketoprofen and 65 % with once daily robenacoxib compared to placebo. Urinary PGE2 excretion decreased with all treatments and excretion was significantly lower with ketoprofen compared to placebo. Urinary TxB2 excretion was significantly increased from baseline only with placebo. NAG increased from baseline with all treatments. Immunohistochemistry on post-mortem renal specimens, obtained from a different group of cats that died naturally of non-renal causes, suggested constitutive COX-1 and COX-2 co-localization in many renal structures including the macula densa (MD).ConclusionsThese data suggest that both COX-1 and COX-2 could generate the signal from the MD to the renin secreting cells in cats exposed to furosemide. Co-localization of COX isoenzymes in MD cells supports the functional data reported here.Electronic supplementary materialThe online version of this article (doi:10.1186/s12917-015-0598-z) contains supplementary material, which is available to authorized users.

Pharmacological characterization of mechanisms involved in the vasorelaxation produced by rosuvastatin in aortic rings from rats with a cafeteria-style diet.

The present study aimed to investigate the possible influence of several inhibitors and blockers on the vascular effect produced by the acute in vitro application of rosuvastatin to phenylephrine-precontracted aortic rings from rats with a semi-solid, cafeteria-style (CAF) diet. It also aimed to examine the effects of rosuvastatin on the expression of endothelial nitric oxide synthase (eNOS), inducible nitric oxide synthase, constitutive cyclooxygenase, and inducible cyclooxygenase in aortic rings from rats with a CAF diet. From comparisons of the effect on phenylephrine-precontracted aortic rings extracted from rats with two different diets (a standard and a CAF diet), it was found that 10−9–10−5-mol/L rosuvastatin produced lower concentration-dependent vasorelaxation on rings from the CAF diet group. The vasorelaxant effect was unaffected by the vehicle, but it was significantly attenuated by 10−5-mol/L NG-nitro-l-arginine methyl ester, 10−2-mol/L tetraethylammonium, 10−3-mol/L 4-aminopyridine, 10−7-mol/L apamin plus 10−7-mol/L charybdotoxin, 10−5-mol/L indomethacin, or 10−5-mol/L cycloheximide. Moreover, in aortic rings from rats with a CAF diet, rosuvastatin enhanced the expression of eNOS, inducible nitric oxide synthase, constitutive cyclooxygenase, and inducible cyclooxygenase. The acute in vitro application of rosuvastatin to phenylephrine-precontracted aortic rings from rats with a CAF diet had a vasorelaxant effect. Overall, the present results suggest that the stimulation of eNOS, the opening of Ca2+-activated and voltage-activated K+ channels, the stimulation of prostaglandin synthesis and enhanced protein levels of eNOS, inducible nitric oxide synthase, constitutive cyclooxygenase, and inducible cyclooxygenase are involved in this relaxant effect.

The cardiovascular and gastrointestinal adverse effects of cyclooxygenase inhibitors seems to be a major concern that restricts their use in the treatment of urinary bladder dysfunction.

The cardiovascular and gastrointestinal adverse effects of cyclooxygenase inhibitors seems to be a major concern that restricts their use in the treatment of urinary bladder dysfunction.

Frataxin deficiency increases cyclooxygenase 2 and prostaglandins in cell and animal models of Friedreich's ataxia.

An inherited deficiency of the mitochondrial protein frataxin causes Friedreich's ataxia (FRDA); the mechanism by which this deficiency triggers neuro- and cardio-degeneration is unclear. Microarrays of neural tissue of animal models of the disease showed decreases in antioxidant genes, and increases in inflammatory genes. Cyclooxygenase (COX)-derived oxylipins are important mediators of inflammation. We measured oxylipin levels using tandem mass spectrometry and ELISAs in multiple cell and animal models of FRDA. Mass spectrometry revealed increases in concentrations of prostaglandins, thromboxane B2, 15-HETE and 11-HETE in cerebellar samples of knockin knockout mice. One possible explanation for the elevated oxylipins is that frataxin deficiency results in increased COX activity. While constitutive COX1 was unchanged, inducible COX2 expression was elevated over 1.35-fold (P < 0.05) in two Friedreich's mouse models and Friedreich's lymphocytes. Consistent with higher COX2 expression, its activity was also increased by 58% over controls. COX2 expression is driven by multiple transcription factors, including activator protein 1 and cAMP response element-binding protein, both of which were elevated over 1.52-fold in cerebella. Taken together, the results support the hypothesis that reduced expression of frataxin leads to elevation of COX2-mediated oxylipin synthesis stimulated by increases in transcription factors that respond to increased reactive oxygen species. These findings support a neuroinflammatory mechanism in FRDA, which has both pathomechanistic and therapeutic implications.

Identification of the molecular pathways that drive constitutive renal COX‐2 expression: implications for novel COX‐2‐targetted therapies that spare the cardiovascular system (837.9)

Drugs such as diclofenac and celecoxib inhibit cyclo-oxygenase (COX)-2 to produce anti-inflammatory benefit, but also produce renal/cardiovascular toxicity by inhibition of constitutive COX-2 in the kidney. During inflammation COX-2 is rapidly induced by well-defined pathways including nuclear factor κB (NFκB) and nuclear factor of activated T-cells (NFAT) but it is not known if these also drive physiological renal expression of COX-2. To explore this we compared the distribution of constitutive COX-2 expression with constitutive NFκB and NFAT activity using luciferase reporter mice. COX-2 was highly expressed in the medulla of the kidney. This closely matched a ‘hotspot’ of constitutive NFAT activity but did not correlate with NFκB activity (Figure A). To determine if constitutive renal COX-2 was driven by this NFAT activity we treated mice with cyclosporine A (CsA) to inhibit calcineurin-dependent NFAT activation. CsA reduced constitutive COX-2 expression in the renal medulla but had no effect on endoto...

Cultured Preadipocytes Undergoing Stable Transfection with Cyclooxygenase-1 in the Antisense Direction Accelerate Adipogenesis During the Maturation Phase of Adipocytes

The arachidonate cyclooxygenase (COX) pathway is involved in the generation of several types of endogenous prostaglandins (PGs) with opposite effects on adipogenesis at different life stages of adipocytes. However, the specific role of COX isoforms, the rate-limiting enzymes for the pathway, remains elusive in the regulation of the endogenous synthesis of PGs. This study was aimed at the selective suppression of the constitutive COX-1 in cultured preadipocytes by the isolation of cloned preadipocytes transfected stably with a mammalian expression vector harboring cDNA encoding mouse COX-1 in the antisense direction. The gene expression analysis revealed that the transcript and protein levels of the constitutive COX-1 were substantially suppressed in the isolated cloned transfectants with antisense COX-1. By contrast, the expression of the inducible COX-2 was not affected in the stable transfectants with antisense COX-1. All of the cloned stable transfectants with antisense COX-1 exhibited a significant reduction in the immediate synthesis of PGE2 serving as an anti-adipogenic factor. The sustained expression of COX-1 in the antisense direction induced the appreciable stimulation of fat storage in adipocytes during the maturation phase, which was associated with the higher expression levels of adipocyte-specific genes, indicating the positive regulation of adipogenesis program. Moreover, the up-regulation of adipogenesis is accompanied by a higher production of J2 series PGs including 15-deoxy-Δ(12,14)-PGJ2 and Δ(12)-PGJ2, known as pro-adipogenic factors by the transfectants with antisense COX-1. The results suggest that the inducible COX-2 can contribute to the endogenous synthesis of PGJ2 derivatives acting as autocrine mediators to simulate adipogenesis during the maturation phase by way of compensation for the suppressed expression of the constitutive COX-1.

Cyclooxygenase and cytokine regulation in lung fibroblasts activated with viral versus bacterial pathogen associated molecular patterns

Cyclooxygenase (COX) is required for prostanoid (e.g. prostaglandin PGE2) production. Constitutive COX-1 and inducible COX-2 are implicated in lung diseases, such as idiopathic pulmonary fibrosis (IPF). Using lung fibroblasts from humans and wild type, COX-1−/− and COX-2−/− mice, we investigated how COX activity modulates cell growth and inflammatory responses induced by activators of Toll-like receptors (TLRs) 1–8. In mouse tissue, PGE2 release from fresh lung was COX-1 driven, in lung in culture (24h) COX-1 and COX-2 driven, and from proliferating lung fibroblasts exclusively COX-2 driven. COX-2 limited proliferation in lung fibroblasts and both isoforms limited KC release induced by a range of TLR agonists. Less effect of COX was seen on TLR-induced IP-10 release. In human lung fibroblasts inhibition of COX with diclofenac was associated with increased release of IL-8 and IP-10. Our results may have implications for the treatment of IPF.

Abstract LB-154: COX-2 as a mediator of oncogenic PKCε in prostate cancer.

Abstract Protein kinase C epsilon (PKCε), a member of the PKC family of phorbol ester/diacylglycerol receptors, is up-regulated in many human cancers, including prostate cancer. We recently demonstrated that PKCε is an essential mediator of NF-κB activation in prostate cancer (Garg et al., JBC, 287 (44), 37570–37582, 2012). Cyclooxygenase-2 (COX-2), a well-known NF-κB regulated gene, has been reported to be up-regulated in a number of human cancers, including metastatic prostate cancer. As PKCε plays an important role in prostate cancer cell survival and cooperates with other oncogenic insults, herein we aim to determine if PKCε regulates COX-2 activation during prostate tumorigenesis. PKCε RNAi depletion diminishes TNFα- or LPS-induced COX-2 mRNA expression in LNCaP prostate cancer cells and constitutive COX-2 levels from PC3 cells. Conversely, PKCε overexpression by adenoviral means potentiates TNFα or LPS-induced COX-2 expression in LNCaP cells. Notably, transgenic overexpression of PKCε in the mouse prostate causes preneoplastic lesions with elevated COX-2 levels. Interestingly, when we intercrossed the prostate-specific PKCε transgenic mice with mice haploinsufficient for Pten, a common genetic alteration in human prostate cancer, the resulting compound mutant mice (PB-PKCε;Pten+/− mice) developed fully invasive adenocarcinoma with NF-κB hyperactivation and high COX-2 levels. Likewise, stable overexpression of PKCε in mouse prostate epithelial cell lines that are either heterozygous (P8) or homozygous (CaP8) for Pten deletion, led to significant enhancements in cell proliferation, motility and invasiveness as well as in LPS-induced COX-2 mRNA expression compared to the respective control cells, and this effect is more pronounced in CaP8 cells. Studies using human prostate tumors revealed a co-existence of PKCε overexpression, NF-κB hyperactivation, and COX-2 up-regulation. Lastly, treatment of PKCε overexpressing P8 or CaP8 cells with the selective COX-2 inhibitor NS398 caused a pronounced growth inhibition. Overall, our study identified COX-2 as a PKCε-regulated gene. Our results suggest that COX-2 as a potential mediator of PKCε oncogenesis in prostate cancer, particularly in the context of Pten loss. Citation Format: Rachana Garg, Priti Lal, Jorge Blando, Fernando J. Benavides, Michael D. Feldman, Emer M. Smyth, Marcelo G. Kazanietz. COX-2 as a mediator of oncogenic PKCε in prostate cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-154. doi:10.1158/1538-7445.AM2013-LB-154

Antiproliferative effect of two novel COX-2 inhibitors on human keratinocytes

Selective COX-2 inhibitors (COXib) belonging to the class of diaryl heterocycles (e.g., celecoxib, rofecoxib, etc.), are devoid of the undesirable effects due to their capacity to inhibit selectively inducible (COX-2), responsible for inflammatory effects but not constitutive cyclooxygenase-1 (COX-1)(COX); responsible for cytoprotective effects on gastric mucosa. In addition, several reports have identified an increased risk of cardiovascular events associated with the use of COXib. We have developed a new series of anti-inflammatory agents (1,5-diarylpyrrole-3-alkoxyethyl esters and ethers).To evaluate the effect of two 1,5-diarylpyrrole-3-alkoxyethyl ethers, VA441 and VA428 (up to 100μM), respectively, in comparison with two well known COXib, celecoxib and rofecoxib, on HaCaT cell (keratinocytes) proliferation and toxicity.Crucial molecules in cell cycle progression, i.e. NFκB and ERK as targets/mediators and cyclin D1 and p21 Cip1/Kip as final effectors were evaluated by Western blot, immunohystochemistry and q–PCR analysis.Both compounds, VA441 and VA428, showed a strong inhibition of cell proliferation, and did not exhibit cytotoxicity. The anti-proliferative effect was accompanied by a strong activation of ERK and induction of the cell cycle inhibitor p21. In addition, there was a clear inhibition of the transcription factor NF-κB and downregulation of cyclin D1, with enforced inhibition of the HaCaT cell cycle progression.These data suggest that compounds VA441 and VA428, along with their role in inhibiting COX-2 and inflammation, could have a possible therapeutic (topical and systemic) use against skin proliferative disorders, such as psoriasis.

Role of Phospholipids in Protection of the GI Mucosa

The focus of peptic ulcer disease research has evolved over the past 50 years. In the past, the greatest interest understandably has been in studying the regulation of gastric acid secretion, following the century-old Schwarz’s dictum of ‘‘no acid, no ulcer’’ [1]. As a consequence, understanding the regulation of gastric acid section and the potential causes of gastric acid hypersecretion became a central research focus until 1970, with major scientific advances including the elucidation of the role of gastrin, acetylcholine, and histamine as physiological stimulants of acid secretion and the identification of luminal acid and somatostatin as physiological inhibitors [2]. Afterwards, the emphasis was on the development of pharmacological inhibitors of gastric acid secretion, with the clinically important discovery of highly effective H2 receptor antagonists (H2RAs) followed by the even more efficacious proton pump inhibitors (PPIs) [3–7]. During this same period came the major discovery by Robert and associates of the role of prostaglandins in inhibiting gastric acid secretion, and more importantly in protecting the gastric mucosa from a damaging agents and ulcerogenic conditions (e.g., stress), a remarkable finding at the time, termed ‘‘cytoprotection’’ [8]. The multi-factorial mechanism that underlies prostaglandin’s cytoprotective action appears to involve increasing and/or maintaining tight junctional integrity, increasing intestinal mucus and bicarbonate section, and increasing mucosal hydrophobicity, mitochondrial integrity, and blood flow [9, 10]. The development of prostaglandins as clinical cytoprotective agents, however, has been disappointing due to the side-effect profile of these biologically active lipids to stimulate uterine and intestinal smooth muscle contraction and intestinal secretion, which may result in miscarriages, bloating, and diarrhea [11]. Lastly, the discovery of Helicobacter pylori by Marshall and Warren [12] revolutionized the field of peptic ulcer disease by implicating a micro-organism in its pathogenesis, leading to novel antibiotic combination therapeutic approaches which not only remarkably reduced the incidence of peptic ulcer disease but of gastric cancer as well [13, 14]. The mechanism by which H. pylori causes peptic ulcer disease surprisingly has yet to be fully resolved, but appears to be dependent on the gastric localization of the infection (antrum vs. body), gastric acid hypersecretion (mostly linked to antral infection), and defects in gastroduodenal barrier function [15, 16]. Nonsteroidal anti-inflammatory drugs (NSAIDs), which are regularly consumed by a large percentage of our populace, represent the other major cause of GI ulceration, with gastroduodenal erosions and/or ulcers affecting between 15 and 40 % of those regularly taking NSAIDs [17], and lower gut pathology being present in [50 % of chronic NSAID users [18]. The mechanism by which aspirin and related NSAIDs injure the GI mucosa was once thought to be strictly due to disruption of the biosynthesis of cytoprotective prostaglandins via inhibition of constitutive cyclooxygenase1 (COX-1) [19], leading to the development of highly selective COX-2 inhibitors (coxibs) as safer anti-inflammatory drugs. However, over time this concept has been challenged, with the demonstration that NSAIDs can induce GI injury by COX-independent mechanisms [20], and the demonstration that a number of highly selective coxibs have unanticipated and potentially L. M. Lichtenberger (&) Department of Integrative Biology and Pharmacology, The University of Texas Health Science Center, Houston, TX 77030, USA e-mail: lenard.m.lichtenberger@uth.tmc.edu

Selective Inos and COX-2 inhibition of Morinda Citrifolia (Noni) fruit extract in rat gastric ulcerated mucosa

Gastric nitric oxide synthase (nNOS, iNOS) and cyclooxygenase (COX-1 and COX-2) inhibitory activity of Morinda citrifolia (noni) aqueous fruit extract (AFE) and ethanolic fruit extract (EFE), two common forms of noni fruit extract used in traditional medicine, was studied in rats treated with topical application of acetic acid. The gastric ulcerated area was excised and the mRNA expression of NOS and COX was analyzed using one-step RT-PCR analysis. Treatment with AFE and EFE at the dose that contain 0.5–1mg/kg of active ingredient: scopoletin for 10d after ulcer induction markedly accelerated ulcer healing that related to their inhibitory activity on an increased mRNA expression level of both pro-inflammatory enzymes (iNOS and COX-2) at the transcription level and at only the excess expression level, but had no markedly inhibitory effect on the mRNA expression level of constitutive nNOS and COX-1 which produce gastric mucosal protective NO and PGs respectively. This indicated that both the fruit extracts at the dose use exerted an optimal oral dose for further clinical study on treatment of human chronic gastric ulcer.

Lack of a chondroprotective effect of cyclooxygenase 2 inhibition in a surgically induced model of osteoarthritis in mice

To investigate the chondroprotective effect of cyclooxygenase 2 (COX-2) inhibition in experimental osteoarthritis (OA). The expression of prostaglandin E2 synthetic enzymes was examined by immunostaining of tibial cartilage from mice with surgically induced knee joint instability and from OA patients undergoing total knee arthroplasty. The effect of orally administered celecoxib (10 mg/kg/day and 30 mg/kg/day) or vehicle alone in mice was examined 12 weeks after the induction of OA. To investigate the involvement of COX-1 and COX-2 in OA development, we also created the model in COX-1-homozygous-knockout (Ptgs1-/-) mice and COX-2-homozygous-knockout (Ptgs2-/-) mice. OA severity was assessed using a grading system developed by our group and by the Osteoarthritis Research Society International scoring system. In mouse and human OA cartilage, the expression of the inducible enzymes COX-2 and microsomal prostaglandin E synthase 1 (mPGES-1) was enhanced, while that of the constitutive enzymes COX-1, cytosolic PGES, and mPGES-2 was suppressed. Daily celecoxib treatment did not prevent cartilage degradation or osteophyte formation during OA development in the mouse model. Furthermore, neither Ptgs1-/- mice nor Ptgs2-/- mice exhibited any significant difference in OA development as compared to wild-type littermates. The two COX enzymes differ in terms of regulation of their expression during OA development. Nevertheless, experiments using inhibitor and genetic deficiency demonstrated a lack of chondroprotective effect of COX-2 inhibition in the mouse surgical OA model.

Berberine ameliorates COX-2 expression in rat small intestinal mucosa partially through PPARγ pathway during acute endotoxemia

Berberine hydrochloride (BBR), a plant alkaloid, has been used to treat intestinal inflammation or infection for years. Cyclooxygenase-2 (COX-2) is pro-inflammatory mediator and involved in the induction of gut inflammation. The expression of COX-2 in small bowel mucosa was determined and the mechanism by which BBR modulated COX-2 expression was explored in a rat model of endotoxemia induced by lipopolysaccharide (LPS). The results showed that without LPS stimulation COX-2 was constitutively expressed at low levels in control rats. LPS challenge rapidly induced COX-2 gene transcription resulting in high levels of inducible COX-2 expression in endotoxemic rats. BBR pre- and post-treatment had no marked effect on constitutive COX-2 expression but inhibited inducible COX-2 overexpression. LPS challenge increased the expression and phosphorylation of peroxisome proliferator-activated receptor gamma (PPARγ), p38 and activating transcription factor 2 and 3 (ATF2, ATF3), but the effects of LPS were inhibited by BBR treatment. GW9662 did not influence constitutive COX-2 expression but enhanced inducible COX-2 overproduction. Besides, GW9662 abolished the inhibitory effect of BBR on inducible COX-2, p38, ATF2, 3 expression and phosphorylation. Collectively, these results indicated that BBR gavage could attenuate the overexpression of inducible COX-2, not constitutive COX-2, in ileal mucosa during acute endotoxemia in part via activation of PPARγ pathway, which negatively interfered with p38/ATFs cascade.