Cyclooxygenase Enzyme Activity Research Articles

Inhibition of NF-κB-mediated transcription and induction of apoptosis by melampolides and repandolides

Nuclear factor-kappaB (NF-kappaB) plays a crucial role in the regulation of inflammatory processes, cell proliferation, and apoptosis. Blocking NF-kappaB signaling may represent a therapeutic strategy in cancer and inflammation therapy. The aim of this study was to investigate the effects of sesquiterpenes isolated from Asteraceae, namely melampolides (enhydrin, tetraludin A) and repandolides (repandins A, B, D and E) on the activation of NF-kappaB, cell growth of cancer cells, cell cycle progression and apoptosis. In addition, their effects on the activity of cyclooxygenase-2 (COX-2) enzyme were also evaluated. Cell-based reporter gene assay was conducted in SW1353 cells. COX-2 enzyme activity and cell growth inhibition was determined by enzyme immunoassay and MTT assay respectively. Cell cycle analysis was carried out by flow cytometry and apoptosis was observed by DAPI staining assay. In SW1353 cells, transcription mediated by NF-kappaB was inhibited by enhydrin, tetraludin A and repandins A, B, D and E, while Sp-1 mediated transcription was not affected. COX-2 enzyme activity was inhibited by enhydrin, repandin A and E, but not by tetraludin A, repandin B and D. These compounds were effective in inhibiting the growth of a panel of human tumor cell lines in a concentration-dependent manner. Cell cycle analysis and DAPI staining indicated cell cycle arrest in G(2)/M phase and induction of apoptosis. Enhydrin, tetraludin A and repandins A, B, D and E inhibited tumor cell growth and induced cell cycle arrest and apoptosis. These effects may be related to inhibition of NF-B activation.

Interaction between inhibitors of inducible nitric oxide synthase and cyclooxygenase in adjuvant-induced arthritis in female albino rats: An isobolographic study

We studied the interaction of S-methylisothiourea (a selective inducible nitric oxide synthase inhibitor) with rofecoxib (a selective cyclooxygenase-2 inhibitor) and mefenamic acid (a non-selective cyclooxygenase inhibitor) in adjuvant-induced arthritis in female albino Wistar rats, applying the isobolographic analysis. Each drug was effective in reducing the progressive increase in paw volume less than 50% except rofecoxib, when used alone. Log dose–response curve was obtained for each drug along with the corresponding ED 25. Following isobolographic analysis, combination of S-methylisothiourea with rofecoxib and mefenamic acid revealed supra-additive or synergistic interaction. Experimental ED 25 of the combinations was significantly lower than the theoretical ED 25 of the corresponding drug combination which substantiated the synergistic type of interaction between inducible nitric oxide synthase and cyclooxygenase in adjuvant-induced arthritis in female albino rats. Results suggest that NO regulates the cyclooxygenase enzyme activity as the activity of cyclooxygenase enzymes in the LPS-stimulated leukocyte lysates was significantly low or hardly detectable in the presence of varying concentrations of S-methylisothiourea. Simultaneous inhibition of inducible nitric oxide synthase and cyclooxygenase appears to offer an alternative approach for ameliorating the progression of arthritis.

Why a Diet Rich in Seafood Is Healthy

Omega-3 fatty acids, which are found in marine organisms, are known for their beneficial health effects. One mechanism for their antiinflammatory effects is competitive inhibition of the enzymatic activity of cyclooxygenase (COX), which is the rate-limiting enzyme in the biosynthesis of prostaglandins. Massaro et al . report that exposure of endothelial cells to the omega-3 fatty acid docosahexaenoate (DHA) for periods long enough to allow incorporation of this lipid into cellular membranes inhibits the activation of nuclear factor κB (NF-κB) and subsequently COX-2 expression and prostaglandin production in response to proinflammatory signals. Treatment of human saphenous vein endothelial cells (HSVECs) with DHA for up to 48 hours maximally inhibited COX activation in response to subsequent exposure of the cells to interleukin 1α (IL-1α) or a phorbol ester. Western blot and Northern analysis showed that DHA treatment inhibited the IL-1α- or phorbol ester-stimulated expression and abundance of COX-2 (the inducible isoform) without affecting that of COX-1 (the constitutively expressed isoform). Reporter gene analysis demonstrated that DHA-mediated inhibition of COX-2 required the NF-κB binding site in the COX-2 promoter, and DHA pretreatment decreased IL-1α stimulation of NF-κB DNA binding (measured by electrophoretic mobility shift assay) and nuclear translocation (measured by cell fractionation followed by Western blotting). DHA treatment inhibited several steps in the pathway activated by IL-1α that leads to NF-κB activation. In cells treated with DHA, subsequent responses to IL-1α showed (i) decreased extracellular signal-regulated kinase 1 and 2 (ERK1/2) activation but no change in activation of p38 mitogen-activated protein kinase (p38 MAPK), (ii) decreased reactive oxygen species production through inhibition of membrane association of the p47 phox subunit of NADPH oxidase, and (iii) decreased membrane association of protein kinase Cε (PKCε) but not PKCα or PKCζ. Thus, it appears that the long-term beneficial effects of omega-3 fatty acids may be due to alteration of membrane lipid composition leading to reduced signaling in response to inflammatory stimuli. M. Massaro, A. Habib, L. Lubrano, S. Del Turco, G. Lazzerini, T. Bourcier, B. B. Weksler, R. De Caterina, The omega-3 fatty acid docosahexaenoate attenuates endothelial cyclooxygenase-2 induction through both NADP(H) oxidase and PKCε inhibition. Proc. Natl. Acad. Sci. U.S.A. 103 , 15184-15189 (2006). [Abstract] [Full Text]

Role of Prostanoids in the Regulation of Cerebral Blood Flow During Normoxia and Hypoxia in the Fetal Sheep

The fetal cardiovascular responses to hypoxia include decreased peripheral blood flow and increased cerebral, cardiac, and adrenal blood flow. Prostanoids, metabolites of cyclooxygenase enzyme activity, have potent effects on vascular tone in both the adult and the fetus. To examine the role of prostanoids in the regulation of fetal cerebral blood flow (CBF) during acute hypoxic stress, eight near term fetal sheep were studied after infusing vehicle or diclofenac, a cyclooxygenase inhibitor, followed by a 30-min period of hypoxia (arterial Po(2) 12 Torr). In the control experiments, CBF, measured continuously with laser Doppler flowmetry, increased to 148% of baseline values (p < 0.01) and cerebral vascular resistance decreased to 70% of baseline values after 30 min of hypoxic stress. During diclofenac infusion, hypoxia resulted in a CBF increase to only 129% of baseline, a significant attenuation (p < 0.05), accompanied by decreased plasma prostanoid concentrations. Increases in mean arterial blood pressure during hypoxia were also attenuated by diclofenac infusion. Flow and pressure responses were not accompanied by changes in cerebral vascular resistance. These results indicate that prostanoids indirectly modulate fetal CBF responses to hypoxia, but that their effects are mediated through modulation of systemic rather than cerebral vascular tone.

Cyclooxygenase-2 directly regulates gene expression of P450 Cyp19 aromatase promoter regions pII, pI.3 and pI.7 and estradiol production in human breast tumor cells

The present studies evaluated the direct effects of the presence of human cyclooxygenase-2 (Cox-2) on gene expression of specific promoter regions of the P450 Cyp19 enzyme aromatase enzyme and its product, estradiol, in Cox-2 null estrogen-dependent MCF-7 breast tumor cells and in a stable clone of MCF-7 cells containing transfected Cox-2 cDNA, designated as MCF-7/Cox-2 Clone 10. Clone 10 human breast tumor cells have significantly increased gene expression of total mRNA of the P450 Cyp19 enzyme aromatase, with high levels of gene expression of specific aromatase promoter (p) regions pII, pI.3, and p1.7, with no significant change in mRNA levels of p1.4. Clone 10 human breast tumor cells produced significantly increased amounts of both prostaglandin E 2 (PGE 2) derived from Cox-2 enzyme activity and estradiol derived from aromatase enzyme activity ( p < 0.01), compared to MCF-7/vector control cells. The greatest inhibition of PGE 2 or estradiol production was observed by the combination of the selective Cox-2 inhibitor celecoxib (25 μM) and the aromatase inhibitor, formestane (10 nM) ( p < 0.01). The greatest anti-proliferative effect in Cox-2 null MCF-7/vector control cells was observed with the combination of 25 μM celecoxib and 10 nM formestane but not with 10 μM celecoxib, suggesting that there are Cox-2-independent mechanisms involved in the anti-proliferative effect of this agent at doses greater than 10 μM. Celecoxib (25 μM) also significantly inhibited proliferation of MCF-7/Cox-2 Clone 10 human breast tumor cells, with no further anti-proliferative activity with the addition of 10 nM formestane observed at either 24 or 48 h of treatment. These studies demonstrate that Cox-2 directly regulates gene expression of specific aromatase promoter regions and regulates aromatase enzyme activity. Agents that inhibit Cox-2 or block the biological effects of PGE 2 may be useful in significantly limiting aromatase activity and proliferation of human breast tumor cells regardless of the presence of Cox-2. In addition, the unique human breast tumor cell model used in these studies may be a useful tool in identifying the spectrum of activities of agents that block the biological effects of PGE 2 and estradiol.

Mixed Tocopherols Inhibit Azoxymethane-Induced Aberrant Crypt Foci in Rats

: Gamma (γ) tocopherol, but not alpha (α) tocopherol (vitamin E), has previously been reported as an effective inhibitor of cyclooxygenase (COX) enzyme activity. In a pilot study of 17 rats, mixed tocopherols containing more than 50% γ-tocopherol, added at 0.1% to an AIN-76A diet, produced a significant inhibition (about 55%) of azoxymethane-induced aberrant crypt foci in the colon of rats. Mixed tocopherols also reduced tetradecanoyl phorbol acetate–induced ear inflammation in mice when topically applied.

Natural COX-2 inhibitors and effects on colon cancer cells

The cyclooxygenase-2 enzyme (COX-2) is involved in prostaglandin biosynthesis and thus plays a significant role in the process of inflammation. Different types of natural compounds can affect the COX-2 enzymatic activity [1]. In recent years it has also been suggested that COX-2 is involved in cancer development [2]. As many natural compounds are present in the human diet this study has focused on discovery of natural COX-2 inhibitors present in food. A number of human fecal water samples (the aqueous phase of feces) from vegetarians were collected and assessed for effects on COX-2 enzymatic activity and also for effects on protein expression level in human colon cancer cells (HT-29). Of 14 samples analyzed, 13 decreased COX-2 protein levels in the cells (19–63% inhibition) [3]. Most samples also significantly decreased prostaglandin (PGE2) production in HT-29 cells [3]. Solid phase extraction was performed to trace the activity of the fecal water samples. The resulting water fraction was found to be responsible for the inhibitory activity of the samples, suggesting that the active compounds are most likely polar in nature. The chemical content of fecal water was analyzed using chromatographic techniques. Gas chromatography-mass spectrometry was used to study the content of the fecal water, with an emphasis on finding phenolic compounds, whereas nuclear magnetic resonance (NMR) was used to obtain an overview of the total fecal water content. The NMR metabolite profiles of vegetarian fecal water samples were very similar, suggesting that the colonic content of the individuals in the study population were similar in composition. A variety of compounds were identified including several phenolic compounds, amino acids and fatty acids. To the best of our knowledge, the use of NMR as a tool in studying colonic contents represents a new approach that would potentially be very useful in colon cancer research.

Cryptotanshinone inhibits cyclooxygenase-2 enzyme activity but not its expression

Cyclooxygenase-2 (COX-2) is a key enzyme that catalyzes the biosynthesis of prostaglandins from arachidonic acid and plays a critical role in some pathologies including inflammation, neurodegenerative diseases and cancer. Cryptotanshinone is a major constituent of tanshinones, which are extracted from the medicinal herb Salvia miltiorrhiza Bunge, and has well-documented antioxidative and anti-inflammatory effects. This study confirmed the remarkable anti-inflammatory effect of cryptotanshinone in the carrageenan-induced rat paw edema model. Since the action of cryptotanshinone on COX-2 has not been previously described, in the present study, we further examined the effect of cryptotanshinone on cyclooxygenase activity in the exogenous arachidonic acid-stimulated insect sf-9 cells, which highly express human COX-2 or human COX-1, and on cyclooxygenases expression in human U937 promonocytes stimulated by lipopolysaccharide (LPS) plus phorbolmyristate acetate (PMA). Cryptotanshinone reduced prostaglandin E 2 synthesis and reactive oxygen species generation catalyzed by COX-2, without influencing COX-1 activity in cloned sf-9 cells. In PMA plus LPS-stimulated U937 cells, cryptotanshinone had negligible effects on the expression of COX-1 and COX-2, at either a mRNA or protein level. These results demonstrate that the anti-inflammatory effect of cryptotanshinone is directed against enzymatic activity of COX-2, not against the transcription or translation of the enzyme.

A New Wrinkle on Topical Vitamin E and Photo-inflammation: Mechanistic Studies of a Hydrophilic γ-Tocopherol Derivative Compared with α-Tocopherol

The antioxidant function of vitamin E is thought to mediate its photo-protective effects. Cyclooxygenase-2 (COX-2) is an important mediator of early photo-inflammation. Thus, the ability of gamma-tocopherol to inhibit COX-2 activity independently of its antioxidant function raises important questions regarding potential roles that this form of vitamin E plays in photo-protection and skin cancer chemoprevention.

Cardamonin inhibits COX and iNOS expression via inhibition of p65NF-κB nuclear translocation and Iκ-B phosphorylation in RAW 264.7 macrophage cells

Cardamonin, a chalcone isolated from the fruits of a local plant Alpinia rafflesiana, has demonstrated anti-inflammatory activity in cellular models of inflammation. In this report, we evaluated the ability of cardamonin to suppress both NO and PGE 2 synthesis, iNOS and COX-2 expression and enzymatic activity, and key molecules in the NF-κB pathway in order to determine its molecular target. Cardamonin suppressed the production of NO and PGE 2 in interferon-γ (IFN-γ)- and lipopolysaccharide (LPS)-induced RAW 264.7 cells. This inhibition was demonstrated to be caused by a dose-dependent down-regulation of both inducible enzymes, iNOS and COX-2, without direct effect upon iNOS or COX-2 enzyme activity. Subsequently we determined that the inhibition of inducible enzyme expression was due to a dose-dependent inhibition of phosphorylation and degradation of I-κBα, which resulted in a reduction of p65NF-κB nuclear translocation. We conclude that cardamonin is a potential anti-inflammatory drug lead that targets the NF-κB pathway.

Regulation of cyclooxygenase activity in cultured endometrial stromal cells by granulocyte-macrophage colony-stimulating factor

To assess the ability of granulocyte-macrophage colony-stimulating factor (GM-CSF) to regulate cyclooxygenase (COX) enzyme activity and prostaglandins (PGs) synthesis, specifically PGE2 production in stromal cells, neither of which have been addressed in the literature. Prospective study. Department of obstetrics and gynecology at a university hospital. Human luteal phase endometrium was obtained from surgical specimens (n = 6) for clinical indications. Confluent stromal cells were stimulated with GM-CSF. Expression of COX mRNA, COX enzyme activity, and PGE2 level in cultured stromal cells. Confluent stromal cell cultures treated with P and E2 for 9 days were stimulated with GM-CSF. After treatment of 12 hours, low-dose GM-CSF (0.001-0.1 ng/mL) increased COX-2 mRNA levels in stromal cell, whereas high dose GM-CSF (1-100 ng/mL) decreased COX-1 and COX-2 mRNA levels. After treatment of 48 hours, low concentrations of GM-CSF (0.001-0.1 ng/mL) increased total COX and COX-2 enzyme activity, whereas high concentrations of GM-CSF (1-100 ng/mL) inhibited COX and COX-2 activity; The PGE2 levels decreased by 31% to 393.3 pg/mL (P < .05) with concentrations of GM-CSF increasing from 1 ng/mL to 100 ng/mL. There appeared to be a biphasic pattern of COX-2 enzyme response to GM-CSF with low concentrations increasing activity and high concentrations inhibiting activity. It is possible that GM-CSF may provide critical regulation of PG production in the preimplantation period.

Induction of HSP70 is dispensable for anti-inflammatory action of heat shock or NSAIDs in mast cells

It is well known that nonsteroidal anti-inflammatory drugs (NSAIDs), such as acetylsalicylic acid, ibuprofen, and indomethacin, induce anti-inflammatory effects through inhibition of cyclooxygenase enzyme activity. However, it has also been established that a variety of their anti-inflammatory effects are independent of cyclooxygenase. In the search for alternative modes of action, it was found that NSAIDs share some cellular effects with heat shock treatment. This prompted us to investigate whether NSAIDs modulate production of proinflammatory cytokines by mast cells through the heat shock response. In mouse mast cells, derived from a culture of bone marrow cells of male BALB/cBy and null HSF-1(-/-) mice, responsiveness to heat shock and NSAIDs was monitored by measuring tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6) production and signaling pathways. In bone marrow-derived mast cells (BMMC), we found that heat shock and a number of NSAIDs induced heat shock protein 70 (HSP70), which was closely paralleled with inhibition of IL-6 and TNF-alpha production. Surprisingly, in BMMC from HSF-1(-/-)mice, heat shock and selected NSAIDs were still able to suppress cytokine production in the absence of HSP70 induction. In this article, we provide evidence that inhibition of release of proinflammatory cytokines by NSAIDs and heat shock may be attributed to inhibition of the inhibitory nuclear factor kappaB (NF-kappaB) kinase activity, extracellular signal-regulated kinases 1/2, and p38 pathways, resulting in decreased transcriptional activity of the NF-kappaB pathway.

Differential modulation of cyclooxygenase-mediated prostaglandin production by the putative cancer chemopreventive flavonoids tricin, apigenin and quercetin

Diet-derived flavonoids possess cancer chemopreventive properties in preclinical models. The knowledge of the pharmacology of most flavonoids is insufficient to warrant their advancement to clinical evaluation. Here the three flavonoids tricin from rice bran, apigenin from leafy vegetables, and quercetin from onions and apples, were compared in terms of their ability to modulate cyclooxygenase- (COX-) catalyzed prostaglandin E-2 (PGE-2) generation. Specifically their effects on the following parameters were studied: (1) COX enzyme activity, (2) COX-2 expression in human-derived colon cancer cells HCA-7, which express COX-2 constitutively, (3) phorbol ester-mediated COX-2 induction in human colon epithelial cells (HCEC), and (4) PGE-2 levels in cellular incubations. Tricin and quercetin inhibited enzyme activity in purified COX-1 and -2 preparations with IC50 values of near 1 (tricin) and 5 microM (quercetin). Apigenin at up to 25 microM did not affect COX enzyme activity. Flavonoids were incubated with cells for 6 or 24 h and COX-2 protein expression and PGE-2 levels were assessed by Western blot and competitive immunoassay, respectively. None of the agents affected constitutive COX-2 expression in HCA-7 cells. Apigenin, but not tricin or quercetin, down-regulated inducible COX-2 expression in HCEC cells on 6 h incubation. All three flavonoids reduced cellular levels of PGE-2 in the supernatant of HCA-7 cells at both time points and of HCEC cells at 6 h. The results demonstrate that these structurally similar flavonoids regulate COX-mediated PGE-2 production in different fashions. Their ability to attenuate prostanoid levels may contribute to their cancer chemopreventive efficacy.

Regulation of the NAD + ‐dependent histone deacetylase Sirt1 in conditions of muscle use and disuse

Caloric restriction promotes longevity in mammals. This effect is mediated by the NAD+-dependent histone deacetylase Sirt1, which is sensitive to metabolic alterations and interacts with the regulatory protein of mitochondrial biogenesis PGC-1α. Conditions of muscle use and disuse such as endurance training and denervation are known to produce alterations in mitochondrial biogenesis as well as energy metabolism. To determine whether Sirt1 is regulated under those conditions, Sirt1 deacetylase activity was evaluated in muscles of rats subjected to chronic electrical stimulation for 7 days (10 Hz; 3 hrs/day), or denervation for 14 days. Mitochondrial biogenesis was determined by measuring the protein expression of PGC-1α and cytochrome c, and COX enzyme activity. Sirt1 activity was measured using a fluorimetric deacetylation assay and its reliability was assessed as a function of sample protein concentration, incubation time, and inhibitor (nicotinamide) concentration. In chronically-stimulated extensor digitorum longus (EDL) muscle, PGC-1α, cytochrome c and COX enzyme activity were increased by 30–50% compared to non-stimulated muscle. In denervated EDL, those markers of mitochondrial biogenesis were decreased to the same extent (30–50%) compared to the control muscle. In contrast, Sirt1 activity was increased by 20% and 76% in chronically-stimulated and denervated EDL, respectively. Our data suggest that 1) SIRT1 and PGC-1α expression are independently regulated, and 2) mitochondrial biogenesis and SIRT1 function are involved in separate metabolic pathways in conditions of muscle use and disuse. Support from CIHR.

Anti‐inflammatory mechanism of rho‐iso‐alpha acids from hop extract

Prostaglandin E2 (PGE2) has been implicated in many inflammatory diseases including rheumatoid arthritis, angiogenesis and cancer. Rho-iso-alpha acids (RIAA, modified hop extract from Humulus lupulus) inhibits PGE2 production in lipopolysaccharide (LPS) activated murine macrophage cell line RAW 264.7. Analysis of the mechanism shows that RIAA inhibits PGE2 by inhibiting cyclooxygenase-2 (COX-2) protein expression in RAW 264.7 cells. In vitro and cell-free enzymatic assays reveal that RIAA does not inhibit PGE2 production by inhibiting COX-2 enzymatic activity. RIAA inhibits NF-□B mediated signaling pathway, as evidenced by reductions in I□B□ degradation, NF-□B p50 nuclear translocation and binding, and NF-□B driven promoter activity. Moreover, the dose dependent inhibition of LPS activated murine COX-2 promoter activity by RIAA is attenuated by mutations at two NF-□B binding sites (−401 and −668) on the COX-2 promoter. Inducible nitric oxide synthase (iNOS), a known NF□B dependent gene, was also inhibited by RIAA. It appears that the anti-inflammatory properties of RIAA are independent of ERK-1/2, p38 and JNK signaling pathways. These results demonstrate that RIAA from hop extract inhibit inflammation through multiple mechanisms, including the NF-□B signaling pathway. (Supported by Metagenics Inc.)

Novel Targeting Of COX‐2 pre‐mRNA Using Antisense Morpholino Oligonucleotides: Suppression Of Cox‐2 Activity In Human Myometrial cells And Amnion‐Derived WISH Cells

Increased expression of COX-2 has been implicated in the onset of both term and preterm labour. In this context both selective and non-selective COX-2 inhibitors have been used in clinical trials to determine their efficacy in delaying preterm labour. However recent evidence indicates that these tocolytics may have potential adverse fetal and maternal side effects. Consequently, the development of more specific and non-toxic agents to inhibit COX-2 needs to be considered. We have evaluated whether antisense morpholino oligonucleotides have therapeutic potential in inhibiting COX-2. Confocal microscopy on ‘live’ cells illustrated high levels of penetrance of antisense morpholino oligonucleotides using the new Endo-Porter formula (Gene-Tools, LLC) with delivery efficiencies of 82% and 78% respectively in amnion-derived WISH and myometrial cells. Substantial inhibition by the morpholino oligonucleotides of COX-2 expression, induced by LPS administration, was observed at both the mRNA and protein level. Loss of enzymic activity of COX-2 was confirmed using a sensitive COX enzyme activity assay which reflects the rate of conversion of arachidonic acid to PGH2. Our results indicate that antisense morpholino oligonucleotides significantly inhibit expression and activity of this enzyme in in vitro cultures of amnion-WISH and myometrial cells. The potential thus exists that a similar approach can be mimicked in vivo to produce a highly specific and non-toxic strategy to inhibit COX-2 activity with its subsequent effects on the better management of preterm labour and other inflammatory conditions.

Novel Targeting of Cyclooxygenase-2 (COX-2) Pre-mRNA Using Antisense Morpholino Oligonucleotides Directed to the 3′ Acceptor and 5′ Donor Splice Sites of Exon 4: Suppression of COX-2 Activity in Human Amnion-Derived WISH and Myometrial Cells

Increased expression of cyclooxygenase-2 (COX-2) has been implicated in the onset of both term and preterm labor. In this context, both selective and nonselective COX-2 inhibitors have been used in clinical trials to determine their efficacy in delaying preterm labor. However, recent evidence indicates that these tocolytics may have potentially adverse fetal and maternal side effects. Therefore, the development of more specific and nontoxic agents to inhibit COX-2 needs to be considered. We have evaluated whether antisense morpholino oligonucleotides have therapeutic potential in inhibiting COX-2 by specifically targeting both the 3' and 5' acceptor and donor sites of exon 4 of COX-2's pre-mRNA sequence. Confocal microscopy on "live" cells illustrated high levels of penetrance of antisense morpholino oligonucleotides using the Endo-Porter formula (Gene-Tools, LLC, Philomath, OR), with delivery efficiencies of 82 and 78%, respectively, in amnion-derived WISH and myometrial cells. Substantial inhibition by the morpholino oligonucleotides of COX-2 expression, induced by lipopolysaccharide administration, was observed at both the mRNA and protein levels. Loss of enzymic activity of COX-2 was confirmed using a sensitive COX enzyme activity assay, which reflects the rate of conversion of arachidonic acid to prostaglandin H2. Our results indicate that antisense morpholino oligonucleotides significantly inhibit expression and activity of this enzyme in in vitro cultures of amnion-WISH and myometrial cells. The potential thus exists that a similar approach can be mimicked in vivo to produce a highly specific and nontoxic strategy to inhibit COX-2 activity with its subsequent effects on the better management of preterm labor and other inflammatory conditions.

Human Fecal Water Inhibits COX-2 in Colonic HT-29 Cells: Role of Phenolic Compounds

The inducible enzyme cyclooxygenase-2 (COX-2) plays a major role in the regulation of inflammation and possibly in the development of colon cancer. The aim of the present study was to screen for COX-2 inhibitors in samples of fecal water (the aqueous phase of feces) and investigate whether phenolic compounds are responsible for any observed effects on COX-2. Volunteers (n = 20) were recruited and asked to supply a 24-h stool sample. Fecal water samples were prepared and analyzed by GC-MS for their content of phenolic compounds. These samples were also evaluated for their effects on COX-2 protein levels (Western blot) and prostaglandin (PG)E2 production in tumor necrosis-alpha-stimulated HT-29 cells and pure enzymatic activity in a COX-2-catalyzed prostaglandin biosynthesis in vitro assay. The major phenolic compounds identified were phenylpropionic acid, phenylacetic acid, cinnamic acid, and benzoic acid derivatives. Of 13 fecal water samples analyzed, 12 significantly decreased PGE2 production (range 5.4-39.7% inhibition, P-value < 0.05) compared with control cells and 13 of 14 samples analyzed decreased COX-2 protein levels in HT-29 cells (19-63% inhibition). Of the 20 fecal water samples, 2 also weakly inhibited enzymatic activity of purified COX-2 (22-24% inhibition). Three compounds identified in fecal water, 3-phenylpropionic acid, 3-hydroxyphenylacetic acid, and 3-(4-hydroxyphenyl)-propionic acid, decreased the protein level at 250 micromol/L (15-62% inhibition). This study shows for the first time that human fecal water contains components that can affect both the COX-2 protein level and enzymatic activity.

Dual inhibitory effects of furonaphthoquinone compound on enzyme activity and lipopolysaccharide-induced expression of cyclooxygenase-2 in macrophages

2-Methyl-2-(2-methylpropenyl)-2,3-dihydronaphtho[2,3- b]furan-4,9-dione (NFD-37) is a synthetic furonaphthoquinone compound. In the present study, the NFD-37 compound was found to inhibit prostaglandin (PG) E 2 production in lipopolysaccharide (LPS)-stimulated macrophages RAW 264.7. NFD-37 compound exhibited a preferred inhibition on enzyme activity of cyclooxygenase (COX)-2 over COX-1. Further, NFD-37 compound attenuated LPS-induced synthesis of both mRNA and protein of COX-2, and suppressed LPS-induced COX-2 promoter activity in the macrophages, indicating that the furonaphthoquinone compound could down-regulate LPS-induced COX-2 expression at the transcription level. Even though COX-2 promoter behaves as a sophisticated biosensor for host defense, nuclear factor (NF)-κB activation has been evidenced to play a major mechanism for LPS-induced COX-2 expression in macrophages. NFD-37 compound exhibited a dose-dependent inhibitory effect on LPS-induced phosphorylation of inhibitory κBα (IκBα) protein, and subsequently inhibited IκBα degradation, DNA binding activity of NF-κB complex as well as NF-κB transcriptional activity in macrophages RAW 264.7. In another experiment, NFD-37 compound inhibited both COX-2 promoter activity and GST-IκBα phosphorylation elicited by an expression vector encoding IκB kinase β. Taken together, NFD-37 compound inhibited enzyme activity of COX-2 but also suppressed COX-2 expression depending on NF-κB activation, and thus could provide an invaluable tool to investigate pharmacological potential in the excess PG-related disorders.

Cyr61 Induces Gastric Cancer Cell Motility/Invasion via Activation of the Integrin/Nuclear Factor-κB/Cyclooxygenase-2 Signaling Pathway

Cysteine-rich 61 (Cyr61/CCN1) is involved in many different types of tumor development and progression. Nonetheless, the role of Cyr61 in human gastric cancer has not yet been fully characterized. We addressed the issue by immunohistochemical staining of 81 gastric adenocarcinoma specimens. Liposome-mediated transfection was used to introduce a Cyr61 expression vector into gastric cancer AGS cell lines. Transfectants were tested in invasion assay by a Boyden chamber. Furthermore, a cyclooxygenase-2 (COX-2) reporter assay and gel mobility shift assay were done to investigate the potential signal pathway of Cyr61. Patients with gastric adenocarcinoma whose tumor displayed high expression of Cyr61 correlated well with aggressive lymph node metastasis, more advanced tumor stage, histologic diffuse type, and early recurrence. Stable transfection of Cyr61 into the AGS cell line strongly enhanced its invasive activity. The overexpression of Cyr61 into AGS cells significantly increased the expression of COX-2 mRNA, protein, and enzymatic activity. Gel mobility shift assays further showed that the nuclear factor-kappaB (NF-kappaB) pathway was evidently activated in Cyr61-expressing AGS cells. Function-neutralizing antibody to alphavbeta3 but not alphavbeta5 effectively suppressed Cyr61-mediated NF-kappaB activation, COX-2 gene expression, and cell invasiveness. Cyr61 may contribute to the malignant progression of gastric cancer by promoting tumor cell motility/invasion through up-regulation of the functional COX-2 via an integrin alphavbeta3/NF-kappaB-dependent pathway.