Cyclooxygenase Enzyme Activity Research Articles

In vitro Anti-inflammatory Activity of Ligustrum vulgare Extracts and Their Analytical Characterization

Interest in the anti-inflammatory effects of Ligustrum vulgare L., which has been used traditionally in China and Japan prompted us to determine antiinflammatory effects of the plant's compounds in leukocytes. The leaves of L. vulgare were used to prepare a decoction which was successively extracted with organic solvents (dichloromethane (DCM), n-butanol, ethyl acetate) using liquid-liquid partition. Extracts were tested for inhibition of LTB4, resp. PGE2 biosynthesis. Each extract was evaluated for its in vitro cyclooxygenase-1/2 (COX-1/2) inhibitory activity using assays with purified COX-1 and COX-2 enzymes, as well as for their LTB4 formation inhibitory activity using an assay with activated human neutrophil granulocytes. All extracts reported inhibitory actions against COXs in comparison with the synthetic inhibitors NS-398 (IC50 = 2.6 microM) and indomethacin (IC50 = 0.9 microM). The dichloromethane extract of privet leaves showed a considerable inhibitory effect against COX-1 and COX-2 enzyme activity. The DCM extract revealed 2.7 times higher inhibitory activity against LTB4 formation in comparison with the known specific LT inhibitor zileuton (IC50 = 5.0 microM). Additionally, oleuropein and echinacoside were detected by HPLC-DAD and LC-MS in the Ligustrum vulgare leaves. Both compounds exhibited weak inhibitory activity on cyclooxygenases and leukotriene formation.

Seizure susceptibility in immature brain due to lack of COX-2-induced PGF2α

The immature brain is prone to seizure; however, the mechanism underlying this vulnerability has not been clarified. Febrile seizure is common in young children, and the use of non-steroidal anti-inflammatory drugs for febrile seizure is not recommended. In previous studies, we established that prostaglandin (PG) F2α, a product of cyclooxygenase (COX), acts as an endogenous anticonvulsant in the adult mouse. Therefore, we assumed that COX-2 activity was involved with seizure susceptibility in early life. In the present study, immature mice (postnatal day 9) were far more prone to kainic acid (KA)-induced seizures than mature mice (after postnatal day 35). Seizure activity began later in immature mice, but was more severe and was unaffected by a potent COX inhibitor, indomethacin; in contrast, indomethacin aggravated seizure activity in mature mice. Immature mouse brains exhibited little basal COX-2 expression and little KA-induced COX-2 induction, while KA-induced COX-2 expression and PGF2α release were prominent in mature brains. During brain development, COX expression was increased and glycosylated in an age-dependent manner, which was necessary for COX enzyme activity. Intracisternal PGF2α administration also reduced KA-induced seizure activity and mortality. Taken together, low COX activity and the resulting deficiency of PGF2α may be an essential cause of increased seizure susceptibility in the immature brain.

In vitro anti-inflammatory effect of picrasmalignan A by the inhibition of iNOS and COX-2 expression in LPS-activated macrophage RAW 264.7 cells

Picrasma quassioides (P. quassioides) has been widely used as a traditional Chinese medicine for clearing fever and detoxification. Previous phytochemical studies have identified a novel dihydrobenzofuran-type neolignan, picrasmalignan A, isolated from the stems of P. quassioides. In the present study, the in vitro anti-inflammatory effects and molecular mechanisms of picrasmalignan A have been investigated in cultured RAW 264.7 cells, a mouse macrophage‑like cell line. A Griess assay was used to demonstrate the inhibitory effect of picrasmalignan A on the overproduction of nitric oxide (NO). An enzyme-linked immunosorbent assay was used to determine the levels of pro-inflammatory cytokines, including tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6). The inhibitory effect on the enzymatic activity of cyclooxygenase 2 (COX‑2) and inducible nitric oxide synthase (iNOS) was observed by colorimetric and fluorimetric assays, respectively. Western blotting was conducted to detect the expression of iNOS and COX-2. Results showed that picrasmalignan A suppressed lipopolysaccharide-stimulated NO production and pro-inflammatory cytokine secretion, including TNF-α and IL-6, in a dose-dependent manner. It also significantly inhibited the expression and enzymatic activity of iNOS and COX-2. These results may demonstrate the anti-inflammatory mechanism of picrasmalignan A.

Effect of ibuprophen administration before embryo transfer on IVF outcome: a randomized controlled trial

To examine the effect of ibuprophen(Moment®) administration before embryo transfer on the pregnancy outcome of IVF-embryo transfer programs. Prospective, randomized, double-blinded placebo-controlled clinical study, conducted from september 2010 to january 2011. Patient(s): One hundred sixty-six consecutive cycles of fresh IVF-ET. The patients underwent IVF because of tubal, male infertility, unexplained, or endometriosis factors. They were randomly randomly divided into treatment and control group. Intervention(s): in the treatment group, 83 patients included in fresh ET cycles received an oral dose of 200mg of ibuprophen (10 drops).In the control group, the same number cycles corresponding to the treatment group were treated with placebo. Both groups started ibuprophen or placebo treatment 90minutes before embryo transfer. Patients and staff were blinded to the treatment. Main outcome measure(s): pregnancy rate and implantation rate. Ibuprophen increases significantly pregnancy rate 34.93 %) and implantation rate (15.10 %) compared to the control group(20.48 % and 9.60 % respectively; p<0.01). Moreover abortion rate was significantly reduced in the treatment group compared to the placebo group (13.79 % and 29.41 % respectively; p <0.001). To our knowledge, this is the first study to investigate the possible consequence of oral ibuprophen before embryo transfer for improving IVF-ET results. Our data indicate that ibuprophen improves IVF results and so may be an effective adjunctive treatment for IVF. Further studies are needed to clarify wether this effect is associated with the reduction of cyclooxygenase enzyme activity during embryo transfer or wether other mechanisms are involved.

Abstract 325: COX2 Expression is Increased in Human and Mouse Calcific Aortic Valve Disease and COX2 Inhibition Blocks the Induction of Osteogenic Gene Expression in Valve Interstitial Cells

Calcific aortic valve disease (CAVD) is considered a disease of aging and is characterized by calcific nodule formation, stenosis of the valve, and induction of osteogenic gene expression. The klotho deficient mouse model of premature aging exhibits CAVD evident in calcific lesions in the aortic valve annulus. Klotho deficient mice develop calcification by 6 weeks of age, with minimal immune cell infiltration and increased osteogenic gene expression. Microarray analyses performed on aortic valve tissues of wild type and klotho deficient mice demonstrate that cyclooxygenase 2 (COX2) expression is increased in klotho deficient mice. We hypothesize that increased COX2 expression contributes to aortic valve calcification and that COX2 inhibition prevents osteogenic gene expression in aortic valve interstitial cells (aVICs). In klotho deficient mice, COX2 protein expression is increased throughout regions of valve calcification relative to wild type controls. Prior to the formation of calcified valve lesions, increased COX2 expression is localized to areas at risk of developing calcification in klotho deficient mice. Thus increased COX2 expression precedes valve calcification. In human explanted calcific aortic valves, COX2 expression also is increased in mineralized regions compared to controls. In bone, COX2 deficiency inhibits osteogenic gene induction. The ability of COX2 inhibition to block osteogenic gene expression was examined using porcine aVICs. Treatment of aVICs with osteogenic media induces bone markers, including osteocalcin, in addition to increasing COX2 gene expression. Furthermore, inhibition of COX2 activity in cells cultured under osteogenic conditions results in significantly decreased expression of osteogenic genes compared to controls. In vivo studies in progress provide initial evidence that loss of COX2 activity reduces aortic valve calcification in klotho deficient mice. In conclusion, COX2 expression is increased in regions of aortic valve calcification in both human and mouse CAVD tissues, and COX2 inhibition suppresses osteogenic gene expression in cultured aVICs. Targeting COX2 enzyme activity may be a potential therapeutic option for preventing or reducing aortic valve calcification in the future.

Abstract 3286: CLEFMA synergizes with celecoxib in suppression of lung cancer cell growth.

Abstract Background: Cyclooxygenase-2 (COX-2) is frequently over-expressed in various malignancies, often associated with poor clinical outcome. Celecoxib, a selective COX-2 inhibitor induces apoptosis in cancer cells. However, at higher concentrations needed for chemoprevention or treatment, celecoxib targets COX-2 independent drug targets resulting in undesired toxicity. CLEFMA or 4-[3, 5-bis (2-chlorobenzylidene-4-oxo-piperidine-1-yl)-4-oxo-2-butenoic acid], a novel molecule synthesized in our laboratory potently inhibits the proliferation of various cancer cells while sparing normal cells. In this study we have tested a hypothesis that CLEFMA would reduce the celecoxib dose required for effective suppression of cancer cell proliferation. Methods: We determined IC50 values of CLEFMA and celecoxib in lung adenocarcinoma (H441 and A549) and small cell lung cancer (H1650) cells. Further, we tested their toxicity in normal lung fibroblast CCL151 cells at 1x IC50 and 2x IC50 dose levels required for H441 cells. Drug concentrations at ratio 1:3 (Mixture 1), 1:1 (Mixture 2) and 3:1 (Mixture 3) of respective IC50 values of CLEFMA and celecoxib were examined for synergism. The data was analyzed with the Calcusyn software. Following synergy experiments, we determined the effect of CLEFMA treatment on total COX and COX-2 enzymatic activity in cultured A549 cells and in tumor tissue lysates obtained from H441 xenografts in mice. Expression of anti-apoptotic (BCL-2, Bcl-XL and surviving) and apoptotic proteins (PARP, caspase-3 and caspase-7) were investigated by immunobloting. Results: We observed that CLEFMA was more effective in suppressing the proliferation of all three cancer cell lines than celecoxib. Celecoxib showed significant toxicity in normal lung fibroblasts at 2x IC50 dose, whereas CLEFMA was non-toxic to the normal cells. From the fractional effect-combination index (Fa-CI) plots, we found that Mixture 1 (1:3:: CLEFMA:celecoxib) was synergistic (CI &amp;lt; 1.0) in all three cell lines; in H441 cells mixture 2 (1:1) was synergistic at lower concentration of the drugs, Mixture 3 (3:1) was synergistic only in A549 cells. CLEFMA does not reduce COX-2 activity/unit enzyme, but reduced the expression levels of COX-2 protein; the combination of CLEFMA with celecoxib synergistically suppressed COX-2 expression. CLEFMA also potentiated the activity of celecoxib in down-regulating Bcl-2, Bcl-XL and survivin (anti-apoptotic proteins) and up-regulating PARP, caspase-3 and caspase-7 (pro-apoptotic proteins). Conclusions: From the results we conclude that CLEFMA synergistically augments the growth inhibitory effect of celecoxib in human lung cancer cell lines without being toxic to normal cells. It appears that the synergy depends on the different mode of actions of the two drugs- CLEFMA suppressing COX-2 protein expression, whereas celecoxib reducing COX-2 activity. Citation Format: Kaustuv K. Sahoo, Vivek R. Yadav, Vibhudutta Awasthi. CLEFMA synergizes with celecoxib in suppression of lung cancer cell growth. [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 3286. doi:10.1158/1538-7445.AM2013-3286

Cellular senescence involves an intracrine prostaglandin E2 pathway in human fibroblasts

Cyclooxygenase 2 and release of prostaglandin E2 are involved in many responses including inflammation and are upregulated during cellular senescence. However, little is known about the role of lipid inflammatory mediators in senescence. Here, we investigated the mechanism by which the COX-2/PGE2 axis induces senescence. Using the NS398 specific inhibitor of COX-2, we provide evidence that reactive oxygen species by-produced by the COX-2 enzymatic activity are negligible in front of the total senescence-associated oxidative stress. We therefore investigated the role of PGE2 by invalidating the PGE2 synthases downstream of COX-2, or the specific PGE2 receptors, or by applying PGE2 or specific agonists or antagonists. We evaluated the effect on senescence by evaluating the senescence-associated proliferation arrest, the percentage of senescence-associated β-galactosidase-positive cells, and the expression of senescent molecular markers such as IL-6 and MCP1. We show that PGE2 acting on its EP specific receptors is able to induce both the onset of senescence and the maintenance of the phenotype. It did so only when the PGE2/lactate transporter activity was enhanced, indicating that PGE2 acts on senescence more via the pool of intracellular EP receptors than via those localized at the cell surface. Treatment with agonists, antagonists and silencing of the EP receptors by siRNA revealed that EP3 was the most involved in transducing the intracrine effects of PGE2. Immunofluorescence experiments confirmed that EP3 was more localized in the cytoplasm than at the cell surface. Taken together, these results suggest that COX-2 contributes to the establishment and maintenance of senescence of normal human fibroblasts via an independent-ROS and a dependent-PGE2/EPs intracrine pathway.

In Vitro Inhibition of the Transcription Factor NF‐κB and Cyclooxygenase by Bamboo Extracts

Several bamboo species have been used in traditional medicine for the treatment of inflammatory conditions. The present study evaluates the in vitro anti-inflammatory properties of the traditionally used bamboo species Phyllostachys nigra (Lodd.) Munro and Sasa veitchii (Carr.) Rehder to explore their future research opportunities and therapeutic potential as anti-inflammatory agents. The extracts were evaluated for their potential inhibitory activity at the level of NF-κB-induced gene expression and suppression of cyclooxygenase (COX)-1 and COX-2 enzyme activities, representative pharmacological targets for the anti-inflammatory action of glucocorticoids and non-steroidal anti-inflammatory drugs, respectively. The activity of P. nigra (Lodd.) Munro and S. veitchii (Carr.) Rehder was compared with bamboo species without traditional anti-inflammatory indications. High-performance liquid chromatography with diode-array detection and liquid chromatography-tandem mass spectrometry analyses were performed to phytochemically characterize the extracts. P. nigra (Lodd.) Munro leaf extract potently inhibited NF-κB-induced gene expression, while S. veitchii (Carr.) Rehder leaf extract exerted a selective COX-2 inhibition. The crude extracts consistently showed a more potent bioactivity than the solid phase extraction fractions. P. nigra (Lodd.) Munro and S. veitchii (Carr.) Rehder both exert anti-inflammatory properties, but act via a different molecular mechanism.

NAD(P)H Quinone Oxidoreductase 1 (NQO1)-Bioactivated Pronqodine A Regulates Prostaglandin Release from Human Synovial Sarcoma Cells

Natural products have contributed to the elucidation of biological mechanisms as well as drug discovery research. Even now, the expectation for natural products is undiminished. We screened prostaglandin release inhibitors that had no effect on in vitro cyclooxygenase activity derived from natural product sources and discovered pronqodine A. Using spectral analysis and total synthesis, the structure of pronqodine A was shown to be a benzo[d]isothiazole-4,7-dione analogue. Evaluation of the biological activity of pronqodine A revealed that the NAD(P)H dehydrogenase quinone 1 (NQO1) converted pronqodine A into a two-electron reductive form. The reductive form underwent autoxidation and reversed to its native form immediately with the generation of reactive oxygen species. Further investigations proved that pronqodine A inhibited cyclooxygenase enzyme activity only in the presence of NQO1. Pronqodine A acts as a potential bioreductive compound, inhibiting prostaglandin release in selectively activated NQO1-expressing cells.

The complex effects of the slow‐releasing hydrogen sulfide donor GYY4137 in a model of acute joint inflammation and in human cartilage cells

The role of hydrogen sulfide (H2S) in inflammation remains unclear with both pro- and anti-inflammatory actions of this gas described. We have now assessed the effect of GYY4137 (a slow-releasing H2S donor) on lipopolysaccharide (LPS)-evoked release of inflammatory mediators from human synoviocytes (HFLS) and articular chondrocytes (HAC) in vitro. We have also examined the effect of GYY4137 in a complete Freund's adjuvant (CFA) model of acute joint inflammation in the mouse. GYY4137 (0.1–0.5 mM) decreased LPS-induced production of nitrite (NO2−), PGE2, TNF-α and IL-6 from HFLS and HAC, reduced the levels and catalytic activity of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) and reduced LPS-induced NF-κB activation in vitro. Using recombinant human enzymes, GYY4137 inhibited the activity of COX-2, iNOS and TNF-α converting enzyme (TACE). In the CFA-treated mouse, GYY4137 (50 mg/kg, i.p.) injected 1 hr prior to CFA increased knee joint swelling while an anti-inflammatory effect, as demonstrated by reduced synovial fluid myeloperoxidase (MPO) and N-acetyl-β-D-glucosaminidase (NAG) activity and decreased TNF-α, IL-1β, IL-6 and IL-8 concentration, was apparent when GYY4137 was injected 6 hrs after CFA. GYY4137 was also anti-inflammatory when given 18 hrs after CFA. Thus, although GYY4137 consistently reduced the generation of pro-inflammatory mediators from human joint cells in vitro, its effect on acute joint inflammation in vivo depended on the timing of administration.

Abstract P3-08-03: Urinary levels of prostaglandin E2 metabolite and postmenopausal breast cancer

Abstract Estrogens are considered a key player in the mammary carcinogenesis. In postmenopausal women, peripheral aromatization of androgens is the predominant source of estrogens. Given that prostaglandin E2 (PGE2) is a potent activator of aromatase expression, we hypothesized that endogenous level of PGE2 would influence estrogen bioavailability and subsequent breast cancer risk in postmenopausal women. In addition, because PGE2 synthesis is suppressed by treatment of non-steroidal anti-inflammatory drugs (NSAIDs) via inhibition of cyclooxygenase enzyme activity, we further hypothesized that regular use of NSAIDs may modify the association between PGE2 and postmenopausal breast cancer risk. We carried out a case-cohort analysis within the NIEHS Sister Study, a prospective cohort study of 50,884 women. Postmenopausal women who were aged 50 or older and did not report current use of hormones were eligible for the present analysis (N = 11,338). Urinary levels of a major PGE2 metabolite (PGE-M), which is generally accepted as the most accurate index of endogenous PGE2 formation, were quantified using liquid chromatography/tandem mass spectrometry in 302 incident breast cancer cases and 305 subcohort members who were randomly selected by frequency matching for age among cases. Hazard ratios (HR) and 95% confidence intervals (95% CI) were estimated using Prentice's pseudo-likelihood approach with Barlow's weighting scheme. Several factors were associated with higher levels of urinary PGE-M in the multivariable analysis, including current smoking, obesity, high caloric intake from saturated fat and surgical menopause. No associations were observed for dietary intake of omega 3 fatty acids, use of vitamin supplements and physical activity. Overall, there was no association between urinary PGE-M and postmenopausal breast cancer risk. After including the interaction terms between NSAID use and urinary levels of PGE-M in the model, however, high levels of urinary PGE-M were associated with an increased risk of breast cancer in postmenopausal women who did not regularly use NSAIDs (likelihood ratio test p &amp;lt;0.01). Taking into account use of NSAIDs 1 day prior to urine collection did not change results. NSAID use may modify the conversion of androgens to estrogen and thereby lower breast cancer risk. Expected data on estrogen and estrogen metabolites will help us better understand these findings. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P3-08-03.

In vitro Anti-Inflammatory Effects of Beta-Carboline Alkaloids, Isolated from Picrasma quassioides, through Inhibition of the iNOS Pathway

In our previous study, fourty-eight compounds have been isolated and identified from the roots of Picrasma quassioides, which have been widely used as a traditional Chinese medicine for clearing heat and for detoxification as described in the Chinese Pharmacopoeia. β-Carboline alkaloids are commonly considered as main active constituents of P. quassioides, but the molecular mechanism remains yet unknown. In the present paper, one new β-carboline alkaloid together with two known β-carboline alkaloids have been investigated for their anti-inflammatory effect and mechanism of action in cultured macrophage RAW 264.7 cells. Griess assay was used to evaluate the inhibitory effect on the overproduction of nitric oxide. ELISA was used to determine the level of proinflammatory cytokines including TNF-α and IL-6. The inhibitory effect on the enzymatic activity of COX-2 and inducible nitric oxide synthase were tested by colorimetric and fluorimetric methods, respectively. Western blot was used to detect the expression of inducible nitric oxide synthase and COX-2. All three β-carboline alkaloids suppressed LPS-stimulated nitric oxide production and proinflammatory cytokines secretion, including TNF-α and IL-6 in a dose-dependent manner. They also strongly inhibited the expression of inducible nitric oxide synthase and inducible nitric oxide synthase enzymatic activity, whereas the expression of COX-2 and COX-2 enzymatic activity were not affected. These results indicated that potent inhibition of nitric oxide, TNF-α, and IL-6, but not COX-2 expression and COX-2 activity, might constitute the anti-inflammatory mechanism of β-carboline alkaloids. β-Carboline alkaloids suppressed the overproduction of nitric oxide through downregulation of inducible nitric oxide synthase expression and inducible nitric oxide synthase enzymatic activity in an LPS-stimulated macrophage.

Effects of Meloxicam and Phenylbutazone on Equine Gastric Mucosal Permeability

Newer NSAIDs that more selectively target the induced isoform of the cyclooxygenase enzyme (COX2) activity might reduce adverse effects while preserving therapeutic benefits of these drugs. To compare the effect of oral administration of multiple dose rates of meloxicam and phenylbutazone (PBZ) on gastric mucosal integrity in horses. Twenty-five light breed horses. In vivo toxicity study. Horses were randomly assigned to 5 treatment groups, receiving placebo, PBZ (4.4 mg/kg PO q12h day 1, 2.2 mg/kg PO q12h for 4 days, 2.2 mg/kg PO q24h for 9 days), or 3 dose rates of meloxicam (0.6 mg/kg q24h, 1.8 mg/kg q24h, 3.0 mg/kg q24h) for 14 days. Sucrose permeability testing was performed on Day 0 (before treatment) and on Day 13. All personnel involved with data collection or analysis were blinded to treatment. Administration of PBZ at the above dose rate significantly increased gastric permeability to sucrose, evidenced by increased peak serum sucrose concentrations (280-1,580 pg/μL, P = .001) after treatment. Similar changes were not evident after administration of meloxicam at any dose rate tested, or in control horses (P > .05). Treatment was not associated with significant differences in ulceration of the squamous or glandular mucosa. Peak sucrose concentrations were not correlated with serum total protein or albumin concentrations (R(2) = -0.07, P = .61, R(2) = -0.08, P = .58, respectively). These results suggest that PBZ was associated with greater compromise to gastric mucosal integrity than meloxicam.

Activation of endogenous opioid gene expression in human keratinocytes and fibroblasts by pulsed radiofrequency energy fields

BackgroundPulsed radiofrequency energy (PRFE) fields are being used increasingly for the treatment of pain arising from dermal trauma. However, despite their increased use, little is known about the biological and molecular mechanism(s) responsible for PRFE-mediated analgesia. In general, current therapeutics used for analgesia target either endogenous factors involved in inflammation, or act on endogenous opioid pathways.Methods and ResultsUsing cultured human dermal fibroblasts (HDF) and human epidermal keratinocytes (HEK), we investigated the effect of PRFE treatment on factors, which are involved in modulating peripheral analgesia in vivo. We found that PRFE treatment did not inhibit cyclooxygenase enzyme activity, but instead had a positive effect on levels of endogenous opioid precursor mRNA (proenkephalin, pro-opiomelanocortin, prodynorphin) and corresponding opioid peptide. In HEK cells, increases in opioid mRNA were dependent, at least in part, on endothelin-1. In HDF cells, additional pathways also appear to be involved. PRFE treatment was also followed by changes in endogenous expression of several cytokines, including increased levels of interleukin-10 mRNA and decreased levels of interleukin-1β mRNA in both cell types.ConclusionThese findings provide a new insight into the molecular mechanism underlying PRFE-mediated analgesia reported in the clinical setting.

The different inhibitory effects of Huang-Lian-Jie-Du-Tang on cyclooxygenase 2 and 5-lipoxygenase

BackgroundHuang-Lian-Jie-Du-Tang (HLJDT), a famous traditional Chinese prescription with wide anti-inflammatory applications, is an aqueous extract of four herbal materials: Rhizoma coptidis, Radix scutellariae, Cortex phellodendri, and Fructus gardeniae. Its effects on the cyclooxygenase (COX)-2 and 5-lipoxygenase (5-LOX) pathways are thought to be responsible for its anti-inflammatory activity. However, our previous work found that the inhibitory effects of HLJDT act on the 5-LOX pathway but not on the COX pathway. The possibility that HLJDT inhibits COX-2- or 5-LOX-catalyzed eicosanoid generation by downregulating enzyme expression requires further investigation. Aim of the studyTo observe the effects of HLJDT and its four major components (baicalin, baicalein, berberine and geniposide) on COX-2- or 5-LOX-catalyzed eicosanoid generation and to distinguish the effects of HLJDT on enzyme activity from those on enzyme expression. MethodsThe topical anti-inflammatory activities and inhibition of eicosanoid formation of HLJDT and its components were observed in an arachidonic acid (AA)-induced mouse ear edema model. Macrophage-based systems were established to observe the effects of the drugs on enzyme activity and enzyme expression of COX-2 and 5-LOX. Further experiments were carried out to confirm these effects at the mRNA and protein levels. ResultsTopical treatment of HLJDT significantly inhibited AA-induced mouse ear edema and reduced PGE2 and LTB4 release in the edematous ears. Baicalein, geniposide, and berberine also ameliorated the symptoms and suppressed eicosanoid generation with varying efficacies. Cell-based assays showed that HLJDT and baicalein inhibited the PGE2 levels by decreasing COX-2 enzyme expression without affecting COX-2 enzyme activity in RAW 246.7 murine macrophages. The other experiments on rat peritoneal macrophages indicated that HLJDT and baicalein exerted significant inhibition on LTB4 production by decreasing 5-LOX enzyme activity. The real-time PCR and western blotting data demonstrated that HLJDT and baicalein reduced COX-2 expression at the mRNA and protein levels, whereas no inhibition on 5-LOX expression was observed. ConclusionsHLJDT can suppress eicosanoid generation via both the COX and LOX pathways, which definitely contributes to its topical anti-inflammatory activity. We have confirmed that its dual inhibition on the COX and LOX pathways mainly result from the downregulation of COX-2 expression and direct inhibition of 5-LOX activity, respectively. Baicalein worked as a potent active component in most of the tests. These findings about the different inhibitory effects of HLJDT on COX-2 and 5-LOX help to better understand the mechanism of HLJDT and promote safer applications of drug.

Immunolocalization, gene expression, and enzymatic activity of cyclooxygenases, prostaglandin e2‐9‐ketoreductase, and nitric oxide synthases in mediterranean buffalo (bubalus bubalis) corpora lutea during diestrus

Immunopresence, gene expression, and enzymatic activity of cyclooxygenase 1 (COX1), COX2, PGE2-9-ketoreductase (PGE2-9-K), endothelial (eNOS), and inducible nitric oxide synthases (iNOS), and hormone in vitro production were examined in early, mid, late, and regressive buffalo corpora lutea (CL). COX1 immunosignals were detected in the cytoplasm of small luteal cells, COX2 in large luteal cells, and PGE2-9-K in all luteal cells. COX2 and PGE2-9-K immunosignals were greater in late CL. Immunopresence of both NOS types were evidenced in the nuclei and cytoplasm of all luteal cells, as well as in the nuclei of endothelial cells, during all stages studied. The eNOS and iNOS immunosignals increased during the early stage. COX1 transcripts were lower in late and regressive CL, COX2 in late, PGE2-9-K higher in regressive, and iNOS higher in early and lower in regressive CL. COX1 enzymatic activity was lower in regressive CL, COX2 increased in mid and late stages, and PGE2-9-K was higher in late CL. Endothelial NOS activity was higher during mid and late stages and lower in regressive, whereas iNOS was greater in late and lower in early. Progesterone in vitro release was higher in mid and lower in late phase, while PGF2α synthesis was higher in late CL and lower in regressive, and PGE2 was higher during regressive stage. These results support the idea that COX, NOS, and PGE2-9-K regulate buffalo CL life span. In particular, regressive CL seems involved in the development of the contralateral early CL, through the production of the luteotrophic PGE2.

Abstract 3215: Metabolic alterations of cancer cell as potential anti-cancer target

Abstract In cancer cells, energy generating pathways are markedly altered like many other processes. Warburg described this effect as increase the rate of glycolysis in the expense of oxidative phosphorylation. Dysfunctional Mitochondria, not Oxygen insufficiency, cause cancer cells to produce inordinate amounts of Lactic acid. The fact that most cancers have poor vascular systems has led to assume that such cells are deprived of a normal supply of oxygen, and thus it was forced to switch for anaerobic glycolysis. Recently many experiments illustrated that, cancer cells keep using anaerobic glycolysis even under conditions where Oxygen is plentiful, resulting in high rates of lactate formation. This indicates; cancer cell switches to anaerobic glycolysis because it's in need for Lactic acid. In comparison to Morula's cell has higher concentration of Lactic acid, than other fully differentiated adult cells, at the same time it has less concentration than cancer cells. This means that Lactic acid is associated with poor differentiation and high proliferation of cells in both cancer and health situations. But after the cell has been specialized and became fully differentiated, Lactic acid starts losing this property. In this context, Lactic acid playing a pivotal role in cell transformation and p53 de-activation, at the same time it's interfere with signaling pathway to activate or enhance the activity of ERK 1/2, result in cell proliferation. Since there is direct link between p53 and COX by showing p53 induction correlated with an up regulation of COXI subunit expression at the mRNA level. So loss of p53 resulted in decreased COXII protein expression with concomitant decreases in COX enzyme activity, and thus halts Mitochondrial oxidative phosphorylation, at the same time force cells to become entirely dependent on glycolysis to meet energy demands. In response, cells start importing more Glucose into the Cytosol by increase the activity of GLUT, which it's under the control of Atypical PKC isozymes, these isozymes control insulin-stimulated glucose transport by promoting the translocation of glucose transporters to the plasma membrane. PKC isozymes are involved in a wide array of diverse cellular functions. Most isozymes (PKC-beta) are involved in proliferation, and differentiation, whereas (PKC-epsilon) is anti-apoptotic. PKC results in activation of Ras, this in turn results in activation of ERK 1/2, and PI3K. PI3K responsible for activation of MDM2, which results in inhibition p53 activity. And thus, over activation of PKC by Lactic acid results in halting p53, and enhancing the activity of Ras. So obviously Lactic acid has binding site in PKC's domain to activate it. But the difference in its activation in cancer and normal ES cells is that in ES cells is controlled by the amount of Lactic acid produced. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3215. doi:1538-7445.AM2012-3215

Licofelone Inhibits Interleukin‐18‐Induced Pro‐Inflammatory Cytokine Release and Cellular Proliferation in Human Mesangial Cells

Licofelone, a novel dual anti-inflammatory drug that inhibits 5-lipoxygenase (5-LOX) and cyclooxygenase (COX), has recently been defined to have therapeutic effects in osteoarthritis. Both 5-LOX and COX play functional roles in the pathogenesis of glomerulonephritis in children as well. Interleukin-18 is a pro-inflammatory cytokine. It remains unclear whether licofelone can ameliorate inflammatory response of human mesangial cells (HMC) exposed to interleukin-18. In this study, HMC were cultured and exposed to interleukin-18 with or without pre-treatment of licofelone. COX-2 and 5-LOX enzyme activities in mesangial cells were determined with chromometry or high-performance liquid chromatography. Prostaglandin E2, cysteinyl leukotriene, monocyte chemotactic protein-1 and interferon-γ concentrations in culture medium were measured using an enzyme-linked immunosorbent assay. Western blotting was employed to detect phosphorylated mitogen-activated protein kinases ERK1/2, p38 and JNK1/2 in HMC. It was found that licofelone attenuated interleukin-18-induced COX-2 enzyme activity in HMC and prostaglandin E2 release in a dose-dependent manner. Similarly, licofelone inhibited interleukin-18-induced 5-LOX enzyme activity and leukotriene release. Licofelone reduced interleukin-18-induced phosphorylation of p38 mitogen-activated protein kinase and suppressed monocyte chemotactic protein-1 and interferon-γ synthesis. Moreover, licofelone inhibited IL-18-induced proliferation of mesangial cells. We conclude that licofelone inhibits interleukin-18-induced pro-inflammatory cytokine release and cellular proliferation in HMC, which may represent a really interesting therapeutic approach for glomerulonephritis in children.

Cyclooxygenase-2 contributes to VX-induced cell death in cultured cortical neurons

The link between cell death and increased cyclooxygenases-2 (COX-2) activity has not been clearly established. In this study, we examined whether COX-2 activation contributed to the mechanism of neurotoxicity produced by an organophosphorous nerve agent in cultured rat cortical neurons. Exposure of neuronal cells to the nerve agent, VX resulted in an increase in COX enzyme activity in the culture media. A concentration dependent increase in the activity levels of COX-2 enzyme was observed while there was little to no effect on COX-1. In addition, COX-2 mRNA and protein levels increased several hours post-VX exposure. Pre-treatment of the cortical cells with the COX-2 selective inhibitor, NS 398 resulted in a decrease in both the enzyme activity and prostaglandin (PGE2 and PGF2α) release, as well as in a reduction in cell death. These findings indicate that the increase in COX-2 activity may contribute to the mechanism of VX-induced neurotoxicity in cultured rat cortical neuron.

The inhibition of lipopolysaccharide-induced macrophage inflammation by 4 compounds in Hypericum perforatum extract is partially dependent on the activation of SOCS3

Our previous studies found that 4 compounds, namely pseudohypericin, amentoflavone, quercetin, and chlorogenic acid, in Hypericum perforatum ethanol extract synergistically inhibited lipopolysaccharide (LPS)-induced macrophage production of prostaglandin E2 (PGE2). Microarray studies led us to hypothesize that these compounds inhibited PGE2 production by activating suppressor of cytokine signaling 3 (SOCS3). In the current study, siRNA was used to knockdown expression of SOCS3 in RAW 264.7 macrophages and investigated the impact of H. perforatum extract and the 4 compounds on inflammatory mediators and cytokines. It was found that the SOCS3 knockdown significantly compromised the inhibition of PGE2 and nitric oxide (NO) by the 4 compounds, but not by the extract. The 4 compounds, but not the extract, decreased interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α), while both lowered interleukine-1β. SOCS3 knockdown further decreased IL-6 and TNF-α. Pseudohypericin was the major contributor to the PGE2 and NO inhibition in cells treated with the 4 compounds, and its activity was lost with the SOCS3 knockdown. Cyclooxygenase-2 (COX-2) and inducible NO synthase protein expression were not altered by the treatments, while COX-2 activity was decreased by the extract and the 4 compounds and increased by SOCS3 knockdown. In summary, it was demonstrated that the 4 compounds inhibited LPS-induced PGE2 and NO through SOCS3 activation. The reduction of PGE2 can be partially attributed to COX-2 enzyme activity, which was significantly elevated with SOCS3 knockdown. At the same time, these results also suggest that constituents in H. perforatum extract were alleviating LPS-induced macrophage response through SOCS3 independent mechanisms.