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Background: The study investigates the antioxidant properties of Catharanthus roseus, focusing on identifying its antioxidant compounds and chemical constituents. We compare antioxidant activities across its root, stem, flower, and leaf and examine the inhibition of reactive oxygen species (ROS)–generating enzymes by the plant's phytocompounds. Methods: We conducted a comprehensive analysis that included proximate analysis, mineral content assessment, and in vitro antioxidant characterization of various plant parts—root, stem, flower, and leaf. The levels of bioactive phytochemicals in both ethanol and mixed‐solvent extracts of Catharanthus roseus were quantified using high‐performance liquid chromatography with a diode array detector (HPLC‐DAD). Additionally, we performed molecular docking studies to explore the interactions of quantified phytocompounds. Results: HPLC‐DAD analysis quantified catechin hydrate, catechol, (−) epicatechin, rutin hydrate, trans‐cinnamic acid, quercetin, vanillic acid, kaempferol, and trans‐ferulic acid in Catharanthus roseus. Despite the ethanol extract having higher total antioxidant properties and flavonoid content, the mixed‐solvent extract exhibited higher EC50 for reducing power and lower IC50 for ABTS, 2,2‐diphenyl‐1‐picrylhydrazyl (DPPH), and metal chelating activities. Molecular docking studies indicated that compounds such as catechin, rutin, epicatechin, quercetin, and kaempferol significantly inhibit the ROS‐generating enzyme microsomal prostaglandin E synthase 1 (mPGES‐1). Conclusions: The mixed‐solvent extract had higher levels of catechin hydrate, rutin hydrate, trans‐ferulic acid, and vanillic acid, whereas the ethanol extract contained more (−) epicatechin, catechol, kaempferol, quercetin, and trans‐cinnamic acid. While the extracts displayed antioxidant activity, the phytoconstituents also inhibited ROS‐generating mPGES‐1. These results identify key compounds with potential for developing new chemotherapeutic agents against ROS. [ABSTRACT FROM AUTHOR]

Some novel triazole-bearing ketone and oxime derivatives were synthesized from Ibuprofen. In vitro cytotoxic activities of all synthesized molecules against five cancer lines (human breast cancer MCF-7, human lung cancer A549, human prostate cancer PC-3, human cervix cancer HeLa, and human chronic myelogenous leukemia K562 cell lines) were evaluated by MTT assay. In addition, mouse embryonic fibroblast cells (NIH/3T3) were also evaluated to determine the selectivity. Compounds 18, 36, and 45 were found to be the most cytotoxic, and their IC50 values were in the range of 17.46–68.76 µM, against the tested cancer cells. According to the results, compounds 7 and 13 demonstrated good anti-inflammatory activity against the microsomal enzyme prostaglandin E2 synthase-1 (mPGES-1) enzyme at IC50 values of 13.6 and 4.95 µM. The low cytotoxicity and non-mutagenity of these compounds were found interesting. Also, these compounds significantly prevented tube formation in angiogenesis studies. In conclusion, the anti-inflammatory and angiogenesis inhibitory activities of these compounds without toxicity suggested that they may be promising agents in anti-inflammatory treatment and they may be supportive agents for the cancer treatment. [ABSTRACT FROM AUTHOR]

Studies on the development of safe anti-inflammatory agents targeting the inhibition of the mPGES-1 enzyme responsible for PGE2 production are increasing day by day. Moreover, selective inhibition of the mPGES-1 enzyme which modulates the tumor microenvironment and inhibits tumor growth, making the mPGES-1 enzyme one of the important macromolecular targets in cancer therapy. The aim of our study was to develop selective mPGES-1 inhibitors and to determine their in silico mPGES-1 enzyme inhibition potential. In this study, the binding affinities of 14 novel designed 5-benzylidene-2-(arylsulfonylhydrazono)thiazolidine-4-one derivatives were investigated against mPGES-1 and COX-2 enzymes by computer-aided molecular modeling studies. Among the designed compounds 1-14, it was presented with in silico data that compound 8-14, which does not interact with the active site of the COX-2 enzyme, exhibited selective binding with mPGES-1 enzyme. Moreover, compounds 10-13 have been suggested as selective mPGES-1 inhibitors with a better in silico binding energy than the first discovered mPGES-1 inhibitor MK886. Finally, ADMET profiles of compounds 1-14 were calculated. None of these compounds violated the Lipinski and Veber rules. [ABSTRACT FROM AUTHOR]

mPGES-1 is an enzyme, which, when activated by inflammatory factors, can cause prostaglandin E synthesis. Traditional non-steroidal anti-inflammatory drugs are capable of inhibiting prostaglandin production, yet they can also cause gastrointestinal reactions and coagulation disorders. mPGES-1, the enzyme at the conclusion of prostaglandin production, does not cause any adverse reactions when inhibited. Numerous studies have demonstrated that mPGES-1 is more abundant in cancerous cells than in healthy cells, indicating that decreasing the expression of mPGES-1 could be a potential therapeutic strategy for cancer. Consequently, the invention of mPGES-1 inhibitors presents a fresh avenue for the treatment of inflammation and cancer. Incorporating a database of TCM compounds, we collected a batch of compounds that had an inhibitory effect on mPGES-1 and possessed IC50 value. Firstly, a pharmacophore model was constructed, and the TCM database was screened, and the compounds with score cut-off values of more than 1 were retained. Then, the compounds retained after being screened via the pharmacodynamic model were screened for docking at the mPGES-1 binding site, followed by high-throughput virtual screening [HTVS] and standard precision [SP] and super-precision [XP] docking, and the compounds in the top 20% of the XP docking score were selected to calculate the total free binding energy of MM-GBSA. The best ten compounds were chosen by comparing their score against the reference ligand 4U9 and the MM-GBSA_dG_Bind score. ADMET analysis resulted in the selection of ten compounds, three of which had desirable medicinal properties. Finally, the binding energy of the target protein mPGES-1 and the candidate ligand compound was analyzed using a 100 ns molecular dynamics simulation of the reference ligand 4U9 and three selected compounds. After a gradual screening study and analysis, we identified a structure that is superior to the reference ligand 4U9 in all aspects, namely compound 15643. Taken together, the results of this study reveal a structure that can be used to inhibit mPGES-1 compound 15643, thereby providing a new option for anti-inflammatory and anti-tumor drugs. [ABSTRACT FROM AUTHOR]

Background and Purpose: Heart failure with reduced ejection fraction (HFrEF) is a major consequence of myocardial infarction (MI). The microsomal prostaglandin E synthase‐1 (mPGES‐1)/PGE2 pathway has been shown to constrain reperfusion injury after acute myocardial ischaemia. However, it is unknown whether pharmacological inhibition of mPGES‐1, a target with lower risk of thrombosis compared with selective inhibition of cyclooxygenase‐2, affects chronic cardiac remodelling after MI. Experimental Approach: Mice were subjected to left anterior descending coronary artery ligation, followed by intraperitoneal treatment with the mPGES‐1 inhibitor compound III (CIII) or 118, celecoxib (cyclooxygenase‐2 inhibitor) or vehicle, once daily for 28 days. Urinary prostanoid metabolites were measured by liquid chromatography–tandem mass spectrometry. Key Results: Chronic administration of CIII improved cardiac function in mice after MI compared with vehicle or celecoxib. CIII did not affect thrombogenesis or blood pressure. In addition, CIII reduced infarct area, augmented scar thickness, decreased collagen I/III ratio, decreased the expression of fibrosis‐related genes and increased capillary density in the ischaemic area. Shunting to urinary metabolites of PGI2, not thromboxane B2 or PGD2, after inhibition of mPGES‐1 was positively correlated with cardiac function after MI. CIII administration significantly increased urinary PGI2/PGE2 metabolite ratio compared to vehicle or celecoxib. The PGI2/PGE2 metabolite ratio correlated positively with ejection fraction, fractional shortening and scar thickness. Treatment with 118 also improved cardiac function. Conclusion and Implications: Inhibition of mPGES‐1 prevented chronic adverse cardiac remodelling via an augmented PGI2/PGE2 metabolite ratio and therefore represents a potential therapeutic strategy for development of HFrEF after MI. [ABSTRACT FROM AUTHOR]

Aims Microsomal prostaglandin E synthase-1 (mPGES-1)/prostaglandin E2 (PGE2) induces angiogenesis through the prostaglandin E2 receptor (EP1–4). Among immune cells, regulatory T cells (Tregs), which inhibit immune responses, have been implicated in angiogenesis, and PGE2 is known to modulate the function and differentiation of Tregs. We hypothesized that mPGES-1/PGE2-EP signalling could contribute to recovery from ischaemic conditions by promoting the accumulation of Tregs. Methods and results Wild-type (WT), mPGES-1-deficient (mPges-1−/−), and EP4 receptor-deficient (Ep4−/−) male mice, 6–8 weeks old, were used. Hindlimb ischaemia was induced by femoral artery ligation. Recovery from ischaemia was suppressed in mPges-1−/− mice and compared with WT mice. The number of accumulated forkhead box protein P3 (FoxP3)+ cells in ischaemic muscle tissue was decreased in mPges-1−/− mice compared with that in WT mice. Expression levels of transforming growth factor-β (TGF-β) and stromal cell derived factor-1 (SDF-1) in ischaemic tissue were also suppressed in mPges-1−/− mice. The number of accumulated FoxP3+ cells and blood flow recovery were suppressed when Tregs were depleted by injecting antibody against folate receptor 4 in WT mice but not in mPges-1−/− mice. Recovery from ischaemia was significantly suppressed in Ep4−/− mice compared with that in WT mice. Furthermore, mRNA levels of Foxp3 and Tgf-β were suppressed in Ep4−/− mice. Moreover, the number of accumulated FoxP3+ cells in ischaemic tissue was diminished in Ep4−/− mice compared with that in Ep4+/+ mice. Conclusion These findings suggested that mPGES-1/PGE2 induced neovascularization from ischaemia via EP4 by promoting the accumulation of Tregs. Highly selective EP4 agonists could be useful for the treatment of peripheral artery disease. [ABSTRACT FROM AUTHOR]

A deep in silico investigation of various microsomal prostaglandin E2 synthase‐1 (mPGES‐1) protein systems is here reported using molecular dynamics (MD) simulations. Firstly, eight different proteins models (Models A−H) were built, starting from the active enzyme trimer system (Model A), namely that bound to three glutathione (GSH) cofactor molecules, and then gradually removing the GSHs (Models B−H), simulating each of them for 100 ns in explicit solvent. The analysis of the obtained data disclosed the structural role of GSH in the chemical architecture of mPGES‐1 enzyme, thus suggesting the unlikely displacement of this cofactor, in accordance with experimentally determined protein structures co‐complexed with small molecule inhibitors. Afterwards, Model A was submitted to microsecond‐scale molecular dynamics simulation (total simulation time=10 μs), in order to shed light about the dynamical behaviour of this enzyme at atomic level and to obtain further structural features and protein function information. We confirmed the structural stability of the enzyme machinery, observing a conformational rigidity of the protein, with a backbone RMSD of ∼3 Å along the simulation time, and highlighting the strong active contribution of GSH molecules due to their active role in packing the protein chains through a tight binding at monomer interfaces. Furthermore, the focused analysis on R73 residue disclosed its role in solvent exchange events, probably excluding its function as route for GSH to enter towards the endoplasmic reticulum membrane, in line with the recently reported function of cap domain residues F44‐D66 as gatekeeper for GSH entrance into catalytic site. [ABSTRACT FROM AUTHOR]

Background/Aim: Microsomal prostaglandin E synthase-1 (mPGES-1) is an enzyme, which catalyzes the final step of prostaglandin E2 (PGE2) synthesis. PGE2 in involved in wound-induced angiogenesis. Regulatory T cells (Tregs) regulate not only immune tolerance but also tissue repair and angiogenesis. We examined whether the mPGES-1/PGE2 axis contributes to wound-induced angiogenesis and granulation tissue formation through Treg accumulation. Materials and Methods: The dorsal subcutaneous tissues of male mPGES-1-deficient (mPGES-1-/-) and C57BL/6 wildtype (WT) mice were implanted with polyurethane sponge disks. Angiogenesis was estimated by determining the wet weight of sponge tissues and the expression of proangiogenic factors including CD31, vascular endothelial growth factor (VEGF), and transforming growth factor ß (TGF-ß) in granulation tissues. Results: Angiogenesis was suppressed in mPGES-1-/- mice compared with WT mice, which was associated with attenuated forkhead box P3 (Foxp3) expression and Foxp3+ Treg accumulation. The number of cells double-positive for Foxp3/TGFß and Foxp3/VEGF were lower in mPGES-1-/- mice than in WT mice. Neutralizing Tregs with antibodies (Abs) against CD25 or folate receptor 4 (FR4) inhibited the Foxp3+ Treg angiogenesis and accumulation in WT mice, but not in mPGES-1-/- mice. The topical application of PGE2 into the implanted sponge enhanced angiogenesis and accumulation of Tregs expressing TGFß and VEGF in WT and mPGES-1-/- mice. Conclusion: Tregs producing TGFß and VEGF accumulate in wounds and contribute to angiogenesis through mPGES-1-derived PGE2. mPGES-1 induction may control angiogenesis in skin wounds by recruiting Tregs. [ABSTRACT FROM AUTHOR]

Inhibition of microsomal prostaglandin E2 synthase-1 (mPGES-1) is promising for designing novel nonsteroidal anti-inflammatory drugs, as they lack side-effects associated with inhibition of cyclooxygenase enzymes. Azole compounds are nitrogen-containing heterocycles and have a wide use in medicine and are considered as promising compounds in medicinal chemistry. Various computer-aided drug design strategies are incorporated in this study. Structure-based virtual screening was performed employing various docking programs. Receiver operator characteristic (ROC) curves were used to evaluate the selectivity of each program. Furthermore, scoring power of Autodock4 and Autodock Vina was assessed by Pearson's correlation coefficients. Pharmacophore models were generated and Güner-Henry score of the best model was calculated as 0.89. Binding modes of the final 10 azole compounds were analyzed and further investigation of the best binding (− 8.38 kcal/mol) compound was performed using molecular dynamics simulation, revealing that furazan1224 (ZINC001142847306) occupied the binding site of the substrate, prostaglandin H2 (PGH2) and remained stable for 100 ns. Continuous hydrogen bonds and hydrophobic interactions with amino acids in the active site supported the stability of furazan1224 throughout the trajectory. Pharmacokinetic profile showed that furazan1224 lacks the risks of inhibiting cytochrome P450 3A4 enzyme and central nervous system-related side-effects. [ABSTRACT FROM AUTHOR]

Background and Purpose: Microsomal prostaglandin E synthase‐1 (mPGES‐1) is an inducible isomerase responsible for prostaglandin E2 production in inflammatory conditions. We evaluated the role of mPGES‐1 in the development and the metabolic and cardiovascular alterations of obesity. Experimental Approach mPGES‐1+/+ and mPGES‐1−/− mice were fed with normal or high fat diet (HFD, 60% fat). The glycaemic and lipid profile was evaluated by glucose and insulin tolerance tests and colorimetric assays. Vascular function, structure and mechanics were assessed by myography. Histological studies, q‐RT‐PCR, and western blot analyses were performed in adipose tissue depots and cardiovascular tissues. Gene expression in abdominal fat and perivascular adipose tissue (PVAT) from patients was correlated with vascular damage. Key Results: Male mPGES‐1−/− mice fed with HFD were protected against body weight gain and showed reduced adiposity, better glucose tolerance and insulin sensitivity, lipid levels and less white adipose tissue and PVAT inflammation and fibrosis, compared with mPGES‐1+/+ mice. mPGES‐1 knockdown prevented cardiomyocyte hypertrophy, cardiac fibrosis, endothelial dysfunction, aortic insulin resistance, and vascular inflammation and remodelling, induced by HFD. Obesity‐induced weight gain and endothelial dysfunction of resistance arteries were ameliorated in female mPGES‐1−/− mice. In humans, we found a positive correlation between mPGES‐1 expression in abdominal fat and vascular remodelling, vessel stiffness, and systolic blood pressure. In human PVAT, there was a positive correlation between mPGES‐1 expression and inflammatory markers. Conclusions and Implications: mPGES‐1 inhibition might be a novel therapeutic approach to the management of obesity and the associated cardiovascular and metabolic alterations. [ABSTRACT FROM AUTHOR]

Background and Purpose: Recent biochemical and pharmacological studies have reported that in several tissues and cell types, microsomal PGE2 synthase (mPGES) and PPAR-γ expression are modulated by a variety of inflammatory factors and stimuli. Considering that very little is known about the biological effects promoted by IL-17 in the context of mPGES-1/PPAR-γ modulation, we sought to investigate the contribution of this unique cytokine on this integrated pathway during the onset of inflammation.Experimental Approach: We evaluated effects of PF 9184 (mPGES-1 inhibitor) and troglitazone (PPAR-γ agonist) in vitro, using the mouse macrophage cell line J774A.1. In vivo, the dorsal air pouch model in CD1 mice was used, and inflammatory infiltrates were analysed by flow cytometry. Locally produced cyto-chemokines and PGs were assessed using elisa assays. Western blots were also employed to determine the activity of various enzymes involved in downstream signalling pathways.Key Results: PF 9184 and troglitazone, in a time- and dose-dependent manner, modulated leukocyte infiltration, myeloperoxidase activity, and the expression of COX-2/mPGES-1, NF-кB/IкB-α, and mPTGDS-1/PPAR-γ, induced by IL-17. Moreover, both PF 9184 and troglitazone modulated PG (PGE2 , PGD2 , and PGJ2 ) production, the expression of different pro-inflammatory cyto-chemokines, and the recruitment of inflammatory monocytes, in response to IL-17.Conclusions and Implications: Our data suggest that IL-17 may constitute a specific modulator of inflammatory monocytes during later phases of the inflammatory response. The results of this study show, for the first time, that the IL-17/mPGES-1/PPAR-γ pathway could represent a potential therapeutic target for inflammatory-based and immune-mediated diseases.Linked Articles: This article is part of a themed issue on Inflammation, Repair and Ageing. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v179.9/issuetoc. [ABSTRACT FROM AUTHOR]

Background: The microvasculature is a target organ for the early manifestations of cardiovascular disease. Therefore, a better understanding of the prostaglandin system and characterising the effects of mPGES‐1 inhibition and concomitant reduction of PGE2 in vascular beds are of interest. Experimental Approach The effects of mPGES‐1 inhibition on constriction and relaxation of resistance arteries (diameter: 100–400 μm) from patients with end stage kidney disease (ESKD) and controls (Non‐ESKD) were studied using wire‐myography in combination with immunological and mass‐spectrometry based analyses. Key Results: Inhibition of mPGES‐1 in arteries from ESKD patients and Non‐ESKD controls significantly reduced adrenergic vasoconstriction, which was unaffected by the COX‐2 inhibitors NS‐398 and Etoricoxib, or by the COX‐1/COX‐2 inhibitor Indomethacin tested in Non‐ESKD controls. However, a significant increase of acetylcholine‐induced dilatation was observed for mPGES‐1 inhibition. In IL‐1β treated arteries, inhibition of mPGES‐1 significantly reduced PGE2 levels while PGI2 levels remained unchanged. In contrast, COX‐2 inhibition blocked the formation of both prostaglandins. Blockade of PGI2 signalling with an IP receptor antagonist did not restore the reduced adrenergic constriction, neither did blocking PGE2‐EP4 or signalling through PPARγ. A biphasic effect was observed for PGE2, inducing dilatation at nanomolar and constriction at micromolar concentrations. Immunohistochemistry demonstrated expression of mPGES‐1, COX‐1, PGIS, weak expression for COX‐2, as well as receptor expression for PGE2 (EP1–4), thromboxane (TP) and PGI2 (IP) in ESKD and Non‐ESKD. Conclusion: Our study demonstrates vasodilating effects following mPGES‐1 inhibition in human microvasculature and suggests that several pathways besides shunting to PGI2 are involved. [ABSTRACT FROM AUTHOR]

Chronic periodontitis (CP) is a bacteria-driven inflammatory disease characterized by the breakdown of gingival tissue, the periodontal ligament, and alveolar bone, leading ultimately to tooth loss. We previously reported the pleckstrin gene (PLEK) to be highly upregulated in gingival tissue of patients with CP and the only gene concurrently upregulated in other inflammatory diseases including rheumatoid arthritis and cardiovascular diseases. Using saliva from 169 individuals diagnosed with CP and healthy controls, we investigated whether pleckstrin could serve as a novel biomarker of periodontitis. Additionally, we explored signal pathways involved in the regulation of PLEK using human gingival fibroblasts (HGFs). Pleckstrin levels were significantly higher (p < 0.001) in the saliva samples of patients with CP compared to controls and closely associated with CP severity. Immunohistochemical analysis revealed the expression of pleckstrin in inflammatory cells and gingival fibroblasts of CP patients. To explore the signal pathways involved in pleckstrin regulation, we stimulated HGFs with either interleukin-1β (IL-1β) or lipopolysaccharides (LPS) alone, or in combination with inhibitors targeting c-Jun N-terminal kinase, tyrosine kinase, protein kinase C, or p38 MAP kinase. Results showed that IL-1β and LPS significantly increased PLEK mRNA and pleckstrin protein levels. VX-745, the p38 MAP kinase inhibitor significantly decreased IL-1β- and LPS-induced pleckstrin levels at both the mRNA and the protein level. Together, these findings show that pleckstrin could serve as a salivary biomarker for the chronic inflammatory disease periodontitis and a regulator of inflammation via the p38 MAP kinase pathway. [ABSTRACT FROM AUTHOR]

Background: To examine the effects of BI 1029539 (GS-248), a novel selective human microsomal prostaglandin E synthase-1 (mPGES-1) inhibitor, in experimental models of acute lung injury (ALI) and sepsis in transgenic mice constitutively expressing the mPGES1 (Ptges) humanized allele.Methods: Series 1: Lipopolysaccharide (LPS)-induced ALI. Mice were randomized to receive vehicle, BI 1029539, or celecoxib. Series 2: Cecal ligation and puncture-induced sepsis. Mice were randomized to receive vehicle or BI 1029539.Results: Series 1: BI 1029539 or celecoxib reduced LPS-induced lung injury, with reduction in neutrophil influx, protein content, TNF-ɑ, IL-1β and PGE2 levels in bronchoalveolar lavage (BAL), myeloperoxidase activity, expression of mPGES-1, cyclooxygenase (COX)-2 and intracellular adhesion molecule in lung tissue compared with vehicle-treated mice. Notably, prostacyclin (PGI2) BAL concentration was only lowered in celecoxib-treated mice. Series 2: BI 1029539 significantly reduced sepsis-induced BAL inflammatory cell recruitment, lung injury score and lung expression of mPGES-1 and inducible nitric oxide synthase. Treatment with BI 1029539 also significantly prolonged survival of mice with severe sepsis. Anti-inflammatory and anti-migratory effect of BI 1029539 was confirmed in peripheral blood leukocytes from healthy volunteers.Conclusions: BI 1029539 ameliorates leukocyte infiltration and lung injury resulting from both endotoxin-induced and sepsis-induced lung injury. [ABSTRACT FROM AUTHOR]

Inflammation is a protective response that develops against tissue injury and infection. Chronic inflammation, on the other hand, is the key player in the pathogenesis of many inflammatory disorders including cancer. The cytokine storm, an inflammatory response flaring out of control, is mostly responsible for the mortality in COVID-19 patients. Anti-inflammatory drugs inhibit cyclooxygenases (COX), which are involved in the biosynthesis of prostaglandins that promote inflammation. The conventional non-steroidal anti-inflammatory drugs (NSAIDs) are associated with gastric and renal side-effects, as they inhibit both the constitutive COX-1 and the inducible COX-2. The majority of selective COX-2 inhibitors (COXIBs) are without gastric side-effects but are associated with cardiac side-effects on long-term use. The search for anti-inflammatory drugs without side-effects, therefore, has become a dream and ongoing effort of the Pharma companies. As PGE2 is the key mediator of inflammatory disorders, coming up with a strategy to reduce the levels of PGE2 alone without affecting other metabolites may form a better choice for the development of next generation anti-inflammatory drugs. In this direction the options being explored are on synthesis of PGE2-mPGES-1; PGE2 degradation through a specific PG dehydrogenase, 15-PGDH, and by blocking its activity mediated through a specific PGE receptor, EP4. As leukotrienes formed via the 5-lipoxygenase (5-LOX) pathway also play an important role in the mediation of inflammation, efforts are also being made to target both COX and LOX pathways. This review focuses on addressing the following three points: 1) How NSAIDs and COXIBs are associated with gastric, renal and cardiac side-effects; 2) Should the focus be on the targets upstream or downstream of PGE2; and 3) the status of alternative targets being explored for the discovery and development of anti-inflammatory drugs without side-effects. [ABSTRACT FROM AUTHOR]

Background and Purpose: mPGES1 catalyses the production of PGE2 , the most abundant prostanoid related to inflammation and pain in arthritis. mPGES1 is suggested to be a safer and more selective drug target in inflammatory conditions compared to the COX enzymes inhibited by NSAIDs. In the present study, we investigated the effects of the selective mPGES1 inhibitor MF63 on gene expression in primary human chondrocytes from patients with osteoarthritis (OA).Experimental Approach: Chondrocytes were isolated from articular cartilage obtained from osteoarthritis patients undergoing knee replacement surgery. The effects of MF63 were studied in the primary chondrocytes with RNA-sequencing based genome-wide expression analysis. The main results were confirmed with qRT-PCR and compared with the effects of the NSAID ibuprofen. Functional analysis was performed with the GO database and interactions between the genes were studied with STRING.Key Results: MF63 enhanced the expression of multiple metallothionein 1 (MT1) isoforms as well as endogenous antagonists of IL-1 and IL-36. The expression of IL-6, by contrast, was down-regulated. These genes were also essential in functional and interaction network analyses. The effects of MF63 were consistent in qRT-PCR analysis, whereas the effects of ibuprofen overlapped only partly with MF63. There were no evident findings of catabolic effects by MF63.Conclusion and Implications: Metallothionein 1 has been suggested to have anti-inflammatory and protective effects in cartilage. Up-regulation of the antagonists of IL-1 superfamily and down-regulation of the pro-inflammatory cytokine IL-6 also support novel anti-inflammatory and possibly disease-modifying effects of mPGES1 inhibitors in arthritis. [ABSTRACT FROM AUTHOR]

Background: Dual inhibitors of the 5-lipoxygenase-activating protein (FLAP) and the microsomal prostaglandin E2 synthase-1 (mPGES-1) may exert better anti-inflammatory efficacy and lower risks of adverse effects versus non-steroidal anti-inflammatory drugs. Despite these advantages, many dual FLAP/mPGES-1 inhibitors are acidic lipophilic molecules with low solubility and strong tendency for plasma protein binding that limit their bioavailability and bioactivity. Here, we present the encapsulation of the dual FLAP/mPGES-1 inhibitor BRP-187 into the biocompatible polymers acetalated dextran (Acdex) and poly(lactic-co-glycolic acid) (PLGA) via nanoprecipitation. Results: The nanoparticles containing BRP-187 were prepared by the nanoprecipitation method and analyzed by dynamic light scattering regarding their hydrodynamic diameter, by scanning electron microscopy for morphology properties, and by UV–VIS spectroscopy for determination of the encapsulation efficiency of the drug. Moreover, we designed fluorescent BRP-187 particles, which showed high cellular uptake by leukocytes, as analyzed by flow cytometry. Finally, BRP-187 nanoparticles were tested in human polymorphonuclear leukocytes and macrophages to determine drug uptake, cytotoxicity, and efficiency to inhibit FLAP and mPGES-1. Conclusion: Our results demonstrate that encapsulation of BRP-187 into Acdex and PLGA is feasible, and both PLGA- and Acdex-based particles loaded with BRP-187 are more efficient in suppressing 5-lipoxygenase product formation and prostaglandin E2 biosynthesis in intact cells as compared to the free compound, particularly after prolonged preincubation periods. [ABSTRACT FROM AUTHOR]

MicroRNAs (miRs) are one of the most important post-transcriptional repressors of gene expression. However, miR-574-5p has recently been shown to positively regulate the expression of microsomal prostaglandin E-synthase-1 (mPGES-1), a key enzyme in the prostaglandin E2 (PGE2) biosynthesis, by acting as decoy to the RNA-binding protein CUG-RNA binding protein 1 (CUGBP1) in human lung cancer. miR-574-5p exhibits oncogenic properties and promotes lung tumor growth in vivo via induction of mPGES-1-derived PGE2 synthesis. In a mass spectrometry-based proteomics study, we now attempted to characterize this decoy mechanism in A549 lung cancer cells at a cellular level. Besides the identification of novel CUGBP1 targets, we identified that the interaction between miR-574-5p and CUGBP1 specifically regulates mPGES-1 expression. This is supported by the fact that CUGBP1 and miR-574-5p are located in the nucleus, where CUGBP1 regulates alternative splicing. Further, in a bioinformatical approach we showed that the decoy-dependent mPGES-1 splicing pattern is unique. The specificity of miR-574-5p/CUGBP1 regulation on mPGES-1 expression supports the therapeutic strategy of pharmacological inhibition of PGE2 formation, which may provide significant therapeutic value for NSCLC patients with high miR-574-5p levels. [ABSTRACT FROM AUTHOR]

mPGES-1 is a terminal rate-limiting enzyme responsible for inflammation-induced PGE2 production. The inhibition of mPGES-1 has been considered as a safe and effective target for the treatment of inflammation and cancer. However, a specific, efficient, and simple method for high-throughput screening of mPGES-1 inhibitors is still lacking. In this study, we developed a fluorescence imaging strategy to monitor the expression of mPGES-1 via CRISPR/Cas9 knock-in system. Immunofluorescence colocalisation, Sanger sequencing, RNAi, and IL-1β treatment all confirmed the successful construction of mPGES-1 reporter cells. The fluorescence signal intensity of the reporter cells treated with four conventional mPGES-1 inhibitors was considerably attenuated via flow cytometry and fluorescent microplate reader, demonstrating that the reporter cells can be used as an efficient and convenient means for screening and optimising mPGES-1 inhibitors. Moreover, it provides a new technical support for the development of targeted small molecule compounds for anti-inflammatory and tumour therapy. [ABSTRACT FROM AUTHOR]

The tumor immune landscape gained considerable interest based on the knowledge that genetic aberrations in cancer cells alone are insufficient for tumor development. Macrophages are basically supporting all hallmarks of cancer and owing to their tremendous plasticity they may exert a whole spectrum of anti-tumor and pro-tumor activities. As part of the innate immune response, macrophages are armed to attack tumor cells, alone or in concert with distinct T cell subsets. However, in the tumor microenvironment, they sense nutrient and oxygen gradients, receive multiple signals, and respond to this incoming information with a phenotype shift. Often, their functional output repertoire is shifted to become tumor-supportive. Incoming and outgoing signals are chemically heterogeneous but also comprise lipid mediators. Here, we review the current understanding whereby arachidonate metabolites derived from the cyclooxygenase and lipoxygenase pathways shape the macrophage phenotype in a tumor setting. We discuss these findings in the context of cyclooxygenase-2 (COX-2) and microsomal prostaglandin E synthase-1 (mPGES-1) expression and concomitant prostaglandin E2 (PGE2) formation. We elaborate the multiple actions of this lipid in affecting macrophage biology, which are sensors for and generators of this lipid. Moreover, we summarize properties of 5-lipoxygenases (ALOX5) and 15-lipoxygenases (ALOX15, ALOX15B) in macrophages and clarify how these enzymes add to the role of macrophages in a dynamically changing tumor environment. This review will illustrate the potential routes how COX-2/mPGES-1 and ALOX5/-15 in macrophages contribute to the development and progression of a tumor. [ABSTRACT FROM AUTHOR]

Abstract: The single‐target drugs against the arachidonic acid inflammatory pathway are associated with serious side effects, hence, as a first step towards multi‐target drugs, we have studied the pharmacophoric features common to the inhibitors of 5‐lipoxygenase‐activating protein (FLAP), microsomal prostaglandin E‐synthase 1 (mPGES‐1) and leukotriene A4 hydrolase (LTA4H). FLAP and mPGES‐1 shared subfamily‐specific positions (SSPs) and four mPGES‐1 inhibitors binding to them mapped onto the pharmacophore derived from FLAP inhibitors (Ph‐FLAP). The reactions of mPGES‐1 and LTA4H had high structural similarity. The pharmacophore derived from two substrate mimic inhibitors of LTA4H (Ph‐LTA4H) also mapped onto three mPGES‐1 inhibitors. Screening of in‐house database for Ph‐FLAP and Ph‐LTA4H identified one compound, C1. It inhibited the production of the mPGES‐1 product, prostaglandin E2 (PGE2) by 97.8±1.6 % at 50 μM in HeLa cells and can be a starting point for designing molecules inhibiting all three targets simultaneously. [ABSTRACT FROM AUTHOR]

Phosphodiesterase-4 (PDE4) inhibitors have recently been introduced to the treatment of COPD and psoriatic arthritis. Microsomal prostaglandin E synthase-1 (mPGES-1) is an inducible enzyme synthesizing PGE2, the most abundant prostanoid related to inflammation and inflammatory pain. mPGES-1 is a potential drug target for novel anti-inflammatory treatments aiming at an improved safety profile as compared to NSAIDs. Here we investigated the effect of the PDE4 inhibitor rolipram on the expression of mPGES-1 in macrophages; and a potential mediator role in the process for MAP kinase phosphatase-1 (MKP-1) which is an endogenous factor limiting the activity of the proinflammatory MAP kinases p38 and JNK. The expression of mPGES-1 was decreased, whereas that of MKP-1 was enhanced by rolipram in wild-type murine macrophages. Interestingly, rolipram did not reduce mPGES-1 expression in peritoneal macrophages from MKP-1-deficient mice. A reduced phosphorylation of JNK, but not p38 MAP kinase, was specifically associated with the decreased expression of mPGES-1. Accordingly, mPGES-1 expression was suppressed by JNK but not p38 inhibitor. These findings underline the significance of the increased MKP-1 expression and decreased JNK phosphorylation associated with the downregulated expression of mPGES-1 by PDE4 inhibitors in inflammation. [ABSTRACT FROM AUTHOR]

Due to the absence of safety of the antipyretics to patients with cardiovascular dysfunction, new targets to treat inflammation have been pursued. mPGES-1 is a promising target because its inhibition would not cause the side-effects related to COX inhibition. To identify novel inhibitors of mPGES-1, we developed a ligand-based pharmacophore model that differentiates true inhibitors from decoys and enlightens the structure-activity relationships for known mPGES-1 inhibitors. The model (four hydrophobic centers, two hydrogen bond acceptor and two hydrogen bond donor points) was employed to select lead-like compounds from ZINC database for in vivo evaluation. Among the 18 compounds selected, five inhibited the fever induced by LPS. The most potent compound (5-(4-fluorophenyl)-3-({6-methylimidazo[1,2-a]pyridin-2-yl}methyl)-2,3dihydro-1,3,4-oxadiazol-2-one) is active peripherally ( i.v.) or centrally ( i.c.v.) (82.18% and 112% reduction, respectively) and reduces (69.13%) hypothalamic PGE2 production, without significant COX-1/2 inhibition. In conclusion, our in silico approach leads to the selection of a compound that presents the chemical features to inhibit mPGES-1 and reduces fever induced by LPS. Furthermore, the in vivo and in vitro results support the hypothesis that its mechanism of action does not depend on COX inhibition. Hence, it can be considered a promising lead compound for antipyretic development, once it would not have the side-effects of COX-1/2 inhibitors. [ABSTRACT FROM AUTHOR]

We previously reported that microsomal prostaglandin E synthase-1 (mPGES-1) contributed to adriamycin (Adr)-induced podocyte apoptosis. However, the molecular mechanism remains unclear. Here we studied the role of mPGES-1/PGE2 cascade in activating Stat3 signaling and the contribution of Stat3 in PGE2- and Adr-induced podocyte apoptosis. In murine podocytes, PGE2 dose- and time-dependently increased the phosphorylation of Stat3 in line with the enhanced cell apoptosis and reduced podocyte protein podocin. In agreement with the increased Stat3 phosphorylation, Stat3-derived cytokines including IL-6, IL-17, MCP-1, and ICAM-1 were significantly upregulated following PGE2 treatment. By application of a specific Stat3 inhibitor S3I-201, PGE2-induced podocyte apoptosis was largely abolished in parallel with a blockade of podocin reduction. Next, we observed that Adr treatment also enhanced p-Stat3 and activated mPGES-1/PGE2 cascade. Blockade of Stat3 by S3I-201 significantly ameliorated Adr-induced cell apoptosis and podocin reduction. More interestingly, silencing mPGES-1 in podocytes by mPGES-1 siRNA blocked Adr-induced increments of Stat-3 phosphorylation, PGE2 production, and Stat3-derived inflammatory cytokines. Taken together, this study suggested that mPGES-1-derived PGE2 could activate Stat3 signaling to promote podocyte apoptosis. Targeting mPGES-1/PGE2/Stat3 signaling might be a potential strategy for the treatment of podocytopathy. [ABSTRACT FROM AUTHOR]

Background/Aims: We previously reported that indoxyl sulfate (IS) could cause mesangial cell (MC) proliferation via a cyclooxygenase (COX)-2-dependent mechanism. However, the specific prostaglandin contributing to COX-2 effect on IS-induced MC proliferation remained unknown. Thus, the present study was undertaken to examine the role of microsomal prostaglandin E synthase-1 (mPGES-1)-derived Prostaglandin E2 (PGE2) in IS-induced MC proliferation. Methods: IS was administered to the MCs with or without mPGES-1 siRNA pretreatment to induce the MC proliferation which was determined by cell cycle analysis, DNA synthesis, and the expressions of cyclins. In another experimental setting, PGE2 was applied to the MCs to examine its direct effect on MC proliferation, as well as the regulation of prostaglandin E receptors (EPs) by qRT-PCR. Results: With the administration of IS, mPGES- 1(not mPGES-2 and cytosolic PGES) was significantly upregulated at both protein and mRNA levels in line with a promoted MC proliferation. Interestingly, silencing mPGES-1 reduced cell number in S and G2 phases and blocked the upregulation of cyclin A2 and cyclin D1 in parallel with blunted PGE2 release after IS treatment, indicating that mPGES-1-derived PGE2 could contribute to MC proliferation. Furthermore, we confirmed that exogenous PGE2 could directly trigger the proliferative response in MCs. Lastly, we observed a selective upregulation of EP2 after PGE2 treatment and enhanced phosphorylation of NF-κB following IS administration in MCs, suggesting the potential involvements of EP2 and NF-κB in this pathological process. Conclusion: mPGES-1-derived PGE2 contributed to IS-induced mesangial cell proliferation. [ABSTRACT FROM AUTHOR]

Background/Aims: We previously reported that indoxyl sulfate (IS) could cause mesangial cell (MC) proliferation via a cyclooxygenase (COX)-2-dependent mechanism. However, the specific prostaglandin contributing to COX-2 effect on IS-induced MC proliferation remained unknown. Thus, the present study was undertaken to examine the role of microsomal prostaglandin E synthase-1 (mPGES-1)-derived Prostaglandin E2 (PGE2) in IS-induced MC proliferation. Methods: IS was administered to the MCs with or without mPGES-1 siRNA pretreatment to induce the MC proliferation which was determined by cell cycle analysis, DNA synthesis and the expressions of cyclins. In another experimental setting, PGE2 was applied to the MCs to examine its direct effect on MC proliferation, as well as the regulation of prostaglandin E receptors (EPs) by qRT-PCR. Results: With the administration of IS, mPGES- 1(not mPGES-2 and cytosolic PGES) was significantly upregulated at both protein and mRNA levels in line with a promoted MC proliferation. Interestingly, silencing mPGES-1 reduced cell number in S and G2 phases and blocked the upregulation of cyclin A2 and cyclin D1 in parallel with blunted PGE2 release after IS treatment, indicating that mPGES-1-derived PGE2 could contribute to MC proliferation. Furthermore, we confirmed that exogenous PGE2 could directly trigger the proliferative response in MCs. Lastly, we observed a selective upregulation of EP2 after PGE2 treatment and enhanced phosphorylation of NF-кB following IS administration in MCs, suggesting the potential involvements of EP2 and NF-кB in this pathological process. Conclusion: mPGES-1-derived PGE2 contributed to IS-induced mesangial cell proliferation. [ABSTRACT FROM AUTHOR]

Microsomal prostaglandin E synthase-1 (mPGES-1) is an inducible enzyme situated downstream of cyclo-oxygenase-2, promoting the excessive PGE2 production in inflammation. Dexamethasone is known to suppress mPGES-1 but the mechanisms regulating mPGES-1 expression remain poorly known. MKP-1 is a phosphatase controlling the proinflammatory MAP kinase pathways p38 and JNK, thus limiting the inflammatory responses. We have now investigated the role of MKP-1 and MAP kinases p38 and JNK in the regulation of mPGES-1 expression by dexamethasone. Dexamethasone increased MKP-1 and decreased mPGES-1 expression in J774 macrophages and in peritoneal macrophages from wild-type but not from MKP-1 deficient mice. Dexamethasone also reduced p38 and JNK phosphorylation along with enhancement of MKP-1, while inhibition of JNK reduced mPGES-1 expression. These findings were also translated to in vivo conditions as dexamethasone downregulated mPGES-1 expression in paw inflammation in wild-type but not in MKP-1 deficient mice. In conclusion, dexamethasone was found to downregulate mPGES-1 expression through enhanced MKP-1 expression and reduced JNK phosphorylation in inflammatory conditions. The results extend the understanding on the regulation of mPGES-1 expression and highlight the potential of MKP-1 as an anti-inflammatory drug target. [ABSTRACT FROM AUTHOR]

Background: Recent studies have suggested that the tumor necrosis factor-α (TNF-α) pathway is a potential target for the management of osteoarthritis (OA). Transforming growth factor (TGF)-β-activated kinase 1 (TAK1) is essential in several cytokine-mediated cascades, including the TNF-α, interleukin-1 (IL-1), and TGF-β pathways. The role of TAK1 in synovial tissue in OA is not fully understood. Using synovial cells harvested from OA patients during surgery, we investigated whether TAK1 inhibition suppresses production of TNF-α-induced extracellular matrix degrading enzymes and expression of pain-related molecules.Methods: Synovial tissues were harvested from ten subjects with radiographic evidence of osteoarthritis (OA) during total knee arthroplasty. Synovial cells were cultured and stimulated with control (culture media), 10 ng/mL human recombinant TNF-α, or 10 ng/mL TNF-α and 10 μM of the TAK1 inhibitor (5Z)-7-oxozeaenol for 24 h. Real-time polymerase chain reaction (PCR) analysis was used to monitor expression of mRNA of the extracellular matrix degrading enzymes matrix metalloproteinase-3 (MMP-3) and a disintegrin-like and metalloprotease (reprolysin type) with thrombospondin type 1 motif, 4 (ADAMTS-4); and of the pain-related molecules cyclooxygenase-2 (COX-2), microsomal prostaglandin E synthase-1 (mPGES-1), and nerve growth factor (NGF). MMP-3 and NGF protein concentrations in cell supernatant were measured by enzyme-linked immunosorbent assay (ELISA). COX-2, mPGES-1 and ADAMTS-4 protein expression was also evaluated by western blotting.Results: TNF-α stimulated increases in ADAMTS-4 and MMP3 mRNA (2.0-fold and 1.6-fold, respectively, p < 0.05) and protein expression (21.5-fold and 2.0-fold, respectively). Treatment with the TAK1 inihibitor (5Z)-7-oxozeaenol reduced ADAMTS-4 and MMP3 mRNA (0.5-fold and 0.6-fold, respectively) and protein expression (1.4-fold and 0.5-fold, respectively) in OA synovial cells. COX-2, mPGES-1 and NGF mRNA (11.2-fold, 3.1-fold and 2.7-fold, respectively) and protein expression (3.0-fold, 2.7-fold and 2.2-fold, respectively) were increased by TNF-α. (5Z)-7-oxozeaenol treatment reduced mPGES1 and NGF mRNA (1.5-fold and 0.8-fold, respectively) and protein (1.5-fold and 0.5-fold, respectively).Conclusion: TAK1 plays an important role in the regulation of TNF-α induced extracellular matrix degrading enzymes and pain-related molecule expression. TAK1 may be a potential target for therapeutic strategies aimed at preventing osteoarthritis progression and pain. [ABSTRACT FROM AUTHOR]

Stress and glucocorticoids suppress adult neurogenesis in the hippocampus. However, the molecular mechanisms underlying stress-induced impairment of adult neurogenesis are poorly understood. We previously suggested that cyclooxygenase (COX)-2 is a common mediator of stresses in the brain. Here, using a lipopolysaccharide (LPS)-induced acute infectious stress model, we evaluated the roles of COX-2 and its major downstream product prostaglandin E2 (PGE2) in adult neurogenesis and the influence of glucocorticoids on COX-2-related signaling. Treatment of rats with LPS significantly decreased neurogenesis in the dentate gyrus (DG) of the hippocampus, and this inhibitory effect of LPS on neurogenesis was reversed by the glucocorticoid receptor antagonist RU486. Moreover, RU486 significantly enhanced the increase in messenger RNA (mRNA) levels of COX-2 and microsomal prostaglandin E synthase (mPGES)-1 in the hippocampus following LPS stimulation. Administration of AH6809, a selective antagonist of the PGE2 EP2 receptor, as well as NS398, a COX-2 selective inhibitor, exacerbated the suppression of proliferation of neural progenitor cells (NPCs) in the DG. Gene expression of EP1, EP2, and EP3, but not EP4, receptors was also increased following LPS stimulation. Immunohistochemical studies indicated that NPCs expressed EP2 receptor, whereas the majority of cells expressing COX-2 and mPGES-1 were mature neurons in the DG. These results suggest that acute infectious stress upregulates COX-2-related signaling in neurons in the DG, which plays a protective role in neurogenesis through EP2 receptor at least partially. In addition, LPS-induced glucocorticoids suppress this COX-2-related signaling, resulting in decreased neurogenesis. [ABSTRACT FROM AUTHOR]

COX-2 inhibitors exhibit anticancer effects in various cancer models but due to the adverse side effects associated with these inhibitors, targeting molecules downstream of COX-2 (such as mPGES-1) has been suggested. Even after calls for mPGES-1 inhibitor design, to date there are only a few published inhibitors targeting the enzyme and displaying anticancer activity. In the present study, we have deployed both ligand and structure-based drug design approaches to hunt novel drug-like candidates as mPGES-1 inhibitors. Fifty-four compounds with tested mPGES-1 inhibitory value were used to develop a model with four pharmacophoric features. 3D-QSAR studies were undertaken to check the robustness of the model. Statistical parameters such asr2= 0.9924,q2= 0.5761 andFtest = 1139.7 indicated significant predictive ability of the proposed model. Our QSAR model exhibits sites where a hydrogen bond donor, hydrophobic group and the aromatic ring can be substituted so as to enhance the efficacy of the inhibitor. Furthermore, we used our validated pharmacophore model as a three-dimensional query to screen the FDA-approved Lopac database. Finally, five compounds were selected as potent mPGES-1 inhibitors on the basis of their docking energy and pharmacokinetic properties such as ADME and Lipinski rule of five. [ABSTRACT FROM PUBLISHER]

Candida is an important opportunistic fungal pathogen, especially in biofilm associated infections. The formation of a Candida biofilm can decrease Candida sensitivity to antifungal drugs and cause drug resistance. Although many effective antifungal drugs are available, their applications are limited due to their high toxicity and cost. Seeking new antifungal agents that are effective against biofilm-associated infection is an urgent need. Many research efforts are underway, and some progress has been made in this field. It has been shown that the arachidonic acid cascade plays an important role in fungal morphogenesis and pathogenicity. Notably, prostaglandin E2 (PGE2) can promote the formation of a Candida biofilm. Recently, the inhibition of PGE2 has received much attention. Studies have shown that cyclooxygenase (COX) inhibitors, such as aspirin, ibuprofen, and indomethacin, combined with fluconazole can significantly reduce Candida adhesion and biofilm development and increase fluconazole susceptibility; the MIC of fluconazole can be decrease from 64 to 2 µg/ml when used in combination with ibuprofen. In addition, in vivo studies have also confirmed the antifungal activities of these inhibitors. In this article, we mainly review the relationship between PGE2 and Candida biofilm, summarize the antifungal activities of COX inhibitors and analyze the possible antifungal activity of microsomal prostaglandin E synthase-1 (MPGES-1) inhibitors; additionally, other factors that influence PGE2 production are also discussed. Hopefully this review can disclose potential antifungal targets based on the arachidonic acid cascade and provide a prevailing strategy to alleviate Candida albicans biofilm formation. [ABSTRACT FROM AUTHOR]

Prostaglandin (PG) signalling is involved in human and animal cancer development. PG E2 (PGE2) tumour-promoting activity has been confirmed and its production is controlled by Cyclooxygenase-2 (COX-2) and microsomal PGE synthase-1 (mPGES-1). Evidence suggests that mPGES-1 and COX-2 contribute to carcinogenesis through the EP2 receptor. The aim of our study was to detect by immunohistochemistry COX-2, mPGES-1 and EP2 receptor expression in canine ( n = 46) and feline ( n = 50) mammary tumours and in mammary non-neoplastic tissues. COX-2 positivity was observed in 83% canine and 81% feline mammary carcinomas, mPGES-1 in 75% canine and 66% feline mammary carcinomas and the EP2 receptor expression was observed in 89% canine and 54% feline carcinomas. The frequency of COX-2, EP2 receptor and mPGES-1 expression was significantly higher in carcinomas than in non-neoplastic tissues and adenomas. COX-2, mPGES-1 and EP2 receptor expression was strongly associated. These findings support a role of the COX-2/PGE2 pathway in the pathogenesis of these tumours. [ABSTRACT FROM AUTHOR]

We present here the development of a novel virtual screening protocol combining Structure-based and Ligand-based drug design approaches for the identification of mouse mPGES-1 inhibitors. We used the existing 3D structural data of the murine enzyme to hypothesize the inhibitors binding mode, which was the starting point for docking simulations, shape screening, and pharmacophore hypothesis screening. The protocol allowed the identification of 16 mouse mPGES-1 inhibitors with low micromolar activity, which, notably, also inhibit the human enzyme in the same concentration range. The inhibitors predicted binding mode is expected to be the base for the rational drug design of new potent dual species inhibitors of human and murine mPGES-1. [ABSTRACT FROM AUTHOR]

Lipocalin-2 (Lcn2) is a small glycoprotein involved in a number of biological processes such as inflammation and antibacterial response. In our study, Lcn2 is expressed in the subluminal stromal cells at implantation site on day 5 of pregnancy. The expression of Lcn2 in stromal cells is under the control of progesterone through Akt-c-Myc signaling pathway. Data from Lcn2 knockdown and recombinant protein treatments indicate that Lcn2 promotes mPGES-1 expression in stromal cells. The expression of Lcn2 and mPGES-1 is strongly stimulated by lipopolysaccharide ( LPS), indicating that Lcn2 mediates LPS-induced inflammation. These findings shed light on the role of Lcn2 during decidualization. [ABSTRACT FROM AUTHOR]

Microsomal prostaglandin E synthase-1 (mPGES-1) is the terminal enzyme for the synthesis of prostaglandin E2 (PGE2), a proproliferative and antiapoptotic lipid molecule important for tissue regeneration and injury repair. In this study, we developed transgenic (Tg) mice with targeted expression of mPGES-1 in the liver to assess Fas-induced hepatocyte apoptosis and acute liver injury. Compared with wild-type (WT) mice, the mPGES-1 Tg mice showed less liver hemorrhage, lower serum alanine transaminase (ALT) and aspartate transaminase (AST) levels, less hepatic necrosis/apoptosis, and lower level of caspase cascade activation after intraperitoneal injection of the anti-Fas antibody Jo2. Western blotting analysis revealed increased expression and activation of the serine/threonine kinase Akt and associated antiapoptotic molecules in the liver tissues of Jo2-treated mPGES-1 Tg mice. Pretreatment with the mPGES-1 inhibitor (MF63) or the Akt inhibitor (Akt inhibitor V) restored the susceptibility of the mPGES-1 Tg mice to Fas-induced liver injury. Our findings provide novel evidence that mPGES-1 prevents Fas-induced liver injury through activation of Akt and related signaling and suggest that induction of mPGES-1 or treatment with PGE2 may represent important therapeutic strategy for the prevention and treatment of Fas-associated liver injuries. [ABSTRACT FROM AUTHOR]

Microsomal prostaglandin E2 synthase type 1 (mPGES-1) is responsible for the formation of the potent lipid mediator prostaglandin E2 under proinflammatory conditions, and this enzyme has received considerable attention as a drug target. Recently, a high-resolution crystal structure of human mPGES-1 was presented, with Ser-127 being proposed as the hydrogen-bond donor stabilizing thiolate anion formation within the cofactor, glutathione (GSH).We have combined site-directed mutagenesis and activity assays with a structural dynamics analysis to probe the functional roles of such putative catalytic residues.We found that Ser-127 is not required for activity, whereas an interaction between Arg-126 and Asp-49 is essential for catalysis. We postulate that both residues, in addition to a crystallographicwater, serve critical roles within the enzymatic mechanism. After characterizing the size or charge conservative mutations Arg-126-Gln, Asp-49-Asn, and Arg- 126-Lys, we inferred that a crystallographic water acts as a general base during GSH thiolate formation, stabilized by interaction with Arg-126, which is itself modulated by its respective interaction with Asp-49. We subsequently found hidden conformational ensembles within the crystal structure that correlate well with our biochemical data. The resulting contact signaling network connects Asp-49 to distal residues involved in GSH binding and is ligand dependent. Our work has broad implications for development of efficientmPGES-1 inhibitors, potential anti-inflammatory and anticancer agents. [ABSTRACT FROM AUTHOR]

Background: Microglia recognize pathogen-associated molecular patterns such as double-stranded RNA (dsRNA) present in some viruses. Polyinosinic-polycytidylic acid [poly(I:C)] is a synthetic analog of dsRNA that activates different molecules, such as retinoic acid-inducible gene I, melanoma differentiation-associated gene 5, and toll-like receptor-3 (TLR3). Poly(I:C) increases the expression of different cytokines in various cell types. However, its role in the regulation of the production of inflammatory mediators of the arachidonic acid pathway by microglia is poorly understood. Methods: In the present study, we evaluated the effect of poly(I:C) on the production of prostaglandin E2 (PGE2) and the inducible enzymes cyclooxygenase-2 (COX-2) and microsomal prostaglandin E synthase-1 (mPGES-1) in primary rat microglia. Microglia were stimulated with different concentrations of poly(I:C) (0.1-10 μg/ml), and the protein levels of COX-2 and mPGES-1, as well as the release of PGE2, were determined by western blot and enzyme immunoassay (EIA), respectively. Values were compared using one-way ANOVA with post hoc Student-Newman-Keuls test. Results: Poly(I:C) increased the production of PGE2, as well as mPGES-1 and COX-2 synthesis. To investigate the mechanisms involved in poly(I:C)-induced COX-2 and mPGES-1, we studied the effects of various signal transduction pathway inhibitors. Protein levels of COX-2 and mPGES-1 were reduced by SB203580, SP600125, and SC514 (p38 mitogen-activated protein kinase (MAPK), c-Jun N-terminal kinase (JNK), and IκB kinase (IKK) inhibitors, respectively), as well as by PD98059 and PD0325901 (mitogen-activated protein kinase kinase (MEK) inhibitors). Rapamycin, a mammalian target of rapamycin (mTOR) inhibitor, enhanced the synthesis of COX-2. Inhibition of phosphatidylinositol 3-kinase (PI3K) by LY294002 or dual inhibition of PI3K/mTOR (with NVP-BEZ235) enhanced COX-2 and reduced mPGES-1 immunoreactivity. To confirm the data obtained with the inhibitors, we studied the phosphorylation of the blocked kinases by western blot. Poly(I:C) increased the phosphorylation of p38 MAPK, extracellular signal-regulated kinase (ERK), JNK, protein kinase B (Akt), and IκB. Conclusions: Taken together, our data demonstrate that poly(I:C) increases the synthesis of enzymes involved in PGE2 synthesis via activation of different signaling pathways in microglia. Importantly, poly(I:C) activates similar pathways also involved in TLR4 signaling that are important for COX-2 and mPGES-1 synthesis. Thus, these two enzymes and their products might contribute to the neuropathological effects induced in response to dsRNA, whereby the engagement of TLR3 might be involved. [ABSTRACT FROM AUTHOR]

There is evidence that an inflammatory microenvironment is associated with the development and progression of prostate cancer (PCa), although the determinants of intrinsic inflammation in PCa cells are not completely understood. Here we investigated whether expression of intrinsic microsomal PGE synthase-1 (mPGES-1) enhanced aggressiveness of PCa cells and might be critical for epidermal growth factor receptor (EGFR)-mediated tumour progression. In PCa, overexpression of EGFR promotes metastatic invasion and correlates with a high Gleason score, while prostaglandin E2 (PGE2) has been reported to modulate oncogenic EGFR-driven oncogenicity. Immunohistochemical studies revealed that mPGES-1 in human prostate tissues is correlated with EGFR expression in advanced tumours. In DU145 and PC-3 cell lines expressing mPGES-1 (mPGES-1SC cells), we demonstrate that silencing or 'knock down' of mPGES-1 (mPGES-1KD) or pharmacological inhibition by MF63 strongly attenuates overall oncogenic drive. Indeed, mPGES-1SC cells express stem-cell-like features (high CD44, β1-integrin, Nanog and Oct4 and low CD24 and a6-integrin) as well as mesenchymal transition markers (high vimentin, high fibronectin, low E-cadherin). They also show increased capacity to survive irrespective of anchorage condition, and overexpress EGFR compared to mPGES-1KD cells. mPGES-1 expression correlates with increased in vivo tumour growth and metastasis. Although EGFR inhibition reduces mPGES-1SC and mPGES-1KD cell xenograft tumour growth, we show that mPGES-1/PGE2 signalling sensitizes tumour cells to EGFR inhibitors. We propose mPGES-1 as a possible new marker of tumour aggressiveness in PCa. [ABSTRACT FROM AUTHOR]

The increase in PGE production by microsomal PGE synthase-1 (mPGES-1) in CNS contributes to the severity of the inflammatory and pain responses in the model of edema formation and hyperalgesia induced by carrageenan. PGI alike to PGE, plays an important role in the inflammation. Low-level laser therapy (LLLT) has been used in the treatment of inflammatory pathologies, reducing both pain and the acute inflammatory process. In this work, we studied the effect of LLLT on the expression of both mPGES-1 and IP messenger RNA (mRNA), in either subplantar or total brain tissues obtained from rats submitted to model of edema formation and hyperalgesia induced by carrageenan administration. The test sample consisted of 30 rats divided into five groups: A1 (control-saline), A2 (carrageenan-0.5 mg/paw), A3 (carrageenan-0.5 mg/paw + LLLT), A4 (carrageenan-1.0 mg/paw), and A5 (carrageenan-1.0 mg/paw + LLLT). The animals from groups A3 and A5 were irradiated 1 h after induction of inflammation by carrageenan injection. Continuous-wave red laser with wavelengths of 660 nm and dose of 7.5 J/cm was used. Six hours after carrageenan-induced inflammation, mPGES-1 and prostacyclin receptor (IP) mRNA expression were significantly increased both in subplantar and brain tissues. LLLT was able to reduce both mPGES-1 and IP mRNA expression in subplantar and brain tissues. We suggest that LLLT is able to reduce both inflammation and hyperalgesia observed in the model of edema formation and hyperalgesia induced by carrageenan, by a mechanism involving the decrease in the expression of both mPGES-1 and IP. [ABSTRACT FROM AUTHOR]

Oxicams are a class of nonsteroidal anti-inflammatory drugs (NSAIDs) structurally related to the enolic acid class of 4-hydroxy-1,2-benzothiazine carboxamides. They are used clinically to treat both acute and chronic inflammation by inhibiting the activity of the two cyclooxygenase (COX) isoforms, COX-1 and COX-2. Oxicams are structurally distinct from all other NSAIDs, exhibiting a novel binding pose in the COX active site. The 4-hydroxyl group on the thiazine ring partners with Ser-530 via hydrogen bonding while two coordinated water molecules mediate a polar interaction between the oxicam and COX. The rotation of Leu-531 in the complex opens a new pocket, which is not used for binding other NSAIDs to the enzyme. This structure provides the basis for understanding documented structure-activity relationships within the oxicam class. In addition, from the oxicam template, a series of potent microsomal prostaglandin E synthase-1 (mPGES-1) inhibitors represents a new direction for drug development. Here, we review the major route of oxicam synthesis and structure-activity for COX inhibition, as well as recent advances in oxicam-mediated mPGES-1 inhibition. © 2014 IUBMB Life, 66(12):803-811, 2014 [ABSTRACT FROM AUTHOR]

Fever is a hallmark of inflammatory and infectious diseases. The febrile response is triggered by prostaglandin E2 synthesis mediated by induced expression of the enzymes cyclooxygenase-2 (COX-2) and microsomal prostaglandin E synthase 1 (mPGES-1). The cellular source for pyrogenic PGE2 remains a subject of debate; several hypotheses have been forwarded, including immune cells in the periphery and in the brain, as well as the brain endothelium. Here we generated mice with selective deletion of COX-2 and mPGES1 in brain endothelial cells. These mice displayed strongly attenuated febrile responses to peripheral immune challenge. In contrast, inflammationinduced hypoactivity was unaffected, demonstrating the physiological selectivity of the response to the targeted gene deletions. These findings demonstrate that PGE2 synthesis in brain endothelial cells is critical for inflammation-induced fever. [ABSTRACT FROM AUTHOR]

Prostaglandin E2 (PGE) involves in progression of various chronic inflammation-related cancers including cholangiocarcinoma (CCA). This study aimed to determine the role of PGE signaling, its biosynthesis-related enzymes in a clinical prognosis, and their targeted inhibition in CCA progression. The immunohistochemical staining of cyclooxygenase (COX)-1, COX-2, mPGES-1, EP1, and EP4 was examined in CCA tissues, and their expressions were compared with clinicopathological parameters. The effect of PGE on levels of its signaling molecules was examined in CCA cell lines using proteome profiler array. The suppression of mPGES-1 using a small-molecule inhibitor (CAY10526) and small interfering RNA (siRNA) was determined for growth and migration ability in CCA cells. The results indicated that strong expressions of COX-1, COX-2, mPGES-1, EP1, and EP4 were found in CCA tissues as 87.5, 47.5, 52.5, 55, and 80 % of frequencies, respectively. High mPGES-1 expression was significantly correlated with tumor stages III-IV ( p = 0.001), lymph node metastasis ( p = 0.004), shorter survival ( p = 0.009), and prognostic indicator of CCA patients (HR = 2.512, p = 0.041). Expressions of COX-1, COX-2, and EP receptors did not correlate with data tested from patients. PGE markedly enhanced protein levels of integrinα6, VE-cadherin, Jagged1, and Notch3, and CAY10526 suppressed those protein levels as well as PGE production in CCA cells. CAY10526 and siRNA mPGES-1 markedly suppressed mPGES-1 protein levels, growth, and migration abilities of CCA cell lines. In conclusion, PGE signaling strongly promotes CCA progression. Therefore, inhibition of PGE synthesis by suppression of its biosynthesis-related enzymes could be useful for prevention and treatment of CCA. [ABSTRACT FROM AUTHOR]

Cyclooxygenase (COX)-2-derived prostaglandin E2 (PGE2) supports the growth of a spectrum of cancers. The potential benefit of COX-2-inhibiting non-steroidal anti-inflammatory drugs (NSAIDs) for cancer treatment is however limited by their well-known cardiovascular side-effects. Therefore, targeting microsomal PGE synthase 1 (mPGES-1), the downstream enzyme in the COX-2-dependent pathway of PGE2 production might be attractive, although conflicting data regarding a potential tumor-supporting function of mPGES-1 were reported. We determined the impact of mPGES-1 in human DU145 prostate cancer cell growth. Surprisingly, knockdown of mPGES-1 did not alter growth of DU145 monolayer cells, but efficiently inhibited the growth of DU145 multicellular tumor spheroids (MCTS). Opposed to MCTS, monolayer cells did not secrete PGE2 due to a lack of COX-2 expression, which was induced during spheroid formation. Pharmacological inhibition of COX-2 and mPGES-1 supported the crucial role of PGE2 for growth of MCTS. The functionality of spheroid-derived PGE2 was demonstrated by its ability to inhibit cytotoxic T cell activation. When investigating mechanisms of spheroid-induced COX-2 induction, we observed that among microenvironmental factors neither glucose deprivation, hypoxia nor tumor cell apoptosis enhanced COX-2 expression. Interestingly, interfering with apoptosis in spheroids triggered a shift towards necrosis, thus augmenting COX-2 expression. We went on to demonstrate that necrotic cells induced COX-2 mRNA expression and PGE2 secretion from live tumor cells. In conclusion, necrosis-dependent COX-2 upregulation in MCTS promoted PGE2-dependent tumor growth and inhibited activated cytotoxic T cells. Hence, blocking mPGES-1 as a therapeutic option may be considered for COX-2/mPGES-1-positive solid cancers. [ABSTRACT FROM AUTHOR]

Prostaglandin E (PGE) is a bioactive lipid that elicits a wide range of biological effects associated with inflammation and cancer. PGE exerts diverse effects on cell proliferation, apoptosis, angiogenesis, inflammation, and immune surveillance. This review concentrates primarily on gastrointestinal cancers, where the actions of PGE are most prominent, most likely due to the constant exposure to dietary and environmental insults and the intrinsic role of PGE in tissue homeostasis. A discussion of recent efforts to elucidate the complex and interconnected pathways that link PGE signaling with inflammation and cancer is provided, supported by the abundant literature showing a protective effect of NSAIDs and the therapeutic efficacy of targeting mPGES-1 or EP receptors for cancer prevention. However, suppressing PGE formation as a means of providing chemoprotection against all cancers may not ultimately be tenable, undoubtedly the situation for patients with inflammatory bowel disease. Future studies to fully understand the complex role of PGE in both inflammation and cancer will be required to develop novel strategies for cancer prevention that are both effective and safe. [ABSTRACT FROM AUTHOR]

Accumulating evidence suggests that sphingosine kinase 1 (SphK1) plays a key role in carcinogenesis by regulating cyclooxygenase-2 (COX-2) expression. Recent clinical studies have revealed that COX-2 inhibitors cause adverse cardiovascular side effects, likely due to inhibition of prostacyclin (PGI2). In this work, we investigated the roles of SphK1 inhibition on blood pressure (BP). The results show that lack of SphK1 expression did not exacerbate angiotensin II (Ang II)-induced acute hypertension, whereas celecoxib, a COX-2 inhibitor, augmented and sustained higher BP in mice. Interestingly, SphK1-knockout mice inhibited prostaglandin E2 (PGE2) but not PGI2 production in response to Ang II, whereas celecoxib blocked both PGE2 and PGI2 production. Mechanistically, SphK1 down-regulation by siRNA in human umbilical vein endothelial cells decreased cytokine-induced PGE2 production primarily through inhibition of microsomal PGE synthase-1 (mPGES-1), not COX-2. SphK1 down-regulation also decreased MKK6 expression, which phosphorylates and activates P38 MAPK, which, in turn, regulates early growth response-1 (Egr-1), a transcription factor of mPGES-1. Together, these data indicate that SphK1 regulates PGE2 production by mPGES-1 expression via the p38 MAPK pathway, independent of COX-2 signaling, in endothelial cells, suggesting that SphK1 inhibition may be a promising strategy for cancer chemoprevention with lack of the adverse cardiovascular side effects associated with coxibs [ABSTRACT FROM AUTHOR]

PGE2 is a natriuretic factor whose production is elevated after water deprivation (WD) but its role in dehydration natriuresis is not well-defined. The goal of the present study was to investigate the role of microsomal prostaglandin E synthase-1 (mPGES-1) in dehydration natriuresis. After 24-h WD, wild-type (WT) mice exhibited a significant increase in 24-h urinary Na+ excretion accompanied with normal plasma Na+ concentration and osmolality. In contrast, WD-induced elevation of urinary Na+ excretion was completely abolished in mPGES-1 knockout (KO) mice in parallel with increased plasma Na+ concentration and a trend increase in plasma osmolality. WD induced a 1.8-fold increase in urinary PGE2 output and a 1.6-fold increase in PGE2 content in the renal medulla of WT mice, both of which were completely abolished by mPGES-1 deletion. Similar patterns of changes were observed for urinary nitrate/nitrite and cGMP. The natriuresis in dehydrated WT mice was associated with a significant downregulation of renal medullary epithelial Na channel-α mRNA and protein, contrasting to unaltered expressions in dehydrated KO mice. By quantitative RT-PCR, WD increased the endothelial nitric oxide synthase (eNOS), inducible NOS, and neuronal NOS expressions in the renal medulla of WT mice by 3.9-, 1.48-, and 2.6-fold, respectively, all of which were significantly blocked in mPGES-1 KO mice. The regulation of eNOS expression was further confirmed by immunoblotting. Taken together, our results suggest that mPGES-1- derived PGE2 contributes to dehydration natriuresis likely via NO/ cGMP. [ABSTRACT FROM AUTHOR]

An increasing level of prostaglandin ( PG) E2 is involved in the progression of neuroinflammation induced by ischemia and bacterial infection. Although an imbalance in the rates of production and clearance of PGE2 under these pathological conditions appears to affect the concentration of PGE2 in the cerebrospinal fluid ( CSF), the regulatory system remains incompletely understood. The purpose of this study was to investigate the cellular system of PGE2 production via microsomal PGE synthetase-1 ( mPGES-1), the inducible PGE2-generating enzyme, and PGE2 elimination from the CSF via the blood- CSF barrier ( BCSFB). Immunohistochemical analysis revealed that mPGES-1 was expressed in the soma and perivascular sheets of astrocytes, pia mater, and brain blood vessel endothelial cells, suggesting that these cells are local production sites of PGE2 in the CSF. The in vivo PGE2 elimination clearance from the CSF was eightfold greater than that of d-mannitol, which is considered to reflect CSF bulk flow. This process was inhibited by the simultaneous injection of unlabeled PGE2 and β-lactam antibiotics, such as benzylpenicillin, cefazolin, and ceftriaxone, which are substrates and/or inhibitors of organic anion transporter 3 ( OAT3). The characteristics of PGE2 uptake by the isolated choroid plexus were at least partially consistent with those of OAT3. OAT3 was able to mediate PGE2 transport with a Michaelis-Menten constant of 4.24 μM. These findings indicate that a system regulating the PGE2 level in the CSF involves OAT3-mediated PGE2 uptake by choroid plexus epithelial cells, acting as a cerebral clearance pathway via the BCSFB of locally produced PGE2. [ABSTRACT FROM AUTHOR]

J. Neurochem. (2012) 122, 952-961. Abstract While free radicals and inflammation constitute major routes of neuronal injury occurring in amyotrophic lateral sclerosis (ALS), neither antioxidants nor non-steroidal anti-inflammatory drugs have shown significant efficacy in human clinical trials. We examined the possibility that concurrent blockade of free radicals and prostaglandin E2 (PGE2)-mediated inflammation might constitute a safe and effective therapeutic approach to ALS. We have developed 2-hydroxy-5-[2-(4-trifluoromethylphenyl)-ethylaminobenzoic acid] (AAD-2004) as a derivative of aspirin. AAD-2004 completely removed free radicals at 50 nM as a potent spin-trapping molecule and inhibited microsomal PGE2 synthase-1 (mPGES-1) activity in response to both lipopolysaccharide-treated BV2 cell with IC50 of 230 nM and recombinant human mPGES-1 protein with IC50 of 249 nM in vitro. In superoxide dismutase 1G93A transgenic mouse model of ALS, AAD-2004 blocked free radical production, PGE2 formation, and microglial activation in the spinal cords. As a consequence, AAD-2004 reduced autophagosome formation, axonopathy, and motor neuron degeneration, improving motor function and increasing life span. In these assays, AAD-2004 was superior to riluzole or ibuprofen. Gastric bleeding was not induced by AAD-2004 even at a dose 400-fold higher than that required to obtain maximal therapeutic efficacy in superoxide dismutase 1G93A. Targeting both mPGES-1-mediated PGE2 and free radicals may be a promising approach to reduce neurodegeneration in ALS and possibly other neurodegenerative diseases. [ABSTRACT FROM AUTHOR]

Microsomal prostaglandin E synthase-1 (mPGES-1) represents an attractive target for the treatment of rheumatoid arthritis and pain, being upregulated in response to inflammatory stimuli. Biochemical assays for prostaglandin E synthase activity are complicated by the instability of the substrate (PGH2) and the challenge of detection of the product (PGE2). A coupled fluorescent assay is described for mPGES-1where PGH2 is generated in situ using the action of cyclooxygenase 2 (Cox-2) on arachidonic acid. PGE2 is detected by coupling through 15-prostaglandin dehydrogenase (15-PGDH) and diaphorase. The overall coupled reaction was miniaturized to 1536-well plates and validated for high-throughput screening. For compound progression, a novel high-throughput mass spectrometry assay was developed using the RapidFire platform. The assay employs the same in situ substrate generation step as the fluorescent assay, after which both PGE2 and a reduced form of the unreacted substrate were detected by mass spectrometry. Pharmacology and assay quality were comparable between both assays, but the mass spectrometry assay was shown to be less susceptible to interference and false positives. Exploiting the throughput of the fluorescent assay and the label-free, direct detection of the RapidFire has proved to be a powerful lead discovery strategy for this challenging target. [ABSTRACT FROM PUBLISHER]