Green tea epigallocatechin-3-gallate inhibits microsomal prostaglandin E2 synthase-1

Prostaglandin (PG)E₂ is a critical lipid mediator connecting chronic inflammation to cancer. The anti-carcinogenic epigallocatechin-3-gallate (EGCG) from green tea (Camellia sinensis) suppresses cellular PGE₂ biosynthesis, but the underlying molecular mechanisms are unclear. Here, we investigated the interference of EGCG with enzymes involved in PGE₂ biosynthesis, namely cytosolic phospholipase (cPL)A₂, cyclooxygenase (COX)-1 and -2, and microsomal prostaglandin E₂ synthase-1 (mPGES-1). EGCG failed to significantly inhibit isolated COX-2 and cPLA₂ up to 30 μM and moderately blocked isolated COX-1 (IC₅₀ > 30 μM). However, EGCG efficiently inhibited the transformation of PGH₂ to PGE₂ catalyzed by mPGES-1 (IC₅₀ = 1.8 μM). In lipopolysaccharide-stimulated human whole blood, EGCG significantly inhibited PGE₂ generation, whereas the concomitant synthesis of other prostanoids (i.e., 12(S)-hydroxy-5-cis-8,10-trans-heptadecatrienoic acid and 6-keto PGF_1α) was not suppressed. Conclusively, mPGES-1 is a molecular target of EGCG, and inhibition of mPGES-1 is seemingly the predominant mechanism underlying suppression of cellular PGE₂ biosynthesis by EGCG.     

Prostaglandin E2 produced by microsomal prostaglandin E synthase-1 regulates the onset and the maintenance of wakefulness

This study examined the effect of prostaglandin E₂ (PGE₂) produced by microsomal prostaglandin E synthase-1 (mPGES-1) on circadian rhythm. Using wild-type mice (WT) and mPGES-1 knockout mice (mPGES-1^−/−), I recorded and automatically analyzed the natural behavior of mice in home cages for 24 h and measured brain levels of PGE₂. The switch to wakefulness was not smooth, and sleepiness and the total duration of sleep were significantly longer in the mPGES-1^−/− mice. Moreover, the basal concentration of PGE₂ was significantly lower in the mPGES-1^−/− mice. These findings suggest that PGE₂ produced by mPGES-1 regulates the onset of wakefulness and the maintenance of circadian rhythm.     

Prostaglandin E synthases: Understanding their pathophysiological roles through mouse genetic models

Prostaglandin E synthase (PGES), which converts cyclooxygenase (COX)-derived prostaglandin H₂ (PGH₂) to PGE₂, is known to comprise a group of at least three structurally and biologically distinct enzymes. Two of them are membrane-bound and have been designated as mPGES-1 and mPGES-2. mPGES-1 is a perinuclear protein that is markedly induced by proinflammatory stimuli and downregulated by anti-inflammatory glucocorticoids as in the case of COX-2. It is functionally coupled with COX-2 in marked preference to COX-1. mPGES-2 is synthesized as a Golgi membrane-associated protein, and the proteolytic removal of the N-terminal hydrophobic domain leads to the formation of a mature cytosolic enzyme. This enzyme is rather constitutively expressed in various cells and tissues and is functionally coupled with both COX-1 and COX-2. Cytosolic PGES (cPGES) is constitutively expressed in a wide variety of cells and is functionally linked to COX-1 to promote immediate PGE₂ production. Recently, mice have been engineered with specific deletions in each of these three PGES enzymes. In this review, we summarize the current understanding of the in vivo roles of PGES enzymes by knockout mouse studies and provide an overview of their biochemical properties.     

The prostaglandin transporter PGT transports PGH2

Prostaglandin H₂ not only serves as the common precursor of all other PGs, but also directly triggers signals (e.g. platelet aggregation), depending on its location and translocation. The prostaglandin carrier PGT mediates the transport of several prostanoids, such as PGE₂, and PGF_2α. Here we used PGT in the plasma membrane as a model system to test the hypothesis that PGT also transports PGH₂. Using wild-type and PGT-expressing MDCK cells, we show that PGH₂ uptake is mediated both by simple diffusion and by PGT. The PGH₂ influx permeability coefficient for diffusion is (5.66 ± 0.63) × 10⁻⁶ cm/s. The kinetic parameters of PGH₂ transport by PGT are K_m = 376 ± 34 nM and V_max = 210.2 ± 11.4 fmol/mg protein/s. PGH₂ transport by PGT can be inhibited by excess PGE₂ or by a PGT inhibitor. We conclude that PGT may play a role in transporting PGH₂ across cellular membranes.     

Photo-crosslinking of proteins in intact cells reveals a dimeric structure of cyclooxygenase-2 and an inhibitor-sensitive oligomeric structure of microsomal prostaglandin E2 synthase-1

We have characterized the structures of cyclooxygenase-2 (COX-2) and microsomal prostaglandin E₂ synthase-1 (mPGES-1) in intact cells using bifunctional and photo-activatable crosslinking agents. A dimeric complex was detected for COX-2 by both crosslinking approaches, consistent with the crystal structure of the enzyme. For mPGES-1, treatment of A549 cells with disuccinimidyl suberate yielded immunoreactive protein bands corresponding to a dimer (33 kDa) and a trimer (45 kDa), as observed for the isolated enzyme. Photo-crosslinking with photoactivatable methionine in intact cells generated complexes with molecular weights corresponding to the dimer (33 kDa) and two putative trimer forms (50 and 55 kDa). Treatment with the selective mPGES-1 inhibitor MF63 prevented the formation of the 50 and 55 kDa crosslinked complexes, while an inactive structural analogue had no effect. Our data indicate that COX-2 forms a dimer in intact cells and that mPGES-1 has an oligomeric structure that can be disrupted by a selective inhibitor.     

Microsomal prostaglandin E synthase-1 aggravates inflammation and demyelination in a mouse model of multiple sclerosis

Microsomal prostaglandin synthetase-1 (mPGES-1) is an inducible terminal enzyme required for prostaglandin E₂ (PGE₂) biosynthesis. In this study, we examined the role of mPGES-1 in the inflammation and demyelination observed in experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS). We induced EAE with myelin oligodendrocyte glycoprotein_35–55 peptide in mPGES-1-deficient (mPGES-1^−/−) and wild-type (WT) mice. First, we examined the histopathology in the early and late phases of EAE progression. Next, we measured the concentration of PGE₂ in the spinal cord and investigated the expression of mPGES-1 using immunohistochemistry. In addition, we examined the progression of the severity of EAE using an EAE score to investigate a correlation between pathological features and paralysis. In this paper, we demonstrate that WT mice showed extensive inflammation and demyelination, whereas mPGES-1^−/− mice exhibited significantly smaller and more localized changes in the perivascular area. The mPGES-1 protein was induced in vascular endothelial cells and microglia around inflammatory foci, and PGE₂ production was increased in WT mice but not mPGES-1^−/− mice. Furthermore, mPGES-1^−/− mice showed a significant reduction in the maximum EAE score and improved locomotor activity. These results suggest that central PGE₂ derived from non-neuronal mPGES-1 aggravates the disruption of the vessel structure, leading to the spread of inflammation and local demyelination in the spinal cord, which corresponds to the symptoms of EAE. The inhibition of mPGES-1 may be useful for the treatment of human MS.     

Prostaglandin E2 produced by inducible COX-2 and mPGES-1 promoting cancer cell proliferation in vitro and in vivo

Aim

Many cancers originate and flourish in a prolonged inflammatory environment. Our aim is to understand the mechanisms of how the pathway of prostaglandin E₂ (PGE₂) biosynthesis and signaling can promote cancer growth in inflammatory environment at cellular and animal model levels.

Main methods

In this study, a chronic inflammation pathway was mimicked with a stable cell line that over-expressed a novel human enzyme consisting of cyclooxygenase isoform-2 (COX-2) linked to microsomal (PGE₂ synthase-1 (mPGES-1)) for the overproduction of pathogenic PGE₂. This PGE₂-producing cell line was co-cultured and co-implanted with three human cancer cell lines including prostate, lung, and colon cancers in vitro and in vivo, respectively.

Key findings

Increases in cell doubling rates for the three cancer cell types in the presence of the PGE₂-producing cell line were clearly observed. In addition, one of the four human PGE₂ subtype receptors, EP₁, was used as a model to identify PGE₂-signaling involved in promoting the cancer cell growth. This finding was further proven in vivo by co-implanting the PGE₂-producing cells line and the EP₁-positive cancer cells into the immune deficient mice, after that, it was observed that the PGE₂-producing cells promoted all three types of cancer formation in the mice.

Significance

This study clearly demonstrated that the human COX-2 linked to mPGES-1 is a pathway that, when mediated by the EP, is linked to promoting cancer growth in a chronic inflammatory environment. The identified pathway could be used as a novel target for developing and advancing anti-inflammation and anti-cancer interventions.     

mPGES-1 as a target for cancer suppression : A comprehensive invited review “Phospholipase A2 and lipid mediators”

Prostaglandin E₂ (PGE₂) is a bioactive lipid that can elicit a wide range of biological effects associated with inflammation and cancer. The physiological roles of PGE₂ are diverse, mediated in part through activation of key downstream signaling cascades via transmembrane EP receptors located on the cell surface. Elevated levels of COX-2 and concomitant overproduction of PGE₂ are often found in human cancers. These observations have led to the use of non-steroidal anti-inflammatory drugs (NSAIDs) as chemopreventive agents, particularly for colorectal cancer (CRC). Their long-term use, however, may be associated with gastrointestinal toxicity and increased risk of adverse cardiovascular events, prompting the development of other enzymatic targets in this pathway. This review will focus on recent efforts to target the terminal synthase, mPGES-1, for cancer chemoprevention. The role of mPGES-1 in the pathogenesis of various cancers is discussed. In addition, an overview of recent efforts to develop small molecule inhibitors that target the protein with high selectivity is also be reviewed.     

Differential role of cyclooxygenase-1 and -2 on renal vasoconstriction to α1-adrenoceptor stimulation in normotensive and hypertensive rats

Aims

Hypertension is associated with the impairment of renal cyclooxygenase (COX) activity, which regulates vascular tone, salt and water balance and renin release. We aimed to evaluate the functional role of COX isoforms in kidneys isolated from spontaneously hypertensive rats (SHR) after α₁-adrenoceptor (α₁-AR) stimulation.

Main methods

Male six-month-old SHR and normotensive Wistar-Kyoto rats (WKY) were used. The kidneys were isolated to measure perfusion pressure and COX-1- or COX-2-derived prostanoids in response to α₁-AR activation.

Key findings

The basal perfusion pressure was higher in SHR kidneys compared with WKY kidneys (95 ± 11 vs. 68 ± 6 mm Hg, P < 0.05). Phenylephrine induced a greater vasopressor response in SHR kidneys (EC₅₀ of 1.89 ± 0.58 nmol) than WKY kidneys (EC₅₀ of 3.30 ± 0.54 nmol, P < 0.05 vs. SHR). COX-1 inhibition decreased the α₁-AR-induced vasoconstrictor response in WKY but did not affect SHR response, while COX-2 inhibition diminished the response in SHR. Both basal prostacyclin (PGI₂) and thromboxane A₂ (TxA₂) values were higher in SHR kidney perfusates (P < 0.05) and were reduced by COX-1 and COX-2 inhibitors in both strains. Furthermore, phenylephrine increased PGI₂ through COX-2 in WKY and through COX-1 in SHR, but the agonist did not significantly modify TxA₂ in both strains.

Significance

The data suggest that COX-1contributes to vasoconstrictor effects in WKY kidneys and that COX-2 has the same effect in SHR kidneys. The results also suggest that basal release of COX-2-derived vasoconstrictor prostanoids is involved in renal vascular hypersensitivity in SHR.     

The effects of microsomal prostaglandin E synthase-1 deletion in acute cardiac ischemia in mice

The goal of the present study was to assess how genetic loss of microsomal prostaglandin E₂ synthase-1 (mPGES-1) affects acute cardiac ischemic damage after coronary occlusion in mice. Wild type (WT), heterozygous (mPGES-1^+/−), and homozygous (mPGES-1^−/−) knockout mice were subjected to left coronary artery occlusion. At 24 h, myocardial infarct (MI) volume was measured histologically. Post-MI survival, plasma levels of creatine phosphokinase (CPK) and cardiac troponin-I, together with MI size, were similar in WT, mPGES-1^+/− and mPGES-1^−/− mice. In contrast, post-MI survival was reduced in mPGES-1^−/− mice pretreated with I prostanoid receptor (IP) antagonist (12/16) compared with vehicle-treated controls (13/13 mPGES-1^−/−) together with increased CPK and cardiac troponin-I release. The deletion of mPGES-1 in mice results in increased prostacyclin I₂ (PGI₂) formation and marginal effects on the circulatory prostaglandin E₂ (PGE₂) level. We conclude that loss of mPGES-1 results in increased PGI₂ formation, and in contrast to inhibition of PGI₂, without worsening acute cardiac ischemic injury.     

Fragment-based discovery of novel and selective mPGES-1 inhibitors Part 1: Identification of sulfonamido-1,2,3-triazole-4,5-dicarboxylic acid

Microsomal prostaglandin E synthase-1 (mPGES-1) is an inducible prostaglandin E synthase that catalyzes the conversion of prostaglandin PGH₂ to PGE₂ and represents a novel target for therapeutic treatment of inflammatory disorders. It is essential to identify mPGES-1 inhibitor with novel scaffold as new hit or lead compound for the purpose of the next-generation anti-inflammatory drugs. Herein we report the discovery of sulfonamido-1,2,3-triazole-4,5-dicarboxylic derivatives as a novel class of mPGES-1 inhibitors identified through fragment-based virtual screening and in vitro assays on the inhibitory activity of the actual compounds. 1-[2-(N-Phenylbenzenesulfonamido)ethyl]-1H-1,2,3-triazole-4,5-dicarboxylic acid (6f) inhibits human mPGES-1 (IC₅₀ of 1.1 μM) with high selectivity (ca.1000-fold) over both COX-1 and COX-2 in a cell-free assay. In addition, the activity of compound 6f was again tested at 10 μM concentration in presence of 0.1% Triton X-100 and found to be reduced to 1/4 of its original activity without this detergent. Compared to the complete loss of activity of nuisance inhibitor with the detergent, therefore, compound 6f would be regarded as a partial nuisance inhibitor of mPGES-1 with a novel scaffold for the optimal design of more potent mPGES-1 inhibitors.     

Expression of prostaglandin E synthases in the bovine oviduct

The oviduct is a specialized organ responsible for the storage and the transport of male and female gametes. It also provides an optimal environment for final gamete maturation, fertilization, and early embryo development. Prostaglandin (PG) E₂ is involved in many female reproductive functions, including ovulation, fertilization, implantation, and parturition. However, the control of its synthesis in the oviduct is not fully understood. Cyclooxygenases (COXs) are involved in the first step of the transformation of arachidonic acid to PGH_2. The prostaglandin E synthases (PGESs) constitute a family of enzymes that catalyze the conversion of PGH₂ to PGE₂, the terminal step in the formation of this bioactive prostaglandin. Quantitative real-time PCR was used to determine the expression of COX-1, COX-2, microsomal prostaglandin E synthase-1 (mPGES-1), microsomal prostaglandin E synthase-2 (mPGES-2), and cytosolic prostaglandin E synthase (cPGES) mRNA in various sections of the oviduct, both ipsilateral and contralateral (to the ovary on which ovulation occurred) at various stages of the estrous cycle. Furthermore, protein expression and localization of cPGES, mPGES-1, and mPGES-2 were determined by Western blot and immunohistochemistry. All three PGESs were detected at both mRNA and protein levels in the oviduct. These PGESs were mostly concentrated in the oviductal epithelial layer and primarily expressed in the ampulla section of the oviduct and to a lesser extent in the isthmus and the isthmic-ampullary junction. The mPGES-1 protein was highly expressed in the contralateral oviduct, which contrasted with mPGES-2 mostly expressed in the ipsilateral oviduct. This is apparently the first report documenting that the three PGESs involved in PGE₂ production were present in the Bos taurus oviduct.     

Spectroelectrochemical characterization of pain biomarkers

This article reports the first electrochemical characterization of pain biomarkers that include arachidonic acid (AA), prostaglandin G₂ (PGG₂), and cyclooxygenase 2 (COX-2). These biomarkers are mediators of pathophysiology of pain, inflammation, and cell proliferation in cancer. The article also reports the development of an electrochemical immunosensor for monitoring these pain biomarkers. The results revealed that direct electron transfer between AA metabolites and the electrode could be easily monitored and that an enzyme-modified electrode dramatically enhanced bioelectrocatalytic activity toward AA. Cyclic voltammetric analysis of AA revealed a concentration-dependent anodic current with a slope of 2.37 and a limit of detection (LOD) of 0.25 nM. This unique AA/gold electrode electron transfer provides a good electrochemical sensing platform for prostaglandin H₂ (PGH₂) as the basis for quantitation of pain. An amperometric signal intensity of a COX-2 antibody-modified gold electrode was linear with COX-2 concentration in the range of 0.1-0.5 μg/ml and an LOD of 0.095 μg/ml. The results also revealed a linear correlation of the concentration of PGG₂ with an LOD of 0.227 μM.     

Heterotypic contact reveals a COX-2-mediated suppression of osteoblast differentiation by endothelial cells: A negative modulatory role for prostanoids in VEGF-mediated cell: cell communication?

In bone, angiogenesis must be initiated appropriately, but limited once remodelling or repair is complete. Our recent findings have supported a role for prostaglandins (PG), known modulators of osteoblast (OB) and endothelial cell (EC) behaviour, in facilitating VEGF-mediated paracrine communication from OBs to ‘remotely located’ ECs, but the mechanism(s) regulating OB:EC crosstalk when these cells are closely opposed are undefined. In this study we have examined: (i) the effects of exogenous PGE₂ on VEGF-driven events in ECs, and (ii) the role of endogenous COX-2-derived prostanoids in mediating communication between intimately opposed OBs and ECs in direct contact. Exposure of ECs to PGE₂ increased ERK1/2 phosphorylation, COX-2 induction, 6-keto-PGF_1α release and EC proliferation. In contrast, PGE₂ attenuated VEGF₁₆₅-induced VEGFR2/Flk1 phosphorylation, ERK1/2 activation and proliferation of ECs, suggesting that exogenous PGE₂ restricts the actions of VEGF. However, the COX-2-selective inhibitor, NS398, also attenuated VEGF-induced proliferation, implying a distinct role for endogenous COX-2 activity in regulating EC behaviour. To examine the effect of OB:EC proximity and the role of COX-2 products further, we used a confrontational co-culture model. These studies showed that COX-2 blockade with NS398 enhanced EC-dependent increases in OB differentiation, that this effect was reversed by exogenous PGH₂ (immediate COX-2 product), and that exogenous VEGF did not influence EC-dependent OB differentiation under these conditions. Our findings indicate that locally produced prostanoids may serve distinct roles depending on OB:EC proximity and negatively modulate VEGF-mediated changes in EC behaviour when these cells are closely opposed to control angiogenesis during bone (re)modelling.     

Microsomal prostaglandin E synthase-2 is not essential for in vivo prostaglandin E2 biosynthesis

Prostaglandin E₂ (PGE₂) plays an important role in the normal physiology of many organ systems. Increased levels of this lipid mediator are associated with many disease states, and it potently regulates inflammatory responses. Three enzymes capable of in vitro synthesis of PGE₂ from the cyclooxygenase metabolite PGH₂ have been described. Here, we examine the contribution of one of these enzymes to PGE₂ production, mPges-2, which encodes microsomal prostaglandin synthase-2 (mPGES-2), by generating mice homozygous for the null allele of this gene. Loss of mPges-2 expression did not result in a measurable decrease in PGE₂ levels in any tissue or cell type examined from healthy mice. Taken together, analysis of the mPGES-2 deficient mouse lines does not substantiate the contention that mPGES-2 is a PGE₂ synthase.     

Prostaglandin E2 modulates components of the Wnt signaling system in bone and prostate cancer cells

Both Wnt signaling and prostaglandin E₂ (PGE₂) play pivotal roles in bone development, remodeling, osteoporosis and prostate cancer (PCa) bone metastases. We investigated the effects of PGE₂ on Wnt signaling in osteoblast-lineage cells and Wnt-inhibitor expression in PCa cells. We demonstrate that low dose PGE₂ (0.1 μM) promotes Wnt signaling while higher doses of PGE₂ (1.0-10 μM) inhibit these same parameters in osteoblast-lineage cells. The differential effects of low vs high-dose PGE₂ on pre-osteoblasts may be attributed to dose-dependent modulation of prostaglandin receptor (EP) subtype expression; with lower doses increasing the expression the cAMP-stimulatory EP4 receptor subtype and higher doses increasing the expression of the cAMP-inhibitory EP3 receptor subtype. Moreover, we demonstrate that high expression levels of COX-2 and PGE₂ promote the secretion of Wnt inhibitors from prostate cancer cells. These data demonstrate that there are dose-dependent effects of PGE₂ on Wnt activation in osteoblast-lineage cells and Wnt-inhibitor expression in PCa cells which may have clinical implications in the management.     

Studies on membrane-associated prostaglandin E synthase-2 with reference to production of 12L-hydroxy-5,8,10-heptadecatrienoic acid (HHT)

Membrane-associated prostaglandin (PG) E synthase (mPGE synthase)-2 catalyzes the conversion of PGH₂ primarily to PGE₂. The enzyme is activated by various sulfhydryl reagents including dithiothreitol, dihydrolipoic acid, and glutathione, and it is different from mPGE synthase-1 and cytosolic PGE synthase, both of which require specifically glutathione. Recently, other investigators reported that their preparation of mPGE synthase-2 containing heme converted PGH₂ to 12L-hydroxy-5,8,10-heptadecatrienoic acid (HHT) rather than to PGE₂ [T. Yamada, F. Takusagawa, Biochemistry 46 (2007) 8414-8424]. As we examined presently, the heme-bound enzyme expressed and purified according to their method synthesized HHT from PGH₂, but also PGE₂ in a decreased amount. Whereas the PGE synthase activity was completely lost at 50 °C for 5 min, the HHT synthase activity remained even at 100 °C for 5 min. In contrast, when the heme-bound enzyme was purified in the presence of dithiothreitol, only PGE₂ was produced, but essentially no HHT was detected. Thus, native mPGE synthase-2 enzymatically catalyzes only the conversion of PGH₂ to PGE₂, but not to HHT, and heme is not involved in this reaction.     

miR-143 decreases COX-2 mRNA stability and expression in pancreatic cancer cells

Small non-coding RNAs, microRNAs (miRNA), inhibit the translation or accelerate the degradation of message RNA (mRNA) by targeting the 3′-untranslated region (3′-UTR) in regulating growth and survival through gene suppression. Deregulated miRNA expression contributes to disease progression in several cancers types, including pancreatic cancers (PaCa). PaCa tissues and cells exhibit decreased miRNA, elevated cyclooxygenase (COX)-2 and increased prostaglandin E₂ (PGE₂) resulting in increased cancer growth and metastases. Human PaCa cell lines were used to demonstrate that restoration of miRNA-143 (miR-143) regulates COX-2 and inhibits cell proliferation. miR-143 were detected at fold levels of 0.41 ± 0.06 in AsPC-1, 0.20 ± 0.05 in Capan-2 and 0.10 ± 0.02 in MIA PaCa-2. miR-143 was not detected in BxPC-3, HPAF-II and Panc-1 which correlated with elevated mitogen-activated kinase (MAPK) and MAPK kinase (MEK) activation. Treatment with 10 μM of MEK inhibitor U0126 or PD98059 increased miR-143, respectively, by 187 ± 18 and 152 ± 26-fold in BxPC-3 and 182 ± 7 and 136 ± 9-fold in HPAF-II. miR-143 transfection diminished COX-2 mRNA stability at 60 min by 2.6 ± 0.3-fold in BxPC-3 and 2.5 ± 0.2-fold in HPAF-II. COX-2 expression and cellular proliferation in BxPC-3 and HPAF-II inversely correlated with increasing miR-143. PGE₂ levels decreased by 39.3 ± 5.0% in BxPC-3 and 48.0 ± 3.0% in HPAF-II transfected with miR-143. Restoration of miR-143 in PaCa cells suppressed of COX-2, PGE₂, cellular proliferation and MEK/MAPK activation, implicating this pathway in regulating miR-143 expression.