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Plant phytoprostanes (PhytoPs) are lipid oxidative stress mediators that share structural similarities with mammal prostaglandins (PGs). They have been demonstrated to modulate inflammatory processes mediated by prostaglandins. The present study aims to test the effects of the most abundant oxylipin from Gracilaria longissima, ent-9-D1t-Phytoprostane (9-D1t-PhytoP), on platelet activation and vascular cells as well as clarify possible interactions with platelets and the endothelial EP3 receptor Platelet and monocyte activation was assessed by flow cytometry in the presence of purified 9-D1t-PhytoP. Cell migration was studied using the human Ea.hy926 cell line by performing a scratch wound healing assay. The RNA expression of inflammatory markers was evaluated by RT-PCR under inflammatory conditions. Blind docking consensus was applied to the study of the interactions of selected ligands against the EP3 receptor protein. The 9D1t-PhytoP exerts several pharmacological effects; these include prothrombotic and wound-healing properties. In endothelial cells, 9D1t-PhytP mimics the migration stimulus of PGE2. Computational analysis revealed that 9D1t-PhytP forms a stable complex with the hydrophobic pocket of the EP3 receptor by interaction with the same residues as misoprostol and prostaglandin E2 (PGE2), thus supporting its potential as an EP3 agonist. The potential to form procoagulant platelets and the higher endothelial migration rate of the 9-D1t-PhytoP, together with its capability to interact with PGE2 main target receptor in platelets suggest herein that this oxylipin could be a strong candidate for pharmaceutical research from a multitarget perspective.

Aims Pathological cardiac hypertrophy induced by activation of the renin–angiotensin–aldosterone system (RAAS) is one of the leading causes of heart failure. However, in current clinical practice, the strategy for targeting the RAAS is not sufficient to reverse hypertrophy. Here, we investigated the effect of prostaglandin E1 (PGE1) on angiotensin II (AngII)-induced cardiac hypertrophy and potential molecular mechanisms underlying the effect. Methods and results Adult male C57 mice were continuously infused with AngII or saline and treated daily with PGE1 or vehicle for two weeks. Neonatal rat cardiomyocytes were cultured to detect AngII-induced hypertrophic responses. We found that PGE1 ameliorated AngII-induced cardiac hypertrophy both in vivo and in vitro. The RNA sequencing (RNA-seq) and expression pattern analysis results suggest that Netrin-1 (Ntn1) is the specific target gene of PGE1. The protective effect of PGE1 was eliminated after knockdown of Ntn1. Moreover, Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed that the PGE1-mediated signaling pathway changes are associated with the mitogen-activated protein kinase (MAPK) pathway. PGE1 suppressed AngII-induced activation of the MAPK signaling pathway, and such an effect was attenuated by Ntn1 knockdown. Blockade of MAPK signaling rescued the phenotype of cardiomyocytes caused by Ntn1 knockdown, indicating that MAPK signaling may act as the downstream effector of Ntn1. Furthermore, inhibition of the E prostanoid (EP)3 receptor, as opposed to the EP1, EP2, or EP4 receptor, in cardiomyocytes reversed the effect of PGE1, and activation of EP3 by sulprostone, a specific agonist, mimicked the effect of PGE1. Conclusion In conclusion, PGE1 ameliorates AngII-induced cardiac hypertrophy through activation of the EP3 receptor and upregulation of Ntn1, which inhibits the downstream MAPK signaling pathway. Thus, targeting EP3, as well as the Ntn1–MAPK axis, may represent a novel approach for treating pathological cardiac hypertrophy. Funding Acknowledgement Type of funding sources: None.

Prostaglandin E2 (PGE2)-induced coughs in vivo and vagal nerve depolarization in vitro are inhibited by systemic and local administration of TRPV1 (JNJ 17203212) and prostaglandin EP3 receptor antagonists (L-798106) respectively. These results indicate an interaction of TRPV1 and EP3 receptor in cough responses to PGE2 likely through the vagal sensory nerve. This study aimed to determine whether 1) inhalation of aerosolized JNJ 17203212 and L-798106 affected cough responses to citric acid (CA to mainly stimulate TRPV1) and PGE2; 2) TRPV1 and EP3 receptor morphologically co-expressed and electrophysiologically functioned in the individual of vagal pulmonary C-neurons (cell bodies of bronchopulmonary C-fibers in the nodose/jugular ganglia); and 3) the interaction of the two receptors occurred in these neurons. To this end, aerosolized CA or PGE2 was inhaled by unanesthetized guinea pigs pretreated without or with JNJ 17203212 or L-798106 given in aerosol. Immunofluorescence was applied to identify the expression of the two receptors in vagal pulmonary C-neurons (retrogradely traced by DiI). Whole-cell patch clamp approach was used to detect capsaicin (CAP)- and PGE2-induced currents in the same neurons and determine the effects of the TRPV1 and EP3 receptor antagonists on the currents. We found that PGE2-evoked cough was attenuated by JNJ 17203212 or L-798106 and CA-evoked cough greatly suppressed only by JNJ 17203212. EP3-labeled neurons always co-expressed TRPV1 with a cell size 2-induced currents could be recorded in the individuals of some vagal pulmonary C-neurons. The former was largely inhibited only by JNJ 17203212, while the latter was suppressed by JNJ 17203212 or L-798106. The similarity of the interaction in cough and vagal pulmonary C-neural activity suggests that a subgroup of vagal pulmonary C-neurons co-expressing TRPV1 and EP3 receptor is, at least in part, responsible for the interaction of the two receptors in the cough response to PGE2.

Fever occurs upon binding of prostaglandin E2(PGE2) to EP3receptors in the median preoptic nucleus of the hypothalamus, but the origin of the pyrogenic PGE2has not been clearly determined. Here, using mice of both sexes, we examined the role of local versus generalized PGE2production in the brain for the febrile response. In wild-type mice and in mice with genetic deletion of the prostaglandin synthesizing enzyme cyclooxygenase-2 in the brain endothelium, generated with an inducible CreERT2under theSlco1c1promoter, PGE2levels in the CSF were only weakly related to the magnitude of the febrile response, whereas the PGE2synthesizing capacity in the hypothalamus, as reflected in the levels of cyclooxygenase-2 mRNA, showed strong correlation with the immune-induced fever. Histological analysis showed that the deletion of cyclooxygenase-2 in brain endothelial cells occurred preferentially in small- and medium-sized vessels deep in the brain parenchyma, such as in the hypothalamus, whereas larger vessels, and particularly those close to the neocortical surface and in the meninges, were left unaffected, hence leaving PGE2synthesis largely intact in major parts of the brain while significantly reducing it in the region critical for the febrile response. Furthermore, injection of a virus vector expressing microsomal prostaglandin E synthase-1 (mPGES-1) into the median preoptic nucleus of fever-refractive mPGES-1 knock-out mice, resulted in a temperature elevation in response to LPS. We conclude that the febrile response is dependent on local release of PGE2onto its target neurons and not on the overall PGE2production in the brain.SIGNIFICANCE STATEMENTBy using mice with selective deletion of prostaglandin synthesis in brain endothelial cells, we demonstrate that local prostaglandin E2(PGE2) production in deep brain areas, such as the hypothalamus, which is the site of thermoregulatory neurons, is critical for the febrile response to peripheral inflammation. In contrast, PGE2production in other brain areas and the overall PGE2level in the brain do not influence the febrile response. Furthermore, partly restoring the PGE2synthesizing capacity in the anterior hypothalamus of mice lacking such capacity with a lentiviral vector resulted in a temperature elevation in response to LPS. These data imply that the febrile response is dependent on the local release of PGE2onto its target neurons, possibly by a paracrine mechanism.

Mast cells (MCs) recognize antigens (Ag) via IgE-bound high affinity IgE receptors (Fc&epsilon, RI) and trigger type I allergic reactions. Fc&epsilon, RI-mediated MC activation is regulated by various G protein-coupled receptor (GPCR) agonists. We recently reported that ionotropic P2X4 receptor (P2X4R) stimulation enhanced Fc&epsilon, RI-mediated degranulation. Since MCs are involved in Ag-independent hypersensitivity, we investigated whether co-stimulation with ATP and GPCR agonists in the absence of Ag affects MC degranulation. Prostaglandin E2 (PGE2) induced synergistic degranulation when bone marrow-derived MCs (BMMCs) were co-stimulated with ATP, while pharmacological analyses revealed that the effects of PGE2 and ATP were mediated by EP3 and P2X4R, respectively. Consistently, this response was absent in BMMCs prepared from P2X4R-deficient mice. The effects of ATP and PGE2 were reduced by PI3 kinase inhibitors but were insensitive to tyrosine kinase inhibitors which suppressed the enhanced degranulation induced by Ag and ATP. MC-dependent PGE2-triggered vascular hyperpermeability was abrogated in a P2X4R-deficient mouse ear edema model. Collectively, our results suggest that P2X4R signaling enhances EP3R-mediated MC activation via a different mechanism to that involved in enhancing Ag-induced responses. Moreover, the cooperative effects of the common inflammatory mediators ATP and PGE2 on MCs may be involved in Ag-independent hypersensitivity in vivo.

Prostaglandin E2 (PGE2) EP receptor subtypes EP3 and EP4 are present in the heart and signal via decreased and increased cAMP production, respectively. Previously we reported that cardiomyocyte-specific EP4 KO mice develop a phenotype of dilated cardiomyopathy with reduced ejection fraction, that PGE2 decreases contractility via EP3 in vivo, in vitro and ex vivo; and that EP3 expression in the heart increases when mice are subjected to Angiotensin (Ang) II-hypertension (HTN). We therefore hypothesized that overexpression of EP3 in the heart would exacerbate Ang II-HTN. To test this hypothesis, we used 10-12 week old male EP3 transgenic (Tg) mice with EP3 overexpression in cardiomyocytes and their wild type (WT) controls infused with either vehicle or 1.4 mg/kg/d Ang II for 2 weeks. Echocardiography was performed on conscious mice at the end of the experiment and systolic blood pressure (SBP) was measured every 2 days by tail cuff. Results: Table 1. All data are reported as means ± SEM. N= 3-6 mice per group. Statistical significance: * and *** p < 0.05 and p < 0.005 vs WT + vehicle, ++ p < 0.01 vs WT + Ang II, §§§ p < 0.005 vs Tg + vehicle. As shown in the table, cardiac function was unaffected in WT mice after Ang II infusion. Baseline cardiac function was reduced in EP3 Tg mice receiving vehicle and in contrast to WT mice, was further diminished after Ang II infusion. These differences were not due to altered systolic blood pressure. In conclusion, EP3 Tg mice present with reduced cardiac function at baseline and in support of our hypothesis, exhibit an enhanced response to Ang II. Antagonism of the EP3 receptor may be a novel treatment for heart failure associated with hypertension.

A novel series of prostaglandin analogues with a seven-membered ring scaffold was designed, synthesized, and evaluated for the functional activation of prostaglandin receptors to identify potent and subtype-selective FP and EP3 dual agonists. Starting from the prostacyclin derivative 5b, a nonselective agonist for prostaglandin receptors, replacement of the core structure with an octahydro-2H-cyclopenta[b]oxepine scaffold led to the discovery of the potent and selective FP and EP3 dual agonist 11b as a lead compound for the development of an antiglaucoma agent.

The mechanisms by which prostanoids contribute to the maintenance of whole body water homeostasis are complex and not fully understood. The present study demonstrates that an EP3-dependent feedback mechanism contributes to the regulation of water homeostasis under high-salt conditions. Rats on a normal diet and tap water were placed in metabolic cages and given either sulprostone (20 μg·kg−1·day−1) or vehicle for 3 days to activate EP3 receptors in the thick ascending limb (TAL). Treatment was continued for another 3 days in rats given either 1% NaCl in the drinking water or tap water. Sulprostone decreased expression of cyclooxygenase 2 (COX-2) expression by ∼75% in TAL tubules from rats given 1% NaCl concomitant with a ∼60% inhibition of COX-2-dependent PGE2levels in the kidney. Urine volume increased after ingestion of 1% NaCl but was reduced ∼40% by sulprostone. In contrast, the highly selective EP3 receptor antagonist L-798106 (100 μg·kg−1·day−1), which increased COX-2 expression and renal PGE2production, increased urine volume in rats given 1% NaCl. Sulprostone increased expression of aquaporin-2 (AQP2) in the inner medullary collecting duct plasma membrane in association with an increase in phosphorylation at Ser269 and decrease in Ser261 phosphorylation; antagonism of EP3 with L-798106 reduced AQP2 expression. Thus, although acute activation of EP3 by PGE2in the TAL and collecting duct inhibits the Na-K-2Cl cotransporter and AQP2 activity, respectively, chronic activation of EP3 in vivo limits the extent of COX-2-derived PGE2synthesis, thereby mitigating the inhibitory effects of PGE2on these transporters and decreasing urine volume.

Prostaglandin E2 (PGE2) signals differently through its 4 receptor subtypes (EP1-EP4) to elicit diverse physiologic/pathologic effects. We previously reported that PGE2 via its EP3 receptor reduces cardiac contractility and male mice with cardiomyocyte-specific deletion of the EP4 receptor develop dilated cardiomyopathy. To determine pathway(s) responsible for the phenotype, we performed gene array on left ventricles from these mice followed by Ingenuity Pathway Analysis (IPA) which demonstrated that genes differentiating WT mice and EP4 KO mice were significantly overrepresented in mitochondrial (p=2.51x10 -28 ) and oxidative phosphorylation (p=3.16 x10 -30 ) pathways. Importantly, carnitine palmitoyltransferase 2 (cpt 2), an enzyme that transports fatty acids across the inner mitochondrial membrane was down-regulated in EP4 KO mice. We therefore hypothesized that PGE2 via EP3 decreases cpt 2 to impair mitochondrial function. To test this hypothesis, we used isolated mouse cardiomyocytes (AVM) from 16-18 week old male C57Bl/6 mice and treated them with either 1 μM of the EP3 agonist sulprostone (sulp), or PGE2. Treatment of AVM with sulp for 24 hrs. decreased cpt 2 levels from a control level of 1.0 to 0.57 ± 0.15, p < 0.05 and treatment with PGE2 decreased cpt 2 levels to 0.74 ± 0.27. Since cpt 2 is regulated by the transcription factor NR4A2 in other cell types, we investigated whether NR4A2 was regulated by PGE2 or sulp. NR4A2 levels were reduced after 4 hr. treatment with either PGE2 or by treatment with sulp; from a control value of 1.0 to 0.70 ± 0.16 and to 0.86 ± 0.01 respectively, p< 0.05 for sulp, n = 3. To associate these changes with mitochondrial function, we measured complex I activity with a spectrophotometric assay. Complex I activity was reduced by 50% (p < 0.05) by 4 hr. treatment with PGE2, from 1.32 ± 0.36 to 0.66 ± 0.08 mOD/min, and this was associated with reduced expression of multiple genes from mitochondrial pathways including sub units of mitochondrial NADH dehydrogenase ubiquinone flavoprotein (Nduf), a component of complex I. In conclusion, these results suggest that increased PGE2 and its EP3 receptor in heart disease may contribute to impaired mitochondrial function and provide yet another link between inflammation and cardiac dysfunction.

The EP3 receptor (EP3r) is a member of the EP receptor subfamily, which facilitate a broad range of physiological and pathological prostaglandin E2 (PGE2) actions. Multiple reports have demonstrated that infusion of PGE2 blunts glucose-stimulated insulin secretion (GSIS) in healthy subjects and that inhibition of PGE2 production in noninsulin dependent diabetes mellitus (NIDDM) patients can partially restore impaired GSIS. The suppression of GSIS by PGE2 has been attributed to EP3r, resulting in the suggestion that EP3r antagonism may be an attractive strategy for treatment of NIDDM. We sought to investigate the therapeutic potential of EP3r antagonists in preclinical models. EP3r-mediated suppression of GSIS was validated in vivo using a dose-range (3-30 µg/kg/minutes) of the EP3r-specific agonist sulprostone in conscious rat ivGTTs. Using this model, we defined the EP3r antagonist plasma exposure required to oppose maximal EP3r-mediated GSIS suppression. Efficacy of an EP3r-specific antagonist was then evaluated in the GK, ZDF, and DIO/streptozotocin diabetic rat models. Despite achieving plasma concentrations at least 2-fold higher than the efficacious exposure predicted by the ivGTT model, an EP3r antagonist did not impact GSIS nor glucose homeostasis in these models. We further interrogated the ability of EP3r to suppress GSIS in conscious non-human primates (NHPs) using infusions of both sulprostone and an EP3r-specific antagonist during ivGTT. In NHPs sulprostone resulted in a significant reduction in both the fasting insulin and acute insulin response compared to vehicle. Surprisingly, EP3r agonism also significantly suppressed fasting plasma glucagon. Given the combined effect on islet hormone secretion in NHPs, and the lack of EP3r antagonist efficacy across multiple diabetic rodent models, these results call into question the therapeutic potential of EP3r antagonists for NIDDM patients. Disclosure M.M. Rankin: Employee; Self; Janssen Research & Development. L. Guo: None. I. Bakaj: None. G. Ho: None. B. Rady: Employee; Self; Janssen Research & Development. S. Zhao: Employee; Self; Janssen Research & Development. P.L. Stahle: Employee; Self; Janssen Research & Development. A. Ghosh: None. Y. Shi: Employee; Self; Janssen Research & Development. Employee; Spouse/Partner; Bristol-Myers Squibb Company. S. Patel: None. V. Tryputsen: None. M. Lubomirski: None. X. Zhang: None. B. Zhu: Employee; Self; Janssen Research & Development. M.P. Winters: None. M.J. Macielag: Employee; Self; Janssen Research & Development. L.D. Norquay: Employee; Self; Janssen Research & Development.

The inflammatory response is a fundamental driving force in the pathogenesis of Alzheimer’s disease (AD). In the setting of accumulating immunogenic Aß peptide assemblies, microglia, the innate immune cells of the brain, generate a non-resolving immune response and fail to adequately clear accumulating Aß peptides, accelerating neuronal and synaptic injury. Pathological, biomarker, and imaging studies point to a prominent role of the innate immune response in AD development, and the molecular components of this response are beginning to be unraveled. The inflammatory cyclooxygenase-PGE2 pathway is implicated in pre-clinical development of AD, both in epidemiology of normal aging populations and in transgenic mouse models of Familial AD. The cyclooxygenase-PGE2 pathway modulates the inflammatory response to accumulating Aß peptides through actions of specific E-prostanoid G-protein coupled receptors.

We recently identified a pathway underlying immune activation in hypertension. Proteins oxidatively modified by reactive γ-ketoaldehydes (isoketals) accumulate in dendritic cells (DCs). These are immunogenic and lead to subsequent T lymphocytes activation. The local signals that stimulate DCs to accumulate isoketal adducts remain undefined. Prostaglandin E 2 (PGE 2 ) has been implicated in the inflammation associated with hypertension. We hypothesized that PGE 2 via its EP3 receptor contributes to DC activation in hypertension. EP3 -/- mice and wild type (WT) littermates were exposed to sequential hypertensive stimuli involving an initial 2-week exposure to the NOS inhibitor L-NAME (LN) in drinking water, a 2 week washout period, and a subsequent 4% high salt diet (HS) for 3 weeks. In WT mice, this protocol increased systolic pressure from 123±2 to 148±8 mmHg (p+ and CD8 + effector memory T cells by 2 to 3 fold. This was associated with a striking accumulation of isoketal protein adducts in splenic DCs. However, the increases in blood pressure, renal T cell infiltration and DC isoketal formation were completely prevented in EP3 -/- mice. We further hypothesized that EP3 receptors contribute to oxidative stress production in the kidney. As measured by dihydroethidium with confocal microscopy, the LNHS protocol induced marked increases in superoxide production in WT mice, but not in EP3 -/- mice. To examine the direct effects of PGE 2 , splenic DCs were incubated with PGE 2 in vitro for 24 hours. PGE 2 dose-dependently increased isoketal-adduct formation in DCs (vehicle: 8.8±5.1% vs. 50 nM PGE 2 : 41.4±11.7%, p2 in DC activation in hypertension. In vivo, PGE 2 has a predominant effect on EP3 receptors to enhance renal vascular ROS production, which likely leads to isoketal-adduct formation and accumulation in DCs. PGE 2 also acts directly on DCs via its EP1 receptors to stimulate intracellular isoketal formation. Together, these findings provide additional information as to how PGE 2 modulates inflammation in hypertension.

Prostaglandin EP3 receptors in the central nervous system (CNS) may exert an excitatory effect on urinary bladder function via modulation of bladder afferent pathways. We have studied this action, using two EP3 antagonists, (2 E)-3-{1-[(2,4-dichlorophenyl)methyl]-5-fluoro-3-methyl-1 H-indol-7-yl}- N-[(4,5-dichloro-2-thienyl)sulfonyl]-2-propenamide (DG041) and (2 E)- N-{[5-bromo-2-(methyloxy)phenyl] sulfonyl}-3-[2-(2-naphthalenylmethyl)phenyl]-2-propenamide (CM9). DG041 and CM9 were proven to be selective EP3 antagonists with radioligand binding and functional fluorescent imaging plate reader (FLIPR) assays. Their effects on volume-induced rhythmic bladder contraction and the visceromotor reflex (VMR) response to urinary bladder distension (UBD) were evaluated in female rats after intrathecal or intracerebroventricular administration. Both DG041 and CM9 showed a high affinity for EP3 receptors at subnanomolar concentrations without significant selectivity for any splice variants. At the human EP3C receptor, both inhibited calcium influx produced by the nonselective agonist PGE2. After intrathecal or intracerebroventricular administration both CM9 and DG041 dose-dependently reduced the frequency, but not the amplitude, of the bladder rhythmic contraction. With intrathecal administration DG041 and CM9 produced a long-lasting and robust inhibition on the VMR response to UBD, whereas with intracerebroventricular injection both compounds elicited only a transient reduction of the VMR response to bladder distension. These data support the concept that EP3 receptors are involved in bladder micturition at supraspinal and spinal centers and in bladder nociception at the spinal cord. A centrally acting EP3 receptor antagonist may be useful in the control of detrusor overactivity and/or pain associated with bladder disorders.

A peptide constrained to a conformation of second extracellular loop of human prostaglandin-E(2) (PGE(2)) receptor subtype3 (hEP3) was synthesized. The contacts between the peptide residues at S211 and R214, and PGE(2) were first identified by NMR spectroscopy. The results were used as a guide for site-directed mutagenesis of the hEP3 protein. The S211L and R214L mutants expressed in HEK293 cells lost binding to [(3)H]PGE(2). This study found that the non-conserved S211 and R214 of the hEP3 are involved in PGE(2) recognition, and implied that the corresponding residues in other subtype receptors could be important to distinguish the different configurations of PGE(2) ligand recognition sites.

Background Activity of cyclooxygenase 2 (COX-2) in mouse oligodendrocyte precursor cells (OPCs) modulates vulnerability to excitotoxic challenge. The mechanism by which COX-2 renders OPCs more sensitive to excitotoxicity is not known. In the present study, we examined the hypothesis that OPC excitotoxic death is augmented by COX-2-generated prostaglandin E2 (PGE2) acting on specific prostanoid receptors which could contribute to OPC death. Methods Dispersed OPC cultures prepared from mice brains were examined for expression of PGE2 receptors and the ability to generate PGE2 following activation of glutamate receptors with kainic acid (KA). OPC death in cultures was induced by either KA, 3′-O-(Benzoyl) benzoyl ATP (BzATP) (which stimulates the purinergic receptor P2X7), or TNFα, and the effects of EP3 receptor agonists and antagonists on OPC viability were examined. Results Stimulation of OPC cultures with KA resulted in nearly a twofold increase in PGE2. OPCs expressed all four PGE receptors (EP1–EP4) as indicated by immunofluorescence and Western blot analyses; however, EP3 was the most abundantly expressed. The EP3 receptor was identified as a candidate contributing to OPC excitotoxic death based on pharmacological evidence. Treatment of OPCs with an EP1/EP3 agonist 17 phenyl-trinor PGE2 reversed protection from a COX-2 inhibitor while inhibition of EP3 receptor protected OPCs from excitotoxicity. Inhibition with an EP1 antagonist had no effect on OPC excitotoxic death. Moreover, inhibition of EP3 was protective against toxic stimulation with KA, BzATP, or TNFα. Conclusion Therefore, inhibitors of the EP3 receptor appear to enhance survival of OPCs following toxic challenge and may help facilitate remyelination.

Objectives. To carry out a descriptive analysis of the expression of the EP3 receptors of PGE2 in different histological grades of OSCC and adjacent normal epithelium. Material and Methods. A total of 46 patients presenting with various histological subtypes and grades of OSCC were recruited from Maxillofacial Surgery Department of Nishtar Institute of Dentistry Multan. Microscopically tumour subtyping and histological grading according to Anneroth’s grading system were carried out. Immunohistochemical staining with rabbit polyclonal EP3 receptor antibody was performed and sections were scored for intensity and proportion of positive adjacent squamous epithelial and tumour cells. Results. Out of 46 patients n=28 (60.9%) were well differentiated, n=15 (32.6%) were moderately differentiated, and only n=3 (6.5%) were poorly differentiated. All n=46 cases of OSCC were positive for EP3 receptor antibody, n=14 (30.4%) cases had strong intensity of anti EP3 antibody staining in tumour tissue, n=17 (37%) cases showed moderate intensity, and n=15 (32.6%) cases showed weak intensity. Conclusion. Prostanoid EP3 receptors are widely but variably expressed in OSCC. Most of well differentiated OSCC cases show a moderate to strong expression of EP3 receptors. However, insignificant statistical relation to histological grades of OSCC has been observed. This might be due to small sample size of the study.

Nonsteroidal antiinflammatories are known to suppress incidence and progression of malignancies including colorectal cancers. However, the precise mechanism of this action remains unknown. Using prostaglandin (PG) receptor knockout mice, we have evaluated a role of PGs in tumor-associated angiogenesis and tumor growth, and identified PG receptors involved. Sarcoma-180 cells implanted in wild-type (WT) mice formed a tumor with extensive angiogenesis, which was greatly suppressed by specific inhibitors for cyclooxygenase (COX)-2 but not for COX-1. Angiogenesis in sponge implantation model, which can mimic tumor-stromal angiogenesis, was markedly suppressed in mice lacking EP3 (EP3−/−) with reduced expression of vascular endothelial growth factor (VEGF) around the sponge implants. Further, implanted tumor growth (sarcoma-180, Lewis lung carcinoma) was markedly suppressed in EP3−/−, in which tumor-associated angiogenesis was also reduced. Immunohistochemical analysis revealed that major VEGF-expressing cells in the stroma were CD3/Mac-1 double-negative fibroblasts, and that VEGF-expression in the stroma was markedly reduced in EP3−/−, compared with WT. Application of an EP3 receptor antagonist inhibited tumor growth and angiogenesis in WT, but not in EP3−/−. These results demonstrate significance of host stromal PGE2-EP3 receptor signaling in tumor development and angiogenesis. An EP3 receptor antagonist may be a candidate of chemopreventive agents effective for malignant tumors.

We know that one of the main risk factors for cervical cancer is an infection with high-risk human papillomavirus (HR-HPV). Prostaglandins and their receptors are very important for the tumour growth and tumour-associated angiogenesis. Little is known about the expression of the Prostaglandin E receptor type 3 (EP3) or the Prostaglandin (PG)E2-EP3 signalling in cervical cancer, so the aim of the study was to analyse the expression of the EP3 receptor in cervical cancer and find prognostic factors in relation to survival; EP3 immunohistological staining of 250 cervical cancer slides was performed and analysed with a semi-quantitative score. The statistical evaluation was performed with Statistical Package for the Social Sciences (SPSS) to evaluate the staining results and the survival analyses of the cervical cancer cases. A significant difference was observed in EP3 expression in Fédération Internationale de Gynécologie et d’Obstétrique (FIGO) stadium I versus FIGO stadium II–IV cases. High expression of EP3 (IRS ≥ 1.5) in cervical cancer patients was correlated with poor prognosis in overall survival rates. Survival in adenocarcinoma (AC) of the cervix was lower than in squamous cell carcinoma (SCC). Cox regression analysis shows that EP3 is an independent prognosticator. In this study we could show that the membrane-bound prostaglandin receptor EP3 is an independent prognosticator for cervical cancer patient survival. Targeting the EP3 receptor seems to be an interesting candidate for endocrine therapy. Therefore, more research is needed on the influence of the receptor system and its influence on cervical cancer growth.

The reusable sulfoximine directing-group-assisted Ru(II)-catalyzed chemo- and regioselective ortho-C–H alkenylation of arenes and heteroarenes with acrylates and α,β-unsaturated ketones/vinyl sulfone is shown. The N-aroyl sulfoximine undergoes annulation with diphenylacetylene, delivering isoquinolinones and methyl phenyl sulfoxide. The present protocol is successfully employed for the synthesis of the EP3 receptor antagonist analogue.

We tested the hypothesis that inhibition of EP3receptors enhances cyclooxygenase (COX)-2 expression in the thick ascending limb (TAL) induced by hypertonic stimuli. COX-2 protein expression in the outer medulla increased approximately twofold in mice given free access to 1% NaCl in the drinking water for 3 days. The increase was associated with an approximate threefold elevation in COX-2 mRNA accumulation and an increase in PGE2production by isolated medullary (m)TAL tubules from 77.3 ± 8.4 to 165.7 ± 10.8 pg/mg protein. Moreover, administration of NS-398 abolished the increase in PGE2production induced by 1% NaCl. EP3receptor mRNA levels also increased approximately twofold in the outer medulla of mice that ingested 1% NaCl. The selective EP3receptor antagonist L-798106 increased COX-2 mRNA by twofold in mTAL tubules, and the elevation in COX-2 protein induced by 1% NaCl increased an additional 50% in mice given L-798106. COX-2 mRNA in primary mTAL cells increased twofold in response to media made hypertonic by the addition of NaCl (400 mosmol/kg H2O). L-798106 increased COX-2 mRNA twofold in isotonic media and fourfold in cells exposed to 400 mosmol/kg H2O. PGE2production by mTAL cells increased from 79.3 ± 4.6 to 286.7 ± 6.3 pg/mg protein after challenge with 400 mosmol/kg H2O and was inhibited in cells transiently transfected with a lentivirus short hairpin RNA construct targeting exon 5 of COX-2 to silence COX-2. Collectively, the data suggest that local hypertonicity in the mTAL is associated with an increase in COX-2 expression concomitant with elevated EP3receptor expression, which limits COX-2 activity in this segment of the nephron.

High-throughput screening and subsequent optimization led to the discovery of novel 3-oxazolidinedione-6-aryl-pyridinones exemplified by compound 2 as potent and selective EP3 antagonists with excellent pharmacokinetic properties. Compound 2 was orally active and showed robust in vivo activities in overactive bladder models. To address potential bioactivation liabilities of compound 2, further optimization resulted in compounds 9 and 10, which maintained excellent potency, selectivity, and pharmacokinetic properties and showed no bioactivation liability in glutathione trapping studies. These highly potent, selective, and orally active EP3 antagonists are excellent tool compounds for investigating and validating potential therapeutic benefits from selectively inhibiting the EP3 receptor.