Effects of selective cyclooxygenase-2 inhibitor and non-selective NSAIDs on Helicobacter pylori-induced gastritis in Mongolian gerbils

Background. It is still a point of controversy whether Helicobacter pylori‐infected patients are more likely to develop mucosal damage while taking NSADIs. Selective cyclooxygenase (COX‐2) inhibitors may be associated with less severe gastric mucosal damage than conventional NSAIDs, but this association is undefined in H. pylori‐induced gastritis. The aim of this study was to evaluate the effects of selective COX‐2 and nonselective NSAIDs on H. pylori‐induced gastritis. Methods. After intragastric administration of indomethacin, NS‐398 or vehicle alone, once daily for 5 days in H. pylori‐infected and uninfected Mongolian gerbils, we evaluated gastric mucosal damage, inflammatory cell infiltration and prostaglandin E₂ (PGE₂) concentration. We investigated whether H. pylori infection induced the COX‐2 expression. Results. In H. pylori‐uninfected groups, the indomethacin‐treated group showed the highest mucosal damage score and the lowest PGE₂ concentration. There was no difference in mucosal damage scores and PGE₂ concentration between NS‐398 and vehicle‐alone treated group. In H. pylori‐infected groups, there was no difference in mucosal damage scores, irrespective of the type of drugs administered. The indomethacin‐treated group showed the lowest PGE₂ concentration, similar to that of the NS‐398 and vehicle‐alone treated groups, both without H. pylori infection. Gastric neutrophil and monocyte infiltration scores were higher in H. pylori‐infected groups than in uninfected groups. However, there was no difference in these scores according to the type of drugs administered, within H. pylori‐infected or uninfected groups. COX‐2 protein expression was observed in H. pylori‐infected Mongolian gerbils but not in uninfected ones. Conclusions. Our animal study showed that H. pylori infection induced COX‐2 expression and increased prostaglandin concentration. Administration of NSAIDs decreased the prostaglandin concentration, but did not increase mucosal damage in H. pylori‐induced gastritis. Selective COX‐2 inhibitors, instead of conventional NSIADs, had no beneficial effect on preventing mucosal damage in H. pylori‐induced gastritis.     

Transforming Growth Factor β1 Regulates the Expression of Cyclooxygenase in Cultured Cortical Astrocytes and Neurons

Abstract: The hypothesis that transforming growth factor β1 (TGFβ1) regulates the synthesis of prostaglandins by CNS tissue was tested by using purified cultures of cortical astrocytes or neurons that were obtained from rat pups on postnatal day 4 or 5 or fetuses on gestational day 16, respectively. The cells were exposed to TGFβ1 for 2 days. The synthesis of prostaglandins depends upon the production and conversion of arachidonic acid, steps that are catalyzed by phospholipase A₂ (PLA₂) and cyclooxygenase (COX), respectively. Prostaglandin E₂ (PGE₂) concentration was determined by radioimmunoassay. The expression of cytosolic PLA₂ and COX (the constitutive COX1 and the inducible COX2) was assessed by using immunohistochemical and quantitative immunoblotting procedures. Astrocytes produced much more PGE₂ than neurons, suggesting that glial cells are an important source of PGE₂ in the CNS. TGFβ1 increased the production of PGE₂ by astrocytes and neurons in a concentration-dependent manner. Furthermore, TGFβ1 enhanced COX activity; the inhibitor indomethacin completely blocked TGFβ1-mediated PGE₂ synthesis. Cultured astrocytes and neurons expressed the three enzymes: cytosolic PLA₂, COX1, and COX2. Cytosolic PLA₂ expression was unaffected by TGFβ1 treatment. In contrast, COX expression was altered by TGFβ1 treatment in a concentration-dependent fashion. COX1 was increased by TGFβ1, but only in astrocytes. TGFβ1 increased COX2 expression in astrocytes and neurons. Thus, TGFβ1-induced increases in PGE₂ concentration are regulated by COX. This study suggests that TGFβ1 is an important regulator of immune and inflammatory processes in the CNS.     

Promotion of the prehierarchical follicle growth by postovulatory follicles involving PGE2–EP2 signaling in chickens

The postovulatory follicle (POF) in birds is an enigmatic structure, the function of which remains largely unknown. Previous studies on chickens have shown that removal of POFs leads to the postponement of oviposition and the disturbance of broody behavior. One suggestion is that POFs may secrete some crucial hormones or cytokines to act on reproductive organs. However, such secretions and their specific target organs remain to be identified. Here, we investigate the putative functions of POFs in promoting the development of prehierarchical follicles in chickens and explore the possible signaling mechanisms controlling these processes. Results show that POFs express steroidogenic acute regulatory protein (STAR), cholesterol side‐chain cleavage enzyme (CYP11A1), cyclooxygenase 1 (COX1), and COX2 in granulosa cells (GCs), and, most notably, that POF1 produces more prostaglandin E2 (PGE₂) or prostaglandin F2α than do the F1 follicle or the other POFs. Using coculture systems, we also found that POF1 or GCs from POF1 (POF1‐GCs) significantly promote the proliferation of theca externa cells of small white follicles (SWFs, one phase of the prehierarchical follicle). Treatment with PGE₂ significantly facilitates theca externa cell proliferation in SWFs. This POF‐stimulating effect on SWF growth was prevented by treatment with indomethacin (COX inhibitor) or TG6‐10‐1 (PGE₂ type 2 receptor [EP2] antagonist). Therefore, POF1 may secrete PGE₂ to stimulate the progression of SWF by PGE₂–EP2 signaling. These results indicate that POF1 may serve as a transient supplementary endocrine gland in the chicken ovary that stimulates the development of the prehierarchical follicles through PGE₂–EP2 signaling.     

Endosomal TLR3 signaling in stromal osteoblasts induces prostaglandin E2–mediated inflammatory periodontal bone resorption

Toll-like receptors (TLRs) are pattern recognition receptors that play a critical role in innate immune diseases. TLR3, which is localized in the endosomal compartments of hematopoietic immune cells, is able to recognize double-stranded RNA (dsRNA) derived from viruses and bacteria and thereby induce innate immune responses. Inflammatory periodontal bone resorption is caused by bacterial infections, which initially is regulated by innate immunity; however, the roles of TLR3 signaling in bone resorption are still not known. We examined the roles of TLR3 signaling in bone resorption using poly(I:C), a synthetic dsRNA analog. In cocultures of mouse bone marrow cells and stromal osteoblasts, poly(I:C) clearly induced osteoclast differentiation. In osteoblasts, poly(I:C) increased PGE₂ production and upregulated the mRNA expression of PGE₂-related genes, Ptgs2 and Ptges, as well as that of a gene related to osteoclast differentiation, Tnfsf11. In addition, we found that indomethacin (a COX-2 inhibitor) or an antagonist of the PGE₂ receptor EP4 attenuated the poly(I:C)-induced PGE₂ production and subsequent Tnfsf11 expression. Poly(I:C) also prolonged the survival of the mature osteoclasts associated with the increased mRNA expression of osteoclast marker genes, Nfatc1 and Ctsk. In ex vivo organ cultures of periodontal alveolar bone, poly(I:C) induced bone-resorbing activity in a dose-dependent manner, which was attenuated by the simultaneous administration of either indomethacin or an EP4 antagonist. These data suggest that TLR3 signaling in osteoblasts controls PGE₂ production and induces the subsequent differentiation and survival of mature osteoclasts. Endogenous TLR3 in stromal osteoblasts and osteoclasts synergistically induces inflammatory alveolar bone resorption in periodontitis.     

Regulatory T cells in colorectal cancer patients suppress anti-tumor immune activity in a COX-2 dependent manner

Objective Naturally occurring regulatory T (T_R) cells suppress autoreactive T cells whereas adaptive T_R cells, induced in the periphery, play an important role in chronic viral diseases and cancer. Several studies indicate that cyclooxygenase (COX) inhibitors prevent cancer development of colon adenomas and delay disease progression in patients with colorectal cancer (CRC). We have shown that adaptive T_R cells express COX-2 and produce PGE₂ that suppress effector T cells in a manner that is reversed by COX-inhibitors. Methods and results Here we demonstrate that CRC patients have elevated levels of PGE₂ in peripheral blood, and CRC tissue samples and draining lymph nodes display increased numbers of FOXP3+ T_R cells. Depletion of T_R cells from PBMC enhanced anti-tumor T-cell responses to peptides from carcinoembryonic antigen. Furthermore, the COX inhibitor indomethacin and the PKA type I antagonist Rp-8-Br-cAMPS significantly improved the anti-tumor immune activity. Conclusion We suggest that adaptive T_R cells contribute to an immunosuppressive microenvironment in CRC and inhibit effector T cells by a COX-2–PGE₂-dependent mechanism and thereby facilitate tumor growth. Therapeutic strategies targeting T_R cells and the PGE₂–cAMP pathway may be interesting to pursue to enhance anti-tumor immune activity in CRC patients.     

Distinct Innate Immune Phagocyte Responses to Aspergillus fumigatus Conidia and Hyphae in Zebrafish Larvae

Aspergillus fumigatus is the most common filamentous fungal pathogen of immunocompromised hosts, resulting in invasive aspergillosis (IA) and high mortality rates. Innate immunity is known to be the predominant host defense against A. fumigatus; however, innate phagocyte responses to A. fumigatus in an intact host and their contributions to host survival remain unclear. Here, we describe a larval zebrafish A. fumigatus infection model amenable to real-time imaging of host-fungal interactions in live animals. Following infection with A. fumigatus, innate phagocyte populations exhibit clear preferences for different fungal morphologies: macrophages rapidly phagocytose conidia and form aggregates around hyphae, while the neutrophil response is dependent upon the presence of hyphae. Depletion of macrophages rendered host larvae susceptible to invasive disease. Moreover, a zebrafish model of human leukocyte adhesion deficiency with impaired neutrophil function also resulted in invasive disease and impaired host survival. In contrast, macrophage-deficient but not neutrophil-deficient larvae exhibited attenuated disease following challenge with a less virulent (ΔlaeA) strain of A. fumigatus, which has defects in secondary metabolite production. Taking these results together, we have established a new vertebrate model for studying innate immune responses to A. fumigatus that reveals distinct roles for neutrophils and macrophages in mediating host defense against IA.     

Short-term therapy with celecoxib and lansoprazole modulates Th1/ Th2 immune response in human gastric mucosa

Background: Selective cyclooxygenase‐2 (COX‐2) inhibitors and proton pump inhibitors may exert immune‐mediated effects in human gastric mucosa. T‐cell immune response plays a role in Helicobacter pylori‐induced pathogenesis. This study evaluated effects of celecoxib and lansoprazole on T‐helper (Th) 1 and Th2 immune response in human gastric mucosa. Methods: Dyspeptic patients with or without osteoarticular pain were given one of the following 4‐week therapies: celecoxib 200 mg, celecoxib 200 mg plus lansoprazole 30 mg, and lansoprazole 30 mg daily. Expression of COX‐2, T‐bet, and pSTAT6 and production of prostaglandin E₂ (PGE₂), interferon (IFN)‐γ, and interleukin (IL)‐4 were determined in gastric biopsies before and after therapy. Histology was evaluated. Results: Cyclooxygenase‐2 expression and PGE₂ production was higher, and Th1 signaling pathway was predominant in H. pylori‐infected vs. uninfected patients. T‐bet expression and IFN‐γ production increased, while STAT6 activation and IL‐4 production decreased following therapy with celecoxib and celecoxib plus lansoprazole, respectively. Th1 and Th2 signaling pathways down‐regulated after therapy with lansoprazole, and this was associated with an improvement of gastritis. Effect of therapy was not affected by H. pylori status. Conclusion: Celecoxib and lansoprazole modulate Th1/Th2 immune response in human gastric mucosa. The use of these drugs may interfere with long‐term course of gastritis.     

Prostaglandin E<Subscript>2 </Subscript>production by high and low virulent strains of <Emphasis Type="Italic">Paracoccidioides brasiliensis</Emphasis>

The production of prostaglandins (PGs) during fungal infections could be an important suppressor factor of host immune response. Host cells are one source of prostaglandin E₂ (PGE₂); however another potential source of PGE₂ is the fungal pathogen itself. Thus, both host and fungal PGE2 production is theorized to play a role in pathogenesis, being critical for growth of the fungus and to modulate the host immune response. The purpose of this work was to investigate if high and low virulent strains of Paracoccidioides brasiliensis have the capacity to produce PGE₂ in vitro, and if this production was related to the fungal growth. The results demonstrated that both strains of P. brasiliensis produce high levels of PGE₂ and the treatment with indomethacin, a cyclooxygenase inhibitor, significantly reduced the production of this mediator, as well as the viability of the fungus. Thus, our data indicate that PGE₂ is produced by P. brasiliensis by a cyclooxygenase–dependent metabolic pathway, and its production is required for fungal survival. This discovery reveals an important factor that has potentially great implications for understanding the mechanisms of immune deviation during infection.     

UCP1 Induction during Recruitment of Brown Adipocytes in White Adipose Tissue Is Dependent on Cyclooxygenase Activity

Background

The uncoupling protein 1 (UCP1) is a hallmark of brown adipocytes and pivotal for cold- and diet-induced thermogenesis.

Methodology/Principal Findings

Here we report that cyclooxygenase (COX) activity and prostaglandin E₂ (PGE₂) are crucially involved in induction of UCP1 expression in inguinal white adipocytes, but not in classic interscapular brown adipocytes. Cold-induced expression of UCP1 in inguinal white adipocytes was repressed in COX2 knockout (KO) mice and by administration of the COX inhibitor indomethacin in wild-type mice. Indomethacin repressed β-adrenergic induction of UCP1 expression in primary inguinal adipocytes. The use of PGE₂ receptor antagonists implicated EP₄ as a main PGE₂ receptor, and injection of the stable PGE₂ analog (EP_3/4 agonist) 16,16 dm PGE₂ induced UCP1 expression in inguinal white adipose tissue. Inhibition of COX activity attenuated diet-induced UCP1 expression and increased energy efficiency and adipose tissue mass in obesity-resistant mice kept at thermoneutrality.

Conclusions/Significance

Our findings provide evidence that induction of UCP1 expression in white adipose tissue, but not in classic interscapular brown adipose tissue is dependent on cyclooxygenase activity. Our results indicate that cyclooxygenase-dependent induction of UCP1 expression in white adipose tissues is important for diet-induced thermogenesis providing support for a surprising role of COX activity in the control of energy balance and obesity development.     

Cyclooxygenase-2 and Prostaglandin E2 Signaling through Prostaglandin Receptor EP-2 Favor the Development of Myocarditis during Acute Trypanosoma cruzi Infection

Inflammation plays an important role in the pathophysiology of Chagas disease, caused by Trypanosoma cruzi. Prostanoids are regulators of homeostasis and inflammation and are produced mainly by myeloid cells, being cyclooxygenases, COX-1 and COX-2, the key enzymes in their biosynthesis from arachidonic acid (AA). Here, we have investigated the expression of enzymes involved in AA metabolism during T. cruzi infection. Our results show an increase in the expression of several of these enzymes in acute T. cruzi infected heart. Interestingly, COX-2 was expressed by CD68⁺ myeloid heart-infiltrating cells. In addition, infiltrating myeloid CD11b⁺Ly6G^- cells purified from infected heart tissue express COX-2 and produce prostaglandin E₂ (PGE₂) ex vivo. T. cruzi infections in COX-2 or PGE₂-dependent prostaglandin receptor EP-2 deficient mice indicate that both, COX-2 and EP-2 signaling contribute significantly to the heart leukocyte infiltration and to the release of chemokines and inflammatory cytokines in the heart of T. cruzi infected mice. In conclusion, COX-2 plays a detrimental role in acute Chagas disease myocarditis and points to COX-2 as a potential target for immune intervention.     

Hypoxia enhances innate immune activation to Aspergillus fumigatus through cell wall modulation

Infection by the human fungal pathogen Aspergillus fumigatus induces hypoxic microenvironments within the lung that can alter the course of fungal pathogenesis. How hypoxic microenvironments shape the composition and immune activating potential of the fungal cell wall remains undefined. Herein we demonstrate that hypoxic conditions increase the hyphal cell wall thickness and alter its composition particularly by augmenting total and surface-exposed β-glucan content. In addition, hypoxia-induced cell wall alterations increase macrophage and neutrophil responsiveness and antifungal activity as judged by inflammatory cytokine production and ability to induce hyphal damage. We observe that these effects are largely dependent on the mammalian β-glucan receptor dectin-1. In a corticosteroid model of invasive pulmonary aspergillosis, A. fumigatus β-glucan exposure correlates with the presence of hypoxia in situ. Our data suggest that hypoxia-induced fungal cell wall changes influence the activation of innate effector cells at sites of hyphal tissue invasion, which has potential implications for therapeutic outcomes of invasive pulmonary aspergillosis.     

Induction of autophagy correlates with increased parasite load of Leishmania amazonensis in BALB/c but not C57BL/6 macrophages

We investigated the role of autophagy in infection of macrophages by Leishmania amazonensis. Induction of autophagy by IFN-γ or starvation increased intracellular parasite load and the percentages of infected macrophages from BALB/c but not from C57BL/6 mice. In contrast, starvation did not affect the replication of either Leishmania major or Trypanosoma cruzi in BALB/c macrophages. In BALB/c macrophages, starvation resulted in increased monodansylcadaverine staining and in the appearance of double-membrane and myelin-like vesicles characteristic of autophagosomes. Increased parasite load was associated with a reduction in NO levels and was attenuated by wortmannin, an inhibitor of autophagy. In infected macrophages from BALB/c, but not from C57BL/6 mice, starvation increased the number of lipid bodies and the amounts of PGE₂ produced. Exogenous PGE₂ increased parasite load in macrophages from BALB/c, but not C57BL/6 mice. The cyclooxygenase inhibitor indomethacin prevented the increase of parasite load in starved BALB/c macrophages, and actually induced parasite killing. These results suggest that autophagy regulates the outcome of L. amazonensis infection in macrophages in a host strain specific manner.     

Effect of chlorambucil and indomethacin on growth and prostaglandin levels in sensitive and resistant walker carcinoma

An analysis of the effect of combinations of chlorambucil and indomethacin, or chlorambucil and prostaglandin E₂ (PGE₂) on the growth of alkylating agent sensitive and resistant Walker carcinoma in vitro has been made by the isobologram approach. Indomethacin alone acts as a growth inhibitor of the Walker carcinoma. High concentrations of indomethacin (5 μg/ml) act to inhibit the growth of the resistant line sub-additively with chlorambucil, whereas low concentrations act additively. For the sensitive line indomethacin acts either additively or supra-additively with chlorambucil at all concentrations employed. Both indomethacin and low concentrations of chlorambucil alone inhibit PGE₂ secretion into the culture medium of both cell lines and an enhanced inhibition is seen with the combination. PGE₂ itself acts as a growth inhibitor of both cell lines, although it causes greater growth inhibition of chlorambucil resistant Walker carcinoma (LD₅₀ 1.8 μg/ml) than of the sensitive line. This correlates with a greater PGE₂ secretion capacity by the resistant cell line (40 pg PGE₂/ml medium/10⁵ cells for the resistant tumour and 17 pg PGE₂/ml medium/10⁵ cells for the sensitive tumour). Combinations of PGE₂ with chlorambucil inhibit growth either additively or sub-additively. It seems unlikely that inhibition of PGE₂ secretion is responsible for the interactive effects of chlorambucil and indomethacin, since growth inhibition produced by the combination is not reversed by PGE₂ at any of the concentrations employed. Possible mechanisms of the interactive effects are discussed.     

Prostaglandin E2 at new glance: Novel insights in functional diversity offer therapeutic chances

Prostaglandin E₂ (PGE₂) is the most abundant eicosanoid and a very potent lipid mediator. PGE₂ is produced predominantly from arachidonic acid by its tightly regulated cyclooxygenases (COX) and prostaglandin E synthases (PGES). Secreted PGE₂ acts in an autocrine or paracrine manner through its four cognate G protein coupled receptors EP1 to EP4. Under physiological conditions, PGE₂ is key in many biological functions, such as regulation of immune responses, blood pressure, gastrointestinal integrity, and fertility. Deregulated PGE₂ synthesis or degradation is associated with severe pathological conditions like chronic inflammation, Alzheimer's disease, or tumorigenesis. Therefore, pharmacological inhibition of COX enzymes and PGE₂ receptor antagonism is of great therapeutic interest.     

Impaired UTP-induced relaxation in the carotid arteries of spontaneously hypertensive rats

Uridine 5′-triphosphate (UTP) has an important role as an extracellular signaling molecule that regulates inflammation, angiogenesis, and vascular tone. While chronic hypertension has been shown to promote alterations in arterial vascular tone regulation, carotid artery responses to UTP under hypertensive conditions have remained unclear. The present study investigated carotid artery responses to UTP in spontaneously hypertensive rats (SHR) and control Wistar Kyoto rats (WKY). Accordingly, our results found that although UTP promotes concentration-dependent relaxation in isolated carotid artery segments from both SHR and WKY after pretreatment with phenylephrine, SHR exhibited significantly lower arterial relaxation responses compared with WKY. Moreover, UTP-induced relaxation was substantially reduced by endothelial denudation and by the nitric oxide (NO) synthase inhibitor N^G-nitro-L-arginine in both SHR and WKY. The difference in UTP-induced relaxation between both groups was abolished by the selective P2Y₂ receptor antagonist AR-C118925XX and the cyclooxygenase (COX) inhibitor indomethacin but not by the thromboxane-prostanoid receptor antagonist SQ29548. Furthermore, we detected the release of PGE₂, PGF_2α, and PGI₂ in the carotid arteries of SHR and WKY, both at baseline and in response to UTP. UTP administration also increased TXA₂ levels in WKY but not SHR. Overall, our results suggest that UTP-induced relaxation in carotid arteries is impaired in SHR perhaps due to impaired P2Y₂ receptor signaling, reductions in endothelial NO, and increases in the levels of COX-derived vasoconstrictor prostanoids.

     

Retinoic acid increases hypoxia-inducible factor-1α through intracrine prostaglandin E2 signaling in human renal proximal tubular cells HK-2

We have previously shown in HK-2 cells that ATRA (all-trans-retinoic acid) up-regulates HIF-1α (hypoxia-inducible factor-1α) in normoxia, which results in increased production of renal protector VEGF-A (vascular endothelial growth factor-A). Here we investigated the role of COXs (cyclooxygenases) in these effects and we found that, i) ATRA increased the expression of COX-1 and COX-2 mRNA and protein and the intracellular levels (but not the extracellular ones) of PGE₂. Furthermore, inhibitors of COX isoenzymes blocked ATRA-induced increase in intracellular PGE₂, HIF-1α up-regulation and increased VEGF-A production. Immunofluorescence analysis found intracellular staining for EP1-4 receptors (PGE₂ receptors). These results indicated that COX activity is critical for ATRA-induced HIF-1α up-regulation and suggested that intracellular PGE₂ could mediate the effects of ATRA; ii) Treatment with PGE₂ analog 16,16-dimethyl-PGE₂ resulted in up-regulation of HIF-1α and antagonists of EP1-4 receptors inhibited 16,16-dimethyl-PGE₂- and ATRA-induced HIF-1α up-regulation. These results confirmed that PGE₂ mediates the effects of ATRA on HIF-1α expression; iii) Prostaglandin uptake transporter inhibitor bromocresol green blocked the increase in HIF-1α expression induced by PGE₂ or by PGE₂-increasing cytokine interleukin-1β, but not by ATRA. Therefore only intracellular PGE₂ is able to increase HIF-1α expression. In conclusion, intracellular PGE₂ increases HIF-1α expression and mediates ATRA-induced HIF-1α up-regulation.     

Role of contractile prostaglandins and Rho-kinase in growth factor-induced airway smooth muscle contraction

Background

In addition to their proliferative and differentiating effects, several growth factors are capable of inducing a sustained airway smooth muscle (ASM) contraction. These contractile effects were previously found to be dependent on Rho-kinase and have also been associated with the production of eicosanoids. However, the precise mechanisms underlying growth factor-induced contraction are still unknown. In this study we investigated the role of contractile prostaglandins and Rho-kinase in growth factor-induced ASM contraction.

Methods

Growth factor-induced contractions of guinea pig open-ring tracheal preparations were studied by isometric tension measurements. The contribution of Rho-kinase, mitogen-activated protein kinase (MAPK) and cyclooxygenase (COX) to these reponses was established, using the inhibitors Y-27632 (1 μM), U-0126 (3 μM) and indomethacin (3 μM), respectively. The Rho-kinase dependency of contractions induced by exogenously applied prostaglandin F_2α (PGF_2α) and prostaglandin E₂ (PGE₂) was also studied. In addition, the effects of the selective FP-receptor antagonist AL-8810 (10 μM) and the selective EP₁-antagonist AH-6809 (10 μM) on growth factor-induced contractions were investigated, both in intact and epithelium-denuded preparations. Growth factor-induced PGF_2α-and PGE₂-release in the absence and presence of Y-27632, U-0126 and indomethacin, was assessed by an ELISA-assay.

Results

Epidermal growth factor (EGF)-and platelet-derived growth factor (PDGF)-induced contractions of guinea pig tracheal smooth muscle preparations were dependent on Rho-kinase, MAPK and COX. Interestingly, growth factor-induced PGF_2α-and PGE₂-release from tracheal rings was significantly reduced by U-0126 and indomethacin, but not by Y-27632. Also, PGF_2α-and PGE₂-induced ASM contractions were largely dependent on Rho-kinase, in contrast to other contractile agonists like histamine. The FP-receptor antagonist AL-8810 (10 μM) significantly reduced (approximately 50 %) and the EP₁-antagonist AH-6809 (10 μM) abrogated growth factor-induced contractions, similarly in intact and epithelium-denuded preparations.

Conclusion

The results indicate that growth factors induce ASM contraction through contractile prostaglandins – not derived from the epithelium – which in turn rely on Rho-kinase for their contractile effects.     

Pharmacological inhibition of microsomal prostaglandin E synthase-1 suppresses epidermal growth factor receptor-mediated tumor growth and angiogenesis

Background

Blockade of Prostaglandin (PG) E₂ production via deletion of microsomal Prostaglandin E synthase-1 (mPGES-1) gene reduces tumor cell proliferation in vitro and in vivo on xenograft tumors. So far the therapeutic potential of the pharmacological inhibition of mPGES-1 has not been elucidated. PGE₂ promotes epithelial tumor progression via multiple signaling pathways including the epidermal growth factor receptor (EGFR) signaling pathway.

Methodology/Principal Findings

Here we evaluated the antitumor activity of AF3485, a compound of a novel family of human mPGES-1 inhibitors, in vitro and in vivo, in mice bearing human A431 xenografts overexpressing EGFR. Treatment of the human cell line A431 with interleukin-1beta (IL-1β) increased mPGES-1 expression, PGE₂ production and induced EGFR phosphorylation, and vascular endothelial growth factor (VEGF) and fibroblast growth factor-2 (FGF-2) expression. AF3485 reduced PGE₂ production, both in quiescent and in cells stimulated by IL-1β. AF3485 abolished IL-1β-induced activation of the EGFR, decreasing VEGF and FGF-2 expression, and tumor-mediated endothelial tube formation. In vivo, in A431 xenograft, AF3485, administered sub-chronically, decreased tumor growth, an effect related to inhibition of EGFR signalling, and to tumor microvessel rarefaction. In fact, we observed a decrease of EGFR phosphorylation, and VEGF and FGF-2 expression in tumours explanted from treated mice.

Conclusion

Our work demonstrates that the pharmacological inhibition of mPGES-1 reduces squamous carcinoma growth by suppressing PGE₂ mediated-EGFR signalling and by impairing tumor associated angiogenesis. These results underscore the potential of mPGES-1 inhibitors as agents capable of controlling tumor growth.