Involvement of cyclooxygenase-1, prostaglandin E2 and EP1 receptors in acid-induced HCO3- secretion in stomach.

We investigated the cyclooxygenase (COX) isoforms as well as prostaglandin E receptor EP subtypes responsible for acid-induced gastric HCO(3)(-) secretion in rats and EP receptor-knockout (-/-) mice. Under urethane anesthesia, a chambered stomach (in the presence of omeprazole) was perfused with saline, and HCO(3)(-) secretion was measured at pH 7.0 using a pH-stat method and by adding 2 mM HCl. Mucosal acidification was achieved by exposing the stomach for 10 min to 50 or 100 mM HCl. Acidification of the mucosa increased the secretion of HCO(3)(-) in the stomach of both rats and WT mice, in an indomethacin-inhibitable manner. The acid-induced gastric HCO(3)(-) secretion was inhibited by prior administration of indomethacin and SC-560 but not rofecoxib in rats and mice. Acidification increased the PGE(2) content of the rat stomach, and this response was significantly attenuated by indomethacin and SC-560 but not rofecoxib. This response was also attenuated by ONO-8711 (EP1 antagonist) but not AE3-208 (EP4 antagonist) in rats and disappeared in EP1 (-/-) but not EP3 (-/-) mice. PGE(2) increased gastric HCO(3)(-) secretion in both rats and WT mice, and this action was inhibited by ONO-8711 and disappeared in EP1 (-/-) but not EP3 (-/-) mice. These results support a mediator role for endogenous PGs in the gastric response induced by mucosal acidification and clearly indicate that the enzyme responsible for production of PGs in this process is COX-1. They further show that the presence of EP1 receptors is essential for the increase in the secretion of HCO(3)(-) in response to mucosal acidification in the stomach.     

Role of the different isoforms of cyclooxygenase and nitric oxide synthase during gastric ulcer healing in cyclooxygenase-1 and -2 knockout mice

Traditional NSAIDs, selective cyclooxygenase (COX)-2 inhibitors, and inhibitors of nitric oxide synthase (NOS) impair the healing of preexisting gastric ulcers. However, the role of COX-1 (with or without impairment of COX-2) and the interaction between COX and NOS isoforms during healing are less clear. Thus we investigated healing and regulation of COX and NOS isoforms during ulcer healing in COX-1 and COX-2 deficiency and inhibition mouse models. In this study, female wild-type COX-1(-/-) and COX-2(-/-) mice with gastric ulcers induced by cryoprobe were treated intragastrically with vehicle, selective COX-1 (SC-560), COX-2 (celecoxib, rofecoxib, and valdedoxib), and unselective COX (piroxicam) inhibitors. Ulcer healing parameters, mRNA expression, and activity of COX and NOS were quantified. Gene disruption or inhibition of COX-1 did not impair ulcer healing. In contrast, COX-2 gene disruption and COX-2 inhibitors moderately impaired wound healing. More severe healing impairment was found in dual (SC-560 + rofecoxib) and unselective (piroxicam) COX inhibition and combined COX impairment (in COX-1(-/-) mice with COX-2 inhibition and COX-2(-/-) mice with COX-1 inhibition). In the ulcerated repair tissue, COX-2 mRNA in COX-1(-/-) mice, COX-1 mRNA in COX-2(-/-) mice, and, remarkably, NOS-2 and NOS-3 mRNA in COX-impaired mice were more upregulated than in wild-type mice. This study demonstrates that COX-2 is a key mediator in gastric wound healing. In contrast, COX-1 has no significant role in healing when COX-2 is unimpaired but becomes important when COX-2 is impaired. As counterregulatory mechanisms, mRNA of COX and NOS isoforms were increased during healing in COX-impaired mice.     

Inhibition of both COX-1 and COX-2 is required for development of gastric damage in response to nonsteroidal antiinflammatory drugs.

We examined the gastric ulcerogenic property of selective COX-1 and/or COX-2 inhibitors in rats, and investigated whether COX-1 inhibition is by itself sufficient for induction of gastric damage. Animals fasted for 18 h were given various COX inhibitors p.o., either alone or in combination, and they were killed 8 h later. The nonselective COX inhibitors such as indomethacin, naproxen and dicrofenac inhibited PG production, increased gastric motility, and provoked severe gastric lesions. In contrast, the selective COX-2 inhibitor rofecoxib did not induce any damage in the stomach, with no effect on the mucosal PGE(2) contents and gastric motility. Likewise, the selective COX-1 inhibitor SC-560 also did not cause gastric damage, despite causing a significant decrease in PGE(2) contents. The combined administration of SC-560 and rofecoxib, however, provoked gross damage in the gastric mucosa, in a dose-dependent manner. SC-560 also caused a marked gastric hypermotility, whereas rofecoxib had no effect on basal gastric motor activity. On the other hand, the COX-2 mRNA was expressed in the stomach after administration of SC-560, while the normal gastric mucosa expressed only COX-1 mRNA but not COX-2 mRNA. These results suggest that the gastric ulcerogenic property of conventional NSAIDs is not accounted for solely by COX-1 inhibition and requires the inhibition of both COX-1 and COX-2. The inhibition of COX-1 up-regulates the COX-2 expression, and this may counteract the deleterious influences, such as gastric hypermotility and the subsequent events, due to a PG deficiency caused by COX-1 inhibition.     

Temporal expression of the PGE2 synthetic system in the kidney is associated with the time frame of renal developmental vulnerability to cyclooxygenase-2 inhibition

Pharmacological blockade of cyclooxygenase-2 (COX-2) causes impairment of kidney development. The present study was aimed at determining temporal expression pattern and activity of the PGE(2) synthetic pathway during postnatal nephrogenesis in mice and its association to the time window sensitive to COX-2 inhibition. During the first 10 days after birth, we observed transient induction of mRNA and protein for microsomal PGE synthase (mPGES)-1 between postnatal days 4 (P4) and P8, but not for mPGES-2 or cytosolic PGE synthase (cPGES). PGE(2) synthetic activity using arachidonic acid and PGH(2) as substrates and also urinary excretion of PGE(2) were enhanced during this time frame. In parallel to the PGE(2) system, COX-2 but not COX-1 expression was also transiently induced. Studying glomerulogenesis in EP receptor knockout mice revealed a reduction in glomerular size in EP1(-/-), EP2(-/-), and EP4(-/-) mice, supporting the developmental role of PGE(2). The most vulnerable time window to COX-2 inhibition by SC-236 was found closely related to the temporal expression of COX-2 and mPGES-1. The strongest effects of COX-2 inhibition were achieved following 8 days of drug administration. Similar developmental damage was caused by application of rofecoxib, but not by the COX-1-selective inhibitor SC-560. COX-2 inhibition starting after P10 has had no effect on the size of glomeruli or on the relative number of superficial glomeruli; however, growth of the renal cortex was significantly diminished, indicating the requirement of COX-2 activity after P10. Effects of COX-2 inhibition on renal cell differentiation and on renal fibrosis needed a prolonged time of exposition of at least 10 days. In conclusion, temporal expression of the PGE(2) synthetic system coincides with the most vulnerable age interval for the induction of irreversible renal abnormalities. We assume that mPGES-1 is coregulated with COX-2 for PGE(2) synthesis to orchestrate postnatal kidney development and growth.     

Anti-inflammatory effect of two isoforms of COX in H. pylori-induced gastritis in mice: possible involvement of PGE2

Neutrophil infiltration mediated by TNF-alpha is associated with various types of gastric injury, whereas PGs play a crucial role in gastric defense. We examined roles of two isoforms of cyclooxygenase (COX) and PGE2 in Helicobacter pylori-induced gastritis in mice. Mice infected with H. pylori were given selective COX-1 inhibitor SC-560 (10 mg/kg), selective COX-2 inhibitor NS-398 (10 mg/kg), or nonselective COX inhibitor indomethacin (2 mg/kg) with or without 16,16-dimethyl PGE2 for 1 wk. H. pylori infection increased levels of mRNA for COX-1 and -2 in gastric tissue by 1.2-fold and 3.3-fold, respectively, accompanied by a significant increase in PGE2 production by gastric tissue. H. pylori infection significantly elevated MPO activity, a marker of neutrophil infiltration, and epithelial cell apoptosis in the stomach. SC-560 augmented MPO activity and epithelial cell apoptosis with associated reduction in PGE2 production, whereas NS-398 had the same effects without affecting PGE2 production. Inhibition of both COX-1 and -2 by indomethacin or concurrent treatment with SC-560 and NS-398 resulted in a stronger increase in MPO activity and apoptosis than inhibition of either COX-1 or -2 alone. H. pylori infection elevated TNF-alpha mRNA expression in the stomach, which was further increased by indomethacin. Effects of COX inhibitors on neutrophil infiltration, apoptosis, and TNF-alpha expression in H. pylori-infected mice were abolished by exogenous 16,16-dimethyl PGE2. In conclusion, PGE2 derived from either COX-1 or -2 is involved in regulation of gastric mucosal inflammation and contributes to maintenance of mucosal integrity during H. pylori infection via inhibition of TNF-alpha expression.     

Cyclooxygenase-2 selective and nitric oxide-releasing nonsteroidal anti-inflammatory drugs and gastric mucosal responses.

Occurrence of gastrointestinal damage and delayed healing of pre-existing ulcer are commonly observed in association with clinical use of nonsteroidal antiinflammatory drugs (NSAIDs). We examined the effects of NS-398, the cyclooxygenase (COX)-2 selective inhibitor, and nitric oxide (NO)- releasing aspirin (NCX-4016) on gastric mucosal ulcerogenic and healing responses in experimental animals, in comparison with those of nonselective COX inhibitors such as indomethacin and aspirin. Indomethacin and aspirin given orally were ulcerogenic by themselves in rat stomachs, while either NS-398 or NCX-4016 was not ulcerogenic at the doses which exert the equipotent antiinflammatory action with indomethacin or aspirin. Among these NSAIDs, only NCX-4016 showed a dose-dependent protection against gastric lesions induced by HCl/ethanol in rats. On the other hand, the healing of gastric ulcers induced in mice by thermal-cauterization was significantly delayed by repeated administration of these NSAIDs for more than 7 days, except NCX-4016. Gastric mucosal prostaglandin contents were reduced by indomethacin, aspirin and NCX-4016 in both normal and ulcerated mucosa, while NS-398 significantly decreased prostaglandin generation only in the ulcerated mucosa. Oral administration of NCX-4016 in pylorus-ligated rats and mice increased the levels of NO metabolites in the gastric contents. In addition, both NS-398 and NCX-4016 showed an equipotent anti-inflammatory effect against carrageenan-induced paw edema in rats as compared with indomethacin and aspirin. These results suggest that both indomethacin and aspirin are ulcerogenic by themselves and impair the healing of pre-existing gastric ulcers as well. The former action is due to inhibition of COX-1, while the latter effect may be accounted for by inhibition of COX-2 and mimicked by NS-398, the COX-2 selective NSAID. NCX-4016, despite inhibiting both COX-1 and COX-2, protects the stomach against damage and preserves the healing response of gastric ulcers, probably because of the beneficial action of NO.     

Involvement of prostaglandin E receptor EP3 subtype and prostacyclin IP receptor in decreased acid response in damaged stomach.

We investigated the roles of cyclooxygenase (COX) isozymes and prostaglandin E (PGE) receptor EP1 and EP3 subtypes or prostacyclin IP receptors in the decrease in acid secretion in the damaged mouse stomach. Male C57/BL6 mice, both wild type and animals lacking EP1, EP3, or IP receptors, were used after 18 h of fasting. Under urethane anesthesia, the stomach was mounted on an ex-vivo chamber and perfused with saline, and acid secretion as well as transmucosal potential difference (PD) was measured before and after exposure to 20 mM taurocholate Na (TC) for 20 min. Indomethacin, SC-560 or rofecoxib was given i.d. 30 min before TC. Mucosal exposure to TC in wild-type mice caused a reduction in PD, followed by decrease in acid secretion. Indomethacin attenuated the decrease in acid secretion after exposure to TC in wild-type mice, an effect mimicked by SC-560 but not rofecoxib, yet none of these drugs affected the decrease in PD. An altered acid response after exposure to TC was similarly observed in EP1 (-/-) mice but mitigated in mice lacking either EP3 or IP receptors, although a decrease in PD was observed in all groups. Furthermore, the decreased acid response was also attenuated by prior administration of the EP3- but not EP1- antagonist. Mucosal levels of PGE(2) and 6-keto PGF(1a) increased after exposure to TC in all groups of mice. In conclusion, the decrease in acid secretion in the damaged stomach is mediated by endogenous PGs derived from COX-1, through PGE(2)/EP3 receptors and prostacyclin/IP receptors.     

ChTX induces oscillatory contraction in guinea pig trachea: role of cyclooxygenase-2 and PGE2

We examined the possible role of cyclooxygenase (COX) in charybdotoxin (ChTX)-induced oscillatory contraction in guinea pig trachea. Involvement of prostaglandin E(2) (PGE(2)) in ChTX-induced oscillatory contraction was also investigated. ChTX (100 nM) induced oscillatory contraction in guinea pig trachea. The mean oscillatory frequency induced by ChTX was 10.7 +/- 0.8 counts/h. Maximum and minimum tensions within ChTX-induced oscillatory contractions were 68.4 +/- 1.8 and 14.3 +/- 1.7% compared with K(+) (72.7 mM) contractions. ChTX-induced oscillatory contraction was completely inhibited by indomethacin, a nonselective COX inhibitor. Valeryl salicylate, a selective COX-1 inhibitor, did not significantly inhibit this contraction, whereas N-(2-cyclohexyloxy-4-nitro-phenyl)-methanesulfonamide, a selective COX-2 inhibitor, abolished this contraction. Exogenously applied arachidonic acid enhanced ChTX-induced oscillatory contraction. SC-51322, a selective PGE receptor subtype EP(1) antagonist, significantly inhibited ChTX-induced oscillatory contraction. Exogenously applied PGE(2) induced only a slight phasic contraction in guinea pig trachea, but PGE(2) induced strong oscillatory contraction after pretreatment with indomethacin and ChTX. Moreover, ChTX time-dependently stimulated PGE(2) generation. These results suggest that ChTX specifically activates COX-2 and stimulates PGE(2) production and that ChTX-induced oscillatory contraction in guinea pig trachea is mediated by activation of EP(1) receptor.     

Cyclooxygenase-2-regulated vascular endothelial growth factor release in gastric fibroblasts

VEGF is a highly specific stimulator of endothelial cells and may play an important role in angiogenesis in the process of tissue regeneration. We previously showed that cyclooxygenase-2 (COX-2) expressed in mesenchymal cells of the ulcer bed is involved in the ulcer repair process. To clarify the role of COX-2 in angiogenesis during gastric ulcer healing, we investigated the relation between COX-2 expression and VEGF production in human gastric fibroblasts in vivo and in vitro. Gastric fibroblasts were cultured in RPMI 1640 with and without IL-1alpha or IL-1beta in the presence or absence of NS-398, a selective COX-2 inhibitor. Supernatant VEGF and PGE(2) concentrations were measured by enzyme-linked immunosorbent assay. COX-2 expression in fibroblasts was determined by Western blot analysis. VEGF and COX-2 expression in surgical resections of human gastric ulcer tissue was examined immunohistochemically. IL-1 dose dependently enhanced VEGF release in cultured gastric fibroblasts after a 24-h stimulation. IL-1 also stimulated PGE(2) production in gastric fibroblasts via COX-2 induction. NS-398 significantly suppressed VEGF and PGE(2) release from IL-1-stimulated gastric fibroblasts; concurrent addition of PGE(2) restored NS-398-inhibited VEGF release. COX-2 and VEGF immunoreactivity were colocalized in fibroblast-like cells in the ulcer bed of gastric tissues. These results suggest that COX-2 plays a key role in VEGF production in gastric fibroblasts stimulated by IL-1 in vitro and that angiogenesis induced by the COX-2-VEGF pathway might be involved in gastric ulcer healing.     

Effects of selective PGE2 receptor antagonists in esophageal adenocarcinoma cells derived from Barrett's esophagus

Accumulating evidence suggests that COX-2-derived prostaglandin E(2) (PGE(2)) plays an important role in esophageal adenocarcinogenesis. Recently, PGE(2) receptors (EP) have been shown to be involved in colon cancer development. Since it is not known which receptors regulate PGE(2) signals in esophageal adenocarcinoma, we investigated the role of EP receptors using a human Barrett's-derived esophageal adenocarcinoma cell line (OE33). OE33 cells expressed COX-1, COX-2, EP(1), EP(2) and EP(4) but not EP(3) receptors as determined by real time RT-PCR and Western-blot. Treatment with 5-aza-dC restored expression, suggesting that hypermethylation is involved in EP(3) downregulation. Endogenous PGE(2) production was mainly due to COX-2, since this was significantly suppressed with COX-2 inhibitors (NS-398 and SC-58125), but not COX-1 inhibitors (SC-560). Cell proliferation ((3)H-thymidine uptake) was significantly inhibited by NS-398 and SC-58125, the EP(1) antagonist SC-51322, AH6809 (EP(1)/EP(2) antagonist), and the EP(4) antagonist AH23848B, but was not affected by exogenous PGE(2). However, treatment with the selective EP(2) agonist Butaprost or 16,16-dimethylPGE(2) significantly inhibited butyrate-induced apoptosis and stimulated OE33 cell migration. The effect of exogenous PGE(2) on migration was attenuated when cells were first treated with EP(1) and EP(4) antagonists. These findings suggest a potential role for EP selective antagonists in the treatment of esophageal adenocarcinoma.     

Prostaglandin E prevents indomethacin-induced gastric and intestinal damage through different EP receptor subtypes

Gastrointestinal ulcerogenic effect of indomethacin is causally related with an endogenous prostaglandin (PG) deficiency, yet the detailed mechanism remains unknown. We examined the effect of various PGE analogues specific to EP receptor subtypes on these lesions in rats and mice, and investigated which EP receptor subtype is involved in the protective action of PGE(2). Fasted or non-fasted animals were given indomethacin s.c. at 35 mg/kg for induction of gastric lesions or 10-30 mg/kg for intestinal lesions, and they were killed 4 or 24 h later, respectively. Various EP agonists were given i.v. 10 min before indomethacin. Indomethacin caused hemorrhagic lesions in both the stomach and intestine. Prior administration of 16,16-dimethyl PGE(2) (dmPGE(2)) prevented the development of damage in both tissues, and the effect in the stomach was mimicked by 17-phenyl PGE2 (EP1), while that in the small intestine was reproduced by ONO-NT-012 (EP3) and ONO-AE-329 (EP4). Butaprost (EP2) did not have any effect on either gastric or intestinal lesions induced by indomethacin. Similar to the findings in rats, indomethacin caused gastric and intestinal lesions in both wild-type and knockout mice lacking EP1 or EP3 receptors. However, the protective action of dmPGE(2) in the stomach was observed in wild-type and EP3 receptor knockout mice but not in mice lacking EP1 receptors, while that in the intestine was observed in EP1 knockout as well as wild-type mice but not in the animals lacking EP3 receptors. These results suggest that indomethacin produced damage in the stomach and intestine in a PGE(2)-sensitive manner, and exogenous PGE(2) prevents gastric and intestinal ulcerogenic response to indomethacin through different EP receptor subtypes; the protection in the stomach is mediated by EP1 receptors, while that in the intestine mediated by EP3/EP4 receptors.     

Regulation of vascular endothelial cell growth factor expression in mouse mammary tumor cells by the EP2 subtype of the prostaglandin E2 receptor

Prostaglandin E(2) (PGE(2)), a major metabolite of the cyclooxygenase pathway in the mammary gland, induces angiogenesis during mammary tumor progression. To better define the molecular mechanisms involved, we examined the role of the G protein-coupled receptors (GPCR) for PGE(2) in mammary tumor cell lines isolated from MMTV-cyclooxygenase-2 (COX-2) transgenic mice. Expression of the EP2 subtype of the PGE(2) receptor was correlated with the tumorigenic phenotype and the ability to induce vascular endothelial growth factor (VEGF). Overexpression of EP2 by adenoviral transduction into EP2-null cells resulted in the induction of VEGF expression in response to PGE(2) and CAY10399, an EP2 receptor agonist. The induction of VEGF by the EP2 receptor did not require the hypoxia inducible factor (HIF)-1alpha pathway, MAP kinase pathway, or phosphoinositide-3-kinase/Akt pathway, but required the cAMP/protein kinase A pathway. These results suggest that EP2 receptor is a critical element for PGE(2) mediated VEGF induction in mouse mammary tumor cells.     

Endothelin-1-induced prostaglandin E2-EP2, EP4 signaling regulates vascular endothelial growth factor production and ovarian carcinoma cell invasion

Cyclooxygenase (COX)-1- and COX-2-derived prostaglandins are implicated in the development and progression of several malignancies. We have recently demonstrated that treatment of ovarian carcinoma cells with endothelin-1 (ET-1) induces expression of both COX-1 and COX-2, which contributes to vascular endothelial growth factor (VEGF) production. In this study, we show that in HEY and OVCA 433 ovarian carcinoma cells, ET-1, through the binding with ETA receptor (ETAR), induces prostaglandin E2 (PGE2) production, as the more represented PG types, and increases the expression of PGE2 receptor type 2 (EP2) and type 4 (EP4). The use of pharmacological EP agonists and antagonists indicates that ET-1 and PGE2 stimulate VEGF production principally through EP2 and EP4 receptors. At the mechanistic level, we prove that the induction of PGE2 and VEGF by ET-1 involves Src-mediated epidermal growth factor receptor transactivation. Finally, we demonstrate that ETAR-mediated activation of PGE2-dependent signaling participates in the regulation of the invasive behavior of ovarian carcinoma cells by activating tumor-associated matrix metalloproteinase. These results implicate EP2 and EP4 receptors in the induction of VEGF expression and cell invasiveness by ET-1 and provide a mechanism by which ETAR/ET-1 can promote and interact with PGE2-dependent machinery to amplify its proangiogenic and invasive phenotype in ovarian carcinoma cells. Pharmacological blockade of ETAR can therefore represent an additional strategy to control PGE2 signaling, which has been associated with ovarian carcinoma progression.     

The role of COX-2/PGE2 in gossypol-induced apoptosis of colorectal carcinoma cells

Our previous study showed that gossypol (GOS) exhibits potent cytotoxic effects via apoptosis induction against human colorectal carcinoma cells; however, the role of cyclooxygenase (COX)-2/prostaglandin (PG)E(2) on GOS-induced apoptosis is still unknown. In the present study, 12-O-tetradecanoylphorbol-13-acetate (TPA) addition significantly inhibited GOS-induced apoptosis in human colorectal carcinoma HT-29 cells in accordance with inducing COX-2 protein/PGE(2) production. TPA inhibition of GOS-induced apoptosis was blocked by adding protein kinase (PK)C inhibitors including staurosporine (ST), GF109203X (GF), and H7, characterized by the occurrence of cleaved caspase 3 proteins and a decrease in COX-2 protein/PGE(2) production in HT-29 cells. The addition of COX activity inhibitors, including NS398 (NS), aspirin (AS), diclofenac (DI), and indomethacin (IN), suppressed TPA protection of GOS-induced apoptosis with decreased PGE(2) production in HT-29 cells. Application of PGE(2), but not it analogs PGD(2), PGJ(2), or PGF(2α), protected HT-29 cells from GOS-induced DNA ladders, and the E-prostanoid (EP(1)) receptor agonist, 17PT-PGE(2), mimicked the protection induced by PGE(2), whereas the selective EP(2) receptor agonist, butaprostol (BUT), the EP(3) receptor agonist, sulprostol (SUL), and the EP(4) receptor agonist, PGE(1) alcohol (PGE(1)), showed no significant effects on GOS-induced apoptosis in HT-29 cells. PGE(2) 's protection against GOS-induced apoptosis was reversed by adding the selective EP(1) receptor antagonist, SC-19220. Furthermore, GOS had an effective apoptotic effect on COLO205 colorectal carcinoma cells which expressed undetectable level of endogenous COX-2 protein than HT-29 cells, and the decreased COX-2 protein level via COX-2 siRNA or addition of COX-2 activity inhibitor NS significantly elevated GOS-induced cell death in HT-29 cells. COLO205-T cells were established through sustained TPA incubation of COLO205 cells, and COLO205-T cells showed a lower sensitivity to GOS-induced cell death with increased COX-2 (not Bcl-2 and Mcl-1) protein than parental COLO-205 cells. A decrease in COX-2 protein expression in COLO205-T cells by COX-2 siRNA transfection or enhanced GOS-induced cell death according to MTT assay and DNA integrity assay. The notion of COX-2/PGE(2) activation against GOS-induced apoptosis in colon carcinoma cells was demonstrated, and the combination of GOS and COX-2 inhibitors to treat colon carcinoma possesses clinical potential worthy of further investigation.     

Effect of misoprostol and indomethacin on cyclooxygenase induction and eicosanoid production in carrageenan-induced air pouch inflammation in rats

Effects of misoprostol, a synthetic prostaglandin E1 (PGE1) analogue, on cyclooxygenase-2 (COX-2) protein level and exudate prostaglandin E2 (PGE2) and thromboxane B2 (TXB2) level were investigated in acute carrageenan-induced air pouch inflammation in rats. Treatment with misoprostol (12.5, 25, and 50 microg/kg) has been started in separated groups, 30 min and 2 days before carrageenan injection and it was given twice a day (total of five doses) by orogastric route. Indomethacin, in doses of 0.5 and 5 mg/kg, and specific COX-2 inhibitor SC-58236, in doses of 5, 10, and 20 mg/kg were given 1 h before carrageenan injection by the orogastric route. Misoprostol increased the levels of PGE2 and COX-2 protein at all doses applied. Despite indomethacin and SC-58236 increased the level of COX-2 protein when they used alone, these drugs partially inhibited misoprostol-induced increase in the level of COX-2 protein. Partial inhibition of misoprostol-induced increase in the level of COX-2 protein by indomethacin or SC-58236 may indicate the modulatory roles of endogenous prostaglandins (PGs, especially, PGE2) on the COX-2 expression.     

Induced microsomal PGE synthase-1 is involved in cyclooxygenase-2-dependent PGE2 production in gastric fibroblasts

We have previously shown that the cyclooxygenase (COX)-2/PGE2 pathway plays a key role in VEGF production in gastric fibroblasts. Recent studies have identified three PGE synthase (PGES) isozymes: cytosolic PGES (cPGES) and microsomal PGES (mPGES)-1 and -2, but little is known regarding the expression and roles of these enzymes in gastric fibroblasts. Thus we examined IL-1beta-stimulated mPGES-1 and cPGES mRNA and protein expression in gastric fibroblasts by quantitative PCR and Western blot analysis, respectively, and studied both their relationship to COX-1 and -2 and their roles in PGE2 and VEGF production in vitro. IL-1beta stimulated increases in both COX-2 and mPGES-1 mRNA and protein expression levels. However, COX-2 mRNA and protein expression were more rapidly induced than mPGES-1 mRNA and protein expression. Furthermore, MK-886, a nonselective mPGES-1 inhibitor, failed to inhibit IL-1beta-induced PGE2 release at the 8-h time point, while totally inhibiting PGE2 at the later stage. However, MK-886 did inhibit IL-1beta-stimulated PGES activity in vitro by 86.8%. N-(2-cyclohexyloxy-4-nitrophenyl)-methanesulfonamide (NS-398), a selective COX-2 inhibitor, totally inhibited PGE2 production at both the 8-h and 24-h time points, suggesting that COX-2-dependent PGE2 generation does not depend on mPGES-1 activity at the early stage. In contrast, NS-398 did not inhibit VEGF production at 8 h, and only partially at 24 h, whereas MK-886 totally inhibited VEGF production at each time point. These results suggest that IL-1beta-induced mPGES-1 protein expression preferentially coupled with COX-2 protein at late stages of PGE2 production and that IL-1beta-stimulated VEGF production was totally dependent on membrane-associated proteins involved in eicosanoid and glutathione metabolism (MAPEG) superfamily proteins, which includes mPGES-1, but was partially dependent on the COX-2/PGE2 pathway.     

Endothelin-1, an ulcer inducer, promotes gastric ulcer healing via mobilizing gastric myofibroblasts and stimulates production of stroma-derived factors

Endothelin (ET)-1 is a potent inducer of peptic ulcers. The roles of ET-1 in ulcer healing, however, have remained unclear, and these were investigated in mice. Gastric ulcers were induced in mice by serosal application of acetic acid. Three days later, mice were given a neutralizing ET-1 antibody or nonimmunized serum. The ulcer size, amount of fibrosis and myofibroblasts, and localization of ET-1 and ET(A/B) receptors were analyzed. To elucidate the mechanisms underlying the effects of ET-1, we examined the proliferation, migration, and release of growth and angiogenic factors in gastric myofibroblasts with or without ET-1. The expression of prepro-ET-1 (an ET-1 precursor) and ET-converting enzyme-1 was examined in gastric myofibroblasts using RT-PCR. Immunoneutralization of ET-1 delayed gastric ulcer healing. The areas of fibrosis and myofibroblasts were smaller in the anti-ET-1 antibody group than in the control. ET-1 was expressed in the gastric epithelium, myofibroblasts, and other cell types. ET(A) receptors, but not ET(B) receptors, were present in myofibroblasts. ET-1 increased proliferation and migration of gastric myofibroblasts. ET-1 stimulated the release of hepatocyte growth factor, VEGF, PGE(2), and IL-6 from gastric myofibroblasts. mRNA for prepro-ET-1 and ET-converting enzyme-1 was also expressed. ET-1 promotes the accumulation of gastric myofibroblasts and collagen fibrils at gastric ulcers. ET-1 also stimulates migration and proliferation of gastric myofibroblasts and enhances the release of growth factors, angiogenic factors, and PGE(2). Thus ET-1 has important roles not only in ulcer formation but also in ulcer healing via mobilizing myofibroblasts and inducing production of stroma-derived factors.     

Role of prostaglandin E2 receptors in migration of murine and human breast cancer cells

Aberrant upregulation of COX-2 enzyme resulting in accumulation of PGE2 in a cancer cell environment is a marker for progression of many cancers, including breast cancer. Four subtypes of cell surface receptors (EP1, EP2, EP3, and EP4), which are coupled with different G-proteins, mediate PGE2 actions. Since migration is an essential step in invasion and metastasis, in the present study we defined the expression of EP receptors and their roles in migratory function of breast cancer cells of murine (C3L5) and human (MDA-MB-231 and MCF-7) origin. Highly metastatic C3L5 and MDA-MB-231 cells, found to be highly migratory in a Transwell migration assay, were shown to accumulate much higher levels of PGE2 in culture media in comparison with nonmetastatic and poorly migrating MCF-7 cells; the levels of PGF2alpha and 6-keto-PGF1alpha were low in all cases. The elevated PGE2 production by metastatic cancer cells was due to COX-2 activity since dual COX-1/2 inhibitor indomethacin and selective COX-2 inhibitor NS-398 equally suppressed both basal and inducible (by IFN-gamma/LPS or Ca2+-ionophores) PGE2 accumulation. RT-PCR analysis revealed that murine C3L5 cells expressed mRNA of EP1, EP3, and EP4 but not EP2 receptors. On the other hand, human MDA-MB-231 and MCF-7 cells expressed all the above receptors. High levels of expression of functional EP4 receptors coupled with Gs-protein was confirmed in C3L5 cells by biochemical assay showing a dose-dependent increase of intracellular cAMP synthesis in response to PGE2. EP receptor antagonists SC-19220, AH-6809, and AH-23848B, having highest affinity for EP1, EP1/EP2/DP, and EP4 receptors, respectively, variably inhibited migration of metastatic breast cancer cells. An autocrine PGE2-mediated migratory activity of these cells appeared to be associated predominantly with EP4 receptor-mediated signaling pathway, which uses cAMP as a second messenger. This conclusion is based on several observations: (1) selective EP4 antagonist AH-23848B effectively inhibited migration of both C3L5 and MDA-MB-231 cells in a dose-dependent manner; (2) exogenous PGE2 and EP4 agonist PGE1 alcohol increased migration of C3L5 cells; (3) forskolin, a potent activator of adenylate cyclase, as well as membrane-permeable analogues of cAMP (8-bromo-cAMP, dibutyryl-cAMP) stimulated migration of C3L5 cells; and (4) Rp-cAMPS, a selective protein kinase A inhibitor, reduced migration of C3L5 cells. Migration of poorly migratory MCF-7 cells remained unaffected with either PGE2 or EP4 antagonist. These findings are relevant for designing therapeutic strategies against breast cancer metastasis.     

Potential involvement of the cyclooxygenase-2 pathway in hepatocellular carcinoma-associated angiogenesis

Angiogenesis plays a crucial role in tumor development and growth. The present study was carried out to investigate the potential involvement of the cyclooxygenase-2 (Cox-2) pathway in the regulation of angiogenesis in hepatocellular carcinoma (HCC). We inhibited Cox-2 expression in HCC cell line HuH-7 by selective Cox-2 inhibitor (SC-58635) or Cox-2 siRNA. Conditioned media (CMs) from HuH-7 cells were used in angiogenic assays in vitro and in vivo. Compared with CMs from untreated and negative siRNA treated HuH-7 cells, CMs from SC-58635 and Cox-2 siRNA treated HuH-7 dramatically suppressed the proliferation, migration, and differentiation of human umbilical vein endothelial cells (HUVECs) in vitro and neovascularization in vivo. These inhibitory effects could be partially reversed by the addition of exogenous PGE2 to CMs. Furthermore, Cox-2 inhibition by SC-58635 resulted in PGE2 reduction accompanied by the down-regulation of four PGE2 receptor (EP receptor) subtypes. Treatment with SC-58635 led to the down-expression of proangiogenic factors such as VEGF, HGF, FGF2, ANGPT1 and ANGPT2 in HCC. An approximately 78% reduction of VEGF level has been found in the CM from SC-58635 treated HuH-7. Our results suggest an involvement of Cox-2 in the control of HCC-associated angiogenesis. PGE2 as a vital angiogenic factor may act directly on endothelial cells to promote HuH-7-stimulated angiogenic process. Moreover, Cox-2/PGE2/EP/VEGF pathway possibly also contributes to tumor angiogenesis in HCC. This study provides the rationale for clinical studies of Cox-2 inhibitors on the treatment or chemoprevention of HCC.