Hypertonic stress induces VEGF production in human colon cancer cell line Caco-2: inhibitory role of autocrine PGE₂

Vascular Endothelial Growth Factor (VEGF) is a major regulator of angiogenesis. VEGF expression is up regulated in response to micro-environmental cues related to poor blood supply such as hypoxia. However, regulation of VEGF expression in cancer cells is not limited to the stress response due to increased volume of the tumor mass. Lipid mediators in particular arachidonic acid-derived prostaglandin (PG)E? are regulators of VEGF expression and angiogenesis in colon cancer. In addition, increased osmolarity that is generated during colonic water absorption and feces consolidation seems to activate colon cancer cells and promote PGE? generation. Such physiological stimulation may provide signaling for cancer promotion. Here we investigated the effect of exposure to a hypertonic medium, to emulate colonic environment, on VEGF production by colon cancer cells. The role of concomitant PGE? generation and MAPK activation was addressed by specific pharmacological inhibition. Human colon cancer cell line Caco-2 exposed to a hypertonic environment responded with marked VEGF and PGE? production. VEGF production was inhibited by selective inhibitors of ERK 1/2 and p38 MAPK pathways. To address the regulatory role of PGE? on VEGF production, Caco-2 cells were treated with cPLA? (ATK) and COX-2 (NS-398) inhibitors, that completely block PGE? generation. The Caco-2 cells were also treated with a non selective PGE? receptor antagonist. Each treatment significantly increased the hypertonic stress-induced VEGF production. Moreover, addition of PGE? or selective EP? receptor agonist to activated Caco-2 cells inhibited VEGF production. The autocrine inhibitory role for PGE? appears to be selective to hypertonic environment since VEGF production induced by exposure to CoCl? was decreased by inhibition of concomitant PGE? generation. Our results indicated that hypertonicity stimulates VEGF production in colon cancer cell lines. Also PGE? plays an inhibitory role on VEGF production by Caco-2 cells exposed to hyperosmotic stress through EP? activation.     

Hypertonic environment elicits cyclooxygenase-2-driven prostaglandin E2 generation by colon cancer cells: Role of cytosolic phospholipase A2-α and kinase signaling pathways

Cyclooxygenase (COX)-2-derived prostaglandin (PG)E₂ controls many aspects of colon cancer development, modulating from apoptosis resistance and cell proliferation to angiogenesis, invasion, and metastasis. Here, we investigated the role of different phospholipases (PL)A₂ in supplying arachidonic acid (AA) for COX-2-dependent PGE₂ generation and signaling pathways involved in activation of colon cancer cells by a physiologically relevant stimulus. To emulate the hypertonic environment found physiologically in colon, the human colon cancer cell line Caco-2 was maintained in hypertonic complete DMEM medium. Human colon cancer cell line Caco-2 exposed to a hypertonic environment responded with marked AA release, COX-2 induction and PGE₂ generation. Selective secretory (s)PLA₂ and calcium-independent (i)PLA₂ inhibitors did not modify PGE₂ generation, while either COX-2 or cytosolic (c)PLA₂ inhibitors completely inhibited PGE₂ generation. cPLA₂-α was responsible for AA supply for PGE₂ generation, but had no role in COX-2 induction. Mitogen-activated protein (MAP) kinases, ERK 1/2, p38, and JNK, participated in the signaling events that lead to PGE₂ generation by modulating AA release, but only ERK 1/2 was involved in COX-2 upregulation. Our results indicate that hypertonic stress activates PGE₂ generation by Caco-2 cells through a mechanism dependent on MAP kinase-regulated AA mobilization, increased cPLA₂-α activity, and COX-2 induction.     

PGE2-induced colon cancer growth is mediated by mTORC1

The inflammatory prostaglandin E₂ (PGE₂) cytokine plays a key role in the development of colon cancer. Several studies have shown that PGE₂ directly induces the growth of colon cancer cells and furthermore promotes tumor angiogenesis by increasing the production of the vascular endothelial growth factor (VEGF). The signaling intermediaries implicated in these processes have however not been fully characterized. In this report, we show that the mechanistic target of rapamycin complex 1 (mTORC1) plays an important role in PGE₂-induced colon cancer cell responses. Indeed, stimulation of LS174T cells with PGE₂ increased mTORC1 activity as observed by the augmentation of S6 ribosomal protein phosphorylation, a downstream effector of mTORC1. The PGE₂ EP₄ receptor was responsible for transducing the signal to mTORC1. Moreover, PGE₂ increased colon cancer cell proliferation as well as the growth of colon cancer cell colonies grown in matrigel and blocking mTORC1 by rapamycin or ATP-competitive inhibitors of mTOR abrogated these effects. Similarly, the inhibition of mTORC1 by downregulation of its component raptor using RNA interference blocked PGE₂-induced LS174T cell growth. Finally, stimulation of LS174T cells with PGE₂ increased VEGF production which was also prevented by mTORC1 inhibition. Taken together, these results show that mTORC1 is an important signaling intermediary in PGE₂ mediated colon cancer cell growth and VEGF production. They further support a role for mTORC1 in inflammation induced tumor growth.     

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

Accumulating evidence suggests that COX-2-derived prostaglandin E₂ (PGE₂) plays an important role in esophageal adenocarcinogenesis. Recently, PGE₂ receptors (EP) have been shown to be involved in colon cancer development. Since it is not known which receptors regulate PGE₂ 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, EP₁, EP₂ and EP₄ but not EP₃ receptors as determined by real time RT-PCR and Western-blot. Treatment with 5-aza-dC restored expression, suggesting that hypermethylation is involved in EP₃ downregulation. Endogenous PGE₂ 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 (³H-thymidine uptake) was significantly inhibited by NS-398 and SC-58125, the EP₁ antagonist SC-51322, AH6809 (EP₁/EP₂ antagonist), and the EP₄ antagonist AH23848B, but was not affected by exogenous PGE₂. However, treatment with the selective EP₂ agonist Butaprost or 16,16-dimethylPGE₂ significantly inhibited butyrate-induced apoptosis and stimulated OE33 cell migration. The effect of exogenous PGE₂ on migration was attenuated when cells were first treated with EP₁ and EP₄ antagonists. These findings suggest a potential role for EP selective antagonists in the treatment of esophageal adenocarcinoma.     

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

Aim

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

Main methods

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

Key findings

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

Significance

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

Prostaglandin E2 inhibits the proliferation of human gingival fibroblasts via the EP2 receptor and Epac

Elevated levels of prostaglandins such as PGE₂ in inflamed gingiva play a significant role in the tissue destruction caused by periodontitis, partly by targeting local fibroblasts. Only very few studies have shown that PGE₂ inhibits the proliferation of a gingival fibroblast (GF) cell line, and we expanded this research by using primary human GFs (hGFs) and looking into the mechanisms of the PGE₂ effect. GFs derived from healthy human gingiva were treated with PGE₂ and proliferation was assessed by measuring cell number and DNA synthesis and potential signaling pathways were investigated using selective activators or inhibitors. PGE₂ inhibited the proliferation of hGFs dose‐dependently. The effect was mimicked by forskolin (adenylate cyclase stimulator) and augmented by IBMX (a cAMP‐breakdown inhibitor), pointing to involvement of cAMP. Indeed, PGE₂ and forskolin induced cAMP generation in these cells. Using selective EP receptor agonists we found that the anti‐proliferative effect of PGE₂ is mediated via the EP₂ receptor (which is coupled to adenylate cyclase activation). We also found that the effect of PGE₂ involved activation of Epac (exchange protein directly activated by cAMP), an intracellular cAMP sensor, and not PKA. While serum increased the amount of phospho‐ERK in hGFs by ～300%, PGE₂ decreased it by ～50%. Finally, the PGE₂ effect does not require endogenous production of prostaglandins since it was not abrogated by two COX‐inhibitors. In conclusion, in human gingival fibroblasts PGE₂ activates the EP₂—cAMP—Epac pathway, reducing ERK phosphorylation and inhibiting proliferation. This effect could hamper periodontal healing and provide further insights into the pathogenesis of inflammatory periodontal disease. J. Cell. Biochem. 108: 207–215, 2009. © 2009 Wiley‐Liss, Inc.     

Prostaglandin E2 Prevents Hyperosmolar-Induced Human Mast Cell Activation through Prostanoid Receptors EP2 and EP4

Background

Mast cells play a critical role in allergic and inflammatory diseases, including exercise-induced bronchoconstriction (EIB) in asthma. The mechanism underlying EIB is probably related to increased airway fluid osmolarity that activates mast cells to the release inflammatory mediators. These mediators then act on bronchial smooth muscle to cause bronchoconstriction. In parallel, protective substances such as prostaglandin E₂ (PGE₂) are probably also released and could explain the refractory period observed in patients with EIB.

Objective

This study aimed to evaluate the protective effect of PGE₂ on osmotically activated mast cells, as a model of exercise-induced bronchoconstriction.

Methods

We used LAD2, HMC-1, CD34-positive, and human lung mast cell lines. Cells underwent a mannitol challenge, and the effects of PGE₂ and prostanoid receptor (EP) antagonists for EP_1–4 were assayed on the activated mast cells. Beta-hexosaminidase release, protein phosphorylation, and calcium mobilization were assessed.

Results

Mannitol both induced mast cell degranulation and activated phosphatidyl inositide 3-kinase and mitogen-activated protein kinase (MAPK) pathways, thereby causing de novo eicosanoid and cytokine synthesis. The addition of PGE₂ significantly reduced mannitol-induced degranulation through EP₂ and EP₄ receptors, as measured by beta-hexosaminidase release, and consequently calcium influx. Extracellular-signal-regulated kinase 1/2, c-Jun N-terminal kinase, and p38 phosphorylation were diminished when compared with mannitol activation alone.

Conclusions

Our data show a protective role for the PGE₂ receptors EP₂ and EP₄ following osmotic changes, through the reduction of human mast cell activity caused by calcium influx impairment and MAP kinase inhibition.     

Targeting COX-2/PGE2 Pathway in HIPK2 Knockdown Cancer Cells: Impact on Dendritic Cell Maturation

Background

Homeodomain-interacting protein kinase 2 (HIPK2) is a multifunctional protein that exploits its kinase activity to modulate key molecular pathways in cancer to restrain tumor growth and induce response to therapies. For instance, HIPK2 knockdown induces upregulation of oncogenic hypoxia-inducible factor-1 (HIF-1) activity leading to a constitutive hypoxic and angiogenic phenotype with increased tumor growth in vivo. HIPK2 inhibition, therefore, releases pathways leading to production of pro-inflammatory molecules such as vascular endothelial growth factor (VEGF) or prostaglandin E2 (PGE₂). Tumor-produced inflammatory mediators other than promote tumour growth and vascular development may permit evasion of anti-tumour immune responses. Thus, dendritic cells (DCs) dysfunction induced by tumor-produced molecules, may allow tumor cells to escape immunosurveillance. Here we evaluated the molecular mechanism of PGE₂ production after HIPK2 depletion and how to modulate it.

Methodology/Principal findings

We show that HIPK2 knockdown in colon cancer cells resulted in cyclooxygenase-2 (COX-2) upregulation and COX-2-derived PGE₂ generation. At molecular level, COX-2 upregulation depended on HIF-1 activity. We previously reported that zinc treatment inhibits HIF-1 activity. Here, zinc supplementation to HIPK2 depleted cells inhibited HIF-1-induced COX-2 expression and PGE₂/VEGF production. At translational level, while conditioned media of both siRNA control and HIPK2 depleted cells inhibited DCs maturation, conditioned media of only zinc-treated HIPK2 depleted cells efficiently restored DCs maturation, seen as the expression of co-stimulatory molecules CD80 and CD86, cytokine IL-10 release, and STAT3 phosphorylation.

Conclusion/Significance

These findings show that: 1) HIPK2 knockdown induced COX-2 upregulation, mostly depending on HIF-1 activity; 2) zinc treatment downregulated HIF-1-induced COX-2 and inhibited PGE₂/VEGF production; and 3) zinc treatment of HIPK2 depleted cells restored DCs maturation.     

Prostaglandin E2-EP1 and EP2 receptor signaling promotes apical junctional complex disassembly of Caco-2 human colorectal cancer cells

Background

The apical junctional complex (AJC) is a dynamic structure responsible to maintain epithelial cell-cell adhesions and it plays important functions such as, polarity, mechanical integrity, and cell signaling. Alteration of this complex during pathological events leads to an impaired epithelial barrier by perturbation of the cell-cell adhesion system. Although clinical and experimental data indicate that prostaglandin E₂ (PGE₂) plays a critical function in promoting cell motility and cancer progression, little is known concerning its role in AJC disassembly, an event that takes place at the beginning of colorectal tumorigenesis. Using Caco-2 cells, a cell line derived from human colorectal cancer, we investigated the effects of prostaglandin E₂ (PGE₂) treatment on AJC assembly and function.

Results

Exposition of Caco-2 cells to PGE₂ promoted differential alteration of AJC protein distribution, as evidenced by immunofluorescence and immunoblotting analysis and impairs the barrier function, as seen by a decrease in the transepithelial electric resistance and an increase in the permeability to ruthenium red marker. We demonstrated the involvement of EP1 and EP2 prostaglandin E₂ receptor subtypes in the modulation of the AJC disassembly caused by prostanoid. Furthermore, pharmacological inhibition of protein kinase-C, but not PKA and p38MAPK significantly prevented the PGE₂ effects on the AJC disassembly.

Conclusion

Our findings strongly suggest a central role of Prostaglandin E2-EP1 and EP2 receptor signaling to mediate AJC disassembly through a mechanism that involves PKC and claudin-1 as important target for the TJ-related effects in human colorectal cancer cells (Caco-2).     

Prostaglandin E<Subscript>2</Subscript> blocks menadione-induced apoptosis through the Ras/Raf/Erk signaling pathway in promonocytic leukemia cell lines

Altered oxidative stress has long been observed in cancer cells, and this biochemical property of cancer cells represents a specific vulnerability that can be exploited for therapeutic benefit. The major role of an elevated oxidative stress for the efficacy of molecular targeted drugs is under investigation. Menadione is considered an attractive model for the study of oxidative stress, which can induce apoptosis in human leukemia HL-60 cell lines. Prostaglandin E₂ (PGE₂) via its receptors not only promotes cell survival but also reverses apoptosis and promotes cancer progression. Here, we present evidence for the biological role of PGE₂ as a protective agent of oxidative stress-induced apoptosis in monocytic cells. Pretreatment of HL-60 cells with PGE₂ markedly ameliorated the menadione-induced apoptosis and inhibited the degradation of PARP and lamin B. The EP₂ receptor antagonist AH6809 abrogated the inhibitory effect of PGE₂, suggesting the role of the EP₂/cAMP system. The PKA inhibitor H89 also reversed apoptosis and decreased the PKA activity that was elevated 10-fold by PGE₂. The treatment of HL-60 cells with NAC or zinc chloride showed a similar protective effect as with PGE₂ on menadione-treated cells. Furthermore, PGE₂ activated the Ras/Raf/MEK pathway, which in turn initiated ERK activation, and ultimately protected menadione-induced apoptosis. These results imply that PGE₂ via cell survival pathways may protect oxidative stress-induced apoptosis in monocytic cells. This study warrants further pre-clinical investigation as well as application towards leukemia clinics.     

Effect of eicosapentaenoic acid-derived prostaglandin E3 on intestinal epithelial barrier function

Prostaglandins (PG) are inflammatory mediators derived from arachidonic or eicosapentaenoic acid giving rise to the 2-series or the 3-series prostanoids, respectively. Previously, we have observed that PGE₂ disrupts epithelial barrier function. Considering the beneficial effect of fish oil consumption in intestinal inflammatory processes, the aim of this study was to assess the role of PGE₃ on epithelial barrier function assessed from transepithelial electrical resistance and dextran fluxes in Caco-2 cells. The results indicate that PGE₃ increased paracellular permeability (PP) to the same extent as PGE₂, through the interaction with EP₁ and EP₄ receptors and with intracellular Ca²⁺ and cAMP as the downstream targets. Moreover, we observed a redistribution of tight junction proteins, occludin and claudin-4. In conclusion, PGE₃ is able to increase PP thus leading to reconsider the role of PGE₂/PGE₃ ratio in the beneficial effects of dietary fish oil supplementation in the disruption of barrier function.     

Regulatory interconnections of cyclooxygenase and inducible NO-Synthase in urinary bladder epithelial cells of the frog <Emphasis Type="Italic">Rana temporaria</Emphasis> under effect of bacterial stimuli

Earlier we have shown that in epithelial cells of the frog urinary bladder under action of bacterial lipopolysaccharides (LPS) there is activated expression of inducible NO-synthase (iNOS) and there is increased the NO production, which can play an important role in providing protective cell reactions form pathogens. The goal of the present work consisted in study of cyclooxygenase (cOG) products and mechanisms of their regulatory effect on expression of iNOS under action of LPS. In experiments on urinary bladder epithelial cells on the frog Rana temporaria it has been shown that incubation of the cells for 21 h with LPS leads to a rise in production of PGE₂ and nitrites, stable NO metabolites. Inhibitor of iNOS 1400W decreased sharply production of nitrites, but did not affect the PGE₂ level. Both the basal and the LPS-stimulated level of PGE₂ and nitrites were inhibited in the presence of selective cOG inhibitors SC-560 (cOG-1) and NS-398 (cOG-2). The IC₅₀ value amounted to 90, 220 and 470 μM for NS-398, SC-560, and diclofenac (unspecific inhibitor of both isoforms), respectively. PGE₂ and butaprost, the EP₂-receptor agonist, but not agonists of EP₁/EP₃ or EP₁ receptors, partially eliminated the inhibitory action of diclofenac on production of nitrites. Action of PGE₂ was accompanied by an increase in the intracellular cAMP. Analysis of expression of iNOS mRNA in the epithelial cells incubated with LPS or LPS + inhibitor of cPG has shown the LPS-stimulated rise in expression of iNOS mRNA to decrease sharply in the presence of SC-560 or NS-398. Thus, the epithelial cells of the frog urinary bladder have the effectively functioning system of the congenital immune protection against bacterial pathogens, the most important component of this system being PGE₂ and NO. Analysis of mechanisms of regulatory interactions of cOG and iNOS indicates that in this cell type the main regulators of iNOS expression and of the nitrogen oxide level are products of the cOG catalytic activity.     

Upregulation of the S1P3 receptor in metastatic breast cancer cells increases migration and invasion by induction of PGE2 and EP2/EP4 activation

Breast cancer is one of the most common and devastating malignancies among women worldwide. Recent evidence suggests that malignant progression is also driven by processes involving the sphingolipid molecule sphingosine 1-phosphate (S1P) and its binding to cognate receptor subtypes on the cell surface. To investigate the effect of this interaction on the metastatic phenotype, we used the breast cancer cell line MDA-MB-231 and the sublines 4175 and 1833 derived from lung and bone metastases in nude mice, respectively. In both metastatic cell lines expression of the S1P₃ receptor was strongly upregulated compared to the parental cells and correlated with higher S1P-induced intracellular calcium ([Ca^2 +]_i), higher cyclooxygenase (COX)-2 and microsomal prostaglandin (PG) E₂ synthase expression, and consequently with increased PGE₂ synthesis. PGE₂ synthesis was decreased by antagonists and siRNA against S1P₃ and S1P₂. Moreover, in parental MDA-MB-231 cells overexpression of S1P₃ by cDNA transfection also increased PGE₂ synthesis, but only after treatment with the DNA methyltransferase inhibitor 5-aza-2-deoxycytidine, indicating reversible silencing of the COX-2 promoter. Functionally, the metastatic sublines showed enhanced migration and Matrigel invasion in adapted Boyden chamber assays, which further increased by S1P stimulation. This response was abrogated by either S1P₃ antagonism, COX-2 inhibition or PGE₂ receptor 2 (EP₂) and 4 (EP₄) antagonism, but not by S1P₂ antagonism. Our data demonstrate that in breast cancer cells overexpression of S1P₃ and its activation by S1P has pro-inflammatory and pro-metastatic potential by inducing COX-2 expression and PGE₂ signaling via EP₂ and EP₄.     

Role of intracellular prostaglandin E2 in cancer-related phenotypes in PC3 cells

Prostaglandin E₂ (PGE₂) and hypoxia-inducible factor-1α (HIF-1α) affect many mechanisms that have been shown to play a role in prostate cancer. In PGE₂-treated LNCaP cells, up-regulation of HIF-1α requires the internalization of PGE₂, which is in sharp contrast with the generally accepted view that PGE₂ acts through EP receptors located at the cell membrane. Here we aimed to study in androgen-independent PC3 cells the role of intracellular PGE₂ in several events linked to prostate cancer progression. To this end, we used bromocresol green, an inhibitor of prostaglandin uptake that blocked the immediate rise in intracellular immunoreactive PGE₂ following treatment with 16,16-dimethyl-PGE₂. Bromocresol green prevented the stimulatory effect of 16,16-dimethyl-PGE on cell proliferation, adhesion, migration and invasion and on HIF-1α expression and activity, the latter assessed as the HIF-dependent activation of (i) a hypoxia response element-luciferase plasmid construct, (ii) production of angiogenic factor vascular endothelial growth factor-A and (iii) in vitro angiogenesis. The basal phenotype of PC3 cells was also affected by bromocresol green, that substantially lowered expression of HIF-1α, production of vascular endothelial growth factor-A and cell proliferation. These results, and the fact that we found functional intracellular EP receptors in PC3 cells, suggest that PGE₂-dependent intracrine mechanisms play a role in prostate cancer Therefore, inhibition of the prostaglandin uptake transporter might be a novel therapeutic approach for the treatment of prostate cancer.     

Effects of pro-inflammatory cytokines,lipopolysaccharide and COX-2 mediators on human colonic neuromuscular function and epithelial permeability

Chronic colitis is associated with decreased colonic muscle contraction and loss of mucosal barrier function. Pro-inflammatory cytokines and bacterial lipopolysaccharide (LPS) are important in the generation and maintenance of inflammation. While colitis is associated with upregulated COX-2 -derived prostanoids and nitric oxide (NO), the direct activity of pro-inflammatory cytokines on human colonic neuromuscular function is less clear. This study investigated the effects of IBD-associated pro-inflammatory cytokines IL-17, TNF-α, IL-1β and LPS on human colonic muscle strip contractility, alone and following inhibition of COX-2 or nitric oxide production. In addition, human colonic epithelial Caco-2 cell monolayers were treated with LPS or COX-2 mediators including prostaglandins (PGE₂, PGF_2α) or their corresponding ethanolamides (PGE₂-EA or PGF_2α-EA) over 48 h and trans-epithelial electrical resistance used to record permeability changes. Longitudinal muscle strips were obtained from healthy colonic resection margins and mounted in organ baths following IL-17, TNF-α, IL-1β and bacterial LPS incubations in an explant setting over 20 h. Contraction in response to acetylcholine (ACh) was then measured, before and after either COX-2 inhibition (nimesulide; 10⁻⁵ M) or nitric oxide synthase (NOS) inhibition (l-NNA; 10⁻⁴ M). None of the cytokine or LPS explant incubations affected the potency or maximum cholinergic contraction in vitro, and subsequent COX-2 blockade with nimesulide revealed a significant but similar decrease in potency of ACh-evoked contraction in control, LPS and cytokine-incubated muscle strips. Pre-treatment with l-NNA provided no functional differences in the potency or maximum contractile responses to ACh in cytokine or LPS-incubated colonic longitudinal smooth muscle. Only PGE₂ transiently increased Caco-2 monolayer permeability at 24 h, while LPS (10 μg/ml) increased permeability over 24–48 h.These findings indicate that cholinergic contractility in the human colon can be decreased by the blockade of COX-2 generated excitatory prostanoids, but major pro-inflammatory cytokines or LPS do not alter the sensitivity or amplitude of this contraction ex vivo. While PGE₂ transiently increase epithelial permeability, LPS generates a significant and sustained increase in permeability indicative of an important role on barrier function at the mucosal interface.     

Inhibition of cyclooxygenase-2 and EP1 receptor antagonism reduces human colonic longitudinal muscle contractility in vitro

We investigated the contribution of cyclo-oxygenase enzyme inhibition and prostamide agonism on human colonic contractility in vitro. The effects of the non-specific COX inhibitor diclofenac were compared against selective COX-2 inhibition via nimesulide, the prostanoid EP₁ receptor antagonist SC19220 or the prostaglandin prodrug/prostamide receptor agonist bimatoprost, on potency of contraction to acetylcholine in human colonic circular and longitudinal muscle strips. Pre-treatment with either nimesulide (10^?5 M) or diclofenac (10^?6 M) caused a significant decrease in the potency of acetylcholine-evoked longitudinal muscle contraction, but did not inhibit acetylcholine-evoked circular muscle contraction. Pre-treatment with the EP₁ receptor antagonist SC19220 (10^?5 M) similarly decreased cholinergic potency in longitudinal muscle, without influence on circular muscle contraction. The prostamide agonist bimatoprost (10^?6 M) increased basal circular and longitudinal muscle tone, but did not alter cholinergic potency in either muscle layer. In conclusion, colonic longitudinal muscle contraction is augmented by COX-2 activity, most likely via PGE₂ acting at EP₁ receptors. While colonic contraction is tonically modulated by bimatoprost, it does not share the same functional properties attributed to other endogenous COX-2 metabolites on colonic contractile function.     

EP3 prostanoid receptor isoforms utilize distinct mechanisms to regulate ERK 1/2 activation

Prostaglandin-E₂ (PGE₂) is a hormone derived from the metabolism of arachidonic acid whose functions include regulation of platelet aggregation, fever and smooth muscle contraction/relaxation. PGE₂ mediates its physiological and pathophysiological effects through its binding to four G-protein coupled receptor subtypes, named EP₁, EP₂, EP₃ and EP₄. The EP₃ prostanoid receptor is unique in that it has multiple isoforms generated by alternative mRNA splicing. These splice variants display differences in tissue expression, constitutive activity and regulation of signaling molecules. To date there are few reports identifying differential activities of EP₃ receptor isoforms and their effects on gene regulation. We generated HEK cell lines expressing the human EP_3-Ia, EP_3-II or EP_3-III isoforms. Using immunoblot analysis we found that nM concentrations of PGE₂ strongly stimulated the phosphorylation of ERK 1/2 by the EP_3-II and EP_3-III isoforms; whereas, ERK 1/2 phosphorylation by the EP_3-Ia isoform was minimal and only occurred at μM concentrations of PGE₂. Furthermore, the mechanisms of the PGE₂ mediated phosphorylation of ERK 1/2 by the EP_3-II and EP_3-III isoforms were different. Thus, PGE₂ stimulation of ERK 1/2 phosphorylation by the EP_3-III isoform involves activation of a Gα_i/PI3K/PKC/Src and EGFR-dependent pathway; while for the EP_3-II isoform it involves activation of a Gα_i/Src and EGFR-dependent pathway. These differences result in unique differences in the regulation of reporter plasmid activity for the downstream effectors ELK1 and AP-1 by the EP_3-II and EP_3-III prostanoid receptor isoforms.     

EP2 receptor activation by prostaglandin E2 leads to induction of HO-1 via PKA and PI3K pathways in C6 cells

Recently we proposed that COX-2 induction precedes expression of HO-1 in ischemic preconditioned rat brain. In the current study, we investigated the molecular mechanism by which prostaglandin E₂, one of COX-2 metabolites, induces HO-1 in rat C6 brain cells. We demonstrated that concentration of PGE₂ increased HO-1 expression in C6 cells in vitro. The effects of PGE₂ were mimicked by PGE₂ receptor EP₂ agonists, 11-deoxy PGE₂, and cAMP analog, dibutyl-cAMP. HO-1 expression by PGE₂ was inhibited by LY294002, PI3K inhibitor and H89, PKA inhibitor. The EP₂-specific antagonist, AH8006 also inhibited PGE₂-mediated HO-1 expression in a concentration-dependent manner. Finally, PGE₂ inhibited GOX-induced apoptosis as assayed by FACS analysis or DNA strand breaks assay, and this cell death was reversed by ZnPPIX, HO-1 inhibitor. In addition to HO-1 induction, PGE₂ also increased phosphorylation of Bad by PKA- and PI3K-depednent manner. Taken together, we conclude that PGE₂ induces HO-1 protein expression through PKA and PI3K signaling pathways via EP₂ receptor in C6 cells. The induction of HO-1 along with increase of p-Bad by PGE₂ is responsible for anti-apoptosis against oxidant stress.     

Effects of adrenaline in human colon adenocarcinoma HT-29 cells

AimsStress has been implicated in the development of cancers. Adrenaline levels are increased in response to stress. The effects of adrenaline on colon cancer are largely unknown. The aims of the study are to determine the effects of adrenaline in human colon adenocarcinoma HT-29 cells and the possible underlying mechanisms involved.Main methodsThe effect of adrenaline on HT-29 cell proliferation was determined by [³H] thymidine incorporation assay. Expression of cyclooxygenase-2 (COX-2) and vascular endothelial growth factor (VEGF) were detected by Western blot. Matrix metalloproteinase-9 (MMP-9) activity and prostaglandin E₂ (PGE₂) release were determined by zymography and enzyme immunoassay, respectively.Key findingsAdrenaline stimulated HT-29 cell proliferation. This was accompanied by the enhanced expression of COX-2 and VEGF in HT-29 cells. Adrenaline also upregulated MMP-9 activity and PGE₂ release. Adrenaline stimulated HT-29 cell proliferation which was reversed by COX-2 inhibitor sc-236. COX-2 inhibitor also reverted the action of adrenaline on VEGF expression and MMP-9 activity. Further study was performed to determine the involvement of β-adrenoceptors. The stimulatory action of adrenaline on colon cancer growth was blocked by atenolol and ICI 118,551, a β₁- and β₂-selective antagonist, respectively. This signified the role of β-adrenoceptors in this process. In addition, both antagonists also abrogated the stimulating actions of adrenaline on COX-2, VEGF expression, MMP-9 activity and PGE₂ release in HT-29 cells.SignificanceThese results suggest that adrenaline stimulates cell proliferation of HT-29 cells via both β₁- and β₂-adrenoceptors by a COX-2 dependent pathway.