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.     

Atorvastatin reduces lipopolysaccharide-induced expression of cyclooxygenase-2 in human pulmonary epithelial cells

Objective

To explore the effects of atorvastatin on expression of cyclooxygenase-2 (COX-2) in human pulmonary epithelial cells (A549).

Methods

A549 cells were incubated in DMEM medium containing lipopolysaccharide (LPS) in the presence or absence of atorvastatin. After incubation, the medium was collected and the amount of prostaglandin E₂ (PGE₂) was measured by enzyme-linked immunosorbent assay (ELISA). The cells were harvested, and COX-2 mRNA and protein were analyzed by RT-PCR and western-blot respectively.

Results

LPS increased the expression of COX-2 mRNA and production of PGE₂ in a dose- and time-dependent manner in A549. Induction of COX-2 mRNA and protein by LPS were inhibited by atorvastatin in a dose-dependent manner. Atorvastatin also significantly decreased LPS-induced production of PGE₂. There was a positive correlation between reduced of COX-2 mRNA and decreased of PGE₂ (r = 0.947, P < 0.05).

Conclusion

Atorvastatin down-regulates LPS-induced expression of the COX-2 and consequently inhibits production of PGE₂ in cultured A549 cells.     

Flurbiprofen,A Cyclooxygenase Inhibitor,Reduces the Brain Arachidonic Acid Signal in Response to the Cholinergic Muscarinic Agonist,Arecoline, in Awake Rats

Cholinergic muscarinic receptors, when stimulated by arecoline, can activate cytosolic phospholipase A₂ (cPLA₂) to release arachidonic acid (AA) from membrane phospholipid. This signal can be imaged in the brain in vivo using quantitative autoradiography following the intravenous injection of radiolabeled AA, as an increment in a regional brain AA incorporation coefficient k*. Arecoline increases k* significantly in brain regions having muscarinic M_1,3,5 receptors in wild-type but not in cyclooxygenase (COX)-2 knockout mice. To further clarify the roles of COX enzymes in the AA signal, in this paper we imaged k* following arecoline (5 mg/kg i.p.) or saline in each of 81 brain regions of unanesthetized rats pretreated 6 h earlier with the non-selective COX inhibitor flurbiprofen (FB, 60 mg/kg s.c.) or with vehicle. Baseline values of k* were unaffected by FB treatment, which however reduced by 80% baseline brain concentrations of prostaglandin E₂ (PGE₂) and thromboxane B₂ (TXB₂), eicosanoids preferentially derived from AA via COX-2 and COX-1, respectively. In vehicle-pretreated rats, arecoline increased the brain PGE₂ but not TXB₂ concentration, as well as values for k* in 77 of the 81 brain regions. FB-pretreatment prevented these arecoline-provoked changes. These results and those reported in COX-2 knockout mice suggest that the AA released in brain following muscarinic receptor-mediated activation is lost via COX-2 to PGE₂ but not via COX-1 to TXB₂, and that increments in k* following arecoline largely represent replacement by unesterified plasma AA of this loss.     

Prostaglandin E2-stimulated glandular ion and water secretion in isolated frog skin (Rana esculenta)

Summary Prostaglandins are known to stimulate the active sodium absorption in frog skin. In this paper it is shown that prostaglandin E₂ (PGE₂) stimulates an active secretion of Cl^–, Na⁺, and K⁺ from the skin glands inRana esculenta. The active Cl secretion is enhanced more than the Na and K secretion. Therefore, in skins where the Na absorption is inhibited by amiloride, the addition of PGE₂ results in an increase in the short-circuit current (SCC). The PGE₂-stimulated Cl secretion could be inhibited by the presence of ouabain or furosemide in the basolateral solution or diphenylamine-2-carboxylate in the apical solution. The PGE₂-stimulated Cl secretion was enhanced by the phosphodiesterase inhibitor, theophylline, indicating that the effect of PGE₂ was caused by an increase in the intracellular cAMP level in the gland cells. The calcium ionophore A23187, which increases the PGE₂ synthesis in frog skin, stimulated the glandular Cl secretion. This secretion could be blocked by the prostaglandin synthesis inhibitor indomethacin, indicating that A23187 acts by increasing the prostaglandin synthesis and not by a direct action of Ca²⁺ ionsper se. The net water flow (J _w) and the Cl secretion were measured simultaneously under the conditions outlined above. The stimulation, inhibition, and the time-course of the outward-directedJ _w were similar to the change observed for the Cl secretion. These results show that PGE₂ stimulates a glandular secretion of Cl and water in frog skin, probably by increasing the cAMP level in the gland cells.     

Rat epiphyseal cells in culture: Responsiveness to bone-seeking hormones

Summary Cell cultures derived from young rat epiphyseal cartilage were grown for approximately 2 wk in BGJ _b medium supplemented with 10% fetal bovine serum to reach confluence. These cells were identified as chondrocytes as checked by morphology, the presence of alkaline phosphatase, and a positive type II collagen antibody reaction. The cells also responded to different hormonal treatment. Parathyroid hormone (PTH) increased cyclic AMP production by 50% within 15 min of treatment, whereas prostaglandin E₂ (PGE₂) caused an increase of 160%. Calcitonin (CT) did not affect cAMP production in these cells. DNA synthesis 24 h after hormonal treatment was increased by PTH (2.5-fold) and PGE₂ (2-fold), but not by CT. Among the vitamin D metabolites, 24,25(OH)₂D₃ increased significantly the [³H]thymidine incorporation into DNA, whereas 1,25(OH)₂D₃ effect was minimal. These results provide evidence for the use of these cell cultures as a model for cartilage in vitro when studying biological and hormonal responsiveness.     

PGE2 involvement in experimental infection with Trypanosoma cruzi subpopulations

PGE₂ involvement in experimental Trypanosoma cruzi infection depends on the lethal capacity of the parasite subpopulation used. Mice acutely infected with non-lethal K98 displayed an enhancement in PGE₂ serum levels during the acute period, while those infected with lethal T. cruzi subpopulations (RA or K98-2) showed levels not different from normal mice. The enhancement detected in K98 group could be related both to an increased number of CD8+ T cell number and to enhanced PGE₂ release per cell by CD8+; values of PGE₂ release by adherent cells were not altered in this group. Treatment with cyclooxygenase inhibitors enhanced mortality rates of mice infected with K98, and administration of 16,16-dimethyl PGE₂ (dPGE) reversed this effect. However, mice infected with RA did not reduce their mortality rates by administration of diverse doses of dPGE. These findings suggest that PGE₂ could play a role in resistance in mice infected with K98.     

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.     

Dietary arachidonic acid suppresses bone turnover in contrast to low dosage exogenous prostaglandin E2 that elevates bone formation in the piglet

This study was designed to compare the effects of dietary arachidonic acid (AA) versus prostaglandin E₂ (PGE₂) on bone cell metabolism and bone mass. Twenty-eight piglets from 7 litters were randomized to 1 of 4 treatments for 15 days: fatty acid supplemented formula (FA: 0.8% of total fatty acids as AA and 0.1% of total fatty acids as DHA)+PGE₂ injections (0.1 mg/kg/day), FA+saline injections, standard formula (STD: n-6:n-3 of 8:1) + PGE₂ injections or STD+saline injections. PGE₂ resulted in elevated osteoblast activity as indicated by plasma osteocalcin and also reduced urinary calcium excretion. Dietary FA resulted in reduced bone resorption as indicated by urinary N-telopeptide and reduced bone PGE₂. Both PGE₂ and FA treatments independently lead to elevated femur mineral content, but the combined treatment caused a reduction. Thus the mechanisms by which PGE₂ and FA lead to enhanced bone mass are distinct.     

Production of leukotriene B4 and prostaglandin E2 by turbot (Scophthalmus maximus) leukocytes

Production of two eicosanoids derived from lipoxygenase and cyclooxygenase activities: leukotriene B₄ (LTB₄) and prostaglandin E₂ (PGE₂), respectively, have been simultaneously determined in turbot (Scophthalmus maximus) blood leucocyte and kidney macrophage supernatants by a reverse phase high performance liquid chromatography (HPLC) system coupled with a Diode–Array detector. Levels of LTB₄ after calcium ionophore challenge were 4.08 ng ml⁻¹ in blood leukocyte supernatants and 0.25 ng ml⁻¹ in kidney macrophage supernatants. The levels found for PGE₂ were 428.23 and 606.67 ng ml⁻¹ in blood leukocytes and kidney macrophage supernatants, respectively. When blood leukocytes were treated with the respective inhibitors for the enzymes implicated on the synthesis of both compounds an inhibition of 90.35% was observed for PGE₂ and 76.44% for LTB₄. The detection limit of the method was 0.15 ng ml⁻¹ for LTB₄ and 50 ng ml⁻¹ for PGE₂.     

Chronobiological Studies on the Hypotensive Effect of Prostaglandin E2 and Arachidonic Acid in the Rat

The present study was designed to determine whether biological rhythm variations could be detected in the hypotensive action of prostaglandin E₂ (PGE₂) and arachidonic acid (AA) in normal rats. Doses of 1.0 μg kg^?1 of PGE₂ or 0.5 mg kg^?1 of AA were administered to pentobarbital-anesthetized rats at 6 times of the day. Maximal reduction of systolic and diastolic blood pressures was obtained when PGE₂ or AA were administered to rats between 0930 and 1200. The lowest falls in blood pressure were found when the same doses of the two substances were injected between 0300 and 0500. Mechanisms to explain these circadian variations are suggested.     

Prostaglandin E2 receptors in asthma and in chronic rhinosinusitis/nasal polyps with and without aspirin hypersensitivity

Chronic rhinosinusitis with nasal polyps (CRSwNP) and asthma frequently coexist and are always present in patients with aspirin exacerbated respiratory disease (AERD). Although the pathogenic mechanisms of this condition are still unknown, AERD may be due, at least in part, to an imbalance in eicosanoid metabolism (increased production of cysteinyl leukotrienes (CysLTs) and reduced biosynthesis of prostaglandin (PG) E₂), possibly increasing and perpetuating the process of inflammation. PGE₂ results from the metabolism of arachidonic acid (AA) by cyclooxygenase (COX) enzymes, and seems to play a central role in homeostasis maintenance and inflammatory response modulation in airways. Therefore, the abnormal regulation of PGE₂ could contribute to the exacerbated processes observed in AERD. PGE₂ exerts its actions through four G-protein-coupled receptors designated E-prostanoid (EP) receptors EP1, EP2, EP3, and EP4. Altered PGE₂ production as well as differential EP receptor expression has been reported in both upper and lower airways of patients with AERD. Since the heterogeneity of these receptors is the key for the multiple biological effects of PGE₂ this review focuses on the studies available to elucidate the importance of these receptors in inflammatory airway diseases.

Electronic supplementary material

The online version of this article (doi:10.1186/s12931-014-0100-7) contains supplementary material, which is available to authorized users.     

Prostaglandin E2 is crucial in the response of podocytes to fluid flow shear stress

Podocytes play a key role in maintaining and modulating the filtration barrier of the glomerulus. Because of their location, podocytes are exposed to mechanical strain in the form of fluid flow shear stress (FFSS). Several human diseases are characterized by glomerular hyperfiltration, such as diabetes mellitus and hypertension. The response of podocytes to FFSS at physiological or pathological levels is not known. We exposed cultured podocytes to FFSS, and studied changes in actin cytoskeleton, prostaglandin E₂ (PGE₂) production and expression of cyclooxygenase-1 and–2 (COX-1, COX-2). FFSS caused a reduction in transversal F-actin stress filaments and the appearance of cortical actin network in the early recovery period. Cells exhibited a pattern similar to control state by 24 h following FFSS without significant loss of podocytes or apoptosis. FFSS caused increased levels of PGE₂ as early as 30 min after onset of shear stress, levels that increased over time. PGE₂ production by podocytes at post-2 h and post-24 h was also significantly increased compared to control cells (p < 0.039 and 0.012, respectively). Intracellular PGE₂ synthesis and expression of COX-2 was increased at post-2 h following FFSS. The expression of COX-1 mRNA was unchanged. We conclude that podocytes are sensitive and responsive to FFSS, exhibiting morphological and physiological changes. We believe that PGE₂ plays an important role in mechanoperception in podocytes.     

An accessory role for ceramide in interleukin-1β induced prostaglandin synthesis

Interleukin-1 (IL-1) is a potent inducer of prostaglandin E₂ (PGE₂) synthesis. We previously showed that ceramide accumulates in fibroblasts treated with IL-1 and that it enhances IL-1-induced PGE₂ production. The present study was undertaken to determine the mechanism(s) by which ceramide and IL-1 interact to enhance PGE₂ production by examining their respective effects on the rate-limiting enzymes in PGE₂ synthesis, cyclooxygenase-1 (COX-1), cyclooxygenase-2 (COX-2), and cytosolic phospholipase A₂ (cPLA₂). IL-1-induced PGE₂ synthesis required 8 h even though COX-1 was constitutively expressed (both mRNA and protein) and enzymatically active in untreated cells. Conversely, COX-2 mRNA was barely detectable in untreated cells but within 2 h, ceramide or IL-1 alone induced a 5 and 20 fold increase in COX-2 mRNA, respectively. However, IL-1 induced COX-2 protein synthesis was only detectable 6-7 h after maximal COX-2 mRNA induction; COX-2 protein accumulation was not induced by ceramide alone. Ceramide however, reduced the length of time required for IL- 1 to induce COX-2 protein accumulation and increased COX-2 protein accumulation. IL-1 induced a 15 fold increase in COX-1 mRNA including an alternatively spliced form of COX-1. IL-1, but not ceramide induced cPLA₂ mRNA and protein expression which corresponded with the initiation of PGE₂ synthesis. These observations indicate that, (1) while either ceramide or IL-1 rapidly induced COX-2 mRNA, COX-2 protein only accumulated in IL- 1 treated cells after a delay of 6-7 h, (2) IL-1-induced PGE₂ synthesis required both COX-2 and cPLA₂ protein synthesis and, (3) ceramide enhanced (temporally and quantitatively) IL-1-induced COX-2 protein accumulation resulting in enhanced PGE₂ production.     

Prostaglandin E1 or E2 inhibits an oxytocin-induced premature luteolysis in ewes when oxytocin is given early in the estrous cycle

The objective of this study was to determine whether PGE₁ or PGE₂ prevents a premature luteolysis when oxytocin is given on Days 1 to 6 of the ovine estrous cycle. Oxytocin given into the jugular vein every 8 hours on Days 1 to 6 postestrus in ewes decreased (P ≤ 0.05) luteal weights on Day 8 postestrus. Plasma progesterone differed (P ≤ 0.05) among the treatment groups; toward the end of the experimental period, concentrations of circulating progesterone in the oxytocin-only treatment group decreased (P ≤ 0.05) when compared with the other treatment groups. Plasma progesterone concentrations in ewes receiving PGE₁ or PGE₁ + oxytocin were greater (P ≤ 0.05) than in vehicle controls or in ewes receiving PGE₂ or PGE₂ + oxytocin and was greater (P ≤ 0.05) in all treatment groups receiving PGE₁ or PGE₂ than in ewes treated only with oxytocin. Chronic intrauterine treatment with PGE₁ or PGE₂ also prevented (P ≤ 0.05) oxytocin decreases in luteal unoccupied and occupied LH receptors on Day 8 postestrus. Oxytocin given alone on Days 1 to 6 postestrus in ewes advanced (P ≤ 0.05) increases in PGF_2α in inferior vena cava or uterine venous blood. PGE₁ or PGE₂ given alone did not affect (P ≥ 0.05) concentrations of PGF_2α in inferior vena cava and uterine venous blood when compared with vehicle controls or oxytocin-induced PGF_2α increases (P ≤ 0.05) in inferior vena cava or uterine venous blood. We concluded that PGE₁ or PGE₂ prevented oxytocin-induced premature luteolysis by preventing a loss of luteal unoccupied and occupied LH receptors.     

Decidual adenylate cyclase and prostaglandin production in vitro

Human decidua contains an active adenylate cyclase, and a number of studies indicate that adenylate cyclase is functionally linked to increased in vitro prostaglandin synthesis. Increased decidual prostaglandin synthesis is associated with parturition, and therefore activation of adenylate cyclase may be involved in the control of human parturition. In this study, third trimester human decidual cells were preincubated for no more than 24 h prior to stimulation with a number of reagents which increase cellular cyclic AMP levels. Forskolin rapidly increased intracellular and extracellular cyclic AMP levels, but there was no increase in prostaglandin E₂ biosynthesis during incubations ranging from 5 min up to 24 h. Dibutyryl cyclic AMP or 8-bromo-cyclic AMP were also without effect on PGE₂ production, which suggests that the adenylate cyclase was not linked to the mechanisms regulating prostaglandin production. Cholera toxin increased basal cyclic AMP and PGE₂ synthesis, and was without effect on IL-1β-stimulated PGE₂ levels. PGE₂ synthesis was increased by 24 h culture with IL-1β in all the cell preparations, indicating that the cells were biologically active, and that the lack of effect of changes in cyclic AMP synthesis on PGE₂ levels could not be attributed to a defect in the prostaglandin synthetic pathway. Our findings did not agree with earlier work which showed that changes in cyclic AMP were correlated with changes in PGE₂ production by human decidual cells. It is clear that in the previous studies the decidual cells were preincubated for 4–7 days prior to stimulation, in contrast with 24 h in our investigation. We suggest that the functional link between cyclic AMP and PGE₂ synthesis reported previously may develop during culture, and not be a part of normal decidual cell function, but further studies are needed to test this hypothesis.     

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

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

The effect of PGE2 on the activation of quiescent lung fibroblasts

The effect of prostaglandin E₂ (PGE₂) on fibroblast proliferation was examined. The presence of PGE₂ for 24 h inhibited the growth of quiescent cells stimulated with serum, platelet-derived growth factor and macrophage-derived factors. Maximal inhibition of nuclear labeling with [³H]thymidine occurred at concentrations greater than 10⁻⁷ M. The inhibitory effect of PGE₂ was less potent in exponentially growing cells and was not the result of conversion of PGE₂ to PGA₂ during incubation in growth medium. The G₁ phase was determined to be 12–14 h in untreated cultures. The extent of growth inhibition by PGE₂ was similar with addition of PGE₂ at 0, 3, 6, or 9 h following restimulation of quiescent cell cultures. Approximately 25% of the cells that enter S phase are refractory to PGE₂-induced growth inhibition. Short-term exposure to PGE₂ (5 min and 30 min) caused substantial growth inhibition. The serum-induced proliferation was also inhibited by the cAMP analogue, dibutyrl cAMP. Our results suggest that PGE₂ affects a distinct subpopulation of cells. Restimulation of quiescent cells treated with PGE₂ for 24 h, indicated that release from PGE₂ exposure is associated with prolongation of the G₁ phase of the cell cycle.     

A combination of aspirin and gamma-tocopherol is superior to that of aspirin and alpha-tocopherol in anti-inflammatory action and attenuation of aspirin-induced adverse effects

Nonsteroidal anti-inflammatory drugs such as aspirin are used for pain relief and chemoprevention against cancer, but frequently cause gastric mucosal injury. We examined whether combinations of aspirin and α-tocopherol (αT) or aspirin and γ-tocopherol (γT), with αT and γT being the two major forms of vitamin E, are better anti-inflammatory agents than aspirin alone, and whether these combinations alleviate aspirin-associated side effects. In the carrageenan-induced air-pouch inflammation model in the rat, aspirin (150 mg/kg) or a combination of aspirin and γT (33 mg/kg) inhibited proinflammatory prostaglandin E₂ (PGE₂) by 70% (P<.02) at the inflammation site 6 h after inflammation was initiated. However, at 18 h, only the combination decreased exudate volume (15%; P<.05) and showed modest inhibition of PGE₂ (40%; P<.07) and lactate dehydrogenase activity (30%; P=.07) in the fluid collected at the inflammation site. γT, but not αT, spared aspirin-induced reduction in food intake, partially reversed aspirin-depressed gastric PGE₂ and attenuated stomach lesions. Surprisingly, the combination of aspirin and αT (33 mg/kg) did not show more benefits than aspirin alone, but worsened gastric injury and food intake reduction. Our study demonstrated that a combination of aspirin and γT, but not a combination of aspirin and αT, has some advantage over aspirin alone in terms of anti-inflammatory effects and attenuation of aspirin-induced adverse effects. This combination may be useful in complementing aspirin in the treatment of chronic inflammatory conditions and cancer.