Prostaglandin E(2) regulates wound closure in airway epithelium

Repair of the airway epithelium after injury is critical for the maintenance of barrier function and the limitation of airway hyperreactivity. Airway epithelial cells (AECs) metabolize arachidonic acid to biologically active eicosanoids via the enzyme cyclooxygenase (COX). We investigated whether stimulating or inhibiting COX metabolites would affect wound closure in monolayers of cultured AECs. Inhibiting COX with indomethacin resulted in a dose-dependent inhibition of wound closure in human and feline AECs. Specific inhibitors for both COX-1 and COX-2 isoforms impaired wound healing. Inhibitors of 5-lipoxygenase did not affect wound closure in these cells. The addition of prostaglandin E(2) (PGE(2)) eliminated the inhibition due to indomethacin treatment, and the exogenous application of PGE(2) stimulated wound closure in a dose-dependent manner. Inhibition of COX with indomethacin only at initial time points resulted in a sustained inhibition of wound closure, indicating that prostanoids are involved in early wound repair processes such as spreading and migration. These differences in wound closure may be important if arachidonic acid metabolism and eicosanoid concentrations are altered in disease states such as asthma.     

Colon carcinoma cell growth is associated with prostaglandin E2/EP4 receptor-evoked ERK activation

Cyclooxygenase (COX) and its prostanoid metabolites have been implicated in the control of cell survival; however, their role as mitogens remains undefined. To better understand the role of prostanoids on cell growth, we used mouse colon adenocarcinoma (CT26) cells to investigate the role of prostaglandin E(2) (PGE(2)) in cell proliferation. CT26 cells express both COX1 and COX2 and metabolize arachidonic acid to PGE(2.) Treatment with indomethacin, or COX-selective inhibitors, prevents PGE(2) biosynthesis and CT26 cell proliferation. The anti-proliferative effects of COX inhibition are rescued specifically by treatment with PGE(2) or the EP4 receptor-selective agonist PGE(1)-OH via phosphatidylinositol 3-kinase/extracellular signal-regulated kinase (ERK) activation, thus providing a functional link between PGE(2)-induced cell proliferation and EP4-mediated ERK signaling. Indomethacin or COX2 inhibitors, but not COX1 inhibitors, reduced the size and number of CT26-derived tumors in vivo. These inhibitory effects are paralleled by marked declines in the levels of tumor PGE(2), suggesting that their anti-tumor effects are directly associated with the inhibition of COX2 enzymatic activity. The described anti-tumor effects of indomethacin are evident whether it is administered at the time of, or 7 days after, tumor cell injection, suggesting that it has tumor preventive and therapeutic actions. Furthermore, the observation that indomethacin increases the survival rates of tumor-bearing mice, even after withdrawal of the drug, indicates that its effects are long lasting and that it may be potentially useful for the prevention and the clinical management of human cancers.     

Proinflammatory actions of thromboxane receptors to enhance cellular immune responses

Metabolism of arachidonic acid by the cyclo-oxygenase (COX) pathway generates a family of prostanoid mediators. Nonsteroidal anti-inflammatory drugs (NSAIDs) act by inhibiting COX, thereby reducing prostanoid synthesis. The efficacy of these agents in reducing inflammation suggests a dominant proinflammatory role for the COX pathway. However, the actions of COX metabolites are complex, and certain prostanoids, such as PGE(2), in some circumstances actually inhibit immune and inflammatory responses. In these studies, we examine the hypothesis that anti-inflammatory actions of NSAIDs may be due, in part, to inhibition of thromboxane A(2) synthesis. To study the immunoregulatory actions of thromboxane A(2), we used mice with a targeted disruption of the gene encoding the thromboxane-prostanoid (TP) receptor. Both mitogen-induced responses and cellular responses to alloantigen were substantially reduced in TP(-/-) spleen cells. Similar attenuation was observed with pharmacological inhibition of TP signaling in wild-type splenocytes, suggesting that reduced responsiveness was not due to subtle developmental abnormalities in the TP-deficient mice. The absence of TP receptors reduced immune-mediated tissue injury following cardiac transplant rejection, an in vivo model of intense inflammation. Taken together, these findings show that thromboxane augments cellular immune responses and inflammatory tissue injury. Specific inhibition of the TP receptor may provide a more precise approach to limit inflammation without some of the untoward effects associated with NSAIDs.     

Eicosanoid-mediated proinflammatory activity of Pseudomonas aeruginosa ExoU

As Pseudomonas aeruginosa ExoU possesses two functional blocks of homology to calcium-independent (iPLA(2)) and cytosolic phospholipase A(2) (cPLA(2)), we addressed the question whether it would exhibit a proinflammatory activity by enhancing the synthesis of eicosanoids by host organisms. Endothelial cells from the HMEC-1 line infected with the ExoU-producing PA103 strain exhibited a potent release of arachidonic acid (AA) that could be significantly inhibited by methyl arachidonyl fluorophosphonate (MAFP), a specific PLA(2) inhibitor, as well as significant amounts of the cyclooxygenase (COX)-derived prostaglandins PGE(2) and PGI(2). Cells infected with an isogenic mutant defective in ExoU synthesis did not differ from non-infected cells in the AA release and produced prostanoids in significantly lower concentrations. Infection by PA103 induced a marked inflammatory response in two different in vivo experimental models. Inoculation of the parental bacteria into mice footpads led to an early increase in the infected limb volume that could be significantly reduced by inhibitors of both COX and lipoxygenase (ibuprofen and NDGA respectively). In an experimental respiratory infection model, bronchoalveolar lavage (BAL) from mice instilled with 10(4) cfu of PA103 exhibited a marked influx of inflammatory cells and PGE(2) release that could be significantly reduced by indomethacin, a non-selective COX inhibitor. Our results suggest that ExoU may contribute to P. aeruginosa pathogenesis by inducing an eicosanoid-mediated inflammatory response of host organisms.     

The induction of cyclooxygenase-2 in IL-1beta-treated endothelial cells is inhibited by prostaglandin E2 through cAMP

Prostaglandins (PGs) have numerous cardiovascular and inflammatory effects. Cyclooxygenase (COX), which exists as COX-1 and COX-2 isoforms, is the first enzyme in the pathway in which arachidonic acid is converted to PGs. Prostaglandin E2 (PGE2) exerts a variety of biological activities for the maintenance of local homeostasis in the body. Elucidation of PGE2 involvement in the signalling molecules such as COX could lead to potential therapeutic interventions. Here, we have investigated the effects of PGE2 on the induction of COX-2 in human umbilical vein endothelial cells (HUVEC) treated with interleukin-1beta (IL-1beta 1 ng/ml). COX activity was measured by the production of 6-keto-PGF1alpha, PGE2, PGF2alpha and thromboxane B2 (TXB2) in the presence of exogenous arachidonic acids (10 microM for 10 min) using enzyme immunoassay (EIA). COX-1 and COX-2 protein was measured by immunoblotting using specific antibody. Untreated HUVEC contained only COX-1 protein while IL-1beta treated HUVEC contained COX-1 and COX-2 protein. PGE2 (3 microM for 24h) did not affect on COX activity and protein in untreated HUVEC. Interestingly, PGE2 (3 microM for 24h) can inhibit COX-2 protein, but not COX-1 protein, expressed in HUVEC treated with IL-1beta. This inhibition was reversed by coincubation with forskolin (100 microM). The increased COX activity in HUVEC treated with IL-1beta was also inhibited by PGE2 (0.03, 0.3 and 3 microM for 24h) in a dose-dependent manner. Similarly, forskolin (10, 50 or 100 microM) can also reverse the inhibition of PGE2 on increased COX activity in IL-1beta treated HUVEC. The results suggested that (i) PGE2 can initiate negative feedback regulation in the induction of COX-2 elicited by IL-1beta in endothelial cells, (ii) the inhibition of PGE2 on COX-2 protein and activity in IL-1beta treated HUVEC is mediated by cAMP and (iii) the therapeutic use of PGE2 in the condition which COX-2 has been involved may have different roles.     

Biosynthesis of prostaglandins and thromboxane B2 by fetal lung homogenates

The conversion of arachidonic acid to prostaglandins (PG's) and thromboxane B2 (TXB2) was investigated in homogenates from fetal and adult bovine and rabbit lungs. Adult bovine lungs were very active in converting arachidonic acid (100 microgram/g tissue) to both PGE2 (10.7 microgram/g tissue) and TXB2 (6.2 microgram/g tissue. Smaller amounts of PGF2alpha (0.9 microgram/g) and 6-oxoPGF1alpha were formed. Homogenates from fetal calf lungs during the third trimester of pregnancy were quite active in converting arachidonic acid to PGE2, but formed very little TXB2, PGF2alpha or 6-oxoPGF1alpha. Homogenates from rabbit lungs converted arachidonic acid (100 microgram/g) mainly to PGE2, both before and after birth. The amount of PGE2 formed increased during gestation to a maximum of about 6 microgram/g tissue at 28 days of gestation. It then decreased to a minimum (1.5 microgram/g) which was observed 8 days after birth, followed by an increase to about 4 microgram/g in older rabbits.     

Prostaglandin synthases: recent developments and a novel hypothesis

Cells are continuously exposed to cues, which signal cell survival or death. Fine-tuning of these conflicting signals is essential for tissue development and homeostasis, and defective pathways are linked to many disease processes, especially cancer. It is well established that prostaglandins (PGs), as signalling molecules, are important regulators of cell proliferation, differentiation and apoptosis. PG production has been a focus of many researchers interested in the mechanisms of parturition. Previously, investigators have focussed on the committed step of PG biosynthesis, the conversion by prostaglandin H synthase (PGHS; also termed cyclo-oxygenase, COX) of arachidonic acid (AA) (substrate) to PGH2, the common precursor for biosynthesis of the various prostanoids. However, recently the genes encoding the terminal synthase enzymes involved in converting PGH2 to each of the bioactive PGs, including the major uterotonic PGs, PGE2 (PGE synthase) and PGF2alpha (PGF synthase), have been cloned and characterized. This review highlights how the regulation of the expression and balance of key enzymes can produce, from a single precursor, prostanoids with varied and often opposing effects.     

Early genotoxic effects in gill cells and haemocytes of Dreissena polymorpha exposed to cadmium, B[a]P and a combination of B[a]P and Cd

The aim of this study was to assess the genotoxic potential of environmentally relevant concentrations of Cd on the zebra mussel, an important freshwater sentinel organism, and to determine the stability of DNA damage in gill cells and haemocytes. The oxidative DNA damage and the co-genotoxicity of Cd in combination with B[a]P were investigated. We measured DNA damage in haemocytes and gill cells of zebra mussels exposed for 11 days to a constant concentration of Cd (10μg/L), B[a]P (10μg/L) or the two combined chemicals (10μg/L+1μg/L). Enzymatic dissociation of gills with dispase gave the lower percentage DNA in tail, compared with collagenase/dispase or collagenase. Bioaccumulation of cadmium in the soft tissues of mussels exposed to CdCl(2) or CdCl(2)+B[a]P increased in a time-dependent manner indicating that both exposures were effective. Cd (10μg/L) is genotoxic only during the first 3 days of exposure in gill cells, while in haemocytes the genotoxicity of Cd was observed later. B[a]P (10μg/L) induced an early increase of DNA damage in gill cells (after 10h and 1 day), while in both gill cells and haemocytes, B[a]P caused a marked increase of DNA damage after 3 days of exposure. The Cd+B[a]P mixture decreased the DNA-damaging effect of Cd and B[a]P in both cell types. Cd induced an increase of DNA damage in Fpg-treated slides, indicating that Cd contributed to oxidative DNA damage. Cadmium induced a cytogenetic effect in gill cells, assessed by the number of micronuclei, throughout the duration of the exposure, while B[a]P did not induce any cytogenetic effect. B[a]P, Cd and Cd+B[a]P induced a transient increase in the number of bi-nucleated cells. Our data clearly show that gills are more sensitive to Cd and B[a]P, which makes them more suitable for future bio-monitoring studies.     

PGE(2) receptors and synthesis in human gastric mucosa: perturbation in cancer

Recent evidence suggests that prostanoids are an important participant in the pathobiology of gastric adenocarcinoma, but the location and identity of cells in tumor-adjacent gastric mucosa able to synthesize and/or bind specific prostanoids is not clear. Using probes for cyclooxygenase 1 and 2 mRNA and protein as well as for the EP family of PGE(2) receptors, we sought to define the biology of prostanoids in adjacent human gastric mucosa at the site of tumor invasion.In mucosa adjacent to an invasive gastric adenocarcinoma, expression of cyclooxygenase was prominent, with COX 1 primarily in mucosal T lymphocytes surrounding nests of tumor cells. Densitometry showed these tumor-adjacent cells had substantial levels of COX 1 immunoreactive protein (relative intensity, 3.2). Cyclooxygenase 2 was newly expressed among these cells as well, but was limited in number (<25% of cyclooxygenase-positive T lymphocytes) in tumor-adjacent mucosa. Further, CD3(+) mononuclear cells, adjacent to tumor, strongly expressed prostanoid receptor EP(4) (relative intensity, 8.0), but cells with this receptor were not evident in the tumor itself. In contrast, normal gastric mucosa showed a consistent and structured expression of cyclooxygenase and PGE(2) receptor immunoreactive protein among mucosal cells. Cyclooxygenase 1 and PGE(2) receptor EP(4) were expressed on mucosal CD3(+) T lymphocytes in the lumenal (upper) third of gastric mucosa; and prostanoid receptors EP(2), EP(3) and EP(4), on gastric epithelia lining gastric pits. In situ hybridization with COX cDNAs confirmed these findings, and neither COX 2-specific mRNA nor protein was detected in normal gastric tissue. Our studies suggest that synthetic machinery and receptors for PGE(2), prominently expressed by T lymphocytes in gastric mucosa at the boundary of normal mucosa with tumor cells, may play a central role in prostanoid-driven tumorigenesis of this tissue.     

Roles of cyclooxygenase (COX)-1 and COX-2 in prostanoid production by human endothelial cells: selective up-regulation of prostacyclin synthesis by COX-2

The two cyclooxygenase (COX) isoforms, COX-1 and COX-2, both metabolize arachidonic acid to PGH(2), the common substrate for thromboxane A(2) (TXA(2)), prostacyclin (PGI(2)), and PGE(2) synthesis. We characterized the synthesis of these prostanoids in HUVECs in relation to COX-1 and COX-2 activity. Untreated HUVEC expressed only COX-1, whereas addition of IL-1beta caused induction of COX-2. TXA(2) was the predominant COX-1-derived product, and TXA(2) synthesis changed little with up-regulation of COX-2 by IL-1beta (2-fold increase). By contrast, COX-2 up-regulation was associated with large increases in the synthesis of PGI(2) and PGE(2) (54- and 84-fold increases, respectively). Addition of the selective COX-2 inhibitor, NS-398, almost completely abolished PGI(2) and PGE(2) synthesis, but had little effect on TXA(2) synthesis. The up-regulation of COX-2 by IL-1beta was accompanied by specific up-regulation of PGI synthase and PGE synthase, but not TX synthase. An examination of the substrate concentration dependencies showed that the pathway of TXA(2) synthesis was saturated at a 20-fold lower arachidonic acid concentration than that for PGI(2) and PGE(2) synthesis. In conclusion, endothelial prostanoid synthesis appears to be differentially regulated by the induction of COX-2. The apparent PGI(2) and PGE(2) linkage with COX-2 activity may be explained by a temporal increase in total COX activity, together with selective up-regulation of PGI synthase and PGE synthase, and different kinetic characteristics of the terminal synthases. These findings have particular importance with regard to the potential for cardiovascular consequences of COX-2 inhibition.     

A dietary conjugated linoleic acid treatment that slows renal disease progression alters renal cyclooxygenase-2-derived prostanoids in the Han: SPRD-cy rat

A mixture of dietary conjugated linoleic acid (CLA) isomers reduces inflammation and mitigates disease progression in the Han:SPRD-cy rat model of chronic kidney disease. Since cyclooxygenase (COX) activities and prostanoid levels are higher in diseased kidneys in this rat, and dietary CLA can inhibit COX2 and prostanoid production in other tissues, the effects of dietary CLA were investigated. Kidney homogenates from normal and diseased Han:SPRD-cy rats were analyzed for prostanoid levels under various conditions: endogenous levels, steady-state levels (60-min incubations) and produced by COX isoforms. Thromboxane B(2) (TXB(2); TXA(2) metabolite), 6-keto-prostaglandin F(1α) (6-keto-PGF(1α); PGI(2) metabolite) and PGE(2) levels under these conditions were two- to ninefold higher in diseased kidneys. Dietary CLA resulted in ～32%-53% lower levels of prostanoids produced by total COX and COX2 activities in normal and diseased kidneys and partially mitigated alterations in COX2 protein levels associated with disease. The COX1 protein and activity were higher in renal disease, resulting in increased production of TXB(2) and 6-ketoPGF(1α), but not PGE(2). Dietary CLA had no effect on COX1, however. Disease resulted in up to twofold higher ratios of TXB(2)/6-ketoPGF(1α), TXB(2)/PGE(2) and 6-ketoPGF(1α) /PGE(2), and dietary CLA partially mitigated these increases under several conditions. Elevated levels of renal membrane associated cytosolic phospholipase A(2) in diseased kidneys also were reduced by 50% with CLA feeding. The effects of CLA feeding on COX2 protein levels and activity indicate that the beneficial effect of dietary CLA in this renal disorder is mediated in part via effects on COX2-derived prostanoids.     

Evaluating the toxicity of environmental concentrations of waterborne chromium (VI) to a model teleost, Oncorhynchus mykiss: a comparative study of in vivo and in vitro

Toxic effects of environmental concentrations (50, 100, and 200μg/L) of waterborne chromium (VI) were evaluated in rainbow trout by comparison of in vitro and in vivo assays. Multiple biomarkers were measured including oxidative stress indices and antioxidant response parameters in liver and brain, as well as Na(+)-K(+)-ATPase in gill. Superoxide dismutase (SOD) and glutathione reductase (GR) activities were significantly induced (1.54-fold and 1.37-fold, respectively) in fish brain in vivo, but no significant differences were observed in any other biomarker or in vivo test group. Oxidative stress was apparent in vitro as significantly higher levels of oxidative indices, with the highest induction of TBARS and CP found in brain at 200μg/L Cr(VI) (2.41-fold and 1.95-fold, respectively), and SOD and GR activities and reduced glutathione in brain were significantly inhibited (65%, 44%, and 36%, respectively). In vitro Na(+)-K(+)-ATPase activity in gill was also significantly inhibited at concentrations of 100 and 200μg/L (69% and 45%, respectively). Short-term exposure to environmental concentrations of Cr(VI) does not therefore evoke marked effects in fish in vivo. Based on the present results, a set of in vitro tests with tissue homogenate can be evoked more remarkable effects by the lower concentrations of Cr(VI) than in vivo, which could provide some useful information and might be a potential alternative approach for monitoring heavy metal pollution in aquatic environments. However, it needs more detailed studies in other area, such as hormonal response or genotoxicity, before these findings could be applied in the field investigation.     

Tissue prostanoids as biomarkers for chemoprevention of colorectal neoplasia: correlation between prostanoid synthesis and clinical response in familial adenomatous polyposis

Recent studies indicate that sulindac, a nonsteroidal anti-inflammatory drug (NSAID), lowers mucosal prostanoid levels and regresses colorectal adenomas in patients with familial adenomatous polyposis (FAP). To determine whether they are biomarkers for sulindac-mediated chemoprevention of colorectal adenomas, levels of 5 prostanoids [prostaglandin (PG) D2, PGE2, PGF2alpha, thromboxane B2, and 6-keto-PGF1alpha] in the normal-appearing rectal mucosa from 7 FAP patients with a history of subtotal colectomy and ileorectal anastomosis and 4 FAP patients without surgery, were measured in the absence or presence of exogenously added arachidonic acid before the initiation and at the end of 3 months of sulindac treatment. The addition of arachidonic acid resulted in a uniform increase in the levels of all 5 prostanoids although this increase was selectively attenuated in patients with ileorectal anastomosis who took sulindac. In the latter patients, arachidonic acid also augmented the inhibition of prostanoid synthesis by sulindac. In contrast, sulindac failed to attenuate the increase in prostanoid levels resulting from arachidonic acid in patients without previous surgery. Importantly, when measured in the presence of arachidonic acid, the reduction in the levels of all 5 prostanoids due to sulindac was statistically correlated with a reduction in the size and number of adenomas in the two groups of patients combined. These results suggest that tissue prostanoids measured in the presence of arachidonic acid may serve as sensitive and reliable biomarkers in monitoring the clinical responsiveness of FAP patients undergoing chemoprevention for colorectal neoplasia with NSAIDs.     

Prostanoid synthesis and vascular responses to exogenous arachidonic acid following cerebral ischemia in piglets

In newborn pigs, cerebral ischemia abolishes both increased cerebral prostanoid production and cerebral vasodilation in response to hypercapnia and hypotension. Attenuation of prostaglandin endoperoxide synthase activity could account for the failure to increase prostanoid synthesis and loss of responses to these stimuli. To test this possibility, arachidonic acid (3, 6, or 30 micrograms/ml) was placed under cranial windows in newborn pigs that had been exposed to 20 min of cerebral ischemia. The conversion to prostanoids and pial arteriolar responses to the arachidonic acid were measured. At all three concentrations, arachidonic acid caused similar increases in pial arteriolar diameter in sham control piglets and piglets 1 hr postischemia. Topical arachidonic acid caused dose-dependent increases of PGE2 in cortical periarachnoid cerebral spinal fluid. 6-keto-PGF1 alpha and TXB2 only increased at the highest concentration of arachidonic acid (30 micrograms/ml). Cerebral ischemia did not decrease the conversion of any concentration of arachidonic acid to PGE2, 6-keto-PGF1 alpha, or TXB2. We conclude that ischemia and subsequent reperfusion do not result in inhibition of prostaglandin endoperoxide synthase in the newborn pig brain. Therefore, the mechanism for the impaired prostanoid production in response to hypercapnia and hypotension following cerebral ischemia appears to involve reduction in release of free arachidonic acid.     

Mechanism of steroid action in inflammation: Inhibition of prostaglandin synthesis and release

Acute arthritis was induced by injection of cell-free extract of group A Streptococci into the knee joints of mature male rats. Slices of control and inflamed synovia were incubated for 30 to 240 minutes and the rate of prostaglandin E (PGE) released into the medium was measured by radioimmunoassay. PGE release from inflamed synovia was 5–8 fold higher than that in normal tissue. Incubation of inflamed synovia with corticosterone acetate, dexamethasone or prednisone (100 μg/ml) for one or four hours reduced PGE release by 33% and 55% respectively. Lower concentrations of corticosterone (10 – 30 μg/ml) were ineffective. Aldosterone and progesterone (100 μg/ml) had no effect on PGE release throughout the incubation period. Chloroquine (10 μg/ml) inhibited PGE release from inflamed synovia by 50%. Indomethacin (1 μg/ml) abolished PGE release by 90%. Corticosterone, dexamethasone and prednisone reduced PGE content of inflamed synovia by approximately 45% during a 4-h incubation period. Aldosterone and progesterone were ineffective, while indomethacin reduced PGE content by 70%. The suppressive action of corticosterone on PGE release was prevented by addition to the medium of arachidonic acid (2 μg/ml). By contrast, the inhibitory action of indomethacin was not affected by provision of exogenous substrate. We suggest that glucocorticosteroids reduce PGE release by limiting the availability of the substrate for prostaglandin biosynthesis, and this may well explain some of their anti-inflammatory properties.     

Extracellular histones disarrange vasoactive mediators release through a COX‐NOS interaction in human endothelial cells

Extracellular histones are mediators of inflammation, tissue injury and organ dysfunction. Interactions between circulating histones and vascular endothelial cells are key events in histone‐mediated pathologies. Our aim was to investigate the implication of extracellular histones in the production of the major vasoactive compounds released by human endothelial cells (HUVECs), prostanoids and nitric oxide (NO). HUVEC exposed to increasing concentrations of histones (0.001 to 100 μg/ml) for 4 hrs induced prostacyclin (PGI2) production in a dose‐dependent manner and decreased thromboxane A2 (TXA2) release at 100 μg/ml. Extracellular histones raised cyclooxygenase‐2 (COX‐2) and prostacyclin synthase (PGIS) mRNA and protein expression, decreased COX‐1 mRNA levels and did not change thromboxane A2 synthase (TXAS) expression. Moreover, extracellular histones decreased both, eNOS expression and NO production in HUVEC. The impaired NO production was related to COX‐2 activity and superoxide production since was reversed after celecoxib (10 μmol/l) and tempol (100 μmol/l) treatments, respectively. In conclusion, our findings suggest that extracellular histones stimulate the release of endothelial‐dependent mediators through an up‐regulation in COX‐2‐PGIS‐PGI2 pathway which involves a COX‐2‐dependent superoxide production that decreases the activity of eNOS and the NO production. These effects may contribute to the endothelial cell dysfunction observed in histone‐mediated pathologies.     

Intracellular measurement of prostaglandin E2: effect of anti-inflammatory drugs on cyclooxygenase activity and prostanoid expression.

Cyclooxygenase (COX) converts arachidonic acid to prostaglandin (PG) H2, which is further metabolized to various prostaglandins, prostacyclin and thromboxane A2. COX exists in at least two different isoforms. COX-1 is constitutively expressed, whereas COX-2 is induced by proinflammatory stimuli. Prostaglandin E2 is a major metabolite of COX activation. In order to compare the activity of target ligands and COX inhibitors on PGE2 synthesis and release, the responsiveness of several cell lines to the calcium ionophore A23187, bacterial lipopolysaccharide (LPS), nonsteroidal anti-inflammatory drugs (NSAIDs), and the glucocorticoid, dexamethasone, were investigated. For intracellular measurements, the culture supernatant was aspirated, and the cells were thoroughly washed and lysed with dodecyltrimethylammonium bromide. Intracellular and secreted PGE2 were measured with an enzyme immunoassay. A23187 and LPS increased intracellular PGE2 in a dose-dependent manner. Kinetic experiments with A23187-stimulated mouse 3T3 fibroblast cells revealed a distinct biphasic response in COX activity. In the presence of NSAIDs or dexamethasone, there was a dose-dependent inhibition in intracellular PGE2 with A23187-stimulated 3T3 cells. Inhibitory studies demonstrated an apparent increased sensitivity of COX activity to the action of inhibitors when measuring intracellular PGE2 compared with using cell culture supernatants. Indeed, intracellular PGE2 levels were comprehensively reduced in the presence of low concentrations of inhibitor. The utilization of cell culture lysates and, in particular, measurement of intracellular PGE2 should prove useful for identifying new COX inhibitors.     

A plant steroid, diosgenin, induces apoptosis, cell cycle arrest and COX activity in osteosarcoma cells

Cyclooxygenases (COXs) are key enzymes in the conversion of arachidonic acid into prostanoids which are involved in apoptosis and inflammation. Two distinct COXs have been identified: COX-1 which is constitutively expressed and COX-2 which is induced by different products such as tumor promoters or growth factors. Previously, we demonstrated that a plant steroid, diosgenin, was a new megakaryocytic differentiation inducer of human erythroleukemia cells. In our study, we investigated the effect of diosgenin on the proliferation rate, cell cycle distribution and apoptosis in the human osteosarcoma 1547 cell line. The effects of this compound were also tested on COX expression and COX activities. Diosgenin treatment caused an inhibition of 1547 cell growth with a cycle arrest in G1 phase and apoptosis induction. Moreover, we found a correlation between p53, p21 mRNA expression and nuclear factor-kappaB activation and we observed a time-dependent increase in PGE2 synthesis after diosgenin treatment.     

Effects of prostaglandins E1, E2, and F2alpha on the growth of leukaemia cells in culture.

Prostaglandins E1, E2, and F2alpha (PGE1, PGE2, and PGF2alpha) were shown to inhibit the growth of mouse leukaemia lymphoblasts L5178Y in culture. The effects of PGE1 and PGE2 were greater than that of PGF2alpha. PGE1 and PGE2, at the concentration of 100 mug per ml showed significant inhibitory effects on the rates of incorporation of tritiated thymidine, uridine and leucine. At concentrations of 50 and 25 mug per ml, there was significant inhibition of thymidine and uridine incorporation, but not of leucine, PGF2alpha showed significant inhibition of thymidine and uridine incorporation but not leucine incorporation, in all 3 concentrations studied (100, 50, and 25 mug/ml). The ability of the cells to form colonies in soft agar was significantly inhibited by PGE1 and PGE2 at concentrations as low as 1-8 mug/ml. For F2alpha, however, a concentration as high as 56mug/ml was required to show inhibitory effect, but at 1-8 mug/ml it was found to be stimulatory.     

Cyclooxygenase inhibitors not inhibit resting lung cancer A549 cell proliferation

Cyclooxygenase (COX) inhibitors were regarded as anticarcinogenic agents for lung cancer at least partly via PGE2; but these were based on cytokin stimulation experiment on A549 cell. In order to clarify whether COX inhibitors directly inhibit A549 cell, three COX inhibitors, NS398 (selective COX-2 inhibitor), SC560 (selective COX-1 inhibitor), and acetyl salicylic acid (ASA, non-selective COX inhibitor), were studied. NS398, and ASA, can inhibit PGE2 generation via COX-2 inhibition. The viability of A549 cell was assayed by MTT. However, without cytokin stimulation, all the three inhibitors (NS398 0.2-20 microM; SC560 1.0-100 nM; ASA 0.01-1.0 mM) were not able to inhibit A549 cell proliferation, in the other way round, NS398 promoted cell growth. And arachidonic acid (AA) and lipopolysaccharide (LPS) did not disturb the property of its growth. These data suggested that without cytokin stimulation, COX and PGE2 may not be the kernel molecules involved in A549 cell proliferation, and COX inhibitors could not inhibit A549 cell growth directly.