Piroxicam and NS-398 rescue neurones from hypoxia/reoxygenation damage by a mechanism independent of cyclo-oxygenase inhibition

We studied whether NS-398, a selective cyclo-oxygenase-2 (COX-2) enzyme inhibitor, and piroxicam, an inhibitor of COX-2 and the constitutively expressed COX-1, protect neurones against hypoxia/reoxygenation injury. Rat spinal cord cultures were exposed to hypoxia for 20 h followed by reoxygenation. Hypoxia/reoxygenation increased lactate dehydrogenase (LDH) release, which was inhibited by piroxicam (180-270 microM) and NS-398 (30 microM). Cell counts confirmed the neuroprotection. Western blotting revealed no COX-1 or COX-2 proteins even after hypoxia/reoxygenation. Production of prostaglandin E2 (PGE2), a marker of COX activity, was barely measurable and piroxicam and NS-398 had no effect on the negligible PGE2 production. Hypoxia/reoxygenation increased nuclear factor-kappa B (NF-kappaB) binding activity, which was inhibited by piroxicam but not by NS-398. AP-1 binding activity after hypoxia/reoxygenation was inhibited by piroxicam but strongly enhanced by NS-398. However, both COX inhibitors induced activation of extracellular signal-regulated kinase (ERK) in neurones and phosphorylation of heavy molecular weight neurofilaments, cytoskeletal substrates of ERK. It is concluded that piroxicam and NS-398 protect neurones against hypoxia/reperfusion. The protection is independent of COX activity and not solely explained by modulation of NF-kappaB and AP-1 binding activity. Instead, piroxicam and NS-398-induced phosphorylation through ERK pathway may contribute to the increased neuronal survival.     

Effects of selective and non-selective COX inhibitors on antigen-induced release of prostanoid mediators and bronchoconstriction in the isolated perfused and ventilated guinea pig lung

The contribution of cycloxygenase (COX)-1 and COX-2 in antigen-induced release of mediators and ensuing bronchoconstriction was investigated in the isolated perfused guinea pig lung (IPL). Antigen challenge with ovalbumin (OVA) of lungs from actively sensitised animals induced release of thromboxane (TX)A(2), prostaglandin (PG)D(2), PGF(2)(alpha), PGI(2) and PGE(2), measured in the lung effluent as immunoreactive TXB(2), PGD(2)-MOX, PGF(2)(alpha), 6-keto PGF(1)(alpha) and PGE(2), respectively. This release was abolished by the non-selective COX inhibitor flurbiprofen (10 microM). In contrast, neither the selective COX-1 inhibitor FR122047 nor the selective COX-2 inhibitor celecoxib (10 microM each) significantly inhibited the OVA-induced bronchoconstriction or release of COX products, except for PGD(2). Another non-selective COX inhibitor, diclofenac (10 microM) also significantly inhibited antigen-induced bronchoconstriction. The data suggest that both COX isoenzymes, COX-1 and COX-2 contribute to the immediate antigen-induced generation of prostanoids in IPL and that the COX-1 and COX-2 activities are not associated with different profiles of prostanoid end products.     

Lipoteichoic acid-induced nitric oxide synthase expression in RAW 264.7 macrophages is mediated by cyclooxygenase-2, prostaglandin E2, protein kinase A, p38 MAPK, and nuclear factor-kappaB pathways

We recently reported that lipoteichoic acid (LTA), a cell wall component of the gram-positive bacterium Staphylococcus aureus, stimulated inducible nitric oxide synthase (iNOS) expression, nitric oxide (NO) release, and cyclooxygenase-2 (COX-2) expression in RAW 264.7 macrophages. This study was carried out to further investigate the roles of COX-2 and prostaglandin E2 (PGE2) in LTA-induced iNOS expression and NO release in RAW 264.7 macrophages. Treatment of RAW 264.7 macrophages with LTA caused a time-dependent increase in PGE2 release. LTA-induced iNOS expression and NO release were inhibited by a non-selective COX inhibitor (indomethacin), a selective COX-2 inhibitor (NS-398), an adenylyl cyclase (AC) inhibitor (dideoxyadenosine, DDA), and a protein kinase A (PKA) inhibitor (KT-5720). Furthermore, both PGE2 and the direct PKA activator, dibutyryl-cAMP, also induced iNOS expression in a concentration-dependent manner. Stimulation of RAW 264.7 macrophages with LTA, PGE2, and dibutyryl-cAMP all caused p38 MAPK activation in a time-dependent manner. LTA-mediated p38 MAPK activation was inhibited by indomethacin, NS-398, and SB 203580, but not by PD 98059. The PGE2-mediated p38 MAPK activation was inhibited by DDA, KT-5720, and SB 203580, but not by PD 98059. LTA caused time-dependent activation of the nuclear factor-kappaB (NF-kappaB)-specific DNA-protein complex formation. The LTA-induced increase in kappaB-luciferase activity was inhibited by indomethacin, NS-398, KT-5720, and a dominant negative mutant of p38 alphaMAPK (p38 alphaMAPK DN). These results suggest that LTA-induced iNOS expression and NO release involve COX-2-generated PGE2 production, and AC, PKA, p38 MAPK, and NF-kappaB activation in RAW 264.7 macrophages.     

Expression of proteinase-activated receptor 2 on human primary gastrointestinal myofibroblasts and stimulation of prostaglandin synthesis

It is known that subepithelial myofibroblast-derived prostaglandin (PG)E2 can regulate intestinal epithelial cell functions, and that proteinase-activated receptor-2 (PAR2) is abundantly expressed in the gastrointestinal tract. Since PAR2 activation has previously been associated with stimulation of PGE2 synthesis, we hypothesized that PAR2 expressed on primary human gastrointestinal myofibroblasts regulates PGE2 synthesis via cyclooxygenase (COX)-1 and (or) COX-2, and associated PGE synthases. Primary human myofibroblasts were isolated from the resection tissue of the esophagus, small intestine, and colon. Expression of functional PAR2 was determined by RT-PCR and by calcium mobilization in Fura-2/AM-loaded cells. Trypsin and the selective PAR2-activating peptide (PAR2-AP) SLIGRL-NH2 stimulated PGE2 synthesis in a concentration-dependent manner, as measured by enzyme immunoassay. Selective COX inhibition showed PAR2-induced PGE2 synthesis to be COX-1 dependent in esophageal myofibroblasts and both COX-1 and COX-2 dependent in colonic cells, consistent with the distribution of COX-1 and COX-2 expression. Although both cytosolic and microsomal PGE synthases were expressed in cells from all tissues, microsomal PGE synthases were expressed at highest levels in the colonic myofibroblasts. Activation of PAR2 on gastrointestinal myofibroblasts stimulates PGE2 synthesis via different pathways in the colon than in the esophagus and small intestine.     

Tumor necrosis factor-alpha-induced cyclooxygenase-2 expression in human tracheal smooth muscle cells: involvement of p42/p44 and p38 mitogen-activated protein kinases and nuclear factor-kappaB

This study was to determine the mechanism of tumor necrosis factor-alpha (TNF-alpha)-enhanced cyclooxygenase (COX)-2 expression associated with prostaglandin E2 (PGE2) synthesis in human tracheal smooth muscle cells (HTSMCs). TNF-alpha markedly increased COX-2 expression and PGE2 synthesis in a time- and concentration-dependent manner, whereas COX-1 remained unaltered. Tyrosine kinase inhibitor (genistein), phosphatidylcholine-specific phospholipase C (PC-PLC) inhibitor (D-609) and PKC inhibitor (GF109203X) attenuated TNF-alpha-induced COX-2 expression and PGE2 synthesis in HTSMCs. TNF-alpha-induced COX-2 expression and PGE2 synthesis were also inhibited by PD98059 (an inhibitor of MEK1/2) and SB203580 and SB202190 (inhibitors of p38 MAPK), respectively, suggesting the involvement of p42/p44 and p38 MAPKs in these responses. This hypothesis was further supported by that TNF-alpha induced a transient activation of p42/p44 and p38 MAPKs in a time-and concentration-dependent manner. Furthermore, TNF-alpha-induced activation of nuclear factor-kappaB (NF-kappaB) reversely correlated with the degradation of IkappaB-alpha in HTSMCs. TNF-alpha-induced COX-2 expression and PGE2 synthesis was also inhibited by NF-kappaB inhibitor pyrrolidinedithiocarbamate (PDTC). These findings suggest that the increased expression of COX-2 correlates with the release of PGE2 from TNF-alpha-challenged HTSMCs, at least in part, mediated through p42/p44 and p38 MAPKs as well as NF-kappaB signaling pathways in HTSMCs.     

Chemoprevention of DMH-induced rat colon carcinoma initiation by combination administration of piroxicam and C-phycocyanin

Cancer research illustrated that combinatorial studies can provide significant improvement in safety and effectiveness over the monotherapy regimens. A combination of two drugs may restrain precancerous colon polyps, opening a new possible opportunity for chemoprevention of colon cancer. In this context, chemopreventive efficacy of a combination regimen of C-phycocyanin, a biliprotein present in Spirulina platensis, a cyanobacterium, which is a selective cycloxygenase-2 (COX-2) inhibitor and piroxicam, a traditional non-steroidal anti-inflammatory drug was considered in 1,2 dimethylhyadrazine (DMH)-induced colon carcinogenesis in rats. Western blotting, immunohistochemistry, DNA fragmentation, fluorescent staining, PGE(2) enzyme immunoassay, and carrageenan-induced paw edema test were performed along with morphological and histological analysis. DMH treatment showed a rich presence of preneoplastic lesions such as multiple plaque lesions, aberrant crypt foci, and well-characterized dysplasia. These features were reduced with piroxicam and C-phycocyanin administration. The number of apoptotic cells was featured prominently in all the groups compared with DMH. DMH treatment revealed intact high molecular weight genomic DNA with no signs of laddering/DNA fragmentation while it was noticeable significantly in control and DMH + piroxicam + C-phycocyanin. DMH group showed highest COX-2 expression and PGE(2) level in comparison with other groups. Doses of piroxicam and C-phycocyanin used in the present study were established at an anti-inflammatory range. A combination regimen of piroxicam and C-phycocyanin, rather than individually has the much greater potential for reduction of DMH-induced colon cancer development and COX-2 being the prime possible target in such chemoprevention.     

11,12-epoxyeicosatrienoic acid attenuates synthesis of prostaglandin E2 in rat monocytes stimulated with lipopolysaccharide

Cytochrome P-450 monooxygenase (epoxygenase)-derived arachidonic acid (AA) metabolites, including 11,12-epoxyeicosatrienoic acid (11,12-EET), possess anti-inflammatory and antipyretic properties. Prostaglandin E2 (PGE2), a cyclooxygenase (COX)-derived metabolite of AA, is a well-defined mediator of fever and inflammation. We have tested the hypothesis that 11,12-EET attenuates synthesis of PGE2 in monocytes, which are the cells that are indispensable for induction of fever and initiation of inflammation. Monocytes isolated from freshly collected rat blood were stimulated with lipopolysaccharide (LPS; 100 ng/2 x 10(5) cells) to induce COX-2 and stimulate generation of PGE2. SKF-525A, an inhibitor of epoxygenases, significantly augmented the lipopolysaccharide-provoked synthesis of PGE2 in cell culture in a concentration-dependent manner. It did not affect, however, elevation of the expression of COX-2 protein in monocytes stimulated with LPS. 11,12-EET also did not affect the induction of COX-2 in monocytes incubated with lipopolysaccharide. However, 11,12-EET suppressed, in a concentration-dependent fashion, the generation of PGE2 in incubates. Preincubation of a murine COX-2 preparation for 0-5 min with three concentrations of 11,12-EET (1, 5, and 10 microM) inhibited the oxygenation of [14C]-labeled AA by the enzyme. The inhibitory effect of 11,12-EET on COX-2 was time-and-concentration-dependent, suggesting a mechanism-based inhibition. Based on these data, we conclude that 11,12-EET suppresses generation of PGE2 in monocytes via modulating the activity of COX-2. These data support the hypothesis that epoxygenase-derived AA metabolites constitute a negative feedback on the enhanced synthesis of prostaglandins upon inflammation.     

Effect of cyclooxygenase inhibitors on the vasopressin induced ACTH and corticosterone response during crowding stress.

The aim of the present study was to compare the effect of social stress on the corticotropin releasing hormone (CRH) and arginine vasopressin (AVP)-induced pituitary-adrenocortical activity. Also the significance of prostaglandins (PG) generated by constitutive and inducible cyclooxygenase (COX-1 and COX-2) in the stimulation of hypothalamic-pituitary-adrenal (HPA) axis by AVP under basal and crowding stress conditions was investigated. The control rats were housed 7 in a standard cage and stressed rats were crowded 24 in a cage of the same size during 7 days. The activity of HPA axis was determined by measuring plasma ACTH and serum corticosterone levels 1 h after i.p. AVP administration. Indomethacin (2.0 mg/kg i.p.), a non-selective COX inhibitor, piroxicam (0.2, 2.0, and 5.0 mg/kg), a more potent COX-1 than COX-2 inhibitor, and compound NS-398 (0.2 and 2.0 mg/kg) a selective COX-2 inhibitor, were administered i.p. 15 min prior to AVP (5.0 microg/kg i.p.) to control or crowded rats. The obtained results indicate that social stress for 7 days considerably inhibits the stimulatory action of AVP on ACTH secretion, while it intensifies the CRH-induced ACTH secretion. Indomethacin, piroxicam and NS-398 significantly diminished the AVP-elicited ACTH and corticosterone secretion in non-stressed rats. None of these COX antagonist induced any significant inhibition of the AVP-induced ACTH and corticosterone secretion in stressed rats. Therefore, PG generated by COX-1 or COX-2 do not participate to a significant extent in the HPA stimulation by AVP during crowding stress. These results suggest that social crowding stress desensitizes the PG stimulatory mechanism which considerably mediates the AVP-induced HPA stimulation under basal conditions. The results contrast with a lack of any involvement of PG in the CRH-induced stimulation of HPA response under basal or crowding stress conditions.     

Novel di-tertiary-butyl phenylhydrazones as dual cyclooxygenase-2/5-lipoxygenase inhibitors: Synthesis,COX/LOX inhibition,molecular modeling,and insights into their cytotoxicities

Although dual inhibition of Cyclooxygenase-2 (COX-2) and 5-Lipoxygenase (5-LOX) enzymes is highly effective than targeting COX or LOX alone, there are only a few reports of examining such compounds in case of colorectal cancers (CRC). In the present work we report that the novel di-tert-butyl phenol-based dual inhibitors DTPSAL, DTPBHZ, DTPINH, and DTPNHZ exhibit significant cytotoxicity against human CRC cell lines. Molecular docking studies revealed a good fit of these compounds in the COX-2 and 5-LOX protein cavities. The inhibitors show significant inhibition of COX-2 and 5-LOX activities and are effective against a panel of human colon cancer cell lines including HCA-7, HT-29, SW480 and intestinal Apc10.1 cells as well as the hyaluronan synthase-2 (Has2) enzyme over-expressing colon cancer cells, through inhibition of the Hyaluronan/CD44v6 cell survival pathway. Western blot analysis and qRT-PCR analyses indicated that the di-tert-butyl phenol-based dual inhibitors reduce the expression of COX-2, 5-LOX, and CD44v6 in human colon cancer HCA-7 cells, while the combination of CD44v6shRNA and DTPSAL has an additional inhibitory effect on CD44v6 mRNA expression. The synergistic inhibitory effect of Celecoxib and Licofelone on CD44v6 mRNA expression suggests that the present dual inhibitors down-regulate cyclooxygenase and lipoxygenase enzymes through CD44v6. The compounds also exhibited enhanced antiproliferative potency compared to standard dual COX/LOX inhibitor, viz. Licofelone. Importantly, the HA/CD44v6 antagonist CD44v6shRNA in combination with synthetic compounds had a sensitizing effect on the cancer cells which enhanced their antiproliferative potency, a finding which is crucial for the anti-proliferative potency of the novel synthetic di-tert-butyl phenol based dual COX–LOX inhibitors in colon cancer cells.     

Effects of oxicam inhibitors of cyclooxygenase on oxidative stress generation in rat gastric mucosa. A comparative study

The aim of this study was to compare the effects of two nonsteroidal anti-inflammatory drugs (NSAID), members of the same family with a different cyclooxygenase (COX) inhibition selectivity, meloxicam, preferent COX-2 inhibitor, and piroxicam, preferent COX-1 inhibitor, on oxygen radical generation in rat gastric mucosa. Therefore, the activity of oxidative stress-related enzymes such as xanthine oxidase (XO), superoxide dismutase (SOD) and glutathione (GSH) homeostasis were studied in rats. Gastric prostaglandins (PG) were also assessed as a measure of COX-1 inhibition. Both oxicams produced a similar extent of the gastric mucosal damage and a significant decrease in PGE2 synthesis, however only piroxicam induced an increase of both myeloperoxidase (MPO) activity and tumor necrosis factor (TNF)-alpha content in the gastric mucosa, indicating that neutrophil-derived free radicals were involved in gastric injury. Furthermore, both compounds reduced SOD activity and increased XO activity in gastric mucosa. Our results also revealed modifications in GSH metabolism: although glutathione peroxidase (GSH-px) activity was unaffected by meloxicam or piroxicam administration, both glutathione reductase (GSSG-rd) activity and total GSH content were significantly decreased after dosing. These results suggest that under our experimental conditions, meloxicam, preferential COX-2 inhibitor causes rates of gastric lesion in rats comparable to those seen with the traditional NSAID piroxicam, preferential COX-1 inhibitor. In addition to suppression of systemic COX activity, oxygen radicals, probably derived via the XO, and neutrophils play an important role in the production of damage induced by both oxicams. Moreover, the decrease in SOD activity and changes in glutathione homeostasis in gastric mucosa may also contribute to pathogenesis of meloxicam- or piroxicam-induced gastropathy.     

Effects of Oxicam Inhibitors of Cyclooxygenase on Oxidative Stress Generation in Rat Gastric Mucosa. A Comparative Study

The aim of this study was to compare the effects of two nonsteroidal anti-inflammatory drugs (NSAID), members of the same family with a different cyclooxygenase (COX) inhibition selectivity, meloxicam, preferent COX-2 inhibitor, and piroxicam, preferent COX-1 inhibitor, on oxygen radical generation in rat gastric mucosa. Therefore, the activity of oxidative stress-related enzymes such as xanthine oxidase (XO), superoxide dismutase (SOD) and glutathione (GSH) homeostasis were studied in rats. Gastric prostaglandins (PG) were also assessed as a measure of COX-1 inhibition. Both oxicams produced a similar extent of the gastric mucosal damage and a significant decrease in PGE 2 synthesis, however only piroxicam induced an increase of both myeloperoxidase (MPO) activity and tumor necrosis factor (TNF)- &#102 content in the gastric mucosa, indicating that neutrophil-derived free radicals were involved in gastric injury. Furthermore, both compounds reduced SOD activity and increased XO activity in gastric mucosa. Our results also revealed modifications in GSH metabolism: although glutathione peroxidase (GSH-px) activity was unaffected by meloxicam or piroxicam administration, both glutathione reductase (GSSG-rd) activity and total GSH content were significantly decreased after dosing. These results suggest that under our experimental conditions, meloxicam, preferential COX-2 inhibitor causes rates of gastric lesion in rats comparable to those seen with the traditional NSAID piroxicam, preferential COX-1 inhibitor. In addition to suppression of systemic COX activity, oxygen radicals, probably derived via the XO, and neutrophils play an important role in the production of damage induced by both oxicams. Moreover, the decrease in SOD activity and changes in glutathione homeostasis in gastric mucosa may also contribute to pathogenesis of meloxicam- or piroxicam-induced gastropathy.     

PGE(2) is generated by specific COX-2 activity and increases VEGF production in COX-2-expressing human pancreatic cancer cells

In some cancers cyclooxygenase (COX) inhibition appears to be anti-mitogenic and anti-angiogenic, but the actions of COX-derived prostaglandins in pancreatic cancer (PaCa) are unknown. In this study COX-2 was detected in three of six PaCa cell lines while COX-1 was identified in all cell lines. COX-2 expression correlated with basal and arachidonic acid (AA) stimulated PGE(2) production. PGE(2) production was inhibited by the COX-2 inhibitor nimesulide. In COX-2 expressing cells, exogenous AA and PGE(2) increased VEGF synthesis via the EP(2) receptor. Whereas PGE(2) stimulated intracellular cAMP formation in COX-2 positive and negative cells, 8-bromo cAMP stimulated VEGF production only in COX-2 expressing cells. Stimulating COX-2 expressing PaCa cell lines with AA enhanced migration of endothelial cells, an effect which was inhibited by a COX-2 inhibitor and EP(2) receptor antagonist. These data identify a subset of human PaCa cell lines that express functional COX-2 enzyme. PGE(2) generated by specific COX-2 activity increases VEGF secretion in human PaCa cells through an autocrine mechanism.     

Nitric oxide and prostaglandin systems in the stimulation of hypothalamic-pituitary-adrenal axis by neurotransmitters and neurohormones.

The review presents our results on the regulatory role of prostaglandins (PG) and nitric oxide (NO) in the activation of hypothalamic-pituitary-adrenal (HPA) axis by cholinergic, adrenergic and histaminergic systems and by neurohormones: corticotropin-releasing hormone (CRH) and arginine vasopressin (AVP) under basal conditions. The synthesis of endogenous PG or NO was inhibited by non-selective and selective cyclooxygenase (COX) antagonists and nitric oxide synthase (NOS) blockers given 15 min before the respective receptor agonist and HPA axis activity was assessed 1 h later by measuring plasma ACTH and serum corticosterone levels. The muscarinic agent - carbachol-induced HPA response was considerably supressed by piroxicam, a predominantly constitutive cyclooxygenase (COX-1) inhibitor and significantly diminished by indomethacin, a non-selective COX blocker, but was unaffected by compound NS-398, an inducible cyclooxygenase (COX-2) antagonist. A non-selective NOS antagonist L-NAME and neuronal NOS blocker L-NNA significantly intensified the carbachol-induced corticosterone secretion. The nicotine-induced increase in ACTH and corticosterone response was significantly supressed by piroxicam, and diminished by indomethacin, but was significantly augmented by L-NAME and L-NNA. The inhibition of PG synthesis by indomethacin totally abolished or reversed the increase of nicotine-induced hormone responses to both NOS blockers. The i.c.v. phenylephrine, an alpha(1)-adrenergic receptor agonist - evoked HPA response was significantly impaired by piroxicam and compound NS-398 and more potently reduced by L-NAME. The i.c.v. clonidine, an alpha(2)-adrenergic agonist - elicited HPA response was also considerably decreased by piroxicam, compound NS-398 and L-NAME. By contrast, the stimulatory effect of i.c.v. isoprenaline, a non-selective beta-adrenergic agonist, was not altered by either COX or NOS inhibitors. The i.c.v. histamine- and HTMT, a histamine H(1)-agonist-induced ACTH and corticosterone response were significantly diminished by piroxicam and indomethacin, respectively. Compound NS-398, did not markedly alter the HPA response to HTMT or amthamine, a histamine H(2) receptor agonist. Inhibition of endogenous NO synthesis by a neuronal NOS inhibitor 7-nitroindazole markedly enhanced the histamine-induced hormone secretion, abolished the HTMT-induced response and did not substantially alter the amthamine-evoked ACTH and corticosterone secretion. COX blockers did not significantly affect the CRH-induced HPA response and the inhibition of NO synthesis by L-NNA markedly intensified ACTH response. The vasopressin-stimulated increase in HPA response, was considerably reduced by the inhibition of PG synthesis by both COX antagonists while inhibition of NO synthesis by NOS blockers greatly enhanced this response. The involvement of PG and NO in the neurohormonal regulation of HPA activity depends mainly on greatly complex and tightly regulated mechanisms at the level of second messengers IP(3) and adenylyl cyclase systems.     

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.     

Regulation and function of COX-2 gene expression in isolated gastric parietal cells

We examined expression, function, and regulation of the cyclooxygenase (COX)-2 gene in gastric parietal cells. COX-2-specific mRNA was isolated from purified (>95%) canine gastric parietal cells in primary culture and measured by Northern blots using a human COX-2 cDNA probe. Carbachol was the most potent inducer of COX-2 gene expression. Gastrin and histamine exhibited minor stimulatory effects. Carbachol-stimulated expression was inhibited by intracellular Ca(2+) chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-AM (90%), protein kinase C (PKC) inhibitor GF-109203X (48%), and p38 kinase inhibitor SB-203580 (48%). Nuclear factor (NF)-kappaB inhibitor 1-pyrrolidinecarbodithioic acid inhibited carbachol-stimulated expression by 80%. Similar results were observed in the presence of adenoviral vector Ad.dom.neg.IkappaB, which expresses a repressor of NF-kappaB. Addition of SB-203580 with Ad.dom.neg.IkappaB almost completely blocked carbachol stimulation of COX-2 gene expression. We examined the effect of carbachol on PGE(2) release by enzyme-linked immunoassay. Carbachol induced PGE(2) release. Ad.dom.neg.IkappaB, alone or with SB-203580, produced, respectively, partial (70%) and almost complete (>80%) inhibition of carbachol-stimulated PGE(2) production. Selective COX-2 inhibitor NS-398 blocked carbachol-stimulated PGE(2) release without affecting basal PGE(2) production. In contrast, indomethacin inhibited both basal and carbachol-stimulated PGE(2) release. Carbachol induces COX-2 gene expression in the parietal cells through signaling pathways that involve intracellular Ca(2+), PKC, p38 kinase, and activation of NF-kappaB. The functional significance of these effects seems to be stimulation of PGE(2) release.