Substance P augments PGE2 and IL-6 production in titanium particles-stimulated fibroblasts from hip periprosthetic membrane

Aseptic loosening remains the primary cause of failure in total joint arthroplasty. Implant-derived particles are thought to be a main cause of osteolysis that leads to failure of total joint arthroplasty. The nervous system has been implicated in the etiology and pathogenesis of joint diseases. Substance P (SP) immunoreactive nerve fibers have been detected in the pseudomembrane and pseudocapsular tissues of aseptic loose hip prostheses, suggesting that SP might be involved in the process of aseptic loosening. Fibroblasts are abundant in periprosthetic membrane. Neuropeptides are able to modulate cytokine production by fibroblasts. In this study, we isolated fibroblasts from periprosthetic membrane at the time of revision hip arthroplasty performed because of aseptic loosening. Fibroblasts were stimulated with titanium (Ti) particles or SP. Prostaglandin (PG) E2 and interleukin-6 (IL-6) assays were performed using enzyme-linked immunosorbent assay kit. PGE2 and IL-6 secretion by fibroblasts have been significantly increased in the presence of Ti particles or SP. Moreover SP caused significant increase in PGE2 and IL-6 production by Ti particles-stimulated fibroblasts. Thus, SP and Ti particles acted synergistically to increase PGE2 and IL-6 secretion in fibroblasts from periprosthetic membrane.     

NS-398对肝癌细胞HepG2增殖和凋亡的影响

目的:探讨选择性环氧合酶II(COX-2)抑制剂NS-398对人肝癌细胞HepG2增殖和凋亡的影响。方法: 应用MTT法研究不同浓度NS-398对HepG2细胞增殖的影响,DNA梯状电泳(DNA ladder)检测凋亡的发生,流式细胞仪检测细胞周期的改变及凋亡百分率的变化,竞争性RT-PCR检测环氧合酶II COX-2 mRNA及抑凋亡基因bcl-2 mRNA表达的改变。结果: NS-398呈剂量依赖性抑制HepG2细胞增殖,并诱导其凋亡,细胞周期分析表明随着浓度增大S期细胞明显减少,有G₀/G₁期细胞累积现象,并伴有Bcl-2 mRNA表达的下调,而对COX-2 mRNA表达改变无明显影响,且COX-2表达改变与NS-398引起的HepG2细胞的增殖和凋亡均无相关性(相关系数分别为:r=0.056,P>0.05和r=0.119,P>0.05)。结论: NS-398能明显抑制HepG2细胞增殖并诱导其凋亡,与细胞G₀/G₁期阻滞以及bcl-2基因表达下调有关,而非依赖于抑制COX-2基因的表达。     

低强度高频率振动对成骨细胞生物学特性的影响

目的　研究低强度高频率振动（low-magnitude high-frequency vibration, LMHFV）对成骨细胞生物学特性的影响。 方法　建立LMHFV加载MC3T3-E1细胞模型,观察不同频率LMHFV对MC3T3-E1细胞OPG/RANKL浓度比的影响,获得OPG/RANKL浓度比最高的频率（F）为后续研究频率;以0 Hz为对照,观察LMHFV对MC3T3-E1细胞碱性磷酸酶 (ALP)、骨钙素(OCN) mRNA和蛋白活性,及钙化结节形成的影响;LMHFV加载形成的条件培养液（CMF）孵育RAW264.7细胞,观察CMF对破骨细胞抗酒石酸酸性磷酸酶（TRAP）染色、多核破骨细胞形成、TRAP mRNA及蛋白活性的影响;观察LMHFV对MC3T3-E1细胞环氧化酶2(COX-2)蛋白水平的表达及COX-2抑制剂NS-398对LMHFV影响MC3T3-E1细胞分化的作用。 结果　30 Hz LMHFV获得OPG/RANKL浓度比最高,促进ALP、OCN mRNA及蛋白活性增加,增加钙化结节形成。30 Hz LMHFV形成的CM抑制RAW264.7细胞向多核破骨细胞分化,抑制TRAP mRNA及活性;LMHFV可诱导COX-2蛋白水平增加,NS-398能抑制LMHFV促进成骨细胞分化。 结论　30 Hz的LMHFV对MC3T3-E1细胞OPG/RANKL浓度比及成骨分化具有积极的影响,通过调控成骨细胞OPG/RANKL浓度比间接抑制骨吸收,COX-2通路参与了LMHFV对成骨细胞生物学特性的调节作用。     

Inhibition of cyclooxygenase-2 suppresses the invasiveness of oral squamous cell carcinoma cell lines via down-regulation of matrix metalloproteinase-2 production and activation

Increased cyclooxygenase (COX-2) expression in tumors is known to be correlated with tumor invasion, angiogenesis, resistance to apoptosis, and suppression of host immunity. We previously reported that the invasiveness of human oral squamous cell carcinoma (OSCC) cell lines NA and HSC-4 was suppressed by treatment with either NS-398, a selective COX-2 inhibitor, or COX-2 antisense oligonucleotide (AS). In the present study, to explore the effects of COX-2 inhibition on the interaction between cancer cells and fibroblasts, we examined the effects of these anti-COX-2 reagents on the expression of matrix metalloproteinases (MMPs) in fibroblast cell lines WI-38 and MRC-5. Western blotting and enzyme-linked immunosorbent assay revealed that NS-398 and COX-2 AS down-regulated the expression and secretion of MMP-2 and the tissue inhibitor of matrix metalloproteinase-2 (TIMP-2) in human fibroblast cell lines. Furthermore, invasion activity of OSCC cells was down-regulated by the addition of culture supernatant from fibroblasts treated with anti-COX-2 reagents in a Matrigel^® invasion assay. These results suggest that selective COX-2 inhibition suppresses the invasion activity of OSCC cells via down-regulation of an MMP-2-activating mechanism involving TIMP-2 and production of the MMP-2 protein by an interaction between cancer cells and stromal fibroblasts. Genetic or pharmacological inhibition of COX-2 may therefore be a beneficial strategy in the treatment of OSCC.     

环氧合酶-2选择性抑制剂NS-398抑制肿瘤细胞的作用机制

目的:探讨COX-2选择性抑制剂NS-398对肿瘤细胞抑制的作用机制。方法:选择持续表达COX-2的人食管癌细胞株EC9706和不表达COX-2的肝癌细胞株SMMC7721作为研究对象，采用［3H］-TdR掺入法检测不同浓度（10、20、50、100 μmol/L）NS-398和作用24 h、48 h、72 h对细胞的增殖抑制效应；流式细胞术（FCM）及DNA片段分析法检测NS-398诱导的细胞凋亡情况；免疫细胞化学方法检测NS-398作用后细胞内survivin表达变化。结果:经不同浓度的NS-398处理，EC9706及SMMC7721的［3H］-TdR掺入量均明显少于空白对照组（P<0.05或P<0.01），具有时间和剂量-效应关系。FCM检测发现EC9706在G1期前出现亚倍体凋亡峰，细胞凋亡率达(45.23±1.08)%，并出现典型的细胞凋亡梯带；而SMMC7721无凋亡峰出现，细胞凋亡率为(3.05±0.15)%，两组比较有显著性差异（P<0.01）。经100 μmol/L NS398作用24 h后，EC9706细胞内survivin表达与未经NS-398作用的EC9706细胞表达明显减少，而SMMC7721表达无变化。结论:NS398通过不同作用机制对肿瘤细胞发挥抑制作用，对COX-2表达阳性的肿瘤细胞具有诱导凋亡作用，与抑制survivin表达有关，而对无表达COX-2的肿瘤细胞无诱导凋亡作用。     

Inhibition of cyclooxygenase-2 suppresses invasiveness of oral squamous cell carcinoma cell lines via down-regulation of matrix metalloproteinase-2 and CD44

Expression of cyclooxygenase-2 (COX-2) in tumors is known to be associated with enhanced angiogenesis, suppression of host immunity, and tumor invasion. In the present study, human oral squamous cell carcinoma (OSCC) cell lines NA and HSC-4 were used to evaluate the effects of NS-398, a selective inhibitor of COX-2, and COX-2 antisense oligonucleotide (COX-2 AS) on the invasion activity of OSCC cells. Matrigel invasion assay revealed that the invasiveness of NA and HSC-4 was suppressed by treatment with either NS-398 or COX-2 AS. These reagents down-regulated the secretion of matrix metalloproteinase-2 (MMP-2) to culture supernatant as well as the expression of MMP-2 mRNA and protein. Membrane-type 1 matrix metalloproteinase (MT1-MMP), an activator of proMMP-2, was also down-regulated by treatment with these reagents. Furthermore, expression of CD44 on the surface of these cells was reduced by treatment with either NS-398 or COX-2 AS. In addition, MMP-2 antisense oligonucleotides reduced the expression of CD44 on the surface of both OSCC cell lines. These findings suggest that NS-398 and COX-2 AS suppress the invasiveness of OSCC cells via down-regulation of MMP-2 and CD44. Genetic or pharmacological inhibition of COX-2 may therefore be a beneficial strategy in the treatment of OSCC patients.     

Inhibition of cyclooxygenase-2 suppresses invasiveness of oral squamous cell carcinoma cell lines via down-regulation of matrix metalloproteinase-2 and CD44

Expression of cyclooxygenase-2 (COX-2) in tumors is known to be associated with enhanced angiogenesis, suppression of host immunity, and tumor invasion. In the present study, human oral squamous cell carcinoma (OSCC) cell lines NA and HSC-4 were used to evaluate the effects of NS-398, a selective inhibitor of COX-2, and COX-2 antisense oligonucleotide (COX-2 AS) on the invasion activity of OSCC cells. Matrigel invasion assay revealed that the invasiveness of NA and HSC-4 was suppressed by treatment with either NS-398 or COX-2 AS. These reagents down-regulated the secretion of matrix metalloproteinase-2 (MMP-2) to culture supernatant as well as the expression of MMP-2 mRNA and protein. Membrane-type 1 matrix metalloproteinase (MT1-MMP), an activator of proMMP-2, was also down-regulated by treatment with these reagents. Furthermore, expression of CD44 on the surface of these cells was reduced by treatment with either NS-398 or COX-2 AS. In addition, MMP-2 antisense oligonucleotides reduced the expression of CD44 on the surface of both OSCC cell lines. These findings suggest that NS-398 and COX-2 AS suppress the invasiveness of OSCC cells via down-regulation of MMP-2 and CD44. Genetic or pharmacological inhibition of COX-2 may therefore be a beneficial strategy in the treatment of OSCC patients.This revised version was published online in August 2005 with a corrected cover date.     

Cyclooxygenase-2 mediates the delayed cardioprotection induced by hydrogen sulfide preconditioning in isolated rat cardiomyocytes

We previously reported that hydrogen sulfide (H₂S) preconditioning (SP) produces cardioprotection in isolated rat cardiomyocytes. The present study was designed to determine the involvement of cyclooxygenase-2 (COX-2) in the SP-induced delayed cardioprotection. Isolated cardiac myocytes were treated with NaHS (100 μM, a H₂S donor) for 30 min and then cultured for 20 h followed by ischemia/reperfusion insults. SP significantly increased cell viability, percentage of rod-shaped cells, and myocyte contractility after 10 min of reperfusion. Given 30 min before and during lethal ischemia, two selective COX-2 inhibitors, NS-398 and celebrex, abrogated SP-induced cardioprotective effects. Moreover, SP upregulated the expression of COX-2 and increased PGE₂ production in the cardiac myocytes. These effects were significantly attenuated by glibenclamide, an ATP-sensitive K⁺ channel (K_ATP) blocker, and chelerythrine, a selective protein kinase C (PKC) inhibitor, suggesting that activation of both K_ATP and PKC is required for the stimulation of COX-2. Additionally, NG-nitro-l-arginine methyl ester, a nitric oxide synthase inhibitor, failed to regulate COX-2 protein expression but inhibited SP-enhanced COX-2 activity and PGE₂ production. In conclusion, we provided the first evidence that SP may produce delayed cardioprotection via K_ATP/PKC dependent induction of COX-2 expression and via nitric oxide-induced COX-2 activation.     

NF-κB与COX-2的正相互作用诱导子宫颈癌细胞生长及抗凋亡

目的探讨NF—κB和COX-2通路之间的相互作用对人子宫颈癌细胞株（Hela细胞）的生长及细胞凋亡的影响。方法应用细胞计数盒（CCK-8）检测细胞存活率;Hoechst33258核染色检测凋亡细胞的形态及数量的改变：Westernblot法检测Caspase-3、NF-κB和COX-2蛋白的表达。结果应用NF．KB抑制剂（PDTC）或COX-2抑制剂（NS-398）处理Hela细胞36h能明显地抑制细胞存活率,PDTC或NS-398处理Hela细胞24h能明显地促进Caspase-3表达,并增加凋亡细胞数量。PDTC处理Hela细胞能显著地抑制COX-2表达,另方面,NS-398处理Hela细胞能抑制NF-κB的表达。结论NF-κB和COX-2通路之间的正相互作用诱导Hela细胞生长及抑制细胞凋亡。     

Interleukin-1 stimulates human uterine prostaglandin production through induction of cyclooxygenase-2 expression

PROBLEM: Uterine infection occurs in as much as 20% of preterm labor and results in increased decidual cytokines. The objective of this study was to examine the effect of interleukin-1 (IL-1) and the cyclooxygenase-2 (COX-2) inhibitor, NS-398, on myometrial prostaglandin (PG) production and COX-2 expression. METHOD OF STUDY: Human uterine myocytes were stimulated with IL-1 (0-50 ng/mL) over 24 hr. PGE2, PGF2alpha, and 6-keto F1alpha were measured by enzyme-linked immunosorbent assay. Both COX-1 and COX-2 proteins and mRNA were measured by western and northern blot, respectively. RESULTS: IL-1 increased PG production beginning at 6 hr, COX-2 protein increased beginning at 4 hr and continued to increase at 24 hr. COX-2 mRNA increased at 2 hr and peaked at 4 hr. NS-398 blocked PG production but had no effect on COX-2 protein or mRNA. CONCLUSIONS: IL-1 increases PG production by myometrium by increased COX-2 expression. NS-398 completely blocks IL-1-induced PG production. With intrauterine infection, IL-1 may induce labor through the autocrine production of uterotonic PGs.     

Prostaglandin production via induction of cyclooxygenase-2 by human gingival fibroblasts stimulated with lipopolysaccharides

The purpose of the present study was to investigate the involvement of cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2) in PGE₂ production by human gingival fibroblasts stimulated with lipopolysaccharides (LPS) from periodondopathogenic bacteria. LPS were isolated fromPorphyromonas gingivalis (P. gingivalis), Actinobacillus actinomycetemcomitans (A. actinomycetemcomitans) andEschericia coli (E. coli) by the phenol-water procedure. The three LPS preparations produced PGE₂ up to 48 h in a time-dependent manner in human gingival fibroblasts.P. gingivalis-LPS was the most potent stimulator of PGE₂ production and, to a lesser extent,A. actinomycetemcomitans- andE. coli-LPS. Treatment of the cells with indomethacin, a non selective COX-1/COX-2 inhibitor and NS-398, a selective COX-2 inhibitor, completely depressed PGE₂ production. Treatment of dexamethasone, known to inhibit COX-2 expression, also significantly prevented PGE₂ production. Immunohistochemical staining of COX-2 protein demonstrated that expression of COX-2 protein was increased at 24 h afterP. gingivalis-LPS stimulation, while expression of COX-1 protein was not affected byP. gingivalis-LPS. In order to investigate the regulation of PGE₂ production,P. gingivalis-LPS-stimulated cells were treated with herbimycin A and genistein, both inhibitors of tyrosine kinases. Both the inhibitors significantly inhibited PGE₂ production. Herbimycin A treatment depressed expression of COX-2 protein. These data suggest that human gingival fibroblasts stimulated with LPS from periodontopathogenic bacteria mainly produce PGE₂ not by COX-1, but by COX-2, induction of which may be regulated by tyrosine kinase and that the produced PGE₂ may be involved in the pathogenesis of periodontal diseases.     

The long-term exposure of rat cultured dorsal root ganglion cells to bradykinin induced the release of prostaglandin E2 by the activation of cyclooxygenase-2

The effects of long-term exposure of primary cultured rat dorsal root ganglion (DRG) cells to bradykinin (BK), compared to short-term exposure, were investigated to establish whether BK could induce prostaglandin E2 (PGE2) release from DRG cells. Short-term exposure (30 min) resulted in a small but significant amount of PGE2 release which was mainly inhibited by a selective COX-1 inhibitor, SC-560 but only partially by a selective COX-2 inhibitor, NS-398, and did not induce COX-2 protein as determined by Western blotting. In contrast, long-term exposure (3 h) induced a large amount of PGE2 release, which was completely abolished by indomethacin or NS-398. The level of COX-2 mRNA began to be detected by ribonuclease protection assay after 30 min of 100 nM BK exposure, maintained maximal expression for 1 h, and subsequently declined to the basal level. The level of COX-2 protein was expressed to follow the time course of COX-2 mRNA induction by BK in a delayed but similar kinetic manner. The expression of COX-2 induced by BK in DRG cells was inhibited by a BK B2 receptor antagonist, HOE140, but not a B1 receptor antagonist, Lys-des-Arg9, (Leu8)-BK. Thus, BK has been shown to induce COX-2 protein by B2 receptor, which may cause prostanoid generation in rat DRG cells, which may play an important role in the pathogenesis of inflammatory pain and hyperalgesia around the primary sensory neurons.     

COX-2抑制剂(NS-398)对结肠癌HT-29细胞体外侵袭力的作用及CD44v6、nm23-H1基因的调节

目的：研究NS-398对结肠癌HT-29细胞体外侵袭力的作用及CD44v6、nm23-H1基因的调节。 方法： 通过流式细胞仪检测COX-2和CD44v6的表达，MTT检测细胞活性，改良的Boyden小室法观察HT-29细胞侵袭重组基底膜的能力，RT-PCR观察nm23-H1 mRNA的表达。 结果： HT-29细胞COX-2表达阳性，0.1、1.0、10 μmol/L NS-398可显著抑制HT-29细胞侵袭重组基底膜的能力，且上述作用与NS-398的毒性作用无关。NS-398可下调CD44v6的表达，上调nm23-H1 mRNA的表达。 结论： NS-398具有抑制结肠癌细胞HT-29体外侵袭力的作用，下调CD44v6的表达和上调nm23-H1 mRNA的表达可能是其作用机制。     

Eicosanoid release in the endotoxin-primed isolated perfused rat lung and its pharmacological modification

OBJECTIVE: Recent observations have demonstrated a central role of the "inducible" isoform of the cyclooxygenase (COX), COX-2, in the rat lung. Therefore, the reported capacity of selective COX-2 inhibitors to potentiate the formation of leukotriene (LT) B4 may raise concern about pro-inflammatory side effects of such drugs in the respiratory system. The present study was aimed at determining the effects of the COX-2 inhibitor NS-398 on the release of COX and 5-lipoxygenase (LOX) metabolites of arachidonic acid in isolated perfused lungs obtained from endotoxin-treated rats before and after stimulation with the leukocyte secretagogue N-formyl-methionyl-leucyl-phenylalanine (FMLP). METHODS: Two hours after rats had received endotoxin i.v., the lung was dissected and perfused via the pulmonary artery with physiological salt solution. After an equilibration period of 20 min the outflow was collected (5-min fractions). In the respective treatment groups, indomethacin, NS-398, or the 5-LOX inhibitor MK886 were present throughout the experiment, while FMLP was added to the perfusate during a single 5-min period. The concentration of eicosanoids in the outflow was determined by radioimmunoassay. RESULTS: Endotoxin treatment of rats resulted in increased expression of COX-2 mRNA in lung tissue, and an elevated basal release of the prostaglandin (PG)I2 metabolite 6-keto PGF1alpha, without a detectable increase of leukotriene (LT) formation. In-vitro exposure to FMLP stimulated LT and prostanoid release, which was significantly enhanced in endotoxin-primed lungs, and was suppressed by the 5-LOX inhibitor MK-886 (3 microM) and the COX-inhibitor indomethacin (5 microM), respectively. Either compound showed selective inhibition of the respective pathway of arachidonic acid metabolism. In endotoxin-primed lungs, the COX-2 inhibitor NS-398 (0.3-1.0 microM) depressed basal as well as FMLP-stimulated release of 6-keto PGF1alpha, but did not cause a significant increase of LTB4 or cysteinyl-LT release. CONCLUSIONS: These results suggest that FMLP, presumably acting on inflammatory cells trapped in the pulmonary circulation of endotoxin treated rats, induced prostanoid formation mainly via the COX-2 pathway, and that its inhibition by NS-398 had no detectable potentiating effect on LTB4 or cysteinyl-LT biosynthesis.     

Selective COX-2 inhibitor, NS-398, inhibits the replicative senescence of cultured dermal fibroblasts

Cyclooxygenase 2 (COX-2) is known to be increased in aged cells. Recent studies suggest that the increased expression of COX-2 may be involved in the pathogenesis of age-associated diseases such as rheumatoid arthritis and cancer. We investigated the role of COX-2 in cell cycle arrest and collagen deficiency during the aging process. Using the replicative senescence model of dermal fibroblasts, we demonstrated the increased expression of COX-2 and increased PGE(2) levels associated with replicative senescence. Replicative senescent cells showed a decreased ability to induce cell proliferation, probably due to the increased expression of the p53 protein and the decreased expression of the PCNA protein, and also showed increased expression of MMP-1, and decreased expression of tissue inhibitor of metalloproteinase-1 (TIMP-1) and procollagen. The selective COX-2 inhibitor, NS-398, can inhibit the senescence-associated increases of COX-2, PGE(2), p53 and MMP-1 expression, and the senescence-associated decreases of PCNA, TIMP-1 and procollagen expression. These results suggest that the increased level of COX-2 and higher level of PGE(2) in aged cells may play an important role in cellular senescence, and that selective COX-2 inhibitors may be useful for the intervention of skin aging.     

Protection against titanium particle-induced osteoclastogenesis by cyclooxygenase-2 selective inhibitor

Wear particle-induced osteoclastogenesis is the most common cause of aseptic loosening in total joint arthroplasty. Although cyclooxygenase (COX)-2, an inducible regulator of prostaglandin E2 (PGE2) synthesis, is known to be involved in osteoclast differentiation, its effect on osteoclastogenesis in response to wear particles remains unclear. In this study, we investigated the role of COX-2 in the regulation of osteoclast differentiation in the osteoclast precursor cell line RAW264.7 stimulated with titanium (Ti) particles. The results showed COX-2 expression in the early stages of RAW264.7 differentiation when stimulated with receptor activator of nuclear factor kappa B ligand (RANKL) and Ti particles. Blockade of COX-2 by celecoxib, a COX-2 selective inhibitor, effectively reduced the expression of PGE2 and inhibited differentiation of RAW264.7 cells into tartrate-resistant acid phosphatase-positive (TRAP+) osteoclastic cells. Quantitative real-time polymerase chain reaction revealed that celecoxib inhibited mRNA expression of RANK, cathepsin K (CPK), TRAP, and the nuclear factor of activated T cells c1 (NFATc1) in RAW264.7 cells stimulated by Ti particles and RANKL. Moreover, exogenous PGE2 reversed the inhibitory effects of celecoxib. These results provide direct evidence that COX-2 dependent PGE2 induced by RANKL and Ti particles is required for osteoclastogenesis and suggests that reduced production of PGE2 by inactivation of COX-2 would provide a promising therapeutic target for the treatment of osteoclastogenesis induced by wear particles.     

Nitric oxide regulates stretch-induced proliferation in C2C12 myoblasts

Mechanical stretch of skeletal muscle activates nitric oxide (NO) production and is an important stimulator of satellite cell proliferation. Further, cyclooxygenase (COX) activity has been shown to promote satellite cell proliferation in response to stretch. Since COX-2 expression in skeletal muscle can be regulated by NO we sought to determine if NO is required for stretch-induced myoblast proliferation and whether supplemental NO can counter the effects of COX-2 and NF-κB inhibitors. C2C12 myoblasts were cultured for 24 h, then switched to medium containing either the NOS inhibitor, l-NAME (200 μM), the COX-2 specific inhibitor NS-398 (100 μM), the NF-κB inhibiting antioxidant, PDTC (5 mM), the nitric oxide donor, DETA-NONOate (10–100 μM) or no supplement (control) for 24 h. Subgroups of each treatment were exposed to 1 h of 15% cyclic stretch (1 Hz), and were then allowed to proliferate for 24 h before fixing. Proliferation was measured by BrdU incorporation during the last hour before fixing, and DAPI stain. Stretch induced a twofold increase in nuclear number compared to control, and this effect was completely inhibited by l-NAME, NS-398 or PDTC (P < 0.05). Although DETA-NONOate (10 μM) did not affect basal proliferation, the NO-donor augmented the stretch-induced increase in proliferation and rescued stretch-induced proliferation in NS-398-treated cells, but not in PDTC-treated cells. In conclusion, NO, COX-2, and NF-κB are necessary for stretch-induced proliferation of myoblasts. Although COX-2 and NF-κB are both involved in basal proliferation, NO does not affect basal growth. Thus, NO requires the synergistic effect of stretch in order to induce muscle cell proliferation.     

Effects of COX-1 and COX-2 inhibitors on eicosanoid biosynthesis and the release of substance P from the guinea-pig isolated perfused lung

OBJECTIVE: In the guinea-pig isolated perfused lung, co-administration of bradykinin (BK) and histamine causes the release of pro-inflammatory neuropeptides, an effect that is largely dependent on BK-induced formation of prostaglandins. Since it is known that at least two isoenzymes, cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2) catalyse the conversion of arachidonic acid to prostaglandins (PGs) and thromboxanes, the present study aimed at investigating the effect of selective COX-1 and COX-2 inhibitors on the evoked release of substance P (SP). MATERIAL AND METHODS: Lungs were vascularly perfused with oxygenated physiological salt solution containing peptidase inhibitors. BK (0.1 microM) and histamine (100 microM) were added to the perfusate for 10 min and 5 min, respectively. The concentrations of 6-keto-PGF1alpha, cysteinyl-leukotriene (LT), and SP were determined in the outflow by radioimmunoassay. RESULTS: In non-stimulated preparations, indomethacin (2 microM) and the selective COX-1 inhibitor SC-560 (0.03-1 microM) reduced basal release of 6-keto-PGF1alpha, without significantly affecting the release of cysteinyl-LT and SP. The selective COX-2 inhibitors NS-398 (1 microM) or DFU (10 microM) had no significant effect on the basal release of eicosanoids or SP. Co-administration of BK and histamine caused a pronounced increase in the concentration of 6-keto-PGF1alpha and cysteinyl-LT, and SP in the effluate. Under these conditions, indomethacin as well as SC-560 reduced the release of 6-keto-PGF1alpha, enhanced cysteinyl-LT release, and attenuated the release of SP. In contrast, the selective COX-2 inhibitors NS 398 and DFU had no significant effect on the stimulated release of eicosanoids or SP. CONCLUSIONS: These results suggest that in the isolated guinea-pig lung, basal prostanoid biosynthesis as well as BK-induced stimulation of prostanoid formation and subsequent facilitation of histamine-induced SP release is primarily mediated by COX-1 without detectable involvement of COX-2.     

CD40 ligation triggers COX-2 expression in endothelial cells: evidence that CD40-mediated IL-6 synthesis is COX-2-dependent

OBJECTIVE: To investigate whether CD40-CD154 interactions on HUVEC can trigger COX-2 synthesis as well as PGE2 and PGI2 secretion in vitro and explore whether the CD40-triggered prostanoids provide costimulatory signals for IL-6 secretion in this cell type. MATERIALS AND METHODS: COX-2 protein expression was examined in HUVEC using Western blot analysis. ELISAs were employed to assess PGE2, PGI2 and IL-6 synthesis. RESULTS: We found that COX-2 expression is upregulated when HUVEC are cultured with CD154+ D1.1 cells but not CD154- B2.7 cells. This effect was specifically inhibited by anti-CD154 mAb, and was amplified by the presence of IFNgamma. Analysis of cell supernatants showed a concomitant rise in PGE2 and PGI2 secretion triggered by CD154+ D1.1 cells, or rsCD154. Use of selective (NS-398) and non-selective (ibuprofen) COX-2 inhibitors effectively inhibited prostanoid synthesis triggered by CD40 ligation. Reduction in prostanoid levels by NS-398 was accompanied by a reduction in IL-6 secretion levels triggered by CD40 ligation. Furthermore, exogenously added PGE2 triggered a dose-dependent IL-6 secretion, which was unaffected by NS-398. CONCLUSIONS: These studies demonstrate that CD40 ligation upregulates HUVEC COX-2 expression and function. Moreover, the data strongly suggest that CD154-induced IL-6 secretion in HUVEC is dependent on COX-2 activity.     

Helicobacter pylori promotes apoptosis, activates cyclooxygenase (COX)-2 and inhibits heat shock protein HSP70 in gastric cancer epithelial cells

Objective

Apoptosis plays an important role in the regulation of gastric epithelial cell number and gastrointestinal disorders induced by Helicobacter pylori (Hp). Heat shock proteins (HSPs) are involved in cell integrity, cell growth and in gastric mucosa colonized by Hp. COX-2 was implicated in Hp-induced carcinogenesis but the effects of this germ and CagA cytotoxin on HSP70, COX-2, Bax and Bcl-2 in gastric cancer epithelial cells have been little studied.

Material and methods

We determined the expression for HSP70, Bax and Bcl-2 in human gastric epithelial MKN7 cells incubated with live strain Hp (cagA?+?vacA+) with or without co-incubation with exogenous CagA and NS-398, the selective COX-2 inhibitor. After 3–48?h of incubation, the expression of HSP70, COX-2, Bax and Bcl-2 mRNA and proteins were determined by RT-PCR and immunoprecipitation.

Results

Hp inhibited expression for HSP70 and this was significantly potentiated by exogenous CagA. Co-incubation of epithelial cells with Hp, without or with CagA increased Bax expression and simultaneously decreased expression for Bcl-2. The increase in COX-2 mRNA and Bax expression were significantly inhibited by NS-398. We conclude that Hp promotes apoptosis in adenocarcinoma gastric epithelial cells in vitro and this is associated with activation of COX-2 and inhibition of HSP70.