Selective COX-2 Inhibitor NS-398 Research Articles

The selective cyclooxygenase-2 inhibitor NS398 ameliorates cisplatin-induced impairments in mitochondrial and cognitive function.

Chemobrain is a condition that negatively affects cognition in cancer patients undergoing active chemotherapy, as well as following chemotherapy cessation. Chemobrain is also known as chemotherapy-induced cognitive impairment (CICI) and has emerged as a significant medical contingency. There is no therapy to ameliorate this condition, hence identification of novel therapeutic strategies to prevent CICI is of great interest to cancer survivors. Utilizing the platinum-based chemotherapy cisplatin in an investigative approach for CICI, we identified increased expression of cyclooxygenase-2 (COX-2) and prostaglandin E2 (PGE2) in the adult mouse hippocampus, and in human cortical neuron cultures derived from induced pluripotent stem cells (iPSCs). Notably, administration of NS398, a selective COX-2 inhibitor, prevented CICI in vivo without negatively affecting the antitumor efficacy of cisplatin or potentiating tumor growth. Given that dysfunctional mitochondrial bioenergetics plays a prominent role in CICI, we explored the effects of NS398 in cisplatin-induced defects in human cortical mitochondria. We found that cisplatin significantly reduces mitochondrial membrane potential (MMP), increases matrix swelling, causes loss of cristae membrane integrity, impairs ATP production, as well as decreases cell viability and dendrite outgrowth. Pretreatment with NS398 in human cortical neurons attenuated mitochondrial dysfunction caused by cisplatin, while improving cell survival and neurite morphogenesis. These results suggest that aberrant COX-2 inflammatory pathways may contribute in cisplatin-induced mitochondrial damage and cognitive impairments. Therefore, COX-2 signaling may represent a viable therapeutic approach to improve the quality of life for cancer survivors experiencing CICI.

Anoxia/reoxygenation enhances spontaneous contractile activity via TRPA1 channel and COX2 activation in isolated rat whole bladder.

Bladder ischemia/reperfusion is an important etiologic factor for overactive bladder disease. The occurrence of this disease is closely associated with enhanced spontaneous contractile activity of the bladder. However, the relationship between bladder ischemia/reperfusion and altered spontaneous bladder contractions (SBC) remains poorly studied. Therefore, the present study investigated whether ischemia/reperfusion affects SBC ex vivo. SBC was measured using isolated whole bladder preparations from rats. The preparations were exposed to anoxia (95% N2 ) for 0.5-6 h, followed by reoxygenation (95% O2 ) in Krebs medium. Anoxia followed by reoxygenation significantly enhanced the amplitude of SBC without affecting its frequency in an anoxic duration-dependent manner. The 5 h anoxia/reoxygenation-induced enhancement of SBC amplitude was completely suppressed by an antioxidant combination of L(+)-ascorbate/D, L-α-tocopherol, or N-acetyl cysteine. Additionally, the enhanced SBC amplitude was inhibited in a concentration-dependent manner by the nonselective TRP antagonist ruthenium red, or selective TRPA1 antagonists HC-030031 or AP-18. A similar inhibitory effect was obtained after repeated treatment with the TRPA1 agonist allyl isothiocyanate, as it induced acute desensitization of TRPA1 channels. Further, the enhanced SBC amplitude was significantly diminished by the nonselective cyclooxygenase (COX) inhibitor indomethacin or selective COX2 inhibitor NS-398, but not by the selective COX1 inhibitor SC-560 and 5-lipoxygenase inhibitor MK-886. The study findings reveal that the spontaneous contractile activity of the bladder is significantly enhanced in response to anoxia/reoxygenation, and that oxidative stress and activation of TRPA1 and COX2 (the resulting production of prostaglandins) are involved in the enhanced SBC activity.

Effects of different cyclooxygenase inhibitors on prostaglandin E2 production, steroidogenesis and ovulation of bovine preovulatory follicles.

Ovulation is an inflammation-like process, and cyclooxygenase-2 (COX-2)-dependent production of prostaglandin E2 (PGE2) is its key mediator. Balanced regulation of inflammatory processes in high-yielding dairy cows may be essential for physiological ovulation and fertility. This study aimed to elucidate the mechanisms underlying ovulation failure and cyst development after disturbing intrafollicular inflammatory cascades. Therefore, nonselective (indomethacin and flunixin-meglumine), COX-2 selective (meloxicam), and highly COX-2 selective (NS-398) inhibitors were injected into preovulatory follicles 16 h after administration of GnRH, and ovulation was monitored via ultrasound examination. Additionally, follicular fluid was collected after injection of indomethacin, meloxicam, and NS-398. Moreover, primary granulosa cell cultures from preovulatory follicles were prepared and treated with indomethacin, meloxicam, and NS-398. The concentrations of 17β-estradiol, progesterone, and prostaglandin E2 (PGE2) in the follicular fluid and cell supernatant were estimated. Indomethacin and flunixin-meglumine blocked ovulation, even at low doses, and led to ovarian cyst development. The selective and highly selective COX-2 inhibitors meloxicam and NS-398 were not effective in blocking ovulation. However, indomethacin, meloxicam, and NS-398 significantly and comparably reduced PGE2 concentration in vivo and in vitro (P < 0.05) but had no effect on estradiol or progesterone production. This may contradict the generally accepted hypothesis that PGE2 is a key mediator of ovulation and progesterone production. Our results suggest a connection between ovarian disorders and inflammatory actions in early postpartum cows.

Paeonol induces antitumor effects in hepatocellular carcinoma cells through survivin via the cyclooxygenase-2/prostaglandin E2 signaling pathway.

BackgroundHepatocellular carcinoma (HCC) is one of the most life-threatening malignancies worldwide due to the lack of significant improvement in therapeutic methods. This study aimed to unravel the effects of paeonol, the main active component of Paeonia suffruticosa, on survivin, a key molecule in tumorigenesis, and elucidate the mechanisms by which paeonol exerts antineoplastic effects in human HCC cells.MethodsImmunohistochemistry (IHC) was used to study the expression levels of survivin and cyclooxygenase-2 (COX-2) in 57 human HCC tissue samples. Human HCC cell lines (HepG2 and SMMC-7721) were treated with prostaglandin E2 (PGE2). Subsequently, the cells were treated with paeonol and NS-398, and the expression levels of survivin, COX-2, and PGE2 were evaluated by Western blotting and enzyme-linked immunosorbent assay (ELISA), respectively. Fluorescence-activated cell sorting (FACS) and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining were performed to analyze cell proliferation and apoptosis.ResultsSurvivin was expressed in 47 of 57 human HCC tissue samples, as observed by IHC, and its expression was correlated with COX-2 activity. Furthermore, Western blotting showed that the expression of survivin was increased in HepG2 and SMMC-7721 cells treated with PGE2, the biosynthesis of which is mainly regulated by COX-2. Interestingly, FACS and TUNEL staining demonstrated that paeonol significantly inhibited the proliferation of HepG2 and SMMC-7721 cells and induced apoptosis, concomitant with the downregulation of survivin. The levels of COX-2 and PGE2 were also reduced by paeonol, as confirmed by Western blotting and ELISA, respectively. To determine the mechanism by which paeonol inhibited survivin in HCC cells the effects of COX-2 expression on surviving were studied. Treatment with the COX-2 selective inhibitor NS398 effectively decreased the levels of PGE2 and survivin, inducing apoptosis in a manner similar to that of paeonol. Survivin expression was increased by PGE2 treatment but was blocked by paeonol, which suggests that paeonol inhibits survivin by inhibiting the COX-2/PGE2 signaling pathway.ConclusionsTo the best of our knowledge, this is the first study to demonstrate that paeonol can exert antitumor effects on HCC cells by targeting survivin via the COX-2/PGE2 signaling pathway. Paeonol could therefore be considered as a potential therapeutic candidate for HCC.

The Characterization of Ground Raspberry Seeds and the Physiological Response to Supplementation in Hypertensive and Normotensive Rats.

This study aimed to evaluate the protective role of ground raspberry seeds (RBS) as a source of polyphenols and essential fatty acids on blood plasma enzymatic antioxidant status, lipid profile, and endothelium-intact vasodilation during physiological and pathological conditions. Young normotensive Wistar–Kyoto rats (WKYs) and spontaneously hypertensive rats (SHRs) at ten weeks of age were fed with either a control diet or were supplemented with added 7% RBS for six weeks (n = 6). The main component of RBS was dietary fiber (64%) and the main polyphenols were ellagitannins (1.2%) and flavan-3-ols (0.45%). Irrespective of the rat model, ground RBS decreased liver enzyme aspartate aminotransferase (0.9-fold) and hydrogen peroxide scavenging capacity (Catalase, 0.9-fold). In supplemented SHRs, preincubation with inducible nitric oxide synthase (iNOS) inhibitor 1400W, nonselective cyclooxygenase (COX) inhibitor indomethacin, selective COX-2 inhibitor NS-398, prostacyclin (PGI2) synthesis inhibitor tranylcypromine (TCP), thromboxane receptor (TP) antagonist SQ-29548, thromboxane synthesis inhibitor furegrelate, and 20-HETE synthesis inhibitor HET0016 induced the same relaxant response to acetylcholine as in the nonsupplemented control group. In supplemented WKYs, atherogenic index was decreased (0.8-fold), while iNOS and COX-2-derived PGI2 increased acetylcholine-induced vasodilation. These effects of ground RBS may constitute a potential mechanism for preventing cardiovascular diseases.

Biological effects of selective COX-2 inhibitor NS398 on human glioblastoma cell lines

BackgroundCyclooxygenase-2 (COX-2), an inflammation-associated enzyme, has been implicated in tumorigenesis and progression of glioblastoma (GBM). The poor survival of GBM was mainly associated with the presence of glioma stem cells (GSC) and the markedly inflammatory microenvironment. To further explore the involvement of COX-2 in glioma biology, the effects of NS398, a selective COX-2 inhibitor, were evaluated on GSC derived from COX-2 expressing GBM cell lines, i.e., U87MG and T98G, in terms of neurospheres’ growth, autophagy, and extracellular vesicle (EV) release.MethodsNeurospheres’ growth and morphology were evaluated by optical and scanning electron microscopy. Autophagy was measured by staining acidic vesicular organelles. Extracellular vesicles (EV), released from neurospheres, were analyzed by transmission electron microscopy. The autophagic proteins Beclin-1 and LC3B, as well as the EV markers CD63 and CD81, were analyzed by western blotting. The scratch assay test was used to evaluate the NS398 influence on GBM cell migration.ResultsBoth cell lines were strongly influenced by NS398 exposure, as showed by morphological changes, reduced growth rate, and appearance of autophagy. Furthermore, the inhibitor led to a functional change of EV released by neurospheres. Indeed, EV secreted by NS398-treated GSC, but not those from control cells, were able to significantly inhibit adherent U87MG and T98G cell migration and induced autophagy in recipient cells, thus leading to effects quite similar to those directly caused by NS398 in the same cells.ConclusionDespite the intrinsic diversity and individual genetic features of U87MG and T98G, comparable effects were exerted by the COX-2 inhibitor NS398 on both GBM cell lines. Overall, our findings support the crucial role of the inflammatory-associated COX-2/PGE2 system in glioma and glioma stem cell biology.

COX-2/PGE2 facilitates fracture healing by activating the Wnt/β-catenin signaling pathway.

The aim of this study was to explore the influence of cyclooxygenase-2 (COX-2)/prostaglandin E2 (PGE2) on fracture healing by activating the Wnt/β-catenin signaling pathway. In this study, 36 adult Sprague-Dawley (SD) rats raised in our laboratory were selected as research objects. The rats were subjected to fracture surgery on the middle part of the right femoral shaft. Subsequently, they were randomly divided into the control group and experimental groups (including experimental group A and experimental group B). Rats in experimental group A were injected with PGE 2 or COX-2 selective inhibitor NS-398, while rats in experimental group B were injected with PGE2 (5 μmol/L). Meanwhile, rats in the control group were injected with the same amount of normal saline. After that, the transcriptional levels of PEG2, COX-2, vascular endothelial growth factor (VEGF) and β-catenin in rats of the experimental group A, experimental group B and control group were detected via fluorescence quantitative Polymerase Chain Reaction (PCR) assay. Enzyme-linked immunosorbent assay (ELISA) and Western blotting were conducted to determine the changes in protein levels of PEG2, COX-2, VEGF and β-catenin in rats of the experimental group A, experimental group B and control group. The expression level of VEGF in bone tissues at fracture ends of rats in the experimental group A, experimental group B and control group was observed through the hematoxylin-eosin (HE) staining. Furthermore, micro-computed tomography (CT) was employed to evaluate callus formation. The transcriptional and translational levels of COX-2, β-catenin and VEGF in rats of experimental group A treated with COX-2 inhibitors were significantly down-regulated when compared with those of the control group, showing statistically significant differences (p<0.05). However, the levels of these genes were markedly elevated in the experimental group B treated with PGE2 in comparison with those in the control group, and the differences were statistically significant (p<0.05). After 6 weeks, HE staining showed that the expression level of VEGF in rats of the experimental group B was remarkably higher than that of the experimental group A (p<0.05). Micro-CT results revealed that the mean trabecular plate density (MTPD) of rats in the experimental group B (73.29±5.4) was markedly higher than the number of osteoblasts (49.6±3.9) in the experimental group A, showing a statistically significant difference (p<0.05). COX-2/PGE2 facilitates fracture healing by activating the Wnt/β-catenin signaling pathway.

JmjC domain-containing protein 8 (JMJD8) represses Ku70/Ku80 expression via attenuating AKT/NF-κB/COX-2 signaling

Jumonji C (JmjC) domain-containing proteins have been shown to regulate cellular processes by hydroxylating or demethylating histone and non-histone targets. JMJD8 is a Jumonji C domain-containing protein localized in the lumen of the endoplasmic reticulum and was recently shown to be involved in endothelial differentiation and cellular inflammation response. However, other physiological functions of JMJD8 remain to be elucidated. In this research, we found that knockdown of JMJD8 in cancer cells significantly increased cell proliferation, and attenuated ionizing irradiation or etoposide treatment-induced DNA double-strand breaks (DSBs) level through enhancing the expression of Ku70 and Ku80 which are key participants in the non-homologous end-joining repair of DSBs. We also provided evidence to show that knockdown of JMJD8 up-regulated cyclooxygenase-2 (COX-2) expression which contributed to the enhanced expression of Ku70/Ku80 as shown by the results that pre-treatment of JMJD8 knockdown cells with COX-2 selective inhibitor NS-398 inhibited the induction of Ku70/Ku80. Furthermore, we identified that the up-regulation of COX-2 in JMJD8 knockdown cells was partially due to the increased activation of AKT/NF-κB signaling, and LY294002 (an inhibitor of the PI3K/AKT signaling pathway) repressed the induction of COX-2 and Ku70/Ku80. In conclusion, our research provided data to establish the role of JMJD8 in regulating tumor cell proliferation and their sensitivity to ionizing irradiation or chemo-therapy drug, and the AKT/NF-κB/COX-2 signaling mediated expression of Ku70/Ku80 was involved. The results of this research indicated that JMJD8 is a potential target for enhancing the efficacy of tumor radio- and chemo-therapies.

The effect of docosahexaenoic acid combined with cyclooxygenase-2 selective inhibitor NS-398 on the apoptosis of bile duct cancer cells QBC939

Objective To investigate the effect of docosahexaenoic acid (DHA) combined with cyclooxygenase-2 (COX-2) selective inhibitor NS-398 on the apoptosis of cholangiocarcinoma QBC939 cells and the mechanism. Methods In vitro, cholangiocarcinoma QBC939 cells were treated with 0, 15, 30, 45, 60 and 75 μg/ml DHA with 0, 25, 50, 100, 150 and 200 μmol/L NS-398, respectively. The absorbances of the QBC939 cells were measured by CCK8 and its growth inhibition ratios were analyzed. Flow cytometry was applied to detect cell apoptosis. The level of β-catenin and COX-2 mRNA and protein were measured by real-time PCR, immunocytochemistry and enzyme-linked immunoadsordent assay, respectively. Results DHA combined with NS-398 could significantly suppress the growth of QBC939 cells (P<0.05). When the concentration of DHA went up to 45 μg/ml and NS-398 was 100 μmol/L, the relative growth inhibition rate of QBC939 cells was 90.0%. If the concentrations were increased, the result showed no significant differences. Furthermore, flow cytometry analysis indicated that DHA combined with NS-398 could induce QBC939 cells apoptosis at the early stage, and the apoptosis rate was significantly different between the experimental and control groups (P<0.01). Real-time PCR showed low β-catenin and COX-2 expression in QBC939 cells disposed by DHA combined with NS-398, and their expression were significantly different between the experimental and control groups (P<0.01). Immunocytochemistry and ELISA demonstrated that DHA combined with NS-398 could decrease β-catenin and COX-2 protein expression in QBC939 cells. Conclusion DHA combined with NS-398 induced apoptosis and inhibited proliferation of cholangiocarcinoma cells QBC939 in vitro through targeting β-catenin and COX-2. Key words: Docosahexaenoic acid (DHA); NS-398; Cholangiocarcinoma; Apoptosis; β-catenin; Cyclooxygenase-2 (COX-2)

Selective cyclooxygenase-2 inhibitor NS-398 attenuates myocardial fibrosis in mice after myocardial infarction via Snail signaling pathway.

The role of NS-398 in Snail pathway of myocardial cells in mice after myocardial infarction and its effect on myocardial fibrosis were investigated in this study. C57BL/6 mice were selected to establish mouse models of myocardial infarction with permanent ligation of anterior descending branch and sham-operation models without ligation. After successful establishment of models, 30 mice were randomly divided into sham-operation group, myocardial infarction group and drug intervention group. The drug intervention group was treated with intraperitoneal injection of NS-398 (5 mg/kg) at 1 week after modeling for 3 weeks. The survival status of mice after operation was monitored, the cardiac function was detected via echocardiography, the collagen levels in heart tissue pathological sections were detected via Masson staining and Sirius red staining. Moreover, the expressions of Snail and type I collagen levels were detected via immunohistochemistry, and the Snail protein expression level and the activity and expression level of E-cadherin protein were detected via Western blotting. At 4 weeks after establishment of myocardial infarction model, the fibrosis reaction was obvious, and the cardiac function was decreased, accompanied with Snail activation. The administration of NS-398 for 3 weeks inhibited the Snail activity expression and significantly improved the fibrosis degree after infarction. However, it did not improve the cardiac function. Inhibiting Snail improved the fibrosis reaction after infarction, in which Snail/E-cadherin signaling pathway was involved. NS-398 improves the myocardial fibrosis in mice after myocardial infarction through inhibiting the Snail signaling pathway.

Cyclooxygenase 2 contributes to bradykinin-induced microvascular responses in peripheral arterioles after cardiopulmonary bypass

BackgroundDiabetic patients are associated with impaired peripheral microvascular function after cardiopulmonary bypass (CPB) and cardiac surgery. We hypothesized that upregulation of the inducible cyclooxygenase 2 (COX-2) contributes to altered microvascular reactivity of peripheral arterioles in diabetic patients undergoing CPB and cardiac surgery. MethodsSkeletal muscle samples of nondiabetic (ND) patients and patients with diabetes mellitus (DM; n = 8 per group) undergoing cardiac surgery were harvested before and after CPB. The protein expression/localization of COX-2 was assayed by Western blotting and immunohistochemistry. Peripheral arterioles were dissected from the harvested skeletal muscle tissue samples, the isolated arterioles (80-180 μm) were cannulated and pressurized, and changes in diameter were measured with video microscopy. In-vitro relaxation responses of precontracted arterioles were examined in the presence of the endothelium-dependent vasodilator bradykinin (10−10 to 10−6M) and in the presence or absence of the selective COX-2 inhibitor NS398 (10−5M). ResultsThe post-CPB protein levels of the inducible COX-2 were significantly increased compared with pre-CPB values in both the ND and DM groups (P < 0.05), whereas, this increase was higher in DM than that of ND (P < 0.05). In the DM arterioles, not the ND vessels, bradykinin-induced relaxation response was inhibited in the presence of the specific COX-2 inhibitor NS398 at baseline (P < 0.05). After CPB, bradykinin-induced relaxation response of the ND and DM arterioles was inhibited in the presence of the specific COX-2 inhibitor NS398, but this effect was more pronounced in the diabetic patients (P < 0.05). ConclusionsDiabetes and CPB are associated with upregulation in COX-2 expression/activation in human peripheral microvasculature. This alteration may lead to altered peripheral microvascular reactivity in diabetic patients undergoing cardiac surgery.

Diabetes Upregulation of Cyclooxygenase 2 Contributes to Altered Coronary Reactivity After Cardiac Surgery

We hypothesized that upregulation of inducible cyclooxygenase 2 (COX-2) contributes to altered coronary arteriolar reactivity early after cardioplegic arrest and cardiopulmonary bypass (CP/CPB) in patients with diabetes mellitus who are undergoing cardiac surgery. The right atrial tissue samples of nondiabetes (ND), controlled diabetes (CDM), and uncontrolled diabetes (UDM) patients undergoing cardiac surgery were harvested before and after CP/CPB. Coronary arterioles (80 to 150 μm) were dissected from the harvested atrial tissue samples, cannulated, and pressurized. The changes in diameter were measured with video microscopy. The protein expression and localization of COX-1 and COX-2 were assayed by Western blot and immunohistochemistry. In the diabetes arterioles, bradykinin-induced relaxation response was inhibited by the selective COX-2 inhibitor NS398 at baseline (p < 0.05). This effect was more pronounced in UDM arterioles than CDM (p < 0.05). After CP/CPB, bradykinin-induced responses in all groups were inhibited by NS398, but this effect was more pronounced in the UDM patients (p < 0.05). The intensities of COX-2 staining of coronary arterioles and COX-2 protein levels in myocardium were higher in diabetes than nondiabetes at baseline (p < 0.05). The post-CP/CPB protein levels of the inducible COX-2 were significantly increased compared with pre-CP/CPB values in all groups (p < 0.05), whereas this increase was higher with diabetes than with ND (p < 0.05). Furthermore, these effects were more profound in UDM than CDM (p < 0.05). Diabetes and CP/CPB are associated with upregulation in COX-2 expression in human coronary vasculature. Upregulation of COX-2 expression may contribute to bradykinin-induced coronary arteriolar relaxation in diabetic patients undergoing cardiac surgery.

Augmented oxidative stress and preserved vasoconstriction induced by hydrogen peroxide in coronary arteries in obesity: role of COX-2.

Oxidative stress plays a key role in the vascular and metabolic abnormalities associated with obesity. Herein, we assessed whether obesity can increase coronary vasoconstriction induced by hydrogen peroxide (H2 O2 ) and the signalling pathways involving COX-2 and superoxide (O2.- ) generation. Contractile responses to H2 O2 and O2.- generation were measured in coronary arteries from genetically obese Zucker rats (OZR) and compared to lean Zucker rats (LZR). Both basal and H2 O2 -stimulated O2.- production were enhanced in coronary arteries from OZR, but H2 O2 -induced vasoconstriction was unchanged. The selective COX-2 inhibitor NS398 significantly reduced H2 O2 -induced contractions in endothelium-denuded arteries from LZR and OZR, but only in endothelium-intact arteries from LZR. PGI2 (IP) receptor antagonism modestly reduced the vasoconstrictor action of H2 O2 while antagonism of the PGE2 receptor 4 (EP4 ) enhanced H2 O2 contractions in arteries from OZR but not LZR. Basal release of COX-2-derived PGE2 was higher in coronary arteries from OZR where the selective agonist of EP4 receptors TCS 2519 evoked potent relaxations. COX-2 was up-regulated after acute exposure to H2 O2 in coronary endothelium and vascular smooth muscle (VSM) and inhibition of COX-2 markedly reduced H2 O2 -elicited O2.- generation in coronary arteries and myocardium. Expression of Nox subunits in VSM and NADPH-stimulated O2.- generation was enhanced and contributed to H2 O2 vasoconstriction in arteries from obese rats. COX-2 contributes to cardiac oxidative stress and to the endothelium-independent O2.- -mediated coronary vasoconstriction induced by H2 O2 in obesity, which is offset by the release of COX-2-derived endothelial PGE2 acting on EP4 vasodilator receptors.

Cyclooxygenase-1 suppresses lipopolysaccharide-induced changes in rat gastric inducible nitric oxide synthase.

Both nitric oxide synthase (NOS) and cyclooxygenase (COX) have inducible isoforms that are up-regulated during inflammatory states. However, the interaction between these enzymes is not clearly understood. The objective was to clarify the interactions between NOS and COX in the rat gastric mucosa in the presence and absence of lipopolysaccharide. Laboratory study. Medical school laboratory. Female Sprague-Dawley rats. We used nonselective and selective COX inhibitors to determine the role of COX on inducible NOS (iNOS) expression in the gastric mucosa. The nonselective COX inhibitors salicylate and indomethacin enhanced the expression of iNOS in the rat gastric mucosa and exacerbated gastric injury in the presence of lipopolysaccharide, effects reversed by exogenous prostaglandin E2. Selective COX-1 inhibition with SC560 similarly increased gastric iNOS expression and exacerbated gastric injury, while the selective COX-2 inhibitor NS398 had no effect on iNOS expression or gastric injury in the presence of lipopolysaccharide. These data suggest that COX-1 derived prostaglandins exert an inhibitory effect on gastric iNOS during endotoxemia, and this may represent a potential cytoprotective mechanism not previously recognized for this enzyme, since up-regulation of iNOS is deleterious in some tissues.

Mono-ethylhexyl phthalate stimulates prostaglandin secretion in human placental macrophages and THP-1 cells.

BackgroundDiethylhexyl phthalate (DEHP) is widely used as a plasticizer in polyvinyl chloride products. DEHP exposure, which is widespread in the US, increases preterm birth risk; however, the mechanisms driving this relationship are unclear. Because cyclooxygenase-2 (COX-2) dependent prostaglandin synthesis is implicated in preterm birth, we evaluated effects of mono-2-ethylhexyl phthalate (MEHP), the active metabolite of DEHP, on prostaglandin E2 (PGE2) synthesis and COX expression in human placental macrophages (PM). In addition, responses in PM were compared to those in a human macrophage-like cell line, THP-1.MethodsPM and THP-1 cells were treated for 2, 4, 8, or 24 h with MEHP concentrations ranging from 10 to 180 micromolar. PGE2 concentrations were assessed in culture medium using ELISA, and COX expression was determined by western blot.ResultsTreatment of PM and THP-1 cells with 180 micromolar MEHP for 24 h significantly increased PGE2 release. Co-treatment of PMs or THP-1 cells with 180 micromolar MEHP and the non-selective COX inhibitor indomethacin reduced MEHP-stimulated PGE2 production. Similarly, co-treatment of PM and THP-1 cells with the COX-2 selective inhibitor NS-398 resulted in a significant decrease in PGE2, suggesting that MEHP-stimulated PGE2 is dependent specifically on increased COX-2 expression. Western blot analysis revealed a significant increase in COX-2 expression in PM and THP-1 cells treated with 180 micromolar MEHP, and no changes in COX-1 expression, supporting the role of COX-2 in MEHP-stimulated PGE2 synthesis.ConclusionsThe findings from this study are the first to demonstrate phthalate-stimulated PGE2 synthesis in PM and warrant future studies into COX-2-dependent prostaglandin synthesis as a mechanism of toxicant-associated preterm birth.Electronic supplementary materialThe online version of this article (doi:10.1186/s12958-015-0046-8) contains supplementary material, which is available to authorized users.

The SK-N-AS human neuroblastoma cell line develops osteolytic bone metastases with increased angiogenesis and COX-2 expression

Neuroblastoma (NB), which arises from embryonic neural crest cells, is the most common extra-cranial solid tumor of childhood. Approximately half of NB patients manifest bone metastasis accompanied with bone pain, fractures and bone marrow failure, leading to disturbed quality of life and poor survival. To study the mechanism of bone metastasis of NB, we established an animal model in which intracardiac inoculation of the SK-N-AS human NB cells in nude mice developed osteolytic bone metastases with increased osteoclastogenesis. SK-N-AS cells induced the expression of receptor activator of NF-κB ligand and osteoclastogenesis in mouse bone marrow cells in the co-culture. SK-N-AS cells expressed COX-2 mRNA and produced substantial amounts of prostaglandin E2 (PGE2). In contrast, the SK-N-DZ and SK-N-FI human NB cells failed to develop bone metastases, induce osteoclastogenesis, express COX-2 mRNA and produce PGE2. Immunohistochemical examination of SK-N-AS bone metastasis and subcutaneous tumor showed strong expression of COX-2. The selective COX-2 inhibitor NS-398 inhibited PGE2 production and suppressed bone metastases with reduced osteoclastogenesis. NS-398 also inhibited subcutaneous SK-N-AS tumor development with decreased angiogenesis and vascular endothelial growth factor-A expression. Of interest, metastasis to the adrenal gland, a preferential site for NB development, was also diminished by NS-398. Our results suggest that COX2/PGE2 axis plays a critical role in the pathophysiology of osteolytic bone metastases and tumor development of the SK-NS-AS human NB. Inhibition of angiogenesis by suppressing COX-2/PGE2 may be an effective therapeutic approach for children with NB.

Inflammation and MiR-21 pathways functionally interact to downregulate PDCD4 in colorectal cancer.

Inflammation plays a direct role in colorectal cancer (CRC) progression; however the molecular mechanisms responsible for this effect are unclear. The inflammation induced cyclooxygenase 2 (COX-2) enzyme required for the production of Prostaglandin E2 (PGE2), can promote colorectal cancer by decreasing expression of the tumour suppressor gene Programmed Cell Death 4 (PDCD4). As PDCD4 is also a direct target of the oncogene microRNA-21 (miR-21) we investigated the relationship between the COX-2 and miR-21 pathways in colorectal cancer progression. Gene expression profile in tumour and paired normal mucosa from 45 CRC patients demonstrated that up-regulation of COX-2 and miR-21 in tumour tissue correlates with worse Dukes' stage. In vitro studies in colonic adenocarcinoma cells revealed that treatment with the selective COX-2 inhibitor NS398 significantly decreased miR-21 levels (p = 0.0067) and increased PDCD4 protein levels (p<0.001), whilst treatment with PGE2 up-regulated miR-21 expression (p = 0.019) and down-regulated PDCD4 protein (p<0.05). These findings indicate that miR-21 is a component of the COX-2 inflammation pathway and that this pathway promotes worsening of disease stage in colorectal cancer by inducing accumulation of PGE2 and increasing expression of miR-21 with consequent downregulation of the tumour suppressor gene PDCD4.

The Role of Cyclooxygenase-1 and -2 in Sevoflurane-Induced Postconditioning Against Myocardial Infarction

Cyclooxygenase (COX)-2 mediates ischemic pre- and postconditioning as well as anesthetic-induced preconditioning. However, the role of COX-1 and -2 in anesthetic-induced postconditioning has not been investigated. We evaluated the role of COX-1 and -2 in sevoflurane-induced postconditioning in vivo. Pentobarbital-anaesthetized male C57BL/6 mice were subjected to 45 minutes of coronary artery occlusion and 3 hours of reperfusion. Animals received either no intervention, the vehicle dimethyl sulfoxide (DMSO, 10 µL/g intraperitoneally), acetylsalicylic acid (ASA, 5 µg/g intraperitoneally), the selective COX-1 inhibitor SC-560 (10 µg/g intraperitoneally), or the selective COX-2 inhibitor NS-398 (5 µg/g intraperitoneally). 1.0 MAC (minimum alveolar concentration) sevoflurane was administered for 18 minutes during early reperfusion either alone or in combination with ASA, SC-560, and NS-398. Infarct size was determined with triphenyltetrazolium chloride. Statistical analysis was performed using 1-way and 2-way analyses of variance with post hoc Duncan testing. The infarct size in the control group was 44% ± 9%. DMSO (42% ± 7%), ASA (36% ± 6%), and NS-398 (44% ± 18%) had no effect on infarct size. Sevoflurane (17% ± 4%; P < .05) and SC-560 (26% ± 10%; P < .05) significantly reduced the infarct size compared with control condition. Sevoflurane-induced postconditioning was not abolished by ASA (16% ± 5%) and SC-560 (22% ± 4%). NS-398 abolished sevoflurane-induced postconditioning (33% ± 14%). It was concluded that sevoflurane induces postconditioning in mice. Inhibition of COX-1 elicits a myocardial infarct size reduction and does not abolish sevoflurane-induced postconditioning. Blockade of COX-2 abolishes sevoflurane-induced postconditioning. These results indicate that sevoflurane-induced postconditioning is mediated by COX-2.

Urocortin increased endothelial ICAM1 by cPLA2-dependent NF-κB and PKA pathways in HUVECs

Urocortin (Ucn1), a member of the corticotrophin-releasing hormone (CRH) family, has been reported to participate in inflammation. The increased expression of intercellular adhesion molecule 1 (ICAM1) plays important roles in inflammation and immune responses. Our previous results demonstrated that Ucn1 significantly enhanced the expression of ICAM1. However, the underlying mechanisms are still unknown. The purpose of this study is to investigate the detailed mechanisms of Ucn1-induced upregulation of ICAM1. Here, we characterized the mechanisms of Ucn1 usage to regulate ICAM1 expression in human umbilical vein endothelial cells (HUVECs). Our data revealed that Ucn1 increased ICAM1 and cyclooxygenase 2 (COX2) expressions in a time-dependent manner via CRH receptor 2 (CRHR2). In addition, COX2 was involved in ICAM1 upregulation. Furthermore, Ucn1 could increase the expression and phosphorylation of cytosolic phospholipases A2 (cPLA2) in a time-dependent manner via CRHR2 and CRHR1. Moreover, ablation of cPLA2 by the inhibitor pyrrophenone or siRNA attenuated the ICAM1 increase induced by Ucn1. In addition, nuclear factor κB (NF-κB) was activated, indicated by the increase in nuclear p65NF-κB expression and phosphorylation of p65NF-κB, depending on cPLA2 and CRHR2 activation. Pyrrolidinedithiocarbamic acid, an inhibitor of NF-κB, abolished the elevation of ICAM1 but not COX2. Also, Ucn1 increased the production of prostaglandin E2 (PGE2) which further activated protein kinase A (PKA)-CREB pathways dependent of cPLA2 via CRHR2. Moreover, the increase in NF-κB phosphorylation was not affected by the selective COX2 inhibitor NS-398 or the PKA inhibitor H89. In conclusion, these data indicate that Ucn1 increase the ICAM1 expression via cPLA2-NF-κB and cPLA2-COX2-PGE2-PKA-CREB pathways by means of CRHR2.

Induction but not inhibition of COX-2 confers human lung cancer cell apoptosis by celecoxib

The antitumorigenic mechanism of the selective cyclooxygenase-2 (COX-2) inhibitor celecoxib is still a matter of debate. Among different structurally related COX-2 inhibitors, only celecoxib was found to cause apoptosis and cell death of human lung cancer cells (IC₅₀ values of 19.96 µM [A549], 12.48 µM [H460], and 41.39 µM [H358]) that was paralleled by a time- and concentration-dependent upregulation of COX-2 and peroxisome proliferator-activated receptor γ (PPARγ) at mRNA and protein levels. Apoptotic death of celecoxib-treated cancer cells was suppressed by the PPARγ antagonist GW9662 and by siRNA targeting PPARγ and, surprisingly, also by the selective COX-2 inhibitor NS-398 and siRNA targeting COX-2. NS-398 (1 µM) was shown to suppress celecoxib-induced COX-2 activity. Among the COX-2-dependent prostaglandins (PG) induced upon celecoxib treatment, PGD₂ and 15-deoxy-Δ¹²,¹⁴-PGJ₂ were found to induce a cytosol-to-nucleus translocation of PPARγ as well as a PPARγ-dependent apoptosis. Celecoxib-elicited PPARγ translocation was inhibited by NS-398. Finally, a COX-2- and PPARγ-dependent cytotoxic action of celecoxib was proven for primary human lung tumor cells. Together, our data demonstrate a proapoptotic mechanism of celecoxib involving initial upregulation of COX-2 and PPARγ and a subsequent nuclear translocation of PPARγ by COX-2-dependent PGs.