Effect of Propyl Gallate on Activity of Cyclooxygenase 1 and 2 in Mice's Peritoneal Macrophages*

Objective: To investigate the effect of Red Peony 801 (propyl gallate,PrG) on cyclooxygenase (COX) activity in murine peritoneal macrophages. Methods: A screening model for COX inhibitors in vitro based on murine peritoneal macrophages was used. COX-1 activity was reflected by the level of 6-ketoprostaglandin F1α(6-keto-PGF1α) in supernatants of cultured macrophages which were stimulated with calcium ionophore A23187 for a short-term, while COX-2 activity was reflected by the level of prostaglandin E2 (PGE2) in supernatants of cultured macrophages which were stimulated with lipopolysaccharide (LPS) for a long-term. Results: PrG did not affect A23187-induced, COX-1-derived 6-keto-PGF1α synthesis at the concentrations of 1×10-5, 5×10-6 mol/L (P>0.05), but enhanced 6-keto-PGF1α synthesis at the concentrations of 1×10-6, 5×10-7, 1×10-7 mol/L (P<0.01) in vitro, and showed a good dose-dependent manner. It inhibited LPS-induced, COX-2-derived PGE2 synthesis at the concentrations of 1×10-5,1×10-6 mol/L (P< 0. 05). Conclusion: Within the range of 1×10-5 to 1×10-7 mol/L, PrG activated COX-1 at lower concentrations and inhibited COX-2 at higher concentrations in murine peritoneal macrophages.

Selective Cyclooxygenase-2 Inhibitors: Design and Synthesis

The discovery of COX-2 provides a novel target developing more effective NSAIDs with fewer side effects. On the basis of results from the structure-activity relationships (SAR) of selective COX-2 inhibitors, we have designed and synthesized some promising compounds.

Cyclooxygenase 2 polymorphism and colorectal cancer:-765G>C variant modifies risk associated with smoking and body mass index

AIM： To explore whether cyclooxygenase 2 （COX-2） -765G〉C polymorphism is associated with susceptibility of colorectal cancer （CRC） and to evaluate the risk of colorectal cancer in relation to environmental exposures and polymorphism. METHODS： We conducted a case-control study of 137 patients with colorectal cancer and 199 cancerfree controls in northeast China. Multivariate logistic regression analysis was performed to calculate the adjusted odds ratio （OR） and 95% confidence interval （95% CI）. RESULTS： The -765G〉C polymorphism was not independently associated with CRC risk. However, risk associated with the polymorphism differed by smoking and body mass index （BMI）. Smoking and BMI associated risks were stronger among those with -765GG genotype, showing that smokers had a 2.682-fold greater risk of CRC than nonsmokers （51/43 vs 68/126, P = 0.006）. Compared to those with a normal body mass index （BMI 18.5-22.9）, those with overweight （BMI 23-24.9） had a 3.909-fold higher risk of CRC （OR = 3.909, 95% CI = 2.081-7.344; P 〈 0.001）, while those with obesity （BMI 〉 25） had a 2.031- fold higher risk of CRC （OR = 1.107, 95% CI = 1.107-3.726; P = 0.022）. is not associated with an increased risk of CRC, -765GG genotype appears to be related to an increased risk in the presence of smoking and higher BMI.

Expression of inducible nitric oxide synthase and cyclooxygenase-2 in pancreatic adenocarcinoma:Correlation with microvessel density

AIM:Cydooxygenases (COX) are key enzymes for conversion of arachidonic acid to prostaglandins.Nitric oxide synthase (NOS) is the enzyme responsible for formation of nitric oxide. Both have constitutive and inducible isoforms.The inducible isoforms (iNOS and COX-2) are of great interest as regulators of tumor angiogenesis,tumorigenesis and inflammatory processes.This study was to clarify their role in pancreatic adenocarcinomas. METHODS:We investigated the immunohistochemical iNOS and COX-2 expression in 40 pancreatic ductal adenocardnomas of different grade and stage.The results were compared with microvessel density and dinicopathological data. RESULTS:Twenty-one (52.5%) of the cases showed iNOS expression,15 (37.5%) of the cases were positive for COX-2. The immunoreaction was heterogeneously distributed within the tumors.Staining intensity was different between the tumors.No correlation between iNOS and COX-2 expression was seen.There was no relationship with microvessel density. However,iNOS positive tumors developed more often distant metastases and the more malignant tumors showed a higher COX-2 expression.There was no correlation with other clinicopathological data. CONCLUSION:Approximately half of the cases expressed iNOS and COX-2.These two enzymes do not seem to be the key step in angiogenesis or carcinogenesis of pancreatic adenocarcinomas.Due to a low prevalence of COX-2 expression,chemoprevention of pancreatic carcinomas by COX-2 inhibitors can only achieve a limited success.

Relationship between expression and distribution of cyclooxygenase-2 and bcl-2 in human gastric adenocarcinoma

AIM: To explore expression and distribution features of COX-2 and bcl-2 in human gastric adenocarcinoma tissues and to study its biological significance.METHODS: Totally 36 human gastric carcinoma samples were enrolled in this study (cardiac adenocarcinoma 16 cases, distal gastric adenocarcinoma 20 cases). The expressions of COX-2 and bcl-2 in cancerous tissues and corresponding para-cancerous tissues were investigated by immunohistochemistry using COX-2 polyclonal antibody and bcl-2 monoclonal antibody. The normal gastric mucosa tissues were used as control.RESULTS: The expressions of COX-2 and bcl-2 in gastric carcinoma were significantly higher than that in the paracancerous tissues (77.8% vs 47.2%, P＜0.01, 80.56% vs 58.33%, P＜0.05). The expression of COX-2 in cardiac adenocarcinoma was remarkably higher than that in the distal gastric carcinoma (93.8% vs 65.0%, P＜0.01). The expression of COX-2 was mainly localized in the cytoplasm of tumor cells and partly in the nucleus. There is a transition of the COX-2 cytoplasmic positivity to nucleic in tumor cells with the increase of gastric carcinoma pathological grade. Interstitial macrophages, fibroblasts and vascular endothelial cells also expressed COX-2. The tissues with higher expression of COX-2 also expressed high level of bcl-2 protein.CONCLUSION: Abnormal expression pattern of COX-2within the tissues of human gastric cancer is correlated with tumor location and lymph node metastasis. COX-2may regulate expression of apoptosis suppressor gene (bcl-2) through interaction of tumor cells and stromal cells and play an important role in the generation and development of tumors, which will be of great help in developing new methods for antitumor therapy.

Expression of cyclooxygenase-1 and -2 in extra-hepatic cholangiocarcinoma

Objective: To investigate the expression of cyclooxy- genase-1 (COX-1) and cyclooxygenase-2 (COX-2) in extra-hepatic cholangiocarcinoma and the relation- ship between their expression and clinicopathological parameters. Methods: COX-1 and COX-2 were detected in 56 ex- tra-hepatic cholangiocarcinomas, including 31 matched tissues originating from non-tumorous bile ductal tissue adjacent to tumours and 6 normal bile ductal tissues, by immunohistochemistry strept avi- din-biotin complex using isozyme selective antibod- ies. Results: There was no difference in expression of COX-1 between carcinomas (96%, 54/56) and non- cancerous specimens (94%, 29/31, P>0.05) or normal bile ductal tissues (100%, 6/6, P>0.05). The positive rate of COX-2 expression in extra-hepat- ic cholangiocarcinomas (86%, 48/56) was signifi- cantly higher than their matched tissues (39%, 12/ 31, P<0.01) and normal bile ductal tissues (0%, 0/6, P<0.01). Overexpression of COX-2 in extra- hepatic cholangiocarcinoma was related to the metas- tasis of lymph nodes, distant organs or tissues (P< 0.05) as well as the degree of tumour differentiation (P<0.05). Conclusions: The overexpression of COX-2 plays a crucial role in the carcinogenesis and development of extra-hepatic cholangiocarcinoma, indicating that COX-2 may serve as a target for chemoprevention of extra-hepatic cholangiocarcinoma.

Cyclooxygenase-2 promotes angiogenesis by increasing vascular endothelial growth factor and predicts prognosis in gallbladder carcinoma

AIM: To investigate the relationships between the expression of cyclooxygenase-2 (COX-2), vascular endothelial growth factor (VEGF) and the degree of vascularization, clinicopathologic feature, survival time of patients with gallbladder carcinomas. METHODS: Sixty-four gallbladder carcinoma specimens were evaluated for COX-2, VEGF expression by immunohi stochemical methods. Microvessel counts (MVC) were determined using CD34. The relationships between COX-2, VEGF expression, CD34-stained MVC, clinicopathologic features and survival time were analyzed. The correlations between COX-2 and VEGF expression, CD34-stained MVC were also investigated. RESULTS: COX-2, VEGF immunoreactivity were observed in 71.9% (46/64) and 54.7% (35/64) specimens, respectively. The average MVC in 64 cases of gallbladder carcinoma was 57±14 per high power vision field. The status of MVC was closely correlated with Nevin staging, tumor differentiation and lymph node metastasis (P<0.01, 0.002, and 0.003, 0.000, respectively). Increased VEGF expression was significantly correlated with tumor differentiation (poorly and moderately>well differentiated, P<0.05, P = 0.016). Clinical stages had no relation with the expression of VEGF (P>0.05, P = 0.612). There was a positive correlation between COX-2 expression and clinical stages. The positive rate of COX-2 was higher in cases of Nevin stages S4-S5 (81.8%) than in those of Nevin stages S1-S3 (50.0%) with a statistical significance (P0.01, P = 0.009). The expression of COX-2 did not vary with differentiation (P>0.05, P= 0.067). Statistically significant differences were also observed according to lymph node metastasis, COX-2 expression and VEGF expression (P<0.01,0.000, and 0.001, respectively). There was no relation between VEGF, COX-2 expression, MVC and the age and sex of patients. MVC and VEGF positive rate in the COX-2 positive gallbladder carcinoma tissue was higher than that in the COX-2 negative tissue (P<0.05, 0.000, and 0.032, respectively). Patients with VEGF, COX-2 positive tumors had a significantly shorter survival time than those with negative tumors (P<0.05,0.004, 0.01, respectively). CONCLUSION: Augmented tumor neovascularization induced by VEGF may be one of the several effects of COX-2 responsible for poor prognosis of human gallbladder carcinoma. COX-2 inhibitor, either in combination therapy with other agents, or for chemoprevention, may be effective via suppression of angiogenesis in this fatal disease.

Haplotype of prostaglandin synthase 2/cyclooxygenase 2 is involved in the susceptibility to inflammatory bowel disease

AIM： Prostaglandin G/H synthase 2 （PTGS2 or COX2） is one of the key factors in the cellular response to inflammation. PTGS2 is expressed in the affected intestinal segments of patients with inflammatory bowel diseases （IBD）. In IBD patients, non-steroidal anti-inflammatory drugs, which have been shown to reduce both the production and activity of PTGS2, may activate IBD and aggravate the symptoms. We aimed at examining genetic variants of PTGS2 that may be risk factors for IBD. METHODS： We genotyped 291 individuals diagnosed with IBD and 367 controls from the Dutch population for the five most frequent polymorphisms of the PTG52 gene. Clinical data were collected on all patients. DNA was extracted via normal laboratory methods. Genotyping was carried out using multiplex PCR followed by the Invader Assay and the 5＇ exonuclease assay （TaqMan）. New polymorphism screening was performed by pre-screening with denaturing high-performance liquid chromatography, followed by fluorescent sequencing. RESULTS： Allele 5209G was weakly associated with Crohn＇s disease （odds ratio [OR] 1.63, 95% confidence interval [CI] 1.03-2.57）, and allele 8473T with ulcerativecolitis （OR 1.50, 95%CI 1.00-2.27）. The haplotype including both alleles showed a strong association with IBD （OR 13.15, 95%CI 3.17-116.15）. This haplotype, while rare （-0.3%） in the general population, is found more frequently in patients （3.5%）. CONCLUSION： Our data suggest that this haplotype of PTGS2 contributes to the susceptibility of IBD.

Comparative analysis of clinicopathological correlations of cyclooxygenase-2 expression in resectable pancreatic cancer

AIM:To perform a comparative analysis of clinicopathological correlations of cyclooxygenase2 (COX2) expression in pancreatic cancer, examined by monoclonal and polyclonal antibodies.METHODS: The COX2 expression in 85 resection specimens of pancreatic ductal adenocarcinoma was immunohistochemically examined using both monoclonal and polyclonal antibodies. The final immunoscores were obtained by multiplying the percentage of positive cells with the numeric score reflecting the staining intensity.COX2 expression levels were classified into three categories (0, 1+, and 2+) and the clinicopathological correlations were statistically evaluated and analyzed.RESULTS: The positive tumor expression rates of COX2 were 80.5% using monoclonal antibody and 69.4% using polyclonal antibody. In the KaplanMeier analysis, no significant correlations were found between levels of COX2 expression and overall survival (OS), but trends to longer OS were found in COX2 negative cases using monoclonal antibody. Significantly longer disease free survival was revealed in COX2 negative cases using monoclonal antibody (P = 0.019). No correlations between COX2 expression levels and grade (G), tumor (T) status and nodal (N) status were demonstrated. Low histological grade showed a strong association with a longer OS (P < 0.001). Correlation of survival and T status revealed a shorter OS in T3 tumors, but the results reached only marginal statistical significance (P = 0.070). In the multivariate Cox proportional hazards regression model, histological grade, T and N status remained valuable predictors of a worse survival with borderline significance for T [hazards ratio (HR) = 4.18 for G (if G = 3, P < 0.001); HR = 1.64 for T (if T = 3, P = 0.065); HR = 2.53 for N (if N = 1, P = 0.006)]. Higher grade, T or N status was associated with a worse OS. CONCLUSION: The immunohistochemically assessed level of COX2 expression does not seem to represent a valuable independent prognostic factor and is not superior to the conventional prognostic factors.

Expression of cyclooxygenase in hyperdynamic portal hypertensive rats

BACKGROUND: By detecting hemodynamic changes, concentration of plasm prostacyclin ( PGI2) and expression of cyclooxygenase( COX) in vasculature and splanchnic tissues, we evaluated the relative contributions of PGI2 and COX mRNA expression to the hyperdynamic circulatory state in chronic portal hypertensive rats. METHODS: Fifty male Sprague-Dawley rats were divided into 3 groups: intrahepatic portal hypertension (IHPH, n = 18) by injection of CCl4, prehepatic portal hypertension (PHPH, n = 18) by partial stenosis of the portal vein, and sham-operated controls (SO, n =14). Splanchnic hemodynamics was measured by radioactive microsphere techniques and the concentration of PGI2 was detected by specific radioimmunoassay for its stable hydrolysis product 6-keto-PGF1α. Semi-quantitive reverse transcriptase-polyme-rase chain reaction (RT-PCR) was performed to measure the levels of COX-1 mRNA and COX-2 mRNA in the thoracic aorta, superior mesenteric artery( SMA),and small intestine of IHPH, PHPH and SO rats, respectively. RESULTS: Hyperdynamic circulatory state was characterized by increased splanchnic blood flow and decreased splanchnic vascular resistance in IHPH and PHPH rats. The concentration of plasma 6-keto-PGF1α ( pg/ml) in IHPH (1093.75 ± 142.15) and PHPH (897. 42 ± 53. 29) rats was significantly higher than that in SO rats (730.13 ± 98. 67) (P <0.05). The expression of COX-1 mRNA in the thoracic aorta, SMA and small intestine was enhanced, whereas COX-2 mRNA expression was not detected in either of these vessels or the small intestine. The plasma 6-keto-PGF1α concentration and the expression of COX-1 mRNA in these vessels and the small intestine were closely correlated with such hemodynamic parameters as portal venous inflow (PVI), splanchnic vascular resistance (SVR) and free portal venous pressure (FPP) (P<0.05). CONCLUSION: The expression of COX-1 mRNA and the levels of PGI2 were closely related to the hyperdynamic circulatory state of portal hypertensive rats.

Indoleamine-2,3-dioxygenase 1/cyclooxygenase 2 expression prediction for adverse prognosis in colorectal cancer

AIM To evaluate indoleamine-2,3-dioxygenase 1/cyclooxygenase 2(IDO1/COX2) expression as an independent prognostic biomarker for colorectal cancer(CRC) patients.METHODS We retrospectively studied the medical records of 95 patients who received surgical resection from August 2008 to January 2010. All patients were randomly assigned to adjuvant treatment with or without celecoxib groups after surgery. We performed standard immunohistochemistry to assess the expression levels of IDO1/COX2 and evaluated the correlation of IDO1/COX2 with clinicopathological factors and overall survival(OS) outcomes.RESULTS The expression of nuclear IDO1 was significantly correlated with body mass index(P < 0.001), and IDO1 expression displayed no association with sex, age, tumor differentiation, T stage, N stage, carcinoembryonic antigen, cancer antigen 19-9, CD3+ and CD8+ tumor infiltrating lymphocytes, and COX2. In univariate analysis, we found that nuclear IDO1(P = 0.039), nuclear/cytoplasmic IDO1 [hazard ratio(HR) = 2.044, 95% confidence interval(CI): 0.871-4.798, P = 0.039], nuclear IDO1/COX2(HR = 3.048, 95%CI: 0.868-10.7, P = 0.0049) and cytoplasmic IDO1/COX2(HR = 2.109, 95%CI: 0.976-4.558, P = 0.022) all yielded significantly poor OS outcomes. Nuclear IDO1(P = 0.041), nuclear/cytoplasmic IDO1(HR = 3.023, 95%CI: 0.585-15.61, P = 0.041) and cytoplasmic IDO1/COX2(HR = 2.740, 95%CI: 0.764-9.831, P = 0.038) have significantly poor OS outcomes for the CRC celecoxib subgroup. In our multivariate Cox model, high coexpression of cytoplasmic IDO1/COX2 was found to be an independent predictor of poor outcome in CRC(HR = 2.218, 95%CI: 1.011-4.48, P = 0.047) and celecoxib subgroup patients(HR = 3.210, 95%CI: 1.074-9.590, P = 0.037).CONCLUSION Our results showed that cytoplasmic IDO1/COX2 coexpression could be used as an independent poor predictor for OS in CRC.

Influence of nitric oxide synthase and cyclooxygenase blockade on expression of cyclooxygenase and hemodynamics in rats with portal hypertension

BACKGROUND : The importance of nitric oxide (NO) in the pathogenesis of portal hypertension (PHT) has been extensively studied, but whether or not prostacyclin (PGI2) plays a role in formation and development of hyperdynamic circutatory state in PHT has not been verified. The present study was undertaken to investigate the possible interaction between prostacyclin (PGI2) and nitric oxide (NO) in the hyperdynamic circulatory state of rats with chronic portal hypertension (PHT), by measuring the hemodynamic changes and expression of cyclooxygenase (COX) mRNA in vessels and small intestine after administration of Nω- nitro-L-arginine (L-NNA) or indomethacin (INDO) either in the short-term (7 days) or long-term (15 days). METHODS: Ninety-seven male Sprague-Dawley rats were divided into three groups: intrahepatic portal hypertension (IHPH) induced by injection of CCl4, prehepatic portal hypertension (PHPH) induced by partial stenosis of the portal vein, and sham-operated controls (SO). Animals of each group received L-NNA or INDO either for 7 or 15 days, with saline as control. Splanchnic hemodynamics was measured by the radioactive microsphere technique. The concentration of NO in serum was determined as the nitrate; nitrite ratio (NO2-/NO3-, μmol/L) by a colorometric method, and that of PGI2 was measured by specific radioimmunoassay for its stable hydrolysis product 6-keto-PGF1α (pg/ml). The reverse transcription- polymerase chain reaction measured the levels of COX-1 mRNA in the superior mesenteric artery, thoracic aorta, and small intestine of these rats.RESULTS: Compared with SO rats, COX-1 mRNA expression and the concentrations of plasma 6-keto- PGF1α and serum NO2-/NO3- were enhanced in both IHPH and PHPH rats; splanchnic vascular resistance (SVR) decreased, but portal venous inflow (PVI) markedly increased (P<0.05). Seven or 15 days of L-NNA treatment reduced COX-1 mRNA expression in these vessels and the small intestine, concomitant with a significant decrease in the concentration of plasma PGI2 and serum NO in IHPH and PHPH rats (P<0.05). At the same time, PVI decreased but SVR increased significantly (P<0.05). In both IHPH and PHPH rats, the COX-1 mRNA expression and the concentration of plasma PGI2 after No synthase (NOS) blockade for 15 days were higher than those for 7 days, whereas the hyperdynamic circulatory state was improved after NOS blockade for 15 days compared with 7 days. The concentration of PGI2 treated by INDO for 15 days was not significantly different from that after 7-day COX blockade, and hemodynamics restored hyperdynamic circulatory state. CONCLUSIONS: The hyperdynamic circulatory state in rats with PHT is correlated with the concentration of serum NO. There is a possible interaction between PGI2 and NO in the hyperhemodynamics of PHT. PGI2 is probably not the mediator in the formation and development of the hyperdynamic circulatory state in rats with chronic PHT.

Aspirin, cyclooxygenase inhibition and colorectal cancer

Colorectal cancer(CRC)is the third most common type of cancer worldwide.Screening measures are far from adequate and not widely available in resourcepoor settings.Primary prevention strategies therefore remain necessary to reduce the risk of developing CRC.Increasing evidence from epidemiological studies,randomized clinical trials and basic science supports the effectiveness of aspirin,as well as other non-steroidal anti-inflammatory drugs,for chemoprevention of several types of cancer,including CRC.This includes the prevention of adenoma recurrence and reduction of CRC incidence and mortality.The detectable benefit of daily low-dose aspirin(at least 75 mg),as used to prevent cardiovascular disease events,strongly suggests that its antiplatelet action is central to explaining its antitumor efficacy.Daily low-dose aspirin achieves complete and persistent inhibition of cyclooxygenase(COX)-1 in platelets(in pre-systemic circulation)while causing alimited and rapidly reversible inhibitory effect on COX-2and/or COX-1 expressed in nucleated cells.Aspirin has a short half-life in human circulation(about 20 minutes);nucleated cells have the ability to resynthesize acetylated COX isozymes within a few hours,while platelets do not.COX-independent mechanisms of aspirin have been suggested to explain its chemopreventive effects but this concept remains to be demonstrated in vivo at clinical doses.

Interplay between post-translational cyclooxygenase-2 modifications and the metabolic and proteomic profile in a colorectal cancer cohort

BACKGROUND Colorectal cancer(CRC) is the second most common cause of cancer death worldwide. It is broadly described that cyclooxygenase-2(COX-2) is mainly overexpressed in CRC but less is known regarding post-translational modifications of this enzyme that may regulate its activity, intracellular localization and stability. Since metabolic and proteomic profile analysis is essential for cancer prognosis and diagnosis, our hypothesis is that the analysis of correlations between these specific parameters and COX-2 state in tumors of a high number of CRC patients could be useful for the understanding of the basis of this cancer in humans.AIM To analyze COX-2 regulation in colorectal cancer and to perform a detailed analysis of their metabolic and proteomic profile.METHODS Biopsies from both healthy and pathological colorectal tissues were taken under informed consent from patients during standard colonoscopy procedure in the University Hospital of Bellvitge(Barcelona, Spain) and Germans Trias i Pujol University Hospital(Campus Can Ruti)(Barcelona, Spain). Western blot analysis was used to determine COX-2 levels. Deglycosylation assays were performed in both cells and tumor samples incubating each sample with peptide N-glycosidase F(PNGase F). Prostaglandin E2(PGE2) levels were determined using a specific ELISA. 1 H high resolution magic angle spinning(HRMAS) analysis was performed using a Bruker AVIII 500 MHz spectrometer and proteomic analysis was performed in a nano-liquid chromatography-tandem mass spectrometer(nano LC-MS/MS) using a QExactive HF orbitrap MS.RESULTS Our data show that COX-2 has a differential expression profile in tumor tissue of CRC patients vs the adjacent non-tumor area, which correspond to a glycosylated and less active state of the protein. This fact was associated to a lesser PGE2 production in tumors. These results were corroborated in vitro performing deglycosylation assays in HT29 cell line where COX-2 protein profile was modified after PNGase F incubation, showing higher PGE2 levels. Moreover,HRMAS analysis indicated that tumor tissue has altered metabolic features vs non-tumor counterparts, presenting increased levels of certain metabolites such as taurine and phosphocholine and lower levels of lactate. In proteomic experiments, we detected an enlarged number of proteins in tumors that are mainly implicated in basic biological functions like mitochondrial activity,DNA/RNA processing, vesicular trafficking, metabolism, cytoskeleton and splicing.CONCLUSION In our colorectal cancer cohort, tumor tissue presents a differential COX-2 expression pattern with lower enzymatic activity that can be related to an altered metabolic and proteomic profile.

Tolerance of neurite outgrowth to Rho kinase inhibitors decreased by cyclooxygenase-2 inhibitor

In this study, PC12 Adh cells and Neuro-2a cells were treated with Rho-associated kinase inhibitors （Y27632 and Fasudil）, a cyclooxygenase-1 selective inhibitor （SC560）, and a cyclooxygenase-2 inhibitor （NS398）. We found that these cells became tolerant to Rho-associated kinase inhibitors, as neurite outgrowth induced by these inhibitors diminished following more than 3 days of exposure in either cell line. The proteins cyclooxygenase-2 and cytosolic prostaglandin E synthetase were upregulated at day 3. NS398 decreased the tolerance to neurite outgrowth induction in both cell lines, whereas SC560 had almost no effect. These findings indicate that cells become tolerant to neurite outgrowth induced by Rho-associated kinase inhibitors, this is at least partly associated with upregulation of proteins involved in the cyclooxygenase-2 pathway, and cyclooxygenases-2 inhibition prevents this tolerance.

Novel functional association of rat testicular membrane-associated cytosolic glutathione S transferases and cyclooxygenase in vitro

Aim:To analyze the role of cytosolic glutathione S-transferases (cGSTs) and membrane-associated cytosolic GSTs (macGSTs) in prostaglandin biosynthesis and to evaluate the possible interaction between glutathione S-transferases (GSTs) and cyclooxygenase (COX) in vitro.Methods:SDS-PAGE analysis was undertaken for characterization of GSTs,thin layer chromatography (TLC) to monitor the effect of GSTs on prostaglandin biosynthesis from arachi- donic acid (AA) and spectrophotometric assays were done for measuring activity levels of COX and GSTs.Results: SDS-PAGE analysis indicates that macGSTs have molecular weights in the range of 25-28 kDa.In a coupled assay involving GSTs,arachidonic acid and cyclooxygenase-1,rat testicular macGSTs produced prostaglandin E2 and F2~, while the cGSTs caused the generation of prostaglandin D2,E2 and F_(2α).In vitro interaction studies on GSTs and COX at the protein level have shown dose-dependent inhibition of COX activity by macGSTs and vice versa.This effect, however,is not seen with cGSTs.The inhibitory effect of COX on macGST activity was relieved with increasing concentrations of reduced glutathione (GSH) but not with 1-chloro 2,4-dinitrobenzene (CDNB).The inhibition of COX by macGSTs,on the other hand,was potentiated by glutathione.Conclusion:We isolated and purified macGSTs and cGSTs from rat testis and analyzed their involvement in prostaglandin biosynthesis.These studies reveal a revers- ible functional interaction between macGSTs and COX in vitro,with possible interactions between them at the GSH binding site of macGSTs.

Expression and significance of carcinoembryonic antigen, cancer antigen 153, and cyclooxygenase-2 in breast cancer

Objective This study aimed to evaluate serum and nipple discharge levels of carcinoembryonic antigen(CEA) and cancer antigen 153(CA153) and tissue cyclooxygenase-2(COX-2) expression in breast cancer cases and associations of these proteins with breast cancer metastasis.Methods The immunohistochemical Ultra Sensitive^(TM) S-P method was used to detect COX-2 expression in 77 cases of invasive breast carcinoma. Of these cases, 52 exhibited CEA and CA153 in both serum and nipple discharge(electrochemiluminescence method), and associations of these biomarkers with breast cancer prognosis were studied. Sixty cases of benign breast lesion were selected as a control group. Overall survival of breast carcinoma patients was evaluated. COX-2 expression was evaluated relative to clinicopathological features and CEA and CA153 levels, and its role in invasiveness was investigated.Results Among cases of invasive breast cancer, 72.7%(56/77) were COX-2 immunopositive, compared to 16.7% of benign lesions(χ2 = 66.745, P = 0.000) percentage of positive cells. COX-2 overexpression in breast cancer correlated positively with histological grade(II vs III; χ2 = 4.064, P = 0.043), lymph node metastasis(χ2 = 9.135, P = 0.003), and distant metastasis(χ2 = 8.021, P = 0.003). However, COX-2 expression did not correlate with age(≤ 50 vs 50 years) or tumor size(≤ 5 vs > 5 cm)(χ2 = 0.081, P = 0.776 and χ2 = 3.702, P = 0.054, respectively). Among breast cancer patients, COX-2 overexpression in tumors also correlated with shorter overall survival(P < 0.05). In brief, increased COX-2 expression correlates with worse prognosis and shorter overall survival. Malignant lesions were associated with significantly higher serum and nipple discharge levels of biomarkers, relative to benign lesions(P < 0.05). These biomarkers were present at significantly higher levels in nipple discharge than in serum(P < 0.05). Furthermore, significantly higher nipple discharge levels of CEA and CA153 were observed in COX-2-positive breast carcinoma patients, compared to COX-2-negative patients(P <0.05). Shorter overall survival in cancer patients group related to COX-2 overexpression in tumors(P < 0.05).Conclusion The study suggests that COX-2 overexpression correlates with poor clinicopathological parameters in breast cancers and might be an important biological marker of invasion and metastasis. The findings of the present study suggest that combined detection of COX-2 tissue expression and CEA and CA153 in serum and nipple discharge could facilitate clinical monitoring and diagnosis of metastasis in patients with breast cancer.

Cyclooxygenase Inhibition in Cancer Immunotherapy: Combination of Indomethacin with Cancer Vaccines Is Not Always Beneficial

Cyclooxygenase (COX)-1, but preferable COX-2 catalyzes the synthesis of PGE2 in several tumors, promoting angiogenesis and a suppressive inflammation in their microenvironments. Different types of cancer vaccines have been combined with COX-2 inhibitors, assuming that its particular mechanism of action will not influence the overall results of the combination. In this research, a possible relationship between the type of cancer vaccine and the outcome of the combination with a COX inhibitor was experimentally addressed. We investigated whether nonsteroidal anti-inflammatory drugs (NSAIDs) affect the immune response to vaccination. Three adjuvants were evaluated for humoral and cellular response using ovalbumin (OVA) as antigen. We evaluated also the impact of indomethacin in five tumor models and the correlation of this effect with the secretion of prostaglandin E2 (PGE2) of these cells. We finally studied the combination of indomethacin with two cancer vaccines in three different experimental settings. COX inhibitor did not interfere with dendritic cells maturation in vitro and did not affect the frequency of splenic immune cell populations in mice. However, the induction of OVA-specific antibodies is affected by the COX inhibitor but its impact on cytotoxic CD8+ T cell response is adjuvant-dependent. In contrast, the antitumor effect of the COX inhibitor in the 3LL-D122 tumor model is not mediated by CD4+ or CD8+ T cells. Interestingly, the in vivo effect observed in this model and others didn’t correlate with levels of PGE2 secretion by the tumor cell lines in vitro. Finally, the combination of a COX inhibitor with cancer vaccines may depend on the type of the cancer vaccine.

Effects of cyclooxygenase 2 inhibitor on growth-associated protein 43 and nerve growth factor expression in dorsal root ganglion during neuropathic pain development

BACKGROUND： Inflammatory responses in injured nerves have been recognized as important factors for initially sensitizing nociceptive neurons. Cyclooxygenase （COX） is the rate-limiting enzyme in prostaglandin synthesis, and COX-2 inhibitor is involved in mechanisms of analgesia and anti-inflammation. OBJECTIVE： To investigate the effects of COX-2 inhibitor on thermal and mechanical hyperalgesia, as well as expression of growth associated protein 43 （GAP-43） and nerve growth factor （NGF） in dorsal root ganglion, in a rat model of neuropathic pain due to chronic constriction injury. DESIGN, TIME AND SETTING： A randomized, controlled, comparison study that was performed at the Surgical Department and Pathological Laboratory, Second Affiliated Hospital of Shantou University Medical College from September 2006 to September 2007. MATERIALS： COX-2 inhibitor, Iornoxicam, was purchased from Nycomed Pharmaceutical （Austria）; rabbit anti-GAP-43, and rabbit anti-NGF polyclonal antibodies were purchased from Boster, Wuhan, China. METHODS： A total of 50 adult, Wistar rats were randomly assigned to four groups： normal control （n = 5）, model （n = 15）, normal saline control （n = 15）, and Iornoxicam treatment （n =15）. With exception of the control group, the sciatic nerve of all rats was loosely ligated to establish a model of chronic constriction injury. The model rats were divided into three subgroups according to varying post-operative survival periods： 3, 7 and 14 days （n = 5）, respectively. Rats in the Iornoxicam treatment group were intraperitoneally injected with 1.3 mg/kg lornoxicam every 12 hours throughout the entire experimental procedure. Rats in the normal saline control group were intraperitoneally injected with 1.3 mL/kg saline. MAIN OUTCOME MEASURES： Immunohistochemistry revealed expression of GAP-43 and NGF in the L5 dorsal root ganglions. Mechanical withdrawal threshold and thermal withdrawal latency were used to observe neurological behavioral changes in rats. RESULTS： The relative gray values of GAP-43- and NGF-positive neurons in the model group were remarkably increased compared with the normal control rats （P 〈 0.01）, while the relative gray values in the Iomoxicam treatment group were significantly less than the model and normal saline control groups （P 〈 0.01）. Mechanical withdrawal threshold and thermal withdrawal latency gradually decreased with increasing injury time in the model, normal saline control, and Iornoxicam treatment groups, and were significantly less than the normal control group （P 〈 0.05）. In addition, mechanical withdrawal threshold and thermal withdrawal latency were significantly greater in the Iornoxicam treatment group compared with the model and normal saline control groups （P 〈 0.05）. CONCLUSION： Intraperitoneal injection of the COX-2 inhibitor Iornoxicam attenuated mechanical and thermal hyperalgesia induced by sciatic nerve chronic constriction injury and inhibited the increased expression of GAP-43 and NGF.

Spatiotemporal expression of inducible nitric oxide synthase and cyclooxygenase 2 in the spinal cord during early stage sciatic nerve crush injury

BACKGROUND： Previous studies have shown that inducible nitric oxide synthase （iNOS） and cyclooxygenase 2 （COX-2） participate in inflammatory immune responses and neuropathic pain following peripheral nerve injury. However, few reports have addressed time-dependent expression of iNOS and COX-2 following peripheral nerve injury. OBJECTIVE： To investigate spatiotemporal expression of iNOS and COX-2 during early stage sciatic nerve crush injury.DESIGN, TIME AND SETTING： The randomized, controlled, animal experiment was performed at the Laboratory of Applied Anatomy, Department of Human Anatomy and Neurobiology, Central South University, China from September 2006 to September 2007.MATERIALS： Mouse anti-rat iNOS monoclonal antibody and goat anti-rat COX-2 monoclonal antibody （Transduction Laboratory, USA）, as well as biotinylated rabbit anti-mouse lgG and biotinylated rabbit anti-goat IgG （Santa Cruz Biotechnology, USA） were used in the present study.METHODS： A total of 48 healthy, adult, Sprague Dawley rats were randomly assigned to three groups. In the model group （n = 32）, crush injury to the right sciatic nerve was established using an artery clamp. The model group was further assigned to four subgroups according to survival time （6,12, 24, and 72 hours）, respectively （n = 8）. Sham surgery （n = 8） and normal control （n = 8） groups were also established.MAIN OUTCOME MEASURES： iNOS and COX-2 expression was detected in the L4-6 spinal cord with immunohistochemistry. Gray values of iNOS- and COX-2-postive cells in the anterior horn and posterior horn of spinal cord, as well as quantification of iNOS- and COX-2-positive cells in the anterior horn of spinal cord, were measured.RESULTS： iNOS and COX-2 expression gradually increased in the anterior horn and posterior horn of the spinal cord on the damaged side over time from 6 hours following sciatic nerve injury （P〈0.05） and peaked at 72 hours. Simultaneously, the number of iNOS- and COX-2-positive cells similarly increased in the anterior horn of spinal cord on the damaged side （P〈 0.05）.CONCLUSION： iNOS and COX-2 expression increased in the spinal cord during early stage sciatic nerve crush, which suggested that iNOS and COX-2 participate in occurrence and development of inflammatory immune responses following peripheral nerve injury.