Overexpression Of Cyclooxygenase Research Articles

Modeling of bacterial lipopolysaccharide-induced endometritis in rats and the possibility of its therapy using plasma enriched with soluble platelet factors

We presents results of a study on endometritis modeling in rats with double intravaginal administration of bacterial lipopolysaccharide (LPS). The established endometrial inflammatory process was accompanied by a decrease in the weight of animals, an increase in the weight of the uterus, neutrophilic infiltration, edema, and overexpression of cyclooxygenase in endometrium on 30-45 days after the start of endometritis induction. Local intravaginal use of plasma enriched with soluble platelet factors/platelet-rich plasma (PORFT/PRP) caused a decrease in the intensity of the inflammatory process and the rate of neutrophil infiltration of the endometrium, a decrease in the intensity of cyclooxygenase, interleukin-6 and transforming growth factor-β expression, and an increase in the body weight of animals. The conclusion was made on the possibility of creating model of endometritis in rats by double administration of LPS and the ability of local administration of PORFT/PRP to have a therapeutic effect on the inflammatory process in the endometrium.

EP3 (prostaglandin E2 receptor 3) expression is a prognostic factor for progression-free and overall survival in sporadic breast cancer

BackgroundIn various cancers, overexpression of cyclooxygenase (COX)-2 and elevated prostaglandin (PG) E2 synthesis have been associated with tumor development and progression. The potential of COX-2 inhibitors in cancer prevention and treatment has been shown repeatedly; however, their clinical use is limited due to toxicity. PGE2 signals via EP receptors 1–4, whose functions are analyzed in current research in search for targeted anti-PG therapies. EP2 and EP4 rather promote tumorigenesis, while the role of EP3, especially in breast cancer, is not yet clear and both pro- and anti-tumorigenic effects have been described. Our study evaluates EP3 receptor expression in sporadic breast cancer and its association with clinicopathological parameters, progression-free and overall survival.MethodsTwo hundred eighty-nine sporadic breast cancer samples without primary distant metastasis were immunohistochemically analyzed for EP3 receptor expression. Tissue was stained with primary anti-EP3-antibodies. Immunoreactivity was quantified by the immunoreactivity-score (IRS); samples with an IRS ≥ 2 scored as EP3 positive. Chi-squared and Mann-Whitney-U test were used for comparison of data; Kaplan-Meier estimates and Cox-regression were used for survival analyses.ResultsEP3 receptor was expressed in 205 of 289 samples analyzed (70.9%). EP3 receptor expression was not associated with clinicopathological parameters (e. g. tumor size, hormone receptors, lymph node status). Kaplan-Meier estimates showed a significant association of EP3 positivity with improved progression-free survival (p = 0.002) and improved overall survival (p = 0.001) after up to 10 years. Cox regression analysis confirmed EP3 positivity as a significant prognostic factor even when other known prognosticators were accounted for.ConclusionsIn sporadic breast cancer, EP3 receptor expression is not significantly associated with clinicopathological parameters but is a significant prognostic factor for improved progression-free and overall survival. However, the functional aspects of EP3 receptor in breast cancer and the way how EP3 may oppose the pro-tumorigenic effects of PGE2 elevation and COX-2 overexpression are not fully understood so far. Further studies aiming at identification of the factors regulated by EP3 are necessary to evaluate the possibility of targeting EP3 in future anti-tumor therapy in breast cancer.

Abstract 847: Anti-cancer effects of selective Cox-2 and EP2 inhibition through suppression of EMT and the clinical implications of overexpression of Cox-2 and downregulation of E-cadherin in pharyngeal squamous cell carcinoma

Abstract Background: Overexpression of cyclooxygenase 2 (Cox-2), an inducible prostaglandin (PG) synthetase, is assumed to promote cancer progression through its multifaceted function, including induction of angiogenesis, stimulation of cell proliferation, restraint on apoptosis, and immunosuppression. Furthermore, recently its inverse relationship with E-cadherin expression was reported. The epithelial-to-mesenchymal transition (EMT) accompanied by the downregulation of E-cadherin is supposed to promote metastasis. However, neither the anti-cancer effect of selective Cox-2 inhibitors on the regulation of E-cadherin nor its specific mechanism has been examined in pharyngeal squamous cell carcinoma (PhSCC). In addition, the anti-tumor effect of EP2 antagonist, a possibly more selective inhibitor of PGE2, remains to be elucidated. Methods: We used quantitative real-time PCR to examine the effects of a selective Cox-2 inhibitors, Celecoxib, and a selective EP2 antagonist, PF-04418948, on the gene expressions of CDH-1, its transcriptional repressors (snail, ZEB1, twist), and vimentin in the human PhSCC cell lines BICR6 and FaDu. Alteration in E-cadherin expression on the cell surface was evaluated by immunofluorescent staining. In vitro proliferation and migration assays were performed to assess cellular behaviors. Clinicopathological factors and immunohistochemical expressions of Cox-2 and E-cadherin were evaluated using surgical specimens from 54 patients with PhSCC who underwent transoral resection. Results: Both Celecoxib and PF-04418948 upregulated the CDH-1 expression and the membranous E-cadherin expression and downregulate the vimentin expression in the PhSCC cells through the suppression of snail, ZEB1, and twist expression. The extent of this effect depended on the baseline expression levels of both E-cadherin and Cox-2 in each cell line. Additionally, the cell migration was attenuated by both Celecoxib and PF-04418948, whereas the cell proliferation was not affected. Univariate analysis showed that differentiation, overexpression of Cox-2, and decreased expression of E-cadherin were significantly correlated with lymph node metastasis. Multivariate logistic regression revealed that overexpression of Cox-2 (odds ratio [OR]=53.49, P<0.001) and decreased expression of E-cadherin (OR=0.06, P=0.016) were the independent risk factors affecting lymph node metastasis. Conclusions: These findings suggest that the appropriately selective administration of the Cox-2 inhibitor and EP2 antagonist may have an anti-metastatic effect through suppression of EMT by restoring E-cadherin expression. In addition, overexpression of Cox-2 and downregulation of E-cadherin may be closely implicated in lymph node metastasis in PhSCC. Citation Format: Yoshihiro Watanabe, Yorihisa Imanishi, Hiroyuki Ozawa, Kaori Kameyama, Koji Sakamoto, Ryoichi Fujii, Seiji Shigetomi, Noboru Habu, Kuninori Otsuka, Yoichiro Sato, Mariko Sekimizu, Fumihiro Ito, Yuichi Ikari, Shin Saito, Kaoru Ogawa. Anti-cancer effects of selective Cox-2 and EP2 inhibition through suppression of EMT and the clinical implications of overexpression of Cox-2 and downregulation of E-cadherin in pharyngeal squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 847. doi:10.1158/1538-7445.AM2017-847

Cleavage and polyadenylation specific factor 4 targets NF-κB/cyclooxygenase-2 signaling to promote lung cancer growth and progression

Overexpression of cyclooxygenase 2 (COX-2) is frequently found in early and advanced lung cancers. However, the precise regulatory mechanism of COX-2 in lung cancers remains unclear. Here we identified cleavage and polyadenylation specific factor 4 (CPSF4) as a new regulatory factor for COX-2 and demonstrated the role of the CPSF4/COX-2 signaling pathway in the regulation of lung cancer growth and progression. Overexpression or knockdown of CPSF4 up-regulated or suppressed the expression of COX-2 at mRNA and protein levels, and promoted or inhibited cell proliferation, migration and invasion in lung cancer cells. Inhibition or induction of COX-2 reversed the CPSF4-mediated regulation of lung cancer cell growth. Cancer cells with CPSF4 overexpression or knockdown exhibited increased or decreased expression of p-IKKα/β and p-IκBα, the translocation of p50/p65 from the cytoplasm to the nucleus, and the binding of p65 on COX-2 promoter region. In addition, CPSF4 was found to bind to COX-2 promoter sequences directly and activate the transcription of COX-2. Silencing of NF-κB expression or blockade of NF-κB activity abrogated the binding of CPSF4 on COX-2 promoter, and thereby attenuated the CPSF4-mediated up-regulation of COX-2. Moreover, CPSF4 was found to promote lung tumor growth and progression by up-regulating COX-2 expression in a xenograft lung cancer mouse model. CPSF4 overexpression or knockdown promoted or inhibited tumor growth in mice, while such regulation of tumor growth mediated by CPSF4 could be rescued through the inhibition or activation of COX-2 signaling. Correspondingly, CPSF4 overexpression or knockdown also elevated or attenuated COX-2 expression in tumor tissues of mice, while treatment with a COX-2 inducer LPS or a NF-κB inhibitor reversed this elevation or attenuation. Furthermore, we showed that CPSF4 was positively correlated with COX-2 levels in tumor tissues of lung cancer patients. Simultaneous high expression of CPSF4 and COX-2 proteins predicted poor prognosis of patients with lung cancers. Our results therefore demonstrated a novel mechanism for the transcriptional regulation of COX-2 by CPSF4 in lung cancer, and also offer a potential therapeutic target for lung cancers bearing aberrant activation of CPSF4/COX-2 signaling.

Correlation Between Cyclo-oxygenase-2 and Vascular Endothelial Growth Factor Expression in Canine and Feline Squamous Cell Carcinomas

Overexpression of cyclo-oxygenase (COX)-2 is involved in tumour growth and spread by modulating the production of angiogenic factors such as vascular endothelial growth factor (VEGF). Expression of COX-2 and VEGF was investigated immunohistochemically in 51 canine and feline cutaneous and non-cutaneous squamous cell carcinomas (SCCs) and the correlation between expression of these molecules and clinicopathological variables was evaluated. COX-2 and VEGF expression was not observed in normal skin keratinocytes. COX-2 overexpression occurred in 53% and 61% of the canine and feline SCCs, respectively. The expression of both markers was higher in cutaneous compared with non-cutaneous SCCs. In both species COX-2 and VEGF expression was correlated with the progression of the disease, but not with the presence of lymphatic invasion, tumour grading or tumour classification in the cutaneous tumours. Further study will be required to understand the role of the COX-2 pathway in angiogenesis in SCC.

Effects of Nrf2 silencing on oxidative stress-associated intestinal carcinogenesis in mice.

To assess the risk of colorectal cancer in humans with inactivation of NRF2, Nrf2‐proficient (Nrf2 +/+) and ‐deficient (Nrf2 −/−) mice were exposed to potassium bromate (KBrO3) at concentrations of 750 or 1500 ppm for 52 weeks. Neoplastic proliferative lesions were observed in the small intestine and exhibited accumulations of β‐catenin and cyclin D1. The lesions had characteristics similar to those in experimental models of human hereditary colorectal cancer. An additional 13‐week study was performed to examine the role of Nrf2 in the effects of oxidative stress. Significant increase in combined incidences of preneoplastic and neoplastic lesions in Nrf2 −/− mice administered high‐dose KBrO3. In the short‐term study, although 8‐hydroxydeoxyguanosine (8‐OHdG) levels in the epithelial DNA of Nrf2 −/− mice at the high dose were significantly lower than those of the corresponding Nrf2 +/+ mice, the difference was very small. mRNA levels of Nrf2‐regulated genes were increased in Nrf2 +/+ mice. Overexpression of cyclooxygenase 2 (COX2) and increased numbers of proliferating cell nuclear antigen (PCNA)‐positive cells in the jejunal crypts were observed in Nrf2 −/− mice administered high‐dose KBrO3. Overall, these data suggested that individuals having single‐nucleotide polymorphisms in NRF2 may have a risk of colorectal cancer to some extent.

Loss of p53 in stromal fibroblasts enhances tumor cell proliferation through nitric-oxide-mediated cyclooxygenase 2 activation

Overexpression of cyclooxygenase 2 (COX-2) by stromal fibroblasts plays a critical role in the early stage of carcinogenesis. COX-2 expression is thought to be positively or negatively regulated by inflammatory chemical mediators or tumor suppressors. In this study, the contributions of inducible nitric oxide synthase (iNOS) and p53 to COX-2 expression were examined using mouse embryonic fibroblasts (MEFs) from wild-type, p53-deficient, iNOS-deficient, and p53/iNOS-deficient mice. These MEFs were treated with 1 μg/mL of lipopolysaccharide and 100 IU/mL of interferon gamma for up to 72 h. iNOS and COX-2 expression were analyzed by Western blotting. iNOS was induced earlier (16 h) in p53-deficient MEFs than in wild-type MEFs (48 h). Elevated expression of COX-2 was sustained for a longer duration in the p53-deficient MEFs. In contrast, COX-2 expression was reduced earlier in the iNOS-deficient MEFs. Addition of an exogenous NO donor (0.8 mM of S-nitroso-l-glutathione) to the iNOS-deficient MEFs augmented COX-2 expression. Co-culture with stimulated p53-deficient MEFs promoted cell proliferation of mouse rectal polyploid carcinoma CMT93 cells, but treatment with a COX-2-specific inhibitor counteracted this effect. These results suggest that loss of function of the p53 gene in stromal fibroblasts enhances COX-2 expression by enhancing iNOS expression and the resultant production of NO, contributing to the promotion of tumor growth.

Cyclooxygenase-2 single-nucleotide polymorphisms and hepatocellular carcinoma in Egypt

Hepatocellular carcinoma (HCC) is a worldwide neoplasm for which early diagnosis is difficult and the prognosis is usually poor. Overexpression of cyclooxygenase 2 (COX-2) has been suggested to be associated with hepatocarcinogenesis. Although several COX-2 inhibitors have been used in hepatoma therapy, the genetic background between COX-2 and HCC remains largely unknown. In this study, the association of genotypic polymorphisms in COX-2 with HCC was investigated. 25 healthy individuals served as control (group I), group II: 50 HCV infection patients without any complications, group III: 50 HCV infected patients complicated with cirrhosis and group IV: 75 HCV infected patients complicated with (45 localized and 30 metastatic) HCC from Zagazig University Hospital in Egypt were genotyped by a PCR-RFLP method. The results showed that, no differences in distribution between the HCC and other groups were found. We found -1195A allele carriers had a higher risk of HCC with HCV infection. As regard the obtained results of serum AFU, a significant increase was detected in HCC as compared with cirrhosis, hepatitis and healthy control groups (p<0.001). Concerning the obtained results of serum AFP, when HCC group was compared with cirrhosis, hepatitis and healthy controls, a significant increase was observed (p<0.001). identification of SNP in COX-2 gene promoter and evaluation of serum AFU and AFP give a red light in early detection of HCC which may be reduce its fatal incidence.

Transcriptional and translational regulation of COX-2 expression by cadmium in C6 glioma cells

High exposure to cadmium is a risk factor for many neuronal diseases. Overexpression of cyclooxygenase (COX)-2 is linked to many neuroinflammatory and neoplastic diseases. We, herein, investigated the effect of cadmium on the expression of COX-2 in C6 rat glioma cells. Treatment with cadmium sulfate (cadmium) increased the expression of COX-2 mRNA. Remarkably, cadmium treatment further increased expression of not only the N-glycosylated COX-2 protein of 72 kDa but also the unglycosylated COX-2 of 66 kDa, as assessed by the unglycosylated COX-2 induced by tunicamycin or glucosamine, known inhibitors of COX-2 N-glycosylation. Of note, when translation was blocked in the presence of cycloheximide (CHX), levels of both N-glycosylated and unglycosylated COX-2 proteins induced by cadmium rapidly declined but the decline was prevented by MG132, a 26S proteasomal inhibitor. However, in the absence of CHX, cadmium induced and maintained expression of the unglycosylated COX-2 proteins. Pharmacological inhibition studies importantly demonstrated that the cadmium-mediated COX-2 transcriptional upregulation in C6 cells was not shown by exogenous glutathione (GSH) supplementation or treatment with inhibitors of extracellular signal-regulated protein kinase-1/2 (ERK-1/2), p38 MAPK and c-Jun N-terminal protein kinase-1/2 (JNK-1/2), respectively. Expression of COX-2 was not noted in C6 cells exposed to other heavy metals (cobalt or manganese). These results demonstrate that cadmium specifically induces expression of COX-2 through both transcriptional and co-translational (N-glycosylation) regulation in C6 cells in which the cadmium-induced COX-2 transcriptional upregulation is closely related to oxidative stress-dependent activation of the family of MAPKs and the cadmium-induced expression of both N-glycosylated and unglycosylated COX-2 proteins is proteasome- and translation-dependent.

Abstract 139: The role of miRNAs in cyclooxygenase-2-mediated breast cancer progression

Abstract We had shown that over-expression of cyclo-oxygenase (COX)-2 in human, as well as murine breast cancer cells, promotes tumor progression and metastasis by multiple mechanisms: host immune cell inactivation and a stimulation of cancer cell migration, invasion, tumor-associated angiogenesis and lymphangiogenesis, which support blood-borne and lymph-borne metastasis. Most of these events resulted from activation of the prostanoid receptor EP4 by endogenous PGE2. Recently, by stable transfection of COX-2 cDNA into a non-metastatic, COX-2 negative human breast cancer cell line MCF-7, we showed that COX-2 induces all the phenotypic properties of stem-like or “tumor initiating cells” (TIC) in MCF-7-COX-2 cells, as defined by in vitro studies and validated in vivo. Through combined gene expression and microRNA (miRNA) micro array analysis, we identified two miRNAs (miR-526b and miR-655) that are up-regulated in MCF-7-COX-2 cells, associated with a down-regulation of 14 target genes linked with tumor-suppressor functions. We hypothesize that these miRNAs are important for COX-2 mediated TIC associated functions in human breast cancer. As a first step, we validated their expression in several COX-2 disparate human breast cancer cell lines: MCF-7 (COX-2 negative), MCF-7-COX-2, SKBR-3 (HER-2 over-expressing but COX-2 negative) and SKBR-3-COX-2 (COX-2 over-expressing following stable transfection of COX-2). The expression levels of miR-655 were strongly correlated with COX-2 mRNA (quantified RT-PCR) expression in these cell lines. Furthermore, the migratory and invasive capacities of the cell lines went hand in hand with miR-655 expression. Expression of miR-655 was markedly inhibited by treating MCF-COX-2 cells with a COX-2 inhibitor NS398 or an EP4 antagonist ONO-AE3-208, indicating that the expression depends on both COX-2 and EP4 activity. Finally, we discovered that cells derived from tumorspheres (in vitro correlate of TIC growth) produced by various human breast cancer cell lines (Hs578T, T47D, MDA-MB-231, SKBR-3, SKBR-3-COX-2, and MCF-7-COX-2) grown on low attachment plates, exhibited a dramatic increase in COX-2 expression in comparison to the cells grown as monolayer. We also found that the tumorspheres over-express miR-655. These findings, taken together, fortify the notion that COX-2, EP4 and COX-2 induced miR-655 expression, play important roles in promoting and maintaining the TIC phenotype in breast cancer cells. In support, COX-2 inhibitors and EP4 antagonists were highly effective in abrogating tumor growth, tumor- associated angiogenesis, lymphangiogenesis and metastasis to the lungs and lymph nodes in our mouse breast cancer model. Currently we are testing whether EP4 and miR-655 serve as useful prognostic markers and therapeutic targets in human breast cancer. (Supported by the CBCF, Ontario chapter and the OICR funds to PKL. LD is a TBCRU scholar.) Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 139. doi:1538-7445.AM2012-139

Association of Cyclooxygenase 2 Single-Nucleotide Polymorphisms and Hepatocellular Carcinoma in Taiwan

Hepatocellular carcinoma (HCC) is a worldwide neoplasm for which early diagnosis is difficult and the prognosis is usually poor. Overexpression of cyclooxygenase 2 (COX-2) has been suggested to be associated with hepatocarcinogenesis. Although several COX-2 inhibitors have been used in hepatoma therapy, the genetic background between COX-2 and HCC remains largely unknown. In this study, the association of genotypic polymorphisms in COX-2 with HCC was investigated. 298 patients with HCC and 298 healthy controls recruited from the China Medical Hospital in Taiwan were genotyped by a PCR-RFLP method. We have investigated six polymorphic variants of COX-2, including A-1195G, G- 765C, T+8473C, and variants in introns 1, 5 and 6, and analyzed the association of specific genotype(s) with susceptibility to HCC. The results showed that, for each of the six genotypes, no differences un distribution between the HCC and control groups were found. There was neither obvious joint effect of COX-2 G-765C/intron 6 haplotype nor its genotypes with smoking or alcohol consumption on HCC risk. Environmental factors, other than smoking and alcohol drinking, may affect the post-natal expression of COX-2 in the etiology of HCC, which is an outcome of complex genetic and environmental interactions. Moreover , our immunohistochemistrical results indicated that the COX-2 protein was significantly over-expressed in well-differentiated HCC, but not significantly increased in expression in poorly-differentiated HCC. We suggest that COX-2 may be a determinant of the differentiation grade of HCC.

Interaction of Cyclooxygenase‐2 promoter polymorphisms with Helicobacter pylori infection and risk of gastric cancer

Overexpression of cyclooxygenase (COX)-2 has been implicated in the development of cancer. This study aimed to evaluate the relationship between genetic variants in COX-2 promoter interacting with Helicobacter pylori and the susceptibility to gastric cancer (GC). Three COX-2 polymorphisms -1290A>G (rs689465), -1195G>A (rs689466), and -765G>C (rs20417) were genotyped in 323 GC patients and 944 controls. Odds ratios (ORs) and 95% confidence intervals (CIs) were calculated by logistic regression model. In GC patients, the ORs were 2.33 (95% CI = 1.50-3.63) and 2.70 (95% CI = 1.68-4.33) for -1195AA and -765CG genotype carriers, respectively. Haplotype analysis showed all -1195A allele-containing haplotypes, except G(-1290)-A(-1195)-G(-765), were associated with increased risk for GC, compared with the A(-1290)-G(-1195)-G(-765) haplotype. Moreover, significant multiplicative and additive interactions were observed between H. pylori infection and all these three polymorphisms, and H. pylori-infected subjects carrying the variant allele of -1290A>G, -1195G>A, or -765G>C had increased risk of GC compared with non-H. pylori-infected subjects with wild-type allele (OR = 4.10, 95% CI = 1.90-8.83; OR = 3.46, 95% CI = 1.31-9.11; and OR = 3.32, 95% = 1.27-8.73, respectively). Our results suggested that the COX-2 promoter polymorphisms were associated with increased risk of GC, especially interacting with H. pylori infection.

COX2 Expression Predicts Resistance to Chemoradiotherapy in Esophageal Squamous Cell Carcinoma

The overexpression of cyclooxygenase (COX)2 is correlated with carcinogenesis, tumor progression, and prognosis, and increased COX2 expression is correlated with radiation resistance. However, no correlation between the COX2 expression and resistance to chemoradiotherapy for esophageal squamous cell carcinoma has been characterized. The purpose of the present study was to evaluate whether COX2 expression is an indicator of resistance to chemoradiotherapy in esophageal squamous cell carcinoma and the feasibility of COX2 as a biomarker for CRT. Fifty-eight patients who were diagnosed with esophageal squamous cell carcinoma from biopsy samples were enrolled in the present series. All patients underwent concurrent chemoradiotherapy in a neoadjuvant setting, followed by radical esophagectomy. COX2 expression was evaluated by immunohistochemical staining and statistically compared with the histopathologic findings in surgically resected specimens. The rate of responders was 87% for weak expression of COX2, 62% for moderate expression, and 30% for strong expression, and there was a close correlation between COX2 expression and the response rate (Kendall's τb = 0.396, P = 0.001). In the univariate analysis, negative or weak expression of COX2 was found to correlate significantly with CRT response (odds ratio, 6.296; 95% confidence interval (CI), 1.58-25.096; P = 0.010). In the multivariate analysis, weak expression of COX2 (30% or less) was found to be an independent prognostic factor (odds ratio, 6.534; 95% CI, 1.535-27.803; P = 0.011). The COX2 expression predicts resistance to chemoradiotherapy in esophageal squamous cell carcinoma, and it also is a feasible biomarker for evaluating the CRT response.

A role for COX2-derived PGE2 and PGE2-receptor subtypes in head and neck squamous carcinoma cell proliferation

The overexpression of cyclooxygenase (COX)-2 is a frequent event in squamous cell carcinomas of the head and neck (HNSCC), and non-steroidal anti-inflammatory drugs, which are potent inhibitors of COX-1 and COX-2, exert chemopreventive effects on HNSCC cancer development. COX-2 promotes the release of the pro-inflammatory mediator prostaglandin E2 (PGE2), which acts on its cell surface G protein-coupled receptors EP1, EP2, EP3, and EP4. Here, we investigated the role of PGE2 and its receptors in cellular proliferation in HNSCC. The expression of COX-2 and EP1-4 was examined in immortalized oral epithelial cells and in a representative panel of HNSCC cell lines, and based on these data EP1-EP3 and COX-2 expression were evaluated by immunohistochemistry in a large clinical sample collection using HNSCC tissue microarrays. The ability of selective COX-2 inhibition to block PGE2 secretion was measured by ELISA specific assays. The effects of PGE2 on cell proliferation were evaluated using PGE2, its stable analog, and EP2 and EP3-specific synthetic agonists. The results presented here show that HNSCC tumoral lesions and their derived cell lines constitutively express COX-2 and the EP1, EP2 and EP3 receptors for PGE2. HNSCC cells secrete PGE2, which can be suppressed by low concentrations of COX-2 selective inhibitors, without inhibiting cell proliferation. Exogenously added stable PGE2 and EP3-specific agonists induce DNA synthesis in all HNSCC cell lines tested. Overall, our study supports the emerging notion that PGE2 produced in the tumor microenvironment by the overexpression of COX-2 in tumoral and inflammatory cells may promote the growth of HNSCC cells in an autocrine and paracrine fashion by acting on PGE2 receptors that are widely expressed in most HNSCC cancer cells. In particular, our findings suggest that EP3 receptor may play a more prominent role in HNSCC cell growth promotion, thus providing a rationale for the future evaluation of this PGE2 receptor as a target for HNSCC prevention strategies.

A plant flavonoid fisetin induces apoptosis in colon cancer cells by inhibition of COX2 and Wnt/EGFR/NF- B-signaling pathways

Overexpression of cyclooxygenase 2 (COX2) and uncontrolled wingless and Int (Wnt)-signaling pathway have long been suggested to play crucial roles in colorectal cancer. Studies show that selective COX2 inhibitors possess great potential as chemopreventive agents for colon cancer. Recent studies suggest that targeting COX2 and epidermal growth factor receptor (EGFR) may provide better therapeutic strategy than inhibiting either single target and that this may alleviate the problem of COX2 inhibitor-associated side effects. Therefore, there have been intensive efforts to develop novel dietary substances that target COX2 and EGFR activation. Fisetin is a naturally occurring flavonoid commonly found in various vegetables and fruits. We found that the treatment of COX2-overexpressing HT29 human colon cancer cells with fisetin (30-120 microM) resulted in induction of apoptosis, downregulation of COX2 protein expression without affecting COX1 and inhibited the secretion of prostaglandin E2. Treatment of cells with fisetin also inhibited Wnt-signaling activity through downregulation of beta-catenin and T cell factor 4 and decreased the expression of target genes such as cyclin D1 and matrix metalloproteinase 7. Fisetin treatment of cells also inhibited the activation of EGFR and nuclear factor-kappa B (NF-kappaB). Finally, the formation of colonies in soft agar was suppressed by fisetin treatment. Taken together, we provide evidence that the plant flavonoid fisetin can induce apoptosis and suppress the growth of colon cancer cells by inhibition of COX2- and Wnt/EGFR/NF-kappaB-signaling pathways. We suggest that fisetin could be a useful agent for prevention and treatment of colon cancer.

In vitro and in vivo inhibitory effect evaluation of cyclooxygenase-2 inhibitors, antisense cyclooxygenase-2 cDNA, and their combination on the growth of human bladder cancer cells

Overexpression of cyclooxygenase (COX)-2 is associated with the progression of various malignancies, but the contribution of COX-2 expression, bioactivity or their cooperation to bladder cancer growth calls for further clarification. In this study, we investigated the inhibitory effect of COX-2 inhibitors, antisense COX-2 nucleotide, and their combination on the growth of bladder cancer cells (5637, 5637-P and 5637-AS). Suppression of either COX-2 expression or activity caused reduced cell proliferation, enhanced cell numbers in G 1 phase, and increased apoptosis; the joint suppression of COX-2 expression and bioactivity enhanced the degree of cell growth inhibition. COX-2 antisense-expressing 5637-AS tumors showed a 41.42 ± 3.08% growth inhibition as compared with 5637 controls. Oral administration of indomethacin (3 mg/kg) or celecoxib (15 mg/kg) caused tumor growth inhibition by 31.5 ± 14.87% or 83.17 ± 1.17%, respectively. When COX-2 antisense cDNA and COX-2 inhibitor celecoxib were combined, the tumor growth inhibition rate was further increased up to 88.78 ± 3.10%. These results provide evidence that celecoxib has potential therapeutic effect on bladder cancer, and the joint use of COX-2 antisense cDNA with celecoxib may improve their individual therapeutic effect, especially significantly increase the growth inhibitory effect of COX-2 antisense cDNA.

Vitamin C suppresses proliferation of the human melanoma cell SK‐MEL‐2 through the inhibition of cyclooxygenase‐2 (COX‐2) expression and the modulation of insulin‐like growth factor II (IGF‐II) production

Vitamin C plays a crucial role in the suppression of proliferation of several types of cancer. Over-expression of cyclooxygenase (COX)-2 and type I insulin-like growth factor (IGF) receptor are important for proliferation and protection from apoptosis in malignancies. However, its specific mechanisms, especially the interaction between COX-2 expression and IGF-I axis mediated by vitamin C, remain yet to be clarified. Therefore, we investigated the effects of vitamin C on the proliferation of melanoma cells via the modulation of COX-2 expression and IGF-I axis. As a result, we found that 1.0 mM vitamin C inhibits the proliferation of SK-MEL-2 without induction of apoptosis. At that moment, IGF-II production was decreased, followed by the inhibition of COX-2 activity. IGF-IR expression was also down-regulated by vitamin C treatment. It coincided with the result from the inhibition of COX-2 by NS-398 and COX-2 siRNA. In addition, the decreased IGF-IR expression by vitamin C was restored by the treatment of recombinant prostaglandin E2. Finally, we determined whether the signal pathway would be involved in vitamin C-induced IGF-II and IGF-IR down-regulation. When the cells were exposed to SB203580, a specific inhibitor of p38 MAPK, COX-2 expression was dramatically recovered. In addition, phosphorylated p38 MAPK was increased after vitamin C treatment. Taken together, vitamin C suppresses proliferation of the human melanoma cell line SK-MEL2 via the down-regulation of IGF-II production and IGF-IR expression, which is followed by the activation of p38 MAPK and the inhibition of COX-2 expression.

Aspirin inhibits the proliferation of tobacco-related esophageal squamous carcinomas cell lines through cyclooxygenase 2 pathway

Cigarette smoking has been verified as the risk factor of esophageal squamous cell carcinoma (ESCC). Overexpression of cyclooxygenase 2 (COX-2) is shown in ESCC. The objective of this study was to investigate the effects of cigarette smoking ethanol extract (EE) on the proliferation of the human ESCC cell lines, and to explore the correlation between the proliferation rate of human ESCC cell lines and the expression pattern of COX-2. Whether aspirin can inhibit the proliferation of the ESCC cell lines pretreated with EE, and regulate the mRNA expression levels of COX-2 are also examined. Two human ESCC cell lines were selected. EC109 was poorly differentiated and EC9706 was highly differentiated. EC109 and EC9706 were treated with EE and aspirin for different time course. The cell growth of ESCC was measured by MTT reduction assay and the expression of COX-2 was measured by RT-PCR and Western blot analysis. EE promoted the proliferation of EC109 and EC9706 in dose- and time-dependent manners. In the concentration range (10 - 100 microg/ml for EE) and in the time range (24 - 72 hours) after addition of EE, the cell proliferation was prominent in an up-scaled manner respectively. Aspirin could inhibit the proliferation of cell lines EC109 and EC9706, pretreated with EE for 5 hours, in a dose-dependent manner. In the concentration range (0.5 - 8.0 mmol/L for aspirin), the cell growth inhibition was prominent in an up-scaled manner accordingly (P < 0.05). The effect of EE on cell proliferation was correlated with the up-regulation of COX-2 gene. However, the cell growth inhibition of aspirin was correlated with the down-regulation of COX-2 gene. EE can stimulate the proliferation of human ESCC cell lines EC109 and EC9706, most likely through up-regulating the expression of COX-2. Aspirin can inhibit the proliferation of ESCC cell lines induced by EE, which suggests it may be advantageous in the chemoprevention and therapy of human tobacco-related ESCC. And its effect is likely to be related with modulating COX-2 activity.

Effects of long‐term cyclo‐oxygenase 2 selective and acid inhibition on Barrett’s oesophagus

There is an overexpression of cyclo-oxygenase 2 (COX-2) in Barrett's oesophagus (BO). To determine the long-term effect of a COX-2 inhibitor on cellular mechanisms involved in BO. A randomized controlled trial was conducted in BO patients allocated to continue the usual proton pump inhibitor (PPI) alone treatment, or PPI combined with rofecoxib (25 mg/day) for 6 months. Cell proliferation index and COX-2 expression in BO glands was determined in biopsy specimens at baseline and after treatment. Cell apoptosis, cyclin D1, p53 and vascular endothelial growth factor (VEGF) expression was also explored in a subset of patients. Student-t test and the U-Mann-Whitney test were used for quantitative and ordinal variables. Of 62 patients, 58 completed the study. A higher proportion of patients on rofecoxib + PPI exhibited a decrease in COX-2 expression compared to those treated with PPI alone, but cell proliferation index was not affected. Unlike PPI alone, rofecoxib + PPI was associated with an increase in the apoptotic cell index, a decrease in p53 cell staining and VEGF expression in mucosal vessels. No effect on low-grade dysplasia or cyclin D1 was observed. The addition of rofecoxib to PPI therapy does not affect cell proliferation index in BO cells after 6 months of therapy, but does reduce COX-2 and VEGF expression and increases cell apoptosis.

Overexpression of cyclooxygenase‐2 correlates with cytoplasmic HuR expression in salivary mucoepidermoid carcinoma but not in pleomorphic adenoma

The overexpression of cyclooxygenase (COX)-2 in several human carcinomas suggests that COX-2 is related to carcinogenesis. Although COX-2 expression has been shown to be up-regulated in carcinomas of the salivary gland, its mechanisms are not completely understood. HuR is an mRNA-binding protein that controls the stability of certain transcripts including COX-2. The expression of COX-2 and HuR was determined by immunohistochemistry in 28 cases of salivary pleomorphic adenoma and 18 cases of salivary mucoepidermoid carcinoma. 28.6% and 72.2% of the pleomorphic adenomas and mucoepidermoid carcinomas showed high COX-2 expression respectively. 35.7% of pleomorphic adenomas and 72.2% of mucoepidermoid carcinomas were tested positive for HuR in the cytoplasm of tumor cells. There was a correlation between a high COX-2 immunoreactivity and cytoplasmic HuR expression in mucoepidermoid carcinomas but not in pleomorphic adenomas. This study suggests that cytoplasmic HuR is correlated with COX-2 expression in salivary mucoepidermoid carcinomas. In addition, the immunoreactivity of COX-2 and cytoplasmic HuR might be used to evaluate the nature of a borderline malignancy in the salivary glands.