Cyclooxygenase-2 Promoter Region Research Articles

Downregulation of LHCGR Attenuates COX-2 Expression and Induces Luteinized Unruptured Follicle Syndrome in Endometriosis.

An association between endometriosis and luteinized unruptured follicle syndrome (LUFs) has long been identified. Although inactivating mutation of luteinizing hormone/choriogonadotropin receptor (LHGCR) results in LUFs, whether LHCGR contributes to promoting LUFs in endometriosis remains elusive. To investigate the effect of LHCGR signaling in the development of endometriosis-associated LUFs and dissect the underlying mechanism in vivo mouse endometriosis model was established to measure the effect on ovarian folliculogenesis. In vitro cultures of primary human GCs collected from patients undergoing in vitro fertilization were performed and treated with human chorionic gonadotropin (hCG), dibutyryl cyclic-AMP (db-cAMP), LHCGR or CCAAT/enhancer binding protein-α (C/EBPα) small interfering RNA to identify the potential mechanisms. KGN cell line was used to investigate the mechanistic features of transcriptional regulation. Results showed an increased incidence of LUFs was observed in mice with endometriosis. The expression of LHCGR was decreased in the GCs of endometriosis mice. In in vitro cell models, LHCGR signaling increased the expression of C/EBPα and cyclooxygenase-2(COX-2), while inhibiting C/EBPα mitigated the induced COX-2 expression. Mechanically, C/EBPα bounded to the promoter region of COX-2 and increased the transcriptional activity under the stimulation of hCG or db-cAMP. Taken together, this study demonstrated that the LHCGR signaling was reduced in GCs of endometriosis and resulted in a decrease in gonadotropin-induced COX-2 expression. Our study might provide new insights into the dysfunction of GCs in endometriosis.

Hypoxia-induced HIF1α dependent COX2 promotes ovarian cancer progress.

Hypoxia can promote the progression and metastasis of ovarian cancer, while the underlying mechanisms are still unclear. Hypoxia culture or CoCl2 induced-oxygen deprivation condition could promote SKOV3 cells to express cyclooxygenase-2 (COX2). Luciferase assay indicates that hypoxia-inducible factor 1α (HIF1α) could bind directly with the promoter region of COX2 to promote the transcription. COX2 over-expressed SKOV3 cells show up-regulated stemness-related markers expression, proinflammatory gene expression, and increased tumor sphere formation. The inflammatory molecules (interleukin-6, C-X-C motif chemokine ligand 12, interleukin-1B, interleukin-10, and C-C motif chemokine ligand 2) and COX2 expression show positive correlations in the Cancer Genome Atlas data. COX2 over-expression could promote SKOV3 cell proliferation in the subcutaneous tumor model and metastasis in the transfer model. In conclusion, hypoxia-induced HIF-1α mediated COX2 expression could promote the proliferation, inflammation, and metastasis of ovarian cancer.

LncRNA-PACER upregulates COX-2 and PGE2 through the NF-κB pathway to promote the proliferation and invasion of colorectal-cancer cells.

Backgroundp50-associated cyclooxygenase-2 extragenic RNA (PACER) is a recently identified antisense long non-coding RNA (lncRNA) located on the upstream of the promoter region of cyclooxygenase-2 (COX-2). Preliminary studies have suggested that PACER is involved in the regulation of COX-2 expression in macrophagocyte and osteosarcoma cells. However, the role of this lncRNA in colorectal cancer (CRC) remains elusive. Here, we investigated the expression of PACER and its effect on cell proliferation and invasion to explore the role of PACER in CRC.MethodsReal-time quantitative PCR (RT-qPCR) analysis was used to evaluate the expression of PACER in CRC tissues and cells. Methyl thiazolyl tetrazolium (MTT) analysis was then used to investigate the inhibition effect of PACER knock-down in cell proliferation. The promoting role of this lncRNA on invasion by CRC cells was analysed by wound-healing assays, colony-formation assay, and transwell assays. We then used fluorescence in situ hybridization (FISH) to establish the subcellular localization of PACER. COX-2 protein levels were quantified by Western blot analysis and grayscale scanning analysis following the knock-down of PACER. Luciferase assay was carried out to monitor the modulation of the COX-2 promoter region by PACER. Tumor xenografts models were used to investigate the impact of PACER on the tumorigenesis of CRC cells in vivo. Enzyme-linked immunosorbent assay (ELISA) was then used to quantify prostaglandin E2 (PGE2) production upon knock-down of PACER.ResultsRT-qPCR analysis revealed that PACER was highly expressed in CRC tissues and cells, and a high PACER-expression level was associated with poor prognosis. MTT assay, wound-healing assay, colony-formation assay, and transwell assay revealed that PACER enhanced CRC-cell proliferation, invasion, and metastasis in vitro. Analysis of lncRNA localization by FISH showed that it mainly resided in the nucleus. RT-qPCR showed that PACER increased mRNA levels of COX-2. Western blot analysis demonstrated, under normal circumstances, that knock-down of PACER decreased the COX-2 protein level. In the case of p50 absence, COX-2 protein increased rapidly and remained highly expressed after knocking down PACER. Luciferase assay revealed that PACER modulated the COX-2 promoter region. Mouse xenograft models of CRC revealed that PACER promoted colorectal tumorigenesis in vivo. ELISA revealed that PACER knock-down inhibited PGE2 production.ConclusionsPACER modulates COX-2 expression through the nuclear factor kappa B (NF-κB) pathway in CRC. An increased level of PACER enhances proliferation, migration, and invasion of tumor cells by increasing COX-2 and PGE2 synthesis.

Correlation analysis between COX-2 gene polymorphism and eclampsia.

The aim of this study was to investigate the expression of cyclooxygenase-2 (COX-2) in eclampsia patients, and to explore the correlation between COX-2 polymorphism and incidence of eclampsia. From January 2016 to January 2018, a total of 280 pregnant women diagnosed in the Obstetrics and Gynecology Department of our hospital were selected for this study. All patients were divided into two groups, including normal pregnancy control group (n=120) and eclampsia group (n=160). The expression of COX-2 in placenta and umbilical cord tissues of eclampsia group and normal group was detected via Reverse Transcription-Polymerase Chain Reaction (RT-PCR), Western blotting and immunohistochemical staining. The single-nucleotide polymorphisms (rs1526172, rs1245231 and rs2198532) in the promoter region of the COX-2 gene were typed via conformation difference gel electrophoresis. Whether the distribution frequency of COX-2 genotypes met Hardy-Weinberg equilibrium law was detected via the Chi-square test. Meanwhile, the correlation between COX-2 alleles and gene polymorphisms and the incidence of eclampsia was analyzed. The messenger ribonucleic acid (mRNA) and protein expression levels of COX-2 in the eclampsia group were significantly higher than those of the normal group (p<0.05). According to the analysis, three polymorphisms of COX-2 gene were all in line with Hardy-Weinberg equilibrium distribution (p>0.05). Gene association analysis revealed that only polymorphisms (rs1526172 and rs1245231) and alleles were correlated with the incidence of eclampsia (p<0.05). However, polymorphism rs2198532 and alleles were not correlated with the incidence of eclampsia (p>0.05). Rs1526172 and rs1245231 in the promoter region of COX-2 are correlated with the incidence of eclampsia, while rs2198532 has no correlation with eclampsia.

Yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ) mediate cell density–dependent proinflammatory responses

A proper inflammatory response is critical to the restoration of tissue homeostasis after injury or infection, but how such a response is modulated by the physical properties of the cellular and tissue microenvironments is not fully understood. Here, using H358, HeLa, and HEK293T cells, we report that cell density can modulate inflammatory responses through the Hippo signaling pathway. We found that NF-κΒ activation through the proinflammatory cytokines interleukin-1β (IL-1β) and tumor necrosis factor α (TNFα) is not affected by cell density. However, we also noted that specific NF-κΒ target genes, such as cyclooxygenase 2 (COX-2), are induced much less at low cell densities than at high cell densities. Mechanistically, we observed that the transcriptional coactivators Yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ) are localized to the nucleus, bind to TEA domain transcription factors (TEADs), recruit histone deacetylase 7 (HDAC7) to the promoter region of COX-2, and repress its transcription at low cell density and that high cell density abrogates this YAP/TAZ-mediated transcriptional repression. Of note, IL-1β stimulation promoted cell migration and invasion mainly through COX-2 induction, but YAP inhibited this induction and thus cell migration and invasion. These results suggest that YAP/TAZ-TEAD interactions can repress COX-2 transcription and thereby mediate cell density-dependent modulation of proinflammatory responses. Our findings highlight that the cellular microenvironment significantly influences inflammatory responses via the Hippo pathway.

Quantitative analysis of COX-2 promoter methylation in gastric carcinoma.

PurposeTo determine the occurrence of COX-2 methylation in gastric carcinoma (GC), the status and level of CpG methylation in the promoter region of cyclooxygenase-2 (COX-2) were analyzed in early and advanced GCs, as well as in normal gastric tissues.MethodsThe extent of promoter methylation of the COX-2 gene was assessed quantitatively using pyrosequencing in 60 early and 60 advanced GCs samples harvested upon gastrectomy, and 40 normal gastric mucosa samples from patients with benign gastric diseases as controls.ResultsThe methylation frequency for the COX-2 gene was significantly higher in early than in advanced GCs (40.0% vs. 20.0%, P < 0.05). A significant difference was found in COX-2 methylation between GCs and normal gastric tissues (30.0% vs. 10.0%, by PS; P < 0.05). COX-2 gene methylation was significantly associated with the depth of invasion (P = 0.003), lymph node metastasis (P = 0.009), distant metastasis (P = 0.036), and TNM staging (P = 0.007). The overall survival of patients with COX-2 methylation was significantly lower than that of patients without COX-2 methylation (P = 0.005).ConclusionThese results demonstrated that COX-2 promoter methylation was significantly higher in tumor tissues, and was an early event for GC, thus, COX-2 gene methylation may be important in the initial development of gastric carcinogenesis. Thus, GCs with methylation in COX-2 may not be good candidates for treatment with COX-2 inhibitors. Furthermore, COX-2 methylation could be a significant prognostic factor predicting a favorable effect on GC patient outcome when downregulated.

COX-2 gene expression and methylation profile in Sapajus apella as an experimental model for gastric adenocarcinoma.

Gastric cancer (GC) remains one of the main causes of cancer-related death worldwide. There are two distinct histological types of GC: diffuse and intestinal. The latter is characterized by the presence of pre-neoplastic lesions. One of the most frequently altered enzymes in intestinal GC is COX-2, an important lesion marker. This work aimed to study COX-2 methylation and expression in N-methyl-N-Nitrosurea (MNU)-induced intestinal GC in six Sapajus apella animals. The partial promoter sequence of S. apella COX-2 gene was obtained and used to identify transcription factors and cis-regulatory element binding sites. The COX-2 methylation pattern was assessed using Methylation-Specific PCR (MSP), and expression was analyzed by immunohistochemistry (IHQ). A total of 20 samples were obtained. A 675 bp fragment of the S. apella COX-2 promoter region was obtained, and it was 99.2% and 68.2% similar to H. sapiens and S. boliviensis, respectively. Similar to humans, several transcription factors and cis-regulatory element binding sites were identified in the S. apella sequence. MSP revealed that all samples were methylated. However, IHQ results demonstrated positive COX-2 expression in all pre-neoplastic and tumoral samples. The results suggest that the analyzed fragment is not crucial in COX-2 regulation of GC in S. apella.

Alantolactone, a natural sesquiterpene lactone, has potent antitumor activity against glioblastoma by targeting IKK\u03b2 kinase activity and interrupting NF-\u03baB/COX-2-mediated signaling cascades

BackgroundGlioblastoma multiforme (GBM) is one of the most refractory and palindromic central nervous system (CNS) neoplasms, and current treatments have poor effects in GBM patients. Hence, the identification of novel therapeutic targets and the development of effective treatment strategies are essential. Alantolactone (ATL) has a wide range of pharmacological activities, and its anti-tumor effect is receiving increasing attention. However, the molecular mechanism underlying the anti-GBM activity of ATL remains poorly understood.MethodsThe biological functions of ATL in GBM cells were investigated using migration/invasion, colony formation and cell cycle/apoptosis assays. The localization of nuclear factor kappa B (NF-κB) p50/p65 and its binding to the cyclooxygenase 2 (COX-2) promoter were determined using confocal immunofluorescence, a streptavidin-agarose pulldown assay and a chromatin immunoprecipitation (ChIP) assay. IKKβ kinase activity was determined using a cell IKKβ kinase activity spectrophotometry quantitative detection kit and a molecular docking study. LC-MS/MS analysis was performed to determine the ability of ATL to traverse the blood-brain barrier (BBB). The in vivo anti-tumor efficacy of ATL was also analyzed in xenografted nude mice. Western blot analysis was performed to detect the protein expression levels.ResultsATL significantly suppressed the growth of GBM in vivo and in vitro. ATL significantly reduced the expression of COX-2 by inhibiting the kinase activity of IKKβ by targeting the ATP-binding site and then attenuating the binding of NF-κB to the COX-2 promoter region. Furthermore, ATL induced apoptosis by activating the cytochrome c (cyt c)/caspase cascade signaling pathway. Moreover, ATL could penetrate the BBB.ConclusionsATL exerts its anti-tumor effects in human GBM cells at least in part via NF-κB/COX-2-mediated signaling cascades by inhibiting IKKβ kinase activity. ATL, which is a natural small molecule inhibitor, is a promising candidate for clinical applications in the treatment of CNS tumors.

Cyclooxygenase-2 (COX-2) gene polymorphism in patients with differentiated thyroid carcinomas in the Turkish population

Background/aim: The most common thyroid malignancies are papillary and follicular thyroid carcinomas. They account for approximately 85%-90% of all thyroid cancers. Recent studies have reported the relevance of cyclooxygenase-2 (COX-2) gene polymorphism in human carcinogenesis. The aim of this study was to investigate the relationship between thyroid carcinoma and COX-2 gene polymorphism in the Turkish population.Materials and methods: We included a total of 96 differentiated thyroid cancer patients (mean age: 46.9 ± 10.3 years; 14 males, 82 females) and 83 healthy control subjects (mean age: 46.0 ± 10.6 years; 39 males, 44 females). The frequency of -765G>C, -8473T>C, and 1195A>G gene polymorphisms in the COX-2 promoter region was investigated in thyroid cancer patients and the control group using the high-resolution melting method. Results: COX-2-765G>C and COX-2-1195A>G gene polymorphisms were similar between thyroid cancer patients and the control group. There was a statistically significant difference between COX-2-8473T>C gene polymorphism in the thyroid cancer group and the control group (P = 0.012).Conclusion: The single nucleotide gene polymorphism COX-2-8473T>C might contribute to genetic susceptibility to differentiated thyroid cancer in the Turkish population.

Epigenetic blockade of neoplastic transformation by bromodomain and extra-terminal (BET) domain protein inhibitor JQ-1

The neoplastic transformation of cells and inflammation are processes that contribute to tumor initiation. Recently, emerging evidence has suggested that epigenetic alterations are also implicated in the early stages of carcinogenesis. Therefore, potent small molecules targeting epigenetic regulators have been developed as novel cancer therapeutic and preventive strategies. Bromodomain and extraterminal domain (BET) proteins are epigenetic readers that play key roles at the interface between chromatin modification and transcriptional regulation. In this study, we investigated the effect of the BET inhibitor JQ-1 on malignant transformation induced by 12-O-tetradecanoylphorbol-13-acetate (TPA) in mouse skin epidermal JB6 P+ cells. Treatment with JQ-1 effectively impaired TPA-induced colony formation in vitro. At the molecular level, the expression of several key TPA-induced pro-survival and pro-proliferative genes (Bcl2, Cyclin D1, and c-Myc) decreased rapidly after BET inhibition. In addition, JQ-1 treatment attenuated the activation of inflammatory NF-κB signaling triggered by TPA. Luciferase reporter assays using plasmids carrying different elements from the COX2 or IL6 promoters demonstrated that JQ-1 does not directly inhibit interactions between NF-κB and its binding sequence; rather, it affects CRE-element-associated transcriptional enhancement. Through siRNA gene silencing, we found that JQ-1 inhibits the p300-dependent transcriptional activation of COX2, which correlates with the results of the luciferase assay. Chromatin immunoprecipitation assays showed that TPA elevated H3K27Ac enrichment in the COX2 promoter region, which is mediated by p300, and Brd4. JQ-1 treatment did not change H3K27Ac levels but decreased the recruitment of Brd4 and RNA Polymerase II. Collectively, our study reveals that the BET inhibitor JQ-1 exerts potent anti-cancer and anti-inflammatory effects by interfering with the core transcriptional program of neoplastic transformation.

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.

Protein Phosphatase 2A in Lipopolysaccharide-Induced Cyclooxygenase-2 Expression in Murine Lymphatic Endothelial Cells.

The lymphatic endothelium plays an important role in the maintenance of tissue fluid homeostasis. It also participates in the pathogenesis of several inflammatory diseases. However, little is known about the underlying mechanisms by which lymphatic endothelial cell responds to inflammatory stimuli. In this study, we explored the mechanisms by which lipopolysaccharide (LPS) induces cyclooxygenase (COX)-2 expression in murine lymphatic endothelial cells (SV-LECs). LPS caused increases in cox-2 mRNA and protein levels, as well as in COX-2 promoter luciferase activity in SV-LECs. These actions were associated with protein phosphatase 2A (PP2A), apoptosis signal-regulating kinase 1 (ASK1), JNK1/2 and p38MAPK activation, and NF-κB subunit p65 and C/EBPβ phosphorylation. PP2A-ASK1 signaling blockade reduced LPS-induced JNK1/2, p38MAPK, p65 and C/EBPβ phosphorylation. Transfection with PP2A siRNA reduced LPS’s effects on p65 and C/EBPβ binding to the COX-2 promoter region. Transfected with the NF-κB or C/EBPβ site deletion of COX-2 reporter construct also abrogated LPS’s enhancing effect on COX-2 promoter luciferase activity in SV-LECs. Taken together, the induction of COX-2 in SV-LECs exposed to LPS may involve PP2A-ASK1-JNK and/or p38MAPK-NF-κB and/or C/EBPβ cascade.

Histone acetyltransferase p300 is induced by p38MAPK after photodynamic therapy: the therapeutic response is increased by the p300HAT inhibitor anacardic acid

Oxidative stress mediated by photodynamic therapy (PDT) mediates the tumoricidal effect, but has also been shown to induce the expression of prosurvival molecules, such as cyclooxygenase-2 (COX-2), which is involved in tumor recurrences after PDT. However, the molecular mechanism is still not fully understood. In this study, we found that activated p38MAPK could significantly up-regulate the activity and expression of histone acetyltransferase p300 (p300HAT) in A375 and C26 cells treated with ALA-and chlorin e6 (Ce6)-mediated photodynamic treatment. A colony-formation assay showed that PDT-induced cytotoxicity was dramatically elevated in the presence of the p300HAT inhibitor anacardic acid (AA). Further studies showed that increased p300HAT acetylates histone H3 and NF-κB p65 subunit to up-regulate the COX-2 expression, which was reduced by AA or p300HAT shRNA. Using chromatin immunoprecipitation analysis, we found that the augmented acetylation of histone H3 and NF-κB increases their binding to the COX-2 promoter region. These in vitro findings were further verified in mice bearing murine C26 and human A375 tumors treated with liposomal Ce6 mediated PDT. Meanwhile, the combination of PDT and AA resulted in greater tumor regression in BALB/c mice bearing C26 tumors, compared with PDT only or combined with COX-2 inhibitor. Finally, we demonstrated that suppression of the PDT-induced p300HAT activity also resulted in the decreased expression of survivin, restoring caspase-3 activity and sensitizing PDT-treated cells from autophagy to apoptosis due to the Becline-1 cleavage. This study demonstrates for the first time the molecular mechanisms involved in histone modification induced by PDT-mediated oxidative stress, suggesting that HAT inhibitors may provide a novel therapeutic approach for improving PDT response.

WMJ-S-001, a novel aliphatic hydroxamate derivative, exhibits anti-inflammatory properties via MKP-1 in LPS-stimulated RAW264.7 macrophages.

Hydroxamate derivatives have attracted considerable attention because of their broad pharmacological properties. Recent studies reported their potential use in the treatment of cardiovascular diseases, arthritis and infectious diseases. However, the mechanisms of the anti-inflammatory effects of hydroxamate derivatives remain to be elucidated. In an effort to develop a novel pharmacological agent that could suppress abnormally activated macrophages, we investigated a novel aliphatic hydroxamate derivative, WMJ-S-001, and explored its anti-inflammatory mechanisms. RAW264.7 macrophages were exposed to LPS in the absence or presence of WMJ-S-001. COX-2 expression and signalling molecules activated by LPS were assessed. LPS-induced COX-2 expression was suppressed by WMJ-S-001. WMJ-S-001 inhibited p38MAPK, NF-κB subunit p65 and CCAAT/enhancer-binding protein (C/EBP)β phosphorylation in cells exposed to LPS. Treatment of cells with a p38MAPK inhibitor (p38MAPK inhibitor III) markedly inhibited LPS-induced p65 and C/EBPβ phosphorylation and COX-2 expression. LPS-increased p65 and C/EBPβ binding to the COX-2 promoter region was suppressed in the presence of WMJ-S-001. In addition, WMJ-S-001 suppression of p38MAPK, p65 and C/EBPβ phosphorylation, and subsequent COX-2 expression were restored in cells transfected with a dominant-negative (DN) mutant of MAPK phosphatase-1 (MKP-1). WMJ-S-001 also caused an increase in MKP-1 activity in RAW264.7 macrophages. WMJ-S-001 may activate MKP-1, which then dephosphorylates p38MAPK, resulting in a decrease in p65 and C/EBPβ binding to the COX-2 promoter region and COX-2 down-regulation in LPS-stimulated RAW264.7 macrophages. The present study suggests that WMJ-S-001 may be a potential drug candidate for alleviating LPS-associated inflammatory diseases.

Cyclooxygenase-2 polymorphisms and breast cancer associated risk in Pakistani patients.

Prostaglandins produced by Cyclooxygenase-2 enzyme have been implicated to have a role in breast carcinogenesis. Several single nucleotide polymorphisms (SNPs) linked to COX-2 enzyme are reported to modulate its expression. The aim of the present study was to examine association of these SNPs to breast cancer risk in Pakistani patients. In this case-control study, three sequence variants rs689465, rs689466, rs20417 in the promoter region of COX-2 were screened to evaluate the association with breast cancer risk. A total of 150 breast cancer patients and 101 healthy control genomic DNA were genotyped for rs689456, rs689466, rs20417 and their genotypes distribution in cases and control were compared using Pearson chi square test. Risk association was analyzed through odd ratio calculated by logistic regression. A screening analysis of COX-2 SNPs in 101 healthy controls showed distribution of Minor allelic frequency distribution of SNPs as follows : rs689465 (0.12), rs689466 (0.15), rs20417 (0.23). Further analyses revealed that their observed genotype frequencies were consistent with Hardy Weinberg equilibrium and strong linkage disequilibrium was identified between rs20417, rs689465 and rs689466. The Combined allele variants analysis showed that Haplotype rs68965G- 689466A-20417C (OR 2.909; CI 95 %1.3776.327; P = 0.007) was significantly associated with breast cancer. Our results indicate no strong association between three most frequent COX-2 SNPs rs689465 rs689466, rs20417 studied with breast cancer risk in the single locus analysis. However, our data suggested that combined COX-2 SNP haplotype have a role in breast cancer associated risk in Pakistani patients.

P50-associated COX-2 extragenic RNA (PACER) activates COX-2 gene expression by occluding repressive NF-κB complexes.

Deregulated expression of COX-2 has been causally linked to development, progression, and outcome of several types of human cancer. We describe a novel fundamental level of transcriptional control of COX-2 expression. Using primary human mammary epithelial cells and monocyte/macrophage cell lines, we show that the chromatin boundary/insulator factor CTCF establishes an open chromatin domain and induces expression of a long non-coding RNA within the upstream promoter region of COX-2. Upon induction of COX-2 expression, the lncRNA associates with p50, a repressive subunit of NF-κB, and occludes it from the COX-2 promoter, potentially facilitating interaction with activation-competent NF-κB p65/p50 dimers. This enables recruitment of the p300 histone acetyltransferase, a domain-wide increase in histone acetylation and assembly of RNA Polymerase II initiation complexes. Our findings reveal an unexpected mechanism of gene control by lncRNA-mediated repressor occlusion and identify the COX-2-lncRNA, PACER, as a new potential target for COX-2-modulation in inflammation and cancer.DOI: http://dx.doi.org/10.7554/eLife.01776.001.

Valproic acid suppresses lipopolysaccharide-induced cyclooxygenase-2 expression via MKP-1 in murine brain microvascular endothelial cells

Inflammation and vascular perturbations are increasingly implicated in the pathogenesis of neurodegenerative diseases. Prevailing evidence suggests that valproic acid (VPA), an antiepileptic and mood stabilizer, exhibits not only neuro-protective effects, but also anti-inflammatory effects in neurodegenerative diseases. However, the underlying mechanism contributing to VPA's suppression of inflammatory responses remains unclear. In this study, we explored the inhibitory action of VPA on cyclooxygenase (COX)-2 expression in bEnd.3 mouse brain microvascular endothelial cells exposed to lipopolysaccharide (LPS), a pro-inflammatory stimulus. The LPS-induced increases in COX-2 protein level and COX-2 promoter-luciferase activity were significantly suppressed by VPA. VPA inhibited p38MAPK and JNK phosphorylation in LPS-stimulated bEnd.3 cells. Treatment of cells with a p38MAPK inhibitor (p38MAPK inhibitor III) or a JNK signaling inhibitor (JNK inhibitor II) significantly inhibited LPS-induced COX-2 expression. VPA inhibited LPS-induced NF-κB subunit p65 phosphorylation and κB-luciferase activity. LPS-increased p65 and C/EBPβ binding to the COX-2 promoter region was attenuated in the presence of VPA. In addition, VPA suppression of p38MAPK, JNK and p65 phosphorylation, and subsequent COX-2 expression was restored in cells transfected with mitogen-activated protein kinase phosphatase-1 (MKP-1) dominant negative (DN) mutant. VPA also caused increases in MKP-1 acetylation and MKP-1 phosphatase activity in bEnd.3 cells. In conclusion, VPA may cause MKP-1 activation to dephosphorylate p38MAPK and JNK, leading to decrease in p65 and C/EBPβ binding to the COX-2 promoter region and COX-2 down-regulation in LPS-stimulated bEnd.3 cells. The present study therefore supports the therapeutic value of VPA in alleviating brain inflammatory processes.

High-density lipoprotein induces cyclooxygenase-2 expression and prostaglandin I-2 release in endothelial cells through sphingosine kinase-2

High-density lipoprotein (HDL) has a significant cardioprotective effects. HDL induces cyclooxygenase-2 (COX-2) expression and prostacyclin I-2 (PGI-2) release in vascular endothelial cells, which contributes to its anti-atherogenic effects. However, the underlying mechanisms are not fully understood. In the present study, we observed that HDL-stimulated COX-2 expression and PGI-2 production in human umbilical vein endothelial cells (HUVECs) in a time- and dose-dependent manner. These effects triggered by HDL were inhibited by pertussis toxin (PTX), protein kinase C (PKC) inhibitor GF109203X, and ERK inhibitor PD98059, suggesting that Gαi/Gαo-coupled GPCR, PKC, and ERK pathways are involved in HDL-induced COX-2/PGI-2 activation. More importantly, we found that silencing of sphingosine kinase 2 (SphK-2) also blocked HDL-induced COX-2/PGI-2 activation. In addition, HDL-activated SphK-2 phosphorylation accompanied by increased S1P level in the nucleus. Our ChIP data demonstrated that SphK-2 is associated with CREB at the COX-2 promoter region. Collectively, these results indicate that HDL induces COX-2 expression and PGI-2 release in endothelial cells through activation of PKC, ERK1/2, and SphK-2 pathways. These findings implicate a novel mechanism underlying anti-atherothrombotic effects of HDL.

The −1195G allele increases the transcriptional activity of cyclooxygenase‐2 gene (COX‐2) in colon cancer cell lines

Up-regulation of cyclooxygenase-2 (COX-2) is an early and key event in human colorectal carcinogenesis (CRC). Nevertheless, the molecular mechanisms leading to this over-expression are largely unknown. We previously reported an association between the -1195G allele and higher predisposition for CRC in a Caucasian population. The biological explanation for the involvement of this polymorphism in CRC remains elusive. We aimed to functionally characterize the influence of the -1195A>G promoter region polymorphism on COX-2 transcription activity in colon cancer cell lines. Luciferase reporter assays were performed to assess whether the -1195A/G alleles influenced COX-2 transcription. The COX-2 promoter's region containing either the -1195A or -1195G alleles was cloned into pGL3-basic reporter vector. The reporter vectors were transiently co-transfected with the pGL4.73 control plasmid to HCT-116 and HCA-7 colon cancer cell lines. The levels of reporter gene expression driven by the -1195G allele-containing COX-2 promoter were significantly higher in both colon cancer cell lines. A 2.2-fold increase in promoter activity was observed in the HCT-116 cell line (P < 0.001), and this over-expression was even more noticeable in the HCA-7 COX-2 expressing cell line with a threefold higher transcriptional activity (P = 0.001). The -1195G allele appeared to enhance COX-2 transcription, providing a molecular basis underlying the increased susceptibility for CRC and potentially a new mechanism for COX-2 overexpression.

Polymorphism −765G&gt;C in Cyclooxygenase-2 and Risk of Colorectal Cancer

Colorectal cancer is the third-most-prevalent form of cancer in the world. Several studies report that prostaglandins are mediators of carcinogenesis. Cyclooxygenase, also known as prostaglandin endoperoxidase H synthase, is a pro-inflammatory enzyme that intercedes in the formation of eicosanoids from arachidonic acid. Polymorphisms in the COX-2 gene have been associated with increased risk of different tumors, including colorectal. Genetic variations in COX-2 may affect enzyme activity or expression, thereby altering the production of prostaglandins and potentially modulating an individual’s inflammatory response and risk of colorectal cancer. A functional polymorphism in the COX-2 promoter region (–765G>C), located at a putative stimulatory protein-1 (SP1) binding site, suggests the involvement of a complex array of factors regulating its expression. This study was designed to evaluate the status of –765G>C polymorphism (rs20417) and risk of sporadic colorectal cancer in an Iranian population.