Cyclooxygenase-2 Gene Promoter Research Articles

The Anesthetic Modality but Not the Mode of Delivery Seem to Modulate the Methylation Status of Cyclooxygenase-2 Promoter of the Newborns.

Cesarean section (CS) rates are high. Epidemiological data supports increased risk of inflammatory conditions in the offspring born by CS. Epigenetic alterations occurring during the perinatal period may account for this risk. Cyclooxygenase-2 (COX2) has strong implications for inflammatory diseases. The methylation of COX2 of newborn infants was compared with respect to their mode of delivery. Ninety healthy term infants born by vaginal delivery (VD), planned cesarean section (PCS), or emergency CS (ECS) were recruited (30 infants in each group). For obstetric anesthesia, local (LA), regional (RA), or general (GA) anesthesia were used. Carefully selected exclusion criteria were implemented to eliminate any confounders with potential epigenetic effects. Umbilical artery blood samples were collected. Demographic and clinical characteristics, folate and CRP levels, and mean methylation levels of the COX2 gene promoter were determined. Except the birth weight and maternal age parameters, VD, PCS, and ECS were similar. The methylation percentage of COX2 was higher in ECS (16.9 ± 5.1) than VD (14.5 ± 4.1) and PCS (14.8 ± 2.9), albeit p was 0.064. Because of the dual anesthetic modality populations (RA and GA) in PCS and ECS and the recent literature on anesthetics and epigenetics, the anesthetic modality groups were also analyzed. The methylation percentage of COX2 was significantly different between LA, RA, and GA groups (14.5 ± 4.1, 13.9 ± 2.8, and 17.0 ± 4.6, respectively, p = 0.012). When the mode of delivery is the question of debate, the anesthetic modality should be remembered as an important epigenetic modulator. · Perinatal period is a vulnerable time period for epigenetic modulations.. · The mode of delivery is influential in any potential epigenetic alterations occurring perinatally.. · The obstetric anesthetic modality should be remembered as an important epigenetic modulator..

Research on the prevalence of COX-2 gene promoter -765G>C polymorphism, inflammation, and serum levels of PGE2 in coronary artery disease

Research on the prevalence of COX-2 gene promoter -765G>C polymorphism, inflammation, and serum levels of PGE2 in coronary artery disease

Epigenetic Regulation of the COX-2 Pathway Modulates Macrophage Inflammation in Diabetic Wound Repair

Abstract Macrophage plasticity is critical for normal tissue repair to ensure transition from the inflammatory to proliferative phase of healing. In pathologic conditions, such as type 2 diabetes (T2D), wounds fail to heal due to impaired resolution of inflammation. There is increasing evidence that epigenetic-based mechanisms control macrophage function. The cyclooxygenase (COX)-2/Prostaglandin E2 (PGE2) axis, as well as upstream pathways including cytosolic phospholipase A2 (c(PL)A2), have been associated with chronic inflammation, however the underlying mechanisms of COX-2/PGE2 regulation in wound repair have not been examined. Here, we find in human and murine diabetic wounds, that PGE2 production is elevated in wound macrophages and regulates inflammation. Further, epigenetic modification of the COX-2/PGE2 pathway was regulated by MLL1, a methyltransferase, that increased H3K4 trimethylation on the cPLA2 promoter in diabetic wound macrophages and human T2D monocytes resulting in increased cPLA2 expression. Myeloid-specific deletion of MLL1 restored basal cPLA2 levels. Separately, increased TGFβ1 in diabetic wounds augmented miR-29b and DNMT 3a/3b in wound macrophages, resulting in hypomethylation of the Cox-2 gene promoter and overexpression of COX-2/PGE2. Inhibition of the PGE2 pathway, with a macrophage-specific nanocarrier containing a COX-2 inhibitor improved diabetic healing. Taken together, our results indicate the COX-2/PGE2 pathway is a critical regulator of macrophage-mediated inflammation in diabetic wounds and therapeutic manipulation of this pathway improves pathologic wound repair.

Sa1622 - Role of Cox-2 Gene Promoter Methylation and Helicobacter Pylori Infection in Gastric Ulcer Healing

Sa1622 - Role of Cox-2 Gene Promoter Methylation and Helicobacter Pylori Infection in Gastric Ulcer Healing

XRCC5 cooperates with p300 to promote cyclooxygenase-2 expression and tumor growth in colon cancers.

Cyclooxygenase (COX) is the rate-limiting enzyme in prostaglandins (PGs) biosynthesis. Previous studies indicate that COX-2, one of the isoforms of COX, is highly expressed in colon cancers and plays a key role in colon cancer carcinogenesis. Thus, searching for novel transcription factors regulating COX-2 expression will facilitate drug development for colon cancer. In this study, we identified XRCC5 as a binding protein of the COX-2 gene promoter in colon cancer cells with streptavidin-agarose pulldown assay and mass spectrometry analysis, and found that XRCC5 promoted colon cancer growth through modulation of COX-2 signaling. Knockdown of XRCC5 by siRNAs inhibited the growth of colon cancer cells in vitro and of tumor xenografts in a mouse model in vivo by suppressing COX-2 promoter activity and COX-2 protein expression. Conversely, overexpression of XRCC5 promoted the growth of colon cancer cells by activating COX-2 promoter and increasing COX-2 protein expression. Moreover, the role of p300 (a transcription co-activator) in acetylating XRCC5 to co-regulate COX-2 expression was also evaluated. Immunofluorescence assay and confocal microscopy showed that XRCC5 and p300 proteins were co-located in the nucleus of colon cancer cells. Co-immunoprecipitation assay also proved the interaction between XRCC5 and p300 in nuclear proteins of colon cancer cells. Cell viability assay indicated that the overexpression of wild-type p300, but not its histone acetyltransferase (HAT) domain deletion mutant, increased XRCC5 acetylation, thereby up-regulated COX-2 expression and promoted the growth of colon cancer cells. In contrast, suppression of p300 by a p300 HAT-specific inhibitor (C646) inhibited colon cancer cell growth by suppressing COX-2 expression. Taken together, our results demonstrated that XRCC5 promoted colon cancer growth by cooperating with p300 to regulate COX-2 expression, and suggested that the XRCC5/p300/COX-2 signaling pathway was a potential target in the treatment of colon cancers.

Leptin induces CREB-dependent aromatase activation through COX-2 expression in breast cancer cells

Leptin plays a key role in the control of adipocyte formation, as well as in the associated regulation of energy intake and expenditure. The goal of this study was to determine if leptin-induced aromatase enhances estrogen production and induces tumor cell growth stimulation. To this end, breast cancer cells were incubated with leptin in the absence or presence of inhibitor pretreatment, and changes in aromatase and cyclooxygenase-2 (COX-2) expression were evaluated at the mRNA and protein levels. Transient transfection assays were performed to examine the aromatase and COX-2 gene promoter activities and immunoblot analysis was used to examine protein expression. Leptin induced aromatase expression, estradiol production, and promoter activity in breast cancer cells. Protein levels of phospho-STAT3, PKA, Akt, ERK, and JNK were increased by leptin. Leptin also significantly increased cAMP levels, cAMP response element (CRE) activation, and CREB phosphorylation. In addition, leptin induced COX-2 expression, promoter activity, and increased the production of prostaglandin E2. Finally, a COX-2 inhibitor and aromatase inhibitor suppressed leptin-induced cell proliferation in MCF-7 breast cancer cells. Together, our data show that leptin increased aromatase expression in breast cancer cells, which was correlated with COX-2 upregulation, mediated through CRE activation and cooperation among multiple signaling pathways.

Induction of cyclooxygenase-2 gene by Candida albicans through EGFR, ERK, and p38 pathways in human urinary epithelium.

In the present data, we found that Candida albicans (C. albicans) caused bladder epithelial cell morphology alteration, cell damage, and inflammatory responses, including cyclooxygenase-2 (COX-2) gene and protein expression as well as prostaglandin E2 accumulation. In addition, the molecular pathway underlying C. albicans-induced urothelial COX-2 gene expression was examined. Among MAPK pathways, phosphorylation of ERK1/2, p38, and JNK each increased following C. albicans infection for 12h. However, C. albicans-induced COX-2 protein expression was inhibited by specific inhibitors of ERK and p38 (U0126 and SB203580) but not by JNK inhibitor SP600125. Additional evidence came from the increased amount of phosphorylated RSK that is the mutual downstream molecule of ERK1/2 and p38. Furthermore, phosphorylation of RSK protein was reduced by the ERK and p38 inhibitor, suggesting that the urothelial COX-2 gene was induced majorly though the ERK/p38-RSK pathway by C. albicans infection. We also found transcription factor CREB-1 showed increased binding to the COX-2 gene promoter by chromatin immunoprecipitation assay. Next, we used receptor inhibitors including Toll-like receptor (TLR)-Myd88 inhibitor ST2825, Dectin-Syk inhibitor Syk inhibitor, and epidermal growth factor receptor (EGFR) inhibitor PD168393 to identify which one was the main target associated with C. albicans binding. The results revealed that it was EGFR, recognized by C. albicans, that mostly mediated the ERK/p38-RSK pathway activation to induce COX-2 gene expression, but this was not the case for TLRs and Dectin receptors. In summary, these results demonstrated the EGFR-ERK/p38-RSK-CREB-1 pathway was involved significantly in the C. albicans-induced COX-2 expression in human urothelium.

Cyclical DNA Methylation and Histone Changes Are Induced by LPS to Activate COX-2 in Human Intestinal Epithelial Cells.

Bacterial lipopolysaccharide (LPS) induces release of inflammatory mediators both in immune and epithelial cells. We investigated whether changes of epigenetic marks, including selected histone modification and DNA methylation, may drive or accompany the activation of COX-2 gene in HT-29 human intestinal epithelial cells upon exposure to LPS. Here we describe cyclical histone acetylation (H3), methylation (H3K4, H3K9, H3K27) and DNA methylation changes occurring at COX-2 gene promoter overtime after LPS stimulation. Histone K27 methylation changes are carried out by the H3 demethylase JMJD3 and are essential for COX-2 induction by LPS. The changes of the histone code are associated with cyclical methylation signatures at the promoter and gene body of COX-2 gene.

TNFα up-regulates COX-2 in chronic progressive nephropathy through nuclear accumulation of RelB and NF-κB2

Objective: The pathogenesis of progressive nephropathies involves inflammatory factors. The inhibition of cyclooxygenase-2 (COX-2) can limit renal damage and inflammation. However, the mechanism of up-regulation of COX-2 in nephropathy is poorly defined. Materials and methods: Here we found that tumor necrosis factor alpha (TNFα) was involved in expression of COX-2 in normal rat kidney (NRK) cell line. Results: TNFα stimulated COX-2 production in a time-dependent manner in NRK cells by inducing nuclear accumulation of RelB and nuclear factor kappa B2 (NF-κB2) and their association with COX-2 gene promoter. Depletion of IκB-inducing kinase alpha, a positive regulator of activation of p100 processing to active p52, attenuated TNFα-induced COX-2 production. Furthermore, TNFα induced COX-2 production and nuclear import in anti-thymocyte serum (ATS) nephropathy. Discussion and conclusion: These data suggest that TNFα-RelB/p52 pathway may be involved in the early stages of renal damage, in part by stimulating COX-2 and inflammatory responses.

Abstract 1059: Analysis of promoter methylation levels of COX2 gene in Turkish patients with head and neck cancer

Abstract Purpose: Methylation of CpG islands in the promoter regions of genes plays an important role in head and neck carcinogenesis. Overexpression of COX2 gene was recorded in various types of cancer in literature. In addition, the promoter region of the COX2 gene displayed high methylation in normal colorectal tissue and normal gastric tissue samples. On the other hand, it has recently been reported in two different gastrointestinal cancer studies, that low expression of COX2 was observed in some colorectal/gastric cancers and hypermethylation/related transcriptional silencing of COX2 gene is common in colorectal cancer. There is no study investigating the COX2 methylation status on head and neck cancer (HNC) in the literature. In our study we evaluated the COX2 gene promoter methylation in patients with HNC. Experimental Design: Methylation of the COX2 was investigated by bisulfite modification/methylation-specific polymerase chain reaction in tumors and matched normal tissue samples from 99 Turkish patients with HNC. Results: The promoter region of the COX2 was methylated in 53.5% and 65.6% of the primary tumors and the corresponding normal tissue samples, respectively [p = 0.082, χ2 = 3.02, OR(95%CI) = 0.602 (0.34-1.06)]. Considering the methylation levels observed in our previous studies, and published data on different cancer types, methylation levels <10% were considered unmethylated, whereas levels between 10% to 25% and 25% to 100% were classified as moderate and high methylation, respectively. Based on this information, methylation ratio was observed less than 10% in 46.5% and 34.3% of tumor and normal samples, respectively. The moderate methylation levels (10-25%) were found in 23.2% of the tumors and 7.1% of the adjacent normal tissues. High methylation levels (>25%) were 30.3% and 58.6% in the tumor and normal tissues, respectively (p = 0.00006, χ2 = 19.24). The results were similar when a subgroup of 60 patients with larynx cancer from this cohort, were analyzed separately (p = 0.0006, χ2 = 14.87). Conclusions: Methylation may play an important role in HNC carcinogenesis. Although we found that there was no significant association between the methylation levels of tumor and adjacent normal tissues, high methylation levels of the COX2 gene were observed (>25%) in normal matched tissues when compared to the levels in HNC tumors. Citation Format: Semra Demokan, Cansu Ozkoklesen, Zubeyde Yalniz, Necati Enver, Murat Ulusan, Nejat Dalay. Analysis of promoter methylation levels of COX2 gene in Turkish patients with head and neck cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1059. doi:10.1158/1538-7445.AM2015-1059

COX-2 gene promoter DNA methylation status in eutopic and ectopic endometrium of Egyptian women with endometriosis

The pathophysiology of COX-2 expression in endometriosis is a matter of debate. The aim was to investigate the role of DNA methylation of the NF-IL6 site within the promoter of COX-2 gene in the pathogenesis of endometriosis. The endometrial tissues (ectopic and eutopic) were collected from 60 women with endometriosis and 30 women without endometriosis (control group). The methylation status of COX-2 was examined by methylation-specific PCR. Quantitative real-time PCR (RT-PCR) was performed to measure COX-2 mRNA levels in endometrial tissues. We found significantly higher levels of COX-2 in ectopic endometriotic tissue compared with eutopic tissue. Also, we found that the frequencies of methylation status of the NF-IL6 site within the COX-2 promoter in the eutopic and ectopic endometrial tissues of endometriosis groups were significantly decreased in comparison to controls (P=0.002, P=0.000 respectively). Our study demonstrated that DNA hypomethylation of the NF-IL6 site within the promoter of COX-2 gene could be a key mechanism for its elevated expression in the eutopic and ectopic tissues of endometriosis.

Ku80 cooperates with CBP to promote COX-2 expression and tumor growth.

Cyclooxygenase-2 (COX-2) plays an important role in lung cancer development and progression. Using streptavidin-agarose pulldown and proteomics assay, we identified and validated Ku80, a dimer of Ku participating in the repair of broken DNA double strands, as a new binding protein of the COX-2 gene promoter. Overexpression of Ku80 up-regulated COX-2 promoter activation and COX-2 expression in lung cancer cells. Silencing of Ku80 by siRNA down-regulated COX-2 expression and inhibited tumor cell growth in vitro and in a xenograft mouse model. Ku80 knockdown suppressed phosphorylation of ERK, resulting in an inactivation of the MAPK pathway. Moreover, CBP, a transcription co-activator, interacted with and acetylated Ku80 to co-regulate the activation of COX-2 promoter. Overexpression of CBP increased Ku80 acetylation, thereby promoting COX-2 expression and cell growth. Suppression of CBP by a CBP-specific inhibitor or siRNA inhibited COX-2 expression as well as tumor cell growth. Tissue microarray immunohistochemical analysis of lung adenocarcinomas revealed a strong positive correlation between levels of Ku80 and COX-2 and clinicopathologic variables. Overexpression of Ku80 was associated with poor prognosis in patients with lung cancers. We conclude that Ku80 promotes COX-2 expression and tumor growth and is a potential therapeutic target in lung cancer.

Lack of association between the cyclooxygenase 2 -765G>C polymorphism and prostate cancer risk: a meta-analysis.

The aim of this study was to investigate the association between the cyclooxygenase 2 (COX2) -765G>C (rs20417) polymorphism and prostate cancer (PC) risk using meta-analysis. A systematic literature search was performed using the PubMed, Embase, Cochrane Library, and Google Scholar databases by using the terms "cyclooxygenase-2/COX-2/PTGs2", "polymorphism" or "variation", and "prostate" and "cancer" or "carcinoma" to identify relevant articles up to June 14, 2014. Crude odds ratios (ORs) with 95% confidence intervals (CIs) were assessed for PC risk associated with COX2 -765G>C polymorphism using fixed- and random-effect models. We identified a total of nine publications, including 5952 cases and 5078 controls, to investigate the effect of COX2 -765G>C on PC risk, and found no significant association in any genetic model tested (CC vs GG: OR = 0.993, 95%CI = 0.923-1.068; GC+CC vs GG: OR = 1.041, 95%CI = 0.931-1.103; CC vs GC+GG: OR = 0.858, 95%CI = 0.689-1.067; CC vs GG: OR = 0.871, 95%CI = 0.689-1.086; GC vs GG: OR = 1.032, 95%CI = 0.945-1.127). Power analysis and tests for publication bias ensured the reliability of our results. This meta-analysis suggested that the functional COX2 -765G>C polymorphism, located in the COX2 gene promoter, is unlikely to be associated with PC risk. However, additional larger, well-designed studies are still required to reach a conclusive result on this issue.

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.

Transcription factor Ets-1 inhibits glucose-stimulated insulin secretion of pancreatic β-cells partly through up-regulation of COX-2 gene expression

Increased cyclooxygenase-2 (COX-2) expression is associated with pancreatic β-cell dysfunction. We previously demonstrated that the transcription factor Ets-1 significantly up-regulated COX-2 gene promoter activity. In this report, we used the pancreatic β-cell line INS-1 and isolated rat islets to investigate whether Ets-1 could induce β-cell dysfunction through up-regulating COX-2 gene expression. We investigated the effects of ETS-1 overexpression and the effects of ETS-1 RNA interference on endogenous COX-2 expression in INS-1 cells. We used site-directed mutagenesis and a dual luciferase reporter assay to study putative Ets-1 binding sites in the COX-2 promoter. The effect of ETS-1 1 overexpression on the insulin secretion function of INS-1 cells and rat islets and the potential reversal of these effects by a COX-2 inhibitor were determined in a glucose-stimulated insulin secretion (GSIS) assay. ETS-1 overexpression significantly induces endogenous COX-2 expression, but ETS-1 RNA interference has no effect on basal COX-2 expression in INS-1 cells. Ets-1 protein significantly increases COX-2 promoter activity through the binding site located in the -195/-186 region of the COX-2 promoter. ETS-1 overexpression significantly inhibited the GSIS function of INS-1 cells and islet cells and COX-2 inhibitor treatment partly reversed this effect. These findings indicated that ETS-1 overexpression induces β-cell dysfunction partly through up-regulation of COX-2 gene expression. Moreover, Ets-1, the transcriptional regulator of COX-2 expression, may be a potential target for the prevention of β-cell dysfunction mediated by COX-2.

Cyclooxygenase 2 genetic variant interacting with tobacco smoking and the risk of lung cancer

To explore the association of -1195G > A genetic variant in the promoter region of cyclooxygenase 2 genetic (COX2) with the genetic susceptibility of lung cancer and its interaction with smoking. Totally, 956 lung cancer patients recruited between January 2000 and December 2008 at Cancer Hospital, Chinese Academy of Medical Science as the case group, and 994 frequency-matched controls were randomly selected from a pool of cancer-free subjects recruited from a nutritional survey. All subjects were ethnic Han Chinese. There was no sex, age restrictions. Case group and control group were matched. Informed consent was obtained and 2 ml peripheral blood was collected from each subject. All samples were genotyped by polymerase chain reaction-restriction fragment length polymorphism method, smoking status of the subjects was surveyed.While the OR and 95% CI were estimated by logistic regression to evaluate the relation of COX2 -1195G > A variant and the risk of lung cancer. The genetic allele COX2 -1195AA of control group and case group were 24.9% (247/994) and 28.3% (271/956) . Case-control analysis showed an increased risk of developing lung cancer for -1195AA genotype carriers (OR = 1.36, 95% CI: 1.03-1.79), compared with -1195GG carriers. When stratified by smoking status, the significant increased risk of lung cancer was found among smokers with COX2-1195AA genotype, with the OR (95%CI) was 1.56 (1.08-2.25); while among non-smokers, difference of lung cancer risk was not found among different genotypes (OR = 1.17; 95%CI: 0.77-1.61). Among heavy smokers (pack-year >20), -1195AA and -1195AG genotype carriers have significant increased risk of lung cancer with 1.85 (1.16-2.95) and 1.62(1.08-2.43) of OR (95%CI), respectively; among light smokers (pack-year ≤ 20), the OR (95%CI) of lung cancer risk in -1195AG and -1195AA genotype carriers were 0.78 (0.47-1.30) and 1.08 (0.60-1.94), respectively. Genetic polymorphism in the promoter of COX2 gene interacting with smoking factor plays an important role in the development of lung cancer.

Abstract 3119: MUC1 transcriptionally regulates COX-2 gene in pancreatic cancer cells.

Abstract Introduction: Pancreatic Cancer (PC) is the fourth leading cause of cancer-related deaths in the United States. Over 80% of human pancreatic ductal adenocarcinomas (PDA) overexpress MUC1, a transmembrane mucin glycoprotein. MUC1high tumors are aggressive and patients with such tumors have poor prognosis. Interestingly, MUC1high PDA tumors overexpress cyclooxygenase-2 (COX-2), an inducible pro-inflammatory enzyme known to promote tumor progression and metastasis. Since the cytoplasmic domain of MUC1 (MUC1-C) is critically involved in the signal transduction cascade that promotes tumor progression and metastasis, we hypothesize that MUC1 induces its effect via regulating COX-2 expression and function. Thus, we aim to establish the relationship between MUC1 and COX-2 expression and determine the mechanism by which MUC1 regulates COX-2 expression. Cell lines: Human BxPC3 and mouse Panc02 PC cell lines that express low endogenous MUC1 were stably transfected with full length MUC1 (MUC1), empty vector (Neo), or full length MUC1 with all 7 tyrosine residues of MUC1 CT mutated to Phenylalanine (Y0). Other cell lines: human HPAFII and Capan-1 and mouse KCM cells that express high endogenous MUC1; and mouse KCKO cells that genetically lack Muc1. Method: We measured the level of COX-2 mRNA and protein by RT-PCR and Western blot respectively. MUC1 was transiently knocked down using a smart pool of MUC1 specific siRNA in MUC1high cells. Immunohistochemistry (IHC) was performed on tumor sections to determine the in situ expression of COX-2 and MUC1. Chromatin immunoprecipitation (ChIP) assay was conducted to evaluate binding of MUC1 CT to the promoter of COX-2 gene. Results: Significantly higher levels of COX-2 mRNA and protein were detected in PC cells expressing high MUC1 compared to cells with low MUC1. This was recapitulated in vivo with higher COX-2 expression in MUC1high versus MUC1low tumors. A subsequent decrease in COX-2 protein was observed upon MUC1 knock down in the MUC1high PC cells. Thus, a direct correlation exists between MUC1 and COX-2 expression in PC cells. Importantly, this correlation was lost in the Y0 cells in which COX-2 expression was significantly decreased even though there was high MUC1 expression. Data clearly suggests that signaling through the tyrosine residues of MUC1 CT is essential for MUC1 induced COX-2 gene expression. Further, in the MUC1high cells, MUC1 CT associates with the COX-2 promoter around 1000 bp upstream of the transcription start site. This association does not occur in the Y0 cells suggesting that an intact CT is necessary for this interaction. Interestingly, MUC1 CT interacts with the COX-2 promoter around the same gene locus where NF-kB associates with the COX-2 promoter. This raises the possibility that MUC1 CT associates with NF-kB via its tyrosine residues and the MUC1/NFkB complex acts as a transcriptional regulator of the COX-2 gene. Citation Format: Sritama Nath, Lopamudra Das Roy, Shanti Rao, Priyanka Grover, Pinku Mukherjee. MUC1 transcriptionally regulates COX-2 gene in pancreatic cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3119. doi:10.1158/1538-7445.AM2013-3119

COX-2 Gene Promoter Methylation in Patients Infected with Helicobacter Pylori.

Cyclooxygenase (COX) plays a critical role in peptic ulcer development. COX-2 contains CpG islands in promoter area, which suggests possible epigenetic mechanisms of gene silencing. We evaluated COX-2 gene promoter methylation levels in the gastric mucosa of patients with various gastric diseases. DNA was extracted from endoscopic biopsy materials collected from the gastric mucosa. The methylation levels of the COX-2 gene promoter were measured quantitatively by using pyrosequencing. COX-2 mRNA expression in Kato III and AGS cells was measured using real-time PCR. COX-2 gene promoter methylation levels were significantly higher in Helicobacter pylori (HP)-positive cases than in HP-negative cases (27.5% vs. 8.1%, respectively, P < 0.001). COX-2 gene promoter methylation levels in patients in whom HP was successfully eradicated were significantly lower than those in HP-positive cases (18.7% vs. 27.5%, respectively, P < 0.01). We then investigated the effects of COX-2 gene promoter methylation on its mRNA expression in vitro. COX-2 mRNA expression was not observed in Kato III cells, despite the addition of the protein kinase C stimulator α-phorbol 12,13-dibutyrate (PDBu). COX-2 expression was observed after the addition of the demethylating agent 5-Aza-dC and was enhanced by PDBu. HP infection caused a significant increase in the methylation levels of the COX-2 gene promoter in the gastric mucosa. In addition to transcriptional regulation, COX-2 expression is regulated through epigenetic mechanisms.

The ETS-Domain Transcription Factor Elk-1 Regulates COX-2 Gene Expression and Inhibits Glucose-Stimulated Insulin Secretion in the Pancreaticβ-Cell Line INS-1

Cyclooxygenase-2 (COX-2) expression is associated with many aspects of physiological and pathological conditions, including pancreatic β-cell dysfunction. Prostaglandin E2 (PGE2) production, as a consequence of COX-2 gene induction, has been reported to impair β-cell function. The molecular mechanisms involved in the regulation of COX-2 gene expression are not fully understood. We previously demonstrated that transcription factor Elk-1 significantly upregulated COX-2 gene promoter activity. In this report, we used pancreatic β-cell line (INS-1) to explore the relationships between Elk-1 and COX-2. We first investigated the effects of Elk-1 on COX-2 transcriptional regulation and expression in INS-1 cells. We thus undertook to study the binding of Elk-1 to its putative binding sites in the COX-2 promoter. We also analysed glucose-stimulated insulin secretion (GSIS) in INS-1 cells that overexpressed Elk-1. Our results demonstrate that Elk-1 efficiently upregulates COX-2 expression at least partly through directly binding to the −82/−69 region of COX-2 promoter. Overexpression of Elk-1 inhibits GSIS in INS-1 cells. These findings will be helpful for better understanding the transcriptional regulation of COX-2 in pancreatic β-cell. Moreover, Elk-1, the transcriptional regulator of COX-2 expression, will be a potential target for the prevention of β-cell dysfunction mediated by PGE2.

Human VRK2 (Vaccinia-related Kinase 2) Modulates Tumor Cell Invasion by Hyperactivation of NFAT1 and Expression of Cyclooxygenase-2

Human VRK2 (vaccinia-related kinase 2), a kinase that emerged late in evolution, affects different signaling pathways, and some carcinomas express high levels of VRK2. Invasion by cancer cells has been associated with NFAT1 (nuclear factor of activated T cells) activation and expression of the COX-2 (cyclooxygenase 2) gene. We hypothesized that VRK proteins might play a regulatory role in NFAT1 activation in tumor cells. We demonstrate that VRK2 directly interacts and phosphorylates NFAT1 in Ser-32 within its N-terminal transactivation domain. VRK2 increases NFAT1-dependent transcription by phosphorylation, and this effect is only detected following cell phorbol 12-myristate 13-acetate and ionomycin stimulation and calcineurin activation. This NFAT1 hyperactivation by VRK2 increases COX-2 gene expression through the proximal NFAT1 binding site in the COX-2 gene promoter. Furthermore, VRK2A down-regulation by RNA interference reduces COX-2 expression at transcriptional and protein levels. Therefore, VRK2 down-regulation reduces cell invasion by tumor cells, such as MDA-MB-231 and MDA-MB-435, upon stimulation with phorbol 12-myristate 13-acetate plus ionomycin. These findings identify the first reported target and function of human VRK2 as an active kinase playing a role in regulation of cancer cell invasion through the NFAT pathway and COX-2 expression.