Transcriptional Regulation Of Cyclooxygenase-2 Research Articles

Abstract 5103: Crosstalk between osteoprotegerin (OPG), fatty acid synthase (FASN), and cyclooxygenase-2 (COX-2) in breast cancer: implications in carcinogenesis

Abstract The cross-talk between malignant and nonmalignant cells in the tumor microenvironment, as maneuvered by cytokines/chemokines, drives breast cancer progression. We demonstrated that OPG is expressed and secreted at very high levels from the highly invasive breast cancer cell lines SUM149PT and SUM1315MO2 as compared to human normal mammary epithelial HMEC cells (1). OPG was involved in modulating aneuploidy, cell proliferation, and angiogenesis in breast cancer (1). Mass spectrometry analysis performed in this study revealed OPG interacts with fatty acid synthase (FASN), which is a key enzyme of the fatty acid biosynthetic pathway in breast cancer cells. Further, electron microscopy, immunofluorescence, and fluorescence quantitation assay highlighted the presence of a large number of lipid bodies (lipid droplets) in SUM149PT and SUM1315MO2 cells in comparison to HMEC. We recently showed upregulation of COX-2 inflammatory pathway and its metabolite PGE2 secretion in SUM149PT and SUM1315MO2 breast cancer cells (2). Interestingly, human breast cancer tissue samples displayed high expression of OPG, PGE2 and FASN. Immunofluorescence staining exposed the co-existence of COX-2 and FASN in the lipid bodies of breast cancer cells. We reasoned that there might be cross-talk between OPG, FASN, and COX-2 that sustains the inflammatory pathways in breast cancer. Here, we identified cis-acting elements involved in the transcriptional regulation of COX-2 and FASN by recombinant human OPG (rhOPG). Treatment with FASN inhibitor C75 decreased the number of lipid body/cell, downregulated phosphorylation of ERK, GSK3β, and induced apoptosis by caspase-3 and caspapse-9 activation. Collectively, the novel biological cross-talk between OPG, FASN, and COX-2 advocates for combinatorial drug treatment to block these players of carcinogenesis as a promising therapeutic to target highly invasive breast cancer.

COX-2 is involved in ET-1-induced hypertrophy of neonatal rat cardiomyocytes: Role of NFATc3

Endothelin-1 (ET-1) is a critical molecule that involved in heart failure. It has been proved that ET-1 stimulation results in cardiac hypertrophy both in vitro and in vivo, but the mechanisms underlying remain largely unknown. In this study, we reported that cyclooxygenase-2 (COX-2) might be an important mediator of hypertrophic responses to ET-1 stimulation. In the cultured rat neonatal cardiomyocytes, ET-1 significantly upregulated the expression and activity of COX-2, which was accompanied by increase in cell surface area and BNP mRNA level. In contrast, ET-1-dependent cardiomyocyte hypertrophy was abolished by COX-2 selective inhibitors, NS-398 and celecoxib, or by COX-2 RNA interference, but the inhibitory effects could be diminished by pretreatment with PGE2. Furthermore, cyclosporin A (CsA) and knockdown of nuclear factor of activated T-cells c3 (NFATc3) inhibited the expression of COX-2 induced by ET-1, and NFATc3 could also bound to the -GGAAA- sequence in the promoter region of rat COX-2 gene, indicating that calcineurin/NFATc3 signaling participated in the transcriptional regulation of COX-2 following ET-1 treatment. These findings provided further insight into the roles of ET-1 in cardiac hypertrophy and suggested a potential therapeutic strategy against cardiac hypertrophy by inhibiting COX-2.

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.

Identification of PARP-1 as one of the transcription factors binding to the repressor element in the promoter region of COX-2

Cyclooxygenase-2 (COX-2) plays important roles in the development of many disease conditions, including pancreatic β-cell dysfunction. Although the processes involved in the transcriptional regulation of COX-2 are well documented, some key elements, especially inhibitory elements, are still unknown. In our previous study, we identified a novel repressor element located in promoter region of mouse COX-2. In this study, we isolated several DNA-binding proteins from NIT-1 cells via DNA affinity chromatography; the most prominent among these proteins was poly (ADP-ribose) polymerase-1 (PARP-1). In this study, gel-supershift assays and chromatin immunoprecipitation assays showed that PARP-1 can bind to the inhibitory element −655/−632 in the promoter region of mouse COX-2 both in vitro and in vivo. Furthermore, overexpression of PARP-1 significantly inhibited promoter activity and decreased COX-2 expression. Conversely, repression of PARP-1 by RNAi upregulated COX-2 expression. These data suggest that PARP-1 plays an important role in the regulation of COX-2 expression via binding to the inhibitory element. Collectively, our findings provide new important information on the transcriptional regulation of COX-2 in pancreatic β-cells.

Abstract 5432: Transcriptional regulation of cyclooxygenase-2 by histone deacetylase inhibitors in non-small cell lung cancer cells

Abstract Cyclooxygenases (COXs), particularly inducible COX-2 catalyze the synthesis of pro-proliferative and pro-angiogenic prostaglandin E2 (PGE2) and NAD+-dependent 15-hydroxyprostaglandin dehydrogenase (15-PGDH) is responsible for its metabolic inactivation. Down-regulation of 15-PGDH has been found in gastric, colorectal, breast and prostate cancers and is at least in part mediated by epigenetic silencing which we hypothesized also mediates 15-PGDH silencing observed in about half of all non-small cell lung cancers. Epigenetic strategies, in particular HDAC inhibitors have shown promise in combination with chemotherapy in the treatment of advanced non-small cell lung cancer. Therefore, we treated lung cancer cell lines with 5-aza-2′ deoxycytidine and/or the specific HDAC inhibitor, trichostatin A (TSA) in order to reverse epigenetic silencing and assess modulation of the Cox-2/15-PGDH axis. While 15-PGDH re-activation could be readily detected on the transcriptional level upon TSA but not DAC treatment, such changes could not be seen on the protein level questioning the functional significance of epigenetic regulation of 15-PGDH expression. However, TSA markedly induces COX-2 expression on both the transcriptional and translational level in all seven non-small cell lung cancer cell lines studied. The levels of COX-2 mRNA and protein were maximal at 24h after treatment with TSA and were also confirmed under hypoxic conditions better mimicking in vivo conditions. Five of these seven NSCLC cell lines indeed produced significantly more PGE2 than untreated cells did as determined by enzyme-linked immunosorbent assay under both normoxic and hypoxic conditions following TSA treatment, indicating that the induced COX-2 is functionally active. To confirm that PGE2 production was associated with the catalytic activity of COX-2, three NSCLC cell lines were selected and cultured in the presence of both TSA and indomethacin, a non-selective COX inhibitor and PGE2 levels were measured after 24 hours of co-treatment. Non-cytotoxic, 10uM concentration of indomethacin potently blocked PGE2 production induced by TSA, namely decreased PGE2 to the level of the control group in all three NSCLC cell lines examined. In conclusion, COX-2 is an inducible gene in NSCLC cells and appears to be transcriptionally regulated by a unique mechanism associated with histone acetylation. Histone deacetylase inhibitors (HDACi) are a new class of promising anti-tumor agents inhibiting cell proliferation and survival in tumor cells with very low toxicity toward normal cells. Induction of Cox-2 and as a result increased PGE2 production by HDAC inhibition could be an untoward side effect of clinical relevance. Our results suggest that combination therapy of TSA and indomethacin could be a potential strategy to overcome this undesirable effect of HDAC inhibitor therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5432.

Transcriptional Regulation of Cyclooxygenase-2 in Response to Proteasome Inhibitors Involves Reactive Oxygen Species-mediated Signaling Pathway and Recruitment of CCAAT/Enhancer-binding Protein δ and CREB-binding Protein

Inhibition of ubiquitin-proteasome pathway has been shown to be a promising strategy for the treatment of inflammation and cancer. Here, we show that proteasome inhibitors MG132, PSI-1, and lactacystin induce COX-2 expression via enhancing gene transcription rather than preventing protein degradation in the human alveolar NCI-H292 and A549, and gastric AGS epithelial cells. NF-IL6 and CRE, but not NF-kappaB elements on the COX-2 promoter were involved in the gene transcription event. The binding of CCAAT/enhancer binding protein (C/EBP)beta and C/EBPdelta to the CRE and NF-IL6 elements, as well as the recruitment of CBP and the enhancement of histone H3 and H4 acetylation on the COX-2 promoter was enhanced by MG132. However, it did not affect the total protein levels of C/EBPbeta and C/EBPdelta. MG132-induced DNA-binding activity of C/EBPdelta, but not C/EBPbeta was regulated by p38, PI3K, Src, and protein kinase C. Small interfering RNA of C/EBPdelta suppressed COX-2 expression, further strengthening the role of C/EBPdelta in COX-2 gene transcription. In addition, the generation of intracellular reactive oxygen species (ROS) in response to MG132 contributed to the activation of MAPKs and Akt. These findings reveal that the induction of COX-2 transcription induced by proteasome inhibitors requires ROS-dependent protein kinases activation and the subsequent recruitments of C/EBPdelta and CBP.

Selenomethionine regulates cyclooxygenase-2 (COX-2) expression through nuclear factor-kappa B (NF-kB) in colon cancer cells

Previously, we showed that selenomethionine (Se-Met) inhibits growth of colon cancer cells via suppressing COX-2 expression at both mRNA and protein level. However, the molecular mechanism by which Se-Met suppresses COX-2 expression remains to be elucidated. To this end, we transiently transfected HCA7 cells with different COX-2 promoter constructs followed by Se-Met treatment (90 µM) for 12 h. The results suggested the role of nuclear factor-kappa B (NF-kB) in transcriptional regulation of COX-2. We also observed complete inhibition of DNA binding activity of NF-kB in Se-Met (90 µM) treated HCA7 cells as shown by electrophoretic mobility shift assay (EMSA). Supershift assays with anti-p65 antibody identified p65 subunit in the protein complex. We further demonstrate dose-dependent inhibition of nuclear translocation of NF-kB/p65 in Se-Met treated HCA7 cells, which could explain the observed reduction in DNA binding of NF-kB/p65. These results suggest that Se-Met regulates COX-2 at transcriptional level by modulating the activity of NF-kB transcription factor.

Effect of flavonoids and Vitamin E on cyclooxygenase-2 (COX-2) transcription

Cyclooxygenase-2 (COX-2)-catalysed synthesis of prostaglandin E2 plays a key role in inflammation and its associated diseases, such as cancer and cardiovascular disease. There are numerous reports demonstrating that flavonoids inhibit COX-2 activity. However, transcriptional regulation of COX-2 can also be important. Nobiletin, amentoflavone, quercetin, quercetin penta-acetate, flavone, resveratrol, apigenin, chrysin, kaempferol, galangin, and genistein have been reported to modulate COX-2 transcription in a wide variety of systems. Here, we briefly review the literature on regulation of COX-2 transcription by flavonoids, and report some new preliminary data on Vitamin E and quercetin conjugates. Quercetin, quercetin 3-glucuronide, quercetin 3′-sulfate and 3′methylquercetin 3-glucuronide reduced COX-2 mRNA expression in both unstimulated and interleukin-1β stimulated colon cancer (Caco2) cells. Quercetin and quercetin 3′-sulfate, unlike quercetin 3-glucuronide and 3′methylquercetin 3-glucuronide, also inhibited COX-2 activity. In contrast, tocopherols (α-tocopherol, α-tocopherol acetate, and γ-tocopherol at 10 μM) did not affect COX-2 mRNA expression in unstimulated Caco2 cells. However, the tocopherols inhibited COX-2 activity showing that the tocopherols act post-transcriptionally on activity, whereas quercetin and some quercetin conjugates affect both the transcription and activity of COX-2. Flavonoid modulation of COX-2 transcription may therefore be an important mechanism in anti-carcinogenesis.

Dynamic Regulation of Cyclooxygenase-2 Promoter Activity by Isoforms of CCAAT/Enhancer-binding Proteins

To elucidate the mechanism by which isoforms of CCAAT/enhancer-binding proteins regulate cyclooxygenase-2 expression, we determined by a novel technique binding of six isoforms of this transactivator to two sequence-specific CCAAT/enhancer-binding protein (-132/-125) and cyclic AMP (-59/-53) regulatory elements in human foreskin fibroblasts treated with phorbol 12-myristate 13-acetate for 4 h. The delta isoform bound to these two elements at basal state, which was displaced by full-length as well as two truncated beta isoforms, a 41-kDa liver-enriched activating protein and a 16-kDa liver-enriched inhibitory protein, after phorbol ester stimulation. Kinetic analysis shows time-dependent changes in beta and delta binding that were concordant with time-dependent increase in cyclooxygenase-2 induction. Overexpression of the 16-kDa beta isoform blocked the promoter activity and protein level induced by phorbol ester. Paradoxically, it increased binding of beta isoforms to the sequence-specific promoter DNA but suppressed cyclooxygenase-2 promoter activation by p300 cotransfection. These findings provide new insight into the regulation of cyclooxygenase-2 promoter by an interplay between two opposite beta isoforms and p300 co-activator.

Cyclooxygenase-2 is a neuronal target gene of NF-κB

BackgroundNF-κB is implicated in gene regulation involved in neuronal survival, inflammmatory response and cancer. There are relatively few neuronal target genes of NF-κB characterized.ResultsWe have identified the neuronal cyclooxygenase-2 (COX-2) as a NF-κB target gene. In organotypic hippocampal slice cultures constitutive NF-κB activity was detected, which was correlated with high anti-COX-2 immunoreactivity. Aspirin a frequently used painkiller inhibits neuronal NF-κB activity in organotypic cultures resulting in a strong inhibition of the NF-κB target gene COX-2. Based on these findings, the transcriptional regulation of COX-2 by NF-κB was investigated. Transient transfections showed a significant increase of COX-2 promoter activity upon stimulation with PMA, an effect which could be obtained also by cotransfection of the NF-κB subunits p65 and p50. In the murine neuroblastoma cell line NB-4, which is characterized by constitutive NF-κB activity, COX-2 promoter activity could not be further increased with PMA or TNF. Constitutive promoter activity could be repressed upon cotransfection of the inhibitory subunit IκB-α. EMSA and mutational analysis conferred the regulatory NF-κB activity to the promoter distal κB-site in the human COX-2 promoter.ConclusionsNF-κB regulates neuronal COX-2 gene expression, and acts as an upstream target of Aspirin. This extends Aspirin's mode of action from a covalent modification of COX-2 to the upstream regulation of COX-2 gene expression in neurons.

Transcriptional regulation of cyclooxygenase-2 in the human microvascular endothelial cell line, HMEC-1: control by the combinatorial actions of AP2, NF-IL-6 and CRE elements.

Interleukin-1beta (IL-1) is a potent inducer of cyclooxygenase-2 (COX-2) and prostaglandin biosynthesis in many types of cells, yet little is known about the molecular mechanisms regulating IL-1 mediated prostanoid biosynthesis in the endothelium of the microvasculature. Therefore, we examined the cis- and trans-acting factors regulating IL-1-induced COX-2 expression in the human microvascular endothelial cell line, HMEC-1. IL-1 enhanced steady state levels of COX-2 protein and mRNA synthesis by approximately 2-fold which preceded a 2-fold increase in PGF(alpha) biosynthesis. Expression of a series of COX-2 promoter-luciferase constructs in IL-1 treated HMEC-1 cells revealed that the 'full length' (-1432/+59 bp) promoter was 10 times more active than the SV-40 promoter/enhancer and that it could be further activated by IL-1. Surprisingly however, all except for the shortest COX-2 promoter construct retained the ability to respond to IL-1 and luciferase activity driven by -191/+59 bp COX-2 promoter was as responsive to IL-1 as the full-length promoter. Moreover, site-directed promoter mutagenesis and electophoretic mobility shift assays (EMSA) indicate that the combinatorial actions of AP2, NF-IL6, and CRE elements are critical for both constitutive and IL-1-inducible COX-2 promoter activity. Understanding the mechanism(s) regulating COX-2 gene expression and prostaglandin biosynthesis in the microvasculature has important implications with regard to inflammation and angiogenesis in vivo.