Inhibition Of Cyclooxygenase-2 Expression Research Articles

Anti-inflammatory and anti-nociceptive properties of Prunus padus

Ethnopharmacological relevancePrunus padus Linne has been widely used as a traditional medicine, with beneficial effects in numerous diseases, including stroke, neuralgia and hepatitis. In this study, we demonstrated anti-inflammatory and anti-nociceptive activities of the methylene chloride fraction of P. padus (MPP). Materials and methodsIn vitro studies, the anti-inflammatory effects of MPP were examined using IFN-γ/LPS-activated murine peritoneal macrophage model. To confirm the anti-inflammatory effects of MPP in vivo, trypsin-induced paw edema test was also conducted. The anti-nociceptive activities of MPP were measured using various experimental pain models including thermal nociception methods such as the tail immersion test and the hot plate test as well as chemical nociception methods like acetic acid-induced writhing test and formalin test. To determine whether analgesic activity of MPP is connected with the opioid receptor, we carried out combination test with naloxone, a nonselective opioid receptor antagonist. ResultsIn the current study, MPP showed potent inhibitory effect on IFN-γ/LPS-induced NO production. MPP also suppressed not only iNOS enzyme activity but also iNOS expression. Moreover, MPP inhibited COX-2 expression dose dependently. IFN-γ/LPS stimulation induced the translocation of NF-κB to nucleus but it was attenuated in the presence of MPP. In vivo study revealed that MPP could reduce paw volume after subplantar injection of trypsin. In addition, MPP showed potent analgesic activities both thermal and chemical nociception compared to tramadol and indomethacin. Furthermore, pre-treatment of naloxone slightly suppress the analgesic activity of MPP indicating that MPP acts as a partial opioid receptor agonist. ConclusionsIn the present study, MPP showed potent anti-inflammatory properties through not only by suppressing various inflammatory mediators in vitro, but reducing the inflammatory edema in vivo. MPP also exhibited strong anti-nociceptive activities via both central and peripheral mechanism by acting as a partial opioid agonist. Based on these results we suggest that P. padus has the potential to provide a therapeutic approach to inflammation-mediated chronic diseases as an effective anti-inflammatory agent and painkiller.

Inibição da expressão de ciclooxigenase 2 em feridas cutâneas de camundongos NOD submetidos à terapia a laser de baixa intensidade

CONTEXTO: A terapia a laser de baixa intensidade (LLLT) tem sido relatada como importante moduladora da cicatrização de feridas cutâneas aumentando a proliferação fibroblástica associada ao aumento da expressão da citocina fator transformador de crescimento- β2 (TGF-βB2). OBJETIVO: No presente estudo foram avaliados os efeitos da LLLT sobre a expressão da enzima ciclooxigenase 2 (COX2) no sítio do reparo tecidual utilizando o modelo experimental com camundongos diabéticos não obesos (NOD) para estudar a cicatrização de feridas cutâneas. MÉTODOS: Foram utilizados 30 camundongos NOD, destes 14 ficaram diabéticos e foram divididos em dois grupos: o grupo I (n=7) foi submetido a um procedimento cirúrgico de feridas cutâneas e o grupo II (n=7) foi submetido a um procedimento cirúrgico de feridas cutâneas e tratados com LLLT. O grupo II foi submetido à LLLT nos seguintes parâmetros: 15 mW de potência, dose de 3,8 J/cm² e tempo de aplicação de 20 segundos. Após sete dias do ato cirúrgico e após aplicação do laser, os animais foram eutanasiados com sobredose de anestesia e amostras das feridas foram colhidas para posterior análise histopatológica, histomorfométrica e imuno-histoquímica. RESULTADOS: A LLLT promoveu a inibição da expressão da COX2 em feridas cutâneas de camundongos diabéticos. CONCLUSÃO: Em conjunto, os resultados sugeriram que a LLLT é capaz de modular negativamente a expressão da enzima COX2 contribuindo para o controle da resposta inflamatória em feridas cutâneas de camundongos NOD.

Baicalin improved the spatial learning ability of global ischemia/reperfusion rats by reducing hippocampal apoptosis

Objective: The pathophysiological mechanism of global ischemia damage is complex. Recent studies indicate that the excessive inflammatory response induced by cerebral ischemia/reperfusion plays an important role in ischemic damage. Baicalin (5,6-dihydroxy-7-O-glucuronide flavonoid glycosides) is extracted from the dry dicotyledonous skullcap root, and belongs to one of the flavonoid compounds. Baicalin has a neuroprotective effect in a variety of brain injury animal models, although the mechanism remains unclear. We suggest that baicalin prevents deterioration in rats’ spatial learning abilities associated with acute global cerebral ischemia by inhibiting the inflammatory reaction and reducing apoptosis. Methods: Forty-two Sprague Dawley rats were divided into three groups: 14 rats in a sham (S) group; 14 in a global cerebral ischemia/reperfusion (I/R) group; and 14 in a global cerebral ischemia/reperfusion+baicalin treatment (I/RB) group. A Morris water maze test was used to assess learning and memory, HE staining was conducted for pathomorphology, a terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay was used to determine neuronal apoptosis, and Western blot for cyclooxygenase-2 (COX-2) expression in the hippocampus CA1 region. Results: Compared to the I/R group, Baicalin improved the learning and memory of I/RB rats. There was decreased hippocampal apoptosis and a reduction in the level of COX-2 expression in the I/RB group. In addition, the pathomorphological changes were worse in the I/R than in the I/RB group. Conclusion: Baicalin improved the detrimental effects on spatial memory associated with global cerebral ischemia by reducing hippocampal apoptosis via the inhibition of COX-2 expression.

Clausenamide improves COX-2 expression in diabetic rats

This study aims to investigate the effects of clausenamide on streptozotocin (STZ)-induced diabetes in rats. The diabetic rat model was established via an intraperitoneal STZ injection. The rats were randomly divided into five groups, the normal control group, the model group, the insulin treatment group, the high-dose clausenamide treatment group, and the low-dose clausenamide treatment group. Three months after drug administration, reverse transcription polymerase chain reaction (RT-PCR) and immunohistochemistry were carried out to measure the Cyclooxygenase-2 (COX-2) expression in the hippocampus of the rats in each group. The RT-PCR results indicate that the hippocampal COX-2 mRNA levels significantly increased in diabetic rats compared with those in the other groups. However, the hippocampal mRNA COX-2 levels in the clausenamide treatment groups significantly decreased, which indicates an inhibitory role of clausenamide on COX-2 mRNA in the diabetic rats. The immunohistochemistry results demonstrate that COX-2 expression in the hippocampus of the diabetic rats significantly increased, which suggests that an abnormal COX-2 expression is involved in the pathogenesis of learning and memory deficiencies, and that clausenamide exerts a protective role by inhibiting COX-2 expression in diabetic rats. Clausenamide has several protective effects on the brain injury induced by long-term diabetes, and significantly reduces diabetes-related learning and memory deficiency. The mechanism correlated well with the inhibition of COX-2 expression in the hippocampus. Key words: Clausenamide, diabetes, hippocampus, cycloxygenase-2 (COX-2).

한약의 다양한 항암기전

ABSTRACT Objectives : Recently there have been encouraging results, from a western perspective, in the cancer research field regarding the anticancer effects of herbal medicine. This paper was aimed to review herbal medicine playing its anticancer role in terms of apoptosis, inflammation control, differentiation and telomerase.Methods : New studies for tang, medicinal herb itself or effective ingr adients of medicinal herb showing anti-cancer effectiveness were reviewed and summarized in terms of pharmacological action.Results : Ethanol extracts of Spatholobus suberectus greatly inhibited cancer cell growth inducing cell apoptosis and cytotoxic effects. Scutellaria baicalensis may be responsible for its anticancer activity showing inhibit ion of PGE 2 synthesis via suppression of COX-2 expression. Saikosaponins i solated from Bupleurum induced the differentiation of C6 glioma cells, cancer cells, into astrocyt es, normal cells. Acetone extract of Bupleurum scorzonerifolium inhibited proliferation of human lung cancer cells via inducing apoptosis and suppressing telomerase activity.Conclusions : Herbal medicine inhibited cancer cell growth inducing cell ap optosis and cytotoxic effects. Inflammation persisting for a decade eventually elevates the ri sk of cancer sufficiently that it is discernible in case control epidemiological studies. Differentiation therapy i s defined as a therapy to treat cancers by inducing differentiation of the stem cells. Telomerase expression is a h allmark of cancer. Nearly the complete spectrum of human tumors has been shown to be telomerase positive.Key words : Korean traditional medicines ; anti-cancer; apoptosis; infla mmation; differentiation therapy

Oroxylin A improves the sensitivity of HT-29 human colon cancer cells to 5-FU through modulation of the COX-2 signaling pathway

5-Fluorouracil (5-FU) is a principal drug for the treatment of colorectal cancer. Due to its low response and high toxicity, synergistic effects of 5-FU in combination with other drugs have been widely researched. This study investigated whether oroxylin A improved the sensitivity of HT-29 human colon cancer cells to 5-FU. A correlation between COX-2 inhibition by oroxylin A and a synergistic effect of 5-FU on the growth of HT-29 cells was observed, and a COX-2 pathway for this effect was recognized; oroxylin A evidently elevated the level of reactive oxygen species in HT-29 cells, which subsequently inhibited COX-2 expression and enhanced the susceptibility of HT-29 cells to 5-FU. Likely also related to COX-2 inhibition, oroxylin A decreased PGE(2) levels in HT-29 cells. The synergistic effect of 5-FU induced by oroxylin A was also found in the suppression of Bcl-2 and in the activation of P53, Bax, PARP, and procaspase-3 proteins in HT-29 cells. Ultimately, a combination of 5-FU with oroxylin A significantly reduced the growth of HT-29 tumors in nude mice compared with treatment with 5-FU or oroxylin A alone. In conclusion, a combination of 5-FU and oroxylin A has a significant synergistic effect in the inhibition of HT-29 cell proliferation in vitro and controls HT-29 tumor growth in vivo. This synergistic effect may be mainly related to COX-2 inhibition by oroxylin A in HT-29 cells.

Additive Effects of Ulinastatin and Docetaxel on Growth of Breast Cancer Xenograft in Nude Mice and Expression of PGE2, IL-10, and IL-2 in Primary Breast Cancer Cells

Ulinastatin is a broad-spectrum enzyme inhibitor extracted from urine. Previous data from our group suggested that ulinastatin could significantly inhibit proliferation of human breast MDA-MB-231 cells, growth of tumor xenograft in nude mice, and expression of interleukin (IL)-6 and IL-8. In the present study, we investigated whether there is an additive effect of ulinastatin and docetaxel on growth of breast cancer xenografts in nude mice and its possible mechanisms. Nude mice and primary human breast cancer cells were treated with phosphate buffered saline (PBS), ulinastatin, docetaxel, or ulinastatin plus docetaxel, respectively. Their effects on xenograft growth; expressions of cyclooxygenase-2 (COX2), prostaglandin E2 receptor 2 (EP2), IL-10, and IL-2; and secretion of prostaglandin E2 (PGE2) were examined using variety of methods, including semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR), Western blot, enzyme-linked immunosorbent (ELISA) assay, and immunohistochemistry SP method. The treatment with ulinastatin, docetaxel, or ulinastatin plus docetaxel could significantly (1) inhibit COX2 and IL-10 expression in primary tumor cells at both mRNA and protein levels, (2) reduce PGE2 secretion in culture supernatant (p<0.05), (3) inhibit COX2, EP2, and IL-10 protein levels in primary xenograft of nude mice, and (4) increase IL-2 expression (p<0.05) in primary xenografts of nude mice. In addition, ulinastatin and docetaxel had additive effects. We suggest that ulinastatin had similar effects of docetaxel and can enhance docetaxel's anticancer effects possibly by inhibiting COX2 expression, reducing PGE2 and EP2 expression and their binding, upregulating IL-2, and downregulating IL-10.

Abstract 2949: miR-16 is an in vivo suppressor of gastrointestinal tumorigenesis

Abstract Commonly observed in colorectal cancer (CRC) is elevated expression of the prostaglandin synthase cyclooxygenase-2 (COX-2). In normal intestinal epithelium, the COX-2 mRNA is normally targeted for rapid decay through RNA elements present within its 3′-untranslated region (3′UTR), whereas in tumors rapid mRNA decay is compromised. To understand the mechanisms controlling COX-2 mRNA fate, we show that the COX-2 3′UTR can mediate mRNA degradation through microRNA-mediated regulation. MicroRNAs (miRNAs) are small non-coding RNAs that bind within the 3′UTR of target mRNAs and function as post-transcriptional regulators of gene expression. MiR-16 was identified to bind the COX-2 3′UTR and inhibit COX-2 expression by promoting rapid mRNA decay. Given the importance of miR-16 in cancer development, we examined miR-16 expression in CRC tumor specimens and cancer cells and observed a 2-fold decrease in miR-16 levels. Additionally, expression of miR-16 in cancer cells attenuated COX-2 expression and prostaglandin synthesis. The in vivo role of miR-16 in the initiation and development of colorectal cancer was examined using ApcMin/+ mice, a mouse model of intestinal neoplasia. To accomplish this, endogenous miR-16 was inhibited in the GI tract using an anti-miR oligonucleotide (AMO) against miR-16 containing 2′-O-(2-Methoxyethyl) modification with unmodified PO backbone. 50 mg/kg of AMO-16 or control AMO containing 4 nucleotide-mismatch of the AMO miR-16 sequence were administered by oral gavage to 8 week old ApcMin/+ and C57Bl/6 control mice twice a week over a 3-week period. Specific inhibition of miR-16 in intestinal tissue was observed. Over the treatment course, control mice displayed normal growth characteristics. Gross and histological analysis did not reveal any morphological changes in the GI tract of AMO-16-treated C57Bl/6 mice. In contrast, a significant 2-fold increase in small intestinal tumor burden was observed in ApcMin/+ mice treated with AMO-16 compared to control AMO (55.7 +/− 5.0 vs. 99.1 +/− 7.9, P = 0.0001). Gene expression analysis showed increased COX-2 and several miR-16 target cell cycle regulatory genes including cyclin D1, cyclin D3, cyclin E1, and CDK6 in AMO-16-treated small intestinal tissue from ApcMin/+ mice compared with AMO control treated mice, with the greatest level of altered expression observed in the proximal portion of the small intestine. These results identify miR-16 as a central post-transcriptional regulator of COX-2 along with other transcripts associated with promoting cancer cell proliferation, and reveal miR-16 to have tumor-suppressor properties in GI tumorigenesis downstream of a tumor-initiating event. 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 2949. doi:1538-7445.AM2012-2949

Abstract 4098: COX-2 expression in human pancreatic cancer cells is associated with an epithelial phenotype

Abstract Epithelial to mesenchymal transition is a developmental pathway that has been shown to have a direct role in tumor progression and drug resistance. Increasing evidence suggests that EMT (and MET) leads to global changes in chromatin architecture that modifies expression of numerous genes. Interestingly, reports in the literature indicate a possible association between cyclooxygenase-2 (COX-2) expression and EMT. COX-2 is upregulated in response to inflammation, and is known to be overexpressed in multiple malignancies, including pancreatic cancer. As a critical mediator of prostaglandin production, COX-2 has proangiogenic properties associated with increased tumor growth and invasion, and is also thought to play a role in the carcinogenic process. In this study, we report that COX-2 expression is associated with an epithelial phenotype among a panel of human pancreatic cancer cells. Using chromatin immunoprecipitation (ChIP), we demonstrate increased acetylated H3 at the COX-2 promoter region in the epithelial cell lines versus mesenchymal cell lines. Treatment with a Type-I selective HDAC inhibitor SNDX-275 increased levels of acetylation at the COX-2 promoter region in mesenchymal cell lines and subsequently promoted COX-2 gene expression. Overall, these data point to an epigenetic suppression of COX-2 expression in mesenchymal pancreatic cancer cells that can be reversed by Type-I HDAC inhibitors. Our results suggest that COX2 inhibitors will preferentially target “epithelial” pancreatic cancers and that combinations of Cox2 and HDAC inhibitors may display increased anti-tumor activity. 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 4098. doi:1538-7445.AM2012-4098

Abstract 4663: The cyclooxygenase-2 inhibitor, celecoxib, suppresses proliferation and induces apoptosis in endometrial cancer cells

Abstract INTRODUCTION: Obesity is associated with increased risk and worse outcomes for women with endometrial cancer. Inflammation may play a critical role in obesity-driven cancers; thus, the anti-inflammatory effects of COX-2 inhibitors may be a novel treatment strategy that is particularly beneficial in endometrial cancer patients. As a result, our objective was to evaluate the effect of COX-2 inhibitors on proliferation and apoptosis in endometrial cancer cell lines. METHODS: Two endometrial cancer cell lines (ECC-1 and Ishikawa) were used in these studies. Cell proliferation was assessed by MTT assay after exposure to the COX-2 inhibitor, celecoxib. Cell cycle progression was evaluated by flow cytometry. Apoptosis was assessed by ELISA for caspase-3. hTERT mRNA expression was assessed by real-time PCR. Western immunoblotting was performed to determine the effect of celecoxib on COX-2 expression in the endometrial cancer cell lines. RESULTS: Celecoxib significantly inhibited proliferation in a dose-dependent manner in both endometrial cancer cell lines (IC50 5-10 μM for ECC-1, 10-20 μM for Ishikawa; p&amp;lt;0.005 for each). Treatment with celecoxib resulted in G1 cell cycle arrest, induction of apoptosis and inhibition of hTERT mRNA expression. Western immunoblot analysis demonstrated that celecoxib treatment suppresses COX-2 expression in both endometrial cancer cell lines. CONCLUSION: Celecoxib potently inhibits cell growth via G1 arrest, decreases telomerase activity and increases apoptotic cell death in endometrial cancer cells. This work suggests that celecoxib may be a novel chemotherapeutic agent for endometrial cancer prevention and treatment, especially given the interplay between obesity, inflammation and cancer risk. 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 4663. doi:1538-7445.AM2012-4663

Abstract 3083: Dual-specificity phosphatase-2 (DUSP2) negatively control cyclooxygenase-2 (COX-2) expression in cancer cells

Abstract Cyclooxygenase-2 (COX-2), an inducible form of cyclooxygenase, plays a crucial role in the development of cancer via its enzymatic product, prostaglandin E2 (PGE2). Clinical studies reveal that COX-2 is overexpressed in numerous types of cancers, and is associated with increase of angiogenesis, metastasis, and drug resistance. Although nonsteroidal anti-inflammatory drugs, COX-2 inhibitors, have been used for cancer therapy; they have serious side effects in many aspects. Therefore, to explore the underlying mechanism of COX-2 overexpression during cancer progression is critical for developing more efficient and safer way to block COX-2 function. Previously, we have found that the expression of dual-specificity phosphatase-2 (DUSP2), a MAPK-specific phosphatase, is markedly reduced in many kinds of cancers. Here, we identified that COX-2 was a potential downstream target gene of DUSP2. In vitro studies demonstrate that knockdown of DUSP2 significantly increases COX-2 expression and PGE2 production while overexpression of DUSP2 reduces those in cancer cells. Xenografted mouse study further reveals that levels of DUSP2 are reduced during cancer development, which is inversely correlated with COX-2 expression. Re-expression of DUSP2 during tumor progression inhibits COX-2 expression and suppresses tumor growth while knockdown of DUSP2 increases COX-2 expression and enhances tumor growth. These results implied that COX-2 overexpression was due to loss of DUSP2 expression in cancer. Furthermore, we found that hypoxia represses DUSP2 expression resulting in COX-2 overexpression. Taken together, we have demonstrated for the first time that aberrant expression of COX-2 during tumor development is regulated by hypoxia-mediated DUSP2 downregulation. Our data also suggest that DUSP2 is a good molecular target for cancer therapy. 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 3083. doi:1538-7445.AM2012-3083

CFTR negatively regulates cyclooxygenase‐2‐PGE2 positive feedback loop in inflammation

Cystic fibrosis (CF) is an autosomal recessive disorder caused by mutations of the cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP-dependent anion channel mostly expressed in epithelia. Accumulating evidence suggests that CF airway epithelia are overwhelmed by excessive inflammatory cytokines and prostaglandins (PGs), which eventually lead to the over-inflammatory condition observed in CF lung disease. However, the exact underlying mechanism remains elusive. In this study, we observed increased cyclooxygenase-2 (COX-2) expression and over-production of prostaglandin E(2) (PGE(2)) in human CF bronchial epithelia cell line (CFBE41o--) with elevated NF-κB activity compared to a wild-type airway epithelial cell line (16HBE14o--). Moreover, we demonstrated that CFTR knockout mice had inherently higher levels of COX-2 and NF-κB activity, supporting the notion that lack of CFTR results in hyper-inflammatory signaling. In addition, we identified a positive feedback loop for production of PGE(2) involving PKA and transcription factor, CREB. More importantly, overexpression of wild-type CFTR significantly suppressed COX-2 expression in CFBE41o- cells, and wild-type CFTR protein expression was significantly increased when 16HBE14o-- cells were challenged with LPS as well as PGE(2), indicating possible involvement of CFTR in negative regulation of COX-2/PGE(2). In conclusion, CFTR is a negative regulator of PGE(2)-mediated inflammatory response, defect of which may result in excessive activation of NF-κB, leading to over production of PGE(2) as seen in inflammatory CF tissues.

Effect of propofol on cyclooxygenase-2 expression in hippocampal neurons in depressed rats after electroconvulsive therapy

Objective To investigate the effect of propofol on cyclooxygenase-2 (COX-2) expression in hippocampal neurons in depressed rats after electroconvulsive therapy (ECT).Methods Fifty 2-3 months old male SD rats weighing 200-250 g were randomly divided into 5 groups (n =10 each):group control (group C) ; group depression (group D); group propofol (group P); group ECT (group E) and group propofol + ECT (group PE).Depression was induced by separation and chronic unpredictable mild stres in groups D,P,E and PE.Groups P and PE received intraperitoneal pro pofol 80 mg/kg.Groups E and PE received ECT at 5 min after IP normal saline 8 ml/kg and propofol 80 mg/kg respectively once a day for 7 consecutive days.The learning and memory function was assessed by using Morris water maze test before (baseline) and after depression was induced and ECT.The animals were then sacrificed and their brains removed for detection of COX-2 mRNA expression in hippocampus (by RT-PCR).Results In group D depression significantly prolonged evasive latency and decreased swimming time percentage in platform quadrant and up-regulated COX-2 mRNA expression as compared with group C.In group E ECT further prolonged evasive latency and up-regulated COX-2 mRNA expression in depressed rats.In group PE propofol pretreatment attenuated ECT-induced impairment of learning-memory function and increase in COX-2 mRNA expression as compared with group E.Conclusion Propofol can ameliorate the decrease in learning and memory function induced by ECT in depressed rats by inhibiting COX-2 expression in hippocampal neurons. Key words: Propofol; Depressive disorder; Electroconvulsive therapy; Cyclooxygenase 2; Hippocampus

Simultaneous modulation of COX‐2, p300, Akt, and Apaf‐1 signaling by melatonin to inhibit proliferation and induce apoptosis in breast cancer cells

Melatonin exhibits anti-inflammatory and anticancer effects and could be a chemopreventive and chemotherapeutic agent against cancers, but the precise mechanisms involved remain largely unresolved. In this study, we evaluated the mechanism of action of melatonin in human MDA-MB-361 breast cancer cells. Melatonin at pharmacological concentrations (10(-3) m) significantly suppressed cell proliferation and induced apoptosis in a dose-dependent manner. The observed suppression of proliferation was accompanied by the melatonin-mediated inhibition of COX-2, p300, and NF-κB signaling. Melatonin significantly inhibited COX-2 expression and prostaglandin E(2) (PGE2) production, abrogated p300 histone acetyltransferase activity and p300-mediated NF-κB acetylation, thereby blocking NF-κB binding and p300 recruitment to COX-2 promoter. Pretreatment with a COX-2- or p300-selective inhibitor abrogated the melatonin-induced inhibition of cell proliferation, whereas PGE2 treatment or COX-2 transfection reversed the inhibition by melatonin. Moreover, melatonin markedly inhibited phosphorylation of PI3K, Akt, PRAS40, and GSK-3 proteins, thereby inactivating the PI3K/Akt signaling pathway. Pretreatment with a PI3K- or an Akt-selective inhibitor or an Akt-specific siRNA blocked the melatonin-mediated inhibition of cell proliferation. Conversely, gene delivery of a constitutively active Akt effectively reversed the inhibition by melatonin. Furthermore, melatonin induced Apaf-1 expression, triggered cytochrome C release, and stimulated caspase-3 and caspase-9 activities and cleavage, leading to an activation of the Apaf-1-dependent apoptotic pathway. Pretreatment with an Apaf-1-specific siRNA effectively attenuated the melatonin-induced apoptosis. These results therefore indicate that melatonin inhibits cell proliferation and induces apoptosis in MDA-MB-361 breast cancer cells in vitro by simultaneously suppressing the COX-2/PGE2, p300/NF-κB, and PI3K/Akt/signaling and activating the Apaf-1/caspase-dependent apoptotic pathway.

Anti‐inflammatory effects of selective glucocorticoid receptor modulators are partially dependent on up‐regulation of dual specificity phosphatase 1

It is thought that the anti-inflammatory effects of glucocorticoids (GCs) are largely due to GC receptor (GR)-mediated transrepression of NF-κB and other transcription factors, whereas side effects are caused by activation of gene expression (transactivation). Selective GR modulators (SGRMs) that preferentially promote transrepression should retain anti-inflammatory properties whilst causing fewer side effects. Contradicting this model, we found that anti-inflammatory effects of the classical GC dexamethasone were partly dependent on transactivation of the dual specificity phosphatase 1 (DUSP1) gene. We wished to determine whether anti-inflammatory effects of SGRMs are also mediated by DUSP1. Dissociated properties of two SGRMs were confirmed using GR- and NF-κB-dependent reporters, and capacity to activate GC-responsive elements of the DUSP1 gene was tested. Effects of SGRMs on the expression of DUSP1 and pro-inflammatory gene products were assessed in various cell lines and in primary murine Dusp1(+/+) and Dusp1(-/-) macrophages. The SGRMs were able to up-regulate DUSP1 in several cell types, and this response correlated with the ability of the compounds to suppress COX-2 expression. Several anti-inflammatory effects of SGRMs were ablated or significantly impaired in Dusp1(-/-) macrophages. Like dexamethasone, SGRMs appear to exert anti-inflammatory effects partly via the up-regulation of DUSP1. This finding has implications for how potentially therapeutic novel GR ligands are identified and assessed.

P300 Exerts an Epigenetic Role in Chronic Neuropathic Pain through its Acetyltransferase Activity in Rats following Chronic Constriction Injury (CCI)

BackgroundNeuropathic pain is detrimental to human health; however, its pathogenesis still remains largely unknown. Overexpression of pain-associated genes and increased nociceptive somato-sensitivity are well observed in neuropathic pain. The importance of epigenetic mechanisms in regulating the expression of pro- or anti-nociceptive genes has been revealed by studies recently, and we hypothesize that the transcriptional coactivator and the histone acetyltransferase E1A binding protein p300 (p300), as a part of the epigenetic mechanisms of gene regulation, may be involved in the pathogenesis of neuropathic pain induced by chronic constriction injury (CCI). To test this hypothesis, two different approaches were used in this study: (I) down-regulating p300 with specific small hairpin RNA (shRNA) and (II) chemical inhibition of p300 acetyltransferase activity by a small molecule inhibitor, C646.ResultsUsing the CCI rat model, we found that the p300 expression was increased in the lumbar spinal cord on day 14 after CCI. The treatment with intrathecal p300 shRNA reversed CCI-induced mechanical allodynia and thermal hyperalgesia, and suppressed the expression of cyclooxygenase-2 (COX-2), a neuropathic pain-associated factor. Furthermore, C646, an inhibitor of p300 acetyltransferase, also attenuated mechanical allodynia and thermal hyperalgesia, accompanied by a suppressed COX-2 expression, in the spinal cord.ConclusionsThe results suggest that, through its acetyltransferase activity in the spinal cord after CCI, p300 epigenetically plays an important role in neuropathic pain. Inhibiting p300, using interfering RNA or C646, may be a promising approach to the development of new neuropathic pain therapies.

The mRNA Stability Factor HuR Inhibits MicroRNA-16 Targeting of COX-2

Commonly observed in colorectal cancer is the elevated expression of the prostaglandin (PG) synthase COX-2. In normal intestinal epithelium, the COX-2 mRNA is targeted for rapid decay through the 3'-untranslated region (3'-UTR) adenylate- and uridylate (AU)-rich element (ARE), whereas in tumors ARE-mediated decay is compromised. Here we show that the COX-2 ARE can mediate degradation through microRNA (miRNA)-mediated regulation. We identified miR-16 to bind the COX-2 3'-UTR and inhibit COX-2 expression by promoting rapid mRNA decay. In colorectal cancer cells and tumors, miR-16 levels were decreased approximately twofold and miR-16 expression in cancer cells attenuated COX-2 expression and PG synthesis. The COX-2 ARE is also bound by the RNA-binding protein HuR. In colorectal cancer tumors, HuR is overexpressed and localized within the cytoplasm, where it promotes ARE-mRNA stabilization. Under conditions of HuR overexpression, miR-16 was unable to promote rapid mRNA decay through the COX-2 ARE. Ribonucleoprotein immunoprecipitation of HuR showed direct association with miR-16 that was reversed when cytoplasmic trafficking of HuR was inhibited. Furthermore, this interaction between HuR and miR-16 promoted the downregulation of miR-16. These new results identify miR-16 as a central posttranscriptional regulator of COX-2 and show the ability of elevated levels of HuR to antagonize miR-16 function. Along with insight into altered ARE-mediated mRNA decay observed in colorectal cancer, these findings provide a new explanation for tumor-derived loss of miR-16.

Phenethyl Isothiocyanate가 Toll-like Receptor Agonists에 의해 유도된 Nuclear Factor-κB 활성과 Cyclooxygenase-2, Inducible Nitric Oxide Synthase 발현에 미치는 효과

염증의 중요한 분자학적 기전에는 cyclooxygenase-2 (COX-2)에 의한 prostaglandins (PGs) 생성과 inducible nitric oxide synthase (iNOS)에 의한 nitric oxide (NO) 생성이 있다. 많은 종류의 박테리아나 바이러스가 전사요소인 nuclear factor-<TEX>${\kappa}$</TEX>B(NF-<TEX>${\kappa}$</TEX>B)를 활성화시켜 여러 타깃 유전자의 발현을 조절해 PGs나 NO와 같은 염증물질을 유도하게 된다. 우리는 이번 실험을 통하여 phenethyl isothiocyanate (PEITC)가 toll-like receptor(TLR) agonists에 의해 유도된 NF-<TEX>${\kappa}$</TEX>B활성과 COX-2, iNOS 발현에 어떠한 영향을 미치는지 알아 보았다. PEITC는 lipopolysaccharide (LPS)와 polyinosinic-polycytidylic acid (poly[I:C])에 의해 유도된 NF-<TEX>${\kappa}$</TEX>B활성을 억제시켰다. 또한 PEITC는 LPS와 Poly[I:C]에 의해 유도된 iNOS의 발현도 억제시켰다. 하지만 PEITC는 TLR agonists들인 LPS, Poly[I:C], 2 kDa macrophage-activating lipopeptide (MALP-2), oligodeoxynucleotide 1668 (ODN1668)에 의한 COX-2 발현은 억제시키지 못하였다. 즉 PEITC가 TRIF-dependent 신호전달체계만을 조절하여 TRIF-dependent 신호전달체계에 의해 조절되는 iNOS는 억제하지만 MyD88-dependent 신호전달 체계에 의해 조절되는 COX-2는 억제하지 못한다는 것을 설명해준다. 이러한 결과는 iNOS와 COX-2가 서로 다른 메커니즘에 의해 조절된다는 것을 암시하며, PEITC가 여러 병원균들로부터 유도되는 염증반응이나 만성적인 질병들을 조절할 수 있음을 제시하는 중요한 결과이다. Toll-like receptors (TLRs) play an important role in induction of innate immune responses. The activation of TLRs triggers inflammatory responses that are essential for host defense against invading pathogens. Phenethyl isothiocyanate (PEITC) extracted from cruciferous vegetables has an effect on anti-inflammatory therapy. Dysregulated activation of nuclear factor-<TEX>${\kappa}$</TEX>B (NF-<TEX>${\kappa}$</TEX>B), inducible nitric oxide synthase (iNOS), and cyclooxygenase-2 (COX-2) has been shown to play important roles in the development of certain inflammatory disease. To evaluate the therapeutic potential of PEITC, NF-<TEX>${\kappa}$</TEX>B activation and COX-2 and iNOS expression induced by lipopolysaccharide (LPS, TLR4 agonist), polyinosinic-polycytidylic acid (Poly[I:C], TLR3 agonist), 2 kDa macrophageactivating lipopeptide (MALP-2, TLR2 and TLR6 agonist) or oligodeoxynucleotide 1668 (ODN1668, TLR9 agonist) were examined. PEITC inhibits the activation of NF-<TEX>${\kappa}$</TEX>B induced by LPS or Poly[I:C] but not by MALP-2 or ODN1668. PEITC also suppressed the iNOS expression induced by LPS or Poly[I:C]. However, PEITC did not suppress COX-2 expression induced by LPS, Poly[I:C], MALP-2, or ODN1668. These results suggest that PEITC has the specific mechanism for antiinflammatory responses.

Triptolide inhibits COX-2 expression by regulating mRNA stability in TNF-α-treated A549 cells

Cyclooxygenase-2 (COX-2) over-expression is frequently associated with human non-small-cell lung cancer (NSCLC) and involved in tumor proliferation, invasion, angiogenesis and resistance to apoptosis. In the present study, the effects of triptolide on COX-2 expression in A549 cells were investigated and triptolide was found to inhibit TNF-α-induced COX-2 expression. In our further studies, it was found that triptolide decreased the half-life of COX-2 mRNA dramatically and that it inhibited 3′-untranslated region (3′-UTR) fluorescence reporter gene activity. Meanwhile, triptolide inhibited the HuR shuttling from nucleus to cytoplasm. After triptolide treatment, decreased COX-2 mRNA in pull-down experiments with anti-HuR antibodies was observed, indicating that the decreased cytoplasmic HuR is responsible for the decreased COX-2 mRNA. Taken together, our results provided evidence for the first time that triptolide inhibited COX-2 expression by COX-2 mRNA stability modulation and post-transcriptional regulation. These results provide a novel mechanism of action for triptolide which may be important in the treatment of lung cancer.

NS398 induces apoptosis in non-small cell lung cancer cells

Non-steroidal anti-inflammatory drugs (NSAIDs) can induce the apoptosis of many tumor cells and inhibit their growth. NS398 is an NSAID that inhibits COX-2 expression and induces tumor apoptosis via other pathways. The current study aims to observe the effects of NS398 on A549 cell apoptosis and investigate the apoptosis mechanism. The A549 cells were treated with different NS398 concentrations. The growth inhibition of A549 cell was analyzed via a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, and morphologic alterations were observed to detect apoptosis. The expression of survivin and caspase-3 mRNA was quantified via reverse transcriptase polymerase chain reaction, and the expression of caspase-3 and p-AKT protein was detected via western blot analysis. The MTT results show that NS398 inhibits A549 cell growth. The inhibition rate of NS398 (400 μmol/L) on A549 cells is up to 66.95% after 48 h of treatment. Simultaneously, the morphology experiment revealed significant apoptotic characteristics in A549 cells, such as green nuclear plasmid and different degrees of nuclear fragmentation. The expression of survivin mRNA was significantly reduced (P < 0.05, P < 0.01, and P < 0.001) and that of caspase-3 mRNA was significantly increased (P < 0.05 and P < 0.001) in the group treated with NS398 for 24 h in a dose-dependent manner. On the other hand, survivin and p-AKT were expressed at low levels (P < 0.01 and P < 0.001) and caspase-3 was increased significantly (P < 0.05 and P < 0.001) in the group treated with NS398 for 48 h in a dose-dependent manner. The current study proves that NS398 induces apoptosis in A549 cells, thereby inhibiting tumor growth. This function of NS398 may be related to the inhibition of AKT phosphorylation and survivin protein downregulation.