Elevated Levels Of Cyclooxygenase-2 Research Articles

Cyclooxygenase-2 Blockade Is Crucial to Restore Natural Killer Cell Activity before Anti-CTLA-4 Therapy against High-Grade Serous Ovarian Cancer.

Chronic inflammation influences the tumor immune microenvironment (TIME) in high-grade serous ovarian cancer (HGSOC). Specifically, cyclooxygenase-2 (COX-2) overexpression promotes cytotoxic T-lymphocyte-associated protein-4 (CTLA-4) expression. Notably, elevated COX-2 levels in the TIME have been associated with reduced response to anti-CTLA-4 immunotherapy. However, the precise impact of COX-2, encoded by PTGS2, on the immune profile remains unknown. To address this, we performed an integrated bioinformatics analysis using data from the HGSOC cohorts (TCGA-OV, n = 368; Australian cohort AOCS, n = 80; GSE26193, n = 62; and GSE30161, n = 45). Employing Gene Set Variation Analysis (GSVA), MIXTURE and Ecotyper cell deconvolution algorithms, we concluded that COX-2 was linked to immune cell ecosystems associated with shorter survival, cell dysfunction and lower NK cell effector cytotoxicity capacity. Next, we validated these results by characterizing circulating NK cells from HGSOC patients through flow cytometry and cytotoxic assays while undergoing COX-2 and CTLA-4 blockade. The blockade of COX-2 improved the cytotoxic capacity of NK cells against HGSOC cell lines. Our findings underscore the relevance of COX-2 in shaping the TIME and suggest its potential as a prognostic indicator and therapeutic target. Increased COX-2 expression may hamper the effectivity of immunotherapies that require NK cell effector function. These results provide a foundation for experimental validation and clinical trials investigating combined therapies targeting COX-2 and CTLA-4 in HGSOC.

Glial-derived Neuroinflammation induced with Amyloid-beta-peptide Plus Fibrinogen Injection in Rat Hippocampus.

The present study has examined microglial and astrocyte activation in association with neuronal degeneration in an animal model using an injection of amyloid-beta peptide Aβ1-42 (Aβ42) plus fibrinogen into rat hippocampus. The combination of stimuli is suggested as a novel and potent perturbation to induce gliosis and the production of glial-derived neurotoxic factors in an animal model exhibiting a leaky BBB (blood-brain barrier). Specifically, Aβ42 + fibrinogen stimulation elevated levels of COX-2 (cyclooxygenase-2) and iNOS (inducible nitric oxide synthase) with a considerable extent of neuronal loss associated with microglia and astrocyte activation. Treatment of injected rats with the broad spectrum anti-inflammatory agent, minocycline or the iNOS inhibitor, 1400 W inhibited gliosis, reduced levels of COX-2 and iNOS, and demonstrated efficacy for neuroprotection. The findings suggest the utility of combining amyloid beta peptide plus fibrinogen as a potent and understudied neuroinflammatory stimulus for the induction of glial-derived neurotoxic factors in BBB-compromised AD brain.

Molecular Imaging of Inflammation in Osteoarthritis Using a Water-Soluble Fluorocoxib.

Clinical imaging approaches to detect inflammatory biomarkers, such as cyclooxygenase-2 (COX-2), may facilitate the diagnosis and therapy of inflammatory diseases. To this end, we report the discovery of N-[(rhodamin-X-yl)but-4-yl]-2-[1-(4-chlorobenzoyl)-5-methoxy-2-methyl-1H-indol-3-yl]acetamide chloride salt (fluorocoxib D), a hydrophilic analog of fluorocoxib A. Fluorocoxib D inhibits COX-2 selectively in purified enzyme preparations and cells. It exhibits adequate photophysical properties to enable detection of COX-2 in intact cells, in a mouse model of carrageenan-induced acute footpad inflammation and inflammation in a mouse model of osteoarthritis. COX-2-selectivity was verified either by blocking the enzyme's active site with celecoxib or by molecular imaging with nontargeted 5-carboxy-X-rhodamine dye. These data indicate that fluorocoxib D is an ideal candidate for early detection of inflammatory or neoplastic lesions expressing elevated levels of COX-2.

Cyclooxygenase-2 Induced the β-Amyloid Protein Deposition and Neuronal Apoptosis Via Upregulating the Synthesis of Prostaglandin E2 and 15-Deoxy-Δ12,14-prostaglandin J2.

Elevated levels of cyclooxygenase-2 (COX-2) and prostaglandins (PGs) have been shown to be involved in the pathogenesis of Alzheimer's disease. Analysis of the underlying mechanisms elucidated a function of sequential PGE2 and PGD2 synthesis in regulating β-amyloid protein (Aβ) deposition by modulating tumor necrosis factor α (TNF-α)-dependent presenilin (PS)1/2 activity in COX-2 and APP/PS1 crossed mice. Specifically, COX-2 overexpression accelerates the expression of microsomal PGE synthase-1 (mPGES-1) and lipocalin-type prostaglandin D synthase (L-PGDS), leading to the synthesis of PGE2 and 15-deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2) in 6-month-old APP/PS1 mice. Consequently, PGE2 has the ability to increase Aβ production by enhancing the expression of PS1/2 in a TNF-α-dependent manner, which accelerates the cognitive decline of COX-2/APP/PS1 mice. More interestingly, low concentrations of 15d-PGJ2 treatment facilitate the effects of PGE2 on the deposition of Aβ via TNF-α-dependent PS1/2 mechanisms. In contrast, high concentrations of 15d-PGJ2 treatment inhibit the deposition of Aβ via suppressing the expression of TNF-α-dependent PS1/2. In this regard, a high concentration of 15d-PGJ2 appears to be a therapeutic agent against Alzheimer's disease. However, the high 15d-PGJ2 concentration treatment induces neuronal apoptosis via increasing the protein levels of Bax, cleaved caspase-3, and DFF45, which further impairs the learning ability of APP/PS1 mice.

Subacute and subchronic oral toxicity assessments of Acridocarpus smeathmannii (DC.) Guill. & Perr. root in Wistar rats

Recent adverse herb reactions have stimulated interest documenting the safety profile of medicinal agents. Thus, subacute and subchronic oral toxicity of the hydroethanolic extract of Acridocarpus smeathmannii root (HEASR) in Wistar rats was investigated. In the 28 and 90-day subacute and subchronic toxicity tests, sixty-four rats (n = male: female = 1:1 = 32) were divided into four of eight/group and ninety-six (n = male: female = 1:1 = 48) into twelve/group respectively. Distilled water (10 mL/kg) or HEASR4, HEASR5 and HEASR6 (250, 500 and 1000 mg/kg/day) respectively were administered via oral gavage. Animals were killed humanely 24 h after the last administration. Using standard methods, acute oral toxicity dose of HEAR (2000 mg/kg) was non-lethal in rodents. Subacute administration of HEASR6 increased total bilirubin (p < 0.05) in female rats. HEASR moderately altered both haematological and biochemical indices in rats. HEASR6 administration reduced ovary weight in both studies while follicle stimulating hormone level in male was reduced at all doses used. HEASR modulated lipid peroxidation, sperm quality and elevated cyclooxygenase-2 levels in rats. Histology revealed gastritis and congestions in vital organs. The low-observed adverse effect level for HEASR was below 250 mg/kg for both sexes. Overall, HEASR demonstrated inherent toxicity evidenced by our current findings. The exaggeration of its folklore medicine applications calls for cautions.

Integrated communications between cyclooxygenase-2 and Alzheimer's disease.

Elevated levels of cyclooxygenase-2 (COX-2) and prostaglandins (PGs) are involved in the pathogenesis of Alzheimer's disease (AD), which is characterized by the accumulation of β-amyloid protein (Aβ) and tau hyperphosphorylation. However, the gaps in our knowledge of the roles of COX-2 and PGs in AD have not been filled. Here, we summarized the literature showing that COX-2 dysregulation obviously influences abnormal cleavage of β-amyloid precursor protein, aggregation and deposition of Aβ in β-amyloid plaques and the inclusion of phosphorylated tau in neurofibrillary tangles. Neuroinflammation, oxidative stress, synaptic plasticity, neurotoxicity, autophagy, and apoptosis have been assessed to elucidate the mechanisms of COX-2 regulation of AD. Notably, an imbalance of these factors ultimately produces cognitive decline. The current review substantiates our understanding of the mechanisms of COX-2-induced AD and establishes foundations for the design of feasible therapeutic strategies to treat AD.-Guan, P.-P., Wang, P. Integrated communications between cyclooxygenase-2 and Alzheimer's disease.

Effect of uric acid on inflammatory COX-2 and ROS pathways in vascular smooth muscle cells

Hyperuricemia is thought to play a role in cardiovascular diseases (CVD), including hypertension, coronary artery disease and atherosclerosis. However, exactly how uric acid contributes to these pathologies is unknown. An underlying mechanism of inflammatory diseases, such as atherosclerosis, includes enhanced production of cyclooxygenase-2 (COX-2) and superoxide anion. Here, we aimed to examine the effect of uric acid on inflammatory COX-2 and superoxide anion production and to determine the role of losartan. Primarily cultured vascular smooth muscle cells (VSMCs) were time and dose-dependently induced by uric acid and COX-2 and superoxide anion levels were measured. COX-2 levels were determined by ELISA, and superoxide anion was measured by the superoxide dismutase (SOD)-inhibitable reduction of ferricytochrome c method. Uric acid elevated COX-2 levels in a time-dependent manner. Angiotensin-II receptor blocker, losartan, diminished uric-acid-induced COX-2 elevation. Uric acid also increased superoxide anion level in VSMCs. Uric acid plays an important role in CVD pathogenesis by inducing inflammatory COX-2 and ROS pathways. This is the first study demonstrating losartan’s ability to reduce uric-acid-induced COX-2 elevation.

Upregulation of Cyclooxygenase-2/Prostaglandin E2 (COX-2/PGE2) Pathway Member Multiple Drug Resistance-Associated Protein 4 (MRP4) and Downregulation of Prostaglandin Transporter (PGT) and 15-Prostaglandin Dehydrogenase (15-PGDH) in Triple-Negative Breast Cancer.

Elevated levels of cyclooxygenase-2 (COX-2) and prostaglandin E2 (PGE2) are indicators of a poor prognosis in breast cancer. Using several independent publicly available breast cancer gene expression databases, we investigated other members of the PGE2 pathway. PGE2 is produced by COX-2 and actively exported by multiple drug resistance-associated protein 4 (MRP4) into the extracellular microenvironment, where PGE2 can bind four cognate EP receptors (EP1–EP4) and initiate diverse biological signaling pathways. Alternatively, PGE2 is imported via the prostaglandin transporter (PGT) and metabolized by 15-prostaglandin dehydrogenase (15-PGDH/HPGD). We made the novel observation that MRP4, PGT, and 15-PGDH are differentially expressed among distinct breast cancer molecular subtypes; this finding was confirmed in independent datasets. In triple-negative breast cancer, the observed gene expression pattern (high COX-2, high MRP4, low PGT, and low 15-PGDH) would favor high levels of tumor-promoting PGE2 in the tumor microenvironment that may contribute to the overall poor prognosis of triple-negative breast cancer.

Esophageal COX-2 expression is increased in Barrett's esophagus, obesity, and smoking.

Increased esophageal cyclooxygenase-2 (COX-2) expression has been associated with Barrett's esophagus (BE); however, it is unknown whether COX-2 expression varies among patient groups with different clinical or socio-demographic factors. We conducted a case-control study among eligible patients scheduled for elective esophagogastroduodenoscopy and patients eligible for screening colonoscopy recruited from primary clinics. We compared 39 BE tissue samples and 47 squamous tissue samples from BE cases and 240 squamous tissue samples from controls. Clinical and socio-demographic data were prospectively collected. Immunohistochemical staining for esophageal COX-2 was performed and scored. The median COX-2 score was significantly higher in BE tissue than squamous tissue from cases or controls (p < 0.001). Median COX-2 expression levels were higher in tissue samples from participants with a waist-to-hip ratio (WHR) in the 2nd tertile [unadjusted odds ratio (OR) 2.04; 95 % confidence interval (95 % CI) 1.17-3.57] and 3rd tertile (unadjusted OR 2.24; 95 % CI 1.20-4.16) compared with the 1st tertile and from current smokers compared with former or non-smokers (unadjusted OR 1.68; 95 % CI 1.03-2.75). In the multivariate analysis, WHRs in the 2nd tertile (OR 1.92; 95 % CI 1.07-3.45) and the 3rd tertile (OR 2.14; 95 % CI 1.10-4.16) were associated with high COX-2 compared with the 1st tertile, as was current smoking (OR 1.78; 95 % CI 1.06-2.97) compared with former and non-smoking. We found a significant association between elevated esophageal mucosa COX-2 levels and the presence of BE tissue, as well as between elevated COX-2 levels and high WHR and current tobacco smoking. This information may assist in identifying patients likely to benefit from chemoprevention with COX-2 inhibitors.

Underwater trauma causes a long-term specific increase in the expression of cyclooxygenase-2 in the ventral CA1 of the hippocampus

The pro-inflammatory enzyme cyclooxygenase-2 (COX-2) is regularly expressed in the hippocampal neurons, but its role in emotional trauma is not known. Here we show that a single acute stress caused by a near-drowning experience results in heightened anxiety-like behavior one month after the trauma. Biochemical analyses of dorsal and ventral hippocampal CA1, CA3 and dentate gyrus revealed decreased ubiquitination and elevated levels of COX-2 in the traumatized animals only in the ventral CA1. To reveal the identity of the ubiquitin E3 ligase that targets COX-2, we tested the effect of several representative E3 ligases on COX-2 expression in vitro. We found that while AIP4 and Nedd4 had no effect, Mdm2 lowered COX-2 expression by nearly 50%, an effect that was not observed by its dominant negative form. To test whether this also occurs in the hippocampus, we immunoprecipitated Mdm2 from dorsal and ventral CA1 of traumatized and control animals and probed for the presence of COX-2. Our results showed that the levels of Mdm2 were not affected by the trauma but there was significantly less COX-2 associated with Mdm2 in the ventral but not dorsal CA1 of the traumatized animals. Together these data propose that an increase in COX-2 expression in ventral CA1 following trauma is likely due to its attenuated degradation. Unraveling the pathways and mechanisms that control hippocampal COX-2 degradation is important to boost the development of novel therapeutic approaches designed to treat stress-related pathologies.

Abstract LB-154: COX-2 as a mediator of oncogenic PKCε in prostate cancer.

Abstract Protein kinase C epsilon (PKCε), a member of the PKC family of phorbol ester/diacylglycerol receptors, is up-regulated in many human cancers, including prostate cancer. We recently demonstrated that PKCε is an essential mediator of NF-κB activation in prostate cancer (Garg et al., JBC, 287 (44), 37570–37582, 2012). Cyclooxygenase-2 (COX-2), a well-known NF-κB regulated gene, has been reported to be up-regulated in a number of human cancers, including metastatic prostate cancer. As PKCε plays an important role in prostate cancer cell survival and cooperates with other oncogenic insults, herein we aim to determine if PKCε regulates COX-2 activation during prostate tumorigenesis. PKCε RNAi depletion diminishes TNFα- or LPS-induced COX-2 mRNA expression in LNCaP prostate cancer cells and constitutive COX-2 levels from PC3 cells. Conversely, PKCε overexpression by adenoviral means potentiates TNFα or LPS-induced COX-2 expression in LNCaP cells. Notably, transgenic overexpression of PKCε in the mouse prostate causes preneoplastic lesions with elevated COX-2 levels. Interestingly, when we intercrossed the prostate-specific PKCε transgenic mice with mice haploinsufficient for Pten, a common genetic alteration in human prostate cancer, the resulting compound mutant mice (PB-PKCε;Pten+/− mice) developed fully invasive adenocarcinoma with NF-κB hyperactivation and high COX-2 levels. Likewise, stable overexpression of PKCε in mouse prostate epithelial cell lines that are either heterozygous (P8) or homozygous (CaP8) for Pten deletion, led to significant enhancements in cell proliferation, motility and invasiveness as well as in LPS-induced COX-2 mRNA expression compared to the respective control cells, and this effect is more pronounced in CaP8 cells. Studies using human prostate tumors revealed a co-existence of PKCε overexpression, NF-κB hyperactivation, and COX-2 up-regulation. Lastly, treatment of PKCε overexpressing P8 or CaP8 cells with the selective COX-2 inhibitor NS398 caused a pronounced growth inhibition. Overall, our study identified COX-2 as a PKCε-regulated gene. Our results suggest that COX-2 as a potential mediator of PKCε oncogenesis in prostate cancer, particularly in the context of Pten loss. Citation Format: Rachana Garg, Priti Lal, Jorge Blando, Fernando J. Benavides, Michael D. Feldman, Emer M. Smyth, Marcelo G. Kazanietz. COX-2 as a mediator of oncogenic PKCε in prostate cancer. [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 LB-154. doi:10.1158/1538-7445.AM2013-LB-154

Regulation of COX2 Expression in Mouse Mammary Tumor Cells Controls Bone Metastasis and PGE2-Induction of Regulatory T Cell Migration

BackgroundThe targeting of the immune system through immunotherapies to prevent tumor tolerance and immune suppression are at the front lines of breast cancer treatment and research. Human and laboratory studies have attributed breast cancer progression and metastasis to secondary organs such as the bone, to a number of factors, including elevated levels of prostaglandin E2 (PGE2) and the enzyme responsible for its production, cyclooxygenase 2 (COX2). Due to the strong connection of COX2 with immune function, we focused on understanding how variance in COX2 expression manipulates the immune profile in a syngeneic, and immune-competent, mouse model of breast cancer. Though there have been correlative findings linking elevated levels of COX2 and Tregs in other cancer models, we sought to elucidate the mechanisms by which these immuno-suppressive cells are recruited to breast tumor and the means by which they promote tumor tolerance.Methodology/Principal FindingsTo elucidate the mechanisms by which exacerbated COX2 expression potentiates metastasis we genetically manipulated non-metastatic mammary tumor cells (TM40D) to over-express COX2 (TM40D-COX2). Over-expression of COX2 in this mouse breast cancer model resulted in an increase in bone metastasis (an observation that was ablated following suppression of COX2 expression) in addition to an exacerbated Treg recruitment in the primary tumor. Interestingly, other immune-suppressive leukocytes, such as myeloid derived suppressor cells, were not altered in the primary tumor or the circulation. Elevated levels of PGE2 by tumor cells can directly recruit CD4+CD25+ cells through interactions with their EP2 and/or EP4 receptors, an effect that was blocked using anti-PGE2 antibody. Furthermore, increased Treg recruitment to the primary tumor contributed to the greater levels of apoptotic CD8+ T cells in the TM40D-COX2 tumors.Conclusion/SignificanceDue to the systemic effects of COX2 inhibitors, we propose targeting specific EP receptors as therapeutic interventions to breast cancer progression.

Enhancement of Anti-Inflammatory Activity of PEP-1-FK506 Binding Protein by Silk Fibroin Peptide

Silk fibroin (SF) peptide has been traditionally used as a treatment for flatulence, spasms, and phlegm. In this study, we examined whether SF peptide enhanced the antiinflammatory effect of PEP-1-FK506 binding protein (PEP-1-FK506BP) through comparing the anti-inflammatory activities of SF peptide and/or PEP-1-FK506BP. In the presence or absence of SF peptide, transduction levels of PEP-1-FK506BP into HaCaT cells and mice skin and anti-inflammatory activities of PEP-1-FK506BP were identified by Western blot and histological analyses. SF peptide alone effectively reduced both mice ear edema and the elevated levels of cyclooxygenase-2, interleukin-6 and -1beta, and tumor necrosis factor-alpha, showing similar anti-inflammatory effect to that of PEP-1-FK506BP. Furthermore, co-treatment with SF peptide and PEP-1- FK506BP exhibited more enhanced anti-inflammatory effects than the samples treated with SF peptides or PEP- 1-FK506BP alone, suggesting the possibility that SF peptide and PEP-1-FK506BP might interact with each other. Moreover, the transduction data demonstrated that SF peptide did not affect the transduction of PEP-1- FK506BP into HaCaT cells and mice skin, indicating that the improvement of anti-inflammatory effect of PEP-1- FK506BP was not caused by enhanced transduction of PEP-1-FK506BP. Thus, these results suggest the possibility that co-treatment with SF peptide and PEP-1-FK506BP may be exploited as a useful therapy for various inflammationrelated diseases.

Arachidonoyl ethanolamide (AEA)‐induced apoptosis is mediated by J‐series prostaglandins and is enhanced by fatty acid amide hydrolase (FAAH) blockade

The endocannabinoid arachidonoyl ethanolamide (AEA) is a potent inducer of tumor cell apoptosis however its mechanism of cytotoxicity is unclear. A previous report from our laboratory showed that AEA induced cell death in a cyclooxygenase-2 (COX-2)-dependent manner and in this report our data indicate that AEA-induced apoptosis is mediated by COX-2 metabolic products of the J-series. In experiments conducted with JWF2 keratinocytes which over-express COX-2, AEA caused a concentration-regulated increase in J-series prostaglandin production and apoptosis. Similarly, cell treatment with exogenously added J-series prostaglandins (15-deoxy, Δ(12,14) PGJ(2) and PGJ(2)) induced apoptosis. AEA-induced apoptosis was inhibited by the antioxidant, N-acetyl cysteine, indicating that reactive oxygen species generation was required for apoptosis. Using antagonists of cannabinoid receptor 1, cannabinoid receptor 2, or transient receptor potential cation channel, subfamily V, member 1, it was observed that cannabinoid receptor inhibition did not block AEA-mediated cell death. In contrast, an inhibitor of fatty acid amide hydrolase (FAAH) potentiated AEA-induced J-series PG synthesis and apoptosis. These results suggest that the metabolism of AEA to J-series PGs regulates the induction of apoptosis in cells with elevated COX-2 levels. Our data further indicate that the proapoptotic activity of AEA can be enhanced by combining it with an inhibitor of FAAH. As such, AEA may be an effective agent to eliminate tumor cells that over-express COX-2.

Shear-induced Interleukin-6 Synthesis in Chondrocytes: ROLES OF E PROSTANOID (EP) 2 AND EP3 IN cAMP/PROTEIN KINASE A- AND PI3-K/Akt-DEPENDENT NF-κB ACTIVATION

Mechanical overloading of cartilage producing hydrostatic stress, tensile strain, and fluid flow can adversely affect chondrocyte function and precipitate osteoarthritis (OA). Application of high fluid shear stress to chondrocytes recapitulates the earmarks of OA, as evidenced by the release of pro-inflammatory mediators, matrix degradation, and chondrocyte apoptosis. Elevated levels of cyclooxygenase-2 (COX-2), prostaglandin (PG) E(2), and interleukin (IL)-6 have been reported in OA cartilage in vivo, and in shear-activated chondrocytes in vitro. Although PGE(2) positively regulates IL-6 synthesis in chondrocytes, the underlying signaling pathway of shear-induced IL-6 expression remains unknown. Using the human T/C-28a2 chondrocyte cell line as a model system, we demonstrate that COX-2-derived PGE(2) signals via up-regulation of E prostanoid (EP) 2 and down-regulation of EP3 receptors to raise intracellular cAMP, and activate protein kinase A (PKA) and phosphatidylinositol 3-kinase (PI3-K)/Akt pathways. PKA and PI3-K/Akt transactivate the NF-kappaB p65 subunit via phosphorylation at Ser-276 and Ser-536, respectively. Binding of p65 to the IL-6 promoter elicits IL-6 synthesis in sheared chondrocytes. Selective knockdown of EP2 or ectopic expression of EP3 blocks PKA- and PI3-K/Akt-dependent p65 activation and markedly diminishes shear-induced IL-6 expression. Similar inhibitory effects on IL-6 synthesis were observed by inhibiting PKA, PI3-K, or NF-kappaB using pharmacological and/or genetic interventions. Reconstructing the signaling network regulating shear-induced IL-6 expression in chondrocytes may provide insights for developing therapeutic strategies for arthritic disorders and for culturing artificial cartilage in bioreactors.

COX-2 expression in malignant melanoma: a novel prognostic marker?

Several studies illustrated considerably elevated levels of cyclooxygenase-2 (COX-2) protein in various types of human cancer including malignant melanoma. Recently, it was reported that COX-2 is strongly expressed in malignant melanoma and may be correlated with the development and progression of disease. In contrast, other groups did not detect COX-2 protein in primary melanoma cells but did in infiltrating inflammatory cells or metastases. However, there are no reports about patterns or alterations of COX-2 expression in melanoma cells during disease progression or of a correlation between COX-2 expression and overall survival. The aim of this study was to investigate whether there is a correlation between expression of COX-2 protein and disease prognosis in malignant melanoma. We therefore analyzed the expression of COX-2 protein by immunohistochemistry in 101 primary malignant melanomas and 28 metastases and correlated our data with Breslow tumor thickness, Clark levels, different melanoma subtypes, metastases, and overall survival. We detected a strong COX-2 expression in 95% of all primary melanomas, primarily restricted to melanoma cells as shown by various immunohistochemical methods. Levels of COX-2 expression in primary melanoma and corresponding metastases remained stable. A significant correlation between immunohistochemical staining intensity and tumor thickness was demonstrated. Furthermore, Kaplan-Meier curves illustrated a significant correlation between staining intensity and disease-specific survival. Our findings emphasize that the COX-2 protein might be a novel prognostic marker. Owing to its strong expression in melanoma cells it might also be a reasonable therapeutic target.

Snail Up-regulates Proinflammatory Mediators and Inhibits Differentiation in Oral Keratinocytes

The transcriptional repressor Snail2 is overexpressed in head and neck squamous cell carcinomas (HNSCC) relative to nonmalignant head and neck mucosal epithelium, and in locally recurrent relative to nonrecurrent HNSCCs. We investigated the mechanisms by which Snails might contribute to the pathogenesis of HNSCCs using cell biological and molecular analyses. Oral keratinocytes that expressed Snails acquired an enhanced ability to attract monocytes and to invade a dense interstitial collagen matrix. They were also found to up-regulate production of proinflammatory cytokines and cyclooxygenase-2 (COX2), which have previously been shown to correlate with malignancy. Induction of nuclear factor-kappaB transcriptional activity by Snails was weak and not sufficient to account for the elevated levels of COX2, interleukin (IL)-6, IL8, or CXCL1. In addition, expression of Snails in oral keratinocytes impaired desquamation in vitro and strongly repressed expression of both ELF3 and matriptase-1, which play important roles in the terminal differentiation of keratinocytes. Reexpression of matriptase-1 in Snail-expressing cells partially rescued desquamation. This implicates Snails as contributing to malignancy both at the early stages, by impeding terminal differentiation, and at later stages, when invasion and inflammation are important.

Bioindicators of Immune Function in Creosote-adapted Estuarine Killifish, Fundulus heteroclitus

Several populations of the estuarine killifish, Fundulus heteroclitus, also known as the mummichog, exhibit characteristics of adaptation to priority pollutants. One such population of mummichog inhabits the Elizabeth River in Virginia at the Atlantic Wood site (AW), a U.S. Environmental Protection Agency (EPA) Superfund site heavily contaminated with creosote containing a mixture of polyaromatic hydrocarbons (PAHs). Although PAHs are known to be immunotoxic in experimental animals, resident AW mummichogs seem to thrive. Mummichogs from the AW site and a reference site were subsequently examined over a 2-yr period for total immunoglobin (IgM), as well as circulating antibody levels against 5 ubiquitous marine bacteria. Expression profiles of circulating and lymphoid lysozyme and lymphoid cyclooxygenase-2 (COX-2) were also examined. Compared to relatively high total IgM and specific antibody responses in reference fish, AW mummichogs had lower circulating IgM and lower specific antibody levels against all bacteria examined, however they had higher levels of circulating lysozyme. Lymphoid cells in the AW mummichogs also expressed higher levels of lysozyme, as well as COX-2, which may indicate a state of macrophage activation. Elevated COX-2 levels may be associated with enhanced metabolism of PAHs through cooxidation-peroxidase pathways. Additional studies attempted to immunize AW mummichogs reared in uncontaminated water to compare their antibody responses to that of reference fish. AW mummichogs did not survive 40 d post culture, while reference fish thrived. Our findings suggest that the chemical environment at the AW site may be vicariously enhancing components of innate immunity, probably through oxidative stress pathways, in resident mummichogs, while actively suppressing humoral immune responses.

Jaceosidin, a Pharmacologically Active Flavone Derived from Artemisia argyi, Inhibits Phorbol‐Ester‐Induced Upregulation of COX‐2 and MMP‐9 by Blocking Phosphorylation of ERK‐1 and ‐2 in Cultured Human Mammary Epithelial Cells

Elevated levels of cyclooxygenase-2 (COX-2) and matrix metalloproteinases (MMPs) are frequently found in various types of cancerous and transformed cells, with recent studies implicating the upregulation of COX-2 and MMPs in the development of breast cancer. This article investigated the effects of jaceosidin (4',5,7-trihydroxy-3',6-dimethoxyflavone) isolated from Artemisia argyi on the upregulation of COX-2 and MMP-9 induced by the tumor promotor 12-O-tetradecanoylphorbol-13-acetate (TPA) in MCF10A human breast epithelial cells (MCF10A cells). Treatment of MCF10A cells with TPA induced the upregulation of COX-2 and MMP-9, and this was attenuated by jaceosidin treatment. Jaceosidin also inhibited the invasive and migrative phenotypes of MCF10A cells induced by TPA. Furthermore, jaceosidin blocked the TPA-induced phosphorylation of extracellular signal-regulated protein kinase-1 and -2 (ERK-1 and -2), which is one of the signaling molecules regulating COX-2 and MMP. These results suggest that jaceosidin inhibits the TPA-induced upregulation of COX-2 and MMP-9 by blocking ERK-1 and -2 phosphorylation in human breast epithelial cells, which may be indicative of its chemopreventive potential.

Curcumin Inhibits Phorbol Ester-Induced Up-Regulation of Cyclooxygenase-2 and Matrix Metalloproteinase-9 by Blocking ERK1/2 Phosphorylation and NF-κB Transcriptional Activity in MCF10A Human Breast Epithelial Cells

Elevated levels of cyclooxygenase-2 (COX-2) and matrix metalloproteinases (MMPs) are often observed in various types of cancerous and transformed cells, and hence recognized as potential molecular targets for the chemoprevention. In the present study, we investigated the possible inhibitory effects of curcumin on the expression of COX-2 and MMP-9 induced by the tumor promoter 12-O-tetradecanoylphorbol 13-acetate (TPA) in MCF10A human breast epithelial (MCF10A) cells and the underlying mechanisms. Curcumin inhibited the TPA-induced COX-2 expression at both transcriptional and post-transcriptional levels, and reduced the synthesis of prostaglandin E(2), one of the major products of COX-2. Likewise, curcumin attenuated invasiveness and motility of MCF10A cells stimulated with TPA through suppression of MMP expression. Curcumin blocked TPA-induced activation of extracellular signal-regulated protein kinase (ERK1/2) and nuclear factor kappaB (NF-kappaB) transcriptional activity. Overexpression of the dominant negative forms of ERK2 abrogated the TPA-induced NF-kappaB transcriptional activity. Treatment of MCF10A cells with U0126, which is a pharmacological inhibitor of ERK1/2, reduced TPA-induced up-regulation of COX-2 and MMP-9. Taken together, these findings suggest that curcumin inhibits the TPA-induced up-regulation of COX-2 and MMP-9 by suppressing ERK1/2 phosphorylation and NF-kappaB trans-activation in human breast epithelial cells, which may contribute to its chemopreventive potential.