High Levels Of COX-2 Research Articles

Nickel and aluminium mixture elicit memory impairment by activation of oxidative stress, COX-2, and diminution of AChE, BDNF and NGF levels in cerebral cortex and hippocampus of male albino rats

This study evaluated nickel and aluminium-induced neurotoxicity, as a binary metal mixture. Twenty-eight male Sprague Dawley albino rats were weight-matched and divided into four groups. Group 1 (control) received deionized water. Group 2 and 3 received Aluminium (1 mg/kg) and Nickel (0.2 mg/kg) respectively, while Group 4 received Ni and Al mixture HMM three times a week orally for 90 days. Barnes maze tests was performed. Rats were sacrificed under pentobarbital anaesthesia, cerebral cortex and hippocampus were separated, and metal levels were measured using Atomic Absorption Spectroscopy (AAS). Malondialdehyde (MDA), catalase (CAT), glutathione content (GSH), superoxide dismutase (SOD), glutathione peroxidase (GPx), Brain Derived Neurotrophic Factor (BDNF), Nerve growth factor NGF, cyclo-oxygenase COX-2 and Acetylcholinesterase (AChE) were assayed using ELISA kits. Ni/Al binary mixture exposed rats showed a shorter latency period (though not significant) of 3.21 ± 1.40 s in comparison to 3.77 ± 1.11 (Ni only) and 3.99 ± 1.16(Al only). Ni/Al mixture gp had the lowest levels of Mg in both the hippocampus and frontal cortex when compared with the individual metals. In the hippocampus Al only exposed rats significantly showed p < 0.05 higher iron and Ca levels in comparison to Ni/Al mixture. Al alone significantly showed p < 0.05 lower levels of Fe but higher Ca than the Ni/Al mixture group. Exposure to Al only showed lower levels of BDNF in comparison to Ni/Al combination, whereas Ni/Al mixture gp had lower levels of NGF in comparison to the individual metals in the hippocampus. In the frontal cortex Ni only, group showed significantly lower BDNF in comparison to Ni/Al mixture whereas the mixture showed significantly lower NGF when compared with Al only group. There were higher levels of COX-2 in the Ni/Al mixture than individual metal treated rats in both hippocampus and frontal cortex. AChE levels in the Ni/Al mixture group was higher than Ni or Al only gps in the hippocampus whereas in the frontal cortex, Ni/Al exposed rats showed significantly lower AChE levels in comparison to Al only group. Ni, Al and Ni/Al mixture exhibited memory impairment by activation of oxidative stress, COX-2, and diminution of AChE, BDNF and NGF levels in cerebral cortex and hippocampus. The BDNF-COX-2 AChE signalling pathway may be involved in the neurotoxicity of Ni and Al.

The Ketogenic Diet Reduces the Harmful Effects of Stress on Gut Mitochondrial Biogenesis in a Rat Model of Irritable Bowel Syndrome.

Functional alterations in irritable bowel syndrome have been associated with defects in bioenergetics and the mitochondrial network. Effects of high fat, adequate-protein, low carbohydrate ketogenic diet (KD) involve oxidative stress, inflammation, mitochondrial function, and biogenesis. The aim was to evaluate the KD efficacy in reducing the effects of stress on gut mitochondria. Newborn Wistar rats were exposed to maternal deprivation to induce IBS in adulthood. Intestinal inflammation (COX-2 and TRL-4); cellular redox status (SOD 1, SOD 2, PrxIII, mtDNA oxidatively modified purines); mitochondrial biogenesis (PPAR-γ, PGC-1α, COX-4, mtDNA content); and autophagy (Beclin-1, LC3 II) were evaluated in the colon of exposed rats fed with KD (IBD-KD) or standard diet (IBS-Std), and in unexposed controls (Ctrl). IBS-Std rats showed dysfunctional mitochondrial biogenesis (PPAR-γ, PGC-1α, COX-4, and mtDNA contents lower than in Ctrl) associated with inflammation and increased oxidative stress (higher levels of COX-2 and TLR-4, SOD 1, SOD 2, PrxIII, and oxidatively modified purines than in Ctrl). Loss of autophagy efficacy appeared from reduced levels of Beclin-1 and LC3 II. Feeding of animals with KD elicited compensatory mechanisms able to reduce inflammation, oxidative stress, restore mitochondrial function, and baseline autophagy, possibly via the upregulation of the PPAR-γ/PGC-1α axis.

2D Nanomaterial, Ti3C2 MXene-Based Sensor to Guide Lung Cancer Therapy and Management.

Major advances in cancer control can be greatly aided by early diagnosis and effective treatment in its pre-invasive state. Lung cancer (small cell and non-small cell) is a leading cause of cancer-related deaths among both men and women around the world. A lot of research attention has been directed toward diagnosing and treating lung cancer. A common method of lung cancer treatment is based on COX-2 (cyclooxygenase-2) inhibitors. This is because COX-2 is commonly overexpressed in lung cancer and also the abundance of its enzymatic product prostaglandin E2 (PGE2). Instead of using traditional COX-2 inhibitors to treat lung cancer, here, we introduce a new anti-cancer strategy recently developed for lung cancer treatment. It adopts more abundant omega-6 (ω-6) fatty acids such as dihomo-γ-linolenic acid (DGLA) in the daily diet and the commonly high levels of COX-2 expressed in lung cancer to promote the formation of 8-hydroxyoctanoic acid (8-HOA) through a new delta-5-desaturase (D5Di) inhibitor. The D5Di does not only limit the metabolic product, PGE2, but also promote the COX-2 catalyzed DGLA peroxidation to form 8-HOA, a novel anti-cancer free radical byproduct. Therefore, the measurement of the PGE2 and 8-HOA levels in cancer cells can be an effective method to treat lung cancer by providing in-time guidance. In this paper, we mainly report on a novel sensor, which is based on a newly developed functionalized nanomaterial, 2-dimensional nanosheets, or Ti3C2 MXene. The preliminary results have proven to sensitively, selectively, precisely, and effectively detect PGE2 and 8-HOA in A549 lung cancer cells. The capability of the sensor to detect trace level 8-HOA in A549 has been verified in comparison with the traditional gas chromatography–mass spectrometry (GC–MS) method. The sensing principle could be due to the unique structure and material property of Ti3C2 MXene: a multilayered structure and extremely large surface area, metallic conductivity, and ease and versatility in surface modification. All these make the Ti3C2 MXene-based sensor selectively adsorb 8-HOA molecules through effective charge transfer and lead to a measurable change in the conductivity of the material with a high signal-to-noise ratio and excellent sensitivity.

Discovery of Novel Pyridazine-Based Cyclooxygenase-2 Inhibitors with a Promising Gastric Safety Profile.

Cyclooxygenase-2 (COX-2) is implicated in the development of chronic inflammatory diseases. Recently, pyridazine derivatives have emerged as a novel prototype to develop COX-2 inhibitors. Accordingly, some pyridazine-based COX-2 inhibitors are reported herein. The reaction of aldehyde 3 and different hydrazines yielded the corresponding hydrazones. The hydrazones were further derivatized to the title compounds, which were assessed for COX-1 and COX-2 inhibitory action, gastric ulcerogenic effects, and lipid peroxidation properties. Molecular docking studies and determination of the physicochemical parameters were also carried out. The allocated structures of the reported compounds were coherent with their spectroscopic data. The compounds 9a (IC50 = 15.50 nM, 114.77%), 9b (IC50 = 17.50 nM, 101.65%), 12 (IC50 = 17.10 nM, 104.03%), 16b (IC50 = 16.90 nM, 105.26%), and 17 (IC50 = 17.70 nM, 100.5%) displayed better COX-2 inhibition than celecoxib (IC50 = 17.79 nM, 100%). These outcomes were harmonious with the molecular docking studies of 9a, 9b, 12, 16b, and 17. These compounds also displayed comparable onset and the duration of action concerning celecoxib and indomethacin in the in vivo studies. No ulcerogenic effects were observed for 9a and 12, whereas 9b, 16b, and 17 showed an insignificant ulcerogenic effect compared to celecoxib. The compounds 9a, 9b, 12, 16b, and 17 displayed a better lipid peroxidation profile than celecoxib and indomethacin. The compounds 9a (%ABS = 84.09), 9b (%ABS = 84.09), 12 (%ABS = 66.87), 16b (%ABS = 75.02), and 17 (%ABS = 81.42) also displayed appreciable calculated absorption compared to celecoxib (%ABS = 82.09). The compounds 9a, 9b, 11, 16b, and 17 have been recognized and postulated as non-ulcerogenic COX-2 inhibitors with promising physicochemical parameters and gastric safety profile. These compounds may be useful candidates to combat diseases caused by higher levels of COX-2.

Upregulation of Cytokines and Differentiation of Th17 and Treg by Dendritic Cells: Central Role of Prostaglandin E2 Induced by Mycobacterium bovis

Mycobacterium bovis (M. bovis) is a zoonotic pathogen that causes bovine and human tuberculosis. Dendritic cells play a critical role in initiating and regulating immune responses by promoting antigen-specific T-cell activation. Prostaglandin E2 (PGE2)-COX signaling is an important mediator of inflammation and immunity and might be involved in the pathogenesis of M. bovis infection. Therefore, this study aimed to reveal the character of PGE2 in the differentiation of naïve CD4+ T cells induced by infected dendritic cells (DCs). Murine bone marrow-derived DCs were pre-infected with M. bovis and its attenuated strain M. bovis bacillus Calmette-Guérin (BCG). Then, the infected DCs were co-cultured with naïve CD4+ T cells with or without the cyclooxygenase (COX) inhibitor indomethacin. Quantitative RT-PCR analysis and protein detection showed that PGE2/COX-2 signaling was activated, shown by the upregulation of PGE2 production as well as COX-2 and microsomal PGE2 synthase (mPGES1) transcription in DCs specifically induced by M. bovis and BCG infection. The further co-culture of infected DCs with naïve CD4+ T cells enhanced the generation of inflammatory cytokines IL-17 and IL-23, while indomethacin suppressed their production. Following this, the differentiation of regulatory T cells (Treg) and Th17 cell subsets was significantly induced by the infected DCs rather than uninfected DCs. Meanwhile, M. bovis infection stimulated significantly higher levels of IL-17 and IL-23 and the differentiation of Treg and Th17 cell subsets, while BCG infection led to higher levels of TNF-α and IL-12, but lower proportions of Treg and Th17 cells. In mice, M. bovis infection generated more bacterial load and severe abnormalities in spleens and lungs, as well as higher levels of COX-2, mPGE2 expression, Treg and Th17 cell subsets than BCG infection. In conclusion, PGE2/COX-2 signaling was activated in DCs by M. bovis infection and regulated differentiation of Treg and Th17 cell subsets through the crosstalk between DCs and naive T cells under the cytokine atmosphere of IL-17 and IL-23, which might contribute to M. bovis pathogenesis in mice.

Targeting Cyclooxygenase-2 in Pheochromocytoma and Paraganglioma: Focus on Genetic Background.

Cyclooxygenase 2 (COX-2) is a key enzyme of the tumorigenesis-inflammation interface and can be induced by hypoxia. A pseudohypoxic transcriptional signature characterizes pheochromocytomas and paragangliomas (PPGLs) of the cluster I, mainly represented by tumors with mutations in von Hippel–Lindau (VHL), endothelial PAS domain-containing protein 1 (EPAS1), or succinate dehydrogenase (SDH) subunit genes. The aim of this study was to investigate a possible association between underlying tumor driver mutations and COX-2 in PPGLs. COX-2 gene expression and immunoreactivity were examined in clinical specimens with documented mutations, as well as in spheroids and allografts derived from mouse pheochromocytoma (MPC) cells. COX-2 in vivo imaging was performed in allograft mice. We observed significantly higher COX-2 expression in cluster I, especially in VHL-mutant PPGLs, however, no specific association between COX-2 mRNA levels and a hypoxia-related transcriptional signature was found. COX-2 immunoreactivity was present in about 60% of clinical specimens as well as in MPC spheroids and allografts. A selective COX-2 tracer specifically accumulated in MPC allografts. This study demonstrates that, although pseudohypoxia is not the major determinant for high COX-2 levels in PPGLs, COX-2 is a relevant molecular target. This potentially allows for employing selective COX-2 inhibitors as targeted chemotherapeutic agents and radiosensitizers. Moreover, available models are suitable for preclinical testing of these treatments.

Fluorescent indomethacin-dansyl conjugates utilize the membrane-binding domain of cyclooxygenase-2 to block the opening to the active site

Many indomethacin amides and esters are cyclooxygenase-2 (COX-2)-selective inhibitors, providing a framework for the design of COX-2-targeted imaging and cancer chemotherapeutic agents. Although previous studies have suggested that the amide or ester moiety of these inhibitors binds in the lobby region, a spacious alcove within the enzyme's membrane-binding domain, structural details have been lacking. Here, we present observations on the crystal complexes of COX-2 with two indomethacin-dansyl conjugates (compounds 1 and 2) at 2.22-Å resolution. Both compounds are COX-2-selective inhibitors with IC50 values of 0.76 and 0.17 μm, respectively. Our results confirmed that the dansyl moiety is localized in and establishes hydrophobic interactions and several hydrogen bonds with the lobby of the membrane-binding domain. We noted that in both crystal structures, the linker tethering indomethacin to the dansyl moiety passes through the constriction at the mouth of the COX-2 active site, resulting in displacement and disorder of Arg-120, located at the opening to the active site. Both compounds exhibited higher inhibitory potency against a COX-2 R120A variant than against the WT enzyme. Inhibition kinetics of compound 2 were similar to those of the indomethacin parent compound against WT COX-2, and the R120A substitution reduced the time dependence of COX inhibition. These results provide a structural basis for the further design and optimization of conjugated COX reagents for imaging of malignant or inflammatory tissues containing high COX-2 levels.

Epinephrine promotes COX-2-dependent immune suppression in myeloid cells and cancer tissues

Activation of the sympathetic nervous system (e.g., due to stress) has been implicated in cancer progression and recurrence, but its cancer-promoting effects have been variable between different studies. Here, we report that although catecholamines, mediators of systemic sympathetic activity, display only weak immunosuppressive impact on their own, their combination with inflammatory signals leads to the induction of COX-2 and multiple COX-2-dependent suppressive factors in human myeloid cells and cancer tissues. Human macrophages exposed to epinephrine and TNFα, or macrophages generated in 6day cultures in the presence of epinephrine, expressed high levels of COX-2, IDO and IL-10, and strongly suppressed both the proliferation and IFNγ production of CD8+ T cells. These suppressive effects of epinephrine were counteracted by celecoxib, a selective inhibitor of COX-2 activity, which inhibited the induction of immunosuppressive factors (including the elevated expression of COX-2 itself) and the ability of epinephrine-exposed macrophages to suppress CD8+ T cell responses. The activation of the COX-2/PGE2 system and COX-2-dependent suppressive events were also observed in ex vivo human breast and colon cancer explant cultures and were similarly counteracted by celecoxib. Our preliminary data also indicate elevated COX-2 expression in mammary tumors of chronic stress-exposed mice. The current demonstration of the interplay between inflammation and the induction of immunosuppressive factors by catecholamines suggest a contextual impact of stress, helping to explain variable results of epidemiologic studies of the link between sympathetic activity and cancer progression, and implicating COX-2 blockade as a potential means to mitigate stress-related immune suppression.

Abstract P6-06-01: High-grade human cytomegalovirus IEA is associated with expression of COX-2 and 5-LO in human breast cancer samples

Abstract Background: The role of cyclooxygenase-2 (COX-2) in breast cancer development and progression has been supported by an increasing number of studies that show the overexpression of COX-2 in all the stages of the disease but in particular, in the metastatic phase. Besides COX-2 and its derived metabolites, 5-lipoxygenase (5-LO) and leukotrienes have also been associated with cancer progression. Human Cytomegalovirus (HCMV) detection in samples from primary BC, sentinel lymph nodes and brain metastases obtained from breast cancer patients' suggests that viral infection may also have a critical role in the development of breast cancer metastasis. Interestingly, in vitro studies showed that HCMV infections induce COX-2 in human fibroblasts, which augments viral replication through a prostaglandin dependent pathway. Thus, our main objective was to investigate whether there is a correlation between HCMV infection and overexpression of COX-2 and 5-LO in breast cancer. If so, HCMV could be an important additional target for breast cancer treatment. Material and Methods: Paraffin embedded breast cancer biopsies (n=48), ductal carcinoma in-situ (DCIS, n=14) and adjacent, benign breast tissue samples (n=29) were retrospectively examined for HCMV-immediate early (IE), HCMV-Late (LA) proteins, COX2 and 5LO by using immunohistochemical techniques established in our laboratory. Clinical data were available from the patients´ hospital files provided from the departments of oncology and pathology at Akershus University Hospital, Norway. All patients underwent direct surgery in 2011. All patients received standard adjuvant treatment according to the Norwegian guidelines. For in vitro studies, breast cancer cell lines (MCF-7, MB-MDA-231 and SKBR3) were infected with HCMV VR1814 strain and levels of COX-2 and 5-LO were determined by qPCR and western blot and immunofluorescence. Results: High levels of COX-2, 5-LO and HCMV-IE were detected mainly in breast cancer samples. High grade HCMV-IE (defined as &amp;gt;50% positive cells in the tumor tissues) was detected in 72% of infiltrating BC and in 28% of DCIS, but it was detected only in 7% of benign, adjacent breast tissue samples. Similarly, high grade COX-2 and 5-LO were detected in 58% and 53% of BC, in 21% and 8% of DCIS, and in 4% and 7% of benign, adjacent breast tissue samples, respectively. We found a statistically significant positive correlation for the levels of HCMV-IE and COX-2 (p=0.001) as well as for HCMV-IE and5-LO (p=0.0002) in infiltrating breast cancer. Furthermore, induction of COX-2 and 5-LO was confirmed in breast cancer cell lines following infection with HCMV was shown at both mRNA and protein level. Conclusion: Our findings confirm a positive correlation of HCMV-IE protein synthesis and overexpression of COX-2 and 5-LO in infiltrating breast cancer, DCIS and benign, adjacent breast tissue samples, which is consistent with the up-regulation of these enzymes in breast cancer cells infected with HCMV. These results suggest that the inflammation driven by COX-2 and 5-LO in human breast cancer might be induced by HCMV infections and lead to tumor progression. Thus, anti-viral therapy should be considered as an additional experimental treatment in selected breast cancer patients. Citation Format: Costa H, Touma J, Davoudi B, Geisler J, Vetvik K, Söderberg Naucler C, Rahbar A. High-grade human cytomegalovirus IEA is associated with expression of COX-2 and 5-LO in human breast cancer samples [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P6-06-01.

Clinicopathological and prognostic significance of COX-2 immunohistochemical expression in breast cancer: a meta-analysis.

The prognostic significance of COX-2 in patients with breast cancer remains controversial. The aims of our meta-analysis are to evaluate its association with clinicopathological characteristics and prognostic value in patients with breast cancer. PubMed, EMBASE, Web of Science, the Ovid Database and Grey literature were systematically searched up to May 2016. Twenty-one studies including 6739 patients with breast cancer were analyzed. The meta-analysis indicated that the incidence difference of COX-2 expression was significant when comparing the lymph node positive group to negative group (OR = 1.76, 95% CI [1.30, 2.39]) and the tumor size ≥ 2cm group to the tumor size < 2cm group (OR = 1.71, 95% CI [1.22, 2.39]). None of other clinicopathological parameters such as the ER status, PR status, HER2 status and the vascular invasion status were associated with COX-2 overexpression. The detection of COX-2 was significantly correlated with the disease-free survival (DFS) of patients (HR = 1.58, 95% CI [1.23, 2.03]) and the overall survival (OS) of patients (HR = 1.51, 95% CI [1.31, 1.72]). Our meta-analysis demonstrates that the presence of high levels of COX-2 is associated with poor prognosis for breast cancer patients and predicts bigger tumor size and lymph node metastasis.

310 - Knocking Down Delta-5 Desaturase and Exploiting High Expression Level of COX-2 to Inhibit Cancer Migration and Invasion

Our recent research has shown that knockdown of delta-5 desaturase (D5D, a key enzyme that converts dihomo-γ-linolenic acid to arachidonic acid) and treatment dihomo-γ-linolenic exerted growth inhibitory effect on both colon and pancreatic cancer cells (Xu Y et al, FRBM 96: 67-77, 2016 and Yang X et al, FRBM 97: 342-350, 2016) due to formation of an anti-cancer byproduct 8-hydroxyoctanoic acid (8-HOA) from COX-catalyzed peroxidation. Here, we further tested whether knockdown of D5D could also be used to inhibit cancer migration and invasion using colon cancer cell line HCA-7 colony 29 and pancreatic cancer cell line BxPC-3 (both express high levels of COX-2). Cell migration and invasion were assessed by wound healing assay and transwell assay along with western blot to investigate the possible molecular mechanism. Our results showed that promoted 8-HOA formation in cancer cells via knocking down D5D (shRNA transfection) can significantly inhibit cell migration. D5D knockdown can also significantly improve the efficacies of 5-flurouracil and gemcitabine on colon and pancreatic cancer cell migration and invasion. The underlying mechanism might be that 8-HOA, serving as histone deacetylase inhibitor, downregulates MMP-2 and 9, consequently leading to altered expression in key proteins involved in epithelial mesenchymal transition (EMT) e.g. E-cadherin and vimentin. For the first time, we demonstrated high COX expression in cancers could be taken advantage to inhibit cancer cell migration and invasion. The research outcome may provide us a promising strategy for cancer metastasis by taking advantage of abundant ω-6 fatty acids in daily diet and exploiting the high COX expression level in cancer (a paradigm shifting concept). This work supported by NIH R15CA195499 and Sanford Health-NDSU Collaborative Research Seed Grant Program.

Abstract 761: Specific inhibition of prostaglandin E2 production in medulloblastoma

Abstract Introduction: Prostaglandin E2 (PGE2) is a key lipid mediator of inflammation and carcinogenesis. Apart from promoting direct growth of cancer cells, PGE2 has been shown to be crucial in maintaining the immunosuppressive tumor microenvironment. Drugs targeting cyclooxygenase (COX)-1 and -2, enzymes upstream in the production of PGE2 and other prostanoids, have successfully reduced tumor growth in many cancer models but these drugs are all associated with adverse long-term effects. The aim of this study is to demonstrate an anti-tumor effect in medulloblastoma by selective inhibition of microsomal prostaglandin E1 (mPGES-1), the enzyme downstream of COX-1/2 specifically required for the synthesis of PGE2. Experimental procedures: Human medulloblastoma cell lines (DAOY and D283) were treated with a selective small-molecule inhibitor against mPGES-1 or COX-2 in the presence or absence of pro-inflammatory interleukin (IL)-1â. Prostaglandins in supernatants were extracted and quantified with LC-MS/MS. Drug efficacy was determined and cell toxicity was measured with cell viability assay. Mice with human medulloblastoma xenograft will function as proof-of-concept for the anti-tumor activities associated with inhibition of PGE2 production. Key inflammatory enzymes were quantified with Western blot. Results: Medulloblastoma cells express high levels of COX-2, mPGES-1, and produce PGE2. The increased production of PGE2 by IL-1â was completely blocked when cells were co-treated with inhibitor for mPGES-1 or COX-2. Both inhibitors showed cell toxicity. We did not observe any shunting towards other prostanoids. Conclusion: We investigate the effect of selective inhibition of mPGES-1 or COX-2 on medulloblastoma growth. We aim to further characterize the mPGES-1 inhibitor and compare to selective COX-2 inhibitor in preclinical medulloblastoma models. Specific inhibition of PGE2 production, rather than general inhibition of prostanoid production, would potentially prove useful as a complement to current medulloblastoma treatments. Citation Format: Filip Bergqvist, Linda Ljungblad, Malin Wickström, Karin Larsson, Anna Kock, Marina Korotkova, John Inge Johnsen, Per Kogner, Per-Johan Jakobsson. Specific inhibition of prostaglandin E2 production in medulloblastoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 761.

Abstract 2454: Omega-3 fatty acids DHA and EPA decrease oncogenic PGE2 in medulloblastoma in vitro and in vivo

Abstract Background: The embryonic brain tumor medulloblastoma is a highly malignant tumor of the cerebellum and the most common CNS malignancy of childhood. By combining neurosurgery, chemotherapy and radiation overall survival is about 70% but the intensive treatment regimen often leads to severe neurological sequelae and the risk of resistant relapses is significant. Hence, there is a need to improve treatment to reduce and alleviate late side effects. Inflammation plays an important role in the tumor microenvironment by suppressing immune response, promoting angiogenesis, facilitating tumor invasion, and metastasis. Prostaglandin E2 (PGE2) is a pro-inflammatory lipid mediator derived from the omega-6 fatty acid arachidonic acid (AA) through conversion by the key enzymes COX-2 and microsomal prostaglandin E synthase-1 (mPGES-1). Previous work from our group showed that medulloblastomas express high levels of COX-2 and mPGES-1 as well as all four PGE2 receptors (EP1-4) and most importantly that PGE2 promotes tumor proliferation. The ω-3 fatty acids docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) show anti-inflammatory characteristics in general and exert anti-tumoral properties in several cancer types. Here we investigate the potential role of DHA and EPA in treatment of medulloblastoma and their effect on prostaglandins in vitro and in vivo. Methods: In six medulloblastoma cell lines the cytotoxic activity of DHA and EPA was analyzed using cell viability assays. PGE2 levels were assessed in three medulloblastoma cell supernatants after 24 hrs of DHA treatment by ELISA. The prostaglandins were evaluated in human medulloblastoma xenografts in nude mice treated with DHA or a combination of DHA and EPA for 30 days from time of tumor take. Control mice were fed with normal chow. The levels of prostaglandins in the tumor tissue were analyzed with LC-MS/MS. Furthermore, the incorporation of fatty acids in tumors, erythrocytes and brain tissue was analyzed with GC-MS/MS. Results: Both DHA and EPA exerted cell toxicity in a dose dependent manner in vitro. DHA reduced the PGE2 production in all three cell lines investigated. In vivo, the treatment with DHA alone and the combination of DHA and EPA significantly increased the omega-3 index in tumors, erythrocytes and normal brain. The PGE2, and prostacyclin was significantly reduced in both treatment groups while thromboxane levels remained unchanged. The in vivo treatment was non-toxic. Conclusions: Lowering the pro-inflammatory PGE2 levels could mitigate the ongoing inflammation in the tumor microenvironment. We show that the omega-3 fatty acids DHA and EPA exert toxic effects on medulloblastoma cells in vitro and reduce PGE2 both in vitro and in vivo. We propose that DHA and EPA are interesting candidates to improve current medulloblastoma therapy. Citation Format: Linda Ljungblad, Filip Bergqvist, Teodora Andonova, Per-Johan Jakobsson, John Inge Johnsen, Per Kogner, Malin Wickström. Omega-3 fatty acids DHA and EPA decrease oncogenic PGE2 in medulloblastoma in vitro and in vivo. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2454.

Abstract 3927: The presence of COX-2 and tumor-associated macrophages as a prognostic marker for invasive breast carcinoma patients

Abstract Inflammation is an important mediator of tumor progression. The objective of this study was to assess whether the tissue localization of COX-2 and tumor-associated macrophages were associated with clinicopathological features of invasive carcinoma, including collagen deposition and patient survival outcome. A tumor microarray (TMA) of 371 biopsy specimens from patients with invasive breast carcinoma was analyzed for expression of high levels of COX-2, and the macrophage markers CD68 and CD163 in either the tumor nest (TN) or the tumor-associated stroma (TS). The study population for this TMA was female patients, 18 to 80 years of age with a median follow up of 8.4 years. Survival tables were calculated according to the Kaplan-Meier method. We found that elevated collagen deposition was associated with high stromal expression of COX-2 (P &amp;lt; 0.0001); however, the amount of collagen deposition was not a predictor for survival outcome. In order to better analyze patient data, samples were divided into quartiles based on their levels of COX-2 expression and/or macrophage infiltration. Tumor nests with high COX-2 expression had worse patient outcome (P = 0.011). One possible mechanism for this is COX-2 dependent recruitment of macrophages to the tumor microenvironment, supported by our finding of high infiltration of CD68+ macrophages in the TS, and CD163+ macrophages in both TN and TS. This recruitment of macrophages was associated with worse overall survival (OS). Furthermore, patient survival was worsened even more if macrophages expressed COX-2, suggesting positive amplification of COX-2 signaling via macrophage recruitment. This notion is further established by the finding of high presence of CD163+ macrophages in the TN as an independent prognostic factor as revealed by multivariate analysis (P &amp;lt; 0.0001, HR = 4.67). These results suggest that in invasive carcinoma the localization of inflammatory markers within the tumor are biomarkers for patient survival outcome. Therefore, we propose that these patients may benefit from therapy with a selective COX-2 inhibitor such as celecoxib. Citation Format: Karla Esbona, Sandeep Saha, Yanyao Yi, Menggang Yu, Kari Wisinski, Lee G. Wilke, Patricia J. Keely. The presence of COX-2 and tumor-associated macrophages as a prognostic marker for invasive breast carcinoma patients. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3927.

Abstract P1-03-04: Response to cyclooxygenase-2 inhibition is regulated by collagen dense stroma

Abstract Women with dense breast have over four-fold risk for breast cancer and high breast density is correlated to increased collagen. In our previously characterized mouse model of MMTV-PyVT x Col1a1tm1jae, increased collagen levels in mammary tumors lead to enhanced tumor formation and progression. Additionally, high collagen density in vitro elevated expression of PTGS2, the gene for cyclooxygenase-2 (COX-2), by over four-fold. Because COX-2 over-expression is observed in 40% of invasive breast carcinoma cases and correlates with poor prognosis, we hypothesized that inhibition of COX-2 may be an effective target in the context of mammary tumors arising in dense tissue. Col1a1tm1jae (HD) or wild-type (wt) tumor mice were randomly assigned at 11 weeks of age to daily treatment with vehicle or celecoxib for 21 days. Tumors in HD mice were larger (p &amp;lt; 0.05) and expressed higher levels of COX-2 (p &amp;lt; 0.05) and PGE2 (p &amp;lt; 0.01). COX-2 and PGE2 expression levels were both decreased with COX-2 inhibition (wt and HD = p &amp;lt; 0.0001, for both markers). Cell proliferation decreased in both wt and HD tumors when COX-2 was inhibited (Epithelium wt and HD p &amp;lt; 0.0001; stroma wt = p &amp;lt; 0.05 and HD = p &amp;lt; 0.01). Notably, several cytokines, immune and stromal cell counts were elevated in HD tumors alone, and COX-2 inhibition reversed this effect. These findings indicate that COX-2 has a direct role in modulating tumor progression in dense matrices and it may be an effective therapeutic target for women with dense breast tissue. Citation Format: Esbona K, Inman DR, Saha S, Wilke LG, Keely PJ. Response to cyclooxygenase-2 inhibition is regulated by collagen dense stroma. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P1-03-04.

Cyclooxygenase-2 expression is associated with initiation of hepatocellular carcinoma, while prostaglandin receptor-1 expression predicts survival.

AIMTo determine whether cyclooxygenase-2 (COX-2) and prostaglandin E1 receptor (EP1) contribute to disease and whether they help predict prognosis.METHODSWe retrospectively reviewed the records of 116 patients with hepatocellular carcinoma (HCC) who underwent surgery between 2008 and 2011 at our hospital. Expression of COX-2 and EP1 receptor was examined by immunohistochemistry of formalin-fixed, paraffin-embedded tissues using polyclonal antibodies. Possible associations between immunohistochemical scores and survival were determined.RESULTSFactors associated with poor overall survival (OS) were alpha-fetoprotein > 400 ng/mL, tumor size ≥ 5 cm, and high EP1 receptor expression, but not high COX-2 expression. Disease-free survival was not significantly different between patients with low or high levels of COX-2 or EP1. COX-2 immunoreactivity was significantly higher in well-differentiated HCC tissues (Edmondson grade I-II) than in poorly differentiated tissues (Edmondson grade III-IV) (P = 0.003). EP1 receptor immunoreactivity was significantly higher in poorly differentiated tissue than in well-differentiated tissue (P = 0.001).CONCLUSIONCOX-2 expression appears to be linked to early HCC events (initiation), while EP1 receptor expression may participate in tumor progression and predict survival.

Abstract B55: Elevation of oxidative expression proteins in colorectal adenomatous polyps

Abstract The optimal management of patients with colorectal adenomatous polyps depends on the accuracy of appropriate staging strategies because patients with similar colorectal adenocarcinoma architecture display heterogeneity in the course and outcome of the disease. Chronic oxidative stress may play a critical role in the pathogenesis of colorectal cancers, given that a major promutagenic threat faced by colonic epithelial cells is that of chronic oxidative stress. Although reactive oxygen species arise in most cells as a result of normal metabolic processes, additional sources of oxidative stress in colonic cells may include enzymes such as cyclooxygenase 2 (COX-2) and ATPase 6 and 8. High levels of COX-2 and ATPase protein expression have been detected in human colorectal polyps, which are lesions that can progress to colorectal carcinomas. This study applied spectrophotometric, dinitrophenylhydrazone (DNPH) assay, two dimensional gel electrophoresis, and Western blot analyses to assess the levels of oxidatively modified carbonyl proteins in 37 pairs of normal/surrounding and colorectal adenomatous polyps and cancer tissues. Our data showed that the levels of reactive protein carbonyl groups increased as level of malignancy increased. The level of carbonyl protein was 58% and 26% less in N/S tissues as compare to tumor tissue in adenocarcinoma, and colorectal adenomatous polyps tissues respectably. The differences between the level of carbonyl potion in N/S and tumor tissues was more pronounced in African American patient than in Caucasians. Also the levels of carbonyl proteins were notably higher in tissue contained higher number of tumor cells in different tissues blocks obtained from same patient. Our results may suggest that oxidative stress is involved in oxidatively modification of carbonyl proteins, leading to increased aggressiveness of colorectal polyps. This study was supported by NIGMS grant # GM099663- awarded to Dr. Felix O. Aikhionbare Citation Format: LaShanale M. Wallace, Sharifeh Mehrabi, Shakeria Cohen, Felix O. Aikhionbare. Elevation of oxidative expression proteins in colorectal adenomatous polyps. [abstract]. In: Proceedings of the Seventh AACR Conference on The Science of Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; Nov 9-12, 2014; San Antonio, TX. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2015;24(10 Suppl):Abstract nr B55.

Co-expression of COX-2 and 5-LO in primary glioblastoma is associated with poor prognosis.

Cyclooxygenase-2 (COX-2) and 5-lipoxygenase (5-LO) are important factors in tumorigenesis and malignant progression; however, studies of their roles in glioblastoma have produced conflicting results. To define the frequencies of COX-2 and 5-LO expression and their correlation with clinicopathological features and prognosis, tumor tissues from 76 cases of newly diagnosed primary ordinary glioblastoma were examined for COX-2 and 5-LO expression by immunohistochemistry. The expression levels of COX-2 and 5-LO and the relationships between the co-expression of COX-2/5-LO and patient age and gender, edema index (EI), Karnofsky Performance Scale and overall survival (OS) were analyzed. COX-2 and 5-LO were expressed in 73.7 % (56/76) and 92.1 % (70/76) of the samples, respectively. Among the clinicopathological characteristics, only age (>60 years) exhibited a significant association with the high expression of COX-2. No statistically significant correlations were found in the 5-LO cohort. A significant positive correlation was revealed between the COX-2 and 5-LO scores (r = 0.374; p = 0.001). The elevated co-expression of COX-2 and 5-LO was observed primarily in the patients over the age of 60 years. Patients with a high expression of COX-2 had a significantly shorter OS (p < 0.01), whereas the immunoexpression of 5-LO was not associated with the OS of patients with glioblastoma. Survival analysis indicated that simultaneous high levels of COX-2 and 5-LO expression were significantly correlated with poor OS and, conversely, that a low/low expression pattern of these two proteins was significantly associated with better OS (p < 0.05). Moreover, the Cox multivariable proportional hazard model showed that a high expression of COX-2, high co-expression of COX-2 and 5-LO, and a high Ki-67 index were significant predictors of shorter OS in primary glioblastoma, independent of age, gender, EI, 5-LO expression and p53 status. The hazard ratios for OS were 2.347 (95 % CI 1.30-4.25, p = 0.005), 1.900 (95 % CI 1.30-2.78, p = 0.001), and 2.210 (95 % CI 1.19-4.09, p = 0.011), respectively. These results suggest that COX-2 and 5-LO play roles in tumorigenesis and the progression of primary glioblastoma and that the co-expression pattern of COX-2/5-LO may be used as an independent prognostic factor in this disease.

COX-2 and EGFR: Partners in Crime Split by Aspirin

COX-2 and EGFR: Partners in Crime Split by Aspirin

Association of miR-21 and miR-155 with regulation of 15-HPGD mRNA in human breast cancer cells

Breast cancer (BC) is the most common form of cancer, leading to high mortality rates among women worldwide. In this study we have analyzed mRNA expression of 15-hydroxy-prostaglandin-dehydrogenases (15-HPGD), cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2) and miRNAs (miR-21, miR-155) in three cell lines of estrogen-positive human breast carcinomas (MCF-7, BT-474, ZR-75-1). Results of three independent experiments have demonstrated significantly higher levels of COX-2 and COX-1 mRNAs in the cell line ZR-75-1 cells than in MCF-7 and BT-474 cells. mRNA levels of total 15-HPGD and functional-15-HPGD were lower in BT-474 than in MCF-7 and in ZR-75-1 cells. Synthesis of the 15-HPGD enzyme in BT-474 cells was blocked at the level of nuclear processing of an immature pre-mRNA. High expression of miR-21 was detected in all the tumor cell lines (MCF-7, ZR-75-1 and BT-474). In the breast cancer cell lines, the expression level of miR-155 was significantly lower than that of miR-21. Correlations have been found between dysregulation of miR-21, miR-155 and mRNA levels of 15-HPGD, COX-1, COX-2. The results obtained in this study showed that miR-21 and miR-155 regulate activity of several genes in the tumor cell and on some genes they exhibited a cumulative effect. Based on these results, we concluded that the miR-21 and miR-155 inhibit activity of the tumor suppressor gene 15-HPGD and induce a potential oncogene COX-2, which contributes to malignancy and metastasis of breast cancer cells.