Thromboxane Biosynthesis Research Articles

Long-term pharmacodynamic and clinical effects of twice- versus once-daily low-dose aspirin in essential thrombocythemia: The ARES trial.

Patients with essential thrombocythemia (ET) are treated with once-daily low-dose aspirin to prevent thrombosis, but their accelerated platelet turnover shortens the antiplatelet effect. The short-term Aspirin Regimens in EsSential Thrombocythemia trial showed that twice-daily aspirin dosing restores persistent platelet thromboxane (TX) inhibition. However, the long-term pharmacodynamic efficacy, safety and tolerability of twice-daily aspirin remain untested. We performed a multicenter, randomized, open-label, blinded-endpoint, phase-2 trial in which 242 patients with ET were randomized to 100 mg aspirin twice- or once-daily and followed for 20 months. The primary endpoint was the persistence of low serum TXB2, a surrogate biomarker of antithrombotic efficacy. Secondary endpoints were major and clinically relevant non-major bleedings, serious vascular events, symptom burden assessed by validated questionnaires, and in vivo platelet activation. Serum TXB2 was consistently lower in the twice-daily versus once-daily regimen on 10 study visits over 20 months: median 3.9 ng/mL versus 19.2 ng/mL, respectively; p < .001; 80% median reduction; 95% CI, 74%-85%. No major bleeding occurred. Clinically relevant non-major bleedings were non-significantly higher (6.6% vs. 1.7%), and major thromboses lower (0.8% vs. 2.5%) in the twice-daily versus once-daily group. Patients on the twice-daily regimen had significantly lower frequencies of disease-specific symptoms and severe hand and foot microvascular pain. Upper gastrointestinal pain was comparable in the two arms. In vivo platelet activation was significantly reduced by the twice-daily regimen. In patients with ET, twice-daily was persistently superior to once-daily low-dose aspirin in suppressing thromboxane biosynthesis and reducing symptom burden, with no detectable excess of bleeding and gastrointestinal discomfort.

Thromboxane biosynthesis and future events in diabetes: the ASCEND trial.

Thromboxane (TX) A2, released by activated platelets, plays an important role in atherothrombosis. Urinary 11-dehydro-TXB2 (U-TXM), a stable metabolite reflecting the whole-body TXA2 biosynthesis, is reduced by ∼70% by daily low-dose aspirin. The U-TXM represents a non-invasive biomarker of in vivo platelet activation and is enhanced in patients with diabetes. This study assessed whether U-TXM is associated with the risk of future serious vascular events or revascularizations (SVE-R), major bleeding, or cancer in patients with diabetes. The U-TXM was measured pre-randomization to aspirin or placebo in 5948 people with type 1 or 2 diabetes and no cardiovascular disease, in the ASCEND trial. Associations between log U-TXM and SVE-R (n = 618), major bleed (n = 206), and cancer (n = 700) during 6.6 years of follow-up were investigated by Cox regression; comparisons of these associations with the effects of randomization to aspirin were made. Higher U-TXM was associated with older age, female sex, current smoking, type 2 diabetes, higher body size, urinary albumin/creatinine ratio of ≥3 mg/mmol, and higher estimated glomerular filtration rate. After adjustment for these, U-TXM was marginally statistically significantly associated with SVE-R and major bleed but not cancer [hazard ratios per 1 SD higher log U-TXM (95% confidence interval): 1.09 (1.00-1.18), 1.16 (1.01-1.34), and 1.06 (0.98-1.14)]. The hazard ratio was similar to that implied by the clinical effects of randomization to aspirin for SVE-R but not for major bleed. The U-TXM was log-linearly independently associated with SVE-R in diabetes. This is consistent with the involvement of platelet TXA2 in diabetic atherothrombosis.

Thromboxane biosynthesis in cancer patients and its inhibition by aspirin: a sub-study of the Add-Aspirin trial

BackgroundPre-clinical models demonstrate that platelet activation is involved in the spread of malignancy. Ongoing clinical trials are assessing whether aspirin, which inhibits platelet activation, can prevent or delay metastases.MethodsUrinary 11-dehydro-thromboxane B2 (U-TXM), a biomarker of in vivo platelet activation, was measured after radical cancer therapy and correlated with patient demographics, tumour type, recent treatment, and aspirin use (100 mg, 300 mg or placebo daily) using multivariable linear regression models with log-transformed values.ResultsIn total, 716 patients (breast 260, colorectal 192, gastro-oesophageal 53, prostate 211) median age 61 years, 50% male were studied. Baseline median U-TXM were breast 782; colorectal 1060; gastro-oesophageal 1675 and prostate 826 pg/mg creatinine; higher than healthy individuals (~500 pg/mg creatinine). Higher levels were associated with raised body mass index, inflammatory markers, and in the colorectal and gastro-oesophageal participants compared to breast participants (P < 0.001) independent of other baseline characteristics. Aspirin 100 mg daily decreased U-TXM similarly across all tumour types (median reductions: 77–82%). Aspirin 300 mg daily provided no additional suppression of U-TXM compared with 100 mg.ConclusionsPersistently increased thromboxane biosynthesis was detected after radical cancer therapy, particularly in colorectal and gastro-oesophageal patients. Thromboxane biosynthesis should be explored further as a biomarker of active malignancy and may identify patients likely to benefit from aspirin.

Novel thiazolopyridine derivatives of diflapolin as dual sEH/FLAP inhibitors with improved solubility

Inflammatory responses are orchestrated by a plethora of lipid mediators, and perturbations of their biosynthesis or degradation hinder resolution and lead to uncontrolled inflammation, which contributes to diverse pathologies. Small molecules that induce a switch from pro-inflammatory to anti-inflammatory lipid mediators are considered valuable for the treatment of chronic inflammatory diseases. Commonly used non-steroidal anti-inflammatory drugs (NSAIDs) are afflicted with side effects caused by the inhibition of beneficial prostanoid formation and redirection of arachidonic acid (AA) into alternative pathways. Multi-target inhibitors like diflapolin, the first dual inhibitor of soluble epoxide hydrolase (sEH) and 5-lipoxygenase-activating protein (FLAP), promise improved efficacy and safety but are confronted by poor solubility and bioavailability. Four series of derivatives bearing isomeric thiazolopyridines as bioisosteric replacement of the benzothiazole core and two series additionally containing mono- or diaza-isosteres of the phenylene spacer were designed and synthesized to improve solubility. The combination of thiazolo[5,4-b]pyridine, a pyridinylen spacer and a 3,5-Cl2-substituted terminal phenyl ring (46a) enhances solubility and FLAP antagonism, while preserving sEH inhibition. Moreover, the thiazolo[4,5-c]pyridine derivative 41b, although being a less potent sEH/FLAP inhibitor, additionally decreases thromboxane production in activated human peripheral blood mononuclear cells. We conclude that the introduction of nitrogen, depending on the position, not only enhances solubility and FLAP antagonism (46a), but also represents a valid strategy to expand the scope of application towards inhibition of thromboxane biosynthesis.

Regulatory Phenomena in the Glutathione Peroxidase Superfamily.

Significance: The selenium-containing Glutathione peroxidases (GPxs)1-4 protect against oxidative challenge, inhibit inflammation and oxidant-induced regulated cell death. Recent Advances: GPx1 and GPx4 dampen phosphorylation cascades predominantly via prevention of inactivation of phosphatases by H2O2 or lipid hydroperoxides. GPx2 regulates the balance between regeneration and apoptotic cell shedding in the intestine. It inhibits inflammation-induced carcinogenesis in the gut but promotes growth of established cancers. GPx3 deficiency facilitates platelet aggregation likely via disinhibition of thromboxane biosynthesis. It is also considered a tumor suppressor. GPx4 is expressed in three different forms. The cytosolic form proved to inhibit interleukin-1-driven nuclear factor κB activation and leukotriene biosynthesis. Moreover, it is a key regulator of ferroptosis, because it reduces hydroperoxy groups of complex lipids and silences lipoxygenases. By alternate substrate use, the nuclear form contributes to chromatin compaction. Mitochondrial GPx4 forms the mitochondrial sheath of spermatozoa and, thus, guarantees male fertility. Out of the less characterized GPxs, the cysteine-containing GPx7 and GPx8 are unique in contributing to oxidative protein folding in the endoplasmic reticulum by reacting with protein isomerase as an alternate substrate. A yeast 2-Cysteine glutathione peroxidase equipped with CP and CR was reported to sense H2O2 for inducing an adaptive response. Critical Issues: Most of the findings compiled are derived from tissue culture and/or animal studies only. Their impact on human physiology is sometimes questionable. Future Directions: The expression of individual GPxs and GPx-dependent regulatory phenomena are to be further investigated, in particular in respect to human health.

Measurement of Thromboxane Biosynthesis in Health and Disease.

Thromboxane (TX) A2 is a chemically unstable lipid mediator involved in several pathophysiologic processes, including primary hemostasis, atherothrombosis, inflammation, and cancer. In human platelets, TXA2 is the major arachidonic acid derivative via the cyclooxygenase (COX)-1 pathway. Assessment of platelet TXA2 biosynthesis can be performed ex vivo through measurement of serum TXB2, an index of platelet COX-1 activity, as well as in vivo through measurement of urinary enzymatic metabolites, a non-invasive index of platelet activation. This article reviews the main findings of four decades of clinical investigation based on these analytical approaches, focusing on the measurement of TXA2 metabolites to characterize the pathophysiologic role of transiently or persistently enhanced platelet activation and to describe the clinical pharmacology of COX-1 inhibition in health and disease.

The role of immunohistochemistry expression of COX-2 in differentiating pigmented benign and malignant skin neoplasms.

Background: Skin cancer is one of the most common types of cancer and its annual mortality rate is increasing. The induction enzyme of cyclooxygenase COX-2 causes biosynthesis of prostaglandin and thromboxane during inflammation of the body. Increasing the expression of COX-2 has an important role in the development and progression of malignant epithelial cancers and other types of cancers. Considering the diagnostic status of the marker, this study aimed to evaluate the expression of COX-2 for diagnosis and differentiation of benign skin pigmented neoplastic lesions from malignant melanoma types. Methods: In this diagnostic study, the immunohistochemistry of COX-2 maker in 82 paraffin blocks of pigmented benign and malignant skin neoplasms of patients (49 men; 33 women) and its association with clinicopathological features of the tumor was evaluated. Data were analyzed using chi-squared and t test in SPSS18. Significance level was set at less than 5%. Results: The findings showed that 20 patients (24.3%) had malignant melanoma and 13 had significant COX-2 (3+ High), while COX-2 marker was not detected in other benign and malignant pigmented skin neoplasms (p<0.001). A significant association was found between COX-2 marker and grade (p<0.001), but there was no significant correlation with other clinicopathological tumor criteria. Sensitivity, specificity, PPV and NPV value of the COX-2 marker were 65%, 100%, 89.9%, and 100%, respectively. Conclusion: Because of the high level of COX-2 in malignant melanoma skin marker, it can be used to distinguish benign and malignant neoplastic lesions (SCC and BCC) from melanoma and to provide effective therapeutic strategies through specific COX-2 enzyme inhibitors.

Effects of anti-inflammatory and adaptogenic herbal extracts on gene expression of eicosanoids signaling pathways in isolated brain cells

IntroductionThe adaptogens modulate expression of genes playing key roles in development of aging-related disorders, which are considered as low-grade systemic inflammatory conditions characterized by an imbalance between pro-and anti-inflammatory eicosanoids. Aim of the StudyWe compared the effects of anti-inflammatory and adaptogenic plant extracts on the expression of genes involved in biosynthesis of eicosanoids with the purpose to find those plants, which selectively upregulated the expression of anti-inflammatory lipoxins signaling pathways and inhibited pro-inflammatory signaling pathways associated with biosynthesis of leukotrienes, prostaglandins and thromboxanes. Materials and MethodsWe conducted transcriptome-wide RNA sequencing to profile gene expression alterations in T98G neuroglia cells upon treatment of plant extract and analyzed the relevance of deregulated genes to eicosanoids signaling pathways using in silico models. ResultsFor the first time, we demonstrated that Rhodiola rosea, Withania somnifera and Eleutherococcus senticosus downregulate the expression of key genes (ALOX5AP, DPEP2, LTC4S) involved biosynthesis of leukotrienes A, B, C, D and E, resulting in inhibition of leukotriene signaling pathway suggesting their potential benefits in Alzheimer disease. The common feature for all tested anti-inflammatory plants extracts was related to downregulation of ALOX12, which was also associated with neuroprotective action of these medicinal plants as well as their potential benefits in neurodegenerative diseases. None of tested anti-inflammatory and adaptogenic plants selectively activated the ALOX15-mediated signaling pathway, which is associated with generation anti-inflammatory lipoxins. Almost all tested plants upregulated the expression of the prostaglandin E receptor 3 gene (PTGER3) suggesting their potential benefits in the treatment of cancer. ConclusionEvery single plant tested in this study revealed a specific “signature” on eicosanoid signaling-related gene expression, regardless of their common features as anti-inflammatory or adaptogenic activity. Further studies of the combination of Rhodiola with Withania (Adaptra) for the treatment of Alzheimer disease are required.

Abstract 360: Rivaroxaban Enhances the Antiplatelet Activity of Aspirin via Inhibition of Nox2-mediated Thromboxane A 2 and Isoprostane Biosynthesis

Background: A recent study demonstrated that a combination of rivaroxaban, an inhibitor of factor Xa, plus aspirin (100mg/day) reduces cardiovascular events in patients with stable vascular disease, compared to aspirin alone but the underlying mechanism is still unknown. Methods: In vitro study was performed consisting in measuring agonist-induced platelet aggregation, thromboxane (Tx)B 2 and isoprostane biosynthesis, soluble Nox 2 -dp, a marker of Nox2 activation, and PLA 2 activation in platelets from healthy subjects (n=5) added with or without scalar doses of aspirin (25-100 uM) and/or rivaroxaban (15-120 ng/ml). Furthermore, we measured urinary excretion of isoprostanes, and soluble Nox2 before and after 3 months of 20 mg rivaroxaban (n=25) or warfarin (n=25) treatment in atrial fibrillation(AF) patients. Results: In vitro study showed that rivaroxaban significantly reduced platelet aggregation, TxB 2 and isoprostane biosynthesis and Nox2 and PLA 2 activation in a dose-dependent fashion. Also, rivaroxaban dose-dependently inhibited aggregation of platelets treated with low-(25uM) as well as high-(100uM) aspirin doses. In low-dose aspirin-treated samples an incremental inhibition of platelet TxB 2 biosynthesis was achieved; conversely, in platelets treated with high doses of aspirin a significant inhibition of isoprostanes and Nox2 was observed. Inhibition of platelet aggregation by rivaroxaban was detected also in platelets stimulated with convulxin suggesting that its antiplatelet effect was GPVI-dependent. In addition, convulxin elicited PLA 2 activation, that was inhibited by rivaroxaban as well as by a Nox2 inhibitor. Finally, in AF patients treated with rivaroxaban a significant reduction of urinary isoprostanes and soluble Nox2 and GPVI as compared to warfarin-treated ones was detected. Conclusion: Here we show that rivaroxaban inhibits platelet aggregation via GPVI interaction and eventually Nox2-mediated thromboxane and isoprostane biosynthesis in aspirin-treated platelets so providing a novel mechanistic insight into the beneficial effects of combining rivaroxaban with aspirin.

3-Substituted 1,5-Diaryl-1 H-1,2,4-triazoles as Prospective PET Radioligands for Imaging Brain COX-1 in Monkey. Part 1: Synthesis and Pharmacology.

Cyclooxygenase-1 (COX-1) is a key enzyme in the biosynthesis of proinflammatory thromboxanes and prostaglandins and is found in glial and neuronal cells within brain. COX-1 expression is implicated in numerous neuroinflammatory states. We aim to find a direct-acting positron emission tomography (PET) radioligand for imaging COX-1 in human brain as a potential biomarker of neuroinflammation and for serving as a tool in drug development. Seventeen 3-substituted 1,5-diaryl-1 H-1,2,4-triazoles were prepared as prospective COX-1 PET radioligands. From this set, three 1,5-(4-methoxyphenyl)-1 H-1,2,4-triazoles, carrying a 3-methoxy (5), 3-(1,1,1-trifluoroethoxy) (20), or 3-fluoromethoxy substituent (6), were selected for radioligand development, based mainly on their high affinities and selectivities for inhibiting human COX-1, absence of carboxyl group, moderate computed lipophilicities, and scope for radiolabeling with carbon-11 ( t1/2 = 20.4 min) or fluorine-18 ( t1/2 = 109.8 min). Methods were developed for producing [11C]5, [11C]20, and [ d2-18F]6 from hydroxy precursors in a form ready for intravenous injection for prospective evaluation in monkey with PET.

Determinants of thromboxane biosynthesis in patients with moderate to severe chronic kidney disease

BackgroundMechanisms of accelerated atherothrombosis in patients with chronic kidney disease (CKD) are only partly characterized. The aims of this study were to evaluate the extent of thromboxane (TX)-dependent platelet activation in patients with CKD, and to characterize the determinants of altered TX biosynthesis in this setting, with particular reference to enhanced lipid peroxidation, low grade inflammation and CKD-related anemia. Patients and methodsA cross sectional comparison between urinary 8-iso-PGF2α and 11-dehydro-TXB2, in vivo markers of oxidative stress and platelet activation, respectively, was performed in 115 patients with stage 1–4 CKD. ResultsLevels of both urinary 11-dehydro-TXB2 and 8-iso-PGF2α increased sequentially across the four CKD stages (P<0.0001, Kruskal–Wallis test). Both urinary prostanoids were inversely associated with either estimated glomerular filtration rate (eGFR, P<0.0001) or hemoglobin levels (P<0.0001). A significant direct correlation was also observed between urinary 11-dehydro-TXB2 and 8-iso-PGF2α (Rho=0.620, P<0.0001). On multivariate analysis, urinary 8-iso-PGF2α (β=0.459, P<0.0001), hemoglobin levels (β=− 0.261, P=0.002) and eGFR (β=−0.172, P=0.032) were independent predictors of urinary 11-dehydro-TXB2 (adjusted R2=0.488). ConclusionsThis study provides biochemical evidence of persistent platelet activation in patients with CKD. This condition occurs early in the natural history of the disease and is related to kidney function and oxidative stress. Moreover, we found an independent inverse relationship between hemoglobin levels and TX-dependent platelet activation. This finding may provide a mechanistic link between CKD-related anemia and increased cardiovascular risk.

Aspirin prevents colorectal cancer metastasis in mice by splitting the crosstalk between platelets and tumor cells.

We investigated whether platelets prime colon cancer cells for metastasis and whether pharmacological inhibition of platelet function may prevent it. Coculturing HT29 human colon carcinoma cells with human platelets led to the induction of mesenchymal-like cancer cells characterized by downregulation of E-cadherin and upregulation of Twist1, enhanced cell mobility and a proaggregatory action on platelets. These changes were prevented by different antiplatelet agents, aspirin[an inhibitor of cyclooxygenase(COX)-1], DG-041[an antagonist of prostaglandin(PG)E2 EP3 receptor] and ticagrelor (a P2Y12 receptor antagonist). The injection of HT29 cells, exposed to platelets in vitro, into the tail vein of humanized immunodeficient mice led to higher incidence of lung metastasis compared to the injection of untreated HT29 cells. This effect was associated with enhanced systemic biosynthesis of thromboxane(TX)A2 and PGE2 in vivo. Platelet COX-1 inhibition by aspirin administration to mice prevented the increased rate of metastasis as well as the enhanced production of TXA2 and PGE2 induced by the in vitro priming of HT29 cells by platelets. In conclusion, targeting platelet COX-1 with low-dose aspirin exerts an antimetastatic action by averting the stem cell mimicry of cancer cells associated with enhanced proaggregatory effects induced by platelet-tumor cell interactions. These effects may be shared by other antiplatelet drugs.

Oxidative stress and thromboxane-dependent platelet activation in inflammatory bowel disease: effects of anti-TNF-α treatment.

Patients with inflammatory bowel disease (IBD) are at higher risk of venous thromboembolism and coronary artery disease despite having a lower burden of traditional risk factors. Platelets from IBD patients release more soluble CD40 ligand (CD40L), and this has been implicated in IBD platelet hyper-activation. We here measured the urinary F2-isoprostane 8-iso-prostaglandin (PG)2α (8-iso-PGF2α), urinary 11-dehydro-thromboxane (TX) B2 (11-dehydro-TXB2) and plasma CD40L in IBD patients, and explored the in vitro action of anti-tumour necrosis factor (TNF)-α antibody infliximab on IBD differentiating megakaryocytes. Urinary and blood samples were collected from 124 IBD patients and 37 healthy subjects. Thirteen IBD patients were also evaluated before and after 6-week infliximab treatment. The in vitro effect of infliximab on patient-derived megakaryocytes was evaluated by immunoflorescence microscopy and by flow cytometry. IBD patients had significantly (p<0.0001) higher urinary 8-iso-PGF2α and 11-dehydro-TXB2 as well as plasma CD40L levels than controls, with active IBD patients displaying higher urinary and plasma values when compared to inactive patients in remission. A 6-week treatment with infliximab was associated with a significant reduction of the urinary excretion of 8-iso-PGF2α and 11-dehydro-TXB2 (p=0.008) and plasma CD40L (p=0.001). Infliximab induced significantly rescued pro-platelet formation by megakaryocytes derived from IBD patients but not from healthy controls. Our findings provide evidence for enhanced in vivo TX-dependent platelet activation and lipid peroxidation in IBD patients. Anti-TNF-α therapy with infliximab down-regulates in vivo isoprostane generation and TX biosynthesis in responder IBD patients. Further studies are needed to clarify the implication of infliximab induced-proplatelet formation from IBD megakaryocytes.

Abstract POSTER-BIOL-1350: Dissecting cellular and molecular consequences of disrupting cyclooxygenase-1 activity in ovarian cancer

Abstract Introduction: Cyclooxygenase-1 (COX-1) and COX-2 catalyze the rate-limiting step in the biosynthesis of prostaglandins and thromboxanes, which contribute to tumorigenesis by promoting tumor proliferation, escape from apoptosis, and angiogenesis. COX-1 is overexpressed in multiple human and mouse models of ovarian cancer and that COX-1 inhibition reduces ovarian cancer cell viability in preclinical models; in contrast COX-2 is the principle COX isoform expressed in most solid tumors. Our group has an ongoing interest in developing COX-1-targeted molecules for molecular imaging and therapeutic use. Hypothesis: We hypothesize that COX-1 is a viable molecular target for the treatment of ovarian cancer. To test this hypothesis, we (i) compared expression of COX-1 and COX-2 in The Cancer Genome Atlas (TCGA) ovarian tumors and in an ovarian cancer tissue microarray (TMA) generated in our laboratory, and (ii) determined cellular and molecular effects of disrupting COX-1 activity in ovarian cancer cells. Methods: RNA-seq mRNA data for COX-1 and COX-2 in the PANCAN 12 tumor panel of TCGA was accessed through the Firehose repository (Broad Institute, Cambridge, MA). COX-1 and COX-2 protein expression in our TMA was determined by immunohistochemistry, and percentage of COX-1 and COX-2-positive tumor cells quantified. An ovarian cancer cell line model of disrupted COX-1 activity was generated by the stable integration of shRNA targeting COX-1 in OVCAR-3 cells (ShCOX1). Cell growth in ShCOX1 cells compared to control cells expressing scrambled shRNA (ShSCR) was determined by sulphorhodamine B assay. Differential gene expression between ShSCR and ShCOX1 cells was determined by RNA-seq. Results: TCGA RNA-seq data revealed that COX-1 mRNA levels was significantly higher than COX-2 expression in ovarian cancer and higher than 11 other solid tumor types. We confirmed these findings at the protein level in our TMA. Specific COX-1 knockdown in OVCAR-3 cells significantly reduced growth compared to control cells. Pathway analysis from RNA-seq data revealed that COX-1 knockdown significantly downregulates genes involved in cell proliferation, cell migration/invasion, angiogenesis and epithelial-mesenchymal transition. Conclusions: Our findings confirm that COX-1 is over-expressed in ovarian cancer, and is likely to play a key role in tumor progression through promoting oncogenic tumor growth and progression. Therefore, exploiting high COX-1 expression in ovarian cancer with novel COX-1-targeted molecules has the potential to improve the detection and treatment of this deadly disease. Citation Format: Andrew J. Wilson, Brian D. Lehmann, Qi Liu, MD Jashim Uddin, Anna Elleman, Brenda Crews, Jeanette Saskowski, Jennifer Pietenpol, Lawrence J. Marnett, Dineo Khabele. Dissecting cellular and molecular consequences of disrupting cyclooxygenase-1 activity in ovarian cancer [abstract]. In: Proceedings of the 10th Biennial Ovarian Cancer Research Symposium; Sep 8-9, 2014; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(16 Suppl):Abstract nr POSTER-BIOL-1350.

Abstract 2068: Cyclooxygenase-1 gene expression contributes to multiple oncogenic pathways in high grade serous ovarian cancer

Abstract Ovarian cancer is the most lethal gynecologic malignancy. Most women diagnosed with high grade serous ovarian cancer present with advanced disease characterized by widespread peritoneal carcinomatosis. Recent epidemiological results indicate that cyclooxygenase (COX) inhibitors aspirin and other non-steroidal anti-inflammatory drugs are a promising preventive strategy to reduce the risk of developing ovarian cancer. COX-1 and COX-2 catalyze the rate-limiting step in the biosynthesis of prostaglandins and thromboxanes, which contribute to tumorigenesis by promoting proliferation, escape from apoptosis, and angiogenesis. Emerging evidence from our group and others suggests that COX-1 is an important molecular target in ovarian cancer, distinguishing ovarian cancer from most other solid tumors, where COX-2 is the dominant isoform. However, the precise roles of COX-1 and COX-2 in human ovarian cancer remain controversial. Therefore, the aim of this study was to gain additional insight into COX-1 gene expression and function in human ovarian cancer. First, we extracted RNA-seq V2 mRNA expression data for COX-1 and COX-2 in the PANCAN12 tumor panel of The Cancer Genome Atlas. COX-1 mRNA levels were significantly higher than those of COX-2 in high grade serous ovarian tumors, and higher than COX-1 expression in 11 other solid tumor types. We confirmed these findings in a tissue microarray generated from 209 ovarian cancer patients. COX-1 protein was highly expressed in a large subset of serous tumors, with significantly lower COX-2 expression present. In contrast, there was markedly lower COX-1 expression in endometrioid, mucinous and clear cell ovarian tumors compared to COX-2. Second, stable COX-1 knockdown in serous-representative OVCAR-3 cells significantly reduced cell viability compared to control cells. Pathway analysis from RNA-seq data revealed that COX-1 knockdown induced a coordinated transcriptional response consistent with reduced cell proliferation (eg. reduced cyclin D1 and increased p21 expression), cell migration/invasion (eg. reduced TGF-β signaling and expression of MMPs), angiogenesis (eg. reduced VEGF expression), epithelial-mesenchymal transition (eg. increased E-cadherin expression and reduced BMP signaling), and NF-κB signaling. Gene expression changes were validated by western blot. In SKOV-3 ovarian cancer cells stably overexpressing COX-1, opposite effects on cell viability and on these genes/pathways were observed. Our findings confirm that COX-1 is over-expressed in serous ovarian cancer, and likely plays a key role in tumor progression. Exploiting high COX-1 expression in ovarian cancer with COX-1-targeted molecules has the potential to improve the treatment and detection of this deadly disease. Citation Format: Andrew J. Wilson, Brian D. Lehmann, Jeanette Saskowski, MD Jashim Uddin, Cristina Daniel, Brenda C. Crews, Qi Liu, Shilin Zhao, Deok-Soo Son, Jennifer A. Pietenpol, Lawrence J. Marnett, Dineo Khabele. Cyclooxygenase-1 gene expression contributes to multiple oncogenic pathways in high grade serous ovarian cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2068. doi:10.1158/1538-7445.AM2015-2068

Abstract 4928: Cyclooxygenase-1 as a target for molecular imaging of high grade serous ovarian cancer

Abstract Ovarian cancer is the most lethal gynecologic malignancy. Most women diagnosed with high grade serous ovarian cancer, the most common subtype, present with advanced disease characterized by widespread peritoneal carcinomatosis. Non-invasive imaging plays a critical role in the management of ovarian cancer by confirming sites of disease, informing treatment interventions, and determining the effectiveness of treatment. However, current imaging methods are limited by relatively high false positive and false negative rates. The development of novel molecular imaging agents is urgently required. Emerging evidence from our group and others suggests that cyclooxygenase-1 (COX-1) is an important molecular target in ovarian cancer and distinguishes ovarian cancer from most other solid tumors, where COX-2 is the dominant isoform. COX-1 and COX-2 catalyze the rate-limiting step in the biosynthesis of prostaglandins and thromboxanes, which contribute to tumorigenesis by promoting tumor proliferation, escape from apoptosis, and angiogenesis. In this study, we examined COX-1 expression in high grade serous ovarian tumors using data extracted from The Cancer Genome Atlas (TCGA) and a tissue microarray (TMA) generated in our laboratory from 209 patients. Raw mRNA RNA-seq expression data from TCGA revealed that PTGS1, the gene encoding COX-1, is in the top 10% of genes expressed in ovarian cancer. Furthermore, mRNA expression of the COX-2-encoding gene, PTGS2, in ovarian tumors was significantly lower than COX-1 mRNA levels (p&amp;lt;0.00001, Mann-Whitney test), and COX-1 expression was selectively higher in ovarian tumors than in 11 other solid cancers in the TCGA PANCAN12 panel (p&amp;lt;0.00001 compared to COX-1 levels in ovarian tumors, Mann-Whitney test). We confirmed these findings at the protein level in our TMA. There was high COX-1 expression in a large subset of high grade serous tumors, but markedly lower expression in other epithelial ovarian cancer subtypes (endometrioid, mucinous and clear cell, p&amp;lt;0.0001, Mann-Whitney test). Having confirmed COX-1 as an attractive molecular target in high grade serous ovarian cancer, newly synthesized COX-1-selective compounds were screened in COX-1-expressing OVCAR-3 ovarian cancer cells. Two novel small molecule inhibitors of COX-1 (LM-9954 and LM-9955) inhibited COX-1 in biochemical assays and reduced prostaglandin synthesis in OVCAR-3 cells at nanomolar concentrations. The highly selective expression of COX1 in serous ovarian carcinoma provides a unique molecular screening opportunity and we are currently synthesizing 18F-conjugated forms of these agents to act as radiotracers in positron emission tomography (PET) imaging. Exploiting high COX-1 expression in high grade serous ovarian cancer with novel COX-1-targeted molecules has the potential to improve detection of this deadly disease. Citation Format: Andrew J. Wilson, Brian D. Lehmann, MD Jashim Ussin, Anna Elleman, Brenda Crews, Jeanette Saskowski, Jennifer Pietenpol, Lawrence J. Marnett, Dineo Khabele. Cyclooxygenase-1 as a target for molecular imaging of high grade serous ovarian cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4928. doi:10.1158/1538-7445.AM2014-4928

Relationship between a Common Variant in the Fatty Acid Desaturase (FADS) Cluster and Eicosanoid Generation in Humans

Dramatic shifts in the Western diet have led to a marked increase in the dietary intake of the n-6 polyunsaturated fatty acid (PUFA), linoleic acid (LA). Dietary LA can then be converted to arachidonic acid (ARA) utilizing three enzymatic steps. Two of these steps are encoded for by the fatty acid desaturase (FADS) cluster (chromosome 11, 11q12.2-q13) and certain genetic variants within the cluster are highly associated with ARA levels. However, no study to date has examined whether these variants further influence pro-inflammatory, cyclooxygenase and lipoxygenase eicosanoid products. This study examined the impact of a highly influential FADS SNP, rs174537 on leukotriene, HETE, prostaglandin, and thromboxane biosynthesis in stimulated whole blood. Thirty subjects were genotyped at rs174537 (GG, n = 11; GT, n = 13; TT, n = 6), a panel of fatty acids from whole serum was analyzed, and precursor-to-product PUFA ratios were calculated as a marker of the capacity of tissues (particularly the liver) to synthesize long chain PUFAs. Eicosanoids produced by stimulated human blood were measured by LC-MS/MS. We observed an association between rs174537 and the ratio of ARA/LA, leukotriene B4, and 5-HETE but no effect on levels of cyclooxygenase products. Our results suggest that variation at rs174537 not only impacts the synthesis of ARA but the overall capacity of whole blood to synthesize 5-lipoxygenase products; these genotype-related changes in eicosanoid levels could have important implications in a variety of inflammatory diseases.

Role of Indomethacin in Acute Pain and Inflammation Management: A Review of the Literature

Nonsteroidal anti-inflammatory drugs are a class of analgesics that includes traditional nonselective and selective cyclooxygenase-2 inhibitors that block the biosynthesis of prostaglandins and thromboxane. Indomethacin is a nonsteroidal anti-inflammatory drug with potent antipyretic, analgesic, and anti-inflammatory activity that has been effectively used in the management of mild-to-moderate pain since the mid-1960s. It is commonly prescribed for the relief of acute gouty arthritis pain, but has demonstrated efficacy in the treatment of various other painful conditions. Numerous comparative studies have affirmed the clinical utility of indomethacin relative to other widely used analgesics. This review provides an historic overview of indomethacin and its efficacy compared with other commonly used analgesics, and discusses new indomethacin drug products.

Nonsteroidal Anti-Inflammatory Drugs and the Heart

Aspirin has been on the market for 115 years. Beginning with the marketing of indomethacin for the treatment of rheumatoid arthritis in 1963, at least 20 other nonsteroidal anti-inflammatory drugs (NSAIDs) with aspirin-like actions have been developed over the past 50 years,1 culminating with the introduction of a new class of selective inhibitors of cyclooxygenase (COX)-2, the coxibs, approximately 15 years ago.2 The NSAIDs represent the single most crowded family of drugs sharing the same therapeutic activities and mechanism of action, perhaps reflecting the unmet therapeutic need in the area of pain management and the large interindividual variability in response to these agents. NSAIDs provide symptomatic relief of pain and inflammation associated with a variety of human disorders, including the rheumatic diseases. Their shared therapeutic actions (ie, analgesic, anti-inflammatory, and antipyretic) are usually accompanied by mechanism-based adverse effects that can, at least in part, be attenuated as a function of individual pharmacokinetic or pharmacodynamic properties.1 Nuances in the tolerability of different NSAIDs had been described in the precoxib era on the basis of clinical trials of a few hundred patients treated for up to a few months with soft end points. More recently, however, important differences in safety have been demonstrated in head-to-head randomized comparisons of individual coxibs and 1 or more traditional NSAIDs that involved tens of thousands of patients treated for up to a few years with hard end points. Even more significantly, the interpretation of the effects of NSAIDs has been greatly enhanced by the availability, for the first time, of large, placebo-controlled trials that aimed to assess the potential for chemoprevention of colorectal cancer by rofecoxib or celecoxib. As a result of these developments, the whole field of NSAIDs has been illuminated during the past 15 years. Although epidemiological studies had previously associated …

Cyclooxygenase-2 in Endothelial and Vascular Smooth Muscle Cells Restrains Atherogenesis in Hyperlipidemic Mice

Placebo-controlled trials of nonsteroidal anti-inflammatory drugs selective for inhibition of cyclooxygenase-2 (COX-2) reveal an emergent cardiovascular hazard in patients selected for low risk of heart disease. Postnatal global deletion of COX-2 accelerates atherogenesis in hyperlipidemic mice, a process delayed by selective enzyme deletion in macrophages. In the present study, selective depletion of COX-2 in vascular smooth muscle cells and endothelial cells depressed biosynthesis of prostaglandin I2 and prostaglandin E2, elevated blood pressure, and accelerated atherogenesis in Ldlr knockout mice. Deletion of COX-2 in vascular smooth muscle cells and endothelial cells coincided with an increase in COX-2 expression in lesional macrophages and increased biosynthesis of thromboxane. Increased accumulation of less organized intimal collagen, laminin, α-smooth muscle actin, and matrix-rich fibrosis was also apparent in lesions of the mutants. Although atherogenesis is accelerated in global COX-2 knockouts, consistent with evidence of risk transformation during chronic nonsteroidal anti-inflammatory drug administration, this masks the contrasting effects of enzyme depletion in macrophages versus vascular smooth muscle cells and endothelial cells. Targeting delivery of COX-2 inhibitors to macrophages may conserve their efficacy while limiting cardiovascular risk.