COX Activity Research Articles

Effect of interleukin-1β on ICAM-1 expression of dental pulp cells: role of PI3K/Akt, MEK/ERK, and cyclooxygenase

Interleukin-1β (IL-1β) is an important inflammatory mediator of the dental pulp. IL-1β stimulates cyclooxygenase-2 (COX-2) expression and prostanoid production of pulp cells and affects the inflammatory and healing processes of the dental pulp. There are two interleukin-1 (IL-1) receptors, IL-1RI and IL-1RII, with opposing effect after activation. However, the expression of IL-1Rs, the effects of IL-1β on intercellular adhesion molecule-1 (ICAM-1) of dental pulp cells, and its relation to protein kinase B (Akt) signaling and COX activation are not clear. Human dental pulp cells were treated with IL-1β with/without pretreatment and co-incubation by LY294002 (a PI3K/Akt inhibitor), U0126 [a mitogen-activated protein kinase kinase/extracellular signal-regulated kinase (MEK/ERK) inhibitor], aspirin (a COX inhibitor), or eugenol (a COX inhibitor) for different time periods. The expression of ICAM-1, IL-1RI, and IL-1RII messenger RNA (mRNA) was evaluated by reverse transcriptase-polymerase chain reaction (RT-PCR). ICAM-1 protein expression was examined by western blotting. Soluble ICAM-1 (sICAM-1) level in the culture medium was determined by enzyme-linked immunosorbent assay (ELISA). Activation of Akt and ERK by IL-1β was measured by Pathscan p-Akt ELISA or western blot. Viable cell number was evaluated by 3-(4,5-dimethyldiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. Dental pulp cells expressed IL-1RI, but little IL-1RII. IL-1β stimulated COX-2 and ICAM-1 mRNA and protein expression as well as sICAM-1 production of pulp cells. Aspirin and eugenol enhanced the IL-1β-induced sICAM-1 production and ICAM-1 expression. IL-1β rapidly activated Akt and ERK. LY294002 and U0126 attenuated IL-1β-induced ICAM-1 expression and sICAM-1 production. These results reveal that IL-1β may be involved in the pulpal inflammatory processes by stimulating ICAM-1 expression and secretion. These events are associated with IL-1RI expression and differential activation of PI3K/Akt and MEK/ERK and COX. Pharmacological inhibition of IL-1β, IL-1RI, COX-2, ICAM-1, and related signaling pathways (MEK/ERK and PI3K/Akt) may be useful for the control of pulpal inflammation.

A series of NSAID-induced anaphylactic response to immunotherapy and a proposal to include NSAIDS avoidance in future immunotherapy guidelines

A review of anaphylaxis in a clinic over three years was performed. A co-factor for anaphylaxis stood out as a commonality to all reactions. All five patients had a non steroidal anti-inflammatory drug (NSAID) 24 hours prior to immunotherapy. Among symptoms, urticaria was the commonest; others included cough, angioedema, dyspnea, nausea, vomiting, hypotension and tachycardia. The symptoms appeared within 5 minutes to two hours post-injection. All reactions resolved after few hours. In all cases, NSAID use pre-injection was the only common factor. Two patients also had moderate exercise post-injection, but previous immunotherapy was without incident. All but one patient were administered epinephrine in clinic and recovered without significant morbidity, some were also given cetirizine as adjunct treatment and all were observed until symptoms resolved. While two patients stopped immunotherapy, three patients continued without incident and are now on maintenance dose. The one patient who did not receive epinephrine presented to a walk in clinic for her reaction and received antihistamine treatment alone. NSAID use, although overlooked in the literature, is a common cofactor in anaphylaxis in response to immunotherapy. At Torontoallergists™, a clinic with three allergists in practice, 5 such cases among approximately 3 600 injections in the last three years were noted after extensive chart review. Therefore an NSAID was associated with anaphylaxis in 0.0014% of the immunotherapy injections, whereas two cases of anaphylaxis involved NSAID and exercise involvement. No cases implicating other risk factors or co-factors for anaphylaxis during immunotherapy, such as dosage errors, or injection during asthma exacerbation were present [1]. It is speculated that aspirin and other NSAIDS lower the threshold for anaphylaxis after allergen injections through their COX-inhibiting mechanism of action [2]. The COX pathway synthesizes, from arachidonic acids, prostaglandin D2 and E2, are repressors of inflammatory mediator release from basophils and mast cells [3]. Therefore, NSAIDS increases the likelihood of anaphylaxis after immunotherapy by suppression of prostaglandin D2 and E2, which would normally inhibit histamine release. Supporting this mechanism is Marone et al’s study where higher concentration of NSAIDS and more inhibition of COX activity correlated with higher release of histamine [2]. However, in spite of the data about NSAID acting as a co-factor for anaphylaxis, NSAID avoidance around immunotherapy cannot be found in practice parameters from the AAAAI, ACAAI, and CSACI [4,5]. Therefore, as a patient safety precaution, we highly encourage NSAIDS avoidance to be included for future immunotherapy practice guidelines. We welcome other centers to corroborate.

Running performance at high running velocities is impaired but V'O(₂max) and peripheral endothelial function are preserved in IL-6⁻/⁻ mice.

It has been reported that IL-6 knockout mice (IL-6−/−) possess lower endurance capacity than wild type mice (WT), however the underlying mechanism is poorly understood. The aim of the present work was to examine whether reduced endurance running capacity in IL-6−/− mice is linked to impaired maximal oxygen uptake (V′O2max), decreased glucose tolerance, endothelial dysfunction or other mechanisms. Maximal running velocity during incremental running to exhaustion was significantly lower in IL-6−/− mice than in WT mice (13.00±0.97 m.min−1 vs. 16.89±1.15 m.min−1, P<0.02, respectively). Moreover, the time to exhaustion during running at 12 m.min−1 in IL-6−/− mice was significantly shorter (P<0.05) than in WT mice. V′O2max in IL-6−/− (n = 20) amounting to 108.3±2.8 ml.kg−1.min−1 was similar as in WT mice (n = 22) amounting to 113.0±1.8 ml.kg−1.min−1, (P = 0.16). No difference in maximal COX activity between the IL-6−/− and WT mice in m. soleus and m. gastrocnemius was found. Moreover, no impairment of peripheral endothelial function or glucose tolerance was found in IL-6−/− mice. Surprisingly, plasma lactate concentration during running at 8 m.min−1 as well at maximal running velocity in IL-6−/− mice was significantly lower (P<0.01) than in WT mice. Interestingly, IL-6−/− mice displayed important adaptive mechanisms including significantly lower oxygen cost of running at a given speed accompanied by lower expression of sarcoplasmic reticulum Ca2+-ATPase and lower plasma lactate concentrations during running at submaximal and maximal running velocities. In conclusion, impaired endurance running capacity in IL-6−/− mice could not be explained by reduced V′O2max, endothelial dysfunction or impaired muscle oxidative capacity. Therefore, our results indicate that IL-6 cannot be regarded as a major regulator of exercise capacity but rather as a modulator of endurance performance. Furthermore, we identified important compensatory mechanism limiting reduced exercise performance in IL-6−/− mice.

Xanthoceraside ameliorates mitochondrial dysfunction contributing to the improvement of learning and memory impairment in mice with intracerebroventricular injection of aβ1-42.

The effects of xanthoceraside on learning and memory impairment were investigated and the possible mechanism associated with the protection of mitochondria was also preliminarily explored in Alzheimer's disease (AD) mice model induced by intracerebroventricular (i.c.v.) injection of Aβ1-42. The results indicated that xanthoceraside (0.08–0.32 mg/kg) significantly improved learning and memory impairment in Morris water maze test and Y-maze test. Xanthoceraside significantly reversed the aberrant decrease of ATP levels and attenuated the abnormal increase of ROS levels both in the cerebral cortex and hippocampus in mice injected with Aβ1-42. Moreover, xanthoceraside dose dependently reversed the decrease of COX, PDHC, and KGDHC activity in isolated cerebral cortex mitochondria of the mice compared with Aβ1-42 injected model mice. In conclusion, xanthoceraside could improve learning and memory impairment, promote the function of mitochondria, decrease the production of ROS, and inhibit oxidative stress. The improvement effects on mitochondria may be through withstanding the damage of Aβ to mitochondrial respiratory chain and the key enzymes in Kreb's cycle. Therefore, the results from present study and previous study indicate that xanthoceraside could be a competitive candidate for the treatment of AD.

Abstract P6-12-11: Celecoxib inhibits the growth of IBC tumors by suppressing the regulation of cancer stem-like cells by nodal

Abstract Background: Inflammatory breast cancer (IBC) is an aggressive type of breast cancer with no known molecular targets for treatment. Although erythema is commonly associated with IBC, the molecular mechanism of inflammation in the pathogenesis of IBC remains unknown. We have previously shown that EGFR is an emerging target in IBC (Zhang D. et al Clin Can Res 2009). As crosstalk between EGFR and COX-2 plays an important role in the inflammatory response in several cancers, including breast cancer, we hypothesized that COX-2 promotes the tumorigenesis and metastasis of IBC cells. Methods: Using clinically derived IBC and non-IBC tumor samples, a Spearman's Rank correlation coefficient analysis was performed to analyze the expression levels of COX-2 and EGFR in IBC and non-IBC. The levels of COX-2 metabolites, prostaglandins (PGs) PGE2 and PGF2α, were measured in IBC and non-IBC cell lines by HPLC/MS method. Cell migration and invasion assays were performed using SUM149 and KPL-4 IBC cell lines treated with PGs or the COX-2 inhibitor, celecoxib. We evaluated the epithelial to mesenchymal transition (EMT)-like phenotype in 3D culture of SUM149 cells treated with celecoxib, and the stem-like population by mammosphere formation, and CD44+/CD24− and aldefluor+ population by FACS. We treated preclinical IBC xenograft mice with celecoxib and measured tumor growth, PGs levels, and the expression of EMT protein markers. Nodal, a stem cell regulator and potential biomarker for breast cancer progression, was evaluated in IBC cells following treatment with celecoxib and recombinant Nodal or transfection with Nodal cDNA. Results: EGFR and COX-2 expression levels positively correlated within IBC, but not non-IBC tumors. Elevated levels of PGE2 and PGF2α were identified in multiple IBC cell lines suggesting that COX activity is elevated within IBC compared to non-IBC cells. PGs altered EMT protein markers and promoted cell migration and invasion, while Celecoxib inhibited EMT and migration and invasion in SUM149 and KPL-4 cells. Celecoxib treatment inhibited tumor growth in mice, and downregulated the expression of EMT protein markers, including Nodal. Celecoxib decreased the stem-like CD44+/CD24−, and aldefluor+ population and the formation of mammospheres. Exogenous Nodal mitigated the effects of celecoxib on cell migration and invasion and the stem-like population in SUM149 cells. Conclusion: We conclude that activation of the COX-2 inflammatory signaling pathway is critical in the development and progression of IBC. This study provides a novel insight into how inflammation may regulate cancer stem cells via Nodal, and will guide future research into the development of stem cell targeted therapies for IBC. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P6-12-11.

Crosstalk between nitric oxide synthases and cyclooxygenase 2 in the adrenal cortex of rats under lipopolysaccharide treatment

The effect of lipopolysaccharide on the modulation of steroid production by adrenal cells has been recently acknowledged. The purpose of this study was to determine the in vivo effects of LPS on adrenal cyclooxygenase 2 (COX-2) expression, analyze its crosstalk with the nitric oxide synthase (NOS) system, and assess its involvement on the modulation of glucocorticoid production. Male Wistar rats were injected with LPS and with specific inhibitors for NOS and COX activities. PGE2 and corticosterone levels were determined by RIA. Protein levels were analyzed by immunoprecipitation and western blotting. Transfection assays were performed in murine adrenocortical Y1 cells. Results show that LPS treatment increases PGE2 production and COX-2 protein levels in the rat adrenal cortex. Systemic inhibition of COX-2 blunted the glucocorticoid response to ACTH, as well as the increase in NOS activity and the NOS-2 expression levels induced by LPS. Conversely, NOS inhibition prevented the LPS-dependent increase in PGE2 production, COX-2 protein levels, and the nitrotyrosine modification of COX-2 protein. Treatment of adrenocortical cells with a NO-donor significantly potentiated the LPS-dependent increase in NFκB activity and COX-2 expression levels. In conclusion, our results show a significant crosstalk between COX-2 and NOS in the adrenal cortex upon LPS stimulation, in which each activity has a positive impact on the other. In particular, as both the activities differently affect adrenal steroid production, we hypothesize that this kind of fine modulation enables the gland to adjust steroidogenesis to prevent either an excessive or an insufficient response to the endotoxin challenge.

Cardioprotective Effects of Sour Cherry Seed Extract (SCSE) on the Hypercholesterolemic Rabbit Heart

The present study evaluates the hypothesis that sour cherry seed extract (SCSE) protects against cardiovascular disease and inflammation in hypercholesterolemic rabbits, and that this protection correlates with SCSE-induced activity of heme oxygenase- 1 (HO-1), a cytoprotective enzyme contributing to oxidative stress responses. 18 New Zealand white rabbits were divided into three groups receiving: I. cholesterol-free rabbit chow; II. chow containing 2% cholesterol; or III. 2% cholesterol plus SCSE for 16 weeks. Heart functions were monitored by echocardiography 0, 4, and 16 weeks after the initiation of cholesterol-supplemented feeding. At the 16-week time-point, isolated hearts were subjected to ischemia-reperfusion (I/R), followed by measurement of heart rate (HR), aortic flow (AF), coronary flow (CF), aortic pressure (AoP), and left ventricular developed pressure (LVDP). Myocardial infarct size was determined using triphenyl tetrazolium chloride (TTC). Quantification of fatty streaks was assessed using Sudan-III staining. Western blot analysis was used to determine the content of cytochrome c oxidase III (COX III), vascular endothelial growth factor (VEGF), and HO-1 in the myocardium. Relative to cholesterol-treated animals not receiving SCSE, SCSE-treated animals exhibited significantly improved cardiac function and improved peak early diastolic velocity to peak atrial velocity ratio (E'/A'), along with decreased atherosclerotic plaque formation and infarct size. Increased HO-1 and COX III protein expression and COX activity were also noted in hearts from SCSE-treated rabbits. This study demonstrates SCSE cardioprotective effects on hypercholesterolemic hearts. Correlation of these outcomes with HO-1 expression suggests that the effect may be mediated by activity of this enzyme. However, definitive proof of HO-1 dependence requires further investigation.

Aspirin Therapy for Colorectal Cancer With PIK3CA Mutation: Simply Complex!

Prospective observational studies and randomized clinical trials have demonstrated that regular use of aspirin and selective cyclooxygense-2 (COX-2) inhibitors reduces the risk of developing colorectal cancer (CRC) and adenoma. More recently, large observational studies have suggested that regular aspirin use may also be associated with improved survival among patients with established CRC. The mechanisms by which aspirin influences CRC risk and possibly survival are poorly understood. COX-2 (or prostaglandinendoperoxide synthase 2 [PTGS2]) promotes inflammation and cell proliferation, and most adenomas and CRCs overexpress this enzyme. Previous studies have reported that aspirin may preferentially reduce the risk of COX-2–expressing CRC and also preferentially improve the survival of patients with CRC with COX-2–expressing tumors. Nonetheless, in laboratory models, aspirin seems to decrease proliferation and increase apoptosis in CRC cell lines without detectable COX activity. Ongoing studies have sought to define the molecular basis for the effect of aspirin on CRC risk and progression. In a large observational study of patients with CRC, Liao et al found that aspirin conferred improved survival in patients with PIK3CA-mutated CRC, but not among those with PIK3CA–wild-type tumors. In the report accompanying our article in Journal of Clinical Oncology, Domingo et al similarly observe a preferential benefit for aspirin in PIK3CA-mutated CRC in a large randomized trial of rofecoxib in patients with stage II/III CRC (VICTOR [Vioxx in Colorectal Cancer Therapy: Definition of Optimal Regime] trial), potentially moving us closer to clinical use of PIK3CA mutation as a predictive biomarker for aspirin use in patients with CRC. Here, we review some of the relevant complex relationships between aspirin, phosphatidylinositol 3-kinase (PI3K) signaling, and other cellular interactions.

Modulation of COX, LOX and NFκB activities by Xanthium spinosum L. root extract and ziniolide

Xanthium spinosum L. (Asteraceae) is a medicinal weed distributed worldwide. Many of its diverse ethnopharmacological uses – namely diarrhoea, inflammation, liver disorders, snake bite and fever – are linked – at least in part – to an uncontrolled release of arachidonic acid metabolites. The crude extract of X. spinosum roots from Jordanian origin dose-dependently inhibited the 5-LOX (IC50≅10μg/mL), COX-1(IC50≅50μg/mL), and 12-LOX (IC50≅170μg/mL) enzymatic pathways in intact pro-inflammatory cells. A direct activity at the level of PLA2 is not probable, but the extract induced the synthesis of the anti-inflammatory eicosanoid 15(S)-HETE, which may in turn inhibit this enzyme. 5-LOX bioguided fractionation of the crude extract led to the isolation of ziniolide, a known 12,8-guaianolide sesquiterpene lactone, from the hydro-alcoholic fraction of the n-hexane extract (IC50=69μM). Both the plant extract and ziniolide are in vitro inhibitors of the phorbol-induced NFκB activation, a key regulator of the arachidonic pathway.

Effect of Phenylbutazone, Flunixin Meglumine and Firocoxib on ex vivo Cyclo‐Oxygenase Activity in Horses Undergoing Elective Surgery

AimsNonsteroidal anti‐inflammatory drugs (NSAIDs) inhibit the production of prostaglandins and other inflammatory mediators by inhibiting the activity of the cyclo‐oxygenase enzymes (COX). Two major isoforms of COX enzymes exist: COX‐2, which is expressed during the inflammatory response, and COX‐1, which is responsible for the physiological production of prostaglandin that regulates tissue homeostasis. The study aims to evaluate the effect of firocoxib ex vivo in the horse as, to the authors' knowledge, published studies assess its effect only in vitro.MethodsHorses (n = 18) undergoing elective surgery were recruited and allocated to treatment groups depending on clinician preference (1) phenylbutazone (4.4 mg/kg bwt i.v. b.i.d.), (2) flunixin meglumine (FM, 1.1 mg/kg bwt i.v. b.i.d.) and (3) firocoxib (FIR, 0.1 mg/kg bwt i.v. s.i.d.). Residual blood samples were collected prior to NSAIDs (T0), 2 h after NSAIDs (T2), and 24 h following surgery (T24). The COX activity was measured using validated immune‐enzymatic assays. A Kruskall–Wallis test was used to determine the effect of time and treatment on COX‐1 and COX‐2 activity. Bonferroni corrections were used to identify the level of significance accounting for multiple comparisons (P&lt;0.017).ResultsAt T2 and T24, the relative COX‐1 activity was significantly greater in horses receiving firocoxib compared with horses receiving either phenylbutazone (P&lt;0.008) or flunixin meglumine (P&lt;0.005). At T2 and T24, COX‐1 activity was reduced (compared with baseline) in horses receiving phenylbutazone or flunixin meglumine. The effect on COX‐2 activity was not significantly different between drugs (P = 0.471).Conclusions and practical significanceCyclo‐oxygenase selectivity of firocoxib is demonstrated ex vivo. Firocoxib is as effective as phenylbutazone or flunixin meglumine in modulating the production of prostaglandins by COX‐2 isoenzyme, whilst the physiological action of COX‐1 isoenzyme is preserved with firocoxib, but not with phenylbutazone and flunixin ex vivo.Ethical animal researchStudy approved by Ethics and Welfare Committee ‐ School of Veterinary Medicine, College of Medical, Veterinary and Life Sciences, University of Glasgow. Sources of funding: Funding provided by John Crawford Endowment Fund, and Mrs I.J. Gates Charity Fund, University of Glasgow. Competing interests: None.

Enhanced Production of Cholesterol Oxidase from Bacillus sp. COX-T&lt;sub&gt;3&lt;/sub&gt; in a Biphasic System

Cholesterol oxidase (COX, E.C.1.1.3.6), catalyses the oxidation of cholesterol to 4-cholesten-3-one with the reduction of oxygen to hydrogen peroxide. Qualitative analysis of bacterial strain (Bacillus sp. BT COX-T3) gave Gram positive rod-shaped colonies with high cholesterol degradation. Organic solvent tolerant microorganisms are novel group of extremophilic microorganisms that have developed resistance to withstand solvent toxicity. These organisms play an important role in biotransansformation and bioconversion of organic compounds. An organic solvent-tolerant Bacillus sp. BT COX-T3 obtained after primary screening for production of extracellular COX transformed cholesterol to 4-cholesten- 3-one in toluene, and resulted in a higher COX production in a biphasic medium due to an increase in solubility of cholesterol in the medium making it easily available to be metabolized by the organism. A simple screening method for 6β-hydroperoxycholest-4-en-3-one formed by Bacillus sp. COX-T3 was devised in this study and bioconversion was monitored spectrophotometerically. Presence of cholesterol in 20% (v/v) toluene was observed to be most effective biotransformation system for bacterial COX production. A maximum COX activity of 0.892 U/ml was obtained at 45°C by using 8% (v/v) inoculum, 0.1% (w/v) cholesterol as the main carbon source and 0.5% (w/v) yeast extract. Amongst various salt ions only Co2+ ions improved the COX production by Bacillus sp. COX-T3. Moreover, the cholesterol provided in the bioconversion system was completely transformed by Bacillus sp. COX-T3. Bacillus sp COX-T3 effectively degraded cholesterol in the presence of toluene and the extracellular COX produced by it was highly stable at the end of 144 h. Keywords: Cholesterol, cholesterol oxidase, bioconversion, solvent tolerant.

A stabilizing factor for mitochondrial respiratory supercomplex assembly regulates energy metabolism in muscle

The mitochondrial respiratory chain is essential for oxidative phosphorylation and comprises multiple complexes, including cytochrome c oxidase, assembled in macromolecular supercomplexes. Little is known about factors that contribute to supercomplex organization. Here we identify COX7RP as a factor that promotes supercomplex assembly. Cox7rp-knockout mice exhibit decreased muscular activity and heat production failure in the cold due to reduced COX activity. In contrast, COX7RP-transgenic mice exhibit increased exercise performance with increased cytochrome c oxidase activity. Two-dimensional blue native electrophoresis reveals that COX7RP is a key molecule that promotes assembly of the III2/IVn supercomplex with complex I. Our study identified COX7RP as a protein that functions in I/III2/IVn supercomplex assembly and is required for full activity of mitochondrial respiration.

Effects of environmental enrichment on anxiety responses, spatial memory and cytochrome c oxidase activity in adult rats

We have studied the effect of an environmental enrichment (EE) protocol in adult Wistar rats on the activity in the elevated zero-maze (EZM), performance in the radial-arm water maze (RAWM) and we have also examined the changes in the neuronal metabolic activity of several brain regions related to anxiety response and spatial memory through cytochrome c oxidase histochemistry (COx). Our EE protocol had anxiolytic effect in the EZM; the animals spent more time and made more entries into the open quadrants, they had lower latency to enter into the open quadrant and lower levels of defecation. Also, the EE group showed fewer working memory and reference memory errors, as well as lesser distance travelled in the first day of the spatial training. In relation to the neuronal metabolic activity, EE reduced the COx activity in brain regions related to anxiety response, such as the infralimbic cortex, the paraventricular thalamic and hypothalamic nucleus, the basolateral amygdala, and the ventral hippocampus. Interestingly, there were no significant differences between groups in the dorsal hippocampus, more related to spatial cognition. These results suggest a beneficial effect of EE on spatial memory as a result of reducing anxiety levels and the COx activity in brain regions involved in anxiety response. We also found a differential pattern of activation inside the hippocampus, suggesting that the dorsal hippocampus has a preferential involvement in spatial learning and memory, whereas the ventral hippocampus has a role in anxiety response.

Cysteinyl leukotriene-receptor-1 antagonists interfere with PGE2 synthesis by inhibiting mPGES-1 activity

Because of their favourable safety profile and beneficial anti-inflammatory properties, the CysLT1 receptor antagonists (LTRA), montelukast, zafirlukast and pranlukast are approved for the treatment of asthma and are frequently prescribed as add-on therapeutics to reduce the amount of inhaled glucocorticoids and β2-agonists. There is evidence that some of these anti-inflammatory properties might be of a secondary nature and therefore, unrelated to the CysLT1 antagonism. Here, we show that LTRA inhibit PGE2 formation in cytokine-stimulated Hela and A549 carcinoma cells and in lipopolysaccharide (LPS)-stimulated human leukocyte preparations (IC50∼20μM). Neither expression of enzymes involved in PGE2 synthesis nor arachidonic acid release and COX activities were inhibited by the compounds. In contrast, mPGES-1 activity was suppressed at low micromolar levels (IC50 between 2 and 4μM). This suppression was specific for PGE2 synthesis, since PGD2 and PGI2 levels in LPS-stimulated leukocyte preparations were not negatively affected. PGF2α levels were concomitantly inhibited, probably due to its direct synthesis from PGE2. Several major conclusions can be drawn from this study: (A) clinical trials investigating elevated doses of the compounds are helpful to confirm suppression of PGE2 synthesis in vivo; (B) studies investigating the role of CysLTs in cell culture or animal models of inflammation and cancer have to be reassessed carefully, if higher doses of LTRA were applied or serum levels in cell culture assays were low; and (C) LTRA may serve as new scaffolds for the development of potent, selective and well tolerated mPGES-1 inhibitors.

Increased Levels of Urinary PGE-M, a Biomarker of Inflammation, Occur in Association with Obesity, Aging, and Lung Metastases in Patients with Breast Cancer

Elevated levels of COX-derived prostaglandin E2 (PGE2) occur in inflamed tissues. To evaluate the potential links between inflammation and breast cancer, levels of urinary prostaglandin E metabolite (PGE-M), a stable end metabolite of PGE2, were quantified. We enrolled 400 patients with breast cancer: controls with early breast cancer (n = 200), lung metastases (n = 100), and metastases to other sites (n = 100). Patients completed a questionnaire, provided urine, and had measurements of height and weight. Urinary PGE-M was quantified by mass spectrometry. Ever smokers with lung metastasis who had not been exposed to nonsteroidal anti-inflammatory drugs (NSAIDs) had the highest PGE-M levels. PGE-M levels were increased in association with elevated body mass index (BMI; P < 0.001), aging (P < 0.001), pack-year smoking history (P = 0.02), lung metastases (P = 0.02), and recent cytotoxic chemotherapy (P = 0.03). Conversely, use of NSAIDs, prototypic inhibitors of COX activity, was associated with reduced PGE-M levels (P < 0.001). On the basis of the current findings, PGE-M is likely to be a useful biomarker for the selection of high-risk subgroups to determine the use of interventions that aim to reduce inflammation and possibly the development and progression of breast cancer, especially in overweight and obese women.

Cyclooxygenase-2 in newborn hyperoxic lung injury

Supraphysiological O2 concentrations, mechanical ventilation, and inflammation significantly contribute to the development of bronchopulmonary dysplasia (BPD).Exposure of newborn mice to hyperoxia causes inflammation and impaired alveolarization similar to that seen in infants with BPD.Previously, we demonstrated that pulmonary cyclooxygenase-2 (COX-2) protein expression is increased in hyperoxia-exposed newborn mice.The present studies were designed to define the role of COX-2 in newborn hyperoxic lung injury.We tested the hypothesis that attenuation of COX-2 activity would reduce hyperoxia-induced inflammation and improve alveolarization.Newborn C3H/HeN micewere injected daily with vehicle, aspirin (nonselective COX-2 inhibitor), or celecoxib (selective COX-2 inhibitor) for the first 7 days of life.Additional studies utilized wild-type (C57Bl/6, COX-2+/+), heterozygous (COX-2+/-), and homozygous (COX-2-/-) transgenic mice.Micewere exposed to room air (21% O2) or hyperoxia (85% O2) for 14 days.Aspirin-injected and COX-2-/- pups had reduced levels of monocyte chemoattractant protein (MCP-1) in bronchoalveolar lavage fluid (BAL).Both aspirin and celecoxib treatment reduced macrophage numbers in the alveolar walls and air spaces.Aspirin and celecoxib treatment attenuated hyperoxia-induced COX activity, including altered levels of prostaglandin (PG)D2 metabolites.Decreased COX activity, however, did not prevent hyperoxia-induced lung developmental deficits.Our data suggest thatincreased COX-2 activity may contribute to proinflammatory responses, including macrophage chemotaxis, during exposure to hyperoxia.Modulation of COX-2 activity may be a useful therapeutic target to limit hyperoxia-induced inflammation in preterm infants at risk of developing BPD.

Abstract 3286: CLEFMA synergizes with celecoxib in suppression of lung cancer cell growth.

Abstract Background: Cyclooxygenase-2 (COX-2) is frequently over-expressed in various malignancies, often associated with poor clinical outcome. Celecoxib, a selective COX-2 inhibitor induces apoptosis in cancer cells. However, at higher concentrations needed for chemoprevention or treatment, celecoxib targets COX-2 independent drug targets resulting in undesired toxicity. CLEFMA or 4-[3, 5-bis (2-chlorobenzylidene-4-oxo-piperidine-1-yl)-4-oxo-2-butenoic acid], a novel molecule synthesized in our laboratory potently inhibits the proliferation of various cancer cells while sparing normal cells. In this study we have tested a hypothesis that CLEFMA would reduce the celecoxib dose required for effective suppression of cancer cell proliferation. Methods: We determined IC50 values of CLEFMA and celecoxib in lung adenocarcinoma (H441 and A549) and small cell lung cancer (H1650) cells. Further, we tested their toxicity in normal lung fibroblast CCL151 cells at 1x IC50 and 2x IC50 dose levels required for H441 cells. Drug concentrations at ratio 1:3 (Mixture 1), 1:1 (Mixture 2) and 3:1 (Mixture 3) of respective IC50 values of CLEFMA and celecoxib were examined for synergism. The data was analyzed with the Calcusyn software. Following synergy experiments, we determined the effect of CLEFMA treatment on total COX and COX-2 enzymatic activity in cultured A549 cells and in tumor tissue lysates obtained from H441 xenografts in mice. Expression of anti-apoptotic (BCL-2, Bcl-XL and surviving) and apoptotic proteins (PARP, caspase-3 and caspase-7) were investigated by immunobloting. Results: We observed that CLEFMA was more effective in suppressing the proliferation of all three cancer cell lines than celecoxib. Celecoxib showed significant toxicity in normal lung fibroblasts at 2x IC50 dose, whereas CLEFMA was non-toxic to the normal cells. From the fractional effect-combination index (Fa-CI) plots, we found that Mixture 1 (1:3:: CLEFMA:celecoxib) was synergistic (CI &amp;lt; 1.0) in all three cell lines; in H441 cells mixture 2 (1:1) was synergistic at lower concentration of the drugs, Mixture 3 (3:1) was synergistic only in A549 cells. CLEFMA does not reduce COX-2 activity/unit enzyme, but reduced the expression levels of COX-2 protein; the combination of CLEFMA with celecoxib synergistically suppressed COX-2 expression. CLEFMA also potentiated the activity of celecoxib in down-regulating Bcl-2, Bcl-XL and survivin (anti-apoptotic proteins) and up-regulating PARP, caspase-3 and caspase-7 (pro-apoptotic proteins). Conclusions: From the results we conclude that CLEFMA synergistically augments the growth inhibitory effect of celecoxib in human lung cancer cell lines without being toxic to normal cells. It appears that the synergy depends on the different mode of actions of the two drugs- CLEFMA suppressing COX-2 protein expression, whereas celecoxib reducing COX-2 activity. Citation Format: Kaustuv K. Sahoo, Vivek R. Yadav, Vibhudutta Awasthi. CLEFMA synergizes with celecoxib in suppression of lung cancer cell growth. [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 3286. doi:10.1158/1538-7445.AM2013-3286

Abstract 3025: Receptor tyrosine kinase ErbB2/HER2 translocates into mitochondria and regulates cellular metabolism.

Abstract It is well accepted that ErbB2, an important oncoprotein, localizes on the plasma membrane as a receptor tyrosine kinase. This study describes a novel observation that ErbB2 localizes in mitochondria of multiple cancer cell lines and patient samples. We identified an endogenous mitochondrial targeting sequence in ErbB2 and found that ErbB2 translocates into mitochondria through the association with mtHSP70, a key player in the canonical mitochondrial protein importation pathway. Additionally, we observed that mitochondrial ErbB2 (mtErbB2) negatively regulates mitochondrial respiratory functions. Oxygen consumption and activities of complex I, II and IV of the mitochondrial electron transport chain were decreased in mtErbB2-overexpressing cells. Mitochondrial membrane potential and the cellular ATP level also were decreased by mtErbB2. In contrast, mtErbB2 enhanced cellular glycolysis. The translocation of ErbB2 and its impact on mitochondrial function are kinase dependent. Mitochondrial ErbB2 regulates the phosphorylation and activity of COX II and consequently the cytochrome c release and apoptosis. Additional studies showed that cancer cells with higher levels of mtErbB2 were more resistant to Trastuzumab and deletion of the mitochondrial targeting sequence of ErbB2 sensitized the cells to Trastuzumab. Our study provides a novel perspective on the metabolic regulatory functions of ErbB2 and reveals that mtErbB2 plays an important role in the regulation of cellular metabolism and cancer cell resistance to therapeutics. Since ErbB2 plays important roles in multiple organs and multiple types of cancers, the study may have broad impact on the fields of cancer biology. The work was supported by The Vincent F. Kilborn Jr. Cancer Research Foundation and National Institutes of Health Grant RO1CA149646. Citation Format: Yan Ding, Zixing Liu, Shruti Desai, Lewis Pannell, Hong Yi, Elizabath Wright, Laurie Owen, Windy Dean-Colomb, Oystein Fodstad, Jianrong Lu, Ming Tan. Receptor tyrosine kinase ErbB2/HER2 translocates into mitochondria and regulates cellular metabolism. [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 3025. doi:10.1158/1538-7445.AM2013-3025

Effect of resveratrol and caloric restriction on mitochondrial regulation within different adipose tissues in aged rats

It is well established that impairments in mitochondrial function/regulation contribute to tissue decline with aging. Caloric restriction (CR) and resveratrol (RSV) treatment in rodents induces beneficial mitochondrial adaptations in tissues such as heart and skeletal muscle but whether similar benefits occur in fat is unknown. Thus, we investigated whether RSV (50 mg/kg/day; 6 weeks) and/or CR (20% reduced AL; 6 weeks) could alter mitochondrial regulation/biogenesis in aged rodent adipose tissues (visceral; VIS, epididymal; EPI, brown adipose tissue; BAT). Aged F344xBN rats (26 mo) were divided into 4 groups (n=4): ad libitum (AL), CR, RSV, RSV+CR and mitochondrial content (cyto c, COX activity, and COX I) and mitochondrial signaling/regulation (AMPK, PGC‐1α) were assessed. Expectedly, mitochondrial content (cyto c, COX activity) and PGC‐1α were significantly elevated (~2–3‐fold) in the BAT compared to EPI and VIS fat. CR and RSV tended to increase COX activity (~20–50%) in all adipose depots but showed inconsistencies with other mitochondrial content/signaling markers. Interestingly, combined CR and RSV treatment had no effect and/or suppressed COX activity in all adipose tissues. Our data indicates that short‐term CR and RSV treatment when applied independently appears to enhance mitochondrial content in adipose tissue and may contribute to improvements in health associated with these paradigms.

Gallic acid: Molecular rival of cancer

Gallic acid, a predominant polyphenol, has been shown to inhibit carcinogenesis in animal models and in vitro cancerous cell lines. The inhibitory effect of gallic acid on cancer cell growth is mediated via the modulation of genes which encodes for cell cycle, metastasis, angiogenesis and apoptosis. Gallic acid inhibits activation of NF-κB and Akt signaling pathways along with the activity of COX, ribonucleotide reductase and GSH. Moreover, gallic acid activates ATM kinase signaling pathways to prevent the processes of carcinogenesis. The data so far available, both from in vivo and in vitro studies, indicate that this dietary polyphenol could be promising agent in the field of cancer chemoprevention.