COX-2 Dependent Mechanism Research Articles

Positive crosstalk between EGFR and the TF-PAR2 pathway mediates resistance to cisplatin and poor survival in cervical cancer.

Cisplatin-based chemoradiation is the standard treatment for cervical cancer, but chemosensitizing strategies are needed to improve patient survival. EGFR (Epidermal Growth Factor Receptor) is an oncogene overexpressed in cervical cancer that is involved in chemoresistance. Recent studies showed that EGFR upregulates multiple elements of the coagulation cascade, including tissue factor (TF) and the protease-activated receptors (PAR) 1 and 2. Moreover, many G protein-coupled receptors, including PARs, have been implicated in EGFR transactivation. However, the role of coagulation proteins in the progression of cervical cancer has been poorly investigated. Herein we employed cervical cancer cell lines and The Cancer Genome Atlas (TCGA) database to evaluate the role of EGFR, TF and PAR2 in chemoresistance. The SLIGKL-NH2 peptide (PAR2-AP) and coagulation factor VIIa (FVIIa) were used as PAR2 agonists, while cetuximab was used to inhibit EGFR. The more aggressive cell line CASKI showed higher expression levels of EGFR, TF and PAR2 than that of C33A. PAR2 transactivated EGFR, which further upregulated cyclooxygenase-2 (COX2) expression. PAR2-AP decreased cisplatin-induced apoptosis through an EGFR- and COX2-dependent mechanism. Furthermore, treatment of CASKI cells with EGF upregulated TF expression, while treatment with cetuximab decreased the TF protein levels. The RNA-seq data from 309 TCGA samples showed a strong positive correlation between EGFR and TF expression (P = 0.0003). In addition, the increased expression of EGFR, PAR2 or COX2 in cervical cancer patients was significantly correlated with poor overall survival. Taken together, our results suggest that EGFR and COX2 are effectors of the TF/FVIIa/PAR2 signaling pathway, promoting chemoresistance.

Electroacupuncture prevents endothelial dysfunction induced by ischemia-reperfusion injury via a cyclooxygenase-2-dependent mechanism: A randomized controlled crossover trial.

ObjectiveExploring clinically effective methods to reduce ischemia-reperfusion (IR) injury in humans is critical. Several drugs have shown protective effects, but studies using other interventions have been rare. Electroacupuncture (EA) has induced similar protection in several animal studies but no study has investigated how the effects could be translated and reproduced in humans. This study aimed to explore the potential effect and mechanisms of EA in IR-induced endothelial dysfunction in humans.MethodsThis is a prospective, randomized, crossover, sham-controlled trial consisting of two protocols. Protocol 1 was a crossover study to investigate the effect of EA on IR-induced endothelial dysfunction. Twenty healthy volunteers were randomly assigned to EA or sham EA (sham). Flow mediated dilation (FMD) of the brachial artery (BA), nitroglycerin-mediated endothelial independent dilation, blood pressure before and after IR were measured. In protocol 2, seven volunteers were administered COX-2 inhibitor celecoxib (200 mg orally twice daily) for five days. After consumption, volunteers underwent FMD before and after IR identical to protocol 1.ResultsIn protocol 1, baseline BA diameter, Pre-IR BA diameter and FMD were similar between the two groups (p = NS). After IR, sham group showed significantly blunted FMD (Pre-IR: 11.41 ± 3.10%, Post-IR: 4.49 ± 2.04%, p < 0.001). However, EA protected this blunted FMD (Pre-IR: 10.96 ± 5.30%, Post-IR: 9.47 ± 5.23%, p = NS, p < 0.05 compared with sham EA after IR). In protocol 2, this protective effect was completely abolished by pre-treatment with celecoxib (Pre-IR: 11.05 ± 3.27%; Post-IR: 4.20 ± 1.68%, p = 0.001).ConclusionEA may prevent IR-induced endothelial dysfunction via a COX-2 dependent mechanism.

Abstract A08: Lymphangiogenesis drives breast tumor cell spread in postpartum women and in models of postpartum breast cancer

Abstract Although tumor cell dissemination via the lymphatic vasculature is thought to be a common pathway of metastasis for solid human cancers, the mechanisms of lymphatic mediated metastasis remain poorly understood1. For breast cancer patients, lymph node involvement remains a very important indicator of patient prognosis and is utilized clinically for therapeutic choices2,3. A recent study has shown that the incidence of being diagnosed with stage IV breast cancer, i.e. breast cancer that has spread outside the breast, is increasing in young women4. Furthermore, young women diagnosed with breast cancer within 5 years postpartum are ∼3 times as likely to have metastatic disease5-7. Given that 35-45% of young women's breast cancers are likely to be postpartum8, i.e. diagnosed within 5 years of giving birth, we predict that postpartum breast cancers may be partially driving the observed increase in metastasis of young women's breast cancer. Recently, we have shown that postpartum breast cancer patients have increased tumor associated lymphatic vessel density and increased incidence of lymph node metastasis9. We hypothesized that postpartum tumors promote lymphangiogenesis, which leads to breast tumor metastasis. To test this hypothesis we utilized xenograft and isograft mouse models of postpartum breast cancer, which show increased metastasis in postpartum animals10,11. We utilized these rodent models, along with human breast tissues, to investigate lymphangiogenesis and lymph vessel invasion in the postpartum period. We showed that 1) lymphangiogenesis is enhanced in the postpartum period, 2) that postpartum tumors display increased lymphatic vessel density and lymphatic vessel invasion in the tumor periphery, and 3) that postpartum tumor cells from our rodent models promote lymphangiogenesis via secretion of PGE2 ex vivo9. Recently, we extended our observations to show that lymphatic vessels in breast tissues from postpartum women are more dilated, and in rodent models more leaky, suggesting the lymphatics as a favorable route for breast tumor metastasis in the postpartum period. To test this, we orthotopically injected tumor cells into postpartum mice, or nulliparous controls, and harvested distant lymph nodes at 4 and 6 days post-injection. We observed evidence for tumor cells in distant lymph nodes at higher frequency in the postpartum group at days 4 and 6 compared to the nulliparous controls. Furthermore, in postpartum rodents we also observed increased lymph node size and lymph node vessel density suggesting that expansion of the lymphatic vasculature in postpartum mice may be systemic. Importantly, we have shown that both normal and tumor associated mammary lymphangiogenesis in vivo are dependent upon COX-2 and that COX-2 inhibitors can block metastasis of postpartum tumors in our rodent models. Thus, we are also investigating COX-2 dependent mechanisms that result in alteration of mammary lymphatics and overall lymph node biology. Overall, our results suggest that COX-2 inhibitor, or NSAID, use may reduce lymphogenous tumor cell spread in postpartum women. 1. Stacker, S.A., Baldwin, M.E. &amp; Achen, M.G. The role of tumor lymphangiogenesis in metastatic spread. Faseb J 16, 922-934 (2002). 2. Alitalo, K. &amp; Carmeliet, P. Molecular mechanisms of lymphangiogenesis in health and disease. Cancer Cell 1, 219-227 (2002). 3. Pepper, M.S., Tille, J.C., Nisato, R. &amp; Skobe, M. Lymphangiogenesis and tumor metastasis. Cell Tissue Res 314, 167-177 (2003). 4. Johnson, R.H., Chien, F.L. &amp; Bleyer, A. Incidence of breast cancer with distant involvement among women in the United States, 1976 to 2009. Jama 309, 800-805 (2013). 5. Callihan, E.B., et al. Postpartum diagnosis demonstrates a high risk for metastasis and merits an expanded definition of pregnancy-associated breast cancer. Breast Cancer Res Treat (2013). 6. Johansson, A.L., Andersson, T.M., Hsieh, C.C., Cnattingius, S. &amp; Lambe, M. Increased Mortality in Women with Breast Cancer Detected during Pregnancy and Different Periods Postpartum. Cancer Epidemiol Biomarkers Prev 20, 1865-1872 (2011). 7. Stensheim, H., Moller, B., van Dijk, T. &amp; Fossa, S.D. Cause-specific survival for women diagnosed with cancer during pregnancy or lactation: a registry-based cohort study. J Clin Oncol 27, 45-51 (2009). 8. Schedin, P. Pregnancy-associated breast cancer and metastasis. Nat Rev Cancer 6, 281-291 (2006). 9. Lyons, T.R., et al. Cyclooxygenase-2-dependent lymphangiogenesis promotes nodal metastasis of postpartum breast cancer. The Journal of clinical investigation 124, 3901-3912 (2014). 10. McDaniel, S.M., et al. Remodeling of the mammary microenvironment after lactation promotes breast tumor cell metastasis. Am J Pathol 168, 608-620 (2006). 11. Lyons, T.R., et al. Postpartum mammary gland involution drives progression of ductal carcinoma in situ through collagen and COX-2. Nat Med 17, 1109-1115 (2011). Citation Format: Sarah Black, Virginia F. Borges, Pepper Schedin, Traci R. Lyons. Lymphangiogenesis drives breast tumor cell spread in postpartum women and in models of postpartum breast cancer. [abstract]. In: Proceedings of the AACR Special Conference: Tumor Angiogenesis and Vascular Normalization: Bench to Bedside to Biomarkers; Mar 5-8, 2015; Orlando, FL. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl):Abstract nr A08.

Testosterone induces leucocyte migration by NADPH oxidase-driven ROS- and COX2-dependent mechanisms.

The mechanisms whereby testosterone increases cardiovascular risk are not clarified. However, oxidative stress and inflammation seem to be determinants. Herein, we sought to determine whether exogenous testosterone, at physiological levels, induces leucocyte migration, a central feature in immune and inflammatory responses and the mediating mechanisms. We hypothesized that testosterone induces leucocyte migration via NADPH oxidase (NADPHox)-driven reactive oxygen species (ROS) and cyclooxygenase (COX)-dependent mechanisms. Sixteen-week-old Wistar rats received an intraperitoneal injection (5 ml) of either testosterone (10(-7) mol/l) or saline. Rats were pre-treated with 5 ml of sodium salicylate (SS, non-selective COX inhibitor, 1.25 × 10(-3) mol/l, 1 h prior to testosterone or saline), flutamide (androgen receptor antagonist, 10(-5) mol/l), apocynin (NADPHox inhibitor, 3 × 10(-4) mol/l), N-[2-Cyclohexyloxy-4-nitrophenyl]methanesulfonamide (NS398, COX2 inhibitor, 10(-4) mol/l) or saline, 4 h before testosterone or saline administration. Leucocyte migration was assessed 24 h after testosterone administration by intravital microscopy of the mesenteric bed. Serum levels of testosterone were measured by radioimmunoassay. NADPHox activity was assessed in membrane fractions of the mesenteric bed by dihydroethidium (DHE) fluorescence and in isolated vascular smooth muscle cells (VSMC) by HPLC. NADPHox subunits and VCAM (vascular cell adhesion molecule) expression were determined by immunoblotting. Testosterone administration did not change serum levels of endogenous testosterone, but increased venular leucocyte migration to the adventia, NADPHox activity and expression (P < 0.05). These effects were blocked by flutamide. SS inhibited testosterone-induced leucocyte migration (P<0.05). Apocynin and NS398 abolished testosterone-induced leucocyte migration and NADPHox activity (P<0.05). Testosterone induces leucocyte migration via NADPHox- and COX2-dependent mechanisms and may contribute to inflammatory processes and oxidative stress in the vasculature potentially increasing cardiovascular risk.

Enhanced NF-κB Activity Impairs Vascular Function Through PARP-1–, SP-1–, and COX-2–Dependent Mechanisms in Type 2 Diabetes

Type 2 diabetes (T2D) is associated with vascular dysfunction. We hypothesized that increased nuclear factor-κB (NF-κB) signaling contributes to vascular dysfunction in T2D. We treated type 2 diabetic (db−/db−) and control (db−/db+) mice with two NF-κB inhibitors (6 mg/kg dehydroxymethylepoxyquinomicin twice a week and 500 μg/kg/day IKK-NBD peptide) for 4 weeks. Pressure-induced myogenic tone was significantly potentiated, while endothelium-dependent relaxation (EDR) was impaired in small coronary arterioles and mesenteric resistance artery from diabetic mice compared with controls. Interestingly, diabetic mice treated with NF-κB inhibitors had significantly reduced myogenic tone potentiation and improved EDR. Importantly, vascular function was also rescued in db−/db−p50NF-κB−/− and db−/db−PARP-1−/− double knockout mice compared with db−/db− mice. Additionally, the acute in vitro downregulation of NF-κB–p65 using p65NF-κB short hairpin RNA lentivirus in arteries from db−/db− mice also improved vascular function. The NF-κB inhibition did not affect blood glucose level or body weight. The RNA levels for Sp-1 and eNOS phosphorylation were decreased, while p65NF-κB phosphorylation, cleaved poly(ADP-ribose) polymerase (PARP)-1, and cyclooxygenase (COX)-2 expression were increased in arteries from diabetic mice, which were restored after NF-κB inhibition and in db−/db−p50NF-κB−/− and db−/db−PARP-1−/− mice. In the current study, we provided evidence that enhanced NF-κB activity impairs vascular function by PARP-1–, Sp-1–, and COX-2–dependent mechanisms in male type 2 diabetic mice. Therefore, NF-κB could be a potential target to overcome diabetes-induced vascular dysfunction.

Amniotic fluid stem cells improve survival and enhance repair of damaged intestine in necrotising enterocolitis via a COX-2 dependent mechanism

ObjectiveNecrotising enterocolitis (NEC) remains one of the primary causes of morbidity and mortality in neonates and alternative strategies are needed. Stem cells have become a therapeutic option for other intestinal...

Abstract A12: A novel in vitro model to study the role of phagocytic mammary epithelial cells in postpartum breast cancer promotion

Abstract Women diagnosed with breast cancer within 5-10 years of pregnancy have significantly poorer prognosis than age-matched nulliparous patients. This poor prognosis is specific to postpartum cases, suggesting that an event related to the completion of pregnancy may drive the poor prognosis of postpartum breast cancer. We have identified postpartum mammary gland involution as a tumor-promotional window following pregnancy/lactation. During postpartum involution, 50-80% of the milk-producing mammary epithelial cells (MECs) undergo apoptosis. Additionally, during involution, the MECs themselves become phagocytic and clear the gland of their dying neighbors. Peak phagocytosis precedes an influx of M2 activated macrophages with immunosuppressive characteristics which are thought to directly contribute to the tumor-promotional microenvironment of the postpartum gland. We hypothesize that clearance of apoptotic cells during involution induces secretion of immune-suppressive signals by phagocytic MECs, promoting M2 macrophage maturation and local immune suppression. These host events are anticipated to facilitate breast tumor cell immune evasion and tumor progression. This hypothesis is based on data that demonstrate clearance of apoptotic cells by professional phagocytes promotes M2 macrophages and immune suppression through TGF-β and COX-2. Here, we characterize the phenotype of phagocytic MECs and determine if phagocytic MECs induce tumor-promotional, M2 macrophage activation. To study the role of phagocytic MECs in promoting M2 macrophages, we developed an in vitro model in which MECs are grown to monolayers with high electrical resistance, modeling the tight cell-cell junctions unique to the lactating epithelium. To mimic involution, these monolayers are then treated with TGF-β3, which disrupts cell junctions and induces phagocytosis. We provide evidence that disruption of cell junctions is required for MECs to switch from a secretory to a phagocytic phenotype. We observe reorganization of cell junctions and E-cadherin cleavage in our in vitro model, and the extent of junction disruption positively correlates with phagocytosis. Similar adherens junction reorganization, E-cadherin cleavage, and β-catenin nuclear localization are observed upon the switch from lactation to involution in vivo in rodents and humans, consistent with a role for junction disruption in the phagocytic switch. To assess whether phagocytic MECs may contribute to M2 macrophage activation and local immune suppression during involution, bone marrow-derived macrophages (BMDM) were treated with conditioned media from phagocytic or control MEC cultures and expression of the M2 marker arginase-1 (Arg-1) was evaluated. Phagocytic cell conditioned media induced Arg-1, indicating M2 macrophage activation. Importantly, Arg-1 expression in BMDM correlated with COX-2 expression by phagocytic MECs, which suggests phagocytic MECs may promote M2 macrophages through a COX-2 dependent mechanism, as observed in the promotion of M2 macrophages by professional phagocytes. In vivo, COX-2 expression detected by IHC increases in MECs during involution, consistent with a role for phagocytic MEC COX-2 in promoting M2 macrophages. Additionally, CD86, a co-stimulatory molecule that promotes T cell activation, was decreased in BMDM treated with phagocytic MEC conditioned media, further supporting a role for phagocytic MECs in promotion of an immune-suppressive microenvironment. By further understanding the role of phagocytic MECs in regulating the immune environment in the breast during involution, we hope to identify novel pathways of metastasis, as well as new targets for postpartum breast cancer therapy. Citation Format: Jaime Fornetti, Peter Henson, Virginia Borges, Pepper Schedin. A novel in vitro model to study the role of phagocytic mammary epithelial cells in postpartum breast cancer promotion. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr A12.

Hyaluronic Acid Induces Radioprotection in the Intestine by a TLR-4 and COX-2 Dependent Mechanism

Background: Hyaluronic acid (HA), a glycosaminoglycan that forms a part of the extracellular matrix, affects inflammation and wound repair through binding to its receptors CD44, TLR2 and TLR4. HA expression is increased in dextran sodium sulfate (DSS) colitis and exogenous HA is protective in DSS colitis through a mechanism that was both MyD88 and COX-2 dependent. PGE2 produced by COX-1 and COX-2 is radioprotective in the intestine. Here we sought to determine if radiation induces HA expression and if exogenous HA induces radioprotection in the intestine. Methods: All experiments were done in wild type, MyD88-/, TLR4-/-, and COX-2-/mice on a C57Bl/6 background. To determine if radiation induces HA expression, mice were irradiated (12Gy), six hours later they were sacrificed. Small intestinal HA expression was assessed by immunohistochemistry. Plasma HA was assessed by ELISA. To determine if exogenous HA is radioprotective, mice were given either vehicle or HA (30mg/kg) intraperitoneally and were irradiated (12Gy). Six hours later the mice were sacrificed and radiation-induced apoptosis was assessed by histology. Mice were given vehicle or HA and irradiated, 84 hours later they were sacrificed and crypt survival was assessed. Using immunohistochemistry for surface markers we identified a population of constitutively COX-2 positive lamina propria mononuclear cells as mesenchymal stem cells (MSCs). We followed the effects of HA on the number and distribution of the MSCs. Results: Radiation increased HA expression in the small intestine with all the expression in the lamina propria. Plasma HA levels six hours after radiation increased six fold (p<.001). Administration of HA prior to irradiation induced a 60% decrease in radiation induced apoptosis at positions 4-12 (p<.01). Crypt survival was 27 crypts per cross section in the mice receiving HA compared with 15 crypts per cross section in mice receiving vehicle (p<.001). The effects of HA on radiation induced apoptosis and crypt survival were MyD88, TLR4, and COX-2dependent. The total number of intestinal MSCs in Wild Type mice changed little after HA treatment, but the distribution changed from 20% in crypts and 80% in villi in controls to 40% in crypts and 60% in villi at 8 hours after HA treatment. In TLR4-/mice, the total number of cells COX-2 expressing decreased after HA treatment, but the distribution remained unchanged with 32% in crypts and 68% in villi. Conclusion: Radiation induces HA expression in the small intestine and increases plasma HA. Intraperitoneal HA induces radioprotection in the small intestine through a COX-2, MyD88, and TLR4 dependent pathway. Intraperitoneal HA induces the migration of COX-2 positive MSCs from the lamina propria in the villi to the lamina propria near the crypt epithelial cells. This migration may relate to the radioprotective effects of HA.

Osteoblast response to fluid induced shear depends on substrate microarchitecture and varies with time

Osteoblasts are exposed to fluid shear in vivo but the effects are not well understood, particularly how substrate properties or length of exposure modify the response. Short exposure (1 h) to shear reduces the stimulatory effect of micron-scale surface structure on osteoblast differentiation, but the effects of longer term exposures are not known. To test the hypothesis that substrate-dependent responses of osteoblasts to shear depend on the length of exposure to fluid flow, MG63 osteoblasts were grown on tissue culture glass, which has an average roughness (Ra) < 0.2 microm; machined Ti disks (PT, Ra < 0.6 microm); Ti disks with a complex microarchitecture [sand blasted acid etched (SLA), Ra = 4-5 microm); and Ti plasma-sprayed surfaces [Ti via plasma spray (TPS), Ra = 7 microm]. Confluent cultures were exposed to pulsatile flow at shear forces of 0, 1, and 14 dynes/cm(2) for 0, 6, 12, and 24 h. Shear reduced cell number on all surfaces, with greatest effects on TPS. Shear had no effect on alkaline phosphatase on smooth surfaces but increased enzyme activity on SLA and TPS in a time-dependent manner. Its effects on osteocalcin, TGF-beta1, and PGE(2) in the conditioned media were greatest on these surfaces as well. Responses to fluid-induced shear were blocked by the general Cox inhibitor indomethacin and the Cox-2 inhibitor meloxicam, indicating that response to shear is mediated by prostaglandin produced via a Cox-2 dependent mechanism. These results show that the effects of fluid induced shear change with time and are substrate dependent, suggesting that substrate microarchitecture regulates the osteoblast phenotype and effects of shear are determined by the maturation state of the responding population.

Differentiation of Cyclooxygenase 1- and 2–Derived Prostanoids in Mouse Kidney and Aorta

Accumulating evidence indicates cyclooxygenase (COX) 1 and COX2 differentially regulate cardiovascular and renal function. We have demonstrated previously in mice that COX2 inhibition enhances angiotensin II-induced hypertension, and COX1 inhibition attenuates the pressor effect of angiotensin II. To further elucidate the mechanism underlying the functional difference of COX1 versus COX2 inhibition, the present studies examined the prostaglandin (PG) profiles derived in COX1- or COX2-inhibited mouse kidney and aorta using gas chromatographic/mass spectrometric assays. PGE2 is the most abundant prostanoid in both renal cortex and medulla in normal C57BL/6J mice, followed by PGI2, PGF2alpha and thromboxane A2. In contrast PGI2 was most abundant in aorta followed by thromboxane A2, PGE2, and PGF2alpha. PGD2 was undetectable in control kidney or aorta. At baseline, inhibition of COX1 decreased total prostaglandins in renal cortex, medulla, and aorta, whereas COX2 inhibition decreased total prostaglandins only in renal medulla. Angiotensin II infusion significantly increased COX2-dependent/COX1-independent PGE2 and PGI2 in renal cortex and medulla. Angiotensin II also significantly increased renal PGF2alpha in cortex, but not in medulla, through both COX1- and COX2-dependent mechanisms. These studies demonstrate that although COX1 primarily contributes to basal prostanoid production in the kidney and aorta, angiotensin II increases renal vasodilator prostanoids predominately via COX2 activity. These effects may contribute to the specific effect of COX2 inhibitors to increase blood pressure.

Stretch-induced myoblast proliferation is dependent on the COX2 pathway

Skeletal muscle increases in size due to weight bearing loads or passive stretch. This growth response is dependent in part upon myoblast proliferation. Although skeletal muscles are responsive to mechanical forces, the effect on myoblast proliferation remains unknown. To investigate the effects of mechanical stretch on myoblast proliferation, primary myoblasts isolated from Balb/c mice were subjected to 25% cyclical uniaxial stretch for 5 h at 0.5 Hz. Stretch stimulated myoblast proliferation by 32% and increased cell number by 41% 24 and 48 h after stretch, respectively. COX2 mRNA increased 3.5-fold immediately poststretch. Prostaglandin E2 and F2alpha increased 2.4- and 1.6-fold 6 h after stretch, respectively. Because COX2 has been implicated in regulating muscle growth and regeneration, we hypothesized that stretched myoblasts may proliferate via a COX2-dependent mechanism. We employed two different models to disrupt COX2 activity: (1) treatment with a COX2-selective drug, and (2) transgenic mice null for COX2. Treating myoblasts with a COX2-specific inhibitor blocked stretch-induced proliferation. Likewise, stretched COX2-/- myoblasts failed to proliferate compared to controls. However, supplementing stretched, COX2-/- myoblasts with prostaglandin E2 or fluprostenol increased proliferation. These data suggest that the COX2 pathway is critical for myoblast proliferation in response to stretch.

Adiponectin protects against myocardial ischemia-reperfusion injury through AMPK- and COX-2–dependent mechanisms

Obesity-related disorders are associated with the development of ischemic heart disease. Adiponectin is a circulating adipose-derived cytokine that is downregulated in obese individuals and after myocardial infarction. Here, we examine the role of adiponectin in myocardial remodeling in response to acute injury. Ischemia-reperfusion in adiponectin-deficient (APN-KO) mice resulted in increased myocardial infarct size, myocardial apoptosis and tumor necrosis factor (TNF)-alpha expression compared with wild-type mice. Administration of adiponectin diminished infarct size, apoptosis and TNF-alpha production in both APN-KO and wild-type mice. In cultured cardiac cells, adiponectin inhibited apoptosis and TNF-alpha production. Dominant negative AMP-activated protein kinase (AMPK) reversed the inhibitory effects of adiponectin on apoptosis but had no effect on the suppressive effect of adiponectin on TNF-alpha production. Adiponectin induced cyclooxygenase (COX)-2-dependent synthesis of prostaglandin E(2) in cardiac cells, and COX-2 inhibition reversed the inhibitory effects of adiponectin on TNF-alpha production and infarct size. These data suggest that adiponectin protects the heart from ischemia-reperfusion injury through both AMPK- and COX-2-dependent mechanisms.

Selenomethionine Inhibits Growth and Suppresses Cyclooxygenase-2 (COX-2) Protein Expression in Human Colon Cancer Cell Lines

Currently, selenium (in the form of high selenium containing yeast or selenomethionine) is being evaluated for anticancer effects against both human colon polyp recurrence and human prostate cancer, respectively. Chemical speciation analysis of the high selenium containing yeast indicates that selenomethionine (SeMet) is a major constituent of selenized yeast. We tested the hypothesis that SeMet might affect colon cancer cell growth by mechanisms involving cyclooxygenases (COX). The growth of all four-colon cancer cell lines tested was inhibited by selenomethionine. Furthermore, selenomethionine decreased COX-2 protein and PGE2 levels in HCA-7 cells. Selenomethionine suppressed COX-2 RNA levels in HCA-7 cells which could account for decreased COX-2 protein levels. Finally, the addition of PGE2 protected cells from the antiproliferative effects of selenomethionine in a concentration dependent manner. Selenomethionine might regulate COX-2 at the transcriptional level. These data suggests that Se-Met-induced cell growth inhibition may be, in part, mediated by COX-2 dependent mechanisms. The results of this study support the use of selenium agents in colon cancer chemoprevention trials.

Tumor Necrosis Factor-Alpha Dependent Stimulation of Cyclooxygenase Involves Nitric Oxide

P68 The aim of this study was to establish the interaction of nitric oxide (NO) and cyclooxygenase (COX) isoforms in vascular smooth muscle cells (VSMC) of rat aorta. We hypothesize that increased NO levels derived from inducible nitric oxide synthase (iNOS) after challenge with tumor necrosis factor-alpha (TNF) stimulates COX-2. TNF [1ng/ml] induced a transient increase in VSMC COX-2 and iNOS mRNA accumulation, without affecting COX-1 mRNA levels. The kinetics of COX-2 and iNOS mRNA accumulation were similar after challenge with TNF; mRNA peaked at 2 hr (COX-2: ∼8 fold and iNOS: ∼2 fold) and decreased to control levels by approximately 6 hr. COX-2 and iNOS protein expression also increased after TNF challenge (COX-2: ∼8-fold and iNOS: ∼1.5fold). Stimulation with TNF for 24 hrs increased nitrite levels by 4-fold (control: 5.26±0.37 vs. TNF: 18.33±0.66 pmol/μg protein; p&lt;0.001). Aminogunidine (AG; [1mM]), a specific iNOS inhibitor, abolished this stimulation. TNF-mediated PGI 2 production increased 4.5-fold (control: 120±14.47 vs. TNF: 541±55.05 pg/μg protein; p&lt;0.001) and 2-fold for TXA 2 (control 30.67±4.17 vs. TNF: 59+4.0 pg/μg protein; p&lt;0.01). AG pretreatment attenuated TNF mediated increase in PGI 2 by 45% (TNF: 541±55.05 vs. TNF+AG: 296±29 pg/μg protein; p&lt;0.001) and TXA 2 by 34% (TNF: 59±4.0 vs. TNF+AG: 39±3 pg/μg protein; p&lt;0.01). Sodium Nitroprusside (SNP), an NO donor increased COX-2 protein expression by ∼3 fold after 1 hr of treatment. PGI 2 levels also increased after incubation with SNP (control: 136±4.95 vs. SNP: 188±10.39 pg/μg protein; p&lt;0.01); TXA 2 levels were unaffected. The data indicate, that TNF-mediated NO production in VSMC is iNOS dependent. Moreover, increased NO levels after exposure to TNF stimulates prostanoid production, possibly via a COX-2 dependent mechanism. We conclude that stimulation of the COX pathway by TNF may involve iNOS-mediated increases in NO levels. This interaction may be relevant to the regulation of hyperplasia/hypertrophy/apoptosis in diseases such as arteriosclerorsis and hypertension, as recent evidence has shown that TNF-mediated increases in VSMC proliferation are COX-2 dependent.