The cytochrome P450 arachidonic acid epoxygenase metabolites, the epoxyeicosatrienoic acids (EETs) are powerful, nonregioselective, stimulators of cell proliferation. In this study we compared the ability of the four EETs (5,6-, 8,9-, 11,12-, and 14,15-EETs) to regulate endothelial cell proliferation in vitro and angiogenesis in vivo and determined the molecular mechanism by which EETs control these events. Inhibition of the epoxygenase blocked serum-induced endothelial cell proliferation, and exogenously added EETs rescued cell proliferation from epoxygenase inhibition. Studies with selective ERK, p38 MAPK, or PI3K inhibitors revealed that whereas activation of p38 MAPK is required for the proliferative responses to 8,9-and 11,12-EET, activation of PI3K is necessary for the cell proliferation induced by 5,6-and 14,15-EET. Among the four EETs, only 5,6-and 8,9-EET are capable of promoting endothelial cell migration and the formation of capillary-like structures, events that are dependent on EET-mediated activation of ERK and PI3K. Using subcutaneous sponge models, we showed that 5,6-and 8,9-EET are pro-angiogenic in mice and that their neo-vascularization effects are enhanced by the co-administration of an inhibitor of EET enzymatic hydration, presumably because of reduced EET metabolism and inactivation. These studies identify 5,6-and 8,9-EET as powerful and selective angiogenic lipids, provide a functional link between the EET proliferative chemotactic properties and their angiogenic activity, and suggest a physiological role for them in angiogenesis and de novo vascularization.

Prostaglandin E2 (PGE 2 ), a major product of cyclooxygenase, exerts its functions by binding to four G protein-coupled receptors (EP1-4) and has been implicated in modulating angiogenesis. The present study examined the role of the EP4 receptor in regulating endothelial cell proliferation, migration, and tubulogenesis. Primary pulmonary microvascular endothelial cells were isolated from EP4 flox/flox mice and were rendered null for the EP4 receptor with adenoCre virus. Whereas treatment with PGE 2 or the EP4 selective agonists PGE 1 -OH and ONO-AE1-329 induced migration, tubulogenesis, ERK activation and cAMP production in control adenovirus-transduced endothelial EP4 flox/flox cells, no effects were seen in adenoCre-transduced EP4 flox/flox cells. The EP4 agonist-induced endothelial cell migration was inhibited by ERK, but not PKA inhibitors, defining a functional link between PGE 2 -induced endothelial cell migration and EP4-mediated ERK signaling. Finally, PGE 2 , as well as PGE 1 -OH and ONO-AE1-329, also promoted angiogenesis in an in vivo sponge assay providing evidence that the EP4 receptor mediates de novo vascularization in vivo.Angiogenesis, the process of new blood vessel formation from pre-existing vessels, is a multistep event that requires endothelial cell proliferation, migration, and tube formation. Angiogenesis is controlled by diverse factors, including cytokines, growth factors, as well as cyclooxygenase-2-derived eicosanoids (1, 2). The pro-angiogenic effects of cyclooxygenase-2 are mediated primarily by three products of arachidonic acid metabolism: thromboxane A 2 , prostaglandin E 2 (PGE 2 ), 2 and prostaglandin I 2 . These pro-angiogenic eicosanoids directly stimulate the synthesis of angiogenic factors, promote vascular sprouting, migration, tube formation, as well as enhance endothelial cell survival (1, 2).PGE 2 exerts its cellular effects by binding to four distinct E-prostanoid receptors (EP1-4) that belong to the family of seven transmembrane G protein-coupled rhodopsin-type receptors (3). Even though there is similar signaling mechanisms among these receptors, it is clear that each receptor has different and often opposing biological effects (4). For example, although the EP2 and EP4 receptors are both Gs coupled receptors and up-regulate intracellular cAMP levels, they mediate differential phosphorylation of cAMP response element-binding proteins (5). In addition, following activation, these two receptors exert different downstream effects on important intracellular mediators, including the PI3K and ERK pathways (6, 7). Moreover, the EP3 receptor usually counteracts EP2-and EP4-mediated up-regulation of cAMP by preferentially coupling to G i proteins (3).Some information regarding the role of PGE 2 in angiogenesis has been obtained using cancer models in mice where the receptors have been deleted by homologous recombination. In this context, mice lacking the EP2 receptor produce significantly fewer and less vascularized tumors than wild type mice in a two-stage skin carcinogenesi...

Cyclooxygenase (COX) and its prostanoid metabolites have been implicated in the control of cell survival; however, their role as mitogens remains undefined. To better understand the role of prostanoids on cell growth, we used mouse colon adenocarcinoma (CT26) cells to investigate the role of prostaglandin E 2 (PGE 2 ) in cell proliferation. CT26 cells express both COX1 and COX2 and metabolize arachidonic acid to PGE 2. Treatment with indomethacin, or COXselective inhibitors, prevents PGE 2 biosynthesis and CT26 cell proliferation. The anti-proliferative effects of COX inhibition are rescued specifically by treatment with PGE 2 or the EP4 receptor-selective agonist PGE 1 -OH via phosphatidylinositol 3-kinase/extracellular signal-regulated kinase (ERK) activation, thus providing a functional link between PGE 2 -induced cell proliferation and EP4-mediated ERK signaling. Indomethacin or COX2 inhibitors, but not COX1 inhibitors, reduced the size and number of CT26-derived tumors in vivo. These inhibitory effects are paralleled by marked declines in the levels of tumor PGE 2 , suggesting that their anti-tumor effects are directly associated with the inhibition of COX2 enzymatic activity. The described anti-tumor effects of indomethacin are evident whether it is administered at the time of, or 7 days after, tumor cell injection, suggesting that it has tumor preventive and therapeutic actions. Furthermore, the observation that indomethacin increases the survival rates of tumor-bearing mice, even after withdrawal of the drug, indicates that its effects are long lasting and that it may be potentially useful for the prevention and the clinical management of human cancers.The identification of cyclooxygenase (COX) 1 as the target for non-steroidal anti-inflammatory drugs (NSAIDs) led to new understandings of its pathophysiological role and of the mechanisms of action of these drugs (1, 2). The discovery of COX2, an isoform that, although catalytically identical to COX1, shows inducible tissue-selective expression, suggested physiological and pathophysiological roles for the constitutively expressed (COX1) and inducible (COX2) isoforms, respectively (1-4). Studies showing a correlation between NSAIDs and decreased colon cancer incidence, and the demonstration of up-regulated COX2 expression in colon carcinoma, suggested a role for COX2 in the pathophysiology of colon cancer and created new paradigms for the study of the role of prostanoids in cancer (1-3). Nevertheless, despite extensive supporting evidence, direct links between the anti-tumor effects of NSAIDs and COX inhibition are yet to be established (4 -9). The potential for non-COX-dependent anti-tumor effects of NSAIDs (6 -9), as well as limited evidence available identifying a specific prostanoid(s) as the mediator responsible for the anti-tumor effects of the COX inhibitors (5), raised questions regarding the mechanism of action of NSAIDs and of the role of arachidonic acid, prostanoids, and COXs in human cancer (4 -9).During studies of arachidonic acid metabolism ...

The collagen IV binding receptor integrin A1B1 has been shown to regulate lung cancer due to its proangiogenic properties; however, it is unclear whether this receptor also plays a direct role in promoting primary lung tumors. To investigate this possibility, integrin A1-null mice were crossed with KrasLA2 mice that carry an oncogenic mutation of the Kras gene (G12D) and develop spontaneous primary tumors with features of non-small cell lung cancer. We provide evidence that KrasLA2/A1-null mice have a decreased incidence of primary lung tumors and longer survival compared with KrasLA2/A1 wild-type controls. Tumors from KrasLA2/A1-null mice were also smaller, less vascularized, and exhibited reduced cell proliferation and increased apoptosis, as determined by proliferating cell nuclear antigen and terminal deoxynucleotidyl-transferase-mediated dUTP nick-end staining, respectively. Moreover, tumors from the KrasLA2/A1-null mice showed diminished extracellular signal-regulated kinase (ERK) but enhanced p38 mitogenactivated protein kinase activation. Primary lung tumor epithelial cells isolated from KrasLA2/A1-null mice showed a significant decrease in anchorage-independent colony formation, collagen-mediated cell proliferation, ERK activation, and, most importantly, tumorigenicity when injected into nude mice compared with KrasLA2/A1 wild-type tumor cells. These results indicate that loss of the integrin A1 subunit decreases the incidence and growth of lung epithelial tumors initiated by oncogenic Kras, suggesting that both Kras and integrin A1B1 cooperate to drive the growth of non-small cell lung cancer in vivo. [Cancer Res 2008;68(15):6127-9]

Chronic lymphocytic leukemia (CLL) remains an incurable B-cell malignancy with many unanswered questions. While the cell of origin and etiology are still unknown, significant scientific progress has revealed numerous molecular targets for novel therapeutic interventions. Phosphatidylinositol 3-kinases (PI3K) regulate key cellular functions, including growth, survival and migration, by integrating and transmitting signals from diverse surface molecules including the B-cell receptor (BCR). In lymphocytes, the PI3Kδ isoform plays a critical role in B-cell homeostasis and function. In CLL, the PI3K pathway is constitutively active and dependent on PI3Kδ. GS-1101 is a highly selective PI3Kδ inhibitor that in CLL patients causes a rapid and sustained reduction in lymphadenopathy, accompanied by transient lymphocytosis. This article will review new insights into the pathophysiology of CLL, the preclinical rationale of a PI3Kδ inhibitor in CLL, and the clinical evidence supporting this first-in-class therapeutic target for CLL patients.

Prostaglandin E 2 , which exerts its functions by binding to four G protein-coupled receptors (EP1-4), is implicated in tumorigenesis. Among the four E-prostanoid (EP) receptors, EP3 is unique in that it exists as alternatively spliced variants, characterized by differences in the cytoplasmic C-terminal tail. Although three EP3 variants, ␣, ␤, and ␥, have been described in mice, their functional significance in regulating tumorigenesis is unknown. In this study we provide evidence that expressing murine EP3 ␣, ␤, and ␥ receptor variants in tumor cells reduces to the same degree their tumorigenic potential in vivo. In addition, activation of each of the three mEP3 variants induces enhanced cell-cell contact and reduces cell proliferation in vitro in a Rho-dependent manner. Finally, we demonstrate that EP3-mediated RhoA activation requires the engagement of the heterotrimeric G protein G 12 . Thus, our study provides strong evidence that selective activation of each of the three variants of the EP3 receptor suppresses tumor cell function by activating a G 12 -RhoA pathway.Colorectal cancer is the second most frequent cause of cancer-related death in the western world. An estimated 153,760 cases will be diagnosed and 52,180 people will die from this disease in the United States in 2007 (1). Overexpression of cyclooxygenase-2 (2-5) and subsequent prostaglandin production (6, 7) have been widely documented in colorectal cancer, and epidemiologic studies have shown that nonsteroidal antiinflammatory drugs reduce the incidence of colorectal cancer mortality by decreasing the formation of PGE 2 , 2 the primary prostaglandin generated in colorectal tumors (8,9). PGE 2 exhibits a broad range of biological actions in diverse tissues by binding to its four distinct G protein-coupled receptor subtypes designated EP1, EP2, EP3, and EP4 (10). The EP1 receptor induces mobilization of intracellular calcium; the EP2 and EP4 receptors couple to stimulatory G proteins (G s ) and signal by increasing intracellular cAMP level, whereas the EP3 receptor couples to an inhibitory G protein (G i ) and reduces cAMP synthesis (11). In the mouse, alternative splicing generates three EP3 variants, ␣, ␤, and ␥, which contain carboxyl tails of 30, 26, and 29 amino acids that do not share any structural motifs or hydrophobic features. The amino acid sequence of the tails is thought to modulate signal transduction. In this context, EP3␣ and EP3␤ couple to G i and inhibit adenylate cyclase (12, 13), whereas the EP3␥ couples to G s , in addition to G i , and evokes cAMP production (13). Moreover, EP3 has been demonstrated to activate the small GTPase Rho in various cell types, resulting in stress fiber formation and neurite retraction (14 -16).At present, little is known on the contribution of the EP3 splice variants to tumor cell function. Unlike the other EP receptors, EP3 expression is decreased in colon cancer in mice, rats, and humans when compared with normal mucosa (17). Similar results were found in mammary tumors, suggesting that EP3 down-...

The incidence of NAARs was lower in patients receiving acetylcysteine in a two-bag regimen compared to the traditional Danish three-bag regimen without an apparent reduction in efficacy.

Objectives: To evaluate the efficacy of intravenous (IV) ibuprofen (Caldolor) administration in the management of acute pain in orthopedic trauma patients and to minimize opioid use. Design: Randomized controlled trial, double-blind, parallel, placebo-controlled. Setting: Level 1 Trauma Center. Patients: A total of 99 consecutive orthopedic trauma patients with fractures of the ribs, face, extremities, and/or pelvis were randomized to receive either 800 mg IV ibuprofen (53 patients) or placebo (44 patients) administered every 6 hours for a total of 8 doses within 48 hours of admission and the same PRN medications along with 20-mg IV/PO Pepcid twice a day. To establish pain reduction efficacy, the analysis was consequently performed in the modified intent-to-treat group that included 74 randomized subjects with a baseline pain score greater than 2. The primary outcomes were reduction in opioid consumption and decrease in pain intensity (PI). Intervention: Administration of study medications. Outcome Measurements: PI measured by Numerical Rating Scale, opioid consumption adjusted to morphine equivalent dose, and time to first narcotic administration. Results: The 2 groups had comparable baseline characteristics: age, sex distribution, mechanism of injury, type of injury, injury severity score, and PI. IV ibuprofen statistically significantly reduced opioid consumption compared with placebo during the initial 48-hour period (P = 0.017). PI calculated as PI differences was statistically different only at 8-hour interval after Caldolor administration. Time to first narcotic medication was significantly longer in the Caldolor group (hazard ratio: 1.640; 95% confidence interval, 1.009–2.665; P = 0.046). Conclusions: IV ibuprofen provided adequate analgesia, prolonged time to first narcotic administration, and was opioid-sparing for the treatment of pain in orthopedic trauma patients, which makes Caldolor a recommended candidate for managing acute pain in the diverse orthopaedic trauma population. Level of Evidence: Therapeutic Level I. See Instructions for Authors for a complete description of levels of evidence.