Kevin R. Kozak

Oxygenation of the Endocannabinoid, 2-Arachidonylglycerol, to Glyceryl Prostaglandins by Cyclooxygenase-2

Cyclooxygenases (COX) play an important role in lipid signaling by oxygenating arachidonic acid to endoperoxide precursors of prostaglandins and thromboxane. Two cyclooxygenases exist which differ in tissue distribution and regulation but otherwise carry out identical chemical functions. The neutral arachidonate derivative, 2-arachidonylglycerol (2-AG), is one of two described endocannabinoids and appears to be a ligand for both the central (CB1) and peripheral (CB2) cannabinoid receptors. Here we report that 2-AG is a substrate for COX-2 and that it is metabolized as effectively as arachidonic acid. COX-2-mediated 2-AG oxygenation provides the novel lipid, prostaglandin H2 glycerol ester (PGH2-G), in vitro and in cultured macrophages. PGH2-G produced by macrophages is a substrate for cellular PGD synthase, affording PGD2-G. Pharmacological studies reveal that macrophage production of PGD2-G from endogenous sources of 2-AG is calcium-dependent and mediated by diacylglycerol lipase and COX-2. These results identify a distinct function for COX-2 in endocannabinoid metabolism and in the generation of a new family of prostaglandins derived from diacylglycerol and 2-AG.

IκB Kinase, a Molecular Target for Inhibition by 4-Hydroxy-2-nonenal

In unstimulated cells, transcription factor NF-κB is retained in the cytoplasm by interaction with the inhibitory protein, IκBα. Appropriate cellular stimuli inactivate IκBα by phosphorylation, ubiquination, and proteolytic degradation, which allows NF-κB to translocate to the nucleus and modulate gene expression. 4-Hydroxy-2-nonenal (HNE), a major lipid peroxidation product, inhibits activation of NF-κB in the human colorectal carcinoma cell line (RKO) and human lung carcinoma cell line (H1299). Pretreatment of cells with HNE dose-dependently suppresses tetradecanoylphorbol acetate (TPA)/ionomycin (IM)-induced NF-κB DNA binding activity and transactivation of luciferase-based reporter constructs. HNE pretreatment has no affect on TPA/IM-induced AP-1 DNA binding activity. HNE inhibits TPA/IM-induced degradation of IκBα in both H1299 and Jurkat T cells. The accumulation of IκBα parallels the inhibition of its phosphorylation. At doses that inhibit IκBα degradation, HNE inhibits IκB kinase (IKK) activity by direct reaction with IKK. Covalent adducts of HNE to IKK are detected on Western blots using antibodies against IKK or HNE-protein conjugates. Addition of dithiothreitol prevents HNE modification of IKK. Thus, HNE is an endogenous inhibitor of NF-κB activation that acts by preventing IKK activation and subsequent IκBα degradation.

15-Lipoxygenase Metabolism of 2-Arachidonylglycerol GENERATION OF A PEROXISOME PROLIFERATOR-ACTIVATED RECEPTOR α AGONIST

The recent demonstrations that cyclooxygenase-2 and leukocyte-type 12-lipoxygenase (LOX) efficiently oxygenate 2-arachidonylglycerol (2-AG) prompted an investigation into related oxygenases capable of metabolizing this endogenous cannabinoid receptor ligand. We evaluated the ability of six LOXs to catalyze the hydroperoxidation of 2-AG. Soybean 15-LOX, rabbit reticulocyte 15-LOX, human 15-LOX-1, and human 15-LOX-2 oxygenate 2-AG, providing 15(S)-hydroperoxyeicosatetraenoic acid glyceryl ester. In contrast, potato and human 5-LOXs do not efficiently metabolize this endocannabinoid. Among a series of structurally related arachidonyl esters, arachidonylglycerols serve as the preferred substrates for 15-LOXs. Steady-state kinetic analysis demonstrates that both 15-LOX-1 and 15-LOX-2 oxygenate 2-AG comparably or preferably to arachidonic acid. Furthermore, 2-AG treatment of COS-7 cells transiently transfected with human 15-LOX expression vectors or normal human epidermal keratinocytes results in the production and extracellular release of 15-hydroxyeicosatetraenoic acid glyceryl ester (15-HETE-G), establishing that lipoxygenase metabolism of 2-AG occurs in an eukaryotic cellular environment. Investigations into the potential biological actions of 15-HETE-G indicate that this lipid, in contrast to its free-acid counterpart, acts as a peroxisome proliferator-activated receptor α agonist. The results demonstrate that 15-LOXs are capable of acting on 2-AG to provide 15-HETE-G and elucidate a potential role for endocannabinoid oxygenation in the generation of peroxisome proliferator-activated receptor α agonists.

Oxidative Metabolism of Endocannabinoids by COX-2

The last decade has witnessed a rapid expansion in our understanding of the mammalian endogenous cannabinoid system. In just a few short years since the discovery of endogenous lipids that serve as cannabinoids in vivo, these molecules have been shown to participate in a broad array of physiological and pathological processes. Consequently, attention has been directed at defining the proteins responsible for endocannabinoid synthesis, transport, and metabolism. Recently, multiple fatty acid oxygenases including, most notably, cyclooxygenase-2 (COX-2), have been implicated in endocannabinoid metabolism. This review will highlight connections between COX-2 and the endogenous cannabinoid system. The available biochemical evidence supporting a role for COX-2 in endocannabinoid metabolism will be presented. Finally, the potential biological consequences of COX-2-mediated endocannabinoid oxygenation will be discussed.

The glyceryl ester of prostaglandin E2 mobilizes calcium and activates signal transduction in RAW264.7 cells

Glyceryl prostaglandins (PG-Gs) are generated by the oxygenation of the endocannabinoid, 2-arachidonylglycerol, by cyclooxygenase 2. The biological consequences of this selective oxygenation are uncertain because the cellular activities of PG-Gs have yet to be defined. We report that the glyceryl ester of PGE2, PGE2-G, triggers rapid, concentration-dependent Ca2+ accumulation in a murine macrophage-like cell line, RAW264.7. Ca2+ mobilization is not observed after addition of PGE2, PGD2-G, or PGF2α-G but is observed after addition of PGF2α. Moreover, PGE2-G, but not PGE2, stimulates a rapid but transient increase in the levels of inositol 1,4,5-trisphosphate (IP3) as well as the membrane association and activation of PKC. PGE2-G induces a concentration-dependent increase in the levels of phosphorylated extracellular signal regulated kinases 1 and 2 through a pathway that requires the activities of PKC, IP3 receptor, and phospholipase C β. The results indicate that PGE2-G triggers Ca2+ mobilization, IP3 synthesis, and activation of PKC in RAW264.7 macrophage cells at low concentrations. These responses are independent of the hydrolysis of PGE2-G to PGE2.

Selective oxygenation of N-arachidonylglycine by cyclooxygenase-2

Nonsteroidal anti-inflammatory drugs prevent hyperalgesia and inflammation by inhibiting the cyclooxygenase-2 (COX-2) catalyzed oxygenation of arachidonic acid to prostaglandin (PG) H(2). The lipoamino acid N-arachidonylglycine (NAGly) has also been shown to suppress tonic inflammatory pain and is naturally present at significant levels in many of the same mammalian tissues that express COX-2. Here, we report that COX-2 selectively metabolizes NAGly to PGH(2) glycine (PGH(2)-Gly) and hydroxyeicosatetraenoic glycine (HETE-Gly). Site-directed mutagenesis experiments identify the side pocket residues of COX-2, especially Arg-513, as critical determinants of the COX-2 selectivity towards NAGly. This is the first report of a charged arachidonyl derivative that is a selective substrate for COX-2. These results suggest a possible role for COX-2 in the regulation of NAGly levels and the formation of a novel class of eicosanoids from NAGly metabolism.

Giant cell glioblastoma: A glioblastoma subtype with distinct epidemiology and superior prognosis

Giant cell glioblastoma (GC) is an uncommon subtype of glioblastoma multiforme (GBM). Consequently, the epidemiology, natural history, and factors associated with outcome are not well defined. Patients diagnosed with GC from 1988 through 2004 were identified in the Surveillance, Epidemiology, and End Results (SEER) database. Outcomes were examined with Kaplan-Meier survival analysis and Cox models. For comparison, similar analyses were conducted for patients diagnosed with GBM. GC was identified in 1% of 16,430 patients diagnosed with either GC or GBM. Compared with GBM, GC showed similar gender and racial distributions. Likewise, tumor size and location were not significantly different between the two histologies. GC tended to occur in younger patients with a median age at diagnosis of 51 years, compared with 62 years for GBM. Additionally, patients with GC were more likely to undergo complete resection compared with patients with GBM. For both histologies, young age, tumor size, extent of resection, and the use of adjuvant radiation therapy (RT) were associated with improved survival. Cox modeling suggests the prognosis for GC is significantly superior to that for GBM (hazard ratio = 0.76; 95% confidence interval, 0.59-0.97) even after adjustment for factors affecting survival. GC is an uncommon GBM subtype that tends to occur in younger patients. Prospective data defining optimal treatment for GC are unavailable; however, these retrospective findings suggest that resection, as opposed to biopsy only, and adjuvant RT may improve survival. The prognosis of GC is superior to that of GBM, and long-term survival is possible, suggesting aggressive therapy is warranted.

Structural Basis of Enantioselective Inhibition of Cyclooxygenase-1 by S-α-Substituted Indomethacin Ethanolamides

The modification of the nonselective nonsteroidal anti-inflammatory drug, indomethacin, by amidation presents a promising strategy for designing novel cyclooxygenase (COX)-2-selective inhibitors. A series of α-substituted indomethacin ethanolamides, which exist as R/S-enantiomeric pairs, provides a means to study the impact of stereochemistry on COX inhibition. Comparative studies revealed that the R- and S-enantiomers of the α-substituted analogs inhibit COX-2 with almost equal efficacy, whereas COX-1 is selectively inhibited by the S-enantiomers. Mutagenesis studies have not been able to identify residues that manifest the enantioselectivity in COX-1. In an effort to understand the structural impact of chirality on COX-1 selectivity, the crystal structures of ovine COX-1 in complexes with an enantiomeric pair of these indomethacin ethanolamides were determined at resolutions between 2.75 and 2.85Å. These structures reveal unique, enantiomer-selective interactions within the COX-1 side pocket region that stabilize drug binding and account for the chiral selectivity observed with the (S)-α-substituted indomethacin ethanolamides. Kinetic analysis of binding demonstrates that both inhibitors bind quickly utilizing a two-step mechanism. However, the second binding step is readily reversible for the R-enantiomer, whereas for the S-enantiomer, it is not. These studies establish for the first time the structural and kinetic basis of high affinity binding of a neutral inhibitor to COX-1 and demonstrate that the side pocket of COX-1, previously thought to be sterically inaccessible, can serve as a binding pocket for inhibitor association.

Enantiospecific, selective cyclooxygenase-2 inhibitors.

Cyclooxygenase inhibition studies with novel indomethacin alkanolamides demonstrate the potential for dramatic differences in inhibitor properties conferred by subtle structural modifications. The transformation of non-selective alpha-(S)-substituted indomethacin ethanolamides to potent, COX-2 selective inhibitors by simple stereocenter inversion highlights this property.

Refusal of radiation therapy and its associated impact on survival.

Objectives:Despite evidence that radiation therapy (RT) improves outcome in multiple malignancies, some patients with strong clinical indications still refuse RT. Data on factors associated with RT refusal are limited. Furthermore, the effect of RT refusal on outcome has not been clearly defined. Methods:Patients with nonmetastatic cancer, diagnosed between 1988 and 2005, were identified in the Surveillance, Epidemiology, and End Results database. Univariate and multivariate methods were used to identify factors associated with RT refusal and the impact of refusal on outcomes. Results:On univariate analysis, age, sex, marital status, tumor site, and tumor stage were associated with RT refusal (P < 0.001). On multivariate analysis, sex and tumor stage were not found to be associated with RT refusal. In contrast, age, race, marital status, and tumor location were significantly associated with RT refusal. The median survival of compliant patients was 171 months compared with just 96 months among patients who refused RT. Conclusions:A significant percentage of patients continue to refuse RT despite medical advice and evidence. Subgroups at particular risk of RT refusal include elderly, black and widowed patients. RT refusal is associated with markedly worse clinical outcomes.