Frank A. Fitzpatrick

Cyclooxygenase-2 and carcinogenesis.

Numerous investigations have shown that COX-2 is a participant in the pathway of colon carcinogenesis, especially when mutation of the APC tumor suppressor is the initiating event. Moreover, it seems that the amount of COX-2 is important, since there is a correlation between its level of expression and the size of the tumors and their propensity to invade underlying tissue [40]. Inhibiting COX-2 at an early stage blocks the development of malignant tumors, causes pre-malignant tumors to regress and may improve the outcome once the cancer is completely established. This set of findings seems to link very strongly with the traditional observation that chronic inflammation is a precursor to a variety of types of cancer. By this formulation, inflammatory stimuli increase COX-2 and the downstream events that it induces promote tumor formation. All of these finding suggest that existing NSAIDs will be useful for the prophylaxis of colon cancer and polyps and we eagerly await clinical investigations that will generate guidelines that suggest those individuals that are the most appropriate recipients for such therapy. Although this field has progressed rapidly in the last few years, many important questions remain.

Regulated formation of eicosanoids

Between 1965 and the mid-1980s, investigators laid a durable foundation for understanding the regulated formation and metabolic disposition of eicosanoids. First, they sought, and found in arachidonic acid, the biosynthetic precursor for the prostaglandins. Second, they identified phospholipids as the cellular compartment that harbored arachidonic acid, and they identified phospholipases as the enzymes essential for its liberation and for the ensuing biosynthesis of prostaglandins (1). Third, they deduced the existence of transient intermediates in the prostanoid biosynthetic pathway, leading to the eventual discovery of the prostaglandin endoperoxides, PGG2 and PGH2 (2, 3). Fourth, they established that the enzyme PGH synthase, or cyclooxygenase (COX), was a molecular target of great medical significance in reproductive, cardiovascular, and inflammatory disorders (4). Fifth, they established that rapid, comprehensive pulmonary metabolism limited the steady-state levels and duration of action of prostanoids, implying that they acted as autocoid lipid mediators, not hormones. Finally, they identified new prostanoids (5, 6) and their biosynthetic enzymes, as well as new lipoxygenase enzymatic pathways and their leukotriene products (7, 8), as medically significant molecules. Over this period, the most prominent themes in eicosanoid research were the identification of novel eicosanoid mediators, the determination of their molecular structures, and the establishment of their pharmacological activities. In a typical study of the time, investigators exposed tissues or cells to 50–100 μM of exogenous arachidonic acid, a concentration five- to tenfold greater than the Km for COX or lipoxygenases (Km ≈ 10 μM) (see Brash, this Perspective series, ref. 9). The contemporary model for eicosanoid biosynthesis, circa 1985, asserted that liberation of free arachidonic acid by phospholipase was the rate-limiting step in biosynthesis. Accordingly, providing saturating amounts of exogenous arachidonic acid ought to reveal all biosynthetic products and pathways. By 1985 or thereabouts, the quest for novel eicosanoid mediators of medical significance had reached the point of diminishing returns. Simultaneously, more and more investigators sought to understand the role of eicosanoid biosynthesis in disease processes. Accordingly, investigators shifted from exogenous arachidonic acid and Ca2+ ionophore as preferred tools and embraced natural ligands, relevant to disease, to initiate receptor-coupled activation of eicosanoid formation. By the late 1980s, two lines of experimental inquiry began to provoke a reconsideration and refinement of the accepted model of eicosanoid biosynthesis. First, kinetic and quantitative aspects of eicosanoid biosynthesis initiated by growth factors or cytokines on mitotically competent cells suggested that it was an oversimplification to regard availability of arachidonic acid as the sole, rate-limiting step in cellular eicosanoid biosynthesis. Second, the discovery of the 5-lipoxygenase activating protein (FLAP) in 1990 proved unambiguously that some eicosanoids (leukotrienes) originated under conditions that were not rate-limited by arachidonic acid availability. The discovery of novel regulatory processes, particularly Ca2++-dependent redistribution of 5-lipoxygenase (5-LO) and the interaction between 5-LO and FLAP (10), offered a new framework on which to reconstruct the earlier model of eicosanoid biosynthesis. Here, we comment on several mechanisms through which cells attain more subtle control of eicosanoid biosynthesis than would be possible by simply limiting the availability of arachidonic acid. We first discuss the coordinated action of specific phospholipases, the enzymes that generate this substrate, with specific COXs and PGH isomerase enzymes. We then consider the restricted expression of eicosanoid biosynthetic enzymes and, finally, turn to the “suicide” inactivation of these biosynthetic enzymes, a general mechanism that may help terminate their proinflammatory function.

Inflammation, carcinogenesis and cancer

To fulfill their role in host-defense, granulocytes secrete chemically reactive oxidants, radicals, and electrophilic mediators. While this is an effective way to eradicate pathogenic microbes or parasites, it inevitably exposes epithelium and connective tissue to certain endogenous genotoxic agents. In ordinary circumstances, cells have adequate mechanisms to reduce the genotoxic burden imposed by these agents to a negligible level. However, inflammation persisting for a decade eventually elevates the risk of cancer sufficiently that it is discernible in case control epidemiological studies. Advances in our understanding of tumor suppressors and inflammatory mediators offer an opportunity to assess the molecular and cellular models used to guide laboratory investigations of this phenomenon. Disappointing results from recent clinical trials with anti-oxidant interventions raise questions about the risks from specific endogenous agents such as hydrogen peroxide and oxy radicals. Simultaneously, the results from the anti-oxidant trials draw attention to an alternate hypothesis, favoring epigenetic inactivation of key tumor suppressors, such as p53, and the consequent liability this places on genomic integrity.

Cyclooxygenase Enzymes: Regulation and Function

The cyclooxygenase isoenzymes, COX-1 and COX-2, catalyze the formation of prostaglandins, thromboxane, and levuloglandins. The prostaglandins are autocoid mediators that affect virtually all known physiological and pathological processes via their reversible interaction with G-protein coupled membrane receptors. The levuloglandins are a newer class of products that appear to act via irreversible, covalent attachment to numerous proteins. COX enzymes are clinically important because they are inhibited by aspirin and numerous other non-steroidal anti-inflammatory drugs. This inhibition of COX confers relief from inflammatory, pyretic, thrombotic, neurodegenerative and oncological maladies. About one hundred years have elapsed since Hoffman designed and synthesized acetylsalicylic (aspirin) as an agent intended to lessen the gastrointestinal irritation of salicylates while maintaining their efficacy. During the past forty years systematic advances in our understanding of the structure, regulation and function of COX isoenzymes have enabled the design and synthesis of COX-2 selective inhibitors as agents intended to lessen the gastrointestinal irritation of aspirin and non-selective NSAIDs. This review discusses: 1) how two separate catalytic processes in COX - peroxidase and prostaglandin synthase - act in an integrated fashion manner to generate prostaglandins; 2) why irreversible inactivation of COX is important constitutively and pharmacologically; 3) how cells have managed to use two closely related, almost identical enzymes in ways that discriminate their physiological versus pathological roles; 4) how investigators have used these advances to formulate and test medically important uses for old drugs (i.e. aspirin) and create new ones that still seek to achieve Hoffmans original goal.

Redox Signaling, Alkylation (Carbonylation) of Conserved Cysteines Inactivates Class I Histone Deacetylases 1, 2, and 3 and Antagonizes Their Transcriptional Repressor Function

Cells use redox signaling to adapt to oxidative stress. For instance, certain transcription factors exist in a latent state that may be disrupted by oxidative modifications that activate their transcription potential. We hypothesized that DNA-binding sites (response elements) for redox-sensitive transcription factors may also exist in a latent state, maintained by co-repressor complexes containing class I histone deacetylase (HDAC) enzymes, and that HDAC inactivation by oxidative stress may antagonize deacetylase activity and unmask electrophile-response elements, thus activating transcription. Electrophiles suitable to test this hypothesis include reactive carbonyl species, often derived from peroxidation of arachidonic acid. We report that α,β-unsaturated carbonyl compounds, e.g. the cyclopentenone prostaglandin, 15-deoxy-Δ12,14-PGJ2 (15d-PGJ2), and 4-hydroxy-2-nonenal (4HNE), alkylate (carbonylate), a subset of class I HDACs including HDAC1, -2, and -3, but not HDAC8. Covalent modification at two conserved cysteine residues, corresponding to Cys261 and Cys273 in HDAC1, coincided with attenuation of histone deacetylase activity, changes in histone H3 and H4 acetylation patterns, derepression of a LEF1·β-catenin model system, and transcription of HDAC-repressed genes, e.g. heme oxygenase-1 (HO-1), Gadd45, and HSP70. Identification of particular class I HDACs as components of the redox/electrophile-responsive proteome offers a basis for understanding how cells stratify their responses to varying degrees of pathophysiological oxidative stress associated with inflammation, cancer, and metabolic syndrome.

Biological activity and metabolism of 20-hydroxyeicosatetraenoic acid in the human platelet.

1 The cytochrome P‐450 metabolite of arachidonic acid, 20‐hydroxyeicosatetraenoic acid (20‐HETE), was found to be a potent, dose‐dependent inhibitor of platelet aggregation and inhibitor of thromboxane biosynthesis induced by arachidonic acid (IC50 5.2 ± 1.5 μm), A23187 (IC50 16.2 ± 5.4 μm), and U46619 (IC50 7.8 ± 2.4 μm). 20‐HETE did not inhibit thrombin‐induced aggregation. 2 The human platelet metabolized 20‐HETE to a series of novel metabolites formed by cyclo‐oxygenase as well as lipoxygenase pathways. The structures of the metabolites were identified by mass spectrometry as 20‐hydroxy‐thromboxane B2, 12,17‐dihydroxyheptadecatrienoic acid, 12,20‐dihydroxyeicosatetraenoic acid, and 11,20‐dihydroxyeicosatetraenoic acid. 3 The identification of the 11‐hydroxy metabolite of 20‐HETE suggests that 20‐HETE is less efficiently cyclized to an endoperoxide intermediate by cyclo‐oxygenase than is arachidonate. 4 Although some biological activity of 20‐HETE may be related to competition with endogenous arachidonate for cyclo‐oxygenase metabolism, the predominant mechanism of action of 20‐HETE appears to be through antagonism of the prostaglandin H2/thromboxane A2 receptor.

Conditional Expression of 15-Lipoxygenase-1 Inhibits the Selenoenzyme Thioredoxin Reductase MODULATION OF SELENOPROTEINS BY LIPOXYGENASE ENZYMES

The selenoenzyme thioredoxin reductase regulates redox-sensitive proteins involved in inflammation and carcinogenesis, including ribonucleotide reductase, p53, NFκB, and others. Little is known about endogenous cellular factors that modulate thioredoxin reductase activity. Here we report that several metabolites of 15-lipoxygenase-1 inhibit purified thioredoxin reductase in vitro. 15(S)-Hydroperoxy-5,8,11-cis-13-trans-eicosatetraenoic acid, a metastable hydroperoxide generated by 15-lipoxygenase-1, and 4-hydroxy-2-nonenal, its non-enzymatic rearrangement product inhibit thioredoxin reductase with IC50 = 13 ± 1.5 μm and 1 ± 0.2 μm, respectively. Endogenously generated metabolites of 15-lipoxygenase-1 also inhibit thioredoxin reductase in HEK-293 cells that harbor a 15-LOX-1 gene under the control of an inducible promoter complex. Conditional, highly selective induction of 15-lipoxygenase-1 caused an inhibition of ribonucleotide reductase activity, cell cycle arrest in G1, impairment of anchorage-independent growth, and accumulation of the pro-apoptotic protein BAX. All of these responses are consistent with inhibition of thioredoxin reductase via 15-lipoxygenase-1 overexpression. In contrast, metabolites of 5-lipoxygenase were poor inhibitors of isolated thioredoxin reductase, and the overexpression of 5-lipoxygenase did not inhibit thioredoxin reductase or cause a G cell cycle arrest. The influences of 15-lipoxygenase-1 on 1inflammation, cell growth, and survival may be attributable, in part, to inhibition of thioredoxin reductase and several redox-sensitive processes subordinate to thioredoxin reductase.

Oxidation of 2-Cys-peroxiredoxins by arachidonic acid peroxide metabolites of lipoxygenases and cyclooxygenase-2.

Human peroxiredoxins serve dual roles as anti-oxidants and regulators of H2O2-mediated cell signaling. The functional versatility of peroxiredoxins depends on progressive oxidation of key cysteine residues. The sulfinic or sulfonic forms of peroxiredoxin lose their peroxidase activity, which allows cells to accumulate H2O2 for signaling or pathogenesis in inflammation, cancer, and other disorders. We report that arachidonic acid lipid hydroperoxide metabolites of 5-, 12-, 15-lipoxygenase-1, and cyclooxygenase-2 oxidize the 2-Cys-peroxiredoxins 1, 2, and 3 to their sulfinic and sulfonic forms. When added exogenously to cells, 5-, 12- and 15-hydroperoxy-eicosatetraenoic acids also over-oxidized peroxiredoxins. Our results suggest that lipoxygenases and cyclooxygenases may affect 2-Cys peroxiredoxin signaling, analogous to NADPH oxidases in the “floodgate” model (Wood, Z. A., Poole, L. B, and Karplus P. A. (2003) Science 300, 600–653). Peroxiredoxin-dependent mechanisms may modulate the receptor-dependent actions of autocoids derived from cellular lipoxygenase and cyclooxygenase catalysis.

Genomics and the discovery of new drug targets.

Molecular medicine and genomics technologies are inseparable for defining new molecular targets. cDNA databases and elementary informatic tools provide instantaneous glimpses of gene families or tissue-restricted expression patterns as a means of new target identification. In addition, cDNA microarrays and two-dimensional gel electrophoresis unmask the expression of genes with unassigned or unexpected functions. Depletion of mRNA with ribozymes or neutralization of proteins with intracellular antibodies enable investigators to reject or embrace new molecular hypotheses about the determinants of disease, pharmacology or toxicology.

Arachidonic acid metabolites do not mediate modulation of neurotransmitter release by adenosine in rat hippocampus or striatum

The possible involvement of arachidonic acid metabolites as mediators of the modulation of neurotransmitter release by adenosine, acetylcholine, and GABA was examined in brain slices of rat hippocampus and striatum. The synaptic modulatory effects of these 3 agents on excitatory transmission in the CA1 region of hippocampus were completely unaffected by a phospholipase inhibitor (p-bromophenacyl bromide, BPB; 10-50 microM), a lipoxygenase inhibitor (nordihydroguaiaretic acid; 5-50 microM), the cyclooxygenase inhibitor indomethacin (10-20 microM), and a cyclooxygenase/lipoxygenase inhibitor (U53059; 5-10 microM). BPB was also found to be ineffective in altering the modulation of transmission by adenosine in the perforant path, and the adenosine inhibition of electrically stimulated release of endogenous dopamine from striatal slices. Arachidonic acid itself also had no effect on synaptic transmission. While these experiments do not rule out such a role for arachidonic acid or its metabolites in mammalian brain, they suggest that in a number of systems the inhibition of transmitter release must occur through an entirely independent mechanism.