Joseph T. O’Flaherty

The Coupling of 5-Oxo-Eicosanoid Receptors to Heterotrimeric G Proteins

5-Oxo-eicosatetraenoic acid (5-oxoETE) stimulated human neutrophil (PMN) and eosinophil chemotaxis, PMN hexose uptake, and PMN membrane GTP/GDP exchange. Pertussis toxin (PT), a blocker of heterotrimeric G proteins (GP), completely inhibited these responses, but proved far less effective on the same responses when elicited by leukotriene B4, C5a, FMLP, platelet-activating factor, IL-8, or RANTES chemotactic factors. 5-OxoETE also specifically bound to the membrane preparations that conducted GTP/GDP exchange. This binding was down-regulated by GTPγS, but not ADPγS, and displaced by 5-oxoETE analogues, but not by leukotriene B4, lipoxin A4, or lipoxin B4. Finally, PMN expressed PT-sensitive GP αι2 and PT-resistant GP αq/11- and α13-chains; eosinophils expressed only αi2 and αq/11. We conclude that 5-oxoETE activates granulocytes through a unique receptor that couples preferentially to PT-sensitive GP. The strict dependency of this putative receptor on PT-sensitive GP may underlie the limited actions of 5-oxoETE, compared with other CF, and help clarify the complex relations between receptors, GP, cell signals, and cell responses.

15-lipoxygenase metabolites of docosahexaenoic acid inhibit prostate cancer cell proliferation and survival.

A 15-LOX, it is proposed, suppresses the growth of prostate cancer in part by converting arachidonic, eicosatrienoic, and/or eicosapentaenoic acids to n-6 hydroxy metabolites. These metabolites inhibit the proliferation of PC3, LNCaP, and DU145 prostate cancer cells but only at ≥1–10 µM. We show here that the 15-LOX metabolites of docosahexaenoic acid (DHA), 17-hydroperoxy-, 17-hydroxy-, 10,17-dihydroxy-, and 7,17-dihydroxy-DHA inhibit the proliferation of these cells at ≥0.001, 0.01, 1, and 1 µM, respectively. By comparison, the corresponding 15-hydroperoxy, 15-hydroxy, 8,15-dihydroxy, and 5,15-dihydroxy metabolites of arachidonic acid as well as DHA itself require ≥10–100 µM to do this. Like DHA, the DHA metabolites a) induce PC3 cells to activate a peroxisome proliferator-activated receptor-γ (PPARγ) reporter, express syndecan-1, and become apoptotic and b) are blocked from slowing cell proliferation by pharmacological inhibition or knockdown of PPARγ or syndecan-1. The DHA metabolites thus slow prostate cancer cell proliferation by engaging the PPARγ/syndecan-1 pathway of apoptosis and thereby may contribute to the prostate cancer-suppressing effects of not only 15-LOX but also dietary DHA.

Fatty Acid Metabolites in Rapidly Proliferating Breast Cancer

Purpose Breast cancers that over-express a lipoxygenase or cyclooxygenase are associated with poor survival possibly because they overproduce metabolites that alter the cancer’s malignant behaviors. However, these metabolites and behaviors have not been identified. We here identify which metabolites among those that stimulate breast cancer cell proliferation in vitro are associated with rapidly proliferating breast cancer. Experimental Design We used selective ion monitoring-mass spectrometry to quantify in the cancer and normal breast tissue of 27 patients metabolites that stimulate (15-, 12-, 5-hydroxy-, and 5-oxo-eicosatetraenoate, 13-hydroxy-octadecaenoate [HODE]) or inhibit (prostaglandin [PG]E2 and D2) breast cancer cell proliferation. We then related their levels to each cancer’s proliferation rate as defined by its Mib1 score. Results 13-HODE was the only metabolite strongly, significantly, and positively associated with Mib1 scores. It was similarly associated with aggressive grade and a key component of grade, mitosis, and also trended to be associated with lymph node metastasis. PGE2 and PGD2 trended to be negatively associated with these markers. No other metabolite in cancer and no metabolite in normal tissue had this profile of associations. Conclusions Our data fit a model wherein the overproduction of 13-HODE by 15-lipoxygenase-1 shortens breast cancer survival by stimulating its cells to proliferate and possibly metastasize; no other oxygenase-metabolite pathway, including cyclooxygenase-PGE2/D2 pathways, uses this specific mechanism to shorten survival.

15-Lipoxygenase-1-mediated metabolism of docosahexaenoic acid is required for syndecan-1 signaling and apoptosis in prostate cancer cells

Fatty acid metabolism impacts multiple intracellular signaling pathways in many cell types, but its role in prostate cancer cells is still unclear. Our previous studies have shown that the n-3 polyunsaturated fatty acid docosahexaenoic acid (DHA) induces apoptosis in human prostate cancer cells by a syndecan-1 (SDC-1)-dependent mechanism. Here, we examined the contribution of lipoxygenase (LOX)- and cyclooxygenase (COX)-mediated DHA metabolism to this effect. Pan-LOX inhibitor (nordihydroguaiaretic acid), 15-LOX inhibitor (luteolin) or 15/12-LOX inhibitor (baicalein) blocked the induced effect of DHA on SDC-1 expression and apoptosis in human prostate cancer cells, whereas 5-LOX inhibitor, AA861, was ineffective. Human prostate cancer cells lines (PC3, LNCaP and DU145 cells) expressed two 15-LOX isoforms, 15-LOX-1 and 15-LOX-2, with higher 15-LOX-1 and lower 15-LOX-2 expressions compared with human epithelial prostate cells. Knockdown of 15-LOX-1 blocked the effect of DHA on SDC-1 expression and caspase-3 activity, whereas silencing 15-LOX-2, 5-LOX, COX-1, COX-2 or 12-LOX had no effect. Moreover, the ability of DHA to inhibit the activity of the PDK/Akt (T308) signaling pathway was abrogated by silencing 15-LOX-1. These findings demonstrate that 15-LOX-1-mediated metabolism of DHA is required for it to upregulate SDC-1 and trigger the signaling pathway that elicits apoptosis in prostate cancer cells.

Platelet-Activating Factor: Mechanisms of Cellular Activation

Platelet-activating factor (PAF) is a phosphatidylcholine containing a long-chain alkyl ether at position 1 and an acetate ester at position 2 (Blank et al., 1979; Demopoulos et al., 1979; Polonsky et al., 1980): Open image in new window Various cell types form and secrete this product when stimulated. PAF is found in the blood of animals undergoing experimentally induced toxic reactions such as anaphylaxis, serum sickness, and endotoxemia. It can mimic these reactions when injected intravenously into healthy animals. In particular, PAF infusion produces the activation of intravascular leukocytes and platelets, contraction of pulmonary airways, vascular instability, enhanced capillary permeability, and cardiac abnormalities. Deposited locally into tissues, PAF promotes edema and leukocyte accumulation. These results implicate PAF as a mediator of allergic and inflammatory reactions. However, even the most complex, systemic effects of PAF must result from fundamental interactions with target cells. Some understanding of these interactions has been obtained using in vitro studies on tissues containing one or only a few cell types.

ω-3 PUFAs, Breast and Prostate Cancer: Experimental Studies

Although human epidemiological and clinical studies to date have failed to provide conclusive data on a protective effect of ω-3 polyunsaturated fatty acids (PUFAs) on breast and prostate cancer, cell culture and animal studies present a more positive story. Experimental models investigated include various human cancer cell lines, rats with chemically induced tumors, mice with transplantable and human xenograft tumors, and, more recently, transgenic models. They have suggested a number of biological targets for ω-3 PUFAs that impact cell proliferation, survival, apoptosis, angiogenesis, invasiveness, and metastasis, i.e., ω-3 PUFAs may have multifactorial properties in preventing and inhibiting cancer. These models have uncovered numerous mechanisms for the anti-cancer activity of ω-3 PUFAs with the most frequently cited being their ability to block the metabolism of ω-6 PUFAs into agents that promote many facets of the malignant behavior of cancer cells.

The Targeting of Leukocytes by 5-Oxo-Eicosanoids

The identities and roles of arachidonic acid (AA) metabolites in human diseases are often elusive. For example, allergens cause tissues to secrete agents that attract eosinophils (Eo) and induce these cells to release granule enzymes, Superoxide anion, and bioactive molecules that lead to the organ dysfunctions seen in allergy1. Zileuten, an anti-lipoxygenase drug, relaxes the airways of asthmatics, perhaps by blocking the synthesis of an Eo-targeting eicosanoid2-4. The drug clearly inhibits allergen-induced Eo fluxes to lung as well as bronchospasm in primates and lower mammals5,6. Thus, an eicosatetraenoate (ETE) may link Eo to allergy. Among the eicosanoids occupying human allergen-reactive sites, 15(S)-hydroxy-ETE (15-HETE), prostaglandins, and thromboxanes are not chemotactic for Eo; leukotriene (LT)B4 attracts Eo but is more active on polymorphonuclear neutrophils (PMN); and peptido-LTs, lipoxins, and 5(S)-hydroxy-ETE (5-HETE) act weakly on both cell types1,7-10. These agents are unlikely to mediate Eo-based lesions in vivo. However, a newly defined 5-HETE analog, 5-oxoETE, has in vitro actions suggesting that it could be such a mediator.

Mechanism of Hexose Transport in Human Polymorphonuclear Leukocytes

Cellular functions of human polymorphonuclear leukocytes (PMNL) including phagocytosis, motility, and bactericidal activity require energy derived from glucose. Whereas insulin does not stimulate hexose transport in human PMNL, we have recently reported that chemotactic factors such as complement-derived C5a, the synthetic oligopeptide N-formyl-methionyl-leucyl-phenylalanine (fMLP), or the calcium ionophore A23187 stimulates transport of deoxyglucose (DOG) into human PMNL1,2. FMLP with an EC50 of 1.2 nM, C5a with an EC50 of 1 nM, and A23187 with an EC50 of 10 nM all cause at least a 5-fold stimulation of DOG uptake.

ω-3 PUFAs: Interventional Trials for the Prevention and Treatment of Breast and Prostate Cancer

Due to global changes in dietary sources and habits, the human diet has shifted to include an unfavorable ratio of ω-6 to ω-3 PUFAs. This is believed to have drastic consequences on health, increasing the risk for cardiovascular and inflammation-related diseases as well as cancer. Epidemiology and experimental studies suggest that ω-3 PUFAs are protective against certain types of cancer, including colon, breast, and prostate cancer. The available data have motivated the recent implementation of interventional clinical trials where cancer patients or individuals at high risk of developing cancer receive dietary ω-3 PUFA supplementation. In this review, we summarize the objectives of ongoing and recently closed clinical trials of ω-3 PUFA supplements for breast and prostate cancer prevention and treatment that are listed in the ClinicalTrials.gov database, a registry of federally and privately supported clinical trials conducted in the United States and worldwide (http://clinicaltrials.gov/).