Marc E. Surette

Mechanisms of the priming effect of lipopolysaccharides on the biosynthesis of leukotriene B4 in chemotactic peptide-stimulated human neutrophils

The goal of this study was to explain the priming effect of lipopolysaccharides (LPS) in human polymorphonuclear leukocytes on leukotriene B4 (LTB4) biosynthesis after stimulation with the receptor‐mediated agonist formyl‐methionyl‐leucylphenylalanine (fMLP). This priming effect for LTB4 biosynthesis was maximal after a 30 min preincubation with LPS but was lost when incubations were extended to 90 min or longer. Priming with LPS resulted in an enhanced maximal activation of 5‐lipoxygenase (5‐ to 15‐fold above unprimed cells) as well as a prolonged activation of the enzyme after stimulation with fMLP compared to that measured in unprimed cells. The activation of 5‐lipoxygenase was associated with its translocation to the nuclear fraction of the cell after stimulation of LPS‐primed cells but not of unprimed cells. Priming of cells with LPS also resulted in an enhanced capacity (fivefold increase) for arachidonic acid (AA) release after stimulation with fMLP compared to unprimed cells as measured by mass spectrometry. This release of AA was very efficiently blocked in a dose‐dependent manner by the 85 kDa cytosolic phospholipase A2 (PLA2) inhibitor MAFP (IC50 = 10nM) but not by the 14 kDa secretory PLA2 inhibitor SB 203347 (up to 5 µM), indicating that the 85 kDa cPLA2 is the PLA2 responsible for AA release in response to receptor‐mediated agonists. In accord with inhibitor studies, the LPS‐mediated phosphorylation of cPLA2 followed the same kinetics as the priming for AA release, and a measurable fMLP‐induced translocation of cPLA2 was observed only in primed cells. As with AA release and LTB4 biosynthesis, both the phosphorylation and capacity to translocate cPLA2 were reversed when the preincubation period with LPS was extended to 120 min. These results explain some of the cellular events responsible for the potentiation and subsequent decline of functional responses of human polymorphonuclear leukocytes recruited to inflammatory foci.—Surette, M. E., Dallaire, N., Jean, N., Picard, S., Borgeat, P. Mechanisms of the priming effect of lipopolysaccharides on the biosynthesis of leukotriene B4 in chemotactic peptide‐stimulated human neutrophils. FASEB J. 12, 1521–1531 (1998)

Dietary omega‐3 PUFA and health: Stearidonic acid‐containing seed oils as effective and sustainable alternatives to traditional marine oils

The daily consumption of dietary omega-3 PUFA is recommended by governmental agencies in several countries and by a number of health organizations. The molecular mechanisms by which these dietary PUFA affect health involve the enrichment of cellular membranes with long-chain 20- and 22-carbon omega-3 PUFA that impacts tissues by altering membrane protein functions, cell signaling, and gene expression profiles. These changes are recognized to have health benefits in humans, especially relating to cardiovascular outcomes. Cellular membrane enrichment and health benefits are associated with the consumption of long-chain omega-3 PUFA found in marine oils, but are not generally linked with the consumption of alpha-linolenic acid, the 18-carbon omega-3 PUFA found in plant seed oils. However, the supply of omega-3 PUFA from marine sources is limited and may not be sustainable. New plant-derived sources of omega-3 PUFA like stearidonic acid-soy oil from genetically modified soybeans and Ahiflower oil from Buglossoides arvensis seeds that are enriched in the 18-carbon omega-3 PUFA stearidonic acid are being developed and show promise to become effective as well as sustainable sources of omega-3 PUFA. An example of changes in tissue lipid profiles associated with the consumption of Ahiflower oil is presented in a mouse feeding study.

Arachidonic Acid Regulates the Translocation of 5-Lipoxygenase to the Nuclear Membranes in Human Neutrophils

Elevation of the intracellular cAMP concentration in agonist-activated human neutrophils (PMN) leads to the concomitant inhibitions of arachidonic acid (AA) release, 5-lipoxygenase (5-LO) translocation, and leukotriene (LT) biosynthesis. We report herein that exogenous AA completely prevents cAMP-dependent inhibition of 5-LO translocation and LT biosynthesis in agonist-activated PMN. Moreover, the group IVA phospholipase A2 inhibitor pyrrophenone and the MEK inhibitor U-0126 inhibited AA release and 5-LO translocation in activated PMN, and these effects were also prevented by exogenous AA, demonstrating a functional link between AA release and 5-LO translocation. Polyunsaturated fatty acids of the C18 and C20 series containing at least three double bonds located from carbon 9 (or closer to the carboxyl group) were equally effective as AA in restoring 5-LO translocation in pyrrophenone-treated agonist-activated PMN. Importantly, experiments with the 5-LO-activating protein inhibitor MK-0591 and the intracellular Ca2+ chelator BAPTA-AM demonstrated that the AA-regulated 5-LO translocation is FLAP- and Ca2+-dependent. Finally, the redox and competitive 5-LO inhibitors L-685,015, L-739,010, and L-702,539 (but not cyclooxygenase inhibitors) efficiently substituted for AA to reverse the pyrrophenone inhibition of 5-LO translocation, indicating that the site of regulation of 5-LO translocation by AA is at or in the vicinity of the catalytic site. This report demonstrates that AA regulates the translocation of 5-LO in human PMN and unravels a novel mechanism of the cAMP-mediated inhibition of LT biosynthesis.

Mechanisms that account for the selective release of arachidonic acid from intact cells by secretory phospholipase A2

The current study examined mechanisms that account for the selective release of arachidonic acid (AA) from cells by secretory phospholipase A2 (sPLA2). Initial studies demonstrated that low concentrations of group I and group III PLA2 isotypes and an sPLA2-enriched extract from bone marrow-derived mast cells (BMMC) selectively released AA from mast cells. Much higher concentrations of group II PLA2 were required to release comparable quantities of AA. Group I PLA2 also selectively released AA from another mast cell line (CFTL-15) and a monocytic cell line (THP-1). In contrast, high concentrations of group I PLA2 were required to release fatty acids from a promyelocytic cell line (HL-60) and this release was not selective for AA. Binding studies revealed that cell types (BMMC, CFTL-15 and THP-1) which selectively released AA also had the capacity to specifically bind group I PLA2. However, group II PLA2, which did not selectively release AA from cells, also did not specifically bind to these same cell types. Additional studies revealed that sPLA2 binding to the mast cell receptor was attenuated after stimulation with antigen or ionophore A23187. Reverse transcriptase-polymerase chain reaction analyses indicated the presence of mRNA for the sPLA2 receptor in BMMC, CFTL-15 and THP-1 and the absence of this mRNA in HL-60. Final studies demonstrated that p-aminophenyl-alpha-D-mannopyranoside BSA, a known ligand of the sPLA2 receptor, also selectively released AA from mast cells but not from HL-60 cells. These experiments indicated that receptor occupancy alone (without PLA2 activity) is sufficient to induce the release of AA from mast cells. Together, these data reveal that specific isotypes of sPLA2 have the capacity to selectively release AA from certain cells by their capacity to bind to sPLA2 receptors on the cell surface.

Inhibition of Leukotriene biosynthesis by a novel dietary fatty acid formulation in patients with atopic asthma: A randomized, placebo-controlled, parallel-group, prospective trial

BACKGROUND Leukotriene inhibitors and leukotriene-receptor antagonists are effective in the treatment of inflammatory diseases such as asthma. A search of the entirety of MEDLINE using the terms diet plus leukotrienes identified numerous studies that have explored dietary-management strategies to reduce leukotriene levels through supplementation with polyunsaturated fatty acids such as gamma-linolenic acid (GLA) and eicosapentaenoic acid (EPA). However, the search found no studies on the use of combinations of these fatty acids in patients with asthma. OBJECTIVE The goal of this study was to determine the effect of daily intake of an emulsion (PLT 3514) containing dietary GLA and EPA on ex vivo stimulated whole blood leukotriene biosynthesis in patients with atopic asthma. METHODS This was a randomized, double-blind, placebo-controlled, parallel-group, prospective trial in patients with mild to moderate atopic asthma. Patients consumed 10 g PLT 3514 emulsion (containing 0.75 g GLA + 0.5 g EPA), 15 g PLT 3514 emulsion (containing 1.13 g GLA + 0.75 g EPA), or placebo (olive oil) emulsion daily for 4 weeks. Plasma fatty acids were measured by gas chromatography, and stimulated whole blood leukotrienes were measured by reverse-phase high-performance liquid chromatography with ultraviolet detection using a diode array detector. RESULTS Forty-three patients (33 women, 10 men) participated in the study. Leukotriene biosynthesis was significantly decreased in patients consuming 10 or 15 g PLT 3514 compared with placebo (P < 0.05, analysis of covariance). No clinically significant changes in vital signs were observed throughout the study, and there were no significant between-group differences in treatment-emergent adverse events or mean clinical laboratory values. CONCLUSION Daily consumption of dietary GLA and EPA in a novel emulsion formulation inhibited leukotriene biosynthesis in this population of patients with atopic asthma and was well tolerated.

Synthesis and Antiradical/Antioxidant Activities of Caffeic Acid Phenethyl Ester and Its Related Propionic, Acetic, and Benzoic Acid Analogues

Caffeic acid phenethyl ester (CAPE) is a bioactive component isolated from propolis. A series of CAPE analogues was synthesized and their antiradical/antioxidant effects analyzed. The effect of the presence of the double bond and of the conjugated system on the antioxidant effect is evaluated with the analogues obtained from 3-(3,4-dihydroxyphenyl) propanoic acid. Those obtained from 2-(3,4-dihydroxyphenyl) acetic acid and 3,4-dihydroxybenzoic acid allow the evaluation of the effect of the presence of two carbons between the carbonyl and aromatic system.

Antiproliferative, antiandrogenic and cytotoxic effects of novel caffeic acid derivatives in LNCaP human androgen-dependent prostate cancer cells.

Caffeic acid and its naturally occurring derivative caffeic acid phenethyl ester (CAPE) have antiproliferative and cytotoxic properties in a variety of cancer cell lines without displaying significant toxicity toward healthy cells, and are considered to be potential anticancer agents. However, little is known about their effects on prostate cancer cells. We synthesized and evaluated the effects of caffeic acid, CAPE (2) and 18 synthetic derivatives on cell viability and androgen-dependent cell proliferation, subcellular localisation and expression of androgen receptor (AR) and secretion of prostate-specific antigen (PSA) in LNCaP human hormone-dependent prostate cancer cells. Several synthetic derivatives of CAPE were strong, concentration-dependent cytotoxic agents in LNCaP cells with IC50 values in the 6.8-26.6 μM range, potencies that were up to five-fold greater than that of CAPE (33.7±4.0 μM). A number of caffeic acid derivatives were inhibitors of androgen-stimulated LNCaP cell proliferation with concomitant inhibition of DHT-stimulated PSA secretion. Compound 24 was the most cytotoxic and antiproliferative caffeic acid derivative (IC50 values of 6.8±0.3 and 2.4±0.8 μM, respectively) inhibiting DHT-stimulated cell proliferation and PSA secretion statistically significantly at concentrations as low as 0.3 μM. Exposure to DHT increased cytoplasmic and nuclear AR levels and co-treatment with increasing concentrations of compound 24 or CAPE (2), notably, further increased these levels. In conclusion, a number of synthetic derivatives of caffeic acid are potent inhibitors of androgen-dependent prostate cancer cell proliferation and viability, acting, at least in part, via an antiandrogenic mechanism that involves increased nuclear accumulation of (presumably inactive) AR.

Maternal Iron Deficiency Alters Essential Fatty Acid and Eicosanoid Metabolism and Increases Locomotion in Adult Guinea Pig Offspring

Iron deficiency (ID) is the most prevalent worldwide nutritional deficiency. Groups at risk of developing ID anemia are infants and pregnant women, even in industrialized countries. Our goal in this study was to evaluate the long-term consequences of maternal ID on the offsprings fatty acid and eicosanoid metabolism, behavior, and spatial memory. Female guinea pigs consumed iron-sufficient (IS) and -deficient (ID) diets for 14 d before mating and throughout pregnancy and lactation. Dietary iron restriction resulted in ID in pregnant females. On postnatal d 9, all offspring (ID and IS) were weaned to the IS diet and at 42 d, all offspring were iron replete. Locomotion was tested in pups on postnatal d 24 and 40 and spatial memory from d 25 to 40. Pups from the ID group were significantly more active in the open field at both times of testing, whereas spatial memory, tested in a Morris water maze, was comparable in both groups. On postnatal d 42, liver, RBC, and brain fatty acid composition were measured. Dihomogammalinolenic [20:3(n-6)], docosapentaenoic [22:5(n-3)], and docosahexaenoic [22:6(n-3)] acid contents were significantly higher in brain phospholipids of offspring born to ID dams. Prostaglandin E(2) and F(2alpha) concentrations were also significantly higher in brains of offspring born to ID dams. This demonstrates that moderate ID during gestation and lactation results in alterations of brain fatty acid and eicosanoid metabolism and perturbation in behavior in adult offspring.

Synthesis and 5-lipoxygenase inhibitory activity of new cinnamoyl and caffeoylclusters.

Novel cinnamoyl and caffeoyl clusters were synthesized by multiple Cu(I)-catalyzed [1,3]-dipolar cycloadditions and their anti-5-lipoxygenase inhibitory activity was tested. Caffeoyl cluster showed an improved 5-lipoxygenase inhibitory activity compared to caffeic acid, with caffeoyl trimer 16 and tetramer 19 showing the best 5-lipoxygenase inhibitory activity.

NMR Metabolomics Analysis of the Effects of 5-Lipoxygenase Inhibitors on Metabolism in Glioblastomas

Changes across metabolic networks are emerging as an integral part of cancer development and progression. Increasing comprehension of the importance of metabolic processes as well as metabolites in cancer is stimulating exploration of novel, targeted treatment options. Arachidonic acid (AA) is a major component of phospholipids. Through the cascade catalyzed by cyclooxygenases and lipoxygenases, AA is also a precursor to cellular signaling molecules as well as molecules associated with a variety of diseases including cancer. 5-Lipoxygenase catalyzes the transformation of AA into leukotrienes (LT), important mediators of inflammation. High-throughput analysis of metabolic profiles was used to investigate the response of glioblastoma cell lines to treatment with 5-lipoxygenase inhibitors. Metabolic profiling of cells following drug treatment provides valuable information about the response and metabolic alterations induced by the drug action and give an indication of both on-target and off-target effects of drugs. Four different 5-lipoxygenase inhibitors and antioxidants were tested including zileuton, caffeic acid, and its analogues caffeic acid phenethyl ester and caffeic acid cyclohexethyl ester. A NMR approach identified metabolic signatures resulting from application of these compounds to glioblastoma cell lines, and metabolic data were used to develop a better understanding of the mode of action of these inhibitors.