Role of omega-3 polyunsaturated fatty acids on cyclooxygenase-2 metabolism in brain-metastatic melanoma

Cyclooxygenase-2 (COX-2) is important in the progression of epithelial tumors. Evidence indicates that omega-6 PUFAs such as arachidonic acid (AA) promote the growth of tumor cells; however, omega-3 fatty acids [eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA)] inhibit tumor cell proliferation. We investigated the effects of omega-3 PUFA on the expression and function of COX-2 in 70W, a human melanoma cell line that metastasizes to the brain in nude mice. We show that 1) tumor necrosis factor-alpha upregulates the expression of both COX-2 mRNA and prostaglandin E2 (PGE2) production, and 2) omega-3 and omega-6 PUFA regulate COX-2 mRNA expression and PGE2 production. AA increased COX-2 mRNA expression and prostaglandin production in omega-6-stimulated 70W cells. Conversely, COX-2 mRNA expression decreased in cells incubated with EPA or DHA. AA increased Matrigel invasion 2.4-fold, whereas EPA or DHA did not. Additionally, PGE2 increased in vitro invasion 2.5-fold, whereas exposure to PGE3 significantly decreased invasion. Our results demonstrate that incubation of 70W cells with either AA or PGE2 increased invasiveness, whereas incubation with EPA or DHA downregulated both COX-2 mRNA and protein expression, with a subsequent decrease in Matrigel invasion. Taken together, these results indicate that omega-3 PUFA regulate COX-2-mediated invasion in brain-metastatic melanoma.     

Treatment with n-3 polyunsaturated fatty acids reverses endothelial dysfunction and oxidative stress in experimental menopause

Menopause is associated with endothelial dysfunction and oxidative stress. In this condition, reduced n-3 polyunsaturated fatty acids (n-3 PUFAs) contribute to cardiovascular disease. We investigated whether treatment with n-3 PUFA reverses endothelial dysfunction and oxidative stress in experimental menopause. Thirty female rats underwent either sham-surgery or bilateral ovariectomy or bilateral ovariectomy+oral n-3 PUFA (0.8 g kg^-1 day^-1 for 2 months).Ovariectomy caused endothelial dysfunction to acetylcholine, which was reversed by superoxide scavenger Tiron. Erythrocyte membrane lipid composition was characterized by reduced n-3 PUFA total content and omega-3 index, and by concomitant increase in n-6:n-3 PUFA ratio. Ovariectomy-related oxidative stress, demonstrated by both enhanced superoxide production and 3-nitrotyrosine expression in aorta, was associated with increased nicotinamide adenine dinucleotide phosphate (NADPH) oxidase subunit NOX-4 protein expression. Endothelial nitric oxide synthase (eNOS) functional inhibition by l-NG-nitroarginine methyl ester, protein expression and activity did not change.In ovariectomized rats, treatment with n-3 PUFA increased n-3 PUFA total content and omega-3 index and decreased n-6:n-3 PUFA ratio in erythrocyte membrane, reversed vascular oxidative stress, endothelial dysfunction, aortic 3-nitrotyrosine and markedly lowered NOX-4 protein expression; eNOS protein expression also increased, paralleled by reversal of inhibitory binding to Caveolin-1, while ex-vivo functional inhibition and NOS synthesis were unchanged.These findings demonstrate in vivo a therapeutic benefit of n-3 PUFA on menopause-associated endothelial dysfunction by reversal of alterations in membrane lipid composition induced by ovariectomy and by reduction of vascular oxidative stress. In this setting they also identify NOX-4 as a potential target to reduce oxidative stress-mediated vascular complications.     

Effect of omega-3 polyunsaturated fatty acids on activity of glutathione-dependent enzymes in the liver cytosol and blood erythrocytes in rats with experimental chronic bronchitis and in the norm

The influence of omega-3 polyunsaturated fatty acids (omega-3 PUFA) on the activity of glutathione reductase, glutathione transferase and glutathione peroxidase in the liver cytosole and red blood cells of normal rats and animals with experimental chronic bronchitis. omega-3 PUFA ("Tekom" medication) activate glutathione reductase of liver cytosole and glutathionperoxidase in the red blood cells in rats. In the rats with chronic inflammatory process in bronchia omega-3 PUFA corrects the glutathione-dependent systems of detoxication. Effects were more expressed in the liver cytosole in comparison with the red blood cells. The using of omega-3 PUFA as a means for treatment and prophylaxis was more effective than for treatment only.     

Interference of retinoic acid binding to its binding protein by omega-6 fatty acids

Cellular retinoic acid-binding protein (CRABP) is the putative mediator of the biological effects of retinoic acid in the control of epithelial differentiation and tumorigenesis. Omega-6 fatty acids such as linoleic acid and arachidonic acid, precursors of prostaglandin synthesis, caused inhibition of retinoic acid binding to CRABP. These fatty acids, however, possessed lower affinity than retinoic acid for the binding protein. Omega-3 fatty acids, such as eicosapentaenoic acid and docosohexaenoic acid, did not cause such inhibition in the binding of retinoic acid. Whereas retinoic acid was a potent modulator of differentiation of F9 embryonal carcinoma cells, neither omega-3 nor omega-6 fatty acids showed any significant differentiation potential. Competition by omega-6 fatty acids with retinoic acid for CRABP may neutralize the binding protein-mediated biological functions of retinoic acid, and could thereby enhance tumor production.     

Dietary polyunsaturated fatty acids enhance hepatic AMP-activated protein kinase activity in rats

Polyunsaturated fatty acids (PUFA) and a number of drugs (metformin, thiazolidinediones) and hormones (leptin, adiponectin) that activate AMP-activated protein kinase (AMPK) have been reported to improve insulin sensitivity. To determine whether PUFA activate AMPK, Sprague-Dawley rats were adapted to a 3h meal-feeding regimen using a fat-free diet (FFD) supplemented with fish oil (n-3) or triolein (n-9) for 7 days. No differences in hepatic AMPK activity were observed between the groups after 21h of fasting. On the other hand, hepatic AMPK phosphorylation was decreased in rats refed the FFD, the FFD+triolein, and the FFD+PUFA by 80%, 75%, and 50%, respectively, when assessed 2h after completion of a meal. In keeping with these changes, decreases in acetyl-CoA carboxylase phosphorylation and carnitine palmitoyl transferase-1 mRNA and increases in fatty acid synthase gene expression were greatest in rats fed the FFD and least in the PUFA-fed rats. The results indicate that dietary PUFA enhance hepatic AMPK activity in vivo, and implicate AMPK as a component of the nutrient-sensing mechanism through which dietary fatty acids and especially PUFA influence the regulation of hepatic lipid metabolism and gene expression.     

Effect of long-chain fatty acids in the culture medium on fatty acid composition of WEHI-3 and J774A.1 cells

As a first step in determining the mechanism of action of specific fatty acids on immunological function of macrophages, a comparative study of the effect of long-chain polyunsaturated fatty acids (PUFA) in the medium was conducted in two macrophage cell lines, J774A.1 and WEHI-3. The baseline fatty-acid profiles of the two cell lines differed in the % distribution of saturated (SFA) and unsaturated fatty acids (UFA). J774A.1 cells had a higher % of SFA (primarily palmitic acid) than WEHI-3 cells. Conversely, WEHI-3 cells had a higher % of UFA (primarily oleic acid) than J774A.1 cells. Neither cell line had detectable amounts of alpha-linolenic acid (ALA) or eicosapentaenoic acid (EPA). The most abundant polyunsaturated fatty acid in both cells lines was arachidonic acid (AA). The efficiency of transport of fatty acids from the medium to the macrophages by two delivery vehicles (BSA complexes and ethanolic suspensions) was compared. Overall, fatty acids were transported satisfactorily by both delivery systems. Alpha-linolenic acid and doscosahexenoic acid (DHA) were transported more efficiently by the ethanolic suspension system. Linoleic acid (LA) was taken up more completely than ALA, and DHA was taken up more completely than EPA by both cell cultures and delivery systems. A dose-response effect was demonstrated for LA, ALA, EPA and DHA in both J774A.1 and WEHI-3 cells. Addition of polyunsaturated fatty acids (PUFA) to the cell cultures modified the total lipid fatty acid composition of the cells. The presence of ALA in the culture medium resulted in a significant decrease in AA in both cell lines. The omega-3/omega-6 fatty acid ratio (omega-3/omega-6), polyunsaturated/saturated fatty acid ratio (P/S), and unsaturation index (UI) increased directly with the amount of PUFA and omega-3 fatty acid provided in the medium. The results indicate that the macrophage cell lines have similar, but not identical, fatty acid profiles that may be the result of differences in fatty acid metabolism. These distinctions could in turn produce differences in immunological function. The ethanol fatty-acid delivery system, when compared with the fatty acid-BSA complex system, is preferable for measurement of dose-response effects, because the cellular fatty acid content increased in proportion to the amount of fatty acid provided in the medium. Similar dose-response results were observed in a previous in vivo study using flaxseed, rich in ALA, as a source of PUFA.     

Effect of saturated, omega-3 and omega-6 polyunsaturated fatty acids on myocardial infarction

Dietary fatty acids have cholesterol lowering, antiatherogenic, and antiarrhythmic properties that decrease the risk of myocardial infarction (MI). This study was designed to study the effects of various oils rich in either polyunsaturated (omega-3 or omega-6) fatty acids (PUFA) or saturated fatty acids (SFA) on the severity of experimentally induced MI. Male albino Sprague-Dawley rats (100-150 g; n = 20) were fed diets enriched with fish oil (omega-3 PUFA), peanut oil (omega-6 PUFA), or coconut oil (SFA) for 60 days. Experimental MI was induced with isoproterenol. Mortality rates; serum enzymes aspartate amino transferase; alanine amino transferase; creatine phosphokinase (CPK); lipid profiles in serum, myocardium, and aorta; peroxide levels in heart and aorta; activities of catalase and superoxide dismutase; and levels of glutathione were measured. The results demonstrated that mortality rate, CPK levels, myocardial lipid peroxides, and glutathione levels were decreased in the omega-3 PUFA treated group. Maximum increase in parameters indicative of myocardial damage was seen in the coconut oil group. These findings suggest that dietary omega-3 PUFA offers maximum protection in experimentally induced MI in comparison to omega-6 PUFA and SFA enriched diets. SFA was found to have the least protective effect.     

Impact of polyunsaturated fatty acids on cytoskeletal linkage of L-selectin

Polyunsaturated fatty acid [omega-3 polyunsaturated fatty acids (omega-3PUFAs)] incorporation into cell membranes has been shown to have potent anti-inflammatory activity, though the mechanisms involved are only partially characterized. Here, we show that PUFA enrichment of T cell membranes decreased the overall expression of L-selectin as well as a highly conserved epitope on L-selectin that may serve as a marker for optimal protein function. Additionally, PUFA enrichment inhibited L-selectin cytoskeletal association, which is thought to be important for optimal functional activity. In support of this, PUFA enrichment of gammadelta T cell membranes reduced L-selectin-dependent rolling interactions under conditions mimicking physiological flow. Taken together, these data suggest that the anti-inflammatory activity of omega-3 polyunsaturated fatty acids may be due, in part, to a novel effect on L-selectin, namely PUFA reduction or prevention of cytoskeletal association of L-selectin.     

Addition of an N-terminal epitope tag significantly increases the activity of plant fatty acid desaturases expressed in yeast cells

Saccharomyces cerevisiae shows great potential for development of bioreactor systems geared toward the production of high-value lipids such as polyunsaturated omega-3 fatty acids, the yields of which are largely dependent on the activity of ectopically expressed enzymes. Here, we show that the addition of an N-terminal epitope tag sequence (either Myc or hemagglutinin) to oleate desaturase (FAD2) or omega-3 linoleate desaturase (FAD3) enzymes from plants, which catalyze consecutive reactions in the production of long chain omega-3 fatty acids, significantly increases their activity up to fourfold when expressed in yeast cells. Quantitative protein blotting using an antibody specific for native FAD2 revealed that the steady-state amount of the epitope-tagged FAD2 protein was also approximately fourfold higher than that of its untagged counterpart, demonstrating a direct relationship between the epitope tag-induced increase in enzyme amount and fatty acid product formation. Protein half-life and RNA blotting experiments indicated that the half-lives and mRNA content of the tagged and untagged FAD2 proteins were essentially the same, suggesting that the epitope tags increased protein abundance by improving translational efficiency. Taken together, these results indicate that the addition of an epitope tag sequence to a plant fatty acid desaturase (FAD) not only provides a useful means for protein immunodetection using highly specific, commercially available antibodies, but that it also significantly increases FAD activity and the production of polyunsaturated fatty acids in yeast cells.     

Formation of highly reactive cyclopentenone isoprostane compounds (A3/J3-isoprostanes) in vivo from eicosapentaenoic acid

Omega-3 (omega-3) polyunsaturated fatty acids (PUFAs) found in marine fish oils are known to suppress inflammation associated with a wide variety of diseases. Eicosapentaenoic acid (EPA) is one of the most abundant omega-3 fatty acids in fish oil, but the mechanism(s) by which EPA exerts its beneficial effects is unknown. Recent studies, however, have demonstrated that oxidized EPA, rather than native EPA, possesses anti-atherosclerotic, anti-inflammatory, and anti-proliferative effects. Very few studies to date have investigated which EPA oxidation products are responsible for this bioactivity. Our research group has previously reported that anti-inflammatory prostaglandin A(2)-like and prostaglandin J(2)-like compounds, termed A(2)/J(2)-isoprostanes (IsoPs), are produced in vivo by the free radical-catalyzed peroxidation of arachidonic acid and represent one of the major products resulting from the oxidation of this PUFA. Based on these observations, we questioned whether cyclopentenone-IsoP compounds are formed from the oxidation of EPA in vivo. Herein, we report the formation of cyclopentenone-IsoP molecules, termed A(3)/J(3)-IsoPs, formed in abundance in vitro and in vivo from EPA peroxidation. Chemical approaches coupled with gas chromatography/mass spectrometry (GC/MS) and liquid chromatography/mass spectrometry (LC/MS) were used to structurally characterize these compounds as A(3)/J(3)-IsoPs. We found that levels of these molecules increase approximately 200-fold with oxidation of EPA in vitro from a basal level of 0.8 +/- 0.4 ng/mg EPA to 196 +/- 23 ng/mg EPA after 36 h. We also detected these compounds in significant amounts in fresh liver tissue from EPA-fed rats at basal levels of 19 +/- 2 ng/g tissue. Amounts increased to 102 +/- 15 ng/g tissue in vivo in settings of oxidative stress. These studies have, for the first time, definitively characterized novel, highly reactive A/J-ring IsoP compounds that form in abundance from the oxidation of EPA in vivo.     

Retroviral-based gene therapy with cyclooxygenase-2 promotes the union of bony callus tissues and accelerates fracture healing in the rat

BACKGROUND: An in vivo gene therapy strategy was developed to accelerate bone fracture repair. METHODS: Direct injection of a murine leukemia virus-based vector targeted transgene expression to the proliferating periosteal cells arising shortly after fracture. Cyclooxygenase-2 (Cox-2) was selected because the transgene for its prostaglandin products that promote angiogenesis, bone formation and bone resorption, are all required for fracture healing. The human (h) Cox-2 transgene was modified to remove AU-rich elements in the 3'-untranslated region and to improve protein translation. RESULTS: In vitro studies revealed robust and sustained Cox-2 protein expression, prostaglandin E(2) and alkaline phosphatase production in rat bone marrow stromal cells and osteoblasts transgenic for the hCox-2 gene. In vivo studies in the rat femur fracture revealed that Cox-2 transgene expression produced bony union of the fracture by 21 days post-fracture, a time when cartilage persisted within the fracture tissues of control animals and approximately 1 week earlier than the healing normally observed in this model. None of the ectopic bone formation associated with bone morphogenetic protein gene therapy was observed. CONCLUSIONS: This study represents the first demonstration that a single local application of a retroviral vector expressing a single osteoinductive transgene consistently accelerated fracture repair.     

The importance of the omega-6/omega-3 fatty acid ratio in cardiovascular disease and other chronic diseases

Several sources of information suggest that human beings evolved on a diet with a ratio of omega-6 to omega-3 essential fatty acids (EFA) of approximately 1 whereas in Western diets the ratio is 15/1-16.7/1. Western diets are deficient in omega-3 fatty acids, and have excessive amounts of omega-6 fatty acids compared with the diet on which human beings evolved and their genetic patterns were established. Excessive amounts of omega-6 polyunsaturated fatty acids (PUFA) and a very high omega-6/omega-3 ratio, as is found in today's Western diets, promote the pathogenesis of many diseases, including cardiovascular disease, cancer, and inflammatory and autoimmune diseases, whereas increased levels of omega-3 PUFA (a lower omega-6/omega-3 ratio), exert suppressive effects. In the secondary prevention of cardiovascular disease, a ratio of 4/1 was associated with a 70% decrease in total mortality. A ratio of 2.5/1 reduced rectal cell proliferation in patients with colorectal cancer, whereas a ratio of 4/1 with the same amount of omega-3 PUFA had no effect. The lower omega-6/omega-3 ratio in women with breast cancer was associated with decreased risk. A ratio of 2-3/1 suppressed inflammation in patients with rheumatoid arthritis, and a ratio of 5/1 had a beneficial effect on patients with asthma, whereas a ratio of 10/1 had adverse consequences. These studies indicate that the optimal ratio may vary with the disease under consideration. This is consistent with the fact that chronic diseases are multigenic and multifactorial. Therefore, it is quite possible that the therapeutic dose of omega-3 fatty acids will depend on the degree of severity of disease resulting from the genetic predisposition. A lower ratio of omega-6/omega-3 fatty acids is more desirable in reducing the risk of many of the chronic diseases of high prevalence in Western societies, as well as in the developing countries.     

Modulation of gene expression in autoimmune disease and aging by food restriction and dietary lipids

Several recent observations carried out by many investigators have offered some clues in understanding the mechanism of how food restriction (FR) acts in the prolongation of life-span, but the precise mechanisms involved in modulating the immune system have not been clearly understood. Our own ongoing studies indicate that FR may act at the molecular level and may extend the life-span by modulating functional activities of several genes in various target tissues. For instance, while cytochrome P-450 IIB1 and IIB2 expression is known to decline with age in ad libitum-fed rats, FR prevented the loss of (drug-inducible) P-450 enzymes in liver tissues. In addition, both alpha 2u-globulin and senescence marker protein 2 expressions, which are regulated by hormones, were also modulated during aging by FR in Fischer 344 male rats. In short-lived autoimmune-prone mice, both FR and omega-3 (n-3) fatty acids diet lowered the severity of autoimmune disease both in lupus-prone (NZB x NZW)F1 mice and in mice prone to develop lymphoproliferative and renal diseases, whereas saturated (n-9) and polyunsaturated (n-6) dietary lipids not only exacerbated autoimmune disease, but also significantly enhanced expression of several oncogenes in lymphoid tissues. FR and omega-3 fatty acids decreased the expression of certain oncogenes. Both FR and omega-3 fatty acids may modulate the aging and autoimmune disease processes by not only altering the fatty acid composition, membrane fluidity, and signal transduction, but also by modulating the lymphokine hormone receptors and their functions and thereby modulating expression of several genes in various tissues during the aging process.     

Protein kinase C betaII and TGFbetaRII in omega-3 fatty acid-mediated inhibition of colon carcinogenesis

Increasing evidence demonstrates that protein kinase C betaII (PKCbetaII) promotes colon carcinogenesis. We previously reported that colonic PKCbetaII is induced during colon carcinogenesis in rodents and humans, and that elevated expression of PKCbetaII in the colon of transgenic mice enhances colon carcinogenesis. Here, we demonstrate that PKCbetaII represses transforming growth factor beta receptor type II (TGFbetaRII) expression and reduces sensitivity to TGF-beta-mediated growth inhibition in intestinal epithelial cells. Transgenic PKCbetaII mice exhibit hyperproliferation, enhanced colon carcinogenesis, and marked repression of TGFbetaRII expression. Chemopreventive dietary omega-3 fatty acids inhibit colonic PKCbetaII activity in vivo and block PKCbetaII-mediated hyperproliferation, enhanced carcinogenesis, and repression of TGFbetaRII expression in the colonic epithelium of transgenic PKCbetaII mice. These data indicate that dietary omega-3 fatty acids prevent colon cancer, at least in part, through inhibition of colonic PKCbetaII signaling and restoration of TGF-beta responsiveness.     

Anti-inflammatory effects of α-linolenic acid in M1-like macrophages are associated with enhanced production of oxylipins from α-linolenic and linoleic acid

Chronic inflammation, mediated in large part by proinflammatory macrophage populations, contributes directly to the induction and perpetuation of metabolic diseases, including obesity, insulin resistance and type 2 diabetes. Polyunsaturated fatty acids (PUFAs) can have profound effects on inflammation through the formation of bioactive oxygenated metabolites called oxylipins. The objective of this study was to determine if exposure to the dietary omega-3 PUFA α-linolenic acid (ALA) can dampen the inflammatory properties of classically activated (M1-like) macrophages derived from the human THP-1 cell line and to examine the accompanying alterations in oxylipin secretion. We find that ALA treatment leads to a reduction in lipopolysaccharide (LPS)-induced interleukin (IL)-1β, IL-6 and tumor necrosis factor-α production. Although ALA is known to be converted to longer-chain PUFAs eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), DHA oxylipins were reduced overall by ALA treatment, as was LPS-induced secretion of EPA oxylipins. In contrast, we observed profound increases in oxylipins directly derived from ALA. Lipoxygenase products of linoleic acid were also dramatically increased, and LPS-induced production of AA oxylipins, particularly prostaglandin D2, was reduced. These results suggest that ALA may act to dampen the inflammatory phenotype of M1-like macrophages by a unique set of mechanisms distinct from those used by the long-chain omega-3 fatty acids EPA and DHA. Thus, there is strong rationale for investigating the functions of ALA oxylipins and lesser-known LA oxylipins since they hold promise as anti-inflammatory agents.     

Stimulation of anaphylaxis in the mouse footpad by dietary fish oil fatty acids

The effect of fish oil-derived omega-3 (omega-3) fatty acids on anaphylaxis, Arthus and delayed type hypersensitivity reactions in mice has been investigated. Mice on a normal chow diet were fed eicosapentaenoic acid and docosahexaenoic acid at a dose of 500 and 333 mg/kg/day, respectively, by a gastric tube over a period of 61 days. Control groups were given water, safflower oil or oleic acid. Anaphylactic and Arthus type reactions were induced in the mouse footpad using bovine serum albumin as an antigen. Carrageenin was utilized to produce a delayed type hypersensitivity reaction. The animals fed omega-3 fatty acids induced a more anaphylactic foodpad reaction. There was no significant effect of the diet on Arthus and delayed type hypersensitivity responses. There was no effect of the fish oil-supplemented diet on production of antibodies to bovine serum albumin. Synthesis of prostaglandin E2 by peritoneal macrophages was significantly inhibited in the animals fed omega-3 fatty acid-enriched fish oil, while leukotriene B4 production was not affected. These results suggest that a diet enriched in omega-3 fatty acids modulates production of arachidonic acid metabolites and this may influence anaphylaxis, but not Arthus and cellular mediated hypersensitivity responses.