Tricia A. Babcock

Eicosapentaenoic acid (EPA): an antiinflammatory ω-3 fat with potential clinical applications.

Evaluation of Eskimos, whose diets derive a large portion of dietary calories from v-3‐ containing fish, demonstrated a significantly lower rate of cardiovascular diseaseassociated mortality and malignancies compared to Western populations.2 Further evidence in support of v-3 fatty acid dietary consumption is derived from experimental studies demonstrating eicosapentaenoic acid (EPA)-mediated antiinflammatory and anticachexiogenic benefits. 3,4 Proinflammatory cytokines (specifically tumor necrosis factor [TNF] and interleukin-1 [IL-1]) are the proximal secondary pathway mediators of the multistep cascade that regulates the metabolic response to inflammation after a primary pathway stimulus, (injury, infection, malignancy, etc.).5 It is generally accepted that the prolonged and inappropriate production and release of proinflammatory cytokines result in clinically evident metabolic abnormalities of anorexia, weight loss, and lean body mass catabolism that as a whole describe the syndrome of cachexia.5,6 A logical approach to attenuate the development of cachexia, and in particular cancer-associated cachexia, would be to reduce or inhibit the propagation of secondary pathway intermediates such as proinflammatory cytokines and arachidonic acid (AA) metabolism intermediates (prostaglandins, prostacyclins, thromboxanes, etc.). Therefore, in our evaluation of new therapies for the treatment of patients with cancerassociated cachexia it would be ideal to use a readily available low-cost and non-toxic agent that would reduce the production and release of inflammatory mediators. In support of this effort, several experimental and clinical studies have demonstrated a benefit for consuming a diet supplemented specifically with EPA, one of the primary v-3 fatty acids found in fish oils. In animal models and for patients with progressive malignancy, EPA supplementation promotes weight maintenance, improves food intake, inhibits tumor growth, and may improve survival. These observations suggest a potential role for EPA in the clinical management of patients with progressive malignancy and justify the need for clinical trials. Exciting as these early observation have been, much work remains to be done. In the present paper the significant evidence supporting the role for EPA as a potential agent for the treatment of cancer-associated cachexia is presented.

Omega-3 Fatty Acid Lipid Emulsion Reduces LPS-Stimulated Macrophage TNF-α Production

Background: Omega-3 (ω-3) fatty acids (FA), specifically eicosapentaenoic acid (EPA), attenuate cytokine-mediated inflammation. Currently, in the United States, there is no commercial source of ω-3 lipid for clinical use. A clinically used European lipid emulsion, Omegaven®, has been shown to have beneficial antiinflammatory effects; however, the mechanisms of its action are not well defined. In the present work, this ω-3 FA emulsion has been evaluated in order to define its effects on TNF-α production in a model of LPS-stimulated macrophages. Materials and Methods: RAW 264.7 cells (1 × 106 cell/well) were incubated with DMEM, Omegaven®, or an isoenergetic ω-6 lipid emulsion, Lipovenos® for 4 h. Cells were washed and then stimulated with LPS (1 μg/mL) or media alone for 3 h. Plate well supernatants were collected and assayed for TNF-α production by ELISA. Statistical analysis was performed by ANOVA and post-hoc analyses; the significance was defined as p < 0.05. Results: Unstimulated RAW cell TNF-α produc...

Lipopolysaccharide-Stimulated RAW 264.7 Macrophage Inducible Nitric Oxide Synthase and Nitric Oxide Production Is Decreased by an Omega-3 Fatty Acid Lipid Emulsion

BACKGROUND Omega-3 fatty acids (omega-3 FA) have been demonstrated to have anti-inflammatory properties, postulated to occur through several principal mechanisms, including (1) displacement of arachidonic acid from the cellular membrane; (2) shifting of prostaglandin E(2) and leukotriene B(4) production; and (3) molecular level alterations including decreased activation of nuclear factor kappa B and activator protein-1. An additional regulator that is likely associated is the production of nitric oxide (NO) by nitric oxide synthetase. NO is a short-lived free radical involved in many biological functions. However, excessive NO production can lead to complications, suggesting that decreased NO production is a potential target for some inflammatory diseases. We hypothesized that pretreating with an omega-3 FA lipid emulsion would decrease the production of NO in macrophages and that this effect would occur through alterations in inducible nitric oxide synthetase (iNOS). MATERIALS AND METHODS Greiss reagent was used to assess NO production in RAW 264.7 macrophages following omega-3 or omega-6 FA treatment alone or in combination with lipopolysaccharide (LPS) stimulation for 12 h/24 h. iNOS levels were determined by Western blot. Tumor necrosis factor-alpha levels were determined by enzyme-linked immunosorbent assay. RESULTS Following LPS-stimulation, omega-3 FA pretreatment at 12 and 24 h produced significantly less NO (P < 0.05) compared to omega-6 FA or media-only conditions. omega-3 FA pretreatment at 12 and 24 h also had less iNOS protein expression compared to omega-6 FA or media-only conditions. Tumor necrosis factor-alpha production was significantly decreased with omega-3 FA treatment compared to omega-6 FA treatment (P < 0.05) after 24 h LPS stimulation. CONCLUSION These experiments demonstrate that, in addition to other anti-inflammatory effects, omega-3 FA lipid emulsions also significantly lower NO production in LPS-stimulated macrophages through altered iNOS protein expression.

Omega-3 fatty acid supplementation reduces tumor growth and vascular endothelial growth factor expression in a model of progressive non-metastasizing malignancy.

BACKGROUND Omega-3 fatty acids, the principal component of fish oil, have been demonstrated to have antiinflammatory properties. The role of eicosapentaenoic acid (EPA) supplementation for cancer patients is currently under investigation; however, the mechanisms of EPA activity have not been defined. The purpose of this study was to characterize tumor-specific and treatment-specific effects of supplemental dietary EPA in an animal model of progressive malignancy. METHODS Fischer 344 rats (200-250 g) underwent flank implantation of the methycholanthrene (MCA)-induced fibrosarcoma on day 0. Rats were randomly divided into 3 treatment groups on day 13: EPA (1 mL, 5.0 g/kg per day) + 10 IU vitamin E; corn oil (1 mL) + 10 IU vitamin E, and saline (1 mL) + 10 IU vitamin E (vitamin E was used to prevent fatty acid oxidation). On day 14, gavage feeding was started and was continued through day 28. The animals were killed on day 29, and the tumors were removed. The tumors were weighed and divided by the tumor-free carcass weight to obtain percentage of tumor volume, and the livers were flash frozen. Vascular endothelial growth factor-alpha (VEGF-alpha) and cyclo-oxygenase 2 (COX-2) mRNA were measured by reverse transcription-polymerase chain reaction (RT-PCR). RESULTS EPA rats had significant reductions in tumor volume compared with isocaloric corn oil and control saline animals (25%, p < .01 and 33%, p < .01, respectively). Rats receiving EPA demonstrated decreased VEGF-alpha mRNA levels (0.023 +/- 0 0.001) compared with those receiving corn oil (0.129 +/- 0.047) or saline (0.150 +/- 0.026; p < .05). CONCLUSIONS These data demonstrate that EPA supplementation inhibits tumor growth, potentially through alterations in the expression of the pro-angiogenic VEGF-alpha. The mechanism(s) of EPA as an inhibitor of tumor-related angiogenic growth factors may be associated with COX-2 enzyme fatty acid metabolism and merits further study.

Eicosapentaenoic acid supplementation reduces tumor volume and attenuates cachexia in a rat model of progressive non-metastasizing malignancy

BACKGROUND Eicosapentaenoic acid (EPA) has been shown to have anti-inflammatory and tumor growth inhibitory effects clinically and experimentally; evidence also supports the role of EPA in attenuating cancer-associated weight loss, but the mechanisms of these effects remain to be defined. As the liver plays a central role in modulating nutritional status and the cachexia syndrome, we examined the liver and nutritional parameters indicative of cachexia along with the tumor volume in response to oral EPA supplementation in a rat model of progressive non-metastasizing malignancy. METHODS Fischer 344 rats implanted with the methylcholanthrene-induced fibrosarcoma (MCA) were trained to meal-feed with access to food from 8:00 PM to 8:00 AM and water ad libitum. On day 13, rats were randomly divided into 3 study groups: (1) 5.0 g/kg per day EPA plus 10 IU vitamin E/g fat, (2) 5.0 g/kg per day corn oil plus 10 IU vitamin E/g fat, and (3) 5.0 g/kg per day saline plus 10 IU vitamin E/g saline. The treatment was delivered via oral gavage twice daily. The animals were killed on day 29, and serum plus tissues (ie, liver and lung) were collected and frozen for analysis. Parameters evaluated include the following: tumor volume, weight loss, liver weight, total liver protein, liver lipid content, serum albumin content, and macrophage inflammatory protein-2 (MIP-2) levels. RESULTS EPA-treated rats showed a reduction in tumor volume compared with corn oil (25% reduction, p < .01) and saline (33% reduction, p < .01) animals. EPA rats also demonstrated increased liver weight (p < .01) and total liver protein levels (p < .03) over saline-treated animals. EPA- and corn oil-treated rats received more calories than the saline group because of the dietary fat treatments (p < .01) and had elevated lipid content in their livers (p = .05 and p = .04, respectively) compared with saline rats. Serum albumin (a marker of liver function) and MIP-2 levels (a marker of the hepatic acute phase response) were not different between treatment groups. CONCLUSIONS EPA supplementation resulted in a dramatic reduction of tumor volume and mild improvements in weight maintenance. In addition, EPA-treated animals demonstrated increased total liver protein and serum protein levels. Regression analyses showed that the weight and protein differences between treatment groups were not correlated with individual tumor volumes. The increase in liver and serum protein was not explained by differences in albumin or MIP-2. We conclude that the tumor growth inhibitory effects and anticachexiogenic effects of EPA are independent phenomena, and the effects on cachexia may be related to increased levels of undefined protein(s) in the liver and serum. To our knowledge, this is the first study to demonstrate the effects of EPA in the MCA fibrosarcoma model and is also novel in its evaluation of EPA as an anticachexiogenic therapy in progressive non-metastasizing malignancy. Further studies may identify the protein(s) elevated in the liver and the mechanisms for the development of EPA nutritional therapies for the treatment of progressive malignancies.

Metabolism/nutritionLipopolysaccharide-Stimulated RAW 264.7 Macrophage Inducible Nitric Oxide Synthase and Nitric Oxide Production Is Decreased by an Omega-3 Fatty Acid Lipid Emulsion

BACKGROUND Omega-3 fatty acids (omega-3 FA) have been demonstrated to have anti-inflammatory properties, postulated to occur through several principal mechanisms, including (1) displacement of arachidonic acid from the cellular membrane; (2) shifting of prostaglandin E(2) and leukotriene B(4) production; and (3) molecular level alterations including decreased activation of nuclear factor kappa B and activator protein-1. An additional regulator that is likely associated is the production of nitric oxide (NO) by nitric oxide synthetase. NO is a short-lived free radical involved in many biological functions. However, excessive NO production can lead to complications, suggesting that decreased NO production is a potential target for some inflammatory diseases. We hypothesized that pretreating with an omega-3 FA lipid emulsion would decrease the production of NO in macrophages and that this effect would occur through alterations in inducible nitric oxide synthetase (iNOS). MATERIALS AND METHODS Greiss reagent was used to assess NO production in RAW 264.7 macrophages following omega-3 or omega-6 FA treatment alone or in combination with lipopolysaccharide (LPS) stimulation for 12 h/24 h. iNOS levels were determined by Western blot. Tumor necrosis factor-alpha levels were determined by enzyme-linked immunosorbent assay. RESULTS Following LPS-stimulation, omega-3 FA pretreatment at 12 and 24 h produced significantly less NO (P < 0.05) compared to omega-6 FA or media-only conditions. omega-3 FA pretreatment at 12 and 24 h also had less iNOS protein expression compared to omega-6 FA or media-only conditions. Tumor necrosis factor-alpha production was significantly decreased with omega-3 FA treatment compared to omega-6 FA treatment (P < 0.05) after 24 h LPS stimulation. CONCLUSION These experiments demonstrate that, in addition to other anti-inflammatory effects, omega-3 FA lipid emulsions also significantly lower NO production in LPS-stimulated macrophages through altered iNOS protein expression.

Synergistic anti-inflammatory activity of omega-3 lipid and rofecoxib pretreatment on macrophage proinflammatory cytokine production occurs via divergent NF-kappaB activation.

UNLABELLED Omega-3 lipid pretreatment significantly decreases TNF-alpha production in LPS-stimulated Mphis; however, this response is only a partial inhibition, suggesting that other nonsubstrate- (lipid) dependent mechanisms are involved. The cyclooxygenase (COX)-2 enzyme is principally responsible for lipid metabolism; thus, a selective COX-2 inhibitor (Rofecoxib) would clarify if it is an omega-3 lipid direct effect or a COX-2 enzyme-associated modulated reduction in TNF-alpha. Moreover, potential synergy between omega-3 lipids and selective COX-2 inhibition is postulated. HYPOTHESIS Through divergent regulatory mechanisms, omega-3 lipids in combination with Rofecoxib will synergistically decrease the LPS-stimulated Mphi inflammatory response. METHODS RAW 264.7 cells were pretreated with omega-3 lipids, Rofecoxib, or combination treatment and then washed and exposed to LPS. Supernatants were collected for ELISA, total proteins were obtained to determine COX-2 protein expression by Western blot, and nuclear extracts were isolated to determine NF-kappaB activation by electromobility shift assay. RESULTS TNF-alpha and PGE2 production was significantly decreased with omega-3 and Rofecoxib pretreatment, and with combination treatment a further decrease in TNF-alpha production was observed. COX-2 protein expression was demonstrated to increase in omega-3, Rofecoxib, and combination groups stimulated with LPS. No alteration in NF-kappaB activation was observed with Rofecoxib or combination pretreatment compared with LPS-stimulated control cells. Repletion of prostaglandin (PGE2) in the Mphi model significantly decreased TNF-alpha in all groups. CONCLUSIONS Omega-3 lipids and Rofecoxib independently decrease TNF-alpha and PGE2 production in LPS-stimulated Mphi, yet in combination a synergistic reduction in TNF-alpha production is observed. Although the anti-inflammatory effects observed from omega-3 lipids are known to occur partially through decreasing NF-kappaB activation, we demonstrated that Rofecoxib or even a combination of omega-3 and Rofecoxib does not alter NF-kappaB activation, as seen with omega-3 lipids alone. These data support that combination treatment may result in decreased Mphi inflammation, yet this occurs via divergent mechanisms.

A Priori Dietary Ω-3 Lipid Supplementation Results in Local Pancreatic Macrophage and Pulmonary Inflammatory Response Attenuation in a Model of Experimental Acute Edematous Pancreatitis (AEP)

BACKGROUND Acute pancreatitis is often complicated by multiorgan dysfunction, which is postulated to occur in part by macrophage infiltration into the pancreas. Eicosapentaenoic acid (EPA), an ω-3 fatty acid, is the principal biologic component of fish oil and has clinically and experimentally been demonstrated to be anti-inflammatory. We hypothesized that dietary EPA supplementation before the induction of pancreatitis would attenuate both Mϕ-mediated local pancreatic and systemic pulmonary inflammatory response in an in vivo model of acute edematous pancreatitis (AEP). METHODS Male Sprague-Dawley (SD) rats were pretreated 2 times per day with oral gavage with EPA (ω-3 fatty acid; 5 mg/kg/dose) or ω-6 fatty acid control (5 mg/kg/dose) or saline (equal volume) for 2 weeks. AEP was induced in ω-3, ω-6, and saline pretreated rats by 5 hourly subcutaneous (SC) injections of cerulein. Pancreas, lung, and serum were harvested 3 hours after the last cerulein injection. Severity of pancreatitis was confirmed by serum amylase and by histopathologic score. Pancreatic macrophage infiltration was assessed by confocal fluorescent microscopy, and pulmonary leukocyte respiratory burst (LRB) analysis was performed on mononuclear cells obtained from bronchioalveolar lavage (BAL). RESULTS All animals demonstrated acute pancreatitis through hyperamylasemia and histopathologic examination. Confocal analysis demonstrated significantly lower macrophage infiltration, and BAL analysis by flow cytometry demonstrated significantly lower ( p < .05) LRB in the ω-3-treated group compared with theω -6 and the saline pancreatitis group. CONCLUSIONS Attenuation of both pancreatic MΦ inflammatory response and pulmonary leukocyte respiratory burst in AEP by EPA supports further investigation into the potential role for EPA dietary supplementation in the progression of pancreatitis-associated sequelae.

Experimental Studies Defining ω-3 Fatty Acid Antiinflammatory Mechanisms and Abrogation of Tumor-Related Syndromes

Clinical and experimental evidence has supported a benefit for the inclusion of fish oils (a primary source of ω-3 fatty acids) as a component of a normal healthy diet. Polyunsaturated ω-3 fatty acids have been demonstrated to be of benefit in a number of inflammation-associated disease states, including atherosclerosis, autoimmune disorders, malignancy, and sepsis. The beneficial effects of ω-3 fatty acids are thought to occur through the postulated antiinflammatory actions of ω-3 fats; however, the specific mechanism(s) of action has not been completely defined. In this review, we discuss the recent progress made in our laboratory on defining the mechanisms of ω-3 fatty acids activity.

Modulation of the ubiquitin-proteasome proteolytic pathway by eicosapentaenoic acid supplementation in a model of progressive malignancy

BACKGROUND A benefit for eicosapentaenoic acid (EPA) supplementation for protein maintenance in cancer patients exists, although specific mechanisms are unknown. As the ubiquitin-proteasome proteolytic (UPP) pathway has been implicated in protein use in malignancy, we determined mRNA levels for UPP components in the liver and muscles from EPA-treated rats bearing the methylcholanthrene (MCA) fibrosarcoma. METHODS Rats implanted with MCA tumor were divided into 3 groups on day 13: EPA (5 g/kg per day plus 10 IU vitamin E/g fat), corn oil (5 g/kg per day plus 10 IU vitamin E/g fat), and saline (5 g/kg per day plus 10 IU E/g saline). On day 29, tumor volume (TV) was determined; liver and quadriceps muscles were also excised to determine gene expression of C2, C3, E2(14k), and E3alpha by reverse transcription-polymerase chain reaction (RT-PCR). RESULTS EPA-treated rats demonstrated a reduced TV of 21% compared with the 28% and 30% TV of corn oil- and saline-treated rats, respectively. Muscle mRNA levels of E2(14k) and E3alpha in EPA-treated animals were decreased compared with corn oil- and saline-treated animals. EPA treatment also decreased hepatic C2, C3, and E2(14k) mRNA levels compared with saline treatment. CONCLUSION EPA supplement decreased skeletal muscle E2(14k), E3alpha, and hepatic C2 mRNA levels compared with the isocaloric, isonitrogenous corn oil supplement, supporting a treatment-specific effect. The decrease in hepatic C3 and E2(14k) mRNA levels induced by EPA were partly because of caloric benefit and partly attributable to a treatment-specific effect. Additionally, differences in the hepatic and muscle gene expressions of UPP components suggested an organ-specific effect for omega-3 fatty acid activity.