Feasibility evaluation of emodin (rhubarb extract) as an inhibitor of pancreatic cancer cell proliferation in vitro.

Emodin is a commonly used traditional herbal treatment in China, including use for pancreatic malignancy. In this study, the potential for emodin to inhibit pancreatic cancer cell proliferation was examined using 4 human pancreatic adenocarcinoma cell lines: Mia Paca-2, BxPC-3, Panc-1, and L3.6pl. WST-1 proliferation, propidium iodide flow cytometry cell cycle analysis, and poly-ADP-ribose polymerase (PARP) Western blot analysis were performed. Forty-eight-hour treatment with 50 muM emodin inhibited proliferation in Mia Paca-2 cells by 42%, BxPc-3 by 38%, L3.6pl by 56%, and Panc-1 by 18% (all P < .01). In three-fourths of the cell lines, emodin treatment resulted in an increase (from 4.7% to 22%) in the cell population number in apoptosis when measured by flow cytometric analysis. Mia Paca-2 revealed a significant PARP cleavage product when compared with control. These feasibility experiments provide initial evidence that emodin exerts an antiproliferative effect, likely through apoptosis induction-related mechanism(s), that is reproducible in various human pancreatic cancer cell lines.

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.

Attenuation of iNOS in an LPS-stimulated macrophage model by omega-3 fatty acids is independent of COX-2 derived PGE2.

BACKGROUND: Omega-3 fatty acids (n-3 FA) demonstrate significant anti-inflammatory properties thought to occur through three principal mechanisms; (1) displacement of arachidonic acid from the cellular membrane, (2) differential prostaglandin E2 (PGE2) and LTB4 production, and (3) molecular level alterations such as diminished nuclear factor kappa B and AP-1 activation. Recently, n-3 FA have been demonstrated to significantly decrease nitric oxide (NO) production in a lipopolysaccharide (LPS)-stimulated M Phi model. We hypothesized that decreased NO production by n-3 FA occurs through inhibition of cyclooxygenase-2 (COX-2) derived PGE2 and that repletion of the system with PGE2 would obliterate these effects. Selective COX-2 inhibitor (L-748,731) experiments and separate PGE2 repletion studies were used to test this hypothesis. METHODS: NO production was assessed following 24 h with or without LPS/PGE2 in the presence of n-3 FA, L-748,731 (a selective COX-2 inhibitor), or combination (n-3 FA + L-748,731) treatment. Western blots were used to assess inducible NO synthase protein expression. RESULTS: Independently or in the presence of LPS, treatment with a COX-2 inhibitor significantly increased NO production compared with control, n-3 FA, and combination treatment. NO production in combination treatment is slightly increased compared to n-3 FA treatment. In control cells treated with LPS, PGE2 repletion resulted in a significant decrease in NO. All other treatment groups repleted with PGE2 demonstrated no significant alterations in NO production. Inducible NO synthase protein expression levels were similar to NO production across all treatments. CONCLUSION: These experiments disproved our original hypothesis that the decrease in NO production associated with n-3 FA treatment occurs through a COX-2 derived PGE2 dependent mechanism. Eliminating COX-2 derived PGE2 by a selective inhibitor actually increased NO production. Exogenous PGE2 repletion did not restore the system. Therefore, mechanisms other than n-3 FA associated alterations in COX-2 derived PGE2 are likely involved in decreasing NO production in LPS stimulated M Phi.

Inhibition of proliferation by omega-3 fatty acids in chemoresistant pancreatic cancer cells.

BACKGROUND: Pancreatic cancer-gemcitabine (GEM) chemoresistance has been demonstrated to be associated with enhanced NF-kB activation and antiapoptotic protein synthesis. The well-known capacity of omega-3 fatty acids (n-3 FAs) to inhibit NF-kB activation and promote cellular apoptosis has the potential to restore or facilitate gemcitabine chemosensitivity. METHODS: Four pancreatic cancer cell lines (MIA PaCa-2, BxPC-3, PANC-1, and L3.6), each with distinct basal NF-kB and differing GEM sensitivity profiles, were administered: 100 uM of (1) n-3FA, (2) n-6FA, (3) GEM, (4) n-3FA + GEM, or (5) n-6FA + GEM for 24 and 48 hours. Proliferation was assessed using the WST-1 assay. To define the mechanism(s) of altered proliferation, electron mobility shift assay for NF-kB activity, western blots of phoshoStat3, phosphoIkappaB, and poly(ADP-ribose) polymerase (PARP) cleavage were performed in the MIA PaCa-2 cell line. RESULTS: All cell lines demonstrated a time/dose-dependent inhibition of proliferation in response to n-3FA. For MIA PaCa-2 cells, n-3FA and n-3FA + GEM treatment resulted in reduction of I-kB phosphorylation and NF-kB activation when compared with n-6FA control. n-3FA and combination treatment also significantly decreased Stat3 phosphorylation, whereas GEM alone had no effect. n-3FAs and n-3FA + GEM groups demonstrated increased PARP cleavage, mirroring NF-kB activity and Stat3 phosphorylation. CONCLUSIONS: n-3 FA treatment is specifically associated with inhibition of proliferation in these four pancreatic cell lines irrespective of varied gemcitabine resistance. An experimental paradigm to screen for potential contributory mechanism(s) in altered pancreatic cancer cellular proliferation was defined, and using this approach the co-administration of n-3 FA with GEM inhibited GEM-induced NF-kB activation and restored apoptosis in the MIA PaCa-2 cell-line.

G2/M cell-cycle arrest and apoptosis by n-3 fatty acids in a pancreatic cancer model.

BACKGROUND: n-3 fatty acids (n-3FA) have anti-inflammatory and anti-proliferative effects including modulation of pro-inflammatory cascade mediators and cytokine elaboration (i.e., TNF-alpha, IL-10 and PGE(2)) in many cell lines. However, mechanisms of anti-proliferative effects have not been clearly defined. MATERIALS AND METHODS: MIA PaCa-2 pancreatic cancer cells were treated either with n-3FA (treatment), media (control), or n-6FA (control) for all experiments. Cellular proliferation was evaluated with WST-1 reagent. Cells were stained with propidium iodide and analyzed by flow cytometry for cell-cycle arrest, which was further analyzed by cdc2 expression. Membrane and media lipid concentrations were analyzed by high-performance liquid chromatography. Apoptosis was evaluated by AnnexinV-FITC flow cytometry and reconfirmed by poly (ADP-ribose) polymerase (PARP) cleavage and B(cl)-2 expression. RESULTS: Propidium iodide flow cytometry of MIA PaCa-2 dosed with n-3FA showed a decrease in cells in G1 phase (11-17%) and an increase cells in G2 phase (7-13%) from controls. cdc2 expression was also decreased at 24 h compared to controls. Annexin-V staining of n-3FA-treated cells demonstrated time-dependent increased apoptosis and PARP cleavage was present only in the n-3FA treatment group. Phospho-B(cl)-2 was also decreased in the n-3FA-treated cells compared to controls. CONCLUSIONS: Co-incubation of MIA PaCa-2 cells with n-3FA results in both dose- and time-dependent cell-cycle arrest. Cells also progress to cell death via apoptosis. These data support the potential applicability for n-3FA as an antiproliferative and pro-apoptotic strategy.

Experimental studies defining omega-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 omega-3 fatty acids) as a component of a normal healthy diet. Polyunsaturated omega-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 omega-3 fatty acids are thought to occur through the postulated antiinflammatory actions of omega-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 omega-3 fatty acids activity.

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.

Inhibition of activator protein-1 transcription factor activation by omega-3 fatty acid modulation of mitogen-activated protein kinase signaling kinases.

BACKGROUND: Lipopolysaccharide (LPS)-stimulated macrophages (Mphi) produce excess tumor necrosis factor (TNF), and the direct inhibition of IkappaB phosphorylation and its subsequent separation from the nuclear factor kappaB (NFkappaB)-IkappaB complex has been experimentally supported as a mechanism for omega-3 fatty acid (FA) inhibition of this TNF response. However, TNF production is a "late" event in the LPS-induced Mpsi inflammatory cascade, and in addition to NFkappaB-associated pathways, a separate transcription factor, activator protein-1 (AP-1) is an important pathway for Mpsi proinflammatory cytokine production. The mitogen-activated protein kinase (MAPK) cascade regulates both NFkappaB-IkappaB--and AP-1-associated gene transcription through several cross-amplifying phosphorylation kinases, specifically p44/42 [ie, extracellular signal-regulated kinase (ERK) 1/2], p38, and c//jun N-terminal kinase (JNK)/stress-activated protein kinase (SAPK). The activation of these kinases occurs in the proximal MAPK cascade and activation modulates AP-1 activation. In this set of experiments, it was hypothesized that inhibition of MAPK signaling phosphorylation kinases by omega-3 fatty acids in a model of LPS-stimulated Mphi(s) would alter the activation of the proinflammatory cytokine transcription factor AP-1. METHODS: RAW 264.7 cells were pretreated with a sterile, commercially available, pharmaceutical grade omega-3 FA emulsion, equivalent grade omega-6 FA emulsion, or Dulbecco's modified eagles medium (media alone) for 4 hours. Cells were washed twice and exposed to LPS for 15 minutes. Total cell lysates were collected, and both total and phosphorylated portions of the p44/42, p38, and JNK/SAPK proteins were determined by Western blotting. AP-1 nuclear translocation was determined by electromobility shift assay. RESULTS: Phosphorylation of p44/42 and JNK/SAPK proteins of the MAPK pathways in LPS-stimulated Mpsi(s) was significantly reduced by omega-3 FA treatment compared with Mphi treated with omega-6 FA or media alone. In contrast, phosphorylation of p38 was not inhibited in the presence of omega-3 or (omega-6 FA treatment compared with media alone. Omega-3 FA pretreatment inhibited AP-1 activation. CONCLUSIONS: omega-3 FA inhibited p44/42 and JNK/SAPK phosphorylation; however, p38 remained unchanged. Phosphorylation of p44/42 and JNK/SAPK are the immediate prior steps in AP-1 activation. Attenuated AP-1 activation and subsequent attenuated gene-level proinflammatory cytokine elaboration is anticipated after inhibition of these MAPK intermediates and is confirmed by the reduction in AP-1 activity. These results provide further evidence for the transcriptional level regulation in the elaboration of proinflammatory cytokines by omega-3 FA in this Mphi model.

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.

Omega-3 fatty acids: implications for the treatment of tumor-associated inflammation.

Our in vivo and in vitro studies using omega-3 fatty acids (FA) have provided insight into the biological effects and mechanisms of their anti-inflammatory action(s). The implications for this research are profound because there are few nutritional therapies available that have the potential to be clinically effective in malignancies and other chronic inflammatory conditions as omega-3 FA. In this summary of experiments the biological effects of omega-3 FA are discussed and the potential mechanisms of action presented.

NF-kappa B inhibition by omega -3 fatty acids modulates LPS-stimulated macrophage TNF-alpha transcription.

Omega-3 fatty acid (FA) emulsions reduce LPS-stimulated murine macrophage TNF-alpha production, but the exact mechanism has yet to be defined. The purpose of this study was to determine the mechanism for omega-3 FA inhibition of macrophage TNF-alpha production following LPS stimulation. RAW 264.7 cells were pretreated with isocaloric emulsions of omega-3 FA (Omegaven), omega-6 FA (Lipovenos), or DMEM and subsequently exposed to LPS. IkappaB-alpha and phospho-IkappaB-alpha were determined by Western blotting. NF-kappaB binding was assessed using the electromobility shift assay, and activity was measured using a luciferase reporter vector. RT-PCR and ELISA quantified TNF-alpha mRNA and protein levels, respectively. Pretreatment with omega-3 FA inhibited IkappaB phosphorylation and significantly decreased NF-kappaB activity. Moreover, omega-3-treated cells demonstrated significant decreases in both TNF-alpha mRNA and protein expression by 47 and 46%, respectively. These experiments demonstrate that a mechanism for proinflammatory cytokine inhibition in murine macrophages by omega-3 FA is mediated, in part, through inactivation of the NF-kappaB signal transduction pathway secondary to inhibition of IkappaB phosphorylation.

Modulation of lipopolysaccharide-stimulated macrophage tumor necrosis factor-alpha production by omega-3 fatty acid is associated with differential cyclooxygenase-2 protein expression and is independent of interleukin-10.

BACKGROUND: The role of omega-3 fatty acids (FA) as anti-inflammatory agents involves the inhibition of macrophage (Mphi) cytokine production, but the mechanisms involved are not well defined. The effects of omega-3 FA on the transcription and translation of cyclooxygenase-2 (COX-2), the production of prostaglandin E(2) (PGE(2)), and the production of interleukin-10 (IL-10) were investigated as potential mechanisms for the down-regulation of lipopolysaccharide (LPS)-induced tumor necrosis factor-alpha production. METHODS: RAW 264.7 Mphi were incubated with Omegaven (10 mg% omega-3 FA), Lipovenos (10 mg% omega-6 FA), or DMEM for 4 h of pretreatment. The cells were then exposed to LPS (1 microg/ml) or medium alone for 3 h. COX-2 mRNA levels were determined by semi-quantitative reverse transcriptase polymerase chain reaction, and COX-2 protein levels were determined by Western blotting. The levels of PGE(2) and IL-10 proteins secreted into the medium were quantified using enzyme-linked immunosorbent assays. RESULTS: Pretreatment with omega-3 FA increased Mphi COX-2 protein expression levels without altering the levels of COX-2 mRNA in response to LPS stimulation. In addition, pretreatment with omega-3 FA dramatically decreased the PGE(2) and IL-10 production induced by LPS, whereas pretreatment with an equivalent dose of omega-6 FA only resulted in a modest increase in PGE(2) and a slight decrease in IL-10 production compared to controls. CONCLUSION: As COX-2 protein levels were increased without a change in COX-2 mRNA levels with omega-3 FA pretreatment, this suggested that omega-3 FA did not upregulate COX-2 at the transcriptional level. The omega-3 FA may instead posttranscriptionally stabilize existing COX-2 mRNA. The increased COX-2 expression may thus be explained by increased translation of COX-2 and/or decreased COX-2 degradation. The decreased PGE(2) production could be attributed to the replacement of Mphi membrane omega-6 FA substrates by omega-3 FA and the competitive inhibition of COX-2 enzyme by omega-3 FA. The reduction of active COX-2 product associated with an increase in COX-2 enzyme implies the existence of a negative feedback mechanism. Surprisingly, IL-10 production was decreased by omega-3 FA pretreatment, indicating that the reduced IL-10 inhibition of Mphi cytokine production was superceded by the other actions of omega-3 FA.

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.

Omega-3 fatty acid lipid emulsion reduces LPS-stimulated macrophage TNF-alpha production.

BACKGROUND: Omega-3 (omega-3) fatty acids (FA), specifically eicosapentaenoic acid (EPA), attenuate cytokine-mediated inflammation. Currently, in the United States, there is no commercial source of omega-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 omega-3 FA emulsion has been evaluated in order to define its effects on TNF-alpha production in a model of LPS-stimulated macrophages. MATERIALS AND METHODS: RAW 264.7 cells (1 x 10(6) cell/well) were incubated with DMEM, Omegaven, or an isoenergetic omega-6 lipid emulsion, Lipovenos for 4 h. Cells were washed and then stimulated with LPS (1 microg/mL) or media alone for 3 h. Plate well supernatants were collected and assayed for TNF-alpha 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-alpha production was similar in all groups and < 60 pg/mL. Lipovenos pretreatment did not alter TNF-alpha production from that of baseline compared to LPS-stimulated cells. Four-hour Omegaven pretreatment significantly reduced TNF-alpha production in LPS-stimulated cells, with a 46% reduction in TNF-alpha from baseline observed. CONCLUSION: Four-hour omega-3 FA emulsion pretreatment significantly attenuated LPS-stimulated macrophage TNF-alpha production. These data support the contention that antiinflammatory effects of omega-3 FA occur at least in part through the inhibition of macrophage TNF-alpha production in response to endotoxin. Further studies to define the antiinflammatory mechanisms of omega-3 FA on macrophages are warranted. The availability of Omegaven as an experimental treatment and Lipovenos as an equivalent control will be useful for future studies.