Lindsay E. Robinson

Mechanisms of omega-3 fatty acid-induced growth inhibition in MDA-MB-231 human breast cancer cells

SummaryThe omega-3 fatty acids, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), inhibit the growth of human breast cancer cells in animal models and cell lines, but the mechanism by which this occurs is not well understood. In order to explore possible mechanisms for the modulation of breast cancer cell growth by omega-3 fatty acids, we examined the effects of EPA and DHA on the human breast cancer cell line MDA-MB-231. Omega-3 fatty acids (a combination of EPA and DHA) inhibited the growth of MDA-MB-231 cells by 30–40% (p<0.05) in both the presence and absence of linoleic acid, an essential omega-6 fatty acid. When provided individually, DHA was more potent than EPA in inhibiting the growth of MDA-MB-231 cells (p<0.05). EPA and DHA treatment decreased tumor cell proliferation (p<0.05), as estimated by decreased [methyl−3H]-thymidine uptake and expression of proliferation-associated proteins (proliferating cell nuclear antigen, PCNA, and proliferation-related kinase, PRK). In addition, EPA and DHA induced apoptosis, as indicated by a loss of mitochondrial membrane potential, increased caspase activity and increased DNA fragmentation (p<0.05). Cells incubated with omega-3 fatty acids demonstrated decreased Akt phosphorylation, as well as NFκB DNA binding activity (p<0.05). The results of this study indicate that omega-3 fatty acids decrease cell proliferation and induce apoptotic cell death in human breast cancer cells, possibly by decreasing signal transduction through the Akt/NFκB cell survival pathway.

Eicosapentaenoic Acid and Rosiglitazone Increase Adiponectin in an Additive and PPARγ‐Dependent Manner in Human Adipocytes

Adiponectin, an anti‐inflammatory and insulin‐sensitizing protein secreted from adipose tissue, may be modulated by dietary fatty acids, although the mechanism is not fully known. Our objective was to investigate the effect of long‐chain n‐3 polyunsaturated fatty acids (PUFAs) on adiponectin in cultured human adipocytes, and to elucidate the role of peroxisome proliferator‐activated receptor‐γ (PPARγ) in this regulation. Isolated human adipocytes were cultured for 48 h with 100 µmol/l eicosapentaenoic acid (C20:5n‐3, EPA), docosahexaenoic acid (C22:6n‐3, DHA), palmitic acid (C16:0), 100 µmol/l EPA plus 100 µmol/l DHA, or bovine serum albumin (control). Additionally, adipocytes were treated for 48 h with a PPARγ antagonist (BADGE) or agonist (rosiglitazone) in isolation or in conjunction with either EPA or DHA. At 48 h, EPA and DHA increased (P < 0.05) adiponectin secretion by 88 and 47%, respectively, while EPA, but not DHA, also increased (136%, P < 0.001) cellular adiponectin protein. Interestingly, PPARγ antagonism completely abolished the DHA‐mediated increase in secreted adiponectin, but only partially attenuated the EPA‐mediated response. Thus, EPAs effects on adiponectin do not appear to be entirely PPARγ mediated. Rosiglitazone increased (P < 0.001) the secreted and cellular adiponectin protein (90 and 582%, respectively). Finally, the effects of EPA and rosiglitazone on adiponectin secretion were additive (+230% at 48 h combined, compared to 121 and 124% by EPA or rosiglitazone alone, respectively). Overall, our findings emphasize the therapeutic importance of long‐chain n‐3 PUFA alone, or in combination with a PPARγ agonist, as a stimulator of adiponectin, a key adipokine involved in obesity and related diseases.

Effect of providing a formula supplemented with long-chain polyunsaturated fatty acids on immunity in full-term neonates

To determine the effect of feeding formula containing long-chain PUFA (LCP) on immune function, healthy term infants were randomised at age 2 weeks to either a standard term formula (Formula; n 14) or the same formula supplemented with the LCP 20 : 4n-6 and 22 : 6n-3 (Formula+LCP; n 16). Peripheral blood was collected at 2 and 6 weeks to measure immune cell response (the rate of [3H]thymidine uptake and cytokine production after stimulation with phytohaemagglutinin (PHA)). Compared with cells from infants receiving only human milk (HM), the rate of [3H]thymidine uptake in response to PHA, but not IL-2 production, was lower for Formula+LCP infants (P < 0.05). Compared with HM-fed infants, Formula-fed infants (but not Formula+LCP infants) produced more TNF-alpha (unstimulated) and had a fewer CD3+CD44+ cells before stimulation and fewer CD11c+ cells post-stimulation (P < 0.05). However, compared with Formula-fed infants, the Formula+LCP infants had an immune cell distribution (higher percentage CD3+CD44+ and CD4+CD28+ cells) and cytokine profile (lower production of TNF-alpha post-stimulation) that did not differ from HM infants. Additionally, it was found that feeding infants formula during the first 10 d of life influenced immune function. These infants had a higher percentage of CD3+, CD4+CD28+, and lower percentage of CD14+ cells and produced more TNF-alpha and interferon-gamma after PHA stimulation than HM-fed infants (P < 0.05). These results demonstrate that early diet influences both the presence of specific cell types and function of infant blood immune cells. Since many diseases have a strong immunological component, these immune changes may be of physiological importance to the developing infant.

Inflammation, obesity, and fatty acid metabolism: influence of n-3 polyunsaturated fatty acids on factors contributing to metabolic syndrome

Metabolic syndrome (MetS) comprises an array of metabolic risk factors including abdominal obesity, dyslipidemia, hypertension, and glucose intolerance. Individuals with MetS are at elevated risk for diabetes and cardiovascular disease. Central to the etiology of MetS is an interrelated triad comprising inflammation, abdominal obesity, and aberrations in fatty acid metabolism, coupled with the more recently recognized changes in metabolism during the postprandial period. We review herein preliminary evidence regarding the role of dietary n-3 polyunsaturated fatty acids in modulating each of the components of the triad of adiposity, inflammation, and fatty acid metabolism, with particular attention to the role of the postprandial period as a contributor to the pathophysiology of MetS.

N-3 Polyunsaturated Fatty Acids: Relationship to Inflammation in Healthy Adults and Adults Exhibiting Features of Metabolic Syndrome

Individuals with metabolic syndrome (MetS) have a higher risk of type 2 diabetes and cardiovascular disease, therefore, research has been directed at reducing various components that contribute to MetS and associated metabolic impairments, including chronic low-grade inflammation. Epidemiological, human, animal and cell culture studies provide evidence that dietary n-3 polyunsaturated fatty acids (n-3 PUFA), including alpha-linolenic acid (18:3n-3, ALA), eicosapentaenoic acid (20:5n-3, EPA) and/or docosahexaenoic acid (22:6n-3, DHA) may improve some of the components associated with MetS. The current review will discuss recent evidence from human observational and intervention studies that focused on the effects of ALA, EPA or DHA on inflammatory markers in healthy adults and those with one or more features of MetS. Observational studies in healthy adults support the recommendation that a diet rich in n-3 fatty acids may play a role in preventing and reducing inflammation, whereas intervention studies in healthy adults have yielded inconsistent results. The majority of intervention studies in adults with features of MetS have reported a benefit for some inflammatory measures; however, other studies using high n-3 fatty acid doses and long supplementation periods have reported no effect. Overall, the data reviewed herein support recommendations for regular fatty fish consumption and point toward health benefits in terms of lowering inflammation in adults with one or more features of MetS.

Trans-fatty acids and cancer: a mini-review.

The association between trans-fatty acids (TFA) and cancer risk is poorly understood and remains controversial. It is recognised that unique biological effects are associated with specific isoforms within families of fatty acids such as those belonging to the n-3 fatty acids. Furthermore, the interactions between diet and genetic polymorphisms are increasingly recognised for their potential risk-modifying effects on human health and disease. Therefore, the aim of the present review is to evaluate whether specific TFA isomers and genetic polymorphisms differentially modify cancer risk in prostate, colon and breast cancers in animal and human models. Potential mechanisms of action by which TFA may affect cancer development are also reviewed. Overall, across a number of experimental models and human studies, there is insufficient and inconsistent evidence linking specific TFA isomers to cancers of the prostate, colon and breast. A number of methodological limitations and experimental considerations were identified which may explain the inconsistencies observed across these studies. Therefore, further research is warranted to accurately assess the relationship between TFA and cancer risk.

The acute impact of ingestion of breads of varying composition on blood glucose, insulin and incretins following first and second meals.

Structural characteristics and baking conditions influence the metabolic responses to carbohydrate-containing foods. We hypothesized that consumption of whole wheat or sourdough breads would have a favourable effect on biomarkers of glucose homeostasis after first and second meals, compared with those for white bread. Ten overweight volunteers consumed 50 g available carbohydrate of each of the four breads (white, whole wheat, sourdough, whole wheat barley) followed 3 h later by a standard second meal. Blood was sampled for 3 h following bread ingestion and a further 2 h after the second meal for determination of glucose, insulin, paracetamol (indirect marker of gastric emptying), glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1 (GLP-1). Glucose and GLP-1 responses to sourdough bread were lower (P < 0.05) than whole wheat and whole wheat barley breads. Glucose area under the curve (AUC) for sourdough bread was lower than those for whole wheat (P < 0.005) and whole wheat barley (P < 0.03) breads for the entire study. GIP AUC after sourdough bread ingestion was lower compared to white (P < 0.004) and whole wheat barley (P < 0.002) breads following the second meal. There were no significant differences in insulin and paracetamol concentrations among the test breads. Ultra-fine grind whole wheat breads did not result in postprandial responses that were lower than those of white bread, but sourdough bread resulted in lower glucose and GLP-1 responses compared to those of these whole wheat breads following both meals.

Docosahexaenoic acid decreases pro-inflammatory mediators in an in vitro murine adipocyte macrophage co-culture model.

Paracrine interactions between adipocytes and macrophages contribute to chronic inflammation in obese adipose tissue. Dietary strategies to mitigate such inflammation include long-chain polyunsaturated fatty acids, docosahexaenoic (DHA) and eicosapentaenoic (EPA) acids, which act through PPARγ-dependent and independent pathways. We utilized an in vitro co-culture model designed to mimic the ratio of macrophages:adipocytes in obese adipose tissue, whereby murine 3T3-L1 adipocytes were cultured with RAW 264.7 macrophages in direct contact, or separated by a trans-well membrane (contact-independent mechanism), with 125 µM of albumin-complexed DHA, EPA, palmitic acid (PA), or albumin alone (control). Thus, we studied the effect of physical cell contact versus the presence of soluble factors, with or without a PPARγ antagonist (T0070907) in order to elucidate putative mechanisms. After 12 hr, DHA was the most anti-inflammatory, decreasing MCP1 and IL-6 secretion in the contact system (−57%, −63%, respectively, p≤0.05) with similar effects in the trans-well system. The trans-well system allowed for isolation of cell types for inflammatory mediator analysis. DHA decreased mRNA expression (p<0.05) of Mcp1 (−7.1 fold) and increased expression of the negative regulator, Mcp1-IP (+1.5 fold). In macrophages, DHA decreased mRNA expression of pro-inflammatory M1 polarization markers (p≤0.05), Nos2 (iNOS; −7 fold), Tnfα (−4.2 fold) and Nfκb (−2.3 fold), while increasing anti-inflammatory Tgfβ1 (+1.7 fold). Interestingly, the PPARγ antagonist co-administered with DHA or EPA in co-culture reduced (p≤0.05) adiponectin cellular protein, without modulating other cytokines (protein or mRNA). Overall, our findings suggest that DHA may lessen the degree of MCP1 and IL-6 secreted from adipocytes, and may reduce the degree of M1 polarization of macrophages recruited to adipose tissue, thereby decreasing the intensity of pro-inflammatory cross-talk between adipocytes and macrophages in obese adipose tissue.

Fish oil prevents high-saturated fat diet-induced impairments in adiponectin and insulin response in rodent soleus muscle

High saturated fatty acid (SFA) diets contribute to the development of insulin resistance, whereas fish oil-derived n-3 polyunsaturated fatty acids (PUFA) increase the secretion of adiponectin (Ad), an adipocyte-derived protein that stimulates fatty acid oxidation (FAO) and improves skeletal muscle insulin response. We sought to determine whether fish oil could prevent and/or restore high SFA diet-induced impairments in Ad and insulin response in soleus muscle. Sprague-Dawley rats were fed 1) a low-fat control diet (CON group), 2) high-SFA diet (SFA group), or 3) high SFA with n-3 PUFA diet (SFA/n-3 PUFA group). At 4 wk, CON and SFA/n-3 PUFA animals were terminated, and SFA animals were either terminated or fed SFA or SFA/n-3 PUFA for an additional 2 or 4 wk. The effect of diet on Ad-stimulated FAO, insulin-stimulated glucose transport, and expression of Ad, insulin and inflammatory signaling proteins was determined in the soleus muscle. Ad stimulated FAO in CON and 4 wk SFA/n-3 PUFA (+36%, +39%, respectively P ≤ 0.05) only. Insulin increased glucose transport in CON, 4 wk SFA/n-3 PUFA, and 4 wk SFA + 4 wk SFA/n-3 PUFA (+82%, +33%, +25%, respectively P ≤ 0.05); this effect was lost in all other groups. TLR4 expression was increased with 4 wk of SFA feeding (+24%, P ≤ 0.05), and this was prevented in 4 wk SFA/n-3 PUFA. Suppressor of cytokine signaling-3 expression was increased in SFA and SFA/n-3 PUFA (+33 and +18%, respectively, P ≤ 0.05). Our results demonstrate that fish oil can prevent high SFA diet-induced impairments in both Ad and insulin response in soleus muscle.

Modifying the n-6/n-3 polyunsaturated fatty acid ratio of a high–saturated fat challenge does not acutely attenuate postprandial changes in inflammatory markers in men with metabolic syndrome

Metabolic syndrome (MetS) features chronic inflammation and exaggerated postprandial triacylglyceride (TAG) responses. Fasting concentrations of interleukin-6 (IL-6) and C-reactive protein (CRP), key inflammatory mediators, decrease after sustained n-3 polyunsaturated fatty acid (PUFA) intake; however, the ability of n-3 PUFA to attenuate postprandial inflammatory responses is not well studied. Thus, we examined the acute effect of modifying the n-6/n-3 PUFA ratio of a high-saturated fatty acid (SFA) oral fat tolerance test (OFTT) on postprandial TAG and inflammatory responses in men with MetS. Men (n = 8, > or = 45 years old) with MetS ingested 2 high-SFA OFTTs (1 g fat per kilogram body weight), with either a 20:1 (low n-3) or 2:1 (high n-3) n-6/n-3 PUFA ratio, and a water control in a randomized crossover design. Blood samples were collected for 8 hours after treatment to measure postprandial TAG, free fatty acids, IL-6, soluble IL-6 receptor, and CRP. Postprandial TAG increased at the same rate after ingestion of the low-n-3 and high-n-3 OFTTs; however, both OFTTs were significantly different from the water control. There were no differences in the rate at which IL-6 concentrations increased after ingestion of either of the OFTTs compared with water. Furthermore, neither time nor treatment affected circulating soluble IL-6 receptor or CRP concentrations. Thus, increasing the n-3 PUFA content of a high-SFA OFTT does not acutely change postprandial TAG or inflammatory responses in men with MetS.