Cell signal mechanisms,conjugated linoleic acids (CLAs) and anti-tumorigenesis

Fats have been adversely implicated in the aetiology of many forms of cancer yet evidence is accumulating that certain types of fatty acids have anticancer properties. This is well documented for fish-oil fatty acids of the n-3 family. Recently, fatty acids found to occur naturally in ruminant-derived food products were found to have anticancer properties. These fatty acids were identified as conjugated linoleic acids (CLAs) derived from the parent linoleic acid by its partial hydrogenation by rumen bacteria. Studies with tumour-bearing animals have shown that consumption of CLAs particularly with regard to breast and prostate cancer is beneficial. Studies with cancer cells have also shown that these fatty acids can inhibit cell proliferation and induce cell death. However, little is known regarding the mechanisms of action of these CLAs. In particular, which cellular signal mechanisms are regulated by CLAs which can explain their anticancer properties. We have shown that CLAs specifically up-regulate cell signal systems at the level of gene expression (mRNA, protein) in human breast and prostate cancer cells which are responsible for the induction of apoptosis or programmed cell death. These findings support the anticancer effects of CLA found in animal models and indicate similar effects could occur in man.     

改变细胞膜的脂肪酸组成可促进乳腺癌细胞凋亡

目的: 研究n-6脂肪酸脱氢酶 fat-1基因在人乳腺癌细胞内的表达,改变细胞膜脂肪酸组成,对乳腺癌细胞的凋亡作用。方法: 构建含有fat-1 基因的重组腺病毒载体 (Ad.GFP.fat-1),通过包装细胞系（293）产生的腺病毒,感染人乳腺癌细胞MCF-7。提取细胞的总RNA,以fat-1的反义mRNA 作探针,用Northern Blot检测fat-1 基因在MCF-7细胞内的表达。MTT法分析fat-1 基因对MCF-7细胞增殖的影响,凋亡染色试剂盒检测细胞的凋亡。气相色谱仪分析对MCF-7细胞的n-6 PUFAs/n-3 PUFAs含量影响。结果: 通过基因重组技术,得到预期的重组病毒;fat-1 基因在人乳腺癌细胞MCF-7 中能有效异源表达,2天后,可检测到fat-1 mRNA的条带。与对照细胞相比,fat-1基因有效地抑制了MCF-7细胞的增殖（23%,p<0.05）,促进了凋亡（增加35%）;同时降低了人乳腺癌细胞MCF-7细胞膜n-6 PUFAs/n-3 PUFAs的比率。结论: 腺病毒介导的fat-1 基因能在人乳腺癌细胞MCF-7内有效异源表达,且抑制了MCF-7细胞的增殖。机理为降低了细胞膜的n-6 PUFAs/n-3 PUFAs的比率。     

Fatty acids and breast cancer: The role of stem cells

Studies with animal models in vivo as well as with animal and human tumor cells in vitro suggest that specific fatty acids could reduce breast tumorigenesis. The most striking dietary fatty acid studies in animal models that show promise for reduction of breast cancer risk in humans are with conjugated linoleic acids (CLA) and n-3 fatty acids. Although a number of mechanisms have been proposed, the specific target of those fatty acids is not yet known. We sought to determine whether the effects of those fatty acids on terminally differentiated tumor cell seen could be due to alteration of breast cancer stem cells. The isomers, cis9, trans11-CLA and trans10, cis12-CLA, and the n-3 fatty acids, docosahexaenoic and eicosapentaenoic, reduced the proliferation of, and had increased toxicity towards, mammary tumor initiating cells. One mechanism involved in the effect of n-3 fatty acids may be due to alteration of the profile of prostaglandins. These results indicate that select fatty acids may be useful for preventing or reducing the risk of breast cancer as they may target the tumor initiating cell.     

Fatty acid modulation of MCF-7 human breast cancer cell proliferation,apoptosis and differentiation

Epidemiological studies suggest that dietary polyunsaturated fatty acids (PUFA) may influence breast cancer progression and prognosis. In order to study potential mechanisms of action of fatty acid modulation of tumor growth, we studied, in vitro, the influence of n-3 and n-6 fatty acids on proliferation, cell cycle, differentiation and apoptosis of MCF-7 human breast cancer cells. Both eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) inhibited the MCF-7 cell growth by 30% and 54%, respectively, while linoleic acid (LA) had no effect and arachidonic acid (AA) inhibited the cell growth by 30% (p < 0.05). The addition of vitamin E (10uM) to cancer cells slightly restored cell growth. The incubation of MCF-7 cells with PUFAs did not alter the cell cycle parameters or induce cell apoptosis. However, the growth inhibitory effects of EPA, DHA and AA were associated with cell differentiation as indicated by positive Oil-Red-O staining of the cells. Lipid droplet accumulation was increased by 65%, 30% and 15% in the presence of DHA, EPA and AA, respectively; (p < 0.05). These observations suggest that fatty acids may influence cellular processes at a molecular level, capable of modulating breast cancer cell growth.     

Protective properties of n-3 fatty acids and implications in obesity-associated breast cancer

Obesity is well documented as a risk factor for developing breast cancer, especially in postmenopausal women. Adipose tissue in the breast under obese conditions induces inflammation by increasing macrophage infiltration and pro-inflammatory cytokines that in turn up-regulates genes and signaling pathways, resulting in increased inflammation, cell proliferation and tumor growth in the breast. Due to their potent anti-inflammatory effects, n-3 polyunsaturated fatty acids (n-3 PUFA) are a promising and safe dietary intervention in reducing breast cancer risk. Here, we briefly review current status of breast cancer and its relationship with obesity. We then review in depth, current research and knowledge on the role of n-3 PUFA in reducing/preventing breast cancer cell growth in vitro, in vivo and in human studies, and how n-3 PUFA may modulate signaling pathways mitigating their effects on breast cancer development.     

Fatty acid regulation of protein kinase C isoforms in prostate cancer cells

Polyunsaturated fatty acids influence the aetiology of prostate cancer. Their effects on cellular mechanisms regulating prostate tumorigenesis are unclear. Using prostate cancer cells (LNCaP), we determined effects of n-9-OA, n-6-LA, and n-3-EPA on total PKC and its isoforms in relation to cell proliferation and PSA production. PKC-alpha, delta, gamma, iota, mu, and zeta were present in LNCaP cells; PKC-beta, epsilon, eta, and theta isoforms were not. PKC-alpha was detected only in cytosol; PKC-delta, iota, gamma, and mu were present in cytosol and in membranes. Fatty acids increased cell proliferation, total PKC activity and elicited pro-proliferative effects on specific PKC isoforms (PKC-delta and -iota). EPA and LA increased total PKC activity and reduced membrane-abundance of PKC-delta. OA reduced cytosolic and membrane PKC-delta. Only EPA reduced PKC-gamma membrane abundance. Fatty acids enhanced cytosolic PKC-iota abundance but only EPA and to a lesser extent LA increased its membrane content. Changes in PKC-delta, -iota, and -gamma did not affect PSA production.     

N-3 Polyunsaturated Fatty Acids of Marine Origin and Multifocality in Human Breast Cancer

Objective

The microenvironment of breast epithelial tissue may contribute to the clinical expression of breast cancer. Breast epithelial tissue, whether healthy or tumoral, is directly in contact with fat cells, which in turn could influence tumor multifocality. In this pilot study we investigated whether the fatty acid composition of breast adipose tissue differed according to breast cancer focality.

Methods

Twenty-three consecutive women presenting with non-metastatic breast cancer underwent breast-imaging procedures including Magnetic Resonance Imaging prior to treatment. Breast adipose tissue specimens were collected during breast surgery. We established a biochemical profile of adipose tissue fatty acids by gas chromatography. We assessed whether there were differences according to breast cancer focality.

Results

We found that decreased levels in breast adipose tissue of docosahexaenoic and eicosapentaenoic acids, the two main polyunsaturated n-3 fatty acids of marine origin, were associated with multifocality.

Discussion

These differences in lipid content may contribute to mechanisms through which peritumoral adipose tissue fuels breast cancer multifocality.     

Influence of testosterone on polyunsaturated fatty acid biosynthesis in Sertoli cells in culture

Sertoli cells play a central role in spermatogenesis, its development and regulation. They are target cells for androgen action in the seminiferous tubules. The Sertoli cell is considered to be the most important cell type in the testis with regard to essential fatty acid metabolism. We studied the response to testosterone of cultured Sertoli cells from immature rats by determining the fatty acid composition of total cellular lipids as well as by the biosynthesis of polyunsaturated fatty acids. Fatty acid methyl esters were analysed by gas liquid chromatography and radiochromatography. Two doses of testosterone were tested (150 and 300 ng ml(-1)). Significant differences were found in fatty acids derived from total cellular lipids after 8 days in culture in the presence of testosterone (300 ng ml(-1), for 48 h). Compared to controls, the hormone produced a significant increase of 16:1 and 18:1 n-9, and of 18:2 n-6, and a decrease of 20:4 and 22:5 n-6 in total cellular lipids. The decrease in the n-6 fatty acid ratios 20:4/20:3, 20:4/18:2 and 24:5/24:4, and the increase in 18:1n-9/18:0 and 16:1n-9/16:0 ratios were taken as an indirect signal of testosterone effects on Delta5, Delta6 and Delta9 desaturase activities. The drop in Delta5 and Delta6 desaturase activities was corroborated by analysing the transformation of [1-14C]20:3 n-6 into its higher homologues. We concluded that testosterone modifies the fatty acid pattern of cultured Sertoli cells, and this hormone is involved in polyunsaturated fatty acid biosynthesis, modulating Delta5 and Delta6 desaturases activity.     

A maternal high n-6 fat diet with fish oil supplementation during pregnancy and lactation in rats decreases breast cancer risk in the female offspring

The timing of dietary fat intake may modify breast cancer risk. In addition, n-3 fatty acids reduce, and n-6 fatty acids increase, the risk of breast cancer and a maternal high n-6 fat diet results in a greater risk of breast cancer in the female offspring. We hypothesized that the timing of n-3 fatty acid-enriched fish oil supplementation would be important for reducing the risk of breast cancer. Female rats were fed to a high n-6 fat diet containing 20% of the sunflower oil by weight during pregnancy and lactation, and the female offspring were exposed to fish oil by oral gavage either during the perinatal period via maternal intake or during puberty or adulthood. Exposure during the perinatal period to a maternal high n-6 fat diet with fish oil supplementation significantly reduced the incidence of carcinogen-induced mammary tumors in the female offspring compared to a maternal high n-6 fat diet with no fish oil supplementation or fish oil supplementation later in life (P=.0228 by Cox proportional hazards model). We found that a maternal high n-6 fat diet during pregnancy is more important in increasing the risk of mammary tumors in the female offspring than a maternal high n-6 fat diet during lactation. This study suggests that fish oil supplementation during the perinatal period decreases the effect of a maternal high n-6 fat diet on subsequent carcinogen-induced mammary tumor risk, whereas fish oil supplementation during puberty or adulthood does not.     

Fish oil and inflammatory status alter the n-3 to n-6 balance of the endocannabinoid and oxylipin metabolomes in mouse plasma and tissues

It is well established that dietary intake of n-3 fatty acids is associated with anti-inflammatory effects, and this has been linked to modulation of the oxylipin and endocannabinoid metabolomes. However, the amount of data on specific tissue effects is limited, and it is not known how inflammation affects this relation. In the present study we systematically explored the combined effects of n-3 fatty acid diets and inflammation on the in vivo endocannabinoid and oxylipin metabolomes using a multicompartment, detailed targeted lipidomics approach. Male C57BL/6 mice received diets containing 0, 1, or 3?% w/w fish oil (FO) for 6?weeks, after which 2?mg/kg LPS or saline was administered i.p. Levels of endocannabinoids/N-acylethanolamines (NAEs) and oxylipins, covering n-3 and n-6 fatty acid derived compounds, were determined in plasma, liver, ileum and adipose tissue using LC?CMS/MS. FO generally increased ??n-3?? NAEs and oxylipins at the expense of compounds derived from other fatty acids, affecting all branches of the oxylipin metabolome. LPS generally increased levels of endocannabinoids/NAEs and oxylipins, with opposing effects across plasma and tissues. Multivariate data analysis revealed that separation between diet groups in the saline treated groups was primarily explained by decreases in other than n-3 derived compounds. In the LPS treated groups, the separation was primarily explained by increases in n-3 derived compounds. In conclusion, FO caused marked changes in the n-3 to n-6 balance of the endocannabinoid and oxylipin metabolomes, with specific effects depending on inflammatory status.     

Glycine uptake by cultured human Y79 retinoblastoma cells: effect of changes in phospholipid fatty acid unsaturation

Glycine uptake was investigated in cultured Y79 retinoblastoma cells containing different degrees of phospholipid fatty acid unsaturation. The modifications were produced by growing the retinoblastoma cells in medium supplemented with various unsaturated fatty acids. Glycine was taken up by the retinoblastoma cells through two kinetically distinguishable process. The high-affinity system is totally dependent upon extracellular Na+ and partially dependent upon Ca2+. Of the glycine taken up by retinoblastoma cells, 85-90% remains as free intracellular glycine and less than 30% is incorporated into cellular protein. When the cells are grown in a medium containing 10% fetal bovine serum as the only source of fatty acids, the phospholipids contained 23% polyunsaturated fatty acids. Under these conditions the high-affinity system has a K'm of 34.2 +/- 3.7 micrometers and a V'max of 91.2 +/- 16.2 pmol min-1 mg protein -1. The low-affinity system has a K'm of 2.7 +/- 0.4 mM and a V'max of 4.1 +/- 0.5 nmol min-1 mg protein-1. When the polyunsaturated fatty acid content of the phospholipids was increased by supplementing the medium with linolenic or docosahexaenoic acids (n-3 polyunsaturates) or linoleic or arachidonic acids (n-6 polyunsaturates), the K'm and V'max of the high-affinity glycine uptake system were increased three- to fourfold. By contrast, supplementing the medium with oleic acid, and n-9 monounsaturate, did not significantly alter the K'm or V'max for glycine uptake. The results with this model system suggest that one of the effects of the high polyunsaturated fatty acid content normally present in neural cell membranes may be a modulation of the high-affinity transport system so that it functions more efficiently in regulating glycine uptake.     

Fatty acid uptake by breast cancer cells (MDA-MB-231): Effects of insulin,leptin, adiponectin,and TNFα

In order to exert metabolic effects, fatty acids must be taken up by cells and metabolize effectively to different classes of cellular lipids (triacylglycerols, phospholipids, etc.) for incorporation into different cellular and intracellular compartments. Therefore, the main aim of the present study is to investigate the uptake and metabolism of fatty acids representing three different series of fatty acids such as oleic acid, 18:1n-9 (OA), arachidonic acid, 20:4n-6 (AA), and eicosapentaneoic acid, 20:5n-3 (EPA) by breast cancer cells, MDA-MB-231. Moreover, we investigated the effects of insulin and several adipokines on the fatty acid uptake by these cells as obesity and insulin resistance syndrome have been suggested to affect breast cancer risk. We report for the first time that AA was predominantly taken up by these cells compared with EPA and OA. Pre-incubation of these cells with TNFα stimulated most of the uptake of EPA (30%), whereas uptake of OA and AA was stimulated only 10–15% compared with the controls. Insulin, leptin, and adiponectin had no effect on fatty acid uptake by these cells. Together these results demonstrate that preferential uptake of AA in MDA-MB-231 cells, and the fatty acid uptake activity of these cells is influenced by TNFα.     

n-3 and n-6 polyunsaturated fatty acids have different effects on acyl-CoA:cholesterol acyltransferase in J774 macrophages.

The effects of incubating J774 mouse macrophages with different fatty acids on cholesterol esterification were investigated. In cells incubated with n-3 polyunsaturated fatty acids, the rate of cholesterol esterification was significantly reduced compared with cells incubated with n-6 polyunsaturated fatty acids or with oleic acid. This change in cholesterol esterification appears to be the result of reductions in the activity of acyl-CoA:cholesterol acyltransferase (ACAT) in the endoplasmic reticulum of the macrophages incubated with the n-3 polyunsaturated fatty acids. No differences in microsomal cholesterol were observed among cells incubated with different fatty acids. However, cellular cholesterol levels were lower in cells incubated with n-3 polyunsaturated fatty acids. In microsomes from cells incubated with n-3 polyunsaturated fatty acids, both the Km and the Vmax of ACAT were lower than in microsomes from cells incubated with n-6 fatty acids or oleic acid. These findings may explain some of the reduction in atherosclerotic lesions that are observed with dietary fish oils that contain high levels of n-3 polyunsaturated fatty acids.     

Dietary omega-3 fatty acids and ionizing irradiation on human breast cancer xenograft growth and angiogenesis

Background  

The effects of an omega-3 (n-3) fatty acid enriched diet alone and in combination with gamma irradiation (IR) therapy in nude mice bearing a human MDA-MB231 breast cancer xenograft were tested. The cancer cells were injected into the mammary fat pad of young female mice. Six weeks later, mice were randomly divided into two diet groups: 1) mice with 10% corn oil (rich in omega 6 fatty acids) in their food, 2) mice consuming a 10% fat diet that was enriched in n-3 fatty acids. After two weeks on the diet, treatment with 200 cGy of IR every second day for four treatments (total 800 cGy) was initiated on half of the mice from each diet group. Some mice in each of the 4 groups were euthanized 24 hours after the end of IR while the remaining mice were followed for 3 additional weeks. Tumor sections were stained for endothelial cells with CD31 and PAS and for hypoxia inducible factor 1α (HIF-α).     

Comparison of stearidonic acid and alpha-linolenic acid on PGE2 production and COX-2 protein levels in MDA-MB-231 breast cancer cell cultures

The bioactivity of stearidonic acid (SDA, 18:4n-3) and alpha-linolenic acid (LNA, 18:3n-3) on cyclooxygenase-2 (COX-2) enzyme expression and prostaglandin E2 (PGE2) production has not been evaluated. This investigation examined the effects of SDA and LNA on PGE2 biosynthesis and COX-2 protein and mRNA levels in MDA-MB-231 human breast cancer cells. Cells were supplemented with SDA, LNA, linoleic acid and arachidonic acid (AA) at concentrations ranging from 10 to 200 microM. At 50 and 200 microM, both SDA and LNA treatments and their combinations reduced PGE2 production as compared with AA. At 50 microM, SDA treatment also lowered the COX-2 protein level as compared with the vehicle, but this reduction was not observed with the LNA treatment. Gas chromatographic analysis of fatty acids in cellular lipids of breast cancer cells revealed that SDA led to significantly greater concentrations of 20:5n-3 and other long-chain (LC) n-3 polyunsaturated fatty acids (PUFAs) (20:4n-3, 22:4n-3 and 22:5n-3) as compared with the LNA treatment. Both SDA and LNA reduced the level of 20:4n-6; however, SDA was more effective than LNA in decreasing the ratio of n-6/n-3 PUFAs in cells. In addition, SDA was more potent than LNA in suppressing the expression of the COX-2 gene, which was associated with the reduction in the levels of nuclear factor kappa B and peroxisome proliferator-activated receptor gamma mRNA. This study showed that although PGE2 production in MDA-MB-231 breast cancer cells was not significantly different between the SDA and LNA treatments, SDA was more effective than LNA in converting into LC n-3 PUFAs and in reducing COX-2 protein and mRNA levels.     

The opposing effects of n-3 and n-6 fatty acids

Polyunsaturated fatty acids (PUFAs) can be classified in n-3 fatty acids and n-6 fatty acids, and in westernized diet the predominant dietary PUFAs are n-6 fatty acids. Both types of fatty acids are precursors of signaling molecules with opposing effects, that modulate membrane microdomain composition, receptor signaling and gene expression. The predominant n-6 fatty acid is arachidonic acid, which is converted to prostaglandins, leukotrienes and other lipoxygenase or cyclooxygenase products. These products are important regulators of cellular functions with inflammatory, atherogenic and prothrombotic effects. Typical n-3 fatty acids are docosahexaenoic acid and eicosapentaenoic acid, which are competitive substrates for the enzymes and products of arachidonic acid metabolism. Docosahexaenoic acid- and eicosapentaenoic acid-derived eicosanoids antagonize the pro-inflammatory effects of n-6 fatty acids. n-3 and n-6 fatty acids are ligands/modulators for the nuclear receptors NFkappaB, PPAR and SREBP-1c, which control various genes of inflammatory signaling and lipid metabolism. n-3 Fatty acids down-regulate inflammatory genes and lipid synthesis, and stimulate fatty acid degradation. In addition, the n-3/n-6 PUFA content of cell and organelle membranes, as well as membrane microdomains strongly influences membrane function and numerous cellular processes such as cell death and survival.     

Metabolism of n-6 fatty acids by NIH-3T3 cells transfected with the ras oncogene

N-6 fatty acid metabolism was compared in NIH-3T3 cells and DT cells, which differ only in the presence of the v-Ki-ras oncogene. Non-dividing cells were incubated with [1-¹⁴C]-labelled fatty acids (18:2n-6, 18:3n-6, 20:3n-6 and 20:4n-6) at different time intervals (2–24 h) and concentration (0–120 M). In both cells lines, the uptake of different fatty acids from the medium was similar and reached a maximum at 6–8 h. All fatty acids reached the same maximum level in DT cells, whereas, the relative uptake of added fatty acids by NIH-3T3 cells was different: 20:4n-6>20:2n-6>18:2n-6=18:3n-6. Throughout the incubation (2–24 h), desaturation and elongation of n-6 fatty acids was more active in DT cells than in NIH-3T3 cells. However, in both cell lines, incubated with different n-6 fatty acid precursors, the levels of radiolabelled 20:4n-6 were relatively constant. In DT cells, phosphatidylcholine was found to be the major fraction labelled with n-6 fatty acids precursors and those of endogenous synthesis, whereas, in NIH-3T3 cells the neutral lipid fraction, particularly triglycerides, was also strongly labelled. In concentration dependent studies, phospholipid labelling by fatty acids was saturable. At lower concentrations, especially in DT cells, phospholipids were labelled predominantly. As the concentration increased there was an overflow into the triglyceride fraction. Since the differences in fatty acid metabolism between the two cell lines cannot be related to the growth rate, it is suggested that they were a consequence of the expression of the v-Ki-ras oncogene.Abbreviations BSA bovine serum albumin - CE cholesterol ester - DG diglyceride - DMEM Dulbecco's modification of Eagle's medium - EL ether lipids (glyceryl ether diesters) - FAME fatty acid methyl ester - FCS fetal calf serum - FFA free fatty acids - HEPES N-2-(hydroxyethyl)piperazine-N-2-ethanesulphonic acid - MG monoglyceride - NL neutral lipid - PC phosphatidylcholine - PE phosphatidylethanolamine - PI phosphatidylinositol - PL phospholipid - s.a specific activity - TG triglyceride - TLC thin layer chromatography     

Effect of polyunsaturated fatty acids on the growth of fish cells in culture

• 1.1. The effects on growth of supplementing the medium with (n-3) and (n-6) polyunsaturated fatty acids (PUFA) were investigated in Atlantic salmon (AS) and turbot (TF) cell lines.
• 2.2. Neither cell line grew in the absence of serum, and addition of increasing percentages of serum resulted in graded increases in cell growth in both cell lines.
• 3.3. The growth of AS cells was stimulated by supplementing the medium with both (n-6)PUFA and (n-3)PUFA at 5–25 μM, especially 18:3(n-3) and 20:5(n-3).
• 4.4. Intermediate concentrations (15–20 μM) of 18:2(n-6) and 18:3(n-3) increased cell growth in TF cells, although only after 8 days in culture.
• 5.5. In contrast, both (n-3) and (n-6)PUFA at 25 μM tended to inhibit the growth of TF cells, and in longer incubations caused cell death.
• 6.6. The inhibition of TF cell growth rate and, in particular, the cell death induced by 25 μM PUFA could be abolished by the addition of vitamin E to the medium.