Evidence for potential mechanisms for the effect of conjugated linoleic acid on tumor metabolism and immune function: lessons from n–3 fatty acids

¹ From the Department of Agricultural, Food and Nutritional Science, University of Alberta, Edmonton, Canada

² Presented at the workshop "The Role of Conjugated Linoleic Acid in Human Health," held in Winnepeg, Canada, March 13–15, 2003.

³ Supported by the Natural Sciences and Engineering Research Council of Canada and 3 members of the Alberta Agriculture Funding Consortium (Alberta Agriculture Research Institute, Alberta Livestock Industry Development Fund, and Agriculture and Agri-Food Canada; CARD). PS is a recipient of scholarships from the Natural Sciences and Engineering Research Council of Canada and the Alberta Heritage Foundation for Medical Research.

⁴ Address reprint requests to CJ Field, Department of Agricultural, Food and Nutritional Science, University of Alberta, 3-18e Agriculture/Forestry Centre, Edmonton, Alberta, Canada T6G 2P5. E-mail: catherine.field{at}ualberta.ca.

ABSTRACT

Conjugated linoleic acid (CLA) and the long-chain polyunsaturated n–3 fatty acids have been shown in vivo and in vitro to reduce tumor growth. Tumor growth could occur by slowing or stopping cell replication (by interfering with transition through the cell cycle), increasing cell death (via necrosis and/or apoptosis), or both. The anticancer effects of fatty acids, shown in vivo, could also be mediated by effects on the host’s immune system. Although it is widely recognized that n–3 fatty acids can alter immune and inflammatory responses, considerably less is known about CLA. For n–3 fatty acids, several candidate mechanisms have been proposed for their immune effects, including changes in 1) membrane structure and composition, 2) membrane-mediated functions and signals (eg, proteins, eicosanoids), 3) gene expression, and 4) immune development. Considerable work has been done that shows the potential importance of CLA as an anticancer treatment; however, many questions remain as to how this effect occurs. This review summarizes the CLA and cancer literature and then uses the evidence for the anticancer immune and tumor properties of the long-chain n–3 fatty acids docosahexaenoic and eicosapentaenoic acids to suggest future research directions for mechanistic studies on CLA and cancer.

Key Words: Docosahexaenoic acid • eicosapentaenoic acid • cancer • mammary tumors • apoptosis • necrosis • cell cycle • rodents

INTRODUCTION

Fats are adversely implicated in the etiology of many cancers, yet evidence is accumulating that certain fatty acids, such as the highly polyunsaturated n–3 fish oil fatty acids, docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), have potential anticancer activity [reviewed by Bartsch et al (1), Rose and Connolly (2), and Hardman (3)]. More recently, anticancer activity was demonstrated for conjugated linoleic acid (CLA) in both human tumor cell lines and in well-accepted rodent models of carcinogenesis (4-10). Human epidemiologic data support the anticancer potential of n–3 fatty acids as an inverse association between dietary n–3 intake (and the intake of fish) and the incidence of several forms of cancer, including breast and colorectal cancers [reviewed by de Deckere (11)]. Although an inverse relation was observed between CLA accumulation in breast tissue and the incidence of breast cancer in postmenopausal women (12), estimated CLA intake was reported to demonstrate a positive (albeit weak) relation with breast cancer incidence in the Netherlands Cohort Study (13). Thus, there is insufficient evidence from epidemiologic studies in humans, at this time, to support the anticarcinogenic properties of CLA demonstrated in animal and tissue culture studies.

The biochemical mechanisms whereby dietary CLA and n–3 fatty acids inhibit carcinogenesis are not established. Fatty acids could influence tumor growth by way of a direct effect on the tumor or by way of their effects on immunosurveillance in the host. This report uses the literature about n–3 fatty acid to explore mechanisms by which CLA might influence tumor growth and immune function.

EFFECTS OF FATTY ACIDS ON TUMORIGENESIS

It is well established that feeding DHA and EPA (from 5% to 20% wt:wt, as fish oil) reduces the growth of tumors in rodents, including tumors of the mammary gland (2, 14), colon (2), prostate (15), liver (16) and pancreas (17). Work in human cancer cell lines has convincingly demonstrated that both the long-chain polyunsaturated n–3 fatty acids DHA and/or EPA can reduce the growth of many different human tumor types, including breast (18), colon (19), pancreatic (20), chronic myelogenous leukemic (21), and melanoma (22) cell lines.

Considerable evidence demonstrates that dietary CLA inhibits the initiation, after initiation, or promotion stages of carcinogenesis, as well as some evidence that it can influence cancer progression [reviewed by Belury (23)]. Feeding a synthetic mixture of CLA isomers (45% c9,t11-CLA, 42% t10,c12-CLA, with several other remaining isomers comprising minor amounts) at 0.5–1.5% (wt:wt) in nutritionally complete semipurified diets either during or after chemical carcinogen treatment inhibited tumorigenesis in the mammary gland, colon, skin, and prostate (5-7, 24, 25). Although there is considerable evidence of the antitumor growth effects of CLA on tumor cell lines and rodent models, CLA was not demonstrated to inhibit tumor growth in all animal models. Feeding CLA did not impede the development of aberrant crypt foci after azoxymethane treatment (26), the occurrence of liver metastasis in ductal pancreatic cancer in rodents (27), tumorigenesis in the Apc^Min mouse (28), or the growth of an aggressive mammary (29) or prostatic (30) tumor. Recently, it was reported that the t10,c12-CLA might even act as a cancer promoter in colon carcinogenesis in the Min mouse, possibly through pathways affecting nuclear factor-êB and cyclin D1 (31). Both the c9,t11- and the t10,c12-CLA isomers and mixtures of these isomers were demonstrated to reduce the proliferation of many tumor cell lines in culture (32, 33), and adding either of the major isomers of CLA to the diet results in a similar inhibition of mammary tumorigenesis (34, 35). In vivo CLA was also demonstrated to inhibit the growth of transplanted prostatic (10) and mammary (9) tumor cell lines. Feeding CLA (1.0 g/100 g diet) reduced the proliferation of terminal-end bud and lobuloalveolar bud structures in the mammary gland of rats (36). These are the sites at which tumors form in both rat and human mammary cancers. Evidence also suggests that CLA could influence the progression (metastasis) of mammary rodent tumors (5, 37).

Fatty acids and their effect on tumor cell growth
As illustrated in Figure 1, fatty acids could alter the growth of tumor cells by 1) influencing cell replication by interfering with components of the cell cycle or 2) increasing cell death, either by way of necrosis or apoptosis.

View larger version (25K):
FIGURE 1.. Potential ways in which fatty acids could influence tumor cell growth. Fatty acids could interfere with cell replication by altering the distribution of tumor cells in the cell cycle, by affecting the expression of cell cycle regulatory proteins, or both. Alternatively, fatty acids could increase cell death via necrosis or apoptosis.

 
Effects on cell replication
Cellular replication is composed of several distinct phases (Figure 1): G₁ is an initial growth phase that leads to DNA synthesis (S phase), followed by a gap phase (G₂), and finally by mitosis (M phase), the actual segregation of chromosomes and cytoplasms (38). Two important families of regulatory molecules promote progression through the cell cycle, the cyclins and the cyclin-dependent kinases (cdks) (38). Normal cells progress through the cell cycle after stimulation of these regulatory molecules by exogenous agents such as growth factors, hormones, or cytokines (38). Cancerous cells, however, appear to lose their dependency on these external signals and often progress, unregulated, through many cell cycles (39). Multiple specific mutations in the genes encoding proteins that normally play a role in regulating the progression of cells through the cell cycle are identified in tumor cells (40).

Some evidence exists that n–3 fatty acids have an effect on tumor cell progression through the cell cycle, but the evidence in vivo is still preliminary. In vitro DHA treatment arrested progression through the cell cycle in human-derived MCF-7 breast cancer (41) and malignant melanoma (22) cell lines. Similarly, EPA treatment in vitro is reported to arrest the growth of K-562 human leukemic (21), pancreatic (42), and colon (19) cancer cell lines in different phases of the cell cycle, correlating with a downregulation of cyclin protein expression in some instances (21). In vivo, fish oil fed to rats implanted with a mammary tumor cell line prolonged the DNA replication time of the tumor cells, supporting the hypothesis that n–3 fatty acids could slow down progression through S phase (43).

Considerable in vitro work suggests that incubating tumor cells with CLA alters the expression of key proteins that regulate the cell cycle [reviewed by Belury (23)]. An in vitro study suggests c9,t11-CLA affects cyclins, cdk inhibitors, and check point proteins (44). Work by Ip et al (45) demonstrated that feeding CLA or c9,t11-CLA-rich butter fat for 4 wk reduces the expression of cyclins D and A in the terminal-end buds and alveolar clusters of the mammary epithelium. Cyclins D and A are key proteins involved in facilitating entry of the cells into the cell cycle and progression through S phase, respectively (46). CLA feeding was also shown to up-regulate the expression of p53 [reviewed by Belury (23)], the protein product of a tumor suppressor gene that is frequently mutated in many tumor cells (47). p53 Is involved in monitoring the quality of DNA after G₁ phase and, if DNA is damaged, will block entry of the cell into S phase (Figure 1) by altering the expression of genes involved in growth arrest and promotion (38). p53 Can also induce genes belonging to a family of regulatory molecules known as cdk inhibitors (46). In the study by Ip et al (45), there was a trend toward an increase, although not statistically significant, in the proportion of cells from the CLA-fed rats that expressed the p16 and p27 cdk inhibitors. Together, these data suggest that CLA could reduce tumor cell proliferation by modifying cell cycle proteins that regulate this process.

Effects on cell death
Cell death can occur by way of necrosis or apoptosis. Necrosis generally results from an insult or toxicity reaction and triggers inflammation, whereas apoptosis describes the distinct energy-requiring process of programmed cell death, characterized by DNA fragmentation, chromosome condensation, nuclear membrane fragmentation, formation of apoptotic bodies, and inversion of phosphatidylserine in the plasma membrane.

Necrosis.
It was reported that many tumor cells do not possess sufficient antioxidant defense systems when compared with healthy cells, and so they are more susceptible to oxidative and peroxidative damage (48). Polyunsaturated fatty acids (PUFAs) are the main intracellular substrates for lipid peroxidation; thus, PUFA-derived reactive lipid compounds could damage cell membranes, change the cellular composition or cytoskeletal assembly, modify membrane transport systems or enzymes, or inhibit polymerase reactions and/or polyamine synthesis (49). Therefore, it is reasonable to expect that PUFA-enriched tumor cells might have an increased susceptibility to oxidant stress. There is evidence for an oxidative effect of n–3 fatty acids from both in vitro (20, 50, 51) and in vivo studies (49, 52). However, it is not entirely clear that lipid peroxidation is cytotoxic to cells (51), and, recently, the specificity of this effect in the in vitro work was questioned (53). Studies in our own laboratory demonstrated that the addition of an antioxidant (vitamin E) to the culture media does not abrogate the growth-inhibitory effects of n–3 fatty acids on breast cancer cell growth (unpublished data, 2004), suggesting that the growth inhibition observed with n–3 fatty acids cannot be attributed to lack of oxidative defense.

Early studies suggested that an oxidative mechanism was involved in the growth-suppressive effects of CLA. Supplementation of cell culture medium with mixed isomer CLA (17–71.5 µmol/L) was reported to increase the susceptibility of tumor cells to lipid peroxidation (54-56). Despite this early interest in oxidative stress on tumor growth, subsequent studies suggested that CLA does not act directly as a pro-oxidant (57). However, CLA enrichment in membranes can result in the production of conjugated diene hydroperoxides (58). These compounds could result in cytotoxic effects or could simply contribute to generating an internal cellular pro-oxidant milieu that influences growth-regulatory signals (59). This indirect oxidative function is supported in vivo in which feeding CLA to healthy subjects was reported to induce both nonenzymatic and enzymatic lipid peroxidation (60). Contrary to the tumor peroxidation hypothesis, CLA enrichment in nontumor tissues was reported to increase these tissues’ oxidative stability (54). This stability is suggested to be due to the decrease in linoleic acid metabolites (particularly arachidonic acid) when CLA concentrations are increased in tissues (61). It was suggested that the different isomers could have different oxidative properties (in healthy tissues), and the proportion of c9,t11-CLA to other CLA isomers, in particular t10,c12-CLA, could alter the balance between anti- and pro-oxidant susceptibility (54).

Apoptosis. In vivo, feeding DHA, EPA, or a mixture was demonstrated to increase the rate of apoptosis of tumor cells in rodent models, including tumors of the mammary gland (62, 63), liver (16), and colon (64, 65). Similarly, adding EPA or DHA to culture media was demonstrated to induce apoptosis in breast (66), colon (19, 64, 67, 68), lymphoma (69), leukemic (70, 71), pancreatic (20, 42), and melanoma (22) human cancer cell lines. The mechanism of induction of apoptosis by n–3 fatty acids is unknown but was suggested to involve n–3-mediated changes in membrane fluidity or structure; products of PUFA metabolism such as lipid peroxides, aldehydes, prostaglandins, or leukotrienes; or synthesis of reactive oxygen species (20).

Similarly, feeding CLA was reported to induce apoptosis in mammary (72), colon (73), and adipose (74) tissues. Providing CLA in vitro induced apoptosis in breast (75), SGC-7901 (46), and HT-29 (76) tumor cells. Although most studies used a mixture of isomers, the effects of CLA on breast or forestomach tumors were shown for the c9,t11-CLA (44, 75, 77) and t10,c12-CLA (75, 77) isomers. Recently, it was suggested that a 50:50 mixture of the 2 main CLA isomers was more effective than individual isomers at inducing apoptosis in breast cancer cell lines (75). As with n–3 fatty acids, the mechanism for the effects of CLA on apoptosis is not established. Data suggest that CLA could down-regulate ErbB3 signaling and the phosphoinositide 3-kinase and Akt pathway (76) and that it can decrease expression of bcl-2, a gene involved in suppression of apoptosis (72). Recently, feeding CLA (as the 2 major isomers or as a mixture) was demonstrated to inhibit the expression of extracellular-regulated kinase 1 protein and to promote the expression of mitogen-activated protein kinase phosphatase-1 protein in a rodent model of forestomach neoplasia (77). Only a small effect of CLA was reported on induction of the apoptosis-promoting Bax protein (23). Recently, it was reported that the apoptosis induced by c9,t11-CLA in SGC-7901 cells could be due to the ability of this isomer to block progression through the cell cycle (44). Considerable data support that CLA can increase peroxisome-proliferator activated receptor- (PPAR) expression in tissues [reviewed by Belury (78)], and PPAR is reported to promote apoptosis in many tumor cell lines (79).

Fatty acids and their effect on the immune system
Immune surveillance, the ability to detect and destroy tumor cells, is an important role of the cellular arm of the immune system (80). T helper (CD4⁺) and cytotoxic T lymphocytes (CD8⁺) play a central role in tumor surveillance (80). There is a progressive decrease in many immune surveillance defenses in animal models of cancer (81) and humans with cancer (82). The influence of various fatty acids, most extensively n–3 PUFAs, on the functional responses of cells of the immune system was examined in vitro, in animal feeding studies, and in human intervention studies.

n–3 Fatty acids and immune function
Although it is well established that long-chain n–3 PUFAs can up-regulate anticancer defenses such as natural killer cell cytotoxicity and humoral and T cell responses [reviewed by Yaqoob (83)], the application of studies in healthy humans and animals to cancer may not be as straightforward. For example, our research has demonstrated that the influence of dietary n–3 PUFAs on the immune response differs between healthy animals and animals with suppressed immune systems (14, 84). Additionally, the amount and the mixture of fatty acids in the diet, particularly the content of n–6 fatty acids, influences the immune effect that results after feeding n–3 fatty acids. Tumor-bearing rats fed long-chain n–3 PUFAs as part of a low-PUFA diet had significantly increased natural killer cell cytotoxicity, a higher proportion of CD8⁺ and CD28⁺ cells that were activated (ie, expressing CD25) and increased nitric oxide and interleukin 2 (IL-2) production after mitogen stimulation, whereas these immune enhancements were not found when n–3 PUFA was supplemented in a high-PUFA diet (84).

Conjugated linoleic acid isomers and immune function
Several studies reported immunologic effects of mixtures of CLA isomers in poultry, rodents, guinea pigs, and pigs [reviewed by Sebedio (85)]. Although not directly related to anticancer defense, there are reports of beneficial effects of feeding CLA to animals and rodents on inflammatory-induced growth suppression (86), endotoxin-induced anorexia (87), mucosal damage and growth failure in experimental colitis (88), and antigen-induced type 1 hypersensitivity response (89, 90). However, feeding mixtures of CLA did not affect the resistance of mice to infection with Listeria monocytogenes (91). The effects on various immune characteristics from feeding and in vitro experiments are presented in Table 1.

View this table:
TABLE 1. Effect of conjugated linoleic acid (CLA) on immune defenses of importance in immune surveillance¹

 
Although it is not always possible to translate in vitro measures specifically to in vivo function against a tumor, all of the findings reported in Table 1 would generally be regarded as beneficial to cancer prevention. Interestingly, in the one study, the effects of CLA on cellular immunity were found to remain for some time beyond the period of dietary supplementation (96). To our knowledge no studies examined the effect of feeding CLA on immune function in the presence of a tumor. As evident in Table 1, few studies were conducted with use of single isomers. However, the results of a recently published randomized double-blinded clinical trial suggest that, unlike most of the tumor studies, the individual isomers could act differently on components of the immune system. In that study, providing 1.6 g/d CLA for 12 wk, a 50:50 mixture of CLA, but not an 80:20 (c9,t11:t10,c12-CLA) mixture improved the proportion of individuals producing a protective antibody titer to hepatitis B vaccination. Interestingly, in those healthy subjects, other aspects of immune function (delayed-type hypersensitivity responses, natural killer cell activity, lymphocyte proliferation, and production of tumor necrosis factor , IL-1Â, IL-6, interferon-ã, IL-2, IL-4, and prostaglandin E₂) were not affected (100). These results are consistent with an earlier report (101).

MECHANISMS TO EXPLAIN THE EFFECTS OF FATTY ACIDS ON IMMUNE AND TUMOR CELLS

Although it is widely recognized that dietary fatty acids can potentially alter immune and inflammatory responses and tumorigenesis, current understanding of the cellular mechanisms is incomplete. Several candidate mechanisms are proposed, including alterations in membrane structure and composition, changes in membrane-mediated functions and signals (eg, proteins, eicosanoids), changes in gene expression, and effects on the development of the immune system. As the evidence for the potential mechanisms for n–3 fatty acids on tumor growth and immune function is the subject of several excellent recent reviews (83, 102-104), this section uses that information to explore the evidence for CLA.

Changes in membrane composition
Immune cell activation [cell proliferation, phagocytosis (105)] and tumor growth [malignancy (106)] result in an increased rate of novo synthesis and turnover of membrane phospholipids. These processes require a constant supply of fatty acids, the main supply being those consumed in the diet. It is well established that both the amount and type of fat consumed in the diet influence the lipid composition of immune (107, 108) and tumor (16, 109-111) cell membranes. Changes in membrane composition would affect growth, interaction with other cells (immune system), and the function of proteins and other components that are in the membrane. The function of the immune system depends on interactions between different cell types and through effects on membrane composition; dietary fatty acids have the potential to influence these interactions (83). Considerable evidence supports this mechanism for n–3 fatty acids [reviewed by Yaqoob (83)]. Although little work demonstrates the incorporation of CLA isomers into immune cell membranes, it can likely be assumed, as it is well established, that feeding CLA isomers is associated with accumulation of CLA and its metabolites in many other tissues and cell types (61, 112). Our work (33) and that of others (59) demonstrated that CLA is rapidly incorporated into the functionally important tumor cell membrane lipids (phospholipids). Our results suggest that CLA isomers (both major isomers) are specifically replacing the essential fatty acids arachidonic and linoleic acids in phospholipids (33). Data exist to suggest that the different isomers might be incorporated at different rates (4, 5, 33).

Lipid rafts are dynamic microenvironments in the exoplasmic leaflets of the phospholipid bilayer of plasma membranes, which are thought to preferentially group transmembrane proteins according to their function (113). Several proteins involved in signaling are commonly found in lipid rafts, and many of these proteins are palmitoylated (114). Activation of the proteins within rafts by an extracellular ligand can result in rapid clustering, which appears to be important for signal transduction. A couple of studies examine n–3 fatty acid incorporation into lipid rafts (115, 116), offering a logical yet unexplored link between changes in the CLA content of cell membranes and changes in cellular function.

Changes in membrane-mediated signals (signal transduction) and proteins
Changes in plasma membrane structural characteristics in mammalian cells can change the activity of proteins that serve as ion channels (117), transporters (117), receptors (118), signal transducers (119), or enzymes (120). Dietary lipids were demonstrated to influence the pattern of fatty acids released from lymphocytes (ie, arachidonic acid) (121), which would ultimately influence the synthesis of eicosanoids (prostaglandins, leukotrienes, thromboxanes). In addition to their role in regulation of immune and inflammatory responses (83), eicosanoids may also be needed to sustain growth of tumor cells (122).

Long-chain n–3 fatty acids were shown in immune cells to alter cell surface costimulatory and activation markers or molecules (123, 124), calcium signaling (125), and protein kinase C translocation in the membrane (126). Similarly, in other cell types, membrane incorporation of n–3 fatty acids can alter membrane permeability (127), membrane fluidity (128, 129), and hormone and growth factor binding (130).

Compared with the amount of work in tumor cells, less work was done on the membrane-mediated effects of CLA on immune cells. In tumors, studies showed that incubation with CLA isomers (either c9,t11- and t10,c12-CLA or a mixture) altered lipid (57) and phospholipid metabolism (78), changed the amount of the membrane protein stearoyl-CoA desaturase (131) and reduced arachidonic acid release from phospholipids (33, 73, 132) in several different tumors or cell lines. Evidence suggests that CLA could also inhibit both the constitutive cyclooxygenase-1 and the inducible form of this enzyme, cyclooxygenase-2 (99, 133). These in vitro studies indirectly suggest that the mechanism by which CLA inhibits tumor growth could involve the modulation of arachidonate-derived eicosanoids (prostaglandin E₂, prostaglandin F₂, leukotriene B₄, and leukotriene C₄). Support for CLA-altering eicosanoid synthesis comes from work in bone and macrophages (134). Although it was suggested in the literature, it is unlikely that conjugated-eicosatetraenoate (20:4; c5,c8,c11,t13) can act as a substrate for cyclooxygenase (78). The studies that found that phospholipid-associated arachidonate concentrations were not altered after feeding CLA (28, 135) open the door for other possible mechanisms.

Effect of fatty acids on gene expression
n–3 Polyunsaturated fatty acids
Considerable evidence indicates that n–3 PUFAs are capable of inducing changes in gene expression in several different cell types, including tumor and immune cells. The list of genes whose expression appears to be affected by fish oil or purified n–3 fatty acids continues to grow, and excellent reviews have been published (136-138). Although the exact way in which n–3 fatty acids alter gene transcription is not known, there is considerable speculation and new evidence that this alteration might involve a class of nuclear receptors called PPARs. PPARs are ligand-activated transcription factors present in a variety of cell types, with diverse actions, mainly in lipid metabolism (83, 139). Activators of both PPAR and PPAR were shown to inhibit the activation of several inflammatory genes [reviewed by Berger and Moller (140)]. n–3 Fatty acids can activate PPARs by directly binding to them (141) or by binding their cyclooxygenase and lipoxygenase metabolites (142).

Conjugated linoleic acid
Evidence supports an effect of CLA on gene expression in tumor cells, because CLA was demonstrated to influence the expression of genes of the cycle (described in Effects on cell replication), thereby regulating cell growth and differentiation (78). Recently, a mixture of CLA isomers was reported to regulate the expression of major oncogenes involved in cell survival and programmed cell death signaling in human mammary cell lines [MCF-7, MDA-MB-231, and MCF-10a cells (75)]. Isomers of CLA have moderate affinity, compared with n–3 fatty acids, for PPARs [reviewed by Belury (78)]. Evidence is accumulating that activators of PPARs are protective against cancers arising in the mammary gland, colon, and prostate (143). It was suggested that CLA could both change the level and alter the activation of several PPARs (78). Although there is not a great deal of experimental support at the present time for CLA modulation of PPARs on immune metabolism, data do suggest that the anti-inflammatory effects of CLA in a macrophage cell line are mediated, at least in part, through changes in PPAR expression (99).

Effect of fatty acids on development of the immune system
Adult immune defenses develop during the first few years of life (144) and are influenced to some extent by the intake of polyunsaturated fats (108, 144). Epidemiologic data suggest that diet, particularly lipids, early in life influences cancer incidence (145, 146). To our knowledge no work is aimed at determining the effects of feeding CLA on immune development. Exciting data suggest that feeding CLA early in life alters the development of the mammary gland in rodents (147). Feeding CLA during mammary gland development in rats resulted in diminished mammary epithelial branching, possibly contributing to the reduction in mammary cancer risk in these rats (45). Thus, in rats, optimal CLA nutrition during pubescence could conceivably control the population of cancer-sensitive target sites in the mammary gland.

CONCLUSIONS AND REMAINING QUESTIONS

Considerable work has been done to demonstrate the potential importance of CLA as an anticancer treatment. There are many clues as to how this molecule might mediate its effects on the tumor and immune system. Many of these effects parallel our current understanding of the anticancer effects of long-chain polyunsaturated n–3 fatty acids. Despite the growth in research in this area, many questions remain (Table 2). Answers to these questions are required to provide the rationale to move CLA and cancer research from the culture plate and animal model to human trials.

View this table:
TABLE 2. Remaining questions on the potential mechanisms for the effect of conjugated linoleic acid (CLA) on tumor metabolism and immune function¹

ACKNOWLEDGMENTS

The authors had no conflict of interest with the financial sponsor or the organization sponsoring or supporting the writing of this review article.

REFERENCES

1. Bartsch H, Nair J, Owen RW. Dietary polyunsaturated fatty acids and cancers of the breast and colorectum: emerging evidence for their role as risk modifiers. Carcinogenesis1999;20:2209–18.
2. Rose DP, Connolly JM. Omega-3 fatty acids as cancer chemopreventive agents. Pharmacol Ther1999;83:217–44.
3. Hardman WE. Omega-3 fatty acids to augment cancer therapy. J Nutr2002;132:3508S–12S.
4. Ha YL, Storkson J, Pariza MW. Inhibition of benzo(a)pyrene-induced mouse forestomach neoplasia by conjugated dienoic derivatives of linoleic acid. Cancer Res1990;50:1097–101.
5. Ip C, Chin SF, Scimeca JA, et al. Mammary cancer prevention by conjugated dienoic derivative of linoleic acid. Cancer Res1991;51:6118–24.
6. Liew C, Schut HA, Chin SF, et al. Protection of conjugated linoleic acids against 2-amino-3-methylimidazo[4,5-f]quinoline-induced colon carcinogenesis in the F344 rat: a study of inhibitory mechanisms. Carcinogenesis1995;16:3037–43.
7. Belury MA, Nickel KP, Bird CE, et al. Dietary conjugated linoleic acid modulation of phorbol ester skin tumor promotion. Nutr Cancer1996;26:149–57.
8. Futakuchi M, Cheng JL, Hirose M, et al. Inhibition of conjugated fatty acids derived from safflower or perilla oil of induction and development of mammary tumors in rats induced by 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP). Cancer Lett2002;178:131–9.
9. Visonneau S, Cesano A, Tepper SA, et al. Conjugated linoleic acid suppresses the growth of human breast adenocarcinoma cells in SCID mice. Anticancer Res1997;17:969–73.
10. Cesano A, Visonneau S, Scimeca JA, et al. Opposite effects of linoleic acid and conjugated linoleic acid on human prostatic cancer in SCID mice. Anticancer Res1998;18:1429–34.
11. de Deckere EAM. Possible beneficial effect of fish and fish n–3 polyunsaturated fatty acids in beast and colorectal cancer. Eur J Cancer Prev1999;8:213–21.
12. Aro A, Mannisto S, Salminen I, et al. Inverse association between dietary and serum conjugated linoleic acid and risk of breast cancer in postmenopausal women. Nutr Cancer2000;38:151–7.
13. Voorrips LE, Brants HA, Kardinaal AF, et al. Intake of conjugated linoleic acid, fat, and other fatty acids in relation to postmenopausal breast cancer: the Netherlands Cohort Study on Diet and Cancer. Am J Clin Nutr2002;76:873–82.
14. Robinson LE, Clandinin MT, Field CJ. R3230AC rat mammary tumor and dietary long-chain (n–3) fatty acids change immune cell composition and function during mitogen activation. J Nutr2001;131:2021–7.
15. Karmali RA, Terano T, Cohen LA, et al. The effects of diteary -3 fatty acids on the DU-145 transplantable human prostatic tumor. Anticancer Res1987;7:1173–80.
16. Calviello G, Palozza P, Piccioni E, et al. Dietary supplementation with eicosapentaenoic and docosahexaenoic acid inhibits growth of Morris hepatocarcinoma 3924A in rats: effects on proliferation and apoptosis. Int J Cancer1998;75:699–705.
17. O’Connor TP, Roebuck BD, Peterson FJ, et al. Effect of dietary omega-3 and omega-6 fatty acids on development of azaserine-induced preneoplastic lesions in rat pancreas. J Natl Cancer Inst1989;81:858–63.
18. Rose DP, Connolly JM. Effects of fatty acids and inhibitors of eicosanoid synthesis on the growth of a human breast cancer cell line in culture. Cancer Res1990;50:7139–44.
19. Clarke RG, Lund EK, Latham P, et al. Effect of eicosapentaenoic acid on the proliferation and incidence of apoptosis in the colorectal cell line HT29. Lipids1999;34:1287–95.
20. Hawkins RA, Sangster K, Arends MJ. Apoptotic death of pancreatic cancer cells induced by polyunsaturated fatty acids varies with double bond number and involves an oxidative mechanism. J Pathol1998;185:61–70.
21. Chiu LCM, Ooi VEC, Wan JMF. Eicosapentaenoic acid modulates cyclin expression and arrests cell cycle progression in human leukemic K-562 cells. Int J Oncol2001;19:845–9.
22. Albino AP, Juan G, Traganos F, et al. Cell cycle arrest and apoptosis of melanoma cells by docosahexaenoic acid: association with decreased pRb phosphorylation. Cancer Res2000;60:4139–45.
23. Belury MA. Inhibition of carcinogenesis by conjugated linoleic acid: potential mechanisms of action. J Nutr2002;132:2995–8.
24. Ip C, Briggs SP, Haegele AD, et al. The efficacy of conjugated linoleic acid in mammary cancer prevention is independent of the level or type of fat in the diet. Carcinogenesis1996;17:1045–50.
25. Yang H, Holcroft J, Glickman BW, et al. Conjugated linoleic acid inhibits mutagenesis by 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine in the prostate of Big Blue (R) rats. Mutagenesis2003;18:195–200.
26. Ealey KN, El Sohemy A, Archer MC. Conjugated linoleic acid does not inhibit development of aberrant crypt foci in colons of male Sprague-Dawley rats. Nutr Cancer2001;41:104–6.
27. Kilian M, Mautsch I, Gregor JI, et al. Influence of conjugated vs. conventional linoleic acid on liver metastasis and hepatic lipidperoxidation in BOP-induced pancreatic cancer in Syrian hamster. Prostaglandins Leukot Essent Fatty Acids2002;67:223–8.
28. Petrik MBH, McEntee MF, Johnson BT, et al. Highly unsaturated (n–3) fatty acids, but not -linolenic, conjugated linoleic or -linolenic acids, reduce tumorigenesis in Apc(Min/+) mice. J Nutr2000;130:2434–43.
29. Wong MW, Chew BP, Wong TS, et al. Effects of dietary conjugated linoleic acid on lymphocyte function and growth of mammary tumors in mice. Anticancer Res1997;17:987–93.
30. Cohen LA, Zhao Z, Pittman B, et al. Effect of soy protein isolate and conjugated linoleic acid on the growth of Dunning R-3327-AT-1 rat prostate tumors. Prostate2003;54:169–80.
31. Rajakangas J, Basu S, Salminen I, et al. Adenoma growth stimulation by the trans-10, cis-12 isomer of conjugated linoleic acid (CLA) is associated with changes in mucosal NF-kappaB and cyclin D1 protein levels in the Min mouse. J Nutr2003;133:1943–8.
32. Masso-Welch PA, Zangani D, Ip C, et al. Inhibition of angiogenesis by the cancer chemopreventive agent conjugated linoleic acid. Cancer Res2002;62:4383–9.
33. Ma DW, Field CJ, Clandinin MT. An enriched mixture of trans-10,cis-12-CLA inhibits linoleic acid metabolism and PGE2 synthesis in MDA-MB-231 cells. Nutr Cancer2002;44:203–12.
34. Ip C, Dong Y, Ip MM, et al. Conjugated linoleic acid isomers and mammary cancer prevention. Nutr Cancer2002;43:52–8.
35. Hubbard NE, Lim D, Erickson KL. Effect of separate conjugated linoleic acid isomers on murine mammary tumorigenesis. Cancer Lett2003;190:13–9.
36. Thompson H, Zhu Z, Banni S, et al. Morphological and biochemical status of the mammary gland as influenced by conjugated linoleic acid: implication for a reduction in mammary cancer risk. Cancer Res1997;57:5067–72.
37. Hubbard NE, Lim D, Summers L, et al. Reduction of murine mammary tumor metastasis by conjugated linoleic acid. Cancer Lett2000;150:93–100.
38. Leake R. The cell cycle and regulation of cancer cell growth. Ann N Y Acad Sci1996;784:252–62.
39. Hanahan D, Weinberg RA. The hallmarks of cancer. Cell2000;100:57–70.
40. Dictor M, Ehinger M, Mertens F, et al. Abnormal cell cycle regulation in malignancy. Am J Pathol1999;112:S40–S52.
41. Kachhap SK, Dange PP, Santani RH, et al. Effect of ù-3 fatty acid (docosahexaenoic acid) on BRCA1 gene expression and growth in MCF-7 cell line. Cancer Biother Radiochem2001;16:257–63.
42. Lai PBS, Ross JA, Fearon KCH, et al. Cell cycle arrest and induction of apoptosis in pancreatic cancer cells exposed to eicosapentaenoic acid in vitro. Br J Cancer1996;74:1375–83.
43. Istfan NW, Wan J, Chen Z-Y. Fish oil and cell proliferation kinetics in a mammary carcinoma tumor model. Adv Exp Med Biol1995;375:149–56.
44. Liu JR, Chen BQ, Yang YM, et al. Effect of apoptosis on gastric adenocarcinoma cell line SGC-7901 induced by cis-9, trans-11-conjugated linoleic acid. World J Gastroenterol2002;8:999–1004.
45. Ip C, Dong Y, Thompson HJ, et al. Control of rat mammary epithelium proliferation by conjugated linoleic acid. Nutr Cancer2001;39:233–8.
46. Johnson DG, Walker CL. Cyclins and cell cycle checkpoints. Annu Rev Pharmacol Toxicol1999;39:295–312.
47. Schafer KA. The cell cycle: a review. Vet Pathol1998;35:461–78.
48. Sun Y. Free radicals, antioxidant enzymes, and carcinogenesis. Free Radic Biol Med1990;8:583–99.
49. Gonzalez MJ, Schemmel RA, Dugan L Jr, et al. Dietary fish oil inhibits human breast carcinoma growth: a function of increased lipid peroxidation. Lipids1993;28:827–32.
50. Begin ME, Ells G, Horrobin DF. Polyunsaturated fatty acid-induced cytotoxicity against tumor cells and its relationship to lipid peroxidation. J Natl Cancer Inst1988;80:188–94.
51. Chajes V, Sattler W, Stranzl A, et al. Influence of n–3 fatty acids on the growth of human breast cancer cells in vitro: relationship to peroxides and vitamin-E. Breast Cancer Res Treat1995;34:199–212.
52. Cognault S, Jourdan ML, Germain E, et al. Effect of an -linolenic acid-rich diet on rat mammary tumor growth depends on the dietary oxidative status. Nutr Cancer2000;36:33–41.
53. Diggle CP. In vitro studies on the relationship between polyunsaturated fatty acids and cancer: tumour or tissue specific effects? Prog Lipid Res2002;41:240–53.
54. Devery R, Miller A, Stanton C. Conjugated linoleic acid and oxidative behaviour in cancer cells. Biochem Soc Trans2001;29:341–4.
55. O’Shea M, Stanton C, Devery R. Antioxidant enzyme defence responses of human MCF-7 and SW480 cancer cells to conjugated linoleic acid. Anticancer Res1999;19:1953–9.
56. Schonberg S, Krokan HE. The inhibitory effect of conjugated dienoic derivatives (CLA) of linoleic acid on the growth of human tumor cell lines is in part due to increased lipid peroxidation. Anticancer Res1995;15:1241–6.
57. Igarashi M, Miyazawa T. The growth inhibitory effect of conjugated linoleic acid on a human hepatoma cell line, HepG2, is induced by a change in fatty acid metabolism, but not the facilitation of lipid peroxidation in the cells. Biochim Biophys Acta2001;1530:162–71.
58. Miller A, Stanton C, Devery R. Modulation of arachidonic acid distribution by conjugated linoleic acid isomers and linoleic acid in MCF-7 and SW480 cancer cells. Lipids2001;36:1161–8.
59. O’Shea M, Devery R, Lawless F, et al. Milk fat conjugated linoleic acid (CLA) inhibits growth of human mammary MCF-7 cancer cells. Anticancer Res2000;20:3591–601.
60. Basu S, Smedman A, Vessby B. Conjugated linoleic acid induces lipid peroxidation in humans. FEBS Lett2000;468:33–6.
61. Banni S, Angioni E, Casu V, et al. Decrease in linoleic acid metabolites as a potential mechanism in cancer risk reduction by conjugated linoleic acid. Carcinogenesis1999;20:1019–24.
62. Connolly JM, Gilhooly EM, Rose DP. Effects of reduced dietary linoleic acid intake, alone or combined with an algal source of docosahexaenoic acid, on MDA-MB-231 breast cancer cell growth and apoptosis in nude mice. Nutr Cancer1999;35:44–9.
63. Senzaki H, Iwamoto S, Ogura E, et al. Dietary effects of fatty acids on growth and metastasis of KPL-1 human breast cancer cells in vivo and in vitro. Anticancer Res1998;18:1621–8.
64. Latham P, Lund EK, Brown JC, et al. Effects of cellular redox balance on induction of apoptosis by eicosapentaenoic acid in HT29 colorectal adenocarcinoma cells and rat colon in vivo. Gut2001;49:97–105.
65. Calviello G, Palozza P, Maggiano N, et al. Cell proliferation, differentiation, and apoptosis are modified by n–3 polyunsaturated fatty acids in normal colonic mucosa. Lipids1999;34:599–604.
66. Yamamoto D, Kiyozuka Y, Adachi Y, et al. Synergistic action of apoptosis induced by eicosapentaenoic acid and TNP-470 on human breast cancer cells. Breast Cancer Res Treat1999;55:149–60.
67. Narayanan BA, Narayanan NK, Reddy BS. Docosahexaenoic acid regulated genes and transcription factors inducing apoptosis in human colon cancer cells. Int J Oncol2001;19:1255–62.
68. Chen ZY, Istfan NW. Docosahexaenoic acid is a potent inducer of apoptosis in HT-29 colon cancer cells. Prostaglandins Leukot Essent Fatty Acids2000;63:301–8.
69. Heimli H, Finstad HS, Drevon CA. Necrosis and apoptosis in lymphoma cell lines exposed to eicosapentaenoic acid and antioxidants. Lipids2001;36:613–21.
70. Chiu LC, Wan JM. Induction of apoptosis in HL-60 cells by eicosapentaenoic acid (EPA) is associated with downregulation of bcl-2 expression. Cancer Lett1999;145:17–27.
71. Chiu LC, Wan JM, Ooi VE. Induction of apoptosis by dietary polyunsaturated fatty acids in human leukemic cells is not associated with DNA fragmentation. Int J Oncol2000;17:789–96.
72. Ip C, Ip MM, Loftus T, et al. Induction of apoptosis by conjugated linoleic acid in cultured mammary tumor cells and premalignant lesions of the rat mammary gland. Cancer Epidemiol Biomarkers Prev2000;9:689–96.
73. Park HS, Ryu JH, Ha YL, et al. Dietary conjugated linoleic acid (CLA) induces apoptosis of colonic mucosa in 1,2-dimethylhydrazine-treated rats: a possible mechanism of the anticarcinogenic effect by CLA. Br J Nutr2001;86:549–55.
74. Tsuboyama-Kasaoka N, Takahashi M, Tanemura K, et al. Conjugated linoleic acid supplementation reduces adipose tissue by apoptosis and develops lipodystrophy in mice. Diabetes2000;49:1534–42.
75. Majumder B, Wahle KW, Moir S, et al. Conjugated linoleic acids (CLAs) regulate the expression of key apoptotic genes in human breast cancer cells. FASEB J2002;16:1447–9.
76. Cho HJ, Kim WK, Kim EJ, et al. Conjugated linoleic acid inhibits cell proliferation and ErbB3 signaling in the HT-29 human colon cell line. Am J Physiol Gastrointest Liver Physiol2003;284:G996–G1005.
77. Chen BQ, Xue YB, Liu JR, et al. Inhibition of conjugated linoleic acid on mouse forestomach neoplasia induced by benzo (a) pyrene and chemopreventive mechanisms. World J Gastroenterol2003;9:44–9.
78. Belury MA. Dietary conjugated linoleic acid in health: physiological effects and mechanisms of action. Annu Rev Nutr2002;22:505–31.
79. McCarty MF. Activation of PPARgamma may mediate a portion of the anticancer activity of conjugated linoleic acid. Med Hypotheses2000;55:187–8.
80. Foss FM. Immunologic mechanisms of antitumor activity. Semin Oncol2002;29:5–11.
81. Shewchuk LD, Baracos VE, Field CJ. Reduced splenocyte metabolism and immune function in rats implanted with the Morris Hepatoma 7777. Metab1996;45:848–55.
82. Salih HR, Nussler V. Commentary: immune escape versus tumor tolerance: how do tumors evade immune surveillance? Eur J Med Res2001;6:323–32.
83. Yaqoob P. Lipids and the immune response: from molecular mechanisms to clinical applications. Curr Opin Clin Nutr Metab Care2003;6:133–50.
84. Robinson LE, Clandinin MT, Field CJ. The role of dietary long-chain n–3 fatty acids in anti-cancer immune defense and R3230AC mammary tumor growth in rats: influence of diet fat composition. Breast Cancer Res Treat2002;73:145–60.
85. Sebedio JL, Gnaedig S, Chardigny JM. Recent advances in conjugated linoleic acid research. Curr Opin Clin Nutr Metab Care1999;2:499–506.
86. Cook ME, Miller CC, Park Y, et al. Immune modulation by altered nutrient metabolism: nutritional control of immune-induced growth depression. Poult Sci1993;72:1301–5.
87. Miller CC, Park Y, Pariza MW, et al. Feeding conjugated linoleic acid to animals partially overcomes catabolic responses due to endotoxin injection. Biochem Biophys Res Commun1994;198:1107–12.
88. Hontecillas R, Wannemeulher MJ, Zimmerman DR, et al. Nutritional regulation of porcine bacterial-induced colitis by conjugated linoleic acid. J Nutr2002;132:2019–27.
89. Whigham LD, Cook EB, Stahl JL, et al. CLA reduces antigen-induced histamine and PGE2 release from sensitized guinea pig tracheae. Am J Physiol Regul Integr Comp Physiol2001;280:R908–12.
90. Whigham LD, Higbee A, Bjorling DE, et al. Decreased antigen-induced eicosanoid release in conjugated linoleic acid-fed guinea pigs. Am J Physiol Reg Integr Comp Physiol2002;282:R1104–12.
91. Turnock L, Cook M, Steinberg H, et al. Dietary supplementation with conjugated linoleic acid does not alter the resistance of mice to Listeria monocytogenes infection. Lipids2001;36:135–8.
92. Pariza MW, Park Y, Cook ME. The biologically active isomers of conjugated linoleic acid. Prog Lipid Res2001;40:283–98.
93. Hayek MG, Han SN, Wu D, et al. Dietary conjugated linoleic acid influences the immune response of young and old C57BL/6NCrlBR mice. J Nutr1999;129:32–8.
94. Kelley DS, Warren JM, Simon VA, et al. Similar effects of c9,t11-CLA and t10,c12-CLA on immune cell functions in mice. Lipids2002;37:725–8.
95. Yamasaki M, Chujo H, Hirao A, et al. Immunoglobulin and cytokine production from spleen lymphocytes is modulated in C57BL/6J mice by dietary cis-9, trans-11 and trans-10, cis-12 conjugated linoleic acid. J Nutr2003;133:784–8.
96. Bassaganya-Riera J, Hontecillas R, Zimmerman DR, et al. Long-term influence of lipid nutrition on the induction of CD8+ responses to viral or bacterial antigens. Vaccine2002;20:1435–44.
97. Bassaganya-Riera J, Hontecillas-Magarzo R, Bregendahl K, et al. Effects of dietary conjugated linoleic acid in nursery pigs of dirty and clean environments on growth, empty body composition, and immune competence. J Anim Sci2001;79:714–21.
98. Yamasaki M, Kishihara K, Mansho K, et al. Dietary conjugated linoleic acid increases immunoglobulin productivity of Sprague-Dawley rat spleen lymphocytes. Biosci Biotech Biochem2000;64:2159–64.
99. Yu Y, Correll PH, Vanden Heuvel JP. Conjugated linoleic acid decreases production of pro-inflammatory products in macrophages: evidence for a PPAR-dependent mechanism. Biochim Biophys Acta2002;1581:89–99.
100. Albers R, Van Der Wielen RP, Brink EJ, et al. Effects of cis-9, trans-11 and trans-10, cis-12 conjugated linoleic acid (CLA) isomers on immune function in healthy men. Eur J Clin Nutr2003;57:595–603.
101. Kelley DS, Taylor PC, Rudolph IL, et al. Dietary conjugated linoleic acid did not alter immune status in young healthy women. Lipids2000;35:1065–71.
102. Hwang D. Fatty acids and immune responses-a new perspective in searching for clues to mechanism. Annu Rev Nutr2000;20:431–56.
103. Miles EA, Calder PC. Modulation of immune function by dietary fatty acids. Proc Nutr Soc1998;57:277–92.
104. Yaqoob P. Lipids and the immune response. Curr Opin Clin Nutr Metab Care1998;1:153–61.
105. Resch K, Gelfand EW, Hansen K, et al. Lymphocyte activation: rapid changes in the phospholipid metabolism of plasma membranes during stimulation. Eur J Immunol1972;2:598–601.
106. Campanella R. Membrane lipids modifications in human gliomas of different degree of malignancy. J Neurosurg Sci1992;36:11–25.
107. Peterson LD, Jeffery NM, Thies F, et al. Eicosapentaenoic and docosahexaenoic acids alter rat spleen leukocyte fatty acid composition and prostaglandin E₂ production but have different effects on lymphocyte functions and cell-mediated immunity. Lipids1998;33:171–80.
108. Field CJ, Thomson CA, Van Aerde JE, et al. Lower proportion of CD45R0+ cells and deficient interleukin-10 production by formula-fed infants, compared with human-fed, is corrected with supplementation of long-chain polyunsaturated fatty acids. J Pediatr Gastroenterol Nutr2000;31:291–9.
109. Noguchi M, Minami M, Yagasaki R, et al. Chemoprevention of DMBA-induced mammary carcinogenesis in rats by low-dose EPA and DHA. Br J Cancer1997;75:348–53.
110. Rose DP, Connolly JM, Rayburn J, et al. Influence of diets containing eicosapentaenoic or docosahexaenoic acid on growth and metastasis of breast cancer cell in nude mice. J Natl Cancer Inst1995;87:587–92.
111. Karmali RA, Marsh J, Fuchs C. Effect of omega-3 fatty acids on growth of a rat mammary tumor. J Natl Cancer Inst1984;73:457–61.
112. Kramer JKG, Sehat N, Dugan MER, et al. Distributions of conjugated linoleic acid (CLA) isomers in tissue lipid classes of pigs fed a commercial CLA mixture determined by gas chromatography and silver ion-high-performance liquid chromatography. Lipids1998;33:549–58.
113. Simons K, Toomre D. Lipid rafts and signal transduction. Nat Rev Mol Cell Biol2000;1:31–9.
114. Brown DA, London E. Functions of lipid rafts in biological membranes. Annu Rev Cell Dev Biol1998;14:111–36.
115. Stulnig TM, Huber J, Leitinger N, et al. Polyunsaturated eicosapentaenoic acid displaces proteins from membrane rafts by altering raft lipid composition. J Biol Chem2001;276:37335–40.
116. Fan YY, McMurray DN, Ly LH, et al. Dietary (n–3) polyunsaturated fatty acids remodel mouse T-cell lipid rafts. J Nutr2003;133:1913–20.
117. Stubbs CD, Smith AD. The modification of mammalian membrane polyunsaturated fatty acid composition in relation to membrane fluidity and function. Biochim Biophys Acta1984;779:89–137.
118. Grimble RF, Tappia PS. Modulatory influence of unsaturated fatty acids on the biology of tumour necrosis factor-. Biochem Soc Trans1995;23:282–7.
119. Jolly CA, Jiang Y-H, Chapkin RS, et al. Dietary (n–3) polyunsaturated fatty acids suppress murine lymphoproliferation, interleukin-2 secretion, and the formation of diacylglycerol and ceramide. J Nutr1997;127:37–43.
120. de Pablo MA, Alvarez DC. Modulatory effects of dietary lipids on immune system functions. Immunol Cell Biol2000;78:31–9.
121. Sanderson P, Thies F, Calder PC. Extracellular release of free fatty acids by rat T lymphocytes is stimulus-dependent and is affected by dietary lipid manipulation. Cell Biochem Funct2000;18:47–58.
122. Bandyopadhyay GK, Imagawa W, Wallace D, et al. Linoleate metabolites enhance the in vitro proliferative response of mouse mammary epithelial cells to epidermal growth factor. J Biol Chem1987;262:2750–6.
123. Hughes DA, Pinder AC. n–3 polyunsaturated fatty acids inhibit the antigen-presenting function of human monocytes. Am J Clin Nutr2000;71:357S–60S.
124. Robinson LE, Field CJ. Dietary long-chain (n–3) fatty acids facilitate immune cell activation in sedentary, but not exercise-trained rats. J Nutr1998;128:498–504.
125. Bonin A, Khan NA. Regulation of calcium signalling by docosahexaenoic acid in human T-cells. Implication of CRAC channels. J Lipid Res2000;41:277–84.
126. Denys A, Hichami A, Khan NA. Eicosapentaenoic acid and docosahexaenoic acid modulate MAP kinase (ERK1/ERK2) signaling in human T cells. J Lipid Res2001;42:2015–20.
127. Stillwell W, Ehringer W, Jenski LJ. Docosahexaenoic acid increases permeability of lipid vesicles and tumor cells. Lipids1993;28:103–8.
128. Hashimoto M, Hossain MS, Yamasaki H, et al. Effect of eicosapentaenoic acid and docosahexaenoic acid on plasma membrane fluidity of aortic endothelial cells. Lipids1999;34:1297–304.
129. Lund EK, Harvey LJ, Ladha S, et al. Effects of dietary fish oil supplementation on the phospholipid composition and fluidity of cell membranes from human volunteers. Ann Nutr Metab1999;43:290–300.
130. Terano T, Shiina T, Tamura Y. Eicosapentaenoic acid suppressed the proliferation of vascular smooth muscle cells through modulation of various steps of growth signals. Lipids1996;31:S301–4.
131. Choi Y, Park Y, Storkson JM, et al. Inhibition of stearoyl-CoA desaturase activity by the cis-9,trans-11 isomer and the trans-10,cis-12 isomer of conjugated linoleic acid in MDA-MB-231 and MCF-7 human breast cancer cells. Biochem Biophys Res Commun2002;294:785–90.
132. Kavanaugh CJ, Liu KL, Belury MA. Effect of dietary conjugated linoleic acid on phorbol ester-induced PGE2 production and hyperplasia in mouse epidermis. Nutr Cancer1999;33:132–8.
133. Bulgarella JA, Patton D, Bull AW. Modulation of prostaglandin H synthase activity by conjugated linoleic acid (CLA) and specific CLA isomers. Lipids2001;36:407–12.
134. Li Y, Watkins BA. Conjugated linoleic acids alter bone fatty acid composition and reduce ex vivo prostaglandin biosynthesis in rats fed n–6 or n–3 fatty acids. Lipids1998;33:417–25.
135. Sugano M, Tsujita A, Yamasaki M, et al. Conjugated linoleic acid modulates tissue levels of chemical mediators and immunoglobulins in rats. Lipids1998;33:521–7.
136. Clarke SD, Gasperikova D, Nelson C, et al. Fatty acid regulation of gene expression: a genomic explanation for the benefits of the mediterranean diet. Ann N Y Acad Sci2002;967:283–98.
137. Jump DB. The biochemistry of n–3 polyunsaturated fatty acids. J Biol Chem2002;277:8755–8.
138. Price PT, Nelson CM, Clarke SD. Omega-3 polyunsaturated fatty acid regulation of gene expression. Curr Opin Lipidol2000;11:3–7.
139. Kliewer SA, Willson TM. The nuclear receptor PPAR - bigger than fat. Curr Opin Genet Dev1998;8:576–81.
140. Berger J, Moller DE. The mechanisms of action of PPARs. Annu Rev Med2002;53:409–35.
141. Krey G, Braissant O, L’Horset F, et al. Fatty acids, eicosanoids, and hypolipidemic agents identified as ligands of peroxisome proliferator-activated receptors by co-activator-dependent receptor ligand assay. Mol Endocrinol1997;11:779–91.
142. Kliewer SA, Lehmann JM, Willson TM. Orphan nuclear receptors: shifting endocrinology into reverse. Science1999;284:757–60.
143. Sporn MB, Suh N, Mangelsdorf DJ. Prospects for prevention and treatment of cancer with selective PPARgamma modulators (SPARMs). Trends Mol Med2001;7:395–400.
144. Field CJ, Clandinin MT, Van Aerde JE. Polyunsaturated fatty acids and T-cell function: implications for the neonate. Lipids2001;36:1025–32.
145. Terry PD, Rohan TE, Wolk A. Intakes of fish and marine fatty acids and the risks of cancers of the breast and prostate and of other hormone-related cancers: a review of the epidemiologic evidence. Am J Clin Nutr2003;77:532–43.
146. Kushi L, Giovannucci E. Dietary fat and cancer. Am J Med2002;113(suppl 9B):63S–70S.
147. Ip C, Singh M, Thompson HJ, et al. Conjugated linoleic acid suppresses mammary carcinogenesis and proliferative activity of the mammary gland in the rat. Cancer Res1994;54:1212–5.