Article pp.44-52 du Vol.28 n°1-2 (2008)

(1)

Milk lipids: their role as potential anti-cancer agents

P. W. Parodi

RÉSUMÉ

Lipides du lait : un effet anti-carcinogène potentiel

Certains composés de la matière grasse laitière ont montré des propriétés potentielle- ment bénéfiques sur des modèles animaux de cancers chimio-induits. L’acide rumé- nique et son précurseur l’acide vaccénique sont de puissants inhibiteurs des tumeurs mammaires. L’acide butyrique, Inhibiteur de l’histone déacétylase, présent dans la matière grasse laitière, inhibe le développement des tumeurs mammaires. De nou- veaux arguments suggèrent que les acides gras branchés iso et anteiso ont un effet anti-carcinogène potentiel. La sphyngomyéline et d’autres sphyngolipides du lait inhi- bent également le développement tumoral dans différents modèles murins. Des étu- des in vitro et in vivo montrent que la co-administration de sphyngomyéline potentialise l’effet des chimiothérapies sur l’apoptose.

Mots clés

matière grasse laitière, acide linoléique conjugué, acide butyrique, acides gras bran- chés, sphingolipides.

SUMMARY

A number of components in milk fat have demonstrated anti-cancer potential in animal models of carcinogenesis. Dietary rumenic acid and its precursor vaccenic acid are potent inhibitors of mammary tumorigenesis. The histone deacetylase inhibitor, butyric acid, which is uniquely present in milk fat, also inhibits mammary tumour development. There is emerging evidence that the monomethyl iso- and anteiso- branched-chain fatty acids in milk fat have anti-cancer potential. Sphingomyelin and other sphingolipids of milk inhibit colon tumour development in several rodent models. In vitro and in vivo studies show co-administration of sphingomyelin with chemotherapy drugs potentate ceramide-induced apoptotic cell death.

Keywords

milk fat, cancer, conjugated linoleic acid, butyric acid, branched-chain fatty acids, sphingolipids.

Human Nutrition and Health Research, Dairy Australia, Melbourne, Victoria.

Correspondence : Dr Peter W Parodi – 9 Hanbury St. – Chermside, 4032 – Queensland – Australia – Email: peterparodi@uq.net.au

(2)

1 – INTRODUCTION

Diet is believed to play an important role in the etiology of cancer at a number of major sites. Fruit, vegetables and cereal products are considered beneficial because of their content of antioxidants, fibre and a number of other putative anti-cancer compounds. On the other hand, dietary fat, which has received the most attention in animal and epidemiological studies, is considered a causative factor. However, not all fats are similar in composition and milk fat contains a number of components with potential anti- cancer activity. The major components are briefly outlined in this review, greater detail can be found in reviews by Parodi, 1999, 2004, 2006; Ip et al., 2003; Schmelz, 2004; Bau- man et al., 2006.

2 – DIETARY FAT AND THE RISK OF CANCER

Initially it is appropriate to consider the association between total fat consumption and the risk of the most prevalent non-smoking related cancers of the colon, breast and prostate. The belief that fat intake is associated with cancer is a result of early interna- tional comparison (ecological) studies that showed strong correlations between assumed per capita daily fat intake and cancer mortality rates in a large number of countries. How- ever, this epidemiological format has many shortcomings, it tells nothing about the diet of individuals who develop cancer and those who do not. There is also substantial confounding by many lifestyle and cultural factors that vary from country to country and can influence malignancy. For these reasons ecological studies cannot provide reliable infor- mation on the role of diet in cancer development.

Early experimental studies with animals suggested that a high fat diet increased the incidence of chemically induced tumours. However, it was shown later that in studies where the effect of calorie intake was separated from the effect of fat content, the fat content did not significantly influence tumour development. Thus it is essential in epidemiological studies to adequately adjust for energy intake and related confounding such as weight, BMI and physical activity. Results from case-control studies, which have the potential for recall and selection bias, provide inconsistent evidence for an association between fat intake and risk of cancer. On the other hand, evidence from the more rigor- ously conducted prospective (cohort) studies and pooled analyses of these studies do not support an association between fat intake and the risk of colon, breast and prostate cancer (Willett, 2001; Kushi and Giovannucci, 2002; Parodi, 2004, 2005, 2006).

There is no convincing evidence from epidemiological studies that any individual fatty acid is implicated in cancer at the major sites. However, this format cannot preclude the possibility that some of the multiple fatty acids present in the diet are exerting opposing effects (Parodi, 2006).

Epidemiological associations cannot be used to ascribe causality. For this, well-conducted randomised control trials are required. The Women’s Health Initiative Dietary Modification Trial is the first randomised control trial to directly assess the health benefits and risks of promoting a low-fat dietary pattern. The goal for the intervention group was to reduce total fat intake to 20% of energy and increase consumption of fruit, vegetables and grains. After 8.1 years of follow-up the low-fat dietary pattern did not result in a sta- tistically significant reduction in invasive breast cancer (Prentice et al., 2006) or colorectal cancer (Beresford et al., 2006) compared to women who consumed a diet with less fruit, vegetables and grains and a fat intake that represented about 35% of energy.

(3)

3 – CONJUGATED LINOLEIC ACID

Conjugated linoleic acid (CLA) is the most important anti-cancer component in milk fat. Indeed, in 1996 the US National Academy of Sciences report on carcinogens and antioxidants in food pointed out “CLA is the only fatty acid shown unequivocally to inhibit carcinogenesis in experimental animals” (Bauman et al., 2006). CLA is the descriptor for all possible positional and geometric isomers of octadecadienoic acid with conjugated double bonds. Milk fat is the richest natural source of CLA with the cis-9, trans-11 isomer, now named rumenic acid (RA), representing about 90% of isomers present. The reported range of CLA in milk fat is 2 to 37 mg/g. The major contributor to this variation is diet with highest levels occurring when cows graze fresh lush pasture or with specific diets (Parodi, 2003).

Pariza and colleagues at the University of Wisconsin were the first to demonstrate the anti-cancer properties of CLA (Ha et al., 1987). Since then there have been multiple in vitro and in vivo studies demonstrating the anti-cancer effect of CLA at a number of sites.

Most of these studies, especially the early ones, used synthetic CLA that contained a number of isomers but usually the cis-9, trans-11- and trans-10, cis-12 isomer predomi- nated in a ratio of around 1:1. It is now known that although these two isomers may have similar biological properties in tissue from some organs, they may also exert dissimilar effects, and even when the outcome is the same the pathways determining the outcome can be different. For this reason it is essential to know what isomers are involved in any study with CLA. In this review CLA refers to mixed isomers, use of individual isomers will be indicated.

3.1 CLA and mammary tumourigenesis

CLA is a potent inhibitor of mammary tumour development. In a landmark study Ip et al., (1991) fed rats a basal diet or that diet supplemented with 0.5, 1.0 and 1.5% CLA two weeks prior to and following administration of the chemical carcinogen 7,12-dimethyl- benz[a]anthracene (DMBA). At the conclusion of the trial the total number of mammary adenocarcinomas in the rats fed 0.5, 1.0 and 1.5% CLA was reduced by 32, 56 and 60%, respectively, compared to rats fed the basal diet. Tumour incidence (% of rats with tumours), tumour multiplicity (number of tumours per rat) and total tumour weight were reduced similarly.

Subsequently, Ip and colleagues conducted a number of other studies that confirmed dietary CLA inhibited mammary tumourigenesis and provided insight into its mechanisms (Scimeca, 1999; Ip et al., 2003; Parodi, 2004, 2006). When the dose of carcinogen was halved, and the tumours took longer to develop, as little as 0.05% of dietary CLA inhibited mammary tumour development. CLA was equally effective with the direct acting carcinogen methylnitrosourea as with DMBA that requires metabolic activation. The amount and type of fat in the diet of rodents can influence tumourigenesis. However, tumour inhibition was similar when CLA was part of a low-fat (5%) or high-fat (20%) diet. Inhibition of tumour development was also similar when CLA was included in a 20% unsaturated-fat diet as corn oil or a 20% saturated-fat diet as lard. Although a diet containing 12% linoleic acid produced more mammary tumours than a 2% linoleic acid diet, CLA inclusion reduced tumour development to the same extent. CLA was preferentially incorporated into the neutral lipids of mammary tissue. When CLA was removed from the diet its dis- appearance from the neutral lipids paralleled the rate of appearance of new tumours. CLA can induce apoptosis or programmed cell death, the mechanism whereby damaged cells, which may later develop into tumours, are removed from the system.

The age of the rat at the time of CLA administration can influence outcome. When rats were fed CLA from weaning at 21 days of age until day 51 only, then administered a carcinogen at day 57, they were protected from subsequent tumour development. However, when CLA entered the diet for the same period of time after carcinogen administration

(4)

and when the animals were older, there was no protection against tumour development.

A continuous intake of CLA was then necessary to obtain equivalent protection. The period from age 21 to 51 days corresponds to development of the mammary gland to adult stage morphology. Further studies showed that during the pubescent period, CLA reduced the development and branching of the expanding mammary ductal tree. There was also a reduction in the density and rate of proliferation of terminal end bud (TEB) cells. TEBs are the least differentiated and most actively growing glandular ductal struc- tures, which are most abundant from weaning to puberty and are the site of chemically induced tumours (Ip et al., 2003). These observations could be important because there is evidence that in humans the risk of breast cancer associated with certain environmental events increased with decreasing age at which exposure occurred or commenced (Parodi, 2005).

Because of the difficulty in obtaining pure RA for experimental studies, techniques for the production of RA enhanced butter were developed. Using this product Ip et al., (1999) demonstrated that in the pubescent mammary gland RA reduced epithelial mass, decreased the size of the TEB population, suppressed the proliferation of TEB cells and decreased tumour incidence and the number of tumours to the same extent as an equivalent amount of synthetic RA. The rats fed butter-derived RA had higher levels of RA in the liver, mammary fat pad, peritoneal fat and blood than rats fed synthetic RA. This increase was due to endogenous Δ⁹-desaturation of vaccenic acid (VA) that was concom- itantly increased in the RA-enhanced butter. VA (trans-11-18:1) is the predominant trans monounsaturated fatty acid in milk fat and studies in animals and humans have demonstrated its bioconversion to RA (Palmquist, 2005).

Banni et al., (2001) showed that 2% dietary VA inhibited chemically induced premalignant lesions in the mammary gland to the same extent as 1% RA. Lock et al., (2005) demonstrated that the anti-tumour action of VA was blocked by a Δ⁹-desaturase inhibitor suggesting that the anti-tumorigenic action of VA is probably exclusively through its conversion to RA. However VA may still be considered an anti-cancer agent.

RA can beneficially modulate a number of important mechanisms involved in carcinogenesis. RA induced apoptosis in chemically induced preneoplastic mammary gland lesions that was associated with reduced expression of the anti-apoptotic regulatory protein bcl-2 (Ip et al., 2000). Masso-Welch et al., (2004) showed dietary RA inhibited angiogenesis in mouse mammary tissue and was associated with a reduction in vascular endothelial growth factor (VEGF). Angiogenesis is the process for generation of new blood capillaries through sprouting of pre-existing vessels and is an absolute requirement for tumour growth. Hubbard et al., (2003) found that dietary RA reduced pulmonary tumour metastasis in mice when mammary tumour cells were either transplanted into mammary fat pads or injected intravenously via the tail vein. Cyclooxygenase 2 (COX-2), one of its major products prostaglandin E₂(PGE₂) and a PGE₂receptor EP2 are over- expressed in breast cancer. Wang et al., (2006) showed that RA inhibited malignant canine mammary cell growth and suppressed COX-2 and EP2 expression.

The ErbB2 (HER-2/new) oncogene is over-expressed in about one-third of all breast cancers and is associated with poor clinical outcome. Recently, Ip et al., (2007) fed transgenic mice over-expressing ErbB2 a control diet or that diet supplemented with 0.5% of the trans-10, cis-12 isomer or RA from weaning. Trans-10, cis-12, but not RA, stimulated lobular hyperplasia of the mammary epithelium, accelerated mammary tumour development and decreased tumour latency. The percentage of tumour-bearing mice with lung metastasis was 31% for the control group, decreased to 14% for RA-fed mice, but increased to 73% for the trans-10, cis-12-fed mice. The trans-10, cis-12-isomer also increased proliferation in mammary epithelium of wild-type mice and increased the number of TEBs 30-fold. Ip et al., (2007) cautioned against the use of trans-10, cis-12 supplements in women. This study accentuates the necessity to specify the CLA isomer used in all experimental studies.

A few epidemiological studies have examined the association between RA intake and the risk of breast cancer. The initial case-control study (Aro et al., 2000) found that postmenopausal Finnish women in the lowest quintile of dietary RA had a 3-fold greater risk of breast cancer compared to women in the highest quintile of intake. Similarly women in

(5)

the lowest quintile of both serum RA and serum VA levels had a 5-fold greater risk of breast cancer. No advantage was noted for premenopausal women. A US study found that although RA was not related to overall breast cancer risk, the risk of estrogen receptor negative breast cancer in premenopausal women, which is the most aggressive form, was reduced with increasing RA intake (Mc Cann et al., 2004). Three other studies did not find an advantage for RA intake, but there were methodological problems with these studies and the range of intake was narrow (Parodi, 2004).

3.2 Cancer at other sites

Studies with RA at other sites are few. Chen et al., (2003) showed RA increased apoptosis and decreased tumour incidence in a chemically induced mouse forestomach model. Recently, Park et al., (2006) reported dietary RA decreased chemically induced total colon tumour numbers and the number of tumours per rat. RA treatment decreased COX-2 levels, increased the level of pro-apoptotic Bax protein, decreased anti-apoptotic Bcl-2 expression and apoptosis. Fifteen-year follow-up data from a prospective study with Swedish women showed that a high intake of RA and also a high intake of dairy fat were associated with a reduced incidence of colorectal cancer (Larsson et al., 2005).

4 – BUTYRIC ACID

Butyric acid (BA) is a potent anti-cancer agent that induces cell cycle arrest, differentiation and apoptosis in various cancer cell lines including colon, breast and prostate (Davie, 2003; Parodi, 2004). BA is classed as a histone deacetylase inhibitor. Acetylation of histone plays an important role in the regulation of gene expression. Deacetylation of histone causes compaction of chromatin that denies transcription factors access to DNA and results in gene silencing. Histone deacetylase inhibitors, on the other hand, allow acetylation of histone and chromatin is decondensed leading to transcription of genes whose expression causes inhibition of tumour cell growth, like p21Waf 1/Cip 1 and induction of apoptosis (Davie, 2003).Histone deacetylase inhibitors are emerging as a new class of potential anti-cancer agents for treatment of solid and haematological malignancies.

BA induces expression of p21Waf 1/Cip 1, which stops cells entering the S phase of the cell cycle so they may undergo differentiation or apoptosis (Davie, 2003). In addition to this inhibition of proliferation BA exerts other anti-cancer effects including induction of differentiation and apoptosis, inhibition of angiogenesis associated with down-regulation of VEGF, anti-inflammatory action and up-regulation of immunosurveillance. There is also enhanced expression of glutathione S-transferase that inactivates carcinogens and sup- pression of nuclear factor κB that regulates genes involved in control of cell proliferation, cell death and immune and inflammatory responses (Davie, 2003; Williams et al., 2003;

Parodi, 2006).

Use of BA in cancer therapy was unsuccessful initially because of a short half-life in the circulation. However, the later use of butyrate derivatives or prodrugs, like tributyrin, extended the half-life. In milk fat about one-third of all milk triglycerides contain one mol of BA (Parodi, 2004). It is interesting to note that BA acts synergistically with a number of common food components and common drugs, such as retinoic acid, 1α, 25-dihydroxy- vitamin D₃, the plant polyphenol resveratrol, HMG CoA reductase inhibitors like the statin drugs and aspirin to inhibit growth of cancer cells. This synergy can reduce the level of plasma BA required to produce an anti-cancer effect (Parodi, 2004, 2006).

Two studies demonstrated that dietary BA inhibited chemically induced mammary tumour development in rats. In the first study the addition of 6% sodium butyrate to a base diet containing 20% fat supplied as a safflower oil-based margarine significantly reduced the incidence of chemically induced mammary tumours (Yanagi et al., 1993). In

(6)

the second study, addition of tributyrin at a level of 1% (BA content equivalent to milk fat) or 3% to a sunflower oil-based diet reduced chemically induced tumour incidence by 20 and 52%, respectively. In this study a milk fat diet produced fewer tumours than a sunflower oil-based diet (Belobrajdic and McIntosh, 2000).

5 – BRANCHED-CHAIN FATTY ACIDS

Milk fat contains a range of monomethyl iso- and anteiso- branched- chain fatty acids (BCFAs) that originate from structural lipids of rumen bacteria. They represent up to 2.5%

of total fatty acids and have carbon chain-lengths of 13 to 22 (Parodi, 2004). Yang et al., (2000) reported that 13-methyltetradecanoic acid induced apoptotic cell death in a range of human cancer cells including colon, breast and prostate. 12-Methyltetradecanoic acid also inhibited proliferation in a range of human cancer cell lines via apoptosis (Yang et al., 2003). Human prostate and lung cancer cells grown in athymic mice were harvested and orthotopically implanted into the prostate and lung, respectively, of a nude mouse model.

Dietary supplementation with 13-methyltetradecanoic acid significantly inhibited subsequent prostate and lung tumour growth (Yang et al., 2000).

Wongtangtintharn et al., (2004) showed that a series of iso- and anteiso-BCFAs were cytotoxic to breast cancer cells. Highest activity was observed for iso-C 16:0 and activity decreased as the chain length increased or decreased from C16:0. It is interesting to note that the cytotoxicity of BCFA was comparable to that of RA. RA and to a lesser extent 13- methyltetradecanoic acid inhibited fatty acid synthase, which is over-expressed in cancer cells from a number of sites.

The multi-BCFAs, phytanic and pristanic acids, present in milk fat in small amounts activate peroxisome proliferator-activated receptors, and agonists for these receptors are known to inhibit chemically induced tumours in animals (Parodi, 2004). Hydroxy fatty acids, also present in small quantities, may exert anti-cancer properties (Abe and Sugiyama, 2005).

6 – SPHINGOLIPIDS

Sphingolipids are structural components of cell membranes and in milk they are mainly associated with the milk fat globule membrane. Milk sphingolipids, which are pre- dominantly sphingomyelin, are digested throughout the small intestine and colon to produce the bioactive metabolites ceramide and sphingosine. These metabolites are absorbed by intestinal cells and utilized for re-synthesis of sphingolipids (Parodi, 2004;

Schmelz, 2004; Duan, 2005).

Cell culture studies with a number of human cancer cells suggest ceramide and sphingosine can inhibit proliferation and induce differentiation and apoptosis (Parodi, 2004). It is now realised that ceramide plays an important role in carcinogenesis. Radia- tion therapy and several common cancer chemotherapy drugs act by induction of apoptotic cell death that results from activation of sphingomyelinase that hydrolyses sphingomyelin to generate ceramide. On the other hand, reduction of intracellular ceramide levels leads to drug resistance (Modrak et al., 2006).

Studies with human colon carcinoma tissue show that sphingomyelinase and ceramide levels are considerably less than in normal tissue (Parodi, 2006). Modrak et al., (2006) reviewed their studies that demonstrated sphingomyelin could enhance the cyto-

(7)

toxic effect of several chemotherapy drugs on cancer cells. In vitro, sphingomyelin co- administered with 5-flurouracil and with doxorubicin in both cases significantly increased the cytotoxicity of the drug alone in four of seven colon cancer cell lines. Similarly, a syn- ergistic interaction between sphingomyelin and gemcitabine in pancreatic cancer cells was related to ceramide-mediated apoptosis. In vivo, intravenous co-administration of sphingomyelin with 5-fluorourical to athymic nude mice bearing human colon tumour xenographs improved efficacy over 5-flurourical alone. Investigation of techniques to increase the ceramide level of target cells, including the use of dietary sphingomyelin is intensifying (Modrak et al., 2006).

Schmelz and her colleagues pioneered the use of dietary sphingomyelin to inhibit colon tumour development in animal models (Schmelz, 2004; Parodi, 2006). Initially it was demonstrated that mice fed as little as 0.025 to 0.1% milk-derived sphingomyelin had less than half the incidence of chemically induced colon tumours than non-supplemented mice. Sphingomyelin also inhibited the formation of aberrant crypt foci (ACF, preneoplastic lesions that may develop into colon tumours) in mice. The complex sphingolipids, glu- cosylceramide, lactosylceramade and ganglioside GD₃, isolated from milk, all reduced the number of chemically induced ACF in mice to the same extent as sphingomyelin.

Apc ^Min/+mice carry a germline mutation in the adenomatous polyposis coli (Apc) gene that is found in 40 to 80% of sporadic human colon cancers. In this model mice develop spontaneous tumours throughout the intestinal tract. Apc ^Min/+mice fed diets supplemented with 0.1% of a mixture of complex sphingolipids with composition resembling that of milk reduced tumour development in all segments of the intestines by about half.

In another study with mice sphingomyelin was equally effective in inhibiting tumour development when supplementation commenced before administration of the carcinogen or after tumour initiation. This effect suggests both a chemopreventive and chemotherapeu- tic benefit.

Dietary sphingomyelin also reduced ACF formation and size in an aged rat model (Exon and South, 2003). Dietary sphingomyelin may also have an anti-cancer effect in peripheral organs. Silins et al., (2003) demonstrated that feeding a diet supplemented with 0.1% sphingomyelin to rats bearing chemically induced preneoplastic liver lesions significantly reduced the number and area of the lesions.

REFERENCES

ABE A., SUGIYAMA K., 2005. Growth Inhibition and apoptosis induction of human melanoma cells by ω-hydroxy fatty acids. Anti-cancer Drugs, 16, 543-549.

ARO A., MANNISTO S., SALMINEN I., OVASKAI- NEN M-L., KATAJA V., UUSITUPA M., 2000.

Inverse association between dietary and serum conjugated linoleic acid and risk of breast cancer in postmenopausal women.

Nutr. Cancer, 38, 151-157.

BANNI S., ANGIONI E., MURRU E., CARTA G., MELIS M.P., BAUMAN D., DONG Y., IP, C., 2001. Vaccenic acid feeding increases tissue levels of conjugated linoleic acid and sup- presses development of premalignant lesions in rat mammary gland. Nutr. Cancer, 41, 91- 97.

BAUMAN D.E., LOCK A.L., CORL B.A., SALTER A.M., IP C., PARODI, P.W., 2006. Milk fatty acids and human health, potential role of

conjugated linoleic acid and trans fatty acids.

In SEJRSEN K., HVELPLUND T. NIELSON M.O. (eds), Ruminant Physiology: Digestion, Metabolism and impact of Nutrition on Gene Expression, Immunology and Stress. 523- 555, Wageningen Academic Publishers, Wageningen, The Netherlands.

BELOBRAJDIC D.P., MCINTOSH G.H., 2000.

Dietary butyrate inhibits NMU-induced mammary cancer in rats. Nutr. Cancer, 36, 217- 223.

BERESFORD S.A.A., JOHNSON K.C., RITEN- BAUGH C., LASSER, N.L., SNETSELAAR L.G., et al., 2006. Low-fat dietary pattern and risk of colorectal cancer. JAMA, 295, 643- 654.

CHEN B.-Q., XUE Y.-B., LIU J.-R., YANG Y.-M., ZHENG Y.-M., WANG X.-L. LIU R.-H., 2003.

Inhibition of conjugated linoleic acid on mouse forestomach neoplasia induced by

(8)

benzo(a)pyrene and chemopreventive mechamisms. World J. Gastroenterol. 9, 44- 49.

DAVIE J.R., 2003. Inhibition of histone deacetylase activity by butyrate. J. Nutr. 133, 2485S- 2493S.

DUAN R.-D., 2005. Anticancer compounds and sphingolipid metabolism in the colon. In vivo, 19, 293-300.

EXON J.H., SOUTH E.H., 2003. Effects of sphingomyelin on aberrant colonic crypt foci development, colon crypt cell proliferation and immune function in an aging rat model. Food Chem. Toxicol. 41, 471-476.

HA Y.L., GRIMM N.K. PARIZA M.W., 1987. Anti- carcinogens from fried ground beef: heat altered derivatives of linoleic acid. Carcino- genesis 8, 1881-1887.

HUBBARD N.E., LIM D., ERICKSON K.L., 2003.

Effect of separate conjugated linoleic acid isomers on murine mammary tumorigenesis.

Cancer Lett. 190,13-19.

IP C., CHIN S.F., SCIMECA J.A., PARIZA M.W., 1991. Mammary cancer prevention by conjugated dienoic derivative of linoleic acid. Can- cer Research, 51, 6118-6124.

IP C., BANNI S., ANGIONI E., CARTA G., McGIN- LEY J., THOMPSON H.J., BARBANO D.

BAUMAN D.E., 1999. Conjugated linoleic acid-enriched butter fat alters mammary gland morphogenesis and reduces cancer risk in rats. J. Nutr. 129, 2135-2142.

IP C., IP M.M., LOFTUS T., SHOEMAKER S., SHEA-EATON W., 2000. Induction of apoptosis by conjugated linoleic acid in cultured mammary tumour cells and premalignant lesions of the rat mammary gland. Cancer Epidemiol. Biomark. Prev. 9, 689-696.

IP M.M., MASSO-WELCH P.A. IP C., 2003. Pre- vention of mammary cancer with conjugated linoleic acid: role of stroma and the epithelium. J. Mammary Gland Biol. Neoplasia., 8, 103-118.

IP M.M., McGEE S.O., MASSO-WELCH P.A., IP C., MENG X., OU L., SHOEMAKER S.F., 2007. The t10,c12 isomer of conjugated linoleic acid stimulates mammary tumorigenesis in transgenic mice over-expressing erbB2 in the mammary epithelium. Carcinogenesis, 28, 1269-1276.

KUSHI L., GIOVANNUCCI E. 2002. Dietary fat and cancer. Am. J. Med, 113, 63S-70S.

LARSSON S.C., BERGKVIST L., WOLK A. 2005.

High-fat dairy food and conjugated linoleic acid intakes in relation to colorectal cancer incidence in the Swedish Mammography cohort. Am. J. Clin. Nutr, 82, 894-900.

LOCK A.L., CORL B.A., BARBANO D.M., BAU- MAN,D.E., IP C., 2005. The anticarcinogenic effect of trans-11 18:1 is dependent on its

conversion to cis-9, trans-11 CLA by Δ9- desaturase in rats. J. Nutr, 134, 2698-2704.

MASSO-WELCH P.A., ZANGANI D., IP C., VAU- GHAN M.M., SHOEMAKER S.F., McGEE S.O., IP M.M., 2004. Isomers of conjugated linoleic acid differ in their effects on angiogenesis and survival of mouse mammary adi- pose vasculature. J. Nutr, 134, 299-307.

McCANN S.E., IP C., IP M.M., McGUIRE M.K., MUTI P., EDGE S.B., TREVISAN M., FREU- DENHEIM J.L., 2004. Dietary intake of conjugated linoleic acid and risk of premenopausal and postmenopausal breast cancer, Western New York Exposures and Breast Cancer Study (WEB Study). Cancer Epidemiol. Bio- mark. Prev, 13, 1480-1484.

MODRAK D.E., GOLD D.V., GOLDENBERG D.M., 2006. Sphingolipid targets in cancer therapy.

Mol. Cancer Ther, 5, 200-2008.

PALMQUIST D.L., LOCK A.L., SHINGFIELD K.J., BAUMAN D.E., 2005. Biosynthesis of conjugated linoleic acid in ruminants and humans.

Adv. Food Nutr. Res, 50, 179-217.

PARK H.S., CHUN C.S., KIM S., HA Y.L., PARK J.H.Y., 2006. Dietary trans-10,cis-12 and cis- 9, trans-11 conjugated linoleic acids induce apoptosis in the colonic mucosa of rats trea- ted with 1,2-dimethyl hydrazine. J. Med.

Food, 9, 22-27.

PARODI P.W., 1999. Conjugated linoleic acid and other anticarcinogenic agents of bovine milk fat. J. Dairy Sci, 82, 1339-1349.

PARODI P.W., 2003. Conjugated linoleic acid in food. In: SEBEDIO J-L., CHRISTIE W.W., ADLOF R.O. (eds.), Advances in Conjugated Linoleic Acid Research Vol. 2, 101-122, AOCS Press, Champaign, Illinois.

PARODI P.W., 2004. Milk fat in human Nutrition.

Aust. J. Dairy Technol, 59, 3-59.

PARODI P.W., 2005. Dairy product consumption and the risk of breast cancer. J. Am. Coll.

Nutr, 24, 556S-568S.

PARODI P.W., 2006. Nutritional significance of milk lipids. In: FOX P.F., McSWEENEY P.L.H.

(eds.) Advanced Dairy Chemistry, Vol 2. 3^rd ed. 601-639, Springer, New York.

PRENTICE R.L., CAAN B., CHLEBOSKI R.T., PATTERSON R., KULLER L.H., et al., 2006.

Low-fat dietary pattern and risk of invasive breast cancer. JAMA, 295, 629-642.

SCHMELZ E.M., 2004. Sphingolipids in the che- moprevention of colon cancer. Front. Biosci, 9, 2632-2639.

SCIMECA J.A., 1999. Cancer inhibition in animals. In: YURAWECZ M.P., MOSSOBA M.M., KRAMER J.K.G., PARIZA M.W., Nel- son G.J. (eds.), Advances in Conjugated Linoleic Acid Research Vol. 1, 420-443, AOCS Press, Champaign, Illinois.

SILINS I., NORDSTRAND M., HOGBERG J., STE- NIUS U., 2003. Sphingolipids suppress pre-

(9)

vivo. Carcinogenesis 24, 1077-1083.

WANG L.-S., HUANG Y.-W., LIU S., CHANG H.- L., YE W., et al., 2006. Conjugated linoleic acid (CLA) modulates prostaglandin E₂ (PGE₂) signaling in canine mammary cells.

Anticancer Res, 26, 889-898.

WILLETT W.C., 2001. Diet and cancer: one view at the start of the millennium. Cancer Epide- miol. Biomark. Prev, 10, 3-8.

WILLIAMS E.A., COXHEAD J.M., MATHERS J.C., 2003. Anti-cancer effects of butyrate: use of micro-array technology to investigate mechanisms. Proc. Nutr. Soc, 62, 107-115.

WONGTANGTINTHARN S., OKU H., IWASAKI H., TODA T. 2004. Effect of branched-chain fatty

acid biosynthesis of human breast cancer cells. J. Nutr. Sci. vitaminol, 50, 137-143.

YANAGI S., YAMASHITA M., IMAI S. 1993.

Sodium butyrate inhibits the enhancing effect of high fat diet on mammary tumorigenesis.

Oncology, 50, 201-204.

YANG P., COLLIN P., MADDEN T., CHAN D., SWEENEY-GOTSCH B., et al., 2003. Inhibi- tion of proliferation of PC3 cells by the branched-chain fatty acid, 12-methyltetradecanoic acid, is associated with induction of 5-lipoxi- dase. Prostate 55, 281-291.

YANG Z., LIU S., CHEN X., CHEN H., HUANG M., ZHENG J., 2000. Induction of apoptotic cell death and in vivo growth inhibition of human cancer cells by a saturated branched- chain fatty acid, 13-methyltetradecanoic acid. Cancer Res, 60, 505- 509.