Importance of the cell membrane

The study of biological membranes regarding function and structure is important as they are the very boundaries within which life exists, separating the vast array of biochemical reactions that define a living cell from the extracellular world. Within the cell, membranes also organise and separate these biochemical reactions from each other, generating compositionally and morphologically distinct compartments. The membrane of a cell is important in maintaining a homoeostatic environment in the cell, serving as a gateway for nutrients and creating a signalling pathway for cellular communication. The properties and functionality of the cell membrane is dependent on particular constituent lipid components such as the phospholipids, cholesterol, proteins and FA. Phospholipids are not symmetrically distributed between the outer and inner membrane leaflets and this feature appears to be integral for correct functioning. Studies involving the erythrocyte membrane indicate that most of the phosphatidylethanolamine (PE) and phosphatidylserine are situated within the inner or cytoplasmic membrane leaflet, while most of the phosphatidylcholine (PC) and sphingomyelin are found in the outer leaflet⁽ Reference Calder and Yaqoob ^{8 ,} Reference Gordesky and Marinetti ^{9 )}.

This characteristic asymmetric lipid distribution of cell membranes is important in the physiological functioning of the membrane. Events such as translocation of phosphatidylserine from the inner to the outer membrane leaflet of erythrocytes or vascular endothelium promotes blood coagulation and translocation of phosphatidylserine and PE to the outer membrane leaflet is a signal for apoptosis and removal of an injured or damaged cell⁽ Reference Daleke ^{10 ,} Reference Pomorski, Hrafnsdóttir and Devaux ^{11 )}. Therefore, disruption of the membrane phospholipid distribution or the PC:PE ratio as well as FA content can change the responsiveness of the cell to normal physiological control mechanisms.

As components of cellular phospholipids, FA are the main structural building blocks of all cell membranes⁽ Reference Rudolph, Kelley and Klasing ^{12 ,} Reference Funari, Barceló and Escribá ^{13 )}. Amongst the FA, the SFA, mono-unsaturated FA and PUFA as well as interaction between these classes play important roles in maintaining the integrity and functioning of the cell membrane. Changes in the FA content, especially with regard to PUFA and SFA, can affect the fluidity of cell membranes, ultimately affecting membrane function⁽ Reference Brenner, Vergroesen and Crawford ^{14 )}. Certain FA are also substrates for the synthesis of eicosanoids which are involved in the regulation of cell growth, cell activity and cell function⁽ Reference Shohet, Beutler, Williams, Beutler, Ersley and Lichtman ^{15 )}. Functionally, FA also affect gene expression by binding to and regulating the activity of nuclear receptors controlling the major regulatory networks that impact cell metabolism and signalling mechanisms⁽ Reference Calder ^{16 )}.

As cell membranes are susceptible to dietary modulation, particularly of the n-3 family, the chemopreventive mechanisms of these FA are multiple. Mechanisms include the suppression of C₂₀ : 4n-6-derived eicosanoid production, enhancement of apoptosis, inhibition of tumour cell proliferation and an increase in anti-angiogenic activity⁽ Reference Berquin, Edwards and Chen ^{17 )}. The incorporation and association of n-6 and n-3 FA with membrane dynamics involving cholesterol, phospholipids and membrane proteins, also significantly impact on signal transduction pathways and cell-to-cell communication in the regulation of cell growth. Therefore, changes in the cellular lipid composition can affect the structural and functional properties of membranes, thereby altering the growth characteristics of neoplastic cells. Membrane components such as a cholesterol and FA are also of particular importance to certain membrane substructures such as lipid ‘rafts’ or ‘domains’ which play important roles in cell signalling⁽ Reference Rose and Connolly ^{18 )}.

Dietary fatty acids and cancer in human subjects

In 1956, Hugh Sinclair⁽ Reference Sinclair ^{19 )} claimed that most of the ‘diseases of civilisation’ such as CHD, thrombosis, cancer, diabetes, inflammation and skin diseases are caused by a disturbance in fat metabolism. The main reason for the occurrence of these diseases was associated with the increased intake of processed foods rich in SFA and trans FA and a subsequent alteration in essential FA intake, such as a shift towards a higher n-6 FA intake. However, the clarity surrounding the association of saturated fats and CVD is being questioned⁽ Reference Malhotra ^{20 )}. During the 1970s the beneficial health effects of n-3 FA gained recognition and since then a growing body of evidence from in vitro studies in cell cultures, animal cancer models, epidemiological and clinical studies in human subjects has provided evidence to support their use in the prevention of cancers such as in the colon, breast and prostate⁽ Reference Berquin, Edwards and Chen ^{17 )}. In contrast, a high dietary intake of n-6 FA is associated with an increased risk for the development of cancer⁽ Reference Lawrence ^{6 )}. Research into the beneficial health effects of the n-3 PUFA were fuelled by the ‘classical’ studies undertaken by Dyerberg, Bang and Hjorne which indicated that high-fish-consuming populations such as the Inuits and Danes have low cancer incidence rates⁽ Reference Dyerberg, Bang and Hjorne ^{21 )}. Further epidemiological studies have shown a protective association regarding n-3 PUFA and cancer risk. In a Japanese population-based study, the n-3 PUFA (EPA, C₂₀ : 5n-3; docosapentaenoic acid, C₂₂ : 5n-3; DHA, C₂₂ : 6n-3) were associated with protection against developing hepatocellular carcinoma⁽ Reference Sawada, Inoue and Iwasaki ^{22 )}, whereas another population-based prospective study undertaken by the Japanese Public Health Center demonstrated an inverse relationship between the intake of marine n-3 PUFA and the risk of colorectal cancer, even though the association was only significant in the proximal site of the colon⁽ Reference Sasazuki, Inoue and Iwasaki ^{23 )}. Large studies, such as the European Prospective Investigation into Cancer and the United States’ Physicians Health study, involving thousands of participants have confirmed the protection against cancer afforded by n-3 PUFA, particularly signifying a decrease in the risk of colorectal cancers⁽ Reference Norat, Bingham and Ferrari ^{24 ,} Reference Hall, Chavarro and Lee ^{25 )}. In a case–control study, erythrocyte levels of EPA and DHA were inversely associated with breast cancer risk⁽ Reference Kuriki, Hirose and Wakai ^{26 )}. Further detailed reviews concerning n-3 PUFA and cancer are given by Gerber⁽ Reference Gerber ^{27 )} and Stephenson et al.⁽ Reference Stephenson, Al-Taan and Arshad ^{28 )}.

Fatty acid and cancer: mechanisms of control

FA may influence carcinogenesis through various mechanisms and it is therefore important to understand the control of FA over these processes, thereby determining possible intervention points.

Diets containing high levels of n-6 FA, such as in corn oil, have been shown to enhance the development of tumours⁽ Reference Lawrence ^{6 ,} Reference Jump ^{29 ,} Reference Reddy ^{30 )}. Most of these effects have been attributed to the metabolism and cell signalling properties of arachidonic acid (C₂₀ : 4n-6). The deregulation or disturbance in C₂₀ : 4n-6 metabolism has been connected to a large number of pathological disorders, indicating the need for tight control over the metabolism of this FA⁽ Reference Hong, Chang and Chapkin ^{31 )}. Therefore control over C₂₀ : 4n-6 metabolism may be an important factor in controlling cellular proliferation and apoptosis and therefore cancer cell survival. In this regard, the effect of n-3 PUFA on chemoprevention has been shown to be effective in vivo and in vitro. Mechanisms involved have been demonstrated to involve the competitive incorporation of n-3 FA in cell membranes, to be involved in signal transduction by acting as ligands of certain nuclear receptors, competing in the synthesis of eicosanoids via the cyclooxygenase-2 pathway, enhancing apoptosis and cell differentiation, and altering the cellular oxidative status by enhancing lipid peroxidation⁽ Reference Marks, Müller-Decker and Fürstenberger ^{32 )}.

Altered lipid profile in liver cancerous tissue

Understanding the alterations in the lipid profile of cancerous tissue will enable possible modulation strategies utilising PUFA in chemoprevention. Studies from animal cancer models and human cancer biopsy tissue demonstrate that a characteristic lipid profile is associated with the growth and development of neoplastic lesions⁽ Reference Diggle ^{33 ,} Reference Abel, Smuts and De Villiers ^{34 )}. This profile entails alterations in membrane cholesterol, phospholipid and PUFA metabolism resulting in a shift in C₂₀ : 4n-6 phospholipid distribution and a decreased cellular oxidative status. Cancerous tissue from a rat liver cancer model and biopsy tissue from human patients with liver cancer showed similarities in the altered lipid profile associated with liver cancer development⁽ Reference Abel, Smuts and De Villiers ^{34 ,} Reference Abel, De Kock and Van Schalkwyk ^{35 )}.

Alterations in the PUFA content of liver cancerous tissue, encompass a decrease in the n-3 PUFA resulting in an increased n-6:n-3 PUFA ratio, increased C₁₈ : 1n-9 (oleic acid), increase in C₂₀ : 4n-6, increased C₂₀ : 4n-6/C₂₀ : 5n-3 and decreased C₂₀ : 4n-6 PC:PE ratios. A shift in the C₂₀ : 4n-6 PC:PE ratio could indicate a higher availability of C₂₀ : 4n-6 for eicosanoid synthesis and cell signalling or a compensatory shift in phospholipid distribution to maintain membrane stability and functionality. Cholesterol was increased, whereas the phospholipid PC:PE ratio decreased. Alterations in the membrane phospholipid and cholesterol levels, affecting the membrane cholesterol:phospholipid and PC:PE ratios, will influence membrane structure, fluidity and functionality. Disruption of the typical phospholipid and cholesterol asymmetrical arrangement may affect cellular signalling influencing events such as apoptosis⁽ Reference Pomorski, Hrafnsdóttir and Devaux ^{11 ,} Reference Rudolph, Kelley and Klasing ^{12 )}. Changes in these parameters are also known to regulate the function of Δ6-desaturase enzyme, a key rate-limiting enzyme in FA metabolism⁽ Reference Leikin and Shinitzky ^{36 )}. Various studies have demonstrated an impairment of the Δ6-desaturase in cancer tissue affecting PUFA synthesis⁽ Reference Gardiner and Duncan ^{37 ,} Reference Hrelia, Bordoni and Biagi ^{38 )}. Lipid peroxidation was decreased in conjunction with altered levels of the antioxidant glutathione (reduced form) as well as altered activity of the antioxidant enzymes, i.e. glutathione reductase and peroxidase, superoxide dismutase and catalase. These changes result in the maintenance of a low cellular oxidative status thereby creating a protective environment for cancer cell survival. The level of lipid peroxidation has also been found to influence tumour growth⁽ Reference Gonzalez and Riordan ^{39 )} and, together with changes in the membrane lipid status, is likely to play an important role in the abnormal cellular growth which prevails in cancerous tissue. These events are highlighted as a driving force sustaining the altered growth characteristics of cancerous lesions together with a diet high in n-6 PUFA playing an underlying stimulating role. The integrity of the cellular membrane is therefore important in the normal functioning of the cell and its responses to external growth stimulatory and/or inhibitory factors.

In the rat liver cancer model, the altered lipid parameters associated with hepatocyte nodule development mimicked normal cellular proliferation in regenerating liver⁽ Reference Abel, Smuts and De Villiers ^{34 )}. However, a major difference between the two proliferating tissue compartments was the persistence of the lipid changes in the hepatocyte nodules suggesting that nodule lipid metabolism escapes the regulatory mechanisms required for normal cellular homoeostasis during proliferation. This indicates that the impaired regulation of lipid metabolism may be responsible for the enhanced proliferation and altered growth pattern in hepatocyte nodules.

Modulation of lipid profile by dietary PUFA and effect on cancer cell development

It can be questioned whether the characteristic lipid profile in cancerous tissue can be modulated by dietary PUFA thereby affecting cancer cell development. Utilising the rat liver cancer model it was demonstrated, dependent on the dietary PUFA composition and stage of intervention, that the development of early preneoplastic cancer lesions (nodules) can be modulated.

Interaction between dietary PUFA and food-borne contaminants

Of interest with regard to carcinogenesis is the interplay between dietary FA and toxins present in foods and effect on carcinogenesis. In this regard, a rat hepatocarcinogenesis model was used to investigate the interactive effect of a toxin and dietary fats on the initiation and promotion stages of carcinogenesis⁽ Reference Riedel ^{44 )}. The mycotoxin, fumonisin B₁ (FB₁), acts as a weak, non-genotoxic cancer initiator and expresses strong cancer promoting activity resulting in the induction and promotion of pre-neoplastic hepatic foci in rat liver. The mechanisms responsible involve alteration of the cell membrane structure resulting from changes in lipid metabolism, more specifically phospholipids, sphingolipids and cell membrane FA content⁽ Reference Gelderblom, Riedel, Burger, Siantar, Trucksess, Scott and Herman ^{45 )}.

To investigate the effect on cancer initiation, weaned male Fischer F344 rats were fed the AIN 93 diet with different oils as fat sources; either S with an n-6:n-3 FA ratio of 700 : 1, or a mixture of SGF to achieve an n-6:n-3 ratio of 6 : 1.

Two different initiation/promotion protocols were used: (i) male Fischer F344 rats were treated with dietary FB₁ (250 mg/kg diet) for 6 weeks to induce initiation and promotion ((−) DEN) and (ii) rats were implanted with minipumps releasing DEN (200 mg/kg body weight) over 7 d as initiation ((+) DEN) and dietary FB₁ (250 mg/kg diet) over 6 weeks as cancer promotion treatment. In the (+) DEN study, rats were weaned on the S diet and switched to the SGF diet 15 d after initiation to prevent any dietary effect during initiation. On termination, liver sections were removed and stained for glutathione-S-transferase (placental) positive (GSTP⁺) areas which represent the pre-neoplastic nodules/lesions in the liver.

In the FB₁ initiation study ((−) DEN), GSTP⁺ lesions were increased in the S/FB₁ group, but no significant increase observed in the SGF and SGF/FB₁-fed group, demonstrating inhibition of FB₁ initiation by the GLA-80 and fish oil combination (Fig. 1a). This could result from decreased hepatotoxicity and increased apoptosis stimuli to remove altered cells (initiated cells) presumably via increased oxidative stress. The reduced FB₁ associated hepatotoxicity by the SGF diet ((−) DEN initiation), as indicated by decreased serum liver function markers (total bilirubin, alanine aminotransferase, aspartate aminotransferase andalkaline phosphatase), could be related to a decreased inflammatory response resultant from a decreased n-6:n-3 FA ratio, decreased C₂₀ : 4n-6 and increased C₂₀ : 3n-6 and C₂₂ : 6n-3 content. The shift in C₂₀ : 4n-6 between the major phospholipids, indicated by an increased C₂₀ : 4n-6-PC:PE phospholipid ratio, resulting from the SGF diet has implications for cell survival. A higher content of this FA in PC is thought to indicate a shift towards apoptosis, which counteracted the decrease in the ratio due to FB₁ promotion associated with an increase in cell proliferation known to selectively increase pre-neoplastic development⁽ Reference Gelderblom, Riedel, Burger, Siantar, Trucksess, Scott and Herman ^{45 ,} Reference Surette, Winkler and Fonteh ^{46 )}. Supplementation with n-3 FA, whose anti-inflammatory properties are established in animal models⁽ Reference Brash ^{47 ,} Reference Jho, Babcock and Helton ^{48 )}, can alleviate FB₁-induced inflammation and therefore reduce liver toxicity.

Fig. 1. (colour online) (a) With fumonisin B₁ (FB₁) as initiator in rat liver, the glutathione-S-transferase placental positive (GSTP+) lesions were higher in the sunflower (S)/FB₁ group, but not significantly different in the sunflower/GLA-80/fish oil (SGF) and SGF/FB₁-fed group compared with the S-fed group, demonstrating GLA-80 and fish oil inhibition of FB₁ initiation⁽ Reference Riedel ^{44 )}. (b) With diethylnitrosamine (DEN) as initiator in rat liver, the SGF diet prevented development in the number and size of GSTP+ lesions. However, the combination of dietary FB₁ and SGF enhanced tumour promotion⁽ Reference Riedel ^{44 )}.

In the group with DEN as initiator ((+) DEN), the SGF diet prevented development in the number and size of GSTP⁺ lesions, once again implicating an inhibitory effect on cancer initiation via apoptosis due to an increased oxidative stress (Fig. 1b). However, combination of FB₁ and SGF in the diet enhanced tumour promotion probably due to the selective resistance of DEN-initiated hepatocytes towards oxidative stress and the apoptotic stimulus. In this scenario of the initiation/promotion model with DEN and FB₁, the SGF diet enhanced hepatotoxicity indicated by increases in serum liver markers and stimulated promotion reflected by the increased appearance of GSTP⁺ lesions. The increased oxidative stress and hepatotoxicity resultant from feeding the SGF diet appeared to create a growth-stimulating environment promoting DEN-initiated foci, i.e. the proliferation stimulus overwhelmed apoptotic effects in the pre-neoplastic lesions thereby increasing the number and size of GSTP⁺ foci.

Therefore, cancer initiation and promotion can be selectively altered by dietary FA which, depending on their interactive roles, provides important information regarding the dietary modulation of food-borne hepatocarcinogenesis.

Points to consider

However, many questions still need to be resolved with regard to which specific dietary factors are most closely linked to cancer prevention and the mechanisms by which food components exert their anti-cancer effects. A compounding effect is inter-individual variations in susceptibility arising from common polymorphisms in genes governing the metabolism of exogenous substances which can modify the carcinogenic or anti-carcinogenic effects of food components and, thus, add an extra level of difficulty to the interpretation of studies. Further complications with regard to cancer prevention are the lack of dose recommendations to date. However, a generalised guide can be deduced from guidelines for other diseases in which PUFA have demonstrated a beneficial component, such as the recommendations from the American Heart Association for patients without CHD of ‘at least two (preferable oily) fish meals per week’ or approximately 500 mg EPA + DHA/d; while for patients with CHD an EPA + DHA intake of 1 g, preferably from oily fish is recommended⁽ Reference Arterburn, Hall and Oken ^{52 )}.