Early studies focused on initiation stages of anticarcinogenic effects of CLA. As an anti-initiator, CLA may modulate events such as free radical-induced oxidation, carcinogen metabolism, and/or carcinogen-DNA adduct formation in some tumor models.50 CLA increases the activity of antioxidant-enzymes (superoxide dismutase, catalase, and glutathione peroxidase) in human breast cancer cells (MCF-7) and colon cancer cells (SW480), suggesting that CLA may shift the pro-oxidant/antioxidant balance.22,27 It has been postulated that, due to the conjugated structure of CLA, there is more efficient trapping of electrons in its double bonds than in methylene-interrupted double bonds, and that antioxidant enzymes are induced as an adaptation of oxidant exposure.50
Isomers and metabolites of CLA are readily incorporated into phospholipid and neutral lipid fractions of numerous tissues.35,3640 51 52 When radiolabeled tracers were used to study the kinetics of 14C-CLA uptake into keratinocytes or hepatoma cells, 14C-CLA was incorporated to the same extent and at a similar rate as 14C-linoleate, and the levels of incorporation of 14C-CLA and 14C-linoleate into epidermal phospholipids and neutral lipid fractions were similar.53 As the CLA content in the diet increased over a range of 0.5 to 2%, there was a progressive increase in the CLA content of rat mammary and peritoneal fat pads, liver, and plasma, and in the level of CLA metabolites represented by 18:3 and 20:3, and a decrease in the linoleic acid metabolites, in particular arachidonic acid.34 However, another study of rats fed a diet containing CLA-rich butter (and linoleate) showed that CLA preferentially accumulated in neutral lipids (~79%) with less incorporation into phosphatidylcholine (~10%), the major phospholipids of lever cells, while linoleate preferentially accumulated in phosphatidylcholine (~50%), with less in neutral lipids (~17%).54 This led to the conclusion that CLA may attenuate lipid peroxidation in neutral lipid-rich tissues by interfering with the formation of linoleic acid-derived arachidonic acid, a fatty acid that is most susceptible to lipid oxidation and formation of malondialdehyde. The ability of CLA to decrease malondialdehyde formation in skeletal muscle and liver microsomes was recently shown to be a reflection of its ability to decrease levels of polyenoic fatty acid such as arachidonic acid.55 In addition, adding 1% CLA to the diet of female rats exposed to dimethylbenz[a]anthracene results in lower levels of mammary tissue malondialdehyde (an end product of lipid peroxidation), but fails to change the levels of 8-hydroxydeoxyguanosine (a marker of oxida-tively damaged DNA),18 Although CLA does not appear to act directly as an antioxidant, it is not known if a decrease in lipid peroxidation contributes to the cancer-protective effect of CLA seen in the mammary gland.55 The data suggest that CLA may have some antioxidant function in vivo in suppressing lipid peroxidation, but its anticarcinogenic activity cannot be accounted for by protecting the target cell DNA against oxidative damage.40 To further weaken the hypothesis that CLA exerts anticancer effects as a result of lipid peroxidation, dietary CLA does not increase lipid peroxidation in mice with transplanted metastatic murine mammary tumors.48 These studies dealing with oxidation have not identified any oxidation products of CLA. While no definitive conclusions can be made, it is unlikely that CLA-inducing lipid peroxidation is the sole mechanism of action.19,21,44
On the promotion stage, recent studies of the anticarcinogenic mechanisms of CLA have focused particularly in the mammary and skin carcinogenesis models.41 This stage of carcinogenesis represents a premalignant state in which tumors arise from cells that have increased cell proliferation, reduced programmed cell death (or apoptosis), and/or dysregulated differentiation. In cultured cells, CLA reduced proliferation of mammary tumor cells in vitro25,56 and in vivo.51 Rats initiated with methylnitrosourea and then fed a diet with CLA (1%) exhibited reduced proliferation of terminal end bud and lobuloalveolar bud structures of mammary epithelium.58 The terminal end bud is the site of tumor formation for both rat and human breast cancer and CLA inhibits proliferation by a reduction in density of the terminal end bud.58 Ip and colleagues59 demonstrated that CLA- or c9,t11-CLA-rich butter fat reduces the incorporation of bromodeoxyuridine and the expression of cyclins A and D.59 Because these two cyclins regulate the conversion of the G^S phase of the cell cycle,5960 CLA reduces cell proliferation in terminal end bud structures by regulating the cell cycle in the rat mammary epithelium59 and in the SGC-7901 cell, with reduced expressions of cyclin A, B, and D and enhanced expressions of cyclin-dependent kinase inhibitors and p21.61 Furthermore, mammary adenocarcinomas induced by PhlP contained significantly fewer proliferating cell nuclear antigen-positive cells in rats fed dietary CLA (0.1 g/100 g).62 Both studies showed that CLA moderately increased levels of p16 and p27 proteins.5962 In MCF-7 breast cancer cells, CLA also inhibited cell proliferation and enhanced the accumulation of p53 and pRb, although t10,c12-CLA isomer was more effective than c9,t11-CLA.63 These studies suggest that CLA modulates molecular signaling events and blocks DNA synthesis that affects the cell cycle, ultimately regulating cell proliferation.
However, there was no relationship between dietary CLA and markers of cell proliferation of other models. CLA had no effect on phorbol ester-induced hyperplasia, ornithine decarboxylase activity, or c-myc mRNA expression in the mouse epidermis,64 but increased cell proliferation in diethylnitrosamine-induced focal lesions in rat livers.15 These data demonstrate that the ability of CLA to reduce cell proliferation may be tissue-specific and/or tumor-model-specific.
In contrast to proliferation, CLA induced apoptosis in numerous tissues including mammary,60 liver,15 colon,65 and adipose66 tissues, and in cultured mammary epithelial cells,67 human breast,68 SGC-7901,6970 and HT-2971 tumor cells. Although most studies used a mixture of isomers, a 50:50 mixture of c9,t11-CLA and t10,c12-CLA was more effective than individual isomers at inducing apop-tosis in breast cancer cell lines.68 In mammary tissue initiated with methylni-trosourea, CLA (64 yM) or c9,t11-CLA (128 yM) induced apoptosis of cells in the terminal end bud and premalignant lesions.60 In these studies, CLA induction of apoptosis was associated with a reduction of Bcl-2. The Bcl-2 gene family is a signaling protein and has differential effects on apoptosis; for example, Bcl-2 and Bcl-xL suppress apoptosis, whereas others, such as Bax and Bak, promote apoptosis. Because CLA reduces Bcl-2 and moderately induces Bax, it appears that CLA elevates apoptosis primarily by reducing the suppressor of apoptosis, Bcl-2. In 1,2-dimenthylhydrazine-treated colon mucosa, CLA decreases the incidence of colon cancer by decreasing cellular proliferation and inducing apoptosis of the colonic mucosa by decreased prostaglandin E2 (PGE2) levels and increased Bax/Bcl-2 ratios.72
CLA has been shown to modulate the protein kinase C (PKC) abundance/activity in membranes from prostate cancer cells (LNCaP) to produce an apoptotic profile with increased PKC-8, -a, and -Z and a decreased PKC-i,73 but Ip and colleagues60 did not observe any changes in the isoforms in mammary tissue for rats in the study. On the other hand, CLA downregulated ErbB3 signaling, the phosphoinositide 3-kinase, and the Akt pathway in human colon cell line,71 and inhibited the expression of extracellular-mitogen-activated protein kinase phosphatase-1 protein in a rodent model of forestomach neoplasia.74 These data suggest that CLA inhibits tumor promotion by inducing signaling events leading to enhanced apoptosis.
CLA induces markers of differentiation in the noncancer model adipose tissue;7576 it is possible that CLA inhibits carcinogenesis by induction of differentiation. CLA fed during the time of mammary gland development and maturation has long-lasting protective effects on mammary carcinogenesis,4158 likely by this very means.
Cell adhesion molecules are important ingredients in maintaining cell-cell adhesion and cell-matrix interactions. The abnormality of cell adhesion molecules closely correlates with neoplastic transformation and metastasis.77 Recent study has shown that c9,t11-CLA increased the level of expression of E-cadherin and alpha-catenin, while decreasing the level of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 in human gastric carcinoma cell line SGC-7901.78 The levels of the adhesion molecules ICAM-1 and E-selection also were reduced by 50% in human umbilical vein endothelial cells when incubated with 10 mM CLA.79 These studies suggest that CLA is involved in metastatic processes and the invasion of tumor cells.
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