CLA isomers inhibit TNFα-induced eicosanoid release from human vascular smooth muscle cells via a PPARγ ligand-like action

Conjugated linoleic acids (CLAs) were reported to have anti-atherogenic properties in animal feeding experiments. In an attempt to elucidate the molecular mechanisms of these anti-atherogenic effects, the modulatory potential of CLA on cytokine-induced eicosanoid production from smooth muscle cells (SMCs), which contributes to the chronic inflammatory response associated with atherosclerosis, has been investigated in the present study. cis-9, trans-11 CLA and trans-10, cis-12 CLA were shown to reduce proportions of the eicosanoid precursor arachidonic acid in SMC total lipids and to inhibit cytokine-induced NF-κB DNA-binding activity, mRNA levels of inducible enzymes involved in eicosanoid formation (cPLA₂, COX-2, mPGES), and the production of the prostaglandins PGE₂ and PGI₂ by TNFα-stimulated SMCs in a dose-dependent manner. The effect of 50 μmol/L of either CLA isomer was as effective as 10 μmol/L of the PPARγ agonist troglitazone in terms of inhibiting the TNFα-stimulated eicosanoid production by SMCs. PPARγ DNA-binding activity was increased by both CLA isomers compared to control cells. Moreover, it was shown that the PPARγ antagonist T0070907 partially abrogated the inhibitory action of CLA isomers on cytokine-induced eicosanoid production and NF-κB DNA-binding activity by vascular SMCs suggesting that PPARγ signalling is at least partially involved in the action of CLA in human vascular SMCs. With respect to the effects of CLA on experimental atherosclerosis, our findings suggest that the anti-inflammatory effect of CLA is at least partially responsible for the anti-atherogenic effects of CLA observed in vivo.     

Formation of conjugated linoleic acid metabolites in human vascular endothelial cells

Conjugated linoleic acids (CLAs) are bioactive lipid compounds showing anti-atherogenic actions in cell culture experiments and animal models of atherosclerosis without exact knowledge about the underlying mechanisms. CLAs were recently reported to be further metabolized to bioactive conjugated metabolites indicating that these metabolites are possibly involved in mediating the anti-atherogenic actions of CLA. Regarding the lack of information with respect to the formation of CLA metabolites in the vascular endothelium, which is strongly involved in the process of atherosclerosis, the present study aimed to explore the potential formation of CLA metabolites in vascular endothelial cells. The results from the present study show for the first time that the CLA isomers cis-9, trans-11 CLA and trans-10, cis-12 CLA are metabolized within endothelial cells to beta-oxidation products such as CD16:2c7t9 and CD16:2t8c10 and elongation products such as CD20:2c11t13, CD20:2t12c14 as well as CD22:2c13t15 and CD22:2t14c16. Different CD16:2/CLA ratios observed between cells treated with different CLA isomers indicate that the metabolism of CLAs depends on the configuration of the conjugated double bonds. In conclusion, regarding the biological activity reported for CD20:2t12c14 and other metabolites of CLA, the present results indicate that metabolites of CLA are possibly also involved in mediating the anti-atherogenic actions of CLA.     

Troglitazone but not conjugated linoleic acid reduces gene expression and activity of matrix-metalloproteinases-2 and -9 in PMA-differentiated THP-1 macrophages

Gene expression and activity of matrix-metalloproteinases (MMP)-2 and -9 in macrophages are reduced through peroxisome proliferator-activated receptor gamma (PPARgamma)-dependent inhibition of NF-kappaB. Since conjugated linoleic acids (CLAs) are PPARgamma ligands and known to inhibit NF-kappaB via PPARgamma, we studied whether CLA isomers are capable of reducing gene expression and gelatinolytic activity of MMP-2 and -9 in PMA-differentiated THP-1 macrophages, which has not yet been investigated. Incubation of PMA-differentiated THP-1 cells with either c9t11-CLA, t10c12-CLA or linoleic acid (LA), as a reference fatty acid, resulted in a significant incorporation of the respective fatty acids into total cell lipids relative to control cells (P<.05). Treatment of PMA-differentiated THP-1 cells with 10 and 20 mumol/L troglitazone but not with 10 or 100 mumol/L c9t11-CLA, t10c12-CLA or LA reduced relative mRNA concentrations and activity of MMP-2 and MMP-9 compared to control cells (P<.05). DNA-binding activity of NF-kappaB and PPARgamma and mRNA expression of the NF-kappaB target gene cPLA(2) were not influenced by treatment with CLA. In contrast, treatment of PMA-differentiated THP-1 cells with troglitazone significantly increased transactivation of PPARgamma and decreased DNA-binding activity of NF-kappaB and relative mRNA concentration of cPLA(2) relative to control cells (P<.05). In conclusion, the present study revealed that CLA isomers, in contrast to troglitazone, did not reduce gene expression and activity of MMP-2 and -9 in PMA-differentiated THP-1 macrophages, which is probably explained by the observation that CLA isomers neither activated PPARgamma nor reduced DNA-binding activity of NF-kappaB. This suggests that CLA isomers are ineffective in MMP-associated extracellular matrix degradation which is thought to contribute to the progression and rupture of advanced atherosclerotic plaques.     

The biologically active isomers of conjugated linoleic acid.

Numerous physiological effects are attributed to conjugated linoleic acid (CLA). The purpose of this presentation is to consider these effects with respect to the cis-9,trans-11 and trans-10,cis-12 CLA isomers. We review previously published data and present new findings that relate to underlying biochemical mechanisms of action. Both isomers are natural products. The cis-9,trans-11 isomer is the principal dietary form of CLA, but the concentrations of this isomer and the trans-10,cis-12 isomer in dairy products or beef vary depending on the diet fed to cows or steers, respectively. The trans-10,cis-12 CLA isomer exerts specific effects on adipocytes, in particular reducing the uptake of lipid by inhibiting the activities of lipoprotein lipase and stearoyl-CoA desaturase. The trans-10,cis-12 CLA isomer also affects lipid metabolism in cultured Hep-G2 human liver cells, whereas both the cis-9,trans-11 and trans-10,cis-12 CLA isomers appear to be active in inhibiting carcinogenesis in animal models. We present new findings indicating that the cis-9,trans-11 CLA isomer enhances growth and probably feed efficiency in young rodents. Accordingly, the effects of CLA on body composition (induced by trans-10,cis-12 CLA) and growth/feed efficiency (induced by cis-9,trans-11 CLA) appear to be due to separate biochemical mechanisms. We also show that a 19-carbon CLA cognate (conjugated nonadecadienoic acid, CNA) inhibits lipoprotein lipase activity as effectively as CLA in cultured 3T3-L1 adipocytes. Presumably, CNA is metabolized differently than the 18-carbon CLA isomers, so this finding indicates direct activity of the administered compound as opposed to acting via a metabolite.     

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

Conjugated linoleic acid (CLA) is a collective term for a group of positional and geometric conjugated dienoic isomers of linoleic acid. CLA has been shown to have strong inhibitory effects on mammary carcinogenesis both in vitro and in vivo. In this study, we investigated the regulation of human stearoyl-CoA desaturase (SCD, EC 1.14.99.5) expression by CLA in human breast cancer cell lines, MDA-MB-231 and MCF-7. Treatment of the cells with the cis-9,trans-11 and trans-10,cis-12 CLA isomers (45 microM) did not repress SCD mRNA in both MDA-MB-231 and MCF-7 cells. However, the cis-9,trans-11 and trans-10,cis-12 CLA isomers significantly decreased SCD protein levels and SCD activity in MDA-MB-231 cells. In MCF-7 cells, both isomers did not affect protein levels, but they inhibited SCD activity. These results suggest that in MDA-MB-231 cells the cis-9,trans-11 and trans-10,cis-12 CLA isomers regulate human SCD by reducing SCD protein levels, while in MCF-7 cells both isomers have a direct inhibitory effect on SCD enzyme activity.     

Mechanisms of action of conjugated linoleic acid: evidence and speculation

Conjugated linoleic acid (CLA) has been shown to inhibit carcinogenesis and atherosclerosis, enhance immunologic function while protecting against the catabolic effects of immune stimulation, affect body composition change (reducing body fat gain while enhancing lean body mass gain), and stimulate the growth of young rats. We discuss possible biochemical mechanisms that underlie these physiological effects. We emphasize the importance of considering the effects, both individually and combined, of the two CLA isomers (cis-9, trans-11 CLA and trans-10, cis-12 CLA) that have been shown to exhibit biological activity and which appear to exert their effects via different biochemical mechanisms.     

Effect of conjugated linoleic acid isomers on lipoproteins and atherosclerosis in the Syrian Golden hamster

Conjugated linoleic acid (CLA) is a group of positional and geometric isomers of linoleic acid (LA, C18:2 cis-9, cis-12) that are reported to have important biological activities, including protection against atherosclerosis. In this study, the potential role of the individual cis-9, trans-11 and trans-10, cis-12 isomers of CLA in atherogenesis were compared with LA in the Syrian Golden hamster. Supplementation of a high-fat, high-cholesterol diet (HFHC) with 1% (w/w) cis-9, trans-11 CLA or trans-10, cis-12 CLA did not significantly affect plasma cholesterol levels compared to supplementation with 1% (w/w) LA. Very low density lipoprotein cholesterol (VLDL-C) was lower and plasma triglycerides (TG) were higher in diets where C18:2 fatty acid was added to the HFHC diet, but neither the cis-9, trans-11 CLA group nor trans-10, cis-12 CLA group was significantly different from the LA control group. CLA supplementation did not significantly affect low density lipoprotein cholesterol (LDL-C). Trans-10, cis-12 CLA increased high density lipoprotein cholesterol (HDL-C) levels compared to LA or cis-9, trans-11 CLA (P<0.02), and although the ratio of non-HDL-C:HDL-C in the cis-9, trans-11 CLA group (1.11+/-0.54) and the trans-10, cis-12 CLA group (1.11+/-0.21) was lower than the LA group (1.29+/-0.45), the reduction did not reach statistical significance. Atherosclerosis was assessed in the ascending aorta by measuring the number of aortic cross-sections containing Oil Red O-stained intimal lesions. Compared to the LA group (60+/-11%), both the cis-9, trans-11 CLA group (38+/-8%) and the trans-10, cis-12 CLA group (28+/-7%) had fewer sections displaying a fatty streak lesion, although the differences did not reach statistical significance. These results suggest that individual CLA isomers may reduce atherosclerotic lesion development in the hamster, but when compared to LA, the apparent atheroprotective effects do not correlate with beneficial changes in lipoprotein profile.     

Effects of conjugated linoleic acid isomers on monocyte, macrophage and foam cell phenotype in atherosclerosis

Conjugated linoleic acid (CLA) is a generic term denoting a group of naturally occurring isomers of linoleic acid (18:2, n6) that differ in the position or geometry (i.e. cis or trans) of their double bonds. The predominant isomers in ruminant fats are cis-9,trans-11 CLA (c9,t11-CLA), and trans-10,cis-12 CLA (t10,c12-CLA). The biological activities of CLA have received considerable attention because of its protective effects in cancer, immune function, obesity and atherosclerosis. Importantly, dietary administration of a blend of the two most abundant isomers of CLA, has been shown to inhibit the progression and induce the regression of pre-established atherosclerosis in the ApoE?/? murine model. Studies investigating the mechanisms involved in CLA induced protective effects are continually emerging with results from both in vitro and in vivo models yielding confounding and often inconsistent results depending on both the isomer of CLA and the species under investigation. The purpose of this review is to comprehensively discuss the effects of CLA on monocyte/macrophage function in atherosclerosis. This review also discusses the possible mechanisms through which CLA mediates its atheroprotective effects with a particular emphasis on the migratory capacity of the monocyte and the inflammatory and cholesterol homeostasis of the macrophage.     

Linoleic acid partially restores the triglyceride content of conjugated linoleic acid-treated cultures of 3T3-L1 preadipocytes

We have previously demonstrated that a crude mixture of commercially available conjugated linoleic acid (CLA) isomers suppressed triglyceride (TG) content and induced apoptosis in post-confluent cultures of murine 3T3-L1 preadipocytes. Furthermore, we found that 100 μM of trans-10, cis-12 isomer of CLA had a greater TG-lowering and apoptotic effect than the crude mixture of CLA isomers. Therefore, the purpose of this study was to: 1) compare the potencies of the two main isomers found in the crude mixture of CLA isomers, e.g. cis-9, trans-11 (41%) and trans-10, cis-12 (44%); and 2) determine if the TG-reducing actions of CLA could be attenuated by the addition of increasing levels of linoleic acid to the cultures. Preadipocyte differentiation was assessed on day 7 of the differentiation protocol by measuring TG content (per 10⁶ cells), cell size, and lipid staining. In experiment 1, post-confluent cultures of 3T3-L1 preadipocytes treated for the first 6 d of differentiation with 100 μM of a crude mixture of CLA isomers or 44 μM of trans-10, cis-12 CLA had less TG content than all other cultures. In contrast, cultures supplemented with 41 μM of the cis-9, trans-11 CLA isomer had the same amount of TG as the BSA controls. In experiment 2, post-confluent cultures of 3T3-L1 preadipocytes treated for the first 6 d of differentiation with 50 μM trans-10, cis-12 CLA had less TG content and a greater number of smaller cells (10–12.5 microns) compared to all other treatments. CLA-treated cultures supplemented with increasing levels of linoleic acid (50–200 μM) had greater TG contents and greater numbers of larger cells (15–20 microns) than cultures treated with 50 μM of the trans-10, cis-12 CLA isomer alone. These data demonstrate that: 1) the TG-lowering effects of the crude mixture of CLA isomers is due almost exclusively to the trans-10, cis-12 isomer; and 2) linoleic acid partially reverses CLA’s attenuation of TG content, suggesting that these unsaturated fatty acids may compete for incorporation into TG or phospholipid-derived eicosanoids that regulate preadipocyte differentiation.     

Trans-10, cis-12, but not cis-9, trans-11 CLA isomer,inhibits brown adipocyte thermogenic capacity

Conjugated linoleic acid (CLA) is reported to have health benefits, including reduction of body fat. Previous studies have shown that brown adipose tissue (BAT) is particularly sensitive to CLA-supplemented diet feeding. Most of them use mixtures containing several CLA isomers, mainly cis-9, trans-11 and trans-10, cis-12 in equal concentration. Our aim was to characterize the separate effects of both CLA isomers on thermogenic capacity in cultured brown adipocytes. The CLA isomers showed opposite effects. Hence, on the one hand, trans-10, cis-12 inhibited uncoupling protein (UCP) 1 induction by norepinephrine (NE) and produced a decrease in leptin mRNA levels. These effects were associated with a blockage of CCAAT-enhancer-binding protein-alpha and peroxisome proliferator-activated receptor-gamma(2) mRNA expression. On the other hand, cis-9, trans-11 enhanced the UCP1 elicited by NE, an effect reported earlier for polyunsaturated fatty acids and also observed here for linoleic acid. These findings could explain, at least in part, the effects observed in vivo when feeding a CLA mixture supplemented diet as a result of the combined action of CLA isomers (reduction of adipogenesis and defective BAT thermogenesis that could be through trans-10, cis-12 and enhanced UCP1 thermogenic capacity through cis-9, trans-11).     

Detection of conjugated dienoic fatty acids in human vascular smooth muscle cells treated with conjugated linoleic acid

Conjugated linoleic acids (CLA) have attracted scientific interest due to their potential beneficial effects on atherosclerosis. Recent studies demonstrated that conjugated metabolites of CLA are found in tissues of CLA-fed animals and cultured cells treated with CLA. This observation has gained in importance since it has recently been shown that these metabolites of CLA exert specific biological activities. Therefore, the present study aimed to explore the potential formation of metabolites of cis-9, trans-11 CLA, trans-10, cis-12 CLA and trans-9, trans-11 CLA in cells of the vascular wall, which has not yet been shown. Examination of fatty acid composition of total cell lipids using Ag+-HPLC, GC-FID and GC-MS analysis revealed a significant isomer-specific formation of conjugated metabolites of CLA such as CD16:2, CD20:2 and CD22:2 in human coronary artery smooth muscle cells treated with various CLA isomers. Different CD16:2/CLA ratios between various CLA isomers as observed in the present study indicate that fatty acid metabolism is differently affected by the configuration of the double bonds. In conclusion, the observation from the present study suggests that the effects of CLA in vascular cells might not only be mediated by CLA itself but also by its conjugated metabolites. Future studies using highly purified conjugated metabolites of CLA are necessary to study their role in mediating biological effects of CLA in cell culture systems.     

Incorporation profiles of conjugated linoleic acid isomers in cell membranes and their positional distribution in phospholipids

Although the conjugated linoleic acids (CLA) have several isomer-specific biological effects including anti-carcinogenic and anti-adipogenic effects, their mechanisms of action remain unclear. To determine their potential effects on membrane structure and function, we studied the incorporation profiles of four CLA isomers (trans-10 cis-12 (A), trans-9 trans-11 (B), cis-9 trans-11 (C), and cis-9 cis-11 (D)) in CHO and HepG2 cells. All four isomers were incorporated into cellular lipids as efficiently as linoleic acid (LA), with the majority of the incorporated CLA present in membrane rafts. Of the four isomers, only CLA-A increased the cholesterol content of the raft fraction. Over 50% of the incorporated CLAs were recovered in phosphatidylcholine of CHO cells, but in HepG2 the neutral lipids contained the majority of CLA. The desaturation index (18:1/18:0 and 16:1/16:0) was reduced by CLA-A, but increased by CLA-B, the effects being apparent mostly in raft lipids. The Δ? desaturase activity was inhibited by CLAs A and C. Unlike LA, which was mostly found in the sn-2 position of phospholipids, most CLAs were also incorporated significantly into the sn-1 position in both cell types. These studies show that the incorporation profiles of CLA isomers differ significantly from that of LA, and this could lead to alterations in membrane function, especially in the raft-associated proteins.     

Conjugated linoleic acids promote human fat cell apoptosis.

Conjugated linoleic acids (CLAs) are conjugated dienoic isomers of linoleic acid. Some isomers have been shown to reduce fat mass in animal and cell culture models. However, controversial results were obtained in studies of supplementation of CLAs in human subjects. In order to get more insights into the direct effects of CLAs on human fat cells, we have studied the influence of cis-9, trans-11 CLA and trans-10, cis-12 CLA on the biology of human SGBS preadipocytes and adipocytes. Both CLA isomers equally inhibited the proliferation of preadipocytes in a dose-dependent manner. Continuous treatment with 1-10 microM trans-10, cis-12 CLA, and to a weaker extent cis-9, trans-11 CLA, inhibited accumulation of lipids during adipogenic differentiation. Treatment with higher doses of CLA induced apoptosis in preadipocytes, in differentiating cells, and adipocytes. The trans-10, cis-12 isomer had a higher apoptotic potency in adipocytes than cis-9, trans-11 CLA. Taken together, the treatment of human preadipocytes and adipocytes with physiological relevant concentrations of CLAs resulted in an impairment of proliferation and differentiation and induction of apoptosis. The trans-10, cis-12 isomer was more potent than the cis-9, trans-11 isomer. Further clinical studies are needed to evaluate the effects of CLAs on human fat mass and metabolism in vivo.     

Inhibition of hepatic stearoyl-CoA desaturase activity by trans-10, cis-12 conjugated linoleic acid and its derivatives

Conjugated linoleic acid (CLA) has been reported to decrease stearoyl-CoA desaturase (SCD) activity by decreasing mRNA expression. This investigation was designed to determine whether structurally related compounds of CLA have a direct inhibitory effect on SCD activity. Trans-10,cis-12 CLA had strong inhibitory activity on SCD while cis-9,trans-11, and trans-9,trans-11 isomers had no effect. Trans-10 octadecenoate was not inhibitory, whereas cis-12 octadecenate was inhibitory, but not as effective as trans-10,cis-12 CLA. Of the oxygenated derivatives, 9-peroxy-cis/trans-10, trans-12 octadecadienoate was a more effective inhibitor than trans-10,cis-12 CLA, whereas 9-hydroxy-trans-10, cis-12 octadecadienoate was less effective. Interestingly, cis-11 octadecadienoate and cis-12 octadecen-10-ynoate were slightly inhibitory. However, trans-9 and trans-11 octadecenoates, and trans-9,cis-12 octadecadienoate were all inactive under test condition, as were linoleate, oleate, and arachidonate. Derivatives of CLA acid modified to alcohol, amide or chloride were all inactive. A cis-12 double bond appears to be a key structural feature for inhibiting SCD activity, especially when coupled with a trans-10 double, whereas a cis-11 double bond is less effective.     

Dietary conjugated linoleic acid has limited effects on tissue protein anabolism in sedentary and exercising adult rats

The effects of conjugated linoleic acid isomers (CLA) and endurance training on lean body mass are expected to result from their action on tissue protein metabolism. The aim of this study was to analyze their effects on protein metabolism in 2 muscles, the small intestine and liver of adult rats. Four-month-old male Wistar rats were fed diets containing either no CLA, cis-9, trans-11 CLA isomer (1 g.100 g(-1)), trans-10, cis-12 CLA isomer (1 g.100 g(-1)) or both isomers (1 g.100 g(-1) each) for 6 weeks. Half of the rats were subjected to endurance training by running on a treadmill. At the end of this period, the rats were injected with a flooding dose of (13)C-valine to determine protein synthesis rates in the post-absorptive (experiment 1) and in the post-prandial (experiment 2) states. No effect of CLA or endurance training were detected in the small intestine. Training reduced food intake and protein synthesis rates in the liver but no effect was found on the protein synthesis rates in muscles. In the post-absorptive state, protein synthesis rate was increased by feeding the trans-10, cis-12 CLA isomer alone in the liver (+9%) or in combination with the cis-9, trans-11 isomer in the gastrocnemius (+30%), mostly in sedentary rats. In the post-prandial state, the cis-9, trans-11 CLA isomer tended to reduce the protein synthesis rate in the gastrocnemius muscle. However, no effect of CLA was found on muscle protein amounts. In conclusion, CLA isomers would have limited but differential effects on tissue protein metabolism in adult rats.     

Dietary conjugated linoleic acid does not adversely affect bone mass in obese fa/fa or lean Zucker rats

Conjugated linoleic acid (CLA) elevates body ash in healthy animals. The objective of the present study was to determine if single or mixed CLA isomers improve bone mass in an obese and hyperinsulinemic state. Male (n = 120) lean and obese fa/fa Zucker rats (age, 6 weeks) were randomized to 8 weeks on a control diet or to 0.4% (w/w) cis-9, trans-11 CLA (Group 1); 0.4% (w/w) trans-10, cis-12 CLA (Group 2); 0.4% (w/w) cis-9, trans-11 CLA and 0.4% (w/w) trans-10, cis-12 CLA (Group 3); 0.4% (w/w) cis-9, trans-11 CLA, 0.4% (w/w) trans-10, cis-12 CLA, and traces of other CLA isomers (Group 4); and 0.4% (w/w) cis-9, trans-11 CLA, 0.4% (w/w) trans-10, cis-12 CLA, and 0.3% (w/w) other CLA isomers (Group 5). Bone area (BA), bone mineral content (BMC), and bone mineral density (BMD) of the whole body, spine, and femur were measured at baseline (6 weeks) and at 14 weeks of age. Effects of genotype, diet, and genotype x diet interactions were assessed using factorial analysis of variance. At 6 and 14 weeks, whole-body BA and BMC were lower in lean rats compared with fa/fa rats. Similarly, at 14 weeks, fa/fa rats had a higher spine and femur BMD despite a lower femur weight. The fa/fa rats in Groups 4 and 5 had higher adjusted whole-body BMC compared with Group 3, but not with Group 1, Group 2, or the control. In lean rats, Group 3 had a greater adjusted whole-body BMC than Groups 1 and 2, but not Group 4, Group 5, or the control. Thus, commercially available CLA mixtures and single CLA isomers do not affect bone mass in a hyperinsulinemic, obese state.     

Conjugated linoleic acid accumulation via 10-hydroxy-12-octadecaenoic acid during microaerobic transformation of linoleic acid by Lactobacillus acidophilus

Specific isomers of conjugated linoleic acid (CLA), a fatty acid with potentially beneficial physiological and anticarcinogenic effects, were efficiently produced from linoleic acid by washed cells of Lactobacillus acidophilus AKU 1137 under microaerobic conditions, and the metabolic pathway of CLA production from linoleic acid is explained for the first time. The CLA isomers produced were identified as cis-9, trans-11- or trans-9, cis-11-octadecadienoic acid and trans-9, trans-11-octadecadienoic acid. Preceding the production of CLA, hydroxy fatty acids identified as 10-hydroxy-cis-12-octadecaenoic acid and 10-hydroxy-trans-12-octadecaenoic acid had accumulated. The isolated 10-hydroxy-cis-12-octadecaenoic acid was transformed into CLA during incubation with washed cells of L. acidophilus, suggesting that this hydroxy fatty acid is one of the intermediates of CLA production from linoleic acid. The washed cells of L. acidophilus producing high levels of CLA were obtained by cultivation in a medium containing linoleic acid, indicating that the enzyme system for CLA production is induced by linoleic acid. After 4 days of reaction with these washed cells, more than 95% of the added linoleic acid (5 mg/ml) was transformed into CLA, and the CLA content in total fatty acids recovered exceeded 80% (wt/wt). Almost all of the CLA produced was in the cells or was associated with the cells as free fatty acid.     

Potentiation of the anti-tumour effect of docetaxel by conjugated linoleic acids (CLAs) in breast cancer cells in vitro

Response rates of tumours to docetaxel (DOCT) are 45-60% in advanced breast cancer but problems associated with side effects, drug resistance and high costs occur. Conjugated linoleic acids (CLAs) also have anti-tumorigenic activity that elicits similar changes in oncogene expression to DOCT and could augment DOCT efficacy. CLA isomers appear to differ in cytotoxicity toward cancer cells. Effects of two CLA isomers on cytotoxicity of DOCT in breast cancer cells (MCF-7; MDA-MB-231) in vitro were assessed. Cells were incubated up to 72 h with 40 microM each of LA or CLA isomers (cis-9, trans-10 CLA, or trans-10, cis-12 CLA) or a 50:50 isomer mix, alone or with DOCT (0-64 microM); a pilot study determined IC(50) and IC(70) concentrations. Treatments were concurrent (CLA and DOCT together) or sequential (CLA then DOCT). MTT assay determined cell viability. Trans-10, cis-12 CLA was the most effective fatty acid (P<0.001) and increased with treatment time. IC(50) and IC(70) concentrations of DOCT were determined, concurrently or sequentially, with and without fatty acids, in the two cell types. Concurrent treatment with trans-10, cis-12 CLA and DOCT augmented inhibition of cell growth in one or both cell lines (decreased IC(50) and IC(70) in MCF-7; P<0.05 but only IC(50) in MDA-MB-231; P<0.05). CLA mix reduced IC(50) and IC(70) in MDA-MB-231 (P<0.001) but not in MCF-7. Cis-9, trans-11 CLA and LA had no effect. Sequential treatment with CLAs then DOCT reduced IC(50) and IC(70) in MCF-7 but not in MDA-MB-231. The latter had increased IC(50) and IC(70) with LA treatment (P<0.05) and increased IC(70) with cis-9, trans-11 CLA (P<0.05) with sequential but not concurrent treatment. Longer pre-incubation times with trans-10, cis-12 CLA (24-72 h) elicited greater reductions in IC(50) and IC(70) in MCF-7 cells. Results show that CLA isomers augment anti-tumour effects of docetaxel in breast cancer cells and suggest possible dual treatment regimens.