Potent inhibitory effect of trans9, trans11 isomer of conjugated linoleic acid on the growth of human colon cancer cells

This study compared the growth inhibitory effects of pure conjugated linoleic acid (CLA) isomers [cis(c)9,c11-CLA, c9,trans(t)11-CLA, t9,t11-CLA, and t10,c12-CLA] on human colon cancer cell lines (Caco-2, HT-29 and DLD-1). When Caco-2 cells were incubated up to 72 h with 200 μM, each isomer, even in the presence of 10% fetal bovine serum (FBS), cell proliferation was inhibited by all CLA isomers in a time-dependent manner. The strongest inhibitory effect was shown by t9,t11-CLA, followed by t10,c12-CLA, c9,c11-CLA and c9,t11-CLA, respectively. The strongest effect of t9,t11-CLA was also observed in other colon cancer cell lines (HT-29 and DLD-1). The order of the inhibitory effect of CLA isomer was confirmed in the presence of 1% FBS. CLA isomers supplemented in the culture medium were readily incorporated into the cellular lipids of Caco-2 and changed their fatty acid composition. The CLA contents in cellular lipids were 26.2±2.7% for t9,t11-CLA, 35.9±0.3% for c9,t11-CLA and 46.3±0.8% for t10,c12-CLA, respectively. DNA fragmentation was clearly recognized in Caco-2 cells treated with t9,t11-CLA. This apoptotic effect of t9,t11-CLA was dose- and time-dependent. DNA fragmentation was also induced by 9c,11t-CLA and t10,c12-CLA. However, fragmentation levels with both isomers were much lower than that with t9,t11-CLA. t9t11-CLA treatment of Caco-2 cells decreased Bcl-2 levels in association with apoptosis, whereas Bax levels remained unchanged. These results suggest that decreased expression of Bcl-2 by t9t11-CLA might increase the sensitivity of cells to lipid peroxidation and to programmed cell death, apoptosis.     

Differential incorporation of dietary conjugated linolenic and linoleic acids into milk lipids and liver phospholipids in lactating and suckling rats

Interest in health benefits of conjugated fatty acids is growing. The present study compared the incorporation pattern of dietary conjugated linolenic acids (CLnA) into milk with that of conjugated linoleic acids (CLA). Lactating Sprague-Dawley rats (Day 1) were divided into five groups fed the control diet (n=4) or one of four experimental diets supplemented with 1–2% CLA or CLnA mixture (n=8 each). Supplementation of 1% and 2% CLA led to enrichment of 4.17% and 8.57% CLA, respectively, while supplementation of 1% and 2% CLnA resulted in enrichment of only 0.98% and 1.71% CLnA in the milk lipids, demonstrating the transfer of CLnA from maternal diet to milk was discriminated. When the lactating rats were given a diet containing a CLnA mixture of 9t,11t,13t-, 9c,11t,13t- and 9c,11t,13c-CLnA isomers, two CLA isomers, namely, 9t,11t (0.59–0.90%) and 9c,11t (1.21–1.96%), were found in the milk, suggesting that three CLnA isomers were Δ-13 saturated. Dietary CLnA at 1–2% had no effect on liver phospholipid (PL) fatty acid composition of both maternal and suckling rats, whereas dietary CLA increased docosahexaenoic acid (4c,7c,10c,13c,16c,19c-22:6) and palmitic acid (16:0) proportionally in the PL of maternal rats, but it suppressed 16:0 in the PL of suckling rats. It is concluded that maternal rats incorporate CLnA isomers into milk differently from that of CLA isomers. Most interesting is that maternal rats can metabolically convert CLnA to CLA.     

Conversion of t11t13 CLA into c9t11 CLA in Caco-2 cells and inhibition by sterculic oil

Background

Conjugated linoleic acids (CLA), and principally c9t11 CLA, are suspected to have numerous preventive properties regarding non-infectious pathologies such as inflammatory diseases, atherosclerosis and several types of cancer. C9t11 CLA is produced in the rumen during biohydrogenation of linoleic acid, but can also be synthesized in mammalian tissues from trans-vaccenic acid (C18:1 t11) through the action of delta-9 desaturase (D9D). For several years, it is also known that c9t11 CLA can be synthesized from conjugated linolenic acids (CLnA), i.e. c9t11c13 CLnA and c9t11t13 CLnA. This study aimed at investigating to which extent and by which route c9t11 CLA can be produced from another isomer of CLA, the t11t13 CLA that is structurally very similar to c9t11t13 CLnA, in Caco-2 cells.

Methodology/Principal Findings

Caco-2 cells were incubated for 24 h with 20 µmol/l of t11t13 CLA in the absence or presence of sterculic oil used as an inhibitor of D9D. Caco-2 cells were able to convert t11t13 CLA into c9t11 CLA, and c9t11t13 CLnA was formed as an intermediate compound. In the presence of sterculic oil, the production of this intermediate was decreased by 46% and the formation of c9t11 CLA was decreased by 26%. No other metabolite was detected.

Conclusions/Significance

These results not only highlight the conversion of t11t13 CLA into c9t11 CLA but demonstrate also that this conversion involves first a desaturation step catalysed by D9D to produce c9t11t13 CLnA and then the action of another enzyme reducing the double bond on the Δ13 position.     

植物乳杆菌ZS2058在磷酸盐缓冲液体系中生物转化共轭亚油酸

植物乳杆菌ZS2058是从泡菜中筛选到一株具有转化共轭亚油酸能力的乳酸菌。该菌株在MRS培养基中经0.5mg/mL的亚油酸诱导培养后,所获得的菌体细胞具有较强的转化能力。文中就植物乳杆菌ZS2058水洗细胞在磷酸盐缓冲液体系中生物转化共轭亚油酸进行了深入研究。在非厌氧条件下,植物乳杆菌ZS2058在亚油酸浓度为1mg/mL,湿细胞质量浓度约为150mg/mL,120r/min、37℃的条件下反应24h后,能将亚油酸转化为共轭亚油酸和羟基脂肪酸,其中c9,t11-CLA占所产生的CLA总量的96.4%,产量可高达312.4μg/mL,说明该菌株有很强的专一性。随着反应进一步进行,反应至36h时,c9,t11-CLA含量逐渐减少,伴随着大量羟基脂肪酸的产生;并且,以CLA(c9,t11-CLA和t10,c12-CLA的混合样品)为底物进行反应时,c9,t11-CLA被转化为羟基脂肪酸。由此可知,c9,t11-CLA可能是该菌株生物转化LA过程中的一个中间产物。     

Dietary trans-10,cis-12 conjugated linoleic acid induces hyperinsulinemia and fatty liver in the mouse

Conjugated linoleic acids (CLA) are a class of positional, geometric, conjugated dienoic isomers of linoleic acid (LA). Dietary CLA supplementation results in a dramatic decrease in body fat mass in mice, but also causes considerable liver steatosis. However, little is known of the molecular mechanisms leading to hepatomegaly. Although c9,t11- and t10,c12-CLA isomers are found in similar proportions in commercial preparations, the respective roles of these two molecules in liver enlargement has not been studied. We show here that mice fed a diet enriched in t10,c12-CLA (0.4% w/w) for 4 weeks developed lipoatrophy, hyperinsulinemia, and fatty liver, whereas diets enriched in c9,t11-CLA and LA had no significant effect. In the liver, dietary t10,c12-CLA triggered the ectopic production of peroxisome proliferator-activated receptor gamma (PPARgamma), adipocyte lipid-binding protein and fatty acid transporter mRNAs and induced expression of the sterol responsive element-binding protein-1a and fatty acid synthase genes. In vitro transactivation assays demonstrated that t10,c12- and c9,t11-CLA were equally efficient at activating PPARalpha, beta/delta, and gamma and inhibiting liver-X-receptor. Thus, the specific effect of t10,c12-CLA is unlikely to result from direct interaction with these nuclear receptors. Instead, t10,c12-CLA-induced hyperinsulinemia may trigger liver steatosis, by inducing both fatty acid uptake and lipogenesis.     

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.     

Differential effects of conjugated linoleic acid isomers on macrophage glycerophospholipid metabolism

Conjugated linoleic acids (CLA) are dietary fatty acids. Whereas cis-9,trans-11-(c9,t11)-CLA can be found in meat and dairy products, trans-9,trans-11-(t9,t11)-CLA is a constituent of vegetable oils. Previous studies showed that these two isomers activate different nuclear receptors and, thus, expression of genes related to lipid metabolism. Here we show that these CLA isomers are differentially elongated and desaturated in primary monocyte-derived macrophages isolated from healthy volunteers by using gas chromatography-mass spectrometry (GC-MS). We further demonstrate that c9,t11-CLA incorporates in phosphatidylcholine (PC) and phosphatidylethanolamine (PE) species and activates de novo glycerophospholipid synthesis by quantitative electrospray ionization-tandem mass spectrometry (ESI-MS/MS). c9,t11-CLA leads to strong shifts of the species profiles to PC 18:2/18:2 and PE 18:2/18:2, which are due to de novo synthesis and fatty acid remodeling. In contrast, t9,t11-CLA is preferentially bound to neutral lipids, including triglycerides and cholesterol esters. Taken together our results show that c9,t11-CLA and t9,t11-CLA have differential effects on PC and PE metabolism. Moreover, these data demonstrate that the structure of fatty acids not only determines their incorporation into lipid classes but also modulates the kinetics of lipid metabolism, particularly PC synthesis.     

cis-9,trans-11 conjugated linoleic acid stimulates expression of angiopoietin like-4 in the placental extravillous trophoblast cells

A number of studies have been carried out to examine the biological function of conjugated linoleic acid (CLA) and its potential health benefits. However, not much is known about how CLA isomers mediate their effect on angiogenesis and vascularization during early placentation. In this paper we demonstrate that cis-9,trans-11(c9,t11)-CLA stimulated the expression of angiopoietin like-4 (ANGPTL4) mRNA and protein accompanied by tube formation in first trimester placental trophoblast cells, HTR8/SVneo whereas the other CLA isomer, trans-10,cis-12 (t10,c12)-CLA had no such effects. c9,t11-CLA however did not stimulate expression of the most potent angiogenic factor, vascular endothelial growth factor (VEGF) in these cells. Silencing ANGPTL4 in these cells significantly reduced the stimulatory effect of c9,t11-CLA on tube formation, indicating the involvement of ANGPTL4. In addition, c9,t11-CLA increased the mRNA expression of several pro-angiogenic factors such as fatty acid binding protein-4 (FABP4), cyclooxygenase-2 (COX-2) and adipose differentiation-related protein (ADRP) in HTR8/SVneo cells. c9,t11-CLA also induced the uptake of docosahexaenoic acid, 22:6n − 3 (DHA), a stimulator of tube formation in these cells. Triacsin C, an acylCoA synthetase inhibitor, attenuated c9,t11-CLA induced DHA uptake, tube formation and cellular proliferation in HTR8/SVneo cells.     

t10c12-CLA maintains higher bone mineral density during aging by modulating osteoclastogenesis and bone marrow adiposity

Conjugated linoleic acid (CLA) has been shown to positively influence calcium and bone metabolism. Earlier, we showed that CLA (equal mixture of c9t11-CLA and t10c12-CLA) could protect age-associated bone loss by modulating inflammatory markers and osteoclastogenesis. Since, c9t11-CLA and t10c12-CLA isomers differentially regulate functional parameters and gene expression in different cell types, we examined the efficacy of individual CLA isomers against age-associated bone loss using 12 months old C57BL/6 female mice fed for 6 months with 10% corn oil (CO), 9.5% CO + 0.5% c9t11-CLA, 9.5% CO + 0.5% t10c12-CLA or 9.5% CO + 0.25% c9t11-CLA + 0.25% t10c12-CLA. Mice fed a t10c12-CLA diet maintained a significantly higher bone mineral density (BMD) in femoral, tibial and lumbar regions than those fed CO and c9t11-CLA diets as measured by dual-energy-X-ray absorptiometry (DXA). The increased BMD was accompanied by a decreased production of osteoclastogenic factors, that is, RANKL, TRAP5b, TNF-alpha and IL-6 in serum. Moreover, a significant reduction of high fat diet-induced bone marrow adiposity was observed in t10c12-CLA fed mice as compared to that of CO and c9t11-CLA fed mice, as measured by Oil-Red-O staining of bone marrow sections. In addition, a significant reduction of osteoclast differentiation and bone resorbing pit formation was observed in t10c12-CLA treated RAW 264.7 cell culture stimulated with RANKL as compared to that of c9t11-CLA and linoleic acid treated cultures. In conclusion, these findings suggest that t10c12-CLA is the most potent CLA isomer and it exerts its anti-osteoporotic effect by modulating osteoclastogenesis and bone marrow adiposity.     

Incorporation of cis-9,trans-11 or trans-10,cis-12 conjugated linoleic acid into plasma and cellular lipids in healthy men

This study investigated the incorporation of cis-9,trans-11 conjugated linoleic acid (c9,t11 CLA) and trans-10,cis-12-CLA (t10,c12 CLA) into plasma and peripheral blood mononuclear cell (PBMC) lipids when consumed as supplements highly enriched in these isomers. Healthy men (n = 49, age 31 +/- 8 years) consumed one, two, and four capsules containing approximately 600 mg of either c9,t11 CLA or t10,c12 CLA per capsule for sequential 8 week periods followed by a 6 week washout before consuming the alternative isomer. Both isomers were incorporated in a dose-dependent manner into plasma phosphatidylcholine (PC) (c9,t11 CLA r = 0.779, t10,c12 CLA r = 0.738; P < 0.0001) and cholesteryl ester (CE) (c9,t11 CLA r = 0.706, t10,c12 CLA r = 0.788; P < 0.0001). Only t10,c12 CLA was enriched in plasma nonesterified fatty acids. Both c9,t11 CLA and t10,c12 CLA were incorporated linearly into PBMC total lipids (r = 0.285 and r = 0.273, respectively; P < 0.0005). The highest concentrations of c9,t11 CLA and t10,c12 CLA in PBMC lipids were 3- to 4-fold lower than those in plasma PC and CE. These data suggest that the level of intake is a major determinant of plasma and PBMC CLA content, although PBMCs appear to incorporate both CLA isomers less readily.     

Conjugated linoleic acid can prevent tumor necrosis factor gene expression by inhibiting nuclear factor binding activity in peripheral blood mononuclear cells from weaned pigs challenged with lipopolysaccharide

Twenty-four weanling barrows were fed corn-soybean diets supplemented with 2% conjugated linolenic acid (CLA) or soybean oil. On day 14 and 21, pigs were injected intraperitoneally with lipopolysaccharide (LPS) or sterile saline. Plasma samples were collected 2h after injection. Peripheral blood mononuclear cells (PBMC) were also collected on day 21, 2 h after injection to determine tumor necrosis factor-alpha (TNF-alpha) production and its mRNA expression. The results indicate that dietary CLA inhibited the production of TNF-alpha by pig PBMC both at the protein and mRNA expression level. In a second experiment, PBMC, collected from a healthy pig, were incubated with either c9,t11-CLA or t10,c12-CLA, or without CLA and stimulated with LPS. Both CLA isomers inhibited LPS-stimulated TNF-alpha production and expression, which may be partially due to inhibition of the binding activity of nuclear factor-kappaB. The t10,c12 isomer was more effective than the c9,t11-CLA isomer in reducing TNF-alpha levels and nuclear factor-kappaB activation.     

The effect of conjugated linoleic acid on the viability and metabolism of human osteoblast-like cells

Studies in experimental animals and murine osteoblast cells in culture have produced conflicting findings on the effect of conjugated linoleic acid (CLA) on bone formation. The present study investigated the influence of CLA on viability and metabolism of two human osteoblast-like cell lines (SaOS2 and MG63). Both cell lines were exposed to increasing concentrations (0-50 microM) of CLA either as pure cis (c) 9: trans (t) 11 and t10:c12 CLA isomers or a blend of isomers, or linoleic acid (C18:2). Cell cytotoxicity and degree of DNA fragmentation were unaffected by any fatty acid treatment. PGE2 biosynthesis by both cell lines was variably reduced by CLA isomer blend and t10:c12 CLA, but not c9:t11 CLA. Alkaline phosphatase activity was variably increased by all CLA treatments. These results suggest a lack of cytotoxic effect of CLA on human osteoblast-like cells and tentatively suggest a possible beneficial effect on bone formation in humans.     

Effect of silage type and concentrate level on conjugated linoleic acids, trans-C18:1 isomers and fat content in milk from dairy cows

The objective of the study was to examine how the fatty acid composition of milk especially concentrations of conjugated linoleic acids (CLA) and trans-C18:1 isomers and milk fat percentage were affected by silage type and concentrate level. Forty dairy cows were blocked and randomly assigned to one of four diets in a 2 x 2 factorial arrangement of treatments and a six week experimental period. Treatments were total mixed rations with maize (M) or grass (G) silage differing in polyunsaturated fatty acid (PUFA) profile and starch content, combined with a high (H) or a low (L) level of concentrate (with or without grain). Treatments had no significant effect on milk, protein and lactose yield, but energy corrected milk yield, milk fat percentage and fat yield was lower and protein percentage higher for maize compared with grass silage diets. Overall, maize silage diets resulted in higher concentrations of CLA isomers compared with grass silage diets, but there was a significant interaction between silage type and concentrate level for concentrations of cis9,trans11-CLA; trans10,cis12-CLA; trans11-C18:1 and trans10-C18:1. A high level of concentrate increased trans10,cis12-CLA and trans10-C18:1 and reduced cis9,trans11-CLA and trans11-C18:1 when maize but not grass silage was provided. The results suggest that high levels of concentrate (grain) do not significantly alter the pattern of PUFA biohydrogenation in the rumen, the concentration of CLA and trans-C18:1 isomers in milk or cause milk fat depression unless combined with forage naturally high in starch and C18:2n-6 such as maize silage.     

Isomer-specific actions of conjugated linoleic acid on muscle glucose transport in the obese Zucker rat

The fatty acid-conjugated linoleic acid (CLA) enhances glucose tolerance and insulin action on skeletal muscle glucose transport in rodent models of insulin resistance. However, no study has directly compared the metabolic effects of the two primary CLA isomers, cis-9,trans-11-CLA (c9,t11-CLA) and trans-10,cis-12-CLA (t10,c12-CLA). Therefore, we assessed the effects of a 50:50 mixture of these two CLA isomers (M-CLA) and of preparations enriched in either c9,t11-CLA (76% enriched) or t10,c12-CLA (90% enriched) on glucose tolerance and insulin-stimulated glucose transport in skeletal muscle of the insulin-resistant obese Zucker (fa/fa) rat. Animals were treated daily by gavage with either vehicle (corn oil), M-CLA, c9,t11-CLA, or t10,c12-CLA (all CLA treatments at 1.5 g total CLA/kg body wt) for 21 consecutive days. During an oral glucose tolerance test, glucose responses were reduced (P < 0.05) by 10 and 16%, respectively, in the M-CLA and t10,c12-CLA animals, respectively, whereas insulin responses were diminished by 21 and 19% in these same groups. There were no significant alterations in these responses in the c9,t11-CLA group. Insulin-mediated glucose transport activity was enhanced by M-CLA treatment in both type I soleus (32%) and type IIb epitrochlearis (58%) muscles and by 36 and 48%, respectively, with t10,c12-CLA. In the soleus, these increases were associated with decreases in protein carbonyls (index of oxidative stress, r = -0.616, P = 0.0038) and intramuscular triglycerides (r = -0.631, P = 0.0028). Treatment with c9,t11-CLA was without effect on these variables. These results suggest that the ability of CLA treatment to improve glucose tolerance and insulin-stimulated glucose transport activity in insulin-resistant skeletal muscle of the obese Zucker rat are associated with a reduction in oxidative stress and muscle lipid levels and can be specifically ascribed to the actions of the t10,c12 isomer. In the obese Zucker rat, the c9,t11 isomer of CLA is metabolically neutral.     

Distribution of conjugated linoleic acid and metabolites in different lipid fractions in the rat liver.

Conjugated linoleic acid (CLA) is known to provide certain health benefits in experimental animal models. The major CLA isomer in food is c 9,t11-CLA. A primary objective of this study was to investigate the uptake of c 9,t11-CLA and its downstream metabolites into various lipid fractions in the liver of rats fed either a high or low CLA diet (containing 0.1 or 0.8 g CLA/100 g diet, respectively). As expected, the levels of all conjugated diene (CD) fatty acids (CD 18:2 + CD 18:3 + CD 20:3 + CD 20:4) were elevated about 8-fold in the high CLA diet group. However, there was no change in the distribution of CLA and CLA metabolites into various lipid fractions due to CLA intake. Unlike linoleic acid or gamma-linolenic acid, which were distributed mainly in phospholipids, CD 18:2, CD 18:3, and CD 20:3 were incorporated primarily in neutral lipid. Furthermore, the incorporation of all nonconjugated unsaturated fatty acids was not perturbed by CLA. Regardless of the level of CLA in the diet, CD 20:4 was predominantly enriched in phosphatidylserine and phosphatidylinositol. In contrast, arachidonic acid was primarily enriched in phosphatidylcholine and less so in phosphatidylethanolamine. The above findings may have potential implication regarding the role of CLA in modulating eicosanoid metabolism.     

共轭亚油酸生理功能及微生物合成调控研究进展

共轭亚油酸(conjugated linoleic acid,CLA)是一种新型功能性油脂,顺9,反11-十八碳二烯酸(c9,t11-CLA)和反10,顺12-十八碳二烯酸(t10,c12-CLA)由于具有比其他异构体更强的生理功能得到广泛关注和研究.微生物合成CLA具有安全性高、选择特异性强等特点,研究CLA产量提高...     

Trans-10, cis-12 conjugated linoleic acid induced cell death in human colon cancer cells through reactive oxygen species-mediated ER stress

Dietary conjugated linoleic acids (CLA) are fatty acid isomers with anticancer activities produced naturally in ruminants or from vegetable oil processing. The anticancer effects of CLA differ upon the cancer origin and the CLA isomers. In this study, we carried out to precise the effects of CLA isomers, c9,t11 and t10,c12 CLA, on mechanisms of cell death induction in colon cancer cells. We first showed that only t10,c12 CLA treatment (25 and 50 μM) for 72 h triggered apoptosis in colon cancer cells without affecting viability of normal-derived colon epithelial cells. Exposure of colon cancer cells to t10,c12 CLA activated ER stress characterized by induction of eIF2α phoshorylation, splicing of Xbp1 mRNA and CHOP expression. Furthermore, we evidenced that inhibition of CHOP expression and JNK signaling decreased t10,c12 CLA-mediated cancer cell death. Finally, we showed that CHOP induction by t10,c12 CLA was dependent on ROS production and that the anti-oxidant N-acetyl-cysteine reduced CHOP induction-dependent cell death. These results highlight that t10,c12 CLA exerts its cytotoxic effect through ROS generation and a subsequent ER stress-dependent apoptosis in colon cancer cells.     

Cis-9,trans-11-conjugated linoleic acid affects lipid raft composition and sensitizes human colorectal adenocarcinoma HT-29 cells to X-radiation

Background

Investigations concerned the mechanism of HT-29 cells radiosensitization by cis-9,trans-11-conjugated linoleic acid (c9,t11-CLA), a natural component of human diet with proven antitumor activity.

Methods

The cells were incubated for 24 h with 70 μM c9,t11-CLA and then X-irradiated. The following methods were used: gas chromatography (incorporation of the CLA isomer), flow cytometry (cell cycle), cloning (survival), Western blotting (protein distribution in membrane fractions), and pulse-field gel electrophoresis (rejoining of DNA double-strand breaks). In parallel, DNA-PK activity, γ-H2AX foci numbers and chromatid fragmentation were estimated. Gene expression was analysed by RT-PCR and chromosomal aberrations by the mFISH method. Nuclear accumulation of the EGF receptor (EGFR) was monitored by ELISA.

Results and conclusions

C9,t11-CLA sensitized HT-29 cells to X-radiation. This effect was not due to changes in cell cycle progression or DNA-repair-related gene expression. Post-irradiation DSB rejoining was delayed, corresponding with the insufficient DNA-PK activation, although chromosomal aberration frequencies did not increase. Distributions of cholesterol and caveolin-1 in cellular membrane fractions changed. The nuclear EGFR translocation, necessary to increase the DNA-PK activity in response to oxidative stress, was blocked. We suppose that c9,t11-CLA modified the membrane structure, thus disturbing the intracellular EGFR transport and the EGFR-dependent pro-survival signalling, both functionally associated with lipid raft properties.

General Significance

The results point to the importance of the cell membrane interactions with the nucleus after injury inflicted by X -rays. Compounds like c9,t11-CLA, that specifically alter membrane properties, could be used to develop new anticancer strategies.     

Cytotoxic effects of the conjugated linoleic acid isomers t10c12, c9t11-CLA and mixed form on rat hepatic stellate cells and CCl4-induced hepatic fibrosis

Rat hepatic stellate cells (HSC-T6) were incubated for 24 h with 10-180 microM of t10c12 (98%), c9t11 (96%) and a mixed form (c9,t11:t10,c12; 41%:44%) of conjugated linoleic acid (CLA). The MTS dye reduction was measured to verify cell viability in a dose-dependent manner. Among the three CLAs, c9,t11-CLA exhibited the most intense cytotoxic effect on HSCs, the survival rate of which was reduced to 60% under 80 microM of treatment, while cell survival was slightly affected by the mixed form. Three CLA-induced cell deaths were determined by measuring DNA fragmentation using 4',6-diamidino-2-phenylindole staining. The degrees of DNA fragmentation were the most severe in HSC treated with 80 microM of c9,t11-CLA. The mitogen-activated protein kinase/extracellular signal-regulated kinase-kinase and mitogen-activated or extracellular signal-regulated protein kinase (MEK) 1 and 2 were not activated in the t10,c12-CLA treatment. This suggests that the MEK-dependent apoptosis signal is crucial in HSC, which is induced by c9,t11 and mixed CLA. In order to evaluate the protective effect of CLA on carbon tetrachloride (CCl4)-induced hepatic fibrosis in vivo, animals were treated with 10% CCl4 to induce hepatic fibrosis during all experimental periods. Rats were divided into two treatment groups: (1) control diet with tap water ad libitum (n=15) and (2) 1% CLA diet with tap water ad libitum (n=15). In the CLA-supplemented rat livers, alpha-smooth muscle actin-positive cells were significantly reduced around the portal vein. In addition, collagen fibers were not detected in the CLA-treated group. These results suggest that 9c,11t-CLA influences cytotoxic effect on HSC in an MEK-dependent manner and preserving liver from fibrosis.