Preventive effect of t,t-conjugated linoleic acid on 12-O-tetradecanoylphorbol-13-acetate-induced inhibition of gap junctional intercellular communication in human mammary epithelial MCF-10A cells.

The anti-tumor promotional effects of t9,t11-conjugated linoleic acid (t9,t11-CLA) and t10,t12-CLA were evaluated on the 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced inhibition of gap junctional intercellular communication (GJIC) in the human mammary epithelial cell line MCF-10A. The results were compared to those obtained from c9,t11-CLA, which is a more effective anti-tumor promoter on TPA-induced GJIC inhibition in MCF-10A cells than t10,c12-CLA. Cells were treated with 20 µM t9,t11-CLA, t10,t12-CLA, or c9,t11-CLA for 24 h followed by 60 nM TPA for 1 h. Both t9,t11-CLA and t10,t12-CLA equally protected MCF-10A cells from TPA-induced inhibition of GJIC with inferior efficacy to c9,t11-CLA.The protection was due to the ameliorated phosphorylation of connexin43 via suppression of extracellular signal-regulated kinases (ERK1/2) activation. Suppression of TPA-induced reactive oxygen species (ROS) generation by t9,t11-CLA and t10,t12-CLA was less effective, relative to c9,t11-CLA. The results suggest that the anti-promotional activities of t9,t11-CLA and t10,t12-CLA are equal but less potent than c9,t11-CLA in TPA-treated MCF-10A cells. The activity might be mediated by the attenuation of ROS production in MCF-10A cells by preventing the downregulation of GJIC during the cancer promotion stage.

Attenuation of 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced gap junctional intercellular communication (GJIC) inhibition in MCF-10A cells by c9,t11-conjugated linoleic acid.

The protective effect of c9,t11-conjugated linoleic acid (CLA) on the inhibition of gap junctional intercellular communication (GJIC) was examined in a human mammary epithelial cell line (MCF-10A) treated with 12-O-tetradecanoylphorbol-13-acetate (TPA), relative to t10,c12-CLA isomer. TPA inhibited GJIC in a dose-dependent and reversible manner and was associated with connexin 43 phosphorylation. Pretreatment of 20 µM c9,t11-CLA for 24 h prior to 60 nM TPA for 1 h prevented the inhibition of GJIC by reducing the phosphorylation of connexin 43 via suppressing extracellular signal-regulated kinases (ERK1/2) activation. Reactive oxygen species (ROS) accumulation by TPA was attenuated by c9,t11-CLA. The efficacy of c9,t11-CLA in protecting inhibition of GJIC, connexin 43 phosphorylation, and ROS production was superior to that of t10,c12-CLA. These results suggest that c9,t11-CLA, including t10,c12-CLA, prevents the carcinogenesis of MCF-10A cells by protecting down-regulation of GJIC during the cancer promotion stage, and lack of their toxicities could be an excellent indicator for the chemoprevention of breast cancer.

Superior anticarcinogenic activity of trans,trans-conjugated linoleic acid in N-methyl-N-nitrosourea-induced rat mammary tumorigenesis.

The anticarcinogenic activity of a mixture of trans,trans-conjugated linoleic acid (trans,trans-CLA) was investigated in rat mammary tumorigenesis induced by N-methyl-N-nitrosourea (MNU), with references to cis-9,trans-11-CLA and trans-10,cis-12-CLA isomers. Female, 7-week-old Sprague-Dawley rats were intraperitoneally injected with MNU (50 mg/kg of body weight) and then subjected to one of five diets (control, 1% trans,trans-CLA, 1% cis-9,trans-11-CLA, 1% trans-10,cis-12-CLA, and 1% linoleic acid; 8 rats/group) for 16 weeks. Food and water were made available ad libitum. trans,trans-CLA significantly (p < 0.05) reduced tumor incidence, number, multiplicity, and size and significantly (p < 0.05) increased apoptosis, relative to cis-9,trans-11-CLA and trans-10,cis-12-CLA. The molecular mechanism of trans,trans-CLA was elucidated by measuring apoptosis-related gene products and fatty acid composition in tumors. trans,trans-CLA led to increases in the p53 protein and Bax protein levels but suppressed the expression of Bcl-2 protein. The activation of caspase-3 led to the cleavage of poly(ADP-ribose) polymerase, which resulted in the execution of apoptosis. In addition, trans,trans-CLA reduced cytosolic phospholipase A2, cyclooxygenease-2, and peroxisome proliferator-activated receptor gamma protein levels. These results suggest that the trans,trans-CLA inhibits MNU-induced rat mammary tumorigenesis through the induction of apoptosis in conjunction with the reduction of arachidonic acid metabolites and that the efficacy of trans,trans-CLA is superior to cis-9,trans-11-CLA and trans-10,cis-12-CLA.

Semipurified fractions from the submerged-culture broth of Agaricus blazei Murill reduce blood glucose levels in streptozotocin-induced diabetic rats.

Hypoglycemic action of semipurified fractions from hot-water extracts of the submerged-culture broth of Agaricus blazei Murill was examined in streptozotocin (60 mg/kg, intraperitoneal)-induced diabetic male Sprague-Dawley rats, relative to the diabetes drug metformin. The hot-water extract, treated with ethanol to remove beta-glucans and glycoproteins, was freeze-dried, and fractionated into hexane, chloroform, ethyl acetate (EA), and butanol fractions. The EA fraction (EAF; 200 mg/kg body weight) reduced (p < 0.05) the blood glucose level in the oral glucose tolerance test, relative to the other fractions and control. In a 14 day-treatment study, diabetic rats treated with the EAF displayed a suppressed blood glucose level and elevated plasma insulin and glucose transport-4 proteins; the reactions occurred in a dose-dependent manner (200 and 400 mg/kg body weight) compared to those in control animals. The EAF reduced the levels of triglyceride and cholesterol in plasma, the activity of glutamate-oxaloacetate transaminase and glutamate-pyruvate transaminase in blood, and the content of thiobarbituric acid reactive substance in the liver and kidney. The hypoglycemic efficacy of the EAF (400 mg/kg body weight) was similar to that of metformin (500 mg/kg body weight). The EAF contained substantial amounts of isoflavonoids including genistein, genistin, daidzein, and daidzin, which could have contributed to the fraction's hypoglycemic action. These results indicate that the hot-water extract of the submerged-culture broth of Agaricus blazei contains an EAF having potent hypoglycemic action, which could be useful in the treatment of diabetes mellitus.

Differential inhibitory effects of conjugated linoleic acid isomers on mouse forestomach neoplasia induced by benzo(a)pyrene.

The differential anticarcinogenic activity of conjugated linoleic acid (CLA) isomers, including c9,t11-CLA, t10,c12-CLA, and t,t-CLA, was examined in a mouse forestomach carcinogenesis regimen induced by benzo(a)pyrene (BP). Female ICR mice (6-7 weeks of age, 26 +/- 1 g) were divided into six groups (30 mice/group, 5 mice/cage): control, linoleic acid, CLA, c9,t11-CLA, t10,c12-CLA, and t,t-CLA. Each mouse was orally given 0.1 mL of sample and 0.1 mL of olive oil on Monday and Wednesday and BP (2 mg in 0.2 mL of olive oil) on Friday. This cycle was repeated four times. Twenty-three weeks later, the experiment was terminated for tumor analysis. t,t-CLA significantly reduced (p < 0.05) both tumor number and tumor size per mouse, relative to CLA and c9,t11-CLA, but similar to t10,c12-CLA. Reduction in tumor incidence by t,t-CLA (84.6%) was similar to that by CLA, c9,t11-CLA, and t10,c12-CLA, but it was significantly reduced (p < 0.05), relative to 100% linoleic acid and control. t,t-CLA elevated the apoptotic index to 35%, relative to 23% for CLA, 21% for c9,t11-CLA, 29% for t10,c12-CLA, 7% for linoleic acid, and 4% for control. t,t-CLA up-regulated the expression of the Bax gene and activated caspase-3 enzymes but down-regulated expression of the Bcl-2 gene. Cytosolic phospholipase A(2) activity was not affected by the CLA isomers tested. These results suggest that t,t-CLA has superior anticarcinogenic potential on BP-induced mouse forestomach neoplasia to CLA, c9,t11-CLA, and t10,c12-CLA, via the induction of apoptosis through mitochondrial dysfunction.

A mixture of trans, trans conjugated linoleic acid induces apoptosis in MCF-7 human breast cancer cells with reciprocal expression of Bax and Bcl-2.

The growth inhibitory effect of a mixture of trans, trans conjugated linoleic acid isomers (t, t CLA) was investigated in a human breast cancer cell line, MCF-7, with references to c9, t11 CLA, t10, c12 CLA, and linoleic acid. The t, t CLA treatment effectively induced a cytotoxic effect in a time-dependent (0-6 days) and concentration-dependent (0-40 microM) manner, as compared to the reference and control treatments. The apoptotic parameters were measured on cells treated with 40 microM t, t CLA for 4 days. The occurrence of the characteristic morphological changes and DNA fragmentation confirmed apoptosis. The t, t CLA treatment led to an increase in the level of p53 tumor suppressor protein and Bax protein, but suppressed the expression of Bcl-2 protein. In addition, cytochrome c was released from the mitochondria into the cytosol, and the activation of caspase-3 led to the cleavage of poly(ADP-ribose) polymerase (PARP). Moreover, the composition of the linoleic and arachidonic acids was decreased in cellular membranes. These findings suggest that incorporation of t, t CLA in the membrane induces a mitochondria-mediated apoptosis that can enhance the antiproliferative effect of t, t CLA in MCF-7 cells.

Production of silkworms with conjugated linoleic acid (CLA) incorporated into their lipids by dietary CLA.

Silkworms with conjugated linoleic acid (CLA) incorporated into their lipids (designated CLA silkworms) were produced to enhance the quality of silkworms having a synergistic effect with CLA functions by dietary synthetic CLA. Silkworm larvae were fed fresh mulberry leaves (control diet) until the third instar stage and were then subjected to various levels (0%, 0.1%, 1%, 5%, and 10%) of CLA-sprayed mulberry leaves (designated CLA diet) beginning on the first day of the fourth instar stage and continuing to the third day of the fifth instar stage. CLA contents in CLA silkworms increased proportionally with increasing CLA levels of CLA diets. CLA silkworms on a 1% CLA diet contained 2.2 g CLA/100 g lipid without body weight reduction, whereas CLA silkworms on a 10% CLA diet contained 14.8 g CLA/100 g lipid with a significant reduction of body weight, relative to the control silkworms. The CLA content in the lipids of CLA silkworms on a 10% CLA diet was significantly higher than that of CLA silkworms on a 5% CLA diet. A 0.1% CLA diet was not sufficient to accumulate CLA in the silkworms. Most of the CLA (approximately 99%) of silkworm lipids was present in triglyceride (TG) with a similar ratio of c9,t11 and t10,c12 CLA isomers. These results suggest that a 1% CLA diet was suitable for the production of CLA silkworms.

Dietary conjugated linoleic acid increases the mRNA ratio of Bax/Bcl-2 in the colonic mucosa of rats.

Previously we have shown that dietary conjugated linoleic acid (CLA) significantly decreased colon tumor incidence in rats injected with 1,2-dimenthylhydrazine (DMH). The present study was performed to explore the mechanisms responsible for the anticarcinogenic effect of CLA. Four groups of rats received either vehicle or intramuscular injections of DMH at the dose of 15 mg/kg body weight twice per week for 6 weeks and were fed a diet containing either 0% or 1.0% CLA ad libitum for 14 weeks. Dietary CLA decreased cellular proliferation and induced apoptosis in the colonic mucosa of both vehicle and DMH-treated rats. Mucosal levels of prostaglandin (PG) E(2), thromboxane B(2), and 1,2-diacylglycerol decreased in rats fed the 1% CLA diet, whereas cyclooxygenase-2 levels were not affected. Arachidonate content of mucosal phospholipids decreased significantly in rats fed the 1% CLA diet. Reverse transcriptase-polymer chain reaction analysis revealed that the Bax/Bcl-2 transcript ratio was significantly increased in rats fed 1% CLA. To examine whether the 1% CLA diet reduces tumor incidence, the DMH-treated rats were continuously fed the assigned diets for 30 weeks. Tumor incidence was significantly decreased in the CLA-fed group. In conclusion, our findings are consistent with the hypothesis that CLA decreases the incidence of colon cancer by decreasing cellular proliferation and inducing apoptosis of the colonic mucosa. These effects may be due in part to decreased PGE(2) levels and increased Bax/Bcl-2 ratios.

Preparation of a t,t conjugated linoleic acid methylester (CLA-Me) isomers mixture from synthetic CLA by methylation with BF3/methanol.

Mixtures of t,t conjugated linoleic acid methylester (t,t CLA-Me) isomers were prepared from synthetic CLA, consisting of 47.8% t10,c12 CLA; 45.5% c9,t11 CLA; 2.0% t,t CLA; and 4.7% others, by methylation with BF(3)/methanol (designated TT-TC/CT) in conjunction with purification at -68 degrees C for 24 h. The amount or composition of the TT-TC/CT was greatly affected by the concentration of BF(3) in methanol and the duration of methylation. The methylation of 50 mg of synthetic CLA for 30 min with 1 mL of 7.0% BF(3)/methanol produced a TT-TC/CT (21.54 mg) with the composition of 1.3% t12,t14; 5.9% t11,t13; 42.7% t10,t12; 44.0% t9,t11; 5.0% t8,t10; and 1.1% t 7,t9 CLA, whereas the methylation for 60 min with 14.0% BF(3)/methanol produced a TT-TC/CT (28.62 mg) with the composition of t,t CLA isomers different from that of TT-TC/CT by methylation for 30 min with 7.0% BF(3)/methanol. A large quantity of TT-TC/CT (14.15 g) with the composition similar to that of TT-TC/CT prepared from 50 mg of synthetic CLA was also prepared from 25 g of synthetic CLA. The purity of TT-TC/CT samples was greater than 98%. These results suggest that TT-TC/CT with a purity greater than 98% was easily prepared from synthetic CLA by BF(3)-catalyzed methylation, and the amount and composition of t,t CLA isomers of TT-TC/CT samples could be controlled by methylation conditions.

Trans-10,cis-12-conjugated linoleic acid inhibits Caco-2 colon cancer cell growth.

A commercially available mixture of conjugated linoleic acid (CLA) isomers decreases colon cancer cell growth. We compared the individual potencies of the two main isomers in this mixture [cis-9,trans-11 (c9t11) and trans-10,cis-12 (t10c12)] and assessed whether decreased cell growth is related to changes in secretion of insulin-like growth factor II (IGF-II) and/or IGF-binding proteins (IGFBPs), which regulate Caco-2 cell proliferation. Cells were incubated in serum-free medium with different concentrations of the individual CLA isomers. t10c12 CLA dose dependently decreased viable cell number (55 +/- 3% reduction 96 h after adding 5 microM t10c12 CLA). t10c12 CLA induced apoptosis and decreased DNA synthesis, whereas c9t11 CLA had no effect. Immunoblot analysis of 24-h serum-free conditioned medium using a monoclonal anti-IGF-II antibody revealed that Caco-2 cells secreted both a mature 7,500 molecular weight (M(r)) IGF-II and higher M(r) forms of IGF-II. The levels of the higher M(r) and the mature form of IGF-II were decreased 50 +/- 3% and 22 +/- 2%, respectively, by 5 microM t10c12 CLA. c9t11 CLA had no effect. Ligand blot analysis of conditioned medium using 125I-labeled IGF-II revealed that t10c12 CLA slightly decreased IGFBP-2 production; c9t11 CLA had no effect. Exogenous IGF-II reversed t10c12 CLA-induced growth inhibition and apoptosis. These results indicate that CLA-inhibited Caco-2 cell growth is caused by t10c12 CLA and may be mediated by decreasing IGF-II secretion in Caco-2 cells.

Inclusion complex of conjugated linoleic acid (CLA) with cyclodextrins.

Conjugated linoleic acid (CLA) inclusion complexes with alpha-cyclodextrin (alpha-CD), beta-cyclodextrin (beta-CD), and gamma-cyclodextrin (gamma-CD) (designated CLA/CDs inclusion complexes) were prepared to determine the mole ratio of CLA complexed with CDs and the oxidative stability of CLA in the CLA/CDs inclusion complexes. When measured by GC, (1)H NMR, and T(1) value analyses, 1 mole of CLA was complexed with 5 mol of alpha-CD, 4 mol of beta-CD, and 2 mol of gamma-CD. The oxidation of CLA induced at 35 degrees C for 80 h was completely prevented by the formation of CLA/CDs inclusion complexes.

Methylation methods for the quantitative analysis of conjugated linoleic acid (CLA) isomers in various lipid samples.

Precise methylation methods for various chemical forms of conjugated linoleic acid (CLA), which minimize the formation of t,t isomers and allylmethoxy derivatives (AMD) with the completion of methylation, were developed using a 50 mg lipid sample, 3 mL of 1.0 N H(2)SO(4)/methanol, and/or 3 mL of 20% tetramethylguanidine (TMG)/methanol solution(s). Free CLA (FCLA) was methylated with 1.0 N H(2)SO(4)/methanol (55 degrees C, 5 min). CLA esterified in safflower oil (CLA-SO) was methylated with 20% TMG/methanol (100 degrees C, 5 min), whereas CLA esterified in phospholipid (CLA-PL) was methylated with 20% TMG/methanol (100 degrees C, 10 min), followed by an additional reaction with 1.0 N H(2)SO(4)/methanol (55 degrees C, 5 min). Similarly, CLA esterified in egg yolk lipid (CLA-EYL) was methylated by base hydrolysis, followed by reaction with 1.0 N H(2)SO(4)/methanol (55 degrees C, 5 min). These results suggest that for the quantitative analysis of CLA in lipid samples by GC, proper methylation methods should be chosen on the basis of the chemical forms of CLA in samples.