石榴酸和α-酮酸对人恶性黑色素瘤A375细胞的细胞毒性作用

<正>石榴酸(punicic acid,PA,9c,11t,13c-18:3)和α-酮酸(α-eleostearic acid,α-ESA,9c,11t,13t-18:3)是两种最重要的共轭亚麻酸(conjugated linolenic acid,CLN)异构体CLN异构体[1]。体内和体外研究表明,共轭亚麻酸(conjugated linolenic acid,CLN)有较强体内和体外抗肿瘤作用[2-3],且CLN单体是抑制各种肿瘤细胞生长强效抑制剂[4]。石榴籽油是一种安全     

共轭亚油酸对肿瘤细胞亚油酸代谢途径中限速酶的影响

目的采用体外细胞培养方法,研究不同浓度c9,t11-共轭亚油酸(c9,t11-CLA)对人胃腺癌细胞(SGC-7901)中亚油酸代谢途径的限速酶的影响。方法用200、100、50和25μmol／L浓度的c9,t11-CLA处理SGC-7901细胞24h,四甲基偶氮唑盐实验检测c9,t11-CLA对SGC-7901细胞增殖的抑制作用,采用逆转录聚合酶链反应检测c9,t11-CLA对SGC-7901细胞中亚油酸代谢途径的Δ6-脱氢酶、△5-脱氢酶、环氧合酶(COX)-1、COX-2和5-脂氧合酶(5-LOX)mRNA表达的影响。结果在200、100、50和25μmol／L浓度时,c9,t11-CLA对SGC-7901增殖的抑制率分别为54．3％、20．5％、10．5％、2．93％;均可下调COX-2mRNA的表达,上调Δ6-脱氢酶、COX-1mRNA的表达,但对Δ5-脱氢酶和5-LOXmRNA表达的影响不显著。结论c9,t11-CLA可通过调节Δ6-脱氢酶和COX的表达抑制肿瘤细胞的增殖,说明c9,t11-CLA通过影响亚油酸代谢途径中限速酶的基因表达而改变类二十碳烷酸的形成,推测c9,t11-CLA影响肿瘤细胞中亚油酸代谢途径的限速酶是其发挥抗癌活性的另一作用机制。     

环氧合酶-2在c9,t11-共轭亚油酸抑制胃腺癌细胞增殖中的作用

目的　采用体外细胞培养方法,研究不同浓度c9,t11 共轭亚油酸对人胃腺癌细胞(SGC- 790 1)的亚油酸代谢途径中限速酶———环氧合酶(COX -2 )及其产物前列腺素E2 (PGE2 )的影响。方法　采用逆转录聚合酶链反应(RT- PCR)和Westernblot方法检测不同浓度c9,t11 CLA处理后的SGC 790 1细胞中COX- 2mRNA和蛋白的表达,放射免疫方法检测细胞分泌的PGE2 浓度。结果　在2 5、5 0、10 0和2 0 0 μmol L浓度时,c9,t11 CLA均可下调COX 2mRNA和蛋白的表达,同时降低细胞中PGE2 的分泌。结论　环氧合酶2是c9,t11 CLA抑制肿瘤细胞增殖的作用靶点。     

t10,c12-共轭亚油酸对骨骼肌细胞脂肪酸代谢的影响

目的通过研究t10,c12-共轭亚油酸(t10,c12-CLA)对高游离脂肪酸环境中骨骼肌细胞脂肪酸代谢的影响,探讨t10,c12-CLA改善脂代谢的作用机制。方法体外培养成熟的C2C12骨骼肌细胞,通过施加软脂酸在体外模拟高游离脂肪酸环境,再给予不同浓度的t10,c12-CLA(20、30和40μmol/L),通过荧光免疫法利用激光共聚焦显微镜观察t10,c12-CLA对细胞脂肪酸转运蛋白(FAT/CD36)的影响,应用Western blot法检测t10,c12-CLA对C2C12细胞脂肪酸代谢相关酶及AMP激活的蛋白激酶(AMPK)的作用。结果t10,c12-CLA剂量依赖性的增加了高游离脂肪酸环境中细胞对脂肪酸的摄取,抑制了乙酰辅酶A羧化酶(ACC)的活性,增加了肉碱酰基转移酶-1(CPT-1)的表达,并且激活了AMPK。结论t10,c12-CLA可通过激活AMPK增强细胞对脂肪酸的氧化代谢。     

共轭亚油酸对人胃腺癌细胞中环氧合酶的影响

目的采用体外细胞培养方法,研究不同浓度c9,t11-共轭亚油酸(CLA)对人胃腺癌细胞(SGC-7901)的亚油酸代谢途径中限速酶-环氧合酶(COX)表达的影响.方法采用逆转录聚合酶链反应(RT-PCR)和蛋白免疫印迹(Western blot)方法检测不同浓度c9,t11-CLA处理后的SGC-7901细胞中环氧合酶(COX)-1、(COX)-2mRNA和蛋白的表达.结果在200,100,50和25μmol/L浓度时,c9,t11-CLA均可下调COX-2 mRNA和蛋白的表达,与共轭亚油酸(CLA)浓度呈负相关;上调COX-1 mRNA的表达,并与CLA浓度呈正相关.结论 c9,t11-CLA的抗癌活性与其影响COX的表达有关.     

市售牛奶中c9,t11-共轭亚油酸测定的气相色谱法研究

目的：用气相色谱内标法检测部分市售牛奶中c9，t11-共轭亚油酸含量（CLA），分析季节因素对牛奶中e9，t11-共轭亚油酸含量的影响。方法：采用气相色谱内标法，内标物为C17：00甲酯，程序升温，将样品提取、甲酯化后，测定并比较夏季和非夏季牛奶样品中c9，t11-共轭亚油酸的含量。结果：实验方法的回收率为83．67％，RSD=3．45％；精密度为RSD=2．09％。夏季和非夏季牛奶样品中c9，t11-共轭亚油酸含量均值范围分别为0～14．9me,／g脂肪和0～7．3mg／g脂肪。夏季牛奶样品中c9，t11-共轭亚油酸含量显著高于非夏季牛奶样品（P〈0．05）。结论：所建立的实验条件准确可靠，季节变化对牛奶样品中c9，t11-共轭亚油酸含量有显著影响。本研究对在国内开展共轭亚油酸强化乳制品的研究具有指导意义。     

气相色谱分析法测定酸奶和奶油中c9,t11-共轭亚油酸的含量

目的：建立用程序升温气相色谱法测定奶制品中c9,t11-共轭亚油酸含量的方法。方法：首先建立本实验方法的气相色谱分析条件；采用内标法，将样品提取、甲酯化后，进行气相色谱分析；同时对实验方法的回收率、精密度也进行了分析。结果：能够将甲酯化亚油酸和c9,t11-共轭亚油酸很好地分离；内标物十七烷酸甲酯与其它脂肪酸甲酯能够完全分离，并不受样品中其它组分的影响；c9,t11-共轭亚油酸的含量分别为：帕玛拉特天然酸奶饮品10．78mg/g，万家宝原味酸奶（搅拌型）5．03mg/g，奶油22．54mg/g脂肪。实验方法的回收率为84．74％，RSD＝3．73％；精密度RSD＝2．58％。结论：所建立的实验条件准确可靠，这对于分析奶及奶制品来源的c9,t11-共轭亚油酸含量具有指导意义。     

共轭亚油酸诱导人乳腺癌细胞(MCF-7)凋亡的作用

目的　为了研究共轭亚油酸单体 (c9,t11 CLA)对人乳腺癌细胞 (MCF 7)凋亡的影响。方法　采用细胞生长曲线 ,凋亡细胞的荧光显微镜和电镜观察、流式细胞光度术的检测以及p5 3蛋白表达的方法 ,观察了用不同浓度c9,t11 CLA(2 5、5 0、10 0和 2 0 0 μmol L)诱导MCF 7细胞凋亡作用。结果　c9,t11 CLA可明显抑制MCF 7细胞生长 ,8天的抑制率分别为 -6 0 %、4 5 2 %、99 0 %和 99 4 %。通过荧光显微镜和电镜技术观察到了发生凋亡的MCF 7细胞 ;用流式细胞光度术也检测到c9,t11 CLA可诱导MCF 7细胞产生凋亡 ,并随着c9,t11 CLA浓度的增加 ,细胞凋亡率逐渐增加 ;而p5 3蛋白表达则随着c9,t11 CLA浓度的增加呈现逐渐降低的趋势。结论　c9,t11 CLA可诱导MCF 7细胞产生凋亡 ,这可能是c9,t11 CLA抑制肿瘤细胞生长的机制之一。     

环氧合酶-2在c9,t11-共轭亚油酸抑制肿瘤细胞转移中的作用

目的 研究c9,t11-共轭亚油酸（c9,t11-CLA）抑制人胃腺癌细胞（SGC-7901）转移作用与亚油酸代谢途径中限速酶环氧合酶-2（COX-2）的关系。方法采用体外细胞培养方法,用COX-2的选择性抑制剂NS．398抑制SGC-7901细胞中COX-2的活性,再加入200、100、50和25μmol／L浓度的c9,t11-CLA,利用重组基底膜侵袭实验、黏附实验、趋向运动实验检测c9,t11-CLA抑制肿瘤细胞转移的作用与COX-2的关系。结果与NS-398组比较,NS-398＋100Ixmol／Lc9,t11-CLA和NS-398＋200μmol／L c9,t11-CLA对SGC-7901细胞的侵袭有明显的抑制作用,穿过膜的细胞数分别为48．7±1．5（F=14．309,P=0．000）和43．7±4．0（F=19．005,P=0．000）;NS-398＋200μmol／L c9,t11-CLA组能显著降低SGC-7901细胞对基质成分层粘连蛋白、纤维粘连蛋白和基质胶的黏附作用,所测吸光度值（A值）分别为0．052±0．011,0．058±0．008（F=3．063,P=0．021）和0．042±0．004（F=6．692,P=0．001;F=11．999,P=0．000）;NS-398＋200ixmol／L c9,t11-CLA对SGC-7901细胞的趋向运动能力无明显的抑制作用（F=1．380,P=0．276）,其中NS-398＋2001xmol／L c9,t11-CLA组细胞数为26．6±3．4。结论 c9,t11-CLA具有抑制SGC-7901细胞体外侵袭能力、黏附能力和趋向运动能力,这种抑制作用可能与COX-2途径有关。     

共轭亚油酸对人胃腺癌细胞侵袭及转移相关基因表达的影响

目的 研究c9,t11－共轭亚油酸（CLA）对人胃腺癌细胞（SGC－7901）侵袭能力的影响,探讨其抑制肿瘤转移的可能机制。方法 用重组基底膜侵袭实验评价癌细胞侵袭能力;用逆转录聚合酶链反应（RT－PCR）检测SGC－7901细胞中组织基质金属蛋白酶抑制剂（TIMP）－1、TIMP－2和nm23-H1mRNA的表达。结果 在200、100和50μmol/L浓度时,c9,t11-CLA对SGC－7901细胞侵袭重组基底膜的抑制率分别为53.7%、40.9%和29.3%。c9,t11-CLA可诱导SGC－7901细胞中TIMP－1、TIMP－2和nm23-H1mRNA的表达。结论 c9,t11-CLA抑制SGC-7901细胞侵袭重组基底膜。c9,t11-CLA的抗侵袭活性与诱导肿瘤细胞中TIMP－1、TIMP－2和nm23-H1mRNA的表达等有关。     

Punicic Acid Triggers Ferroptotic Cell Death in Carcinoma Cells

Plant-derived conjugated linolenic acids (CLnA) have been widely studied for their preventive and therapeutic properties against diverse diseases such as cancer. In particular, punicic acid (PunA), a conjugated linolenic acid isomer (C18:3 c9t11c13) present at up to 83% in pomegranate seed oil, has been shown to exert anti-cancer effects, although the mechanism behind its cytotoxicity remains unclear. Ferroptosis, a cell death triggered by an overwhelming accumulation of lipid peroxides, has recently arisen as a potential mechanism underlying CLnA cytotoxicity. In the present study, we show that PunA is highly cytotoxic to HCT-116 colorectal and FaDu hypopharyngeal carcinoma cells grown either in monolayers or as three-dimensional spheroids. Moreover, our data indicate that PunA triggers ferroptosis in carcinoma cells. It induces significant lipid peroxidation and its effects are prevented by the addition of ferroptosis inhibitors. A combination with docosahexaenoic acid (DHA), a known polyunsaturated fatty acid with anticancer properties, synergistically increases PunA cytotoxicity. Our findings highlight the potential of using PunA as a ferroptosis-sensitizing phytochemical for the prevention and treatment of cancer.     

Dietary conjugated α-linolenic acid did not improve glucose tolerance in a neonatal pig model

Purpose

There is an increased interest in the benefits of conjugated α-linolenic acid (CLNA) on obesity-related complications such as insulin resistance and diabetes. The aim of the study was to investigate whether a 1 % dietary supplementation of mono-CLNA isomers (c9-t11-c15-18:3 + c9-t13-c15-18:3) improved glucose and lipid metabolism in neonatal pigs.

Methods

Since mono-CLNA isomers combine one conjugated two-double-bond system with an n-3 polyunsaturated fatty acid (PUFA) structure, the experimental protocol was designed to isolate the dietary structural characteristics of the molecules by comparing a CLNA diet with three other dietary fats: (1) conjugated linoleic acid (c9-t11-18:2 + t10-c12-18:2; CLA), (2) non-conjugated n-3 PUFA, and (3) n-6 PUFA. Thirty-two piglets weaned at 3 weeks of age were distributed among the four dietary groups. Diets were isoenergetic and food intake was controlled by a gastric tube. After 2 weeks of supplementation, gastro-enteral (OGTT) and parenteral (IVGTT) glucose tolerance tests were conducted.

Results

Dietary supplementation with mono-CLNA did not modify body weight/fat or blood lipid profiles (p > 0.82 and p > 0.57, respectively) compared with other dietary groups. Plasma glucose, insulin, and C-peptide responses to OGTT and IVGTT in the CLNA group were not different from the three other dietary groups (p > 0.18 and p > 0.15, respectively). Compared to the non-conjugated n-3 PUFA diet, CLNA-fed animals had decreased liver composition in three n-3 fatty acids (18:3n-3; 20:3n-3; 22:5n-3; p < 0.001).

Conclusions

These results suggest that providing 1 % mono-CLNA is not effective in improving insulin sensitivity in neonatal pigs.     

Trans-11-18 : 1 is effectively Delta9-desaturated compared with trans-12-18 : 1 in humans

The aim of this human intervention study was to evaluate the Delta9-desaturation of trans-11-18 : 1 (trans-vaccenic acid; tVA) to cis-9,trans-11-18 : 2 (c9,t11 conjugated linoleic acid; CLA) and of trans-12-18 : 1 (t12) to cis-9,trans-12-18 : 2 after a short-term (7 d) and a long-term (42 d) supplementation period. The conversion rates of both trans-18 : 1 isomers were estimated by lipid analysis of serum and red blood cell membranes (RBCM). Subjects started with a 2-week adaptation period without supplements. During the 42 d intervention period, the diet of the test group was supplemented with 3 g/d of tVA and 3 g/d of t12. The diet of the control group was supplemented with a control oil. Serum tVA and t12 levels in the test group increased by fivefold and ninefold after 7 d, respectively, and by eight- and 12-fold after 42 d, respectively, when compared with the adaptation period (P< or =0.002). The serum c9,t11 CLA levels increased by 1.7- and 2.0-fold after 7 d and 42 d, respectively (P< or =0.001). After 42 d, the test group's RBCM c9,t11 CLA content was elevated by 20 % (P=0.021), whereas in the control group it was decreased by 50 % (P=0.002). The conversion rate of tVA was estimated at 24 % by serum and 19 % by RBCM. No increase in c9,t12-18 : 2 was observed in the serum and RBCM, and thus no conversion of t12 could be determined. In conclusion, the endogenous conversion of dietary tVA to c9,t11 CLA contributes approximately one quarter to the human CLA pool and should be considered when determining the CLA supply.     

Effects of dietary cis 9, trans 11-18:2, trans 10, cis 12-18:2, or vaccenic acid (trans 11-18:1) during lactation on body composition, tissue fatty acid profiles, and litter growth in mice

Cis 9, trans 11 (c 9, t11)-18:2 and trans 10, cis 12 (t10, c12)-18:2 are the major conjugated linoleic acid (CLA) isomers in dietary supplements which reduce milk fat content in nursing women. The present study evaluated the effects of each CLA isomer or vaccenic acid on body composition and tissue fatty acids during lactation in mice. Dams were fed 30 g rapeseed oil (control)/kg diet or 20 g control plus 10 g 18:0, trans 11-18:1 (t11-18:1), c 9, t11-18:2, or t10, c12-18:2. Dietary t10, c12-18:2 reduced food intake by 18 % and carcass fat weight of the dams by 49 % compared with the other treatments. Milk fat percentage ranked by treatment was 18:0>t11-18:1=c 9, t11-18:2>t10, c12-18:2. The sum of saturated 12:0 to 16:0 in milk fat was lower when c 9, t11-18:2 was fed compared with the control, 18:0, or t11-18:1 treatments. Dietary t10, c12-18:2 caused further reductions in milk fat 12:0 to 16:0. The proportion of CLA isomers was 3-fold greater in milk fat than in the carcasses of the dams. The pups nursing from the dams fed t10, c12-18:2 had the lowest body weights and carcass fat, protein, and ash contents. Nursing from the dams fed c 9, t11-18:2 also resulted in lower carcass fat compared with the 18:0 or t11-18:1 treatments. The ratios of cis 9-16:1:16:0 or cis 9-18:1:18:0, proxies for Delta(9)-desaturase activity, were markedly lower in the carcasses of the dams and pups fed t10, c12-18:2. The ratio of 20:4n-6:18 : 2n-6, a proxy for Delta(6)- and Delta(5)-desaturase and elongase activity, in the liver of the dams and pups fed t10, c12-18:2 also was lower. Dietary t11-18:1 enhanced the content of c 9, t11-18:2 in milk fat and carcasses. As in previous studies, the reduction in food intake by t10, c12-18:2 could not entirely account for the marked decrease in carcass fat content and milk fat concentration. T10, c12-18:2 probably had a negative effect on Delta(9)-desaturase and mammary de novo fatty acid synthesis. Although these effects need to be confirmed in lactating women, the results suggest that the consumption of supplements containing t10, c12-18:2 should be avoided during the nursing period.     

Dietary effect of pomegranate seed oil rich in 9cis, 11trans, 13cis conjugated linolenic acid on lipid metabolism in obese,hyperlipidemic OLETF Rats

Conjugated fatty acid, the general term of positional and geometric isomers of polyunsaturated fatty acids with conjugated double bonds, has attracted considerable attention because of its potentially beneficial biological effects. In the present study, dietary effect of pomegranate seed oil rich in punicic acid (9 cis, 11 trans, 13 cis -conjugated linolenic acid; 9c, 11t, 13c-CLNA) on lipid metabolism was investigated in obese, hyperlipidemic Otsuka Long-Evans Tokushima Fatty (OLETF) rats. After 2 weeks feeding period, OLETF rats revealed obesity and hyperlipidemia compared with their progenitor LETO rats. Feeding of the diet supplemented with 9% safflower oil and 1% pomegranate seed oil (9c, 11t, 13c-CLNA diet) did not affect abdominal white adipose tissue weights and serum lipid levels compared with the diet supplemented with 10% safflower oil (control diet) in OLETF rats. However, the accumulated hepatic triacylglycerol was markedly decreased by 9c, 11t, 13c-CLNA diet in OLETF rats. Activities of hepatic enzymes related to fatty acid synthesis and fatty acid β-oxidation were not altered by 9c, 11t, 13c-CLNA diet. Levels of monounsaturated fatty acid (MUFA), major storage form of fatty acid, in serum triacylglycerol were markedly higher in obese, hyperlipidemic OLETF rats than in lean LETO rats. In addition, 9c, 11t, 13c-CLNA diet significantly decreased MUFA levels in OLETF rats. This is the first study showing that 9c, 11t, 13c-CLNA suppresses delta-9 desaturation in vivo, and we suggest that the alleviation of hepatic triacylglycerol accumulation by 9c, 11t, 13c-CLNA diet was, at least in part, attributable to the suppression of delta-9 desaturation in OLETF rats.     

An enriched mixture of trans-10,cis-12-CLA inhibits linoleic acid metabolism and PGE2 synthesis in MDA-MB-231 cells

Conjugated linoleic acid (CLA) isomers are potent inhibitors of mammary tumor cell growth. Evidence suggests that CLA modulates essential fatty acid (EFA) metabolism; however, it is not clear which parts of this pathway are important regulatory points modulated by CLA. Enriched mixtures of D9-cis,11-trans (D9c,11t)- and D10-trans,12-cis (D10t,12c)-18:2 were used to assess outcome measures of EFA metabolism pertaining to membrane phospholipid incorporation, tumor cell growth, and prostaglandin E2 (PGE2) synthesis in the MDA-MB-231 mammary tumor cell line. Tumor cells were treated with linoleic acid (LA), an equal mixture (Mix), or enriched preparations of D9c,11t- or D10t,12c-18:2. Treatment with Mix or the enriched mixture of D10t,12c-18:2 significantly inhibited the synthesis of arachidonic acid (AA) from LA, resulting in increased levels of LA and decreased levels of AA in membrane phosphatidylcholine and phosphatidylethanolamine (P < 0.05). LA and AA levels were not altered in cells treated with enriched D9c,11t-18:2 and were similar to those in LA control treated cells. All CLA treatments reduced [3H]thymidine uptake, an indicator of tumor cell growth, by more than one-half relative to LA controls. MDA-MB-231 cells challenged with AA in the presence of all CLA mixtures resulted in significantly reduced PGE2 synthesis relative to controls treated with LA (P < 0.05). It is evident that individual isomers exert inhibitory effects at specific steps of EFA metabolism, which correspondingly leads to a reduction in PGE2 synthesis and, ultimately, tumor growth.     

共轭亚油酸对正常动物细胞和人体肿瘤细胞间通讯功能的影响

为探讨c9,t11-共轭亚油酸 (CLA)的抑癌作用可能机制 ,在促癌物 (TPA)存在下 ,对正常细胞(CHL)以及人体肿瘤细胞 (SGC 790 1细胞和MCF 7细胞 )的细胞间隙连接通讯功能 (GJIC)的影响 ,采用划痕标记染料示踪技术 (SLDT) ;c9,t11 CLA剂量为 2 5 (mol L ,5 0 (mol L ,10 0 (mol L和 2 0 0 (mol L ,阴性对照为乙醇。结果显示 ,c9,t11 CLA可明显地提高TPA对CHL细胞的GJIC的抑制效应 ,当c9,t11 CLA浓度为 2 0 0 (mol L作用 48h时 ,细胞间隙通讯功能基本上与阴性对照组相近 ;当用c9,t11 CLA作用SGC 790 1细胞和MCF 7细胞2 4h和 48h时 ,可见 (2 4h 10 0 μmol L的MCF 7细胞 ) 2 4h 2 0 0 μmol L和 48h 10 0、2 0 0 μmol L剂量组的肿瘤细胞有一定的细胞间隙通讯功能。提示c9,t11 CLA可提高SGC 790 1细胞和MCF 7细胞的GJIC的功能 ,并且不同程度的拮抗TPA对CHL细胞GJIC的抑制效应