115. 共轭亚油酸对正常动物细胞和肿瘤细胞间通讯传递的影响

细胞间隙连接通讯(gap junctional intercellular communication，GJIC）对细胞的正常增殖分化和代谢功能以及组织自身稳定等起着重要调节作用。而肿瘤组织和肿瘤细胞间的GJIC表达减弱或消失。本文目的：采用c9，t11-CLA来探讨其对人体肿瘤细胞（SGC-7901细胞和MCF-7细胞）GJIC的影响以及在促癌物（TPA）存在下，对CHL细胞GJIC的影响。方法：采用划痕标记染料示踪技术（SLDT）； c9，t11-CLA剂量为25 μmol／L， 50 μmol／1， 100 μmol／1和200 μmol／L，阴性对照为96％乙醇（0．1％v／v）。结果：①荧光染料荧光黄（luciffer yellow，LY)划痕标记SGC-7901细胞和MCF-7细胞3 min后，在阴性对照组中，LY黄色荧光仅分布在伤沿细胞列内，说明SGC-7901细胞无间隙通讯功能。当用c9，t11-CLA作用SGC-7901细胞和MCF-7细胞24 h和48 h时，可见（24 h 100 μmol／L的MCF-7细胞）24 h 200 μmol／L和48 h 100 μmol／L，200 μmol／L剂量组的肿瘤细胞有一定的细胞间隙通讯功能，可见LY沿伤沿列传向邻近的细胞；②LY划痕标记CHL细胞3 min后，在阴性对照组中，LY黄色荧光分布在伤沿细胞列和邻近的2～3列细胞内，表示CHL细胞有间隙通讯功能，而单独使用TPA剂量组，LY仅分布伤沿细胞内，表示CHL细胞间隙通讯功能被完全阻滞。在TPA存在时，c9，t11-CLA作用CHL细胞24 h和48 h时，各个剂量组不同程度地恢复CHL细胞间隙通讯功能，当CLA剂量为200 μmol／L 24 h时，LY荧光染料已从伤沿传递至2列细胞，48 h时，细胞间隙通讯功能基本上与阴性对照组相近。结论：c9，t11-CLA可提高SGC-7901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。     

共轭亚油酸单体诱导乳腺癌细胞MCF-7凋亡及其作用机制的研究

目的:研究2种共轭亚油酸(conjugated linoleic acid,CLA)单体--顺9,反11-CLA(cis 9,trans11-CLA, c 9,t11-CLA)和反10,顺12- CLA(trans10,cis12-CLA, t10,c12-CLA) 诱导乳腺癌细胞MCF-7凋亡及其作用机制.方法:采用MTT法检测CLA对MCF-7细胞的生长抑制作用,锥虫蓝染色绘制CLA作用后MCF-7细胞的生长曲线;荧光显微镜观察及FCM检测MCF-7细胞的凋亡和细胞周期的改变;RT-PCR和Western印迹法检测MCF-7细胞PPARγ、Bcl-xL和Bcl-xS mRNA以及PPARγ、Bcl-2、Bax和caspase-3的蛋白表达.结果:2种CLA单体均可抑制MCF-7细胞增殖并诱导细胞凋亡,与对照组相比差异有统计学意义(P<0.05);RT-PCR和Western印迹法检测结果显示,2种CLA单体均可以提高PPARγ、Bcl-xS mRNA和PPARγ、Bax、caspase-3蛋白的表达,降低Bcl-xL mRNA和Bcl-2蛋白的表达,与对照组比较差异有统计学意义(P<0.05);且2种CLA单体对PPARγ与凋亡相关蛋白Bax、Bcl-2和caspase-3的表达影响呈剂量和时间依赖性及同步相关性.结论:c 9,t11-CLA和t10,c12-CLA对乳腺癌MCF-7细胞具有抑制生长和促凋亡的作用,CLA可能作为PPARγ的配体通过激活PPARγ-Bcl-2-caspase-3细胞凋亡信号通路而实现抑制肿瘤细胞生长的作用.     

共轭亚油酸对正常动物细胞和肿瘤细胞间通讯传递的影响

细胞间隙连接通讯（gap junctional intercellular communication,GJIC)对细胞的正常增殖分化和代谢功能以及组织自身稳定等起着重要调节作用。而肿瘤组织和肿瘤细胞的GJIC表达减弱或消失。本文目的：采用c9,tll-CLA来探讨其对人体肿瘤细胞（SGC－7901细胞的MCF－7细胞GJIC的影响以及在促癌物（TPA）存在下,对CHL细胞GJIC的影响。方法：采用划痕标记染料示踪技术（SLDT）;c9,t11-CLA剂量为25μmol/L,50μmol/l,100μmol/L,阴性对照为96％乙醇（0.1%v/v) 。结果：（1）荧光染料荧光黄（luciffer yellow,LY)划痕标记SGC－7901细胞和MCF－7细胞3min后,在阴性对照组中,LY黄色荧光仅分布在伤沿细胞列内,说明SGC－7901细胞无间隙通讯功能。当用c9,tll-CLA作用SGC－7901细胞和MCF－7细胞24h t 48h时,可见（24h 100μmol/L的MCF－7细胞）24h 200μmol/L和48h100μmol/L,200μmol/L剂量组的肿瘤细胞有一定的细胞间隙通讯功能,可见LY沿伤沿列传向邻近的细胞;（2）LY划痕标记CHL细胞3min后,在阴性对照组中,LY黄色荧光分布在伤沿细胞列和邻近的2－3列细胞内,表示CHL细胞有间隙通讯功能,而单独使用TPA剂量组,LY仅分布伤沿细胞内,表示CHL细胞间隙通讯功能被完全阻滞。在TPA存在时,c9,tll-CLA作用CHL细胞24h和48h时,各个剂量组不同程度地恢复CHL细胞间隙通讯功能,当CLA剂量为200μmol/L24h时,LY荧光染料已从伤沿传递至2列细胞,48h时,细胞间隙通讯功能基本上与阴性对照组相近。结论：c9,tll-CLA可提高SGC－7901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。     

熊果酸抗肿瘤作用研究进展

熊果酸分布广泛，生物学效应多样，其抗肿瘤作用是多方面和全方位的，有望成为一种高效低毒的抗肿瘤新药。     

力尔凡抗肿瘤分子机理研究(2)

本文以 BAIB/C艾氏腹水癌荷瘤小鼠为模型,用[~3H]-甲基掺入法测定了各增殖时相的艾氏腹水癌细胞内DNA甲基化酶活力水平,DNA甲基化作用是生物体内普遍存在的一种基因修饰作用,这种修饰作用发生在DNA复制之后,转录之前,其过程为在DNA甲基化酶作用下,以S-腺苷酞-甲硫氨酸为甲基供体,使DNA特异序列上的胞嘧啶(5mc)甲基化.由于甲基的移位,故影响基因的表达,DNAA甲基化作用在基因表达与调控中起重要作用,DNA甲基化酶活力大小可反应出DNA甲其化水平的高低.是从基因表达与调控角度阐明抗肿瘤药物作用分子机理的重要手段之一.     

力尔凡抗肿瘤分子机理研究(1)

力尔凡药是吉林大学生命科学技术研究所张德安教授等用基因工程方法研制出来的抗肿瘤新药,为了评价力尔凡抗肿瘤作用的有效性,为临床治疗提供科学依据,我们以8BC小鼠U14腹水癌荷瘤动物为模型,用[3~H]-TIP掺入法观察了该药对8BC小鼠UM4癌细胞增殖数,DNA聚合酶活性的影响的研究.现将结果报告如下.     

阿魏菇抗肿瘤作用的研究进展

阿魏菇是一种有药用价值的名贵珍稀食用菌, 具有抗肿瘤的生物活性.本文从诱导肿瘤细胞凋亡、抑制肿瘤细胞增殖及核酸和蛋白质合成等方面对阿魏菇的抗肿瘤机制研究进展作一综述.     

JS-K的抗肿瘤作用研究进展

肿瘤已经成为当今社会的重大健康问题,作为抗肿瘤药物开发的一氧化氮供体药JS-K在这方面引人注目,其具有广谱的抗肿瘤活性.到目前为止,已有研究表明JS-K在白血病、多发性骨髓瘤、肝癌、肺癌和乳腺癌等多种肿瘤细胞中有良好的抗肿瘤作用,也有研究表明JS-K具有对正常细胞几乎没有杀伤作用的特点,本课题组前期研究发现JS-K也具有抗病毒的作用,并对乙肝病毒转染的HepG2.2.15细胞有很好的生长抑制作用.本文通过查阅国内外相关文献,对JS-K的抗肿瘤研究进展作一综述,以期为JS-K对抗病毒引起的肝肿瘤的研究提供一定的参考和借鉴.     

苏木的抗肿瘤作用研究进展

中医认为肿瘤后期多有血瘀证的表现,所以活血化瘀法成为后期治疗恶性肿瘤的重要方法。全文从体内实验和体外实验两个方面,分别介绍目前国内外苏木抗肿瘤的研究进展,分析苏木抑制肿瘤的可能作用机制,并为临床活血化瘀药抗肿瘤应用提供依据。     

Retention of conjugated linoleic acid in the mammary gland is associated with tumor inhibition during the post-initiation phase of carcinogenesis

Conjugated linoleic acid (CLA) has been reported to have significant activity in inhibiting mammary carcinogenesis. A major objective of this study was to evaluate how changes in the concentration of CLA in mammary tissue as a function of CLA exposure/withdrawal were correlated with the rate of occurrence of mammary carcinomas. Rats treated with a single dose of dimethylbenz[a]anthracene (DMBA) at 50 days of age were given 1% CLA in the diet for either 4 weeks, 8 weeks or continuously following carcinogen administration. No cancer protection was evident in the 4 or 8 week-CLA treatment groups. Significant tumor inhibition was observed only in rats that were given CLA for the entire duration of the experiment (20 weeks). Analysis of CLA in the mammary gland showed that the incorporation of CLA was much higher in neutral lipids than in phospholipids. When CLA was removed from the diet, neutral lipid- and phospholipid-CLA returned to basal values in about 4 and 8 weeks, respectively. The rate of disappearance of neutral lipid-CLA (rather than phospholipid-CLA) subsequent to CLA withdrawal paralleled more closely the rate of occurrence of new tumors in the target tissue. It appears that neutral lipid-CLA may be a more sensitive marker of tumor protection than phospholipid-CLA. However, the physiological relevance of CLA accumulation in mammary lipids is unclear and remains to be determined. A secondary goal of this study was to investigate whether CLA might selectively inhibit clonal expansion of DMBA- initiated mammary epithelial cells with wild-type versus codon 61 mutated Ha-ras genes. Approximately 16% of carcinomas in the control group (without CLA) were found to express codon 61 ras mutation. Although continuous treatment with CLA reduced the total number of carcinomas by 70%, it did not alter the proportion of ras mutant versus wild-type carcinomas, suggesting that CLA inhibits mammary carcinogenesis irrespective of the presence or absence of the ras mutation.      

Effect of separate conjugated linoleic acid isomers on murine mammary tumorigenesis

Recent studies have linked conjugated linoleic acid (CLA) to altered tumorigenesis of several sites. We showed recently that a mixture of CLA isomers was able to significantly decrease mammary tumor metastasis in mice. That effect was seen with as little as 0.1% CLA in the diet. Other studies with dietary CLA have shown that various isomers may have differential effects. The purpose of this work was to assess which individual CLA isomers had similar effects in alteration of mouse mammary tumor metastasis. For that, we fed six 20% (w/w) total fat diets which contained either no CLA, low (0.1%, w/w) or high (0.25%, w/w) levels of cis9,trans11-CLA (c9,t11), trans10,cis12-CLA (t10,c12) or a mixture of the 2 isomers (0.125% of each, w/w) as free fatty acids. Neither the separate isomers nor the mixture had an effect on the latency or growth of primary line 4526 tumors when compared to the group without CLA. However, all diets containing CLA significantly decreased the total tumor burden (volume of tumor, mm(3)) in lungs of mice from both spontaneous metastasis (reduced by 42-73%) as well as implantation and survival of the metastatic cell (reduced by 46-61%) when compared with diets containing no CLA. Diets containing a greater concentration of either c9,t11 or t10,c12 had a significantly greater effect compared to the lower concentrations of the respective isomers when metastatic nodule size and total tumor burden were assessed. The diet containing both isomers decreased total tumor burden similarly to the diets containing the lower concentration of each of the isomers. Thus, the effects of c9,t11 and t10,c12 may not be additive and possibly share similar mechanisms for decreasing metastatic tumor burden in mice transplanted with mammary tumor cells.     

Anti-angiogenic activity of conjugated linoleic acid on basic fibroblast growth factor-induced angiogenesis

Conjugated linoleic acid (CLA) is a potent inhibitor of mammary carcinogenesis. Cancer cells produce various angiogenic factors which stimulate host vascular endothelial cell mitogenesis and chemotaxis for their growth and metastasis. Basic fibroblast growth factor (bFGF) is a potent angiogenic factor that is expressed in many tumors. In this study, we found that CLA decreased bFGF-induced endothelial cell proliferation and DNA synthesis in a dose-dependent manner. However, CLA did not inhibit endothelial cell migration. Furthermore, CLA showed a potent inhibitory effect on embryonic vasculogenesis and bFGF-induced angiogenesis in vivo. Collectively, these results suggest that CLA selectively inhibits the active proliferating endothelial cells induced by bFGF, which may explain its anti-carcinogenic properties in vivo.     

Mammary cancer prevention by conjugated dienoic derivative of linoleic acid

Conjugated dienoic derivative of linoleic acid (CLA) is a collective term which refers to a mixture of positional and geometric isomers of linoleic acid. It is a naturally occurring substance in food and is present at higher concentrations in products from animal sources. The present study reports that synthetically prepared CLA is an effective agent in inhibiting the development of mammary tumors induced by dimethylbenz(a)anthracene. Rats were fed either the AIN-76A basal diet or the same diet supplemented with 0.5, 1, or 1.5% CLA by weight. These diets were started 2 weeks before carcinogen administration and continued until the end of the experiment. The total number of mammary adenocarcinomas in the 0.5, 1, and 1.5% CLA groups was reduced by 32, 56, and 60%, respectively. The final tumor incidence and cumulative tumor weight were similarly diminished in rats fed the CLA-containing diets. In general, there appeared to be a dose-dependent protection at levels of 1% CLA and below, but no further beneficial effect was evident at levels above 1%. Chronic feeding of up to 1.5% CLA produced no adverse consequences in the animals. Analysis of the phospholipid fraction from liver and mammary tumor extracts showed that only the c9,t11 isomer of CLA was incorporated and that the level of incorporation increased with dietary intake. An interesting property of CLA is its ability to suppress peroxide formation from unsaturated fatty acid in a test-tube model (Cancer Res., Ha et al. 50: 1097-1101, 1990). In view of this information, the amount of thiobarbituric acid-reactive substances (lipid peroxidation products) present endogenously in liver and mammary gland was quantitated. The feeding of CLA (for either 1 or 6 months) resulted in a decrease in the extent of lipid peroxidation in the mammary gland, but such a suppressive effect was not detected in the liver. It should be noted that maximal antioxidant activity was observed with only 0.25% CLA in the diet, whereas maximal tumor inhibition was achieved at about 1% CLA. Hence there is a discrepancy between the antioxidant efficacy of CLA and its anticarcinogenic potency, suggesting that some other mechanisms might be involved in cancer protection. Unlike the stimulatory effect of linoleic acid in carcinogenesis (Cancer Res., Ip et al., 45: 1997-2001, 1985), the reaction of CLA in cancer prevention is specific, and CLA is more powerful than any other fatty acid in modulating tumor development.     

Reduction of murine mammary tumor metastasis by conjugated linoleic acid

Recent studies have shown that conjugated linoleic acid (CLA) can inhibit the initiation and thus, incidence of mammary tumors in rodents. The concentration of CLA required for these effects was as low as 0.1% of the diet, with no increased effects above 1%. To date, there is little evidence that CLA has any effect on growth or metastasis of mammary tumors. In this report, we demonstrate that CLA, at the concentrations used in previous studies, had a significant effect on the latency, metastasis, and pulmonary tumor burden of transplantable murine mammary tumors grown in mice fed 20% fat diets. The latency of tumors from mice fed CLA was significantly increased when compared with the 0% CLA control diet. The volume of pulmonary tumor burden, as a result of spontaneous metastasis, decreased proportionately with increasing concentrations of dietary CLA. With 0.5 and 1% CLA, pulmonary tumor burden was significantly decreased compared to mice treated with the eicosanoid inhibitor, indomethacin and fed diets containing no CLA. Tumors of mice fed as little as 0.1% CLA and as much as 1% had significantly decreased numbers of pulmonary nodules when compared with diets containing no CLA. The decrease in the number of pulmonary nodules by CLA was nearly as effective as indomethacin, a known suppressor of tumor growth and metastasis in this malignant model. These data suggest that effects of CLA on mammary tumorigenesis may go beyond the reported alterations in tumor incidence and effect later stages, especially metastasis.     

Decrease in linoleic acid metabolites as a potential mechanism in cancer risk reduction by conjugated linoleic acid.

Previous research suggested that conjugated linoleic acid (CLA) feeding during the period of pubescent mammary gland development in the rat resulted in diminished mammary epithelial branching which might account for the reduction in mammary cancer risk. Terminal end buds (TEB) are the primary sites for the chemical induction of mammary carcinomas in rodents. One of the objectives of the present study was to investigate the modulation of TEB density by increasing levels of dietary CLA and to determine how this might affect the risk of methylnitrosourea-induced mammary carcinogenesis. The data show a graded and parallel reduction in TEB density and mammary tumor yield produced by 0.5 and 1% CLA. No further decrease in either parameter was observed when CLA in the diet was raised to 1.5 or 2%. Thus, optimal CLA nutrition during pubescence could conceivably control the population of cancer-sensitive target sites in the mammary gland. Since both CLA and linoleic acid are likely to share the same enzyme system for chain desaturation and elongation, it is possible that increased CLA intake may interfere with the further metabolism of linoleic acid. Fatty acid analysis of total lipid showed that CLA and CLA metabolites continued to accumulate in mammary tissue in a dose-dependent manner over the range 0.5-2% CLA. There was no perturbation in tissue linoleic acid, however, linoleic acid metabolites (including 18:3, 20:3 and 20:4) were consistently depressed by up to 1% CLA. Of particular interest was the significant drop in 20:4 (arachidonic acid), which is the substrate for the cyclooxygenase and lipoxygenase pathways of eicosanoid biosynthesis. Thus the CLA dose-response effect on arachidonic acid suppression corresponded closely with the CLA dose-response effect on cancer protection in the mammary gland. This information is critical in providing new insights regarding the biochemical action of CLA.     

Inhibition of angiogenesis by the cancer chemopreventive agent conjugated linoleic acid

Dietary conjugated linoleic acid (CLA) has been shown previously to inhibit rat mammary carcinogenesis. In addition to direct effects on mammary epithelial cells,including decreased proliferation and induction of apoptosis, CLA may exert its effects indirectly by inhibiting the differentiation of mammary stromal cells to an endothelial cell type. Specifically, CLA was found to decrease the ability of mammary stromal cells to form complex anastomosing microcapillary networks in vitro on Engelbreth-Holm-Swarm-derived reconstituted basement membrane. This suggested that CLA might inhibit angiogenesis in vivo. To test this possibility, CD2/F(1) mice were placed on synthetic diets containing 0, 1, or 2% CLA for 6 weeks, before angiogenic challenge by s.c. injection with an angiogenic gel substrate (Matrigel pellet assay). After 7 days, the pellets from animals fed the control diet were infiltrated by abundant branching networks of blood vessels with patent lumen-containing RBCs. In contrast, pellets from the CLA-fed animals contained fewer infiltrating cells, which formed limited branching cellular networks, the majority of which had collapsed lumen and no RBCs. Both levels of dietary CLA showed similar effects, with the number of RBC-containing vessels per 20x field decreased to a third of that seen in control. Dietary CLA decreased serum levels of vascular endothelial growth factor (VEGF) and whole mammary gland levels of VEGF and its receptor Flk-1. Both cis-9, trans-11 and trans-10, cis-12 CLA isomers were effective in inhibiting angiogenesis in vitro in a dose-dependent fashion. The ability of CLA to inhibit angiogenesis may contribute to its efficacy as a chemopreventive agent.     

Differential inhibition of fatty acid transport in tissue-isolated steroid receptor negative human breast cancer xenografts perfused in situ with isomers of conjugated linoleic acid

The peroxisome proliferator-activated receptor (PPARgamma) is a nuclear receptor that plays a regulatory role in cell differentiation and proliferation. PPARgamma was first detected in adipocytes, however, it has been shown that this receptor is also expressed in normal as well as tumor cells including malignant colonic epithelial cells. In this study, the effect of the PPARgamma agonist RO205-2349, a recently developed thiazolidinedione, on tumor growth was evaluated. For this purpose, human colon cancer cells (HT29) were grown in severe combined immunodeficient mice. Under daily RO205-2349 treatment (50 mg/kg/day) a significantly reduced tumor weight became evident after 3 weeks. In the control (n = 10) and treatment (n = 10) groups the mean tumor weights were 0.45 and 0.16 g, respectively. The mean percentages of apoptotic cells were 0.8 and 2.7% in the control and treatment groups, respectively, and the cell diameter measured on average 11.4 and 9.4 microm. In contrast, cell proliferation and differentiation, which are considered to be influenced by the PPARgamma, remained unaffected as could be seen by Ki-67 and carcinoembryonic antigen immunoreactivity indicating that increased rate of apoptosis and cell shrinkage are responsible for the differences in tumor growth. Hence, in this human/mouse xenograft model, mechanisms other than the classical activation of PPARgamma are likely reasons causing limited tumor growth.     

Effect of conjugated linoleic acid isomers on growth factor-induced proliferation of human breast cancer cells

We evaluated the effect of conjugated linoleic acid (CLA) isomers on the growth factor-induced proliferation of human breast cancer MCF-7 cells. When MCF-7 cells were cultured in RPMI 1640 medium supplemented with 1% fetal bovine serum (FBS), CLA inhibited the proliferation and notably cis9, trans11 (c9,t11)-CLA showed the strongest effect. However, cells barely grew when cultured with 1% charcoal-treated FBS (cFBS). Proliferation was promoted in cFBS cultured cells by the addition of 17beta-estradiol (E2), insulin, and epidermal growth factor (EGF). Trans10, cis12 (T10,c12)-CLA inhibited cell proliferation induced by E2 and insulin, but not by EGF. T10,c12-CLA also exhibited cell-killing activity when cells were induced with insulin. On the other hand, c9,t11-CLA was shown to have no effect on MCF-7 cell proliferation induced by and of these three growth factors. In conclusion, although both c9, t11 and t10, c12-CLA can inhibit the proliferation of MCF-7 cells, our results suggested that they have separate mechanisms and different targets of actions.     

PPARgamma-dependent effects of conjugated linoleic acid on the human glioblastoma cell line (ADF)

Conjugated linoleic acid (CLA) has been shown to exert beneficial effects against carcinogenesis, atherosclerosis and diabetes. It has been demonstrated that CLA modulates lipid metabolism through the activation of peroxisome proliferator-activated receptors (PPARs). The PPAR family comprises 3 closely related gene products, PPAR alpha, beta/delta and gamma, differing for tissue distribution, developmental expression and ligand specificity. It has also been demonstrated that activated PPARgamma results in growth inhibition and differentiation of transformed cells. These observations stimulated a great interest toward PPARgamma ligands as potential anticancer drugs to be used in a differentiation therapy. Glioblastomas are the most commonly diagnosed primary tumors of the brain in humans. The prognosis of patients with high-grade gliomas is poor and only marginally improved by chemotherapy. The aim of this work was to study the effects of CLA and of a specific synthetic PPARgamma ligand on cell growth, differentiation and death of a human glioblastoma cell line as well as on parameters responsible for the metastatic behavior of this tumor. We demonstrate here that CLA and PPARgamma agonist strongly inhibit cell growth and proliferation rate and induce apoptosis. Moreover, both treatments decrease cell migration and invasiveness. The results obtained show that CLA acts, directly or indirectly, as a PPARgamma activator, strongly suggesting that this naturally occurring fatty acid may be used as brain antitumor drug and as a chemopreventive agent. Moreover, the gamma-agonist, once experimented and validated on man, may represent a useful coadjuvant in glioblastoma therapy and in the prevention of recurrences.