Insulin receptor substrate-1 expression is regulated by estrogen in the MCF-7 human breast cancer cell line

Estrogens can stimulate the proliferation of estrogen-responsive breast cancer cells by increasing their proliferative response to insulin-like growth factors. The mechanism underlying the increased proliferation could involve the induction of components of the insulin-like growth factor signal transduction pathway by estrogen. In this study we have examined the regulation of the expression of insulin receptor substrate-1, a major intracellular substrate of the type I insulin-like growth factor receptor tyrosine kinase. Estradiol increased insulin receptor substrate-1 mRNA and protein levels at concentrations consistent with a mechanism involving the estrogen receptor. Insulin receptor substrate-1 was not induced significantly by the antiestrogens tamoxifen and ICI 182,780, but they inhibited the induction of insulin receptor substrate-1 by estradiol. Analysis of tyrosine-phosphorylated insulin receptor substrate-1 showed that the highest levels were found in cells stimulated by estradiol and insulin-like growth factor-I, whereas low levels were found in the absence of estradiol irrespective of whether type I insulin-like growth factor ligands were present. Insulin receptor substrate-2, -3, and -4 were not induced by estradiol. These results suggest that estrogens and antiestrogens may regulate cell proliferation by controlling insulin receptor substrate-1 expression, thereby amplifying or attenuating signaling through the insulin-like growth factor signal transduction pathway.     

TFF1 is membrane-associated in breast carcinoma cell line MCF-7

Trefoil factor family (TFF) domain peptides, products of mucin-secreting epithelial cells, are thought to influence mucosal integrity. Molecular studies revealed that mammalian TFFs lack transmembrane domains. Using immunocytochemistry and FACS analysis we demonstrated the association of TFF1 with the cell membrane in MCF-7 (a breast adenocarcinoma cell line), and tested the hypothesis that glycosylphosphatidylinositol (GPI) linkage is the mechanism for this association. Cleavage of GPI anchorage using phospholipase C did not affect TFF1 binding to the cell membrane. Our results demonstrate for the first time that TFF1 is associated with the cell membrane of MCF-7 cells and is not linked via a GPI anchor.     

The effect of Topotecan on oxidative stress in MCF-7 human breast cancer cell line

PURPOSE: Topotecan, a semisynthetic water-soluble derivative of camptothecin exerts its cytotoxic effect by inhibiting topoisomerase I and causes double-strand DNA breaks which inhibit DNA function and ultimately lead to cell death. In previous studies it was shown that camptothecin causes ROS formation. The aim of this study was to investigate if Topotecan like camptotecin causes oxidative stress in MCF-7 human breast cancer cell line. Determining the oxidant effect of Topotecan may elucidate a possible alternative mechanism for its cytotoxicity. EXPERIMENTAL DESIGN: MCF-7 cells were cultured and exposed to Topotecan for 24 h at 37 degrees C. The viability of the cells (% of control) was measured using the colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. Lipid peroxidation (TBARS), protein oxidation (carbonyl content), sulfhydryl, glutathione (GSH) levels, superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) activities were determined in MCF-7 cells with and without Topotecan incubation. RESULTS: We found the IC(50) concentration of Topotecan as 0.218 microM in MCF-7 cells. This concentration of Topotecan was used in the incubations of the cells. Our data indicated increased oxidative status, as revealed by increased lipid peroxidation and protein oxidation, and decreased GSH and sulfhydryl levels in MCF-7 cells exposed to Topotecan compared to control cells. In contrast, there was a slight increase in SOD and a significant increase in GPx and catalase activity in MCF-7 cells incubated with Topotecan compared to the control. CONCLUSIONS: These results support our hypothesis that Topotecan increases oxidative stress in MCF-7 cells.     

乳腺癌细胞株MCF-7-PC-1-46的建立

以人前列腺癌C4-2细胞基因组DNA为模板,扩增出PC-1基因N端编码46个氨基酸残基及其上游非编码区共599bp的DNA序列,将其正向克隆到真核表达载体pIRES2中,并在脂质体介导下,转染人乳腺癌细胞MCF-7,经G418筛选获得阳性单克隆,细胞扩大培养后,进行PCR和RT-PCR分析,检测外源PC-1基因在靶细胞中的整合与转录,PCR和RT-PCR结果表明,稳定转梁细胞株MCF-7-PC-1-46具有外源目的基因的整合和相应mRNA的高表达,说明成功建立了稳定表达外源PC-1基因N端46个氨基酸的人乳腺癌细胞株,为进一步研究PC-1基因的生物学功能提供了实验材料。     

Methionine cytotoxicity in the human breast cancer cell line MCF-7

Summary Among the first nutrients to be linked to cancer were methyl group containing nutrients including methionine. Methionine and its metabolic derivatives are essential components in several indispensable biological reactions including protein synthesis, polyamine synthesis, and many transmethylation reactions. The purpose of this study was to determine the extent to which methionine excess affects the proliferation and gene expression of the human breast cancer cell line MCF-7. Cells were first grown in control medium; the medium was then replaced with either control or methionine-supplemented treatment media. We found that 5 and 10 g/L methionine significantly suppressed cell growth on day 1, and no further growth was detected after 3 d of treatment. Cell, proliferation in the methionine treated group was significantly lower than that of the control group. Northern analysis revealed that expression of p53 in methionine-treated MCF-7 cells was approximately 70% lower than that of control cells. p53 is a key cell cycle regulatory, protein that has been implicated in tumorigenesis and cancer progression. Alteration of the p53 tumor suppressor gene is the most common genetic change found in a wide variety of malignancies, including cancer. This study shows that excess methionine (5 g/L) inhibited proliferation of MCF-7 breast cancer cells, and down regulation of p53 is correlated with this inhibition. These findings may aid in the development of nutritional strategies for breast cancer therapy.     

An insulin effect on cytoplasmic estrogen receptor in the human breast cancer cell line MCF-7

It has recently been reported (Horwitz, K. B., Zava, D. T., Thilagar, A. K., Jensen, E. M., and McGuire, W. L. (1978) Cancer Res. 38, 2434-2437) than the human breast cancer-derived cell line MCF-7 from EG&G Mason Research Institute contains no 8 S and very little 4 S cytoplasmic estrogen receptor. Even so, we have found significant levels of cytoplasmic estrogen receptor in MCF-7 cells from this source. The receptor was found at a maximum level of 132 fmol/mg of cytoplasmic protein, and had an apparent dissociation constant at 30 degrees C of 7.3 X 10(-10) M and at 4 degrees C of 1.2 X 10(-10) M. In sucrose gradients without KCl, the receptor migrated at 6-7 S, and with 0.4 M KCl, at 3-4 S. The receptor was specific for estrogen, in that a 100-fold excess of diethylstilbestrol eliminated binding of radiolabeled estrogen, whereas hydrocortisone, aldosterone, progesterone, and testosterone had no effect. It was further demonstrated that at least part of the reason for the discrepancy between our data and those of Horwitz et al. is that the high insulin level (10 microgram/ml) used by Horwitz et al. dramatically lowers the assayable level of receptor. These results may have important implications for steroid receptor assays in other cell lines in tissue culture and in human breast cancer patients as well.     

Proteomic analysis of the effect of retinoic acids on the human breast cancer cell line MCF-7

Breast cancer is the most common type of cancer in women in many areas and is increasing found in developing countries, where the majority of cases are diagnosed in late stages. Retinoic acids, through their associated nuclear receptors, exert intoxicating effects on cell growth, differentiation and apoptosis, and hold significant promise in relation to cancer therapy and chemoprevention. To enhance our understanding of the molecular mechanisms associated with retinoic acids in the breast cancer cell line MCF-7 in a time-dependent manner, we conducted a proteomic analysis of MCF-7 cells using the 2-DE couple with high-throughput mass spectrometry and bioinformatics tools. In the 2-DE patterns of MCF-7 cells treated with retinoic acid in a time-dependent manner, 35 protein spots were found to be differentially expressed. These were 17 increased, 4 decreased, and 14 unevenly expressed protein spots, all of which were analyzed using LTQ-FTICR mass spectrometry. Furthermore, five candidate proteins, up-regulated, were validated by western blotting. These were nucleoredoxin, latexin, aminomethyltransferase, translationally controlled one tumor protein, and rab GDP dissociation inhibitor β. These observations represent novel findings leading to new insight into the exact mechanism behind the effect of retinoic acids in MCF-7 cells while also identifying possible therapeutic targets for breast cancer diagnosis and novel drug development paths for the treatment of this disease.     

Altered regulation of P-450IA1 expression in a multidrug-resistant MCF-7 human breast cancer cell line

MCF-7 human breast cancer cells, selected for resistance to adriamycin (AdrR), exhibit the phenotype of multidrug resistance (MDR). Previous studies have shown that resistance in AdrR MCF-7 cells is associated with several biochemical changes that are similar to those induced in rat hyperplastic nodules, preneoplastic liver lesions which display broad spectrum resistance to carcinogens and hepatotoxins. In this report, we show that these changes in the AdrR MCF-7 cells are also associated with the development of cross-resistance to the procarcinogen benzo(a)pyrene (BP) and are associated with a marked defect in the conversion of BP to its cytotoxic, carcinogenic metabolites by AdrR cells. Since aryl hydrocarbon hydroxylase is the principle enzyme activity which converts benzo(a)pyrene to toxic hydroxylated forms, the regulation of cytochrome P-450IA1 expression, the gene encoding this enzyme activity in MCF-7 cells, was examined. Incubation with 100 nM 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) for 24 h results in a marked increase in aryl hydrocarbon hydroxylase activity in wild type (WT) but not AdrR MCF-7 cells. The alteration in aryl hydrocarbon hydroxylase expression in the AdrR cells is not overcome by incubation either with higher concentrations of TCDD (1 microM) or for longer periods of time (4 days). Northern blot analysis indicates that this defect in AdrR MCF-7 cells involves a regulatory defect at the level of P-450IA1 RNA. Following transfection of a construct containing the normal mouse P-450IA1 promoter fused to a reporter gene (bacterial chloramphenicol acetyltransferase) into WT and AdrR MCF-7 cells, TCDD induced chloramphenicol acetyltransferase activity in WT MCF-7 cells only. Furthermore, TCDD also induces both DT-diaphorase and UDP-glucuronyltransferase activities in WT, but not AdrR cells. These data suggest that the defect in the AdrR MCF-7 cells is not due to a structural P-450IA1 gene mutation, but rather involves a product regulating the polycyclic hydrocarbon-inducible expression of several drug-metabolizing enzyme activities. This defect in the AdrR MCF-7 cells is also associated with the development of resistance to ellipticine, an anticancer agent which is converted to more toxic hydroxylated species by aryl hydrocarbon hydroxylase or a similar mixed function oxidase. The WT and AdrR MCF-7 cells represent a useful model to study the regulation of the P-450IA1 gene in human cells.     

Lipotropes regulate bcl-2 gene expression in the human breast cancer cell line,MCF-7

Lipotropes, a methyl group containing nutrients, including choline, methionine, folic acid, and vitamin B(12), are essential nutrients for humans. They are important methyl donors that interact in the metabolism of one-carbon units and are essential for the synthesis and methylation of deoxyribonucleic acid. The purpose of this study was to examine the effects of excess lipotropes on the growth of a human breast cancer cell line, MCF-7, and normal mammary cells, MCF-10A, in culture. Both cell lines were grown in basal culture medium for 24 h and then switched to medium supplemented with 50 times the amount of each lipotrope as basal culture medium (control). Although there were no significant differences in growth between treatments in either cell line, gene array and Northern analysis revealed that expression of bcl-2 was decreased in lipotrope-treated MCF-7 cells. The ability to induce tumor cell death could have many uses in the prevention and treatment of cancer. Bcl-2 regulates apoptosis and has been shown to directly affect the sensitivity of cancer cells to chemotherapy agents, and it is suggested that strategies designed to block Bcl-2 might prove useful in sensitizing tumor cells to chemotherapy-induced apoptosis. This study shows that although excess lipotropes do not inhibit the growth of breast cancer cells, they can down-regulate the bcl-2 gene, suggesting that lipotropes may increase the susceptibility of breast cancer cells to anticancer drugs.     

白细胞介素-2受体在乳腺癌MCF-7细胞的表达

目的 :观察乳腺癌MCF 7细胞上白细胞介素 2受体 (IL 2R)α、β和γ链的表达、IL 2对MCF 7细胞增殖的作用及雌激素对三条链表达的影响。方法 :使用特异性IL 2R多克隆抗体以免疫细胞化学方法和流式免疫荧光法检测MCF 7细胞上IL 2R的表达 ,以MTT法及3 H TdR掺入法检测细胞增殖情况。结果 :MCF 7细胞上存在IL 2Rα、β、γ的免疫阳性物质 ,其中IL 2Rγ的表达要强于IL 2Rα、β的表达 ;10 -6mol/L浓度的雌二醇可促进IL 2Rα、β的阳性细胞数及IL 2Rγ的免疫阳性物质的含量 ;IL 2在 10 0U/ml至 10 0 0U/ml的浓度范围内可显著促进MCF 7细胞的增殖。结论 :MCF 7细胞上存在IL 2R且其表达受雌二醇的调节 ,IL 2可能通过IL 2R影响MCF 7细胞的增殖     

Additive growth-inhibitory effects of DL-alpha-difluoromethylornithine and antiestrogens on MCF-7 breast cancer cell line

We studied the growth inhibitory effects of DL-alpha-difluoromethylornithine, and antiestrogens (tamoxifen, 4-hydroxytamoxifen, trioxifene, keoxifene, and LY117018) as single agents and in combinations on the proliferation of a breast cancer cell line, MCF-7. At 0.1 mM difluoromethylornithine, the proliferation of MCF-7 cells was inhibited to 75 +/- 6% of the controls. Treatment of the cells with 0.1 microM 4-hydroxytamoxifen reduced cell growth to 72 +/- 4%. Combination of 0.1 mM difluoromethylornithine and 0.1 microM 4-hydroxytamoxifen reduced cell growth to 38 +/- 5%, indicating additive growth inhibitory effects. Similar additive effects were observed with all 5 antiestrogens in combination with difluoromethylornithine.     

Apoptosis-like cell death of human breast cancer cell line MCF-7 induced by buprenorphine hydrochloride

The analgesic buprenorphine hydrochloride (Bph) induced apoptosis-like cell death in the caspase-3-deficient human breast cancer cell line, MCF-7. This apoptosis-like cell death activated key molecules in the mitochondrial apoptotic pathway: cytochrome c, caspase-9, caspase-7, and caspase-6. Bph caused the release of fluorescent protein from the mitochondria of MCF-7 cells transfected with the pDsRed2-Mito-vector in a time-dependent manner, suggesting disruption of the mitochondrial membrane. Zn(2+) as high as 2 mM did not inhibit the DNase that took part in this apoptosis. Thus, this unidentified DNase might resemble other DNases involved in apoptosis-like cell death whose activity is not inhibited by zinc ion.     

The 5-HT2A serotoninergic receptor is expressed in the MCF-7 human breast cancer cell line and reveals a mitogenic effect of serotonin

Serotonin (5-hydroxytryptamine, 5-HT) has been described as a mitogen in a variety of cell types and carcinomas. It exerts its mitogenic effect by interacting with a wide range of 5-HT receptor types. Certain studies suggest that some selective serotonin re-uptake inhibitors promote breast cancer in animals and humans. This study attempts to clarify the role of serotonin in promoting the growth of neoplastic mammary cells. Expression of the 5-HT(2A) serotoninergic receptor subtype in MCF-7 cells was determined by RT-PCR, Western blotting, and immunofluorescence analysis. The mitogenic effect of 5-HT on MCF-7 cells was determined by means of the MTT proliferation assay. We have demonstrated that the 5-HT(2A) receptor subtype is fully expressed in the MCF-7 human breast cancer cell line, in terms of encoding mRNA and receptor protein. Automated sequencing has confirmed that the 5-HT(2A) receptor present in this cell line is identical to the 5-HT(2A) receptor found in human platelets and in human cerebral cortex. Furthermore, this receptor was found by immunofluorescence to be on the plasma membrane. MTT proliferation assays revealed that 5-HT and DOI, a selective 5-HT(2A) receptor subtype agonist, stimulated MCF-7 cell. These results indicate that 5-HT plays a mitogenic role in neoplastic mammary cells. Our data also indicate that 5-HT exerts this positive growth effect on MCF-7 cells through, in part, the 5-HT(2A) receptor subtype, which is fully expressed in this cell line.