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.     

Cloning of cDNA sequences of a progestin-regulated mRNA from MCF7 human breast cancer cells.

A cDNA clone corresponding to an mRNA regulated by the progestin R5020, has been isolated by differential screening of a cDNA library from the MCF7 breast cancer cell line, which contains estrogen and progesterone receptors. This probe hybridized with a single species of poly A + RNA of 8-kb molecular weight as shown by Northern blot analysis and could also be used to total RNA preparation. This recombinant clone hybridized specifically to an mRNA coding for a 250,000 daltons protein when translated in vitro. This protein was identical to the 250 kDa progestin-regulated protein that we previously described (Biochem. Biophys. Res. Commun. 121, 421-427, 1984) as shown by immunoprecipitation with specific rabbit polyclonal antibodies. Dose-response curve and specificity studies show that the accumulation of the Pg8 mRNA and that of the 250-kDa protein was increased by 5 to 30-fold following progestin treatment and that this effect was mediated by the progesterone receptor. Time course of induction indicated that the accumulation of mRNA was rapid and preceded that of the protein. This is the first report on a cloned cDNA probe of progestin-regulated mRNA in human cell lines.     

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.     

Stable expression of cytochrome P450IIIA7 cDNA in human breast cancer cell line MCF-7 and its application to cytotoxicity testing.

A mammalian cell expression plasmid containing cytochrome P450IIIA7 complementary DNA was constructed. Breast cancer cells (MCF-7) were transfected with the plasmid and neomycin-resistant selection marker plasmid. We established three cell lines, termed M13, M21, and M27, which expressed the cytochrome P450IIIA7 as examined by RNA blot and immunoblot analyses. These cell lines showed 8- to 10-fold higher sensitivity against aflatoxin B1 compared to parental MCF-7 cells, suggesting that cytochromes P450IIIA7 expressed in the cells were responsible for the production of the cytotoxic metabolite of aflatoxin B1.     

Connective tissue growth factor induces apoptosis in human breast cancer cell line MCF-7

Connective tissue growth factor (CTGF) is a member of an emerging CCN gene family that is implicated in various diseases associated with fibro-proliferative disorder including scleroderma and atherosclerosis. The function of CTGF in human cancer is largely unknown. We now show that CTGF induces apoptosis in the human breast cancer cell line MCF-7. CTGF mRNA was completely absent in MCF-7 but strongly induced by treatment with transforming growth factor beta (TGF-beta). TGF-beta by itself induced apoptosis in MCF-7, and this effect was reversed by co-treatment with CTGF antisense oligonucleotide. Overexpression of CTGF gene in transiently transfected MCF-7 cells significantly augmented apoptosis. Moreover, recombinant CTGF protein significantly enhanced apoptosis in MCF-7 cells as evaluated by DNA fragmentation, Tdt-mediated dUTP biotin nick end-labeling staining, flow cytometry analysis, and nuclear staining using Hoechst 33258. Finally, recombinant CTGF showed no effect on Bax protein expression but significantly reduced Bcl2 protein expression. Taken together, these results suggest that CTGF is a major inducer of apoptosis in the human breast cancer cell line MCF-7 and that TGF-beta-induced apoptosis in MCF-7 cells is mediated, in part, by CTGF.     

Existence of GPR40 functioning in a human breast cancer cell line, MCF-7

GPR40, which has recently been identified as a G-protein-coupled cell-surface receptor for long-chain fatty acids, was assessed in a human breast cancer cell line (MCF-7). We detected GPR40 mRNA by RT-PCR and found that oleate and linoleate, but not palmitate or stearate, caused an increase in cellular Ca(2+) concentrations, which was partially blocked by the pertussis toxin (PTX) treatment. We examined the expression of GPR40 mRNA by quantitative RT-PCR in the relation to cell number. It was significantly increased at the beginning and at the end of cell proliferation. These results indicate the possibility that GPR40 for long-chain fatty acids may be involved in cellular function such as cell proliferation, providing a new perspective for the action of long-chain fatty acids on mammary epithelial cells.     

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.     

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.     

17beta-Estradiol inhibits osteoprotegerin production by the estrogen receptor-alpha-positive human breast cancer cell line MCF-7

Estrogen regulates various cytokines and growth factors in estrogen receptor (ER)-positive human breast cancer. Receptor activator of NF-κB ligand (RANKL) is an essential cytokine for osteoclasts, whereas osteoprotegerin (OPG) is a soluble inhibitor for RANKL. We analyzed the regulation of the RANKL/OPG system by estrogens and androgens in the ER-positive breast cancer cell line MCF-7 and the ER-negative breast cancer cell line MDA-MB-231. In MCF-7 cells, which predominantly express ER-α, 17β-estradiol and testosterone dose-dependently decreased OPG mRNA levels and protein secretion by 70 and 65%, respectively (p < 0.0001 by ANOVA). The inhibition of OPG production by 17β-estradiol and testosterone was specifically prevented by the pure anti-estrogen ICI 182,780, and the testosterone effect was prevented by an aromatase inhibitor. In conclusion, 17β-estradiol suppressed OPG production by human breast cancer cell lines in a dose-dependent and specific manner, indicating that the RANKL/OPG cytokine system is an estrogen-responsive target in breast cancer.     

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.     

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.     

Serum and insulin inhibit cell death induced by cycloheximide in the human breast cancer cell line MCF-7

Summary Continuous exposure of cells to cycloheximide (CHM) terminates in cell death. This may result from CHM’s inhibition of protein synthesis. In the present study we investigated the effect of serum and insulin on cell death induced by CHM in the human breast cancer cell line MCF-7, and correlated this effect to the inhibition of protein synthesis. Cell death was evaluated by measuring either dead cells by the trypan blue dye exclusion test or by the release of lactic dehydrogenase into the culture medium. CHM (0.1 to 50 μg/ml) was shown to induce cell death in a time- and concentration-dependent manner. Including either fetal bovine serum or insulin in the culture medium inhibited this cell death in a concentration-dependent manner. Protein synthesis as measured by [³H]leucine incorporation was inhibited by the increasing concentration of CHM, However, fetal bovine serum and insulin did not alter the protein synthesis inhibition rate induced by CHM. These results indicate that inhibition of protein synthesis is not enough for cell death to proceed. Insulin or factors present in serum may stabilize some crucial cell proteins (key enzymes, cytoskeletal or membrane components) which are vital for cell life.     

Estrogen-like properties of brominated analogs of bisphenol A in the MCF-7 human breast cancer cell line

Tetrabromobisphenol A (TeBBPA) is a four-meta-brominated variant of bisphenol A (BPA) and is one of the most commonly used brominated flame retardants worldwide. We compared the estrogenic potency of TeBBPA, BPA and the brominated analogs mono- (MBBPA), di- (DBBPA), and tribromobisphenol A (TrBBPA) in the estrogen-dependent human breast cancer cell line MCF-7. All of the compounds competed with 17β-estradiol for binding to the estrogen receptor, although the affinity of the test chemicals to the estrogen receptor was much lower than that of 17β-estradiol. TrBBPA and TeBBPA showed a considerably lower access to the estrogen receptors within intact MCF-7 cells incubated in 100% serum compared to incubation in serum-free medium, indicating a strong binding to serum proteins. BPA, MBBPA, and DBBPA showed only a slightly reduced access to the receptors. All of the test compounds induced proliferation in MCF-7 cells, the potential decreasing with increasing number of bromo-substitutions. TeBBPA did not induce maximal cell growth, indicating cytotoxic effects at high concentrations. BPA and the brominated analogs, except TeBBPA, induced progesterone receptor and pS2 to the same extent as 17β-estradiol, although at much higher concentrations. Our studies demonstrate that compared to 17β-estradiol, BPA and the brominated analogs have much lower estrogenic potencies for all of the endpoints tested, TeBBPA being the least estrogenic compound. This revised version was published online in July 2006 with corrections to the Cover Date.     

Purification and characterization of an alpha 1-antichymotrypsin-like 66 kDa protein from the human breast cancer cell line, MCF-7.

The synthesis of a 66 kDa protein immunoreactive with antibodies to human alpha 1-antichymotrypsin (alpha 1-ACT) is induced by estradiol (E2) in the human breast cancer cell line MCF-7. We have purified this alpha 1-ACT-like 66 kDa protein from medium conditioned by MCF-7 cells, performed a comparative physico-chemical characterization with serum alpha 1-ACT, and analysed its presumed positive regulatory effect on growth of MCF-7 cells. The 66 kDa protein is a functional antiproteinase which is antigenically identical to serum alpha 1-ACT. The 66 kDa protein does however deviate from serum alpha 1-ACT with respect to mol. wt. and pattern of microheterogeneity, the molecular mechanism for this is probably an incomplete glycoprotein processing in the MCF-7 cells. The results of our growth experiments suggest that the 66 kDa protein is a minor positive growth regulatory factor, which may contribute to breast carcinoma cell proliferation in a cooperative manner.     

Styrylpyrone derivative induces apoptosis through the up-regulation of Bax in the human breast cancer cell line MCF-7

In the fight against cancer, novel chemotherapeutic agents are constantly being sought to complement existing drugs. Various studies have presented evidence that the apoptosis that is induced by these anticancer agents is implicated in tumor regression, and Bcl-2 family genes play a part in apoptosis following treatment with various stimuli. Here, we present data that a styrylpyrone derivative (SPD) that is extracted from the plant Goniothalamus sp. showed cytotoxic effects on the human breast cancer cell line MCF-7. SPD significantly increased apoptosis in MCF-7 cells, as visualized by phase contrast microscopy and evaluated by the Tdt-mediated dUTP nick end-labeling assay and nuclear morphology. Western blotting and immunostaining revealed up-regulation of the proapoptotic Bax protein expression. SPD, however, did not affect the expression of the anti-apoptotic protein, Bcl-2. These results, therefore, suggest SPD as a potent cytotoxic agent on MCF-7 cells by inducing apoptosis through the modulation of Bax levels.