Estrogen and progesterone up-regulate glucose transporter expression in ZR-75-1 human breast cancer cells

Breast cancer incidence increases in women receiving combined estrogen and progesterone therapy. Breast tumors show increased expression of the glucose transporter GLUT1. We determined the effect of these hormones on GLUT1-4 expression and deoxyglucose transport in ZR-75-1 breast cancer cells. Immunoblotting, immunocytochemistry, flow cytometry, and RT-PCR showed that GLUT1 expression is up-regulated by progesterone and, to a greater degree, combined therapy. GLUT2 expression is unaffected by hormonal treatment. GLUT3 protein and RNA is up-regulated by progesterone and combined therapy, and GLUT4 protein expression is up-regulated by all hormonal treatments. Deoxyglucose transport studies revealed the presence of three transport components with characteristics corresponding to GLUT1/4, GLUT2, and GLUT3. 17beta-Estradiol produced a slight increase in transport at the Michaelis constant (Km) corresponding to GLUT3. Progesterone produced a small increase in transport at the Km corresponding to GLUT1/4, and combined 17beta-estradiol and progesterone produced a small increase in transport at the Km corresponding to GLUT3 and a large increase in transport at the Km corresponding to GLUT1/4. This indicates that 17beta-estradiol and progesterone differentially regulate GLUT1-4 expression and that these changes correlate to changes in glucose uptake. We postulate that combined hormone replacement therapy provides a survival advantage to developing ZR-75 breast cancer cells.     

Demonstration of proteinase inhibitors cystatin A, B and C in breast cancer and in cell lines MCF-7 and ZR-75-1

Polyclonal rabbit antibodies to endogenous natural inhibitors of cysteine proteinases (cystatins) were used for their immunohistochemical demonstration in samples of breast cancers and in two breast cancer derived cell lines (MCF-7 and ZR-75-1). The influence of estrogen stimulation on the expression of these inhibitors was also studied. The results showed that all cystatins tested can be found equally inside carcinomatous cells or in surrounding connective tissue, as well as in both cell lines examined. Further, the expression of inhibitors can be regulated by estrogen, and a certain role of cystatins in the regulation of the mitotic activity and differentiation of mammary cells can be supposed.     

Hormonal control of plasminogen activator secretion in ZR-75-1 human breast cancer cells in culture

Activity of secreted plasminogen activator (PA) by ZR-75-1 human breast cancer cells in culture is shown to be altered by the addition of physiologically relevant concentrations of the hormones 17 beta-estradiol (E2), insulin, and dexamethasone. After 48 h, E2 stimulated PA activity 6-fold at concentrations as low as 10(-12) M. This stimulation was prevented by the addition of actinomycin D and cycloheximide. The antiestrogen tamoxifen reduced estrogen stimulation of PA, but had slight stimulatory effects on PA secretion by itself. Insulin (5 X 10(-10) M) induced a 2-fold increase in PA activity. Effects of insulin and E2 were additive, suggesting independent sites of control of PA production. Dexamethasone (10(-8) M) decreased PA activity by 20%, but did not inhibit cell growth at the concentration tested. These data suggest that secreted PA activity is differentially regulated by hormones and that effects of PA and growth do not occur in parallel.     

Down-regulation of estrogen receptors by androgens in the ZR-75-1 human breast cancer cell line

Much clinical evidence indicates that androgens have beneficial effects in the treatment of breast cancer in women. Physiological concentrations of androgens strongly inhibit both basal and estrogen-induced cell proliferation in the human breast cancer cell line ZR-75-1 through their interaction with the androgen receptor. The present study shows that androgens strongly suppress estrogen receptor (ER) and progesterone receptor contents in this model, as measured by radioligand binding and anti-ER monoclonal antibodies. Similar inhibitory effects are observed on the levels of ER messenger RNA (mRNA) measured by ribonuclease protection assay. The androgenic effect is observed at subnanomolar concentrations of the nonaromatizable androgen 5 alpha-dihydrotestosterone, regardless of the presence of estrogens, and is competitively reversed by the antiandrogen hydroxyflutamide. Such data on ER expression provide an explanation for at least part of the antiestrogenic effects of androgens on breast cancer cell growth and moreover suggest that the specific inhibitory effects of androgen therapy could be additive to the standard treatment limited to blockade of estrogens by antiestrogens.     

Melatonin augments the sensitivity of MCF-7 human breast cancer cells to tamoxifen in vitro.

Cultured MCF-7 human breast cancer cells were pre-exposed to either melatonin (232 ng/mL) or vehicle for 24 hrs prior to being washed and then re-exposed to either ethanol-vehicle or varying concentrations of tamoxifen (37.1 ng/mL, 3.71 micrograms/mL, 371 micrograms/mL) or melatonin (2.32 pg/mL, 232 ng/mL, 23.2 ng/mL) for 5 additional days. Only 371 ng/mL tamoxifen caused a 38% growth inhibition of cells pre-exposed to vehicle whereas all concentrations of tamoxifen inhibited the growth of melatonin pre-exposed cells by 28% to 61% in a dose-dependent manner. Melatonin pre-exposure, potentiated the inhibitory effect of only 232 ng/mL melatonin. Comparison of IC50 values indicate that tamoxifen is approximately a 100 times more potent inhibitor of breast cancer cell growth following the pretreatment of cells with a physiological concentration of melatonin. These results indicate that melatonin has the capability to augment the inhibitory actions of tamoxifen, and to a lesser extent itself, on human breast cancer cell growth.     

Inducible upregulation of oestrogen receptor-beta1 affects oestrogen and tamoxifen responsiveness in MCF7 human breast cancer cells

To investigate the effect of altered oestrogen receptor (ER)alpha and ERbeta expression on oestrogen and anti-oestrogen action in breast cancer, we have stably expressed an inducible ERbeta1 in MCF7 breast cancer cells. Stably expressing clones were isolated and over-expression of ERbeta1 correlated with increased levels of specific radiolabelled oestradiol (E2) binding. Increased ERbeta1 did not affect endogenous levels of ERalpha but increased progesterone receptor (PR) levels. Over-expression of ERbeta1 reduced growth responses to E2 in contrast to little if any effect of over-expression of ERalpha. In oestrogen-replete conditions, over-expression of ERbeta1 but not ERalpha reduced proliferation. Over-expression of ERbeta1 did not result in anti-oestrogen resistance but was associated with increased sensitivity to 4-hydroxytamoxifen. Our results suggested that over-expression of ERbeta1 in the presence of an endogenously expressed ERalpha was associated with tamoxifen sensitivity but may negatively modulate ERalpha-mediated growth. However, not all ERalpha activities were inhibited since endogenous PR expression was increased by both ERalpha and ERbeta1 over-expression. These data paralleled those seen in some in vivo studies showing a relationship between PR and ERbeta expression as well as ERbeta expression and tamoxifen sensitivity of ER-positive breast cancer patients. These models are relevant and will be useful for dissecting the role of ERbeta1 expression in ER-positive breast cancer.     

Opposite effects of estrogen and the progestin R5020 on cell proliferation and GCDFP-15 expression in ZR-75-1 human breast cancer cells

We have recently demonstrated that physiological concentrations of androgens caused a marked inhibition of basal and 17 beta-estradiol (E2)-induced cell growth in ZR-75-1 human breast cancer cells. Moreover, these steroids exert effects on GCDFP-15 (gross cystic disease fluid protein-15) expression that are opposite to their above-indicated actions on cell proliferation. The synthetic progestin R5020 (17.21-dimethyl-19-nor-4,9-pregnadiene-3,20-dione), on the other hand, causes a potent inhibition of E2-induced ZR-75-1 cell growth. In order to further characterize the hormonal regulation of GCDFP-15 expression and to better understand the antagonism between progestin and estrogen action in breast cancer cells, we have studied the effect of R5020 on both GCDFP-15 expression and cell growth in ZR-75-1 cells. After a 10-day incubation, the 4-fold stimulatory effect of 1 nM E2 on cell growth was 60% decreased by maximal effective concentrations of R5020 (greater than 1 nM) while, in the absence of E2, R5020 had no effect. The mitogenic action of E2 was accompanied by a 75% inhibition of GCDFP-15 secretion while nanomolar concentrations of R5020 induced 1.4- and 5.2-fold increases in GCDFP-15 secretion in control and E2-treated ZR-75-1 cells, respectively. While E2 caused a marked inhibition of GCDFP-15 mRNA levels, R5020 induced a maximal 2- to 3-fold increase (above control) in GCDFP-15 mRNA accumulation in cells simultaneously incubated with E2.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of sulphation on the oestrogen agonist activity of the phytoestrogens genistein and daidzein in MCF-7 human breast cancer cells

The phytoestrogens genistein, daidzein and the daidzein metabolite equol have been shown previously to possess oestrogen agonist activity. However, following consumption of soya diets, they are found in the body not only as aglycones but also as metabolites conjugated at their 4'- and 7-hydroxyl groups with sulphate. This paper describes the effects of monosulphation on the oestrogen agonist properties of these three phytoestrogens in MCF-7 human breast cancer cells in terms of their relative ability to compete with [(3)H]oestradiol for binding to oestrogen receptor (ER), to induce a stably transfected oestrogen-responsive reporter gene (ERE-CAT) and to stimulate cell growth. In no case did sulphation abolish activity. The 4'-sulphation of genistein reduced oestrogen agonist activity to a small extent in whole-cell assays but increased the relative binding affinity to ER. The 7-sulphation of genistein, and also of equol, reduced oestrogen agonist activity substantially in all assays. By contrast, the position of monosulphation of daidzein acted in an opposing manner on oestrogen agonist activity. Sulphation at the 4'-position of daidzein resulted in a modest reduction in oestrogen agonist activity but sulphation of daidzein at the 7-position resulted in an increase in oestrogen agonist activity. Molecular modelling and docking studies suggested that the inverse effects of sulphation could be explained by the binding of daidzein into the ligand-binding domain of the ER in the opposite orientation compared with genistein and equol. This is the first report of sulphation enhancing activity of an isoflavone and inverse effects of sulphation between individual phytoestrogens.     

Endogenously expressed estrogen receptor and coactivator AIB1 interact in MCF-7 human breast cancer cells

Coactivators are believed to mediate estrogen-induced gene responses via interaction with estrogen receptors (ER). Currently, a major challenge is to determine the importance of each coactivator in a specific cell type and promoter context in response to a particular ligand. The potential of ER to interact with a growing list of coactivators has been shown in a variety of in vitro and gene transfer assays, yet very few data have demonstrated the interaction of endogenous coactivators with ER in intact cells. We report here a ligand-specific interaction of endogenous human ER (hER) and the AIB1 coactivator in MCF-7 human breast cancer cells by using immunoprecipitation analyses. Complexes between endogenously expressed hER and AIB1 were detected in estradiol-treated cells and to a much lesser extent in cells treated with the partial agonist, monohydroxytamoxifen. We were unable to detect an hER-SRC-1 complex in our immunoprecipitations from MCF-7 cells. The in vitro-binding affinity for mouse ER interaction with AIB1 was estimated to be 40-120 nM. We conclude that AIB1 is a major coactivator for hER in MCF-7 human breast cancer cells.     

Let-7a对人乳腺癌MCF-7细胞的作用及机制探讨

目的观察Let-7a对体外生长的人乳腺癌MCF-7细胞株的抑制作用,并探讨其作用机制。方法分别用Lipofectamine^TM2000介导的Let-7a和siRNA转染人乳腺癌细胞株MCF-7（分别为Let-7a组、空白对照组和阴性对照组）,半定量RT—PCR法检测两组C—myc mRNA表达;Western blot法测定转染24h后C-myc蛋白的表达水平;CCK-8法检测细胞增殖抑制率。结果Let-7a组的C-myc mRNA表达水平明显低于空白对照组和阴性对照组,P均〈0．01;在MCF-7细胞中存在相对分子质量62kD的特异性条带,与C-myc相对分子质量相符,Let-7a组特异性条带明显弱于对照组;Let-7a组细胞增殖抑制率明显高于阴性对照组（P〈0．05）,且具有时间和浓度依赖性。转染24h后,Let-7a组凋亡率明显高于对照组（P〈0．05）。结论Let-7a对体外生长的人乳腺癌MCF-7细胞增殖有抑制作用;其机制可能为抑制C-myc基因的表达。     

Progesterone increases tissue factor gene expression, procoagulant activity, and invasion in the breast cancer cell line ZR-75-1

Progesterone in hormonal preparations increases the incidence of breast cancer. Tissue factor (TF), the initiator of the extrinsic coagulation pathway, is associated with metastasis in a wide variety of cancers. We demonstrate herein that TF mRNA and protein are up-regulated by progesterone in the breast cancer cell line ZR-75. Epidermal growth factor, also associated with increased breast cancer risk, did not regulate TF. The increase in TF is both rapid and transient; increasing after 6 h, reaching a maximum at 24 h, before decreasing to basal levels at 72 h. Sucrose gradient experiments demonstrated that TF is located in the heavy fraction of the plasma membrane, although caveolin-1 is not expressed in ZR-75. To understand the physiological implications of an increase in TF, we performed coagulation and invasion assays. An increase in TF corresponded to an increase in procoagulant activity. Furthermore, progesterone increased the invasion of ZR-75 cells through a matrigel, an effect that was blocked by an antibody against TF. Because TF expression is associated with an enhanced risk of metastasis, we postulate that the progesterone-dependent up-regulation of TF provides a survival advantage to burgeoning breast cancer cells and may contribute to the increased risk of cancer associated with combined hormone replacement therapy.     

Melatonin effects on intercellular junctional communication in MCF-7 human breast cancer cells

Melatonin exerts a direct antiproliferative effect on estrogen-responsive MCF-7 cells in culture. Recently, the importance of the anti-invasive actions of melatonin as a part of the oncostatic action of this indolamine has been reported. Gap junctional intercellular communication is known to be involved in controlling cell proliferation and differentiation, and a decrease in intercellular junctional communication has been described in highly invasive mammary cancer cells. Because melatonin at physiological doses (1 nM) shifts MCF-7 cells to a lower invasive status, we postulate that melatonin could modulate the levels of gap junctional intercellular communication in these tumor cells. To test our hypothesis, we studied gap junctional intercellular communication in MCF-7 human breast cancer cells previously (7-8 days) treated, or not, with melatonin (10 microM or 1 nM). Using the scrape-loading assay dye-transfer technique to introduce 0.05% Lucifer yellow into cells, we measured the ability of the tumor cells to transfer dye to adjacent cells. Rhodamine dextran (0.05%) was used as a control dye to verify that dye-transfer occurs through intercellular junctions. The presence of melatonin (10 microM or 1 nM) in the culture medium significantly increased (P < 0.01) the transfer of the dye to adjacent cells through gap junctions. This increase was greater at 10 microM melatonin, and averaged scan profiles of cells treated with melatonin 10 microM showed a statistically significant increase (P < 0.01) in the integrated optical density values, and a broadening of the densitometric scan. These findings suggest that melatonin could exert its antitumor action, at least in part, by increasing regulatory signals that are passed between adjacent epithelial cells through intercellular junctions.     

Estrogen regulation of transferrin gene expression in MCF-7 human breast cancer cells

Transferrin (Tf) is an iron transport protein expressed in MCF-7 human breast cancer cells. In nuclear run-on assays, 17beta-estradiol (E2) increased the rate of Tf gene expression approximately 3-fold within 1 h after treatment and reporter gene activity was also induced in MCF-7 cells transfected with a construct containing a -3600 to +39 Tf gene promoter insert. Deletion and mutation analysis identified an E2-responsive promoter region between -811 and -762, which was GC-rich (80%) and contained two nonconsensus estrogen response elements (EREs). E2-responsiveness of this region was associated with a GGACA(N)(3)TGGCC motif (-803 to -791) which bound human estrogen receptor alpha (hERalpha) in gel mobility shift assays. In Drosophila Schneider SL-2 cells, the -811 to -752 was E2-responsive after cotransfection with hERalpha expression plasmid plus E2, whereas Sp1 protein did not induce transactivation. These studies confirm that E2 induces Tf gene expression through a nonconsensus distal ERE.     

Phosphorylation of the estradiol receptor in MCF-7 human breast cancer cells in culture

Double labelling and Western blot techniques were used to demonstrate phosphorylation of estradiol receptor. Cells in monolayer culture were incubated with [32P]orthophosphate for 18 h followed by covalent whole cell labelling of the estradiol receptor with tritiated tamoxifen aziridine [( 3H]TA). Labelled receptor was precipitated with the monoclonal antibodies H222 or JS 34/32, coupled to protein A-Sepharose, and purified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), or transferred to nitrocellulose paper. Receptor protein was detected on the Western blot with the monoclonal antibody H222 and rabbit anti-rat peroxidase conjugate. Phosphorylated receptor was visualized by autoradiography. Tritium and 32P activities were monitored in the gels. Two phosphorylated forms of the receptor (molecular weights 67 and 50 kDa) have been detected in MCF-7 cells. Estradiol treatment of the cells was found to increase phosphorylation of the receptor. In estradiol-treated cells both phosphorylated receptor forms were present mainly in the nuclear extract. Both forms bound [3H]TA as evidence by SDS-PAGE. [3H]TA binding was abolished by excess non-radioactive estradiol. In addition two phosphorylated proteins of approximately 120 and 90 kDa were regularly coprecipitated with receptor in cytosol. These proteins did not bind [3H]TA. The 90 kDa phosphorylated protein was identified as a heat shock protein (hsp-90).