MCF-7; a human breast cancer cell line with estrogen, androgen, progesterone, and glucocorticoid receptors.

We have identified receptors for glucocorticoids, progestins, and androgens in a human breast tumor cell line (MCF-7) known to have estrogen receptor. Sucrose density gradients show that MCF-7 cytosol contains approximately 100 fm/mg protein estradiol (E2-3H) receptor, more than 300 fm/mg protein progesterone receptor (measured with R5020-3H), about 40 fm/mg protein 5alpha-dihydrotestosterone (5alpha-DHT-3H) receptor, and 800 fm/mg glucocorticoid receptor (measured with dexamethasone-3H). Dissociation constants obtained by Scatchard analyses were approximately 0.6 x 10(-10)M (E2), 1 x 10(-9)M (R5020), 2.8 x 10(-10)M (5alpha-DHT) and 8 x 10(-9)M (dexamethasone). No cross competition was found for estrogen receptor, but progestins competed for androgen and glucocorticoid binding. The androgen, but not the glucocorticoid, partially competed for R5020 binding to progesterone receptor. This first demonstration of 4 classes of steroid receptors in human breast cancer means that MCF-7 may be an excellent in vitro model for studying the mechanism of tumor response to endocrine therapy as well as the complex relationships between binding and biological actions of these hormones.     

Androgens inhibit estrogen action in MCF-7 human breast cancer cells

We have observed that the estrogen-dependent augmentation of cytoplasmic progesterone receptor (PR_c) content in MCF-7 human breast cancer cells is completely inhibited in the presence of testosterone (T) or dihydrotestosterone (DHT)¹. This effect is neither a result of altered PR_c affinity for test ligand nor a result of the direct interaction of either androgen with PR_c. Furthermore, the antiestrogenic activity of DHT is blocked in the presence of the antiandrogen, cyproterone, indicating that it is mediated through the androgen receptor. Further investigations of this cell line may provide important insights into the effects of sex steroids upon the hormonal regulation of a variety of healthy and malignant human tissues.     

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.     

槟榔碱对人乳腺癌MCF-7细胞增殖和凋亡的影响

目的：观察槟榔碱对人乳腺癌细胞（MCF-7）增殖和凋亡的影响,并探讨其机制。方法：采用四甲基偶氮唑盐（MTT）法检测不同浓度（0、10、30、50、100、300、500μmol/L）槟榔碱对MCF-7细胞增殖的影响,Hoechst 33342染色和流式细胞术检测细胞凋亡,Western blot法检测Bax,Bcl-2和P53蛋白表达。结果：低浓度（0、10、30、50μmol/L）槟榔碱不影响细胞的增殖和凋亡;而高浓度（100、300、500 μmol/L）槟榔碱呈浓度依赖性抑制MCF-7细胞增殖、诱导MCF-7细胞凋亡、提高P53和Bax蛋白表达、降低Bcl-2蛋白表达。结论：高浓度槟榔碱抑制MCF-7细胞增殖、诱导凋亡,其机制可能与提高P53和Bax蛋白表达,降低Bcl-2蛋白表达有关。     

Effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin, 12-O-tetradecanoylphorbol-13-acetate and 17β-estradiol on estrogen receptor regulation in MCF-7 human breast cancer cells

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) exhibits remarkably potent antiestrogenic activity. To further elucidate the role of estrogen receptor (ER) regulation in this response, we examined the effects of exposure to TCDD in MCF-7 human breast cancer cells on ER mRNA levels by using an RNase protection assay, on ER accumulation by using an ER immunocytochemical essay (ER-ICA), and on ER function by competitive binding assays under conditions of saturating 17β-estradiol (E₂). Comparative studies were conducted with E₂ and 12-O-tetradecanoylphorbol-13-acetate (TPA), as both compounds are known to suppress ER expression. Our results indicate that 1 nM E₂ and 100 nM TPA both suppress ER mRNA levels as early as 4 h after exposure and to 33.6% and 16.5% of control levels, respectively, after 72 h. In contrast, no significant effect on ER mRNA levels was attributed to exposure to 10 nM TCDD. A greater than 50% reduction in positive staining was observed by ER-ICA after 72 h exposure to 1 nM E₂ and to 100 nM TPA, while only an 11% reduction in positive staining was observed with 10 nM TCDD. Specific binding of [³H]E₂ under saturating conditions (10 nM E₂) in whole cells was reduced by 50% in cultures exposed to 100 nM TPA, although no effect on binding was observed with exposure to 10 nM TCDD. In contrast, specific binding using subsaturating 1 nM [³H]E₂ was depressed by 49% in MCF-7 cells exposed to 10 nM TCDD for 72 h. This depression was inhibited by a 1-h treatment with 5 μM α-naphthoflavone, which inhibits TCDD-induced, P450-mediated, E₂ metabolism, and subsequent E₂ depletion. In conclusion, while TPA and E₂ effectively down-regulate ER expression, TCDD, under antiestrogenic conditions, has little if any effect on total ER levels in MCF-7 cells, and thus ER modulation is probably not necessary for the suppression of estrogenic activity in MCF-7 cells by TCDD. © 1996 Wiley-Liss, Inc.     

Relationship between estrogen receptors, 17 beta-hydroxysteroid dehydrogenase and estrogen content in human breast cancer.

Estrone and estradiol levels in tumor tissue cytosols were determined in 11 premenopausal and 20 postmenopausal women at the same time that 17 beta-hydroxysteroid dehydrogenase and estrogen receptors (ER) were carried out on their breast cancers. Estrogen receptor positive tumors showed significantly higher levels of estrone and estradiol. However, all ER negative tumors contained measurable amounts of both estradiol and estrone. Higher levels of estrone were observed in ER negative tumors which correlates well with high 17 beta-hydroxysteroid dehydrogenase activity. These results suggest that false negative receptor assays in the premenopausal women is not likely to be due to occupancy of receptors by endogenous estrogens. Furthermore, the higher estrone content in the ER negative group is probably due to high 17 beta-hydroxysteroid dehydrogenase activity inherent to these tumor cells.     

The covalent labeling of proteins by 17 beta-estradiol, retinoic acid, and progesterone in the human breast cancer cell lines MCF-7 and MCF-7/AdrR.

In this study we analyzed the covalent binding to proteins of 17 beta-estradiol (E2), retinoic acid (RA), and progesterone in MCF-7 and MCF-7/AdrR cells. MCF-7 cells have receptors for E2 and progesterone. MCF-7/AdrR cells do not have these receptors. After a 1-day incubation period with either [3H]E2, [3H]progesterone, or [3H]RA the levels of covalently bound radioactivity was between 1.4- to 2-fold greater in MCF-7 cells than in MCF-7/AdrR cells. We analyzed the labeled proteins with two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and fluorography. About 40 proteins were labeled by E2 in MCF-7 cells and about 10 of these proteins were the only proteins labeled by E2 in MCF-7/AdrR cells. We saw that the same 8 proteins were labeled by RA in both cell lines. Progesterone labeled 2 proteins with M(r) values of 37,000 and 20,000 in MCF-7 cells. These 2 proteins had mobilities that were the same as proteins that were labeled by either E2 or RA in both MCF-7 and MCF-7/AdrR cells. Besides these 2 proteins, we saw proteins of M(r) 51,000 (p51) and 55,000 that were covalently labeled by E2 in MCF-7 cells and by RA in both MCF-7 and MCF-7/AdrR cells. The p51 had the same mobility on 2D-PAGE as an 8-azido-[32P]cAMP-labeled protein. This protein is probably RII alpha, the type II cAMP-binding regulatory subunit of type II cAMP-dependent protein kinase. These results suggest that the estrogen receptor, while not obligatory, might still modulate the covalent linkage of E2 to protein. In addition, our results raise the possibility that some effects of some ligands of the thyroid/steroid hormone receptor family may involve the covalent linking of these hormones to proteins, including RII alpha.     

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     

Visfatin stimulates proliferation of MCF-7 human breast cancer cells

Obesity, a condition characterized by increased fat content and altered secretion of adipokines, is a risk factor for postmenopausal breast cancer. Visfatin has recently been established as a novel adipokine that is highly enriched in visceral fat. Here we report that visfatin regulated proliferation of MCF-7 human breast cancer cells. Exogenous administration of recombinant visfatin increased cell proliferation and DNA synthesis rate in MCF-7 cells. Furthermore, visfatin activated G1-S phase cell cycle progression by upregulation of cyclin D1 and cdk2 expression. Visfatin also increased the expression of matrix metalloproteinases 2, matrix metalloproteinases 9, and vascular endothelial growth factor genes, suggesting that it may function in metastasis and angiogenesis of breast cancer. Taken together, these findings suggest that visfatin plays an important role in breast cancer progression.     

Effects of estradiol concentration on levels of nuclear estrogen receptors in MCF-7 breast tumor cells

We have measured the time-course of estrogen receptor levels in nuclei of the estrogen-responsive breast tumor cell line MCF-7 during 90-120 min exposure of the cells to estradiol at physiologic (10(-10)M), pharmacologic (10(-6)M), and an intermediate (10(-8)M) concentration. Cells were preincubated for one week in a serum-free defined medium resembling that of Barnes and Sato, and then incubated in estradiol-containing medium. Nuclei were isolated at various times during the incubation, and filled and unfilled nuclear estrogen receptor levels were assayed. Increasing the concentration of estradiol in the incubation medium from 10(-10)M to 10(-8)M yielded increasing levels of filled nuclear receptor at all times studied, while further increase of the estradiol concentration of 10(-6)M decreased filled receptor levels from 10(-8)M values. Unfilled receptor levels dropped rapidly to zero under 10(-6)M and 10(-8)M estradiol incubation, but remained unchanged under 10(-10)M estradiol incubation. Together these results suggest that high-concentration estradiol may lead to "down-regulation" of filled nuclear receptors, which may be a contributing factor in inhibition of tumor growth. On the other hand, the continued presence of unfilled receptors only under physiological concentrations of estradiol may suggest a role for these receptors in sustaining tumor growth.     

Effect of nomegestrol acetate on estrogen biosynthesis and transformation in MCF-7 and T47-D breast cancer cells

Although ovaries serve as the primary source of estrogen for pre-menopausal women, after menopause estrogen biosynthesis from circulating precursors occurs in peripheral tissues by the action of several enzymes, 17beta-hydroxysteroid dehydrogenase 1 (17beta-HSD1), aromatase and estrogen sulfatase. In the breast, both normal and tumoral tissues have been shown to be capable of synthesizing estrogens, and this local estrogen production can be implicated in the development of breast tumors. In these tissues, estradiol (E(2)) can be synthesized by three pathways: (1) estrone sulfatase transforms estrogen sulfates into bioactive estrogens, (2) 17beta-HSD1 converts estrone (E(1)) into E(2), (3) aromatase which converts androgens into estrogens is also present and contributes to the in situ synthesis of active estrogens but to a far lesser extent than estrone sulfatase. Quantitative assessment of E(2) formation in human breast tumors indicates that metabolism of estrone sulfate (E(1)S) via the sulfatase pathway produces 100-500 times more E(2) than androgen aromatization. Breast tissue also possesses the estrogen sulfotransferase involved in the conversion of estrogens into their sulfates that are biologically inactive. In the present review, we summarized the action of the 19-nor-progestin nomegestrol acetate (NOMAC) on the sulfatase, 17beta-HSD1 and sulfotransferase activities in the hormone-dependent MCF-7 and T47-D human breast cancer cell lines. Using physiological doses of substrates NOMAC blocks very significantly the conversion of E(1)S to E(2). It inhibits the transformation of E(1) to E(2). NOMAC has a stimulatory effect on sulfotransferase activity in both cell lines, with a strong stimulating effect at low doses but only a weak effect at high concentrations. The effects on the three enzymes are always stronger in the progesterone-receptor rich T47-D cell line as compared with the MCF-7 cell line. Besides, no effect is found for NOMAC on the transformation of androstenedione to E(1) in the aromatase-rich choriocarcinoma cell line JEG-3. In conclusion, the inhibitory effect provoked by NOMAC on the enzymes involved in the biosynthesis of E(2) (sulfatase and 17HSD pathways) in estrogen-dependent breast cancer, as well as the stimulatory effect on the formation of the inactive E(1)S, can open attractive perspectives for future clinical trials.     

Antiestrogen action of 2-hydroxyestrone on MCF-7 human breast cancer cells

The estrogen responsive human breast cancer MCF-7 cell culture was examined for its response to 2-hydroxyestrone a principal metabolite of estradiol. Addition of 2-hydroxyestrone to the cell cultures in concentration of 10(-9) - 10(-6) M had no effect on cell growth and proliferation because of rapid O-methylation of the catechol estrogen by catechol O-methyltransferase which is highly active in these cells. In the presence of quinalizarin, a potent catechol O-methyltransferase inhibitor which reduces the O-methylation of the steroid, 10(-7) M and 10(-8) M 2-hydroxyestrone markedly suppresses the growth and proliferation of the cells. The tumor cell growth-inhibitory action of the catechol estrogen was neutralized by the presence of 10(-9) M estradiol. The catechol estrogen inhibition of cell growth is not observed in the estrogen receptor-negative human breast cancer cell lines MDA-MB-231 and MDA-MB-330 providing evidence that the inhibition is specific and is estrogen receptor-mediated. In contrast, the 16 alpha-hydroxylated metabolites of estradiol, estriol and 16 alpha-hydroxyestrone, are effective stimulators of MCF-7 cell proliferation with the latter exhibiting potency in excess of that expected from its estrogen receptor affinity. The present results represent the first observation of a specific receptor-mediated antiestrogenic action of 2-hydroxyestrone and suggest that the physiological regulation of the agonist activity of the primary estrogen may involve in situ generation of catechol estrogen.     

Estrogen-stimulated glucuronidation of dihydrotestosterone in MCF-7 human breast cancer cells

The non-aromatizable androgen dihydrotestosterone (DHT) has been shown to exert a potent inhibitory effect on the proliferation of some human breast cancer cell lines. DHT, however, has little or no significant inhibition on MCF-7 cell proliferation in either the presence or absence of estradiol (E₂). Since the metabolism of DHT into non-active compounds may be responsible for the observed lack of androgenic effect in this cell line, we have investigated the metabolic fate of labeled DHT in MCF-7 cells. A time course incubation was performed with 1 nM [³H]DHT and analysis of the various metabolites formed revealed a time-dependent increase in glucuronidated steroids which was stimulated more than 4-fold by 0.1 nM E₂. The major glucuronidated steroid was androstane-3, 17β-diol in both control and E₂-stimulated cells, comprising 22 ± 1.2% and 30 ± 0.6% of the total radioactivity in the medium, respectively. Other steroid glucuronides observed included DHT, androstane-3β, 17β-diol, and androsterone, all of which were elevated in the E₂-treated cells relative to control values. The present data show that E₂ exerts a stimulatory effect on the glucuronidation of androgens and their metabolites in the estrogen-dependent breast cancer celll line MCF-7. Since glucuronidation is an effective means of cellular elimination of active steroids, such a pathway may be considered as a possible site of regulation of breast cancer cell growth by hormones.