Induction of epidermal growth factor-related polypeptides by 17 beta-estradiol in MCF-7 human breast cancer cells

MCF-7 human breast cancer cells release polypeptides related to insulin-like growth factor-I (IGF-I) and epidermal growth factor (EGF) into serum-free culture medium (CM). Treatment of MCF-7 cells with 17 beta-estradiol, which is required in vivo for MCF-7 tumor growth in the nude mouse and stimulates the MCF-7 growth rate in vitro, resulted in selectively enhanced growth factor activities in CM. Autostimulatory growth-promoting activity was elevated at least 2-fold, and EGF-like polypeptides were elevated 5-fold but IGF-I immunoreactivity was not elevated. Several species of estrogen-induced receptor-reactive EGF-like polypeptides, suggestive of high molecular weight transforming growth factor alpha, were detected after gel exclusion chromatography of CM extracted with 1 M acetic acid. A 30,000 mol wt peak of EGF receptor competing activity comigrated with a peak of autostimulatory and fibroblast-transforming activity. It is possible that estradiol stimulation of MCF-7 growth and/or tumor formation may depend on induction of EGF-related polypeptide growth factors. EGF-I- and EGF-related polypeptides may act together as autocrine or paracrine growth factors in breast cancer.     

Transcriptional activation of insulin-like growth factor-binding protein-4 by 17beta-estradiol in MCF-7 cells: role of estrogen receptor-Sp1 complexes.

Insulin-like growth factor-binding protein-4 (IGFBP-4) is expressed in MCF-7 human breast cancer cells, and treatment of these cells with 17beta-estradiol (E2) resulted in induction of IGFBP-4 gene expression (>3-fold) and protein secretion (>6-fold). To identify genomic sequences associated with E2 responsiveness, the 5'-promoter region (-1214 to +18) of the IGFBP-4 gene was cloned into a vector upstream from the firefly luciferase reporter gene, and E2 induced a 10-fold increase in luciferase activity in MCF-7 cells transiently transfected with this construct. Deletion analysis of this region of the IGFBP-4 gene promoter identified two GC-rich sequences at -559 to -553 and -72 to -64 that were important for E2-induced trans-activation. Gel mobility shift assays using 32P-labeled -569 to -540 and -83 to -54 oligonucleotides from the IGFBP-4 gene promoter showed that Sp1 protein bound these oligonucleotides to form a retarded band, and the intensity of the band was competitively decreased after coincubation with unlabeled IGFBP-4-derived and consensus Sp1 oligonucleotides. Mutation of the GC-rich sites within these sequences resulted in loss of the retarded band formation. Wild-type human estrogen receptor did not bind directly to the IGFBP-4 oligonucleotides; however, human estrogen receptor enhanced Sp1-DNA binding in a concentration-dependent manner. The results of this study demonstrate that at least two GC-rich sequences at -559 to -553 and -72 to -64 are required for induction of IGFBP-4 gene expression by E2 in MCF-7 cells.     

2,3,7,8-Tetrachlorodibenzo-p-dioxin causes an extensive alteration of 17 beta-estradiol metabolism in MCF-7 breast tumor cells.

MCF-7 breast tumor cells form multicellular foci in vitro when supplemented with 17 beta-estradiol (E2). In the presence of E2 and the aryl hydrocarbon-receptor agonist 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), MCF-7 cells grow to confluence but do not form foci. To investigate the role of E2 metabolism in this antiestrogenic effect of TCDD, analyses were performed by capillary GC/MS. The results revealed that pretreatment of MCF-7 cultures with TCDD (10 nM) rapidly depletes E2. In untreated cultures supplemented with 10 nM E2, the concentration of free E2 decreased to 4 nM in the first 12 hr, followed by a slower rate of decline. After 3 days most E2 in the medium was in conjugated form(s); 1.7 nM was present as free E2, and 2.9 nM was released by treatment with glucuronidase/sulfatase. In TCDD-treated cultures, E2 declined to 290 pM in 12 hr and after 2 days was not detected (less than 100 pM) either as free steroid or after treatment with glucuronidase/sulfatase. Intracellular E2 and estrone were likewise depleted by pretreatment with TCDD. Microsomes from TCDD-treated cells showed highly elevated aryl hydrocarbon-hydroxylase activity and catalyzed hydroxylations of E2 at C-2, C-4, C-15 alpha, and C-6 alpha with a combined rate of 0.85 nmol/min per nmol of cytochrome P-450 at saturating E2. These results suggest that depletion of E2 by enhanced metabolism accounts for the antiestrogenic activity of TCDD in MCF-7 cells.     

Synergistic up-regulation of prostaglandin E synthase expression in breast cancer cells by 17beta-estradiol and proinflammatory cytokines

Inflammatory mediators, such as cytokines and prostaglandins, play a fundamental role in estrogen-dependent breast cancer through their ability to up-regulate aromatase expression and subsequent local production of estrogens in the breast. To study the link between estrogens and inflammation further, we examined the regulation of prostaglandin E synthase (PTGES), a key enzyme in the production of prostaglandin E2. We found that 17beta-estradiol (E2) rapidly and robustly up-regulates PTGES mRNA and protein levels in estrogen receptor (ER)-positive breast cancer cells through ER recruitment to an essential estrogen response element located in the 5' flanking region of the PTGES gene. PTGES is also up-regulated by the proinflammatory cytokines TNFalpha or IL-1beta. Surprisingly, the combination of E2 and cytokines leads to a synergistic up-regulation of PTGES in an ER and nuclear factor-kappaB (NFkappaB)-dependent manner. This is in contrast to the mutual transrepression between ER and NFkappaB that has been well characterized in other cell types. Furthermore, we found enhanced recruitment of ERalpha as well as the NFkappaB family member, p65, to the PTGES estrogen response element by the combination of E2 and TNFalpha compared with either E2 or TNFalpha alone. The synergistic up-regulation of PTGES may result in enhanced prostaglandin E2 production, which in turn may further enhance aromatase expression and production of local estrogens. Our findings suggest that a finely tuned positive feedback mechanism between estrogens and inflammatory factors may exist in the breast and contribute to hormone-dependent breast cancer growth and progression.     

Mechanism of transcriptional regulation of LRP16 gene expression by 17-beta estradiol in MCF-7 human breast cancer cells

LRP16 gene expression is induced by 17-betaestradiol (E2) via estrogen receptor alpha (ERalpha) in MCF-7 human breast cancer cells. A previous study also demonstrated that ectopic expression of LRP16 gene promoted MCF-7 cell proliferation. To explore the mechanism of hormone-induced LRP16 gene expression, the LRP16 gene promoter region (-2600 to -24 bp upstream of the LRP16 gene translation starting site) was analyzed in the present study by using different 5'-truncated constructs, and a luciferase reporter. The 5'-flanking sequence of -676 to -24 bp (pGL3-S5) was found to be E2-responsive. After exchange of the fragment from -213 to -24 bp with the TK gene proximal promoter region in pGL3-S5, E2 still induced reporter gene activity in MCF-7 and HeLa cells. Sequence analysis showed that the pGL3-S6 (-676 to -214) sequence contains two motifs that may contribute to E2-induced transactivation; namely, an estrogen-responsive element (ERE) half-site/Sp1 at -246 to -227 bp and an E-box site at -225 to -219 bp. Further deletion and mutation analysis of these two motifs indicated that both the 1/2 ERE and Sp1 binding sites were required for E2 action, while E-box deletion did not affect the luciferase activity in MCF-7 and HeLa cells. The results of gel mobility shift and chromatin immunoprecipitation assays confirmed that both ERalphaand Sp1 were required for hormone-induced transactivation, which involved both ERalphaand Sp1 directly binding to DNA. Taken together, these findings suggest that ERalphaand Sp1 play a role in activation of the human LRP16 gene promoter.     

A multidrug resistance transporter from human MCF-7 breast cancer cells

MCF-7/AdrVp is a multidrug-resistant human breast cancer subline that displays an ATP-dependent reduction in the intracellular accumulation of anthracycline anticancer drugs in the absence of overexpression of known multidrug resistance transporters such as P glycoprotein or the multidrug resistance protein. RNA fingerprinting led to the identification of a 2.4-kb mRNA that is overexpressed in MCF-7/AdrVp cells relative to parental MCF-7 cells. The mRNA encodes a 663-aa member of the ATP-binding cassette superfamily of transporters that we term breast cancer resistance protein (BCRP). Enforced expression of the full-length BCRP cDNA in MCF-7 breast cancer cells confers resistance to mitoxantrone, doxorubicin, and daunorubicin, reduces daunorubicin accumulation and retention, and causes an ATP-dependent enhancement of the efflux of rhodamine 123 in the cloned transfected cells. BCRP is a xenobiotic transporter that appears to play a major role in the multidrug resistance phenotype of MCF-7/AdrVp human breast cancer cells.     

Modulation of intracellular calcium and calmodulin by melatonin in MCF-7 human breast cancer cells

The pineal hormone, melatonin, has been shown to inhibit the proliferation of the estrogen receptor alpha (ERalpha)-positive macrophage chemotactic factor (MCF)-7 human breast cancer cells. Previous studies from other systems indicate that melatonin modulates the calcium (Ca2+)/calmodulin (CaM) signaling pathway either by changing intracellular calcium concentration ([Ca2+]i) via activation of its G-protein coupled membrane receptors, or through a direct interaction with CaM. In this study, although melatonin alone had no effect on basal [Ca2+]i in MCF-7 cells, it significantly enhanced the elevation of [Ca2+]i induction by extracellular adenosine triphosphate (ATP), which increases [Ca2+]i via the G protein-coupled P2y-purinoceptor and the phospholipase C (PLC) pathway. Pretreatment of MCF-7 cells with 10(-7) M melatonin increased the 10(-5) M ATP-induced [Ca2+]i peak change from 79.4 +/- 11.6 nM to 146.2 +/- 22.3 nM. Furthermore, without changing total cellular CaM levels, melatonin markedly increased the amount of membrane-bound CaM to 237 and 162% of control levels after I and 6 hr of treatment, respectively. Cytosolic CaM levels were also elevated to 172% of control after 6 hr of melatonin treatment. Correlative growth studies demonstrated that ATP (10(-5) M) can stimulate MCF-7 cell growth, that melatonin can suppress MCF-7 cell proliferation, but that pretreatment of MCF-7 cells with melatonin followed by ATP(10(-5) M), like 10(-4) M ATP can further suppress MCF-7 cell proliferation; this indicates that melatonin's potentiation of ATP induced [Ca2+]i may be above the threshold for cell growth. Given the important role of [Ca2+]i and CaM in tumor cell homeostasis and proliferation and melatonin's modulation of [Ca2+]i, melatonin's effects on the Ca2+/CaM signaling pathway may play an important role in mediating the growth-inhibitory effect of melatonin on MCF-7 human breast cancer cells.     

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.     

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.     

The hydrolysis of estrone sulfate and dehydroepiandrosterone sulfate by MCF-7 human breast cancer cells

Reports of estrone (E1) and dehydroepiandrosterone (DHEA) sulfatase (sulfohydrolase) activities within many human breast cancers have prompted us to undertake the identification and partial characterization of these enzyme activities within MCF-7 human breast cancer cells. Enzyme assays were performed within subcellular preparations and intact cultures by quantifying the total nonpolar 3H-labeled metabolites formed from [3H]E1 sulfate (E1S) and [3H]DHEA sulfate (DHEAS). The results have shown that the hydrolysis of each steroid sulfate is mediated by different particulate enzymes, which demonstrate optimal activity between pH 6.0-7.0. The analysis of enzyme kinetic data showed the Km values of E1S and DHEAS for their enzymes to be approximately 6.3 and 3.6 microM/L, respectively. Neither enzyme was subject to product inhibition. Androsterone sulfate and pregnenolone sulfate produced significant inhibition of E1, but not DHEA, sulfatase activity. E1S inhibited DHEA sulfatase competitively, with an approximate Ki of 11 microM, whereas DHEAS inhibited E2 sulfatase in a noncompetitive fashion, demonstrating an approximate Ki of 0.6 microM. Studies carried out with intact MCF-7 cultures using physiological concentrations of 3H-labeled E1S (2 nM) or DHEAS (1 microM) showed the accumulation of nonpolar metabolites during a 20-h incubation period. When cultures were incubated with similar concentrations of both steroid sulfates the apparent intracellular activity of E1 sulfatase was reduced by approximately 70%, whereas DHEA sulfatase activity remained unchanged. The results of these studies confirm the ability of MCF-7 cells to hydrolyze extracellular E1S and DHEAS, indicate that these reactions are mediated by different enzymes, and demonstrate that DHEAS is a potent inhibitor of MCF-7 E1 sulfatase. Circulating DHEAS, therefore, may substantially limit the ability of most postmenopausal breast cancers to use E1S as a substrate for intracellular estrogen biosynthesis.     

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).     

Effects of antioestrogens on the proliferation of MCF-7 human breast cancer cells

Non-steroidal antioestrogens, such as tamoxifen, inhibit the growth of human breast cancer cells. The experiments described here compare and contrast the efficacy of tamoxifen and the 'pure' antioestrogen, ICI 164384, on the inhibition of proliferation of MCF-7 cells. Previous studies have shown that ICI 164384 has a greater maximal inhibitory effect than conventional antioestrogens on the growth of MCF-7 cells. Both types of compound block progression of cells through the cell cycle in the early G1 phase. These studies have been extended to measure the population distribution of antioestrogen-treated cells by the use of two-parameter flow cytometry. ICI 164384 proved to be more effective than tamoxifen in decreasing the proportion of actively growing cells in an asynchronous population. In cells grown in the complete absence of exogenous oestrogens, growth was stimulated by oestradiol, insulin, insulin-like growth factor-I (IGF-I) or transforming growth factor-alpha (TGF-alpha). The potent metabolite of tamoxifen, trans 4'-hydroxytamoxifen (4'-OHT), alone also stimulated growth, whereas ICI 164384 did not. Oestradiol and insulin added together demonstrated a clear synergistic enhancement of cell growth. Correspondingly, the stimulatory effect of 4'-OHT on growth was magnified in the presence of insulin, and a combination of ICI 164384 with insulin revealed a much weaker stimulatory action of the 'pure' antagonist. For both compounds the interaction with insulin was complex and characterized by a bell-shaped dose-response curve. However, for 4'-OHT at all concentrations in the range 1 pM-1 microM in the presence of insulin, cell numbers were greater than in cultures exposed to insulin alone. This was not the case for ICI 164384 which suggested that differences in efficacy may be due to interactions between oestrogen and growth factor-mediated mechanisms. Furthermore, ICI 164384 was more effective in inhibiting the action of IGF-I and TGF-alpha alone or in combination, although both antioestrogens produced a partial blockade of growth factor responses in the complete absence of oestradiol. It is concluded that the difference in efficacy between partial agonist and 'pure' antagonist antioestrogens to inhibit growth in vitro is consistent with the difference in the pharmacological profile of these compounds. The absence of stimulatory activity of ICI 164384 is of particular significance in reducing to a minimum the synergistic interaction between oestrogens and insulin.(ABSTRACT TRUNCATED AT 400 WORDS)     

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。     

Estrogen-like activity of metals in MCF-7 breast cancer cells

The ability of metals to activate estrogen receptor-alpha (ERalpha) was measured in the human breast cancer cell line, MCF-7. Similar to estradiol, treatment of cells with the divalent metals copper, cobalt, nickel, lead, mercury, tin, and chromium or with the metal anion vanadate stimulated cell proliferation; by d 6, there was a 2- to 5-fold increase in cell number. The metals also decreased the concentration of ERalpha protein and mRNA by 40-60% and induced expression of the estrogen-regulated genes progesterone receptor and pS2 by1.6- to 4-fold. Furthermore, there was a 2- to 4-fold increase in chloramphenicol acetyltransferase activity after treatment with the metals in COS-1 cells transiently cotransfected with the wild-type receptor and an estrogen-responsive chloramphenicol acetyltransferase reporter gene. The ability of the metals to alter gene expression was blocked by an antiestrogen, suggesting that the activity of these compounds is mediated by ERalpha. In binding assays the metals blocked the binding of estradiol to the receptor without altering the apparent binding affinity of the hormone (K(d) = 10(-10) M). Scatchard analysis employing either recombinant ERalpha or extracts from MCF-7 cells demonstrated that (57)Co and (63)Ni bind to ERalpha with equilibrium dissociation constants of 3 and 9.5 x 10(-9) and 2 and 7 x 10(-9) M, respectively. The ability of the metals to activate a chimeric receptor containing the hormone-binding domain of ERalpha suggests that their effects are mediated through the hormone-binding domain. Mutational analysis identified amino acids C381, C447, E523, H524, N532, and D538 as potential interaction sites, suggesting that divalent metals and metal anions activate ERalpha through the formation of a complex within the hormone-binding domain of the receptor.