Control of sulfatase and sulfotransferase activities by medrogestone in the hormone-dependent MCF-7 and T-47D human breast cancer cell lines.

In the present study, we explored the effect of the progestin medrogestone on the sulfatase and sulfotransferase activities in the hormone-dependent MCF-7 and T-47D human breast cancer cell lines. After 24 h incubation at 37 degrees C of physiological concentrations of estrone sulfate ([3H]-E1S: 5x10(-9) mol/l), it was observed that this estrogen was converted in a great proportion to E2 in both cell lines. Medrogestone significantly inhibits this transformation, at all the concentrations tested (5x10(-8) to 5x10(-5) mol/l), in both cell lines. The IC50 values were 1.93 micromol/l and 0.21 micromol/l in MCF-7 and T-47D cells, respectively. In another series of studies, after 24 h incubation at 37 degrees C of physiological concentrations of estrone ([3H]-E1: 5x10(-9) mol/l), the sulfotransferase activity was detectable in both cell lines. Estrogen sulfates (ES) are found exclusively in the culture medium, which suggests that as soon as they are formed they are excreted into the medium. Medrogestone has a biphasic effect on sulfotransferase activity in both cell lines. At low doses: 5x10(-8) and 5x10(-7) mol/l, this compound stimulates the enzyme by +73.5 and 52.7%, respectively, in MCF-7, and by 84.5 and 62.6% in T-47D cells. At high concentrations: 5x10(-6) and 5x10(-5) mol/l, medrogestone has no effect on MCF-7 cells, but inhibits the sulfotransferase activity in T-47D cells by -31.4% at 5x10(-5) mol/l. In conclusion, the inhibitory effect provoked by medrogestone on the enzyme involved in the biosynthesis of E2 (sulfatase pathway) in estrogen-dependent breast cancer, as well as the stimulatory effect on the formation of the inactive ES, support a probable anti-proliferative effect of this progestin in breast tissue. Clinical applications of these findings can open new therapeutic possibilities for this disease.     

Norelgestromin as selective estrogen enzyme modulator in human breast cancer cell lines. Effect on sulfatase activity in comparison to medroxyprogesterone acetate

Human breast cancer tissue contains enzymes (estrone sulfatase, 17beta-hydroxysteroid dehydrogenase, aromatase) involved in the last steps of estradiol (E(2)) formation. In this tissue, E(2) can be synthesized by two main pathways: (1) sulfatase-transforms estrogen sulfates into bioactive E(2), and the (2) aromatase-converts androgens into estrogens. 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.In the present study, we demonstrated in T-47D and MCF-7 human breast cancer cells that norelgestromin (NGMN) (a metabolite of norgestimate) is a potent inhibitory agent of the estrone sulfatase activity. After 24h incubation of physiological concentrations of E(1)S (5 x 10(-9)mol/l) the inhibitory effect of NGMN at concentrations of 5 x 10(-9), 5 x 10(-7) and 5 x 10(-5)mol/l was 43+/-7, 74+/-4 and 97+/-2%, respectively, in T-47D cells; 25+/-4, 57+/-5 and 96+/-2% respectively, in MCF-7 cells. Comparative studies using medroxyprogesterone acetate (MPA) showed that this progestin also has an inhibitory effect on sulfatase activity, but significantly less intense than that of NGMN. The inhibition for MPA at concentrations of 5 x 10(-9), 5 x 10(-7) and 5 x 10(-5)mol/l was 31+/-5, 47+/-3 and 61+/-3%, respectively, for T-47D cells; 6+/-3, 20+/-3 and 63+/-4%, respectively, for MCF-7 cells.In conclusion, the present data show that NGMN is a very potent inhibitory agent for sulfatase activity in the hormone-dependent breast cancer cells, resulting in decreased tissue concentration of E(2). The clinical significance of this finding remains to be elucidated.     

Estradiol as an anti-aromatase agent in human breast cancer cells

Estradiol (E(2)) is an important risk factor in the development and progression of breast cancer. However, a "direct effect" of E(2) in breast cancerization has not yet been demonstrated. The estrogen receptor complex can mediate the activation of oncogens, proto-oncogens, nuclear proteins and other target genes that can be involved in the transformation of normal to cancerous cells. Breast cancer cells possess all the enzymes (sulfatase, aromatase, 17beta-hydroxysteroid dehydrogenase (17beta-HSD)) necessary for the local bioformation of E(2). In the last years, many studies have shown that treatment of breast cancer patients using anti-aromatase agents has beneficial therapeutic effects. The aromatase activity is very low in most breast cancer cells but was significantly increased in a hormone-dependent breast cancer cell line: the MCF-7aro, using the aromatase cDNA transfection and G-418 (neomycin) selection. In the present study, we explore the effect of E(2) on the aromatase activity of this cell line. The MCF-7aro cell line was a gift from Dr. S. Chen (Beckman Research Institute, Duarte, U.S.A.). For experiments the cells were stripped of endogenous steroids and incubated with physiological concentrations of [(3)H]-testosterone (5 x 10(-9)mol/l) alone or in the presence of E(2) (5 x 10(-5), 5 x 10(-7) and 5 x 10(-9)mol/l) for 24h at 37 degrees C. The cellular radioactivity uptake was determined in the ethanolic supernatant and the DNA content in the remaining pellet. [(3)H]-E(2), [(3)H]-estrone ([(3)H]-E(1)) and [(3)H]-testosterone were characterized by thin layer chromatography and quantified using the corresponding standard. It was observed that [(3)H]-testosterone is converted mainly into [(3)H]-E(2) and not to E(1), which suggests very low or absence of oxidative 17beta-HSD (type 2) activity in these experimental conditions. The aromatase activity, corresponding to the conversion of [(3)H]-testosterone to [(3)H]-E(2) after 24h, is relatively high, since the concentration of E(2) was 2.74+/-0.11pmol/mg DNA in the non-treated cells. E(2) inhibits this conversion by 77, 57 and 21%, respectively, at the concentrations of 5 x 10(-5), 5 x 10(-7) and 5 x 10(-9)mol. In previous studies, it was demonstrated that E(2) exerts a potent anti-sulfatase activity in the MCF-7 and T-47D breast cancer cells. The present data show that E(2) can also block the aromatase activity. The dual inhibition of the aromatase and sulfatase activities, two crucial enzymes for the biosynthesis of E(2) by E(2) itself in breast cancer add interesting and attractive information for the use of estrogen therapeutic treatments.     

Effect of nomegestrol acetate on estrone-sulfatase and 17β-hydroxysteroid dehydrogenase activities in human breast cancer cells

It is well recognized that estradiol (E₂) is one of the most important hormones supporting the growth and evolution of breast cancer. Consequently, to block this hormone before it enters the cancer cell or in the cell itself, has been one of the main targets in recent years. In the present study we explored the effect of the progestin, nomegestrol acetate, on the estrone sulfatase and 17β-hydroxy-steroid dehydrogenase (17β-HSD) activities of MCF-7 and T-47D human breast cancer cells. Using physiological doses of estrone sulfate (E₁S: 5 × 10⁻⁹ M), nomegestrol acetate blocked very significantly the conversion of E₁S to E₂. In the MCF-7 cells, using concentrations of 5 × 10⁻⁶ M and 5 × 10⁻⁵ M of nomegestrol acetate, the decrease of E₁S to E₂ was, respectively, −43% and −77%. The values were, respectively, −60% and −71% for the T-47D cells. Using E₁S at 2 × 10⁻⁶ M and nomegestrol acetate at 10⁻⁵ M, a direct inhibitory effect on the enzyme of −36% and −18% was obtained with the cell homogenate of the MCF-7 and T-47D cells, respectively. In another series of studies, it was observed that after 24 h incubation of a physiological concentration of estrone (E₁: 5 × 10⁻⁹ M) this estrogen is converted in a great proportion to E₂. Nomegestrol acetate inhibits this transformation by −35% and −85% at 5 × 10⁻⁷ M and 5 × 10⁻⁵ M, respectively in T-47D cells; whereas in the MCF-7 cells the inhibitory effect is only significant, −48%, at 5 × 10⁻⁵ M concentration of nomegestrol acetate. It is concluded that nomegestrol acetate in the hormone-dependent MCF-7 and T-47D breast cancer cells significantly inhibits the estrone sulfatase and 17β-HSD activities which converts E₁S to the biologically active estrogen estradiol. This inhibition provoked by this progestin on the enzymes involved in the biosynthesis of E₂ can open new clinical possibilities in breast cancer therapy.     

Paradoxical effect of estradiol: it can block its own bioformation in human breast cancer cells

The great majority of breast cancers are in their early stage hormone-dependent and it is well accepted that estradiol (E(2)) plays an important role in the genesis and evolution of this tumor. Human breast cancer tissues contain all the enzymes: estrone sulfatase, 17 beta-hydroxysteroid dehydrogenase (17 beta-HSD), aromatase, involved in the last steps of E(2) bioformation in this tissue. Quantitative data show that the 'sulfatase pathway', which transforms estrogen sulfates into the bioactive unconjugated E(2), is 100-500 times higher than the 'aromatase pathway' which converts androgens into estrogens. In this paper we explore the effect of E(2) on the sulfatase activity using two hormone-dependent human breast cancer cells: MCF-7 and T-47D. The action of E(2) on the sulfatase activity was evaluated by the conversion of estrone sulfate (E(1)S) into E(2). The cells were incubated in Minimal Essential Medium (MEM) containing 5% steroid-depleted fetal calf serum and incubated with physiological concentrations of [(3)H]E(1)S (5 x 10(-9) M) alone (control) or in the presence of E(2) (5 x 10(-10) to 5 x 10(-5) M) for 24 h at 37 degrees C. It was found that E(2) is a potent inhibitory agent of the estrone sulfatase activity in both cell lines. A low concentration of E(2): 5 x 10(-9) M decreases the sulfatase activity by 67% in MCF-7 cells and 57% in T-47D cells. More than 80% of the decrease in the formation of E(2) was obtained with the dose of 5 x 10(-7) M in both cell lines. It is concluded that this paradoxical effect of E(2) adds a new biological response of this hormone and could be related to estrogen replacement therapy in which it was observed to have either no effect or to decrease breast cancer mortality in postmenopausal women. Preliminary results are indicated in the Proceedings of the 14th International Symposium of the Journal of Steroid Biochemistry & Molecular Biology (Quebec, Canada, 24-27 June 2000) [J. Steroid Biochem. Molec. Biol. 76 (2001) 95-104](1) and presented at the 83rd Annual Meeting of the Endocrine Society (Denver, USA, 20-23 June 2001 (abstract no. P2-615).     

Estradiol and estrone C-16 derivatives as inhibitors of type 1 17beta-hydroxysteroid dehydrogenase: blocking of ER+ breast cancer cell proliferation induced by estrone

Estrogens play an important role in the development of breast cancer. Inhibiting 17beta-hydroxysteroid dehydrogenase type 1 (17beta-HSD1)--the enzyme responsible for the last step in the biosynthesis of the most potent estrogen, estradiol (E2)--would thus allow hindering the growth of estrogen-sensitive tumors. Based on a previous study identifying 16beta-benzyl-E2 (1) as a lead compound for developing inhibitors of the transformation of estrone (E1) into E2, we modified the benzyl group of 1 to improve its inhibitory activity. Three strategies were also devised to produce compounds with less residual estrogenic activity: (1) replacing the hydroxy group by a hydrogen at position 3 (C3); (2) adding a methoxy at C2; and (3) adding an alkylamide chain known to be antiestrogenic at C7. In order to test the inhibitory potency of the new compounds, we used the human breast cancer cell line T-47D, which exerts a strong endogenous 17beta-HSD1 activity. In this intact cell model, 16beta-m-carbamoylbenzyl-E2 (4m) emerged as a potent inhibitor of 17beta-HSD1 with an IC50 value of 44 nM for the transformation of [14C]-E1 (60 nM) into [14C]-E2 (24-h incubation). In another assay aimed at assessing the unwanted estrogenic activity, a 10-day treatment with 4m at a concentration of 0.5 microM induced some proliferation (38%) of T-47D estrogen-sensitive (ER+) breast cancer cells. Interestingly, when 4m (0.5 microM) was given with E1 (0.1 nM) in a 10-day treatment, it blocked 62% of the T-47D cell proliferation induced by E1 after its reduction to E2 by 17beta-HSD1. Thus, in addition to generating useful structure-activity relationships for the development of 17beta-HSD1 inhibitors, our study demonstrates that using such inhibitors is a valuable strategy for reducing the level of E2 and consequently its proliferative effect in T-47D ER+ breast cancer cells.     

Prolactin-dependent up-regulation of BRCA1 expression in human breast cancer cell lines.

We report experimental evidence that BRCA1, a breast and ovarian cancer susceptibility gene, is up-regulated in response to prolactin (PRL) stimulation. Expression of the BRCA1 gene was monitored in 2 human breast cancer cell lines (MCF-7 and T-47D) and in the normal mammary epithelial cell line MCF10a. Using competitive RT-PCR, we have shown that PRL induced an increase in BRCA1 mRNA level in MCF-7 and T-47D cell lines at a dose resulting in the maximal enhancement of cell proliferation. The up-regulation was 12-fold in MCF-7 cells and 2-fold in T-47D cells. No increase in BRCA1 mRNA level was observed in the MCF10a cell line. The level of BRCA1 protein was quantified using an affinity chromatography strategy. At the protein level, PRL treatment induced a 4-fold increase of BRCA1 protein expression in MCF-7 and a 6-fold increase in T-47D cells, whereas BRCA1 protein expression was not affected by PRL in MCF10a.     

CCAAT/Enhancer binding protein delta (c/EBPdelta) regulation and expression in human mammary epithelial cells: I. "Loss of function" alterations in the c/EBPdelta growth inhibitory pathway in breast cancer cell lines

"Loss of function" alterations in growth inhibitory signal transduction pathways are common in cancer cells. In this study, we show that growth arrest (GA) treatments--serum and growth factor withdrawal and growth inhibitory IL-6 family cytokines (Interleukin-6 and Oncostatin M (OSM))--increase STAT3 phosphorylation (pSTAT3), increase CCAAT enhancer binding protein delta (C/EBPdelta) gene expression and induce GA of primary, finite-lifespan human mammary epithelial cells (HMECs), and immortalized breast cell lines (MCF-10A and MCF-12A). In contrast, serum and growth factor withdrawal from human breast cancer cell lines (MCF-7, SK-BR-3, T-47D, and MDA-MB-231) for up to 48 h induced a relatively modest increase in pSTAT3 levels and C/EBPdelta gene expression and resulted in varying levels of GA. In most breast cancer cell lines, IL-6 family cytokine treatment increased pSTAT3 levels and C/EBPdelta gene expression, however, growth inhibition was cell line dependent. In addition to "loss of function" alterations in growth inhibitory pathways, breast cancer cell lines also exhibit "gain of function" alterations in growth signaling pathways. The Akt growth/ survival pathway is constitutively activated in T-47D and MCF-7 breast cancer cells. The Akt inhibitor LY 294,002 significantly enhanced T-47D growth inhibition by serum and growth factor withdrawal or IL-6 family cytokine treatment. Finally, we show that activation of the pSTAT3/C/EBPdelta growth control pathway is independent of estrogen receptor status. These results demonstrate that "loss of function" alterations in the pSTAT3/C/EBPdelta growth inhibitory signal transduction pathway are relatively common in human breast cancer cell lines. Defective activation of the pSTAT3/ C/EBPdelta growth inhibitory signal transduction pathway, in conjunction with constitutive activation of the Akt growth stimulatory pathway, may play a synergistic role in the etiology or progression of breast cancer.     

Epigenetic regulation of bone morphogenetic protein-6 gene expression in breast cancer cells

Bone morphogenetic protein-6 (BMP-6) is closely correlated with tumor differentiation and skeletal metastasis. Our previous research found that BMP-6 gene expression can be activated dose-dependently by estrogen in estrogen receptor positive (ER⁺) breast cancer cell line MCF-7, but not in ER negative (ER⁻) cell line MDA-MB-231. This experiment is designed to investigate the epigenetic regulatory mechanism of the BMP-6 gene expression in breast cancer cell lines MDA-MB-231, MCF-7 and T47D with regard to the methylation status in the 5′ flanking region of the human BMP-6 gene. The endogenous level of BMP-6 mRNA in ER⁻ cell line MDA-MB-231 was relatively lower than that in ER⁺ MCF-7 and T47D cell lines. After the treatment with 5-aza-2′-deoxycytidine (5-aza-dC, especially in the concentration of 10 μM), the BMP-6 mRNA expression in MDA-MB-231 was obviously up-regulated. However, 5-aza-dC treatment failed to regulate the expression of BMP-6 in MCF-7 and T47D cells. Using enzyme restriction PCR (MSRE-PCR), as well as bisulfite sequencing (BSG), methylation of human BMP-6 gene promoter was detected in MDA-MB-231; while in MCF-7 and T47D, BMP-6 gene promoter remained demethylated status. In 33 breast tumor specimens, promoter methylation of BMP-6 was detected by methylation-specific PCR, hypermethylation of BMP-6 was observed in ER negative cases (16 of 16 cases (100%)), while obviously lower methylation frequency were observed in ER positive cases (3 of 17 cases (18%)), indicating that BMP-6 promoter methylation status is correlated with ER status in breast cancer.     

C6-(N,N-butyl-methyl-heptanamide) derivatives of estrone and estradiol as inhibitors of type 1 17beta-hydroxysteroid dehydrogenase: Chemical synthesis and biological evaluation

A series of estrone and estradiol derivatives having an N-butyl,methyl heptanamide side chain at C6-position were synthesized, tested as inhibitors of type 1 17beta-HSD and assessed for their possible estrogenic activity. A better type 1 17beta-HSD inhibition was obtained for the 6beta-side chain orientation over 6alpha; the C17-alcohols are more potent inhibitors than the corresponding ketones; introducing a 2-methoxy group decreased the inhibitory potency; and the replacement of a C-S bond by a C-C bond in the C6beta-side chain is not detrimental to inhibition. Interestingly, the new inhibitors were also found less estrogenic than the lead compound in two breast cancer cell lines, T-47D and MCF-7.     

A progestin effect on lactate dehydrogenase in the human breast cancer cell line T-47D

The human breast cancer cell line T-47D has high levels of progesterone receptor even in the absence of exogenously added estrogen. Because of this it is a good line in which to study aspects of progestin action. It has been shown by others that lactate dehydrogenase in MCF-7 cells is responsive to estrogen but not to progesterone. Other proteins in other systems have been found to be responsive to both estrogen and progesterone, often requiring priming by estrogen, presumably to produce sufficiently high quantities of progesterone receptor. Reasoning that lactate dehydrogenase in T-47D cells might be stimulated by progestins alone at physiological levels since these cells already have high levels of progesterone receptor, we now report that this is indeed the case.     

Estrogenic constituents of the heartwood of Dalbergia parviflora

From the heartwood of Dalbergia parviflora, five compounds, dalparvin A (1), B (2), C (3), dalparvinol C (4), and neokhriol A (5), along with 11 known compounds, kenusanone G (6), cajanin (7), sophorol (8), alpinetin (9), hesperetin (10), 3'-O-methylorobol, odoratin, (2R)(3R)-2,3-trans 7-hydroxy-5-methoxydihydroflavonol, (6aR, 11aR)-3,8-dihydroxy-9-methoxypterocarpan, (6aR, 11aR)- vesticarpan, and methyl-3,4-dihydroxy-2-methoxybenzoate were isolated and characterized. Isolates were evaluated for their cell proliferation stimulatory activity against MCF-7, T-47D, and BT20 human breast cancer cell lines. Along with 7-10, two compounds 2 and 3 stimulated not only MCF-7, but also T-47D human breast cancer cell proliferation. Compound 6 had activity only against MCF-7 cells, and the activity of 7 was more than equivalent to that of daidzein. On the other hand, none of the isolates had any significant effects on BT20 cell proliferation, and these results indicated that the stimulative activity of these compounds was not general to any cell proliferations. Furthermore, these compounds were tested in the estrogen-responsive transient luciferase reporter assay.     

Expression of aromatase and 17beta-hydroxysteroid dehydrogenase types 1, 7 and 12 in breast cancer. An immunocytochemical study

It is known that there is a local biosynthesis of estradiol (E2) in breast carcinoma. The steroidogenic enzymes involved in E2 formation are aromatase which transforms testosterone into E2 and androstenedione into estrone (E1) and reductive 17beta-hydroxysteroid dehydrogenases (17beta-HSDs) which convert E1 into E2. Using immunocytochemistry, we have studied the expression of aromatase and the three reductive 17beta-HSDs 17beta-HSD types 1, 7 and 12 in 41 specimens of female human breast carcinoma and adjacent non-malignant tissues. These results were correlated with the estrogen receptor alpha (ERalpha) and beta (ERbeta), progesterone receptor, androgen receptor, CDC47 and c-erb B-2 expressions and with the tumor stages. Aromatase was found in 58%, 17beta-HSD type 7 in 47% and 17beta-HSD type 12 in 83% of the breast cancer specimens. The 17beta-HSD type 1 could be detected in only one tumor. A significant correlation was observed between the aromatase, 17beta-HSD type 7 and 17beta-HSD type 12 expression, as well as between each of the two enzymes 17beta-types 7 and 12 and the ERbeta expression. The expression of 17beta-HSD type 12 was significantly higher in breast carcinoma specimens than in normal tissue. There was also a significant association of CDC 47 expression with ERbeta, AR and 17beta-HSD type 12. The results indicate that aromatase, 17beta-HSD type 7 and 17beta-HSD type 12, but not 17beta-HSD type 1, are commonly expressed in human breast cancer. Moreover, the high expression of both 17beta-HSD type 12 and ERbeta in breast carcinoma cells may play a role in the development and/or progression of breast cancer.     

Inhibition of mitogen-activated protein kinase kinase selectively inhibits cell proliferation in human breast cancer cells displaying enhanced insulin-like growth factor I-mediated mitogen-activated protein kinase activation.

Mitogen-activated protein (MAP) kinase mediates cell proliferation, cell differentiation, and cell survival by regulating signaling pathways activated by receptor protein tyrosine kinases (RPTKs), including the insulin-like growth factor 1 receptor (IGF-IR). We analyzed the upstream signaling components of the MAP kinase pathway, including RPTKs, in human breast cancer cell lines and found that some of those components were overexpressed. Importantly, signaling molecules such as IGF-IR, insulin receptor, and insulin receptor substrate 1, leading to the MAP kinase pathway, were found to be concomitantly overexpressed within certain tumor lines, i.e., MCF-7 and T-47D. When compared with the nonmalignant and other breast tumor lines examined, MCF-7 and T-47D cells displayed a more rapid, robust, and sustained MAP kinase activation in response to insulin-like growth factor I (IGF-I) stimulation. By contrast, IGF-I treatment led to a sustained down-regulation of MAP kinase in those lines overexpressing ErbB2-related RPTKs. Interestingly, blocking the MAP kinase pathway with PD098059 had the greatest antiproliferative effect on MCF-7 and T-47D among the normal and tumor lines tested. Furthermore, addition of an IGF-IR blocking antibody to growth medium attenuated the ability of PD098059 to suppress the growth of MCF-7 and T-47D cells. Thus, our study suggests that concomitant overexpression of multiple signaling components of the IGF-IR pathway leads to the amplification of IGF-I-mediated MAP kinase signaling and resultant sensitization to PD098059. The enhanced sensitivity to PD098059 implies an increased requirement for the MAP kinase pathway in those breast cancer cells, making this pathway a potential target in the treatment of selected breast malignancies.     

Expression of transforming growth factor alpha and its messenger ribonucleic acid in human breast cancer: its regulation by estrogen and its possible functional significance

We have studied the estrogenic regulation and the potential autocrine role of transforming growth factor alpha (TGF alpha) in the human breast cancer cell line MCF-7. A biologically active apparent mol wt 30 k TGF alpha was identified by gel filtration chromatography in medium conditioned by MCF-7 breast cancer cells. We previously reported induction of TGF alpha levels in medium by 17 beta-estradiol. We now report correlated increases in TGF alpha mRNA, by Northern and slot blot analysis, after estrogen treatment of MCF-7 cells in vitro. In vivo experiments confirmed these data: estrogen withdrawal from MCF-7 tumor-bearing nude mice resulted in a decline in tumor size and TGF alpha mRNA levels. To explore the functional significance of TGF alpha in MCF-7 cells, anti-TGF alpha antibody was added to MCF-7 soft agar cloning assays. Inhibition of MCF-7 growth resulted, supporting an autocrine role for TGF alpha. Further experiments using an anti-EGF receptor antibody expanded this data, demonstrating inhibition of estrogen-stimulated monolayer MCF-7 cell growth. Examining the generality of TGF alpha expression, 4.8 kilobase TGF alpha mRNAs were seen in three other human breast cancer cell lines, MDA-MB-231, ZR 75B, and T47D. Expression of TGF alpha mRNA was detected in 70% of estrogen receptor positive and negative primary human breast tumors from 40 patients when examined by slot blot and Northern analysis. Thus, we have demonstrated broad expression of TGF alpha in human breast cancer, its hormonal regulation in an estrogen-responsive cell line, and its possible functional significance in MCF-7 cell growth.     

Ligand-modulated regulation of progesterone receptor messenger ribonucleic acid and protein in human breast cancer cell lines

We have examined the effects of estrogen and progestin agonist and antagonist ligands on regulation of progesterone receptor (PR) protein and mRNA levels in a variety of human breast cancer cell lines. By Northern blot analysis, using human PR cDNA probes, PR mRNA in T47D and MCF-7 cells appears as five species of approximately 11.4, 5.8, 5.3, 3.5, and 2.8 kilobases. PR mRNA species are not detected in the PR protein-negative breast cancer cell lines MDA-MB-231 and LY2. T47D cells contain high levels of PR mRNA and protein (detected by hormone binding assay or Western blot analysis), and the PR protein and mRNA content of T47D cells are reduced to about 10% of the control level within 48 h of treatment with 10 nM promegestone; 17, 21-dimethyl-19-nor-pregna-4,9-diene-3, 20-dione (R5020) or 16 alpha-ethyl-21-hydroxy-19-nor-pregn-4-ene-3,20-dione (ORG2058), both potent progestins. In contrast, treatment of T47D cells with the antiprogestin 17 beta-hydroxy-11 beta-[4-dimethylaminophenyl]-17 alpha-(1-propynyl)-estra- 4, 9-dien-3-one) (RU38486) reduces PR protein and mRNA levels only transiently. PR protein and mRNA are virtually undetectable in control MCF-7 cells grown in the absence of estrogens. When estradiol is administered to MCF-7 cells, the PR mRNA and protein levels increase gradually and proportionately (10- or 40-fold, respectively, in 3 days).(ABSTRACT TRUNCATED AT 250 WORDS)     

Growth inhibition of estrogen receptor-positive and aromatase-positive human breast cancer cells in monolayer and spheroid cultures by letrozole, anastrozole, and tamoxifen

Two third-generation aromatase inhibitors, letrozole and anastrozole, and the antiestrogen tamoxifen, were compared for growth-inhibiting activity in two estrogen receptor (ER)-positive aromatase-overexpressing human breast cancer cell lines, MCF-7aro and T-47Daro. Inhibition of hormone (1 nM testosterone)-stimulated proliferation was evaluated in both monolayer cultures and in three-dimensional spheroid cultures. Letrozole and anastrozole were also compared for effectiveness of aromatase inhibition, and relative affinity for aromatase, under both monolayer and spheroid growth conditions. Letrozole was an effective inhibitor of MCF-7aro monolayer cell proliferation, with an estimated 50% inhibitory concentration (IC50) of 50-100 nM, whereas an IC50 was not reached with anastrozole at any concentration tested (100-500 nM). An IC50 of tamoxifen was 1000 nM. Proliferation of T-47Daro monolayer cells was more sensitive to inhibition by all three agents; as with MCF-7aro cells, letrozole was the most effective inhibitor. MCF-7aro spheroids were slightly less sensitive than monolayer cells proliferation-inhibiting effects of letrozole (IC50 about 200 nM), and there was no significant inhibition with 100-200 nM anastrozole or 200-1000 nM tamoxifen. Letrozole and anastrozole significantly inhibited T-47Daro spheroid cell proliferation, at 15-25 and 50 nM, respectively, consistent with the greater sensitivity of T-47Daro monolayer cells to inhibition of proliferation by these agents. Tamoxifen failed to significantly inhibit T-47Daro spheroid cell proliferation over a 100-500 nM concentration range. Determination of aromatase inhibition in monolayers of both cell lines by a direct-access microsomal assay and an intact-cell assay revealed that letrozole was more active than anastrozole in monolayers of both cell lines and in both assays. In MCF-7aro spheroids following cell lysis, only letrozole significantly inhibited aromatase activity, supporting the conclusion that letrozole binds stronger to aromatase than anastrozole does. Our results demonstrate that MCF-7aro and T-47Daro spheroids could be a suitable model for evaluation of growth-inhibitory effects of agents used in hormonal therapy of breast cancer.     

Estrogen regulation of nuclear matrix-intermediate filament proteins in human breast cancer cells

The tissue matrix consists of linkages and interactions of the nuclear matrix, cytoskeleton, and extracellular matrix. This system is a dynamic structural component of the cell that organizes and processes structural and functional information to maintain and coordinate cell function and gene expression. We have studied estrogen regulation of nuclear matrix associated proteins, including the intimately connected cytoskeletal intermediate filaments, in T-47D5 human breast cancer cells. Three proteins (identified as cytokeratins 8, 18, and 19) present in the nuclear matrix-intermediate filament fraction (NM-IF) of cells grown in estrogen-replete conditions were dramatically reduced when the cells were grown in acute (1 week) estrogen-depleted conditions. Replacing estrogen in the medium of acute estrogen-depleted cells restored expression of these proteins. T-47D5 cells that are chronically depleted of estrogen (T5-PRF) are estrogen-nonresponsive in culture. These cells overexpressed these three proteins, compared to parent cells grown in the presence of estrogen. Treatment of the T5-PRF cells with estrogen did not lead to further up-regulation of these proteins. Treating T-47D5 cells in estrogen-replete conditions with the antiestrogens 4-hydroxytamoxifen and ICI 164 384 (100 nM, 3 days) resulted in a significant reduction in these proteins, while no effect was seen in long-term chronic estrogen-depleted T-47D5 cells. In conclusion, we have identified NM-IF proteins (cytokeratins 8, 18, and 19) in human breast cancer cells that are estrogen regulated and may play a role in estrogen action in human breast cancer cells. © 1996 Wiley-Liss, Inc.     

Retinol conversion to retinoic acid is impaired in breast cancer cell lines relative to normal cells

The bioactivity of retinol (vitamin A) is in part dependent on its metabolism to retinoic acid (RA). We investigated the ability of breast epithelial cells to synthesize RA when challenged with a physiological retinol dose (2 microM). Normal human mammary epithelial cells (HMEC) cultured from reduction mammoplasties were competent in RA synthesis and the ability to synthesize RA was retained by immortal, nontumorigenic breast epithelial cell lines (MTSV1.7, MCF-10F, and 184B5). In contrast, most (five of six) breast cancer cell lines could not synthesize RA or did so at low rates relative to normal cells. A notable exception was the MDA-MB-468 cell line, which was fully competent in RA synthesis. Most (>/=68%) of the RA synthesized by breast cells was recovered from the culture medium. Cellular retinol binding protein and cellular RA binding protein II, both expressed in HMEC, had various expression patterns in the cell lines that did not correlate with the observed differences in RA synthesizing ability. Strong RA induction of the RA hydroxylase P450RAI (CYP26) was confined to ERalpha-positive T47D and MCF-7 breast cancer cells and did not appear to explain the lack of detectable RA levels in these cells since RA remained undetectable when the cells were treated with 5-10 microM liarozole, a P450RAI inhibitor. We hypothesize that retinol bioactivity is impaired in breast cancer cells that cannot synthesize RA. In preliminary support of this hypothesis, we found that retinol (0.5-2 microM) inhibited MCF-10F but not T47D or MCF-7 cell growth.