E2-stimulated Cell Research Articles

17β-Estradiol (E2) Activates Matrix Mineralization through Genomic/Nongenomic Pathways in MC3T3-E1 Cells.

Estrogen plays an important role in osteoporosis prevention. We herein report the possible novel signaling pathway of 17β-estradiol (E2) in the matrix mineralization of MC3T3-E1, an osteoblast-like cell line. In the culture media-containing stripped serum, in which small lipophilic molecules such as steroid hormones including E2 were depleted, matrix mineralization was significantly reduced. However, the E2 treatment induced this. The E2 effects were suppressed by ICI182,780, the estrogen receptor (ER)α, and the ERβ antagonist, as well as their mRNA knockdown, whereas Raloxifene, an inhibitor of estrogen-induced transcription, and G15, a G-protein-coupled estrogen receptor (GPER) 1 inhibitor, had little or no effect. Furthermore, the E2-activated matrix mineralization was disrupted by PMA, a PKC activator, and SB202190, a p38 MAPK inhibitor, but not by wortmannin, a PI3K inhibitor. Matrix mineralization was also induced by the culture media from the E2-stimulated cell culture. This effect was hindered by PMA or heat treatment, but not by SB202190. These results indicate that E2 activates the p38 MAPK pathway via ERs independently from actions in the nucleus. Such activation may cause the secretion of certain signaling molecule(s), which inhibit the PKC pathway. Our study provides a novel pathway of E2 action that could be a therapeutic target to activate matrix mineralization under various diseases, including osteoporosis.

Heat shock factor 1 (HSF1) cooperates with estrogen receptor α (ERα) in the regulation of estrogen action in breast cancer cells.

Heat shock factor 1 (HSF1), a key regulator of transcriptional responses to proteotoxic stress, was linked to estrogen (E2) signaling through estrogen receptor α (ERα). We found that an HSF1 deficiency may decrease ERα level, attenuate the mitogenic action of E2, counteract E2-stimulated cell scattering, and reduce adhesion to collagens and cell motility in ER-positive breast cancer cells. The stimulatory effect of E2 on the transcriptome is largely weaker in HSF1-deficient cells, in part due to the higher basal expression of E2-dependent genes, which correlates with the enhanced binding of unliganded ERα to chromatin in such cells. HSF1 and ERα can cooperate directly in E2-stimulated regulation of transcription, and HSF1 potentiates the action of ERα through a mechanism involving chromatin reorganization. Furthermore, HSF1 deficiency may increase the sensitivity to hormonal therapy (4-hydroxytamoxifen) or CDK4/6 inhibitors (palbociclib). Analyses of data from The Cancer Genome Atlas database indicate that HSF1 increases the transcriptome disparity in ER-positive breast cancer and can enhance the genomic action of ERα. Moreover, only in ER-positive cancers an elevated HSF1 level is associated with metastatic disease.

Modulatory effect of aquaporin 5 on estrogen-induced epithelial-mesenchymal transition in prostate epithelial cells.

BackgroundEstrogen is involved in the pathophysiological process of benign prostatic hyperplasia (BPH), in which epithelial-mesenchymal transition (EMT) plays an important role. Upregulation of aquaporin (AQP) 5, which is directly activated by estrogen, has been reported to promote EMT in multiple cells. This study aimed to examine the effects of AQP5 on estrogen-induced EMT in the prostate.MethodsNormal prostate (NP) tissue samples without any histopathological changes and BPH tissue samples with pathologically confirmed hyperplasia were obtained. An EMT cell model was subsequently established by adding estradiol (E2) to RWPE-1 cells, after which AQP5 knockdown was performed. Tissue morphological and immunohistochemical features were examined using hematoxylin-eosin and immunohistochemical staining. Western blot analysis was performed to determine the expression of AQPs, estrogen receptors, and EMT-related proteins. Cell proliferation was assessed and supernatants were collected for enzyme-linked immunosorbent assay to determine transforming growth factor-β1 (TGF-β1) concentrations. Immunofluorescence staining was performed to assess protein expressions in RWPE-1 cells.ResultsBPH tissues exhibited greater EMT (TGF-β1: 1.362 ± 0.196 vs. 0.107 ± 0.067, P = 0.003; vimentin: 1.581 ± 0.508 vs. 0.221 ± 0.047, P < 0.001; E-cadherin: 0.197 ± 0.188 vs. 1.344 ± 0.088, P < 0.001), higher AQP5 (1.268 ± 0.136 vs. 0.227 ± 0.055, P < 0.001) and estrogen receptor (ER) α (1.250 ± 0.117 vs. 0.329 ± 0.134, P < 0.001) expression but lower ERβ (0.271 ± 0.184 vs. 1.564 ± 0.130, P < 0.001) expression than NP tissues. E2-stimulated cells had higher AQP5 expression (1.298 ± 0.058 vs. 1.085 ± 0.104, P = 0.049), increased cell proliferation (1.510 ± 0.089 vs.1.000 ± 0.038, P < 0.001), and EMT (TGF-β1 concentration: 0.352 ± 0.021 ng/mL vs. 0.125 ± 0.014 ng/mL, P < 0.001; vimentin: 1.641 ± 0.120 vs. 0.188 ± 0.020, P = 0.002; E-cadherin: 0.075 ± 0.030 vs. 0.843 ± 0.046, P < 0.001) than controls. E2-stimulated cells with AQP5 knockdown exhibited decreased EMT (TGF-β1 concentration: 0.223 ± 0.041 ng/mL vs. 0.352 ± 0.021 ng/mL, P = 0.016; vimentin: 0.675 ± 0.056 vs. 1.641 ± 0.120, P = 0.001; E-cadherin: 0.159 ± 0.037 vs. 0.075 ± 0.030, P = 0.040) than E2-stimulated cells with non-related small interfering RNA (siRNA).ConclusionOur findings suggest that estrogen induces BPH possibly by promoting AQP5 expression. Hence, AQP5 might be a novel target for modulating EMT in prostate epithelial cells.

Overcoming Taxol-resistance in A549 cells: A comprehensive strategy of targeting P-gp transporter, AKT/ERK pathways, and cytochrome P450 enzyme CYP1B1 by 4-hydroxyemodin

Taxol-based chemotherapy is widely used as the first-line treatment for non-small cell lung cancer (NSCLC), however, the subsequent development of taxol-resistance is a major concern and challenge, resulting in tumor relapse and poor prognosis. Given the complex nature of taxol-resistance, we further delved into its mechanisms and demonstrated that CYP1B1 was associated to taxol response in taxol-resistant A549/Taxol cells. Compared to its parent A549 counterpart, A549/Taxol presented much higher level of CYP1B1, which was paralleled by increased aryl hydrocarbon receptor (AhR) expressions likely due to the long term taxol exposure and thereby allowed a subsequent up-regulation of CYP1B1. Inhibition of CYP1B1 by TMS [(E)-2,3′,4,5′-tetramethoxystilbene], the specific CYP1B1 inhibitor, remarkably enhanced the sensitivity of A549/Taxol to taxol. Moreover, pre-incubation of taxol with human recombinant CYP1B1 did not affect drug toxicity in A549 cells, precluding the possibility of drug resistance ascribed to CYP1B1 due to directly inactivating taxol. Indeed, CYP1B1 is responsible for bio-transforming estrogen (E2) into the carcinogenetic metabolite that would inhibit microtubule stabilization induced by taxol and thereby compromising treatment efficacy. Remarkably, our data revealed potent CYP1B1 inhibition efficacy of 4-hydroxyemodin (HEM) as reflected by both molecular docking simulations and EROD assay, which posed HEM the advantage of breaking the vicious circle between E2 and CYP1B1, not only favoring to overcome taxol-resistance, but also offering long term benefit via circumventing carcinogenesis and tumor progression induced by E2. In addition to CYP1B1 inhibition, HEM notably inhibited P-gp activity and expression, a common feature of drug resistance, as well as significantly inactivated AKT/ERK pathways that contributed to the cell proliferation, migration, and drug resistance. Thus, HEM may act in concert to overcome taxol-resistance through comprehensive targeting three considered arms of drug-resistance mechanisms. Moreover, HEM profoundly resisted E2-stimulated cell migration in both A549 and A549/Taxol cells, a primary reason for tumor patients’ mortality, as well as inflicted selective injury to A549/Taxol cells rather than normal lung cells, supporting HEM to be a promising agent for overcoming taxol-resistance in A549 cells.

Role of metformin in inhibiting estrogen-induced proliferation and regulating ERα and ERβ expression in human endometrial cancer cells.

Diabetes mellitus (DM) is an important factor that contributes to the development of type I endometrial cancer (EC). Previous studies have demonstrated that metformin decreases mortality and risk of neoplasms in patients with DM. Since estrogen and estrogen receptor (ER) expression has been associated with the development of EC, the present study aimed to investigate the effects of metformin on cell proliferation and ER expression in EC cell lines that are sensitive to estrogen. The viability and proliferation of Ishikawa and HEC-1-A cells were measured following treatment with metformin and/or a 5' AMP-activated protein kinase (AMPK) inhibitor (compound C) with or without treatment with estradiol (E2). In addition, the levels of ERα, ERβ, AMPK, ribosomal protein S6 kinase β-1 (p70S6K), myc proto-oncogene protein (c-myc) and proto-oncogene c-fos (c-fos) were measured following treatment. Metformin significantly decreased E2-stimulated cell proliferation; an effect that was rescued in the presence of compound C. Metformin treatment markedly increased the phosphorylation of AMPK while decreasing p70S6K phosphorylation, indicating that metformin exerts its effects through stimulation of AMPK and subsequent inhibition of the mammalian target of rapamycin (mTOR) signaling pathway. In addition, metformin significantly inhibited ERα expression while increasing ERβ expression, whereas treatment with compound C reversed these effects. Reverse transcription-quantitative polymerase chain reaction analysis demonstrated that c-fos and c-myc expression were attenuated by metformin, an effect that was rescued in the presence of compound C. Therefore, metformin regulates the expression of ERs, and inhibits estrogen-mediated proliferation of human EC cells through the activation of AMPK and subsequent inhibition of the mTOR signaling pathway.

Abstract S4-01: A novel BRD4 inhibitor enhances endocrine therapy efficacy and circumvents endocrine-resistance in estrogen receptor-positive breast cancer models

Abstract Background: The selective estrogen receptor (ER) down-regulator (SERD) fulvestrant (Ful) antagonizes ER activity and degrades ER protein in a dose-dependent manner in the preclinical and clinical settings, though its efficacy is limited by an incomplete abolition of ER protein levels. Therefore additional strategies are needed to achieve a more complete suppression of ER level and activity. The bromodomain-containing protein 4 (BRD4), a member of the BET family that is required both for ESR1 gene expression and for ER-mediated gene transcription, represents an attractive therapeutic target for ER+ breast cancer (BC). Here, we investigated the efficacy of the novel BRD4 inhibitor GS-626510 (GS-6510) in a panel of ER+ BC parental and endocrine-resistant (EndoR) cell lines and in a patient-derived xenograft (PDX) model. Materials and Methods: The effects of GS-6510 (25nM – 290nM) alone or in combination with endocrine therapies were tested in ER+ MCF7, T47D, and ZR75-1 cell lines, as well as in their derivatives made resistant to estrogen (E2) deprivation (EDR), tamoxifen (TamR), or Ful (FulR). Cell growth (by methylene blue) after 6 days of GS-6510 and protein levels (by Western blot) after 2 days of GS-6510 were assessed. The in vivo efficacy of GS-6510, Ful, and the combination was tested in the ER+ HCBx34 PDX model. The mRNA levels of genes associated with cell cycle, ER signaling, and endocrine-resistance were assessed using the NanoString platform. Results: A dose-dependent inhibitory effect of GS-6510 was observed in all of the experimental settings. At a clinically relevant dose, GS-6510 reduced E2-stimulated cell growth and enhanced the efficacy of endocrine therapies in all parental cell lines. In the endocrine-sensitive HCBx34 PDX model, GS-6510 reduced tumor growth and, in combination with Ful, induced tumor regression and inhibited the expression of ER-dependent and cell cycle related genes including CCND1, MYC, and BCL2. Notably, the addition of GS-6510 to EndoR cell models that continue to rely on and express ER (MCF7 EDR and TamR) led to a substantial inhibition in cell growth (85%; 98%, respectively). Though the combination of GS-6510 and Ful was not associated with a significantly greater cell growth inhibition compared to GS-6510 alone in these models, a better suppression of ER levels was observed. Interestingly, GS-6510 also remained effective in EndoR models that exhibited an ER-independent growth, including all FulR lines, though its efficacy varied among the different cell lines and resistant derivatives. Conclusion: Our findings suggest that the epigenetic regulator BRD4 is a suitable target for therapeutic intervention in ER+ BC. The anti-tumor efficacy of GS-6510 in endocrine sensitive and especially in ER-dependent EndoR models is worthy of further clinical investigation. The growth inhibitory effects observed in some of the ER-independent EndoR models suggests that additional genes/pathways involved in endocrine resistance could be affected by GS-6510. Identifying these pathways and determining their predictive role are needed to guide patient selection for future clinical trials. Citation Format: De Angelis C, Nardone A, Cataldo ML, Fu X, Trivedi M, Yi S, Breckenridge D, Chamnsess GC, Vitorino P, Osborne CK, Schiff R. A novel BRD4 inhibitor enhances endocrine therapy efficacy and circumvents endocrine-resistance in estrogen receptor-positive breast cancer models [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr S4-01.

CaM kinase activation and cancer cell growth are controlled by AKAP7 (613.4)

Estrogen (E2) activates the calcium/calmodulin‐dependent protein kinases (CaM Kinases) in breast cancer cells. The CaM Kinases regulate ERK leading to transcription and cell growth. CaM Kinase activation of ERK may be blocked by PKA phosphorylation and inhibition of CaM KK. The ability of PKA to phosphorylate its cellular targets may be dictated by protein kinase A anchoring proteins (AKAPs). Hormones that activate PKA, such as vitamin D (VitD) and epinephrine (Epi), may utilize AKAPs to facilitate the assembly of protein complexes and signal transduction. AKAP7 participates in intracellular calcium signaling and therefore may regulate CaM Kinases. Our goal was to evaluate the role of AKAP7 in regulating E2 activation of the CaM KK and CaM KI cell growth pathway in MCF‐10, MCF‐7 and MDA‐MB‐231 cells. Our results suggest that E2 treatment of MCF‐7 cells activates CaM KI, an effect that is blocked by the CaM KK‐inhibitor STO‐609 but not by the ERK‐inhibitor U0126. Interestingly, purified GST‐RII subunit of PKA pulled down both CaM KK and CaM KI from MCF‐7 cells. Similarly, precipitation with STag‐AKAPs showed that CaM Kinases bind to AKAP7 but not AKAP5, an effect that is enhanced with E2. Purified AKAP7 directly binds purified CaM KK, and endogenous AKAP7 is expressed in MCF‐10, MCF‐7, and MDA‐MB‐231 cells. Precipitation of endogenous AKAP7 revealed a robust association with CaM KK in E2‐, but not in STO‐609‐ or VitD‐treated cells. Similarly, VitD pre‐treatment blocked E2’s ability to increase CaM KI phosphorylation and activation. Interestingly, siRNA knockdown of AKAP7 increased basal ERK phosphorylation and prevented E2’s activation of ERK. Finally, E2‐stimulated cell growth was inhibited by pre‐treatment with VitD, STO‐609, and the AKAP inhibitor, Ht‐31. Taken together, these results suggest that AKAP7 directly associates with CaM KK in a complex with CaM KI, an effect that is blocked by VitD via PKA’s inhibition of CaM KK. Finally, VitD and Ht‐31 inhibit growth in breast cancer cells.Grant Funding Source: Supported by M.J.Murdock Charitable Trust #2011267

P66Shc longevity protein regulates the proliferation of human ovarian cancer cells

p66Shc functions as a longevity protein in murine and exhibits oxidase activity in regulating diverse biological activities. In this study, we investigated the role of p66Shc protein in regulating ovarian cancer (OCa) cell proliferation. Among three cell lines examined, the slowest growing OVCAR-3 cells have the lowest level of p66Shc protein. Transient transfection with p66Shc cDNA expression vector in OVCAR-3 cells increases cell proliferation. Conversely, knock-down of p66Shc by shRNA in rapidly growing SKOV-3 cells results in decreased cell growth. In estrogen (E2)-treated CaOV-3 cells, elevated p66Shc protein level correlates with ROS level, ErbB-2 and ERK/MAPK activation, and cell proliferation. Further, the E2-stimulated proliferation of CaOV-3 cells was blocked by antioxidants and ErbB-2 inhibitor. Additionally, in E2-stimulated cells, the tartrate-sensitive, but not the tartrate-resistant, phosphatase activity decreases; concurrently, the tyrosine phosphorylation of ErbB-2 increases. Conversely, inhibition of phosphatase activity by L(+)-tartrate treatment increases p66Shc protein level, ErbB-2 tyrosine phosphorylation, ERK/MAPK activation, and cell growth. Further, inhibition of the ERK/MAPK pathway by PD98059 blocks E2-induced ERK/MAPK activation and cell proliferation in CaOV-3 cells. Moreover, immunohistochemical analyses showed that the p66Shc protein level was significantly higher in cancerous cells than in noncancerous cells in archival OCa tissues (n = 76; P = 0.00037). These data collectively indicate that p66Shc protein plays a critical role in up-regulating OCa progression.

Membrane-receptor initiated proliferative effects of dienogest in human breast cancer cells

Objectives: Dienogest (DNG) is already used in hormone therapy, since recently being also the progestogenic component of the first estradiol based contraceptive pill. Data on breast cancer risk are currently not available. Progesterone receptor membrane component 1 (PGRMC1) is highly expressed in tissues of breast cancer patients and has already been proposed as a predictor for breast cancer risk. Methods: MCF-7 cells overexpressing PGRMC1 were stimulated with DNG, medroxyprogesterone acetate (MPA), norethisterone (NET) and progesterone (P) as well as sequentially and continuously combined with estradiol (E2). Results: DNG and MPA alone elicited a significant proliferation at 10–6 and 10–5 M. NET increased cell proliferation at all concentrations tested whereas P showed no effect. E2 alone elicited a significant increase at 10−10 M, no effect was seen at 10–12 M. Addition of the progestins (10–6 M) to E2 at 10−10 M had, compared to E2 only, no additional proliferating effect. However, at the low E2 concentration, DNG, MPA and NET significantly increased the E2-stimulated cell proliferation. Conclusion: DNG increased proliferation alone and in combination with low E2 concentrations. Thus a progestogen-derived breast cancer risk in the presence of low E2 concentrations cannot be excluded at least in women overexpressing PGRMC1.

Prolactin and estradiol utilize distinct mechanisms to increase serine-118 phosphorylation and decrease levels of estrogen receptor α in T47D breast cancer cells

Potential interactions between prolactin (PRL) and estradiol (E2) in breast cancer cells were explored by examining the effect of PRL on estrogen receptor (ER) serine-118 phosphorylation, ER down-regulation, and E2-stimulated cell proliferation. Both E2 and PRL resulted in prolonged ERalpha serine-118 phosphorylation, but used different signaling pathways to achieve this end. Both hormones also decreased the amount of ERalpha, but the mechanisms were different: for E2, the decrease was rapid and resulted from proteasomic degradation, whereas for PRL the decrease was slow and resulted from an effect on levels of ERalpha mRNA. PRL alone had no effect on cell number, but enhanced the increase in number in response to E2. These results are the first to demonstrate similar effects of PRL and E2 on parameters considered key to E2's effects. This suggests heretofore unrecognized and potentially important interactions between these two hormones in the natural history of breast cancer.

Novel actions of estrogen to promote proliferation: Integration of cytoplasmic and nuclear pathways

Both steroids and growth factors stimulate proliferation of steroid-dependent tumor cells, and interaction between these signaling pathways occurs at several levels. Steroid receptors are classified as ligand-activated transcription factors, and steps by which they activate target gene transcription are well understood. Several steroid responses have now been functionally linked to other intracellular signaling pathways, including c-Src or tyrosine kinase receptors. Steroids such as 17β-estradiol (E2), via binding to cytoplasmic or membrane-associated receptors, were also shown to rapidly activate intracellular signaling cascades such as ERK, PI3K and STATs. These E2-stimulated phosphorylations can then contribute to altered tumor cell function. ER-positive breast cancer cells, in which proliferation is stimulated by E2 and suppressed by antiestrogens, have been of particular interest in dissecting nuclear and cytoplasmic roles of estrogen receptors (ER). In some cell contexts, ER interacts directly with the intracellular tyrosine kinase c-Src and other cytoplasmic signaling and adaptor molecules, such as Shc, PI3K, MNAR, and p130 Cas. Although the hierarchy among these associations is not known, it is clear that c-Src plays a fundamental role in both growth factor and E2-stimulated cell growth, and this may also require other growth factor receptors such as those for EGF or IGF-1. STAT transcription factors represent one pathway to integrate E2 cytoplasmic and nuclear signaling. STAT5 is phosphorylated in the cytoplasm at an activating tyrosine in response to E2 or EGF, and then is translocated to the nucleus to stimulate target gene transcription. E2 stimulates recruitment of STAT5 and ER to the promoter of several proliferative genes, and STAT5 knockdown prevents recruitment of either protein to these promoters. STAT5 activation by E2 in breast cancer cells requires c-Src and EGF receptor, and inhibition of c-Src or EGFR, or knockdown of STAT5, prevents E2 stimulation of several genes and breast cancer cell proliferation. Hyperactivation of the growth factor receptor-c-Src pathway can in some contexts decrease growth responses to E2, or render cells and tumors resistant to suppressive actions of endocrine therapies. Crosstalk between growth factors and steroids in both the cytoplasm and nucleus may thus have a profound impact on complex biological processes such as cell growth, and may play a significant role in the treatment of steroid-dependent breast cancers.

Genomic analysis of estrogen cascade reveals histone variant H2A.Z associated with breast cancer progression

We demonstrate an integrated approach to the study of a transcriptional regulatory cascade involved in the progression of breast cancer and we identify a protein associated with disease progression. Using chromatin immunoprecipitation and genome tiling arrays, whole genome mapping of transcription factor-binding sites was combined with gene expression profiling to identify genes involved in the proliferative response to estrogen (E2). Using RNA interference, selected ERα and c-MYC gene targets were knocked down to identify mediators of E2-stimulated cell proliferation. Tissue microarray screening revealed that high expression of an epigenetic factor, the E2-inducible histone variant H2A.Z, is significantly associated with lymph node metastasis and decreased breast cancer survival. Detection of H2A.Z levels independently increased the prognostic power of biomarkers currently in clinical use. This integrated approach has accelerated the identification of a molecule linked to breast cancer progression, has implications for diagnostic and therapeutic interventions, and can be applied to a wide range of cancers.

P38 Mitogen-activated protein kinases is required for counteraction of 2-methoxyestradiol to estradiol-stimulated cell proliferation and induction of apoptosis in ovarian carcinoma cells via phosphorylation Bcl-2

2-Methoxyestradiol (2ME2), a natural endogenous product of estradiol (E2) metabolism, has been shown to be a selective apoptotic agent for cancer cells but not for normal cells. In this study, we determined that 2ME2 counteracts E2-stimulated cell growth and induces apoptosis in ovarian carcinoma cells. In addition, we demonstrate that 2ME2 induces apoptosis via p38 and phospho-Bcl2 pathway. 2ME2 and/or E2 were administered to the OVCAR-3 (human ovarian cancer) cell line. Cell growth inhibition was analyzed by [3H] Thymidine incorporation assay and DNA fluorometric assay. Cell apoptosis was tested by DNA fragmentation analysis and FACS. The signaling pathway was determined by a series of biochemical assays. 2ME2 inhibited estradiol-stimulated cell growth and induced apoptosis in an ovarian carcinoma cell line. MAPK and p38, but not JNK, were found to be critical mediators in this process. Expression of a dominant negative mutant of p38 kinase or p38 specific inhibitor, SB 203580, almost completely blocked the process. Furthermore, Bcl-2 phosphorylation was required for 2ME2-induced effects. Our data suggest that 2ME2 inhibits E2-stimulated proliferation and induces apoptosis in ovarian carcinoma cells. Furthermore, activation of p38 and phosphorylation of Bcl-2 plays a critical role in the mechanism. 2ME2 therefore, may have a clinical application for the treatment of ovarian cancer.

Tyrosine kinase inhibitors as antiproliferative agents against an estrogen-dependent breast cancer cell line in vitro.

Receptor tyrosine kinase (RTK) activation is critical for growth factor-mediated cell proliferation. Blockade of RTK activation inhibits growth factor-induced cell proliferation. A panel of RTK inhibitors (tyrphostins) have been tested and compared for their antiproliferative effects on the hormone-dependent human breast cancer cell line, MCF-7, in vitro. MCF-7 cells (10(4)/well) were seeded into 96 well plates and maintained in DMEM with 1% bovine serum albumin (BSA), 200-pg/mL estrogen, or 10% fetal bovine serum. After a defined time interval, the cells were exposed to RTK inhibitors and a non-RTK-inhibitory analog of tyrphostins (0 to 400 microM). After 3 days, the number of viable cells in each well was estimated by an MTT assay and the results expressed as percent of controls. Using a representative tyrphostin, A47, the validity of MTT assay as a measure of cell proliferation was tested by a colony formation assay and by immunostaining with Ki-67 antibodies. MCF-7 cells maintained in DMEM containing 1% BSA without E2 or serum showed a minimal increase in cell number. Supplementation with E2 stimulated cell proliferation in a dose-dependent manner. This E2-mediated growth stimulation was completely inhibited (cytostatic effects) by the epidermal growth factor receptor (EGFR)-selective tyrphostins A47, B48, RG13022, and B50. These same tyrphostins also decreased the cell numbers to below control numbers in cultures maintained in 1% BSA or in serum containing medium (cytostatic/cytotoxic effects). B44 (EGFR-selective tyrphostin), AG1295 (platelet-derived growth factor receptor [PDGFR]-selective tyrphostin), and A1 had no inhibitory effects on cells with or without E2 treatments. However, A1 inhibited cell growth under serum supplementation. Genistein, a phytoestrogen, stimulated the autonomous, E2-induced as well as serum-induced growth of MCF-7 cells. Cell proliferation results derived from the MTT assay were corroborated by both the colony formation assay as well as the Ki-67 assay. Of the agents tested, only EGFR-selective tyrphostins blocked E2-stimulated tumor cell proliferation, as opposed to the PDGFR-selective tyrphostin, RTK noninhibitory agent, or the phytoestrogen, genistein, which did not exert such an effect. These findings suggest that epidermal growth factor (EGF) is an important mediator of E2-induced proliferation of MCF-7 cells. Thus, tyrphostins may be selectively used to prevent the growth of hormone-dependent breast cancers, particularly regrowth of residual tumor in postmenopausal breast cancer survivors receiving estrogen replacement therapy.