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.     

Effect of insulin receptor down regulation on insulin-stimulated thymidine incorporation in cultured human fibroblasts and tumor cell lines.

Insulin receptors in transformed tissue are relatively resistant to down regulation by insulin, and although receptor downregulation reduces rapid onset biologic responses to insulin in normal tissue, this is not observed in tumor cells. The present study compares longterm insulin responses (thymidine incorporation and cell growth) in normal human fibroblasts with responses in human tumor cell lines (MCF-7, T-47D and HCT-8) to determine whether these responses are also resistant to the effects of receptor down regulation. Thymidine incorporation into fibroblasts was more responsive to insulin than was incorporation into tumor cells, although stimulation of uptake into fibroblasts was not paralleled by changes in cell replication. In contrast, physiological insulin concentrations inhibited, and high concentrations of insulin stimulated, thymidine incorporation and cell replication in MCF-7 and T-47D cells. All insulin concentrations inhibited thymidine incorporation in HCT-8 cells without affecting cell replication. The responsiveness of fibroblasts, MCF-7 and HCT-8 cells to insulin was unaltered by down regulation of insulin receptors prior to measuring thymidine incorporation, whereas receptor down regulation paradoxically increased the responsiveness of T-47D cells to insulin. Exposure of fibroblasts to 5 x 10(-8) M dexamethasone for 24h increased their responsiveness to insulin but did not influence the response of MCF-7 or HCT-8 cells, whereas insulin-stimulated incorporation of thymidine in T-47D cells was inhibited. Thus, receptor down regulation does not influence the longterm biologic response to insulin in normal cells, and paradoxically increases responsiveness in one of three tumor cell lines. These changes may contribute to the well-described stimulatory effects of insulin on tumor cell growth and inhibition of this response with dexamethasone may be relevant to cancer treatment programs.     

Protein kinase C desensitization by phorbol esters and its impact on growth of human breast cancer cells

Active phorbol esters such as TPA (12-0-tetra-decanoylphorbol-13-acetate) inhibited growth of mammary carcinoma cells (MCF-7 greater than BT-20 greater than MDA-MB-231 greater than = ZR-75-1 greater than HBL-100) with the exception of T-47-D cells presumably by interacting with the phospholipid/Ca2+-dependent protein kinase (PKC). The nonresponsive T-47-D cells exhibited the lowest PKC activity. A rapid (30 min) TPA-dependent translocation of cytosolic PKC to membranes was found in the five TPA-sensitive cell without affecting cell growth. However, TPA-treatment of more than 10 hours inhibited reversibly the growth of TPA-responsive cells. This effect coincided with the complete loss of cellular PKC activity due to the proteolysis of the translocated membrane-bound PKC holoenzyme (75K) into 60K and 50K PKC fragments. Resumption of cell growth after TPA-removal was closely related to the specific reappearance of the PKC holoenzyme activity (75K) in the TPA-responsive human mammary tumor cell lines suggesting an involvement of PKC in growth regulation.     

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.     

Role of thrombin in the proliferative response of T-47D mammary tumor cells : Mitogenic action and pleiotropic modifications induced together with epidermal growth factor and insulin

The growth of the human metastatic cell line (T-47D) in a chemically defined medium (DM) is shown to be dependent on the presence of three traditional growth factors: epidermal growth factor, insulin, and transferrin. The addition of thrombin further stimulates its growth. The mitogenic action on a human mammary tumor cell line from epithelial origin is a novel action of thrombin. Cells in the DM show striking morphological changes which are dramatically enhanced by the addition of thrombin. These observations are part of a pleiotropic response to the growth factors: the protein content of the cells increases in the defined medium; the 2DG gels of the 35S- and 32P-labeled proteins show important changes in spots, several of which are probably of cytoskeletal origin. It is also shown that cells in a semisolid growth factor-supplemented medium have growth advantages over their counterparts grown with serum. All the phenotypic changes mentioned above reveal the important role of growth factors in the growth and behavior of this mammary cell line. The results obtained with thrombin indicate a new site of action of this enzyme which may be important in the metastatic spread of human mammary tumor cells.     

Epidermal growth factor protects epithelial cells against Fas-induced apoptosis. Requirement for Akt activation.

Chemotherapeutic drugs that damage DNA kill tumor cells, in part, by inducing the expression of a death receptor such as Fas or its ligand, FasL. Here, we demonstrate that epidermal growth factor (EGF) stimulation of T47D breast adenocarcinoma and embryonic kidney epithelial (HEK293) cells protects these cells from Fas-induced apoptosis. EGF stimulation of epithelial cells also inhibited Fas-induced caspase activation and the proteolysis of signaling proteins downstream of the EGF receptor, Cbl and Akt/protein kinase B (Akt). EGF stimulation of Akt kinase activity blocked Fas-induced apoptosis. Expression of activated Akt in MCF-7 breast adenocarcinoma cells was sufficient to block Fas-mediated apoptosis. Inhibition of EGF-stimulated extracellular signal-regulated kinase (ERK) activity did not affect EGF protection from Fas-mediated apoptosis. The findings indicate that EGF receptor stimulation of epithelial cells has a significant survival function against death receptor-induced apoptosis mediated by Akt.     

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.     

Retinoids arrest breast cancer cell proliferation: retinoic acid selectively reduces the duration of receptor tyrosine kinase signaling

Retinoic acid (RA) induces cell cycle arrest of hormone-dependent human breast cancer (HBC) cells. Previously, we demonstrated that RA-induced growth arrest of T-47D HBC cells required the activity of the RA-induced protein kinase, protein kinase Calpha (PKCalpha) [J. Cell Physiol. 172 (1997) 306]. Here, we demonstrate that RA treatment of T-47D cells interfered with growth factor signaling to downstream, cytoplasmic and nuclear targets. RA treatment did not inhibit epidermal growth factor (EGF) receptor activation but resulted in rapid inactivation. The lack of sustained EGFR activation was associated with transient rather than sustained association of the EGFR with the Shc adaptor proteins and activation of Erk 1/2 and with compromised induction of expression of immediate early response genes. Inhibiting the activity of PKCalpha, a retinoic acid-induced target gene, prevented the effects of RA on cell proliferation and EGF signaling. Constitutive expression of PKCalpha, in the absence of RA, decreased cell proliferation and decreased EGF signaling. RA treatment increased steady-state levels of the protein tyrosine phosphatase PTP-1C and all measured effects of RA on EGF receptor function were reversed by the tyrosine phosphate inhibitor orthovanadate. These results indicate that RA-induced target genes, particularly PKCalpha, prevent sustained growth factor signaling, uncoupling activated receptor tyrosine kinases and nuclear targets that are required for cell cycle progression.     

The cytosolic phorboid receptor correlates with hormone dependency in six mammary carcinoma cell lines

Potent, structurally different tumor promoters inhibited growth of 6 human mammary carcinoma cell lines (ROOS et al, PNAS in press). This growth inhibition was investigated by measuring the phorboid receptor binding using [3H] PDBu (4 beta-phorbol 12, 13 dibutyrate). Specific, high affinity receptors were found in all six cell lines. [3H] PDBu binding affinities were higher in the cytosolic fractions than in the corresponding intact cells (K alpha = app. 1nM vs K alpha = app. 15nM). The hormone-independent cell lines (BT-20, HBL-100 and MDA-MB-231) exhibited significantly higher levels of cytosolic [3H] PDBu receptors than the hormone-dependent cells (MCF-7, T-47-D and ZR-75-1). The subcellular distribution of the [3H] PDBu binding correlated well with the distribution of the protein kinase C activity (r = 0.95).     

RRR-alpha-tocopheryl succinate induces MDA-MB-435 and MCF-7 human breast cancer cells to undergo differentiation.

RRR-alpha-Tocopheryl succinate (vitamin E succinate, VES) is a potent antitumor agent, inducing DNA synthesis arrest, differentiation, and apoptosis. Because little is known about VES-induced differentiation, studies reported here characterize VES effects on the differentiation status of human breast cancer cell lines and investigate possible molecular mechanisms involved. VES-induced differentiation of human MCF-7 and MDA-MB-435 breast cancer cells was characterized by morphological changes, induction of lipid droplets, induction of beta-casein mRNA expression, and down-regulation of Her2/neu protein. In contrast, VES treatment of normal human mammary epithelial cells, MCF-10A cells, and T-47D cells did not induce differentiation. Studies addressing mechanisms showed that neither antibody neutralization of the transforming growth factor-beta signaling pathway nor expression of a dominant-negative mutant of c-Jun N-terminal kinase blocked the ability of VES to induce differentiation; however, treatment of cells with PD 98059, a chemical inhibitor of mitogen-activated protein kinase kinase (MEK1/2), blocked the ability of VES to induce differentiation.     

Role of extracellular electrolytes in the activation of ribosomal protein S6 kinase by epidermal growth factor, insulin-like growth factor 1, and insulin in ZR-75-1 cells

Activation of ribosomal protein S6 kinase by epidermal growth factor (EGF), insulin, and insulin-like growth factor 1 (IGF1) was studied in the human mammary tumor cell line ZR-75-1 in isotonic buffers. In contrast to growth factor-dependent S6 phosphorylation which is strongly dependent on extracellular pH (Chambard, J. C., and J. Pouyssegur. 1986. Exp. Cell Res. 164:282-294.) preincubation of cells in buffers with different pH values ranging from 7.5 to 6.5 had no effect on basal or EGF-stimulated S6 kinase activity. Replacement of extracellular Na+ with choline or replacement of extracellular Ca++ with EGTA also did not inhibit stimulation of S6 kinase by EGF. When intracellular Ca++ was buffered with the permeable Ca++ chelator quin2, EGF stimulation was reduced 50%. A similar inhibition of the EGF response was observed when cells were incubated in buffers with high K+ concentrations or in the presence of the K+ ionophore valinomycin. Insulin and IGF1 stimulation of S6 kinase were also inhibited by high K+ concentrations and by buffering intracellular Ca++. In contrast to the responses to EGF, insulin- and IGF1-activation of S6 kinase was enhanced when glucose was present and depended on the presence of bicarbonate in the medium. The results indicate that ionic signals generated by growth factors and insulin, such as increases in intracellular pH or Na+, do not seem to be involved in the activation of S6 kinase. However, effects of growth factors or insulin on membrane potential and/or K+ fluxes and redistribution of intracellular Ca++ may play a role in the activation process. Furthermore, the mechanism of insulin activation of S6 kinase is distinct from the growth factors by its dependency on extracellular bicarbonate.     

Characterization of a sheep pituitary-derived growth factor for rat and human mammary tumor cells

A growth factor for rat and human mammary tumor cells (MTGF-Pit) was isolated from lyophilized powders of whole sheep pituitaries by a rapid four-step procedure utilizing acetic acid extraction, heating at 93 degrees C, and sequential chromatography in 0.10 M acetic acid on sulphopropyl Sephadex and Sephadex G-50. From 10 g of pituitary powder, 8-10-mg amounts of MTGF-Pit were isolated. By 8 M urea, 0.1% SDS-12.5% polyacrylamide gel electrophoresis analysis followed by Coomassie blue staining, this preparation was shown to be one major stained band. When assayed for growth effects on cells maintained in serum-free medium, 5.1-19.2 nM MTGF-Pit half replaced the growth of MTW9/PL rat and MCF-7 and T-47D human mammary tumor cells in response to 2% to 10% serum. MTGF-Pit shows mitogenic activity toward normal human diploid fibroblasts only at concentrations in excess of 2.5 X 10(-4) M, while rat fibroblasts are unresponsive even at this high concentration. From data available, we conclude that a mitogenic activity for epithelial-type mammary cells has been isolated, and this growth factor appears to be a previously undetected acid- and heat-stable activity that is highly abundant (estimated at 0.16% or more of the total dry weight of the pituitary powder). The isolated ovine MTGF-Pit (3,900 +/- 200 daltons) does not share the molecular weight of native prolactin (24,000 daltons), "cleaved" prolactin (16,000 daltons), or growth hormone (22,000 daltons), and by all tests applied cannot be replaced with other known hormones and purified growth factors. We conclude a potent new mammary tumor cell mitogenic activity has been identified from sheep pituitaries.     

Secretion of an epidermal growth factor-like growth factor by epidermal growth factor-independent rat mammary carcinoma cells.

Rat mammary carcinoma (RMC) cells derived from serially transplantable mammary tumors are independent of epidermal growth factor (EGF) for long-term growth in serum-free medium. This phenotype is in contrast to that of normal mammary epithelial cells or cells derived from nontransplantable tumors that express an absolute requirement for EGF for growth in culture. The results of the experiments reported here indicate that EGF-independent RMC cells secrete a growth factor with potent EGF-like mitogenic activity. Conditioned media obtained from these cells can substitute for EGF for the growth of the EGF-dependent cell line MCF-10. This growth factor is neither EGF nor transforming growth factor alpha and does not compete with 125I-EGF for binding to EGF receptors. Phosphotyrosine Western blot analysis of lysates obtained from EGF-independent RMC cells revealed the presence of a 190 kilodalton (kDa) protein that was distinct from the EGF receptor. Similarly, growth of MCF-10 cells to confluence in serum-free medium supplemented with conditioned medium growth factor in place of EGF resulted in the disappearance of the EGF receptor band and appearance of the 190 kDa band in phosphotyrosine Western blots. The 190 kDa tyrosine-phosphorylated protein detected in cells stimulated by the conditioned medium factor is unlikely to be the c-erbB-2 protein, as indicated by negative results in immunoprecipitation experiments and in vitro kinase assays. In summary, EGF-independent RMC cells secrete a factor with potent EGF-like mitogenic activity. This suggests that an autocrine loop involving this growth factor mediates EGF independence in these cells.     

Demonstration of insulin-like growth factor (IGF-I and -II) receptors and binding protein in human breast cancer cell lines

The insulin like growth factors (IGFs), potent mitogens for a variety of normal and transformed cells, have been reported to be secreted by several human breast cancer cell lines (BC). We have investigated the binding characteristics of IGF-I and -II in four human BC: MCF-7, T-47D, MDA 231 and Hs578T. Binding studies in microsomal membrane preparations detected high specific binding for both IGF in all four BC studied. Cross-linking with 125I-IGF-I, followed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) under reduced conditions, revealed the presence of an alpha subunit of apparent Mr = 130,000 in MCF-7, T-47D and MDA 213 cells. When 125I-IGF-II was cross-linked, a major band of apparent Mr = 260,000 was seen in all BC. This band was inhibited by IGF-II, but not by insulin. Cross-linking of 125I-IGF-I to conditioned media from BC demonstrated the presence of three binding proteins of apparent Mr = 45,000, 36,000 and 29,000 in all BC but T-47D, in which the 36,000 band was not seen. These data demonstrate that BC possess classical receptors for both IGF-I and -II and, furthermore, that BC produce specific binding proteins for these growth factors.     

Morphological and proliferative characteristics of human breast tumor cells cultured on plastic and in collagen matrix

Summary Collagen, a major component of the extracellular matrix, is important in maintaining the in vivo characteristics of epidermal cells in vitro. In the present study, the morphological and proliferative characteristics of two human mammary epithelial cell lines (T-47D and MCF-7) cultured in cowhide collagen (Vitrogen 100) were studied. When grown in collagen, the tumor cells displayed a spherical shape and formed multilayered, tumorlike aggregates; desmosomes were observed between cells. In contrast, both cell lines grew as monolayers on plastic substratum; cells were characteristically flat and polygonal. When grown in collagen matrix, the human breast cancer cells became more dependent on serum for growth: cells proliferated in the presence of 10% fetal bovine serum (FBS) but failed to grow in 1% serum. On the other hand, these cells proliferated rapidly in 1% serum when they were grown on plastic. Even in 10% serum the doubling time of cells cultured in collagen was longer than that of cells maintained on plastic. In addition, cells cultured in collagen proliferated rapidly in a serum-free medium containing insulin, epidermal growth factor (EGF), estrogen, and transferrin. The collagen gel system may be useful for characterizing physiologically important trophic factors that regulate the proliferation and other functions of human breast tumor cells. The advice of Drs. J. A. Paterson and B. Dronzek in the electron microscopy studies is appreciated. This research was supported by the Medical Research Council of Canada. Clement K. H. Leung was supported by a University of Manitoba graduate fellowship. Portions of this work were reported at the Twentieth Annual Meeting of the American Society for Cell Biology held in Cincinnati, Ohio, November 14–18, 1980.     

Effect of Medrogestone on 17beta-hydroxysteroid dehydrogenase activity in the hormone-dependent MCF-7 and T-47D human breast cancer cell lines

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 Medrogestone (Prothil) on 17beta-hydroxysteroid dehydrogenase (17beta-HSD) activities of the hormone-dependent MCF-7 and T-47D human breast cancer cell lines. Using physiological doses of estrone ([3H]-E1: 5 x 10(-9) mol/l) this estrogen is converted in a great proportion to E2 in both cell lines. After 24 h of the cell culture, Medrogestone significantly inhibits this transformation in a dose-dependent manner by 39% and 80% at 5 x 10(-8) M and 5 x 10(-5) M, respectively in T-47D cells; the effect is less intense in MCF-7 cells: 25% and 55% respectively. The IC50 values are 0.45 micromol/l in T-47D and 17.36 micromol/l in MCF-7 cells. It is concluded that the inhibition provoked by Medrogestone on the reductive 17beta-HSD activity involved in the local biosynthesis of the biologically active estrogen estradiol, may constitute a new therapeutic approach for the treatment of breast cancer.     

HIV-1 Tat peptide immunoconjugates differentially sensitize breast cancer cells to selected antiproliferative agents that induce the cyclin-dependent kinase inhibitor p21WAF-1/CIP-1

In this study, we evaluated the ability of anti-p21 antibodies conjugated to 17-mer peptides [GRKKRRQRRRPPQGYGC] harboring the membrane-translocating and nuclear import sequences [underlined] of HIV-1 tat protein to inhibit the cyclin-dependent kinase inhibitor, p21(WAF-1/Cip-1) (p21) and differentially sensitize MDA-MB-468 and MCF-7 human breast cancer (BC) cells to the antiproliferative effects of treatments that induce or do not induce p21. BC cells were treated with increasing concentrations of epidermal growth factor (EGF; 0.5-10 nM), the topoisomerase I inhibitor, camptothecin (CPT; 0.1-4 muM), or increasing doses of gamma-radiation (2-20 Gy). Western blot was used to evaluate p21 expression. The effect of treatment on cell cycle distribution was studied. Growth inhibition was measured by the WST-1 assay. Expression of p21 was increased in MDA-MB-468 cells treated with EGF or CPT but not by gamma-irradiation. MCF-7 cells exhibited p21 upregulation following exposure to CPT and gamma-radiation but not EGF. EGF caused cell cycle arrest in G(1) phase for MDA-MB-468 cells. CPT caused G(1)-phase arrest in MDA-MB-468 cells and prolonged S phase in MCF-7 cells. gamma-Radiation caused an increase in cells in G(2)/M phase for MDA-MB-468 and MCF-7. MDA-MB-468 cells were growth-inhibited by EGF, CPT, and gamma-radiation. MCF-7 cells were growth-stimulated by EGF and inhibited by CPT and gamma-radiation. Combining EGF with tat-anti-p21 immunoconjugates (ICs) amplified the growth-inhibitory effect on MDA-MB-468 cells 1.2-fold to 2.3-fold, but had no effect on the growth stimulation of MCF-7 cells by EGF. Tat-anti-p21 ICs sensitized MCF-7 cells 1.4-fold to gamma-radiation but had no effect on the growth of gamma-irradiated MDA-MB-468 cells. Tat-anti-p21 ICs sensitized both MDA-MB-468 and MCF-7 cells 1.7-fold to CPT. We conclude that tat-anti-p21 ICs are promising sensitizers for cytotoxic cancer therapies and that their sensitization is dependent on treatment-related p21 expression. This general approach could potentially be extended to other growth-regulatory molecules that are associated with tumor growth and progression.     

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.