The effects of TGF-α and 17β-estradiol on polyphosphoinositide metabolism in MCF-7 breast cancer cells

Summary The mechanism by which transforming growth factor-alpha (TGF-) stimulates breast cancer cell proliferation is largely unknown. Furthermore, its potential role as an autocrine effector of estradiol-17 (E₂)-stimulated growth of hormone-dependent mammary tumors remains controversial. Transient changes in phosphatidylinositol (PI) turnover have been demonstrated in several tissues in response to growth factors. In these experiments, we tested the effects of TGF- and E₂ on PI metabolism in three MCF-7 breast cancer cell sublines (MCF-7B, MCF-7I, and MCF-7J). Although TGF- was mitogenic in MCF-7I and MCF-7J cells, PI hydrolysis was stimulated by the growth factor only in the MCF-7I cells. In addition, the TGF- effect was relatively modest, ranging from 23% to 42%. E₂ effects on PI turnover were tested in the MCF-7B cells, which were the most sensitive to the proliferative effect of the hormone. E₂ did not stimulate PI hydrolysis, whether or not the cells were labelled in the presence of the hormone. On the other hand, E₂ did seem to stimulatede novo synthesis of phosphatidylinositol and induce activation of PI kinases. These results demonstrate that TGF--stimulated PI hydrolysis is modest and cell type dependent. At least under certain conditions, PI metabolism is not involved in the proliferative effects of TGF- (MCF-7J) or E₂ (MCF-7B). The role of increased PI synthesis in E₂-stimulated MCF-7 cell growth remains to be established.This work is supported by a grant from the National Cancer Institute, POI CA40011.     

Polyamine involvement in the secretion and action of TGF-α in hormone sensitive human breast cancer cells in culture

These experiments were designed to test polyamine (PA)* involvement in the secretion and action of transforming growth factor (TGF-) in hormone responsive MCF-7 breast cancer cells in liquid culture. At the same time, we evaluated the influence of culture conditions (with serum vs. serum depleted) and subclonality of MCF-7 cells on PA involvement in estrogen (E₂) and TGF- stimulated cell proliferation. Despite inducing a profound suppression of cellular PA levels and inhibiting basal and E₂-stimulated growth, administration of the PA synthesis inhibitor -difluoromethylornithine (DFMO) did not influence either basal or E₂-induced TGF- secretion. In the same experiments, on the other hand, addition of DFMO completely blocked the growth stimulatory effect of exogenous TGF-. However, when the culture conditions were changed to serum-free medium, TGF- and E₂-induced cell proliferation was affected modestly or not at all by DFMO administration, despite similar suppression of cellular ornithine decarboxylase (ODC) activity and PA levels. In addition, different clones of MCF-7 cells differed in their sensitivity to the antiproliferative effect of DFMO as well as in basal levels of ODC activity and PA. We conclude that PAs are not involved in basal or E₂-stimulated TGF- secretion in MCF-7 breast cancer cells. On the other hand, PAs do seem to be important mediators of TGF- and E₂-induced breast cancer cell proliferation, though the degree of such involvement appears to be influenced by serum factors and clonal variability of MCF-7 cells.     

Growth factor involvement in the multihormonal regulation of MCF-7 breast cancer cell growth in soft agar

Summary The hormone dependency of the MCF-7 breast cancer cell line, while extensively tested in liquid culture, has not been previously evaluated under conditions of anchorage-independent growth in serum-free media. Using the soft agar clonogenic assay, we demonstrate that physiologically relevant concentrations of estradiol (E₂), progesterone (Pg), and prolactin (PRL) similarly stimulated MCF-7 cell colony formation in the absence of serum. Addition of an anti-insulin-like growth factor-I (IGF-I) antibody inhibited E₂- and Pg-stimulated growth, while PRL action was not affected. Similar results were obtained with an anti-IGF-I receptor antibody, except that its inhibitory effect on Pg-induced colony formation was modest and not statistically significant. Administration of either an anti-transforming growth factor- (TGF-) antibody or an anti-epidermal growth factor (EGF) receptor antibody similarly inhibited E₂-stimulated MCF-7 cell growth in soft agar, while neither antibody influenced Pg or PRL effects. Addition of TGF-₁, -₂, -₃ similarly suppressed MCF-7 cell colony formation in a dose dependent manner to a degree comparable to that observed with 4-OH-tamoxifen (4-OH-T). Furthermore, the growth inhibitory effect of 4-OH-T was completely reversed by an anti-TGF- antibody. We conclude that IGFs and TGF- are important mediators of E₂-stimulated MCF-7 cell growth in soft agar. IGFs may also be playing a role in Pg action, while neither growth factor is involved in PRL-stimulated colony formation. Finally, TGF- appears to be an important mediator of antiestrogen-induced inhibition of tumor growth.Abbreviations IGF insulin-like growth factor - TGF transforming growth factor - E₂ estradiol-17 - Pg progesterone - PRL prolactin - 4-OH-T 4-hydroxy-tamoxifen - IMEM improved minimal essential medium     

Regulation of estrogen sulfotransferase by estrogen in MCF-7 human mammary cancer cells

Summary The importance of the steroid hormone microenvironment within cells is now recognised in studies on endocrine-related neoplasms such as breast cancer. This focuses attention on ezymes which control the intracellular levels of estradiol-17 (E₂). One such enzyme, estrogen sulfotransferase, which converts E₂ to inactive E₂-3 sulfate, has now been shown to be regulated by estrogen in MCF-7 human mammary cancer cells. Hydroxysteroid sulfotransferase, which sulfurylates the adrenal-derived estrogen 5-androstene-3,17-diol, is also under estrogen control. Evidence is provided which shows that one function of these enzymes may involve elimination of estrogen from the cell following processing of the ligand-charged estrogen receptor (ER).     

Transformation of estrone and estradiol in hormone-dependent and hormone-independent human breast cancer cells

Summary Using different hormone-dependent (MCF-7, T-47D) and hormone-independent (MDA-MB-231, Hs-578S, MDA-MB-436) human breast cancer cells, the interconversion estrone (E₁) estradiol (E₂) was explored. The data show very clearly that in the hormone-dependent cells the tendency is to form E₂ after incubation with E₁, whereas after incubation with E₂ most of this estrogen remains unchanged. In the hormone-independent cells, in contrast most of E₁ remains E₁, while E₂ is converted into E₁. The tendency of the reductive oxidative direction is supported by the analysis of estrogens in the culture medium. To explore the possible action of different drugs on the 17-hydroxysteroid dehydrogenase (17-HSD) activity, it was observed that the potent antiestrogen ICI 164,384 inhibits the conversion of E₁ to E₂, while a lesser effect is observed with Danazol and only weak inhibition is obtained with the progestagen Promegestone (R-5020). It is concluded that the orientation of 17-HSD activity for the interconversion E₁ E₂ in hormone-dependent and -independent cells is related to the hormonal status of the cells.     

Acquired antiestrogen resistance in MCF-7 human breast cancer sublines is not accomplished by altered expression of receptors in the ErbB-family

Development of acquired resistance against antiestrogen treatment is a serious problem in human breast cancer, and knowledge of alterations resulting in resistance is important for selection of further treatment. To mimic the clinical situation we have established a series of MCF-7 human breast cancer cell lines by long term treatment with the antiestrogens tamoxifen, ICI 164,384, and ICI 182,780. Common for these cell lines is a decreased expression of the estrogen receptor (ER). In human breast cancer, lack of response to endocrine therapy is often associated with decreased expression of the estrogen receptor and increased expression of epidermal growth factor receptor (EGFR) and/or HER-2/neu (ErbB-2). Our antiestrogen resistant cell lines did not express altered levels of EGFR, HER-2/neu, ErbB-3, or ErbB-4. Estrogen and antiestrogen regulation of HER-2/neu expression was essentially similar in parent and resistant MCF-7 cells. Treatment with antibodies to HER-2/neu (Herceptin^TM) did not affect growth of MCF-7 cells or resistant cells, indicating that in this in vitro model system, acquired antiestrogen resistance does not emerge from activation of the HER-2/neu signaling pathway. In MCF-7 cells transfected with HER-2/neu and/or ErbB-3, overexpression alone did not result in resistance. However, addition of heregulinl-1 abolished the inhibitory activity of ICI 182,780 on both vector and HER-2/neu/ErbB-3 transfected MCF-7 cells, demonstrating that activation of the HER-2/neu receptor signaling pathway can override the growth inhibitory effect of ICI 182,780.     

Human alpha – Fetoprotein peptides bind estrogen receptor and estradiol, and suppress breast cancer

Alpha-fetoprotein (AFP) is a transporter of various serum ligands and regulator of cellular growth during pregnancy. Estrogens modify AFP to exhibit growth suppressive properties. We recently synthesized a peptide (P149) from human AFP that suppresses the growth of mouse uterus and MCF-7 breast cancer cells. Here it is shown that molar excess treatment of native AFP with estradiol-17 (E₂) exposes the P149 site on AFP. The anti-estrogenic and anti-tumor activities of AFP-peptides were tested in vivo in the immature mouse uterine assay and mammary tumor (6WI-101)-induced ascites assay, and in vitro in a cytostatic assay using five different human breast tumor cell lines. AFP-peptide P149, and fragments of P149, P149A and P149C but not P149B, suppressed the growth in both in vivo assays. P149 also suppressed the in vitro growth of MCF-7, MDA-MB-231, MDA-MB435 breast cancer cells by more than 75%. P149 and P149A bound the estrogen receptor- (ER) with low affinities compared to E₂ and tamoxifen, while P149B bound ³H-E₂ with 10⁵ fold less affinity compared to ER. The recent epidemiologic observation that high AFP levels in young pregnant women reduce their subsequent risk of postmenopausal breast cancer may be related to the growth suppressive property of AFP with the exposed P149 epitope.     

Effect of ligands of nuclear hormone receptors on sodium/iodide symporter expression and activity in breast cancer cells

Iodide uptake by normal and cancerous thyroid cells is an active process mediated by the sodium/iodide symporter (NIS). Using quantitative real-time RT-PCR, we found that all 22 fresh human breast cancer samples had very low NIS expression similar to levels in untreated MCF-7 breast cancer cells. 9-cis retinoic acid (9-cis RA), a ligand for both retinoic acid receptor (RAR)/retinoic X receptor (RXR) heterodimers as well as RXR/RXR homodimers, markedly induced NIS mRNA expression in MCF-7 breast cancer cells in a dose- and time-dependent fashion, with maximal levels occurring at 12 h. All-trans retinoic acid, ATRA, a RAR specific ligand had a similar potency. Among eight breast cancer cell lines, three out of four estrogen receptor (ER)-positive and zero of four ER-negative cell lines responded to 9-cis RA by increasing their expression of NIS. Combining a RAR with a RXR selective ligand enhanced both NIS mRNA expression and iodide uptake in MCF-7 cells. Similarly, a ligand for proliferator-activated receptor (PPAR) when combined with 9-cis RA synergistically increased both NIS mRNA levels and iodide uptake in these MCF-7 cells. The iodide uptake was blocked by KClO₄. In conclusions, these findings suggest that selected combinations of NHR ligands should be examined in a limited trial to determine if their administration to patients allows the use of radioactive iodine for diagnosis and possibly treatment of metastatic breast cancer.     

Involvement of breast epithelial-stromal interactions in the regulation of protein tyrosine phosphatase-γ (PTPγ) mRNA expression by estrogenically active agents

Background. Protein tyrosine phosphatase (PTP) has been implicated as a tumor suppressor gene in kidney and lung cancers. Our previous results indicate that estradiol-17 (E₂)-induced suppression of PTP may play a role in mammary tumorigenesis. Zeranol (Z), a nonsteroidal growth promoter with estrogenic activity that is used by the US meat industry, induces estrogenic responses in primary cultured breast cells and breast cancer cell lines. Methods. PTP mRNA expression in human breast tissues and cells isolated from surgical specimens of mammoplasty and breast cancer patients were detected and quantified by RT-PCR. Immunohistochemical staining was used to localize PTP in human breast tissues. Breast epithelial and stromal cells were isolated and co-cultured to determine the involvement of cell–cell interaction in the regulation of PTP mRNA expression by E₂ and Z. Results. PTP mRNA expression was lower in cancerous than in normal breast tissues. Both E₂ and Z suppressed PTP mRNA levels in cultured normal breast tissues by 80%, but had a lesser effect in cultured epithelial cells isolated from normal breast tissues. In the co-culture system, both E₂ and Z suppressed PTP mRNA to a greater degree in epithelial cells than in stromal cells. In whole breast tissues, PTP was immunolocalized to the epithelium. Treatment with E₂ or Z diminished PTP staining indicating reductions in PTP at the protein level. Conclusions. The results indicate that both E₂ and Z regulate PTP expression in human breast and that epithelial–stromal cells interaction is important in the regulation of PTP expression by estrogenically active agents.     

Autocrine and paracrine growth regulation of human breast cancer

Summary We consider the hypothesis that estrogen control of hormone dependent breast cancer is mediated by autocrine and paracrine growth factors secreted by the breast cancer cells themselves. Though we show direct, unmediated effects of estrogen on specific cell functions, we also provide evidence that human breast cancer cells secrete a collection of growth factors (IGF-I, TGF, TGF, a PDGF-like competency factor, and at least one new epithelial colony stimulating factor). Some of these are estrogen-regulated in hormone dependent cells, and are constitutively increased in cells which acquire independence either spontaneously or byras transfection. Collectively, the secreted growth factors are capable of promoting tumor formation by MCF-7 cells in nude mice, though not to the same extent as estrogens. There would seem to be potential for clinical intervention in the autocrine and paracrine control of breast cancer cells, including some cells which are no longer dependent on estrogens.     

Role of transforming growth factor beta in the growth inhibition of human breast cancer cells by basic fibroblast growth factor

Recent studies from our laboratory have revealed that basic fibroblast growth factor (bFGF) selectively inhibits the proliferation of human MCF-7 breast cancer cells. It has also been shown to enhance cis-platinum-induced apoptosis, decrease levels of the anti-apoptotic gene product bcl-2, and increase levels of the cyclin-dependent protein kinase inhibitor p21/WAF1/Cip1. Transforming growth factor beta-1 (TGF₁), a cell growth regulator has been found to have an inhibitory effect on breast cancer cells. The aim of the present study was to evaluate the possible role of TGF₁ in the antiproliferative effects of bFGF in MCF-7 breast cancer cells. We found that exogenous, as well as endogenous (overexpressed) bFGF increased TGF₁ mRNA expression in the cells and enhanced the secretion of TGF₁ into culture medium. However, exogenous addition of TGF₁ neither led to a decrease in bcl-2 nor induced an increase in the levels of p21/WAF1/Cip1 and neutralizing antibodies to TGF₁, did not reverse bFGF-induced G₁ arrest nor the increase in p21/WAF1/Cip1 level. In contrast, antisense oligonucleotides to TGF₁ abrogated the antiproliferative effects and inhibited the induction of p21/WAF1/Cip1 by bFGF in MCF-7 cells. These data suggest that the anti-proliferative effects of bFGF in human MCF-7 breast cancer cells are mediated by endogenous TGF₁, while exogenous TGF₁ does not mimic all the effects of bFGF on these breast cancer cells. These findings provide an important basis for further investigations into the autocrine and paracrine processes that control the growth of breast cancer cells.     

A novel approach of targeted ablation of mammary carcinoma cells through luteinizing hormone receptors using Hecate-CGbeta conjugate

Recent studies have shown that human and animal mammary gland carcinoma cell line express luteinizing hormone receptors (LHRs). We have examined the cytotoxic effect of Hecate-CG conjugate, that is, fusion of a lytic peptide (Hecate) and a 15-amino acid fragment of the CG-chain in vitro. To test the hypothesis that the Hecate-CG conjugate selectively abolishes cells possessing LHR, estrogen dependent and independent human breast cancer cell lines (MCF-7; MDA-MB-231) and a mouse Leydig tumor cell line (BLT-1) were treated in vitro with Hecate-CG conjugate and Hecate alone. Cytotoxic effects of the Hecate-CG conjugate and the Hecate alone was measured by lactate dehydrogenase (LDH) release immediately after treatment. We observed that the Hecate-CG conjugate selectively, in dose-dependent manner destroys cells possessing LHR in lower concentrations of preparate comparing to the Hecate alone and that the cytotoxic effect is strongly correlated with the number of LHR. Using Western blot analysis we characterized the LHR on membranes of MDA-MB-231, MCF-7 and BLT-1 tumor cell lines. In addition, we showed the evaluation of inhibition potential of the Hecate-CG conjugate to LHR. At a concentration of 33 µM the conjugate inhibited (50%; IC₅₀) the binding of CG to LHR.We suggest further development of this novel approach for the treatment of breast cancer by the Hecate-CG for in vivo trials.     

Effects of secretory products of breast cancer cells on osteoblast-like cells

Summary The pathogenesis of breast cancer-induced osteolysis remains largely unknown. To evaluate the potential role of osteoblasts as target cells during this process, we incubated SaOS-2 human osteoblast-like cells (OBL) with culture media conditioned by proliferative (PM, Proliferation Media) or confluent (CfM, Confluence Media) MCF-7 human breast cancer cells. CfM decreased the growth of OBL by 26% (P < 0.01) while PM was without significant effect on this parameter. In contrast, both PM and CfM obtained from MCF-7 cultures increased the cyclic AMP (cAMP) response of OBL to the osteolytic agents PTH (10^–8 M) and PTH-related peptide (PTHrP, 10^–8 M) by a factor of about 3 (P < 0.001), and to prostaglandin E₂ (PGE₂, 10^–6 M) by a factor of about 2 (P < 0.01). No significant modulation of OBL growth or sensitivity to PTH, PTHrP, or PGE2 was induced by media obtained from HBL-100 non-malignant immortalized breast epithelial cell cultures. 17-estradiol (E₂, 10^–8 M) and the antiestrogen tamoxifen (Tam, 10^–7 M) added for 48 h to MCF-7 cultures before collecting conditioned media attenuated and potentiated, respectively, the PM- but not the CfM-induced increase in the response of OBL to PTH or PTHrP. Along the same line, the addition to MCF-7 conditioned media of a polyclonal anti-transforming growth factor- (TGF-) antibody attenuated by about 25% (P < 0.01) the PM-induced increase in OBL response to PTH and PTHrP while abrogating the modulatory effects of E₂ and Tam on that response. Together, our results indicate that MCF-7 breast cancer cells secrete factors which inhibit the growth of OBL and increase their sensitivity to various osteolytic agents. TGF- was only partly responsible for these effects, and accounts for their modulation by E₂ and Tam. The identification of other osteoblast-modulatory factor(s) should contribute to a better understanding and treatment of breast cancer-induced osteolysis.     

Role of transforming growth factor-α-related peptides in the autocrine/paracrine control of experimental breast cancer growthin vitro by estradiol,prolactin, and progesterone

Summary We have recently suggested that estradiol (E₂), prolactin (oPrl), and progesterone (Pg) support the growth of the hormone-responsive N-nitrosomethylurea (NMU) rat mammary tumor in soft agar through autocrine/paracrine mechanisms. To gain insight into the nature of these hormonally regulated growth factors, we tested the ability of two monoclonal antibodies (MAb-425 and 528) directed against the epidermal growth factor receptor (EGF-R) to inhibit the colony-stimulating effects of conditioned media (CM) obtained from E₂, oPrl, and Pg-treated NMU rat mammary tumors. Since both MAbs are specific for human EGF-R, MCF-7 breast cancer cells grown in soft agar in the absence of serum were used as our indicator system. Both MAb-425 and 528 totally abolished the colony-stimulating effect of genuine EGF, while having no agonistic/antagonistic action when added alone. Both MAb-425 and 528 markedly inhibited the colony-stimulating effect of rat mammary tumor E₂-CM in a dose-dependent fashion. MAb-425 was also found to inhibit the growth-promoting action of Pg-CM, although this effect appeared to be somewhat less consistent and pronounced than that observed with E₂-CM. In contrast, the colony-stimulating effect of Prl-CM was only rarely and, usually, modestly affected by the addition of either MAb-425 or 528. Our data suggest that in the NMU mammary tumor grown in soft agar, EGF/TGF-related peptides are produced upon exposure to E₂ and possibly Pg but only rarely following Prl administration. The possible role of these growth factors as mediators of hormonal effects in our experimental system remains to be established.     

Effects of human breast fibroblasts on growth and 17β-estradiol dehydrogenase activity of MCF-7 cells in culture

Summary The effect of conditioned medium from human breast fibroblasts on growth and 17-estradiol dehydrogenase (E2DH) activity of the MCF-7 human breast cancer cell line has been investigated. Fibroblasts were derived from normal and tumorous (benign and malignant) breast tissue. Conditioned medium from normal derived fibroblasts inhibited the growth of MCF-7 cells, the effect being statistically significant by day 3 of culture. In contrast, conditioned medium from benign and malignant derived fibroblasts significantly enhanced the growth of MCF-7 cells by day 9 of culture. When added to MCF-7 cells for three days, conditioned medium from all three types of fibroblasts increased E2DH activity in the reductive direction (estrone (E1) estradiol (E2)) 2–3 fold. There was little or no effect on the oxidative direction (E2 E1). After 12 days, enzyme activity in the reductive direction was still markedly increased, the effect being greatest in conditioned medium from fibroblasts derived from malignant breast tissue, and least in conditioned medium from fibroblasts derived from benign breast tumors. These results demonstrate that human breast fibroblasts may have paracrine influences on neighbouring epithelial cellsin vivo.     

The T61 human breast cancer xenograft: An experimental model of estrogen therapy of breast cancer

Summary Endocrine therapy is one of the principal treatment modalities of breast cancer, both in an adjuvant setting and in advanced disease. The T61 breast cancer xenograft described here provides an experimental model of the effects of estrogen treatment at a molecular level. T61 is an estrogen receptor positive tumor which was originally derived from a T1N0M0 invasive ductal cancer and has been carried as a serially transplanted xenograft in nude mice. T61 is a hormone sensitive tumor whose growth is suppressed by both estrogen and tamoxifen, in contrast to other estrogen receptor positive tumors such as MCF-7 which are stimulated by estrogen.Molecular studies have demonstrated that T61 expresses easily detectable levels of mRNA for a number of peptide growth factors, including transforming growth factor alpha (TGF-) and insulin-like growth factors I and II (IGF-I and IGF-II), but not transforming growth factor beta-1 (TGF-1). Of these, IGF-II is the only peptide whose expression is altered by endocrine therapy. Treatment of T61-bearing nude mice with physiologic doses of estrogen is accompanied by loss of IGF-II mRNA expression within 24 hours, and rapid regression of tumor. T61 tumor growth is also inhibited in animals treated with a monoclonal antibody which blocks binding of ligand to the IGF-I receptor, which mediates the mitogenic signal of bound IGF-II through autophosphorylation of its intracellular tyrosine kinase domain.These results demonstrate the utility of the T61 model in the study of the molecular mechanism of estrogen therapy in breast cancer, and suggest that in this system, modulation of a specific growth factor (IGF-II) by endocrine therapy can have profound effects on tumor growth.     

Urinary 2-hydroxyestrone/16alpha-hydroxyestrone ratio and family history of breast cancer in premenopausal women

It has been hypothesized that the ratio of urinary 2-hydroxyestrone to 16-hydroxyestrone (2-OHE₁/16-OHE₁ represents a biomarker for breast cancer risk; the lower the ratio the higher the risk. We obtained early morning urine samples from 70 'high risk' premenopausal women who had a first degree family history of breast cancer and 27 'low risk' women with no such history. Five estrogen metabolites in urine were determined: 2-OHE₁, 16-OHE₁, estrone (E₁), estradiol (E₂), and estriol (E₃) conjugates. We compared geometric mean levels of each metabolite adjusted for age and weight. 'High risk' women did not have elevated levels of any of these metabolites. Instead, we observed decrements of 3–27% in women with a family history of breast cancer compared with women without such history, this difference was statistically significant for E₂, 2- OHE₁, and 16-OHE₁. The ratio of 2-OHE₁/16-OHE₁ was identical in women with and without a family history of breast cancer. These results were unchanged, when additionally adjusted for recent intake of alcohol or cruciferous vegetables. Our data suggest that among premenopausal women, family history is not associated with higher urinary estrogen levels or a lower ratio of urinary 2-OHE₁/16-OHE₁.     

Inhibitory effects of medroxyprogesterone acetate (MPA) and the pure antiestrogen EM-219 on estrone (E1)-stimulated growth of dimethylbenz(a)anthracene (DMBA)-induced mammary carcinoma in the rat

Summary Estrogens are well known to play a predominant role in promoting the growth of DMBA-induced mammary tumors in the rat. Estrone (E₁), a steroid having weak estrogenic activity, is one of most important estrogens in post-menopausal women, where it is converted into the potent estrogen estradiol (E₂) by 17-hydroxysteroid dehydrogenase (17-HSD) in many peripheral tissues, including the mammary gland. In this report, we have studied the effect of a new antiestrogen (EM-219) (N-butyl, N-methyl-11-(3, 17-dihydroxy-17-ethinylestra-135(10), 14-tetraen-7-yl) undecanamide) on E₁-stimulated growth of DMBA-induced mammary tumors and compared its effect with that of medroxyprogesterone acetate (MPA) alone or in combination. After 18 days, ovariectomy (OVX) reduced total tumor area to 29.6 ± 7.1% of the original size, while E₁ (1.0 µg, twice daily) caused a 139 ± 21% increase in tumor size in OVX animals. MPA (1.5 mg, twice daily) partially reversed the stimulatory effect of E₁ to 66.0 ± 9.0%, while the antiestrogen EM-219 (40 µg, twice daily) decreased tumor size to 70.0 ± 10%. Combination of these two compounds led to a further inhibition of tumor size to 30.7 ± 7.4% of the value found in OVX animals treated with E₁. Tumor E₂ levels decreased from 1688 ± 155 pmoles/kg tissue in OVX animals receiving E₁ to 709 ± 92, 1347 ± 98, and 184 ± 11 pmoles/kg tissue in MPA-, EM-219-, and MPA + EM-219-treated OVX-E₁ animals, respectively. Treatment of OVX animals with E₁ increased by 69% the reductive activity of 17-hydroxysteroid dehydrogenase (17-HSD) while MPA abolished completely this effect of E₁. In the oxidative direction, treatment with E₁, E₁ + MPA, or E₁ + EM-219 had minimal or no significant effect on the activity of 17-HSD (vs OVX), while the combined treatment with MPA + EM-219 induced a 2-fold increase in 17-HSD activity, thus leading to an increased conversion of E₂ into E₁. The present data show that combination of the pure antiestrogen EM-219 with MPA exerts a greater reduction in DMBA-induced mammary tumor growth and intratumoral E₂ levels stimulated by E₁ than either compound used alone. This interactive effect of the antiestrogen and MPA could at least partially be related to the increased inactivation of E₂ into E₁. The present data suggest that such a combination could be a useful approach for the treatment of breast cancer, especially in post-menopausal women.     

Combined effects of 1,25-dihydroxyvitamin D3 and tamoxifen on the growth of MCF-7 and ZR-75-1 human breast cancer cells

Summary In the present study we assessed the effect of combined treatment with 1,25-dihydroxyvitamin D₃ (1,25-(OH)₂D₃) and tamoxifen (TAM) on the growth of estrogen-responsive (MCF-7) and estrogen-dependent (ZR-75-1) human breast cancer cells. Both basal and 17-estradiol (17-E₂)-stimulated growth were studied. 1,25-(OH)₂D₃ (10^–10 – 10^–7 M) time- and dose-dependently inhibited basal growth of MCF-7 cells, with growth arrest at 10^–7 M. Also, 17-E₂-stimulated growth of MCF-7 and ZR-75-1 cells was inhibited by 1,25-(OH)₂D₃ in a time- and dose-dependent manner. TAM inhibited 17-E₂-stimulated growth of both cell lines and at high concentration (10^–6 M) it also inhibited basal growth of MCF-7 cells. 10^–6 M TAM together with 1,25-(OH)₂D₃ resulted in a further inhibition of basal (MCF-7 cells) as well as 17-E₂-stimulated proliferation (MCF-7 and ZR-75-1 cells) compared to the inhibition by these agents alone. TAM in combination with 10^–7 M 1,25-(OH)₂D₃ resulted in growth arrest of 17-E₂-stimulated growth of MCF-7 cells. The inhibition of basal and 17-E₂-stimulated growth of MCF-7 cells was additive at early time points (4 days), but less than additive at later time points (8–10 days). It was demonstrated that with co-treatment of MCF-7 cells an equipotent inhibition of basal growth could be reached with lower concentrations of 1,25-(OH)₂D₃, compared to treatment with 1,25-(OH)₂D₃ alone. Studies with low concentrations (< 10^–7 M) of TAM revealed a partial estrogenic effect, i.e. stimulation of MCF-7 proliferation in the absence of 17-E₂. This effect, which may resemble TAM-induced tumor flare, was completely prevented by co-treatment with a low concentration of 1,25-(OH)₂D₃ (10^–9 M). Together, these results demonstrate the potent inhibition of breast cancer cell proliferation by 1,25-(OH)₂D₃ combined with TAM and indicate a potential benefit of combining these agents for the treatment of breast cancer.     

Aromatase and prostaglandin inter-relationships in breast adipose tissue: significance for breast cancer development

The role of prostaglandins in the development of breast cancer is a topic of growing interest. Stimulation of aromatase expression within the breast has been proposed as a mechanism whereby prostaglandins could influence breast cancer growth. In the present study, we show that PGE₂ is a powerful stimulator of aromatase expression in human breast adipose stromal cells. Moreover, TNF, which also stimulates aromatase expression in breast adipose stromal cells, acts to increase the secretion of PGE₂ by these cells, as well as the expression of COX 2 and PGE synthase, but not that of COX 1. On the other hand, class I cytokines had no effect, either by themselves or in the presence of estradiol. These factors had little influence on secretion of 15-deoxy-^12,14-PGJ₂, which is inhibitory of aromatase expression by breast adipose stromal cells. These results further substantiate an important role for PGE₂ to stimulate estrogen biosynthesis within the local environment of the breast.