Hormone-dependent Human Breast Cancer Cell Research Articles

Secosteroid thiosemicarbazides and secosteroid–1,2,4-triazoles as antiproliferative agents targeting breast cancer cells: Synthesis and biological evaluation

A convenient and selective approach to 13,17-secoestra-1,3,5(10)-trien-17-oic acid [N’-arylcarbothioamido]hydrazides and hybrid molecules containing secosteroid and 1,2,4-triazole fragments was disclosed and these novel types of secosteroids were screened for cytotoxicity against hormone-dependent human breast cancer cell line MCF-7. Most of secosteroid–1,2,4-triazole hybrids showed significant cytotoxic effect comparable or superior to that of the reference drug cisplatin. Hit secosteroid–1,2,4-triazole hybrids 4b and 4h were characterized by high cytotoxicity and good selectivity towards MCF-7 breast cancer cells. PARP cleavage (marker of apoptosis) and ERα and cyclin D1 downregulation were discovered in MCF-7 cells treated with lead secosteroid–1,2,4-triazole hybrid 4b. The synthesized secosteroids may be considered as new promising anticancer agents.

Synthesis and Characterization of Folic Acid-Functionalized DPLA-co-PEG Nanomicelles for the Targeted Delivery of Letrozole.

An effective treatment for hormone-dependent breast cancer is chemotherapy using cytotoxic agents such as letrozole (LTZ). However, most anticancer drugs, including LTZ, are classified as class IV biopharmaceuticals, which are associated with low water solubility, poor bioavailability, and significant toxicity. As a result, developing a targeted delivery system for LTZ is critical for overcoming these challenges and limitations. Here, biodegradable LTZ-loaded nanocarriers were synthesized by solvent emulsification evaporation using nanomicelles prepared with dodecanol-polylactic acid-co-polyethylene glycol (DPLA-co-PEG). Furthermore, cancer cell-targeting folic acid (FA) was conjugated into the nanomicelles to achieve a more effective and safer cancer treatment. During our investigation, DPLA-co-PEG and DPLA-co-PEG-FA displayed a uniform and spherical morphology. The average diameters of DPLA-co-PEG and DPLA-co-PEG-FA nanomicelles were 86.5 and 241.3 nm, respectively. Our preliminary data suggest that both nanoformulations were cytocompatible, with ≥90% cell viability across all concentrations tested. In addition, the amphiphilic nature of the nanomicelles led to high drug loading and dispersion in water, resulting in the extended release of LTZ for up to 50 h. According to the Higuchi model, nanomicelles functionalized with FA have a greater potential for the controlled delivery of LTZ into target cells. This model was confirmed experimentally, as LTZ-containing DPLA-co-PEG-FA was significantly and specifically more cytotoxic (up to 90% cell death) toward MCF-7 cells, a hormone-dependent human breast cancer cell line, when compared to free LTZ and LTZ-containing DPLA-co-PEG. Furthermore, a half-maximal inhibitory concentration (IC50) of 87 ± 1 nM was achieved when MCF-7 cells were exposed to LTZ-containing DPLA-co-PEG-FA, whereas higher doses of 125 ± 2 and 100 ± 2 nM were required for free LTZ and LTZ-containing DPLA-co-PEG, respectively. Collectively, DPLA-co-PEG-FA represents a promising nanosized drug delivery system to target controllably the delivery of drugs such as chemotherapeutics.

Secosteroidal hydrazides: Promising scaffolds for anti-breast cancer agents

A convenient and selective approach to 13,17-secoestra-1,3,5(10)-trien-17-oic acid hydrazides and their N’-(het)arylmethylene derivatives was disclosed and these novel types of secosteroids were screened for cytotoxicity against hormone-dependent human breast cancer cell line MCF-7. A number of 13,17-secoestra-1,3,5(10)-trien-17-oic acid [N’-(het)arylmethylene]hydrazides show significant cytotoxic effect comparable or superior to that for reference drug cisplatin. Compound 3l exhibits the highest activity with the IC50 value of about 2 μM and is 2.8 times more active than cisplatin. Hit 13,17-secoestra-1,3,5(10)-trien-17-oic acid [N’-(het)arylmethylene]hydrazides 3d, 3l and 3q are characterized by high cytotoxicity and good selectivity towards MCF-7 breast cancer cells. The synthesized secosteroids may be considered as new promising antitumor agents.

Selective synthesis of the two main progesterone metabolites, 3α-hydroxy-5α-pregnanolone (allopregnanolone) and 3α-hydroxypregn-4-en-20-one, and an assessment of their effect on proliferation of hormone-dependent human breast cancer cells

A directed synthesis of two progesterone metabolites, allopregnanolone and 3a-hydroxy-pregn-4-en-20-one, from Δ16-pregnanolone and progesterone, respectively, was carried out by a reduction of the carbonyl groups in positions 3 and subsequent inversion of the configuration of the resulting alcohols by the Mitsunobu reaction. The selectivity of the reduction of the conjugated carbonyl group in position 3 of progesterone with sodium borohydride in the presence of cerium(III) chloride (Luche reduction) was demonstrated. The ef ect of the obtained metabolites on the proliferation of breast cancer cells of the MCF-7 and T47D lines under normal and steroid-free conditions was studied. It is shown that the ef ect of these compounds on the proliferation depends on the presence of additional steroids in the culture medium. Metabolites exerted small cytostatic ef ects on the growth of the MCF-7 cells under standard conditions, while the transfer of the cells to a steroid-free medium weakened these cytotoxic ef ects. In the experiments with the T47D line cells, the cell growth was stimulated under both standard and steroid-free conditions. Allopregnanolone and progesterone stimulate the growth to a greater extent under steroid-free conditions than under standard ones.

Cholesterol biosynthesis inhibitor RO 48-8071 reduces progesterone receptor expression and inhibits progestin-dependent stem cell-like cell growth in hormone-dependent human breast cancer cells.

Clinical trials and studies have shown that postmenopausal women undergoing combination hormone replacement therapy containing estrogen and progestin have an increased risk of breast cancer compared with women taking estrogen or placebo alone. Using animal models, we have previously shown that synthetic progestins, including medroxyprogesterone acetate (MPA), which is widely used clinically, accelerate breast cancer tumor growth and promote metastasis. Furthermore, we have found that MPA elevates CD44 protein expression and aldehyde dehydrogenase (ALDH) activity, two markers of cancer stem cells (CSCs), and increases mammosphere formation, another hallmark of stem cells, in hormone-dependent T47-D human breast cancer cells. Herein, we show that RO 48-8071 (RO), an inhibitor of cholesterol synthesis, reduced MPA-induced CD44 protein expression in two hormone-dependent human breast cancer cell lines, T47-D and BT-474. Because we have previously shown that MPA induction of CD44 is progesterone receptor (PR) dependent, we examined RO’s effects on PR protein and mRNA expressions in T47-D cells. PR mRNA levels remained unchanged after RO treatment; however, RO significantly reduced the protein expression of both PR receptor isoforms, PR-A and PR-B. Using the proteasome inhibitor MG-132, we demonstrated that RO decreases PR protein expression in T47-D cells via the proteasomal degradation pathway. Importantly, treatment of T47-D cells with RO abolished MPA-induced mammosphere formation. Based on our observations, we contend that RO may represent a novel means of preventing MPA-induced CSC expansion. RO could be used clinically to both treat and prevent hormone-dependent breast cancers, which represent the majority of human breast cancers. RO may also have clinical utility in reducing resistance to antihormone therapy.

Design, synthesis, molecular docking, and biological studies of novel phytoestrogen-tanaproget hybrids

ABSTRACTA diverse range of novel and highly functionalized flavonoid-based tanaproget hybrids were synthesized and evaluated in vitro for their antimicrobial and antiproliferative activities. Novel products were synthesized in good yields (81–95%) under Pd-catalyzed reaction from bromo flavones and tanaproget boronic acids within 18–20 min at 60 °C. Bioassay results exhibited excellent activities against both hormone-dependent and hormone-independent human breast cancer cells (MCF-7, MDA-MB-231, DU-145, PC-3, and HeLa). Among them, compounds 4e, 9a, 9c, 9e, 9 g, 9 h, 9 m, and 9n displayed excellent activity. Compounds 4d, 4o, and 9o were found equally potent against C. albicans compared to fluconazole. Compound 5c showed better antibacterial activity against S. aureus. Compounds 5a, 9i, 9o, and 10c have shown admirable antibacterial activity against E. coli.

Cholesterol biosynthesis inhibitors as potent novel anti-cancer agents: suppression of hormone-dependent breast cancer by the oxidosqualene cyclase inhibitor RO 48-8071.

In most human breast cancers, tumor cell proliferation is estrogen dependent. Although hormone-responsive tumors initially respond to anti-estrogen therapies, most of them eventually develop resistance. Our goal was to identify alternative targets that might be regulated to control breast cancer progression. Sulforhodamine B assay was used to measure the viability of cultured human breast cancer cell lines exposed to various inhibitors. Protein expression in whole-cell extracts was determined by Western blotting. BT-474 tumor xenografts in nude mice were used for in vivo studies of tumor progression. RO 48-8071 ([4'-[6-(Allylmethylamino)hexyloxy]-4-bromo-2'-fluorobenzophenone fumarate]; RO), a small-molecule inhibitor of oxidosqualene cyclase (OSC, a key enzyme in cholesterol biosynthesis), potently reduced breast cancer cell viability. In vitro exposure of estrogen receptor (ER)-positive human breast cancer cells to pharmacological levels of RO or a dose close to the IC50 for OSC (nM) reduced cell viability. Administration of RO to mice with BT-474 tumor xenografts prevented tumor growth, with no apparent toxicity. RO degraded ERα while concomitantly inducing the anti-proliferative protein ERβ. Two other cholesterol-lowering drugs, Fluvastatin and Simvastatin, were less effective in reducing breast cancer cell viability and were found not to induce ERβ. ERβ inhibition or knockdown prevented RO-dependent loss of cell viability. Importantly, RO had no effect on the viability of normal human mammary cells. RO is a potent inhibitor of hormone-dependent human breast cancer cell proliferation. The anti-tumor properties of RO appear to be in part due to an off-target effect that increases the ratio of ERβ/ERα in breast cancer cells.

Abstract 449: Progesterone-induced immunosuppression thwarts immunosurveillance to tumors and promotes lung metastasis in a breast cancer model.

Abstract Exposure to sexual hormones is one of the strongest risk factors for breast cancer. Increased risk is entwined with a greater number of reproductive cycles and the use of progestin-containing hormone replacement therapy. In addition, the importance of progesterone in dampening immune response was illustrated in several models of infection and pregnancy. Based on the tolerogenic properties of progesterone (Pg) and its promoting role in breast cancer, in this work we investigated whether progesterone may thwart immunosurveillance and promote tumor metastasis by fostering an immunosuppressive microenvironment in breast cancer. Exogenously added Pg: (10−8M) or its synthetic analogue medroxiprogesterone acetate (MPA: 10−8M) induced the expression of the immunosuppressive protein galectin-1 (Gal1) in the hormone-dependent human breast cancer cell lines T47D and MCF-7 and in the mouse mammary adenocarcinoma C4HD. This effect was abrogated by pre-treatment with RU486 (10−7M) indicating that the progesterone receptor (PR) was involved. Interestingly, in vitro MPA treatment of human PBMC or mouse splenocytes controlled the expansion of regulatory T cells (Tregs) (CD4+CD25+Foxp3+) and skewed the balance toward a Th2-type cytokine profile. Furthermore, differentiation from naive T-cells to Tregs cells was enhanced in the presence of MPA, IL-2 and suboptimal concentrations of TGF-β. In addition, in vivo treatment with MPA (15mg 30-day release depot) of C4HD tumor-bearing mice increased both the expression of Gal1 and the frequency of Treg cells in tumor-draining lymph nodes and the tumor microenvironment. While the increase in Treg cell frequency was associated with an effect on Treg cell SMAD4 expression that potentiates TGF-β signalling, homing of Treg cells to tumor sites was supported through the induction of tumor-derived CCL22. Finally, we used a PR negative breast tumor model (4T1) to evaluate the effect of MPA on the tumor microenvironment and the consequences of this effect on tumor progression. In vivo treatment with MPA promoted lung metastasis by inducing an invasive phenotype in breast cancer cells. Co-culture of Treg cells differentiated in vivo in the presence of MPA with 4T1 cells promoted its invasive activity and modulate several genes associated with EMT, including SNAIL. In summary, these results demonstrate that progesterone hierarchically fosters an immunosuppressive tumor microenvironment that promotes metastasis by co-ordinately regulating Gal1 expression, stimulating the TGF-β pathway and augmenting the frequency of Treg cells. Our findings provide new insights into the role of progesterone in breast cancer as a crucial promoter of tumor-immune escape. Citation Format: Mariana Salatino, Tomás Dalotto, Diego O. Croci, Santiago P. Mendez Huergo, Sebastian L. Dergan Dylon, Juan P. Cerliani, Marta A. Toscano, Gabriel A. Rabinovich. Progesterone-induced immunosuppression thwarts immunosurveillance to tumors and promotes lung metastasis in a breast cancer model. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 449. doi:10.1158/1538-7445.AM2013-449

S3-4: ER Downregulation with Fulvestrant in Combination with pan-PI3K Inhibitor BKM120 Synergizes Against ER+/PI3K-Mutant Breast Cancer Xenografts In Vivo.

Abstract ER+ breast cancers typically respond to aromatase inhibitors (AIs), but a significant fraction exhibit de novo or acquired resistance. To discover mechanisms of resistance to AIs, we maintained four ER+, hormone-dependent human breast cancer cell lines in hormone-depleted conditions for several months until estrogen-independent populations emerged (termed long-term estrogen-deprived, LTED). A siRNA kinome screen identified 42 kinases that suppressed hormone-independent MCF-7/LTED cell growth ≥33% (p<0.05). Pathway analysis revealed phosphatidylinositol 3-kinase (PI3K) as a central hub which integrates signaling from, or emits signaling to many of these 42 kinases. Further, treatment with the PI3K/mTOR dual inhibitor BEZ235 or the pan-PI3K inhibitor BKM120 suppressed LTED cell growth, and prevented the emergence of hormone-independent cells. Activating mutations in PIK3CA, the gene encoding the p110α catalytic subunit of PI3K, are present in about 35% of ER+ breast cancers. We investigated whether PIK3CA mutations modify the tumor response to estrogen deprivation with letrozole in a pre-surgical clinical trial (NCT00651976). Twenty-one patients with stage I-II operable ER+/HER2− breast cancer were treated with letrozole for 10–21 days up to 24 h prior to surgery. Core tumor biopsies were obtained before treatment and at the time of surgical resection. Ki67 immunohistochemistry (IHC) was scored in pre- and post-letrozole specimens. The change in natural log of the Ki67 score between pre-and post-treatment samples was stratified by PIK3CA mutant vs. wildtype (WT) status. PIK3CA-mutant tumors exhibited a statistically lower reduction in Ki67 score compared to tumors with WT PIK3CA (p=0.03). These findings suggest that mutational activation of PI3K attenuates the response to estrogen deprivation. In some LTED cell lines, downregulation of ER with fulvestrant or siRNA inhibited estrogen-independent growth, suggesting that the unliganded ER can maintain proliferation of cells adapted to estrogen deprivation. Prior work suggested that PI3K-ER crosstalk promotes resistance to estrogen deprivation. However, inhibition of PI3K/AKT/TORC1 signaling did not alter ERS167 phosphorylation in LTED cells, and ER downregulation with fulvestrant did not inhibit PI3K/AKT, suggesting that PI3K and ER modulate non-overlapping pathways in cells adapted to estrogen deprivation. Treatment with BKM120 synergized with fulvestrant to inhibit the growth of 6/8 ER+ breast cancer cell lines in estrogen-depleted conditions. Treatment with single-agent fulvestrant or BKM120 in athymic mice devoid of estrogen supplementation partially inhibited the growth of ER+/PI3K-mutant MCF-7 xenografts, while the combination induced near-complete tumor regression. IHC analysis showed increased cleaved caspase-3+ cells in BKM120-treated tumors and decreased Ki67+ cells in fulvestrant-treated tumors, implying that the combination induced tumor regression by both inhibition of tumor cell proliferation and induction of apoptosis. These data suggest that upon progression on an aromatase inhibitor, patients with ER+/PI3K-mutant breast cancer would benefit from a combination of an ER downregulator and a PI3K pathway inhibitor. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr S3-4.

Resistance to Endocrine Therapy in Estrogen Receptor-Positive (ER+) Breast Cancer Is Dependent upon Phosphatidylinositol-3 Kinase (PI3K) Signaling.

Abstract ER+ breast cancers typically respond to treatment with aromatase inhibitors (AIs), but a significant fraction exhibit de novo or acquired resistance. To model AI resistance, we cultured four ER+, hormone-dependent human breast cancer cell lines under hormone-depleted conditions for several months until hormone-independent populations emerged (termed long-term estrogen-deprived, LTED). LTED cells outgrew parental counterparts under hormone-depleted conditions. While two LTED lines showed increased ER levels and response to estradiol, two LTED lines showed the opposite. Therefore, ER+ breast cancer cells did not consistently overcome hormone deprivation by increasing sensitivity to low estrogen levels.We analyzed protein lysate arrays with 625 antibodies against signaling proteins to discover common mechanisms of hormone-independent growth. All LTED lines showed increased phosphorylation of p70S6K, an mTOR substrate, compared to parental controls. An siRNA library screen targeting 779 kinases revealed that downregulation of kinases linked to the phosphatidylinositol-3 kinase (PI3K) signaling pathway (serum/glucocorticoid-regulated kinase 1; insulin receptor; p110α/PI3K) inhibited the growth of MCF-7/LTED but not parental MCF-7 cells under hormone-depleted conditions. Immunoblot analysis for P-AKT and P-p70S6K showed that PI3K/AKT/mTOR signaling was increased in all LTED lines. Pathway analysis of gene expression microarray data showed significant alteration of genes involved in insulin-like growth factor-I (IGF-I) signaling in 3/4 LTED lines. Receptor tyrosine kinase array analysis revealed increased activation of receptors for IGF-I (IGF-IR) and/or insulin (InsR) in 3/4 LTED lines. These receptors transduce signals to potently activate PI3K. Treatment with the IGF-IR/InsR kinase inhibitor AEW541 decreased P-AKT in 3/4 LTED lines. Treatment with inhibitors of IGF-IR/InsR (AEW541), PI3K/mTOR (BEZ235), or mTOR (RAD001) suppressed the monolayer and anchorage-independent growth of 3/4, 4/4, and 4/4 LTED lines, respectively, and prevented the emergence of hormone-independent cells. Treatment with PI3K and mTOR inhibitors induced apoptosis in 3/4 LTED lines. Finally, we analyzed levels of PI3K pathway markers (P-AKT, P-S6, P-GSK3α/β) in primary tumor lysates from 65 ER+ breast cancer patients using reverse-phase protein arrays. Hierarchical clustering yielded two groups of tumors with high or low PI3K activation. Following adjuvant anastrozole therapy, PI3K-high patients had significantly shorter disease-free survival compared to PI3K-low patients. With a median follow-up of 9.4 months, recurrence rates were 16% and 0%, respectively (p<0.05). These data suggest that upon the acquisition of hormone independence and resistance to endocrine therapy, ER+ breast cancer cells increase their dependence on the PI3K pathway. Thus, resistance to endocrine therapy may be abrogated by combination therapies targeting both the ER and PI3K pathways, which may sometimes be achieved by blocking IGF-IR/InsR signaling upstream of PI3K. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 403.

Re-activation of the p53 pathway inhibits in vivo and in vitro growth of hormone-dependent human breast cancer cells

Mutations in wild-type p53 (wtp53) protein lead to loss of its tumor suppressor function in breast cancer cells, facilitating uncontrolled tumor growth. Consequently, procedures to repair defective p53 functions in tumor cells are being actively pursued. We sought to determine whether expression of wtp53 protein, or conversion of endogenous mutant p53 (mtp53) into a functional p53 protein with small molecule PRIMA-1, can override the tumor-promoting effects of naturally occurring mtp53 protein in hormone-responsive T47-D human breast cancer cells. We show that transfection of wtp53 gene into T47-D cells suppresses their proliferation in regular media, and inhibits estrogen-dependent cell proliferation in media containing dextran-coated charcoal treated serum. Growth inhibition was not due to the absence of estrogen receptor-alpha or estrogen receptor-beta though receptor levels for estrogen receptor-alpha were drastically reduced in wtp53 expressing cells. Focused microarray analysis of wtp53 expressing cells revealed suppression of PCNA cell-cycle regulatory mRNA and protein. Wild-type p53 transfected T47-D cells also failed to grow in vivo in estrogen supplemented nude mice. Furthermore, xenografts obtained with parental T47-D cells expressing mtp53 grew poorly in nude mice treated with PRIMA-1. PRIMA-1 treated tumors exhibited a low proliferation index, even though mice were estrogen-supplemented. PRIMA-1 treatment of tumor cells suppressed VEGF and induced expression of estrogen receptor-beta though expression of estrogen receptor-alpha and progesterone receptors was unaffected. These data indicate that alteration of the p53 signal transduction pathway by re-expression of wtp53 protein in T47-D cells, or treatment of parental cells with PRIMA-1, can prevent in vivo and in vitro proliferation of T47-D breast cancer cells.

JNK pathway regulates estradiol-induced apoptosis in hormone-dependent human breast cancer cells

Estrogen is known to stimulate breast cancer development in humans. Ironically, high doses of estrogen can induce regression of hormone-dependent breast cancer in postmenopausal women. The mechanism by which estrogen induces tumour regression in breast cancer is still unknown. We found that under low growth-stimulated conditions, high concentrations of 17-beta-estradiol (estradiol) induces apoptosis and concomitantly increases phosphorylation of c-jun in estrogen receptor (ER)-positive breast cancer cell line, MCF-7, but not in ER-negative breast cancer cell line MDA-MB 231 suggesting an ER-mediated event. Interestingly, when the c-jun NH2-terminal kinase (JNK) signalling pathway was disrupted by the JNK inhibitor SP600125, the ability of estradiol to inhibit the growth of MCF-7 cells and to induce apoptosis was completely blocked. These data suggest that JNK plays a central role in mediating the anticancer effect of high concentrations of estradiol in MCF-7 cells. Our data showing the apoptotic effect of estradiol in low growth-stimulated conditions suggest potential implications for the pharmacological control of breast cancer with high dose estrogen in postmenopausal women. Furthermore, our results indicate that augmenting JNK activity could be an efficient novel approach for treating 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 Cα (PKCα) [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 PKCα, a retinoic acid-induced target gene, prevented the effects of RA on cell proliferation and EGF signaling. Constitutive expression of PKCα, 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 PKCα, prevent sustained growth factor signaling, uncoupling activated receptor tyrosine kinases and nuclear targets that are required for cell cycle progression.

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, 17β-hydroxysteroid dehydrogenase (17β-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 1S) 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 [ 3H]E 1S (5×10 −9 M) alone (control) or in the presence of E 2 (5×10 −10 to 5×10 −5 M) for 24 h at 37° 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: 5×10 −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 5×10 −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 1 [1]cited in abstract. and presented at the 83rd Annual Meeting of the Endocrine Society (Denver, USA, 20–23 June 2001 (abstract no. P2-615).

CSF-1 activates MAPK-dependent and p53-independent pathways to induce growth arrest of hormone-dependent human breast cancer cells.

The CSF-1 receptor (CSF-1R) is expressed in >50% of human breast cancers. To investigate the consequence of CSF-1R expression, hormone-dependent human breast cancer cell lines, MCF-7 and T-47D, were transfected with CSF-1R. Unexpectedly, CSF-1 substantially inhibited estradiol (E2) and insulin-dependent proliferation of MCF-7 transfectants (MCF-7fms) and prevented cyclin E/cdk2 and cyclin A/cdk2 activation, consistent with a G1 arrest. In contrast, CSF-1 increased DNA synthesis in T-47D transfectants (T-47Dfms) alone and with E2 or insulin. In response to CSF-1, there was a marked and sustained upregulation of the cyclin-dependent kinase inhibitor, p21Waf1/Cip1, in MCF-7fms but not T-47Dfms. CSF-1 also markedly upregulated cyclin D1 in MCF-7fms. The coordinate increase in cyclin D1 and p21 had the effect of decreasing the specific but not absolute activity of cyclin D1/cdk4. p53 was not involved since CSF-1 induction of p21 was unaffected by dominant-negative p53 expression. ERK activation by CSF-1 was robust and sustained in MCF-7fms and to a much lesser extent in T-47Dfms. Using pharmacological and transient transfection approaches, we showed that ERK activation was necessary and sufficient for p21 induction in MCF-7fms. Moreover, activated MEK inhibited E2-stimulated cdk2 activity. Our findings indicate that the consequence of CSF-1R-mediated signals in human breast cancer cells is dependent on the genetic background of the particular tumor.

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.

Steroid metabolism in the hormone dependent MCF-7 human breast carcinoma cell line and its two hormone resistant subpopulations [formula omitted] and [formula omitted

Androgen and estrogen metabolism was investigated in the hormone-dependent human breast cancer cell line MCF-7 and its two hormone-resistant sublines MCF-7 LCC1 and MCF-7 LCC2 . Using the product isolation method, the activity of aromatase, 5α-reductase, 3α β - hydroxysteroid oxidoreductase and 17β-hydroxysteroid oxidoreductase were investigated isolating the following steroids: estriol (E 3), estradiol (E 2), estrone (E 1), 3α β - androstanediol (A-diol), testosterone (T), dihydrotestosterone (DHT), androsterone (AND), androstenedion (4-AD) and androstanedione (A-dion). For all experiments, cells were preincubated with cortisol and subsequently incubated with [ 14C]T or [ 14C]4-AD as the substrate in medium without phenol red and with serum charcoal stripped of steroids. The results showed no aromatase activity in any of the cell lines under the experimental conditions used, and preincubation with cortisol had no effect on the enzyme activity. With [ 14C]T as the substrate, the metabolized level of DHT was very similar in the three cell lines, though MCF-7 LCC1 and MCF-7 LCC2 utilized the substrate to a much lesser extent. The amount of DHT and 4-AD produced were comparable in the two hormone-resistant cell lines, while the amount of 4-AD was significantly higher in MCF-7 cells. No differences in enzyme activity were found in the three cell lines when [ 14C]4-AD was used as the substrate. This study showed an altered androgen metabolism in the MCF-7 LCC1 and MCF-7 LCC2 sublines compared to the parent MCF-7. However, since treatment with DHT and T inhibited cell growth equally well in all three tumor cell lines, it is unlikely that the found differences in steroid metabolism was involved in the acquisition of the endocrine resistance of the two MCF-7 sublines.

Tumour necrosis factor and interferon are selectively cytostatic in vitro for hormone-dependent and hormone-independent human breast cancer cells.

Since experimental studies have shown that tumour necrosis factor-alpha (TNF-alpha) has potent anti-tumour activity that can be potentiated with cytokines, we tested the efficacy of TNF-alpha with interferon-gamma (IFN-gamma) on different human breast cancer cell lines, particularly comparing hormone-dependent and -independent phenotypes. TNF-alpha inhibited the growth of hormone-dependent human MCF-7, ZR-75-1 and T47-D breast cancer cells with a half maximal concentration of 0.25 nM. In contrast, the growth of hormone-independent cells MDA-MB-231 and HS578T was not affected by TNF-alpha alone, but a synergistic inhibition was observed when using IFN-gamma and TNF-alpha together. The mRNA for the proto-oncogene C-MYC, as an intracellular indicator of cell activation, was significantly increased in MCF-7 cells in the presence of TNF-alpha. In MDA-MB-231 cells this mRNA was increased only in the presence of both TNF-alpha and IFN-gamma, without a change in the number of surface TNF receptors. These findings indicate that TNF-alpha treatment in combination with IFN-gamma may provide a successful approach to overcome the cellular heterogeneity of advanced breast tumours.

C- myc gene chromatin of estrogen receptor positive and negative breast cancer cells

Expression of the c- myc protooncogene is estrogen regulated in estrogen receptor (ER) positive, hormone-dependent human breast cancer cells, but it is constitutively active in ER negative, hormone-independent breast cancer cells. To determine whether these differences are reflected in c- myc chromatin, DNase I hypersensitive sites (DHS) were mapped. Six DHS were detected in all cell lines studied, with DHS 3 2 being more prominent than DHS 3 1. The accessibility of DHS 2 was markedly greater in ER negative cells than in ER positive cells, and this relative accessibility remained unchanged when cells were grown in estrogen free medium. DHS 2, 3 1 and 3 2 map near the P0, P1 and P2 promoters, respectively. An analysis of promoter usage demonstrated that P2 was the preferred promoter. Thus, the differences in the accessibility of DHS 2 in c- myc chromatin of ER positive and negative cells likely reflects alterations in DNA-protein interactions in this region.

Comparative effect of embryonic mouse fibroblasts (Balb/c-3T3) on the proliferation of hormone-dependent (T-47D) and hormone-independent (MDA-MB-231) human breast cancer cell lines

The effects of cellular extracts (CE) and conditioned medium (CM) of embryonic mouse BALB/c-3T3 (clone A 31) cells on the proliferation and DNA content of hormone-dependent T-47D and hormone-independent MDA-MB-231 breast cancer cell lines were explored. After 6 days of culture, CE and CM provoke an intense proliferative effect in T-47D cells which correlates with DNA content. In contrast, in the MDA-MB-231 cells a significant inhibitory effect was observed. For both CE and CM the action was dose-dependent. In the T-47D cells, the CM can abolish the inhibitory effect provoked by the potent antiestrogen ICI 164,384. It is concluded that mouse embryonic BALB/c-3T3 cells contain factors which can stimulate or inhibit the growth of human mammary cancer cells.