MCF-7 Human Breast Tumor Research Articles

Estrogen's Effects on Mitochondrial Gene Expression: Mechanisms and Potential Contributions to Estrogen Carcinogenesis

Estrogen receptor (ER)alpha and ERbeta are localized in the nucleus and involved in the regulation of nuclear estrogen-responsive genes by 17beta estradiol (E2). In addition, recently others have shown that upon E2 binding, ERalpha localizes to the plasma membrane and initiates mitogen-activated protein kinase (MAPK)-mediated signal transduction. Previously, we reported that in liver, cultured rat hepatocytes and human HepG2 cells, estrogen treatment enhanced mitochondrial DNA (mtDNA)-encoded gene transcript levels. These effects were blocked by a specific antiestrogen, suggesting a role for the ER. Others have reported the presence of putative estrogen-responsive elements in mtDNA. These observations suggested the hypothesis that the ER localized in mitochondria and functioned directly to enhance the levels of mtDNA-encoded transcripts, analogous to what has been observed for the glucocorticoid hormone receptor. Using Western blot analysis, confocal immunofluorescence, immunogold electron microscopy, and gel electrophoresis mobility shift assays, we have demonstrated the estrogen-dependent presence of ERbeta and ERalpha within mitochondria of HepG2 and MCF-7 human breast tumor cells. Together, these results suggest that the ERs may act as transcription factors directly involved in the regulation by E2 of mtDNA transcription.

Attenuation of telomerase activity by hammerhead ribozyme targeting human telomerase RNA induces growth retardation and apoptosis in human breast tumor cells

Ribozyme possesses specific endoribonuclease activity and catalyzes the hydrolysis of specific phosphodiester bonds, which results in the cleavage of target RNA sequences. Here, we evaluated the ability of hammerhead ribozymes targeting human telomerase RNA (hTR) to inhibit the catalytic activity of telomerase and the proliferation of cancer cells. Hammerhead ribozymes were designed against 7 NUX sequences located in open loops of the hTR secondary structure. We verified the ribozyme specificity by in vitro cleavage assay by using a synthetic RNA substrate. Subsequently, we introduced ribozyme expression vector into human breast tumor MCF-7 cells and assessed the biologic effects of ribozyme. Hammerhead ribozyme R1 targeting the template region of hTR efficiently cleaved hTR in vitro, and stable transfectants of this ribozyme induced the degradation of target hTR RNA and attenuated telomerase activity in MCF-7 cells. Moreover, the ribozyme R1 transfectant displayed a significant telomere shortening and a lower proliferation rate than parental cells. Clones with reduced proliferation capacity showed enlarged senescence-like shapes or highly differentiated dendritic morphologies of apoptosis. In conclusion, the inhibition of telomerase activity by hammerhead ribozyme targeting the template region of the hTR presents a promising strategy for inhibiting the growth of human breast cancer cells.

Benzoxepin-Derived Estrogen Receptor Modulators: A Novel Molecular Scaffold for the Estrogen Receptor

We present and examine the efficacy of a novel benzoxepin-based scaffold for modulation of the human estrogen receptor. Receptor tolerance of this new molecular scaffold is examined through presentation of experimentally determined antiproliferative effects on human MCF-7 breast tumor cells and measured binding affinities. The effect of functional group substitution on the benzoxepin scaffold is explored through a brief computational structure-activity relationship investigation with molecular simulation.

Combined effect of epirubicin and lymphokine-activated killer cells on the resistant human breast cancer cells

Accumulating evidence suggests the concept that epirubicin and lymphokine-activated killer (LAK) cells cytotoxicity may be mediated by free radicals generation and P-glycoprotein-positive (Pg-p+) cancer cells are more sensitive for LAK cells than their drug-sensitive parental lines. We tested this hypothesis further by exposing drug-sensitive (WT) and epirubicin-resistant MCF-7 human breast tumor cells to epirubicin and LAK cells. Subsequently, we monitored cell proliferation as a measure of cytotoxicity. The cytotoxicity of epirubicin, LAK, and LAK + epirubicin (1/10 of IC50) was evaluated in 400-fold epirubicin resistant MCF-7 EPI(R) (P-glycoprotein overexpressing) and drug-sensitive MCF-7 WT cells. IC50 values were measured using the MTT cytotoxicity test. The MCF-7 EPI(R) cells exhibited an increased susceptibility to LAK cells than did the MCF-7 WT cells. P-gp+ MCF-7 EPI(R) cells were lysed by human LAK cells to a greater extend than were their drug-sensitive counterparts. LAK + epirubicin combined treatment increased susceptibility of MCF-7 WT and MCF-7 EPI(R) cells to LAK cells cytotoxicity. For both cell lines, cytotoxicity was dependent upon the concentration of the epirubicin and effector cell/target cell (E/T) ratio. The resistance of MCF-7 EPI(R) cells to epirubicin appears to be associated with a developed tolerance to superoxide, most likely because of a tree-fold increase in superoxide dismutase (SOD) activity and 13-fold augmented selenium dependent glutathione peroxidase (GSH-Px) activity. Acting in concert, these two enzymes would decrease the formation of hydroxyl radical from reduced molecular oxygen intermediates. The addition of SOD decreased cytotoxicity of epirubicin and LAK cells. Taken together, these observations support the role of oxygen radicals in the cytotoxicity mechanism of epirubicin and suggest further that the development of resistance to this drug by the MCF-7 EPI(R) tumor cells may have a component linked to oxygen free radicals. It is proposed that production of reactive oxygen species by the treatment of epirubicin and LAK cells can cause cytotoxicity of MCF-7 WT and MCF-7 EPI(R) cells. SOD, catalase, GSH-Px, GST (glutathione S-transferase), and GSH (reduced glutathione) must be considered as part of the intracellular antioxidant defense mechanism of MCF-7 WT and MCF-7 EPI(R) cells against reactive oxygen species.

Differentiation-inducing quinolines as experimental breast cancer agents in the MCF-7 human breast cancer cell model

The purpose of this work is to develop agents for cancer differentiation therapy. We showed that five antiproliferative quinoline compounds in the National Cancer Institute database stimulated cell differentiation at growth inhibitory concentrations (3–14 μM) in MCF-7 human breast tumor cells in vitro. The differentiation-inducing quinolines caused lipid droplet accumulation, a phenotypic marker of differentiation, loss of Ki67 antigen expression, a cell cycle marker indicative of exit into G 0, and reduced protein levels of the G 1 − S transcription factor, E2F1. The antimalarial quinolines, chloroquine, hydroxychloroquine and quinidine had similar effects in MCF-7 cells, but were 3–10 times less potent than the NSC compounds. NSC3852 and NSC86371 inhibited histone deacetylase (HDAC) activity in vitro and caused DNA damage and apoptosis in MCF-7 cells, consistent with their differentiation and antiproliferative activities. However, the HDAC assay results showed that for other compounds, direct HDAC enzyme inhibition was not required for differentiation activity. E2F1 protein was suppressed by all differentiation quinolines, but not by non-differentiating analogs, quinoline and primaquine. At equivalent antiproliferative concentrations, NSC69603 caused the greatest decrease in E2F1 protein (90%) followed by antimalarials quinidine and hydroxychloroquine. NSC69603 did not cause DNA damage. The other NSC compounds caused DNA damage and apoptosis and reduced E2F1 levels. The physicochemical properties of NSC3852, NSC69603, NSC86371, and NSC305819 predicted they are drug candidates suitable for development as experimental breast tumor cell differentiation agents. We conclude DNA damage and reductions in E2F1 protein are mechanistically important to the differentiation and antiproliferative activities of these quinoline drug candidates.

RNA expression of human telomerase subunits TR and TERT is differentially affected by melatonin receptor agonists in the MCF-7 tumor cell line

The RNA expression levels of human catalytic subunit (TERT) and the RNA subunit (TR) of telomerase were analysed after treatment with the agonists of the membrane receptor (S 20098) and the nuclear receptor (CGP 52608) for melatonin in the MCF-7 human breast tumor cell line. Neither membrane nor nuclear signalling affected the RNA steady-state levels of the TR subunit of telomerase. On the contrary, we observed a significant decreased in the RNA levels of TERT after treatment with CGP 52608 while S 20098 produced a significant increase in the RNA levels of TERT. These results support a cross-talk between membrane and nuclear melatonin signalling and provide new data on the hormonal regulation of telomerase function.

Invasive and angiogenic phenotype of MCF-7 human breast tumor cells expressing human cyclooxygenase-2

To evaluate the direct effect of human cyclooxygenase-2 (hCox-2) on human breast tumor cell proliferation, invasion, and angiogenesis, hCox-2 cDNA was transfected into slow growing, non-metastatic MCF-7 human breast tumor cells that express low levels of Cox-2. Two stable transfectant clones, designated MCF-7/hCox-2 clones 8 and 10, had significantly decreased (P<0.05) doubling time, with two-fold greater number of cells during exponential growth compared to the MCF-7/vector control. Proliferation of both of the MCF-7/hCox-2 clones was significantly inhibited in a time- and dose-dependent manner by celecoxib. The MCF-7/hCox-2 clones 8 and 10 formed larger and greater numbers of colonies in soft agar than the MCF-7/vector control, with a corresponding increased invasion across an artificial Matrigel basement membrane in response to recombinant human epidermal growth factor (hEGF). The MCF-7/hCox-2 clones 8 and 10 had higher mRNA levels of two splice variants of vascular endothelial growth factor (VEGF), V145 and V165. These results demonstrate that hCox-2 directly increases breast tumor cell proliferation, stimulates invasion across a basement membrane, and induces synthesis of specific heparin binding splice variants of VEGF.

Invasive and angiogenic phenotype of MCF-7 human breast tumor cells expressing human cyclooxygenase-2

To evaluate the direct effect of human cyclooxygenase-2 (hCox-2) on human breast tumor cell proliferation, invasion, and angiogenesis, hCox-2 cDNA was transfected into slow growing, non-metastatic MCF-7 human breast tumor cells that express low levels of Cox-2. Two stable transfectant clones, designated MCF-7/hCox-2 clones 8 and 10, had significantly decreased ( P<0.05) doubling time, with two-fold greater number of cells during exponential growth compared to the MCF-7/vector control. Proliferation of both of the MCF-7/hCox-2 clones was significantly inhibited in a time- and dose-dependent manner by celecoxib. The MCF-7/hCox-2 clones 8 and 10 formed larger and greater numbers of colonies in soft agar than the MCF-7/vector control, with a corresponding increased invasion across an artificial Matrigel basement membrane in response to recombinant human epidermal growth factor (hEGF). The MCF-7/hCox-2 clones 8 and 10 had higher mRNA levels of two splice variants of vascular endothelial growth factor (VEGF), V 145 and V 165. These results demonstrate that hCox-2 directly increases breast tumor cell proliferation, stimulates invasion across a basement membrane, and induces synthesis of specific heparin binding splice variants of VEGF.

2-Methoxyestradiol Exhibits a Biphasic Effect on VEGF-A in Tumor Cells and Upregulation Is Mediated Through ER-α: A Possible Signaling Pathway Associated with the Impact of 2-ME2 on Proliferative Cells

2-Methoxyestradiol (2-ME2) was reported to elicit both stimulation and inhibition of tumor angiogenesis and growth depending on the dosage used. However, the mechanism(s) of the biphasic action of 2-ME2 has been elusive. Here we describe a regulatory role of vascular endothelial growth factor-A (VEGF-A) in the biphasic effects on estrogen receptor (ER)+ GH3 rat pituitary tumor cells and MCF-7 human breast tumor cells depending on the dosage of 2-ME2 used. We observed that acute exposure to 2-ME2, irrespective of dosage, did not alter cellular proliferation, but enhanced the VEGF-A mRNA level. As the treatment duration increased, biphasic effect was elicited. A concentration of 1 μM 2-ME2 increased both cell proliferation and VEGF-A levels in these cells, whereas higher doses exhibited reversed impact. A low dose of 2-ME2 also increased the VEGF-A mRNA expression in ER-α-transfected human mammary epithelial cells (HMECs). The effect was reversed in ER- cells. The enhanced expression of VEGF-A mRNA could be blocked by the pure estrogen antagonist, ICI 182,780, reveal that the upregulation of VEGF-A expression by 2-ME2 is mediated through ER-α. Furthermore, the biphasic effect of 2-ME2 on cell proliferation can be modulated by administrating VEGF-A antibodies or VEGF-A proteins. Studies also demonstrate that the VEGF-A protein, induced by 2-ME2, is functionally active and upregulates the proliferation of adjacent endothelial cells.

Determination of chemotherapeutic activity in vivo by luminescent imaging of luciferase-transfected human tumors.

Human DU-145 prostate and MCF-7 breast tumor cell lines were stably transfected with plasmid pcDNA3.1-Luc expressing firefly luciferase. Studies were performed with the transfected cell lines to evaluate luminescent imaging for measuring the efficacy of anti-cancer agents. In vitro experiments demonstrated a dose response of both cell lines to topotecan (Hycamtin) with an IC50 of 0.013 microM for MCF-7 Luc cells and 0.002 microM for DU-145 Luc cells. In vivo imaging experiments were performed using athymic nude mice inoculated i.p. with 5 x 10(6) MCF-7 cells or s.c. with 5 x 10(6) DU-145 cells and then treated with topotecan at 2.5 mg/kg body weight. Tumor progression and regression were monitored for 27 days. Animals inoculated s.c. with DU-145 Luc cells and then treated with topotecan demonstrated significant tumor growth and regression as measured with calipers and luminescent imaging. High correlation was observed between caliper and imaging results. The correlation coefficient was 0.75 for the control untreated group and 0.93 for the topotecan-treated group. Similarly, tumor progression and regression were measurable using luminescent imaging for untreated and topotecan-treated mice inoculated i.p. with MCF-7 Luc cells. These data indicate that luminescent imaging is a useful tool for evaluating anti-cancer drugs in vivo and may prove to be particularly useful for the development of novel agents. Luminescent imaging could also be used to locate and harvest residual tumors in drug-treated animals in order to study mechanisms of drug resistance.

Imaging of luciferase and GFP-transfected human tumours in nude mice.

Studies were performed to compare green fluorescent protein (GFP)-transfected and fi re fl y luciferase (Luc)-transfected MCF-7 human breast tumour cells both in vitro and in vivo. For in vitro studies, cells were serially diluted in 96-well microplates and analysed using a NightOwl LB 981 Molecular Light Imager and a Victor multilabel reader. For in vivo studies, nude mice were injected either intraperitoneally, intravenously or subcutaneously with transfected cells and then imaged using the NightOwl Imager after intraperitoneal injection of d-luciferin for Luc tumours, or excitation at 470 nm for GFP tumours. In vitro imaging studies revealed that both GFP and Luc transfectants were quantifiable. However, the Luc-transfected cells were detectable at a significantly lower concentration compared to GFP transfectants. In vivo studies demonstrated that GFP-transfected tumours were detectable as subcutaneous and intraperitoneal tumours but not as deep tissue lesions, whereas Luc-transfected tumours were detectable as subcutaneous and intraperitoneal tumours and as deep tissue lesions resulting from intraperitoneal or intravenous inoculation. These findings demonstrate that GFP-transfected cells may be useful for imaging studies of superficial tumours where both excitation and emission wavelengths are able to penetrate tissues, whereas luciferase-transfected cells appear superior for imaging studies of primary and metastatic tumours in distant sites and deep tissues.

Salicylate enhances necrosis and apoptosis mediated by the mitochondrial permeability transition.

Onset of the mitochondrial permeability transition (MPT) causes both necrotic and apoptotic cell death in cultured hepatocytes. Salicylate lowers the threshold for onset of the MPT. In this study, our aim was to determine whether nontoxic concentrations of salicylate potentiate MPT-mediated cell killing. In necrotic killing models to rat hepatocytes, salicylate (1 mM) enhanced calcium ionophore (Br-A23187)- and tert-butylhydroperoxide (t-BuOOH)-induced cell death, which was blocked or delayed by cyclosporin A (CsA, 2 microM), a specific inhibitor of the MPT. In hepatocyte apoptosis induced by tumor necrosis factor-alpha (TNF-alpha), salicylate accelerated cell killing after low-dose TNF-alpha (1 ng/ml), which by itself induced little apoptosis. Salicylate enhancement of apoptosis was associated with onset of the MPT and accelerated caspase 3 activation. Salicylate also augmented killing of MCF-7 human breast tumor cells by etoposide and PLC/PRF/5 human hepatoma cells by tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). In conclusion, salicylate potentiates both necrotic and apoptotic cell killing by promoting onset of the MPT. Enhancement by salicylate of MPT-dependent apoptosis may play a role in protection by aspirin and other nonsteroidal anti-inflammatory drugs against colon, lung, and breast cancer.

Synthesis and secretion of Muc4/sialomucin complex: implication of intracellular proteolysis.

Muc4/sialomucin complex (SMC) is a heterodimeric glycoprotein complex implicated in epithelial protection and overexpressed in some tumours. It is encoded by a single gene, and the two subunits are produced by proteolytic cleavage at a time before substantial O-glycosylation, near the time of transit from the endoplasmic reticulum to the Golgi. Although Muc4/SMC is translated as a membrane protein, it is produced as a soluble form in many epithelia. Transfection experiments using Cos-7, HBL-100 human epithelial, MCF-7 human breast tumour and HC11 mouse mammary cell lines showed that soluble rat Muc4/SMC is produced by a proteolytic cleavage mechanism and released by secretion from multiple cell lines, including both human and mouse normal epithelial cells and tumour cells. Similar transfection experiments demonstrated the same mechanism for the human analogue MUC4. Gel electrophoresis analyses of deglycosylated membrane and soluble transmembrane subunits and of the membrane-associated cleavage fragment indicated a released cleavage product of 25 kDa, resulting from cleavage between two epidermal growth factor-like domains. Further evidence for this site was obtained from deletion mutants removing this region of the protein, which blocked secretion. Finally, pulse-chase analyses of Muc4/SMC biosynthesis indicated no kinetic difference between the timing of the cleavage to release the soluble form and that to produce the two subunits, indicating that the soluble form is created early in transit to the cell surface. These studies provide the first clear evidence that membrane mucins can be released from cells by an intracellular proteolytic mechanism that leads to secretion of the soluble form of the mucin.

5-Fluoroindole-3-acetic acid: a prodrug activated by a peroxidase with potential for use in targeted cancer therapy

Indole-3-acetic acid and some derivatives are oxidized by horseradish peroxidase, forming a radical-cation that rapidly fragments (eliminating CO 2) to form cytotoxic products. No toxicity is seen when either indole-3-acetic acid or horseradish peroxidase is incubated alone at concentrations that together form potent cytotoxins. Unexpectedly, 5-fluoroindole-3-acetic acid, which is oxidized by horseradish peroxidase compound I 10-fold more slowly than indole-3-acetic acid, is much more cytotoxic towards V79 hamster fibroblasts in the presence of peroxidase than the unsubstituted indole. The fluorinated prodrug/peroxidase combination also shows potent cytotoxic activity in human and rodent tumor cell lines. Cytotoxicity is thought to arise in part from the formation of 3-methylene-2-oxindole (or analogues) that can conjugate with thiols and probably DNA or other biological nucleophiles. Levels of the fluorinated prodrug in the murine carcinoma NT after intraperitoneal administration of 50 mg/kg were about 200 μM. Although these were 4–5-fold lower than plasma levels (which reached 1 mM), the integrated area under the concentration/time curve in tumors over 2 hr was ∼20 mM min, almost double the exposure needed to achieve ∼90–99% cell kill in human MCF7 breast or HT29 colon tumor cell lines and CaNT murine cells in vitro, although the human bladder T24 carcinoma cell line was more resistant. The high cytotoxicity of 5-fluoroindole-3-acetic acid after oxidative activation suggests its further evaluation as a prodrug for targeted cancer therapy involving antibody-, polymer-, or gene-directed delivery of horseradish peroxidase or similar activating enzymes.

Forkhead Homologue in Rhabdomyosarcoma Functions as a Bifunctional Nuclear Receptor-interacting Protein with Both Coactivator and Corepressor Functions

In a search for novel transcriptional intermediary factors for the estrogen receptor (ER), we used the ligand-binding domain and hinge region of ER as bait in a yeast two-hybrid screen of a cDNA library derived from tamoxifen-resistant MCF-7 human breast tumors from an in vivo athymic nude mouse model. Here we report the isolation and characterization of the forkhead homologue in rhabdomyosarcoma (FKHR), a recently described member of the hepatocyte nuclear factor 3/forkhead homeotic gene family, as a nuclear hormone receptor (NR) intermediary protein. FKHR interacts with both steroid and nonsteroid NRs, although the effect of ligand on this interaction varies by receptor type. The interaction of FKHR with ER is enhanced by estrogen, whereas its interaction with thyroid hormone receptor and retinoic acid receptor is ligand-independent. In addition, FKHR differentially regulates the transactivation mediated by different NRs. Transient transfection of FKHR into mammalian cells dramatically represses transcription mediated by the ER, glucocorticoid receptor, and progesterone receptor. In contrast, FKHR stimulates rather than represses retinoic acid receptor- and thyroid hormone receptor-mediated transactivation. Most intriguingly, overexpression of FKHR dramatically inhibits the proliferation of ER-dependent MCF-7 breast cancer cells. Therefore, FKHR represents a bifunctional NR intermediary protein that can act as either a coactivator or corepressor, depending on the receptor type.

Certain Styrene Oligomers Have Proliferative Activity on MCF-7 Human Breast Tumor Cells and Binding Affinity for Human Estrogen Receptor a

Certain Styrene Oligomers Have Proliferative Activity on MCF-7 Human Breast Tumor Cells and Binding Affinity for Human Estrogen Receptor a

Certain styrene oligomers have proliferative activity on MCF-7 human breast tumor cells and binding affinity for human estrogen receptor.

To examine the estrogenic activities of styrene oligomers, we carried out cell proliferation assays with estrogen-sensitive MCF-7 cells and competitive binding assays to human estrogen receptor [alpha] (hER[alpha]). The styrene oligomers tested were 1,3-diphenyl propane (SD-1), 2,4-diphenyl-1-butene (SD-2), cis-1,2-diphenyl cyclobutane (SD-3), trans-1,2-diphenyl cyclobutane (SD-4), 2,4,6-triphenyl-1-hexene (ST-1), 1a-phenyl-4a-(1'-phenylethyl)tetralin (ST-2), 1a-phenyl-4e-(1'-phenylethyl)tetralin (ST-3), 1e-phenyl-4a-(1'-phenylethyl)tetralin (ST-4), 1e-phenyl-4e-(1'-phenylethyl)tetralin (ST-5), 1e,3e,5a-triphenylcyclohexane (ST-6), and 1e,3e,5e-triphenylcyclohexane (ST-7). In the MCF-7 cell proliferation assay, styrene trimers (ST-1, ST-3, ST-4, and ST-5) had the highest proliferative activities of the compounds tested. The relative potency of these chemicals was 0.0002-0.0015%, which was comparable with that of bisphenol A (0.0001-0.0025%), and their relative proliferative effect was 51-104%. Styrene dimers (SD-3 and SD-4) also significantly increased the cell yields. However, SD-1, SD-2, ST-2, ST-6, and ST-7 had insignificant proliferative activities. The competitive binding assay revealed the binding affinity of some styrene oligomers for hER[alpha]. The order of their binding potency for hER[alpha] was as follows: ST-4 > ST-2 > ST-3 > ST-5 > ST-1 > SD-3 > SD-4 > SD-2 > SD-1. ST-6 and ST-7 did not appear to bind to hER[alpha]. The present studies indicate that styrene dimers SD-3 and SD-4 and styrene trimers ST-1, ST-3, ST-4, and ST-5 have estrogenic activity on MCF-7 cells and binding affinity for hER[alpha]. These compounds might be endocrine disrupters.

Flexible Estrogen Receptor Modulators: Design, Synthesis, and Antagonistic Effects in Human MCF-7 Breast Cancer Cells

Although many series of estrogen receptor antagonists continue to be produced, the majority are direct structural analogues of existing modulators. To examine the tolerance of the estrogen receptor toward flexible ligands, a series of novel flexible estrogen receptor antagonists were prepared and their antiproliferative effects on human MCF-7 breast tumor cells investigated. Each of these compounds deviated from the traditional triphenylethylene backbone associated with common tamoxifen analogues through the introduction of a flexible methylene (benzylic) spacing group between one of the aryl rings and the ethylene group and through variations in the basic side chain moiety. The compounds prepared, when assayed in conjunction with a tamoxifen standard, demonstrated high potency in antiproliferative assays against an MCF-7 human breast cancer cell line with low cytotoxicity and high binding affinity. A computational study was undertaken to investigate the compounds' potential interactions with specific residues within the human estrogen receptor alpha ligand-binding domain (ER-LBD), predicting these compounds bind in an antiestrogenic fashion within the ER-LBD and interact with those important residues previously identified in the structures of ER-LBD agonist/antagonist cocrystals. These compounds further illustrate the eclectic nature of the estrogen receptor in terms of ligand flexibility tolerance.

Contraceptive progestins. Various 11-substituents combined with four 17-substituents: 17α-ethynyl, five- and six-membered spiromethylene ethers or six-membered spiromethylene lactones

Norethisterone (NET) is a 19-nortestosterone derivative with progestagenic and some androgenic activity, which was used in the first generation of contraceptives. NET was succeeded by levonorgestrel (LNG) and later on by desogestrel (DSG) and gestodene (GSD). Although these latter two progestins had increased potency, there was still androgenicity with gestodene and to a lesser extent with desogestrel. New progestins were synthesized in order to further enhance progestagenic and to reduce androgenic activity. Four different chemical moieties were introduced in position 17 of 19-nortestosterone, viz. 17α-ethynyl, five- and six-membered spiromethylene ethers, and a six-membered-spiromethylene lactone. In combination with these structures seven different substituents were added at position 11, i.e. methylene, methyl, ethyl, ethenyl, ethynyl, 2-propenyl and 1-propynyl. All substituents except for methylene occupied the 11β-position. All these 32 compounds were synthesized and analysed in vitro and in vivo against etonogestrel (ETG, 3-keto-desogestrel), the biologically active metabolite of desogestrel. Their relative binding potency to progesterone (PR), androgen (AR) and estrogen (ER) receptors were determined in cell lysates of human breast tumor MCF-7 cells and to glucocorticoid (GR) receptors in that of human leukemic IM-9 cells. Moreover, their relative agonistic activities were assessed in Chinese hamster ovary cell-based transactivation assays. All in vivo activities were determined in McPhail (progestagenic), ovulation inhibition (progestagenic and estrogenic), Hershberger (androgenic), hormone screening (glucocorticoid and estrogen) and Allen-Doisy (estrogenic) tests after oral and for the McPhail test also after subcutaneous administration. The progestagenic binding and transactivation potencies of all compounds in the three 17-spiro series were higher than those of the corresponding analogues in the 17α-ethynyl series. None of the compounds showed estrogenic or clear androgenic binding and transactivation potential except for a six-membered-spiromethylene lactone with a propynyl group. This compound showed strong androgenic binding. The glucocorticoid binding and transactivation were very low for the compounds with the 17α-ethynyl and the five-membered-spiromethylene ether groups, whereas both six-membered-spiro series showed, clearly with methyl and ethynyl substituents, and less pronounced with methylene and ethenyl, higher binding and transactivation values. For the 17α-ethynyl series, the McPhail test showed high potencies with methylene, methyl and ethenyl substituents after oral treatment or with propenyl after subcutaneous administration. The introduction of the spiro substituents in position 17 led to high potencies for other 11-substituents as well. Besides methyl, also ethyl, ethynyl and propynyl were potent substituents. With ovulation inhibition tests, the ethyl, ethenyl and ethynyl substituents were the more potent compounds in all four series. However, compounds with methyl or ethynyl additions appeared to be glucocorticoidal in the hormone screening test irrespective of the 17-substituent, while with the three spiro series even methylene and ethenyl groups became active. Androgenicity was only observed at dose levels at or above 5 mg/kg, which is 2.5-fold weaker than ETG. Moreover, estrogenicity appeared negligible with the three spiro series, while with the 17α-ethynyl series methyl, ethyl, ethenyl and ethynyl substituents, a very high estrogenic potential was assessed. Based on the high efficacy and low side-effects, the following compounds show a high selectivity: 17α-ethynyl with ethyl, ethenyl and 2-propenyl substituents, six-membered spiromethylene ether with ethyl and six-membered-spiromethylene lactone with ethyl, 2-propenyl or 1-propynyl substituents. These compounds have relatively high binding and transactivation values for PR, and have high biopotencies in the McPhail and ovulation inhibition tests, while showing very weak androgenic and glucocorticoid activities. These compounds may be very useful for contraception for either oral and/or subcutaneous administration.

Effect of ploidy, recruitment, environmental factors, and tamoxifen treatment on the expression of sigma-2 receptors in proliferating and quiescent tumour cells.

Recently, we demonstrated that sigma-2 receptors may have the potential to be a biomarker of tumour cell proliferation (Mach et al (1997) Cancer Res57: 156–161). If sigma-2 receptors were a biomarker of tumour cell proliferation, they would be amenable to detection by non-invasive imaging procedures, thus eliminating many of the problems associated with the flow cytometric measures of tumour cell proliferation presently used in the clinic. To be a good biomarker of tumour cell proliferation, the expression of sigma-2 receptors must be essentially independent of many of the biological, physiological, and/or environmental properties that are found in solid tumours. In the investigation reported here, the mouse mammary adenocarcinoma lines, 66 (diploid) and 67 (aneuploid), 9L rat brain tumour cells, and MCF-7 human breast tumour cells were used to study the extent and kinetics of expression of sigma-2 receptors in proliferative (P) and quiescent (Q) tumour cells as a function of species, cell type, ploidy, pH, nutrient depletion, metabolic state, recruitment from the Q-cell compartment to the P-cell compartment, and treatment with tamoxifen. In these experiments, the expression of sigma-2 receptors solely reflected the proliferative status of the tumour cells. None of the biological, physiological, or environmental properties that were investigated had a measurable effect on the expression of sigma-2 receptors in these model systems. Consequently, these data suggest that the proliferative status of tumours and normal tissues can be non-invasively assessed using radiolabelled ligands that selectively bind sigma-2 receptors. © 1999 Cancer Research Campaign