piperidines and afimoxifene

The general pharmacology of tamoxifen in animals and man is reviewed with particular reference to the long-term adjuvant therapy of node-positive breast cancer. Rats with dimethylbenzanthracene (DMBA)-induced mammary carcinomata have been used extensively as a laboratory model to study hormone-dependent cancer. The administration of a 30-day course of tamoxifen (50 micrograms daily) starting 5, 15, 30, or 50 days after DMBA caused a delay in tumor appearance and decrease in the cumulative number of tumors that were induced by 200 days. Similarly, the administration of increasing doses of tamoxifen (0.2, 3, 50, and 800 micrograms daily) between 30 and 60 days after DMBA produced a dose-related delay in tumor appearance and a decrease in the cumulative number of tumors at 200 days. Since the tumors that were induced after tamoxifen still responded to ovariectomy, tamoxifen appears to act as an inhibitor of the tumor cell cycle rather than as a tumoricidal agent in this model. This principle was exemplified by comparing a short course (30 day) with a continuous course (170 day) of tamoxifen initiated 30 days after DMBA. The short course of therapy only delayed tumor appearance whereas continuous therapy maintained 90% of the animals in a tumor-free state. These data strongly support the use of long-term (up to five-year) adjuvant therapy with tamoxifen in patients as a suppressive therapy for hormone-sensitive metastases.

Topics: 9,10-Dimethyl-1,2-benzanthracene; Animals; Antineoplastic Combined Chemotherapy Protocols; Breast Neoplasms; Combined Modality Therapy; Disease Models, Animal; Dose-Response Relationship, Drug; Estrogen Antagonists; Female; Half-Life; Humans; Mammary Neoplasms, Experimental; Models, Biological; Piperidines; Pyrrolidines; Raloxifene Hydrochloride; Rats; Rats, Inbred Strains; Receptors, Estrogen; Tamoxifen; Thiophenes

The fallopian tube epithelium is one of the potential sources of high-grade serous ovarian cancer (HGSC). The use of estrogen only hormone replacement therapy increases ovarian cancer (OVCA) risk. Despite estrogen's influence in OVCA, selective estrogen receptor modulators (SERMs) typically demonstrate only a 20 % response rate. This low response could be due to a variety of factors including the loss of estrogen receptor signaling or the role of estrogen in different potential cell types of origin. The response of fallopian tube epithelium to SERMs is not known, and would be useful when determining therapeutic options for tumors arising from this cell type, such as HGSC.. Using normal murine derived oviductal epithelial cells (mouse equivalent to the fallopian tube) estrogen receptor expression was confirmed and interaction with its ligand, estradiol, triggered mRNA and protein induction of progesterone receptor (PR). The SERMs 4-hydroxytamoxifen, raloxifene and desmethylarzoxifene, functioned as estrogen receptor antagonists in oviductal cells. Cellular proliferation and migration assays suggested that estradiol does not significantly impact cellular migration and increased proliferation. Further, using RNAseq, the oviduct specific transcriptional genes targets of ER when stimulated by estradiol and 4-hydroxytamoxifen signaling were determined and validated. The RNA-seq revealed enrichment in proliferation, anti-apoptosis, calcium signaling and steroid signaling processes. Finally, the ER and PR receptor status of a panel of HGSC cell lines was investigated including Kuramochi, OVSAHO, OVKATE, OVCAR3, and OVCAR4. OVSAHO demonstrated receptor expression and response, which highlights the need for additional models of ovarian cancer that are estrogen responsive.. Overall, the fallopian tube has specific gene targets of estrogen receptor and demonstrates a tissue specific response to SERMs consistent with antagonistic action.

Topics: Animals; Antineoplastic Agents, Hormonal; Carcinoma in Situ; Cell Line, Tumor; Drug Resistance, Neoplasm; Estradiol; Estrogen Antagonists; Estrogens; Fallopian Tubes; Female; Gene Expression Regulation, Neoplastic; Genome; Humans; Mice; Neoplasms, Cystic, Mucinous, and Serous; Ovarian Neoplasms; Piperidines; Raloxifene Hydrochloride; Receptors, Estrogen; Tamoxifen; Thiophenes; Transcriptome

The risk of developing hormone-dependent cancers with long-term exposure to estrogens is attributed both to proliferative, hormonal actions at the estrogen receptor (ER) and to chemical carcinogenesis elicited by genotoxic, oxidative estrogen metabolites. Nontumorigenic MCF-10A human breast epithelial cells are classified as ER(-) and undergo estrogen-induced malignant transformation. Selective estrogen receptor modulators (SERM), in use for breast cancer chemoprevention and for postmenopausal osteoporosis, were observed to inhibit malignant transformation, as measured by anchorage-independent colony growth. This chemopreventive activity was observed to correlate with reduced levels of oxidative estrogen metabolites, cellular reactive oxygen species (ROS), and DNA oxidation. The ability of raloxifene, desmethylarzoxifene (DMA), and bazedoxifene to inhibit this chemical carcinogenesis pathway was not shared by 4-hydroxytamoxifen. Regulation of phase II rather than phase I metabolic enzymes was implicated mechanistically: raloxifene and DMA were observed to upregulate sulfotransferase (SULT 1E1) and glucuronidase (UGT 1A1). The results support upregulation of phase II metabolism in detoxification of catechol estrogen metabolites leading to attenuated ROS formation as a mechanism for inhibition of malignant transformation by a subset of clinically important SERMs.

Topics: Cell Transformation, Neoplastic; Cells, Cultured; Cytoprotection; Estradiol; Humans; Inactivation, Metabolic; Indoles; Mammary Glands, Human; MCF-7 Cells; Oxidants; Oxidative Stress; Piperidines; Raloxifene Hydrochloride; Reactive Oxygen Species; Selective Estrogen Receptor Modulators; Tamoxifen; Thiophenes; Up-Regulation

The existing drugs for benign prostatic hyperplasia (BPH) are partially effective with undesirable side-effects; hence new agents acting by different mechanism(s) are required as supplements. Modulation of estrogen receptor signaling using selective estrogen receptor modulators (SERMs) offers an alternative approach for BPH management. Using human BPH-derived stromal cells and tissue explants in culture we evaluated two SERMs, DL-2-[4-(2-piperidinoethoxy)phenyl]-3-phenyl-2 H-1-benzopyran (BP) and Ormeloxifene (Orm) in comparison to Tamoxifen (Tam) and 4-hydroxytamoxifen (OHT). BP, OHT and Tam were more effective than Orm in reducing stromal cell proliferation of human BPH. BP was either equipotent or more effective than OHT and Tam in increasing estrogen receptor(ER)-ß, TGFß1, Fas and FasL, and in decreasing ER-α, AR, EGF-R and IGF-I expressions in BPH stromal cells. BP, Tam and Orm (1.0 mg/Kg) reduced rat prostate weights by almost same extent as Finasteride (Fin, 5.0 mg/Kg); however combination treatment (SERM+Fin) was more effective. BP was exceptionally efficient in reducing IGF-1 and cleaving PARP while combination treatments more effectively increased bax:bcl-2 ratio. Fin reduced acinar diameter and prostatic DHT level but increased testosterone, estradiol (E(2)) and E(2)/T+DHT ratio. SERMs, especially BP, reduced epithelial cell height drastically without significantly altering steroid hormone levels and E(2)/T+DHT ratio. Combination treatment reduced both acinar diameter and epithelial cell height with modest increase in E(2), T and E(2)/T+DHT. The study reveals the potential of SERMs per se for BPH management, and more effectively in combination with a 5α-reductase inhibitor. BP appears promising for further evaluation as a drug candidate for BPH and prostate cancer.

Topics: 5-alpha Reductase Inhibitors; Animals; Apoptosis; Aromatase; Benzopyrans; Cell Proliferation; Cell Survival; Cells, Cultured; Dihydrotestosterone; Dose-Response Relationship, Drug; Drug Therapy, Combination; Estradiol; Estrogen Receptor alpha; Estrogen Receptor beta; Finasteride; Gene Expression Regulation; Humans; Male; Piperidines; Prostate; Prostatic Hyperplasia; Rats; Rats, Sprague-Dawley; Receptors, Androgen; RNA, Messenger; Selective Estrogen Receptor Modulators; Signal Transduction; Stromal Cells; Tamoxifen; Testosterone; Tissue Culture Techniques

Estrogen receptor (ER) biology reflects the actions of estrogens through the two receptors, ERα and ERβ, although little is known regarding the preference for formation of ER homo- vs. heterodimers, and how this is affected by the level of ligand occupancy and preferential ligand affinity for one of the ER subtypes. In this report, we use a split optical reporter-protein complementation system to demonstrate the physical interaction between ERα and ERβ in response to different ER ligands in cells and, for the first time, by in vivo imaging in living animals. The genetically encoded reporter vectors constructed with the ligand-binding domains of ERα and ERβ, fused to split firefly or Renilla luciferase (Fluc or hRluc) fragments, were used for this study. This molecular proteomic technique was used to detect ERα/ERα or ERβ/ERβ homodimerization, or ERα/ERβ heterodimerization induced by ER subtype-selective and nonselective ligands, and selective ER modulators (SERM), as well as in dimers in which one mutant monomer was unable to bind estradiol. The SERM-bound ERα and ERβ form the strongest dimers, and subtype-preferential homodimerization was seen with ERα-selective ligands (methyl piperidino pyrazole/propyl pyrazole triol) and the ERβ-selective ligands (diarylpropionitrile/tetrahydrochrysene/genistein). We also demonstrated that a single ligand-bound monomer can form homo- or heterodimers with an apo-monomer. Xenografts of human embryonic kidney 293T cells imaged in living mice by bioluminescence showed real-time ligand induction of ERα/ERβ heterodimerization and reversal of dimerization upon ligand withdrawal. The results from this study demonstrate the value of the split luciferase-based complementation system for studying ER-subtype interactions in cells and for evaluating them in living animals by noninvasive imaging. They also probe what combinations of ERα and ERβ dimers might be the mediators of the effects of different types of ER ligands given at different doses.

Topics: Animals; Estrogen Receptor alpha; Estrogen Receptor beta; Female; Gene Expression Regulation; Genes, Reporter; HEK293 Cells; Humans; Luciferases, Firefly; Luciferases, Renilla; Mice; Mice, Nude; Molecular Imaging; Piperidines; Protein Multimerization; Protein Stability; Protein Structure, Tertiary; Pyrazoles; Raloxifene Hydrochloride; Recombinant Fusion Proteins; Selective Estrogen Receptor Modulators; Tamoxifen; Transplantation, Heterologous

Rhabdoid Tumors (RTs) are highly aggressive pediatric malignancies with poor prognosis. There are currently no standard or effective treatments for RTs in part because treatments are not designed to specifically target these tumors. Our previous studies indicated that targeting the cyclin/cdk pathway is a novel therapeutic strategy for RTs and that a pan-cdk inhibitor, flavopiridol, inhibits RT growth. Since the toxicities and narrow window of activity associated with flavopiridol may limit its clinical use, we tested the effect of combining flavopiridol with 4-hydroxy-Tamoxifen (4OH-Tam) in order to reduce the concentration of flavopiridol needed for inhibition of RTs.. The effects of flavopiridol, 4OH-Tam, and their combination on RT cell cycle regulation and apoptosis were assessed by: i) cell survival assays, ii) FACS analysis, iii) caspase activity assays, and iv) immunoblot analysis. Furthermore, the role of p53 in flavopiridol- and 4OH-Tam-mediated induction of cell cycle arrest and apoptosis was characterized using RNA interference (siRNA) analysis. The effect of p53 on flavopiridol-mediated induction of caspases 2, 3, 8 and 9 was also determined.. We found that the combination of flavopiridol and 4OH-Tam potently inhibited the growth of RT cells. Low nanomolar concentrations of flavopiridol induced G₂ arrest, which was correlated to down-modulation of cyclin B1 and up-regulation of p53. Addition of 4OH-Tam did not affect flavopiridol-mediated G₂ arrest, but enhanced caspase 3,7-mediated apoptosis induced by the drug. Abrogation of p53 by siRNA abolished flavopiridol-induced G₂ arrest, but enhanced flavopiridol- (but not 4OH-Tam-) mediated apoptosis, by enhancing caspase 2 and 3 activities.. Combining flavopiridol with 4OH-Tam potently inhibited the growth of RT cells by increasing the ability of either drug alone to induce caspases 2 and 3 thereby causing apoptosis. The potency of flavopiridol was enhanced by abrogation of p53. Our results warrant further studies investigating the combinatorial effects of flavopiridol and 4OH-Tam as a novel therapeutic strategy for RTs and other tumors that have been shown to respond to flavopiridol.

Topics: Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Caspases; Cell Cycle; Cell Survival; Drug Screening Assays, Antitumor; Flavonoids; Flow Cytometry; G2 Phase; Humans; Piperidines; Rhabdoid Tumor; RNA Interference; Tamoxifen; Tumor Suppressor Protein p53

Tamoxifen is currently used as adjuvant therapy for estrogen receptor (ER) positive breast cancer patients and as a chemopreventative agent. Although ER is a predictive marker for tamoxifen response, ER status fails to predict tamoxifen response in a significant number of patients highlighting the need to identify new pathways for tamoxifen sensitivity/resistance. To identify novel proteins induced by tamoxifen in breast cancer cells sensitive to tamoxifen growth inhibition, two-dimensional (2D) gel electrophoresis was used to profile proteins in T47D breast cancer cells. Six proteins were identified that were differentially regulated by 17beta-estradiol, 4-hydroxytamoxifen and the pure antagonist acolbifene (EM-652); calreticulin, synapse associated protein 1 (SYAP1), CD2 antigen binding protein 2 (CD2BP2), nucleosome assembly protein 1 like 1 (NAP1L1), d-3-phosphoglycerate dehydrogenase (3-PHGDH) and pyridoxine 5' phosphate oxidase (PNPO). At the mRNA level, these ligands differentially regulated expression of mRNAs encoding the identified proteins in T47D and MCF7 cells but had no effect on mRNA in ERalpha-negative MDA-MB-231 breast cancer cells. These novel SERM-regulated proteins may participate in new or existing pathways for sensitivity or resistance to SERMs.

Topics: Amino Acid Sequence; Breast Neoplasms; Cell Line, Tumor; Estradiol; Estrogen Antagonists; Estrogen Receptor alpha; Female; Humans; Molecular Sequence Data; Neoplasm Proteins; Piperidines; Tamoxifen

Estrogen receptor (ER)alpha and -beta interact with a variety of coactivator proteins, most notably members of the steroid receptor coactivator (SRC) family, and these interactions have been shown to be regulated by estrogenic ligands and growth factor signaling. Here, using fluorescence resonance energy transfer (FRET), the selectivity of different stimulants on ERalpha and -beta interactions with coactivator receptor interaction domains (RIDs) were examined in living cells. We first show that ERalpha and ERbeta homo- and heterodimers form in vivo independently of the presence of 17beta-estradiol (E2) or antiestrogens. We then demonstrate that E2 enhances interactions between ERalpha and the RIDs of SRC-1 and SRC-3, whereas the interaction between ERalpha with the SRC-2 RID is ligand independent. The transcriptionally inactive mutant ERalphaL539A showed no interaction with all three SRC RIDs. Similarly, treatment with the antagonists 4-hydroxytamoxifen and EM-652 abolished all interactions between ERalpha and the SRC RIDs. FRET data also demonstrate that, in contrast to ERalpha, ERbeta interacts with all three SRC RIDs in a ligand-independent manner. However, these interactions were further enhanced or stabilized by E2, whereas the antiestrogen EM-652 abolished all interactions. In the presence of both ERalpha and ERbeta, E2 treatment led to the recruitment of SRC RIDs to the nuclei. Finally, expression of the oncogenic activated ErbB-2/Neu protein specifically enhanced ERalpha but not ERbeta interactions with SRC RIDs to an extent similar to E2-stimulated interactions. In summary, using FRET, we demonstrated preferential interactions between ER isoforms and coactivators upon hormonal treatment and activation of a growth factor signal transduction pathway in living cells.

Topics: Acetyltransferases; Bacterial Proteins; Cells, Cultured; Dimerization; Estradiol; Estrogen Receptor alpha; Estrogen Receptor beta; Fluorescence Resonance Energy Transfer; Green Fluorescent Proteins; Histone Acetyltransferases; Humans; Luminescent Proteins; Mutation; Nuclear Receptor Coactivator 1; Nuclear Receptor Coactivator 3; Oncogene Proteins; Piperidines; Protein Isoforms; Protein Structure, Tertiary; Receptor, ErbB-2; Receptors, Estrogen; Recombinant Proteins; Signal Transduction; Tamoxifen; Trans-Activators; Transcription Factors

Arzoxifene (Arzox) is a novel benzothiophene analogue with selective estrogen receptor modulator activity similar to raloxifene. Arzox is being developed as a treatment for breast cancer and has a predominantly antiestrogenic effect on the rodent uterus. Our objectives were to verify whether the novel selective estrogen receptor modulator, Arzox, can be a good first-line agent and also be effective at controlling the growth of endometrial cancer after exposure to tamoxifen (Tam).. We compared the effects of Tam and Arzox on the growth of estrogen responsive ECC-1 endometrial cancer cells in vitro, and we determined their antitumor effects on ECC-1 and EnCa101 endometrial carcinoma growth in athymic mice.. We observed that estrogen receptor protein expression is down-regulated by Arzox to the same extent as raloxifene, whereas 4-hydroxytamoxifen, the active metabolite of Tam, does not affect estrogen receptor protein levels. Tam and Arzox inhibit the growth of Tam-naïve ECC-1 tumors in athymic mice. However when Tam-stimulated or estrogen-stimulated (which had been treated with Tam previously) EnCa101 endometrial tumors were treated with Tam or Arzox, we observed a stimulatory effect of both compounds in these models.. The results indicate that Arzox may be a good first-line agent, but it may be ineffective at controlling the growth of endometrial cancer after exposure to Tam. Our data suggest that Arzox stimulates endometrial tumor growth to at least the same extent as Tam, thereby suggesting a limited role as a second-line agent for the patient on Tam who develops occult endometrial cancer.

Topics: Animals; Cell Division; Down-Regulation; Endometrial Neoplasms; Estrogen Antagonists; Female; Humans; Mice; Mice, Nude; Piperidines; Receptors, Estrogen; Tamoxifen; Thiophenes; Transplantation, Heterologous; Tumor Cells, Cultured

Recent data indicate that insulin-like growth factor I (IGF-I) may have a function in mediating the mitogenic effects of 17beta-estradiol (E2) in the uterus and in regulating the growth of uterine neoplasms. This study was designed to determine whether synthetic and plant-derived chemicals that interact with estrogen receptor-alpha (ERalpha) and elicit estrogenic responses also mimic E2 by activating the uterine IGF-I signaling pathway. Ovariectomized adult female mice were treated with both environmental and clinically relevant chemicals previously reported to display estrogenic and/or antiestrogenic properties, and their uteri were evaluated for an activated IGF-I signaling pathway. Diethylstilbestrol, 4-hydroxytamoxifen, the raloxifene analog LY353381, 2,2-bis(p-hydroxyphenyl)-1,1,1-trichloroethane (HPTE), bisphenol A, and genistein were shown to mimic E2 in the uterus by increasing the level of IGF-I messenger RNA, inducing IGF-I receptor (IGF-IR) tyrosine phosphorylation, stimulating the formation of IGF-IR signaling complexes, and increasing both proliferating cell nuclear antigen expression and the number of mitotic cells in the epithelium. The dose of chemical necessary to activate IGF-I signaling varied, with the order of potency: E2 = diethylstilbestrol > LY353381 > 4-hydroxytamoxifen > genistein > HPTE > bisphenol A. Administration of the chemicals to ERalpha knockout mice did not activate IGF-IR, indicating that ERalpha is required for activation of uterine IGF-IR by these diverse chemicals. This study demonstrates that several chemicals shown previously to display estrogenic activities also mimic E2 by activating uterine IGF-I signaling.

Topics: Animals; Benzhydryl Compounds; Blotting, Northern; Estradiol Congeners; Estrogen Antagonists; Estrogen Receptor alpha; Estrogens; Estrogens, Non-Steroidal; Female; Insulin-Like Growth Factor I; Mice; Mice, Knockout; Mitosis; Ovariectomy; Phenols; Piperidines; Precipitin Tests; Proliferating Cell Nuclear Antigen; Receptors, Estrogen; Ribonucleases; RNA, Messenger; Signal Transduction; Tamoxifen; Thiophenes; Uterus

The budding yeast Saccharomyces cerevisiae has been used extensively as a biological 'test tube' to study the regulation of the human estrogen receptor (ER) alpha. However, anti-estrogens, which are of great importance as therapeutic agents and research tools, fail to antagonize the activation by estrogen in yeast. Here, we have surveyed the antagonistic potential of five different anti-estrogens of diverse chemical nature. While they all act as agonists for wild-type ERalpha, we have established a novel yeast assay system for anti-estrogens, in which at least the commonly used anti-estrogen hydroxytamoxifen is a potent antagonist.

Topics: Dose-Response Relationship, Drug; Drug Evaluation, Preclinical; Estradiol; Estrogen Antagonists; Estrogen Receptor alpha; Humans; Indoles; Nafoxidine; Piperidines; Polyunsaturated Alkamides; Receptors, Estrogen; Saccharomyces cerevisiae; Sulfones; Tamoxifen; Two-Hybrid System Techniques

We have shown that 4-hydroxytamoxifen (4-OHT) has estrogen-like effects on induction of TGFalpha mRNA in estrogen receptor (ER)-negative MDA-MB-231 human breast cancer cells, transfected with either wildtype (S30 cells) or a codon 351asp-->tyr mutant ER (BC-2 cells). The mutant receptor used to produce the stable transfectants was identified in a tamoxifen-stimulated human breast tumor. We have also demonstrated that raloxifene exhibits a gene-specific estrogen-like effect with mutant ER (BC-2 cells) but not with wildtype ER (S30 cells) (Levenson, A.S., Catherino, W.H. and Jordan, V.C. (1997) Estrogenic activity is increased for an antiestrogen by a natural mutation of the estrogen receptor. J. Steroid Biochem. Mol. Biol., 60, 261-268). We now describe the regulation of urokinase plasminogen activator (uPA) and plasminogen activator inhibitor type 1 (PAI-1) endogenous gene expression by estradiol (E2) and different antiestrogens in BC-2 cells. Northern blot analyses revealed that 4-OHT and raloxifene have concentration-dependent agonistic (E2-like) effects on the regulation of these genes. In contrast, the pure antiestrogen ICI 182780 alone had no effect but could block the action of E2, 4-OHT and raloxifene. The E2-like effects of non-steroidal antiestrogens in this model system cannot be explained by the mutation in the ER alone because 4-OHT acts as an agonist with wildtype receptor as well. We propose that the clear cut biological expression of estrogen-like qualities with different antiestrogens will in the future serve as an important model to dissect the signal transduction pathway.

Topics: Breast Neoplasms; Estrogen Antagonists; Female; Gene Expression Regulation, Neoplastic; Humans; Piperidines; Plasminogen Activator Inhibitor 1; Raloxifene Hydrochloride; Receptors, Estrogen; RNA, Messenger; Tamoxifen; Tumor Cells, Cultured; Urokinase-Type Plasminogen Activator

Activation of the estrogen receptor (ER) by hormone involves at least two steps. First, hormone binding initially relieves repression, a property imposed on ER in cis by its ligand-binding domain (EBD). Subsequently, the derepressed ER binds specific genomic sites and regulates transcription. In addition to the natural hormone, ER binds a broad range of ligands that evoke a spectrum of responses ranging from full ER activation by agonists to partial activation and inhibition by partial or complete antagonists. How these different ligands evoke different ER responses remains unclear. To address this issue, we have developed a nontranscriptional assay for ER ligand responsiveness based on Flp recombinase/human EBD protein chimeras. These fusion proteins transduce the transient event of ligand binding into a permanent DNA change in a human cell line system. A fusion protein including ER D, E, and F domains was activated by all the ER ligands tested, demonstrating that both agonists and antagonists serve to relieve initial repression, and that differences between them lie downstream in the activation pathway. Mutant variants of the Flp-ER protein that distinguish between agonists and antagonists, and a mutant EBD that selectively lost the ability to respond to 17beta,-estradiol but not to other ligands, were also identified. Thus, agonists and antagonists can be functionally distinguished in a nontranscriptional assay.

Topics: Diethylstilbestrol; Dimerization; DNA Nucleotidyltransferases; Estradiol; Estrogen Antagonists; Fulvestrant; Hexestrol; Humans; Mutation; Piperidines; Raloxifene Hydrochloride; Receptors, Estrogen; Recombinant Fusion Proteins; Tamoxifen

The ligand-binding domains of steroid receptors convey ligand-dependent regulation to certain proteins to which they are fused. Here we characterize fusion proteins between a site-specific recombinase, FLP, and steroid receptor ligand-binding domains. These proteins convert ligand binding into DNA recombination. Thus, ligand binding is directly coupled to an enzyme activity that is easily measured by DNA rearrangements or heritable genetic changes in marker gene expression, as opposed to the multiple events leading to transcription. Recombination by a FLP-estrogen receptor (FLP-EBD) fusion is activated by all tested estrogens, whether agonists or antagonists, indicating that all induce EBD release from the 90-kDa heat shock protein complex. Altering the distance between FLP and the EBD domain in the fusion proteins, by reducing the included length of the estrogen receptor D domain, affects ligand efficacy. A FLP-EBD with no D domain shows reduced inducibility by agonists and, unexpectedly, complete insensitivity to induction by all antagonists tested. A FLP-EBD including some D domain shows a ligand-inducible phenotype intermediate to those displayed by FLP-EBDs containing all or none of the D domain. Thus, we observed a tethered interference between FLP and the EBD domains that differs depending on the distance between the two domains, the conformations induced by agonists or antagonists, and which presents a previously undetectable distinction between estrogen agonists and antagonists in yeast.

Topics: Amino Acid Sequence; Bacterial Outer Membrane Proteins; Diethylstilbestrol; DNA Nucleotidyltransferases; Dose-Response Relationship, Drug; Estradiol; Estrogen Antagonists; Estrogens, Non-Steroidal; Fulvestrant; Hexestrol; HSP90 Heat-Shock Proteins; Humans; Ligands; Molecular Sequence Data; Nafoxidine; Piperidines; Raloxifene Hydrochloride; Receptors, Estrogen; Recombinant Fusion Proteins; Tamoxifen; Time Factors; Yeasts

We have previously demonstrated sex-specific stimulation of creatine kinase specific activity (CK) in bone cells both in vivo and in vitro, in primary culture cells derived from rat and human bone and in established human bone-derived cell lines. We found that the female-derived cell line, SaOS-2, responded to 17 beta-estradiol (E2) by increased CK specific activity. The effects of E2 on the CK activity in SaOS-2 cells was inhibited by 100-fold excess of 4-hydroxytamoxifen (Tam) as well as by the other antiestrogen, ICI 164,384. Tam by itself had some stimulatory effect whereas ICI 164,384 showed no estrogenic activity. We also demonstrated the estrogenic-like effect of another anti-estrogen, raloxifene (Ral), which is agonist only in the SaOS-2 osteoblast-like cells but not in the human endometrial, Ishikawa cell line. Ishikawa cells respond to E2 and to Tam by increased CK activity. In both osteoblasts and endometrial cell lines, Ral and Tam were inhibitory in the presence of E2. The effects of E2 on SaOS-2 cells are at least partially mediated by the estrogen receptor (ER) at the level of transcription as demonstrated by transient transfection experiments using the human creatine kinase promoter chloramphenicol acetyltransferase in these cells. Pretreatment of SaOS-2 with calcitropic hormones, either 1,25 dihydroxyvitamin D3 (1,25(OH)2D3) or human parathyroid hormone (1-34) (hPTH(1-34)) increased the stimulation of CK by E2 by 40-60% relative to E2 alone and significantly increased the sensitivity of the cells to E2 by lowering the effective hormonal dose needed for stimulation of CK by E2 by 100-fold. This stimulatory effect of pretreatment of the cells with 1,25(OH)2D3 was due to a 2.5-fold increase in the level of ER expression as measured directly by enzyme immunoassay in the SaOS-2/1 subline. The increase in the responsiveness to E2 by hPTH(1-34) was not due to an increase in ER level in the cells. We can conclude that in cell cultures as in vivo, Ral shows different effects depending on the cell type, namely estrogenic-like activity in skeletal cells but not in uterine cells. We can also conclude that as with rat-derived cells, in bone cells derived from human bone 1,25(OH)2D3 increased the sensitivity to E2 due to an increase in the number of ER in the cells, whereas PTH(1-34) augmented the response to E2 without increasing ER, by another, as yet unknown, mechanism. These studies suggest that the treatment of pathological bone disorders may be i

Topics: Calcitriol; Creatine Kinase; Endometrium; Estradiol; Estrogen Antagonists; Female; Humans; Osteoblasts; Piperidines; Plasmids; Polyunsaturated Alkamides; Raloxifene Hydrochloride; Recombinant Proteins; Stimulation, Chemical; Tamoxifen; Teriparatide; Tumor Cells, Cultured

We have developed a series of in vitro models with which to evaluate the biological activity of estrogen receptor (ER) agonists and antagonists. Using a protease digestion assay we show that the conformational changes induced within ER are distinct for agonists and antagonists. However, this assay is unable to discriminate between pure antagonists like ICI164,384 and partial agonists such as 4-OH tamoxifen or keoxifene. Using a chimeric ER-VP16 construct, we demonstrate that both pure antagonists and partial agonists deliver ER to its DNA target within cells. However, the ability of the DNA-bound receptor to activate transcription in the presence of a given antagonist is dependent on cell and promoter context. These data, suggesting functional differences among ER antagonists, were confirmed by additional experiments demonstrating that their ability to modulate the transcriptional activity of a series of ER mutants is dramatically different. Depending on the cell and promoter context and the particular ER form expressed, 4-OH tamoxifen and the related compound, keoxifene, functioned as partial agonists. Importantly, the transcriptional profiles of these two compounds were dissimilar, suggesting that they are functionally different from each other and from ICI164,384, which does not display agonist activity under any context examined. Our results reveal functional differences between these clinically important antiestrogens and suggest that the distinct biologies manifest by these compounds in vivo relate to their ability to differentially regulate ER function.

Topics: Animals; Cell Line; Chlorocebus aethiops; DNA; Estradiol; Estrogen Antagonists; Gene Expression Regulation; Kidney; Molecular Structure; Piperidines; Polyunsaturated Alkamides; Protein Binding; Protein Conformation; Raloxifene Hydrochloride; Receptors, Estrogen; Recombinant Proteins; Structure-Activity Relationship; Tamoxifen; Transfection

The agonistic/antagonistic properties of two non-steroidal antiestrogens, namely trans-4-monohydroxytamoxifen (OH-TAM) and keoxifene (LY156758), and the new steroidal antiestrogen ICI164384, a 7 alpha-alkylamide derivative of estradiol (E2), were assessed by measuring their effect on the proliferation of ZR-75-1 cells, an estrogen-responsive human breast cancer cell line. While subnanomolar concentrations of both OH-TAM and LY156758 had significant estrogenic stimulatory activity on cell growth in the absence of estrogens and higher concentrations were inhibitory, ICI164384 behaved exclusively as a growth inhibitor and more potently so than the two other compounds. The three antiestrogens had similar potency to inhibit the mitogenic effect of E2 and at 300 nM, all antiproliferative effects were completely reversible by the estrogen. ICI164384 was a weaker competitor of 3H-labeled E2 or R2858 (moxestrol) uptake in intact ZR-75-1 cells in a 1-hour assay, partly because of a slower intracellular access to estrogen specific binding sites. Moreover, ICI164384 interacted in a rapidly (approximately 6 h) reversible manner with estrogen-specific binding sites, while the non-steroidal antiestrogens induced a longer-acting (greater than 24 h) down-regulation of specific [3H]R2858 uptake. The present data indicate that, among the antiestrogens studied, ICI164384 is the only compound acting as a pure antiestrogen in ZR-75-1 breast cancer cells, while LY156758 and OH-TAM behave as antiestrogens endowed with partial agonistic activity in this system.

Topics: Binding, Competitive; Breast Neoplasms; Cell Division; Dexamethasone; Dihydrotestosterone; Dose-Response Relationship, Drug; Drug Interactions; Estradiol; Estrogen Antagonists; Humans; Piperidines; Polyunsaturated Alkamides; Raloxifene Hydrochloride; Receptors, Estrogen; Tamoxifen; Time Factors; Tumor Cells, Cultured

The non-steroidal antioestrogens tamoxifen, 4-hydroxytamoxifen, trioxifene, LY 117018 and LY 139481 have widely divergent affinities for oestrogen receptors from rat mammary tumours. The latter two compounds have much reduced partial agonist activity in rat uterus, compared to tamoxifen, but were less effective antitumour agents than tamoxifen. No direct correlation was established between receptor affinity and biological response in rat uterus or rat mammary carcinoma. However, in in vitro studies of growth inhibition of human breast cancer cells (MCF7), the order of potency was the same as the order of relative binding affinity. Differences in in vivo activity of these antioestrogens may be related to biological "half-life" which is dependent on the dose, route of administration and metabolic stability of the antioestrogens. Growth inhibition in MCF 7 cells did not correlate with affinity for tamoxifen-specific binding sites, nor was there any evidence for differences between antioestrogens in their mechanism of action on the rat uterus. It is concluded that the primary effects of antioestrogens are mediated by binding to oestrogen receptors.

Topics: Animals; Breast Neoplasms; Cell Line; Estrogen Antagonists; Female; Humans; Mammary Neoplasms, Experimental; Piperidines; Pyrrolidines; Raloxifene Hydrochloride; Rats; Receptors, Estrogen; Tamoxifen; Thiophenes; Uterus