Antiestrogen Binding Sites Research Articles

Antiestrogen-Binding Sites in 7,12-Dimethylbenz(a)anthracene-Induced Rat Mammary Tumors: Relation to Growth

We have detected specific high-affinity binding sites for nonsteroidal antiestrogens in 98% of 7,12-dimethylbenz(a)anthracene-induced rat mammary tumors. Since recent studies have suggested that these binding sites may be involved in the regulation of cell growth and proliferation, we attempted to define a possible relationship between the growth of these hormone-dependent tumors and their antiestrogen-binding site content. Rats bearing such tumors were either treated with haloperidol (to increase prolactin secretion and stimulate tumor growth) or oophorectomized (to reduce circulating estrogen concentration and suppress tumor growth). Compared with controls, haloperidol treatment clearly enhanced tumor growth while oophorectomy induced tumor regression, but neither procedure had any effect on the antiestrogen-binding site concentration. Furthermore, tumors which responded to endocrine manipulation had similar antiestrogen-binding site concentrations as tumors which did not respond. We conclude that (1) the alterations in tumor growth induced by these endocrine manipulations are probably not mediated through a change in antiestrogen-binding site concentration, and (2) the tumor concentration of these binding sites is not under estrogen or prolactin control.

Comparison of the effects of tamoxifen and of a tamoxifen analogue that does not bind the estrogen receptor on serum lipid profiles in the cockerel

Administration of the nonsteroidal antiestrogen tamoxifen to cockerels results in dose- and time-dependent decreases in the levels of free and esterified cholesterol, phospholipids, and triglycerides in serum and in very low density and low density lipoprotein fractions. Similar changes can be elicited using a tamoxifen analogue, N,N-diethyl-2-[(4-phenylmethyl)phenoxy]ethanamine.HCl (DPPE). Like tamoxifen, this compound is capable of binding antiestrogen binding sites and exhibits a relative binding affinity of 90% compared with tamoxifen (Ki approximately 4-5 nM). Unlike tamoxifen, DPPE shows no measureable affinity for the cockerel liver nuclear estrogen receptor. Further, DPPE exhibits no estrogen agonist or antagonist activity as measured at the level of synthesis of apolipoprotein II of very low density lipoprotein by liver, synthesis of ovalbumin by oviduct, or growth of the oviduct. Although it is possible that the lipid-lowering effects of tamoxifen result from the opposition of endogenous estrogen action in the cockerel, the similarity of the effects of tamoxifen and DPPE on the lipid profiles suggests common mechanisms that do not involve the estrogen receptor.

Cytostatic effect of antiestrogens in lymphoid cells: relationship to high affinity antiestrogen-binding sites and cholesterol

The antiproliferative effect of 10(-6) M antiestrogens in an estrogen receptor-negative lymphoid cell line (K36) was enhanced in lipoprotein-poor growth medium. The enhancement was not due to increased bioavailability because cellular uptake of [3H]tamoxifen was not increased and the lipoprotein fraction of serum had negligible [3H]tamoxifen-binding capacity. Cholesterol and lipoproteins, but not mevalonate, reversed the cytostatic effect of antiestrogens. Reversal by cholesterol was dose-related (10(-7) M to 10(-5) M), while that by lipoproteins could also be demonstrated in medium undepleted of lipoproteins. The cytostatic efficacy of a series of ten compounds correlated well with their relative binding affinities for solubilized antiestrogen-binding sites from K36 cells when log IC50 values (concentration required to reduce [3H]thymidine incorporation by 50%) were plotted against log RBA50 values (concentration required to reduce [3H]tamoxifen binding by 50%) (correlation coefficient 0.94). Transmission electron microscopy of antiestrogen-treated cells showed evidence of disordered cytokinesis which was partially reversed by cholesterol. These observations implicate the antiestrogen-binding protein in the antiproliferative effect of antiestrogens in nonestrogen target cells.

125I]Iodotamoxifen cytotoxicity in cultured human (MCF-7) breast cancer cells

We evaluated the uptake and radiotoxicity of [ 125I]iodotamoxifen ( 125ITAM) in MCF-7 human breast cancer cells in the presence or absence of excess non-radioactive estradiol (E2) or iodotamoxifen (ITAM). Studies in cells under wash-out conditions and in nuclei isolated from previously exposed cells showed that 125ITAM binds estrogen receptor (ER) and antiestrogen binding sites (AEBS) and has the capacity for considerable non-specific binding. The radiotoxicity of 125ITAM was a complex function related to ER content and/or function as well as interactions with ER, AEBS and non-specific binding. Addition of E2 or ITAM abolished ER mediated cell killing. ITAM but not E2 abolished AEBS mediated cytotoxicity. Non-specific binding accounted for considerable cytotoxicity. Although these studies confirm the radiotoxicity of nuclear bound 125I, multiple nuclear binding sites, variability in ER content and function and non-specific binding will all adversely influence ultimate clinical efficacy.

Characterization of mcf 7 breast cancer cell growth inhibition by the antiestrogen nitromifene (CI 628) and selected metabolites

Besides undergoing O-demethylation in vivo the triarylethylene antiestrogen nitromifene [1(4-(2-pyrrolidinylethoxy)phenyl)-1( 4-methoxy)-phenyl-2-phenyl-2-nitroethene, 1] undergoes biotransformation via nitroreduction, ethene bond cleavage, and pyrrolidine ring oxidation affording ketone metabolites 2 and 3 and a lactam metabolite 4. Estrogen receptor (ER) affinities of 1, 2, and 4 were, in turn, 1.7, 0. l, and 3.8% that of estradiol in MCF 7 human breast cancer cells, and these compounds inhibited by 50% the proliferation of MCF 7 cells at respective concentrations of 1.1, 5.6, and 2.0 μM The inhibitory effect of 4 was fully reversible by estradiol, but that of 2 was only partially reversible. Also 3, which did not interact with ER, inhibited proliferation by 44% at a concentration of 10 μM. These results suggested that in constrast to 4, the effects of 2 and 3 were due in part to interaction with sites distinct from ER. Antiestrogen binding sites and calmodulin have been suggested to mediate antiproliferative effects of drugs. Interaction of ligands with the former sites has been proposed to antagonize the growth promoting effect of histamine. Although 2 and 3 had high affinities for these sites, their inhibitory effects on MCF 7 cell growth were largely unaffected by the presence of histidine, the source of intracellular histamine. Thus, the relationship between antiestrogen binding site affinity and antiproliferative effects of 2 and 3 was not clarified. In contrast, MCF 7 cell growth suppression potencies paralleled calmodulin antagonist potencies of 1 and 2 suggesting that interaction of 1 and 2 with calmodulin may contribute to their anticancer effects.

Effect of triphenylacrylonitrile derivatives on estradiol-receptor binding and on human breast cancer cell growth

In a study of a series of 26 triphenylacrylonitrile derivatives (TPEs), we investigated the influence of several possibly interrelated factors on the proliferation of human breast cancer cell lines. (1) Chemical substituents: the test compounds were for the most part para-hydroxylated with increasingly bulky hydrophobic and/or basic side chains [isopropyloxy or (diethylamino)ethoxy] or standard reference compounds. (2) Relative binding affinities (RBAs): they competed diversely for [3H]estradiol (E2) binding to calf uterus cytosol and little, if at all, for binding to the [3H]tamoxifen-labeled antiestrogen binding site (AEBS) in lower speed supernatant. A multiparametric comparison of RBAs recorded for calf, rat, and mouse uterus cytosol estrogen receptor (ER) revealed a possible influence of species-specific receptor conformation and/or environment on binding. (3) Estrogen/antiestrogen potency: their stimulation and inhibition of the proliferation of the ER-positive human breast cancer cell line (MCF7) was measured. Compounds with only hydroxy substituents stimulated proliferation more markedly than methylated derivatives and had a maximum effect at 10(-11)-10(-6) M. Stimulation was related to the RBA for ER. Compounds with isopropyloxy or (diethylamino)ethoxy side chains only weakly stimulated MCF7 cell growth and more powerfully antagonized E2-promoted growth. The extent of inhibition depended upon the bulk of the side chain and could be reversed by 10(-7) M E2. Within the same concentration ranges, the test compounds were without effect on the BT20 ER-negative cell line. (4) Cytostatic and/or cytolytic activity: most compounds could arrest the proliferation of both MCF7 and BT20 cells at concentrations above 3 x 10(-6) M. This activity was thus independent of ER. Nevertheless, those compounds with a charged hydrophobic side chain, which were the most powerful antagonists of E2-promoted cell growth, were also the most cytotoxic. The overall results for all molecules on all parameters were submitted to a multivariate analysis (correspondence analysis) which revealed the progressive influence of increasing substitution by hydroxy and more bulky groups on the generation of antagonist activity and cytotoxicity.

Interactions of sterols with antiestrogen-binding sites: structural requirements for high-affinity binding.

Animal and human tissues contain a microsomal protein that binds nonsteroidal antiestrogens with high affinity and specificity. The functions of these binding sites and the identity of their natural ligands are unknown. Following a report that certain sterols inhibit [3H]tamoxifen binding to this site, we attempted to define the structural requirements for maximal inhibition of [3H]tamoxifen binding to rat liver antiestrogen-binding sites. Our studies identified 5 alpha-cholestan-3 beta-ol-7-one (7-ketocholestanol) as the most potent sterol, having an inhibitory activity that was 12% that of unlabeled tamoxifen and an equilibrium dissociation constant of 6.3 nM. Structural features that appeared important for the inhibitory activity of this sterol include the presence of i) a hydrocarbon side chain at C17; ii) an oxygen function at C7; iii) a hydroxyl group at C3; and iv) the absence of a double-bond between C5 and C6. Saturation analysis and kinetic studies of [3H]tamoxifen binding in the presence of varying concentrations of 7-ketocholestanol clearly indicated that this sterol competed directly with tamoxifen for the antiestrogen-binding site. Unlike tamoxifen, this sterol did not bind to the estrogen receptor. These features make 7-ketocholestanol a potentially valuable tool for studying the properties and functions of this site.

Role of estrogen receptors and antiestrogen binding sites in an early effect of antiestrogens, the inhibition of cholesterol biosynthesis

The effects of estradiol (E 2), 4-hydroxy-tamoxifen (OH-Tam), and LY117018 on cholesterogenesis were investigated in two human breast cancer cell lines (MCF-7 and BT20), and in rat hepatoma (HTC) and fibroblastic (NRK-49F) cell lines. It was found that 10 −10 M E 2 stimulated and 10 −8 M OH-Tam inhibited cholesterol synthesis in the estrogen-sensitive MCF-7 cell line. The OH-Tam effect occurred in less than 15 min whereas E 2 only stimulated after 8 h. The inhibition of cholesterol synthesis was not reversed by E 2 · E 2 was without effect in the HTC and estrogenresistant BT20 cell lines whereas OH-Tam was as effective as in the MCF-7 cells. LY117018 had nearly as much effect on cholesterol synthesis as OH-Tam, in both MCF-7 and BT20 cells. Neither E 2 nor OH-Tam had any effect on the NRK-49F cell line, even at micromolar concentrations. The three lines (MCF-7, BT20, HTC), whose cholesterol synthesis has been shown to be OH-Tam sensitive, appeared to contain high-affinity antiestrogen binding sites (AEBS); since the OH-Tamresistant line (NRK) only contained low-affinity AEBS, there appears to be some relationship between OH-Tam sensitivity and high-affinity AEBS content. This suggests that the cholesterogenesis inhibition induced by antiestrogens is ER-independent and may involve AEBS. The cholesterogenesis stimulation induced by E 2 occurred via a different pathway that appears to be related to the presence of ER in the cells.

Antiestrogen binding sites: General and comparative properties

Nonsteroidal antiestrogens, such as tamoxifen, bind estrogen receptors with relatively high affinity. They also bind with high affinity and distinctive specificity to another class of intracellular sites, often termed “antiestrogen binding sites” or “AEBS”. These sites do not bind estrogens and their function is unknown. In this article we compare and contrast the binding specificity, intracellular localization, concentration, tissue distribution and detergent solubilization characteristics of AEBS from a number of species. In general the sites are found in highest concentration in microsomal fractions from liver. AEBS from chicken liver can be solubilized with CHAPS detergent giving rise to a detergent-associated complex with an apparent molecular mass of about 265 kDa.

Antiulcerogenic and antisecretory effects of a novel diphenylmethane derivative and antiestrogen binding site ligand.

N,N-Diethyl-2-[4-(phenylmethyl)-phenoxy]-ethanamine hydrochloride (DPPE) is a para-diphenylmethane derivative that binds selectively and with high affinity to the microsomal antiestrogen binding site (AEBS). Recent studies with DPPE indicate that AEBS is closely related to a lower affinity non-H1, non-H2 histamine site that may be associated with calcium channels; the DPPE-AEBS site is different from that which verapamil binds, however, DPPE, but not verapamil, demonstrates antiproliferative effects in vitro and is antiestrogenic in vivo. We now show that DPPE profoundly inhibits restraint and cold stress and ethanol-induced gastric ulcer formation, accelerates ulcer healing, attenuates the stress-induced rise in plasma corticosterone level, and significantly reduces basal and H2 agonist (dimaprit)-stimulated and, to a lesser extent, bethanechol-stimulated gastric acid output in conscious rats. A nonulcerogenic but prostaglandin-depleting dose of indomethacin completely blocks the inhibitory effects of DPPE on stress ulcer formation. Conversely, verapamil only slightly attenuates dimaprit-stimulated gastric acid secretion and exacerbates ethanol-induced gastric ulcers; its anti-stress ulcer effects are only partially attenuated by indomethacin. These findings support the likelihood that the site of action of DPPE is different from that of verapamil, and that an effect on prostaglandins may, at least in part, contribute to its antiulcer and apparent cytoprotective effects.

Further evidence for a biological role of anti-estrogen-binding sites in mediating the growth inhibitory action of diphenylmethane derivatives

Several diphenylmethane derivatives have been synthesized with variable affinities for Anti-estrogen Binding Sites (ABS) but not for the estrogen receptor. Using these molecules as probes it is shown that their binding affinities for ABS correlate with their abilities to inhibit the growth of MCF-7 human breast cancer cells. In contrast they have no influence on the proliferation of tamoxifen-resistant variant cells (RTx 6) in which ABS are undetectable. These data support the conclusion that ABS has a functional role in the anti-proliferative effect of triphenylethylene anti-estrogens and structurally related compounds.

A novel non-H1, non-H2 histamine antagonist protects against cysteamine-induced duodenal ulcers in rats.

A newly synthesized para-diphenylmethane derivative, N,N-diethyl-2-[4-(phenylmethyl)phenoxy]ethanamine-HCl (DPPE), binds with high affinity to the microsomal anti-estrogen binding site (AEBS). Recent data suggest that the DPPE/AEBS binding site is closely related to a novel low-affinity, non-H1, non-H2 histamine site which may be associated with a calcium channel. We previously have shown that DPPE markedly reduces stress-induced and ethanol-induced gastric ulcers and attenuates gastric acid secretion. We now show that DPPE also profoundly reduces cysteamine-induced duodenal ulcers in rats.

Roles of antiestrogen binding sites in human endometrial cancer cells

Estrogen-noncompatible antiestrogen binding sites (AEBS) as well as estrogen receptors (ER), and the growth-inhibitory effect of tamoxifen were investigated in two human endometrial cancer cell lines, IK-90 and HEC-IA cells. IK-90 cells contained specific AEBS, but no ER was found in these cells. Scatchard plot analysis of AEBS in 12,000 g supernatant from IK-90 cells showed a high affinity binding site for tamoxifen ( K d :5.6 ± 1.0nM) with the maximum binding site of 457 ± 47 fmol/mg protein. However, no measurable ER or AEBS was found in HEC-IA cells. The effect of tamoxifen on the growth of cells was found to be identical in both cell lines; the addition of 10 μ M tamoxifen to culture medium was cytocidal whereas tamoxifen at lower concentrations (1 nM-1μM) did not significantly affect the growth of both IK-90 and HEC-IA cells. These results demonstrate for the first time the presence of AEBS in human endometrial cancer cells. The present results also suggest that AEBS does not play a fundamental role in mediating the growth:inhibitory effect of tamoxifen in endometrial cancer cells.

Triphenylethylene antiestrogen binding sites (TABS) specificity

The relative binding affinities (RBA) of various compounds for the triphenylethylene antiestrogen binding sites (TABS) were examined. The ability of tamoxifen to inhibit the binding of [ 3H]tamoxifen to salt extracted (0.4 M KCl) TABS from rat liver nuclei was used as a standard by which other compounds were compared (tamoxifen RBA, 100; K d ~ 1 nM). Nafoxidine was the most effective triphenylethylene compound used (RBA 333; K d ~ 0.3 nM) whereas the RBA of zuclomiphene and enclomiphene was not different from tamoxifen. MER-29 was the weakest inhibitor of the triphenylethylene derivatives (RBA 10; K d ~ 10 nM). Trifluoperazine, chlorpromazine and the anti-calmodulin drugs W-13 and W-12 had RBA's of 25, 1, 1 and 0.1 respectively. The binding affinities of cholesterol and 7-ketocholesterol were significant ( K d ~ 22 nM) while the steroid hormones, estradiol, testosterone, progesterone and corticosterone displayed not observable affinity. Various compounds obtained from Merrill Dow Pharmaceuticals and the Eli Lilly Company which contained alklaminoethoxy side chains linked to aromatic ring structures had RBA's ranging from 1−0.3. We conclude, as other investigators have also concluded, that the similar binding affinities of various triphenylethylene antiestrogens for TABS and their divergent activities as antiestrogens makes it unlikely that TABS are directly involved in estrogen antagonism. The moderate but significant affinity of TABS for trifluoperazine and other drugs thought to be involved in calmodulin regulation indicates that TABS may be a linked in some way to calmodulin function. The binding of cholesterol and 7-ketocholesterol is also significant and may indicate that TABS are involved in cholesterol metabolism.

Antiestrogen binding sites in microsomal fractions of malignant and nonmalignant human breast tissues.

Antiestrogen binding sites (AEBS) were measured in microsomal fractions of 102 malignant breast tumors and 24 nonmalignant breast tissues. The number of AEBS was determined by Scatchard analysis. The cytosol contents of estrogen and progesterone receptors were also analyzed in these tissues. Overall, 50% of the malignant tumors and 33% of the nonmalignant normal breast tissues had detectable contents of AEBS. No correlation was observed between cytosol estrogen receptor (ER) content and microsomal AEBS in the tumors. Detailed data analysis in patients at Stage IV disease revealed that 60% of estrogen receptor positive tumors had no detectable microsomal AEBS contents whereas in the remaining 40% tumors, high affinity AEBS were observed. On the other hand, AEBS were also detected in 35% of ER-poor tumors. Anti-estrogen binding sites were higher in tumors obtained from premenopausal women than in those of postmenopausal women. The incidence of AEBS-positive tumors or AEBS concentration was not influenced by either the patients' obesity or their disease stage.

Interactions of tamoxifen in the chicken

The triphenylethylene antiestrogens are very potent antagonists of estrogen action in the chicken and manifest little agonist activity compared to their action in other species. The estrogen antagonism is most probably mediated by the estrogen receptor, to which tamoxifen binds with a K i of 2.6 nM. Tamoxifen is readily metabolized by liver to 4-hydroxytamoxifen, which binds the liver nuclear estrogen receptor with a K i , of 0.1 nM. The K d of the receptor is 0.7 nM. Estrogen receptor concentrations in liver from immature chickens are relatively low both in nuclear and cytosol fractions. Treatment with estradiol results in 10-fold up-regulation of the nuclear levels to give a total receptor concentration of about 2 pmol/g tissue. Tamoxifen can promote this up-regulation to a limited extent, but interpretation of experimental results is compromised by difficulties with exchange assays in the face of the very high binding affinity of 4-hydroxytamoxifen. Tamoxifen also binds with high affinity ( K d 2–4 nM) and distinctive specificity to antiestrogen binding sites (AEBS) present in a wide variety of chicken tissues and in the highest concentration in the liver (800 pmol/g tissue). Liver and serum contain ether-soluble components which can compete for binding of [ 3H]tamoxifen to the AEBS. The serum AEBS inhibitory activity is chromatographically heterogeneous and is associated with a sterol-like fraction as well as with a fatty-acid-containing fraction. Tamoxifen treatment of cockerels results in dose- and time-dependent decreases in serum free and esterified cholesterol, and in phospholipids and triglycerides. These changes may reflect estrogenreceptor-independent interactions of tamoxifen.

Antiestrogens, different sites of action than the estrogen receptor?

Nonsteroidal antiestrogens display antagonistic as well as agonistic properties when compared with estrogens. These observations and studies of their molecular interactions suggest that part of their activity is mediated through estrogen receptor (ER)-mediated pathways. However, other data, concerning mainly effects on growth and cellular proliferation, cannot be explained on this basis. Recent investigations have shown binding of these molecules to a number of other intracellular binding sites beside ER. Using different cell variants of the human breast cancer cell line MCF-7 we could confirm the peculiar role of one of these sites, the specific antiestrogen-binding site (ABS). Published data on ABS are critically reviewed.

Anti-estrogen binding sites [AEBS] in human and rat breast and liver tissues

Anti-estrogen binding sites [AEBS] in human and rat breast and liver tissues

Determination of antiestrogen-binding sites (AEBS) in breast cancer specimens

Determination of antiestrogen-binding sites (AEBS) in breast cancer specimens

Subcellular localization of triphenylethylene antiestrogen binding sites (TABS) in rat liver

The subcellular distribution of triphenylethylene anti-estrogen binding sites (TABS) was examined in the rat liver. Nuclear, mitochondrial and microsomal fractions were prepared by differential centrifugation, extracted with 0.5 M KCl and bound [ 3H]tamoxifen was determined by the dextran coated charcoal method. The relative concentration of TABS in each fractions were: nuclear 30.2; mitochondrial, 14.8 and microsomal, 10.2 pmol/g tissues. No TABS were detected in the high speed cytosol. The dissociation constants of nuclear and mitochondrial TABS were similar (1–2 nM); however, a higher number was obtained for microsomal TABS (5–6 mM). The ability of other triphenylethylene drugs to compete for [ 3H]tamoxifen binding to TABS was similar to tamoxifen for mitochondrial and microsomal sites. In contrast, nafoxidine was a more potent inhibitory for nuclear TABS. Exposure of high salt nuclear extracts to charcoal prior to assay did not reveal any evidence for an endogenous ligand of high affinity. We conclude that TABS are present in nuclear, mitochondrial and microsomal fractions of rat liver and that the nuclear fraction contains the highest concentration of these sites.