Journal of Toxicology and Environmental Health
ISSN: 0098-4108 (Print) (Online) Journal homepage: http://www.tandfonline.com/loi/uteh19
Nonsteroidal antiestrogens: Their biological effects and potential mechanisms of action V. Craig Jordan , Clive J. Dix , Karen E. Naylor , Graham Prestwich & Linda Rowsby To cite this article: V. Craig Jordan , Clive J. Dix , Karen E. Naylor , Graham Prestwich & Linda Rowsby (1978) Nonsteroidal antiestrogens: Their biological effects and potential mechanisms of action, Journal of Toxicology and Environmental Health, 4:2-3, 363-390, DOI: 10.1080/15287397809529666 To link to this article: http://dx.doi.org/10.1080/15287397809529666
Published online: 19 Oct 2009.
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NONSTEROIDAL ANTIESTROGENS: THEIR BIOLOGICAL EFFECTS AND POTENTIAL MECHANISMS OF ACTION V. Craig Jordan, Clive J. Dix, Karen E. Naylor, Graham Prestwich, Linda Rowsby
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Department of Pharmacology, School of Medicine, Leeds, England
The uterotropic and ontiuterotropic effects of a variety of structural derivatives of the nonsteroidal antiestrogen tamoxifen have been determined in the rat and the mouse. One derivative, monohydroxytamoxifen, was found to be a potent antiestrogen in the rat, with a high affinity for the estrogen receptor. Various techniques of sucrose density gradient analysis were used to demonstrate that estradiol and tamoxifen bind to the rat uterine cytoplasmic estrogen receptor. Estrogens and antiestrogens provoke the translocation of estrogen receptors to the nucleus and deplete the cytoplasmic estrogen receptor pool for short or long periods depending on the dose administered. Estradiol stimulates endometrial hyperplasia with an increase in total uterine DNA content, whereas tamoxifen stimulates endometrial hypertrophy with only a slight increase in uterine DNA content. It is concluded that the molecular shape of the ligand that binds to the estrogen receptor determines antiestrogenlc activity.
INTRODUCTION The report by Lerner et al. (1958) that the nonsteroidal compound MER-25 (ethamoxytriphetol) inhibits the effects of estradiol in laboratory animals and the subsequent finding that MER-25 is useful for the clinical control of endometrial hyperplasia and carcinoma (Kistner and Smith, 1961) stimulated a search for more potent derivatives. Clomiphene (chloramiphene or MRL-41) was found to be an antiestrogen with antifertility properties in laboratory animals (Holtkamp et al., 1960; Van Maanen et al., 1961), but ironically clinical trials We would like to thank Dr. Dora Richardson, Imperial Chemical Industries Ltd. (Pharmaceuticals Division), for the synthesis of compounds used in this study, and Mr. John Burn, Imperial Chemical Industries Ltd. (Pharmaceuticals Division), for the synthesis and purification of tritiated tamoxifen. We would also like to thank Mr. R. R. MacDonald, Department of Obstetrics and Gynaecology, Leeds Maternity Hospital, for his cooperation in obtaining suitable tissue samples for the investigations involving human estrogen receptors. This study was supported in part by generous grants from Imperial Chemical Industries Ltd. (Pharmaceuticals Division) and the Yorkshire Cancer Research Campaign. Requests for reprints should be sent to V. C. Jordan, Department of Pharmacology, School of Medicine, Leeds LS2 9NL, England.
363 Journal of Toxicology and Environmental Health, 4:363-390, 1978 Copyright © 1978 by Hemisphere Publishing Corporation 0098-4108/78/0402-0363 $2.25
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demonstrated that the drug stimulated ovulation in the human (Greenblatt et al., 1961) and it is now available as a profertility agent. In its crude form, clomiphene is a mixture of cis (zuclomiphene) and trans (enclomiphene) geometric isomers (Palopoli et al., 1967), but the discovery that only the trans isomers of triphenylethylenes are antiestrogenic (Harper and Walpole, 1966) provided a basis for future structure-activity relationship studies. Of all the compounds that have been screened for antiestrogenic and antifertility effects in laboratory animals, perhaps four—enclomiphene (Dipietro et al., 1969), tamoxifen (Harper and Walpole, 1967a, 1967b), CI-628 (Callantine et al., 1966), and nafoxidine (Duncan et al., 1963; Lednicer et al., 1967) (Fig. 1)—have received the most attention as tools in endocrinologic research. The discovery of potent nonsteroidal antiestrogens, however, has stimulated a new search for appropriate clinical applications. The knowledge that advanced breast cancer will respond to endocrine ablation has focused attention on the use of antihormone therapy as an alternative to surgery. In the laboratory MER-25 (Terenius, 1971a) and tamoxifen (Jordan, 1974, 1976a) inhibit the initiation, and nafoxidine (Terenius, 1971b), enclomiphene (Schultz et al., 1971), CI-628 (DeSombre and Arbogast, 1974), and tamoxifen (Nicholson and Golder, 1975; Jordan and Dowse, 1976; Jordan and Jaspan, 1976; Jordan and Koerner, 1976) inhibit the growth, of hormone-dependent rat mammary carcinomas. Similarly, in clinical trials nafoxidine (Heuson et al., 1972; Bloom and Boesen, 1974), clomiphene (mixed isomers) (Herbst et al., 1964; Hecker et al., 1974), and tamoxifen (Cole et al., 1971; Ward, 1973) have been shown to be effective in provoking objective tumor regression in approximately 30% of patients with advanced breast cancer. Tamoxifen has become the antiestrogen of choice in the treatment of advanced breast cancer because of the reported low incidence of side effects. Clearly, with the wider clinical application of nonsteroidal antiestrogen CH3 C2H5
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FIGURE 1. Structures of some nonsteroidal antiestrogens.
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TAMOXIFEN C2H5
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C2H5
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FIGURE 2. Structures of tamoxifen and its mono- and dihydroxylated derivatives.
therapy, it has now become essential to understand the mechanism (or mechanisms) of action of these drugs so that they can be used to their best advantage. Furthermore, the study of these compounds may provide additional insight into the control of estrogen-dependent events and potentially facilitate the design of more effective therapeutic agents. The present paper reviews our recent laboratory studies with nonsteroidal estrogens and antiestrogens and reviews the potential mechanisms whereby antiestrogens may prohibit estradiol-stimulated growth. METHODS Tamoxifen [1-(4-/3-dimethylaminoethoxyphenyl)-l,2-diphenylbut-'Iene], monohydroxytamoxifen [1-(4-|3-dimetnylaminoethoxyphenyl)-1-(4hydroxyphenyl)-2-phenylbut-1-ene], dihydroxytamoxifen [l-(4-]3-dimethylaminoethoxyphenyl)-1-(3,4-dihydroxyphenyl)-2-phenyl-2-phenylbut-1-ene] (Fig. 2), and ICI 3188 [1,1,2-(4-hydroxyphenyl)prop-1-ene] (Fig. 3) were
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ZUCLOMIPHENE
FIGURE 3. Structures of some nonsteroidal estrogens.
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gifts from Imperial Chemical Industries Ltd. (Pharmaceuticals Division). Estradiol-17/3 was obtained from Sigma Chemicals and diethylstilbestrol (DES) from British Drug Houses. All sc injections to immature rats were in 0.1 ml peanut oil and to ovariectomized mice in 0.05 ml peanut oil. Injections were prepared by taking aliquots from freshly made ethanolic solutions of compounds, adding the required volume of peanut oil, and evaporating the ethanol under N2 on a warm-water bath (60°C). [6,7-3H]-17/3-Estradiol (specific activity, 41 Ci/mmol) was obtained from the Radiochemical Centre, Amersham, 98% pure and was used without further purification. [1,2,6,7-3H] Progesterone (114 Ci/mmol) was obtained from New England Nuclear Corp. [ 3 H]Tamoxifen (19.47 Ci/mmol), with two tritium atoms ortho to the aminoethoxy side chain, was obtained from ICI Ltd. (Pharmaceuticals Division). Immature Rat and Ovariectomized Mouse Uterine Weight Test Immature female rats (35-45 g, Alderley Park Strain) were randomized into groups and injected (sc) with test compounds on three consecutive days and killed on d 4. Uteri were dissected free of adhering fat, the uterine fluid was expelled, and the blotted uteri were weighed wet on a torsion balance. Mature female mice (25-30 g, Tuck No. 1 strain) were ovariectomized under ether anesthesia and used for experiments 7 d later. Animals were randomized into groups and injected (sc) with test compounds on three consecutive days and killed on d 4. Uteri were weighed as described above. Steroid Receptor Assays in the Cytoplasm and Nucleus Cytoplasmic progesterone and estrogen receptors were measured by adaptations of the methods described by Liskowski et al. (1976) and Katzenellenbogen et al. (1973), respectively. Details of the methods have been published elsewhere (Jordan and Prestwich, 1978; Jordan et al., 1977a). Nuclear estrogen receptor levels were measured using the technique established by Anderson et al. (1970); the method has been published previously (Jordan et al., 1977a). DNA determinations were undertaken using the method described by Burton (1956) using calf thymus DNA standards. Interaction of Estradiol and Antiestrogens in Vitro The dextran-coated charcoal technique used to determine the inhibition by antiestrogen of the binding of [3H]estradiol to rat uterine estrogen receptors has been published elsewhere (Jordan et al., 1977b). The method used for sucrose density gradient analysis was an adaptation of the method reported by Jensen et al. (1971). The detailed methodology of swinging bucket rotor (SBR) and vertical tube rotor (VTR) sucrose
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density gradient analysis has been described in earlier reports (Jordan et al., 1977b; Jordan and Prestwich, 1977a, 1977b). Exchange assay techniques of SBR sucrose gradients were undertaken by incubating (30°C for 3 h) 200-/xl fractions of the gradient with a final concentration of 30 nmol/l, [3H]estradiol or 30 nmol/l [3H]estradiol and 3 jumol/l DES. Free ligand was removed by adding 300 jul of a 1% suspension of dextran-coated charcoal in Tris buffer and leaving for 25 min at 4°C. Tubes were centrifuged for 10 min at 2000 Xg (4°C) and a 400-jul sample of the supernatant was added to 10 ml tritium scintillator (6 g butyl PBD, 135 ml toluene, 720 ml dioxane, 100 g naphthalene, 45 ml methanol) and counted for 5 min in a Beckman LS3133T liquid scintillation spectrometer. Binding of [ 3 H]Estradiol in Vivo Immature female rats were injected with various doses of monohydroxytamoxifen or tamoxifen on three consecutive days. [ 3 H] Estradiol (0.08 ng) was injected sc in 0.1 ml arachis oil 24 h after the last dose of antiestrogen. Two hours later animals were killed and the uteri were dissected out, dried overnight in a 60°C oven, and burned in a Packard Tricarb tissue oxidizer. Radioactive water was collected into 10 ml tritium scintillator and counted as described above. Results were represented as femtomoles [3H]estradiol per uterus. RESULTS AND DISCUSSION Biological Effects of Nonsteroidal Antiestrogens in Laboratory Animals Tamoxifen is known to possess partial uterotropic activity in the rat (Harper and Walpole, 1967a) while being able to antagonize the uterotropic effects of concomitantly administered estradiol. By contrast, tamoxifen is fully estrogenic in the mouse (Harper and Walpole, 1967a; Terenius, 1971c) although large doses have been shown to induce a prolonged refractory state in the vagina, which is subsequently unable to respond to administered estradiol (Emmens, 1971; Jordan, 1975). The species differences are further illustrated by the recent report that tamoxifen is apparently a pure antiestrogen in the chick oviduct and possesses little or no detectable estrogenic activity (Sutherland et al., 1977). The aim of the present investigations was to compare the pharmacological effects of various derivatives of tamoxifen in laboratory animals so that structure-activity relationships could be established. The doseresponse curves of tamoxifen, monohydroxytamoxifen, and dihydroxytamoxifen were compared with those of estradiol-17(3 in the 3-d immature rat uterine weight test (Fig. 4). ICI 3188 was included in the test as a
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FIGURE 4. Uterotropic effect of estradiol, ICI 3188, tamoxifen, monohydroxytamoxifen, and dihydroxytamoxifen in the 3-d immature rat uterine weight test. Control animals received peanut oil alone. At least eight rats were used' per group. Results are means ± SEM. Data are from Jordan et al. (1977b) and Jordan (1976b).
hydroxylated triphenylethylene that did not possess the alkylaminoethoxy side chain that was common to the other derivatives of tamoxifen. In the uterotropic test, monohydroxytamoxifen appeared to be more potent than tamoxifen, while dihydroxytamoxifen was unable to induce any uterotropic response at the dose levels employed. Estradiol-17/3 and ICI 3188 both produced sigmoidal dose-response curves, associated with full agonist activity, whereas tamoxifen and monohydroxytamoxifen were only partial agonists in that the maximum uterine growth achieved was only approximately 50% of that obtained by the full estrogens. To directly compare the effects of an antiestrogen and a long-acting estrogen on the resulting uterine growth in the rat, we administered tamoxifen (25 fig) or estradiol benzoate (25 jug) to groups of immature rats and sacrificed the animals at various times up to 72 h. The uterine responses are illustrated in Fig. 5. Tamoxifen produced a partial rise in uterine wet weight compared with the response observed after the administration of estradiol benzoate. The rise in uterine weight provoked by tamoxifen, however, was associated with only a small rise in uterine DNA content compared with the doubling of total uterine DNA content observed in animals treated with estradiol benzoate. This result is consistent with our previous reports that tamoxifen does not provoke a
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consistent rise in ovariectomized (Jordan, 1976b) or immature (Jordan et al.,. 1977a) rat total uterine DNA content. An earlier study by Kang et al. (1975) compared the effects of CI-628 and estradiol on the growth and histology of the immature rat uterus. They showed that CI-628 caused the endometrial cells to hypertrophy with virtually no increase in the incorporation of [ 3 H]thymidine, whereas estradiol stimulated endometrial hyperplasia, which was associated with an increased incorporation of [ 3 H]thymidine. Recently, we suggested (Clark et al., 1978) that the partial rise in immature rat uterine wet weight that occurs during tamoxifen stimulation may be accounted for by uterine hypertrophy because the endometrial cells increase in size without undergoing mitosis. In the immature rat uterine weight test for antiestrogenic activity, monohydroxytamoxifen was found to be more potent than tamoxifen (Fig. 6). Dihydroxytamoxifen was found to inhibit the uterotropic activity of estradiol, although the activity was less than that observed with tamoxifen (Jordan et al., 1977b). ICI 3188 was found to possess no antiestrogenic activity (V. C. Jordan and L. Rowsby, unpublished observation). The importance of the presence of the aminoethoxy side chain in a particular position in space, for effective antiestrogenic activity, has previously been illustrated among a group of structural derivatives of nafoxidine (Lednicer et al., 1967). Similarly, we have shown that
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FIGURES. Effect of tamoxifen (25 jig) or estradiol benzoate (25 Mg) on (A) uterine wet weight and (B) uterine DNA content. Control animals were treated with saline; 72 rats per group. Results represent means ± SEM. Data are from Clark et al. (1978).
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FIGURE 6. Antiestrogenic effect of monohydroxytamoxifem and tatnoxifen given with estradiol (0.08 /jg/d) in the 3-d immature rat uterine weight test. Control animals received peanut oil alone; 10 rats per group. Results represent means ± SEM. Data are from Jordan et al. (1978).
restriction of the positions that the aminoethoxy side chain can adopt in space, by the introduction of two methyl groups ortho to the ether oxygen in the phenyl ring, reduces tamoxifen's ability to compete with [3H]-17j3estradiol for mouse uterine estrogen receptors in vitro (Abbot et al., 1976), and a similar substitution in MER-25 destroys its antiestrogenic activity in vivo (Clark and Jordan, 1976).
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u 40 MONOHYOROXYTAMOXtfEN
DIHYDROXYTAMOXIFEN
04 1« DAILY DOSE OF COMPOUNDS
FIGURE 7. Uterotropic effect of estradiol, tamoxifen, monohydroxytamoxifen, and dihydroxytamoxifen in the 3-d ovariectomized mouse uterine weight test. Control animals received peanut oil alone; seven mice per group. Results represent means ± SEM.
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In the 3-d ovariectomized mouse uterine weight test, tamoxifen and monohydroxytamoxifen were fully uterotropic when compared with estradiol-17j3 while dihydroxytamoxifen was only partially uterotropic (Fig. 7). In antiestrogenic tests in the ovariectomized mouse, neither tamoxifen nor monohydroxytamoxifen possessed any antiuterotropic activity, whereas daily doses of 16, 32, and 64 jug dihydroxytamoxifen were effective in antagonizing the uterotropic effects of 0.024 ng estradiol per day. Even though the biological responses to tamoxifen, monohydroxytamoxifen, and dihydroxytamoxifen were different in the rat and mouse uterus, the relative potencies of the compounds were similar in both species. In general, the mouse appeared to be less sensitive to the effects of the triphenylethylenes and it is interesting to note that the antiestrogen MER-25 also appears to be less active in the mouse than in the rat (Emmens et al., 1960). Since monohydroxytamoxifen was very active as an antiestrogen in the immature rat uterine weight test, we compared this potent new compound with tamoxifen in our subsequent experiments. Subcellular Effects of Nonsteroidal Antiestrogens Estradiol is believed to stimulate uterine growth by selectively binding to estrogen receptor proteins located in the cell cytoplasm, which are then translocated in association with the steroid to the nucleus, where estrogeninduced synthetic events are initiated (Jensen and DeSombre, 1973). Nonsteroidal antiestrogens have been shown to competitively antagonize the binding of [3H]estradiol to the cytoplasmic estrogen receptor in vitro (Korenman, 1970; Skidmore et al., 1972). Furthermore, by using sucrose density gradient analysis to identify the binding protein, nonsteroidal antiestrogens have been found to inhibit the binding of [ 3 H ] estradiol to the 8S estrogen receptor derived from human (Jensen et al., 1972; Hunter and Jordan, 1975; Jordan and Koerner, 1975) and animal (Powell-Jones et al., 1975; Jordan and Dowse, 1976) estrogen target tissues. In the present study we have compared the ability of estradiol-17/3, monohydroxytamoxifen, and tamoxifen to inhibit the binding of [ 3 H ] estradiol to rat uterine estrogen receptors in vitro, using a dextran-coated charcoal assay. The introduction of the hydroxyl group into tamoxifen had a dramatic effect on its ability to inhibit the binding of [ 3 H] estradiol. Monohydroxytamoxifen was almost equipotent with nonradioactive estradiol (Fig. 8). Similarly, sucrose density gradient analysis (SBR) showed that tamoxifen and monohydroxytamoxifen (one-tenth the concentration of tamoxifen) were effective in inhibiting the binding of [ 3 H] estradiol to the 8S estrogen receptor derived from human endometrium (Fig. 9). This result has been compared with the dose-related ability of tamoxifen to inhibit the binding of [3H]estradiol to the human endometrium 85 estrogen receptor, determined by using a VTR.
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LIGAND CONCENTRATION (mol/l)
FIGURE 8. Effect of increasing concentrations of estradiol, monohydroxytamoxifen, or tamoxifen on the specific binding of [ 3 H]estradiol (2 X 10"' mol/l) to uterine cytosol from immature rats. Data are from Jordan et al. (1977b).
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FIGURE 9. Sucrose density gradient analysis [5-20% in (A) and 10-30% in (B)]. (A) The effect of (o) tamoxifen (1 X IO" 6 mol/l) or (*) monohydroxytamoxifen (1 X IO" 1 mol/l) on the binding of (•) [3 H] estradiol (2 X 1 0 " ' mol/l) in human endometrial cytosol using a swinging bucket rotor (15 h, 225,000 X g, 4°C). (B) The effect of increasing concentrations of tamoxifen at (o) 2X 10" 8 mol/l, (A) 8X 10" 8 mol/l, and (•) 3.2 X 10" 7 mol/l on the binding of [ 3 H]estradiol (5 X 1 0 " ' mol/t) in human endometrial cytosol using a vertical tube rotor (2 h, 400,000 X g, 4°C). BSA was run in a separate tube as a sedimentation standard (4.6S). Data in (A) are from Jordan et al. (1977b).
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The high affinity of monohydroxytamoxifen for the estrogen receptor suggests that the inclusion of a hydroxyl group at the para position in the C-1 phenyl in tamoxifen may result in an interaction of this hydroxyl at the same site on the estrogen receptor normally occupied by the C-3 phenolic hydroxyl of estradiol. It is perhaps relevant to consider that when CI-680 (a structural analog of CI-628) is demethylated to expose a free hydroxyl group at the same position as in monohydroxytamoxifen, there is also an increased affinity for the estrogen receptor (Ferguson and Katzenellenbogen, 1977). Both nafoxidine. and CI-628 (Fig. 1) have methoxy groups substituted at the same position in the phenyl ring, but whether demethylation to the free hydroxylated compounds has a major role to play in the mechanism of action of nonsteroidal antiestrogens must await the completion of pharmacokinetic studies to determine the relationship between the parent drug and its metabolites. Certainly it is tempting to speculate by analogy with the estrogen mestranol, whose affinity for the estrogen receptor is increased by demethylation to ethinylestradiol (Eisenfeld, 1974), that demethylation of antiestrogens may account for their long biological half-lives. Monohydroxytamoxifen has been detected in the serum of laboratory animals (Fromson et al., 1973a) and patients (Fromson et al., 1973b) treated with tamoxifen, which suggests that this potent metabolite may contribute to the maintenance of the antiestrogenic effect and be beneficial in the long-term control of hormone-dependent cancers. The nonsteroidal antiestrogens nafoxidine (Clark et al., 1973; Capony and Rochefort, 1975; Katzenellenbogen and Ferguson, 1975), enclomiphene (Ruh and Baudendistel, 1977), and tamoxifen (Jordan et al., 1977a) have each been shown to translocate estrogen receptors from the cytoplasm to the nucleus in the rat uterus. In general, each of these studies has compared estradiol-17/3 with a nonsteroidal antiestrogen and found that the time course of retention of estrogen receptors in the nucleus after estradiol was a few hours, whereas after nonsteroidal antiestrogens, estrogen receptors were present in the nucleus for several days. We have now compared the effects of estradiol (5 jug), monohydroxytamoxifen (5 fig), and tamoxifen (25 jug) on the measurement of estrogen receptors in the rat uterine nucleus for 48 h after administration (Fig. 10). Tamoxifen maintained a consistently high level of estrogen receptors within the nuclear compartment for up to 48 h, whereas the initial rise produced by estradiol had returned to control levels by 24 h. In contrast to tamoxifen, monohydroxytamoxifen produced a pattern of nuclear estrogen receptor concentrations similar to that seen after estradiol. Ferguson and Katzenellenbogen (1977) recently reported a similar finding with a structurally related nonsteroidal antiestrogen, 9411 X 27 (94X). Presumably as a result of the translocation of estrogen receptors to the
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FIGURE 10. Levels of estrogen receptors determined by nuclear exchange assay (Anderson et al., 1970) in the nucleus of immature female rats at various times after the administration of tamoxifen (25 jig), monohydroxytamoxifen (5 /ig), and estradiol (5 /jg). Control animals received peanut oil alone; 12 rats per group. Results represent means ± SEM.
nucleus, nonsteroidal antiesfrogens have been found to stimulate an increase in the concentration of progesterone receptors in the hamster uterus (Leavitt et a!., 1977) and in human hormone-dependent breast cancer cells grown in tissue culture (Horwitz and McGuire, 1977). Since it has been reported that the nonsteroidal antiestrogen nafoxidine can increase the activity of rat uterine RNA polymerase (Hardin et al., 1976), we have determined whether the increase in uterine weight in response to tamoxifen and monohydroxytamoxifen is associated with an increase in protein synthetic capacity, as demonstrated by an increase in progesterone receptor content. Tamoxifen (50 /ig), estradiol (5 jug), or monohydroxytamoxifen (50 jug) was administered as a single injection to ovariectomized rats. Tamoxifen produced a slow rise in uterine progesterone receptor concentration, whereas estradiol and monohydroxytamoxifen both produced a rapid rise in uterine progesterone receptor concentrations at 6 h (Fig. 11). Thus estrogen receptors that are translocated to the nucleus by nonsteroidal antiestrogens are capable of provoking protein synthesis and cellular hypertrophy but, as already discussed in the previous section, they are unable to affect an efficient endometrial hyperplasia. Whether the antiestrogen-estrogen receptor complex is incapable of interacting with an as yet undetermined nuclear acceptor site, which is necessary to provoke DNA synthesis, or whether the cytoplasmic protein synthesis that is
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FIGURE 11. Effect of monohydroxytamoxifen (50 jig), tamoxifen (50 ii%), and estradiol (5 Mg) on the progesterone receptor content of ovariectomized rat uteri. Control animals received peanut oil; five rats per group. Results represent means ± SEM. Data are from Jordan and Prestwich (1978).
stimulated by antiestrogens is out of phase with the requirements of cell division remains to be determined. In the following section we will consider some of the current theories of antiestrogen action in order to formulate a strategy for future investigations. Potential Mechanisms of Action of Nonsteroidal Antiestrogens Based on a generalized model of estrogen action in target tissues (Fig. 12), nonsteroidal antiestrogens can potentially disrupt this scheme of events at one or more stages (or at an as yet undetermined site): competition with estradiol for the cytoplasmic estrogen receptor and subsequent INHIBITION OF RECEPTOR RESYNTHESIS
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FIGURE 12. Potential mechanisms of action of antiestrogens in the target tissue cell.
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inhibition of receptor transformation and translocation, or alternatively specific inhibition of estrogen receptor resynthesis, thus rendering the tissue refractory to subsequent estrogenic stimuli. Antiestrogens can inhibit the binding of estradiol to the estrogen receptor in vitro (Fig. 8), and similarly the administration of the antiestrogen nafoxidine (Jensen et al., 1972), clomiphene (Roy et al., 1964), CI-628 (Perry et al., 1973), or tamoxifen (Jordan, 1976a; Jordan and Dowse, 1976) can prevent the binding of [3H]estradiol target tissues in vivo. Before the development of nuclear exchange assays to determine ligand-filled sites (Anderson et al., 1970), it was conceivable that the estrogen receptor could be neutralized in the cytoplasm by nonsteroidal antiestrogens. This is now known to be untrue since the levels of exchangeable estrogen receptors rise in the nucleus after the administration of nonsteroidal antiestrogens (Clark et al., 1973) (Fig. 10). Once estrogen binds to the cytoplasmic estrogen receptor, the complex is transformed to a higher-molecular-weight unit (Brecher et al., 1970). Studies in vitro using sucrose density gradient analysis have shown that the AS estrogen-receptor complex that is present in buffer containing 0.4 M KG becomes converted to a 55 estrogen-receptor complex on gentle warming (Notides and Nielsen, 1974). The precise significance of this reaction is unknown, although it has been suggested that this step is necessary before estrogen receptors can activate RNA polymerase in the nucleus (Jensen et al., 1973). Similar attempts to investigate this reaction when antiestrogens are the binding ligands have been hampered by the lack of high-specific-activity radiolabeled antiestrogens with a high binding affinity for the estrogen receptor. At present, therefore, it is not known whether nonsteroidal antiestrogens do permit the transformation reaction prior to nuclear entry. When estradiol translocates estrogen receptors to the nucleus, the cytoplasmic estrogen receptor pool is replenished during the following 24 h by a process of resynthesis (Sarff and Gorski, 1971). Since nonsteroidal antiestrogens deplete the cytoplasmic estrogen receptor pool for longer than 24 h (Clark et al., 1973; Katzenellenbogen and Ferguson, 1975), it has been suggested that nonsteroidal antiestrogens specifically inhibit the resynthesis of cytoplasmic estrogen receptors, thus rendering the tissue refractory to subsequent estrogenic stimuli (Clark et al., 1974). In the present study we have addressed the question of cytoplasmic estrogen receptor replenishment in response to estrogenic and antiestrogenic stimuli in order to determine whether the prolonged depletion of the cytoplasmic estrogen receptor pool is an exclusive characteristic of nonsteroidal antiestrogens. First, we compared the antiestrogen ic activity of tamoxifen and monohydroxytamoxifen in the 3-d immature rat uterine weight test and also determined the levels of available cytoplasmic estrogen receptors 24 h after the last dose of antiestrogens. It is clear from Fig. 13 that the
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DAILY D O S E OF C O M P O U N D ((ifl)
FIGURE 13. Effect of daily administration of estradiol (0.08 jug), estradiol and various doses of monohydroxytamoxifen (•), or estradiol and various doses of tamoxifen (•) for 3 d on the uterine levels of available cytoplasmic estrogen receptors determined 24 h after the last dose. Control animals received peanut oil; 10 rats per group. Results represent means ± SEM.
depletion of cytoplasmic estrogen receptor levels is a dose-related phenomenon, and antiestrogenic effects are apparent (Fig. 6) before a depletion of estrogen receptor levels below those of control uteri. The observation that estrogen receptors are apparently available for binding estradiol during an antiestrogenic uterine weight test with tamoxifen is consistent with our earlier report (Jordan et al., 1977a). To ensure that the available uterine estrogen receptors determined in vitro were truly available to bind estradiol in vivo, we determined the effect of the administration of different daily doses of tamoxifen (2, 8, and 32 jug) or monohydroxytamoxifen (0.32, 1.28, and 5.12 /ig) on the uterine binding of [3H]estradiol (0.08 jug) administered 24 h after the last dose of the antiestrogens. Again, the effect of the antiestrogens was related to the dose administered, but at doses of tamoxifen (8 jug/d) or monohydroxytamoxifen (1.28 jug/d) that had previously been found to be antiestrogenic, there was no significant difference (determined by Student's Mest) between the levels of [3H]estradiol in treated and control uteri (Fig. 14). Therefore, the depletion of estrogen receptors in the rat uterine cytoplasm is related to the dose of the antiestrogen administered, and the
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FIGURE 14. Effect of different daily doses of monohydroxytamoxifen or tamoxifen for 3 d on the binding of [ 3 H]estradiol (0.08 ti%) injected 24 h after the last dose of antiestrogen. Levels of radioactivity in the uterus were determined 2 h after the administration of [3 H] estradiol. Six rats per group. Results represent means ± SEM.
antagonism of estrogen action apparently occurs before estrogen binding capacity is completely denied. To extend this observation, we determined the effects of increasing doses of nonsteroidal estrogens on the available cytoplasmic estrogen receptor pool. ICI 3188 was administered on three consecutive days to groups of immature female rats, and 24 h after the last dose of the estrogen available cytoplasmic estrogen receptor levels were determined. ICI 3188 produced a full uterotropic response, which plateaued at maximal stimulation, whereas increasing doses of the compound produced a dose-related decrease in the levels of cytoplasmic estrogen receptors (Fig. 15). We also found that increasing doses of tamoxifen's estrogenic cis isomer, ICI 47699, produced a dose-related decrease in cytoplasmic estrogen receptor content with a corresponding rise in the concentration of estrogen receptors within the nucleus (Jordan et al., 1978). Since the depletion of cytoplasmic estrogen receptors appears to be a function of the dose of any compound that will translocate receptors to the.nucleus, then the differences observed between the effects of estradiol and nonsteroidal antiestrogens on the measurement of cytoplasmic estrogen receptors are probably related to differences in their respective biological half-lives. Blood levels of estradiol are maintained for only a few hours after administration (Castracane and Jordan, 1975), whereas nonsteroidal antiestrogens like tamoxifen have a biological half-life of several
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days (Fromson et al., 1973a). Therefore, it is possible that estrogen receptor resynthesis does occur after antiestrogen administration, but the newly synthesized receptors are never allowed to accumulate within the cytoplasm since binding ligands from the blood immediately provoke translocation to the nucleus. This interpretation would account for the reports of high levels of estrogen receptors within the nucleus for prolonged periods after antiestrogen administration (Clark et al., 1973; Katzenellenbogen and Ferguson, 1975) and would suggest that these nuclear estrogen receptors are a dynamic pool that are being processed in order to initiate biosynthetic events in the cytoplasm, and are then immediately replenished by resynthesized receptors from the cytoplasm. The short-term retention of estrogen receptors in the nucleus after monohydroxytamoxifen (Fig. 10) could be caused by a shorter biological half-life since it would be expected that this compound would be readily conjugated in the liver and the products rapidly excreted (see Ferguson and Katzenellenbogen, 1977). Since both estrogens and nonsteroidal antiestrogens can translocate estrogen receptors from the cytoplasm to the nucleus, but only estrogens
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ISSN: 0098-4108 (Print) (Online) Journal homepage: http://www.tandfonline.com/loi/uteh19
Nonsteroidal antiestrogens: Their biological effects and potential mechanisms of action V. Craig Jordan , Clive J. Dix , Karen E. Naylor , Graham Prestwich & Linda Rowsby To cite this article: V. Craig Jordan , Clive J. Dix , Karen E. Naylor , Graham Prestwich & Linda Rowsby (1978) Nonsteroidal antiestrogens: Their biological effects and potential mechanisms of action, Journal of Toxicology and Environmental Health, 4:2-3, 363-390, DOI: 10.1080/15287397809529666 To link to this article: http://dx.doi.org/10.1080/15287397809529666
Published online: 19 Oct 2009.
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NONSTEROIDAL ANTIESTROGENS: THEIR BIOLOGICAL EFFECTS AND POTENTIAL MECHANISMS OF ACTION V. Craig Jordan, Clive J. Dix, Karen E. Naylor, Graham Prestwich, Linda Rowsby
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Department of Pharmacology, School of Medicine, Leeds, England
The uterotropic and ontiuterotropic effects of a variety of structural derivatives of the nonsteroidal antiestrogen tamoxifen have been determined in the rat and the mouse. One derivative, monohydroxytamoxifen, was found to be a potent antiestrogen in the rat, with a high affinity for the estrogen receptor. Various techniques of sucrose density gradient analysis were used to demonstrate that estradiol and tamoxifen bind to the rat uterine cytoplasmic estrogen receptor. Estrogens and antiestrogens provoke the translocation of estrogen receptors to the nucleus and deplete the cytoplasmic estrogen receptor pool for short or long periods depending on the dose administered. Estradiol stimulates endometrial hyperplasia with an increase in total uterine DNA content, whereas tamoxifen stimulates endometrial hypertrophy with only a slight increase in uterine DNA content. It is concluded that the molecular shape of the ligand that binds to the estrogen receptor determines antiestrogenlc activity.
INTRODUCTION The report by Lerner et al. (1958) that the nonsteroidal compound MER-25 (ethamoxytriphetol) inhibits the effects of estradiol in laboratory animals and the subsequent finding that MER-25 is useful for the clinical control of endometrial hyperplasia and carcinoma (Kistner and Smith, 1961) stimulated a search for more potent derivatives. Clomiphene (chloramiphene or MRL-41) was found to be an antiestrogen with antifertility properties in laboratory animals (Holtkamp et al., 1960; Van Maanen et al., 1961), but ironically clinical trials We would like to thank Dr. Dora Richardson, Imperial Chemical Industries Ltd. (Pharmaceuticals Division), for the synthesis of compounds used in this study, and Mr. John Burn, Imperial Chemical Industries Ltd. (Pharmaceuticals Division), for the synthesis and purification of tritiated tamoxifen. We would also like to thank Mr. R. R. MacDonald, Department of Obstetrics and Gynaecology, Leeds Maternity Hospital, for his cooperation in obtaining suitable tissue samples for the investigations involving human estrogen receptors. This study was supported in part by generous grants from Imperial Chemical Industries Ltd. (Pharmaceuticals Division) and the Yorkshire Cancer Research Campaign. Requests for reprints should be sent to V. C. Jordan, Department of Pharmacology, School of Medicine, Leeds LS2 9NL, England.
363 Journal of Toxicology and Environmental Health, 4:363-390, 1978 Copyright © 1978 by Hemisphere Publishing Corporation 0098-4108/78/0402-0363 $2.25
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demonstrated that the drug stimulated ovulation in the human (Greenblatt et al., 1961) and it is now available as a profertility agent. In its crude form, clomiphene is a mixture of cis (zuclomiphene) and trans (enclomiphene) geometric isomers (Palopoli et al., 1967), but the discovery that only the trans isomers of triphenylethylenes are antiestrogenic (Harper and Walpole, 1966) provided a basis for future structure-activity relationship studies. Of all the compounds that have been screened for antiestrogenic and antifertility effects in laboratory animals, perhaps four—enclomiphene (Dipietro et al., 1969), tamoxifen (Harper and Walpole, 1967a, 1967b), CI-628 (Callantine et al., 1966), and nafoxidine (Duncan et al., 1963; Lednicer et al., 1967) (Fig. 1)—have received the most attention as tools in endocrinologic research. The discovery of potent nonsteroidal antiestrogens, however, has stimulated a new search for appropriate clinical applications. The knowledge that advanced breast cancer will respond to endocrine ablation has focused attention on the use of antihormone therapy as an alternative to surgery. In the laboratory MER-25 (Terenius, 1971a) and tamoxifen (Jordan, 1974, 1976a) inhibit the initiation, and nafoxidine (Terenius, 1971b), enclomiphene (Schultz et al., 1971), CI-628 (DeSombre and Arbogast, 1974), and tamoxifen (Nicholson and Golder, 1975; Jordan and Dowse, 1976; Jordan and Jaspan, 1976; Jordan and Koerner, 1976) inhibit the growth, of hormone-dependent rat mammary carcinomas. Similarly, in clinical trials nafoxidine (Heuson et al., 1972; Bloom and Boesen, 1974), clomiphene (mixed isomers) (Herbst et al., 1964; Hecker et al., 1974), and tamoxifen (Cole et al., 1971; Ward, 1973) have been shown to be effective in provoking objective tumor regression in approximately 30% of patients with advanced breast cancer. Tamoxifen has become the antiestrogen of choice in the treatment of advanced breast cancer because of the reported low incidence of side effects. Clearly, with the wider clinical application of nonsteroidal antiestrogen CH3 C2H5
"
Ng-j/
ENCIOMIPHENE
CH
CH 3
TAMOXIFEN
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NAFOXIDINE
FIGURE 1. Structures of some nonsteroidal antiestrogens.
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TAMOXIFEN C2H5
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C2H5
MONOHYDROXYTAMOXIFEN
DIHYDROXYTAMOXIFEN
FIGURE 2. Structures of tamoxifen and its mono- and dihydroxylated derivatives.
therapy, it has now become essential to understand the mechanism (or mechanisms) of action of these drugs so that they can be used to their best advantage. Furthermore, the study of these compounds may provide additional insight into the control of estrogen-dependent events and potentially facilitate the design of more effective therapeutic agents. The present paper reviews our recent laboratory studies with nonsteroidal estrogens and antiestrogens and reviews the potential mechanisms whereby antiestrogens may prohibit estradiol-stimulated growth. METHODS Tamoxifen [1-(4-/3-dimethylaminoethoxyphenyl)-l,2-diphenylbut-'Iene], monohydroxytamoxifen [1-(4-|3-dimetnylaminoethoxyphenyl)-1-(4hydroxyphenyl)-2-phenylbut-1-ene], dihydroxytamoxifen [l-(4-]3-dimethylaminoethoxyphenyl)-1-(3,4-dihydroxyphenyl)-2-phenyl-2-phenylbut-1-ene] (Fig. 2), and ICI 3188 [1,1,2-(4-hydroxyphenyl)prop-1-ene] (Fig. 3) were
ICI 47699
ZUCLOMIPHENE
FIGURE 3. Structures of some nonsteroidal estrogens.
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gifts from Imperial Chemical Industries Ltd. (Pharmaceuticals Division). Estradiol-17/3 was obtained from Sigma Chemicals and diethylstilbestrol (DES) from British Drug Houses. All sc injections to immature rats were in 0.1 ml peanut oil and to ovariectomized mice in 0.05 ml peanut oil. Injections were prepared by taking aliquots from freshly made ethanolic solutions of compounds, adding the required volume of peanut oil, and evaporating the ethanol under N2 on a warm-water bath (60°C). [6,7-3H]-17/3-Estradiol (specific activity, 41 Ci/mmol) was obtained from the Radiochemical Centre, Amersham, 98% pure and was used without further purification. [1,2,6,7-3H] Progesterone (114 Ci/mmol) was obtained from New England Nuclear Corp. [ 3 H]Tamoxifen (19.47 Ci/mmol), with two tritium atoms ortho to the aminoethoxy side chain, was obtained from ICI Ltd. (Pharmaceuticals Division). Immature Rat and Ovariectomized Mouse Uterine Weight Test Immature female rats (35-45 g, Alderley Park Strain) were randomized into groups and injected (sc) with test compounds on three consecutive days and killed on d 4. Uteri were dissected free of adhering fat, the uterine fluid was expelled, and the blotted uteri were weighed wet on a torsion balance. Mature female mice (25-30 g, Tuck No. 1 strain) were ovariectomized under ether anesthesia and used for experiments 7 d later. Animals were randomized into groups and injected (sc) with test compounds on three consecutive days and killed on d 4. Uteri were weighed as described above. Steroid Receptor Assays in the Cytoplasm and Nucleus Cytoplasmic progesterone and estrogen receptors were measured by adaptations of the methods described by Liskowski et al. (1976) and Katzenellenbogen et al. (1973), respectively. Details of the methods have been published elsewhere (Jordan and Prestwich, 1978; Jordan et al., 1977a). Nuclear estrogen receptor levels were measured using the technique established by Anderson et al. (1970); the method has been published previously (Jordan et al., 1977a). DNA determinations were undertaken using the method described by Burton (1956) using calf thymus DNA standards. Interaction of Estradiol and Antiestrogens in Vitro The dextran-coated charcoal technique used to determine the inhibition by antiestrogen of the binding of [3H]estradiol to rat uterine estrogen receptors has been published elsewhere (Jordan et al., 1977b). The method used for sucrose density gradient analysis was an adaptation of the method reported by Jensen et al. (1971). The detailed methodology of swinging bucket rotor (SBR) and vertical tube rotor (VTR) sucrose
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density gradient analysis has been described in earlier reports (Jordan et al., 1977b; Jordan and Prestwich, 1977a, 1977b). Exchange assay techniques of SBR sucrose gradients were undertaken by incubating (30°C for 3 h) 200-/xl fractions of the gradient with a final concentration of 30 nmol/l, [3H]estradiol or 30 nmol/l [3H]estradiol and 3 jumol/l DES. Free ligand was removed by adding 300 jul of a 1% suspension of dextran-coated charcoal in Tris buffer and leaving for 25 min at 4°C. Tubes were centrifuged for 10 min at 2000 Xg (4°C) and a 400-jul sample of the supernatant was added to 10 ml tritium scintillator (6 g butyl PBD, 135 ml toluene, 720 ml dioxane, 100 g naphthalene, 45 ml methanol) and counted for 5 min in a Beckman LS3133T liquid scintillation spectrometer. Binding of [ 3 H]Estradiol in Vivo Immature female rats were injected with various doses of monohydroxytamoxifen or tamoxifen on three consecutive days. [ 3 H] Estradiol (0.08 ng) was injected sc in 0.1 ml arachis oil 24 h after the last dose of antiestrogen. Two hours later animals were killed and the uteri were dissected out, dried overnight in a 60°C oven, and burned in a Packard Tricarb tissue oxidizer. Radioactive water was collected into 10 ml tritium scintillator and counted as described above. Results were represented as femtomoles [3H]estradiol per uterus. RESULTS AND DISCUSSION Biological Effects of Nonsteroidal Antiestrogens in Laboratory Animals Tamoxifen is known to possess partial uterotropic activity in the rat (Harper and Walpole, 1967a) while being able to antagonize the uterotropic effects of concomitantly administered estradiol. By contrast, tamoxifen is fully estrogenic in the mouse (Harper and Walpole, 1967a; Terenius, 1971c) although large doses have been shown to induce a prolonged refractory state in the vagina, which is subsequently unable to respond to administered estradiol (Emmens, 1971; Jordan, 1975). The species differences are further illustrated by the recent report that tamoxifen is apparently a pure antiestrogen in the chick oviduct and possesses little or no detectable estrogenic activity (Sutherland et al., 1977). The aim of the present investigations was to compare the pharmacological effects of various derivatives of tamoxifen in laboratory animals so that structure-activity relationships could be established. The doseresponse curves of tamoxifen, monohydroxytamoxifen, and dihydroxytamoxifen were compared with those of estradiol-17(3 in the 3-d immature rat uterine weight test (Fig. 4). ICI 3188 was included in the test as a
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002
0-1
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DAILY DOSE OF COMPOUNDS (|ig)
FIGURE 4. Uterotropic effect of estradiol, ICI 3188, tamoxifen, monohydroxytamoxifen, and dihydroxytamoxifen in the 3-d immature rat uterine weight test. Control animals received peanut oil alone. At least eight rats were used' per group. Results are means ± SEM. Data are from Jordan et al. (1977b) and Jordan (1976b).
hydroxylated triphenylethylene that did not possess the alkylaminoethoxy side chain that was common to the other derivatives of tamoxifen. In the uterotropic test, monohydroxytamoxifen appeared to be more potent than tamoxifen, while dihydroxytamoxifen was unable to induce any uterotropic response at the dose levels employed. Estradiol-17/3 and ICI 3188 both produced sigmoidal dose-response curves, associated with full agonist activity, whereas tamoxifen and monohydroxytamoxifen were only partial agonists in that the maximum uterine growth achieved was only approximately 50% of that obtained by the full estrogens. To directly compare the effects of an antiestrogen and a long-acting estrogen on the resulting uterine growth in the rat, we administered tamoxifen (25 fig) or estradiol benzoate (25 jug) to groups of immature rats and sacrificed the animals at various times up to 72 h. The uterine responses are illustrated in Fig. 5. Tamoxifen produced a partial rise in uterine wet weight compared with the response observed after the administration of estradiol benzoate. The rise in uterine weight provoked by tamoxifen, however, was associated with only a small rise in uterine DNA content compared with the doubling of total uterine DNA content observed in animals treated with estradiol benzoate. This result is consistent with our previous reports that tamoxifen does not provoke a
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consistent rise in ovariectomized (Jordan, 1976b) or immature (Jordan et al.,. 1977a) rat total uterine DNA content. An earlier study by Kang et al. (1975) compared the effects of CI-628 and estradiol on the growth and histology of the immature rat uterus. They showed that CI-628 caused the endometrial cells to hypertrophy with virtually no increase in the incorporation of [ 3 H]thymidine, whereas estradiol stimulated endometrial hyperplasia, which was associated with an increased incorporation of [ 3 H]thymidine. Recently, we suggested (Clark et al., 1978) that the partial rise in immature rat uterine wet weight that occurs during tamoxifen stimulation may be accounted for by uterine hypertrophy because the endometrial cells increase in size without undergoing mitosis. In the immature rat uterine weight test for antiestrogenic activity, monohydroxytamoxifen was found to be more potent than tamoxifen (Fig. 6). Dihydroxytamoxifen was found to inhibit the uterotropic activity of estradiol, although the activity was less than that observed with tamoxifen (Jordan et al., 1977b). ICI 3188 was found to possess no antiestrogenic activity (V. C. Jordan and L. Rowsby, unpublished observation). The importance of the presence of the aminoethoxy side chain in a particular position in space, for effective antiestrogenic activity, has previously been illustrated among a group of structural derivatives of nafoxidine (Lednicer et al., 1967). Similarly, we have shown that
no
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FIGURES. Effect of tamoxifen (25 jig) or estradiol benzoate (25 Mg) on (A) uterine wet weight and (B) uterine DNA content. Control animals were treated with saline; 72 rats per group. Results represent means ± SEM. Data are from Clark et al. (1978).
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FIGURE 6. Antiestrogenic effect of monohydroxytamoxifem and tatnoxifen given with estradiol (0.08 /jg/d) in the 3-d immature rat uterine weight test. Control animals received peanut oil alone; 10 rats per group. Results represent means ± SEM. Data are from Jordan et al. (1978).
restriction of the positions that the aminoethoxy side chain can adopt in space, by the introduction of two methyl groups ortho to the ether oxygen in the phenyl ring, reduces tamoxifen's ability to compete with [3H]-17j3estradiol for mouse uterine estrogen receptors in vitro (Abbot et al., 1976), and a similar substitution in MER-25 destroys its antiestrogenic activity in vivo (Clark and Jordan, 1976).
X o
IUJ
u 40 MONOHYOROXYTAMOXtfEN
DIHYDROXYTAMOXIFEN
04 1« DAILY DOSE OF COMPOUNDS
FIGURE 7. Uterotropic effect of estradiol, tamoxifen, monohydroxytamoxifen, and dihydroxytamoxifen in the 3-d ovariectomized mouse uterine weight test. Control animals received peanut oil alone; seven mice per group. Results represent means ± SEM.
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In the 3-d ovariectomized mouse uterine weight test, tamoxifen and monohydroxytamoxifen were fully uterotropic when compared with estradiol-17j3 while dihydroxytamoxifen was only partially uterotropic (Fig. 7). In antiestrogenic tests in the ovariectomized mouse, neither tamoxifen nor monohydroxytamoxifen possessed any antiuterotropic activity, whereas daily doses of 16, 32, and 64 jug dihydroxytamoxifen were effective in antagonizing the uterotropic effects of 0.024 ng estradiol per day. Even though the biological responses to tamoxifen, monohydroxytamoxifen, and dihydroxytamoxifen were different in the rat and mouse uterus, the relative potencies of the compounds were similar in both species. In general, the mouse appeared to be less sensitive to the effects of the triphenylethylenes and it is interesting to note that the antiestrogen MER-25 also appears to be less active in the mouse than in the rat (Emmens et al., 1960). Since monohydroxytamoxifen was very active as an antiestrogen in the immature rat uterine weight test, we compared this potent new compound with tamoxifen in our subsequent experiments. Subcellular Effects of Nonsteroidal Antiestrogens Estradiol is believed to stimulate uterine growth by selectively binding to estrogen receptor proteins located in the cell cytoplasm, which are then translocated in association with the steroid to the nucleus, where estrogeninduced synthetic events are initiated (Jensen and DeSombre, 1973). Nonsteroidal antiestrogens have been shown to competitively antagonize the binding of [3H]estradiol to the cytoplasmic estrogen receptor in vitro (Korenman, 1970; Skidmore et al., 1972). Furthermore, by using sucrose density gradient analysis to identify the binding protein, nonsteroidal antiestrogens have been found to inhibit the binding of [ 3 H ] estradiol to the 8S estrogen receptor derived from human (Jensen et al., 1972; Hunter and Jordan, 1975; Jordan and Koerner, 1975) and animal (Powell-Jones et al., 1975; Jordan and Dowse, 1976) estrogen target tissues. In the present study we have compared the ability of estradiol-17/3, monohydroxytamoxifen, and tamoxifen to inhibit the binding of [ 3 H ] estradiol to rat uterine estrogen receptors in vitro, using a dextran-coated charcoal assay. The introduction of the hydroxyl group into tamoxifen had a dramatic effect on its ability to inhibit the binding of [ 3 H] estradiol. Monohydroxytamoxifen was almost equipotent with nonradioactive estradiol (Fig. 8). Similarly, sucrose density gradient analysis (SBR) showed that tamoxifen and monohydroxytamoxifen (one-tenth the concentration of tamoxifen) were effective in inhibiting the binding of [ 3 H] estradiol to the 8S estrogen receptor derived from human endometrium (Fig. 9). This result has been compared with the dose-related ability of tamoxifen to inhibit the binding of [3H]estradiol to the human endometrium 85 estrogen receptor, determined by using a VTR.
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O £40
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LIGAND CONCENTRATION (mol/l)
FIGURE 8. Effect of increasing concentrations of estradiol, monohydroxytamoxifen, or tamoxifen on the specific binding of [ 3 H]estradiol (2 X 10"' mol/l) to uterine cytosol from immature rats. Data are from Jordan et al. (1977b).
23M2,
FRACTION
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FIGURE 9. Sucrose density gradient analysis [5-20% in (A) and 10-30% in (B)]. (A) The effect of (o) tamoxifen (1 X IO" 6 mol/l) or (*) monohydroxytamoxifen (1 X IO" 1 mol/l) on the binding of (•) [3 H] estradiol (2 X 1 0 " ' mol/l) in human endometrial cytosol using a swinging bucket rotor (15 h, 225,000 X g, 4°C). (B) The effect of increasing concentrations of tamoxifen at (o) 2X 10" 8 mol/l, (A) 8X 10" 8 mol/l, and (•) 3.2 X 10" 7 mol/l on the binding of [ 3 H]estradiol (5 X 1 0 " ' mol/t) in human endometrial cytosol using a vertical tube rotor (2 h, 400,000 X g, 4°C). BSA was run in a separate tube as a sedimentation standard (4.6S). Data in (A) are from Jordan et al. (1977b).
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The high affinity of monohydroxytamoxifen for the estrogen receptor suggests that the inclusion of a hydroxyl group at the para position in the C-1 phenyl in tamoxifen may result in an interaction of this hydroxyl at the same site on the estrogen receptor normally occupied by the C-3 phenolic hydroxyl of estradiol. It is perhaps relevant to consider that when CI-680 (a structural analog of CI-628) is demethylated to expose a free hydroxyl group at the same position as in monohydroxytamoxifen, there is also an increased affinity for the estrogen receptor (Ferguson and Katzenellenbogen, 1977). Both nafoxidine. and CI-628 (Fig. 1) have methoxy groups substituted at the same position in the phenyl ring, but whether demethylation to the free hydroxylated compounds has a major role to play in the mechanism of action of nonsteroidal antiestrogens must await the completion of pharmacokinetic studies to determine the relationship between the parent drug and its metabolites. Certainly it is tempting to speculate by analogy with the estrogen mestranol, whose affinity for the estrogen receptor is increased by demethylation to ethinylestradiol (Eisenfeld, 1974), that demethylation of antiestrogens may account for their long biological half-lives. Monohydroxytamoxifen has been detected in the serum of laboratory animals (Fromson et al., 1973a) and patients (Fromson et al., 1973b) treated with tamoxifen, which suggests that this potent metabolite may contribute to the maintenance of the antiestrogenic effect and be beneficial in the long-term control of hormone-dependent cancers. The nonsteroidal antiestrogens nafoxidine (Clark et al., 1973; Capony and Rochefort, 1975; Katzenellenbogen and Ferguson, 1975), enclomiphene (Ruh and Baudendistel, 1977), and tamoxifen (Jordan et al., 1977a) have each been shown to translocate estrogen receptors from the cytoplasm to the nucleus in the rat uterus. In general, each of these studies has compared estradiol-17/3 with a nonsteroidal antiestrogen and found that the time course of retention of estrogen receptors in the nucleus after estradiol was a few hours, whereas after nonsteroidal antiestrogens, estrogen receptors were present in the nucleus for several days. We have now compared the effects of estradiol (5 jug), monohydroxytamoxifen (5 fig), and tamoxifen (25 jug) on the measurement of estrogen receptors in the rat uterine nucleus for 48 h after administration (Fig. 10). Tamoxifen maintained a consistently high level of estrogen receptors within the nuclear compartment for up to 48 h, whereas the initial rise produced by estradiol had returned to control levels by 24 h. In contrast to tamoxifen, monohydroxytamoxifen produced a pattern of nuclear estrogen receptor concentrations similar to that seen after estradiol. Ferguson and Katzenellenbogen (1977) recently reported a similar finding with a structurally related nonsteroidal antiestrogen, 9411 X 27 (94X). Presumably as a result of the translocation of estrogen receptors to the
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FIGURE 10. Levels of estrogen receptors determined by nuclear exchange assay (Anderson et al., 1970) in the nucleus of immature female rats at various times after the administration of tamoxifen (25 jig), monohydroxytamoxifen (5 /ig), and estradiol (5 /jg). Control animals received peanut oil alone; 12 rats per group. Results represent means ± SEM.
nucleus, nonsteroidal antiesfrogens have been found to stimulate an increase in the concentration of progesterone receptors in the hamster uterus (Leavitt et a!., 1977) and in human hormone-dependent breast cancer cells grown in tissue culture (Horwitz and McGuire, 1977). Since it has been reported that the nonsteroidal antiestrogen nafoxidine can increase the activity of rat uterine RNA polymerase (Hardin et al., 1976), we have determined whether the increase in uterine weight in response to tamoxifen and monohydroxytamoxifen is associated with an increase in protein synthetic capacity, as demonstrated by an increase in progesterone receptor content. Tamoxifen (50 /ig), estradiol (5 jug), or monohydroxytamoxifen (50 jug) was administered as a single injection to ovariectomized rats. Tamoxifen produced a slow rise in uterine progesterone receptor concentration, whereas estradiol and monohydroxytamoxifen both produced a rapid rise in uterine progesterone receptor concentrations at 6 h (Fig. 11). Thus estrogen receptors that are translocated to the nucleus by nonsteroidal antiestrogens are capable of provoking protein synthesis and cellular hypertrophy but, as already discussed in the previous section, they are unable to affect an efficient endometrial hyperplasia. Whether the antiestrogen-estrogen receptor complex is incapable of interacting with an as yet undetermined nuclear acceptor site, which is necessary to provoke DNA synthesis, or whether the cytoplasmic protein synthesis that is
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• — • MONOHYDROXYTAMOXIFEN •—»TAMOXIFEN c—oESTRADIOL
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6 24 48 72 HOURS AFTER ADMINISTRATION
FIGURE 11. Effect of monohydroxytamoxifen (50 jig), tamoxifen (50 ii%), and estradiol (5 Mg) on the progesterone receptor content of ovariectomized rat uteri. Control animals received peanut oil; five rats per group. Results represent means ± SEM. Data are from Jordan and Prestwich (1978).
stimulated by antiestrogens is out of phase with the requirements of cell division remains to be determined. In the following section we will consider some of the current theories of antiestrogen action in order to formulate a strategy for future investigations. Potential Mechanisms of Action of Nonsteroidal Antiestrogens Based on a generalized model of estrogen action in target tissues (Fig. 12), nonsteroidal antiestrogens can potentially disrupt this scheme of events at one or more stages (or at an as yet undetermined site): competition with estradiol for the cytoplasmic estrogen receptor and subsequent INHIBITION OF RECEPTOR RESYNTHESIS
COMPETITIVE ANTAGONISM
r
SUBSEOUENT * SUBSEOUENT INHIBITION INHIBITION OF OF TRANSLOCATION TRANSFORMATION
FIGURE 12. Potential mechanisms of action of antiestrogens in the target tissue cell.
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inhibition of receptor transformation and translocation, or alternatively specific inhibition of estrogen receptor resynthesis, thus rendering the tissue refractory to subsequent estrogenic stimuli. Antiestrogens can inhibit the binding of estradiol to the estrogen receptor in vitro (Fig. 8), and similarly the administration of the antiestrogen nafoxidine (Jensen et al., 1972), clomiphene (Roy et al., 1964), CI-628 (Perry et al., 1973), or tamoxifen (Jordan, 1976a; Jordan and Dowse, 1976) can prevent the binding of [3H]estradiol target tissues in vivo. Before the development of nuclear exchange assays to determine ligand-filled sites (Anderson et al., 1970), it was conceivable that the estrogen receptor could be neutralized in the cytoplasm by nonsteroidal antiestrogens. This is now known to be untrue since the levels of exchangeable estrogen receptors rise in the nucleus after the administration of nonsteroidal antiestrogens (Clark et al., 1973) (Fig. 10). Once estrogen binds to the cytoplasmic estrogen receptor, the complex is transformed to a higher-molecular-weight unit (Brecher et al., 1970). Studies in vitro using sucrose density gradient analysis have shown that the AS estrogen-receptor complex that is present in buffer containing 0.4 M KG becomes converted to a 55 estrogen-receptor complex on gentle warming (Notides and Nielsen, 1974). The precise significance of this reaction is unknown, although it has been suggested that this step is necessary before estrogen receptors can activate RNA polymerase in the nucleus (Jensen et al., 1973). Similar attempts to investigate this reaction when antiestrogens are the binding ligands have been hampered by the lack of high-specific-activity radiolabeled antiestrogens with a high binding affinity for the estrogen receptor. At present, therefore, it is not known whether nonsteroidal antiestrogens do permit the transformation reaction prior to nuclear entry. When estradiol translocates estrogen receptors to the nucleus, the cytoplasmic estrogen receptor pool is replenished during the following 24 h by a process of resynthesis (Sarff and Gorski, 1971). Since nonsteroidal antiestrogens deplete the cytoplasmic estrogen receptor pool for longer than 24 h (Clark et al., 1973; Katzenellenbogen and Ferguson, 1975), it has been suggested that nonsteroidal antiestrogens specifically inhibit the resynthesis of cytoplasmic estrogen receptors, thus rendering the tissue refractory to subsequent estrogenic stimuli (Clark et al., 1974). In the present study we have addressed the question of cytoplasmic estrogen receptor replenishment in response to estrogenic and antiestrogenic stimuli in order to determine whether the prolonged depletion of the cytoplasmic estrogen receptor pool is an exclusive characteristic of nonsteroidal antiestrogens. First, we compared the antiestrogen ic activity of tamoxifen and monohydroxytamoxifen in the 3-d immature rat uterine weight test and also determined the levels of available cytoplasmic estrogen receptors 24 h after the last dose of antiestrogens. It is clear from Fig. 13 that the
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ESTRADIOLJ
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DAILY D O S E OF C O M P O U N D ((ifl)
FIGURE 13. Effect of daily administration of estradiol (0.08 jug), estradiol and various doses of monohydroxytamoxifen (•), or estradiol and various doses of tamoxifen (•) for 3 d on the uterine levels of available cytoplasmic estrogen receptors determined 24 h after the last dose. Control animals received peanut oil; 10 rats per group. Results represent means ± SEM.
depletion of cytoplasmic estrogen receptor levels is a dose-related phenomenon, and antiestrogenic effects are apparent (Fig. 6) before a depletion of estrogen receptor levels below those of control uteri. The observation that estrogen receptors are apparently available for binding estradiol during an antiestrogenic uterine weight test with tamoxifen is consistent with our earlier report (Jordan et al., 1977a). To ensure that the available uterine estrogen receptors determined in vitro were truly available to bind estradiol in vivo, we determined the effect of the administration of different daily doses of tamoxifen (2, 8, and 32 jug) or monohydroxytamoxifen (0.32, 1.28, and 5.12 /ig) on the uterine binding of [3H]estradiol (0.08 jug) administered 24 h after the last dose of the antiestrogens. Again, the effect of the antiestrogens was related to the dose administered, but at doses of tamoxifen (8 jug/d) or monohydroxytamoxifen (1.28 jug/d) that had previously been found to be antiestrogenic, there was no significant difference (determined by Student's Mest) between the levels of [3H]estradiol in treated and control uteri (Fig. 14). Therefore, the depletion of estrogen receptors in the rat uterine cytoplasm is related to the dose of the antiestrogen administered, and the
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FIGURE 14. Effect of different daily doses of monohydroxytamoxifen or tamoxifen for 3 d on the binding of [ 3 H]estradiol (0.08 ti%) injected 24 h after the last dose of antiestrogen. Levels of radioactivity in the uterus were determined 2 h after the administration of [3 H] estradiol. Six rats per group. Results represent means ± SEM.
antagonism of estrogen action apparently occurs before estrogen binding capacity is completely denied. To extend this observation, we determined the effects of increasing doses of nonsteroidal estrogens on the available cytoplasmic estrogen receptor pool. ICI 3188 was administered on three consecutive days to groups of immature female rats, and 24 h after the last dose of the estrogen available cytoplasmic estrogen receptor levels were determined. ICI 3188 produced a full uterotropic response, which plateaued at maximal stimulation, whereas increasing doses of the compound produced a dose-related decrease in the levels of cytoplasmic estrogen receptors (Fig. 15). We also found that increasing doses of tamoxifen's estrogenic cis isomer, ICI 47699, produced a dose-related decrease in cytoplasmic estrogen receptor content with a corresponding rise in the concentration of estrogen receptors within the nucleus (Jordan et al., 1978). Since the depletion of cytoplasmic estrogen receptors appears to be a function of the dose of any compound that will translocate receptors to the.nucleus, then the differences observed between the effects of estradiol and nonsteroidal antiestrogens on the measurement of cytoplasmic estrogen receptors are probably related to differences in their respective biological half-lives. Blood levels of estradiol are maintained for only a few hours after administration (Castracane and Jordan, 1975), whereas nonsteroidal antiestrogens like tamoxifen have a biological half-life of several
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days (Fromson et al., 1973a). Therefore, it is possible that estrogen receptor resynthesis does occur after antiestrogen administration, but the newly synthesized receptors are never allowed to accumulate within the cytoplasm since binding ligands from the blood immediately provoke translocation to the nucleus. This interpretation would account for the reports of high levels of estrogen receptors within the nucleus for prolonged periods after antiestrogen administration (Clark et al., 1973; Katzenellenbogen and Ferguson, 1975) and would suggest that these nuclear estrogen receptors are a dynamic pool that are being processed in order to initiate biosynthetic events in the cytoplasm, and are then immediately replenished by resynthesized receptors from the cytoplasm. The short-term retention of estrogen receptors in the nucleus after monohydroxytamoxifen (Fig. 10) could be caused by a shorter biological half-life since it would be expected that this compound would be readily conjugated in the liver and the products rapidly excreted (see Ferguson and Katzenellenbogen, 1977). Since both estrogens and nonsteroidal antiestrogens can translocate estrogen receptors from the cytoplasm to the nucleus, but only estrogens
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