antiestrogenic properties of keoxifene,trans-4-hydroxytamoxifen, and ici 164384, a new steroidal...

Breast Cancer Research and Treatment 14: 65-76, 1989. © 1989 Kluwer Academic Publishers. Printed in the Netherlands.

Report

Antiestrogenic properties of keoxifene, trans-4-hydroxytamoxifen, and ICI 164384, a new steroidal antiestrogen, in ZR-75-1 human breast cancer cells

Richard Poulin, Yves Merand, Donald Poirier, Charles Levesque, Jean-Marc Dufour and Fernand Labrie Medical Research Council Group in Molecular Endocrinology, Laval University Medical Center, Quebec GIV 4G2, Canada

Key words: antiestrogens, estrogen receptor, ICI 164384, keoxifene, tamoxifen, breast cancer, mixed agonist-antagonist

Abstract

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

Introduction

Since the establishment of the role of estrogens in the growth of human breast tumors [1], consid- erable attention has been given to the mechanisms of action involved and to the development of inhib- itors of estrogen action at the target cell level. The existing ablative procedures, either surgical or medical, do not permit complete elimination of

estrogens [2--4]. Consequently, antiestrogens or compounds blocking estrogen action at the receptor level, remain the most rational approach to the therapy of estrogen-responsive breast cancer.

However, until very recently, no agent with pure antiestrogenic activity under in vivo conditions was available. The antiestrogen currently used in endocrine therapy, namely the triphenylethylene derivative tamoxifen (TAM) [5, 6], behaves as a

Address for offprints: F. Labrie, MRC Group in Molecular Endocrinology, CHUL Research Center, Laval University Medical Center, 2705 Boulevard Laurier, Sainte-Foy (Quebec), Canada, G1V 4G2

66 Richard Poulin et al.

mixed agonist/antagonist of estrogen action, the relative importance of the two activities depending on the tissue and/or endpoint examined [7, 8]. Al- though TAM has a weak affinity for the estrogen receptor (ER), as compared with E2, it is extensive- ly metabolized in peripheral tumor tissues, being transformed, among other metabolites, into the trans-4-hydroxylated form which is a much stronger antiestrogen and has greater affinity for the ER than its precursor [9, 10]. Nevertheless, the metabolically activated antiestrogen, e.g. trans-4-monohydroxytamoxifen (OH-TAM), has about equal agonistic activity in various tests to that found with tamoxifen [11]. Even the potent benzothiophene derivatives LYl17018 [12] and keoxifene (LY 156758) [13] display significant uterotrophic activity in the rat, although they have somewhat higher relative antiuterotrophic activity than triphenylethylene derivatives [12, 13].

Recently, Wakeling et al. have reported that a series of 7a-alkylamide derivatives of 1715-estradiol derived from structures originally developed for the affinity chromatography of the ER [14] dis- played significant antagonistic activity in various systems, including human breast cancer cells [15- 17]. One of these compounds, namely ICI164384 [(N-n-butyl-ll- (3, 1715 -dihydroxy-estra- 1, 3, 5 (10) trien-7a-yl)-N-methyl undecanamide] exhibited no agonistic activity, while strongly and competitively antagonizing estrogen action in a number of in vivo and in vitro bioassays. We now report that ICI 164384 indeed behaves as a pure antiestrogen in estrogen-responsive ZR-75-1 human breast cancer cells [18] in culture, under conditions where the other potent antiestrogenic compounds, OH-TAM and LY156758, exert significant significant partial estrogenic activity. We have also characterized competition by OH-TAM, LY156758, and ICI 164384 for the specific uptake of 3H-labeled estrogens in intact cells, and have found that as compared with the non-steroidal antiestrogens, ICI 164384 apparently interacts with the ER in a more rapidly reversible manner.

Materials and methods

Chemicals

ICI164384 and trans-4-monohydroxytamoxifen (OH-TAM) [a-(415-N-dimethylaminoethoxy)phenyl-hydroxy-a'-ethyl-transtilbene] were generous- ly provided by Drs. A.E. Wakeling and G.F. Cos- tello, respectively (Imperial Chemical Industries PLC, Pharmaceuticals Division, Macclesfield, England). LY156758 [(6-hydroxy-2-(4-hydroxy- phenyl)benzo(b)thien-3-yl),(4-(2-1-piperidinyl)- ethoxy)phenyl)methanone hydrochloride] was a gift from Dr J.A. Clemens (Lilly Research Lab- oratories, Indianapolis, IN).

[2,4,6,7-3H]1715-estradiol ([3H] E2) (sp. act. 85 Ci mmo1-1) was purchased from Amersham (Arling- ton Heights, IL). Unlabeled moxestrol (R2858) [1113-methoxy-19-nor-17a-pregna-l,3,5(10)-trien- 20-yne-3,1715-diol] and [l115-methoxy-3H]R2858 were obtained from New England Nuclear (La- chine, Quebec, Canada), while 1715-estradiol (E2), 5-androstene-313,1713-diol (AS-diol) and 5a-dihydrotestosterone (DHT) were from Steraloids (Pawling, NY). Dexamethasone, diethylstilbestrol, and all media and supplements for cell culture were purchased from Sigma (St. Louis, MO), except for fetal bovine serum, which was from Hy- Clone (Logan, UT) and Flow Laboratories.

Cell culture and growth experiments

The ZR-75-1 (18) and MDA-MB-231 [19] human breast cancer cell lines at their 83rd and 24th passages, respectively, were obtained from the Amer- ican Type Culture Collection (Rockville, MD), and routinely cultured in phenol red-free [20] RPMI 1640 medium supplemented with 10 nM E2, 1 mM sodium pyruvate, 2 mM L-glutamine, 15 mM Hepes, 100 IU penicillin per ml, 100/.~g streptomy- cin sulfate per ml, and 10% (v/v) fetal bovine serum, as described [21]. The addition of E2 to the medium had no effect on the growth of MDA- MB-231 cells which do not possess ER [22] but was added solely for comparative purposes. ZR-75-1

and MDA-MB-231 cells were subcultured every 8 days and every 4--5 days, respectively.

Growth experiments

Cells in late logarithmic growth phase were harvested with 0.05% trypsin 0.02% EDTA (w/v) and resuspended in phenol red-free RPMI 1640 medium with the indicated supplements, except that E2 was omitted and the serum supplement was replaced with 5% (v/v) dextran-coated charcoal- treated fetal bovine serum and 500 ng of bovine insulin per ml (SD medium). Prior to experiments, ZR-75-1 cells were deprived of estrogens by sub- cultivation in SD medium for a total 12-day period. Cells were seeded in Linbro 24-well plastic culture plates (2 cm2/well) at 4 × 103 cells/cm 2 and incubated for 48 h in SD medium to allow for adhesion to substrate and completion of growth lag. Additions of steroids and/or antiestrogens were then made in fresh SD medium from 1000- to 10,000-fold concen- trated stock solutions in 99% redistilled EtOH. The total EtOH concentration did not exceed 0.12% (v/v) and had no significant effect on either cell growth or morphology. Cells were incubated for the indicated time periods under the given conditions and then harvested by a 10-min treatment with 200 ~1 of 0.05% trypsin/0.02% EDTA (w/v). Following the addition of 200/~1 of chicken ovomu- coid trypsin inhibitor [0.1% (w/v) in phosphate- buffered saline (Sigma) and thorough dissociation of clumps by 2 passages though a 181/2-gauge nee- dle, cell suspensions were added to 20ml Isoton (Coulter Electronics, Hialeah FA), and cell number was determined with a Coulter Counter (model ZM).

ER competition studies

The interference of antiestrogens on the specific binding of [3H]E2 and [3H]R2858 in intact cells was determined by a whole-cell uptake method as described [21, 23] with minor modifications. Briefly, ZR-75-1 cells were grown in steroid-free SD medium in 24-well Linbro plastic culture plates to a

Antiestrogenic properties 67

density of approximately 1.5-2.5 x 105 cells/cm 2 with or without treatment, as indicated in the 'Re- suits' section. Medium was then removed, and cells were washed twice with RPMI 1640 medium and layered with 500/xl of SD medium, from which serum was omitted and replaced with fatty acid- free bovine serum albumin. (fraction V, 0.1%, w/v; Sigma) plus the indicated concentrations of [3H]E2 and competitor. For uptake measurements over periods exceeding 1 hour, incubation medium was complete SD medium (without bovine serum albumin) and the radioligand used was [3H]R2858. Cells were incubated with radioligand for the indicated time periods at 37 ° C. Medium was then removed and cell monolayers were rapidly rinsed with 3 × l ml of ice-cold buffer (5mM sodium phosphate, pH 7.4,250 mM sucrose, 10% glycerol, 0.5% (w/v) fatty acid-free bovine serum albumin). One ml of EtOH was then added to wells and transferred to scintillation vials after a 30-min incubation at room temperature. Cell monolayers were rinsed with a further 0.5 ml EtOH, and radioactivity was determined in the pooled extracts by liquid scintillation spectrometry. Non-specific uptake was determined in parallel cell cultures incubated in identical media plus a 200-fold excess of diethylstilbestrol (for [3H]E2 incubations) or unlabeled R2858 (for [3H]R2858 incubations). Specif- ic uptake was calculated as the difference (+ SEM) between the mean radioactivity measured in cells incubated in the absence (total uptake), and that recovered in cells incubated with an excess of ra- dioinert ligand (non-specific uptake).

Statistical analyses and calculations

Apparent IC50 values were calculated using an iter- ative least square regression method [24]. Appar- ent Ki values for receptor-mediated growth-inhibition and for competition for the whole-cell uptake of estrogens were calculated according to Cheng and Prusoff [25] and Munson and Rodbard [26], respectively. Statistical significance was measured according to the multiple-range test of Duncan- Kramer [27]. Values are presented as means


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Fig. 1. Effects of a 15-day incubation with increasing concentrations of 3 antiestrogens in the ZR-75-1 human breast cancer cell line. Cells were deprived of steroids for a total 12-day period prior to incubation with the indicated concentrations of ICI164384 (O), LY156758 (0) or OH-TAM (ff]) added to SD medium. Points, means of triplicate determinations from a rep- resentative experiment, bars, SEM. Doubling time in the control ceils was 74 h.

+ SEM of triplicate measurements. When no bar is shown, the SEM was smaller than the symbol used.

R e s u l t s

As shown in Fig. 1, a 15-day incubation of ZR-75-1 cells with increasing concentrations of the two non- steroidal antiestrogens LY156758 and OH-TAM led to two types of effects on ZR-75-1 cell growth, i.e. a mitogenic effect at subnanomolar concentrations and a biphasic pattern of inhibition at higher concentrations. Growth stimulation was observed at about 3-fold higher concentrations for OH-TAM

than for LY156758, both antiestrogens having a net mitogenic effect in a 2-log range of concentrations. Growth inhibition overcame mitogenic effects at concentrations greater than 0.1 and l nM for LY156758 and OH-TAM, respectively. Gross cytotoxic effects were evident at micromolar concentrations of OH-TAM, as seen by the cell killing induced by 10/xM OH-TAM whicla was complete after 3 to 4 days of incubation.

On the other hand, it can be seen that increasing concentrations of 1CI164384 exclusively had inhibitory effects on ZR-75-1 cell proliferation, with a dearly biphasic pattern. Of special interest is the fact that ICI164384 was reproducibly more potent than either non-steroidal antiestrogen in the amplitude of its growth-inhibitory effects. The sigmoidal shape of the effect seen up to 300 nM (about 50% inhibition) allowed the estimation of a half-maximal inhibitory concentration (IC50) of about 0.3nM. Higher concentrations of ICI164384 further decreased cell growth down to 15-20% of control at the maximal concentration used (10~M), a plateau being observed between 1 and 1000nM (Fig. 1).

Non-steroidal antiestrogens have been shown to exert ER-related and ER-independent antiproliferative effects in breast cancer cells [28-30]. We therefore examined the influence of the compounds under study on growth of the MDA- MB-231 human breast cancer cells, which are ER- negative [22]. As illustrated in Fig. 2, concentrations of antiestrogens lower than about 100 nM had no significant effect on the proliferation of these cells, which had reached saturation densities within approximately 6 days. However, a dramatic decrease of cell number was exerted by all antiestrogens at concentrations exceeding 100nM, OH- TAM being the most potent. Killing of MDA- MB-231 cells by 10~M OH-TAM was complete after about 72 h of incubation (data not shown). Thus, ICI164384 did not differ from the two non- steroidal antiestrogens in its ability at micromolar concentrations to exert effects unrelated to the ER. This was further confirmed by the total lack of effect of E2 in MDA-MB-231 cells, either alone or in the presence of increasing concentrations of ICI164384.

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Fig. 2. Effects of a 7-day incubation with increasing concentrations of three antiestrogens on the growth of the MDA-MB-231 human breast cancer cell line. Cells were treated exactly as ZR-75-1 cells in the experiment shown in Fig. 1, except for the total growth period at harvesting time, which was shorter because of the about 2-fold higher growth rate of MDA-MB-231 cells. Cells were incubated with ICI164384 alone (O) or in the presence of E2 ( lnM, O), with LY156758 (rq), or with OH- TAM ( • ) , dissolved in SD medium. Points, means of triplicate determinations; bars, SEM.


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Fig. 3. Reversal of antiestrogen-induced growth inhibition by incubation with increasing concentrations of E2 in ZR-75-1 cells. Cells were deprived of steroids for a total 12-day period prior to a 15-day incubation with the indicated concentrations of E2 in the absence of antiestrogen (O) or in the presence of 300 nM of either ICI164384 (0) , LY156758 ([q), or OH-TAM ( • ) , dissolved in SD medium. Points, means of triplicate determina- tion; bars, SEM.

The ability of the antiestrogens to antagonize the mitogenic effect of E2 is shown in Fig. 3. Increasing concentrations of the estrogen up to 10 nM induced a 2.5-fold increase in cell number with a half-maximal effect (ECs0) observed at approximately 25 pM. Concentrations of E2 exceeding 10 nM were suboptimally mitogenic, with about only 55% of the maximal growth stimulation being observed at 1/zM E2. OH-TAM, LY156758, and ICI164384 (all added at 300 nM) decreased basal cell number by about 50, 55, and 65%, respectively, and reversed the effect of increasing concentrations of E2 in a

competitive manner. The EC50 values of interference with E 2 action were calculated at about 15, 10, and 4nM in the presence of 300nM of OH- TAM, LY156758, and ICI164384, respectively. The much sharper, mitogenic response to E2 pro- duced in ZR-75-1 cells incubated with ICI164384 was observed in successive experiments. That at least part of the decrease in the potency of E2 to stimulate cell proliferation over 10 nM was mediated by the ER, was suggested by the fact that all antiestrogens caused an upward shift in the mitogenic response to E2 in this range of concentrations.

70 R i c h a r d P o u l i n et al.

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Fig. 4. Additive inhibition of ZR-75-1 cell proliferation by the steroidal antiestrogen ICI164384 in the presence of maximally effective concentrations of androgen and glucocorticoid. Cells were deprived of steroids for a total 12-day period prior to incubation for 15 days in SD medium containing either 10nM 5ct-dihydrotestosterone (DHT, @), 100nM dexamethasone (DEX, [3), or no steroid (O). Points, means of triplicate determinations; bars, SEM.

In order to further assess the specificity of the antiestrogenic activity of ICI164384, we studied the ability of this steroid to interfere with the growth inhibition induced by maximally effective concentrations of 5a-dihydrotestosterone (DHT) and dexamethasone (DEX). Androgens [21] and glucocorticoids [31] have been shown to decrease ZR-75-1 cell growth rates through specific interaction with their respective receptors, their effects being clearly additive (Hat ton AC and Labrie F, unpublished results). As shown in Fig. 4, increasing concentrations of ICI164384 did not reverse to any extent the inhibitory effects of either D H T (10nM) or D E X (100nM), which themselves caused a 55 and 45% decrease in basal cell prolifer-

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Fig. 5. Competition for the uptake of [3H]E2 by estrogens and antiestrogens. As described in 'Materials and Methods', confluent ZR-75-1 cell cultures were incubated for 60 min with 5 nM [3H]E2 plus increasing concentrations of E2 (O), androst-5- ene-313-diol (0, AS-diol), LY156758 ([3), OH-TAM (11), or ICI164384 (A). The radioactivity associated with cell-bound [3H]E2 was then determined. Points, means of triplicate determinations; bars, SEM.

ation, respectively. Most interestingly, the antiestrogen further inhibited ZR-75-1 cell growth under all conditions with little, if any, change in potency.

We next examined the ability of the antiestrogens and androst-5-ene-3[3,17[3-diol (AS-diol), a C-19 steroid of adrenal origin which exhibits pure estrogenic activity [32], to compete for the specific uptake of [3H]E2 in intact ZR-75-1 cells (Fig. 5). Both LY156758 and OH-TAM have a very similar affinity for estrogen specific binding sites (ESBS), with calculated Ki values [24] of 0.89 and 1.6 nM, respectively, assuming a Kd value of 0.6 nM measured for E2 under similar conditions [32]. AS-diol had 9-fold less relative affinity for ESBS than E2

( K i - 5.4 nM), in reasonable agreement with published values [32, 33]. Quite surprisingly, ICI164384 competed with much lower affinity for [3H]E 2 specific uptake (extrapolated Ki value, 1.14/zM) in the 60-min assay. Since ICI164384 had been shown [15] to have relative binding affinity (= 19%) close to that of E2 (= 100%), we speculat- ed that the amphipathic, 7a-aliphatic side-chain of ICI164384 might somewhat hinder its rapid intracellular transport because of non-specific hydrophobic interactions, thus slowing its kinetics of ex- change with the ER in the intact cell. However, this question could not be addressed by extending incubation time for competition, the extensive me- tabolism of [3H]E2 in these cells becoming unduly important (Poulin R, Th6riault C, Labrie F, unpublished results).

We thus took two separate approaches in order to further investigate the kinetics of antiestrogen competition for ESBS in intact ZR-75-1 cells. First, cell monolayers were preincubated for various time periods with the antiestrogens (1/zM) and then allowed to interact with [3H]E2 in the continuing presence of the antiestrogen at the same fixed concentration. As illustrated in Fig. 6, a preincubation period of 2 hours was sufficient to induce a complete suppression of subsequent [3H]E2 specific uptake for LY156758 and OH-TAM, which had virtually identical time course patterns. On the other hand, a similar inhibition of [3H]E2 uptake required a 6-hour preincubation in the case of ICI164384.

Although these results suggest that kinetic factors limit the rapid access of ICI164384 to ESBS, the preceding approach was not a truly competitive assay, inasmuch as the interaction of some antiestrogens with the ER in intact systems may be partly irreversible [34, 35]. Direct competition experiments were thus performed using the high-affinity ER ligand [3H]R2858, which is expected to be metabolically more stable than E2 [36]. As shown in Fig. 7, [3H]R2858 behaved in a manner very similar to the action of [3H]E 2 in our routine assay, yielding virtually identical maximal numbers of ESBS after a 1-hour incubation and subsequently exhibiting a rapid and relatively constant loss of such sites, or 'processing' [37], at least up to 24h after ligand addition.


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Fig. 6. Time dependence for the maximal occupancy of E 2 binding sites (ESBS) by antiestrogens under non-competitive conditions. Confluent ZR-75-1 cell monolayers were incubated with 1 laM of either ICI164384 (O), OH-TAM (O), or LY156758 (11) for the indicated time periods, and then incubated with 5 nM [3H]E2 in the continuing presence of the respective antiestrogen (1 laM), plus (non-specific uptake) or minus (total uptake) 1 laM diethylstilbestrol. Other conditions are as described in 'Materials and Methods'. Specific uptake is the mean total uptake minus mean non-specific uptake. Points, differences between means of triplicate determinations of total uptake and of non-specific uptake; bars, SEM of the difference between mean total and non-specific uptake.

Under such conditions, the two non-steroidal antiestrogens, as well as E2, competed well with [3H]R2858 for the early binding to ESBS, suppression of detectable radioligand uptake being essen- tially maximal 1 h after the onset of incubation (Fig. 8A). ICI164384 (1/zM) was a much better competitor of [3H]R2858 than of [3H]E2 specific uptake after 60min (cf. Fig. 5). However, while E2 kept blocking the specific uptake of [3H]R2858 for at least 6 hours, there was a temporary loss of compet-


itive ability for the 3 antiestrogens, since parallel increases in radioligand binding to ESBS were measured up to 4 hours (ICI164384) or 6 hours (LY156758 and OH-TAM) after ligand addition. These fluctuations eventually disappeared for the non-steroidal antiestrogens, which had completely suppressed [3H]R2858 specific uptake after 24 hours. Likewise, the relative binding affinity of ICI164384 for ESBS stabilized after 6 to 8h, although, as for E2, at the concentrations used, a small but significant number of ESBS were still accessible to [3H]R2858 up to 24 h after the onset of incubation.

It can be seen in Fig. 8B that there were marked differences in the apparent reversibility of the interaction of these ligands with ESBS. In this assay, ZR-75-1 cells were preincubated for 24 hours with competitors to allow equilibrium conditions to be reached, and were then exposed to 3 nM [3H]R2858 in the absence of unlabeled ligand. Although similar low specific uptake of [3H]R2858 was initially detectable under all conditions, only ICI164384 had apparent dissociation characteristics compat- ible with a fast and fully reversible mechanism of interaction with the ER. Thus, nearly complete restoration of ESBS for [3H]R2858 binding oc- curred 6 h after removal of ICI164384. Preincuba- tion of ZR-75-1 cells with both non-steroidal antiestrogens, especially LY156758, induced a sus- tained decrease in the apparent number of ESBS accessible to ligand binding. In the case of E2- pretreated cells, there was again evidence of a time-dependent increase in the cellular capacity for [3H]R2858 specific uptake.

Discussion

The present data dearly indicate that, among the compounds studied, ICI164384 is the only one having the antagonistic properties expected from a pure antiestrogen on the growth of the estrogen- responsive human breast cancer cell line ZR-75-1. Thus, in these cells, ICI164384 exhibits no detectable agonistic (i. e. mitogenic) effect on basal cell growth, while two non-steroidal antiestrogens exert significant estrogenic activity at subnanomolar

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concentrations. In a preliminary report of our characterization of the effects of ICI164384 on ZR-75-1 cell growth [38], shorter incubation periods and the lack of preliminary estrogen deprivation might have explained the absence of a significant mitogenic effect for OH-TAM (but not LY156758). While TAM [39, 40] and OH-TAM [40-42] had already been reported to have some mitogenic activity in breast cancer cells in culture when present at similar concentrations, especially under phenol red-free conditions, we are not aware of compara- ble effects having been reported for the benzothiophene derivatives, which have behaved as pure antiestrogens, or very nearly so in vitro [42, 43]. The present evidence showing that LY156758 be-


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Fig. 8. A . Time course of competition by F_~ and 3 antiestrogens for the specific uptake of [3H]R2858. Cells were incubated for the indicated time periods in the presence of 3 nM [3H]R2858 as well as 3 laM of either E2 (O), ICI164384 (O), LY156758 (D), or OH-TAM (11), in SD medium, and the specific uptake of [3H]R2858 was determined as described in Fig. 7. B. Time course of [3H]R2858 specific uptake in ZR-75-1 cells preloaded with E2 or antiestrogens. Confluent cell monolayers were preincubated for 24 h with 3 laM of either F_~ (O), ICI164384 (O), LY156758 (Q), or OH-TAM ( I ) , in SD medium, washed twice with SD medium, and then incubated for the time periods indicated with 3 nM [3H]R2858 (dissolved in SD medium) without any further addition. Specific uptake was determined as indicated above. Points, differences between total and non-specific uptake measured in triplicate; bars, SEM. The corresponding time course of control cells (without addition of competitor) is the same as shown in Fig. 7.

haves almost identically to OH-TAM could suggest that the phenomenon of estrogenic activity already described for triphenylethylene derivatives in vitro might well apply to other non-steroidal antiestrogens as well.

The ER-independent inhibitory effects of triphenylethylene antiestrogens on growth-related cellular activities have been attributed, among other hypotheses, to their activity as calmodulin [44] and Ca 2÷ channel antagonists [45]. The data obtained with MDA-MB-231 cells, as well as the biphasic pattern of growth inhibition induced by the other antiestrogens studied in ZR-75-1 cells, suggest that ICI164384 might also exert similar cytotoxic effects at micromolar concentrations. Whether this non- antiestrogenic, antiproliferative activity may be- come significant in vivo, as postulated for the tri- phenylethylenes, deserves further study.

It is important to mention that ICI164384 be-

haves like LY156758 [21; Poulin R and Labrie F, unpublished observations) concerning the additiv- ity of its inhibitory action to that resulting from the addition of androgens and glucocorticoids. More- over, the present experiments show no evidence indicating that ICI164384 might antagonize the action of pure agonists of androgen and glucocorticoid action such as DHT and DEX. The mechanisms through which antiestrogens decrease cell proliferation in estrogen target cells under steroid- free conditions have received much attention since the original hypothesis that unoccupied ER might exert a constitutive, basal activation of estrogen- regulated gene elements [46]. In this respect, ICI164384 behaves qualitatively like the non-steroidal antiestrogens, and its strong antiproliferative activity does not appear to be modified following prolonged estrogen deprivation (Tremblay S, Poulin R, and Labrie F, unpublished data). Since


the experiments herein described were all conduct- ed in the presence of insulin (500 ng/ml), the inhibition of estrogen-free cell growth by antiestrogens in ZR-75-1 cells may be directed against the mitogenic effect of this hormone [41]. Preliminary evidence suggests that this could be the case, since little effect of ICI164384 was found in ZR-75-1 cells grown in insulin- and estrogen-free media (Poulin R, Baker D, and Labrie F, unpublished results). Among a number of explanations for the stronger amplitude of ICI164384-induced growth inhibition observed in estrogen-free media, as compared with the non-steroidal antiestrogens, the apparent absence of intrinsic agonistic activity of ICI164384 could play a major role. Thus, partial agonists/ antagonists such as the non-steroidal antiestrogens studied in the present system, would have sub- optimal, so-called ER-mediated cytostatic and/or cytotoxic effects [47] as controlled by their relative estrogenic and antiestrogenic activities.

The present study also suggests that ICI164384 might interact with the ER differently from estrogens and non-steroidal antiestrogens in an intact cell system. Estrogens as well as high concentrations of OH-TAM induce ER processing in intact MCF-7 breast cancer cells for periods similar to those used in the present study to preload intracellular ESBS [23, 35, 48, 49]. Moreover, ER is con- verted from a fast to a more slowly dissociating form upon ligand-induced activation [50], regard- less of the extent of processing [51]. Such consid- erations have led to the validation of the whole-cell uptake method as a measurement of the ER-activa- ring potential of a given competitor [52]. These observations suggest that ICI164384 does not acti- vate the ER and thus dissociates much faster than the non-steroidal antiestrogens from the ER. Ac- cordingly, this steroidal antiestrogen has a lower ability to compete for estrogen specific uptake despite its high affinity for the ER [15], and a much extended period of time is required to enable the compound to reach equilibrium under competitive binding conditions.

One cannot exclude, however, that ICI164384 also has a slower access to ESBS than non-steroidal antiestrogens (Fig. 6), possibly due to non-specific interactions of the long 7~t-alkylamide chain with

membrane and/or intracellular hydrophobic structures. On the other hand, the non-steroidal antiestrogens appear to induce a time-stable decrease in the exchangeability of the ER which is still maximal 24 hours after ligand removal. The nature of these changes cannot be identified at the present time, but is highly suggestive of processing and possibly of other nuclear events modifying ER ex- pression, turnover, or binding characteristics. How ICI164384 is nevertheless a more potent estrogen antagonist than non-steroidal antiestrogens in various bioassays, despite its apparently unfavorable kinetic properties observed in intact cells, remains to be determined. Further studies using labeled compounds should help clarify the marked differences observed between the interaction of non- steroidal antiestrogens and ICI164384 with the ER.

The time-dependent loss of competitive potency for E2 binding to ESBS in the presence of [3H]R2858 likely results from its metabolic inacti- vation through the high 17[3-hydroxysteroid dehy- drogenase activity present in ZR-75-1 cells.

In conclusion, we have extended the characterization of the antagonistic properties of ICI164384, and compared them with those of the most widely studied non-steroidal antiestrogens. A benzothiophene derivative (LY156758) is shown for the first time to exert as marked estrogenic activity as a triphenylethylene derivative (OH-TAM) in an in vitro system, while ICI164384 has pure antiestrogenic activity. The clinical interest of using compounds such as ICI164384 to specifically and more completely block estrogen action cannot be over- emphasized, and the unique features of interaction of this compound with ESBS should lead to a better understanding of the mechanism of action of antiestrogens.

Acknowledgements

We wish to express our sincere gratitude to Dr A.E. Wakeling for interest in our work, and to Ms. Elaine Leclerc for her excellent secretarial help in the preparation of this report. This work has been supported in part by a Group Grant from the Med- ical Research Council of Canada.

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antiestrogenic properties of keoxifene,trans-4-hydroxytamoxifen, and ici 164384, a new steroidal...

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