Biological response of the anti-estrogen ICI 164,384 in human hormone-dependent and hormone-independent mammary cancer cell lines

Cancer Letters, 50 (1990) 133- 139 Elsevier Scientific Publishers

133

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Biological response of the anti-estrogen ICI 164,384 in human hormone-dependent and hormone-independent mammary cancer cell lines J.R. Pasqualini C.N.R.S. (France)

and C. Gelly

Steroid Hormone

Research Unit, Foundation for Hormone

Research, 26 Boulevard Brune, 75014 Paris

(Received 10 October 1089) (Revision received 5 December 1989) (Accepted 6 December 11989)

Summary The biological response on proliferation and progesterone receptor (PR) of the anti-estrogen ICI 164,384 [N-n-butyl-N-methyl-ll(3,17(3-dihydroxyestra-1,3,5(10trien7a-yl))undecanamide] w,as studied in different mammary cancer cell lines. In the hormonedependent cancer
Correspondence to: J.R. Pasqualini. 0 1990 Elsevier Scientific Publishers 0304-3835/90/$03.50 Published and Printed in Irelend

significantly decreases the PR concentrations in both the non-treated and estradiol-treated cells. It is concluded that ICI 164,384 is a full antagonist in the hormone-dependent breast cancer cells, but it has no effect in the antiestrogen-resistant or in hormone-independent cell lines. Keywords: breast cancer; anti-estrogen; gesterone receptor; mammary cancer lines.

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Introduction The anti-estrogen tamoxifen, due to its lack of toxicity, is one of the most frequently used drugs as additive hormonal therapy in breast cancer [6,26]. In experimental models tamoxifen can act as a weak or a full estrogen [9,20] depending on the animal species, the target organ or the experimental conditions [lo, 12,201. In clinical applications tamoxifen is predominantly anti-estrogenic [8,15] with the exception of a very limited number of patients with breast cancer who developed endometrial adenocarcinoma after treatment with this drug [141. Recently, ICI Pharmaceuticals (Macclesfield, England) synthesized an estradiol deriva-

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tive, ICI 164,384, with full anti-estrogenic properties [24]. In the present work we describe the biological effects of this antiestrogen in different hormone-dependent and hormone-independent mammary cancer cell lines. Materials and methods Cell culture The following human breast cancer cell lines were used: MCF-7 [22], T-47D, another hormone-dependent cell line which contains high levels of progesterone receptors and low levels of estrogen receptors [13], the LY-2 which is a variant clone derived from the MCF7 cells and is resistant to the antiestrogen LY117018 [3] and the MDA-MB-436, which is hormone-independent [5]. These cell lines were kindly provided by Dr M.E. Lippman (Georgetown University, Washington, U.S.A.). The cells were grown routinely as monolayer cultures in Eagle’s Minimal Essential Medium (MEM) containing 10 mM HEPES, supplemented with 2 mM L-glutamine, 100 units/ml penicillin-streptomycin (Gibco Europe Ltd., Paisley, Sctoland) and 5% fetal calf serum (FCS) (Flow Laboratories Inc., U.K.) in a humidified atmosphere of 5% CO, in air at 37 OC. The cells were passed every 8-10 days and replated in 75-cm2 flasks (C.M.L., Nemours, France) at 3 x lo6 cells/flask for MCF-7, T-47D and LY-2 cells and at 0.8 x lo6 cells/flask for MDA-MB-436 cell line. Cell growth and DNA measurement MCF-7 cells were plated in quadruplicate in 24-well dishes at a density of 75,000 cells/well for MCF-7, T-47D and LY-2 and of 25,000 cells/well for MDA-MB-436 cell line. The cells were grown in MEM with 5% steroid-depleted FCS, prepared by mixing the dextran-coated charcoal (DCC-FCS) . Estradiol was purchased from Steraloids (Touzart et Matignon, Vitrysur-Seine, France) and the anti-estrogen ICI 164,384 [N-n-butyl-N-methyl-ll-(3,17p-dihydroxyestra-1,3,5(10-trien-7a-yl))undecana-

mide] was a gift from Dr A. Wakeling (ICI Ltd, Macclesfield, England). The compounds were added in ethanol (final concentration < 0.1%) . At various times, DNA was measured in each well as described by Taylor et al. [23]. Briefly, the medium was removed and the cells were stored at - 20°C until assayed. Eight hundred microliters 0.5 N PCA were added to each well and the plates were heated at 90°C in a dry oven for 30 min. Standards of calf thymus DNA (2-60 pg) were made in 0.5 N PCA and treated in the same manner. After cooling at room temperature, 1.6 ml diphenylamine reagent [4] were added to each well. The optical density was measured at 595 nM after an overnight incubation at room temperature. The coefficient of correlation between DNA and cell number counted in hemocytometer was about 0.95. rl-f]Thymidine incorporation experiments Cells were cultured in the same manner as that described for cell growth experiments. The cells were labeled with 0.5 &i/well of [3H]thymidine (CEA, Gif-sur-Yvette, France). After 2 h incubation at 37OC, the cells were washed 3 times with 2 ml ice-cold HBSS and 1 ml 0.5 N PCA added. Acid-insoluble counts were extracted by a 30-min incubation at 9o”c. Progesterone receptor assay At confluence, MCF-7 cells were trypsinized and replated at 3 x lo6 cells/flask into MEM containing 5% DCC-FCS. Five days later, estradiol (E,) (lo-lo M) and ICI 164,384 (10m9 M 10m8 M) were added for 6 days with a change of medium every 2-3 days. Cells were harvested by a 15-min incubation at 37 OC with 1 mM EDTA prepared in HBSS. The suspension of harvested cells from two flasks were homogenized twice in 1 ml of TED buffer (0.01 M Tris-HCI, 1.5 mM dithiothreitol, pH 7.4) with centrifugation at 900 x g for 10 min. The supernatants were saved and centrifuged at 200,000 x g for 30 min to obtain the cytosol fraction. The 900 x g pellet was extracted twice with 1 ml 0.6 M KCI in TED

135

Results

buffer (pH 8.5) and also centrifuged at 200,000 x g for 30 min to obtain a clear nuclear myofibrillar extract. The progesterone binding was determined using the synthetic progestin [3H]R5020 (spec. act. 56 Ci/mmol, NEN, France). All the operations were carried out at 4OC. Cytosol and nuclear extracts were incubated with 1O-:3M [3H]R5020 with or without a lOO-fold molar excess of unlabeled R5020 at 4OC overnight. Bound and unbound R5020 were separated by adsorption with DCC (0.025%: 0.25% final concentration). Progesterone receptor concentrations are expressed as the sum of the binding found in both the cytosol and the 0.6 M KC1 nuclear extract. DNA concentrations were determined according to Burton [4].

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Comparative studies of the effect of the antiestrogen ICI 164,384 on the growth of different breast cancer cell lines are shown in Fig. lA-D. When MCF-7 (Fig. 1A) or T-47D (Fig. 1B) were cultured for 6 days in the presence of the anti-estrogen ICI 164,384, the cell proliferation was inhibited in a concentration-dependent manner. However, the MCF-7 cells were more sensitive to the anti-estrogen treatment. In the LY-2 subline (Fig. lC), selected for its resistance to the growth inhibitory effects of the anti-estrogen LY-117018 131, the cell

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Fig. 1. Dose response of the effect of ICI 164,384 on growth and 13H]thymidine incorporation in various breast cancer cells. MCF-7 (A), T-471) (B), LY-2 (C) and MDA-MB-436 (D) cells were plated in triplicate in 24.well dishes in MEM + 5% DCC-FCS. Increasing concentrations of ICI 164,384 (10-l’ M to lo-’ M) were added at 0 time. The DNA concentrations and the incorporated radioactivity were determined in each well 6 days later. Cellular proliferation ( n -W) (in pg DNA/well) and [3H]thymidine incorporation (O- - -Cl) (in dpm/well) were recorded as % of those in non-treated wells considered as control. Data shown on A, B, C and D are the average of two experiments.

136

Effect of ICI 164,384 on the progesterone receptor concentrations The basic progesterone receptor (PR) levels in MCF-7 cells growing in MEM + 5% DCCFCS are of 0.5-1.2 pmol/mg DNA, and the values for T-47D cells are 11.2---21.2 pmol/ mg DNA. In previous studies in this laboratory, it was observed that maximal induction of PR in MCF-7 or T-47D cells is obtained with lo-lo M E, [ 19, unpublished data]. Table 1 shows that in MCF-7 cells this basal level of PR was increased 4- to 7-fold after 6 days treatment with lo-lo M estradiol, whereas in T-47D cells, in which the basal levels of PR are very high, an increase of about 50% is observed after E, treatment. Using various concentrations of ICI 164,384 ( 10m8- lo-lo M) it is observed that alredy from a concentration of 10m9M this anti-estrogen decreased the PR induction and completely blocked it with a concentration of lob8 M. It is to be remarked that the cells are grown in medium containing phenol red, a compound which has been demonstrated to have a weak estrogenic activity [Z]. In the present experiments, when T47D cells were cultured in the absence of

proliferation was not significantly affected by the presence of ICI 164,384, only the antiproliferative effect is observed at high doses (lo+ M). There is also no effect of this antiestrogen on the cell growth of the estrogenindependent cell line MDA-MB-436 (Fig. 1D) up to a 10m6 M concentration which provoked a decrease of about 30% of the proliferation. Studies of the effect of ICI 164,384 on [3H]thymidine incorporation agree with the effect on cell proliferation in the different cell lines. Time course experiments with MCF-7 cells showed that a 10m7M concentration of ICI 164,384 completely inhibited cell proliferation (Fig. ZA). In contrast, time course experiments with the other estrogen dependent cell line, T47D (Fig. ZB), showed a smaller effect with only a ~30% decrease even after 14 days of treatment. Figure 2 shows also that ICI 164,384 in the MCF-7 cells (Fig. 2A) at the dose of 10m7 M completely inhibits the proliferative effect of E . In the T-47D cells at the dose studied, E, ( 10WIO M) has a limited effect on growth and ICI 164,384 significantly decreases this effect (Fig. 2B) .

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DAYS IN CULTURE Fig. 2. Time course of the growth inhibition by ICI 164.384 in MCF-7 and T-47D breast cancer cells. MCF-7 (A) and T-47D (B) cells were grown in MEM + 5% DCC-FCS for increasing periods of time. After 3 days compounds were added to the medium: E, (lo-I’M) (A), ICI 164.384 (10m7M) (m), E, (lo-l0 M) + ICI 164,384 (10m7M) (17). Results are expressed as the average of two experiments.

137 Table 1.

Effect of the anti-estrogen

ICI 164,384 on PR concentrations

in MCF-7 and T-47D mammary

Total PR (cytosol + 0.6 M KC1 nuclear extracts) (pmol/mg

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Five days after plating in 5% DCC-FCS, MCF-7 and T-47D cells were treated for 6 days with the anti-estrogen ICI 164,384 ( 10m9- lo-’ M) or with estradiol (lo-” M) alone or in the presence of ICI 164,384 (lo+lo+ M) The cytosol and nuclear extracts were prepared as described in Materials and methods. The results are expressed as the mean + S.E. of 4-7 determinations. lP< 0.005 vs. control values. 'P< 0.05 vs. E,-treated cells. l

phenol red, lower concentrations of PR (4.6 pmol/mg DNA) in non-treated cells were observed. Therefore, it is suggested that part of the decrease in basal PR concentrations observed after treatment of the cells with lo+ M ICI 164,384 alone could be due to the suppression by the anti-estrogen of the weak phenol red estrogenic activity. Discussion The present de.ta show significant differences in the antiproliferative effect of the antiestrogen ICI 164,384 in the various mammary cancer cell lines studied. In the MCF-7 cells at very low concentrations (lo-l1 M) an effect on cell growth can already be observed and 50% of the inhibition is obtained with a concentration of 10e9 M. However, in T-47D cells a concentration of lo+ M is necessary to reach 50% of growth inhibition. Related to the effect in MCF-7 cells, the present data are in agreement with that obtained by Wakeling and Bowler 1241 who also observed a similar effect in ZR75-l mammary cancer cells [25]. As the T47D cells contain very low concentrations of estrogen receptor [ 1131,this could explain why ICI 164,384 provokes a much less intense

effect on cell proliferation in T-47D than in MCF-7 cells. Interesting information is obtained with the growth effect in LY-2 cells which are resistant to the anti-estrogen LY-117018. In these cells very little or no effect was observed still at very high doses (10S6 M) of ICI 164,384. As these cells contain significant quantities of estrogen receptors, the lack of activity of this anti-estrogen can be explained by the possibility of different mechanisms in the various steps of the anti-estrogen action (e.g., in the process of activation of the estrogen receptor). Finally, no effect on proliferation was observed with the hormone-independent MDA-MB-436 cell line. In the different cell lines studied it was observed that this effect of ICI 164,384 on cell proliferation agrees with the [3H]thymidine incorporation. Another attractive aspect of the present work is obtained in the effect of ICI 164,384 on PR in MCF-7 and T-47D cells. E, in the MCF-7 cells at the concentration of lo-lo M stimulates PR by 5-7 times and ICI 164,384 can block this effect at the dose of 10m9M and completely at lo-* M. This is important because it is well known that PR is controlled by E, in different target tissues [7,16,18] and it

138

has been postulated that PR might serve as a marker of estrogen action in hormone-dependent breast cancer [II]. It was shown that in the MCF-7 cells tamoxifen related to PR can have a dual dose-dependent effect; at low concentrations (10-8-10-7 M) it acts as a potent estrogen and induces PR, but at 10m6M PR is inhibited [lo]. Recent data have also demonstrated that tamoxifen can provoke a dual effect in breast cancer patients. Noguchi et al. [17] observed that after treatment with tamoxifen (20 mg twice daily) PR increased 4.6-fold in postmenopausal patients and 2-fold in pre-menopausal patients treated for 3-7 days, however after a longer treatment (14 days) the stimulatory effect on PR is abolished. It is to remark that, in another hormonedependent mammary cancer cell line, the Zr75-1, tamoxifen has no effect on PR [l]. In the T-47D cells which contained 18-25 times the concentration values of PR than in the MCF-7 cells, ICI 164,384 also provoked a significant decrease of PR in both the nontreated and &-treated cells. The most probable mechanism of the action of ICI 164,384 in these biological responses is through the estrogen receptor. This is argued because [3H]ICI 164,384 binds estrogen receptor with high affinity (K = 0.69 nM) which is similar to the binding of estradiol to estrogen receptor [27], and second no response was found with the mammary cancer cell line MDA 436 which does not contain estrogen receptor. the anti-estrogen ICI In conclusion, 164,384 acts as a full antagonist in hormonedependent mammary cancer cells (MCF-7 and T-47D) for both responses, proliferation and progesterone receptor, has no effect in an antiestrogen resistant cell, the LY-2, and in a hormone-independent cell line (MDA-MB-436). Consequently, the utilization of the anti-estrogen ICI 164,384 in experimental models can open new possibilities in the knowledge of the different steps of the mechanism of action of the anti-estrogens in breast cancer.

Acknowledgements Part of the expenses of this work was National de la defrayed by the “Centre Recherche Scientifique”, France (C.N.R.S. UA-548), and by the “Fondation pour la Recherche Medicale Francaise”. We would like to express our deep thanks to Dr M.E. Lippman (Washington, U.S.A.) for the different mammary cancer cell lines, and to Dr A.E. Wakeling (Macclesfield, England, U.K.) for the gift of ICI 164,384. References 1

2

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