Effects of Liarozole, a New Antitumoral Compound, on Retinole Acid

[CANCER RESEARCH 52, 2841-2846. May 15, 1992] Effects of Liarozole, a New Antitumoral Compound, on Retinole Acid-induced Inhibition of Cell Growth and on Retinoic Acid Metabolism in MCF-7 Human Breast Cancer Cells Walter Wouters,1 Jacky van Dun, Ann Dillen, Marie-Claire Coene, Willy Cools, and Roland De Coster

Departments of Endocrinology and Oncologi' Â¡W.W., J. v. D., A. D., R. D. C.J and Immunology /M-C. C., W. C.J, Janssen Research Foundation, Turnhoutseweg 30, B-2340 Beerse, Belgium

ABSTRACT decreased by at least 50% in more than half of the patients (1, 3). Liarozole Â¡sa new imidazole derivative with antitumoral properties. The antitumoral effect of liarozole in androgen-independent Effects of the compound alone and in combination with all-frans-retinoic acid on proliferation of MCF-7 human breast cancer cells were examined rat prostate tumor models as well as in androgen refractory prostate carcinoma in men suggests the involvement of a non- using a 3-(4,5-dimethylthiazoI-2-yl)-2,5-diphenyl-2//-tetrazolium bro androgen-related mechanism of action. mide assay. Following 9 days of drug exposure, MCF-7 cell growth was concentra Retinoic acid is known to play a key role in proliferation and tion dependent!) inhibited by all-fra/u-retinoic acid (drug concentration differentiation of epithelial tissues and has been shown to have resulting in 50% growth inhibition, 2 x 1(1KM), while liarozole at III'" anticarcinogenic and antitumoral properties (4-7). Retinoic M inhibited cell growth by only 35%. acid is, however, rapidly metabolized, which results in its deac- When MCF-7 cells were incubated with a combination of all-m/;iv- tivation (8, 9). The first enzymatic reaction in the biodÃ©grada retinoic acid and Harozole, the antiproliferative effect of all-frani-retinoic tion of retinoic acid is its conversion to the less active 4-hydroxy acid was clearly enhanced. This enhancement was dependent on the metabolite. This reaction is catalyzed by the cytochrome P-450- liarozole concentration and was more than 10-fold. A combination of 10s M Â¡ill-rn/nv-ri'tumiracid and IO1' M liarozole resulted in a greater dependent 4-hydroxylase (10-13) which has been shown to be antiproliferative effect than that obtained with 10' M all-fra/iÃ®-retinoic present mainly in the liver. However, retinoic acid metabolism was also found in F9 teratocarcinoma cells (14) and LCC-PK, acid alone. When MCF-7 cells were incubated for 4 h with |'H|all-rrani-retinoic cells (15), as well as in yV-nitroso-/V-methylurea-induced rat acid, the radioactivity in the supernatant consisted of unaltered retinoid. mammary tumors (16). However, when cells had been pretreated with 10 '' M all-Ãrans-retinoic Liarozole inhibits several cytochrome P-450-dependent en acid overnight, they were able to substantially metabolize |'ll|Â¡tll-m/n\- zymes (17, 18), and among these is 4-hydroxylase, which me retinoic acid during a subsequent 4-h incubation. High-performance liquid tabolizes retinoic acid. Oral administration of liarozole to ex chromatography analysis of the supernatants revealed that the reaction perimental animals results in increased plasma retinoic acid products consisted mainly of very polar metabolites. Liarozole inhibited the metabolism of all-rrans-retinoic acid in MCF-7 cells with KP" M levels (19), and this may contribute to the antitumoral effect of the compound. liarozole reducing the amount of polar metabolites by 87%. The present study was undertaken to examine the effects of It is concluded that the enhancement by liarozole of the antiprolifera tive effects of retinoic acid on MCF-7 human breast cancer cells is liarozole in relation to retinoic acid on cancer cells in tissue probably due to inhibition of retinoic acid metabolism. Further research culture. For this purpose, the MCF-7 human breast cancer cell into these effects in MCF-7 cells as well as in other cancer cell lines will line was chosen since it is known that retinoids have profound provide more information concerning the exact mechanism of action of effects on proliferation of this cell line (20, 21). liarozole and the use of inhibitors of retinoid metabolism in cancer treatment. MATERIALS AND METHODS Drugs and Chemicals. Liarozole |5-[(3-chlorophenyl)(l//-imidazol- INTRODUCTION l-yl)methyl]-l//-benzimidazole| (chemical structure depicted in Fig. 1), ketoconazole, miconazole, and vorozole (R 83 842) were synthesized Liarozole (R 75 251) is a new imidazole derivative with by and obtained from the Janssen Research Foundation. Aminoglute- antitumoral properties. The compound reduces tumor growth thimide and metyrapone were obtained from Sigma Chemical Co. (St. in the androgen-dependent Dunning G and H rat prostate Louis, MO). The compounds were dissolved at a concentration of 10~2 adenocarcinoma models as well as in the androgen-independent M in ethanol or dimethyl sulfoxide. All-frani-retinoic acid (Serva, Matlu, PIF-1, and H* sublines. The antitumoral effect of lia Heidelberg, Germany) was dissolved at an initial concentration of 4 x IO"3 M in ethanol. [1l,12-3H(N)]All-ÃranÃ-retinoicacid (1824 GBq/ rozole in the androgen-dependent Dunning G model cannot be mmol, 49.3 Ci/mmol) was obtained from NEN (Dupont de Nemours, reversed by supplementation with testosterone (1,2). BrÃ¼ssel,Belgium). Retinoid stock solutions were regularly checked for In an open pilot study of 31 patients with actively progressing purity using HPLC2 analysis. Retinoid manipulations were carried out stage D prostate carcinoma after castration, liarozole induced in a dark room with yellow illumination. Further dilutions of all test subjective responses (marked improvement in pain and per compounds were made in culture medium. Final solvent concentrations formance) in the majority of Ã©valuablepatients and partial during incubation were always <0.25% (v/v). These concentrations had remission (50% reduction in primary tumor or metastatic no effect on cell growth. lymph node volume) in 2 of 2 patients with measurable disease. Cell Cultivation. The MCF-7 cell line was obtained from the Ameri After 3 months of treatment, prostate-specific antigen levels can Type Culture Collection (Rockville, MD). Cells were routinely cultured as adherent monolayers at 37" in 5% CO2-95% air at 100%

Received 8/8/91; accepted 3/11/92. relative humidity in Falcon tissue culture flasks (Becton Dickinson, The costs of publication of this article were defrayed in part by the payment Aalst, Belgium). The culture medium was Dulbecco's modified Eagle's of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 2The abbreviations used are: HPLC, high-performance liquid chromatogra ' To whom requests for reprints should be addressed. phy; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2//-tetrazolium bromide. 2841

Brussels, Belgium). Samples were eluted with methanol:water:formic acid (65:35:0.05) containing 10 mM ammonium acetate at a flow rate of 2 ml/min. After 20 min, the solvent was changed to 100% methanol in order to elute retinole acid. Statistical Analysis. Where appropriate, data were analyzed using the two-tailed Student's t test. Significance was defined at the level of P < 0.05. Fig. 1. Chemical structure of liarozole.

RESULTS medium with 4.5 g/liter glucose and 3.7 g/liter sodium bicarbonate supplemented with 2 mM glutamine, 1 mM sodium pyruvate, 100 IU/ The effects of all-fra/is-retinoic acid and liarozole on prolif ml penicillin, 100 ^g/ml streptomycin sulfate (all reagents from Life eration of MCF-7 cells are shown in Fig. 2. Continuous expo Technologies, Gent, Belgium), and 10% fetal calf serum (Sera-Lab, International Medical Products, BrÃ¼ssel,Belgium). Cells were subcul- sure of the cells to all-iraws-retinoic acid for 9 days led to concentration-dependent inhibition of cell growth with >90% tured once weekly at a split ratio of 1:10 using trypsin/EDTA solution inhibition achieved at the highest retinoid concentration (10~5 and were regularly checked for Mycoplasma contamination. For the experiments described in this study, the MCF-7 cell line was used at M). The calculated drug concentration resulting in 50% growth passages between 158 and 174. inhibition was 2 x 10~8M. Liarozole up to 10~6M had no effect Growth Experiments. Stock cultures were trypsinized, and cells were on cell proliferation, and at IO"5 M a 35% inhibition was seen. counted using a hemocytometer and plated at 2000 cells/well in 96- To study possible interrelations between liarozole and all- well microtest tissue culture plates (Falcon; Becton Dickinson, Aalst, fra/is-retinoic acid, MCF-7 cells were continuously incubated Belgium) in 150 uI medium. For each experimental condition, 8 repli with combinations of both substances during 9 days. Two low cate wells were used. The cells were allowed to attach for 24 h. Then, concentrations of all-fra/w-retinoic acid, which had no (10~9 M) the test compounds or the solvent were added to a final volume of 200 or low (10~8 M) antiproliferative effect when used alone, were n\ (day 0), and the cells were further incubated for the indicated times. Medium and test compounds were renewed on days 2, 5, and 7. combined with increasing concentrations of liarozole (Fig. 3). Following the appropriate incubation period, the amount of metaboli- This resulted in an enhancement of the antiproliferative effect cally active cells was measured using a tetrazolium-based (MTT) assay that was dependent on the liarozole concentration. When 10~9 (22). M all-rrans-retinoic acid (producing no inhibition when used MTT Assay. MTT (Serva) was dissolved at 5 mg/ml in phosphate- alone) was used, addition of 10~6 M liarozole significantly buffered saline. At the end of the growth experiments, 25 n\ of this inhibited cell growth by 52% and, at 10~5 M liarozole, an 85% solution was added to each well without removing the medium, and the inhibition was obtained. With IO"8 M all-/rans-retinoic acid cells were further incubated for 2 h at 37Â°C. The medium was then (producing 28% inhibition when used alone), growth was sig carefully aspirated, and the blue MTT-formazan product was solubi- nificantly inhibited by 53, 83, and 93% with 10~7, Ifr6, and lized by addition of 100 M' dimethyl sulfoxide. The microtest plates 10~5M liarozole, respectively. were shaken for 10 min on a microplate shaker, and the absorbance at 540 nm was read using an automatic plate reader (Titertek Multiskan The effects of all-fra/is-retinoic acid, liarozole, and combina MCC/340; Flow, Asse-Relegem, Belgium). The MTT assay was vali tions of both substances on MCF-7 cell growth were also dated for use with MCF-7 cells by comparing the MTT results obtained 3 h after plating different numbers of cells with those obtained by hemocytometer counting (data not shown). The method was linear up Cell proliferation to the highest cell concentration tested (40,000 cells/well). (% of control) Retinole Acid Metabolism. A 175-cm2 confluent MCF-7 culture, 140-1 growing under normal conditions, was treated for 18 h with IO"6M all- rrani-retinoic acid. Cells were then washed twice with 20 ml culture medium and trypsinized. Cells were suspended at 6 x IO6 cells/ml in 120- Dulbecco's modified Eagle's medium without phenol red, containing 1 g/liter glucose and supplemented with 2 mM glutamine, 1 mM sodium 100- pyruvate, 100 ID/ml penicillin, 100 Mg/ml streptomycin sulfate, 2.5 Mg/ml Fungizone, and 10% heat-inactivated fetal calf serum (all re agents from Life Technologies). 80- Aliquots (900 n\) of this cell suspension in glass test tubes were preincubated for 5 min at 37Â°Cwith 50 IJL\liarozole(final concentration 10~5 M) or its solvent. Incubation was then continued for 4 h with 60- occasional shaking in the presence of 50 n\ [3H]all-Ãra/iÃ-retinoicacid (final concentration 10~7M). At the end of incubation, the cell suspen 40- sion was centrifuged at 3000 rpm for 5 min. The cell pellet was extracted with I ml methanol. Aliquots of this extract and of the supernatant were counted for recovery; the rest was dried in vacuo (Savant Speed 20- Vac Concentrator) and used for HPLC analysis. HPLC Analysis. Reverse-phase HPLC analysis was carried out as described previously (23) with several modifications. A Varian 5560 10' 10 " io- 10' 10Â° 10' 10" liquid Chromatograph equipped with a Perkin-Elmer ISS 100 automatic injector, a UV-200 variable wavelength detector set at 340 nm, and a Drug concentration (M) Vista 401 data system was used. Radioactivity in the eluate was moni Fig. 2. Effect of all-/rans-retinoic acid (â€¢)and liarozole (O) on proliferation of tored on-line by /3-counting (Berthold LB504 radioactivity monitor) MCF-7 cells. Cells were incubated with the compounds for 9 days with medium using Pico-Aqua (Canberra-Packard, BrÃ¼ssel,Belgium) as the scintil changes on days 2, 5, and 7. Cell proliferation was estimated using an MTT assay. Results are expressed as percentages of a control incubation with solvent lation solvent. Samples were analyzed on a 10-Mm CiÂ«^Bondapak and are means (points) Â±SD (bars) of three independent experiments with eight column (3.9 mm inside diameter x 300 mm) (Waters Associates, replicates for each experimental condition. 2842

Cell proliferation Several different inhibitors of cytochrome P-450 were tested (% of control) for their ability to enhance the antiproliferative effects of 10~9 120n _._ M all-Ãrans-retinoic acid; the results are shown in Table 1. The drugs were used at 10~6 M. At this concentration when used 100- alone, they had no antiproliferative effect. Liarozole, ketoconazole, and miconazole were able to enhance the effect of all- fra/is-retinoic acid, resulting in cell growth inhibition by 46, 26, 80 and 19%, respectively. The effects of liarozole and ketoconazole ; l were statistically different. 60 Since liarozole and ketoconazole are known to inhibit the 4-hydroxylation of retinoic acid, we next tried to assess retinoic 40- acid metabolism in MCF-7 cells. When cells growing under normal conditions were incubated for 4 h with 10~7 M ['HJall- 20- /ra/is-retinoic acid, the radioactivity recovered from the super natant consisted of [JH]retinoic acid (results not shown). Therefore, cells were pretreated overnight with 10~6M all-Ãraws 10-8 10'7 106 10-5 retinoic acid, washed twice, collected, and subsequently incu bated with ['Hjall-fra/is-retinoic acid. Fig. 5A shows a repre [Liarozole] (M) sentative example of the radioactivity profile obtained by HPLC Cell proliferation analysis of the supernatant of cells treated in this way. The (% of control) quantitative analysis of this experiment is given in Table 2. 120 Following a 4-h incubation, >85% of the radioactivity was B consistently recovered in the supernatant. Only 47% of this 100

Absorbance (540 nm) 1.4-1

1.2-

1.0

0.8 10'7 10-6 1Q5 [Liarozole] (M) 0.6- Fig. 3. Effects of 10"' (A) and I0~* M (B) all-frani-retinoic acid alone or combined with liarozole on proliferation of MCF-7 cells. A, effect of liarozole 0.4- alone. Cells were incubated with the compounds for 9 days with medium changes on days 2, 5, and 7. Cell proliferation was estimated using an Mil assay. Results are expressed as percentages of a control incubation with solvent and are the 0.2 means (columns) Â±SD (bars) of three independent experiments with eight replicates for each experimental condition. * = P < 0.05 versus all-Ãrans-retinoic 8 10 acid alone. Time (days) Fig. 4. MCF-7 growth curves in the presence of solvent (O), 10 * M liarozole (â€¢),10~' M all-franj-retinoic acid (A), 10~*M all-frans-retinoic acid (D). 10~7 M studied as a function of incubation time. Fig. 4 shows the results all-franj-retinoic acid (x), 10~9 M all-franj-retinoic acid combined with IO"6 M liarozole (A), or 10~! M all-frans-retinoic acid combined with 10~* M liarozole of a representative experiment (similar results were obtained in (â€¢).Cells were incubated with the compounds for 9 days with medium changes two other experiments). Cells were continuously cultured with on days 2, 5, and 7. On days 2, 5, 7. and 9, cell proliferation was estimated using the test compounds, and growth was assessed on days 2, 5, 7, an MTT assay. A representative experiment; point, mean of eight replicates. and 9. Under control conditions, cells grew exponentially until day 9, and incubation with 10~6M liarozole or 10~9M all-trans- Table 1 Effects of different cytochrome P-450 inhibitors (10 ' M) combined with retinoic acid did not alter the growth curve. Effects of 10~8 M I0~g M all-trans-retinoic acid on the growth of MCF-7 cells all-mzHs-retinoic acid alone or of a combination of 10~9 M all- Results are expressed as percentages of control growth in the presence of 10~9 fra/w-retinoic acid with 10~6 M liarozole were apparent on day M all-Ãrans-retinoic acid alone and are means Â±SD. The number of experiments is given in parentheses (eight replicates were used within each experiment). 9 only. The antiproliferative effect of 10~7 M all-fraws-retinoic proliferation acid alone or a combination of 10~8 M all-fraws-retinoic acid DrugsLiarozole (% ofcontrol)54 with 10~6 M liarozole was already visible on day 5. Evaluation Â±8(4) of the data on day 9 revealed that the combination of IO"9 M Ketoconazole 74 Â±9(3)Â° 81 Â±6(2)Â° all-frfl/js-retinoic acid with 10~6 M liarozole was more effective Miconazole Vorozole 92 Â±6(4)Â°-* than 10~8 M all-frflrts-retinoic acid alone. Similarly, a combi Aminogluthetimidc 101 Â±7(3)Â°-* nation of 10~8 M all-fraws-retinoic acid with 10~6 M liarozole MetyraponeCell 90 Â±2(2)"-* was more effective than 10~7 M all-Ãrans-retinoic acid alone. ' Statistically different versus liarozole. *Not statistically different versus control. Thus, liarozole enhanced the retinoid effect about 10-fold. 2843

Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 1992 American Association for Cancer Research. EFFECTS OF LIAROZOLE AND RETINOIC ACID IN MCF-7 CELLS DTE: sentially similar results. Liarozole (10 5M) reduced the amount of polar metabolites in the supernatant by 94 Â±6% (mean Â± U SD).

DISCUSSION

The present study was undertaken to gain more insight into I the as yet incompletely understood antitumoral mechanism of action of liarozole, a new compound showing antiproliferative g effects in both steroid-dependent and steroid-independent ani OC mal tumor models. Liarozole by itself had nearly no effect on the growth of MCF-7 cells but significantly potentiated the antiproliferative effects of all-/rfl/is-retinoic acid > 10-fold. This potentiation was clearly a synergistic interaction since concentrations of all- /rans-retinoic acid and liarozole, which by themselves were inactive, showed antiproliferative effects when combined. Moreover, this synergy is seen at physiologically relevant con B centrations of both substances. Indeed, a concentration of 1- 10 nM of all-frans-retinoic acid correlates with a normal human plasma level, and the active concentrations of liarozole (10~7- 10~5M) are within the range of the drug plasma levels obtained after oral intake of a standard liarozole dose (300 mg). Very few other compounds have been reported to act syner- gistically with retinoids in MCF-7 cells. Valette and Botanch rc (24) showed synergistic potentiation of the antiproliferative o effects of all-frartÃ®-retinoicacid by transforming growth factor- ÃŸ.Although interactions between 7-interferon or a-interferon and retinoic acid were also described (25, 26), these effects were additive rather than synergistic and were obtained with retinoic acid concentrations 2-3 orders of magnitude higher than those used in the present study. The structure and pharmacological profile of liarozole do not suggest any similarity with these cytokines. 10 20 30 On the other hand, liarozole inhibits several cytochrome P- Retention time (min) 450-dependent enzymes. The two most prominent enzymes for Fig. 5. Metabolism of all-ira/tj-retinoic acid in MCF-7 cells. Cells were which inhibition by liarozole has been shown are 4-hydroxylase preincubated with IO"6 M all-franÃ-retinoic acid for 18 h, washed twice, and (4-hydroxylation of retinoic acid) and aromatase (conversion of collected. Cells were then incubated for 4 h with 10~7 M |3H]all-frani- retinoic acid in the absence (A) or presence (B) of 10"' M liarozole. and the supernatants androgens to estrogens) (17, 19). Therefore, we compared the were analyzed by reverse-phase HPLC. I lutimi positions: peak I, very polar effects of liarozole to those of several other known P-450 metabolites: peak II, moderately polar metabolites; peak III, M-lrans- and 13-ris- blockers. Vorozole (R 83 842), a potent and selective aromatase retinoic acid; peak Ã•V,apolar metabolite. The quantitative analysis of this exper inhibitor (27), had no effect on MCF-7 cell growth when used iment is shown in Table 2. alone or combined with all-fra/is-retinoic acid. The same was true for aminoglutethimide, an inhibitor of aromatase, 11- consisted of retinoic acid (all-trans + 13-c/s) (peak III), while hydroxylase, and side-chain cleavage (28), and metyrapone, a 48% was recovered as very polar metabolites (peak I) and 3% 11-hydroxylase inhibitor (29). Only ketoconazole and micona- as metabolites with intermediate polarity (peak II). One metab zole showed results similar to those obtained with liarozole, but olite with less polarity than retinoic acid was also found, but the potentiation of the effect of all-/raws-retinoic acid was less this consisted of only 2% of total radioactivity (peak IV). In the pronounced than with liarozole. Ketoconazole, which derives cell extract, the same overall metabolite profile was found, but its antifungal properties from blocking 14Â«-demethylase in 29% of total radioactivity was recovered in the apolar peak (Table 2). Table 2 Quantitative analysis off'HJall-trans-retinoic add metabolism In the presence of 10"* M liarozole, distribution of radioac in MCF-7 cells tivity between the cell supernatant and the cell pellet was Roman numerals in parentheses refer to the HPLC elution positions indicated in Fig. 6. unchanged. The radioactivity profile obtained by HPLC analy Radioactivity (% of total) sis, however, was completely different (Fig. 5B, Table 2). In the supernatant, only 6% of total radioactivity was recovered as Supernatant Cell extract very polar metabolites (peak I), and another 7% was found in HPLC peaks Control + 10 * M liarozole Control -I-10"* M liarozole the apolar peak (peak IV). The rest (87%) eluted in the retinoic Retinoic acid (all-rran; 47 87 24 52 acid peak (peak III). In the cell extract, the amount of polar MetabolitesVery metabolites was reduced to 3%. The rest of the radioactivity polar(1)Moderately eluted as unchanged retinoic acid and in the apolar peak. (II)Apolar polar This metabolism experiment was repeated 4 times with es (IV)4832607470293045 2844

Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 1992 American Association for Cancer Research. EFFECTS OF LIAROZOLE AND RETINOIC ACID IN MCF-7 CELLS yeasts and fungi, also inhibits 17a-hydroxylase/17,20-lyase these compounds have potential use in cancer therapy by en (conversion of progestins to androgens) and aromatase, as well hancing endogenous plasma retinoic acid levels through inhi as the 4-hydroxylation of retinole acid (23, 30). Miconazole is bition of liver retinoic acid metabolism (18). If tumor tissue an antifungal which is also a weak aromatase inhibitor (31). also metabolizes retinoic acid, treatment with metabolism in The results of this comparative experiment were in agreement hibitors may result in higher local (tumor) retinoic acid levels. with the activity of these compounds for inhibition of in vivo In this way, the full antineoplastic activity of retinoic acid might metabolism of all-fraÂ«i-retinoic acid as described by Van be exploited while minimizing the side effects related to high Wauwe et al. (19) and with the data of Williams and Napoli systemic retinoic acid levels. (32) obtained for retinoic acid metabolism in F9 cells. This suggested that the effects of liarozole on MCF-7 cells could be related to inhibition of 4-hydroxylase, an enzyme activity not ACKNOWLEDGMENTS previously described in these cells. The authors wish to thank Dr. Jean Van Wauwe for his critical We were unable to detect under basal conditions any retinoic review of the manuscript. acid metabolism in MCF-7 cells. However, pretreatment with all-fraws-retinoic acid induced a very active conversion of all- mzns-retinoic acid to more polar metabolites with most of the REFERENCES metabolized retinoic acid recovered as very polar metabolites. 1. De Coster, R., Van Ginckel, R., van Moorselaar, R. J. A., Schalken, J. A., A similar induction by retinoic acid of its own metabolism was Debruyne, F. M. J., Bruynseels, J., and Denis, L. Antitumoral effect of R previously reported for some embryonal carcinoma cell lines 7S2S1 in prostatic carcinoma: experimental and clinical studies. Proc. Am. Assoc. Cancer Res., 32: 213, 1991. (33). The overall HPLC profile was similar to that found in F9 2. 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Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 1992 American Association for Cancer Research. Effects of Liarozole, a New Antitumoral Compound, on Retinoic Acid-induced Inhibition of Cell Growth and on Retinoic Acid Metabolism in MCF-7 Human Breast Cancer Cells

Walter Wouters, Jacky van Dun, Ann Dillen, et al.

Cancer Res 1992;52:2841-2846.

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