Autoinduction of Retinoic Acid Metabolism to Polar Derivatives with Decreased Biological Activity in Retinoic Acid-sensitive, but Not in Retinoic Acid-resistant Human Breast Cancer Cells
1997; Elsevier BV; Volume: 272; Issue: 29 Linguagem: Inglês
10.1074/jbc.272.29.17921
ISSN1083-351X
AutoresBas‐jan M. van der Leede, C.E. van den Brink, W.W.M. Pim Pijnappel, Edwin Sonneveld, Paul T. van der Saag, Bart van der Burg,
Tópico(s)Antioxidant Activity and Oxidative Stress
ResumoPrevious studies have shown that all-trans-retinoic acid (RA) inhibits in vitroproliferation of hormone-dependent human breast cancer cells but not the growth of hormone-independent cells. Here we report on RA metabolism in breast cancer cells as examined by high performance liquid chromatography analysis and found a correlation with sensitivity to growth inhibition by RA. RA-sensitive T-47D and MCF-7 cells exhibited high rate metabolism to polar metabolites, whereas RA-resistant MDA-MB-231 and MDA-MB-468 cells metabolized RA to a much lesser extent, and almost no polar metabolites could be detected. The high metabolic rate in RA-sensitive cells appears to be the result of autoinduction of RA metabolism, whereas RA-resistant cells showed no such induction of metabolism. We observed furthermore that transfection with retinoic acid receptor-α expression vectors in RA-resistant MDA-MB-231 cells resulted in increased RA metabolism and inhibition of cell proliferation. Metabolism of RA, however, seems not to be required to confer growth inhibition of human breast cancer cells. The biological activity of the polar metabolites formed in RA-sensitive cells was found to be equal or lower than that of RA, indicating that RA itself is the most active retinoid in these cells. Together our data suggest that RA-sensitive cells contain mechanisms to activate strongly the catabolism of RA probably to protect them from the continuous exposure to this active retinoid. Previous studies have shown that all-trans-retinoic acid (RA) inhibits in vitroproliferation of hormone-dependent human breast cancer cells but not the growth of hormone-independent cells. Here we report on RA metabolism in breast cancer cells as examined by high performance liquid chromatography analysis and found a correlation with sensitivity to growth inhibition by RA. RA-sensitive T-47D and MCF-7 cells exhibited high rate metabolism to polar metabolites, whereas RA-resistant MDA-MB-231 and MDA-MB-468 cells metabolized RA to a much lesser extent, and almost no polar metabolites could be detected. The high metabolic rate in RA-sensitive cells appears to be the result of autoinduction of RA metabolism, whereas RA-resistant cells showed no such induction of metabolism. We observed furthermore that transfection with retinoic acid receptor-α expression vectors in RA-resistant MDA-MB-231 cells resulted in increased RA metabolism and inhibition of cell proliferation. Metabolism of RA, however, seems not to be required to confer growth inhibition of human breast cancer cells. The biological activity of the polar metabolites formed in RA-sensitive cells was found to be equal or lower than that of RA, indicating that RA itself is the most active retinoid in these cells. Together our data suggest that RA-sensitive cells contain mechanisms to activate strongly the catabolism of RA probably to protect them from the continuous exposure to this active retinoid. Retinoids are a group of naturally occurring (e.g.all-trans-retinoic acid; RA 1The abbreviations used are: RA, all-trans-retinoic acid; 4-oxo-RA, 4-oxo-all-trans-retinoic acid; 4-hydroxy-RA, 4-hydroxy-all-trans-retinoic acid; 9-cis-RA, 9-cis-retinoic acid; 13-cis-RA, 13-cis-retinoic acid; RAR, retinoic acid receptor; RXR, retinoid X receptor; CRABP, cellular retinoic acid-binding protein; FCS, fetal calf serum; DCC, dextran-coated charcoal; HPLC, high performance liquid chromatography. ) and synthetic analogs of vitamin A which play an important role in cellular growth and differentiation (1De Luca L.M. FASEB J. 1991; 5: 2924-2932Crossref PubMed Scopus (816) Google Scholar, 2Gudas L.J. Sporn M.B. Roberts A.B. Sporn M.B. Roberts A.B. Goodman D.S. The Retinoids: Biology, Chemistry, and Medicine. Raven Press, New York1994: 443-520Google Scholar). The actions of retinoids are mediated by two types of receptors, the retinoic acid receptors (RARs) and retinoid X receptors (RXRs) (3Mangelsdorf D.J. Umesono K. Evans R.M. Sporn M.B. Roberts A.B. Goodman D.S. The Retinoids: Biology, Chemistry, and Medicine. Raven Press, New York1994: 319-350Google Scholar, 4Chambon P. FASEB J. 1996; 10: 940-954Crossref PubMed Scopus (2604) Google Scholar). Both receptor types belong to the steroid-thyroid hormone receptor superfamily and regulate transcription in the presence of their ligands. RARs can be activated both by RA and 9-cis-RA, whereas only 9-cis-RA binds to RXRs (5Heyman R.A. Mangelsdorf D.J. Dyck J.A. Stein R.B. Eichele G. Evans R.M. Thaller C. Cell. 1992; 68: 397-406Abstract Full Text PDF PubMed Scopus (1568) Google Scholar, 6Levin A.A. Sturzenbecker L.J. Kazmer S. Bosakowski T. Huselton C. Allenby G. Speck J. Kratzeisen C. Rosenberger M. Lovey A. Grippo J.F. Nature. 1992; 355: 359-361Crossref PubMed Scopus (1101) Google Scholar). Retinoids are highly effective in preventing chemically induced carcinogenesis in experimental animals (7Moon R.C. Mehta R.G. Rao K.V.N. Sporn M.B. Roberts A.B. Goodman D.S. The Retinoids: Biology, Chemistry, and Medicine. Raven Press, New York1994: 573-595Google Scholar) and can inhibit proliferation of a large variety of normal and neoplastic cell typesin vitro (8Lotan R. Biochim. Biophys. Acta. 1980; 605: 33-91Crossref PubMed Scopus (1041) Google Scholar). More recently the effectiveness of retinoids in the treatment and prevention of a number of human cancers has been established (9Huang M.E. Yu-Chen Y. Shu-Rong C. Jin-Ren C. Jia-Xiang L. Long-Jun G. Zhen-Yi W. Blood. 1988; 72: 567-572Crossref PubMed Google Scholar, 10Castaigne S. Chomienne C. Daniel M.T. Ballerini P. Berger R. Fenaux P. Degos L. Blood. 1990; 76: 1704-1709Crossref PubMed Google Scholar, 11Hong W.K. Endicott J. Itri L.M. Doos W. Batsakis J.G. 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Oncol. 1993; 11: 1216-1222Crossref PubMed Scopus (325) Google Scholar). Unfortunately, lack of response to retinoid treatment and relapse of tumors are commonly observed. It is becoming increasingly clear that variations in metabolic rates of retinoids may be involved in the differences in retinoid response. Interindividual variation in the pharmacokinetics of retinoids has been reported for several malignancies, and a recent study suggested that high rate metabolism of RA is linked to an increased risk of squamous or large cell lung cancer (16Adamson P.C. Pitot H.C. Balis F.M. Rubin J. Murphy R.F. Poplack D.G. J. Natl. Cancer Inst. 1993; 85: 993-996Crossref PubMed Scopus (52) Google Scholar, 17Rigas J.R. Francis P.A. Muindi J.R.F. Kris M.G. Huselton C. DeGrazia F. Orazem J.P. Young C.W. Warrell R.P. J. Natl. Cancer Inst. 1993; 85: 1921-1926Crossref PubMed Scopus (93) Google Scholar, 18Rigas J.R. Miller V.A. Zhang Z.-F. Klimstra D.S. Tong W.P. Kris M.G. Warrell R.P. Cancer Res. 1996; 56: 2692-2696PubMed Google Scholar). In acute promyelocytic leukemia patients, relapse and resistance to RA have been associated with a rapid and marked decrease of retinoid levels in the plasma (19Muindi J.R.F. Frankel S.R. Huselton C. DeGrazia F. Garland W.A. Young C.W. Warrell R.P. Cancer Res. 1992; 52: 2138-2142PubMed Google Scholar, 20Takitani K. Tamai H. Morinobu T. Kawamura N. Miyake M. Fujimoto T. Mino M. J. Nutr. Sci. Vitaminol. 1995; 41: 493-498Crossref PubMed Scopus (6) Google Scholar). Retinoid resistance has also been documented in vitro in cell lines derived from various tumor types, including breast cancer (21Geradts J. Chen J.-Y. Russell E.K. Yankaskas J.R. Nieves L. Minna J.D. Cell Growth & Differ. 1993; 4: 799-809PubMed Google Scholar, 22van der Leede B.M. van den Brink C.E. van der Saag P.T. Mol. Carcinogen. 1993; 8: 112-122Crossref PubMed Scopus (43) Google Scholar). We and others have demonstrated that RA strongly inhibits proliferation of estrogen receptor-positive human breast cancer cell lines but not the growth of estrogen receptor-negative cell lines (23Marth C. Mayer I. Daxenbichler G. Biochem. Pharmacol. 1984; 33: 2217-2221Crossref PubMed Scopus (51) Google Scholar, 24Fontana J.A. Miranda D. Burrows Mezu A. Cancer Res. 1990; 50: 1977-1982PubMed Google Scholar, 25van der Burg B. van der Leede B.M. Kwakkenbos-Isbrücker L. Salverda S. de Laat S.W. van der Saag P.T. Mol. Cell. Endocrinol. 1993; 91: 149-157Crossref PubMed Scopus (136) Google Scholar). To investigate whether the RA resistance of human breast cancer cells is associated with enhanced retinoid breakdown, we examined metabolism in two estrogen receptor-positive and RA-sensitive (T-47D and MCF-7) and two estrogen receptor-negative and RA-resistant (MDA-MB-468 and MDA-MB-231) cell lines. We found that the RA-sensitive cells exhibited high rate metabolism of RA to polar metabolites, whereas the RA-resistant cells metabolized RA to a much lesser extent, and almost no polar metabolites were detected. We furthermore observed that transfection with RARα expression vectors in RA-resistant MDA-MB-231 cells resulted in increased RA metabolism and inhibition of cell proliferation. These observations suggested that metabolism of RA may be required to confer growth inhibition of human breast cancer cells. To investigate this possibility we examined the biological activity of the polar metabolites formed in RA-sensitive cells. By using retinoid-sensitive reporter cells, we found that only some RA derivatives (e.g. 4-oxo-RA) are active, whereas most others are inactive catabolic products, indicating that RA itself is the most active retinoid in these cells. Together our data suggest that RA-sensitive cells contain mechanisms to activate catabolism of RA to protect them from the continuous exposure to this active retinoid. DF medium (a 1:1 mixture of Dulbecco's modified Eagle's medium and Ham's F-12, buffered with 44 mmNaHCO3) and fetal calf serum (FCS) were obtained from Life Technologies, Inc. DCC-FCS was prepared by treatment of FCS with dextran-coated charcoal (DCC) to remove retinoids and steroids, as described previously (26van der Burg B. Rutteman G.R. Blankenstein M.A. de Laat S.W. van Zoelen J.J. J. Cell. Physiol. 1988; 134: 101-108Crossref PubMed Scopus (224) Google Scholar). Liarozole fumarate (27Wouters W. van Dun J. Dillen A. Coene M.-C. Cools W. De Coster R. Cancer Res. 1992; 52: 2841-2846PubMed Google Scholar) was a kind gift from Dr. W. Wouters (Janssen Research Foundation, Beerse, Belgium), dissolved in ethanol at a concentration of 10 mm and stored at −80 °C. [11,12-3H]RA (50 Ci/mmol) was obtained from NEN Life Science Products. RA was purchased from Sigma, and 4-hydroxy-RA and 4-oxo-RA were kindly provided by Drs. M. C. Hsu and L. H. Foley (Hoffman-LaRoche Laboratories, Nutley, NJ). Retinoids were dissolved in dimethyl sulfoxide at a concentration of 10 mmand stored in the dark at −80 °C. MCF-7 cells were supplied by Dr. C. Quirin-Stricker (Strasbourg, France) and T-47D cells by Dr. R. L. Sutherland (Sydney, Australia). MDA-MB-231 and MDA-MB-468 cells were purchased from the American Type Culture Collection (Rockville, MD). The RARα-transfected MDA-MB-231 cells have been described previously (28van der Leede B.M. Folkers G.E. van den Brink C.E. van der Saag P.T. van der Burg B. Mol. Cell. Endocrinol. 1995; 109: 77-86Crossref PubMed Scopus (59) Google Scholar). The F9–1.8 cell line is a derivative of F9 embryonal carcinoma cells stably transfected with a 1.8-kilobase mouse RARβ2 promoter-lacZ reporter construct as described (29Nikawa T. Schulz W.A. van den Brink C.E. Hanusch M. van der Saag P. Stahl W. Sies H. Arch. Biochem. Biophys. 1995; 316: 1-8Crossref PubMed Scopus (54) Google Scholar). All cell lines were maintained in DF medium supplemented with 7.5% FCS. In case of F9–1.8 cells, geneticin (200 μg/ml) was added to the medium. Cells were seeded into a 24-well plate in DF medium supplemented with 7.5% FCS. After 24 h of attachment, cells were treated with test compounds for the indicated times. Control cells were treated with solvent alone. The total DNA content/well was determined by fluorescent staining with Hoechst as described previously (26van der Burg B. Rutteman G.R. Blankenstein M.A. de Laat S.W. van Zoelen J.J. J. Cell. Physiol. 1988; 134: 101-108Crossref PubMed Scopus (224) Google Scholar). Experiments were performed in triplicate. Cell lines were cultured in 100-mm dishes in DF medium supplemented with 7.5% FCS at different densities (ranging from 4 to 7 × 106 cells) to obtain equal amounts of protein/dish at the start of RA treatment. Cells were then treated with 10 nm [3H]RA or 1 μm RA for the indicated times. After incubation, medium was removed, and plates were rinsed with ice-cold phosphate-buffered saline. Cells were scraped in 1 ml of ice-cold phosphate-buffered saline and collected by centrifugation. Cell pellets were stored at −80 °C until extraction. Retinoids were extracted essentially as described previously (30Pijnappel W.W.M. Hendriks H.F.J. Folkers G.E. van den Brink C.E. Dekker E.J. Edelenbosch C. van der Saag P.T. Durston A.J. Nature. 1993; 366: 340-344Crossref PubMed Scopus (246) Google Scholar). In short, cells were lysed in 0.8 ml of distilled water, and the lysate was added to 3 ml of methanol:dichloromethane (2:1). After addition of the internal standard Ro10–1670 (31Pilkington T. Brogden R.N. Drugs. 1992; 43: 597-627Crossref PubMed Scopus (95) Google Scholar) (Hoffmann-LaRoche Laboratories), the mixture was vortexed for 1 min and filtered through a glass sinter. The residue was washed once with 3 ml of methanol:dichloromethane (2:1) and once with 5 ml of dichloromethane. The filtrate and washes were combined, and 1.75 ml of 0.9% NaCl was added. The mixture was vortexed for 1 min, and the aqueous phase was removed. The organic phase was evaporated using a stream of nitrogen, and the lipids were dissolved in 100 μl of methanol, 60 mm ammonium acetate, pH 5.75 (9:1). In some experiments retinoids were extracted from the medium by using the same method. To examine the biological activity of RA metabolites present in T-47D extracts, near confluent cultures of 10 150-mm dishes were treated with 1 μm RA for 4 h. One dish was incubated also with 10 nm [3H]RA for 4 h to check whether peaks detected by UV measurement corresponded to derivatives of [3H]RA. Cell pellets of the dishes were pooled and stored at −80 °C. Retinoid extracts were prepared and analyzed by HPLC. One-min fractions were collected and extracted by partition between distilled water (0.9 volume), methanol (1 volume), and dichloromethane (1 volume) to remove mobile phase salts. The organic phase was evaporated, and the residue was dissolved in dimethyl sulfoxide. Aliquots of the fractions were tested in the reporter cell system described below. Retinoid extracts were injected into a reverse phase HPLC system containing a Spherisorb S50DS2 column (25 × 0.46 cm; Phase Separations Inc., Franklin, MA) and separated by gradient elution with solvent A (60 mmammonium acetate, pH 5.75) and solvent B (methanol). The gradient program with a flow rate of 1 ml/min was 5 min isocratic at 65% B followed by a convex gradient (no. 4; Waters Associates, Brussel, Belgium) to 85% B in 15 min, a linear gradient to 99% B in 10 min, and 10 min isocratic at 99% B. Retinoids and the internal standard were detected by measuring the absorbance at 350 nm in a model 481 UV flow spectrometer (Waters Associates). After UV measurement radiolabeled retinoids were detected on-line with a LB506 radiochromatography monitor (Berthold, Bad Wildbad, Germany) equipped with a Z-1000 flow cell and a scintillant flow rate of 2 ml/min. Radioinert retinoids were run as standards. F9–1.8 cells were seeded into a 96-wells plate in DF medium supplemented with 10% DCC-FCS. The following day, retinoid fractions were added to the wells. After 24 h of incubation, cells were rinsed twice with phosphate-buffered saline and lysed by overnight shaking in 50 μl of buffer containing 100 mm potassium phosphate, pH 7.8, 0.2% Triton X-100, and 1 mm dithiothreitol. The β-galactosidase activity in the cell lysates was determined by using the Galacto-Light chemiluminescent reporter assay system (Tropic Inc., Bedford, MA). Chemiluminescence was measured in a Topcount scintillation counter (Packard Instrument Company, Meriden, CT). Whole cell extracts were prepared as described previously (28van der Leede B.M. Folkers G.E. van den Brink C.E. van der Saag P.T. van der Burg B. Mol. Cell. Endocrinol. 1995; 109: 77-86Crossref PubMed Scopus (59) Google Scholar). Fifteen μg of protein was run on a 10% sodium dodecyl sulfate-polyacrylamide gel and transferred electrophoretically to a nitrocellulose sheet. Membranes were treated with blocking buffer containing 4% non-fat powdered milk, 10 mm Tris-HCl, pH 8.0, and 150 mm NaCl and then incubated for 2 h with anti-RARα rabbit polyclonal antiserum RPα(F) (32Gaub M.P. Lutz Y. Ruberte E. Petkovich M. Brand N. Chambon P. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 3089-3093Crossref PubMed Scopus (62) Google Scholar) diluted at 1/500 in blocking buffer. The RPα(F) antiserum was kindly provided by Drs. M. P. Gaub and C. Rochette-Egly (Institut de Génétique et de Biologie Moleculaire et Cellulaire, Illkirch, France). After washing in 10 mmTris-HCl, pH 8.0, 150 mm NaCl, and 0.05% Tween 20, the membranes were immunostained using the ECL Western blotting system (Amersham International plc, Little Chalfont, Bucks, U. K.). Our previousin vitro experiments have demonstrated that T-47D and MCF-7 human breast cancer cells are sensitive and that MDA-MB-468 and MDA-MB-231 cells are resistant to growth inhibition by 1 μm RA (25van der Burg B. van der Leede B.M. Kwakkenbos-Isbrücker L. Salverda S. de Laat S.W. van der Saag P.T. Mol. Cell. Endocrinol. 1993; 91: 149-157Crossref PubMed Scopus (136) Google Scholar). To investigate the role of RA metabolism in RA sensitivity of these cell lines, cells were incubated with 10 nm [3H]RA for 1–24 h, and cell extracts were analyzed by HPLC. Fig. 1 shows a representative example of the HPLC profiles of radioactive retinoids in the cell lines after an incubation period of 4 h. The RA-sensitive T-47D and MCF-7 cells demonstrated extensive conversion to polar metabolites (retention times 1–20), including 4-oxo-RA (peak 2) and 4-hydroxy-RA (peak 3). MCF-7 cells furthermore showed conversion to apolar RA derivatives (retention times 30–40). By contrast, the HPLC profiles of RA-resistant MDA-MB-468 and MDA-MB-231 cells showed no significant conversion of RA to polar or apolar derivatives. Fig. 2 shows that the RA-sensitive cell lines metabolized RA at a high rate. Most of the cell-associated RA was converted within 4 h to polar metabolites detected in the organic phase and very polar derivatives present in the aqueous phase of cell extracts. The majority of the very polar metabolites were discharged by the cells into the medium. After 24 h of treatment more than 80% of the initial amount of RA was recovered in the medium as derivatives with very high polarity. In Fig. 3 the rate of metabolism in RA-sensitive (T-47D and MCF-7) and RA-resistant (MDA-MB-468 and MDA-MB-231) cells is compared. In contrast to the RA-sensitive cell lines, the RA-resistant cell lines metabolized trace amounts of RA (open circles) at a much slower rate. The amount of cell-associated RA in MDA-MB-231 cells remained almost at a constant level during 24 h of treatment, and MDA-MB-468 cells exhibited a low rate metabolism. Although most of the intracellular RA in MDA-MB-468 cells was metabolized after 16 h, the amount of polar metabolites was very low because of conversion to very polar derivatives that were discharged into the medium (data not shown). To investigate RA metabolism at the same conditions under which RA-mediated growth inhibition has been studied, the human breast cancer cell lines were also incubated with 1 μm RA for 1 to 24 h (Fig. 3, closed circles). Both RA-sensitive cell lines show a strong reduction of cell-associated RA with kinetics similar to that observed during incubation with 10 nm[3H]RA. After 24 h, the amounts of RA in T-47D and MCF-7 cells were decreased to undetectable levels. The RA-resistant cell lines exhibited no rapid depletion of intracellular RA levels. MDA-MB-231 cells retained more than 50% of the initial amount of RA even after 24 h of treatment as has been found during incubation with 10 nm [3H]RA. MDA-MB-468 cells showed a decrease of intracellular RA to about 45% of the initial amount after 24 h of incubation. This is in contrast with the observations in experiments with 10 nm treatment, where most of the intracellular RA was cleared from the MDA-MB-468 cells after 24 h. In summary, RA-sensitive breast cancer cells show a high rate of retinoid metabolism, whereas RA-resistant cells metabolize RA to a much lesser extent. Formation of the metabolites 4-oxo-and 4-hydroxy-RA have long been considered as an inactivating pathway of RA (33Frolik C.A. Roberts A.B. Tavela T.E. Roller P.P. Newton D.L. Sporn M.B. Biochemistry. 1979; 18: 2092-2097Crossref PubMed Scopus (98) Google Scholar). However, recent studies have demonstrated that these metabolites exhibit strong biological activity (29Nikawa T. Schulz W.A. van den Brink C.E. Hanusch M. van der Saag P. Stahl W. Sies H. Arch. Biochem. Biophys. 1995; 316: 1-8Crossref PubMed Scopus (54) Google Scholar, 30Pijnappel W.W.M. Hendriks H.F.J. Folkers G.E. van den Brink C.E. Dekker E.J. Edelenbosch C. van der Saag P.T. Durston A.J. Nature. 1993; 366: 340-344Crossref PubMed Scopus (246) Google Scholar, 34Reynolds N.J. Fisher G.J. Griffiths C.E.M. Tavakkol A. Talwar H.S. Rowse P.E. Hamilton T.A. Voorhees J.J. J. Pharmacol. Exp. Ther. 1993; 266: 1636-1642PubMed Google Scholar, 35Gaemers I.C. van Pelt A.M.M. van der Saag P.T. de Rooij D.G. Endocrinology. 1996; 137: 479-485Crossref PubMed Scopus (69) Google Scholar), suggesting that their formation could also be an activation step. Since we have found that RA-sensitive and not RA-resistant cells convert RA to 4-oxo-and 4-hydroxy-RA (Fig. 1), it can be argued that conversion of RA to these metabolites may be required to confer growth inhibition. To investigate the effect of 4-oxo and 4-hydroxy-RA on breast cancer cell proliferation, we treated the cell lines with various concentrations of retinoid for 3 days. Fig. 4 shows that both retinoids could inhibit the proliferation of RA-sensitive T-47D and MCF-7 cells in a concentration-dependent fashion and were equipotent in growth inhibition compared with RA. By contrast, the RA-resistant MDA-MB-231 and MDA-MB-468 cells were not inhibited in their growth by 4-oxo or 4-hydroxy-RA, demonstrating that the resistant phenotype is not due to the inability of the cells to convert RA to these polar metabolites. Enzymes of the cytochrome P450 system play an active role in the oxidative metabolism of RA (33Frolik C.A. Roberts A.B. Tavela T.E. Roller P.P. Newton D.L. Sporn M.B. Biochemistry. 1979; 18: 2092-2097Crossref PubMed Scopus (98) Google Scholar, 36Leo M.A. Iida S. Lieber C.S. Arch. Biochem. Biophys. 1984; 234: 305-312Crossref PubMed Scopus (143) Google Scholar). It has been demonstrated that P450 inhibitors, such as ketoconazole and liarozole, inhibit RA metabolism in various in vivo andin vitro systems, including human breast cancer cells (27Wouters W. van Dun J. Dillen A. Coene M.-C. Cools W. De Coster R. Cancer Res. 1992; 52: 2841-2846PubMed Google Scholar,37van Wauwe J.P. Coene M.C. Goossens J. Van Nijen G. Cools W. Lauwers W. J. Pharmacol. Exp. Ther. 1988; 245: 718-722PubMed Google Scholar, 38van Wauwe J.P. Coene M.C. Goossens J. Cools W. Monbaliu J. J. Pharmacol. Exp. Ther. 1990; 252: 365-369PubMed Google Scholar). To investigate whether inhibition of RA metabolism by liarozole alters the sensitivity of breast cancer cells to growth inhibition by RA, MCF-7 cells were treated with various concentrations RA for 7 days, in the presence or absence of 10 μmliarozole. Fig. 5 shows that the combination of liarozole and RA significantly enhanced the growth-inhibitory effect of RA, particularly at lower concentrations. When 10 nm RA was used in combination with liarozole, an effect was found similar to that when cells were treated with 100 nm RA alone. Treatment with 10 μm liarozole alone had no effect on the proliferation of MCF-7 cells (data not shown). Since 10 μm liarozole strongly inhibited the proliferation of T-47D cells, we could not examine the effect of liarozole on retinoid-mediated growth inhibition in these breast cancer cells. We also examined the effect of 4-oxo-RA on the proliferation of MCF-7 cells in the presence or absence of 10 μm liarozole. Fig.5 shows that liarozole significantly increased the growth-inhibitory effect of 4-oxo-RA. This increase, however, was less pronounced than the enhancement of growth inhibition by RA. We furthermore observed that 4-oxo-RA was somewhat less potent than RA to inhibit the growth of MCF-7 cells in this 7-day proliferation assay. This is in contrast with the results in the 3-day proliferation assay, where 4-oxo-RA was almost equipotent to RA (Fig. 4). To check whether RA metabolism indeed was inhibited by liarozole in the proliferation experiments, MCF-7 cells were pretreated with 10 μm liarozole for 24 h and subsequently incubated with 10 nm [3H]RA and 10 μmliarozole for 4 h. HPLC analysis of the MCF-7 extracts revealed that conversion of RA to polar metabolites was reduced by more than 75% (data not shown). Together the results indicate that enhancement by liarozole of the antiproliferative effects of RA and 4-oxo-RA is due to inhibition of their metabolism. The above results suggest that RA does not require prior metabolism for its antiproliferative effects on breast cancer cells, even though some of the metabolites formed are active as well and can contribute to growth inhibition (e.g. 4-oxo-RA, Fig. 4). To investigate further to what extent RA metabolites formed in breast cancer cells are bioactive retinoids, we incubated T-47D cells with 1 μm RA for 4 h and tested the extracted retinoids for their ability to transactivate the retinoid-inducible 1.8-kilobase RARβ2 promoter stably transfected into F9–1.8 reporter cells. This reporter system has been demonstrated to be very useful in determining biological activity of retinoids, and the response of the RARβ2 promoter correlated well with other effects of retinoids in the F9–1.8 cells (29Nikawa T. Schulz W.A. van den Brink C.E. Hanusch M. van der Saag P. Stahl W. Sies H. Arch. Biochem. Biophys. 1995; 316: 1-8Crossref PubMed Scopus (54) Google Scholar). Fig. 6 A shows the HPLC profile of retinoids extracted from T-47D cells after 4 h of incubation. At this time point, T-47D cells contain high levels of polar metabolites, as has been found previously (Fig. 2). Fig. 6 B shows that most of the fractions containing polar metabolites were unable to activate the RARβ2 promoter. Only fractions 13, 14, 15, and 17 showed activation of the RARβ2 promoter. Activity in fraction 13 and fraction 14 is very probably due to the presence of 4-oxo- and 4-hydroxy-RA, respectively, as these are the positions where the respective retinoids coelute under these conditions. The identities of the active retinoids present in fraction 15 and 17, however, are presently unknown. The RA-containing fractions (fractions 26 and 27) exhibited most of the activity in the T-47D cell extract. The formation of a limited amount of biological active metabolites, as measured in this assay system, again suggests that in T-47D cells metabolism of RA to polar derivatives is not required for antiproliferative activity. The strong increase of RA metabolism in T-47D and MCF-7 cells observed after 4 h of incubation with either 10 nm or 1 μm RA (Fig. 3) suggests that RA metabolism in these cell lines is induced by RA. To investigate this possibility, cells were pretreated with either 10 nm or 1 μm RA for various times and subsequently incubated with 10 nm [3H]RA for 2 h. Fig.7 shows that RA metabolism in T-47D cells was induced already after 2 h of pretreatment, with no further increase by prolonged pretreatment. A stronger induction of RA metabolism was observed by pretreatment with 1 μm than with 10 nm RA. The difference in induction of RA metabolism is most pronounced after 24 h of pretreatment, where strongly increased metabolism was seen in the presence 1 μm RA but not in the presence of 10 nm RA. Similar effects of pretreatment with RA on metabolism of RA were obtained in MCF-7 cells (data not shown). By contrast, RA could not induce RA metabolism in RA-resistant MDA-MB-231 (Fig. 7) and MDA-MB-468 cells (data not shown). These results suggest that the low rate of RA metabolism in RA-resistant cells is due to their inability to induce metabolism in the presence of RA. Retinoid action is mediated by the nuclear retinoid receptors (RARs and RXRs), which are known to activate transcription of target genes in response to nanomolar concentrations of retinoids (4Chambon P. FASEB J. 1996; 10: 940-954Crossref PubMed Scopus (2604) Google Scholar). The RA-mediated induction of RA
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