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*

(Received for publication, January 2, 1997, and in revised form, April 21, 1997)

Bas-jan M. van der Leede Dagger , Christina E. van den Brink , Wilhelmus W. M. Pijnappel , Edwin Sonneveld , Paul T. van der Saag and Bart van der Burg §

From the Hubrecht Laboratory, Netherlands Institute for Developmental Biology, 3584 CT Utrecht, The Netherlands

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES


ABSTRACT

Previous studies have shown that all-trans-retinoic acid (RA) inhibits in vitro proliferation 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-alpha 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.


INTRODUCTION

Retinoids are a group of naturally occurring (e.g. all-trans-retinoic acid; RA1) and synthetic analogs of vitamin A which play an important role in cellular growth and differentiation (1, 2). The actions of retinoids are mediated by two types of receptors, the retinoic acid receptors (RARs) and retinoid X receptors (RXRs) (3, 4). 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 (5, 6).

Retinoids are highly effective in preventing chemically induced carcinogenesis in experimental animals (7) and can inhibit proliferation of a large variety of normal and neoplastic cell types in vitro (8). More recently the effectiveness of retinoids in the treatment and prevention of a number of human cancers has been established (9-15).

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 (16-18). 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 (19, 20).

Retinoid resistance has also been documented in vitro in cell lines derived from various tumor types, including breast cancer (21, 22). 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 (23-25). 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 RARalpha 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.


EXPERIMENTAL PROCEDURES

Materials

DF medium (a 1:1 mixture of Dulbecco's modified Eagle's medium and Ham's F-12, buffered with 44 mM NaHCO3) 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 (26). Liarozole fumarate (27) 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 mM and stored in the dark at -80 °C.

Cell Culture

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 RARalpha -transfected MDA-MB-231 cells have been described previously (28). The F9-1.8 cell line is a derivative of F9 embryonal carcinoma cells stably transfected with a 1.8-kilobase mouse RARbeta 2 promoter-lacZ reporter construct as described (29). 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.

Cell Proliferation Experiments

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 (26). Experiments were performed in triplicate.

Retinoid Extraction and Fractionation

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 (30). 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 (31) (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.

HPLC Analysis

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 mM ammonium 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.

Reporter Cell System

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 beta -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).

Western Blotting

Whole cell extracts were prepared as described previously (28). 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-RARalpha rabbit polyclonal antiserum RPalpha (F) (32) diluted at 1/500 in blocking buffer. The RPalpha (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 mM Tris-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.).


RESULTS

Metabolism of RA in Human Breast Cancer Cells

Our previous in 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 (25). 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. 1. Reverse phase HPLC analysis of [3H]RA metabolites extracted from cultures of human breast cancer cell lines. Cells were incubated with 10 nM [3H]RA for 4 h. Retinoids were extracted and analyzed by HPLC as described under "Experimental Procedures." Indicated elution positions: 1, polar metabolites; 2, 4-oxo-RA; 3, 4-hydroxy-RA; 4, 13-cis-RA; 5, RA.
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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.


Fig. 2. Conversion of [3H]RA to radioactive products with different polarity by RA-sensitive breast cancer cells. Cells were incubated with 10 nM [3H]RA for the indicated times. Cells (panel A) and medium (panel B) were harvested, and extracts were prepared as described under "Experimental Procedures." Aliquots of the aqueous and organic phase of the extracts were examined for radioactivity. The organic phase was subsequently analyzed by HPLC. Very polar RA derivatives were recovered in the aqueous phase (black-down-triangle ), whereas polar metabolites (black-triangle) and RA and its isomers (bullet ) were recovered in the organic phase of extracts. Results are expressed as percentages of the sum of the radioactivity in cells and medium at the same time point. The figure shows the data of T-47D cells, but similar results were obtained with MCF-7 cells.
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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).


Fig. 3. Kinetics of RA metabolism in RA-sensitive and -resistant human breast cancer cell lines. Cells were incubated with 10 nM [3H]RA or 1 µM RA for the indicated times. Cells were harvested, and retinoid extracts were analyzed by HPLC. The kinetics of RA metabolism of 10 nM [3H]RA is shown; the levels of [3H]RA (open circle ) are expressed as counts/min (CPM). The kinetics of metabolism of 1 µM RA is shown; the levels of RA (bullet ) are expressed as absorbance units at 350 nm (OD350 nm units). The data represent one of two independent experiments with similar results.
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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.

Effect of 4-Oxo- and 4-Hydroxy-RA on the Proliferation of Breast Cancer Cells

Formation of the metabolites 4-oxo-and 4-hydroxy-RA have long been considered as an inactivating pathway of RA (33). However, recent studies have demonstrated that these metabolites exhibit strong biological activity (29, 30, 34, 35), 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.


Fig. 4. Effect of different retinoids on the proliferation of human breast cancer cell lines. Cells were treated with various concentrations of RA (bullet ), 4-oxo- (black-triangle), or 4-hydroxy-RA (black-down-triangle ) for 3 days. Cell proliferation was estimated as described under "Experimental Procedures." Results are expressed as percentages of the control incubation with solvent and represent the means (± S.E.) of three independent experiments.
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Liarozole Increases the Sensitivity of Breast Cancer Cells to Growth Inhibition by Retinoids

Enzymes of the cytochrome P450 system play an active role in the oxidative metabolism of RA (33, 36). It has been demonstrated that P450 inhibitors, such as ketoconazole and liarozole, inhibit RA metabolism in various in vivo and in vitro systems, including human breast cancer cells (27, 37, 38). 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 µM liarozole. 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.


Fig. 5. Effect of RA (bullet , open circle ), and 4-oxo-RA (black-triangle, triangle ) on the proliferation of MCF-7 cells, in the absence (bullet , black-triangle) or presence (open circle , triangle ) of 10 µM liarozole. Cells were treated with the compounds for 7 days. Medium and compounds were refreshed on days 2 and 5. Cell proliferation was measured as described under "Experimental Procedures." Results are expressed as percentages of the control incubation with solvent and represent the means (±S.E.) of three independent experiments.
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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 µM liarozole 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.

Biological Activity of RA Metabolites Extracted from Breast Cancer Cells

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 RARbeta 2 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 RARbeta 2 promoter correlated well with other effects of retinoids in the F9-1.8 cells (29). Fig. 6A 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. 6B shows that most of the fractions containing polar metabolites were unable to activate the RARbeta 2 promoter. Only fractions 13, 14, 15, and 17 showed activation of the RARbeta 2 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.


Fig. 6. Biological activity of RA metabolites extracted from T-47D incubated with 1 µM RA for 4 h. Cultures of T-47D cells were incubated with 1 µM RA for 4 h. Cells were harvested and pooled. Retinoids were extracted and analyzed by HPLC. One-min fractions were collected and extracted. Fractions were tested for biological activity in F9-1.8 reporter cells as described under "Experimental Procedures." Panel A, reverse phase HPLC analysis of the T-47D cell extract. Indicated elution positions: 1, polar metabolites; 2, 4-oxo-RA; 3, 4-hydroxy-RA; 4, 13-cis-RA; 5, RA. Panel B, transcriptional activation of the 1.8-kilobase RARbeta 2 promoter-lacZ construct in F9-1.8 reporter cells by fractions. The beta -galactosidase activity is expressed as fold induction over solvent-treated cells. Results represent the means (± S.D.) of two independent experiments.
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RA Treatment Induces RA Metabolism in RA-sensitive Breast Cancer Cells

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.


Fig. 7. RA induction of RA metabolism in human breast cancer cell lines. Cells were pretreated with 10 nM (solid bars) or 1 µM RA (hatched bars) for the indicated times. Subsequently cells were rinsed twice with medium and incubated with 10 nM [3H]RA for 2 h. Control cells were incubated with 10 nM [3H]RA for 2 h without RA pretreatment (open bars). Cells were harvested, and retinoid extracts were analyzed by HPLC. The levels of [3H]RA are expressed as counts/min (CPM).
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Increased RARalpha Expression Enhances the Rate of RA Metabolism in MDA-MB-231 cells

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 (4). The RA-mediated induction of RA metabolism by low doses of RA in breast cancer cells suggests a receptor-mediated process. Since T-47D and MCF-7 cells express higher levels of RARalpha than MBA-MD-468 and MDA-MB-231 cells (25), we investigated whether increased expression of RARalpha in MDA-MB-231 cells could influence the rate of RA metabolism. RARalpha -transfected derivatives of MDA-MB-231 cells, clones alpha 8, alpha 18, and alpha 26, expressing different levels of RARalpha , were incubated with 1 µM RA for 2-24 h, and retinoid extracts were analyzed by HPLC. Fig. 8 shows that clones alpha 8 and alpha 18 containing high levels of RARalpha protein demonstrated an increased rate of RA metabolism, whereas clone alpha 26 with low level expression of RARalpha showed a metabolic rate similar to that of parental MDA-MB-231 cells.


Fig. 8. RA metabolism in MDA-MB-231 and RARalpha -transfected derivatives (alpha 8, alpha 18, and alpha 26). Cells were incubated with 1 µM RA for the indicated times. Cells and media were harvested, and retinoids extracts were analyzed by HPLC. Panel A, kinetics of RA metabolism in the cells. The levels of RA are shown as OD350 nm units. Panel B, levels of RA in the media after 24-h incubation with 1 µM RA. Results are expressed as percentage (±S.D.) of initial concentration of RA present in the media of cultures. Panel C, RARalpha protein expression, determined by Western blot analysis on whole cell extracts as described under "Experimental Procedures." MCF-7 extract was used as positive control (C).
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DISCUSSION

We investigated RA metabolism in human breast cancer cells and found a correlation with sensitivity to growth inhibition by RA. The time course experiments showed that most of the RA added to the cultures of RA-sensitive T-47D and MCF-7 cells was converted to more polar derivatives within 4 h, whereas RA-resistant MDA-MB-231 and MDA-MB-468 cells retained more than 45% of the initial amount of RA after 24 h of incubation. We could demonstrate that RA induces its own metabolism in RA-sensitive cells. Two hours of pretreatment with RA dramatically increased the rate of metabolism, whereas no further enhancement was observed after prolonged pretreatment. These observations were made during incubation with both 10 nM and 1 µM RA. RA-induced metabolism of RA was not observed in the RA-resistant cells, suggesting that the low rate of RA metabolism in these cells may at least in part be explained by the lack of this autoinduction mechanism.

RA-induced metabolism of RA appears to be regulated at the transcriptional level. It was shown recently that actinomycin D prevented the induction of RA metabolism in T-47D cells (39). We have repeated these experiments with similar results (data not shown). Since RA induced metabolism in RA-sensitive cells within 2 h, transcription and translation of the appropriate target genes must occur rapidly. In an efficient cloning strategy used to identify RA-responsive genes in embryonal carcinoma cells, it was found that some of the cloned genes were rapidly induced by RA (40). An increase in RNA levels of these genes was detectable 30 min after the RA addition, and the RNA levels further increased up to 6 h and subsequently decreased between 12 and 24 h. Genes involved in the induction of RA metabolism in breast cancer cells presumably belong to this class of early RA-responsive genes. So far individual candidate genes have not been identified.

A category of proteins relevant to RA metabolism is the cytochrome P450 enzyme system. The P450 enzymes play an active role in RA metabolism, and ketoconazole and liarozole were shown to inhibit the P450-dependent oxidation of RA (27, 37, 38). Various purified isozymes of microsomal cytochrome P450, such as P450 1A2, 2C7, and 2C8, have been shown to be capable of metabolizing RA (36, 41, 42) and other retinoids (43) in a reconstituted system. More recently, it has been reported that expression of P450 enzymes can be regulated by retinoids (44-46). Furthermore P450 enzymes have been shown to be expressed in normal and tumor breast tissue (47). Although the identities of the P450s involved in metabolism of RA in the breast are unknown, it is clear from our experiments with liarozole that RA-metabolizing P450 enzymes are present in the RA-sensitive cells.

Takatsuka et al. (48) recently reported a similar relationship between RA metabolism and inhibition cell proliferation in various cell lines, including breast cancer cells. However, the investigators obtained these results using serum-free assay conditions and could not obtain similar results in 10% serum-containing medium. Under the latter conditions, RA was not rapidly taken up by the cells, and no difference in metabolism between RA-sensitive and -resistant cells nor an effect on cell growth was detected (48). We performed our experiments in 7.5% serum-containing medium but did not encounter these problems. The authors furthermore suggested that a metabolite of RA, rather than RA itself, may be responsible for the observed growth inhibition. However, our data do not support this idea as will be discussed below.

We demonstrated that in contrast to RA-sensitive breast cancer cells, the RA-resistant cell lines exhibit no significant metabolism of RA, and polar metabolites were present in very low amounts. The most simple explanation for the RA resistance of MDA-MB-231 and MDA-MB-468 cells would be that metabolism of RA to polar metabolites is required for the antiproliferative effect of RA on breast cancer cells. Although it has been found that metabolites of RA exhibit strong biological activity in vivo and in vitro (29, 30, 34, 35, this study), our data do not support this hypothesis. In the first place, examination of the biological activity of retinoid fractions from metabolizing T-47D cells revealed that RA-containing fractions exhibited more activity than fractions containing polar metabolites. Some of the polar metabolites may have a higher transactivation capacity than RA, but their concentration in the fractions is too low to give strong activation of the RARbeta 2 promoter. Second, we demonstrated that the RA-resistant breast cancer cells were not growth-inhibited by the metabolites 4-oxo- and 4-hydroxy-RA, whereas these retinoids were potent growth inhibitors of RA-sensitive cells. These results show that the resistant phenotype of breast cancer cells is not due to their inability to convert RA to the 4-oxygenated metabolites. Third, we demonstrated that the enhancement by liarozole of the antiproliferative effects of RA on MCF-7 cells appears to be due to decreased metabolism. We also found that growth inhibition by 4-oxo-RA was enhanced by liarozole, suggesting that the breakdown of 4-oxo-RA is also inhibited. It has been reported recently that liarozole indeed is capable of inhibiting both the metabolism of RA and 4-oxo-RA (49). Finally, we demonstrated that 4-oxo-RA is somewhat less potent than RA in growth inhibition of MCF-7 cells during a 7-day proliferation experiment, whereas treatment for 3 days appeared to be too short to observe the difference between the growth-inhibitory activity of these retinoids. This difference may be explained by the fact that RA is converted first to metabolites, such as 4-oxo-RA, prior to its catabolism.

A second possible explanation for RA resistance of breast cancer cells is that impairment in the retinoid signaling has occurred at the level of the cellular RA-binding proteins (CRABPs). CRABPs are thought to play a negative role in retinoid signaling by sequestering RA in the cytoplasm and facilitating its catabolism such that lower RA concentrations reach the nucleus (50-52). It has been demonstrated that cells from patients with acute promyelocytic leukemia at relapse contained high levels of CRABP-I, whereas this protein was not detected prior to RA administration (53). Furthermore, overexpression of CRABP-I in F9 embryonal carcinoma cells resulted in decreased sensitivity to RA (51). Our preliminary data show that both RA-sensitive and -resistant breast cancer cells display low or no expression of CRABP-I mRNA (data not shown). Similar observations have recently been reported for other breast cancer cell lines (54). Together these results indicate that the RA-resistant phenotype of human breast cancer cells seems not to be due to high levels of CRABP-I.

The third possibility to explain RA resistance of breast cancer cells is that impairment in the retinoid signaling occurs at the level of nuclear receptors. We and others have demonstrated that RAR expression is of crucial importance for the cellular response of breast cancer cells and that RA-resistant cells expressed low levels of RARalpha compared with RA-sensitive cells (25, 55). It was furthermore shown that retinoic acid-responsive element-dependent transcription induced by RA was higher in RA-sensitive than in -resistant cells, indicating that resistance could be due to underexpression of functional RARs (25). More recently, our laboratory and others demonstrated that elevation of RAR expression levels in RA-resistant MDA-MB-231 and MDA-MB-468 cells by stable transfection with RARalpha or RARbeta expression vectors restored growth inhibition by RA (27, 56-59). Here we demonstrated that RARalpha -transfected MDA-MB-231 cells showed increased RA metabolism, which indicates that at least RARalpha is involved in RA-mediated growth inhibition and in retinoid metabolism. Retinoid receptors are known to activate transcription in response to nanomolar concentrations of RA. Since RA metabolism can be induced by 10 nM RA and appears to be directly at the transcriptional level, it is tempting to speculate that RA may directly regulate transcription of the metabolizing enzyme(s) through RARalpha . The exquisite sensitivity of this (negative) regulatory loop probably emphasizes the necessity for the cell to control strictly the intracellular levels of RA as a potential growth and differentiation regulatory molecule. Another autoregulatory loop has been described recently in human keratinocytes, whereby RA regulates its own biosynthesis from retinol through regulation of retinol esterification, indicating that the availability of RA in these cells is controlled strictly (60).


FOOTNOTES

*   This work was supported by Grant HUBR 93-558 from the Dutch Cancer Society (Koningin Wilhelmina Fonds).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Dagger    Present address: Janssen Pharmaceutica N.V., Turnhoutseweg 30, B-2340 Beerse, Belgium.
§   To whom correspondence should be addressed: Hubrecht Laboratory, Netherlands Institute for Developmental Biology, Uppsalalaan 8, 3584 CT Utrecht, The Netherlands. Tel.: 31-30-251-0211; Fax: 31-30-251-6464; E-mail: bvdb{at}N10B.KNAW.NL.
1   The 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.

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