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Reversal of MRP-Mediated Multidrug Resistance in Human Lung Cancer Cells by the Antiprogestatin Drug RU486
Biochemical and Biophysical Research Communications 258, 513–518 (1999) Article ID bbrc.1999.0671, available online at http://www.idealibrary.com on
Reversal of MRP-Mediated Multidrug Resistance in Human Lung Cancer Cells by the Antiprogestatin Drug RU486 Le´a Payen, Laurence Delugin, Arnaud Courtois, Yolande Trinquart, Andre´ Guillouzo, and Olivier Fardel 1 Unite´ INSERM U456 De´toxication et Re´paration Tissulaire, Faculte´ des Sciences Pharmaceutiques et Biologiques, 2 Avenue du Pr L. Bernard, 35043 Rennes, France
Received April 8, 1999
Multidrug resistance-associated protein (MRP) and P-glycoprotein (P-gp) are drug efflux pumps conferring multidrug resistance to tumor cells. RU486, an antiprogestatin drug known to inhibit P-gp function, was examined for its effect on MRP activity in MRPoverexpressing lung tumor GLC4/Sb30 cells. In such cells, the antihormone compound was found to increase intracellular accumulation of calcein, a fluorescent compound transported by MRP, in a dosedependent manner, through inhibition of cellular export of the dye ; in contrast, it did not alter calcein levels in parental GLC4 cells. RU486, when used at 10 mM, a concentration close to plasma concentrations achievable in humans, strongly enhanced the sensitivity of GLC4/Sb30 cells towards two known cytotoxic substrates of MRP, the anticancer drug vincristine and the heavy metal salt potassium antimonyl tartrate. Vincristine accumulation levels were moreover up-regulated in RU486-treated GLC4/Sb30 cells. In addition, such cells were demonstrated to display reduced cellular levels of glutathione which is required for MRP-mediated transport of some anticancer drugs. These findings therefore demonstrate that RU486 can down-modulate MRP-mediated drug resistance, in addition to that linked to P-gp, through inhibition of MRP function. © 1999 Academic Press
Multidrug resistance is a major cause of failure in the treatment of human malignancies. Such a resistance, usually defined as cross-resistance to structurally and functionally unrelated anticancer drugs, is often linked to overexpression of membrane proteins effluxing antitumor compounds out of cells and therefore lowering their intracellular accumulation (1). The first described of such transporters was P-glycoprotein 1
Corresponding author. Fax: 33 2 99 33 62 42. E-mail: olivier. [email protected].
(P-gp), a 170 kDa protein encoded by the MDR1 gene (2); another transporter more recently characterized is the 190 kDa multidrug resistance-associated protein (MRP, also known as MRP1) (3). Both P-gp and MRP belong to the ATP-binding cassette protein superfamily (2, 4). They share numerous substrates including anthracyclins, vincristine and epipodophyllotoxins. However, the two efflux pumps also display some functional differences. Thus, paclitaxel is handled by P-gp, but not by MRP, whereas the latter pump, unlike the former, mediates the transport of anionic compounds such as glutathione S-conjugates and of some heavy metals such as antimony (4 – 6). In addition, MRP, unlike P-gp, has been demonstrated to cotransport some anticancer drugs such as vincristine in an unmodified form with reduced glutathione (GSH), thus outlining the role of GSH in some MRP-mediated transport processes (7). Increased levels of P-gp and/or MRP have been detected in tumor samples from patients suffering from different kinds of cancers and have been correlated, at least for P-gp, with lack of response to chemotherapy and poor survival in some malignancies (1, 5, 8, 9). P-gp activity can be blocked by a wide variety of drugs including calcium channel blockers such as verapamil and immunosuppressive agents such as cyclosporin A (10). These compounds, termed modulators or chemosensitizers, increase intracellular accumulation of anticancer drugs in P-gp-overexpressing cells and thus circumvent drug resistance (11). Some of them have now entered clinical trials (11, 12). In contrast to P-gp-mediated transport, that linked to MRP has been demonstrated to be inhibited only by few agents which, moreover, often act at elevated and toxic doses, therefore precluding their use in reversing experiments (4, 13, 14). There is therefore a need to identify new MRP modulators. To this aim, the analysis of P-gp inhibitors may be useful since some of these compounds have
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been demonstrated to also interact with MRP (15, 16). In the present study, RU486, an steroid compound with antiprogestatin properties already known to inhibit P-gp-related transport at concentrations thought to have no serious side effects (17, 18), was therefore tested for its potential inhibitory effects on MRP activity. Our results demonstrate that this anti-hormone drug markedly down-modulated drug resistance of MRP-overexpressing human lung GLC4/Sb30 cells through inhibition of MRP function. MATERIALS AND METHODS Chemicals. Calcein acetoxymethoxyl ester (calcein AM) and [ 3H] vincristine (specific activity 5.8 Ci/mmol) were purchased from Molecular Probes (Eugene, OR) and Amersham (Les Ulis, France), respectively. Progesterone, vincristine, dexamethasone, potassium antimonyl tartrate (PAT), b-estradiol, probenecid and buthionine sulfoximine (BSO) were obtained from Sigma (St Louis, MO). RU486 was kindly provided by Roussel-Uclaf (Romainville, France). Cell culture. The human lung cancer cell line GLC4 and its antimony-selected subline GLC4/Sb30 were cultured in RPMI medium supplemented with 10% fetal calf serum at 37°C in a 5% CO 2 atmosphere. GLC4/Sb30 cells overexpress MRP but not P-gp and are cross-resistant to anticancer drugs such as doxorubicin and vincristine when compared to their parental counterparts (19); they were routinely maintained in the presence of PAT (30 mg/ml) which was however withdrawn for at least 3 days before the use of the cells for experiments. Calcein accumulation and efflux assays. Calcein is an anionic fluorescent probe substrate for MRP and determination of its cellular accumulation and efflux allow to investigate MRP activity (20). For calcein accumulation experiments, cells were incubated with 0.5 mM calcein AM for 90 min in the absence or the presence of RU486 or one of the other potential MRP chemosensitizer tested. The non-fluorescent esterified form calcein AM freely diffuses into cells where it is cleaved by intracellular esterases to give the fluorescent anionic dye calcein (21) which can be exported out of cells through MRP1 activity (20). Cells were then washed three times with ice-cold phosphate-buffered saline (PBS) and lyzed in distilled water by ultrasonication. Amounts of intracellular calcein were next determined by fluorimetry using a Titertek Fluoroscan spectrofluorometer (Flow Laboratories, Puteaux, France); excitation and emission wavelenghts were 485 nm and 538 nm, respectively. An aliquot of cell lysate was used in parallel to determine cellular protein concentration by the Bio-Rad assay (22). Results were expressed as fluorescence arbitrary units after normalization to cellular protein content. For calcein efflux assays, calcein-preloaded cells were incubated in calcein AM-free medium for 90 min in the absence or presence of RU486. Cells were then washed three times with PBS and intracellular calcein retained was then determined by fluorimetry as described above. Results were expressed as percentages of initial calcein staining. Intracellular vincristine accumulation. Cells were incubated with 20 nM [ 3H] vincristine for 90 min in the presence or the absence of RU486. Cells were then washed three times with ice-cold PBS and lyzed in distilled water by ultrasonication. Radioactivity of [ 3H] vincristine in the cell extract was then determined by scintillation counting and normalized to cellular protein content. Drug-sensitivity assay. The effects of cytotoxic compounds on cell proliferation was evaluated using the 3-[4,5-dimethylthiazol-2-yl]2,5-diphenyl tetrazolium bromide (MTT) as previously described (23). Briefly, cells were cultured with various concentrations of vin-
FIG. 1. Effect of various concentrations of RU486 on calcein accumulation in drug-resistant GLC4/Sb30 and drug-sensitive GLC4 cells. Cells were incubated with 0.5 mM calcein AM in the presence of various doses of RU486 (from 0 to 100 mM) for 90 min. Intracellular calcein accumulation was then determined by fluorimetry. The values are expressed as fluorescence arbitrary units (FAU) normalized to protein content and are the mean 6 SD of at least three independent experiments in triplicate. *, P , 0.05 when compared to cells not treated by RU486.
cristine or PAT for 96 h in the presence or the absence of RU486 in 96-well microplates. 100 ml of a 1 mg/ml MTT solution was then added to each well for 120 min. The medium was next discarded and replaced by 100 ml of dimethyl sulfoxide. The blue formazan product formed was further quantified by its absorbance at 540 nm using a Titertek Multiskan MCC/340 (Flow Laboratories). Growth inhibition was evaluated as IC 50, i.e. the drug concentration providing a 50% reduction in cell number as compared to controls cultured in parallel without cytotoxic compound. Cellular GSH content. Total cellular GSH content was determined using a colorimetric assay (Bioxytech GSH-400) supplied by Oxis (Portland, OR); the values were then normalized to cellular protein content. Statistical analysis. Data were analyzed by the Student’s t-test. The criterion of significance of the differences between the means (6 standard deviation) was P , 0.05.
RESULTS The effects of several concentrations of RU486 on the accumulation of the MRP substrate calcein in MRPoverexpressing GLC4/Sb30 cells and in their parental GLC4 counterparts were firstly investigated. As indicated in Fig. 1, the antiprogestatin compound increased calcein levels in GLC4/Sb30 cells in a dosedependent manner. Indeed, RU486 used at 10 mM was found to enhance cellular dye-related fluorescence by approximately 2-fold whereas the use of higher concentrations (25-100 mM) allowed to increase calcein levels by approximately 3-fold and thus to reach the dye levels observed in parental GLC4 cells (Fig. 1). In contrast, 1 mM RU486 was found to have no effect whereas 5 mM only slightly increased intracellular dye accumulation in GLC4/Sb30 cells. None of the RU486 concentrations tested over the 90-min incubation period af-
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FIG. 2. Effect of dexamethasone, progesterone, b-estradiol, and probenecid on calcein accumulation in drug-resistant GLC4/Sb30 cells. GLC4/Sb30 cells were incubated with 0.5 mM calcein AM in the absence (UNT) or the presence of 100 mM dexamethasone (DEX), 50 mM progesterone (PRG), 20 mM b-estradiol (EST), or 2 mM probenecid (PRB) for 90 min. Intracellular calcein accumulation was then determined by fluorimetry. The values are expressed as fluorescence arbitrary units (FAU) normalized to protein content and are the mean 6 SD of at least three independent experiments in triplicate. *, P , 0.05 when compared to cells not treated by RU486.
fected cell viability as assessed by MTT assays and determination of cellular protein content (data not shown). In contrast to GLC4/Sb30 cells, GLC4 cells did not show any alteration in calcein-related fluorescence in response to RU486 used at 50 or 100 mM (Fig. 1). In order to determine whether other steroids distinct from RU486 might also up-regulate calcein levels in GLC4/Sb30 cells, cellular accumulation of the dye was investigated in the presence of dexamethasone, progesterone and b-estradiol. Results indicated that 50 mM progesterone increased calcein levels in GLC4/Sb30 cells (Fig. 2). In contrast, 100 mM dexamethasone and 20 mM b-estradiol had no effect whereas 2 mM probenecid, a well-known inhibitor of MRP activity (4), markedly enhanced calcein-related cellular fluorescence. Action of RU486 on calcein export out of GLC4/Sb30 and GLC4 cells was next determined by dye efflux experiments. As indicated in Fig. 3, GLC4/Sb30 cells poorly retained calcein when compared to their parental counterparts and such a loss of dye was found to be strongly inhibited by the antiprogestatin compound. In contrast, RU486 did not alter retention levels of calcein in GLC4 cells. To determine whether RU486 may also alter intracellular retention of MRP substrates distinct from calcein, intracellular accumulation of the anticancer drug vincristine was further determined in the presence or the absence of the antiprogestatin drug (Fig. 4). RU486 was found to markedly enhance vincristine accumula-
FIG. 3. Effect of RU486 on calcein efflux from GLC4/Sb30 and GLC4 cells. Calcein-loaded GLC4/Sb30 and GLC4 cells were incubated in calcein AM-free medium for 90 min in the absence or the presence of 50 mM RU486. Intracellular retained calcein was then determined by fluorimetry and expressed relative to initial dye staining. The values are the mean 6 SD of three independent experiments in triplicate. *, P , 0.05 when compared to cells not treated by RU486.
tion in GLC4/Sb30 cells whereas it had no major effect in GLC4 cells. Cellular levels of the anticancer agent were thus similar in RU486-treated GLC4/Sb30 cells
FIG. 4. Effect of RU486 on [ 3H] vincristine accumulation in GLC4/Sb30 and GLC4 cells. GLC4/Sb30 and GLC4 cells were incubated for 90 min with 20 nM [ 3H] vincristine in the absence or the presence of 50 mM RU486. Intracellular [ 3H] vincristine accumulation was then determined by scintillation counting. The values are the mean 6 SD of three independent experiments in triplicate. *, P , 0.05 when compared to cells not treated by RU486.
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Effects of RU486 on PAT and Vincristine Cytotoxicity PAT IC 50 (mg/ml)
Vincristine IC 50 (ng/ml)
Cells
Control
1 RU486
Control
1 RU486
GLC4/Sb30 GLC4
40.7 6 5.1 0.8 6 0.3
2.2 6 0.8 (18.5) 0.3 6 0.1 (2.7)
3.5 6 0.2 0.4 6 0.1
0.4 6 0.1 (8.7) 0.2 6 0.03 (2)
Note. The values of potassium antimonyl tartrate (PAT) and vincristine IC 50 were determined in the absence or in the presence of 10 mM RU486 using the MTT assay and are the means 6 SD of at least three independent experiments. Numbers in parentheses indicate the sensitization factor, i.e., the ratio of the IC 50 in the absence of RU486 versus the IC 50 in the presence of RU486.
and in GLC4 cells whereas the resistant cells poorly accumulated vincristine in the absence of the antiprogestatin compound when compared to their parental drug-sensitive cells (Fig. 4). The effects of prolonged RU486 exposure (96 h) on cell proliferation were then monitored using the MTT dye assay. RU486 IC 50 values were found to be 18 6 3 mM and 20 6 2 mM for GLC4/Sb30 cells and GLC4 cells, respectively. Interestingly, the dose of 10 mM was found to not obviously alter growth of both GLC4/Sb30 cells and GLC4 cells; this RU486 concentration active on MRP-mediated transport (Fig. 1) and in the range of the plasma concentrations observed in humans after a single oral dose (24) was further retained for reversing experiments. In such assays, the antiprogestatin drug was evidenced to strongly down-modulate resistance of GLC4/Sb30 cells towards vincristine and PAT. Indeed, PAT and vincristine IC 50 values for GLC4/Sb30 cells in the presence of RU486 were reduced by 18.5- and 8.7fold, respectively, and therefore became close to those observed for parental GLC4 cells (Table 1). The antiprogestatin was also found to enhance the cytotoxicity of PAT and vincristine towards GLC4 cells, but only slightly, thus likely reflecting the inhibition of the MRP constitutively present at low, but detectable levels, in these cells as already reported (19). Since reversion of MRP-mediated resistance has been linked to down-regulation of cellular GSH content by some chemosensitizers (25), we next determined cellular GSH levels in the presence or the absence of RU486. As indicated in Table 2, exposure to 10 mM RU486 for 96 h was found to result in a strong reduction of cellular GSH levels in GLC4 Sb30 cells and also, although to a less extent, in parental GLC4 cells; the GSH content in RU486-treated GLC4/Sb30 cells thus represented 11% of that found in their untreated counterparts. GSH depletion was also achieved by shorter exposure to the antiprogestatin compound; indeed, GLC4/Sb30 cells exposed to 10 mM RU486 for 90 min displayed a reduction in GSH content by approximately a 2.2-fold factor (data not shown). To further investigate the relationships between GSH levels and
membrane transport of MRP substrates in GLC4/Sb30 cells, intracellular accumulation of calcein and vincristine were determined in GLC4/Sb30 cells exposed to 25 mM BSO, a well-known inhibitor of GSH synthesis, for 24 h. Such a BSO treatment was found to reduce cellular GSH content to 18 6 3% of that observed in untreated GLC4/Sb30 cells. It did not alter calcein accumulation since determination of intracellular dye labelling after a 90 min-incubation with 0.5 mM calcein AM indicated that calcein found in BSO-treated cells corresponded to 95 6 8% of that found in untreated cells. In contrast, GLC4/Sb30 cells exposed to the GSHdepleting agent accumulated more vincristine than their untreated counterparts; indeed, vincristine levels in BSO-treated cells measured after a 90-min incubation with 20 nM vincristine represented 173 6 16% of those found in cells not exposed to BSO. DISCUSSION The antiprogestatin compound RU486 has been previously shown to block P-gp-mediated drug transport and therefore to reverse P-gp-related multidrug resistance (17, 18, 26). The results reported in the present study demonstrated for the first time to the best of our knowledge that RU486 also inhibits activity of MRP, a drug efflux pump distinct from P-gp and also involved in drug resistance of tumor cells. Indeed, the antiprogestatin drug was shown to increase intracellular accumulation of calcein and vincristine, two known substrates of MRP, in MRP-overexpressing GLC4/Sb30 cells. Intracellular levels of these two compounds in RU486-treated GLC4/Sb30 cells thus reached those observed in parental GLC4 cells. Such an effect was most likely related to inhibition of MRP-related drug efflux since RU486 was demonstrated to strongly down-modulate calcein export out of GLC4/Sb30 cells. In contrast, the antiprogestatin compound did not obviously alter calcein and vincristine accumulation in parental GLC4 cells; it also did not modify calcein export out of these cells, therefore indicating that its effects on drug levels in GLC4/Sb30 cells are rather
TABLE 2
Effects of RU486 on Cellular GSH Content GSH levels (nM/mg protein) Cells
Control
1 RU486
GLC4/Sb30 GLC4
7.6 6 1.0 8.2 6 1.3
0.8 6 0.2 (0.11) 3.5 6 0.7 (0.43)
Note. Cellular GSH contents were determined in untreated control cells and in cells exposed to 10 mM RU486 for 96 h. Values are means 6 SD of three independent experiments. Numbers in parentheses correspond to the ratio of GSH levels in the presence of RU486 versus those in the absence of the RU486.
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specific. In addition, it is noteworthy that GLC4/Sb30 cells have been demonstrated to not overexpress P-gp (19), thus suggesting that RU486-mediated alteration of P-gp activity cannot account for its effects in GLC4/ Sb30 cells. Increased drug accumulation in GLC4/Sb30 cells in response to RU486 treatment was associated with enhanced cytotoxicity of MRP substrates such as vincristine and PAT as demonstrated in reversing experiments. Interestingly, the 10 mM concentration of RU486 used in such drug sensitivity assays is in the range of plasma concentrations achievable in patients and is thought to be devoid of major adverse effects (17, 24). These data therefore indicate that RU486 may lead to an effective inhibition of MRP activity in vivo without toxicity. This potential clinical relevance of the antiprogestatin compound may be emphasized since many known MRP modulators such as genistein, acrolein, chloroacetaldehyde and brefeldin A usually act at elevated doses which preclude their use in reversing assays (13, 14, 27). It is also noteworthy that the 10 mM concentration of RU486 capable of inhibiting MRP activity is also sufficient for blocking P-gp activity in several P-gp overexpressing cell lines (17, 18); the antiprogestatin drug may therefore be considered as a bifonctional chemosensitizer acting on both P-gp- and MRP-mediated drug resistance. In addition, the interest of using RU486 as a modulator agent in the treatment of malignancies may likely be reinforced by the fact that the antiprogestatin drug has own antiproliferative properties against some kinds of cancer cells (28). The exact mechanism by which RU486 interferes with MRP activity remains to be fully determined. Previous studies have demonstrated that downregulation of MRP-mediated drug transport may involve a depletion of cellular GSH content or a direct interaction of chemosensitizers with the efflux pump, possibly via competition for its drug-binding sites (4). RU486-treated GLC4/Sb30 cells were shown to display a strong reduction in cellular GSH levels when compared to their untreated counterparts; a similar GSH depletion was also achieved by a 24 h-treatment by BSO. Interestingly, BSO-treated GLC4/Sb30 cells were found to accumulate more vincristine than untreated cells; this result agrees with previous data indicating that MRP-mediated transport of some drugs, including vincristine, requires GSH (7, 25). Therefore, decreased levels of GSH occurring in GLC4/Sb30 cells in response to RU486 may be considered as a factor contributing to the reversing effects of the antiprogestatin drug, especially with respect to vincristine. On the other hand, calcein accumulation was not impaired in BSO-treated GLC4/Sb30 cells. Alteration in GSH levels can thus not account for the RU486-related inhibition of calcein export out of GLC4/Sb30 cells. It can therefore be hypothetized that the antiprogestatin drug alters MRP-
mediated transport of the dye, not through GSH depletion, but via direct interaction with the efflux pump. Such an hypothesis is also supported by the fact that RU486 has already been reported to be able to interfere with drug binding sites of efflux pump such as P-gp (17). Taken together, all these data favor the idea that RU486 may be considered as an uncommon MRP chemosensitizer acting through both GSH depletion and direct inhibition of the membrane pump. In addition to RU486, progesterone was also shown to enhance calcein accumulation in GLC4/Sb30 cells whereas other steroids such as dexamethasone and b-estradiol had no effect at the doses tested. This suggests that structural features restricted to progesterone and RU486 are involved in inhibition of MRPmediated drug transport. A similar conclusion can also be applied to down-modulation of P-gp activity by some steroids since, in addition to RU486 and unlike dexamethasone, progesterone inhibits P-gp function (29). Progesterone and RU486 are however not themselves substrates of P-gp (29, 30). In the same way, RU486 is most likely not transported by MRP since MRPoverexpressing GLC4/Sb30 cells were not crossresistant to the antiprogestatin drug as demonstrated by MTT assays. In summary, our data demonstrated that the antiprogestatin drug RU486 inhibits MRP-mediated drug transport and enhances the sensitivity of MRPoverexpressing cells to cytotoxic compounds such as vincristine. RU486, which is also known to block P-gp activity, may therefore be considered as a promising chemosensitizer likely allowing to overcome both MRPand P-gp-related multidrug resistance of tumor cells. ACKNOWLEDGMENTS Le´a Payen and Arnaud Courtois are recipients of fellowships from the Ligue Nationale contre le Cancer (Comite´ du Morbihan) and the Association pour la recherche sur le Cancer, respectively. We thank Dr. L. Vernhet for the critical reading of the manuscript.
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Reversal of MRP-Mediated Multidrug Resistance in Human Lung Cancer Cells by the Antiprogestatin Drug RU486 Le´a Payen, Laurence Delugin, Arnaud Courtois, Yolande Trinquart, Andre´ Guillouzo, and Olivier Fardel 1 Unite´ INSERM U456 De´toxication et Re´paration Tissulaire, Faculte´ des Sciences Pharmaceutiques et Biologiques, 2 Avenue du Pr L. Bernard, 35043 Rennes, France
Received April 8, 1999
Multidrug resistance-associated protein (MRP) and P-glycoprotein (P-gp) are drug efflux pumps conferring multidrug resistance to tumor cells. RU486, an antiprogestatin drug known to inhibit P-gp function, was examined for its effect on MRP activity in MRPoverexpressing lung tumor GLC4/Sb30 cells. In such cells, the antihormone compound was found to increase intracellular accumulation of calcein, a fluorescent compound transported by MRP, in a dosedependent manner, through inhibition of cellular export of the dye ; in contrast, it did not alter calcein levels in parental GLC4 cells. RU486, when used at 10 mM, a concentration close to plasma concentrations achievable in humans, strongly enhanced the sensitivity of GLC4/Sb30 cells towards two known cytotoxic substrates of MRP, the anticancer drug vincristine and the heavy metal salt potassium antimonyl tartrate. Vincristine accumulation levels were moreover up-regulated in RU486-treated GLC4/Sb30 cells. In addition, such cells were demonstrated to display reduced cellular levels of glutathione which is required for MRP-mediated transport of some anticancer drugs. These findings therefore demonstrate that RU486 can down-modulate MRP-mediated drug resistance, in addition to that linked to P-gp, through inhibition of MRP function. © 1999 Academic Press
Multidrug resistance is a major cause of failure in the treatment of human malignancies. Such a resistance, usually defined as cross-resistance to structurally and functionally unrelated anticancer drugs, is often linked to overexpression of membrane proteins effluxing antitumor compounds out of cells and therefore lowering their intracellular accumulation (1). The first described of such transporters was P-glycoprotein 1
Corresponding author. Fax: 33 2 99 33 62 42. E-mail: olivier. [email protected].
(P-gp), a 170 kDa protein encoded by the MDR1 gene (2); another transporter more recently characterized is the 190 kDa multidrug resistance-associated protein (MRP, also known as MRP1) (3). Both P-gp and MRP belong to the ATP-binding cassette protein superfamily (2, 4). They share numerous substrates including anthracyclins, vincristine and epipodophyllotoxins. However, the two efflux pumps also display some functional differences. Thus, paclitaxel is handled by P-gp, but not by MRP, whereas the latter pump, unlike the former, mediates the transport of anionic compounds such as glutathione S-conjugates and of some heavy metals such as antimony (4 – 6). In addition, MRP, unlike P-gp, has been demonstrated to cotransport some anticancer drugs such as vincristine in an unmodified form with reduced glutathione (GSH), thus outlining the role of GSH in some MRP-mediated transport processes (7). Increased levels of P-gp and/or MRP have been detected in tumor samples from patients suffering from different kinds of cancers and have been correlated, at least for P-gp, with lack of response to chemotherapy and poor survival in some malignancies (1, 5, 8, 9). P-gp activity can be blocked by a wide variety of drugs including calcium channel blockers such as verapamil and immunosuppressive agents such as cyclosporin A (10). These compounds, termed modulators or chemosensitizers, increase intracellular accumulation of anticancer drugs in P-gp-overexpressing cells and thus circumvent drug resistance (11). Some of them have now entered clinical trials (11, 12). In contrast to P-gp-mediated transport, that linked to MRP has been demonstrated to be inhibited only by few agents which, moreover, often act at elevated and toxic doses, therefore precluding their use in reversing experiments (4, 13, 14). There is therefore a need to identify new MRP modulators. To this aim, the analysis of P-gp inhibitors may be useful since some of these compounds have
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been demonstrated to also interact with MRP (15, 16). In the present study, RU486, an steroid compound with antiprogestatin properties already known to inhibit P-gp-related transport at concentrations thought to have no serious side effects (17, 18), was therefore tested for its potential inhibitory effects on MRP activity. Our results demonstrate that this anti-hormone drug markedly down-modulated drug resistance of MRP-overexpressing human lung GLC4/Sb30 cells through inhibition of MRP function. MATERIALS AND METHODS Chemicals. Calcein acetoxymethoxyl ester (calcein AM) and [ 3H] vincristine (specific activity 5.8 Ci/mmol) were purchased from Molecular Probes (Eugene, OR) and Amersham (Les Ulis, France), respectively. Progesterone, vincristine, dexamethasone, potassium antimonyl tartrate (PAT), b-estradiol, probenecid and buthionine sulfoximine (BSO) were obtained from Sigma (St Louis, MO). RU486 was kindly provided by Roussel-Uclaf (Romainville, France). Cell culture. The human lung cancer cell line GLC4 and its antimony-selected subline GLC4/Sb30 were cultured in RPMI medium supplemented with 10% fetal calf serum at 37°C in a 5% CO 2 atmosphere. GLC4/Sb30 cells overexpress MRP but not P-gp and are cross-resistant to anticancer drugs such as doxorubicin and vincristine when compared to their parental counterparts (19); they were routinely maintained in the presence of PAT (30 mg/ml) which was however withdrawn for at least 3 days before the use of the cells for experiments. Calcein accumulation and efflux assays. Calcein is an anionic fluorescent probe substrate for MRP and determination of its cellular accumulation and efflux allow to investigate MRP activity (20). For calcein accumulation experiments, cells were incubated with 0.5 mM calcein AM for 90 min in the absence or the presence of RU486 or one of the other potential MRP chemosensitizer tested. The non-fluorescent esterified form calcein AM freely diffuses into cells where it is cleaved by intracellular esterases to give the fluorescent anionic dye calcein (21) which can be exported out of cells through MRP1 activity (20). Cells were then washed three times with ice-cold phosphate-buffered saline (PBS) and lyzed in distilled water by ultrasonication. Amounts of intracellular calcein were next determined by fluorimetry using a Titertek Fluoroscan spectrofluorometer (Flow Laboratories, Puteaux, France); excitation and emission wavelenghts were 485 nm and 538 nm, respectively. An aliquot of cell lysate was used in parallel to determine cellular protein concentration by the Bio-Rad assay (22). Results were expressed as fluorescence arbitrary units after normalization to cellular protein content. For calcein efflux assays, calcein-preloaded cells were incubated in calcein AM-free medium for 90 min in the absence or presence of RU486. Cells were then washed three times with PBS and intracellular calcein retained was then determined by fluorimetry as described above. Results were expressed as percentages of initial calcein staining. Intracellular vincristine accumulation. Cells were incubated with 20 nM [ 3H] vincristine for 90 min in the presence or the absence of RU486. Cells were then washed three times with ice-cold PBS and lyzed in distilled water by ultrasonication. Radioactivity of [ 3H] vincristine in the cell extract was then determined by scintillation counting and normalized to cellular protein content. Drug-sensitivity assay. The effects of cytotoxic compounds on cell proliferation was evaluated using the 3-[4,5-dimethylthiazol-2-yl]2,5-diphenyl tetrazolium bromide (MTT) as previously described (23). Briefly, cells were cultured with various concentrations of vin-
FIG. 1. Effect of various concentrations of RU486 on calcein accumulation in drug-resistant GLC4/Sb30 and drug-sensitive GLC4 cells. Cells were incubated with 0.5 mM calcein AM in the presence of various doses of RU486 (from 0 to 100 mM) for 90 min. Intracellular calcein accumulation was then determined by fluorimetry. The values are expressed as fluorescence arbitrary units (FAU) normalized to protein content and are the mean 6 SD of at least three independent experiments in triplicate. *, P , 0.05 when compared to cells not treated by RU486.
cristine or PAT for 96 h in the presence or the absence of RU486 in 96-well microplates. 100 ml of a 1 mg/ml MTT solution was then added to each well for 120 min. The medium was next discarded and replaced by 100 ml of dimethyl sulfoxide. The blue formazan product formed was further quantified by its absorbance at 540 nm using a Titertek Multiskan MCC/340 (Flow Laboratories). Growth inhibition was evaluated as IC 50, i.e. the drug concentration providing a 50% reduction in cell number as compared to controls cultured in parallel without cytotoxic compound. Cellular GSH content. Total cellular GSH content was determined using a colorimetric assay (Bioxytech GSH-400) supplied by Oxis (Portland, OR); the values were then normalized to cellular protein content. Statistical analysis. Data were analyzed by the Student’s t-test. The criterion of significance of the differences between the means (6 standard deviation) was P , 0.05.
RESULTS The effects of several concentrations of RU486 on the accumulation of the MRP substrate calcein in MRPoverexpressing GLC4/Sb30 cells and in their parental GLC4 counterparts were firstly investigated. As indicated in Fig. 1, the antiprogestatin compound increased calcein levels in GLC4/Sb30 cells in a dosedependent manner. Indeed, RU486 used at 10 mM was found to enhance cellular dye-related fluorescence by approximately 2-fold whereas the use of higher concentrations (25-100 mM) allowed to increase calcein levels by approximately 3-fold and thus to reach the dye levels observed in parental GLC4 cells (Fig. 1). In contrast, 1 mM RU486 was found to have no effect whereas 5 mM only slightly increased intracellular dye accumulation in GLC4/Sb30 cells. None of the RU486 concentrations tested over the 90-min incubation period af-
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FIG. 2. Effect of dexamethasone, progesterone, b-estradiol, and probenecid on calcein accumulation in drug-resistant GLC4/Sb30 cells. GLC4/Sb30 cells were incubated with 0.5 mM calcein AM in the absence (UNT) or the presence of 100 mM dexamethasone (DEX), 50 mM progesterone (PRG), 20 mM b-estradiol (EST), or 2 mM probenecid (PRB) for 90 min. Intracellular calcein accumulation was then determined by fluorimetry. The values are expressed as fluorescence arbitrary units (FAU) normalized to protein content and are the mean 6 SD of at least three independent experiments in triplicate. *, P , 0.05 when compared to cells not treated by RU486.
fected cell viability as assessed by MTT assays and determination of cellular protein content (data not shown). In contrast to GLC4/Sb30 cells, GLC4 cells did not show any alteration in calcein-related fluorescence in response to RU486 used at 50 or 100 mM (Fig. 1). In order to determine whether other steroids distinct from RU486 might also up-regulate calcein levels in GLC4/Sb30 cells, cellular accumulation of the dye was investigated in the presence of dexamethasone, progesterone and b-estradiol. Results indicated that 50 mM progesterone increased calcein levels in GLC4/Sb30 cells (Fig. 2). In contrast, 100 mM dexamethasone and 20 mM b-estradiol had no effect whereas 2 mM probenecid, a well-known inhibitor of MRP activity (4), markedly enhanced calcein-related cellular fluorescence. Action of RU486 on calcein export out of GLC4/Sb30 and GLC4 cells was next determined by dye efflux experiments. As indicated in Fig. 3, GLC4/Sb30 cells poorly retained calcein when compared to their parental counterparts and such a loss of dye was found to be strongly inhibited by the antiprogestatin compound. In contrast, RU486 did not alter retention levels of calcein in GLC4 cells. To determine whether RU486 may also alter intracellular retention of MRP substrates distinct from calcein, intracellular accumulation of the anticancer drug vincristine was further determined in the presence or the absence of the antiprogestatin drug (Fig. 4). RU486 was found to markedly enhance vincristine accumula-
FIG. 3. Effect of RU486 on calcein efflux from GLC4/Sb30 and GLC4 cells. Calcein-loaded GLC4/Sb30 and GLC4 cells were incubated in calcein AM-free medium for 90 min in the absence or the presence of 50 mM RU486. Intracellular retained calcein was then determined by fluorimetry and expressed relative to initial dye staining. The values are the mean 6 SD of three independent experiments in triplicate. *, P , 0.05 when compared to cells not treated by RU486.
tion in GLC4/Sb30 cells whereas it had no major effect in GLC4 cells. Cellular levels of the anticancer agent were thus similar in RU486-treated GLC4/Sb30 cells
FIG. 4. Effect of RU486 on [ 3H] vincristine accumulation in GLC4/Sb30 and GLC4 cells. GLC4/Sb30 and GLC4 cells were incubated for 90 min with 20 nM [ 3H] vincristine in the absence or the presence of 50 mM RU486. Intracellular [ 3H] vincristine accumulation was then determined by scintillation counting. The values are the mean 6 SD of three independent experiments in triplicate. *, P , 0.05 when compared to cells not treated by RU486.
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Effects of RU486 on PAT and Vincristine Cytotoxicity PAT IC 50 (mg/ml)
Vincristine IC 50 (ng/ml)
Cells
Control
1 RU486
Control
1 RU486
GLC4/Sb30 GLC4
40.7 6 5.1 0.8 6 0.3
2.2 6 0.8 (18.5) 0.3 6 0.1 (2.7)
3.5 6 0.2 0.4 6 0.1
0.4 6 0.1 (8.7) 0.2 6 0.03 (2)
Note. The values of potassium antimonyl tartrate (PAT) and vincristine IC 50 were determined in the absence or in the presence of 10 mM RU486 using the MTT assay and are the means 6 SD of at least three independent experiments. Numbers in parentheses indicate the sensitization factor, i.e., the ratio of the IC 50 in the absence of RU486 versus the IC 50 in the presence of RU486.
and in GLC4 cells whereas the resistant cells poorly accumulated vincristine in the absence of the antiprogestatin compound when compared to their parental drug-sensitive cells (Fig. 4). The effects of prolonged RU486 exposure (96 h) on cell proliferation were then monitored using the MTT dye assay. RU486 IC 50 values were found to be 18 6 3 mM and 20 6 2 mM for GLC4/Sb30 cells and GLC4 cells, respectively. Interestingly, the dose of 10 mM was found to not obviously alter growth of both GLC4/Sb30 cells and GLC4 cells; this RU486 concentration active on MRP-mediated transport (Fig. 1) and in the range of the plasma concentrations observed in humans after a single oral dose (24) was further retained for reversing experiments. In such assays, the antiprogestatin drug was evidenced to strongly down-modulate resistance of GLC4/Sb30 cells towards vincristine and PAT. Indeed, PAT and vincristine IC 50 values for GLC4/Sb30 cells in the presence of RU486 were reduced by 18.5- and 8.7fold, respectively, and therefore became close to those observed for parental GLC4 cells (Table 1). The antiprogestatin was also found to enhance the cytotoxicity of PAT and vincristine towards GLC4 cells, but only slightly, thus likely reflecting the inhibition of the MRP constitutively present at low, but detectable levels, in these cells as already reported (19). Since reversion of MRP-mediated resistance has been linked to down-regulation of cellular GSH content by some chemosensitizers (25), we next determined cellular GSH levels in the presence or the absence of RU486. As indicated in Table 2, exposure to 10 mM RU486 for 96 h was found to result in a strong reduction of cellular GSH levels in GLC4 Sb30 cells and also, although to a less extent, in parental GLC4 cells; the GSH content in RU486-treated GLC4/Sb30 cells thus represented 11% of that found in their untreated counterparts. GSH depletion was also achieved by shorter exposure to the antiprogestatin compound; indeed, GLC4/Sb30 cells exposed to 10 mM RU486 for 90 min displayed a reduction in GSH content by approximately a 2.2-fold factor (data not shown). To further investigate the relationships between GSH levels and
membrane transport of MRP substrates in GLC4/Sb30 cells, intracellular accumulation of calcein and vincristine were determined in GLC4/Sb30 cells exposed to 25 mM BSO, a well-known inhibitor of GSH synthesis, for 24 h. Such a BSO treatment was found to reduce cellular GSH content to 18 6 3% of that observed in untreated GLC4/Sb30 cells. It did not alter calcein accumulation since determination of intracellular dye labelling after a 90 min-incubation with 0.5 mM calcein AM indicated that calcein found in BSO-treated cells corresponded to 95 6 8% of that found in untreated cells. In contrast, GLC4/Sb30 cells exposed to the GSHdepleting agent accumulated more vincristine than their untreated counterparts; indeed, vincristine levels in BSO-treated cells measured after a 90-min incubation with 20 nM vincristine represented 173 6 16% of those found in cells not exposed to BSO. DISCUSSION The antiprogestatin compound RU486 has been previously shown to block P-gp-mediated drug transport and therefore to reverse P-gp-related multidrug resistance (17, 18, 26). The results reported in the present study demonstrated for the first time to the best of our knowledge that RU486 also inhibits activity of MRP, a drug efflux pump distinct from P-gp and also involved in drug resistance of tumor cells. Indeed, the antiprogestatin drug was shown to increase intracellular accumulation of calcein and vincristine, two known substrates of MRP, in MRP-overexpressing GLC4/Sb30 cells. Intracellular levels of these two compounds in RU486-treated GLC4/Sb30 cells thus reached those observed in parental GLC4 cells. Such an effect was most likely related to inhibition of MRP-related drug efflux since RU486 was demonstrated to strongly down-modulate calcein export out of GLC4/Sb30 cells. In contrast, the antiprogestatin compound did not obviously alter calcein and vincristine accumulation in parental GLC4 cells; it also did not modify calcein export out of these cells, therefore indicating that its effects on drug levels in GLC4/Sb30 cells are rather
TABLE 2
Effects of RU486 on Cellular GSH Content GSH levels (nM/mg protein) Cells
Control
1 RU486
GLC4/Sb30 GLC4
7.6 6 1.0 8.2 6 1.3
0.8 6 0.2 (0.11) 3.5 6 0.7 (0.43)
Note. Cellular GSH contents were determined in untreated control cells and in cells exposed to 10 mM RU486 for 96 h. Values are means 6 SD of three independent experiments. Numbers in parentheses correspond to the ratio of GSH levels in the presence of RU486 versus those in the absence of the RU486.
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specific. In addition, it is noteworthy that GLC4/Sb30 cells have been demonstrated to not overexpress P-gp (19), thus suggesting that RU486-mediated alteration of P-gp activity cannot account for its effects in GLC4/ Sb30 cells. Increased drug accumulation in GLC4/Sb30 cells in response to RU486 treatment was associated with enhanced cytotoxicity of MRP substrates such as vincristine and PAT as demonstrated in reversing experiments. Interestingly, the 10 mM concentration of RU486 used in such drug sensitivity assays is in the range of plasma concentrations achievable in patients and is thought to be devoid of major adverse effects (17, 24). These data therefore indicate that RU486 may lead to an effective inhibition of MRP activity in vivo without toxicity. This potential clinical relevance of the antiprogestatin compound may be emphasized since many known MRP modulators such as genistein, acrolein, chloroacetaldehyde and brefeldin A usually act at elevated doses which preclude their use in reversing assays (13, 14, 27). It is also noteworthy that the 10 mM concentration of RU486 capable of inhibiting MRP activity is also sufficient for blocking P-gp activity in several P-gp overexpressing cell lines (17, 18); the antiprogestatin drug may therefore be considered as a bifonctional chemosensitizer acting on both P-gp- and MRP-mediated drug resistance. In addition, the interest of using RU486 as a modulator agent in the treatment of malignancies may likely be reinforced by the fact that the antiprogestatin drug has own antiproliferative properties against some kinds of cancer cells (28). The exact mechanism by which RU486 interferes with MRP activity remains to be fully determined. Previous studies have demonstrated that downregulation of MRP-mediated drug transport may involve a depletion of cellular GSH content or a direct interaction of chemosensitizers with the efflux pump, possibly via competition for its drug-binding sites (4). RU486-treated GLC4/Sb30 cells were shown to display a strong reduction in cellular GSH levels when compared to their untreated counterparts; a similar GSH depletion was also achieved by a 24 h-treatment by BSO. Interestingly, BSO-treated GLC4/Sb30 cells were found to accumulate more vincristine than untreated cells; this result agrees with previous data indicating that MRP-mediated transport of some drugs, including vincristine, requires GSH (7, 25). Therefore, decreased levels of GSH occurring in GLC4/Sb30 cells in response to RU486 may be considered as a factor contributing to the reversing effects of the antiprogestatin drug, especially with respect to vincristine. On the other hand, calcein accumulation was not impaired in BSO-treated GLC4/Sb30 cells. Alteration in GSH levels can thus not account for the RU486-related inhibition of calcein export out of GLC4/Sb30 cells. It can therefore be hypothetized that the antiprogestatin drug alters MRP-
mediated transport of the dye, not through GSH depletion, but via direct interaction with the efflux pump. Such an hypothesis is also supported by the fact that RU486 has already been reported to be able to interfere with drug binding sites of efflux pump such as P-gp (17). Taken together, all these data favor the idea that RU486 may be considered as an uncommon MRP chemosensitizer acting through both GSH depletion and direct inhibition of the membrane pump. In addition to RU486, progesterone was also shown to enhance calcein accumulation in GLC4/Sb30 cells whereas other steroids such as dexamethasone and b-estradiol had no effect at the doses tested. This suggests that structural features restricted to progesterone and RU486 are involved in inhibition of MRPmediated drug transport. A similar conclusion can also be applied to down-modulation of P-gp activity by some steroids since, in addition to RU486 and unlike dexamethasone, progesterone inhibits P-gp function (29). Progesterone and RU486 are however not themselves substrates of P-gp (29, 30). In the same way, RU486 is most likely not transported by MRP since MRPoverexpressing GLC4/Sb30 cells were not crossresistant to the antiprogestatin drug as demonstrated by MTT assays. In summary, our data demonstrated that the antiprogestatin drug RU486 inhibits MRP-mediated drug transport and enhances the sensitivity of MRPoverexpressing cells to cytotoxic compounds such as vincristine. RU486, which is also known to block P-gp activity, may therefore be considered as a promising chemosensitizer likely allowing to overcome both MRPand P-gp-related multidrug resistance of tumor cells. ACKNOWLEDGMENTS Le´a Payen and Arnaud Courtois are recipients of fellowships from the Ligue Nationale contre le Cancer (Comite´ du Morbihan) and the Association pour la recherche sur le Cancer, respectively. We thank Dr. L. Vernhet for the critical reading of the manuscript.
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