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Originally published In Press as doi:10.1074/jbc.M404466200 on July 19, 2004

J. Biol. Chem., Vol. 279, Issue 39, 40412-40418, September 24, 2004
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Inhibition of Glucosylceramide Synthase Does Not Reverse Drug Resistance in Cancer Cells*

Edward Norris-Cervetto{ddagger}§, Richard Callaghan§, Frances M. Platt{ddagger}, Raymond A. Dwek{ddagger}, and Terry D. Butters{ddagger}

From the {ddagger}Oxford Glycobiology Institute, University of Oxford, South Parks Road, Oxford OX1 3QU and the §Nuffield Department of Clinical Laboratory Sciences, Level 4, John Radcliffe Hospital, Oxford OX1 3QU, United Kingdom

Received for publication, April 22, 2004 , and in revised form, July 16, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The multidrug-resistant cancer cell lines NCI/AdRRES and MES-SA/DX-5 have higher glycolipid levels and higher P-glycoprotein expression than the chemosensitive cell lines MCF7-wt and MES-SA. Inhibiting glycolipid biosynthesis by blocking glucosylceramide synthase has been proposed to reverse drug resistance in MDR cells by causing an increased accumulation of proapoptotic ceramide during treatment of cells with cytotoxic drugs. We treated both multidrug-resistant cell lines with the glucosylceramide synthase inhibitors PDMP (D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol), C9DGJ (N-nonyl-deoxygalactonojirimycin) or C4DGJ (N-butyl-deoxygalactonojirimycin). PDMP achieved a significant reversal of drug resistance in agreement with previous reports. However, the N-alkylated iminosugars C9DGJ and C4DGJ, which are more selective glucosylceramide synthase inhibitors than PDMP, failed to cause any reversal of drug resistance despite depleting glycolipids to the same extent as PDMP. Our results suggest that (a) inhibition of glucosylceramide synthase does not reverse multidrug resistance and (b) the chemosensitization achieved by PDMP cannot be caused by inhibition of glucosylceramide synthase alone.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
A major limitation in chemotherapy for cancer is multidrug resistance (MDR),1 an innate or acquired phenotype, which allows cancer cells to resist a broad spectrum of chemotherapeutic drugs. One of the most extensively studied and clinically relevant mechanisms of drug resistance is the overexpression by cancer cells of the transmembrane multidrug transporter P-glycoprotein (MDR1, ABCB1, EC 3.6.3.44 [EC] ) (1). However, a drug resistance phenotype comprises many, often interacting, mechanisms of resistance (2). These include increased DNA repair, altered target sensitivity, decreased apoptotic response and numerous aberrant signal transduction pathways. Elevated levels of glycolipids have been correlated with multidrug resistance in cancer (3, 4). Manipulation of glycolipid levels in some MDR cancer cells was able to reverse drug resistance (5–12). This led to the hypothesis that elevated glucosylceramide synthase (GCS, EC 2.4.1.80 [EC] ) activity is a novel form of multidrug resistance and that inhibition of GCS is a promising therapeutic strategy for combating multidrug resistance. The biochemical basis is that an elevated GCS activity prevents the accumulation of ceramide, which is thought to precede, and trigger, apoptosis in response to some cytotoxic drugs (13–16). Therefore, inhibition of GCS activity will promote the accumulation of pro-apoptotic ceramide and enhance cell death in response to cytotoxic agents (17–21).

This hypothesis rests largely on three lines of evidence. The first is that multidrug resistance correlates with elevated glycolipid levels (4), which is unfortunately hindered by the small number of samples analyzed and the fact that variability of glycolipid levels in cancers remains unknown.

Second, genetic manipulation of the GCS enzyme in MCF7 cells can affect their sensitivity to various cytotoxic drugs (5–7). However, manipulation of GCS levels in Jurkat and GM95 cells had no effect on their sensitivity to cytotoxic challenge (22, 23).

Third, PDMP (D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol) and its derivatives (24) are able to sensitize drug-resistant cancer cells in vitro (8–12). However, there is evidence that these compounds can (i) inhibit enzymes other than GCS (25, 26), (ii) cause cell cycle arrest (27), and (iii) even increase the resistance to some cytotoxic drugs (28).

The aim of this study was to use a different class of GCS inhibitors to try and sensitize MDR cancer cells to chemotherapy. N-Alkylated iminosugars (29) were chosen because they are well tolerated and appear to have fewer side effects in cells than PDMP and its derivatives (25, 26) Specifically, N-alkyldeoxygalactonojirimycin (N-alkyl-DGJ) compounds were selected because they are even more selective for GCS than N-alkyl-deoxynojirimycin (N-alkyl-DNJ) compounds which also inhibit {alpha}-glucosidases I and II, albeit with much lower potency (30, 31).

The N-alkylated iminosugars C9DGJ (N-nonyl-deoxygalactonojirimycin) and C4DGJ (N-butyl-deoxygalactonojirimycin) did not sensitize MDR cells to chemotherapy despite achieving a comparable reduction in glycolipid levels to PDMP, which did sensitize MDR cells. Additionally, the P-glycoprotein inhibitor XR9576, which had no effect on glycolipid levels, was able to reverse drug resistance in both cell lines completely. We conclude that the effect of PDMP and its derivatives on drug resistance cannot be explained by inhibition of GCS alone and we agree with other recent reports (22, 23) that GCS activity has no effect on MDR.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials—The FITC-labeled, mouse anti-human-P-glycoprotein monoclonal antibody I5D3 and the FACSCalibur instrument were from BD Biosciences (Oxford, UK). Quantum 1000 FITC-labeled, medium level microbeads were from Flow Cytometry Standards Corporation (San Juan). The BCA assay kit was from Sigma. [14C]Palmitate (55 mCi/mmol) was from ICN Pharmaceuticals (Basingstoke, UK). Doxorubicin HCl and vinblastine sulfate were from CNBiosciences (Nottingham, UK). The C9DGJ (N-nonyl-deoxygalactonojirimycin) and C4DGJ (N-butyl-deoxygalactonojirimycin) were from Toronto Research Chemicals (Toronto, Canada). PDMP was from Sigma. The P-glycoprotein inhibitor XR9576 was a gift from Xenova (Slough, UK). The CellTiter-96 AQueous Cellular Proliferation Assay was from Promega (Southampton, UK). Aminopropyl cartridges were Supelclean LC-NH2 SPE Tubes (1 ml capacity) from Supelco (Poole, UK). C18 cartridges were Sep-Pak Vac 1cc (100 mg) Cartridges from Waters Corporation (Hoddesdon, UK). The DPA-6S columns were from Sigma. Silica gel 60 HP-TLC aluminum plates (20 x 20 cm) and silica gel 60 TLC glass plates (10 x 20 cm) were from Merck, Sharpe & Dohme. The HPLC instruments were a Waters 2695 Separation Module with a Waters 474 Scanning Fluorescence Detector from Waters Corporation (Hoddesdon, UK). The HPLC column was a TSK gel-Amide 80 column from Anachem (Luton, UK). The glycolipids glucosylceramide (GlcCer), lactosylceramide (Lac-Cer), globotriaosylceramide (Gb3), globotetraosylceramide (Gb4), monosialoganglioside GM1, monosialoganglioside GM2, and monosialoganglioside GM3 were from Sigma. Tissue culture materials were from Invitrogen. Drug-sensitive MCF7-wt human breast cancer cells were obtained from the NCI-Frederick Cancer DCTD tumor cell repository (Bethesda, MD). Multidrug-resistant NCI/AdRRES cells were obtained from Professor Cowan and were generated by selection in adriamycin as described (32). Drug-sensitive MES-SA human uterine sarcoma cells and multidrug-resistant MES-SA/DX-5 cells were obtained from ATCC. All other compounds were HPLC grade from Sigma.

Tissue Culture—MCF7-wt and multidrug-resistant NCI/AdRRES cancer cells were grown as previously described (33) in Dulbecco's DMEM-F12 medium supplemented with 10% (v/v) fetal bovine serum, 2 mML-glutamine, 100 units/ml penicillin, and 100 µg/ml streptomycin at 37 °C and 5% CO2. NCI/AdRRES stock cells were supplemented with 5 µM doxorubicin every third passage but cells used in experiments were grown in media devoid of doxorubicin for at least 7 days. Both MES cell lines were grown in McCoy's medium supplemented with 10% (v/v) fetal bovine serum, 2 mM L-glutamine, 100 units/ml penicillin, and 100 µg/ml streptomycin at 37 °C and 5% CO2.

Quantification of P-glycoprotein by Flow Cytometry—P-glycoprotein expression in cells was determined by flow cytometry as previously described (34) using an FITC-labeled, mouse anti-human-P-glycoprotein monoclonal antibody I5D3 and Quantum 1000 FITC-labeled microbeads.

Extraction of Cellular Lipids—Cells (8 x 106) were harvested using phosphate-buffered saline + 0.02% (w/v) EDTA. An aliquot of 90 µl of cells was mixed with 10 µl of 10% SDS and passed repeatedly through a thin gauge needle. The protein concentration of this solubilized cellular preparation was determined using a BCA assay according to the manufacturer's instructions. Cellular lipids were then extracted from 500 µg (protein) of cells using the method of Svennerholm (35) and dried down under N2 at 37 °C.

Purification of GlcCer—Silicic acid that had been dried overnight at 80 °C was mixed in chloroform to obtain a 10% (w/v) solution and then 2 ml of this solution was placed in a disposable polypropylene column. The column was pre-equilibrated with 5 x 1 ml chloroform and the lipid extract loaded in 1 ml of chloroform. The column was washed consecutively with 2x 1 ml of chloroform, 2x 1 ml of 99:1 (v/v) CHCl3:MeOH, 2x 1 ml of 98:2 (v/v) CHCl3:MeOH, and 2x 97:3 (v/v) CHCl3:MeOH. Glucosylceramides (GlcCer) were eluted with 2x 1 ml of 96:4 (v/v) CHCl3:MeOH, 2x 1 ml of 95:4 (v/v) CHCl3:MeOH, and 2x 1 ml of 94:6 (v/v) CHCl3:MeOH and dried down under N2 at 37 °C.

Quantification of GlcCer by Thin Layer Chromatography—Purified GlcCer was resuspended in 10 µl of 2:1 (v/v) CHCl3:MeOH and spotted onto a silica gel 60 glass TLC plate, which had been presoaked in 2.5% (w/v) H3BO3 in MeOH and dried at 80 °C before use. The plate was first run in chloroform, then developed in CHCl3:MeOH:H2O:25% (v/v) NH4OH 65:35:4:4 before being sprayed with 0.2% (w/v) orcinol in 1 M H2SO4 and dried at 80 °C. TLC plates were scanned using an Agfa Arcus II scanner and the relative optical densities analyzed using NIH ImageJ for Mac OS X.

Purification of Glycolipids and Ceramides—Ceramides, neutral glycolipids, and gangliosides were purified from this lipid extract using a modified version of the method of Bodennec et al. (36).2 The lipid extract was dried under N2 at 37 °C and redissolved in 500 µl of CHCl3. This was loaded onto a Supelclean LC-NH2 SPE cartridge (pre-equilibrated with 2 ml of hexane). The column was then eluted consecutively with 2 ml of diethyl ether (fraction 1), 1.6 ml of CHCl3:MeOH 23:1 (v/v) (fraction 2), 1.8 ml of diisopropyl ether:acetic acid 98:4 (v/v) (fraction 3), 2 ml of acetone:MeOH 9:1.2 (v/v) (fraction 4), 2 ml of CHCl3:MeOH 2:1 (v/v) (fraction 5), 2 ml of 0.2 M NaCOOCH3 in MeOH, and 2 ml of H2O (fraction 6). Fraction 6 was then loaded onto a C18-column (pre-equilibrated with 1 ml of MeOH and 2 ml of MeOH:PBS 1:10 (v/v)), washed with 2 ml of H2O, and then eluted with 2 ml of MeOH and 2 ml of CHCl3:MeOH 1:1 (v/v) (fraction 7). Fraction 4 (neutral glycolipids) and fraction 7 (gangliosides) were combined to form the purified glycolipid extract, whereas fraction 2 contained ceramide.

Quantification of Glycolipids by Ceramide Glycanase Digestion, Anthranilic Acid (2-AA) Labeling, and NP-HPLC—The purified glycolipid extract was digested by ceramide glycanase as previously described (37). The oligosaccharides released by ceramide glycanase were analyzed using the method of Neville et al. (38). Briefly, oligosaccharides were labeled with 2-AA, purified using Discovery DPA-6S columns, separated by normal phase high performance liquid chromatography (NP-HPLC) using a 4.6 x 250-mm TSK gel-Amide 80 column and detected by fluorescence ({lambda}ex = 360 nm; {lambda}em = 425 nm). All chromatography was controlled and data collected and processed using Waters Millennium or Empower software. Glucose unit values were determined, following comparison with a 2-AA-labeled glucose oligomer ladder (derived from a partial hydrolysate of dextran) external standard, using Peak Time software (developed in-house) and identified by comparison to glucose unit values of 2-AA-labeled oligosaccharides prepared from commercially available glycolipids as described above. Glycolipids extracted from the cell types used in this study and purified as described above were also analyzed by HP-TLC with orcinol staining and compared with commercial glycolipid standards to support the identification of glycolipid species made by NP-HPLC (data not shown).

Quantification of Ceramide by Radiolabeling—NCI/AdRRES cells (50% confluent 75-cm2 flasks) were grown for 48 h in the presence of media supplemented with either 2.5 µM PDMP, 25 µM C9DGJ, or 0.02% (v/v) Me2SO (control) and containing 0.5 µCi/ml of [14C]palmitate sonicated in 500 µl of fetal calf serum. After 48 h, the media was replaced with fresh media with or without 60 µM doxorubicin, supplemented with 2.5 µM PDMP, 25 µM C9DGJ, or 0.02% (v/v) Me2SO (control). Cells were grown for a further 1 h or 48 h, harvested and the ceramide extracted and purified as described above (fraction 2). To remove any glyceride esters from this fraction, base hydrolysis was performed using the method of Butters (39) except that the hydrolysis was performed at 57 °C for 1 h. The ceramide fraction was then resuspended in 10 ml of CHCl3:MeOH 2:1 (v/v) and analyzed by HP-TLC using 10 mg of type III ceramides in CHCl3:MeOH 2:1 (v/v) as standard. The plate was first run in a chloroform tank and then in a CHCl3:CH3COOH 9:1 (v/v) tank. Radiolabeled bands were visualized by exposure to a Molecular Dynamics PhosphorScreen for 4 days. Non-radiolabeled ceramide was visualized by spraying the plate with 3% (w/v) Cu(CH3COO)2 in 8% (w/v) H3PO4 and developing at 80 °C for 10 min. The HPTLC plates were scanned using an Agfa Arcus II scanner, and the relative optical densities of ceramide bands were determined using NIH ImageJ.

Cytotoxicity Assays, Toxicity of GCS Inhibitors and P-glycoprotein Inhibitors—Cells were seeded at densities of 500 cells/well in 96-well plates in 200 µl of supplemented media containing either 0.01% Me2SO (control), 2.5 µM PDMP, 25 µM C9DGJ, 100 µM C4DGJ, 2 mM C4DGJ, or 25 nM XR9576. A different column of the 96-well plate was assayed daily for cell viability using the Cell Titer-96 AQueous cellular proliferation assay kit according to the manufacturer's instructions. The media was changed every 72 h, and cells were grown until full growth curves had been obtained.

Cytotoxicity Assays, Reversal of Drug Resistance—Cells were seeded in 96-well plates at densities of 1250 MCF7-wt/well, 625 NCI/AdRRES/well, 1500 MES-SA/well, and 1500 MES-SA/DX-5/well in 200 µl of supplemented media. After overnight incubation, the media replaced with fresh media containing either 0.01% (v/v) Me2SO (control), 2.5 µM PDMP, 25 µM C9DGJ, 100 µM C4DGJ, 2 mM C4DGJ, or 25 nM XR9576 as well as doxorubicin or vinblastine (from 10–10 to 10–4 M). Cells were grown for 4 days and their media replaced with 100 µl of fresh supplemented media. Cellular viability was then assayed using the MTS and PMS reagents supplied with the Cell Titer-96 AQueous cellular proliferation assay kit according to the manufacturer's instructions. The viability was calculated as shown below in Equation 1 and plotted as a function of drug concentration,

(Eq. 1)
where A490 is MTS absorbance at 490 nm of cells in the presence of drug and A490° is MTS absorbance at 490 nm of untreated cells. Cells treated with either C9DGJ or C4DGJ during cytotoxicity assays had been preincubated for 7 days in these compounds to guarantee maximal depletion of glycolipids. A dose-response curve in Equation 2 was fitted to the data to determine the toxicity (IC50 value) of a particular drug,

(Eq. 2)
where Y is viability and [M] = concentration of cytotoxic agent (doxorubicin or vinblastine), IC50 is the concentration of cytotoxic agent that causes 50% cell death, and n is the slope factor (Hill coefficient).

Effect of Glycolipid Depletion on [3H]Vinblastine Accumulation in NCI/AdRRES Cells—NCI/AdRRES cells were grown for 7 days in supplemented media with or without 25 µM C9DGJ or 2 mM C4DGJ. Cells were then seeded in 24-well plates at cell densities of 105 cells/well in 200 µl of supplemented media with or without 25 µM C9DGJ or 2 mM C4DGJ, as appropriate. After overnight incubation, one well from each treatment was counted for cell number. The media from all wells was then replaced with 500 µl of fresh, unsupplemented media containing 0.125 µCi of [3H]vinblastine with or without 25 µM C9DGJ, 2 mM C4DGJ, or 30 µM nicardipine as appropriate. Cells were then incubated at 37 °C, 5% CO2 for 3 h. The media was removed, and cells washed twice in 500 µl of ice-cold phosphate-buffered saline. The contents of each well were solubilized with 250 µl of 2% SDS and transferred to a scintillation vial. Each well was further washed with 250 µl of H2O, also transferred to the same scintillation vial. After addition of 3 ml of scintillation fluid, vials were counted for 3H on a Beckman Scintillation Counter and the amount of [3H]vinblastine accumulated per cell determined.

Data Analysis—All curve fitting and statistical analyses were done using the software Prism 4.0 (from GraphPad). Data are presented as means ± S.E. Differences between groups of data were tested for significance using one-way analysis of variance and Dunnett's post-hoc test to compare data to control values.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
P-glycoprotein and Glycolipid Levels in MDR Cell Lines— Flow cytometry analysis of the cells using the anti-P-glycoprotein I5D3 antibody showed that NCI/AdRRES cells had (92 ± 2) x 104 I5D3 molecules bound per cell, compared with only (1.4 ± 0.4) x 104 I5D3 molecules bound per MCF7-wt cell. The cell line MES-SA/DX-5 had (295 ± 3) x 104 I5D3 molecules bound per cell, with no expression of P-glycoprotein being detectable in the chemosensitive MES-SA cell line.

The glycolipid levels of the various cell lines used in this study were quantified by either TLC (GlcCer) or by ceramide glycanase digestion, 2-AA labeling, and NP-HPLC (all other glycolipids) and the results are summarized in Table I. GlcCer was quantified by TLC because ceramide glycanase is not 100% efficient at digesting this glycolipid and because of the abundance of glucose not derived from glycolipids that always remains in the sample (37). Results are summarized in Table I and are expressed as the percentage of the untreated value in the corresponding MDR cell line. Although we now know NCI/AdRRES cells (formerly MCF7-AdR) are not derived from MCF7-wt cells (40, 41), this pair was compared for consistency with previous publications on this subject. The NCI/AdRRES cell line showed significantly higher levels of GlcCer, LacCer, Gb3, GM3, and significantly lower levels of GM1 than MCF7-wt cells. The MES-SA/Dx-5 cell line showed significantly higher levels of GlcCer, Gb3, and GM2 than the parental, chemosensitive MES-SA cells.


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  		TABLE I
Effect of GCS inhibitors and P-glycoprotein inhibitors on glycolipid levels

Cells were grown for 7 days in media containing either 0.02 % Me2SO (untreated), 25 µM C9DGJ, 2 mM or 100 µM C4DGJ, 2.5 µM PDMP, or 25 nM XR9576. Cellular lipids were then extracted and glycolipids purified. Glucosylceramide was analysed by TLC followed by staining and densitometry. All other glycolipids were digested with ceramide glycanase, labelled with 2AA, and examined by NP-HPLC as described under "Experimental Procedures." Glycolipid species are abbreviated as: GlcCer (glucosylceramide), LacCer (lactosylceramide), Gb3 (globotriosylceramide), GM3 (monosialoganglioside GM3), GM2 (monosialoganglioside GM2) and GM1 (monosialoganglioside GM1). Data are presented as percentatges of the value found in the corresponding untreated MDR cell line. Data represent the mean ± S.E. of at least four independent experiments. All treatments were compared to the untreated MDR cell line using ANOVA with Dunnett's post-test.

 
Treatment of either MDR cell line for 7 days with 25 µM C9DGJ, 2 mM C4DGJ, or 2.5 µM PDMP achieved a similar, and maximal, reduction of glycolipid levels in that cell line. Thus, all three treatments reduced GlcCer levels by at least 33% and LacCer levels by at least 80% in NCI/AdRRES cells, and reduced GM2 levels in MES-SA/DX-5 cells by at least 79%. The reduction in GlcCer in NCI/AdRRES cells achieved by PDMP was similar to that reported previously (12). In contrast, treatment with the P-glycoprotein inhibitor XR9576 had no significant effect on glycolipid levels in either NCI/AdRRES or MES-SA/DX-5 cells. It is important to stress that treatment of all cells with either 25 µM C9DGJ, 2 mM C4DGJ, 2.5 µM PDMP, or 25 nM XR9576 was not toxic given that identical growth curves were obtained for treated and untreated cells (data not shown).

Effect of GCS Inhibitors and P-glycoprotein Inhibitors on Drug Resistance—NCI/AdRRES cells are 40-fold more resistant to doxorubicin and 1095-fold more resistant to vinblastine than MCF7-wt cells. Similarly, MES-SA/DX-5 cells are 7-fold more resistant to doxorubicin and 385-fold more resistant to vinblastine than MES-SA cells (Tables II, III). To study the relative contributions of elevated glycolipid levels and increased P-glycoprotein expression to drug resistance, the toxicity of doxorubicin and vinblastine was assayed using MDR cells that had been treated with either GCS inhibitors or the P-glycoprotein inhibitor XR9576 (42). An example is shown in Fig. 1, demonstrating that cytotoxicity assays were performed over a wide enough range of cytotoxic drug for accurate determination of the IC50 value. P-glycoprotein is a well documented contributor to drug resistance, and thus it was no surprise to find that inhibition of P-glycoprotein with XR9576 completely reversed drug resistance in both NCI/AdRRES and MES-SA/DX-5 cells, to both doxorubicin and vinblastine (Fig. 1, Tables II and III). The P-glycoprotein inhibitor XR9576 had no effect at all on the sensitivity of MCF7-wt or MES-SA cells to either doxorubicin or vinblastine (data not shown).


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  		TABLE II
Effect of various drugs on the cytotoxicity of doxorubicin

Cytotoxicity assays for MCF7-wt and NCI/AdRRES cells were carried out in the presence of 25 µM C9DGJ, 2 mM C4DGJ, 2.5 µM PDMP, and 25 nM XR9576 and the cytotoxicity of doxorubicin determined as described under "Experimental Procedures." MES cells were treated identically except that 100 µM C4DGJ was used. Results represent the mean ± S.E. of the IC50 values obtained from independent cytotoxicity assays. The number of independent experiments (n) is given. Data sets were compared to the untreated MDR values using an a one-way ANOVA test with Dunnett's post-test calculations. The resistance is the mean IC50 of a particular drug treatment divided by the mean IC50 of the untreated MDR cell line times 100.

 


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  		TABLE III
Effect of various drugs on the cytotoxicity of vinblastine

MCF7-wt and NCI/AdRRES cells were pretreated with 25 µM C9DGJ (7 days), 2 mM C4DGJ (7 days), 2.5 µM PDMP (4 days), and 25 nM XR9576 (0 days) and then the cytotoxicity of vinblastine determined in the presence of these compounds as described under "Experimental Procedures." MES cells were treated identically except that 100 µM C4DGJ (7 days) was used. Results represent the mean ± S.E. of the IC50 values obtained from independent cytotoxicity assays. The number of independent experiments (n) is given. Data sets were compared to the untreated MDR values using an a one-way ANOVA test with Dunnett's post-test calculations. The resistance is the mean IC50 of a particular drug treatment divided by the mean IC50 of the untreated MDR cell line times 100.

 



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  		FIG. 1.
Representative example of a cytotoxicity assay. Results are for the effect of the P-glycoprotein inhibitor XR9576 on the cytotoxicity of doxorubicin in NCI/AdRRES cells. Cytotoxicity assays were carried out as described under "Experimental Procedures." Results are represented as ({circ}, thin solid line) MCF7-wt untreated; (, thick solid line) NCI/AdRRES untreated; ({Delta}, dashed line) NCI/AdRRES + 25 nM XR9576. Results for MCF7-wt cells treated with 25 nM XR9576 were superimposable with those of untreated MCF7-wt cells (data not shown). Data represent the mean ± S.E. of at least four independent experiments.

 
Previous reports (8–12) have already shown that GCS inhibitors of the PDMP family are capable of achieving a partial reversal of drug resistance in MDR cells. Our results confirmed this by finding that treatment of NCI/AdRRES cells during the cytotoxicity assay with PDMP at a non-toxic concentration (2.5 µM) reduced the resistance of NCI/AdRRES cells to doxorubicin by 73% and their resistance to vinblastine by 96% (Table II and Table III). Similarly, PDMP reduced the resistance of MES-SA/DX-5 cells to vinblastine by 99%. However, PDMP failed to produce a statistically significant reversal of resistance of MES-SA/DX-5 cells to doxorubicin (Table II and Table III). Previous reports conclude that the mechanism of PDMP mediated reversal of drug resistance in these cells is inhibition of glucosylceramide synthase (GCS) (8–12). If so, one would expect other inhibitors of this enzyme such as N-alkylated iminosugars to similarly reverse drug resistance.

However, treatment of the MDR cells with either C9DGJ or C4DGJ failed to achieve any significant reversal of drug resistance in either NCI/AdRRES or MES-SA/DX-5 cells, to either doxorubicin or vinblastine (Tables II and III). This is despite the fact that C9DGJ and C4DGJ achieved a statistically similar reduction in glycolipid levels to PDMP (Table I). Furthermore, to rule out the possibility that the N-alkylated iminosugars were taking longer than PDMP to inhibit GCS, cells treated with C9DGJ or C4DGJ during the cytotoxicity assays had been preincubated for 7 days in these compounds to ensure maximal depletion of glycolipids. We even tried using C4DGJ in NCI/AdRRES cells at the incredibly high (but non-toxic) concentration of 2 mM, and yet no significant effect on drug resistance was observed.

The P-glycoprotein inhibitor XR9576 could still reverse drug resistance fully in the presence of either C9DGJ or C4DGJ, ruling out the unlikely scenario that the N-alkylated iminosugars had introduced a mechanism of drug resistance of their own. This data suggests that the sensitization of MDR cells achieved by PDMP cannot be explained by inhibition of the enzyme glucosylceramide synthase alone.

Effect of Glycolipid Depletion on [3H]Vinblastine Accumulation in NCI/AdRRES Cells—It is possible that the N-alkylated iminosugars are causing a membrane alteration leading to a P-glycoprotein independent decrease in drug accumulation because of their chaotropic, amphiphilic properties. Therefore, we examined whether treatment with N-alkylated iminosugars altered the accumulation of [3H]vinblastine in NCI/AdRRES cells. Cells that had been grown for 7 days in either 25 µM C9DGJ or 2 mM C4DGJ, to reduce glycolipid levels, were incubated for 3 h in the presence of 100 nM [3H]vinblastine and 25 µM C9DGJ, 2 mM C4DGJ, or 30 µM nicardipine (a P-glycoprotein inhibitor). Untreated NCI/AdRRES cells accumulated 9.9 ± 1.8 pmol of [3H]vinblastine/106 cells whereas untreated MCF7-wt cells accumulated 23.8 ± 7.3 pmol of [3H]vinblastine/106 cells. Treatment of NCI/AdRRES cells with the P-glycoprotein inhibitor nicardipine caused the accumulation of vinblastine to rise to 21.4 ± 3.2 pmol of [3H]vinblastine/106 cells, an accumulation comparable to that seen in MCF7-wt cells. By contrast, treatment of NCI/AdRRES cells with 25 µM C9DGJ or 2 mM C4DGJ did not change vinblastine accumulation, which remained at 7.9 ± 2.1 pmol [3H]vinblastine/106 cells and 10.3 ± 0.3 pmol [3H]vinblastine/106 cells, respectively. These results, summarized in Fig. 2, confirm that treatment with N-alkylated iminosugars does not lead to a decreased accumulation of cytotoxic drugs in cells.



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  		FIG. 2.
Effect of glycolipid depletion on the accumulation of [3H]vinblastine in NCI/AdRRES cells. Cells were treated for 7 days with 25 µM C9DGJ or 2 mM C4DGJ and the accumulation of [3H]vinblastine in these cells measured as described under "Experimental Procedures." As a positive control, cells were incubated with 30 µM nicardipine during the assay. Results for NCI/AdRRES cells are shown in gray bars. Results for MCF7-wt cells are shown in the white bar. Data represent the mean ± S.E. of at least three independent experiments. Statistical significance compared with untreated NCI/AdRRES cells is shown as (*) with p < 0.05.

 
Effect of GCS Inhibitors on Ceramide Levels in MDR Cells—In an attempt to explain why PDMP may be able to reverse drug resistance when N-alkylated iminosugars cannot, we examined if PDMP by itself was causing a greater elevation in ceramide than the N-alkylated iminosugars, as this could predispose cells to apoptosis. NCI-AdRRES cells treated for 72 h with 2.5 µM PDMP had 74% higher ceramide (p < 0.01) than untreated cells, and cells treated for 72 h with 25 µM C9DGJ had 76% higher ceramide levels (p < 0.01) than untreated cells, despite the fact that neither drug was toxic at these concentrations. Cells treated for 72 h with 2 mM C4DGJ did not have significantly higher ceramide levels than untreated cells. Thus, the different effect that PDMP and N-alkylated iminosugars have on multidrug resistance is not caused by their effect on ceramide levels prior to exposure to cytotoxic agents, given that both C9DGJ and PDMP caused similar rises in ceramide and yet only PDMP had any effect on multidrug resistance.

To determine whether PDMP or N-alkylated iminosugars altered drug induced increases in ceramide, NCI/AdRRES cells that had been pretreated for 72 h with either 2.5 µM PDMP or 25 µM C9DGJ were incubated with doxorubicin. Surprisingly, we found that treatment of NCI/AdRRES cells with 60 µM doxorubicin alone (a concentration which is 10 times greater than the IC50 of doxorubicin in these cells and is thus considerably toxic, see Fig. 1) for 1 or 48 h did not cause any significant rise in ceramide levels. This agrees with recent reports that also used NCI/AdRRES cells and doxorubicin (43), but contradicts other reports suggesting that ceramide levels should rise following exposure to cytotoxic agents (14, 16). It may be possible to reconcile these differences if the rise in ceramide was transient and occurred before or after our time points, as suggested by other authors (15). After 48 h treatment with doxorubicin, ceramide levels remained unchanged in untreated cells, but were 56% higher (p < 0.05) in cells pretreated with C9DGJ. Ceramide levels were 123% higher (p < 0.01) in cells pretreated with PDMP, which is significantly different (p < 0.05) from cells pretreated with C9DGJ. It is tempting to suggest that the greater rise in ceramide upon exposure to doxorubicin observed in cells pretreated with PDMP compared with cells pretreated with C9DGJ may explain why PDMP alone has an effect on multidrug resistance.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
A role for GCS in multidrug resistance has been suggested in numerous reviews. The hypothesis suggests that an elevated GCS activity in cancer cells can prevent the accumulation of ceramide, which is thought to precede, and trigger, apoptosis in response to some cytotoxic drugs. This hypothesis rests on three lines of evidence: 1) A correlation between elevated GlcCer levels and the multidrug resistance phenotype (3, 4). 2) The observation that genetic manipulation of GCS levels in cells can affect their resistance to various cytotoxic drugs (5–7,43). 3) The observation that GCS inhibitors of the PDMP family can reverse drug resistance in some multidrug-resistant cell lines (8–12).Go


View this table:
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  		TABLE IV
Ceramide levels in NCI/AdRRES cells treated with various drugs

Cells were radiolabelled with [14C]palmitate for 48 h and then treated with various compounds. Ceramides were extracted, purified, and resolved by thin layer chromatography before being exposed to a phosphorimager plate. Ceramide levels are shown as the percent increase in ceramide levels with respect to untreated NCI/AdRRES cells. Results represent the mean ± S.E. of at least three independent experiments. Statistical significance was obtained from comparing results to the "untreated" values using an ANOVA test with Dunnett's post-hoc analysis.

 
Unfortunately, there are problems with all three lines of evidence. First, elevated levels of the glycolipid GlcCer have only been reported in a small number of multidrug-resistant cancer cell lines or clinical specimens (3, 4), and the relationship between glycolipid levels and cancers (both general and drug resistant) is yet to be established.

Secondly, Liu et al. (5–7, 43) have presented data showing that drug sensitive MCF7-wt cells transfected to overexpress glucosylceramide synthase (GCS) were more resistant to drugs than untransfected cells, and that multidrug resistant NCI/AdRRES cells transfected with either antisense GCS cDNA or antisense GCS oligonucleotides were less resistant to drugs than untransfected NCI/AdRRES cells. However, transfection of Jurkat cells or GM95 cells with plasmids expressing GCS had no effect on their sensitivity to cytotoxic drugs (22, 23). Furthermore, the antisense oligonucleotides used by Liu et al. were pro-apoptotic and anti-proliferative by themselves in NCI/AdRRES cells despite only causing a 25% reduction in GlcCer levels (43). This conflicts with our data showing that NCI/AdRRES are perfectly viable in the presence of various GCS inhibitors, which achieve greater reductions in glycolipid levels.

Third, various authors have shown that GCS inhibitors of the PDMP family can achieve reversal of drug resistance in different types of multidrug-resistant cancer cells (8–12). These authors concluded that the reversal of drug resistance achieved by PDMP and its analogues was due to inhibition of the enzyme glucosylceramide synthase. However, PDMP has been reported to inhibit other enzymes involved in glycolipid metabolism (20, 25, 26), cause cell cycle arrest in cells (27) and even protect cells against some cytotoxic agents (28). For this reason, we decided to evaluate the effect of a different class of GCS inhibitors on multidrug resistance. We chose to use N-alkylated iminosugars because they are better tolerated than PDMP and its analogues (25, 26), and we specifically used N-alkyl-deoxygalactonojirimycin (N-alkyl-DGJ) compounds because they more selective inhibitors of GCS than the older N-alkylated-deoxynojirimycin (N-alkyl-DNJ) compounds (31). Veldman et al. (23) have already reported that treatment of B16 melanoma cells with the GCS inhibitor C4DNJ does not make these cells more sensitive to cytotoxic drugs. However, this may not be surprising given that B16 melanoma cells are not resistant to cytotoxic drugs in the first place. After all, the P-glycoprotein inhibitor XR9576 is a potent chemosensitizer in NCI/AdRRES cells but not in MCF7-wt cells, because the latter are not drug resistant. Thus, we chose to examine the effect of two N-alkyl-DGJ compounds on the resistance of the two well established, multidrug-resistant cell lines NCI/AdRRES (32, 40) and MES-SA/DX-5 (44, 45).

We confirmed that PDMP was able to achieve a significant reversal of drug resistance to doxorubicin and vinblastine in NCI/AdRRES cells and to vinblastine in MES-SA/DX-5 cells. However, we found that neither of the N-alkylated iminosugars used (C9DGJ and C4DGJ) had any effect on the resistance to either doxorubicin or vinblastine, in either NCI/AdRRES or MES-SA/DX-5 cells. Analysis of glycolipid levels in these cells confirmed that PDMP, C9DGJ, and C4DGJ were all achieving a similar (and maximal) inhibition of glycolipid biosynthesis at the concentrations that were used. N-Alkylated iminosugars are not introducing resistance by themselves because the P-glycoprotein inhibitor XR9576 (42) was still able to reverse drug resistance completely in the presence of both C9DGJ and C4DGJ. Furthermore, treatment of cells with C9DGJ or C4DGJ caused no difference in the accumulation of [3H]vinblastine.

Our data also show that inhibition of P-glycoprotein with XR9576 in NCI/AdRRES and MES-SA/Dx5 cells can completely reverse their resistance to doxorubicin and vinblastine. This contradicts a recent study (43) showing that the P-glycoprotein inhibitors verapamil and cyclosporin A were unable to reverse the resistance of NCI/AdRRES cells to doxorubicin, but the concentration of verapamil and cyclosporin A used in that study (1 µM) was too low to cause any significant inhibition of P-glycoprotein (46, 47). The lack of any residual resistance in the presence of XR9576 argues against any contribution of increased GCS activity to multidrug resistance in these cells. We confirmed that XR9576 has no effect on glycolipid levels in NCI/AdRRES nor MES-SA/Dx5 cells. This contradicts previous reports showing that P-glycoprotein inhibitors can affect glycolipid levels in cells (48) and suggests that the contribution of P-glycoprotein to glycolipid levels may be cell type-specific or that the inhibitors used in those studies may also be inhibiting GCS (8).

Our data suggest that the effect of PDMP on multidrug resistance cannot be attributed to inhibition of GCS alone. It is possible that PDMP is also directly affecting P-glycoprotein activity. However, previous reports suggest this is not the case (10), as does our own data.3 Another possibility is that PDMP per se is causing a rise in ceramide levels which predisposes cells to apoptosis, something observed by previous authors (20, 25–27, 49). However, we found that at the non-toxic concentrations used in these studies, both PDMP and C9DGJ by themselves caused similar increases in ceramide levels, despite only PDMP having any effect on drug resistance. We did find that ceramide levels after 48 h exposure to 60 µM doxorubicin were significantly higher in cells that had been pretreated with PDMP than those pretreated with C9DGJ and it is possible that this difference holds they key to explaining why PDMP can sensitize multidrug-resistant cancer cells.

In summary, numerous investigators have suggested that inhibition of glucosylceramide synthase is a promising strategy for reversing multidrug resistance. However, there is a lack of data showing a clinically relevant correlation between glycolipid levels and multidrug resistance, and our results presented here suggest that reversal of drug resistance by PDMP and its analogues is not due to inhibition of glucosylceramide synthase alone. We conclude that inhibition of glucosylceramide synthase alone does not reverse multidrug resistance in cancer cells.


   FOOTNOTES
 
* This research was funded by the Medical Research Council (UK), Cancer Research (UK), and Oxford GlycoSciences PLC (UK). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

To whom correspondence should be addressed. Tel.: 44-0-1865-275725; E-mail: terry.butters{at}bioch.ox.ac.uk.

1 The abbreviations used are: MDR, multidrug resistance; GCS, glucosylceramide synthase; PDMP, D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol; C9DGJ, N-nonyl-deoxygalactonojirimycin; C4DGJ, N-butyl-deoxygalactonorijimycin; DGJ, deoxygalactonojirimycin; DNJ, deoxynojirimycin; 2-AA, anthranilic acid; GlcCer, glucosylceramide; LacCer, lactosylceramide; Gb3, globotriaosylceramide, ceramide trihexoside; Gb4, globotetraosylceramide, globoside; GM1, monosialoganglioside GM1; GM2, monosialoganglioside GM2; GM3, monosialoganglioside GM3; FITC, fluorescein isothiocyanate. Back

2 Dr. Iuliana Popa, Laboratoire de Dermatologie, Hopital Edouard Herriot, 69003 Lyon, France, personal communication. Back

3 E. Norris-Cervetto, R. Callaghan, F. M. Platt, R. A. Dwek, and T. D. Butters, unpublished results. Back


   ACKNOWLEDGMENTS
 
We thank D. C. A. Neville for developing the HPLC method, I. Popa for developing the glycolipid purification method, C. A. Martin for help with the cytotoxicity assays, and G. Reinkensmeier for her assistance with the flow cytometry. We also thank H. R. Mellor, S. Modok, A. J. Rothnie, J. Storm, and P. T. O. Amo for their critical reading of the manuscript. The FACSCalibur instrument was purchased with a grant from The Wellcome Trust (UK), Prism 4.0 was a kind gift from GraphPad, and XR9576 was a kind gift from Xenova Ltd.



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