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Experimental Therapeutics, Preclinical Pharmacology |
Groningen Utrecht Institute for Drug Exploration, Department of Pediatric Oncology, Beatrix Children’s Hospital, [H. S., W. K.] and Department of Hematology [D. v. d. K., E. V.] University Hospital Groningen, 9713 GZ Groningen, and Department of Physiological Chemistry, University of Groningen [H. S., R. J. V., B. A., W. N., J. W. K.], 9713 AV Groningen, the Netherlands
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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PDMP is a well-known inhibitor of Cer glycosylation, resulting in decreased GlcCer levels and often an accumulation of Cer (14) . In addition, PDMP has been shown to inhibit protein transport through the Golgi complex and from the Golgi to the plasma membrane (15) , possibly as a result of elevated levels of Cer (16) . Recently, we have shown that PDMP blocks membrane transport in the endoplastic reticulum /Golgi system, not through its effects on sphingolipid metabolism, but rather by modulation of calcium homeostasis. Interestingly, this effect of PDMP could be mimicked by the MRP1 inhibitor MK571 (17) . These results indicate that disruption of intracellular membrane transport may contribute to decreased drug resistance in MDR cells.
In addition to decreasing levels of GlcCer, long-term PDMP treatment results in the reduction of complex glycolipids, including gangliosides. Neuroblastoma cells contain relatively high amounts of these sialic acid-containing glycosphingolipids (18) . Recently, we have shown that gangliosides shed by these and other tumor cells have inhibitory effects on the hemopoietic system (19 , 20) , whereby blocking the biosynthesis of gangliosides and their shedding with PDMP results in abrogation of their inhibitory effects on hemopoiesis (19) . The present study was aimed to establish PDMP also as a direct reducer of neuroblastoma cell survival. We show that PDMP highly sensitizes neuroblastoma cells to treatment with microtubule-affecting cytostatics and provides evidence for the involvement of sustained Cer accumulation and possibly increased drug accumulation in this process.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Measurement of Cellular Sensitivity to Cytotoxic Drugs.
Drug sensitivities of the Neuro-2a and C1300 cells were determined with
the tetrazolium salt WST-1 test (Boehringer Mannheim, Almere,
the Netherlands). Neuro-2a cells (1000/well) or C1300 (5000/well) were
seeded into 96-microtiter plates (six replicate) and incubated for
24 h to adhere. Thereafter, cytotoxic drugs
(Taxol/vincristine/VP16) in various concentrations, either with or
without the inhibitors MK571 (a kind gift from Professor A.W.
Ford-Hutchinson; Merck-Frosst Inc., Kirkland, Canada) or PSC833
(provided by Novartis Pharma Inc., Basel, Switzerland) and either with
or without PDMP (Matreya Inc., Pleasant Gap, PA), were added to a total
volume of 100 µl. After 72 h of incubation, 10 µl of WST-1 was
added to each well. WST-1 tetrazolium salt is cleaved to formazan by
the succinate-tetrazolium reductase system, which belongs to the
respiratory chain of the mitochondria, and this only occurs in viable
cells. The formazan dye was quantified using an ELISA reader (450 nm).
MRP1 and P-gp Expression Measured by Flow Cytometry.
Cells (0.5 x 106) were permeabilized in
10% (v/v) lysing solution (Becton Dickinson Medical Systems, Sharon,
MA) in H2O and incubated for 15 min in PBS/BSA
(2%) containing 1% normal goat serum. The cells were incubated for
1 h at 4°C in 200 µl PBS/BSA (2%) with the monoclonal
antibody UIC2 (1 µg/ml; IgG2; Immunotech, Mijdrecht, the Netherlands)
or MRPm6 (10 µg/ml; IgG1; kindly donated by Professor R. J.
Scheper, Amsterdam, the Netherlands) or the mouse isotype-matched
control monoclonal antibodies. Antibody binding was detected with
R-Phycoerythrin-labeled rabbit antimouse immunoglobulins (Dako,
Glostrup, Denmark). Fluorescence was measured by flow cytometry
(FACStar, Becton Dickinson Medical Systems, Sharon, MA). For each
sample, 10,000 events were collected. Protein expressions are given as
the ratio of mean fluorescence of UIC2/IgG2 control or MRPm6/IgG1
control.
Flow Cytometric Detection of P-gp and MRP1-mediated Drug Efflux.
The efflux by tumor cells of Rho123 (Sigma Chemical Co., St. Louis, MO)
or CF, in the absence or presence of the P-gp inhibitor PSC833 or the
MRP1 inhibitor MK571, respectively, was measured flow cytometrically,
as previously described (21)
. Neuro-2a or C1300 cells
(1 x 106) were incubated for 20 min at
37°C in RPMI 1640 medium, containing either 200 ng/ml Rho123 or 0.1
µM CFDA (Sigma), with or without inhibitor (2 µg/ml
PSC833 or 20 µM MK571, respectively). Cells were washed
with ice-cold medium, resuspended in medium with or without inhibitor,
and incubated for 60 min at 37°C (efflux). Efflux was stopped by the
substitution with ice-cold medium, followed by the measurement of
Rho123/CF fluorescence by flow cytometry (FACStar, Becton Dickinson
Medical Systems, Sharon, MA). The Rho123/CF fluorescence of 10,000
events was logarithmically measured at a wavelength of 488 nm through a
530-nm band-pass filter. These signals were converted into linear
values and expressed as relative fluorescence units (rel. F.U.) using
win-list 2.0 software (Verity Software House Inc. Topsham, ME). Values
from cells incubated with Rho123/CFDA only, i.e., without
inhibitors, served as baseline Rho123/CFDA uptake. The BF was defined
as follows:
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[14C]Taxol or [3H]vincristine Efflux
Studies.
Neuro-2a cells were incubated with 3 ml of Hanks’ solution, containing
either 0.01 µCi [14C]Taxol (44.5 mCi/mmol;
Sigma Chemical Co., St. Louis, MO) or 1 µCi
[3H]vincristine (5.7 Ci/mmol; Amersham,
Buckinghamshire, England) for 20 min at 37°C, followed by washing
with ice-cold Hanks’ solution. Thereafter, the cells were incubated
for 60 min at 37°C in the absence (control) or presence of PDMP (5
µM), PSC833 (2 µg/ml), MK571 (20 µM), or
combinations of these drugs. Cells and incubation medium were then
separated, and the radioactive content was measured in a scintillation
counter. The efflux was expressed as a percentage and defined as
(radioactivity in efflux medium)/total radioactivity x 100%.
Radiolabeling and Analysis of Cellular Cer Levels.
The total pool of sphingolipids was radiolabeled by growing the cells
in the presence of 3 µCi/ml
[3H]L-serine (30 Ci/mmol;
Amersham), a precursor for sphingolipid biosynthesis. After 48 h
of incubation, the medium was removed, and the cells were fixed in
ice-cold CH3OH, followed by lipid extraction from
the cells (23)
. Aliquots of the lipid extracts were taken
for the determination of the total amount of lipid-incorporated
radioactivity. Acylglycerolipids were hydrolyzed during 1 h of
incubation at 37°C in
CHCl3/CH3OH (1:1,
v/v) containing 0.1 M KOH. The remaining lipids were
re-extracted and applied on high performance thin-layer chromatography
plates. Plates were developed in
CHCl3/CH3OH/H2O
(14:6:1, v/v) in the first dimension and in
CHCl3/CH3COOH (9:1,
v/v) in the second dimension to resolve Cer. Cer-containing
spots were scraped and subjected to scintillation counting. The effects
of cytotoxic agents and PDMP on Cer levels were determined by growing
the cells, after overnight adherence, for 4 days in medium containing
[3H]serine. Drugs were present for the entire
4-day period or only the final 6 h before the end of the
incubation. Treatment with 0.1 units/ml bacterial sphingomyelinase
(Sigma) for the final 2 h of the incubation served as positive
control (maximal Cer formation from sphingomyelin; see Ref.
23
).
Statistical Analysis.
The unpaired two tailed Student’s t test was used for
statistical analysis. Values of P < 0.05 were
considered to indicate statistically significant differences between
data sets.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. The IC50 value
for Taxol was well above 100 nM, but shifted to 46.4 and
22.4 nM in the presence of 2.5 and 5 µM PDMP,
respectively.
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). Cell survival decreased
from 100% (±9; n > 3) to 32% (±5;
n > 3) with the addition of 5
µM PDMP at a low dose of vincristine (5 ng/ml).
The IC50 value for vincristine decreased from
21.3 to 3.7 ng/ml at 5 µM PDMP. In contrast to
Taxol and vincristine, PDMP had no additional effect on VP16-induced
cytotoxicity of Neuro-2a cells. The IC50 value
for VP16 was 260 ng/ml in both VP16- and VP16/PDMP-treated cells (Fig. 2B
).
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33% when PDMP was present (Table 1)
. No synergistic effect of PDMP in
combination with either Taxol or VP16 on the Cer level was observed. On
the other hand, long-term Taxol treatment resulted in a marginally
significant increase of 22% (P = 0.058) in Cer levels,
whereas it decreased by 27% in the case of VP16. Interestingly,
although PDMP was ineffective alone, when it was in combination with
Taxol, a synergistic effect was observed on increasing Cer
levels. In contrast, PDMP did not affect the reduction of Cer
levels as a consequence of VP16 treatment. Thus, the chemosensitizing
effect of PDMP on Taxol cytotoxicity correlated with a synergistic
effect on increasing Cer levels. In the case of VP16, neither
chemosensitization nor Cer increase was observed after PDMP
cotreatment.
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with a cell survival of 37% (± 5; n > 3) at a
concentration of 50 nM Taxol, whereas Neuro-2a
cells remained insensitive at this concentration (cell survival of 95%
± 5%; n > 3; see Fig. 1
). Still, PDMP acted as a
chemosensitizer for Taxol cytotoxicity in C1300 cells. The
IC50 value for Taxol decreased from 43.3 to 16.7
nM at 5 µM PDMP (Fig. 3)
.
Similar to Neuro-2a cells, a synergistic effect of PDMP on vincristine
cytotoxicity was observed in C1300 cells, whereas PDMP again had no
effect on VP16 cytotoxicity (data not shown).
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. Rho123/CFDA efflux studies showed
that functional activities of P-gp and MRP were low as well based on
the low BF values in both Neuro-2a and C1300 cells as compared to
2780AD (BF = 93 ± 10.8) and GLC4/ADR cells (BF =
45.9 ± 5.8; Table 3
).
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The efflux of [14C]Taxol was reduced by all
compounds, with PSC833 being most effective. Similar results were
obtained with [3H]vincristine, although in this
case, MK571 was most effective. Additive effects of PDMP and MDR
inhibitors were not observed.
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. There is a
clear relation between Cer levels and cell survival. Increased Cer
levels were measured with the use of the diglyceride kinase assay. A
10-fold increase compared to the control was found in the combined
treatment of Taxol, PDMP, and PSC833, whereas the combination of Taxol
and PSC833 resulted in a 7-fold increase (data not shown).
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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PDMP, an inhibitor of Cer glucosylation, is known to result in the accumulation of Cer (14) , at least in the initial period following treatment. At longer term, cells are capable of removing excess Cer through diverse metabolic pathways (23 , 26) . Cer is a well-known mediator of programmed cell death (24 , 25) . Daunorubicin-induced cell death has been shown to be mediated by Cer generated either through sphingomyelin hydrolysis (11) or by de novo biosynthesis (10) . Recently, Myrick et al. (27) demonstrated a potentiation of Taxol-mediated apoptosis in leukemic cells by Cer. Therefore, we investigated whether Cer accumulation occurred only under those conditions resulting in the negative synergistic effect of PDMP on cell survival. Our results show that PDMP elevates Cer levels after relatively short-term (6 h) incubations, but Cer levels have returned to normal after long-term (4 days) treatment. The short-term increase in Cer levels also occurs in the presence of VP16 (as well as Taxol). Clearly, if Cer accumulation is involved in the synergistic negative effect of combined treatment with PDMP/Taxol on cell survival, a short-term increase in the level of this sphingolipid is not sufficient. Interestingly, Neuro-2a cells treated with both PDMP and Taxol showed a significant long-term accumulation of Cer. On the other hand, long-term VP16 treatment did not raise the level of Cer, but rather caused a decrease, which was similar in the absence or presence of PDMP. These results are in agreement with the lack of synergistic effects on cell survival with PDMP/VP16 in contrast to PDMP/Taxol. Taken together, short-term treatment with PDMP results in a Cer excess, which upon metabolic removal does not result in long-term loss of cell viability. However, in the case of cotreatment with the microtubule-affecting agent, Taxol, the decrease in cell survival correlated with long-term Cer accumulation.
PDMP also inhibits the formation of gangliosides in neuroblastoma cells (19) . Gangliosides are implicated in the regulation of proliferation, differentiation, tumor progression, immunosuppression, and MDR (28, 29, 30) . Although heterogeneity in ganglioside composition in drug-resistant and drug-sensitive cells has been reported, no direct correlation with drug resistance has been demonstrated (31) . We consider the involvement of specific gangliosides in chemosensitization of neuroblastoma cells by PDMP unlikely for two reasons. First, the ganglioside composition of Neuro-2a and C1300 cells is very different (19) , and second, both cells are susceptible to PDMP-enhanced Taxol cytotoxicity.
In addition to a role for long-term Cer accumulation, the
chemosensitizing effect of PDMP could (partly) be mediated by increased
accumulation of the cytotoxic drugs. This effect could result from
PDMP-inhibited drug efflux mediated by the MDR proteins P-gp and/or
MRP. Flow-cytometric assays employing a fluorescent P-gp or MRP
substrate revealed that the functional activity of these transporters
was low in Neuro-2a/C1300 cells as compared to cell lines abundantly
expressing P-gp (2780AD) or MRP1 (GLC4/ADR). This argues against a
major impact on cell survival of inhibition of the activity of these
transporters. However, PDMP did appear to cause some inhibition of
[14C]Taxol and
[3H]vincristine efflux, similar to the P-gp and
MRP inhibitors PSC833 and MK571, respectively. Yet, we consider the
contribution of such an effect to the observed long-term synergistic
action of PDMP on cell survival limited for the following reasons:
(a) If PDMP treatment resulted in a significant increase in
the intracellular concentration of Taxol by modulation of MDR activity,
it would also be expected to occur for VP16 because removal of
structurally unrelated compounds is a characteristic of MDR proteins;
(b) PDMP did not have an additional effect on
[14C]Taxol efflux in PSC833-treated cells
(Table 4)
. However, under similar conditions, PDMP further reduced cell
survival (Fig. 4)
.This decreased cell survival correlated with
increased Cer levels (data not shown).
In summary, we have obtained evidence for a chemosensitizing effect of PDMP on neuroblastoma cells treated with the microtubule-affecting antimitotic agents, Taxol and vincristine. Although a contribution of drug efflux inhibition by PDMP cannot be excluded, this synergistic effect correlates with long-term Cer accumulation. After an initial rise of Cer (6 h) in PDMP-treated cells, the levels return to normal at the long term (4 days). This most likely involves metabolism of Cer to other sphingolipids, such as galactosylceramide and sphingomyelin, and possibly breakdown via sphingosine. However, when microtubules were stabilized by Taxol or disrupted by vincristine, Cer levels remained elevated. This suggests that due to blocking of transport processes in the cell, which require dynamic microtubular function, Cer is less efficiently metabolized. In the case of VP16, which reduces cell viability by an entirely different mechanism, long-term Cer accumulation does not occur even after cotreatment with PDMP. Thus, the present study provides evidence for a chemosensitizing effect of PDMP in neuroblastoma cells and opens new perspectives in improving the therapeutic efficacy of cytotoxic agents against malignancies. In fact, PDMP exerts a dual effect: In addition to strongly reducing the viability of Taxol/vincristine-treated neuroblastoma cells, it diminishes the adverse effects of neuroblastoma-derived gangliosides on hemopoiesis by inhibiting ganglioside biosynthesis and shedding.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported by a grant from the Groningen
Foundation of Pediatric Oncology (KOCG 94–03). Jan Willem Kok was
supported by a fellowship of the Royal Netherlands Academy of Arts and
Sciences. 
2 To whom requests for reprints should be
addressed, at Department of Physiological Chemistry, Faculty of Medical
Sciences, University of Groningen, A. Deusinglaan 1, 9713 AV Groningen,
the Netherlands. Phone: 31-50-3632725/3632743; Fax: 31-50-3632728;
E-mail: j.w.kok{at}med.rug.nl
3 The abbreviations used are: MDR, multidrug
resistance; BF, efflux-blocking factor; Cer, ceramide; GlcCer,
glucosylceramide; CF, 5-carboxyfluorescein; CFDA, CF diacetate;
MRP, multidrug resistance protein; PDMP, D,
L-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol;
P-gp, P-glycoprotein; Rho123, rhodamine 123.
Received 7/ 9/99; revised 12/ 6/99; accepted 12/ 6/99.
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REFERENCES |
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, 1
µM; •, 2.5 µM; 
, 5 µM;
, 10 µM). Cell survival was determined with a cell
proliferation assay (WST-1 test), as described in "Materials and
Methods". Data represents the mean ± SD of more than three
experiments, each consisting of six replicate determinations.
