(Received for publication, June 6, 1995; and in revised form, August 24, 1995)
From the
Safingol is a lysosphingolipid protein kinase C (PKC) inhibitor
that competitively interacts at the regulatory phorbol binding domain
of PKC. We investigated the effects of safingol on antineoplastic drug
sensitivity and PKC activity of MCF-7 tumor cell lines. Safingol
treatment of P-labeled MCF-7 WT and MCF-7 DOX
cells inhibited phosphorylation of the myristoylated alanine-rich
protein kinase C substrate in both cell lines, suggesting inhibition of
cellular PKC. However, only in MCF-7 DOX
cells did safingol
treatment increase accumulation of [
H]vinblastine
and enhance toxicity of Vinca alkaloids and anthracyclines.
Drug accumulation changes in MCF-7 DOX
cells treated with
safingol were accompanied by inhibition of basal and phorbol
12,13-dibutyrate-stimulated phosphorylation of P-glycoprotein (P-gp).
Expression of P-gp and levels of mdr1 message in MCF-7
DOX
cells were not altered by safingol treatment alone or
in combination with vinblastine. Treatment of MCF-7 DOX
cell membranes with safingol did not inhibit
[
H]vinblastine binding or
[
H]azidopine photoaffinity labeling of P-gp.
Furthermore, safingol did not stimulate P-gp ATPase activity in
membranes prepared from MCF-7 DOX
cells. We conclude that
enhanced drug accumulation and sensitivity in MCF-7 DOX
cells treated with safingol are correlated with inhibition of PKC
rather than competitive interference with P-gp drug binding through
direct interaction with P-glycoprotein.
Exposure of cancer cells to a single natural product
antineoplastic drug can give rise to cells that also exhibit
cross-resistance to other unrelated natural cytotoxins. This phenotype
is known as multidrug resistance and is characterized by the reduced
intracellular accumulation of these drugs, due to drug efflux by
P-glycoprotein (P-gp), ()a 150-180-kDa plasma membrane
transport ATPase encoded by the human mdr1 gene (reviewed in (1) and (2) )). A number of agents have been shown to
inhibit multidrug resistance(3) . Most modulators of multidrug
resistance inhibit specific binding of drugs to P-gp in membranes
derived from MDR cells (4, 5, 6, 7) and photoaffinity
labeling of P-gp by photoactive drug
analogs(4, 8, 9) . These results suggest that
competitive inhibition of P-gp drug binding is a mechanism by which
P-gp function and multidrug resistance may be inhibited.
P-gp is subject to phosphorylation (10, 11) and changes in P-gp phosphorylation have been associated with inhibition of P-gp drug transport by modulators of the MDR phenotype(12, 13) . Although other kinases have been shown to phosphorylate P-gp in vitro(11, 14) , there is considerable experimental evidence that suggests that phosphorylation of P-gp by protein kinase C (PKC) may play a role in regulating multidrug resistance.
PKC is a family of serine/threonine kinases, composed of
calcium-dependent and calcium-independent isotypes, which are dependent
upon phospholipid for activation(15) . In vitro, P-gp
is phosphorylated by PKC (13, 16) at serine residues (13, 17) primarily in the linker region of
P-gp(18, 19) . Increases in PKC activity have been
noted in many MDR cell lines (20, 21, 22) and
in some instances have been associated with changes in the expression
of specific PKC isotypes(23, 24, 25) .
Treatment of MDR cells with PKC activators has been reported to
increase phosphorylation of the P-gp (13, 17, 25, 26) and enhance P-gp
function(12, 20, 24, 26, 27) .
Conversely, treatment of MDR cells with PKC inhibitors has been shown
to decrease drug transport activity and phosphorylation of
P-gp(13, 26, 28) . Most PKC inhibitors that
modulate P-gp function have also been reported to inhibit labeling of
P-gp by the photoactive calcium channel blocker
[H]azidopine, suggesting that these compounds
also competitively inhibit drug binding to
P-gp(26, 29, 30) . In studies that use PKC
inhibitors that also inhibit drug binding to P-gp, the following
question is raised: Does inhibition of P-glycoprotein function by PKC
inhibitors bound by P-gp result from inhibition of drug binding or from
inhibition of P-glycoprotein phosphorylation by PKC?
Recently, the
calcium channel blocker SR33557 was shown to increase cellular levels
of sphingosine leading to inhibition of protein kinase C and drug
resistance(31) . SR33557 did not inhibit P-gp drug binding or
photolabeling of P-gp by [H]azidopine, and the
photoactive analog [
H]SR33557 did not photolabel
P-gp. These results suggested that SR33557 did not directly interact
with P-gp and also raised the possibility that lysosphingolipids could
modulate MDR without competing for drug binding to P-gp(31) .
The PKC inhibitor safingol is a saturated analog of sphingosine, the
naturally occurring lysosphingolipid. PKC inhibition by
lysosphingolipids results from competitive interaction with the
regulatory diacylglycerol/phorbol binding domain of PKC(32) .
Lysosphingolipids have been shown to inhibit growth (33) and
differentiation of cancer cells(34, 35) , gastric
cancer cell invasion (36) , and tumor promotion (37) by
inhibiting PKC activity. In the present study we have investigated
effects of safingol on phosphorylation of the myristoylated
alanine-rich protein kinase C substrate (MARCKS), a prominent cellular
substrate of PKC, to demonstrate inhibition of PKC activity by safingol
in drug-sensitive and MDR cells. The antitumor activity of safingol,
when given alone and in combination with other antineoplastic drugs,
was characterized, and the effects of safingol on phosphorylation and
drug binding of P-gp were studied. The results indicate that safingol
inhibits protein kinase C and partially inhibits the MDR phenotype of
MCF-7 DOX cells but does not exhibit properties of a
substrate for P-gp or alter P-gp expression.
To evaluate
the effect of agents on antineoplastic drug toxicity, cells were
treated with 50 µl of media containing either vehicle, safingol, or
verapamil for 30 min. The cells were then treated with 40 µl of
media containing graded concentrations of antineoplastic drugs and 10
µl of media containing vehicle, safingol, or verapamil to maintain
final concentrations of 3-5 µM safingol or 5
µM verapamil. Percentages were calculated relative to cell
groups treated with sensitizer alone, and IC values were
calculated as above.
Cells (5
10
/assay) or membranes (100 µg/assay) were incubated in
the presence of drugs or vehicle (Me
SO) for 30 min before
the addition of 0.5 µM [
H]azidopine
and incubating an additional 20 min. The samples were photolabeled on
ice by UV irradiation for 10 min, diluted in 2
sample
buffer(42) , and analyzed by SDS-PAGE. Labeling of P-gp bands
was detected by autofluorography and quantitated by densitometry.
Figure 1: Structure of safingol. (2S,3S)-2-amino-1,3-octadecanediol; L-threo-dihydrosphingosine.
To determine
whether safingol could inhibit PKC activity in cells, the effect of
safingol treatment on basal phosphorylation of MARCKS in MCF-7 WT and
MCF-7 DOX cells was evaluated. Antisera to MARCKS
immunoprecipitated an 87-kDa protein from MCF-7 WT (Fig. 2A)
and MCF-7 DOX
cells (Fig. 2B) labeled
with
P. Safingol was shown to inhibit MARCKS
phosphorylation in a concentration-dependent manner in both cell types,
demonstrating that safingol treatment inhibits PKC activity of both
drug-sensitive and MCF-7 DOX
cells.
Figure 2:
Inhibitory effects of safingol on
phosphorylation of MARCKS in MCF-7 WT and MCF-7 DOX cells.
MCF-7 WT (A) and MCF-7 DOX
(B) cells were
treated with the indicated concentrations of safingol and labeled with
[
P]orthophosphate for 2.5 h in 1 ml of
phosphate-free RPMI 1640 containing 0.1 mM BSA. The MARCKS
protein was immunoprecipitated with a polyclonal antibody from
detergent lysates, resolved by SDS-PAGE using 6 or 7.5% acrylamide
gels. Film was exposed for 30 h. with an intensifying screen in panel A and overnight in panel B. The positions of
molecular weight standards are shown on the left, and the
MARCKS protein is identified with an arrow. Similar results
were obtained in two (MCF-7 WT) or three (MCF-7 DOX
)
additional experiments.
Figure 3:
Inhibitory effects of safingol on basal
phosphorylation of P-gp in MCF-7 DOX cells. A,
P-gp was immunoprecipitated with C-219 antibody from an aliquot of the
MCF-7 DOX
cell lysate used for immunoprecipitation of
MARCKS shown in Fig. 2B. P-gp was immunoprecipitated
and resolved by SDS-PAGE on a discontinuous 6% acrylamide gel. Similar
results were observed in two additional experiments. B, MCF-7
DOX
cells were treated with the indicated concentrations of
safingol as described above, and P-gp was immunoprecipitated with mouse
C-219 antibody. After discontinuous SDS-PAGE (7.5% acrylamide minigels)
the samples were transferred, and immunoblot analysis was performed
using ASP-54 antibody to mdr1 P-gp and enhanced
chemiluminescence for detection of P-gp. Similar results were obtained
in an additional experiment.
Figure 4:
Inhibitory effects of safingol on phorbol
dibutyrate-stimulated phosphorylation of P-gp in MCF-7 DOX cells and P-gp phosphopeptides. MCF-7 DOX
cells were
labeled with
P for 4 h and washed three times to remove
unincorporated label. One ml of media containing 50 µM safingol or vehicle (100 µM BSA, 0.2% ethanol) was
added to the wells at timed intervals. After 90 s, 100 nM PDBu
or vehicle (0.01% dimethyl sulfoxide) was added, and cells were
incubated for 10 min. P-gp was immunoprecipitated and resolved by
SDS-PAGE as described under ``Experimental Procedures.'' (A). The bands were excised, digested with S. Aureus V8 protease, and analyzed on 12.5% acrylamide gels as described
under ``Experimental Procedures'' (B). Data shown
are representative of two experiments.
Figure 5:
Stimulatory effects of safingol and
verapamil on [H] vinblastine accumulation in
MCF-7 DOX
cells. MCF-7 DOX
cells were incubated
with a vehicle control (50 µM BSA, 0.25% ethanol; open
bar), 10 µM verapamil (hatched bar), or the
indicated concentration of safingol (filled bars) for 30 min
before the addition of [
H]vinblastine to a final
concentration of 100 nM. Net accumulation of vinblastine was
measured at 2 h as described under ``Experimental
Procedures'' and is expressed as pmol/mg of protein. Basal
accumulation of [
H]vinblastine in MCF-7 WT cells
was approximately 15-fold greater than in untreated MCF-7 DOX
cells in this assay and was not altered by safingol or verapamil
treatment (not shown). Data shown are the averages and standard
deviations of three experiments.
To verify that these increases in vinblastine accumulation were
related to inhibition of PKC, MCF-7 DOX cells were treated
with PDBu 5 min prior to addition of safingol and
[
H]vinblastine. Safingol has much lower affinity
for PKC than PDBu; thus, it is difficult for this drug to displace PDBu
from the regulatory domain. PDBu treatment was found to decrease
vinblastine accumulation 70 (± 15)% in cells treated with 50
µM safingol but only 10-15% in vehicle- and
verapamil-treated cells (n = 3). The partial reversal
of safingol-mediated increases in drug accumulation in response to
prior activation of PKC by PDBu implicates PKC in these increases. The
correlation of increased drug accumulation and inhibition of MARCKS (Fig. 2B) and P-gp phosphorylation (Fig. 3A) in MCF-7 DOX
cells treated with
safingol suggests an association between inhibition of PKC by safingol
and enhanced accumulation of vinblastine in these MDR cells. Safingol
inhibition of MARCKS phosphorylation in MCF-7 WT cells was not
associated with increases in drug accumulation, a finding consistent
with the idea that inhibition of P-gp phosphorylation by PKC was
central to the changes in drug accumulation.
Figure 6:
Effects of vinblastine, verapamil, and
safingol on specific binding of [H] vinblastine
to membrane vesicles of MCF-7 DOX
cells. Specific binding
of [
H]vinblastine to MCF-7 DOX
cell
membranes was measured in a rapid filtration assay as described under
``Experimental Procedures.'' Membrane vesicles were incubated
in the presence of vehicle (1% ethanol), or the indicated
concentrations of vinblastine (open circles), verapamil (filled circles), or safingol (open squares) for 20
min in binding assay buffer. The vesicles were collected by rapid
filtration and washed twice. Specific binding of
[
H]vinblastine was calculated by subtracting
nonspecific binding from total binding. Nonspecific binding was
measured in the presence of a 1000-fold molar excess of unlabeled
vinblastine and constituted 15-20% of total binding of
vehicle-treated vesicles. Data shown are the averages and standard
deviations from a representative experiment in which effects of
vinblastine, verapamil, and safingol were directly compared. Similar
results have been observed for each drug in at least two additional
experiments.
Figure 7:
Effects of safingol on
[H]azidopine photoaffinity labeling of P-gp in
membrane vesicles of MCF-7 DOX
cells.
[
H]Azidopine photolabeling of membranes prepared
from sensitive (WT) and MCF-7 DOX
cells was performed on
membranes preincubated in the presence of a vehicle control or with the
indicated concentrations of drugs. The samples were analyzed by
SDS-PAGE, and autofluorography was performed as described under
``Experimental Procedures.'' Similar results were observed in
experiments in which [
H]azidopine photolabeling
of MCF-7 DOX
cells was studied.
Vehicle-treated
vesicles (1% ethanol) specifically bound 32.4 pmol of
[H]vinblastine/mg of protein. Unlabeled
vinblastine inhibited specific binding by 73-96% at
concentrations between 1 and 100 µM. In a similar fashion
verapamil inhibited specific drug binding, while safingol did not
appreciably reduce drug binding even when present in a 1000-fold molar
excess over [
H]vinblastine (Fig. 6). MCF-7
WT membrane vesicles did not exhibit specific binding of
[
H]vinblastine (data not shown).
The major
[H]azidopine photolabeled band in membrane
vesicles from MCF-7 DOX
cells corresponded with P-gp and
was absent from MCF-7 WT membrane vesicles (Fig. 7).
Densitometry revealed greater than 50% inhibition of photolabeling in
the presence of 10 µM nicardipine and 50 µM verapamil, which corresponded to 20- and 100-fold molar excess
over [
H]azidopine, respectively. In contrast,
treatment of membranes with 50 or 100 µM safingol did not
inhibit photoaffinity labeling of P-gp, although it was present in a
100-200-fold molar excess over [
H]azidopine (Fig. 7). In situ [
H]azidopine
photoaffinity labeling experiments gave similar results (data not
shown).
Figure 8:
Effects of vinblastine, verapamil, and
safingol on the ATPase activity of P-gp in MCF-7 DOX cell
membranes. Vanadate-sensitive ATPase activity was measured as described
under ``Methods.'' Verapamil (filled circles),
vinblastine (open circles), and safingol (filled
squares) were dissolved in ethanol and diluted 100-fold to the
indicated concentrations. Similar results were obtained in two other
experiments.
The biological activities of
amphipathic compounds such as lysosphingolipids are affected by
partitioning in the lipid bilayer. In vitro and in cells
lysosphingolipids have been reported to exhibit surface dilution
kinetics. The in vitro potency of sphingosine as a PKC
inhibitor has been shown to be primarily determined by its surface
concentration (i.e. its molar ratio to other lipid assay
components)(48) . For example, increasing the concentration of
lipids in mixed micelles has been reported to decrease the in vitro PKC inhibitory activity of sphingosine (48) . It has been
determined that in cellular systems the ratio of the total mass of
lysosphingolipid present in solution to cell number provides an
estimate of effective lysosphingolipid concentration in cell
membranes(38, 48) . The total safingol mass was
calculated by multiplying the concentration of safingol by the assay
volume, and the effective concentration of safingol was calculated from
a formula describing surface dilution kinetics of safingol: effective
[safingol] ∝ safingol mass (mol)/cell number or
[safingol] assay volume/cell number. The ratio of
safingol to cells in the 72-h MTT proliferation assays was
approximately 16-fold greater than in drug accumulation assays due to
decreased cell number and an increase in the ratio of assay volume to
cell number in MTT proliferation versus drug accumulation
assays. An equivalent ratio of safingol to cells (80-100 fmol
safingol/cell) was present at 50 µM safingol in drug
accumulation assays and at 3 µM in MTT proliferation
assays, respectively. On the basis of these calculations one would
predict that surface dilution kinetics of safingol could account for
these differences in cytotoxicity. A series of surface dilution
experiments were performed to evaluate this hypothesis.
The effects
of varying safingol mass by decreasing cell number or assay volume on
toxicity of safingol were evaluated in 72-h proliferation assays (Fig. 9A). Using the same stock solutions of safingol,
increasing the ratio of safingol to cells by plating half the number of
cells in the assay (3000 cells/well) was found to shift the curve to
the left and increase the toxicity approximately 2-fold. Conversely,
decreasing safingol mass by reducing the volume of the assay 50%, from
200 to 100 µl, was found to decrease toxicity of safingol, shift
the curve to the right, and approximately double the IC value of safingol. A comparison of MTT absorbance indicated that
volume differences did not alter cell growth. When the number of cells
plated was reduced by half an approximately 50% reduction in absorbance
was obtained. The cytotoxicity curves in 72-h MTT assays are identical
when effective concentrations of safingol (calculated from the ratio of
safingol mass to cell number) are plotted (Fig. 9B). An
equivalent IC
value of approximately 200 fmol of
safingol/cell was found for MCF-7 DOX
cells cultured under
the varied assay conditions described above. These results illustrate
the validity of the surface dilution kinetic model for safingol.
Additional 24-h MTT proliferation experiments verified that 50
µM safingol was a nontoxic concentration under conditions
when cell density and volume were standardized to mimic the higher cell
density and decreased media volume/cell ratio present in drug
accumulation assays (data not shown). The variation of assay volume in
24-h exposures did not alter the final cell number. These results
suggested that surface dilution kinetics were responsible for the
differences in the molar concentrations of safingol that produced
cytotoxicity in drug accumulation and MTT assays.
Figure 9:
Effects of surface dilution kinetics on
safingol toxicity. MCF-7 DOX cells were plated in 96-well
plates for 24 h. The cells were exposed to the indicated concentrations
of safingol for 72 h under the following conditions: 200 µl of
media, 6000 cells/well (standard conditions; open circles),
100 µl of media, 6000 cells/well (filled circles), or 200
µl of media, 3000 cells/well (open squares). After 72 h
the medium was replaced with 200 µl of medium containing 5 mg/ml
MTT and incubated for 4 h. The formazan reduction product was
quantitated as described under ``Methods.'' The panel A
abscissa is the extracellular concentration of safingol. The panel B abscissa is the effective safingol concentration in
cells calculated from the ratio of safingol mass to cell number. Data
shown are the average of two experiments performed in
quadruplicate.
A general agreement between the relative potency of safingol
to verapamil in these different assays was noted. The increase in drug
accumulation elicited by treatment of MDR cells with 10 µM verapamil was not significantly different than the increase
elicited by treatment with 50 µM safingol, indicating that
verapamil was approximately five times more potent than safingol in
[H]vinblastine accumulation assays. Verapamil was
also noted to be approximately 4-fold more potent than safingol when
compared at 5 µM in MTT assays that evaluated effects on
vinblastine resistance (Table 1).
To verify the association of inhibition of P-gp phosphorylation by safingol with modulation of drug resistance, phosphorylation of P-gp at 2.5 h was examined under conditions of the MTT assay. Fig. 10shows that P-gp phosphorylation was inhibited by 5 µM safingol under these conditions, thus establishing a correlation between inhibition of P-gp phosphorylation by safingol with partial reversal of multidrug resistance. As suggested by the similar effective concentrations of safingol, 80-100 fmol/cell, the inhibition of P-gp phosphorylation was similar at both 5 and 50 µM safingol under conditions of MTT and drug accumulation assays, respectively.
Figure 10:
Effects of safingol on P-gp
phosphorylation under MTT assay conditions. To mimic the conditions of
the MTT assays, MCF-7 DOX cells were plated at a density of
1.7
10
cells/well in 6-well plates and cultured
overnight. The cells were washed three times before the addition of 5
µM safingol in 5.9 ml of phosphate-free RPMI containing
0.2 mCi of
P-orthophosphate/ml and 0.1 mM BSA.
After labeling for 2.5 h the cells were washed and lysed in detergent
buffer, and P-gp was immunoprecipitated from aliquots of the lysate
matched by trichloroacetic acid-precipitated counts as described under
``Experimental Procedures.'' Immunblots of aliquots of the
immunoprecipitated P-gp showed that equal amounts of P-gp were present
in samples matched by trichloroacetic acid-precipitated counts (not
shown). Data shown are representative of three
experiments.
Figure 11:
Effects of safingol on P-gp expression
under MTT assay conditions. To mimic the conditions of the MTT assays,
2.8 10
MCF7 DOX
cells were plated in
148-cm
culture dishes and exposed to the indicated
concentrations of drugs for 24, 48, or 72 h in a final volume of 92.5
ml. Membranes were prepared from three to six plates of monolayers
containing approximately 3-4
10
cells. 10
µg of membrane protein was resolved by SDS-PAGE, and detection of
P-gp was performed as described under ``Experimental
Procedures'' using C219 antibody and enhanced chemiluminescence.
Data shown are representative of two
experiments.
An assessment of multiple criteria was used to demonstrate
that inhibition of PKC-mediated phosphorylation of P-gp by safingol was
associated with modulation of the drug accumulation defect of MCF-7
DOX cells, a hallmark of the MDR phenotype.
Inhibition
of MARCKS phosphorylation in MCF-7 WT and MCF-7 DOX cells
by safingol demonstrated inhibition of PKC in these cells. This
prominent cellular substrate of protein kinase C is known to be rapidly
phosphorylated subsequent to PKC activation by growth factor
stimulation in fibroblasts(50) , by ligands which stimulate
phagocytic inflammatory cells (51, 52) and by
neurosecretory stimuli(53) . Previously, phosphorylation of
MARCKS was shown to correlate with enhanced expression of PKC
in
MCF-7 DOX
cells (25) and with overproduction of PKC
I in the rat embryo fibroblast cell line R6-PKC3(54) .
Although safingol treatment of MCF-7 WT and MCF-7 DOX
decreased MARCKS phosphorylation in both cell lines, safingol treatment
enhanced drug accumulation only in MDR MCF-7 cells. Furthermore, the
enhanced accumulation of vinblastine and decreased phosphorylation of
P-gp and MARCKS in cells treated with safingol occurred with similar
concentration dependence. These findings suggest that safingol
inhibition of PKC-mediated P-gp phosphorylation is associated with
inhibition of P-gp function.
Safingol treatment was also shown to
inhibit phosphorylation of P-gp in MDR MCF-7 cells stimulated by PDBu,
suggesting that safingol inhibited PKC-mediated phosphorylation of
P-gp. Partial V8 protease digestion and analysis of P-gp
phosphopeptides showed that PDBu stimulated phosphorylation of three
major preexisting phosphorylated sites and that safingol inhibited
phosphorylation of these three sites equally. These results support the
notion that PKC is the major kinase that phosphorylates P-gp in MCF-7
DOX cells under basal conditions and that safingol inhibits
P-gp phosphorylation at specific PKC phosphorylation sites. These
phosphopeptides may represent individual sites of phosphorylation or
overlapping peptides containing common phosphorylation sites. Serine
residues in the linker region of P-gp have been identified as the major in vitro PKC and PKA phosphorylation sites of both the human
P-gp (19) and the murine mdr1b P-gp(18) .
Previously, activators of PKC have been shown to stimulate
phosphorylation of P-gp at distinct tryptic P-gp
phosphopeptides(17) . However, in some MDR cell lines, PKC
activators are reported to enhance phosphorylation of P-gp tryptic
peptides, which are also phosphorylated in unstimulated
cells(16, 46) . Variability in expression of PKC
isotypes in MDR cells (23, 24, 25) may be
associated with the differences in patterns of phosphorylation of
tryptic P-gp peptides.
In MCF-7 DOX cells, reciprocal
changes in calcium-dependent and calcium-independent PKC activity have
been described(25) . Decreased expression of PKC
and
isoenzymes and calcium-independent PKC activity were found in
MCF-7 DOX
cells relative to MCF-7 WT cells(25) . In
contrast, increased expression of PKC
isoenzyme in MDR MCF-7
cells was associated with increased calcium-dependent PKC activity and
enhanced phosphorylation of MARCKS and P-gp(25) . A role for
the PKC
isoenzyme as a positive regulator of P-gp function in
MCF-7 DOX
cells has also been suggested by transfection
studies with antisense cDNA for PKC
. Expression of PKC
antisense cDNA decreased PKC activity, P-gp phosphorylation, and P-gp
function(55) . Additionally, transfection of MCF-7 cells
expressing mdr1 with PKC
has been shown to increase
resistance to natural product drugs in association with reduced
accumulation and enhanced phosphorylation of P-gp(16) .
Safingol was found to inhibit human PKC
activity at approximately
the same concentration as it inhibited PDBu binding and enzymatic
activity of rat brain PKC.
Stimulation of PKC in MCF-7 DOX cells by phorbol dibutyrate partially reversed increased drug
accumulation in cells treated with safingol. The ability to reverse
lysosphingolipid-mediated changes using phorbol esters or
diacylglycerol has previously been used to discern PKC-dependent and
PKC-independent activities of D-erythro-sphingosine
in platelets (49) , in quiescent Swiss 3T3 cells(56) ,
and in rat adipocytes(57) . The partial reversal of
safingol-mediated increases in drug accumulation in response to
activation of PKC by PDBu suggests that these increases are
PKC-dependent.
Other PKC inhibitors including
staurosporine(29) , calphostin C(26) , and NA-382, a
staurosporine derivative(30) , have been reported to inhibit
[H]azidopine photolabeling of P-gp. For these
compounds it is not clear if inhibition of P-gp function is a result of
PKC inhibition, inhibition of drug binding, or inhibition of both
activities. In contrast, safingol did not inhibit
[
H]vinblastine binding to P-gp or
[
H]azidopine photoaffinity labeling of P-gp and
did not alter P-gp ATPase activity in MCF-7 DOX
membrane
vesicles. These findings suggest that safingol does not directly
interact with these P-gp drug binding site(s) as a means of enhancing
vinblastine accumulation. In view of the possible dual mechanisms in
the previously studied PKC inhibitors above, this finding is novel.
Currently, the number and characteristics of drug binding sites on P-gp are not well defined. Structure-activity studies of thioxanthenes(58) , and reserpine analogs (59) have described a general pharmacophore for modulator binding to P-gp, which contains planar aromatic domains and a basic nitrogen side chain. Lysosphingolipids lack aromatic rings but have a positively charged nitrogen at physiologic pH, which is a characteristic of most (but not all) inhibitors of multidrug resistance (2) . The hypothesis that lysosphingolipids do not directly interact with P-gp is consistent with studies using the calcium channel blocker SR33557. This MDR reversal agent did not inhibit P-gp drug binding but increased levels of sphingosine in MDR leukemia cells, leading to inhibition of cellular PKC activity(31) .
Although less potent than verapamil,
safingol at nontoxic concentrations was shown to partially sensitize
MCF-7 DOX cells to natural product drugs (anthracyclines
and Vinca alkaloids) but not to the antimetabolite 5-FU.
Results of phosphorylation studies, Western blots, and Northern blots
indicated that safingol treatment decreased P-gp phosphorylation rather
than stability of P-gp or message for P-gp. Growth, viability, and
metabolic assessments indicated that inhibition of P-gp phosphorylation
and modulation of the MDR phenotype by safingol was not associated with
general toxicity. The ability of safingol to inhibit P-gp
phosphorylation and drug resistance without significant growth
inhibition may reflect a transitory inhibition of PKC due to metabolism
of safingol into more complex sphingolipids. Thus, the moderate
relative efficiency of safingol as an MDR inhibitor could result from
transitory inhibition of PKC and P-gp function due to safingol
metabolism. Alternatively, P-gp phosphorylation may have only a limited
role in modulating drug resistance in these cells. Verapamil, which
inhibits drug binding to P-gp, also only partially inhibited the MDR
phenotype of MCF-7 DOX
cells, perhaps because the
drug-resistant phenotype of MCF-7 DOX
cells may be
multifactorial.
Overexpression of glutathione detoxification enzymes (61) and increased activity of other drug-metabolizing enzymes
in this cell line have been reported(62) . Safingol inhibition
of PKC may alter activities of other phosphorylated drug
resistance-associated enzymes such as topoisomerase II, which is known
to be phosphorylated by PKC in vitro(63) .
Hyperphosphorylation of topoisomerase II has been associated with
decreased sensitivity to topoisomerase II inhibitors in human
etoposide-resistant KB cancer cells(64) . However, to the best
of our knowledge there are no reports correlating altered
phosphorylation of topoisomerase II with activity changes in the MCF-7
DOX cell line. The reported absence of detectable message
for the multidrug resistance-associated protein, MRP in MCF-7 DOX
cells(66) , suggests that this member of the ABC
transport protein family, which shares homology with P-gp(65) ,
is unlikely to contribute to the MDR phenotype of these cells. Although
non-P-gp-mediated mechanisms may contribute to resistance to a given
drug in MCF-7 DOX
cells, P-gp is generally thought to be
the major mechanism of MDR in this cell line. Although activities of
other phosphorylated drug resistance proteins unrelated to P-gp may be
altered by safingol inhibition of PKC, the pattern of partial
inhibition of natural product drug toxicity versus unaltered
5-FU toxicity suggests an association of decreased phosphorylation of
P-gp and specificity for the MDR phenotype.
A marked difference in the molar concentrations of safingol required to effectively enhance drug accumulation and inhibit resistance was noted. The data indicate that surface dilution of safingol probably accounts for the differences in concentration dependence between drug accumulation and MTT assays. This premise is supported by the finding that when assay conditions were standardized inhibition of P-gp phosphorylation was associated with both partial inhibition of drug resistance and enhanced drug accumulation. Surface dilution kinetics of sphingosine and other long chain sphingoid bases has been previously observed both in vitro and in situ(32, 33, 34, 39, 49) .
Calmodulin antagonists, cyclosporins, hormonal analogs, calcium channel blockers, and antiarrythmic drugs can all modulate P-gp activity by directly binding to the P-gp. The evidence presented here, however, indicates that safingol may modulate P-gp activity by a different mechanism than these previously described agents, namely via inhibition of PKC-mediated phosphorylation of P-gp.