(Received for publication, June 13, 1995; and in revised form, October 30, 1995)
From the
The predominant characteristics of multidrug resistant (MDR)
cancer cells are broad spectrum resistance to chemotherapeutic agents
and a pronounced defect in intracellular accumulation of the drugs, in
association with overexpression of the drug efflux pump P-glycoprotein.
Protein kinase C (PKC) phosphorylates the linker region of
P-glycoprotein. Evidence has been presented that the isozyme PKC-
may contribute to the drug resistance phenotype of human breast cancer
MCF7-MDR cells. PKC-
is markedly overexpressed in MCF7-MDR cells,
and artificial overexpression of PKC-
in MCF7 constructs that
overexpress P-glycoprotein significantly enhances the MDR phenotype of
the cells in association with increased P-glycoprotein phosphorylation.
Verapamil, cyclosporin A, and a number of other agents that compete
with cytotoxic drugs for binding sites on P-glycoprotein can potently
reverse MDR, but this is accompanied by severe toxicity in
vivo. In this report, we demonstrate that an N-myristoylated peptide that contains a sequence corresponding
to the pseudosubstrate region of PKC-
(P1) partially reverses
multidrug resistance in MCF7-MDR cells by a novel mechanism that
involves inhibition of PKC-
. P1 and two related PKC inhibitory N-myristoylated peptides restored intracellular accumulation
of chemotherapeutic drugs in association with inhibition of the
phosphorylation of three PKC-
substrates in MCF7-MDR cells:
PKC-
, Raf-1 kinase, and P-glycoprotein. A fourth N-myristoylated peptide substrate analog of PKC, P7, did not
affect drug accumulation in the MCF7-MDR cells and failed to inhibit
the phosphorylation of the PKC-
substrates. The effects of P1 and
verapamil on drug accumulation in MCF7-MDR cells were additive. P1 did
not affect P-glycoprotein expression. MCF7-MDR cells were not
cross-resistant to P1, which suggests that the peptide was not
transported by P-glycoprotein. Furthermore, P1 was distinguished from
MDR reversal agents such as verapamil and cyclosporin A by its
inability to inhibit [
H]azidopine photoaffinity
labeling of P-glycoprotein. P1 actually increased
[
H]azidopine photoaffinity labeling of
P-glycoprotein in MCF7-MDR cells, providing evidence that the effects
of P1 on P-glycoprotein in MCF7-MDR cells are not restricted to
inhibition of the phosphorylation of the pump. P1 may provide a basis
for developing a new generation of MDR reversal agents that function by
a novel mechanism that involves inhibition of PKC-
-catalyzed
P-glycoprotein phosphorylation.
Resistance to chemotherapy is a major obstacle to successful
cancer treatment, and it often accounts for the failure of aggressive
chemotherapy to eradicate malignant disease(1) . Most
metastatic cancers are either innately resistant to chemotherapy or
acquire drug resistance during the course of chemotherapy(1) .
Multidrug resistant (MDR) ()cancer cells are characterized
by broad spectrum resistance to chemotherapeutic drugs, markedly
reduced intracellular accumulation of the drugs, and overexpression of
the drug efflux pump P-glycoprotein(1, 2) . The
relevance of MDR to clinical drug resistance in cancer therapy is
indicated by the abundant expression of P-glycoprotein and its message mdr1 in specimens of human cancer that are intrinsically
resistant to chemotherapy and in malignant tumors from patients who
have relapsed during or after
chemotherapy(1, 3, 4) .
Protein kinase C
(PKC) is an isozyme family with at least ten mammalian
members(5) . Highly selective phorbol-ester PKC activators
induce resistance in cancer cells to multiple cytotoxic drugs that are
P-glycoprotein substrates in association with a sharp reduction in the
intracellular accumulation of the drugs, providing evidence that PKC
activation contributes to MDR. The phorbol-ester effects on
chemosensitivity and drug accumulation have been observed in several
drug-sensitive and MDR cancer cell lines including human breast cancer
MCF7-WT and MCF7-MDR, and the magnitude of the effect is generally a
2-6-fold increase in the IC values of cytotoxic
drugs (6, 7, 8, 9, 10, 11, 12) .
PKC phosphorylates the linker region of P-glycoprotein in MDR human
KB-V1 cancer cells(9, 13, 14) , and this is
tightly coupled to the effects of PKC on intracellular drug
accumulation and MDR, providing evidence that PKC regulates the
function of P-glycoprotein in this system(11) . The isozyme
PKC-
has been shown to be overexpressed in several MDR cancer cell
lines including
MCF7-MDR(8, 15, 16, 17, 18) .
Evidence that PKC-
activation contributes to MDR has been provided
by observations that MDR is induced by the selective activation of
PKC-
in human colon cancer cells (12) and by transfection
of an mdr1-transfected human breast cancer MCF7 subline with
PKC-
(19) .
Verapamil, cyclosporin A, and related MDR
reversal agents that compete with chemotherapeutic drugs for binding
sites on P-glycoprotein (20, 21, 22, 23) generally cause
severe toxicity in vivo at therapeutic concentrations, and
this precludes their use in the treatment of drug-resistant
cancer(24, 25) . In a previous report, we
characterized the mechanism of PKC inhibition by an N-myristoylated peptide corresponding to the autoinhibitory
pseudosubstrate sequence of PKC- (P1) that had been shown to
selectively inhibit PKC in human fibroblasts(26, 27) .
In this report, we show that the peptide P1 reverses MDR by a novel
mechanism that is associated with a sharp increase in intracellular
drug accumulation and inhibition of the phosphorylation of
P-glycoprotein and two other PKC-
substrates, Raf-1 kinase and
PKC-
itself. P1 was distinguished from verapamil and related MDR
reversal agents(20, 21, 22, 23) in
that it did not inhibit photoaffinity labeling of drug-binding sites on
P-glycoprotein by [
H]azidopine. P1 did not affect
P-glycoprotein expression in MCF7-MDR cells. MCF7-MDR cells were not
cross-resistant to P1, providing indirect evidence that the peptide was
not transported by P-glycoprotein. The N-myristoylated
PKC-
pseudosubstrate peptide P1 may be a valuable starting point
for developing a new generation of MDR reversal agents that function by
a novel mechanism that involves inhibition of PKC-
-catalyzed
P-glycoprotein phosphorylation.
The
procedures used to immunoprecipitate P-glycoprotein, PKC-, and
Raf-1 kinase were based on previously described methods(18) .
For P-glycoprotein immunoprecipitation, the supernatant (100 µg of
protein) was incubated with 5 µg of C219 monoclonal antibody by
rotation for 16 h at 4 °C. In the case of Raf-1 kinase and
PKC-
immunoprecipitation, the supernatant was incubated with Raf-1
kinase mAb (5 µg) (Oncogene Science, Uniondale, NY) or PKC-
mAb (5 µg) (Upstate Biotechnology, Inc., Lake Placid, NY) by
rotation for 2 h at 4 °C, and rotation was continued in the
presence of 5 µg rabbit antimouse IgG for an additional 30 min. at
4 °C. Next, 0.2 ml of a 25% (v/v) suspension of protein A-Sepharose
was added to the incubation mixture followed by rotation for 30 min at
4 °C. After a 1-min centrifugation in a microcentrifuge, the beads
were recovered as a pellet, and they were washed successively with 1 M NaCl, 1% Nonidet P-40, and lysis buffer containing 1 M urea. To recover immune complexes from the beads, the beads were
incubated for 10 min at 30 °C with 0.1 ml of SDS-PAGE sample
buffer, and then they were pelleted in a microcentrifuge. The
supernatant was recovered and subjected to SDS-PAGE. Gels were silver
stained, and autoradiography was done.
Table 1shows the structures of the peptides under
investigation for modulatory effects on the intracellular accumulation
and growth-inhibitory activity of cytotoxic anticancer drugs in human
breast cancer MCF7-MDR cells. P2 contains the core sequence of the
pseudosubstrate region of PKC- (PKC[20-28]), P4
contains a more extensive PKC-
pseudosubstrate sequence
(PKC[19-31])(26, 27) , and P6 contains
the sequence of the PKC phosphorylation site of the epidermal growth
factor receptor at Thr
(27) . P1, P3, and P5 are N-myristoylated analogs of P2, P4, and P6, respectively. Each
of these N-myristoylated peptides inhibits the histone kinase
reaction of purified PKC (Table 1); the corresponding
nonmyristoylated peptides do not inhibit PKC-catalyzed histone
phosphorylation (Table 1). P7 is an N-myristoylated
peptide with a sequence that corresponds to a PKC phosphorylation site
in P-glycoprotein (residues 656-666)(13) . Although P7
does inhibit the histone kinase activity of PKC, it is weaker than P1,
P3, and P5 in this regard, and, at concentrations
10
µM, P7 actually stimulates the histone kinase reaction (Table 1). In a previous report, we showed that the mechanism of
PKC inhibition by several N-myristoylated peptide substrate
analogs of PKC, including P1 and P5, entailed binding interactions of
the inhibitor peptides with the active site of PKC and with
phospholipid vesicles(27) . In control experiments that
monitored the absorbance of the Tyr-containing P1 analog NmYARKGALRQ,
we have found that the phospholipid-interacting peptide does not bind
to plastic-, glass-, or collagen-coated surfaces (data not shown).
P1 has been shown to inhibit phosphorylation of the PKC substrate MARCKS in intact rat fibroblasts(26) , and the truncated P5 analog N-myristoyl-KRTLR antagonizes PKC-requiring pathways in T-lymphocytes(31, 32) . The demonstrated ability of PKC-inhibitory N-myristoylated peptide substrate analogs to inhibit PKC activity in nonpermeabilized mammalian cells (26, 31, 32) provides indirect evidence that the peptides enter mammalian cells, which is consistent with their membrane-active nature(27) . P5 partially reverses adriamycin resistance in murine fibrosarcoma cells(33) . Specific activation of PKC is correlated with enhancement of MDR in tumor cells, and inhibition of the enzyme is often associated with partial reversal of MDR(10, 11) . We hypothesized that P1 and related PKC-inhibitory N-myristoylated peptides might antagonize MDR in human breast cancer MCF7-MDR cells.
Fig. 1shows the
effects of the peptides P1-P6 on the intracellular accumulation
of [C]ADR and [
H]VLB,
which are P-glycoprotein
substrates(1, 2, 10) , and
[
H] 5FU, which is not a P-glycoprotein
substrate(1, 2, 10) , in MCF7-MDR cells. All
assays of drug accumulation in MCF7-MDR and MCF7-WT cells described in
this report were done under conditions where cell viability was >95%
at the end of the assay period, according to measurements of trypan
blue exclusion. The N-myristoylated peptides P1, P3, and P5
markedly and significantly increased the accumulation of
[
C]ADR (Fig. 1A) and
[
H]VLB (Fig. 1B) in the MCF7-MDR
cells, whereas the nonmyristoylated peptides P2, P4, and P6 had no
effect on their accumulation (Fig. 1, A and B). The N-myristoylated peptides enhanced drug
accumulation in the MCF7-MDR cells from 3-9-fold, which is
comparable with the enhancement of drug accumulation that was achieved
by the potent MDR reversal agent verapamil (VP) (Fig. 1, A and B). For comparison, the levels of intracellular
[
C]ADR and [
H]VLB
accumulation in MCF7-WT were respectively 5- and 10-fold higher than
those in MCF7-MDR. The enhancement of drug uptake was most pronounced
when the N-myristoylated peptide concentration was 100
µM. However, at a concentration of 50 µM, P1
and P3 each significantly enhanced [
C]ADR
uptake, and P1 and P5 significantly enhanced
[
H]VLB uptake in the MCF7-MDR cells (Fig. 1, A and B). In parallel experiments, we
found that like VP, P1-P6 did not significantly affect
[
H]5FU accumulation in MCF7-MDR cells (Fig. 1C). This demonstrates a degree of specificity in
the effects of the N-myristoylated peptides on drug
accumulation in MCF7-MDR cells. [
C]ADR and
[
H]VLB accumulation in MCF7-MDR cells were
enhanced by less than 5% by myristic acid (25-100
µM) and by N,N-dimethylmyristamide
(25-100 µM), which is a nonpeptidic amide of
myristic acid. Thus, specificity is also indicated by the lack of
effect of the acyl head group of the peptides on
[
C]ADR and [
H]VLB
accumulation. In addition, P7 (25-100 µM) failed to
enhance [
C]ADR and [
H]VLB
accumulation in the cells, indicating that not all N-myristoylated cationic peptides can enhance the retention of
these cytotoxic drugs in MCF7-MDR cells.
Figure 1:
Effects of synthetic peptide
substrate analogs of PKC on cytotoxic drug accumulation in MCF7-MDR
cells. Effects of the N-myristoylated peptides P1, P3, and P5
and the corresponding nonmyristoylated peptides P2, P4, and P6 at
concentrations of 25, 50, and 100 µM on drug accumulation
in MCF7-MDR cells are shown. Drug accumulation was assayed as described
under ``Materials and Methods.'' Under each experimental
condition examined in A-C, >95% cell viability was
observed at the end of the drug accumulation period by measurements of
trypan blue exclusion. Each experimental value shown is the average
value from three experiments that were done in triplicate. Values that
differ significantly from the untreated control are identified by asterisks.**, p < 0.015; *, p < 0.05. A, [C]ADR accumulation in MCF7-MDR
cells is shown. % of Control, the amount of
[
C]ADR accumulation observed in the presence of
the indicated peptide expressed as a percentage of
[
C]ADR accumulation observed in untreated
control (CTRL) MCF7-MDR cells. Treatment with 10 µM VP served as a positive control. The [
C]ADR
concentration was 0.1 µM, and accumulation was measured
after a 2-h incubation period. Untreated cells retained 2.2 ±
0.2 pmol [
C]ADR/10
cells. B, [
H]VLB accumulation in MCF7-MDR cells
is shown. Assays were done with 10 nM [
H]VLB, and accumulation was measured after
incubating the cells with drugs for 30 min. Untreated MCF7-MDR cells
retained 0.066 ± 0.005 pmol
[
H]VLB/10
cells. For other details,
see the description of A. C,
[
H]5FU accumulation in MCF7-MDR cells is shown.
Where indicated, peptides (P1-P6) were present at 100
µM; [
H]5FU was at 10 nM.
Drug accumulation was measured after 30 min and 2 h, as shown.
[
H]5FU accumulation in control (CTRL)
cells was 0.058 ± 0.002 (30 min) and 0.058 ± 0.004 pmol
[
H] 5FU/10
cells (2 h). For other
details, see the description of A.
Table 2shows that
P1-P6 increased [C]ADR and
[
H]VLB accumulation in the drug-sensitive cell
line MCF7-WT only marginally (<1.8-fold) (P2, P3, P4) or not at all
(P1, P5, P6), and they had no detectable effect on
[
H]5FU accumulation in the cells. Of the N-myristoylated peptides, only P3 detectably increased
[
C]ADR and [
H]VLB
accumulation in the MCF7-WT cells (Table 2). Thus, the marked
enhancement of [
C]ADR and
[
H]VLB uptake achieved by the N-myristoylated peptides P1, P3, and P5 but not by their
nonmyristoylated counterparts (Fig. 1) were specific to the MDR
subline.
Having established that the N-myristoylated
peptides significantly increase [C]ADR and
[
H]VLB accumulation in MCF7-MDR cells at 2 h and
30 min, respectively (Fig. 1), we next examined the effects of
P1-P6 on the accumulation of [
C]ADR and
[
H]VLB in MCF7-MDR cells over a 6-h time course.
In these experiments, cells were pretreated with peptide for 30 min,
and the time course was initiated by the addition of drug, as described
under ``Materials and Methods.'' The enhancement of drug
accumulation by the N-myristoylated peptides (P1, P3, and P5)
was observed within 5 and 15 min, respectively, for
[
H]VLB (Fig. 2, C and D)
and [
C]ADR (Fig. 2, A and B). The enhancement of [
C]ADR
accumulation was sustained at a maximal or nearly maximal level over a
6-h time course in each case, as was the enhancement of
[
H]VLB accumulation by P1 (Fig. 2, A-C), but the effects of P3 and P5 on
[
H]VLB accumulation peaked within 2 h and then
gradually declined throughout the remainder of the time course (Fig. 2D). No enhancement of
[
C]ADR or [
H]VLB
accumulation was observed with the nonmyristoylated peptides (P2, P4,
and P6) (Fig. 2). The relatively slow uptake of
[
C]ADR induced by the N-myristoylated
peptides allowed estimation of the effects of the peptides on the
[
C]ADR efflux rate of MCF7-MDR cells by
measuring the net efflux of [
C]ADR from
preloaded cells. Across a 30-min time course, a decline in the net
[
C]ADR efflux rate was observed in MCF7-MDR
cells with each N-myristoylated peptide (100 µM P1, P3, and P5), and the mean value of the decline achieved by the
peptides was 13 ± 4%, which was comparable with the decline of
19 ± 2% observed with 10 µM verapamil.
Figure 2:
Effects of synthetic peptide substrate
analogs of PKC on the kinetics of net drug uptake in MCF7-MDR cells.
Intracellular accumulation of [C]ADR (A and B) and [
H]VLB (C and D) was measured in MCF7-MDR cells at the indicated time
intervals. Where specified, assays were done in the presence of 100
µM peptide (A and C, P1 and P2; B and D, P3, P4, P5, and P6) or 10 µM VP (A and C). In these experiments, cell viability was
>95% according to trypan blue exclusion measurements. For definition
of % of Control, assay conditions, and other experimental
details, see the legend to Fig. 1and ``Materials and
Methods.'' Each experimental value represents an average of
triplicate determinations, and the results shown were determined to be
reproducible in a duplicate experiment.
At a
concentration of 50 µM, the N-myristoylated
pseudosubstrate peptide P1 significantly enhanced
[C]ADR and [
H]VLB
accumulation in the MCF7-MDR cells, but in each case the degree of
enhancement was very modest when compared with 10 µM VP (Fig. 1). Likewise, at concentrations of
2.5
µM, VP had little effect on [
C]ADR
accumulation (Table 3). In an attempt to demonstrate efficacy of
P1 at concentrations of
50 µM, we measured the
enhancement of [
C]ADR uptake in MCF7-MDR cells
by combinations of P1 (
50 µM) and VP (
2.5
µM). We found that 2.5 µM VP in combination
with 25 µM P1 enhanced [
C]ADR
accumulation in MCF7-MDR cells approximately as effectively as 10
µM VP, and the degree of enhancement was significantly
greater than that achieved by either 25 µM P1 or 2.5
µM VP alone (Table 3). We observed a similar but
more marked statistically significant effect by 50 µM P1
in combination with 2.5 µM VP (Table 3). These
results demonstrate that the effects of P1 and VP on drug accumulation
in MCF7-MDR cells are additive, and they show that P1 can modulate drug
accumulation in the MDR breast cancer cells at concentrations as low as
25 µM. (5 µM P1 was ineffective in
combination with VP in enhancing [
C]ADR
accumulation) (data not shown).
The restoration of
[C]ADR and [
H]VLB
accumulation by the N-myristoylated pseudosubstrate peptide P1
in MCF7-MDR cells suggested that the peptide might also sensitize
MCF7-MDR cells to the cytotoxic drugs. To test this, we measured the
effect of a 1-h exposure to P1 on MCF7-MDR cell growth using a 96-h
assay system and found that under these conditions, P1 was not
growth-inhibitory (Fig. 3). Thus, P1 could be tested for MDR
reversal activity in this system under conditions where it potently
stimulated drug accumulation in MCF7-MDR cells (Fig. 2, A and C). Under these conditions, P1 significantly reduced
the ADR concentration required for 50% MCF7-MDR cell growth inhibition
(IC
) approximately 2-fold, from a value of 26.33 ±
0.40 µg/ml to 14.11 ± 0.41 µg/ml (p < 0.001, n = 6). Representative results obtained in one
experiment are shown in Fig. 3A. Similarly, the
IC
of VLB was reduced by P1 approximately 2-fold in
MCF7-MDR cells, from 2.56 ± 0.32 µg/ml to 1.18 ± 0.08
µg/ml VLB (p < 0.02, n = 5);
representative data are shown in Fig. 3B. In contrast,
P1 was without effect on 5FU cytotoxicity in MCF7-MDR cells. Fig. 3C shows that the percentage of MCF7-MDR cell
growth inhibition achieved by 5FU was approximately the same in the
presence (open circles) and in the absence of P1 (closed
circles); the results shown are an average of three experiments.
Taken together with the effects of P1 on drug accumulation in MCF7-MDR
cells ( Fig. 1and Fig. 2), these results provide evidence
that P1-mediated chemosensitization of MCF7-MDR cells involves
inhibition of P-glycoprotein-mediated drug transport.
Figure 3:
Chemosensitization of MCF7-MDR cells by
the N-myristoylated PKC- pseudosubstrate peptide P1.
Effects of 100 µM P1 on the growth-inhibitory activity of
ADR (A), VLB (B), and 5FU (C) in MCF7-MDR
cells are shown. In these experiments, cells were treated with P1 for 1
h followed by a 96-h exposure to the cytotoxic drug. Under these
conditions, the direct growth-inhibitory effects of P1 were negligible.
For other experimental details, see ``Materials and
Methods.'' In A and B, results of a single
experiment are shown; each point represents an average of eight
determinations. In C, the data shown are an average of three
experiments, in which each data point was an average of eight
determinations.
We next tested
whether restoration of drug accumulation in MCF7-MDR cells by P1 and
related N-myristoylated PKC-inhibitory peptides was associated
with inhibition of P-glycoprotein phosphorylation. Fig. 4A shows that exposure of MCF7-MDR cells to P1 (25-100
µM) during a 3-h P-labeling period was
associated with a concentration-dependent inhibition of P-glycoprotein
phosphorylation; maximal inhibition was achieved with 100 µM P1. Similarly, P3 and P5 inhibited P-glycoprotein phosphorylation
in MCF7-MDR cells, although P5 was considerably less effective than
either P1 or P3 in this respect (Table 4). In contrast, the
effects of the nonmyristoylated peptides P2, P4, P6, and the N-myristoylated peptide P7 on P-glycoprotein phosphorylation
in MCF7-MDR cells were negligible (Table 4). In control
experiments, we determined by immunoblot analysis of P-glycoprotein
with C219 mAb that P1, P3, and P5 had only minor or negligible effects
on P-glycoprotein expression under the conditions of the P-glycoprotein
phosphorylation experiments (Fig. 4B; Table 4).
Figure 4:
Inhibition of P-glycoprotein
phosphorylation in MCF7-MDR cells by the N-myristoylated
PKC- pseudosubstrate peptide P1. A, effects of P1 on
P-glycoprotein phosphorylation in MCF7-MDR cells were determined by
exposing the cells to P1 during a 3-h
[
P]P
-labeling period and recovering
P-labeled P-glycoprotein from lysates of P1-treated cells
by immunoprecipitation with the mAb C219. The immunoprecipitated
protein was subjected to SDS-PAGE analysis followed by autoradiography
of gels, as described under ``Materials and Methods.''
MCF7-WT (lane A), MCF7-MDR treated with 100 µM P1 (lane B), 50 µM P1 (lane C), 25
µM P1 (lane D), and 0 µM P1 (lane E) are shown. P-glycoprotein is the 160-kDa band. B, the effect of 100 µM P1 on P-glycoprotein
expression in MCF7-MDR cells was measured under the conditions of the
P-glycoprotein phosphorylation experiments by immunoblot analysis of
cell lysates with the mAb C219 (500 ng/ml), as described under
``Materials and Methods.'' Immunoblots corresponding to
untreated (lane 1) and P1-treated MCF7-MDR cells (lane
2) (25 µg of protein/lane) are shown. P-glycoprotein is the
band migrating at 160 kDa.
To determine whether the PKC-inhibitory effects of the N-myristoylated peptides in MCF7-MDR cells were restricted to
integral membrane protein PKC substrates such as P-glycoprotein, we
examined the phosphorylation state of the PKC substrate Raf-1 kinase (34) , which shuttles between the cytoplasmic compartment and
the plasma membrane of mammalian cells(35) . Raf-1 kinase has
been implicated in MDR(36) , and it plays a pivotal role in
PKC--mediated signal transduction(34) . Activation of
cellular PKC-
triggers a protein kinase cascade that begins with
PKC-
-catalyzed phosphorylation and activation of Raf-1 kinase and
ultimately results in the phosphorylation of nuclear
proteins(37) . Raf-1 kinase phosphorylation was analyzed by
immunoprecipitation of the protein from lysates of
P-labeled cells with a Raf-1 kinase-specific mAb. We found
that the phosphorylation of Raf-1 kinase was at least 10 times greater
in the MCF7-MDR line compared with the drug-sensitive line MCF7-WT (Fig. 5A, lanes F and G), when the
phosphorylation data in Fig. 5A were subjected to
densitometric analysis and then normalized for the approximately 2-fold
increase in Raf-1 kinase expression that we observed in the MDR line by
Western analysis (Fig. 5B). This increase in the
phosphorylation of the PKC-
substrate Raf-1 kinase in MCF7-MDR is
consistent with the reported 30-fold overexpression of PKC-
in the
cells(18) . As in the case of P-glycoprotein phosphorylation,
100 µM P1, P3, and P5 each potently inhibited Raf-1 kinase
phosphorylation in the MCF7-MDR cells, whereas 100 µM P7
was noninhibitory (Fig. 5A, lanes B-E).
In contrast, the peptides had little or no effect on Raf-1 kinase
expression in MCF7-MDR cells, according to immunoblot analysis of cell
lysates (Fig. 5B). Densitometric analysis revealed that
100 µM P3 and P5 each inhibited Raf-1 kinase
phosphorylation in MCF7-MDR cells by more than 95% (Fig. 5A, lanes C and D), and 200
µM P1 achieved a similar degree of inhibition (Fig. 5A, lane A). The inhibition of Raf-1
kinase phosphorylation achieved by 100 µM P1 was
approximately 80% (Fig. 5A, lane B).
Figure 5:
Inhibition of Raf-1 kinase phosphorylation
in MCF7-MDR cells by N-myristoylated synthetic peptide
substrate analogs of PKC. A, MCF7-WT or MCF7-MDR cells were
labeled with P for 3 h prior to detergent lysis of cells
and recovery of Raf-1 kinase by immunoprecipitation with a Raf-1 kinase
mAb as described under ``Materials and Methods.'' Where
indicated, MCF7-MDR cells were treated with N-myristoylated
synthetic peptide substrate analogs of PKC for the duration of the 3-h
labeling period. Immunoprecipitated Raf-1 kinase was analyzed by
SDS-PAGE followed by autoradiography. The same exposure period was used
for each lane. MCF7-MDR cells were treated with 200 µM P1 (lane A), 100 µM P1 (lane B), 100
µM P3 (lane C), 100 µM P5 (lane
D), or 100 µM P7 (lane E). In control lanes,
untreated MCF7-WT (lane F) and MCF7-MDR (lane G)
cells are shown. Raf-1 kinase migrated as a 72-kDa band. B,
immunoblot analysis of Raf-1 kinase expression in MCF7 cell lysates (50
µg of cell lysate protein/lane) using 10 µg/ml Raf-1 kinase
monoclonal antibody as the primary antibody is shown in the upper
panel; the lower panel shows the results of a control
immunoblot analysis done in the absence of primary antibody. The arrows indicate the positions of MCF7-MDR Raf-1 kinase (72
kDa); MCF7-WT Raf-1 kinase migrated slightly more rapidly (68 kDa).
Cells were treated with peptides as described for A. Lane
1, MCF7-WT; lane 2, MCF7-MDR; lane 3, P1-treated
MCF7-MDR; lane 4, P3-treated MCF7-MDR; lane 5,
P5-treated MCF7-MDR; lane 6, P7-treated MCF7-MDR. Peptides
were present at 100 µM. The results shown in A and B were reproducible in separate
experiments.
PKC--catalyzed Raf-1 kinase phosphorylation is preceded by
PKC-
activation and autophosphorylation(38, 39) .
To test whether the alterations in Raf-1 kinase phosphorylation shown
in Fig. 5A were reflective of changes in PKC-
activity, we analyzed the MCF7-WT and MDR cells for comparable changes
in PKC-
phosphorylation. Like Raf-1 kinase, PKC-
is localized
in the plasma membrane and the cytoplasmic compartment of mammalian
cells(17, 40) . Following a 3-h labeling period, a
major 82-kDa radiolabeled band corresponding to phosphorylated
PKC-
was detected in
P-labeled MCF7-MDR but not in
MCF7-WT by immunoprecipitation of the protein from the cell lysates
with a PKC-
-specific mAb (Fig. 6A, lanes A and F). Similarly, PKC-
was readily detected in an
MCF7-MDR cell lysate but not in an MCF7-WT cell lysate by immunoblot
analysis (Fig. 6B, lanes 1 and 2).
Densitometric analysis of Fig. 6A showed that as in the
case of Raf-1 kinase phosphorylation, PKC-
phosphorylation was
inhibited in MCF7-MDR cells >95% by 100 µM P3 (lane
C) and 100 µM P5 (lane D) and approximately
80% by 100 µM P1 (lane B); 100 µM P7
affected <25% inhibition of PKC-
phosphorylation (lane
E). The peptides had little or no effect on the expression of
PKC-
in MCF7-MDR cells under these experimental conditions
according to immunoblot analysis of cell lysates (Fig. 6B). At 200 µM, P1 inhibited
PKC-
phosphorylation in MCF7-MDR cells >95%, but P7 still
achieved <25% inhibition (data not shown). Thus, the N-myristoylated peptides P1, P3, and P5 potently inhibited the
phosphorylation of three PKC-
substrates (P-glycoprotein, Raf-1
kinase, and PKC-
) in MCF7-MDR cells under conditions where the
peptides restored intracellular drug accumulation, whereas the N-myristoylated peptide P7 inhibited the phosphorylation of
these PKC-
substrates very weakly or not at all and was without
effect on the uptake of cytotoxic drugs in the MDR breast cancer cells.
In most cases, the peptide concentration required for potent induction
of drug uptake by P1, P3, and P5 (100 µM) (Fig. 1)
caused
80% inhibition of the phosphorylation of P-glycoprotein,
Raf-1-kinase, and PKC-
in the MCF7-MDR cells (Fig. 4Fig. 5Fig. 6; Table 4), suggesting
that nearly complete inhibition of PKC-
catalysis may be required
for substantial reversal of MDR by the peptides.
Figure 6:
Inhibition of PKC- phosphorylation in
MCF7-MDR cells by N-myristoylated synthetic peptide-substrate
analogs of PKC. A, MCF7-WT or MCF7-MDR cells were incubated
with [
P]P
in phosphate-free medium
for 3 h as described under ``Materials and Methods.'' Where
indicated, MCF7-MDR cells were treated with synthetic peptides (P1, P3,
P5, and P7) for the duration of the labeling period.
P-labeled cells were lysed with detergent, and PKC-
was immunoprecipitated from the cell lysates, as described under
``Materials and Methods.'' Immunoprecipitated PKC-
was
detected by SDS-PAGE and autoradiography of the gel; the autoradiogram
was subjected to densitometric analysis. Results are shown for MCF7-WT (lane A), MCF7-MDR treated with 100 µM P1 (lane B), 100 µM P3 (lane C), 100
µM P5 (lane D), 100 µM P7 (lane
E), and untreated MCF7-MDR (lane F). PKC-
is the
radiolabeled band migrating at 82 kDa. B, immunoblot analysis
of PKC-
expression in MCF7 cell lysates (50 µg of
protein/lane) using 1 µg/ml PKC-
monoclonal antibody as the
primary antibody (12) is shown in the upper panel; a
control analysis done in the absence of the primary antibody is shown
in the lower panel. The arrows indicate the positions
of PKC-
(82 kDa). Cells were treated with peptides as described
for A. Lane 1, MCF7-WT; lane 2, MCF7-MDR; lane 3, P1-treated MCF7-MDR; lane 4, P3-treated
MCF7-MDR; lane 5, P5-treated MCF7-MDR; lane 6,
P7-treated MCF7-MDR. Peptides were present at 100 µM. The
results shown in A and B were reproducible in
separate experiments.
Prenylcysteine
methyl esters and several cyclic peptides have been shown to compete
for drug-binding sites on P-glycoprotein in studies of photoaffinity
labeling of the transporter with
[H]azidopine(41, 42) , and
several hydrophobic linear peptides have been reported to serve as
P-glycoprotein substrates (42, 43, 44, 45) . In contrast, the
amphiphilic linear peptides melittin and alamethicin do not interact
with P-glycoprotein(42) . To test whether the mechanism of MDR
reversal by the amphiphilic peptide P1 could involve direct
interactions between P1 and drug-binding sites of P-glycoprotein in
addition to inhibition of P-glycoprotein phosphorylation, we analyzed
the effects of P1 and related peptides on the photoaffinity labeling of
P-glycoprotein with [
H]azidopine in MCF7-MDR
cells. Results of photoaffinity labeling experiments done in whole MCF7
cells are shown in Fig. 7. A single prominent
[
H]azidopine-labeled band that corresponded to
P-glycoprotein (160 kDa) was observed in the lane corresponding to
untreated MCF7-MDR cells (lane B); the absence of this band in
the MCF7-WT sample (lane A) confirmed its identity as
P-glycoprotein. Exposure of the MCF7-MDR cells to vinblastine achieved
about 50% inhibition of the labeling of P-glycoprotein according to
densitometric analysis (lane C). Exposure to 100 µM P1, P3, and P5 (lanes D-F) had no inhibitory effect
on photoaffinity labeling of P-glycoprotein in MCF7-MDR cells. In fact,
the peptides actually enhanced the labeling of P-glycoprotein by
[
H]azidopine.
Figure 7:
Effects of N-myristoylated
synthetic peptide substrate analogs of PKC on photoaffinity labeling of
P-glycoprotein by [H] azidopine in MCF7-MDR
cells. Photoaffinity labeling of P-glycoprotein in MCF7-MDR cells was
done following preincubation of the cells with
[
H]azidopine and the peptide under investigation
in the dark, as described under ``Materials and Methods.''
Peptides were employed at a concentration of 100 µM.
Photoaffinity labeling was followed by extraction and
immunoprecipitation of P-glycoprotein and SDS-PAGE analysis.
[
H]Azidopine-labeled P-glycoprotein, which
migrated as a 160-kDa band, was quantitated by autoradiography and
computerized densitometry. Samples were untreated MCF7-WT (lane
A), untreated MCF7-MDR (lane B), MCF7-MDR treated with 30
µM VLB (lane C), 100 µM P1 (lane
D), 100 µM P3 (lane E), and 100 µM P5 (lane F).
Comparisons of the chemosensitivities of MCF7-MDR and MCF7-WT cells to cytotoxic drugs have shown that the relative resistances of MCF7-MDR cells to the P-glycoprotein substrates ADR and VLB are, respectively, 610- and 360-fold(18) . As a test of whether P1 could serve as a P-glycoprotein substrate in MCF7-MDR cells, we analyzed the MCF7-MDR cells for cross-resistance to P1 by comparing the growth-inhibitory activity of P1 and related peptides against MCF7-WT and MCF7-MDR cells. In these experiments, cells were exposed to the N-myristoylated peptides during the entire growth inhibition assay period (96 h; the 1-h exposure period employed in Fig. 3was not used because it would have required very high peptide concentrations to achieve >50% cell growth inhibition). Although statistically significant cross-resistance was observed in MCF7-MDR cells with each peptide, the very modest degree of cross-resistance to the pseudosubstrate peptides P1 and P3 (<1.5-fold) (Table 5) provided evidence that P1 and P3 did not serve effectively as P-glycoprotein substrates in the cells. In contrast, the cross-resistance of the cells to P5, which contains a sequence that corresponds to a PKC phosphorylation site in the EGF receptor, was pronounced (10-fold) (Table 5), providing evidence that P5 may be transported by P-glycoprotein. Ideally, an MDR reversal agent should be equipotent against drug-sensitive and MDR cancer cells in its direct growth-inhibitory effects, because cross-resistance of the MDR cells to the agent could necessitate its use at concentrations that are directly toxic to nontransformed cells to accomplish reversal of MDR in cancer cells in vivo. Thus, the minor degree of cross-resistance of the MCF7-MDR cells to the N-myristoylated pseudosubstrate peptides P1 and P3 (Table 5) further indicates the potential value of the pseudosubstrate peptides as MDR reversal agents. In contrast, the pronounced cross-resistance of the MCF7-MDR cells to the EGF receptor-related peptide P5 indicates that it is not appropriate for MDR reversal.
Previous reports have shown that the isozyme PKC- is
selectively overexpressed in human breast cancer MCF7-MDR
cells(18) , and artificial overexpression of PKC-
in MCF7
constructs that overexpress P-glycoprotein increases the drug
resistance of the cells in association with increased P-glycoprotein
phosphorylation(19) , providing evidence that
PKC-
-catalyzed P-glycoprotein phosphorylation may contribute to
MDR in MCF7 cells. In this report, we demonstrate that an N-myristoylated PKC-
pseudosubstrate peptide, N-myristoyl-FARKGALRQ(P1), partially reverses drug resistance
in MCF7-MDR by a novel mechanism that involves PKC-
inhibition. P1
induced cytotoxic drug accumulation in MCF7-MDR cells just as
effectively as the potent MDR reversal agent verapamil (Fig. 1)
in association with potent inhibition of P-glycoprotein phosphorylation (Fig. 4). P1 also inhibited the phosphorylation of two other
PKC-
substrates in MCF7-MDR cells, Raf-1 kinase (Fig. 5),
and PKC-
(Fig. 6) under these conditions. Thus, induction
of drug accumulation by P1 was associated with PKC-
inhibition in
MCF7-MDR cells. Based on the evidence described above (18, 19) that PKC-
-catalyzed P-glycoprotein
phosphorylation may be a contributing factor in the MDR phenotype of
MCF7-MDR cells, it is evident that the mechanism of MDR reversal by P1
most likely involves inhibition of P-glycoprotein phosphorylation.
The mechanism of P1-mediated MDR reversal clearly does not involve
competitive binding at [H]azidopine binding sites
on P-glycoprotein (Fig. 7). This distinguishes P1 from MDR
reversal agents such as PKC-inhibitory
staurosporines(47, 48) , verapamil, and cyclosporin A,
which are highly effective MDR reversal agents in vitro but
cannot be used to reverse MDR in vivo due to severe toxic
effects at therapeutic concentrations(24, 47) . It is
also evident that MDR reversal by P1 does not involve altered
P-glycoprotein expression and that it is not compromised by
cross-resistance in the MDR cells (Fig. 4B, Table 5). It
should be noted that modulation of the ATPase activity of isolated
P-glycoprotein and the [
H]VLB binding activity of
P-glycoprotein-containing membrane vesicles by phospholipid-interacting
peptides, such as melittin (42) and the N-myristoylated peptides described here(27) , cannot
be used to characterize interactions between the peptides and
P-glycoprotein because of the pronounced nonspecific effects of
phospholipid-interacting peptides in these assay systems (42) .
It is also worthwhile to note that because treatment of breast cancer
patients with tamoxifen, which is PKC-inhibitory at therapeutic
concentrations(49) , is associated with little toxicity, it
appears that PKC-inhibitory MDR reversal agents such as P1 could
potentially give rise to a new generation of MDR reversal agents that
are associated with acceptably low toxicity.
The inability of P1 to
antagonize [H]azidopine labeling of
P-glycoprotein in MCF7-MDR cells and the lack of cross-resistance of
MCF7-MDR cells to P1 suggest that P1 is not a P-glycoprotein substrate.
However, these results do not exclude the possibility that linear
myristoylated peptides such as P1 may interact with a P-glycoprotein
site that is nonoverlapping and distinct from the azidopine binding
site. In fact, the enhancement of [
H]azidopine
labeling of P-glycoprotein by P1 is suggestive of such interactions.
The enhanced labeling affected by P1 cannot be explained simply by the
inhibitory activity of P1 against P-glycoprotein phosphorylation,
because PKC-
-catalyzed P-glycoprotein phosphorylation increases
azidopine-labeling of the pump (46) . Nor can it be explained
by the amphiphilicity of P1, because other linear amphiphilic peptides
do not enhance azidopine labeling of P-glycoprotein(42) .
In
this study, we compared the effects of P1 and other N-myristoylated peptide-substrate analogs of PKC in MCF7-MDR
cells. In general, the ability of the N-myristoylated
peptide-substrate analogs to inhibit the phosphorylation of endogenous
PKC- substrates (P-glycoprotein, Raf-1 kinase, and PKC-
) in
MCF7-MDR cells correlated with restoration of drug uptake in the cells
by the peptides, i.e. P1, P3, and P5 were active, whereas P7
was inactive. Discrepancies within this general trend may be due to the
pronounced cross-resistance of the MCF7-MDR cells to P5, effects of the N-myristoylated peptides on P-glycoprotein in addition to
inhibition of the phosphorylation of the pump (these putative effects
are inferred from the enhancement of photoaffinity labeling of
P-glycoprotein by the peptides as discussed above), differences among
the amphiphilic peptides in their interactions with cell membranes,
etc. Furthermore, because P1 and P3 contain PKC-
pseudophosphorylation sequences(26, 27) , they are
likely to be recognized not only by PKC-
but also by other
proteins that interact with naturally occurring PKC-
phosphorylation sites, e.g. protein phosphatases. It is also
important to note that because of the overlapping substrate
specificities of PKC isozymes(50) , P1 and P3 may also
antagonize the function of several other PKC isozymes in MCF7-MDR
cells, and these inhibitory effects may contribute to their MDR
reversal activity. Finally, complexity is also introduced by the
existence of multiple mechanisms of drug resistance in drug-selected
MDR cancer cells that overexpress P-glycoprotein, such as MCF7-MDR.
Contributing factors to MDR can include multidrug resistance protein
(MRP), glutathione S-transferase, topoisomerases,
etc.(25) , and some of these drug resistance mechanisms might
also be affected by the peptides.
Studies of the inhibition of purified PKC by N-myristoylated peptide substrate analogs demonstrated that the N-myristoylated peptides interacted with the phospholipid cofactor, whereas their nonmyristoylated counterparts did not(27) . The membrane-active nature of the N-myristoylated peptides most likely accounts for their ability to access cellular PKC(26, 31, 33) . As the phospholipid cofactor concentration is increased in the PKC assay system, the inhibitory potency of the N-myristoylated peptides declines(27) , providing evidence that their inhibitory effects are subject to surface dilution. This may account for the sharp increase in the potencies of P1, P3, and P5 as inducers of drug uptake in MCF7-MDR cells when the bulk peptide concentration was increased from 50 to 100 µM.
Bioactive peptides
such as P1 are subject to proteolytic degradation, which often limits
their potency. This may explain the superior potency of P1 in inducing
drug uptake in short-term assays (6 h) compared with its potency in
reversing MDR over a 96-h time course. In some cases, this problem can
be overcome by designing retro-inverso analogs of the peptides. In a
retro-inverso analog, the sequence of the parent peptide is reversed,
and the residues are replaced by the corresponding D-enantiomers(51) . The side chain surfaces of parent
and retro-inverso peptides are similar or identical, but the topologies
of their backbones differ(51) . Thus, superior bioactivity can
be achieved by retro-inverso analogs, when the side chain surface
prevails in the interaction of the bioactive peptide with its
target(52) . A retro-inverso analog of a PKC-
pseudosubstrate peptide has been shown to potently inhibit
phosphorylation of a synthetic peptide-substrate by PKC(53) .
Efforts are now underway to design retro-inverso analogs of P1 and
related N-myristoylated peptides that are superior to the
parent peptides in the reversal of MDR in human breast cancer cells. In
a recent report, computer-based algorithms were successfully used to
predict MDR reversal activity based on structural features of compounds
entered into the data base (54) ; this type of strategy may
also be useful for designing peptidic or peptidomimetic P1 analogs with
optimized MDR reversal activity.