Cisplatin induces renal expression of P-glycoprotein and
canalicular multispecific organic anion transporter
Michel
Demeule,
Mathieu
Brossard, and
Richard
Béliveau
Laboratoire de Médecine Moléculaire, Département
de Chimie-Biochimie, Université du Québec à
Montréal, Montreal, Quebec H3C 3P8; and Hôpital
Sainte-Justine, Montreal, Quebec, Canada H3T 1C5
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ABSTRACT |
The expression of two members of the ATP-binding cassette family
of transport proteins, P-glycoprotein (P-gp) and the canalicular multispecific organic anion transporter (cMOAT or Mrp2), was evaluated in renal brush-border membranes (BBM) and various rat tissues after
cisplatin treatment. One administration of cisplatin (5 mg/kg)
increased P-gp expression by >200-300% in renal BBM and in
crude membranes from liver and intestine. The increase in P-gp expression in the kidney was also detected in photolabeling
experiments, suggesting the induction of functional P-gp. cMOAT
expression was increased by >10-fold in renal BBM after cisplatin
administration, although it had no effect on liver cMOAT expression.
The increase in the levels of both proteins was maximal at 2 days after
cisplatin treatment and lasted for at least 8 days. These results
indicate that a single administration of cisplatin induces
overexpression of P-gp and cMOAT in specific tissues. This may be of
significant relevance to the design of clinical trials using cisplatin
as a single chemotherapeutic agent or in combination with other drugs.
multidrug resistance; Mrp2; renal brush-border
membranes
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INTRODUCTION |
CANCER CELLS MAY BECOME resistant to a range of drugs
that have different structures and cellular targets during
chemotherapeutic treatment with a single drug (14, 23). In humans,
multidrug resistance (MDR) is associated with membrane proteins that
are members of the ATP-binding cassette (ABC) family. Two members of
this family, P-glycoprotein (P-gp) and canalicular multispecific organic anion transporter (cMOAT), seem very important for the establishment of the MDR phenotype in cancer cells (14, 35).
P-gp was identified as an ATP-dependent transporter that excludes from
cells a wide variety of unmodified hydrophobic substrates, including
Vinca alkaloids, colchicine, antibiotics, and anthracyclines (12, 14,
23). P-gp is expressed in a variety of normal secretory tissues such as
kidney, intestines, liver and at high levels in the endothelial cells
of brain capillaries (5, 14, 19).
A high ATP-dependent transport activity for organic anions was reported
in the canalicular membrane of hepatocytes and was identified as the
cMOAT (Mrp2; see Refs. 16-18, 24, and 29). This transporter may
contribute to drug resistance by transporting a wide range of
glutathione, glucoronate, and sulfate conjugates out of cells by an
ATP-dependent mechanism. This GS-X pump, a 190-kDa membrane
glycoprotein, is mainly expressed in the canalicular membrane of
hepatocytes but was also shown to be present in renal brush-border
membrane (BBM), intestines, and in several multidrug-resistant cell
lines selected for cisplatin resistance (9, 35).
Cisplatin
[cis-dichlorodiammineplatinum(II)]
is an effective antineoplastic agent used in the treatment of various
solid tumors. However, cancer cells may acquire resistance during
cisplatin treatment by limiting its cellular concentration and by
altering its interaction with DNA. Cancer cells may also have the
ability to sequester or inactivate cisplatin and hence reduce its
toxicity. The resistance to cisplatin, which is not a P-gp substrate,
is not associated with P-gp or MRP1 expression (15). However, it was
also shown that cisplatin may increase the expression of P-gp in some
cisplatin-resistant cell lines (27). A direct interaction between
cisplatin and glutathione (1:2 molar ratio) has also been reported
(35). This cisplatin-glutathione conjugate was transported in an
ATP-dependent manner by cMOAT, and a striking correlation was obtained
between cisplatin resistance and cMOAT expression in
cisplatin-resistant cell lines.
In the present study, the expression of P-gp and cMOAT was evaluated in
rat tissues after cisplatin treatment. P-gp and cMOAT levels were
evaluated by Western blot analysis using the monoclonal antibody (MAb)
C219, which recognizes P-gp, and with a polyclonal antibody (PAb)
directed against the COOH-terminal portion of cMOAT. Our results
suggest that cisplatin, a nonstandard P-gp substrate, can induce P-gp
expression in specific tissues. Furthermore, we are also reporting that
a single administration of cisplatin induces cMOAT expression in renal
BBM. Because cisplatin is widely used, therapies using this agent must
therefore be carefully designed to be efficient.
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MATERIALS AND METHODS |
Materials. Cisplatin was obtained from
Bristol Myers Squibb (Montreal, PQ) and Sigma Chemical (Oakville, ON).
A Mini-Protean II apparatus for electrophoresis and electrophoresis
reagents were from Bio-Rad (Mississauga, ON). Polyvinylidene difluoride membranes and a Milliblot-Graphite electroblotter I were from Millipore
(Mississauga, ON). MAb C219 directed against P-gp and MAbs directed
against MRP1 (QCRL-1 and MRPr1) were from ID Laboratories (London, ON).
MAb 6/1C, which is specifically directed against human MDR1, was
purchased from Kamiya Biomedical (Seattle, WA). Anti-mouse and
anti-rabbit IgG horseradish peroxidase-linked whole antibody and
enhanced chemiluminescence (ECL) reagents were purchased from Amersham
(Oakville, ON).
[125I]iodoaryl
azidoprazosin (IAAP) was purchased from DuPont-New England Nuclear
(Markham, ON). All other reagents were from Sigma Chemical (Oakville, ON).
Cisplatin treatments. Male
Sprague-Dawley rats weighing 300-350 g were treated with a single
subcutaneous injection of cisplatin (5 mg/kg) diluted in sterile
saline. For each treatment, each group of animals comprised five rats.
The control and the treated groups received single injections of saline
or cisplatin, respectively. Rats were killed 1, 2, 4, 6, 8, and 15 days
after cisplatin administration, and tissue sampling was performed.
Isolation of tissue crude membrane fractions, renal
BBM, and brain capillaries. Crude membrane fractions
were prepared from intestine, liver, heart, lung, and stomach of
control and treated rats. Tissues from individual animals were pooled
and homogenized in a buffer containing 250 mM sucrose and 5 mM
HEPES-Tris (pH 7.4), with a Polytron tissue homogenizer (Brinkmann
Instruments, Rexdale, ON), and the homogenates were centrifuged at
3,000 g for 10 min. The supernatants
were then centrifuged at 33,000 g for
30 min, and the pellets containing the crude membrane fractions were
resuspended in 50 mM mannitol and 20 mM HEPES-Tris, pH 7.5, and were
stored at
80°C. Renal BBM were prepared by the
MgCl2 precipitation method of
Booth and Kenny (2). Purified membranes were resuspended in 50 mM
mannitol and 20 mM HEPES-Tris, pH 7.5, and were stored at
80°C. Brain capillaries of control and treated rats were
purified from brain cortex by the method of Dallaire et al. (6).
Protein content was determined in all experiments with the method of
Bradford (3).
Detection of P-gp and cMOAT. P-gp and
cMOAT were detected by Western blot analysis. SDS-PAGE was performed
according to the method of Laemmli (22). Membranes were resuspended in
sample buffer to a final concentration of 1 mg/ml and were loaded on 6.25 or 7.5% acrylamide-bisacrylamide (29.1:0.9) gels without prior
heating. P-gp was detected using MAb C219 as described previously (19),
whereas cMOAT was evaluated using a PAb directed against its
COOH-terminal portion. Horseradish peroxidase-conjugated antibodies directed against mouse and rabbit IgGs were used as secondary antibodies. Detection was made with ECL reagents according to the
manufacturer's instructions. The blots were exposed to preflashed Fuji films.
Photoaffinity labeling with
[125I]IAAP.
Renal BBM proteins (100 µg) from control and cisplatin-treated rats
were incubated with 20 nM of
[125I]IAAP in 20 mM
Tris · HCl, pH 7.5, supplemented with proteinase inhibitors (2 µg/ml aprotinin, 10 µg/ml pepstatin, and 100 µg/ml bacitracin). The incubation was carried out for 1 h at 25°C in the
dark and was followed by cross-linking under a Spectroline ultraviolet
lamp (Fisher Scientific, Montreal, PQ) at 254 nm for 5 min at 4°C.
The labeled P-gp was recovered by immunoprecipitation with MAb C219 and
protein A-Sepharose beads, as described in Jetté et al. (19).
Enzyme assays. Leucineaminopeptidase
activity in membrane samples was measured spectrophotometrically at 820 nm after 10 min at 37°C using leucine
p-nitroanilide (1 mM) as the substrate
(11).
-Glutamyltranspeptidase activity was assayed using
L-
-glutamyl-p-nitroanilide as the substrate. The reaction was performed at 37°C for 5-15 min as described previously (19).
Immunization and antibody
purification. The peptide EAGIENVNHTEL, corresponding
to a COOH-terminal portion of cMOAT (30), was synthesized using
multiple antigenic peptide system (MAPS) chemistry and was
obtained from Service de Séquence de Peptides de l'Est du
Québec (Centre Hospitalier de l'Université Laval). On
day 0, two rabbits were injected
subcutaneously at two sites with a total of 400 µg of the cMOAT
peptide mixed with Freund's complete adjuvant (Pierce, Rockford, IL)
in a 1:1 volume ratio, i.e., 0.5 ml adjuvant and 0.5 ml of solution
containing 400 µg peptide. Booster injections with 100 µg of
antigen were administered on day 28 using Freund's incomplete adjuvant. The final injection (day 50) consisted of 150 µg of
peptide adsorbed to aluminum hydroxide (Imject Alum; Pierce),
administered subcutaneously at two sites. The rabbits were bled 18 days
after the final injection. The blood was allowed to coagulate at room
temperature for 1 h and was stored overnight at 4°C to allow the
clot to retract. The serum was collected after removal of blood cells
by centrifugation at 10,000 g for 10 min. The serum was passed through a 22-ml protein A-Sepharose 4 Fast
Flow column (Pharmacia, Dorval, PQ) equilibrated with 50 mM
Tris · HCl, pH 8.6, using a Fast Protein Liquid
Chromatography apparatus (FPLC; Pharmacia). The IgGs were eluted with
0.2 M glycine, pH 2.2. The fractions (5 ml) were collected in 0.8 ml of
1 M Tris · HCl, pH 9. The antibodies were further
purified by affinity chromatography on a peptide-Sepharose column
prepared as previously described. The IgGs were passed through this
column equilibrated with 100 mM NaCl and 20 mM
Tris · HCl, pH 7.5 (buffer
A). After extensive washing, the antibodies were eluted
from the column with 0.2 M glycine, pH 2.2. The neutralized fractions
that contained IgGs directed against cMOAT peptide were pooled and
dialyzed overnight against buffer A and adjusted
to the desired concentration by ultrafiltration.
Densitometric and statistical
analyses. The intensities of the bands obtained from
Western blot analysis and the photolabeling studies were estimated with
a Personal densitometer SI (Molecular Dynamics, Sunnyvale, CA).
Statistical analyses were made with the Student's paired
t-test using Microsoft Excel.
P < 0.05 was considered significant.
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RESULTS |
Detection of P-gp in tissues from cisplatin-treated
rats. Rats were treated with cisplatin to study its
effects on P-gp expression in normal tissues. Tissues were isolated
from rats 4 days after a single subcutaneous injection of cisplatin (5 mg/kg). P-gp expression was evaluated by Western blot analysis with MAb
C219 (Fig. 1). P-gp was detected by MAb
C219 as a 140- to 180-kDa protein in membranes isolated from control
and treated rats. After cisplatin administration, an increase in the
expression of P-gp was detected in specific tissues, including
intestine, kidney, and liver. The increase in P-gp expression was
quantified by laser densitometry, and levels of P-gp were expressed as
a percentage of the corresponding control groups (Fig.
2). In liver, kidney, and intestine, the amount of P-gp detected was increased to 330, 270, and 250% compared with control groups, respectively. Western blot analysis using a MAb
directed against human MDR1 (MAb 6/1C) was performed in an attempt to
identify the P-gp isoform induced by cisplatin. Unfortunately, no
reliable signal was obtained for membrane preparations from control and
cisplatin-treated rats.

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Fig. 1.
Immunodetection of P-glycoprotein (P-gp) in normal tissues 4 days after
cisplatin treatment. Rats were treated with saline or 5 mg/kg of
cisplatin. Western blot analysis: protein samples from control (C) and
cisplatin-treated (T) tissues were resolved by SDS-PAGE.
Immunodetection was performed with monoclonal antibody (MAb) C219. One
representative experiment is shown. BBM, brush-border membrane.
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Fig. 2.
Quantification of P-gp expression in normal tissues 4 days after
cisplatin treatment. Immunoreactive protein bands detected by Western
blot analysis in Fig. 1 were evaluated by laser densitometry. P-gp
levels are expressed as a percentage of the total amount of
immunoreactive protein present in control tissues. Values represent
means ± SE obtained from 6 (liver, kidney, intestine) or 3 (intestine, heart, lung, brain capillaries) independent experiments.
* P < 0.05.
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Photolabeling of P-gp by
[125I]IAAP.
Photoaffinity labeling experiments were performed on BBM isolated from
control and treated groups (Fig. 3) to
measure the binding activity of P-gp. The amount of photolabeled P-gp
in BBM from treated rats was increased by 2.75-fold. The increase in P-gp measured by photolabeling is very similar to the value measured by
Western blot analysis. Photoaffinity labeling experiments were also
performed on intestine and liver isolated from control and treated
groups. We were unable to detect photolabeled P-gp in these tissues,
probably because the expression of P-gp is too low (data not shown).

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Fig. 3.
[125I]iodoaryl
azidoprazosin (IAAP) photolabeling of P-gp in kidney after cisplatin
treatment. CHRC5 membranes (C5), which overexpress P-gp,
and kidney BBM isolated from control and cisplatin-treated rats on
day 4 were incubated with IAAP and
cross-linked with ultraviolet light. P-gp was immunoprecipitated with
MAb C219, and gels were exposed to preflashed Kodak films for 1 h
(CHRC5 membranes) or 2 wk (renal BBM);
n = 3 rats.
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Detection of cMOAT in tissues from cisplatin-treated
rats. An antibody directed against the EAGIENVNHTEL
peptide of cMOAT was produced in rabbit and purified on a protein
A-Sepharose column followed by a peptide-affinity column. In Western
blot analysis, this antibody recognized a 190-kDa protein in normal
liver (Fig. 4A). In
the kidney, a faint band was immunodetected at the same molecular
weight. Aside from cMOAT, this antibody also detected other proteins
with molecular weights of 155, 110, and 68 kDa. After cisplatin
treatment, there was a strong increase in the cMOAT level in renal BBM,
whereas all other detected proteins were unaffected. The 190-kDa band
corresponding to cMOAT was scanned by laser densitometry. Cisplatin
induced a 7 ± 1.2-fold increase of cMOAT expression in renal BBM,
whereas its level was unchanged in liver. When the samples were boiled
before SDS-PAGE, the band corresponding to cMOAT in renal BBM and liver
on Western blots completely disappeared, whereas the other nonspecific
bands were still detected (Fig. 4A).
This experiment strongly suggests that the 190-kDa band induced by
cisplatin in renal BBM is indeed cMOAT, since it was previously
detected by Western blots in unheated samples (30), indicating its heat
sensitivity.

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Fig. 4.
Immunodetection of canalicular multispecific organic anion transporter
(cMOAT) after cisplatin and lipopolysaccharide (LPS) treatments. Rats
were treated with a single administration of saline or 5 mg/kg of
cisplatin and were killed on day 4.
A: Western blot analysis: protein
samples (50 µg) of renal BBM and liver membranes from control and
cisplatin-treated rats were loaded onto SDS-PAGE, with (+) or without
( ) boiling, and were subjected to electrophoresis. Immunoblots
were performed using PAb directed against cMOAT or preimmune serum and
enhanced chemiluminescence (ECL) reagents, as described in
MATERIALS AND METHODS. B:
Western blots were also performed on protein samples (50 µg) of brain
capillaries and heart and lung membranes from control and
cisplatin-treated rats. cMOAT in renal BBM from cisplatin-treated rats
was detected as a positive control. C:
rats were treated with a single intraperitoneal injection of saline or
LPS (4 mg/kg) and were killed 24 h later as previously described (38).
cMOAT was immunodetected by Western blots in protein samples (50 µg)
of crude membranes isolated from saline ( ) or LPS-treated (+)
rats. Kidney BBM from cisplatin-treated rats (+) was used again as a
positive control
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In addition to cMOAT, the PAb recognized a number of lower
molecular weight bands. However, these bands were also observed using
preimmune serum (Fig. 4A), suggesting that they were not related to cMOAT. In addition, negative controls were performed using
heart membranes, brain capillaries, and lung membranes from control and
cisplatin-treated animals (Fig. 4B).
In these tissues, cMOAT was undetected in control and cisplatin-treated
samples. Lower nonspecific molecular weight bands were still detected
in these tissues.
Because previous studies reported that lipopolysaccharides (LPS)
downregulate cMOAT in liver (30, 38), rats were treated with LPS to
confirm that the 190-kDa band was cMOAT (Fig.
4C). As expected, the detection of
the 190-kDa protein decreased in liver crude membranes isolated
from LPS-treated rats, whereas nonspecific bands of lower molecular
weight were unaffected by the treatment. These results presented in
Fig. 4 show that the 190-kDa band corresponds to cMOAT and that
cisplatin induced its expression in the kidney. Attempts were also made
to detect MRP1 in these membrane preparations by Western blot analysis
using two MAbs (QCRL-1 and MRPr1) that are commercially available. No signal was detected with either MAb because of their low sensitivity or
due to the low expression of MRP1 in these tissues (data not shown).
Effect of cisplatin treatment on membrane
markers. At 4 days after cisplatin treatment, the
activity of various membrane marker enzymes was measured on
preparations from kidney, liver, brain capillaries, and intestine
(Table 1). Leucineaminopeptidase and
-glutamyltranspeptidase were unaffected by cisplatin, suggesting that the increased levels of P-gp in these membranes are specific to
this protein. The enrichment of these membranes from control and
treated rats was also similar for all cisplatin treatments (data not
shown), suggesting that cisplatin does not affect those physicochemical
properties of membranes that are used for their isolation. Alkaline
phosphatase activity in intestine was reduced by 45% by cisplatin,
whereas it remained unaffected by the drug in renal BBM and liver. In
addition, cytochrome P-450 was
measured in homogenates from liver, kidney, and intestine by Western
blot using a PAb that recognizes the 3A1 and 3A2 isoforms (Fig.
5). Levels of cytochrome
P-450 were unaffected by cisplatin in
liver and kidney homogenates. This latter result indicates that there is no correlation between the increase in P-gp and cMOAT expression and
the cytochrome P-450 isoforms in the
tissues studied.

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Fig. 5.
Immunodetection of cytochrome P-450
3A,1/2 (Cyt P450) isoforms after cisplatin treatment. Rats were treated
with a single administration of cisplatin (5 mg/kg) and were killed on
day 4. Western blot analysis: protein
samples (40 µg) of homogenates from liver and kidney isolated from
control and cisplatin-treated rats were resolved by SDS-PAGE.
Immunoblots were performed using polyclonal antibody (PAb) directed
against cytochrome P-450 3A,1/2
isoforms and ECL reagents, as described in MATERIALS AND
METHODS. One representative experiment is shown; 4 experiments
were performed.
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Time course of the induction of P-gp
expression. Western blot analysis was used to follow
P-gp expression in membranes from liver (Fig.
6A),
kidney (Fig. 6B), and intestine
(Fig. 6C) isolated 1, 2, 4, 6, 8, and 15 days after a single administration of cisplatin. The density of
the band corresponding to P-gp was estimated by laser densitometry.
Levels of P-gp were described as a percentage of the values obtained
from animals injected with saline (Fig. 7).
The increase in P-gp levels in these three tissues increased rapidly
and was detectable 1 day after cisplatin injection. In these tissues,
the increased P-gp expression was maximal between days
2 and 6. Levels of
P-gp remained elevated for a longer time in liver than in kidney and
intestine. In intestine, many bands of lower molecular weight than P-gp
were detected (Fig. 1). Because this tissue contains a high
concentration of proteases, these bands could be P-gp degradation
products. Western blot studies of crude membranes from intestine were
difficult to perform due to these high protease levels. Nevertheless,
the elevated P-gp expression returned to control values at 8 and 15 days after cisplatin treatment in intestine and kidney, respectively,
whereas it was still twofold higher in liver after 15 days.

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Fig. 6.
Time course of P-gp expression after cisplatin treatment. Rats were
treated with one administration of saline or 5 mg/kg of cisplatin and
were killed 1, 2, 4, 8, and 15 days after the treatment. Protein
samples (50 µg) of membranes from liver
(A), kidney
(B), and intestine
(C) isolated from control and
cisplatin-treated rats were resolved by SDS-PAGE with a 6.25%
acrylamide gel. Immunoblots were performed with MAb C219, as described
in MATERIALS AND METHODS.
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Fig. 7.
Quantification of P-gp expression after cisplatin treatment.
Immunoreactive bands detected by Western blot analysis in Fig. 6 were
evaluated by laser densitometry. P-gp levels are expressed as a
percentage of the total amount of immunoreactive protein present in
liver (A), kidney
(B), and intestine
(C) membranes isolated from control
rats. Values represent means ± SE for 2 independent experiments for
liver and kidney and means ± SD for 1 experiment for intestine.
Immunodetection of P-gp for each group of rats was performed in
duplicate at least.
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Time course of the induction of cMOAT
expression. cMOAT expression in liver and kidney was
also estimated at 1, 2, 4, 6, 8, and 15 days after cisplatin treatment
(Fig. 8). In liver, cMOAT expression was
unaffected by this cisplatin treatment (Fig.
8A). In contrast, cMOAT expression
was strongly increased in renal BBM isolated from cisplatin-treated
rats (Fig. 8B). The 190-kDa band
corresponding to cMOAT in the samples was scanned, and the results were
expressed as a percentage where 100% equalled the value from control
rats (Fig. 9). Although cMOAT levels
remained similar in liver from control and cisplatin-treated rats, its expression in renal BBM increased by at least 10-fold 2 days after cisplatin administration. This increase in cMOAT expression was even
stronger than that measured in previous treatments (Fig. 4). The cMOAT
level in renal BBM remained high for at least 8 days and then returned
to control values by 15 days after cisplatin treatment.

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Fig. 8.
Time course of cMOAT expression after cisplatin treatment. Rats were
treated with one administration of saline or 5 mg/kg of cisplatin and
were killed 1, 2, 4, 8, and 15 days after the treatment. Protein
samples (50 µg) of membranes from liver
(A) and kidney
(B) isolated from control and
cisplatin-treated rats were resolved by SDS-PAGE with a 6.25%
acrylamide gel. Immunoblots were performed with PAb directed against
cMOAT, as described in MATERIALS AND METHODS.
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Fig. 9.
Quantification of cMOAT expression after cisplatin treatment.
Immunoreactive bands detected by Western blot analysis in Fig. 7 were
evaluated by laser densitometry. cMOAT levels are expressed as a
percentage of the total amount of immunoreactive protein present in
liver crude membranes ( ) and renal BBM ( ) isolated from control
rats. Values represent means ± SE for 2 independent experiments.
Immunodetection of cMOAT after each treatment was performed at least in
duplicate.
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DISCUSSION |
We investigated the effect of a single injection of cisplatin (5 mg/kg)
on the endogenous expression of P-gp and cMOAT in normal tissues. This
dose of cisplatin corresponds to chemotherapeutic levels known to
induce mild renal failure in rats (13, 34). In addition, because renal
insufficiency and both morphological and functional alterations in
renal cortex mitochondria have also been reported to be maximal on
days 3-5 after cisplatin
treatment (13, 33), we chose day 4 after cisplatin treatment for collecting tissues. We used an
immunoblotting procedure to detect the expression of both proteins.
After this injection, P-gp levels were increased in liver, kidney, and
intestine, whereas cMOAT level was increased only in kidney. These
results show that cisplatin can modulate the expression of both
proteins in specific tissues in vivo.
Alkaline phosphatase,
-glutamyltranspeptidase, and
leucineaminopeptidase activities in the kidney were shown to be
inhibited by cisplatin in vitro, whereas they were unaffected by
cisplatin in vivo (7). As was reported by the latter study, our results indicate that these three enzyme activities were similar after in vivo
treatment with cisplatin, not only in the kidney but also in liver. The
only enzyme affected by the drug was intestinal alkaline phosphatase.
Cisplatin, which causes profound gastrointestinal symptoms for many
days after drug administration, was also shown to produce morphological
changes in the small intestinal mucosa and to affect the activities of
intestinal enzymes (1, 20). Nevertheless, our results suggest that the
effects of cisplatin treatment on P-gp and cMOAT expression are rather
specific, since the two other marker enzymes were unaffected in these
three tissues.
Overlaps in substrate specificities between cytochrome
P-4503A and P-gp have been reported,
suggesting that these enzymes have complementary roles in the
pharmacokinetics of drug absorption and elimination that are
particularly relevant to cancer chemotherapy (37). Changes in
cytochrome P-4503A and P-gp levels in
rat liver were also observed after administration of hydroxyethyl
cyclosporin A (39). Altered levels of these two enzymes could
drastically affect the delivery of cancer chemotherapeutic agents.
Cisplatin treatment was shown to increase the level of
P-4502C3 in renal microsomes and of
P-4502A1, 2C7, 2E1, 4A2, and 4A3 in
hepatic microsomes and to decrease the levels of
P-4502C11 and 3A2 in rat liver
(28). In the present study, the cytochrome
P-450 3A1 and 3A2 isoforms in
homogenates from liver, kidney, and intestine were unaffected after
cisplatin administration. Thus our results indicate that there is no
parallel between the induction of P-gp or cMOAT caused by cisplatin and
the total levels of cytochrome P-450
3A,1/2 isoforms.
In the present study, the increase in P-gp levels caused by cisplatin,
which is not a classic substrate for P-gp, suggests that P-gp is
induced in specific tissues in response to cisplatin treatment.
Intracellular events leading to the overexpression of P-gp by cisplatin
remain to be established. However, previous studies reported that the
human Y-Box binding protein (YB-1), a member of a DNA-binding protein
family, was directly involved in MDR1 gene activation in response to
genotoxic stress (27). The YB-1 concentration was shown to be much
higher in cisplatin-resistant cell lines than in equivalent
cisplatin-sensitive cell lines (26, 27). Furthermore, transfectants
with lower levels of YB-1 presented increased sensitivity to cisplatin,
mitomycin, and ultraviolet light, suggesting that YB-1 may protect
cells from the cytotoxic effects of agents that induce cross-linking of
DNA (26, 27). Thus cisplatin may induce P-gp expression as do many of
its substrates, by a mechanism that involves YB-1. These studies
suggest that the induction of P-gp in specific tissues (liver, kidney,
and intestine) may be related to tissue toxicity and also to YB-1 distribution. Because it was previously reported that cisplatin is not
a P-gp substrate (15), we can speculate that the overexpression of P-gp
by cisplatin will not modify the pharmacokinetics of this drug.
However, the pharmacokinetics and the distribution of coadministered drugs that are P-gp substrates may be strongly changed after the induction of P-gp in these secretory tissues.
The increase of P-gp expression in the kidney after cisplatin treatment
may be related to its renal toxicity, which is morphologically characterized by tubular necrosis, loss of microvilli, alterations in
number and size of lysosomes, and mitochondrial vacuolization in
proximal tubular epithelial cells (21). The wide tissue distribution of
P-gp suggests that this protein may be involved in protective mechanisms against occasionally encountered toxins or commonly encountered toxic natural products (5, 10). In mdr1a knockout mice,
P-gp was shown to play an important role in the defense of the
organism, since its absence affected the pharmacokinetics and tissue
distribution of vinblastine and ivermectin (31). Modulation of P-gp
expression in vitro by cytotoxic drugs was reported (4). Chronic
administration of the chemosensitizer cyclosporin A (CsA) to rats also
induced overexpression of P-gp in various tissues such as kidney,
intestine, liver, stomach, heart, lungs, testis, and spleen (19). The
increased P-gp expression in kidney after cisplatin treatment may be
involved in the defense of the kidney against the stress caused by this
drug. In addition, the increased P-gp expression in kidney caused by
cisplatin was also detected by photoaffinity labeling, suggesting the
induced P-gp is functional. In intestine and liver, the induction of
P-gp expression was undetected with photoaffinity labeling experiments, indicating that P-gp levels in these tissues are too low to be labeled
by IAAP.
cMOAT (Mrp2), a novel ATP-dependent export pump for amphiphilic anionic
conjugates, which has been cloned from liver, was also identified in
rat kidney and was localized to the apical membrane domain of proximal
tubule epithelia (30). In rat, mrp2 mRNA was highly expressed in liver and at lower levels in kidney, duodenum, and ileum (9, 29). Using the amino acid sequence EAGIENVNHTEL
at the COOH-terminal region of the rat Mrp2 protein, as described
previously (8, 30, 38), we raised a PAb directed against this protein.
As previously reported, the level of cMOAT immunodetected was higher in
liver than in the renal BBM of normal rats. Previous studies
demonstrated that cisplatin-resistant cell lines contained increased
levels of cMOAT (9, 35). However, its expression does not seem
essential for all cases of cisplatin resistance, since some highly
resistant cell lines do not contain detectable levels of cMOAT.
Nevertheless, data reported by various groups suggested that cMOAT
could contribute to cisplatin resistance by exporting the
cisplatin-glutathione complex, which is formed by one molecule of
cisplatin and two molecules of glutathione (9, 18, 35). cMOAT function
is also impaired in various experimental models of intrahepatic and
obstructive cholestasis (36) or in Groningen yellow/transport-deficient
and Eisai hyperbilirubinemic rats (18). Steady-state levels of mRNA for
cMOAT were also examined in human lung cancer specimens in relation to
platinum drug resistance (25). It was found that cMOAT expression
levels did not correlate with platinum drug exposure but that MRP and
-glutamylcysteine synthetase genes correlated with the exposure of
human lung cancers to platinum drugs. Unfortunately, we were unable to
detect cMOAT in lung, even after cisplatin treatment, suggesting that
its expression in this tissue in rats may be too low for Western
blotting analysis.
In normal rats, ~47% of the initial cisplatin dose is excreted by
the kidney, whereas 1-5% is excreted by the liver. Urinary excretion of cisplatin involves glomerular filtration and tubular secretion (32). In the present study, only one injection of cisplatin
was necessary to induce the expression of cMOAT in renal BBM. In
addition, the large increase in the expression of cMOAT in renal BBM
suggests that this transporter may be involved in the handling or
excretion of cisplatin by the kidney. In contrast, the lack of
induction of cMOAT expression in liver by cisplatin could be a
consequence of the already high endogenous levels of cMOAT (16, 24, 29)
or may be because this tissue is not the principal one involved in the
elimination of the drug.
In conclusion, there was a rapid modulation of P-gp and cMOAT
expression in specific tissues. The increase in expression for both
proteins is rapid (1 day of treatment). In comparison, 5 days of
administration of cyclosporin A, a P-gp substrate that is also
nephrotoxic, were needed to increase P-gp expression by 50-100%
in various rat tissues (19), whereas one administration of cisplatin
increased P-gp expression by >200-300% in liver, kidney, and
intestine. Furthermore, in the present study, cMOAT expression
increased >10-fold in renal BBM after cisplatin treatment. cMOAT and
P-gp levels remained high for many days after the treatment. These
results suggest that the increase of both protein levels depends on
early events induced by cisplatin rather than on later responses caused
by irreversible binding of cisplatin to macromolecules (32). The return
of both proteins to control values may depend on cisplatin elimination
and also on the turnover of these proteins. Because cisplatin is widely
administered in different situations, the determination of the
molecular mechanisms involved in the modulation of these two important
ABC transporters is crucial for future clinical trials using this drug.
 |
ACKNOWLEDGEMENTS |
We thank Pascale Chamberland and Malika Robichaud for technical
support and Dr. É. Beaulieu for scientific advice.
 |
FOOTNOTES |
This work was supported by grants from the Natural Sciences and
Engineering Research Council of Canada and the Foundation Charles-Bruneau.
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: R. Béliveau, Laboratoire de Médecine moléculaire,
Département de chimie-biochimie, Université du Québec
à Montréal, C.P. 8888, Succursale centre-ville,
Montréal, Québec, Canada H3C 3P8 (E-mail:
oncomol{at}er.uqam.ca).
Received 22 October 1998; accepted in final form 8 June 1999.
 |
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