[Cancer Research 61, 1693-1698, February 15, 2001]
© 2001 American Association for Cancer Research
Drug Resistance Induced by Ouabain via the Stimulation of MDR1 Gene Expression in Human Carcinomatous Pulmonary Cells1
Franck Brouillard,
Danielle Tondelier,
Aleksander Edelman and
Maryvonne Baudouin-Legros2
Institut National de la Santé et de la Recherche Médicale U.467, Faculté de Médecine Necker, 75015 Paris, France
 |
ABSTRACT
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The inhibition of the Na+/K+-ATPase by
cardiotonic drugs like ouabain deeply perturbs both the properties of
the cell membrane and the ionic composition of the cytoplasm and hence
alters fundamental cell reactions. These three types of reactions may
be involved in the stimulation of multidrug resistance 1
(MDR-1) gene expression and the synthesis of
permeability glycoprotein [P-glycoprotein (P-gp)]. We have determined
whether ouabain, which binds to an extracellular motif of the
Na+/K+-ATPase, stimulates MDR-1
gene expression by measuring both mRNA and protein and whether the
resulting P-gp extrudes hydrophobic compounds and causes resistance to
antimitotic agents. The experiments were performed on Calu-3 cells, a
human cell line from a pulmonary carcinoma. Northern blotting showed
that treating the cells with submicromolar concentrations of ouabain
stimulated MDR-1 gene expression within 24 h. The
ouabain-induced stimulation of MDR-1 expression was not
restricted to Calu-3 cells but also occurred in human carcinomatous
colon (T-84 and HT-29) and hepatic (H7V3) cells. However, it is not
ubiquitous because it was not found in HeLa cells. The stimulation was
reproduced by other Na+/K+-ATPase inhibitors
and occurred via enhanced gene transcription, apparently due to the
increased cytosolic calcium concentration. Ouabain also increased the
membrane content of P-gp, as detected by immunoblotting and
immunohistology. We have developed a microvideo assay based on the
properties of acetoxymethyl ester calcein and calcein to show
that this P-gp extruded the hydrophobic acetoxymethyl ester
calcein. Ouabain also caused the Calu-3 cells to become
resistant to doxorubicin and vinblastine. Thus, although ouabain acts
extracellularly, it may stimulate MDR-1 gene expression
and P-gp synthesis and make cells resistant to hydrophobic cytotoxic
compounds.
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INTRODUCTION
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The development of multidrug resistance is a major obstacle to the
success of chemotherapy of neoplastic diseases.
P-gp3
is a transmembrane protein that acts as an energy-dependent efflux pump
to remove natural drugs from cells. It is encoded by the
MDR-1 gene. P-gp is normally present in the apical membrane
of some epithelial cells in renal proximal tubules, hepatic bile ducts,
and colon villi, but its function in these cells is not clear
(1)
. Its clinical deleterious role arises from the
multidrug resistance caused by its production triggered by
carcinogenesis and antimitotic treatments (2)
. The
overproduction of P-gp is usually due to enhanced transcription of the
MDR-1 gene, but the molecular mechanisms involved in the
reaction are not clear. Various agents, such as differentiating
compounds or environmental stresses (UV radiation and acid external
pH), stimulate MDR-1 gene transcription, and many
transcription factors can bind to and activate the gene promoter
(3)
. Recent studies demonstrate that the stimulation of
MDR-1 gene transcription by UV irradiation is due to the
combined actions of several transcription factors and histone
modification (4
, 5)
. Several molecular processes may also
be involved in the multidrug resistance produced by P-gp. P-gp is a
multifunctional protein that not only acts as an efflux pump for
various hydrophobic compounds but also regulates membrane composition
via its flippase properties and ion transport by modulating the
activity of the cell swelling-stimulated Cl-
conductance (6
, 7)
. The relationship between its various
properties is not clear. The multidrug cell resistance produced by P-gp
may occur because the protein alters intracellular ion concentrations
(8)
.
We have therefore examined the question of whether inhibiting
Na+/K+-ATPase, a major
membrane protein responsible for maintaining the membrane electrical
potential and cell homeostasis, modulates MDR-1 gene
expression. Na+/K+-ATPase
is specifically inhibited by cardiotonics, the best known of which is
ouabain. Ouabain also inhibits the cytotoxicity of various anticancer
agents (9
, 10)
, but this quick-acting effect of ouabain is
not related to P-gp activity, even when it acts against doxorubicin, a
P-gp substrate (9)
. The effect of prolonged administration
of ouabain has not been studied in terms of MDR-1 mRNA and
P-gp. Ouabain, which can induce gene transcription (11)
,
may exert such an action, and the phenomenon might have practical
implications because cardiotonics are often administered to prevent
cardiac damage during anticancer treatment. We have treated human
pulmonary cancerous Calu-3 cells (12)
with ouabain and
monitored MDR-1 gene expression and P-gp concentration. We
have also explored P-gp function by monitoring the transport of CAL-AM,
a P-gp substrate, and cell resistance to doxorubicin and vinblastine
toxicity.
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MATERIALS AND METHODS
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Cell Culture and Treatment
Calu-3 cells were obtained from the ATCC and cultured in DMEM
containing 1 mM sodium pyruvate, nonessential amino acids,
and 10% FCS at 37°C in 5% CO2-enriched
atmosphere. Cells were cultured on plastic for RNA and protein analysis
and on glass slides for measuring the P-gp function. They were
incubated with freshly prepared ouabain (Sigma-Aldrich) for the
indicated time (generally, 24 h for RNA analysis and 48 h for
protein detection and functional studies). Ouabain toxicity was
assessed by treating the confluent cell cultures with various
concentrations of the drug for 48 h and then counting the living
cells (trypan blue exclusion). The capacity of ouabain to modulate the
MDR-1 mRNAs in other human cell lines was also studied. The
cells were T-84 and HT-29 cells derived from colon carcinoma (obtained
from the ATCC), HeLa cells (obtained from the ATCC), and hepatic
carcinomatous HuH7 cells (13)
. The T-84 cells were
cultured in DMEM/Ham’s F-12; the HT-29, HeLa, and HuH7 cells were
cultured in DMEM.
RNA Extraction and Analysis
MDR-1 mRNAs were measured in confluent cells placed in
serum-free medium for 24 h before being treated with ouabain in
the absence of FCS. Total RNAs were isolated with phenol/chloroform
using the Trizol reagent (Life Technologies, Inc.) according to the
manufacturer’s instructions, fractionated on 0.9% agarose gels (15
µg/well), and transferred to nylon membranes (Promega)
(14)
. The membranes were hybridized with
32P-labeled cDNA probes (specific activity >
109 cpm/µg) with the Quik Hyb protocol provided by
Stratagene. The MDR-1 probe was the 1.5-kb
EcoRI-EcoRI fragment of human MDR-1 cDNA probe
(generously supplied by Dr. J. P. Marie; Institut National de la
Santé et de la Recherche Médicale E.9912, Paris, France),
and the human ß-actin cDNA probe was purchased from Oncogene Science.
The mRNAs were quantified by densitometry using an ImageMaster
VSD (Pharmacia-Biotech-Amersham, Orsay, France), and the amounts
of MDR-1 mRNA were normalized to those of ß-actin. All
experiments were repeated at least four times.
Protein Analysis
Western Blot Analysis.
Newly synthesized P-gp was detected in membranes extracted from cells
treated for 48 h with ouabain by Western blotting
(15)
using the monoclonal antibody C-219 against P-gp
(Valbiotech). The 100,000 g membrane extracts were prepared from
cells lysed in hypotonic buffer enriched with a classical antiprotease
mixture. The extract was then electrophoresed in a denaturing 6%
SDS-polyacrylamide gel run at 15–25 mV, with 30 µg membrane
protein/lane. The separated proteins were transferred to a
nitrocellulose membrane (0.45 µm; Bio-Rad), which was probed with the
anti-Pgp monoclonal antibody C-219. The secondary antibody was a
horseradish peroxidase-conjugated antimouse IgG. The blots were
developed with the enhanced chemiluminescence reagent (ECL kit;
Amersham) and exposed to Hyperfilm (Amersham).
Immunocytochemistry.
Cells grown on glass slides under basal conditions or after treatment
with 0.2 µM ouabain for 48 h were fixed in PBS/3%
formaldehyde. They were then washed with PBS and incubated for
2 h at room temperature with a monoclonal antibody directed
against an extracellular epitope of human P-gp, mAb MRK-16 (Valbiotech;
diluted 1:10). The secondary FITC-conjugated antibody (Nordic
Immunology; diluted 1:500) was used to detect mAb MRK-16. The slides
were washed in PBS, mounted on a horizontal stage, and imaged using a
Zeiss confocal microscope. Images were collected using a x40
plan-apochromatic oil immersion objective. Serial scans were collected
using a 495 nm laser line to excite the Nordic fluorochrome.
|
Functional Assays
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P-gp-mediated Transport of CAL-AM.
We monitored the function of newly synthesized P-gp by microvideo
imaging using the properties of CAL-AM and CAL. The nonfluorescent
hydrophobic CAL-AM enters the cells by diffusion and is de-esterified
to form fluorescent CAL in the cytoplasm (16)
. CAL
fluorescence is insensitive to ion concentration, such as
[Ca2+]i or
pHi, which may vary during many cell stimulation
(17)
. Therefore, the increase in fluorescence of cells
incubated with CAL-AM represents CAL accumulation. P-gp in the membrane
extrudes a portion of CAL-AM before it is de-esterified to fluorescent
CAL, so that cell fluorescence increases more slowly (
F/t is
smaller than in controls). The assay was done using cells subcultured
on glass slides with or without ouabain (0.2 µM) for 2
days. The cells were then placed in a perfusion chamber on the stage of
an inverted microscope (Diaphot, Nikon, France) and perfused with
physiological saline at 37°C containing 0.25 µM CAL
acetylmethyl ester (Molecular Probes), the membrane-permeable
nonfluorescent form of the dye. The CAL fluorescence (excitation, 390
nm; emission, 410 nm) was measured in single cells with a digital
imaging system and a CDD camera (Photonic Sci, Millham, United
Kingdom). The results were analyzed using Imstar software
(Paris, France), which calculates the mean fluorescence of the 10–12
cells present in the field at a given time (every 30 or 60 s). The
slopes of the curves (F/t) reflecting the rate of formation of
intracellular CAL were determined using Microsoft Excel software.
CAL-AM efflux by P-gp was antagonized by adding mAb MRK-16 (10
µg/ml), which inhibits P-gp function (2)
, to the
superfusing medium.
Analysis of the Cell Sensitivity to Doxorubicin and Vinblastine.
Confluent Calu-3 cells were cultured under control conditions or with
0.2 µM ouabain for 2 days and then cultured for 4 h
with doxorubicin and vinblastine (0.5 or 1 µM) alone or
with the P-gp inhibitor verapamil (50 µM), or in normal
medium with or without verapamil (18)
. The cells were
washed twice with culture medium, trypsinized, seeded at
105/cm2 in normal culture
medium, and allowed to grow for 3 days. The toxicity of doxorubicin and
vinblastine was then assessed by counting the living cells (trypan blue
test). For each experimental condition (pretreatment or no pretreatment
with ouabain; the presence or absence of verapamil), the number of
living cells found in the cultures of doxorubicin- or
vinblastine-treated cells was expressed as a percentage of the number
found in the cultures of cells that were not incubated with the
anticancer agent.
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RESULTS
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MDR-1 mRNA
Serum-deprived Calu-3 cells treated with ouabain for 24 h
showed dose-dependent increases in MDR-1 mRNA from 0.05 to
0.5 µM ouabain. There was very little
MDR-1 mRNA in control cells, and 0.1
µM ouabain produced a significant increase
(Fig. 1)
. The ouabain effect was time dependent; it was maximal at 24 h
and half-maximal after 12 h (Fig. 2
, A1 and B). The effect of ouabain was reversible
(Fig. 2
A2); cells treated for 24 h with 0.2
µM ouabain that were rinsed and allowed to
recover for 24 h in normal medium had the same MDR-1
mRNA content as untreated cells.
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Fig. 1. Concentration dependence of the ouabain effect.
Serum-deprived Calu-3 cells were incubated in serum-free culture medium
containing various amounts of ouabain for 24 h. A,
typical Northern blot obtained with 15 µg of total RNAs extracted
from cells incubated with the indicated amounts of ouabain.
B, Northern blot data expressed by the experimental
MDR-1:ß-actin mRNA ratios expressed as percentages of the mean ratio
found in untreated cells. Each value is the mean ± SE
of six experiments. *, P < 0.05.
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Fig. 2. Time course of the ouabain-induced increase in MDR-1 mRNA.
The results were obtained and expressed as described in the Fig. 1
legend. A1, typical Northern blot obtained after
incubating the cells with 0.2 µM ouabain for the
indicated times. A2, on this Northern blot, the signal
+24 was obtained after a 24-h incubation followed by
24 h of recovery without ouabain. B, each
MDR-1:ß-actin ratio is the mean ± SE of six
experiments. *, P < 0.05.
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High concentrations of ouabain also decreased the
ß-actin mRNA in Calu-3 cells (Figs. 1
and 2)
,
and this decrease was also reversed by washing out the ouabain. Despite
their opposing changes, the increase in MDR-1 mRNA and the
decrease in ß-actin transcripts triggered by
ouabain may both be due to stress caused by the drug. There were
significantly fewer living cells in cultures treated with 0.5 or 1
µM ouabain for 48 h than in controls
(Table 1)
. Therefore, all subsequent experiments were performed on cells treated
with 0.2 µM ouabain.
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Table 1 Ouabain-induced cell death
Confluent cells were treated for 48 h with ouabain, washed twice,
trypsinized, centrifuged, and resuspended in trypan blue in
physiological saline. The living cells were counted on a Malassez cell.
Three different wells were treated in each experiment, and each of them
was counted three times. The values are the means ± SE
of six experiments. Each experimental value was compared with that of
the control untreated culture, using Student’s t test.
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Ouabain also stimulated MDR-1 gene expression in human colic
T-84 and HT-29 cells and in hepatic HuH7 cells, but not in HeLa cells
(Fig. 3)
.
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Fig. 3. Effect of ouabain on MDR-1 mRNA in four different cell
lines. Like the Calu-3 cells, the T-84, HT-29, HuH7, and HeLa cells
were grown to confluence, deprived of serum for 24 h, and then
treated with ouabain (0.1 or 0.2 µM) for 24 h. The
Northern blots were performed on 15 µg of total RNA. Each value is
representative of four experiments.
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Digoxin and palitoxin, two other
Na+/K+-ATPase inhibitors
chemically distinct from ouabain (15)
, had the same the
effect as ouabain on Calu-3 cells (Fig. 4A)
. Increased MDR-1 gene transcripts thus appeared
to be triggered by inhibiting the
Na+/K+ pump. The
ouabain-induced increase in MDR-1 mRNA still occurred in the
presence of cycloheximide (6 µM; Fig. 4B
), indicating that the phenomenon does not require protein
synthesis. The inhibition of the ouabain effect by actinomycin D (5
µg/ml added to the medium 30 min before ouabain) suggests that
ouabain stimulates MDR-1 gene transcription (Fig. 4B)
. The stimulation of MDR-1 gene transcription
by ouabain was enhanced by thapsigargin, which increases cytosolic
calcium concentration, and suppressed when intracellular calcium was
chelated by BAPTA (Fig. 4C)
. These results indicate that the
increase in cytosolic calcium concentration produced by inhibiting the
Na+/K+-ATPase participates
in the stimulation of MDR-1 gene transcription.
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Fig. 4. Properties of the ouabain-induced increase in MDR-1 mRNA.
All of the Northern blots were obtained with 15 µg of total RNAs
extracted from serum-deprived cells treated for 24 h as indicated.
A, concentration dependence of the effects of digoxin
and palitoxin (the figure is representative of four experiments).
B, cycloheximide (CX; 6 µM)
and actinomycin D (act. D; 5 µg/ml) were added to the
cultures 30 min before ouabain (0.2 µM) and remained in
the medium during the ouabain treatment (the figure is representative
of three experiments). C, thapsigargine
(Thaps.; 2 µM) and BAPTA-AM at the
indicated concentrations were added to the medium 15 min before ouabain
(0.2 µM) for the incubation time.
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P-gp Synthesis
Western blot analysis with mAb C-219 showed a single
immunoreactive band of about Mr
170,000, compatible with P-gp, in the microsomes from Calu-3
cells. It was very faint in control membranes and dose-dependently
increased by ouabain (0.05 to 0.2 µM; Fig. 5
).
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Fig. 5. Changes in membrane P-gp. The Western blots were performed
as indicated in "Materials and Methods" on membranes extracted from
cells incubated for 48 h with the indicated concentrations of
ouabain. The proteins were separated by electrophoresis (30 µg/lane)
and transferred to the membrane. They were detected by incubation with
mAb C-219 directed against human P-gp. The figure is representative of
three experiments.
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Immunocytochemistry (Fig. 6)
also revealed a surface immunoreactivity to mAb MRK-16; it was very
faint in control cells (Fig. 6
B1) and enhanced by treating
the cells with 0.2 µM ouabain for 48 h
(Fig. 6
C1). Labeling was specific because it did not exist
when the cells were incubated with the secondary antibody directly
(Fig. 6A1). Ouabain thus stimulates
MDR-1 gene transcription and P-gp synthesis.
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Fig. 6. Immunocytochemistry. The pictures on the
left show the immunological staining with mAb MRK-16.
The cells, either under basal conditions (A and
B) or after incubation for 48 h with 0.2
µM ouabain (C), were incubated with mAb
MRK-16 for 3 h (B and C).
A, cells incubated with the second antibody directly.
The pictures on the right show the same cells observed
under a phase-contrast microscope.
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P-gp Function
CAL Efflux.
When control Calu-3 cells were incubated with CAL-AM (0.25
µM), the intracellular de-esterification of the dye led
to increased cell CAL fluorescence
[(F/t)basal = 0.198 ± 0.056 (n = 14)] (Fig. 7)
. Pretreating the cells with 0.2 µM ouabain
considerably reduced the rate of increase of fluorescence
[(f/t)ouab = 0.066 ± 0.022 (n = 14)], which went
on for 30 min (data not shown). Incubation with the specific anti-P-gp
mAb MRK-16 (10 µg/ml added 15 min before CAL-AM) significantly
reduced the difference between the treated and untreated cells. The
rate of increase of fluorescence in the ouabain-treated cells in
the presence of MRK-16
[(F/t)MRK16-ouab =
0.125 ± 0.037 (n = 14)] was very close to that of controls
[(F/t)MRK16-control =
0.145 ± 0.066 (n = 14)], and
the maximal fluorescence was the same in both cases. This indicates
that the low fluorescence of the ouabain-treated cells not incubated
with MRK-16 could be due to a rapid extrusion (before its
de-esterification) of the CAL-AM from these treated cells. Verapamil
(50 µM) reproduced the effect of mAb MRK-16 and
increased the rate of fluorescence of the ouabain-treated cells
(results not shown), but it also decreased the responses of the control
cells at this concentration. These results show that the
ouabain-induced P-gp extrudes hydrophobic compounds.
Resistance to Doxorubicin and Vinblastine.
The toxicity of doxorubicin and vinblastine, estimated as indicated in
"Materials and Methods" 3 days after treating the cells with the
anticancer drugs, showed that treating the cells with ouabain (0.2
µM for 48 h) made them resistant to a 4-h exposure
with 0.5 µM of both drugs (Fig. 8)
. Three days after the exposure, control cells treated with 0.5
µM doxorubicin or vinblastine were less numerous
[-38 ± 8% (n = 6) and
-42 ± 6% (n = 4),
respectively] than those that were not incubated with the anticancer
agents; verapamil had no significant effect. However, ouabain-treated
cells were significantly resistant to doxorubicin, which decreased
their numbers by only 18 ± 10% (n = 6), and to vinblastine, which decreased their numbers by
19 ± 9% (n = 4). In both
cases, the resistance was inhibited by verapamil; with verapamil,
doxorubicin and vinblastine decreased the number of living
ouabain-pretreated cells by 47 ± 9%
(n = 6) and 56 ± 9%
(n = 4), respectively. The ouabain treatment
did not significantly modify the effect of 1 µM
doxorubicin (however, verapamil significantly increased
doxorubicin-induced cell death in ouabain-treated cells), but it still
protected the cells against 1 µM vinblastine.
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DISCUSSION
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We have shown that submicromolar concentrations of ouabain
stimulate MDR-1 gene expression and P-gp synthesis in human
tumoral cells. We believe that this is the first demonstration of a
P-gp-mediated drug efflux that is pharmacologically induced by a
hydrophylic glycoside that acts on an extracellular site of a
transmembrane protein, the
Na+/K+-ATPase. The
microscopic CAL-AM/CAL assay developed to demonstrate this P-gp
function at the cellular level may be suitable for clinical use on
small tissue samples.
The concentration dependence of the increase in MDR-1
transcripts caused by ouabain corresponds to the parameters of the
Na+/K+-ATPase inhibition.
The changes in MDR-1 mRNA occurred at 0.1–0.5
µM ouabain, whereas the drug binds to the
Na+/K+ pump with an
affinity constant of about 0.01 or 0.1 µM,
depending on the form of the 
protein subunit. Half-maximal
inhibition of the human
Na+/K+ pump occurs at 0.1
µM in nonmyocardial human cells
(19)
. The fact that both digoxin, another
Na+/K+-ATPase inhibitor
that is a lipid compound and acts intracellularly, and palitoxin, which
decreases the pump activity by a different mechanism (19)
,
also increase MDR-1 transcripts further demonstrates that
inhibition of Na+/K+-ATPase
activity triggers MDR-1 gene expression in our experimental
model. This MDR-1 gene overexpression is not limited to
Calu-3 cells because it also occurred in T-84 and HT-29 colon cells,
which differ in differentiation, and in HuH7 carcinomatous hepatic
cells. However, the fact that it was not found in HeLa cells
demonstrates that it is not a ubiquitous cell response to the
cardiotonic.
The inhibition of the
Na+/K+-ATPase by ouabain is
immediate, leading to gradual changes in cytosolic ion concentrations.
Various ion transports are activated during the cell adaptation that
follows these changes. The time dependence of the reversible
ouabain-induced increase in MDR-1 gene expression agrees
with the participation of P-gp in the adaptation of the cell to the
Na+/K+ pump blockade. The
increase in MDR-1 transcripts (and the decrease in ß-actin
mRNA) in Calu-3 cells may be due to ouabain-induced changes in cell ion
concentrations. It appears to start with enhanced transcription and
does not depend on the synthesis of any intermediate protein. The
contrast between the stimulation of MDR-1 gene expression
triggered by low concentration of ouabain in Calu-3, T-84, HT-29, and
HuH7 cells and the absence of response of HeLa cells may result from
cell-to-cell differences in either cytoplasmic or nuclear reactions.
Further work is needed to define these cell characteristics, which may
be responsible for the differing sensitivities of cancerous cells to
multidrug resistance. The elevation in cytosolic calcium concentration
that follows the increase in intracellular Na+
caused directly by the
Na+/K+-ATPase blockade
(20)
may be involved in the phenomenon. For example,
ouabain stimulates early-activated gene (c-fos and
c-jun) transcription via increased cytosolic calcium
concentration in both rat cardiac myocytes and various human cell lines
(10
, 21
, 22)
. The reaction (which is triggered in HeLa
cells by micromolar concentrations of ouabain; Ref. 22
)
does not require protein synthesis but results from the stimulation of
mitogen-activated protein kinases (21)
, which control
various gene expressions in several cell types (23)
. In
Calu-3 cells, an increased intracellular calcium concentration also
appears to be the link between
Na+/K+-ATPase inhibition
and the increase in MDR-1 mRNA stimulated by ouabain because
the ouabain effect was reinforced by thapsigargin and suppressed by the
calcium chelator BAPTA. Because it was detected with low concentrations
of ouabain (0.1 µM), it cannot be a consequence
of the cell damage caused by higher concentrations of ouabain via
elevated cytosolic calcium and activation of kinases. Ouabain-induced
cell detachment, which is due to phosphorylation of proteins devoted to
cell support or cell-cell contacts and which leads to cell death
(24)
, may occur in our model with 0.5 and 1
µM ouabain, but it is preceded by
MDR-1 gene overexpression, which is maximally induced by 0.2
µM ouabain.
Enhancement of MDR-1 gene expression by ouabain led to
synthesis of P-gp, which was immunodetected in the cell membranes. The
efflux of hydrophobic drugs is the major function of P-gp, and this was
why we looked for it in the ouabain-treated Calu-3 cells. Because P-gp
extrudes the nonfluorescent hydrophobic CAL-AM before its
de-esterification to fluorescent CAL, we measured the rate of increase
of fluorescence in cells incubated with CAL-AM to detect functional
P-gp in the cell membrane. This technique was first developed to assess
the P-gp-related multidrug resistance in large numbers of tumoral cells
by flow cytometry (16)
. These results show that the rate
of increase of the fluorescence was slower in cells from patients who
had multidrug resistance or in cultured cells treated for a long time
with high concentrations of drugs such as doxorubicin or
colchicine that induce MDR-1 gene expression.
However, this method can only be used with large numbers of cells
producing large amounts of P-gp. We detected activities of P-gp in
isolated cells or clusters of cells (50 cells) in our
experiments using low CAL-AM concentrations (up to 0.25
µM) and a sensitive videomicroscopy system. The
difference between control and treated cells was very significant under
these conditions. Although CAL fluorescence is insensitive to variation
in ionic concentration (17)
, this difference could be due
to some alteration in the cytoplasm induced by ouabain. Its inhibition
by mAb MRK-16, which allows the maximal fluorescence of ouabain-treated
cells to reach control level, clearly indicates that the inhibition of
CAL accumulation in ouabain-treated cells in the absence of the P-gp
antagonist is linked to P-gp function. These results agree with our
data on the resistance of ouabain-treated cells to doxorubicin and
vinblastine cytotoxicity. The cell resistance was not complete and was
seen only with low concentrations of the anticancer drugs. However, it
was significant, especially versus vinblastine, and was
inhibited by verapamil. Hence, our results show that inhibiting
Na+/K+-ATPase may cause
multidrug resistance by stimulating MDR-1 gene expression
and drug efflux mediated by P-gp.
The stimulation by ouabain of the synthesis of P-gp able to extrude
hydrophobic compounds appears to result from disturbed cell ion
concentrations. The data show that cells do not need to be exposed to
cytotoxic agents to make P-gp able to extrude such drugs, but the cell
damage caused by ouabain via the ionic disorders that stimulate
MDR-1 gene expression cannot be ruled out. The P-gp-mediated
CAL-AM efflux and the resistance of the ouabain-treated Calu-3 cells to
both doxorubicin and vinblastine are not very large. Low
resistance to chemotherapeutic drugs was shown to be a characteristic
of cells transfected with MDR-1, and the alterations in
pHi and membrane potential caused by the induced P-gp were
sufficient to trigger this reduced multidrug resistance
(8)
. Additional studies are now required to determine
whether the ouabain-treated cells behave as a pharmacological
equivalent of these "pure transfects." This would mean that P-gp is
essential for the integrated control of cell ion concentrations. The
ouabain-induced multidrug cell resistance might also be important in
clinical situations because cardiotonics may be prescribed together
with certain anticancer drugs.
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ACKNOWLEDGMENTS
|
|---|
We thank Dr. Jeanine Fritsch for help with immunohistology and
Owen Parkes for correcting the manuscript.
|
FOOTNOTES
|
|---|
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 Supported by the Institut National de la
Santé et de la Recherche Médicale and by a grant from the
Association Française de Lutte contre la Mucoviscidose. 
2 To whom requests for reprints should be
addressed, at Institut National de la Santé et de la Recherche
Médicale U.467, Faculté de Médecine Necker, 156 Rue
de Vaugirard, 75015 Paris, France. Phone: 33-1-40-61-56-28; Fax:
33-1-40-61-55-91; E-mail: legros{at}necker.fr
3 The abbreviations used are: P-gp,
P-glycoprotein; CAL-AM, acetoxymethyl ester calcein; CAL, calcein;
ATCC, American Type Culture Collection; BAPTA,
[1,2-bis-(o-aminophenoxy)ethane-N,N,N',N'-tetraaceticacid].
Received 7/13/00.
Accepted 12/13/00.
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Top
ABSTRACT
INTRODUCTION
) and cells treated with ouabain
(0.2 µM; 48 h; 
) were incubated for 4 h with
doxorubicin or vinblastine (0.5 and 1 µM) with
(hatched columns) or without verapamil (50
µM), washed, counted, and incubated for an additional 3
days. They were then counted, and the results for doxorubicin- and
vinblastine-treated cells were expressed as percentages of those for
controls. Each figure is the mean ± SE of four
(vinblastine) or six (doxorubicin) experiments. The means were compared
using Student’s unpaired t test. *,
P < 0.05.