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Cancer Therapy Vol 5, page 67
67
Cancer Therapy Vol 5, 67-76, 2007
Overcoming K562Dox resistance to STI571
(Gleevec) by downregulation of P-gp expression
using siRNAs Research Article
Raquel T. Lima1,2, José Eduardo Guimarães1,2,3, M. Helena Vasconcelos1,4* 1Cancer Biology Group, IPATIMUP – Institute of Molecular Pathology and Immunology of the University of Porto, Porto,
Portugal 2Faculty of Medicine of the University of Porto, Porto, Portugal 3 Hospital São João, Porto, Portugal 4Department of Microbiology, Faculty of Pharmacy of the University of Porto, Porto, Portugal
__________________________________________________________________________________
*Correspondence: Maria Helena Vasconcelos, IPATIMUP, Rua Dr. Roberto Frias, s/n, 4200-465 Porto, Portugal. Tel: +351 22
5570700; Fax: +351 22 5570799; E-mail: [email protected]
Keywords: P-gp, MDR, STI571, Gleevec, Imatinib, RNAi, siRNA, CML
Abbreviations: chronic myeloid leukemia, (CML); control siRNA, (CRNAi); multidrug resistance, (MDR); P-glycoprotein, (P-gp);
RNA interference, (RNAi); short-hairpin RNAs, (shRNAs)
Received: 23 June 2006; Revised: 22 December 2006
Accepted: 27 February 2007; electronically published: March 2007
Summary Resistance to STI571 is possibly due to several mechanisms, including the overexpression of P-glycoprotein (P-gp).
The objective of the present study was to verify if downregulation of P-gp expression with specific siRNAs, in the
K562Dox overexpressing P-gp cell line, would allow to overcome resistance of these cells to this drug. Uptake of
fluorescently-labelled siRNAs was verified by fluorescence microscopy and confirmation of siRNAs efficiency in
reducing P-gp protein expression was carried out by Western Blot. Transfection of the K562Dox cells with the
siRNAs prior to treatment with STI571 enhanced the effects of this drug, as confirmed by counting the number of
viable cells. This increase in the sensitivity was due to an increase in cellular apoptosis, as verified by the TUNEL
assay. This data indicates that P-gp downregulation increased sensitivity of CML cells to STI571, by means of
promoting apoptosis.
I. Introduction More than 90% of chronic myeloid leukemia (CML)
cases are associated with the presence of an acquired
genetic abnormality, the Philadelphia chromosome
(Deininger et al, 2000a; Kurzrock et al, 2003). This is a
shortened chromosome 22, which results from a reciprocal
translocation between chromosomes 9 and 22. As a result
of the translocation, the Philadelphia chromosome has the
BCR-ABL fusion gene, which codes for a 210KDa
chimeric protein with the same name. This protein has a
deregulated tyrosine kinase activity resulting in the
activation of several transduction pathways in the cell,
shown to be responsible for the CML phenotype
(Deininger et al, 2000a,b; Shet et al, 2002; Calabretta and
Perrotti 2004).
Recently, a potent tyrosine kinase inhibitor named
STI571 mesylate has been developed, which has been
shown to specifically bind to the catalytic pocket of the
BCR-ABL, avoiding the association of ATP to this protein
and thereby blocking its tyrosine kinase activity (Savage
and Antman 2002). This drug has been shown to be highly
effective in the treatment of CML, especially in the
chronic phase of the disease. However, several reports
already state that this drug is not as effective in the more
advanced phases of the disease, indicating that resistance
to this drug is arising (Gorre et al, 2001; Kano et al, 2001;
O'Dwyer et al, 2002; Weisberg and Griffin 2003).
Resistance to STI571 has been attributed to several
mechanisms, including amplification and mutations of the
BCR-ABL gene (Gorre et al, 2001; Roche-Lestienne et al,
2002), increase in !"1-acid glycoprotein levels
(Gambacorti-Passerini et al, 2000) and overexpression of
multidrug resistance (MDR) genes, namely the MDR1
gene that codifies for P-gp (Mahon et al, 2000; Druker
Lima et al: Overcoming K562Dox resistance to (Gleevec)
68
2002; Nimmanapalli and Bhalla 2002; Knight and
McLellan 2004).
P-gp is a 170KDa transmembrane glycoprotein
involved in the cellular efflux of several drugs. It is known
to confer MDR to cells and shown to have a high
expression in cancers that are intrinsically resistant to
therapy, such as blastic phase CML (Goldstein et al, 1989;
Fardel et al, 1996). Several studies have shown that
downregulation of P-gp expression results in the
sensitization of cells to several chemotherapeutical drugs.
Indeed, the use of antisense or rybozime technologies
targeting the MDR1 gene has allowed to increase the
sensitivity of human hepatocarcinoma cells (Wang et al,
2003), CML (Sola and Colombani 1996; Motomura et al,
1998), ovarian (Pan et al, 2001), colon and breast cancer
cells to doxorubicin (Ramachandran and Wellham 2003)
as well as to increase the sensitivity of chronic and acute
myeloid leukemia cells to daunorubicin (Sola and
Colombani 1996; Motomura et al, 1998). Furthermore,
downregulation of P-gp expression by RNA interference
(RNAi) has allowed the resistance of several cell lines to
be overcome (Nieth et al, 2003; Wu et al, 2003; Celius et
al, 2004), including of CML to several drugs (Yague et al,
2004).
The involvement of P-gp in resistance to STI571 has
been previously described by other authors. Indeed, P-gp
overexpressing cell lines showed increased resistance to
STI571 (Che et al, 2002; Kotaki et al, 2003; Mahon et al,
2003; Illmer et al, 2004). Furthermore, in the course of this
work, by downregulating P-gp expression using short-
hairpin RNAs (shRNAs), Rumpold and collaborators were
able to overcome the resistance of CML cells to STI571
(Rumpold et al, 2005). However, other studies reported
that the overexpression of P-gp in K562 cells did not
confer resistance to STI571 (Ferrao et al, 2003; Zong et al,
2005). Furthermore, mdr1a/1b-null CML mice did not
respond better to STI571 treatment (Zong et al, 2005).
Therefore, these conflicting data illustrate the need to
validate the relevance of P-gp in the resistance of CML to
STI571. The purpose of the present study was to carry out
such validation, by downregulating P-gp expression in
CML cells that overexpress P-gp and that are resistant to
STI571. Furthermore, the work intended to verify if
downregulation of P-gp expression with siRNAs increased
sensitivity of CML cells to STI571 by means of promoting
apoptosis in these cells. This would allow to validate P-gp
as a possible molecular therapeutic target, for adjuvant
therapy in the treatment of CML with STI571.
II. Materials and Methods A. Cell lines and siRNAs Two CML cell lines in blast phase were used in this study,
K562 (ECACC, European Collection of Cell Cultures, UK) and
K562Dox (a kind gift from Professor J.P.Marie, Paris, France).
Both these cell lines were routinely cultured in RPMI
1640 medium (GIBCO) containing 10% fetal bovine serum
(FBS) and maintained at 37ºC in a humidified atmosphere
containing 5% CO2. The K562Dox cell line, which overexpresses
P-gp, had previously been obtained by others, by selection of the
K562 cell line after exposure to doxorubicin. To maintain P-gp
expression in the K562Dox cell line, 1µM doxorubicin was
added to the cells every two weeks. In order to maintain equal
levels of P-gp expression throughout the experiments, all
experiments with the K562Dox cells were carried out 6 days
after this treatment with doxorubicin.
Two different siRNAs targeting the MDR1 mRNA,
previously designed by Wu et al, (2003) and Nieth et al, (2003),
were used and named MDR-CR and MDR-FE siRNAs,
respectively. Thus, the MDR-CR siRNA had the following
sequences: sense – 5’GGA AAA GAA ACC AAC UGU
CdTdT3’ and antisense –5’GAC AGU UGG UUU CUU UUC
CdTdT3’. The MDR-FE siRNA had the following sequences:
sense– 5’AAU GUU GUC UGG ACA AGC AdTdT3’ and
antisense –5’UGC UUG UCC AGA CAA CAU UdTdT3’. A
negative control siRNA was used with the following sequences
(designed by Qiagen): sense – 5’UUC UCC GAA CGU GUC
ACG UdTdT3’ and antisense –5’ACG UGA CAC GUU CGG
AGA AdTdT3’. A negative control siRNA (with the same
sequence) labelled with Alexa Fluor 488 fluorochrome (Qiagen)
was used in some experiments. Treatments with the control
siRNA are referred to as CRNAi. All siRNAs were purchased
from Qiagen and resuspended in siRNA suspension buffer,
according to the manufacturer’s instructions.
B. Transfection of K562Dox cells with
siRNAs and verification of uptake of the siRNAs
and of downregulation of P-gp protein expression K562Dox cells (5x105 cells/well in 24-well plates) were
transfected with siRNAs using jetSI# Reagent (Qbiogene). The
manufacturer’s instructions were followed, using siRNA
concentrations of 200nM and no FBS during the initial 4 hours of
transfection. Following 4 hours of incubation, 400µl of medium
containing 25% FBS was added to each well and cells were
further incubated. The uptake of the siRNAs by the K562Dox
cell line was verified by transfecting this cell line with 200nM
control siRNA labelled with Alexa Fluor 488 and examining
cells by fluorescence microscopy.
To analyze P-gp and BCR-ABL protein expression, cells
were lysed in Winman’s buffer (1% NP-40, 0.1M Tris-HCl pH
8.0, 0.15M NaCl and 5mM EDTA) with EDTA-free protease
inhibitor cocktail (Boehringer Mannheim) and proteins were
quantified and separated: i) in 4-20% Tris-Glycine gel (Novex)
in the case of P-gp analysis or ii) in 10% Bis-Tris gels
(Sambrook et al, 1989) in the case of BCR-ABL. Proteins were
then transferred to a nitro-cellulose membrane (Amersham) with
the Novex Electrophoresis System. The membranes were
incubated with mouse anti-P-gp (F4 clone; 1:2500, Sigma) or
with mouse anti-BCR-ABL (1:50; Santa Cruz Biotechnology)
and then incubated with goat anti-mouse IgG -HRP (1:2000,
Santa Cruz Biotechnology). The signal was detected with the
ECL Amersham kit (Amersham), the Hyperfilm ECL
(Amersham) and the Kodak GBX developer and fixer twin pack
(Sigma) as previously described (Lima et al, 2004). The intensity
of the bands obtained in each film was further analyzed using the
software Quantity One – 1D Analysis (Bio-Rad, USA).
C. Response of K562 and K562Dox cells to
treatments with STI571, in terms of viable cell
number and programmed cell death In the experiments to determine the response of both cell
lines to STI571 mesylate (Novartis), cells were plated in 24-well
plates at 5x105 cells/well using serum free medium and incubated
at 37ºC in a humidified atmosphere containing 5% CO2. After 4
hours of incubation, 400 µl of medium containing 25% FBS was
added to each well and cells were further incubated. Different
concentrations of STI571 mesylate (0, 0.25, 0.5, 0.75, 1.0 and
1.25µM) were added to the cells 24 hours after plating. Viability
was assessed 24 hours and 48 hours after drug exposure, with the
Trypan Blue exclusion assay.
Cancer Therapy Vol 5, page 69
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In the experiments to investigate sensitization to STI571
after transfection with siRNAs, STI571 was added only at the
concentrations of 0.5µM or 1µM, 24 hours after transfecting the
cells as previously described. As control, the equivalent volume
of solvent of the drug (water) was also added to the cells at the
same time point. Viability was assessed 48 hours after treatment
with STI571, using the Trypan Blue exclusion assay.
Apoptosis was also analysed in these cells, 48 hours after
treatment with STI571, using the “in situ cell death detection kit”
(Roche). In brief, cytospins were prepared and fixed in 4%
paraformaldehyde solution. Cells were permeabilised (0.1%
Triton X-100 in 0.1% sodium citrate) and incubated with
TUNEL reaction mix, according to the optimized procedure
recommended by the manufacturer (enzyme dilution 1:20). Cells
were observed in a DM IRE 2 microscope (LEICA) and a semi-
quantitative evaluation was performed by counting a minimum of
500 cells per slide.
D. Statistical analysis Results were expressed as mean ± SE. Differences
between treatments with STI571 and the respective
treatments with solvent were analyzed using a two-tailed
paired Student’s t-test (Statview for PC), as appropriate.
Significance was defined as P$ 0.05.
III. Results A. P-gp and BCR-ABL basal protein
expression in K562 and K562Dox cells The K562Dox cell line was chosen to carry out the
present study, since it had been shown to have an
increased expression of P-gp, responsible for conferring
the multiple drug resistance (MDR) phenotype. In order to
confirm that the levels of P-gp expression were higher in
K562Dox than in K562 cells, total protein extracts of these
two cell lines were analyzed by Western Blot. It was
observed that K562Dox cells express P-gp while K562
cells do not (Figure 1A). The levels of BCR-ABL protein
expression in both K562 and K562Dox cell lines were also
verified by Western Blot, since STI571 acts by blocking
BCR-ABL protein function. Results show that there are
similar levels of this protein in both cell lines (Figure 1B).
Furthermore, since resistance to STI571 may be due to
mutations in the BCR-ABL gene, the K562Dox cell line
was analyzed (Centro Genética Clínica, Portugal) and no
mutations were detected (data not shown).
B. Cellular response to STI571 To verify the response of both K562 and K562Dox cells to
STI571, both cell lines were treated with different
concentrations of this drug. The effects of STI571 were
analyzed 24h and 48h later, by counting the number of
viable cells and analyzing the results as a percentage of
control cells (cells that were not treated with STI571). In
what concerns the K562 cell line, a decrease in the number
of viable cells was verified with the increase in STI571
concentration, as expected (Figure 2 - A and B). The
IC50 concentration was only achieved at 48h and with the
highest drug concentration tested, 1.25µM (Figure 2B).
On the other hand, and also as expected, the response of
the K562Dox cell line was different from that observed in
the K562 cell line. Indeed, treatment of K562Dox cells
with STI571 concentrations up to 0.5µM did not affect
their viability. A modest decrease in the number of viable
cells was observed, but only with concentrations of
STI571 greater than 0.5µM (Figure 2 – A and B).
Furthermore, the 1.25µM STI571 concentration
(determined to be the IC50 concentration in the K562 cell
line) only caused a decrease in the number of the
K562Dox viable cells to 76% of the control (Figure 2B).
Since the IC50 was only achieved 48h following treatment
with STI571, this length of treatment with this drug was
chosen for the consecutive experiments.
To further confirm that the effect of STI571 was
different in the two cell lines, the levels of apoptosis were
analysed by the TUNEL assay 48h following treatment of
the cells with 1µM STI571. The results (Table 1)
confirmed that the K562Dox cells are resistant to the
apoptosis inducing effects of STI571.
C. Verification of the effect of the siRNAs: uptake of the siRNAs and
downregulation of P-gp protein expression The optimum conditions for transfection using the
jetSI# transfection reagent were previously established
(data not shown). Uptake of the siRNAs was confirmed by
using fluorescently-labelled siRNAs and by verifying the
presence of small green fluorescent aggregates localized
near the DAPI-stained nuclei, as visualized by
fluorescence microscopy (Figure 3). Indeed, it was
verified that 24 hours after transfection, 53% of the cells
presented the green fluorescent aggregates, indicating a
successful transfection of the siRNAs.
Figure 1. Basal levels of P-gp and
BCR-ABL protein expression in K562
and K562Dox cell lines. (A) Western
Blots (20 µg protein per lane) were
probed for P-gp and reprobed for
Actin. (B) Western Blots (30 µg
protein per lane) were probed for
BCR-ABL and reprobed for Actin.
Lima et al: Overcoming K562Dox resistance to (Gleevec)
70
Figure 2. Response of the K562 and K562Dox cell lines to different STI571 concentrations. This analysis was performed at 24 hours
(A) and at 48 hours (B) after the treatment with the drug. The full line indicates the response of the K562 cell line and the dashed line
indicates the response of the K562Dox cell line, both represented as % of viable cells in relation to control cells (cells without treatment
with STI571).The graph represents the mean ± SE of 3 independent experiments for 0.25µM, 0.75µM and 1.25µM STI571
concentrations and of 4 independent experiments for the remaining concentrations (0µM, 0.5µM and 1µM). The vertical bars represent
the standard errors.
Table 1. Induction of apoptosis in K562 and K562Dox cells by STI571. The values represent the mean ± SE of the % of
apoptotic cells from 3 independent experiments.
H2O 1µM STI571
K562 2%±0% 7%±0%
K562Dox 3%±0% 4%±0%
To confirm if the siRNAs were efficient, the
expression of P-gp protein was analyzed 24 and 48 hours
after transfection. It was verified that 24 hours after
transfection with either of the siRNAs for P-gp (MDR-CR
or MDR-FE), there was a specific downregulation of P-gp
expression (Figure 4A-left panel). When carrying out
semi-quantitative analysis of Western Blots from 3
independent experiments, it was possible to confirm that
this downregulation was more pronounced with the MDR-
CR siRNA than with the MDR-FE siRNA, 24 hours after
transfection (Figure 4B-left panel). Indeed, this semi-
quantitative analysis showed that, 24 hours after
transfection, the levels of P-gp expression in the cells
transfected with the MDR-FE or MDR-CR siRNAs
decreased to 67% or 43% of the levels of the cells
transfected with the control siRNAs (CRNAi),
respectively (Figure 4B -left panel). When analyzing the
results 48 hours after transfection, it was possible to verify
that P-gp protein levels in the treatments with siRNAs for
MDR1 had returned to levels similar to the treatments with
the control siRNAs (Figures 4A,B-right panels).
Figure 3. Uptake of Alexa Fluor 488-
labelled control siRNA in the
K562Dox cell line. Uptake was
verified by fluorescence microscopy,
24 hours after transfection of the
siRNA with jetSI# reagent. Nuclei
were stained with Dapi and green
fluorescence resulted from siRNA
internalization. The mean ± SE of the
percentage of internalization of the
siRNA is represented in the bottom left
corner (obtained from 3 independent
experiments). The bar in the image
represents 100µm.
Cancer Therapy Vol 5, page 71
71
Figure 4. Analysis of P-gp protein levels after transfection with siRNAs. (A) Protein expression analysis by Western Blot. Proteins
were extracted at 24h and 48h after transfection of K562Dox cells with control siRNA (CRNAi) or with the MDR-FE or MDR-CR
siRNAs. Blots (10 µg protein per lane) were probed for P-gp and reprobed for actin. (B) Semi-quantitative analysis of the intensity of the
bands obtained by Western Blot. Each bar represents the mean ± SE of the ratios P-gp/actin, obtained from the intensity of the bands
from 3 independent experiments.
Figure 5. Effects of downregulation of P-gp on the sensitization to 0.5µM STI571. Results are represented as a % of the K562Dox
Control cells treated with the solvent of STI571 (water), analysed 48 hours after this treatment, considering this as 100% in all
experiments. For each group of columns, the filled bar represents the number of viable cells after treatment with solvent and the dashed
bar represents the treatment with 0.5µM STI571. The results are the mean ± SE of 3 independent experiments. The first group of
columns represents the effect of STI571 or its solvent in the number of viable control cells (Control). The remaining groups of columns
represent the effects of the treatment with STI571 or its solvent in cells previously transfected with control siRNA (CRNAi) or siRNAs
for P-gp (MDR-FE or MDR-CR). * Represents P! 0.05 between the treatment with STI571 and the respective treatment with solvent,
analysed individually for each group of columns.
Lima et al: Overcoming K562Dox resistance to (Gleevec)
72
Figure 6. Effects of downregulation of P-gp on the sensitization to 1µM STI571. Results are represented as a % of the K562Dox
control cells treated with the solvent of STI571 (water), analysed 48 hours after treatment, considering this as 100% in all experiments.
For each group of columns, the filled bar represents the treatment with solvent and the dashed bar represents the number of viable cells
after treatment with 1"M STI571. The results are the mean ± SE of 4 independent experiments. The first group of columns represents the
effect of STI571 or its solvent in the number of viable control cells (Control). The remaining groups of columns represent the effects of
the treatment with STI571 or its solvent in cells previously transfected with control siRNA (CRNAi) or siRNAs for P-gp (MDR-FE or
MDR-CR). * Represents P! 0.05 between the treatment with STI571 and the respective treatment with solvent, analysed individually for
each group of columns.
D. Effects of downregulation of P-gp expression in the cellular response to STI571:
effects on the number of viable and apoptotic
cells To investigate whether downregulation of P-gp
expression in the K562Dox cells was sufficient to sensitize
these resistant cells to treatment with STI571, cells
previously transfected with siRNAs for P-gp were treated
with 0.5 or 1µM STI571 and the number of viable cells
was counted 48 hours after treatment. The choice of
STI571 concentrations and length of treatment to be used
was based on the above mentioned response of both cells
lines (K562 and K562Dox) to this drug (Figure 2).
Indeed, it had been observed that these concentrations
affected the sensitive cell line (K562) reducing its viable
cell number to 58% or to 54% of the control cells 48h after
treatment with 0.5 or 1µM STI571 respectively, but did
not strongly affect the viable cell number of the resistant
cell line (K562Dox). Results were analyzed as a
percentage of the viable Control cell number (treated only
with water) and are presented in Figure 5 (for 0.5µM
STI571) and Figure 6 (for 1µM STI571). When analysing
the results obtained from treating the cells with 0.5µM
STI571 (Figure 5) it was observed that the addition of
STI571 to the Control cells (not transfected) did not
significantly affect their viability, as expected. Indeed,
only a small reduction in the viable cell number was
observed, to 88% of its control with solvent (Figure 5-first
group of columns). It was also observed that transfection
of the cells with the control siRNA (CRNAi) did not affect
their response to STI571, since a similar viable cell
number was obtained after the addition of solvent or
STI571 (Figure 5-second group of columns). On the other
hand, when cells were transfected with the MDR-CR or
MDR-FE siRNAs, there was an increase in the effects of
the STI571. This was verified, in the case of the MDR-FE
siRNA treatment, by the decrease in the viable cell
number from 88% (after treatment with solvent) to 75%
(after treatment with 0.5µM STI571). The effect of STI571
was even stronger in the cells previously transfected with
the MDR-CR siRNA. In this case, there was a statistically
significant decrease in the viable cell number from 94%
(after treatment with solvent) to 71% (after treatment with
0.5µM STI571).
Analysing the results from the treatment with 1µM
STI571 (Figure 6), the addition of STI571 to the Control
cells (not transfected) did not significantly affect their
viability, as expected (Figure 6–first group of columns).
Transfection of the cells with control siRNA (CRNAi) did
also not affect their response to 1µM STI571 (Figure 6–
second group of columns). In both these cases a small
decrease in the viable cell number was observed after
treatment with STI571, but was not considered statistically
significant. However, when cells treated with 1µM STI571
had previously been transfected with the MDR-FE or
MDR-CR siRNAs, a significant decrease in the viable cell
number was verified. In both cases there was an
accentuated decrease from 86% (after solvent treatment) to
58% (after STI571 treatment), which was considered
statistically significant (Figure 6– last 2 groups of
columns).
This sensitization effect was seen when cells were
treated for 48h with STI571. No effect was seen when
cells were treated for 24h only (results not shown)
possibly due to the fact that the effect of STI571 was only
Cancer Therapy Vol 5, page 73
73
evident in these cell lines 48h after treatment (see Figure
2).
In order to clarify if this reduction in the viable cell
number, observed after transfection with the siRNAs for
P-gp and treatment with STI571, was due to an increase in
apoptosis, the TUNEL assay was carried out 48 hours after
drug treatment. Results from this assay were analyzed by
fluorescence microscopy and the percentage of apoptotic
cells in the different treatments was determined (Figure
7). Transfection of the cells with any of the siRNAs did
not cause, on its own, a significant increase in the levels of
apoptosis, since these levels only increased from 2% in the
Control treatment to 3% in the transfected treatments
(Figure 7-top panel). Furthermore, when 1µM STI571 was
Figure 7. Apoptosis in K562Dox cells following treatment with 1µM STI571, detected by the TUNEL assay.
Apoptosis was determined 48h after treatment with the STI571. The values represent the mean ± SE of the % of apoptotic cells,
determined after counting at least 500 cells per experiment in 3 independent experiments. A typical apoptotic cell is indicated with the
arrow A and a typical non-apoptotic cell is indicated with the arrow B. Nuclei are labelled with DAPI. * Represents P! 0.05 between the
treatment with 1"M STI571 and the respective treatment with solvent. The bar represents 200µm.
added to the Control cells or to the CRNAi cells, there was
no significant increase in the % of apoptotic cells.
However, in the cells in which the P-gp expression had
previously been downregulated by RNAi, the treatment
with 1µM STI571 led to a significant increase in the levels
of apoptosis, in comparison to the respective treatment
with its solvent. Indeed, in cells transfected with either the
MDR-FE or the MDR-CR siRNAs, the % of apoptosis
significantly increased from 3% (after treatment with
solvent) to 7% (after treatment with 1µM STI571).
IV. Discussion In order to validate the importance of P-gp in the
resistance of CML cells to STI571, two blastic-phase
CML cell lines, one expressing and the other not
expressing P-gp, were used as a model. It was important
that the only difference between these cells, that could
justify a difference in response to STI571, was at the level
of P-gp expression. Therefore, it was imperative to
confirm that one cell line had a high expression level of P-
gp, whereas the other cell line did not express P-gp (at the
limit of detection of the technique used-Western Blot).
Following the same line of thought, it was confirmed that
there were no differences in the levels of BCR-ABL
expression between the two cell lines, and that there were
no mutations of BCR-ABL in the resistant cell line, that
could justify that resistance.
The two cell lines responded differently to various
concentrations of STI571 suggesting that P-gp was
responsible for resistance to STI571. Such results were
already anticipated since they are in agreement with the
results from previously published studies carried out by
other authors, in which cells derived from the K562 cell
line, selected for resistance to other drugs and
overexpressing P-gp, were resistant to STI571 when
compared to the parental cell line, K562 (Che et al, 2002;
Kotaki et al, 2003; Mahon et al, 2003; Illmer et al, 2004).
However, the results contradict those from other studies in
which P-gp overexpression in the K562 cell line did not
increase its resistance to STI571 (Ferrao et al, 2003; Zong
et al, 2005). The reason for such contradicting data may be
the difference in the experimental models used, the first
two and the present work based on cell lines which had
high levels of P-gp expression obtained by exposure to a
drug, whereas in the last two studies the high levels of P-
gp were obtained by transfection of cDNA. It is known
that cells that are selected by resistance to drug treatment
may have other alterations, apart from P-gp
overexpression (Zong et al, 2005). Such alterations could
justify the results observed in the present study and in the
studies from the other authors (Che et al, 2002; Kotaki et
al, 2003; Mahon et al, 2003; Illmer et al, 2004). On the
other hand, studies in which cells are engineered to
overexpress P-gp are based upon an “aberrant” model and
therefore the lack of resistance to the STI571 observed in
these studies (Ferrao et al, 2003; Zong et al, 2005) may be
due to this unnatural model.
These existent conflicting data deriving from a
diversity of experimental models show that it is important
Lima et al: Overcoming K562Dox resistance to (Gleevec)
74
to complement these studies, in order to further validate
the relevance of P-gp in the resistance to STI571. The
experimental model used in this study was based on a
recently discovered biotechnology, which relies on a
natural mechanism existent in cells to silence gene
expression, RNAi. We aimed at using one cell line that
overexpressed P-gp, which had been selected by drug
resistance and therefore could have other alterations that
could justify resistance to STI571, and reducing P-gp
expression in this cell line using the natural RNAi
methodology, in order to determine if P-gp was the
responsible alteration for such resistance.
The RNAi technology with siRNAs relies on the
uptake of the siRNAs by the cells. The optimized
conditions showed relatively low levels of uptake of the
siRNAs (53%). However, this was considered to be
acceptable since these cells are known to be very difficult
to transfect. Once the accepted mechanism for RNAi is
based on specific degradation of the target mRNA and
therefore reduction in the targeted protein expression, the
analysis of the protein levels are necessary in order to
confirm if and when the siRNAs are active. The data here
presented indicate that the siRNAs were capable of
reducing P-gp expression. The reduction was pronounced
at 24h and was greater in the cells transfected with the
MDR-CR siRNA than with the MDR-FE siRNA.
The difference in the effect of these siRNAs is
probably due to the fact that they target different regions
of the mRNA. In fact, previous knowledge that some
siRNAs hybridize with the target mRNA better than
others, was the reason to work from the outset with two
different siRNAs, targeting the same mRNA. Indeed, it is
known that the efficacy of hybridization between siRNAs
and its target mRNA depends on several factors, such as
the thermodynamic structure of the siRNAs themselves
and the structure of the target mRNA (Schubert et al,
2005). It was also verified that none of the siRNAs used
was capable of totally silencing P-gp expression. This was
already expected and is possibly due to the low percentage
of transfection in this cell line, as verified by the uptake of
the fluorescent siRNA, and also to the transient effect of
the transfections, as verified by the Western Blot results.
One possible way to overcome this transient RNAi effect
is to create stable cell lines through the transfection of
vectors cloned with shRNAs, allowing for the continuous
expression of siRNAs in the cells and therefore a
permanent RNAi effect. This approach has already been
carried out by others in order to downregulate P-gp
expression in several cell lines (Celius et al, 2004; Stege et
al, 2004; Yague et al, 2004), including the one used in this
study (Rumpold et al, 2005). However, the use of siRNAs
has the advantage of yielding faster results. Furthermore, it
is possible that siRNAs themselves could become
therapeutic weapons in the future. In fact, there are already
ongoing clinical trials with the use of siRNAs for other
diseases (Hede 2005).
The transfections with the control siRNA caused
some toxicity in the K562Dox cell line (considered as non-
specific effects since there was no observed reduction of
P-gp in the Western Blot, in the CRNAi treatment). This
toxicity, observed in the cells transfected with the control
siRNA and treated with solvent (Figures 5 and 6–second
groups of columns-filled bars) could justify the apparent
lack of effect of 0.5µM STI571 in these cells, when
comparing to the control treatment and analysing the
results of STI571 in relation to its solvent. This toxicity
could be possibly avoided if different control siRNA
sequences had been designed. Some authors indicate that
other controls should be used in RNAi experiments, such
as siRNAs that have already been confirmed to participate
in the RNAi machinery but that are not known to interfere
with the protein expression of mammalian cells (Hannon
and Rossi 2004).
Pre-treatment of cells with siRNAs for P-gp
enhanced their sensitivity to STI571. The fact that the
increase in the sensitivity to 0.5µM STI571 was only
considered statistically significant when cells were treated
with the MDR-CR siRNA, but not when they were treated
with the MDR-FE siRNA (Figure 5), is in agreement with
the data shown above in which the MDR-CR siRNA
decreased P-gp expression to a higher extent than the
MDR-FE siRNA (Figure 4) and is therefore probably due
to the different potency of the two siRNAs. Increasing
STI571 concentration from 0.5µM to 1µM caused a more
pronounced increase in sensitivity to STI571, considered
statistically significant and similar for cells transfected
with the MDR-FE or the MDR-CR siRNAs. Indeed, the
increase in the sensitivity of these cells was of such an
order that their response to 1µM STI571 (viable cell
number = 56%) became similar to the response of the
K562 cell line (sensitive cell line) to that same STI571
concentration (viable cell number = 54%). This proved
that it is possible to revert STI571 drug resistance, in this
cell line, by downregulating MDR1 gene expression.
STI571 has been shown to induce apoptosis in the
K562 cell line (Jacquel et al, 2003) and the results here
presented prove that this is also the case for the K562Dox
cell line. Indeed, results from the TUNEL assay with 1µM
STI571 confirmed that once STI571 is able to reduce the
viable cell number, it does so by means of increasing
apoptosis.
The data here presented allows to conclude that
downregulation of P-gp protein expression with siRNAs
permitted sensitization to STI571, of CML cells which
overexpressed P-gp. This suggests that P-gp may be
considered a good molecular therapeutic target, as an
adjuvant to therapy with STI571. Furthermore, the
enhancement of the effect of STI571 was due to an
increase in apoptosis.
Acknowledgements The authors would like to thank Novartis Oncology
Portugal for financial support and Novartis Pharma for the
STI571 used in this study. We would also like to thank
Prof. Clara Sambade and Prof. Paula Soares for general
advice and finally Patrícia Pontes, Joana Figueiredo and
Hugo Seca for technical assistance.
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