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Cancer Therapy Vol 5, page 67

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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)

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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.


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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.


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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.


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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.


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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


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


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.

References Calabretta B and Perrotti D (2004) The biology of CML blast

crisis. Blood 103, 4010-4022.


75

Celius T, Garberg P and Lundgren B (2004) Stable suppression

of MDR1 gene expression and function by RNAi in Caco-2

cells. Biochem Biophys Res Commun 324, 365-371.

Che XF, Nakajima Y, Sumizawa T, Ikeda R, Ren XQ, Zheng

CL, Mukai M, Furukawa T, Haraguchi M, Gao H, Sugimoto

Y and Akiyama S (2002) Reversal of P-glycoprotein

mediated multidrug resistance by a newly synthesized 1,4-

benzothiazipine derivative, JTV-519. Cancer Lett 187, 111-

119.

Deininger MW, Goldman JM and Melo JV (2000a) The

molecular biology of chronic myeloid leukemia. Blood 96,

3343-3356.

Deininger MW, Vieira S, Mendiola R, Schultheis B, Goldman

JM and Melo JV (2000b) BCR-ABL tyrosine kinase activity

regulates the expression of multiple genes implicated in the

pathogenesis of chronic myeloid leukemia. Cancer Res 60,

2049-2055.

Druker BJ (2002) Imatinib and chronic myeloid leukemia:

validating the promise of molecularly targeted therapy. Eur

J Cancer 38 (Suppl5),70-76.

Fardel O, Lecureur V and Guillouzo A (1996) The P-

glycoprotein multidrug transporter. Gen Pharmacol 27,

1283-1291.

Ferrao PT, Frost MJ, Siah SP and Ashman LK (2003)

Overexpression of P-glycoprotein in K562 cells does not

confer resistance to the growth inhibitory effects of imatinib

(STI571) in vitro. Blood 102, 4499-4503.

Gambacorti-Passerini C, Barni R, le Coutre P, Zucchetti M,

Cabrita G, Cleris L, Rossi F, Gianazza E, Brueggen J,

Cozens R, Pioltelli P, Pogliani E, Corneo G, Formelli F and

D'Incalci M (2000) Role of alpha1 acid glycoprotein in the in

vivo resistance of human BCR-ABL(+) leukemic cells to the

abl inhibitor STI571. J Natl Cancer Inst 92, 1641-1650.

Goldstein LJ, Galski H, Fojo A, Willingham M, Lai SL, Gazdar

A, Pirker R, Green A, Crist W, Brodeur GM and et al, (1989)

Expression of a multidrug resistance gene in human cancers.

J Natl Cancer Inst 81, 116-124.

Gorre ME, Mohammed M, Ellwood K, Hsu N, Paquette R, Rao

PN and Sawyers CL (2001) Clinical resistance to STI-571

cancer therapy caused by BCR-ABL gene mutation or

amplification. Science 293, 876-880.

Hannon GJ and Rossi JJ (2004) Unlocking the potential of the

human genome with RNA interference. Nature 431, 371-

378.

Hede K (2005) Blocking cancer with RNA interference moves

toward the clinic. J Natl Cancer Inst 97, 626-628.

Illmer T, Schaich M, Platzbecker U, Freiberg-Richter J,

Oelschlagel U, von Bonin M, Pursche S, Bergemann T,

Ehninger G and Schleyer E (2004) P-glycoprotein-mediated

drug efflux is a resistance mechanism of chronic

myelogenous leukemia cells to treatment with imatinib

mesylate. Leukemia 18, 401-408.

Jacquel A, Herrant M, Legros L, Belhacene N, Luciano F, Pages

G, Hofman P and Auberger P (2003) Imatinib induces

mitochondria-dependent apoptosis of the Bcr-Abl-positive

K562 cell line and its differentiation toward the erythroid

lineage. Faseb J 17, 2160-2162.

Kano Y, Akutsu M, Tsunoda S, Mano H, Sato Y, Honma Y and

Furukawa Y (2001) In vitro cytotoxic effects of a tyrosine

kinase inhibitor STI571 in combination with commonly used

antileukemic agents. Blood 97, 1999-2007.

Knight GW and McLellan D (2004) Use and limitations of

imatinib mesylate (Glivec), a selective inhibitor of the

tyrosine kinase Abl transcript in the treatment of chronic

myeloid leukaemia. Br J Biomed Sci 61, 103-111.

Kotaki M, Motoji T, Takanashi M, Wang YH and Mizoguchi H

(2003) Anti-proliferative effect of the abl tyrosine kinase

inhibitor STI571 on the P-glycoprotein positive K562/ADM

cell line. Cancer Lett 199, 61-68.

Kurzrock R, Kantarjian HM, Druker BJ and Talpaz M (2003)

Philadelphia chromosome-positive leukemias: from basic

mechanisms to molecular therapeutics. Ann Intern Med

138, 819-830.

Lima RT, Martins LM, Guimaraes JE, Sambade C and

Vasconcelos MH (2004) Specific downregulation of bcl-2

and xIAP by RNAi enhances the effects of chemotherapeutic

agents in MCF-7 human breast cancer cells. Cancer Gene

Ther 11, 309-316.

Mahon FX, Belloc F, Lagarde V, Chollet C, Moreau-Gaudry F,

Reiffers J, Goldman JM and Melo JV (2003) MDR1 gene

overexpression confers resistance to imatinib mesylate in

leukemia cell line models. Blood 101, 2368-2373.

Mahon FX, Deininger MW, Schultheis B, Chabrol J, Reiffers J,

Goldman JM and Melo JV (2000) Selection and

characterization of BCR-ABL positive cell lines with

differential sensitivity to the tyrosine kinase inhibitor

STI571: diverse mechanisms of resistance. Blood 96, 1070-

1079.

Motomura S, Motoji T, Takanashi M, Wang YH, Shiozaki H,

Sugawara I, Aikawa E, Tomida A, Tsuruo T, Kanda N and

Mizoguchi H (1998) Inhibition of P-glycoprotein and

recovery of drug sensitivity of human acute leukemic blast

cells by multidrug resistance gene (mdr1) antisense

oligonucleotides. Blood 91, 3163-3171.

Nieth C, Priebsch A, Stege A and Lage H (2003) Modulation of

the classical multidrug resistance (MDR) phenotype by RNA

interference (RNAi). FEBS Lett 545, 144-150.

Nimmanapalli R and Bhalla K (2002) Novel targeted therapies

for Bcr-Abl positive acute leukemias: beyond STI571.

Oncogene 21, 8584-8590.

O'Dwyer ME, Mauro MJ and Druker BJ (2002) Recent

advancements in the treatment of chronic myelogenous

leukemia. Annu Rev Med 53369-381.

Pan L, Tong Y, Jin Y, Zhou S, Zhang Y, Yang X and Mao N

(2001) Reversing drug resistance in the ovarian carcinoma

cell line SKOV3/mdr1 in vitro by antisense

oligodeoxynucleotides. Chin Med J 114

Ramachandran C and Wellham LL (2003) Effect of MDR1

phosphorothioate antisense oligodeoxynucleotides in

multidrug-resistant human tumor cell lines and xenografts.

Anticancer Res 23, 2681-2690.

Roche-Lestienne C, Soenen-Cornu V, Grardel-Duflos N, Lai JL,

Philippe N, Facon T, Fenaux P and Preudhomme C (2002)

Several types of mutations of the Abl gene can be found in

chronic myeloid leukemia patients resistant to STI571, and

they can pre-exist to the onset of treatment. Blood 100, 1014-

1018.

Rumpold H, Wolf AM, Gruenewald K, Gastl G, Gunsilius E and

Wolf D (2005) RNAi-mediated knockdown of P-

glycoprotein using a transposon-based vector system durably

restores imatinib sensitivity in imatinib-resistant CML cell

lines. Exp Hematol 33, 767-775.

Sambrook J, Fritsh EF, Maniatis T. (1989) Detection and

analysis of proteins expressed from cloned genes. In: Ford N,

Nolan C and Ferguson M, eds.

Savage DG and Antman KH (2002) Imatinib mesylate--a new

oral targeted therapy. N Engl J Med 346, 683-693.

Schubert S, Grunweller A, Erdmann VA and Kurreck J (2005)

Local RNA target structure influences siRNA efficacy:

systematic analysis of intentionally designed binding regions.

J Mol Biol 348, 883-893.

Shet AS, Jahagirdar BN and Verfaillie CM (2002) Chronic

myelogenous leukemia: mechanisms underlying disease

progression. Leukemia 16, 1402-1411.


76

Sola JE and Colombani PM (1996) Modulation of multidrug

resistance with antisense oligodeoxynucleotide to mdr1

mRNA. Ann Surg Oncol 3, 80-85.

Stege A, Priebsch A, Nieth C and Lage H (2004) Stable and

complete overcoming of MDR1/P-glycoprotein-mediated

multidrug resistance in human gastric carcinoma cells by

RNA interference. Cancer Gene Ther 11, 699-706.

Wang H, Chen XP and Qiu FZ (2003) Overcoming multi-drug

resistance by anti-MDR1 ribozyme. World J Gastroenterol

9, 1444-1449.

Weisberg E and Griffin JD (2003) Resistance to imatinib

(Glivec): update on clinical mechanisms. Drug Resist Updat

6, 231-238.

Wu H, Hait WN and Yang JM (2003) Small interfering RNA-

induced suppression of MDR1 (P-glycoprotein) restores

sensitivity to multidrug-resistant cancer cells. Cancer Res

63, 1515-1519.

Yague E, Higgins CF and Raguz S (2004) Complete reversal of

multidrug resistance by stable expression of small interfering

RNAs targeting MDR1. Gene Ther 11, 1170-1174.

Zong Y, Zhou S and Sorrentino BP (2005) Loss of P-

glycoprotein expression in hematopoietic stem cells does not

improve responses to imatinib in a murine model of chronic

myelogenous leukemia. Leukemia 19, 1590-1596.

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