ORIGINAL PAPER

Combined Use of Anticancer Drugs and an Inhibitor of MultipleDrug Resistance-Associated Protein-1 Increases Sensitivityand Decreases Survival of Glioblastoma Multiforme Cells In Vitro

Lilia Peignan • Wallys Garrido • Rodrigo Segura •

Romulo Melo • David Rojas • Juan Guillermo Carcamo •

Rody San Martın • Claudia Quezada

Accepted: 26 March 2011 / Published online: 5 May 2011

� Springer Science+Business Media, LLC 2011

Abstract Glioblastoma multiforme (GBM) is a brain

tumour characterised by a remarkably high chemoresis-

tance and infiltrating capability. To date, chemotherapy

with temozolomide has contributed only poorly to

improved survival rates in patients. One of the most

important mechanisms of chemoresistance comes about

through the activity of certain proteins from the ATP-

binding cassette superfamily that extrudes antitumour

drugs, or their metabolites, from cells. We identify an

increased expression of the multiple drug resistance-asso-

ciated protein 1 (Mrp1) in glioblastoma multiforme biop-

sies and in T98G and G44 cell lines. The activity of this

transporter was also confirmed by measuring the extrusion

of the fluorescent substrate CFDA. The sensitivity of GBM

cells was low upon exposure to temozolomide, vincristine

and etoposide, with decreases in cell viability of below

20% seen at therapeutic concentrations of these drugs.

However, combined exposure to vincristine or etoposide

with an inhibitor of Mrp1 efficiently decreased cell via-

bility by up to 80%. We conclude that chemosensitization

of cells with inhibitors of Mrp1 activity might be an effi-

cient tool for the treatment of human GBM.

Keywords Glioblastoma multiforme � Multiple drugs

resistance � Mrp1

Introduction

Resistance of tumour cells to anticancer drugs is one of the

reasons that cancer chemotherapy fails. One of the mech-

anisms involved in the multi-drug resistant (MDR) phe-

notype, is the expression of one or more proteins belonging

to the ABC transporter gene family (ATP-binding cassette)

in tumour cells [1]. These proteins function as pumps for

removal of different metabolites and xenobiotics, including

antitumour drugs from cells [2]. To date, only 10 members

of this superfamily and two proteins non-related to ABC

transporters have been recognised as having a substrate

selectivity that can extrude antitumour drugs or their

derivatives from cells [2–6]. In peripheral tumours,

increased expression of one or more of these resistance

proteins has been demonstrated. This increased expression

is a requirement for insensitivity to chemotherapy [2, 7, 8].

Mortality from brain tumours represents 2.4% of cancer

deaths in the USA. In children, it is the second specific

cause of death after hematopoietic malignancies [9]. Glio-

blastoma multiforme cells (WHO glioma grade IV) are

characterised by insensitivity to the majority of anticancer

treatments, highly infiltrating tumours and consequent

dramatic low survival of patients [10]. Standard treatment

of glioblastoma multiforme is with temozolomide after

resection of the tumour. It is the only drug which offers a

modest improvement—of no more than 20% increase—in

patient survival in clinical trials [11]. Thus, major advances

in the chemotherapeutic treatment of high-grade brain

tumours will require a comprehensive understanding of the

mechanisms of specific- and multiple-drug resistance. Glial

cells normally express low levels of some drug-resistant

Electronic supplementary material The online version of thisarticle (doi:10.1007/s11064-011-0464-8) contains supplementarymaterial, which is available to authorized users.

L. Peignan � W. Garrido � J. G. Carcamo �R. San Martın � C. Quezada (&)

Instituto de Bioquımica, Facultad de Ciencias, Universidad

Austral de Chile, Campus Isla Teja s/n, P.O. box 567,

Valdivia, Chile

e-mail: claudiaquezada@uach.cl

R. Segura � R. Melo � D. Rojas

Servicio de Neurocirugıa, Instituto de Neurocirugıa Dr. Asenjo,

Santiago, Chile

123

Neurochem Res (2011) 36:1397–1406

DOI 10.1007/s11064-011-0464-8

transporters [12]. But there are currently only a few

studies in high-grade primary brain tumours that correlate

the activity of any drug-resistant transporters with

chemoresistance.

Among the transporters that confer multiple resistance

to antitumour drugs, P-glycoprotein (P-gp, MDR1 or

ABCB1), belonging to the ABC gene family, was the first

protein transporter identified and that which has been most

extensively studied [13–15]. A predominant role has been

attributed to P-gp in chemoresistance in several tumour

types [2, 7]. Thus, a P-gp inhibitor combination has been

included in chemotherapy protocols in order to increase the

intracellular bioavailability of antitumour drugs, and in this

way, improve the efficiency of cytotoxicity [2, 13, 16].

However, this approach has not proven to be generally

beneficial for cancer treatment, because P-gp is not the

only transporter whose expression and activity is important

for extruding drugs in all tumour types [7, 17]. Studies

using cytofluorometric analysis and immunohistochemistry

of glioma samples correlate a high expression of MDR

proteins Mrp1, Mrp3 and Mrp5 with high-grade gliomas,

while P-gp expression was found to be insignificant

[18–21]. In a similar way, studies in glioblastoma cell lines

corroborate the predominant content of MRP subfamily

members, particularly Mrp1 [22]. Recent studies to detect

the mRNA content of the ABCG2 transporter indicated that

this transporter was up-regulated in both glioblastoma

vessels and parenchymal tissue, suggesting a role for brain

endothelial ABCG2 transporter in modulating drug deliv-

ery to the brain or in conferring drug resistance to glio-

blastoma [23].

We measured the expression of the three major drug

resistance transporters (P-gp, Mrp1 and ABCG2) in two

cell lines and in human glioblastoma multiforme tissue. In

this study we demonstrate that the activity of Mrp1 in

human GBM is relevant in conferring resistance to vin-

cristine and etoposide and that its inhibition, in combina-

tion with chemotherapy, provides a better cytotoxic effect

on these cells compared to current treatment in vitro with

temozolomide. It suggests that inhibitors of Mrp1 activity

might be efficient chemosensitizers for the treatment of

glioblastoma multiforme.

Methods

Cell Cultures and Drugs

The human glioblastoma multiforme T98G cell line was

acquired from ATCC Company (ATCC CRL-1690) and

the G44 cell line was kindly provided by Dr. Manuel Grez.

The cells were grown in DMEM-F12 medium supple-

mented with 10% foetal bovine serum, 1% penicillin/

streptomycin, at 37�C in an atmosphere humidified with

5% CO2. Antitumour drugs, MK571 and probenecid were

from Tocris Bioscience.

Brain Tumours

Tissue samples were obtained from resection procedures in

brain tumour patients by therapeutic indication at the

Department of Neurosurgery, Institute of Neurosurgery

Asenjo, Santiago, Chile. All procedures were carried out

with the approval of the Bioethics Committee of the Uni-

versidad Austral de Chile. Part of each sample was intro-

duced immediately into RNAlater solution or was fixed for

immunohistochemistry.

Reverse Transcription and PCR

Total cellular RNA was isolated using Trizol� Isolation

Reagent (Invitrogen). First strand cDNA was synthesised

from 1 lg of total RNA using 50 U of MMLV Reverse

Transcriptase (Stratagene) and oligo(dT)18 according to

manufacturers’ recommendations. PCR amplifications

were carried out in a Personal Thermal Cycler (Eppendorf)

using 1 ll of cDNA, 19 PCR buffer, 1.5 mM Mg2?,

0.4 mM dNTP’s, 1.25 U Taq DNA polymerase (Invitro-

gen, USA) and 0.5 lM of gene-specific oligonucleotides

[24]. PCR amplifications were performed for 30 cycles,

each of which consisted of denaturing at 92�C for 60 s,

annealing to 55�C for 45 s and extension to 72�C for 30 s.

The PCR products were separated by 2% agarose gel

electrophoresis. EtBr fluorescence intensities were quanti-

fied by densitometry analysis using a Molecular Analyst

Software (BioRad). The relative abundance of transcripts

from each gene was estimated by comparison with b-actin

ratios.

Western Blot Analysis

Total protein extracts from T98G and G44 cell lines or

brain tumours were obtained in buffer 10 mM Tris–HCl,

2% SDS, 10% glycerol, 1 mM PMSF and protease inhib-

itors (Complete, Roche). Aliquots (50 lg) were fraction-

ated in 10% SDS–PAGE followed by transfer to

nitrocellulose membranes (BioRad). The upper fraction of

the membranes were incubated with primary antibodies

anti-human Mrp1 (sc-18835), anti-human P-gp (sc-55510)

and anti-human Abcg2 (sc-58222) from Santa Cruz Bio-

technology at 1:1000 dilutions for 1 h and the lower frac-

tion of membranes with an anti-human b-actin antisera

(sc-47778, Santa Cruz Biotech) at 1:5000 dilution. After

washing, the blots were further incubated with horseradish

peroxidase-conjugated anti-mouse IgG antibody (DAKO)

at 1:5000 dilution for 1 h at RT. The immune detections

1398 Neurochem Res (2011) 36:1397–1406

123

were revealed using the chemiluminescent reagent

ECL-plus (Amersham Pharmacia).

Cell Viability Assays

1.0 9 104 cells per well were cultured in 96-well plates for

24 h and then exposed to antitumour drugs alone, 100 nM

vincristine or 2 lM etoposide, and in combination with

inhibitors 20 lM MK571 or 1 mM probenecid for 24 h.

Cells were then incubated with 5 mg/ml MTT reagent

(thiazol blue tetrazolium) in culture medium for 1 h and

formazan crystals were lysed using 100 ll DMSO. The

absorbance at 550 nm was read using a microplate reader.

Absorbance values are expressed as a percentage relative to

control cells without treatment. Cell viability was also

evaluated using temozolomide 100–400 lM. The effect of

inhibitors MK571 and probenecid on cell proliferation was

assayed in cells (60% of confluence) grown in 3H-thymi-

dine at 0.5 lCi/ml for 24 h. The resulting trichloroacetic

acid-insoluble materials were collected on 0.45 lm PVDF

filters and radioactivity was counted in a liquid scintillation

counter. Data were normalized with respect to the protein

content of each culture well. Apoptosis by inhibitors of

Mrp1 was evaluated in total protein extracts using anti-

cleaved caspase-3 (Asp175) antibody in western blot.

Immunohistochemistry

Brain tissues were fixed in formalin, paraffin embedded

and 5 lm sections were mounted on xylanized slides. For

immune detection, the sections were sequentially deparaf-

fined and rehydrated, incubated with 10 mM sodium citrate

(pH 6.0) for 30 min, hydrogen peroxide (70% methanol,

3% perhydrol) for 5 min and blocked with PBS1x con-

taining 1% bovine serum albumin, 0.3% Triton X-100

and 5% fat free milk for 30 min at room temperature.

Sections were incubated with primary antibodies (anti-

Mrp1 sc-18835, anti-Pgp sc-55510, anti-Abcg2 sc-58222,

anti-Ki-67 sc-56320 and anti-GFAP sc-52333 from Santa

Cruz Biotechnology) in blocking solution overnight at 4�C.

They were then washed three times with PBS1x for 5 min

and immune signals were revealed using the LSAB?

System-HRP system (Dako).

Immunofluorescence Laser Confocal Microscopy

Cells grown on circular coverslips at semi-confluence were

washed with 0.1 M phosphate buffer (pH 7.4), fixed with

Histochoice (Sigma) and permabilised using 0.3% Triton

X-100 in 1XPBS. Preparations were blocked with 1% BSA

and incubated with monoclonal antibody anti-Mrp1

(QCRL-1, sc-18835, Santa Cruz Biotechnology). A sec-

ondary antibody tagged with Alexa fluor 488 was also used

(Molecular Probes). The subcellular distribution of Mrp1

was visualised using confocal microscopy (Olympus

Fluoview 1000).

Mrp1 Functional Assays

The cells (2 9 105) were pre-incubated in serum-free

medium DMEM for 6 h at 37�C in 24-well plates. Then they

were loaded with 500 nM CFDA for 15 min. After that, they

were washed three times with 1XPBS and incubated for

15 min in serum-free DMEM medium. When required, cells

were exposed to MK571 and probenecid after loading. Next,

cells were washed three times with ice-cold PBS and lysed

in PBS containing 0.4% Triton X-100. The fluorescence in

cell extracts was measured with a spectrofluorimeter (Perkin

Elmer LS-55) at excitation 488 nm and emission 530 nm

[25]. The fraction of substrate extruded by Mrp1 was esti-

mated from the equation: [substrates accumulated in the

presence of selective inhibitors] - [substrates accumulated

in the absence of inhibitors].

RNA Interference

siRNA specific to human Mrp1 was expressed using the

pSilencer adeno 1.0 System (Ambion) targeting the

sequence of the mRNA 50-GATGACACCTCTCAACAA

ATT-30 [26]. Glioblastoma cells (T98G and G44) were

seeded in 6-well plates (80% confluence). Once attached to

the plate, cells were transfected with siRNA Mrp1 plasmid

(0.5 mg) using Lipofectamine 2000. Expression levels of

Mrp1 were analysed 48 h after transfection by Western

blot analysis. Extrusion activity was also evaluated.

Statistical Analysis

Analyses were carried out on raw data using the Peritz’ F

multiple means comparison. Student’s t test was applied for

unpaired data and P \ 0.05 was considered statistically

significant.

Results

With RT–PCR, we detected transcripts from the expression

of genes that encode for multidrug resistance proteins

Mrp1, P-gp and Abcg2 in RNA isolated from tumour tissue

samples as well as from human glioblastoma multiforme

cell lines T98G and G44 (Fig. 1a). Worthy of note, an

observed higher relative abundance of transcript coding for

the transporter Mrp1, compared to that of P-gp and Abcg2.

In contrast to described in lower grade brain tumours, the

abundance of messenger RNA coding for the transporter

P-gp was low. The protein contents of transporters were

Neurochem Res (2011) 36:1397–1406 1399

123

perfectly correlated to abundance of transcripts in tumour

tissue samples as well as in glioblastoma multiforme cell

lines (Fig. 1b).

In accordance with given data for the abundance of

transporters in cell cultures, the immune signal of Mrp1

was clearly abundant in the tumour zone and detected

slightly in the peritumour tissue. The signal of Mrp1

colocalised with the glial cell marker (GFAP) and cell

proliferation marker (Ki-67) in immunohistochemistry

studies (Fig. 2). In contrast, the signals for P-gp and Abcg2

were distributed uniformly through the human biopsies, in

both peritumour and tumour tissue, though at a lower

intensity compared to Mrp1 (Fig. 2). Besides, a strong

immunosignal for Mrp1 and Abcg2 was widely distributed

in brain tissue blood vessels from both tumour and non-

tumour biopsies.

With respect to functional characterisation of Mrp1 in

GBM cells, the subcellular allocation of this protein was

found to be distributed within plasma membrane and per-

inuclear regions (Fig. 3a). Its activity was assayed by

measuring the extrusion of the substrate CFDA from cells

[27]. At 15 min after loading, the extrusion of CFDA from

T98G and G44 cells was 78.4 and 68.0%, respectively,

which represent the activity of Mrp1 in these cells. The

remnant CFDA in T98G and G44 cells was increased by

the selective Mrp1 inhibitors MK571 and probenecid [22,

28] or by knocking-down expression using siRNA (Fig. 3b,

c). We found no effect of inhibitor MK571 or probenecid

on cell viability, proliferation or apoptosis (Fig. 3d).

Human GBM cell lines have shown to be insensitive to

some antitumour drugs such as vincristine and etoposide

[22, 29]. This suggests that the mechanism of chemore-

sistance may be mediated by the activity of overexpressed

Mrp1 in this tumour. Current chemotherapy for recurrent

GBM uses temozolomide [11]. However, the survival of

patients is only modestly prolonged. In order to find a more

effective treatment for this type of tumour, we evaluated in

vitro the increase in the sensitivity of GBM cells to vin-

cristine or etoposide when exposed together with Mrp1

inhibitors. The effect of the combined treatment on cell

viability was compared to those obtained using temozolo-

mide. For these assays, we used antitumour drugs at con-

centration that have been seen to arise in peripheral plasma

in clinical studies [10, 30–32]. Temozolomide caused a

minor decrease in cell viability upon exposure for 24 h.

Higher temozolomide doses of up to 400 lM did not

produce an additional decrease in cell viability (Fig. 4c).

Cells also appear insensitive to etoposide and treatment

using vincristine has only a small effect on cell viability in

T98G (Fig. 4a, b). However, when we assayed the cell

viability in GBM cells under combined treatment condi-

tions using vincristine or etoposide plus an Mrp1 inhibitor,

we found a decreased viability compared to treatment with

antitumour drugs alone or with temozolomide alone

(Fig. 4a, b). Similarly, T98G and G44 cells were respon-

sive to vincristine and etoposide when the expression of

Mrp1 was decreased using a specific siRNA (Fig. 4a, b).

Discussion

Cancer is a leading cause of death worldwide. Despite

efforts to develop better treatment, patient survival remains

low, especially for high-grade brain tumours such as GBM.

Until now, chemotherapy has not made a significant con-

tribution to treatment of patients with high-grade malignant

brain tumours, even though this method has the advantage

of being non-invasive and with far fewer complications

compared to surgery and radiotherapy. New generations of

Fig. 1 Expression of ABC transporters in glioblastoma multiforme

cells. a The products of PCR amplifications using gene specific

primers and total cDNA from G44 and T98G cell lines or

glioblastoma multiforme tissue samples were fractionated by elec-

trophoresis in agarose gel and stained using Et-Br. St, molecular

weight standard 100 bp leader; negative, PCR amplification reaction

using b-actin primers and devoid of template. b Western blot analysis

of total protein extracts (50 lg) from G44, T98G and primary

cultured glioblastoma multiforme cells were performed using specific

primary antibodies against ABC transporters (See ‘‘Methods’’). The

membranes were also used for the detection of b-actin as internal

control. The immune detections were revealed by chemiluminescence

1400 Neurochem Res (2011) 36:1397–1406

123

clinical drugs, including temozolomide, have limited effi-

ciency (*20% increase in survival) in the treatment of

high-grade tumours. Temozolomide effectiveness only

extends GBM patient survival by up to 15 months when

used in conjunction with radiotherapy versus a 12-month

extension with radiotherapy alone [11, 33]. The low

effectiveness of chemotherapy has been attributed to the

phenomenon known as ‘‘specific resistance’’, such as that

which occurs with the high activity of O6-methyguanine-

DNA-methyltransferase (MGMT) [34] and expression of

Tyrosinase-related protein (TRP)-2 [35] in GBM confer-

ring chemoresistance to temozolomide, or ‘‘multiple

resistance’’, which is conferred by the activity of MDR

proteins in these cells [1, 5, 6].

Our studies established an overexpression of Mrp1 in

T98G and G44 GBM cells compared to other transporters,

such as P-gp and Abcg2 and localised this protein in

tumour growth areas. Previous reports have shown that

Fig. 2 Expression of MRP1 in

human glioblastoma

multiforme. The tissue samples

were obtained from surgical

resection of glioblastoma

multiforme and processed for

immunohistochemistry (See

‘‘Methods’’). Representative

immune detections are shown.

Primary antibodies anti-GFAP

and Ki-67 were used as markers

of proliferation of glial cells.

The expression of Mrp1, P-gp

and Abcg2 were also assessed in

the brain sections as indicated in

each image. The immune

detections were revealed using

diaminobenzidine as substrate

for peroxidase. The samples

were counterstained using

hematoxiline. Control for IgG1

isotype of anti-Mrp1 antibody

or lacking primary antibody are

shown. Original Magnifications

2009. Scale bars 100 lm.

Asterisks indicates the area of

the tumour. Inserts show

expression in vascular beds

(magnification 10009)

Neurochem Res (2011) 36:1397–1406 1401

123

MDR1 gene expression was not detected, or was only

weakly detected in glioma cells, suggesting that P-gp

contributes to cellular resistance in a mere small subgroup

of gliomas, but contributes frequently in neuroblastomas

and meningiomas. Other studies indicate that among glial

tumours, grade I and II astrocytomas expressed the highest

levels of P-gp, Mrp3 and lung resistance-related protein

(LRP), and that these high levels of expression may be

related to the primary resistance of low-grade gliomas to

chemotherapy [36]. In contrast, Mrp1 was suggested to

Fig. 3 Mrp1 activity in GBM cell lines. a Immunocytochemical

detection of Mrp1 in T98G cells using a specific primary monoclonal

antibody (See ‘‘Methods’’). Nuclear staining was carried out using

propidium iodide. Fluorescent images were captured using a confocal

microscope. Original magnifications were obtained using an objective

409. b The activity of Mrp1 in T98G (left) and G44 (right) cells was

evaluated by the extrusion of the fluorescent substrate CFDA. The

graphs depict the intracellular fluorescence that remains following

15 min post loaded. MK571 and probenecid (Pb) are inhibitors of

Mrp1 activity. *P \ 0.05, **P \ 0.01, n = 5. c The accumulation of

CFDA was assayed in T98G cells following 48 h of transfected with a

plasmid that expresses a Mrp1 siRNA or an empty plasmid (control).

**P \ 0.01, n = 7. The upper figure shows a *70% decreasing in

Mrp1 expression using the specific siRNA. d The effect of Mrp1

inhibitors on cell viability, proliferation and apoptosis were assayed in

T98G and G44 cells. n = 5. No statistically significances were found

1402 Neurochem Res (2011) 36:1397–1406

123

play a constitutive role in the intrinsic chemoresistance of

glioblastoma multiforme cells [18, 19, 22, 37]. Further-

more, sections from high-grade gliomas were more fre-

quently positive for Mrp1, Mrp3 and Mrp5 compared to

samples from low-grade gliomas [19]. Abe et al. [29] have

shown Mrp1 expression at both protein and mRNA levels

in surgical specimens from patients with gliomas and have

detected a positive correlation with the grade of glioma.

Recently, was suggested that the hypoxia inducible factor-

1a (HIF-1a) plays a role in mediating chemotherapeutic

drug resistance by regulation of Mrp1 expression in a

glioblastoma multiforme cells line [38].

Although Mrp1 is widely expressed in normal tissue,

numerous studies have shown upregulation of Mrp1 in a

variety of solid tumours. Such is the cases of medullo-

blastomas and ependymomas [36]. Similarly, Mrp1 was

frequently found to be overexpressed in a large proportion

of non-small cell lung carcinoma (NSCLC) tumours prior

to treatment exposure. Moreover, Mrp1 expression was

found to be a highly significant indicator of poor response

to chemotherapy and poor overall survival in NSCLC [39,

40] and in small cell lung carcinoma (SCLC) [41, 42].

Mrp1 expression also constitutes a negative prognostic

marker for early-stage breast cancer, with several studies

revealing a strong association between expression level and

reductions in time to relapse and overall survival [43–45].

In prostate cancer, the levels of Mrp1 tend to increase with

disease stage and invasiveness [46]. Furthermore, Mrp1

overexpression confers chemoresistance in prostate cancer

cell lines exposed to the DNA intercalating agent doxoru-

bicin [47]. In addition, a high-level of Mrp1 expression was

associated with poor clinical outcome in a prospective

study of primary neuroblastoma [48].

Standard treatment of recurrent glioblastoma multiforme

is with temozolomide [11]. Temozolomide is an alkylating

agent that has replaced other drugs for treatment of glio-

blastoma multiforme because its attributes are well toler-

ated, it has good oral bioavailability and it easily penetrates

the haematoencephalic barrier [11, 32]. Advantages over

other nitrosourea-derived drugs were demonstrated for

temozolomide in clinical trials in patients with relapsed

GBM [49–51] and improved survival was also demon-

strated in patients with newly diagnosed GBM [11].

However, the beneficial effects seen on progression-free

survival rates remain poor, less than 20% in GBM patients.

In vitro evaluation of the sensitivity of primary cultured

Fig. 4 Chemosensitization of GBM cells using inhibitors of Mrp1

activity. The effect of antitumour drugs vincristine (Vc) 100 nM and

etoposide (ET) 2 lM on cell viability was assayed alone or in

combination with the inhibitors of Mrp1 activity MK571 (MK) and

probenecid (Pb) following 24 h of treatments. The antitumours drugs

were also assayed in cells transfected with a plasmid expressing a

specific Mrp1 siRNA. The effects on cell viability were compared to

temozolomide (TMZ) 100 lM in T98G (A) or G44 (B) cells.

*P \ 0.05 versus control, **P \ 0.01 versus control, #P \ 0.01

versus TMZ. n = 15. C. Effect of temozolomide (TMZ) on cell

viability. The T98G cells were exposed for 24 h to increasing

concentrations of temozolomide from 100 to 400 lM and cell

viability was assayed. *P \ 0.05, n = 6

c

Neurochem Res (2011) 36:1397–1406 1403

123

glioblastoma cells indicated that viability was slightly

decreased (*15%) upon exposure to equivalent concen-

trations to therapeutic doses of temozolomide (100 lM).

Only, a tenfold increase in this dose (1,000 lM) rendered

toxicity that ranged from 24 to 81%, with responses being

different between samples of different patients [52]. Fur-

ther, temozolomide did not show a clear benefit compared

to a standard regime of procarbazine, lomustine and vin-

cristine (PCV) in cohorts of patients suffering recurrent

high-grade glioma [51]. Vincristine is a vinca alkaloid,

widely used in several types of cancer [53]. It binds to

tubulin dimers inhibiting assembly of microtubule struc-

tures and therefore affecting all rapidly dividing cell types

[54]. GBM cell lines GL15 and 8MG have shown a very

poor response to vincristine [55] or low accumulation of

this drug [22]. Similarly, T98G cells were insensitive to

etoposide, an inhibitor of topoisomerase II [38]. We dem-

onstrate here that in the T98G and G44 GBM cell lines, the

sensitivity to vincristine or etoposide is low but similar to

that seen with temozolomide. However, sensitivity to vin-

cristine and etoposide can be notably increased in vitro

using inhibitors of Mrp1 activity such as MK571, pro-

benecid or by knocking down with interfering RNA. This

effect was not seen when temozolomide was assayed in

conjunction with Mrp1 inhibitors, because temozolomide

appear not to be a substrate for this ABC transporter (see

supplementary data). Further, despite the fact that vincris-

tine is also a substrate for P-gp our results suggest that this

transporter shows to play only a minor role in high-grade

glioma cells. Therefore, we reinforce the role of Mrp1 in

conferring multiple-drug resistance to GBM cells because

an inhibition in Mrp1 activity was sufficient to notably

increase the sensitivity of these cells to chemotherapy. On

the other hand, we observed differences of effectiveness

between the inhibitors used, reaching a strongest decreas-

ing on cell viability when probenecid was assayed in

conjunction with antitumour drugs in spite to be MK571

the best inhibitor of the Mrp1 activity, as we shown in

Fig. 3. This could be explained because probenecid could

also inhibit other members of the MRP subfamily of ABC

transporters, particularly the activity of Mrp3 which

exhibits some overlapping substrate specificity with Mrp1

and is expressed in GBM cells [56, 57].

It is important to note that in high-grade brain cancer,

such as glioblastoma multiforme, the greatest resistance

comes from MDR transporters expressed in glioma because

the blood brain barrier (BBB) becomes more permeable

through break-up of the tight junctions between constituent

endothelial cells [58, 59]. However, because resection of

tumours in GBM patients is limited by neurological dam-

age, that therapeutic concentrations may be reached in

distal seemingly intact areas that are also known to contain

infiltrating tumour cells, remains an enormous challenge.

Penetration of the BBB in infiltrating zones or newly

vascularised recurrent GBM might also be increased using

Mrp1 inhibitors, because it is an ABC transporter that is

highly expressed in microvascular endothelial cells, as

shown from immunohistochemical analyses of brain

tissue [60].

Molecules developed for chemosensitization in patients

are mainly designed with P-gp inhibition in mind. How-

ever, CBT-1 [61] and biricodar [62] are drugs which are

characterised for their dual inhibition of P-gp and Mrp1

and they have been deemed safe in clinical trials. We

conclude that chemosensitization using Mrp1 inhibitors in

conjunction with antitumour drug substrates for the Mrp1

transporter may be a valuable tool for the treatment of

GBM.

Acknowledgments This work was supported by grants from

FONDECYT-Chile 11080226, DID UACH SB2007-68 and PIA

ANILLO ACT73 from CONICYT Chile.

References

1. Sharom FJ (2008) ABC multidrug transporters: structure, func-

tion and role in chemoresistance. Pharmacogenomics 9:105–127

2. Ozben T (2006) Mechanisms and strategies to overcome multiple

drug resistance in cancer. FEBS Lett 580:2903–2909

3. Thomas H, Coley HM (2003) Overcoming multidrug resistance

in cancer: an update on the clinical strategy of inhibiting p-gly-

coprotein. Cancer Control 10:159–165

4. Gottesman MM, Fojo T, Bates SE (2002) Multidrug resistance in

cancer: role of ATP-dependent transporters. Nat Rev Cancer

2:48–58

5. Awasthi S, Singhal S, Singhal J et al (2007) Role of RLIP76 in

lung cancer doxorubicin resistance: III. Anti-RLIP76 antibodies

trigger apoptosis in lung cancer cells and synergistically increase

doxorubicin cytotoxicity. Int J Oncol 22:721–732

6. Steiner E, Holzmann K, Elbling L et al (2006) Cellular functions

of vaults and their involvement in multidrug resistance. Curr

Drug Targets 7:923

7. Paredes A, Blanco JL, Echenique-Elizondo M (2006) Expression

of multidrug resistance (MDR) associated proteins in solid

tumors. Cir Esp 79:202–214

8. Choi CH (2005) ABC transporters as multidrug resistance

mechanisms and the development of chemosensitizers for their

reversal. Cancer Cell Int 5:30

9. Ahmedin J, Siegel R, Xu J et al (2010) Cancer statistics, 2010.

CA Cancer J Clin 65:277–300

10. Chang SM, Butowski NA, Sneed PK et al (2006) Standard

treatment and experimental targeted drug therapy for recurrent

glioblastoma multiforme. Neurosurg Focus 20:E4

11. Stupp R, van den Bent MJ, Hegi ME (2005) Optimal role of

temozolomide in the treatment of malignant gliomas. Curr Neurol

Neurosci Rep 5:198–206

12. Loscher W, Potschka H (2005) Drug resistance in brain diseases

and the role of drug efflux transporters. Nat Rev Neurosci

6:591–602

13. Yuan H, Li X, Wu J et al (2008) Strategies to overcome or

circumvent P-glycoprotein mediated multidrug resistance. Curr

Med Chem 15:470–476

1404 Neurochem Res (2011) 36:1397–1406

123

14. Hennessy M, Spiers JP (2007) A primer on the mechanics of

P-glycoprotein the multidrug transporter. Pharmacol Res 55:1–15

15. Mizutani T, Hattori A (2005) New horizon of MDR1 (P-glyco-

protein) study. Drug Metab Rev 37:489–510

16. Mealey KL (2004) Therapeutic implications of the MDR-1 gene.

J Vet Pharmacol Ther 27:257–264

17. Liscovitch M, Lavie Y (2002) Cancer multidrug resistance: a

review of recent drug discovery research. IDrugs 5:349–355

18. Benyahia B, Huguet S, Decleves X et al (2004) Multidrug

resistance-associated protein MRP1 expression in human glio-

mas: chemosensitization to vincristine and etoposide by indo-

methacin in human glioma cell lines overexpressing MRP1.

J Neuro-oncol 66:65–70

19. Calatozzolo C, Gelati M, Ciusani E et al (2005) Expression of

drug resistance proteins Pgp, MRP1, MRP3, MRP5 and GST-pi

in human glioma. J Neuro-oncol 74:113–121

20. Decleves X, Amiel A, Delattre JY et al (2006) Role of ABC

transporters in the chemoresistance of human gliomas. Curr

Cancer Drug Targets 6:433–445

21. Matsumoto Y, Miyake K, Kunishio K et al (2004) Reduction of

expression of the multidrug resistance protein (MRP)1 in glioma

cells by antisense phosphorothioate oligonucleotides. J Med

Invest 51:194–201

22. Decleves X, Fajac A, Lehmann-Che J et al (2002) Molecular and

functional MDR1-Pgp and MRPs expression in human glioblas-

toma multiforme cell lines. Int J Cancer 98:173–180

23. Zhang W, Mojsilovic-Petrovic J, Andrade MF et al (2003) The

expression and functional characterization of ABCG2 in brain

endothelial cells and vessels. FASEB J 17:2085–2087

24. Quezada CA, Garrido WX, Gonzalez-Oyarzun MA et al (2008)

Effect of tacrolimus on activity and expression of P-glycoprotein

and ATP-binding cassette transporter A5 (ABCA5) proteins in

hematoencephalic barrier cells. Biol Pharm Bull 31:1911–1916

25. Echevarria-Lima J, Kyle-Cezar F, Leite DFP et al (2005)

Expression and activity of multidrug resistance protein 1 in a

murine thymoma cell line. Immunology 114:468–475

26. Hammond CL, Marchan R, Krance SM et al (2007) Glutathione

export during apoptosis requires functional multidrug resistance-

associated proteins. J Biol Chem 282:14337–14347

27. Dogan AL, Legrand O, Faussat AM et al (2004) Evaluation and

comparison of MRP1 activity with three fluorescent dyes and

three modulators in leukemic cell lines. Leuk Res 28:619–622

28. Gekeler V, Ise W, Sanders KH et al (1995) The leukotriene LTD4

receptor antagonist MK571 specifically modulates MRP associated

multidrug resistance. Biochem Biophys Res Commun 208:345–352

29. Abe T, Mori T, Wakabayashi Y, Nakagawa M et al (1998)

Expression of multidrug resistance protein gene in patients with

glioma after chemotherapy. J Neuro-oncol 40:11–18

30. Bredel M, Zentner J (2002) Brain-tumour drug resistance: the

bare essentials. Lancet Oncol 3:397–406

31. Patel M, McCully C, Godwin K et al (2003) Plasma and cere-

brospinal fluid pharmacokinetics of intravenous temozolomide in

non-human primates. J Neuro-oncol 61:203–207

32. Ostermann S, Csajka C, Buclin T et al (2004) Plasma and cere-

brospinal fluid population pharmacokinetics of temozolomide in

malignant glioma patients. Clin Cancer Res 10:3728–3736

33. Chang JE, Robins HI, Mehta MP (2007) Therapeutic advances in

the treatment of brain metastases. Clin Adv Hematol Oncol 5:

54–64

34. Hegi ME, Liu L, Herman JG et al (2008) Correlation of O6-

methylguanine methyltransferase (MGMT) promoter methylation

with clinical outcomes in glioblastoma and clinical strategies to

modulate MGMT activity. J Clin Oncol 26:4189–4199

35. Liu G, Akasaki Y, Khong HT et al (2005) Cytotoxic T cell tar-

geting of TRP-2 sensitizes human malignant glioma to chemo-

therapy. Oncogene 24:5226–5234

36. Valera ET, Machado HR, Scrideli CA et al (2007) Drug-resis-

tance in central nervous system tumors: from the traditional cell-

resistance model to the genetically driven approaches on therapy.

Curr Pharm Biotechnol 8:105–113

37. Spiegl-Kreinecker S, Buchroithner J, Elbling L et al (2002)

Expression and functional activity of the ABC-transporter pro-

teins P-glycoprotein and multidrug-resistance protein 1 in human

brain tumor cells and astrocytes. J Neuro-oncol 57:27–36

38. Chen L, Feng P, Li S et al (2009) Effect of hypoxia-inducible

factor-1alpha silencing on the sensitivity of human brain glioma

cells to doxorubicin and etoposide. Neurochem Res 34:984–990

39. Ota E, Abe Y, Oshika Y, Ozeki Y et al (1995) Expression of the

multidrug resistance-associated protein (MRP) gene in non-

small-cell lung cancer. Br J Cancer 72:550–554

40. Berger W, Setinek U, Hollaus P et al (2005) Multidrug resistance

markers P-glycoprotein, multidrug resistance protein 1, and lung

resistance protein in non-small cell lung cancer: prognostic

implications. J Cancer Res Clin Oncol 131:355–363

41. Hsia TC, Lin CC, Wang JJ et al (2002) Relationship between

chemotherapy response of small cell lung cancer and P-glyco-

protein or multidrug resistance-related protein expression. Lung

180:173–179

42. Kuo TH, Liu FY, Chuang CY et al (2003) To predict response

chemotherapy using technetium-99 m tetrofosmin chest images in

patients with untreated small cell lung cancer and compare with

p-glycoprotein, multidrug resistance related protein-1, and lung

resistance-related protein expression. Nucl Med Biol 30:627–632

43. Kartenbeck J, Leuschner U, Mayer R et al (1996) Absence of the

canalicular isoform of the MRP gene-encoded conjugate export

pump from the hepatocytes in Dubin-Johnson syndrome. Hepa-

tology 23:1061–1066

44. Nooter K, de la Riviere GB, Klijn J et al (1997) Multidrug resis-

tance protein in recurrent breast cancer. Lancet 349:1885–1886

45. Rudas M, Filipits M, Taucher S et al (2003) Expression of MRP1,

LRP and Pgp in breast carcinoma patients treated with preoper-

ative chemotherapy. Breast Cancer Res Treat 81:149–157

46. Sullivan GF, Amenta PS, Villanueva JD et al (1998) The

expression of drug resistance gene products during the progres-

sion of human prostate cancer. Clin Cancer Res 4:1393–1403

47. Zalcberg J, Hu XF, Slater A et al (2000) MRP1 not MDR1 gene

expression is the predominant mechanism of acquired multidrug

resistance in two prostate carcinoma cell lines. Prostate Cancer

Prostatic Dis 3:66–75

48. Haber M, Smith J, Bordow SB et al (2006) Association of high-level

MRP1 expression with poor clinical outcome in a large prospective

study of primary neuroblastoma. J Clin Oncol 24:1546–1553

49. Yung WK, Albright RE, Olson J et al (2000) A phase II study of

temozolomide vs. procarbazine in patients with glioblastoma

multiforme at first relapse. Br J Cancer 83:588–593

50. Brada M, Hoang-Xuan K, Rampling R et al (2001) Multicenter

phase II trial of temozolomide in patients with glioblastoma

multiforme at first relapse. Ann Oncol 12:259–266

51. Brada M, Stenning S, Gabe R et al (2010) Temozolomide versus

procarbazine, lomustine, and vincristine in recurrent high-grade

glioma. J Clin Oncol 28:4601–4608

52. Pedeboscq S, L’Azou B, Liguoro D et al (2007) Interindividual

differences in anticancer drug cytotoxicity in primary human

glioblastoma cells. Exp Toxicol Pathol 58:247–253

53. Akan I, Akan S, Akca H et al (2005) Multidrug resistance-

associated protein 1 (MRP1) mediated vincristine resistance:

effects of N-acetylcysteine and Buthionine sulfoximine. Cancer

Cell Int 5:22

54. Loe DW, Deeley RG, Cole SP (1998) Characterization of vin-

cristine transport by the M(r) 190, 000 multidrug resistance

protein (MRP): evidence for cotransport with reduced glutathi-

one. Cancer Res 58:5130–5136

Neurochem Res (2011) 36:1397–1406 1405

123

55. Styczynski J, Olszewska-Slonina D, Kolodziej B et al (2006) Activity

of bortezomib in glioblastoma. Anticancer Res 26:4499–4503

56. Kuan CT, Wakiya K, Herndon JE II et al (2010) MRP3: a

molecular target for human glioblastoma multiforme immuno-

therapy. BMC Cancer 10:468–482

57. Bodo A, Bakos E, Szeri F et al (2003) Differential modulation of

the human liver conjugate transporters MRP2 and MRP3 by bile

acids and organic anions. J Biol Chem 278:23529–23537

58. Lee SW, Kim WJ, Park JA et al (2006) Blood-brain barrier

interfaces and brain tumors. Arch Pharm Res 29:265–275

59. Hawkins BT, Davis TP (2005) The blood-brain barrier/neuro-

vascular unit in health and disease. Pharmacol Rev 57:173–185

60. Wang F, Zhou F, Kruh GD et al (2010) Influence of blood-brain

barrier efflux pumps on the distribution of vincristine in brain and

brain tumors. Neuro Oncol 12:1043–1049

61. Robey RW, Shukla S, Finley EM et al (2008) Inhibition of

P-glycoprotein (ABCB1)- and multidrug resistance-associated

protein 1 (ABCC1)-mediated transport by the orally administered

inhibitor, CBT-1(R). Biochem Pharmacol 75:1302–1312

62. Peck RA, Hewett J, Harding MW et al (2001) Phase I and

pharmacokinetic study of the novel MDR1 and MRP1 inhibitor

biricodar administered alone and in combination with doxorubi-

cin. J Clin Oncol 19:3130–3141

1406 Neurochem Res (2011) 36:1397–1406

123