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World J Urol

DOI 10.1007/s00345-014-1469-0

ORIGINAL ARTICLE

Differential expression of the multidrug resistance 1 (MDR1) protein in prostate cancer cells is independent from anticancer drug treatment and Y box binding protein 1 (YB-1) activity

Madeleine Saupe · Lisa Rauschenberger · Melanie Preuß · Stefan Oswald ·

Sebastian Fussek · Uwe Zimmermann · Reinhard Walther · Cornelius Knabbe ·

Martin Burchardt · Matthias B. Stope

Received: 27 November 2014 / Accepted: 18 December 2014

© Springer-Verlag Berlin Heidelberg 2014

presence of the first-line chemotherapeutic agent docetaxel.

Incubation of 22Rv1 cells with docetaxel, cabazitaxel, and

abiraterone did not significantly alter MDR1 expression

levels. Furthermore, overexpression of the MDR1 control-

ling factor YB-1 showed no impact on MDR1 expression

levels.

Conclusions MDR1 was detectable in the PC cell line

22Rv1. However, this study suggests that MDR1 is of less

importance for drug resistance in PC cells than in other

types of solid cancer. Furthermore, in contrast to YB-1

properties in other malignancies, MDR1 regulation through

YB-1 seems to be unlikely.

Keywords Prostate cancer · Chemoresistance · Multidrug

resistance protein 1 · Y box binding protein 1

Abbreviations

PC Prostate cancer

MDR1 Multidrug resistance 1

YB-1 Y box binding protein 1

CYP17A1 Cytochrome P450 17A1

GAPDH Glyceraldehyde 3-phosphate dehydrogenase

LC–MS/MS Liquid chromatography–tandem mass

spectrometry

ABC ATP-binding cassette transporter

MRP1 Multidrug resistance-associated protein 1

Introduction

The development of a multidrug-resistant phenotype of

tumor cells represents one of the major obstacles regarding

the treatment of castration-resistant prostate cancer (PC).

Patients with advanced PC are treated with cytotoxic agents

all characterized by limited response duration. Allowing a

Abstract

Purpose The development of a drug-resistant phenotype

is the major challenge during treatment of castration-resist-

ant prostate cancer (PC). In solid cancer entities, one of

the major contributors to chemoresistance is the multidrug

resistance 1 (MDR1) protein. Believed to be involved in the

induction of MDR1 expression is the presence of antican-

cer drugs as well as the Y box binding protein 1 (YB-1).

Methods Basal as well as drug-induced expression of

MDR1 in established PC cell lines was assessed by West-

ern blotting and mass spectrometry. Subsequently, the

influence of YB-1 on MDR1 expression was examined via

transient overexpression of YB-1.

Results While LNCaP and PC-3 cells showed no detect-

able amounts of MDR1, the resistance factor was found

to be expressed in 22Rv1 cells. Despite this difference, all

three cell lines demonstrated similar growth behavior in the

M. Saupe · L. Rauschenberger · M. Preuß · S. Fussek ·

U. Zimmermann · M. Burchardt · M. B. Stope (*)

Department of Urology, University Medicine Greifswald,

Ferdinand-Sauerbruch-Straße, 17475 Greifswald, Germany

e-mail: matthias.stope@uni-greifswald.de

S. Oswald

Department of Pharmacology, Center of Drug Absorption

and Transport (C_DAT), University Medicine Greifswald,

Felix-Hausdorf-Straße 3, 17487 Greifswald, Germany

R. Walther

Department of Medical Biochemistry and Molecular Biology,

University Medicine Greifswald, Ferdinand-Sauerbruch-Straße,

17475 Greifswald, Germany

C. Knabbe

Institute for Laboratory and Transfusion Medicine, Heart

and Diabetes Center North Rhine-Westphalia, Ruhr University

Bochum, Georgstraße 11, 32545 Bad Oeynhausen, Germany

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median survival of 19.2 months, the first-line chemotherapy

of advanced PC with docetaxel inevitably ends in relapse

and tumor progression [1]. Believed to be implicated in

chemoresistance is the multidrug resistance 1 (MDR1) pro-

tein. MDR1 represents an efflux pump which is capable of

lowering the intracellular concentration of a vast range of

anticancer compounds [2]. MDR1 gene expression is pri-

marily controlled by the transcription and translation factor

Y box binding protein 1 (YB-1), which is known to be a

predictor of tumor progression and poor prognosis [3, 4].

Experimental data concerning MDR1 regulation and

expression in PC cells are, however, far from conclusion.

While some studies describe a higher MDR1 expression

in healthy prostate tissue and benign prostate hyperpla-

sia than in PC tissue [5], opposite findings have been

reported as well [6]. Furthermore, control of MDR1

expression in distinct phases of PC progression remains

unanswered.

With the hope of being able to overcome tumor resist-

ance one day, the objective of this study was to advance

the knowledge of the role and regulation of MDR1 in PC.

For this purpose, we primarily focused on examinations of

the established PC cell lines 22Rv1, LNCaP, and PC-3. For

anticancer drug incubation approaches, the approved PC

therapeutics docetaxel, cabazitaxel, and abiraterone were

applied. Docetaxel and cabazitaxel are both members of

the taxane group; however, docetaxel has been described

as a substrate of MDR1, whereas the second-generation

taxane cabazitaxel demonstrated a low affinity toward the

MDR1 transporter [7, 8]. Abiraterone efficacy is in con-

trast based on the inhibition of the steroid synthesis enzyme

cytochrome P450 17A1 (CYP17A1), reflecting a com-

pletely different molecular mode of action [9]. Finally, a

putative regulation of MDR1 expression by the multifunc-

tional factor YB-1 was assessed by modulating the YB-1

expression.

Materials and methods

Antibodies, chemicals, and plasmids

Primary antibodies directed against MDR1 (Santa Cruz

Biotechnology, Heidelberg, Germany), YB-1 (Abcam, Cam-

bridge, UK), glyceraldehyde 3-phosphate dehydrogenase

(GAPDH; Abfrontier, Seoul, Korea) and mouse-specific

and rabbit-specific secondary antibodies (Cell Signaling

Technology, Danvers, MA, USA) were used. Cabazitaxel

(3.0 × 10−10 M) was kindly provided by Sanofi-Aventis

(Frankfurt/M., Germany), abiraterone (3.0 × 10−5 M) was

kindly provided by Janssen-Cilag (Neuss, Germany), and

docetaxel (1.0 × 10−8 M) was obtained from Sigma-Aldrich

(Deisenhofen, Germany). pcDNA3.1(+)-YB-1 was applied

for YB-1 overexpression [10] using pcDNA3.1 (Invitrogen,

Karlsruhe, Germany) as empty control vector.

Cell culture

All cell lines were acquired from Cell Lines Service (CLS,

Heidelberg, Germany) and cultured in 1640 RPMI medium

supplemented with 5 % pyruvate, 10 % fetal bovine serum,

and 5 % penicillin/streptomycin (all PAN Biotech, Aiden-

bach, Germany) at 37 °C and 5 % CO2.

Western blotting

Protein preparation was done using TRIzol reagent (Life

Technologies, Darmstadt, Germany) according to the

manufacturer’s protocol. A total amount of 200 µg of

protein was separated by sodium dodecyl sulfate poly-

acrylamide gel electrophoresis and transferred to a

nitrocellulose membrane (Whatman, Dassel, Germany).

Immobilized protein was specifically detected by antibod-

ies and visualized by LumiGLO reagent (Cell Signaling

Technology) in a ChemiDoc XRS System (Bio-Rad). Pro-

tein signals were compared to GAPDH signals as internal

reference.

Protein quantification by liquid chromatography–tandem

mass spectrometry (LC–MS/MS)

Sample preparation and MDR1 quantification was done

by MS-based targeted proteomics using a validated LC–

MS/MS method as recently described [11]. Final protein

bCell line

MDR1 [pmol]

Total protein [mg]

MDR1

GAPDH

22Rv1 LNCaP PC-3Cell line

35

40

100

130

170

a kDa

22Rv1

0.230

LNCaP

0.003

PC-3

0.041

HEK-293

0.268

Fig. 1 Basal expression of MDR1 in PC cell lines 22Rv1, LNCaP,

and PC-3. a Detection of MDR1 via Western blotting using an

MDR1-specific antibody. GAPDH was used as loading control. b

Detection of MDR1 by LC–MS/MS analysis. A cell lysate of the cell

line HEK-293 was used as a positive control. The measurements in

the LNCaP and PC-3 cells were found to be under the lower limit of

quantification

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expression data were calculated by normalization to total

protein content.

Cell viability assay

1.0 × 104 cells in a 96-well plate were incubated after

indicated time points with 15 µl of 3-(4,5-dimethylthia-

zol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; Sigma-

Aldrich) solution for 2 h at 37 °C. After removal of the

supernatant, cells were lysed with 120 µl of a lysis reagent

and analyzed in a microplate reader (Infinite M200 Pro,

Tecan, Switzerland) at 550 nm.

Transfection experiments

One day prior to transfection, 1.5 × 105 cells were plated

into 6-well cell culture plates. Transfection was achieved

by 1.5 µl of lipofectamine (Life Technologies) and 1 µg of

plasmid DNA.

Statistical analysis

Statistical analysis was performed by using GraphPad

Prism 5.0 software. Data shown in the figures are either

representative experiments or expressed as mean ± SD of

at least three independent experiments.

Results

MDR1 basal expression is detectable only in the PC cell

line 22Rv1

MDR1 expression in the established PC cell lines 22Rv1,

LNCaP, and PC-3 was determined via Western blotting.

Detectable amounts of MDR1 could be found only in

22Rv1 cells, whereas Western blotting analysis of LNCaP

and PC-3 cells failed to show MDR1 signals (Fig. 1a).

These data were evaluated by MS analysis again validating

Fig. 2 Assessment of sensitiv-

ity toward docetaxel in 22Rv1

cells, LNCaP cells, and PC-3

cells. Cells were treated with

three different concentrations

of docetaxel as indicated and

cell viability was assessed after

24, 48, 72, and 96 h applying a

MTT viability assay in a 22Rv1

cells, b LNCaP cells, and c

PC-3 cells. Despite the presence

of MDR1 in 22Rv1 cells, no

advantage of survival could be

detected

240

20

40

60

80

100

Incubation (h)

Docetaxel (M) 10-11

9689 86

57

48 72 96

10-9

83

67

51

30

10-7

90

45

28

12

48 72 96 48 72 9624 24

Via

bilit

y (

% C

on

tro

l)a

240

20

40

60

80

100

Incubation (h)

Docetaxel (M)

48 72 96

10-11

96

80

62

39

48 72 96

10-9

97

64

48

29

48 72 96

10-7

85

5647

19

24 24

Via

bilit

y (

% C

on

tro

l)c

240

20

40

60

80

100

Incubation (h)

Docetaxel (M) 10-11

98

86 8390

48 72 96

10-9

97

77

64

45

10-7

94

6758

26

48 72 96 48 72 9624 24

Via

bilit

y (

% C

on

tro

l)b

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MDR1 protein expression exclusively in 22Rv1 cells

(0.230 pmol MDR1/mg total protein) and HEK-293 con-

trol cell lysate (0.268 pmol MDR1/mg total protein) with

MDR1 concentrations under the lower limit of quantifica-

tion in LNCaP (0.003 pmol MDR1/mg total protein) and

PC-3 (0.041 pmol MDR1/mg total protein) cells (Fig. 1b).

MDR1 expression in 22Rv1 cells confers no survival

advantage in the presence of docetaxel compared to MDR1

negative LNCaP and PC-3 cells

Using the MTT viability assay, cell survival of 22Rv1,

LNCaP, and PC-3 cells were examined in the presence

of 1.0 × 10−11, 1.0 × 10−9, and 1.0 × 10−7 M docetaxel,

respectively, over a period of 96 h. Notably, cellular via-

bility of 22Rv1 cells (Fig. 2a), LNCaP cells (Fig. 2b), and

PC-3 cells (Fig. 2c) revealed a similar pattern of growth

inhibition. For instance, the incubation with 1.0 × 10−7 M

docetaxel for 96 h led to a reduction of living 22Rv1 cells

of 12.3 ± 1.7 % compared with untreated control cells,

while 26.4 ± 3.0 % LNCaP cells and 19.4 ± 2.2 % PC-3

cells survived docetaxel incubation. Despite the presence

of MDR1, 22Rv1 cells showed an equal and by trend a

slightly increased sensitivity toward docetaxel compared

with the other PC cell lines.

MDR1 expression in 22Rv1 is not induced in the presence

of PC chemotherapeutics

The development of an MDR1 phenotype is believed to be

the consequence of tumor cell stimulation with anticancer

drugs. Hence, 22Rv1 cells were incubated with docetaxel

(Fig. 3a), cabazitaxel (Fig. 3b), and abiraterone (Fig. 3c)

for 24 and 72 h, after which intracellular MDR1 protein

was determined. The treatment of 22Rv1 cells with chem-

otherapeutics did not lead to a significant modulation of

MDR1 protein compared to vehicle treated cells for any of

the three tested compounds.

The transcription and translation factor YB-1 does not

control MDR1 expression in 22Rv1 cells

Aside from having been reported to be up-regulated dur-

ing PC progression, YB-1 is discussed as a drug-inducible

putative regulator of the MDR1 gene expression. Perform-

ing drug incubation approaches as aforementioned, YB-1

protein expression appeared to not be linked to the pres-

ence of docetaxel, cabazitaxel, and abiraterone, respec-

tively, for 24 h (Fig. 4a) and 72 h (Fig. 4b). Furthermore,

overexpression of YB-1 as shown in Fig. 4c did not lead

to a modulation of MDR1 after 24 and 72 h (Fig. 4d), sug-

gesting that YB-1 is not responsible for the regulation of

MDR1 expression.

Discussion

While it is widely acknowledged that MDR1 is capable of

actively extruding toxic compounds out of cancer cells, lit-

tle is known about the role of MDR1 in PC chemotherapy.

Even though Henrique et al. [5] have reported low MDR1

expression levels in untreated PC-3 and LNCaP cells,

numerous studies demonstrated a lack of basal MDR1

expression on mRNA and protein level [6, 12–15], con-

firming the data of our recent study. A possible explana-

tion for the inconsistencies in demonstrating the presence

of MDR1 in PC cell lines might be the fact that MDR1

100

130170

Vehicle

-

+

-

-

+ +Abiraterone

Incubation (h) 24 72

GAPDH

GAPDH

MDR1

MDR1

GAPDH

MDR1

Vehicle

-

+

-

-

+

+ -

+Cabazitaxel

Incubation (h) 24 72

Vehicle

-

+

-

-

+

+ -

+ -

+Docetaxel

Incubation (h) 24 72

35

40

100

130170

35

40

100

130170

35

40

a

c

b

kDa

kDa

kDa

Fig. 3 MDR1 expression modulation in the presence of drugs. West-

ern blot analysis of MDR1 expression in 22Rv1 cells after incubation

with a docetaxel, b cabazitaxel, and c abiraterone for 24 and 72 h,

respectively. DMSO served used as a vehicle control and GAPDH as

a loading control

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is a stress-responsive gene. Aside from cytostatic agents,

MDR1 expression can be induced by various other stimuli

such as environmental changes, infections, and ultraviolet

radiation [2]. Additionally, Corcoran and co-workers have

specified an exosomal MDR1 protein transfer from doxo-

rubicin-resistant PC cells to drug-sensitive maternal cells,

remarkably, pointing to MDR1 regulation accomplished

independently of cellular gene expression machinery [16].

However, the PC cell line 22Rv1, which is derived from a

xenograft model that relapsed during antihormonal treat-

ment [17], weakly exhibited basal MDR1 protein expres-

sion as demonstrated for the first time by Western blotting

as well as MS analysis. Notably, even though 22Rv1 cells

clearly express detectable amounts of MDR1, incuba-

tion experiments with docetaxel as a model compound for

chemotherapy manifested no differences in growth inhibi-

tion compared to MDR1-negative LNCaP and PC-3 cells.

This is especially interesting, considering the fact that

docetaxel is a well-known substrate of MDR1 [7]. On the

other hand, there are no data available yet on the MDR1

gene sequence in 22Rv1 tumor cells. Consequently, we

cannot exclude that a dominant negative mutation may cir-

cumvent an effective detoxification in the presence of anti-

cancer compounds. A growing body of the literature has

shown that members of the ATP-binding cassette (ABC)

transporter superfamily are regulated at the expression level

and are frequently induced by anticancer drugs, and that

the presence of these transporters is linked to poor progno-

sis and chemoresistance in a wide variety of malignancies

[2]. MDR1 expression rates in drug-resistance PC cell lines

appear to be regulated by promoter methylation [18].

Since former studies have reported a correlation between

YB-1 and drug resistance in PC [19–21] and overexpres-

sion of YB-1 has been described to increase the efflux of

the MDR1 substrate vinblastine [3], we hypothesized YB-1

to be an inductor of MDR1 expression in 22Rv1 cells.

However, experiments applying the PC drugs docetaxel,

cabazitaxel, and abiraterone to 22Rv1 cells demonstrated

no significant alterations of MDR1 as well as YB-1 in none

of the three incubation approaches. Subsequent transfec-

tion experiments with the goal of overexpressing YB-1 in

22Rv1 cells served as proof-of-principle; however, elevated

levels of YB-1 did not modulate intracellular MDR1 pro-

tein concentrations. Since experimental evidence for direct

binding of YB-1 to the promoter of the MDR1 gene is still

missing, some authors discussed a parallel induction/activa-

tion of YB-1 and MDR1 as part of a general stress response

[22, 23]. Moreover, Kaszubiak et al. [24] demonstrated that

down-regulation of YB-1 in drug-resistant gastrointestinal

cells did not lead to a shift of the MDR1 expression pattern.

While YB-1 has been shown to play an important part

in the progression from androgen-dependent PC into

Fig. 4 YB-1 expression modu-

lation in the presence of drugs

and impact of YB-1 overexpres-

sion on the expression levels

of MDR1. Detection of MDR1

and YB-1 in 22Rv1 cells after

incubation with docetaxel,

cabazitaxel and abiraterone for

a 24 h and b 72 h. DMSO was

used as a vehicle control and

GAPDH as a loading control. c

YB-1 was transiently overex-

pressed in 22Rv1 cells with the

empty vector pcDNA3.1(−) as

control. d MDR1 expression

levels in 22Rv1 cells overex-

pressing YB-1, compared to

control transfected cells. The

experiment was conducted after

an incubation time of 24 and

72 h. GAPDH served as a load-

ing control

35

40

40

5570

-+ -Vehicle -

Docetaxel

-

-

-

+ -

+

-

Cabazitaxel -

Abiraterone +

40

5570

40

35

YB-1

GAPDH

-+ - -

-

-

-

+ -

+

-

-

- - - - - - +

Incubation (h) 24 72

100130

170

YB-1

GAPDH

Incubation (h) 24 72

40

5570

40

35

-+ +pcDNA3.1(-) -

pcDNA3.1(+)-YB-1 - + - +

-+ + -

- + - +

MDR1

GAPDH

24 72

40

35

aDkaDk

d

ba

c

aDkaDk

YB-1

GAPDH

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castration-resistant PC, our data suggest that this is not

caused through up-regulation of MDR1. In fact, this study

supports the increasing evidence that MDR1 expression is

independent from YB-1. While we conclude that MDR1

regulation is one of the lesser important genetic alterations

taking place during PC progression, the role of YB-1 in this

type of cancer is subject of further studies. Moreover, our

recent results support the hypothesis that MDR1 is unlikely

to be a main actor to cause PC cells to acquire a drug-

induced chemoresistant phenotype and that other factors,

for instance the multidrug resistance-associated protein 1

(MRP1) [14], seem more likely to be responsible for drug

resistance in PC.

Acknowledgments The authors thank Anne Brandenburg and Katja

Wittig for excellent technical assistance.

Conflict of interest By way of disclosure of conflict of interest, the

compound abiraterone acetate was kindly provided by Janssen-Cilag

GmbH, Neuss, Germany, and the compound cabazitaxel was kindly

provided by Sanofi-Aventis, Frankfurt/M., Germany.

Ethical standard The manuscript does not contain clinical studies

or patient data.

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