Biochemical Pharmacology 84 (2012) 1070–1077

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Biochemical Pharmacology
journal homepage: www.elsevier.com/locate/biochempharmEnhanced expression of organic anion transporting polypeptides (OATPs)in androgen receptor-positive prostate cancer cells: Possible role of OATP1A2
in adaptive cell growth under androgen-depleted conditions
Hiroshi Arakawa a, Takeo Nakanishi a, Chihiro Yanagihara a, Tomohiro Nishimoto a,
Tomohiko Wakayama b, Atsushi Mizokami c, Mikio Namiki c, Keiichi Kawai d, Ikumi Tamai a,*a

Department of Membrane Transport and Biopharmaceutics, Faculty of Pharmaceutical Science, Kanazawa University, Kakuma-machi, Kanazawa, 920-1192, Japan
Department of Histology and Embryonology, Faculty of Medicine, Graduate School of Medical Science, Kanazawa University, 13-1 Takara-machi, Kanazawa 920-8640, Japanc
Integrative Cancer Therapy and Urology, Faculty of Medicine, Graduate School of Medical Science, Kanazawa University, 13-1 Takara-machi, Kanazawa 920-8640, Japand
Department of Quantum Medical Technology, Faculty of Health Sciences, Graduate School of Medical Sciences, Kanazawa University, 5-11-80 Kodatsuno, Kanazawa 920-0942, Japanb

A R T I C L E I N F O

A B S T R A C T

Article history:
Received 2 June 2012
Accepted 25 July 2012
Available online 1 August 2012

The biological mechanisms underlying castration resistance of prostate cancer are not fully understood.
In the present study, we examined the role of organic anion transporting polypeptides (OATPs) as
importers of dehydroepiandrosterone sulfate (DHEAS) into cells to support growth under androgendepleted conditions. Cell growth and mRNA expression of OATP genes were studied in human prostatecancer LNCaP and 22Rv1 cells under androgen-depleted conditions. The stimulatory effect of DHEAS oncell growth was investigated in LNCaP cells in which OATP1A2 had been silenced. Growth of both celllines was stimulated by DHEAS and the effect was attenuated by STX64, an inhibitor of steroid sulfatasewhich can covert DHEAS to DHEA. OATP1A2 mRNA expression was increased most prominently among
various genes tested in LNCaP cells grown in androgen-depleted medium. Similar results were obtainedwith 22Rv1 cells. Furthermore, the characteristics of [3H]DHEAS uptake by LNCaP cells were consistent
with those of OATP-mediated transport. Knockdown of OATP1A2 in LNCaP cells resulted in loss of the
DHEAS sensitivity of cell growth. Our results suggest that enhanced OATP1A2 expression is associatedwith adaptive cell growth of prostate cancer cells under androgen-depleted conditions. Thus, OATP1A2
may be a pharmacological target for prostate cancer treatment.
ß 2012 Elsevier Inc. All rights reserved.

Keywords:
Prostate cancer
OATP1A2
DHEAS
Androgen receptor
Steroid sulfatase

1. Introduction
Gonadal androgens play a critical role in protein synthesis andcell survival in prostate tumors [1]. Therefore, androgen deprivation therapy (ADT) to remove gonadal testosterone or toantagonize androgen receptors is currently a mainstream treatment for prostate cancer. Once the disease progresses toAbbreviations: AR, androgen receptor; STX64, 6-oxo-6,7,8,9,10,11-hexahydrocyclohepta[c]chromen-3-yl sulfamate; CRPC, castration-resistant prostate cancer;
STS, steroid sulfatase; OATP, organic anion transporting polypeptide; DHEA,
dehydroepiandrosterone; DHEAS, dehydroepiandrosterone sulfate; DHT,
dihydrotestosterone; ADT, androgen deprivation therapy; SRB, sulforhodamine B;
PSA, prostate speciﬁc antigen; HPRT1, hypoxanthine guaninephosphoribosyltransferase; HSD, hydroxysteroid dehydrogenase; AP-1, activator protein-1; NTCP,
Na+-dependent taurocholate cotransporting polypeptide; E3S, estrone 3-sulfate;
BSP, bromosulfophthalein; TCA, taurocholate; PRO, probenecid; SAL, salicylate;
PAH, p-aminohippurate; TEA, tetra-ethylammonium; MPP+, 1-methyl-4-phenylpyridinium.
* Corresponding author. Tel.: +81 76 234 4479; fax: +81 76 264 6284.
E-mail address: tamai@p.kanazawa-u.ac.jp (I. Tamai).
0006-2952/$ – see front matter ß 2012 Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.bcp.2012.07.026

castration-resistant prostate cancer (CRPC), it no longer responds
to ADT. CRPC tumor progression is considered to involve enhanced
androgen receptor (AR) function, mainly due to AR gene
ampliﬁcation/overexpression [2], stabilization of AR protein andincreased sensitivity of AR to androgens [3], and constitutive,
ligand-independent activation [4].
The organic anion transporting polypeptide (OATP/SLCO) family
members generally mediate Na+-independent transport of amphipathic organic anion compounds, including bile salts, steroid
conjugates, thyroid hormones, and oligopeptides [5,6]. The
physiological roles of these transporters are not yet fully
understood, but there is compelling evidence that certain
members of the OATP family promote cell proliferation andsurvival of human malignant tissues. We have previously shownthat transport of estrone 3-sulfate via OATPs sustains the growth ofhormone-dependent human breast cancer cells [7,8], because
estrone 3-sulfate can be hydrolyzed by steroid sulfatase (STS) toestrone and eventually estradiol. We recently showed thatOATP1B3 contributes to estrone 3-sulfate uptake in estrogen
receptor-positive human breast cancer MCF-7 cells [9]. In addition

H. Arakawa et al. / Biochemical Pharmacology 84 (2012) 1070–1077

to estrone 3-sulfate, these OATPs translocate dehydroepiandrosterone sulfate (DHEAS) [10,11]. DHEAS is a thousand fold moreabundant than testosterone in human serum [12], and isessentially unaffected by ADT. DHEAS is hydrolyzed to DHEA by
STS [13], and DHEA can be converted to androstenedione inprostate cancer [14], resulting in activation of AR function [15].
More recently, clinical observations have suggested that several
OATPs are upregulated in castration-resistant metastatic prostate
tumor tissues derived from human patients [16]; however, itremains unclear whether OATPs play a role in prostate cancer cell
survival under androgen-depleted conditions. We hypothesizedthat increased expression of OATPs does contribute to cell survivalof androgen-dependent prostate cancer cells under androgendeprivation, by providing the cells with an increased supply of
DHEAS as a precursor of active androgen.
In the present study, we investigated the expression offunctional OATPs in, and the growth of, androgen-dependent
prostate cancer cells. Among the OATP family members tested,OATP1A2 was found to be remarkably upregulated in AR-positive
human prostate cancer cells cultured under androgen-depleted
conditions. We propose that enhanced expression of OATP1A2 inthese cells plays a role in progression of prostate cancer byincreasing the availability of DHEAS as a precursor of active
androgen, under conditions of androgen depletion.
2. Materials and methods
2.1. Materials
DHEAS, dihydrotestosterone (DHT), and [3H]DHEAS sodium salt
(3.50 TBq/mmol) were purchased from Tokyo Chemical Industry(Tokyo, Japan), Wako Pure Chemical Industries (Osaka, Japan), andPerkinElmer Life Science (Boston, MA), respectively. Human
prostate cancer LNCaP and 22Rv1 cell lines were purchased from
American Type Culture Collection (Manassas, VA).
2.2. Cell culture and growth assays
LNCaP and 22Rv1 cells were cultured in RPMI1640 (Wako Pure
Chemical Industries) with penicillin (100 units/mL, Nakalai
Tesque, Kyoto, Japan), streptomycin (100 mg/mL, Wako Pure
Chemical Industries) and 10% fetal bovine serum (FBS, Invitrogen).
Cells were cultured in a humidiﬁed atmosphere containing 5% CO2
at 37 8C. For the cell growth assay, cells were cultured in RPMI1640
with penicillin (100 units/mL), streptomycin (100 mg/mL) supplemented with FBS or charcoal-stripped FBS (CSS, Invitrogen) in thepresence or absence of androgen and/or STX64 (also known as 667
coumate or 6-oxo-6,7,8,9,10,11-hexahydrocyclohepta[c]chromen3-yl sulfamate, Sigma Aldrich, St. Louis, MO), which is a tricylic
coumarin-based sulfamate that inhibits steroid sulfatase. The
growth-stimulatory effect of androgens, including DHEAS, wasevaluated by measuring cell growth after cells were plated at adensity of 1.5–2.4  104 cells/cm2 on a 96-well tissue culture plate.
In general, once cells became attached to the plate, a test androgenor DMSO (at 0.1% of the ﬁnal concentration) was added to the well,and cell growth was monitored for up to 9 days by means ofsulforhodamine B (SRB, Sigma Aldrich) assay as previously
described [17].
2.3. Gene expression proﬁling assay
LNCaP cells were cultured in RPMI1640 with penicillin(100 units/mL), streptomycin (100 mg/mL) and 10% CSS for 6
days. Then, total RNA was prepared using Isogen reagent (Nippon
Gene, Tokyo, Japan). Microarray experiments were performed anddata were analyzed according to the manufacturer’s instructions

1071(Agilent Technologies, Santa Clara, CA). In brief, 300 ng of total RNA
was subjected to linear ampliﬁcation and Cy3 labeling using the
Low RNA Input Linear Ampliﬁcation Kit (Agilent Technologies), andthen hybridized to a 44 K Whole Human Genome Microarray(Agilent Technologies). Data were extracted using Feature Extraction software (version 9.5.3) and analyzed using GeneSpring GX
software (version 11.0).
2.4. Reverse transcription-polymerase chain reaction (RT-PCR)
Total RNA was prepared from cells using Isogen reagent(Nippon Gene). mRNA expression of OATP1A2, OATP1B1,
OATP1B3, OATP2B1, OAT2, OAT3, and OAT4 was measured byquantitative RT-PCR (qRT-PCR), using Brilliant SYBR Green QPCR
Master Mix (Agilent Technologies). The fold change in mRNA
expression of transporter genes was normalized to hypoxanthine
guaninephosphoribosyl-transferase 1 (HPRT1) using the 2-DD
threshold cycles (Ct) method [18]. Primer sequences speciﬁc totransporter genes tested are listed in Table 1.
2.5. [3H]DHEAS cellular uptake assay
Intracellular accumulation of DHEAS was evaluated by meansof uptake assays as described previously [17]. Brieﬂy, cells wereplated at a density of 1.0  105 cells/cm2 on a 24-well tissueculture plate in culture medium supplemented with 10% FBS or
CSS in the absence or presence of DHT (1 nM) two days before
assay containing 0.1% DMSO of the ﬁnal concentration. The assay
was initiated by adding transport buffer (125 mM NaCl, 4.8 mMKCl, 5.6 mM D-glucose, 1.2 mM CaCl2, 1.2 mM KH2PO4,1.2 mM,
MgSO4, and 25 mM HEPES, adjusted to pH 7.4) containing
0.5 mCi/mL [3H]DHEAS (10 nM) with or without inhibitors at
37 8C. Sodium dependence of uptake was evaluated by replacing
Na+ with Li+, K+ or N-methylglucamine (NMG+). At the end of theassay, cells were washed with ice-cold transport buffer andsolubilized in 1% (v/v) Triton X-100 (Wako Pure Chemical
Industries). The radioactivity in the resultant cell lysate wasmeasured using a liquid scintillation counter (Aloka, Tokyo,
Japan). Intracellular [3H]DHEAS accumulation was normalized byprotein content determined with a Bio-Rad protein assay kit (BioRad, Hercules, CA), and is shown as the ratio of accumulation to
the initial concentration of DHEAS, i.e., cell-to-medium (C/M)
ratio (mL/mg).
2.6. Western blot
To determine protein expression of OATP1A2, cells werecollected and lysed in RIPA buffer (150 mM sodium chloride,
1.0% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, and 50 mM Tris
at pH 8.0) by sonication. A 50 mg aliquot of total cell lysate wassubjected to SDS polyacrylamide gel electrophoresis, and thenelectrotransferred onto a polyvinylidene diﬂuoride membrane(Millipore, Billerica, MA). The blots were probed with anti-human
OATP1A2 rabbit polyclonal antibody (Assay Biotechnology Company, San Francisco, CA) at 1 to 500 dilution or with antiglyceraldehyde-3-phosphate dehydrogenase (GAPDH) rabbit
monoclonal antibody (Cell Signaling Technology, Danvers, MA)
at 1 to 2000 dilution, followed by appropriate secondary antibodies
conjugated to horseradish peroxidase (Amersham Pharmacia
Biotech, Piscataway, NJ).
2.7. Construction of OATP1A2-transfected LNCaP cells
LNCaP cells were transfected with pcDNA3.1/OATP1A2 or
pcDNA3.1 alone (Invitrogen), and then selected with 800 mg/mL
of G418 (Wako Pure Chemicals). Enhanced OATP1A2 expression

H. Arakawa et al. / Biochemical Pharmacology 84 (2012) 1070–1077

1072
Table 1
Gene speciﬁc primers for RT-PCR.
SymbolGene

Protein

SLCO1A2

OTAP1A2

SLCO2A1OATP2A1

SLCO1B1OATP1B1

SLCO2B1OATP2B1

SLCO5A1OATP5A1

SLCO1B3OATP1B3

SLCO3A1OATP3A1

SLCO4A1OATP4A1

STS

STS

KLK3

PSA

HPRT1

HPRT

Fw
Rev
Fw
Rev
Fw
Rev
Fw
Rev
Fw
Rev
Fw
Rev
Fw
Rev
Fw
Rev
Fw
Rev
Fw
Rev
Fw
Rev

Primer sequence (50 to 30 )Length (bp)Amplicon (bp)

AAGACCAACGCAGGATCCATGAGTTTCACCCATTCCACGTACA
CTGTGGAGACAATGGAATCGAGCACGATCCTGTCTTTGCTGAAGGAATGCCCAAGAGATGATGCTT
AACCCAGTGCAAGTGATTTCAATCGCACTCACTGATTCCTACACATT
AAGCCATACCATCACTCATTACAC
TCATTGGAAYGGGCTCCACTGAAACGAGGGTCATTCTGG
GTCCAGTCATTGGCTTTGCACAACCCAACGAGAGTCCTTAGGTACGACAATGTGGTCTACCGATACCAGAGTTCAGCCCTCCTTTAGTCAGCACCAGTTGAAGGACAGC
GAGCCAGATGGAGAGAGGGGAGAAATTCATGGCGGAAGGTGGGCTCATCAATCTTCTGG
AGAGGAGTTCTTGACCCCAAAGGCACACCATTACAGACAAGTGGTGACACTGGCAAAACAATGCAGGTCCTTTTCACCAGCAAGCT

20
23
22
22
22
23
24
24
19
20
20
22
24
24
19
18
20
21
22
22
21
21

101

was veriﬁed by uptake study of [3H]DHEAS (0.5 mCi/mL) for up to
5 min and Western blotting using anti-human OATP1A2 rabbit IgG.

147
154
278
213
111
309
88
134
182
194

(Fig. 1C). In parallel, DHEAS induced mRNA expression of prostate
speciﬁc antigen (PSA) in LNCaP cells, reﬂecting the effect of DHEAS on
the cell growth (Fig. 1D).

2.8. OATP1A2 knockdown in LNCaP cells
To develop prostate cancer cell lines with knockdown of
OATP1A2, double-stranded DNA containing a hairpin sequence forthe targeted site (50 -tattctttggctgccatacct-30 ) and (50 -aagtgtcatctacctggtgaa-30 ) of OATP1A2 mRNA was inserted into the microRNA-adapted vector pcDNA6.2-GW/EmGFP-miR (Invitrogen).
Knockdown of OATP1A2 was performed using the BLOCK-iT Pol
II miR RNAi expression vector kit according to the manufacturer’s
instructions (Invitrogen). The pcDNA6.2-GW/EmGFP-miR-negative control plasmid contains an oligonucleotide not related to
OATP1A2, which serves as a negative control. LNCaP cells weretransfected with these plasmid DNAs using Lipofectamine 2000
reagent (Invitrogen). Two days later, selection of transfected cells
was started with blasticidin (2 mg/mL, Invitrogen). After about 3
weeks, blasticidin-resistant and EmGFP-positive cells were used
for the proliferation studies and subjected to RT-PCR analysis.
3. Results
3.1. DHEAS-induced cell growth and PSA expression in androgen
receptor-positive prostate cancer cells
In order to determine whether DHEAS can stimulate cell growthof androgen receptor-positive human prostate cancer LNCaP cells,
cell growth was measured in androgen-depleted medium. The cell
growth was stimulated by DHEAS in a concentration-dependent
manner. At a physiologically relevant serum concentration of
DHEAS (5 mM), the growth increased about 174% compared to thatin the absence of DHEAS, corresponding to about half of the effectof DHT, an active androgen, at 1 nM (Fig. 1A). DHEAS also exhibiteda stimulatory effect on cell growth of 22Rv1 cells (16%  1.82% on
Day 9) compared to that in the absence of DHEAS (data not shown).
The stimulatory effect observed in LNCaP cells was signiﬁcantly
attenuated in the presence of STX64, a speciﬁc inhibitor of STS(Fig. 1B), suggesting that activation of cell growth by DHEAS requires
its conversion to DHEA mediated by STS. Expression of STS showed nochange in LNCaP cells grown in normal and CSS-containing medium

3.2. Alteration in organic anion transporter levels in androgen
receptor-positive prostate cancer cells
DHEAS is an organic anion at physiological pH, and its uptake
requires carrier-mediated transport across plasma membranes.
We therefore performed a microarray analysis to compare the gene
expression proﬁles of transporters that might be involved in
DHEAS uptake between LNCaP cells grown in normal andandrogen-depleted culture medium. Table 2 summarizes thegenes whose expression altered by more than 1.5 times under the
androgen-depleted condition; there were signiﬁcant increases inseveral OATP genes, including OATP5A1, 1A2, and 1B1. This was
conﬁrmed by qRT-PCR (Fig. 2A). OATP1A2 mRNA expression wasincreased most markedly among the genes tested, and was 3.6and 6.4-fold greater in LNCaP cells cultured in CSS-containing
medium on Days 3 and 7, respectively, than on Day 1; however, nosuch increase was observed in LNCaP cells cultured for 7 days innormal medium (data not shown). Similarly, mRNA expression
levels of OATP2A1 and 1B1 on Day 7 were increased 3.4- and 6.4fold, respectively (Fig. 2A). Table 3 summarizes the fold differencein mRNA expression of OATP genes tested on Days 3 and 7. Western
blotting analysis indicated a time-dependent increase of OATP1A2

Table 2
Alterations of transporter gene expression in LNCaP cells under androgen-depleted
conditions, as determined by microarray assay.
Gene

Protein

Major substrate

Fold change

SLC10A4
SLCO5A1
SLCO1A2
SLCO1B1
SLCO2B1
SLC22A6
SLC10A1
SLC22A13
SLC04A1P4
OATP5A1OATP1A2OATP1B1OATP2B1
OAT1
NTCP
OAT10
OATP4A1

Bile acid
Organic anion
Organic anion
Organic anion
Organic anion
Organic anion
Taurocholate
Organic anion
Organic Anion

4.85
4.39
3.87
2.47
1.74
0.654
0.606
0.486
0.411

H. Arakawa et al. / Biochemical Pharmacology 84 (2012) 1070–1077

1073

Fig. 1. DHEAS-induced cell proliferation and PSA expression in LNCaP cells. (A) Growth of LNCaP cells was monitored by means of SRB assay for up to 7 days. LNCaP cells
cultured in RPMI1640 with 10% CSS for 7 days were seeded at 8000 cells each in the presence or absence of androgen. Each bar represents the mean  S.E. (n = 6), and * indicatesa signiﬁcant difference from the control (p < 0.05) by Student’s t-test. (B) Effect of STS inhibitor STX64 (10 mM) on stimulation of LNCaP cell growth by DHEAS. LNCaP cells cultured
in RPMI1640 with 5% CSS for up to 9 days were seeded at 8000 cells. Their growth was monitored in the absence (open, control) and presence (closed) of DHEAS (5 mM) with (square)or without (circle) STX64 (10 mM). Experiments were done in quadruplicate and repeated at least twice. Each point represents the mean  S.E. (n = 4), and * indicates a signiﬁcantdifference from the control (p < 0.05) by Student’s t-test. (C) Expression change of STS or (D) PSA in LNCaP cells was measured by qRT-PCR. LNCaP cells were cultured in RPMI1640
supplemented with 10% FBS or 10% CSS in the presence or absence of androgen. Each bar represents the mean  S.E. (n = 3), and * indicates a signiﬁcant difference from the control(p < 0.05) by Student’s t-test.

protein expression (Fig. 2B). More interestingly, the stimulation of
OATP1A2 mRNA expression under the androgen-depleted condition was almost completely abolished by the addition of DHT(Fig. 2C), suggesting that OATP1A2 gene transcription is negatively
regulated by androgens. Expression of DHEAS transporters was
also studied in 22Rv1 cells grown in both normal and androgendepleted medium, and qualitative PCR results are shown in Fig. 2D.
Signiﬁcant mRNA expression of OATP2A1, 2B1, 3A1, 4A1 and
SLC10A4 was detected in 22Rv1 cells as well. Similarly, transcription of OATP1A2 and SLC10A4 appeared to be upregulated whenthe medium was deprived of androgen. Further qRT-PCR analysis

showed that OATP1A2 mRNA expression was increased 3.7-fold in
22Rv1 cells, conﬁrming DHT-mediated negative regulation of thetranscription (Fig. 2E).
3.3. Enhanced cellular uptake of DHEAS under androgen deprivation
To study the contribution of OATPs to cellular uptake of DHEAS
in LNCaP cells, initial uptake of [3H]DHEAS was measured in theabsence or presence of several inhibitors of organic anion transportsystems. The uptake was signiﬁcantly reduced in the presence of
unlabeled DHEAS, estrone 3-sulfate and BSP, which are inhibitors

1074

H. Arakawa et al. / Biochemical Pharmacology 84 (2012) 1070–1077
Table 3
Quantitative changes in mRNA expression of OATP transporters in LNCaP cells
under androgen-depleted condition, as determined by qRT-PCR.
Protein nameOATP1A2OATP2A1OATP5A1OATP3A1OATP1B1OATP1B3
OATP2B1OATP4A1

Fold change relative to Day 1
Day 3

Day 7

3.6  0.20*
1.7  0.31*
0.96  0.30*
0.49  0.15*
1.9  0.59*
0.72  0.20
1.7  0.25*
1.0  0.21

6.4  1.2*
3.4  0.23*
0.93  0.36
2.3  1.2
7.4  2.9*0.80  0.37
2.2  0.38*
0.98  0.88

All data are presented as mean  S.E. (n = 5 or 6).
*
Indicates a signiﬁcant difference from Day 1 (p < 0.05) by Student’s t-test.

of OATPs, but inhibitors of OAT (salicylate and p-aminohippouricacid) and organic cation transporter inhibitors (tetra-ethylammonium and 1-methyl-4-phenylpyridinium) had no effect (Fig. 3A).
The uptake was tolerant of Na+ replacement with Li+, K+ and NMG+
(Fig. 3B). Accordingly, [3H]DHEAS uptake by LNCaP cells has thecharacteristics of OATP-mediated transport. Furthermore,[3H]DHEAS uptake was signiﬁcantly enhanced in LNCaP cells
cultured with CSS-containing medium for 2 days, but addition of
DHT (1 nM) to the medium signiﬁcantly decreased the uptake,
implying that cellular uptake of DHEAS is regulated by OATPs
whose expression is negatively regulated by androgen, such as
OATP1A2 (Fig. 3C).
3.4. Contribution of OATP1A2 to DHEAS-induced cell growth in LNCaP
cells

Fig. 2. Changes of expression of organic anion transporters in androgen receptorpositive prostate cancer cells. (A) mRNA expression of OATP transporter genes in
LNCaP cells was measured by qRT-PCR. LNCaP cells were cultured in RPMI1640 with
10% CSS for up to 6 days. Each bar represents the mean  S.E. (n = 5 or 6), and *indicates a signiﬁcant difference from the control (p < 0.05) by Student’s t-test. (B)OATP1A2 protein expression was measured by Western blotting. LNCaP cells were
cultured in RPMI1640 supplemented with 10% CSS in the presence or absence of DHT(1 nM) for up to 6 days. A typical Western blot showing increased OATP1A2 expression ispresented. Each bar (sum of the two bands shown) represents the mean value of
densitometric analysis of three individual blots  S.E. (n = 3), and * indicates a signiﬁcantdifference from the control (p < 0.05) by Student’s t-test. (C) Effect of DHT (1 nM) on
mRNA expression of OATP1A2 in LNCaP and (E) 22Rv1 cells cultured in RPMI1640
supplemented with 10% FBS or 10% CSS in the presence or absence of DHT (1 nM) for 2days. Each bar represents the mean  S.E. (n = 3). * and y indicate a signiﬁcant difference
from the expression of OATP1A2 in LNCaP cultured in RPMI1640 supplemented with

To further examine the role of OATP1A2 in DHEAS-induced
growth of prostate cancer cells, growth of OATP1A2-transfected
LNCaP cells was examined in CSS-containing medium in theabsence or presence of 5 mM DHEAS. In the presence of DHEAS,
growth of OATP1A2-transfected cells was stimulated by 41%,
whereas that of cells transfected with an empty-vector plasmid
increased by only 11%. Furthermore, the stimulatory effect of
DHEAS was evaluated in LNCaP cell lines in which OATP1A2 hadbeen silenced, designated as KD16 and KD34, compared with
control cell lines (C3 and C9). As shown in Fig. 4A, mRNA
expression of OATP1A2 in KD16 and KD34 cells was almost
completely knocked down. The effect of DHEAS on growth of thecell lines tested was normalized to that in the absence of DHEAS(1.00) on Day 3 through Day7, respectively (Fig. 4B). In the twocontrol cells lines, DHEAS signiﬁcantly stimulated cell growth withthe highest stimulation on Day 5 or 6, even though there weredifferences in degree of stimulation, whereas there was no
statistically signiﬁcant effect on growth of KD16 and KD34 cells
during the experiments (p-value of 0.207 for KD16 and 0.056 forKD34 by one-way ANOVA). The ﬁnding that growth of OATP1A2knockdown cells was insensitive to DHEAS suggests that OATP1A2
plays a predominant role in DHEAS-induced cell growth under
androgen-depleted conditions.
4. Discussion
Our present ﬁndings indicate that OATP-mediated DHEAS
transport plays a major role in survival and proliferation of

10% FBS or 10% CSS (p < 0.05) by Student’s t-test, respectively. (D) mRNA expression oforganic anion transporters and HPRT in 22Rv1 cells was determined by RT-PCR. 22Rv1
cells were cultured in RPMI1640 supplemented with 10% FBS (top panel) or 10% CSS(bottom) for 2 days. PCR products were visualized on 2% agarose gel.

H. Arakawa et al. / Biochemical Pharmacology 84 (2012) 1070–1077

0

Log2 Fold Change

-0.5
-1
-1.5
-2
-2.5
-3
-3.5
-4
-4.5

C9

C3

KD16

KD34

B 3.5
Fold Change in Cell Growth by DHEAS

A 0.5

1075

3

2.5

2

1.5

1

0.5
3

4

5

6

7

Day

Fig. 4. Impact of OATP1A2-mediated DHEAS transport on proliferation of LNCaP
cells. (A) Quantiﬁcation of OATP1A2 mRNA expression in OATP1A2-knockdown(KD16 and KD34, closed bars) cells, compared with that in control (C3 and C9, openbars). Results are shown as the mean fold change (log2)  S.E. Quantiﬁcation ofmRNA expression by qRT-PCR was repeated three times in duplicate. (B) Stimulatory
effect of DHEAS on growth of OATP1A2-knockdown (open) and control cells (closed) in
SRB assay. Cells were plated at 8000 cells in each well. The ratio of cell growth to thatwithout DHEAS (5 mM) was calculated for C3 (circle), C9 (square), KD16 (circle), andKD34 (square) each day. Each value is the mean of 7 or 8 individual results with S.E.
(n = 4).

Fig. 3. Characterization of [3H]DHEAS uptake by LNCaP cells. (A) Cellular uptake of[3H]DHEAS (10 nM) was measured in the presence or absence of the indicatedcompounds at pH 7.4 for 5 min in LNCaP cells cultured in RPMI1640 with 10% CSS
for 2 days. Each bar represents the mean  S.E. (n = 3 or 4), and * indicates a signiﬁcantdifference from the control (p < 0.05) by Student’s t-test. E3S; Estrone 3-sulfate, BSP;
bromosulfophthalein, TCA; taurocholate, PRO; probenecid, SAL; salicylate, PAH; paminohippurate, TEA; tetra-ethylammonium, MPP+; 1-methyl-4-phenylpyridinium.
(B) Cellular uptake of [3H]DHEAS (10 nM) was measured in the presence or absence of
extracellular Na+. Extracellular Na+ was replaced with K+, Li+, or N-methylglucamine(NMG+) at pH 7.4 for 5 min in LNCaP cells cultured in RPMI1640 with 10% CSS for 2days. Each value represents the mean  S.E. (n = 3 or 4). (C) Cellular uptake of[3H]DHEAS (10 nM) in LNCaP cells was measured. LNCaP cells were cultured in
RPMI1640 with 10% FBS (open, circle) or 10% CSS (closed) in the absence (closed circle)
or the presence (closed diamond) of DHT (1 nM) for 2 days. Triangle symbols represent
uptake of [3H]DHEAS (10 nM) in the presence of DHEAS (1 mM). Each point represents
the mean  S.E. (n = 3 or 4), and * indicates a signiﬁcant difference from the uptake rateof [3H]DHEAS in LNCaP cells cultured with 10% CSS (p < 0.05) by Student’s t-test.

androgen-receptor-positive prostate cancer cells under conditionsof androgen depletion. In other words, enhanced import of DHEAS
mediated by increased expression of OATPs, most likely predominantly OATP1A2, can be utilized to provide an alternative source of
androgen under conditions of androgen depletion, allowing
prostate cancer cells scope to acquire castration resistance,
because the high serum concentration of DHEAS is little alteredduring the course of ADT.
To date, little is known about the role of OATPs in prostatecancer cells, although enhanced expression of OATPs in prostate
tumors has been reported [16,19]. Among them, OATP1B3 may
be an important player for driving cell proliferation of prostatecancer cells, because it facilitates testosterone uptake into cells[19]. A more recent study with patient-derived prostate tumor
specimens indicated that mRNA expression of six SLCO genes,
including SLCO1B3 and 2B1, was enhanced several-fold in CRPC
metastases, compared to untreated prostate cancer, implying

their association with prostate cancer-speciﬁc motility [16]. In
the present study, we observed remarkable upregulation of
OATP1A2 mRNA expression, as well as signiﬁcant increases of
OATP2A1 and 1B1 on Day 7 in CSS-containing culture medium,
whereas expression of OATP2B1 and OATP1B3 was increased
only moderately and slightly, respectively (Fig. 2). We also
found a stimulatory effect of DHEAS on growth of LNCaP and
22Rv1 cells (Fig. 1); therefore, we focused here on DHEAS
transport by OATPs, even though several other substrates arealso commonly transported by the OATPs whose expression isenhanced.
Several classes of SLC transporters have been shown tomediate DHEAS uptake, including OATP1A2, 1B1, 1B3, and 2B1[10,11], OAT2, 3, and 4 [20–22], and Na+-dependent taurocholate cotransporting polypeptide (NTCP) [23]. Hence, we conducted gene expression proﬁling of human prostate cancer
LNCaP cells cultured under androgen-depleted conditions (Table
2A and Table 3). Microarray analysis suggests that OATP
molecules are preferentially upregulated in LNCaP cells cultured
in the absence of androgens, and this idea was supported byevidence that the functional characteristics of DHEAS transport
in LNCaP cells resemble those of OATP-mediated transport(Fig. 3A and B). Among several OATPs whose expression isenhanced by androgen deprivation, OATP1A2 showed thegreatest increase in expression, and interestingly, its expression
is negatively regulated by androgen signaling (Fig. 2C). The
increase of OATP1A2 mRNA was conﬁrmed by Western blotting,showing two distinct bands around 75 and 100 kDa. This result
agrees with two bands previously shown for OATP1A2 expressedin breast cancer cells in Western blotting, suggesting thatOATP1A2 is glycosylated [24]. We found that expression of
OATP1A2 was increased in LNCaP and 22Rv1 cells cultured
under conditions of hormone deprivation, and the increase wasconsistent with the increase of initial uptake of DHEAS in LNCaP
cells (Fig. 3C). Ligand-bound nuclear hormone receptors activatetranscription in the classical mode of receptor action, by binding
to the regulatory sequence of the target gene promoter in dimer
form. However, they also silence gene expression in various
ways, such as through recruiting co-repressor, changing
chromatin structure and accessibility, or inhibiting signaling1076

H. Arakawa et al. / Biochemical Pharmacology 84 (2012) 1070–1077

pathways via cross-talk. For instance, androgen receptor has
been shown to act as a ligand-dependent trans-repressor of
activator protein-1 (AP-1) activity, resulting in attenuation ofAP-1-activated gene expression [25]. The proximal promoter
region of OATP1A2 has been characterized, and contains active
cis-elements for several hepatocyte nuclear factors, a TAATAT
box, and a putative AP-1 binding site [26]; it was therefore
speculated that androgen-dependent negative regulation may
be due to inhibition of AP1 by ligand-bound androgen receptor
in LNCaP cells. Further study is needed to clarify the precise
mechanism of repression of OATP1A2 expression by active
androgens.
Because DHEAS is an inactive precursor of DHEA, it has to be
activated by desulfation. It was reported that DHEAS ishydrolyzed by STS in LNCaP cells [27] and the expression of
STS is increased in prostate cancer cells, compared to nonmalignant prostate tissues, as determined by immunohistochemical staining [28]. In the present study, expression of STS
mRNA was detected in LNCaP cells, and this expression was notaffected under androgen-depleted conditions (Fig. 1C). Furthermore, cell growth of LNCaP cells was signiﬁcantly reduced in thepresence of STX64 (Fig. 1B), while PSA mRNA expression in
LNCaP cells was increased in the presence of DHEAS (Fig. 1D).
These results suggest that DHEAS undergoes metabolism at least
to DHEA, mediated by STS. DHEA thus generated may have aweak androgenic potential to activate AR directly, and may be
converted to T or DHT via 3b-hydroxysteroid dehydrogenase(3bHSD) since cell proliferation of LNCaP cells in the presence of
DHEA was reported to be suppressed by inhibition of 3bHSD[15]. Although DHEA is present in human serum (5 nM), the
DHEAS concentration in serum (3–5 mM) is almost a thousand
fold greater [12]; therefore, DHEAS may be taken up by prostatecancer cells via OATPs as a source of DHEA to compensate for the
shortage of androgen in serum. This notion is supported by theexperimental ﬁnding that knockdown of OATP1A2 in LNCaP cells
resulted in loss of the cell growth response to DHEAS (Fig. 4).
Thus, concomitant use of a blocker of OATP1A2 may bepharmacologically effective to eradicate prostate cancer cells
during the course of ADT. It is also noteworthy that, among nonsynonymous polymorphisms which have been reported in
SLCO1A2 gene, decreased transport of estrone 3-sulfate wasreported in c.516 A > C (rs11568563, E172D) and c.404 A > T(rs45502302, N135I) [29,30]. Therefore, the efﬁcacy of ADT
might be affected in prostate cancer patients with thesegenotypes, of which c.516 A > C is relatively frequent inEuropeans and Americans (5.3%).
5. Conclusion
In conclusion, our results indicate that OATP1A2 plays a rolein the growth of prostate cancer cells under androgen-depleted
conditions by supplying DHEAS as an alternative source of
androgen, so that the cells can survive to acquire castration
resistance. This is a novel mechanism underpinning the
progression of advanced prostate cancer to the castrationresistant state. Hence, OATPs (especially OATP1A2) may serve asa pharmacological target for more effective treatment of
prostate cancer. In other words, pharmacological inhibition of
OATP1A2-mediated inﬂux transport of DHEAS in conjunction
with ADT might provide a clinical beneﬁt for prostate cancer
patients.
Conﬂict of interest
There are no conﬂicts of interest.

Acknowledgments
This research was carried out with the support of a Grant-in-Aid
for Scientiﬁc Research (KAKENHI) to T. N. (23590176) from theJapan Society for the Promotion of Science.

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