HHS Public Access
Author manuscript
Author Manuscript

Mol Imaging Biol. Author manuscript; available in PMC 2017 October 19.
Published in final edited form as:
Mol Imaging Biol. 2014 December ; 16(6): 756–764. doi:10.1007/s11307-014-0756-x.

Accumulation of Trans-1-Amino-3[18F]Fluorocyclobutanecarboxylic Acid in Prostate Cancer due toAndrogen-Induced Expression of Amino Acid Transporters

Author Manuscript

Hiroyuki Okudaira1, Shuntaro Oka1, Masahiro Ono1,2, Takeo Nakanishi3, David M.
Schuster4, Masato Kobayashi2, Mark M. Goodman4, Ikumi Tamai3, Keiichi Kawai2,5, andYoshifumi Shirakami1
1Research Center, Nihon Medi-Physics Co., Ltd., 3-1 Kitasode, Sodegaura, Chiba, 299-0266,

Japan
2Division of Health Sciences, Graduate School of Medical Science, Kanazawa University,

Ishikawa, Japan
3Department of Membrane Transport and Biopharmaceutics, Faculty of Pharmaceutical Sciences,

Kanazawa University, Ishikawa, Japan
4Division of Nuclear Medicine and Molecular Imaging, Department of Radiology and Imaging

Sciences, Emory University, Atlanta, GA, USA
5Biomedical Imaging Research Center, University of Fukui, Fukui, Japan

Author Manuscript

Abstract
Purpose—Androgens play a crucial role in prostate cancer progression, and trans-1-amino-3[18F]fluorocyclobutanecarboxylic acid (anti-[18F]FACBC) are used for visualization of prostatecancer. We examined the effect of androgen on the expression of amino acid transporters related toanti-[18F]FACBC transport and uptake of trans-1-amino-3-fluoro-[1-14C]cyclobutanecarboxylic
acid (anti-[14C]FACBC).
Procedures—Expression of amino acid transporters and uptake of anti-[14C]FACBC in
androgen receptor (AR)-positive LNCaP and AR-negative DU145 human prostate cancer cells
cultured with/without 5α-dihydrotestosterone (DHT) and the effect of bicalutamide, an AR
antagonist, on DHT-associated changes were investigated.

Author Manuscript

Results—DHT stimulated the expression of amino acid transporters ASCT2, SNAT5, 4F2 heavy
chain, and LAT3 in LNCaP but not in DU145 cells. Anti-[14C]FACBC uptake was enhanced, in a
DHT-dependent manner, in LNCaP cells only.

Correspondence to: Hiroyuki Okudaira; hiroyuki_okudaira@nmp.co.jp.
Electronic supplementary material The online version of this article (doi:10.1007/s11307-014-0756-x) contains supplementarymaterial, which is available to authorized users.

Conflict of Interest. Hiroyuki Okudaira, Shuntaro Oka, Masahiro Ono, and Yoshifumi Shirakami are employees of Nihon MediPhysics Co., Ltd. Mark M. Goodman and Emory University have patent rights for anti[18F]FACBC and are eligible to receive
royalties on anti-[18F]FACBC from Nihon Medi-Physics Co., Ltd. Mark M. Goodman, David M. Schuster, and Keiichi Kawai haveongoing research collaborations with Nihon Medi-Physics Co., Ltd.

Okudaira et al.

Page 2

Author Manuscript

Conclusions—DHT enhanced the expression of ASCT2, the transporter responsible foranti[18F]FACBC uptake, thereby increasing anti-[14C]FACBC uptake in AR-positive LNCaP cells.
Androgen-mediated induction may contribute to the distinct anti-[18F]FACBC accumulation
pattern in prostate cancer.
Keywords

anti-FACBC; Prostate cancer; Androgen; Amino acid transporter; ASCT2Introduction

Author Manuscript

Prostate cancer is the second most frequently diagnosed cancer among men worldwide. Its
incidence and mortality rates are rising in several Asian and European countries, including
Japan [1]. 2-Deoxy-2-[18F]fluoro-D-glucose ([18F]FDG), 18F- and 11C-labeled choline,
and 18F-and 11C-labeled acetate are the most-studied positron emission tomography (PET)
radiotracers for use in prostate cancer detection and visualization. These tracers are trappedin prostate cancer cells due to the high glycolytic and metabolic rates of choline and fatty
acids, respectively [2]. Recently, these probes have been used clinically for prostate cancer
imaging. For example, [18F]FDG PET/computed tomography (CT) has been used toefficiently evaluate the extent and treatment responsiveness of metastatic castration-resistant
prostate cancer (CRPC) [3, 4]. However, the present imaging techniques are not ideal for
detecting primary prostate cancer or lymph-node metastases, especially in patients withlower prostate-specific antigen (PSA) levels.

Author Manuscript
Author Manuscript

Trans-1-amino-3-[18F] fluorocyclobutanecarboxylic acid (anti-[18F]FACBC) is a synthetic
amino acid that accumulates in prostate cancer cells [5]. A recent clinical study [6, 7]
showed that anti-[18F]FACBC accurately detected prostate cancer within the prostate and in
pelvic lymph nodes with greater specificity and sensitivity than [111In]capromab single
photon emission computed tomography (SPECT)/CT, which targets prostate-specificmembrane antigens. A recent phase I clinical study of anti-[18F]FACBC reported favorable
characteristics for prostate cancer imaging, such as slow urinary excretion and high in vivo
stability [8]. We reported that two amino acid transporters, system ASC amino acidtransporter 2 (ASCT2; encoded by SLC1A5) and system L amino acid transporter 1 (LAT1;
encoded by SLC7A5), were important determinants of anti-FACBC uptake by prostatecancer cells [9, 10]. We further demonstrated that their affinities for anti-FACBC weresimilar to the cognate amino acid substrates (e.g., glutamine) [11]. Therefore, thepreferential accumulation of anti-FACBC in prostate cancer may result from cancer cells’
increased requirement for amino acids essential for growth. Because ASCT2 and LAT1 areoften upregulated in various human cancer cells and their expression is associated with poor
prognosis, they have been considered potential targets for chemotherapy and radiodiagnosis[12, 13].
Male gonadal hormones (androgens) and the androgen receptor (AR) constitute an essential
axis for promoting the development and progression of AR-positive prostate cancer [14, 15].
Although serum androgen concentrations significantly decrease after androgen deprivation
therapy, low levels of intraprostatic androgens and circulating conjugated androgen are

Mol Imaging Biol. Author manuscript; available in PMC 2017 October 19.

Okudaira et al.

Page 3

Author Manuscript

sufficient to activate the AR and stimulate tumor growth [16, 17]. Therefore, even inadvanced-stage prostate cancer, AR activity is a key determinant of therapeutic response.
Although many androgen-regulated genes have been identified, limited data are available on
their mechanisms of regulation. PSA is the protein product of the most-studied androgenregulated gene [18]. In prostate cancer patients treated with endocrine or other therapies,
elevated serum PSA levels are considered evidence for prostate cancer recurrence. In
addition, androgen regulates the expression of some amino acid transporters. For example, inhuman prostate cancer LNCaP cells, androgen exposure upregulated the expression ofsystem L amino acid transporter 3 (LAT3; encoded by SLC43A1) [19], which was firstdescribed as a gene overexpressed in human prostate cancer [20, 21]. In addition, in
castrated adult mice, androgen exposure upregulated the expression of system N amino acidtransporter 2 (SNAT5; encoded by SLC38A5) [22]. A subsequent study reported increased
expression of system ASC amino acid transporter 1 (ASCT1; encoded by SLC1A4),
ASCT2, and 4F2 heavy chain (4F2hc; encoded by SLC3A2) in the presence of androgen[23]. Therefore, it is hypothesized that androgen induces the expression of amino acidtransporters involved in anti-[18F]FACBC transport. If so, prostate cancer cells are expected
to show increased uptake of anti-[18F]FACBC. Therefore, anti-[18F]FACBC may be aneffective diagnostic tool for prostate cancer.

Author Manuscript

In the present study, we investigated the effect of androgen on the expression of amino acidtransporters that are thought to transport anti-FACBC [9–11] and its effect on anti-FACBC
accumulation using AR-positive and AR-negative human prostate cancer cell lines.
Furthermore, we examined the effects of androgen on the transport of FDG, choline, andacetate, which are commonly used PET tracers for the diagnosis of various cancers.

Author Manuscript

Materials and Methods
Materials
14C-labeled compounds were used because of their longer half-lives (T =5,700 years)
1/2
compared to those of 18F- (T1/2=110 min) or 11C-labeled compounds (T1/2=20 min).
Trans-1-amino-3-fluoro-[1-14C]cyclobutanecarboxylic acid (anti-[14C]FACBC, 2.09 GBq/mmol) was radiolabeled by Sekisui Medical Co. Ltd. as described previously [10]. L-[14C(U)]glutamine ([14C]Gln, 10.17 GBq/mmol) was purchased from PerkinElmer. L[methyl-14C]methionine ([14C]Met, 2.04 GBq/mmol), [14C (U)]2-deoxy-2-fluoro-D-glucose([14C]FDG, 11.10 GBq/mmol), [methyl-14C]choline chloride ([14C]choline, 2.04 GBq/mmol), [1-14C]acetic acid, and sodium salt ([14C]acetate, 1.85 GBq/mmol) were purchasedfrom American Radiolabeled Chemicals.

Author Manuscript

The human prostate cancer cell lines LNCaP-FGC (LNCaP) and DU145 and fetal bovine
serum (FBS) were purchased from American Type Culture Collection (ATCC). Cell culture
medium and antibiotics were purchased from Life Technologies. 5α-Dihydrotestosterone(DHT) and bicalutamide were purchased from Sigma-Aldrich.

Mol Imaging Biol. Author manuscript; available in PMC 2017 October 19.

Okudaira et al.

Page 4Cell Cultures

Author Manuscript

LNCaP and DU145 cells were maintained in RPMI-1640 medium and Dulbecco’s modified
Eagle medium, respectively, and both media were supplemented with 10 % FBS, 100
units/ml penicillin, and 100 μg/ml streptomycin. Cells were cultured at 37 °C in 5 %CO2/95 % air.

Author Manuscript

For experiments, LNCaP and DU145 cells were seeded into 6-well and 24-well plates,
respectively, with phenol red-free RPMI-1640 and phenol red-free Dulbecco’s modified
Eagle medium, respectively. Both media were supplemented with 10 % charcoal-stripped
FBS (Thermo Fisher Scientific), 100 units/ml penicillin, and 100 μg/ml streptomycin. On thefollowing day, cells were exposed to 1 nM DHT alone or 1 nM DHT in combination with 10
μM bicalutamide for 3 days. The experimental media containing DHT with or withoutbicalutamide were changed after 2 days. DHT and bicalutamide were dissolved in ethanol(final concentration of ethanol, 0.1 %). The control cells were treated with the solvent used
to prepare DHT or bicalutamide solutions (final concentration of ethanol, 0.1 %).
Cell Proliferation Assay
After exposure to DHT with or without bicalutamide, cells were counted by Trypan blue
exclusion using a Countess Automated Cell Counter (Life Technologies).
Quantitative Real-Time Polymerase Chain Reaction

Author Manuscript
Author Manuscript

The gene expression of ASCT2, SNAT2, SNAT5, LAT1, 4F2hc, LAT3, B0AT1, B0AT2, and
ATB0,+ was analyzed as described previously [9]. Briefly, total RNA was extracted from
cells using an RNeasy Plus Mini Kit (QIAGEN) after exposure to DHT with or withoutbicalutamide. Complementary DNA (cDNA) was synthesized from 1 μg of total RNA usinga Transcriptor Universal cDNA Master kit (Roche Applied Science) according to the
manufacturer’s protocol. Primers (Supplemental Table 1) were designed using the Roche
Applied Science Website-based Universal ProbeLibrary Assay Design Center (http://
www.roche-applied-science.com) and were synthesized by Nihon Gene ResearchLaboratories. Quantitative real-time polymerase chain reaction (qRT-PCR) was performed
using an Mx3000P QPCR system (Agilent Technologies), FastStart Universal Probe MasterMix (ROX), and the Universal ProbeLibrary (Roche Applied Science). Cycling conditions
were as follows: 1 cycle of enzyme activation at 95 °C for 10 min followed by 60 cycles ofdenaturing at 95 °C for 15 s and extension at 60 °C for 1 min. All reactions were run in
triplicate. The PCR products were analyzed by agarose gel electrophoresis, and nononspecific PCR bands were detected. The PCR products for each gene were subsequently
purified using a High Pure PCR Cleanup Micro Kit (Roche Applied Science) and quantified
based on concentration and base pairs of the amplicons. The messenger RNA (mRNA) copy
number was calculated from standard curves generated by amplifying serial dilutions ofknown quantities of purified amplicons. Expression data were normalized against the copy
number of β-actin.
Uptake Assay
The uptake assay was performed as described previously [9]. Briefly, the sodium-containing
incubation medium was prepared using phosphate-buffered saline (PBS, pH 7.4–7.6), 137
Mol Imaging Biol. Author manuscript; available in PMC 2017 October 19.

Okudaira et al.

Page 5

Author Manuscript

mM NaCl, 2.7 mM KCl, 8 mM Na2HPO4, 1.5 mM KH2PO4, 5.6 mM D-glucose, 0.9 mM
CaCl2, and 0.5 mM MgCl2. After the culture medium was removed, each well was incubatedwith incubation medium for 5 min at 37 °C. The cells were then incubated with the
incubation medium containing the respective 14C-labeled compound (1 μM) for 5 min at
37 °C. At the end of the uptake period, each well was rapidly washed twice with ice-cold
incubation medium. The cells were then solubilized in 0.1 N NaOH, and the radioactivity ofeach aliquot was measured using a liquid scintillation counter (Tri-Carb 2910TR;
PerkinElmer) after addition of a scintillation cocktail (Ultima Gold; PerkinElmer). The
protein content of the cell lysate was determined by a bicinchoninic acid (BCA) assay,
which was performed using a BCA Protein Assay kit (Thermo Fisher Scientific). Data wereexpressed as the percentage of the control values, which were calculated as follows:
percentage of the control=(Vexposed/Vunexposed)×100, where Vexposed and Vunexposed are theuptake levels (pmol/mg protein) in exposed cells and unexposed cells, respectively.

Author Manuscript

Statistical Analysis
Experiments were repeated at least twice. Data have been presented as mean and standard
deviation values. All statistical analyses were performed with SAS software version 5.0(SAS Institute). Statistical significance between two groups was determined using the F test
followed by the Student’s t test for homoscedastic data or the Welch test for nonhomoscedastic data. P values less than 0.05 were considered statistically significant.

Results
Androgen Sensitivity of Human Prostate Cancer Cell Lines

Author Manuscript

To investigate the androgen sensitivity of AR-positive LNCaP and AR-negative DU145
cells, we evaluated the effect of DHT, the most potent gonadal androgen, on cellproliferation (Fig. 1). When LNCaP cells were exposed to 1 nM DHT, their numbers
increased by 45 % relative to that of unexposed cells. In contrast, DHT did not stimulate
proliferation in DU145 cells (cell count, 82 % of the control). Next, we evaluated whether
these effects occurred through an AR-mediated pathway (Fig. 1). Indeed, an AR antagonist,bicalutamide, blocked the effects of DHT on proliferation of LNCaP cells (cell count, 80 %of the control) but this effect was not observed in DU145 cells (cell count, 80 % of thecontrol).
Effect of Androgen on Expression of Amino Acid Transporter Genes

Author Manuscript

In order to determine whether androgen induces the expression of amino acid transporters
involved in anti[18F]FACBC transport, qRT-PCR was performed for nine amino acidtransporters. The mRNA expression of ASCT2, SNAT2, and 4F2hc was relatively high inboth LNCaP and DU145 cells (Supplemental Table 2). Among system L amino acidtransporters, LAT3 was more highly expressed than LAT1 in LNCaP cells, and the inverse
was observed in DU145 cells (Supplemental Table 2).
The observed levels of mRNAs encoding SNAT5 and broad-spectrum neutral (0) amino acidtransporter 1 (B0AT1; encoded by SLC6A19) were negligible when compared with themRNA levels of other transporters in both LNCaP and DU145 cells (Supplemental Table 2).

Mol Imaging Biol. Author manuscript; available in PMC 2017 October 19.

Okudaira et al.

Page 6

Author Manuscript

mRNAs of broad-spectrum neutral (0) amino acid transporter 2 (B0AT2; encoded by
SLC6A15) and amino acid transporter B0,+ (ATB0,+; encoded by SLC6A14) were notdetected in LNCaP cells but were detected at low levels in DU145 cells (Supplemental Table2). These results suggest that SNAT5, system B0 transporters, and ATB0,+ likely make small
contributions to anti-[18F]FACBC transport in prostate cancer cells.

Author Manuscript

Table 1 shows changes in gene expression relative to that in unexposed cells. The levels of
LAT3, one of the AR-regulated genes, were monitored as the positive control [19]. In
LNCaP cells, DHT exposure significantly increased the levels of mRNAs of ASCT2,
SNAT5, LAT3, and 4F2hc, and bicalutamide abolished the DHT-induced increase to thelevel observed in unexposed cells. DHT combined with bicalutamide significantly increased
the expression of LAT3 mRNA; however, compared to DHT alone, bicalutamide
significantly decreased the expression of LAT3 mRNA (P<0.05). DHT exposure also
increased the expression of LAT1 mRNA, but bicalutamide did not abolish this effect. On
the other hand, DHT exposure markedly decreased the expression of B0AT1 mRNA, which
was recovered by the addition of bicalutamide. In AR-negative DU145 cells, neither DHT
nor bicalutamide affected gene expression of the transporters tested.
Effect of Androgen on the Uptake of Amino Acids and PET Tracers
After investigating the increased expression of amino acid transporters, we investigated theeffect of androgen on amino acid uptake by measuring cellular uptake of 14C-labeled aminoacids (Fig. 2). Anti-[14C]FACBC uptake was the highest among all radiotracers tested in
unexposed LNCaP and DU145 cells (Table 2).

Author Manuscript

In LNCaP cells, DHT stimulated anti-[14C]FACBC uptake by 68 % relative to that in
unexposed cells, and the uptake was reduced to the control levels in the presence ofbicalutamide (124 % of the control). On the other hand, DHT or bicalutamide slightly
decreased the uptake of anti-[14C]FACBC in DU145 cells (66 % with DHT and 58 % with
DHT plus bicalutamide). A similar result was observed with [14C]Gln in LNCaP cells(182 % with DHT and 88 % with DHT plus bicalutamide) and DU145 cells (59 % with
DHT and 46 % with DHT plus bicalutamide). Similarly, DHT induced [14C]Met uptake in
LNCaP cells (119 % with DHT and 92 % with DHT plus bicalutamide); however, its uptake
was unchanged by DHT or bicalutamide in DU145 cells (105 % with DHT and 98 % with
DHT plus bicalutamide).

Author Manuscript

For comparison, we examined the uptake of 14C-labeled FDG, choline, and acetate (Fig. 3).
Similar to anti-[14C]FACBC, DHT stimulated the uptake of these tracers (176 % with
[14C]FDG, 138 % with [14C]choline, and 146 % with [14C]acetate), and bicalutamide
suppressed DHT’s effects (100 % with [14C]FDG, 91 % with [14C]choline, and 62 % with
[14C]acetate) in LNCaP cells. Furthermore, neither DHT nor bicalutamide affected theuptake of [14C]FDG (100 % with DHT and 92 % with DHT plus bicalutamide),[14C]choline (92 % with DHT and 91 % with DHT plus bicalutamide), and [14C]acetate(86 % with DHT and 86 % with DHT plus bicalutamide) in DU145 cells.

Mol Imaging Biol. Author manuscript; available in PMC 2017 October 19.

Okudaira et al.

Page 7

Author Manuscript

Discussion
In the present study, DHT stimulated the expression of amino acid transporters (ASCT2,
4F2hc, and LAT3) that are essential for cell proliferation and that mediate transport ofanti[18F]FACBC in LNCaP cells. DHT stimulated the subsequent uptake of anti[14C]FACBC. Because ASCT2 and system L transporters are upregulated in cancer cells andare thought to stimulate tumor growth by regulating the mammalian target of rapamycin
through nutrient pathways [13], accumulation of anti[18F]FACBC in prostate cancer cells
may be proportional to cell growth. Therefore, our data reaffirm the utility ofanti[18F]FACBC for prostate cancer imaging.

Author Manuscript
Author Manuscript

The effects of DHT combined with bicalutamide on anti[14C]FACBC uptake varied with thecell type (AR-positive LNCaP or AR-negative DU145), although DHT combined withbicalutamide inhibited the proliferation of both cells. One possible explanation for thisvariation is that the optimum effective concentration for bicalutamide could depend on theconditions or the cell type, which could result in one of the effects being slightly
predominant. Furthermore, with regard to inhibition of cell proliferation, bicalutamide might
inhibit the effect of marginal androgens present in the culture medium and within the
LNCaP cells. On the other hand, in DU145 cells, inhibition of proliferation by DHT was
similar to that achieved by DHT in combination with bicalutamide. This result is consistent
with a previous finding that DHT inhibited the growth of DU145 cells [24]. In addition,
inhibition of anti-[14C]FACBC uptake could be partly attributed to the change in theexpression of LAT1 and 4F2hc. In LNCaP cells, DHT combined with bicalutamide did notchange the amount of 4F2hc mRNA. However, it increased the amount of LAT1 mRNA.
This could be one of the reasons for the mild increase in anti-[14C]FACBC uptake. In
contrast, both treatments—DHT alone and DHT plus bicalutamide—tended to decrease
4F2hc mRNA levels in DU145 cells; however, the decrease was not significant.
Furthermore, while DHT tended to increase LAT1 mRNA levels, albeit nonsignificantly,
DHT plus bicalutamide did not change the mRNA levels. Although protein turnover could
also contribute to these phenomena, 4F2hc level would be a limiting factor for anti[14C]FACBC uptake by DU145 cells. In addition, 4F2hc is involved not only in amino acidtransport but also in integrin signaling, which is associated with tumor growth andmetastasis [25]. Thus, anti-[14C]FACBC uptake would decrease following reduction in the
4F2hc level in DU145 cells.

Author Manuscript

Figure 4 presents a schematic of the transport mechanism for anti-[18F]FACBC in prostatecancer cells whose cellular proliferation is stimulated by androgen. Anti-[18F]FACBC isneither metabolized nor incorporated into proteins [9]. Our present findings showed thattranscription of ASCT2, the transporter responsible for anti-[18F]FACBC transport [9, 10],was stimulated by DHT in LNCaP cells, as reported recently [23]. With respect to theexpression of system L transporters (LAT1 and LAT3), which may be involved in uptake ofanti-[18F]FACBC, LAT3 was also upregulated by DHT in LNCaP cells. However, theincrease in LAT1 expression by DHT exposure in LNCaP cells varied widely, andbicalutamide did not abolish DHT’s effects, which suggests the effect of DHT on LAT1
expression could be smaller than that on other transporters. The expression of 4F2hc, whichheterodimerizes with LAT1 to form a functional transporter complex, was enhanced by DHT

Mol Imaging Biol. Author manuscript; available in PMC 2017 October 19.

Okudaira et al.

Page 8

Author Manuscript

in LNCaP cells. Upregulation of these amino acid transporters could increase anti[18F]FACBC accumulation in AR-positive prostate cancer cells. However, since the affinityof LAT3 (millimolar range) [21] for its substrate is lower than that of LAT1 (micromolar
range) [11, 26], LAT3 may contribute less to anti-[18F]FACBC transport than LAT1.
Furthermore, ASCT2 was found to be more highly expressed than LAT1 in LNCaP cells,
whereas it was expressed at levels similar to that of LAT1 in DU145 cells (Supplemental
Table 2). In addition, the affinity of anti[14C]FACBC for ASCT2 is higher than that for
LAT1 [11]; therefore, ASCT2 could be a major transporter of anti[18F]FACBC in prostatecancer cells.

Author Manuscript

Although LAT1 and LAT3 transport Met, the mRNA levels of LAT1 and LAT3 were lower
than that of ASCT2 in LNCaP cells. The difference in the expression levels of amino acidtransporters may explain why DHT had a lesser effect on the uptake of [14C]Met than on
anti-[14C]FACBC. Interestingly, DHT’s effects on the proliferation of LNCaP and DU145
cells were similar to its effects on anti-[14C]FACBC uptake by both cell types. Thus, since
the accumulation of anti-[14C]FACBC better reflects androgen-induced cell proliferation
than does [14C]Met, anti[18F]FACBC might be more suitable than S-[methyl-11C]-Lmethionine for prostate cancer imaging.

Author Manuscript

In addition to androgen, some steroid hormones (estrogen and aldosterone) and epidermal
growth factor activate the expression of ASCT2 [27–29]. The ubiquitous transcription
factors, activating transcription factor 4 (ATF4) and c-MYC, also control the expression of
ASCT2 [23, 30]. Thus, since a wide variety of factors involved in cancer cell proliferationinduce ASCT2 expression and because upregulation of ASCT2 has been observed in several
tumors [31], increased accumulation of anti-[18F]FACBC might also occur in other cancer
tissues as well as in prostate cancer.

Author Manuscript

Regarding the representative PET tracers for prostate cancer, DHT stimulated [14C]FDG
uptake similar to that of anti[14C]FACBC. However, [18F]FDG PET/CT imaging has limited
efficacy because [18F]FDG is internalized by both prostate cancer cells and benign prostatic
tissue, such as benign prostatic hyperplasia. Moreover, prostate cancer cells have a relatively
lower metabolic rate and lower expression of glucose transporter than the cells of othercancers, suggesting relatively low [18F]FDG uptake by prostate cancer cells [32]. On theother hand, DHT stimulated [14C]choline and [14C]acetate uptake to slightly lower degrees
than it did the uptake of anti-[14C]FACBC. Furthermore, anti-[14C]FACBC uptake washigher than that of [14C]choline or [14C]acetate. These findings suggest that anti[18F]FACBC more accurately detects prostate cancer cells stimulated by androgen than doradiolabeled choline and acetate. Indeed, a recent study reported that anti-[18F]FACBC may
better identify prostate cancer recurrence than [11C]choline might [33]. Thus, these data
Androgen-deprivation therapy (usually a combination of orchiectomy, luteinizing hormonereleasing hormone analogs, and anti-androgens) is often the first choice among several
therapeutic options for advanced prostate cancer. However, despite an initial response to
androgen-deprivation therapy, most prostate cancers eventually become resistant to therapy
within several years, which has been termed CRPC. Nevertheless, the AR plays a role in the
progression of prostate cancer toward castration resistance, where AR signaling remains

Mol Imaging Biol. Author manuscript; available in PMC 2017 October 19.

Okudaira et al.

Page 9Author Manuscript

intact [15]. Numerous factors contribute to AR reactivation despite castrate serum levels of
androgens, including adrenal and intratumoral androgen synthesis, AR gene amplification,
AR gene mutations, increased expression of AR coactivators, ligand-independent AR splicevariants, and epigenetic modifications [34, 35]. Since AR signaling plays an important rolein cell proliferation in both androgen-dependent prostate cancer and CRPC,
anti[18F]FACBC, whose uptake is stimulated by androgen, might be useful for the diagnosisof recurrent prostate cancer or for therapy evaluation. Our future studies will investigate
anti-FACBC transport in CRPC cells to test this hypothesis.

Conclusions

Author Manuscript

DHT induced ASCT2 gene expression and enhanced the cellular uptake of anti[14C]FACBC in AR-positive LNCaP prostate cancer cells but not in AR-negative DU145
cells. Given the role of androgen in prostate cancer cell proliferation, anti-[18F]FACBC may
be a valuable PET tracer for the diagnosis of prostate cancer.

Supplementary Material
Refer to Web version on PubMed Central for supplementary material.

Acknowledgments
We thank Ms. Sachiko Naito for her excellent technical assistance. We also thank Dr. Atsushi Mizokami for his
helpful suggestions and comments regarding these experiments.

References
Author Manuscript
Author Manuscript

1. Jemal A, Bray F, Center MM, Ferlay J, Ward E, Forman D. Global cancer statistics. CA Cancer J
Clin. 2011; 61:69–90. [PubMed: 21296855]
2. Jadvar H. Prostate cancer: PET with 18F-FDG, 18F- or 11C-acetate, and 18F- or 11C-choline. J Nucl
Med. 2011; 52:81–89. [PubMed: 21149473]
3. Jadvar H, Desai B, Ji L, et al. Baseline 18F-FDG PET/CT parameters as imaging biomarkers ofoverall survival in castrate-resistant metastatic prostate cancer. J Nucl Med. 2013; 54:1195–1201.
[PubMed: 23785174]
4. Jadvar H. Molecular imaging of prostate cancer with PET. J Nucl Med. 2013; 54:1685–1688.
[PubMed: 24084704]
5. Schuster DM, Votaw JR, Nieh PT, et al. Initial experience with the radiotracer anti-1-amino-3-18Ffluorocyclobutane-1-carboxylic acid with PET/CT in prostate carcinoma. J Nucl Med. 2007; 48:56–
63. [PubMed: 17204699]
6. Schuster DM, Savir-Baruch B, Nieh PT, et al. Detection of recurrent prostate carcinoma with anti-1amino-3-18F-fluorocyclobutane-1-carboxylic acid PET/CT and 111In-capromab pendetide
SPECT/CT. Radiology. 2011; 259:852–861. [PubMed: 21493787]
7. Schuster DM, Nieh PT, Jani AB, et al. Anti-3-[18F]FACBC positron emission tomographycomputerized tomography and 111In-capromab pendetide single photon emission computerized
tomography-computerized tomography for recurrent prostate carcinoma: results of a prospective
clinical trial. J Urol. 2014; 191:1446–1453. [PubMed: 24144687]
8. Asano Y, Inoue Y, Ikeda Y, et al. Phase I clinical study of NMK36: a new PET tracer with thesynthetic amino acid analogue anti[18F]FACBC. Ann Nucl Med. 2011; 25:414–418. [PubMed:
21409348]

Mol Imaging Biol. Author manuscript; available in PMC 2017 October 19.

Okudaira et al.

Page 10

Author Manuscript
Author Manuscript
Author Manuscript
Author Manuscript

9. Okudaira H, Shikano N, Nishii R, et al. Putative transport mechanism and intracellular fate oftrans-1-amino-3-18F-fluorocyclobutanecarboxylic acid in human prostate cancer. J Nucl Med. 2011;52:822–829. [PubMed: 21536930]
10. Oka S, Okudaira H, Yoshida Y, Schuster DM, Goodman MM, Shirakami Y. Transport mechanismsof trans-1-amino-3-fluoro[1-14C]cyclobutanecarboxylic acid in prostate cancer cells. Nucl Med
Biol. 2012; 39:109–119. [PubMed: 21958853]
11. Okudaira H, Nakanishi T, Oka S, et al. Kinetic analyses of trans-1-amino-3-[18F]
fluorocyclobutanecarboxylic acid transport in Xenopus laevis oocytes expressing human ASCT2and SNAT2. Nucl Med Biol. 2013; 40:670–675. [PubMed: 23647854]
12. Ganapathy V, Thangaraju M, Prasad PD. Nutrient transporters in cancer: relevance to Warburg
hypothesis and beyond. Pharmacol Ther. 2009; 121:29–40. [PubMed: 18992769]
13. Nakanishi T, Tamai I. Solute carrier transporters as targets for drug delivery and pharmacologicalintervention for chemotherapy. J Pharm Sci. 2011; 100:3731–3750. [PubMed: 21630275]
14. Feldman BJ, Feldman D. The development of androgen-independent prostate cancer. Nat Rev
Cancer. 2001; 1:34–45. [PubMed: 11900250]
15. Taplin ME, Balk SP. Androgen receptor: a key molecule in the progression of prostate cancer tohormone independence. J Cell Biochem. 2004; 91:483–490. [PubMed: 14755679]
16. Dutt SS, Gao AC. Molecular mechanisms of castration-resistant prostate cancer progression.
Future Oncol. 2009; 5:1403–1413. [PubMed: 19903068]
17. Arakawa H, Nakanishi T, Yanagihara C, et al. Enhanced 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. Biochem Pharmacol. 2012;
84:1070–1077. [PubMed: 22864060]
18. Trapman J, Cleutjens KB. Androgen-regulated gene expression in prostate cancer. Semin Cancer
Biol. 1997; 8:29–36. [PubMed: 9299579]
19. Wang Q, Bailey CG, Ng C, et al. Androgen receptor and nutrient signaling pathways coordinate thedemand for increased amino acid transport during prostate cancer progression. Cancer Res. 2011;
71:7525–7536. [PubMed: 22007000]
20. Chuaqui RF, Englert CR, Strup SE, et al. Identification of a novel transcript up-regulated in aclinically aggressive prostate carcinoma. Urology. 1997; 50:302–307. [PubMed: 9255310]
21. Babu E, Kanai Y, Chairoungdua A, et al. Identification of a novel system L amino acid transporter
structurally distinct from heterodimeric amino acid transporters. J Biol Chem. 2003; 278:43838–
43845. [PubMed: 12930836]
22. Chauvin TR, Griswold MD. Androgen-regulated genes in the murine epididymis. Biol Reprod.
2004; 71:560–569. [PubMed: 15115731]
23. Wang Q, Tiffen J, Bailey CG, et al. Targeting amino acid transport in metastatic castration-resistant
prostate cancer: effects on cell cycle, cell growth, and tumor development. J Natl Cancer Inst.
2013; 105:1463–1473. [PubMed: 24052624]
24. Janssen T, Raviv G, Camby I, et al. In-vitro characterization of dihydrotestosterone-induced,
epidermal growth factor-induced and basic fibroblastic growth factor-induced modifications in thegrowth dynamics of the human prostate-cancer cell-line LNCaP, DU145 and PC3. Int J Oncol.
1995; 7:1219–1225. [PubMed: 21552954]
25. Cantor JM, Ginsberg MH. CD98 at the crossroads of adaptive immunity and cancer. J Cell Sci.
2012; 125:1373–1382. [PubMed: 22499670]
26. Yanagida O, Kanai Y, Chairoungdua A, et al. Human L-type amino acid transporter 1 (LAT1):
characterization of function and expression in tumor cell lines. Biochim Biophys Acta. 2001;
1514:291–302. [PubMed: 11557028]
27. Todorova VK, Kaufmann Y, Luo S, Klimberg VS. Tamoxifen and raloxifene suppress theproliferation of estrogen receptor-negative cells through inhibition of glutamine uptake. Cancer
Chemother Pharmacol. 2011; 67:285–291. [PubMed: 20383709]
28. Amaral JS, Pinho MJ, Soares-da-Silva P. Genomic regulation of intestinal amino acid transporters
by aldosterone. Mol Cell Biochem. 2008; 313:1–10. [PubMed: 18347756]

Mol Imaging Biol. Author manuscript; available in PMC 2017 October 19.

Okudaira et al.

Page 11

Author Manuscript
Author Manuscript

29. Wolfgang CL, Lin C, Meng Q, Karinch AM, Vary TC, Pan M. Epidermal growth factor activationof intestinal glutamine transport is mediated by mitogen-activated protein kinases. J Gastrointest
Surg. 2003; 7:149–156. [PubMed: 12559196]
30. Wise DR, DeBerardinis RJ, Mancuso A, et al. Myc regulates a transcriptional program that
stimulates mitochondrial glutaminolysis and leads to glutamine addiction. Proc Natl Acad Sci
USA. 2008; 105:18782–18787. [PubMed: 19033189]
31. Fuchs BC, Bode BP. Amino acid transporters ASCT2 and LAT1 in cancer: partners in crime?
Semin Cancer Biol. 2005; 15:254–266. [PubMed: 15916903]
32. Schwarzenböck S, Souvatzoglou M, Krause BJ. Choline PET and PET/CT in primary diagnosis
and staging of prostate cancer. Theranostics. 2012; 2:318–330. [PubMed: 22448198]
33. Nanni C, Schiavina R, Boschi S, et al. Comparison of 18F-FACBC and 11C-choline PET/CT inpatients with radically treated prostate cancer and biochemical relapse: preliminary results. Eur J
Nucl Med Mol Imaging. 2013; 40:S11–S17. [PubMed: 23591953]
34. Galsky MD, Small AC, Tsao CK, Oh WK. Clinical development of novel therapeutics for
castration-resistant prostate cancer: historic challenges and recent successes. CA Cancer J Clin.
2012; 62:299–308. [PubMed: 22535487]
35. Shafi AA, Yen AE, Weigel NL. Androgen receptors in hormone-dependent and castration-resistant
prostate cancer. Pharmacol Ther. 2013; 140:223–238. [PubMed: 23859952]

Author Manuscript
Author Manuscript
Mol Imaging Biol. Author manuscript; available in PMC 2017 October 19.

Okudaira et al.

Page 12

Author Manuscript
Author Manuscript

Fig. 1.

Quantification of LNCaP and DU145 cells exposed to DHT with or without bicalutamide.
Each bar represents the mean and standard deviation (n=6). *P<0.05, **P<0.01.

Author Manuscript
Author Manuscript
Mol Imaging Biol. Author manuscript; available in PMC 2017 October 19.

Okudaira et al.

Page 13

Author Manuscript
Author Manuscript

Fig. 2.

Uptake of anti-[14C]FACBC, [14C]Gln, and [14C]Met by LNCaP and DU145 cells exposed
to DHT with or without bicalutamide. Each bar represents the mean and standard deviation(n=6–12). *P<0.05, **P<0.01.

Author Manuscript
Author Manuscript
Mol Imaging Biol. Author manuscript; available in PMC 2017 October 19.

Okudaira et al.

Page 14

Author Manuscript
Author Manuscript

Fig. 3.

Uptake of [14C]FDG, [14C]choline, and [14C]acetate by LNCaP and DU145 cells exposed to
DHT with or without bicalutamide. Each bar represents the mean and standard deviation(n=6–9). *P<0.05, **P<0.01.

Author Manuscript
Author Manuscript
Mol Imaging Biol. Author manuscript; available in PMC 2017 October 19.

Okudaira et al.

Page 15

Author Manuscript
Fig. 4.

Author Manuscript

Schematic representation of the transport mechanism of anti-[18F]FACBC in AR-positive
prostate cancer cells. demonstrate the usefulness of anti-[18F]FACBC relative to [18F]FDG,[18F] and [11C]choline, and [18F] and [11C]acetate for prostate cancer imaging.

Author Manuscript
Author Manuscript
Mol Imaging Biol. Author manuscript; available in PMC 2017 October 19.

Author Manuscript

Author Manuscript

Author Manuscript

Mol Imaging Biol. Author manuscript; available in PMC 2017 October 19.

ATB0,+

B0AT2

B0AT1

LAT3

4F2hc

LAT1

SNAT5

SNAT2

−
+

+

+

+

+
−

−

−

−

+

+

+

−

−

+

+

+
−

−

−

−

+

+

+

−

−

+

+

+
−

−

−

−

+

+

+

−

−

+

+

+
−

−

+

−

−

−

+

−

+

+

−

−

ASCT2

Bic

DHT

Transporters

Exposure

Not detected

Not detected

92.8±44.923.1±5.6

100.0±29.7

147.0±65.8

201.7±65.5

100.0±25.9

99.5±23.2

128.5±26.3

100.0±24.4

149.5±87.3

157.6±55.4

100.0±34.6

79.3±48.0

210.5±74.2

100.0±30.5

91.2±31.9

112.8±30.2

100.0±20.0

107.1±28.0

197.4±73.5

100.0±28.2

LNCaP cells

NS

<0.01

<0.01

<0.01

NS

<0.01

<0.05

<0.01

NS

<0.01

NS

NS

NS

<0.01

P

102.5±113.8

144.3±95.8

100.0±63.0

77.3±44.2

98.8±36.3

100.0±27.0

102.6±73.7

158.6±156.2

100.0±53.0

112.4±35.0

112.3±57.3

100.0±54.8

89.9±21.7

94.9±28.9

100.0±15.8

103.6±62.4

128.5±91.1

100.0±37.7

101.2±56.2

85.5±42.6

100.0±77.6

123.0±40.8

117.5±32.9

100.0±35.3

104.0±47.1

119.0±51.5

100.0±48.8

DU145 cells

mRNA expression (% of the control)

NS

NS

NS

NS

NS

NS

NS

NS

NS

NS

NS

NS

NS

NS

NS

NS

NS

NS

P

Relative amino acid transporter gene expression in LNCaP and DU145 cells exposed to DHT with or without bicalutamide

Author Manuscript

Table 1
Okudaira et al.
Page 16

Author Manuscript

Author Manuscript
Data are expressed as mean ± standard deviation (n=6)

Bic bicalutamide, NS not statistically significant

Statistical significance was tested by comparing exposed cells and unexposed cells

Okudaira et al.
Page 17

Author Manuscript

Author Manuscript

Mol Imaging Biol. Author manuscript; available in PMC 2017 October 19.

Okudaira et al.

Page 18

Table 2

Author Manuscript

Radiotracer uptake in LNCaP and DU145 cells that were not exposed to DHT or bicalutamide
Uptake amount (pmol/mg of protein)
Radiotracers

LNCaP cells

DU145 cells

anti-[14C]FACBC

105.9±15.7110.8±14.5[14C]Gln

88.6±14.959.0±6.2[14C]Met

23.0±1.656.7±10.8[14C]FDG

2.8±0.7

1.9±0.5[14C]choline

45.8±12.4

15.6±2.8[14C]acetate14.1±2.4

20.8±3.8

Data are expressed as mean ± standard deviation (n=6–12)

Author Manuscript

Note that the uptake amount of [14C]FDG could not be compared with that of other radiotracers because the incubation medium used in this study
contained D-glucose

Author Manuscript
Author Manuscript
Mol Imaging Biol. Author manuscript; available in PMC 2017 October 19.