Putative transport mechanism and intracellular fate of trans-1-amino-3-18F-fluorocyclobutanecarboxylic acid in human prostate cancer.

Abstract

Trans-1-amino-3-(18)F-fluorocyclobutanecarboxylic acid (anti-(18)F-FACBC) is an amino acid PET tracer that has shown promise for visualizing prostate cancer. Therefore, we aimed to clarify the anti-(18)F-FACBC transport mechanism in prostate cancer cells. We also studied the fate of anti-(18)F-FACBC after it is transported into cells.
For convenience, because of their longer half-lives, (14)C compounds were used instead of (18)F-labeled tracers. Trans-1-amino-3-fluoro-1-(14)C-cyclobutanecarboxylic acid ((14)C-FACBC) uptake was examined in human prostate cancer DU145 cells with the following substrates of amino acid transporters: α-(methylamino) isobutyric acid (a system A-specific substrate) and 2-amino-2-norbornanecarboxylic acid (a system L-specific substrate). The messenger RNA expression of amino acid transporters in human prostate cancer specimens was analyzed by complementary DNA microarray and quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR). Gene expression in DU145 cells was analyzed by qRT-PCR. We also examined the knockdown effect of the amino acid transporters system ASC transporter 2 (ASCT2) and sodium-coupled neutral amino acid transporter 2 (SNAT2) on (14)C-FACBC uptake. In addition, the possibility of (14)C-FACBC incorporation into proteins was examined.
(14)C-FACBC uptake by DU145 cells was markedly decreased to approximately 20% in the absence of Na(+), compared with that in its presence, indicating that Na(+)-dependent transporters are mainly responsible for the uptake of this tracer. Moreover, 2-amino-2-norbornanecarboxylic acid inhibited the transport of (14)C-FACBC to the basal level in Na(+)-free buffer. In contrast, α-(methylamino) isobutyric acid did not inhibit (14)C-FACBC accumulation in DU145 cells. Human prostate tumor specimens and DU145 cells had similar messenger RNA expression patterns of amino acid transporter genes. Although SNAT2 and ASCT2 are 2 major amino acid transporters expressed in prostate tumor tissues and DU145 cells, ASCT2 knockdown using small interfering RNA was more effective in lowering (14)C-FACBC transport than SNAT2. Almost all intracellular (14)C-FACBC was recovered from the nonprotein fraction.
ASCT2, which is a Na(+)-dependent amino acid transporter, and to a lesser extent Na(+)-independent transporters play a role in the uptake of (14)C-FACBC by DU145 cells. Among the Na(+)-independent transporters, system L transporters are also involved in the transport of (14)C-FACBC. Moreover, (14)C-FACBC is not incorporated into proteins in cells. These findings suggest a possible mechanism of anti-(18)F-FACBC PET for prostate cancer.

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Putative Transport Mechanism and Intracellular Fate
of Trans-1-Amino-3-18F-Fluorocyclobutanecarboxylic Acid in
Human Prostate Cancer
Hiroyuki Okudaira1,2, Naoto Shikano3, Ryuichi Nishii4, Tohru Miyagi5, Mitsuyoshi Yoshimoto6, Masato Kobayashi1,
Kazuyo Ohe1, Takeo Nakanishi7, Ikumi Tamai7, Mikio Namiki5, and Keiichi Kawai1,8
1Division of Health Sciences, Graduate School of Medical Science, Kanazawa University, Ishikawa, Japan;2Research Center, Nihon
Medi-Physics Co., Ltd., Chiba, Japan;3Department of Radiological Sciences, Ibaraki Prefectural University of Health Sciences,
Ibaraki, Japan;4Research Institute, Shiga Medical Center, Shiga, Japan;5Department of Integrative Cancer Therapy and Urology,
Graduate School of Medical Science, Kanazawa University, Ishikawa, Japan;6Research Institute, National Cancer Center, Tokyo,
Japan;7Department of Membrane Transport and Biopharmaceutics, School of Pharmaceutical Sciences, Kanazawa University,
Ishikawa, Japan; and8Biomedical Imaging Research Center, University of Fukui, Fukui, Japan
Trans-1-amino-3-18F-fluorocyclobutanecarboxylic acid (anti–18F-
FACBC) is an amino acid PET tracer that has shown promise for
visualizing prostate cancer. Therefore, we aimed to clarify the
anti–18F-FACBC transport mechanism in prostate cancer cells.
We also studied the fate of anti–18F-FACBC after it is trans-
ported into cells. Methods: For convenience, because of
their longer half-lives,14C compounds were used instead of
18F-labeled tracers. Trans-1-amino-3-fluoro-1-14C-cyclobutane-
carboxylic acid (14C-FACBC) uptake was examined in human
prostate cancer DU145 cells with the following substrates of
amino acid transporters: a-(methylamino) isobutyric acid (a sys-
tem A–specific substrate) and 2-amino-2-norbornanecarboxylic
acid (a system L–specific substrate). The messenger RNA
expression of amino acid transporters in human prostate cancer
specimens was analyzed by complementary DNA microarray
and quantitative real-time reverse transcription polymerase
chain reaction (qRT-PCR). Gene expression in DU145 cells
was analyzed by qRT-PCR. We also examined the knockdown
effect of the amino acid transporters system ASC transporter
2 (ASCT2) and sodium-coupled neutral amino acid transporter
2 (SNAT2) on14C-FACBC uptake. In addition, the possibility of
14C-FACBC incorporation into proteins was examined. Results:
14C-FACBC uptake by DU145 cells was markedly decreased to
approximately 20% in the absence of Na1, compared with that
in its presence, indicating that Na1-dependent transporters are
mainly responsible for the uptake of this tracer. Moreover,
2-amino-2-norbornanecarboxylic acid inhibited the transport
of14C-FACBC to the basal level in Na1-free buffer. In contrast,
a-(methylamino) isobutyric acid did not inhibit
accumulation in DU145 cells. Human prostate tumor specimens
and DU145 cells had similar messenger RNA expression
patterns of amino acid transporter genes. Although SNAT2
and ASCT2 are 2 major amino acid transporters expressed
in prostate tumor tissues and DU145 cells, ASCT2 knockdown
using small interfering RNA was more effective in lowering
14C-FACBC
14C-FACBC transport than SNAT2. Almost all intracellular
14C-FACBC was recovered from the nonprotein fraction.
Conclusion: ASCT2, which is a Na1-dependent amino acid
transporter, and to a lesser extent Na1-independent transport-
ers play a role in the uptake of14C-FACBC by DU145 cells.
Among the Na1-independent transporters, system L transport-
ers are also involved in the transport of14C-FACBC. Moreover,
14C-FACBC is not incorporated into proteins in cells. These
findings suggest a possible mechanism of anti–18F-FACBC
PET for prostate cancer.
Key
transporter; ASCT2
Words:
anti-FACBC;prostate cancer;aminoacid
2011; 52:822–829
DOI: 10.2967/jnumed.110.086074
Prostate cancer is one of the most common malignant
neoplasms among men, and its incidence is increasing
worldwide (1). In Japan, because the age-standardized
prostate cancer incidence rate has been rapidly increasing
since 1998 (2), there is an urgent demand for accurate
prostate cancer diagnosis leading to an appropriate treat-
ment strategy. However, the current methodology does not
allow precise identification of the site of the disease within
the prostate.
18F-FDG is most commonly used for whole-body PET
but does not work well for certain tumor tissues, such as
brain tumors and tumors located in the pelvic region,
because of its physiologic accumulation and elimination
(3). In addition to18F-FDG, radiolabeled amino acids have
been studied as potential tumor-seeking PET agents for
clinical use (4). The most frequently used amino acid
PET tracer is11C-methionine because of its easy and fast
radiosynthesis (5). The availability and clinical efficacy of
11C-methionine in patients with brain tumors (4) and pros-
tate cancers (6) have been investigated. Besides PET with
radiolabeled natural amino acids, PETwith synthetic amino
Received Nov. 30, 2010; revision accepted Jan. 31, 2011.
For correspondence or reprints contact: Keiichi Kawai, Graduate School of
Medical Science, Kanazawa University, 5-11-80 Kodatsuno, Kanazawa,
Ishikawa 920-0942, Japan.
E-mail: kei@mhs.mp.kanazawa-u.ac.jp
COPYRIGHT ª 2011 by the Society of Nuclear Medicine, Inc.
822THE JOURNAL OF NUCLEAR MEDICINE • Vol. 52 • No. 5 • May 2011

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acids labeled with11C, such as 1-11C-aminocyclobutanecar-
boxylic acid (11C-ACBC) (7), 1-11C-aminocyclopentane-
carboxylic acid (7), and a-11C-aminoisobutyric acid (8),
exhibit high tumor–to–nontumor concentration ratios.
Among these studies, Washburn et al. (7) show that11C-
ACBC is the most selective radioprobe for tumor imaging
in an animal model. In a clinical study,11C-ACBC is shown
to be a good diagnostic probe specific for astrocytoma,
suggesting that it can be taken up by such tumors, whereas
it is negligibly taken up by normal brain tissue (9). How-
ever, because of the short half-life of11C, the availability of
11C-labeled tracers is limited to facilities equipped with an
in-house cyclotron, thus preventing the widespread use of
these compounds. Therefore,18F-labeled amino acids are
more attractive as PET agents and are widely used in many
facilities.
To improve the availability of11C-ACBC, Shoup et al.
(10) developed trans-1-amino-3-18F-fluorocyclobutanecar-
boxylic acid (anti–18F-FACBC). Anti–18F-FACBC is a syn-
thetic L-leucine analog that exhibits high tumor-specific
accumulation in patients with glioblastoma multiforme
(10), renal papillary cell cancer (11), and prostate cancer
(12). One of the interesting features of anti–18F-FACBC is
that its renal excretion is much slower than that of18F-FDG
(13); this slower excretion could be helpful for prostate
cancer imaging.
Little is known about the transport mechanism of
anti–18F-FACBC into cells, although anti–18F-FACBC is
thought to cross the plasma membrane via amino acid trans-
porters because of its structural similarity to natural amino
acids. Because of the identification and kinetic studies of
amino acid transporters, these transporters are categorized
into at least 17 distinct classes (14). Neutral amino acids
are considered to be mainly transported by 3 systems: A,
ASC, and L (15). Systems A and ASC mainly serve to take
up amino acids with short, polar, or linear side chains such as
L-alanine and L-serine. In contrast, large, branched, and aro-
matic amino acids such as L-tyrosine mainly enter cells via
system L (16). Although several transporters may be involved
in anti–18F-FACBC uptake, previous studies show that the
uptake of anti–18F-FACBC is mediated by the Na1-independ-
ent system L amino acid transporter in rat 9L gliosarcoma cells
(17,18). However, the mechanism of anti–18F-FACBC uptake
by prostate cancer cells is not well understood. Furthermore,
the gene expression of amino acid transporters in prostate
cancer cells has not been fully clarified.
In the present study, we characterized anti–18F-FACBC
transport in human prostate cancer cells using a cell uptake
assay to find candidate transporters. The gene expression
profile of amino acid transporters was examined by comple-
mentary DNA (cDNA) microarray in human prostate biopsy
specimens. Furthermore, we confirmed the gene expression
of amino acid transporters in the human prostate cancer
tissues and DU145 cells by quantitative real-time reverse
transcription polymerase chain reaction (qRT-PCR). In addi-
tion, we examined the role of Na1-dependent amino acid
transporters in anti–18F-FACBC uptake, using the small
interfering RNA (siRNA) transfection method, and the intra-
cellular fate of anti–18F-FACBC after its uptake.
MATERIALS AND METHODS
Materials
For convenience, because of their longer half-lives (5,700 y),
14C-labeled amino acids and analogs were used rather than18F
(half-life, 110 min) or11C (half-life, 20 min). Trans-1-amino-
3-fluoro-1-14C-cyclobutanecarboxylic acid (14C-FACBC; 2.08
GBq/mmol) was radiosynthesized by EaglePicher Pharmaceutical
Services. L-1-14C-methionine (14C-Met, 2.04 GBq/mmol) was
purchased from American Radiolabeled Chemicals. Both
L-14C (U)-alanine (14C-Ala; 5.92 or 6.29 GBq/mmol) and
L-14C (U)-leucine (14C-Leu; 11.99 GBq/mmol) were pur-
chased from both Moravek Biochemicals and PerkinElmer.
The DU145 human prostate cancer cell line was obtained from
the American Type Culture Collection.
Collection of Human Biopsy Specimens
Human prostate biopsy specimens (Table 1) for cDNA micro-
array and qRT-PCR were obtained by needle biopsy from 6
patients with elevated prostate-specific antigen levels at Kanazawa
University Hospital from November 2007 to February 2008. All
patients involved in this study were informed of a complete guar-
antee of confidentiality of their individual records, our ethically
acceptable experiment, and the right to refuse or withdraw. The
biopsy specimens used in this study were obtained from these
patients with their informed consent. The specimens used for
pathologic diagnosis were reviewed by pathologists; the speci-
mens for gene expression analysis were immediately stored in
RNAlater (Ambion) at 4?C and subsequently stored at 280?C
until required. Patients were designated prostate cancer–free if
cancer cells were not detected in their biopsy specimens.
Culture of Human Cell Lines
DU145 cells were cultured in Dulbecco’s modified Eagle’s
medium (Life Technologies) supplemented with 10% fetal bovine
serum (American Type Culture Collection), 100 units of penicillin
per milliliter, and 100 mg of streptomycin per milliliter (Life
Technologies). The cells were cultured at 37?C in 5% CO2/95%
air; these cells were used for the messenger RNA (mRNA) quan-
titation and uptake assay.
TABLE 1
List of Human Prostate Biopsy Specimens
Patient no.
Prostate-specific
antigen (ng/mL) Gleason score
Normal prostate
1
2
3
Adenocarcinoma
4
5
6
3.6
5.1
9.0
—
—
—
55.0
96.0
227.8
4 1 4 5 8
4 1 4 5 8
4 1 4 5 8
TRANSPORT AND FATE OF ANTI–18F-FACBC • Okudaira et al.823

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RNA Extraction
Total RNA was extracted from human prostate biopsy speci-
mens and DU145 cells using the RNeasy Micro and RNeasy Plus
Mini Kits (Qiagen), respectively.
cDNA Microarray Analysis
All reagents, equipment, and software were purchased from
Agilent Technologies. cDNA microarray analysis was performed
according to the manufacturer’s protocol. In brief, total RNA was
subjected to linear amplification and Cy3 labeling using the Low
RNA Input Linear Amplification Kit and a One Color RNA Spike-
In Kit and was subsequently hybridized to a 44-K Whole Human
Genome Microarray using a Gene Expression Hybridization Kit.
Data were extracted using Feature Extraction software (version
9.5.3) and were analyzed using GeneSpring GX software (version
11.0).
qRT-PCR
cDNA was synthesized from 250 ng of total RNA of human
biopsy samples using the WT-Ovation RNA Amplification System
(NuGEN Technologies) according to the manufacturer’s protocol.
Total RNA (1 mg) from DU145 cells was reverse-transcribed using
a Transcriptor First Strand cDNA Synthesis Kit (Roche Applied
Science) by random hexamer priming at 50?C for 1 h according to
the manufacturer’s instructions. The primers (Supplemental Table
1; supplemental materials are available online only at http://jnm.
snmjournals.org) were designed using the Universal ProbeLibrary
Assay Design Center (Roche Applied Science) and were synthe-
sized by Nihon Gene Research Laboratories. qt-PCR was per-
formed using an Mx3005P or Mx3000P QPCR system (Agilent
Technologies), FastStart Universal Probe Master (ROX), and the
Universal ProbeLibrary (Roche Applied Science) with the follow-
ing profile: 1 cycle of enzyme activation at 95?C for 10 min, 40
cycles of denaturing 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 no nonspecific PCR
bands were detected. The PCR products for each gene were sub-
sequently purified using a High Pure PCR Cleanup Micro Kit
(Roche Applied Science) and quantified from the concentration
and base pair numbers of amplicons. The mRNA copy number
was calculated from standard curves generated by amplifying
serial dilutions of a known quantity of purified amplicons. Expres-
sion data were normalized against the copy number of 18S ribo-
somal RNA.
The correlation coefficient of the log ratio between the results
of the cDNA microarray and qRT-PCR was calculated as follows:
log2(mRNA expression in cancer specimens/mRNA expression in
normal specimens).
Measurement of14C-Labeled Amino Acid Transport
Transport assays were based on the methods described by
Shikano et al. (19,20). In brief, DU145 cells were seeded on 24-
well tissue culture plates at 1 · 105cells per well with 1 mL of
culture medium. Transport assays were then conducted on the next
day after inoculation (semiconfluent phase cells).
The sodium-containing incubation medium used was based
on phosphate-buffered saline (pH 7.4–7.6), consisting of 137 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. In the sodium-free
incubation medium (Na1-free buffer), NaCl and Na2HPO4were
replaced with the equivalent concentrations of choline chloride
and K2HPO4, respectively. After the culture medium was re-
moved, each well was incubated with 1 mL of incubation medium
for 10 min at 37?C. The cells were then incubated with 0.3 mL of
incubation medium containing the respective14C-labeled amino
acid (10 mM) for 10 min at 37?C. At the end of uptake, each well
was rapidly washed twice with 1 mL of ice-cold incubation
medium. The cells were then solubilized in 0.5 mL of 0.1N NaOH,
and the radioactivity of each aliquot was measured using a liquid
scintillation counter (LS 6000SC; Beckman Coulter) after scintil-
lation cocktail (Ultima Gold; PerkinElmer) was added. The pro-
tein content of the cell lysate was determined by assay with a BCA
Protein Assay kit (Thermo Fisher Scientific).
For the competitive inhibition assay, the following compounds
were used at a final concentration of 1 mM: a-(methylamino)
isobutyric acid (MeAIB—a substrate specific to system A trans-
porters), 2-amino-2-norbornanecarboxylic acid (BCH—a substrate
specific to system L transporters), p-aminohippurate (PAH—a
substrate specific to organic anion transporters), and tetraethylam-
monium chloride (TEA—a substrate specific to organic cation
transporters). Basal transport activity was defined by the results
of these assays performed on ice.
All experimental conditions were examined in triplicate.
Reproducibility was confirmed by repeating the same experiment.
siRNA Transfection
All siRNAs used in this study were purchased from Ambion. We
used 2 different kinds of siRNAs for each target gene; each
transfection was performed using only one of them. The identi-
fication numbers of siRNAs used are as follows: s12916 and s12918
for system ASC transporter 2 (ASCT2) and s633 and s634 for
sodium-coupled neutral amino acid transporter 2 (SNAT2). Silencer
Select Negative Control 1 and 2 siRNA (catalog nos. 4390843 and
4390846) were used as controls. siRNA transfection was performed
according to the manufacturer’s instructions. DU145 cells were
transfected with 5 nM of each siRNA in antibiotic-free growth
medium using Lipofectamine 2000 (Life Technologies). After
transfection (48 h), the cells were used for amino acid uptake
experiments. The efficiency and specificity of each siRNA were
confirmed by qRT-PCR. We rated the result as the target gene–
specific effect when both siRNAs for the same target gene signifi-
cantly altered the transport of14C-labeled amino acids.
Measurement of14C-Labeled Amino Acid
Incorporation into Proteins
DU145 cells were incubated with 50 mM14C-FACBC at 37?C
for 3 or 24 h. The cells were then washed twice with phosphate-
buffered saline and lysed in CelLytic M cell lysis reagent (Sigma-
Aldrich) containing protease inhibitor cocktail (Sigma-Aldrich).
Cell lysates were centrifuged at 18,000g for 15 min at 4?C, and
the supernatants were subjected to trichloroacetic acid (TCA) pre-
cipitation. The precipitation reaction was performed for 10 min on
ice by adding ice-cold TCA (final concentration, 10%) to the
supernatants, and the TCA precipitates were washed 3 times with
ice-cold 5% TCA. The TCA precipitates and cell debris were
lysed in 0.1N NaOH and neutralized with 0.1N HCl. The radio-
activity of the TCA precipitates, TCA-soluble fraction, and cell
debris was measured using a liquid scintillation counter after scin-
tillation cocktail was added.
Statistical Analysis
Data are presented as means and SDs. P values were calculated
using a 2-tailed paired Student t test for comparison between 2
groups. A P value less than 0.05 was considered significant.
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RESULTS
In Vitro Uptake and Competitive Inhibition Assay
We examined the contribution of individual transport
systems to anti-FACBC uptake by measuring the intracellular
accumulation of14C-FACBC in DU145 cells in the absence
or presence of Na1or various types of inhibitors (Fig. 1). In
Na1-free buffer,14C-FACBC uptake markedly decreased to
approximately 20% of the control. BCH, a substrate specific
to Na1-dependent systems B0and B0,1and Na1-independ-
ent system L, decreased the transport of14C-FACBC to
approximately 75% in Na1-containing buffer and inhibited
its uptake to the basal level in Na1-free buffer. In contrast,
14C-FACBC uptake was unchanged in the presence of
MeAIB (a substrate specific to Na1-dependent system A
and Na1-independent H1-coupled amino acid transport sys-
tem [system PAT]) and TEA. PAH slightly decreased14C-
FACBC uptake to approximately 95% of the control.
Regarding the comparison of uptake mechanisms, other
14C-labeled natural amino acids (i.e., Ala, Leu, and Met)
were also examined (Fig. 1).14C-FACBC uptake in Na1-
containing buffer without inhibitors was higher than the
uptake of other14C-labeled natural amino acids (Fig. 1),
and we found a difference in the Na1dependency of the
uptake of these amino acids by DU145 cells. That is, the
Na1dependency of14C-FACBC and14C-Ala uptake was
relatively larger than that of14C-Leu and14C-Met.14C-Ala
transport was completely abolished in Na1-free buffer, and
BCH, MeAIB, PAH, and TEA did not inhibit its uptake in
Na1-containing buffer.
By contrast, both14C-Leu transport and14C-Met trans-
port were decreased to approximately 85% in Na1-free
buffer, and MeAIB, PAH, and TEA did not inhibit their
accumulation in Na1-containing buffer. Furthermore,
BCH inhibited the uptake of both14C-Leu and14C-Met
to the basal level in both Na1-containing and Na1-free
buffer, because for14C-Leu and14C-Met, results for assays
performed on ice were almost the same as those for assays
performed in the presence of BCH.
Amino Acid Transporter mRNA Expression
To evaluate transporter gene expression in biopsy speci-
mens from patients with prostate tumor tissues, gene
expression was profiled by cDNA microarray (data not
shown). On the basis of the cDNA microarray results, 40
gene expressions of amino acid transporter mRNAs were
quantified by qRT-PCR in the clinical specimens and
DU145 cells (Fig. 2). There was a strong significant corre-
lation between the log ratio determined by the cDNA
microarray and qRT-PCR for the gene expression of amino
acid transporters analyzed in the tissue specimens (r 5
0.71, P , 0.01) (Fig. 3). qRT-PCR revealed that the mRNA
expression of both ASCT2 and SNAT2, which are Na1-
dependent transporters, was remarkably high in both the
human prostate biopsy specimens and DU145 cells (Fig.
2). In addition, several amino acid transporter mRNAs
(i.e., ASCT2, LAT3, xCT, and PAT1) tended to be ex-
pressed more in human prostate cancer samples than in
noncancerous samples (Fig. 2). The mRNA expression pat-
tern of amino acid transporters in DU145 cells was similar
to that in biopsy specimens.
ASCT2 Involvement in14C-FACBC Uptake
To evaluate the involvement of Na1-dependent amino
acid transporters in anti-FACBC uptake by prostate cancer
cells, we examined the transport of14C-FACBC in ASCT2
and SNAT2 knockdown DU145 cells. The expression of
ASCT2 and SNAT2 were silenced with a gene-specific
siRNA in DU145 cells. qRT-PCR showed that ASCT2
and SNAT2 mRNA expression significantly decreased in
the ASCT2 and SNAT2 siRNA-transfected cells, compared
with those of the controls (Fig. 4A).14C-FACBC uptake
was decreased to 69% and 58% of the control in DU145
cells with ASCT2 knocked down by s12916 and s12918
siRNAs, respectively; uptake decreased to 83% of the con-
trol in DU145 cells with SNAT2 knocked down by both
s633 and s634 (Fig. 4B).
For comparison,14C-labeled Ala, Leu, and Met were
examined in the same way (Fig. 4B). The alteration in
14C-Ala uptake was similar to that of14C-FACBC;14C-
Ala uptake decreased to 59% and 54% in DU145 cells with
ASCT2 knocked down by s12916 and s12918 siRNAs,
respectively, and to 79% and 84% in cells with SNAT2
knocked down by s633 and s634, respectively.14C-Leu
uptake was slightly reduced to 93% in ASCT2 knockdown
FIGURE 1.
14C-Ala,14C-Leu, and14C-Met uptake by
DU145 cells. Data are expressed as per-
centage of uptake in cells that were incu-
bated without inhibitors. Uninhibited uptake
of14C-FACBC,14C-Ala,14C-Leu, and14C-
Met was 6,510.71, 3,086.29, 3,193.04, and
4,036.06 pmol/mg of protein, respectively.
Each bar represents mean and SD (n 5 3–
6). *P , 0.05. **P , 0.01.
Comparison of
14C-FACBC,
TRANSPORT AND FATE OF ANTI–18F-FACBC • Okudaira et al.825

Page 5

cells and decreased to 93% (s633) and 86% (s634) of the
control in SNAT2 knockdown cells.14C-Met uptake did not
change significantly in either ASCT2 or SNAT2 knock-
down cells, compared with the controls.
Intracellular Fate of14C-FACBC
Because little is known about the intracellular fate of14C-
FACBC, we measured the radioactivity of TCA precipitate
(protein fraction), TCA-soluble fraction (intracellular sus-
pended fraction), and cell debris (nucleic acid fraction),
which were prepared from DU145 cells incubated with
14C-FACBC or14C-Met. Figure 5A shows that almost all
intracellular14C-FACBC was detected in the TCA-soluble
fraction after a 24-h incubation. In contrast, 67% and 81% of
intracellular14C-Met was detected in the TCA precipitate
after 3- and 24-h incubations, respectively (Fig. 5B). To
further clarify whether14C-FACBC is incorporated into pro-
teins, we analyzed the cell lysates obtained from DU145
cells incubated for 24 h with14C-FACBC or14C-Met by
sodium dodecyl sulfate polyacrylamide gel electrophoresis
(SDS-PAGE). After SDS-PAGE, the distribution of radioac-
tivity of the radiolabeled proteins was analyzed. However, no
radiolabeled bands corresponding to cellular proteins were
detected on the SDS-PAGE gel with lysates from the cells
incubated with14C-FACBC, whereas significant radiolabeled
protein bands were evident with the lysates from the cells
incubated with14C-Met (data not shown).
DISCUSSION
This is the first report, to our knowledge, that demon-
strates the transport mechanism of anti-FACBC that refers
to amino acid transporter subtype. Our competitive inhib-
ition assays clearly demonstrate a major role of Na1-
dependent amino acid transporters for14C-FACBC uptake
by DU145 cells. The mRNA expression analysis results
show that ASCT2 and SNAT2 are 2 major amino acid trans-
porters in prostate tumor tissues and DU145 cells. The
RNA interference studies revealed that ASCT2 knock-
down in DU145 cells leads to a corresponding reduction
of14C-FACBC accumulation. Given the possibility of com-
pensatory uptake, the effect of ASCT2 knockdown on14C-
FACBC uptake is thought to be more pronounced. Thus,
ASCT2 is critically involved in14C-FACBC uptake. In con-
FIGURE 2.
human prostate biopsy specimens (A) and DU145 cells (B) deter-
mined by qRT-PCR. Each bar represents mean and SD (n 5 3).
rRNA 5 ribosomal RNA.
mRNA expression levels of amino acid transporters in
FIGURE 3.
ers in human prostate biopsy specimens determined by cDNA
microarray and qRT-PCR. **P , 0.01.
Correlation between log ratio of amino acid transport-
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Page 6

trast, organic anion and cation transport systems involved in
the uptake of many kinds of drugs do not apparently take
part in14C-FACBC uptake by DU145 cells, also showing
that the amino acid transporters are mainly involved in the
uptake of anti-FACBC.
While considering the transport mechanism of anti-
FACBC, we concluded that anti-FACBC is transported into
prostate cancer cells in a fashion similar to the transport of
L-Ala. Among the amino acids examined in this study, the
characteristics of14C-FACBC uptake were similar to those
of14C-Ala but not those of14C-Met and14C-Leu. L-Ala is a
typical substrate of ASCT2 (14), which is a major trans-
porter involved in14C-FACBC uptake by DU145 cells.
Therefore, it is thought that anti-FACBC shares the binding
sites of ASCT2 with other substrates.
SNAT2 is one of the isoforms of system A amino acid
transporters, and its typical substrate is L-Ala. It is reported
that the expression of this transporter is more widespread
than those of other system A subtypes (21). However, the
contribution of SNAT2 to anti-FACBC uptake by prostate
cancer cells would be lower than that of ASCT2, although
its gene was the most expressed among amino acid trans-
porters in prostate cancer tissues and DU145 cells.14C-
FACBC uptake slightly decreased in SNAT2 knockdown
cells. However, MeAIB did not affect14C-FACBC uptake
in the inhibition assay. Because14C-Ala uptake was also
affected by the knockdown of SNAT2 but not MeAIB, the
inhibitory effect of the knockdown on SNAT2 transport func-
tion might be stronger than that of MeAIB. In addition, it is
known that the affinity of substrate amino acids—particularly
small neutral amino acids—to SNAT2 is lower than that to
ASCT2 (14). Their affinity (Kmvalue) to ASCT2 is less
than 20 mM (22), whereas the affinity of typical substrates,
such as L-Ala and MeAIB, to SNAT2 is 200–400 mM
(23,24). Hence, the affinity of14C-FACBC to SNAT2 may
be lower than that to ASCT2; this difference in the Km
values of anti-FACBC would reflect the contribution to its
transport. However, the extent of the affinity between14C-
FACBC and transporters is still a matter of speculation at
this time; we are currently planning to confirm this conjec-
ture.
System L transporters are also possibly involved in the
Na1-independent transport of14C-FACBC in DU145 cells,
although our data suggest that Na1-dependent transporters
play a major role in14C-FACBC uptake by DU145 cells.
The LAT1 subtype of system L functions as a transporter
for neutral amino acids with large, branched, or aromatic
side chains (e.g., leucine and phenylalanine) when LAT1
heterodimerizes with the 4F2 heavy chain (25). It is
reported that the expression of LAT1 is increased in many
FIGURE 4.
with control siRNA-transfected cells. (A) mRNA expression levels
of amino acid transporters in siRNA-treated DU145 cells. (B) Uptake
of14C-FACBC,14C-Ala,14C-Leu, and14C-Met by each siRNA-
transfected DU145 cell. Each bar represents mean and SD (n 5
6). **P , 0.01.
Effects of ASCT2 or SNAT2 knockdown, compared
FIGURE 5.
Met (B) when DU145 cells were incubated with each tracer for 3 or
24 h. Each bar represents mean and SD (n 5 3), except14C-FACBC
for 3 h, which represents mean (n 5 2).
Intracellular distribution of14C-FACBC (A) and14C-
TRANSPORT AND FATE OF ANTI–18F-FACBC • Okudaira et al.827

Page 7

types of cancer tissues (25) and that LAT1 transports sub-
strate amino acids with a high affinity, with Kmvalues of
15–50 mM (25). Hence, although the mRNA expression of
LAT1 was lower than that of ASCT2 in human biopsy
samples and DU145 cells, LAT1 may be also involved in
anti-FACBC uptake.
Among the Na1-dependent transporters, the system B0, 1
transporter (ATB0, 1) may be involved in the transport of
anti-FACBC. The involvement of other Na1-dependent
transporters in anti-FACBC uptake is also suggested
because
14C-FACBC uptake was inhibited to approxi-
mately 80% in Na1-free buffer, and the transport activity
of14C-FACBC remained at more than 50% of the control
in ASCT2 knockdown cells. The inhibition assay using
BCH implies the participation of systems B0and B0, 1.
Because the affinity of substrates to system B0, 1is higher
than that to system B0(26–28), the contribution of system
B0, 1to anti-FACBC uptake would be higher than that of
system B0.
The anti-FACBC transport mechanism clarified in this
study has important implications for the feasibility of
anti–18F-FACBC PET. This mechanism illustrates the clin-
ical application of anti–18F-FACBC for imaging cancers in
a wide variety of sites, because the expression of ASCT2
and LAT1 are elevated in many kinds of primary human
cancer tissues relative to other neutral amino acid trans-
porters (25). These amino acid transporters have important
roles in anti–18F-FACBC transport. Furthermore, it has
been reported that ASCT2 expression is associated with
aggressive biologic behavior in colorectal adenocarcinomas
(29). Witte et al. (29) showed that the survival of colorectal
adenocarcinoma patients decreases with an increased per-
centage of ASCT2-positive cancer cells. These findings
imply that anti–18F-FACBC PET is effective for the diag-
nosis of other malignancies in addition to prostate cancer
and that anti–18F-FACBC PET might be able to predict
patient prognosis. Further investigations on the expression
profiling of amino acid transporters in cancers and the rela-
tionship between prognosis of cancer patients and expres-
sion level of amino acid transporters would clarify the
target sites that can be effectively diagnosed using
anti–18F-FACBC in addition to the potential ability of this
radiotracer.
Considering how anti-FACBC is used or metabolized
once it is taken up by cells, how it is incorporated into
proteins because of its structural similarity to natural
amino acids should be determined. However, this study
indicates that14C-FACBC is not incorporated into pro-
teins. The intracellular distribution pattern of14C-FACBC
was quite different from that of14C-Met. The intracellular
distribution ratio of TCA precipitate containing
FACBC was 2.08%, even when the cells were incubated
with14C-FACBC for 24 h. The radioactivity of TCA pre-
cipitate including14C-FACBC was not considered signifi-
cant because no protein bands were observed in the lysates
from the cells incubated with14C-FACBC in SDS-PAGE.
14C-
The radioactivity of TCA precipitate might be derived
from the nonspecific binding of14C-FACBC to cellular
proteins. A previous report shows that 1-11C-aminocyclo-
pentanecarboxylic acid, which is a structural analog of
anti-FACBC, is not metabolized and remains intact inside
cells (30). Therefore, anti-FACBC may remain intact
without being incorporated into cellular proteins—an
important feature of PET tracers for clinical use, because
the incorporation of anti-FACBC into enzymes, peptides,
and proteins that are essential for biologic phenomena
might cause significant adverse effects. However, this
study suggests that almost all14C-FACBC in cells was
recovered from the nonprotein fraction. Furthermore, it
is reported that “cold” anti-FACBC does not exhibit any
acute toxic effects on Sprague–Dawley rats, even when the
amount of injected “cold” anti-FACBC was approximately
1 · 106times in excess of the amount that a patient would
receive (10). Thus, these findings indicate that this radio-
tracer would not affect the biologic function of proteins;
these features would enable the safe use of anti–18F-
FACBC in PET examination. In addition, these features
indicate that the images derived from anti–18F-FACBC
PET would simply represent the amount of amino acid
transporters involved in anti-FACBC uptake and their
activity and not protein synthesis or amino acid metabo-
lism.
Our findings are currently limited to 2 amino acid
transporter systems extracted from an inhibition assay,
mRNA expression analysis, and RNA interference study.
To further interpret the transportation mechanism of anti-
FACBC, future studies are necessary to clarify the affinity
of anti-FACBC for the amino acid transporters that are
expressed, to some extent, in cancer cells and the expres-
sion of amino acid transporter proteins in malignancies.
CONCLUSION
We demonstrated that Na1-dependent amino acid trans-
porters predominantly function in14C-FACBC uptake by
DU145 cells. Na1-independent system L transporters are
also involved in
14C-FACBC transport. Gene expression
analysis suggests that the mRNA expression of both ASCT2
and SNAT2 is remarkably high in human prostate specimens
and DU145 cells. ASCT2, which is a Na1-dependent amino
acid transporter, at least in part plays an important role in
the uptake of14C-FACBC by DU145 cells. Moreover,14C-
FACBC is not incorporated into cellular protein. These find-
ings suggest a possible mechanism of anti–18F-FACBC PET
for prostate cancer.
DISCLOSURE STATEMENT
The costs of publication of this article were defrayed in
part by the payment of page charges. Therefore, and solely
to indicate this fact, this article is hereby marked “adver-
tisement” in accordance with 18 USC section 1734.
828THE JOURNAL OF NUCLEAR MEDICINE • Vol. 52 • No. 5 • May 2011

Page 8

ACKNOWLEDGMENTS
We thank Hiroyo Araki, Kodai Nishi, Masato Ogura, and
Fumiya Takahashi for their assistance with the gene
expression analysis. We also acknowledge the valuable
discussion with Dr. Shuntaro Oka and the helpful advice of
Drs. Yasunori Yoshida, Yoshifumi Shirakami, and Kazuhiro
Shiba.
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