Putative Transport Mechanism and Intracellular Fateof 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; and 8Biomedical Imaging Research Center, University of Fukui, Fukui, Japan

14C-FACBCTrans-1-amino-3-18F-fluorocyclobutanecarboxylic acid (anti–18FFACBC) is an amino acid PET tracer that has shown promise forvisualizing prostate cancer. Therefore, we aimed to clarify theanti–18F-FACBC transport mechanism in prostate cancer cells.
We also studied the fate of anti–18F-FACBC after it is transported 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-cyclobutanecarboxylic acid (14C-FACBC) uptake was examined in humanprostate cancer DU145 cells with the following substrates ofamino acid transporters: a-(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 microarrayand 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 transporter2 (SNAT2) on 14C-FACBC uptake. In addition, the possibility of
14C-FACBC incorporation into proteins was examined. Results:
14C-FACBC uptake by DU145 cells was markedly decreased toapproximately 20% in the absence of Na1, compared with thatin its presence, indicating that Na1-dependent transporters aremainly responsible for the uptake of this tracer. Moreover,
2-amino-2-norbornanecarboxylic acid inhibited the transportof 14C-FACBC to the basal level in Na1-free buffer. In contrast,
a-(methylamino) isobutyric acid did not inhibit 14C-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 expressedin prostate tumor tissues and DU145 cells, ASCT2 knockdown
using small interfering RNA was more effective in lowering
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.

822

transport than SNAT2. Almost all intracellular
was recovered from the nonprotein fraction.
Conclusion: ASCT2, which is a Na1-dependent amino acidtransporter, and to a lesser extent Na1-independent transporters play a role in the uptake of 14C-FACBC by DU145 cells.
Among the Na1-independent transporters, system L transporters are also involved in the transport of 14C-FACBC. Moreover,
14C-FACBC is not incorporated into proteins in cells. Thesefindings suggest a possible mechanism of anti–18F-FACBC
PET for prostate cancer.
14C-FACBC

Key Words: anti-FACBC; prostate cancer; amino acidtransporter; ASCT2
2011; 52:822–829
DOI: 10.2967/jnumed.110.086074

P

rostate 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 treatment strategy. However, the current methodology does notallow 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 to 18F-FDG, radiolabeled amino acids havebeen studied as potential tumor-seeking PET agents forclinical use (4). The most frequently used amino acidPET tracer is 11C-methionine because of its easy and fast
radiosynthesis (5). The availability and clinical efficacy of
11C-methionine in patients with brain tumors (4) and prostate cancers (6) have been investigated. Besides PET withradiolabeled natural amino acids, PET with synthetic amino

THE JOURNAL OF NUCLEAR MEDICINE • Vol. 52 • No. 5 • May 2011

acids labeled with 11C, such as 1-11C-aminocyclobutanecarboxylic acid (11C-ACBC) (7), 1-11C-aminocyclopentanecarboxylic acid (7), and a-11C-aminoisobutyric acid (8),exhibit high tumor–to–nontumor concentration ratios.
Among these studies, Washburn et al. (7) show that 11CACBC 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). However, because of the short half-life of 11C, 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 of 11C-ACBC, Shoup et al.
(10) developed trans-1-amino-3-18F-fluorocyclobutanecarboxylic acid (anti–18F-FACBC). Anti–18F-FACBC is a synthetic L-leucine analog that exhibits high tumor-specificaccumulation in patients with glioblastoma multiforme(10), renal papillary cell cancer (11), and prostate cancer(12). One of the interesting features of anti–18F-FACBC isthat its renal excretion is much slower than that of 18F-FDG(13); this slower excretion could be helpful for prostatecancer imaging.
Little is known about the transport mechanism ofanti–18F-FACBC into cells, although anti–18F-FACBC isthought to cross the plasma membrane via amino acid transporters because of its structural similarity to natural aminoacids. Because of the identification and kinetic studies ofamino 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 takeup amino acids with short, polar, or linear side chains such as
L-alanine and L-serine. In contrast, large, branched, and aromatic amino acids such as L-tyrosine mainly enter cells via
system L (16). Although several transporters may be involvedin anti–18F-FACBC uptake, previous studies show that theuptake of anti–18F-FACBC is mediated by the Na1-independent 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 prostatecancer cells has not been fully clarified.
In the present study, we characterized anti–18F-FACBCtransport 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 complementary DNA (cDNA) microarray in human prostate biopsy
specimens. Furthermore, we confirmed the gene expressionof amino acid transporters in the human prostate cancer
tissues and DU145 cells by quantitative real-time reverse
transcription polymerase chain reaction (qRT-PCR). In addition, we examined the role of Na1-dependent amino acidtransporters in anti–18F-FACBC uptake, using the smallinterfering RNA (siRNA) transfection method, and the intracellular 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 than 18F(half-life, 110 min) or 11 C (half-life, 20 min). Trans-1-amino3-fluoro-1- 14C-cyclobutanecarboxylic acid ( 14 C-FACBC; 2.08
GBq/mmol) was radiosynthesized by EaglePicher Pharmaceutical
Services. L -1- 14 C-methionine (14 C-Met, 2.04 GBq/mmol) was
purchased from American Radiolabeled Chemicals. BothL - 14 C (U)-alanine ( 14 C-Ala; 5.92 or 6.29 GBq/mmol) andL - 14 C (U)-leucine ( 14 C-Leu; 11.99 GBq/mmol) were purchased from both Moravek Biochemicals and PerkinElmer.
The DU145 human prostate cancer cell line was obtained fromthe American Type Culture Collection.
Collection of Human Biopsy Specimens
Human prostate biopsy specimens (Table 1) for cDNA microarray 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 guarantee 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 thesepatients with their informed consent. The specimens used forpathologic diagnosis were reviewed by pathologists; the specimens for gene expression analysis were immediately stored in
RNAlater (Ambion) at 4C and subsequently stored at 280C
until required. Patients were designated prostate cancer–free ifcancer 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 (LifeTechnologies). The cells were cultured at 37C in 5% CO2/95%
air; these cells were used for the messenger RNA (mRNA) quantitation and uptake assay.

TABLE 1
List of Human Prostate Biopsy SpecimensPatient no.
Normal prostate12
3
Adenocarcinoma
4
5
6

Prostate-specificantigen (ng/mL)

Gleason score

3.6
5.1
9.0

—
—
—

55.0
96.0
227.8

41458
41458
41458

TRANSPORT AND FATE OF ANTI–18F-FACBC • Okudaira et al.

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RNA Extraction
Total RNA was extracted from human prostate biopsy specimens 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 SpikeIn 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 humanbiopsy 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 usinga Transcriptor First Strand cDNA Synthesis Kit (Roche Applied
Science) by random hexamer priming at 50C 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 synthesized by Nihon Gene Research Laboratories. qt-PCR was performed using an Mx3005P or Mx3000P QPCR system (Agilent
Technologies), FastStart Universal Probe Master (ROX), and the
Universal ProbeLibrary (Roche Applied Science) with the following profile: 1 cycle of enzyme activation at 95C for 10 min, 40
cycles of denaturing at 95C for 15 s, and extension at 60C for
1 min. All reactions were run in triplicate. The PCR products wereanalyzed by agarose gel electrophoresis, and no nonspecific 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 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. Expression data were normalized against the copy number of 18S ribosomal RNA.
The correlation coefficient of the log ratio between the resultsof the cDNA microarray and qRT-PCR was calculated as follows:log2(mRNA expression in cancer specimens/mRNA expression innormal specimens).
Measurement of 14C-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 24well tissue culture plates at 1 · 105 cells per well with 1 mL ofculture medium. Transport assays were then conducted on the next
day after inoculation (semiconfluent phase cells).
The sodium-containing incubation medium used was basedon 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-freeincubation medium (Na1-free buffer), NaCl and Na2HPO4 were
replaced with the equivalent concentrations of choline chloride
and K2HPO4, respectively. After the culture medium was re-824

moved, each well was incubated with 1 mL of incubation medium
for 10 min at 37C. The cells were then incubated with 0.3 mL of
incubation medium containing the respective 14C-labeled aminoacid (10 mM) for 10 min at 37C. 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 scintillation cocktail (Ultima Gold; PerkinElmer) was added. The protein 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 transporters), 2-amino-2-norbornanecarboxylic acid (BCH—a substratespecific to system L transporters), p-aminohippurate (PAH—asubstrate specific to organic anion transporters), and tetraethylammonium chloride (TEA—a substrate specific to organic cationtransporters). Basal transport activity was defined by the resultsof 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; eachtransfection was performed using only one of them. The identification 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 and4390846) were used as controls. siRNA transfection was performed
according to the manufacturer’s instructions. DU145 cells weretransfected with 5 nM of each siRNA in antibiotic-free growth
medium using Lipofectamine 2000 (Life Technologies). Aftertransfection (48 h), the cells were used for amino acid uptake
experiments. The efficiency and specificity of each siRNA wereconfirmed by qRT-PCR. We rated the result as the target gene–
specific effect when both siRNAs for the same target gene significantly altered the transport of 14C-labeled amino acids.
Measurement of 14C-Labeled Amino Acid
Incorporation into Proteins
DU145 cells were incubated with 50 mM 14C-FACBC at 37C
for 3 or 24 h. The cells were then washed twice with phosphatebuffered saline and lysed in CelLytic M cell lysis reagent (SigmaAldrich) containing protease inhibitor cocktail (Sigma-Aldrich).
Cell lysates were centrifuged at 18,000g for 15 min at 4C, andthe supernatants were subjected to trichloroacetic acid (TCA) precipitation. The precipitation reaction was performed for 10 min on
ice by adding ice-cold TCA (final concentration, 10%) to thesupernatants, and the TCA precipitates were washed 3 times with
ice-cold 5% TCA. The TCA precipitates and cell debris werelysed in 0.1N NaOH and neutralized with 0.1N HCl. The radioactivity of the TCA precipitates, TCA-soluble fraction, and celldebris was measured using a liquid scintillation counter after scintillation cocktail was added.
Statistical Analysis
Data are presented as means and SDs. P values were calculatedusing a 2-tailed paired Student t test for comparison between 2
groups. A P value less than 0.05 was considered significant.

THE JOURNAL OF NUCLEAR MEDICINE • Vol. 52 • No. 5 • May 2011

RESULTSIn Vitro Uptake and Competitive Inhibition Assay

We examined the contribution of individual transportsystems to anti-FACBC uptake by measuring the intracellularaccumulation of 14C-FACBC in DU145 cells in the absenceor presence of Na1 or various types of inhibitors (Fig. 1). In
Na1-free buffer, 14C-FACBC uptake markedly decreased toapproximately 20% of the control. BCH, a substrate specificto Na1-dependent systems B0 and B0,1 and Na1-independent system L, decreased the transport of 14C-FACBC toapproximately 75% in Na1-containing buffer and inhibitedits 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 system [system PAT]) and TEA. PAH slightly decreased 14CFACBC 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 Na1containing buffer without inhibitors was higher than the
uptake of other 14C-labeled natural amino acids (Fig. 1),
and we found a difference in the Na1 dependency of theuptake of these amino acids by DU145 cells. That is, theNa1 dependency of 14C-FACBC and 14C-Ala uptake wasrelatively larger than that of 14C-Leu and 14C-Met. 14C-Alatransport was completely abolished in Na1-free buffer, andBCH, MeAIB, PAH, and TEA did not inhibit its uptake inNa1-containing buffer.
By contrast, both 14C-Leu transport and 14C-Met transport were decreased to approximately 85% in Na1-freebuffer, and MeAIB, PAH, and TEA did not inhibit theiraccumulation in Na1-containing buffer. Furthermore,
BCH inhibited the uptake of both 14C-Leu and 14C-Met
to the basal level in both Na1-containing and Na1-freebuffer, because for 14C-Leu and 14C-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 specimens from patients with prostate tumor tissues, gene

expression was profiled by cDNA microarray (data notshown). 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 correlation between the log ratio determined by the cDNA
microarray and qRT-PCR for the gene expression of aminoacid transporters analyzed in the tissue specimens (r 50.71, P , 0.01) (Fig. 3). qRT-PCR revealed that the mRNA
expression of both ASCT2 and SNAT2, which are Na1dependent transporters, was remarkably high in both thehuman 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 expressed more in human prostate cancer samples than in
noncancerous samples (Fig. 2). The mRNA expression pattern of amino acid transporters in DU145 cells was similar
to that in biopsy specimens.
ASCT2 Involvement in 14C-FACBC Uptake

To evaluate the involvement of Na1-dependent aminoacid transporters in anti-FACBC uptake by prostate cancer
cells, we examined the transport of 14C-FACBC in ASCT2and SNAT2 knockdown DU145 cells. The expression of
ASCT2 and SNAT2 were silenced with a gene-specificsiRNA in DU145 cells. qRT-PCR showed that ASCT2and SNAT2 mRNA expression significantly decreased in
the ASCT2 and SNAT2 siRNA-transfected cells, comparedwith 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 control in DU145 cells with SNAT2 knocked down by boths633 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 of 14C-FACBC; 14CAla 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. Comparison of 14C-FACBC,
14C-Ala, 14C-Leu, and 14C-Met uptake by
DU145 cells. Data are expressed as percentage of uptake in cells that were incubated without inhibitors. Uninhibited uptakeof 14C-FACBC, 14C-Ala, 14C-Leu, and 14CMet 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.

TRANSPORT AND FATE OF ANTI–18F-FACBC • Okudaira et al.

825

FIGURE 3. Correlation between log ratio of amino acid transporters in human prostate biopsy specimens determined by cDNA
microarray and qRT-PCR. **P , 0.01.

(protein fraction), TCA-soluble fraction (intracellular suspended fraction), and cell debris (nucleic acid fraction),
which were prepared from DU145 cells incubated with
14C-FACBC or 14C-Met. Figure 5A shows that almost all
intracellular 14C-FACBC was detected in the TCA-soluble
fraction after a 24-h incubation. In contrast, 67% and 81% of
intracellular 14C-Met was detected in the TCA precipitate
after 3- and 24-h incubations, respectively (Fig. 5B). To
further clarify whether 14C-FACBC is incorporated into proteins, we analyzed the cell lysates obtained from DU145
cells incubated for 24 h with 14C-FACBC or 14C-Met bysodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS-PAGE). After SDS-PAGE, the distribution of radioactivity of the radiolabeled proteins was analyzed. However, noradiolabeled bands corresponding to cellular proteins weredetected on the SDS-PAGE gel with lysates from the cells
incubated with 14C-FACBC, whereas significant radiolabeledprotein bands were evident with the lysates from the cells
incubated with 14C-Met (data not shown).
DISCUSSIONFIGURE 2. mRNA expression levels of amino acid transporters inhuman prostate biopsy specimens (A) and DU145 cells (B) determined by qRT-PCR. Each bar represents mean and SD (n 5 3).
rRNA 5 ribosomal RNA.

cells and decreased to 93% (s633) and 86% (s634) of thecontrol in SNAT2 knockdown cells. 14C-Met uptake did notchange significantly in either ASCT2 or SNAT2 knockdown cells, compared with the controls.
Intracellular Fate of 14C-FACBC

Because little is known about the intracellular fate of 14CFACBC, we measured the radioactivity of TCA precipitate

826This is the first report, to our knowledge, that demonstrates the transport mechanism of anti-FACBC that refers
to amino acid transporter subtype. Our competitive inhibition assays clearly demonstrate a major role of Na1dependent amino acid transporters for 14C-FACBC uptake
by DU145 cells. The mRNA expression analysis results
show that ASCT2 and SNAT2 are 2 major amino acid transporters in prostate tumor tissues and DU145 cells. The
RNA interference studies revealed that ASCT2 knockdown in DU145 cells leads to a corresponding reductionof 14C-FACBC accumulation. Given the possibility of compensatory uptake, the effect of ASCT2 knockdown on 14CFACBC uptake is thought to be more pronounced. Thus,
ASCT2 is critically involved in 14C-FACBC uptake. In con-

THE JOURNAL OF NUCLEAR MEDICINE • Vol. 52 • No. 5 • May 2011

FIGURE 4. Effects of ASCT2 or SNAT2 knockdown, comparedwith control siRNA-transfected cells. (A) mRNA expression levelsof amino acid transporters in siRNA-treated DU145 cells. (B) Uptakeof 14C-FACBC, 14C-Ala, 14C-Leu, and 14C-Met by each siRNAtransfected DU145 cell. Each bar represents mean and SD (n 56). **P , 0.01.

FIGURE 5.
Intracellular distribution of 14C-FACBC (A) and 14CMet (B) when DU145 cells were incubated with each tracer for 3 or
24 h. Each bar represents mean and SD (n 5 3), except 14C-FACBC
for 3 h, which represents mean (n 5 2).

trast, organic anion and cation transport systems involved in
the uptake of many kinds of drugs do not apparently takepart in 14C-FACBC uptake by DU145 cells, also showingthat the amino acid transporters are mainly involved in the
uptake of anti-FACBC.
While considering the transport mechanism of antiFACBC, 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, thecharacteristics of 14C-FACBC uptake were similar to thoseof 14C-Ala but not those of 14C-Met and 14C-Leu. L-Ala is atypical substrate of ASCT2 (14), which is a major transporter involved in 14C-FACBC uptake by DU145 cells.
Therefore, it is thought that anti-FACBC shares the bindingsites of ASCT2 with other substrates.
SNAT2 is one of the isoforms of system A amino acidtransporters, and its typical substrate is L-Ala. It is reported
that the expression of this transporter is more widespreadthan those of other system A subtypes (21). However, thecontribution of SNAT2 to anti-FACBC uptake by prostatecancer cells would be lower than that of ASCT2, although
its gene was the most expressed among amino acid transporters in prostate cancer tissues and DU145 cells. 14CFACBC uptake slightly decreased in SNAT2 knockdown
cells. However, MeAIB did not affect 14C-FACBC uptake

in the inhibition assay. Because 14C-Ala uptake was also
affected by the knockdown of SNAT2 but not MeAIB, theinhibitory effect of the knockdown on SNAT2 transport function 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 (Km value) to ASCT2 is lessthan 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 of 14C-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 between 14CFACBC and transporters is still a matter of speculation at
this time; we are currently planning to confirm this conjecture.
System L transporters are also possibly involved in the
Na1-independent transport of 14C-FACBC in DU145 cells,
although our data suggest that Na1-dependent transporters
play a major role in 14C-FACBC uptake by DU145 cells.
The LAT1 subtype of system L functions as a transporter
for neutral amino acids with large, branched, or aromaticside chains (e.g., leucine and phenylalanine) when LAT1
heterodimerizes with the 4F2 heavy chain (25). It isreported that the expression of LAT1 is increased in many

TRANSPORT AND FATE OF ANTI–18F-FACBC • Okudaira et al.

827

types of cancer tissues (25) and that LAT1 transports substrate amino acids with a high affinity, with Km values 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 inanti-FACBC uptake.
Among the Na1-dependent transporters, the system B0, 1
transporter (ATB0, 1) may be involved in the transport ofanti-FACBC. The involvement of other Na1-dependenttransporters in anti-FACBC uptake is also suggested
because 14C-FACBC uptake was inhibited to approximately 80% in Na1-free buffer, and the transport activityof 14C-FACBC remained at more than 50% of the control
in ASCT2 knockdown cells. The inhibition assay using
BCH implies the participation of systems B0 and B0, 1.
Because the affinity of substrates to system B0, 1 is higher
than that to system B0 (26–28), the contribution of system
B0, 1 to anti-FACBC uptake would be higher than that ofsystem B0.
The anti-FACBC transport mechanism clarified in thisstudy has important implications for the feasibility ofanti–18F-FACBC PET. This mechanism illustrates the clinical application of anti–18F-FACBC for imaging cancers ina wide variety of sites, because the expression of ASCT2and LAT1 are elevated in many kinds of primary humancancer tissues relative to other neutral amino acid transporters (25). These amino acid transporters have importantroles in anti–18F-FACBC transport. Furthermore, it has
been reported that ASCT2 expression is associated withaggressive biologic behavior in colorectal adenocarcinomas(29). Witte et al. (29) showed that the survival of colorectal
adenocarcinoma patients decreases with an increased percentage of ASCT2-positive cancer cells. These findings
imply that anti–18F-FACBC PET is effective for the diagnosis of other malignancies in addition to prostate cancer
and that anti–18F-FACBC PET might be able to predictpatient prognosis. Further investigations on the expression
profiling of amino acid transporters in cancers and the relationship between prognosis of cancer patients and expression level of amino acid transporters would clarify thetarget sites that can be effectively diagnosed usinganti–18F-FACBC in addition to the potential ability of thisradiotracer.
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 naturalamino acids should be determined. However, this study
indicates that 14C-FACBC is not incorporated into proteins. The intracellular distribution pattern of 14C-FACBC
was quite different from that of 14C-Met. The intracellular
distribution ratio of TCA precipitate containing 14CFACBC was 2.08%, even when the cells were incubatedwith 14C-FACBC for 24 h. The radioactivity of TCA precipitate including 14C-FACBC was not considered significant because no protein bands were observed in the lysatesfrom the cells incubated with 14C-FACBC in SDS-PAGE.

828

The radioactivity of TCA precipitate might be derived
from the nonspecific binding of 14C-FACBC to cellular
proteins. A previous report shows that 1-11C-aminocyclopentanecarboxylic acid, which is a structural analog ofanti-FACBC, is not metabolized and remains intact inside
cells (30). Therefore, anti-FACBC may remain intact
without being incorporated into cellular proteins—animportant 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, thisstudy suggests that almost all 14C-FACBC in cells wasrecovered 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 theamount of injected “cold” anti-FACBC was approximately
1 · 106 times in excess of the amount that a patient would
receive (10). Thus, these findings indicate that this radiotracer would not affect the biologic function of proteins;these features would enable the safe use of anti–18FFACBC in PET examination. In addition, these featuresindicate that the images derived from anti–18F-FACBC
PET would simply represent the amount of amino acidtransporters involved in anti-FACBC uptake and theiractivity and not protein synthesis or amino acid metabolism.
Our findings are currently limited to 2 amino acidtransporter systems extracted from an inhibition assay,
mRNA expression analysis, and RNA interference study.
To further interpret the transportation mechanism of antiFACBC, future studies are necessary to clarify the affinityof anti-FACBC for the amino acid transporters that are
expressed, to some extent, in cancer cells and the expression of amino acid transporter proteins in malignancies.
CONCLUSION

We demonstrated that Na1-dependent amino acid transporters predominantly function in 14C-FACBC uptake by
DU145 cells. Na1-independent system L transporters arealso involved in 14C-FACBC transport. Gene expression
analysis suggests that the mRNA expression of both ASCT2and SNAT2 is remarkably high in human prostate specimens
and DU145 cells. ASCT2, which is a Na1-dependent aminoacid transporter, at least in part plays an important role in
the uptake of 14C-FACBC by DU145 cells. Moreover, 14CFACBC is not incorporated into cellular protein. These findings suggest a possible mechanism of anti–18F-FACBC PET
for prostate cancer.
DISCLOSURE STATEMENT

The costs of publication of this article were defrayed inpart by the payment of page charges. Therefore, and solely
to indicate this fact, this article is hereby marked “advertisement” in accordance with 18 USC section 1734.

THE JOURNAL OF NUCLEAR MEDICINE • Vol. 52 • No. 5 • May 2011

ACKNOWLEDGMENTS

We thank Hiroyo Araki, Kodai Nishi, Masato Ogura, andFumiya Takahashi for their assistance with the gene
expression analysis. We also acknowledge the valuable
discussion with Dr. Shuntaro Oka and the helpful advice ofDrs. Yasunori Yoshida, Yoshifumi Shirakami, and Kazuhiro
Shiba.
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Supplemental Table 1
Sequences of Primers used in qRT-PCR
Probe
Sequences (5'-3')Official Symbol Alias
GenBank accession numberNo.
Forward
Reverse
SLC38A1
SNAT1
attttgggactcgcctttg
agcaatgtcactgaagtcaaaagt
47 NM_001077484.1 NM_030674.3
SLC38A2
SNAT2
cctatgaaatctgtacaaaagattgg ttgtgtacccaatccaaaacaa
9 NM_018976.3
SLC38A4
SNAT4
ggacaccccactcacacag
tgaacgagacaagccaatca
77 NM_018018.2
SLC38A6
SNAT6
taccaaccatggctttttca
tgcattcttttctttgaaggact
53 NM_153811.1
SLC6A9
GLYT-1gctctccttcggccagttcaccaccatcataccatagcc
65 NM_201649.2
NM_001024845.1 NM_006934.2
SLC6A5
GLYT-2
tgagaatgtggcagaccaag
ggcccagaacggagagag
64 NM_004211.3
SLC1A4
ASCT1
tttgcgacagcatttgctac
gcacttcatcatagagggaagg
78 NM_003038.2
SLC1A5ASCT2gaggaatatcaccggaacca
aggatgttcatcccctcca
43 NM_005628.1
SLC6A7PROT
cttcccctatcgagcgtaca
atgaggaagtagggcacgag
25 NM_014228.3
SLC6A19
gcttcccctacctgtgtcag
ggatgaggaacgggatcat
63 NM_001003841.1
B0AT1
SLC6A15
gtgtcctttggtgaaaaatgc
tcagtgcttccctgtaccagt
77 NM_182767.3
NM_018057.4
B0AT2
SLC6A20
SIT1
cggggaccctgaagtatca
ataggccgggtaatctttgg
81 NM_022405.2
NM_020208.2
SLC38A3
SNAT3
tgttgcagccgagttcag
tggtctcacaccaacagtcag
32 NM_006841.4
SLC38A5
SNAT5
gagttgcggccacttcag
tccattcatctttggatcctg
15 NM_033518.1
SLC6A6TAUT
tctgaggagctcccaaacaa
ttcaggatgtccttgtggaa
49 NM_003043.3
SLC7A7
agctgcttcgccccttat
gcacagttaatgaaggttaagagaca
27 NM_001126105.1 NM_001126106.1 NM_003982.3
y+LAT1
SLC7A6
cctatccctgctttactgttcaa
aagctgaagtagttgataagctgga
88 NM_003983.4
NM_001076785.1
y+LAT2SLC7A5
LAT1
gtggaaaaacaagcccaagt
gcatgagcttctgacacagg
25 NM_003486.5
SLC7A8LAT2
ttgccaatgtcgcttatgtc
ggagcttctctccaaaagtcac
17 NM_182728.1
NM_012244.2
SLC43A1
LAT3
gccctcatgattggctctta
ccggcatcgtagatcagc
29 NM_003627.4
SLC43A2
LAT4
caagtatggcccgaggaag
caatcagcaagcaggaaacc
3 NM_152346.1
SLC16A10
TAT1
ggtgtgaagaaggtttatctacagg agggccccaaagatgcta
6 NM_018593.3
SLC7A10asc-1
ggactacgcctacgtcacaga
ggtggggtacatgatgagga
20 NM_019849.1
SLC6A14
tgaaatgcccgagtttcttc
ttctctgatgaagccgacact
7 NM_007231.1
ATB0.+SLC7A9BAT1
cctcaaatcgttgtgaaatgc
cgcacgctcagtgagttc
70 NM_001126335.1 NM_014270.4
SLC7A1
CAT1
gtctgtctgttcgcgatcct
caaggaaggtttcagaatccaa
70 NM_003045.3
SLC7A2
CAT2
gcagcctgtgttctcatcct
acatttgggctggtcgtaag
64 NM_003046.3
NM_001008539.1
SLC7A3
CAT3
actctgggccttcaccact
gccacactggctgtacca
55 NM_032803.4
NM_001048164.1
SLC1A3EAAT1
ttgaactgaacttcggacaaatta
attccagctgccccaatact
5 NM_004172.3
SLC1A2EAAT2
cctgccaacagaggacatc
gactgaagttctcatcctgtcca
76 NM_004171.3
SLC1A1
EAAT3
agttgaatgacctggacttgg
gcagatgtggccgtgatac
9 NM_004170.4
SLC1A6EAAT4tgcagatgctggtgttacct
gttgtccagggatgccata
19 NM_005071.1
SLC1A7EAAT5
gagtcggtcatgaagatcgtg
tcttacccgcaatgaggaac
76 NM_006671.3
SLC7A11xCT
cccagatatgcatcgtcctt
ccatgaagaggcatgtgaag
33 NM_014331.3
SLC36A1
PAT1
ggtcatgatctaccagttcattgt
gagagggtaggtcttccaagg
10 NM_078483.2
SLC36A2
PAT2
cctggctccagaatcacg
gccaagttgggtgttaataagg
60 NM_181776.1
SLC36A3
PAT3
tgtttatggcagacaatttacaaca
gcagatgttggaggtcacg
31 NM_181774.2
SLC36A4
PAT4
tgatgaaatcaaaggcagca
tcacaaaaatgccaaaggaat
60 NM_152313.2
SLC3A1rBAT
accacactgtgaatgttgatgtc
agtagctcattggcatgaagtaga
26 NM_000341.2
NM_001012661.1 NM_002394.4
NM_001012664.1
SLC3A24F2hc
cagaagtggtggcacacg
gtaatcgagacgccccttc
81
NM_001012662.1 NM_001012663.1 NM_001013251.1
18S rRNA
gcaattattccccatgaacg
gggacttaatcaacgcaagc
48 M10098.1
ACTB
actin，beta ccaaccgcgagaagatga
ccagaggcgtacagggatag
64 NM_001101.2
GAPDH
agccacatcgctcagacac
gcccaatacgaccaaatcc
60 NM_002046.3

THE JOURNAL OF NUCLEAR MEDICINE • Vol. 52 • No. 5 • May 2011

Okudaira et al.