Title:
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.