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Cancer Sci
2015 Mar 01;1063:279-86. doi: 10.1111/cas.12602.
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Boronophenylalanine, a boron delivery agent for boron neutron capture therapy, is transported by ATB0,+, LAT1 and LAT2.
Wongthai P
,
Hagiwara K
,
Miyoshi Y
,
Wiriyasermkul P
,
Wei L
,
Ohgaki R
,
Kato I
,
Hamase K
,
Nagamori S
,
Kanai Y
.
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The efficacy of boron neutron capture therapy relies on the selective delivery of boron carriers to malignant cells. p-Boronophenylalanine (BPA), a boron delivery agent, has been proposed to be localized to cells through transporter-mediated mechanisms. In this study, we screened aromatic amino acid transporters to identify BPA transporters. Human aromatic amino acid transporters were functionally expressed in Xenopus oocytes and examined for BPA uptake and kinetic parameters. The roles of the transporters in BPA uptake were characterized in cancer cell lines. For the quantitative assessment of BPA uptake, HPLC was used throughout the study. Among aromatic amino acid transporters, ATB(0,+), LAT1 and LAT2 were found to transport BPA with Km values of 137.4 ± 11.7, 20.3 ± 0.8 and 88.3 ± 5.6 μM, respectively. Uptake experiments in cancer cell lines revealed that the LAT1 protein amount was the major determinant of BPA uptake at 100 μM, whereas the contribution of ATB(0,+) became significant at 1000 μM, accounting for 20-25% of the total BPA uptake in MCF-7 breast cancer cells. ATB(0,+), LAT1 and LAT2 transport BPA at affinities comparable with their endogenous substrates, suggesting that they could mediate effective BPA uptake in vivo. The high and low affinities of LAT1 and ATB(0,+), respectively, differentiate their roles in BPA uptake. ATB(0,+), as well as LAT1, could contribute significantly to the tumor accumulation of BPA at clinical dose.
Figure 1. Separation of p-boronophenylalanine (BPA) by HPLC. Chromatograms showing the separation of a BPA standard (0.2 pmol) (a), amino acid standards (0.2 pmol each) (b), a sample from the oocyte expressing LAT1 (c), and the control oocyte not expressing LAT1 (d). (a, b) The BPA peak was identified by retention time and spike study (not shown). Similarly, by comparison with amino acid standards and a spike study (not shown), the peaks neighboring BPA in oocyte samples were identified as alanine (Ala) and proline (Pro). (c, d) The LAT1-expressing oocyte and non-expressing control oocyte were incubated in the uptake buffer containing BPA. Samples from the oocytes were separated by HPLC. The increased BPA peak height in (c) showed that the uptake of BPA was mediated by LAT1. Arg, arginine; Asp, aspartic acid; Glu, glutamic acid; His, histidine; Ser, serine.
Figure 2. Chromatograms of p-boronophenylalanine (BPA) taken up by aromatic amino acid transporters. Chromatograms for oocytes expressing each transporter (solid line) and non-expressing control oocytes (dotted line) are overlaid on the same scaling. In ATB0,+ (b), LAT1 (c) and LAT2 (d), the BPA peaks were significantly higher than those in the controls, but not in the others. The endogenous alanine (Ala) and proline (Pro) peaks were frequently found to be lower in the oocytes expressing transporters, probably because of the translational consumption of endogenous amino acids.
Figure 3. Time-dependent uptake of p-boronophenylalanine (BPA). Uptakes of BPA by oocytes expressing the indicated transporter (○) and by non-expressing controls (▾) were measured over 60 min in Na+ uptake buffer containing 50 μM BPA for ATB0,+ (a), and in Na+-free uptake buffer for LAT1 (b) and LAT2 (c). The time-course of the uptake was determined at 50 μM BPA because the measurement became less accurate for the 10- and 20-min time points at lower concentrations. The transporter-mediated uptakes (•) were determined by subtracting the uptake in non-expressing control oocytes (▾) from the uptake in oocytes expressing each transporter (○). Uptake by ATB0,+ and LAT2 linearly increased up to 30 min (a, c). Uptake by LAT1 was linear over 60 min (b). Each data point represents the mean ± SEM of three to five oocytes.
Figure 4. Concentration-dependent uptake of p-boronophenylalanine (BPA). Uptake of BPA was measured for 30 min at 3–1000 μM for ATB0,+ (a), LAT1 (b) and LAT2 (c). The velocity (V) of transporter-mediated uptake was determined by subtracting the uptake in non-expressing control oocytes from the uptake in oocytes expressing each transporter. Representative Michaelis–Menten fitting (left panels) and Lineweaver–Burk plot (right panels) are shown for each transporter. Each data point represents the mean ± SEM of five to seven oocytes.
Figure 5. Transporter expression and p-boronophenylalanine (BPA) uptake in cancer cell lines. (a) Expression level of LAT1 was analyzed by western blotting using crude membrane fraction. The cell lines were ordered in increasing LAT1 amounts. Na+/K+ ATPase was used as loading control. (b) Similarly, the expression of ATB0,+ was analyzed. ATB0,+ was expressed in MCF-7 cells, to a lesser degree in T3M4 cells, and not detected in the other cell lines. (c) Uptake of BPA in cell lines was measured for 5 min in the presence or absence of Na+. The presence of Na+ did not significantly affect the BPA uptakes in the cell lines. A representative result was shown with the mean ± SEM (n = 4).
Figure 6. Differential roles of ATB0,+ and LAT1 in p-boronophenylalanine (BPA) uptake. (a) Uptake of BPA in MCF-7 cells was measured for 10 min at 100 μM (left) and 1000 μM (right) BPA. At 1000 μM, the uptake rate in the presence of Na+ increased by ∽1.4-fold from the Na+-free uptake. (b) MCF-7 cells were transfected with non-targeting control siRNA #1 and #2, and LAT1-targeting siRNA #3, #4 and #5. The siRNAs #3–#5 achieved a ∽80% reduction of LAT1 protein amount. (c) Uptake by ATB0,+ was separated by LAT1 knockdown and lysine inhibition. The uptakes in the presence of Na+ and with mock knockdown were set at 1.0 at each BPA concentration (bars 1 and 4). At 100 μM BPA, the uptake levels with LAT1 knockdown did not differ regardless of Na+ (bars 2 and 3). At 1000 μM, LAT1 knockdown left a significant Na+-dependent component (bars 5 and 6). Overall, although with various statistical significances, the BPA uptake in bar 5 was inhibited by 5 mM lysine to a level similar to bar 6 (bar 7). This Na+-dependent and lysine-inhibitable component accounted for at least 20–25% of the total uptake. siRNA#1 was used for control siRNA. n.s., not significant.
Barth,
Boron neutron capture therapy of cancer: current status and future prospects.
2005, Pubmed
Barth,
Boron neutron capture therapy of cancer: current status and future prospects.
2005,
Pubmed
Barth,
Current status of boron neutron capture therapy of high grade gliomas and recurrent head and neck cancer.
2012,
Pubmed
Coderre,
Boron neutron capture therapy for glioblastoma multiforme using p-boronophenylalanine and epithermal neutrons: trial design and early clinical results.
1997,
Pubmed
Detta,
L-amino acid transporter-1 and boronophenylalanine-based boron neutron capture therapy of human brain tumors.
2009,
Pubmed
Di Pierro,
Determination of boronophenylalanine in biological samples using precolumn o-phthalaldehyde derivatization and reversed-phase high-performance liquid chromatography.
2000,
Pubmed
Fuchs,
Amino acid transporters ASCT2 and LAT1 in cancer: partners in crime?
2005,
Pubmed
Ganapathy,
Nutrient transporters in cancer: relevance to Warburg hypothesis and beyond.
2009,
Pubmed
Gupta,
Upregulation of the amino acid transporter ATB0,+ (SLC6A14) in colorectal cancer and metastasis in humans.
2005,
Pubmed
Haining,
Relation of LAT1/4F2hc expression with pathological grade, proliferation and angiogenesis in human gliomas.
2012,
Pubmed
Henriksson,
Boron neutron capture therapy (BNCT) for glioblastoma multiforme: a phase II study evaluating a prolonged high-dose of boronophenylalanine (BPA).
2008,
Pubmed
Joel,
Effect of dose and infusion time on the delivery of p-boronophenylalanine for neutron capture therapy.
1999,
Pubmed
Kaira,
Fluorine-18-alpha-methyltyrosine positron emission tomography for diagnosis and staging of lung cancer: a clinicopathologic study.
2007,
Pubmed
Kanai,
Heterodimeric amino acid transporters: molecular biology and pathological and pharmacological relevance.
2001,
Pubmed
Kanai,
Expression cloning and characterization of a transporter for large neutral amino acids activated by the heavy chain of 4F2 antigen (CD98).
1998,
Pubmed
,
Xenbase
Karunakaran,
Interaction of tryptophan derivatives with SLC6A14 (ATB0,+) reveals the potential of the transporter as a drug target for cancer chemotherapy.
2008,
Pubmed
,
Xenbase
Karunakaran,
SLC6A14 (ATB0,+) protein, a highly concentrative and broad specific amino acid transporter, is a novel and effective drug target for treatment of estrogen receptor-positive breast cancer.
2011,
Pubmed
Kato,
Effectiveness of BNCT for recurrent head and neck malignancies.
2004,
Pubmed
Khunweeraphong,
Establishment of stable cell lines with high expression of heterodimers of human 4F2hc and human amino acid transporter LAT1 or LAT2 and delineation of their differential interaction with α-alkyl moieties.
2012,
Pubmed
Laurent,
A novel human hepatoma cell line, FLC-4, exhibits highly enhanced liver differentiation functions through the three-dimensional cell shape.
2012,
Pubmed
Müller,
Imaging tumour ATB0,+ transport activity by PET with the cationic amino acid O-2((2-[18F]fluoroethyl)methyl-amino)ethyltyrosine.
2014,
Pubmed
Nakada,
Unique and selective expression of L-amino acid transporter 1 in human tissue as well as being an aspect of oncofetal protein.
2014,
Pubmed
Nawashiro,
L-type amino acid transporter 1 as a potential molecular target in human astrocytic tumors.
2006,
Pubmed
Riemann,
Kinetic parameters of 3-[(123)I]iodo-L-alpha-methyl tyrosine ([(123)I]IMT) transport in human GOS3 glioma cells.
2001,
Pubmed
Sahoo,
Membrane transporters in a human genome-scale metabolic knowledgebase and their implications for disease.
2014,
Pubmed
Segawa,
Identification and functional characterization of a Na+-independent neutral amino acid transporter with broad substrate selectivity.
1999,
Pubmed
,
Xenbase
Sloan,
Cloning and functional expression of a human Na(+) and Cl(-)-dependent neutral and cationic amino acid transporter B(0+).
1999,
Pubmed
,
Xenbase
Smith,
Quantitative imaging and microlocalization of boron-10 in brain tumors and infiltrating tumor cells by SIMS ion microscopy: relevance to neutron capture therapy.
2001,
Pubmed
Uchino,
Transport of amino acid-related compounds mediated by L-type amino acid transporter 1 (LAT1): insights into the mechanisms of substrate recognition.
2002,
Pubmed
,
Xenbase
Uhlen,
Towards a knowledge-based Human Protein Atlas.
2010,
Pubmed
Wiriyasermkul,
Transport of 3-fluoro-L-α-methyl-tyrosine by tumor-upregulated L-type amino acid transporter 1: a cause of the tumor uptake in PET.
2012,
Pubmed
Wittig,
Mechanisms of transport of p-borono-phenylalanine through the cell membrane in vitro.
2000,
Pubmed
Wyss,
Spatial heterogeneity of low-grade gliomas at the capillary level: a PET study on tumor blood flow and amino acid uptake.
2007,
Pubmed
Yokoyama,
Pharmacokinetic study of BSH and BPA in simultaneous use for BNCT.
2006,
Pubmed
Yoshida,
Enhancement of sodium borocaptate (BSH) uptake by tumor cells induced by glutathione depletion and its radiobiological effect.
2004,
Pubmed
Yoshimoto,
Predominant contribution of L-type amino acid transporter to 4-borono-2-(18)F-fluoro-phenylalanine uptake in human glioblastoma cells.
2013,
Pubmed
