L-type Amino Acid Transporter Research Articles

Branched-chain amino acids enhance cyst development in autosomal dominant polycystic kidney disease

Autosomal dominant polycystic kidney disease (ADPKD) is characterized by the progressive development of kidney and liver cysts. The mammalian target of rapamycin (mTOR) cascade is one of the important pathways regulating cyst growth in ADPKD. Branched-chain amino acids (BCAAs), including leucine, play a crucial role to activate mTOR pathway. Therefore, we administered BCAA dissolved in the drinking water to Pkd1flox/flox:Mx1-Cre (cystic) mice from four to 22 weeks of age after polyinosinic-polycytidylic acid-induced conditional Pkd1 knockout at two weeks of age. The BCAA group showed significantly greater kidney/body weight ratio and higher cystic index in both the kidney and liver compared to the placebo-treated mice. We found that the L-type amino acid transporter 1 that facilitates BCAA entry into cells is strongly expressed in cells lining the cysts. We also found increased cyst-lining cell proliferation and upregulation of mTOR and mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) pathways in the BCAA group. Invitro, we cultured renal epithelial cell lines from Pkd1 null mice with or without leucine. Leucine was found to stimulate cell proliferation, as well as activate mTOR and MAPK/ERK pathways in these cells. Thus, BCAA accelerated disease progression by mTOR and MAPK/ERK pathways. Hence, BCAA may be harmful to patients with ADPKD.

Thyroid hormone transport across L-type amino acid transporters: What can molecular modelling tell us?

Thyroid hormones (THs) and their derivatives require transmembrane transporters (TTs) to mediate their translocation across the cell membrane. Among these TTs, the L-type amino acid transporters (LAT) not only transport amino acids (AAs) but also certain THs and their derivatives.This review summarizes available knowledge concerning structure function patterns of the TH transport by LAT1 and LAT2. For example, LAT2 imports 3,3′-T2 and T3, but not rT3 and T4. In contrast to amino acids, THs are not at all exported by LAT2. Homology modelling of LAT1 and LAT2 is based on available crystal structures from the same superfamily the amino acid/polyamine/organocation transporter (APC). Molecular model guided mutagenesis has been used to predict substrate interaction sites. A common recognition feature for amino acid- and TH-derivatives has been suggested in an interior cavity of LAT1 and LAT2. Therein additional distinct molecular determinants that are responsible for the bidirectional AA transport but allowing only unidirectional import of particular THs have been confirmed for LAT2 by mutagenesis. Characterized substrate features that are needed for TH translocation and distinct LAT2 properties will be highlighted to understand the molecular import and export mechanisms of this transporter in more detail.

Inhibition of l-type amino acid transporter 1 activity as a new therapeutic target for cholangiocarcinoma treatment.

Unlike normal cells, cancer cells undergo unlimited growth and multiplication, causing them to require massive amounts of amino acid to support their continuous metabolism. Among the amino acid transporters expressed on the plasma membrane, l-type amino acid transporter-1, a Na+-independent neutral amino acid transporter, is highly expressed in many types of human cancer including cholangiocarcinoma. Our previous study reported that l-type amino acid transporter-1 and its co-functional protein CD98 were highly expressed and implicated in cholangiocarcinoma progression and carcinogenesis. Therefore, this study determined the effect of JPH203, a selective inhibitor of l-type amino acid transporter-1 activity, on cholangiocarcinoma cell inhibition both in vitro and in vivo. JPH203 dramatically suppressed [14C]l-leucine uptake as well as cell growth in cholangiocarcinoma cell lines along with altering the expression of l-type amino acid transporter-1 and CD98 in response to amino acid depletion. We also demonstrated that JPH203 induced both G2/M and G0/G1 cell cycle arrest, as well as reduced the S phase accompanied by altered expression of the proteins in cell cycle progression: cyclin D1, CDK4, and CDK6. There was also cell cycle arrest of the related proteins, P21 and P27, in KKU-055 and KKU-213 cholangiocarcinoma cells. Apoptosis induction, detected by an increase in trypan blue-stained cells along with a cleaved caspase-3/caspase-3 ratio, occurred in JPH203-treated cholangiocarcinoma cells at the highest concentration tested (100 µM). As expected, daily intravenous administration of JPH203 (12.5 and 25 mg/kg) significantly inhibited tumor growth in KKU-213 cholangiocarcinoma cell xenografts in the nude mice model in a dose-dependent manner with no statistically significant change in the animal's body weight and with no differences in the histology and appearance of the internal organs compared with the control group. Our study demonstrates that suppression of l-type amino acid transporter-1 activity using JPH203 might be used as a new therapeutic strategy for cholangiocarcinoma treatment.

Ultrastructural immunohistochemical study of L-type amino acid transporter 1-4F2 heavy chain in tumor microvasculatures of N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN) induced rat bladder carcinoma.

Angiogenesis is essential for tumor growth, and an enhanced vasculature supplying nutrients and oxygen might reflect malignant potential. L-type amino acid transporter 1 (LAT1/4F2hc) comprises a major nutrient transport system responsible for the Na+-independent transport of large neutral amino acids. Seventy five to seventy eight percent N-butyl-N-(4-hydroxybutyl) nitrosamine-induced rat bladder carcinoma cells showed high LAT1/4F2hc expression. While the intracarcinoma microvasculatures of fenestrated endothelial cells highly expressing LAT1/4F2hc might progressively transport essential amino acids from the microvasculatures to the extracellular matrix, non-fenestrated endothelial cells and pericytes did not. The present study revealed that the tumor angiogenesis is one of target anti-L-type amino acid transporter 1 drug.

Structure-activity relationship of a novel series of inhibitors for cancer type transporter L-type amino acid transporter 1 (LAT1)

L-type amino acid transporter 1 (LAT1) is known as a cancer-type amino acid transporter. In cancer cells, LAT1 is responsible for the cellular uptake of many essential amino acids including leucine that activates mechanistic/mammalian target of rapamycin (mTOR), regulating cancer cell growth. In this study, we designed a novel series of LAT1 inhibitors, SKN101–105, based on the structure of triiodothyronine (T3), a known LAT1 blocker. The compounds consist of core structure of 2-amino-3-[3,5-dichloro-4-(naphthalene-1-methoxy)-phenyl]-propanoic acid and different modifications on the naphthalene. Among them, the compounds including SKN103 with a modified phenyl group at C-7 position of naphthalene inhibited LAT1-mediated leucine transport, whereas SKN102 with a phenyl group at C-6 position did not, indicating the importance of the position of substituents on the naphthalene for the interaction with LAT1. SKN103 was suggested to be a non-transportable blocker rather than a substrate of LAT1 and inhibited LAT1 in a competitive manner with the Ki value of 2.1 μM. SKN103 suppressed mTOR activity and the growth of cancer cells. Moreover, SKN103 in combination with cisplatin additively enhanced the growth inhibition in cancer cells. This study provides an additional insight into the structure-activity relationship of LAT1 ligands, which could lead to designing desirable LAT1 inhibitors.

Low uptake of fluorodeoxyglucose in positron emission tomography/computed tomography in ovarian clear cell carcinoma may reflect glutaminolysis of its cancer stem cell-like properties.

The characteristics of ovarian cancers that showed low activation of glycolysis were investigated. Using medical records of patients with ovarian cancers who had undergone fluorodeoxyglucose positron emission tomography/computed tomography(FDG-PET/CT) prior to their primary surgery at the University of Tokyo Hospital between 2010 and2015, we identified cases with a low uptake of FDG in PET/CT. We considered the maximum standardized uptake value(SUVmax) as the degree of glucose uptake. We investigated the properties which may account for the low activation of glycolysis invitro. The expression level of alanine, serine, cysteine-preferring transporter2(ASCT2, a glutamine influx transporter), system L-type amino acid transporter1(LAT1, a glutamine efflux transporter) and glucose transporter1(GLUT1, a glucose influx transporter) were investigated by western blotting. The phosphorylation level of AMP-activated protein kinase(AMPK), which is one of the metabolic sensors, was also investigated. Most of the cases with a low uptake SUVmax were limited to patients with ovarian clear cell carcinoma(CCC). We obtained cancer stem cell(CSC)-like properties from CCC cell lines, and compared the expression levels of transporters between non-CSCs and CSCs. Whereas the expression level of ASCT2 was nearly unchanged between non-CSCs and CSCs, the expression levels of LAT1 and GLUT1 were decreased in CSCs compared to non-CSCs. The phosphorylation level of AMPK was reduced in CSCs compared to non-CSCs. Inconclusion, we suggested that ovarian CCC showed low activation of glycolysis, and this may reflect glutaminolysis of its CSC-like properties.

Molecular features of the L-type amino acid transporter 2 determine different import and export profiles for thyroid hormones and amino acids

The L-type amino acid transporter 2 (LAT2) imports amino acids (AA) and also certain thyroid hormones (TH), e.g. 3,3′-T2 and T3, but not rT3 and T4. We utilized LAT2 mutations (Y130A, N133S, F242W) that increase 3,3′-T2 import and focus here on import and export capacity for AA, T4, T3, BCH and derivatives thereof to delineate molecular features.Transport studies and analysis of competitive inhibition of import by radiolabelled TH and AA were performed in Xenopus laevis oocytes. Only Y130A, a pocket widening mutation, enabled import for T4 and increased it for T3. Mutant F242W showed increased 3,3′-T2 import but no import rates for other TH derivatives. No export was detected for any TH by LAT2-wild type (WT). Mutations Y130A and N133S enabled only the export of 3,3′-T2, while N133S also increased AA export. Thus, distinct molecular LAT2-features determine bidirectional AA transport but only an unidirectional 3,3′-T2 and T3 import.

JPH203, a selective L-type amino acid transporter 1 inhibitor, induces mitochondria-dependent apoptosis in Saos2 human osteosarcoma cells.

Most normal cells express L-type amino acid transporter 2 (LAT2). However, L-type amino acid transporter 1 (LAT1) is highly expressed in many tumor cells and presumed to support their increased growth and proliferation. This study examined the effects of JPH203, a selective LAT1 inhibitor, on cell growth and its mechanism for cell death in Saos2 human osteosarcoma cells. FOB human osteoblastic cells and Saos2 cells expressed LAT1 and LAT2 together with their associating protein 4F2 heavy chain, but the expression of LAT2 in the Saos2 cells was especially weak. JPH203 and BCH, a non-selective L-type amino acid transporter inhibitor, potently inhibited L-leucine uptake in Saos2 cells. As expected, the intrinsic ability of JPH203 to inhibit L-leucine uptake was far more efficient than that of BCH in Saos2 cells. Likewise, JPH203 and BCH inhibited Saos2 cell growth with JPH203 being superior to BCH in this regard. Furthermore, JPH203 increased apoptosis rates and formed DNA ladder in Saos2 cells. Moreover, JPH203 activated the mitochondria-dependent apoptotic signaling pathway by upregulating pro-apoptotic factors, such as Bad, Bax, and Bak, and the active form of caspase-9, and downregulating anti-apoptotic factors, such as Bcl-2 and Bcl-xL. These results suggest that the inhibition of LAT1 activity via JPH203, which may act as a potential novel anti-cancer agent, leads to apoptosis mediated by the mitochondria-dependent intrinsic apoptotic signaling pathway by inducing the intracellular depletion of neutral amino acids essential for cell growth in Saos2 human osteosarcoma cells.

Prognostic impact of LAT1 and CD98 expression in cutaneous angiosarcoma.

L-type amino acid transporter 1 (LAT1) and CD98 are frequently expressed in various human cancers, and closely correlated with tumor aggressiveness and survival. However, little is known about the expression of LAT1 and CD98 in cutaneous angiosarcoma. The aim of this study is to investigate the clinicopathological significance of these markers in the dismal disease. Atotal of 52 patients with cutaneous angiosarcoma were retrospectively reviewed. Immunohistochemical staining of tumor specimens were evaluated using anti-LAT1, CD98 and Ki-67 antibodies. The rates of high expression for LAT1 and CD98 were 56% (29/52) and 79% (41/52), respectively. The frequency of high expression for CD98 was significantly higher than that for LAT1 (p=0.021). The low expression of CD98 was significantly associated with distant metastasis (p=0.044) and was identified as asignificant prognostic predictor by multivariate analysis. The expression level of LAT1 was not significantly correlated with prognosis. The low expression of CD98 is anovel biomarker for predicting poor prognosis in patients with cutaneous angiosarcoma.

AB011. OS03.01. Expression of L-type amino acid transporter 1 is possible diagnostic and prognostic marker of thymic carcinoma

Background: According to the WHO classification, thymic epithelial tumors (TET) are classified from type A thymoma to thymic carcinoma, though for pathological diagnosis a type B3 thymoma can be difficult to distinguish from a thymic carcinoma, which is a rare malignancy and its treatment is challenging. Recent studies have demonstrated that L-type amino acid transporter 1 (LAT1) is a cancer-specific amino acid transporter on the membrane of many different types of cancer cells, while its expression has been associated with poor prognosis in cases of malignancy. The purpose is to investigate the differences in expression of LAT1 among TETs and its impact on prognosis of affected patients.

Novel therapeutic approaches targeting L-type amino acid transporters for cancer treatment.

L-type amino acid transporters (LATs) mainly assist the uptake of neutral amino acids into cells. Four LATs (LAT1, LAT2, LAT3 and LAT4) have so far been identified. LAT1 (SLC7A5) has been attracting much attention in the field of cancer research since it is commonly up-regulated in various cancers. Basic research has made it increasingly clear that LAT1 plays a predominant role in malignancy. The functional significance of LAT1 in cancer and the potential therapeutic application of the features of LAT1 to cancer management are described in this review.

Technetium-99m complexes of l-arginine derivatives for targeting amino acid transporters.

Although relevant from the clinical point of view, radiotracers targeting cationic amino acid transporters are relatively unexplored and, in particular, no metal-based radiotracers are known. The rare examples of complexes recognized by amino acid transporters, namely by the Na+-independent neutral l-type amino acid transporter 1 (LAT1), are 99mTc(i)/Re(i) compounds. Herein, we describe conjugates comprising a pyrazolyl-diamine chelating unit and the cationic amino acid l-arginine (l-Arg) linked by a propyl (L1) or hexyl linker (L2), which allowed the preparation of stable complexes of the type fac-[99mTc(CO)3(k3-L)]+ (Tc1, L = L1; Tc2, L = L2) and of the respective surrogates Re1 and Re2. Interestingly, complex Tc2 exhibited moderate levels of time-dependent internalization in three human tumoural cell lines, with approximately 3% of total applied activity internalized, corresponding to 21% of the cell-associated activity. A putative mechanism of retention in the cytoplasm of cells could be the interaction of the complex with inducible nitric oxide synthase (iNOS), which is the enzyme responsible for the catalytic oxidation of l-Arg to citrulline and nitric oxide. However, the surrogate complex Re2 does not recognize iNOS, as demonstrated by the in vitro assays with purified iNOS and in studies with lipopolysaccharide(LPS)-activated macrophages. Preliminary mechanistic studies suggest that the internalization of Tc2 is linked to the cationic amino acid transporters, namely system y+. This finding might open the way towards the development of novel families of metal-based radiotracers for probing metabolically active cancer cells.

Increased SLC7A8 expression mediates L-DOPA uptake by renal tubular epithelial cells.

The kidney serves a central role in the control of blood pressure through the release of vasoactive substances and the urinary excretion of Na+. Patients with essential hypertension usually exhibit persistent high blood pressure accompanied by Na+ retention. L-dihydroxyphenylalanine (L‑DOPA) is an amino acid, converted by the enzyme aromatic L‑amino acid decarboxylase to dopamine. The uptake of L‑DOPA by cells of the proximal tubular epithelium of the kidney is controlled by the L‑type amino acid transporter 2 (LAT2). LAT2 belongs to the solute carrier family 7 (SLC7) of amino acid transporters and is coded by the SLC7A8 gene. SLC7A8 expression is increased in the second‑order mesenteric arteries and kidneys of spontaneously hypertensive rats. The present study aimed to investigate the physiological role of the SLC7A8 gene in L‑DOPA handling by kidney cells. Selective upregulation of SLC7A8 mRNA and protein levels was achieved by adenoviral transduction of NRK‑52E cells, which retain several properties of proximal tubular epithelial cells. In addition, L‑DOPA uptake was determined using high performance liquid chromatography; NRK‑52E cells expressing SLC7A8 exhibited increased uptake of L‑DOPA. The results of the present study suggested that SLC7A8 may serve a critical role in blood pressure control through regulating L‑DOPA uptake in renal epithelial cells of the proximal tubule.

Gastrin stimulates renal dopamine production by increasing the renal tubular uptake of l-DOPA.

Gastrin is a peptide hormone that is involved in the regulation of sodium balance and blood pressure. Dopamine, which is also involved in the regulation of sodium balance and blood pressure, directly or indirectly interacts with other blood pressure-regulating hormones, including gastrin. This study aimed to determine the mechanisms of the interaction between gastrin and dopamine and tested the hypothesis that gastrin produced in the kidney increases renal dopamine production to keep blood pressure within the normal range. We show that in human and mouse renal proximal tubule cells (hRPTCs and mRPTCs, respectively), gastrin stimulates renal dopamine production by increasing the cellular uptake of l-DOPA via the l-type amino acid transporter (LAT) at the plasma membrane. The uptake of l-DOPA in RPTCs from C57Bl/6J mice is lower than in RPTCs from normotensive humans. l-DOPA uptake in renal cortical slices is also lower in salt-sensitive C57Bl/6J than in salt-resistant BALB/c mice. The deficient renal cortical uptake of l-DOPA in C57Bl/6J mice may be due to decreased LAT-1 activity that is related to its decreased expression at the plasma membrane, relative to BALB/c mice. We also show that renal-selective silencing of Gast by the renal subcapsular injection of Gast siRNA in BALB/c mice decreases renal dopamine production and increases blood pressure. These results highlight the importance of renal gastrin in stimulating renal dopamine production, which may give a new perspective in the prevention and treatment of hypertension.

Effect of branched-chain amino acid ratio on the proliferation, differentiation, and expression levels of key regulators involved in protein metabolism of myocytes

ObjectivesBranched-chain amino acids (BCAAs), including leucine (Leu), isoleucine (Ile), and valine (Val), are key regulators of protein synthesis in muscle. The aim of this study was to investigate the effect of different BCAA ratios (Leu:Ile:Val) on the proliferation, differentiation, and expression levels of the regulators related to protein metabolism of C2 C12 myocytes. MethodsStudies were conducted in C2C12 myocytes exposed to different BCAA ratios (Leu: Ile: Val = 0, 1:0.25:0.25, 1:1:1). ResultsThe ratio of 1:0.25:0.25 increased cell viability and promoted cell cycle progression from G0/G1 phase to S phase, which was an indicator of proliferation enhancement (P < 0.05). Moreover, this optimal ratio (1:0.25:0.25) promoted the differentiation of myocytes into myotubes by upregulating myogenin and interleukin-15 gene expression, and differently regulated the expression of L-type amino acid transporter 1 and 4 and system ASC amino acid transporters 2. Furthermore, the ratio stimulated mTOR expression at the mRNA and phosphorylated protein levels, as well as ribosomal protein S6 kinase and regulatory-associated protein of mTOR (raptor). In contrast, the optimal ratio decreased the amount of ubiquitin ligase muscle-specific RING finger 1 and muscle atrophy F-box during proliferation and differentiation (P < 0.05). No change was observed in the expression of key genes related to energy metabolism except for uncoupling protein 3 (P > 0.05). ConclusionsThe results suggested that appropriate BCAA ratios could enhance proliferation and differentiation of the C2 C12 myocytes, also mediate the key regulators related to protein metabolism including the mTORC1 pathway. A proper utilization of balanced BCAA ratio in food would be beneficial to human and animal nutrition.

May glutamine addiction drive the delivery of antitumor cisplatin-based Pt(IV) prodrugs?

A small series of Pt(IV) prodrugs containing Gln-like (Gln=glutamine) axial ligands has been designed with the aim to take advantage of the increased demand of Gln showed by some cancer cells (glutamine addiction). In complex 4 the Gln, linked through the α-carboxylic group is recognized by the Gln transporters, in particular by the solute carrier transporter SLC1A5. All compounds showed cellular accumulation, as well as antiproliferative activity, related to their lipophilicity, as already demonstrated for the majority of Pt(IV) prodrugs, that enter cells mainly by passive diffusion. On the contrary, when the Gln concentration in cell medium is near or lower to the physiological value, complex 4 acts as a Trojan horse: it enters SLC1A5-overexpressing cells, where, upon reduction, it releases the active metabolite cisplatin and the Gln-containing ligand, thus preventing any possible extrusion by the L-type amino acid transporter LAT1. This selective mechanism could decrease off-target accumulation of 4 and, consequently, Pt-associated side-effects.

Dihydro‐β‐agarofurans from the Australian Endemic Rainforest Plant Denhamia pittosporoides Inhibit Leucine Transport in Prostate Cancer Cells

AbstractTwo previously unknown dihydro‐β‐agarofuran sesquiterpenoids, denhaminol I (1) and denhaminol J (2), together with four related and known metabolites, 1α,2α,6β,15‐tetraacetoxy‐9α‐benzoyloxy‐8‐oxodihydro‐β‐agarofuran (3), wilforsinine F (4), 1α,2α,6β,8α,15‐pentaacetoxy‐9α‐benzoyloxydihydro‐β‐agarofuran (5), and 1α,2α,6β,15‐tetraacetoxy‐9β‐benzoyloxydihydro‐β‐agarofuran (6), were isolated from the leaves of an Australian rainforest plant, Denhamia pittosporoides. The structures of compounds 1 and 2 were determined by analysis of their 1D/2D NMR and MS data. The absolute configuration of compound 1 was established by single‐crystal X‐ray diffraction analysis. Compounds 1 and 4 were shown to inhibit leucine transport in the human prostate cancer cell line LNCaP, with IC50 values of 51.5 and 95.5 μm, respectively. Both compounds 1 and 4 were more potent than the l‐type amino acid transporter (LAT) family inhibitor 2‐aminobicyclo[2.2.1]‐heptane‐2‐carboxylic acid (BCH). This is the first report of dihydro‐β‐agarofurans from D. pittosporoides.

Positive Regulation of Decidualization by l-Type Amino Acid Transporter 1 (lat1) in Pregnant Mice.

Amino acids have an important role in the pre and post implantation of placenta and embryo development. l-type amino-acid transporter 1 (lat1) is responsible for the transportation of large neutral amino acids and is mainly expressed in human fetal liver, placenta, brain, etc. This study is the first to investigate the expression of lat1 in the early pregnancy of mouse uteri and its role in the process of decidualization. Endometrial stromal cells of a mouse model were used to evaluate decidualization from Day 4–8 of pregnancy in vitro followed by lat1 knock down by small interfering RNA and by a competitive inhibitor of Leucine transport 2-aminobicyclo-(2,2,1)-heptane-2-carboxylic acid (BCH). The effects of lat1 on decidualization in vivo were assessed by injecting BCH into the uterine horns. The mRNA and protein expressions of lat1 in the implantation sites were higher than that in the inter-implantation sites and were localized in the luminal and gland epithelium, stromal and decidual cells. Its increased expression (p < 0.05) was associated with artificial decidualization as well as activation of prl expression. Down-regulation of lat1 expression in these cells by siRNA and BCH inhibited the decidual progression in vitro. Inhibition of lat1 transportation by BCH controlled decidual progression in vivo also accompanied the down-regulation of prl, lat1 expression in the decidual area and embryo size on Day 8 of pregnancy. In conclusion, these results revealed that lat1 might play an important role in the decidual progression both in vitro and in vivo.

Involvement of l-type amino acid transporter 1 in the transport of gabapentin into human placental choriocarcinoma cells

Gabapentin (GBP) is a widely used antiepileptic drug, with potential for use in the treatment of epilepsy in pregnant women. Although studies have examined GBP transport mechanisms across the blood-brain barrier, kidney, and intestine, the mechanism in the placenta has not been fully elucidated. We previously reported that GBP accumulates at high concentrations in human placental choriocarcinoma BeWo cells. The purpose of this study was to examine the transport mechanism of GBP in placental choriocarcinoma cells (BeWo and JEG-3), and to identify the carrier involved. High concentrations of intracellular GBP accumulations were also found in JEG-3 cells. A kinetic analysis showed that a single carrier system was involved in the uptake of GBP. Furthermore, substrates for l-type amino acid transporter (LAT) and siRNAs targeted to LAT1 significantly decreased GBP uptake. Our observations from this study suggest that LAT1 is the main contributor to GBP transport in placental choriocarcinoma cells.

Prostate Cancer Cells in Different Androgen Receptor Status Employ Different Leucine Transporters

Leucine stimulates cancer cell proliferation through the mTOR pathway, therefore, inhibiting leucine transporters may be a novel therapeutic target for cancer. L-type amino acid transporter (LAT) 1, a Na+ -independent amino acid transporter, is highly expressed in many tumor cells. However, leucine transporter(s) in different stages of prostate cancer, particularly in the stages of castration resistance with androgen receptor (AR) expression, is unclear. LNCaP and DU145 and PC-3 cell lines were used as a model of androgen dependent, and metastatic prostate cancer. A new "LN-cr" cell line was established after culturing LNCaP cells for 6 months under androgen-free conditions, which is considered a model of castration resistant prostate cancer (CRPC) with androgen AR expression. The expression of leucine transporters was investigated with quantitative PCR and immunofluorescence. Uptake of 14 C Leucine was examined in the presence or absence of BCH (a pan-LAT inhibitor), JPH203 (an LAT1-specific inhibitor), or Na+ . Cell growth was assessed with MTT assay. siRNA studies were performed to evaluate the indispensability of y+ LAT2 on leucine uptake and cell viability in LN-cr. Cell viability showed a 90% decrease in the absence of leucine in all four cell lines. LNCaP cells principally expressed LAT3, and their leucine uptake was more than 90% Na+ -independent. BCH, but not JPH203, inhibited leucine uptake, and cell proliferation (IC50BCH :15 mM). DU145 and PC-3 cells predominantly expressed LAT1. Leucine uptake and cell growth were suppressed by BCH or JPH203 in a dose-dependent manner (IC50BCH : ∼20 mM, IC50JPH203 : ∼5 µM). In LN-cr cells, Na+ -dependent uptake of leucine was 3.8 pmol/mgprotein/min, while, Na+ -independent uptake was only 0.52 (P < 0.05). Leucine uptake of LN-cr was largely (∼85%) Na+ -dependent. y+ LAT2 expression was confirmed in LN-cr. Knockdown of y+ LAT2 lead to significant leucine uptake inhibition (40%) and cell growth inhibition (20%). New CRPC cell line with increased expression of y+ LAT2 as a leucine transporter was established in vitro. Anti-leucine transporter therapy could be an important option against prostate cancer. Prostate 77:222-233, 2017. © 2016 Wiley Periodicals, Inc.