L-type Amino Acid Transporter 1 Expression Research Articles

Correlation of high LAT1 expression with the prognosis of endometrioid carcinoma of the uterine corpus.

The expression of L-type amino acid transporter 1 (LAT1) has been described to play essential roles in cancer cell growth and survival. To determine the significance of LAT1 in the prognosis of endometrial endometrioid carcinoma, we investigated LAT1 expression in 353 endometrioid carcinoma patients by immunohistochemical analysis using tissue microarray. The tumors in which stained tumor cells made up more than 25% of the tumor were graded as high expression. High expression of LAT1 was detected in 29 (8.2%) of patients. The ratio of high LAT1 expression did not significantly differ by age (< 60 vs. ≥ 60), FIGO stage (stage I/II vs. III/IV), histological grade (grade 1 vs. grade 2/3), or lymph node metastasis (positive vs. negative). However, high LAT1 expression in endometrioid carcinoma was associated with a poorer progression-free survival and overall survival, as per the results of the log-rank test (P = 0.0263 and 0.0404, respectively). Cox univariate and multivariate analyses revealed that high LAT1 expression is an independent marker of poor progression-free survival (hazard ratio = 2.598, P = 0.0137), in addition to a higher age (≥ 60years vs. < 60years), FIGO stage (stage III/IV vs. I/II), and histological grade (grade 2/3 vs. grade 1). In conclusion, we demonstrate that LAT1 is associated with a poor prognosis of endometrioid carcinoma of the uterine corpus.

Prenatal dexamethasone exposure exerts sex-specific effect on placental oxygen and nutrient transport ascribed to the differential expression of IGF2.

BackgroundClinical studies have showed that dexamethasone exposure during pregnancy could cause fetal growth retardation, but the mechanism by which prenatal dexamethasone exposure influences placental nutrient transport is still unclear. This study investigated the impacts of prenatal dexamethasone on the placental oxygen and nutrient transport.MethodsPregnant Wistar rats were subcutaneously administered with dexamethasone from day 9 to day 20 of gestation at 0.2 or 0.8 mg/kg·d. Pregnant rats were sacrificed on gestational day 20. The placental tissue was collected for analysis.ResultsPrenatal dexamethasone exposure (PDE) declined the fetal weight and increased the intrauterine growth retardation (IUGR) rate in a dose-dependent manner. The total placental volume and the length, density and surface area of fetal capillaries in the labyrinth zone reduced in a dose-dependent manner. In addition, the thickness of syncytial membrane dose-dependently increased, resulting in a dose-dependent decrease in oxygen diffusion capacity. Furthermore, after PDE, the nutrient transport area and oxygen diffusion capacity of male placenta were lower than that of female placenta. The mRNA and protein expression of placental nutrient transporters including glucose transporter 1 (GLUT1), glucose transporter 3 (GLUT3), L-type amino acid transporter 1 (LAT1), lipoprotein lipase (LPL) and scavenger receptor class B type 1 (SRB1) increased in female placenta. However, in male placenta, the expression of LAT1, LPL and SRB1 was significantly decreased and GLUT1 and GLUT3 have a decrease trend. We further investigated the expression of insulin-like growth factor 1 (IGF1) and insulin-like growth factor 2 (IGF2) related to placental and fetal growth and development. Our study showed that the expression of IGF1 was significantly decreased both in male and female placentas after PDE. But the expression of IGF2 was significantly increased in female placentas while significantly decreased in male placentas.ConclusionsOur study shows prenatal dexamethasone exposure exerts sex-specific influence on the placental oxygen and nutrient transport. This might be ascribed to the differential expression of IGF2 after PDE. These findings provide evidence on the dexamethasone-induced toxicity to the placenta and fetal development.

Expression of L-type amino acid transporter 1 as a molecular target for prognostic and therapeutic indicators in bladder carcinoma

L-type amino acid transporter 1 (LAT1) plays a role in transporting essential amino acids including leucine, which regulates the mTOR signaling pathway. Here, we studied the expression profile and functional role of LAT1 in bladder cancer. Furthermore, the pharmacological activity of JPH203, a specific inhibitor of LAT1, was studied in bladder cancer. LAT1 expression in bladder cancer cells was higher than that in normal cells. SiLAT1 and JPH203 suppressed cell proliferative and migratory and invasive abilities in bladder cancer cells. JPH203 inhibited leucine uptake by > 90%. RNA-seq analysis identified insulin-like growth factor-binding protein-5 (IGFBP-5) as a downstream target of JPH203. JPH203 inhibited phosphorylation of MAPK / Erk, AKT, p70S6K and 4EBP-1. Multivariate analysis revealed that high LAT1 expression was found as an independent prognostic factor for overall survival (HR3.46 P = 0.0204). Patients with high LAT1 and IGFBP-5 expression had significantly shorter overall survival periods than those with low expression (P = 0.0005). High LAT1 was related to the high Grade, pathological T stage, LDH, and NLR. Collectively, LAT1 significantly contributed to bladder cancer progression. Targeting LAT1 by JPH203 may represent a novel therapeutic option in bladder cancer treatment.

Evaluation of D-isomer of 18F-FBPA for oncology PET focusing on the differentiation of glioma and inflammation.

L-4-borono-2-18F-fluoro-phenylalanine (L-[18F]FBPA), a substrate of L-type amino acid transporter 1 (LAT1), is a tumor-specific probe used in positron emission tomography (PET). On the other hand, it has not been examined whether another isomer D-[18F]FBPA accumulates specifically in the tumor. Here, we compared the accumulation of D-[18F]FBPA in C6 glioma and inflammation to evaluate the performance of D-[18F]FBPA as a tumor-specific probe. HEK293-LAT1 and HEK293-LAT2 cells were tested for [14C]-leucine or [14C]-alanine transport, and IC50 values of L- and D-FBPA were evaluated in both cell types. PET was conducted in rat xenograft model of C6 glioma with LAT1 expression and model of turpentine oil-induced subcutaneous inflammation (n=10 for both models). The concentrations of D-[18F]FBPA were compared between glioma and inflammatory lesion using standardized uptake value (SUV). In contrast to L-FBPA, which inhibited substrate uptake in both HEK293-LAT1 and -LAT2 cells, D-FBPA showed no inhibitory effect on both cells, suggesting low transporter selectivity of D-[18F]FBPA against LAT1 and LAT2. Static PET analysis showed low accumulation of D-[18F]FBPA in C6 glioma and inflammatory lesion (SUVmax=0.80±0.16, 0.56±0.09, respectively). Although there was a statistical difference in SUVmax between these tissues, it was difficult to distinguish glioma from inflammation on the PET image due to its low uptake level. Therefore, it was suggested that D-[18F]FBPA is not a suitable tumor-specific probe for oncology PET in contrast to L-[18F]FBPA. This study demonstrated that D-[18F]FBPA is not a LAT1-specific PET probe and shows low uptake in C6 glioma, indicating its unsuitability as a tumor diagnosis PET probe.

NI-11 PATHOLOGICAL ASSESSMENT IN THE IMPACT OF ONE-SHOT BEVACIZUMAB ON UPTAKE OF 11C-METHIONINE

BACKGROUNDThis study aimed to clarify how one-time administration of bevacizumab (BEV) changes histological features in glioblastoma, and how histological changes affect the uptake of 11C-methyl-L-methionine (11C-met) as an amino-acid tracer.MATERIALS AND METHODSSubjects were 18 patients with newly diagnosed glioblastoma who were assigned to 2 groups: BEV group, single intravenous administration of BEV before surgical tumor removal; and control group, surgical tumor removal alone. After surgery, we compared the densities of tumor cells and microvessels, and microvascular structures including vascular pericytes and L-type amino acid transporter-1 (LAT1), between the BEV and control groups. Correlations between 11C-met uptake on positron emission tomography before surgery, microvascular density, and LAT1 expression were assessed in each group.RESULTSBEV induced significant reductions in microvascular density, while tumor cell density and proliferation were retained in the BEV group. Percentages of vessels with pericytes and vascular endothelium with LAT1 expression were lower in the BEV group than in controls. Uptake of 11C-met correlated significantly with microvascular density in the BEV group, but not with LAT1expression.CONCLUSIONSThe present study showed that even one course of BEV administration induced reductions in microvessels, vascular pericytes, and LAT1 expression in glioblastomas. One course of BEV therapy also reduced 11C-met uptake, which might have been largely attributed to reductions in microvessels rather than reductions in LAT1 expression, in addition to reduction of vascular permeability.

Role of L-Type Amino Acid Transporter 1 (LAT1) for the Selective Cytotoxicity of Sesamol in Human Melanoma Cells.

Sesamol is effective against melanoma cells with less damage to normal cells. The underlying selective cytotoxicity of sesamol in melanoma vs. non-cancerous cells is undefined. Melanoma cells differ from normal cells by over-expression of the L-type amino acid transporter 1 (LAT1). We sought to clarify the transport mechanism on selective cytotoxicity of sesamol in melanoma cells. A human melanoma cell line (SK-MEL-2) and African monkey epithelial cell line (Vero) were used to study the cellular uptake and cytotoxicity of sesamol. The intracellular concentration of sesamol was quantified by UV-HPLC. The cytotoxicity was determined by neutral red uptake assay. Sesamol showed a higher distribution volume and uptake clearance in SK-MEL-2 than Vero cells. Sesamol was distributed by both carrier-mediated and passive transport by having greater carrier-mediated transport into SK-MEL-2 cells than Vero cells. Higher mRNA expression and function of LAT1 over LAT2 were evident in SK-MEL-2 cells compared to Vero cells. Sesamol uptake and sesamol cytotoxicity were inhibited by the LAT1 inhibitor, suggesting LAT1 had a role in sesamol transport and its bioactivity in melanoma. The LAT1-mediated transport of sesamol is indicative of how it engages cytotoxicity in melanoma cells with promising therapeutic benefits.

High expression of L-type amino acid transporter 1 is associated with colorectal cancer metastasis

Objective To investigate the expression and significance of L-type amino acid transporter 1 (LAT1) in human colorectal cancer. Methods Sixty pairs of colorectal cancer tissues and paracancerous tissues were collected from sixty advanced colorectal cancer (CRC) patients for detecting LAT1 expression. Real-time quantitative reverse transcriptase-polymerase chain reaction (RT-qPCR) and immunohistochemical analysis were used to analyze the expression of LAT1 in colorectal cancer tissues and adjacent normal tissues. In addition, we also study the relationship between LAT1 expression and the clinical features of sixty patients with colorectal cancer. Results Bioinformatics analysis through GEPIA database suggested that LAT1 mRNA expression was elevated in colorectal cancer. We also observed markedly increased LAT1 mRNA and protein expression in colorectal cancer compared with adjacent normal tissues (P<0.01). In addition, we found high expression of LAT1 protein was closely related to lymphatic metastasis (P<0.05), liver metastasis (P<0.05), and TNM stage (P<0.05). Conclusion LAT1 expression is increased in colorectal cancer and asscciated with tumor metastasis. Key words: L-type amino acid transporter 1; Colorectal cancer; Lymphatic metastasis; Liver metastasis; TNM stage

L-type amino-acid transporter 1 (LAT1) expression is predictive of response to autologous stem cell transplantation for multiple myeloma

L-type amino acid transporter 1 (LAT1) plays a key role in cell growth and survival. Overexpression is a common feature of many malignancies to support increased protein synthesis demand. Among patients with multiple myeloma, high LAT1 expression has been shown to be associated higher risk disease but also a higher response rate following treatment with oral melphalan in clinical and pre-clinical models (Isoda, et al, Cancer Sci, 2014; Hathi, et al, J Nucl Med, 2018). Oral melphalan is infrequently used in today's clinical practice; however, high-dose intravenous melphalan is the standard of care for conditioning prior to autologous stem cell transplantation (ASCT).

Impacts on Histological Features and 11C-Methyl-L-methionine Uptake After “One-Shot” Administration with Bevacizumab Before Surgery in Newly Diagnosed Glioblastoma

BACKGROUND: Bevacizumab (BEV), an antiangiogenic agent, induces dramatic normalization of the tumor vasculature in glioblastoma. This study aimed to clarify how one-time administration of BEV changes histological features in glioblastoma and how histological changes affect the uptake of 11C-methyl-L-methionine (11C-met) as an amino-acid tracer. MATERIALS AND METHODS: Subjects were 18 patients with newly diagnosed glioblastoma who were assigned to two groups: BEV group, single intravenous administration of BEV before surgical tumor removal; and control group, surgical tumor removal alone. After surgery, we compared the densities of tumor cells and microvessels, and microvascular structures including vascular pericytes and L-type amino acid transporter-1 (LAT1) between the BEV and control groups. Correlations between 11C-met uptake on positron emission tomography before surgery, microvascular density, and LAT1 expression were assessed in each group. RESULTS: BEV induced significant reductions in microvascular density, while tumor cell density and proliferation were retained in the BEV group. Percentages of vessels with pericytes and vascular endothelium with LAT1 expression were lower in the BEV group than in controls. Uptake of 11C-met correlated significantly with microvascular density in the BEV group but not with LAT1expression. CONCLUSIONS: The present study showed that even one course of BEV administration induced reductions in microvessels, vascular pericytes, and LAT1 expression in glioblastomas. One course of BEV therapy also reduced 11C-met uptake, which might have been largely attributed to reductions in microvessels rather than reductions in LAT1 expression.

Correlation between 18F-1-amino-3-fluorocyclobutane-1-carboxylic acid (18F-fluciclovine) uptake and expression of alanine-serine-cysteine-transporter 2 (ASCT2) and L-type amino acid transporter 1 (LAT1) in primary prostate cancer

PurposeTo evaluate the expression of alanine-serine-cysteine-transporter 2 (ASCT2) and L-type amino acid transporter1 (LAT1) in prostate cancer (PCa) and their impact on uptake of 18F-1-amino-3-fluorocyclobutane-1-carboxylic acid (18F-fluciclovine) which is approved for the detection of recurrent PCa.MethodsTwenty-five hormone-naïve patients with histologically confirmed PCa underwent PET/CT before prostatectomy. Dynamic imaging was performed immediately after injection of 368 ± 10 MBq of 18F-fluciclovine and the uptake in PCa was expressed as SUVmax at six sequential 4-min time frames and as tracer distribution volume (VT) using Logan plots over 0–24 min. The expression of ASCT2 and LAT1 was studied with immunohistochemistry (IHC) on a tissue microarray (TMA) containing three cores per carcinoma lesion. The TMA slides were scored independently by two trained readers based on visual intensity of ASCT2/LAT1 expression on a four-tiered scale. The correlations between ASCT2/LAT1 staining intensity, SUVmax/VT, and Gleason grade group (GGG) were assessed using Spearman’s rank correlation coefficient (ρ).ResultsForty tumor foci (> 0.5 mm in diameter, max. 3 per patient) were available for TMA. In visual scoring, low, moderate, and high staining intensity of ASCT2 was observed in 4 (10%), 24 (60%), and 12 (30%) tumors, respectively. No tumors showed high LAT1 staining intensity while moderate intensity was found in 10 (25%), 25 (63%) showed low, and the remaining 5 (12%) were negative for staining with LAT1. Tumors with GGG > 2 showed significantly higher uptake of 18F-fluciclovine and higher LAT1 staining intensity (p < 0.05). The uptake of 18F-fluciclovine correlated significantly with LAT1 expression (ρ = 0.39, p = 0.01, for SUVmax at 2 min and ρ = 0.39, p = 0.01 for VT). No correlation between ASCT2 expression and 18F-fluciclovine uptake or GGG was found.ConclusionsOur findings suggest that LAT1 is moderately associated with the transport of 18F-fluciclovine in local PCa not exposed to hormonal therapy. Both high and low Gleason grade tumors express ASCT2 while LAT1 expression is less conspicuous and may be absent in some low-grade tumors. Our observations may be of importance when using 18F-fluciclovine imaging in the planning of focal therapies for PCa.

Role of Amino Acid Transporter Expression as a Prognostic Marker in Patients With Surgically Resected Colorectal Cancer.

L-type amino acid transporter 1 (LAT1) is highly expressed in various human cancers. However, the clinicopathological significance of LAT1 and 4F2 cell surface antigen (4F2hc) in patients with colorectal cancer (CRC) is unknown. The aim of this study was to clarify the prognostic significance of LAT1 expression in CRC patients who underwent surgical resection. Samples from one hundred and forty-seven patients were examined by immunohistochemistry. The expression of LAT1 and 4F2hc, and the Ki-67 labeling index were assessed using resected tumor specimens. The positive expression of LAT1 and 4F2c was 80% (118/147) and 58% (86/147) (p<0.01), respectively. The expression of LAT1 was identified as an independent significant marker linked to worse prognosis in patients with CRC, and was correlated with tumor cell proliferation, tumor aggressiveness, and metastasis. Moreover, LAT1 was closely associated with the expression of 4F2hc and phosphorylation of the mTOR pathway. LAT1 is a significant molecular marker used to predict prognosis after surgical resection of CRC patients.

Bacillus amyloliquefaciens Ameliorates H2O2-Induced Oxidative Damage by Regulating Transporters, Tight Junctions, and Apoptosis Gene Expression in Cell Line IPEC-1

Probiotics have always been considered as a supplementary therapy for many diseases especially gut disorders. The absorption and barrier function of the gut play a vital role in the maintenance of body homeostasis. This study was to investigate the protective effects of Bacillus amyloliquefaciens SC06 (Ba) on H2O2-induced oxidative stress on intestinal porcine epithelial cells (IPEC-1) based on the level of gene expression. We demonstrated that Ba was a safe probiotic strain in the first place. Results showed that treatment with H2O2 significantly increased the mRNA expression of absorptive transporters glucose transporter 2 (GLUT2), Ala/Ser/Cys/Thr transporter 1 (ASCT1), and ASCT2 compared with the control group. Meanwhile, oxidative stress induced a significant improvement in the mRNA expression of occludin (OCLN) and caspase-3, and remarkably inhibited the expression of L-type amino acid transporter 1 (LAT1) or B cell lymphoma-2 (Bcl-2), respectively. Pretreatment with Ba dramatically reversed the disturbance induced by oxidative stress on the mRNA expression of ASCT1, ASCT2, and OCLN, which also significantly prevented H2O2-inhibited LAT1 and Bcl-2 mRNA expression. However, Ba failed to exert any significant protective effect on GLUT2 and caspase-3 mRNA expression. We concluded that pretreatment with Ba could alleviate the damage caused by oxidative stress to a certain extent and conferred a protective effect to the intestine.

Distribution of LAT1-targeting PET tracer was independent of the tumor blood flow in rat xenograft models of C6 glioma and MIA PaCa-2.

L-type amino acid transporter 1 (LAT1) is strongly expressed on the cell membrane in various types of human cancer cells, while being minimally expressed in normal or inflammatory tissues. Therefore, LAT1-targeting PET tracers have been developed for cancer-specific imaging. The purpose of this study was to study the distribution of two LAT1-targeting PET tracers, L-4-borono-2-18F-fluoro-phenylalanine (18F-FBPA) and L-3-18F-alpha-methyl tyrosine (18F-FAMT), in relation to the tumor blood flow, using rat xenograft models. Rat tumor xenograft models of C6 glioma (n = 4; tumors = 8) and MIA PaCa-2 (pancreatic cancer) (n = 4; tumors = 6) were used. The expressions of LAT1 and CD98hc were evaluated by both immunofluorescence staining and western blot analysis. Dynamic PET was performed after injection of 18F-FAMT or 18F-FBPA (scan duration = 70min) following 15O-water PET (scan duration = 10min). The PET data were subjected to kinetic analyses, and the K1, k2, and total distribution volume (Vt) were calculated using the one-tissue compartment model. The accumulation of the LAT1 tracers was expressed in terms of their Vt. Tumor blood flow (TBF) was represented by the K1 value in 15O-water PET. LAT1/CD98hc expression was confirmed in both xenografts by immunofluorescence staining. Western blot analysis showed higher functional expression of LAT1 in the C6 glioma cells as compared to the MIA PaCa-2 cells (C6 glioma/MIA PaCa-2 relative expression ratio = 1.70). The Vt values of both 18F-FBPA and 18F-FAMT were significantly higher in the C6 glioma xenografts than in the MIA PaCa-2 xenografts (C6 glioma: 2.27 ± 0.35 and 2.03 ± 0.23, respectively; MIA PaCa-2: 1.28 ± 0.26 and 1.35 ± 0.15, respectively). Meanwhile, there was no significant correlation of the Vt value of either 18F-FBPA or 18F-FAMT with the TBF, in either the C6 glioma or the MIA PaCa-2 xenografts. This study revealed that total distribution volumes of the LAT1-targeting PET tracers 18F-FBPA and 18F-FAMT were independent of the tumor blood flow and might reflect the functional expression levels of LAT1 in the C6 glioma and MIA PaCa-2 xenograft models.

Significant relationship between the LAT1 expression pattern and chemoresistance in ovarian clear cell carcinoma.

L-type amino acid transporter 1 (LAT1) is a Na+-independent neutral amino acid transporter that plays a key role in cancer cell growth and survival. To determine the significance of LAT1 in prognosis and resistance to chemotherapy in ovarian carcinoma, we investigated the LAT1 expression in 245 ovarian carcinoma patients by immunohistochemistry using tissue microarray. High expression of LAT1 was detected in 85 (34.7%) patients. The ratio of high expression of LAT1 was significantly high in clear cell carcinoma and low in serous carcinoma compared to other histological types (P < 0.0001). High expression of LAT1 in ovarian carcinoma was associated with poorer prognosis as per log-rank test (P = 0.008). Cox's univariate and multivariate analysis revealed that high expression of LAT1 is an independent marker indicating poor prognosis (hazard ratio = 2.810, P < 0.0001) as well as the FIGO stage III/IV (vs. I/II) and suboptimal surgery. High LAT1 expression was also found to be associated with resistance to chemotherapy (P = 0.016) notably in clear cell carcinoma. In conclusion, we demonstrate that LAT1 is not only associated with poor prognosis of ovarian carcinoma, but also associated with chemoresistance in ovarian carcinoma.

Different Response Profiles of Gastrointestinal Cancer Cells to an L-Type Amino Acid Transporter Inhibitor, JPH203.

L-type amino acid transporter 1 (LAT1) is a promising molecular target for cancer therapy. The present study aimed to characterize the anti-cancer effects of JPH203, an LAT1-selective inhibitor, on gastrointestinal cancer cells. Three esophageal, two gastric, and two colon cancer cell lines were used. Cytotoxic effects of JPH203 were assessed by a WST-8 assay. LAT1 mRNA levels were determined by quantitative PCR. The inhibitory property of JPH203 against LAT1 function was examined by a transport assay. JPH203 treatment significantly reduced the viability of all gastric and colon cancer cells. While LAT1 expression levels and inhibitory potencies of JPH203 on LAT1 functions were comparable among the cells, all the esophageal cells were resistant to JPH203. JPH203 was effective in reducing gastric and colon cancer cells. To clarify its cell type-dependent efficacy, identification of the causal factors for JPH203 resistance will be needed.

Expression of amino acid transporter (LAT1 and 4F2hc) in pulmonary pleomorphic carcinoma

Amino acid transporters are necessary for tumor growth, metastasis, and survival of various neoplasms; however, the clinicopathological significance of L-type amino acid transporter 1 (LAT1) and 4F2 cell surface antigen (4F2hc) in patients with pulmonary pleomorphic carcinoma (PPC) remainsunknown. The aim of this study is to clarify the prognostic impact of these amino acid transporters in PPC. One hundred five patients with surgically resected PPC were assessed by immunohistochemistry. The expression of LAT1 and 4F2hc, and Ki-67 labeling index were investigated using specimens of the resected tumors. LAT1 and 4F2hc were highly expressed in 35% and 53% of all patients (n = 105, P < .01), 25% and 48% of patients with an adenocarcinoma component (n = 48, P = .02), and 44% and 58% of patients with a nonadenocarcinoma component (n = 57, P = .18), respectively. A high LAT1 expression was significantly related to advanced disease, lymphatic permeation, tumor cell proliferation, and 4F2hc expression. By multivariate analysis, LAT1 and 4F2hc were identified as significant independent markers for predicting a worse prognosis. LAT1 is highly expressed in PPC, and high LAT1 expression can serve as a significant predictor linked to a worse prognosis in patients with PPC.

Imaging Melphalan Therapy Response in Preclinical Extramedullary Multiple Myeloma with 18F-FDOPA and 18F-FDG PET.

Multiple myeloma (MM) is a debilitating neoplasm of terminally differentiated plasma B cells that resulted in over 13,000 deaths in 2017 alone. Combination therapies involving melphalan, a small-molecule DNA alkylating agent, are commonly prescribed to patients with relapsed or refractory MM, necessitating the stratification of responding patients to minimize toxicities and improve quality of life. Here, we evaluated the use of 3,4-dihydroxy-6-18F-fluoro-l-phenylalanine (18F-FDOPA), a clinically available PET radiotracer with specificity to the L-type amino acid transporter 1 (LAT1), which also mediates melphalan uptake, for imaging melphalan therapy response in a preclinical immunocompetent model of MM. Methods: C57BL/KaLwRij mice were implanted subcutaneously with unilateral murine green fluorescent protein-expressing 5TGM1 tumors and divided into 3 independent groups: untreated, treated beginning week 2 after tumor implantation, and treated beginning week 3 after tumor implantation. The untreated and week 2 treated groups were imaged with preclinical MRI and dynamic 18F-FDG and 18F-FDOPA PET/CT at week 4 on separate, contiguous days, whereas the week 3 treated group was longitudinally imaged weekly for 3 wk. Metabolic tumor volume, total lesion avidity, SUVmax, and total uptake were calculated for both tracers. Immunohistochemistry was performed on representative tissue from all groups for LAT1 and glucose transporter 1 (GLUT1) expression. Results: Melphalan therapy induced a statistically significant reduction in lesion avidity and uptake for both 18F-FDG and 18F-FDOPA. There was no visible effect on GLUT1 expression, but LAT1 density increased in the week 2 treated group. Longitudinal imaging of the week 3 treated group showed variable changes in 18F-FDG and 18F-FDOPA uptake, with an increase in 18F-FDOPA lesion avidity in the second week relative to baseline. LAT1 and GLUT1 surface density in the untreated and week 3 treated groups were qualitatively similar. Conclusion:18F-FDOPA PET/CT complemented 18F-FDG PET/CT in imaging melphalan therapy response in preclinical extramedullary MM. 18F-FDOPA uptake was linked to LAT1 expression and melphalan response, with longitudinal imaging suggesting stabilization of LAT1 levels and melphalan tumor cytotoxicity. Future work will explore additional MM cell lines with heterogeneous LAT1 expression and response to melphalan therapy.

Role of l-Type Amino Acid Transporter 1 at the Inner Blood-Retinal Barrier in the Blood-to-Retina Transport of Gabapentin.

Gabapentin is an antiseizure drug that is known to also have beneficial effects on the retinal cells. To use gabapentin in retinal pharmacotherapy, it is critical to understand gabapentin distribution in the retina. The purpose of this study was to clarify the kinetics of gabapentin influx transport across the inner and outer blood-retinal barrier (BRB), which regulates the exchange of compounds/drugs between the circulating blood and the retina. In vivo blood-to-retina gabapentin transfer was evaluated by the rat carotid artery injection technique. In addition, gabapentin transport was examined using in vitro models of the inner (TR-iBRB2 cells) and outer BRB (RPE-J cells). The in vivo [3H]gabapentin transfer to the rat retina across the BRB was significantly reduced in the presence of unlabeled gabapentin, suggesting transporter-mediated blood-to-retina distribution of gabapentin. Substrates of the Na+-independent l-type amino acid transporter 1 (LAT1), such as 2-aminobicyclo[2.2.1]heptane-2-carboxylic acid (BCH), also significantly inhibited the in vivo [3H]gabapentin transfer. [3H]Gabapentin uptake in TR-iBRB2 and RPE-J cells exhibited Na+-independent and saturable kinetics with a Km of 735 and 507 μM, respectively. Regarding the effect of various transporter substrates/inhibitors on gabapentin transport in these cells, LAT1 substrates significantly inhibited [3H]gabapentin uptake in TR-iBRB2 and RPE-J cells. In addition, preloaded [3H]gabapentin release from TR-iBRB2 and RPE-J cells was trans-stimulated by LAT1 substrates through the obligatory exchange mechanism as LAT1. Immunoblot analysis indicates the protein expression of LAT1 in TR-iBRB2 and RPE-J cells. These results imply that LAT1 at the inner and outer BRB takes part in gabapentin transport between the circulating blood and retina. Moreover, treatment of LAT1-targeted small interfering RNA to TR-iBRB2 cells significantly reduced both the level of LAT1 protein expression and [3H]gabapentin uptake activities in TR-iBRB2 cells. In conclusion, data from the present study indicate that LAT1 at the inner BRB is involved in retinal gabapentin transfer, and also suggest that LAT1 mediates gabapentin transport in the RPE cells.

Effects of heat stress treatment and leucine supplementation on age‐related muscle loss in mice

Acute heat stress treatment activates mammalian target of rapamycin (mTOR) signaling in skeletal muscle, leading to increase skeletal muscle protein synthesis and skeletal muscle mass. Leucine intake also activates mTOR signaling and stimulates protein synthesis. This study investigated the effects of heat stress treatment and leucine supplementation on sarcopenia (muscle loss with aging) in mice. C57BL/6J male mice (14 or 98 weeks old) were assigned randomly to young control (YC, N=6), old control (OC, n=9), heat stress (OH, n=7), leucine supplementation (OL, n=7), and heat stress and leucine supplementation (OLH) groups. Mice in OH and OLH groups were exposed to hot environment chamber (41ºC) for 40 min twice per week. Mice in OL and OLH groups were given ad libitum access to leucine‐supplemented water (114mM leucine). After 12‐week intervention, there were no differences in body weight and grip strength among groups. Body fat percentage in OLH group was significantly lower than that in OC group (29.0 ± 6.4 % vs. 39.1 ± 8.5 %, p &lt; 0.05). The vastus lateralis muscle weight was significantly lower in OC, OH and OL groups compared with that of YC group, but the muscle weight in OLH group was significantly greater than that in OC group (191.9 ± 12.8 mg vs. 167.1 ± 10.1 mg, p &lt; 0.05). Heat stress treatment induced the expression of heat shock protein 72 (HSP72) in muscle of OH and OLH groups (~5 fold, p &lt; 0.05). There were no differences in the muscle mTOR signaling (the phosphorylation of mTOR, p70s6k, S6 and 4E‐BP1) between groups under overnight fasting condition. On the other hand, the expression of L‐type amino acid transporter 1 (LAT1) of muscle was significantly increased in OC, OH and OL groups compared with YC group (~3 fold, p &lt; 0.05), but there were no difference between YC and OLH group. Our current observations show that the combination of heat stress treatment and leucine supplementation appears to be clinically beneficial for age‐related muscle loss. In addition, normal expression of LAT1 in muscle might contribute to suppression of muscle loss.Support or Funding InformationThis work was supported by JSPS KAKENHI Grant Number JP16K16565.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

L-Type Amino Acid Transporter 1 Immunoreactivity as a Possible Diagnostic and Prognostic Marker of Thymic Carcinoma.

L-type amino acid transporter 1 (LAT1) functions to transport large neutral amino acids, such as leucine, isoleucine, valine, phenylalanine, tyrosine, tryptophan, methionine, and histidine. These amino acids are essential for cell growth and proliferation. Many studies have demonstrated LAT1 expression in various types of cancer, and its high expression level was associated with poor prognosis. However, the significance of LAT1 expression in thymic epithelial tumors is controversial. We conducted this retrospective study to investigate the LAT1 immunoreactivity in thymic epithelial tumors and its impact on prognosis. We analyzed 32 patients with thymoma and 14 patients with thymic carcinoma who underwent surgery at our institute. Immunohistochemical analysis was performed using formalin-fixed paraffin-embedded surgical tissues and an anti-LAT1 polyclonal antibody. We thus found that LAT1 immunoreactivity was undetectable in all of the thymoma specimens, regardless of the subtypes of thymoma. By contrast, LAT1 immunoreactivity was consistently detected in the cytosol of thymic carcinoma cells; namely, all 14 thymic carcinoma specimens demonstrated LAT1 immunoreactivity in the cytosol. Among these 14 thymic carcinoma specimens, four carcinoma specimens also showed LAT1 immunoreactivity in the cell membrane. Survival analysis indicated that the thymic carcinoma with the LAT1 membrane signal was associated with poor prognosis, compared with the specimens with the LAT1 cytosol signal. We therefore propose that LAT1 is expressed in the cytosol of thymic carcinoma cells, which could be a diagnostic marker of thymic carcinoma. Moreover, LAT1 expression in the cell membrane is a prognostic marker of thymic carcinoma.