Expression and roles of system L amino acid transporters in human embryonal carcinoma cells.

BACKGROUND: Testicular germ cell tumors (TGCTs) are the most common malignant cancer in young men. Although TGCTs are generally responsive to platinum-based chemotherapy particularly cisplatin, acquired resistance in patients with metastasis still occurs resulting in poor prognosis. Specifically, differentiation of embryonal carcinoma (EC) cells, the stem cells of TGCTs, can lead to the reduction of cisplatin responsiveness. Therefore, novel therapeutic strategies for TGCTs are needed. System L amino acid transporters have been reported to be up-regulated and to play an important role in tumorigenesis. However, expression and role of system L amino acid transporters in TGCTs remain elusive. MATERIALS AND METHODS: Expression of system L amino acid transporters was analyzed in TGCT samples from The Cancer Genome Atlas (TCGA). Expression of LAT1, LAT2, and 4F2hc was examined in human embryonal carcinoma cell line NTERA2. Roles of system L amino acid transporters on NTERA2 cell survival, cell proliferation, pluripotency, and cisplatin sensitivity were evaluated. RESULTS: Based upon TCGA datasets, we found that two isoforms of system L (LAT1 and LAT2) and their chaperone protein 4F2hc are highly expressed in EC samples compared with other groups. Treatment with the system L inhibitor BCH significantly suppressed leucine uptake into the pluripotent EC cell line NTERA2. The malignant phenotypes including cell viability, cell proliferation, and clonal ability were decreased following BCH treatment. Nonetheless, system L inhibition did not alter expression of stemness genes in NTERA2 cells. After NTERA2 differentiation, expressions of LAT1 and LAT2 were decreased. Finally, co-administration of BCH enhanced cisplatin sensitivity in both undifferentiated and differentiated cells. These effects were associated with the reduction in p70S6K phosphorylation. CONCLUSION: Taken together, these results shed light on the roles of system L amino acid transporters in TGCTs. Therefore, system L amino acid transporters could provide novel therapeutic targets for treatment against TGCTs.

Positive correlation of expression of L-type amino-acid transporter 1 with colorectal tumor progression and prognosis: Higher expression in sporadic colorectal tumors compared with ulcerative colitis-associated neoplasia.

The role of L-type amino-acid transporter 1 (LAT1), an oncofetal protein, in tumor progression is not well known, although it is important for the survival and proliferation of cancer cells. LAT1 expression was immunohistochemically analyzed and compared in sporadic (conventional) colorectal tumors and ulcerative colitis (UC)-associated neoplasia development and progression. LAT1 expression showed a significant stepwise increase in the order: conventional low-grade tubular adenoma, high-grade tubular adenoma, and invasive adenocarcinoma. Similarly, the same increasing trend in LAT1 expression was found in UC-associated low-grade dysplasia, high-grade dysplasia, and adenocarcinoma, whereas expression was significantly lower compared with that in an adenoma-adenocarcinoma series. LAT1 expression was predominant in the upper half of mucosal lesions in low-grade adenoma. This localized difference in LAT1 expression between the upper and lower halves of mucosal lesions disappeared in conventional high-grade adenoma and adenocarcinoma. LAT1 expression in the colorectal mucosa was significantly increased in the order: nontumor mucosa, quiescent phase of UC, and active phase of UC. Considering the histological pattern of Ki-67 labeling, LAT1 expression appeared partly related to cell proliferation, but this was not significant. In relation to the prognosis of patients with sporadic phase IV colorectal adenocarcinoma, this was significantly poorer in the group with high LAT1 expression compared with that with low LAT1 expression. This suggests LAT1 expression may be used as a companion biomarker for anti-cancer therapy targeting the LAT1 molecule in colorectal cancers.

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.

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.

The Regulation and Function of the L-Type Amino Acid Transporter 1 (LAT1) in Cancer.

The progression of cancer is associated with increases in amino acid uptake by cancer cells. Upon their entry into cells through specific transporters, exogenous amino acids are used to synthesize proteins, nucleic acids and lipids and to generate ATP. The essential amino acid leucine is also important for maintaining cancer-associated signaling pathways. By upregulating amino acid transporters, cancer cells gain greater access to exogenous amino acids to support chronic proliferation, maintain metabolic pathways, and to enhance certain signal transduction pathways. Suppressing cancer growth by targeting amino acid transporters will require an in-depth understanding of how cancer cells acquire amino acids, in particular, the transporters involved and which cancer pathways are most sensitive to amino acid deprivation. L-Type Amino Acid Transporter 1 (LAT1) mediates the uptake of essential amino acids and its expression is upregulated during the progression of several cancers. We will review the upstream regulators of LAT1 and the downstream effects caused by the overexpression of LAT1 in cancer cells.

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.

LAT1 is a putative therapeutic target in endometrioid endometrial carcinoma.

l-type amino acid transporters (LAT1-4) are expressed in various cancer types and are involved in the uptake of essential amino acids such as leucine. Here we investigated the expression of LAT1-4 in endometrial adenocarcinoma and evaluated the contribution of LATs to endometrial cancer cell growth. Analysis of human gene expression data showed that all four LAT family members are expressed in endometrial adenocarcinomas. LAT1 was the most highly expressed, and showed a significant increase in both serous and endometrioid subtypes compared to normal endometrium. Endometrioid patients with the highest LAT1 levels exhibited the lowest disease-free survival. The pan-LAT inhibitor BCH led to a significant decrease in cell growth and spheroid area in four endometrial cancer cell lines tested in vitro. Knockdown of LAT1 by shRNA inhibited cell growth in HEC1A and Ishikawa cells, as well as inhibiting spheroid area in HEC1A cells. These data show that LAT1 plays an important role in regulating the uptake of essential amino acids such as leucine into endometrial cancer cells. Increased ability of BCH compared to LAT1 shRNA at inhibiting Ishikawa spheroid area suggests that other LAT family members may also contribute to cell growth. LAT1 inhibition may offer an effective therapeutic strategy in endometrial cancer patients whose tumours exhibit high LAT1 expression.

LAT1 acts as a crucial transporter of amino acids in human thymic carcinoma cells.

L-type amino acid transporter 1 (LAT1, SLC7A5) incorporates essential amino acids into cells. Recent studies have shown that LAT1 is a predominant transporter in various human cancers. However, the function of LAT1 in thymic carcinoma remains unknown. Here we demonstrate that LAT1 is a critical transporter for human thymic carcinoma cells. LAT1 was strongly expressed in human thymic carcinoma tissues. LAT1-specific inhibitor significantly suppressed leucine uptake and growth of Ty82 human thymic carcinoma cell lines, suggesting that thymic carcinoma takes advantage of LAT1 as a quality transporter and that LAT1-specific inhibitor might be clinically beneficial in therapy for thymic carcinoma.

Study of LAT1 Expression in Brain Metastases: Towards a Better Understanding of the Results of Positron Emission Tomography Using Amino Acid Tracers.

Positron emission tomography using radiolabeled amino acid (PET-AA) appears to be promising in distinguishing between recurrent tumour and radionecrosis in the follow-up of brain metastasis (BM). The amino acid transporter LAT1 and its cofactor CD98, which are involved in AA uptake, have never been investigated in BM. The aim of our study was to determine and compare the expression of LAT1 and CD98 in BM and in non-tumoral brain tissue (NT). The expression of LAT1 and CD98 were studied by immunohistochemistry in 67 BM, including 18 BM recurrences after radiotherapy, in 53 NT, and in 13 cases of patients with previously irradiated brain tumor and investigated by [18F] FDOPA-PET. LAT1 and CD98 expression were detected in 98.5% and 59.7% of BM respectively and were significantly associated with BM tissue as compared to NT (p<0.001). LAT1 expression in recurrent BM was significantly increased as compared to newly occurring BM. Ten cases investigated by [18F] FDOPA-PET corresponding to recurrent BM displayed significant [18F] FDOPA uptake and LAT1 overexpression whereas three cases corresponding to radionecrosis showed no or low uptake and LAT1 expression. LAT1 expression level and [18F] FDOPA uptake were significantly correlated. In conclusion, we hypothesized that BM may overexpress the AA transporter LAT1. We have shown that LAT1 overexpression was common in BM and was specific for BM as compared to healthy brain. These results could explain the specific BM uptake on PET-AA.

Inhibition of glucose metabolism prevents glycosylation of the glutamine transporter ASCT2 and promotes compensatory LAT1 upregulation in leukemia cells.

Leukemia cells are highly dependent on glucose and glutamine as bioenergetic and biosynthetic fuels. Inhibition of the metabolism of glucose but also of glutamine is thus proposed as a therapeutic modality to block leukemia cell growth. Since glucose also supports protein glycosylation, we wondered whether part of the growth inhibitory effects resulting from glycolysis inhibition could indirectly result from a defect in glycosylation of glutamine transporters. We found that ASCT2/SLC1A5, a major glutamine transporter, was indeed deglycosylated upon glucose deprivation and 2-deoxyglucose exposure in HL-60 and K-562 leukemia cells. Inhibition of glycosylation by these modalities as well as by the bona fide glycosylation inhibitor tunicamycin however marginally influenced glutamine transport and did not impact on ASCT2 subcellular location. This work eventually unraveled the dispensability of ASCT2 to support HL-60 and K-562 leukemia cell growth and identified the upregulation of the neutral amino acid antiporter LAT1/SLC7A5 as a mechanism counteracting the inhibition of glycosylation. Pharmacological inhibition of LAT1 increased the growth inhibitory effects and the inactivation of the mTOR pathway resulting from glycosylation defects, an effect further emphasized during the regrowth period post-treatment with tunicamycin. In conclusion, this study points towards the underestimated impact of glycosylation inhibition in the interpretation of metabolic alterations resulting from glycolysis inhibition, and identifies LAT1 as a therapeutic target to prevent compensatory mechanisms induced by alterations in the glycosylating process.

Overexpression of L-Type Amino Acid Transporter 1 (LAT1) and 2 (LAT2): Novel Markers of Neuroendocrine Tumors.

BACKGROUND: 6-18F-fluoro-L-3,4-dihydroxyphenylalanine (18F-FDOPA) PET is a useful tool in the clinical management of pheochromocytoma (PHEO) and medullary thyroid carcinoma (MTC). 18F-FDOPA is a large neutral amino acid biochemically resembling endogenous L-DOPA and taken up by the L-type amino acid transporters (LAT1 and LAT2). This study was conducted to examine the expression of the LAT system in PHEO and MTC. METHODS: Real-time PCR and Western blot analyses were used to assess LAT1 and LAT2 gene and protein expression in 32 PHEO, 38 MTC, 16 normal adrenal medulla and 15 normal thyroid tissue samples. Immunohistochemistry method was applied to identify the proteins' subcellular localization. RESULTS: LAT1 and LAT2 were overexpressed in both PHEO and MTC by comparison with normal tissues. LAT1 presented a stronger induction than LAT2, and their greater expression was more evident in PHEO (15.1- and 4.1-fold increases, respectively) than in MTC (9.9- and 4.1-fold increases, respectively). Furthermore we found a good correlation between LAT1/2 and GLUT1 expression levels. A positive correlation was also found between urinary noradrenaline and adrenaline levels and LAT1 gene expression in PHEO. The increased expression of LAT1 is also confirmed at the protein level, in both PHEO and MTC, with a strong cytoplasmic localization. CONCLUSIONS: The present study is the first to provide experimental evidence of the overexpression in some NET cancers (such as PHEO or MTC) of L-type amino acid transporters, and the LAT1 isoform in particular, giving the molecular basis to explain the increase of the DOPA uptake seen in such tumor cells.

Clinicopathological significance of LAT1 and ASCT2 in patients with surgically resected esophageal squamous cell carcinoma.

BACKGROUND: Amino acid transporters are highly expressed in various human cancers. L-type amino acid transporter 1 (LAT1) and system alanine-serine-cysteine amino acid transporter-2 (ASCT2) play a crucial role in tumor progression and survival. However, the clinicopathological significance of these transporters in patients with esophageal squamous cell carcinoma (ESCC) remains unclear. METHODS: One hundred and fifty-seven patients with surgically resected ESCC were evaluated. Immunohistochemical analysis was performed for LAT1, ASCT2, CD98, Ki-67, and micro-vessel density (MVD), as determined by CD34 expression. RESULTS: LAT1 and ASCT2 were positively expressed in 59% (93/157) and 48% (76/157) of tumors respectively. LAT1 and ASCT2 expression significantly correlated with T factor, N factor, lymphatic permeation, vascular invasion, and CD98 expression. The 5-year survival rates of LAT1-high and -low and ASCT2-high and -low expressing patients were 62.0% and 69.6% (P < 0.05) and 59.6% and 70.1% (P = 0.068), respectively. The combined positive expression of LAT1 and ASCT2 was a significant prognostic factor in univariate analysis. CONCLUSION: High expression of LAT1 and ASCT2 correlates with metastasis and invasion. Accordingly, these proteins could serve as prognostic biomarkers and therapeutic targets for treating patients with surgically resectable ESCC. J. Surg. Oncol. 2016;113:381-389. © 2016 Wiley Periodicals, Inc.

Up-Regulation of LAT1 during Antiandrogen Therapy Contributes to Progression in Prostate Cancer Cells.

PURPOSE: Cancer cells require massive amounts of amino acids for survival. LAT1 (L-type amino acid transporter 1) transports essential amino acids, including leucine, which trigger the downstream mTOR (mammalian target of rapamycin) pathway. We examined the association between androgen receptor and LAT1, and the association between LAT1 expression and the acquisition of castration resistance. MATERIALS AND METHODS: Western blot and real-time polymerase chain reaction were performed to study protein and mRNA expression. siRNA was used to knock down target genes. A total of 92 prostate biopsy specimens of patients who underwent androgen deprivation therapy were used for immunohistochemical analyses. Cox hazard proportional models and the Kaplan-Meier method were used for statistical analyses. RESULTS: LAT1 was highly expressed in hormone resistant prostate cancer cell lines. Knockdown of LAT1 in LNCaP and C4-2 cells significantly suppressed cell proliferation, migration and invasion. Androgen receptor siRNA or androgen receptor blocking through bicalutamide (10 µM) or MDV3100 (10 µM) significantly increased LAT1 expression (p <0.01). Treatment with dihydrotestosterone (0.1 to 10 nM) reduced LAT1 expression in a dose dependent manner (p <0.01). Bicalutamide/MDV3100 plus siLAT1 synergistically suppressed prostate cancer cell proliferation compared to single inhibition by androgen receptor or LAT1 (p <0.01). High LAT1 expression correlated with significantly shorter prostate specific antigen recurrence-free survival in patients receiving androgen deprivation therapy (p <0.0001). LAT1 expression was an independent predictor of castration resistance on multivariate analysis (HR 3.56, p = 0.0133). CONCLUSIONS: The current data may indicate a novel mechanism to acquire castration resistance through activation of the amino acid transporter LAT1.

L-Type amino acid transporter 1 (LAT1) expression in lymph node metastasis of gastric carcinoma: Its correlation with size of metastatic lesion and Ki-67 labeling.

L-Type amino acid transporter 1 (LAT1) is one of the major amino acid transporters. High levels of LAT1 expression have been reported in various tumors, which can act as a novel prognostic marker. Previously, we demonstrated that LAT1 is highly expressed in advanced gastric carcinoma with lymph node metastasis, and proposed that LAT1 is an independent prognostic factor in non-scirrhous gastric carcinoma. The aim of the present study was to investigate the relationship between LAT1 expression and the size of lymph node metastatic lesions in gastric carcinoma. LAT1 and Ki-67 expression was immunohistochemically analyzed in 64 cases of advanced gastric carcinoma with lymph node metastasis. LAT1 expression in the metastatic lymph nodes was correlated with that in the primary lesions. The high LAT1 expression group showed a larger size of metastatic lesion and a higher Ki-67 labeling index than the low LAT1 expression group. LAT1 expression had a weak association with Ki-67 labeling index and tumor diameter of lymph nodes. These results suggest that LAT1 expression is associated with disease progression in gastric carcinoma. We proposed that LAT1 could be a potential therapeutic target for gastric carcinoma cases with large lymph node metastasis.

L-amino acid transporter 1 may be a prognostic marker for local progression of prostatic cancer under expectant management.

BACKGROUND: Oncocytic L-amino acid transporter (LAT) 1 could be a target of new molecular therapy against malignancies. OBJECTIVE: To assess the correlation between overexpression of LAT1 and local progression (LP) in prostatic carcinoma (PC) patients under expectant management (EM). METHODS: This retrospective study analyzed 109 patients with PC who received EM from 1991 to 2006. The expression of LAT1, LAT2, and CD98, as well as Ki-67 labeling indices (LI), was analyzed immunohistochemically in first biopsy or TUR samples diagnosed as adenocarcinomas. RESULTS: Of the 109 patients, 44 (40%) showed LP on clinical examinations, whereas 65 showed stable disease (SD). LAT1 score and intensity were significantly higher in the LP than in the SD group, as well as among Gleason score (GS)-low (GS < 7) patients who were associated with low-risk. When the LP group was subdivided by D'Amico risk category (low-, intermediate- and high-risk groups), each showed higher LAT1 expression than the SD group. LAT1 expression did not correlate with GS or Ki-67 LI. CONCLUSIONS: Independently of GS, aberrant overexpression of LAT1 in prostatic adenocarcinoma could predict LP under EM. Although prostate biopsy samples are small, LAT1 may be a novel prognostic biomarker of LP.

Biological significance of fluorine-18-α-methyltyrosine (FAMT) uptake on PET in patients with oesophageal cancer.

PURPOSE: (18)F-FAMT as an amino-acid tracer for positron emission tomography (PET) is useful for detecting human neoplasms. (18)F-FAMT is accumulated in tumour cells solely via L-type amino-acid transporter 1 (LAT1). This study was conducted to investigate the biological significance of (18)F-FAMT uptake in patients with oesophageal cancer. METHODS: From April 2008 to December 2011, 42 patients with oesophageal cancer underwent both (18)F-FAMT PET/CT and (18)F-FDG PET/CT before surgical treatment. The immunohistochemical analysis of LAT1, CD98, Ki-67, CD34, p53, p-Akt and p-mTOR was performed on the primary lesions. In vitro experiments were performed to examine the mechanism of (18)F-FAMT uptake. RESULTS: High uptake of (18)F-FAMT was significantly associated with advanced stage, lymph node metastasis and the expression of LAT1, CD98, Ki-67 and CD34. LAT1 expression yielded a statistically significant correlation with CD98 expression, cell proliferation, angiogenesis and glucose metabolism. In vitro experiments revealed that (18)F-FAMT was specifically transported by LAT1. CONCLUSIONS: The uptake of (18)F-FAMT within tumour cells is determined by the LAT1 expression and correlated with cell proliferation and angiogenesis in oesophageal cancer. The present experiments also confirmed the presence of LAT1 as an underlying mechanism of (18)F-FAMT accumulation.

Targeting glutamine transport to suppress melanoma cell growth.

Amino acids, especially leucine and glutamine, are important for tumor cell growth, survival and metabolism. A range of different transporters deliver each specific amino acid into cells, some of which are increased in cancer. These amino acids consequently activate the mTORC1 pathway and drive cell cycle progression. The leucine transporter LAT1/4F2hc heterodimer assembles as part of a large complex with the glutamine transporter ASCT2 to transport amino acids. In this study, we show that the expression of LAT1 and ASCT2 is significantly increased in human melanoma samples and is present in both BRAF(WT) (C8161 and WM852) and BRAF(V600E) mutant (1205Lu and 451Lu) melanoma cell lines. While inhibition of LAT1 by BCH did not suppress melanoma cell growth, the ASCT2 inhibitor BenSer significantly reduced both leucine and glutamine transport in melanoma cells, leading to inhibition of mTORC1 signaling. Cell proliferation and cell cycle progression were significantly reduced in the presence of BenSer in melanoma cells in 2D and 3D cell culture. This included reduced expression of the cell cycle regulators CDK1 and UBE2C. The importance of ASCT2 expression in melanoma was confirmed by shRNA knockdown, which inhibited glutamine uptake, mTORC1 signaling and cell proliferation. Taken together, our study demonstrates that ASCT2-mediated glutamine transport is a potential therapeutic target for both BRAF(WT) and BRAF(V600E) melanoma.

Amino acid transporters expression in acinar cells is changed during acute pancreatitis.

Pancreatic acinar cells accumulate amino acids against a marked concentration gradient to synthesize digestive enzymes. Thus, the function of acinar cells depends on amino acid uptake mediated by active transport. Despite the importance of this process, pancreatic amino acid transporter expression and cellular localization is still unclear. We screened mouse pancreas for the expression of genes encoding amino acid transporters. We showed that the most highly expressed transporters, namely sodium dependent SNAT3 (Slc38a3) and SNAT5 (Slc38a5) and sodium independent neutral amino acids transporters LAT1 (Slc7a5) and LAT2 (Slc7a8), are expressed in the basolateral membrane of acinar cells. SNAT3 and SNAT5, LAT1 and LAT2 are expressed in acinar cells. Additional evidence that these transporters are expressed in mature acinar cells was gained using acinar cell culture and acute pancreatitis models. In the acute phase of pancreatic injury, when acinar cell loss occurs, and in an acinar cell culture model, which mimics changes occurring during pancreatitis, SNAT3 and SNAT5 are strongly down-regulated. LAT1 and LAT2 were down-regulated only in the in vitro model. At protein level, SNAT3 and SNAT5 expression was also reduced during pancreatitis. Expression of other amino acid transporters was also modified in both models of pancreatitis. The subset of transporters with differential expression patterns during acute pancreatitis might be involved in the injury/regeneration phases. Further expression, localization and functional studies will follow to better understand changes occurring during acute pancreatitis. These findings provide insight into pancreatic amino acid transport in healthy pancreas and during acute pancreatitis injury.

Cloning, large scale over-expression in E. coli and purification of the components of the human LAT 1 (SLC7A5) amino acid transporter.

The high yield expression of the human LAT1 transporter has been obtained for the first time using E. coli. The hLAT1 cDNA was amplified from HEK293 cells and cloned in pH6EX3 vector. The construct pH6EX3-6His-hLAT1 was used to express the 6His-hLAT1 protein in the Rosetta(DE3)pLysS strain of E. coli. The highest level of expression was detected 8 h after induction by IPTG at 28 °C. The expressed protein was collected in the insoluble fraction of cell lysate. On SDS-PAGE the apparent molecular mass of the polypeptide was 40 kDa. After solubilization with sarkosyl and denaturation with urea the protein carrying a 6His N-terminal tag was purified by Ni(2+)-chelating affinity chromatography and identified by anti-His antibody. The yield of the over-expressed protein after purification was 3.5 mg/L (cell culture). The human CD98 cDNA amplified from Imagene plasmid was cloned in pGEX-4T1. The construct pGEX-4T1-hCD98 was used to express the GST-hCD98 protein in the Rosetta(DE3)pLysS strain of E. coli. The highest level of expression was detected in this case 4 h after induction by IPTG at 28 °C. The expressed protein was accumulated in the soluble fraction of cell lysate. The molecular mass was determined on the basis of marker proteins on SDS-PAGE; it was about 110 kDa. GST was cleaved from the protein construct by incubation with thrombin for 12 h and the hCD98 was separated by Sephadex G-200 chromatography (size exclusion). hCD98 showed a 62 kDa apparent molecular mass, as determined on the basis of molecular mass markers using SDS-PAGE. The yield of CD98 was 2 mg/L of cell culture.

L-Leucine and L-isoleucine enhance growth of BBN-induced urothelial tumors in the rat bladder by modulating expression of amino acid transporters and tumorigenesis-associated genes.

We investigated the underlying mechanisms of L-leucine and L-isoleucine mediated promotion of bladder carcinogenesis using an initiation-promotion model. Rats were administered N-butyl-N-(4-hydroxybutyl) nitrosamine for 4 weeks and then fed AIN-93G basal diet or diet supplemented with L-leucine or L-isoleucine for 8 weeks followed by the basal diet for another 8 weeks. At the end of the experiment, week 20, there was a significant elevation of papillary and nodular (PN) hyperplasia multiplicity in the amino acid groups. L-Leucine and L-isoleucine transporters were up-regulated in PN hyperplasias and/or bladder tumors compared with concomitant normal-appearing bladder urothelium at weeks 12 and/or 20 in all groups. In addition, in normal-appearing bladder urothelium, significantly increased mRNA levels of y+LAT1, LAT2, LAT4, and 4F2hc were observed in the amino acid groups compared with the BBN control group at both weeks 12 and 20, and increased mRNA levels of LAT1 were observed at week 20. Furthermore, up-regulation of TNF-α, c-fos, ß-catenin, p53, p21(Cip1/WAF1), cdk4, cyclin D1 and caspase 3 in the amino acid groups was detected in normal-appearing bladder urothelium. Overall, our results indicate that supplementation with l-leucine or l-isoleucine enhanced growth of bladder urothelial tumors by triggering expression of amino acid transporters and tumorigenesis-associated genes.