L-amino Acid Transporter Research Articles

Volta Phase Plate Cryo-EM Structure of the Human Heterodimeric Amino Acid Transporter 4F2hc-LAT2.

Heteromeric amino acid transporters (HATs) are protein complexes that catalyze the transport of amino acids across plasma membranes. HATs are composed of two subunits, a heavy and a light subunit, which belong to the solute carrier (SLC) families SLC3 and SLC7. The two subunits are linked by a conserved disulfide bridge. Several human diseases are associated with loss of function or overexpression of specific HATs making them drug targets. The human HAT 4F2hc-LAT2 (SLC3A2-SLC7A8) is specific for the transport of large neutral L-amino acids and specific amino acid-related compounds. Human 4F2hc-LAT2 can be functionally overexpressed in the methylotrophic yeast Pichia pastoris and pure recombinant protein purified. Here we present the first cryo-electron microscopy (cryo-EM) 3D-map of a HAT, i.e., of the human 4F2hc-LAT2 complex. The structure could be determined at ~13 Å resolution using direct electron detector and Volta phase plate technologies. The 3D-map displays two prominent densities of different sizes. The available X-ray structure of the 4F2hc ectodomain fitted nicely into the smaller density revealing the relative position of 4F2hc with respect to LAT2 and the membrane plane.

L-amino acid transporter 1 overexpression in nonmelanocytic skin tumors: an immunohistochemical study

Background Despite the low mortality rate of nonmelanoma skin cancer, the associated morbidity and cost place them as the fifth most expensive cancer to treat. Therefore, development of novel agent to treat is needed, but before new therapeutic strategy can be advised, knowledge regarding its molecular mechanisms is required. Objective To elucidate the role of L-amino acid transporter 1 (LAT1) in nonmelanoma skin cancer etiopathogenesis through LAT1 immunohistochemical evaluation in basal cell carcinoma (BCC) and squamous cell carcinoma (SCC) lesions compared with controls, and to correlate this expression with the proliferative activity of these tumors (assessed by Ki-67 antigen) and with the clinicopathological parameters of the studied patients. Patients and methods In this case–control study, 16 patients having BCC, 16 SCC cases, and 10 healthy controls were subjected to history taking, clinical examination, and skin biopsies (from BCC and SCC lesions and site matched of controls). Biopsies were stained by LAT1 and Ki-67 immunohistochemical stains. Results LAT1 immunstaining in epidermis revealed significant gradual increase from controls (18.70±24.23) to BCC (32.00±27.04) to SCC (53.64±56.26) (P=0.013). Moreover, tumor islands in both BCC and SCC groups and the control epidermis showed significant stepwise increase of LAT1 H score from controls (18.70±24.23), to BCC (41.33±51.70) and finally SCC cases (79.29±68.55) (P=0.001). However, there was no significant correlations between LAT1 and Ki-67 in all groups. Conclusion LAT1, through its amino acids transporting function, may participate actively in BCC and SCC etiopathogenesis. Moreover, molecular LAT1-targeting agents may be a promising therapeutic approach for BCC and SCC management programs.

Developing selective L-Amino Acid Transport 1 (LAT1) inhibitors: A Structure-Activity Relationship overview

System L amino acid transporters are a member of the Solute Carrier transporter Family (SLC). L-Amino acid transporter 1 (LAT1) belong to SLC7 and requires the heavy chained 4F2hc chaperone protein for amino acid transport. LAT1 is expressed in tumor cells and cancerous cells in vivo are strongly linked to LAT1 expression. Herein, we provide historical aspects regarding initial Structure Activity Relationship (SAR) findings reported in 2002 with the oocyte model and in 2008 with S2-LAT1 and S2-LAT2 cell lines. We summarize a series of dichloro- dibromo- and diiodo- tyrosine analogs that were prepared, and tested their potential to inhibit leucine transport in vitro to afford IC50 values with a few being low microM LAT1 inhibitors. We then summarize our efforts regarding novel LAT1 inhibitors with sub-nanomolar (nM) IC50s to produce JPH203 which has completed Phase I clinical trial and is currently in Phase II trial in Japan. We describe differences observed between LAT1 and LAT2. Overall, we provide an expanded SAR model regarding potent and selective LAT1 inhibitors.

Anti-lung Cancer and Anti-angiogenic Activities of New Designed Boronated Phenylalanine Metal Complexes.

Drug design and discovery studies still remain of great importance in the search for more convenient chemotherapeutic to avoid the drug resistance, systemic toxicity or the longterm side effects. A series of mononuclear gold (III) and platinum (II) complexes based on 4-dihydroxyboryl- DL-phenylalanine (BPA) was designed and synthesized, for the first time, by using 2, 2'-dipyridyl (L1) and 4, 4'-diaminobibenzyl (L2) ligands. Characterization of the synthesized complexes was achieved by using 1H-NMR, IR, MS and elemental analyses. MTT cell viability, endothelial tube formation, cancer cell colony formation and TRITCphalloidin cytoskeleton staining assays were performed on human umbilical vein endothelial (HUVEC) and human lung adenocarcinoma (A549) cells to establish the anticancer and anti-angiogenic activities of the complexes. It was determined that the organometallic complexes that include 2, 2'-dipyridyl ligand have higher antiproliferative activity than L2-based complexes in the micromolar range. Colony formation experiments showed that the anchorage-independent growth ability of A549s was significantly affected by the complexes in a concentration-dependent manner though L1-based complexes were more effective than L2-based ones. It was also clearly observed that the complexes have significant anti-angiogenic and cytoskeleton alterative activities. Consequently, the phenylalanine-based organometallic complexes seem to have anti-lung cancer and anti-angiogenic activities depending on the ligand type and a great potential in oncology drug development because phenylalanine amino acid has an ability to cross the cell membrane by using L-amino acid transport system. Design, synthesis and activity studies with amino acid analogs should be therefore increased to discover more efficient drugs to cure cancer diseases.

Abstract 2638: Overexpressions of cortisol, annexin V and Lost in expression of PD-L1 and DARC proteins are associated with aggressive prostate cancer phenotypes in Black men: A CaPTC Cohort Study

Abstract Prostate cancer (CaP) is the most common male gender cancer and present with a 5-year prevalence, incidence and mortality rate in Nigerian Black men. There is disproportionate prevalence and poor understanding of CaP in Black men globally. The Prostate Cancer Transatlantic Consortium (CaPTC) has documented significant CaP burden among Nigerian black men in Nigeria and in the Diaspora. Notably, African ancestry is one of the most important risk factors of CaP globally. Increased levels of cortisol, a stress-related catabolic hormone is significantly associated to prostate specific antigen and poor prognosis in prostate cancer patients. Cortisol causes degradation of muscle proteins into amino-acids. Amino acids such as leucine, serves as a source of fuel for aggressive prostate cancer cells in a Warburg effect model through the L-amino acid transporters (LATs). The annexins are calcium and phospholipid binding proteins implicated in cancer development and progression. Overexpression of annexin 1 is one of the mechanisms by which cortisol inhibit inflammation. Annexin V has a high affinity to phosphatidylserine and play important role of inhibiting engulfment of apoptotic cells by macrophages to increase the immunogenicity of tumor cells undergoing apoptosis. PD-L1 is an important membrane-bound costimulatory molecule that inhibits immune responses through its receptor, PD-1. Overexpression of PD-L1 results to cancer cells aggressively evading the host immune system. The Duffy Antigen/Receptor for Chemokines (DARC) is a 7-transmembrane protein atypical chemokine receptor expressed on erythrocytes and vascular endothelial cells that binds to and clears angiogenic chemokines. Expression pattern of DARC is associated to cancer growth and metastasis. In this study, we report the expression pattern of tissue Cortisol, Annexin V, PD-L1 and DARC proteins in relationship to Gleason score in Nigerian Black men with Prostate cancer using immunohistochemistry and immunofluorescence. In addition, we assessed salivary cortisol levels by enzyme immunoassay and physical activity by a standardized CaPTC-AC3-MADCaP (C.A.M) CaP measures from 500 subjects recruited by via CaPTC cohort. Results showed significant expression of tissue and salivary cortisol in the CaP patients compared to the normal subjects (p > 0.05). The Tissue cortisol protein expression was higher in CaP cases with Gleason score 8. There was significant overexpression of Annexin protein in virtually the prostate cancer samples studied. We found a negative expression of PD-L1 and DARC proteins in all the CaP tissues studied. Cortisol and Annexin may serve as an important biomarker for prostate cancer diagnosis and prognosis in Nigerian Blacks. Citation Format: Faruk Mohammed, Folakemi T. Odedina, Sani Ibrahim, Abdulmumini Hassan Rafindadi, Ahmed Adamu, Abdullahi Adamu, Ahmad Bello, Surajo Mohammed Aminu, John Idoko, Aishatu Maude Suleiman, Solomon O. Rotimi, Ernie Kaninjing, Getachew A. Dagne, Clayton Yates, Yawale Iliyasu, Nissa Askins, Iya Eze Bassey, Renee Reams, Abdullahi Mohammad, Hussaini Yusuf Maitama, Dauda Maigatari, Mohammed Sani Shehu, Cheh Augustine Awasum, Abdulkadir Lawal Rafinadadi, Danladi Amodu Ameh, Serah Adewunmi, Ruth Agaba, Haruna Mohammad Muktar, Ahmad Mai, Saad Aliyu Ahmed, Ahmad Bello Kumo, Kasimu Umar Adoke, Ahmad Tijjani Lawal, Ahmad Muhammad, Omolora Fatiregun, Sunday Atawodi, Shehu Akuyam, Yusuf Abdulrashid, Mubarak Liman, Aliyu Muhammad, Abidemi Omonisi, Rebecca Gali, Hassan Dogo, Nkegoum Blaise, Anthonia Sowunmi, Titilola Akinremi, Emeka J. Iweala, Jigo Dangude Yaro, Badejo Adebukola Catherine, Akinwumi Oluwole Komolafe. Overexpressions of cortisol, annexin V and Lost in expression of PD-L1 and DARC proteins are associated with aggressive prostate cancer phenotypes in Black men: A CaPTC Cohort Study [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2638.

Amino acid-dependent cMyc expression is essential for NK cell metabolic and functional responses in mice

Natural killer (NK) cells are lymphocytes with important anti-tumour functions. Cytokine activation of NK cell glycolysis and oxidative phosphorylation (OXPHOS) are essential for robust NK cell responses. However, the mechanisms leading to this metabolic phenotype are unclear. Here we show that the transcription factor cMyc is essential for IL-2/IL-12-induced metabolic and functional responses in mice. cMyc protein levels are acutely regulated by amino acids; cMyc protein is lost rapidly when glutamine is withdrawn or when system l-amino acid transport is blocked. We identify SLC7A5 as the predominant system l-amino acid transporter in activated NK cells. Unlike other lymphocyte subsets, glutaminolysis and the tricarboxylic acid cycle do not sustain OXPHOS in activated NK cells. Glutamine withdrawal, but not the inhibition of glutaminolysis, results in the loss of cMyc protein, reduced cell growth and impaired NK cell responses. These data identify an essential role for amino acid-controlled cMyc for NK cell metabolism and function.

Assessing Photosensitizer Targeting Using Meso-Tetra(Carboxyphenyl) Porphyrin.

Mesotetra(4-carboxyphenyl)porphyrin (mTCPP) is a commercially available small molecule fluorophore and photosensitizer with four free carboxylic acid groups. mTCPP can readily be conjugated with amines for facile attachment of functional groups. In this work, we synthesized and assessed tetravalent, lysine-conjugated mTCPP, for its potential applications in targeted imaging and photodynamic therapy. Fmoc-protected d-lysine or l-lysine was conjugated to mTCPP via amide coupling with the epsilon amine group of lysine, followed by Fmoc deprotection. The resulting compounds did not dissolve well in aqueous solvent, but could be solubilized with the assistance of surfactants, including cholic acid. The l-amino acid transporter (LAT1) can uptake diverse neutral l-amino acids. In vitro studies with U87 cells revealed a non-specific uptake of the hydrophobic Fmoc-protected lysine-conjugated mTCPP precursors, but not d- or l-lysine mTCPP. Likewise, only the Fmoc-protected compounds induced substantial phototoxicty in cells following incubation and irradiation with blue light. These experimental results do not provide evidence to suggest that lysine-mTCPP is able to specifically target cancer cells. However, they do highlight mTCPP as a convenient and accessible framework for assessing molecular targeting of photosensitizers.

Impact of oxygen status on 10B-BPA uptake into human glioblastoma cells, referring to significance in boron neutron capture therapy.

Boron neutron capture therapy (BNCT) can potentially deliver high linear energy transfer particles to tumor cells without causing severe damage to surrounding normal tissue, and may thus be beneficial for cases with characteristics of infiltrative growth, which need a wider irradiation field, such as glioblastoma multiforme. Hypoxia is an important factor contributing to resistance to anticancer therapies such as radiotherapy and chemotherapy. In this study, we investigated the impact of oxygen status on 10B uptake in glioblastoma cells in vitro in order to evaluate the potential impact of local hypoxia on BNCT. T98G and A172 glioblastoma cells were used in the present study, and we examined the influence of oxygen concentration on cell viability, mRNA expression of L-amino acid transporter 1 (LAT1), and the uptake amount of 10B-BPA. T98G and A172 glioblastoma cells became quiescent after 72 h under 1% hypoxia but remained viable. Uptake of 10B-BPA, which is one of the agents for BNCT in clinical use, decreased linearly as oxygen levels were reduced from 20% through to 10%, 3% and 1%. Hypoxia with <10% O2 significantly decreased mRNA expression of LAT1 in both cell lines, indicating that reduced uptake of 10B-BPA in glioblastoma in hypoxic conditions may be due to reduced expression of this important transporter protein. Hypoxia inhibits 10B-BPA uptake in glioblastoma cells in a linear fashion, meaning that approaches to overcoming local tumor hypoxia may be an effective method of improving the success of BNCT treatment.

Specific alterations in gut microbiota are associated with prognosis of Budd-Chiari syndrome.

Gut microbiota is associated with liver diseases. However, gut microbial characteristics of Budd–Chiari syndrome (B-CS) have not been reported. Here, by MiSeq sequencing, gut microbial alterations were characterized among 37 health controls, 20 liver cirrhosis (LC) patients, 31 initial B-CS patients (B-CS group), 33 stability patients after BCS treatment (stability group) and 23 recurrent patients after BCS treatment (recurrence group). Gut microbial diversity was increased in B-CS versus LC. Bacterial community of B-CS clustered with controls but separated from LC. Operational taxonomic units (OTUs) 421, 502 (Clostridium IV) and 141 (Megasphaera) were unique to B-CS. Genera Escherichia/Shigella and Clostridium XI were decreased in B-CS versus controls. Moreover, nine genera, mainly including Bacteroides and Megamonas, were enriched in B-CS versus LC. Notably, Megamonas could distinguish B-CS from LC with areas under the curve (AUCs) of 0.7904. Microbial function prediction revealed that L-amino acid transport system activity was decreased in B-CS versus both LC and controls. Furthermore, OTUs 27 (Clostridium XI), 137 (Clostridium XIVb) and 40 (Bacteroides) were associated with B-CS stability. Importantly, genus Clostridium XI was enriched in stability group versus both recurrence group and B-CS group. Also, PRPP glutamine biosynthesis was reduced in stability group versus recurrence group, but was enriched in stability group versus B-CS group. In conclusion, specific microbial alterations associated with diagnosis and prognosis were detected in B-CS patients. Correction of gut microbial alterations may be a potential strategy for B-CS prevention and treatment.

Relationship between [14C]MeAIB uptake and amino acid transporter family gene expression levels or proliferative activity in a pilot study in human carcinoma cells: Comparison with [3H]methionine uptake

IntroductionTo clarify the difference between system A and L amino acid transport imaging in PET clinical imaging, we focused on the use of α-[N-methyl-11C]-methylaminoisobutyric acid ([11C]MeAIB), and compared it with [S-methyl-11C]-L-methionine ([11C]MET). The aim of this study was to assess the correlation of accumulation of these two radioactive amino acid analogs with expression of amino acid transporters and cell proliferative activity in carcinoma cells. MethodsAmino acid uptake inhibitor studies were performed in four human carcinoma cells (epidermal carcinoma A431, colorectal carcinoma LS180, and lung carcinomas PC14/GL and H441/GL) using the radioisotope analogs [3H]MET and [14C]MeAIB. MeAIB was used to inhibit the A system and 2-amino-2-norbornane-carboxylic acid (BCH) was used to inhibit the L system. The carcinoma gene expression levels of a number of amino acid transporters were measured by microarray and quantitative polymerase chain reaction. Carcinoma proliferative activity was assessed using accumulation of [methyl-3H]-3'-deoxy-3'-fluorothymidine ([3H]FLT). Results and conclusion[14C]MeAIB uptake occurred principally via a Na+-dependent A type mechanism whereas [3H]MET uptake occurred predominantly via a Na+-independent L type mechanism although other transporters were also utilized depending on cell type. There was no correlation between [3H]MET uptake and total system L amino acid transporter (LAT) expression. In contrast, [14C]MeAIB uptake strongly correlated with total system A amino acid transporter (SNAT) expression and proliferative activity in this preliminary study using four human carcinoma cell lines. Carcinoma proliferative activity also correlated with total SNAT expression.Advances in Knowledge and Implications for Patient Care: Because there is a significant correlation between the accumulation of [14C]MeAIB and the gene expression level of total SNAT as well as the accumulation of [3H]FLT, it is suggested that use of the analog [11C]MeAIB in PET may provide an indication of tumor cell proliferative activity. [11C]MeAIB is therefore expected to be very useful in PET imaging.

A novel role for PHT1 in the disposition of l-histidine in brain: In vitro slice and in vivo pharmacokinetic studies in wildtype and Pht1 null mice

PHT1 (SLC15A4) is responsible for translocating l-histidine (l-His), di/tripeptides and peptide-like drugs across biological membranes. Previous studies have indicated that PHT1 is located in brain parenchyma, however, its role and significance in brain along with effect on the biodistribution of substrates is unknown. In this study, adult gender-matched Pht1-competent (wildtype) and Pht1-deficient (null) mice were used to investigate the effect of PHT1 on l-His brain disposition via in vitro slice and in vivo pharmacokinetic approaches. We also evaluated the serum clinical chemistry and expression levels of select transporters and enzymes in the two genotypes. No significant differences were observed between genotypes in serum chemistry, body weight, viability and fertility. PCR analyses indicated that Pept2 had a compensatory up-regulation in Pht1 null mice (about 2-fold) as compared to wildtype animals, which was consistent in different brain regions and confirmed by immunoblots. The uptake of l-His was reduced in brain slices by 50% during PHT1 ablation. The l-amino acid transporters accounted for 30% of the uptake, and passive (other) pathways for 20% of the uptake. During the in vivo pharmacokinetic studies, plasma concentration-time profiles of l-His were comparable between the two genotypes after intravenous administration. Still, biodistribution studies revealed that, when sampled 5min after dosing, l-His values were 28–48% lower in Pht1 null mice, as compared to wildtype animals, in brain parenchyma but not cerebrospinal fluid. These findings suggest that PHT1 may play an important role in histidine transport in brain, and resultant effects on histidine/histamine homeostasis and neuropeptide regulation.

Efficacy of system l amino acid transporter 1 inhibition as a therapeutic target in esophageal squamous cell carcinoma.

System l amino acid transporter 1 (LAT1) is highly expressed in various types of human cancer, and contributes to cancer growth and survival. Recently, we have shown that LAT1 expression is closely related to the growth and aggressiveness of esophageal cancer, and is an independent marker of poor prognosis. However, it remains unclear whether LAT1 inhibition could suppress esophageal cancer growth. In this study, we investigated the tumor‐suppressive effects of the inhibition of LAT1. Both LAT1 and CD98, which covalently associates to LAT1 on the membrane, were expressed in human esophageal cancer cell lines KYSE30 and KYSE150. Quantitative PCR analysis showed that the expression of LAT1 was much higher than other subtypes of LAT. A selective inhibitor of LAT, 2‐aminobicyclo‐(2,2,1)‐heptane‐2‐carboxylic acid (BCH), suppressed cellular uptake of l‐14C‐leucine and cell proliferation in a dose‐dependent manner. It also suppressed phosphorylation of mammalian target of rapamycin, 4E‐BP1, and p70S6K protein, and induced cell cycle arrest at G1 phase. These results suggest that suppression of both mammalian target of rapamycin signaling and cell cycle progression is involved in BCH‐induced growth inhibition. In tumor‐bearing mice, daily treatment with BCH significantly delayed tumor growth and decreased glucose metabolism, indicating that LAT1 inhibition potentially suppresses esophageal cancer growth in vivo. Thus, our results suggest that LAT1 inhibition could be a promising molecular target for the esophageal cancer therapy.

Stabilization of a prokaryotic LAT transporter by random mutagenesis.

The knowledge of three-dimensional structures at atomic resolution of membrane transport proteins has improved considerably our understanding of their physiological roles and pathological implications. However, most structural biology techniques require an optimal candidate within a protein family for structural determination with (a) reasonable production in heterologous hosts and (b) good stability in detergent micelles. SteT, the Bacillus subtilis L-serine/L-threonine exchanger is the best-known prokaryotic paradigm of the mammalian L-amino acid transporter (LAT) family. Unfortunately, SteT's lousy stability after extracting from the membrane prevents its structural characterization. Here, we have used an approach based on random mutagenesis to engineer stability in SteT. Using a split GFP complementation assay as reporter of protein expression and membrane insertion, we created a library of 70 SteT mutants each containing random replacements of one or two residues situated in the transmembrane domains. Analysis of expression and monodispersity in detergent of this library permitted the identification of evolved versions of SteT with a significant increase in both expression yield and stability in detergent with respect to wild type. In addition, these experiments revealed a correlation between the yield of expression and the stability in detergent micelles. Finally, and based on protein delipidation and relipidation assays together with transport experiments, possible mechanisms of SteT stabilization are discussed. Besides optimizing a member of the LAT family for structural determination, our work proposes a new approach that can be used to optimize any membrane protein of interest.

Spermine modulates the expression of two probable polyamine transporter genes and determines growth responses to cadaverine in Arabidopsis.

Two genes, LAT1 and OCT1 , are likely to be involved in polyamine transport in Arabidopsis. Endogenous spermine levels modulate their expression and determine the sensitivity to cadaverine. Arabidopsis spermine (Spm) synthase (SPMS) gene-deficient mutant was previously shown to be rather resistant to the diamine cadaverine (Cad). Furthermore, a mutant deficient in polyamine oxidase 4 gene, accumulating about twofold more of Spm than wild type plants, showed increased sensitivity to Cad. It suggests that endogenous Spm content determines growth responses to Cad in Arabidopsis thaliana. Here, we showed that Arabidopsis seedlings pretreated with Spm absorbs more Cad and has shorter root growth, and that the transgenic Arabidopsis plants overexpressing the SPMS gene are hypersensitive to Cad, further supporting the above idea. The transgenic Arabidopsis overexpressing L-Amino acid Transporter 1 (LAT1) absorbed more Cad and showed increased Cad sensitivity, suggesting that LAT1 functions as a Cad importer. Recently, other research group reported that Organic Cation Transporter 1 (OCT1) is a causal gene which determines the Cad sensitivity of various Arabidopsis accessions. Furthermore, their results suggested that OCT1 is involved in Cad efflux. Thus we monitored the expression of OCT1 and LAT1 during the above experiments. Based on the results, we proposed a model in which the level of Spm content modulates the expression of OCT1 and LAT1, and determines Cad sensitivity of Arabidopsis.

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

Oncocytic L-amino acid transporter (LAT) 1 could be a target of new molecular therapy against malignancies. To assess the correlation between overexpression of LAT1 and local progression (LP) in prostatic carcinoma (PC) patients under expectant management (EM). 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. 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. 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.

Targeting of AIDS related encephalopathy using phenylalanine anchored lipidic nanocarrier

Transport of the anti-HIV agents across the blood–brain barrier (BBB) is a prerequisite to treat acquired immunodeficiency syndrome (AIDS) related encephalopathy. In the present study, we explored facilitated transport of efavirenz (EFV, a non-nucleoside reverse transcriptase inhibitor) across BBB using phenylalanine anchored solid lipid nanoparticles (PA-SLN). PA (amino acid micro-nutrient) was used as a ligand which facilitated carrier mediated transport (CMT) via l-amino acid transporter i.e. LAT1 to traverse BBB. PA was coupled to SLN via amide linkage using carbodiimide chemistry and coupling was confirmed by comparative infrared spectroscopic analysis. SLNs (SLN and PA-SLN) were nanometric in size (around 150nm) and possessed good entrapment efficiency (around 70%). In vitro drug release revealed controlled release pattern for more than 24h. In vivo studies showed 2–3-folds and 7–8-folds accumulation of PA-SLN in brain as compared to SLN and EFV, respectively. Further, transcytosis studies confirmed capability of PA-SLN to cross BBB i.e. 10-fold higher transcytosis potential as compared to EFV. Fluorescence microscopic imaging reassured enhanced brain localization of PA-SLN. Thus, PA-SLN improved the EFV bioavailability and maintained therapeutic levels in the brain for an extended period of time that can result in significant eradication of the viral load therein. Such nutrient mediated drug targeting could bring forth advances in biocompatible and biodegradable drug delivery systems.

First clinical results of (D)-18F-Fluoromethyltyrosine (BAY 86-9596) PET/CT in patients with non-small cell lung cancer and head and neck squamous cell carcinoma.

(D)-(18)F-fluoromethyltyrosine (d-(18)F-FMT), or BAY 86-9596, is a novel (18)F-labeled tyrosine derivative rapidly transported by the l-amino acid transporter (LAT-1), with a faster blood pool clearance than the corresponding l-isomer. The aim of this study was to demonstrate the feasibility of tumor detection in patients with non-small cell lung cancer (NSCLC) or head and neck squamous cell cancer (HNSCC) compared with inflammatory and physiologic tissues in direct comparison to (18)F-FDG. 18 patients with biopsy-proven NSCLC (n = 10) or HNSCC (n = 8) were included in this Institutional Review Board-approved, prospective multicenter study. All patients underwent (18)F-FDG PET/CT scans within 21 d before d-(18)F-FMT PET/CT. For all patients, safety and outcome data were assessed. No adverse reactions were observed related to d-(18)F-FMT. Fifty-two lesions were (18)F-FDG-positive, and 42 of those were malignant (34 histologically proven and 8 with clinical reference). Thirty-two of the 42 malignant lesions were also d-(18)F-FMT-positive, and 10 lesions had no tracer uptake above the level of the blood pool. Overall there were 34 true-positive, 8 true-negative, 10 false-negative, and only 2 false-positive lesions for d-(18)F-FMT, whereas (18)F-FDG was true-positive in 42 lesions, with 10 false-positive and only 2 false-negative, resulting in a lesion-based detection rate for d-(18)F-FMT and (18)F-FDG of 77% and 95%, respectively, with an accuracy of 78% for both tracers. A high d-(18)F-FMT tumor-to-blood pool ratio had a negative correlation with overall survival (P = 0.050), whereas the (18)F-FDG tumor-to-blood pool ratio did not correlate with overall survival. d-(18)F-FMT imaging in patients with NSCLC and HNSCC is safe and feasible. The presented preliminary results suggest a lower sensitivity but higher specificity for d-(18)F-FMT over (18)F-FDG, since there is no d-(18)F-FMT uptake in inflammation. This increased specificity may be particularly beneficial in areas with endemic granulomatous disease and may improve clinical management. Further clinical investigations are needed to determine its clinical value and relevance for the prediction of survival prognosis.

P13.09 * ADVANCES IN CLINICAL APPLICATION OF BORON NEUTRON CAPTURE THERAPY (BNCT) IN GLIOBLASTOMA

BNCT is a biologically targeted form of enhanced cellular radiotherapy where preferential accumulation of boron in the cancerous as opposed to adjacent normal cells is able to interact with incident neutrons to cause irreversible alpha particle DNA damage. The key to the implementation of this potentially powerful and selective therapy is the delivery of at least 30ppm 10B within the tumour tissue while minimising superfluous 10B in healthy tissue. It is thus an elegant technique for treating infiltrating tumours such as diffuse gliomas. In order to assess its clinical potential we carried out a pharmacokinetic study in glioblastoma patients where we sought to determine the optimal route of delivering a new formulation of the boronated drug (p-boronophenylalanine, BPA), its pharmacokinetic behaviour, toxicity profile, and cellular uptake. Using a number of analytical techniques, including inductively-coupled plasma mass spectrometry, secondary ion mass spectrometry (SIMS) and immunohistochemistry (IHC), boron was measured at various times in blood, urine, cerebrospinal fluid, extracellular fluid (ECF), and tumour-related solid tissue spanning 0.5 h pre- and up to 48 h post-BPA infusion in newly-diagnosed patients (n = 10). Blood was sampled through a central catheter whilst the ECF was sampled by parenchymal microdialysis catheters, placed remotely from the tumour site. Urine was collected over the same time period. Tumour and brain-around tumour (BAT) tissue was sampled stereotactically at 2.5 h and 3.5 h post-infusion. IHC expression levels of the BPA transporter molecule, L-amino acid transporter 1 (LAT-1), were recorded as % LAT-1 positive cells, and cellular boron levels were estimated as spatially resolved pixels in normalised-to-C+ isotopic SIMS images of the biopsies. There were no toxicity-related issues with this new formulation of BPA given at 375 mg/kg as a 2 h intravenous or intracarotid infusion with or without pre-infusion mannitol-induced BBB disruption. The pharmacokinetic profile indicates a high plasma-to-brain concentration gradient from intracarotid infusion and BBB manipulation and reliably places boron in high concentrations in the brain compartment. However, tumour and BAT uptake varied, but a continuing uptake in BAT over time to >30 ppm boron suggests a more favourable capacity to concentrate boron for BNCT after debulking surgery. At the cellular level, the ratio of boron uptake as measured by SIMS in tumour vs BAT was 0.96, 1.85 and 2.40 in the intravenous, intravenous plus mannitol BBB-D, and intracarotid cohorts respectively. Thus improved specific and targeted delivery of boron is possible. These observations conform to a three compartment model where tumoural uptake of boron is governed solely by the abundance and two-way kinetics of tumour LAT-1 activity rather than BBB penetration and may explain some of the variation in clinical results of others.

Association of common variants in PAH and LAT1 with non-syndromic cleft lip with or without cleft palate (NSCL/P) in the Polish population

BackgroundNon-syndromic cleft lip with or without cleft palate (NSCL/P) is a common structural malformation with a complex and multifactorial aetiology. Associations of abnormalities in phenylalanine metabolism and orofacial clefts have been suggested. MethodsEight single nucleotide polymorphisms (SNPs) of genes encoding phenylalanine hydroxylase (PAH) and large neutral l-amino acid transporter type 1 (LAT1), as well as the PAH mutation that is most common in the Polish population (rs5030858; R408W), were investigated in 263 patients with NSCL/P and 270 matched controls using high resolution melting curve analysis (HRM). ResultsWe found that two polymorphic variants of PAH appear to be risk factors for NSCL/P. The odds ratio (OR) for individuals with the rs7485331 A allele (AC or AA) compared to CC homozygotes was 0.616 (95% confidence interval [CI]=0.437–0.868; p=0.005) and this association remains statistically significant after multiple testing correction. The PAH rs12425434, previously associated with schizophrenia, was borderline associated with orofacial clefts. Moreover, haplotype analysis of polymorphisms in the PAH gene revealed a 4-marker combination that was significantly associated with NSCL/P. The global p-value for a haplotype comprised of SNPs rs74385331, rs12425434, rs1722392, and the mutation rs5030858 was 0.032, but this association did not survive multiple testing correction. ConclusionThis study suggests the involvement of the PAH gene in the aetiology of NSCL/P in the tested population. Further replication will be required in separate cohorts to confirm the consistency of the observed association.

Unique and selective expression of L-amino acid transporter 1 in human tissue as well as being an aspect of oncofetal protein.

Dysregulated expression of L-type amino acid transporter 1 (LAT1), which transports large neutral amino acids, is a characteristic of various human cancers and possibly offers a molecular target for chemotherapy. LAT2, in contrast, shows lower expression in neoplasms. LAT1 is presumed to be a biomarker of many cancers, suggesting a kind of oncoprotein. However, no precise analysis of LAT1 and LAT2 expression has been performed in systemic normal tissues. To see characteristics of LAT1 and LAT2, immunohistochemical expression of LAT1 and LAT2 was assessed and compared in normal human systemic organs and tissues from 3 adults, 3 children and 3 fetuses in the present study. Cardiac muscles, hepatocytes, thymic epithelial cells and primitive neuroectodermal cells in fetus were positive with LAT1, whereas no expression was found in the respective adult tissues, indicating an aspect of oncofetal protein. In adult tissues, LAT1 was found to be expressed proximal to proliferative zones in gastrointestinal mucosa by double immunostaining of LAT1 and Ki-67. Testicular Sertoli cells, ovarian follicular cells, and pancreatic islet cells showed strong expression. Although the systemic capillary endothelium did not express LAT1, but did express LAT2, capillaries corresponding to the blood-brain, blood-follicle, and blood-retinal barriers demonstrated strong LAT1 immunoreactions. In conclusion, LAT1 was expressed in gonad tissues and several kinds of cells having special functions, as well as being discovered to be an aspect of oncofetal protein. In addition, ubiquitous LAT2 expression was confirmed immunohistochemically in systemic tissues, indicating constitutional function.