Analysis of L-type amino acid transporter in canine hepatocellular carcinoma

Abstract

ABSTRACT. Analysis of L-type amino acid transport expression of hepatocellular carcinoma cells (HCCs) of the dog was performed. The leucine transport activity of canine HCCs was 0.628 ± 0.018 nmol/mg protein/min. The inhibitor of LAT 2-aminobicyclo[2.2.1]heptane-2-carboxylic acid (BCH) reduced 90% of the activity at 1 mM. The deduced amino acid sequences of canine LAT2, LAT3 and LAT4 were well conserved in mammalians, exhibiting 89, 88 and 77% homology, respectively. RT-PCR revealed distinct LAT1 expression compared with normal hepatocytes. Western blotting analysis confirmed the potent LAT1 expression in canine HCCs but not hepatocytes, and realtime RT-PCR analysis indicated that canine HCCs possessed 28 times higher LAT1 expression than hepatocytes. These results indicated that the leucine transport activity of canine HCCs was due to LAT1. KEY WORDS: canine, hepatoma, LAT, transporter doi: 10.1292/jvms.14-0392; J. Vet. Med. Sci. 77(5): 527-534, 2015 Many amino acid transport systems have been distinguished based on differences in their substrate-selectivity, ion-dependence, pH sensitivity, kinetics and regulatory properties by using membrane vesicle preparations or cultured cells [5,6]. Among them, system L is an amino acid transport system that mediates sodium-independent transport of neutral amino acids. It was first described in Ehrlich ascites tumor cells as a sodium-independent transport system for neutral amino acids that is specifically inhibited by a bicyclic amino acid, 2-aminobicyclo[2.2.1]heptane-2-carboxylic acid (BCH) [28,29,37]. Later, 2 subtypes for system L with distinct characteristics in substrate affinity, selectivity, and transport properties were reported and named systems L1 and L2 [37]. To date, 4 isoforms of system L have been identified at the molecular level. The first cloned transporters with system L activity were LAT1 and LAT2, which are members of the SLC (solute carrier) 7 family of transporters [17,21,34,36]. They mediate sodium-independent amino acid exchange and recognize a wide range of large neutral amino acids as substrates [35], expanding to small neutral amino acids in the case of LAT2 [30,31,33]. These proteins form heteromeric complexes via a disulfide bond with the heavy chain of 4F2 antigen (4F2hc, SLC3A2), a single transmembrane domain protein essential for the functional expression of LAT1 and LAT2.Later, other transporters were identified by expression cloning [1,3], and it was reported that they exhibited Naindependent, BCH-sensitive neutral amino acid transport activity. In spite of possessing the properties of system L, these transporters (LAT3 and LAT4) were structurally different from system L1 (LAT1 and LAT2), and additionally, they did not require co-expression with 4F2hc to elicit transport activity at the plasma membrane. LAT3 and LAT4 transport activities show specific properties: (i) preferential substrate specificity [6], (ii) complex kinetics [6], which are compatible with 2 simultaneous apparent affinities and (iii) inactivation by the thiol reagen...

The L-Type Amino Acid Transporter LAT1—An Emerging Target in Cancer

“…SLC7A5/ LAT1 was found overexpressed in a wide range of solid tumors including the most frequently diagnosed cancers such as breast cancer [13,14], prostate cancer [15,16,17], and lung cancer [18,19,20,21,22,23,24,25,26,27,28,29,30]. Additionally, LAT1 was found overexpressed in bladder cancer [31], bone cancer [32], brain tumors [33,34], gynecological cancers [35,36], esophagus cancers [37,38,39], gastrointestinal cancers [40,41], liver [42,43,44,45], pancreas [46,47], head and neck cancers [48], kidney cancer [49], leukemia [50], skin cancer [51,52,53,54], myeloma [55,56], sarcoma [32], thymic carcinoma [57], and thyroid cancer [58,59]. Most of these studies detected LAT1 at the protein level in patient-derived tissue microarrays and benign tissue was used as the control.…”