Expression and characterization of amino acid transport system L in HTB-41 human salivary gland epidermoid carcinoma cells

Abstract

사람 타액선 편평상피암세포주 HTB-41에서 아미노산 수송계 L의 발현 및 이들 수송계 L을 통한 이미노산 수송특성을 밝히기 위하여,HTB-41 세포에서 RT-PCR, analysis,westernblotanalysis,uptake실험 및 MTT 분석을 시행하였다. HTB-41세포에서 아미노산 수송체 LAT1및 그 보조인자 4F2hc의 발현은 확인 할 수 있었으나,LAT2의 발현은 관찰할 수 없었다.HTB-41세포에서 L-leucine수송 은 Na+-비의존적이었으며,아미노산 수송계 L의 선택적 억제제인 BCH에 의해 완전히 차단되었다.HTB-41 세포에서 [14C]L-leucine의 수송친화력은 Xenopus oocyte에서 시행한 LAT1의 수송친화력과 유사하였으며,아미노산들에 의한 [^(14)C]L-leucine의 수송 억제는 Xenopus oocyte에서 시행한 LAT1의 실험결과와 유사하였다. BCH는 HTB-41세포의 성장을 시간-및 농도-의존적으로 억제하였다. 본 연구의 결과로 사람 타액선 편평상피암세포주 HTB-41에서는 중성아미노산 수 송계 L 중에서 주로 LAT1을 통해 L-leucine을 포함한 중성아미노산의 수송이 이루어 지고 있다는 것을 확인 할 수 있었으며,BCH는 이 LAT1을 차단하여 중성아미노산들 의 세포 내 고갈을 유도함으로서 HTB-41세포성장의 억제를 유도하는 것으로 사료된 다.또한 본 연구의 결과로 LAT1의 연구에 HTB-41 세포 유용성의 제시 및 이 LAT1의 억제제를 이용하여 타액선 암세포의 성장억제에 관한 또 하나의 방향성을 제 시할 수 있을 것으로 사료된다.|Amino acids are required for protein synthesis and energy sources in all living cells. The amino acid transport system L is a major nutrient transport system that is responsible for Na+-independent transport of neutral amino acids including several essential amino acids. The system L is divided into 2 major subgroups, the L-type amino acid transporter 1 (LAT1) and the L-type amino acid transporter 2 (LAT2). In malignant tumors, the LAT1 is highly expressed to support their continuous growth and proliferation. Although the salivary gland squamous cell carcinomas has an extremely poor prognosis, the expression and characterization of amino acid transporters for supplying nutrition to cells in the salivary gland tumors including the salivary gland squamous carcinomas cells are not known at all. In the present study, the expression and functional characterization of amino acid transport system L were, therefore, examined in the HTB-41 human submaxillary salivary gland epidermoid carcinoma cells. The expressions and functions of the system L amino acid transporters in the HTB-41 cells were investigated using RT-PCR analysis, western blot analysis and amino acid transport measurements. RT-PCR analysis and western blot analysis have revealed that the HTB-41 cells expressed the LAT1 together with its associating protein heavy chain of 4F2 antigen (4F2hc) in the plasma membrane, whereas the HTB-41 cells did not express the LAT2. The uptakes of [^(14)C]L-leucine by HTB-41 cells were Na+-independent and completely inhibited by a system L selective inhibitor, 2- aminobicyclo-(2,2,1)-heptane-2-carboxylic acid (BCH). The affinity of [^(14)C]L-leucine uptake and the inhibition profile of [^(14)C]L-leucine uptake by various L-amino acids in the HTB-41 cells were comparable with those for the LAT1 expressed in Xenopus oocytes. The majority of [^(14)C]L-leucine uptake is, therefore, mediated by LAT1 in the HTB-41 cells. In the MTT assay, the BCH inhibited the growth of the HTB-41 cells in the time- and concentration-dependent manners, indicating that the growth inhibition of HTB-41 cells by BCH is induced by the blocking of neutral amino acid transport mediated by LAT1. These results suggest that the transports of neutral amino acids including several essential amino acids into the HTB-41 human submaxillary salivary gland epidermoid carcinoma cells are mediated by LAT1. Therefore, the HTB-41 cell is proposed to be an excellent tool for examine the properties of LAT1. Moreover, the specific inhibition of LAT1 in tumor cells including the salivary gland tumor cells might be a new rationale for anti-tumor therapy.