In vitro characterization of uptake mechanism of L-[methyl-(3)H]-methionine in hepatocellular carcinoma.
In vitro characterization of uptake mechanism of L-[methyl-(3)H]-methionine in hepatocellular carcinoma.
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DOI：
10.1007/s11307-014-0720-9
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发表时间：
2014-08
影响因子：
3.1
通讯作者：
Lee Z
中科院分区：
文献类型：
--
作者：
Kuang Y;Wang F;Corn DJ;Tian H;Lee Z
甲硫氨酸（Met）可能是诊断肝细胞癌（HCC）的有用的成像生物标志物，如L-[甲基-11 C]-Met的PET成像所示。在HCC细胞中，蛋白质合成主要有助于放射性药物摄取。相反，通过磷脂酰乙醇胺（PE）甲基化途径的脂质合成是L-[甲基-11 C]-Met在正常肝细胞中的主要代谢途径，这有助于PET图像中观察到的背景对比度。然而，氨基酸转运的机制和两个关键酶，蛋氨酸腺苷转移酶（MAT）和磷脂酰乙醇胺N-甲基转移酶（PEMT）的作用，尚未完全理解。本研究的目的是探讨氨基酸转运蛋白和这两个关键酶在肝癌细胞摄取L-[甲基-11 C]-Met中的作用。本研究使用了分化良好的土拨鼠肝癌细胞系WCH 17。以WCH 17细胞为研究对象，通过对细胞氨基酸转运蛋白的分析，探讨肝癌细胞中Met的转运过程。用5 mM S-腺苷甲硫氨酸（SAM）处理WCH 17细胞8、16、24和48 h，以下调MAT 2A基因表达。对照或SAM处理的WCH 17细胞用L-[甲基-3H]-Met脉冲5分钟，并用冷培养基追踪以模拟放射性标记的Met的快速血液清除（脉冲追踪实验）。平行地，WCH 17细胞用小鼠肝PEMT 2表达载体转染，并进行脉冲追踪实验以研究放射性标记的Met在HCC细胞中的摄取。随后分别提取和测量来自总摄取的水溶性、蛋白质和脂质相。肝细胞癌细胞内Met的转运是一个易化转运过程，具有L系统和ASC系统的特点，Na+依赖，低亲和力，部分能量依赖。SAM处理的肝癌细胞对L-[甲基-3H]-Met的总摄取量减少。这种减少模式遵循MAT 2A表达的模式（SAM处理的持续时间）。SAM处理的HCC细胞中掺入的3 H主要分布在蛋白质相中，并且在较小程度上通过PE甲基化途径分布在脂质相中。MAT 2A表达下调导致蛋白质和水溶性相的摄取减少。此外，在用PEMT 2表达载体转染的WCH 17细胞中观察到脂质相的摄取增加。氨基酸转运过程可能是负责放射性标记的蛋氨酸后，肝癌显像示踪剂静脉注射的快速积累。HCC中MAT 2A表达上调和PEMT 2活性受损与PET检测到的L-[甲基-11 C]-Met的特定代谢模式相关。
Methionine (Met) could be a useful imaging biomarker for the diagnosis of hepatocellular carcinoma (HCC), as demonstrated by PET imaging with L-[methyl-11C]-Met. In HCC cells, protein synthesis mainly contributes to radiopharmaceutical uptake. In contrast, lipid synthesis via the phosphatidylethanolamine (PE) methylation pathway is the major metabolic route of L-[methyl-11C]-Met in normal hepatocytes, which contributes to the background contrast observed in PET images. However, the mechanisms of amino acid transport and the roles of the two key enzymes, methionine adenosyltransferase (MAT) and phosphatidylethanolamine N-methyltransferase (PEMT), are not yet completely understood. The aim of this study was to investigate the roles of the amino acid transporters and these two key enzymes in the uptake of L-[methyl-11C]-Met in HCC cells. A well-differentiated woodchuck HCC cell line, WCH17, was used for the study. The amino acid transporter of WCH17 cells was assayed to investigate the Met transport process in HCC. WCH17 cells were treated with 5 mM S-adenosylmethionine (SAM) for 8, 16, 24, and 48 h to downregulate MAT2A gene expression. Control or SAM-treated WCH17 cells were pulsed with L-[methyl-3H]-Met for 5 min and chased with cold media to mimic the rapid blood clearance of radiolabeled Met (pulse-chase experiment). In parallel, WCH17 cells were transfected with a mouse liver PEMT2 expression vector, and the pulse-chase experiment was performed to investigate the uptake of the radiolabeled Met in HCC cells. The water-soluble, protein, and lipid phases from the total uptake were subsequently extracted and measured, respectively. Met was transported into HCC cells via a facilitative transport process, which was characterized as system L and ASC-like, Na+ dependent, and low affinity with partial energy dependence. The total uptake of L-[methyl-3H]-Met was decreased in HCC cells with SAM treatment. This reduction pattern followed that of MAT2A expression (the duration of SAM treatment). The incorporated 3H was mostly distributed in the protein phase and, to a lesser degree, in the lipid phase via PE methylation pathway in HCC cells with SAM treatment. The downregulated MAT2A expression led to the decreased uptake in protein and water-soluble phases. In addition, an increased uptake in the lipid phase was observed in WCH17 cells transfected with PEMT2 expression vector. The amino acid transport processes may be responsible for the rapid accumulation of radiolabeled Met after the intravenous injection of tracers for the imaging of HCC. Upregulated MAT2A expression and impaired PEMT2 activities in HCC are associated with the specific metabolic pattern of L-[methyl-11C]-Met detected by PET.