Investigation of biological significance of neutral amino acid transporter expression in human brain tumors and development of novel therapeutic strategies

目标。恶性肿瘤细胞表现出较高的增殖活性，这需要增加细胞内代谢。LAT1在其辅助因子4F2hc的存在下，构成系统L中性氨基酸转运体，负责大多数必需氨基酸的细胞运输。最近的报道表明LAT1在癌细胞中的表达增加，这促使我们研究其在人类胶质瘤细胞中的表达和生物学作用。采用Western blot和RT-PCR检测了13株胶质瘤细胞系中LAT1和4F2hc的表达。用系统L抑制剂BCH处理lat1阳性胶质瘤细胞，分别用BrdU染色和TUNEL法检测其增殖率和凋亡率。用编码人LAT1 cDNA的质粒转染LAT1阴性胶质瘤细胞，高表达LAT1的细胞进行体外生长试验，并皮下注射到裸鼠体内。采用^<14 bb0 c - le0法测定无酸吸收活性。大多数胶质瘤细胞系表达LAT1蛋白。所有细胞系均阳性表达4F2hc mRNA。BCH对lat1阳性LNZ308细胞的增殖有显著抑制作用，而对lat1阴性LN319细胞的增殖无明显抑制作用。BCH处理后LNZ308细胞增殖减少，凋亡增加，细胞周期调节因子表达水平和caspase激活水平发生变化。引入LAT1不影响LN319细胞的体外生长速率。然而，LAT1过表达导致U87MG细胞氨基酸摄取活性增加3.4倍，导致体内致瘤性增强。LAT1及其伴侣4F2hc在大多数人类胶质瘤细胞中表达。我们的研究结果表明，氨基酸转运系统的主要组成部分LAT1可能在胶质瘤的形成中发挥重要作用，可能是恶性胶质瘤的一个新的潜在治疗靶点。少

Objectives. Malignant tumor cells show an elevated proliferative activity which would require increased intracellular metabolism. LAT1, in the presence of its co-factor 4F2hc, constitutes the system L neurtral amino acid transporter which is responsible for cellular transport of most essential amino acids. Recent reports demonstrating increased expression of LAT1 in cancer cells prompted us to investigate its expression and biological roles in human glioma cells.Methods. Expression of LAT1 and 4F2hc was determined in 13 human glioma cell lines using Western blot and RT-PCR analyses. LAT1-positive glioma cells were treated with a system L inhibitor BCH, and their proliferation and apoptosis rates were examined by BrdU staining and TUNEL assay, respectively. LAT1-negative glioma cells were transfected with the plasmid encoding human LAT1 cDNA and the cells expressing high levels of LAT1 were subjected to in vitro growth assays, and were injected into nude mice subcutaneously as well. Ami … More no acid uptake activity was measured using ^<14>C-Leu.Results. Majority of glioma cell lines expressed LAT1 protein. All cell lines were positive for 4F2hc mRNA expression. BCH treatment resulted in significant reduction of proliferation in LAT1-positive LNZ308 cells, whereas it was ineffective in LAT1-negative LN319 cells. LNZ308 cells showed reduced proliferation and increased apoptosis upon BCH treatment, which were accompanied with changes of expression levels of cell-cycle regulators and caspase activation. Introduction of LAT1 did not affect growth rates in LN319 cells in vitro. However, LAT1 overexpression leading to 3.4-fold increase in amino acid uptake activity in U87MG cells resulted in enhanced tumorigenicity in vivo.Conclusions. LAT1 and its partner 4F2hc are expressed in majority of human glioma cells. Our results suggest that LAT1, the major component of the amino acid transport system, may play an important role in gliomagenesis and may be a novel potential therapeutic target for malignant gliomas. Less