In this study, we examined whether the cancer cell-killing effects of boron neutron capture therapy (BNCT) are enhanced by manipulating the expression levels of l-type amino acid transporter 1 (LAT1) of human cancer cells, which transports boronophenylalanine into cells. We transfected pCMV/LAT1-GFP plasmids into a T98G glioblastoma cell line and selected several clones. Confocal laser microscopic observation was performed to confirm the stable overexpression of LAT1 in the plasma membranes of the clones. Western blot was used to analyze the cellular accumulation of LAT1 protein in the clones. Relative intracellular uptake of boronophenylalanine (BPA) was determined by measuring the radioactivity of ¹⁴C-BPA using a radioactive iodine (RI) tracer method. Sensitivity to neutron and gamma (γ)-ray fluences generated by a research reactor facility at Kyoto University was assayed using colony formation assay. Green fluorescent protein (GFP)-tagged LAT1 was observed in the plasma membranes of the LAT1-overexpressing clones and the cellular accumulation of GFP-tagged LAT1 was largely increased in these clones. Intracellular uptake of BPA was 1.5–5.0 times greater among the clones than that in a control clone. The LAT1-overexpressing clones and transiently LAT1-lipofected T98G cells showed clearly enhanced sensitivity to neutron and γ-ray fluences compared to the control clone when they were treated with ¹⁰BPA. The sensitivity of cancer cells to the fluences was well correlated with the expression level of LAT1 in the cells and the level of BPA uptake. These results suggest that overexpression of LAT1 in cancer cells results in enhanced anticancer effects of BNCT, and BNCT combined with gene therapy is beneficial for tumors with low LAT1 expression.

在这项研究中，我们检查了硼中子俘获疗法 (BNCT) 的癌细胞杀伤作用是否通过操纵人类癌细胞的l型氨基酸转运蛋白 1 (LAT1)的表达水平来增强，后者将硼苯丙氨酸转运到细胞中。我们将pCMV / LAT1-GFP质粒转染到 T98G 胶质母细胞瘤细胞系中并选择了几个克隆。进行共聚焦激光显微观察以确认 LAT1 在克隆的质膜中的稳定过表达。使用蛋白质印迹分析克隆中 LAT1 蛋白的细胞积累。硼苯丙氨酸 (BPA) 的相对细胞内摄取通过测量¹⁴C-BPA 使用放射性碘 (RI) 示踪剂方法。使用集落形成测定法测定对京都大学研究反应堆设施产生的中子和伽马 (γ) 射线通量的敏感性。在LAT1 过表达克隆的质膜中观察到绿色荧光蛋白 (GFP)标记的 LAT1，并且这些克隆中 GFP 标记的 LAT1 的细胞积累大大增加。克隆中 BPA 的细胞内摄取量是对照克隆中的 1.5-5.0 倍。在LAT1 -overexpressing克隆和瞬时LAT1-脂转染T98G细胞显示明显增强到与对照相比，克隆和中子γ射线通量的灵敏度，当他们被处理用¹⁰双酚A。癌细胞对通量的敏感性与细胞中 LAT1 的表达水平和 BPA 摄取水平密切相关。这些结果表明，癌细胞中LAT1的过表达导致BNCT的抗癌作用增强，BNCT联合基因治疗有利于LAT1低表达的肿瘤。