State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Department of Drug Metabolism, Beijing Key Laboratory of Non-Clinical Drug Metabolism and PK/PD Study, Beijing Key Laboratory of Active Substances Discovery and Drug Ability Evaluation, Institute of Materia Medica, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, 100050, China.
State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Department of Drug Metabolism, Beijing Key Laboratory of Non-Clinical Drug Metabolism and PK/PD Study, Beijing Key Laboratory of Active Substances Discovery and Drug Ability Evaluation, Institute of Materia Medica, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, 100050, China.
Anal Biochem. 2021 Oct 15;631:114357. doi: 10.1016/j.ab.2021.114357. Epub 2021 Aug 29.
The growth and proliferation of most cancer cells involve the excessive uptake of glucose mediated by glucose transporters. An effective strategy for cancer therapy has been to inhibit the GLUTs that are usually overexpressed in a variety of tumor cells. 2-NBDG is a GLUT1 substrate that can be used as a probe for GLUT1 inhibitors. An accurate and simple assay for 2-NBDG in a HEK293T cell model overexpressing GLUT1 was developed using liquid chromatography-tandem mass spectrometry. Chromatographic separation was achieved using a Xbridge® Amide column (3.5 μm, 2.1 mm × 150 mm, Waters) with acetonitrile-water containing 2 μM ammonium acetate (80:20, v/v) at a flow rate of 0.25 mL/min. Mass detection was conducted in the parallel reaction monitoring (PRM) mode. The calibration curve for 2-NBDG showed good linearity in the concentration range of 5-500 ng/mL with satisfactory precision, a relative standard deviation ranging from 2.92 to 9.59% and accuracy with a relative error ranging from -13.14 to 7.34%. This method was successfully applied to quantify the uptake of GLUT1-mediated 2-NBDG, and the results clearly indicated inhibition of GLUT1 by WZB117 and quercetin (two potent glucose transporter inhibitors) in the GLUT1-HEK293T cell model. This study provides a convenient and accurate method for high-throughput screening of selective and promising GLUT1 inhibitors.
大多数癌细胞的生长和增殖都涉及葡萄糖转运蛋白介导的葡萄糖过度摄取。癌症治疗的一种有效策略是抑制通常在各种肿瘤细胞中过度表达的 GLUTs。2-NBDG 是 GLUT1 的底物，可用作 GLUT1 抑制剂的探针。在过表达 GLUT1 的 HEK293T 细胞模型中，使用液相色谱-串联质谱法开发了一种用于 2-NBDG 的准确且简单的测定方法。采用 Xbridge®酰胺柱（3.5μm，2.1mm×150mm，Waters），以乙腈-水（含 2μM 乙酸铵，80:20，v/v）为流动相，流速为 0.25mL/min，实现色谱分离。采用平行反应监测（PRM）模式进行质量检测。2-NBDG 的校准曲线在 5-500ng/mL 的浓度范围内具有良好的线性，精密度令人满意，相对标准偏差范围为 2.92-9.59%，准确度的相对误差范围为-13.14%-7.34%。该方法成功地应用于定量测定 GLUT1 介导的 2-NBDG 的摄取，结果清楚地表明 WZB117 和槲皮素（两种有效的葡萄糖转运蛋白抑制剂）在 GLUT1-HEK293T 细胞模型中抑制 GLUT1。本研究为高通量筛选选择性和有前途的 GLUT1 抑制剂提供了一种方便、准确的方法。
