Department of Radiation Oncology, Yuan's General Hospital, Kaohsiung 80249, Taiwan.
Department of Biochemistry and Molecular Cell Biology, School of Medicine, College of Medicine, Taipei Medical University, Taipei 11031, Taiwan.
Int J Biol Macromol. 2023 Jan 1;224:927-937. doi: 10.1016/j.ijbiomac.2022.10.178. Epub 2022 Oct 25.
Low-molecular-weight chitosan (LMWCS) damaged cell membranes in zebrafish showed its possibility to release reporter proteins for detection. In this study, we developed a simple fluorometric-based assay for the evaluation of clinical antiangiogenic drugs using LMWCS and Tg(fli1:EGFP) transgenic zebrafish, which expressed green-fluorescence protein (GFP) in the endothelial cells of blood vessel. In vitro stable and transiently transfected cell lines was released luciferase and green fluorescent protein (GFP) for intensity evaluation upon LMWCS fluorometric-based assay. In vivo Tg(fli1:EGFP) transgenic zebrafish was also released GFP from endothelial cells of blood vessels and show an increase of fluorescent intensity upon LMWCS fluorometric-based assay. Treatment with the clinical antiangiogenic drug sorafenib and analyzed by LMWCS fluorometric-based assay showed significantly reduction of angiogenesis. Furthermore, treatment with 2 μM sorafenib showed a significant reduction in angiogenesis of the intersegmental vein (ISV) and dorsal longitudinal anastomotic vessels (DLAV) in Tg(fli1:EGFP) transgenic zebrafish. Fluorescence intensity reduction from 2 μM sorafenib was used as a factor in the LMWCS fluorescence-based assay for relative antiangiogenic evaluation. Relative angiogenesis evaluation of the clinical drugs axitinib, cabozantinib, and regorafenib showed a significant reduction. Collectively, this study provided a simple, convenient, and rapid LMWCS fluorometric-based assay for evaluating angiogenic drugs using transgenic zebrafish.
低相对分子质量壳聚糖（LMWCS）损伤斑马鱼细胞膜，表明其有可能释放报告蛋白进行检测。在这项研究中，我们开发了一种简单的荧光测定法，用于评估临床抗血管生成药物，该方法使用 LMWCS 和 Tg(fli1:EGFP)转基因斑马鱼，后者在血管内皮细胞中表达绿色荧光蛋白（GFP）。在体外，稳定和瞬时转染的细胞系释放荧光素酶和绿色荧光蛋白（GFP），通过 LMWCS 荧光测定法进行强度评估。体内 Tg(fli1:EGFP)转基因斑马鱼也从血管内皮细胞释放 GFP，并在 LMWCS 荧光测定法中显示荧光强度增加。用临床抗血管生成药物索拉非尼进行处理，并通过 LMWCS 荧光测定法进行分析，显示血管生成明显减少。此外，用 2μM 索拉非尼处理 Tg(fli1:EGFP)转基因斑马鱼，可显著减少节间静脉（ISV）和背侧纵向吻合血管（DLAV）的血管生成。荧光强度从 2μM 索拉非尼的降低被用作 LMWCS 荧光测定法中相对抗血管生成评估的一个因素。临床药物阿西替尼、卡博替尼和瑞戈非尼的相对血管生成评估显示明显减少。总之，这项研究提供了一种简单、方便、快速的 LMWCS 荧光测定法，用于使用转基因斑马鱼评估血管生成药物。
