A low-molecular-weight chitosan fluorometric-based assay for evaluating antiangiogenic drugs

Abstract

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.