Drug resistance‑related sunitinib sequestration in autophagolysosomes of endothelial cells

Our previous study demonstrated that the tyrosine kinase receptor inhibitor sunitinib induces acquired drug resistance in endothelial cells. The present study explored the role of lysosomal sequestration of sunitinib in the acquisition of drug resistance in human microcapillary endothelial HMEC1 cells. Resistance was induced by escalating concentrations of sunitinib and a shift in IC50 from 12.8 to >20 microM was detected. The results of timelapse fluorescence microscopy illustrated an instantaneous emergence of fluorescent vesicles in living cells once sunitinib was added. Most of these vesicles emerged in the juxtanuclear area, and exhibited the characteristics of growing autophagosomes and lysosomes. The vesicles were identified as autophagosomes and lysosomes because they colocated with the lysosomal tracers LysoER and LysoNIR, and the protein markers lysosomalassociated membrane protein 1 (LAMP1) and microtubuleassociated protein 1A/1Blight chain 3 (LC3). The results of western blotting demonstrated that sunitinib induced upregulation of LAMP1 and LC3II, and downregulation of sequestosome 1/p62, indicating the activation of autophagy. Bafilomycin A1, which suppresses lysosomal acidification, completely blocked sunitinib sequestration; however, chloroquine, which blocks lysosomal fusion with autophagosomes, exhibited no effect. Notably, bafilomycin A1 and chloroquine significantly counterbalanced HMEC1 drugresistance. These results provided evidence for autophagyfluxassociated sunitinib lysosomal sequestration in endothelial cells, leading to isolation of the drug from the cytoplasm; a key process involved in the development of drug resistance during antiangiogenic therapy. These data supported the notion that inhibiting autophagy may be a potential strategy to prevent drug sequestration and resistance to antiangiogenic therapy.