Drug resistance‑related sunitinib sequestration in autophagolysosomes of endothelial cells

Wu,Shuang; Wu,Shuang; Huang,Limin; Huang,Limin; Shen,Rong; Shen,Rong; Bernard‑Cacciarella,Melanie; Bernard‑Cacciarella,Melanie; Zhou,Pei; Zhou,Pei; Hu,Chaoquan; Hu,Chaoquan; Di Benedetto,Melanie; Di Benedetto,Melanie; Janin,Anne; Janin,Anne; Bousquet,Guilhem; Bousquet,Guilhem; Li,Hong; Li,Hong; He,Zhixu; He,Zhixu; Lu,He; Lu,He

doi:10.3892/ijo.2019.4924

Drug resistance‑related sunitinib sequestration in autophagolysosomes of endothelial cells

Authors:
- Shuang Wu
- Limin Huang
- Rong Shen
- Melanie Bernard‑Cacciarella
- Pei Zhou
- Chaoquan Hu
- Melanie Di Benedetto
- Anne Janin
- Guilhem Bousquet
- Hong Li
- Zhixu He
- He Lu
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Affiliations: West China Second University Hospital, Sichuan University, Chengdu, Sichuan 610065, P.R. China, National Institute of Health and Medical Research, Medical Research Unit 942/Paris University 7 and 13, Avicenne Hospital, 93000 Bobigny, France, INSERM U1234/Rouen University, Faculty of Medicine and Pharmacy, 76000 Rouen, France
Published online on: November 26, 2019 https://doi.org/10.3892/ijo.2019.4924
Pages: 113-122
Copyright: © Wu et al. This is an open access article distributed under the terms of Creative Commons Attribution License.

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Abstract

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 HMEC‑1 cells. Resistance was induced by escalating concentrations of sunitinib and a shift in IC50 from 12.8 to >20 µM was detected. The results of time‑lapse 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 co‑located with the lysosomal tracers Lyso‑ER and Lyso‑NIR, and the protein markers lysosomal‑associated membrane protein 1 (LAMP‑1) and microtubule‑associated protein 1A/1B‑light chain 3 (LC3). The results of western blotting demonstrated that sunitinib induced upregulation of LAMP‑1 and LC3‑II, 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 HMEC‑1 drug‑resistance. These results provided evidence for autophagy‑flux‑associated 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.

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