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Drug resistance?related sunitinib sequestration in autophagolysosomes of endothelial cells.

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.

SUBMITTER: Wu S

PROVIDER: S-EPMC6910192 | biostudies-literature | 2020 Jan

REPOSITORIES: biostudies-literature

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Drug resistance‑related sunitinib sequestration in autophagolysosomes of endothelial cells.

Wu Shuang S Huang Limin L Shen Rong R Bernard-Cacciarella Melanie M Zhou Pei P Hu Chaoquan C Di Benedetto Melanie M Janin Anne A Bousquet Guilhem G Li Hong H He Zhixu Z Lu He H

International journal of oncology 20191126 1

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 instantaneo ...[more]

PMID: 31789391

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Project description:Angiosarcoma is a rare cancer of blood vessel-forming cells with a high patient mortality and few treatment options. Although chemotherapy often produces initial clinical responses, outcomes remain poor, largely due to the development of drug resistance. We previously identified a subset of doxorubicin-resistant cells in human angiosarcoma and canine hemangiosarcoma cell lines that exhibit high lysosomal accumulation of doxorubicin. Hydrophobic, weak base chemotherapeutics, like doxorubicin, are known to sequester within lysosomes, promoting resistance by limiting drug accessibility to cellular targets. Drug synergy between the beta adrenergic receptor (β-AR) antagonist, propranolol, and multiple chemotherapeutics has been documented in vitro, and clinical data have corroborated the increased therapeutic potential of propranolol with chemotherapy in angiosarcoma patients. Because propranolol is also a weak base and accumulates in lysosomes, we sought to determine whether propranolol enhanced doxorubicin cytotoxicity via antagonism of β-ARs or by preventing the lysosomal accumulation of doxorubicin. β-AR-like immunoreactivities were confirmed in primary tumor tissues and cell lines; receptor function was verified by monitoring downstream signaling pathways of β-ARs in response to receptor agonists and antagonists. Mechanistically, propranolol increased cytoplasmic doxorubicin concentrations in sarcoma cells by decreasing the lysosomal accumulation and cellular efflux of this chemotherapeutic agent. Equivalent concentrations of the receptor-active S-(-) and -inactive R-(+) enantiomers of propranolol produced similar effects, supporting a β-AR-independent mechanism. Long-term exposure of hemangiosarcoma cells to propranolol expanded both lysosomal size and number, yet cells remained sensitive to doxorubicin in the presence of propranolol. In contrast, removal of propranolol increased cellular resistance to doxorubicin, underscoring lysosomal doxorubicin sequestration as a key mechanism of resistance. Our results support the repurposing of the R-(+) enantiomer of propranolol with weak base chemotherapeutics to increase cytotoxicity and reduce the development of drug-resistant cell populations without the cardiovascular and other side effects associated with antagonism of β-ARs.

Dataset Information

Drug resistance?related sunitinib sequestration in autophagolysosomes of endothelial cells.

Publications

Drug resistance‑related sunitinib sequestration in autophagolysosomes of endothelial cells.

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