Abstract
Amiodarone is a cationic amphiphilic drug used as an antiarrhythmic agent. It induces phospholipidosis, i.e., the accumulation of phospholipids within organelles of the endosomal–lysosomal system. Extracellular vesicles (EVs) are membrane-enclosed structures released by any type of cell and retrieved in every fluid of the body. EVs have been initially identified as a system to dispose cell waste, but they are also considered to be an additional manner to transmit intercellular signals. To understand the role of EVs in drug-induced phospholipidosis, we investigated EVs release in amiodarone-treated HEK-293 cells engineered to produce fluorescently labelled EVs. We observed that amiodarone induces the release of a higher number of EVs, mostly of a large/medium size. EVs released upon amiodarone treatment do not display significant morphological changes or altered size distribution, but they show a dose-dependent increase in autophagy associated markers, indicating a higher release of EVs with an autophagosome-like phenotype. Large/medium EVs also show a higher content of phospholipids. Drugs inducing lysosomal impairment such as chloroquine and bafilomycin A1 similarly prompt a higher release of EVs enriched in autophagy markers. This result suggests a mechanism associated with amiodarone-induced lysosomal impairment more than a connection with the accumulation of specific undigested substrates. Moreover, the implementation of the lysosomal function by overexpressing TFEB, a master gene regulator of lysosomal biogenesis, prevents the amiodarone-induced release of EVs, suggesting that this could be a feasible target to attenuate drug-induced abnormalities.
Highlights
Phospholipidosis is a major concern for drug development, as intracellular accumulation of undigested phospholipids in lamellar bodies is a major source of side effects for different tissues [1]
We investigated whether they could play a role in the cell response to cationic amphiphilic drugs (CAD)-induced phospholipid accumulation, possibly contributing to discard deposits and alleviate this phenotype
Our cell model was based on the overexpression in HEK cells of the mCherry-CD63 construct, which fuses the Extracellular vesicles (EVs) marker CD63 with the fluorescent protein mCherry and allows one to follow the release of both large/medium (10K fraction) and small (100K fraction) fluorescent EVs upon their separation from the cell culture medium by differential ultracentrifugation (dUC)
Summary
Drug-induced phospholipidosis (DIPL) is an acquired lipid storage disorder due to the massive intracellular accumulation of phospholipids in multilamellar inclusion bodies within the lysosomal–endosomal system [1,2,3]. The molecular mechanisms leading to the prevalent accumulation of phospholipids are unclear. The accumulation of this lipid class could be caused either by the inhibition of phospholipid degradation or enhancement of their biosynthesis. Phospholipids are usually degraded into lysosomes and the direct drug-induced inhibition of lysosomal phospholipases, or the formation of nondegradable drug–phospholipid complexes have been reported as a possible cause of their accumulation [2,4]. Lysosomal enzymes are synthesized in the endoplasmic reticulum (ER) and transported into the
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