Abstract
Purpose :
We previously showed that chloroquine (CQ)-induced retinal toxicity occurs mainly by the inhibition of autophagic flux. To find potential therapeutics against CQ toxicity we used ARPE-19 cells and measured cell death as well as marker for autophagic flux.
Methods :
We selected potential therapeutic drugs against CQ-cytotoxicity in ARPE-19 cell cultures by screening the LOPAC®1280 (the library of pharmacologically active compounds, Sigma). Cell death was quantitatively assessed by measuring lactate dehydrogenase (LDH) activity released into the culture medium. In parallel, cell viability was also assessed using a 3-[4,5-dimethylthiazole-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay. For western blots for autophagy-related markers, cultured ARPE-19 cells were treated with 50 mM CQ for 3 hours. Lysosomal pH was measured by confocal microscopy using a lysosome pH indicator, Lysosensor DND-189.
Results :
ARPE-19 cells developed cytosolic vacuoles within 1 hour after the onset of CQ-treatment. Twenty-four hours later, ARPE-19 cells showed a significant increase of CQ-induced cell death (84.42 ± 3.48 % by LDH assay) compared to non-treated control ARPE-19 cells. For the HTS, 10 mM of each compound from LOPAC®1280 library was added with CQ, and cell death was assessed 24 hours later. Initially, we selected 7 compounds that reduced CQ toxicity by 40% or more by LDH assay. Then, we tested concentration-protection effects of each with the MTT viability assay, and selected 3 compounds as candidates. Western blot analysis showed that LC3-II and p62 accumulated in ARPE-19 cells 3 hours after CQ treatment, likely as results of autophagy arrest. All 3 compounds reduced CQ-induced increases in p62 levels consistent with increases in autophagy flux. Lysosensor (DND-189) confocal microscopy showed that 3 compounds ameliorated CQ-induced alkaline shift of lysosomal lumens.
Conclusions :
CQ induces lysosomal alkalinization, arrested autophagy, and death in ARPE-19 cells. The selected compounds reduced CQ-induced cell death likely via reacidifying lysosomes and consequently enhancing autophagy flux.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.