Casein kinase I inhibitor D4476 influences autophagy and apoptosis in chloroquine-induced adult retinal pigment epithelial-19 cells

• Published in: Experimental eye research Vol. 218; p. 109004
• Main Authors: Nguyen Hoang, Anh Thu, Lee, Haesung, Lee, Sook-Jeong
• Format: Journal Article
• Language: English
• Published: England Elsevier Ltd 01.05.2022
• Subjects: Apoptosis; Autophagy; Beclin-1 - metabolism; Casein kinase 1 inhibitor; Casein Kinase I - metabolism; Chloroquine; Chloroquine - toxicity; Human retinal pigment epithelium; Humans; Lysosomotropic agent; Proto-Oncogene Proteins c-bcl-2 - metabolism; Retinal Diseases - metabolism; Retinal Pigment Epithelium - metabolism; Retinal Pigments - metabolism; Retinal Pigments - pharmacology; Casein kinase 1 inhibitor; Lysosomotropic agent; Autophagy; Human retinal pigment epithelium; Chloroquine
• ISSN: 0014-4835; 1096-0007
• DOI: 10.1016/j.exer.2022.109004

The antimalarial drug chloroquine (CQ) induces retinopathy, a disorder characterized by lysosomotropic alteration. In this study, we examined whether D4476 (4-(4-(2,3-dihydrobenzo [1,4] dioxin-6-yl)-5-pyridin-2-yl-1H-imidazole-2-yl) benzamide), a specific casein kinase 1 inhibitor, alleviate CQ-induced retinopathy in adult retinal pigment epithelial (ARPE-19) cells. Cultured ARPE-19 cells were exposed to CQ with or without D4476 and cell death was quantified using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. To examine autophagy flux, ARPE-19 cells were transfected with green fluorescence protein light chain 3 (GFP-LC3)-red fluorescence protein (RFP)-LC3ΔG plasmid DNA and co-stained with the lysosomal-associated membrane protein (LAMP)-1 antibody. Western blotting and fluorescence-activated cell sorting (FACS) showed apoptosis, whereas the fluorescence intensity of 2′-7′-dichlorofluorescein diacetate revealed levels of cellular oxidative stress. We then confirmed the effect of D4476 on the interaction between Beclin 1 and B-cell lymphoma-2 (Bcl-2) through immunoprecipitation with an anti-Bcl-2 antibody. Following CQ exposure, ARPE-19 cells accumulated autophagosomes because of defective lysosomal degradation. Furthermore, CQ trapped Beclin 1 with Bcl-2, disturbing autophagy initiation and autolysosome formation. However, D4476 alleviated CQ-induced effects by rescuing ARPE-19 cells from CQ-induced toxicity by modulating the association between Beclin 1 and Bcl-2. Therefore, D4476 controls autophagy and apoptosis simultaneously by upregulating autophagy flux, decreasing ROS formation, and triggering the expression of anti-apoptotic proteins through inhibition of mTOR, JNK, and p38 MAPK signals. We conclude that D4476 is a promising treatment strategy for CQ-mediated retinopathy. ●CKI inhibitor D4476 relieves CQ-induced autophagy stress in ARPE-19 cells.●D4476 controls the interaction between Beclin 1 and Bcl-2 in ARPE-19 cells.●D4476 regulates both autophagy and apoptosis in CQ-treated ARPE-19 cells.●D4476 affects phosphorylation of mTOR, p38 MAPK, and JNK in ARPE-19 cells.