Activation of a Caspase-3-Independent Mode of Cell Death Associated with Lysosomal Destabilization in Cultured Human Retinal Pigment Epithelial Cells (ARPE-19) Exposed to 7β-Hydroxycholesterol

Abstract

Purpose: To characterize the possible cytotoxic effects of oxysterols(7β-hydroxycholesterol (7β-OH), 25-hydroxycholesterol (25-OH)) in human retinalpigment epithelial cells (ARPE-19) and to detail the relationships between someof these effects. Methods: ARPE-19 cells were treated with 7β-OH and 25-OH. Cellviability was measured with the MTT assay. Membrane permeability, mitochondrialpotential, and lysosomal integrity were measured by flow cytometry with propidiumiodide, DiOC6(3), and acridine orange, respectively. Cell death was characterizedby staining with Hoechst 33342, transmission electron microscopy, and analysis ofthe DNA fragmentation pattern. Caspase activity was examined with fluorochrome labeledinhibitors of caspases (FLICA) and Western blotting. Immunofluorescencestaining was used to visualize the cellular distribution of cytochrome c (Cyt-c) andapoptosis-inducing factor (AIF). The effect of the cathepsin inhibitor (z-FA-fmk)on oxysterol-induced cell death was evaluated. Results: Cell viability of ARPE-19cells was decreased with 7β-OH, whereas 25-OH had no cytotoxic effects. Lossof mitochondrial potential and lysosomal destabilization was associated with 7β-OH-induced cell death, few morphologically apoptotic cells were identified, andno internucleosomal DNA fragmentation was found. Slight caspase activation wasdetected with FLICA, and no caspase-3 activation was revealed. A pronouncedrelocalization of Cyt-c and AIF was observed. Noteworthy, z-FA-fmk was able toprevent cell death. Conclusion: 7β-OH induced a caspase-3-independent mode ofcell death associated with lysosomal destabilization, which could play a key role inthe signaling pathways leading to cell death, as shown by the ability of z-FA-fmkto counteract the cytotoxic effects of 7β-OH.