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 7beta-hydroxycholesterol.
OBJECTIVE
To characterize the possible cytotoxic effects of oxysterols (7beta-hydroxycholesterol (7beta-OH), 25-hydroxycholesterol (25-OH)) in human retinal pigment epithelial cells (ARPE-19) and to detail the relationships between some of these effects.


METHODS
ARPE-19 cells were treated with 7beta-OH and 25-OH. Cell viability was measured with the MTT assay. Membrane permeability, mitochondrial potential, and lysosomal integrity were measured by flow cytometry with propidium iodide, DiOC6(3), and acridine orange, respectively. Cell death was characterized by staining with Hoechst 33342, transmission electron microscopy, and analysis of the DNA fragmentation pattern. Caspase activity was examined with fluorochrome-labeled inhibitors of caspases (FLICA) and Western blotting. Immunofluorescence staining was used to visualize the cellular distribution of cytochrome c (Cyt-c) and apoptosis-inducing factor (AIF). The effect of the cathepsin inhibitor (z-FA-fmk) on oxysterol-induced cell death was evaluated.


RESULTS
Cell viability of ARPE-19 cells was decreased with 7beta-OH, whereas 25-OH had no cytotoxic effects. Loss of mitochondrial potential and lysosomal destabilization was associated with 7beta-OH-induced cell death, few morphologically apoptotic cells were identified, and no internucleosomal DNA fragmentation was found. Slight caspase activation was detected with FLICA, and no caspase-3 activation was revealed. A pronounced relocalization of Cyt-c and AIF was observed. Noteworthy, z-FA-fmk was able to prevent cell death.


CONCLUSIONS
7beta-OH induced a caspase-3-independent mode of cell death associated with lysosomal destabilization, which could play a key role in the signaling pathways leading to cell death, as shown by the ability of z-FA-fmk to counteract the cytotoxic effects of 7beta-OH.
Malvitte
Laure
L
Faculte des Sciences Gabriel, Centre de Recherche Inserm-Equipe Biochimie Metabolique et Nutritionnelle, Inserm U866-Universite de Bourgogne, Dijon, France.
Montange
Thomas
T
Vejux
Anne
A
Joffre
Corinne
C
Bron
Alain
A
Creuzot-Garcher
Catherine
C
Lizard
Gerard
G
D051033:Apoptosis Inducing Factor / Q000378:metabolism; D015153:Blotting, Western; D053148:Caspase 3 / Q000378:metabolism*; D016923:Cell Death / Q000187:drug effects; D002463:Cell Membrane Permeability / Q000502:physiology; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D045304:Cytochromes c / Q000378:metabolism; D053938:DNA Fragmentation; D004151:Dipeptides / Q000494:pharmacology; D004587:Electrophoresis, Agar Gel; D004789:Enzyme Activation; D005434:Flow Cytometry; D019084:Fluorescent Antibody Technique, Indirect; D006801:Humans; D006888:Hydroxycholesterols / Q000494:pharmacology*; D007659:Ketones / Q000494:pharmacology; D008247:Lysosomes / Q000378:metabolism*; D008564:Membrane Potentials / Q000502:physiology; D051336:Mitochondrial Membranes / Q000502:physiology; D010857:Pigment Epithelium of Eye / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology
D051033:Apoptosis Inducing Factor; D015853:Cysteine Proteinase Inhibitors; D004151:Dipeptides; D006888:Hydroxycholesterols; D007659:Ketones; C057383:MDL 201053; C011724:cholest-5-en-3 beta,7 alpha-diol; C007997:25-hydroxycholesterol; D045304:Cytochromes c; D053148:Caspase 3