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
Current Eye Research. 2008-01-01; 33(9): 769-781
DOI: 10.1080/02713680802337397
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1. Curr Eye Res. 2008 Sep;33(9):769-81. doi: 10.1080/02713680802337397.
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.
Malvitte L(1), Montange T, Vejux A, Joffre C, Bron A, Creuzot-Garcher C, Lizard
G.
Author information:
(1)Faculte des Sciences Gabriel, Centre de Recherche Inserm-Equipe Biochimie
Metabolique et Nutritionnelle, Inserm U866-Universite de Bourgogne, Dijon,
France.
PURPOSE: 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.
CONCLUSION: 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.
DOI: 10.1080/02713680802337397
PMID: 18798080 [Indexed for MEDLINE]