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  • Lysosomal release of cathepsin D precedes relocation of cytochrome c and loss of mitochondrial transmembrane potential during apoptosis induced by oxidative stress.

Lysosomal release of cathepsin D precedes relocation of cytochrome c and loss of mitochondrial transmembrane potential during apoptosis induced by oxidative stress.

Free radical biology & medicine (2000-01-21)
K Roberg, U Johansson, K Ollinger
ABSTRACT

Apoptosis was induced in human foreskin fibroblasts by the redox-cycling quinone naphthazarin (5,8-dihydroxy-1,4-naphthoquinone). Most of the cells displayed ultrastructure typical of apoptosis after 8 h of exposure to naphthazarin. Apoptosis was inhibited in fibroblasts pretreated with the cathepsin D inhibitor pepstatin A. Immunofluorescence analysis of the intracellular distribution of cathepsin D revealed a distinct granular pattern in control cells, whereas cells treated with naphthazarin for 30 min exhibited more diffuse staining that corresponded to release of the enzyme from lysosomes to the cytosol. After 2 h, release of cytochrome c from mitochondria to the cytosol was indicated by immunofluorescence. The membrane-potential-sensitive probe JC-1 and flow cytometry did not detect a permanent decrease in mitochondrial transmembrane potential (delta psi(m)) until after 5 h of naphthazarin treatment. Our findings show that, during naphthazarin-induced apoptosis, lysosomal destabilization (measured as release of cathepsin D) precedes release of cytochrome c, loss of delta psi(m), and morphologic alterations. Moreover, apoptosis could be inhibited by pretreatment with pepstatin A.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
p-Benzoquinone, reagent grade, ≥98%
Supelco
1,4-Benzoquinone, Pharmaceutical Secondary Standard; Certified Reference Material
Supelco
p-Benzoquinone, for spectrophotometric det. of amines, ≥99.5% (HPLC)
Sigma-Aldrich
Cathepsin D from human liver, lyophilized powder, ≥250 units/mg protein (E1%/280)
Sigma-Aldrich
Cathepsin D from bovine spleen, lyophilized powder, ≥2.0 units/mg protein