Original Contributions
Lysosomal release of Cathepsin D precedes relocation of Cytochrome C and loss of mitochondrial transmembrane potential during apoptosis induced by oxidative stress

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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 (ΔΨm) 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 ΔΨm, and morphologic alterations. Moreover, apoptosis could be inhibited by pretreatment with pepstatin A.

Introduction

Apoptosis is a morphologically distinct form of programmed cell death that is responsible for physiologic elimination of cells. Malfunction of the apoptosis mechanism is associated with pathologic conditions such as cancer, autoimmune diseases, and many neurodegenerative diseases. Apoptosis occurs through activation of a cell suicide process that is regulated by many different signals arising from both the intracellular and the extracellular milieu [1]. Apoptosis has a number of morphologic features, such as cell shrinkage, chromatin condensation, nuclear fragmentation, and formation of apoptotic bodies. Changes in the mitochondria, such as loss of transmembrane potential (ΔΨm) and release of cytochrome c (cyt c), together with activation of the caspase cascade, are typical biochemical characteristics of apoptosis. Cyt c is a nuclear DNA-encoded protein that is localized to the intermembrane space and to the surface of the inner membrane in mitochondria [2]. Release of cyt c from mitochondria to the cytosol is a critical event in apoptosis, and it allows complexation of the cyt c with cytosolic Apaf-1 (apoptosis protein-activating factor 1), which, in presence of deoxyadenosine triphosphate or adenosine triphosphate (ATP), could lead to activation of procaspase 9.

The ΔΨm is the result of asymmetric distribution of protons and other ions on both sides of the inner mitochondrial membrane, and it gives rise to a chemical (pH) and an electrical gradient, both of which are essential for mitochondrial function [3], [4]. It has been shown that cells display a reduction in incorporation of ΔΨm-sensitive dyes early in apoptosis, indicating a decline in the ΔΨm. Furthermore, Zamzami et al. [5] reported that disruption of ΔΨm occurs before cells exhibit either nuclear DNA fragmentation or aberrant exposure of phosphatidylserine on the outer cell membrane leaflet. Early disruption has been detected in different types of cells treated with various apoptosis-inducing stimuli [6].

Apoptosis induced by oxidative stress has been observed in several studies [7], [8], [9]. Cell-cycle regulating genes (e.g., p53 and p21), are activated during apoptosis provoked by oxidative stress. Moreover, tumor necrosis factor-α–induced apoptosis can be prevented by antioxidants [10] and by overexpression of the proto-oncogene Bcl-2 [11], thus it has been hypothesized that stimulation with tumor necrosis factor-α causes a rise in the intracellular level of reactive oxygen species. Bcl-2 is located in the outer mitochondrial membrane, the nuclear membrane, and the endoplasmic reticulum. Moreover, it has been suggested that Bcl-2 prevents apoptosis by exerting an antioxidant effect, and functional studies have indicated that Bcl-2 inhibits apoptosis by maintaining the ΔΨm and preventing the release of cyt c [12]. Compared with wild-type cells, cells that overexpress Bcl-2 have a higher ΔΨm [13] and are more resistant to anticancer drugs [14].

The lysosomal compartment is the major site of intracellular protein degradation. Its interior is acidic [15] and contains numerous hydrolytic enzymes that can degrade nearly all cellular components. Among the most powerful hydrolytic enzymes are the cathepsins. Cathepsin D (cat D) is a lysosomal aspartic protease that is present in practically all animal cells [16]. It degrades proteins at low pH and is believed to play important roles in protein catabolism, antigen processing [17], protein targeting [18], and breast cancer progression [19]. In experiments on cultured cells, oxidative stress has been found to cause destabilization of lysosomal membranes seen as decreased uptake of the lysosomotropic weak base acridine orange early in the cell-killing sequence [20], [21], [22]. Using immunochemistry and electron microscopy, we have previously noted [23] that exposure of cardiac myocytes to a free radical–generating quinone resulted in relocation of cat D from lysosomal structures to the cytosol; ensuing incubation under normal culture conditions led to apoptotic cell death. Pretreatment of these cultures with α-tocopherol succinate diminished the cat D relocation and also decreased the number of apoptotic cells. In recent investigations, increased expression of cat D occurred during apoptosis after activation of Fas/APO-1 [24] and exposure to doxorubicin [25]. The lysosomal cysteine protease cathepsin B has also been reported to activate procaspases [26].

Our aims were to investigate the temporal relationship between lysosomal release of cat D, cyt c release from mitochondria, and decrease in ΔΨm, and to clarify the role of cat D during apoptosis induced in fibroblasts by oxidative stress.

Section snippets

Chemicals

Glutamine, penicillin-G, streptomycin, Eagle’s minimal essential medium, and fetal bovine serum were obtained from GIBCO (Paisley, UK). Paraformaldehyde and Epon-812 were purchased from Fluka AG (Buchs, Switzerland); JC-1 (5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethyl-benzimidazolcarbocyanine iodide) was from Molecular Probes (Eugene, OR, USA); and naphthazarin (5,8-dihydroxy-1,4-naphthoquinone) was from Aldrich Chemie (Steinheim, Germany). Polyclonal rabbit antihuman cathepsin D antibodies were

Apoptosis induced by oxidative stress

In cultures exposed to naphthazarin for 8 h, most of the cells showed typical ultrastructural signs of apoptosis: reduced cell size, chromatin condensation, dilated endoplasmic reticulum, and apoptotic bodies (Fig. 1). Pepstatin A is a specific inhibitor of aspartic proteases [28], hence the intracellular effects of this compound have been attributed to inhibition of cat D activity. Control cells pretreated with pep A exhibited a slightly enhanced number of autophagosomes (Fig. 2A), and the

Discussion

We have previously found that lysosomal destabilization is an early event in cellular devitalization caused by oxidative stress [20], [21], [22], [23]. Naphthazarin, a structural analogue of the anticancer drug doxorubicin, is metabolized to its semiquinone form by intracellular reductases and is then reoxidized by molecular oxygen in a redox cycle that produces equimolar amounts of superoxide radicals. We noted that the cytotoxicity of both naphthazarin and doxorubicin could be inhibited by

Acknowledgements

This study was supported by Swedish Cancer Foundation Grant 2703 and the Swedish Society for Medical Research.

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