Caspase inhibition switches the mode of cell death induced by cyanide by enhancing reactive oxygen species generation and PARP-1 activation

Abstract

Execution of apoptosis can involve activation of the caspase family of proteases. Recent studies show that caspase inhibition can switch the morphology of cell death from apoptotic to necrotic without altering the level of death among cell populations. In the present study, the effect of caspase inhibition on cortical (CX) cell death induced by cyanide was investigated. In primary cultured CX cells exposed to cyanide (400 μM), death was primarily apoptotic as indicated by positive TUNEL staining. Reactive oxygen species (ROS) generation and subsequent caspase activation mediated the apoptosis. Inhibition of the caspase cascade with zVAD-fmk switched the apoptotic response to necrotic cell death, as assessed by increased cellular efflux of LDH and propidium iodide uptake by the cells. The change in death mode was accompanied by a marked increase in poly (ADP-ribose) polymerase-1 (PARP-1) activity, reactive oxygen species (ROS) generation, a reduction in the mitochondrial membrane potential (Δψ_m), and reduced cellular ATP. Prior treatment of cells with 3-aminobenzamide (3-AB), a PARP-1 inhibitor, prevented the cells from undergoing necrosis and preserved intracellular ATP levels. These findings indicate that apoptosis and necrosis share common initiation pathways and caspase inhibition can switch the apoptotic response to necrosis. Inhibition of PARP-1 preserves cellular ATP levels and in turn blocks execution of the necrotic death pathway.

Introduction

Cyanide is a rapid-acting neurotoxic compound that enhances intracellular generation of reactive oxygen species (ROS) to produce cell injury and death (Jones et al., 2003). In neuronal cells, cyanide produces two distinct modes of death, apoptosis and necrosis, depending on cell type and the level of oxidative insult (Prabhakaran et al., 2002). These modes of cell death appear to share common initiation stimuli, but divergent intracellular cascades are activated to produce either apoptosis or necrosis Prabhakaran et al., 2002, Shou et al., 2003. In cyanide-induced neuronal death, apoptosis is caspase-dependent and mediated by release of proapoptotic proteins from mitochondria. Cells undergoing necrosis display a more intense level of oxidative stress and experience a rapid breakdown of mitochondrial function characterized by onset of mitochondrial membrane permeability transition (MPT) and ATP depletion (Prabhakaran et al., 2002).

In many cell models, the mode of death produced by an initiation stimulus may be switched by inhibiting control points or key execution steps in the death pathway, such as caspase activation, cytochrome c release, or ROS generation Hartmann et al., 2001, Kalai et al., 2002, Nicotera et al., 1999. Inhibition of caspases or transient inhibition of their expression can switch the mode of death from apoptosis to necrosis (Denecker et al., 2001), and changes in the generation level of ROS and nitric oxide can either inhibit apoptosis or switch to necrosis Fiers et al., 1999, Leist et al., 1999, Mello et al., 2000. Because execution of apoptosis requires ATP, PARP-1 activation leads to ATP depletion and necrosis Ha and Snyder, 1999, Herceg and Wang, 1999. In apoptosis, caspases cleave PARP-1 and thereby conserve ATP. In turn, inhibition of PARP-1 can switch the mode of death from necrosis to apoptosis Los et al., 2002, Walisser and Thies, 1999.

Inhibition of caspase proteases by natural caspase inhibitors or small peptide inhibitors such as zVAD-fmk can modulate apoptotic cell death Lemaire et al., 1999, Vercammen et al., 1998a, Vercammen et al., 1998b. In some cell lines, caspase inhibition may increase cell survival (Ozoren et al., 2000), whereas other cells may respond with an increased level of death by switching the mode of death to oncosis or necrosis Lemaire et al., 1998, Lemaire et al., 1999, Ozoren et al., 2000, Ruemmele et al., 1999, Vercammen et al., 1998b. The caspase family of proteases plays a pivotal role in the intrinsic apoptotic cascade because the level of cellular caspase activity is a dominant factor in the death pathway decision (apoptosis vs. necrosis). The effect of caspase inhibition on the cellular response is cell line specific and involves either maintenance of viability (Ozoren et al., 2000) or a switch to a death mode exhibiting apoptosis-independent characteristics, such as necrosis Ruemmele et al., 1999, Vercammen et al., 1998b. Thus caspase inhibition may not prevent cell death, rather induce a switch in the mode of death to necrosis. This has therapeutic implications in light of the potential use of caspase inhibitors to treat cytotoxicity.

In this study, the role of caspases in regulating the mode of cortical (CX) cell death induced by cyanide was investigated using the pan-caspase inhibitor, zVAD-fmk. Inhibition of caspases reduced the total level of cell death and switched the mode from apoptosis to necrosis.