Isatin, an endogenous monoamine oxidase inhibitor, triggers a dose- and time-dependent switch from apoptosis to necrosis in human neuroblastoma cells

Abstract

Isatin is an endogenous indole that is increased in stress, inhibits monoamine oxidase (MAO) B and improves bradykinesia and striatal dopamine levels in rat models of Parkinson's disease. Consequently, it has been suggested that isatin might be a possible treatment for Parkinson's disease although little is known about its effects on neural cell growth and survival. The aim of this study was to investigate the survival of dopaminergic human neuroblastoma (SH-SY5Y) cells following treatment with increasing concentrations of isatin. SH-SY5Y cells were exposed to isatin for defined time points, after which cell survival was determined by MTT assay. A combination of Annexin V binding and propidium iodide (PI) exclusion was used to distinguish apoptosis from necrosis in flow cytometry experiments and FACS profiles of permeabilised PI-labelled cells were employed for the assessment of cell cycle distribution. Isatin treatment (1–400 μM) for 24 h induced a significant dose-dependent increase in MTT metabolism by SH-SY5Y cells in culture, but this was not due to an increase in cell division. At the higher concentrations (200–400 μm) isatin triggered cell death, although MTT metabolism was still increased in the culture, suggesting that surviving cells were hypermetabolic. Following a longer (48 h) exposure, isatin was found to cause cell death in a dose-dependent manner; at lower concentrations (50 μM), the predominant mode of cell death was apoptosis while at the highest concentration (400 μm) increasing numbers of necrotic cells were also evident. Thus, in dopaminergic SH-SY5Y cells isatin induces cell death in dose- and time-dependent manner. This death occurred as a continuum of survival, apoptosis and necrosis. Our results re-emphasise that caution should be exercised when considering high doses of isatin as a putative anti-Parkinson's disease therapeutic.

Introduction

Isatin is an endogenous indole present in mammalian tissues and fluids (Glover et al., 1988). The substance was initially discovered as a component of endogenous monoamine oxidase (MAO) inhibitory activity, tribulin, and subsequently identified as a selective inhibitor of MAO B (Glover et al., 1980). Further investigations have shown that isatin acts as an antagonist of both atrial natriuretic peptide-stimulated (Glover et al., 1995, Medvedev et al., 1996) and nitric oxide-stimulated guanylate cyclase activity (Medvedev et al., 2002). Isatin has a distinct and discontinuous distribution in rat brain and other tissues; the highest concentrations in the brain are found in the hippocampus and cerebellum (Watkins et al., 1990). Moreover, ³H isatin binding in rat brain slices has demonstrated a restricted regional distribution (Crumeyrolle-Arias et al., 2003), suggesting a specific physiological role for this compound in the brain. Accordingly, isatin administered in vivo exhibits a range of physiological and behavioral effects. In rodent models isatin has been shown to cause a wide spectrum of dose-dependent physiological and biological actions, such as anxiogenic and sedative effects, memory dysfunction and inhibition of food and water intake (Bhattacharya et al., 1991b, Morley et al., 1996, Glover et al., 1998). We and others have shown that isatin output increases following physiological stress in animal models (Tozawa et al., 1998, Igosheva et al., 2004). Similarly, urinary isatin excretion has been shown to increase markedly in patients with PD and the levels correlate with clinical severity of the disease (Hamaue et al., 2000). On the other hand, in rat models of PD, isatin treatment prevented dopamine depletion in the striatum and improved bradykinesia (Ogata et al., 2003, Hamaue et al., 2004), suggesting that isatin could be a possible treatment for PD. In none of these studies was the precise molecular basis of the effects of isatin on the nervous system investigated. The present study was designed to evaluate the specific effects of isatin on neural cell growth and survival, using as a model of human dopaminergic neurons, SH-SY5Y neuroblastoma cells.