Potentiation of okadaic acid-induced ceramide elevation but not apoptosis by inhibition of glucosylceramide synthase in human neuroepithelioma cells

Abstract

Caspase-dependent apoptosis induced by okadaic acid (OA) in CHP-100 neuroepithelioma cells has previously been shown to associate with a rapid and sustained elevation in intracellular ceramide concentration. We now report that treatment of CHP-100 cells with OA also evoked a rapid elevation in glucosylceramide levels that was maintained at steady state as cells underwent apoptosis; moreover, as observed for ceramide, OA-induced glucosylceramide accumulation was not blocked by fumonisin B₁. Remarkably, when cell death was prevented by caspase inhibition, glucosylceramide accumulation was potentiated and ceramide elevation reduced, thus suggesting that, during apoptosis completion, accumulation of ceramide was partly driven by impairment of its glucosylation through a caspase-dependent mechanism. We studied whether ceramide glucosylation provided a mechanism for negative modulation of OA-induced apoptosis. We observed that the blocking of glucosylceramide synthesis markedly potentiated OA-induced ceramide elevation, but neither accelerated apoptosis onset nor potentiated the apoptotic response. These results indicate that modulation of ceramide glucosylation does not affect the apoptotic response to okadaic acid and suggest that caution must be exercised concerning the possibility that ceramide plays a key role in apoptosis induction.

Introduction

¹There is general agreement that ceramide (N-acyl-erythro-sphingosine, Cer) is a key mediator in apoptosis induced by a variety of stimuli, including receptor ligation, irradiation, and drug treatment, on a wide range of cell systems [1], [2]. In this respect, unequivocal evidence has been obtained by studies showing that stimuli evoking both programmed cell death and ceramide elevation lose their apoptotic potential after blocking of the metabolic pathway that leads to Cer generation [3], [4], [5]. In other studies, the role of Cer as a mediator of apoptosis has been largely inferred on the basis of the evidence that programmed cell death associates with intracellular Cer elevation and can be reproduced by administration of exogenous and cell-permeant short-chain Cer [6].

We have reported that OA, a potent inhibitor of serine–threonine PP produced by the marine sponge Halichondria okadai[7], induces apoptosis in CHP-100 human neuroepithelioma cells in a manner that largely involves caspase activation [8]; in addition, caspase-dependent apoptosis associates with an elevation in intracellular Cer levels [8]. These results, together with the finding that administration of short-chain Cer induces caspase-dependent apoptosis in CHP-100 cells [9], suggested that Cer might be the mediator linking sustained PP inhibition to apoptosis. CHP-100 cells actively glucosylate both endogenous and exogenously administered Cer [10]. In line with the notion that glucosylation provides a major pathway by which the apoptogenic pools of Cer are down-regulated [11], [12], [13], [14], we have reported that, in CHP-100 cells, the apoptotic effect of short-chain Cer is potentiated by inhibition of GlcCer synthase [10]. We investigated here the kinetics of GlcCer accumulation, as observed in CHP-100 cells in relationship to OA-induced apoptosis. Moreover, we studied whether the apoptotic response triggered by OA is propagated by inhibition of Cer glucosylation.