Sphingosine mediates TNFα-induced lysosomal membrane permeabilization and ensuing programmed cell death in hepatoma cells

Chiara Ullio,Josefina Casas,Ulf T Brunk,Giuseppina Sala,Gemma Fabriàs,Riccardo Ghidoni,Gabriella Bonelli,Francesco M Baccino,Riccardo Autelli

doi:10.1194/jlr.m022384

Abstract

Normally, cell proliferation and death are carefully balanced in higher eukaryotes, but one of the most important regulatory mechanisms, apoptosis, is upset in many malignancies, including hepatocellular-derived ones. Therefore, reinforcing cell death often is mandatory in anticancer therapy. We previously reported that a combination of tumor necrosis factor-α (TNF) and cycloheximide (CHX) efficiently kill HTC cells, a rat hepatoma line, in an apoptosis-like mode. Death is actively mediated by the lysosomal compartment, although lysosomal ceramide was previously shown not to be directly implicated in this process. In the present study, we show that TNF/CHX increase lysosomal ceramide that is subsequently converted into sphingosine. Although ceramide accumulation does not significantly alter the acidic compartment, the sphingosine therein generated causes lysosomal membrane permeabilization (LMP) followed by relocation of lysosomal cathepsins to the cytoplasm. TNF/CHX-induced LMP is effectively abrogated by siRNAs targeting acid sphingomyelinase or acid ceramidase, which prevent both LMP and death induced by TNF/CHX. Taken together, our results demonstrate that lysosomal accumulation of ceramide is not detrimental per se, whereas its degradation product sphingosine, which has the capacity to induce LMP, appears responsible for the observed apoptotic-like death.

Highlights

Cell proliferation and death are carefully balanced in higher eukaryotes, but one of the most important regulatory mechanisms, apoptosis, is upset in many malignancies, including hepatocellular-derived ones
tumor necrosis factor-␣ (TNF)/CHX elevated sphingosine (Fig. 1B), another sphingolipid known to be increased by TNF to induce cell death [21, 22]
Because this drug is a known functional inhibitor of the acid sphingomyelinase, we investigated whether this protection was secondary to a change in sphingolipid levels

Summary

Introduction

Cell proliferation and death are carefully balanced in higher eukaryotes, but one of the most important regulatory mechanisms, apoptosis, is upset in many malignancies, including hepatocellular-derived ones. Sphingosine mediates TNF␣-induced lysosomal membrane permeabilization and ensuing programmed cell death in hepatoma. A tightly controlled balance between cellular proliferation and death is the basis for tissue homeostasis in multicellular organisms Dysregulation of this poise results in abnormal production or loss of cells, accounting for common diseases such as neurodegenerative disorders and malignancies. The cytotoxic cytokine tumor necrosis factor-␣ (TNF), a member of a family of ligands for a number of death receptors that includes FASL and TRAIL, is of interest for its partial selectivity for malignant cells [4, 5]. The role played by ceramide in TNF cytotoxicity was reevaluated because during TNFinduced PCD, sphingolipids other than ceramide, such as sphingosine, are affected [21,22,23,24,25] As the latter is a split product of ceramide, it is often not easy to discriminate between the effects of these two sphingolipids

Methods

Results

Conclusion