Alteration of the Sphingomyelin/Ceramide Pathway Is Associated with Resistance of Human Breast Carcinoma MCF7 Cells to Tumor Necrosis Factor-α-mediated Cytotoxicity

Zhenzi Cai,Ali Bettaieb,Nour El Mahdani,Luc G Legrès,Rodica Stancou,Joëlle Masliah,Salem Chouaib

doi:10.1074/jbc.272.11.6918

Abstract

The interference of tumor necrosis factor-alpha (TNF) signaling processes with the acquisition of tumor resistance to TNF was investigated using the TNF-sensitive human breast carcinoma MCF7 cell line and its established TNF-resistant variant (R-A1). The resistance of R-A1 cells to TNF correlated with a low level of p55 TNF receptor expression and an absence of TNF signaling through TNF receptors. Stable transfection of wild-type p55 receptor in R-A1 resulted in enhancement of p55 expression and in partial restoration of TNF signaling, including nuclear factor-kappaB (NF-kappaB) activation. However, the transfected cells remained resistant to TNF-induced apoptosis. Northern blot analysis revealed a comparable induction of manganous superoxide dismutase and A20 mRNA expression in p55-transfected cells and in sensitive MCF7 cells, making it unlikely that these genes are involved in the resistance to TNF-mediated cytotoxicity. While TNF significantly stimulated both neutral and acidic sphingomyelinase (SMase) activities with concomitant sphingomyelin (SM) hydrolysis and ceramide generation in MCF7, it failed to trigger these events in TNF-resistant p55-transfected cells. In addition, the basal SM content was significantly higher in sensitive MCF7 as compared to the resistant counterparts. Furthermore, the TNF-resistant cells tested could be induced to undergo cell death after exposure to exogenous SMase or cell-permeable C6-ceramide. This study also shows that TNF failed to induce arachidonic acid release in p55-transfected resistant cells, suggesting that an alteration of phospholipase A2 activation may be associated with MCF7 cell resistance to TNF. Our findings strongly suggest a role of ceramide in the mechanism of cell resistance to TNF-mediated cell death and may be relevant in elucidating the biochemical nature of intracellular messengers leading to such resistance.

Highlights

§ Recipient of a grant from the Institut de Formation Superieure BioMedicale and the Association pour la Recherche sur le Cancer
In contrast to the rapid progress that has been made in defining gene products capable of regulating tumor necrosis factor-␣ (TNF)-induced cell death, the knowledge of the molecular components involved in cell resistance to TNF remains limited
We attempted to delineate the functional role of some second messengers in the acquisition of tumor resistance to TNF by comparing a TNFsensitive human breast cancer cell line MCF7 with its R-A1 variant selected for resistance to TNF

Summary

Introduction

While TNF significantly stimulated both neutral and acidic sphingomyelinase (SMase) activities with concomitant sphingomyelin (SM) hydrolysis and ceramide generation in MCF7, it failed to trigger these events in TNF-resistant p55-transfected cells. This study shows that TNF failed to induce arachidonic acid release in p55-transfected resistant cells, suggesting that an alteration of phospholipase A2 activation may be associated with MCF7 cell resistance to TNF.

Results

Conclusion