Necroptosis is associated with low procaspase-8 and active RIPK1 and -3 in human glioma cells.

Sara Melo-Lima,Faustino Mollinedo,Maria Celeste Lopes

doi:10.18632/oncoscience.89

Abstract

Necroptosis is a regulated necrotic cell death that involves receptor-interacting protein kinases RIPK1 and RIPK3. Here, we report that edelfosine triggers a rapid and massive cell death in human glioblastoma cells with characteristics of necrosis. Only a minor proportion of edelfosine-treated cells underwent caspase-dependent apoptosis. Autophagy and a rapid influx of extracellular calcium into the cells had little impact on cell death. Levels of procaspase-8 were very low in necroptosis-prone glioma cells compared with the levels in other cancer cell types that underwent apoptosis upon edelfosine treatment. The RIPK1-dependent necroptosis inhibitors necrostatin-1 (Nec-1) and Nec-1s as well as siRNA-mediated silencing of RIPK3 inhibited edelfosine-induced necroptosis, resulting in increased caspase-dependent apoptosis in edelfosine-treated glioblastoma U118 cells. Inhibition of the RIPK3 substrate MLKL with necrosulfonamide also increased apoptosis in edelfosine-treated cells. These data support a major role for RIPK1 and RIPK3 in the induction of necrotic cell death and in the switch from necrosis to apoptosis following edelfosine treatment. These results indicate that the ether lipid edelfosine exerts a rapid necroptotic cell death in apoptosis-reluctant glioblastoma cells, suggesting that induction of necroptosis could constitute a new approach for glioblastoma therapy.

Highlights

Malignant glioma or glioblastoma is the most common and the most aggressive malignant primary brain tumor with a very poor prognosis
We found that U118 cells expressed RIPK3, and RIPK3 silencing by using siRNA (Fig. 7A), dramatically reduced (~80%) necrotic phenotype and, to what was observed by using necrostatin-1, induced apoptotic cell death following edelfosine treatment, as assessed by an increase in sub-G1 cell population through cell cycle analysis, and by the presence of morphologic features of apoptosis, including cell surface blebbing and chromatin condensation in DAPI-stained nuclei in edelfosine-treated U118 cells (Fig. 7B and C)
The ether phospholipid edelfosine induces a rapid necrotic cell death in human U118 glioblastoma cells, which is inhibited by the specific receptor-interacting protein kinase-1 (RIPK1) inhibitors Nec-1 and Nec-1s as well as by RIPK3 silencing, and accounts for most of the cell death (~80%) occurring in edelfosine-treated U118 cells, involving both RIPK1 and RIPK3 in the cell death process

Summary

INTRODUCTION

Malignant glioma or glioblastoma is the most common and the most aggressive malignant primary brain tumor with a very poor prognosis. The unfavorable prognosis for glioblastoma patients is strongly correlated to the intrinsic apoptosis resistance of glioblastoma cells [12, 13] On these grounds, induction of alternative types of cell death could be an option for the treatment of glioblastoma. Edelfosine has been shown to be an effective in vitro and in vivo antitumor drug, which acts through the reorganization of membrane domains, termed lipid rafts, as well as through an endoplasmic reticulum stress response, leading to caspase- and mitochondria-mediated apoptosis in different hematological and solid tumor cells [22,23,24,25,26,27,28]. We report that edelfosine induces mainly necroptosis in the U118 (U-118 MG) glioblastoma cell line, used as a brain tumor cell line model, whereas apoptosis and autophagy are relatively minor responses. Edelfosine-induced necroptototic response is very rapid and potent, suggesting a putative therapeutic role for necroptosis in brain tumor therapy

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METHODS