Abstract
BackgroundRecent evidence suggests that co-clustering of Fas/CD95 death receptor and lipid rafts plays a major role in death receptor-mediated apoptosis.Methodology/Principal FindingsBy a combination of genetic, biochemical, and ultrastructural approaches, we provide here compelling evidence for the involvement of lipid raft aggregates containing recruited Fas/CD95 death receptor, Fas-associated death domain-containing protein (FADD), and procaspase-8 in the induction of apoptosis in human T-cell leukemia Jurkat cells by the antitumor drug edelfosine, the prototype compound of a promising family of synthetic antitumor lipids named as synthetic alkyl-lysophospholipid analogues. Co-immunoprecipitation assays revealed that edelfosine induced the generation of the so-called death-inducing signaling complex (DISC), made up of Fas/CD95, FADD, and procaspase-8, in lipid rafts. Electron microscopy analyses allowed to visualize the formation of raft clusters and their co-localization with DISC components Fas/CD95, FADD, and procaspase-8 following edelfosine treatment of Jurkat cells. Silencing of Fas/CD95 by RNA interference, transfection with a FADD dominant-negative mutant that blocks Fas/CD95 signaling, and specific inhibition of caspase-8 prevented the apoptotic response triggered by edelfosine, hence demonstrating the functional role of DISC in drug-induced apoptosis. By using radioactive labeled edelfosine and a fluorescent analogue, we found that edelfosine accumulated in lipid rafts, forming edelfosine-rich membrane raft clusters in Jurkat leukemic T-cells. Disruption of these membrane raft domains abrogated drug uptake and drug-induced DISC assembly and apoptosis. Thus, edelfosine uptake into lipid rafts was critical for the onset of both co-aggregation of DISC in membrane rafts and subsequent apoptotic cell death.Conclusions/SignificanceThis work shows the involvement of DISC clusters in lipid raft aggregates as a supramolecular and physical entity responsible for the induction of apoptosis in leukemic cells by the antitumor drug edelfosine. Our data set a novel framework and paradigm in leukemia therapy, as well as in death receptor-mediated apoptosis.
Highlights
In the last few years, a growing amount of evidence suggests that apoptosis induced by Fas/CD95 death receptor is mediated by the formation of Fas/CD95 aggregates in lipid rafts [1,2,3,4,5,6,7]
We extend these results by finding that Fas/ CD95 as well as downstream signaling molecules Fasassociated death domain-containing protein (FADD) and procaspase-8 were translocated into membrane rafts (Figure 1A), forming the proapoptotic complex death-inducing signaling complex (DISC) in lipid rafts (Figure 1B), upon edelfosine treatment of Jurkat leukemic T-cells
Procaspase-8 was localized in the same region in edelfosine-treated cells, forming DISC aggregates in discrete zones of the plasma membrane enriched in lipid rafts after drugtreated Jurkat cells were labeled with specific antibodies to each DISC component and with cholera toxin (CTx) B subunit to identify membrane rafts (Figure 2D)
Summary
In the last few years, a growing amount of evidence suggests that apoptosis induced by Fas/CD95 death receptor is mediated by the formation of Fas/CD95 aggregates in lipid rafts [1,2,3,4,5,6,7]. Clustering of death receptor Fas/CD95 can be achieved by interaction with its natural ligand FasL/CD95L, but through non-physiological agents independently of its ligand [1,4,8], providing a new framework for novel therapeutic interventions [6]. This ligand-independent activation of Fas/CD95 has a great potential therapeutic utility as it avoids the toxic side effects derived from the use of FasL/CD95L and agonistic anti-Fas/. Recent evidence suggests that co-clustering of Fas/CD95 death receptor and lipid rafts plays a major role in death receptor-mediated apoptosis
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