Involvement of Raft Aggregates Enriched in Fas/CD95 Death-Inducing Signaling Complex in the Antileukemic Action of Edelfosine in Jurkat Cells

BACKGROUND: Recent evidence suggests that co-clustering of Fas/CD95 death receptor and lipid rafts plays a major role in death receptor-mediated apoptosis. METHODOLOGY/PRINCIPAL FINDINGS: By 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/SIGNIFICANCE: This 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

The orally available, synthetic ether lipid edelfosine inhibits T cell proliferation and induces a type I interferon response

The drug edelfosine is a synthetic analog of 2-lysophosphatidylcholine. Edelfosine is incorporated by highly proliferating cells, e.g. activated immune cells. It acts on cellular membranes by selectively aggregating the cell death receptor Fas in membrane rafts and interference with phosphatidylcholine (PC) synthesis with subsequent induction of apoptosis. Edelfosine has been proposed for the treatment of autoimmune diseases like multiple sclerosis (MS). Earlier studies on the animal model of MS, experimental autoimmune encephalomyelitis (EAE), have generated first evidence for the efficacy of edelfosine treatment. However, it is unknown if the previously described mechanisms for edelfosine action, which are derived from in vitro studies, are solely responsible for the amelioration of EAE or if edelfosine may exert additional effects, which may be beneficial in the context of autoimmunity. Since it was the purpose of our studies to assess the potential usefulness of edelfosine for the treatment of MS, we examined its mechanism/s of action on immune functions in human T cells. Low doses of edelfosine led to a decrease in homeostatic proliferation, and further studies of the mechanism/s of action by genome-wide transcriptional profiling showed that edelfosine reduces the expression of MHC class II molecules, of molecules involved in MHC class II-associated processing and presentation, and finally upregulated a series of type I interferon-associated genes. The inhibition of homeostatic proliferation, as well as the effects on MHC class II expression and -presentation, and the induction of type I interferon-associated genes are novel and interesting in the context of developing edelfosine for clinical use in MS and possibly also other T cell-mediated autoimmune diseases

Proliferating or Differentiating Stimuli Act on Different Lipid-dependent Signaling Pathways in Nuclei of Human Leukemia Cells

Previous results have shown that the human promyelocytic leukemia HL-60 cell line responds to either proliferating or differentiating stimuli. When these cells are induced to proliferate, protein kinase C (PKC)-βII migrates toward the nucleus, whereas when they are exposed to differentiating agents, there is a nuclear translocation of the α isoform of PKC. As a step toward the elucidation of the early intranuclear events that regulate the proliferation or the differentiation process, we show that in the HL-60 cells, a proliferating stimulus (i.e., insulin-like growth factor-I [IGF-I]) increased nuclear diacylglycerol (DAG) production derived from phosphatidylinositol (4,5) bisphosphate, as indicated by the inhibition exerted by 1-O-octadeyl-2-O-methyl-sn-glycero-3-phosphocholine and U-73122 (1-[6((17β-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl]-1H-pyrrole-2,5-dione), which are pharmacological inhibitors of phosphoinositide-specific phospholipase C. In contrast, when HL-60 cells were induced to differentiate along the granulocytic lineage by dimethyl sulfoxide, we observed a rise in the nuclear DAG mass, which was sensitive to either neomycin or propranolol, two compounds with inhibitory effect on phospholipase D (PLD)-mediated DAG generation. In nuclei of dimethyl sulfoxide-treated HL-60 cells, we observed a rise in the amount of a 90-kDa PLD, distinct from PLD1 or PLD2. When a phosphatidylinositol (4,5) bisphosphate-derived DAG pool was generated in the nucleus, a selective translocation of PKC-βII occurred. On the other hand, nuclear DAG derived through PLD, recruited PKC-α to the nucleus. Both of these PKC isoforms were phosphorylated on serine residues. These results provide support for the proposal that in the HL-60 cell nucleus there are two independently regulated sources of DAG, both of which are capable of acting as the driving force that attracts to this organelle distinct, DAG-dependent PKC isozymes. Our results assume a particular significance in light of the proposed use of pharmacological inhibitors of PKC-dependent biochemical pathways for the therapy of cancer disease