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

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.

Antitumor alkyl-lysophospholipid analog edelfosine induces apoptosis in pancreatic cancer by targeting endoplasmic reticulum

“…We have previously shown that edelfosine increases cytosolic [Ca 2 þ ] in HeLa cells (Nieto-Miguel et al, 2007). On these grounds, and taking into account the interaction of edelfosine with cell membranes and lipids (Gajate et al, , 2009a(Gajate et al, , 2009bZaremberg et al, 2005;Mollinedo and Gajate, 2006;Torrecillas et al, 2006;Busto et al, 2007Busto et al, , 2008Ausili et al, 2008;, it might be envisaged that the increase in cytosolic [Ca 2 þ ] could be due to an increased permeability of the plasma membrane, resulting in Ca 2 þ entry from the extracellular medium, which it then leads to the herein reported increase in [Ca 2 þ ] ER , well above the steady state levels in control cells. The effect on [Ca 2 þ ] ER may seem puzzling, because ER stress has been rather associated with ER-stored Ca 2 þ release.…”

“…Synthetic alkyl-lysophospholipid analogs (ALPs) are a novel class of unnatural lipids with promising anticancer activity, including clinically relevant drugs such as miltefosine, perifosine and erucylphosphocholine, which act at the cell membrane level (Gajate and Mollinedo, 2002;Mollinedo et al, 2004). Edelfosine (1-O-octadecyl-2-O-methoxyrac-glycero-3-phosphocholine), considered to be the ALP prototype compound, has been shown to induce apoptosis in malignant cells in a rather selective way through the involvement of lipid rafts and ER (Mollinedo et al, 1997;Gajate et al, 2000bGajate et al, , 2004Gajate et al, , 2009aMollinedo, 2001, 2007;Gajate, 2006, 2010;Nieto-Miguel et al, 2007).…”

Endocytosis of Resveratrol via Lipid Rafts and Activation of Downstream Signaling Pathways in Cancer Cells

“…A variety of clathrin-independent endocytic pathways are responsible for taking up large particles or small solutes, together with membrane into cells, and some of them depend on lipid rafts (29)(30)(31), including endocytosis of glycosylphosphatidylinositol (GPI)-anchored proteins (32,33), interleukin-2 linked to its receptors (34), and several ether lipids (e.g., alkyl lysophospholipid; refs. [35][36][37]. After initial insertion in the outer leaflet of the plasma membrane lipid bilayer, resveratrol accumulates in lipid rafts, as described for epigallocatechin (38), and is taken up by lipid raftdependent, clathrin-independent endocytosis.…”

Could Fucoxanthin Interaction with Lipid Rafts Mediate its Cytotoxicity in Cancer Cells?

“…Structure and biological activity of fucoxanthin and carotenoids in cancer cells [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18] Biological functions of carotenoids in cell membranes [19][20][21][22] Interaction of carotenoids with lipid rafts [23] Cell model to study the integration of xenobiotics into lipid rafts [24] Antioxidant and pro-oxidant activity of fucoxanthin in animal cells and tissues [25][26][27] Structure and function of lipid rafts in cell membranes [28,29] Targeting lipid rafts in cancer cells [30][31][32][33][34][35][36] Ion channels, lipid rafts and cancer cells [37,38] Lipid rafts, cytoskeleton, cancer cells invasivity and tumor angiogenesis [30,40] …”

“…It is also demonstrated that cell death can be initiated by activation of death domain receptors, located in lipid rafts [33]. Previous studies have documented that some cancer cells contain an increased level of cholesterol-rich lipid rafts [36] and that a cholesterol-depleting agent such as methyl-β-cyclodextrin triggered BcL-XL down regulation, caspase-3 activation and Akt inactivation [36]. These changes, that correspond to classical observations associated with apoptosis, have also been reported in cancer cells treated with fucoxanthin [3].…”