Abstract
Fas expression is inversely correlated with the metastatic potential of osteosarcoma (OS) cells to the lungs. Fas+ cells are rapidly eliminated when they enter the lungs via their interaction with constitutive Fas ligand (FasL) on the lung epithelium, whereas Fas− OS cells escape this FasL-induced apoptosis and survive in the lung microenvironment. Upregulation of Fas expression in established OS lung metastases results in tumor regression. Here, we demonstrate that treatment of Fas− OS cells with the histone deacetylase inhibitor MS-275 results in the upregulation of Fas mRNA and sensitizes these cells to FasL-induced apoptosis. However, flow cytometry analysis revealed that Fas cell surface protein expression was not significantly increased. Rather, we observed increased levels of Fas within the membrane lipid rafts, as demonstrated by an increase in Fas expression in detergent-insoluble lipid raft fractions and colocalization with GM1+ lipid rafts. We had previously shown that MS-275 treatment inhibited expression of the anti-apoptotic cellular FLICE-inhibitory protein (c-FLIP). Here, we demonstrated that transfection of cells with short hairpin RNA to c-FLIP also resulted in the localization of Fas to lipid rafts. Overall, our studies indicate that MS-275 sensitizes OS cells to FasL by upregulating the expression of Fas in membrane lipid rafts, which correlates with the c-FLIP-dependent distribution of Fas to lipid rafts.
Highlights
We previously demonstrated that the histone deacetylase (HDAC) inhibitor MS-275 sensitized Fas À OS cells to FasLinduced cell death.[15]
Our findings indicate that therapeutic agents that increase Fas localization to lipid rafts or those that decrease cellular FLICE-inhibitory protein (c-FLIP) expression may be useful in sensitizing OS cells to FasL-induced apoptosis
We previously demonstrated that MS-275 downregulates c-FLIP expression in OS cells and in OS lung metastases following oral administration of MS-275.16 We investigated whether the MS-275-induced redistribution of Fas to the lipid rafts was mediated by an effect on c-FLIP
Summary
Fas-induced apoptosis involves the binding of Fas receptor with its ligand, which induces the recruitment of Fasassociated death domain (FADD) and procaspase-8, leading to the activation of caspase-8 by autocatalytic cleavage. Fas-mediated apoptosis is cellular FLICE-inhibitory protein (c-FLIP), which functions by competitively binding to FADD and inhibiting the cleavage of procaspase-8.10 Cancer cell lines and primary cells and tissues from patients have been found to overexpress c-FLIP.[6,24,25,26,27,28,29,30,31] As overexpression of c-FLIP is associated with increased resistance to death receptor pathways, several investigators have found that downregulation of c-FLIP results in the sensitization of tumor cell lines to Fas-mediated apoptosis. These studies suggest that Fas redistribution to lipid rafts occurs upon stimulation and is required for Fas-mediated apoptosis
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