Fenretinide-dependent upregulation of death receptors through ASK1 and p38α enhances death receptor ligand-induced cell death in Ewing's sarcoma family of tumours

Background: Sustained p38 MAPK phosphorylation upregulates p75 neurotrophin (p75 NTR ) and induces apoptosis in Ewing's sarcoma family of tumours (ESFT). As fenretinide induces ESFT death through sustained p38 MAPK phosphorylation, we hypothesised that this may be effected through upregulation of death receptors (DRs) and that treatment of fenretinide plus DR ligands may enhance apoptosis. Methods: DR expression was determined by flow cytometry. Trypan blue exclusion assays, caspase-8 flow cytometry and immunoblotting for Bid were used to measure cell death. Results: Fenretinide upregulated cell surface expression of tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) receptors, FAS and p75 NTR , in an ASK1- and p38 α -dependent manner. Cotreatment with fenretinide and DR ligands resulted in synergistic death compared with either agent alone; caspase-8 and Bid were cleaved in a time-dependent manner. Fenretinide did not increase DR expression in non-malignant cells. Furthermore, fenretinide, TRAIL or a combination of both agents was non-cytotoxic to non-malignant cells. Etoposide and actinomycin D increased expression of all DRs examined, whereas vincristine increased FAS alone. Only actinomycin D and TRAIL, and etoposide with TRAIL or FasL, enhanced death compared with either agent alone. Conclusion: The synergistic death observed with fenretinide and DR ligands suggests that this combination may be an attractive strategy for the treatment of ESFT.