Fenretinide-induced cell death is enhanced by p38^MAPK-dependent upregulation of death receptors and co-treatment with cognate ligand

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Fenretinide initiates cell death in ESFT through generation of ROS and sustained phosphorylation of p38^MAPK (Myatt et al. 2005. Clin. Cancer Res. 11: 3136-48). We have examined the hypothesis that the redox-regulated kinase ASK1 regulates this phosphorylation of p38^MAPK, and investigated the downstream targets of this activation. These studies led to the hypothesis that cell death may be enhanced by fenretinide-induced upregulation of death receptor expression and treatment with the cognate ligand.
 ASK1 activity was measured in ESFT cell lines by immune-complex kinase assay. Changes in cellular RNA after treatment with fenretinide (3μM, 8h) were investigated using the Q series Apoptosis array (SuperArray). Cell surface expression of death receptors and cleavage of caspases 8 and 9 were examined by flow cytometry. Cell viability was determined by the trypan blue exclusion assay.
 Fenretinide (1.5μM) increased ASK1 kinase activity within 5 minutes exposure. This increase preceded phosphorylation of p38^MAPK, which was sustained up to 2 hours. Activation of this pathway led to upregulation of 9% and downregulation of 1% of cellular RNAs. One of the most abundantly upregulated RNAs was the death receptor DR5. Expression of DR5, FAS and p75^NTR was increased following fenretinide treatment (1.5μM, 16-24h). Pharmacological inhibition of p38^MAPK with SB202190 (20μM, 1h) reduced fenretinide-induced death receptor expression, indicating that this is a p38^MAPK-dependent event. Pre-treatment of cells with fenretinide (1.5μM, 16h) and subsequent treatment with the cognate receptor ligand (NGF,TRAIL or FasL; 10-40ng/ml, 24h) significantly decreased viable cell number compared to any agent alone (p<0.01). Fenretinide and FasL further increased fenretinide-induced death by 34%, whereas NGF and TRAIL both enhanced death by 25%. Consistent with activation of the extrinsic death pathway, caspase 8 was cleaved in a time-dependent manner when cells that had been pre-treated with fenretinide were exposed to death receptor ligands (20ng/ml, 2-24h). In contrast, ligands or fenretinide alone did not cause caspase 8 cleavage; fenretinide-induced cell death was effected through cleavage of caspase 9 and activation of the intrinsic mitochondrial death cascade.
 To conclude, fenretinide induces cell death through ASK1-dependent sustained activation of p38^MAPK leading to cleavage of caspase 9 and activation on the intrinsic mitochondrial death cascade. This results in the upregulation of cell surface death receptor expression, which may be exploited to enhance ESFT cell death by co-treatment with the cognate receptor ligand to effect a cleavage of caspase 8 and activation of the extrinsic death cascade.