The majority of high-risk neuroblastomas lack the expression of caspase-8 due to gene silencing which suggest a mechanism for the selection of tumour cells that are refractory to multiple cytotoxic drugs including tumour necrosis factor-related apoptosis-inducing ligand (TRAIL). Inhibitors of DNA methyltransferases and IFN-γ induce expression of caspase-8, and sensitise some neuroblastoma cells to TRAIL-mediated apoptosis. Here we demonstrate that a combination of cytostatic drugs with IFN-γ and TRAIL synergistically induces neuroblastoma cell death, which may have implications for future therapy of children with neuroblastoma. Treatment of neuroblastoma cells with IFN-γ induced caspase-8 expression in all cell lines investigated. In five of the neuroblastoma cell lines (SHEP-1, SK-N-AS, SK-N-FI, SH-SY-5Y and Kelly), IFN-γ promoted TRAIL-mediated cleavage of caspase-8, initiating a caspase cascade involving caspase-7 and PARP followed by apoptosis. IFN-γ-mediated facilitation of apoptosis was inhibited by the pan-caspase inhibitor zVAD-fmk and the caspase-8 specific inhibitor zIEDT-fmk, indicating an important role of caspase-8 in mediating sensitation by IFN-γ in neuroblastoma cells. In three of the cell lines [SK-N-BE(2), SK-N-DZ and IMR-32] caspase-8 expression was induced by IFN-γ, but the cells were still resistant to TRAIL-mediated apoptosis. The pattern of basal TRAIL receptor expression, decoy receptors, FLIP and FADD could not be correlated with resistance or sensitivity to TRAIL-induced apoptosis. Importantly, treatment of neuroblastoma cell lines with cytostatic drugs increased apoptosis in the TRAIL-sensitive cell lines whereas the resistant cell lines were susceptible to TRAIL-mediated apoptosis in the presence of the anticancer drugs. The mechanism of the increased susceptibility to apoptosis might results from drug-mediated up-regulation of the death receptors DR4 and DR5.

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