Sensitizing neuroblastoma for TRAIL-induced apoptosis by Bortezomib

Despite recent advances of therapeutic strategies for treatment of childhood malignancies, the outcome of children with high-risk neuroblastoma (NB) remains poor. So, there is still a need of novel therapeutic approaches to treat aggressive NB. In this study we investigated the potential of the proteasome inhibitor Bortezomib to sensitize NB cells for TNF-related apoptosis inducing ligand (TRAIL)-induced apoptosis. Combination therapy of Bortezomib and TRAIL led to significant enhancement of cell death in comparison to single agent treatment in a synergistic manner and also inhibited longterm survival. This was demonstrated in various NB cell lines as well as in primary cultured NB cells, underscoring the clinical relevance of this finding. Importantly, Bortezomib and TRAIL also acted in concert to suppress tumor growth in an in vivo model of neuroblastoma. Exploring the underlying molecular mechanism of sensitization, we found enhanced cleavage of caspase-8, -3 and Bid by combination treatment compared to single-agent treatment. Furthermore, Bortezomib profoundly enhanced TRAIL-induced cleavage of Bid into tBid, accumulation of tBid in the cytosol and its insertion into mitochondrial membranes, pointing to a concerted effect on Bid cleavage (TRAIL) and stabilization of tBid (Bortezomib), which links the death receptor to the mitochondrial pathway. Also, combination treatment with Bortezomib and TRAIL compared to single agent treatment led to enhanced conformational change of Bax and Bak, loss of the mitochondrial membrane potential and cytochrome c release. By overexpressing the anti-apoptotic protein Bcl-2, which inhibits the activation of the mitochondrial apoptotic pathway, apoptosis induction after combination treatment was partially blocked. Our data demonstrate that the proteasome inhibitor Bortezomib sensitizes NB cells for TRAIL-induced apoptosis by increasing TRAIL-triggered Bid activation, thereby enhancing mitochondrial outer membrane permeabilization. Thus, the combination of Bortezomib and TRAIL represents a promising therapeutic approach for NB treatment.