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A limiting factor in the therapeutic outcome of children with high-risk neuroblastoma is the intrinsic and acquired resistance to common chemotherapeutic treatments Here we investigated the molecular mechanisms by which the hemisynthetic cardiac glycoside UNBS1450 overcomes this limitation and induces differential cell death modalities in both neuroblastic and stromal neuroblastoma through stimulation of a cell-type-specific autophagic response eventually leading to apoptosis or necroptosis In neuroblastic SH-SY5Y cells we observed a time-dependent production of reactive oxygen species that affects lysosomal integrity inducing lysosome-associated membrane protein 2 degradation and cathepsin B and L activation Subsequent mitochondrial membrane depolarization and accumulation of mitochondria in phagophores occurred after 8h of UNBS1450 treatment Results were confirmed by mitochondrial mass analysis electron microscopy and co-localization of mitochondria with GFP-LC3 suggesting the impaired clearance of damaged mitochondria Thus a stress-induced defective autophagic flux and the subsequent lack of clearance of damaged mitochondria sensitized SH-SY5Y cells to UNBS1450-induced apoptosis Inhibition of autophagy with small inhibitory RNAs against ATG5 ATG7 and Beclin-1 protected SH-SY5Y cells against the cytotoxic effect of UNBS1450 by inhibiting apoptosis In contrast autophagy progression towards the catabolic state was observed in stromal SK-N-AS cells here reactive oxygen species ROS generation remained undetectable preserving intact lysosomes and engulfing damaged mitochondria after UNBS1450 treatment Moreover autophagy inhibition determined sensitization of SK-N-AS to apoptosis We identified efficient mitophagy as the key mechanism leading to failure of activation of the apoptotic pathway that increased resistance of SK-N-AS to UNBS1450 triggering rather necroptosis at higher doses Altogether we characterize here the differential modulation of ROS and mitophagy as a main determinant of neuroblastoma resistance with potential relevance for personalized anticancer therapeutic approaches

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Cell type-dependent ROS and mitophagy response leads to apoptosis or necroptosis in neuroblastoma