Promising effects of the 4HPR–BSO combination in neuroblastoma monolayers and spheroids

To enhance the efficacy of fenretinide (4HPR)-induced reactive oxygen species (ROS) in neuroblastoma, 4HPR was combined with buthionine sulfoximine (BSO), an inhibitor of glutathione (GSH) synthesis, in neuroblastoma cell lines and spheroids, the latter being a three-dimensional tumor model. 4HPR exposure (2.5–10 μM, 24 h) resulted in ROS induction (114–633%) and increased GSH levels (68–120%). A GSH depletion of 80% of basal levels was observed in the presence of BSO (25–100 μM, 24 h). The 4HPR–BSO combination resulted in slightly increased ROS levels (1.1- to 1.3-fold) accompanied by an increase in cytotoxicity (110–150%) compared to 4HPR treatment alone. A correlation was observed between the ROS-inducing capacity of each cell line and the increase in cytotoxicity induced by 4HPR–BSO compared to 4HPR. No significant correlation between baseline antioxidant levels and sensitivity to 4HPR or BSO was observed. In spheroids, 4HPR–BSO induced a strong synergistic growth retardation and induction of apoptosis. Our data show that BSO increased the cytotoxic effects of 4HPR in neuroblastoma monolayers and spheroids in ROS-producing cell lines. This indicates that the 4HPR–BSO combination might be a promising new strategy in the treatment of neuroblastoma.