PRIMA-1-induced neuroblastoma cell death is modulated by p53 and mycn through glutathione level.

Background: Neuroblastoma is the most common extracranial solid tumor in children. This cancer has a low frequency of TP53 mutations and its downstream pathway is usually intact. This study assessed the efficacy of the p53 activator, PRIMA-1, in inducing neuroblastoma cell death.

Methods: CellTiter 2.0 was used to study susceptibility and specificity of NB cell lines to PRIMA-1. Real-time PCR and western blot were used to assess the most common p53 transactivation targets. Induction of p53 and Noxa, and inhibition of Cas3/7, were used to assess impact on cell death after PRIMA-1 treatment. Flow cytometry was used to analyze cell cycle phase and induction of apoptosis, reactive oxygen species, and the collapse of mitochondrial membrane potential.

Results: Neuroblastoma cell lines were at least four times more susceptible to PRIMA-1 than were primary fibroblasts and keratinocyte cell lines. PRIMA-1 induced cell death rapidly and in all cell cycle phases. Although PRIMA-1 activated p53 transactivation activity, p53's role is likely limited because its main targets remained unaffected, whereas pan-caspase inhibitor demonstrated no ability to prevent cell death. PRIMA-1 induced oxidative stress and modulated the methionine/cysteine/glutathione axis. Variations of MYCN and p53 modulated intracellular levels of GSH and resulted in increased/decreased sensitivity of PRIMA-1. PRIMA-1 inhibited thioredoxin reductase, but the effect of PRIMA-1 was not altered by thioredoxin inhibition.

Conclusions: PRIMA-1 could be a promising new agent to treat neuroblastoma because it demonstrated good anti-tumor action. Although p53 is involved in PRIMA-1-mediated cell death, our results suggest that direct interaction with p53 has a limited role in neuroblastoma but rather acts through modulation of GSH levels.