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-1MET, in inducing neuroblastoma cell death.
Methods: CellTiter 2.0 was used to study susceptibility and specificity of NB cell lines to PRIMA-1MET. 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-1MET 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-1MET than were primary fibroblasts and keratinocyte cell lines. PRIMA-1MET induced cell death rapidly and in all cell cycle phases. Although PRIMA-1MET 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-1MET 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-1MET. PRIMA-1MET inhibited thioredoxin reductase, but the effect of PRIMA-1MET was not altered by thioredoxin inhibition.
Conclusions: PRIMA-1MET could be a promising new agent to treat neuroblastoma because it demonstrated good anti-tumor action. Although p53 is involved in PRIMA-1MET-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.
Keywords: Glutathione; MYCN; Neuroblastoma; PRIMA-1MET; p53.