Background

Neuroblastoma (NB) is the most common extra cranial solid tumour in childhood. In high risk NB, survival remains poor (< 40 %) despite advances in chemo-radiotherapy and aggressive surgical resection. We hypothesised that activation of the transcription factor, NF-?B may be one of the potential mechanisms underlying the drug resistance observed in NB.

Method

NF-?B activity was measured using reporter gene assay (luminometry with NF-Luc) and real time RT-PCR for endogenous target genes (I?B?, I?Be and A20). Sensitivity of four human NB cell lines and primary NBL cell cultures to cytotoxics (etoposide and doxorubicin) and NF-?B inhibitors were measured using MTT assay. Mechanism of cell death was confirmed by caspase 3/7 assay and confocal microscopy using annexin V-propidium iodide binding. Data were analysed using one way ANOVA test and means comparison by Bonferoni method (Origin v8.0). Significance: p <0.05.

Results

All NB cell lines showed relative drug resistance to cytototics. Reporter gene assay and real-time RT-PCR for I?B?, I?Be and A20 confirmed that the NF-?B pathway was activated by the cytotoxics in S- (stromal) and N-type (neuronal) NB cell lines. Inhibition of NF-?B was observed with chemical (Bay11 7082 and BMS 345541) and biological agents (I?B? and mutant I?B?). NF-?B inhibition resulted in significant cell death in NB cell lines and primary cultures. Cell death was confirmed to be a result of apoptotis. Synergistic cell death was not observed with combination chemotherapy and BMS (p<0.05, ANOVA). BMS was also observed to induce inhibition of the p53 pathway.

Conclusion

NF-?B inhibition may have potential therapeutic application in NB. Given the central role of NF-?B and p53 in cell fate decisions pharmacologic agents will need to be screened for their effects on both pathways, and focus given on agents which would simultaneously inhibit NF-?B and activate p53.