Role of NF-KAPPA B in cell fate  decisions in neuroblstoma


Year:

Session type:

Dhanya Mullassery1, Violaine See3, Dave Spiller3, Barry Pizer2, Carlo Dominici4, Heather McDowell2, Edwin Jesudason1, Paul Losty1, Mike White3
1Academic paediatric Surgery, University of Liverpool, Liverpool, United Kingdom,2Paediatric Oncology, Alder Hey Children’s NHS Foundation Trust, Liverpool, United Kingdom,3Centre for Cell Imaging, University of Liverpool, Liverpool, United Kingdom,4Department of Paediatrics, La Sapienza University, Rome, Italy

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.