We recently reported that radiation (IR) induced NFκB in neuroblastoma cells and treatment with curcumin impedes the activation of NFκB and enhance IR-induced cell death. However, full potential of curcumin is yet to be realized because of its poor bioavailability. This prompted us to investigate the efficacy of a more potent synthetic analogue, EF24 in inhibiting IR-induced NFκB dependent cell survival and tumor progression and delineated the functional mechanism involved. Human SH-SY5Y and IMR-32 cells, either exposed to 2Gy or treated with 100nM EF24 were exposed to IR and examined after 1h, 3, 6, 24, 48 or 72h. IR profoundly inhibited both eNOS phosphorylation and transcriptional activation. Consistently, IR reduced the bioavailability of NO. EMSA analysis showed a robust and persistent NFκB activation at least up to 72h in IR exposed cells. eNOS inhibition (L-NAME) and NO induction (GSNO) studies demonstrated the influence of IR-regulated eNOS and NO in NFκB activation. Furthermore, IR significantly induced transcriptional levels of MMP-1, 2, 3, 7, 9, 11, 13 and 14 in neuroblastoma cells. NFκB knock-out (using RelA siRNA) or forced expression of NF-kB (p50, p65 over expression) exhibits that IR-induced NFκB mediates MMPs activation. Conversely, EF24 treatment significantly reverted IR-negated eNOS activity and enhanced NO availability in the cells. Moreover, EF24 profoundly inhibited IR-induced NFκB and this induced inhibition was sustained at least up to 72h. eNOS inhibition studies with EF24 clearly portraits that EF24 regulates induced NFκB in an eNOS-NO dependent manner. More importantly, EF24 completely suppressed IR-induced MMPs transcript levels in these cells. NFκB over expression studies validates that EF24 regulates IR-induced MMPs by muting IR-activated NFκB. In addition, MTT analysis revealed that EF24 significantly conferred IR-induced inhibition of cell survival in both SH-SY5Y and IMR-32 cells by targeting IR-induced NFκB. Taken together these results suggests that IR-regulated eNOS and reduction in NO availability activates NFκB and NFκB dependent transcriptional activation of MMPs. More importantly, reverting IR-regulated eNOS→NO with EF24 may significantly inhibit NFκB dependent survival advantage and, may serve as a potential deliverable in mitigating neuroblastoma relapse and progression after radiotherapy.

Funding Support: ACS- IRG-05-066-01; Presbyterian Health Foundation

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5547.