R. B. Interiano1,2, J. Yang1, D. Hu1, N. Hinkle1,2, C. Morton1, A. M. Davidoff1,2 1St. Jude Children’s Research Hospital,Surgery,Memphis, TN, USA 2University Of Tennessee Health Science Center,Surgery,Memphis, TN, USA
Introduction: Neuroblastoma is the most common solid, extra-cranial, pediatric malignancy, and MYCN amplification has been associated with poor prognosis in this tumor type. Valinomycin is a potassium ionophore which has been shown to cause significant deregulation of intracellular potassium. In a preliminary comparative drug study, we noted valinomycin to have potential effect on tumors with down-regulated MYCN. We sought to assess the antitumor effect of this drug on the expression of MYCN.
Methods: MYCN-amplified and non-MYCN-amplified neuroblastoma cell lines SK-N-BE2, IMR32, NB1691, SK-N-AS, and SK-N-SH were treated with valinomycin with a range of doses (0, 0.001, 0.01, 0.1, 1, 10 μM) for variable time courses. Cell viability was assessed by Presto Blue assay and protein expression of MYCN was assessed via western blot analysis and quantitative real-time polymerase chain reaction (qRT-PCR). Time courses of treatment with valinomycin at 10nM were then used to assess apoptosis with western blot. Mice with flank tumors were then treated at the maximum tolerated dose of 0.5 mg/kg, and were assessed for therapeutic effect
Results:Valinomycin had a significant antitumor effect with >50% cell death in all neuroblastoma cell lines following treatment at 10nM for 72 hours. There was a significant decrease in protein expression of MYCN and c-myc in non-MYCN-amplified lines, except SK-N-AS, occurring as early as 2-4 hours, prior to PARP cleavage and apoptosis occurring. There was no significant change in MYCN mRNA at 24-hour treatment with valinomycin at 10nM. Valinomycin given to xenograft bearing MYCN-amplified tumors showed slowing of progression in SJ-NB12 (~30% at 3 weeks); however, no therapeutic effect was noted in SK-N-AS or NB1691.
Conclusion: Valinomycin has a significant antitumor effect on neuroblastoma cell lines in vitro. This antitumor effect is in part through the down-regulation of MYCN, prior to apoptotic signaling. Understanding the mechanism of targeting MYCN down-regulation may lead to improved therapeutics. Drug toxicity likely does not allow an appreciable pharmacodynamic effect on in vivo neuroblastoma tumors, although preliminary results suggest it may cause growth delay in some tumors.
