After extensive research on radiochemotherapy, 5-year survival rates of children with high risk neuroblastoma still do not exceed 50%, owing to adverse side-effects exemplified by doxorubicin-induced cardiomyopathy. A promising new approach is the combination of conventional therapies with specific modulation of cell signaling pathways promoting therapeutic resistance, such as inhibition of aberrant kinase activity or re-expression of silenced tumor suppressor genes by means of chromatin remodeling. In this regard, we established a system that allows to identify potential drug targets as well as to validate respective candidate inhibitors in high-risk neuroblastoma model cell lines. Cell culture, drug exposure, shRNA-mediated knockdown and phenotype analysis are integrated into an efficient and versatile single well-based protocol. By utilizing this system, we assessed RG108, SGI-1027 and nanaomycin A, three novel DNA methyltransferase inhibitors that have not been tested in neuroblastoma cell lines so far, for their potential of synergistic anti-tumor activity in combination with doxorubicin. We found that, similarly to azacytidine, SGI-1027 and nanaomycin A mediate synergistic growth inhibition with doxorubicin independently of N-Myc status. However, they display high cytotoxicity but lack global DNA demethylation activity. Secondly, we conducted a lentiviral shRNA screen of F-box proteins, key regulators of protein stability, and identified Fbxw11/β-TrCP2 as well as Fbxo5/Emi1 as potential therapeutic targets in neuroblastoma. These results complement existing studies and underline the reliability and versatility of our single well-based protocol.
Neuroblastoma F-box proteins DNA methyltransferase inhibitors RNA-interference Screening system Doxorubicin
Online Resource 1Effects of azacytidine, RG108, SGI-1027 and nanaomycin A +/-doxorubicin on cell cycle distribution. Cell cycle distribution measured by flow cytometry using propidium iodide fluorescence labeling: DNA content of individual cells is represented by pulse area (PE-A). Pulse width (PE-W) was used to exclude cell doublets. a) SK-N-AS and SH-EP cells (N-Myc single copy).b) LAN-1 and SK-N-BE(2) cells (N-Myc amplified). (PDF 78 kb)
Online Resource 2Effects of azacytidine, RG108, SGI-1027 and nanaomycin A +/-doxorubicin on apoptosis quantified with flow cytometry using Annexin V (AV) and propidium iodide (PI) fluorescence labeling. Viable and dead cells are labeled AV-/PI- and AV+/PI+, while early and late apoptosis are characterized as AV+/PI- and AV+/PI+. a) SK-N-AS and SH-EP cells (N-Myc single copy). b) LAN-1 and SK-N-BE(2) cells (N-Myc amplified). (PDF 101 kb)