Abstract B41: Withaferin A promotes ROS-mediated differentiation of neuroblastoma

Background: Neuroblastoma (NB), the most common extra-cranial solid tumor in children, originates from the precursor neuroblasts of the sympathetic nervous system. NB accounts for approximately 7-10% of childhood cancers and 15% of childhood cancer death. Despite an aggressive treatment regimen, the 5-year survival for high risk NB remains less than 50%. The differentiation of NB cells into mature cells represents a promising strategy for NB therapy. Currently, retinoids are the most commonly used differentiating agents. However, their use can be limited due to intrinsic or acquired resistance, as well as toxicity. We sought to evaluate the potential of the natural product withaferin A (WA), a steroidal lactone derived from the medicinal plant Withania somnifera, to induce NB cell differentiation. Methods: For differentiation studies NB cell lines (NB1691, SMS-KCNR, SH-SY5Y and the primary cell line SVBM15) were exposed to WA (100-500nM) for 7-10 days and evaluated by light microscopy, immunocytochemistry and western blot analysis. NB stem-like cell lines were generated by culturing NB1691 and SVBM15 cells in neurosphere media. To determine the IC50 (concentration needed to reduce viability by 50%), NB stem-like cell lines were exposed to increasing concentrations of WA and viability was assessed at 72 hours using MTS assay. Reactive oxygen species (ROS) were detected using CM-H2DFDA and ROS induction was inhibited with N-acetyl-L- Cysteine (NAC). To determine WA effect on neurosphere formation, NB cells were plated in 96-well plates at 50 cells/well. Cells were grown in increasing concentrations of WA and spheres were counted at 14 days. Results: WA promoted morphologic alterations (neurite outgrowth) and growth inhibition in a dose dependent manner. Immunocytochemistry and western blot analysis indicated an increase in neuronal markers including neurofilament, β-tubulin and MAP2, as well as a decrease in the stem cell markers BMi-1 and musashi. WA promoted ROS induction, which could be prevented with NAC pretreatment. NAC prevented WA-induced morphological changes and inhibited WA-induced changes in stem cell and differentiation marker expression. WA induced NB stem-like cell death in a dose dependent manner (IC50 of NB1691=1.05 μM; SVBM15=1.06 μM), and significantly inhibited neurosphere formation at concentrations as low as 50nM, which did not exhibit cytotoxicity in regular cell culture conditions. Conclusion: Withania somnifera has been used for centuries and is commonly used in ayurvedic medicine. WA has been shown to affect multiple pathways important for cancer progression and induce anti-cancer effects in breast, prostate and pancreatic cancers. Our data indicates that WA induces NB stem-like cell death, and promotes ROS-mediated NB cell differentiation. Differentiation therapy aims at reducing the risk of the tumor regrowth following high-dose chemotherapy and stem cell transplant in patients classified as high-risk. WA holds great promise as a novel alternative NB treatment strategy for children with persistent minimal residual disease. Citation Format: Gregor A. Rodriguez, Claudia P. Zapata, Anthony Sanchez, Nicolas A. De Cordoba, Beatriz E. Hawkins, Nadia G. Myrthil, Sarah A. Samuels, Amelia Bahamonde, Steven Vanni, Regina M. Graham. Withaferin A promotes ROS-mediated differentiation of neuroblastoma. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Pediatric Cancer Research: From Mechanisms and Models to Treatment and Survivorship; 2015 Nov 9-12; Fort Lauderdale, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(5 Suppl):Abstract nr B41.