Glioblastoma (GBM) has a dismal prognosis and successful elimination of GBM stem cells (GSCs) is a high-priority as these cells are responsible for tumor regrowth following therapy and ultimately patient relapse. Natural products and their derivatives continue to be a source for the development of effective anticancer drugs and have been shown to effectively target pathways necessary for cancer stem cell self-renewal and proliferation. We generated a series of curcumin inspired bis-chalcones and examined their effect in multiple patient-derived GSC lines. Of the 19 compounds synthesized, four analogs robustly induced GSC death in six separate GSC lines, with a half maximal inhibitory concentration (IC50) ranging from 2.7–5.8 μM and significantly reduced GSC neurosphere formation at sub-cytotoxic levels. Structural analysis indicated that the presence of a methoxy group at position 3 of the lateral phenylic appendages was important for activity. Pathway and drug connectivity analysis of gene expression changes in response to treatment with the most active bis-chalcone 4j (the 3,4,5 trimethoxy substituted analog) suggested that the mechanism of action was the induction of endoplasmic reticulum (ER) stress and unfolded protein response (UPR) mediated cell death. This was confirmed by Western blot analysis in which 4j induced robust increases in CHOP, p-jun and caspase 12. The UPR is believed to play a significant role in GBM pathogenesis and resistance to therapy and as such represents a promising therapeutic target.

Introduction. Glioblastoma multiforme (GBM) is the most common and aggressive form of primary brain cancer affecting 3 to 4 per 100,000 people annually in the United States. Despite aggressive therapy including surgical resection followed by both radiation and chemotherapy, overall survival remains dismal at approximately 14 months. Recent studies have determined that GBM's contain a small population of stem cells (GSCs), which promote tumor growth through unlimited self-renewal. Since GSCs are chemo- and radio-resistant and drive tumor regrowth following therapy, successful treatment depends on the elimination of these cells. Withaferin A (WA) is a bioactive compound derived from the plant Withonia somnifera which possesses anti-inflammatory and anti-cancer properties. Here we evaluated the effects of WA on patient derived GSCs. Methods. With IRB approval, GSC lines were generated. GSCs were propagated in neurosphere media and examined for the stem cell markers CD133, nestin, musashi and BMi1 by immunocytochemistry and western blot analysis. Effect of WA on cell viability was determined using MTS assay. The effect of WA on cell survival and stress pathways was evaluated by western blot analysis. Results. WA induced a significant decrease in cell viability at low micromolar concentrations. The IC50 for 3 patient derived GSC lines ranged between 0.96-1.78μM. WA induced ER stress and apoptosis in the GSCs as indicated by increased expression of ER stress markers grp78 and CHOP and increased levels of cleaved PARP. Furthermore, WA decreased survivin but increased phospho-ERK levels. However, inhibition of ERK activity with U0126 did not potentiate the effect of WA on cell death. Conclusion. Ashwagandha has been used safely for medicinal purposes for centuries and pharmacokinetic studies in mice indicate that micromolar plasma concentrations of WA are obtainable. Our studies indicate that very low concentrations of WA induce ER stress and cell death in GSC lines. Targeting GSC with WA may increase patient survival by hindering GSC mediated tumor regrowth following surgery. Our data demonstrates the efficacy of WA and supports additional studies toward the use of WA as an adjuvant GBM therapy. Citation Format: Baoyu Zhang, Branko Cuglievan, Nadia G. Myrthil, Steven Vanni, Ricardo J. Komotar, Regina M. Graham. Targeting glioblastoma stem cells with withaferin A. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5497. doi:10.1158/1538-7445.AM2014-5497

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.