ET-71THE ANTITUMOR EFFECTS OF WITHAFERIN A IN GLIOBLASTOMA STEM CELLS

INTRODUCTION: Glioblastoma Multiforme (GBM) is the most common and malignant primary brain tumors. Recent studies indicate that GBM stem cells (GSCs), are chemotherapy and radiotherapy resistant and are responsible for tumor cell invasion into normal brain, tumor growth and tumor reoccurrence following treatment. Withaferin A (WA), a steroidal lactone isolated from Withania somnifera, has demonstrated anti-tumor potential including inhibiting tumor growth, metastasis and angiogenesis. However, effects of WA in GSCs have not been previously explored. METHODS: GSCs were generated from patient's tumors and analyzed for stem cell markers CD133, CD44, GFAP, Nestin, Musashi, SOX2 and BMi1 by immunocytochemistry. Effect of WA on GSC viability was determined by MTS and/or LDH assay. Morphological effects of WA were evaluated by transmission electron microscopy. Effect of WA on cell signaling pathways was evaluated with flow cytometry, western blot analysis and fluorescent microscopy. ROS was detected using MitoSOX Red. RESULTS: WA induced both dose and time dependent increase in cell death in GSC lines (IC 50 average 1.27+ 0.12uM for 6 GSC lines). In contrast, primary neurons were not affected by these concentrations and had a much higher IC 50 (5.78uM). Treatment of GSCs with WA significantly increased the diameter of endoplasmic reticulum (ER) and western blot analysis confirmed ER stress demonstrating increased grp78, IRE1, and CHOP protein levels. WA treatment increased mitochondrial ROS production and induced apoptosis as indicated by increased levels of cleaved PARP, caspase 4/3/7/12, Bax, and bak. Pre-treatment with the antioxidant N-acetyl-L-cysteine (NAC) or caspase inhibitor (z-vad-fmk) attenuated WA-induced apoptosis. Lastly WA increased both velcade and NVP-BEZ235 induced cell death. CONCLUSION: Our data indicates that WA mediates anti-tumor effects via ROS generation, ER stress activation, and apoptosis induction in GSCs and potentially sensitizes these cells to additional chemotherapy. These findings warrant further investigation of WA as an adjuvant therapy for GBM.