DDEL-04AshwaMAX AND WITHAFERIN A INHIBITS GLIOMAS IN CELLULAR AND MURINE ORTHOTOPIC MODELS

Abstract

INTRODUCTION: Glioblastoma multiforme (GBM) is an aggressive, malignant cancer. An extract from the winter cherry plant (Withania somnifera), AshwaMAX, is concentrated (4.3%) for Withaferin A, a steroidal lactone that inhibits cancer cells. We hypothesized that AshwaMAX could treat GBM and that bioluminescent molecular imaging (BLI) could track oral therapy in orthotopic murine models of glioblastoma. METHODS: Human glioblastoma cells (GBM2, GBM39) were isolated from primary tumors while U87-MG was obtained commercially. Proliferative potential of these cells was assessed by the alamar blue bioassay. GBM2 was transduced with lentiviral vectors that express GFP/firefly luciferase fusion proteins. Mutational, expression and proliferative status of GBMs were studied. Intracranial xenografts of glioblastomas were grown in the right parietal regions of female, nude mice (n = 3-5 per experiment). Tumor growth was followed through BLI. RESULTS: Both wild type and EGFRvIII were present in GBM2 and GBM39. GBM39 expressed EGFRvIII 20-fold higher than GBM2 but U87-MG had virtually no EGFRvIII. Neurosphere cultures of U87-MG, GBM2 and GBM39 were inhibited by AshwaMAX at IC50 of 1.4, 0.19 and 0.22 µM equivalent respectively and by Withaferin A with IC50 of 0.31, 0.28 and 0.25 µM respectively. These data led to preclinical studies with pcGBM2 xenografts in nude mice. Oral gavage, every other day, of non-toxic doses of AshwaMAX (40mg/kg per day) significantly reduced bioluminescence signal (n = 3 mice, p < 0.02, four parameter non-linear regression analysis). After 30 days, bioluminescent signal increased suggesting the onset of resistance. BLI signal for control, untreated mice increased and then plateaued. Similar findings were reported for GBM39/luc xenografts. BLI microscopy showed diffuse growth patterns of GBM2 in murine brain. CONCLUSION: With AshwaMAX, GBM neurospheres collapsed at nanomolar concentrations. Pioneering preclinical studies confirmed that AshwaMAX is effective against GBM in orthotopic murine models. AshwaMAX is thus a promising candidate for future clinical translation in GBM.