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AshwaMAX and Withaferin A inhibits gliomas in cellular and murine orthotopic models


Glioblastoma multiforme (GBM) is an aggressive, malignant cancer Johnson and O’Neill (J Neurooncol 107: 359–364, 2012). An extract from the winter cherry plant (Withania somnifera ), AshwaMAX, is concentrated (4.3 %) for Withaferin A; a steroidal lactone that inhibits cancer cells Vanden Berghe et al. (Cancer Epidemiol Biomark Prev 23: 1985–1996, 2014). We hypothesized that AshwaMAX could treat GBM and that bioluminescence imaging (BLI) could track oral therapy in orthotopic murine models of glioblastoma. Human parietal-cortical glioblastoma cells (GBM2, GBM39) were isolated from primary tumors while U87-MG was obtained commercially. GBM2 was transduced with lentiviral vectors that express Green Fluorescent Protein (GFP)/firefly luciferase fusion proteins. Mutational, expression and proliferative status of GBMs were studied. Intracranial xenografts of glioblastomas were grown in the right frontal regions of female, nude mice (n = 3–5 per experiment). Tumor growth was followed through BLI. Neurosphere cultures (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. Oral gavage, every other day, of AshwaMAX (40 mg/kg per day) significantly reduced bioluminescence signal (n = 3 mice, p < 0.02, four parameter non-linear regression analysis) in preclinical models. After 30 days of treatment, bioluminescent signal increased suggesting onset of resistance. BLI signal for control, vehicle-treated mice increased and then plateaued. Bioluminescent imaging revealed diffuse growth of GBM2 xenografts. With AshwaMAX, GBM neurospheres collapsed at nanomolar concentrations. Oral treatment studies on murine models confirmed that AshwaMAX is effective against orthotopic GBM. AshwaMAX is thus a promising candidate for future clinical translation in patients with GBM.

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Bioluminescence Imaging




Human Epidermal Growth Factor


Epidermal Growth Factor Receptor


Epidermal Growth Factor Receptor variant III


Human Fibroblast Growth Factor


Glioblastoma Multiforme


Patient-derived glioblastoma multiforme cell culture (from Stanford University School of Medicine)


GBM2 that is genetically modified to express a fusion protein of firefly luciferase and Green Fluorescence Protein (GFP)


Patient-derived glioblastoma multiforme cell culture (from University of California at San Diego School of Medicine)


Green Fluorescent Protein


Fusion protein of Green Fluorescence Protein and firefly luciferase


Heat Shock Protein 70


Phosphate Buffered Saline


Human Platelet-derived Growth Factor variant AA


Human Platelet-derived Growth Factor variant BB


GBM cell line purchased from ATCC


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GBM39 was the donated to us by Dr. Paul Mischel, MD, PhD while the cell lines of GBM2 and GBM2/gfp/Luc were kindly provided to us by Dr. Michelle Monje, MD, PhD. We thank Dr’s. Laura Pisani, PhD and Timothy Doyle, PhD of the Stanford Small Animal Imaging Facility (SAIF) for their excellent technical support. AshwaMAX was obtained from Dr. Lal Hingorani from Pharmaza Herbal Pvt Ltd. (Gujarat, India). We acknowledge the excellent technical support of Kathryn Li, Tara Thakurta, Alex Serafini and Xiaofan Wu.


We gratefully acknowledge the Ben and Catherine Ivy Foundation for funding of the research.

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Correspondence to Edwin Chang.

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All preclinical studies were approved by the Institutional Animal Care and Use Committee of Stanford University. All ethical guidelines and standards were adhered to.

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Chang, E., Pohling, C., Natarajan, A. et al. AshwaMAX and Withaferin A inhibits gliomas in cellular and murine orthotopic models. J Neurooncol 126, 253–264 (2016). https://doi.org/10.1007/s11060-015-1972-1

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  • Glioblastoma
  • Bioluminescent
  • Preclinical
  • AshwaMAX
  • Withaferin A