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Journal of Neuro-Oncology

, Volume 126, Issue 2, pp 253–264 | Cite as

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

  • Edwin Chang
  • Christoph Pohling
  • Arutselvan Natarajan
  • Timothy H. Witney
  • Jasdeep Kaur
  • Lingyun Xu
  • Gayatri Gowrishankar
  • Aloma L D’Souza
  • Surya Murty
  • Sophie Schick
  • Liyin Chen
  • Nicholas Wu
  • Phoo Khaw
  • Paul Mischel
  • Taher Abbasi
  • Shahabuddin Usmani
  • Parag Mallick
  • Sanjiv S. Gambhir
Laboratory Investigation

Abstract

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.

Keywords

Glioblastoma Bioluminescent Preclinical AshwaMAX Withaferin A 

Abbreviations

BLI

Bioluminescence Imaging

DAPI

4′,6-Diamidino-2-Phenylindole

h-EGF

Human Epidermal Growth Factor

EGFR

Epidermal Growth Factor Receptor

EGFRvIII

Epidermal Growth Factor Receptor variant III

h-FGF

Human Fibroblast Growth Factor

GBM

Glioblastoma Multiforme

GBM2

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

GBM2/gfp/luc

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

GBM39

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

GFP

Green Fluorescent Protein

GFP/Luc

Fusion protein of Green Fluorescence Protein and firefly luciferase

HSP70

Heat Shock Protein 70

PBS

Phosphate Buffered Saline

h-PDGF-AA

Human Platelet-derived Growth Factor variant AA

h-PDGF-BB

Human Platelet-derived Growth Factor variant BB

U87

GBM cell line purchased from ATCC

Notes

Acknowledgments

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.

Funding

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

Compliance with ethical standards

Conflict of interest

The Authors declare no conflict of interests

Animal studies

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

Supplementary material

11060_2015_1972_MOESM1_ESM.pptx (1.7 mb)
Supplementary material 1 (PPTX 1694 kb)
11060_2015_1972_MOESM2_ESM.docx (13 kb)
Supplementary material 2 (DOCX 13 kb)

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Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  • Edwin Chang
    • 1
    • 2
    • 3
    • 4
  • Christoph Pohling
    • 1
    • 2
    • 3
    • 4
  • Arutselvan Natarajan
    • 1
    • 2
    • 3
    • 4
  • Timothy H. Witney
    • 1
    • 2
    • 3
    • 4
  • Jasdeep Kaur
    • 1
    • 2
    • 3
    • 4
  • Lingyun Xu
    • 1
    • 2
    • 3
    • 4
  • Gayatri Gowrishankar
    • 1
    • 2
    • 3
    • 4
  • Aloma L D’Souza
    • 1
    • 2
    • 3
    • 4
  • Surya Murty
    • 1
    • 2
    • 3
    • 4
  • Sophie Schick
    • 1
    • 2
    • 3
    • 4
  • Liyin Chen
    • 1
    • 2
    • 3
    • 4
  • Nicholas Wu
    • 1
    • 2
    • 3
    • 4
  • Phoo Khaw
    • 1
    • 2
    • 3
    • 4
  • Paul Mischel
    • 5
  • Taher Abbasi
    • 6
  • Shahabuddin Usmani
    • 6
  • Parag Mallick
    • 1
    • 2
    • 3
    • 4
  • Sanjiv S. Gambhir
    • 1
    • 2
    • 3
    • 4
  1. 1.Departments of Radiology and BioengineeringStanford UniversityPalo AltoUSA
  2. 2.Molecular Imaging Program at StanfordStanford UniversityPalo AltoUSA
  3. 3.Canary Center at Stanford for Cancer Early DetectionStanford UniversityPalo AltoUSA
  4. 4.Nuclear MedicineStanford UniversityPalo AltoUSA
  5. 5.Department of Medicine, Ludwig Institute for Cancer ResearchUniversity of California at San DiegoLa JollaUSA
  6. 6.Cellworks Group Inc.San JoseUSA

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