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Synergistic inhibition of glioma cell proliferation by Withaferin A and tumor treating fields

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Abstract

Glioblastoma (GBM) is the most aggressive and lethal form of brain cancer. Standard therapies are non-specific and often of limited effectiveness; thus, efforts are underway to uncover novel, unorthodox therapies against GBM. In previous studies, we investigated Withaferin A, a steroidal lactone from Ayurvedic medicine that inhibits proliferation in cancers including GBM. Another novel approach, tumor treating fields (TTFields), is thought to disrupt mitotic spindle formation and stymie proliferation of actively dividing cells. We hypothesized that combining TTFields with Withaferin A would synergistically inhibit proliferation in glioblastoma. Human glioblastoma cells (GBM2, GBM39, U87-MG) and human breast adenocarcinoma cells (MDA-MB-231) were isolated from primary tumors. The glioma cell lines were genetically engineered to express firefly luciferase. Proliferative potential was assessed either by bioluminescence imaging or cell counting via hemocytometer. TTFields (4 V/cm) significantly inhibited growth of the four cancer cell lines tested (n = 3 experiments per time point, four measurements per sample, p < 0.02 at least; 2-way ANOVA, control vs. treatment). The combination of Withaferin A (10–100 nM) with TTFields significantly inhibited the growth of the glioma cells to a degree beyond that of Withaferin A or TTFields alone. The interaction of the Withaferin A and TTFields on glioma cells was found to be synergistic in nature (p < 0.01, n = 3 experiments). These findings were validated by both bioluminescence and hemocytometric measurements. The combination of Withaferin A with TTFields represents a novel approach to treat GBM in a manner that is likely better than either treatment alone and that is synergistic.

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Abbreviations

ANOVA:

Analysis of variance

BLI:

Bioluminescence imaging

DAPI:

4′,6-Diamidino-2-Phenylindole

DMEM:

Dulbecco’s modified eagle’s medium

H-EGF:

Human epidermal growth factor

EGFR:

Epidermal growth factor receptor

EGFRvIII:

Epidermal growth factor receptor variant III

FBS:

Fetal bovine serum

FDA:

Food and drug administration

H-FGF:

Human fibroblast growth factor

GBM:

Glioblastoma

GBM2:

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

GBM2/GFP-Luc:

GBM2 that was genetically modified to express a fusion protein of firefly luciferase and GFP

GBM39:

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

GBM39/Luc:

GBM39 that was genetically modified to express firefly luciferase

GFP:

Green fluorescent protein

GFP/Luc:

Fusion protein of GFP and firefly luciferase

MDA-MB-231:

Human breast adenocarcinoma cancer cell line (from ATCC)

PBS:

Phosphate-buffered saline

H-PDGF-AA:

Human platelet-derived growth factor variant AA

H-PDGF-BB:

Human platelet-derived growth factor variant BB

TTField:

Tumor treating field

U87-MG:

Human-derived GBM cell line that was purchased from ATCC

U87-MG/eGFP-Luc:

U87-MG that was genetically modified to express a fusion protein of firefly luciferase and GFP

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Acknowledgements

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 (SSG) and the R25 Translational Neuroscience Training Grant R25NS065741 (CBP) for their support of our research. We thank Novocure Inc. for providing us with the inovitro™ device as well as technical support. Finally, we thank Dr. Moshe Giladi for all of his thoughts, comments and feedback.

Funding

Ben and Catherine Ivy Foundation (SSG) and the R25 Translational Neuroscience Training Grant R25NS065741 (CBP).

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Correspondence to Sanjiv S. Gambhir.

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Chang, E., Pohling, C., Beygui, N. et al. Synergistic inhibition of glioma cell proliferation by Withaferin A and tumor treating fields. J Neurooncol 134, 259–268 (2017). https://doi.org/10.1007/s11060-017-2534-5

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