Oxidative cytotoxic agent withaferin A resensitizes temozolomide-resistant glioblastomas via MGMT depletion and induces apoptosis through Akt/mTOR pathway inhibitory modulation
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Temozolomide (TMZ) has remained the chemotherapy of choice in patients with glioblastoma multiforme (GBM) primarily due to the lack of more effective drugs. Tumors, however, quickly develop resistance to this line of treatment creating a critical need for alternative approaches and strategies to resensitize the cells. Withaferin A (WA), a steroidal lactone derived from several genera of the Solanaceae plant family has previously demonstrated potent anti-cancer activity in multiple tumor models. Here, we examine the effects of WA against TMZ-resistant GBM cells as a monotherapy and in combination with TMZ. WA prevented GBM cell proliferation by dose-dependent G2/M cell cycle arrest and cell death through both intrinsic and extrinsic apoptotic pathways. This effect correlated with depletion of principle proteins of the Akt/mTOR and MAPK survival and proliferation pathways with diminished phosphorylation of Akt, mTOR, and p70 S6K but compensatory activation of ERK1/2. Depletion of tyrosine kinase cell surface receptors c-Met, EGFR, and Her2 was also observed. WA demonstrated induction of N-acetyl-L-cysteine-repressible oxidative stress as measured directly and through a subsequent heat shock response with HSP32 and HSP70 upregulation and decreased HSF1. Finally, pretreatment of TMZ-resistant GBM cells with WA was associated with O6-methylguanine-DNA methyltransferase (MGMT) depletion which potentiated TMZ-mediated MGMT degradation. Combination treatment with both WA and TMZ resulted in resensitization of MGMT-mediated TMZ-resistance but not resistance through mismatch repair mutations. These studies suggest great clinical potential for the utilization of WA in TMZ-resistant GBM as both a monotherapy and a resensitizer in combination with the standard chemotherapeutic agent TMZ.
KeywordsWithaferin A Glioblastoma multiforme Temozolomide resistance Oxidative stress Heat shock response Akt/mTOR pathway O6-methylguanine-DNA methyltransferase
We would like to recognize Huaping Zhang (University of Kansas) for his preparation of WA. We would also like to thank the KUMC flow core facility for utilization of its resources established by a generous endowment from the Hall Foundation and by NIH Grant Number P20 RR016443 from the COBRE program of the National Center for Research Resources. We are thankful for research support provided by the Departments of Surgery at the University of Kansas Medical Center and University of Michigan as well as the University of Michigan Comprehensive Cancer Center.
Conflict of interest
None related to this work.
Role of funding sources
This work was made possible by support from the National Institutes of Health (NIH-COBRE P20 RR015563 P.I. B. Timmermann), the Institute for Advancing Medical Innovation (PI: MS Cohen), a University of Kansas Cancer Center Summer Student Training Program grant (PT Grogan), the Departments of Surgery at the University of Kansas Medical Center and University of Michigan (MS Cohen), and a University of Michigan Comprehensive Cancer Center CCSG Development award (PI: MS Cohen).
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