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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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Summary

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.

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Acknowledgments

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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Authors and Affiliations

  1. Department of Pharmacology, Toxicology, and Therapeutics, University of Kansas Medical Center, Kansas City, KS, 66160, USA

    Patrick T. Grogan & Mark S. Cohen

  2. Department of Surgery, University of Michigan Hospital and Health Systems, Ann Arbor, MI, 48109, USA

    Patrick T. Grogan & Mark S. Cohen

  3. Department of Radiation Oncology, Mayo Clinic, Rochester, MN, 55905, USA

    Jann N. Sarkaria

  4. Department of Medicinal Chemistry, University of Kansas School of Pharmacy, Lawrence, KS, 66045, USA

    Barbara N. Timmermann

  5. Section of General Surgery, University of Michigan Hospital and Health Systems, 2920K Taubman Center, SPC 5331, 1500 E. Medical Center Drive, Ann Arbor, MI, 48109-5331, USA

    Mark S. Cohen

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  1. Patrick T. Grogan
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Correspondence to Mark S. Cohen.

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Online Resource 1

WA treatment resulted in depletion of total protein levels in both AMPKα and TSC2 at 24-48h in both U251TMZ and U87TMZ cells. U87TMZ cells also demonstrated reduced phosphorylation of AMPKα in contrast to previous findings in U87 parental cells. U251TMZ cells display dose-dependent activating phosphorylation of AMPKα and downstream TSC2. (PPT 402 kb)

Online Resource 2

(a) 24h WA pretreatment did not prevent TMZ efficacy in TMZ-sensitive U251 and U87 parental cells as assessed by a normalized MTS assay, suggesting a lack of inhibition of functional MMR. In U251 cells, WA mildly sensitized cells to TMZ, but no sensitization was observed in U87 cells. (b) Given a described oxidative component of TMZ therapy through activation of AMPKα, markers of oxidation were assessed during combination therapy of WA and TMZ in TMZ-resistant cells. HSP32 and HSP70, known to be upregulated in response to oxidative stress, increased with WA treatment to a maximal level but were not further modulated by the presence of TMZ. Treatment with TMZ only resulted in elevated p-AMPKα in only U138 cells, but WA failed to further increase this phosphorylation in combination with TMZ. This was confirmed downstream by failure to further inhibit mTOR activation with combination therapy, suggesting efficacy of such treatment was not due to potentiated or synergized elevation in oxidation status. **p < 0.01 (PPT 791 kb)

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Grogan, P.T., Sarkaria, J.N., Timmermann, B.N. et al. Oxidative cytotoxic agent withaferin A resensitizes temozolomide-resistant glioblastomas via MGMT depletion and induces apoptosis through Akt/mTOR pathway inhibitory modulation. Invest New Drugs 32, 604–617 (2014). https://doi.org/10.1007/s10637-014-0084-7

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