Cytotoxicity of withaferin A in glioblastomas involves induction of an oxidative stress-mediated heat shock response while altering Akt/mTOR and MAPK signaling pathways


Withaferin A (WA), a steroidal lactone derived from the plant Vassobia breviflora, has been reported to have anti-proliferative, pro-apoptotic, and anti-angiogenic properties against cancer growth. In this study, we identified several key underlying mechanisms of anticancer action of WA in glioblastoma cells. WA was found to inhibit proliferation by inducing a dose-dependent G2/M cell cycle arrest and promoting cell death through both intrinsic and extrinsic apoptotic pathways. This was accompanied by an inhibitory shift in the Akt/mTOR signaling pathway which included diminished expression and/or phosphorylation of Akt, mTOR, p70 S6K, and p85 S6K with increased activation of AMPKα and the tumor suppressor tuberin/TSC2. Alterations in proteins of the MAPK pathway and cell surface receptors like EGFR, Her2/ErbB2, and c-Met were also observed. WA induced an N-acetyl-L-cysteine-repressible enhancement in cellular oxidative potential/stress with subsequent induction of a heat shock stress response primarily through HSP70, HSP32, and HSP27 upregulation and HSF1 downregulation. Taken together, we suggest that WA may represent a promising chemotherapeutic candidate in glioblastoma therapy warranting further translational evaluation.

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  1. 1.

    Wen PY, Kesari S (2008) Malignant gliomas in adults. N Engl J Med 359:492–507

    PubMed  Article  CAS  Google Scholar 

  2. 2.

    Stupp R, Hegi ME, Mason WP, van den Bent MJ, Taphoorn MJ, Janzer RC, Ludwin SK, Allgeier A, Fisher B, Belanger K, Hau P, Brandes AA, Gijtenbeek J, Marosi C, Vecht CJ, Mokhtari K, Wesseling P, Villa S, Eisenhauer E, Gorlia T, Weller M, Lacombe D, Cairncross JG, Mirimanoff RO (2009) Effects of radiotherapy with concomitant and adjuvant temozolomide versus radiotherapy alone on survival in glioblastoma in a randomised phase III study: 5-year analysis of the EORTC-NCIC trial. Lancet Oncol 10:459–466

    PubMed  Article  CAS  Google Scholar 

  3. 3.

    Giese A, Bjerkvig R, Berens ME, Westphal M (2003) Cost of migration: invasion of malignant gliomas and implications for treatment. J Clin Oncol 21:1624–1636

    PubMed  Article  CAS  Google Scholar 

  4. 4.

    Quick A, Patel D, Hadziahmetovic M, Chakravarti A, Mehta M (2010) Current therapeutic paradigms in glioblastoma. Rev Recent Clin Trials 5:14–27

    PubMed  Article  CAS  Google Scholar 

  5. 5.

    Chamberlain MC (2010) Temozolomide: therapeutic limitations in the treatment of adult high-grade gliomas. Expert Rev Neurother 10:1537–1544

    PubMed  Article  CAS  Google Scholar 

  6. 6.

    Samadi AK, Tong X, Mukerji R, Zhang H, Timmermann BN, Cohen MS (2010) Withaferin A, a cytotoxic steroid from Vassobia breviflora, induces apoptosis in human head and neck squamous cell carcinoma. J Nat Prod 73:1476–1481

    PubMed  Article  CAS  Google Scholar 

  7. 7.

    Oh JH, Kwon TK (2009) Withaferin A inhibits tumor necrosis factor-alpha-induced expression of cell adhesion molecules by inactivation of Akt and NF-kappaB in human pulmonary epithelial cells. Int Immunopharmacol 9:614–619

    PubMed  Article  CAS  Google Scholar 

  8. 8.

    Koduru S, Kumar R, Srinivasan S, Evers MB, Damodaran C (2010) Notch-1 inhibition by withaferin-A: a therapeutic target against colon carcinogenesis. Mol Cancer Ther 9:202–210

    PubMed  Article  CAS  Google Scholar 

  9. 9.

    Yu Y, Hamza A, Zhang T, Gu M, Zou P, Newman B, Li Y, Gunatilaka AA, Zhan CG, Sun D (2010) Withaferin A targets heat shock protein 90 in pancreatic cancer cells. Biochem Pharmacol 79:542–551

    PubMed  Article  CAS  Google Scholar 

  10. 10.

    Stan SD, Zeng Y, Singh SV (2008) Ayurvedic medicine constituent withaferin a causes G2 and M phase cell cycle arrest in human breast cancer cells. Nutr Cancer 60(Suppl 1):51–60

    PubMed  Article  CAS  Google Scholar 

  11. 11.

    Oh JH, Lee TJ, Kim SH, Choi YH, Lee SH, Lee JM, Kim YH, Park JW, Kwon TK (2008) Induction of apoptosis by withaferin A in human leukemia U937 cells through down-regulation of Akt phosphorylation. Apoptosis 13:1494–1504

    PubMed  Article  CAS  Google Scholar 

  12. 12.

    Mandal C, Dutta A, Mallick A, Chandra S, Misra L, Sangwan RS (2008) Withaferin A induces apoptosis by activating p38 mitogen-activated protein kinase signaling cascade in leukemic cells of lymphoid and myeloid origin through mitochondrial death cascade. Apoptosis 13:1450–1464

    PubMed  Article  CAS  Google Scholar 

  13. 13.

    Mohan R, Hammers HJ, Bargagna-Mohan P, Zhan XH, Herbstritt CJ, Ruiz A, Zhang L, Hanson AD, Conner BP, Rougas J, Pribluda VS (2004) Withaferin A is a potent inhibitor of angiogenesis. Angiogenesis 7:115–122

    PubMed  Article  CAS  Google Scholar 

  14. 14.

    Singh D, Aggarwal A, Maurya R, Naik S (2007) Withania somnifera inhibits NF-kappaB and AP-1 transcription factors in human peripheral blood and synovial fluid mononuclear cells. Phytother Res 21:905–913

    PubMed  Article  CAS  Google Scholar 

  15. 15.

    Samadi AK, Mukerji R, Shah A, Timmermann BN, Cohen MS (2010) A novel RET inhibitor with potent efficacy against medullary thyroid cancer in vivo. Surgery 148:1228–1236, discussion 1236

    PubMed  Article  Google Scholar 

  16. 16.

    Shah N, Kataria H, Kaul SC, Ishii T, Kaur G, Wadhwa R (2009) Effect of the alcoholic extract of Ashwagandha leaves and its components on proliferation, migration, and differentiation of glioblastoma cells: combinational approach for enhanced differentiation. Cancer Sci 100:1740–1747

    PubMed  Article  CAS  Google Scholar 

  17. 17.

    Yang HJ, Shi GQ, Dou QP (2007) The tumor proteasome is a primary target for the natural anticancer compound withaferin A isolated from “Indian Winter Cherry”. Mol Pharmacol 71:426–437

    PubMed  Article  CAS  Google Scholar 

  18. 18.

    Stan SD, Hahm ER, Warin R, Singh SV (2008) Withaferin A causes FOXO3a- and Bim-dependent apoptosis and inhibits growth of human breast cancer cells in vivo. Cancer Res 68:7661–7669

    PubMed  Article  CAS  Google Scholar 

  19. 19.

    Devi PU, Kamath R, Rao BS (2000) Radiosensitization of a mouse melanoma by withaferin A: in vivo studies. Indian J Exp Biol 38:432–437

    PubMed  CAS  Google Scholar 

  20. 20.

    Samadi AK, Cohen SM, Mukerji R, Chaguturu V, Zhang X, Timmermann BN, Cohen MS, Person EA (2012) Natural withanolide withaferin A induces apoptosis in uveal melanoma cells by suppression of Akt and c-MET activation. Tumor Biol 33:1179–1189

    Article  CAS  Google Scholar 

  21. 21.

    Fong MY, Jin S, Rane M, Singh RK, Gupta R, Kakar SS (2012) Withaferin A synergizes the therapeutic effect of Doxorubicin through ROS-mediated autophagy in ovarian cancer. PLoS One 7:e42265

    PubMed  Article  CAS  Google Scholar 

  22. 22.

    Munagala R, Kausar H, Munjal C, Gupta RC (2011) Withaferin A induces p53-dependent apoptosis by repression of HPV oncogenes and upregulation of tumor suppressor proteins in human cervical cancer cells. Carcinogenesis 32:1697–1705

    PubMed  Article  CAS  Google Scholar 

  23. 23.

    Santagata S, Xu YM, Wijeratne EM, Kontnik R, Rooney C, Perley CC, Kwon H, Clardy J, Kesari S, Whitesell L, Lindquist S, Gunatilaka AA (2012) Using the heat-shock response to discover anticancer compounds that target protein homeostasis. ACS Chem Biol 7:340–349

    PubMed  Article  CAS  Google Scholar 

  24. 24.

    Grogan PT, Samadi AK, Cohen MS (2010) A novel cytotoxic agent induced apoptosis in malignant gliomas in vitro. In: Academic Surgical Congress, Journal of Surgical Research, San Antonio, TX, pp. 341–342

  25. 25.

    Grogan PT, Sleder KD, Stecklein SR, Cohen MS (2011) Vassobia Breviflora Root-Extract Withaferin A As A Novel Cytotoxic And Synergistic Agent Against Malignant Gliomas. In: Academic Surgical Congress, Journal of Surgical Research, Huntington Beach, CA, pp. 311

  26. 26.

    Hahm ER, Moura MB, Kelley EE, Van Houten B, Shiva S, Singh SV (2011) Withaferin A-induced apoptosis in human breast cancer cells is mediated by reactive oxygen species. PLoS One 6:e23354

    PubMed  Article  CAS  Google Scholar 

  27. 27.

    Mayola E, Gallerne C, Esposti DD, Martel C, Pervaiz S, Larue L, Debuire B, Lemoine A, Brenner C, Lemaire C (2011) Withaferin A induces apoptosis in human melanoma cells through generation of reactive oxygen species and down-regulation of Bcl-2. Apoptosis 16:1014–1027

    PubMed  Article  CAS  Google Scholar 

  28. 28.

    Widodo N, Priyandoko D, Shah N, Wadhwa R, Kaul SC (2010) Selective killing of cancer cells by Ashwagandha leaf extract and its component Withanone involves ROS signaling. PLoS One 5:e13536

    PubMed  Article  Google Scholar 

  29. 29.

    Malik F, Kumar A, Bhushan S, Khan S, Bhatia A, Suri KA, Qazi GN, Singh J (2007) Reactive oxygen species generation and mitochondrial dysfunction in the apoptotic cell death of human myeloid leukemia HL-60 cells by a dietary compound withaferin A with concomitant protection by N-acetyl cysteine. Apoptosis 12:2115–2133

    PubMed  Article  CAS  Google Scholar 

  30. 30.

    Samadi AK, Zhang X, Mukerji R, Donnelly AC, Blagg BS, Cohen MS (2011) A novel C-terminal HSP90 inhibitor KU135 induces apoptosis and cell cycle arrest in melanoma cells. Cancer Lett 312:158–167

    PubMed  Article  CAS  Google Scholar 

  31. 31.

    Puputti M, Tynninen O, Sihto H, Blom T, Maenpaa H, Isola J, Paetau A, Joensuu H, Nupponen NN (2006) Amplification of KIT, PDGFRA, VEGFR2, and EGFR in gliomas. Mol Cancer Res 4:927–934

    PubMed  Article  CAS  Google Scholar 

  32. 32.

    Wullich B, Muller HW, Fischer U, Zang KD, Meese E (1993) Amplified met gene linked to double minutes in human glioblastoma. Eur J Cancer 29A:1991–1995

    PubMed  Article  CAS  Google Scholar 

  33. 33.

    Berezowska S, Schlegel J (2011) Targeting ErbB receptors in high-grade glioma. Curr Pharm Des 17:2468–2487

    PubMed  Article  CAS  Google Scholar 

  34. 34.

    Potti A, Forseen SE, Koka VK, Pervez H, Koch M, Fraiman G, Mehdi SA, Levitt R (2004) Determination of HER-2/neu overexpression and clinical predictors of survival in a cohort of 347 patients with primary malignant brain tumors. Cancer Invest 22:537–544

    PubMed  Article  CAS  Google Scholar 

  35. 35.

    Guessous F, Zhang Y, diPierro C, Marcinkiewicz L, Sarkaria J, Schiff D, Buchanan S, Abounader R (2010) An orally bioavailable c-Met kinase inhibitor potently inhibits brain tumor malignancy and growth. Anticancer Agents Med Chem 10:28–35

    PubMed  Article  CAS  Google Scholar 

  36. 36.

    Tew KD, Townsend DM (2011) Redox platforms in cancer drug discovery and development. Curr Opin Chem Biol 15:156–161

    PubMed  Article  CAS  Google Scholar 

  37. 37.

    Antosiewicz J, Ziolkowski W, Kar S, Powolny AA, Singh SV (2008) Role of reactive oxygen intermediates in cellular responses to dietary cancer chemopreventive agents. Planta Med 74:1570–1579

    PubMed  Article  CAS  Google Scholar 

  38. 38.

    Wondrak GT (2009) Redox-directed cancer therapeutics: molecular mechanisms and opportunities. Antioxid Redox Signal 11:3013–3069

    PubMed  Article  CAS  Google Scholar 

  39. 39.

    Deneke SM (2000) Thiol-based antioxidants. Curr Top Cell Regul 36:151–180

    PubMed  Article  CAS  Google Scholar 

  40. 40.

    Ansari N, Khodagholi F, Amini M (2011) 2-Ethoxy-4,5-diphenyl-1,3-oxazine-6-one activates the Nrf2/HO-1 axis and protects against oxidative stress-induced neuronal death. Eur J Pharmacol 658:84–90

    PubMed  Article  CAS  Google Scholar 

  41. 41.

    Qiao S, Lamore SD, Cabello CM, Lesson JL, Munoz-Rodriguez JL, Wondrak GT (2012) Thiostrepton is an inducer of oxidative and proteotoxic stress that impairs viability of human melanoma cells but not primary melanocytes. Biochem Pharmacol 83:12

    Article  Google Scholar 

  42. 42.

    Laurent A, Nicco C, Chereau C, Goulvestre C, Alexandre J, Alves A, Levy E, Goldwasser F, Panis Y, Soubrane O, Weill B, Batteux F (2005) Controlling tumor growth by modulating endogenous production of reactive oxygen species. Cancer Res 65:948–956

    PubMed  CAS  Google Scholar 

  43. 43.

    Cabello CM, Bair WB 3rd, Wondrak GT (2007) Experimental therapeutics: targeting the redox Achilles heel of cancer. Curr Opin Investig Drugs 8:1022–1037

    PubMed  CAS  Google Scholar 

  44. 44.

    Xu W, Trepel J, Neckers L (2011) Ras, ROS and proteotoxic stress: a delicate balance. Cancer Cell 20:281–282

    PubMed  Article  CAS  Google Scholar 

  45. 45.

    Au Q, Zhang Y, Barber JR, Ng SC, Zhang B (2009) Identification of inhibitors of HSF1 functional activity by high-content target-based screening. J Biomol Screen 14:1165–1175

    PubMed  Article  CAS  Google Scholar 

  46. 46.

    Hu Y, Mivechi NF (2011) Promotion of heat shock factor Hsf1 degradation via adaptor protein filamin A-interacting protein 1-like (FILIP-1L). J Biol Chem 286:31397–31408

    PubMed  Article  CAS  Google Scholar 

  47. 47.

    Evans SM, Judy KD, Dunphy I, Jenkins WT, Hwang WT, Nelson PT, Lustig RA, Jenkins K, Magarelli DP, Hahn SM, Collins RA, Grady MS, Koch CJ (2004) Hypoxia is important in the biology and aggression of human glial brain tumors. Clin Cancer Res 10:8177–8184

    PubMed  Article  CAS  Google Scholar 

  48. 48.

    Evans SM, Judy KD, Dunphy I, Jenkins WT, Nelson PT, Collins R, Wileyto EP, Jenkins K, Hahn SM, Stevens CW, Judkins AR, Phillips P, Geoerger B, Koch CJ (2004) Comparative measurements of hypoxia in human brain tumors using needle electrodes and EF5 binding. Cancer Res 64:1886–1892

    PubMed  Article  CAS  Google Scholar 

  49. 49.

    Lee JJ, Kim BC, Park MJ, Lee YS, Kim YN, Lee BL, Lee JS (2011) PTEN status switches cell fate between premature senescence and apoptosis in glioma exposed to ionizing radiation. Cell Death Differ 18:666–677

    PubMed  Article  CAS  Google Scholar 

  50. 50.

    Koul D (2008) PTEN signaling pathways in glioblastoma. Cancer Biol Ther 7:1321–1325

    PubMed  Article  CAS  Google Scholar 

  51. 51.

    Lino MM, Merlo A (2011) PI3Kinase signaling in glioblastoma. J Neurooncol 103:417–427

    PubMed  Article  CAS  Google Scholar 

  52. 52.

    Nogueira V, Park Y, Chen CC, Xu PZ, Chen ML, Tonic I, Unterman T, Hay N (2008) Akt determines replicative senescence and oxidative or oncogenic premature senescence and sensitizes cells to oxidative apoptosis. Cancer Cell 14:458–470

    PubMed  Article  CAS  Google Scholar 

  53. 53.

    Mihaylova MM, Shaw RJ (2011) The AMPK signalling pathway coordinates cell growth, autophagy and metabolism. Nat Cell Biol 13:1016–1023

    PubMed  Article  CAS  Google Scholar 

  54. 54.

    Zhang WB, Wang Z, Shu F, Jin YH, Liu HY, Wang QJ, Yang Y (2010) Activation of AMP-activated protein kinase by temozolomide contributes to apoptosis in glioblastoma cells via p53 activation and mTORC1 inhibition. J Biol Chem 285:40461–40471

    PubMed  Article  CAS  Google Scholar 

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We would like to thank Dr. Jann Sarkaria of the Mayo Clinic (Rochester, MN) and Dr. John Ohlfest of the University of Minnesota (Minneapolis, MN) for generously providing the cell lines utilized. We would also like to thank the KUMC flow core facility for utilization of its resources as 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

Conflict of interest

None declared

Role of funding sources

This work was made possible by grant support from the National Institutes of Health (NIH-COBRE P20 RR015563 P.I. B. Timmermann), the Institute for Advancing Medical Innovation (PI: MS Cohen), and a University of Kansas Cancer Center, Summer Student Training Program grant (PT Grogan).

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Correspondence to Mark S. Cohen.

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Grogan, P.T., Sleder, K.D., Samadi, A.K. et al. Cytotoxicity of withaferin A in glioblastomas involves induction of an oxidative stress-mediated heat shock response while altering Akt/mTOR and MAPK signaling pathways. Invest New Drugs 31, 545–557 (2013).

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  • Withaferin A
  • Glioblastoma multiforme
  • Oxidative stress
  • Heat shock response
  • Akt/mTOR pathway
  • MAPK pathway