Skip to main content

Advertisement

SpringerLink
Log in

ACT001 modulates the NF-κB/MnSOD/ROS axis by targeting IKKβ to inhibit glioblastoma cell growth

  • Original Article
  • Published:
Journal of Molecular Medicine Aims and scope Submit manuscript

Abstract

Glioblastomas are high-grade brain tumors with poor prognoses, and new therapeutic approaches for these tumors are critically needed. This study revealed the underlying mechanisms of a new orphan drug, ACT001, that is currently in clinical trials for the treatment of advanced glioblastoma in Australia and China. ACT001 significantly suppressed glioma cell proliferation and induced apoptosis and cell cycle arrest in vitro, as determined by Cell Counting Kit-8 assays and flow cytometry. In addition, U-118 MG cells with high expression of p-IKKβ were sensitive to ACT001. Changes in the oxidative stress pathway in U-118 MG cells were detected with the isobaric tags for relative and absolute quantitation (iTRAQ) method. We further verified that ACT001 elevated the levels of reactive oxygen species (ROS) by regulating NF-κB-targeted MnSOD. ACT001 markedly inhibited NF-κB activation by directly binding IKKβ and inhibiting its phosphorylation. Overexpression of IKKβ markedly attenuated the changes in MnSOD and NOX1, indicating that ACT001 increased the levels of ROS by reducing the protein expression of p-IKKβ. Furthermore, ACT001 reduced cyclin B1/CDC2 expression and triggered G2/M phase arrest by increasing ROS production. ACT001 also upregulated the expression of Bax and Bim and induced apoptosis in a ROS-dependent manner. ACT001 effectively suppressed the growth of U-118 MG tumors in BALB/c nude mice and GL-261-luciferase tumors in C57BL/6 J mice. Finally, ACT001 downregulated the expression of p-p65, MnSOD, cyclin B1, CDC2, and Ki67 in U-118 MG tumor tissues. Patients with activated NF-κB signaling should thus be given priority for enrollment in future phase II clinical trials.

Key messages

  • ACT001 directly bind to IKKβ and inhibited its phosphorylation.

  • The inhibition of p-IKKβ induced the generation of ROS.

  • ACT001 promoted the generation of ROS by regulating MnSOD expression to induce G2/M phase arrest.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7

Similar content being viewed by others

Abbreviations

GBM:

Glioblastoma

CCK-8:

Cell Counting Kit-8

iTRAQ:

Isobaric tags for relative and absolute quantitation

ROS:

Reactive oxygen species

NF-κB:

Nuclear factor-κB

MnSOD:

Manganese superoxide dismutase

NAC:

N-acetylcysteine

PI:

Propidium iodide

PTL:

Parthenolide

SQLs:

Sesquiterpene lactones

MCL:

Micheliolide

FCM:

Flow cytometry

VCR:

Vincristine

IAM:

Iodoacetamide

DCFH-DA:

Dichlorodihydrofluorescein diacetate.

References

  1. Weller M, Wick W, Aldape K, Brada M, Berger M, Pfister SM et al (2015) Glioma. Nature reviews Disease primers 1:15017

    Article  PubMed  Google Scholar 

  2. Chinot OL, Wick W, Mason W, Henriksson R, Saran F, Nishikawa R, Carpentier AF, Hoang-Xuan K, Kavan P, Cernea D, Brandes AA, Hilton M, Abrey L, Cloughesy T (2014) Bevacizumab plus radiotherapy-temozolomide for newly diagnosed glioblastoma. N Engl J Med 370:709–722

    Article  CAS  PubMed  Google Scholar 

  3. 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, European Organisation for Research and Treatment of Cancer Brain Tumour and Radiation Oncology Groups, National Cancer Institute of Canada Clinical Trials Group (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

    Article  PubMed  CAS  Google Scholar 

  4. Stupp R, Mason WP, van den Bent MJ, Weller M, Fisher B, Taphoorn MJ et al (2005) Radiotherapy plus concomitant and adjuvant temozolomide for glioblastoma. N Engl J Med 352:987–996

    Article  CAS  PubMed  Google Scholar 

  5. Mehta M, Wen P, Nishikawa R, Reardon D, Peters K (2017) Critical review of the addition of tumor treating fields (TTFields) to the existing standard of care for newly diagnosed glioblastoma patients. Crit Rev Oncol Hematol 111:60–65

    Article  PubMed  CAS  Google Scholar 

  6. Gilbert MR, Dignam JJ, Armstrong TS, Wefel JS, Blumenthal DT, Vogelbaum MA, Colman H, Chakravarti A, Pugh S, Won M, Jeraj R, Brown PD, Jaeckle KA, Schiff D, Stieber VW, Brachman DG, Werner-Wasik M, Tremont-Lukats IW, Sulman EP, Aldape KD, Curran WJ Jr, Mehta MP (2014) A randomized trial of bevacizumab for newly diagnosed glioblastoma. N Engl J Med 370:699–708

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  7. Weller M, van den Bent M, Hopkins K, Tonn JC, Stupp R, Falini A et al (2014) EANO guideline for the diagnosis and treatment of anaplastic gliomas and glioblastoma. Lancet Oncol 15:e395–e403

    Article  PubMed  Google Scholar 

  8. Sanai N, Polley MY, McDermott MW, Parsa AT, Berger MS (2011) An extent of resection threshold for newly diagnosed glioblastomas. J Neurosurg 115:3–8

    Article  PubMed  Google Scholar 

  9. Klein M (2012) Neurocognitive functioning in adult WHO grade II gliomas: impact of old and new treatment modalities. Neuro-oncology 14(Suppl 4):iv17-24

    Article  PubMed  Google Scholar 

  10. Panetta JC, Kirstein MN, Gajjar AJ, Nair G, Fouladi M, Stewart CF (2003) A mechanistic mathematical model of temozolomide myelosuppression in children with high-grade gliomas. Math Biosci 186:29–41

    Article  PubMed  Google Scholar 

  11. Singhal N, Selva-Nayagam S, Brown MP (2007) Prolonged and severe myelosuppression in two patients after low-dose temozolomide treatment-case study and review of literature. J Neuro-Oncol 85:229–230

    Article  CAS  Google Scholar 

  12. Sylvester RK, Steen P, Tate JM, Mehta M, Petrich RJ, Berg A et al (2011) Temozolomide-induced severe myelosuppression: analysis of clinically associated polymorphisms in two patients. Anti-Cancer Drugs 22:104–110

    Article  PubMed  CAS  Google Scholar 

  13. Taphoorn MJ, Stupp R, Coens C, Osoba D, Kortmann R, van den Bent MJ, Mason W, Mirimanoff RO, Baumert BG, Eisenhauer E, Forsyth P, Bottomley A, European Organisation for Research and Treatment of Cancer Brain Tumour Group, EORTC Radiotherapy Group, National Cancer Institute of Canada Clinical Trials Group (2005) Health-related quality of life in patients with glioblastoma: a randomised controlled trial. Lancet Oncol 6:937–944

    Article  PubMed  Google Scholar 

  14. Chang S, Zhang P, Cairncross JG, Gilbert MR, Bahary JP, Dolinskas CA, Chakravarti A, Aldape KD, Bell EH, Schiff D, Jaeckle K, Brown PD, Barger GR, Werner-Wasik M, Shih H, Brachman D, Penas-Prado M, Robins HI, Belanger K, Schultz C, Hunter G, Mehta M (2017) Phase III randomized study of radiation and temozolomide versus radiation and nitrosourea therapy for anaplastic astrocytoma: results of NRG Oncology RTOG 9813. Neuro-oncology. 19:252–258

    Article  PubMed  CAS  Google Scholar 

  15. Morgan MJ, Liu ZG (2011) Crosstalk of reactive oxygen species and NF-kappaB signaling. Cell Res 21:103–115

    Article  PubMed  CAS  Google Scholar 

  16. Cahill KE, Morshed RA, Yamini B (2016) Nuclear factor-kappaB in glioblastoma: insights into regulators and targeted therapy. Neuro-oncology. 18:329–339

    Article  PubMed  CAS  Google Scholar 

  17. Karin M (2006) Nuclear factor-kappaB in cancer development and progression. Nature. 441:431–436

    Article  PubMed  CAS  Google Scholar 

  18. Puliyappadamba VT, Hatanpaa KJ, Chakraborty S, Habib AA (2014) The role of NF-kappaB in the pathogenesis of glioma. Mol Cell Oncol 1:e963478

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  19. Bredel M, Bredel C, Juric D, Duran GE, Yu RX, Harsh GR, Vogel H, Recht LD, Scheck AC, Sikic BI (2006) Tumor necrosis factor-alpha-induced protein 3 as a putative regulator of nuclear factor-kappaB-mediated resistance to O6-alkylating agents in human glioblastomas. J Clin Oncol : Off J Am Soc Clin Oncol 24:274–287

    Article  CAS  Google Scholar 

  20. Lavon I, Fuchs D, Zrihan D, Efroni G, Zelikovitch B, Fellig Y, Siegal T (2007) Novel mechanism whereby nuclear factor kappaB mediates DNA damage repair through regulation of O(6)-methylguanine-DNA-methyltransferase. Cancer Res 67:8952–8959

    Article  PubMed  CAS  Google Scholar 

  21. Weaver KD, Yeyeodu S, Cusack JC Jr, Baldwin AS Jr, Ewend MG (2003) Potentiation of chemotherapeutic agents following antagonism of nuclear factor kappa B in human gliomas. J Neuro-Oncol 61:187–196

    Article  Google Scholar 

  22. Djavaheri-Mergny M, Javelaud D, Wietzerbin J, Besancon F (2004) NF-kappaB activation prevents apoptotic oxidative stress via an increase of both thioredoxin and MnSOD levels in TNFalpha-treated Ewing sarcoma cells. FEBS Lett 578:111–115

    Article  PubMed  CAS  Google Scholar 

  23. Kairisalo M, Korhonen L, Blomgren K, Lindholm D (2007) X-linked inhibitor of apoptosis protein increases mitochondrial antioxidants through NF-kappaB activation. Biochem Biophys Res Commun 364:138–144

    Article  PubMed  CAS  Google Scholar 

  24. Dixon SJ, Stockwell BR (2014) The role of iron and reactive oxygen species in cell death. Nat Chem Biol 10:9–17

    Article  PubMed  CAS  Google Scholar 

  25. Jin P, Madieh S, Augsburger LL (2007) The solution and solid state stability and excipient compatibility of parthenolide in feverfew. AAPS PharmSciTech 8:E105

    Article  PubMed  Google Scholar 

  26. Curry EA 3rd, Murry DJ, Yoder C, Fife K, Armstrong V, Nakshatri H et al (2004) Phase I dose escalation trial of feverfew with standardized doses of parthenolide in patients with cancer. Investig New Drugs 22:299–305

    Article  CAS  Google Scholar 

  27. Zhang Q, Lu Y, Ding Y, Zhai J, Ji Q, Ma W et al (2012) Guaianolide sesquiterpene lactones, a source to discover agents that selectively inhibit acute myelogenous leukemia stem and progenitor cells. J Med Chem 55:8757–8769

    Article  PubMed  CAS  Google Scholar 

  28. Yu T, Li G, Dong S, Liu P, Zhang J, Zhao B (2016) Proteomic analysis of maize grain development using iTRAQ reveals temporal programs of diverse metabolic processes. BMC Plant Biol 16:241

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  29. Jain R, Kulkarni P, Dhali S, Rapole S, Srivastava S (2015) Quantitative proteomic analysis of global effect of LLL12 on U87 cell's proteome: an insight into the molecular mechanism of LLL12. J Proteome 113:127–142

    Article  CAS  Google Scholar 

  30. Ji Q, Ding YH, Sun Y, Zhang Y, Gao HE, Song HN, Yang M, Liu XL, Zhang ZX, Li YH, Gao YD (2016) Antineoplastic effects and mechanisms of micheliolide in acute myelogenous leukemia stem cells. Oncotarget. 7:65012–65023

    Article  PubMed  PubMed Central  Google Scholar 

  31. Dong T, Li C, Wang X, Dian L, Zhang X, Li L et al (2015) Ainsliadimer A selectively inhibits IKKalpha/beta by covalently binding a conserved cysteine. Nat Commun 6:6522

    Article  PubMed  CAS  Google Scholar 

  32. Kwok BH, Koh B, Ndubuisi MI, Elofsson M, Crews CM (2001) The anti-inflammatory natural product parthenolide from the medicinal herb Feverfew directly binds to and inhibits IkappaB kinase. Chem Biol 8:759–766

    Article  PubMed  CAS  Google Scholar 

  33. Li J, Li S, Guo J, Li Q, Long J, Ma C et al (2018) Natural product micheliolide (MCL) irreversibly activates pyruvate kinase M2 and suppresses leukemia. J Med Chem

  34. Xu G, Lo YC, Li Q, Napolitano G, Wu X, Jiang X et al (2011) Crystal structure of inhibitor of kappaB kinase beta. Nature. 472:325–330

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  35. Zhou X, Liu J, Zhang J, Wei Y, Li H (2018) Flubendazole inhibits glioma proliferation by G2/M cell cycle arrest and pro-apoptosis. Cell Death Dis 4:18

    Article  CAS  Google Scholar 

  36. Kawabe T (2004) G2 checkpoint abrogators as anticancer drugs. Mol Cancer Ther 3:513–519

    PubMed  CAS  Google Scholar 

  37. Kreuger MR, Grootjans S, Biavatti MW, Vandenabeele P, D'Herde K (2012) Sesquiterpene lactones as drugs with multiple targets in cancer treatment: focus on parthenolide. Anti-Cancer Drugs 23:883–896

    Article  PubMed  CAS  Google Scholar 

  38. Ghantous A, Gali-Muhtasib H, Vuorela H, Saliba NA, Darwiche N (2010) What made sesquiterpene lactones reach cancer clinical trials? Drug Discov Today 15:668–678

    Article  PubMed  CAS  Google Scholar 

  39. Chaturvedi D, Goswami A, Saikia PP, Barua NC, Rao PG (2010) Artemisinin and its derivatives: a novel class of anti-malarial and anti-cancer agents. Chem Soc Rev 39:435–454

    Article  PubMed  CAS  Google Scholar 

  40. Crespo-Ortiz MP, Wei MQ (2012) Antitumor activity of artemisinin and its derivatives: from a well-known antimalarial agent to a potential anticancer drug. J Biomed Biotechnol 2012:247597

    Article  PubMed  CAS  Google Scholar 

  41. Jansen FH, Adoubi I, J CK, T DEC, Jansen N, Tschulakow A et al (2011) First study of oral Artenimol-R in advanced cervical cancer: clinical benefit, tolerability and tumor markers. Anticancer Res 31:4417–4422

    PubMed  CAS  Google Scholar 

  42. Zunino SJ, Ducore JM, Storms DH (2007) Parthenolide induces significant apoptosis and production of reactive oxygen species in high-risk pre-B leukemia cells. Cancer Lett 254:119–127

    Article  PubMed  CAS  Google Scholar 

  43. D'Anneo A, Carlisi D, Lauricella M, Puleio R, Martinez R, Di Bella S et al (2013) Parthenolide generates reactive oxygen species and autophagy in MDA-MB231 cells. A soluble parthenolide analogue inhibits tumour growth and metastasis in a xenograft model of breast cancer. Cell Death Dis 4:e891

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  44. Mathema VB, Koh YS, Thakuri BC, Sillanpaa M (2012) Parthenolide, a sesquiterpene lactone, expresses multiple anti-cancer and anti-inflammatory activities. Inflammation. 35:560–565

    Article  PubMed  CAS  Google Scholar 

  45. Guzman ML, Rossi RM, Neelakantan S, Li X, Corbett CA, Hassane DC, Becker MW, Bennett JM, Sullivan E, Lachowicz JL, Vaughan A, Sweeney CJ, Matthews W, Carroll M, Liesveld JL, Crooks PA, Jordan CT (2007) An orally bioavailable parthenolide analog selectively eradicates acute myelogenous leukemia stem and progenitor cells. Blood. 110:4427–4435

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  46. An Y, Guo W, Li L, Xu C, Yang D, Wang S et al (2015) Micheliolide derivative DMAMCL inhibits glioma cell growth in vitro and in vivo. PloS one 10:e0116202

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  47. Oka D, Nishimura K, Shiba M, Nakai Y, Arai Y, Nakayama M et al (2007) Sesquiterpene lactone parthenolide suppresses tumor growth in a xenograft model of renal cell carcinoma by inhibiting the activation of NF-kappaB. Int J Cancer 120:2576–2581

    Article  PubMed  CAS  Google Scholar 

  48. Huska JD, Lamb HM, Hardwick JM (1877) Overview of BCL-2 family proteins and therapeutic potentials. Methods Mol Biol 2019:1–21

    Google Scholar 

  49. De Stefano D, Nicolaus G, Maiuri MC, Cipolletta D, Galluzzi L, Cinelli MP et al (2009) NF-kappaB blockade upregulates Bax, TSP-1, and TSP-2 expression in rat granulation tissue. J Mol Med 87:481–492

    Article  PubMed  CAS  Google Scholar 

  50. Eberstal S, Sanden E, Fritzell S, Darabi A, Visse E, Siesjo P (2014) Intratumoral COX-2 inhibition enhances GM-CSF immunotherapy against established mouse GL261 brain tumors. Int J Cancer 134:2748–2753

    Article  PubMed  CAS  Google Scholar 

Download references

Funding

This work was supported by the National Natural Science Foundation of China (No. 31770974 to Z.Z.W., No. 81573282 to Y.C. and No. 81872063 to X.J. Y), the Foundation of Tianjin Medical University (No. 2011KY24 to Z.H. J), and the Natural Science Foundation of Tianjin (No. 17JCYBJC24600 to L.Q. Z and No. 14JCYBJC24500 to Y.P. Z).

Author information

Authors and Affiliations

  1. State Key Laboratory of Medicinal Chemical Biology, College of Pharmacy and Tianjin Key Laboratory of Molecular Drug Research, Nankai University, Tianjin, 300353, China

    Qiuying Li, Xin Hao, Weizhi Ge, Yue Chen & Yahui Ding

  2. College of Life Sciences, Nankai University, Tianjin, 300353, China

    Yu Sun, Liqing Zhao & Zhenzhou Wu

  3. Henan Key Laboratory of Immunology and Targeted Drugs, Research Center for Molecular Oncology and Functional Nucleic Acids, Henan Collaborative Innovation Center of Molecular Diagnosis and Laboratory Medicine, School of Laboratory Medicine, Xinxiang Medical University, Xinxiang, 453003, Henan, China

    Bowen Liu

  4. Department of Neurosurgery, Tianjin Medical University General Hospital, Tianjin, 300052, China

    Jiabo Li & Xuejun Yang

  5. Accendatech Co., Ltd., Tianjin, 300384, China

    Xuemei Zhang, Shiqi Bao, Jianmiao Gong & Chuanjiang Qiu

  6. Key Laboratory of Hormones and Development (Ministry of Health), Tianjin Key Laboratory of Metabolic Diseases, Tianjin Metabolic Diseases Hospital & Tianjin Institute of Endocrinology, Tianjin Medical University, Tianjin, 300070, China

    Zhenhuan Jiang

  7. People’s Liberation Army No. 254 Hospital, Tianjin, 300142, China

    Yapu Zhao

Authors
  1. Qiuying Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Yu Sun
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Bowen Liu
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Jiabo Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Xin Hao
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Weizhi Ge
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Xuemei Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Shiqi Bao
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Jianmiao Gong
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Zhenhuan Jiang
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Chuanjiang Qiu
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. Liqing Zhao
    View author publications

    You can also search for this author in PubMed Google Scholar

  13. Yapu Zhao
    View author publications

    You can also search for this author in PubMed Google Scholar

  14. Yue Chen
    View author publications

    You can also search for this author in PubMed Google Scholar

  15. Xuejun Yang
    View author publications

    You can also search for this author in PubMed Google Scholar

  16. Yahui Ding
    View author publications

    You can also search for this author in PubMed Google Scholar

  17. Zhenzhou Wu
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

Zhenzhou Wu, Qiuying Li, Yahui Ding, and Yue Chen designed the study. Zhenzhou Wu, Yue Chen, Xuejun Yang, Zhenhuan Jiang, Liqing Zhao, and Yapu Zhao provided material support. Jiabo Li guided the in situ animal model. Qiuying Li, Xuemei Zhang, and Yahui Ding polished the manuscript. Qiuying Li performed experiments, analyzed the data, and drafted the manuscript. Bowen Liu, Yu Sun, Shiqi Bao, Weizhi Ge, Xuemei Zhang, Shiqi Bao, and Jianmiao Gong performed parts of the experiments. All authors read and approved the final manuscript.

Corresponding authors

Correspondence to Yue Chen, Xuejun Yang, Yahui Ding or Zhenzhou Wu.

Ethics declarations

Conflict of interest

The authors declare that they have no conflict of interest.

Additional information

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Electronic supplementary material

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Li, Q., Sun, Y., Liu, B. et al. ACT001 modulates the NF-κB/MnSOD/ROS axis by targeting IKKβ to inhibit glioblastoma cell growth. J Mol Med 98, 263–277 (2020). https://doi.org/10.1007/s00109-019-01839-0

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00109-019-01839-0

Keywords

Search

Navigation