The sodium pump α1 subunit regulates bufalin sensitivity of human glioblastoma cells through the p53 signaling pathway

Abstract

Bufalin is the primary component of the traditional Chinese medicine “Chan Su,” which has been widely used for cancer treatment at oncology clinics in certain countries. Evidence suggests that this compound possesses potent antitumor activities, although the exact molecular mechanism(s) require further elucidation. Therefore, this study aimed to further clarify the in vitro and in vivo antiglioma effects of bufalin and the molecular mechanism underlying the regulation of drug sensitivity. The anticancer effects of bufalin were determined by colony formation assays, apoptosis assays, and cellular redox state tests of glioma cells. Confocal microscopy was performed to determine the expression changes of the DNA damage biomarker γ-H2AX and the nuclear translocation of p53 in glioma cells. Western blotting and RT-PCR were used to detect the protein and gene expression levels, respectively. Here, we report that bufalin induced glioblastoma cell apoptosis and oxidative stress and triggered DNA damage. The critical roles of the sodium pump α1 subunit (ATP1A1) in mediating the XPO1-targeted anticancer effect of bufalin in human glioma were further confirmed. Mechanistic studies confirmed the important roles of Src and p53 signaling in mediating bufalin-induced apoptosis. Importantly, bufalin also inhibited the growth of glioma xenografts. In conclusion, our study indicated that therapies targeting the ATP1A1 and p53 signaling-mediated mitochondrial apoptotic pathways regulated by bufalin might be potential treatments for human glioma, and these findings will provide molecular bases for developing bufalin into a drug candidate for the treatment of malignant glioma.

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

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

References

  1. Ahmed A, Pitt B, Rahimtoola SH, Waagstein F, White M, Love TE, et al. Effects of digoxin at low serum concentrations on mortality and hospitalization in heart failure: a propensity-matched study of the DIG trial. Int J Cardiol. 2008;123(2):138–46.

    PubMed  Google Scholar 

  2. Bai Y, Wu J, Li D, Morgan EE, Liu J, Zhao X, et al. Differential roles of caveolin-1 in ouabain-induced Na+/K+-ATPase cardiac signaling and contractility. Physiol Genomics. 2016;48(10):739–48.

    CAS  PubMed  PubMed Central  Google Scholar 

  3. Clarke PR, Zhang C. Spatial and temporal coordination of mitosis by Ran GTPase. Nat Rev Mol Cell Biol. 2008;9(6):464–77.

    CAS  PubMed  Google Scholar 

  4. Eskiocak U, Ramesh V, Gill JG, Zhao Z, Yuan SW, Wang M, et al. Synergistic effects of ion transporter and MAP kinase pathway inhibitors in melanoma. Nat Commun. 2016;7:12336.

    CAS  PubMed  PubMed Central  Google Scholar 

  5. Fujii E, Inada Y, Kakoki M, Nishimura N, Endo S, Fujiwara S, et al. Bufalin induces DNA damage response under hypoxic condition in myeloma cells. Oncol Lett. 2018;15(5):6443–9.

    PubMed  PubMed Central  Google Scholar 

  6. Fukuda M, Asano S, Nakamura T, Adachi M, Yoshida M, Yanagida M, et al. CRM1 is responsible for intracellular transport mediated by the nuclear export signal. Nature. 1997;390(6657):308–11.

    CAS  PubMed  Google Scholar 

  7. Garcia DG, de Castro-Faria-Neto HC, da Silva CI, de Souza e Souza KF, Gonçalves-de-Albuquerque CF, Silva AR, et al. Na/K-ATPase as a target for anticancer drugs: studies with perillyl alcohol. Mol Cancer. 2015;14:105.

    PubMed  PubMed Central  Google Scholar 

  8. Gill S, Gill R, Wicks D, Despotovski S, Liang D. Development of an HTS assay for Na+, K+-ATPase using nonradioactive rubidium ion uptake. Assay Drug Dev Technol. 2004;2(5):535–42.

    CAS  PubMed  Google Scholar 

  9. Gjesdal K, Feyzi J, Olsson SB. Digitalis: a dangerous drug in atrial fibrillation? An analysis of the SPORTIF III and V data. Heart. 2008;94(2):191–6.

    CAS  PubMed  Google Scholar 

  10. Grasso CS, Wu YM, Robinson DR, Cao X, Dhanasekaran SM, Khan AP, et al. The mutational landscape of lethal castration resistant prostate cancer. Nature. 2012;487(7406):239–43.

    CAS  PubMed  PubMed Central  Google Scholar 

  11. Hanke JH, Gardner JP, Dow RL, Changelian PS, Brissette WH, Weringer EJ, et al. Discovery of a novel, potent, and Src family-selective tyrosine kinase inhibitor. Study of Lck- and FynT-dependent T cell activation. J Biol Chem. 1996;271(2):695–701.

    CAS  PubMed  PubMed Central  Google Scholar 

  12. Haux J, Klepp O, Spigset O, Tretli S. Digitoxin medication and cancer; case control and internal dose-response studies. BMC Cancer. 2001;1:11.

    CAS  PubMed  PubMed Central  Google Scholar 

  13. Houtgraff JH, Versmissen J, van der Giessen WJ. A concise review of DNA damage check points and repair in mammalian cells. Cardiovasc Revasc Med. 2006;7(3):165–72.

    Google Scholar 

  14. Huang WY, Yue L, Qiu WS, Wang LW, Zhou XH, Sun YJ. Prognostic value of CRM1 in pancreas cancer. Clin Invest Med. 2009;32(6):E315.

    PubMed  Google Scholar 

  15. Jansson K, Nguyen AN, Magenheimer BS, Reif GA, Aramadhaka LR, Bello-Reuss E, et al. Endogenous concentrations of ouabain act as a cofactor to stimulate fluid secretion and cyst growth of in vitro ADPKD models via cAMP and EGFR-Src-MEK pathways. Am J Physiol Renal Physiol. 2012;303(7):F982–90.

    CAS  PubMed  PubMed Central  Google Scholar 

  16. Kau TR, Way JC, Silver PA. Nuclear transport and cancer: from mechanism to intervention. Nat Rev Cancer. 2004;4(2):106–17.

    CAS  PubMed  Google Scholar 

  17. Kurz EU, Lees-Miller SP. DNA damage-induced activation of ATM and ATM-dependent signaling pathways. DNA Repair (Amst). 2004;3(8–9):889–900.

    CAS  Google Scholar 

  18. Lan YL, Wang X, Lou JC, Xing JS, Yu ZL, Wang H, et al. Bufalin inhibits glioblastoma growth by promoting proteasomal degradation of the Na+/K+-ATPase α1 subunit. Biomed Pharmacother. 2018;103:204–15.

    CAS  PubMed  Google Scholar 

  19. Li Z, Zhang Z, Xie JX, Li X, Tian J, Cai T, et al. Na/K-ATPase mimetic pNaKtide peptide inhibits the growth of human cancer cell. J Biol Chem. 2011;286(37):32394–403.

    CAS  PubMed  PubMed Central  Google Scholar 

  20. Liang M, Cai T, Tian J, Qu W, Xie ZJ. Functional characterization of Src-interacting Na/K-ATPase using RNA interference assay. J Biol Chem. 2006;281(28):19709–19.

    CAS  PubMed  Google Scholar 

  21. Liu X, Chong Y, Liu H, Han Y, Niu M. Novel reversible selective inhibitor of CRM1 for targeted therapy in ovarian cancer. J Ovarian Res. 2015;8:35.

    PubMed  PubMed Central  Google Scholar 

  22. Liu X, Chong Y, Tu Y, Liu N, Yue C, Qi Z, et al. CRM1/XPO1 is associated with clinical outcome in glioma and represents a therapeutic target by perturbing multiple core pathways. J Hematol Oncol. 2016;9(1):108.

    CAS  PubMed  PubMed Central  Google Scholar 

  23. Mellman I, Nelson WJ. Coordinated protein sorting, targeting and distribution in polarized cells. Nat Rev Mol Cell Biol. 2008;9(11):833–45.

    CAS  PubMed  PubMed Central  Google Scholar 

  24. Mijatovic T, Roland I, Van Quaquebeke E, Nilsson B, Mathieu A, Van Vynckt F, et al. The alpha1 subunit of the sodium pump could represent a novel target to combat nonsmall cell lung cancers. J Pathol. 2007a;212(2):170–9.

    CAS  PubMed  Google Scholar 

  25. Mijatovic T, Van Quaquebeke E, Delest B, Debeir O, Darro F, Kiss R. Cardiotonic steroids on the road to anti-cancer therapy. Biochim Biophys Acta. 2007b;1776(1):32–57.

    CAS  PubMed  Google Scholar 

  26. Mor A, White MA, Fontoura BM. Nuclear trafficking in health and disease. Curr Opin Cell Biol. 2014;28:28–35.

    CAS  PubMed  Google Scholar 

  27. Moreno Y, Banuls L, Katz A, Miklos W, Cimmino A, Tal DM, et al. Hellebrin and its aglycone form hellebrigenin display similar in vitro growth inhibitory effects in cancer cells and binding profiles to the alpha subunits of the Na+/K+-ATPase. Mol Cancer. 2013;12:33.

    Google Scholar 

  28. Newman RA, Yang P, Pawlus AD, Block KI. Cardiac glycosides as novel cancer therapeutic agents. Mol Interv. 2008;8(1):36–49.

    CAS  PubMed  Google Scholar 

  29. Nigg EA. Nucleocytoplasmic transport: signals, mechanisms and regulation. Nature. 1997;386(6627):779–87.

    CAS  PubMed  Google Scholar 

  30. Noske A, Weichert W, Niesporek S, Röske A, Buckendahl AC, Koch I, et al. Expression of the nuclear export protein chromosomal region maintenance/exportin 1/Xpo1 is a prognostic factor inhuman ovarian cancer. Cancer. 2008;112(8):1733–43.

    CAS  PubMed  Google Scholar 

  31. Ogawa H, Shinoda T, Cornelius F, Toyoshima C. Crystal structure of the sodium-potassium pump (Na+, K+-ATPase) with bound potassium and ouabain. Proc Natl Acad Sci U S A. 2009;106(33):13742–7.

    CAS  PubMed  PubMed Central  Google Scholar 

  32. Pan L, Zhang Y, Zhao W, Zhou X, Wang C, Deng F. The cardiac glycoside oleandrin induces apoptosis in human colon cancer cells via the mitochondrial pathway. Cancer Chemother Pharmacol. 2017;80(1):91–100.

    CAS  PubMed  Google Scholar 

  33. Phesse TJ, Myant KB, Cole AM, Ridgway RA, Pearson H, Muncan V, et al. Endogenous c-Myc is essential for p53-induced apoptosis in response to DNA damage in vivo. Cell Death Differ. 2014;21(6):956–66.

    CAS  PubMed  PubMed Central  Google Scholar 

  34. Reinhard L, Tidow H, Clausen MJ, Nissen P. Na(+), K (+)-ATPase as a docking station: protein-protein complexes of the Na(+), K (+)-ATPase. Cell Mol Life Sci. 2013;70:205–22.

    CAS  PubMed  Google Scholar 

  35. Rogakou EP, Nieves-Neira W, Boon C, Pommier Y, Bonner WM. Initiation of DNA fragmentation during apoptosis induces phosphorylation of H2AX histone at serine 139. J Biol Chem. 2000;275(13):9390–5.

    CAS  PubMed  Google Scholar 

  36. Roy A, Kucukural A, Zhang Y. I-TASSER: a unified platform for automated protein structure and function prediction. Nat Protoc. 2010;5(4):725–38.

    CAS  PubMed  PubMed Central  Google Scholar 

  37. Roy S, Kar M, Roy S, Saha A, Padhi S, Banerjee B. Role of β-catenin in cisplatin resistance, relapse and prognosis of head and neck squamous cell carcinoma. Cell Oncol. 2018;41(2):185–200.

    CAS  Google Scholar 

  38. Shen A, Wang Y, Zhao Y, Zou L, Sun L, Cheng C. Expression of XPO 1 in human gliomas and its significance in p27 expression and clinical prognosis. Neurosurgery. 2009;65(1):153–9 discussion 159-60.

    PubMed  Google Scholar 

  39. Shen S, Zhang Y, Wang Z, Liu R, Gong X. Bufalin induces the interplay between apoptosis and autophagy in glioma cells through endoplasmic reticulum stress. Int J Biol Sci. 2014;10(2):212–24.

    CAS  PubMed  PubMed Central  Google Scholar 

  40. Stenkvist B, Bengtsson E, Eriksson O, Holmquist J, Nordin B, Westman-Naeser S. Cardiac glycosides and breast cancer. Lancet. 1979;1(8115):563.

    CAS  PubMed  Google Scholar 

  41. Takai N, Kira N, Ishii T, Yoshida T, Nishida M, Nishida Y, et al. Bufalin, a traditional oriental medicine, induces apoptosis in human cancer cells. Asian Pac J Cancer Prev. 2012;13(1):399–402.

    PubMed  Google Scholar 

  42. Turcato F, Kim P, Barnett A, Jin Y, Scerba M, Casey A, et al. Sequential combined treatment of pifithrin-α and posiphen enhances neurogenesis and functional recovery after stroke. Cell Transplant. 2018;27(4):607–21.

    PubMed  PubMed Central  Google Scholar 

  43. Turner JG, Sullivan DM. CRM1-mediated nuclear export of proteins and drug resistance in cancer. Curr Med Chem. 2008;15(26):2648–55.

    CAS  PubMed  Google Scholar 

  44. van der Watt PJ, Maske CP, Hendricks DT, Parker MI, Denny L, Govender D, et al. The karyopherin proteins, XPO 1 and karyopherin beta1, are overexpressed in cervical cancer and are critical for cancer cell survival and proliferation. Int J Cancer. 2009;124(8):1829–40.

    PubMed  Google Scholar 

  45. Van Maerken T, Rihani A, Van Goethem A, De Paepe A, Speleman F, Vandesompele J. Pharmacologic activation of wild-type p53 by nutlin therapy in childhood cancer. Cancer Lett. 2014;344(2):157–65.

    PubMed  Google Scholar 

  46. Wang Z, Zheng M, Li Z, Li R, Jia L, Xiong X, et al. Cardiac glycosides inhibit p53 synthesis by a mechanism relieved by Src or MAPK inhibition. Cancer Res. 2009;69(16):6556–64.

    CAS  PubMed  PubMed Central  Google Scholar 

  47. Wang J, Xia Y, Zuo Q, Chen T. Molecular mechanisms underlying the antimetastatic activity of bufalin. Mol Clin Oncol. 2018;8(5):631–6.

    CAS  PubMed  PubMed Central  Google Scholar 

  48. Wood LD, Parsons DW, Jones S, Lin J, Sjöblom T, Leary RJ, et al. The genomic landscapes of human breast and colorectal cancers. Science. 2007;318(5853):1108–13.

    CAS  PubMed  Google Scholar 

  49. Wu SH, Wu TY, Hsiao YT, Lin JH, Hsu SC, Hsia TC, et al. Bufalin induces cell death in human lung cancer cells through disruption of DNA damage response pathways. Am J Chin Med. 2014;42(3):729–42.

    CAS  PubMed  Google Scholar 

  50. Xiao Y, Meng C, Lin J, Huang C, Zhang X, Long Y, et al. Ouabain targets the Na+/K+-ATPase α3 isoform to inhibit cancer cell proliferation and induce apoptosis. Oncol Lett. 2017;14(6):6678–84.

    PubMed  PubMed Central  Google Scholar 

  51. Xie Z, Cai T. Na+-K+-ATPase-mediated signal transduction: from protein interaction to cellular function. Mol Interv. 2003;3(3):157–68.

    CAS  PubMed  Google Scholar 

  52. Yatime L, Laursen M, Morth JP, Esmann M, Nissen P, Fedosova NU. Structural insights into the high affinity binding of cardiotonic steroids to the Na+, K+-ATPase. J Struct Biol. 2011;174(2):296–306.

    CAS  PubMed  Google Scholar 

  53. Yee D, Hao C, Cheung HC, Chen HT, Dabbagh L, Hanson J, et al. Effect of radiation on cytokine and cytokine receptor messenger-RNA profiles in p53 wild and mutated human glioblastoma cell lines. Clin Invest Med. 2001;24(2):76–82.

    CAS  PubMed  Google Scholar 

  54. Yu CH, Kan SF, Pu HF, JeaChien E, Wang PS. Apoptotic signaling in bufalin- and cinobufagin-treated androgen-dependent and -independent human prostate cancer cells. Cancer Sci. 2008;99(12):2467–76.

    CAS  PubMed  Google Scholar 

  55. Yuan J, Adamski R, Chen J. Focus on histone variant H2AX: to be or not to be. FEBS Lett. 2010;584(17):3717–24.

    CAS  PubMed  PubMed Central  Google Scholar 

  56. Zhang K, Wang M, Tamayo AT, Shacham S, Kauffman M, Lee J, et al. Novel selective inhibitors of nuclear export CRM1 antagonists for therapy in mantle cell lymphoma. Exp Hematol. 2013;41(1):67–78.e4.

    PubMed  Google Scholar 

Download references

Funding

This work is supported by grants from the National Natural Science Foundation of China (Nos. 81372714, 81672480), Liaoning Provincial Natural Science Foundation of China (No. 201602244), Distinguished Professor Project of Liaoning Province, Special Grant for Translational Medicine, Dalian Medical University (No. 2015002), and Basic Research Projects in Colleges and Universities of Liaoning Province (No. LQ2017033).

Author information

Affiliations

Authors

Contributions

BZ and YD conceived and supervised the study and revised the manuscript; YLL, YJZ, and JCL designed and performed the experiments, analyzed the data, and drafted the manuscript; and JSX, XW, SZ, and BBM collected data and revised the manuscript.

Corresponding authors

Correspondence to Yan Ding or Bo Zhang.

Ethics declarations

Conflict of interest

The authors declare that they have no competing interests.

Additional information

Publisher’s note

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

Electronic supplementary material

ESM 1

(PDF 329 kb)

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Lan, Y., Zou, Y., Lou, J. et al. The sodium pump α1 subunit regulates bufalin sensitivity of human glioblastoma cells through the p53 signaling pathway. Cell Biol Toxicol 35, 521–539 (2019). https://doi.org/10.1007/s10565-019-09462-y

Download citation

Keywords

  • Glioma
  • Bufalin
  • Sodium pump
  • p53
  • Apoptosis