Molecular and Cellular Biochemistry

, Volume 398, Issue 1–2, pp 95–104 | Cite as

Ouabain elicits human glioblastoma cells apoptosis by generating reactive oxygen species in ERK-p66SHC-dependent pathway

  • Xiaofei Yan
  • FenLi Liang
  • Dongmin Li
  • Jin Zheng


Excessive reactive oxygen species (ROS) generation has been implicated as one of main agents in ouabain-induced anticancer effect. Unfortunately, the signaling pathways under it are not very clarified. In the present study, we investigated the molecular mechanism involved in ouabain-induced ROS generation and cell apoptosis on human U373MG and U87MG glioma cells. Ouabain-induced glioblastoma cells apoptosis and increased ROS generation. Clearance ROS by three different ROS scavenger partly, but not totally, reversed ouabain’s effect on cell apoptosis. Ouabain-induced ROS generation was not regulated by calcium overload, reduced nicotinamide adenine dinucleotide phosphate oxidation, but by p66Shc phosphorylation. Ouabain treatment increased p66Shc Ser36 phosphorylation. Knockdown of p66Shc by siRNA significantly inhibited ROS generations in response to ouabain. Ouabain-induced p66Shc phosphorylation through Src/Ras/extracellular signal-regulated kinase signal pathway. Our results uncovered a novel signaling pathway with p66Shc, ouabain-induced ROS generation, and glioblastoma cell apoptosis.


Glioblastoma Ouabain ROS p66Shc Na+/K+-ATPase 



The project was supported by the Fundamental Research Funds for the Central Universities (No.XJTU-HRT-002) and supported by the National Natural Science Foundation of China (No. 81200545).


  1. 1.
    Maher EA, Furnari FB, Bachoo RM, Rowitch DH, Louis DN, Cavenee WK, DePinho RA (2001) Malignant glioma: genetics and biology of a grave matter. Genes Dev 15:1311–1333PubMedCrossRefGoogle Scholar
  2. 2.
    Friesen C, Hormann I, Roscher M, Fichtner I, Alt A, Hilger R, Debatin KM, Miltner E (2014) Opioid receptor activation triggering downregulation of cAMP improves effectiveness of anti-cancer drugs in treatment of glioblastoma. Cell Cycle 13:1560–1570. doi: 10.4161/cc.28493 PubMedCrossRefGoogle Scholar
  3. 3.
    Prassas I, Diamandis EP (2008) Novel therapeutic applications of cardiac glycosides. Nat Rev Drug Discov 7(11):926–935. doi: 10.1038/nrd2682 PubMedCrossRefGoogle Scholar
  4. 4.
    Newman RA, Yang P, Pawlus AD, Block KI (2008) Cardiac glycosides as novel cancer therapeutic agents. Mol Interv 8(1):36–49. doi: 10.1124/mi.8.1.8 PubMedCrossRefGoogle Scholar
  5. 5.
    Lefranc F, Mijatovic T, Kondo Y, Sauvage S, Roland I, Debeir O, Krstic D, Vasic V, Gailly P, Kondo S, Blanco G, Kiss R (2008) Targeting the alpha 1 subunit of the sodium pump to combat glioblastoma cells. Neurosurgery 62(1):211–221. doi: 10.1227/01.NEU.0000311080.43024.0E PubMedCrossRefGoogle Scholar
  6. 6.
    Mijatovic T, Roland I, Van Quaquebeke E, Nilsson B, Mathieu A, Van Vynckt F, Darro F, Blanco G, Facchini V, Kiss R (2007) The alpha1 subunit of the sodium pump could represent a novel target to combat non-small cell lung cancers. J Pathol 212(2):170–179PubMedCrossRefGoogle Scholar
  7. 7.
    Suzuki K, Nakamura K, Kato K, Hamada H, Tsukamoto T (2007) Exploration of target molecules for prostate cancer gene therapy. Prostate 67(11):1163–1173PubMedCrossRefGoogle Scholar
  8. 8.
    Kometiani P, Liu L, Askari A (2005) Digitalis-induced signaling by Na+/K+-ATPase in human breast cancer cells. Mol Pharmacol 67(3):929–936PubMedCrossRefGoogle Scholar
  9. 9.
    McConkey DJ, Lin Y, Nutt LK, Ozel HZ, Newman RA (2000) Cardiac glycosides stimulate Ca2+ increases and apoptosis in androgen-independent, metastatic human prostate adenocarcinoma cells. Cancer Res 60(14):3807–3812PubMedGoogle Scholar
  10. 10.
    Watabe M, Masuda Y, Nakajo S, Yoshida T, Kuroiwa Y, Nakaya K (1996) The cooperative interaction of two different signaling pathways in response to bufalin induces apoptosis in human leukemia U937 cells. J Biol Chem 271(24):14067–14072PubMedCrossRefGoogle Scholar
  11. 11.
    Kulikov A, Eva A, Kirch U, Boldyrev A, Scheiner-Bobis G (2007) Ouabain activates signaling pathways associated with cell death in human neuroblastoma. Biochim Biophys Acta 1768(7):1691–1702PubMedCrossRefGoogle Scholar
  12. 12.
    Joshi AD, Parsons DW, Velculescu VE, Riggins GJ (2011) Sodium ion channel mutations in glioblastoma patients correlate with shorter survival. Mol Cancer 10:171–179Google Scholar
  13. 13.
    Huang YT, Chueh SC, Teng CM, Guh JH (2004) Investigation of ouabain-induced anticancer effect in human androgen-independent prostate cancer PC-3 cells. Biochem Pharmacol 67(4):727–733PubMedCrossRefGoogle Scholar
  14. 14.
    Stenkvist B (1999) Is digitalis a therapy for breast carcinoma? Oncol Rep 6(3):493–496PubMedGoogle Scholar
  15. 15.
    López-Lázaro M (2007) Digitoxin as an anticancer agent with selectivity for cancer cells: possible mechanisms involved. Expert Opin Ther Targets 11(8):1043–1053PubMedCrossRefGoogle Scholar
  16. 16.
    Chanvorachote P, Pongrakhananon V (2013) Ouabain downregulates Mcl-1 and sensitizes lung cancer cells to TRAIL-induced apoptosis. Am J Physiol Cell Physiol 304(3):C263–C272. doi: 10.1152/ajpcell.00225 PubMedCrossRefGoogle Scholar
  17. 17.
    Newman RA, Yang P, Hittelman WN, Lu T, Ho DH, Ni D, Chan D, Vijjeswarapu M, Cartwright C, Dixon S, Felix E, Addington C (2006) Oleandrin-mediated oxidative stress in human melanoma cells. J Exp Ther Oncol 5(3):167–181PubMedGoogle Scholar
  18. 18.
    Ulivieri C (2010) Cell death: insights into the ultrastructure of mitochondria. Tissue Cell 42(6):339–347. doi: 10.1016/j.tice.2010.10.004 PubMedCrossRefGoogle Scholar
  19. 19.
    Francia P, delli Gatti C, Bachschmid M, Martin-Padura I, Savoia C, Migliaccio E, Pelicci PG, Schiavoni M, Lüscher TF, Volpe M, Cosentino F (2004) Deletion of p66shc gene protects against age-related endothelial dysfunction. Circulation 110(18):2889–2895Google Scholar
  20. 20.
    Trinei M, Giorgio M, Cicalese A, Barozzi S, Ventura A, Migliaccio E, Milia E, Padura IM, Raker VA, Maccarana M, Petronilli V, Minucci S, Bernardi P, Lanfrancone L, Pelicci PG (2002) A p53-p66Shc signalling pathway controls intracellular redox status, levels of oxidation-damaged DNA and oxidative stress-induced apoptosis. Oncogene 21(24):3872–3878PubMedCrossRefGoogle Scholar
  21. 21.
    Napoli C, Martin-Padura I, de Nigris F, Giorgio M, Mansueto G, Somma P, Condorelli M, Sica G, De Rosa G, Pelicci P (2003) Deletion of the p66Shc longevity gene reduces systemic and tissue oxidative stress, vascular cell apoptosis, and early atherogenesis in mice fed a high-fat diet. Proc Natl Acad Sci 100(4):2112–2116PubMedCentralPubMedCrossRefGoogle Scholar
  22. 22.
    Pellegrini M, Pacini S, Baldari CT (2005) p66SHC: the apoptotic side of Shc proteins. Apoptosis 10(1):13–18PubMedCrossRefGoogle Scholar
  23. 23.
    Lin CH, Yu MC, Chiang CC, Bien MY, Chien MH, Chen BC (2013) Thrombin-induced NF-κB activation and IL-8/CXCL8 release is mediated by c-Src-dependent Shc, Raf-1, and ERK pathways in lung epithelial cells. Cell Signal 25(5):1166–1175. doi: 10.1016/j.cellsig.2013.01.018 PubMedCrossRefGoogle Scholar
  24. 24.
    Xi G, Shen X, Clemmons DR (2010) p66shc inhibits insulin-like growth factor-I signaling via direct binding to Src through its polyproline and Src homology 2 domains, resulting in impairment of Src kinase activation. J Biol Chem 285(10):6937–6951. doi: 10.1074/jbc.M109.069872 PubMedCentralPubMedCrossRefGoogle Scholar
  25. 25.
    Clark JS, Faisal A, Baliga R, Nagamine Y, Arany I (2010) Cisplatin induces apoptosis through the ERK-p66shc pathway in renal proximal tubule cells. Cancer Lett 297(2):165–170. doi: 10.1016/j.canlet.2010.05.007 PubMedCrossRefGoogle Scholar
  26. 26.
    Xie Z, Askari A (2002) Na+/K+-ATPase as a signal transducer. Eur J Biochem 269(10):2434–2439PubMedCrossRefGoogle Scholar
  27. 27.
    Xie Z (2003) Molecular mechanisms of Na/K-ATPase-mediated signal transduction. Ann N Y Acad Sci 986:497–503PubMedCrossRefGoogle Scholar
  28. 28.
    Guo C, Liang F, Shah Masood W, Yan X (2014) Hydrogen sulfide protected gastric epithelial cell from ischemia/reperfusion injury by Keap1 s-sulfhydration, MAPK dependent anti-apoptosis and NF-κB dependent anti-inflammation pathway. Eur J Pharmacol 725:70–78. doi: 10.1016/j.ejphar.2014.01.009 PubMedCrossRefGoogle Scholar
  29. 29.
    Xu ZW, Wang FM, Gao MJ, Chen XY, Shan NN, Cheng SX, Mai X, Zala GH, Hu WL, Xu RC (2011) Cardiotonic steroids attenuate ERK phosphorylation and generate cell cycle arrest to block human hepatoma cell growth. J Steroid Biochem Mol Biol 125(3–5):181–191. doi: 10.1016/j.jsbmb.2010.12.016 PubMedCrossRefGoogle Scholar
  30. 30.
    Kim SJ, Kim MS, Lee JW, Lee CH, Yoo H, Shin SH, Park MJ, Lee SH (2006) Dihydroartemisinin enhances radiosensitivity of human glioma cells in vitro. J Cancer Res Clin Oncol 132(2):129–135PubMedCrossRefGoogle Scholar
  31. 31.
    Oh SH, Lim SC (2006) A rapid and transient ROS generation by cadmium triggers apoptosis via caspase-dependent pathway in HepG2 cells and this is inhibited through N-acetylcysteine-mediated catalase upregulation. Toxicol Appl Pharmacol 212(3):212–223PubMedCrossRefGoogle Scholar
  32. 32.
    Wang Z, Li W, Meng X, Jia B (2012) Resveratrol induces gastric cancer cell apoptosis via reactive oxygen species, but independent of sirtuin1. Clin Exp Pharmacol Physiol 39(3):227–232. doi: 10.1111/j.1440-1681.2011.05660.x PubMedCrossRefGoogle Scholar
  33. 33.
    McConkey DJ, Lin Y, Nutt LK, Ozel HZ, Newman RA (2000) Cardiac glycosides stimulate Ca2+ increases and apoptosis in androgen-independent, metastatic human prostate adenocarcinoma cells. Cancer Res 60(14):3807–3812PubMedGoogle Scholar
  34. 34.
    Xu KY, Zhu W, Chen L, DeFilippi C, Zhang J, Xiao RP (2011) Mechanistic distinction between activation and inhibition of (Na(+)+K(+))-ATPase-mediated Ca2+ influx in cardiomyocytes. Biochem Biophys Res Commun 406(2):200–203. doi: 10.1016/j.bbrc.2011.02.013 PubMedCentralPubMedCrossRefGoogle Scholar
  35. 35.
    Khundmiri SJ, Metzler MA, Ameen M, Amin V, Rane MJ, Delamere NA (2006) Ouabain induces cell proliferation through calcium-dependent phosphorylation of Akt (protein kinase B) in opossum kidney proximal tubule cells. Am J Physiol Cell Physiol 291(6):C1247–C1257PubMedCrossRefGoogle Scholar
  36. 36.
    Ning N, Hu JF, Sun JD, Han N, Zhang JT, Chen NH (2012) (−)Clausenamide facilitates synaptic transmission at hippocampal Schaffer collateral-CA1 synapses. Eur J Pharmacol 682(1–3):50–55. doi: 10.1016/j.ejphar.2012.02.004 PubMedCrossRefGoogle Scholar
  37. 37.
    Marí M1, Morales A, Colell A, García-Ruiz C, Fernández-Checa JC (2009) Mitochondrial glutathione, a key survival antioxidant. Antioxid Redox Signal 11(11):2685–2700. doi: 10.1089/ARS.2009.2695
  38. 38.
    Maraldi T, Prata C, Caliceti C, Vieceli Dalla Sega F, Zambonin L, Fiorentini D, Hakim G (2010) VEGF-induced ROS generation from NAD(P)H oxidases protects human leukemic cells from apoptosis. Int J Oncol 36(6):1581–1589PubMedGoogle Scholar
  39. 39.
    Jung J, Kim HY, Kim M, Sohn K, Kim M, Lee K (2011) Translationally controlled tumor protein induces human breast epithelial cell transformation through the activation of Src. Oncogene 30(19):2264–2274. doi: 10.1038/onc.2010.604 PubMedCrossRefGoogle Scholar
  40. 40.
    Giorgio M, Migliaccio E, Orsini F, Paolucci D, Moroni M, Contursi C, Pelliccia G, Luzi L, Minucci S, Marcaccio M, Pinton P, Rizzuto R, Bernardi P, Paolucci F, Pelicci PG (2005) Electron transfer between cytochrome c and p66Shc generates reactive oxygen species that trigger mitochondrial apoptosis. Cell 122(2):221–233PubMedCrossRefGoogle Scholar
  41. 41.
    Danesh SM, Kundu P, Lu R, Stefani E, Toro L (2011) Distinct transcriptional regulation of human large conductance voltage- and calcium-activated K+ channel gene (hSlo1) by activated estrogen receptor alpha and c-Src tyrosine kinase. J Biol Chem 286(36):31064–31071. doi: 10.1074/jbc.M111.235457 PubMedCentralPubMedCrossRefGoogle Scholar
  42. 42.
    Li D, Cui Q, Chen SG, Wu LJ, Tashiro S, Onodera S, Ikejima T (2007) Inactivation of ras and changes of mitochondrial membrane potential contribute to oridonin-induced autophagy in a431 cells. J Pharmacol Sci 105(1):22–33PubMedCrossRefGoogle Scholar
  43. 43.
    Hennion JP, el-Masri MA, Huff MO, el-Mailakh RS (2002) Evaluation of neuroprotection by lithium and valproic acid against ouabain-induced cell damage. Bipolar Disord 4(3):201–206Google Scholar
  44. 44.
    Cuozzo F, Raciti M, Bertelli L, Parente R, Di Renzo L (2012) Pro-death and pro-survival properties of ouabain in U937 lymphoma derived cells. J Exp Clin Cancer Res 31:95. doi: 10.1186/1756-9966-31-95 PubMedCentralPubMedCrossRefGoogle Scholar
  45. 45.
    Song H, Thompson SM, Blaustein MP (2013) Nanomolar ouabain augments Ca2+ signalling in rat hippocampal neurones and glia. J Physiol 591(Pt 7):1671–1689. doi: 10.1113/jphysiol.2012.248336 PubMedCentralPubMedCrossRefGoogle Scholar
  46. 46.
    Fontana JM, Burlaka I, Khodus G, Brismar H, Aperia A (2013) Calcium oscillations triggered by cardiotonic steroids. FEBS J 280(21):5450–5455. doi: 10.1111/febs.12448 PubMedCrossRefGoogle Scholar
  47. 47.
    Santos CX, Tanaka LY, Wosniak J, Laurindo FR (2009) Mechanisms and implications of reactive oxygen species generation during the unfolded protein response: roles of endoplasmic reticulum oxidoreductases, mitochondrial electron transport, and NADPH oxidase. Antioxid Redox Signal 11(10):2409–2427. doi: 10.1089/ARS.2009.2625 PubMedCrossRefGoogle Scholar
  48. 48.
    Marchi S, Giorgi C, Suski JM, Agnoletto C, Bononi A, Bonora M, De Marchi E, Missiroli S, Patergnani S, Poletti F, Rimessi A, Duszynski J, Wieckowski MR, Pinton P (2012) Mitochondria-ros crosstalk in the control of cell death and aging. J Signal Transduct 2012:329–635. doi: 10.1155/2012/329635 Google Scholar
  49. 49.
    Galimov ER, Chernyak BV2, Sidorenko AS, Tereshkova AV, Chumakov PM (2014) Prooxidant properties of p66shc are mediated by mitochondria in human cells. PLoS One 9(3):e86521. doi:  10.1371/journal.pone.0086521
  50. 50.
    Bashir M, Parray AA, Baba RA, Bhat HF, Bhat SS, Mushtaq U, Andrabi KI, Khanday FA (2014) β-Amyloid-evoked apoptotic cell death is mediated through MKK6-p66shc pathway. Neuromol Med 16(1):137–149. doi: 10.1007/s12017-013-8268-4 CrossRefGoogle Scholar
  51. 51.
    Wu J, Akkuratov EE, Bai Y, Gaskill CM, Askari A, Liu L (2013) Cell signaling associated with Na(+)/K(+)-ATPase: activation of phosphatidylinositide 3-kinase IA/Akt by ouabain is independent of Src. Biochemistry 52(50):9059–9067. doi: 10.1021/bi4011804 PubMedCentralPubMedCrossRefGoogle Scholar
  52. 52.
    Kominato R, Fujimoto S, Mukai E, Nakamura Y, Nabe K, Shimodahira M, Nishi Y, Funakoshi S, Seino Y, Inagaki N (2008) Src activation generates reactive oxygen species and impairs metabolism-secretion coupling in diabetic Goto-Kakizaki and ouabain-treated rat pancreatic islets. Diabetologia 51(7):1226–1235. doi: 10.1007/s00125-008-1008-x PubMedCrossRefGoogle Scholar
  53. 53.
    Tian J, Gong X, Xie Z (2001) Signal-transducing function of Na+-K+-ATPase is essential for ouabain’s effect on [Ca2+]i in rat cardiac myocytes. Am J Physiol Heart Circ Physiol 281(5):H1899–H1907PubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2014

Authors and Affiliations

  • Xiaofei Yan
    • 1
    • 2
  • FenLi Liang
    • 3
  • Dongmin Li
    • 1
  • Jin Zheng
    • 2
  1. 1.Department of Biochemistry and Molecular Biology, Medical SchoolXi’an Jiaotong UniversityXi’anPeople’s Republic of China
  2. 2.First Affiliated Hospital of Medical College of Xi’an Jiaotong UniversityXi’anPeople’s Republic of China
  3. 3.Center for Cancer Research, Medical SchoolXi’an Jiaotong UniversityXi’anPeople’s Republic of China

Personalised recommendations