Skip to main content

Advertisement

SpringerLink
Log in

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

  • Published:
Molecular and Cellular Biochemistry Aims and scope Submit manuscript

Abstract

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.

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

Similar content being viewed by others

References

  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 15:1311–1333

    Article  CAS  PubMed  Google Scholar 

  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

    Article  CAS  PubMed  Google Scholar 

  3. Prassas I, Diamandis EP (2008) Novel therapeutic applications of cardiac glycosides. Nat Rev Drug Discov 7(11):926–935. doi:10.1038/nrd2682

    Article  CAS  PubMed  Google Scholar 

  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

    Article  CAS  PubMed  Google Scholar 

  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

    Article  PubMed  Google Scholar 

  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):170–179

    Article  CAS  PubMed  Google Scholar 

  7. Suzuki K, Nakamura K, Kato K, Hamada H, Tsukamoto T (2007) Exploration of target molecules for prostate cancer gene therapy. Prostate 67(11):1163–1173

    Article  CAS  PubMed  Google Scholar 

  8. Kometiani P, Liu L, Askari A (2005) Digitalis-induced signaling by Na+/K+-ATPase in human breast cancer cells. Mol Pharmacol 67(3):929–936

    Article  CAS  PubMed  Google Scholar 

  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):3807–3812

    CAS  PubMed  Google Scholar 

  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):14067–14072

    Article  CAS  PubMed  Google Scholar 

  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):1691–1702

    Article  CAS  PubMed  Google Scholar 

  12. Joshi AD, Parsons DW, Velculescu VE, Riggins GJ (2011) Sodium ion channel mutations in glioblastoma patients correlate with shorter survival. Mol Cancer 10:171–179

    Google Scholar 

  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):727–733

    Article  CAS  PubMed  Google Scholar 

  14. Stenkvist B (1999) Is digitalis a therapy for breast carcinoma? Oncol Rep 6(3):493–496

    CAS  PubMed  Google Scholar 

  15. López-Lázaro M (2007) Digitoxin as an anticancer agent with selectivity for cancer cells: possible mechanisms involved. Expert Opin Ther Targets 11(8):1043–1053

    Article  PubMed  Google Scholar 

  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

    Article  CAS  PubMed  Google Scholar 

  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):167–181

    CAS  PubMed  Google Scholar 

  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

    Article  CAS  PubMed  Google Scholar 

  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):2889–2895

  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):3872–3878

    Article  CAS  PubMed  Google Scholar 

  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):2112–2116

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  22. Pellegrini M, Pacini S, Baldari CT (2005) p66SHC: the apoptotic side of Shc proteins. Apoptosis 10(1):13–18

    Article  CAS  PubMed  Google Scholar 

  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

    Article  CAS  PubMed  Google Scholar 

  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

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  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

    Article  CAS  PubMed  Google Scholar 

  26. Xie Z, Askari A (2002) Na+/K+-ATPase as a signal transducer. Eur J Biochem 269(10):2434–2439

    Article  CAS  PubMed  Google Scholar 

  27. Xie Z (2003) Molecular mechanisms of Na/K-ATPase-mediated signal transduction. Ann N Y Acad Sci 986:497–503

    Article  CAS  PubMed  Google Scholar 

  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

    Article  CAS  PubMed  Google Scholar 

  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

    Article  CAS  PubMed  Google Scholar 

  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):129–135

    Article  CAS  PubMed  Google Scholar 

  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):212–223

    Article  CAS  PubMed  Google Scholar 

  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

    Article  PubMed  Google Scholar 

  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):3807–3812

    CAS  PubMed  Google Scholar 

  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

    Article  PubMed Central  PubMed  Google Scholar 

  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–C1257

    Article  CAS  PubMed  Google Scholar 

  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

    Article  CAS  PubMed  Google Scholar 

  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. 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):1581–1589

    CAS  PubMed  Google Scholar 

  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

    Article  CAS  PubMed  Google Scholar 

  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):221–233

    Article  CAS  PubMed  Google Scholar 

  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

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  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):22–33

    Article  CAS  PubMed  Google Scholar 

  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):201–206

  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

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  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

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  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

    Article  CAS  PubMed  Google Scholar 

  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

    Article  CAS  PubMed  Google Scholar 

  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. 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. 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

    Article  CAS  Google Scholar 

  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

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  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

    Article  CAS  PubMed  Google Scholar 

  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–H1907

    CAS  PubMed  Google Scholar 

Download references

Acknowledgments

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).

Author information

Authors and Affiliations

  1. Department of Biochemistry and Molecular Biology, Medical School, Xi’an Jiaotong University, Xi’an, 710061, Shaanxi, People’s Republic of China

    Xiaofei Yan & Dongmin Li

  2. First Affiliated Hospital of Medical College of Xi’an Jiaotong University, Xi’an, 710061, Shaanxi, People’s Republic of China

    Xiaofei Yan & Jin Zheng

  3. Center for Cancer Research, Medical School, Xi’an Jiaotong University, Xi’an, 710061, Shaanxi, People’s Republic of China

    FenLi Liang

Authors
  1. Xiaofei Yan
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. FenLi Liang
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Dongmin Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Jin Zheng
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Jin Zheng.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Yan, X., Liang, F., Li, D. et al. Ouabain elicits human glioblastoma cells apoptosis by generating reactive oxygen species in ERK-p66SHC-dependent pathway. Mol Cell Biochem 398, 95–104 (2015). https://doi.org/10.1007/s11010-014-2208-y

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11010-014-2208-y

Keywords

Search

Navigation