Molecular and Cellular Biochemistry

, Volume 428, Issue 1–2, pp 23–39 | Cite as

Cytotoxic effects of the cardenolide convallatoxin and its Na,K-ATPase regulation

  • Naira Fernanda Zanchett Schneider
  • Izabella Thais Silva
  • Lara Persich
  • Annelise de Carvalho
  • Sayonarah C. Rocha
  • Lucas Marostica
  • Ana Carolina Pacheco Ramos
  • Alex G. Taranto
  • Rodrigo M. Pádua
  • Wolfgang Kreis
  • Leandro A. Barbosa
  • Fernão C. Braga
  • Cláudia M. O. Simões
Article

Abstract

Cardenolides are cardiac glycosides, mostly obtained from natural sources. They are well known for their inhibitory action on the Na,K-ATPase, an effect that regulates cardiovascular alterations such as congestive heart failure and atrial arrhythmias. In recent years, they have also sparked new interest in their anticancer potential. In the present study, the cytotoxic effects of the natural cardenolide convallatoxin (CON) were evaluated on non-small cell lung cancer (A549 cells). It was found that CON induced cytostatic and cytotoxic effects in A549 cells, showing essentially apoptotic cell death, as detected by annexin V-propidium iodide double-staining, as well as changes in cell form. In addition, it prompted cell cycle arrest in G2/M and reduced cyclin B1 expression. This compound also increased the number of cells in subG1 in a concentration- and time-dependent manner. At a long term, the reduction of cumulative population doubling was shown along with an increase of β-galactosidase positive cells and larger nucleus, indicative of senescence. Subsequently, CON inhibited the Na,K-ATPase in A549 cells at nM concentrations. Interestingly, at the same concentrations, CON was unable to directly inhibit the Na,K-ATPase, either in pig kidney or in red blood cells. Additionally, results of docking calculations showed that CON binds with high efficiency to the Na,K-ATPase. Taken together, our data highlight the potent anticancer effects of CON in A549 cells, and their possible link with non-classical inhibition of Na,K-ATPase.

Keywords

Cardenolides Convallotoxin Convallatoxin Cytotoxic effects A549 cells Apoptosis Na,K-ATPase 

Abbreviations

CON

Convallatoxin

NSCLC

Non-small cell lung cancer

PAC

Paclitaxel

FBS

Fetal bovine serum

TB

Trypan blue

NII

Nuclear irregularity index

SDS

Sodium dodecyl sulfate

DOC

Deoxycholate

EDTA

Ethylenediaminetetraacetic acid

RIPA

Radioimmunoprecipitation assay buffer

PVDF

Polyvinylidene fluoride

DAPI

4′,6-Diamidino-2-phenylindole

EGTA

Ethylene glycol tetraacetic acid

FACS

Fluorescence-activated cell sorting.

Notes

Acknowledgements

The Brazilian authors would like to thank the funding agencies CAPES /MEC (Ministry of Education) and CNPq/MCTI (Ministry of Science, Technology and Innovation) for their research scholarships. This work was also supported by the CNPq [Grants 472544/2013-6 and 490057/2011-0], the Marie Curie Foundation—IRSES/European Community [Grant 295251], and CAPES [Grant PNPD 2257/2011].

Supplementary material

11010_2016_2914_MOESM1_ESM.docx (1.2 mb)
Supplementary material 1 (DOCX 1190 KB)

References

  1. 1.
    Siegel RL, Miller KD, Jemal A (2015) Cancer statistics, 2015. CA Cancer J Clin 65:5–29. doi: 10.3322/caac.21254 CrossRefPubMedGoogle Scholar
  2. 2.
    Koh PK, Faivre-Finn C, Blackhall FH, De Ruysscher D (2012) Targeted agents in non-small cell lung cancer (NSCLC): clinical developments and rationale for the combination with thoracic radiotherapy. Cancer Treat Rev 38:626–640. doi: 10.1016/j.ctrv.2011.11.003 CrossRefPubMedGoogle Scholar
  3. 3.
    Holohan C, Van Schaeybroeck S, Longley DB, Johnston PG (2013) Cancer drug resistance: an evolving paradigm. Nat Rev Cancer 13:714–726. doi: 10.1038/nrc3599 CrossRefPubMedGoogle Scholar
  4. 4.
    Cragg GM, Newman DJ (2013) Natural products: a continuing source of novel drug leads. Biochim Biophys Acta 1830:3670–3695. doi: 10.1016/j.bbagen.2013.02.008 CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Newman DJ, Cragg GM (2016) Natural products as sources of new drugs from 1981 to 2014. J Nat Prod 75:311–335. doi: 10.1021/np200906s CrossRefGoogle Scholar
  6. 6.
    Winnicka K, Bielawski K, Bielawska A (2006) Cardiac glycosides in cancer research. Acta Pol Pharm 63:109–115PubMedGoogle Scholar
  7. 7.
    Newman RA, Yang P, Pawlus AD, Block KI (2008) Cardiac glycosides as novel cancer therapeutic agents. Mol Interv 8:36–49. doi: 10.1124/mi.8.1.8 CrossRefPubMedGoogle Scholar
  8. 8.
    Cerella C, Dicato M, Diederich M (2013) Assembling the puzzle of anti-cancer mechanisms triggered by cardiac glycosides. Mitochondrion 13:225–234. doi: 10.1016/j.mito.2012.06.003 CrossRefPubMedGoogle Scholar
  9. 9.
    Mijatovic T, Van Quaquebeke E, Delest B et al (2007) Cardiotonic steroids on the road to anti-cancer therapy. Biochim Biophys Acta 1776:32–57. doi: 10.1016/j.bbcan.2007.06.002 PubMedGoogle Scholar
  10. 10.
    Wang Y, Qiu Q, Shen J-J et al (2012) Cardiac glycosides induce autophagy in human non-small cell lung cancer cells through regulation of dual signaling pathways. Int J Dev Biol 44:1813–1824. doi: 10.1016/j.biocel.2012.06.028 Google Scholar
  11. 11.
    Felth J, Rickardson L, Rosén J et al (2009) Cytotoxic effects of cardiac glycosides in colon cancer cells, alone and in combination with standard chemotherapeutic drugs. J Nat Prod 72:1969–1974. doi: 10.1021/np900210m CrossRefPubMedGoogle Scholar
  12. 12.
    Cerella C, Muller F, Gaigneaux A et al (2015) Early downregulation of Mcl-1 regulates apoptosis triggered by cardiac glycoside UNBS1450. Cell Death Dis 6:e1782. doi: 10.1038/cddis.2015.134
  13. 13.
    Chanvorachote P, Pongrakhananon V (2013) Ouabain downregulates Mcl-1 and sensitizes lung cancer cells to TRAIL-induced apoptosis. Am J Physiol Cell Physiol 304:263–272. doi: 10.1152/ajpcell.00225.2012 CrossRefGoogle Scholar
  14. 14.
    Elbaz HA., Stueckle TA., Wang HYL et al (2012) Digitoxin and a synthetic monosaccharide analog inhibit cell viability in lung cancer cells. Toxicol Appl Pharmacol 258:51–60. doi: 10.1016/j.taap.2011.10.007 CrossRefPubMedGoogle Scholar
  15. 15.
    Pongrakhananon V, Stueckle TA, Wang HL et al (2014) Monosaccharide digitoxin derivative sensitize human non-small cell lung cancer cells to anoikis through Mcl-1 proteasomal degradation. Biochem Pharmacol 88:23–35. doi: 10.1016/j.bcp.2013.10.027 CrossRefPubMedGoogle Scholar
  16. 16.
    Hong DS, Henary H, Falchook GS et al (2014) First-in-human study of pbi-05204, an oleander-derived inhibitor of akt, fgf-2, nf-κΒ and p70s6k, in patients with advanced solid tumors. Invest New Drugs 32:1204–1212. doi: 10.1007/s10637-014-0127-0 CrossRefPubMedGoogle Scholar
  17. 17.
    Schneider NFZ, Geller FC, Persich L et al (2016) Inhibition of cell proliferation, invasion and migration by the cardenolides digitoxigenin monodigitoxoside and convallatoxin in human lung cancer cell line. Nat Prod Res 30:1327–1331. doi: 10.1080/14786419.2015.1055265 CrossRefPubMedGoogle Scholar
  18. 18.
    Yang SY, Kim NH, Cho YS et al (2014) Convallatoxin, a dual inducer of autophagy and apoptosis, inhibits angiogenesis in vitro and in vivo. PloS ONE 9:e91094. doi: 10.1371/journal.pone.0091094 CrossRefPubMedPubMedCentralGoogle Scholar
  19. 19.
    Levrier C, Kiremire B, Guéritte F, Litaudon M (2012) Toxicarioside M, a new cytotoxic 10β-hydroxy-19-nor-cardenolide from Antiaris toxicaria. Fitoterapia 83:660–664. doi: 10.1016/j.fitote.2012.02.001 CrossRefPubMedGoogle Scholar
  20. 20.
    Prassas I, Karagiannis GS, Batruch I et al (2011) Digitoxin-induced cytotoxicity in cancer cells is mediated through distinct kinase and interferon signaling networks. Mol Cancer Ther 10:2083–2093. doi: 10.1158/1535-7163.MCT-11-0421 CrossRefPubMedGoogle Scholar
  21. 21.
    Babula P, Masarik M, Adam V et al (2013) From Na+/K+-ATPase and cardiac glycosides to cytotoxicity and cancer treatment. Anticancer Agents Med Chem 13:1069–1087. doi: 10.2174/18715206113139990304 CrossRefPubMedGoogle Scholar
  22. 22.
    Jorgensen PL, Håkansson KO, Karlish SJD (2003) Structure and Mechanism of Na, K-ATPase: functional sites and their interactions. Annu Rev Physiol 65:817–849. doi: 10.1146/annurev.physiol.65.092101.142558 CrossRefPubMedGoogle Scholar
  23. 23.
    Blanco G, Mercer RW (1998) Isozymes of the Na-K-ATPase: heterogeneity in structure, diversity in function. Am J Physiol 275:F633–F650. doi: 10.1152/ajprenal.00721.2010
  24. 24.
    Khan MI, Chesney JA, Laber DA, Miller DM (2009) Digitalis, a targeted therapy for cancer? Am J Med Sci 337:355–359. doi: 10.1097/MAJ.0b013e3181942f57 CrossRefPubMedGoogle Scholar
  25. 25.
    Mijatovic T, Kiss R (2013) Cardiotonic steroids-mediated Na+/K+-ATPase targeting could circumvent various chemoresistance pathways. Planta Med 79:189–198. doi: 10.1055/s-0032-1328243 CrossRefPubMedGoogle Scholar
  26. 26.
    Cereijido M, Contreras RG, Shoshani L, Larre I (2012) The Na, K-ATPase as self-adhesion molecule and hormone receptor. Am J Physiol Cell Physiol 302:C473–C481. doi: 10.1152/ajpcell.00083.2011 CrossRefPubMedGoogle Scholar
  27. 27.
    Strober W (2001) Trypan blue exclusion test of cell viability. Curr Protoc Immunol. doi: 10.1002/0471142735.ima03bs21.Google Scholar
  28. 28.
    Franken NAP, Rodermond HM, Stap J et al (2006) Clonogenic assay of cells in vitro. Nat Protoc 1:2315–2319. doi: 10.1038/nprot.2006.339 CrossRefPubMedGoogle Scholar
  29. 29.
    Filippi-Chiela EC, Oliveira MM, Jurkovski B et al (2012) Nuclear morphometric analysis (NMA): screening of senescence, apoptosis and nuclear irregularities. PLoS ONE. doi: 10.1371/journal.pone.0042522 PubMedPubMedCentralGoogle Scholar
  30. 30.
    Riccardi C, Nicoletti I (2006) Analysis of apoptosis by propidium iodide staining and flow cytometry. Nat Protoc 1:1458–1461. doi: 10.1038/nprot.2006.238 CrossRefPubMedGoogle Scholar
  31. 31.
    Henry CM, Hollville E, Martin SJ (2013) Measuring apoptosis by microscopy and flow cytometry. Methods 61:90–97. doi: 10.1016/j.ymeth.2013.01.008 CrossRefPubMedGoogle Scholar
  32. 32.
    Silva AO, Felipe KB, Villodre ES et al (2016) A guide for the analysis of long-term population growth in cancer. Tumor Biol 37:13743–13749. doi: 10.1007/s13277-016-5255-z
  33. 33.
    Stewart JJP (2013) Optimization of parameters for semiempirical methods VI: more modifications to the NDDO approximations and re-optimization of parameters. J Mol Model 19:1–32. doi: 10.1007/s00894-012-1667-x CrossRefPubMedGoogle Scholar
  34. 34.
    Stweart JJP (2012) Stewart computational chemistry. MOPAC2012. http://OpenMOPAC.net
  35. 35.
    Laursen M, Yatime L, Nissen P, Fedosova NU (2013) Crystal structure of the high-affinity Na + K+-ATPase-ouabain complex with Mg2+ bound in the cation binding site. Proc Natl Acad Sci USA 110:10958–10963. doi: 10.1073/pnas.1222308110 CrossRefPubMedPubMedCentralGoogle Scholar
  36. 36.
    Jaghoori MM, Bleijlevens B, Olabarriaga SD (2016) 1001 ways to run AutoDock Vina for virtual screening. J Comput Aided Mol Des 30:237–249. doi: 10.1007/s10822-016-9900-9 CrossRefPubMedPubMedCentralGoogle Scholar
  37. 37.
    Rocha SC, Pessoa MTC, Neves LDR et al (2014) 21-benzylidene digoxin: a proapoptotic cardenolide of cancer cells that up-regulates Na, K-ATPase and epithelial tight junctions. PLoS ONE 9:e108776. doi: 10.1371/journal.pone.0108776 CrossRefPubMedPubMedCentralGoogle Scholar
  38. 38.
    Trott O, Olson A (2010) NIH public access. J Comput Chem 31:455–461. doi: 10.1002/jcc.21334.AutoDock PubMedPubMedCentralGoogle Scholar
  39. 39.
    Accelrys Software Inc (2013) Discovery studio modeling environment, release 4.5.Google Scholar
  40. 40.
    Jorgensen P (1974) Purification and characterization of (Na + plus K+)-ATPase. 3. Purification from the outer medulla of mammalian kidney after selective removal of membrane components by sodium dodecylsulphate. Biochim Biophys Acta 12:36–52CrossRefGoogle Scholar
  41. 41.
    Jensen BYJ, Nrby JG, Ottolenghi P (1984) Binding of sodium and potassium to the sodium pump of pig kidney evaluated from nucleotide-binding behaviour. J Physiol 346:219–241CrossRefPubMedPubMedCentralGoogle Scholar
  42. 42.
    Sousa L, Garcia IJP, Costa TGF et al (2015) Effects of iron overload on the activity of Na, K-ATPase and lipid profile of the human erythrocyte membrane. Plos ONE 10:e0132852. doi: 10.1371/journal.pone.0132852
  43. 43.
    Fiske C, Subbarow Y (1825) The colorimetric determination of phosphorus. J Biol Chem 66:375–400Google Scholar
  44. 44.
    Noël F, Pimenta PHC, Dos Santos AR et al (2011) ∆2,3-ivermectin ethyl secoester, a conjugated ivermectin derivative with leishmanicidal activity but without inhibitory effect on mammalian P-type ATPases. Naunyn–Schmiedeberg’s Arch Pharm 383:101–107. doi: 10.1007/s00210-010-0578-6 CrossRefGoogle Scholar
  45. 45.
    Klaus B (2016) Statistical relevance—relevant statistics, part II†¯: presenting experimental data. EMBO J 35:1–4. doi: 10.15252/embj.201694659 CrossRefGoogle Scholar
  46. 46.
    Liu Q, Tang J-S, Hu M-J et al (2013) Antiproliferative cardiac glycosides from the latex of Antiaris toxicaria. J Nat Prod 76:1771–1780. doi: 10.1021/np4005147 CrossRefPubMedGoogle Scholar
  47. 47.
    Shi LS, Kuo SC, Sun HD et al (2014) Cytotoxic cardiac glycosides and coumarins from Antiaris toxicaria. Bioorg Med Chem 22:1889–1898. doi: 10.1016/j.bmc.2014.01.052 CrossRefPubMedPubMedCentralGoogle Scholar
  48. 48.
    Kuete V, Vouffo B, Mbaveng AT et al (2009) Evaluation of Antiaris africana methanol extract and compounds for antioxidant and antitumor activities. Pharm Biol 47:1042–1049. doi: 10.3109/13880200902988595 CrossRefGoogle Scholar
  49. 49.
    Kaushik V, Yakisich J, Azad N et al (2016) Anti-tumor effects of cardiac glycosides on human lung cancer cells and lung tumorspheres. J Cell Physiol. doi: 10.1002/jcp.25611 Google Scholar
  50. 50.
    Zhou S, Zhao L, Kuang M et al (2012) Autophagy in tumorigenesis and cancer therapy: Dr. Jekyll or Mr. Hyde? Cancer Lett 323:115–127. doi: 10.1016/j.canlet.2012.02.017 CrossRefPubMedGoogle Scholar
  51. 51.
    Leu WJ, Chang HS, Chan SH et al (2014) Reevesioside A, a cardenolide glycoside, induces anticancer activity against human hormone-refractory prostate cancers through suppression of c-myc expression and induction of G1 arrest of the cell cycle. PLoS ONE 9:1–13. doi: 10.1371/journal.pone.0087323 Google Scholar
  52. 52.
    Lapenna S, Giordano A (2009) Cell cycle kinases as therapeutic targets for cancer. Nat Rev Drug Disc 8:547–566. doi: 10.1038/nrd2907
  53. 53.
    Van Quaquebeke E, Simon G, RE A et al (2005) Identification of a novel cardenolide (2′’-oxovoruscharin) from Calotropis procera and the hemisynthesis of novel derivatives displaying potent in vitro antitumor activities and high in vivo tolerance: structure–activity relationship analyses. J Med Chem 48:849–856. doi: 10.1021/jm049405a CrossRefPubMedGoogle Scholar
  54. 54.
    Juncker T, Cerella C, Teiten M et al (2011) UNBS1450, a steroid cardiac glycoside inducing apoptotic cell death in human leukemia cells. Biochem Pharmacol 81:13–23. doi: 10.1016/j.bcp.2010.08.025 CrossRefPubMedGoogle Scholar
  55. 55.
    Feng B, Guo Y-W, Huang C-G et al (2010) 2′-epi-2′-O-acetylthevetin B extracted from seeds of Cerbera manghas L. induces cell cycle arrest and apoptosis in human hepatocellular carcinoma HepG2 cells. Chem Biol Interact 183:142–153. doi: 10.1016/j.cbi.2009.10.012 CrossRefPubMedGoogle Scholar
  56. 56.
    Kulikov A, Eva A, Kirch U et al (2007) Ouabain activates signaling pathways associated with cell death in human neuroblastoma. Biochim Biophys Acta 1768:1691–1702. doi: 10.1016/j.bbamem.2007.04.012 CrossRefPubMedGoogle Scholar
  57. 57.
    Bielawski K, Winnicka K, Bielawska A (2006) Inhibition of DNA topoisomerases I and II, and growth inhibition of breast cancer MCF-7 cells by ouabain, digoxin and proscillaridin A. Biol Pharm Bull 29:1493–1497CrossRefPubMedGoogle Scholar
  58. 58.
    Weigand KM, Laursen M, Swarts HGP et al (2014) Na+,K+-ATPase isoform selectivity for digitalis-like compounds is determined by two amino acids in the first extracellular loop. Chem Res Toxicol 27:2082–2092. doi: 10.1021/tx500290k CrossRefPubMedGoogle Scholar
  59. 59.
    Alves SLG, Paixão N, Ferreira LGR et al (2015) c-Benzylidene digoxin derivatives synthesis and molecular modeling†¯: evaluation of anticancer and the Na, K-ATPase activity effect. Bioorg Med Chem 23:4397–4404. doi: 10.1016/j.bmc.2015.06.028 CrossRefPubMedGoogle Scholar
  60. 60.
    Trenti A (2012) Analysis of the molecular mechanisms of the antineoplastic effect of ouabain. Università degli Studi di PadovaGoogle Scholar
  61. 61.
    Pierre SV, Sottejeau Y, Gourbeau J et al (2008) Isoform specificity of Na-K-ATPase-mediated ouabain signaling. Am J Physiol Renal Physiol 1:859–866. doi: 10.1152/ajprenal.00089.2007.CrossRefGoogle Scholar
  62. 62.
    Xie J, Ye Q, Cui X et al (2015) Expression of rat Na-K-ATPase alpha 2 enables ion pumping but not ouabain-induced signaling in alpha1-deficient porcine renal epithelial cells. Am J Physiol Cell Physiol 309:C373–C382. doi: 10.1152/ajpcell.00103.2015 CrossRefPubMedPubMedCentralGoogle Scholar
  63. 63.
    Slingerland M, Cerella C, Guchelaar HJ et al (2013) Cardiac glycosides in cancer therapy: from preclinical investigations towards clinical trials. Invest New Drugs 31:1087–1094. doi: 10.1007/s10637-013-9984-1 CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2017

Authors and Affiliations

  • Naira Fernanda Zanchett Schneider
    • 1
  • Izabella Thais Silva
    • 1
  • Lara Persich
    • 1
  • Annelise de Carvalho
    • 1
  • Sayonarah C. Rocha
    • 2
  • Lucas Marostica
    • 1
  • Ana Carolina Pacheco Ramos
    • 2
  • Alex G. Taranto
    • 3
  • Rodrigo M. Pádua
    • 4
  • Wolfgang Kreis
    • 5
  • Leandro A. Barbosa
    • 2
  • Fernão C. Braga
    • 4
  • Cláudia M. O. Simões
    • 1
  1. 1.Departamento de Ciências Farmacêuticas, Centro de Ciências da SaúdeUniversidade Federal de Santa Catarina (UFSC)Florianópolis,Brazil
  2. 2.Laboratório de Bioquímica Celular, Campus Centro-Oeste Dona LinduUniversidade Federal de São João del ReiDivinopolisBrazil
  3. 3.Laboratório de Bioinformática, Campus Centro Oeste Dona LinduUniversidade Federal de São João Del ReiDivinópolisBrazil
  4. 4.Departamento de Produtos Farmacêuticos, Faculdade de FarmáciaUniversidade Federal de Minas GeraisBelo HorizonteBrazil
  5. 5.Department of BiologyFriedrich-Alexander UniversitätErlangenGermany

Personalised recommendations