Cancer Chemotherapy and Pharmacology

, Volume 64, Issue 4, pp 793–802 | Cite as

Pro-apoptotic and cytostatic activity of naturally occurring cardenolides

  • Elena Bloise
  • Alessandra Braca
  • Nunziatina De Tommasi
  • Maria Antonietta BelisarioEmail author
Original Article



Cardenoliddes are steroid glycosides which are known to exert cardiotonic effects by inhibiting the Na+/K+-ATPase. Several of these compounds have been shown also to possess anti-tumor potential. The aim of the present work was the characterization of the tumor cell growth inhibition activity of four cardenolides, isolated from Periploca graeca L., and the mechanisms underlying such an effect.


The pro-apoptotic and cytostatic effect of the compounds was tested in U937 (monocytic leukemia) and PC3 (prostate adenocarcinoma). Characterization of apoptosis and cell cycle impairment was obtained by cytofluorimetry and WB.


Periplocymarin and periplocin were the most active compounds, periplocymarin being more effective than the reference compound ouabain. The reduction of cell number by these two cardenolides was due in PC3 cells mainly to the activation of caspase-dependent apoptotic pathways, while in U937 cells to the induction of cell cycle impairment without extensive cell death. Interestingly, periplocymarin, at cytostatic but non-cytotoxic doses, was shown to sensitize U937 cells to TRAIL.


Taken together, our data outline that cardiac glycosides are promising anticancer drugs and contribute to the identification of new natural cardiac glycosides to obtain chemically modified non-cardioactive/low toxic derivatives with enhanced anticancer potency.


Cardenolides PC3 U937 Apoptosis Cell cycle TRAIL 



The student Paola Torre contributed to some experiments and results will be included in her degree thesis. This work was supported in part by research grant from the University of Salerno.


  1. 1.
    Hauptmann PJ, Garg R, Kelly RA (1999) Cardiac glycosides in the next millennium. Prog Cardiovasc Dis 41:247–254CrossRefGoogle Scholar
  2. 2.
    Rahimtoola SH, Tak T (1996) The use of digitalis in heart failure. Curr Probl Cardiol 21:781–853PubMedCrossRefGoogle Scholar
  3. 3.
    Xie Z, Cai T (2003) Na+/K+-ATPase-mediated signal transduction: from protein interaction to cellular function. Mol Interv 3:157–168PubMedCrossRefGoogle Scholar
  4. 4.
    Aperia A (2007) New roles for an old enzyme: Na+/K+-ATPase emerges as an interesting drug target. J Intern Med 261:44–52PubMedCrossRefGoogle Scholar
  5. 5.
    Newman RA, Yang P, Pawlus AD, Block KI (2008) Cardiac glycosides as novel cancer therapeutic agents. Mol Interv 8:36–49PubMedCrossRefGoogle Scholar
  6. 6.
    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 67:727–733PubMedCrossRefGoogle Scholar
  7. 7.
    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 29:1493–1497PubMedCrossRefGoogle Scholar
  8. 8.
    Ramirez-Ortega M, Maldonado-Lagunas V, Melendez-Zajgla J, Carrillo-Hernandez JF, Pastelín-Hernandez G, Picazo-Picazo O, Ceballos-Reyes G (2006) Proliferation and apoptosis of HeLa cells induced by in vitro stimulation with digitalis. Eur J Pharmacol 534:71–76PubMedCrossRefGoogle Scholar
  9. 9.
    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 1768:1691–1702PubMedCrossRefGoogle Scholar
  10. 10.
    Larre I, Ponce A, Fiorentino R, Shoshani L, Contreras RG, Cereijido M (2006) Contacts and cooperation between cells depend on the hormone ouabain. Proc Natl Acad Sci USA 103:10911–10916PubMedCrossRefGoogle Scholar
  11. 11.
    Stenkvist B, Pengtsson E, Dahlqvist B, Eriksson O, Jarkrans T, Nordin B (1982) Cardiac glycosides and breast cancer, revisited. N Engl J Med 306:484PubMedGoogle Scholar
  12. 12.
    Haux J, Klepp O, Spigset O, Tretli S (2001) Digitoxin medication and cancer; case control and internal dose-response studies. BMC Cancer 1:11PubMedCrossRefGoogle Scholar
  13. 13.
    Repke KHR, Benga GH, Tager JM (1988) Biomembranes, basic and medical research. Springer, Berlin, pp 161–173Google Scholar
  14. 14.
    Langenhan JM, Peters NR, Guzei IA, Hoffmann FM, Thorson JS (2005) Enhancing the anticancer properties of cardiac glycosides by neoglycorandomization. Proc Natl Acad Sci USA 102:12305–12310PubMedCrossRefGoogle Scholar
  15. 15.
    Daniel D, Süsal C, Kopp B, Opelz G, Terness P (2003) Apoptosis-mediated selective killing of malignant cells by cardiac steroids: maintenance of cytotoxicity and loss of cardiac activity of chemically modified derivatives. Int Immunopharmacol 3:1791–1801PubMedCrossRefGoogle Scholar
  16. 16.
    López-Lázaro M (2007) Digitoxin as an anticancer agent with selectivity for cancer cells: possible mechanisms involved. Expert Opin Ther Targets 11:1043–1053PubMedCrossRefGoogle Scholar
  17. 17.
    Spera D, Siciliano T, De Tommasi N, Braca A, Vessières A (2007) Antiproliferative cardenolides from Periploca graeca. Planta Med 73:384–387PubMedCrossRefGoogle Scholar
  18. 18.
    Jortani SA, Helm RA, Valdes R Jr (1996) Inhibition of Na+/K+-ATPase by oleandrin and oleandrigenin, and their detection by digoxin immunoassays. Clin Chem 42:1654–1658PubMedGoogle Scholar
  19. 19.
    Nicoletti I, Migliorati G, Pagliacci MC, Grignani F, Riccardi C (1991) A rapid and simple method for measuring thymocyte apoptosis by propidium iodide staining and flow cytometry. J Immunol Methods 139:271–279PubMedCrossRefGoogle Scholar
  20. 20.
    Scaduto RC Jr, Grotyohann LW (1999) Measurement of mitochondrial membrane potential using fluorescent rhodamine derivatives. Biophys J 76:469–477PubMedCrossRefGoogle Scholar
  21. 21.
    Dal Piaz F, Nigro P, Braca A, De Tommasi N, Belisario MA (2007) 13-Hydroxy-15-oxo-zoapatlin, an ent-kaurane diterpene, induces apoptosis in human leukemia cells, affecting thiol-mediated redox regulation. Free Radic Biol Med 43:1409–1422PubMedCrossRefGoogle Scholar
  22. 22.
    Vermes I, Haanen C, Steffens-Nakken H, Reutelingsperger C (1995) A novel assay for apoptosis. Flow cytometric detection of phosphatidylserine expression on early apoptotic cells using fluorescein labelled Annexin V. J Immunol Methods 184:39–51PubMedCrossRefGoogle Scholar
  23. 23.
    Gogvadze V, Orrenius S (2006) Mitochondrial regulation of apoptotic cell death. Chem Biol Interact 163:4–14PubMedCrossRefGoogle Scholar
  24. 24.
    Almasan A, Ashkenazi A (2003) Apo2L/TRAIL: apoptosis signaling, biology, and potential for cancer therapy. Cytokine Growth Factor Rev 14:337–348PubMedCrossRefGoogle Scholar
  25. 25.
    MacFarlane M, Inoue S, Kohlhaas SL, Majid A, Harper N, Kennedy DB, Dyer MJ, Cohen GM (2005) Chronic lymphocytic leukemic cells exhibit apoptotic signaling via TRAIL-R1. Cell Death Differ 12:773–782PubMedCrossRefGoogle Scholar
  26. 26.
    Mijatovic T, De Nève N, Gailly P, Mathieu V, Haibe-Kains B, Bontempi G, Lapeira J, Decaestecker C, Facchini V, Kiss R (2008) Nucleolus and c-Myc: potential targets of cardenolide-mediated antitumor activity. Mol Cancer Ther 7(5):1285–1296PubMedCrossRefGoogle Scholar
  27. 27.
    Ihenetu K, Qazzaz HM, Crespo F, Fernandez-Botran R, Valdes R (2007) Digoxin-like immunoreactive factors induce apoptosis in human acute T-cell lymphoblastic leukemia. J Clin Chem 53:1315–1322CrossRefGoogle Scholar
  28. 28.
    Schoner W (2002) Sodium pump and steroid hormone receptor. Na+/K+-ATPase. Eur J Biochem 269:2423PubMedCrossRefGoogle Scholar
  29. 29.
    Kaplan JH (2002) Biochemistry of Na+/K+-ATPase. Annu Rev Biochem 71:511–535PubMedCrossRefGoogle Scholar
  30. 30.
    Wasserstrom JA, Aistrup GL (2005) Digitalis: new actions for an old drug. Am J Physiol Heart Circ Physiol 289:H1781–H1793PubMedCrossRefGoogle Scholar
  31. 31.
    Gilmore-Hebert M, Schneider JW, Greene AL, Berliner N, Stolle CA, Lomax K, Mercer RW, Benz EJ Jr (1989) Expression of multiple Na+, K+-adenosine triphosphatase isoform genes in human hematopoietic cells. Behavior of the novel A3 isoform during induced maturation of HL60 cells. J Clin Invest 84:347–351PubMedCrossRefGoogle Scholar
  32. 32.
    Duran MJ, Chosseler M, Pressley T (2004) Regulation of Na, K-pump-mediated transport by prolactin in cultured human prostate epithelial cells. Cell Mol Biol 50:809–814PubMedGoogle Scholar
  33. 33.
    Raghavendra PB, Sreenivasan Y, Manna SK (2007) Oleandrin induces apoptosis in human, but not in murine cells: dephosphorylation of Akt, expression of FasL, and alteration of membrane fluidity. Mol Immunol 44:2292–2302PubMedCrossRefGoogle Scholar
  34. 34.
    Kometiani P, Liu L, Askari A (2005) Digitalis-induced signaling by Na+/K+-ATPase in human breast cancer cells. Mol Pharmacol 67:929–936PubMedCrossRefGoogle Scholar
  35. 35.
    Hao C, Song JH, Hsi B, Lewis J, Song DK, Petruk KC, Tyrrell DL, Kneteman NM (2004) TRAIL inhibits tumor growth but is nontoxic to human hepatocytes in chimeric mice. Cancer Res 64:8502–8506PubMedCrossRefGoogle Scholar
  36. 36.
    MacFarlane M, Harper N, Snowden RT, Dyer MJ, Barnett GA, Pringle JH, Cohen GM (2002) Mechanisms of resistance to TRAIL induced apoptosis in primary B cell chronic lymphocytic leukaemia. Oncogene 21:6809–6818PubMedCrossRefGoogle Scholar
  37. 37.
    Todaro M, Lombardo Y, Francipane MG, Alea MP, Cammareri P, Iovino F, Di Stefano AB, Di Bernardo C, Agrusa A, Condorelli G, Walczak H, Stassi G (2008) Apoptosis resistance in epithelial tumors is mediated by tumor-cell-derived interleukin-4. Cell Death Differ 15:762–772PubMedCrossRefGoogle Scholar
  38. 38.
    Frese S, Frese-Schaper M, Andres AC, Miescher D, Zumkehr B, Schmid RA (2006) Cardiac glycosides initiate Apo2L/TRAIL-induced apoptosis in non-small cell lung cancer cells by up-regulation of death receptors 4 and 5. Cancer Res 66:5867–5874PubMedCrossRefGoogle Scholar
  39. 39.
    Rosato RR, Almenara JA, Coe S, Grant S (2007) The multikinase inhibitor sorafenib potentiates TRAIL lethality in human leukemia cells in association with Mcl-1 and cFLIPL down-regulation. Cancer Res 67:9490–9500PubMedCrossRefGoogle Scholar
  40. 40.
    Inoue S, MacFarlane M, Harper N, Wheat LM, Dyer MJ, Cohen GM (2004) Histone deacetylase inhibitors potentiate TNF-related apoptosis-inducing ligand (TRAIL)-induced apoptosis in lymphoid malignancies. Cell Death Differ 11(Suppl 2):S193–S206PubMedCrossRefGoogle Scholar
  41. 41.
    Dumitru CA, Carpinteiro A, Trarbach T, Hengge UR, Gulbins E (2007) Doxorubicin enhances TRAIL-induced cell death via ceramide-enriched membrane platforms. Apoptosis 12:1533–1541PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2009

Authors and Affiliations

  • Elena Bloise
    • 1
  • Alessandra Braca
    • 2
  • Nunziatina De Tommasi
    • 1
  • Maria Antonietta Belisario
    • 1
    Email author
  1. 1.Dipartimento di Scienze FarmaceuticheUniversità di SalernoFiscianoItaly
  2. 2.Dipartimento di Chimica Bioorganica e BiofarmaciaUniversità di PisaPisaItaly

Personalised recommendations