Toxic-dose warfarin-induced apoptosis and its enhancement by gamma ionizing radiation in leukemia K562 and HL-60 cells is not mediated by induction of oxidative stress

  • Ilhan Onaran
  • Sevide Sencan
  • Halil Demirtaş
  • Birsen Aydemir
  • Turgut Ulutin
  • Murat Okutan
Original Article


The purpose of this study was to test the hypothesis that warfarin may enhance free radical production and oxidative damage on cancer cells. We examined the possible concentration-dependent effect of warfarin on cytotoxicity with respect to oxidative stress on leukemia cell lines (K562 and HL-60) and normal human peripheral blood mononuclear cells (PBMC). Gamma radiation was used as a positive control agent for oxidative stress. At all concentrations of warfarin (5–200 μM), 5-amino-2,3-dihydro-1,4-phthalazinedione (luminol)- and bis-N-methylacridinium nitrate (lucigenin)-amplified chemiluminescence responses and lipid peroxidation and protein oxidation were stable after 72 h incubation at 37°C. However, The 2′,7′-dichlorofluorescein diacetate (DCFH-DA) oxidation was increased when cells were incubated with high concentrations (50–200 μM) of warfarin. In these concentration ranges, warfarin reduced cell growth in a dose-dependent manner, producing apoptosis. Our results also revealed that at concentrations above 5 μM, warfarin had a potentiating effect on radiation-mediated growth inhibition and apoptosis. Furthermore, marked effects were observed on leukemic cells compared with PBMC. We report here that the increase of DCFH oxidation might be due to the increase in the release of cytochrome C caused by warfarin, as cytosolic cytochrome C content was significantly elevated in the warfarin-treated cells compared with control cells, and because cotreatment with antioxidants N- acetylcysteine or 4,5-dihydroxy-1,3-benzene-disulfonic acid (Tiron) was unable to prevent cytochrome C release and DCFH oxidation induced by the drug. Taken together, these results suggest that high warfarin concentrations may be toxic to leukemic cells in vitro through apoptosis, although at the pharmacological concentrations (<50 μM), warfarin has no prooxidant or cytotoxic effect on PBMC, K562, and HL-60 cells. In addition, when the treatment of leukemic cells with warfarin at concentrations above 5 μM is combined with radiation, we observed an increase in radiation-induced cytotoxicity. The mechanism by which warfarin potentiates this cytotoxicity is unclear, but it may not be directly due to toxic damage induced by warfarin-generated free radicals.


Warfarin Oxidative stress Apoptosis Human peripheral blood mononuclear cells K562 HL-60 Radiation 



This work was partly supported by Research Fund of The University of Erciyes, project SBT-06-01. The authors acknowledge Dr. Emre Basatemür (Barnet General Hospital, London) for critical revision of the manuscript.


  1. Aitken RJ, Buckingham DW, West KM (1992) Reactive oxygen species and human spermatozoa: analysis of the cellular mechanisms involved in luminol- and lucigenin-dependent chemiluminescence. J Cell Physiol 151:466–477PubMedCrossRefGoogle Scholar
  2. Andersson L, Gahmberg CG, Ehblom P (1985) Gene expression during normal and malignant differentiation. Academic Press, New YorkGoogle Scholar
  3. Aramaki M, Kawano K, Sasaki A, Ohno T, Tahara K, Takeuchi Y, Yoshida T, Kitano S (1999) Potential role of heparin in prevention of liver metastasis from colon cancer. Hepato-gastroenterol 46:3241–3243Google Scholar
  4. Berkarda B, Arda O, Tasyurekli M, Derman U (1992) Mitochondria-lytic action of warfarin in lymphocytes. Int J Clin Pharmacol Ther Toxicol 30:277–279PubMedGoogle Scholar
  5. Bertocchi F, Breviario F, Proserpio P, Wang JM, Ghezzi P, Travagli RA, Prosdocimi M, Dejana E (1989) In vitro inhibition of human polymorphonuclear cell function by cloricromene. Naunyn Schmiedebergs Arch Pharmacol 339:697–703PubMedCrossRefGoogle Scholar
  6. Brubacher JL, Bols NC (2001) Chemically de-acetylated 2′,7′-dichlorodihydrofluorescein diacetate as a probe of respiratory burst activity in mononuclear phagocytes. J Immunol Methods 251:81–91PubMedCrossRefGoogle Scholar
  7. Budzisz E, Brzezinska E, Krajewska U, Rozalski M (2003) Cytotoxic effects, alkylating properties and molecular modelling of coumarin derivatives and their phosphonic analogues. Eur J Med Chem 38:597–603PubMedCrossRefGoogle Scholar
  8. Burkitt MJ, Wardman P (2001) Cytochrome C is a potent catalyst of dichlorofluorescin oxidation: implications for the role of reactive oxygen species in apoptosis. Biochem Bioph Res Com 282:329–333CrossRefGoogle Scholar
  9. Cambiaqqi C, Dominci S, Comporti M, Pompella A (1997) Modulation of human T lymphocyte proliferation by 4-hydroxynonenal, the bioactive product of neutrophil-dependent lipid peroxidation. Life Sci 61:777–785CrossRefGoogle Scholar
  10. Childs AC, Phaneuf SL, Dirks AJ, Phillips T, Leeuwenburgh C (2002) Doxorubicin treatment in vivo causes cytochrome C release and cardiomyocyte apoptosis, as well as increased mitochondrial efficiency, superoxide dismutase activity, and Bcl-2:Bax ratio. Cancer Res 62:4592–4598PubMedGoogle Scholar
  11. Francis JL, Biggerstaff J, Amirkhosravi A (1998) Hemostasis and malignancy. Semin Thromb Hemost 24:93–109PubMedGoogle Scholar
  12. Green DR, Reed JC (1998) Mitochondria and apoptosis. Science 281:1309–1312PubMedCrossRefGoogle Scholar
  13. Gunaydin B, Demiryurek AT (2001) Interaction of lidocaine with reactive oxygen and nitrogen species. Eur J Anaesth 18:816–822CrossRefGoogle Scholar
  14. Kakkar A, Hedges AR, Williamson RCN, Kakkar VV (1995) Peroperative heparin therapy inhibits late death from metastatic cancer. Int J Oncol 6:885–888Google Scholar
  15. Khadir A, Verreault J, Averill DA (1999) Inhibition of antioxidants and hyperthermia enhance bleomycin-induced cytotoxicity and lipid peroxidation in Chinese hamster ovary cells. Arch Biochem Biophys 370:163–175PubMedCrossRefGoogle Scholar
  16. Lawrence A, Jones CM, Wardman P, Burkitt MJ (2003) Evidence for the role of a peroxidase compound I-type intermediate in the oxidation of glutathione, NADH, ascorbate and dichlorofluorescin by cytochrome c/H2O2 Implications for oxidative stress during apoptosis. J Biol Chem 278:29410–29419PubMedCrossRefGoogle Scholar
  17. LeBel CP, Ischiropoulos H, Bondy SC (1992) Evaluation of the probe 2′,7′-dichlorofluorescin as an indicator of reactive oxygen species formation and oxidative stress. Chem Res Toxicol 5:227–231PubMedCrossRefGoogle Scholar
  18. Liebmann J (2004) COX-2 inhibitors as cancer treatment: will they be the new warfarin or trastuzumab? Cancer Invest 22:324–325PubMedCrossRefGoogle Scholar
  19. Levine RL, Garland D, Oliver CN, Amici A, Climent I, Lenz AG, Ahn BW, Shaltiel S, Stadtman ER (1990) Determination of carbonyl content of oxidatively modified proteins. Methods Enzymol 186:64–478Google Scholar
  20. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein measurement with the Folin Phenol Reagent. J Biol Chem 193:269–275Google Scholar
  21. McCulloch P, George WD (1989) Warfarin inhibits metastasis of Mtln3 rat mammary carcinoma without affecting primary tumour growth. Brit J Cancer 59:179–183PubMedGoogle Scholar
  22. Meng TC, Fukada T, Tonks NK (2002) Reversible oxidation and inactivation of protein tyrosine phosphatases in vivo. Mol Cell 9:387–399PubMedCrossRefGoogle Scholar
  23. Murphy ME, Sies H (1990) Visible-range low-level chemiluminescence in biological systems. Methods Enzymol 186:595–610PubMedCrossRefGoogle Scholar
  24. Myhre O, Andersen JM, Aarnes H, Fonnum F (2003) Evaluation of the probes 2′,7′-dichlorofluorescin diacetate, luminol, and lucigenin as indicators of reactive species formation. Biochem Pharmacol 65:1575–1582PubMedCrossRefGoogle Scholar
  25. Neubauer BL, Bemis KG, Best KL, Goode RL, Hoover DM, Smith GF, Tanzer LR, Merriman RL (1986) Inhibitory effect of warfarin on the metastasis of the PAIII prostatic adenocarcinoma in the rat. J Urol 135:163–166PubMedGoogle Scholar
  26. Park JG, Kramer BS, Steinberg SM, Carmichael J, Collins JM, Minna JD, Gazdar AF (1987) Chemosensitivity testing of human colorectal carcinoma cell lines using a tetrazolium-based colorimetric assay. Cancer Res 47:5875–5879PubMedGoogle Scholar
  27. Prandoni P, Piccioli A (1997) Venous thromboembolism and cancer: a two-way clinical association. Front Biosci 2:e12–e20PubMedGoogle Scholar
  28. Sheng-Tanner X, Bump EA, Hedley DW (1998) An oxidative stress-mediated death pathway in irradiated human leukemia cells mapped using multilaser flow cytometry. Radiat Res 150:636–647PubMedCrossRefGoogle Scholar
  29. Sun JS, Hang YS, Huang IH, Lu FJ (1996) A simple chemiluminescence assay for detecting oxidative stress in ischemic limb injury. Free Radical Biol Med 20:107–112CrossRefGoogle Scholar
  30. Zacharski LR (2002) Anticoagulants in cancer treatment: malignancy as a solid phase coagulopathy. Cancer Lett 186:1–9PubMedCrossRefGoogle Scholar
  31. Zacharski LR, Henderson WG, Rickles FR, Forman WB, Cornell Jr CJ, Forcier RJ, Edward RL, Headley E, Kim SH, O, Donnell JF (1984) Effect of warfarin anticoagulation on survival in carcinoma of the lung, colon, head and neck, and prostate. Cancer 53:2046–2052PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2008

Authors and Affiliations

  • Ilhan Onaran
    • 1
  • Sevide Sencan
    • 2
  • Halil Demirtaş
    • 2
  • Birsen Aydemir
    • 3
  • Turgut Ulutin
    • 1
  • Murat Okutan
    • 4
  1. 1.Department of Medical Biology, Cerrahpasa Medical FacultyIstanbul UniversityMecidiyeköy – İstanbulTurkey
  2. 2.Department of Medical Biology and Genetics, Medical FacultyErciyes UniversityKayseriTurkey
  3. 3.Department of Biophysics, Cerrahpaşa Medical Facultyİstanbul UniversityİstanbulTurkey
  4. 4.Oncology InstituteIstanbul UniversityIstanbulTurkey

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