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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

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Abstract

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.

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Acknowledgments

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.

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Authors and Affiliations

  1. Department of Medical Biology, Cerrahpasa Medical Faculty, Istanbul University, Ortaklar Cd. Butan Sk. No:2, 34394, Mecidiyeköy – İstanbul, Turkey

    Ilhan Onaran & Turgut Ulutin

  2. Department of Medical Biology and Genetics, Medical Faculty, Erciyes University, Kayseri, Turkey

    Sevide Sencan & Halil Demirtaş

  3. Department of Biophysics, Cerrahpaşa Medical Faculty, İstanbul University, İstanbul, Turkey

    Birsen Aydemir

  4. Oncology Institute, Istanbul University, Istanbul, Turkey

    Murat Okutan

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  1. Ilhan Onaran
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  2. Sevide Sencan
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Correspondence to Ilhan Onaran.

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Onaran, I., Sencan, S., Demirtaş, H. et al. 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. Naunyn-Schmied Arch Pharmacol 378, 471–481 (2008). https://doi.org/10.1007/s00210-008-0306-7

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  • DOI: https://doi.org/10.1007/s00210-008-0306-7

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