WARFARİN’İN SİTOTOKSİK ETKİSİNİN K562 LÖSEMİK HÜCRE SOYUNDA ÇALIŞILMASI

Öz

Klinik ve hayvan model laboratuvar çalışmalarında antikoagülanların dolaylı olarak primer tümörlerin ve metastazın gelişmesini önlediği belirtilmiştir. Bu antikoagulanlardan biri olan warfarinin normal hücrelerden farklı olarak malignant hücrelerde sitotoksik etkiye sahip olduğu gösterilmiştir. Bu sitotoksik etkinin warfarinin elektron transfer etme özelliğiyle kanser hücrelerinde superoksid ve hidrojen peroksid gibi reaktif oksijen türlerini arttırarak meydana geldiği öne sürülmüştür. Buna rağmen bu görüş hakkında hiçbir literatüre rastlanmamıştır. Çalışmamızda, warfarinin kanser hücrelerinde serbest radikal oluşumunu artırabilir hipotezini test etmek için myeloid löseminin blastik kriz evresinden kaynaklanan K562 hücreleri ile HL-60 promyelositik lösemi hücreleri ve insan lenfositleri 72 saat warfarinin çeşitli konsantrasyonları ile (0-200 µM) inkübe edildi. Warfarinin K562 hücrelerinde oksidatif stres üzerine etkisi lüminol ve lüsigenine bağlı kemilüminesans oluşumu ve 2’, 7’-dichlorofluorescinhidrodiasetat oksidasyonu ile değerlendirildi. İstatistiksel değerlendirme ANOVA testi ile yapıldı ve p<0.05 istatistiksel olarak anlamlı kabul edildi. Sonuçlarımız, warfarinin lösemik hücrelerde farmakolojik dozlara yakın konsantrasyonlarda (<50µM) reaktif oksijen türevlerinin üretiminde doğrudan bir etkisi olmadığını göstermektedir. Ancak yüksek dozlarda warfarin (50-200 µM), 2’, 7’-dichlorofluorescinhidrodiasetat oksidasyonunu lösemik hücrelerde lenfositlere göre anlamlı olarak artırmıştır. Her ne kadar bu artışın nedenin oksidatif stres ile ilişkili olmadığı görülmekle birlikte, bu artışın mekanizmasını açıklamak için daha fazla çalışmaya gerek duyulmaktadır

Anahtar Kelimeler

• Warfarin, K562, kanser, sitotoksite, serbest radikal

Evaluation of the Effect on the Cell Cytotoxicity of Warfarin in K562 Leukemia Cell Line

Öz

In the experimental studies of clinical and animal models, it has been reported that anticoagulants have prevented development of primary tumors and metastasis indirectly. It has been shown that warfarin (which is a well- known oral anticoagulant) have a cytotoxic effect on malignant cells, sparing normal cells. It has been suggested that this cytotoxic effect may be due to reactive oxygen species as superoxide and hydrogen peroxide produced in the malignant cells by warfarin, which is a potent electron transferring substance. However, this view is not encountered in literature. This study examined the oxidative and apoptotic potentials of warfarin on three cell types in vitro, namely, human chronic myelogenous leukemic K562 cells, promyelocytic leukemic HL-60 cells, and normal human peripheral blood mononuclear cells. The effects of warfarin were also compared with acetylsalicylic acid (ASA). The cells were incubated with 0-200 µM concentrations of warfarin and 100 µM ASA for 72 h at 37oC. The luminol and lucigenin-dependent chemiluminescense assay and 2', 7'-dichlorofluorescin diacetate (DCFH-DA) method were used as biomarkers of oxidative stress. Statistical analysis of data was performed by analysis of variance (ANOVA) and p<0.05 was considered statistically significant for all experiments. The present results indicate that the warfarin at the pharmacological concentrations (<50µM) have no prooxidant on K562 cells and HL-60 cells. However, DCFH oxidation was increased when cells were incubated with high concentrations (50-200 µM) of warfarin. On the other hand, our study suggests that oxidative stress does not seem to be involved in warfarin-induced DCFH of both cell lines. Therefore, the specific source(s) responsible to DCFH oxidation in leukemic cells in response to in vitro warfarin require future studies

Anahtar Kelimeler

• Warfarin, K562, cancer, cytotoxicity, free Radicals effect on cleft segments, back and medial alveolar widths

Kaynakça

1. Fuster V, Wayne AR, O’Rourke R. The Heart. Cilt 3 2004, pp 1411
2. Ngui JS, Chen Q, Shou M, Wang RW, Stearns RA, Baillie TA, Tang W. In Vitro Stimulation of Warfarin Metabolism by Quinidine: Increases in the Formation of 4*- and 10- Hydroxywarfarin. Drug Metab Dispos 2001, 29(6): 877-886
3. Bobek V, Kovarik J. Antitumor and antimetastatic effect of warfarin and heparins. Biomed Pharmacother 2004, 58: 213–219
4. Zacharski LR, Prandomi P, Monreal M. Warfarin versus low-molecular weight haparin therapy in cancer. Oncologist 2005, 10(1): 72-79
5. Berkarda B, Arda O, Tasyurekli M, Derman U. Mitochondrial-lytic action of warfarin in lymphocytes. Int J Clin Pharmacol Ther Toxicol 1992, 30(8): 277-279
6. Dias N, Bailly C. Drugs targeting mitochondrial functions to control tumor cell growth. Biochem Pharmacol 2005, 70: 1–12
7. Kınnula VL, Crapo JD. Superoxide dismutases in malignant cells and human tumors. Free Radic Biol Med 2004, 36(6): 718-744
8. Cam Sımsek F, Yuksel M, Turker L, Haklar K, G ve ark. The role of reactive oxygen species and apoptosis in the pathogenesis of varicocele in a rat model and efficiency of vitamin E treatment. Int J Androl 2004, 27: 228–33

9. Ohashi T, Mizutani A, Murakami A, Kojo S, Ishii T, Taketani S. Rapid oxidation of dichlorodihydro£uorescin with heme and hemoproteins: formation of the £uorescein is independent of the generation of reactive oxygen species. FEBS Lett 2002, 511: 21-27
10. Carew JS, Huang P. Mitochondrial defects in cancer. Mol Cancer 2002, 9: 1-9
11. Muller-Hocker J, Aust D, Rohrbach H, Napiwotzky J, Reith A, Link TA, Seibel R, Holzel D, Kadenbach B. Defects of the Respiratory Chain in the Normal Human Liver and in Cirrhosis During Aging. Hepatology 1997, 26(3): 709-719
12. Lawrence A, et al. 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 2003, 278: 29410– 29419