Ankaferd hemostat induces DNA damage, apoptosis and cytotoxic activity by generating reactive oxygen species in melanoma and normal cell lines

Although, Ankaferd hemostat (Ankaferd Blood Stopper, ABS) could be utilized successfully as hemostatic agent, studies demonstrated that it has cytotoxic effects on cells. However, the mechanism(s) of this effect has not been elucidated yet. In this study, cytotoxic, genotoxic, apoptotic and reactive oxygen generating (ROS) activities of ABS were investigated in melanoma and normal cell lines. The cells were incubated with different concentrations of ABS (0.125 to 2%) for 24 h. The cell viability was assessed based on ATP cell viability assay. Intracellular accumulation of reactive oxygen species (ROS) was determined using the fluorescent probes 2’,7’-dichloro-dihydrofluorescein-diacetate (H2DCF-DA). DNA damage was evaluated by alkaline single cell gel electrophoresis assay (Comet Assay) and, apoptosis induction was detected by Acridine Orange/Ethidium Bromide AO/EB double staining method. Our results demonstrated that ABS increases DNA damage, apoptosis and ROS levels in both melanoma and normal cell lines in a dose dependent manner, and all of these activities were significantly higher in melanoma cells than in normal cells. There was a statistically significant positive correlation between DNA damage, apoptosis and ROS levels in ABS treated cell lines. Our results revealed that although ABS commonly used as hemostatic agent, it causes DNA damage and apoptosis by generating ROS in a dose dependent manner. Therefore, it should be removed the unused ABS by cleaning once the hemostasis is achieved to minimize the postoperative side effects. These results could also contribute to the development of new treatment for cancer.