INVESTIGATION OF INTERACTION OF PLATINUM-BASED CYTOSTATIC DRUGS WITH DNA BY SANGER SEQUENCING

Platinum-based cytostatic drugs such as cisplatin, carboplatin and oxaliplatin play an important role in the battle with cancer. The mechanism of their activity is widely studied and the quantification of the drug incorporated in the DNA structure is in the center of attention. In this study we investigated the behavior of the platinum-based cytostatic drug and DNA adducts during the well established Sanger sequencing method involving capillary electrophoretic (CE) separation. Three selected platinum-based cytostatic drugs (cisplatin, carboplatin and oxaliplatin) were incubated with the DNA fragment (498 bp) to create adducts and subsequently sequenced. It was found that the fluorescence signal provided by fluorescently labeled DNA fragments decreased significantly depending on concentration of the drug. Moreover, even though four types of fluorescently labeled fragments are created during the sequencing reaction prior to the CE separation; similar decrease of the signal was observed in all of the fragment types. This suggests that cytostatic drugs do not influence the CE separation itself but the labeling sequencing reaction. Finally, the difference between three types of the cytostatic drugs was found. It follows from the results that to reach the signal decrease of 75% compared to the control DNA sample only 0.3 mu g/ml of cisplatin is required. On the other hand, 7 and 75 mu g/ml of oxaliplatin and carboplatin, respectively are required to reach the same effect. Our hypothesis was verified by electrochemical analysis and the highest amount of platinum was determined in the cisplatinated DNA sample followed by oxaliplatinated and carboplatinated DNA.