Intracellular half-life of cisplatin in malignant cells : A kinetic study of the decline rate of intracellular cisplatin concentration in in-vitro grown cells determined by LC-ICP-MS following exposure to clinically relevant cisplatin doses

Abstract: Cisplatin is a common cancer therapy substance used against several types of malignant tumors and often shows great efficiency. The most important tumour killing mechanism is believed to be formation of Pt-DNA adducts. It is however a common phenomenon that tumors develop resistance causing inefficient treatment. It is generally believed that Pt-DNA adduct formation follows after intracellular “activation” of the drug substances by hydrolysis reactions due to the lower chloride concentration intra- compared to extra-cellular. The kinetics for these activation processes and other potential metabolic reactions for the Pt-drugs in-vivo are virtually unknown. The presence of free intracellular cisplatin in in-vitro grown human malignant melanoma cell lines have been shown previously and the aim of this study is to determine the kinetics of decline in intracellular cisplatin concentration in in-vitro grown cells following exposure to clinically relevant cisplatin doses 2, 20 and100μM with 60 minutes exposure time. Species were separated on a 2.1 x 150mm, 3.5 μm, 200Å ZIC-HILIC column and detected online by selective platinum (m/z 194 and 195) monitoring using ICP-MS. The system was optimized with respect to detection limit (0.5pg) to be able to use low exposure concentration of cisplatin in the cell incubation experiments. The half life and the kinetic curve were determined by using the incubation time of 0 to 30 minutes in cisplatin free medium. A ceramic bead method was used for cell lysis, and cisplatin, total platinum and total protein concentration were measured in the lysates. The, intracellular concentration of cisplatin and total platinum as well as the intracellular half life of cisplatin, were determined in both cisplatin sensitive (T289 wild type) and resistant (T289 DDP) malignant melanoma cells. The half life and total platinum depends on the type of cells and the exposure doses. For sensitive cells, with low exposure doses (2, 20μM), the half life was determined to 12 minutes and with high exposure dose, the half life reduced quickly and was determined to be 6 minutes. For resistant cells, the half life was determined to 16, 14, and10 minutes for 2, 20, and 100μM exposure doses, respectively. The intracellular cisplatin and total platinum in resistant cells were lower than in sensitive cells. The reaction order for the metabolism of intracellular cisplatin was also investigated. The decline in cisplatin concentration did not really follow the first or second order reaction. However, for sensitive cells, the reduction seemed to be close to the first order at low exposure doses (2, 20μM) and the second order with the high exposure dose (100μM) and for resistant cells, the reduction of cisplatin seemed to follow the first order in all exposure doses.