The Electroporation as a Tool for Studying the Role of Plasma Membrane in the Mechanism of Cytotoxicity of Bisphosphonates and Menadione.

In this study, the role of the cell plasma membrane as a barrier in the mechanism of the cytotoxicity of nitrogen-containing bisphosphonates and menadione was studied, and the possibility of increasing the efficiency of bisphosphonates and menadione (vitamin K3) as chemotherapeutic agents by permeabilizing the cell plasma membrane has been investigated in vitro. The plasma membrane barrier was reduced by electropermeabilization with the pulse of strong electric field. Two membrane-impermeant bisphosphonates with different hydrophilicities were chosen as study objects: ibandronate and pamidronate. For the comparison, an amphiphilic vitamin K3, which is able to cross the cell membrane, was studied as well. The impact of nitrogen-containing bisphosphonates and vitamin K3 on MH-22A cells viability was evaluated for the case of long (9 days) and short (20 min) exposure. When cells were cultured in the medium with vitamin K3 for 9-10 days, it exhibited toxicity of 50 % over the control at 6.2 µM for mouse hepatoma MH-22A cells. Ibandronate and pamidronate were capable of reducing drastically the cell viability only in the case of long 9-days incubation and at high concentrations (~20 µM for pamidronate and over 100 µM for ibandronate). Single, square-wave electric pulse with the duration of 100 µs and the field strength of 2 kV/cm was used to electroporate mouse hepatoma MH-22A cells in vitro. The results obtained here showed that the combination of the exposure of cells to membrane-impermeable bisphosphonates pamidronate and ibandronate with electropermeabilization of the cell plasma membrane did not increase their cytotoxicity. In the case of membrane-permeable vitamin K3, cell electropermeabilization did increase vitamin K3 killing efficiency. However, this increase was not substantial, within the range of 20-30 % depending on the duration of the exposure. Electropermeabilization improved cytotoxic effect of vitamin K3 but not of pamidronate and ibandronate.