Increased doxorubicin uptake and toxicity in multicellular tumour spheroids treated with DC electrical fields

Electrochemotherapy (ECT) is a new approach to the treatment of tumours. In the present study, multicellular prostate tumour spheroids were treated with non-lethal direct current (DC) electrical fields, and uptake and toxicity of doxorubicin were investigated. An electrical field with a field strength of 500 Vm(-1) applied for a duration of 90 s resulted in neither reversible nor irreversible membrane breakdown as revealed by fluid phase uptake studies of the membrane impermeant tracer Lucifer yellow. However, treated spheroids showed an increased uptake of doxorubicin and, consequently, an increased toxicity following electrical field exposure. The electrical field raised intracellular reactive oxygen species (ROS) as revealed using 2',7'-dichlorofluorescein diacetate (H2DCFDA) as an indicator. ROS induced membrane lipid peroxidation since the lipid peroxidation end products malondialdehyde (MDA) and 4-hydroxy-2-(E)-nonenal (4-HNE) were detected after electrical field treatment. Moreover, lipid peroxidation decreased the lipid diffusion coefficient D from 4.2 x 10(-10) cm2 s(-1) to 2.7 x 10(-10) cm2 s(-1) in the control and treated sample, respectively, as revealed by fluorescence recovery after photobleaching (FRAP) experiments. The field effects could be mimicked by incubating spheroids with 100 nM hydrogen peroxide and were inhibited by the radical scavengers dehydroascorbate (DHA) and alpha-tocopherol (vitamin E), indicating that the increased uptake of doxorubicin after electrical field treatment is owing to lipid peroxidation and decreased membrane lipid mobility. Treatment of tumours with low intensity electrical fields may be useful to improve the cytotoxic capacity of anthracyclines.