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 wi th non-lethal direct current (DC) electrical fields, and uptake and toxicit y of doxorubicin were investigated. An electrical field with a field streng th of 500 Vm(-1) applied for a duration of 90 s resulted in neither reversi ble nor irreversible membrane breakdown as revealed by fluid phase uptake s tudies of the membrane impermeant tracer Lucifer yellow. However,treated sp heroids showed an increased uptake of doxorubicin and, consequently, an inc reased toxicity following electrical field exposure. The electrical field r aised intracellular reactive oxygen species (ROS) as revealed using 2',7'-d ichlorofluorescein diacetate (H(2)DCFDA) as an indicator. ROS induced membr ane lipid peroxidation since the lipid peroxidation end products malondiald ehyde (MDA) and 4-hydroxy-2-(E)-nonenal (4-HNE) were detected after electri cal field treatment. Moreover, lipid peroxidation decreased the lipid diffu sion coefficient D from 4.2 x 10(-10) cm(2) s(-1) to 2.7 x 10(-10) cm(2) s( -1) in the control and treated sample, respectively, as revealed by fluores cence recovery after photobleaching (FRAP) experiments. The field effects c ould be mimicked by incubating spheroids with 100 nM hydrogen peroxide and were inhibited by the radical scavengers dehydroascorbate (DHA) and alpha-t ocopherol (vitamin E), indicating that the increased uptake of doxorubicin after electrical field treatment is owing to lipid peroxidation and decreas ed membrane lipid mobility. Treatment of tumours with low intensity electri cal fields may be useful to improve the cytotoxic capacity of anthracycline s.