Time-dependent stress responses determine bleomycin electrochemotherapy efficacy in drug-resistant breast Cancer cells.

Electrochemotherapy (ECT), combining pulsed electric fields (PEFs) with cytotoxic drugs, enhances intracellular drug delivery and tumor control; however, cellular stress responses determining long-term efficacy, particularly in drug-resistant cancer cells, remain incompletely understood. Here, we investigated time-dependent responses to microsecond PEF protocols (5 × 50 μs, 100-2000 V/cm, 1 Hz) combined with doxorubicin or bleomycin in drug-sensitive (MCF-7/WT) and doxorubicin-resistant (MCF-7/DOX) breast adenocarcinoma cells, using the ESOPE protocol as a clinical reference. ECT induced field- and time-dependent cytotoxicity, with bleomycin-based protocols producing sustained loss of viability in both cell lines. Treatment was accompanied by increased oxidative stress, reflected by enhanced lipid peroxidation, particularly at higher electric field strengths. Stress adaptation differed between phenotypes: bleomycin-based ECT strongly increased Hsp27 expression, especially in resistant cells, whereas Hsp70 showed divergent regulation, including suppression under combined PEF and doxorubicin exposure. Bleomycin-based ECT was associated with increased apoptotic signaling and limited necrosis, whereas doxorubicin-based protocols showed reduced efficacy in resistant cells. These findings identify oxidative stress and heat shock protein responses as key determinants of ECT outcome and support bleomycin as a preferential agent for electroporation-assisted treatment of chemoresistant breast tumors.