The exposure of cancer cells to hyperthermia, iron oxide nanoparticles, and mitomycin C influences membrane multidrug resistance protein expression levels

Abstract

Karolin Franke, Melanie Kettering, Kathleen Lange, Werner A Kaiser, Ingrid Hilger Institute of Diagnostic and Interventional Radiology, Department of Experimental Radiology, Institute of Diagnostic and Interventional Radiology I, University Hospital Jena, Friedrich-Schiller Universität Jena, Jena, Germany Purpose: The presence of multidrug resistance-associated protein (MRP) in cancer cells is known to be responsible for many therapeutic failures in current oncological treatments. Here, we show that the combination of different effectors like hyperthermia, iron oxide nanoparticles, and chemotherapeutics influences expression of MRP 1 and 3 in an adenocarcinoma cell line. Methods: BT-474 cells were treated with magnetic nanoparticles (MNP; 1.5 to 150 µg Fe/cm2) or mitomycin C (up to 1.5 µg/cm2, 24 hours) in the presence or absence of hyperthermia (43°C, 15 to 120 minutes). Moreover, cells were also sequentially exposed to these effectors (MNP, hyperthermia, and mitomycin C). After cell harvesting, mRNA was extracted and analyzed via reverse transcription polymerase chain reaction. Additionally, membrane protein was isolated and analyzed via sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting. Results: When cells were exposed to the effectors alone or to combinations thereof, no effects on MRP 1 and 3 mRNA expression were observed. In contrast, membrane protein expression was influenced in a selective manner. The effects on MRP 3 expression were less pronounced compared with MRP 1. Treatment with mitomycin C decreased MRP expression at high concentrations and hyperthermia intensified these effects. In contrast, the presence of MNP only increased MRP 1 and 3 expression, and hyperthermia reversed these effects. When combining hyperthermia, magnetic nanoparticles, and mitomycin C, no further suppression of MRP expression was observed in comparison with the respective dual treatment modalities. Discussion: The different MRP 1 and 3 expression levels are not associated with de novo mRNA expression, but rather with an altered translocation of MRP 1 and 3 to the cell membrane as a result of reactive oxygen species production, and with shifting of intracellular MRP storage pools, changes in membrane fluidity, etc, at the protein level. Our results could be used to develop new treatment strategies by repressing mechanisms that actively export drugs from the target cell, thereby improving the therapeutic outcome in oncology. Keywords: magnetic nanoparticles, hyperthermia, chemotherapy, drugs, MDR, MRP, cancer, nanotechnology, iron oxide