Nrf2-Dependent Expression of Glutathione Antioxidant System Genes and Redox Status in Cells of In Vivo Drug-Resistant Murine P388 Leukemia Strains.

We measured the content of ROS and malondialdehyde in cells of in vivo drug-resistant murine P388 leukemia strains. It was found that the strains did not differ by malondialdehyde concentration, but intracellular concentration of ROS in cells of the cyclophosphamide-resistant strain (P388/CP) was higher than in cells of the original (P388) and other studied strains (P388/Rub, P388/cPt). Nuclear localization of the transcription factor Nrf2 in cells of strain P388/CP attested to its constitutive activation. Enhanced relative expression of the GCLM gene was found in all studied drug-resistant strains; the expression of the GSR and GPX1 genes was increased only in cells of the cyclophosphamide-resistant strain. These findings suggest that the mechanism of resistance of strain P388/CP is associated with increased activity of glutathione metabolism that developed as a result of activation of the antioxidant response transcription factor Nrf2 against the background of high intracellular concentration of ROS.

Activity of Antioxidant Enzymes and Content of Reduced Glutathione in Cells of Drug-Resistant Murine Leukemia P388 Strains.

Activities of superoxide dismutase and catalase and content of reduced glutathione in cells of drug-resistant murine leukemia P388 strains were studied without or after administration of antitumor compounds. In the absence of chemotherapeutic agents, no significant differences in activities of the studied enzymes in cells of the initial strain and strains resistant to cyclophosphamide, cisplatin, and rubomycin were observed. Compounds to which resistance was developed did not significantly affect activity of enzymes in cells of drug-resistant strains, while the use of compounds that were not resistance inductors was accompanied by a significant decrease in enzyme activity in cells resistant to cisplatin and rubomycin. In cells of strains resistant to cisplatin and cyclophosphamide, the content of reduced glutathione significantly differed from that in the initial strain. In addition, the concentration of reduced glutathione in cells of cyclophosphamide-resistant strain considerably decreased upon addition of the drug producing a therapeutic effect. Our findings suggest that the mechanism of resistance of in vivo derived cyclophosphamide resistant cell strain is related to increased level of reduced glutathione and activity of its metabolism.