The role of bioreductive activation of doxorubicin in cytotoxic activity against leukaemia HL60-sensitive cell line and its multidrug-resistant sublines

Abstract

Clinical usefulness of doxorubicin (DOX) is limited by the occurrence of multidrug resistance (MDR) associated with the presence of membrane transporters (e.g. P-glycoprotein, MRP1) responsible for the active efflux of drugs out of resistant cells. Doxorubicin is a well-known bioreductive antitumour drug. Its ability to undergo a one-electron reduction by cellular oxidoreductases is related to the formation of an unstable semiquionone radical and followed by the production of reactive oxygen species. There is an increasing body of evidence that the activation of bioreductive drugs could result in the alkylation or crosslinking binding of DNA and lead to the significant increase in the cytotoxic activity against tumour cells. The aim of this study was to examine the role of reductive activation of DOX by the human liver NADPH cytochrome P450 reductase (CPR) in increasing its cytotoxic activity especially in regard to MDR tumour cells. It has been evidenced that, upon CPR catalysis, DOX underwent only the redox cycling (at low NADPH concentration) or a multistage chemical transformation (at high NADPH concentration). It was also found, using superoxide dismutase (SOD), that the first stage undergoing reductive activation according to the mechanism of the redox cycling had the key importance for the metabolic conversion of DOX. In the second part of this work, the ability of DOX to inhibit the growth of human promyelocytic-sensitive leukaemia HL60 cell line as well as its MDR sublines exhibiting two different phenotypes of MDR related to the overexpression of P-glycoprotein (HL60/VINC) or MRP1 (HL60/DOX) was studied in the presence of exogenously added CPR. Our assays showed that the presence of CPR catalysing only the redox cycling of DOX had no effect in increasing its cytotoxicity against sensitive and MDR tumour cells. In contrast, an important increase in cytotoxic activity of DOX after its reductive conversion by CPR was observed against HL60 as well as HL60/VINC and HL60/DOX cells.

Activation of the PI3K/Akt and MAPK signaling pathways antagonizes adriamycin-induced HL-60 leukemia cell apoptosis

“…Several mechanisms have been proposed for the antitumor effects of adM, including dna intercalation, free radical formation with induction of dna damage, inhibition of topoisomerase ii and activation of apoptosis-related signaling pathways (5)(6)(7)(8)(9). However, certain leukemia cells still exhibit insensitivity or resistance to adM, which results in therapy failure and tumor relapse.…”

Role of Drug Metabolism in the Cytotoxicity and Clinical Efficacy of Anthracyclines –

“…The study involved the reductive activation of DOX by CPR and NADPH extracellularly, followed by treatment of the cells, allowing the metabolite to diffuse into the cells. Their findings indicated that NADPH is a necessary cofactor for CPR-dependent reductive conversion (semiquinone formation) and this formation does not occur at NADPH concentrations below 500 M. Only at high NADPH concentrations was CPR effective at both reductive conversion of DOX and achieving between a two and three-fold increase in toxicity in both the DOX-sensitive and DOX-resistant human leukemia cell lines [98]. The study was conducted at clini-cally relevant doses of DOX, since DOX levels can reach 1-2 M in the plasma of patients receiving treatment [14].…”

“…Although the ability of DOX to generate reactive oxygen species in tumors has been well demonstrated, several studies suggest that its ability to create covalent modifications or adducts in cellular materials accounts for the enhanced effect of the semiquinone metabolite relative to the quinone [67,98]. Cummings et al have suggested that within tumors, DOX metabolism is mostly impacted by NADPH-cytochrome P450 reductase [99].…”

“…Kostrzewa-Nowak et al investigated the effects of human liver CPR on reductive activation of DOX during the treatment of human promyelocytic HL60 cells and multi-drug resistant derivatives of these cells overexpressing either P-glycoprotein or MRP-1 [98]. The study involved the reductive activation of DOX by CPR and NADPH extracellularly, followed by treatment of the cells, allowing the metabolite to diffuse into the cells.…”

“…The study was conducted at clini-cally relevant doses of DOX, since DOX levels can reach 1-2 M in the plasma of patients receiving treatment [14]. Interestingly, since drug potentiation occurred in cells overexpressing drug transporters capable of exporting DOX out of the cells, it was further proposed by Kostrzewa-Nowak et al that the reactive metabolite is able to bind to cellular targets and escape MDR protein pumps [98]. These investigators also showed that the addition of superoxide dismutase, an oxygen radical-scavenging enzyme, abrogated reductive conversion of DOX by CPR in the presence of sufficient NADPH [98].…”