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.

Figure 1

Structure of doxorubicin (DOX).

Figure 2

Spectroscopic changes followed during incubation of DOX in enzymatic systems. The selected absorption wavelengths, 340 and 480 nm, represent the maximum absorption wavelengths for NADPH and DOX, respectively. The samples contained the following: (A) 100 μM DOX, 100 μM NADPH and 0.1 mg ml⁻¹ CPR; (B) 100 μM DOX, 500 μM NADPH and 0.1 mg ml⁻¹ CPR; (C) 100 μM DOX, 500 μM NADPH, 0.1 mg ml⁻¹ CPR and 500 U ml⁻¹ SOD. The measurements were carried out in 0.01 M K₂HPO₄/KH₂PO₄ buffer (pH 7.25) at 37°C. The reactions were initiated by the addition of CPR. Data shown are from a representative experiment.

Figure 2

Spectroscopic changes followed during incubation of DOX in enzymatic systems. The selected absorption wavelengths, 340 and 480 nm, represent the maximum absorption wavelengths for NADPH and DOX, respectively. The samples contained the following: (A) 100 μM DOX, 100 μM NADPH and 0.1 mg ml⁻¹ CPR; (B) 100 μM DOX, 500 μM NADPH and 0.1 mg ml⁻¹ CPR; (C) 100 μM DOX, 500 μM NADPH, 0.1 mg ml⁻¹ CPR and 500 U ml⁻¹ SOD. The measurements were carried out in 0.01 M K₂HPO₄/KH₂PO₄ buffer (pH 7.25) at 37°C. The reactions were initiated by the addition of CPR. Data shown are from a representative experiment.

Figure 3

Cytotoxic activity of doxorubicin (DOX) towards (A) HL60 cells, (B) HL60/VINC cells and (C) HL60/DOX cells. The enzymatic samples contained as follows: for DOX ‘recycling’: 100 μM DOX, 100 μM NADPH and 0.1 mg ml⁻¹ CPR; for DOX activated (undergoing reductive conversion): 100 μM DOX, 500 μM NADPH and 0.1 mg ml⁻¹ CPR; for DOX activated+SOD: 100 μM DOX, 500 μM NADPH, 0.1 mg ml⁻¹ CPR and 500 U ml⁻¹ SOD (0.01 M K₂HPO₄/KH₂PO₄ buffer, pH 7.25; 37°C). The appropriate volumes of the enzymatic sample were added directly to the cell suspension to yield the DOX concentration varying in the range of 0.1–100 nM, 5 nM–3 μM and 5 nM–5 μM for HL60 (A), HL60/VINC (B) and HL60/DOX (C) cells, respectively. The cytotoxic effect of DOX was determined by incubating cells (10⁵) with 10 different concentrations of the compound for 72 h. The data points are from a representative experiment.

Figure 4

Schema representing the reduction of DOX by CPR: (A) redox cycling at low NADPH concentration (100–350 μM); (B) reductive conversion at high NADPH concentration (500 μM–2 mM).