The role of bioreductive activation of antitumour anthracycline drugs in cytotoxic activity against sensitive and multidrug resistant leukaemia HL60 cells

European Journal of Pharmacology 674 (2012) 112–125

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European Journal of Pharmacology journal homepage: www.elsevier.com/locate/ejphar

Molecular and Cellular Pharmacology

The role of bioreductive activation of antitumour anthracycline drugs in cytotoxic activity against sensitive and multidrug resistant leukaemia HL60 cells Dorota Kostrzewa-Nowak a, Bohdan Bieg b, Mark J.I. Paine c, C. Roland Wolf c, Jolanta Tarasiuk a,⁎ a b c

Department of Biochemistry, University of Szczecin, 3c Felczaka St, 71-412 Szczecin, Poland Department of Physics, Maritime University, 1/2 Waly Chrobrego St, 70-500 Szczecin, Poland Cancer Research UK Molecular Pharmacology Unit, Biomedical Research Centre, Ninewells Hospital and Medical School, Dundee DD1 9SY, UK

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Article history: Received 4 July 2011 Received in revised form 8 October 2011 Accepted 30 October 2011 Available online 15 November 2011 Keywords: Anthracycline antitumour drug Sugar-modified and quinone-modified derivative NADPH cytochrome P450 reductase HL60 human promyelocytic leukaemia Multidrug resistance

a b s t r a c t Clinical usefulness of anthracyclines belonging to bioreductive antitumour drugs is limited by the occurrence of multidrug resistance (MDR). The aim of this study was to examine the role of structural factors of antitumour anthracycline drugs in the ability to undergo bioreductive activation by NADPH cytochrome P450 reductase (CPR) and determine the impact of this activation on increasing the activity especially in regard to MDR tumour cells. It was evidenced that at high NADPH concentration (500 μM) anthracyclines having non-modified quinone structure: doxorubicin (DOX), daunorubicin (DR) and idarubicin (IDA) were susceptible upon CPR catalysis to undergo a multi-stage chemical transformation concerning their chromophore part. Additionally, it was evidenced that the modification of the sugar moiety of DOX did not disturb the susceptibility of the obtained derivative (4′-O-tetrahydropyranyl-doxorubicin, pirarubicin, PIRA) to undergo CPR reductive activation. It was also evidenced that the derivatives having modified quinone groupment (5-iminodaunorubicin, 5-Im-DR) were not able to undergo reductive activation by CPR. The high impact of CPR-dependent reductive activation of anthracycline drugs on increasing the activity in regard to sensitive leukaemia HL60 cell line and its MDR sublines overexpressing P-glycoprotein (HL60/VINC) and MRP1 (HL60/DOX) was evidenced. Furthermore, significant changes in binding manner of activated compounds to naked DNA and cellular nucleus in comparison to their non-activated forms were also observed. It could prevent the export of formed adducts out of the cell by MDR proteins and may explain significant increases in intracellular accumulation of these compounds in HL60/VINC and HL60/DOX cells. © 2011 Elsevier B.V. All rights reserved.

1. Introduction The anthracycline antitumor agents (e.g. doxorubicin, DOX; daunorubicin, DR; idarubicin, IDA; pirarubicin, PIRA) are among the most effective drugs, currently available for the treatment of various human neoplastic diseases including leukaemias, lymphomas and solid tumours (Lown, 1993; Mandelli et al., 2009). Different mechanisms have been proposed for anthracycline antitumour effects including DNA intercalation with consequent inhibition of DNA biosynthesis, reactive oxygen species formation with induction of DNA damage, alkylation of DNA and DNA cross-linking, inhibition of topoisomerase II, activation of signalling pathways and apoptosis (for recent review, see Minotti et al., 2004). Reactive oxygen species generation by these agents is linked to their one-electron reduction by cellular oxidoreductases (Celik and Arinç, 2008; Garner et al.,

⁎ Corresponding author at: University of Szczecin, Department of Biochemistry, 3a Felczaka St, 71-412 Szczecin, Poland. Tel./fax: + 48 91 444 15 50. E-mail address: [email protected] (J. Tarasiuk). 0014-2999/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.ejphar.2011.10.047

1999; Mordente et al., 2001; Pawłowska et al., 2003; Vasquez-Vivar et al., 1997). Anthracycline-induced generation of O2–• , •OH, H2O2 and 1 O2 was proposed to be a major mechanism of their dose-dependent cardiotoxicity (Koh et al., 2002; Minotti et al., 2004; Myers et al., 1987; Tong et al., 1979). On the other hand, there is an increasing body of evidence that the reductive activation of anthracycline drugs leads to the generation of reactive intermediates capable of alkylation or crosslinking binding to DNA (Bartoszek, 2002; Cullinane et al., 1994; Cummings et al., 1991; Cutts et al., 2005; Kostrzewa-Nowak et al., 2005; Skarka et al., 2010; Skladanowski and Konopa, 1994; Taatjes et al., 1997; Zeman et al., 1998). The relevance of reductive activation of DOX by NADPH cytochrome P450 reductase (CPR) for increasing cytotoxic activity of this drug towards sensitive human breast cell line MCF-7 has been also demonstrated using purified rat CPR (Bartoszek and Wolf, 1992). It was also evidenced that the transfection of human CPR cDNA into Chinese hamster ovary cells increased the cytotoxic activity of DOX by 1.8–3.3-fold (Sawamura et al., 1996). However, the clinical usefulness of anthracycline drugs is limited by the occurrence of multidrug resistance (MDR) associated with the presence of membrane transporters (e.g. P-glycoprotein, MRP1),

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belonging to the ATP-binding cassette protein family (Borst et al., 2000; Gottesman and Pastan, 1993; Martínez-Lacaci et al., 2007). These transporters are responsible for the active ATP-dependent efflux of drugs out of resistant cells resulting in the decreased intracellular accumulation insufficient to inhibit resistant cell proliferation (Martínez-Lacaci et al., 2007; Zaman et al., 1994). In our previous study we have evidenced the important role of bioreductive activation of DOX by exogenously added CPR from human liver and NADPH in cytotoxic activity against 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) (Kostrzewa-Nowak et al., 2005). The aim of this study was to examine the role of structural factors of antitumour anthracycline drugs (modified at the chromophore part as well as at the sugar moiety) in the ability to undergo bioreductive activation by CPR and determine the impact of this activation on increasing their activity against sensitive and MDR leukaemic HL60 cells. 2. Materials and methods 2.1. Reagents Idarubicin (IDA), NADPH, superoxide dismutase (SOD) and vincristine were obtained from Sigma-Aldrich (Saint Louis, USA). Daunorubicin (DR) was kindly provided Drug Development Branch (Bethesda MD, USA). Pirarubicin (PIRA) was a gift from Pharmacia-Upjohn (Milano, Italy). 5-iminodaunorubicin (5-Im-DR) was synthesised according to the procedure published elsewhere (Tong et al., 1979). NADPH cytochrome P450 reductase from human liver (CPR) was obtained in C. Roland Wolf's laboratory, Ninewells Hospital and Medical School (Dundee, UK) according to the procedure described earlier (Smith et al., 1994). 2.2. Cell culture HL60 human promyelocytic leukaemia line (Division of Biology, Kansas State University, Manhattan, Kansas 66506, USA) and its resistant sublines: HL60/VINC (overexpressing P-glycoprotein) (McGrath et al., 1989) and HL60/DOX (overexpressing MRP1) (Krishnamachary and Center, 1993; Marsh et al., 1986) were cultured. The cells were grown in RPMI 1640 (Gibco Limited; Grand Island, USA) medium supplemented with 2 mM glutamine and 10% FBS (Gibco Limited; Grand Island, USA) at 37 °C in a humidified atmosphere of 95% air and 5% CO2. HL60/VINC cells were cultured in the presence of 10 nM vincristine and HL60/DOX cells in the presence of 200 nM DOX. All cultures (HL60, HL60/VINC, HL60/DOX) initiated at a density of 10 5 cells/ml grew exponentially to about 106 cells/ml in 72 h. They were counted before the assay using a Burker hemocytometer. Cell viability was assessed by trypan blue exclusion. 2.3. Enzymatic studies Stock solutions of anthracycline compounds and NADPH (C0 = 10 − 3 M) were prepared just prior to use. Concentrations were determined by diluting stock solutions in water to approximately 50 μM and using an extinction coefficient of ε480 = 11500 M− 1 cm− 1 for DR, IDA and PIRA, ε550 = 13800 M− 1 cm− 1 for 5-Im-DR and ε340 = 6220 M− 1 cm− 1 for NADPH, respectively. The reaction mixtures in 0.01 M K2HPO4/KH2PO4 buffer (pH 7.25) contained: 100 μM anthracycline drug, 100 μM or 500 μM NADPH, 4 μg/ml CPR and 0 or 500 U/ml SOD, respectively. All the reactions were initiated by the addition of CPR and conducted at 37 °C. Absorption spectra of tested anthracycline compounds were recorded at the indicated time points in the visible region (330–800 nm). NADPH oxidation was measured at λ = 340 nm using an extinction coefficient

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of ε = 6220 M − 1 cm − 1. Absorption measurements were made on a Marcel E330 spectrophotometer. 2.4. Cytotoxicity assays For each cell line, the cytotoxic effects of examined anthracyclines were determined by incubating cells (104) with 10 different concentrations of the compound for 72 h in standard 96-well plates. The enzymatic samples contained for the tested anthracycline acting in redox cycling: 100 μM drug, 100 μM NADPH and 4 μg/ml CPR; for the drug incubated in the presence of activating system: 100 μM examined anthracycline, 500 μM NADPH, 4 μg/ml CPR and 0 or 500 U/ml SOD (0.01 M K2HPO4/KH2PO4 buffer, pH 7.25 at 37 °C). The appropriate volumes of the enzymatic sample were added directly to the cell suspensions to yield concentration varying in the ranges: 0.01–500 nM for IDA and PIRA or 10 nM–10 μM in the case of DR and 5-Im-DR for all tested cells. Control assays were carried out for buffer alone at the highest percentage employed in culture medium (2%, v/v) and for NADPH alone (50 μM) or CPR alone (0.4 μg/ml) at the highest concentrations used in in vitro studies. The cell growth was determined by counting the viable cells in the presence of trypan blue using a Burker hemocytometer. 2.5. Cellular drug uptake The cellular uptake of anthracyclines (IDA, PIRA) alone (non-activated), acting in the redox cycling and incubating in the presence of the activating system (without SOD or in the presence of SOD, respectively) was followed by monitoring the changes of the fluorescence signal. This spectrofluorometric method has been largely described previously for anthracycline drugs (Tarasiuk and Garnier-Suillerot, 1992; Tarasiuk et al., 1989, 1993). The incubation of cells with the compound proceeds without compromising cell viability. All experiments were conducted in 1 cm quartz cuvettes containing 2 ml of 20 mM HEPES buffer plus 132 mM NaCl, 3.5 mM KCl, 1 mM CaCl2, 0.5 mM MgCl2, 5 mM glucose (pH 7.25) at 37 °C. In a typical experiment, 2 × 10 6 cells in logarithmic growth phase were suspended in 2 ml of HEPES buffer under vigorous stirring. At t0 20 μl of the stock compound solution (for “drug alone” assays) or 20 μl of an appropriate enzymatic sample was quickly added to this suspension yielding a concentration equal to 1 μM. The decrease in the fluorescence intensity F at 590 nm (λex = 480 nm) was followed as a function of time until the curve F = f(t) reached a plateau. All fluorescence measurements were made on a Perkin-Elmer LS 50B spectrofluorometer. 2.6. Intracellular accumulation of anthracyclines determined by flow cytometry Cells in logarithmic growth phase were suspended in 1 ml of 20 mM HEPES buffer containing 132 mM NaCl, 3.5 mM KCl, 1 mM CaCl2, 0.5 mM MgCl2, and 5 mM glucose (pH 7.25) at 37 °C to the final concentration of 10 6 cells/ml. At t0 10 μl of the stock compound solution (for “drug alone” assays) or 10 μl of an appropriate enzymatic sample was added to this suspension yielding a concentration equal to 1 μM and incubated at 37 °C in a humidified atmosphere of 95% air and 5% CO2 for 15 min in the case of IDA and PIRA or 5 h in the case of DR and 5-Im-DR, respectively. After the indicated incubation time the intensity of the fluorescence signal was measured by flow cytometry (FACSCalibur, Becton Dickinson). The measurements were conducted within the FL-2 fluorescence channel (bandpass filter, λ = 585 ± 24 nm) after an excitation with the argon-ion laser (λex = 488 nm) for DR, IDA, and PIRA or within the FL-4 fluorescence channel (bandpass filter, λ = 661 ± 16 nm) after an excitation with the red-diode laser (λex = 635 nm) for 5-Im-DR. For each experimental point, the fluorescence signal of 1 × 10 4 cells was measured. The data were analysed using BD CellQuest Pro as well as WinMDI (ver. 2.8) software.

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2.7. Interaction of anthracycline compounds with naked DNA High molecular weight calf thymus DNA was dissolved in PBS buffer for 3 h under vigorous stirring. DNA concentration was determined from absorbance measurements at 260 nm using an extinction coefficient ε260 = 13200 M − 1 cm − 1 (bp). The interaction of anthracycline compounds: DR, IDA, PIRA and 5-Im-DR (1 μM) alone (nonactivated), acting in the redox cycling and incubating in the presence of activating system (in the absence or in the presence of SOD) of the drug obtained upon CPR catalysis with naked DNA was examined by the fluorometric titration using a spectrofluorometric method (Tarasiuk et al., 1989). All experiments were conducted in 1 cm quartz cuvettes containing 2 ml of 20 mM HEPES buffer plus 132 mM NaCl, 3.5 mM KCl, 1 mM CaCl2, 0.5 mM MgCl2, and 5 mM glucose (pH 7.25) at 37 °C. At t0 20 μl of the stock compound solution (for “drug alone” assays) or 20 μl of an appropriate enzymatic sample was quickly added to 2 ml of HEPES buffer yielding a concentration equal to 1 μM The binding of anthracycline compounds to DNA was followed by monitoring the decrease of the fluorescence signal at 590 nm (λex = 480 nm) for DR, IDA and PIRA or at 620 nm (λex = 550 nm) for 5-Im-DR after the addition of 2 μM DNA portions. The fluorescence measurements were made on a Perkin-Elmer LS 50B spectrofluorometer. 2.8. Statistical analysis Results are presented as the mean± S.D. of at least five independent experiments. Statistical analysis of the significance level of the differences observed between analysed values found for non-activated and activated anthracycline compounds was carried out using Student's t-test. P b 0.05 was considered as a significant difference. 3. Results 3.1. Reduction of anthracycline drugs by NADPH cytochrome P450 reductase (CPR) — spectroscopic studies The structure of examined anthracycline drugs: daunorubicin (DR), idarubicin (IDA), pirarubicin (PIRA) and 5-iminodaunorubicin (5-Im-DR) is presented in Fig. 1. Fig. 2 illustrates the representative absorption spectra of samples recorded during the incubation of 100 μM anthracycline drug (DR, IDA PIRA or 5-Im-DR respectively) with 4 μg/ml CPR in the presence of NADPH at low concentration (100 μM) or at high concentration (500 μM) (Fig. 2). Fig. 2A–C contains also results found for the samples containing 100 μM drug (DR, IDA or PIRA, respectively), 500 μM NADPH, 4 μg/ml CPR and additionally 500 U/ml SOD. The absorption measurements at selected absorption wavelengths: 340 nm, 480 nm and 550 nm were also carried out continuously for each sample studied. These selected wavelengths represent the maximum absorption wavelengths for NADPH O

(340 nm), anthracycline compounds having non-modified quinine structure: DR, IDA, PIRA (480 nm) and 5-imino-DR (550 nm), respectively. For each examined anthracycline drugs, it was found that at low NADPH concentration (100 μM), a very important decrease in the absorption intensity at 340 nm (A340 nm) was observed immediately after the addition of CPR. It indicates that the drug caused a high stimulation of NADPH oxidation catalysed by CPR. However, no changes were observed in the absorption spectra of studied anthracyclines up to 3 h (the presented data show the absorption spectra recorded for the first 60 min only). In contrast, at high NADPH concentration (500 μM) in the case of anthracycline compounds having nonmodified quinone structure (DR, IDA and PIRA) after the addition of CPR not only the decrease in A340 nm was observed but after about 2 min the important decrease in A480 nm was also observed followed by further modifications of absorption spectra of these compounds. About 5 min after the addition of CPR the important shift of the absorption band of DR, IDA and PIRA was noticed indicating the modifications in the chromophore part of these drugs. During the prolongated incubation (1 h) the aglycone precipitation occurred. It was also found that after the addition of superoxide dismutase (SOD) to the enzymatic samples containing CPR and high concentrations of NADPH (500 μM) only the rapid oxidation of NADPH occurred but no changes were observed in the absorption spectra of DR, IDA and PIRA (Fig. 2A–C). The similar observations were found in our previous studies for DOX (Kostrzewa-Nowak et al., 2005). In the case of quinone-modified derivative of DR (5-Im-DR) at high NADPH concentration (500 μM), immediately after the addition of CPR a significant decrease in the absorption intensity at 340 nm (A340 nm) was observed indicating that 5-Im-DR caused NADPH oxidation catalysed by this enzyme. However, in contrast to results obtained for anthracycline compounds having nonmodified quinone structure (DR, IDA and PIRA), no changes in the absorption spectra of 5-Im-DR were observed in the presence of the activating system (Fig. 2D). 3.2. The activity of anthracycline drugs towards HL60 cell line and its multidrug resistant sublines: HL60/VINC and HL60/DOX The ability of examined anthracycline drugs (DR, IDA, PIRA and 5Im-DR) to inhibit the growth of human promyelocytic 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 or in the absence of exogenously added NADPH and CPR. All control cells proliferated during 72 h. The growth rate of the parent cells (HL60) was comparable with both resistant cells used in the study (HL60/VINC and HL60/DOX). All cultures initiated at a density of 10 5 cells/ml grew to about 10 6 cells/ml (control count) in 72 h. The cytotoxic effect of anthracycline drugs was determined by incubating cells (10 5) with 10 different concentrations of studied compounds for

OH COCH2 R1

R1

R2

R3

R4

DOX

OH

OCH3

O

H

DR

H

OCH3

O

H

IDA

H

H

O

H

PIRA

OH

OCH3

O

5-Im-DR

H

OCH3

NH

OH

R2

R3 CH 3

O H 2N

R4O

O

OH

O

Fig. 1. Structures of examined anthracycline drugs.

H

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72 h. In calculating cell growth (% of control) untreated cells were used as the control. The results obtained for each cell line are illustrated in Fig. 3. Additional assays carried out for buffer alone, NADPH alone or CPR alone at the highest concentrations used in in vitro

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studies showed that these agents had no effect on cell growth. 100% of control HL60, HL60/VINC and HL60/DOX cell growth was observed at the highest percentage of buffer employed in culture medium (2%, v/v) as well as at highest concentrations of NADPH (50 μM) or CPR

100 µM DR + 100 µM NADPH + 4 µg/ml CPR

A

CPR t0 = 0 min t1 = 1 min t2 = 2 min t3 = 5 min t4 = 15 min t5 = 60 min

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100 µM DR + 500 µM NADPH + 4 µg/ml CPR CPR 1.2

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100 µM DR + 500 µM NADPH + 4 µg/ml CPR + 500 U/ml SOD CPR 1.2

2.5

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Fig. 2. Spectroscopic changes followed during incubation of examined anthracycline drugs: A) DR, B) IDA, C) PIRA, D) 5-Im-DR in enzymatic systems. The selected absorption wavelengths: 340 nm, 480 nm and 550 nm represent maximum absorption wavelengths for NADPH, anthracycline compounds having non-modified quinone structure (DR, IDA and PIRA) and 5-Im-DR, respectively. The samples contained: 100 μM drug, indicated amount of NADPH (100 μM or 500 μM, respectively), 4 μg/ml CPR and indicated amount of SOD (0 or 500 U/ml, respectively). 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.

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B

100 µM IDA + 100 µM NADPH + 4 µg/ml CPR

1.0

0.7 1.2 0.6 1.0 0.5 0.8

A480

0.8 0.6

0.4

0.6

0.3 Absorbance at λ = 480 nm

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0.0 330 380 430 480 530 580 630 680 730 780

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CPR

t0 = 0 min t1 = 1 min t2 = 2 min t3 = 5 min t4 = 15 min t5 = 60 min

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100 µM IDA + 500 µM NADPH + 4 µg/ml CPR CPR 2.5 1.2

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100 µM IDA + 500 µM NADPH + 4 µg/ml CPR + 500 U/ml SOD CPR

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Time [min] Fig. 2 (continued).

(0.4 μg/ml) used. Control assays carried out for enzymatic samples containing simultaneously NADPH and CPR at the same highest concentrations showed that they have neither any effect on cell growth (100% of control cell growth was also observed, data not presented). Our results (Fig. 3), similarly to findings reported previously for DOX, showed that the incubation of HL60 sensitive and MDR resistant (HL60/VINC and HL60/DOX) cells with all examined anthracycline compounds (DR, IDA, PIRA and 5-Im-DR) pretreated in the presence

of CPR and low NADPH concentration (100 μM) had no effect in increasing its activity in comparison with the drug alone. In contrast, the incubation of HL60 cells with DR, IDA and PIRA pretreated with CPR at high NADPH concentration (500 μM) resulted in an important increase in cell growth inhibition. The increasing effect in cytotoxic activity of these compounds was conserved even the drug (DR, IDA or PIRA, respectively) was added after 10 min preincubation step with CPR and NADPH (data not presented). This effect was completely

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100 µM PIRA + 100 µM NADPH + 4 µg/ml CPR CPR t0 = 0 min 1.4 t1 = 1 min t2 = 2 min 1.2 t3 = 5 min t4 = 15 min t5 = 60 min 1.0

1.2 1.0

A480

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100 µM PIRA + 500 µM NADPH + 4 µg/ml CPR CPR 1.2 2.4

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100 µM PIRA + 500 µM NADPH + 4 µg/ml CPR + 500 U/ml SOD CPR 1.2

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Wavelength [nm] Fig. 2 (continued).

abolished in the presence of 500 U/ml SOD added to the enzymatic sample. Similar results were reported in our previous study for DOX (Kostrzewa-Nowak et al., 2005). However, in the case of 5-Im-DR, contrary to these results obtained for DOX, DR, IDA and PIRA, it was found that the incubation of this quinone-modified derivative with CPR at high NADPH concentration (500 μM) did not cause the increase in

the cytotoxic effect on HL60 sensitive and MDR resistant (HL60/VINC and HL60/DOX) cells in comparison to the drug alone (Fig. 3D). IC50 values (drug concentrations required to inhibit 50% of cell growth) are summarised in Table 1. As it can be seen, the important decrease in IC50 values was observed for DR, IDA and PIRA activated by CPR in comparison to the drug alone (non-activated) not only for

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D

100 µM 5-Im-DR + 100 µM NADPH + 4 µg/ml CPR t0 = 0 min t1 = 1 min t2 = 2 min t3 = 5 min t4 = 15 min t5 = 60 min

1.6 1.4

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10 12 14 16 18 20

Time [min] Fig. 2 (continued).

sensitive HL60 but also in the case of MDR resistant (HL60/VINC and HL60/DOX) cells examined. Similar results were published for DOX (Kostrzewa-Nowak et al., 2005). In contrast, no statistically significant changes were observed between IC50 values found for 5-Im-DR alone and the compound incubated in the presence of the activating system for each studied cells (HL60, HL60/VINC and HL60/DOX). 3.3. Interaction of anthracycline compounds with intact cells The interaction of anthracycline drugs with intact HL60, HL60/ VINC and HL60/DOX cells was studied for two selected anthracycline drugs (1 μM IDA and 1 μM PIRA) having the high kinetics of cellular influx. These studies were performed using our spectrofluorometric method largely described for anthracycline drugs (Tarasiuk and Garnier-Suillerot, 1992; Tarasiuk et al., 1989, 1993). It allows continuous monitoring of the uptake of fluorescent anthracycline molecules intercalating between the base pairs of nuclear DNA. Control assays performed in the study showed that the activation of examined anthracycline compounds by CPR did not affect their fluorescence properties (no changes in spectra shape as well as signal intensity were observed; data not presented). As it is shown in Fig. 4, during the incubation time of anthracycline drug alone (1 μM IDA or 1 μM PIRA, respectively) with examined cells (HL60, HL60/VINC and HL60/DOX) a significant decrease in the fluorescence signal has been observed until the steady-state was reached.

Similar results were found for 1 μM IDA and 1 μM PIRA operating in the redox cycling (at 100 μM NADPH concentration) during the incubation with sensitive (HL60) as well as resistant (HL60/VINC and HL60/DOX) cells. In contrast, no decrease in the fluorescence signal was observed for 1 μM IDA and 1 μM PIRA undergoing reductive conversion (in the presence of activating system — at 500 μM NADPH concentration). It could suggest the inhibition of the cellular uptake of the generated reactive intermediates or indicate another type of interaction of these metabolites with the intact cells (no intercalation between the base pairs of nuclear DNA). Furthermore, it should be emphasised that in the presence of SOD (500 U/ml) in the activating system, the interaction of examined anthracycline drugs (1 μM IDA and 1 μM PIRA) with cells was very similar to the interaction observed in the case of the drug alone or operating in the redox cycling. 3.4. Intracellular accumulation of anthracycline drugs in HL60, HL60/ VINC and HL60/DOX cells The results concerning the interaction of 1 μM IDA and 1 μM PIRA undergoing the reductive activation presented above showed the important differences in comparison with the drug alone and could suggest the inhibition of the cellular uptake of generated reactive intermediates into examined HL60, HL60/VINC and HL60/DOX cells. Therefore, the intracellular accumulation of anthracycline compounds: DR, IDA, PIRA and 5-Im-DR alone (non-activated), acting in

D. Kostrzewa-Nowak et al. / European Journal of Pharmacology 674 (2012) 112–125

HL60

Inhibition of cell growth [%]

A 100

100

80

80

80

60

60

60

40

40

40

20

20

20

0 100

1

10000

0 1

DR [nM]

Inhibition of cell growth [%]

80

80

80

60

60

60

40

40

20

20

1

100

10000

C

20

0 0.01

1

0 0.01

100

100

80

80

80

60

60

60

40

40

40

20

20

20

100

1000

0 0.01

PIRA [nM]

0 0.1

100

1

PIRA [nM] 100

100

80

80

80

60

60

60

40

40

40

20

20

20

0

0 100

10000

5-Im-DR [nM]

10000

100

1

10

100

1000

PIRA [nM]

100

1

1

IDA [nM]

100

1

10000

40

IDA [nM]

C 100

0 0.01

100

DR [nM] 100

IDA [nM]

Inhibition of cell growth [%]

1

10000

100

0 0.01

Inhibition of cell growth [%]

100

DR [nM]

B 100

D

HL60/DOX

HL60/VINC 100

0

119

0 1

100

10000

5-Im-DR [nM]

1

100

100000

5-Im-DR [nM]

drug alone (non-activated)

drug in the presence of activating system

drug „recycling”

drug in the presence of activating system + SOD

Fig. 3. Cytotoxic activity of examined anthracycline drugs: A) DR, B) IDA, C) PIRA, D) 5-Im-DR towards HL60, HL60/VINC and HL60/DOX cells. The enzymatic samples contained for drug “recycling”: 100 μM drug, 100 μM NADPH and 4 μg/ml CPR; for drug in the presence of the activating system: 100 μM drug, 500 μM NADPH and 4 μg/ml CPR; for drug in the presence of the activating system+ SOD: 100 μM drug, 500 μM NADPH, 4 μg/ml CPR and 500 U/ml SOD (0.01 M K2HPO4/KH2PO4 buffer, pH 7.25; 37 °C). The appropriate volumes of the enzymatic samples were added directly to the cell suspensions to yield the IDA and PIRA concentration varying in the range: 0.01–500 nM and 1 nM–10 μM in the case of DR and 5-Im-DR. The cytotoxic effect of studied drugs was determined by incubating cells (105) with 10 different concentrations of the compound for 72 h. The data points are from a representative experiment.

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the redox cycling and incubating in the presence of the activating system (without or with the addition of SOD) was determined by flow cytometry. Fig. 5 shows histograms obtained in typical experiments for examined samples after 15 min incubation of cells with compounds having a high kinetics of cellular uptake (IDA and PIRA) or 5 h incubation with compounds having a low kinetics of cellular uptake (DR and 5-Im-DR), respectively. The results obtained after the incubation of sensitive HL60 as well as resistant (HL60/VINC and HL60/DOX) cells with 1 μM DR, 1 μM IDA and 1 μM PIRA in the presence of activating system showed an important increase in intracellular drug fluorescence in comparison to the level found for the drug alone (non-activated). In contrast, it was found that the intracellular fluorescence of these anthracyclines operating in the redox cycling (at low NADPH concentration) as well as preincubated in the presence of activating system containing SOD were similar to fluorescence levels observed for drugs alone (Fig. 5A–C). Interestingly, it was found for each examined cells (HL60, HL60/VINC and HL60/DOX) that the intracellular fluorescence level of 5-Im-DR incubated in the presence of the activating system was comparable with the intracellular fluorescence level observed for this quinone modified derivative alone (Fig. 5D).

Table 1 Cytotoxic activity of examined drugs (DR, IDA, PIRA and 5-Im-DR) towards HL60 cell line and its multidrug resistant sublines: HL60/VINC and HL60/DOX. Cell line HL60

HL60/VINC

113 ± 17

381 ± 51

1448 ± 166

58 ± 12b

136 ± 25a

247 ± 24b

24.0 ± 4.0

42.2 ± 6.0

119 ± 35

17.7 ± 4.7b

31.9 ± 7.0a

7.5 ± 1.7

32.5 ± 7.5

45 ± 12

2.04 ± 0.42a

15.2 ± 2.4b

25.1 ± 4.6a

342 ± 74

587 ± 85

1375 ± 182

325 ± 25

579 ± 53

1384 ± 102

Drug

HL60/DOX

IC50 [nM]

DR alone (non-activated) DR in the presence of activating IDA alone (non-activated) IDA in the presence of activating PIRA alone (non-activated) PIRA in the presence of activating 5-Im-DR alone (non-activated) 5-Im-DR in the presence of activating

system

9.5 ± 1.5

b

system

system

system

IC50 is the drug concentration required to inhibit 50% of cell growth. The values represent mean ± S.D. of at least five independent experiments. The significance level of the differences observed (Student's t-test). a P b 0.01. b P b 0.001 versus values found for the drug alone (non-activated).

F590

A

HL60

The results concerning the interaction of 1 μM IDA and 1 μM PIRA undergoing reductive activation showed the important differences in comparison with the drug alone (non-activated). It could be presumed that it results from another type of interaction of generated

HL60/VINC

HL60/DOX

1 μM IDA

1 μM IDA

1 μM IDA

120

120

120

100

100

100

80

80

80

60

60

60

40

40

40

20

20

cells

0 0

5

20

cells

0

10

15

20

25

0

30

5

B

15

20

25

0

30

120

100

100

100

80

80

80

60

60

60

40

40

40

5

10

15

20

25

30

0

20

25

30

25

30

20

20

cells

15

1 μM PIRA

120

20

10

Time [min]

1 μM PIRA

1 μ M PIRA

0

5

Time [min]

120

0

cells

0

10

Time [min]

F590

3.5. Interaction of anthracycline compounds with naked DNA

cells

cells 0

Time [min]

5

10

0 15

20

Time [min]

25

30

0

5

10

15

20

Time [min]

drug alone (non-activated)

drug in the presence of activating system

drug “recycling”

drug in the presence of activating system + SOD

Fig. 4. Cellular uptake of examined anthracycline drugs: A) IDA, B) PIRA by HL60, HL60/VINC and HL60/DOX cells. Cells in logarithmic growth phase were suspended in a cuvette filled with 2 ml of 20 mM HEPES buffer containing 132 mM NaCl, 3.5 mM KCl, 1 mM CaCl2, 0.5 mM MgCl2 and 5 mM glucose (pH 7.25, 37 °C) under vigorous stirring. At t0 20 μl of a drug solution (for “drug alone” assays) or 20 μl of an appropriate enzymatic sample (described in detail in the legend to Fig. 3) was added to the cell suspension yielding a 1 μM drug (IDA or PIRA, respectively) concentration. Fluorescence intensity at 590 nm (λex = 480 nm) was recorded as a function of incubation time until the steady state. The presented figures are from a representative experiment.

D. Kostrzewa-Nowak et al. / European Journal of Pharmacology 674 (2012) 112–125

HL60

HL60/VINC

A

121

HL60/DOX

125

A

C

B

100

101

102

103

A

B C

104

0

0

0

Events

BC

Events

140

A

Events

120

DR

100

101

102

FL2-H

103

104

100

101

FL2-H

102

103

104

FL2-H

c a

300

C

300

300

200

100

B

B

B

FL2-H

FL2-H

200

c

C 100

100

B 0

200

B

B

B

B

B

0

0

IDA

B C

A

C

101

102

103

104

B C

0

0

0 100

A

Events

B

Events

Events

140

B

140

A

140

FL2-H

C

100

101

102

FL2-H

103

104

100

101

FL2-H

102

103

104

FL2-H

c

1200

1200

800 400 0

B

B

B

1600

c

C

1200

FL2-H

1600

FL2-H

FL2-H

C 1600

800 400 0

B

B

B

c

800

C 400

B

B

B

0

Fig. 5. Intracellular accumulation of examined anthracycline drugs: A) DR, B) IDA, C) PIRA, D) 5-Im-DR in HL60, HL60/VINC and HL60/DOX cells. Cells in logarithmic growth phase were suspended in 1 ml of 20 mM HEPES buffer containing 132 mM NaCl, 3.5 mM KCl, 1 mM CaCl2, 0.5 mM MgCl2 and 5 mM glucose (pH 7.25, 37 °C) to the final concentration of 106 cells/ml. At t0 10 μl of a drug solution (for “drug alone” assays) or 10 μl of an appropriate enzymatic sample (described in detail in the legend to Fig. 3.) was added to the cell suspension yielding 1 μM drug (DR, IDA, PIRA or 5-Im-DR, respectively) concentration and incubated at 37 °C in a humidified atmosphere of 95% air and 5% CO2 for 15 min in the case of IDA and PIRA or 5 h in the case of DR and 5-Im-DR, respectively. After the indicated incubation time the intensity of the fluorescence signal was measured within the FL-2 fluorescence channel (bandpass filter, λ = 585 ± 24 nm) after an excitation with the argon-ion laser (λex = 488 nm) for DR, IDA, and PIRA or within the FL-4 fluorescence channel (bandpass filter, λ = 661 ± 16 nm) after an excitation with the red-diode laser (λex = 635 nm) for 5-Im-DR, respectively. For each experimental point, the fluorescence signal of 1 × 104 cells was measured. The experiment was repeated five times and the presented histograms are representative examples. The significance level of the differences observed (Student's t-test): aP b 0.01; bP b 0.001; cP b 0.0001 vs. values found for the drug alone (non-activated).

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HL60

C

HL60/VINC

HL60/DOX

A

101

102

103

104

100

101

102

FL2-H

C

101

102

B

100

C

200

150 100

50

50

0

0

B

B

104

a

C

150 100

B

50

B

B

B

0

5-Im-DR A

150

B, C

100

101

102

103

104

A

B, C

0

0

0

Events

150

B, C

Events

A

Events

150

D

100

101

FL4-H

102

103

104

100

101

FL4-H

600

B

600

C

0

A

control cells

B

104

C

C FL4-H

FL4-H

200

103

600

B 400

102

FL4-H

B

FL4-H

103

FL2-H

FL2-H

B

FL2-H

FL2-H

100

250

200

150

B

104

c

250

200

103

FL2-H

c

250

BC

0

0

0 100

A

Events

BC

Events

130

BC

Events

150

A

130

PIRA

400

400

200

200

0

0 cells + drug alone (non-activated)

C

cells + drug in the presence of activating system

cells + drug “recycling” cells + drug in the presence of activating system + SOD

Fig. 5 (continued).

reactive intermediates with the nucleus. To verify this hypothesis, the interaction of anthracycline compounds (1 μM) with naked DNA was studied by the fluorometric titration with naked DNA at 37 °C in

HEPES buffer (pH 7.25). An important quenching of the fluorescence signal for all anthracyclines studied (1 μM DR, 1 μM IDA, 1 μM PIRA and 1 μM 5-Im-DR) was observed in the case of drugs alone (non-

D. Kostrzewa-Nowak et al. / European Journal of Pharmacology 674 (2012) 112–125

A

B

1 μM DR

1 μM IDA

μ DNA 2 µM

μ DNA 2 µM 120

... ... ...

100

100

80

80

F590

F590

120

... ... ...

60

... ...

60

40

...

40

HEPES buffer 0

5

15

10

C

25

30

0

5

10

15

20

25

20

25

Time [min] 1 μM 5-Im-DR

D

1 μM PIRA

μ DNA 2 µM

μ DNA 2 µM 140

120

120

100

... ...

100

...

80 60

... ......

40

... ... ... ... ... ...

80

F590

F590

......

HEPES buffer

0 20

Time [min]

60 40 20

20 0

...

20

20 0

123

HEPES buffer 0

5

10

15

HEPES buffer

0 20

25

30

Time [min]

0

5

10

15

Time [min]

drug alone (non-activated)

drug in the presence of activating system

drug “recycling”

drug in the presence of activating system + SOD

Fig. 6. Interaction of examined anthracycline drugs: A) DR, B) IDA, C) PIRA, D) 5-Im-DR with naked DNA. At t0 20 μl of a drug solution (for “drug alone” assays) or 20 μl of an appropriate enzymatic sample (described in detail in the legend to Fig. 3) was added to 2 ml HEPES buffer containing 132 mM NaCl, 3.5 mM KCl, 1 mM CaCl2, 0.5 mM MgCl2 and 5 mM glucose (pH 7.25, 37 °C) yielding 1 μM drug (DR, IDA, PIRA or 5-Im-DR, respectively) concentration The binding of anthracycline compounds to DNA (0–50 μM) was followed by monitoring the decrease of the fluorescence signal at 590 nm (λex = 480 nm) for DR, IDA and PIRA or at 620 nm (λex = 550 nm) for 5-Im-DR after the addition of 2 μM DNA portions. Data shown are from a representative experiment.

activated) and operating in the redox cycling after the addition of naked DNA (Fig. 6). However, much less important decrease in the fluorescence signal was observed for DR, IDA and PIRA undergoing reductive conversion whereas in the presence of SOD (500 U/ml) in the activating system, the interaction of examined anthracycline drugs (1 μM DR, 1 μM IDA and 1 μM PIRA, respectively) with naked DNA was very similar to the interaction observed in the case of the drug alone or operating in the redox cycling (Fig. 6A–C). In contrast, the results of our studies made under the same condition for 1 μM 5-Im-DR showed that the preincubation of this quinonemodified derivative in the presence of the activating system did not change its interaction with naked DNA in comparison to the drug alone or operating in the redox cycling (Fig. 6D).

4. Discussion The ability of neoplastic cells to develop multi-drug resistance (MDR) to chemotherapeutic agents (e.g. anthracyclines, vinca alkaloids, podophylotoxins, colchicine), structurally dissimilar and having different intracellular targets constitutes the major problem in cancer therapy (Fekete et al., 2005; Martínez-Lacaci et al., 2007). The MDR transporters (e.g. P-glycoprotein, MRP1) are responsible for the active efflux of drugs out of resistant cells resulting in the decreased

intracellular accumulation insufficient to inhibit resistant cell proliferation (Berger et al., 2005; Ma et al., 2009; Marbeuf-Gueye et al., 1999). In our previous study we have evidenced the important role of bioreductive activation of DOX by exogenously added NADPH cytochrome P450 reductase (CPR) from human liver and NADPH in cytotoxic activity against 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) (Kostrzewa-Nowak et al., 2005). In this work we examine the bioreductive activation of several anthracycline drugs (modified at the chromophore part as well as at the sugar moiety) by CPR in order to identify the role of structural factors in the ability to undergo this activation and determine its impact on increasing the activity especially in regard to MDR tumour cells. Spectroscopic studies performed during incubation of anthracycline drugs in enzymatic systems containing CPR and various amounts of NADPH showed, similarly to results previously found for DOX (KostrzewaNowak et al., 2005) as well as to data reported by Saunders et al. (2000) for tirapazamine, that the availability of NADPH as a cofactor of enzymatic reactions was a crucial factor determining the route of bioreductive drug activation. It suggests that NADPH could participate in forming a coupled interactive system and, in consequence, constitutes a control point in drug activation by cellular oxidoreductases. It was

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evidenced that at high NADPH concentration (500 μM) anthracyclines having non-modified quinone structure: daunorubicin (DR) and idarubicin (IDA) were susceptible upon CPR catalysis to undergo a multistage chemical transformation concerning its chromophore part, whereas at low NADPH concentration (100 μM) they underwent only the redox cycling. Furthermore, it was found using superoxide dismutase (SOD) decomposing O2–• that the formation of superoxide radical in a one-electron reduction pathway had the key importance in further steps of the bioreductive conversion of these compounds by CPR observed in the presence of high concentration of NADPH (500 μM). It indicates that the first stage of the CPR-dependent multi-stage transformations of anthracycline drugs having non-modified quinone structure undergo probably according to the mechanism of the redox cycling. It seems that O2–• could be involved (directly or indirectly) in chemical transformations of these compounds. Similar results were found previously for DOX (Kostrzewa-Nowak et al., 2005). Additionally, it was evidenced that the modification of the sugar moiety of anthracycline drug by attaching the tetrahydropyranyl substituent at 4′-OH position of DOX did not disturb the susceptibility of the obtained derivative (4′O-tetrahydropyranyl-doxorubicin, pirarubicin, PIRA) to undergo CPR multi-stage reductive activation. Simultaneously, in the second part of the work we have evidenced that the presence of CPR catalysing only the redox cycling of these compounds (DR, IDA, PIRA) had no effect in increasing their activity against 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). In contrast, an important increase in the activity of these anthracyclines having non-modified quinone structure after its reductive conversion by CPR was observed against HL60 as well as HL60/VINC and HL60/DOX cells. Similar results were found previously for DOX (Kostrzewa-Nowak et al., 2005). It is worth to note that the data presented in the study for anthracycline having non modified quinone structure: DR, IDA, PIRA (similar increase of about 2–6 fold in cytotoxic activity against resistant HL60/ VINC and HL60/DOX cells as it was observed in the case of sensitive HL60 cell line) suggest that reactive metabolites of these compounds generated extracellulary are able to enter the cell and bind to cellular targets before being extruded by MDR exporting pumps. At this stage of our research the nature of these metabolites is unknown. It is evident that their formation is related to the modifications of the chromophore part of anthracycline molecule resulted in the important changes of absorption spectrum. The data obtained evidenced also that they were relatively long-lived species because the increasing effect in cytotoxic activity of DR, IDA and PIRA, similarly to results previously found for DOX, was conserved even the drug was added after 10 min preincubation step with NADPH and CPR. Furthermore, significant changes in binding manner of activated compounds (DR, IDA, PIRA) to naked DNA and cellular nucleus in comparison to their non-activated forms were also observed suggesting that the generated reactive metabolites would covalently bind to DNA without intercalating between the base pairs. It could prevent the export of formed adducts out of the cell by MDR proteins (P-glycoprotein and MRP1) and may explain significant increases in intracellular accumulation of these compounds in MDR cells: HL60/VINC and HL60/DOX (as it has been demonstrated using flow cytometry technique) and as a consequence, increasing cytotoxic activity of activated compounds against MDR cell lines. In contrast, it was evidenced in the present study that the derivative having modified quinone groupment (5-Im-DR) was not able to undergo reductive activation by CPR (no changes were observed in the absorption spectra of the drug in the presence of the activating system, no effect of exogenously added CPR was observed in modulating the cytotoxicity against sensitive HL60 as well as resistant HL60/VINC and HL60/DOX cells in comparison to 5-Im-DR alone, no increase in the intracellular accumulation of this derivative and no

changes in its binding to naked DNA in the presence of the activating system were observed). It proves that the presence of modified quinone function is the structural factor excluding reductive activation of the compound by CPR. According to the literature data, metabolic activation of drugs can also undergo efficiently inside tumour cells. It is known that intracellular CPR expression involved in the activation of bioreductive agents can be modulated in cells by many internal factors such as oxygen deficiency, intracellular pH changes and by malignant transformations themselves (Adams and Stratford, 1994; Forkert et al., 1996). The development of a gene directed enzyme prodrug therapy (GDEPT) approach targeted toward specific oxidoreductase enzyme for antitumour drug bioactivation is also in progress in many laboratories (Baldwin et al., 2003; Cowen et al., 2003; Schmalix et al., 1996). However, the reductive activation of antitumor drugs in situ in target cells by CPR could be influenced by several cellular factors e.g. bioavailability of NADPH, competitive metabolic pathways of the drug catalysed by other enzymes depending on their cellular levels or rapid decomposition of ROS and drug free radicals by the antioxidant defence system of the cell. In the presented study the metabolic activation of anthracycline compounds having non-modified quinone structure (DR, IDA, PIRA) occurred under well-controlled experimental conditions and the obtained results indicate that reactive metabolites of these bioreductive agents formed extracellularly are able to penetrate into the tumour cell and bind to intracellular targets before being decomposed. Nevertheless, in further steps of the study it would be also interesting to examine the intracellular activation of these drugs in leukaemia cells overexpressing CPR. 5. Conclusions It was evidenced that at high NADPH concentration (500 μM) anthracyclines having non-modified quinone structure: daunorubicin (DR) and idarubicin (IDA) were susceptible upon CPR catalysis to undergo a multi-stage chemical transformation concerning its chromophore part, whereas at low NADPH concentration (100 μM) they underwent only the redox cycling. Additionally, it was evidenced that the modification of the sugar moiety of anthracycline drug by attaching the tetrahydropyranyl substituent at 4′-OH position of doxorubicin (DOX) did not disturb the susceptibility of the obtained derivative (4′-O-tetrahydropyranyl-doxorubicin, pirarubicin, PIRA) to undergo CPR reductive activation. It was also evidenced that the derivative having modified quinone groupment (5-iminodaunorubicin, 5-Im-DR) was not able to undergo reductive activation by CPR. It proves that the presence of modified quinone function is the structural factor excluding reductive activation of the compound by CPR. Furthermore, the data presented in the study suggest that the reductive activation of anthracycline drugs having non-modified quinine structure by exogenously added CPR constitutes a possibility to potentiate the antitumour activity of these clinically important drugs not only towards sensitive leukaemia cells but also against resistant cells overexpressing MDR exporting pumps. Thus, it would be proposed that these reactive metabolites generated by CPR are able to bind to cellular targets before being pumped out of the cell by P-glycoprotein or MRP1. However, the clinical significance of the presented results obtained in the model system remains to be elucidated. Therefore, further studies are needed to prove the potential importance of the presented data in leukaemia therapy. Acknowledgements These studies were supported by the Faculty of Natural Sciences, University of Szczecin, Poland and Ministry of Science and Higher Education, Poland (Grant no. N401 138 31/2952). The authors acknowledge Magdalena Rutkowska for her technical assistance and Bartosz Krefta for typewriting help.

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