Increased resistance of tumor cells to daunorubicin after transfection of cDNAs coding for anthracycline inactivating enzymes

Cancer Letters 255 (2007) 49–56 www.elsevier.com/locate/canlet

Increased resistance of tumor cells to daunorubicin after transfection of cDNAs coding for anthracycline inactivating enzymes Mariann Plebuch a, Michael Soldan a, Christoph Hungerer a, Lutz Koch a, Edmund Maser a,b,* a

Institute of Toxicology and Pharmacology for Natural Scientists, University Medical School Schleswig-Holstein, Campus Kiel, Brunswiker Strasse 10, 24105 Kiel, Germany b Department of Pharmacology and Toxicology, School of Medicine, Philipps-University of Marburg, Karl-von-Frisch-Strasse 1, 35033 Marburg, Germany Received 19 February 2007; received in revised form 22 March 2007; accepted 23 March 2007

Abstract Carbonyl reduction is a main but undesired metabolic pathway of the anti-cancer drug daunorubicin (DRC). The resulting alcohol metabolite daunorubicinol has a far less anti-tumor potency and, in addition, is responsible for the life-threatening cardiac toxicity that limits the clinical use of DRC. Elevated levels of carbonyl-reducing enzymes in cancer cells may therefore contribute to the development of DRC chemoresistance and affect the clinical outcome. In the present investigation, human pancreas carcinoma cells were transfected with three important DRC reductases, namely carbonyl reductase (CBR1), aldehyde reductase (AKR1A1) and aldose reductase (AKR1B1), and levels of resistance towards DCR determined. Overexpression of all three reductases lead to a higher DRC inactivation and to an elevation of chemoresistance (7-fold for CBR1, 4.5-fold for AKR1A1 and 3.7-fold for AKR1B1), when IC50-values were considered. Coadministration of DRC reductase inhibitors in DRC chemotherapy may be desirable since this would reduce the formation of the cardiotoxic alcohol metabolite and prevent drug resistance.  2007 Elsevier Ireland Ltd. All rights reserved. Keywords: Daunorubicin; Daunorubicinol; Drug resistance; Cardiotoxicity; Carbonyl reductase; Aldo–keto reductase

Abbreviations: AKR1A1, human aldehyde reductase; AKR1B1, human aldose reductase; CBR1, human carbonyl reductase; DRC, daunorubicin; DRCOL, daunorubicinol. * Corresponding author. Address: Institute of Toxicology and Pharmacology for Natural Scientists, University Medical School Schleswig-Holstein, Campus Kiel, Brunswiker Strasse 10, 24105 Kiel, Germany. Tel.: +49 431 597 3540; fax: +49 431 597 3558. E-mail address: [email protected] (E. Maser).

1. Introduction Resistance towards anticancer drugs is a major problem in the chemotherapy of malignant tumors. The development of classical multidrug-resistance (MDR) is generally associated with a decrease of intracellular drug concentrations through an

0304-3835/$ - see front matter  2007 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.canlet.2007.03.018

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ATP-dependent efflux of the unmodified drug from the cell [1]. This form of MDR is the result of, at least in part, the activity of efflux transporters of the ATP-binding cassette (ABC) family such as ABCB1 (P-glycoprotein 170, P-gp), the ABCC (multidrug resistance-related protein, MRP) family and ABCG2 (breast cancer resistance protein, BCRP) [2–5]. Another mechanism of resistance towards chemotherapeutic drugs is the induced expression of drug metabolizing enzymes, like aldehyde dehydrogenase [6] or the phase II conjugating enzymes glucuronyl transferase and glutathione transferase [7–10]. Anthracycline antibiotics such as daunorubicin (DRC) and doxorubicin are among the most effective anti-cancer drugs ever developed [11]. Their main antineoplastic effect is topoisomerase II inhibition, DNA intercalation, and RNA synthesis inhibition [12,13]. Other proposed mechanisms of anthracycline action include generation of free radicals and induction of apoptosis [13]. As with any other anticancer agent, however, the clinical use of both doxorubicin and DRC soon proved to be hampered by such serious problems as the development of resistance in tumor cells or toxicity in healthy tissues, most notably in the form of chronic cardiomyopathy and congestive heart failure [13,14]. In addition to ATP-driven effluxes, metabolic inactivation by carbonyl reduction contributes to an acquired resistance of potent anthracycline chemotherapeutics like doxorubicin and DRC [15–20]. Carbonyl reduction is a phase I biotransformation reaction and takes place on the side chain C-13 carbonyl moiety of doxorubicin and DRC, resulting in the formation of the secondary alcohol metabolites doxorubicinol and daunorubicinol (DRCOL), respectively [21–23]. Since the 13-hydroxy metabolites doxorubicinol and DRCOL are significantly less potent than the parent drug in terms of inhibiting tumor cell growth in vitro [18–23], carbonyl-reducing enzymes are considered to constitute an important mechanism in the development of resistance towards these drugs. Moreover, doxorubicinol and DRCOL, but not doxorubicin and DRC, are thought to be responsible for the cardiotoxicity observed upon chemotherapy [19,24–26]. As a consequence, the pharmacologic inhibition of anthracycline carbonyl reduction may be cardioprotective without a loss of antineoplastic potency. Responses to pharmacological agents in the human population are highly variable, owing in

part to variations in gene expression. These differences require identification and characterization of the candidate protein. An important group of enzymes has been described, acting as carbonyl reductases towards the carbonyl group of doxorubicin and DRC. These enzymes belong to two protein superfamilies, the short-chain dehydrogenase/reductases (SDR) [27] and the aldo–keto reductases (AKR) [28]. SDR and AKR enzymes have overlapping substrate specificities including many endogenous and xenobiotic compounds [29–32]. Enzymes that are important for DRC carbonyl reduction to DRCOL have previously been identified in in vitro studies as being carbonyl reductase (CBR1; EC 1.1.1.184), aldehyde reductase (AKR1A1; EC 1.1.1.2) [33] and aldose reductase (AKR1B1; EC 1.1.1.21) [34]. Whereas human CBR1 is a member of the SDRs [29,30], AKR1A1 and AKR1B1 are important human AKRs [31,32]. For the present investigation, CBR1, AKR1A1 and AKR1B1 were cloned from a human liver genomic library and transiently expressed in pancreas carcinoma cells. The overexpression of these DRC reductases resulted in a higher DRC carbonyl reducing activity, which was paralleled by a several fold increase in resistance of the pancreas carcinoma cells towards DRC. 2. Materials and methods 2.1. Chemicals DRC was supplied by Rhoˆne-Poulenc Pharma GmbH and DRCOL was donated by Farmitalia Carlo Erba GmbH. All other chemicals were of highest commercially available grade. 2.2. Cell lines and culture conditions The DRC-sensitive human pancreas adenocarcinoma cell line EPP85-181P was kindly provided by Prof. M. Dietel. The tumor cell line was grown as monolayer culture in Leibovitz L15 medium (PAA Laboratories) completed with the following supplements: 10% fetal bovine serum, 1 mM L-glutamine, 6.25 mg/l fetuin, 80 IE/l insulin, 2.5 mg/l transferrin, 1 g/l glucose, 1.1 g/l NaHCO3, 1% minimal essential vitamins, 20,000 KIU (Kallikrein-Inhibitor-Units)/l Trasylol, 5 mg/l gentamicin and 200 mg/l piperacillin (Wyeth-Pharma) in a humidified atmosphere of 5% CO2 at 37. Mean population doubling times were approximately 10 h. The cells were free of mycoplasma as judged by staining with 4,6-diamino-2phenylindole-dihydrochloride.

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2.3. Cloning and amplification of cDNAs encoding CBR1, AKR1A1 and AKR1B1 The coding sequences of CBR1, AKR1A1 and AKR1B1 were amplified by a PCR standard procedure from a human fetal liver cDNA library (Uni-ZAP XR library) using primers (MWG BIOTECH GmbH) of the following sequences:

CBR1: forward primer: 5 0 -CCG AAT TCA GCC ATG TCG TCC GGC ATC CAT GTA GC-3 0 ; reverse primer: 5 0 -GAG TCT AGA TCA CCA CTG TTC AAC TCT CTT CTC-3 0 . AKR1A1: forward primer: 5 0 -AAG CTA GCA ATG GCG GCT TCC TGT GTT C-3 0 ; reverse primer: 5 0 -AGC TCT AGA CTC AGT ACG GGT CAT TAA AGG-3 0 . AKR1B1: forward primer: 5 0 -ATG CTA GCA TGG CAA GCC GTC TCC TGC TCA AC-3 0 ; reverse primer: 5 0 -AAC TCT AGA TTC AAA ACT CTT CAT GGA AGG GGT A-3 0 . Purification of the PCR-products was performed according to the QIAquick PCR Purification Kit (Qiagen) protocol. Restriction of the cDNAs and the corresponding expression vector was done with restriction enzymes EcoRI and XbaI for CBR1, and NheI and XbaI for both AKR1A1 and AKR1B1. For amplification, the high copy plasmid pCl-neo Mammalian Expression Vector (Promega) was used. cDNA restriction was performed with 30 ll cDNA, 1 ll of restriction enzymes, 5 ll of restriction buffer (AGS, Boehringer) and 13 ll DEPC (diethylpyrocarbonate) water during 3–4 h at 37 C. The expression vector (2 lg) was restricted with 0.5 ll of restriction enzymes, 2 ll of restriction buffer and 15 ll aqua bidest. After ligation into the multiple cloning site, Escherichia coli cells XL1-Blue MRF‘ (Stratagene) were transformed with the expression vector constructs by electroporation in a GenePulserTM (Bio-Rad). After amplification, the cDNAs were purified and their identity realised by a modified Sanger procedure with the SequenaseR 2.0 Kit (Amersham) in a Sequi-Gen sequencer (Bio-Rad). 2.4. Development of cell lines The parental DRC-sensitive human pancreas adenocarcinoma cell line EPP85-181P was transfected with vectors harbouring the different reductases by using the transfection reagent DAC-30 (Eurogentec) in OptiMEM medium according to the manufacturer’s protocol. Cells were plated in 24-well plates at a density of 80,000 cells/ well using 2 lg/ml DAC-30 and 2 lg/ml plasmid-DNA

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each in 200 ll OptiMEM medium. The DNA was allowed to remain on the cells for 6 h and was then replaced by 400 ll of Leibovitz L15 medium with 100 mg/ml pipril. This change of medium was repeated after 24 h, and after 54 h the cells were harvested. 2.5. Determination of enzyme expression by RT-PCR RT-PCR experiments were carried out with a RT-PCR kit (Ready To Go, Amersham). Total cellular RNA was isolated by the RNeasy kit (Qiagen). An amount of cDNA representing 1 lg RNA was subjected to PCR for 35 cycles in a final volume of 50 ll using 15 pmol of each primer. Following an initial RT and denaturation of 30 min at 42 C and 5 min at 95 C, each PCR cycle consisted of 1 min at 95 C, 1 min at 55 C and 1 min at 72 C. The encoding cDNA-specific primers (MWG BIOTECH) used were:

CBR1: forward primer: 5 0 -CTT TGG TAC CCG AGA TGT GTG CAC AG-3 0 ; reverse primer: 5 0 -AGT TTC CTG GCG TGG ATC CTG GAC AG-3 0 . AKR1A1: forward primer: 5 0 -CAT TGA TTG TGC TGC TAT CTA CGG-3 0 ; reverse primer: 5 0 -GCC TTC CAA GTC TCC TTG TAG TGG-3 0 . AKR1B1: forward primer: 5 0 -AGC GAC CTG AAG CTG GAC TAC CTG G-3 0 ; reverse primer: 5 0 -GGT CAC CAC GAT GCC TTT GGA CTG G-3 0 . The expression of b2-microglobulin was used as an internal standard. Primers for b2-microglobulin cDNA were:

forward primer: 5 0 -GTG GAG CAT TCA GAC TTG TCT TTC AGC-3 0 ; reverse primer: 5 0 -TTC ACT CAA TCC AAA TGC GGC ATC TTC-3 0 . Aliquots (10 ll of RT-PCR products) were then subjected to electrophoresis in a 3% agarose gel and visualized by staining with ethidium bromide. Gels were photographed and analysed by scanning densitometry using the ImageMaster VDS (Amersham) detection and analysis system. Expression of all measured gene products (density units) was compared in transfected and non-transfected cell lines after being normalized against b2-microglobulin signals as the internal standard to account for RT-PCR and DNA loading variations.

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2.6. DRC carbonyl reduction assay

3. Results

The preparation of AKR1A1, AKR1B1 and CBR1 containing fractions was performed as described elsewhere [15]. DRC carbonyl reduction was determined in standard assays by incubating 20 ll of enzyme fraction and 15 ll of 50 mM sodium phosphate buffer, pH 7.4, in a final volume of 50 ll at 37 C. After addition of 10 ll of NADPH (final concentration 2 mM), the reaction was started by adding 5 ll of DRC to a final concentration of 0.089 mM. The reaction was stopped after 30 min by adding 150 ll of ice-cold acetonitrile into the incubation mixture and transferring the reaction vessel on ice for 15 min. The samples were centrifuged in an Eppendorf centrifuge at 4 C and 8000g for 6 min, and 20 ll of the supernatant served for HPLC determination of the substrates and their reduced alcohol metabolites. Preliminary examinations proved linearity of the reaction within the chosen incubation time. Control experiments were performed without biological material.

3.1. Production of DRC reductase overexpressing tumor cells

2.7. Determination of DRCOL After enzymatic conversion, DRC and DRCOL were detected on a Bio-Rad reverse-phase HPLC system with a Merck LiChrospher 100 RP18 column (LiChroCart 250– 4). HPLC separation of DRC and DRCOL was achieved using an eluent of 28% acetonitrile in 50 mM ammonium formate buffer, pH 4.0, and a flow rate of 1.3 ml/min. Substances were monitored with a fluorescence detector (Merck Hitachi L-748) at excitation wavelength 470 nm and emission wavelength 550 nm. Metabolite quantification was performed with the aid of calibration curves generated by using known concentrations of authentic DRCOL. Six experiments were performed for each transfected cell line. 2.8. Cytotoxicity assays The MTT cytotoxicity assay involves the conversion of tetrazolium salt to coloured formazan by living cells, serving as an indirect measurement of cell proliferation and viability. The cells were plated in flat-bottom (Sarstedt) microtiter plates at a cell density of 2000 cells/well with 12 replicates for each drug concentration. After a 5–12 h preincubation time, cells were incubated with DRC for 3 h and then tested for viability with 5 mg/ml MTT (exposure time 4 h). The formazan crystals were dissolved in a solution containing 0.1 N HCl in isopropanol. The optical density of the colored product was measured at 570 nm using a Bio-Rad microplate reader. Cytotoxicity (IC80, IC50, IC20) data were determined by analysis with the GraphPad Prism computer software. 2.9. Statistical analysis Differences in enzymatic activities and DRC cytotoxicity in sensitive and transfected pancreas carcinoma cells were analysed by the Student’s t-test.

The parental human pancreas adenocarcinoma cell line EPP85-181P constitutively expresses basal levels of the DRC reductases CBR1, AKR1A1 and AKR1B1. In order to simulate their induced expression and to infer their contribution to DRC resistance, EPP85-181P cells were transiently transfected with the pCl-neo Mammalian Expression Vector harbouring cDNAs coding for CBR1, AKR1A1 and AKR1B1, respectively. Overexpression of the DRC reductases was examined on the mRNA level by RT-PCR. The size of the RTPCR products was 291 bp for CBR1, 309 bp for AKR1A1 and 333 bp for AKR1B1, thus corresponding to the fragments expected according to the respective pair of primers chosen (Fig. 1). After being normalized against b2-microglobulin as internal standard, densitometric quantification of the band intensities was related to basal expression levels which were set as 100%. Compared to non-transfected tumor cells, a roughly twofold higher expression of CBR1, AKR1A1 and AKR1B1 was detected in the transfected tumor cells. 3.2. DRC detoxification in transfected and non-transfected tumor cells DRC reductase activities were measured by direct HPLC fluorescence determination of the product DRCOL (Fig. 2). Non-transfected cells showed a DRC reducing activity of 0.074 lmol/mg protein · min. Levels of DRC reduction in AKR1A1 transfected cells were 1.2-fold elevated to 0.09 lmol/mg protein · min. AKR1B1 transfected cells showed a more than 2.5-fold increase (0.195 lmol/mg protein · min), whereas CBR1 transfected cells had the highest potency of DRC inactivation (0.317 lmol/mg protein · min) which means a 4.3fold elevation compared to that of the parental cells. 3.3. Contribution of DRC reductases to DRC resistance A subsequent MTT cytotoxicity assay demonstrated the resulting increase in resistance towards DRC (Fig. 3). This assay measures the metabolic activity within the cell, and determines the number of live cells left after a given treatment. Each transfected DRC reductase was shown to protect the tumor cells from DRC toxicity, yet to a different extent. CBR1 exhibited the highest effectiveness, whereas those of AKR1A1 and AKR1B1 were somewhat weaker. CBR1 showed a 7-fold increase in the IC50 values compared to the controls. The IC50 values for AKR1A1 and AKR1B1 increased by factor of 4.5 and 3.7, respectively. This pattern of elevated resistance towards DRC was also seen at lower as well as at higher

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500 bp 300 bp

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500 bp 300 bp

CBR1 (tr)

CBR1 (Co)

AKR1B1 (tr)

AKR1B1 (Co)

AKR1A1 (tr)

AKR1A1 (Co)

500 bp

500 bp

200 bp

200 bp I.S. (CBR1 tr)

I.S. (Co)

I.S. (AKR1B1 tr)

I.S. (AKR1A1 tr)

I.S. (Co)

Fig. 1. Overexpression of AKR1A1, AKR1B1 and CBR1 in pancreas carcinoma cells. RT-PCR products of non-transfected (Co) and transfected (tr) EPP85-181P cells were resolved by agarose gel electrophoresis. The intensity of each band was analyzed by densitometry, normalized against b2-microglobulin as internal standard (I.S.) and then related to basal expression levels which were set as 100%. Values of overexpression were 205%, 181% and 196% for AKR1A1, AKR1B1 and CBR1, respectively. All results were reproduced in three separate experiments with SD variations of less than 10%.

4. Discussion

Fig. 2. DRC carbonyl reduction to DRCOL in non-transfected and transfected pancreas carcinoma cells. Enzyme activities are given as lmol DRCOL formed per mg of protein within 30 min. Co, control (not transfected); AKR1A1, human aldehyde reductase, AKR1B1, human aldose reductase; CBR1, human carbonyl reductase. Each bar represents the mean ± SD of N, 6 individual determinations. P < 0.0001.

DRC concentrations, which is reflected by the IC80 and IC20 values, respectively (Fig. 3). The discrepancy between AKR1A1 and AKR1B1 regarding overexpression, DRC detoxification and cytoprotection might be due to a higher catalytic efficiency of AKR1A1 in DRC carbonyl reduction as compared to AKR1B1. The results support the notion that induction of DRC reductases constitute an important mechanism in the development of DRC resistance in tumor cells. From these DRC reductases, CBR1 seems to be the most significant enzyme.

Previous studies revealed that resistance of tumor cells towards anthracycline chemotherapeutics is not only the result of alterations in drug uptake and retention [7,35–37], but also mediated by phase I biotransformation enzymes that catalyze carbonyl reduction of anthracyclines into their inactive alcohol metabolites [15–17]. Since anthracycline detoxifying enzymes have been shown to be upregulated upon exposure to these drugs, carbonyl reduction may be regarded as an additional mechanism in the development of non-classical MDR [15–17]. In a variety of different cancer cell systems, including pancreas carcinoma, breast cancer, stomach cancer and ovarian carcinoma, DRC carbonyl reduction was inducible by the substrate DRC itself [15–17,38,39]. The generation of resistant cell clones with acquired resistance to selected chemotherapeutics is a promising approach to study the clinical problem of multidrug resistance. For the DRC-sensitive human pancreas adenocarcinoma cell line EPP85181P, the anthracycline DRC was reported the most cytotoxic antibiotic compared to epirubicin and

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Fig. 3. Cytotoxicity of DRC in non-transfected and transfected pancreas carcinoma cells. Cytotoxicity of DRC was measured with the MTT test and is given as IC80, IC50, and IC20 values of cell proliferation and viability after 3 h exposure to DRC concentrations ranging from 0.01 to 100 lg/ml. Co, control (not transfected); AKR1A1, human aldehyde reductase; AKR1B1, human aldose reductase, CBR1, human carbonyl reductase. Each bar represents the means ± SD of N = 5–7 individual determinations. P < 0.005.

doxorubicin [40]. Given multiple enzymes with potential carbonyl reducing activity, it is important to demonstrate that specifically overexpressing a single DRC reductase is relevant to an enhanced resistance towards DRC. Therefore, by transient transfection, cell lines overexpressing the most important DRC carbonyl reductases were generated. Transfection of the pancreas carcinoma cell line with cDNAs coding for CBR1, AKR1A1 and AKR1B1 demonstrated that each single DRC reductase confers DRC resistance to cancer cells. Among these, CBR1 turned out to have the highest potency. Overexpression of CBR1 lead to a 7fold increase in DRC-resistance when IC50 values were taken into account. This finding is in accordance with results from DRC resistant human stomach carcinoma cells which showed strongest increase in CBR1 mRNA expression [15]. In another study, CBR1-deficient K562 cells gained protection towards DRC toxicity after transfection with CBR1 [41]. Therefore, CBR1 seems to be the dominant reductase in tumor cells during generation of DRC resistance by induction of phase-I metabolism. Human CBR1 belongs to a group of NADPHdependent cytosolic enzymes of the short chain dehydrogenase/reductase (SDR) superfamily [42]. The enzyme is ubiquitous in nature and catalyses the reduction of a large number of biologically and pharmacologically active carbonyl compounds to their corresponding alcohol metabolites [29,30]. CBR1 was also reported to provide the enzymatic basis of quinone detoxification in man [43]. AKR1A1, previously designated as aldehyde reductase, belongs to the AKR superfamily [31,32] and is a well known cytosolic enzyme participating in DRC carbonyl reduction [33]. In the present investigations it was shown that overexpression of

AKR1A1 resulted in a 4.5-fold elevation of resistance towards DRC when considering the IC50 values. AKR1B1, otherwise named aldose reductase, is a member of the 1B subfamily within the AKRs and an important target to prevent diabetic complications like retinopathy, nephropathy and neuoropathy [44]. AKR1B1 was shown to render cancer cells resistant to various toxic carbonyl compounds [39,45] and also proven to catalyze the carbonyl reduction of DRC [34]. Overexpression of AKR1B1 in the present study was followed by 3.7-fold increase in DRC resistance of the tumor cells. In earlier investigations it was shown that AKR1B1 undergoes upregulation upon exposure to DRC [15]. DRC is a highly effective antineoplastic agent, but it can produce the serious side effects of acute cardiac injury and chronic congestive heart failure. The mechanism by which DRC or its metabolites cause chronic cardiomyopathy is not fully understood. Hypotheses regarding the mechanism of cardiac toxicity include perturbation of calcium homeostasis, formation of iron complexes, mitochondrial dysfunction, and damage to cell membranes [46]. Since the development of chronic cardiomyopathy usually coincides with an accumulation of anthracycline secondary alcohol metabolites in the heart, doxorubicinol and DRCOL are hypothesized to be responsible for this severe sideeffect that limits the clinical use of these drugs [13,22]. It should be noted here that CBR1, AKR1A1 and AKR1B1 have been implicated in the development of doxorubicin-induced cardiotoxicity. For example, transgenic mice that overexpress human CBR1 exclusively in the heart show increased heart damage and decreased survival after doxorubicin

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treatment [24]. On the other hand, significant cardiac protection is seen in mice heterozygous (±) for Cbr1 expression with a 40–50% decrease of CBR1 protein levels [47]. The clinical consequences of our results remain to be established. It is noteworthy that the IC80 values for the three reductases, which reflect lower DRC concentrations, show a much higher extent of resistance compared to the controls (30-fold for CBR1, 19-fold for AKR1A1 and 20-fold for AKR1B1). On the other hand, anthracycline cumulative doses upon chemotherapy of cancer patients must not exceed 500 mg/m2, since otherwise serious side effects in cardiotoxicity (see above) can be expected. Hence, inhibition of DRC carbonyl reduction in a chemotherapeutic regimen is of twofold importance especially at low DRC doses: first, the preservation of the antineoplastic potency of the parent drug and second, the prevention of cardiomyopathy caused by its reduced alcohol metabolite DRCOL. In summary, our results show that the increased expression of one single DRC carbonyl reductase is sufficient to protect cancer cells from DRC toxicity. Thus diminution of DRC reductases using pharmacologic inhibitors may be a useful means of enhancing the antineoplastic potency of DRC. This inhibition should result in an increase in tumor exposure to the parent chemotherapeutic compound, DRC, with a concurrent decrease in cardiac exposure to the hydroxy metabolite, daunorubicinol. Since chronic cardiomyopathy develops by mechanisms other than those mediating their antitumor effectiveness, the concept of preventing DRC carbonyl reduction may be a strategy for protecting the heart while not diminishing tumor response. Acknowledgements The present study was supported by a grants from the Alfred and Ursula Kulemannn-Stiftung, Marburg (to M.P.), the Deutsche Forschungsgemeinschaft MA 1704/3-1, MA 1704/3-2 (to E.M.) and the European Commission (to E.M).

[3]

[4]

[5]

[6]

[7]

[8]

[9]

[10]

[11] [12]

[13]

[14]

[15]

[16]

References [17] [1] J.A. Endicott, V. Ling, The biochemistry of P-glycoproteinmediated multidrug resistance, Annu. Rev. Biochem. 58 (1989) 137–171. [2] G.L. Scheffer, P.L.J. Wijngaard, M.J. Flens, M.A. Izquierdo, M.L. Slovak, H.M. Pinedo, C.J.L.M. Meijer, H.C. Clevers, R.J. Scheper, The drug resistance-related

[18]

55

protein LRP is the human major vault protein, Nature Med. 1 (1995) 578–582. G.J.R. Zaman, M.J. Flens, M.R. van Leusden, M. de Haas, H.S. Mu¨lder, J. Lankelma, H.M. Pinedo, R.J. Scheper, F. Baas, H.J. Broxterman, P. Borst, The human multidrug resistance-associated protein MRP is a plasma membrane drug-efflux pump, Proc. Natl. Acad. Sci. USA 91 (1994) 8822–8826. I. Cascorbi, Role of pharmacogenetics of ATP-binding cassette transporters in the pharmacokinetics of drugs, Pharmacol. Ther. 112 (2006) 457–473. K. Takara, T. Sakaeda, K. Okumura, An update on overcoming MDR1-mediated multidrug resistance in cancer chemotherapy, Curr. Pharm. Des. 12 (2006) 273–286. G.K. Rekha, L. Sreerama, N.E. Sladek, Intrinsic cellular resistance to oxazaphosphorines exhibited by a human colon carcinoma cell line expressing relatively large amounts of a class-3 aldehyde dehydrogenase, Biochem. Pharmacol. 48 (1994) 1943–1952. T. Gessner, L.A. Vaughan, B.C. Beehler, C.J. Bartels, R.M. Baker, Elevated pentose cycle and glucuronyltransferase in daunorubicin-resistant P388 cells, Cancer Res. 50 (1990) 3921–3927. G. Batist, A. Tulpule, B.K. Sinha, A.G. Katki, G.E. Myers, K.H. Cowans, Overexpression of a novel anionic glutathione transferase in multidrug-resistant human breast cancer cells, J. Biol. Chem. 261 (1986) 15544–15549. T.M. Bosch, I. Meijerman, J.H. Beijnen, J.H. Schellens, Genetic polymorphisms of drug-metabolising enzymes and drug transporters in the chemotherapeutic treatment of cancer, Clin. Pharmacokinet. 45 (2006) 253–285. J. Cummings, B.T. Ethell, L. Jardine, G. Boyd, J.S. Macpherson, B. Burchell, J.F. Smyth, D.I. Jodrell, Glucuronidation as a mechanism of intrinsic drug resistance in human colon cancer: reversal of resistance by food additives, Cancer Res. 63 (2003) 8443–8450. R.B. Weiss, The anthracyclines: will we ever find a better doxorubicin? Semin. Oncol. 19 (1992) 670–686. A. Rabbani, R.M. Finn, J. Ausio, The anthracycline antibiotics: antitumor drugs that alter chromatin structure, Bioassays 27 (2005) 50–56. G. Minotti, P. Menna, E. Salvatorelli, G. Cairo, L. Gianni, Anthracyclines: molecular advances and pharmacologic developments in antitumor activity and cardiotoxicity, Pharmacol. Rev. 56 (2004) 185–229. R. Zucchi, R. Danesi, Cardiac toxicity of antineoplastic anthracyclines, Curr. Med. Chem. Anticancer Agents 3 (2003) 151–171. W. Ax, M. Soldan, L. Koch, E. Maser, Development of daunorubicin resistance in tumour cells by induction of carbonyl reduction, Biochem. Pharmacol. 59 (2000) 293–300. M. Soldan, W. Ax, M. Plebuch, L. Koch, E. Maser, Cytostatic drug resistance. Role of phase-I daunorubicin metabolism in cancer cells, Adv. Exp. Med. Biol. 463 (1999) 529–538. M. Soldan, K.J. Netter, E. Maser, Induction of daunorubicin carbonyl reducing enzymes by daunorubicin in sensitive and resistant pancreas carcinoma cells, Biochem. Pharmacol. 51 (1996) 117–123. B. Schott, J. Robert, Comparative activity of anthracycline 13-hydroxymetabolites against rat glioblastoma cells in culture, Biochem. Pharmacol. 38 (1989) 4069–4074.

56

M. Plebuch et al. / Cancer Letters 255 (2007) 49–56

[19] R.D. Olson, P.S. Mushlin, D.E. Brenner, S. Fleischer, B.J. Cusack, B.K. Chang, R.J. Boucek Jr., Doxorubicin cardiotoxicity may be caused by its metabolite, doxorubicinol, Proc. Natl. Acad. Sci. USA 85 (1988) 3585–3589. [20] M.J. Kuffel, J.M. Reid, M.M. Ames, Anthracyclines and their C-13 alcohol metabolites: growth inhibition and DNA damage following incubation with human tumor cells in culture, Cancer Chemother. Pharmacol. 30 (1992) 51–57. [21] S. Licata, A. Saponiero, A. Mordente, G. Minotti, Doxorubicin metabolism and toxicity in human myocardium: role of cytoplasmic deglycosidation and carbonyl reduction, Chem. Res. Toxicol. 13 (2000) 414–420. [22] A. Mordente, G. Minotti, G.E. Martorana, A. Silvestrini, B. Giardina, E. Meucci, Anthracycline secondary alcohol metabolite formation in human or rabbit heart: biochemical aspects and pharmacologic implications, Biochem. Pharmacol. 66 (2003) 989–998. [23] G.L. Forrest, B. Gonzalez, Carbonyl reductase, Chem. Biol. Interact. 129 (2000) 21–40. [24] G.L. Forrest, B. Gonzalez, W. Tseng, X. Li, J. Mann, Human carbonyl reductase overexpression in the heart advances the development of doxorubicin-induced cardiotoxicity in transgenic mice, Cancer Res. 60 (2000) 5158–5164. [25] X. Li, B.J. Cusack, R.J. Boucek Jr., P.S. Mushlin, T.B. Bledsoe, D.E. Brenner, R.D. Olson, Effects of daunorubicin and its primary metabolite, daunorubicinol, on cardiac contractility and calcium loading of sarcoplasmic reticulum, FASEB J. 5 (1991) A1395. Abstract. [26] R.D. Olson, P.S. Mushlin, Doxorubicin cardiotoxicity: analysis of prevailing hypotheses, FASEB J. 4 (1990) 3076– 3086. [27] H. Jo¨rnvall, B. Persson, M. Krook, S. Atrian, R. GonzalezDuarte, J. Jeffery, D. Ghosh, Short-chain dehydrogenases/ reductases (SDR), Biochemistry 34 (1995) 6003–6013. [28] J.M. Jez, T.G. Flynn, T.M. Penning, A new nomenclature for the aldo–keto reductase superfamily, Biochem. Pharmacol. 54 (1997) 639–647. [29] U. Oppermann, Carbonyl reductases: the complex relationships of Mammalian carbonyl- and quinone-reducing enzymes and their role in physiology, Annu. Rev. Pharmacol. Toxicol. 47 (2007) 293–322. [30] F. Hoffmann, E. Maser, Carbonyl reductases and pluripotent hydroxysteroid dehydrogenases of the short-chain dehydrogenase/reductase superfamily, Drug Metab. Rev. 39 (2007) 87–144. [31] Y. Jin, T.M. Penning, Aldo–keto reductases and bioactivation/detoxication, Annu. Rev. Pharmacol. Toxicol. 47 (2007) 263–292. [32] T. Matsunaga, S. Shintani, A. Hara, Multiplicity of mammalian reductases for xenobiotic carbonyl compounds, Drug Metab. Pharmacokinet. 21 (2006) 1–18. [33] H. Ohara, Y. Miyabe, Y. Deyashiki, K. Matsuura, A. Hara, Reduction of drug ketones by dihydrodiol dehydrogenases, carbonyl reductase and aldehyde reductase of human liver, Biochem. Pharmacol. 50 (1995) 221–227.

[34] K.W. Lee, B.C. Ko, Z. Jiang, D. Cao, S.S. Chung, Overexpression of aldose reductase in liver cancers may contribute to drug resistance, Anticancer Drugs 12 (2001) 129–132. [35] G. Toffoli, F. Simone, M. Gigante, M. Boiocchi, Comparison of mechanisms responsible for resistance to idarubicin and daunorubicin in multidrug resistant LoVo cell lines, Biochem. Pharmacol. 48 (1994) 1871–1881. [36] T. McGrath, M.S. Center, Adriamycin resistance in HL60 cells in absence of detectable P-glycoprotein, Biochem. Biophys. Res. Com. 145 (1987) 1171–1176. [37] C. Ramachandran, Z.K. Yuan, X.L. Huang, A. Krishan, Doxorubicin resistance in human melanoma cells: MDR-1 and glutathione S-transferase p gene expression, Biochem. Pharmacol. 45 (1993) 743–751. [38] G.L. Forrest, S. Akman, S. Krutzik, R.J. Paxton, R.S. Sparkes, J. Doroshow, R.L. Felsted, C.J. Glover, T. Mohandas, N.R. Bachur, Induction of a human carbonyl reductase gene located on chromosome 21, Biochim. Biophys. Acta 1048 (1990) 149–155. [39] D.J. Hyndman, R. Takenoshita, N.L. Vera, S.C. Pang, T.G. Flynn, Cloning and sequencing, and enzymatic activity of an inducible aldo–keto reductase from Chinese hamster ovary cells, J. Biol. Chem. 272 (1997) 13286–13291. [40] H. Lage, M. Dietel, Multiple mechanisms confer different drug-resistant phenotypes in pancreatic carcinoma cells, J. Cancer Res. Clin. Oncol. 128 (2002) 349–357. [41] B. Gonzalez, S. Akman, J. Doroshow, H. Rivera, W.D. Kaplan, G.L. Forrest, Protection against daunorubicin cytotoxicity by expression of a cloned human carbonyl reductase cDNA in K562 leukemia cells, Cancer Res. 55 (1995) 4646–4650. [42] B. Wermuth, K.M. Bohren, G. Heinemann, J.P. von Wartburg, K.H. Gabbay, Human carbonyl reductase. Nucleotide sequence analysis of a cDNA and amino acid sequence of the encoded protein, J. Biol. Chem. 263 (1988) 16185–16188. [43] B. Wermuth, K.L. Platt, A. Seidel, F. Oesch, Carbonyl reductase provides the enzymatic basis of quinone detoxication in man, Biochem. Pharmacol. 35 (1986) 1277–1282. [44] S.K. Srivastava, K.V. Ramana, A. Bhatnagar, Role of aldose reductase and oxidative damage in diabetes and the consequent potential for therapeutic options, Endocr. Rev. 26 (2005) 380–392. [45] M. Takahashi, J. Fujii, E. Miyoshi, A. Hoshi, N. Taniguchi, Elevation of aldose reductase gene expression in rat primary hepatoma and hepatoma cell lines: implication in detoxification of cytotoxic aldehydes, Int. J. Cancer 62 (1995) 749– 754. [46] A. Mordente, E. Meucci, G.E. Martorana, B. Giardina, G. Minotti, Human heart cytosolic reductases and anthracycline cardiotoxicity, IUBMB Life 52 (2001) 83–88. [47] L.E. Olson, D. Bedja, S.J. Alvey, A.J. Cardounel, K.L. Gabrielson, R.H. Reeves, Protection from doxorubicininduced cardiac toxicity in mice with a null allele of carbonyl reductase 1, Cancer Res. 63 (2003) 6602–6606.