Curcumin is a tight-binding inhibitor of the most efficient human daunorubicin reductase – Carbonyl reductase 1

Chemico-Biological Interactions xxx (2015) xxx–xxx

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Curcumin is a tight-binding inhibitor of the most efficient human daunorubicin reductase – Carbonyl reductase 1 Jan Hintzpeter ⇑, Jan Hornung, Bettina Ebert, Hans-Jörg Martin, Edmund Maser Institute of Toxicology and Pharmacology for Natural Scientists, University Medical School Schleswig-Holstein, Campus Kiel, Brunswikerstr. 10, D-24105 Kiel, Germany

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Article history: Available online xxxx Keywords: Anthracycline resistance Daunorubicin Carbonyl reductase 1 Tight-binding inhibition Cardiotoxicity Curcumin

a b s t r a c t Curcumin is a major component of the plant Curcuma longa L. It is traditionally used as a spice and coloring in foods and is an important ingredient in curry. Curcuminoids have anti-oxidant and anti-inflammatory properties and gained increasing attention as potential neuroprotective and cancer preventive compounds. In the present study, we report that curcumin is a potent tight-binding inhibitor of human carbonyl reductase 1 (CBR1, Ki = 223 nM). Curcumin acts as a non-competitive inhibitor with respect to the substrate 2,3-hexandione as revealed by plotting IC50-values against various substrate concentrations and most likely as a competitive inhibitor with respect to NADPH. Molecular modeling supports the finding that curcumin occupies the cofactor binding site of CBR1. Interestingly, CBR1 is one of the most effective human reductases in converting the anthracycline anti-tumor drug daunorubicin to daunorubicinol. The secondary alcohol metabolite daunorubicinol has significantly reduced anti-tumor activity and shows increased cardiotoxicity, thereby limiting the clinical use of daunorubicin. Thus, inhibition of CBR1 may increase the efficacy of daunorubicin in cancer tissue and simultaneously decrease its cardiotoxicity. Western-blots demonstrated basal expression of CBR1 in several cell lines. Significantly less daunorubicin reduction was detected after incubating A549 cell lysates with increasing concentrations of curcumin (up to 60% less with 50 lM curcumin), suggesting a beneficial effect in the co-treatment of anthracycline anti-tumor drugs together with curcumin. Ó 2014 Elsevier Ireland Ltd. All rights reserved.

1. Introduction The yellow-orange pigment curcumin from the East Indian plant Curcuma longa has been used for centuries in cooking as well as in traditional Indian and Chinese medicine. A search under the keyword ‘‘curcumin’’ in the Pubmed database of the National Center for Biotechnology Information dates the first entry that reports its antibacterial action from 1949 [1]. Since that time numerous investigations were published describing the beneficial effects of curcumin and related compounds, mostly with respect to inflammation and cancer. Intensive investigations also led to the elucidation of a variety of biological interactions [2–4]. In this study, we present evidence that curcumin is a potent inhibitor of human carbonyl reductase type 1 (CBR1, listed as SDR21C in the SDR-database [5]), an enzyme of the short-chain dehydrogenase/reductase superfamily. CBR1 is known for more than 30 years, but even today its physiological role is not fully understood [6,7]. Expressed in many tissues, endogenous CBR1

⇑ Corresponding author. Tel.: +49 431 597 2967; fax: +49 431 597 3558.

substrates comprise steroids, eicosanoids, cofactors, neurotransmitters and polyols. In addition, a large number of xenobiotics has been identified as substrates for CBR1, including quinones, the tobacco derived carcinogen NNK (4-(methylnitrosamino)-1(3-pyridyl)-1-butanone) and drugs such as warfarin or ketoprofen [8–11]. Also the anthracycline anticancer drugs daunorubicin (DAUN) and doxorubicin (DOX) are reduced by CBR1, resulting in secondary alcohols at the C-13 positions (Fig. 1) [12,13]. Over time, DAUN and DOX have become a gold standard for the treatment of various cancers, such as hematological (leukemia, lymphoma) and solid breast, ovarian, lung and liver tumors [14]. Unfortunately, the clinical success of these agents is overshadowed by serious side effects, such as systemic toxicity, cardiotoxicity or drug resistance. Cardiotoxicity is the main limiting side effect that can ultimately lead to potentially lethal congestive heart failure [15]. Convincing evidence supports the idea that the C-13 hydroxy metabolites of DAUN and DOX, daunorubicinol (DAUNOL) and doxorubicinol (DOXOL), respectively, are the main trigger for chronic cardiotoxicity [16–21]. Thus, inhibition of CBR1 may increase the efficacy and decrease cardiotoxicity of anthracyclines

E-mail address: [email protected] (J. Hintzpeter). http://dx.doi.org/10.1016/j.cbi.2014.12.019 0009-2797/Ó 2014 Elsevier Ireland Ltd. All rights reserved.

Please cite this article in press as: J. Hintzpeter et al., Curcumin is a tight-binding inhibitor of the most efficient human daunorubicin reductase – Carbonyl reductase 1, Chemico-Biological Interactions (2015), http://dx.doi.org/10.1016/j.cbi.2014.12.019

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[7,22,23]. Indeed, in various cell culture models it has been demonstrated that inhibition of CBR1 by known inhibitors, e.g. hydroxy-PP (3-(1-tert-butyl-4-amino-1H-pyrazolo[3,4-d]pyrimidin-3-yl)phenol), enhanced the effectiveness and decreased the cardiotoxicity of the anticancer drug DAUN by preventing its reduction to DAUNOL [24]. Here, we examined the effects of curcumin on CBR1 mediated reactions, determined its IC50-value and inhibition constant Ki, and tested the inhibitory effect of curcumin on A549 cell lysates with regard to DAUNOL formation. Docking experiments were performed to study potential binding sites for curcumin and CBR1. Our results suggest that curcumin strongly inhibits CBR1-catalyzed DAUNOL formation and therefore may enhance the therapeutic effectiveness and decrease the cardiotoxic side effects of antineoplastic drugs like DAUN or DOX.

2. Materials and methods 2.1. Materials Protein and DNA molecular weight standards were purchased from Fermentas GmbH (St. Leon-Rot, Germany). 2,3-Hexandione, curcumin and quercetin were obtained from Sigma–Aldrich (St. Louis, MO, USA). Daunorubicin was purchased from Biomol GmbH (Hamburg, Germany). NADPH, acetonitrile (gradient grade) were obtained from Carl Roth GmbH + Co. KG (Karlsruhe, Germany). Selective primary antibodies against CBR1 (Ab4148) were purchased from Abcam (Cambridge, UK), Anti-beta-actin antibody and anti-rabbit HRP-conjugated secondary antibody were obtained from Neo-Markers (Fremont, CA, USA) (Cat. No. RB-9421-P1). Cell culture media and supplements were purchased from PAA (Coelbe, Germany). Human cell lines (A549, HT-29, Caco-2, SW-480) were obtained from the Deutsche Sammlung für Mikroorganismen und Zellkulturen (DSMZ, Braunschweig, Germany) and HepG2, OVCAR-3, PANC-1, and A431 were purchased from cell lines service

(CLS, Eppelheim, Germany). HCT116 cells were generously provided by J. Abel (IUF, University of Duesseldorf, Germany). 2.2. Methods 2.2.1. Preparation of recombinant carbonyl reductase 1 His-tagged human carbonyl reductase 1 (CBR1) was expressed in Escherichia coli and purified as published previously [25]. 2.2.2. Cell culture and Western blots Treatment of human cell lines, preparation of cell lysates and detection of CBR1 by Western blots was performed as published previously [26]. 2.2.3. Inhibition of daunorubicinol formation by curcumin in A549 cell lysates A549 cells ((3.5  107); 95% confluence) were rinsed 2 times with 0.1 M NaH2PO4 buffer (pH 7.4) and then scraped off with a cell scraper. The cells were resuspended followed by cell disruption using ultrasonication at minimal power for 10 s on ice. The protein concentration of the cell lysates was 3.6 mg/ml. Stock solutions of DAUN and curcumin were prepared in DMSO. Cell lysates (45 lg protein) were incubated for 30 min at 37 °C with 200 lM DAUN and various amounts of curcumin in a total volume of 250 ll. The final DMSO concentration did not exceed 1%. The reaction was stopped by adding 250 ll ice-cold acetonitrile and the samples were analyzed by HPLC (high-performance liquid chromatography). For DAUNOL detection a modified method of Fogli et al. was used [27] (mobile phase, 50 mM sodium phosphate/acetonitrile (75:25), pH 4.0; flow rate, 1.5 ml/min). 2.2.4. Determination of inhibition parameters Catalytic properties were determined by measuring the decrease in absorbance at 340 nm (Cary 100 scan photometer, Varian, California, USA). A reaction mixture without inhibitor consisted of different concentrations of 2,3-hexanedione, 200 lM

Fig. 1. Scheme of curcumin inhibition of CBR1 mediated DAUN and DOXO reduction.

Please cite this article in press as: J. Hintzpeter et al., Curcumin is a tight-binding inhibitor of the most efficient human daunorubicin reductase – Carbonyl reductase 1, Chemico-Biological Interactions (2015), http://dx.doi.org/10.1016/j.cbi.2014.12.019

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NADPH, 0.1 M NaH2PO4 buffer (pH 7.4) and an appropriate amount of enzyme in a total assay volume of 0.8 ml. For inhibition studies stock solutions of inhibitors were prepared in DMSO. The final concentration of DMSO in the assay was 61% and did not affect enzyme activity. When collecting data for dose–response curves initial velocities of 2,3 hexanedione reduction (substrate concentration at Km) in the presence of inhibitors were assayed as described above. The percentage of inhibition was calculated considering the activity in the absence of inhibitor to be 100%. For determination of the inhibition constant Ki, the results were fitted to the Morrison equation by non-linear regression (concentration of CBR1 = 0.096 lM; concentration of 2,3-hexandione = 217 lM; Km = 217 lM). To identify the mode of interaction between CBR1 and tightbinding inhibitors, IC50-values were determined at five different 2,3-hexanedione concentrations. All data obtained were plotted and analyzed using GraphPad Prism6 (GraphPad Software Inc., San Diego, CA). 2.2.5. Inhibition of recombinant human CBR1 mediated daunorubicin reduction by curcumin Recombinant human CBR1 (4 lg) was incubated for 5 min at 37 °C with 200 lM DAUN and various amounts of curcumin in a total volume of 250 ll. A 5 min incubation period was found to be in the linear range of enzymatic activity for the DAUN concentration used to conduct the enzymatic assays. The reaction mixture consisted of 200 lM DAUN, 400 lM NADPH, 0.1 M NaH2PO4 buffer (pH 7.4) and various curcumin concentrations between 31.25 nM and 100 lM. The reaction was stopped by adding 250 ll ice-cold acetonitrile and the samples were analyzed by HPLC (high-performance liquid chromatography). Detection of DAUNOL by fluorescence was performed as mentioned above.

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3.2. Daunorubicin reduction in curcumin-treated A549 cell lysates The incubation of A549 human lung cell lysates with DAUN resulted in significant DAUNOL formation. The latter could be easily detected and quantified by an HPLC-system connected with a fluorescence detector. In incubations containing increasing amounts of curcumin less DAUNOL was detected (Fig. 3). However, inhibition was less than 100% even in the presence of 50 lM curcumin, indicating the presence of other reductases which are not inhibited by curcumin. 3.3. Inhibition of recombinant human CBR1 by curcumin Curcumin is a potent inhibitor of CBR1 with an IC50-value of 382 ± 1.1 nM and a Ki-value of 223 ± 12 nM. To determine the Kivalue, the Morrison equation for tight-binding inhibitors was used [32–34] (Fig. 4). The type of inhibition for tight-binding inhibitors can be studied by plotting IC50-values at different substrate concentrations. This was done for five concentrations of 2,3-hexanedione in the presence of curcumin and also in the presence of the well known CBR1 inhibitor quercetin [7] (Fig. 5). The shape of the resulting curve shows that curcumin inhibition of CBR1 is of the non-competitive type with a > 1 (cf. inset in Fig. 5). Increasing IC50-values with increasing substrate concentrations, which were observed in the case of quercetin, also indicate non-competitive inhibition but with a < 1. Fig. 6 shows a dose-dependent inhibition curve of DAUN reduction mediated recombinant human CBR1 by curcumin. The half maximal inhibitory concentration for curcumin on human CBR1 mediated DAUNOL formation was determined to be IC50 = 2.90 ± 1.12 lM. 3.4. Docking of curcumin and human CBR1

2.2.6. Docking of curcumin into CBR1 For docking calculations the crystal structure of human CBR1, available at the Worldwide Protein Data Bank (wwPDB), PDB code 1wma, was used [24,28]. Calculations were performed by the web service SwissDock. The binding model of CBR1 and curcumin possessing the most favorable energy was taken and pictured by UCSF Chimera package [29–31].

The docking of curcumin and human CBR1 using the SwissDock server shows that curcumin occupies the same space as the co-factor NADPH based on the FullFitness parameter (Fig. 7). This result suggests that curcumin interferes with the binding of the co-factor in a competitive way. 4. Discussion

3. Results 3.1. Basal CBR1 expression in different cell lines CBR1 expression can be observed in many tissues, particular high levels were detected in liver and brain [6,9]. We found CBR1 on the protein level in six out of eight cell lines from five tissues (lung, A549; colon, Caco-2, HT-29, HCT-116, SW-480; pancreas PANC-1; skin, A431; liver, HepG2), (Fig. 2). No signal was detected in HCT-116, one of the colon cell lines. Interestingly, also in the HepG2 cell line from human liver CBR1 was almost absent.

CBR1 is an efficient reductase for the important anti-neoplastic agents DAUN and DOX. Their reduced metabolites (DAUNOL and DOXOL) are most likely responsible for chronic cardiotoxicity [12,13]. This severe side effect limits the clinical use of those otherwise effective drugs. Although carbonyl reduction is conceivable at both carbonyl groups of the anthraquinone moiety, the main metabolites are C-13 alcohols [13]. Here, we report that the natural phenol curcumin found in C. longa is a new and potent inhibitor of human CBR1. The structural configuration responsible for the high binding affinity of curcumin for human CBR1 was determined by

Fig. 2. Basal expression levels of CBR1 protein in different human cancer cell lines: lung (A549), colon (Caco-2, HT-29, HCT-116, SW-480), pancreas (PANC-1), skin (A431), and liver (HepG2). Whole cell lysates were prepared from different human cancer cell lines, and proteins (70 lg) were analyzed by Western blotting using a goat anti-CBR1 primary antibody (abcam, dilution 1:2000). b-Actin served as the loading control.

Please cite this article in press as: J. Hintzpeter et al., Curcumin is a tight-binding inhibitor of the most efficient human daunorubicin reductase – Carbonyl reductase 1, Chemico-Biological Interactions (2015), http://dx.doi.org/10.1016/j.cbi.2014.12.019

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Fig. 3. Effect of curcumin on DAUNOL production in A549 cell lysates with respect to the uninhibited control. A549 cell lysates were incubated for 30 min at 37 °C with 200 lM DAUN, 1 mM NADPH and increasing amounts of curcumin. The formation of DAUNOL was quantified by HPLC. Data are presented as mean ± SD from at least three experiments. A one-way ANOVA, which was followed by a Dunnett’s Multiple Comparison Test was used to assess the statistical significance of experiments that investigated the inhibition potency of curcumin on the reductive anthracycline metabolism mediated by A549 cell lysates. ⁄p < 0.05; ⁄⁄p < 0.01; ⁄⁄⁄p < 0.001 compared with cells without curcumin addition (control).

Fig. 4. Inhibition of the CBR1-catalyzed 2,3-hexanedione reduction by curcumin. Enzymatic activity is expressed as the ratio of inhibited vs. non-inhibited reaction rate. Data were fitted to the Morrison equation for tight-binding inhibitors. The inset shows a dose–response curve fitted to the same data. Data are presented as mean ± SD from at least three experiments.

comparing the docked models of curcumin to the ternary complex of CBR1, NADPH and Hydroxy-PP. The results showed that curcumin positions in the co-factor binding site. Indeed, experiments revealed a low IC50-value of 382 nM and an even lower Ki-value of 223 nM with respect to 2,3-hexanedione, which prompted us to consider curcumin as a tight-binding inhibitor for CBR1. Interestingly, an increase of the IC50 was observed when DAUN was used as the substrate for CBR1 (IC50 = 2.90 ± 1.12 lM). It has been suggested that the steady-state approximations should be abandoned whenever the Ki of an inhibitor is less than 1000-fold greater than the total enzyme concentration [34]. To identify the true mode of interaction between CBR1 and the tight-binding inhibitors, quercetin and curcumin, the IC50-values for the inhibitors were determined at fixed enzyme concentrations and at five different 2,3-hexanedione concentrations. For a non-competitive inhib-

Fig. 5. IC50-values of the CBR1-catalyzed 2,3-hexanedione reduction as a function of substrate concentration in the presence of the inhibitors curcumin (open circles) and quercetin (open triangles). The equilibrium scheme shows enzyme turnover in the presence or absence of an inhibitor (E, enzyme; S, substrate; I, inhibitor; P, product; KS, dissociation constant for the ES complex; Ki, dissociation constant for the EI complex; kP, rate constant for product formation; factor a reflects the influence of the enzyme for the substrate or the inhibitor, respectively) [34]. Data are presented as mean ± SD from at least three experiments.

itor a plot of IC50-values as a function of substrate concentration [S] will curve upward or downward, or be independent of [S], depending on whether the factor a is greater than, less than, or equal to 1 (see scheme in the inset of Fig. 5). The data for both, quercetin and curcumin, are characteristic for non-competitive inhibitors. Interestingly, curcumin has been shown to also act as an inhibitor for other reductases belonging to the aldo–keto reductase superfamily: in a study by Matsunaga et al. the inhibitory effects of dietary plant polyphenols on the aldo–keto reductases AKR1B1 and AKR1B10 were examined [35]. Both enzymes, next to other reductases, participate in the reductive metabolism of DAUN and DOX, with AKR1B10 having a higher catalytic efficiency (AKR1B10

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Fig. 6. Dose-dependent inhibition of daunorubicin reduction of recombinant human CBR1 by curcumin. Results are presented as means ± SD of at least three experiments.

Fig. 7. Curcumin docked into the active site of human CBR1 (PDB-code: 1wma). The amino acids S139, K197, N113 and Y193, which are directly involved in the catalytic mechanism, are depicted as sticks colored by element. The overlapping structures of the co-factor NADPH (purple) and curcumin (turquoise) are also shown as sticks in the vicinity of the catalytic tetrad. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

kcat/Km = 2752 ± 382 s 1 M 1 in contrast to AKR1B1 kcat/Km = 329 ± 14 s 1  M 1) toward DAUN [36]. In the report by Matsunaga et al. bisdemethoxycurcumin showed 3-fold higher inhibition potency than curcumin. Therefore, it cannot be ruled out that in our experiments other curcuminoids are even more potent with respect to CBR1 inhibition, since the product used in this study consisted of P80% curcumin with a total content of P95% curcuminoids. CBR1 is expressed in many tissues and it is also expressed constitutively in many cancer cell lines (Fig. 2). Accordingly, in experiments with cell lysates of A549 cells DAUNOL was detected and a significant decrease in DAUNOL formation was observed in the presence of increasing concentrations of curcumin (Fig. 3). Instead of the cytosolic fraction crude A549 cell lysates have been used to ensure the involvement of all DAUN reductases in this cancer cell line. This also includes the recently discovered microsomal reductase, which seems to be participating in anthracycline metabolism [37]. The presence of other reductases not inhibited by curcumin may explain the fact that despite the low Ki-value for CBR1, residual activity was 40% even in the presence of 50 lM curcumin. C-13 carbonyl reduction of DAUN and DOX is mediated by several members of the SDR and AKR superfamilies, namely CBR1, CBR3, CBR4, AKR1A1, AKR1B1, AKR1B10, AKR1C1, AKR1C2, AKR1C3, AKR1C4 and AKR7A2 [36,38–41], although reports concerning the role of

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CBR3 [41] and AKR1C2 in anthracycline metabolism are inconsistent [40,42]. The two monomeric isoforms of cytosolic carbonyl reductase CBR1 and CBR3 show a high sequence similarity of 85% on the amino acid level, suggesting a functional similarity of both enzymes. However, inconsistant results from different groups of the scientific community led to a controversial debate about the role of CBR3 in anthracycline metabolism. Consequently, we performed experiments with human recombinant CBR3 to test the DAUN reduction activity. In our studies human recombinant CBR3 did not show any detectable DAUN reductase activity. Our results indicate that at least the human form of CBR3 plays no significant role in reductive anthracycline metabolism. We tested the inhibition potency of curcumin with CBR3 on isatin reduction. Curcumin fails to inhibit CBR3 even at relatively high curcumin concentrations (>25 lM). However, it is rather unlikely that CBR3, even if upregulated under specific conditions, plays a major role in the reductive metabolism of anthracyclines. Interestingly, a study of Bains et al. provides evidence that eight aldo–keto reductase and two carbonyl reductase isoforms are upregulated after pre-exposure to DAUN or DOX [20]. Thus, elevated enzymatic reduction of DAUN or DOX may also be part of the mechanisms that cause pharmacokinetic anthracycline resistance in tumors. In addition to that, curcumin also seems to have an influence on the expression of at least some of the above mentioned reductases. Curcumin has been shown to be an effective inducer of many phase II biotransformation enzymes via activation of the Nrf2 transcription factor [43–45]. CBR1 is upregulated by Nrf2 [46], thus potentially increasing the conversion of residual DAUN to DAUNOL. The same is true for AKR1B1, AKR1B10, AKR1C3 and AKR7A2 which are also regulated by Nrf2 [47]. The situation is even more complex, since curcumin seems to suppress other reductase induction pathways as well as transport of anthracyclines out of the cell. It is reported to suppress NF-kappaB activation induced by TNF, phorbor esters, and hydrogen peroxide through suppression of I-kappaB degradation [48–50]. Therefore, curcumin may also suppress the upregulation of NFkappaB regulated reductases, e.g. CBR1 [51]. Another positive effect of curcumin is the efficient inhibition of ABC-transporters, which play a pivotal role in anthracycline resistance. Since anthracyclines like DAUN and DOX are substrates for P-gp (P-glycoprotein 1 or ABCB1) [52] some cancer cells express large amounts of P-gp, which would gain anthracycline related resistance [53,54]. Interestingly, it has been demonstrated that curcumin not only interacts directly with the drug binding site of the ABCB1-transporter [55,56] but also the expression of the multidrug resistance gene and ABCB1-transporter (protein) was significantly suppressed in both, in vitro and in vivo, respectively [57]. In summary, the effects of curcumin depend on both, the upregulation of anthracycline reductases and the inhibition of ABC-transporters and reductases. However, the co-administration of anthracyclines with CBR1 inhibitors on patients or model organisms seems to be promising. Indeed, beneficial effects of curcumin were described by several groups with respect to cancer and cardiotoxicity [58]. Sadzuka et al. observed decreased tumor weight in mice treated with a combination of curcumin and DOX but not with DOX alone [59]. Additionally, positive effects of the co-administration of curcumin with DAUN or DOX have been reported in rat models [60–62] and in numerous human cancer cell lines [63–65]. The effect of CBR1 on anthracycline metabolism was investigated as well: a selective CBR1 inhibitor induced DAUN-mediated A549-cell killing consistent with its ability to inhibit CBR1-mediated DAUN metabolism [24]. Mice with a null allele of CBR1 were protected from DOX-induced cardiac toxicity, whereas the wildtype was not [22]. On the contrary, transgenic mice overexpressing

Please cite this article in press as: J. Hintzpeter et al., Curcumin is a tight-binding inhibitor of the most efficient human daunorubicin reductase – Carbonyl reductase 1, Chemico-Biological Interactions (2015), http://dx.doi.org/10.1016/j.cbi.2014.12.019

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CBR1 exhibited increased cardio-toxicity associated with DOX treatment [7]. The pivotal role of CBR1 is also supported by Plebuch et al. where human pancreas carcinoma cells showed an increased resistance toward DAUN when transfected with cDNA coding for CBR1 [66]. Additionally, one clinical study showed that an increased expression of CBR1 significantly reduced the in vitro cytotoxicity of DAUN and was also positively correlated with intracellular DAUNOL levels in acute myeloid leukemia patients [67]. The latter even suggests that ‘‘inhibition of CBR1 can be an option to improve the efficacy and prevent toxicity’’. Altogether, this provides strong evidence for a critical role of CBR1 in humans receiving anthracycline therapy. Therefore, it might be assumed that the co-treatment of cancer patients receiving anthracyclines and curcumin in combination may protect cardiac tissue from DAUNOL induced damage by inhibiting CBR1 and/or other mentioned reductases. Nevertheless, it has been questioned whether carbonyl reduction and its prevention by enzyme inhibitors plays a role in cardioprotection [68]. A study performed on perfused rat hearts found DAUN to be more cardiotoxic than its metabolite DAUNOL. These conflicting results indicate that the mechanisms underlying the anti-neoplastic and adverse effects of anthracyclines (and their metabolites) are still poorly understood. Among other mechanisms, these include oxidative stress, DNA and RNA damage after intercalation of the drugs, disturbance of calcium and iron homeostasis in myocardial cells and induction of apoptosis [15]. Regardless of the mechanisms involved, our in vitro studies with recombinant CBR1 show that curcumin is a potent tight-binding inhibitor of CBR1 that decreases DAUNOL formation. Curcumin may yield the potential to enhance the therapeutic effectiveness of DAUN by preventing heart tissue damage through the inhibition of CBR1 mediated reduction of DAUN to DAUNOL. Conflict of interest The authors declare that there are no conflicts of interest. Transparency Document The Transparency document associated with this article can be found in the online version.

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