Hans-Jörg Martin

Carbonyl Reductase 1 Is a Predominant Doxorubicin Reductase in the Human Liver

A first step in the enzymatic disposition of the antineoplastic drug doxorubicin (DOX) is the reduction to doxorubicinol (DOX-OL). Because DOX-OL is less antineoplastic but more cardiotoxic than the parent compound, the individual rate of this reaction may affect the antitumor effect and the risk of DOX-induced heart failure. Using purified enzymes and human tissues we determined enzymes generating DOX-OL and interindividual differences in their activities. Human tissues express at least two DOX-reducing enzymes. High-clearance organs (kidney, liver, and the gastrointestinal tract) express an enzyme with an apparent Km of ∼140 μM. Of six enzymes found to reduce DOX, Km values in this range are exhibited by carbonyl reductase 1 (CBR1) and aldo-keto reductase (AKR) 1C3. CBR1 is expressed in these three organs at higher levels than AKR1C3, whereas AKR1C3 has higher catalytic efficiency. However, inhibition constants for DOX reduction with 4-amino-1-tert-butyl-3-(2-hydroxyphenyl)pyrazolo[3,4-d]pyrimidine (an inhibitor that can discriminate between CBR1 and AKR1C3) were identical for CBR1 and human liver cytosol, but not for AKR1C3. These results suggest that CBR1 is a predominant hepatic DOX reductase. In cytosols from 80 human livers, the expression level of CBR1 and the activity of DOX reduction varied >70- and 22-fold, respectively, but showed no association with CBR1 gene variants found in these samples. Instead, the interindividual differences in CBR1 expression and activity may be mediated by environmental factors acting via recently identified xenobiotic response elements in the CBR1 promoter. The variability in the CBR1 expression may affect outcomes of therapies with DOX, as well as with other CBR1 substrates.

Purification and characterization of AKR1B10 from human liver: role in carbonyl reduction of xenobiotics

Members of the aldo-keto reductase (AKR) superfamily have a broad substrate specificity in catalyzing the reduction of carbonyl group-containing xenobiotics. In the present investigation, a member of the aldose reductase subfamily, AKR1B10, was purified from human liver cytosol. This is the first time AKR1B10 has been purified in its native form. AKR1B10 showed a molecular mass of 35 kDa upon gel filtration and SDS-polyacrylamide gel electrophoresis. Kinetic parameters for the NADPH-dependent reduction of the antiemetic 5-HT3 receptor antagonist dolasetron, the antitumor drugs daunorubicin and oracin, and the carcinogen 4-methylnitrosamino-1-(3-pyridyl)-1-butanone (NNK) to the corresponding alcohols have been determined by HPLC. Km values ranged between 0.06 mM for dolasetron and 1.1 mM for daunorubicin. Enzymatic efficiencies calculated as kcat/Km were more than 100 mM–1 min–1 for dolasetron and 1.3, 0.43, and 0.47 mM–1 min–1 for daunorubicin, oracin, and NNK, respectively. Thus, AKR1B10 is one of the most significant reductases in the activation of dolasetron. In addition to its reducing activity, AKR1B10 catalyzed the NADP+-dependent oxidation of the secondary alcohol (S)-1-indanol to 1-indanone with high enzymatic efficiency (kcat/Km = 112 mM–1 min–1). The gene encoding AKR1B10 was cloned from a human liver cDNA library and the recombinant enzyme was purified. Kinetic studies revealed lower activity of the recombinant compared with the native form. Immunoblot studies indicated large interindividual variations in the expression of AKR1B10 in human liver. Since carbonyl reduction of xenobiotics often leads to their inactivation, AKR1B10 may play a role in the occurrence of chemoresistance of tumors toward carbonyl group-bearing cytostatic drugs.

Role of human aldo–keto-reductase AKR1B10 in the protection against toxic aldehydes

Damage of cell membranes by reactive oxygen species results in the formation of toxic lipid peroxides which may ultimately lead to cell death. Among the best characterized intermediates of oxidative stress are the unsaturated aldehydes 4-hydroxynon-2-enal (4-HNE) and its oxidized counterpart 4-oxonon-2-enal (4-ONE). 4-HNE has been linked to various pathological conditions including atherosclerosis, Parkinsons and Alzheimers disease. 4-Methylpentanal (4-MP) is a side-chain cleavage product formed endogenously during steroidogenesis from cholesterol. Like 4-HNE and 4-ONE, 4-MP is capable of binding covalently to and cross-linking of proteins. These aldehydes are also damaging DNA by the formation of adducts. We found that AKR1B10, a cytosolic member of the aldo-keto reductase superfamily, efficiently catalyzes the reduction of 4-HNE (K(m)=0.3mM, k(cat)=43 min(-1)), 4-ONE (K(m)=0.3mM, k(cat)=40 min(-1)) and 4-MP (K(m)=0.05 mM, k(cat)=25 min(-1)). AKR1B10 catalyzed 4-MP reduction with a 30-fold increase in activity using NADPH as cofactor compared with NADH. As was observed for aldose reductase (AKR1B1) 4-ONE rapidly inactivates AKR1B10, while this inactivation is not observed when the enzyme is pre-incubated with NADPH. It was shown that cysteine 298 of aldose reductase was protected by NADPH from the alpha,beta-unsaturated carbonyls of 4-ONE thus rendering resistance towards inactivation. We generated a mutant AKR1B10, changing the respective cysteine on position 299 of AKR1B10 into a serine. This C299S mutant is still active towards 4-HNE and 4-ONE, albeit at a somewhat lower catalytic efficiency. However, it is still inactivated by 4-ONE in the absence of NADPH.While the best substrates for AKR1B10 are retinals, the high catalytic efficiency together with the protection from inactivation by NADPH suggests a role of AKR1B10 in the detoxification of biogenic aldehydes.

Peroxide-induced cell death and lipid peroxidation in C6 glioma cells.

Peroxides are often used as models to induce oxidative damage in cells in vitro. The aim of the present study was to elucidate the role of lipid peroxidation in peroxide-induced cell death. To this end (i) the ability to induce lipid peroxidation in C6 rat astroglioma cells of hydrogen peroxide (H2O2), cumene hydroperoxide (CHP) and t-butyl hydroperoxide (t-BuOOH) (ii) the relation between peroxide-induced lipid peroxidation and cell death in terms of time and concentration dependency and (iii) the capability of the lipid peroxidation chain breaking alpha-tocopherol to prevent peroxide-induced lipid peroxidation and/or cell death were investigated. Lipid peroxidation was characterised by measuring thiobarbituric acid reactive substances (TBARS) and, by HPLC, malondialdehyde (MDA), 4-hydroxynonenal (4-HNE) and hexanal. Within 2 h CHP, t-BuOOH and H2O2 induced cell death with EC50 values of 59+/-9 microM, 290+/-30 microM and 12+/-1.1 mM, respectively. CHP and t-BuOOH, but not H2O2 induced lipid peroxidation in C6 cells with EC50 values of 15+/-14 microM and 130+/-33 microM, respectively. The TBARS measured almost exclusively consisted of MDA. 4-HNE was mostly not detectable. The concentration of hexanal slightly increased with increasing concentrations of organic peroxides. Regarding time and concentration dependency lipid peroxidation preceded cell death. Pretreatment with alpha-tocopherol (10 microM, 24 h) prevented both, peroxide-induced lipid peroxidation and cell death. The results strongly indicate a major role of lipid peroxidation in the killing of C6 cells by organic peroxides but also that lipid peroxidation is not involved in H2O2 induced cell death.

Analysis of the substrate-binding site of human carbonyl reductases CBR1 and CBR3 by site-directed mutagenesis

Human carbonyl reductase is a member of the short-chain dehydrogenase/reductase (SDR) protein superfamily and is known to play an important role in the detoxification of xenobiotics bearing a carbonyl group. The two monomeric NADPH-dependent human isoforms of cytosolic carbonyl reductase CBR1 and CBR3 show a sequence similarity of 85% on the amino acid level, which is definitely high if compared to the low similarities usually observed among other members of the SDR superfamily (15-30%). Despite the sequence similarity and the similar features found in the available crystal structures of the two enzymes, CBR3 shows only low or no activity towards substrates that are metabolised by CBR1. This surprising substrate specificity is still not fully understood. In the present study, we introduced several point mutations and changed sequences of up to 17 amino acids of CBR3 to the corresponding amino acids of CBR1, to gather insight into the catalytic mechanism of both enzymes. Proteins were expressed in Escherichia coli and purified by Ni-affinity chromatography. Their catalytic properties were then compared using isatin and 9,10-phenanthrenequinone as model substrates. Towards isatin, wild-type CBR3 showed a catalytic efficiency of 0.018 microM(-1)min(-1), whereas wild-type CBR1 showed a catalytic efficiency of 13.5 microM(-1)min(-1). In particular, when nine residues (236-244) in the vicinity of the catalytic center and a proline (P230) in CBR3 were mutated to the corresponding residues of CBR1 a much higher k(cat)/K(m) value (5.7 microM(-1)min(-1)) towards isatin was observed. To gain further insight into the protein-ligand binding process, docking simulations were perfomed on this mutant and on both wild-type enzymes (CBR1 and CBR3). The theoretical model of the mutant was ad hoc built by means of standard comparative modelling.

Proteomic identification of aldo-keto reductase AKR1B10 induction after treatment of colorectal cancer cells with the proteasome inhibitor bortezomib

Targeting the ubiquitin-proteasome pathway with the proteasome inhibitor bortezomib has emerged as a promising approach for the treatment of several malignancies. The cellular and molecular effects of this agent on colorectal cancer cells are poorly characterized. This study investigated the antiproliferative effect of bortezomib on colorectal cancer cell lines (Caco-2 and HRT-18). In order to define the proteins potentially involved in the mechanisms of action, proteome profiling was applied to detect the proteins altered by bortezomib. The in vitro efficacy of bortezomib as a single agent in colorectal cancer cell lines was confirmed. Proteome profiling with two-dimensional PAGE followed by mass spectrometry revealed the up-regulation of the major inducible isoform of heat shock protein 70 (hsp72) and lactate dehydrogenase B in both cell lines, as well as the induction of aldo-keto reductase family 1 member B10 (AKR1B10) in HRT-18 cells. Both AKR1B10 and hsp72 exert cell-protective functions. This study shows for the first time a bortezomib-induced up-regulation of AKR1B10. Small interfering RNA–mediated inhibition of this enzyme with known intracellular detoxification function sensitized HRT-18 cells to therapy with the proteasome inhibitor. To further characterize the relevance of AKR1B10 for colorectal tumors, immunohistochemical expression was shown in 23.2% of 125 tumor specimens. These findings indicate that AKR1B10 might be a target for combination therapy with bortezomib. [Mol Cancer Ther 2009;8(7):1995–2006]

Studies on reduction of S-nitrosoglutathione by human carbonyl reductases 1 and 3

Human carbonyl reductases 1 and 3 (CBR1 and CBR3) are monomeric NADPH-dependent enzymes of the short-chain dehydrogenase/reductase superfamily. Despite 72% identity in primary structure they exhibit substantial differences in substrate specificity. Recently, the endogenous low molecular weight S-nitrosothiol S-nitrosoglutathione (GSNO) has been added to the broad substrate spectrum of CBR1. The current study initially addressed whether CBR3 could equally reduce GSNO which was not the case. Neither the introduction of residues which contribute to glutathione binding in CBR1, i.e. K106Q and S97V/D98A, nor the exchange C143S, which prevents a theoretical disulfide bond with C227 in CBR3, could engender activity towards GSNO. However, exchanging amino acids 236-244 in CBR3 to correspond to CBR1 was sufficient to engender catalytic activity towards GSNO. Catalytic efficiency was further improved by the exchanges Q142M, C143S, P230W and H270S. Hence, the same residues previously reported as important for reduction of carbonyl compounds appear to be key to CBR1-mediated reduction of GSNO. Furthermore, for CBR1-mediated reduction of GSNO, considerable substrate inhibition at concentrations >5 K(m) was observed. Treatment of CBR1 with GSNO followed by removal of low molecular weight compounds decreased the GSNO reducing activity, suggesting a covalent modification. Treatment with dithiothreitol, but not with ascorbic acid, could rescue the activity, indicating S-glutathionylation rather than S-nitrosation as the underlying mechanism. As C227 has previously been identified as the reactive cysteine in CBR1, the variant CBR1 C227S was generated, which, in comparison to the wild-type protein, displayed a similar k(cat), but a 30-fold higher K(m), and did not show substrate inhibition. Collectively, the results clearly argue for a physiological role of CBR1, but not for CBR3, in GSNO reduction and thus ultimately in regulation of NO signaling. Furthermore, at higher concentrations, GSNO appears to work as a suicide inhibitor for CBR1, probably through glutathionylation of C227.

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

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 μM curcumin), suggesting a beneficial effect in the co-treatment of anthracycline anti-tumor drugs together with curcumin.

Partial purification and characterization of a new human membrane-bound carbonyl reductase playing a role in the deactivation of the anticancer drug oracin

Carbonyl reducing enzymes play important roles in the biotransformation and detoxification of endo- and xenobiotics. They are grouped into two protein superfamilies, the short-chain dehydrogenases (SDR) and aldo-keto reductases (AKR), and usually are present in the cytoplasm of a cell. So far, only one membraneous carbonyl reductase has been described, 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1), which is located in the endoplasmic reticulum and which significantly contributes to the metabolism of a variety of carbonyl containing drugs and toxicants. Oracin is a new and prospective anticancer drug bearing a prochiral carbonyl moiety. The main metabolic pathway of oracin is carbonyl reduction to 11-dihydrooracin (DHO) which, however, eliminates the therapeutic potential of the drug, because the two DHO enantiomers formed have significantly less anti-tumor activities. Therefore, the oracin inactivating enzymes should urgently be identified to search for specific inhibitors and to enhance the chemotherapeutic efficacy. Interestingly, the calculation of enzyme specific activities and stereospecificities of (+)-DHO and (-)-DHO formation strongly suggested the existence of a second, hitherto unknown microsomal oracin carbonyl reductase in human liver. Therefore, the aim of the present study was to provide proof for the existence of this new enzyme and to develop a purification method for further characterization. First, we succeeded in establishing a gentle solubilization technique which provided a favourable detergent surrounding during the further purification procedure by stabilizing the native form of this fragile protein. Second, we could partially purify this new microsomal carbonyl reductase by a two step separation on Q-sepharose followed by Phenyl-sepharose. The enzyme turned out to be NADPH specific, displaying kinetic values for oracin carbonyl reduction of K(m)=42 microM and V(max)=813 nmol/(30 min x mg protein). Compared to the microsomal fraction, the enzyme specific activity towards oracin could be enhanced 73-fold, while the stereospecificity of (+)-DHO formation shifted from 40% to 86%. Considering these data for 11beta-HSD1, as described in previous reports, it is clear that the microsomal carbonyl reductase investigated in the present study is new and has a great potential to significantly impair the chemotherapy with the new anticancer drug oracin.

The Drosophila carbonyl reductase sniffer is an efficient 4-oxonon-2-enal (4ONE) reductase.

Studies with the fruit-fly Drosophila melanogaster demonstrated that the enzyme sniffer prevented oxidative stress-induced neurodegeneration. Mutant flies overexpressing sniffer had significantly extended life spans in a 99.5% oxygen atmosphere compared to wild-type flies. However, the molecular mechanism of this protection remained unclear. Sequence analysis and database searches identified sniffer as a member of the short-chain dehydrogenase/reductase superfamily with a 27.4% identity to the human enzyme carbonyl reductase type I (CBR1). As CBR1 catalyzes the reduction of the lipid peroxidation products 4HNE and 4ONE, we tested whether sniffer is able to metabolize these lipid derived aldehydes by carbonyl reduction. To produce recombinant enzyme, the coding sequence of sniffer was amplified from a cDNA-library, cloned into a bacterial expression vector and the His-tagged protein was purified by Ni-chelate chromatography. We found that sniffer catalyzed the NADPH-dependent carbonyl reduction of 4ONE (K(m)=24±2 μM, k(cat)=500±10 min(-1), k(cat)/K(m)=350 s(-1) mM(-1)) but not that of 4HNE. The reaction product of 4ONE reduction by sniffer was mainly 4HNE as shown by HPLC- and GC/MS analysis. Since 4HNE, though still a potent electrophile, is less neurotoxic and protein reactive than 4ONE, one mechanism by which sniffer exerts its neuroprotective effects in Drosophila after oxidative stress may be enzymatic reduction of 4ONE.