Adam Skarka

Hydroxysteroid dehydrogenases (HSDs) in bacteria – A bioinformatic perspective

Steroidal compounds including cholesterol, bile acids and steroid hormones play a central role in various physiological processes such as cell signaling, growth, reproduction, and energy homeostasis. Hydroxysteroid dehydrogenases (HSDs), which belong to the superfamily of short-chain dehydrogenases/reductases (SDR) or aldo-keto reductases (AKR), are important enzymes involved in the steroid hormone metabolism. HSDs function as an enzymatic switch that controls the access of receptor-active steroids to nuclear hormone receptors and thereby mediate a fine-tuning of the steroid response. The aim of this study was the identification of classified functional HSDs and the bioinformatic annotation of these proteins in all complete sequenced bacterial genomes followed by a phylogenetic analysis. For the bioinformatic annotation we constructed specific hidden Markov models in an iterative approach to provide a reliable identification for the specific catalytic groups of HSDs. Here, we show a detailed phylogenetic analysis of 3α-, 7α-, 12α-HSDs and two further functional related enzymes (3-ketosteroid-Δ(1)-dehydrogenase, 3-ketosteroid-Δ(4)(5α)-dehydrogenase) from the superfamily of SDRs. For some bacteria that have been previously reported to posses a specific HSD activity, we could annotate the corresponding HSD protein. The dominating phyla that were identified to express HSDs were that of Actinobacteria, Proteobacteria, and Firmicutes. Moreover, some evolutionarily more ancient microorganisms (e.g., Cyanobacteria and Euryachaeota) were found as well. A large number of HSD-expressing bacteria constitute the normal human gastro-intestinal flora. Another group of bacteria were originally isolated from natural habitats like seawater, soil, marine and permafrost sediments. These bacteria include polycyclic aromatic hydrocarbons-degrading species such as Pseudomonas, Burkholderia and Rhodococcus. In conclusion, HSDs are found in a wide variety of microorganisms including bacteria and archaea, suggesting that steroid metabolism is an evolutionarily conserved mechanism that might serve different functions such as nutrient supply and signaling. Article from a special issue on steroids and microorganisms.

Anthracyclines and their metabolism in human liver microsomes and the participation of the new microsomal carbonyl reductase.

Anthracyclines (ANTs) are widely used in the treatment of various forms of cancer. Although their usage contributes to an improvement in life expectancy, it is limited by severe adverse effects-acute and chronic cardiotoxicity. Several enzymes from both AKR and SDR superfamilies have been reported as participants in the reduction of ANTs. Nevertheless all of these are located in the cytosolic compartment. One microsomal reductase has been found to be involved in the metabolism of xenobiotics-11beta-HSD1, but no further information has been reported about its role in the metabolism of ANTs. The aim of this study is to bring new information about the biotransformation of doxorubicin (DOX), daunorubicin (DAUN) and idarubicin (IDA), not only in human liver microsomal fraction, but also by a novel human liver microsomal carbonyl reductase that has been purified by our group. The reduction of ANTs at C-13 position is regarded as the main pathway in the biotransformation of ANTs. However, our experiments with human liver microsomal fraction show different behaviour, especially when the concentration of ANTs in the incubation mixture is increased. Microsomal fraction was incubated with doxorubicin, daunorubicin and idarubicin. DOX was both reduced into doxorubicinol (DOXOL) and hydrolyzed into aglycone DOX and then subsequently reduced. The same behaviour was observed for the metabolism of DAUN and IDA. The activity of hydrolases definitely brings a new look to the entire metabolism of ANTs in microsomal fraction, as formed aglycones undergo reduction and compete for the binding site with the main ANTs. Moreover, as there are two competitive reducing reactions present for all three ANTs, kinetic values of direct reduction and the reduction of aglycone were calculated. These results were compared to previously published data for human liver cytosol. In addition, the participation of the newly determined human liver microsomal carbonyl reductase was studied. No reduction of DOX into DOXOL was detected. Nevertheless, the involvement in reduction of DAUN into DAUNOL as well as IDA into IDAOL was demonstrated. The kinetic values obtained were then compared with data which have already been reported for cytosolic ANTs reductases.

The Influence of Sesquiterpenes from Myrica rubra on the Antiproliferative and Pro-Oxidative Effects of Doxorubicin and Its Accumulation in Cancer Cells

The sesquiterpenes β-caryophyllene, β-caryophyllene oxide (CAO), α-humulene (HUM), trans-nerolidol (NER), and valencene (VAL) are substantial components of the essential oil from Myrica rubra leaves which has exhibited significant antiproliferative effects in several intestinal cancer cell lines, with CaCo-2 cells being the most sensitive. The present study was designed to evaluate the effects of these sesquiterpenes on the efficacy and toxicity of the anticancer drug doxorubicin (DOX) in CaCo-2 cancer cells and in primary culture of rat hepatocytes. Our results showed that HUM, NER, VAL and CAO inhibited proliferation of CaCo-2 cancer cells but they did not affect the viability of hepatocytes. CAO, NER and VAL synergistically potentiated the efficacy of DOX in cancer cells killing. All sesquiterpenes exhibited the ability to selectively increase DOX accumulation in cancer cells and did not affect DOX concentration in hepatocytes. Additionally, CAO and VAL were able to increase the pro-oxidative effect of DOX in CaCo-2 cells. Moreover, CAO mildly ameliorated DOX toxicity in hepatocytes. Based on all results, CAO seems to be the most promising compound for further testing.

Isoquinoline alkaloids as a novel type of AKR1C3 inhibitors

AKR1C3 is an important human enzyme that participates in the reduction of steroids and prostaglandins, which leads to proliferative signalling. In addition, this enzyme also participates in the biotransformation of xenobiotics, such as drugs and procarcinogens. AKR1C3 is involved in the development of both hormone-dependent and hormone-independent cancers and was recently demonstrated to confer cell resistance to anthracyclines. Because AKR1C3 is frequently upregulated in various cancers, this enzyme has been suggested as a therapeutic target for the treatment of these pathological conditions. In this study, nineteen isoquinoline alkaloids were examined for their ability to inhibit a recombinant AKR1C3 enzyme. As a result, stylopine was demonstrated to be the most potent inhibitor among the tested compounds and exhibited moderate selectivity towards AKR1C3. In the follow-up cellular studies, stylopine significantly inhibited the AKR1C3-mediated reduction of daunorubicin in intact cells without considerable cytotoxic effects. This inhibitor could therefore be used as a model AKR1C3 inhibitor in research or evaluated as a possible therapeutic anticancer drug. Furthermore, based on our results, stylopine can serve as a model compound for the design and future development of structurally related AKR1C3 inhibitors.

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.

Pharmacokinetic interactions of breast cancer chemotherapeutics with human doxorubicin reductases

Paclitaxel (PTX), docetaxel (DTX), 5-fluorouracil (5-FU), cyclophosphamide (CYC) or tamoxifen (TMX) are combined with doxorubicin (DOX) in first-line chemotherapy regimens that are indicated for breast cancer patients. Although the efficacies of these drugs in combination treatments have been demonstrated in clinical practice, their possible interference with DOX metabolism has not been described in detail to date. In the present study, we investigated the possible interactions of human carbonyl reducing enzymes with 5-FU, PTX, DTX, CYC and TMX. First, the reducing activities of carbonyl reducing enzymes toward DOX were tested using incubations with purified recombinant enzymes. In the subsequent studies, we investigated the possible effects of the tested anticancer agents on the DOX-reducing activities of the most potent enzymes (AKR1C3, CBR1 and AKR1A1) and on the DOX metabolism driven by MCF7, HepG2 and human liver cytosols. In both of these assays, we observed that CYC and its active metabolites inhibited DOX metabolism. In the final study, we tracked the changes in AKR1C3, CBR1 and AKR1A1 expression levels following exposure to the tested cytostatics in MCF7 and HepG2 cells. Consequently, no significant changes in the expression levels of tested enzymes were detected in either cell line. Based on these findings, it is feasible to presume that inhibition rather than induction plays a role in the interactions of the tested anticancer agents with DOX-reducing enzymes. In conclusion, our results describe important molecular events that occur during combination breast cancer therapies and might modulate pharmacokinetic DOX resistance and/or behaviour.

Molecular and biochemical characterisation of human short-chain dehydrogenase/reductase member 3 (DHRS3).

Dehydrogenase/reductase (SDR family) member 3 (DHRS3), also known as retinal short-chain dehydrogenase/reductase (retSDR1) is a member of SDR16C family. This family is thought to be NADP(H) dependent and to have multiple substrates; however, to date, only all-trans-retinal has been identified as a DHRS3 substrate. The reductive reaction catalysed by DHRS3 seems to be physiological, and recent studies proved the importance of DHRS3 for maintaining suitable retinoic acid levels during embryonic development in vivo. Although it seems that DHRS3 is an important protein, knowledge of the protein and its properties is quite limited, with the majority of information being more than 15 years old. This study aimed to generate a more comprehensive characterisation of the DHRS3 protein. Recombinant enzyme was prepared and demonstrated to be a microsomal, integral-membrane protein with the C-terminus oriented towards the cytosol, consistent with its preference of NADPH as a cofactor. It was determined that DHRS3 also participates in the metabolism of other endogenous compounds, such as androstenedione, estrone, and DL-glyceraldehyde, and in the biotransformation of xenobiotics (e.g., NNK and acetohexamide) in addition to all-trans-retinal. Purified and reconstituted enzyme was prepared for the first time and will be used for further studies. Expression of DHRS3 was shown at the level of both mRNA and protein in the human liver, testis and small intestine. This new information could open other areas of DHRS3 protein research.

Purification and reconstitution of human membrane-bound DHRS7 (SDR34C1) from Sf9 cells

Dehydrogenase/reductase SDR family member 7 (DHRS7, SDR34C1, retSDR4) is one of the many endoplasmic reticulum bound members of the SDR superfamily. Preliminary results indicate its potential significance in human metabolism. DHRS7 containing TEV-cleavable His10 and FLAG-tag expressed in the Sf9 cell line was solubilised, purified, and reconstituted into liposomes to enable the improved characterisation of this enzyme in the future. Igepal CA-630 was determined to be the best detergent for the solubilisation process. The solubilised DHRS7 was purified using affinity chromatography, and the purified enzyme was subjected to TEV cleavage of the affinity tags and then repurified using subtractive Ni-IMAC. The cleaved and uncleaved versions of DHRS7 were successfully reconstituted into liposomes. In addition, using tobacco specific carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) as the substrate, the cleaved liposomal DHRS7 was found to be inactive, whereas the pure and uncleaved liposomal DHRS7 were confirmed as enzymes, which reduce carbonyl group of the substrates.

Enzyme stereospecificity as a powerful tool in searching for new enzymes.

Chirality is a ubiquitous feature present in all biological systems that plays a very important role in many processes. Drug metabolism is one of these and is the subject of this review. Chiral drugs can be metabolized without changes in their chiral characteristics, but also their biotransformation may give rise to a new chiral center. On the other hand, prochiral drugs are always metabolized to chiral metabolites. The ratio of formed enantiomers/diastereoisomers is the constant known as enzyme stereospecificity, and this is as important a characteristic for each enzyme-substrate pair as is the Michaelis constant. Drugs are often substrates for multiple biotransformation enzymes, and all enzymes involved may metabolize a chiral or prochiral drug with different stereospecificity so that variant enantiomer ratios are achieved. Enzyme stereospecificity of whole cell fraction is the sum of the stereospecificities of all enzymes participating in metabolism of a substrate. Differing stereospecificities in the metabolism of a drug between whole cell fraction and enzymes point to the contribution of other enzymes. Using several drugs as examples, this review shows that enzyme stereospecificity can serve as a powerful tool in searching for new biotransformation enzymes. Although it is not often used in this way, it is clear that this is possible. There are today drugs with well-known chiral metabolism, but, inasmuch as many xenobiotics are poorly characterized in terms of chiral metabolism, enzyme stereospecificity could be widely utilized in researching such substances.

The Effects of Selected Sesquiterpenes from Myrica rubra Essential Oil on the Efficacy of Doxorubicin in Sensitive and Resistant Cancer Cell Lines

β-caryophyllene oxide (CAO), α-humulene (HUM), trans-nerolidol (NER) and valencene (VAL) are constituents of the essential oil of Myrica rubra (MEO), which has significant antiproliferative effect in various cancer cell lines. In the present study, we compared the antiproliferative effect of these sesquiterpenes alone and in combination with the cytostatic drug doxorubicin (DOX) in cancer cell lines with different sensitivity to DOX. Two ovarian cancer cell lines (sensitive A2780 and partly resistant SKOV3) and two lymphoblast cancer cell lines (sensitive CCRF/CEM and completely resistant CEM/ADR) were used. The observed effects varied among sesquiterpenes and also differed in individual cell lines, with only VAL being effective in all the cell lines. A strong synergism of DOX with NER was found in the A2780 cells, while DOX acted synergistically with HUM and CAO in the SKOV3 cells. In the CCRF/CEM cells, a synergism of DOX with CAO and NER was observed. In resistant CEM/ADR cells, sesquiterpenes did not increase DOX efficacy, although they significantly increased accumulation of DOX (up to 10-times) and rhodamine-123 (substrate of efflux transporter ABCB1) within cancer cells. In conclusion, the tested sesquiterpenes were able to improve DOX efficacy in the sensitive and partly resistant cancer cells, but not in cells completely resistant to DOX.