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.

Reduction of doxorubicin and oracin and induction of carbonyl reductase in human breast carcinoma MCF-7 cells.

In cancer cells, the drug-metabolizing enzymes may deactivate cytostatics, thus contributing to their survival. Moreover, the induction of these enzymes may also contribute to development of drug-resistance through acceleration of cytostatics deactivation. However, the principal metabolic pathways contributing to deactivation of many cytostatics still remain poorly defined. The main aims of the present study were: (i) to compare the reductive deactivation of cytostatic drugs doxorubicin (DOX) and oracin (ORC) in human breast cancer MCF-7 cells; (ii) to identify major enzyme(s) involved in the carbonyl reduction; and iii) to evaluate the activities and expression of selected carbonyl reducing enzymes in MCF-7 cells upon a short-term (48 h) exposure to either DOX or ORC. We found that MCF-7 cells were able to effectively metabolize both DOX and ORC through reduction of their carbonyl groups. The reduction of ORC was stereospecific, with a preferential formation of + enantiomer of dihydrooracin (DHO). The cytosolic carbonyl reductase CBR1 seemed to be a principal enzyme reducing both drugs, while cytosolic aldo-keto reductase AKR1C3 or microsomal reductases probably did not play important role in metabolism of either DOX or ORC. The exposure of MCF-7 cells to low (nanomolar) concentrations of DOX or ORC caused a significant elevation of reduction rates of both cytostatics, accompanied with an increase of CBR1 protein levels. Taken together, the present results seem to suggest that the accelerated metabolic deactivation of ORC or DOX might contribute to the survival of breast cancer cells during exposure to these cytostatics.

Inactivation of the anticancer drugs doxorubicin and oracin by aldo-keto reductase (AKR) 1C3.

Resistance towards anticancer drugs is a general problem upon chemotherapy. Among the mechanisms of resistance, metabolic inactivation by carbonyl reduction is a major cause of chemotherapy failure that applies to drugs bearing a carbonyl moiety. Oracin is a promising anticancer drug which is presently in phase II clinical trials. Pharmacokinetic studies have revealed that oracin undergoes metabolic inactivation by carbonyl reduction. In the present study, we provide evidence that AKR1C3, a member of the aldo-keto reductase (AKR) superfamily, catalyzes the inactivation of oracin. Moreover, AKR1C3 does also mediate C13 carbonyl reduction of doxorubicin to its inactive hydroxy metabolite doxorubicinol. Doxorubicinol, however, has also been considered responsible for the cardiomyopathy observed upon doxorubicin chemotherapy. Since AKR1C3 is overexpressed in hormone-dependent malignancies like prostate and breast cancer, coadministration of AKR1C3 inhibitors might enhance the chemotherapeutic efficacy of oracin and doxorubicin, and simultaneously reduce the risk of cardiomyopathy upon doxorubicin treatment.

The stereoselective biotransformation of the anti-obesity drug sibutramine in rat liver microsomes and in primary cultures of rat hepatocytes.

Sibutramine is an anti-obesity drug sold as a racemic mixture under the trademark Meridia or Reductil. With the aim of evaluating the stereoselectivity in phase I of sibutramine biotransformation, the formation of the main metabolites from R -sibutramine, S -sibutramine and rac-sibutramine was studied in rat microsomes and primary cultures of hepatocytes. A novel analytical method for the determination of sibutramine and its phase I metabolites in culture medium and microsomal incubates using isocratic reversed-phase liquid chromatography with UV detection was developed. Only two metabolites, mono-desmethylsibutramine (M1) and di-desmethylsibutramine (M2), were found in the rat microsomes incubated with sibutramine and NADPH. The kinetics of M1 and M2 formation slightly differed depending on the enantiomeric form of the sibutramine used. The stereoselectivity in sibutramine biotransformation was much more evident in primary cultures of rat hepatocytes. While R-sibutramine incubation led to the formation of M1 and M2 metabolites only, the incubation of S-sibutramine or rac-sibutramine (to a lesser extent) resulted in four major metabolites (M1, M2, M3 and M4) and 2 or 3 minor metabolites. On the basis of our results, R-sibutramine might represent the more advantageous sibutramine enantiomer from the pharmacokinetic standpoint.