Michael Soldan

Development of daunorubicin resistance in tumour cells by induction of carbonyl reduction

A resistant descendant of the human stomach carcinoma cell line EPG85-257 was selected in the presence of increasing concentrations of daunorubicin (DRC). To avoid the expression and activity of P-glycoprotein (P-gp) and multidrug resistance-associated protein (MRP), cells were cultured in the presence of verapamil. The resulting cells were used to evaluate an induced carbonyl reduction as a new determinant in DRC resistance. The MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl tetrazolium bromide) toxicity assay was performed to estimate sensitivity to DRC in both cell lines. IC50 values of DRC increased almost 8-fold in the resistant descendants compared to the parental cell line. P-gp transcripts were detectable in both cell lines at only very low levels, and no significant alterations between sensitive and resistant cells were observed. MRP mRNA expression was markedly higher compared to P-gp mRNA, but, as was the case with P-gp, MRP mRNA levels in sensitive and resistant cells showed no alteration. This was probably due to the effect of the presence of verapamil during cell selection. Another known drug resistance factor, the lung resistance-related protein (LRP), was not at all detectable. Interestingly, resistant cells possessed 6-fold higher levels of DRC carbonyl-reducing activity, leading to the less toxic 13-hydroxy metabolite daunorubicinol (DRCOL). The 6-fold higher DRCOL formation roughly parallels the 8-fold increase in DRC IC50 values during cell selection, and therefore may account for DRC resistance in these cells. The determination of specific carbonyl reducing enzymes, known to be involved in DRC detoxification, revealed that mRNA expression of carbonyl reductase (EC 1.1.1.184), aldose reductase (EC 1.1.1.21), and dihydrodiol dehydrogenase 2 (EC 1.3.1.20) increased in the resistant descendant. In contrast, the phase II-conjugating enzyme activities of glutathione S-transferases were significantly lower in resistant than in sensitive cells, whereas those of glucuronosyl transferase were not detectable in either cell line. Apparently, conjugating enzymes are not involved in DRC resistance in human stomach carcinoma cells. These studies indicate that DRC resistance in human stomach carcinoma cells may appear as a result of an induction of metabolic DRC inactivation via carbonyl reduction to the less active 13-hydroxy metabolite DRCOL.

Two-dimensional gel electrophoresis: a powerful method to elucidate cellular responses to toxic compounds.

Humans are exposed to a variety of environmental toxicants and combinations thereof, and a large number of interacting factors contribute to an individuals risk for disease. Therefore, new strategies in toxicological research are needed for efficient screening of environmental hazards on complex living systems. The rapidly expanding field of proteomics relies heavily upon the use of two-dimensional gel electrophoresis (2-DE) of protein samples. 2-DE is a key separation technique in proteome analysis due to its advantage of simultaneous separation of thousands of proteins at a time, excellent reproducibility, and ability to exhibit post-translational modifications. Therefore, 2-D proteome analysis is becoming a popular method of choice to detect differentially expressed proteins between proteome profiles after exposure to toxicants. The goal of this study was to examine the response of pancreas carcinoma cells to increasing concentrations of the cytotoxic agent daunorubicin (DRC). The proteomic investigation revealed a number of proteins that were up-regulated by DRC treatment, some in a dose-dependent manner. However, these changes were not seen by reverse transcriptase-polymerase chain reaction. The determination of proteome changes following exposure to xenobiotics will aid our understanding of the mechanisms of their toxicity as well as providing the possibility for the establishment of biomarkers that can be used in risk assessment as well as for the identification of individual susceptibility factors.

Induction of daunorubicin carbonyl reducing enzymes by daunorubicin in sensitive and resistant pancreas carcinoma cells

Daunorubicin (DRC) and other anthracyclines are valuable cytotoxic agents in the clinical treatment of certain malignancies. However, as is the case with virtually all anticancer drugs, tumor cell resistance to these agents is one of the major obstacles to successful chemotherapy. In addition to an increased energy-dependent efflux of chemotherapeutic agents, enzymatic drug-inactivating mechanisms also contribute to multidrug resistance of tumor cells. In the case of DRC, carbonyl reduction leads to 13-hydroxydaunorubicinol (DRCOL), the major metabolite of DRC with a significantly lower antineoplastic potency compared to the parent drug. In the present study, we compared two pancreas carcinoma cell lines (a DRC-sensitive parental line and its DRC-resistant subline) with respect to their capacity of DRC inactivation via carbonyl reduction. In addition, we cultured the two cell lines in the presence of increasing sublethal concentrations of DRC. Evidence is presented that DRC treatment itself leads to a concentration-dependent induction of DRC carbonyl reduction in subcellular fractions of both the sensitive and resistant pancreas carcinoma cells, resulting, surprisingly, in different susceptibilities to DRC. The principal difference between the two cell lines becomes most apparent at high-dose DRC supplementation (1 microgram/mL), at which DRC resistant cells exhibited higher inducibility of DRC-inactivating enzymes, whereas respective sensitive cells already showed an impairment of cellular viability. The use of the diagnostic model substrates metyrapone and p-nitrobenzaldehyde reveals that this adaptive enhancement of DRC inactivation can be attributed to the induction of DRC carbonyl reductases different from known aldehyde and carbonyl reductases. In conclusion, these findings suggest that inactivation of anthracyclines by carbonyl reduction is inducible by the substrate itself, a fact that might be considered as one of the enzymatic mechanisms that contribute to the acquired resistance to these drugs.

Interindividual variability in the expression and NNK carbonyl reductase activity of 11β-hydroxysteroid dehydrogenase 1 in human lung

The balance between metabolic activation and detoxification is critical in determining the susceptibility to lung cancer upon exposure to the tobacco specific nitrosamine, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). Carbonyl reduction of NNK, followed by glucuronidation, is the main detoxification pathway of this lung carcinogen in humans. Recently, we have identified 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD 1) as microsomal NNK carbonyl reductase in liver and lung. In the present study, the interindividual variability of 11beta-HSD 1 expression and NNK-carbonyl reductase activity was examined in human lung by RT-PCR, Western blot analysis and enzyme activity. Levels of 11beta-HSD 1 mRNA varied over an almost 20-fold range among different subjects. Levels of NNK carbonyl reductase activity in lung microsomes closely resembled the relative amounts of immunoreactive protein as determined by Western blot analysis. In view of the large interindividual differences in the susceptibility of tobacco smoke related lung cancer, we present the first data on the variability of 11beta-HSD 1 expression and NNK carbonyl reduction in human lung.

Biotransformation and detoxification of insecticidal metyrapone analogues by carbonyl reduction in the human liver

1. The carbonyl reduction of insecticidal metyrapone analogues to their hydroxyl metabolites by human liver microsomes and cytosol was examined. Metabolite quantification was performed by means of hplc determination and inhibition experiments, using specific carbonyl reductase inhibitors, were conducted. 2. The cytotoxicity of the ketones and their hydroxy metabolites was assessed with the MTT test, using Chang liver cells. 3. It was found that the alcohol derivatives are the major metabolite, both in microsomes and cytosol. The microsomal reductive metabolism, considered to be mediated by 11 beta-hydroxysteroid dehydrogenase (11 beta-HSD) (EC 1.1.1.146), was more extensive than the cytosolic carbonyl reduction. In each case, this metabolism was inhibited significantly by equimolar concentrations of the microsomal 11 beta-HSD inhibitor glycyrrhetinic acid and the cytosolic carbonyl reductase inhibitor quercitrin, respectively. 4. The parent ketones were more cytotoxic than their alcohol metabolites. 5. These results demonstrate that the metyrapone analogues are extensively metabolized by human liver microsomes, presumably by 11 beta-HSD, to the less cytotoxic and readily excretable alcohols. 6. Since the metyrapone analogues can inhibit ecdysone 20-monooxygenase (EC 1.14.99.22), our results indicate potential application of these compounds as insecticides, which would be safer for humans, due to their reductive detoxification, mainly by the hepatic microsomal 11 beta-HSD, to the less toxic hydroxy metabolites.

Modulation of daunorubicin toxicity by liposomal encapsulation and use of specific inhibitors in vitro.

Anthracyclines serve as a valuable tool in chemotherapy, but their usefulness is often limited by the occurrence of resistance mechanisms in tumor cells. Resistance of tumor cells is a multifactorial event, where several mechanisms act concurrently, including drug efflux and enzymatic drug inactivation. Liposomal encapsulation of anthracyclines has been discussed as a successful regimen to overcome drug resistance. Our investigations were carried out on a daunorubicin (DRC) sensitive breast cancer cell line and two DRC resistant sublines generated thereof. In all three cell lines, the extent of DRC detoxification via carbonyl reduction to daunorubicinol (DRCOL) was determined. In addition, rutin, the most effective inhibitor of carbonyl reducing enzymes, was tested to affect DRCOL formation. DRC IC(50) values were determined in relation to several combinations of DRC administration, (a) liposomal encapsulated DRC, (b) addition of verapamil (inhibitor of drug efflux), (c) addition of rutin (inhibitor of DRC carbonyl reduction). We could show that DRC sensitive and resistant breast cancer cell lines are able to catalyze DRC detoxification via carbonyl reduction to DRCOL. Rutin was shown to inhibit this reaction, but could not serve as an enhancer of DRC toxicity in MTT tests. Verapamil was effective only in resistant cells due to the overexpression of P-glycoprotein 170. Liposomal encapsulation of DRC did not show the expected increase in DRC toxicity in the present tumor cell model.

Cytostatic Drug Resistance

Inherent or acquired resistance to multiple anti-cancer drugs is a problem of major importance in chemotherapy, and numerous mechanisms of resistance have been investigated until now. Multidrug resistance phenotype (MDR) is the result of, at least in part, the overexpression of P-glycoprotein 170 (P-gp) and multidrug resistance associated protein (MRP) which act by decreasing intracellular drug concentrations through an ATP-de- pendent, verapamil-sensitive efflux of unmodified drug from the cell (Endicott and Ling, 1989). Drug resistance mediated by P-gp and MRP can be reversed by the chemosensitizier verapamil. Multidrug resistant cell lines frequently overexpress a 110 kDa protein called LRP (lung resistance related protein), identified as the major vault protein. LRP expression at time of diagnosis provides a strong and independent prognostic factor for response to chemotherapy and outcome in different tumor types (Scheffer et al., 1995).

Metabolic inactivation and efflux of daunorubicin as complementary mechanisms in tumor cell resistance.

Multidrug-resistance is a major obstacle in cancer therapy with antineoplastic chemotherapeutics. Drug-resistant tumor cells frequently display overexpression of a plasma membrane phosphoglycoprotein termed P-170. P-170 is thought to act as an energy dependend efflux pump (Juliano and Ling, 1976) that lowers the intracellular accumulation of structurally and functionally unrelated antineoplastic agents such as anthracyclines, vinca alkaloides and taxol, and, thus, confers multidrug resistance. Anthracyclines, like daunorubicin (DRC) are the most valuable cytostatic agents in clinical chemotherapy, but their usefulness is limited by intrinsic or aquired resistance towards these drugs. However, it is known that P-170 cannot fully account for the chemoresistance phenomenon in multidrug resistant tumor cells (Booser and Hortobagyi, 1994). In addition to an increasing efflux of chemotherapeutic agents, enzymatic drug inactivation can also contribute to a higher drug resistance (Deffie et al., 1988). In the case of DRC, carbonyl reduction leads to 13-hydroxydaunorubicinol (DRCOL), the major metabolite of DRC with a significant lower antineoplastic potency compared to the parent drug (Schott and Robert, 1989).

Alterations in the expression of daunorubicin phase-I metabolising enzymes in different carcinoma cell lines.

Drug resistance is a major obstacle in the successful treatment of cancer by chemotherapy. The phenomenon of multidrug resistance is often related to the overexpression of two plasma membrane transporters, termed glycoprotein P-170 (MDR1) and multidrug resistance related protein (MRP). Both proteins are acting as ATP-driven efflux pumps, removing intracellular cytotoxic agents out of tumor cells (Endicott and Ling, 1989). Another protein mediating drug resistance is involved in the nucleo-cytoplasmic transport process. It is termed lung resistance related protein (LRP), because it was found first time in a lung cancer cell line (Scheffer et al., 1995). This resistance mechanism also occurs in several other cancer cells. But also the metabolism of cytostatic drugs could contribute to a lower toxicity and failure of chemotherapy. It is known that elevated levels of drug-inactivating enzymes support tumor cells in acquiring resistance (Deffie et al., 1988; Gessner et al., 1990; Rekha et al., 1994).