Modulation of daunorubicin toxicity by liposomal encapsulation and use of specific inhibitors in vitro
Introduction
Treatment of malignant tumors with cytostatic drugs is one of the proven strategies in chemotherapy, especially if the lesions have spread within the organism. Most often, first line regimens are successful in reducing the tumor size, and sometimes tumor masses are below the detection limit after treatment. These acceptable results are normally of relatively short duration, because single tumor cells survive somewhere in the body and are often a source of recurrent disease. Under the selection pressure of cytostatic drugs, tumor cells are able to develop certain mechanisms to acquire resistance (Patel and Rothenberg, 1994). Anthracyclines, such as daunorubicin (DRC), are major components of combination chemotherapy for a wide range of cancer diseases. Several possibilities exist for tumor cells to acquire resistance against DRC.
Overexpression of membrane transporter proteins may protect tumor cells by preventing intracellular DRC accumulation. P-glycoprotein 170 (P-gp) and the multidrug-resistance related protein (MRP) are ABC-(ATP-binding-cassette)-transporters which act by decreasing intracellular DRC concentrations through an ATP-dependent efflux of unmodified drug out of the cell (Endicott and Ling, 1989). This enhanced efflux can be blocked by specific inhibitors like, e.g. verapamil (Horio et al., 1991).
Other potential resistance mechanisms include elevated levels of drug inactivating enzymes such as glucuronosyl transferase, glutathione transferase and cytosolic class-3 aldehyde dehydrogenase, or alterations in levels and/or activity of topoisomerase II, which can also contribute to anthracycline resistance of tumor cells (Batist et al., 1986, Capranico et al., 1987, Deffie et al., 1988, Kramer et al., 1988, Gessner et al., 1990, Volm et al., 1992, Rekha et al., 1994).
One of the other yet defined mechanisms of DRC resistance is an upregulated enzymatic drug detoxification via DCR carbonyl reduction (Soldan et al., 1996, Ax et al., in press), leading to the less toxic 13-hydroxy metabolite daunorubicinol (DRCOL). Three hitherto known enzymes have been identified to catalyse this reaction. Two of these are members of the aldo-keto reductase (AKR) superfamily, namely aldehyde reductase (EC 1.1.1.2) and dihydrodiol dehydrogenase (EC 1.3.1.20). The other enzyme is a member of the short chain dehydrogenase/reductase (SDR) superfamily termed carbonyl reductase (EC 1.1.1.184) (Ohara et al., 1995).
One of the successful regimens to overcome resistance during DRC treatment is the application of liposomal DRC. DRC encapsulated in small uni-lamellar liposomes (SUV) reduces DRC mediated side effects (Presant et al., 1993) and increases drug accumulation in the tumor cells (Merlin et al., 1995). Both in vivo and in vitro studies revealed that more than a dozen antineoplastic agents showed positive results upon liposomal administration (Kim, 1993). However, failure of liposomal treatment against resistant tumor cells has also been observed (Hu et al., 1995).
Our investigations were carried out in a DRC sensitive breast cancer cell line and two DRC resistant sublines generated thereof. In all three cell lines, the extent of DRC carbonyl reduction was determined. In addition, rutin, the most effective inhibitor of carbonyl reducing enzymes, was tested to affect DRCOL formation. In order to circumvent DRC resistance and to find the most effective mode of treatment to enhance DRC toxicity, we have compared DRC IC50 values in relation to several combinations of DRC administration via the MTT-test: (a) liposomal encapsulated DRC, (b) addition of verapamil (inhibitor of the ABC transporters), (c) addition of rutin (inhibitor of DRC carbonyl reduction).
Section snippets
Chemicals
DRC was supplied by Rhone-Poulenc Pharma GmbH (Köln, Germany) and DRCOL was donated by Farmitalia Carlo Erba GmbH (Milano, Italy). Verapamil was purchased from Sigma Chemie (Deisenhofen, Germany). Culture medium and fetal calf serum were obtained from Gibco BRL (Eggenstein, Germany). All other chemicals were of the highest commercially available grade.
Cell lines
Three human breast cancer cell lines were used in this study: the parental DRC sensitive cell line MaTu/p19, and two DRC resistant sublines
Carbonyl reduction of DRC
Fig. 1 shows the DRC carbonyl reducing activities in the cytosolic fractions of the three breast cancer cell lines. Obviously, all three cell lines have the ability to detoxify DRC to its 13-hydroxymetabolite DRCOL. The descendant line grown in the presence of the highest DRC concentration (MaTu 0.25) had significant higher activities compared to MaTu 0.1 and MaTu/p19. A slight increase of activity, compared to MaTu/p19, could also be observed in the cell line MaTu 0.1.
Inhibition of DRC carbonyl reduction by rutin
Since the three breast
Discussion
DRC carbonyl reduction to DRCOL was detectable in the cytosolic fractions of all three breast cancer cell lines investigated in this study. The resistant cell lines, grown in the presence of DRC, were able to reduce DRC more effectively than the sensitive cells. This phenomenon has already been described in two pancreas carcinoma cell lines (Soldan et al., 1996), where an extensive induction of reductive DRC metabolism after DRC pretreatment was observed. It was discussed that an inhibition of
Acknowledgements
This study was supported by grants from the Deutsche Forschungsgemeinschaft (MA 1704/3-1), the Stiftung Verhalten und Umwelt (München, Germany), and the European Commission (BIO4-97-2123). This paper is dedicated to Professor Karl J. Netter, on the occasion of his retirement from our Department and as European Editor of Toxicology.
References (29)
- et al.
Overexpression of a novel anionic glutathione transferase in multidrug-resistant human breast cancer cells
J. Biol. Chem.
(1986) - et al.
The effects of aldose reductase inhibitors on the metabolism of cultured monkey kidney epithelial cells
Biochem. Pharmacol.
(1981) - et al.
Reexamination and further development of a precise and rapid dye method for measuring cell growth/cell kill
J. Immunol. Methods
(1989) - et al.
Agents which reverse multidrug-resistance are inhibitors of [3H]vinblastine transport by isolated vesicles
Biochim. Biophys. Acta.
(1991) - et al.
Failure of liposomal encapsulation of doxorubicin to circumvent multidrug resistance in an in vitro model of rat glioblastoma cells
Eur. J. Cancer
(1995) - et al.
Cellular detoxification of tripeptidyl aldehydes by an aldo-keto reductase
J. Biol. Chem.
(1993) - et al.
Characterisation of liposomal systems containing doxorubicin entrapped in response to pH gradient
Biochem. Biophys. Acta
(1990) - et al.
Reduction of drug ketones by dihydrodiol dehydrogenases, carbonyl reductase and aldehyde reductase of human liver
Biochem. Pharmacol.
(1995) - et al.
Liposomal daunorubicin treatment of HIV-associated Kaposi's sarcoma
Lancet
(1993) - et al.
Intrinsic cellular resistance to oxazaphosphorines exhibited by a human colon carcinoma cell line expressing relatively large amounts of a class-3 aldehyde dehydrogenase
Biochem. Pharmacol.
(1994)