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

doi:10.1016/S0006-2952(99)00322-6

Biochemical Pharmacology

Volume 59, Issue 3, 1 February 2000, Pages 293-300

https://doi.org/10.1016/S0006-2952(99)00322-6 Get rights and content

Abstract

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.

Section snippets

Chemicals

DRC was supplied by Rhône–Poulenc Pharma GmbH and DRCOL was donated by Farmitalia Carlo Erba GmbH. Leibovits L-15 medium and fetal bovine serum were obtained from GIBCO BRL. All other chemicals were of highest commercially available grade.

Cells and cell culture

A DRC-sensitive human stomach carcinoma cell line (EPG85-257), isolated from liver metastases, was kindly provided by Prof. M. Dietel. Cells were grown in Leibovits L-15 medium completed with 10% fetal bovine serum, insulin 80 I.E./L, transferrin 2.5 mg/mL,

Generation of DRC-resistant stomach carcinoma cells

The generation of a DRC-resistant subline was accomplished by culturing human stomach carcinoma cells in the absence (control) or presence of stepwise increasing concentrations of DRC, ranging from 0.1 ng/mL (starting concentration) to 12.8 ng/mL (final concentration) after 5 months. DRC concentrations were doubled every two weeks, finally resulting in a DRC-resistant subline grown at 12.8 ng/mL DRC (EPG Res). To suppress the overexpression of P-gp or MRP, DRC was co-supplemented with 20 μM

Discussion

In general, well-characterized sensitive and resistant cell lines derived from the same origin represent suitable models to study the mechanisms of acquired resistance to selected chemotherapeutics and to trace these mechanisms to classical and/or non-classical MDR. In this study, we developed a DRC-resistant stomach carcinoma cell line by continuous passage in increasing sublethal concentrations of DRC. After eight passages (five months), cells grown at 12.8 ng/mL DRC had ic₅₀ values for DRC

Acknowledgements

The present study was supported by a grant from the Alfred and Ursula Kulemann-Stiftung, Marburg, and by the European Commission (BIO4-97-2123).

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Chemotherapy and Metabolic InhibitorsDevelopment of daunorubicin resistance in tumour cells by induction of carbonyl reduction

Abstract

Section snippets

Chemicals

Cells and cell culture

Generation of DRC-resistant stomach carcinoma cells

Discussion

Acknowledgements

J Biol Chem

Biochem Pharmacol

J Biol Chem

Biochem Pharmacol

Biochem Biophys Res Commun

Biochem Pharmacol

Biochem Pharmacol

Biochem Pharmacol

Biochem Pharmacol

J Biol Chem

J Immunol Methods

Methods Enzymol

Methods Enzymol

J Chromatograph

J Biol Chem

Biochem Pharmacol

Blood

J Urol

J Biol Chem

Methods Enzymol

Biochim Biophys Acta

Biochem Pharmacol

Biochem Pharmacol

The biochemistry of P-glycoprotein-mediated multidrug resistance

Annu Rev Biochem

The human multidrug resistance-associated protein MRP is a plasma membrane drug-efflux pump

Proc Natl Acad Sci USA

Chemotherapy and Metabolic Inhibitors
Development of daunorubicin resistance in tumour cells by induction of carbonyl reduction