A comparative study of cellular and molecular pharmacology of doxorubicin and MEN 10755, a disaccharide analogue1
Introduction
The anthracycline antibiotic doxorubicin (DOX) has been one of the most extensively used agents in the chemotherapy regimens of cancer patients for the past 30 years [1], [2]. The identification of topoisomerase II as the main molecular target of DOX cytotoxicity has provided a biochemical basis for the identification of more effective anthracyclines [3]. DNA topoisomerase catalyses the change in DNA topology via a concerted mechanism of transient DNA strand cleavage and religation. Anthracyclines stabilise a transient DNA-topoisomerase II complex in which DNA strands are cut and covalently linked to the enzyme subunits [4]. The stabilised complex results in DNA damage that is related to the cytotoxic effect [5], [6]. Treatment with topoisomerase II inhibitors results in arrest of cells in the G1 and G2 phases of the cell cycle [5], [6]. G2 arrest was observed after treatment with topoisomerase-targeted drugs, as with agents inducing other types of DNA damage. The net result of the signalling steps after treatment with topoisomerase-directed agents could be the initiation of cell death with the characteristic feature of apoptosis [5], [6].
A common structural feature of anthracyclines is the presence of an amino sugar as the carbohydrate moiety directly linked to the chromophore [1]. The non-intercalating moiety of the anthracycline molecule (i.e. the sugar residue) is believed to play a role in the stabilisation of the ternary complex DNA-drug-topoisomerase II [7]. Interestingly, it has been shown that disaccharide anthracyclines have peculiar antitumour properties [8]. A disaccharide anthracycline analogue of the series, MEN 10755, has been shown to produce a broader antitumoural effect than DOX in a wide panel of human tumours xenografted in nude mice, and to be active against tumours naturally resistant to DOX [9], [10]. Furthermore, MEN 10755 was found to induce earlier and greater in vivo apoptosis than DOX in a variety of tumour types [9].
The aim of the study was to investigate the cellular and molecular bases of the efficacy of MEN 10755 and DOX in the human A2780 ovarian cancer cell line, with particular reference to cellular drug uptake and localisation, DNA damage, and induction of apoptosis. The results support an increased ability of the disaccharide analogue to induce topoisomerase-mediated DNA damage in spite of a reduced concentration at the nuclear level.
Section snippets
Chemicals
DOX was purchased from Sigma-Aldrich and stock solutions (5 mM) were prepared in distilled water, aliquoted, and stored at −20°. MEN 10755 (7-O-(2,6-dideoxy-4-O-(2,3,6-trideoxy-3-amino-alpha--lyxo-hexopyranosyl)-alpha--lyxo-hexopyranosyl-(4-demethoxy-14-hydroxy daunomycinone hydrochloride) was synthesised at the Chemistry Department of Menarini Ricerche as previously described [9], and stock solutions (5 mM) were prepared in distilled water, aliquoted, and stored at −20°. Both compounds were
Cellular drug accumulation and subcellular distribution
Fig. 1 shows the kinetics of total drug contents in whole cells (panel A) and in nuclear (panel B) and cytoplasmic (panel C) compartments in A2780 cells incubated for 30–240 min with 0.5 μM [14C]MEN 10755 or [14C]DOX, 0.5 μM being the lowest drug concentration necessary for drug detectability in the cytoplasmic compartment. During the observation time (2 hr), DOX was mainly localised inside the nuclei (10.6 ± 2.5 pmol/106 cells), and a lower level of radioactivity was detected in the cytoplasm
Discussion
The results presented in this study indicate that the disaccharide MEN 10755 has a lower cytotoxic potency than DOX. The reduced cytotoxic potency of MEN 10755, more marked following short-term exposure, could be related to reduced intracellular accumulation. This finding is consistent with the pharmacokinetics and better tolerability of the analogue in in vivo studies [9], [10], [15].
Furthermore, the results indicate that MEN 10755 is characterised by: (i) a greater ability than DOX to induce
Acknowledgements
We wish to thank Dr. Sapora (I.S.S., Rome, Italy) for cell irradiation, Dr. M. Binaschi and Dr. M. Grandi for helpful discussion, Mrs. Irrissuto for technical support, and Mrs. Bozzitelli and Ms. Zanesi for typing and editing the manuscript. This study was partially supported by IMI Grant 63216 and by the Associazione Italiana per la Ricerca sul Cancro, Milan.
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Abbreviations: DOX, doxorubicin; DNA-SSB, single-strand breaks; and DNA-DSB, double-strand breaks.