A comparative study of cellular and molecular pharmacology of doxorubicin and MEN 10755, a disaccharide analogue¹

Abstract

MEN 10755 is a disaccharide anthracycline endowed with a broader spectrum of antitumour activity than doxorubicin (DOX). To investigate the cellular and molecular basis of its action, cytotoxic activity, drug uptake, subcellular localisation, induction of DNA damage, and apoptosis were assessed in the human A2780 ovarian carcinoma cell line. Experiments with radiolabelled anthracyclines indicated that MEN 10755 exhibited reduced cellular accumulation and a different subcellular distribution (higher cytoplasmic/nuclear ratio) than DOX. In spite of the lower nuclear concentration, MEN 10755 was as potent as DOX in eliciting DNA single- and double-strand breaks, G2/M cell arrest, and apoptosis. Sequencing of drug-induced topoisomerase II cleavage sites showed a common DNA cleavage pattern for MEN 10755 and DOX. Cleavage sites were always characterised by the presence of adenine in −1 position. However, the extent of DNA cleavage stimulation induced by MEN 10755 was greater than that produced by DOX. Reversibility studies showed that MEN 10755-stimulated DNA cleavage sites were more persistent than those induced by DOX, thus suggesting a more stable interaction of the drug in the ternary complex. As a whole, the study indicated that the cellular pharmacokinetics of MEN 10755 substantially differs from that of DOX, showing a lower uptake and a different subcellular disposition. In spite of the apparently unfavourable cellular pharmacokinetics, MEN 10755 was still as potent as DOX in inducing topoisomerase-mediated DNA damage. Although the extent and persistence of protein-associated DNA breaks may contribute to the cytotoxic effects, the drug’s efficacy as apoptosis inducer and antitumour agent could not be adequately explained on the basis of DNA damage mediated by the known target (i.e. topoisomerase II), thus supporting additional cellular effects that may be relevant in cellular response.

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.