The Ortho-Quinone Metabolite of the Anticancer Drug Etoposide (VP-16) Is a Potent Inhibitor of the Topoisomerase II/DNA Cleavable Complex TSVETAN G. GANTCHEV and DAREL J. HUNTING Medical Research Council Group in the Radiation Sciences, Department of Nuclear Medicine and Radiobiology, Faculty of Medicine, Universite ´ de Sherbrooke, Sherbrooke, Quebec J1H 5N4, Canada Received September 5, 1997; Accepted November 12, 1997 This paper is available online at http://www.molpharm.org ABSTRACT Epipodophyllotoxin derivatives, such as etoposide (VP-16), constitute an important class of anticancer agents, the major cytotoxic effects of which are associated with trapping of the topoisomerase II/DNA cleavable complex and formation of pro- tein-DNA cross-links and nicked DNA. VP-16, however, can be metabolized to several highly reactive products, including an ortho-quinone (VPQ). The inhibitory activity of VPQ against purified human topoisomerase II processing of supercoiled DNA was studied and compared with that of the parent com- pound, VP-16. Our results show that VPQ is a powerful inhibitor of topoisomerase II, which prevents DNA strand passage in the presence of ATP. As with VP-16, trapping of the cleavable complex is highly reversible upon removal of divalent ions, which indicating that VPQ alters the cleavage-reunion equilib- rium of topoisomerase II and DNA mainly by noncovalent inter- actions, as does the parent compound. However, we observed several differences between the effects induced by VP-16 and VPQ, including a strong inhibition of the second DNA strand religation, which implies the involvement of additional (asym- metric) mode(s) of interactions of the VPQ, possibly by inter- ference with ATP binding by the homodimeric enzyme, and/or involving covalent interactions. Reduced or oxidized glutathi- one prevented trapping of the topoisomerase/DNA cleavable complex by VPQ, but not by VP-16, probably by forming co- valent adducts with the former. Type II topoisomerases alter DNA topology by the forma- tion of a double-stranded break, followed by DNA strand passage through an ATP-dependent protein clamp (referred to as a two-gate mechanism) (Liu et al., 1983; Hsieh, 1983; Roca and Wang, 1994). The eukaryotic enzymes function as homodimers and exist in two forms: p170 and p180. Both forms catalyze DNA strand passage, but the reaction mech- anisms are different: the p170 form relaxes SC DNA in a highly distributive manner, whereas the p180 form changes the DNA linking number in a processive way, although the exact mechanistic differences remain obscure (Drake et al., 1989). DNA strand passage is preceded by the formation of a transient DNA-protein (cleavable) complex, anchored by co- valent phosphotyrosil bonds between the active site of the homodimeric enzyme and the 5'-DNA ends of the cleaved strands of the gate-segment of DNA (Hsieh, 1990; Wigley, 1995). The final stages of DNA processing by topoisomerases are the religation of the cleaved strands, followed by release of the DNA. Most of the topoisomerase-targeting anticancer drugs (e.g., epipodophyllotoxins) exert their therapeutic ac- tion by a specific and reversible block of the DNA-strand rejoining steps, resulting in trapping of the covalent topoi- somerase II/DNA cleavable complex (Chen et al., 1984), ac- cumulation of protein-linked DNA breaks, late S and/or G 2 - phase cell-cycle arrest and, ultimately, cell death (Wozniak and Ross, 1983; Ross et al., 1984; Markovits et al., 1987; Gantchev et al., 1996). One of these topoisomerase poisons, used in the treatment of a number of neoplastic disorders, is the epipodophyllotoxin VP-16 [4'- demethyl-epipodophyllotoxin-9- (4, 6-O-ethylidene--D-glucopyranoside)]. VP-16, however, can undergo oxido-reductive transformations in cells. Two major pathways have been identified: O-demethylation cat- alyzed by cytochrome P450-dependent monooxygenases (van Maanen et al., 1987), and one- or two-electron oxidation mediated by some peroxidases (Haim et al., 1986) and tyrosi- nase (Gantchev et al., 1994). These enzymatic transforma- tions affect the pendant dimethoxyphenolic group (E-ring) of the drug, leading to several products, the major one being the ortho-quinone derivative VPQ, as summarized in Fig. 1. This work was supported by the Medical Research Council of Canada. ABBREVIATIONS: VP-16, etoposide; VPQ, ortho-quinone derivative of etoposide; LNR, linear DNA form; NC, nicked circular DNA form; PRLX, partially relaxed DNA form; RLX, relaxed DNA form; SC, supercoiled DNA form; GSH, reduced glutathione; GSSG, oxidized glutathione; SDS, sodium dodecyl sulfate. 0026-895X/98/030422-07$3.00/0 Copyright © by The American Society for Pharmacology and Experimental Therapeutics All rights of reproduction in any form reserved. MOLECULAR PHARMACOLOGY, 53:422–428 (1998). 422 at ASPET Journals on July 20, 2018 molpharm.aspetjournals.org Downloaded from