title: In vitro cytotoxicity and DNA damage production in Chinese hamster ovary cells and topoisomerase II inhibition by 2-[2'-(dimethylamino)ethyl]-1, 2-dihydro-3H-dibenz[de,h]isoquinoline-1,3-diones with substitutions at the 6 and 7 positions (azonafides).
abstract: The mechanism of action of a group of anthracene-containing analogs of amonafide was studied in Chinese hamster ovary (CHO) cells. These agents differ structurally from amonafide by the replacement of the naphthalene chromophore with an anthracene chromophore, the lack of a primary amine moiety in the 5 position, and substitutions at the 6 and 7 positions on the anthracene nucleus. In this study, five analogs with potent growth inhibitory activity and with low cardiotoxicity were chosen. Cytotoxicity analyses with tetrazolium dye assays (MTT) in vitro and continuous drug exposure revealed IC50 values in CHO cells in the nanomolar range. Intracellular scanning laser confocal microscopy of these drug-treated CHO cells showed that all analogs are able to enter cell nuclei with varying nuclear/cytoplasmic distribution: the more potent dimethylaminoethyl substituted analogs, 47 and 104, were primarily localized in the nucleus. Three analogs, including the unsubstituted parent (1), and numbers 35 (6-amino substituted) and 53 (6-aminoethyl substituted) inhibited DNA and RNA synthesis when assayed immediately after a 1 h exposure. In contrast, analogs 47 and 104 required 24 h post-drug exposure for 1 h to inhibit DNA and RNA synthesis. Using alkaline elution techniques, each analog also produced DNA single- and double-stranded breaks, as well as DNA protein cross-links. Interestingly, the most cytotoxic analogs, 47 and 104, produced minimal DNA strand damage in CHO cells at their IC90 concentrations, whereas the three other compounds with lower growth inhibitory potency produced marked and roughly equivalent DNA damage at equitoxic concentrations. Gel shift analysis of SV40 DNA exposed to the compounds demonstrated that these agents do not directly induce DNA strand breaks. However, catalytic studies with purified human topoisomerase II (Topo II) and plasmid DNA demonstrated that these drugs inhibit this enzyme. These results suggest that the azonafides inhibit Topo II to cause protein-associated strand breaks and impaired DNA and RNA synthesis. However, other mechanisms may also be operant, especially with the more potent dimethylamino ethyl substituted analogs.
authors.affiliation: Department of Pharmacology/Toxicology, University of Arizona, Tucson 85724, USA.
authors.forename: C A
authors.initials: CA
authors.lastname: Mayr
authors.forename: S M
authors.initials: SM
authors.lastname: Sami
authors.forename: R T
authors.initials: RT
authors.lastname: Dorr
mesh_terms: D000818:Animals; D000970:Antineoplastic Agents / Q000633:toxicity*; D016466:CHO Cells; D002470:Cell Survival / Q000187:drug effects; D002474:Cell-Free System; D006224:Cricetinae; D004249:DNA Damage; D004273:DNA, Neoplasm / Q000096:biosynthesis / Q000187:drug effects*; D004123:Dimethylamines / Q000633:toxicity*; D004354:Drug Screening Assays, Antitumor; D004791:Enzyme Inhibitors / Q000633:toxicity*; D007546:Isoquinolines / Q000633:toxicity*; D018613:Microscopy, Confocal; D009363:Neoplasm Proteins / Q000096:biosynthesis; D010957:Plasmids / Q000187:drug effects; D013778:Tetrazolium Salts; D013844:Thiazoles; D059005:Topoisomerase II Inhibitors
chemical_list: D000970:Antineoplastic Agents; D004273:DNA, Neoplasm; D004123:Dimethylamines; D004791:Enzyme Inhibitors; D007546:Isoquinolines; D009363:Neoplasm Proteins; D013778:Tetrazolium Salts; D013844:Thiazoles; D059005:Topoisomerase II Inhibitors; C022616:thiazolyl blue