IN-VITRO CYTOTOXICITY AND DNA-DAMAGE PRODUCTION IN CHINESE-HAMSTER OVARY CELLS AND TOPOISOMERASE-II INHIBITION BY 1,2-DIHYDRO-3H-DIBENZ[DE,H]ISOQUINOLINE-1,3-DIONES WITH SUBSTITUTIONS AT THE 6-POSITION AND 7-POSITION (AZONAFIDES)

The mechanism of action of a group of anthracene-containing analogs of amonafide was studied in Chinese hamster ovary (CHO) cells. These age nts differ structurally from amonafide by the replacement of the napht halene chromophore with an anthracene chromophore, the lack of a prima ry amine moiety in the 5 position, and substitutions at the 6 and 7 po sitions on the anthracene nucleus. In this study, five analogs with po tent growth inhibitory activity and with low cardiotoxicity were chose n. Cytotoxicity analyses with tetrazolium dye assays (MTT) in vitro an d continuous drug exposure revealed IC50 values in CHO cells in the na nomolar range. Intracellular scanning laser confocal microscopy of the se drug-treated CHO cells showed that all analogs are able to enter ce ll nuclei with varying nuclear/cytoplasmic distribution: the more pote nt dimethylaminoethyl substituted analogs, 47 and 104, were primarily localized in the nucleus. Three analogs, including the unsubstituted p arent (1), and numbers 35 (6-amino substituted) and 53 (6-aminoethyl s ubstituted) inhibited DNA and RNA synthesis when assayed immediately a fter a 1 h exposure. In contrast, analogs 47 and 104 required 24 h pos t-drug exposure for 1 h to inhibit DNA and RNA synthesis. Using alkali ne elution techniques, each analog also produced DNA single- and doubl e-stranded breaks, as well as DNA-protein cross-links. Interestingly, the most cytotoxic analogs, 47 and 104, produced minimal DNA strand da mage in CHO cells at their IC90 concentrations, whereas the three othe r compounds with lower growth inhibitory potency produced marked and r oughly equivalent DNA damage at equitoxic concentrations. Gel shift an alysis of SV40 DNA exposed to the compounds demonstrated that these ag ents do not directly induce DNA strand breaks. However, catalytic stud ies with purified human topoisomerase II (Topo II) and plasmid DNA dem onstrated 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 eth yl substituted analogs.