Salicylate, a Catalytic Inhibitor of Topoisomerase II, Inhibits DNA Cleavage and Is Selective for the a Isoform

Topoisomerase II (topo II) is a ubiquitous enzyme that is essential for cell survival through its role in regulating DNA topology and chromatid separation. Topo II can be poisoned by common chemotherapeutics (such as doxorubicin and etoposide), leading to the accumulation of cytotoxic enzyme-linked DNA doublestranded breaks. In contrast, nonbreak-inducing topo II catalytic inhibitors have also been described and have more limited use in clinical chemotherapy. These agents, however, may alter the efficacy of regimens incorporating topo II poisons. We previously identified salicylate, the primary metabolite of aspirin, as a novel catalytic inhibitor of topo II. We have now determined the mechanism by which salicylate inhibits topo II. As catalytic inhibitors can act at a number of steps in the topo II catalytic cycle, we used multiple independent, biochemical approaches to interrogate the catalytic cycle. Furthermore, as mammalian cells express two isoforms of topo II (a and b), we examined whether salicylate was isoform selective. Our results demonstrate that salicylate is unable to intercalate DNA, and does not prevent enzyme–DNA interaction, nor does it promote stabilization of topo IIa in closed clamps on DNA. Although salicylate decreased topo IIa ATPase activity in a dose-dependent noncompetitive manner, this was secondary to salicylate-mediated inhibition of DNA cleavage. Surprisingly, comparison of salicylate’s effects using purified human topo IIa and topo IIb revealed that salicylate selectively inhibits the a isoform. These findings provide a definitive mechanism for salicylate-mediated inhibition of topo IIa and provide support for further studies determining the basis for its isoform selectivity.