Inhibition of DNA Topoisomerases by Sanguiin H-6, a Cytotoxic Dimeric Ellagitannin fromSanguisorba officinalis1

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Abstract
Many tannins were previously identified as candidate topoisomerase poisons. Here we report further studies on sanguiin H-6, a dimeric ellagitannin isolated from Sanguisorba officinalis as an inhibitor of DNA topoisomerases. Catalytic strand-passing activities of topoisomerases I and II were inhibited in vitro with IC50 values of 1 µ and 0.01 µM, respectively. This inhibition was not associated with stabilization of covalent enzyme-DNA complexes but rather by a mechanism preventing formation of such covalent intermediates, as measured by interference with drug-induced cleavage in vitro. The IC50 values for topoisomerase I-DNA complexes induced by camptothecin and with topoisomerase II-DNA complexes induced by VP-16 were 0.02 µM and 0.16 µM, respectively. Pre-incubation studies followed by drug-dilution revealed that the in vitro inhibitory effects of sanguiin H-6 were irreversible, and for topoisomerase I, the test compound prevented enzyme-DNA interaction as seen by shifts in mobility on agarosc gels. By measuring interference with drug-induced protein-linked DNA breaks in isolated HeLa nuclei, inhibition of topoisomerases I and II on a natural chromatin template was demonstrated with IC50 values of 5 µM and > 10 µM, respectively. Sanguiin H-6 inhibited HeLa cell growth with an ED50 of 12 µM and also interfered in a dose-dependent fashion with intracellular topoisomerase activities but with lower potencies than those observed using sub-cellular assay systems. Based on these studies, sanguiin H-6 could be broadly classified as a type of poison which does not stimulate the formation of cleavable-complexes, with intracellular activity but without any marked selectivity. The widespread presence of tannins in plant extracts may complicate drug-screening procedures designed to afford other types of novel topoisomerase poison from crude mixtures of phytochemicals.

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