INHIBITION OF DNA TOPOISOMERASES BY SANGUIIN H-6, A CYTOTOXIC DIMERIC ELLAGITANNIN FROM SANGUISORBA-OFFICINALIS

Many tannins were previously identified as candidate topoisomerase poisons. Here we report further studies on sanguiiin 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 lC50 values of 1 muM and 0.01 muM, 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 muM and 0.16 muM, 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 agarose 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 muM and > 10 muM, respectively. Sanguiin H-6 inhibited HeLa cell growth with an ED50 Of 12 muM and also interfered in a dose-dependent fashion with intracellular topoisomerase activities but with lower potencies than those observed using subcellular 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.