The Iron Chelator Dp44mT Causes DNA Damage and Selective Inhibition of Topoisomerase IIα in Breast Cancer Cells

Di-2-pyridylketone-4,4,-dimethyl-3-thiosemicarbazone (Dp44mT) is being developed as an iron chelator with selective anticancer activity. We investigated the mechanism whereby Dp44mT kills breast cancer cells, both as a single agent and in combination with doxorubicin. Dp44mT alone induced selective cell killing in the breast cancer cell line MDA-MB-231 when compared with healthy mammary epithelial cells (MCF-12A). It induces G(1) cell cycle arrest and reduces cancer cell clonogenic growth at nanomolar concentrations. Dp44mT, but not the iron chelator desferal, induces DNA double-strand breaks quantified as S139 phosphorylated historic foci (gamma-H2AX) and Comet tails induced in MDA-MB-231 cells. Doxorubicin-induced cytotoxicity and DNA damage were both enhanced significantly in the presence of low concentrations of Dp44mT. The chelator caused selective poisoning of DNA topoisomerase II alpha (top2 alpha) as measured by an in vitro DNA cleavage assay and cellular topoisomerase-DNA complex formation. Heterozygous Nalm-6 top2 alpha knockout cells (top2 alpha(+/-)) were partially resistant to Dp44mT-induced cytotoxicity compared with isogenic top2 alpha(+/+) or top2 beta(-/-) cells. Specificity for top2 alpha was confirmed using top2 alpha and top2 beta small interfering RNA knockdown in HeLa cells. The results show that Dp44mT is cytotoxic to breast cancer cells, at least in part, due to selective inhibition of top2 alpha. Thus, Dp44mT may serve as a mechanistically unique treatment for cancer due to its dual ability to chelate iron and inhibit top2 alpha activity. [Cancer Res 2009;69(3):948-57]