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
histone 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 IIalpha (top2alpha) as measured by an in vitro DNA cleavage assay and cellular topoisomerase-
DNA complex formation. Heterozygous Nalm-6 top2alpha knockout cells (top2alpha(+/-)) were partially resistant to Dp44mT-induced cytotoxicity compared with isogenic top2alpha(+/+) or top2beta(-/-) cells. Specificity for top2alpha was confirmed using top2alpha and top2beta
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 top2alpha. Thus,
Dp44mT may serve as a mechanistically unique treatment for
cancer due to its dual ability to chelate
iron and inhibit top2alpha activity.