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

¹ Laboratory of Biochemistry, Center for Drug Evaluation and Research, Food and Drug Administration, U.S. Department of Health and Human Services; ² National Cancer Institute-Food and Drug Administration Interagency Oncology Task Force Program; ³ Laboratory of Molecular Pharmacology, Center for Cancer Research, National Cancer Institute, U.S. Department of Health and Human Services, Bethesda, Maryland; and ⁴ Yokohama City University, Yokohama, Japan

Requests for reprints: V. Ashutosh Rao or Emily B. Shacter, 29 Lincoln Drive, Building 29A, Room 2A-11, HFD-122, Bethesda, MD 20892. Phone: 301-827-4487/301-827-1833; Fax: 301-480-3256; E-mail: ashutosh.rao{at}fda.hhs.gov or emily.shacter{at}fda.hhs.gov.

Key Words: Iron chelator • Dp44mT • doxorubicin • topoisomerase • -H2AX • breast cancer

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 G₁ 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 (-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 (top2) as measured by an in vitro DNA cleavage assay and cellular topoisomerase-DNA complex formation. Heterozygous Nalm-6 top2 knockout cells (top2^+/–) were partially resistant to Dp44mT-induced cytotoxicity compared with isogenic top2^+/+ or top2β^–/– cells. Specificity for top2 was confirmed using top2 and top2β 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. Thus, Dp44mT may serve as a mechanistically unique treatment for cancer due to its dual ability to chelate iron and inhibit top2 activity. [Cancer Res 2009;69(3):948–57]