title: The iron chelator Dp44mT causes DNA damage and selective inhibition of topoisomerase IIalpha in breast cancer cells.
abstract: 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.
authors.affiliation: US Department of Health and Human Services, Laboratory of Biochemistry, Center for Drug Evaluation and Research, Food and Drug Administration, Bethesda, Maryland, USA. ashutosh.rao@fda.hhs.gov
authors.forename: V Ashutosh
authors.initials: VA
authors.lastname: Rao
authors.forename: Sarah R
authors.initials: SR
authors.lastname: Klein
authors.forename: Keli K
authors.initials: KK
authors.lastname: Agama
authors.forename: Eriko
authors.initials: E
authors.lastname: Toyoda
authors.forename: Noritaka
authors.initials: N
authors.lastname: Adachi
authors.forename: Yves
authors.initials: Y
authors.lastname: Pommier
authors.forename: Emily B
authors.initials: EB
authors.lastname: Shacter
mesh_terms: D000951:Antigens, Neoplasm; D001943:Breast Neoplasms / Q000188:drug therapy* / Q000201:enzymology / Q000235:genetics / Q000473:pathology; D002453:Cell Cycle / Q000187:drug effects; D048708:Cell Growth Processes / Q000187:drug effects; D045744:Cell Line, Tumor; D004249:DNA Damage; D004250:DNA Topoisomerases, Type II; D004268:DNA-Binding Proteins / Q000037:antagonists & inhibitors*; D006801:Humans; D007502:Iron Chelating Agents / Q000494:pharmacology*; D013882:Thiosemicarbazones / Q000494:pharmacology*; D059005:Topoisomerase II Inhibitors
chemical_list: D000951:Antigens, Neoplasm; D004268:DNA-Binding Proteins; D007502:Iron Chelating Agents; D013882:Thiosemicarbazones; D059005:Topoisomerase II Inhibitors; C539263:di-2-pyridylketone-4,4-dimethyl-3-thiosemicarbazone; D004250:DNA Topoisomerases, Type II