In vitro activity: Dp44mT shows pronounced antiproliferative effects in SK-N-MC, SK-Mel-28, and MCF-7 cells with IC50 of 30 nM, 60 nM, and 60 nM, respectively, while no effects on normal MRC-5 fibroblasts. Dp44mT inhibits cellular Fe uptake from Fe-Tf in SK-N-MC neuroepithelioma and M109 cells, and induces cell apoptosis. In MDA-MB-231 cells, Dp44mT specifically targets topoisomerase topo2α, and thus causes DNA damage. Dp44mT, as a Pgp substrate, also overcomes multidrug resistance by the hijacking of lysosomal P-glycoprotein (Pgp).

Kinase Assay: oligonucleotides are 5'-end labeled with [32P]ATP and T4 polynucleotide kinase. Labeling mixtures are subsequently centrifuged through Mini Quick Spin DNA columns (for pSK fragments) or Oligo columns (for oligonucleotides) to remove the unincorporated label. Annealing to the complementary strand of the oligonucleotides is done by heating the reaction mixture to 95°C and overnight cooling to room temperature in 10 mM Tris-HCl (pH 7.8), 100 mM NaCl, and 1 mM EDTA. DNA substrates (10 pmol/reaction) are incubated with 500 ng of top2a or top2h in the presence or absence of Dp44mT for the indicated times at 25°C in 10 μL of reaction buffer. Reactions are stopped by adding SDS (final concentration 0.5%). Samples are separated on 16% (for pSK DNA) or 20% (for the oligonucleotides) denaturing polyacrylamide gels (7 M urea). Imaging and quantitation are done using a PhosphorImager.

Cell Assay: Cells (SK-N-MC, SK-Mel-28, MCF-7 and MRC-5 cells) are incubated in the presence and absence of DFO, 311, 3-aminopyridine-2-carboxaldehyde thiosemicarbazone, doxorubicin, and the DpT series of chelators (0-25 μM) for 72 hours at 37°C。 The effect of the chelators on proliferation is examined using the MTT assay.