CPX-induced cell death is iron dependent. (A) Leukemia (NB4, MDAY-D2, and OCI-M2) and solid tumor (OVCAR-3 and PPC-1) cells were loaded with the intracellular iron-chelating fluorescent dye calcein-AM. Cells were then treated with CPX (10 μM) or DFX (100 μM) for 2 minutes. Intracellular iron bound by calcein was measured by flow cytometry. Percentage increase ± SD in the geometric mean intracellular calcein fluorescence is represented *P < .05 (Student t test). One of at least 2 representative experiments performed in triplicate is shown. (B) HT-29(i) and HepG2 (ii) cells were treated with increasing concentrations of CPX with or without iron supplementation with iron in a complex with a transferrin-replacement compound (8 μM). Seventy-two hours after incubation, cell viability was measured by MTS. Data represent the mean ± SD percentage of viable cells from 1 of 3 representative experiments. (C) Jurkat and NB4 leukemia cells were treated with increasing concentrations of CPX or DFX. Seventy-two hours after incubation, cell viability was measured by the MTS assay. Data represent the mean ± SD percentage viable cells from 1 of at least 3 representative experiments performed in triplicate. (D) NB4 cells were loaded with the intracellular iron-chelating fluorescent dye calcein-AM (i). Cells were then treated with 10 μM of CPX or the structurally CPX-related analogs DPOH, ADP, and MDP for 2 minutes. Intracellular iron bound by the compounds was measured by flow cytometry as described in panel A. (ii) NB4 cells were treated with increasing concentrations of CPX or the analogs in panel Di. Seventy-two hours after incubation, cell viability was measured by the MTS assay. Data represent the mean ± SD percentage viable cells from 1 of at least 2 representative experiments performed in triplicate.