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Pixantrone dimaleate is a topoisomerase II inhibitor. Pixantrone induces cell death in multiple cancer cell lines independent of cell cycle perturbation, with IC50s of 37.3 nM, 126 nM and 136 nM for T47D, MCF-10A and OVCAR5 cells, respectively. Pixantrone induces DNA damage at high concentrations (500 nM) but not at concentrations (100 nM) sufficient to kill PANC1 cells. Pixantrone (25 or 100 nM) induces severe chromosomal aberrations and mitotic catastrophe in PANC1 cells. Pixantrone (100 nM) may disrupt chromosome segregation because of generating merotelic kinetochore attachments that cause chromosome non-disjunction. Pixantrone potently inhibits growth of human Leukemia K562 cells, etoposide-resistant K/VP.5 cells, MDCK and ABCB1-transfected MDCK/MDR cells, with IC50s of 0.10 μM, 0.56 μM, 0.058 μM and 4.5 μM, respectively. Pixantrone (0.01-0.2 μM) leads to a concentration-dependent formation of linear DNA through acting on topoisomerase IIα. Pixantrone produces semiquinone free radicals in an enzymatic reducing system, although not in a cellular system, most likely due to low cellular uptake. Pixantrone (0.01-10 μM) shows potent inhibitory activities against rat 97-116 peptide-specific T cell proliferation.