In vitro activity: Through a topoisomerase II-mediated reaction, Amonafide treatment produces DNA single-strand breaks (SSB), double-strand breaks (DSB), and DNA-protein cross-links in human myeloid leukemia cells. Amonafide treatment inhibits conlony formation of the leukemic cell lines and the normal human bone marrow GM-CFC in a dose-dependent manner. Amonafide does not produce topoisomerase I-mediated DNA cleavage even at 100 μM. The m-AMSA-resistant line is less than 2-fold resistant to Amonafide. Amonafide interferes with the DNA breakage-reunion activity of mammalian DNA topoisomerase II resulting in DNA cleavage stimulation. Compared with those of other antitumor drugs, Amonafide-stimulated cleavage intensity patterns are markedly different. Amonafide highly prefers a cytosine, and excludes guanines and thymines instead, at position -1, with lower preference for an adenine at position +1. Topoisomerase II-mediated DNA cleavage induced by Amonafide is affected only slightly (less than 3-fold) by 1 mM ATP, suggeting that Amonafide is an ATP-insensitive topoisomerase II inhibitor in contrast to doxorubicin, etoposide, and mitoxantrone. Amonafide significantly inhibits the growth of HT-29, HeLa, and PC3 cells with IC50 of 4.67 μM, 2.73 μM, and 6.38 μM, respectively. Amonafide is unaffected by P-glycoprotein-mediated efflux, unlike those of the classical topoisomerase II inhibitors (daunorubicin, doxorubicin, idarubicin, etoposide, and mitoxantrone).

Cell Assay: All cell lines (HT-29, HeLa, and PC3) are in the logarithmic phase of growth when the assay of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) is carried out. Cells are harvested and seeded into 96-well tissue culture plates at a density of 2.5 × 103 cells/well in 150 μL aliquots of medium. The concentrations tested are serial dilutions of a stock solution (10 μM in DMSO) with phosphate-buffered saline (PBS) and are added 24 hours later. The assay is ended after 72 hours of Amonafide exposure and PBS is used as a negative control. After 72 hours treatment, cells are washed twice with PBS, and then 50 μL/well of MTT reagent (1 mg/mL in PBS) together with 150 μL/well of prewarmed medium are added. The plates are returned to the incubator for 4 hours. Subsequently, DMSO is added as solvent. Absorbance is determined at 570 nm with a Microplate reader. All experiments are performed at least three times, and the average of the percentage absorbance is plotted against concentration. Then, the concentration of Amonafide required to inhibit 50% of cell growth (IC50) is calculated for Amonafide.