Peroxisomicine A(1) (T-514) is a dimeric anthracenone first isolated from the plant Karwinskia humboldtiana. The compound presents a high and selective toxicity toward liver and skin cell cultures and is currently the subject of preclinical studies as an
antitumor drug. To date, the molecular basis for its diverse
biological effects remains poorly understood. To elucidate its mechanism of action, we studied its interaction with
DNA and its effects on human
DNA topoisomerases. Practically no interaction with
DNA was detected. Peroxisomicine was found to inhibit
topoisomerase II but not
topoisomerase I.
DNA relaxation and decatenation assays indicated that the
drug interferes with the catalytic activity of
topoisomerase II but does not stimulate DNA cleavage, in contrast to conventional topoisomerase
poisons such as
etoposide. Two human
leukemia cell lines sensitive or resistant to
mitoxantrone were used to assess the cytotoxicity of the toxin and its effect on the cell cycle. In both cases, peroxisomicine treatment was associated with a loss of cells from every phase of the cell cycle and was accompanied by a large increase in the sub-G1 region which is characteristic of apoptotic cells. The cell cycle changes were more pronounced with the sensitive HL-60 cells than with the resistant HL-60/MX2 cells (with reduced
topoisomerase II activity), in agreement with the cytotoxicity measurements. Treatment of HL-60 cells with
T-514 stimulated the cleavage of the
poly(ADP-ribose) polymerase by intracellular
proteases such as
caspase-3. The cytometry and Western blot analyses reveal that peroxisomicine induces apoptosis in
leukemia cells. In addition, we characterized a catabolite of peroxisomicine, named T-510R, in the form of a highly stable radical metabolite. The electron spin resonance and mass spectrometry data are consistent with the formation of an anionic semiquinonic radical. The oxidized product T-510R inhibits
topoisomerase II with a reduced efficiency compared to the parent toxin and was found to be about 3-4 times less toxic to both the sensitive and resistant
leukemia cell lines than
T-514. Collectively, the results suggest that
topoisomerase II inhibition plays a role in the cytotoxicity of the plant toxin peroxisomicine. Inhibition of
topoisomerase II may serve as an inducing signal triggering the apoptotic cell death of
leukemia cells exposed to the toxin. The dihydroxyanthracenone unit may represent a useful chemotype for the preparation of
topoisomerase II-targeted
anticancer agents.