Ascididemin (ASC) is a pentacyclic
DNA-
intercalating agent isolated from the Mediterranean ascidian Cystodytes dellechiajei. This marine
alkaloid exhibits marked cytotoxic activities against a range of
tumor cells, but its mechanism of action remains poorly understood. We investigated the effects of ASC on DNA cleavage by human topoisomerases I and II. Relaxation assays using
supercoiled DNA showed that ASC stimulated double-stranded cleavage of
DNA by
topoisomerase II, but exerted only a very weak effect on
topoisomerase I. ASC is a conventional
topoisomerase II poison that significantly promoted DNA cleavage, essentially at sites having a C on the 3' side of the cleaved bond (-1 position), as observed with
etoposide. The stimulation of DNA cleavage by
topoisomerase I in the presence of ASC was considerably weaker than that observed with
camptothecin. Cytotoxicity measurements showed that ASC was even less toxic to
P388 leukemia cells than to P388CPT5 cells resistant to
camptothecin. In addition, the marine
alkaloid was found to be equally toxic to HL-60
leukemia cells sensitive or resistant to
mitoxantrone. It is therefore unlikely that topoisomerases are the main cellular targets for ASC. This
alkaloid was found to strongly induce apoptosis in HL-60 and
P388 leukemia cells. Cell cycle analysis showed that ASC treatment was associated with a loss of cells in the G1 phase accompanied with a large increase in the sub-G1 region. Cleavage experiments with
poly(ADP-ribose) polymerase (PARP) revealed that
caspase-3 was a mediator of the apoptotic pathway induced by ASC. The
DNA of ASC-treated cells was severely fragmented. Collectively, these findings indicate that ASC is a potent inducer of apoptosis in
leukemia cells.