Inhibition of topoisomerase II by the marine alkaloid ascididemin and induction of apoptosis in leukemia cells

Abstract

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.