Mechanism of ascididemin-induced cytotoxicity

Some marine animals are rich sources of unique polycyclic aromatic alkaloids that are cytotoxic against tumor cell lines and effective in mouse tumor xenograft models. Ascididemin is a pyridoacridine alkaloid originally derived from a Didemnum sp. tunicate. It has potent cytotoxicity against tumor cells in vitro and in vivo. Preclinical screening at NCI revealed the antineoplastic activities of ascididemin and a synthetic analogue 48. Ascididemin has been reported to inhibit topoisomerase II and induce topoisomerase II-mediated DNA cleavage. This study, however, focuses on the unique ability of ascididemin and two synthetic analogues (48 and 109) to cleave DNA in the absence of topoisomerase I or II. An in vitro assay revealed their concentration-dependent ability to cleave DNA and identified dithiothreitol as the sole requirement for maximal activity. On the basis of shared structural features of the three analogues, a double N-bay region and iminoquinone heterocyclic ring, two possible mechanisms of action were hypothesized: (1) generation of reactive oxygen species facilitated by metal binding to the common phenanthroline bay region, and (2) production of reactive oxygen species by direct reduction of the iminoquinone moiety. Experimental results supported direct iminoquinone reduction and ROS generation as the mechanism of ascididemin cytotoxicity. Antioxidants protected against DNA cleavage in vitro and protected cultured Chinese hamster ovary cells from toxicity. Additionally, it was shown that cells deficient in the ability to repair reactive oxygen species damage to their DNA were more susceptible to ascididemin and analogues than repair competent cells. Ascididemin-treated cells were also shown to induce oxygen-stress related proteins, further implicating the production of reactive oxygen species as the mechanism of cytotoxicity for these molecules.