NK314 is a novel synthetic benzo[c]phenanthridine alkaloid that shows strong antitumor activity. It inhibited topoisomerase II activity and stabilized topoisomerase II-DNA cleavable complexes. The DNA breaks occurred within 1h after treatment with NK314 even without digestion of topoisomerase II by proteinase K, whereas etoposide required digestion of the enzyme protein in cleavable complex to detect DNA breaks. Pretreatment with topoisomerase II catalytic inhibitors, ICRF-193 and suramin, reduced both cleavable complex-mediated DNA breaks and proteinase K-independent DNA breaks, but protease inhibitors and nuclease inhibitors only decreased the latter. These results indicate that NK314 might affect topoisomerase II in the different manner from cleavable complex formation and activate intracellular proteinase and nuclease to produce DNA fragmentation. As a result of this unique mechanism of DNA breakage, NK314 showed substantial growth inhibition of topoisomerase II inhibitor-resistant tumors.