Through the screening of DNA topoisomerase I (Top I) inhibitors, a new cytotoxic agent, BPR0Y007 [2,5-bis(4-hydroxy-3-methoxybenzylidene)cyclopentanone], was identified. BPR0Y007 was less potent than camptothecin (CPT) in the inhibition of Top I in vitro. Also, in vitro data showed that BPR0Y007 induces DNA cleavage in the presence of Top I at micromolar concentrations, with a cleavage specificity similar to that of CPT. High concentrations of BPR0Y007 did not produce detectable DNA unwinding, suggesting that BPR0Y007 is not a DNA intercalator. However, BPR0Y007 displaced Hoechst 33342 dye, suggesting that BPR0Y007 binds to DNA at the Hoechst 33342 binding site. Furthermore, BPR0Y007 generated protein-linked DNA breaks in a cell-based study. Cell cycle analysis demonstrated that the cell cycle effect of BPR0Y007 differs from that of CPT. Cells accumulated in the S-phase when treated with high concentrations of CPT, whereas cells accumulated gradually in the G(2)/M phase when treated with increasing concentrations of BPR0Y007. Further studies showed that BPR0Y007 inhibits tubulin polymerization in vivo and in vitro, and induces apoptosis in a concentration-dependent manner. No cross-resistance with BPR0Y007 was observed in CPT-, VP-16-, or vincristine-resistant cell lines. The IC(50) of BPR0Y007 for various human cancer cell lines ranged from 1 to 8 microM. Taken together, these results suggest that BPR0Y007 acts on both Top I and tubulin. Given its unique biochemical mechanisms of action, BPR0Y007 warrants exploration as an antitumor compound.