OBJECTIVE To explore the potential anti-cancer function and the pathway of Varacin-1 (VCA-1), which is an analog of varacin C, an agent known to induce DNA damage.METHODS VCA1 was synthesized by the chemistry department of NUS, and two pairs of p53-WT/KO human cancer cells , HCT116 and U2OS/Saos2, were used.After treating all cells with designated doses of VCA-1,apoptosis was determined by morphological changes, Hoechst staining, and western blot result of PARP.Intracellular ROS level were measured by CM-H2DCFDA and analyzed by flow cytometry.At the same time, cell protein (p53, caspase-8,caspase-3,XlAP) levels were analysed by Western blot.The transient transfection of XIAP was also performed.RESULTS (1) VCA-1 induces apoptosis in a p53-independent way,which is supported by the facts that the cells with or without p53 displayed similar apoptosis to VCA-1 treatment.(2) VCA-1 induces caspase-8 dependent caspase cascade in apoptosis, because a time-dependent cleavage of caspase-8 and caspase-3 were detected in VCA-1 treated p53-WT/KO cell lines, and pan-caspase inhibitor zVAD was able to suppress PARP cleavage caused by VCA-1 treatment.(3) VCA-1 enhances intracellular ROS production.(4) Evident reduction of XlAP protein level in all four cells lines treated with VCA-1 and significant protection against VCA-1-induced cell death in over-expression of XlAP cells show that XIAP down regulation is critical in VCA-1-induced apoptosis.(5) The fact that a well-known antioxidant NAC completely blocked XIAP down regulation, and hence PARP cleavage, and zVAD successfully blocked VCA-1-induced apoptosis but not XIAP suggests that ROS contribute to XIAP down regulation in VAC-1-induced apoptosis.CONCLUSION VCA-1 induces apoptosis of cancer cells mainly via the extrinsic pathway involving caspase-8 in a p53-independent way,excessive ROS generation plays a fundamental role in VCA-1-induced apoptosis, probably by XIAP down regulation, and consequently promotes caspase activation.