The effect of isolancifolide on the apoptosis in HL-60 cells through caspase-8-dependent and -independent pathways

Isolancifolide is a compound extracted and isolated from Actinodaphne lancifolia, which is a traditional oriental medicine. To determine whether isolancifolide has therapeutic potential as an anticancer molecule, we assessed its apoptotic effects on HL-60 cells, a human leukemia cell line. Apoptotic activities were investigated using DNA fragmentation assay, immunoblotting, and flow cytometry. We found that the inhibitory concentration 50% of isolancifolide was approximately 20 M. The time- and dose-dependent effects of isolancifolide on apoptosis were determined by DNA fragmentation and propidium iodide staining, and the involvement of caspases and the Bcl-2 family in isolancifolide-induced apoptosis was assessed by Western blotting. During exposure to isolancifolide, the pro-forms or full length of caspases-8, -3, and Bid were decreased, as assessed by Western blotting, while the levels of cleaved forms of caspases-8, -3, and PARP were increased. We observed that the release of cytochrome c and Smac/DIABLO from the mitochondria to the cytosol was accompanied by the loss of mitochondrial membrane potential. The caspase specific inhibitors, z-IETD-fmk and z-LEHD-fmk, blocked the accumulation of sub-G1 cells and the release of cytochrome c, but not that of Smac/DIABLO. These results indicate that isolancifolide induces apoptosis of HL-60 cells through both death receptor and mitochondria pathways, in caspase-8-dependent and -independent manners, suggesting that isolancifolide may be useful in anticancer strategies.