Alteration of Bcl-2 family proteins by the dietary compound fisetin in HCT-116 human colon cancer cells: a mechanism for apoptosis induction

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Fisetin, 3,3',4',7-tetrahydroxyflavone, is found in fruits, vegetables and wine, and exhibits anticarcinogenic activity. We have previously observed that fisetin inhibits the proliferation of HT-29 and HCT-116 human colon cancer cells. The present study examined whether fisetin alters protein levels of Bcl-2 family proteins which are known to regulate mitochondrial membrane permeability thereby controlling the fate of cells. HCT-116 cells were cultured with 0, 5, 10, or 20 μmol/L of fisetin. DNA condensations examined by Hoechst 33258 staining and apoptotic cell numbers examined by Annexin V staining were increased in fisetin dose-dependent manners. Western blot analysis of total cell lysates revealed that fisetin increased cleavage of caspsase-9, -7 and -3, and poly(ADP-ribose) polymerase. Fisetin also induced cytochrome c and Smac/Diablo release from the mitochondria to the cytosol and mitochondria membrane depolarization. Fisetin decreased protein levels of the pro-survival members of the Bcl-2 family, Bcl-xL and Bcl-2, and increased pro-apoptotic Bcl-2 family proteins, Bak, Bok and mitochondrial Bax. The protein levels of Bim_EL, Bim_L, Bim_S, Bik, and truncated Bid known as “BH3 only proteins” were increased in fisetin treated cells. Fisetin increased p53 protein levels and decreased MDM2 in HCT-116 cells. In addition, fisetin increased the protein levels of membrane-bound Fas ligand and cleaved caspase-8. Furthermore, the fisetin-induced apoptosis and caspase-3 activation were mitigated by a caspase-8 inhibitor, Z-IETD-FMK. However, the fisetin-induced activation of caspase-9 was not affected by the caspase-8 inhibitor. The present results indicate that the alteration of Bcl-2 family proteins and subsequent increase in mitochondrial membrane permeability contribute the fisetin-induced apoptosis in HCT-116 cells. However, the activation of the death receptor-dependent pathway via induction of Fas ligand/caspase-8 activation also contributes the fisetin-induced activation of caspase-3 and apoptosis in HCT-116 cells.