Casticin induces caspase-mediated apoptosis via activation of mitochondrial pathway and upregulation of DR5 in human lung cancer cells

Objective:To assess if casticin induces caspase-mediated apoptosis via activation of mitochondrial pathway and upregulation of DR5 in human lung cancer ceils.Methods:Human non-small-cell lung carcinoma cell lines H460,AS49 and H157 were cultured in vitro.The cytotoxic activities were determined using MTT assay.The apoptotic cells death was examined by flow cytometry using PI staining and DMA agarose gel electrophoresis.The activities of caspase-3, -8 and -9 were measured via ELISA.Cellular fractionation was determined by flow cytometry to assess release of cytochrome c and the mitochondrial transmembrane potential.Bcl-2/Bcl-XL/ XIAP/Bid/ DR5 and DR4 proteins were analyzed using western blot.Results:The concentrations required for a 50%decrease in cell growth(IC50) ranged from 1.8 to 3.2 Jt M.Casticin induced rapid apoptosis and triggered a series of effects associated with apoptosis by way of mitochondrial pathway,including the depolarization of the mitochondrial membrane,release of cytochrome c from mitochondria,activation of procaspase-9 and -3,and increase of DNA fragments.Moreover, the pan caspase inhibitor zVAD-FMK and the caspase-3 inhibitor zOEVD-FMK suppressed casticin-induced apoptosis.In addition,casticin induced XIAP and Bcl-XL down-regulation, Bax upregulation and Bid clearage.In H157 cell line,casticin increased expression of DRS at protein levels but not affect the expression of DR4.The prelreatmenl with DR5/Fc chimera protein effectively attenuated casticin-induced apoptosis in H157 cells.No correlation was found between cell sensitivity to casticin and that to p53 status,suggesting that casticin induce a p53- independent apoptosis.Conclusions:Our results demonstrate that casticin induces caspase-mediated apoptosis via activation of mitochondrial pathway and upregulation of DRS in human lung cancer cells.