8-bromo-7-methoxychrysin inhibits properties of liver cancer stem cells via downregulation of β-catenin

AIM:To evaluate whether 8-bromo-7-methoxychrysin(BrMC),a synthetic analogue of chrysin,inhibits the properties of cancer stem cells derived from the human liver cancer MHCC97 cell line and to determine the potential mechanisms.METHODS:CD133+cells were sorted from the MHCC97 cell line by magnetic activated cell sorting,and amplified in stem cell-conditioned medium to obtain the enriched CD133+sphere forming cells(SFCs).The stem cell properties of CD133+SFCs were validated by the tumorsphere formation assay in vitro and the xenograft nude mouse model in vivo,and termed liver cancer stem cells(LCSCs).The effects of BrMC on LCSCs in vitro were evaluated by MTT assay,tumorsphere formation assay and transwell chamber assay.The effects of BrMC on LCSCs in vivo were determined using a primary and secondary xenograft model in Balb/c-nu mice.Expressions of the stem cell markers,epithelialmesenchymal transition(EMT)markers andβ-catenin protein were analyzed by western blotting or immunohistochemical analysis.RESULTS:CD133+SFCs exhibited stem-like cell properties of tumorsphere formation and tumorigenesis capacity in contrast to the parental MHCC97 cells.We found that BrMC preferentially inhibited proliferation and self-renewal of LCSCs(P<0.05).Furthermore,BrMC significantly suppressed EMT and invasion of LCSCs.Moreover,BrMC could efficaciously eliminate LCSCs in vivo.Interestingly,we showed that BrMC decreased the expression ofβ-catenin in LCSCs.Silencing ofβ-catenin by small interfering RNA could synergize the inhibition of self-renewal of LCSCs induced by BrMC,while Wnt3a treatment antagonized the inhibitory effects of BrMC.CONCLUSION:BrMC can inhibit the functions and characteristics of LCSCs derived from the liver cancer MHCC97 cell line through downregulation ofβ-catenin expression.

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.