The Effect Of8-bromo-7-methoxychrysin On Self-renewal Of Sphere Forming Cells From Human Hepatocellular Carcinoma Huh-7Cell Line

ObjectiveThe objective of this study was to investigate whether8-bromo-7-methoxychrysin(BrMC), a novel synthetic chrysin analogue, inhibit cell viability, self-renewal anddown-regulate the expression of β-catenin protein in LCSLCs. The sphere formingcells derived from human hepatocellular carcinoma (HCC) cell line Huh-7cultured invitro, was named liver cancer stem cell like cells (LCSLCs).MethodsThe human hepatocellular carcinoma Huh-7cell line was cultured in vitro. Thestem cell conditioned culture system was used to enrich and amplify LCSLCs. Aftertreated with BrMC in various concentrations,Cell viability of LCSLCs was examinedby MTT assay,while the self-renewal of LCSLCs was assessed by tumorsphereformation assay. The expression levels of CD133, a stem cell marker of LCSLCs,was detected using FCM analysis with FITC-CD133. Finally, the expression ofβ-catenin protein in LCSLCs was examined by western blot analysis.ResultsFrom the Huh-7cell line was cultured and amplified in vitro,we isolated thesphere-forming cells namely LCSLCs in the stem cell conditioned culture system for7days. In this condition, cells grew as nonadherent, three-dimensional sphere clusters,called LCSLCs.After treated with BrMC in various concentrations, MTT assayshowed that the cell viability of LCSLCs and the parental Huh-7cells were significantly declined (P＜0.05), in a concentration-dependent manner, while the cellviability of LCSLCs was lower compared to the parental Huh-7cells. The resultsfrom tumorsphere formation assay indicated that BrMC between5.0and20.0μMsignificantly decreased the amount of sphere forming cells, in aconcentration-dependent manner(P＜0.05). The data from FCM analysis withFITC-CD133indicated that BrMC significantly downregulated the expression ofCD133protein in LCSLCs. In addition, BrMC inhibited the expression of β-cateninprotein in LCSLCs by concentration-dependent manner (P＜0.05).Conclusion1. BrMC possesses the inhibitory effect on cell viability and self-renewal of LCSLCs.2. Inhibition of the self-renewal of LCSLCs by BrMC is associated with decrease thelevel of β-catenin protein expression.