Investigation Of Inhibitory Effects Of CAS On Self-renewal Of Liver Cancer Stem-like Cells From MHCC97

Objective：To evaluate the effects of casticin（CAS）on the self-renewalcapacity of liver cancer stem-like cells (LCSLCs) derived from the HCC MHCC97.Methods：The CD133+subpopulation was sorted from the MHCC97bymagnetic activated cell sorting (MACS). The expression of the stem cell marker CD133was examined by Flow cytometry analysis（FCM）; To evaluate the self-renewal of theMHCC97CD133+sphere-forming cells(SFCs) through sphere formation assay; Toexamine the effects of CAS on the ability of proliferative activity and self-renewal ofthe MHCC97CD133+SFCs, MTT assay and sphere formation assay were usedrespectively. To examine the expression of β-catenin, cyclin D1in MHCC97CD133+SFCs and observe the effects on the expression of β-catenin, cyclin D1after treatedwith Casticin and Lithium chloride, Western blotting analysis was used.Results：Using flow cytometry analysis, we find that the subpopulation ofCD133+cells showed a high purity of CD133(56.26%±2.34%),but the purityCD133-and the parental cells were (1.04%±0.27%) and (3.32%±0.38%) respectively.The sphere formation assay not only evaluated that the CD133+subpopulationexhibited larger tumorsphere formation amount and size compared with the parentalcells, but also the single CD133+SFCs perform the ability of forminganchorage-independent3-D spheres. CAS significantly inhibited the proliferation ofCD133+SFCs in a dose-dependent manner (p<0.05), the IC50of parental cells andCD133+SFCs was17.9μmol/L and0.5μmol/L respectively by MTT assay. CAS alsoreduced the primary and secondary sphere-forming capacity. Otherwise, CASdown-regulated β-catenin and cyclin D1protein expression of CD133+SFCs. Lithiumchloride, an agonist, known to activate the wnt/β-catenin pathway, attenuated casticin-induced down-regulation of β-catenin、cyclin D1protein expression andinhibited self-renewal capacity.Conclusions：1. CD133+SFCs of the MHCC97possess the characteristics ofLCSCs.2. CAS may inhibit the self-renewal of LCSLCs through the Wnt/β-cateninpathway.