Casticin Inhibits EMT Of Liver Cancer Stem-like Cells From SMMC-7721Cell Line Through Down-regulating Twist

Objective To investigate the effect and molecular mechanism of casticin onepithelial-mesenchymal transition(EMT) of liver cancer stem-like cells(LCSLCs)derived from SMMC-7721cell line.Methods SMMC-7721cells were cultured in vitro. Both phenotypes of CD133+cells and CD133-cells in SMMC-7721cell line were sorted by MACS. Stemcell-conditioned medium were used to expand CD133+sphere-forming cells(SFCs)from sorted CD133+cells of SMMC-7721cell line. The protein expressions ofE-cadherin, N-cadherin, Twist were detected by Western blot analysis.Twist-expressing stable transfectants were obtained through pcDNA3.1-Twisttransfection.Results Compared with CD133-cells and unsorted ones, CD133+sphere-formingcells(SFCs) sorted from SMMC-7721cell line possessed higher capacity to formtumor spheroids in vitro, indicating those had self-renewal capacity of LCSLCs. AsLCSLCs and parental SMMC-7721cells were cultured adherently inDMEM(Dulbecco’s modification of Eagle’s medium Dulbecco) culture mediumsupplemented with10%fetal bovine serum, LCSLCs exhibited a spindle-like shape,while parental cells displayed a cobble-stone-like phenotype. However, treatment with1.0μM casticin suppressed EMT in LCSLCs as morphological changes from aspindle-like shape to a cobble-stone-like appearance were showed. Compared to theparental cells,CD133+SFCs expressed a higher N-cadherin and Twist protein level,and a lower expression of epithelium-associated E-cadherin protein,demonstrated thatthe CD133+SFCs sorted from SMMC-7721cell line had typical morphologicphenotypes of EMT. Casticin increased the expression of protein E-cadherin and decreased N-cadherin in LCSLCs. Treatment of LCSLCs with casticin for48h alsodecreased the level of EMT-associated transcription factor Twist, in aconcentration-dependent manner. Overexpression of Twist attenuated casticin-inducedregulation of E-cadherin and N-cadherin protein expression and the EMT andself-renewal capacity of LCSLCs.Conclusions1. The CD133+SFCs of SMMC-7721cell line possessed self-renewal capacity ofLCSLCs, and exhibited typical phenotypes of EMT.2. The down-regulation of Twist expression might be one of the mechanisms bywhich casticin reverse EMT and inhibit self-renewal capacity of LCSLCs.