Figure 4

UCN-01 reduces Huh7 cell invasion compared with untreated controls. (A) A Transwell invasion assays were performed in the presence of HGF as the chemoattractant with the different hepatoma cell lines (Huh7, HepG2, and Hep3B) and different doses of UCN-01. After 72 h, the lower layer membrane was fixed and observed using the 400× objective. The areas (labelled with 1–8,) were observed using the 400× objective. The upper line shows that Huh7 cell numbers decreased in a UCN-01 dose-dependent manner (areas 1, 2, 3, and 4 correspond to 0, 30, 100, and 300 nM UCN-01, respectively). The lower line shows that there were no obvious changes in HepG2 (5 and 6 correspond to 0 and 300 nM UCN-01, respectively) and Hep3B (7 and 8 correspond to 0 and 300 nM UCN-01, respectively) cells. (B) The column graph represents the percentage of viable Huh7 cells cultured in serum-free media or 10% FBS after treatment with different UCN-01 concentrations. There were no significant differences between cells using the different concentrations of UCN-01 compared with untreated cells when cultured with serum-free media (P > 0.05). (C) The column graph represents the percentage of Huh7 cells that invaded through the transwell compared with the control. The values from each concentration represent the mean ± SD derived from triplicate tests. Significant differences between treated cells and the untreated cells are indicated by an asterisk (P < 0.05). (D) Total β-catenin, phosphorylated β-catenin, and phosphorylated p53 in Huh7 cells were detected using specific antibodies after treatment with 100 nM UCN-01. β-Actin was used as the loading control. Total β-catenin levels did not change after 24 h of UCN-01 treatment; however, phosphorylated β-catenin was down-regulated between 1 to 8 h of treatment, and the down-regulation was significant during 4–8 h of treatment. Phosphorylated p53 levels were gradually up-regulated from 1 to 24 hr of treatment.(E) Western blots of total β-catenin and phosphorylated β-catenin in the Huh7 cell lines. There was no significant change in total β-catenin or phosphorylated β-catenin after treatment with 0, 30, 100, and 300 nM UCN-01 for 24 h.