FTY720 decreased viability, proliferation, and motility and induced apoptosis in human hepatoblastoma cells.

(A) Following 24 hours of treatment with FTY720, the viability of HuH6 cells measured using the alamarBlue cell viability assay was significantly decreased (LD50 = 8.4 μM, p ≤ 0.05). (B) Following 24 hours of treatment with FTY720, the proliferation of HuH6 cells measured using the CellTiter 96 cell proliferation assay was significantly decreased (IC50 = 6.4 μM, p ≤ 0.05). (C) HuH6 cells were plated and allowed to reach 80% confluence, at which time the media was changed for fresh media (0, 5, or 8 μM FTY720) and a standard scratch wound was made. At 48 hours, there was a significant decrease in motility in the HuH6 cells treated with 8 μM FTY720 compared to control cells. Data were reported as mean fold change in scratch area remaining ± SEM. (D) Immunoblotting was performed on HuH6 lysates for total and cleaved PARP with β-actin controls. Total PARP decreased and cleaved PARP increased with FTY720 treatment, indicating apoptosis. Histograms are representative of densitometry analysis of three or more biologic replicates and are reported as intensity based upon the respective control that is set at 1. Data are reported as mean ± SEM. (E) Cell cycle analysis was utilized to determine the percentage of cells in the sub G1 phase, representing the apoptotic cell population. There was a significant increase in the percent of hepatoblastoma cells in sub G1 with increasing concentrations of FTY720.