Figure 5

In vitro cytotoxic effects of FTY720.

(A) The dose-dependent effect of FTY720 in normal oral fibroblasts (NHOF4 and NHOF6), normal keratinocytes (HaCaT) and OSCC (H357 and H400) cell lines. Cells were treated with FTY720 in complete media for 72 hours. Results are expressed as a percentage of viability of cells treated with vehicle control ( = 100%) and are shown as mean ± SD values of triplicates. (B) H400 cells (50 or 100 cells) were seeded and cultured in complete media for 24-hours and then treated with FTY720 (5 μM). The results represent the mean number of colonies (>50 cells) formed ± SD in three separate dishes for each condition and are expressed as a percentage of colonies formed by cells treated with vehicle control (=100%). (C) The cytotoxic effect of FTY720-P in H357 and H400 cells using MTT assays. Results are expressed as a percentage of viability of cells treated with vehicle control (=100%) and are shown as mean ± SD values of triplicates. (D) H400 cells were treated with vehicle control, 10 or 20 μM FTY720 in complete media. Results show the percentage of total apoptotic cells (early and late) ± SD of duplicates as determined by Annexin V/PI dual staining. (E) DNA laddering observed for H400 cells treated with 10 μM and 20 μM of FTY720 for 48 hours. Cells treated with 5% DMSO were used as a positive control. (F) H400 cells were treated at different concentrations of FTY720 in complete media for 24 hours, prior to measuring caspase 3/7, 8 and 9 activities. The results are expressed as relative luminescence units (RLU) calculated based on the luminescence produced relative to cells treated with vehicle control at 12 hours (=1). *ρ < 0.05 and **ρ < 0.01 (Dunnett’s post-hoc test).