Caspase-9, −3 and −8 activation in the apoptosis of colon cancer cells. Cell lines were treated with betulin (10 µg/ml) for 0, 4, 8, 12 and 24 h. Cell-free caspase-9 activity of (A) HCT116 and (B) HT29 cells was analyzed using N-acetyl-Leu-Glu-His-Asp-7-amido-4-trifluoromethylcoumarin. Caspase-3 activity of (C) HCT116 and (D) HT29 cells was analyzed using N-acetyl-Asp-Glu-Val-Asp-7-amido-4-trifluoromethylcoumarin. Caspase-8 activity of (E) HCT116 and (F) HT29 cells was examined using N-acetyl-Ile-Glu-Thr-Asp-7-amido-4-trifluoromethylcoumarin. Data from the control groups (untreated cells) were not shown. Results are presented as the mean ± standard deviation of three experiments. *P<0.01, **P<0.001, treatment in indicated time point vs. 0 h treatment.