3-O-acetyloleanolic acid dose-dependently inhibited the viability of HCT-116 cells. Apoptosis was characterized by detection of cell surface annexin V and sub-G1 apoptotic cell populations.

Expression of TRAIL-mediated apoptosis signaling-related death receptor DR5 was increased in 3-O-acetyloleanolic acid-treated HCT-116 cells. Activation of caspase-8 and caspase-3, critical mediators of extrinsic apoptosis signaling, was also increased by 3-O-acetyloleanolic acid. 3-O-acetyloleanolic acid induces apoptosis in HCT-116 cells mediated by an extrinsic apoptosis signaling cascade via up-regulation of DR5.

Modulation of phosphatidylserine externalization during apoptosis was measured using annexin V conjugated with the fluorescent dye fluorescein isothiocyanate (FITC). The HCT-116 cells were seeded onto 6-well plates at a density of 3*10^5 cells/well. After treatment with 25 uM 3-O-acetyloleanolic acid for 6 h, the cells were stained with the annexin V-FITC conjugate, and then imaged at a^40 objective magnification using a confocal laser scanning microscope.