In vitro activity: asiatic acid inhibited cell growth in two human breast cancer cell lines in a concentration-dependent manner, with MCF-7 being more sensitive to asiatic acid-induced cell growth inhibition than MDA-MB-231. The IC50 values of asiatic acid were 5.95 μM for MCF-7 and 8.12 μM for MDA-MB-231.

Kinase Assay: Immunoprecipitation/Immunoblot and ERK1/2 and p38 MAPK Kinase Activity Assays. Cells were treated with 10 μM asiatic acid in the absence or presence of MAPK inhibitors for specified time intervals. Mitochondrial and cytoplasmic fractions were separated using cytochrome c releasing apoptosis assay kit (BioVision Inc., Mountain View, CA). For immunoblotting, the cells were lysed on ice for 40 min in a solution containing 50 mM Tris, 1% Triton X-100, 0.1% SDS, 150 mM NaCl, 2 mM Na3VO4, 2 mM EGTA, 12 mM β-glycerol phosphate, 10 mM NaF, 16 μg/ml benzamidine hydrochloride, 10 μg/ml phenanthroline, 10 μg/ml aprotinin, 10 μg/ml leupeptin, 10 μg/ml pepstatin, and 1 mM phenylmethylsulfonyl fluoride. The cell lysate was centrifuged at 14,000g for 15 min, and the supernatant fraction was collected for immunoblotting. Equivalent amounts of protein were resolved by SDS-polyacrylamide gel electrophoresis (10-12%) and transferred to polyvinylidene difluoride membranes. After blocking for 1 h in 5% nonfat dry milk in Tris-buffered saline, the membrane was incubated with the desired primary antibody for 1 to 16 h. The membrane was then treated with appropriate peroxidase-conjugated secondary antibody, and the immunoreactive proteins were detected using an enhanced chemiluminescence kit (Amersham Biosciences Inc., Piscataway, NJ) according to the manufacturer's instructions.

Cell Assay: Inhibition of cell proliferation by asiatic acid was measured by sodium 3′-[1-(phenylamino-carbonyl)-3,4-tetrazolium]-bis(4-methoxy-6-nitro) benzene-sulfonic acid hydrate (XTT) assay. Briefly, cells were plated in 96-well culture plates (1 × 104 cells/well). After 24-h incubation, the cells were treated with asiatic acid (0, 2.5, 5, 10, and 20 μM) for 48 h. Fifty microliters of XTT test solution, which was prepared by mixing 5 ml of XTT-labeling reagent with 100 μl of electron coupling reagent, was then added to each well. After 4-h incubation, absorbance was measured on an ELISA reader (Multiskan EX; Labsystem, Helsinki, Finland) at a test wavelength of 492 nm and a reference wavelength of 690 nm. Data were calculated as the percentage of inhibition by the following formula: inhibition % = [100 - (ODt/ODs) × 100]. ODt and ODs indicated the optical density of the test substances and the solvent control, respectively. The concentration of 50% cellular cytotoxicity of cancer cells (IC50) of test substances was calculated based on 48-h absorbance values.

J Pharmacol Exp Ther. 2005 Apr;313(1):333-44.