Apoptosis and cell cycle in vitro study (mouse mammary carcinoma BJMC3879luc2 cells). (A) Cell viability was significantly lower in mouse mammary carcinoma BJMC3879luc2 cells treated with more than 12 μM α-mangostin for 24 or 48 hours (**P < 0.01). Five samples from each dosage of α-mangostin were examined. The IC50 concentration was determined to be 12 μM; therefore, 12 μM α-mangostin and 24 hour-incubation was used for all in vitro studies. (B) Caspase activities were evaluated using the luminescence assay. Activities of caspase-3, caspase-8 and caspase-9, but not caspase-12, were significantly elevated in BJMC3879luc2 cells treated with 12 μM α-mangostin for 24 hours (*P < 0.05 or **P < 0.01). Six samples from the control and eight samples from α-mangostin-treated cells were used for measurement of caspase-3 activity, and three samples from each group were used for activity measurements of the other caspases. (C) Cytochrome c in the cytosolic fraction, as determined by ELISA, was significantly increased in BJMC3879luc2 cells treated with α-mangostin for 24 hours compared to control levels (*P < 0.05). Three samples each from the control and α-mangostin-treated cell cultures were examined. (D) Western blots of Bid (22 kDa) in BJMC3879luc2 cells treated with or without α-mangostin for 24 hours were similar (upper panel). Cleaved Bid (15 kDa) was not observed after α-mangostin treatment. β-Actin served as the internal control (lower panel). (E) In BJMC3879luc2 cells treated with α-mangostin for 24 hours, cell viability was significantly increased by 10 or 100 μM of the broad-spectrum caspase inhibitor z-VAD-fmk, the caspase-3 specific inhibitor Ac-DNLD-CHO, the caspase-8 specific inhibitor z-IETD-fmk, and the caspase-9 specific inhibitor z-LETD-fmk (**P < 0.01). (F) Cell-cycle analysis showed that α-mangostin induced arrest in the G1-phase and inhibition of cells entering the S-phase in metastatic mouse mammary carcinoma BJMC3879luc2 cells (**P < 0.01). Data are presented as mean ± SD. Ac: acetyl; CHO: aldehyde; ELISA: enzyme-linked immunosorbent assay; fmk: fluoromethyl ketone; z: N-benzyloxycarbonyl.