Figure 2
In vitro raloxifene study. A. Cell viability was significantly decreased in mouse mammary carcinoma BJMC3879luc2 cells (1 × 104 cells and 5 × 104 cells/well) treated with more than 10 μM of raloxifene for 48 h (**P < 0.01). The IC50 concentration was determined to be 20 μM; therefore, 20 μM raloxifene for 48 h-incubation was used for in vitro studies. Ten samples from each dosage of raloxifene were examined. B. Caspase activities were evaluated according to luminescent assay. Activities of caspase-3, caspase-8 and caspase-9 (but not caspase-12) were significantly elevated in BJMC3879luc2 cells treated with 20 μM raloxifene for 48 h (**P < 0.01). Three samples each of control and raloxifene-treated cells were examined. C. Cytochrome c in the cytosolic fraction, as determined by ELISA, was significantly increased in cells treated with raloxifene for 48 h as compared to the control levels (*P < 0.05). Six samples from control cells and five samples from raloxifene-treated cells were examined. D. Western blots of Bid (22 kDa) in control cells and cells treated with raloxifene for 48 h were similar (upper panel). Cleaved Bid was not observed after raloxifene treatment. β-Actin served as an internal control (lower panel). E. In BJMC3879luc2 cells treated with raloxifene for 48 h, cell viabilities were significantly increased by 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 at 10 or 100 μM (*P < 0.05; **P < 0.01). Six samples each of control and raloxifene-treated cells were examined. F. Cell-cycle analysis showed that raloxifene induced arrest in the G1-phase and inhibition of the S-phase in metastatic mouse mammary carcinoma BJMC3879luc2 cells (**P < 0.01). Three samples each of control and raloxifene-treated cells were examined. G. Levels of DNA synthesis, as assessed by BrdU incorporation rates, were significantly decreased in the cells treated with raloxifene for 48 h (**P < 0.01). rlu: relative luminescent unit. Data presented are means ± SD values. Four samples each of control and raloxifene-treated cells were examined.