Figure 1. Carnosic acid sensitizes Caki cells to TRAIL-mediated apoptosis.

(A and B) Caki cells were treated with 50 ng/ml TRAIL in the presence or absence of the indicated concentrations of carnosic acid for 24 h. The sub-G1 fraction was measured by flow cytometry as an indicator of the level of apoptosis. The protein expression levels of PARP and actin were determined by Western blotting. The level of actin was used as a loading control. (C) Isoboles were obtained by plotting the combined concentrations of each drug required to produce 50% cell death. The straight line connecting the IC₅₀ values obtained for two agents when applied alone corresponds to an additivity of their independent effects. Values below this line indicate synergy, whereas values above this line indicate antagonism. (D) Caki cells were treated with 50 ng/ml TRAIL in the presence or absence of the indicated concentrations of carnosic acid for the indicated time periods. Cell viability was analyzed by trypan blue exclusion. (E–G) Caki cells were treated with 50 ng/ml TRAIL in the presence or absence of 20 μM carnosic acid for 24 h. The condensation and fragmentation of the nuclei were detected by 4′,6′-diamidino-2-phenylindole staining (E). The cytoplasmic histone-associated DNA fragments were determined by a DNA fragmentation detection kit (F). Caspase activities were determined with colorimetric assays using caspase-3 (DEVDase) assay kits (G). (H) Caki cells were treated with 20 μM carnosic acid plus 50 ng/ml TRAIL for 24 h in the presence or absence of 20 μM z-VAD-fmk (z-VAD). The sub-G1 fraction was measured by flow cytometry. The protein expression levels of PARP and actin were determined by Western blotting. The level of actin was used as a loading control. The values in (A, D, F, G and H) represent the mean ± SD from three independent samples. *p < 0.01 compared to the carnosic acid treatment alone. #p < 0.01 compared to the co-treatment of carnosic acid and TRAIL.