Figure 1.

Combined treatment with carboplatin and thioridazine induces apoptosis in human head and neck cancer (AMC-HN4) cells. (a) AMC-HN4 cells were treated with 200 nM carboplatin (Car) in the presence or absence of 10 μM thioridazine (Thio) 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 blot. The level of actin was used as a loading control. (b) 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 the two agents, when applied alone, corresponded to the addition of their independent effects. The values below this line indicate synergy, whereas the values above this line indicate antagonism. (c–d) AMC-HN4 cells were treated with 200 nM carboplatin in the presence or absence of 10 μM thioridazine for 24 h. Condensation and fragmentation of the nuclei were detected by 4′,6′-diamidino-2-phenylindole staining (c). The cytoplasmic histone-associated DNA fragments were determined by a DNA fragmentation detection kit (d). Caspase activities were determined with colorimetric assays using caspase-3 (DEVDase) assay kits (e). (f) AMC-HN4 cells were treated with 200 nM carboplatin plus 10 μM thioridazine 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, pro-caspase-3, cleaved caspase-3, and actin were determined by western blotting. The level of actin was used as a loading control. (g) AMC-HN4 cells were treated with 200 nM carboplatin in the presence or absence of 10 μM thioridazine for 24 h. The protein expression levels of cIAP1, cIAP2, XIAP, c-FLIP, Mcl-1, Bcl-xL, Bcl-2, and actin were determined by western blotting. The level of actin was used as a loading control. The values in a, d, e, and f represent the mean±S.D. from three independent samples. *P<0.01 compared with the control. ^#P<0.01 compared with the combined treatment with carboplatin and thioridazine