Fig. 3.

Combined treatment with thioridazine and curcumin induces caspase-mediated apoptosis in human head and neck squamous cell carcinoma (AMC-HN4) cells. (A) 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. Values below this line indicate synergy, whereas values above this line indicate antagonism. (B–D) AMC-HN4 cells were treated with 10 μM thioridazine in the presence or absence of 0.5 μM curcumin for 24 h. The condensation and fragmentation of the nuclei were detected by 4′,6′-diamidino-2-phenylindole staining (B). The cytoplasmic histone-associated DNA fragments were determined by a DNA fragmentation detection kit. Tamoxifen (10 μM, 24 h) was used as a positive control (C). Caspase activities were determined with colorimetric assays using caspase-3 (DEVDase) assay kits (D). (E) AMC-HN4 cells were treated with 10 μM thioridazine plus 0.5 μM curcumin for 24 h in the presence or absence of 20 μM z-VAD-fmk (z-VAD). The sub-G1 fraction and histogram were measured by flow cytometry. The protein expression levels of PARP, pro-caspase-3, cleaved caspase-3 and actin were determined by Western blot. The level of actin was used as a loading control. (F) AMC-HN4 cells were treated with 10 μM thioridazine in the presence or absence of 0.5 μM curcumin 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 blot. The level of actin was used as a loading control. The values in C, D and E represent the mean ± SD from three independent samples. * p < 0.01 compared to the control. # p < 0.01 compared to combined treatment with thioridazine and curcumin.