Fig. 6.

Thioridazine plus curcumin induced up-regulation of PSMA5 expression. (A) 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 PSMA5 and actin were determined by Western blot. The level of actin was used as a loading control. (B and C) AMC-HN4 cells were treated with 10 μM thioridazine plus 0.5 μM curcumin for the indicated time periods. The protein (B) and mRNA (C) expression levels of PSMA5 and actin were determined by Western blot and RT-PCR, respectively. The level of actin was used as a loading control. (D) AMC-HN4 cells were transiently transfected with a plasmid harboring the luciferase gene under the control of the PSMA5/-277 promoter. After transfection, cells were treated with 10 μM thioridazine plus 0.5 μM curcumin for 24 h. The luciferase activity was analyzed. Sulforaphane (200 μM, 24 h) was used as a positive control. (E) AMC-HN4 cells were transiently transfected with a control siRNA or PSMA5 siRNA. Twenty-four hours after transfection, cells were treated with 10 μM thioridazine plus 0.5 μM curcumin for 24 h. The sub-G1 fraction and histogram were measured by flow cytometry as an indicator of the level of apoptosis. The protein expression levels of PARP, c-FLIP, Mcl-1, PSMA5 and actin were determined by Western blot. The level of actin was used as a loading control. The values in D and E represent the mean ± SD from three independent samples. * p < 0.01 compared to control. & p < 0.01 compared to thioridazine plus curcumin-treated control siRNA.