Figure 5.

Reactive oxygen species has a critical role in carboplatin plus thioridazine-mediated PSMA5 expression. (a) AMC-HN4 cells were treated with 200 nM carboplatin plus 10 μM thioridazine for 6 h (left panel) or the indicated time periods (right panel), and the cells were then loaded with the H₂DCF-DA fluorescent dye. The H₂DCF-DA fluorescence intensity was detected by a fluorescence microscope (left panel) and flow cytometry (right panel). (b) AMC-HN4 cells were treated with 200 nM carboplatin plus 10 μM thioridazine for the indicated time periods. The protein expression levels of Prx-SO3 and actin were determined by western blotting. The level of actin was used as a loading control. (c) AMC-HN4 cells were pretreated with 5 mM NAC, 2 mM GEE, and 200 μM trolox for 30 min and were then treated with 200 nM carboplatin plus 10 μM thioridazine for 24 h. After treatment, the nuclear extracts were analyzed for Nrf2 and Ref-1 by western blotting as described in the Materials and Methods. Ref-1 was used as a marker of the nuclear fraction. (d) AMC-HN4 cells were transfected with an ARE-luciferase construct for 24 h. The cells were pretreated with 5 mM NAC, 2 mM GEE, and 200 μM trolox for 30 min and were then treated with 200 nM carboplatin plus 10 μM thioridazine for 24 h. After treatment, the cells were lysed and assayed for luciferase activity. (e) AMC-HN4 cells were transiently transfected with a plasmid harboring the luciferase gene under the control of the PSMA5/-277 promoter. After transfection, the cells were pretreated with 5 mM NAC, 2 mM GEE, and 200 μM trolox for 30 min and were then treated with 200 nM carboplatin plus 10 μM thioridazine for 24 h. The luciferase activity was analyzed. (f) AMC-HN4 cells were pretreated with 5 mM NAC, 2 mM GEE, and 200 μM trolox for 30 min and were then treated with 200 nM carboplatin plus 10 μM thioridazine for 24 h. The sub-G1 fraction was measured by flow cytometry. The protein expression levels of PARP, PSMA5, c-FLIP, Mcl-1, 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 carboplatin plus thioridazine