Fig. 2. Capsaicin decreased the ERCC1 expression in a dose and time-dependent manner. (A) Left panel, A549 or H1975 cells (106) were cultured in complete medium for 18 h and then exposed to capsaicin (50 μM) for 2, 4, 8 12, or 24 h. Right panel, various concentrations of capsaicin (12.5–100 μM) were added to cells for 24 h in complete medium. The total RNA was isolated and subjected to real-time PCR for ERCC1 mRNA expression. The results (mean ± SEM) were from three independent experiments. *p < 0.05, **p < 0.01 using Student's t-test for comparison between the cells treated with or without capsaicin. (B) After treatment as above, the cell extracts were examined by western blot for determination of ERCC1, phospho-AKT(Ser473), phospho-AKT(Thr308), actin, and AKT protein levels. Capsaicin decreased ERCC1 expression via AKT inactivation in A549 and H1975 cells. (C and D) A549 or H1975 cells (5 × 105) were transfected with the AKT-CA expression vector for 24 h prior to treatment with capsaicin in complete medium for 24 h. The results (mean ± SEM) were from 3 independent experiments. **p < 0.01, using Student's t-test for comparison between cells treated with capsaicin in pcDNA3 or AKT-CA-transfected cells. (E and F) LY294002 (10 μM) was added to A549 or H1975 cells for 1 h before capsaicin treatment for 24 h. The results (mean ± SEM) were from four independent experiments. **p < 0.01 using Student's t-test for comparison between the cells treated with capsaicin–DMSO or a capsaicin–LY294002 combination. After treatment, the cell extracts were examined via real-time PCR (C, E) and western blot (D, F) for the determination of ERCC1 mRNA and protein levels, respectively.
