Fig. 7. Enhancement of AKT activity restored the suppressed ERCC1 protein expression and cell survival in capsaicin and erlotinib-exposed A549 and H1975 cells. (A and B) AKT-CA (3 μg) or pcDNA3 (3 μg) expression plasmids were transfected into cells using lipofectamine. After expression for 24 h, the cells were treated with capsaicin (25 μM) and erlotinib (5 μM) for an additional 24 h, and total RNA was isolated and subjected to real-time PCR for ERCC1 mRNA expression, and western blot for determination of ERCC1 protein levels. (C) After treatment as in (A). Cytotoxicity was determined by assessment with the MTS assay. The means ± standard deviation (SD) from four independent experiments. ** denotes p < 0.01, using Student's t-test to compare cells treated with capsaicin and erlotinib in AKT-CA vs. pcDNA3-transfected cells. (D) A549 or H1975 cells were pretreated with LY294002 for 1 h and then co-treated with capsaicin (12.5 μM) and erlotinib (2.5 μM) for 24 h. Total RNA was isolated and subjected to real-time PCR for ERCC1 mRNA expression. (E) After treatment as the above, the whole-cell extracts were collected for western blot analysis. (F) After treatment as in (D). Cytotoxicity was determined by the MTS assay. **p < 0.01 using Student's t-test for comparison between the cells treated with capsaicin/erlotinib–DMSO or a capsaicin/erlotinib–LY294002 combination. (G) Upper panel, inactivation of the AKT kinase and down-regulation of the ERCC1 expression involved in capsaicin-induced cytotoxicity. Lower panel, capsaicin enhanced NSCLC cells sensitivity to erlotinib as a result of AKT inactivation mediated ERCC1 decrease in NSCLC cells.