FOXO-Dependent Regulation of Tumorigenesis Figure 5. Inhibition of PI3K/AKT and MEK/ERK pathways synergistically/additively enhanced resveratrol -induced apoptosis and FOXO transcriptional activity in pancreatic cancer cells. (A), PANC-1 and AsPC-1 cells were transiently transfected with 66 DBE-luciferase construct for 24 h. After transfection, cells were pretreated with AKT inhibitor IV (1 mM) and/or MEK1/2 inhibitor PD98059 (10 mM) for 2 h, followed by treatment with or without resveratrol (10 mM) for 24 h. Cells were harvested for firefly/Renilla luciferase assays using the Dual-Luciferase Reporter Assay System (Promega). Luciferase counts were normalized using Renilla luciferase transfection control (pRL-TK; Promega). Data represent the mean 6 S.D. *, # and ** = significantly different from respective controls, P,0.05. (B), Resveratrol induces translocation of wild type and triple mutant FOXO1 to nucleus. Pancreatic cancer PANC-1 cells were transfected with either GFP-FOXO1 or GFP-FOXO-TM and treated with resveratrol (10 mM) for 24 h. Cells were visualized by fluorescence microscopy. (C), Phosphorylation deficient mutants of FOXO (triple mutants) enhance resveratrol-induced FOXO transcriptional activity in pancreatic cancer. PANC-1 and AsPC-1 cells were transiently transfected with empty vector or constructs encoding FOXO1-TM, FOXO3a-TM, or FOXO4-TM together with 66 DBE-luciferase for 24 h. After transfection, culture medium was replaced with complete RPMI, treated with resveratrol (10 mM) for 24 h, and harvested for firefly/Renilla luciferase assays using the Dual-Luciferase Reporter Assay System (Promega). Luciferase counts were normalized using Renilla luciferase transfection control (pRL-TK; Promega). Data represent the mean 6 S.D. *, % and # = significantly different from control, P,0.05. (D) Left panel, Resveratrol inhibits cytoplasmic phosphorylation of FOXO1 and FOXO3a. PANC-1 cells were treated with resveratrol (0, 10 and 20 mM) for 36 h, and cytoplasmic fractions were prepared. The expressions of p-FOXO1 and p-FOXO3a were measured by the Western blot analysis. Right Panel, FOXO-DNA binding. PANC-1 cells were treated with or without resveratrol (10 mM) for various time points (0–32 h). Nuclear extracts were prepared. Gelshift assay was performed as we described in Material and Methods. Lane 1 = probe only, lane 2 = 506 cold probe, lane 3 = control, lane 4 = cell lysates from control samples treated with SP1 probe, lanes 5–11 = cell lysate from resveratrol treated samples at 4, 8, 12, 16, 20, 24 and 32 h. doi:10.1371/journal.pone.0025166.g005 hypothesis that resveratrol activates FOXO transcription factors by inhibiting ERK and PI3K/AKT pathways in pancreatic cancer cells. Discussion In this paper we have demonstrated, for the first time, that polyphenolic compound resveratrol causes growth arrest and induces apoptosis through activation of FOXO transcription factors. Resveratrol induces apoptosis through caspase-3 activa- tion, and causes growth arrest through induction of p21 and p27 and inhibition of cyclin D1 which are the downstream targets of FOXO. Inhibition of PI3K/AKT and MEK/ERK pathways acts together to enhance the activation of FOXO transcriptional activity, and phosphorylation deficient triple mutants of FOXO further enhance resveratrol-induced FOXO activity and apopto- sis. Inhibition of FOXO1, FOXO3a and FOXO4 by shRNA blocked resveratrol-induced cell cycle arrest and apoptosis. Resveratrol-induced suppression of PANC1 tumor growth in nude mice was associated with inhibition of ERK, PI3K, AKT, FOXO1 and FOXO3a phosphorylation, and induction of apoptosis in tumor tissues. Resveratrol also induced the expression PLoS ONE | www.plosone.org 7 September 2011 | Volume 6 | Issue 9 | e25166