FOXO-Dependent Regulation of Tumorigenesis Figure 3. Effects of resveratrol on the expression of PTEN, AKT and MAP kinases; and the effects of PI3K/AKT and MAPK pathways on resveratrol-induced apoptosis. (A), PANC-1 cells were treated with or without resveratrol (0–20 mM) for 24 h. The cells were harvested and the expression of PTEN, phospho-AKT, AKT, Ras, phospho-ERK, ERK, phospho-JNK, JNK, phospho-p38 and p38 was measured by Western blotting. (B), AKT kinase activity. PANC-1 cells were treated with resveratrol (0–20 mM) for 24 h. AKT kinase activity was measured as we described in materials and methods. Data represent the mean 6 S.D. *, #, %, and @ = significantly different from respective controls, P,0.05. (C), PTEN and dominant negative AKT enhance resveratrol-induced apoptosis. PANC-1 and AsPC-1 cells were transiently transfected with empty vector (pcDNA3.1), PTEN wild type (PTEN-WT) or dominant negative AKT (AKT-DN) along with pCMV-LacZ vector (as transfection control) for 24 h. After medium replacement, cells were treated with resveratrol (10 mM) for 48 h and, apoptosis was measured by TUNEL assay. Data represent the mean 6 S.D. * and # = significantly different from respective controls, P,0.05. (D), PANC-1 and AsPC-1 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 48 h. At the end of incubation period, cells were harvested and apoptosis was measured by TUNEL assay. Data represent mean 6 SD. *, ** and # = significantly different from respective controls, P,0.05. doi:10.1371/journal.pone.0025166.g003 transcription factors are required, at least in part, for induction of apoptosis by resveratrol. Inhibition of PI3K/AKT and MEK/ERK pathways or overexpression of FOXO transcription factor synergistically induces FOXO transcriptional activity in the presence or absence of resveratrol Since inhibition of PI3K/AKT and MEK/ERK pathways synergistically induces apoptosis in pancreatic cancer cells, we next sought to examine whether inhibition of these two pathways act together to regulate FOXO activity. FOXO-luciferase construct (pGL3-6X DBE) have previously been described [24]. AKT inhibitor (AKT Inh-IV) and MEK1/2 inhibitor (PD98059) synergistically induced FOXO transcriptional activity in AsPC-1 and PANC-1 cells (Fig. 5A). AKT Inh-IV or PD98059 enhanced resveratrol-induced FOXO transcriptional activity. Interestingly, the combination of AKT Inh-IV and PD98059 with resveratrol- induced higher FOXO transcriptional activity than AKT Inh-IV plus resveratrol or PD98059 plus resveratrol. These data suggest that inhibition of PI3K/AKT and MEK/ERK pathways acts synergistically/additively to regulate FOXO transcriptional activ- ity in the absence or presence of resveratrol. Since inhibition of FOXO1 (FKHR) transcription factor enhanced resveratrol-induced apoptosis in pancreatic cancer cells, we sought to examine whether resveratrol enhances translocation of wild type and phosphorylation deficient mutant of FOXO1 to nucleus (Fig. 5B). PANC-1 cells were transfected with GFP- FOXO1 or GFP-FOXO1-TM plasmids and treated with resveratrol. The translocation of FOXO1 to nucleus was measured by fluorescence microscopy. Resveratrol induced translocation of FOXO1 to nucleus. However, the translocation of phosphoryla- tion deficient mutant of FOXO1 was much higher than wild type FOXO1. These data suggest that resveratrol can regulate translocation of FOXO transcription factor to the nucleus. We next examined whether resveratrol induces transcriptional activation of FOXO in the presence or absence phosphorylation deficient triple mutants of FOXO proteins (FOXO1-TM, PLoS ONE | www.plosone.org 5 September 2011 | Volume 6 | Issue 9 | e25166