FOXO-Dependent Regulation of Tumorigenesis Figure 2. Effect of resveratrol on apoptosis and cell cycle. (A), Pancreatic cancer PANC-1, MIA PaCa-2, Hs 766T and AsPC-1 cells were treated with resveratrol (0–30 mM) for 48 h and apoptosis was measured by TUNEL assay. Data represent the mean 6 S.D. * = significantly different from respective controls, P,0.05. (B), PANC-1 cells were transfected with either scrambled or FOXO1/FOXO3a/FOXO4 shRNA expressing constructs and treated with resveratrol (10 mM) for 24 h. At the end of incubation period, cells were harvested, and cell cycle analysis was performed by flow cytometry. (C), Effects of resveratrol on cell cycle regulatory genes. PANC-1 cells were treated with resveratrol (0–20 mM) for 24 h. The expression of cyclin D1, p21/CIP1, p27/KIP1 and Bim was measured by Western blotting. Anti b-actin antibody was used as a loading control. doi:10.1371/journal.pone.0025166.g002 with resveratrol further enhanced apoptosis. These data suggest that inhibition of PI3K/AKT pathway enhances resveratrol- induced apoptosis in pancreatic cancer cells. Since inhibition of PI3K/AKT and MEK/ERK pathways together induce apoptosis in an additive or synergistic manner, we sought to examine whether these pathways act together to regulate resveratrol-induced apoptosis. The activations of AKT and MEK/ ERK were inhibited by AKT Inh-IV and PD98059, respectively. AKT inhibitor (AKT Inh-IV) and MEK1/2 inhibitor (PD98059) alone induced apoptosis in both PANC-1 and AsPC-1 cells (Fig. 3D). AKT inhibitor and MEK1/2 inhibitor (PD98059) synergistically/additively induced apoptosis in PANC-1 and AsPC-1 cells. Interestingly, the combination of AKT inhibitor and PD98059 with resveratrol induced more apoptosis than AKT inhibitor plus resveratrol or PD98059 plus resveratrol. Based on these data, it can be suggested that the inhibition of PI3K/AKT and MEK/ERK pathways act together to enhance resveratrol- induced apoptosis. FOXO transcription factors regulate resveratrol-induced apoptosis in pancreatic cancer cells Inhibition of PI3K/AKT and MEK/ERK pathways activates FOXO transcription factors. We therefore examined whether FOXO transcription factors regulate apoptosis induced by resveratrol in pancreatic cancer cells. PANC-1 and AsPC-1 cells were transfected with FOXO1-WT, FOXO1-TM (phosphoryla- tion mutant), FOXO3a or FOXO3a-TM (phosphorylation deficient mutant) (Fig. 4A and B). Wild type and phosphorylation deficient mutants of FOXO expression plasmids have previously been described [24]. Overexpression of FOXO1, FOXO1-TM, FOXO3a and FOXO3a-TM induced apoptosis in both PANC-1 and AsPC-1 cells. However, phosphorylation deficient mutants of FOXO1 and FOXO3a induced more apoptosis than wild type FOXO1 and FOXO3a. Resveratrol also induced apoptosis in both PANC-1 and AsPC-1 cells. Overexpression of FOXO1-WT, FOXO1-TM, FOXO3a or FOXO3a-TM further enhanced resveratrol-induced apoptosis. Phosphorylation deficient mutants of FOXO1 and FOXO3a had more effects on resveratrol-induced apoptosis than wild type counterparts. These data suggest that the phosphorylation of FOXO transcription factors plays a major role in their activities, and FOXO (wild type and triple mutant) can enhance resveratrol-induced apoptosis. We next examined whether inhibition of FOXO transcription factors by shRNA abolishes resveratrol-induced apoptosis (Fig. 4C and D). Pancreatic cancer PANC-1 and MIA PaCa-2 cells were stably transfected with plasmids expressing FOXO1, FOXO3a and FOXO4 shRNA. These expression vectors have been described elsewhere [28]. Resveratrol induced apoptosis in PANC-1 and MIA PaCa-2 cells transfected with scrambled vectors. By comparison, inhibition of FOXO1, FOXO3a and FOXO4 through shRNA inhibited resveratrol induced apoptosis in PANC-1 and MIA PaCa-2 cells. These data suggest that FOXO PLoS ONE | www.plosone.org 4 September 2011 | Volume 6 | Issue 9 | e25166