FOXO-Dependent Regulation of Tumorigenesis Figure 1. Effects of resveratrol on cell viability, colony formation and caspase-3 activity in pancreatic cancer cells. (A), Cell Viability. Pancreatic cancer (PANC-1, MIA PaCa-2, Hs766T and AsPC-1) cells were treated with resveratrol (0–30 mM) for 48 h. Cell viability was measured by XTT assay. Data represent the mean 6 S.D. * = significantly different from respective controls, P,0.05. (B), Colony formation. Pancreatic cancer (PANC-1, MIA PaCa-2, Hs766T and AsPC-1) cells were treated with resveratrol (0–30 mM) for 3 weeks. At the end of incubation period, colonies were fixed and number of colonies were counted. Data represent the mean 6 S.D. * = significantly different from respective controls, P,0.05. (C), Caspase-3 activity. Pancreatic cancer PANC-1, MIA PaCa-2, Hs 766T and AsPC-1 cells were treated with resveratrol (0–40 mM) for 24 h and caspase-3 activity was measured as per manufacturer’s instructions (EMD Biosciences). Data represent the mean 6 S.D. * = significantly different from respective controls, P,0.05. doi:10.1371/journal.pone.0025166.g001 (Control = 48.1%, resveratrol = 83.6%). By comparison, inhibition of FOXO1/FOXO3a/FOXO4 by shRNA blocked resveratrol- induced growth arrest. These data suggest that FOXO transcrip- tion factors are required for resveratrol-induced cell cycle arrest. We next examined the effects of resveratrol on FOXO target genes (Fig. 2C). Resveratrol inhibited the expression of cyclin D1 and induced the expression of cell cycle inhibitors (p21/CIP1 and p27/KIP1) and Bim in PANC-1 cells. Over all, these data suggest that resveratrol causes growth arrest and induces apoptosis through regulation of FOXO target genes such as cyclin D1, p21/CIP1, p27/KIP1 and Bim. Inhibition of PI3K/AKT and MAP kinase pathways enhance resveratrol-induced apoptosis The constitutively AKT has been shown to enhance survival of cancer cells and also to be the cause of drug-resistance [29]. Since PTEN regulates AKT activity, we measured the expression of PTEN and phosphorylation status of AKT in pancreatic cancer cells treated with resveratrol (Fig. 3A). Resveratrol induces PTEN expression and inhibits AKT phosphorylation in PANC-1 cells. By comparison, resveratrol has no effect on total AKT expression. We next confirmed the inhibition of AKT activation by resveratrol with kinase assay (Fig. 3B). Resveratrol inhibited AKT kinase activity in a dose-dependent manner. These data suggest that resveratrol inhibits cell proliferation by regulating PI3K/AKT pathway. Ras/Raf/MAP kinase pathway has been shown to regulate migration, mitosis, metabolism, and apoptosis [20]. We therefore examined the effects of resveratrol on the expression of Ras, and activation of ERK, JNK and p38 MAP kinases. Resveratrol inhibited Ras expression in PANC-1 cells (Fig. 3A). Treatment of PANC-1 cells with resveratrol caused a decrease in ERK phosphorylation, and an increase in JNK phosphorylation. By comparison, treatment of PANC-1 cells with resveratrol resulted in a slight increase in the phosphorylation of p38 MAP kinase. These data suggest that resveratrol inhibits growth and induces apoptosis through regulation of Ras/Raf/MAP kinase pathway. PI3K/AKT pathway has been shown to inhibit apoptosis. We next examined whether resveratrol induces apoptosis through PI3K/AKT pathway (Fig. 3C). Pancreatic cancer cells were transfected with empty vector, wild type PTEN, dominant negative AKT (DN-AKT), and apoptosis was measured. Overex- pression of wild type PTEN or DN-AKT induced apoptosis in AsPC-1 and PANC-1 cells. Treatment of these transfected cells PLoS ONE | www.plosone.org 3 September 2011 | Volume 6 | Issue 9 | e25166