Figure 6. Inhibtion of Sirts by resveratrol in pancreatic cancer cells. (A–C) Pancreatic cancer PANC-1, MIA PaCa-2 and AsPC-1 cells were treated with resveratrol (0–30 mM) for 24 h, and the expression of Sirt1, Sirt2 and Sirt3 was measured by qRT-PCR. Data represent mean 6 SD. * = significantly different from respective control, P,0.05. doi:10.1371/journal.pone.0025166.g006 of FOXO target genes such as Bim, p27/KIP1 and p21/CIP1 in pancreatic tumor tissues. Similarly, others have also demonstrated the antiproliferative and proapoptotic effects of resveratrol in pancreatic cancer [30,31], and here we further added the role of FOXO transcription factors in mediating the effects of resveratrol. Since resveratrol is a nontoxic polyphenolic compound, it can be safely used for the treatment and/or prevention of pancreatic cancer. Resveratrol exerts its anticancer and chemopreventive proper- ties against various types of cancer [32]. Some of the beneficial effects of resveratrol may be due to its ability to suppress autophagy by targeting p70 S6 kinase (S6K1) [33]. Resveratrol suppresses proliferation of various types of cancer cells, including breast, lung, pancreas, liver, stomach, prostate, colon, leukemia, and medulloblastoma [7]. Antitumor activities of resveratrol have also been demonstrated in several mouse models of cancer [7], although the precise mechanisms and pathways involved remain unclear. In the present study, we have discovered a new mechanism by which resveratrol inhibits pancreatic tumor growth i.e. activation of FOXO transcription factors. FOXO-Dependent Regulation of Tumorigenesis FOXO transcription factors have been shown to regulate tissue homeostasis in the pancreas, diabetes and cancer. In recent years, they have emerged as critical transcriptional integrators among pathways regulating proliferation, survival, differentiation and angiogenesis. FOXO regulates apoptotic and cell cycle genes such as Bim [28], Fas ligand [34], p27/KIP1 [28], and Bcl-6 [35]. Our results show that resveratrol induced the expression of PTEN and inhibited the phosphorylation/activation of AKT in PANC-1 cells. We also demonstrate that the inhibition of AKT phosphorylation is correlated with the activation of FOXO transcription factors and induction of Bim, p21/CIP1 and p27/KIP1. In vivo, resveratrol also induced the expression of FOXO target genes such as Bim, p27/KIP1 and p21/CIP1 in pancreatic tumor tissues. In other studies, we have demonstrated that anti-angiogenic effects of chemopreventive agents EGCG, resveratrol and sulforaphane are mediated through activation of FOXO transcription factors [25,36]. Together, these studies suggest that activation of FOXO transcription factors by chemopreventive agents may regulate cell cycle, apoptosis and angiogenesis. The PI3K/AKT and MAPK pathways can be activated by Kras [24,25,37]. AKT and ERK both have been shown to directly phosphorylate and inactivate FOXO transcription factors, resulting in cytoplasmic retention, inactivation, and inhibition of the expression of FOXO-regulated genes, which control various processes such as metabolism, cell cycle, cell death and oxidative stress [38]. Our results show that resveratrol inhibited cytoplasmic phosphorylation of FOXOs, and enhanced FOXO nuclear translocation, FOXO-DNA binding and tran- scriptional activities. The inhibition of PI3K/AKT and MEK/ ERK pathways induced FOXO transcriptional activity and apoptosis. Overexpression of phosphorylation deficient triple mutants of FOXOs induced FOXO activity, and further enhanced resveratrol-induced FOXO activity and apoptosis, whereas deletion of FOXOs genes by shRNA abrogated resveratrol-induced cell cycle arrest and apoptosis. Resveratrol- induced suppression of tumor growth in mice was associated with inhibition of ERK and PI3K/AKT pathway and activation of FOXO1 and FOXO3a. Taken together, these studies demon- strate that the activation of FOXOs has significant implication for the treatment and prevention of pancreatic cancer, where Kras is activated in about 90% patients, and PI3K/AKT and MEK/ ERK pathways are highly activated. In addition to phosphorylation, the regulation of FOXO transcription factors by acetylation has also been demonstrated. The acetylation/deacetylation of FOXO can be regulated by p300, Cbp (CREB-binding protein) and Pcaf (p300/CBP- associated factors) in response to oxidative stress or DNA binding, followed by deacetylation by class I and II histone deacetylases [39], including Sirt1 [40]. Many of the SIRT1 substrates are transcription factors and key regulators described to take part in cancer development, such as the NFkB, the DNA repair factor Ku70, the tumor suppressor gene p53, and the FOXOs. The relationship between tumorigenesis and SIRT1 activity is still open to debate. Although some studies have suggested that SIRT1 may function as a tumor promoter because of its increased expression in some types of cancers, other studies have demonstrated that SIRT1 levels are reduced in some other types of cancers thereby acting as tumor suppressors. With an improved SIRT1 deacetyla- tion assay, it has been reported that polyphenolic stimulators Epigallocatechin galate (EGCG), epicatechin galate (ECG) and myricetin stimulated SIRT1 under stabilizing conditions, whereas without stabilization, these polyphenols strongly inhibited SIRT1, probably due to H2O2 formation [41]. In the present study, resveratrol inhibited the expression of SIRT1, SIRT2 and SIRT3 PLoS ONE | www.plosone.org 8 September 2011 | Volume 6 | Issue 9 | e25166