cell lines, synergized the apoptotic effects of gemcitabine, and inhibited the constitutive activation of NFkB and its gene products [8]. In vivo, resveratrol significantly suppressed the growth of the tumor and this effect was further enhanced by gemcitabine [8]. Resveratrol also suppressed the NFkB activation and expression of cyclin D1, COX-2, ICAM-1, MMP-9 and survivin in tumor tissues [8]. Furthermore, we recently demonstrated that resveratrol induced apoptosis by activating capase-3/7 and inhibiting the expression of Bcl-2 and XIAP in human pancreatic cancer stem cells (CSCs) [9]. Resveratrol inhibited pluripotency maintaining factors (Nanog, Sox-2, c-Myc and Oct-4) and drug resistance gene ABCG2 in CSCs. Inhibition of Nanog by shRNA enhanced the inhibitory effects of resveratrol on self-renewal capacity of CSCs. Finally, resveratrol inhibited CSC’s migration and invasion and markers of epithelial-mesenchymal transition (EMT) such as Zeb- 1, Slug and Snail. Overall, resveratrol inhibited pancreatic CSC characteristics in human and KrasG12D transgenic mice by inhibiting pluripotency maintaining factors and EMT. Transcription factors of the Forkhead box O (FOXO) class are predominantly regulated through phosphoinositide 3-kinase/AKT (also known as PKB) pathway. FOXO1a/FKHR, FOXO3a/ FKHRL1, and FOXO4/AFX are members of FOXO subfamily [10]. The PI3K/AKT pathway phosphorylates all of these FOXO proteins, resulting in impairment of DNA binding ability and inhibition of FOXO-dependent transcription [11]. Inhibition of the PI3K/AKT pathway leads to dephosphorylation and nuclear translocation of active FOXOs, which causes cell cycle arrest and apoptosis [12]. Conversely, loss of PTEN activity results in increased AKT activity leading to inhibition of FOXO activity through phosphorylation and cytoplasmic sequestration [12]. FOXO transcriptional activity controls cellular proliferation and apoptosis downstream of PTEN, PI3K and AKT [13]. Since inactivation and loss of PTEN, and overexpression of AKT are frequently observed in pancreatic cancer [14], targeting a downstream target such as FOXO may be an attractive idea for pancreatic cancer prevention and/or treatment. In addition to PI3K/AKT, MAP kinase pathway has also been shown to regulate FOXO activities. The Ras proteins transduce signals from ligand-activated tyrosine kinase receptors to down- stream effectors which can lead to cellular transformation [15,16]. Constitutive activation of the Ras-ERK/MAPK signaling pathway often leads to promotion of abnormal cell growth and tumorigen- esis [17]. In mammalian cells, ERK [18], p38 MAP kinase [19], and JNK [20,21] regulate mitosis, movement, metabolism and apoptosis. These MAP kinases are activated by the dual phosphorylations of neighboring threonine and tyrosine residues in response to various extracellular stimuli [22]. Specifically, ERKs are often activated by growth signals [23], whereas p38 and JNK are activated by stress-responsive signal [20]. Activation of ERK leads to phosphorylation and inactivation of FOXO proteins. Since FOXO proteins can be phosphorylated by AKT and ERK, inhibition of these pathways could be considered as a novel strategy for the prevention and/or treatment of pancreatic cancer. In support of this hypothesis, we have recently demonstrated that inhibition of PI3K/AKT and MEK/ERK pathways act synergis- tically to regulate anti-angiogenic effects of EGCG and sulforaph- ane through activation of FOXO transcription factors and downstream target genes [24,25]. Although resveratrol has been shown to inhibit pancreatic cancer cell growth, its effects on the regulation of FOXO transcription factors and downstream targets in pancreatic cancer have never been examined. Furthermore, resveratrol, which simultaneously inhibits the activation of PI3K/AKT and MEK/ ERK pathways (resulting in activation of FOXO), could be an FOXO-Dependent Regulation of Tumorigenesis attractive candidate for the management of pancreatic cancer. The objectives of our study were to examine the molecular mechanisms by which resveratrol inhibits pancreatic cancer growth in vitro and in vivo. Our results demonstrate that resveratrol induced the expression of PTEN, phosphorylation of JNK and p38, and inhibited phosphorylation of PI3K, AKT, ERK, FOXO1 and FOXO3a leading to growth arrest and apoptosis. Furthermore, inhibition of FOXOs by shRNA blocked anti-proliferative and proapoptotic effects of resveratrol. Finally, resveratrol-treated mice showed significant inhibition in tumor growth which was associated with reduced phosphorylation of ERK, PI3K, AKT, FOXO1 and FOXO3a, and induction of apoptosis and FOXO target genes in tumor tissues. Results Inhibition of pancreatic cancer cell viability and colony formation by resveratrol We first examined the effects of resveratrol on cell viability of four pancreatic cancer cell lines by XTT assay. These four pancreatic cancer cell lines (MIA PaCa-2, AsPC-1, PANC-1 and Hs766T) were selected due to their different genetic background and pathological stages. As shown in Fig. 1A, resveratrol inhibited cell viability in a dose-dependent manner. PANC-1 and MIA PaCa-2 cell lines were most sensitive, AsPC-1 cell line was moderately sensitive, and Hs 766T cell line was least sensitive. These data demonstrate the anti-proliferative effects of resveratrol on human pancreatic cancer cells. We next examined the effects of resveratrol on colony formation on four pancreatic cancer cell lines by soft agar assay. Resveratrol inhibited colony formation in a dose-dependent manner (Fig. 1B). Colonies formed by PANC-1 and MIA PaCa-2 cells were most sensitive, AsPC-1 cell line was moderately sensitive, and Hs 766T cell line was least sensitive. These data suggest that resveratrol can be used as a potent chemopreventive/therapeutic agent for pancreatic cancer. Mitochondria play a major role in apoptosis which can be induced by active caspase-3 [26]. We therefore examined whether resveratrol induces apoptosis through activation of caspase-3 in pancreatic cancer cell lines (Fig. 1C). Resveratrol induced caspase- 3 activity in PANC-1, MIA PaCa-2, Hs 766T and AsPC-1 cells. However, the activation of caspase-3 was less in Hs766T cells compared to other pancreatic cancer cell lines. These data suggest that resveratrol induces apoptosis through activation of caspase-3 in pancreatic cancer cells. Regulation of apoptosis and cell cycle proteins in pancreatic cancer cells by resveratrol We next examined the effects of resveratrol on apoptosis (Fig. 2A). Resveratrol induced apoptosis in PANC-1, MIA PaCa-2, HS766T and AsPC-1 cells in a dose-dependent manner. PANC-1 and MIA PaCa-2 cell lines were most sensitive, AsPC-1 cell line was moderately sensitive, and Hs 766T cell line was least sensitive. These data suggest that resveratrol can induce apoptosis in pancreatic cancer cells irrespective of their Kras status, and thus can be used as a cancer preventive agent. PI3K/AKT signaling pathway has been shown to regulate cell cycle progression through regulation of FOXO transcription factors [27]. We therefore examined the effects of FOXO transcription factors on resveratrol-induced cell cycle arrest by inhibiting the FOXO transcription factors by shRNA (Fig. 2B). FOXO1/FOXO3a/FOXO4 shRNA expression vectors have been described elsewhere [28]. Treatment of PANC-1/scrambled cells with resveratrol resulted in cell cycle arrest at G1 stage PLoS ONE | www.plosone.org 2 September 2011 | Volume 6 | Issue 9 | e25166