Resveratrol Inhibits Growth of Orthotopic Pancreatic Tumors through Activation of FOXO Transcription Factors Sanjit K. Roy1, Qinghe Chen1¤, Junsheng Fu 2 , Sharmila Shankar2, Rakesh K. Srivastava1* 1 Department of Pharmacology, Toxicology and Therapeutics, and Medicine, The University of Kansas Cancer Center, The University of Kansas Medical Center, Kansas City, Kansas, United States of America, 2 Department of Pathology and Laboratory Medicine, The University of Kansas Cancer Center, The University of Kansas Medical Center, Kansas City, Kansas, United States of America Abstract Background: The forkhead transcription factors of the O class (FOXO) play a direct role in cellular proliferation, oxidative stress response, and tumorigenesis. The objectives of this study were to examine whether FOXOs regulate antitumor activities of resveratrol in pancreatic cancer cells in vitro and in vivo. Methodology/Principal Findings: Pancreatic cancer cell lines were treated with resveratrol. Cell viability, colony formation, apoptosis and cell cycle were measured by XTT, soft agar, TUNEL and flow cytometry assays, respectively. FOXO nuclear translocation, DNA binding and transcriptional activities were measured by fluorescence technique, gelshift and luciferase assay, respectively. Mice were orthotopically implanted with PANC1 cells and orally gavaged with resveratrol. The components of PI3K and ERK pathways, FOXOs and their target gene expressions were measured by the Western blot analysis. Resveratrol inhibited cell viability and colony formations, and induced apoptosis through caspase-3 activation in four pancreatic cancer cell lines (PANC-1, MIA PaCa-2, Hs766T, and AsPC-1). Resveratrol induced cell cycle arrest by up- regulating the expression of p21/CIP1, p27/KIP1 and inhibiting the expression of cyclin D1. Resveratrol induced apoptosis by up-regulating Bim and activating caspase-3. Resveratrol inhibited phosphorylation of FOXOs, and enhanced their nuclear translocation, FOXO-DNA binding and transcriptional activities. The inhibition of PI3K/AKT and MEK/ERK pathways induced FOXO transcriptional activity and apoptosis. Furthermore, deletion of FOXO genes abrogated resveratrol-induced cell cycle arrest and apoptosis. 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. Conclusions: These data suggest that inhibition of ERK and AKT pathways act together to activate FOXO transcription factors which are involved in resveratrol-mediated pancreatic tumor growth suppression. Citation: Roy SK, Chen Q, Fu J, Shankar S, Srivastava RK (2011) Resveratrol Inhibits Growth of Orthotopic Pancreatic Tumors through Activation of FOXO Transcription Factors. PLoS ONE 6(9): e25166. doi:10.1371/journal.pone.0025166 Editor: Dhyan Chandra, Roswell Park Cancer Institute, United States of America Received August 1, 2011; Accepted August 26, 2011; Published September 27, 2011 Copyright: 2011 Roy et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: The authors have no funding or support to declare. Competing Interests: The authors have declared that no competing interests exist. * E-mail: rsrivastava@kumc.edu ¤ Current address: Virginia Bioinformatics Institute, Virginia Polytechnic Institute and State University, Blacksburg, Virginia, United States of America Introduction Cancer of the pancreas is the fourth leading cause of cancer- related mortality in the United States with a 5-year survival less than 5% [1]. Pancreatic ductal adenocarcinoma is a highly aggressive disease that invariably evades early diagnosis [2]. The mean survival time for patients with metastatic disease is only 3–6 months, and only 20–30% of pancreatic cancer cases are alive after 12 months. Several factors are associated with increased risk for pancreatic cancer and these include chronic pancreatitis, smoking, diabetes, prior gastric surgery, exposure to certain classes of organic solvents, radiation, and specific gene polymorphisms [3,4]. Heritable as well as several acquired gene mutations have been identified in pancreatic tumors [5]. The K-Ras oncogene is primarily mutated in codon 12 in .90% of pancreatic tumors and the mutation results in a constitutively active form of ras that can lead to increased cell proliferation. Mutations in the tumor suppressor gene p53, the cyclin-dependent kinase inhibitor p16, and SMAD4, a downstream target of TGFb also exhibit high mutation frequencies in pancreatic tumors. Since pancreatic cancer is frequently detected at an advanced stage, it has limited treatment options and insignificant improve- ments in tumor regression and overall survival [6]. 5-Fluorouracil alone or in combination with other drugs has been extensively used for treatment of advanced pancreatic cancer, and gemcita- bine, a deoxycytidine analog (or antimetabolite), has partially replaced 5-fluorouracil as a treatment for pancreatic cancer. Therefore, effective strategies are urgently needed to prevent pancreatic cancer. Resveratrol is a polyphenolic phytochemical that exhibits a broad spectrum of anticancer activities against multiple tumor types, including pancreatic cancer [7]. In vitro, resveratrol inhibited the proliferation of human pancreatic cancer PLoS ONE | www.plosone.org 1 September 2011 | Volume 6 | Issue 9 | e25166