FOXO-Dependent Regulation of Tumorigenesis Figure 7. Resveratrol inhibits the growth of PANC-1 tumors orthotopically inplanted in Balb C Nude mice by inhibiting FOXO transcription factors. (A) Upper Panel, PANC-1 cells were orthotopically implanted into the pancreas of Balb C nude mice. Tumor bearing mice were treated with resveratrol (0–60 mg/kg body weight) through gavage (Monday to Friday, once daily) for 6 weeks. At the end of the experiment, pancreatic tumor weights were recorded. Data represent the mean 6 S.D. *, #, % = significantly different from control, P,0.05. Lower Panel, Body weight of tumor-bearing mice during the experiment. Data represent the mean 6 S.D. (B), Effects of resveratrol on apoptosis and cell proliferation in tumor tissues. Immunohistochemistry was performed in tumor tissues isolated from control and resveratrol treated mice. Apoptosis was measured by TUNEL assay and cell proliferation was measured by Ki67 staining. Data represent the mean 6 S.D. *, #, % = significantly different from control, P,0.05. (C), Effects of resveratrol on markers of apoptosis and cell cycle in tumor tissues. Western blot analyses were performed to examine the expression of Bim, caspase-3, PCNA, cell cycle inhibitors p27 and p21. The b-actin was used as a loading control. (D), Effects of resveratrol on ERK, PI3K/AKT and FOXO proteins isolated from tumor tissues. Western blot analyses were performed to examine the expression of p-ERK, ERK, p-PI3K (Tyr458), PI3K, p-AKT (Ser473), AKT, p-FOXO (Ser256), FOXO, p-FOXO3a (Ser253), and FOXO3a. The b-actin was used as a loading control. doi:10.1371/journal.pone.0025166.g007 in pancreatic cancer cells, thus suggesting a role of SIRT1 as a tumor suppressor in cancer cells. However, further studies are needed to examine the conse- quences of acetylation/deacetylation of FOXO transcription factors on tumorigenesis and angiogenesis. In summary, we have demonstrated that resveratrol induces cell cycle arrest and apoptosis through regulation of FOXO transcription factors. Pharmacological and genetic inhibitions of PI3K/AKT and MEK/ERK pathways can have synergistic effects on the activation of FOXO transcription factors through dephosphorylation and nuclear retention, resulting in regulation of FOXO-target genes. Our study provides important information regarding the mechanisms by which resveratrol regulates cell cycle, apoptosis and tumor growth through activation of FOXO transcription factors. Thus, our studies 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. Methods Ethics Statement All experiments involving animals were approved by the Institutional Animal Care and Use Committee (IACUC) of the University of Texas Health Science Center at Tyler, protocol # 373. Reagents Antibodies against PTEN, phospho-AKT, AKT, ras, phospho- ERK, ERK, phospho-p38, p38, p21/CIP1, p27/KIP1, cyclin D1, p-FOXO1, FOXO, pFOXO3a, FOXO3a, Bim, PCNA, caspase- 3, and b-actin were purchased from Cell Signaling Technology, Inc. (Danvers, MA). Enhanced chemiluminescence (ECL) Western blot detection reagents were from Amersham Life Sciences Inc. (Arlington Heights, IL). Terminal Deoxynucleotidyl Transferase Biotin-dUTP Nick End Labeling (TUNEL) assay kit was purchased from EMD Biosciences/Calbiochem (San Diego, CA). Resveratrol was purchased from LKT Laboratories, Inc. (St. Paul, MN). Kits for Terminal Deoxynucleotidyl Transferase Biotin- dUTP Nick End Labeling (TUNEL) and caspase-3 assays were purchased from EMD Biosciences/Calbiochem (San Diego, CA). Cell Culture PANC-1, MIA PaCa-2, AsPC-1 and Hs 766T cells were obtained from the American Type Culture Collection (Manassas, VA) and cultured in RPMI 1640 supplemented with 10% fetal bovine serum (FBS) and 1% antibiotic-antimycotic (Invitrogen) at 37uC in a humidified atmosphere of 95% air and 5% CO2. Transient Transfection Cells were plated in 60-mm dishes in RPMI 1640 containing 10% FBS and 1% penicillin-streptomycin mixture at a density of 16106 cells/dish. The next day transfections were performed using LipofectAMINE (Invitrogen Life Technologies, Carlsbad, CA). Cells were transfected with expression constructs encoding wild type PTEN (pSG5L-HA-PTENwt), mutant PTEN (pSG5L- HA-PTEN-G 129E and pSGL5-HA-PTEN-G 129R), wild type- AKT (pUSE-WT-AKT), constitutively active-AKT (pUSE-CA- AKT), dominant negative-AKT (pUSE-DN-AKT), or the corre- sponding empty vectors (pSG5L or pUSE ) in the presence of an PLoS ONE | www.plosone.org 9 September 2011 | Volume 6 | Issue 9 | e25166