Abstract
BackgroundThe 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 FindingsPancreatic 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.ConclusionsThese 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.
Highlights
Cancer of the pancreas is the fourth leading cause of cancerrelated mortality in the United States with a 5-year survival less than 5% [1]
These data suggest that inhibition of ERK and AKT pathways act together to activate Forkhead box O (FOXO) transcription factors which are involved in resveratrol-mediated pancreatic tumor growth suppression
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 synergistically to regulate anti-angiogenic effects of Epigallocatechin galate (EGCG) and sulforaphane through activation of FOXO transcription factors and downstream target genes [24,25]
Summary
Cancer of the pancreas is the fourth leading cause of cancerrelated mortality in the United States with a 5-year survival less than 5% [1]. 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. 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 exhibit high mutation frequencies in pancreatic tumors. The objectives of this study were to examine whether FOXOs regulate antitumor activities of resveratrol in pancreatic cancer cells in vitro and in vivo
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