FOXO-Dependent Regulation of Tumorigenesis Figure 4. Effects of FOXO transcription factors on resveratrol induced apoptosis. (A and B), Phosphorylation deficient mutants of FOXO enhance resveratrol-induced apoptosis in pancreatic cancer cells. PANC-1 and AsPC-1 cells were transiently transfected with empty vector or plasmids encoding FOXO1-TM, FOXO3a-TM, or FOXO4-TM for 24 h. After transfection, culture medium was replaced with complete RPMI medium and treated with resveratrol (10 mM) for 48 h. Apoptosis was measured by TUNEL assay. Data represent the mean 6 S.D. *, #, **, = significantly different from control, P,0.05. (C and D), PANC-1 and MIA PaCa-2 cells were stably transfected with either scrambled or FOXO1, FOXO3a and FOXO4 shRNA plasmids. Transfected cells were treated with resveratrol (10 mM) and apoptosis was measured by TUNEL assay. Data represent the mean 6 S.D. *, # = significantly different from respective controls, P,0.05. doi:10.1371/journal.pone.0025166.g004 FOXO3a-TM, or FOXO4-TM) (Fig. 5C). PANC-1 and AsPC-1 cells were transfected with wild type FOXO promoter linked to a luciferase reporter gene in the presence or absence of plasmids expressing FOXO1-TM, FOXO3a-TM, or FOXO4-TM. After transfection, cells were treated with resveratrol for 24 h, and luciferase activity was measured. Transfection of cells with plasmids expressing FOXO1-TM, FOXO3a-TM, or FOXO4- TM induced FOXO transcriptional activity compared with the empty vector (control). Resveratrol also induced FOXO tran- scriptional activity. Interestingly, resveratrol-induced FOXO transcriptional activity was further enhanced in the presence of FOXO1-TM, FOXO3a-TM, and FOXO4-TM. These data indicate that FOXO transcription factor may play a major role in mediating biological effects of resveratrol in pancreatic cancer cells. Since resveratrol induces apoptosis and FOXO transcriptional activity, we next sought to examine the effects of resveratrol on phosphorylation of FOXO1 and FOXO3a in the cytoplasmic fractions (Fig. 5D, left Panel). Resveratrol inhibited the phosphor- ylation of FOXO1 and FOXO3a in PANC-1 cells. Since resveratrol inhibited the phosphorylation of FOXO1 and FOXO3a and enhanced the nuclear translocation of wild type and mutant FOXO1-TM, we next sought to examine the FOXO- DNA binding activity by gelshift assay (Fig. 5D, right panel). Treatment of PANC-1 cells with resveratrol resulted in enhanced FOXO-DNA binding activity reaching maximum at 24 h. The FOXO-DNA binding in the presence of SP1 (lysate from untreated cells) was similar to untreated control. Overall, these data suggest that resveratrol can inhibit the phosphorylation of FOXO transcription factors, leading to their translocation to nucleus and enhanced FOXO transcriptional activity. Resveratrol inhibits the expression of Sirt1, Sirt2 and Sirt3 in pancreatic cancer cells Since Sirts regulate tumor formation and angiogenesis, we next examined the effects of resveratrol on the expression of Sirt1, Sirt2 and Sirt3 in pancreatic cancer cells by q-RT-PCR. Resveratrol inhibited the expression of Sirt1, Sirt2 and Sirt3 in PANC-1, MIA PaCa-2 and AsPC-1 cells (Fig. 6). These data suggest that anti- proliferative effects of resveratrol may be exerted through Sirts. Resveratrol inhibits the growth of PANC-1 cells orthotopically implanted in Balb C Nude mice by inhibiting FOXO transcription factors We next validated our in vitro data that resveratrol inhibits growth of pancreatic cancer cells by inhibiting ERK and PI3K/ AKT pathways leading to activation of FOXO transcription factors. PANC-1 cells were orthotopically implanted in Balb C nude mice and treated with resveratrol (0–60 mg/kg body weight) through gavage (Monday to Friday, once daily) for 6 weeks. As shown in Fig. 7A, resveratrol inhibited pancreatic tumor growth in Balb C nude mice in a dose-dependent manner. Furthermore, resveratrol has no effect on the body weight of tumor bearing mice, although mice gained weight during the treatment. We next examined the effects of resveratrol on apoptosis and cell proliferation in tumor tissues derived from control and resveratrol treated mice (Fig. 7B). Resveratrol induced apoptosis and inhibited cell proliferation in tumor tissues in a dose- dependent manner. Since resveratrol induced apoptosis and inhibited cell proliferation, we next sought to examine the molecular mechanisms of the effects of resveratrol. Resveratrol induced the expression of Bim, p27/KIP1 and p21/CIP1, cleaved caspase-3, and inhibited the expression of PCNA (Fig. 7C). Since ERK and AKT phosphorylates FOXO, we next sought to examine the effects of resveratrol on the dephosphorylation/ activation FOXOs. 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. As shown in Fig. 7D, resveratrol inhibited the phosphorylation of ERK, PI3K (Tyr458), p-AKT (Ser473), p- FOXO (Ser256) and p-FOXO3a (Ser253). It has no significant effect on the expression of total proteins. These data confirm our PLoS ONE | www.plosone.org 6 September 2011 | Volume 6 | Issue 9 | e25166