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Inhibition of PI3K/AKT and MAPK/ERK pathways causes activation of FOXO transcription factor, leading to cell cycle arrest and apoptosis in pancreatic cancer

Sanjit K Roy, Rakesh K Srivastava, Sharmila Shankar

Journal of Molecular Signaling · 2010 · CC BY

doi:10.1186/1750-2187-5-10

Abstract

Background. Mammalian forkhead members of the class O (FOXO) transcription factors, including FOXO1, FOXO3a, and FOXO4, are implicated in the regulation of several biological processes, including the stress resistance, metabolism, cell cycle, apoptosis and DNA repair. The objectives of this study were to examine the molecular mechanisms by which FOXO transcription factors induced cell cycle arrest and apoptosis and enhanced anti-proliferative effects of sulforaphane (SFN, an active compound in cruciferous vegetables) in pancreatic cancer cells.

Results. Our data demonstrated that SFN inhibited cell proliferation and colony formation, and induced apoptosis through caspase-3 activation in pancreatic cancer cells. The inhibition of PI3K/AKT and MEK/ERK pathways activated FOXO transcription factors. SFN inhibited phosphorylation of AKT and ERK, and activated FOXO transcription factors, leading to cell cycle arrest and apoptosis. Phosphorylation deficient mutants of FOXO proteins enhanced FOXO transcriptional activity, and further enhanced SFN-induced FOXO activity and apoptosis. SFN induced the expression of p21/CIP1 and p27/KIP1, and inhibited the expression of cyclin D1.

Conclusion. These data suggest that inhibition of PI3K/AKT and ERK pathways acts together to activate FOXO transcription factor and enhances SFN-induced FOXO transcriptional activity, leading to cell cycle arrest and apoptosis.

Full text

From PubMed Central, U.S. National Library of Medicine, as current on Oct 17, 2026. Paragraph numbers are ours, so an answer can point at one.

Background

Cancer of the pancreas is the fourth leading cause of cancer death in the United States. This year approximately 32,000 Americans will die from cancer of the pancreas. With an overall 5-year survival rate of 3%, pancreatic cancer has one of the poorest prognoses among all cancers. Only 20% of pancreatic cancer patients are eligible for surgical resection, which currently remains the only potentially curative therapy. Unfortunately, many cancers of the pancreas are not resectable at the time of diagnosis. There are limited treatment options available for this disease because chemo- and radio-therapies are largely ineffective, and metastatic disease frequently redevelops even after surgery. Therefore, developing effective strategies to prevent pancreatic neoplasms are of paramount importance.

Sulforaphane (SFN), a constituent of cruciferous vegetables, is a naturally occurring isothiocyanate with promising chemopreventive activity. Epidemiological studies have shown that people who eat cruciferous vegetables have reduced incidence of breast and prostate cancer. SFN possesses anti-oxidant, anti-proliferative and anti-carcinogenic properties. SFN is effective in preventing chemically induced breast, stomach and colon cancers in rats. We and others have shown that SFN inhibited the growth of prostate, breast, oral and squamous carcinoma xenografts. SFN enhanced radiosensitivity of tumor cells in vitro and in vivo. Furthermore, a pharmacokinetic study has demonstrated that it is rapidly absorbed and 82% bioavailable. SFN induces a phase 2 enzyme, thereby neutralizing carcinogens before they can damage DNA. SFN inhibits benzo[a]pyrene-DNA and 1,6-dinitropyrene-DNA adducts formation, and downregulates PI3K/AKT and NFκB pathways. We have recently demonstrated that SFN induces death receptors (DR4 and DR5) and proapoptotic members of Bcl-2 family, inhibits antiapoptotic Bcl-2 proteins, activates caspase(s), and enhances apoptosis-inducing potential of TRAIL in vitro. In vivo, SFN inhibits growth of PC-3 cells orthotopically implanted in nude mice by inducing apoptosis and inhibiting tumor cell proliferation, metastasis and angiogenesis. These studies strongly suggest that SFN can be developed as a cancer preventive agent.

PTEN (phosphatase and tensin homolog deleted on chromosome 10, also called MMAC1 or TEP1) is a tumor suppressor gene, which is frequently deleted or mutated in a wide range of human cancers, including glioblastoma, melanoma, and prostate, breast, and endometrial cancers. While point mutations in PTEN rarely occur in pancreatic cancer, functional inactivation of PTEN through promoter methylation, loss of protein expression, reduction of mRNA levels, or loss of heterozygocity (LOH) of linked markers occur with high frequency. Phosphatidylinositol 3,4,5-trisphosphate (PIP3) is a substrate of PTEN. AKT is a serine-threonine protein kinase regulated by PIP3 that is implicated in survival signaling in a wide a variety of cells, including fibroblastic, epithelial, and neuronal cells. PTEN increases sensitivity to cell death in response to several apoptotic stimuli by negatively regulating the PI3K/AKT pathway. In addition to its role in regulating the PI3K/AKT cell survival pathway, PTEN also inhibits growth factor-induced Shc phosphorylation and suppresses the MAP kinase signaling pathway, suggesting that PTEN has roles in independent of PI3K/AKT signaling pathway. Hyperactivation of AKT is associated with resistance to apoptosis, increased cell growth, cell proliferation, metastasis, angiogenesis, and cellular energy metabolism. Overexpression of AKT has been reported in a variety of human cancers, including pancreatic cancer, and cells expressing elevated levels of AKT are less sensitive to apoptosis stimuli. Antagonizing PI3K activity negatively regulates AKT activity. Once activated, however, AKT exerts antiapoptotic effects through phosphorylation of substrates such as Bad and caspase-9 that directly regulate the apoptotic machinery, or human telomerase reverse transcriptase subunit, forkhead transcription family members and IB kinases that indirectly inhibit apoptosis. Studies in pancreatic cancer cell lines have demonstrated that PI3K is required for growth and survival of tumor cells. Furthermore, amplification or activation of AKT2 occurs in up to 60% of pancreatic cancer, supporting the participation of an activated PI3K-AKT axis in this disease.

FOXO subfamily of forkhead transcription factors include FOXO1a/FKHR, FOXO3a/FKHRL1, and FOXO4/AFX. The PI3K pathway, via activation of its downstream kinase AKT, phosphorylates each of the FOXO proteins. These phosphorylations result in impairment of DNA binding ability and increased binding affinity for the 14-3-3 protein. Newly formed 14-3-3-FOXO complexes are then exported from the nucleus, thereby inhibiting FOXO-dependent transcription. Inhibition of the PI3K pathway leads to dephosphorylation and nuclear translocation of active FKHRL1, FKHR, and AFX; which induce cells cycle arrest and apoptosis. Conversely, loss of PTEN activity results in increased AKT activity leading to inhibition of FOXO protein activity through phosphorylation and cytoplasmic sequestration. In addition, the data demonstrate that FOXO transcriptional activity controls cellular proliferation and apoptosis downstream of PTEN. FOXO regulates cell cycle and apoptotic genes such as cyclin-dependent kinase inhibitor (CKI) p27KIP1, Bim, Fas ligand, and Bcl-6. Consequently, activation of the PI3K pathway serves to repress FOXO-mediated growth arrest and apoptosis. However, regulation of FOXO target genes is multifactorial, and therefore other transcription factors and post-translation regulatory events will influence the final level of protein expression. Interestingly, overexpression of AKT, and inactivation and loss of PTEN are frequently observed in pancreatic cancer, indicating a potential role for FOXOs in modulating both cell cycle and apoptosis during tumorigenesis and treatment. Together, these results indicate that FOXO proteins are important downstream effectors of PTEN tumor suppressive activity; however, their molecular targets and mechanisms of action in pancreatic cancer are not well understood.

The Ras proteins are small (21 kDa) GTP-binding, membrane-associated proteins. The Ras proteins transduce signals from ligand-activated tyrosine kinase receptors to downstream effectors. Activating mutations can impair GTP hydrolysis and lead to constitutively activated Ras that impacts the cellular phenotype. Oncogenic Ras can lead to cellular transformation, presumably by perturbing its signal transduction pathways. Ras regulates multiple signaling pathways. Three major groups of MAP kinases are found in mammalian cells: extracellular signal-regulated protein kinase (ERK), p38 MAP kinase, and c-Jun N-terminal kinase (JNK). MAP kinases regulate many cellular activities, which range from gene expression to 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. Specifically, p38 and JNK have been implicated in stress-responsive signaling leading to the initiation of adaptive events such as gene expression, differentiation, metabolism, and apoptosis. ERKs are often activated by growth signals, such as epidermal growth factor (EGF) or platelet-derived growth factor. We have recently demonstrated that inhibition of PI3K/AKT and MEK/ERK pathways act synergistically to regulate antiangiogenic effects of EGCG and SFN through activation of FOXO transcription factors.

Furthermore, FOXO transcription factors play a crucial role in the regulation of tissue homeostasis in organs such as the pancreas, and complex diseases such as diabetes and cancer. Unfortunately, the intracellular mechanisms by which SFN inhibits growth and induces apoptosis in pancreatic cancer cells through regulation of FOXO transcription factors have never been examined. The objectives of our study were to examine the molecular mechanisms by which FOXO transcription factors induce cell cycle arrest and apoptosis and enhances the anti-proliferative effects of SFN in pancreatic cancer cells. Our results demonstrate that inhibition of PI3K/AKT and ERK pathways activates FOXO transcription factors. SFN inhibited phosphorylation of AKT and ERK, and dephosphorylated FOXO transcription factors, leading to cell cycle arrest and apoptosis. Phosphorylation deficient mutants of FOXO proteins enhanced FOXO transcriptional activity, and further enhanced SFN-induced FOXO activity.

Sulforaphane (SFN) inhibits cell growth in human pancreatic cancer cells

We first examined the effects of SFN on cell proliferation in four pancreatic cancer cell lines by XTT assay. We have selected four pancreatic cancer cell lines (MIA PaCa-2, AsPC-1, PANC-1 and Hs766T) because they have been derived from different pathological stages and may thus respond differently to SFN. MIA PaCa-2 harbors a point mutation on Kras gene resulting in amino acid sunbstitution from the wild-type glycine to a valine at codon 12. AsPC-1 and PANC-1 harbor a point mutation on Kras gene resulting in amino acid substitution from glycine to aspartate. Hs766T cell line does not possess a point mutation in codon 12 of the Kras gene. SFN inhibited cell viability in a dose dependent manner (Fig. 1). 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 SFN can be a viable agent for inhibiting pancreatic cancer cell proliferation.

Sulforaphane inhibits colony formation in human pancreatic cancer cells

We next examined the effects of SFN on colony formation (a characteristic of cancer) on four pancreatic cancer cell lines by soft agar assay. SFN inhibited colony formation in a dose dependent manner (Fig. 2). 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 SFN can be used as a potent chemopreventive agent for pancreatic cancer.

Sulforaphane induces caspase-3 activation in human pancreatic cancer cell

Most chemopreventive agents induce apoptosis through mitochondrial pathway, which activates caspase-3. We therefore examined whether SFN-induced apoptosis through caspase-3 activation in pancreatic cancer cell lines (Fig. 3). SFN induced caspase-3 activity in PANC-1, MIA PaCa-2, Hs 766T and AsPC-1 cells. However, a relatively high dose of SFN was required to activate caspase-3 in Hs 766T cells compared to other pancreatic cancer cell lines. These data suggest that SFN induced apoptosis through caspase-3 activation and may engage the mitochondria.

Regulation and function of PI3K/AKT and MAP kinase pathways by sulforaphane

In most cancer cells, AKT is constitutively active and enhances cell proliferation. In order to understand a relationship between PTEN and AKT in SFN-induced apoptosis, we measured the expression of PTEN and phosphorylation status of AKT in cells treated with SFN (Fig. 4A). SFN induces PTEN expression and inhibits AKT phosphorylation in pancreatic cancer PANC-1 cells. By comparison, SFN has no effect on total AKT expression. These data suggest that SFN inhibits cell proliferation by regulating PI3K/AKT pathway.

Ras/Raf/MAP kinase pathway regulates many cellular activities, which range from gene expression to mitosis, movement, metabolism, and apoptosis. We therefore examined the effects of SFN on the expression of Ras, and activation of ERK, JNK and p38 MAP kinases. SFN inhibited Ras expression in PANC-1 cells (Fig. 4A). Treatment of PANC-1 cells with SFN caused a decrease in ERK phosphorylation, and an increase in JNK phosphorylation. SFN has no significant effect on p38 MAP kinase activity in PANC-1 cells. These data suggest that SFN inhibits growth and induces apoptosis through regulation of Ras/Raf/MAP kinase pathway.

We next examined whether SFN induces apoptosis through PI3K/AKT pathway (Fig. 4B). Pancreatic cancer cells were transfected with empty vector, wild type PTEN, dominant negative AKT (DN-AKT), and apoptosis was measured. Overexpression of wild type PTEN or DN-AKT induced apoptosis in AsPC-1 and PANC-1 cells. Treatment of transfected cells with SFN further enhanced apoptosis. These data suggest that inhibition of PI3K/AKT pathway enhances SFN-induced apoptosis in pancreatic cancer cells.

We next examined whether inhibition of MEK/ERK pathway enhances SFN-induced apoptosis in pancreatic cancer cells. MEK1/2 inhibitor (PD98059) induced apoptosis in PANC-1 and AsPC-1 cells (Fig. 4C). PD98059 enhanced SFN-induced apoptosis. Overall, these data suggest that inhibition of PI3K/AKT and MEK/ERK pathways enhanced SFN-induced apoptosis.

Sulforaphane induces p21/WAF1/CIP1, and p27/KIP1 and inhibits cyclin D1

PI3K/AKT signaling pathway may be involved in the control of the cell cycle progression most likely through mechanisms involving the activation of FOXO transcription factors. We next examined the effects of SFN on cell cycle regulatory genes. SFN induced the expression cell cycle inhibitors p21/WAF1/CIP1 and p27/KIP1, and inhibited the expression of cyclin D1 in PANC-1 cells (Fig. 5). These data suggest that SFN causes growth arrest by regulating expression of cell cycle genes.

Overexpression of FOXO transcription factors inhibits cell viability and enhances FOXO transcriptional activity in pancreatic cancer cells

In order to examine whether FOXO transcription factors affect the ability of SFN to inhibit cell viability, pancreatic cancer cells were transfected with FOXO1, FOXO3a or FOXO4 (Fig. 6A and 6B). FOXO expression plasmids and FOXO-luciferase construct (pGL3-6X DBE) have previously been described. Overexpression of FOXO1, FOXO3a, and FOXO4 inhibited cell viability in PANC-1 and AsPC-1 cells. The inhibitory effects of SFN on cell viability were further enhanced when pancreatic cancer cells were transfected with FOXO1, FOXO3a, and FOXO4. These data suggest that FOXO transcription factors can enhance the antiproliferative effects of SFN.

We next examined whether SFN induces transcriptional activation of FOXO in the presence or absence phosphorylation deficient triple mutants of FOXO proteins (FOXO1-TM, FOXO3a-TM, or FOXO4-TM). 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 (Fig. 6C and 6D). After transfection, cells were treated with SFN 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). SFN-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 SFN in pancreatic cancer cells.

Inhibition of PI3K/AKT and MEK/ERK pathways synergistically/additively induces FOXO transcriptional activity and apoptosis in the presence or absence of sulforaphane

Since inhibition of PI3K/AKT and MEK/ERK pathways induce apoptosis in pancreatic cancer cells, we sought to examine whether these pathways act together to regulate SFN-induced apoptosis. AKT inhibitor (AKT Inh-IV) and MEK1/2 inhibitor (PD98059) synergistically/additively induced apoptosis in PANC-1 and AsPC-1 cells (Fig. 7A and 7B). AKT inhibitor and PD98059 alone enhanced SFN-induced apoptosis. Interestingly, the combination of AKT inhibitor and PD98059 with SFN induced more apoptosis than AKT inhibitor plus SFN or PD98059 plus SFN. These data suggest that inhibition of PI3K/AKT and MEK/ERK pathways act synergistically/additively to regulate apoptosis in the absence or presence of SFN.

Since inhibition of PI3K/AKT and MEK/ERK pathways synergistically/additively induces apoptosis in pancreatic cancer cells, we sought to examine whether inhibition of these two pathways act together to regulate FOXO activity. AKT inhibitor (AKT Inh-IV) and MEK1/2 inhibitor (PD98059) synergistically induced FOXO transcriptional activity in AsPC-1 and PANC-1 cells (Fig. 7C and 7D). AKT inhibitor or PD98059 enhanced SFN-induced FOXO transcriptional activity. Interestingly, the combination of AKT Inh-IV and PD98059 with SFN induced greater FOXO transcriptional activity than AKT Inh-IV plus SFN or PD98059 plus SFN. These data suggest that inhibition of PI3K/AKT and MEK/ERK pathways acts synergistically/additively to regulate FOXO transcriptional activity in the absence or presence of SFN.

Discussion

Our study demonstrates, for the first time, that cancer preventive effects of SFN are regulated through activation of FOXO transcription factors. Specifically, we have demonstrated that (i) SFN induces apoptosis through caspase-3 activation, and causes growth arrest through induction of p21 and p27 and inhibition of cyclin D1; (ii) SFN induces apoptosis through inhibition of both PI3K/AKT and MEK/ERK pathways, and activation of FOXO transcription factors; (iii) inhibition of PI3K/AKT and MEK/ERK pathways acts together to enhance the activation of FOXO transcription factors; and (iv) phosphorylation deficient mutants of FOXO proteins further enhance SFN-induced FOXO activity and apoptosis. Our data are in agreement with others who demonstrated the anticancer activity of SFN in pancreatic cancer.

FOXO transcription factors play a crucial role in the regulation of tissue homeostasis in organs such as the pancreas and the ovaries and complex diseases such as diabetes and cancer. FOXO transcription factors are emerging as critical transcriptional integrators among pathways regulating differentiation, proliferation, survival, and angiogenesis. FOXO transcription factors regulate angiogenesis and postnatal neovascularization by regulation angiopoietin 2 (Ang2) and eNOS. Gene expression profiling showed that FOXO1 and FOXO3a specifically regulate a nonredundant but overlapping set of angiogenesis- and vascular remodeling-related genes. The FOXO1-deficient mice died around embryonic day 11 because of defects in the branchial arches and remarkably impaired vascular development of embryos and yolk sacs. We have recently demonstrated that inhibition of the MEK/ERK and PI3K/AKT pathways synergistically induced FOXO transcriptional activity and inhibited angiogenesis (cell migration and capillary tube formation); these events were further enhanced in the presence of SFN. Phosphorylation deficient mutants of FOXO enhanced antiangiogenic effects of SFN by activating the FOXO transcription factor. These studies suggest that activation of FOXO transcription factor by SFN could be an important physiological process to inhibit angiogenesis which may ultimately control tumor growth.

Activation of Kras has been shown to activate both PI3K/AKT and MAPK pathways. Oxidative stress and activation of the JNK pathway induce the nucleocytoplasmic translocation of the pancreatic transcription factor Pdx-1, which leads to pancreatic β-cell dysfunction. Furthermore, FOXO1/FKHR plays a role as a mediator between the JNK pathway and Pdx-1. Under oxidative stress conditions, FOXO1 changed its intracellular localization from the cytoplasm to the nucleus in the pancreatic β-cell line HIT-T15. The overexpression of JNK also induced the nuclear localization of FOXO1, but in contrast, suppression of JNK reduced the oxidative stress-induced nuclear localization of FOXO1, suggesting the involvement of the JNK pathway in FOXO1 translocation. In addition, oxidative stress or activation of the JNK pathway decreased the activity of AKT in HIT cells, leading to the decreased phosphorylation of FOXO1 following nuclear localization. Furthermore, adenovirus-mediated FOXO1 overexpression reduced the nuclear expression of Pdx-1, whereas repression of FOXO1 by FOXO1-specific small interfering RNA retained the nuclear expression of Pdx-1 under oxidative stress conditions. Activation of ERK has been shown to phosphorylate FOXO proteins, resulting in nuclear exclusion and transcriptional repression. In addition to ERK, direct phosphorylation of FOXO by AKT results in cytoplasmic retention and inactivation, inhibiting the expression of FOXO-regulated genes, which control the cell cycle, cell death, cell metabolism and oxidative stress. Taken together, these studies demonstrate that dephosphorylation and activation of FOXO by inhibition of PI3K/AKT and MEK/ERK pathways has significant implication for pancreatic cancer treatment and prevention, where Kras is activated in about 90% patients.

In addition to phosphorylation, the acetylation/deacetylation of FOXO can be regulated by p300, Cbp (CREB-binding protein) and Pcaf (p300/CBP-associated factors) in response to oxidative stress or DNA binding, followed by deacetylation by class I and II histone deacetylases, including Sirt1, the NAD+-dependent deacetylase encoded by the ortholog of yeast longevity gene Sir2. Therefore, further studies are needed to examine the consequences of acetylation/deacetylation of FOXO transcription factors on anti-proliferative and anti-angiogenic effects of SFN.

In conclusion, we have demonstrated that SFN 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. Thus, SFN appears to be as an attractive agent for pancreatic cancer prevention and treatment.

Reagents

Antibodies against PTEN, phospho-AKT, AKT, phospho-ERK, ERK, phospho-p38, p38, p21/CIP1, p27/KIP1, cyclin D1, and β-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). Sulforaphane 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 37°C in a humidified atmosphere of 95% air and 5% CO2.

Western Blot Analysis

Western blots were performed as we described earlier. In brief, cells were lysed in RIPA buffer containing 1 × protease inhibitor cocktail, and protein concentrations were determined using the Bradford assay (Bio-Rad, Philadelphia, PA). Proteins were separated by 12.5% SDS/PAGE and transferred to membranes (Millipore, Bedford, MA) in a Tris (20 mM), glycine (150 mM) and methanol (20%) buffer at 55 V for 4 h at 4°C. After blocking in 5% nonfat dry milk in TBS, the membranes were incubated with primary antibodies at 1:1,000 dilution in TBS overnight at 4°C, washed three times with TBS-Tween 20, and then incubated with secondary antibodies conjugated with horseradish peroxidase at 1:5,000 dilution in TBS for 1 hour at room temperature. Membranes were washed again in TBS-Tween 20 for three times at room temperature. Protein bands were visualized on X-ray film using an enhanced chemiluminescence detection system.

Caspase-3 Assay

Cells (3 × 104 per well) were seeded in a 96-well plate with 200 μl culture medium. Approximately 16 h later, cells were treated with various doses of SFN to induce apoptosis. Casapse-3 activity was measured by a fluorometer as per manufacturer's instructions (EMD Biosciences).

Statistical Analysis

The mean and SD were calculated for each experimental group. Differences between groups were analyzed by one or two way ANOVA, followed by Bonferoni's multiple comparison tests using PRISM statistical analysis software (GrafPad Software, Inc., San Diego, CA). Significant differences among groups were calculated at P < 0.05.

List of abbreviations used

ANOVA: Analysis of Variance; PTEN: Phosphatase and Tensin Homolog Deleted on Chromosome 10; RIPA: Radio-Immunoprecipitation Assay; SDS-PAGE: Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis; SFN: Sulforaphane; TBS: Tris Buffer Saline.

Competing interests

The authors declare that they have no competing interests.

Authors' contributions

SKR and SS performed the experiments. SS and RKS designed and wrote the manuscript. All the authors have read and approved the final manuscript.

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    Steck PA Pershouse MA Jasser SA Yung WK Lin H Ligon AH Langford LA Baumgard ML Hattier T Davis T Identification of a candidate tumour suppressor gene, MMAC1, at chromosome 10q23.3 that is mutated in multiple advanced cancersNat Genet1997 15356362

    Not in our library10.1038/ng0497-356

  30. 30.

    Li DM Sun H TEP1, encoded by a candidate tumor suppressor locus, is a novel protein tyrosine phosphatase regulated by transforming growth factor betaCancer Res1997 5721242129

    Not in our library

  31. 31.

    Wang SI Puc J Li J Bruce JN Cairns P Sidransky D Parsons R Somatic mutations of PTEN in glioblastoma multiformeCancer Res1997 5741834186

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  32. 32.

    Guldberg P thor Straten P Birck A Ahrenkiel V Kirkin AF Zeuthen J Disruption of the MMAC1/PTEN gene by deletion or mutation is a frequent event in malignant melanomaCancer Res1997 5736603663

    Not in our library

  33. 33.

    Cairns P Okami K Halachmi S Halachmi N Esteller M Herman JG Jen J Isaacs WB Bova GS Sidransky D Frequent inactivation of PTEN/MMAC1 in primary prostate cancerCancer Res1997 5749975000

    Not in our library

  34. 34.

    Rhei E Kang L Bogomolniy F Federici MG Borgen PI Boyd J Mutation analysis of the putative tumor suppressor gene PTEN/MMAC1 in primary breast carcinomasCancer Res1997 5736573659

    Not in our library

  35. 35.

    Tashiro H Blazes MS Wu R Cho KR Bose S Wang SI Li J Parsons R Ellenson LH Mutations in PTEN are frequent in endometrial carcinoma but rare in other common gynecological malignanciesCancer Res1997 5739353940

    Not in our library

  36. 36.

    Sakurada A Suzuki A Sato M Yamakawa H Orikasa K Uyeno S Ono T Ohuchi N Fujimura S Horii A Infrequent genetic alterations of the PTEN/MMAC1 gene in Japanese patients with primary cancers of the breast, lung, pancreas, kidney, and ovaryJpn J Cancer Res1997 8810251028

    Not in our library10.1111/j.1349-7006.1997.tb00324.x

  37. 37.

    Okami K Wu L Riggins G Cairns P Goggins M Evron E Halachmi N Ahrendt SA Reed AL Hilgers W Analysis of PTEN/MMAC1 alterations in aerodigestive tract tumorsCancer Res1998 58509511

    Not in our library

  38. 38.

    Asano T Yao Y Zhu J Li D Abbruzzese JL Reddy SA The PI 3-kinase/Akt signaling pathway is activated due to aberrant Pten expression and targets transcription factors NF-kappaB and c-Myc in pancreatic cancer cellsOncogene2004 2385718580

    Not in our library10.1038/sj.onc.1207902

  39. 39.

    Altomare DA Tanno S De Rienzo A Klein-Szanto AJ Tanno S Skele KL Hoffman JP Testa JR Frequent activation of AKT2 kinase in human pancreatic carcinomasJ Cell Biochem2003 88470476

    Not in our library10.1002/jcb.10287

  40. 40.

    Ebert MP Fei G Schandl L Mawrin C Dietzmann K Herrera P Friess H Gress TM Malfertheiner P Reduced PTEN expression in the pancreas overexpressing transforming growth factor-beta 1Br J Cancer2002 86257262

    Not in our library10.1038/sj.bjc.6600031

  41. 41.

    Hahn SA Seymour AB Hoque AT Schutte M da Costa LT Redston MS Caldas C Weinstein CL Fischer A Yeo CJ Allelotype of pancreatic adenocarcinoma using xenograft enrichmentCancer Res1995 5546704675

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  42. 42.

    Maehama T Dixon JE The tumor suppressor, PTEN/MMAC1, dephosphorylates the lipid second messenger, phosphatidylinositol 3,4,5-trisphosphateJ Biol Chem1998 2731337513378

    Not in our library10.1074/jbc.273.22.13375

  43. 43.

    Stambolic V Suzuki A de la Pompa JL Brothers GM Mirtsos C Sasaki T Ruland J Penninger JM Siderovski DP Mak TW Negative regulation of PKB/Akt-dependent cell survival by the tumor suppressor PTENCell1998 952939

    Not in our library10.1016/s0092-8674(00)81780-8

  44. 44.

    Tamura M Gu J Matsumoto K Aota S Parsons R Yamada KM Inhibition of cell migration, spreading, and focal adhesions by tumor suppressor PTENScience1998 28016141617

    Not in our library10.1126/science.280.5369.1614

  45. 45.

    Kulik G Weber MJ Akt-dependent and -independent survival signaling pathways utilized by insulin-like growth factor IMol Cell Biol1998 1867116718

    Not in our library10.1128/mcb.18.11.6711

  46. 46.

    Gu J Tamura M Yamada KM Tumor suppressor PTEN inhibits integrin- and growth factor-mediated mitogen-activated protein (MAP) kinase signaling pathwaysJ Cell Biol1998 14313751383

    Not in our library10.1083/jcb.143.5.1375

  47. 47.

    Downward J PI 3-kinase, Akt and cell survivalSemin Cell Dev Biol2004 15177182

    Not in our library10.1016/j.semcdb.2004.01.002

  48. 48.

    Harrington LS Findlay GM Lamb RF Restraining PI3K: mTOR signalling goes back to the membraneTrends Biochem Sci2005 303542

    Not in our library10.1016/j.tibs.2004.11.003

  49. 49.

    Dudek H Datta SR Franke TF Birnbaum MJ Yao R Cooper GM Segal RA Kaplan DR Greenberg ME Regulation of neuronal survival by the serine-threonine protein kinase AktScience1997 275661665

    Not in our library10.1126/science.275.5300.661

  50. 50.

    Kaufmann SH Hengartner MO Programmed cell death: alive and well in the new millenniumTrends Cell Biol2001 11526534

    Not in our library10.1016/s0962-8924(01)02173-0

  51. 51.

    Kennedy SG Wagner AJ Conzen SD Jordan J Bellacosa A Tsichlis PN Hay N The PI 3-kinase/Akt signaling pathway delivers an anti-apoptotic signalGenes Dev1997 11701713

    Not in our library10.1101/gad.11.6.701

  52. 52.

    Khwaja A Akt is more than just a Bad kinaseNature1999 4013334

    Not in our library10.1038/43354

  53. 53.

    Chen X Thakkar H Tyan F Gim S Robinson H Lee C Pandey SK Nwokorie C Onwudiwe N Srivastava RK Constitutively active Akt is an important regulator of TRAIL sensitivity in prostate cancerOncogene2001 2060736083

    Not in our library10.1038/sj.onc.1204736

  54. 54.

    Kandasamy K Srivastava RK Role of the phosphatidylinositol 3'-kinase/PTEN/Akt kinase pathway in tumor necrosis factor-related apoptosis-inducing ligand-induced apoptosis in non-small cell lung cancer cellsCancer Res2002 6249294937

    Not in our library

  55. 55.

    Cicenas J Urban P Vuaroqueaux V Labuhn M Kung W Wight E Mayhew M Eppenberger U Eppenberger-Castori S Increased level of phosphorylated akt measured by chemiluminescence-linked immunosorbent assay is a predictor of poor prognosis in primary breast cancer overexpressing ErbB-2Breast Cancer Res2005 7R394401

    Not in our library10.1186/bcr1015

  56. 56.

    Semba S Moriya T Kimura W Yamakawa M Phosphorylated Akt/PKB controls cell growth and apoptosis in intraductal papillary-mucinous tumor and invasive ductal adenocarcinoma of the pancreasPancreas2003 26250257

    Not in our library10.1097/00006676-200304000-00008

  57. 57.

    Yao Z Okabayashi Y Yutsudo Y Kitamura T Ogawa W Kasuga M Role of Akt in growth and survival of PANC-1 pancreatic cancer cellsPancreas2002 244246

    Not in our library10.1097/00006676-200201000-00006

  58. 58.

    Datta SR Dudek H Tao X Masters S Fu H Gotoh Y Greenberg ME Akt phosphorylation of BAD couples survival signals to the cell-intrinsic death machineryCell1997 91231241

    Not in our library10.1016/s0092-8674(00)80405-5

  59. 59.

    del Peso L Gonzalez-Garcia M Page C Herrera R Nunez G Interleukin-3-induced phosphorylation of BAD through the protein kinase AktScience1997 278687689

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  60. 60.

    Cardone MH Roy N Stennicke HR Salvesen GS Franke TF Stanbridge E Frisch S Reed JC Regulation of cell death protease caspase-9 by phosphorylationScience1998 28213181321

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  61. 61.

    Kang SS Kwon T Kwon DY Do SI Akt protein kinase enhances human telomerase activity through phosphorylation of telomerase reverse transcriptase subunitJ Biol Chem1999 2741308513090

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  62. 62.

    Brunet A Bonni A Zigmond MJ Lin MZ Juo P Hu LS Anderson MJ Arden KC Blenis J Greenberg ME Akt promotes cell survival by phosphorylating and inhibiting a Forkhead transcription factorCell1999 96857868

    Not in our library10.1016/s0092-8674(00)80595-4

  63. 63.

    Kops GJ de Ruiter ND De Vries-Smits AM Powell DR Bos JL Burgering BM Direct control of the Forkhead transcription factor AFX by protein kinase BNature1999 398630634

    Not in our library10.1038/19328

  64. 64.

    Romashkova JA Makarov SS NF-kappaB is a target of AKT in anti-apoptotic PDGF signallingNature1999 4018690

    Not in our library10.1038/43474

  65. 65.

    Ozes ON Mayo LD Gustin JA Pfeffer SR Pfeffer LM Donner DB NF-kappaB activation by tumour necrosis factor requires the Akt serine-threonine kinaseNature1999 4018285

    Not in our library10.1038/43466

  66. 66.

    Perugini RA McDade TP Vittimberga FJ Jr Callery MP Pancreatic cancer cell proliferation is phosphatidylinositol 3-kinase dependentJ Surg Res2000 903944

    Not in our library10.1006/jsre.2000.5833

  67. 67.

    Shah SA Potter MW Hedeshian MH Kim RD Chari RS Callery MP PI-3' kinase and NF-kappaB cross-signaling in human pancreatic cancer cellsJ Gastrointest Surg2001 5603612discussion 612-603

    Not in our library10.1016/s1091-255x(01)80102-5

  68. 68.

    Bondar VM Sweeney-Gotsch B Andreeff M Mills GB McConkey DJ Inhibition of the phosphatidylinositol 3'-kinase-AKT pathway induces apoptosis in pancreatic carcinoma cells in vitro and in vivoMol Cancer Ther2002 1989997

    Not in our library

  69. 69.

    Cheng JQ Ruggeri B Klein WM Sonoda G Altomare DA Watson DK Testa JR Amplification of AKT2 in human pancreatic cells and inhibition of AKT2 expression and tumorigenicity by antisense RNAProc Natl Acad Sci USA1996 9336363641

    Not in our library10.1073/pnas.93.8.3636

  70. 70.

    Ruggeri BA Huang L Wood M Cheng JQ Testa JR Amplification and overexpression of the AKT2 oncogene in a subset of human pancreatic ductal adenocarcinomasMol Carcinog1998 218186

    Not in our library10.1002/(sici)1098-2744(199802)21:2<81::aid-mc1>3.0.co;2-r

  71. 71.
    Incidence, mechanism and prognostic value of activated AKT in pancreas cancer · 2003
    No notice
  72. 72.

    Galili N Davis RJ Fredericks WJ Mukhopadhyay S Rauscher FJ Emanuel BS Rovera G Barr FG Fusion of a fork head domain gene to PAX3 in the solid tumour alveolar rhabdomyosarcomaNat Genet1993 5230235

    Not in our library10.1038/ng1193-230

  73. 73.

    Anderson MJ Viars CS Czekay S Cavenee WK Arden KC Cloning and characterization of three human forkhead genes that comprise an FKHR-like gene subfamilyGenomics1998 47187199

    Not in our library10.1006/geno.1997.5122

  74. 74.

    Hillion J Le Coniat M Jonveaux P Berger R Bernard OA AF6q21, a novel partner of the MLL gene in t(6;11)(q21;q23), defines a forkhead transcriptional factor subfamilyBlood1997 9037143719

    Not in our library

  75. 75.

    Borkhardt A Repp R Haas OA Leis T Harbott J Kreuder J Hammermann J Henn T Lampert F Cloning and characterization of AFX, the gene that fuses to MLL in acute leukemias with a t(X;11)(q13;q23)Oncogene1997 14195202

    Not in our library10.1038/sj.onc.1200814

  76. 76.

    Van Der Heide LP Hoekman MF Smidt MP The ins and outs of FoxO shuttling: mechanisms of FoxO translocation and transcriptional regulationBiochem J2004 380297309

    Not in our library10.1042/bj20040167

  77. 77.

    Guo S Rena G Cichy S He X Cohen P Unterman T Phosphorylation of serine 256 by protein kinase B disrupts transactivation by FKHR and mediates effects of insulin on insulin-like growth factor-binding protein-1 promoter activity through a conserved insulin response sequenceJ Biol Chem1999 2741718417192

    Not in our library10.1074/jbc.274.24.17184

  78. 78.

    Medema RH Kops GJ Bos JL Burgering BM AFX-like Forkhead transcription factors mediate cell-cycle regulation by Ras and PKB through p27kip1Nature2000 404782787

    Not in our library10.1038/35008115

  79. 79.

    Nakamura N Ramaswamy S Vazquez F Signoretti S Loda M Sellers WR Forkhead transcription factors are critical effectors of cell death and cell cycle arrest downstream of PTENMol Cell Biol2000 2089698982

    Not in our library10.1128/mcb.20.23.8969-8982.2000

  80. 80.

    Dijkers PF Medema RH Pals C Banerji L Thomas NS Lam EW Burgering BM Raaijmakers JA Lammers JW Koenderman L Coffer PJ Forkhead transcription factor FKHR-L1 modulates cytokine-dependent transcriptional regulation of p27(KIP1)Mol Cell Biol2000 2091389148

    Not in our library10.1128/mcb.20.24.9138-9148.2000

  81. 81.

    Dijkers PF Birkenkamp KU Lam EW Thomas NS Lammers JW Koenderman L Coffer PJ FKHR-L1 can act as a critical effector of cell death induced by cytokine withdrawal: protein kinase B-enhanced cell survival through maintenance of mitochondrial integrityJ Cell Biol2002 156531542

    Not in our library10.1083/jcb.200108084

  82. 82.

    Cappellini A Tabellini G Zweyer M Bortul R Tazzari PL Billi AM Fala F Cocco L Martelli AM The phosphoinositide 3-kinase/Akt pathway regulates cell cycle progression of HL60 human leukemia cells through cytoplasmic relocalization of the cyclin-dependent kinase inhibitor p27(Kip1) and control of cyclin D1 expressionLeukemia2003 1721572167

    Not in our library10.1038/sj.leu.2403111

  83. 83.

    Burgering BM Kops GJ Cell cycle and death control: long live ForkheadsTrends Biochem Sci2002 27352360

    Not in our library10.1016/s0968-0004(02)02113-8

  84. 84.

    Dijkers PF Medema RH Lammers JW Koenderman L Coffer PJ Expression of the pro-apoptotic Bcl-2 family member Bim is regulated by the forkhead transcription factor FKHR-L1Curr Biol2000 1012011204

    Not in our library10.1016/s0960-9822(00)00728-4

  85. 85.

    Gilley J Coffer PJ Ham J FOXO transcription factors directly activate bim gene expression and promote apoptosis in sympathetic neuronsJ Cell Biol2003 162613622

    Not in our library10.1083/jcb.200303026

  86. 86.

    Tang TT Dowbenko D Jackson A Toney L Lewin DA Dent AL Lasky LA The forkhead transcription factor AFX activates apoptosis by induction of the BCL-6 transcriptional repressorJ Biol Chem2002 2771425514265

    Not in our library10.1074/jbc.m110901200

  87. 87.

    Boguski MS McCormick F Proteins regulating Ras and its relativesNature1993 366643654

    Not in our library10.1038/366643a0

  88. 88.

    Bokoch GM Der CJ Emerging concepts in the Ras superfamily of GTP-binding proteinsFaseb J1993 7750759

    Not in our library10.1096/fasebj.7.9.8330683

  89. 89.

    Gibbs JB Sigal IS Poe M Scolnick EM Intrinsic GTPase activity distinguishes normal and oncogenic ras p21 moleculesProc Natl Acad Sci USA1984 8157045708

    Not in our library10.1073/pnas.81.18.5704

  90. 90.

    Shih C Weinberg RA Isolation of a transforming sequence from a human bladder carcinoma cell lineCell1982 29161169

    Not in our library10.1016/0092-8674(82)90100-3

  91. 91.

    Campbell SL Khosravi-Far R Rossman KL Clark GJ Der CJ Increasing complexity of Ras signalingOncogene1998 1713951413

    Not in our library10.1038/sj.onc.1202174

  92. 92.

    Schaeffer HJ Weber MJ Mitogen-activated protein kinases: specific messages from ubiquitous messengersMol Cell Biol1999 1924352444

    Not in our library10.1128/mcb.19.4.2435

  93. 93.

    Han J Ulevitch RJ Emerging targets for anti-inflammatory therapyNat Cell Biol1999 1E3940

    Not in our library10.1038/10032

  94. 94.

    Davis RJ Signal transduction by the JNK group of MAP kinasesCell2000 103239252

    Not in our library10.1016/s0092-8674(00)00116-1

  95. 95.

    Chang L Karin M Mammalian MAP kinase signalling cascadesNature2001 4103740

    Not in our library10.1038/35065000

  96. 96.

    Robinson MJ Cobb MH Mitogen-activated protein kinase pathwaysCurr Opin Cell Biol1997 9180186

    Not in our library10.1016/s0955-0674(97)80061-0

  97. 97.

    Woessmann W Meng YH Mivechi NF An essential role for mitogen-activated protein kinases, ERKs, in preventing heat-induced cell deathJ Cell Biochem1999 74648662

    Not in our library10.1002/(sici)1097-4644(19990915)74:4<648::aid-jcb14>3.0.co;2-6

  98. 98.

    Kyriakis JM Avruch J Sounding the alarm: protein kinase cascades activated by stress and inflammationJ Biol Chem1996 2712431324316

    Not in our library10.1074/jbc.271.40.24313

  99. 99.

    Ono K Han J The p38 signal transduction pathway: activation and functionCell Signal2000 12113

    Not in our library10.1016/s0898-6568(99)00071-6

  100. 100.

    Lubinus M Meier KE Smith EA Gause KC LeRoy EC Trojanowska M Independent effects of platelet-derived growth factor isoforms on mitogen-activated protein kinase activation and mitogenesis in human dermal fibroblastsJ Biol Chem1994 26998229825

    Not in our library

  101. 101.

    Shankar S Chen Q Srivastava RK Inhibition of PI3K/AKT and MEK/ERK pathways act synergistically to enhance antiangiogenic effects of EGCG through activation of FOXO transcription factorJ Mol Signal2008 37

    Not in our library10.1186/1750-2187-3-7

  102. 102.

    Bardeesy N DePinho RA Pancreatic cancer biology and geneticsNat Rev Cancer2002 2897909

    Not in our library10.1038/nrc949

  103. 103.

    Li D Xie K Wolff R Abbruzzese JL Pancreatic cancerLancet2004 36310491057

    Not in our library10.1016/s0140-6736(04)15841-8

  104. 104.

    Srivastava RK Intracellular mechanisms of TRAIL and its role in cancer therapyMol Cell Biol Res Commun2000 46775

    Not in our library10.1006/mcbr.2001.0265

  105. 105.

    Datta SR Brunet A Greenberg ME Cellular survival: a play in three AktsGenes Dev1999 1329052927

    Not in our library10.1101/gad.13.22.2905

  106. 106.

    Mielke K Herdegen T JNK and p38 stresskinases--degenerative effectors of signal-transduction-cascades in the nervous systemProg Neurobiol2000 614560

    Not in our library10.1016/s0301-0082(99)00042-8

  107. 107.

    McCubrey JA May WS Duronio V Mufson A Serine/threonine phosphorylation in cytokine signal transductionLeukemia2000 14921

    Not in our library10.1038/sj.leu.2401657

  108. 108.

    Camps M Nichols A Arkinstall S Dual specificity phosphatases: a gene family for control of MAP kinase functionFaseb J2000 14616

    Not in our library

  109. 109.

    Farooq A Zhou MM Structure and regulation of MAPK phosphatasesCell Signal2004 16769779

    Not in our library10.1016/j.cellsig.2003.12.008

  110. 110.

    Hutzen B Willis W Jones S Cen L Deangelis S Fuh B Lin J Dietary agent, benzyl isothiocyanate inhibits signal transducer and activator of transcription 3 phosphorylation and collaborates with sulforaphane in the growth suppression of PANC-1 cancer cellsCancer Cell Int2009 924

    Not in our library10.1186/1475-2867-9-24

  111. 111.

    Kallifatidis G Rausch V Baumann B Apel A Beckermann BM Groth A Mattern J Li Z Kolb A Moldenhauer G Sulforaphane targets pancreatic tumor-initiating cells by NF-{kappa}B-induced anti-apoptotic signalingGut2009 5894963

    Not in our library10.1136/gut.2008.149039

  112. 112.

    Pham NA Jacobberger JW Schimmer AD Cao P Gronda M Hedley DW The dietary isothiocyanate sulforaphane targets pathways of apoptosis, cell cycle arrest, and oxidative stress in human pancreatic cancer cells and inhibits tumor growth in severe combined immunodeficient miceMol Cancer Ther2004 312391248

    Not in our library

  113. 113.

    Kitamura T Nakae J Kitamura Y Kido Y Biggs WH Wright CV White MF Arden KC Accili D The forkhead transcription factor Foxo1 links insulin signaling to Pdx1 regulation of pancreatic beta cell growthJ Clin Invest2002 11018391847

    Not in our library10.1172/jci200216857

  114. 114.

    Castrillon DH Miao L Kollipara R Horner JW DePinho RA Suppression of ovarian follicle activation in mice by the transcription factor Foxo3aScience2003 301215218

    Not in our library10.1126/science.1086336

  115. 115.

    Nakae J Biggs WH Kitamura T Cavenee WK Wright CV Arden KC Accili D Regulation of insulin action and pancreatic beta-cell function by mutated alleles of the gene encoding forkhead transcription factor Foxo1Nat Genet2002 32245253

    Not in our library10.1038/ng890

  116. 116.

    Xia SJ Pressey JG Barr FG Molecular pathogenesis of rhabdomyosarcomaCancer Biol Ther2002 197104

    Not in our library10.4161/cbt.51

  117. 117.

    Hu MC Lee DF Xia W Golfman LS Ou-Yang F Yang JY Zou Y Bao S Hanada N Saso H IkappaB kinase promotes tumorigenesis through inhibition of forkhead FOXO3aCell2004 117225237

    Not in our library10.1016/s0092-8674(04)00302-2

  118. 118.

    Furuyama T Kitayama K Shimoda Y Ogawa M Sone K Yoshida-Araki K Hisatsune H Nishikawa S Nakayama K Nakayama K Abnormal angiogenesis in Foxo1 (Fkhr)-deficient miceJ Biol Chem2004 2793474134749

    Not in our library10.1074/jbc.m314214200

  119. 119.

    Dejana E Taddei A Randi AM Foxs and Ets in the transcriptional regulation of endothelial cell differentiation and angiogenesisBiochim Biophys Acta2007 1775298312

    Not in our library10.1016/j.bbcan.2007.05.003

  120. 120.

    Chlench S Mecha Disassa N Hohberg M Hoffmann C Pohlkamp T Beyer G Bongrazio M Da Silva-Azevedo L Baum O Pries AR Zakrzewicz A Regulation of Foxo-1 and the angiopoietin-2/Tie2 system by shear stressFEBS Lett2007 581673680

    Not in our library10.1016/j.febslet.2007.01.028

  121. 121.

    Potente M Urbich C Sasaki K Hofmann WK Heeschen C Aicher A Kollipara R DePinho RA Zeiher AM Dimmeler S Involvement of Foxo transcription factors in angiogenesis and postnatal neovascularizationJ Clin Invest2005 11523822392

    Not in our library10.1172/jci23126

  122. 122.

    Jin S Shen JN Wang J Huang G Zhou JG Oridonin induced apoptosis through Akt and MAPKs signaling pathways in human osteosarcoma cellsCancer Biol Ther2007 6261268

    Not in our library10.4161/cbt.6.2.3621

  123. 123.

    Schwab TS Madison BB Grauman AR Feldman EL Insulin-like growth factor-I induces the phosphorylation and nuclear exclusion of forkhead transcription factors in human neuroblastoma cellsApoptosis2005 10831840

    Not in our library10.1007/s10495-005-0429-y

  124. 124.

    Srivastava RK Unterman TG Shankar S FOXO transcription factors and VEGF neutralizing antibody enhance antiangiogenic effects of resveratrolMol Cell Biochem2010 337201212

    Not in our library10.1007/s11010-009-0300-5

  125. 125.

    Kaneto H Kawamori D Matsuoka TA Kajimoto Y Yamasaki Y Oxidative stress and pancreatic beta-cell dysfunctionAm J Ther2005 12529533

    Not in our library10.1097/01.mjt.0000178773.31525.c2

  126. 126.

    Kaneto H Kawamori D Nakatani Y Gorogawa S Matsuoka TA Oxidative stress and the JNK pathway as a potential therapeutic target for diabetesDrug News Perspect2004 17447453

    Not in our library10.1358/dnp.2004.17.7.863704

  127. 127.

    Kawamori D Kaneto H Nakatani Y Matsuoka TA Matsuhisa M Hori M Yamasaki Y The forkhead transcription factor Foxo1 bridges the JNK pathway and the transcription factor PDX-1 through its intracellular translocationJ Biol Chem2006 28110911098

    Not in our library10.1074/jbc.m508510200

  128. 128.

    Stahl M Dijkers PF Kops GJ Lens SM Coffer PJ Burgering BM Medema RH The forkhead transcription factor FoxO regulates transcription of p27Kip1 and Bim in response to IL-2J Immunol2002 16850245031

    Not in our library10.4049/jimmunol.168.10.5024

  129. 129.

    Uddin S Hussain AR Siraj AK Manogaran PS Al-Jomah NA Moorji A Atizado V Al-Dayel F Belgaumi A El-Solh H Role of phosphatidylinositol 3'-kinase/AKT pathway in diffuse large B-cell lymphoma survivalBlood2006 10841784186

    Not in our library10.1182/blood-2006-04-016907

  130. 130.

    Daitoku H Fukamizu A FOXO transcription factors in the regulatory networks of longevityJ Biochem2007 141769774

    Not in our library10.1093/jb/mvm104

  131. 131.

    van der Heide LP Smidt MP Regulation of FoxO activity by CBP/p300-mediated acetylationTrends Biochem Sci2005 308186

    Not in our library10.1016/j.tibs.2004.12.002

  132. 132.

    Brunet A Sweeney LB Sturgill JF Chua KF Greer PL Lin Y Tran H Ross SE Mostoslavsky R Cohen HY Stress-dependent regulation of FOXO transcription factors by the SIRT1 deacetylaseScience2004 30320112015

    Not in our library10.1126/science.1094637

  133. 133.

    Imai S Armstrong CM Kaeberlein M Guarente L Transcriptional silencing and longevity protein Sir2 is an NAD-dependent histone deacetylaseNature2000 403795800

    Not in our library10.1038/35001622

  134. 134.

    Shankar S Ganapathy S Hingorani SR Srivastava RK EGCG inhibits growth, invasion, angiogenesis and metastasis of pancreatic cancerFront Biosci2008 13440452

    Not in our library10.2741/2691

  135. 135.

    Shankar S Siddiqui I Srivastava RK Molecular mechanisms of resveratrol (3,4,5-trihydroxy-trans-stilbene) and its interaction with TNF-related apoptosis inducing ligand (TRAIL) in androgen-insensitive prostate cancer cellsMol Cell Biochem2007 304273285

    Not in our library10.1007/s11010-007-9510-x

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