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Sample records for akt downstream target

  1. MicroRNA-145 suppresses hepatocellular carcinoma by targeting IRS1 and its downstream Akt signaling

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    Wang, Yelin [Department of Anesthesiology, The First Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou (China); Key Laboratory of Combined Multi-organ Transplantation, Ministry of Public Health, The First Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou (China); Hu, Chen; Cheng, Jun [Department of Surgery, The First Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou (China); Key Laboratory of Combined Multi-organ Transplantation, Ministry of Public Health, The First Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou (China); Chen, Binquan [Department of Anesthesiology, The First Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou (China); Ke, Qinghong; Lv, Zhen; Wu, Jian [Department of Surgery, The First Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou (China); Key Laboratory of Combined Multi-organ Transplantation, Ministry of Public Health, The First Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou (China); Zhou, Yanfeng, E-mail: zyfhdj@yahoo.com [Department of Anesthesiology, The First Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou (China)

    2014-04-18

    Highlights: • MiR-145 expression is down-regulated in HCC tissues and inversely related with IRS1 levels. • MiR-145 directly targets IRS1 in HCC cells. • Restored expression of miR-145 suppressed HCC cell proliferation and growth. • MiR-145 induced IRS1 under-expression potentially reduced downstream AKT signaling. - Abstract: Accumulating evidences have proved that dysregulation of microRNAs (miRNAs) is involved in cancer initiation and progression. In this study, we showed that miRNA-145 level was significantly decreased in hepatocellular cancer (HCC) tissues and cell lines, and its low expression was inversely associated with the abundance of insulin receptor substrate 1 (IRS1), a key mediator in oncogenic insulin-like growth factor (IGF) signaling. We verified IRS1 as a direct target of miR-145 using Western blotting and luciferase reporter assay. Further, the restoration of miR-145 in HCC cell lines suppressed cancer cell growth, owing to down-regulated IRS1 expression and its downstream Akt/FOXO1 signaling. Our results demonstrated that miR-145 could inhibit HCC through targeting IRS1 and its downstream signaling, implicating the loss of miR-145 regulation may be a potential molecular mechanism causing aberrant oncogenic signaling in HCC.

  2. Downstream targets of WRKY33.

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    Petersen, Klaus; Fiil, Berthe Katrine; Mundy, John; Petersen, Morten

    2008-11-01

    Innate immunity signaling pathways in both animals and plants are regulated by mitogen-activated protein kinase (MAPK) cascades. In a recent publication we show that MPK4 and its substrate MKS1 interact with WRKY33 in vivo, and that WRKY33 is released from complexes with MPK4 upon infection. Transcriptome analysis of a wrky33 loss-of-function mutant identified a subset of defense-related genes as putative targets of WRKY33. These genes include PAD3 and CYP71A13, which encode cytochrome P450 monoxygenases required for synthesis of the antimicrobial phytoalexin camalexin. Chromatin immunoprecipitation confirmed that WRKY33 bound the promoter of PAD3 when plants were inoculated with pathogens. Here we further discuss the involvement of two other targets of WRKY33, NUDT6 and ROF2 in defense responses against invading pathogens.

  3. Downstream targets of WRKY33

    DEFF Research Database (Denmark)

    Petersen, Klaus; Fiil, Berthe Katrine; Mundy, John;

    2008-01-01

    Innate immunity signaling pathways in both animals and plants are regulated by mitogen-activated protein kinase (MAPK) cascades. In a recent publication we show that MPK4 and its substrate MKS1 interact with WRKY33 in vivo, and that WRKY33 is released from complexes with MPK4 upon infection....... Transcriptome analysis of a wrky33 loss-of-function mutant identified a subset of defense-related genes as putative targets of WRKY33. These genes include PAD3 and CYP71A13, which encode cytochrome P450 monoxygenases required for synthesis of the antimicrobial phytoalexin camalexin. Chromatin...... immunoprecipitation confirmed that WRKY33 bound the promoter of PAD3 when plants were inoculated with pathogens. Here we further discuss the involvement of two other targets of WRKY33, NUDT6 and ROF2 in defense responses against invading pathogens....

  4. A novel AKT inhibitor, AZD5363, inhibits phosphorylation of AKT downstream molecules, and activates phosphorylation of mTOR and SMG-1 dependent on the liver cancer cell type

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    ZHANG, YUNCHENG; ZHENG, YUANWEN; FAHEEM, ALI; SUN, TIANTONG; LI, CHUNYOU; LI, ZHE; ZHAO, DIANTANG; WU, CHAO; LIU, JUN

    2016-01-01

    Due to frequent phosphoinositide 3-kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) signaling pathway dysregulation, AKT is typically accepted as a promising anticancer therapeutic target. mTOR, in particular, represents a suitable therapeutic target for hepatocellular carcinoma, whilst suppressor with morphogenetic effect on genitalia family member-1 (SMG-1) is believed to serve a potential tumor suppressor role in human cancer. Despite SMG-1 and mTOR belonging to the same PI3K-related kinase family, the interactions between them are not yet fully understood. In the present study, a novel pyrrolopyrimidine-derived compound, AZD5363, was observed to suppress proliferation in liver cancer Hep-G2 and Huh-7 cells by inhibiting the phosphorylation of downstream molecules in the AKT signal pathway, in a dose- and time-dependent manner. AZD5363 activated the phosphorylation of mTOR, dependent on the liver cancer cell type, as it may have differing effects in various liver cancer cell lines. Additionally, AZD5363 also activated SMG-1 within the same liver cancer cells types, which subsequently activated the phosphorylation of mTOR. In conclusion, the present study indicates that AZD5363 inhibited phosphorylation of AKT downstream molecules, and activated phosphorylation of mTOR and SMG-1, dependent on the liver cancer type. PMID:26998062

  5. Targeting FAK Radiosensitizes 3-Dimensional Grown Human HNSCC Cells Through Reduced Akt1 and MEK1/2 Signaling

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    Hehlgans, Stephanie [OncoRay-National Center for Radiation Research in Oncology, Medical Faculty Carl Gustav Carus, Dresden University of Technology, Dresden (Germany); Department of Radiotherapy and Oncology, University of Frankfurt, Frankfurt am Main (Germany); Institute of Radiopharmacy, Helmholtz Center Dresden-Rossendorf, Dresden (Germany); Eke, Iris [OncoRay-National Center for Radiation Research in Oncology, Medical Faculty Carl Gustav Carus, Dresden University of Technology, Dresden (Germany); Cordes, Nils, E-mail: Nils.Cordes@OncoRay.de [OncoRay-National Center for Radiation Research in Oncology, Medical Faculty Carl Gustav Carus, Dresden University of Technology, Dresden (Germany); Institute of Radiopharmacy, Helmholtz Center Dresden-Rossendorf, Dresden (Germany); Department of Radiation Oncology, University Hospital and Medical Faculty Carl Gustav Carus, Dresden University of Technology, Dresden (Germany)

    2012-08-01

    Purpose: Focal adhesion kinase (FAK), a main regulator of integrin signaling and cell migration, is frequently overexpressed and hyperphosphorylated in human head-and-neck squamous cell carcinoma (HNSCC). We have previously shown that pharmacologic FAK inhibition leads to radiosensitization of 3-dimensionally grown HNSCC cell lines. To further evaluate the role of FAK in radioresistance and as a potential cancer target, we examined FAK and FAK downstream signaling in HNSCC cell lines grown in more physiologic extracellular matrix-based 3-dimensional cell cultures. Methods and Materials: Seven HNSCC cell lines were grown in 3-dimensional extracellular matrix and the clonogenic radiation survival, expression, and phosphorylation of FAK, paxillin, Akt1, extracellular signal-regulated kinase (ERK)1/2, and MEK1/2 were analyzed after siRNA-mediated knockdown of FAK, Akt1, MEK1, FAK+Akt1, or FAK+MEK1 compared with controls or stable overexpression of FAK. The role of MEK1/2 for clonogenic survival and signaling was investigated using the MEK inhibitor U0126 with or without irradiation. Results: FAK knockdown moderately or significantly enhanced the cellular radiosensitivity of 3-dimensionally grown HNSCC cells. The FAK downstream targets paxillin, Akt1, and ERK1/2 were substantially dephosphorylated under FAK depletion. FAK overexpression, in contrast, increased radiation survival and paxillin, Akt1, and ERK1/2 phosphorylation. The degree of radiosensitization upon Akt1, ERK1/2, or MEK1 depletion or U0126 was superimposable to FAK knockdown. Combination knockdown conditions (ie, Akt1/FAK, MEK1/FAK, or U0126/FAK) failed to provide additional radiosensitization. Conclusions: Our data provide further evidence for FAK as important determinant of radiation survival, which acts in the same signaling axis as Akt1 and ERK1/2. These data strongly support our hypothesis that FAK is a relevant molecular target for HNSCC radiotherapy.

  6. Targeting the AKT pathway: Repositioning HIV protease inhibitors as radiosensitizers

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    Jayant S Goda

    2016-01-01

    Full Text Available Cellular resistance in tumour cells to different therapeutic approaches has been a limiting factor in the curative treatment of cancer. Resistance to therapeutic radiation is a common phenomenon which significantly reduces treatment options and impacts survival. One of the mechanisms of acquiring resistance to ionizing radiation is the overexpression or activation of various oncogenes like the EGFR (epidermal growth factor receptor, RAS (rat sarcoma oncogene or loss of PTEN (phosphatase and tensin homologue which in turn activates the phosphatidyl inositol 3-kinase/protein kinase B (PI3-K/AKT pathway responsible for radiation resistance in various tumours. Blocking the pathway enhances the radiation response both in vitro and in vivo. Due to the differential activation of this pathway (constitutively activated in tumour cells and not in the normal host cells, it is an excellent candidate target for molecular targeted therapy to enhance radiation sensitivity. In this regard, HIV protease inhibitors (HPIs known to interfere with PI3-K/AKT signaling in tumour cells, have been shown to sensitize various tumour cells to radiation both in vitro and in vivo. As a result, HPIs are now being investigated as possible radiosensitizers along with various chemotherapeutic drugs. This review describes the mechanisms by which PI3-K/AKT pathway causes radioresistance and the role of HIV protease inhibitors especially nelfinavir as a potential candidate drug to target the AKT pathway for overcoming radioresistance and its use in various clinical trials for different malignancies.

  7. Targeting the AKT pathway: Repositioning HIV protease inhibitors as radiosensitizers.

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    Goda, Jayant S; Pachpor, Tejaswini; Basu, Trinanjan; Chopra, Supriya; Gota, Vikram

    2016-02-01

    Cellular resistance in tumour cells to different therapeutic approaches has been a limiting factor in the curative treatment of cancer. Resistance to therapeutic radiation is a common phenomenon which significantly reduces treatment options and impacts survival. One of the mechanisms of acquiring resistance to ionizing radiation is the overexpression or activation of various oncogenes like the EGFR (epidermal growth factor receptor), RAS (rat sarcoma) oncogene or loss of PTEN (phosphatase and tensin homologue) which in turn activates the phosphatidyl inositol 3-kinase/protein kinase B (PI3-K)/AKT pathway responsible for radiation resistance in various tumours. Blocking the pathway enhances the radiation response both in vitro and in vivo. Due to the differential activation of this pathway (constitutively activated in tumour cells and not in the normal host cells), it is an excellent candidate target for molecular targeted therapy to enhance radiation sensitivity. In this regard, HIV protease inhibitors (HPIs) known to interfere with PI3-K/AKT signaling in tumour cells, have been shown to sensitize various tumour cells to radiation both in vitro and in vivo. As a result, HPIs are now being investigated as possible radiosensitizers along with various chemotherapeutic drugs. This review describes the mechanisms by which PI3-K/AKT pathway causes radioresistance and the role of HIV protease inhibitors especially nelfinavir as a potential candidate drug to target the AKT pathway for overcoming radioresistance and its use in various clinical trials for different malignancies.

  8. MicroRNA-184 inhibits neuroblastoma cell survival through targeting the serine/threonine kinase AKT2

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    Murphy Derek M

    2010-04-01

    Full Text Available Abstract Background Neuroblastoma is a paediatric cancer of the sympathetic nervous system. The single most important genetic indicator of poor clinical outcome is amplification of the MYCN transcription factor. One of many down-stream MYCN targets is miR-184, which is either directly or indirectly repressed by this transcription factor, possibly due to its pro-apoptotic effects when ectopically over-expressed in neuroblastoma cells. The purpose of this study was to elucidate the molecular mechanism by which miR-184 conveys pro-apoptotic effects. Results We demonstrate that the knock-down of endogenous miR-184 has the opposite effect of ectopic up-regulation, leading to enhanced neuroblastoma cell numbers. As a mechanism of how miR-184 causes apoptosis when over-expressed, and increased cell numbers when inhibited, we demonstrate direct targeting and degradation of AKT2, a major downstream effector of the phosphatidylinositol 3-kinase (PI3K pathway, one of the most potent pro-survival pathways in cancer. The pro-apoptotic effects of miR-184 ectopic over-expression in neuroblastoma cell lines is reproduced by siRNA inhibition of AKT2, while a positive effect on cell numbers similar to that obtained by the knock-down of endogenous miR-184 can be achieved by ectopic up-regulation of AKT2. Moreover, co-transfection of miR-184 with an AKT2 expression vector lacking the miR-184 target site in the 3'UTR rescues cells from the pro-apoptotic effects of miR-184. Conclusions MYCN contributes to tumorigenesis, in part, by repressing miR-184, leading to increased levels of AKT2, a direct target of miR-184. Thus, two important genes with positive effects on cell growth and survival, MYCN and AKT2, can be linked into a common genetic pathway through the actions of miR-184. As an inhibitor of AKT2, miR-184 could be of potential benefit in miRNA mediated therapeutics of MYCN amplified neuroblastoma and other forms of cancer.

  9. Synthetic sulfoglycolipids targeting the serine-threonine protein kinase Akt.

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    Costa, Barbara; Dangate, Milind; Vetro, Maria; Donvito, Giulia; Gabrielli, Luca; Amigoni, Loredana; Cassinelli, Giuliana; Lanzi, Cinzia; Ceriani, Michela; De Gioia, Luca; Filippi, Giulia; Cipolla, Laura; Zaffaroni, Nadia; Perego, Paola; Colombo, Diego

    2016-08-15

    The serine-threonine protein kinase Akt, also known as protein kinase B, is a key component of the phosphoinositide 3-kinase (PI3K)-Akt-mTOR axis. Deregulated activation of this pathway is frequent in human tumors and Akt-dependent signaling appears to be critical in cell survival. PI3K activation generates 3-phosphorylated phosphatidylinositols that bind Akt pleckstrin homology (PH) domain. The blockage of Akt PH domain/phosphoinositides interaction represents a promising approach to interfere with the oncogenic potential of over-activated Akt. In the present study, phosphatidyl inositol mimics based on a β-glucoside scaffold have been synthesized as Akt inhibitors. The compounds possessed one or two lipophilic moieties of different length at the anomeric position of glucose, and an acidic or basic group at C-6. Docking studies, ELISA Akt inhibition assays, and cellular assays on different cell models highlighted 1-O-octadecanoyl-2-O-β-d-sulfoquinovopyranosyl-sn-glycerol as the best Akt inhibitor among the synthesized compounds, which could be considered as a lead for further optimization in the design of Akt inhibitors.

  10. PI3K and Akt as molecular targets for cancer therapy: current clinical outcomes

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    Ipsita PAL; Mahitosh MANDAL

    2012-01-01

    The PI3K-Akt pathway is a vital regulator of cell proliferation and survival.Alterations in the PIK3CA gene that lead to enhanced PI3K kinase activity have been reported in many human cancer types,including cancers of the colon,breast,brain,liver,stomach and lung.Deregulation of PI3K causes aberrant Akt activity.Therefore targeting this pathway could have implications for cancer treatment.The first generation PI3K-Akt inhibitors were proven to be highly effective with a low IC50,but later,they were shown to have toxic side effects and poor pharmacological properties and selectivity.Thus,these inhibitors were only effective in preclinical models.However,derivatives of these first generation inhibitors are much more selective and are quite effective in targeting the PI3K-Akt pathway,either alone or in combination.These second-generation inhibitors are essentially a specific chemical moiety that helps to form a strong hydrogen bond interaction with the PI3K/Akt molecule.The goal of this review is to delineate the current efforts that have been undertaken to inhibit the various components of the PI3K and Akt pathway in different types of cancer both in vitro and in vivo.Our focus here is on these novel therapies and their inhibitory effects that depend upon their chemical nature,as well as their development towards clinical trials.

  11. Targeting the Akt1 allosteric site to identify novel scaffolds through virtual screening.

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    Yilmaz, Oya Gursoy; Olmez, Elif Ozkirimli; Ulgen, Kutlu O

    2014-02-01

    Preclinical data and tumor specimen studies report that AKT kinases are related to many human cancers. Therefore, identification and development of small molecule inhibitors targeting AKT and its signaling pathway can be therapeutic in treatment of cancer. Numerous studies report inhibitors that target the ATP-binding pocket in the kinase domains, but the similarity of this site, within the kinase family makes selectivity a major problem. The sequence identity amongst PH domains is significantly lower than that in kinase domains and developing more selective inhibitors is possible if PH domain is targeted. This in silico screening study is the first time report toward the identification of potential allosteric inhibitors expected to bind the cavity between kinase and PH domains of Akt1. Structural information of Akt1 was used to develop structure-based pharmacophore models comprising hydrophobic, acceptor, donor and ring features. The 3D structural information of previously identified allosteric Akt inhibitors obtained from literature was employed to develop a ligand-based pharmacophore model. Database was generated with drug like subset of ZINC and screening was performed based on 3D similarity to the selected pharmacophore hypotheses. Binding modes and affinities of the ligands were predicted by Glide software. Top scoring hits were further analyzed considering 2D similarity between the compounds, interactions with Akt1, fitness to pharmacophore models, ADME, druglikeness criteria and Induced-Fit docking. Using virtual screening methodologies, derivatives of 3-methyl-xanthine, quinoline-4-carboxamide and 2-[4-(cyclohexa-1,3-dien-1-yl)-1H-pyrazol-3-yl]phenol were proposed as potential leads for allosteric inhibition of Akt1.

  12. Targets downstream of Cdk8 in Dictyostelium development

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    Skelton Jason

    2011-01-01

    Full Text Available Abstract Background Cdk8 is a component of the mediator complex which facilitates transcription by RNA polymerase II and has been shown to play an important role in development of Dictyostelium discoideum. This eukaryote feeds as single cells but starvation triggers the formation of a multicellular organism in response to extracellular pulses of cAMP and the eventual generation of spores. Strains in which the gene encoding Cdk8 have been disrupted fail to form multicellular aggregates unless supplied with exogenous pulses of cAMP and later in development, cdk8- cells show a defect in spore production. Results Microarray analysis revealed that the cdk8- strain previously described (cdk8-HL contained genome duplications. Regeneration of the strain in a background lacking detectable gene duplication generated strains (cdk8-2 with identical defects in growth and early development, but a milder defect in spore generation, suggesting that the severity of this defect depends on the genetic background. The failure of cdk8- cells to aggregate unless rescued by exogenous pulses of cAMP is consistent with a failure to express the catalytic subunit of protein kinase A. However, overexpression of the gene encoding this protein was not sufficient to rescue the defect, suggesting that this is not the only important target for Cdk8 at this stage of development. Proteomic analysis revealed two potential targets for Cdk8 regulation, one regulated post-transcriptionally (4-hydroxyphenylpyruvate dioxygenase (HPD and one transcriptionally (short chain dehydrogenase/reductase (SDR1. Conclusions This analysis has confirmed the importance of Cdk8 at multiple stages of Dictyostelium development, although the severity of the defect in spore production depends on the genetic background. Potential targets of Cdk8-mediated gene regulation have been identified in Dictyostelium which will allow the mechanism of Cdk8 action and its role in development to be determined.

  13. Integrative analysis of RUNX1 downstream pathways and target genes

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    Liu Marjorie

    2008-07-01

    Full Text Available Abstract Background The RUNX1 transcription factor gene is frequently mutated in sporadic myeloid and lymphoid leukemia through translocation, point mutation or amplification. It is also responsible for a familial platelet disorder with predisposition to acute myeloid leukemia (FPD-AML. The disruption of the largely unknown biological pathways controlled by RUNX1 is likely to be responsible for the development of leukemia. We have used multiple microarray platforms and bioinformatic techniques to help identify these biological pathways to aid in the understanding of why RUNX1 mutations lead to leukemia. Results Here we report genes regulated either directly or indirectly by RUNX1 based on the study of gene expression profiles generated from 3 different human and mouse platforms. The platforms used were global gene expression profiling of: 1 cell lines with RUNX1 mutations from FPD-AML patients, 2 over-expression of RUNX1 and CBFβ, and 3 Runx1 knockout mouse embryos using either cDNA or Affymetrix microarrays. We observe that our datasets (lists of differentially expressed genes significantly correlate with published microarray data from sporadic AML patients with mutations in either RUNX1 or its cofactor, CBFβ. A number of biological processes were identified among the differentially expressed genes and functional assays suggest that heterozygous RUNX1 point mutations in patients with FPD-AML impair cell proliferation, microtubule dynamics and possibly genetic stability. In addition, analysis of the regulatory regions of the differentially expressed genes has for the first time systematically identified numerous potential novel RUNX1 target genes. Conclusion This work is the first large-scale study attempting to identify the genetic networks regulated by RUNX1, a master regulator in the development of the hematopoietic system and leukemia. The biological pathways and target genes controlled by RUNX1 will have considerable importance in disease

  14. Akt mediated ROS-dependent selective targeting of mutant KRAS tumors.

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    Iskandar, Kartini; Rezlan, Majidah; Pervaiz, Shazib

    2014-10-01

    Reactive oxygen species (ROS) play a critical role in a variety of cellular processes, ranging from cell survival and proliferation to cell death. Previously, we reported the ability of a small molecule compound, C1, to induce ROS dependent autophagy associated apoptosis in human cancer cell lines and primary tumor cells (Wong C. et al. 2010). Our ongoing investigations have unraveled a hitherto undefined novel signaling network involving hyper-phosphorylation of Akt and Akt-mediated ROS production in cancer cell lines. Interestingly, drug-induced Akt activation is selectively seen in cell lines that carry mutant KRAS; HCT116 cells that carry the V13D KRAS mutation respond favorably to C1 while HT29 cells expressing wild type KRAS are relatively resistant. Of note, not only does the compound target mutant KRAS expressing cells but also induces RAS activation as evidenced by the PAK pull down assay. Corroborating this, pharmacological inhibition as well as siRNA mediated silencing of KRAS or Akt, blocked C1-induced ROS production and rescued tumor colony forming ability in HCT116 cells. To further confirm the involvement of KRAS, we made use of mutant KRAS transformed RWPE-1 prostate epithelial cells. Notably, drug-induced ROS generation and death sensitivity was significantly higher in RWPE-1-KRAS cells than the RWPE-1-vector cells, thus confirming the results obtained with mutant KRAS colorectal carcinoma cell line. Lastly, we made use of HCT116 mutant KRAS knockout cells (KO) where the mutant KRAS allele had been deleted, thus expressing a single wild-type KRAS allele. Exposure of the KO cells to C1 failed to induce Akt activation and mitochondrial ROS production. Taken together, results show the involvement of activated Akt in ROS-mediated selective targeting of mutant KRAS expressing tumors, which could have therapeutic implications given the paucity of chemotherapeutic strategies specifically targeting KRAS mutant cancers. PMID:26461287

  15. Role of the Phosphoinositide 3-Kinase-Akt-Mammalian Target of the Rapamycin Signaling Pathway in Long-Term Potentiation and Trace Fear Conditioning Memory in Rat Medial Prefrontal Cortex

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    Sui, Li; Wang, Jing; Li, Bao-Ming

    2008-01-01

    Phosphatidylinositol 3-kinase (PI3K) and its downstream targets, including Akt (also known as protein kinase B, PKB), mammalian target of rapamycin (mTOR), the 70-kDa ribosomal S6 kinase (p70S6k), and the eukaryotic initiation factor 4E (eIF4E)-binding protein 1 (4E-BP1), may play important roles in long-term synaptic plasticity and memory in many…

  16. Increased cytotoxicity of ionizing radiation in combination with membrane-targeted apoptosis modulators involves downregulation of protein kinase B/Akt-mediated survival-signaling

    International Nuclear Information System (INIS)

    Background and purpose: The membrane-targeted apoptosis modulators erucylphosphocholine (ErPC) and erucylphosphohomocholine (ErPC3) induce apoptosis in highly apoptosis resistant malignant glioma cell lines and enhance radiation-induced cell death and eradication of clonogenic tumor cells in vitro. Aim of the present study was to elucidate molecular mechanisms of combined action. Materials and methods: Induction of apoptosis was evaluated by determination of nuclear morphology (fluorescence microscopy), alteration of mitochondrial function and caspase-activation (flow cytometry, Western blot). Activity of protein kinase B (PKB/Akt) and key downstream effectors involved in apoptosis regulation was verified by Western blot analysis using activation-specific antibodies. Results: Increased cytotoxicity of the combination was linked to a more efficient activation of the intrinsic apoptosis pathway with increased damage of the mitochondria and caspase-activation. Moreover, activity of the survival kinase PKB/Akt was downregulated upon treatment with ErPC/ErPC3 alone or in combination with ionizing radiation. Inhibition of PKB/Akt was associated with decreased phosphorylation and thus activation of the pro-apoptotic Bcl-2 protein Bad as well as dephosphorylation of the transcription factor FOXO3A (FKHRL1) that may be responsible for the observed increased expression of the pro-apoptotic Bcl-2 protein Bim. Conclusions: Our data suggest a role for inhibition of PKB/Akt-mediated anti-apoptotic signaling in increased efficacy of the combination

  17. Upstream and Downstream Co-inhibition of Mitogen-Activated Protein Kinase and PI3K/Akt/mTOR Pathways in Pancreatic Ductal Adenocarcinoma

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    Matthew H. Wong

    2016-07-01

    Full Text Available BACKGROUND: Extensive cross talk exists between PI3K/Akt/mTOR and mitogen-activated protein kinase (MAPK pathways, and both are upregulated in pancreatic ductal adenocarcinoma (PDAC. Our previous study suggested that epidermal growth factor receptor inhibitor erlotinib which acts upstream of these pathways acts synergistically with PI3K inhibitors in PDAC. Horizontal combined blockade upstream and downstream of these two pathways is therefore explored. METHODS: Erlotinib paired with PI3K inhibitor (BYL719 was tested against erlotinib plus dual PI3K/mTOR inhibitor BEZ-235, and MEK inhibitor (PD98059 plus BEZ235, on five primary PDAC cell lines and on two pairs of parent and erlotinib-resistant (ER cell lines. A range of in vitro assays including cell proliferation, Western blotting, migration, clonogenic, cell cycle, and apopotic assays was used to test for the efficacy of combined blockade. RESULTS: Dual downstream blockade of the MAPK and PAM pathways was more effective in attenuating downstream molecular signals. Synergy was demonstrated for erlotinib and BEZ235 and for PD-98059 and BEZ-235. This resulted in a trend of increased growth cell cycle arrest, apoptosis, cell proliferation, and colony and migration suppression. This combination showed more efficacy in cell lines with acquired resistance to erlotinib. CONCLUSIONS: The additional mTOR blockade provided by BEZ235 in combined blockade resulted in increased anticancer effect. The hypersensitivity of ER cell lines to additional mTOR blockade suggested PAM pathway oncogenic dependence via mTOR. Dual downstream combined blockade of MAPK and PAM pathways with MEK and PI3K/mTOR inhibitor appeared most effective and represents an attractive therapeutic strategy against pancreatic cancer and its associated drug resistance.

  18. Iron deficiency down-regulates the Akt/TSC1-TSC2/mammalian Target of Rapamycin signaling pathway in rats and in COS-1 cells.

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    Ndong, Moussa; Kazami, Machiko; Suzuki, Tsukasa; Uehara, Mariko; Katsumata, Shin-Ichi; Inoue, Hirohumi; Kobayashi, Ken-Ichi; Tadokoro, Tadahiro; Suzuki, Kazuharu; Yamamoto, Yuji

    2009-09-01

    Iron deficiency (ID) is one of the most commonly known forms of nutritional deficiencies. Low body iron is thought to induce neurologic defects but may also play a protective role against cancer development by cell growth arrest. Thus, ID may affect cellular pathways controlling cell growth and proliferation, the mechanism of which is still not fully understood. The serine/threonine protein kinase Akt and its downstream target, the mammalian Target of Rapamycin (mTOR), is known to play a crucial role in the regulation of cell growth and survival. Therefore, we hypothesized that Akt/mTOR pathway could be influenced by ID. Three-week-old male Wistar-strain rats were divided into 3 groups and the 2 groups had free access to a control diet (C group) or an iron-deficient diet (D group). The third group (PF group) were pair-fed the control diet to the mean intake of the D group. After 4 weeks, rats were killed and their brains were sampled. In separate experiments, COS-1 cells were cultured with or without the iron chelator deferoxamine. Western blots of brain samples and COS-1 lysates were used to analyze the expression and phosphorylation state of Akt, TSC2, mTOR, and S6 kinase proteins implicated in the Akt/mTOR pathway. Using 2 different ID models, we show for the first time that iron deficiency depresses Akt activity in rats and in COS-1 cells, leading to a decrease in mTOR activity. PMID:19854379

  19. Targeting the PI3K-AKT-mTOR signaling network in cancer

    Institute of Scientific and Technical Information of China (English)

    Khurum H.Khan; Timothy A.Yap; Li Yan; David Cunningham

    2013-01-01

    The phosphoinositide 3-kinase-AKT-mammalian target of rapamycin (PI3K-AKT-mTOR) pathway is a frequently hyperactivated pathway in cancer and is important for tumor cell growth and survival.The development of targeted therapies against mTOR,a vital substrate along this pathway,led to the approval of allosteric inhibitors,including everolimus and temsirolimus,for the treatment of breast,renal,and pancreatic cancers.However,the suboptimal duration of response in unselected patients remains an unresolved issue.Numerous novel therapies against critical nodes of this pathway are therefore being actively investigated in the clinic in multiple tumour types.In this review,we focus on the progress of these agents in clinical development along with their biological rationale,the need of predictive biomarkers and various combination strategies,which will be useful in counteracting the mechanisms of resistance to this class of drugs.

  20. Quercetin inhibits angiogenesis mediated human prostate tumor growth by targeting VEGFR- 2 regulated AKT/mTOR/P70S6K signaling pathways.

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    Poyil Pratheeshkumar

    Full Text Available Angiogenesis is a crucial step in the growth and metastasis of cancers, since it enables the growing tumor to receive oxygen and nutrients. Cancer prevention using natural products has become an integral part of cancer control. We studied the antiangiogenic activity of quercetin using ex vivo, in vivo and in vitro models. Rat aortic ring assay showed that quercetin at non-toxic concentrations significantly inhibited microvessel sprouting and exhibited a significant inhibition in the proliferation, migration, invasion and tube formation of endothelial cells, which are key events in the process of angiogenesis. Most importantly, quercetin treatment inhibited ex vivo angiogenesis as revealed by chicken egg chorioallantoic membrane assay (CAM and matrigel plug assay. Western blot analysis showed that quercetin suppressed VEGF induced phosphorylation of VEGF receptor 2 and their downstream protein kinases AKT, mTOR, and ribosomal protein S6 kinase in HUVECs. Quercetin (20 mg/kg/d significantly reduced the volume and the weight of solid tumors in prostate xenograft mouse model, indicating that quercetin inhibited tumorigenesis by targeting angiogenesis. Furthermore, quercetin reduced the cell viability and induced apoptosis in prostate cancer cells, which were correlated with the downregulation of AKT, mTOR and P70S6K expressions. Collectively the findings in the present study suggest that quercetin inhibits tumor growth and angiogenesis by targeting VEGF-R2 regulated AKT/mTOR/P70S6K signaling pathway, and could be used as a potential drug candidate for cancer therapy.

  1. Potential Molecular Targeted Therapeutics: Role of PI3-K/Akt/mTOR Inhibition in Cancer.

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    Sokolowski, Kevin M; Koprowski, Steven; Kunnimalaiyaan, Selvi; Balamurugan, Mariappan; Gamblin, T Clark; Kunnimalaiyaan, Muthusamy

    2016-01-01

    Primary liver cancer is one of the most commonly occurring cancers worldwide. Hepatocellular carcinoma (HCC) represents the majority of primary liver cancer and is the 3rd most common cause of cancer-related deaths globally. Survival rates of patients with HCC are dependent upon early detection as concomitant liver dysfunction and advanced disease limits traditional therapeutic options such as resection or ablation. Unfortunately, at the time of diagnosis, most patients are not eligible for curative surgery and have a five-year relative survival rate less than 20%, leading to systemic therapy as the only option. Currently, sorafenib is the only approved systemic therapy; however, it has a limited survival advantage and low efficacy prompting alternative strategies. The inception of sorafenib for HCC systemic therapy and the understanding involved of cancer therapy have led to an enhanced focus of the PI3-k/Akt/mTOR pathway as a potential area of targeting including pan and isoform-specific PI3-K inhibitors, Akt blockade, and mTOR suppression. The multitude, expanding roles, and varying clinical trials of these inhibitors have led to an increase in knowledge and availability for current and future studies. In this review, we provide a review of the literature with the aim to focus on potential targets for HCC therapies as well as an in depth focus on Akt inhibition.

  2. Expression of EGFR and Molecules Downstream to PI3K/Akt, Raf-1-MEK-1-MAP (Erk1/2, and JAK (STAT3 Pathways in Invasive Lung Adenocarcinomas Resected at a Single Institution

    Directory of Open Access Journals (Sweden)

    Alba Fabiola Torres

    2014-01-01

    Full Text Available Therapies targeting EGFR are effective in treating tumors that harbor molecular alterations; however, there is heterogeneity in long-term response to these therapies. We retrospectively analyzed protein expression of EGFR, Stat3, phospho-Akt, and phospho-Erk1/2 by immunohistochemistry in a series of resected cases from a single institution, correlated with clinicopathological variables. There were 96 patients, with the majority of cases being of low stage tumors (17 pT1a, 23 pT1b, 30 pT2a, and 18 pT2b. Histologic subtypes were 45 acinar predominant, 2 cribriform, 25 solid, 7 papillary, 11 lepidic, and 4 mucinous tumors. The EGFR score was higher in tumors with vascular invasion (P=0.013, in solid and cribriform acinar histology, and in high stage tumors (P=0.006 and P=0.01. EGFR was more likely overexpressed in solid compared to lepidic tumors (P=0.02. Acinar tumors had the highest rate of ERK1/2 positivity (19%. There was a strong correlation among positivity for ERCC1 and other markers, including STAT3 (P=0.003, Akt (P=0.02, and ERK1/ERK2 (P=0.0005. Expression of molecules downstream to EGFR varied from 12% to 31% of tumors; however, the expression did not directly correlate to EGFR expression, which may suggest activation of the cascades through different pathways. The correlation of protein expression and the new lung adenocarcinoma classification may help in the understanding of activated pathways of each tumor type, which may act in the oncogenesis and drug resistance of these tumors.

  3. Down-regulation of Akt/mammalian target of rapamycin signaling pathway in response to myostatin overexpression in skeletal muscle.

    OpenAIRE

    Amirouche, Adel; Durieux, Anne-Cécile; Banzet, Sébastien; Koulmann, Nathalie; Bonnefoy, Régis; Mouret, Catherine; Bigard, Xavier; Peinnequin, André; Freyssenet, Damien

    2009-01-01

    Myostatin, a member of the TGF-beta family, has been identified as a master regulator of embryonic myogenesis and early postnatal skeletal muscle growth. However, cumulative evidence also suggests that alterations in skeletal muscle mass are associated with dysregulation in myostatin expression and that myostatin may contribute to muscle mass loss in adulthood. Two major branches of the Akt pathway are relevant for the regulation of skeletal muscle mass, the Akt/mammalian target of rapamycin ...

  4. Uncovering potential downstream targets of oncogenic GRPR overexpression in prostate carcinomas harboring ETS rearrangements.

    Science.gov (United States)

    Santos, Joana; Mesquita, Diana; Barros-Silva, João D; Jerónimo, Carmen; Henrique, Rui; Morais, António; Paulo, Paula; Teixeira, Manuel R

    2015-01-01

    Gastrin-releasing peptide receptor (GRPR) is known to be overexpressed in several human malignancies, including prostate cancer, and has been implicated in multiple important neoplastic signaling pathways. We recently have shown that GRPR is an ERG and ETV1 target gene in prostate cancer, using a genome-wide scale and exon-level expression microarray platform. Due to its cellular localization, the relevance of its function and the availability of blocking agents, GRPR seems to be a promising candidate as therapeutic target. Our present work shows that effective knockdown of GRPR in LNCaP and VCaP cells attenuates their malignant phenotype by decreasing proliferation, invasion and anchorage-independent growth, while increasing apoptosis. Using an antibody microarray we were able to validate known and identify new targets of GRPR pathway, namely AKT1, PKCε, TYK2 and MST1. Finally, we show that overexpression of these GRPR targets is restricted to prostate carcinomas harboring ERG and/or ETV1 rearrangements, establishing their potential as therapeutic targets for these particular molecular subsets of the disease. PMID:26097883

  5. Anti-Breast Cancer Potential of Quercetin via the Akt/AMPK/Mammalian Target of Rapamycin (mTOR) Signaling Cascade

    Science.gov (United States)

    Rivera Rivera, Amilcar; Castillo-Pichardo, Linette; Gerena, Yamil; Dharmawardhane, Suranganie

    2016-01-01

    The Akt/adenosine monophosphate protein kinase (AMPK)/mammalian target of rapamycin (mTOR) pathway has emerged as a critical signaling nexus for regulating cellular metabolism, energy homeostasis, and cell growth. Thus, dysregulation of this pathway contributes to the development of metabolic disorders such as obesity, type 2diabetes, and cancer. We previously reported that a combination of grape polyphenols (resveratrol, quercetin and catechin: RQC), at equimolar concentrations, reduces breast cancer (BC) growth and metastasis in nude mice, and inhibits Akt and mTOR activities and activates AMPK, an endogenous inhibitor of mTOR, in metastatic BC cells. The objective of the present study was to determine the contribution of individual polyphenols to the effect of combined RQC on mTOR signaling. Metastatic BC cells were treated with RQC individually or in combination, at various concentrations, and the activities (phosphorylation) of AMPK, Akt, and the mTOR downstream effectors, p70S6 kinase (p70S6K) and 4E binding protein (4EBP1), were determined by Western blot. Results show that quercetin was the most effective compound for Akt/mTOR inhibition. Treatment with quercetin at 15μM had a similar effect as the RQC combination in the inhibition of BC cell proliferation, apoptosis, and migration. However, cell cycle analysis showed that the RQC treatment arrested BC cells in the G1 phase, while quercetin arrested the cell cycle in G2/M. In vivo experiments, using SCID mice with implanted tumors from metastatic BC cells, demonstrated that administration of quercetin at 15mg/kg body weight resulted in a ~70% reduction in tumor growth. In conclusion, quercetin appears to be a viable grape polyphenol for future development as an anti BC therapeutic. PMID:27285995

  6. Anti-Breast Cancer Potential of Quercetin via the Akt/AMPK/Mammalian Target of Rapamycin (mTOR) Signaling Cascade.

    Science.gov (United States)

    Rivera Rivera, Amilcar; Castillo-Pichardo, Linette; Gerena, Yamil; Dharmawardhane, Suranganie

    2016-01-01

    The Akt/adenosine monophosphate protein kinase (AMPK)/mammalian target of rapamycin (mTOR) pathway has emerged as a critical signaling nexus for regulating cellular metabolism, energy homeostasis, and cell growth. Thus, dysregulation of this pathway contributes to the development of metabolic disorders such as obesity, type 2diabetes, and cancer. We previously reported that a combination of grape polyphenols (resveratrol, quercetin and catechin: RQC), at equimolar concentrations, reduces breast cancer (BC) growth and metastasis in nude mice, and inhibits Akt and mTOR activities and activates AMPK, an endogenous inhibitor of mTOR, in metastatic BC cells. The objective of the present study was to determine the contribution of individual polyphenols to the effect of combined RQC on mTOR signaling. Metastatic BC cells were treated with RQC individually or in combination, at various concentrations, and the activities (phosphorylation) of AMPK, Akt, and the mTOR downstream effectors, p70S6 kinase (p70S6K) and 4E binding protein (4EBP1), were determined by Western blot. Results show that quercetin was the most effective compound for Akt/mTOR inhibition. Treatment with quercetin at 15μM had a similar effect as the RQC combination in the inhibition of BC cell proliferation, apoptosis, and migration. However, cell cycle analysis showed that the RQC treatment arrested BC cells in the G1 phase, while quercetin arrested the cell cycle in G2/M. In vivo experiments, using SCID mice with implanted tumors from metastatic BC cells, demonstrated that administration of quercetin at 15mg/kg body weight resulted in a ~70% reduction in tumor growth. In conclusion, quercetin appears to be a viable grape polyphenol for future development as an anti BC therapeutic. PMID:27285995

  7. Anti-Breast Cancer Potential of Quercetin via the Akt/AMPK/Mammalian Target of Rapamycin (mTOR Signaling Cascade.

    Directory of Open Access Journals (Sweden)

    Amilcar Rivera Rivera

    Full Text Available The Akt/adenosine monophosphate protein kinase (AMPK/mammalian target of rapamycin (mTOR pathway has emerged as a critical signaling nexus for regulating cellular metabolism, energy homeostasis, and cell growth. Thus, dysregulation of this pathway contributes to the development of metabolic disorders such as obesity, type 2diabetes, and cancer. We previously reported that a combination of grape polyphenols (resveratrol, quercetin and catechin: RQC, at equimolar concentrations, reduces breast cancer (BC growth and metastasis in nude mice, and inhibits Akt and mTOR activities and activates AMPK, an endogenous inhibitor of mTOR, in metastatic BC cells. The objective of the present study was to determine the contribution of individual polyphenols to the effect of combined RQC on mTOR signaling. Metastatic BC cells were treated with RQC individually or in combination, at various concentrations, and the activities (phosphorylation of AMPK, Akt, and the mTOR downstream effectors, p70S6 kinase (p70S6K and 4E binding protein (4EBP1, were determined by Western blot. Results show that quercetin was the most effective compound for Akt/mTOR inhibition. Treatment with quercetin at 15μM had a similar effect as the RQC combination in the inhibition of BC cell proliferation, apoptosis, and migration. However, cell cycle analysis showed that the RQC treatment arrested BC cells in the G1 phase, while quercetin arrested the cell cycle in G2/M. In vivo experiments, using SCID mice with implanted tumors from metastatic BC cells, demonstrated that administration of quercetin at 15mg/kg body weight resulted in a ~70% reduction in tumor growth. In conclusion, quercetin appears to be a viable grape polyphenol for future development as an anti BC therapeutic.

  8. Expected signal for the TBID and the ionization chambers downstream of the CNGS target station

    CERN Document Server

    Sarchiapone, L; Gschwendtner, E; Lorenzo-Sentis, M

    2006-01-01

    Downstream of the carbon graphite target of the CNGS (CERN Neutrinos to Gran Sasso) facility at CERN a secondary emission monitor called TBID (Target Beam Instrumentation Downstream) is installed to measure the multiplicities and the left/right as well as up/down asymmetries of secondary particles from the target. Calculations show that the titanium windows used to close off the TBID vacuum tank might not withstand the highest beam intensities with small spot sizes expected at CNGS, in case the proton beam accidentally misses the 4-5 mm diameter target rods. Therefore it has been suggested to place two ionisation chambers as a backup for the TBID, located left and right of the TBID monitor. Monte Carlo simulations with the particle transport code FLUKA were performed firstly to obtain the fluence of charged particles in the region of interest and secondly to estimate the induced radioactivity (background signal) in this area. This allows to assess the actual signal/noise situation and thus to determine the op...

  9. Using mechanistic Bayesian networks to identify downstream targets of the Sonic Hedgehog pathway

    Directory of Open Access Journals (Sweden)

    McMahon Andrew P

    2009-12-01

    Full Text Available Abstract Background The topology of a biological pathway provides clues as to how a pathway operates, but rationally using this topology information with observed gene expression data remains a challenge. Results We introduce a new general-purpose analytic method called Mechanistic Bayesian Networks (MBNs that allows for the integration of gene expression data and known constraints within a signal or regulatory pathway to predict new downstream pathway targets. The MBN framework is implemented in an open-source Bayesian network learning package, the Python Environment for Bayesian Learning (PEBL. We demonstrate how MBNs can be used by modeling the early steps of the sonic hedgehog pathway using gene expression data from different developmental stages and genetic backgrounds in mouse. Using the MBN approach we are able to automatically identify many of the known downstream targets of the hedgehog pathway such as Gas1 and Gli1, along with a short list of likely targets such as Mig12. Conclusions The MBN approach shown here can easily be extended to other pathways and data types to yield a more mechanistic framework for learning genetic regulatory models.

  10. Exploration of FoxM1 and downstream related target molecule expression in cervical cancer tissue

    Institute of Scientific and Technical Information of China (English)

    Yi-Chong Yuan; QiongYang

    2016-01-01

    Objective:To study the expression of FoxM1 and downstream related target molecules in cervical cancer tissue.Methods:Cervical cancer tissue and normal cervical tissue were collected to detect the expression of FoxM1, proliferation-related genes (CDK6 and CDK8) and angiogenesis-related genes (VEGFA, VEGFB and VEGFC); Hela cells were cultured and transfected with FoxM1 siRNA, and then expression of CDK6, CDK8, VEGFA, VEGFB and VEGFC were detected.Results:mRNA contents of FoxM1, CDK6, CDK8, VEGFA, VEGFB and VEGFC in cervical cancer tissue were significantly higher than those in normal cervical tissue; mRNA content of FoxM1 was positively correlated with mRNA contents of CDK6, CDK8, VEGFA, VEGFB and VEGFC; mRNA contents of CDK6, CDK8, VEGFA, VEGFB and VEGFC of FoxM1-siRNA group were significantly lower than those of negative control-siRNA group.Conclusion:FoxM1 expression abnormally increases in cervical cancer tissue, and its downstream target genes include CDK6, CDK8, VEGFA, VEGFB and VEGFC.

  11. Targeting Protein Kinase C Downstream of Growth Factor and Adhesion Signalling

    Energy Technology Data Exchange (ETDEWEB)

    Dowling, Catríona M., E-mail: Catriona.Dowling@ul.ie; Kiely, Patrick A., E-mail: Catriona.Dowling@ul.ie [Department of Life Sciences, Materials and Surface Science Institute and Stokes Institute, University of Limerick, Limerick 78666 (Ireland); Health Research Institute (HRI), University of Limerick, Limerick 78666 (Ireland)

    2015-07-15

    The signaling outputs of Receptor Tyrosine Kinases, G-protein coupled receptors and integrins converge to mediate key cell process such as cell adhesion, cell migration, cell invasion and cell proliferation. Once activated by their ligands, these cell surface proteins recruit and direct a diverse range of proteins to disseminate the appropriate response downstream of the specific environmental cues. One of the key groups of proteins required to regulate these activities is the family of serine/threonine intracellular kinases called Protein Kinase Cs. The activity and subcellular location of PKCs are mediated by a series of tightly regulated events and is dependent on several posttranslational modifications and the availability of second messengers. Protein Kinase Cs exhibit both pro- and anti-tumorigenic effects making them an interesting target for anti-cancer treatment.

  12. Proliferating cell nuclear antigen (Pcna) as a direct downstream target gene of Hoxc8

    Energy Technology Data Exchange (ETDEWEB)

    Min, Hyehyun; Lee, Ji-Yeon; Bok, Jinwoong; Chung, Hyun Joo [Department of Anatomy, Embryology Laboratory, Brain Korea 21 Project for Medical Science, Yonsei University College of Medicine, Seoul 120-752 (Korea, Republic of); Kim, Myoung Hee, E-mail: mhkim1@yuhs.ac [Department of Anatomy, Embryology Laboratory, Brain Korea 21 Project for Medical Science, Yonsei University College of Medicine, Seoul 120-752 (Korea, Republic of)

    2010-02-19

    Hoxc8 is a member of Hox family transcription factors that play crucial roles in spatiotemporal body patterning during embryogenesis. Hox proteins contain a conserved 61 amino acid homeodomain, which is responsible for recognition and binding of the proteins onto Hox-specific DNA binding motifs and regulates expression of their target genes. Previously, using proteome analysis, we identified Proliferating cell nuclear antigen (Pcna) as one of the putative target genes of Hoxc8. Here, we asked whether Hoxc8 regulates Pcna expression by directly binding to the regulatory sequence of Pcna. In mouse embryos at embryonic day 11.5, the expression pattern of Pcna was similar to that of Hoxc8 along the anteroposterior body axis. Moreover, Pcna transcript levels as well as cell proliferation rate were increased by overexpression of Hoxc8 in C3H10T1/2 mouse embryonic fibroblast cells. Characterization of 2.3 kb genomic sequence upstream of Pcna coding region revealed that the upstream sequence contains several Hox core binding sequences and one Hox-Pbx binding sequence. Direct binding of Hoxc8 proteins to the Pcna regulatory sequence was verified by chromatin immunoprecipitation assay. Taken together, our data suggest that Pcna is a direct downstream target of Hoxc8.

  13. Vacuum calculation of the TBID (Target Beam Instrumentation Downstream) for CNGS

    CERN Document Server

    Chiggiato, P

    2006-01-01

    For the TBID (Target Beam Instrumentation Downstream) monitor of CNGS (CERN Neutrinos to Gran Sasso) a design identical to that of the TBIU (Target Beam Instrumentation Upstream) monitor of the SPS targets has been adopted. However the vacuum solution implemented in the SPS, namely ion pumping, cannot be trivially applied to CNGS; in fact the high radioactivity level expected for the latter could damage the electrical cables in the about 10 years of expected activity and hinder or delay any human intervention. In this note the possibility of NEG pumping, without any electrical feeding, is considered. The getter should ensure the demanded pressure limit of 10-4 Torr in conditions that are very harsh: temperature up to 150°C, 0.1 A electron bombardment and proton bombardment during the 10 years of working of the monitor. It is shown that the beam induced CH4 pumping speed is not enough at the beginning of the operation to ensure the pressure requirements. In addition, in the worst scenario when a uniform ele...

  14. Computer-Aided Targeting of the PI3K/Akt/mTOR Pathway: Toxicity Reduction and Therapeutic Opportunities

    Directory of Open Access Journals (Sweden)

    Tan Li

    2014-10-01

    Full Text Available The PI3K/Akt/mTOR pathway plays an essential role in a wide range of biological functions, including metabolism, macromolecular synthesis, cell growth, proliferation and survival. Its versatility, however, makes it a conspicuous target of many pathogens; and the consequential deregulations of this pathway often lead to complications, such as tumorigenesis, type 2 diabetes and cardiovascular diseases. Molecular targeted therapy, aimed at modulating the deregulated pathway, holds great promise for controlling these diseases, though side effects may be inevitable, given the ubiquity of the pathway in cell functions. Here, we review a variety of factors found to modulate the PI3K/Akt/mTOR pathway, including gene mutations, certain metabolites, inflammatory factors, chemical toxicants, drugs found to rectify the pathway, as well as viruses that hijack the pathway for their own synthetic purposes. Furthermore, this evidence of PI3K/Akt/mTOR pathway alteration and related pathogenesis has inspired the exploration of computer-aided targeting of this pathway to optimize therapeutic strategies. Herein, we discuss several possible options, using computer-aided targeting, to reduce the toxicity of molecularly-targeted therapy, including mathematical modeling, to reveal system-level control mechanisms and to confer a low-dosage combination therapy, the potential of PP2A as a therapeutic target, the formulation of parameters to identify patients who would most benefit from specific targeted therapies and molecular dynamics simulations and docking studies to discover drugs that are isoform specific or mutation selective so as to avoid undesired broad inhibitions. We hope this review will stimulate novel ideas for pharmaceutical discovery and deepen our understanding of curability and toxicity by targeting the PI3K/Akt/mTOR pathway.

  15. Black raspberry extracts inhibit benzo(a)pyrene diol-epoxide-induced activator protein 1 activation and VEGF transcription by targeting the phosphotidylinositol 3-kinase/Akt pathway.

    Science.gov (United States)

    Huang, Chuanshu; Li, Jingxia; Song, Lun; Zhang, Dongyun; Tong, Qiangsong; Ding, Min; Bowman, Linda; Aziz, Robeena; Stoner, Gary D

    2006-01-01

    Previous studies have shown that freeze-dried black raspberry extract fractions inhibit benzo(a)pyrene [B(a)P]-induced transformation of Syrian hamster embryo cells and benzo(a)pyrene diol-epoxide [B(a)PDE]-induced activator protein-1 (AP-1) activity in mouse epidermal Cl 41 cells. The phosphotidylinositol 3-kinase (PI-3K)/Akt pathway is critical for B(a)PDE-induced AP-1 activation in mouse epidermal Cl 41 cells. In the present study, we determined the potential involvement of PI-3K and its downstream kinases on the inhibition of AP-1 activation by black raspberry fractions, RO-FOO3, RO-FOO4, RO-ME, and RO-DM. In addition, we investigated the effects of these fractions on the expression of the AP-1 target genes, vascular endothelial growth factor (VEGF) and inducible nitric oxide synthase (iNOS). Pretreatment of Cl 41 cells with fractions RO-F003 and RO-ME reduced activation of AP-1 and the expression of VEGF, but not iNOS. In contrast, fractions RO-F004 and RO-DM had no effect on AP-1 activation or the expression of either VEGF or iNOS. Consistent with inhibition of AP-1 activation, the RO-ME fraction markedly inhibited activation of PI-3K, Akt, and p70 S6 kinase (p70(S6k)). In addition, overexpression of the dominant negative PI-3K mutant delta p85 reduced the induction of VEGF by B(a)PDE. It is likely that the inhibitory effects of fractions RO-FOO3 and RO-ME on B(a)PDE-induced AP-1 activation and VEGF expression are mediated by inhibition of the PI-3K/Akt pathway. In view of the important roles of AP-1 and VEGF in tumor development, one mechanism for the chemopreventive activity of black raspberries may be inhibition of the PI-3K/Akt/AP-1/VEGF pathway. PMID:16397275

  16. Black raspberry extracts inhibit benzo(a)pyrene diol-epoxide-induced activator protein 1 activation and VEGF transcription by targeting the phosphotidylinositol 3-kinase/Akt pathway.

    Science.gov (United States)

    Huang, Chuanshu; Li, Jingxia; Song, Lun; Zhang, Dongyun; Tong, Qiangsong; Ding, Min; Bowman, Linda; Aziz, Robeena; Stoner, Gary D

    2006-01-01

    Previous studies have shown that freeze-dried black raspberry extract fractions inhibit benzo(a)pyrene [B(a)P]-induced transformation of Syrian hamster embryo cells and benzo(a)pyrene diol-epoxide [B(a)PDE]-induced activator protein-1 (AP-1) activity in mouse epidermal Cl 41 cells. The phosphotidylinositol 3-kinase (PI-3K)/Akt pathway is critical for B(a)PDE-induced AP-1 activation in mouse epidermal Cl 41 cells. In the present study, we determined the potential involvement of PI-3K and its downstream kinases on the inhibition of AP-1 activation by black raspberry fractions, RO-FOO3, RO-FOO4, RO-ME, and RO-DM. In addition, we investigated the effects of these fractions on the expression of the AP-1 target genes, vascular endothelial growth factor (VEGF) and inducible nitric oxide synthase (iNOS). Pretreatment of Cl 41 cells with fractions RO-F003 and RO-ME reduced activation of AP-1 and the expression of VEGF, but not iNOS. In contrast, fractions RO-F004 and RO-DM had no effect on AP-1 activation or the expression of either VEGF or iNOS. Consistent with inhibition of AP-1 activation, the RO-ME fraction markedly inhibited activation of PI-3K, Akt, and p70 S6 kinase (p70(S6k)). In addition, overexpression of the dominant negative PI-3K mutant delta p85 reduced the induction of VEGF by B(a)PDE. It is likely that the inhibitory effects of fractions RO-FOO3 and RO-ME on B(a)PDE-induced AP-1 activation and VEGF expression are mediated by inhibition of the PI-3K/Akt pathway. In view of the important roles of AP-1 and VEGF in tumor development, one mechanism for the chemopreventive activity of black raspberries may be inhibition of the PI-3K/Akt/AP-1/VEGF pathway.

  17. Targeting the phosphatidylinositol 3-kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) pathway: an emerging treatment strategy for squamous cell lung carcinoma.

    Science.gov (United States)

    Beck, Joseph Thaddeus; Ismail, Amen; Tolomeo, Christina

    2014-09-01

    Squamous cell lung carcinoma accounts for approximately 30% of all non-small cell lung cancers (NSCLCs). Despite progress in the understanding of the biology of cancer, cytotoxic chemotherapy remains the standard of care for patients with squamous cell lung carcinoma, but the prognosis is generally poor. The phosphatidylinositol 3-kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) pathway is one of the most commonly activated signaling pathways in cancer, leading to cell proliferation, survival, and differentiation. It has therefore become a major focus of clinical research. Various alterations in the PI3K/AKT/mTOR pathway have been identified in squamous cell lung carcinoma and a number of agents targeting these alterations are in clinical development for use as single agents and in combination with other targeted and conventional treatments. These include pan-PI3K inhibitors, isoform-specific PI3K inhibitors, AKT inhibitors, mTOR inhibitors, and dual PI3K/mTOR inhibitors. These agents have demonstrated antitumor activity in preclinical models of NSCLC and preliminary clinical evidence is also available for some agents. This review will discuss the role of the PI3K/AKT/mTOR pathway in cancer and how the discovery of genetic alterations in this pathway in patients with squamous cell lung carcinoma can inform the development of targeted therapies for this disease. An overview of ongoing clinical trials investigating PI3K/AKT/mTOR pathway inhibitors in squamous cell lung carcinoma will also be included.

  18. Targeting AKT with the allosteric AKT inhibitor MK-2206 in non-small cell lung cancer cells with acquired resistance to cetuximab.

    Science.gov (United States)

    Iida, Mari; Brand, Toni M; Campbell, David A; Starr, Megan M; Luthar, Neha; Traynor, Anne M; Wheeler, Deric L

    2013-06-01

    The epidermal growth factor receptor (EGFR) is a central regulator of tumor progression in human cancers. Cetuximab is an anti-EGFR monoclonal antibody that has been approved for use in oncology. Despite clinical success the majority of patients do not respond to cetuximab and those who initially respond frequently acquire resistance. To understand how tumor cells acquire resistance to cetuximab we developed a model of resistance using the non-small cell lung cancer line NCI-H226. We found that cetuximab-resistant (Ctx (R) ) clones manifested strong activation of EGFR, PI3K/AKT and MAPK. To investigate the role of AKT signaling in cetuximab resistance we analyzed the activation of the AKT pathway effector molecules using a human AKT phospho-antibody array. Strong activation was observed in Ctx (R) clones for several key AKT substrates including c-jun, GSK3β, eIF4E, rpS6, IKKα, IRS-1 and Raf1. Inhibition of AKT signaling by siAKT1/2 or by the allosteric AKT inhibitor MK-2206 resulted in robust inhibition of cell proliferation in all Ctx (R) clones. Moreover, the combinational treatment of cetuximab and MK-2206 resulted in further decreases in proliferation than either drug alone. This combinatorial treatment resulted in decreased activity of both AKT and MAPK thus highlighting the importance of simultaneous pathway inhibition to maximally affect the growth of Ctx (R) cells. Collectively, our findings demonstrate that AKT activation is an important pathway in acquired resistance to cetuximab and suggests that combinatorial therapy directed at both the AKT and EGFR/MAPK pathways may be beneficial in this setting. PMID:23760490

  19. Activated Ras alters lens and corneal development through induction of distinct downstream targets

    Directory of Open Access Journals (Sweden)

    Reneker Lixing

    2010-01-01

    results suggest that Ras activation a induces distinct sets of downstream targets in the lens and cornea resulting in distinct cellular responses and b is sufficient for initiation but not completion of lens fiber differentiation.

  20. Akt kinase-interacting protein1, a novel therapeutic target for lung cancer with EGFR-activating and gatekeeper mutations.

    Science.gov (United States)

    Yamada, T; Takeuchi, S; Fujita, N; Nakamura, A; Wang, W; Li, Q; Oda, M; Mitsudomi, T; Yatabe, Y; Sekido, Y; Yoshida, J; Higashiyama, M; Noguchi, M; Uehara, H; Nishioka, Y; Sone, S; Yano, S

    2013-09-12

    Despite initial dramatic response, epidermal growth factor receptor (EGFR) mutant lung cancer patients always acquire resistance to EGFR-tyrosine kinase inhibitors (TKIs). Gatekeeper T790M mutation in EGFR is the most prevalent genetic alteration underlying acquired resistance to EGFR-TKI, and EGFR mutant lung cancer cells are reported to be addictive to EGFR/Akt signaling even after acquired T790M mutation. Here, we focused on Akt kinase-interacting protein1 (Aki1), a scaffold protein of PI3K (phosphoinositide 3-kinase)/PDK1 (3-phosphoinositide-dependent protein kinase)/Akt that determines receptor signal selectivity for non-mutated EGFR, and assessed its role in EGFR mutant lung cancer with or without gatekeeper T790M mutation. Cell line-based assays showed that Aki1 constitutively associates with mutant EGFR in lung cancer cells with (H1975) or without (PC-9 and HCC827) T790M gatekeeper mutation. Silencing of Aki1 induced apoptosis of EGFR mutant lung cancer cells. Treatment with Aki1 siRNA dramatically inhibited growth of H1975 cells in a xenograft model. Moreover, silencing of Aki1 further potentiated growth inhibitory effect of new generation EGFR-TKIs against H1975 cells in vitro. Aki1 was frequently expressed in tumor cells of EGFR mutant lung cancer patients (53/56 cases), including those with acquired resistance to EGFR-TKI treatment (7/7 cases). Our data suggest that Aki1 may be a critical mediator of survival signaling from mutant EGFR to Akt, and may therefore be an ideal target for EGFR mutant lung cancer patients, especially those with acquired EGFR-TKI resistance due to EGFR T790M gatekeeper mutation. PMID:23045273

  1. Targeting EMP3 suppresses proliferation and invasion of hepatocellular carcinoma cells through inactivation of PI3K/Akt pathway.

    Science.gov (United States)

    Hsieh, Yi-Hsien; Hsieh, Shu-Ching; Lee, Chien-Hsing; Yang, Shun-Fa; Cheng, Chun-Wen; Tang, Meng-Ju; Lin, Chia-Liang; Lin, Chu-Liang; Chou, Ruey-Hwang

    2015-10-27

    Epithelial membrane protein-3 (EMP3), a typical member of the epithelial membrane protein (EMP) family, is epigenetically silenced in some cancer types, and has been proposed to be a tumor suppressor gene. However, its effects on tumor suppression are controversial and its roles in development and malignancy of hepatocellular carcinoma (HCC) remain unclear. In the present study, we found that EMP3 was highly expressed in the tumorous tissues comparing to the matched normal tissues, and negatively correlated with differentiated degree of HCC patients. Knockdown of EMP3 significantly reduced cell proliferation, arrested cell cycle at G1 phase, and inhibited the motility and invasiveness in accordance with the decreased expression and activity of urokinase plasminogen activator (uPA) and matrix metalloproteinase 9 (MMP-9) in HCC cells. The in vivo tumor growth of HCC was effectively suppressed by knockdown of EMP3 in a xenograft mouse model. The EMP3 knockdown-reduced cell proliferation and invasion were attenuated by inhibition of phosphatidylinositol 3-kinase (PI3K) or knockdown of Akt, and rescued by overexpression of Akt in HCC cells. Clinical positive correlations of EMP3 with p85 regulatory subunit of PI3K, p-Akt, uPA, as well as MMP-9 were observed in the tissue sections from HCC patients. Here, we elucidated the tumor progressive effects of EMP3 through PI3K/Akt pathway and uPA/MMP-9 cascade in HCC cells. The findings provided a new insight into EMP3, which might be a potential molecular target for diagnosis and treatment of HCC. PMID:26472188

  2. MiR-221 Promotes Capan-2 Pancreatic Ductal Adenocarcinoma Cells Proliferation by Targeting PTEN-Akt

    Directory of Open Access Journals (Sweden)

    Wenzhuo Yang

    2016-05-01

    Full Text Available Background/Aims: MicroRNAs (miRNAs, miRs have emerged as critical regulators of cancer cell proliferation. The effect of miR-221 on cancer cell growth could be significantly changeable in different cell lines. Although miR-221 was reported to promote the cell growth of pancreatic ductal adenocarcinoma (PDAC cells, its role in Capan-2 cell line is largely unknown. Methods: Capan-2 cells were transfected with miR-221 mimics, inhibitors, or negative controls. Cell Counting Kit-8 was used to determine cell viability. EdU staining and cell cycle analysis were used to measure cell proliferation. Western blotting was used to detect the expression levels of PTEN and phospho-Akt. The PI3K-Akt pathway activator SC-79 and inhibitor LY294002 were used to perform the rescue experiment in determining cell proliferation. Results: Overexpressing miR-221 significantly increased cell vitality and promoted cell proliferation and G1-to-S phase transition of the cell cycle in Capan-2 cells, while inhibition of miR-221 decreased that. The protein level of PTEN in Capan-2 cells was downregulated by overexpressing miR-221, while upregulated by inhibiting miR-221. Consistently, enhanced phosphorylation of AktSer473 was observed in miR-221 overexpressed Capan-2 cells, and the opposite result was found in miR-221 inhibited cells. LY294002 restored the pro-proliferation effect of miR-221 on Capan-2 cells, while SC-79 had no additional effect on cell proliferation in Capan-2 cells transfected with miR-221 mimics. Conclusion: Our study indicates that miR-221 is an oncogenic miRNA which promotes Capan-2 cells proliferation by targeting PTEN-Akt pathway.

  3. KNK437, abrogates hypoxia-induced radioresistance by dual targeting of the AKT and HIF-1{alpha} survival pathways

    Energy Technology Data Exchange (ETDEWEB)

    Oommen, Deepu, E-mail: oommen1978@gmail.com [Centre for Cancer Research and Cell Biology, Queen' s University Belfast, Belfast, Northern Ireland (United Kingdom); Prise, Kevin M. [Centre for Cancer Research and Cell Biology, Queen' s University Belfast, Belfast, Northern Ireland (United Kingdom)

    2012-05-11

    Highlights: Black-Right-Pointing-Pointer KNK437, a benzylidene lactam compound, is a novel radiosensitizer. Black-Right-Pointing-Pointer KNK437 inhibits AKT signaling and abrogates the accumulation of HIF-1{alpha} under hypoxia. Black-Right-Pointing-Pointer KNK437 abrogates hypoxia induced resistance to radiation. -- Abstract: KNK437 is a benzylidene lactam compound known to inhibit stress-induced synthesis of heat shock proteins (HSPs). HSPs promote radioresistance and play a major role in stabilizing hypoxia inducible factor-1{alpha} (HIF-1{alpha}). HIF-1{alpha} is widely responsible for tumor resistance to radiation under hypoxic conditions. We hypothesized that KNK437 sensitizes cancer cells to radiation and overrides hypoxia-induced radioresistance via destabilizing HIF-1{alpha}. Treatment of human cancer cells MDA-MB-231 and T98G with KNK437 sensitized them to ionizing radiation (IR). Surprisingly, IR did not induce HSPs in these cell lines. As hypothesized, KNK437 abrogated the accumulation of HIF-1{alpha} in hypoxic cells. However, there was no induction of HSPs under hypoxic conditions. Moreover, the proteosome inhibitor MG132 did not restore HIF-1{alpha} levels in KNK437-treated cells. This suggested that the absence of HIF-1{alpha} in hypoxic cells was not due to the enhanced protein degradation. HIF-1{alpha} is mainly regulated at the level of post-transcription and AKT is known to modulate the translation of HIF-1{alpha} mRNA. Interestingly, pre-treatment of cells with KNK437 inhibited AKT signaling. Furthermore, down regulation of AKT by siRNA abrogated HIF-1{alpha} levels under hypoxia. Interestingly, KNK437 reduced cell survival in hypoxic conditions and inhibited hypoxia-induced resistance to radiation. Taken together, these data suggest that KNK437 is an effective radiosensitizer that targets multiple pro-survival stress response pathways.

  4. Akt1 mediates neuronal differentiation in zebrafish via a reciprocal interaction with notch signaling.

    Directory of Open Access Journals (Sweden)

    Yi-Chuan Cheng

    Full Text Available Akt1 is well known for its role in regulating cell proliferation, differentiation, and apoptosis and is implicated in tumors and several neurological disorders. However, the role of Akt1 in neural development has not been well defined. We have isolated zebrafish akt1 and shown that this gene is primarily transcribed in the developing nervous system, and its spatiotemporal expression pattern suggests a role in neural differentiation. Injection of akt1 morpholinos resulted in loss of neuronal precursors with a concomitant increase in post-mitotic neurons, indicating that knockdown of Akt1 is sufficient to cause premature differentiation of neurons. A similar phenotype was observed in embryos deficient for Notch signaling. Both the ligand (deltaA and the downstream target of Notch (her8a were downregulated in akt1 morphants, indicating that Akt1 is required for Delta-Notch signaling. Furthermore, akt1 expression was downregulated in Delta-Notch signaling-deficient embryos and could be induced by constitutive activation of Notch signaling. In addition, knockdown of Akt1 was able to nullify the inhibition of neuronal differentiation caused by constitutive activation of Notch signaling. Taken together, these results provide in vivo evidence that Akt1 interacts with Notch signaling reciprocally and provide an explanation of why Akt1 is essential for the inhibition of neuronal differentiation.

  5. Wogonin inhibits the proliferation and invasion, and induces the apoptosis of HepG2 and Bel7402 HCC cells through NF‑κB/Bcl-2, EGFR and EGFR downstream ERK/AKT signaling.

    Science.gov (United States)

    Liu, Xiaodong; Tian, Shuo; Liu, Mei; Jian, Lingyan; Zhao, Limei

    2016-10-01

    The anticancer effects of the natural flavonoid, wogonin, have been reported. However, its molecular mechanisms of action have not yet been fully explored. In the present study, we aimed to examine the molecular mechanisms of action of wogonin and its effects on the biological behavior of the HepG2 and Bel7402 hepatocellular carcinoma (HCC) cell lines. We also examined the effects of wogonin on nuclear factor-κB (NF-κB)/Bcl-2 and epidermal growth factor receptor (EGFR) signaling, as well as on downstream pathways of EGFR, namely extracellular signal-regulated kinase (ERK)/AKT signaling. We found that treatment with wogonin inhibited the proliferation and invasion, and induced the apoptosis of the HepG2 and Bel7402 cells. In addition, treatment with wogonin decreased cyclin D1, cyclin E, CDK4/6, Bcl-2 and matrix metalloproteinase 2 (MMP2) expression, and promoted the cleavage of caspase-3 and caspase-9 in a concentration-dependent manner. Further experiments revealed that wogonin inhibited NF-κB/Bcl-2 signaling by decreasing the IκB and p65 phosphorylation levels. Wogonin also inhibited the activation of the EGFR (Tyr845) signaling pathway, and that of downstream pathways of EGFR, namely ERK/AKT/MMP2 signaling. The depletion of EGFR by siRNA partly abolished the inhibitory effects of wogonin on cyclin D1, MMP2 expression. On the whole, our our findings demonstrate that wogonin effectively suppresses the proliferation, invasion and survival of HCC cells through the modulation of the NF-κB and EGFR signaling pathways.

  6. Drosophila Fascin is a novel downstream target of prostaglandin signaling during actin remodeling

    OpenAIRE

    Groen, Christopher M.; Spracklen, Andrew J.; Fagan, Tiffany N.; Tootle, Tina L.

    2012-01-01

    Although prostaglandins (PGs)—lipid signals produced downstream of cyclooxygenase (COX) enzymes—regulate actin cytoskeletal dynamics, their mechanisms of action are unknown. We previously established Drosophila oogenesis, in particular nurse cell dumping, as a new model to determine how PGs regulate actin remodeling. PGs, and thus the Drosophila COX-like enzyme Pxt, are required for both the parallel actin filament bundle formation and the cortical actin strengthening required for dumping. He...

  7. Inhibition of HSP27 alone or in combination with pAKT inhibition as therapeutic approaches to target SPARC-induced glioma cell survival

    Directory of Open Access Journals (Sweden)

    Schultz Chad R

    2012-04-01

    Full Text Available Abstract Background The current treatment regimen for glioma patients is surgery, followed by radiation therapy plus temozolomide (TMZ, followed by 6 months of adjuvant TMZ. Despite this aggressive treatment regimen, the overall survival of all surgically treated GBM patients remains dismal, and additional or different therapies are required. Depending on the cancer type, SPARC has been proposed both as a therapeutic target and as a therapeutic agent. In glioma, SPARC promotes invasion via upregulation of the p38 MAPK/MAPKAPK2/HSP27 signaling pathway, and promotes tumor cell survival by upregulating pAKT. As HSP27 and AKT interact to regulate the activity of each other, we determined whether inhibition of HSP27 was better than targeting SPARC as a therapeutic approach to inhibit both SPARC-induced glioma cell invasion and survival. Results Our studies found the following. 1 SPARC increases the expression of tumor cell pro-survival and pro-death protein signaling in balance, and, as a net result, tumor cell survival remains unchanged. 2 Suppressing SPARC increases tumor cell survival, indicating it is not a good therapeutic target. 3 Suppressing HSP27 decreases tumor cell survival in all gliomas, but is more effective in SPARC-expressing tumor cells due to the removal of HSP27 inhibition of SPARC-induced pro-apoptotic signaling. 4 Suppressing total AKT1/2 paradoxically enhanced tumor cell survival, indicating that AKT1 or 2 are poor therapeutic targets. 5 However, inhibiting pAKT suppresses tumor cell survival. 6 Inhibiting both HSP27 and pAKT synergistically decreases tumor cell survival. 7 There appears to be a complex feedback system between SPARC, HSP27, and AKT. 8 This interaction is likely influenced by PTEN status. With respect to chemosensitization, we found the following. 1 SPARC enhances pro-apoptotic signaling in cells exposed to TMZ. 2 Despite this enhanced signaling, SPARC protects cells against TMZ. 3 This protection can be reduced

  8. Withaferin-A suppress AKT induced tumor growth in colorectal cancer cells.

    Science.gov (United States)

    Suman, Suman; Das, Trinath P; Sirimulla, Suman; Alatassi, Houda; Ankem, Murali K; Damodaran, Chendil

    2016-03-22

    The oncogenic activation of AKT gene has emerged as a key determinant of the aggressiveness of colorectal cancer (CRC); hence, research has focused on targeting AKT signaling for the treatment of advanced stages of CRC. In this study, we explored the anti-tumorigenic effects of withaferin A (WA) on CRC cells overexpressing AKT in preclinical (in vitro and in vivo) models. Our results indicated that WA, a natural compound, resulted in significant inhibition of AKT activity and led to the inhibition of cell proliferation, migration and invasion by downregulating the epithelial to mesenchymal transition (EMT) markers in CRC cells overexpressing AKT. The oral administration of WA significantly suppressed AKT-induced aggressive tumor growth in a xenograft model. Molecular analysis revealed that the decreased expression of AKT and its downstream pro-survival signaling molecules may be responsible for tumor inhibition. Further, significant inhibition of some important EMT markers, i.e., Snail, Slug, β-catenin and vimentin, was observed in WA-treated human CRC cells overexpressing AKT. Significant inhibition of micro-vessel formation and the length of vessels were evident in WA-treated tumors, which correlated with a low expression of the angiogenic marker RETIC. In conclusion, the present study emphasizes the crucial role of AKT activation in inducing cell proliferation, angiogenesis and EMT in CRC cells and suggests that WA may overcome AKT-induced cell proliferation and tumor growth in CRC. PMID:26883103

  9. Mammalian TBX1 preferentially binds and regulates downstream targets via a tandem T-site repeat.

    Directory of Open Access Journals (Sweden)

    Raquel Castellanos

    Full Text Available Haploinsufficiency or mutation of TBX1 is largely responsible for the etiology of physical malformations in individuals with velo-cardio-facial/DiGeorge syndrome (VCFS/DGS/22q11.2 deletion syndrome. TBX1 encodes a transcription factor protein that contains an evolutionarily conserved DNA binding domain termed the T-box that is shared with other family members. All T-box proteins, examined so far, bind to similar but not identical consensus DNA sequences, indicating that they have specific binding preferences. To identify the TBX1 specific consensus sequence, Systematic Evolution of Ligands by Exponential Enrichment (SELEX was performed. In contrast to other TBX family members recognizing palindrome sequences, we found that TBX1 preferentially binds to a tandem repeat of 5'-AGGTGTGAAGGTGTGA-3'. We also identified a second consensus sequence comprised of a tandem repeat with a degenerated downstream site. We show that three known human disease-causing TBX1 missense mutations (F148Y, H194Q and G310S do not alter nuclear localization, or disrupt binding to the tandem repeat consensus sequences, but they reduce transcriptional activity in cell culture reporter assays. To identify Tbx1-downstream genes, we performed an in silico genome wide analysis of potential cis-acting elements in DNA and found strong enrichment of genes required for developmental processes and transcriptional regulation. We found that TBX1 binds to 19 different loci in vitro, which may correspond to putative cis-acting binding sites. In situ hybridization coupled with luciferase gene reporter assays on three gene loci, Fgf8, Bmper, Otog-MyoD, show that these motifs are directly regulated by TBX1 in vitro. Collectively, the present studies establish new insights into molecular aspects of TBX1 binding to DNA. This work lays the groundwork for future in vivo studies, including chromatin immunoprecipitation followed by next generation sequencing (ChIP-Seq to further elucidate the

  10. Mammalian TBX1 preferentially binds and regulates downstream targets via a tandem T-site repeat.

    Science.gov (United States)

    Castellanos, Raquel; Xie, Qing; Zheng, Deyou; Cvekl, Ales; Morrow, Bernice E

    2014-01-01

    Haploinsufficiency or mutation of TBX1 is largely responsible for the etiology of physical malformations in individuals with velo-cardio-facial/DiGeorge syndrome (VCFS/DGS/22q11.2 deletion syndrome). TBX1 encodes a transcription factor protein that contains an evolutionarily conserved DNA binding domain termed the T-box that is shared with other family members. All T-box proteins, examined so far, bind to similar but not identical consensus DNA sequences, indicating that they have specific binding preferences. To identify the TBX1 specific consensus sequence, Systematic Evolution of Ligands by Exponential Enrichment (SELEX) was performed. In contrast to other TBX family members recognizing palindrome sequences, we found that TBX1 preferentially binds to a tandem repeat of 5'-AGGTGTGAAGGTGTGA-3'. We also identified a second consensus sequence comprised of a tandem repeat with a degenerated downstream site. We show that three known human disease-causing TBX1 missense mutations (F148Y, H194Q and G310S) do not alter nuclear localization, or disrupt binding to the tandem repeat consensus sequences, but they reduce transcriptional activity in cell culture reporter assays. To identify Tbx1-downstream genes, we performed an in silico genome wide analysis of potential cis-acting elements in DNA and found strong enrichment of genes required for developmental processes and transcriptional regulation. We found that TBX1 binds to 19 different loci in vitro, which may correspond to putative cis-acting binding sites. In situ hybridization coupled with luciferase gene reporter assays on three gene loci, Fgf8, Bmper, Otog-MyoD, show that these motifs are directly regulated by TBX1 in vitro. Collectively, the present studies establish new insights into molecular aspects of TBX1 binding to DNA. This work lays the groundwork for future in vivo studies, including chromatin immunoprecipitation followed by next generation sequencing (ChIP-Seq) to further elucidate the molecular

  11. Reduced phosphorylation of brain insulin receptor substrate and Akt proteins in apolipoprotein-E4 targeted replacement mice.

    Science.gov (United States)

    Ong, Qi-Rui; Chan, Elizabeth S; Lim, Mei-Li; Cole, Gregory M; Wong, Boon-Seng

    2014-01-17

    Human ApoE4 accelerates memory decline in ageing and in Alzheimer's disease. Although intranasal insulin can improve cognition, this has little effect in ApoE4 subjects. To understand this ApoE genotype-dependent effect, we examined brain insulin signaling in huApoE3 and huApoE4 targeted replacement (TR) mice. At 32 weeks, lower insulin receptor substrate 1 (IRS1) at S636/639 and Akt phosphorylation at T308 were detected in fasting huApoE4 TR mice as compared to fasting huApoE3 TR mice. These changes in fasting huApoE4 TR mice were linked to lower brain glucose content and have no effect on plasma glucose level. However, at 72 weeks of age, these early changes were accompanied by reduction in IRS2 expression, IRS1 phosphorylation at Y608, Akt phosphorylation at S473, and MAPK (p38 and p44/42) activation in the fasting huApoE4 TR mice. The lower brain glucose was significantly associated with higher brain insulin in the aged huApoE4 TR mice. These results show that ApoE4 reduces brain insulin signaling and glucose level leading to higher insulin content.

  12. Targeted deletion of Kif18a protects from colitis-associated colorectal (CAC) tumors in mice through impairing Akt phosphorylation

    Energy Technology Data Exchange (ETDEWEB)

    Zhu, Houbao [State Key Laboratory of Medical Genomics, Research Center for Experimental Medicine, Rui-Jin Hospital and Department of Medical Genetics, E-Institutes of Shanghai Universities, Shanghai Jiao Tong University School of Medicine (SJTUSM), Shanghai 200025 (China); Xu, Wangyang [Department of Clinical Laboratories, Ninth People’s Hospital, SJTUSM, Shanghai 200011 (China); Zhang, Hongxin [State Key Laboratory of Medical Genomics, Research Center for Experimental Medicine, Rui-Jin Hospital and Department of Medical Genetics, E-Institutes of Shanghai Universities, Shanghai Jiao Tong University School of Medicine (SJTUSM), Shanghai 200025 (China); Liu, Jianbing [State Key Laboratory of Medical Genomics, Research Center for Experimental Medicine, Rui-Jin Hospital and Department of Medical Genetics, E-Institutes of Shanghai Universities, Shanghai Jiao Tong University School of Medicine (SJTUSM), Shanghai 200025 (China); Shanghai Research Center for Model Organisms, Shanghai 201203 (China); Xu, Haimin [Department of Pathology, Rui-Jin Hospital, SJTUSM, Shanghai 200025 (China); Lu, Shunyuan; Dang, Suying [State Key Laboratory of Medical Genomics, Research Center for Experimental Medicine, Rui-Jin Hospital and Department of Medical Genetics, E-Institutes of Shanghai Universities, Shanghai Jiao Tong University School of Medicine (SJTUSM), Shanghai 200025 (China); Kuang, Ying [Shanghai Research Center for Model Organisms, Shanghai 201203 (China); Jin, Xiaolong [Department of Pathology, Rui-Jin Hospital, SJTUSM, Shanghai 200025 (China); Wang, Zhugang, E-mail: zhugangw@shsmu.edu.cn [State Key Laboratory of Medical Genomics, Research Center for Experimental Medicine, Rui-Jin Hospital and Department of Medical Genetics, E-Institutes of Shanghai Universities, Shanghai Jiao Tong University School of Medicine (SJTUSM), Shanghai 200025 (China); Shanghai Research Center for Model Organisms, Shanghai 201203 (China)

    2013-08-16

    Highlights: •Kif18A is up-regulated in CAC of mouse model. •Kif18a{sup −/−} mice are protected from CAC. •Tumor cells from Kif18a{sup −/−} mice undergo more apoptosis. •Kif18A deficiency induces poor Atk phosphorylation. -- Abstract: Kinesins are a superfamily of molecular motors involved in cell division or intracellular transport. They are becoming important targets for chemotherapeutic intervention of cancer due to their crucial role in mitosis. Here, we demonstrate that the kinesin-8 Kif18a is overexpressed in murine CAC and is a crucial promoter during early CAC carcinogenesis. Kif18a-deficient mice are evidently protected from AOM–DSS-induced colon carcinogenesis. Kif18A is responsible for proliferation of colonic tumor cells, while Kif18a ablation in mice promotes cell apoptosis. Mechanistically, Kif18a is responsible for induction of Akt phosphorylation, which is known to be associated with cell survival regulation. In conclusion, Kif18a is critical for colorectal carcinogenesis in the setting of inflammation by mechanisms of increased PI3K-AKT signaling. Inhibition of Kif18A activity may be useful in the prevention or chemotherapeutic intervention of CAC.

  13. Targeted deletion of Kif18a protects from colitis-associated colorectal (CAC) tumors in mice through impairing Akt phosphorylation

    International Nuclear Information System (INIS)

    Highlights: •Kif18A is up-regulated in CAC of mouse model. •Kif18a−/− mice are protected from CAC. •Tumor cells from Kif18a−/− mice undergo more apoptosis. •Kif18A deficiency induces poor Atk phosphorylation. -- Abstract: Kinesins are a superfamily of molecular motors involved in cell division or intracellular transport. They are becoming important targets for chemotherapeutic intervention of cancer due to their crucial role in mitosis. Here, we demonstrate that the kinesin-8 Kif18a is overexpressed in murine CAC and is a crucial promoter during early CAC carcinogenesis. Kif18a-deficient mice are evidently protected from AOM–DSS-induced colon carcinogenesis. Kif18A is responsible for proliferation of colonic tumor cells, while Kif18a ablation in mice promotes cell apoptosis. Mechanistically, Kif18a is responsible for induction of Akt phosphorylation, which is known to be associated with cell survival regulation. In conclusion, Kif18a is critical for colorectal carcinogenesis in the setting of inflammation by mechanisms of increased PI3K-AKT signaling. Inhibition of Kif18A activity may be useful in the prevention or chemotherapeutic intervention of CAC

  14. Drosophila Fascin is a novel downstream target of prostaglandin signaling during actin remodeling.

    Science.gov (United States)

    Groen, Christopher M; Spracklen, Andrew J; Fagan, Tiffany N; Tootle, Tina L

    2012-12-01

    Although prostaglandins (PGs)-lipid signals produced downstream of cyclooxygenase (COX) enzymes-regulate actin cytoskeletal dynamics, their mechanisms of action are unknown. We previously established Drosophila oogenesis, in particular nurse cell dumping, as a new model to determine how PGs regulate actin remodeling. PGs, and thus the Drosophila COX-like enzyme Pxt, are required for both the parallel actin filament bundle formation and the cortical actin strengthening required for dumping. Here we provide the first link between Fascin (Drosophila Singed, Sn), an actin-bundling protein, and PGs. Loss of either pxt or fascin results in similar actin defects. Fascin interacts, both pharmacologically and genetically, with PGs, as reduced Fascin levels enhance the effects of COX inhibition and synergize with reduced Pxt levels to cause both parallel bundle and cortical actin defects. Conversely, overexpression of Fascin in the germline suppresses the effects of COX inhibition and genetic loss of Pxt. These data lead to the conclusion that PGs regulate Fascin to control actin remodeling. This novel interaction has implications beyond Drosophila, as both PGs and Fascin-1, in mammalian systems, contribute to cancer cell migration and invasion.

  15. Detection of miRNA-21 content in cervical cancer tissue and preliminary analysis of its downstream target molecules

    Institute of Scientific and Technical Information of China (English)

    Rong Shen; Jian-Wu Gao; Yan-Yu Li; Peng Teng

    2015-01-01

    Objective:To study the miRNA-21 content in cervical cancer tissue and analyze its downstream target molecules.Methods:Patients with different FIGO stages of cervical cancer and healthy subjects were selected, cervical cancer tissue and normal cervical tissue were collected, and contents of miRNA-21 and apoptotic genes were detected; cervical cancer SiHa cells were cultured, miRNA-21 mimics and inhibitors were transfected, and then apoptotic gene contents were detected.Results:miRNA-21 contents in different stages of cervical cancer tissue were all higher than those in normal cervical tissue, mRNA contents of p16ink4a, ASPP1, Fas and GRIM-19 were lower than those in normal tissue, and mRNA contents of p16ink4a, ASPP1, Fas and GRIM-19 were negatively correlated with miRNA-21 contents; after miRNA-21 mimics were transfected, mRNA contents of p16ink4a, ASPP1, Fas and GRIM-19 significantly decreased, and after miRNA-21 inhibitors were transfected, mRNA contents of p16ink4a, ASPP1, Fas and GRIM-19 significantly increased.Conclusion:miRNA-21 contents in cervical cancer tissue significantly increase; downstream target genes of this miRNA may be apoptotic genes p16ink4a, ASPP1, Fas and GRIM-19.

  16. miR-451a is underexpressed and targets AKT/mTOR pathway in papillary thyroid carcinoma.

    Science.gov (United States)

    Minna, Emanuela; Romeo, Paola; Dugo, Matteo; De Cecco, Loris; Todoerti, Katia; Pilotti, Silvana; Perrone, Federica; Seregni, Ettore; Agnelli, Luca; Neri, Antonino; Greco, Angela; Borrello, Maria Grazia

    2016-03-15

    Papillary Thyroid Carcinoma (PTC) is the most frequent thyroid cancer. Although several PTC-specific miRNA profiles have been reported, only few upregulated miRNAs are broadly recognized, while less consistent data are available about downregulated miRNAs. In this study we investigated miRNA deregulation in PTC by miRNA microarray, analysis of a public dataset from The Cancer Genome Atlas (TCGA), literature review and meta-analysis based on a univocal miRNA identifier derived from miRBase v21. A list of 18 miRNAs differentially expressed between PTC and normal thyroid was identified and validated in the TCGA dataset. Furthermore, we compared our signature with miRNA profiles derived from 15 studies selected from literature. Then, to select possibly functionally relevant miRNA, we integrated our miRNA signature with those from two in vitro cell models based on the PTC-driving oncogene RET/PTC1. Through this strategy, we identified commonly deregulated miRNAs, including miR-451a, which emerged also by our meta-analysis as the most frequently reported downregulated miRNA. We showed that lower expression of miR-451a correlates with aggressive clinical-pathological features of PTC as tall cell variant, advanced stage and extrathyroid extension. In addition, we demonstrated that ectopic expression of miR-451a impairs proliferation and migration of two PTC-derived cell lines, reduces the protein levels of its recognized targets MIF, c-MYC and AKT1 and attenuates AKT/mTOR pathway activation.Overall, our study provide both an updated overview of miRNA deregulation in PTC and the first functional evidence that miR-451a exerts tumor suppressor functions in this neoplasia. PMID:26871295

  17. NF-kB activation and its downstream target genes expression after heavy ions exposure

    Science.gov (United States)

    Chishti, Arif Ali; Baumstark-Khan, Christa; Hellweg, Christine; Schmitz, Claudia; Koch, Kristina; Feles, Sebastian

    2016-07-01

    To enable long-term human space flight cellular radiation response to densely ionizing radiation needs to be better understood for developing appropriate countermeasures to mitigate acute effects and late radiation risks for the astronaut. The biological effectiveness of accelerated heavy ions (which constitute the most important radiation type in space) with high linear energy transfer (LET) for effecting DNA damage response pathways as a gateway to cell death or survival is of major concern not only for space missions but also for new regimes of tumor radiotherapy. In the current research study, the contribution of NF-κB in response to space-relevant radiation qualities was determined by a NF-κB reporter cell line (HEK-pNF-κB-d2EGFP/Neo L2). The NF-κB dependent reporter gene expression (d2EGFP) after ionizing radiation (X-rays and heavy ions) exposure was evaluated by flow cytometry. Because of differences in the extent of NF-κB activation after X-irradiation and heavy ions exposure, it was expected that radiation quality (LET) might play an important role in the cellular radiation response. In addition, the biological effectiveness (RBE) of NF-κB activation and reduction of cellular survival was examined for heavy ions having a broad range of LET (˜0.3 - 9674 keV/µm). Furthermore, the effect of LET on NF-κB target gene expression was analyzed by real time reverse transcriptase quantitative PCR (RT-qPCR). In this study it was proven that NF-κB activation and NF-κB dependent gene expression comprises an early step in cellular radiation response. Taken together, this study clearly demonstrates that NF-κB activation and NF-κB-dependent gene expression by heavy ions are highest in the LET range of ˜50-200 keV/μupm. The up-regulated chemokines and cytokines (CXCL1, CXCL2, CXCL10, IL-8 and TNF) might be important for cell-cell communication among hit as well as unhit cells (bystander effect). The results obtained suggest the NF-κB pathway to be a

  18. Transcription Factors Expressed in Lateral Organ Boundaries: Identification of Downstream Targets

    Energy Technology Data Exchange (ETDEWEB)

    Springer, Patricia S

    2010-07-12

    The processes of lateral organ initiation and patterning are central to the generation of mature plant form. Characterization of the molecular mechanisms underlying these processes is essential to our understanding of plant development. Communication between the shoot apical meristem and initiating organ primordia is important both for functioning of the meristem and for proper organ patterning, and very little is known about this process. In particular, the boundary between meristem and leaf is emerging as a critical region that is important for SAM maintenance and regulation of organogenesis. The goal of this project was to characterize three boundary-expressed genes that encode predicted transcription factors. Specifically, we have studied LATERAL ORGAN BOUNDARIES (LOB), LATERAL ORGAN FUSION1 (LOF1), and LATERAL ORGAN FUSION2 (LOF2). LOB encodes the founding member of the LOB-DOMAIN (LBD) plant-specific DNA binding transcription factor family and LOF1 and LOF2 encode paralogous MYB-domain transcription factors. We characterized the genetic relationship between these three genes and other boundary and meristem genes. We also used an ectopic inducible expression system to identify direct targets of LOB.

  19. Optimal Classes of Chemotherapeutic Agents Sensitized by Specific Small-Molecule Inhibitors of Akt In Vitro and In Vivo

    Directory of Open Access Journals (Sweden)

    Yan Shi

    2005-11-01

    Full Text Available Akt is a serine/threonine kinase that transduces survival signals from survival/growth factors. Deregulation and signal imbalance in cancer cells make them prone to apoptosis. Upregulation or activation of Akt to aid the survival of cancer cells is a common theme in human malignancies. We have developed small-molecule Akt inhibitors that are potent and specific. These Akt inhibitors can inhibit Akt activity and block phosphorylation by Akt on multiple downstream targets in cells. Synergy in apoptosis induction was observed when Akt inhibitors were combined with doxorubicin or camptothecin. Akt inhibitor-induced enhancement of topoisomerase inhibitor cytotoxicity was also evident in long-term cell survival assay. Synergy with paclitaxel in apoptosis induction was evident in cells pretreated with paclitaxel, and enhancement of tumor delay by paclitaxel was demonstrated through cotreatment with Akt inhibitor Compound A (A-443654. Combination with other classes of chemotherapeutic agents did not yield any enhancement of cytotoxicity. These findings provide important guidance in selecting appropriate classes of chemotherapeutic agents for combination with Akt inhibitors in cancer treatment.

  20. mTOR Promotes Survival and Astrocytic Characteristics Induced by Pten/Akt Signaling in Glioblastoma

    Directory of Open Access Journals (Sweden)

    Xiaoyi Hu

    2005-04-01

    Full Text Available Combined activation of Ras and Akt leads to the formation of astrocytic glioblastoma multiforme (GBM in mice. In human GBMs, AKT is not mutated but is activated in approximately 70% of these tumors, in association with loss of PTEN and/or activation of receptor tyrosine kinases. Mechanistic justification for the therapeutic blockade of targets downstream of AKT, such as mTOR, in these cancers requires demonstration that the oncogenic effect of PTEN loss is through elevated AKT activity. We demonstrate here that loss of Pten is similar to Akt activation in the context of glioma formation in mice. We further delineate the role of mTOR activity downstream of Akt in the maintenance of Akt+KRas-induced GBMs. Blockade of mTOR results in regional apoptosis in these tumors and conversion in the character of surviving tumor cells from astrocytoma to oligodendroglioma. These data suggest that mTOR activity is required for the survival of some cells within these GBMs, and mTOR appears required for the maintenance of astrocytic character in the surviving cells. Furthermore, our study provides the first example of conversion between two distinct tumor types usually thought of as belonging to specific lineages, and provides evidence for signal transduction-mediated transdifferentiation between glioma subtypes.

  1. A mathematical model of phosphorylation AKT in Acute Myeloid Leukemia

    Science.gov (United States)

    Adi, Y. A.; Kusumo, F. A.; Aryati, L.; Hardianti, M. S.

    2016-04-01

    In this paper we consider a mathematical model of PI3K/AKT signaling pathways in phosphorylation AKT. PI3K/AKT pathway is an important mediator of cytokine signaling implicated in regulation of hematopoiesis. Constitutive activation of PI3K/AKT signaling pathway has been observed in Acute Meyloid Leukemia (AML) it caused by the mutation of Fms-like Tyrosine Kinase 3 in internal tandem duplication (FLT3-ITD), the most common molecular abnormality associated with AML. Depending upon its phosphorylation status, protein interaction, substrate availability, and localization, AKT can phosphorylate or inhibite numerous substrates in its downstream pathways that promote protein synthesis, survival, proliferation, and metabolism. Firstly, we present a mass action ordinary differential equation model describing AKT double phosphorylation (AKTpp) in a system with 11 equations. Finally, under the asumtion enzyme catalyst constant and steady state equilibrium, we reduce the system in 4 equation included Michaelis Menten constant. Simulation result suggested that a high concentration of PI3K and/or a low concentration of phospatase increased AKTpp activation. This result also indicates that PI3K is a potential target theraphy in AML.

  2. Calcium-dependent modulation and plasma membrane targeting of the AKT2 potassium channel by the CBL4/ CIPK6 calcium sensor/protein kinase complex

    Institute of Scientific and Technical Information of China (English)

    Katrin Held; Jean-Baptiste Thibaud; J(o)rg Kudla; Francois Pascaud; Christian Eckert; Pawel Gajdanowicz; Kenji Hashimoto; Claire Corratgé-Faillie; Jan Niklas Offenborn; Beno(i)t Lacombe; Ingo Dreyer

    2011-01-01

    Potassium (K+) channel function is fundamental to many physiological processes. However, components and mechanisms regulating the activity of plant K+ channels remain poorly understood. Here, we show that the calcium (Ca2+)sensor CBL4 together with the interacting protein kinase CIPK6 modulates the activity and plasma membrane (PM)targeting of the K+ channel AKT2 from Arabidopsis thaliana by mediating translocation of AKT2 to the PM in plant cells and enhancing AKT2 activity in oocytes. Accordingly, akt2, cbl4 and cipk6 mutants share similar developmental and delayed flowering pheuotypes. Moreover, the isolated regulatory C-terminal domain of CIPK6 is sufficient for mediating CBL4- and Ca2+-dependent channel translocation from the endoplasmic reticulum membrane to the PM by a novel targeting pathway that is dependent on dual lipid modifications of CBL4 by myristoylation and palmitoylation. Thus, we describe a critical mechanism of ion-channel regulation where a Ca2+ sensor modulates K+ channel activity by promoting a kinase interaction-dependent but phosphorylation-independent translocation of the channel to the PM.

  3. The Emerging Role of the Phosphatidylinositol 3-Kinase/ Akt/Mammalian Target of Rapamycin Signaling Network in Cancer Stem Cell Biology

    Directory of Open Access Journals (Sweden)

    James A. McCubrey

    2010-08-01

    Full Text Available The cancer stem cell theory entails the existence of a hierarchically organized, rare population of cells which are responsible for tumor initiation, self-renewal/maintenance, and mutation accumulation. The cancer stem cell proposition could explain the high frequency of cancer relapse and resistance to currently available therapies. The phosphatidylinositol 3-kinase (PI3K/Akt/mammalian target of rapamycin (mTOR signaling pathway regulates a wide array of physiological cell functions which include differentiation, proliferation, survival, metabolism, autophagy, and motility. Dysregulated PI3K/Akt/mTOR signaling has been documented in many types of neoplasias. It is now emerging that this signaling network plays a key role in cancer stem cell biology. Interestingly, cancer stem cells displayed preferential sensitivity to pathway inhibition when compared to healthy stem cells. This observation provides the proof-of-principle that functional differences in signaling pathways between neoplastic stem cells and healthy stem cells could be identified. In this review, we present the evidence which links the signals emanating from the PI3K/Akt/mTOR cascade with the functions of cancer stem cells, both in solid and hematological tumors. We then highlight how targeting PI3K/Akt/mTOR signaling with small molecules could improve cancer patient outcome.

  4. Long noncoding RNA H19 contributes to gallbladder cancer cell proliferation by modulated miR-194-5p targeting AKT2.

    Science.gov (United States)

    Wang, Shou-Hua; Wu, Xiao-Cai; Zhang, Ming-Di; Weng, Ming-Zhe; Zhou, Di; Quan, Zhi-Wei

    2016-07-01

    Gallbladder cancer (GBC) is a highly malignant cancer with poor prognosis. Although long noncoding RNA (lncRNA) H19 has been reported to play vital role in many human cancers, whether it is involved in GBC proliferation is still unknown. This study was designed to explore the effect of H19 in GBC cell proliferation. The expression of H19 and AKT2 were significantly elevated in GBC tissues, and the level of miR-194-5p is markedly decreased. Moreover, the RNA levels of H19 and AKT2 were positively correlated, and H19 elevation was significantly associated with tumor size. Cell proliferation decreased significantly after knockdown of H19 in GBC-SD and NOZ cells and after knockdown of AKT2 in NOZ cells. Results from cell cycle studies indicated that the S phase were significantly decreased after knockdown of H19 in NOZ cells but significantly elevated after overexpression of H19 in GBC-SD cells. Furthermore, knockdown of H19 upregulated miR-194-5p levels, yet significantly decreased miR-194-5p targeting AKT2 gene expression in NOZ cells. Inhibitor against miR-194-5p reversed these effects. In addition, overexpression of H19 in GBC-SD cells downregulated miR-194-5p and markedly increased AKT2 expression, and miR-194-5p mimic reversed these effects. Eventually, GBC cells were arrested in G0/G1-phase after H19 knockdown, inhibition of miR-194-5p markedly promoted cells into S-phase and co-transfection of siH19, and miR-194-5p inhibitor exerted mutually counter-regulated effects on cell cycle. These results suggested that H19/miR-194-5p/AKT2 axis regulatory network might modulate cell proliferation in GBC. PMID:26803515

  5. Anomalous altered expressions of downstream gene-targets in TP53-miRNA pathways in head and neck cancer.

    Science.gov (United States)

    Mitra, Sanga; Mukherjee, Nupur; Das, Smarajit; Das, Pijush; Panda, Chinmay Kumar; Chakrabarti, Jayprokas

    2014-01-01

    The prevalence of head and neck squamous cell carcinoma, HNSCC, continues to grow. Change in the expression of TP53 in HNSCC affects its downstream miRNAs and their gene targets, anomalously altering the expressions of the five genes, MEIS1, AGTR1, DTL, TYMS and BAK1. These expression alterations follow the repression of TP53 that upregulates miRNA-107, miRNA- 215, miRNA-34 b/c and miRNA-125b, but downregulates miRNA-155. The above five so far unreported genes are the targets of these miRNAs. Meta-analyses of microarray and RNA-Seq data followed by qRT-PCR validation unravel these new ones in HNSCC. The regulatory roles of TP53 on miRNA-155 and miRNA-125b differentiate the expressions of AGTR1 and BAK1in HNSCC vis-à-vis other carcinogenesis. Expression changes alter cell cycle regulation, angiogenic and blood cell formation, and apoptotic modes in affliction. Pathway analyses establish the resulting systems-level functional and mechanistic insights into the etiology of HNSCC.

  6. Identification of cyclins A1, E1 and vimentin as downstream targets of heme oxygenase-1 in vascular endothelial growth factor-mediated angiogenesis

    OpenAIRE

    Bauer, Andrea; Mylroie, Hayley; Thornton, C. Clare; Calay, Damien; Birdsey, Graeme M.; Kiprianos, Allan P.; Garrick K. Wilson; Soares, Miguel P.; Yin, Xiaoke; Mayr, Manuel; Randi, Anna M.; Mason, Justin C.

    2016-01-01

    Angiogenesis is an essential physiological process and an important factor in disease pathogenesis. However, its exploitation as a clinical target has achieved limited success and novel molecular targets are required. Although heme oxygenase-1 (HO-1) acts downstream of vascular endothelial growth factor (VEGF) to modulate angiogenesis, knowledge of the mechanisms involved remains limited. We set out identify novel HO-1 targets involved in angiogenesis. HO-1 depletion attenuated VEGF-induced h...

  7. The Akt switch model: Is location sufficient?

    Science.gov (United States)

    Gray, Catheryn W; Coster, Adelle C F

    2016-06-01

    Akt/PKB is a biochemical regulator that functions as an important cross-talk node between several signalling pathways in the mammalian cell. In particular, Akt is a key mediator of glucose transport in response to insulin. The phosphorylation (activation) of only a small percentage of the Akt pool of insulin-sensitive cells results in maximal translocation of glucose transporter 4 (GLUT4) to the plasma membrane (PM). This enables the diffusion of glucose into the cell. The dysregulation of Akt signalling is associated with the development of diabetes, cancer and cardiovascular disease. Akt is synthesised in the cytoplasm in the inactive state. Under the influence of insulin, it moves to the PM, where it is phosphorylated to form pAkt. Although phosphorylation occurs only at the PM, pAkt is found in many cellular locations, including the PM, the cytoplasm, and the nucleus. Indeed, the spatial distribution of pAkt within the cell appears to be an important determinant of downstream regulation. Here we present a simple, linear, four-compartment ordinary differential equation (ODE) model of Akt activation that tracks both the biochemical state and the physical location of Akt. This model embodies the main features of the activation of this important cross-talk node and is consistent with the experimental data. In particular, it allows different downstream signalling motifs without invoking separate feedback pathways. Moreover, the model is computationally tractable, readily analysed, and elucidates some of the apparent anomalies in insulin signalling via Akt. PMID:26992575

  8. Fluorescence imaging analysis of upstream regulators and downstream targets of STAT3 in melanoma precursor lesions obtained from patients before and after systemic low-dose interferon-alpha treatment.

    Science.gov (United States)

    Smith, Amanda Pfaff; Kirkwood, John M; Edington, Howard D; Jukic, Drazen M; Farkas, Daniel L; Becker, Dorothea

    2003-01-01

    Atypical nevi are the precursors and risk markers of melanoma. Apart from persistently monitoring these nevocytic lesions and resecting them at the earliest signs of clinical changes, there is as yet no systemic clinical treatment available to interfere with their progression to melanoma. To explore clinical treatments that might interfere with and possibly prevent atypical nevus progression, a previous study documented that 3 months systemic low-dose interferon-alpha (IFN-alpha) treatment of patients with a clinical history of melanoma and numerous atypical nevi, led to inactivation of the STAT1 and STAT3 transcription factors in atypical nevi. Based upon this finding, we initiated a second study to determine whether systemic low-dose IFN-alpha treatment also impairs the expression of upstream regulators and downstream targets of STAT1 and STAT3 in atypical nevi. Using cyanine dye-conjugated antibodies, fluorescence imaging analysis revealed expression of JAK2, JNK1, AKT1, NF-kappa B, and IFN-alpha/beta receptor in benign and atypical nevi, and early- and advanced-stage melanomas. To determine possible changes in the level of expression of these molecules in atypical nevi, excised before and after 3 months of systemic low-dose IFN-alpha treatment, newly designed optical imaging software was used to quantitate the captured fluorescent hybridization signals on a cell-by-cell basis and across an entire nevus section. The results of this analysis did not provide evidence that systemic low-dose IFN-alpha treatment alters the level of expression of upstream regulators or downstream targets of STAT1 and STAT3.

  9. MicroRNA-379-5p inhibits tumor invasion and metastasis by targeting FAK/AKT signaling in hepatocellular carcinoma.

    Science.gov (United States)

    Chen, Jing-Song; Li, Hua-Shu; Huang, Jiong-Qiang; Dong, Shi-Hao; Huang, Zhi-Jie; Yi, Wei; Zhan, Gao-Fang; Feng, Ju-Tao; Sun, Jian-Cong; Huang, Xiao-Hui

    2016-05-28

    Some microRNAs (miRNAs) have been implicated in hepatocellular carcinoma (HCC) development and progression. However, the roles and mechanisms of several miRNAs in HCC remain poorly understood. Here, we report that miR-379-5p, which is down-regulated in HCC tissues and cell lines, is associated with advanced TNM stage and metastasis in HCC. The ectopic overexpression of miR-379-5p inhibited HCC cell migration, invasion, epithelial-to-mesenchymal transition (EMT) and metastasis both in vitro and in vivo. Conversely, miR-379 knockdown increased migration, invasion and EMT in HCC cells. Moreover, miR-379-5p exerted this function by directly targeting focal adhesion kinase (FAK) 3'-UTR and repressing FAK expression, thus leading to suppression of AKT signaling. Furthermore, the tumor suppressive effects of miR-379-5p in HCC cells were reversed by activating AKT signaling or restoring FAK expression. In clinical samples of HCC, miR-379-5p negatively correlated with FAK, which was up-regulated in HCC. Taken together, our findings highlight the important role of miR-379-5p in regulating the EMT and metastasis of HCC by targeting FAK/AKT signaling, suggesting that miR-379-5p may represent a novel potential therapeutic target and prognostic marker for HCC. PMID:26944318

  10. Preclinical pharmacology, antitumor activity, and development of pharmacodynamic markers for the novel, potent AKT inhibitor CCT128930.

    Science.gov (United States)

    Yap, Timothy A; Walton, Mike I; Hunter, Lisa-Jane K; Valenti, Melanie; de Haven Brandon, Alexis; Eve, Paul D; Ruddle, Ruth; Heaton, Simon P; Henley, Alan; Pickard, Lisa; Vijayaraghavan, Gowri; Caldwell, John J; Thompson, Neil T; Aherne, Wynne; Raynaud, Florence I; Eccles, Suzanne A; Workman, Paul; Collins, Ian; Garrett, Michelle D

    2011-02-01

    AKT is frequently deregulated in cancer, making it an attractive anticancer drug target. CCT128930 is a novel ATP-competitive AKT inhibitor discovered using fragment- and structure-based approaches. It is a potent, advanced lead pyrrolopyrimidine compound exhibiting selectivity for AKT over PKA, achieved by targeting a single amino acid difference. CCT128930 exhibited marked antiproliferative activity and inhibited the phosphorylation of a range of AKT substrates in multiple tumor cell lines in vitro, consistent with AKT inhibition. CCT128930 caused a G(1) arrest in PTEN-null U87MG human glioblastoma cells, consistent with AKT pathway blockade. Pharmacokinetic studies established that potentially active concentrations of CCT128930 could be achieved in human tumor xenografts. Furthermore, CCT128930 also blocked the phosphorylation of several downstream AKT biomarkers in U87MG tumor xenografts, indicating AKT inhibition in vivo. Antitumor activity was observed with CCT128930 in U87MG and HER2-positive, PIK3CA-mutant BT474 human breast cancer xenografts, consistent with its pharmacokinetic and pharmacodynamic properties. A quantitative immunofluorescence assay to measure the phosphorylation and total protein expression of the AKT substrate PRAS40 in hair follicles is presented. Significant decreases in pThr246 PRAS40 occurred in CCT128930-treated mouse whisker follicles in vivo and human hair follicles treated ex vivo, with minimal changes in total PRAS40. In conclusion, CCT128930 is a novel, selective, and potent AKT inhibitor that blocks AKT activity in vitro and in vivo and induces marked antitumor responses. We have also developed a novel biomarker assay for the inhibition of AKT in human hair follicles, which is currently being used in clinical trials. PMID:21191045

  11. Neurotensin-induced miR-133α expression regulates neurotensin receptor 1 recycling through its downstream target aftiphilin.

    Science.gov (United States)

    Law, Ivy Ka Man; Jensen, Dane; Bunnett, Nigel W; Pothoulakis, Charalabos

    2016-01-01

    Neurotensin (NT) triggers signaling in human colonic epithelial cells by activating the G protein-coupled receptor, the neurotensin receptor 1 (NTR1). Activated NTR1 traffics from the plasma membrane to early endosomes, and then recycles. Although sustained NT/NTR1 signaling requires efficient NTR1 recycling, little is known about the regulation of NTR1 recycling. We recently showed that NT/NTR1 signaling increases expression of miR-133α. Herein, we studied the mechanism of NT-regulated miR-133α expression and examined the role of miR-133α in intracellular NTR1 trafficking in human NCM460 colonocytes. We found that NT-induced miR-133α upregulation involves the negative transcription regulator, zinc finger E-box binding homeobox 1. Silencing of miR-133α or overexpression of aftiphilin (AFTPH), a binding target of miR-133α, attenuated NTR1 trafficking to plasma membrane in human colonocytes, without affecting NTR1 internalization. We localized AFTPH to early endosomes and the trans-Golgi network (TGN) in unstimulated human colonic epithelial cells. AFTPH overexpression reduced NTR1 localization in early endosomes and increased expression of proteins related to endosomes and the TGN trafficking pathway. AFTPH overexpression and de-acidification of intracellular vesicles increased NTR1 expression. Our results suggest a novel mechanism of GPCR trafficking in human colonic epithelial cells by which a microRNA, miR-133α regulates NTR1 trafficking through its downstream target AFTPH. PMID:26902265

  12. T3-induced liver AMP-activated protein kinase signaling: Redox dependency and upregulation of downstream targets

    Science.gov (United States)

    Videla, Luis A; Fernández, Virginia; Cornejo, Pamela; Vargas, Romina; Morales, Paula; Ceballo, Juan; Fischer, Alvaro; Escudero, Nicolás; Escobar, Oscar

    2014-01-01

    AIM: To investigate the redox dependency and promotion of downstream targets in thyroid hormone (T3)-induced AMP-activated protein kinase (AMPK) signaling as cellular energy sensor to limit metabolic stresses in the liver. METHODS: Fed male Sprague-Dawley rats were given a single ip dose of 0.1 mg T3/kg or T3 vehicle (NaOH 0.1 N; controls) and studied at 8 or 24 h after treatment. Separate groups of animals received 500 mg N-acetylcysteine (NAC)/kg or saline ip 30 min prior T3. Measurements included plasma and liver 8-isoprostane and serum β-hydroxybutyrate levels (ELISA), hepatic levels of mRNAs (qPCR), proteins (Western blot), and phosphorylated AMPK (ELISA). RESULTS: T3 upregulates AMPK signaling, including the upstream kinases Ca2+-calmodulin-dependent protein kinase kinase-β and transforming growth factor-β-activated kinase-1, with T3-induced reactive oxygen species having a causal role due to its suppression by pretreatment with the antioxidant NAC. Accordingly, AMPK targets acetyl-CoA carboxylase and cyclic AMP response element binding protein are phosphorylated, with the concomitant carnitine palmitoyltransferase-1α (CPT-1α) activation and higher expression of peroxisome proliferator-activated receptor-γ co-activator-1α and that of the fatty acid oxidation (FAO)-related enzymes CPT-1α, acyl-CoA oxidase 1, and acyl-CoA thioesterase 2. Under these conditions, T3 induced a significant increase in the serum levels of β-hydroxybutyrate, a surrogate marker for hepatic FAO. CONCLUSION: T3 administration activates liver AMPK signaling in a redox-dependent manner, leading to FAO enhancement as evidenced by the consequent ketogenic response, which may constitute a key molecular mechanism regulating energy dynamics to support T3 preconditioning against ischemia-reperfusion injury. PMID:25516653

  13. Akt inhibition promotes ABCA1-mediated cholesterol efflux to ApoA-I through suppressing mTORC1.

    Directory of Open Access Journals (Sweden)

    Fumin Dong

    Full Text Available ATP-binding cassette transporter A1 (ABCA1 plays an essential role in mediating cholesterol efflux to apolipoprotein A-I (apoA-I, a major housekeeping mechanism for cellular cholesterol homeostasis. After initial engagement with ABCA1, apoA-I directly interacts with the plasma membrane to acquire cholesterol. This apoA-I lipidation process is also known to require cellular signaling processes, presumably to support cholesterol trafficking to the plasma membrane. We report here that one of major signaling pathways in mammalian cells, Akt, is also involved. In several cell models that express ABCA1 including macrophages, pancreatic beta cells and hepatocytes, inhibition of Akt increases cholesterol efflux to apoA-I. Importantly, Akt inhibition has little effect on cells expressing non-functional mutant of ABCA1, implicating a specific role of Akt in ABCA1 function. Furthermore, we provide evidence that mTORC1, a major downstream target of Akt, is also a negative regulator of cholesterol efflux. In cells where mTORC1 is constitutively activated due to tuberous sclerosis complex 2 deletion, cholesterol efflux to apoA-I is no longer sensitive to Akt activity. This suggests that Akt suppresses cholesterol efflux through mTORC1 activation. Indeed, inhibition of mTORC1 by rapamycin or Torin-1 promotes cholesterol efflux. On the other hand, autophagy, one of the major pathways of cholesterol trafficking, is increased upon Akt inhibition. Furthermore, Akt inhibition disrupts lipid rafts, which is known to promote cholesterol efflux to apoA-I. We therefore conclude that Akt, through its downstream targets, mTORC1 and hence autophagy, negatively regulates cholesterol efflux to apoA-I.

  14. Identification of aurora kinase B and Wee1-like protein kinase as downstream targets of (V600E)B-RAF in melanoma.

    Science.gov (United States)

    Sharma, Arati; Madhunapantula, SubbaRao V; Gowda, Raghavendra; Berg, Arthur; Neves, Rogerio I; Robertson, Gavin P

    2013-04-01

    BRAF is the most mutated gene in melanoma, with approximately 50% of patients containing V600E mutant protein. (V600E)B-RAF can be targeted using pharmacological agents, but resistance develops in patients by activating other proteins in the signaling pathway. Identifying downstream members in this signaling cascade is important to design strategies to avoid the development of resistance. Unfortunately, downstream proteins remain to be identified and therapeutic potential requires validation. A kinase screen was undertaken to identify downstream targets in the (V600E)B-RAF signaling cascade. Involvement of aurora kinase B (AURKB) and Wee1-like protein kinase (WEE1) as downstream proteins in the (V600E)B-RAF pathway was validated in xenografted tumors, and mechanisms of action were characterized in size- and time-matched tumors. Levels of only AURKB and WEE1 decreased in melanoma cells, when (V600E)B-RAF, mitogen-activated protein kinase 1/2, or extracellular signal-regulated kinase 1/2 protein levels were reduced using siRNA compared with other identified kinases. AURKB and WEE1 were expressed in tumors of patients with melanoma at higher levels than observed in normal human melanocytes. Targeting these proteins reduced tumor development by approximately 70%, similar to that observed when inhibiting (V600E)B-RAF. Furthermore, protein or activity levels of AURKB and WEE1 decreased in melanoma cells when pharmacological agents targeting upstream (V600E)B-RAF or mitogen-activated protein kinase were used to inhibit the (V600E)B-RAF pathway. Thus, AURKB and WEE1 are targets and biomarkers of therapeutic efficacy, lying downstream of (V600E)B-RAF in melanomas.

  15. Akt kinase-interacting protein1, a novel therapeutic target for lung cancer with EGFR-activating and gatekeeper mutations

    OpenAIRE

    Yamada, Tadaaki; Takeuchi, Shinji; Fujita, Naoya; Nakamura, Akito; Wang, Wei; Li, Qi; Oda, Makoto; Mitsudomi, Tetsuya; Yatabe, Yasushi; Sekido, Yoshitaka; Yoshida, Junji; Higashiyama, Masahiko; Noguchi, Masayuki; Uehara, Hisanori; Nishioka, Yasuhiko

    2013-01-01

    Despite initial dramatic response, epidermal growth factor receptor (EGFR) mutant lung cancer patients always acquire resistance to EGFR-tyrosine kinase inhibitors (TKIs). Gatekeeper T790M mutation in EGFR is the most prevalent genetic alteration underlying acquired resistance to EGFR-TKI, and EGFR mutant lung cancer cells are reported to be addictive to EGFR/Akt signaling even after acquired T790M mutation. Here, we focused on Akt kinase-interacting protein1 (Aki1), a scaffold protein of PI3...

  16. Metabonomics applied in exploring the antitumour mechanism of physapubenolide on hepatocellular carcinoma cells by targeting glycolysis through the Akt-p53 pathway

    Science.gov (United States)

    Ma, Ting; Fan, Bo-Yi; Zhang, Chao; Zhao, Hui-Jun; Han, Chao; Gao, Cai-Yun; Luo, Jian-Guang; Kong, Ling-Yi

    2016-01-01

    Metabolomics can be used to identify potential markers and discover new targets for future therapeutic interventions. Here, we developed a novel application of the metabonomics method based on gas chromatography-mass spectrometry (GC/MS) analysis and principal component analysis (PCA) for rapidly exploring the anticancer mechanism of physapubenolide (PB), a cytotoxic withanolide isolated from Physalis species. PB inhibited the proliferation of hepatocellular carcinoma cells in vitro and in vivo, accompanied by apoptosis-related biochemical events, including the cleavage of caspase-3/7/9 and PARP. Metabolic profiling analysis revealed that PB disturbed the metabolic pattern and significantly decreased lactate production. This suggests that the suppression of glycolysis plays an important role in the anti-tumour effects induced by PB, which is further supported by the decreased expression of glycolysis-related genes and proteins. Furthermore, the increased level of p53 and decreased expression of p-Akt were observed, and the attenuated glycolysis and enhanced apoptosis were reversed in the presence of Akt cDNA or p53 siRNA. These results confirm that PB exhibits anti-cancer activities through the Akt-p53 pathway. Our study not only reports for the first time the anti-tumour mechanism of PB, but also suggests that PB is a promising therapeutic agent for use in cancer treatments and that metabolomic approaches provide a new strategy to effectively explore the molecular mechanisms of promising anticancer compounds. PMID:27416811

  17. p300 and p53 levels determine activation of HIF-1 downstream targets in invasive breast cancer

    NARCIS (Netherlands)

    Vleugel, M.M.; Shvarts, D.; Wall, E. van der; Diest, P.J. van

    2006-01-01

    In previous studies, we noted that overexpression of hypoxia-inducible factor (HIF)–1a in breast cancer, especially the diffuse form, does not always lead to functional activation of its downstream genes. Transcriptional activity of HIF-1 may be repressed by p53 through competition for transcription

  18. Tumor suppressor PRSS8 targets Sphk1/S1P/Stat3/Akt signaling in colorectal cancer

    Science.gov (United States)

    Wang, Qian; Li, Zexin; Yang, Yiqiong; Chen, Zhiguo; Wang, Jianguo; Zhao, Weixing; Zhang, Huijuan; Chen, Jiwang; Dong, Huali; Shen, Kui; Diamond, Alan M.; Yang, Wancai

    2016-01-01

    PRSS8 is a membrane-anchored serine protease prostasin and has been shown an association with carcinogenesis. Herein we found that PRSS8 expression was significantly reduced in colorectal adenomas and adenocarcinomas. The decreased PRSS8 was well correlated with clinical stages, poor differentiation and shorter survival time of colorectal cancer. Furthermore, increase of PRSS8 led to the inhibition of colorectal cancer cell proliferation, knockdown of PRSS8 accelerated cell proliferation in vitro, and overexpressing PRSS8 retarded cancer cell growth in nude mice. Mechanistic studies revealed that PRSS8 inhibited Sphk1/S1P/Stat3/Akt signaling pathway, in terms of inverse association between PRSS8 and Sphk1 in human colorectal cancers and in Sphk1-/− mice. In conclusion, PRSS8 acts as a tumor suppressor by inhibiting Sphk1/S1P/Stat3/Akt signaling pathway, and could be used as a biomarker to monitor colorectal carcinogenesis and predict outcomes. PMID:27050145

  19. Constitutively Active Akt1 Cooperates with KRasG12D to Accelerate In Vivo Pancreatic Tumor Onset and Progression

    Directory of Open Access Journals (Sweden)

    Toya M. Albury

    2015-02-01

    Full Text Available BACKGROUND AND AIMS: Pancreatic adenocarcinoma is a deadly disease characterized by metastatic progression and resistance to conventional therapeutics. Mutation of KRAS is the most frequent early event in pancreatic tumor progression. AKT isoforms are frequently activated in pancreatic cancer, and reports have implicated hyperactivation of AKT1, as well as AKT2, in pancreatic tumor formation. The objective here is to delineate the role of AKT in facilitating in vivo pancreatic tumor progression in the context of KRAS mutation and predisposition to pancreatic cancer. METHODS: Mice with Akt1 and KRas mutant alleles expressed using the pancreas Pdx promoter were mated to characterize the incidence and frequency of histologic and genetic alterations known to occur commonly in human pancreatic ductal adenocarcinoma. RESULTS: Active Akt1 (Akt1Myr, containing a myristoylation sequence cooperated with active mutant KRasG12D to accelerate pancreatic carcinoma onset and progression and increase phosphorylation of downstream effectors in the Akt pathway. Mucin and smooth muscle actin expression was found in and around pancreatic intraepithelial neoplasms (PanINs, and accelerated time to metastasis was found in Akt1Myr/KRasG12D mice. CONCLUSIONS: In contrast to prior reports of pancreatic KRas mutant mice mated with mice deficient for various tumor suppressor genes, which resulted in aggressive disease within a few months of age, Akt1Myr/KRasG12D mice enabled the study of PanINs and spontaneous pancreatic transformation more characteristic of human pancreatic progression in elderly individuals. The Akt1Myr/KRasG12D model holds promise for delineating the tumor biology and biomarkers critical for understanding their cooperation in cancer oncogenesis and future targeting in therapeutic strategies.

  20. MiR-34a targets GAS1 to promote cell proliferation and inhibit apoptosis in papillary thyroid carcinoma via PI3K/Akt/Bad pathway

    Energy Technology Data Exchange (ETDEWEB)

    Ma, Yanfei; Qin, Huadong [Department of Fourth Surgery, the Second Affiliated Hospital of Harbin Medical University, 148 Xuefu Road, Nangang District, Harbin 150086 (China); Cui, Yunfu, E-mail: yfma77@126.com [Department of First Surgery, the Second Affiliated Hospital of Harbin Medical University, 148 Xuefu Road, Nangang District, Harbin 150086 (China)

    2013-11-29

    Highlights: •MiR-34a is up- and GAS1 is down-regulated in papillary thyroid carcinoma. •GAS1 is a direct target for miR-34a. •MiR-34a promotes PTC cells proliferation and inhibits apoptosis through PI3K/Akt/Bad pathway. -- Abstract: MicroRNAs (miRNAs) are fundamental regulators of cell proliferation, differentiation, and apoptosis, and are implicated in tumorigenesis of many cancers. MiR-34a is best known as a tumor suppressor through repression of growth factors and oncogenes. Growth arrest specific1 (GAS1) protein is a tumor suppressor that inhibits cancer cell proliferation and induces apoptosis through inhibition of RET receptor tyrosine kinase. Both miR-34a and GAS1 are frequently down-regulated in various tumors. However, it has been reported that while GAS1 is down-regulated in papillary thyroid carcinoma (PTC), miR-34a is up-regulated in this specific type of cancer, although their potential roles in PTC tumorigenesis have not been examined to date. A computational search revealed that miR-34a putatively binds to the 3′-UTR of GAS1 gene. In the present study, we confirmed previous findings that miR-34a is up-regulated and GAS1 down-regulated in PTC tissues. Further studies indicated that GAS1 is directly targeted by miR-34a. Overexpression of miR-34a promoted PTC cell proliferation and colony formation and inhibited apoptosis, whereas knockdown of miR-34a showed the opposite effects. Silencing of GAS1 had similar growth-promoting effects as overexpression of miR-34a. Furthermore, miR-34a overexpression led to activation of PI3K/Akt/Bad signaling pathway in PTC cells, and depletion of Akt reversed the pro-growth, anti-apoptotic effects of miR-34a. Taken together, our results demonstrate that miR-34a regulates GAS1 expression to promote proliferation and suppress apoptosis in PTC cells via PI3K/Akt/Bad pathway. MiR-34a functions as an oncogene in PTC.

  1. PI3K/Akt signalling pathway and cancer.

    Science.gov (United States)

    Fresno Vara, Juan Angel; Casado, Enrique; de Castro, Javier; Cejas, Paloma; Belda-Iniesta, Cristóbal; González-Barón, Manuel

    2004-04-01

    Phosphatidylinositol-3 kinases, PI3Ks, constitute a lipid kinase family characterized by their ability to phosphorylate inositol ring 3'-OH group in inositol phospholipids to generate the second messenger phosphatidylinositol-3,4,5-trisphosphate (PI-3,4,5-P(3)). RPTK activation results in PI(3,4,5)P(3) and PI(3,4)P(2) production by PI3K at the inner side of the plasma membrane. Akt interacts with these phospholipids, causing its translocation to the inner membrane, where it is phosphorylated and activated by PDK1 and PDK2. Activated Akt modulates the function of numerous substrates involved in the regulation of cell survival, cell cycle progression and cellular growth. In recent years, it has been shown that PI3K/Akt signalling pathway components are frequently altered in human cancers. Cancer treatment by chemotherapy and gamma-irradiation kills target cells primarily by the induction of apoptosis. However, the development of resistance to therapy is an important clinical problem. Failure to activate the apoptotic programme represents an important mode of drug resistance in tumor cells. Survival signals induced by several receptors are mediated mainly by PI3K/Akt, hence this pathway may decisively contribute to the resistant phenotype. Many of the signalling pathways involved in cellular transformation have been elucidated and efforts are underway to develop treatment strategies that target these specific signalling molecules or their downstream effectors. The PI3K/Akt pathway is involved in many of the mechanisms targeted by these new drugs, thus a better understanding of this crossroad can help to fully exploit the potential benefits of these new agents. PMID:15023437

  2. ROLE OF PI3K-AKT-mTOR AND Wnt SIGNALING PATHWAYS IN G1-S TRANSITION OF CELL CYCLE IN CANCER CELLS

    Directory of Open Access Journals (Sweden)

    LAKSHMIPATHI eVADLAKONDA

    2013-04-01

    Full Text Available The PI3K–Akt pathway together with one of its downstream targets, the mechanistic target of rapamycin (mTOR is a highly deregulated pathway in cancers. There is a reciprocal relation between the Akt phosphorylation and mTOR complexes. Akt phosphorylated at T308 activates mTORC1 by inhibition of the tuberous sclerosis complex (TSC1/2, where as mTORC2 is recognized as the kinase that phosphorylates Akt at S473. Recent developments in the research on regulatory mechanisms of autophagy places mTORC1 mediated inhibition of autophagy at the central position in activation of proliferation and survival pathways in cells. Autophagy is a negative regulator of Wnt signaling pathway and the downstream effectors of Wnt signaling pathway, cyclin D1 and the c-Myc, are the key players in initiation of cell cycle and regulation of the G1-S transition in cancer cells. Production of reaction oxygen species (ROS, a common feature of a cancer cell metabolism, activates several downstream targets like the transcription factors FoxO, which play key roles in promoting the progression of cell cycle. A model is presented on the role of PI3K -Akt - mTOR and Wnt pathways in regulation of the progression of cell cycle through Go-G1-and S phases.

  3. Development of Drugs Targeting the PI3K Signalling Pathway in Leukaemias and Lymphomas

    OpenAIRE

    Alexandre Arcaro

    2015-01-01

    The phosphoinositide 3-kinase (PI3K) family of signalling enzymes play a key role in the transduction of signals from activated cell surface receptors controlling cell growth and proliferation, survival, metabolism, and migration. The intracellular signalling pathway from activated receptors to PI3K and its downstream targets v-akt murine thymoma viral oncogene homolog (Akt) and mechanistic target of rapamycin (mTOR) is very frequently deregulated by genetic and epigenetic mechanisms in human...

  4. Disulfiram targets cancer stem-like properties and the HER2/Akt signaling pathway in HER2-positive breast cancer.

    Science.gov (United States)

    Kim, Ji Young; Cho, Youngkwan; Oh, Eunhye; Lee, Nahyun; An, Hyunsook; Sung, Daeil; Cho, Tae-Min; Seo, Jae Hong

    2016-08-28

    HER2-positive breast tumors are known to harbor cancer stem-like cell populations and are associated with an aggressive tumor phenotype and poor clinical outcomes. Disulfiram (DSF), an anti-alcoholism drug, is known to elicit cytotoxicity in many cancer cell types in the presence of copper (Cu). The objective of the present study was to investigate the mechanism of action responsible for the induction of apoptosis by DSF/Cu and its effect on cancer stem cell properties in HER2-positive breast cancers in vitro and in vivo. DSF/Cu treatment induced apoptosis, associated with a marked decrease in HER2, truncated p95HER2, phospho-HER2, HER3, phospho-HER3 and phospho-Akt levels, and p27 nuclear accumulation. This was accompanied by the eradication of cancer stem-like populations, concomitant with the suppression of aldehyde dehydrogenase 1 (ALDH1) activity and mammosphere formation. DSF administration resulted in a significant reduction in tumor growth and an enhancement of apoptosis, as well as HER2 intracellular domain (ICD) and ALDH1A1 downregulation. Our results demonstrate that DSF/Cu induces apoptosis and eliminates cancer stem-like cells via the suppression of HER2/Akt signaling, suggesting that DSF may be potentially effective for the treatment of HER2-positive cancers. PMID:27238567

  5. Inhibition of AKT with the orally active allosteric AKT inhibitor, MK-2206, sensitizes endometrial cancer cells to progestin.

    Directory of Open Access Journals (Sweden)

    Alok Pant

    Full Text Available Progestin resistance is a major obstacle to treating early stage, well-differentiated endometrial cancer as well as recurrent endometrial cancer. The mechanism behind the suboptimal response to progestin is not well understood. The PTEN tumor suppressor gene is frequently mutated in type I endometrial cancers and this mutation results in hyperactivation of the PI3K/AKT pathway. We hypothesized that increased activation of AKT promotes an inadequate response to progestins in endometrial cancer cells. Ishikawa cells stably transfected with progesterone receptor B (PRB23 cells were treated with the AKT inhibitor, MK-2206, which effectively decreased levels of p(Ser473-AKT in a dose-dependent (10 nM to 1 uM and time-dependent manner (0.5 h to 24 h. MK-2206 inhibited levels of p(Thr308-AKT and a downstream target, p(Thr246-PRAS40, but did not change levels of p(Thr202/Tyr204ERK or p(Thr13/Tyr185SAPK/JNK, demonstrating specificity of MK-2206 for AKT. Additionally, MK-2206 treatment of PRB23 cells resulted in a significant increase in levels of progesterone receptor B (PRB protein. Microarray analysis of PRB23 cells identified PDK4 as the most highly upregulated gene among 70 upregulated genes in response to R5020. Inhibition of AKT further upregulated progestin-mediated expression of PDK4 but did not affect another progestin-responsive gene, SGK1. Treatment of PRB23 cells with R5020 and MK-2206 independently decreased viability of cells while the combination of R5020 and MK-2206 caused the greatest decrease in cell viability. Furthermore, mice with xenografted tumors treated with MK-2206 alone or with progesterone alone exhibited modest reductions in their tumor volume. The largest decrease in tumor size was observed in the mice treated with both MK-2206 and progesterone; these tumors exhibited the least proliferation (Ki67 and the most apoptosis (cleaved caspase-3 of all the treatment groups. In summary, inhibition of AKT stabilizes the Progesterone

  6. Experimental and numerical study on the flow pattern of the ADS windowless spallation target with a second free surface downstream using model fluid water

    Energy Technology Data Exchange (ETDEWEB)

    Xiong, Zhenqin, E-mail: zqxiong@sjtu.edu.cn; Gu, Hanyang; Gong, Shenjie

    2015-09-15

    Highlights: • A windowless spallation target with a buffer tank is tested. • Shape of the main free surface is recorded. • Streamline is obtained with the planar laser induced fluorescence method. • Stability of free surface is improved by the buffer tank. • Flow structure is simulated using RNG k-e turbulence model and VOF model. - Abstract: The windowless spallation targets are a promising design solution for accelerator driven system (ADS) due to their extended life compared to the spallation targets with a window. Keeping the stability of the free surface and reducing the recirculation zone is one of the key tasks for the design of a windowless spallation target. A windowless spallation target with a second free surface downstream (which is a buffer used to stabilize the main free surface of the flow) is studied experimentally and numerically using water at atmospheric pressure. By using planar laser induced fluorescence technique (LIF), the flow pattern inside the target zone is visualized for Reynolds numbers varying between 3.5 × 10{sup 4} and 7.0 × 10{sup 4} and pressure differences from 100 to 804 Pa. The experimental results reveal that the stability of the free surface is improved by adding a buffer in the downstream thus making it easier to control the height of the surface. The effect of the pressure difference between the void above the second free surface (high pressure side) and beam pipe (low pressure side) on the flow pattern is analyzed, as well as the inlet flow rate. The height of the surface length decreases with an increase in the pressure difference. The formation of the spallation zone is simulated with Fluent using the LES turbulence model and VOF model. The interface predicted agrees well with the experimental results.

  7. Experimental and numerical study on the flow pattern of the ADS windowless spallation target with a second free surface downstream using model fluid water

    International Nuclear Information System (INIS)

    Highlights: • A windowless spallation target with a buffer tank is tested. • Shape of the main free surface is recorded. • Streamline is obtained with the planar laser induced fluorescence method. • Stability of free surface is improved by the buffer tank. • Flow structure is simulated using RNG k-e turbulence model and VOF model. - Abstract: The windowless spallation targets are a promising design solution for accelerator driven system (ADS) due to their extended life compared to the spallation targets with a window. Keeping the stability of the free surface and reducing the recirculation zone is one of the key tasks for the design of a windowless spallation target. A windowless spallation target with a second free surface downstream (which is a buffer used to stabilize the main free surface of the flow) is studied experimentally and numerically using water at atmospheric pressure. By using planar laser induced fluorescence technique (LIF), the flow pattern inside the target zone is visualized for Reynolds numbers varying between 3.5 × 104 and 7.0 × 104 and pressure differences from 100 to 804 Pa. The experimental results reveal that the stability of the free surface is improved by adding a buffer in the downstream thus making it easier to control the height of the surface. The effect of the pressure difference between the void above the second free surface (high pressure side) and beam pipe (low pressure side) on the flow pattern is analyzed, as well as the inlet flow rate. The height of the surface length decreases with an increase in the pressure difference. The formation of the spallation zone is simulated with Fluent using the LES turbulence model and VOF model. The interface predicted agrees well with the experimental results

  8. mTOR-rictor is the Ser473 kinase for AKT1 in mouse one-cell stage embryos.

    Science.gov (United States)

    Zhang, Zhe; Zhang, Guojun; Xu, Xiaoyan; Su, Wenhui; Yu, Bingzhi

    2012-02-01

    Mammalian target of rapamycin (mTOR) controls cell growth and proliferation via the raptor-mTOR (TORC1) and rictor-mTOR (TORC2) protein complexes. The mTORC2 containing mTOR and rictor is thought to be rapamycin insensitive and it is recently shown that both rictor and mTORC2 are essential for the development of both embryonic and extra embryonic tissues. To explore rictor function in the early development of mouse embryos, we disrupted the expression of rictor, a specific component of mTORC2, in mouse fertilized eggs by using rictor shRNA. Our results showed that one-cell stage eggs that were lack of rictor could not enter into the two-cell stage normally. Recent biochemical studies suggests that TORC2 is the elusive PDK2 (3'-phosphoinositide-dependent kinase 2) for AKT/PKB Ser473 phosphorylation, which is deemed necessary for AKT function, so we microinjected AKT-S473A into mouse fertilized eggs to investigate whether AKT-S473A is downstream effector of mTOR.rictor to regulate the mitotic division. Our findings revealed that the rictor induced phosphorylation of AKT in Ser473 is required for TORC2 function in early development of mouse embryos. PMID:22057724

  9. Novel Drugs Targeting the Epidermal Growth Factor Receptor and Its Downstream Pathways in the Treatment of Colorectal Cancer: A Systematic Review

    Directory of Open Access Journals (Sweden)

    Amartej Merla

    2012-01-01

    Full Text Available Colorectal cancer is the second most common malignancy among men and women in the United States, and the 5-year survival rate remains poor despite recent advances in chemotherapy and targeted agents. The mainstay of therapy for advanced disease remains the cytotoxic chemotherapy including 5-FU, irinotecan, and oxaliplatin. The USFDA approval and introduction of targeted therapies, including cetuximab and panitumumab (monoclonal antibodies targeting the epidermal growth factor receptor (EGFR and bevacizumab (monoclonal antibody targeting the vascular epithelial growth factor (VEGF, has improved the median survival of patients with metastatic colorectal cancer to around 24 months. Clearly, better and more efficacious drugs are needed, and target-specific agents remain the future of cancer treatment. On this front, rapid advances are being made, which are likely to change the future of the management of metastatic colorectal cancer. However, absence of specific biomarkers for the use of targeted agents, in the subset of population who will benefit from the treatment, remains a major drawback. In this paper, we review agents that are in phases 1 and 2 clinical development, specifically targeting the EGFR and its subsequent downstream pathways.

  10. DLC-1 induces mitochondrial apoptosis and epithelial mesenchymal transition arrest in nasopharyngeal carcinoma by targeting EGFR/Akt/NF-κB pathway.

    Science.gov (United States)

    Huang, Wei; Liu, Jie; Feng, Xiangling; Chen, Huan; Zeng, Liang; Huang, Guoling; Liu, Weidong; Wang, Lei; Jia, Wei; Chen, Jiawen; Ren, Caiping

    2015-04-01

    Loss of deleted in liver cancer-1 (DLC-1) can induce apoptosis and inhibit the mobility, migration and metastasis in several cancers. Previously, we revealed that ectopic expression of DLC-1 can suppress proliferation, mobility, migration and tumorigenesis in nasopharyngeal carcinoma (NPC). However, the molecular mechanisms accounting for the roles of DLC-1 in NPC are still obscure. In the present work, we attempted to study and uncover the mechanisms underlying the functions of DLC-1 in NPC. The apoptosis of 5-8F-DLC-1 cells, established previously, was analyzed by mitochondrial membrane potentials assay and flow cytometer analysis. And the antibodies involving pathways such as mitochondrial-associated apoptosis, epithelial mesenchymal transition and metastasis were applied to detect and compare the expression level of targeted proteins. The obvious apoptosis of 5-8F-DLC-1 cells was observed reflected by mitochondrial depolarization and lower ratio in cell viability. Subsequently, the activation of mitochondrial apoptosis was verified by the increased expressions of Bax, Apaf1, cleave-caspases and cleave-PARP, etc, and the decreased expressions of Bcl-2, Bcl-xL, Mcl-1, Survivin, etc, in 5-8F-DLC-1 cells. Then, the inhibited epithelial mesenchymal transition of 5-8F-DLC-1 cells was validated by upregulated expression of E-cadherin and downregulated expression of N-cadherin, Snail, Vimentin. Subsequently, downregulated expressions of proteins such as FAK, RhoA, ROCK1 and cdc25 related to cell adhesion and cytoskeleton organization were also observed. And expressions of MMPs were inhibited in 5-8F-DLC-1 cells. At last, the inhibited activity of EGFR/Akt/NF-κB axis was revealed by the decreased expressions of phosho-EGFR, phosho-Akt, phosho-p38MAPK, phosho-IKKα and phosho-p65. Here, we systematically explored the mechanisms underlying the negative roles of DLC-1 in NPC cells. For the first time, we confirmed that the ectopic expression DLC-1 can induce mitochondrial

  11. MiR-128b is down-regulated in gastric cancer and negatively regulates tumour cell viability by targeting PDK1/Akt/NF-κB axis

    Indian Academy of Sciences (India)

    Ling Zhang; Jun Lei; Zi-Ling Fang; Jian-Ping Xiong

    2016-03-01

    Gastric cancer (GC) is the fourth most prevalent type of cancer worldwide, which is usually caused by the interaction between environmental and genetic factors, or epigenetic aspects. Referring to the non-coding RNAs, miR-128b has been reported to be associated with many tumour cases, and exerts distinct functions in different types of cancers. However, the function of miR-128b in GC onset and progression largely remains unknown. In the present study, we found that miR-128b expression was down-regulated in tissues from 18 GC patients and 3 carcinoma cell lines. In turn, over-expression of miR-128b suppressed GC cell proliferation, invasion and promoted apoptosis. Moreover, miR-128b was predicted to bind the 3'UTR of PDK1 gene using bioinformatic target-screening tools. Accordingly, ectopic expression of miR-128b inhibited the PDK1 expression at both transcriptional and post-transcriptional levels, and furthermore, the expression of gene tailed by the 3′UTR of PDK1 gene was significantly decreased in a dualluciferase reporter assay, suggesting that PDK1 was a direct target of miR-128b in GC cells. In the conditon of miR-128b over-expression, we also observed spontaneous inactivation of the Akt/NF-κB signalling, implying PDK1 was a potential regulator of this pathway. In conclusion, our study shed some novel light on miR-128b-PDK1/Akt/NF-κB axis on GC progression.

  12. MicroRNA-143-3p inhibits hyperplastic scar formation by targeting connective tissue growth factor CTGF/CCN2 via the Akt/mTOR pathway.

    Science.gov (United States)

    Mu, Shengzhi; Kang, Bei; Zeng, Weihui; Sun, Yaowen; Yang, Fan

    2016-05-01

    Post-traumatic hypertrophic scar (HS) is a fibrotic disease with excessive extracellular matrix (ECM) production, which is a response to tissue injury by fibroblasts. Although emerging evidence has indicated that miRNA contributes to hypertrophic scarring, the role of miRNA in HS formation remains unclear. In this study, we found that miR-143-3p was markedly downregulated in HS tissues and fibroblasts (HSFs) using qRT-PCR. The expression of connective tissue growth factor (CTGF/CCN2) was upregulated both in HS tissues and HSFs, which is proposed to play a key role in ECM deposition in HS. The protein expression of collagen I (Col I), collagen III (Col III), and α-smooth muscle actin (α-SMA) was obviously inhibited after treatment with miR-143-3p in HSFs. The CCK-8 assay showed that miR-143-3p transfection reduced the proliferation ability of HSFs, and flow cytometry showed that either early or late apoptosis of HSFs was upregulated by miR-143-3p. In addition, the activity of caspase 3 and caspase 9 was increased after miR-143-3p transfection. On the contrary, the miR-143-3p inhibitor was demonstrated to increase cell proliferation and inhibit apoptosis of HSFs. Moreover, miR-143-3p targeted the 3'-UTR of CTGF and caused a significant decrease of CTGF. Western blot demonstrated that Akt/mTOR phosphorylation and the expression of CTGF, Col I, Col III, and α-SMA were inhibited by miR-143-3p, but increased by CTGF overexpression. In conclusion, we found that miR-143-3p inhibits hypertrophic scarring by regulating the proliferation and apoptosis of human HSFs, inhibiting ECM production-associated protein expression by targeting CTGF, and restraining the Akt/mTOR pathway.

  13. Upregulation of AKT3 Confers Resistance to the AKT Inhibitor MK2206 in Breast Cancer.

    Science.gov (United States)

    Stottrup, Casey; Tsang, Tiffany; Chin, Y Rebecca

    2016-08-01

    Acquired resistance to molecular targeted therapy represents a major challenge for the effective treatment of cancer. Hyperactivation of the PI3K/AKT pathway is frequently observed in virtually all human malignancies, and numerous PI3K and AKT inhibitors are currently under clinical evaluation. However, mechanisms of acquired resistance to AKT inhibitors have yet to be described. Here, we use a breast cancer preclinical model to identify resistance mechanisms to a small molecule allosteric AKT inhibitor, MK2206. Using a step-wise and chronic high-dose exposure, breast cancer cell lines harboring oncogenic PI3K resistant to MK2206 were established. Using this model, we reveal that AKT3 expression is markedly upregulated in AKT inhibitor-resistant cells. Induction of AKT3 is regulated epigenetically by the bromodomain and extra terminal domain proteins. Importantly, knockdown of AKT3, but not AKT1 or AKT2, in resistant cells restores sensitivity to MK2206. AKT inhibitor-resistant cells also display an epithelial to mesenchymal transition phenotype as assessed by alterations in the levels of E-Cadherin, N-Cadherin, and vimentin, as well as enhanced invasiveness of tumor spheroids. Notably, the invasive morphology of resistant spheroids is diminished upon AKT3 depletion. We also show that resistance to MK2206 is reversible because upon drug removal resistant cells regain sensitivity to AKT inhibition, accompanied by reexpression of epithelial markers and reduction of AKT3 expression, implying that epigenetic reprogramming contributes to acquisition of resistance. These findings provide a rationale for developing therapeutics targeting AKT3 to circumvent acquired resistance in breast cancer. Mol Cancer Ther; 15(8); 1964-74. ©2016 AACR. PMID:27297869

  14. LRF is an essential downstream target of GATA1 in erythroid development and regulates BIM-dependent apoptosis

    OpenAIRE

    Maeda, Takahiro; Ito, Keisuke; Merghoub, Taha; Poliseno, Laura; Hobbs, Robin M.; Wang, Guocan; Dong, Lin; Maeda, Manami; Dore, Louis C.; Zelent, Arthur; Luzzatto, Lucio; Teruya-Feldstein, Julie; Weiss, Mitchell J.; Pandolfi, Pier Paolo

    2009-01-01

    GATA-1-dependent transcription is essential for erythroid differentiation and maturation. Suppression of programmed cell death is also thought to be critical for this process; however, the link between these two features of erythropoiesis has remained elusive. Here, we show that the POZ-Krüppel family transcription factor, LRF (also known as Zbtb7a/Pokemon), is a direct target of GATA1 and plays an essential anti-apoptotic role during terminal erythroid differentiation. We find that loss of L...

  15. eIF4B is a convergent target and critical effector of oncogenic Pim and PI3K/Akt/mTOR signaling pathways in Abl transformants.

    Science.gov (United States)

    Chen, Ke; Yang, Jianling; Li, Jianning; Wang, Xuefei; Chen, Yuhai; Huang, Shile; Chen, Ji-Long

    2016-03-01

    Activation of eIF4B correlates with Abl-mediated cellular transformation, but the precise mechanisms are largely unknown. Here we show that eIF4B is a convergent substrate of JAK/STAT/Pim and PI3K/Akt/mTOR pathways in Abl transformants. Both pathways phosphorylated eIF4B in Abl-transformed cells, and such redundant regulation was responsible for the limited effect of single inhibitor on Abl oncogenicity. Persistent inhibition of one signaling pathway induced the activation of the other pathway and thereby restored the phosphorylation levels of eIF4B. Simultaneous inhibition of the two pathways impaired eIF4B phosphorylation more effectively, and synergistically induced apoptosis in Abl transformed cells and inhibited the growth of engrafted tumors in nude mice. Similarly, the survival of Abl transformants exhibited a higher sensitivity to the pharmacological inhibition, when combined with the shRNA-based silence of the other pathway. Interestingly, such synergy was dependent on the phosphorylation status of eIF4B on Ser422, as overexpression of eIF4B phosphomimetic mutant S422E in the transformants greatly attenuated the synergistic effects of these inhibitors on Abl oncogenicity. In contrast, eIF4B knockdown sensitized Abl transformants to undergo apoptosis induced by the combined blockage. Collectively, the results indicate that eIF4B integrates the signals from Pim and PI3K/Akt/mTOR pathways in Abl-expressing leukemic cells, and is a promising therapeutic target for such cancers. PMID:26848623

  16. TIM-3 promotes the metastasis of esophageal squamous cell carcinoma by targeting epithelial-mesenchymal transition via the Akt/GSK-3β/Snail signaling pathway.

    Science.gov (United States)

    Shan, Baoen; Man, Hongwei; Liu, Junfeng; Wang, Ling; Zhu, Tienian; Ma, Ming; Xv, Zhili; Chen, Xinran; Yang, Xingxiao; Li, Pengfei

    2016-09-01

    T-cell immunoglobulin and mucin domain-con-taining protein-3 (TIM-3), a negative regulator of antitumor immune response, has been demonstrated to be involved in the onset and progression of several types of malignancies. The present study aimed to determine whether and how TIM‑3 plays such a role in esophageal squamous cell carcinoma (ESCC). TIM-3 expression was analyzed by immunohistochemistry and real‑time fluorescence quantitative PCR (qRT‑PCR) in ESCC and matched adjacent normal tissues. Functional experiments in vitro were performed to elucidate the effect of TIM‑3 knockdown on the proliferation, apoptosis, migration, invasion and epithelial-mesenchymal transition (EMT) in Eca109 and TE‑1 cell lines. Our data revealed that TIM‑3 expression was significantly elevated at both the mRNA and protein levels in ESCC tissues compared with the levels in the matched adjacent normal tissues (both Pdownregulation of matrix metalloproteinase (MMP)-9 and upregulation of tissue inhibitor of metalloproteinase (TIMP)-1, and with reversion of EMT, as reflected by higher levels of the epithelial marker E‑cadherin and lower levels of the mesenchymal markers N‑cadherin and vimentin. Further study found that TIM‑3 depletion suppressed the signaling pathway involving p‑Akt, p‑GSK‑3β and Snail. Taken together, these results suggest that TIM‑3 is a novel therapeutic target and prognostic biomarker for ESCC and promotes metastasis of ESCC by inducing EMT via, at least partially, the Akt/GSK-3β/Snail signaling pathway. PMID:27430162

  17. 靶向 Akt1基因 RNA 干扰对胸主动脉平滑肌细胞增殖的影响%Effect of targeting Akt1gene RNA intervention on proliferation of smooth muscle cell of rabbits

    Institute of Scientific and Technical Information of China (English)

    刘苏健; 范波; 丁明超; 王意忠; 王斌

    2014-01-01

    目的:构建兔Akt1短发卡RNA( shRNA)真核表达载体,探讨靶向Akt1基因RNA干扰对兔胸主动脉平滑肌细胞增殖的影响。方法经预实验挑选2条有效短发卡RNA转染入原代培养日本大耳白兔胸主动脉平滑肌细胞内,设置空白对照组( A组)、阴性对照组( B组)、Akt1-shRNA-1组( C组)、Akt1-shRna-2组( D组)。48、72 h荧光显微镜下观察细胞转染效率,分别在转染平滑肌细胞后24、48、72、96 h使用流式细胞仪和TUNEL法检测凋亡细胞数目。结果平滑肌细胞48 h转染效率为17櫃.8%;流式细胞仪测定C组和D组在48 h凋亡率分别为19.72%和15.43%,抑制增值效果达到最高峰,与A、B组比较,差异有统计学意义( P <0.05)。 TUNEL法测定结果表明,C、D组细胞凋亡水平与A、B组比较明显增多( P <0.05),且以C组较为明显。结论成功构建了2条兔Akt1 shRNA真核表达载体,可有效地抑制平滑肌细胞增殖。%Objective To construct Akt1 shRNA eukaryon expression vector to investigate the effect of targeting Akt1gene RNA intervention on proliferation of smooth muscle cell of rabbits .Methods After preliminary experiment , two strips of effective shRNA were selected to be transfected into thoracic aorta smooth muscle cells ( SMC ) of rabbits (Oryctolagus cuniculus).The experiment was divided into four groups ,blank control group (group A),negative control group (group B),Akt1-shRNA-1group (group C),Akt1-shRna-2 group (group D).The cell transfection rate was detected under fluorescence microscope at 48h,72h after transfection, and the cell apoptosis rates were measured by flow cytometry and TUNEL respectively at 24h,48h,72h,96h after transfection.Results The two sequences of synthesized shRNA named Akt1-shRNA-1 and Akt1-shRNA-2 were exactly the same as the designed ones .The SMC transfection rate at 48h was 17.8%. The cell apoptosis rate was 19.72% and 15.43% at 48h

  18. LRF is an essential downstream target of GATA1 in erythroid development and regulates BIM-dependent apoptosis.

    Science.gov (United States)

    Maeda, Takahiro; Ito, Keisuke; Merghoub, Taha; Poliseno, Laura; Hobbs, Robin M; Wang, Guocan; Dong, Lin; Maeda, Manami; Dore, Louis C; Zelent, Arthur; Luzzatto, Lucio; Teruya-Feldstein, Julie; Weiss, Mitchell J; Pandolfi, Pier Paolo

    2009-10-01

    GATA-1-dependent transcription is essential for erythroid differentiation and maturation. Suppression of programmed cell death is also thought to be critical for this process; however, the link between these two features of erythropoiesis has remained elusive. Here, we show that the POZ-Krüppel family transcription factor, LRF (also known as Zbtb7a/Pokemon), is a direct target of GATA1 and plays an essential antiapoptotic role during terminal erythroid differentiation. We find that loss of Lrf leads to lethal anemia in embryos, due to increased apoptosis of late-stage erythroblasts. This programmed cell death is Arf and p53 independent and is instead mediated by upregulation of the proapoptotic factor Bim. We identify Lrf as a direct repressor of Bim transcription. In strong support of this mechanism, genetic Bim loss delays the lethality of Lrf-deficient embryos and rescues their anemia phenotype. Thus, our data define a key transcriptional cascade for effective erythropoiesis, whereby GATA-1 suppresses BIM-mediated apoptosis via LRF. PMID:19853566

  19. Overexpression of Akt1 enhances adipogenesis and leads to lipoma formation in zebrafish.

    Directory of Open Access Journals (Sweden)

    Che-Yu Chu

    Full Text Available BACKGROUND: Obesity is a complex, multifactorial disorder influenced by the interaction of genetic, epigenetic, and environmental factors. Obesity increases the risk of contracting many chronic diseases or metabolic syndrome. Researchers have established several mammalian models of obesity to study its underlying mechanism. However, a lower vertebrate model for conveniently performing drug screening against obesity remains elusive. The specific aim of this study was to create a zebrafish obesity model by over expressing the insulin signaling hub of the Akt1 gene. METHODOLOGY/PRINCIPAL FINDINGS: Skin oncogenic transformation screening shows that a stable zebrafish transgenic of Tg(krt4Hsa.myrAkt1(cy18 displays severely obese phenotypes at the adult stage. In Tg(krt4:Hsa.myrAkt1(cy18, the expression of exogenous human constitutively active Akt1 (myrAkt1 can activate endogenous downstream targets of mTOR, GSK-3α/β, and 70S6K. During the embryonic to larval transitory phase, the specific over expression of myrAkt1 in skin can promote hypertrophic and hyperplastic growth. From 21 hour post-fertilization (hpf onwards, myrAkt1 transgene was ectopically expressed in several mesenchymal derived tissues. This may be the result of the integration position effect. Tg(krt4:Hsa.myrAkt1(cy18 caused a rapid increase of body weight, hyperplastic growth of adipocytes, abnormal accumulation of fat tissues, and blood glucose intolerance at the adult stage. Real-time RT-PCR analysis showed the majority of key genes on regulating adipogenesis, adipocytokine, and inflammation are highly upregulated in Tg(krt4:Hsa.myrAkt1(cy18. In contrast, the myogenesis- and skeletogenesis-related gene transcripts are significantly downregulated in Tg(krt4:Hsa.myrAkt1(cy18, suggesting that excess adipocyte differentiation occurs at the expense of other mesenchymal derived tissues. CONCLUSION/SIGNIFICANCE: Collectively, the findings of this study provide direct evidence that Akt1

  20. Interplay between FOXO, TOR, and Akt

    OpenAIRE

    Hay, Nissim

    2011-01-01

    FOXO transcription factors have emerged as rheostats that coordinate the activities of Akt and target of rapamycin complexes (TORCs). This review summarizes the regulatory circuits mediated by the activation of FOXO, which in turn modulate Akt and TORCs activities. The biological significance of these regulatory circuits is discussed in this article.

  1. Comparative analyses of downstream signal transduction targets modulated after activation of the AT1 receptor by two β-arrestin-biased agonists.

    Science.gov (United States)

    Santos, Geisa A; Duarte, Diego A; Parreiras-E-Silva, Lucas T; Teixeira, Felipe R; Silva-Rocha, Rafael; Oliveira, Eduardo B; Bouvier, Michel; Costa-Neto, Claudio M

    2015-01-01

    G protein-coupled receptors (GPCRs) are involved in essentially all physiological processes in mammals. The classical GPCR signal transduction mechanism occurs by coupling to G protein, but it has recently been demonstrated that interaction with β-arrestins leads to activation of pathways that are independent of the G protein pathway. Also, it has been reported that some ligands can preferentially activate one of these signaling pathways; being therefore called biased agonists for G protein or β-arrestin pathways. The angiotensin II (AngII) AT1 receptor is a prototype GPCR in the study of biased agonism due to the existence of well-known β-arrestin-biased agonists, such as [Sar(1), Ile(4), Ile(8)]-AngII (SII), and [Sar(1), D-Ala(8)]-AngII (TRV027). The aim of this study was to comparatively analyze the two above mentioned β-arrestin-biased agonists on downstream phosphorylation events and gene expression profiles. Our data reveal that activation of AT1 receptor by each ligand led to a diversity of activation profiles that is far broader than that expected from a simple dichotomy between "G protein-dependent" and "β-arrestin-dependent" signaling. We observed clusters of activation profiles common to AngII, SII, and TRV027, as well as downstream effector activation that are unique to AngII, SII, or TRV027. Analyses of β-arrestin conformational changes after AT1 receptor stimulation with SII or TRV027 suggests that the observed differences could account, at least partially, for the diversity of modulated targets observed. Our data reveal that, although the categorization "G protein-dependent" vs. "β-arrestin-dependent" signaling can be of pharmacological relevance, broader analyses of signaling pathways and downstream targets are necessary to generate an accurate activation profile for a given ligand. This may bring relevant information for drug development, as it may allow more refined comparison of drugs with similar mechanism of action and effects, but with

  2. Comparative analyses of downstream signal transduction targets modulated after activation of the AT1 receptor by two β-arrestin biased agonists

    Directory of Open Access Journals (Sweden)

    Geisa A Santos

    2015-07-01

    Full Text Available G protein-coupled receptors (GPCRs are involved in essentially all physiological processes in mammals. The classical GPCR signal transduction mechanism occurs by coupling to G protein, but it has recently been demonstrated that interaction with β-arrestins leads to activation of pathways that are independent of the G protein pathway. Also, it has been reported that some ligands can preferentially activate one of these signaling pathways; being therefore called biased agonists for G protein or β-arrestin pathways. The angiotensin II (AngII AT1 receptor is a prototype GPCR in the study of biased agonism due to the existence of well-known β-arrestin biased agonists, such as [Sar1,Ile4,Ile8]-AngII (SII, and [Sar1,D-Ala8]-AngII (TRV027. The aim of this study was to comparatively analyze the two above mentioned β-arrestin biased agonists on downstream phosphorylation events and gene expression profiles. Our data reveal that activation of AT1 receptor by each ligand led to a diversity of activation profiles that is far broader than that expected from a simple dichotomy between G protein-dependent and β-arrestin-dependent signaling. We observed clusters of activation profiles common to AngII, SII and TRV027, as well as downstream effector activation that are unique to AngII, SII, or TRV027. Analyses of β-arrestin conformational changes after AT1 receptor stimulation with SII or TRV027 suggests that the observed differences could account, at least partially, for the diversity of modulated targets observed. Our data reveal that, although the categorization G protein-dependent vs. β-arrestin-dependent signaling can be of pharmacological relevance, broader analyses of signaling pathways and downstream targets are necessary to generate an accurate activation profile for a given ligand. This may bring relevant information for drug development, as it may allow more refined comparison of drugs with similar mechanism of action and effects, but with

  3. Targeting TRAP1 as a downstream effector of BRAF cytoprotective pathway: A novel strategy for human BRAF-driven colorectal carcinoma

    Science.gov (United States)

    Sisinni, Lorenza; Lettini, Giacomo; Matassa, Danilo Swann; Piscazzi, Annamaria; Palladino, Giuseppe; Amoroso, Maria Rosaria; Esposito, Franca; Landriscina, Matteo

    2015-01-01

    The HSP90 chaperone TRAP1 is translational regulator of BRAF synthesis/ubiquitination, since BRAF down-regulation, ERK signaling inhibition and delay of cell cycle progression occur upon TRAP1 silencing/inhibition. Since TRAP1 is upregulated in human colorectal carcinomas (CRCs) and involved in protection from apoptosis and as human BRAF-driven CRCs are poorly responsive to anticancer therapies, the relationship between TRAP1 regulation of mitochondrial apoptotic pathway and BRAF antiapoptotic signaling has been further evaluated. This study reports that BRAF cytoprotective signaling involves TRAP1-dependent inhibition of the mitochondrial apoptotic pathway. It is worth noting that BRAF and TRAP1 interact and that the activation of BRAF signaling results in enhanced TRAP1 serine-phosphorylation, a condition associated with resistance to apoptosis. Consistently, a BRAF dominant-negative mutant prevents TRAP1 serine phosphorylation and restores drug sensitivity in BRAFV600E CRC drug-resistant cells with high TRAP1 levels. In addition, TRAP1 targeting by the mitochondria-directed HSP90 chaperones inhibitor gamitrinib induces apoptosis and inhibits colony formation in BRAF-driven CRC cells. Thus, TRAP1 is a downstream effector of BRAF cytoprotective pathway in mitochondria and TRAP1 targeting may represent a novel strategy to improve the activity of proapoptotic agents in BRAF-driven CRC cells. PMID:26084290

  4. Isosilybin A Induces Apoptosis in Human Prostate Cancer Cells via Targeting Akt, NF-κB and Androgen Receptor Signaling

    OpenAIRE

    DEEP, GAGAN; Gangar, Subhash Chander; Oberlies, Nicholas H.; Kroll, David J.; Agarwal, Rajesh

    2010-01-01

    Prostate cancer (PCA) is second most malignancy in American men. Advanced stage PCA cells possess unlimited replication potential as well as resistance to apoptosis. Therefore, targeting survival mechanisms and activating apoptotic machinery in PCA cells using non-toxic phytochemicals is suggested as an attractive strategy against this deadly malignancy. In the present study, we assessed the effect of one such botanical agent, namely isosilybin A, on apoptotic machinery and key members of cel...

  5. Akt is transferred to the nucleus of cells treated with apoptin, and it participates in apoptin-induced cell death

    DEFF Research Database (Denmark)

    Bay, GH; Kroczak, TJ; Ande, SR;

    2007-01-01

    . Downstream of PI3-K, Akt is activated and translocated to the nucleus together with apoptin. Direct interaction between apoptin and Akt is documented. Co-expression of nuclear Akt significantly potentiates cell death induced by apoptin. Thus, apoptin-facilitated nuclear Akt, in contrast to when in its...

  6. The Akt inhibitor MK-2206 enhances the cytotoxicity of paclitaxel (Taxol) and cisplatin in ovarian cancer cells.

    Science.gov (United States)

    Lin, Ying-Hsi; Chen, Bert Yu-Hung; Lai, Wei-Ting; Wu, Shao-Fu; Guh, Jih-Hwa; Cheng, Ann-Lii; Hsu, Lih-Ching

    2015-01-01

    Abnormalities in the phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway are commonly observed in human cancers and contribute to chemotherapy resistance. Combination therapy, involving the use of molecular targeted agents and traditional cytotoxic drugs, may represent a promising strategy to lower resistance and enhance cytotoxicity. Here, we demonstrate the efficacy of an Akt inhibitor, MK-2206, in increasing the cytotoxic effect of either paclitaxel (Taxol) or cisplatin against the ovarian cancer cell lines SKOV3 (with constitutively active Akt) and ES2 (with inactive Akt). Sequential treatment of Taxol or cisplatin, followed by MK-2206, induced a synergistic inhibition of cell proliferation and effectively promoted cell death, either by inhibiting the phosphorylation of Akt and its downstream effectors 4E-BP1 and p70S6K in SKOV3 cells or by restoring p53 levels, which were downregulated after Taxol or cisplatin treatment, in ES2 cells. Combination treatment also downregulated the pro-survival protein Bcl-2 in both SKOV3 and ES2 cells, which may have contributed to cell death. In addition, we discovered that Taxol/MK-2206 or cisplatin/MK-2206 combination treatment resulted in significant enhancement of intracellular reactive oxygen species (ROS) induced by MK-2206, in both SKOV3 and ES2 cells; however, MK-2206-induced growth inhibition was reversed by a ROS scavenger only in ES2 cells. MK-2206 also suppressed DNA repair, particularly in SKOV3 cells. Taken together, our results demonstrate that the Akt inhibitor MK-2206 enhances the efficacy of cytotoxic agents in both Akt-active and Akt-inactive ovarian cancer cells but through different mechanisms.

  7. An integrative genomic and proteomic analysis of PIK3CA, PTEN and AKT mutations in breast cancer

    Energy Technology Data Exchange (ETDEWEB)

    Stemke-Hale, Katherine; Gonzalez-Angulo, Ana Maria; Lluch, Ana; Neve, Richard M.; Kuo, Wen-Lin; Davies, Michael; Carey, Mark; Hu, Zhi; Guan, Yinghui; Sahin, Aysegul; Symmans, W. Fraser; Pusztai, Lajos; Nolden, Laura K.; Horlings, Hugo; Berns, Katrien; Hung, Mien-Chie; van de Vijver, Marc J.; Valero, Vicente; Gray, Joe W.; Bernards, Rene; Mills, Gordon B.; Hennessy, Bryan T.

    2008-05-06

    Phosphatidylinositol-3-kinase (PI3K)/AKT pathway aberrations are common in cancer. By applying mass spectroscopy-based sequencing and reverse phase protein arrays to 547 human breast cancers and 41 cell lines, we determined the subtype specificity and signaling effects of PIK3CA, AKT and PTEN mutations, and the effects of PIK3CA mutations on responsiveness to PI3K inhibition in-vitro and on outcome after adjuvant tamoxifen. PIK3CA mutations were more common in hormone receptor positive (33.8%) and HER2-positive (24.6%) than in basal-like tumors (8.3%). AKT1 (1.4%) and PTEN (2.3%) mutations were restricted to hormone receptor-positive cancers with PTEN protein levels also being significantly lower in hormone receptor-positive cancers. Unlike AKT1 mutations, PIK3CA (39%) and PTEN (20%) mutations were more common in cell lines than tumors, suggesting a selection for these but not AKT1 mutations during adaptation to culture. PIK3CA mutations did not have a significant impact on outcome in 166 hormone receptor-positive breast cancer patients after adjuvant tamoxifen. PIK3CA mutations, in comparison with PTEN loss and AKT1 mutations, were associated with significantly less and indeed inconsistent activation of AKT and of downstream PI3K/AKT signaling in tumors and cell lines, and PTEN loss and PIK3CA mutation were frequently concordant, suggesting different contributions to pathophysiology. PTEN loss but not PIK3CA mutations rendered cells sensitive to growth inhibition by the PI3K inhibitor LY294002. Thus, PI3K pathway aberrations likely play a distinct role in the pathogenesis of different breast cancer subtypes. The specific aberration may have implications for the selection of PI3K-targeted therapies in hormone receptor-positive breast cancer.

  8. 人胚胎干细胞Pten基因表达及PI3K/Akt/mTOR信号通路下游蛋白的磷酸化%Pten gene expression and phosphoproteomic analysis of the downstream of PI3K/Akt/mTOR signaling in human embryonic stem cells

    Institute of Scientific and Technical Information of China (English)

    周睿卿; 龚玉萍; 邢宏运; 杨曦

    2011-01-01

    BACKGROUND: Phosphoprteomic analyses of human embryonic stem cells (hESCs) in undifferentiated state and their differentiated derivatives have become a hotspot. The researchs suggest that phosphorylation events determine hESCs'fate. OBJECTIVE: To investigate the expression of Pten Mrna and the key proteins of PTEN/Akt/Mtor signaling in human embryonic stem cells in order to provide the basic research for stem cells expansion and differentiation. METHODS: The hESCs were cocultured with the murine fetal fibroblasts to maintain undifferentiation and digested by collagenase for detection. The growth morphology of hESCs mere observed. RT-PCR assay was used to determine Pten gene expression, and Western blot assay to detect the expression of p-PTEN, p-Mtor, p-P70S6K. P-4E-BP1 in hESCs. RESULTS AND CONCLUSION: The expression level of Pten was higher in hESCs and feeder cells than in K562. While the expression level of the key proteins in hESCs was lower than K562. Especially p-4E-BP1 in hESCs. Over-activation of the PI3K/Akt/Mtor signaling may accelerate the proliferation of hESCs and deduce the apoptosis, and provide more cells for differentiation and regeneration medicine.%背景:各种磷酸化蛋白质表达水平对人胚胎干细胞维持未分化状态或定向分化的影响逐渐成为人胚胎干细胞的研究热点,研究发现磷酸化蛋白质表达水平可能决定着人胚胎干细胞的命运.目的:对PI3K/Akt途径下游的关键蛋白磷酸化水平进行检测,寻找PI3K/Akt途径中能够维持人胚胎干细胞未分化状态的下游蛋白.方法:用胎鼠成纤维细胞作为饲养层,二维培养的方法培养人胚胎干细胞,胶原酶消化后待测;以饲养层细胞、K562细胞株为对照.观察人胚胎干细胞生长状态;RT-PCR检测Pten基因表达;Western Blot检测p-PTEN、p-mTOR、p-P70S6K、p-4E-BP1 4种蛋白的表达.结果与结论:人胚胎干细胞在未分化状态时Pten mRNA表达高于肿瘤细胞K562,而且Pten抑制

  9. Improved algorithms in the CE-QUAL-W2 water-quality model for blending dam releases to meet downstream water-temperature targets

    Science.gov (United States)

    Rounds, Stewart A.; Buccola, Norman L.

    2015-01-01

    Water-quality models allow water resource professionals to examine conditions under an almost unlimited variety of potential future scenarios. The two-dimensional (longitudinal, vertical) water-quality model CE-QUAL-W2, version 3.7, was enhanced and augmented with new features to help dam operators and managers explore and optimize potential solutions for temperature management downstream of thermally stratified reservoirs. Such temperature management often is accomplished by blending releases from multiple dam outlets that access water of different temperatures at different depths. The modified blending algorithm in version 3.7 of CE-QUAL-W2 allows the user to specify a time-series of target release temperatures, designate from 2 to 10 floating or fixed-elevation outlets for blending, impose minimum and maximum head and flow constraints for any blended outlet, and set priority designations for each outlet that allow the model to choose which outlets to use and how to balance releases among them. The modified model was tested with a variety of examples and against a previously calibrated model of Detroit Lake on the North Santiam River in northwestern Oregon, and the results compared well. These updates to the blending algorithms will allow more complicated dam-operation scenarios to be evaluated somewhat automatically with the model, with decreased need for multiple model runs or preprocessing of model inputs to fully characterize the operational constraints.

  10. PI-103 and Quercetin Attenuate PI3K-AKT Signaling Pathway in T- Cell Lymphoma Exposed to Hydrogen Peroxide

    Science.gov (United States)

    Maurya, Akhilendra Kumar; Vinayak, Manjula

    2016-01-01

    Phosphatidylinositol 3 kinase—protein kinase B (PI3K-AKT) pathway has been considered as major drug target site due to its frequent activation in cancer. AKT regulates the activity of various targets to promote tumorigenesis and metastasis. Accumulation of reactive oxygen species (ROS) has been linked to oxidative stress and regulation of signaling pathways for metabolic adaptation of tumor microenvironment. Hydrogen peroxide (H2O2) in this context is used as ROS source for oxidative stress preconditioning. Antioxidants are commonly considered to be beneficial to reduce detrimental effects of ROS and are recommended as dietary supplements. Quercetin, a ubiquitous bioactive flavonoid is a dietary component which has attracted much of interest due to its potential health-promoting effects. Present study is aimed to analyze PI3K-AKT signaling pathway in H2O2 exposed Dalton’s lymphoma ascite (DLA) cells. Further, regulation of PI3K-AKT pathway by quercetin as well as PI-103, an inhibitor of PI3K was analyzed. Exposure of H2O2 (1mM H2O2 for 30min) to DLA cells caused ROS accumulation and resulted in increased phosphorylation of PI3K and downstream proteins PDK1 and AKT (Ser-473 and Thr-308), cell survival factors BAD and ERK1/2, as well as TNFR1. However, level of tumor suppressor PTEN was declined. Both PI-103 & quercetin suppressed the enhanced level of ROS and significantly down-regulated phosphorylation of AKT, PDK1, BAD and level of TNFR1 as well as increased the level of PTEN in H2O2 induced lymphoma cells. The overall result suggests that quercetin and PI3K inhibitor PI-103 attenuate PI3K-AKT pathway in a similar mechanism. PMID:27494022

  11. Gq-mediated Akt translocation to the membrane: a novel PIP3-independent mechanism in platelets.

    Science.gov (United States)

    Badolia, Rachit; Manne, Bhanu Kanth; Dangelmaier, Carol; Chernoff, Jonathan; Kunapuli, Satya P

    2015-01-01

    Akt is an important signaling molecule regulating platelet aggregation. Akt is phosphorylated after translocation to the membrane through Gi signaling pathways by a phosphatidylinositol-3,4,5-trisphosphate (PIP3)-dependent mechanism. However, Akt is more robustly phosphorylated by thrombin compared with adenosine 5'-diphosphate in platelets. This study investigated the mechanisms of Akt translocation as a possible explanation for this difference. Stimulation of washed human platelets with protease-activated receptor agonists caused translocation of Akt to the membrane rapidly, whereas phosphorylation occurred later. The translocation of Akt was abolished in the presence of a Gq-selective inhibitor or in Gq-deficient murine platelets, indicating that Akt translocation is regulated downstream of Gq pathways. Interestingly, phosphatidylinositol 3-kinase (PI3K) inhibitors or P2Y12 antagonist abolished Akt phosphorylation without affecting Akt translocation to the membrane, suggesting that Akt translocation occurs through a PI3K/PIP3/Gi-independent mechanism. An Akt scaffolding protein, p21-activated kinase (PAK), translocates to the membrane after stimulation with protease-activated receptor agonists in a Gq-dependent manner, with the kinetics of translocation similar to that of Akt. Coimmunoprecipitation studies showed constitutive association of PAK and Akt, suggesting a possible role of PAK in Akt translocation. These results show, for the first time, an important role of the Gq pathway in mediating Akt translocation to the membrane in a novel Gi/PI3K/PIP3-independent mechanism.

  12. 14-Thienyl Methylene Matrine (YYJ18, the Derivative from Matrine, Induces Apoptosis of Human Nasopharyngeal Carcinoma Cells by Targeting MAPK and PI3K/Akt Pathways in Vitro

    Directory of Open Access Journals (Sweden)

    Mao Xie

    2014-05-01

    Full Text Available Background/Aims: Nasopharyngeal carcinoma (NPC is a distinctive type of head and neck cancer with the highest incidence in South China. Previous studies have proved that matrine, a main alkaloid isolated from Sophora flavescens Ait, has antitumor activity against NPC. However, the effect is not so pronounced and the underlying mechanism remains largely unclear. Here we investigated whether 14-thienyl methylene matrine (YYJ18 that was derived from matrine could exert more effective suppression activity on NPC, along with the underlying mechanism. Methods: NPC cell lines CNE1, CNE2 and HONE1 were treated with YYJ18. Cell proliferation and apoptosis were determined by MTT assay and flow cytometry. Activation of mitogen-activated protein kinases (MAPK and phosphatidylinositol 3-kinase/protein kinase B (PI3K/Akt pathways were determined by Western blotting and quantitative RT-PCR. Results: YYJ18 remarkably inhibited proliferation and induced apoptosis of all three NPC cell lines in a dose-dependent manner, especially in CNE2 cells. Furthermore, YYJ18 treatment significantly suppressed phosphorylation of p38 in CNE2 cells, but upregulated phosphorylation of extracellular signal-regulated kinase1/2 (ERK1/2 and Akt. Next, alterations in downstream signaling were found, including activation of BCL2-associated X protein (Bax, caspase-3 and inactivation of B-cell CLL/lymphoma 2 (Bcl-2. Conclusion: We demonstrate the potent inhibitory effects of 14-thienyl methylene matrine on NPC cells for the first time, which could be mediated by modulation of MAPK and PI3K/Akt pathways.

  13. TRAP1 is involved in BRAF regulation and downstream attenuation of ERK phosphorylation and cell-cycle progression: a novel target for BRAF-mutated colorectal tumors.

    Science.gov (United States)

    Condelli, Valentina; Piscazzi, Annamaria; Sisinni, Lorenza; Matassa, Danilo Swann; Maddalena, Francesca; Lettini, Giacomo; Simeon, Vittorio; Palladino, Giuseppe; Amoroso, Maria Rosaria; Trino, Stefania; Esposito, Franca; Landriscina, Matteo

    2014-11-15

    Human BRAF-driven tumors are aggressive malignancies with poor clinical outcome and lack of sensitivity to therapies. TRAP1 is a HSP90 molecular chaperone deregulated in human tumors and responsible for specific features of cancer cells, i.e., protection from apoptosis, drug resistance, metabolic regulation, and protein quality control/ubiquitination. The hypothesis that TRAP1 plays a regulatory function on the BRAF pathway, arising from the observation that BRAF levels are decreased upon TRAP1 interference, was tested in human breast and colorectal carcinoma in vitro and in vivo. This study shows that TRAP1 is involved in the regulation of BRAF synthesis/ubiquitination, without affecting its stability. Indeed, BRAF synthesis is facilitated in a TRAP1-rich background, whereas increased ubiquitination occurs upon disruption of the TRAP1 network that correlates with decreased protein levels. Remarkably, BRAF downstream pathway is modulated by TRAP1 regulatory activity: indeed, TRAP1 silencing induces (i) ERK phosphorylation attenuation, (ii) cell-cycle inhibition with cell accumulation in G0-G1 and G2-M transitions, and (iii) extensive reprogramming of gene expression. Interestingly, a genome-wide profiling of TRAP1-knockdown cells identified cell growth and cell-cycle regulation as the most significant biofunctions controlled by the TRAP1 network. It is worth noting that TRAP1 regulation on BRAF is conserved in human colorectal carcinomas, with the two proteins being frequently coexpressed. Finally, the dual HSP90/TRAP1 inhibitor HSP990 showed activity against the TRAP1 network and high cytostatic potential in BRAF-mutated colorectal carcinoma cells. Therefore, this novel TRAP1 function represents an attractive therapeutic window to target dependency of BRAF-driven tumors on TRAP1 translational/quality control machinery.

  14. CCAAT-enhancer-binding Protein β (C/EBPβ) and Downstream Human Placental Growth Hormone Genes Are Targets for Dysregulation in Pregnancies Complicated by Maternal Obesity*

    Science.gov (United States)

    Vakili, Hana; Jin, Yan; Menticoglou, Savas; Cattini, Peter A.

    2013-01-01

    Human chorionic somatomammotropin (CS) and placental growth hormone variant (GH-V) act as metabolic adaptors in response to maternal insulin resistance, which occurs in “normal” pregnancy. Maternal obesity can exacerbate this “resistance,” suggesting that CS, GH-V, or transcription factors that regulate their production might be targets. The human CS genes, hCS-A and hCS-B, flank the GH-V gene. A significant decrease in pre-term placental CS/GH-V RNA levels was observed in transgenic mice containing the CS/GH-V genes in a model of high fat diet (HFD)-induced maternal obesity. Similarly, a decrease in CS/GH-V RNA levels was detected in term placentas from obese (body mass index (BMI) ≥ 35 kg/m2) versus lean (BMI 20–25 kg/m2) women. A specific decrease in transcription factor CCAAT-enhancer-binding protein β (C/EBPβ) RNA levels was also seen with obesity; C/EBPβ is required for mouse placenta development and is expressed, like CS and GH-V, in syncytiotrophoblasts. Binding of C/EBPβ to the CS gene downstream enhancer regions, which by virtue of their position distally flank the GH-V gene, was reduced in placenta chromatin from mice on a HFD and in obese women; a corresponding decrease in RNA polymerase II associated with CS/GH-V promoters was also observed. Detection of decreased endogenous CS/GH-V RNA levels in human placental tumor cells treated with C/EBPβ siRNA is consistent with a direct effect. These data provide evidence for CS/GH-V dysregulation in acute HFD-induced obesity in mouse pregnancy and chronic obesity in human pregnancy and implicate C/EBPβ, a factor associated with CS regulation and placental development. PMID:23782703

  15. CCAAT-enhancer-binding protein β (C/EBPβ) and downstream human placental growth hormone genes are targets for dysregulation in pregnancies complicated by maternal obesity.

    Science.gov (United States)

    Vakili, Hana; Jin, Yan; Menticoglou, Savas; Cattini, Peter A

    2013-08-01

    Human chorionic somatomammotropin (CS) and placental growth hormone variant (GH-V) act as metabolic adaptors in response to maternal insulin resistance, which occurs in "normal" pregnancy. Maternal obesity can exacerbate this "resistance," suggesting that CS, GH-V, or transcription factors that regulate their production might be targets. The human CS genes, hCS-A and hCS-B, flank the GH-V gene. A significant decrease in pre-term placental CS/GH-V RNA levels was observed in transgenic mice containing the CS/GH-V genes in a model of high fat diet (HFD)-induced maternal obesity. Similarly, a decrease in CS/GH-V RNA levels was detected in term placentas from obese (body mass index (BMI) ≥ 35 kg/m(2)) versus lean (BMI 20-25 kg/m(2)) women. A specific decrease in transcription factor CCAAT-enhancer-binding protein β (C/EBPβ) RNA levels was also seen with obesity; C/EBPβ is required for mouse placenta development and is expressed, like CS and GH-V, in syncytiotrophoblasts. Binding of C/EBPβ to the CS gene downstream enhancer regions, which by virtue of their position distally flank the GH-V gene, was reduced in placenta chromatin from mice on a HFD and in obese women; a corresponding decrease in RNA polymerase II associated with CS/GH-V promoters was also observed. Detection of decreased endogenous CS/GH-V RNA levels in human placental tumor cells treated with C/EBPβ siRNA is consistent with a direct effect. These data provide evidence for CS/GH-V dysregulation in acute HFD-induced obesity in mouse pregnancy and chronic obesity in human pregnancy and implicate C/EBPβ, a factor associated with CS regulation and placental development.

  16. Omentin-1 Stimulates Human Osteoblast Proliferation through PI3K/Akt Signal Pathway

    Directory of Open Access Journals (Sweden)

    Shan-Shan Wu

    2013-01-01

    Full Text Available It has been presumed that adipokines deriving from adipose tissue may play important roles in bone metabolism. Omentin-1, a novel adipokine, which is selectively expressed in visceral adipose tissue, has been reported to stimulate proliferation and inhibit differentiation of mouse osteoblast. However, little information refers to the effect of omentin-1 on human osteoblast (hOB proliferation. The current study examined the potential effects of omentin-1 on proliferation in hOB and the signal pathway involved. Omentin-1 promoted hOB proliferation in a dose-dependent manner as determined by [3H]thymidine incorporation. Western blot analysis revealed that omentin-1 induced activation of Akt (phosphatidylinositol-3 kinase downstream effector and such effect was impeded by transfection of hOB with Akt-siRNA. Furthermore, LY294002 (a selective PI3K inhibitor and HIMO (a selective Akt inhibitor abolished the omentin-1-induced hOB proliferation. These findings indicate that omentin-1 induces hOB proliferation via the PI3K/Akt signaling pathway and suggest that osteoblast is a direct target of omentin-1.

  17. PPAR-γ ligands repress TGFβ-induced myofibroblast differentiation by targeting the PI3K/Akt pathway: implications for therapy of fibrosis.

    Directory of Open Access Journals (Sweden)

    Ajit A Kulkarni

    Full Text Available Transforming growth factor beta (TGFβ induced differentiation of human lung fibroblasts to myofibroblasts is a key event in the pathogenesis of pulmonary fibrosis. Although the typical TGFβ signaling pathway involves the Smad family of transcription factors, we have previously reported that peroxisome proliferator-activated receptor-γ (PPAR-γ ligands inhibit TGFβ-mediated differentiation of human lung fibroblasts to myofibroblasts via a Smad-independent pathway. TGFβ also activates the phosphatidylinositol 3 kinase/protein kinase B (PI3K/Akt pathway leading to phosphorylation of Akt(S473. Here, we report that PPAR-γ ligands, 2-cyano-3,12-dioxooleana-1,9-dien-28-oic acid (CDDO and 15-deoxy-(12,14-15d-prostaglandin J(2 (15d-PGJ(2, inhibit human myofibroblast differentiation of normal and idiopathic pulmonary fibrotic (IPF fibroblasts, by blocking Akt phosphorylation at Ser473 by a PPAR-γ-independent mechanism. The PI3K inhibitor LY294002 and a dominant-negative inactive kinase-domain mutant of Akt both inhibited TGFβ-stimulated myofibroblast differentiation, as determined by Western blotting for α-smooth muscle actin and calponin. Prostaglandin A(1 (PGA(1, a structural analogue of 15d-PGJ(2 with an electrophilic center, also reduced TGFβ-driven phosphorylation of Akt, while CAY10410, another analogue that lacks an electrophilic center, did not; implying that the activity of 15d-PGJ(2 and CDDO is dependent on their electrophilic properties. PPAR-γ ligands inhibited TGFβ-induced Akt phosphorylation via both post-translational and post-transcriptional mechanisms. This inhibition is independent of MAPK-p38 and PTEN but is dependent on TGFβ-induced phosphorylation of FAK, a kinase that acts upstream of Akt. Thus, PPAR-γ ligands inhibit TGFβ signaling by affecting two pro-survival pathways that culminate in myofibroblast differentiation. Further studies of PPAR-γ ligands and small electrophilic molecules may lead to a new generation of

  18. Roles of the PI3K/Akt pathway in Epstein-Barr virus-induced cancers and therapeutic implications.

    Science.gov (United States)

    Chen, Jiezhong

    2012-12-12

    Viruses have been shown to be responsible for 10%-15% of cancer cases. Epstein-Barr virus (EBV) is the first virus to be associated with human malignancies. EBV can cause many cancers, including Burkett's lymphoma, Hodgkin's lymphoma, post-transplant lymphoproliferative disorders, nasopharyngeal carcinoma and gastric cancer. Evidence shows that phosphoinositide 3-kinase/protein kinase B (PI3K/Akt) plays a key role in EBV-induced malignancies. The main EBV oncoproteins latent membrane proteins (LMP) 1 and LMP2A can activate the PI3K/Akt pathway, which, in turn, affects cell survival, apoptosis, proliferation and genomic instability via its downstream target proteins to cause cancer. It has also been demonstrated that the activation of the PI3K/Akt pathway can result in drug resistance to chemotherapy. Thus, the inhibition of this pathway can increase the therapeutic efficacy of EBV-associated cancers. For example, PI3K inhibitor Ly294002 has been shown to increase the effect of 5-fluorouracil in an EBV-associated gastric cancer cell line. At present, dual inhibitors of PI3K and its downstream target mammalian target of rapamycin have been used in clinical trials and may be included in treatment regimens for EBV-associated cancers. PMID:24175221

  19. Eukaryotic initiation factor 2α--a downstream effector of mammalian target of rapamycin--modulates DNA repair and cancer response to treatment.

    Directory of Open Access Journals (Sweden)

    Liron Tuval-Kochen

    Full Text Available In an effort to circumvent resistance to rapamycin--an mTOR inhibitor--we searched for novel rapamycin-downstream-targets that may be key players in the response of cancer cells to therapy. We found that rapamycin, at nM concentrations, increased phosphorylation of eukaryotic initiation factor (eIF 2α in rapamycin-sensitive and estrogen-dependent MCF-7 cells, but had only a minimal effect on eIF2α phosphorylation in the rapamycin-insensitive triple-negative MDA-MB-231 cells. Addition of salubrinal--an inhibitor of eIF2α dephosphorylation--decreased expression of a surface marker associated with capacity for self renewal, increased senescence and induced clonogenic cell death, suggesting that excessive phosphorylation of eIF2α is detrimental to the cells' survival. Treating cells with salubrinal enhanced radiation-induced increase in eIF2α phosphorylation and clonogenic death and showed that irradiated cells are more sensitive to increased eIF2α phosphorylation than non-irradiated ones. Similar to salubrinal--the phosphomimetic eIF2α variant--S51D--increased sensitivity to radiation, and both abrogated radiation-induced increase in breast cancer type 1 susceptibility gene, thus implicating enhanced phosphorylation of eIF2α in modulation of DNA repair. Indeed, salubrinal inhibited non-homologous end joining as well as homologous recombination repair of double strand breaks that were induced by I-SceI in green fluorescent protein reporter plasmids. In addition to its effect on radiation, salubrinal enhanced eIF2α phosphorylation and clonogenic death in response to the histone deacetylase inhibitor--vorinostat. Finally, the catalytic competitive inhibitor of mTOR--Ku-0063794--increased phosphorylation of eIF2α demonstrating further the involvement of mTOR activity in modulating eIF2α phosphorylation. These experiments suggest that excessive phosphorylation of eIF2α decreases survival of cancer cells; making eIF2α a worthy target for

  20. Upregulated WDR26 serves as a scaffold to coordinate PI3K/ AKT pathway-driven breast cancer cell growth, migration, and invasion.

    Science.gov (United States)

    Ye, Yuanchao; Tang, Xiaoyun; Sun, Zhizeng; Chen, Songhai

    2016-04-01

    The phosphatidylinositol 3-kinase (PI3K)/AKT pathway transmits signals downstream of receptor tyrosine kinases and G protein-coupled receptors (GPCRs), and is one of the most dysregulated pathways in breast cancer. PI3Ks and AKTs consist of multiple isoforms that play distinct and even opposite roles in breast cancer cell growth and metastasis. However, it remains unknown how the activities of various PI3K and AKT isoforms are coordinated during breast cancer progression. Previously, we showed WDR26 is a novel WD40 protein that binds Gβγ and promotes Gβγ signaling. Here, we demonstrate that WDR26 is overexpressed in highly malignant breast tumor cell lines and human breast cancer samples, and that WDR26 overexpression correlates with shortened survival of breast cancer patients. In highly malignant cell lines (MDA-MB231, DU4475 and BT549), downregulation of WDR26 expression selectively alleviated GPCR- but not EGF receptor-stimulated PI3K/AKT signaling and tumor cell growth, migration and invasion. In contrast, in a less malignant cell line (MCF7), WDR26 overexpression had the opposite effect. Additional studies indicate that downstream of GPCR stimulation, WDR26 serves as a scaffold that fosters assembly of a specific signaling complex consisting of Gβγ, PI3Kβ and AKT2. In an orthotopic xenograft mouse model of breast cancer, disrupting formation of this complex, by overexpressing WDR26 mutants in MDA-MB231 cells, abrogated PI3K/AKT activation and tumor cell growth and metastasis. Together, our results identify a novel mechanism regulating GPCR-dependent activation of the PI3K/AKT signaling axis in breast tumor cells, and pinpoint WDR26 as a potential therapeutic target for breast cancer.

  1. Inhibition of breast cancer invasion by TIS21/BTG2/Pc3-Akt1-Sp1-Nox4 pathway targeting actin nucleators, mDia genes.

    Science.gov (United States)

    Choi, J-A; Jung, Y S; Kim, J Y; Kim, H M; Lim, I K

    2016-01-01

    The mammalian homolog of Drosophila diaphanous (mDia), actin nucleator, has been known to participate in the process of invasion and metastasis of cancer cells via regulating a number of actin-related biological processes. We have previously reported that tumor suppressor TIS21(/BTG2/Pc3) (TIS21) inhibits invadopodia formation by downregulating reactive oxygen species (ROS) in MDA-MB-231 cells. We herein report that TIS21(/BTG2/Pc3) downregulates diaphanous-related formin (DRF) expression via reducing NADPH oxidase 4 (Nox4)-derived ROS generation by Akt1 activation and subsequently impairs invasion activity of the highly invasive breast cancer cells. Knockdown of Akt1 by RNA interference recovered the TIS21(/BTG2/Pc3)-inhibited F-actin remodeling and ROS generation by recovering Nox4 expression. Furthermore, Sp1-mediated Nox4 transcription was downregulated by TIS21(/BTG2/Pc3)-Akt1 signals, leading to the inhibition of cancer cell invasion via F-actin remodeling by mDia genes. To our best knowledge, this is the first study to show that TIS21(/BTG2/Pc3)-Akt1 inhibited Sp1-Nox4-ROS cascade, subsequently reducing invasion activity via inhibition of mDia family genes.

  2. Akt is negatively regulated by the MULAN E3 ligase

    Institute of Scientific and Technical Information of China (English)

    Seunghee Bae; Jongdoo Kim; Hong-Duck Um; In-Chul Park; Su-Jae Lee; Seon Young Nam; Young-Woo Jin; Jae Ho Lee; Sungkwan An; Sun-Yong Kim; Jin Hyuk Jung; Yeongmin Yoon; Hwa Jun Cha; Hyunjin Lee; Karam Kim; Jongran Kim; In-Sook An

    2012-01-01

    The serine/threonine kinase Akt functions in multiple cellular processes,including cell survival and tumor development.Studies of the mechanisms that negatively regulate Akt have focused on dephosphorylation-mediated inactivation.In this study,we identified a negative regulator of Akt,MULAN,which possesses both a RING finger domain and E3 ubiquitin ligase activity.Akt was found to directly interact with MULAN and to be ubiquitinated by MULAN in vitro and in vivo.Other molecular assays demonstrated that phosphorylated Akt is a substantive target for both interaction with MULAN and ubiquitination by MULAN.The results of the functional studies suggest that the degradation of Akt by MULAN suppresses cell proliferation and viability.These data provide insight into the Akt ubiquitination signaling network.

  3. Targeting downstream transcription factors and epigenetic modifications following Toll-like receptor 7/8 ligation to forestall tissue injury in anti-Ro60 associated heart block.

    Science.gov (United States)

    Clancy, Robert M; Markham, Androo J; Reed, Joanne H; Blumenberg, Miroslav; Halushka, Marc K; Buyon, Jill P

    2016-02-01

    Based on the consistent demonstration of fibrosis of the atrioventricular node surrounded by macrophages and multinucleated giant cells in anti-Ro antibody exposed fetuses dying with heart block, this study focuses on macrophage signaling stimulated by ssRNA associated with the Ro60 protein and the impact of antagonizing innate cell drivers such as TLR7/8. Transcriptome and epigenetic modifications which affect transcription factors, NF-κB and STAT1, were selected to evaluate the phenotype of macrophages in which TLR7/8 was ligated following treatment with either anti-Ro60/Ro60/hY3 RNA immune complexes or transfection with hY3. Based on microarray, TNF and IL6 were among the most highly upregulated genes in both stimulated conditions, each of which was significantly inhibited by preincubation with hydroxychloroquine (HCQ). In contrast, following stimulation of macrophages with either TNF-α or IFN-α, which do not signal through TLR, the resultant gene expression was refractory to HCQ. Ligation of TLR7/8 resulted in increased histone methylation as measured by increased H3K4me2, a requirement for binding of NF-κB at certain promoters, specifically the kB1 region in the TNF promoter (ChIP-qPCR), which was significantly decreased by HCQ. In summary, these results support that the HCQ-sensitive phenotype of hY3 stimulated macrophages reflects the bifurcation of TLR downstream signals involving NF-κB and STAT 1 pathways and for the former dimethylation of H3K4. Accordingly, HCQ may act more as a preventive measure in downregulating the initial production of IFN-α or TNF-α and not affect the resultant autocoid stimulation reflected in TNF-α and IFN-α responsive genes. The beneficial scope of antimalarials in the prevention of organ damage, inclusive of heart block in an anti-Ro offspring or more broadly SLE, may include in part, a mechanism targeting TLR-dependent epigenetic modification.

  4. Analysis of the expression of miRNAs and downstream target genes in gastric cancer tissue and exploration of its relationship with clinicopathologic stage

    Institute of Scientific and Technical Information of China (English)

    Zhen Xiong

    2016-01-01

    Objective:To study and analyze the expression of miRNAs and downstream target genes in gastric cancer tissue and its relationship with clinicopathologic stage.Methods:Patients diagnosed with gastric cancer in our hospital from April 2012 to Decempber 2014 were selected for study, and gastric cancer tissue and paracancer tissue were collected to detect the expression of miRNAs as well as the contents of proteins encoded by drug resistance-related genes, proliferation-related genes and EMT-related genes.Results: miR-21, miR-106a, miR-192, miR-194, miR-210 and miR-215 expression in gastric cancer tissue was significantly up-regulated, miR-30a, miR-125, miR-149, miR-194, miR-205 and miR-365 expression was significantly down-regulated, and the higher the TNM stage of tumor, the more significant the change of the expression of above miRNAs; the trend of miR106 and miR-30a were the most significant, the former was up-regulated by 4.38 times and the latter was down-regulated by 0.23 times; P-gP, GST-π, CACNA2D1, RPL23, Hsp27, ZNF139, Mcmp4, OPCML, N-cadherin and Vimentin contents in gastric cancer tissue were significantly higher than those in paracancer tissue, and E-cadherin content was significantly lower than that in paracancer tissue; miR106 expression level was positively correlated with P-gP, GST-π, CACNA2D1, RPL23, Hsp27, ZNF139, Mcmp4, OPCML, N-cadherin and Vimentin contents and negatively correlated with E-cadherin content; miR-30a expression level was negatively correlated with P-gP, GST-π, CACNA2D1, RPL23, Hsp27, ZNF139, Mcmp4, OPCML, N-cadherin and Vimentin contents and positively correlated with E-cadherin content.Conclusion: miR106 expression significantly increases and miR-30a expression significantly decreases in gastric cancer tissue, and miR106 and miR-30a can regulate the expression of drug resistance genes, proliferation genes and EMT genes.

  5. Curcumin inhibits Akt/mTOR signaling through protein phosphatase-dependent mechanism*

    OpenAIRE

    Yu, Siwang; Shen, Guoxiang; Khor, Tin Oo; Kim, Jung-Hwan; Kong, Ah-Ng

    2008-01-01

    Akt/mTOR signaling plays an important role in tumorigenesis and is dysregulated in many tumors, especially metastatic prostate cancers. Curcumin has been shown to effectively prevent or inhibit prostate cancer in vivo and inhibit Akt/mTOR signaling in vitro, but the mechanism(s) remains unclear. Here we show that curcumin concentration- and time-dependently inhibited the phosphorylation of Akt, mTOR, and their downstream substrates in human prostate cancer PC-3 cells, and this inhibitory effe...

  6. Integrative Analysis of the Acquisition of Pluripotency in PGCs Reveals the Mutually Exclusive Roles of Blimp-1 and AKT Signaling

    Directory of Open Access Journals (Sweden)

    Go Nagamatsu

    2015-07-01

    Full Text Available Primordial germ cells (PGCs are lineage-restricted unipotent cells that can dedifferentiate into pluripotent embryonic germ cells (EGCs. Here we performed whole-transcriptome analysis during the conversion of PGCs into EGCs, a process by which cells acquire pluripotency. To examine the molecular mechanism underlying this conversion, we focused on Blimp-1 and Akt, which are involved in PGC specification and dedifferentiation, respectively. Blimp-1 overexpression in embryonic stem cells suppressed the expression of downstream targets of the pluripotency network. Conversely, Blimp-1 deletion in PGCs accelerated their dedifferentiation into pluripotent EGCs, illustrating that Blimp-1 is a pluripotency gatekeeper protein in PGCs. AKT signaling showed a synergistic effect with basic fibroblast growth factor plus 2i+A83 treatment on EGC formation. AKT played a major role in suppressing genes regulated by MBD3. From these results, we defined the distinct functions of Blimp-1 and Akt and provided mechanistic insights into the acquisition of pluripotency in PGCs.

  7. Neurofilament heavy polypeptide regulates the Akt-beta-catenin pathway in human esophageal squamous cell carcinoma.

    Directory of Open Access Journals (Sweden)

    Myoung Sook Kim

    Full Text Available Aerobic glycolysis and mitochondrial dysfunction are common features of aggressive cancer growth. We observed promoter methylation and loss of expression in neurofilament heavy polypeptide (NEFH in a significant proportion of primary esophageal squamous cell carcinoma (ESCC samples that were of a high tumor grade and advanced stage. RNA interference-mediated knockdown of NEFH accelerated ESCC cell growth in culture and increased tumorigenicity in vivo, whereas forced expression of NEFH significantly inhibited cell growth and colony formation. Loss of NEFH caused up-regulation of pyruvate kinase-M2 type and down-regulation of pyruvate dehydrogenase, via activation of the Akt/beta-catenin pathway, resulting in enhanced aerobic glycolysis and mitochondrial dysfunction. The acceleration of glycolysis and mitochondrial dysfunction in NEFH-knockdown cells was suppressed in the absence of beta-catenin expression, and was decreased by the treatment of 2-Deoxyglucose, a glycolytic inhibitor, or API-2, an Akt inhibitor. Loss of NEFH activates the Akt/beta-catenin pathway and increases glycolysis and mitochondrial dysfunction. Cancer cells with methylated NEFH can be targeted for destruction with specific inhibitors of deregulated downstream pathways.

  8. Piperlongumine promotes autophagy via inhibition of Akt/mTOR signalling and mediates cancer cell death

    OpenAIRE

    Makhov, P; Golovine, K.; Teper, E.; Kutikov, A.; Mehrazin, R.; Corcoran, A; A. Tulin; Uzzo, R G; Kolenko, V M

    2014-01-01

    Background: The Akt/mammalian target of rapamycin (mTOR) signalling pathway serves as a critical regulator of cellular growth, proliferation and survival. Akt aberrant activation has been implicated in carcinogenesis and anticancer therapy resistance. Piperlongumine (PL), a natural alkaloid present in the fruit of the Long pepper, is known to exhibit notable anticancer effects. Here we investigate the impact of PL on Akt/mTOR signalling. Methods: We examined Akt/mTOR signalling in cancer cell...

  9. PIK3R1 targeting by miR-21 suppresses tumor cell migration and invasion by reducing PI3K/AKT signaling and reversing EMT, and predicts clinical outcome of breast cancer.

    Science.gov (United States)

    Yan, Li-Xu; Liu, Yan-Hui; Xiang, Jian-Wen; Wu, Qi-Nian; Xu, Lei-Bo; Luo, Xin-Lan; Zhu, Xiao-Lan; Liu, Chao; Xu, Fang-Ping; Luo, Dong-Lan; Mei, Ping; Xu, Jie; Zhang, Ke-Ping; Chen, Jie

    2016-02-01

    We have previously shown that dysregulation of miR-21 functioned as an oncomiR in breast cancer. The aim of the present study was to elucidate the mechanisms by which miR-21 regulate breast tumor migration and invasion. We applied pathway analysis on genome microarray data and target-predicting algorithms for miR-21 target screening, and used luciferase reporting assay to confirm the direct target. Thereafter, we investigated the function of the target gene phosphoinositide-3-kinase, regulatory subunit 1 (α) (PIK3R1), and detected PIK3R1 coding protein (p85α) by immunohistochemistry and miR-21 by RT-qPCR on 320 archival paraffin-embedded tissues of breast cancer to evaluate the correlation of their expression with prognosis. First, we found that PIK3R1 suppressed growth, invasiveness, and metastatic properties of breast cancer cells. Next, we identified the PIK3R1 as a direct target of miR-21 and showed that it was negatively regulated by miR-21. Furthermore, we demonstrated that p85α overexpression phenocopied the suppression effects of antimiR-21 on breast cancer cell growth, migration and invasion, indicating its tumor suppressor role in breast cancer. On the contrary, PIK3R1 knockdown abrogated antimiR‑21-induced effect on breast cancer cells. Notably, antimiR-21 induction increased p85α, accompanied by decreased p-AKT level. Besides, antimiR-21/PIK3R1-induced suppression of invasiveness in breast cancer cells was mediated by reversing epithelial-mesenchymal transition (EMT). p85α downregulation was found in 25 (7.8%) of the 320 breast cancer patients, and was associated with inferior 5-year disease-free survival (DFS) and overall survival (OS). Taken together, we provide novel evidence that miR-21 knockdown suppresses cell growth, migration and invasion partly by inhibiting PI3K/AKT activation via direct targeting PIK3R1 and reversing EMT in breast cancer. p85α downregulation defined a specific subgroup of breast cancer with shorter 5-year DFS and OS

  10. Mitochondrial Akt Regulation of Hypoxic Tumor Reprogramming.

    Science.gov (United States)

    Chae, Young Chan; Vaira, Valentina; Caino, M Cecilia; Tang, Hsin-Yao; Seo, Jae Ho; Kossenkov, Andrew V; Ottobrini, Luisa; Martelli, Cristina; Lucignani, Giovanni; Bertolini, Irene; Locatelli, Marco; Bryant, Kelly G; Ghosh, Jagadish C; Lisanti, Sofia; Ku, Bonsu; Bosari, Silvano; Languino, Lucia R; Speicher, David W; Altieri, Dario C

    2016-08-01

    Hypoxia is a universal driver of aggressive tumor behavior, but the underlying mechanisms are not completely understood. Using a phosphoproteomics screen, we now show that active Akt accumulates in the mitochondria during hypoxia and phosphorylates pyruvate dehydrogenase kinase 1 (PDK1) on Thr346 to inactivate the pyruvate dehydrogenase complex. In turn, this pathway switches tumor metabolism toward glycolysis, antagonizes apoptosis and autophagy, dampens oxidative stress, and maintains tumor cell proliferation in the face of severe hypoxia. Mitochondrial Akt-PDK1 signaling correlates with unfavorable prognostic markers and shorter survival in glioma patients and may provide an "actionable" therapeutic target in cancer. PMID:27505672

  11. Combination of carmustine and selenite effectively inhibits tumor growth by targeting androgen receptor, androgen receptor-variants, and Akt in preclinical models: New hope for patients with castration resistant prostate cancer.

    Science.gov (United States)

    Thamilselvan, Vijayalakshmi; Menon, Mani; Thamilselvan, Sivagnanam

    2016-10-01

    Despite established androgen receptor (AR) antagonists, AR/AR-variants signaling remain a major obstacle for the successful treatment of castration resistant prostate cancer (CRPC). In addition, CRPC cells adapt to survive via AR-independent pathways to escape next generation therapies. Therefore, there is an urgent need for drugs that can target these signaling pathways in CRPC. In this study, we sought to determine whether carmustine and selenite in combination could induce apoptosis and inhibit growth of CRPC in-vitro and in-vivo. CRPC (22Rv1, VCaP, and PC-3) cell lines in culture and xenograft mouse were used. Combination of carmustine and selenite treatment significantly increased reactive oxygen species, apoptosis and growth inhibition in CRPC cells with down regulation of anti-apoptotic (Bcl-2 and Mcl-1) and proliferative proteins (c-Myc and cyclin-D1). This effect was associated with complete reduction of AR/AR-variants, AR-V7, PSA and significant induction of p27Kip1. Combination treatment substantially abolished phospho-Akt, phospho-GSK-3β, and anchorage-independent growth in AR-positive and AR-negative cells. Consistent with in-vitro results, combination treatment effectively induced apoptosis and completely inhibited xenograft tumor growth and markedly reduced AR/AR-variants, AR-V7, PSA, and Bcl-2 in xenograft tumors without causing genotoxicity in host mice. Individual agent treatment showed only partial effect. The combination treatment showed a significant synergistic effect. The present study is the first to demonstrate that the combination of carmustine and selenite treatment completely suppressed CRPC tumor growth by reducing AR/AR-variants and Akt signaling. Our findings suggest that the combination of carmustine and selenite could constitute a promising next-generation therapy for successful treatment of patients with CRPC. PMID:27198552

  12. A novel signaling pathway associated with Lyn, PI 3-kinase and Akt supports the proliferation of myeloma cells

    Energy Technology Data Exchange (ETDEWEB)

    Iqbal, Mohd S. [Department of Bio-Signal Analysis, Applied Medical Engineering Science, Graduate School of Medicine, Yamaguchi University, Ube, Yamaguchi 755-8505 (Japan); Enteric and Food Microbiology Laboratory, Laboratory Sciences Division, International Center for Diarrhoeal Disease Research, Bangladesh, P.O. Box 128, Dhaka 1000 (Bangladesh); Tsuyama, Naohiro [Department of Analytical Molecular Medicine and Devices, Division of Frontier Medical Science, Graduate School of Medical Sciences, Hiroshima University, Hiroshima, Hiroshima 734-8553 (Japan); Obata, Masanori [Department of Bio-Signal Analysis, Applied Medical Engineering Science, Graduate School of Medicine, Yamaguchi University, Ube, Yamaguchi 755-8505 (Japan); Ishikawa, Hideaki, E-mail: hishika@yamaguchi-u.ac.jp [Department of Bio-Signal Analysis, Applied Medical Engineering Science, Graduate School of Medicine, Yamaguchi University, Ube, Yamaguchi 755-8505 (Japan)

    2010-02-12

    Interleukin-6 (IL-6) is a growth factor for human myeloma cells. We have recently found that in myeloma cells the activation of both signal transducer and activator of transcription (STAT) 3 and extracellular signal-regulated kinase (ERK) 1/2 is not sufficient for the IL-6-induced proliferation, which further requires the activation of the src family kinases, such as Lyn. Here we showed that the Lyn-overexpressed myeloma cell lines had the higher proliferative rate with IL-6 and the enhanced activation of the phosphatidylinositol (PI) 3-kinase and Akt. The IL-6-induced phosphorylation of STAT3 and ERK1/2 was not up-regulated in the Lyn-overexpressed cells, indicating that the Lyn-PI 3-kinase-Akt pathway is independent of these pathways. The PI 3-kinase was co-precipitated with Lyn in the Lyn-overexpressed cells of which proliferation with IL-6 was abrogated by the specific inhibitors for PI 3-kinase or Akt, suggesting that the activation of the PI 3-kinase-Akt pathway associated with Lyn is indeed related to the concomitant augmentation of myeloma cell growth. Furthermore, the decreased expression of p53 and p21{sup Cip1} proteins was observed in the Lyn-overexpressed cells, implicating a possible downstream target of Akt. This study identifies a novel IL-6-mediated signaling pathway that certainly plays a role in the proliferation of myeloma cells and this novel mechanism of MM tumor cell growth associated with Lyn would eventually contribute to the development of MM treatment.

  13. Cadmium Activates Multiple Signaling Pathways That Coordinately Stimulate Akt Activity to Enhance c-Myc mRNA Stability.

    Directory of Open Access Journals (Sweden)

    Jia-Shiuan Tsai

    Full Text Available Cadmium is a known environmental carcinogen. Exposure of Cd leads to the activation of several proto-oncogenes in cells. We investigated here the mechanism of c-Myc expression in hepatic cells under Cd treatment. The c-Myc protein and mRNA levels increased in dose- and time-dependent manners in HepG2 cells with Cd treatment. This increase was due to an increase in c-Myc mRNA stability. To explore the mechanism involved in enhancing the mRNA stability, several cellular signaling factors that evoked by Cd treatment were analyzed. PI3K, p38, ERK and JNK were activated by Cd. However, ERK did not participate in the Cd-induced c-Myc expression. Further analysis revealed that mTORC2 was a downstream factor of p38. PI3K, JNK and mTORC2 coordinately activated Akt. Akt was phosphorylated at Thr450 in the untreated cells. Cd treatment led to additional phosphorylation at Thr308 and Ser473. Blocking any of the three signaling factors resulted in the reduction of phosphorylation level at all three Akt sites. The activated Akt phosphorylated Foxo1 and allowed the modified protein to translocate into the cytoplasm. We conclude that Cd-induced accumulation of c-Myc requires the activation of several signaling pathways. The signals act coordinately for Akt activation and drive the Foxo1 from the nucleus to the cytoplasm. Reduction of Foxo1 in the nucleus reduces the transcription of its target genes that may affect c-Myc mRNA stability, resulting in a higher accumulation of the c-Myc proteins.

  14. Antiproliferative factor decreases Akt phosphorylation and alters gene expression via CKAP4 in T24 bladder carcinoma cells

    Directory of Open Access Journals (Sweden)

    Zhang Chen-Ou

    2010-12-01

    Full Text Available Abstract Background Urinary bladder cancer is a common malignancy worldwide, and outcomes for patients with advanced bladder cancer remain poor. Antiproliferative factor (APF is a potent glycopeptide inhibitor of epithelial cell proliferation that was discovered in the urine of patients with interstitial cystitis, a disorder with bladder epithelial thinning and ulceration. APF mediates its antiproliferative activity in primary normal bladder epithelial cells via cytoskeletal associated protein 4 (CKAP4. Because synthetic asialo-APF (as-APF has also been shown to inhibit T24 bladder cancer cell proliferation at nanomolar concentrations in vitro, and because the peptide segment of APF is 100% homologous to part of frizzled 8, we determined whether CKAP4 mediates as-APF inhibition of proliferation and/or downstream Wnt/frizzled signaling events in T24 cells. Methods T24 cells were transfected with double-stranded siRNAs against CKAP4 and treated with synthetic as-APF or inactive control peptide; cells that did not undergo electroporation and cells transfected with non-target (scrambled double-stranded siRNA served as negative controls. Cell proliferation was determined by 3H-thymidine incorporation. Expression of Akt, glycogen synthase kinase 3β (GSK3β, β-catenin, p53, and matrix metalloproteinase 2 (MMP2 mRNA was determined by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR. Akt, GSK-3β, MMP2, β-catenin, and p53 protein expression, plus Akt, GSK-3β, and β-catenin phosphorylation, were determined by Western blot. Results T24 cell proliferation, MMP2 expression, Akt ser473 and thr308 phosphorylation, GSK3β tyr216 phosphorylation, and β-catenin ser45/thr41 phosphorylation were all decreased by APF, whereas p53 expression, and β-catenin ser33,37/thr41 phosphorylation, were increased by APF treatment in non-electroporated and non-target siRNA-transfected cells. Neither mRNA nor total protein expression of Akt, GSK3β, or

  15. Novel PI3K/AKT targeting anti-angiogenic activities of 4-vinylphenol, a new therapeutic potential of a well-known styrene metabolite.

    Science.gov (United States)

    Yue, Grace Gar-Lee; Lee, Julia Kin-Ming; Kwok, Hin-Fai; Cheng, Ling; Wong, Eric Chun-Wai; Jiang, Lei; Yu, Hua; Leung, Hoi-Wing; Wong, Yuk-Lau; Leung, Ping-Chung; Fung, Kwok-Pui; Lau, Clara Bik-San

    2015-01-01

    The pneumo- and hepato-toxicity of 4-vinylphenol (4VP), a styrene metabolite, has been previously reported. Nevertheless, the present study reported the novel anti-angiogenic activities of 4VP which was firstly isolated from the aqueous extract of a Chinese medicinal herb Hedyotis diffusa. Our results showed that 4VP at non-toxic dose effectively suppressed migration, tube formation, adhesion to extracellular matrix proteins, as well as protein and mRNA expressions of metalloproteinase-2 of human endothelial cells (HUVEC and HMEC-1). Investigation of the signal transduction revealed that 4VP down-regulated PI3K/AKT and p38 MAPK. Besides, 4VP interfered with the phosphorylation of ERK1/2, the translocation and expression of NFkappaB. In zebrafish embryo model, the new blood vessel growth was significantly blocked by 4VP (6.25-12.5 μg/mL medium). The VEGF-induced blood vessel formation in Matrigel plugs in C57BL/6 mice was suppressed by 4VP (20-100 μg/mL matrigel). In addition, the blood vessel number and tumor size were reduced by intraperitoneal 4VP (0.2-2 mg/kg) in 4T1 breast tumor-bearing BALB/c mice, with doxorubicin as positive control. Together, the in vitro and in vivo anti-angiogenic activities of 4VP were demonstrated for the first time. These findings suggest that 4VP has great potential to be further developed as an anti-angiogenic agent. PMID:26053458

  16. Targeting and valuing conservation investments in support of a water fund: linking upstream land management with downstream services in the Upper Tana catchment, Kenya

    Science.gov (United States)

    Bryant, B. P.; Droogers, P.; Hunink, J.; Vogl, A.; Wolny, S.

    2014-12-01

    We apply an integrated modeling framework to both target and value watershed management interventions in the Upper Tana watershed, which provides municipal water, irrigation water, and hydropower services to Nairobi and surrounding areas. The analysis begins by applying an index model approach that incorporates existing land use and land surface characteristics to prioritize the type and location of conservation investments in different subbasins, subject to budget constraints and stakeholder concerns (Resource Investment Optimization System -- RIOS). We then run the Soil and Water Assessment Tool (SWAT) using the RIOS-identified investment scenarios to produce spatially explicit scenarios that simulate changes in water yield and suspended sediment. Finally, we link those biophysical outputs to monetary and non-monetary human well-being metrics for multiple benefit streams, including: Reduced water treatment costs, increased hydropower production, and crop yield benefits for upstream farmers in the conservation area. The viability of a payment for watershed services scheme is discussed, with attention to the various components of value assessed and to dependencies on water management approaches. While other studies have examined links between land use and the provision of hydrologic services, this study is novel in that it presents an integrated analysis that targets interventions in a decision context and then relies on calibrated, process-based, biophysical models to demonstrate the return on those investments considering multiple (and sometimes competing) hydrological services, doing so at a sub-annual time-scale.

  17. Resveratrol suppresses constitutive activation of AKT via generation of ROS and induces apoptosis in diffuse large B cell lymphoma cell lines.

    Directory of Open Access Journals (Sweden)

    Azhar R Hussain

    Full Text Available BACKGROUND: We have recently shown that deregulation PI3-kinase/AKT survival pathway plays an important role in pathogenesis of diffuse large B cell lymphoma (DLBCL. In an attempt to identify newer therapeutic agents, we investigated the role of Resveratrol (trans-3,4', 5-trihydroxystilbene, a naturally occurring polyphenolic compound on a panel of diffuse large B-cell lymphoma (DLBCL cells in causing inhibition of cell viability and inducing apoptosis. METHODOLOGY/PRINCIPAL FINDINGS: We investigated the action of Resveratrol on DLBCL cells and found that Resveratrol inhibited cell viability and induced apoptosis by inhibition of constitutively activated AKT and its downstream targets via generation of reactive oxygen species (ROS. Simultaneously, Resveratrol treatment of DLBCL cell lines also caused ROS dependent upregulation of DR5; and interestingly, co-treatment of DLBCL with sub-toxic doses of TRAIL and Resveratrol synergistically induced apoptosis via utilizing DR5, on the other hand, gene silencing of DR5 abolished this effect. CONCLUSION/SIGNIFICANCE: Altogether, these data suggest that Resveratrol acts as a suppressor of AKT/PKB pathway leading to apoptosis via generation of ROS and at the same time primes DLBCL cells via up-regulation of DR5 to TRAIL-mediated apoptosis. These data raise the possibility that Resveratrol may have a future therapeutic role in DLBCL and possibly other malignancies with constitutive activation of the AKT/PKB pathway.

  18. Melatonin Modulates Endoplasmic Reticulum Stress and Akt/GSK3-Beta Signaling Pathway in a Rat Model of Renal Warm Ischemia Reperfusion

    Directory of Open Access Journals (Sweden)

    Kaouther Hadj Ayed Tka

    2015-01-01

    Full Text Available Melatonin (Mel is widely used to attenuate ischemia/reperfusion (I/R injury in several organs. Nevertheless, the underlying mechanisms remain unclear. This study was conducted to explore the effect of Mel on endoplasmic reticulum (ER stress, Akt and MAPK cascades after renal warm I/R. Eighteen Wistar rats were randomized into three groups: Sham, I/R, and Mel + I/R. The ischemia period was 60 min followed by 120 min of reperfusion. Mel (10 mg/kg was administrated 30 min prior to ischemia. The creatinine clearance, MDA, LDH levels, and histopathological changes were evaluated. In addition, Western blot was performed to study ER stress and its downstream apoptosis as well as phosphorylation of Akt, GSK-3β, VDAC, ERK, and P38. Mel decreased cytolysis and lipid peroxidation and improved renal function and morphology compared to I/R group. Parallely, it significantly reduced the ER stress parameters including GRP 78, p-PERK, XBP 1, ATF 6, CHOP, and JNK. Simultaneously, p-Akt level was significantly enhanced and its target molecules GSK-3β and VDAC were inhibited. Furthermore, the ERK and P38 phosphorylation were evidently augmented after Mel administration in comparison to I/R group. In conclusion, Mel improves the recovery of renal function by decreasing ER stress and stimulating Akt pathway after renal I/R injury.

  19. Macrophage migration inhibitory factor induces phosphorylation of Mdm2 mediated by phosphatidylinositol 3-kinase/Akt kinase: Role of this pathway in decidual cell survival.

    Science.gov (United States)

    Costa, Adriana Fraga; Gomes, Sara Zago; Lorenzon-Ojea, Aline R; Martucci, Mariane; Faria, Miriam Rubio; Pinto, Décio Dos Santos; Oliveira, Sergio F; Ietta, Francesca; Paulesu, Luana; Bevilacqua, Estela

    2016-05-01

    The phosphatidylinositol 3-kinase (PI3K)/Akt pathway has an anti-apoptotic effect through several downstream targets, which includes activation of the transformed mouse 3T3 cell double-minute 2 (Mdm2) protein, its translocation to the nucleus and degradation of the tumor suppressor p53. We show that Mif, the Macrophage Migration Inhibitory Factor, an important cytokine at the maternal fetal interface in several species, triggers phosphorylation of Mdm2 protein in a PI3K/Akt-dependent manner, thereby preventing apoptosis in cultured mouse decidual cells. Inhibition of Akt and PI3K suppresses the pathway. Mif treatment also changes the nuclear translocation of p53 and interferes with the apoptotic fate of these cells when challenged with reactive oxygen species. In conclusion, an important mechanism has been found underlying decidual cell survival through Akt signaling pathway activated by Mif, suggesting a role for this cytokine in decidual homeostasis and in the integrity of the maternal-fetal barrier that is essential for successful gestation. PMID:27208405

  20. Neuroprotection Signaling of Nuclear Akt in Neuronal Cells

    OpenAIRE

    Ahn, Jee-Yin

    2014-01-01

    Akt is one of the central kinases that perform a pivotal function in mediating survival signaling in a wide range of neuronal cell types in response to growth factor stimulation. The recent findings of a number of targets for Akt suggest that it prohibits neuronal death by both impinging on the cytoplasmic cell death machinery and by regulating nuclear proteins. The presence of active Akt in the nuclei of mammalian cells is no longer debatable, and this has been corroborated by the finding of...

  1. Construction of the AKT2 3′-UTR luciferase reporter gene vector and verification of the targeted relationship with miRNA-625%A KT23′-U TR荧光素酶报告基因载体构建及与miRNA-625靶向关系验证

    Institute of Scientific and Technical Information of China (English)

    魏斌; 邓霞; 卞秀娟; 钱粉红

    2016-01-01

    目的:通过构建荧光素酶报告基因重组质粒验证miRNA-625与AKT2基因的靶向调控关系。方法:通过生物信息学软件预测AKT2为miRNA-625靶基因;构建野生型AKT2基因3′端非翻译区荧光素酶报告基因载体及miR-NA-625靶序列突变型载体;将HEK-293T细胞株随机分为野生型+miRNA-625组、野生型+miRNA内参组、突变型+miRNA-625组、突变型+miRNA内参组;Lipofectamine 2000转染相应的质粒与miRNA,双荧光素酶报告基因系统检测荧光素酶活性。结果:构建野生型及突变型荧光素酶报告载体鉴定正确,双荧光素酶报告基因检测系统显示野生型+miRNA-625组相对荧光素酶活性较野生型+miRNA内参组明显降低(t=14.1764,P<0.05)。结论:miRNA-625对野生型AKT2重组质粒荧光活性有明显的抑制作用,证实miRNA-625能够靶向调控AKT2基因。%Objective:To identify the targeted-regulating relationship between miRNA-625 and AKT2 via constructing luciferase reporter gene vector.Methods:The AKT2 was predicted as miRNA-625 target gene by biological software.The 3′-untranslated regions(3′-UTR)of the wild type AKT2 and mutant AKT2 sequence were cloned into luciferase reporter vector pMIR-Report.The HEK-293T cells were divided into four groups randomly,wild-type plasmid +miRNA-625 group,wild-type plasmid +miRNA negative con-trol group,mutant plasmid +miRNA-625 group,mutant plasmid+miRNA negative control group.The rel-evant plasmids and miRNAs were transfected into groups by Lipofectamine 2000.The luciferase activity was detected by dual luciferase reporter gene system.Results:The recombinant plasmids were identified cor-rectly.Dual-luciferase reporter assay system revealed luciferase activity of wild-type plasmid+miRNA-625 group was significantly lower than that of wild-type plasmid+miRNA negative control group(P<0.05 ). Conclusion:The miRNA-625 inhibited the luciferase activity of AKT2 wild type

  2. Platelet-derived growth factor-induced Akt phosphorylation requires mTOR/Rictor and phospholipase C-γ1, whereas S6 phosphorylation depends on mTOR/Raptor and phospholipase D

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    Razmara Masoud

    2013-01-01

    Full Text Available Abstract Mammalian target of rapamycin (mTOR can be found in two multi-protein complexes, i.e. mTORC1 (containing Raptor and mTORC2 (containing Rictor. Here, we investigated the mechanisms by which mTORC1 and mTORC2 are activated and their downstream targets in response to platelet-derived growth factor (PDGF-BB treatment. Inhibition of phosphatidylinositol 3-kinase (PI3K inhibited PDGF-BB activation of both mTORC1 and mTORC2. We found that in Rictor-null mouse embryonic fibroblasts, or after prolonged rapamycin treatment of NIH3T3 cells, PDGF-BB was not able to promote phosphorylation of Ser473 in the serine/threonine kinase Akt, whereas Thr308 phosphorylation was less affected, suggesting that Ser473 in Akt is phosphorylated in an mTORC2-dependent manner. This reduction in Akt phosphorylation did not influence the phosphorylation of the S6 protein, a well established protein downstream of mTORC1. Consistently, triciribine, an inhibitor of the Akt pathway, suppressed PDGF-BB-induced Akt phosphorylation without having any effect on S6 phosphorylation. Thus, mTORC2 does not appear to be upstream of mTORC1. We could also demonstrate that in Rictor-null cells the phosphorylation of phospholipase Cγ1 (PLCγ1 and protein kinase C (PKC was impaired, and the PKCα protein levels strongly reduced. Furthermore, interfering with the PLCγ/Ca2+/PKC pathway inhibited PDGF-BB-induced Akt phosphorylation. In addition, PDGF-BB-induced activation of mTORC1, as measured by phosphorylation of the downstream S6 protein, was dependent on phospholipase D (PLD. It has been shown that Erk1/2 MAP-kinase directly phosphorylates and activates mTORC1; in partial agreement with this finding, we found that a Mek1/2 inhibitor delayed S6 phosphorylation in response to PDGF-BB, but it did not block it. Thus, whereas both mTORC1 and mTORC2 are activated in a PI3K-dependent manner, different additional signaling pathways are needed. mTORC1 is activated in a PLD-dependent manner

  3. The role of PI3K/AKT-related PIP5K1α and the discovery of its selective inhibitor for treatment of advanced prostate cancer.

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    Semenas, Julius; Hedblom, Andreas; Miftakhova, Regina R; Sarwar, Martuza; Larsson, Rikard; Shcherbina, Liliya; Johansson, Martin E; Härkönen, Pirkko; Sterner, Olov; Persson, Jenny L

    2014-09-01

    Nitrogen-containing heterocyclic compounds are an important class of molecules that are commonly used for the synthesis of candidate drugs. Phosphatidylinositol-4-phosphate 5-kinase-α (PIP5Kα) is a lipid kinase, similar to PI3K. However, the role of PIP5K1α in oncogenic processes and the development of inhibitors that selectively target PIP5K1α have not been reported. In the present study we report that overexpression of PIP5K1α is associated with poor prognosis in prostate cancer and correlates with an elevated level of the androgen receptor. Overexpression of PIP5K1α in PNT1A nonmalignant cells results in an increased AKT activity and an increased survival, as well as invasive malignant phenotype, whereas siRNA-mediated knockdown of PIP5K1α in aggressive PC-3 cells leads to a reduced AKT activity and an inhibition in tumor growth in xenograft mice. We further report a previously unidentified role for PIP5K1α as a druggable target for our newly developed compound ISA-2011B using a high-throughput KINOMEscan platform. ISA-2011B was discovered during our synthetic studies of C-1 indol-3-yl substituted 1,2,3,4-tetrahydroisoquinolines via a Pictet-Spengler approach. ISA-2011B significantly inhibits growth of tumor cells in xenograft mice, and we show that this is mediated by targeting PIP5K1α-associated PI3K/AKT and the downstream survival, proliferation, and invasion pathways. Further, siRNA-mediated knockdown of PIP5K1α exerts similar effects on PC3 cells as ISA-2011B treatment, significantly inhibiting AKT activity, increasing apoptosis and reducing invasion. Thus, PIP5K1α has high potential as a drug target, and compound ISA-2011B is interesting for further development of targeted cancer therapy. PMID:25071204

  4. Identification of Pou5f1, Sox2, and Nanog downstream target genes with statistical confidence by applying a novel algorithm to time course microarray and genome-wide chromatin immunoprecipitation data

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    Xin Li

    2008-06-01

    Full Text Available Abstract Background Target genes of a transcription factor (TF Pou5f1 (Oct3/4 or Oct4, which is essential for pluripotency maintenance and self-renewal of embryonic stem (ES cells, have previously been identified based on their response to Pou5f1 manipulation and occurrence of Chromatin-immunoprecipitation (ChIP-binding sites in promoters. However, many responding genes with binding sites may not be direct targets because response may be mediated by other genes and ChIP-binding site may not be functional in terms of transcription regulation. Results To reduce the number of false positives, we propose to separate responding genes into groups according to direction, magnitude, and time of response, and to apply the false discovery rate (FDR criterion to each group individually. Using this novel algorithm with stringent statistical criteria (FDR Pou5f1 suppression and published ChIP data, we identified 420 tentative target genes (TTGs for Pou5f1. The majority of TTGs (372 were down-regulated after Pou5f1 suppression, indicating that the Pou5f1 functions as an activator of gene expression when it binds to promoters. Interestingly, many activated genes are potent suppressors of transcription, which include polycomb genes, zinc finger TFs, chromatin remodeling factors, and suppressors of signaling. Similar analysis showed that Sox2 and Nanog also function mostly as transcription activators in cooperation with Pou5f1. Conclusion We have identified the most reliable sets of direct target genes for key pluripotency genes – Pou5f1, Sox2, and Nanog, and found that they predominantly function as activators of downstream gene expression. Thus, most genes related to cell differentiation are suppressed indirectly.

  5. Discovery of novel AKT inhibitors with enhanced anti-tumor effects in combination with the MEK inhibitor.

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    Melissa Dumble

    Full Text Available Tumor cells upregulate many cell signaling pathways, with AKT being one of the key kinases to be activated in a variety of malignancies. GSK2110183 and GSK2141795 are orally bioavailable, potent inhibitors of the AKT kinases that have progressed to human clinical studies. Both compounds are selective, ATP-competitive inhibitors of AKT 1, 2 and 3. Cells treated with either compound show decreased phosphorylation of several substrates downstream of AKT. Both compounds have desirable pharmaceutical properties and daily oral dosing results in a sustained inhibition of AKT activity as well as inhibition of tumor growth in several mouse tumor models of various histologic origins. Improved kinase selectivity was associated with reduced effects on glucose homeostasis as compared to previously reported ATP-competitive AKT kinase inhibitors. In a diverse cell line proliferation screen, AKT inhibitors showed increased potency in cell lines with an activated AKT pathway (via PI3K/PTEN mutation or loss while cell lines with activating mutations in the MAPK pathway (KRAS/BRAF were less sensitive to AKT inhibition. Further investigation in mouse models of KRAS driven pancreatic cancer confirmed that combining the AKT inhibitor, GSK2141795 with a MEK inhibitor (GSK2110212; trametinib resulted in an enhanced anti-tumor effect accompanied with greater reduction in phospho-S6 levels. Taken together these results support clinical evaluation of the AKT inhibitors in cancer, especially in combination with MEK inhibitor.

  6. Discovery of novel AKT inhibitors with enhanced anti-tumor effects in combination with the MEK inhibitor.

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    Dumble, Melissa; Crouthamel, Ming-Chih; Zhang, Shu-Yun; Schaber, Michael; Levy, Dana; Robell, Kimberly; Liu, Qi; Figueroa, David J; Minthorn, Elisabeth A; Seefeld, Mark A; Rouse, Meagan B; Rabindran, Sridhar K; Heerding, Dirk A; Kumar, Rakesh

    2014-01-01

    Tumor cells upregulate many cell signaling pathways, with AKT being one of the key kinases to be activated in a variety of malignancies. GSK2110183 and GSK2141795 are orally bioavailable, potent inhibitors of the AKT kinases that have progressed to human clinical studies. Both compounds are selective, ATP-competitive inhibitors of AKT 1, 2 and 3. Cells treated with either compound show decreased phosphorylation of several substrates downstream of AKT. Both compounds have desirable pharmaceutical properties and daily oral dosing results in a sustained inhibition of AKT activity as well as inhibition of tumor growth in several mouse tumor models of various histologic origins. Improved kinase selectivity was associated with reduced effects on glucose homeostasis as compared to previously reported ATP-competitive AKT kinase inhibitors. In a diverse cell line proliferation screen, AKT inhibitors showed increased potency in cell lines with an activated AKT pathway (via PI3K/PTEN mutation or loss) while cell lines with activating mutations in the MAPK pathway (KRAS/BRAF) were less sensitive to AKT inhibition. Further investigation in mouse models of KRAS driven pancreatic cancer confirmed that combining the AKT inhibitor, GSK2141795 with a MEK inhibitor (GSK2110212; trametinib) resulted in an enhanced anti-tumor effect accompanied with greater reduction in phospho-S6 levels. Taken together these results support clinical evaluation of the AKT inhibitors in cancer, especially in combination with MEK inhibitor.

  7. EGF‑stimulated AKT activation is mediated by EGFR recycling via an early endocytic pathway in a gefitinib‑resistant human lung cancer cell line.

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    Nishimura, Yukio; Takiguchi, Soichi; Ito, Shigeru; Itoh, Kazuyuki

    2015-04-01

    The receptor tyrosine kinase epidermal growth factor receptor (EGFR) and its ligand epidermal growth factor (EGF) are known to play important roles in malignant tumor cells, and the EGFR signaling pathway is one of the most important targets in various tumors, including non-small cell lung cancer (NSCLC). We reported recently that an aberration in certain steps of EGF-stimulated phosphorylated epidermal growth factor receptor (pEGFR) endocytic trafficking from the early endosomes to the late endosomes occurs in the gefitinib-resistant NSCLC cells, in which large amounts of sorting nexin 1 (SNX1) are colocalized with EGFR in the aggregated early endosomes where the internalized pEGFR is also accumulated of these cells. To further investigate the role of SNX1 in EGF‑stimulated pEGFR endocytosis, followed by downstream signaling leading to the activation of phosphatidylinositol 3-kinase (PI3K)--the serine/threonine kinase AKT pathway, we examined the effect of depletion of SNX1 knock-down expression by siRNA and an inhibition of targeting membrane recycling using monensin. Using immunofluorescence, we observed an efficient endocytic transport of pEGFR from early endosomes to late endosomes/lysosomes after EGF-stimulation in the cells transfected with siRNA‑SNX1, whereas the delayed endocytic delivery of pEGFR was evident in the siRNA-control-transfected cells. Furthermore, a large amount of endocytosed pEGFR was accumulated in the presence of monensin in the early endosomes of the SNX1 knock-down cells. In western blot analysis, EGF stimulation of both control and cells transfected with siRNA-SNX1 resulted in rapid phosphorylation of EGFR and enhanced AKT phosphorylation. Monensin-dependent inhibition of AKT phosphorylation was stronger in SNX1 knock-down cells than in controls. In contrast, however, monensin had no effect on AKT phosphorylation triggered by activation of the MET receptor tyrosine kinase. Collectively, we suggest that EGF-stimulated recycling of

  8. Increased level of phosphorylated akt measured by chemiluminescence-linked immunosorbent assay is a predictor of poor prognosis in primary breast cancer overexpressing ErbB-2

    International Nuclear Information System (INIS)

    Akt1, Akt2 and Akt3 kinases are downstream components of phosphoinositol 3-kinase derived signals from receptor tyrosine kinases, which influence cell growth, proliferation and survival. Akt2 overexpression and amplification have been described in breast, ovarian and pancreatic cancers. The present study was designed to investigate the prognostic significance of activated Akt in primary breast cancer and its association with other tumour biomarkers. Using a two-site chemiluminescence-linked immunosorbent assay, we measured the quantitative expression levels of total phosphorylated (P-S473) Akt (Akt1/Akt2/Akt3) on cytosol fractions obtained from fresh frozen tissue samples of 156 primary breast cancer patients. Akt phosphorylation was not associated with nodal status or ErbB-2 protein expression levels. High levels of phosphorylated Akt correlated (P < 0.01) with poor prognosis, and the significance of this correlation increased (P < 0.001) in the subset of patients with ErbB-2 overexpressing tumours. In addition, phosphorylated Akt was found to be associated with mRNA expression levels of several proliferation markers (e.g. thymidylate synthase), measured using quantitative real-time RT-PCR. Our findings demonstrate that, in breast cancer patients, Akt activation is associated with tumour proliferation and poor prognosis, particularly in the subset of patients with ErbB2-overexpressing tumours

  9. The Akt-inhibitor Erufosine induces apoptotic cell death in prostate cancer cells and increases the short term effects of ionizing radiation

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    Eibl Hans-Jörg

    2010-11-01

    Full Text Available Abstract Background and Purpose The phosphatidylinositol-3-kinase (PI3K/Akt pathway is frequently deregulated in prostate cancer and associated with neoplastic transformation, malignant progression, and enhanced resistance to classical chemotherapy and radiotherapy. Thus, it is a promising target for therapeutic intervention. In the present study, the cytotoxic action of the Akt inhibitor Erufosine (ErPC3 was analyzed in prostate cancer cells and compared to the cytotoxicity of the PI3K inhibitor LY294002. Moreover, the efficacy of combined treatment with Akt inhibitors and ionizing radiation in prostate cancer cells was examined. Materials and methods Prostate cancer cell lines PC3, DU145, and LNCaP were treated with ErPC3 (1-100 µM, LY294002 (25-100 µM, irradiated (0-10 Gy, or subjected to combined treatments. Cell viability was determined by the WST-1 assay. Apoptosis induction was analyzed by flow cytometry after staining with propidium iodide in a hypotonic citrate buffer, and by Western blotting using antibodies against caspase-3 and its substrate PARP. Akt activity and regulation of the expression of Bcl-2 family members and key downstream effectors involved in apoptosis regulation were examined by Western blot analysis. Results The Akt inhibitor ErPC3 exerted anti-neoplastic effects in prostate cancer cells, however with different potency. The anti-neoplastic action of ErPC3 was associated with reduced phosphoserine 473-Akt levels and induction of apoptosis. PC3 and LNCaP prostate cancer cells were also sensitive to treatment with the PI3K inhibitor LY294002. However, the ErPC3-sensitive PC3-cells were less susceptible to LY294002 than the ErPC3-refractory LNCaP cells. Although both cell lines were largely resistant to radiation-induced apoptosis, both cell lines showed higher levels of apoptotic cell death when ErPC3 was combined with radiotherapy. Conclusions Our data suggest that constitutive Akt activation and survival are

  10. The Akt-inhibitor Erufosine induces apoptotic cell death in prostate cancer cells and increases the short term effects of ionizing radiation

    International Nuclear Information System (INIS)

    The phosphatidylinositol-3-kinase (PI3K)/Akt pathway is frequently deregulated in prostate cancer and associated with neoplastic transformation, malignant progression, and enhanced resistance to classical chemotherapy and radiotherapy. Thus, it is a promising target for therapeutic intervention. In the present study, the cytotoxic action of the Akt inhibitor Erufosine (ErPC3) was analyzed in prostate cancer cells and compared to the cytotoxicity of the PI3K inhibitor LY294002. Moreover, the efficacy of combined treatment with Akt inhibitors and ionizing radiation in prostate cancer cells was examined. Prostate cancer cell lines PC3, DU145, and LNCaP were treated with ErPC3 (1-100 µM), LY294002 (25-100 µM), irradiated (0-10 Gy), or subjected to combined treatments. Cell viability was determined by the WST-1 assay. Apoptosis induction was analyzed by flow cytometry after staining with propidium iodide in a hypotonic citrate buffer, and by Western blotting using antibodies against caspase-3 and its substrate PARP. Akt activity and regulation of the expression of Bcl-2 family members and key downstream effectors involved in apoptosis regulation were examined by Western blot analysis. The Akt inhibitor ErPC3 exerted anti-neoplastic effects in prostate cancer cells, however with different potency. The anti-neoplastic action of ErPC3 was associated with reduced phosphoserine 473-Akt levels and induction of apoptosis. PC3 and LNCaP prostate cancer cells were also sensitive to treatment with the PI3K inhibitor LY294002. However, the ErPC3-sensitive PC3-cells were less susceptible to LY294002 than the ErPC3-refractory LNCaP cells. Although both cell lines were largely resistant to radiation-induced apoptosis, both cell lines showed higher levels of apoptotic cell death when ErPC3 was combined with radiotherapy. Our data suggest that constitutive Akt activation and survival are controlled by different different molecular mechanisms in the two prostate cancer cell lines

  11. Protein kinase B (PKB/AKT1) formed signaling complexes with mitochondrial proteins and prevented glycolytic energy dysfunction in cultured cardiomyocytes during ischemia-reperfusion injury.

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    Deng, Wu; Leu, Hsin-Bang; Chen, Yumay; Chen, Yu-Han; Epperson, Christine M; Juang, Charity; Wang, Ping H

    2014-05-01

    Our previous studies showed that insulin stimulated AKT1 translocation into mitochondria and modulated oxidative phosphorylation complex V in cardiac muscle. This raised the possibility that mitochondrial AKT1 may regulate glycolytic oxidative phosphorylation and mitochondrial function in cardiac muscle cells. The aims of this project were to study the effects of mitochondrial AKT1 signaling on cell survival in stressed cardiomyocytes, to define the effect of mitochondrial AKT1 signaling on glycolytic bioenergetics, and to identify mitochondrial targets of AKT1 signaling in cardiomyocytes. Mitochondrial AKT1 signaling played a protective role against apoptosis and necrosis during ischemia-reperfusion stress, suppressed mitochondrial calcium overload, and alleviated mitochondrial membrane depolarization. Activation of AKT1 signaling in mitochondria increased glucose uptake, enhanced respiration efficiency, reduced superoxide generation, and increased ATP production in the cardiomyocytes. Inhibition of mitochondrial AKT attenuated insulin response, indicating that insulin regulation of ATP production required mitochondrial AKT1 signaling. A proteomic approach was used to reveal 15 novel targets of AKT1 signaling in mitochondria, including pyruvate dehydrogenase complex (PDC). We have confirmed and characterized the association of AKT1 and PDC subunits and verified a stimulatory effect of mitochondrial AKT1 on the enzymatic activity of PDC. These findings suggested that AKT1 formed protein complexes with multiple mitochondrial proteins and improved mitochondrial function in stressed cardiomyocytes. The novel AKT1 signaling targets in mitochondria may become a resource for future metabolism research.

  12. Hydrogen peroxide/ceramide/Akt signaling axis play a critical role in the antileukemic potential of sanguinarine.

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    Rahman, Anees; Thayyullathil, Faisal; Pallichankandy, Siraj; Galadari, Sehamuddin

    2016-07-01

    Dysregulation of apoptosis is a prime hallmark of leukemia. Therefore, drugs which restore the sensitivity of leukemic cells to apoptotic stimuli are promising candidates in the treatment of leukemia. Recently, we have demonstrated that sanguinarine (SNG), a benzophenanthridine alkaloid, isolated from Sanguinaria canadensis induces ROS-dependent ERK1/2 activation and autophagic cell death in human malignant glioma cells (Pallichankandy et al., 2015; [43]). In this study, we investigated the antileukemic potential of SNG in vitro, and further examined the molecular mechanisms of SNG-induced cell death. In human leukemic cells, SNG activated apoptotic cell death pathway characterized by activation of caspase cascade, DNA fragmentation and down-regulation of anti-apoptotic proteins. Importantly, we have identified a crucial role for hydrogen peroxide (H2O2)-dependent ceramide (Cer) generation in the facilitation of SNG-induced apoptosis. Additionally, we have found that SNG inhibits Akt, a key anti-apoptotic protein kinase by dephosphorylating it at Ser(473), leading to the dephosphorylation of its downstream targets, GSK3β and mTOR. Interestingly, inhibition of Cer generation, using acid sphingomyelinase inhibitor, significantly reduced the SNG-induced Akt dephosphorylation and apoptosis, whereas, activation of Cer generation using inhibitors of acid ceramidase and glucosylceramide synthase enhanced it. Furthermore, using a group of ceramide activated protein phosphatases (CAPPs) inhibitor (calyculin A, Okadaic acid, and phosphatidic acid), the involvement of protein phosphatase 1 form of CAPP in SNG-induced Akt dephosphorylation and apoptosis was demonstrated. Altogether, these results underscore a critical role for H2O2-Cer-Akt signaling axis in the antileukemic action of SNG. PMID:27154977

  13. Analysis of receptor tyrosine kinases (RTKs) and downstream pathways in chordomas†

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    Tamborini, Elena; Virdis, Emanuela; Negri, Tiziana; Orsenigo, Marta; Brich, Silvia; Conca, Elena; Gronchi, Alessandro; Stacchiotti, Silvia; Manenti, Giacomo; Casali, Paolo G.; Pierotti, Marco A.; Pilotti, Silvana

    2010-01-01

    We have previously demonstrated that chordomas express activated platelet-derived growth factor receptor (PDGFRB) and that treatment with imatinib, which is capable of switching off the activation of various receptor tyrosine kinases (RTKs) including PDGFRB, benefits a number of patients. The aim of this study was to identify the possible presence of other activated RTKs and their downstream signaling effectors. Cryopreserved material from 22 naïve sporadic chordomas was investigated for the presence of activated RTKs and their cognate ligands and downstream signaling effectors by means of human phospho-RTK antibody arrays, Western blotting, and molecular analysis; immunohistochemistry and fluorescence in situ hybridization were used to analyze the corresponding formalin-fixed and paraffin-embedded samples. We detected activated PDGFRB, FLT3, and colony stimulating factor 1 receptor (CSF1R) of the PDGFR family and highly phosphorylated EGFR, HER2/neu, and (to a lesser extent) HER4 of the EGFR family. The detection of PDGFRB/PDGFB confirmed our previous data. The presence of activated EGFR was paralleled by the finding of high levels of epidermal growth factor (EGF) and transforming growth factor α (TGFα) and PDGFB co-expression and PDGFRB co-immunoprecipitation. Of the downstream effectors, the PI3K/AKT and RAS/MAPK pathways were both activated, thus leading to the phosphorylation of mammalian target of rapamycin (mTOR) and 4E-BP1 among the regulators involved in translational control. Taken together, our results (i) provide a rationale for tailored treatments targeting upstream activated receptors, including the PDGFR and EGFR families; (ii) support the idea that a combination of upstream antagonists and mTOR inhibitors enhances the control of tumor growth; and (iii) indicate that the 4E-BP1/eIF4E pathway is a major regulator of protein synthesis in chordoma. PMID:20164240

  14. Ramentaceone, a Naphthoquinone Derived from Drosera sp., Induces Apoptosis by Suppressing PI3K/Akt Signaling in Breast Cancer Cells.

    Science.gov (United States)

    Kawiak, Anna; Lojkowska, Ewa

    2016-01-01

    The phosphoinositide 3-kinase (PI3K) signaling pathway plays an important role in processes critical for breast cancer progression and its upregulation confers increased resistance of cancer cells to chemotherapy and radiation. The present study aimed at determining the activity of ramentaceone, a constituent of species in the plant genera Drosera, toward breast cancer cells and defining the involvement of PI3K/Akt inhibition in ramentaceone-mediated cell death induction. The results showed that ramentaceone exhibited high antiproliferative activity toward breast cancer cells, in particular HER2-overexpressing breast cancer cells. The mode of cell death induced by ramentaceone was through apoptosis as determined by cytometric analysis of caspase activity and Annexin V staining. Apoptosis induction was found to be mediated by inhibition of PI3K/Akt signaling and through targeting its downstream anti-apoptotic effectors. Ramentaceone inhibited PI3-kinase activity, reduced the expression of the PI3K protein and inhibited the phosphorylation of the Akt protein in breast cancer cells. The expression of the anti-apoptotic Bcl-2 protein was decreased and the levels of the pro-apoptotic proteins, Bax and Bak, were elevated. Moreover, inhibition of PI3K and silencing of Akt expression increased the sensitivity of cells to ramentaceone-induced apoptosis. In conclusion, our results indicate that ramentaceone induces apoptosis in breast cancer cells through PI3K/Akt signaling inhibition. These findings suggest further investigation of ramentaceone as a potential therapeutic agent in breast cancer therapy, in particular HER2-positive breast cancer. PMID:26840401

  15. Ramentaceone, a Naphthoquinone Derived from Drosera sp., Induces Apoptosis by Suppressing PI3K/Akt Signaling in Breast Cancer Cells.

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    Anna Kawiak

    Full Text Available The phosphoinositide 3-kinase (PI3K signaling pathway plays an important role in processes critical for breast cancer progression and its upregulation confers increased resistance of cancer cells to chemotherapy and radiation. The present study aimed at determining the activity of ramentaceone, a constituent of species in the plant genera Drosera, toward breast cancer cells and defining the involvement of PI3K/Akt inhibition in ramentaceone-mediated cell death induction. The results showed that ramentaceone exhibited high antiproliferative activity toward breast cancer cells, in particular HER2-overexpressing breast cancer cells. The mode of cell death induced by ramentaceone was through apoptosis as determined by cytometric analysis of caspase activity and Annexin V staining. Apoptosis induction was found to be mediated by inhibition of PI3K/Akt signaling and through targeting its downstream anti-apoptotic effectors. Ramentaceone inhibited PI3-kinase activity, reduced the expression of the PI3K protein and inhibited the phosphorylation of the Akt protein in breast cancer cells. The expression of the anti-apoptotic Bcl-2 protein was decreased and the levels of the pro-apoptotic proteins, Bax and Bak, were elevated. Moreover, inhibition of PI3K and silencing of Akt expression increased the sensitivity of cells to ramentaceone-induced apoptosis. In conclusion, our results indicate that ramentaceone induces apoptosis in breast cancer cells through PI3K/Akt signaling inhibition. These findings suggest further investigation of ramentaceone as a potential therapeutic agent in breast cancer therapy, in particular HER2-positive breast cancer.

  16. Mechanical Stimulation and IGF-1 Enhance mRNA Translation Rate in Osteoblasts Via Activation of the AKT-mTOR Pathway.

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    Bakker, Astrid D; Gakes, Tom; Hogervorst, Jolanda M A; de Wit, Gerard M J; Klein-Nulend, Jenneke; Jaspers, Richard T

    2016-06-01

    Insulin-like growth factor-1 (IGF-1) is anabolic for muscle by enhancing the rate of mRNA translation via activation of AKT and subsequent activation of the mammalian target of rapamycin complex 1 (mTOR), thereby increasing cellular protein production. IGF-1 is also anabolic for bone, but whether the mTOR pathway plays a role in the rate of bone matrix protein production by osteoblasts is unknown. We hypothesized that anabolic stimuli such as mechanical loading and IGF-1 stimulate protein synthesis in osteoblasts via activation of the AKT-mTOR pathway. MC3T3-E1 osteoblasts were either or not subjected for 1 h to mechanical loading by pulsating fluid flow (PFF) or treated with or without human recombinant IGF-1 (1-100 ng/ml) for 0.5-6 h, to determine phosphorylation of AKT and p70S6K (downstream of mTOR) by Western blot. After 4 days of culture with or without the mTOR inhibitor rapamycin, total protein, DNA, and gene expression were quantified. IGF-1 (100 ng/ml) reduced IGF-1 gene expression, although PFF enhanced IGF-1 expression. IGF-1 did not affect collagen-I gene expression. IGF-1 dose-dependently enhanced AKT and p70S6K phosphorylation at 2 and 6 h. PFF enhanced phosphorylation of AKT and p70S6K already within 1 h. Both IGF-1 and PFF enhanced total protein per cell by ∼30%, but not in the presence of rapamycin. Our results show that IGF-1 and PFF activate mTOR, thereby stimulating the rate of mRNA translation in osteoblasts. The known anabolic effect of mechanical loading and IGF-1 on bone may thus be partly explained by mTOR-mediated enhanced protein synthesis in osteoblasts. PMID:26505782

  17. A combination of indol-3-carbinol and genistein synergistically induces apoptosis in human colon cancer HT-29 cells by inhibiting Akt phosphorylation and progression of autophagy

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    Watanabe Hirotsuna

    2009-11-01

    Full Text Available Abstract Background The chemopreventive effects of dietary phytochemicals on malignant tumors have been studied extensively because of a relative lack of toxicity. To achieve desirable effects, however, treatment with a single agent mostly requires high doses. Therefore, studies on effective combinations of phytochemicals at relatively low concentrations might contribute to chemopreventive strategies. Results Here we found for the first time that co-treatment with I3C and genistein, derived from cruciferous vegetables and soy, respectively, synergistically suppressed the viability of human colon cancer HT-29 cells at concentrations at which each agent alone was ineffective. The suppression of cell viability was due to the induction of a caspase-dependent apoptosis. Moreover, the combination effectively inhibited phosphorylation of Akt followed by dephosphorylation of caspase-9 or down-regulation of XIAP and survivin, which contribute to the induction of apoptosis. In addition, the co-treatment also enhanced the induction of autophagy mediated by the dephosphorylation of mTOR, one of the downstream targets of Akt, whereas the maturation of autophagosomes was inhibited. These results give rise to the possibility that co-treatment with I3C and genistein induces apoptosis through the simultaneous inhibition of Akt activity and progression of the autophagic process. This possibility was examined using inhibitors of Akt combined with inhibitors of autophagy. The combination effectively induced apoptosis, whereas the Akt inhibitor alone did not. Conclusion Although in vivo study is further required to evaluate physiological efficacies and toxicity of the combination treatment, our findings might provide a new insight into the development of novel combination therapies/chemoprevention against malignant tumors using dietary phytochemicals.

  18. Mechanical Stimulation and IGF-1 Enhance mRNA Translation Rate in Osteoblasts Via Activation of the AKT-mTOR Pathway.

    Science.gov (United States)

    Bakker, Astrid D; Gakes, Tom; Hogervorst, Jolanda M A; de Wit, Gerard M J; Klein-Nulend, Jenneke; Jaspers, Richard T

    2016-06-01

    Insulin-like growth factor-1 (IGF-1) is anabolic for muscle by enhancing the rate of mRNA translation via activation of AKT and subsequent activation of the mammalian target of rapamycin complex 1 (mTOR), thereby increasing cellular protein production. IGF-1 is also anabolic for bone, but whether the mTOR pathway plays a role in the rate of bone matrix protein production by osteoblasts is unknown. We hypothesized that anabolic stimuli such as mechanical loading and IGF-1 stimulate protein synthesis in osteoblasts via activation of the AKT-mTOR pathway. MC3T3-E1 osteoblasts were either or not subjected for 1 h to mechanical loading by pulsating fluid flow (PFF) or treated with or without human recombinant IGF-1 (1-100 ng/ml) for 0.5-6 h, to determine phosphorylation of AKT and p70S6K (downstream of mTOR) by Western blot. After 4 days of culture with or without the mTOR inhibitor rapamycin, total protein, DNA, and gene expression were quantified. IGF-1 (100 ng/ml) reduced IGF-1 gene expression, although PFF enhanced IGF-1 expression. IGF-1 did not affect collagen-I gene expression. IGF-1 dose-dependently enhanced AKT and p70S6K phosphorylation at 2 and 6 h. PFF enhanced phosphorylation of AKT and p70S6K already within 1 h. Both IGF-1 and PFF enhanced total protein per cell by ∼30%, but not in the presence of rapamycin. Our results show that IGF-1 and PFF activate mTOR, thereby stimulating the rate of mRNA translation in osteoblasts. The known anabolic effect of mechanical loading and IGF-1 on bone may thus be partly explained by mTOR-mediated enhanced protein synthesis in osteoblasts.

  19. Expression and significance of CD44 and p-AKT in pancreatic head cancer

    OpenAIRE

    Xiaoping, Li; Xiaowei, Zhang; Leizhen, Zheng; Weijian, Guo

    2015-01-01

    Background CD44 and phosphorylated AKT (p-AKT) is a potentially interesting prognostic marker and therapeutic target in pancreatic cancer. The expression of CD44 and p-AKT has been reported to correlate with poor prognosis of pancreatic cancer in most literatures. The purpose of this study is to investigate the roles of CD44 and p-AKT in pancreatic head cancer and their correlation with the prognosis of pancreatic head cancer patients. Methods Forty-eight pancreatic head cancer samples were c...

  20. Involvement of PI3K/Akt/FoxO3a and PKA/CREB Signaling Pathways in the Protective Effect of Fluoxetine Against Corticosterone-Induced Cytotoxicity in PC12 Cells.

    Science.gov (United States)

    Zeng, Bingqing; Li, Yiwen; Niu, Bo; Wang, Xinyi; Cheng, Yufang; Zhou, Zhongzhen; You, Tingting; Liu, Yonggang; Wang, Haitao; Xu, Jiangping

    2016-08-01

    The selective serotonin reuptake inhibitor fluoxetine is neuroprotective in several brain injury models. It is commonly used to treat major depressive disorder and related conditions, but its mechanism of action remains incompletely understood. Activation of the phosphatidylinositol-3-kinase/protein kinase B/forkhead box O3a (PI3K/Akt/FoxO3a) and protein kinase A/cAMP-response element binding protein (PKA/CREB) signaling pathways has been strongly implicated in the pathogenesis of depression and might be the downstream target of fluoxetine. Here, we used PC12 cells exposed to corticosterone (CORT) to study the neuroprotective effects of fluoxetine and the involvement of the PI3K/Akt/FoxO3a and PKA/CREB signaling pathways. Our results show that CORT reduced PC12 cells viability by 70 %, and that fluoxetine showed a concentration-dependent neuroprotective effect. Neuroprotective effects of fluoxetine were abolished by inhibition of PI3K, Akt, and PKA using LY294002, KRX-0401, and H89, respectively. Treatment of PC12 cells with fluoxetine resulted in increased phosphorylation of Akt, FoxO3a, and CREB. Fluoxetine also dose-dependently rescued the phosphorylation levels of Akt, FoxO3a, and CREB, following administration of CORT (from 99 to 110, 56 to 170, 80 to 170 %, respectively). In addition, inhibition of PKA and PI3K/Akt resulted in decreased levels of p-CREB, p-Akt, and p-FoxO3a in the presence of fluoxetine. Furthermore, fluoxetine reversed CORT-induced upregulation of p53-upregulated modulator of apoptosis (Puma) and Bcl-2-interacting mediator of cell death (Bim) via the PI3K/Akt/FoxO3a signaling pathway. H89 treatment reversed the effect of fluoxetine on the mRNA level of brain-derived neurotrophic factor, which was decreased in the presence of CORT. Our data indicate that fluoxetine elicited neuroprotection toward CORT-induced cell death that involves dual regulation from PI3K/Akt/FoxO3a and PKA/CREB pathways. PMID:27412469

  1. DMH1 increases glucose metabolism through activating Akt in L6 rat skeletal muscle cells.

    Directory of Open Access Journals (Sweden)

    Xin Xie

    Full Text Available DMH1(4-[6-(4-Isopropoxyphenylpyrazolo [1,5-a]pyrimidin-3-yl] quinoline is a compound C analogue with the structural modifications at the 3- and 6-positions in pyrazolo[1,5-a]pyrimidine backbone. Compound C was reported to inhibit both AMPK and Akt. Our preliminary work found that DMH1 activated Akt. Since Akt was involved in glucose metabolism, we aimed to identify the effects of DMH1 on glucose metabolism in L6 rat muscle cells and the potential mechanism. Results showed that DMH1 increased lactic acid release and glucose consumption in L6 rat muscle cells in a dose-dependent manner. DMH1 activated Akt in L6 cells. Akt inhibitor inhibited DMH1-induced Akt activation and DMH1-induced increases of glucose uptake and consumption. DMH1 had no cytotoxicity in L6 cells, but inhibited mitochondrial function and reduced ATP production. DMH1 showed no effect on AMPK, but in the presence of Akt inhibitor, DMH1 significantly activated AMPK. Compound C inhibited DMH1-induced Akt activation in L6 cells. Compound C inhibited DMH1-induced increase of glucose uptake, consumption and lactic acid release in L6 cells. DMH1 inhibited PP2A activity, and PP2A activator forskolin reversed DMH1-induced Akt activation. We concluded that DMH1 increased glucose metabolism through activating Akt and DMH1 activated Akt through inhibiting PP2A activity in L6 rat muscle cells. In view of the analogue structure of DMH1 and compound C and the contrasting effects of DMH1 and compound C on Akt, the present study provides a novel leading chemical structure targeting Akt with potential use for regulating glucose metabolism.

  2. Inhibition of Adipogenesis by Oligonol through Akt-mTOR Inhibition in 3T3-L1 Adipocytes

    Directory of Open Access Journals (Sweden)

    Jae-Yeo Park

    2014-01-01

    Full Text Available Polyphenols have recently become an important focus of study in obesity research. Oligonol is an oligomerized polyphenol, typically comprised of catechin-type polyphenols from a variety of fruits, which has been found to exhibit better bioavailability and bioreactivity than natural polyphenol compounds. Here, we demonstrated that Oligonol inhibits 3T3-L1 adipocyte differentiation by reducing adipogenic gene expression. During adipogenesis, Oligonol downregulated the mRNA levels of peroxisome proliferator-activated receptor γ (PPARγ, CCAAT/enhancer binding proteins α (C/EBPα, and δ (C/EBPδ in a dose-dependent manner and the expression of genes involved in lipid biosynthesis. The antiadipogenic effect of Oligonol appears to originate from its ability to inhibit the Akt and mammalian target of rapamycin (mTOR signaling pathway by diminishing the phosphorylation of ribosomal protein S6 kinase (p70S6K, a downstream target of mTOR and forkhead box protein O1 (Foxo1. These results suggest that Oligonol may be a potent regulator of obesity by repressing major adipogenic genes through inhibition of the Akt signaling pathway, which induces the inhibition of lipid accumulation, ultimately inhibiting adipogenesis.

  3. PI3K/Akt promotes feedforward mTORC2 activation through IKKα.

    Science.gov (United States)

    Dan, Han C; Antonia, Ricardo J; Baldwin, Albert S

    2016-04-19

    The ser-thr Akt plays a critical role in the regulation of cell survival, cell growth and proliferation, as well as energy metabolism and is dysregulated in many cancers. The regulation of Akt activity depends on the phosphorylation at two sites: (i) Thr308 in the activation loop by phosphoinositide-dependent kinase-1 (PDK1) and (ii) Ser473 hydrophobic motif at the carboxyl terminus by a second activity termed PDK2, which is the mTORC2 complex composed of mTOR, rictor, and Sin1. Previously we demonstrated that IKKα, a component of the IKK complex that controls NF-κB activation, participates in the Akt-dependent regulation of mTORC1. Here we have explored a potential involvement of IKKα in controlling Akt activity and whether this may involve mTORC2. The experiments show that IKKα associates with mTORC2 in several cancer cells in a manner dependent on PI3K/Akt activity and that IKKα positively promotes Akt phosphorylation at Ser473 and at Thr308. Moreover, IKKα enhances mTORC2 kinase activity directed to Akt on Ser473 and Akt-mediated phosphorylation of FOXO3a and GSK3β, but not other Akt-associated targets such as TSC2 and PRAS40, indicating the existence of multiple mechanisms of Akt activation in cells. In addition, loss of IKKα suppresses growth factor-induced Akt activation associated with mTORC1 inhibition. These results indicate that IKKα serves as a feedforward regulator of mTORC2 and that IKKα could serve as a key therapeutic target to block mTORC2 and Akt activation in some cancers. PMID:27027448

  4. Essential role of PH domain and leucine-rich repeat protein phosphatase 2 in Nrf2 suppression via modulation of Akt/GSK3β/Fyn kinase axis during oxidative hepatocellular toxicity.

    Science.gov (United States)

    Rizvi, F; Shukla, S; Kakkar, P

    2014-01-01

    Instances of sustained oxidative activity have been shown to involve dysregulation of Nrf2-mediated transcriptional induction; however, mechanisms warranting Nrf2-repression remain unclear. In this study, using primary rat hepatocytes, we have attempted to identify factors that may negatively influence Nrf2 survival pathway. Though studies indicate a conspicuous association between Akt and Nrf2, a confirmatory link between the two is unaddressed. On inhibiting PI3K/Akt pathway, we observed compromised activities of antioxidant and detoxification enzymes culminating in oxidative cytotoxicity. This was accompanied by reduced nuclear retention of Nrf2 and its ARE binding affinity, increased Nrf2 ubiquitination and concurrent decline in its downstream targets. Moreover, Akt inhibition enhanced nuclear translocation as well as phosphorylation of Fyn kinase, an enzyme linked to Nrf2 degradation, by relieving GSK3β from phosphorylation-mediated repression. The involvement of Akt and Fyn kinase in influencing Nrf2 signaling was further confirmed in oxidatively stressed hepatocytes by using tert-butyl hydroperoxide (tBHP). tBHP-induced decrease in Nrf2 levels was associated with enhanced Fyn kinase phosphorylation, Fyn kinase nuclear translocation and decreased levels of phosphorylated GSK3β(Ser9) in a time-dependent manner. Interestingly, tBHP induced site-specific deactivation of Akt as only Akt(Ser473) phosphorylation was observed to be affected. Further, protein expression as well as nuclear localization of PHLPP2, a phosphatase specific for Akt(Ser473), was found to be significantly enhanced in tBHP-stressed hepatocytes. Silencing of PHLPP2 not only resulted in considerable restoration of Nrf2 signaling, enhanced Nrf2-ARE binding and reduced Nrf2 ubiquitination but also significantly suppressed tBHP-induced ROS generation and alterations in mitochondrial permeability. We infer that cellular PHLPP2 levels may aggravate oxidative toxicity by suppressing Nrf2/ARE

  5. Phosphoinositide 3-kinase/Akt Pathway Mediates Fip1-like1-platelet-derived Growth Factor Receptor α-induced Cell Infiltration and Activation: Possible Molecular Mechanism for the Malignant Phenotype of Chronic Eosinophilic Leukemia

    Directory of Open Access Journals (Sweden)

    Bin Li

    2015-01-01

    Full Text Available The fip1-like1/platelet-derived growth factor receptor-α fusion gene (F/P is responsible for 14-60% cases of hypereosinophilia syndrome (HES, also known as F/P-positive chronic eosinophilic leukemia (F/P(+ CEL. The major pathogenesis of F/P(+ CEL is known to involve migration and activation of mast cells and eosinophils, leading to severe multi-organ dysfunction, but the mechanism was still unclear. Phosphoinositide 3-kinase (PI3K and serine-threonine protein kinase Akt have been reported to be targets of F/P in the F/P-promoted cell proliferation. They are extensively involved in the migration and adhesion of hematopoietic stem/progenitor cells, and also control cell invasion in some leukemias. The PI3K/Akt pathway is involved in eosinophil/neutrophil activation and infiltration; its possible role in regulating F/P induced cytotoxicity and upregulation of A4-integrin in eosinophils, and inducing eosinophil activation through controlling F/P-induced Nuclear factor-kB activity. Akt was recently shown to be stimulated by F/P, synergistically with stem cell factor, resulting in the induction of MCs migration and excessive activation. PI3K/Akt pathway is also a principal mediator of interleukin-5 (IL-5-induced signal transduction promoting eosinophil trafficking and degranulation, whereas IL-5 is a necessary cytokine for F/P-mediated CEL development. We, therefore, propose the hypothesis that the PI3K/Akt pathway might be vital downstream of F/P to induce target cell activation and tissue infiltration, resulting in the malignant phenotype seen in F/P(+ CEL.

  6. Downstream in Mawrth Valles

    Science.gov (United States)

    2005-01-01

    [figure removed for brevity, see original site] The THEMIS VIS camera is capable of capturing color images of the Martian surface using five different color filters. In this mode of operation, the spatial resolution and coverage of the image must be reduced to accommodate the additional data volume produced from using multiple filters. To make a color image, three of the five filter images (each in grayscale) are selected. Each is contrast enhanced and then converted to a red, green, or blue intensity image. These three images are then combined to produce a full color, single image. Because the THEMIS color filters don't span the full range of colors seen by the human eye, a color THEMIS image does not represent true color. Also, because each single-filter image is contrast enhanced before inclusion in the three-color image, the apparent color variation of the scene is exaggerated. Nevertheless, the color variation that does appear is representative of some change in color, however subtle, in the actual scene. Note that the long edges of THEMIS color images typically contain color artifacts that do not represent surface variation. This false color image is from further downstream in Mawrth Valles than yesterday's image. The channel here is at the end of the vallis. This image was collected during the Northern Spring season. Image information: VIS instrument. Latitude 26.7, Longitude 340.2 East (19.8 West). 37 meter/pixel resolution. Note: this THEMIS visual image has not been radiometrically nor geometrically calibrated for this preliminary release. An empirical correction has been performed to remove instrumental effects. A linear shift has been applied in the cross-track and down-track direction to approximate spacecraft and planetary motion. Fully calibrated and geometrically projected images will be released through the Planetary Data System in accordance with Project policies at a later time. NASA's Jet Propulsion Laboratory manages the 2001 Mars Odyssey

  7. The Akt1/IL-6/STAT3 pathway regulates growth of lung tumor initiating cells.

    Science.gov (United States)

    Malanga, Donatella; De Marco, Carmela; Guerriero, Ilaria; Colelli, Fabiana; Rinaldo, Nicola; Scrima, Marianna; Mirante, Teresa; De Vitis, Claudia; Zoppoli, Pietro; Ceccarelli, Michele; Riccardi, Miriam; Ravo, Maria; Weisz, Alessandro; Federico, Antonella; Franco, Renato; Rocco, Gaetano; Mancini, Rita; Rizzuto, Antonia; Gulletta, Elio; Ciliberto, Gennaro; Viglietto, Giuseppe

    2015-12-15

    Here we report that the PI3K/Akt1/IL-6/STAT3 signalling pathway regulates generation and stem cell-like properties of Non-Small Cell Lung Cancer (NSCLC) tumor initiating cells (TICs). Mutant Akt1, mutant PIK3CA or PTEN loss enhances formation of lung cancer spheroids (LCS), self-renewal, expression of stemness markers and tumorigenic potential of human immortalized bronchial cells (BEAS-2B) whereas Akt inhibition suppresses these activities in established (NCI-H460) and primary NSCLC cells. Matched microarray analysis of Akt1-interfered cells and LCSs identified IL-6 as a critical target of Akt signalling in NSCLC TICs. Accordingly, suppression of Akt in NSCLC cells decreases IL-6 levels, phosphorylation of IkK and IkB, NF-kB transcriptional activity, phosphorylation and transcriptional activity of STAT3 whereas active Akt1 up-regulates them. Exposure of LCSs isolated from NSCLC cells to blocking anti-IL-6 mAbs, shRNA to IL-6 receptor or to STAT3 markedly reduces the capability to generate LCSs, to self-renew and to form tumors, whereas administration of IL-6 to Akt-interfered cells restores the capability to generate LCSs. Finally, immunohistochemical studies in NSCLC patients demonstrated a positive correlative trend between activated Akt, IL-6 expression and STAT3 phosphorylation (n = 94; p cells in NSCLC.

  8. Protein kinase B/Akt1 inhibits autophagy by down-regulating UVRAG expression

    Energy Technology Data Exchange (ETDEWEB)

    Yang, Wonseok; Ju, Ji-hyun; Lee, Kyung-min; Nam, KeeSoo; Oh, Sunhwa [Department of Life Science, College of Natural Science, Hanyang University, 17 Haengdang-dong, Seongdong-gu, Seoul 133-791 (Korea, Republic of); Shin, Incheol, E-mail: incheol@hanyang.ac.kr [Department of Life Science, College of Natural Science, Hanyang University, 17 Haengdang-dong, Seongdong-gu, Seoul 133-791 (Korea, Republic of)

    2013-02-01

    Autophagy, or autophagocytosis, is a selective intracellular degradative process involving the cell's own lysosomal apparatus. An essential component in cell development, homeostasis, repair and resistance to stress, autophagy may result in either cell death or survival. The targeted region of the cell is sequestered within a membrane structure, the autophagosome, for regulation of the catabolic process. A key factor in both autophagosome formation and autophagosome maturation is a protein encoded by the ultraviolet irradiation resistance-associated gene (UVRAG). Conversely, the serine/threonine-specific protein kinase B (PKB, also known as Akt), which regulates survival in various cancers, inhibits autophagy through mTOR activation. We found that Akt1 may also directly inhibit autophagy by down-regulating UVRAG both in a 293T transient transfection system and breast cancer cells stably expressing Akt1. The UVRAG with mutations at putative Akt1-phosphorylation sites were still inhibited by Akt1, and dominant-negative Akt1 also inhibited UVRAG expression, suggesting that Akt1 down-regulates UVRAG by a kinase activity-independent mechanism. We showed that Akt1 overexpression in MDA-MB-231 breast cancer cells down-regulated UVRAG transcription. Cells over-expressing Akt1 were more resistant than control cells to ultraviolet light-induced autophagy and exhibited the associated reduction in cell viability. Levels of the autophagosome indicator protein LC3B-II and mRFP-GFP-LC3 were reduced in cells that over-expressing Akt1. Inhibiting Akt1 by siRNA or reintroducing UVRAG gene rescued the level of LC3B-II in UV-irradiation. Altogether, these data suggest that Akt1 may inhibit autophagy by decreasing UVRAG expression, which also sensitizes cancer cells to UV irradiation.

  9. Ape1/Ref-1 Stimulates GDNF/GFRalpha1-mediated Downstream Signaling and Neuroblastoma Proliferation.

    Science.gov (United States)

    Kang, Mi-Young; Kim, Kweon Young; Yoon, Young; Kang, Yoonsung; Kim, Hong Beum; Youn, Cha Kyung; Kim, Dong-Hui; Kim, Mi-Hwa

    2009-10-01

    We previously reported that glial cell line-derived neurotropic factor (GDNF) receptor alpha1 (GFRalpha1) is a direct target of apurinic/apyrimidinic endonuclease 1 (Ape1/Ref-1). In the present study, we further analyzed the physiological roles of Ape1/Ref-1-induced GFRalpha1 expression in Neuro2a mouse neuroblastoma cells. Ape1/Ref-1 expression caused the clustering of GFRalpha1 immunoreactivity in lipid rafts in response to GDNF. We also found that Ret, a downstream target of GFRalpha1, was functionally activated by GDNF in Ape1/Ref-1-expressing cells. Moreover, GDNF promoted the proliferation of Ape1/Ref-1-expressing Neuro2a cells. Furthermore, GFRalpha1-specific RNA experiments demonstrated that the downregulation of GFRalpha1 by siRNA in Ape1/Ref-1-expressing cells impaired the ability of GDNF to phosphorylate Akt and PLCgamma-1 and to stimulate cellular proliferation. These results show an association between Ape1/Ref-1 and GDNF/GFRalpha signaling, and suggest a potential molecular mechanism for the involvement of Ape1/Ref-1 in neuronal proliferation.

  10. AKT3 controls mitochondrial biogenesis and autophagy via regulation of the major nuclear export protein CRM-1.

    Science.gov (United States)

    Corum, Daniel G; Tsichlis, Philip N; Muise-Helmericks, Robin C

    2014-01-01

    Our previous work has shown that Akt3 is required for mitochondrial biogenesis in primary human endothelial cells (ECs) and in Akt3-null mice; Akt3 affects subcellular localization of peroxisome proliferator-activated receptor γ coactivator-1 (PGC-1α), the master regulator of mitochondrial biogenesis. The purpose of this study is to determine the mechanism by which Akt3 controls the subcellular distribution of PGC-1α and to explore the effect on mitochondrial biogenesis and turnover during angiogenesis. Here we use standard biochemical analyses and Akt3-knockdown strategies to show that Akt3 controls the stabilization of chromosome maintenance region-1 (CRM-1), the major nuclear export receptor. Site-directed mutagenesis and association analyses show that PGC-1α nuclear export is CRM-1 dependent. Akt3 knockdown and CRM-1 overexpression cause 3-fold reductions in PGC-1α target gene expression, compared to control levels. Akt3 inhibition causes autophagy, as measured by autophagosome formation, in a CRM-1-dependent, Akt1/mTOR-independent pathway. In vivo, Akt3-null and heterozygous mice show dose-dependent decreases in angiogenesis compared to wild-type littermates (~5- and 2.5-fold decreases, respectively), as assessed by Matrigel plug assays. This correlates with an ~1.5-fold decrease in mitochondrial Cox IV expression. Our studies suggest that Akt3 is a regulator of mitochondrial dynamics in the vasculature via regulation of CRM-1-dependent nuclear export.

  11. Downstream product marketing

    International Nuclear Information System (INIS)

    Petro-Canada's views regarding energy deregulation were presented. Their experience with gasoline retailing and their response to competition was also discussed. Petro-Canada regards deregulation as a good thing in terms of its promotion of fair competition. Industrial and residential consumers of energy will benefit from more efficient and innovative service. Today's consumers are very well informed about existing energy products and while they are incredibly price sensitive, they still want to buy value. The relevance of brand names in a competitive market was examined. One reaction to the demanding customer has been to augment the base product (the fuel) with other things, such as convenience store items, car washes, specialized bay businesses and promotional offerings. Petro-Canada has been successful in improving sales volumes with these sales strategies, and profits achieved indicate good target audience acceptance. Shareholders and the board of directors also appear to be in agreement with the Corporation's performance to date

  12. Molecular characterization of the encoding regions and tissue expression analyses for 3 novel buffalo AKT genes, AKT1, AKT2, and AKT3

    OpenAIRE

    Wu, Chunfeng; LIU, Lixian; HUO, Jinlong; Li, Dalin; YUAN, Yueyun; Yuan,Feng; Miao, Yongwang

    2014-01-01

    The objective of this study was to obtain the complete coding sequences (CDSs) of 3 buffalo genes (AKT1, AKT2, and AKT3) using reverse-transcriptase polymerase chain reaction and to depict their molecular characterizations and tissue expression patterns in buffalo. The buffalo AKT1, AKT2, and AKT3 CDSs were 1443 bp, 1446 bp, and 1440 bp in length and encoded 480, 481, and 479 amino acids, respectively. Nine, 13, and 3 nucleotide differences were found in the CDSs between buffalo and other bov...

  13. Akt1 in osteoblasts and osteoclasts controls bone remodeling.

    Directory of Open Access Journals (Sweden)

    Naohiro Kawamura

    Full Text Available Bone mass and turnover are maintained by the coordinated balance between bone formation by osteoblasts and bone resorption by osteoclasts, under regulation of many systemic and local factors. Phosphoinositide-dependent serine-threonine protein kinase Akt is one of the key players in the signaling of potent bone anabolic factors. This study initially showed that the disruption of Akt1, a major Akt in osteoblasts and osteoclasts, in mice led to low-turnover osteopenia through dysfunctions of both cells. Ex vivo cell culture analyses revealed that the osteoblast dysfunction was traced to the increased susceptibility to the mitochondria-dependent apoptosis and the decreased transcriptional activity of runt-related transcription factor 2 (Runx2, a master regulator of osteoblast differentiation. Notably, our findings revealed a novel role of Akt1/forkhead box class O (FoxO 3a/Bim axis in the apoptosis of osteoblasts: Akt1 phosphorylates the transcription factor FoxO3a to prevent its nuclear localization, leading to impaired transactivation of its target gene Bim which was also shown to be a potent proapoptotic molecule in osteoblasts. The osteoclast dysfunction was attributed to the cell autonomous defects of differentiation and survival in osteoclasts and the decreased expression of receptor activator of nuclear factor-kappaB ligand (RANKL, a major determinant of osteoclastogenesis, in osteoblasts. Akt1 was established as a crucial regulator of osteoblasts and osteoclasts by promoting their differentiation and survival to maintain bone mass and turnover. The molecular network found in this study will provide a basis for rational therapeutic targets for bone disorders.

  14. IL-17 induces AKT-dependent IL-6/JAK2/STAT3 activation and tumor progression in hepatocellular carcinoma

    Directory of Open Access Journals (Sweden)

    Gu Fang-Ming

    2011-12-01

    Full Text Available Abstract Background The Th17 subset and IL-17 have been found in increased frequencies within certain tumors. However, their relevance in cancer biology remains controversial. This study aimed to clarify the biological action of IL-17 on hepatocellular carcinoma (HCC. Methods Effects and underlying molecular mechanisms of IL-17 on human HCC were explored in vitro using exogenous IL-17 stimulation and in nude mice by implanting IL-17 overexpressed HCC cells. The clinical significance of IL-17 was investigated in tissue microarrays containing HCC tissues from 323 patients following hepatectomy using immunohistochemistry. Results Although exogenous IL-17 showed no direct effect on the growth rate of HCC cells in vitro, PCR and ELISA showed that IL-17 selectively augmented the secretion of diverse proinvasive factors and transwell showed a direct promotion of invasion of HCC cells by IL-17. Furthermore, transfection of IL-17 into HCC cells significantly promoted neoangiogenesis, neutrophil recruitment and tumor growth in vivo. Using siRNA mediated knockdown of AKT and STAT3, we suggested that the effects of IL-17 were operated through activation of the AKT signaling in HCC, which resulted in IL-6 production. Then, IL-6 in turn activated JAK2/STAT3 signaling and subsequently up-regulated its downstream targets IL-8, MMP2, and VEGF. Supporting these findings, in human HCC tissues, immunostaining indicated that IL-17 expression was significantly and positively associated with STAT3 phosphorylation, neutrophil infiltration and increased tumor vascularity. The clinical significance of IL-17 was authenticated by revealing that the combination of intratumoral IL-17+ cells and phospho-STAT3 served as a better prognosticator for postoperative tumor recurrence than either marker alone. Conclusions IL-17 mediated tumor-promoting role involves a direct effect on HCC cells through IL-6/JAK2/STAT3 induction by activating the AKT pathway.

  15. Notch信号在增生性瘢痕表皮中的表达%Expression of Notch receptors, ligands and downstream target genes in epidermis of hypertrophic scar

    Institute of Scientific and Technical Information of China (English)

    夏炜; 潘宝华; 刘宾; 张曦; 马福成; 王映梅; 杨晓婷; 刘丹; 郭树忠

    2009-01-01

    Objective To study the expression of Notch receptors, ligands and downstream target genes in hypertrophic scar and normal skin, and to investigate its role in the development of hypertrophic scar. Methods By immunohistochemistry, the expression of epidermal differentiation markers-β1 integrin, keratin 14 (K14) and keratin 19 (K19), as well as Notch 1-4 and Jagged1 were examined in hypertrophic scars and normal skins. The expression of Notch downstream genes- P21 and P63 was analyzed with real-time quantitative PCR and immtmohistechemistry staining. Results Histological analysis revealed a significant epidermal thickening in the hypertrophic scars, with excessive cell layers above the basal layer. Compared to the normal epidermis, the expression of β1 integrin, K19 and K14 decreased in hypertrophic scars (P<0.05). Positive expression rate of Notch1 and Jagged1 in keratinocytes was significantly higher in hypertrophic scar than in normal skin (P<0.05), while there was no difference in Notch2 and 3 positive expression rate. Furthermore, the expression of P21 was significantly up-regulated, while the expression of P63 was down-regulated in keratinocytes of hypertrophic scar (P<0.05). Conclusions Notch signal may play an important role in hypertrophic scar pathogenesis. Over-defferentiation of Keratinocytes in hypertrophic scar may be related to the overexpression of Notch1 and Jagged1, up-regalation of P21 gene and down-regulation of P63 gene.%目的 研究Notch信号相关分子在增生性瘢痕表皮中的表达情况,探讨其是否参与增生性瘢痕的形成. 方法 收集年龄、性别、部位互为对照的增生性瘢痕和健康皮肤组织各8例.行免疫组织化学检测:①表皮分化标志物,包括整合素β1、角蛋白14(K14)和19(K19);②Notch受体1~4以及配体Jagged1.行Real-time PCR和免疫组织化学检测Notch下游基因P21和P63的表达以及定位. 结果 组织学检测发现增生性瘢痕表皮较健康表皮明显增厚,

  16. Aging-associated dysfunction of Akt/protein kinase B: S-nitrosylation and acetaminophen intervention.

    Directory of Open Access Journals (Sweden)

    Miaozong Wu

    Full Text Available BACKGROUND: Aged skeletal muscle is characterized by an increased incidence of metabolic and functional disorders, which if allowed to proceed unchecked can lead to increased morbidity and mortality. The mechanism(s underlying the development of these disorders in aging skeletal muscle are not well understood. Protein kinase B (Akt/PKB is an important regulator of cellular metabolism and survival, but it is unclear if aged muscle exhibits alterations in Akt function. Here we report a novel dysfunction of Akt in aging muscle, which may relate to S-nitrosylation and can be prevented by acetaminophen intervention. PRINCIPAL FINDINGS: Compared to 6- and 27-month rats, the phosphorylation of Akt (Ser473 and Thr308 was higher in soleus muscles of very aged rats (33-months. Paradoxically, these increases in Akt phosphorylation were associated with diminished mammalian target of rapamycin (mTOR phosphorylation, along with decreased levels of insulin receptor beta (IR-beta, phosphoinositide 3-kinase (PI3K, phosphatase and tensin homolog deleted on chromosome 10 (PTEN and phosphorylation of phosphoinositide-dependent kinase-1 (PDK1 (Ser241. In vitro Akt kinase measurements and ex vivo muscle incubation experiments demonstrated age-related impairments of Akt kinase activity, which were associated with increases in Akt S-nitrosylation and inducible nitric oxide synthase (iNOS. Impairments in Akt function occurred parallel to increases in myocyte apoptosis and decreases in myocyte size and the expression of myosin and actin. These age-related disorders were attenuated by treating aged (27-month animals with acetaminophen (30 mg/kg body weight/day for 6-months. CONCLUSIONS: These data demonstrate that Akt dysfunction and increased S-nitrosylation of Akt may contribute to age-associated disorders in skeletal muscle and that acetaminophen may be efficacious for the treatment of age-related muscle dysfunction.

  17. Isoliquiritigenin induces growth inhibition and apoptosis through downregulating arachidonic acid metabolic network and the deactivation of PI3K/Akt in human breast cancer

    Energy Technology Data Exchange (ETDEWEB)

    Li, Ying; Zhao, Haixia [Department of Pharmacology, School of Medicine, Wuhan University, Wuhan 430071 (China); Wang, Yuzhong [Key Laboratory for Oral Biomedical Engineering of Ministry of Education, School and Hospital of Stomatology, Wuhan University, Wuhan 430079 (China); Zheng, Hao; Yu, Wei; Chai, Hongyan [Department of Pharmacology, School of Medicine, Wuhan University, Wuhan 430071 (China); Zhang, Jing [Animal Experimental Center of Wuhan University, Wuhan 430071 (China); Falck, John R. [Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, TX 75390,USA (United States); Guo, Austin M. [Department of Pharmacology, School of Medicine, Wuhan University, Wuhan 430071 (China); Department of Pharmacology, New York Medical College, Valhalla, NY 10595 (United States); Yue, Jiang; Peng, Renxiu [Department of Pharmacology, School of Medicine, Wuhan University, Wuhan 430071 (China); Yang, Jing, E-mail: yangjingliu2013@163.com [Department of Pharmacology, School of Medicine, Wuhan University, Wuhan 430071 (China); Research Center of Food and Drug Evaluation, Wuhan University, Wuhan 430071 (China)

    2013-10-01

    Arachidonic acid (AA)-derived eicosanoids and its downstream pathways have been demonstrated to play crucial roles in growth control of breast cancer. Here, we demonstrate that isoliquiritigenin, a flavonoid phytoestrogen from licorice, induces growth inhibition and apoptosis through downregulating multiple key enzymes in AA metabolic network and the deactivation of PI3K/Akt in human breast cancer. Isoliquiritigenin diminished cell viability, 5-bromo-2′-deoxyuridine (BrdU) incorporation, and clonogenic ability in both MCF-7 and MDA-MB-231cells, and induced apoptosis as evidenced by an analysis of cytoplasmic histone-associated DNA fragmentation, flow cytometry and hoechst staining. Furthermore, isoliquiritigenin inhibited mRNA expression of multiple forms of AA-metabolizing enzymes, including phospholipase A2 (PLA2), cyclooxygenases (COX)-2 and cytochrome P450 (CYP) 4A, and decreased secretion of their products, including prostaglandin E{sub 2} (PGE{sub 2}) and 20-hydroxyeicosatetraenoic acid (20-HETE), without affecting COX-1, 5-lipoxygenase (5-LOX), 5-lipoxygenase activating protein (FLAP), and leukotriene B{sub 4} (LTB{sub 4}). In addition, it downregulated the levels of phospho-PI3K, phospho-PDK (Ser{sup 241}), phospho-Akt (Thr{sup 308}), phospho-Bad (Ser{sup 136}), and Bcl-x{sub L} expression, thereby activating caspase cascades and eventually cleaving poly(ADP-ribose) polymerase (PARP). Conversely, the addition of exogenous eicosanoids, including PGE{sub 2}, LTB{sub 4} and a 20-HETE analog (WIT003), and caspase inhibitors, or overexpression of constitutively active Akt reversed isoliquiritigenin-induced apoptosis. Notably, isoliquiritigenin induced growth inhibition and apoptosis of MDA-MB-231 human breast cancer xenografts in nude mice, together with decreased intratumoral levels of eicosanoids and phospho-Akt (Thr{sup 308}). Collectively, these data suggest that isoliquiritigenin induces growth inhibition and apoptosis through downregulating AA metabolic

  18. PPARβ Regulates Liver Regeneration by Modulating Akt and E2f Signaling.

    Directory of Open Access Journals (Sweden)

    Hui-Xin Liu

    Full Text Available The current study tests the hypothesis that peroxisome proliferator-activated receptor β (PPARβ has a role in liver regeneration due to its effect in regulating energy homeostasis and cell proliferation. The role of PPARβ in liver regeneration was studied using two-third partial hepatectomy (PH in Wild-type (WT and PPARβ-null (KO mice. In KO mice, liver regeneration was delayed and the number of Ki-67 positive cells reached the peak at 60 hr rather than at 36-48 hr after PH shown in WT mice. RNA-sequencing uncovered 1344 transcriptomes that were differentially expressed in regenerating WT and KO livers. About 70% of those differentially expressed genes involved in glycolysis and fatty acid synthesis pathways failed to induce during liver regeneration due to PPARβ deficiency. The delayed liver regeneration in KO mice was accompanied by lack of activation of phosphoinositide-dependent kinase 1 (PDK1/Akt. In addition, cell proliferation-associated increase of genes encoding E2f transcription factor (E2f 1-2 and E2f7-8 as well as their downstream target genes were not noted in KO livers 36-48 hr after PH. E2fs have dual roles in regulating metabolism and proliferation. Moreover, transient steatosis was only found in WT, but not in KO mice 36 hr after PH. These data suggested that PPARβ-regulated PDK1/Akt and E2f signaling that controls metabolism and proliferation is involved in the normal progression of liver regeneration.

  19. PI3K-Akt pathway: its functions and alterations in human cancer.

    Science.gov (United States)

    Osaki, M; Oshimura, M; Ito, H

    2004-11-01

    Phosphatidylinositol-3-kinase (PI3K) is a lipid kinase and generates phosphatidylinositol-3,4,5-trisphosphate (PI(3, 4, 5)P3). PI(3, 4, 5)P3 is a second messenger essential for the translocation of Akt to the plasma membrane where it is phosphorylated and activated by phosphoinositide-dependent kinase (PDK) 1 and PDK2. Activation of Akt plays a pivotal role in fundamental cellular functions such as cell proliferation and survival by phosphorylating a variety of substrates. In recent years, it has been reported that alterations to the PI3K-Akt signaling pathway are frequent in human cancer. Constitutive activation of the PI3K-Akt pathway occurs due to amplification of the PIK3C gene encoding PI3K or the Akt gene, or as a result of mutations in components of the pathway, for example PTEN (phosphatase and tensin homologue deleted on chromosome 10), which inhibit the activation of Akt. Several small molecules designed to specifically target PI3K-Akt have been developed, and induced cell cycle arrest or apoptosis in human cancer cells in vitro and in vivo . Moreover, the combination of an inhibitor with various cytotoxic agents enhances the anti-tumor efficacy. Therefore, specific inhibition of the activation of Akt may be a valid approach to treating human malignancies and overcoming the resistance of cancer cells to radiation or chemotherapy. PMID:15505410

  20. Hypoxia activates Akt and induces phosphorylation of GSK-3 in PC12 cells.

    Science.gov (United States)

    Beitner-Johnson, D; Rust, R T; Hsieh, T C; Millhorn, D E

    2001-01-01

    Akt is a serine/threonine kinase that has been shown to play a central role in promoting cell survival and opposing apoptosis. We evaluated the effect of hypoxia on Akt in rat pheochromocytoma (PC12) cells. PC12 cells were exposed to varying levels of hypoxia, including 21%, 15%, 10%, 5%, and 1% O(2). Hypoxia dramatically increased phosphorylation of Akt (Ser(473)). This effect peaked after 6 h exposure to hypoxia, but persisted strongly for up to 24 h. Phosphorylation of Akt was paralleled with a progressive increase in phosphorylation of glycogen synthase kinase-3 (GSK-3), one of its downstream substrates. The effect of hypoxia on phosphorylation of Akt was completely blocked by pretreatment of the cells with wortmannin (100 nM), indicating that this effect is mediated by phosphatidylinositol 3-kinase (P13K). In contrast, whereas hypoxia also strongly induced phosphorylation of the transcription factors CREB and EPAS1, these effects persisted in the presence of wortmannin. Thus, hypoxia regulates both P13K-dependent and P13K-independent signaling pathways. Furthermore, activation of the P13K and Akt signaling pathways may be one mechanism by which cells adapt and survive under conditions of hypoxia. PMID:11257444

  1. Shikonin Inhibits the Migration and Invasion of Human Glioblastoma Cells by Targeting Phosphorylated β-Catenin and Phosphorylated PI3K/Akt: A Potential Mechanism for the Anti-Glioma Efficacy of a Traditional Chinese Herbal Medicine

    Directory of Open Access Journals (Sweden)

    Feng-Ying Zhang

    2015-10-01

    Full Text Available Shikonin is an anthraquinone derivative extracted from the root of lithospermum. Shikonin is traditionally used in the treatment of inflammatory and infectious diseases such as hepatitis. Shikonin also inhibits proliferation and induces apoptosis in various tumors. However, the effect of shikonin on gliomas has not been fully elucidated. In the present study, we aimed to investigate the effects of shikonin on the migration and invasion of human glioblastoma cells as well as the underlying mechanisms. U87 and U251 human glioblastoma cells were treated with shikonin at 2.5, 5, and 7.5 μmol/L and cell viability, migration and invasiveness were assessed with CCK8, scratch wound healing, in vitro Transwell migration, and invasion assays. The expression and activity of matrix metalloproteinase-2 (MMP-2 and matrix metalloproteinase-9 (MMP-9 and the expression of phosphorylated β-catenin (p-β-catenin and phosphorylated PI3K/Akt were also checked. Results showed that shikonin significantly inhibited the cell proliferation, migration, invasion, and expression of MMP-2 and MMP-9 in U87 and U251 cells. The expression of p-β-catenin showed contrary trends in two cell lines. It was significantly inhibited in U87 cells and promoted in U251 cells. Results in this work indicated that shikonin displayed an inhibitory effect on the migration and invasion of glioma cells by inhibiting the expression and activity of MMP-2 and -9. In addition, shikonin also inhibited the expression of p-PI3K and p-Akt to attenuate cell migration and invasion and MMP-2 and MMP-9 expression in both cell lines, which could be reversed by the PI3K/Akt pathway agonist, insulin-like growth factor-1 (IGF-1.

  2. Shikonin Inhibits the Migration and Invasion of Human Glioblastoma Cells by Targeting Phosphorylated β-Catenin and Phosphorylated PI3K/Akt: A Potential Mechanism for the Anti-Glioma Efficacy of a Traditional Chinese Herbal Medicine.

    Science.gov (United States)

    Zhang, Feng-Ying; Hu, Yi; Que, Zhong-You; Wang, Ping; Liu, Yun-Hui; Wang, Zhen-Hua; Xue, Yi-Xue

    2015-10-09

    Shikonin is an anthraquinone derivative extracted from the root of lithospermum. Shikonin is traditionally used in the treatment of inflammatory and infectious diseases such as hepatitis. Shikonin also inhibits proliferation and induces apoptosis in various tumors. However, the effect of shikonin on gliomas has not been fully elucidated. In the present study, we aimed to investigate the effects of shikonin on the migration and invasion of human glioblastoma cells as well as the underlying mechanisms. U87 and U251 human glioblastoma cells were treated with shikonin at 2.5, 5, and 7.5 μmol/L and cell viability, migration and invasiveness were assessed with CCK8, scratch wound healing, in vitro Transwell migration, and invasion assays. The expression and activity of matrix metalloproteinase-2 (MMP-2) and matrix metalloproteinase-9 (MMP-9) and the expression of phosphorylated β-catenin (p-β-catenin) and phosphorylated PI3K/Akt were also checked. Results showed that shikonin significantly inhibited the cell proliferation, migration, invasion, and expression of MMP-2 and MMP-9 in U87 and U251 cells. The expression of p-β-catenin showed contrary trends in two cell lines. It was significantly inhibited in U87 cells and promoted in U251 cells. Results in this work indicated that shikonin displayed an inhibitory effect on the migration and invasion of glioma cells by inhibiting the expression and activity of MMP-2 and -9. In addition, shikonin also inhibited the expression of p-PI3K and p-Akt to attenuate cell migration and invasion and MMP-2 and MMP-9 expression in both cell lines, which could be reversed by the PI3K/Akt pathway agonist, insulin-like growth factor-1 (IGF-1).

  3. Arabic downstream crushes competition; Arabische downstream verpletterd concurrentie

    Energy Technology Data Exchange (ETDEWEB)

    Widdershoven, C. [Capgemini International, Utrecht (Netherlands)

    2007-07-01

    Oil producers in the Middle East have enormous advantages while the costs for resources are very low. The petrochemical industry in this region shows a considerable growth. The question, however, is whether this situation will continue for a long time. [Dutch] Veel downstream-bedrijven in het Westen hebben hun activiteiten afgebouwd wegens zeer lage of negatieve winstmarges. Op hetzelfde moment is er een exponentiele groei van de downstream-sector te zien in het Midden-Oosten, West-Afrika en Azie. De komende jaren wordt er wereldwijdvoor meer dan zeshonderd miljard dollar aan nieuwe downstream-projecten op poten gezet. Een substantieel deel van deze projecten is in het Midden-Oosten gepland, waarbij Saudi-Arabie, Qatar, Verenigde Arabische Emiraten, Iran en Egypte het voortouw nemen. De Arabische nationale olie- en gasmaatschappijen - Saudi Aramco, Qatargas, NIOC and EGPC - onderkennen dat het strategisch en financieel interessant is om deze sector in handen te krijgen.

  4. Sensitization of Cancer Cells through Reduction of Total Akt and Downregulation of Salinomycin-Induced pAkt, pGSk3β, pTSC2, and p4EBP1 by Cotreatment with MK-2206

    Directory of Open Access Journals (Sweden)

    Ae-Ran Choi

    2014-01-01

    Full Text Available MK-2206 is an inhibitor of Akt activation. It has been investigated as an anticancer drug in clinical trials assessing the potential of pAkt targeting therapy. The purpose of this study was to identify conditions that increase the sensitivity of cancer cells to MK-2206. We found that the treatment of cancer cells with a high concentration of salinomycin (Sal reduced total Akt protein levels but increased activated Akt levels. When cancer cells were cotreated with MK-2206 and Sal, both pAkt and total Akt levels were reduced. Using microscopic observation, an assessment of cleaved PARP, FACS analysis of pre-G1 region, and Hoechst staining, we found that Sal increased apoptosis of MK-2206-treated cancer cells. These results suggest that cotreatment with MK-2206 and Sal sensitizes cancer cells via reduction of both pAkt and total Akt. Furthermore, cotreatment of cancer cells with Sal and MK-2206 reduced pp70S6K, pmTOR, and pPDK1 levels. In addition, Sal-induced activation of GSK3β, TSC2, and 4EBP1 was abolished by MK-2206 cotreatment. These results suggest that cotreatment using MK-2206 and Sal could be used as a therapeutic method to sensitize cancer cells through targeting of the PI3K/Akt/mTOR pathway. Our findings may contribute to the development of MK-2206-based sensitization therapies for cancer patients.

  5. Akt Inhibitor A-443654 Interferes with Mitotic Progression by Regulating Aurora A Kinase Expression

    Directory of Open Access Journals (Sweden)

    Xuesong Liu

    2008-08-01

    Full Text Available Both Akt and Aurora A kinase have been shown to be important targets for intervention for cancer therapy. We report here that Compound A (A-443654, a specific Akt inhibitor, interferes with mitotic progression and bipolar spindle formation. Compound A induces G2/M accumulation, defects in centrosome separation, and formation of either monopolar arrays or disorganized spindles. On the basis of gene expression array studies, we identified Aurora A as one of the genes regulated transcriptionally by Akt inhibitors including Compound A. Inhibition of the phosphatidylinositol 3-kinase (PI3K/Akt pathway, either by PI3K inhibitor LY294002 or by Compound A, dramatically inhibits the promoter activity of Aurora A, whereas the mammalian target of rapamycin inhibitor has little effect, suggesting that Akt might be responsible for up-regulating Aurora A for mitotic progression. Further analysis of the Aurora A promoter region indicates that the Ets element but not the Sp1 element is required for Compound A-sensitive transcriptional control of Aurora A. Overexpression of Aurora A in cells treated with Compound A attenuates the mitotic arrest and the defects in bipolar spindle formation induced by Akt inhibition. Our studies suggest that that Akt may promote mitotic progression through the transcriptional regulation of Aurora A.

  6. The restructuring of Shell Downstream; La restructuration de Shell Downstream

    Energy Technology Data Exchange (ETDEWEB)

    Maxwell, F.

    2005-01-15

    To facing a more and more competitive environment, the Group Shell began a restructuring. While the group was organized on horizontal national lines, it is creating today an integrated downstream activity. The word of this restructuring is profit. (A.L.B.)

  7. Suppression of SPIN1-mediated PI3K-Akt pathway by miR-489 increases chemosensitivity in breast cancer.

    Science.gov (United States)

    Chen, Xu; Wang, Ya-Wen; Xing, Ai-Yan; Xiang, Shuai; Shi, Duan-Bo; Liu, Lei; Li, Yan-Xiang; Gao, Peng

    2016-08-01

    Drug resistance is one of the major obstacles for improving the prognosis of breast cancer patients. Increasing evidence has linked the association of aberrantly expressed microRNAs (miRNAs) with tumour development and progression as well as chemoresistance. Despite recent advances, there is still little known about the potential role and mechanism of miRNAs in breast cancer chemoresistance. Here we describe that 16 miRNAs were found to be significantly down-regulated and 11 up-regulated in drug-resistant breast cancer tissues compared with drug-sensitive tissues, using a miRNA microarray. The results also showed miR-489 to be one of the most down-regulated miRNAs in drug-resistant tissues and cell lines, as confirmed by miRNA microarray screening and real-time quantitative PCR. A decrease in miR-489 expression was associated with chemoresistance as well as lymph node metastasis, increased tumour size, advanced pTNM stage and poor prognosis in breast cancer. Functional analysis revealed that miR-489 increased breast cancer chemosensitivity and inhibited cell proliferation, migration and invasion, both in vitro and in vivo. Furthermore, SPIN1, VAV3, BCL2 and AKT3 were found to be direct targets of miR-489. SPIN1 was significantly elevated in drug-resistant and metastatic breast cancer tissues and inversely correlated with miR-489 expression. High expression of SPIN1 was associated with higher histological grade, lymph node metastasis, advanced pTNM stage and positive progesterone receptor (PR) status. Increased SPIN1 expression enhanced cell migration and invasion, inhibited apoptosis and partially antagonized the effects of miR-489 in breast cancer. PIK3CA, AKT, CREB1 and BCL2 in the PI3K-Akt signalling pathway, demonstrated to be elevated in drug-resistant breast cancer tissues, were identified as downstream effectors of SPIN1. It was further found that either inhibition of SPIN1 or overexpression of miR-489 suppressed the PI3K-Akt signalling pathway. These data

  8. Pharmacophore Modeling, Virtual Screening, and Molecular Docking Studies for Discovery of Novel Akt2 Inhibitors

    Directory of Open Access Journals (Sweden)

    Jia Fei, Lu Zhou, Tao Liu, Xiang-Yang Tang

    2013-01-01

    Full Text Available Akt2 is considered as a potential target for cancer therapy. In order to find novel Akt2 inhibitors which have different scaffolds, structure-based pharmacophore model and 3D-QSAR pharmacophore model were built and validated by different methods. Then, they were used for chemical databases virtual screening. The selected compounds were further analyzed and refined using drug-like filters and ADMET analysis. Finally, seven hits with different scaffolds were picked out for docking studies. These seven hits were predicted to have high inhibitory activity and good ADMET properties, they may act as novel leads for Akt2 inhibitors designing.

  9. The Metastasis Suppressor, N-MYC Downstream-regulated Gene-1 (NDRG1), Down-regulates the ErbB Family of Receptors to Inhibit Downstream Oncogenic Signaling Pathways.

    Science.gov (United States)

    Kovacevic, Zaklina; Menezes, Sharleen V; Sahni, Sumit; Kalinowski, Danuta S; Bae, Dong-Hun; Lane, Darius J R; Richardson, Des R

    2016-01-15

    N-MYC downstream-regulated gene-1 (NDRG1) is a potent growth and metastasis suppressor that acts through its inhibitory effects on a wide variety of cellular signaling pathways, including the TGF-β pathway, protein kinase B (AKT)/PI3K pathway, RAS, etc. To investigate the hypothesis that its multiple effects could be regulated by a common upstream effector, the role of NDRG1 on the epidermal growth factor receptor (EGFR) and other members of the ErbB family, namely human epidermal growth factor receptor 2 (HER2) and human epidermal growth factor receptor 3 (HER3), was examined. We demonstrate that NDRG1 markedly decreased the expression and activation of EGFR, HER2, and HER3 in response to the epidermal growth factor (EGF) ligand, while also inhibiting formation of the EGFR/HER2 and HER2/HER3 heterodimers. In addition, NDRG1 also decreased activation of the downstream MAPKK in response to EGF. Moreover, novel anti-tumor agents of the di-2-pyridylketone class of thiosemicarbazones, namely di-2-pyridylketone 4,4-dimethyl-3-thiosemicarbazone and di-2-pyridylketone 4-cyclohexyl-4-methyl-3-thiosemicarbazone, which markedly up-regulate NDRG1, were found to inhibit EGFR, HER2, and HER3 expression and phosphorylation in cancer cells. However, the mechanism involved appeared dependent on NDRG1 for di-2-pyridylketone 4,4-dimethyl-3-thiosemicarbazone, but was independent of this metastasis suppressor for di-2-pyridylketone 4-cyclohexyl-4-methyl-3-thiosemicarbazone. This observation demonstrates that small structural changes in thiosemicarbazones result in marked alterations in molecular targeting. Collectively, these results reveal a mechanism for the extensive downstream effects on cellular signaling attributed to NDRG1. Furthermore, this study identifies a novel approach for the treatment of tumors resistant to traditional EGFR inhibitors. PMID:26534963

  10. Salinomycin induces cell death and differentiation in head and neck squamous cell carcinoma stem cells despite activation of epithelial-mesenchymal transition and Akt

    International Nuclear Information System (INIS)

    Cancer stem cells (CSC) are believed to play a crucial role in cancer recurrence due to their resistance to conventional chemotherapy and capacity for self-renewal. Recent studies have reported that salinomycin, a livestock antibiotic, selectively targets breast cancer stem cells 100-fold more effectively than paclitaxel. In our study we sought to determine the effects of salinomycin on head and neck squamous cell carcinoma (HNSCC) stem cells. MTS and TUNEL assays were used to study cell proliferation and apoptosis as a function of salinomycin exposure in JLO-1, a putative HNSCC stem cell culture. MTS and trypan blue dye exclusion assays were performed to investigate potential drug interactions between salinomycin and cisplatin or paclitaxel. Stem cell-like phenotype was measured by mRNA expression of stem cell markers, sphere-forming capacity, and matrigel invasion assays. Immunoblotting was also used to determine expression of epithelial-mesenchymal transition (EMT) markers and Akt phosphorylation. Arrays by Illumina, Inc. were used to profile microRNA expression as a function of salinomycin dose. In putative HNSCC stem cells, salinomycin was found to significantly inhibit cell viability, induce a 71.5% increase in levels of apoptosis, elevate the Bax/Bcl-2 ratio, and work synergistically with cisplatin and paclitaxel in inducing cell death. It was observed that salinomycin significantly inhibited sphere forming-capability and repressed the expression of CD44 and BMI-1 by 3.2-fold and 6.2-fold, respectively. Furthermore, salinomycin reduced invasion of HNSCC stem cells by 2.1 fold. Contrary to expectations, salinomycin induced the expression of EMT markers Snail, vimentin, and Zeb-1, decreased expression of E-cadherin, and also induced phosphorylation of Akt and its downstream targets GSK3-β and mTOR. These results demonstrate that in HNSCC cancer stem cells, salinomycin can cause cell death and decrease stem cell properties despite activation of both EMT and

  11. Downstream fining - a literature review -

    NARCIS (Netherlands)

    Frings, R.M.

    2004-01-01

    In this report a detailed overview is given of the present knowledge about downstream fining. The influence of abrasion (chapter 2), selective transport (chapter 3), sediment addition, extraction and redistribution (chapter 4) is described and discussed. Then some attention is attained to a very str

  12. Mutual inhibition of insulin signaling and PHLPP-1 determines cardioprotective efficiency of Akt in aged heart.

    Science.gov (United States)

    Xing, Yuan; Sun, Wanqing; Wang, Yishi; Gao, Feng; Ma, Heng

    2016-05-01

    Insulin protects cardiomyocytes from myocardial ischemia/reperfusion (MI/R) injury through activating Akt. However, phosphatase PHLPP-1 (PH domain leucine-rich repeat protein phosphatase-1) dephosphorylates and inactivates Akt. The balanced competitive interaction of insulin and PHLPP-1 has not been directly examined. In this study, we have identified the effect of mutual inhibition of insulin signaling and PHLPP-1 on the cardioprotective efficiency of Akt in aged heart. Young (3 months) and aged (20 months) Sprague Dawley (SD) rats were subjected to MI/Rin vivo. The PHLPP-1 level was higher in aged vs. young hearts at base. But, insulin treatment failed to decrease PHLPP-1 level during reperfusion in the aged hearts. Consequently, the cardioprotection of insulin-induced Akt activation was impaired in aged hearts, resulting in more susceptible to MI/R injury. In cultured rat ventricular myocytes, PHLPP-1 knockdown significantly enhanced insulin-induced Akt phosphorylation and reduced simulated hypoxia/reoxygenation-induced apoptosis. Contrary, PHLPP-1 overexpression terminated Akt phosphorylation and deteriorated myocytes apoptosis. Using in vivo aged animal models, we confirmed that cardiac PHLPP-1 knockdown or enhanced insulin sensitivity by exercise training dramatically increased insulin-induced Akt phosphorylation. Specifically, MI/R-induced cardiomyocyte apoptosis and infarct size were decreased and cardiac function was increased. More importantly, we found that insulin regulated the degradation of PHLPP-1 and insulin treatment could enhance the binding between PHLPP-1 and β-transducin repeat-containing protein (β-TrCP) to target for ubiquitin-dependent degradation. Altogether, we have identified a new mechanism by which insulin suppresses PHLPP-1 to enhance Akt activation. But, aged heart possesses lower insulin effectiveness and fails to decrease PHLPP-1 during MI/R, which subsequently limited Akt activity and cardioprotection. PHLPP-1 could be a

  13. Methylglyoxal mediates adipocyte proliferation by increasing phosphorylation of Akt1.

    Directory of Open Access Journals (Sweden)

    Xuming Jia

    Full Text Available Methylglyoxal (MG is a highly reactive metabolite physiologically presented in all biological systems. The effects of MG on diabetes and hypertension have been long recognized. In the present study, we investigated the potential role of MG in obesity, one of the most important factors to cause metabolic syndrome. An increased MG accumulation was observed in the adipose tissue of obese Zucker rats. Cell proliferation assay showed that 5-20 µM of MG stimulated the proliferation of 3T3-L1 cells. Further study suggested that accumulated-MG stimulated the phosphorylation of Akt1 and its targets including p21 and p27. The activated Akt1 then increased the activity of CDK2 and accelerated the cell cycle progression of 3T3-L1 cells. The effects of MG were efficiently reversed by advanced glycation end product (AGE breaker alagebrium and Akt inhibitor SH-6. In summary, our study revealed a previously unrecognized effect of MG in stimulating adipogenesis by up-regulation of Akt signaling pathway and this mechanism might offer a new approach to explain the development of obesity.

  14. AKT induces erythroid-cell maturation of JAK2-deficient fetal liver progenitor cells and is required for Epo regulation of erythroid-cell differentiation.

    Science.gov (United States)

    Ghaffari, Saghi; Kitidis, Claire; Zhao, Wei; Marinkovic, Dragan; Fleming, Mark D; Luo, Biao; Marszalek, Joseph; Lodish, Harvey F

    2006-03-01

    AKT serine threonine kinase of the protein kinase B (PKB) family plays essential roles in cell survival, growth, metabolism, and differentiation. In the erythroid system, AKT is known to be rapidly phosphorylated and activated in response to erythropoietin (Epo) engagement of Epo receptor (EpoR) and to sustain survival signals in cultured erythroid cells. Here we demonstrate that activated AKT complements EpoR signaling and supports erythroid-cell differentiation in wild-type and JAK2-deficient fetal liver cells. We show that erythroid maturation of AKT-transduced cells is not solely dependent on AKT-induced cell survival or proliferation signals, suggesting that AKT transduces also a differentiation-specific signal downstream of EpoR in erythroid cells. Down-regulation of expression of AKT kinase by RNA interference, or AKT activity by expression of dominant negative forms, inhibits significantly fetal liver-derived erythroid-cell colony formation and gene expression, demonstrating that AKT is required for Epo regulation of erythroid-cell maturation.

  15. Impaired Striatal Akt Signaling Disrupts Dopamine Homeostasis and Increases Feeding

    OpenAIRE

    Nicole Speed; Christine Saunders; Davis, Adeola R.; W Anthony Owens; Heinrich J.G. Matthies; Sanaz Saadat; Jack P Kennedy; Vaughan, Roxanne A.; Neve, Rachael L.; Lindsley, Craig W.; Russo, Scott J.; Daws, Lynette C.; Niswender, Kevin D.; Aurelio Galli

    2011-01-01

    BACKGROUND: The prevalence of obesity has increased dramatically worldwide. The obesity epidemic begs for novel concepts and therapeutic targets that cohesively address "food-abuse" disorders. We demonstrate a molecular link between impairment of a central kinase (Akt) involved in insulin signaling induced by exposure to a high-fat (HF) diet and dysregulation of higher order circuitry involved in feeding. Dopamine (DA) rich brain structures, such as striatum, provide motivation stimuli for fe...

  16. Activation of Akt by advanced glycation end products (AGEs: involvement of IGF-1 receptor and caveolin-1.

    Directory of Open Access Journals (Sweden)

    Su-Jung Yang

    Full Text Available Diabetes is characterized by chronic hyperglycemia, which in turn facilitates the formation of advanced glycation end products (AGEs. AGEs activate signaling proteins such as Src, Akt and ERK1/2. However, the mechanisms by which AGEs activate these kinases remain unclear. We examined the effect of AGEs on Akt activation in 3T3-L1 preadipocytes. Addition of AGEs to 3T3-L1 cells activated Akt in a dose- and time-dependent manner. The AGEs-stimulated Akt activation was blocked by a PI3-kinase inhibitor LY 294002, Src inhibitor PP2, an antioxidant NAC, superoxide scavenger Tiron, or nicotinamide adenine dinucleotide phosphate (NAD(PH oxidase inhibitor DPI, suggesting the involvement of Src and NAD(PH oxidase in the activation of PI3-kinase-Akt pathway by AGEs. AGEs-stimulated Src tyrosine phosphorylation was inhibited by NAC, suggesting that Src is downstream of NAD(PH oxidase. The AGEs-stimulated Akt activity was sensitive to Insulin-like growth factor 1 receptor (IGF-1R kinase inhibitor AG1024. Furthermore, AGEs induced phosphorylation of IGF-1 receptorβsubunit (IGF-1Rβ on Tyr1135/1136, which was sensitive to PP2, indicating that AGEs stimulate Akt activity by transactivating IGF-1 receptor. In addition, the AGEs-stimulated Akt activation was attenuated by β-methylcyclodextrin that abolishes the structure of caveolae, and by lowering caveolin-1 (Cav-1 levels with siRNAs. Furthermore, addition of AGEs enhanced the interaction of phospho-Cav-1 with IGF-1Rβ and transfection of 3T3-L1 cells with Cav-1 Y14F mutants inhibited the activation of Akt by AGEs. These results suggest that AGEs activate NAD(PH oxidase and Src which in turn phosphorylates IGF-1 receptor and Cav-1 leading to activation of IGF-1 receptor and the downstream Akt in 3T3-L1 cells. AGEs treatment promoted the differentiation of 3T3-L1 preadipocytes and addition of AG1024, LY 294002 or Akt inhibitor attenuated the promoting effect of AGEs on adipogenesis, suggesting that IGF-1

  17. AKT regulates NPM dependent ARF localization and p53mut stability in tumors.

    Science.gov (United States)

    Hamilton, Garth; Abraham, Aswin G; Morton, Jennifer; Sampson, Oliver; Pefani, Dafni E; Khoronenkova, Svetlana; Grawenda, Anna; Papaspyropoulos, Angelos; Jamieson, Nigel; McKay, Colin; Sansom, Owen; Dianov, Grigory L; O'Neill, Eric

    2014-08-15

    Nucleophosmin (NPM) is known to regulate ARF subcellular localization and MDM2 activity in response to oncogenic stress, though the precise mechanism has remained elusive. Here we describe how NPM and ARF associate in the nucleoplasm to form a MDM2 inhibitory complex. We find that oligomerization of NPM drives nucleolar accumulation of ARF. Moreover, the formation of NPM and ARF oligomers antagonizes MDM2 association with the inhibitory complex, leading to activation of MDM2 E3-ligase activity and targeting of p53. We find that AKT phosphorylation of NPM-Ser48 prevents oligomerization that results in nucleoplasmic localization of ARF, constitutive MDM2 inhibition and stabilization of p53. We also show that ARF promotes p53 mutant stability in tumors and suppresses p73 mediated p21 expression and senescence. We demonstrate that AKT and PI3K inhibitors may be effective in treatment of therapeutically resistant tumors with elevated AKT and carrying gain of function mutations in p53. Our results show that the clinical candidate AKT inhibitor MK-2206 promotes ARF nucleolar localization, reduced p53(mut) stability and increased sensitivity to ionizing radiation in a xenograft model of pancreatic cancer. Analysis of human tumors indicates that phospho-S48-NPM may be a useful biomarker for monitoring AKT activity and in vivo efficacy of AKT inhibitor treatment. Critically, we propose that combination therapy involving PI3K-AKT inhibitors would benefit from a patient stratification rationale based on ARF and p53(mut) status.

  18. Continuous downstream processing of biopharmaceuticals.

    Science.gov (United States)

    Jungbauer, Alois

    2013-08-01

    Continuous manufacturing has been applied in many different industries but has been pursued reluctantly in biotechnology where the batchwise process is still the standard. A shift to continuous operation can improve productivity of a process and substantially reduce the footprint. Continuous operation also allows robust purification of labile biomolecules. A full set of unit operations is available to design continuous downstream processing of biopharmaceuticals. Chromatography, the central unit operation, is most advanced in respect to continuous operation. Here, the problem of 'batch' definition has been solved. This has also paved the way for implementation of continuous downstream processing from a regulatory viewpoint. Economic pressure, flexibility, and parametric release considerations will be the driving force to implement continuous manufacturing strategies in future.

  19. Activation of AKT negatively regulates the pro-apoptotic function of death-associated protein kinase 3 (DAPK3) in prostate cancer.

    Science.gov (United States)

    Das, Trinath P; Suman, Suman; Papu John, A M Sashi; Pal, Deeksha; Edwards, Angelena; Alatassi, Houda; Ankem, Murali K; Damodaran, Chendil

    2016-07-28

    The activation of AKT governs many signaling pathways and promotes cell growth and inhibits apoptosis in human malignancies including prostate cancer (CaP). Here, we investigated the molecular association between AKT activation and the function of death-associated protein kinase 3 (DAPK3) in CaP. An inverse correlation of pAKT and DAPK3 expression was seen in a panel of CaP cell lines. Inhibition of AKT by wortmannin/LY294002 or overexpression of DAPK3 reverts the proliferative function of AKT in CaP cells. On the other hand, ectopic expression of AKT inhibited DAPK3 function and induced proliferation of CaP cells. In addition, AKT over-expressed tumors exhibit aggressive growth when compared to control vector in xenograft models. The immunohistochemistry results revealed a down-regulation of DAPK3 expression in AKT over-expressed tumors as compared to control tumors. Finally, we examined the expression pattern of AKT and DAPK3 in human CaP specimens - the expected gradual increase and nuclear localization of pAKT was seen in higher Gleason score samples versus benign hyperplasia (BPH). On the contrary, reduced expression of DAPK3 was seen in higher Gleason stages versus BPH. This suggests that inhibition of DAPK3 may be a contributing factor to the carcinogenesis of the prostate. Understanding the mechanism by which AKT negatively regulates DAPK3 function may suggest whether DAPK3 can be a therapeutic target for CaP. PMID:27126362

  20. Modulation of the Akt Pathway Reveals a Novel Link with PERK/eIF2α, which Is Relevant during Hypoxia

    Science.gov (United States)

    Sánchez, Manuel Alejandro; Urrutia, Carolina; Grande, Alicia; Risso, Guillermo; Srebrow, Anabella; Alfaro, Jennifer; Colman-Lerner, Alejandro

    2013-01-01

    The unfolded protein response (UPR) and the Akt signaling pathway share several regulatory functions and have the capacity to determine cell outcome under specific conditions. However, both pathways have largely been studied independently. Here, we asked whether the Akt pathway regulates the UPR. To this end, we used a series of chemical compounds that modulate PI3K/Akt pathway and monitored the activity of the three UPR branches: PERK, IRE1 and ATF6. The antiproliferative and antiviral drug Akt-IV strongly and persistently activated all three branches of the UPR. We present evidence that activation of PERK/eIF2α requires Akt and that PERK is a direct Akt target. Chemical activation of this novel Akt/PERK pathway by Akt-IV leads to cell death, which was largely dependent on the presence of PERK and IRE1. Finally, we show that hypoxia-induced activation of eIF2α requires Akt, providing a physiologically relevant condition for the interaction between Akt and the PERK branch of the UPR. These data suggest the UPR and the Akt pathway signal to one another as a means of controlling cell fate. PMID:23922774

  1. Modulation of the Akt pathway reveals a novel link with PERK/eIF2α, which is relevant during hypoxia.

    Directory of Open Access Journals (Sweden)

    Matías Blaustein

    Full Text Available The unfolded protein response (UPR and the Akt signaling pathway share several regulatory functions and have the capacity to determine cell outcome under specific conditions. However, both pathways have largely been studied independently. Here, we asked whether the Akt pathway regulates the UPR. To this end, we used a series of chemical compounds that modulate PI3K/Akt pathway and monitored the activity of the three UPR branches: PERK, IRE1 and ATF6. The antiproliferative and antiviral drug Akt-IV strongly and persistently activated all three branches of the UPR. We present evidence that activation of PERK/eIF2α requires Akt and that PERK is a direct Akt target. Chemical activation of this novel Akt/PERK pathway by Akt-IV leads to cell death, which was largely dependent on the presence of PERK and IRE1. Finally, we show that hypoxia-induced activation of eIF2α requires Akt, providing a physiologically relevant condition for the interaction between Akt and the PERK branch of the UPR. These data suggest the UPR and the Akt pathway signal to one another as a means of controlling cell fate.

  2. Protein S blocks the extrinsic apoptotic cascade in tissue plasminogen activator/N-methyl D-aspartate-treated neurons via Tyro3-Akt-FKHRL1 signaling pathway

    Directory of Open Access Journals (Sweden)

    Freeman Robert S

    2011-02-01

    Full Text Available Abstract Background Thrombolytic therapy with tissue plasminogen activator (tPA benefits patients with acute ischemic stroke. However, tPA increases the risk for intracerebral bleeding and enhances post-ischemic neuronal injury if administered 3-4 hours after stroke. Therefore, combination therapies with tPA and neuroprotective agents have been considered to increase tPA's therapeutic window and reduce toxicity. The anticoagulant factor protein S (PS protects neurons from hypoxic/ischemic injury. PS also inhibits N-methyl-D-aspartate (NMDA excitotoxicity by phosphorylating Bad and Mdm2 which blocks the downstream steps in the intrinsic apoptotic cascade. To test whether PS can protect neurons from tPA toxicity we studied its effects on tPA/NMDA combined injury which in contrast to NMDA alone kills neurons by activating the extrinsic apoptotic pathway. Neither Bad nor Mdm2 which are PS's targets and control the intrinsic apoptotic pathway can influence the extrinsic cascade. Thus, based on published data one cannot predict whether PS can protect neurons from tPA/NMDA injury by blocking the extrinsic pathway. Neurons express all three TAM (Tyro3, Axl, Mer receptors that can potentially interact with PS. Therefore, we studied whether PS can activate TAM receptors during a tPA/NMDA insult. Results We show that PS protects neurons from tPA/NMDA-induced apoptosis by suppressing Fas-ligand (FasL production and FasL-dependent caspase-8 activation within the extrinsic apoptotic pathway. By transducing neurons with adenoviral vectors expressing the kinase-deficient Akt mutant AktK179A and a triple FKHRL1 Akt phosphorylation site mutant (FKHRL1-TM, we show that Akt activation and Akt-mediated phosphorylation of FKHRL1, a member of the Forkhead family of transcription factors, are critical for FasL down-regulation and caspase-8 inhibition. Using cultured neurons from Tyro3, Axl and Mer mutants, we show that Tyro3, but not Axl and Mer, mediates

  3. Sporoderm-Broken Spores of Ganoderma lucidum Inhibit the Growth of Lung Cancer: Involvement of the Akt/mTOR Signaling Pathway.

    Science.gov (United States)

    Chen, Yali; Lv, Jing; Li, Kun; Xu, Jing; Li, Mingyan; Zhang, Wen; Pang, Xiufeng

    2016-10-01

    The sporoderm-broken spores of Ganoderma lucidum (SBGS) and their extracts exhibited a wide range of biological activities. In the present study, we prepare ethanol/ethanol extract (E/E-SBGS) and ethanol/aqueous extract (E/A-SBGS) from SBGS and examine their antitumor activities against human lung cancer. Our results showed that E/E-SBGS, not E/A-SBGS, inhibited the survival and migration of lung cancer cells in a dose-dependent manner. E/E-SBGS arrested cell cycle at G2/M phase and triggered apoptosis by decreasing the expression and activity of cell cycle regulators, cyclin B1 and cdc2, as well as anti-apoptotic proteins, Bcl-2 and Bcl-xl. Consequently, colony formation of lung cancer cells was markedly blocked by E/E-SBGS at subtoxic concentrations. Oral administration of both E/E-SBGS and SBGS significantly suppressed tumor volume and tumor weight without gross toxicity in mice. Mechanism study showed that E/E-SBGS dose-dependently suppressed the activation of Akt, the mammalian target of rapamycin (mTOR) and their downstream molecules S6 kinase and 4E-BP1 in treated tumor cells. Taken together, these results indicate that the ethanol extract of sporoderm-broken spores of G. lucidum suppresses the growth of human lung cancer, at least in part, through inhibition of the Akt/mTOR signaling pathway, suggesting its potential role in cancer treatments.

  4. Targeting the Metastasis Suppressor, N-Myc Downstream Regulated Gene-1, with Novel Di-2-Pyridylketone Thiosemicarbazones: Suppression of Tumor Cell Migration and Cell-Collagen Adhesion by Inhibiting Focal Adhesion Kinase/Paxillin Signaling.

    Science.gov (United States)

    Wangpu, Xiongzhi; Lu, Jiaoyang; Xi, Ruxing; Yue, Fei; Sahni, Sumit; Park, Kyung Chan; Menezes, Sharleen; Huang, Michael L H; Zheng, Minhua; Kovacevic, Zaklina; Richardson, Des R

    2016-05-01

    Metastasis is a complex process that is regulated by multiple signaling pathways, with the focal adhesion kinase (FAK)/paxillin pathway playing a major role in the formation of focal adhesions and cell motility. N-myc downstream regulated gene-1 (NDRG1) is a potent metastasis suppressor in many solid tumor types, including prostate and colon cancer. Considering the antimetastatic effect of NDRG1 and the crucial involvement of the FAK/paxillin pathway in cellular migration and cell-matrix adhesion, we assessed the effects of NDRG1 on this important oncogenic pathway. In the present study, NDRG1 overexpression and silencing models of HT29 colon cancer and DU145 prostate cancer cells were used to examine the activation of FAK/paxillin signaling and the formation of focal adhesions. The expression of NDRG1 resulted in a marked and significant decrease in the activating phosphorylation of FAK and paxillin, whereas silencing of NDRG1 resulted in an opposite effect. The expression of NDRG1 also inhibited the formation of focal adhesions as well as cell migration and cell-collagen adhesion. Incubation of cells with novel thiosemicarbazones, namely di-2-pyridylketone 4,4-dimethyl-3-thiosemicarbazone and di-2-pyridylketone 4-cyclohexyl-4-methyl-3-thiosemicarbazone, that upregulate NDRG1 also resulted in decreased phosphorylation of FAK and paxillin. The ability of these thiosemicarbazones to inhibit cell migration and metastasis could be mediated, at least in part, through the FAK/paxillin pathway. PMID:26895766

  5. Histone deacetylase inhibitor reverses multidrug resistance by attenuating the nucleophosmin level through PI3K/Akt pathway in breast cancer.

    Science.gov (United States)

    Chen, Si-Ying; Zheng, Xiao-Wei; Cai, Jiang-Xia; Zhang, Wei-Peng; You, Hai-Sheng; Xing, Jian-Feng; Dong, Ya-Lin

    2016-07-01

    The development of multidrug resistance (MDR) is the major obstacle in the chemotherapy of breast cancer, and it restricts the application of antitumor drugs in the clinic. Therefore it is urgent to search for ways to reverse MDR and restore sensitivity to chemotherapeutics in breast carcinoma. Currently, histone deacetylase inhibitors (HDACIs) offer a promising strategy for tumor therapy as the effective anticancer drugs. Based on the potential resistant target of nucleophosmin (NPM), the purpose of this study was to explore the reversal effect of a new synthetic histone deacetylase inhibitor, FA17, on MDR in methotrexate-resistant breast cancer cells (MCF-7/MTX) and xenograft tumors. It was shown that the abnormal expression of NPM induced MDR and inhibited downstream mitochondrial apoptotic pathway by activating PI3K/Akt signaling pathway in MCF-7/MTX cells. The reversal effect and molecular mechanism of FA17 were investigated both in vitro and in vivo. We found that FA17 could significantly reverse resistance and sensitize MCF-7/MTX cells to methotrexate. FA17 obviously enhanced resistant cell apoptosis, inhibited expressions of NPM and efflux transporters. Additionally, FA17 could reverse MDR via inactivating PI3K/Akt pathway and accelerating mitochondrial apoptotic pathway both in MCF-7/MTX cells and in xenograft tumors. Taken together, the novel histone deacetylase inhibitor could effectively reverse drug resistance due to suppressing the activity of NPM and drug efflux pumps by PI3K/Akt and mitochondrial apoptotic pathway. The above not only indicated the potential applied value of FA17 in reversing MDR and enhancing the sensitivity of chemotherapy, but also confirmed the role of NPM in the development of MDR in breast cancer. PMID:27211281

  6. Multiple roles and therapeutic implications of Akt signaling in cancer

    Directory of Open Access Journals (Sweden)

    Emiliano Calvo

    2009-06-01

    Full Text Available Emiliano Calvo1, Victoria Bolós2, Enrique Grande21Centro Integral Oncológico Clara Campal (CiOCC, Madrid. Spain; 2Pfizer Oncology, Alcobendas-Madrid, SpainAbstract: The prominence of the PI3K-Akt signaling pathway in several tumors indicates a relationship with tumor grade and proliferation. Critical cellular processes are driven through this pathway. More detailed knowledge of the pathogenesis of tumors would enable us to design targeted drugs to block both membrane tyrosine kinase receptors and the intracellular kinases involved in the transmission of the signal. The newly approved molecular inhibitors sunitinib (an inhibitor of vascular endothelial growth factor receptor, platelet-derived growth factor receptor, and other tyrosine kinase receptors, sorafenib (a serine–threonine kinase inhibitor that acts against B-Raf and temsirolimus (an mTOR inhibitor shown clinical activity in advanced kidney cancer. Chronic myeloid leukemia has changed its natural history thanks to imatinib and dasatinib, both of which inhibit the intracellular bcr/abl protein derived from the alteration in the Philadelphia chromosome. Intracellular pathways are still important in cancer development and their blockade directly affects outcome. Cross-talk has been observed but is not well understood. Vertical and horizontal pathway blockade are promising anticancer strategies. Indeed, preclinical and early clinical data suggest that combining superficial and intracellular blocking agents can synergize and leverage single-agent activity. The implication of the Akt signaling pathway in cancer is well established and has led to the development of new anticancer agents that block its activation.Keywords: Akt, cancer, therapeutic target, Akt inhibitors

  7. Anti-malarial drug artesunate attenuates experimental allergic asthma via inhibition of the phosphoinositide 3-kinase/Akt pathway.

    Directory of Open Access Journals (Sweden)

    Chang Cheng

    Full Text Available BACKGROUND: Phosphoinositide 3-kinase (PI3K/Akt pathway is linked to the development of asthma. Anti-malarial drug artesunate is a semi-synthetic derivative of artemisinin, the principal active component of a medicinal plant Artemisia annua, and has been shown to inhibit PI3K/Akt activity. We hypothesized that artesunate may attenuate allergic asthma via inhibition of the PI3K/Akt signaling pathway. METHODOLOGY/PRINCIPAL FINDINGS: Female BALB/c mice sensitized and challenged with ovalbumin (OVA developed airway inflammation. Bronchoalveolar lavage fluid was assessed for total and differential cell counts, and cytokine and chemokine levels. Lung tissues were examined for cell infiltration and mucus hypersecretion, and the expression of inflammatory biomarkers. Airway hyperresponsiveness was monitored by direct airway resistance analysis. Artesunate dose-dependently inhibited OVA-induced increases in total and eosinophil counts, IL-4, IL-5, IL-13 and eotaxin levels in bronchoalveolar lavage fluid. It attenuated OVA-induced lung tissue eosinophilia and airway mucus production, mRNA expression of E-selectin, IL-17, IL-33 and Muc5ac in lung tissues, and airway hyperresponsiveness to methacholine. In normal human bronchial epithelial cells, artesunate blocked epidermal growth factor-induced phosphorylation of Akt and its downstream substrates tuberin, p70S6 kinase and 4E-binding protein 1, and transactivation of NF-κB. Similarly, artesunate blocked the phosphorylation of Akt and its downstream substrates in lung tissues from OVA-challenged mice. Anti-inflammatory effect of artesunate was further confirmed in a house dust mite mouse asthma model. CONCLUSION/SIGNIFICANCE: Artesunate ameliorates experimental allergic airway inflammation probably via negative regulation of PI3K/Akt pathway and the downstream NF-κB activity. These findings provide a novel therapeutic value for artesunate in the treatment of allergic asthma.

  8. Operational optimization in the downstream; Otimizacao operacional no downstream

    Energy Technology Data Exchange (ETDEWEB)

    Silberman, Luis; Cunha, Filipe Silveira Ramos da [Petroleo Ipiranga, Porto Alegre, RS (Brazil)

    2004-07-01

    On the present competitive down stream's market, there is a great necessity of optimization aiming to guarantee the best price and quality of our clients. Our goal is to attend these expectations while we guarantee an efficient operation. The greatest question is how far we are from the ideal model. This way, a lot of projects have been executed during the last years aiming the operational optimization of all our activities. We divide the projects in 4 areas: Logistic (new modals distribution), Transport (transport optimization - quality and more deliveries with less trucks), Client Support (Internet Ipiranga and Support Center), Distribution Terminals Productivity (automation and environment). This work intend to present our ideal, perfect and complete Downstream Operation model. We will talk about how close we are of this ideal model and we will present the projects that we had already developed and implanted on the automation of the terminals and the logistics area. (author)

  9. Protein kinase B/Akt activates c-Jun NH(2)-terminal kinase by increasing NO production in response to shear stress

    Science.gov (United States)

    Go, Y. M.; Boo, Y. C.; Park, H.; Maland, M. C.; Patel, R.; Pritchard, K. A. Jr; Fujio, Y.; Walsh, K.; Darley-Usmar, V.; Jo, H.

    2001-01-01

    Laminar shear stress activates c-Jun NH(2)-terminal kinase (JNK) by the mechanisms involving both nitric oxide (NO) and phosphatidylinositide 3-kinase (PI3K). Because protein kinase B (Akt), a downstream effector of PI3K, has been shown to phosphorylate and activate endothelial NO synthase, we hypothesized that Akt regulates shear-dependent activation of JNK by stimulating NO production. Here, we examined the role of Akt in shear-dependent NO production and JNK activation by expressing a dominant negative Akt mutant (Akt(AA)) and a constitutively active mutant (Akt(Myr)) in bovine aortic endothelial cells (BAEC). As expected, pretreatment of BAEC with the PI3K inhibitor (wortmannin) prevented shear-dependent stimulation of Akt and NO production. Transient expression of Akt(AA) in BAEC by using a recombinant adenoviral construct inhibited the shear-dependent stimulation of NO production and JNK activation. However, transient expression of Akt(Myr) by using a recombinant adenoviral construct did not induce JNK activation. This is consistent with our previous finding that NO is required, but not sufficient on its own, to activate JNK in response to shear stress. These results and our previous findings strongly suggest that shear stress triggers activation of PI3K, Akt, and endothelial NO synthase, leading to production of NO, which (along with O(2-), which is also produced by shear) activates Ras-JNK pathway. The regulation of Akt, NO, and JNK by shear stress is likely to play a critical role in its antiatherogenic effects.

  10. Blunted Akt/FOXO signalling and activation of genes controlling atrophy and fuel use in statin myopathy.

    Science.gov (United States)

    Mallinson, Joanne E; Constantin-Teodosiu, Dumitru; Sidaway, James; Westwood, F Russell; Greenhaff, Paul L

    2009-01-15

    Statins are used clinically for cholesterol reduction, but statin therapy is associated with myopathic changes through a poorly defined mechanism. We used an in vivo model of statin myopathy to determine whether statins up-regulate genes associated with proteasomal- and lysosomal-mediated proteolysis and whether PDK gene expression is simultaneously up-regulated leading to the impairment of muscle carbohydrate oxidation. Animals were dosed daily with 80 mg kg(-1) day(-1) simvastatin for 4 (n = 6) and 12 days (n = 5), 88 mg kg(-1) day(-1) simvastatin for 12 days (n = 4), or vehicle (0.5% w/v hydroxypropyl-methylcellulose and 0.1% w/v polysorbate 80; Control, n = 6) for 12 days by oral gavage. We found, in biceps femoris muscle, decreased Akt(Ser473), FOXO1(Ser253) and FOXO3a(Ser253) phosphorylation in the cytosol (P PDK2 (P < 0.05) and PDK4 (P < 0.05). These changes were accompanied by increased PPARalpha (P < 0.05), TNFalpha (P < 0.01), IL6 (P < 0.01), Mt1A (P < 0.01) mRNA and increased muscle glycogen (P < 0.05) compared to Control. RhoA activity decreased after 4 days simvastatin (P < 0.05); however, activity was no different from Control after 12 days. Simvastatin down-regulated PI3k/Akt signalling, independently of RhoA, and up-regulated FOXO transcription factors and downstream gene targets known to be implicated in proteasomal- and lysosomal-mediated muscle proteolysis, carbohydrate oxidation, oxidative stress and inflammation in an in vivo model of statin-induced myopathy. These changes occurred in the main before evidence of extensive myopathy or a decline in the muscle protein to DNA ratio. PMID:19001041

  11. Phenylbutyric acid induces the cellular senescence through an Akt/p21{sup WAF1} signaling pathway

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Hag Dong [Laboratory of Biochemistry, School of Life Sciences and Biotechnology, and BioInstitute, Korea University, Seoul 136-701 (Korea, Republic of); Jang, Chang-Young [Research Center for Cell Fate Control, College of Pharmacy, Sookmyung Women' s University, Seoul 140-742 (Korea, Republic of); Choe, Jeong Min [Laboratory of Biochemistry, School of Life Sciences and Biotechnology, and BioInstitute, Korea University, Seoul 136-701 (Korea, Republic of); Department of Biochemistry, Korea University College of Medicine, Seoul 136-705 (Korea, Republic of); Korean Institute of Molecular Medicine and Nutrition, Seoul 136-705 (Korea, Republic of); Sohn, Jeongwon, E-mail: biojs@korea.ac.kr [Department of Biochemistry, Korea University College of Medicine, Seoul 136-705 (Korea, Republic of); Korean Institute of Molecular Medicine and Nutrition, Seoul 136-705 (Korea, Republic of); Kim, Joon, E-mail: joonkim@korea.ac.kr [Laboratory of Biochemistry, School of Life Sciences and Biotechnology, and BioInstitute, Korea University, Seoul 136-701 (Korea, Republic of)

    2012-06-01

    Highlights: Black-Right-Pointing-Pointer Phenylbutyric acid induces cellular senescence. Black-Right-Pointing-Pointer Phenylbutyric acid activates Akt kinase. Black-Right-Pointing-Pointer The knockdown of PERK also can induce cellular senescence. Black-Right-Pointing-Pointer Akt/p21{sup WAF1} pathway activates in PERK knockdown induced cellular senescence. -- Abstract: It has been well known that three sentinel proteins - PERK, ATF6 and IRE1 - initiate the unfolded protein response (UPR) in the presence of misfolded or unfolded proteins in the ER. Recent studies have demonstrated that upregulation of UPR in cancer cells is required to survive and proliferate. Here, we showed that long exposure to 4-phenylbutyric acid (PBA), a chemical chaperone that can reduce retention of unfolded and misfolded proteins in ER, induced cellular senescence in cancer cells such as MCF7 and HT1080. In addition, we found that treatment with PBA activates Akt, which results in p21{sup WAF1} induction. Interestingly, the depletion of PERK but not ATF6 and IRE1 also induces cellular senescence, which was rescued by additional depletion of Akt. This suggests that Akt pathway is downstream of PERK in PBA induced cellular senescence. Taken together, these results show that PBA induces cellular senescence via activation of the Akt/p21{sup WAF1} pathway by PERK inhibition.

  12. Mechanism of Akt1 inhibition of breast cancer cell invasionreveals a protumorigenic role for TSC2

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Hong; Radisky, Derek C.; Nelson, Celeste M.; Zhang, Hui; Fata, Jimmie; Roth, Richard A.; Bissell, Mina J.

    2006-02-07

    Akt1 is frequently upregulated in human tumors, and has been shown to accelerate cell proliferation and to suppress programmed cell death; consequently, inhibiting the activity of Akt1 has been seen as an attractive target for therapeutic intervention. Paradoxically, hyperactivation of the Akt1 oncogene can also prevent the invasive behavior that underlies progression to metastasis. Here we show that overexpression of activated myr-Akt1 in human breast cancer cells phosphorylates and thereby targets the tumor suppressor tuberous sclerosis complex 2 (TSC2) for degradation, leading to reduced Rho-GTPase activity, decreased actin stress fibers and focal adhesions, and reduced motility and invasion. Overexpression of TSC2 rescues the migration phenotype of myr-Akt1-expressing tumor cells, and high levels of TSC2 in breast cancer patients correlate with increased metastasis and reduced survival. These data indicate that the functional properties of genes designated as oncogenes or tumor suppressor genes depends on the context of the cell type and the tissues studied, and suggest the need for caution in designing therapies targeting the function of individual genes in epithelial tissues.

  13. Sea urchin akt activity is Runx-dependent and required for post-cleavage stage cell division

    KAUST Repository

    Robertson, Anthony J.

    2013-03-25

    In animal development following the initial cleavage stage of embryogenesis, the cell cycle becomes dependent on intercellular signaling and controlled by the genomically encoded ontogenetic program. Runx transcription factors are critical regulators of metazoan developmental signaling, and we have shown that the sea urchin Runx gene runt-1, which is globally expressed during early embryogenesis, functions in support of blastula stage cell proliferation and expression of the mitogenic genes pkc1, cyclinD, and several wnts. To obtain a more comprehensive list of early runt-1 regulatory targets, we screened a Strongylocentrotus purpuratus microarray to identify genes mis-expressed in mid-blastula stage runt-1 morphants. This analysis showed that loss of Runx function perturbs the expression of multiple genes involved in cell division, including the pro-growth and survival kinase Akt (PKB), which is significantly underexpressed in runt-1 morphants. Further genomic analysis revealed that Akt is encoded by two genes in the S. purpuratus genome, akt-1 and akt-2, both of which contain numerous canonical Runx target sequences. The transcripts of both genes accumulate several fold during blastula stage, contingent on runt-1 expression. Inhibiting Akt expression or activity causes blastula stage cell cycle arrest, whereas overexpression of akt-1 mRNA rescues cell proliferation in runt-1 morphants. These results indicate that post-cleavage stage cell division requires Runx-dependent expression of akt.

  14. Akt Regulates Axon Wrapping and Myelin Sheath Thickness in the PNS

    Science.gov (United States)

    Baloui, Hasna; Meng, Xiaosong; Zhang, Yanqing; Deinhardt, Katrin; Dupree, Jeff L.; Einheber, Steven; Chrast, Roman

    2016-01-01

    The signaling pathways that regulate myelination in the PNS remain poorly understood. Phosphatidylinositol-4,5-bisphosphate 3-kinase 1A, activated in Schwann cells by neuregulin and the extracellular matrix, has an essential role in the early events of myelination. Akt/PKB, a key effector of phosphatidylinositol-4,5-bisphosphate 3-kinase 1A, was previously implicated in CNS, but not PNS myelination. Here we demonstrate that Akt plays a crucial role in axon ensheathment and in the regulation of myelin sheath thickness in the PNS. Pharmacological inhibition of Akt in DRG neuron-Schwann cell cocultures dramatically decreased MBP and P0 levels and myelin sheath formation without affecting expression of Krox20/Egr2, a key transcriptional regulator of myelination. Conversely, expression of an activated form of Akt in purified Schwann cells increased expression of myelin proteins, but not Krox20/Egr2, and the levels of activated Rac1. Transgenic mice expressing a membrane-targeted, activated form of Akt under control of the 2′,3′-cyclic nucleotide 3′-phosphodiesterase promoter, exhibited thicker PNS and CNS myelin sheaths, and PNS myelin abnormalities, such as tomacula and myelin infoldings/outfoldings, centered around the paranodes and Schmidt Lanterman incisures. These effects were corrected by rapamycin treatment in vivo. Importantly, Akt activity in the transgenic mice did not induce myelination of nonmyelinating Schwann cells in the sympathetic trunk or Remak fibers of the dorsal roots, although, in those structures, they wrapped membranes redundantly around axons. Together, our data indicate that Akt is crucial for PNS myelination driving axonal wrapping by unmyelinated and myelinated Schwann cells and enhancing myelin protein synthesis in myelinating Schwann cells. SIGNIFICANCE STATEMENT Although the role of the key serine/threonine kinase Akt in promoting CNS myelination has been demonstrated, its role in the PNS has not been established and remains

  15. V-akt murine thymoma viral oncogene homolog 3 (AKT3) contributes to poor disease outcome in humans and mice with pneumococcal meningitis.

    Science.gov (United States)

    Valls Serón, Mercedes; Ferwerda, Bart; Engelen-Lee, JooYeon; Geldhoff, Madelijn; Jaspers, Valery; Zwinderman, Aeilko H; Tanck, Michael W; Baas, Frank; van der Ende, Arie; Brouwer, Matthijs C; van de Beek, Diederik

    2016-05-18

    Pneumococcal meningitis is the most common and severe form of bacterial meningitis. Fatality rates are substantial, and long-term sequelae develop in about half of survivors. Here, we have performed a prospective nationwide genetic association study using the Human Exome BeadChip and identified gene variants in encoding dynactin 4 (DCTN4), retinoic acid early transcript 1E (RAET1E), and V-akt murine thymoma viral oncogene homolog 3 (AKT3) to be associated with unfavourable outcome in patients with pneumococcal meningitis. No clinical replication cohort is available, so we validated the role of one of these targets, AKT3, in a pneumococcal meningitis mouse model. Akt3 deficient mice had worse survival and increased histopathology scores for parenchymal damage (infiltration) and vascular infiltration (large meningeal artery inflammation) but similar bacterial loads, cytokine responses, compared to wild-type mice. We found no differences in cerebrospinal fluid cytokine levels between patients with risk or non-risk alleles. Patients with the risk genotype (rs10157763, AA) presented with low scores on the Glasgow Coma Scale and high rate of epileptic seizures. Thus, our results show that AKT3 influences outcome of pneumococcal meningitis.

  16. Blocking the PI3K/AKT pathway enhances mammalian reovirus replication by repressing IFN-stimulated genes

    Science.gov (United States)

    Tian, Jin; Zhang, Xiaozhan; Wu, Hongxia; Liu, Chunguo; Li, Zhijie; Hu, Xiaoliang; Su, Shuo; Wang, Lin-Fa; Qu, Liandong

    2015-01-01

    Many host cellular signaling pathways were activated and exploited by virus infection for more efficient replication. The PI3K/Akt pathway has recently attracted considerable interest due to its role in regulating virus replication. This study demonstrated for the first time that the mammalian reovirus strains Masked Palm Civet/China/2004 (MPC/04) and Bat/China/2003 (B/03) can induce transient activation of the PI3K/Akt pathway early in infection in vitro. When UV-treated, both viruses activated PI3K/Akt signaling, indicating that the virus/receptor interaction was sufficient to activate PI3K/Akt. Reovirus virions can use both clathrin- and caveolae-mediated endocytosis, but only chlorpromazine, a specific inhibitor of clathrin-mediated endocytosis, or siRNA targeting clathrin suppressed Akt phosphorylation. We also identified the upstream molecules of the PI3K pathway. Virus infection induced phosphorylation of focal adhesion kinase (FAK) but not Gab1, and blockage of FAK phosphorylation suppressed Akt phosphorylation. Blockage of PI3K/Akt activation increased virus RNA synthesis and viral yield. We also found that reovirus infection activated the IFN-stimulated response element (ISRE) in an interferon-independent manner and up-regulated IFN-stimulated genes (ISGs) via the PI3K/Akt/EMSY pathway. Suppression of PI3K/Akt activation impaired the induction of ISRE and down-regulated the expression of ISGs. Overexpression of ISG15 and Viperin inhibited virus replication, and knockdown of either enhanced virus replication. Collectively, these results demonstrate that PI3K/Akt activated by mammalian reovirus serves as a pathway for sensing and then inhibiting virus replication/infection. PMID:26388843

  17. MiR-145 functions as a tumor suppressor targeting NUAK1 in human intrahepatic cholangiocarcinoma

    Energy Technology Data Exchange (ETDEWEB)

    Xiong, Xinkui; Sun, Daoyi; Chai, Hao; Shan, Wengang [Liver Transplantation Center, First Affiliated Hospital of Nanjing Medical University, Nanjing, Jiangsu Province (China); Key Laboratory of Living Donor Liver Transplantation, Ministry of Public Health, Nanjing, Jiangsu Province (China); Yu, Yue [Key Laboratory of Living Donor Liver Transplantation, Ministry of Public Health, Nanjing, Jiangsu Province (China); Pu, Liyong [Liver Transplantation Center, First Affiliated Hospital of Nanjing Medical University, Nanjing, Jiangsu Province (China); Key Laboratory of Living Donor Liver Transplantation, Ministry of Public Health, Nanjing, Jiangsu Province (China); Cheng, Feng, E-mail: docchengfeng@njmu.edu.cn [Liver Transplantation Center, First Affiliated Hospital of Nanjing Medical University, Nanjing, Jiangsu Province (China); Key Laboratory of Living Donor Liver Transplantation, Ministry of Public Health, Nanjing, Jiangsu Province (China)

    2015-09-18

    The dysregulation of micro (mi)RNAs is associated with cancer development. The miRNA miR-145 is downregulated in intrahepatic cholangiocarcinoma (ICC); however, its precise role in tumor progression has not yet been elucidated. Novel (nua) kinase family (NUAK)1 functions as an oncogene in various cancers and is a putative target of miR-145 regulation. In this study, we investigated the regulation of NUAK1 by miR-145 in ICC. We found that miR-145 level was significantly decreased in ICC tissue and cell lines, which corresponded with an increase in NUAK1 expression. NUAK1 was found to be a direct target of miR-145 regulation. The overexpression of miR-145 in ICC cell lines inhibited proliferation, growth, and invasion by suppressing NUAK1 expression, which was associated with a decrease in Akt signaling and matrix metalloproteinase protein expression. Similar results were observed by inhibiting NUAK1 expression. These results demonstrate that miR-145 can prevent ICC progression by targeting NUAK1 and its downstream effectors, and can therefore be useful for clinical diagnosis and targeted therapy of ICC. - Highlights: • MiR-145 suppresses ICC proliferation and invasion abilities. • We demonstrated that miR-145 directly targets NUAK1 in ICC. • MiR-145 expression in ICC was associated with Akt signaling and MMPs expression.

  18. Pancreas-specific Pten deficiency causes partial resistance to diabetes and elevated hepatic AKT signaling

    Institute of Scientific and Technical Information of China (English)

    Zan Tong; Yan Fan; Weiqi Zhang; Jun Xu; Jing Cheng; Mingxiao Ding; Hongkui Deng

    2009-01-01

    PTEN, a negative regulator of the phosphatidylinositol-3-kinase/AKT pathway, is an important modulator of insu-lin signaling. To determine the metabolic function of pancreatic Pten, we generated pancreas-specific Pten knockout (PPKO) mice. PPKO mice had enlarged pancreas and elevated proliferation of acinar cells. They also exhibited hy-poglycemia, hypoinsulinemia, and altered amino metabolism. Notably, PPKO mice showed delayed onset of strepto-zotocin (STZ)-induced diabetes and sex-biased resistance to high-fat-diet (HFD)-induced diabetes. To investigate the mechanism for the resistance to HFD-induced hyperglycemia in PPKO mice, we evaluated AKT phosphorylation in major insulin-responsive tissues: the liver, muscle, and fat. We found that Pten loss in the pancreas causes the eleva-tion of AKT signaling in the liver. The phosphorylation of AKT and its downstream substrate GSK3β was increased in the liver of PPKO mice, while PTEN level was decreased without detectable excision of Pten allele in the liver of PPKO mice. Proteomics analysis revealed dramatically decreased level of 78-kDa glucose-regulated protein (GRP78) in the liver of PPKO mice, which may also contribute to the lower blood glucose level of PPKO mice fed with HFD. Together, our findings reveal a novel response in the liver to pancreatic defect in metabolic regulation, adding a new dimension to understanding diabetes resistance.

  19. AKT1 E17K mutation profiling in breast cancer: prevalence, concurrent oncogenic alterations, and blood-based detection

    OpenAIRE

    Rudolph, Marion; Anzeneder, Tobias; Schulz, Anke; Beckmann, Georg; Byrne, Annette T.; Jeffers, Michael; Pena, Carol; Politz, Oliver; Köchert, Karl; Vonk, Richardus; Reischl, Joachim

    2016-01-01

    Background: The single hotspot mutation AKT1 [G49A:E17K] has been described in several cancers, with the highest incidence observed in breast cancer. However, its precise role in disease etiology remains unknown.Methods: We analyzed more than 600 breast cancer tumor samples and circulating tumor DNA for AKT1 E17K and alterations in other cancer-associated genes using Beads, Emulsions, Amplification, and Magnetics digital polymerase chain reaction technology and targeted exome sequencing.Resul...

  20. AKT1 (E17K) mutation profiling in breast cancer: prevalence, concurrent oncogenic alterations, and blood-based detection.

    OpenAIRE

    Rudolph, Marion; Anzeneder, Tobias; Schulz, Anke; Beckmann, Georg; Byrne, Annette T.; Jeffers, Michael; Pena, Carol; Politz, Oliver; Köchert, Karl; Vonk, Richardus; Reischl, Joachim

    2016-01-01

    BACKGROUND: The single hotspot mutation AKT1 [G49A:E17K] has been described in several cancers, with the highest incidence observed in breast cancer. However, its precise role in disease etiology remains unknown. METHODS: We analyzed more than 600 breast cancer tumor samples and circulating tumor DNA for AKT1 (E17K) and alterations in other cancer-associated genes using Beads, Emulsions, Amplification, and Magnetics digital polymerase chain reaction technology and targeted exome sequencing...

  1. Combination treatment with MEK and AKT inhibitors is more effective than each drug alone in human non-small cell lung cancer in vitro and in vivo.

    Directory of Open Access Journals (Sweden)

    Jieru Meng

    Full Text Available AZD6244 and MK2206 are targeted small-molecule drugs that inhibit MEK and AKT respectively. The efficacy of this combination in lung cancer is unknown. Our previous work showed the importance of activated AKT in mediating resistance of non-small cell lung cancer (NSCLC to AZD6244. Thus we hypothesized that dual inhibition of both downstream MEK and AKT pathways would induce synergistic antitumor activity. In this study, we evaluated the efficacy of AZD6244 and MK2206 individually on a large panel of lung cancer cell lines. Then, we treated 28 human lung cancer cell lines with a combination of AZD6244 and MK2206 at clinically applicable drug molar ratios. The AZD6244-MK2206 combination therapy resulted in a synergistic effect on inhibition of lung cancer cell growth compared to the results of single drug treatment alone. MK2206 enhanced AZD6244-induced Bim overexpression and apoptosis in A549 and H157 cells. When we tested the combination of AZD6244 and MK2206 at ratios of 8∶1, 4∶1, 2∶1, and 1∶8, we found that the synergistic effect of the combination therapy was ratio-dependent. At ratios of 8∶1, 4∶1, and 2∶1, the drug combination consistently demonstrated synergy, whereas decreasing the ratio to 1∶8 resulted in a loss of synergy and produced an additive or antagonistic effect in most cell lines. Furthermore, the AZD6244-MK2206 combination therapy showed synergy in the suppression of A549 and H157 xenograft tumor growth and increased mean animal survival time. The AZD6244-MK2206 combination therapy resulted in effective inhibition of both p-ERK and p-AKT expression in tumor tissue. In addition, a significant increase of apoptosis was detected in tumor tissue from mice treated with AZD6244-MK2206 compared with that from the single agent treated mice. Our study suggests that the combination of AZD6244 and MK2206 has a significant synergistic effect on tumor growth in vitro and in vivo and leads to increased survival rates in mice

  2. SIN1/MIP1 maintains rictor-mTOR complex integrity and regulates Akt phosphorylation and substrate specificity.

    Science.gov (United States)

    Jacinto, Estela; Facchinetti, Valeria; Liu, Dou; Soto, Nelyn; Wei, Shiniu; Jung, Sung Yun; Huang, Qiaojia; Qin, Jun; Su, Bing

    2006-10-01

    Mammalian target of rapamycin (mTOR) controls cell growth and proliferation via the raptor-mTOR (TORC1) and rictor-mTOR (TORC2) protein complexes. Recent biochemical studies suggested that TORC2 is the elusive PDK2 for Akt/PKB Ser473 phosphorylation in the hydrophobic motif. Phosphorylation at Ser473, along with Thr308 of its activation loop, is deemed necessary for Akt function, although the regulatory mechanisms and physiological importance of each phosphorylation site remain to be fully understood. Here, we report that SIN1/MIP1 is an essential TORC2/PDK2 subunit. Genetic ablation of sin1 abolished Akt-Ser473 phosphorylation and disrupted rictor-mTOR interaction but maintained Thr308 phosphorylation. Surprisingly, defective Ser473 phosphorylation affected only a subset of Akt targets in vivo, including FoxO1/3a, while other Akt targets, TSC2 and GSK3, and the TORC1 effectors, S6K and 4E-BP1, were unaffected. Our findings reveal that the SIN1-rictor-mTOR function in Akt-Ser473 phosphorylation is required for TORC2 function in cell survival but is dispensable for TORC1 function. PMID:16962653

  3. Impaired striatal Akt signaling disrupts dopamine homeostasis and increases feeding.

    Directory of Open Access Journals (Sweden)

    Nicole Speed

    Full Text Available BACKGROUND: The prevalence of obesity has increased dramatically worldwide. The obesity epidemic begs for novel concepts and therapeutic targets that cohesively address "food-abuse" disorders. We demonstrate a molecular link between impairment of a central kinase (Akt involved in insulin signaling induced by exposure to a high-fat (HF diet and dysregulation of higher order circuitry involved in feeding. Dopamine (DA rich brain structures, such as striatum, provide motivation stimuli for feeding. In these central circuitries, DA dysfunction is posited to contribute to obesity pathogenesis. We identified a mechanistic link between metabolic dysregulation and the maladaptive behaviors that potentiate weight gain. Insulin, a hormone in the periphery, also acts centrally to regulate both homeostatic and reward-based HF feeding. It regulates DA homeostasis, in part, by controlling a key element in DA clearance, the DA transporter (DAT. Upon HF feeding, nigro-striatal neurons rapidly develop insulin signaling deficiencies, causing increased HF calorie intake. METHODOLOGY/PRINCIPAL FINDINGS: We show that consumption of fat-rich food impairs striatal activation of the insulin-activated signaling kinase, Akt. HF-induced Akt impairment, in turn, reduces DAT cell surface expression and function, thereby decreasing DA homeostasis and amphetamine (AMPH-induced DA efflux. In addition, HF-mediated dysregulation of Akt signaling impairs DA-related behaviors such as (AMPH-induced locomotion and increased caloric intake. We restored nigro-striatal Akt phosphorylation using recombinant viral vector expression technology. We observed a rescue of DAT expression in HF fed rats, which was associated with a return of locomotor responses to AMPH and normalization of HF diet-induced hyperphagia. CONCLUSIONS/SIGNIFICANCE: Acquired disruption of brain insulin action may confer risk for and/or underlie "food-abuse" disorders and the recalcitrance of obesity. This molecular

  4. Effects of D-Pinitol on Insulin Resistance through the PI3K/Akt Signaling Pathway in Type 2 Diabetes Mellitus Rats.

    Science.gov (United States)

    Gao, Yunfeng; Zhang, Mengna; Wu, Tianchen; Xu, Mengying; Cai, Haonan; Zhang, Zesheng

    2015-07-01

    D-pinitol, a compound isolated from Pinaceae and Leguminosae plants, has been reported to possess insulin-like properties. Although the hypoglycemic activity of D-pinitol was recognized in recent years, the molecular mechanism of D-pinitol in the treatment of diabetes mellitus remains unclear. In this investigation, a model of type 2 diabetes mellitus (T2DM) with insulin resistance was established by feeding a high-fat diet (HFD) and injecting streptozocin (STZ) to Sprague-Dawley (SD) rats, targeting the exploration of more details of the mechanism in the therapy of T2DM. D-pinitol was administrated to the diabetic rats as two doses [30, 60 mg/(kg·body weight·day)]. The level of fasting blood glucose (FBG) was decreased 12.63% in the high-dosage group, and the ability of oral glucose tolerance was improved in D-pinitol-treated groups. The biochemical indices revealed that D-pinitol had a positive effect on hypoglycemic activity. Western boltting suggested that D-pinitol could promote the expression of the phosphatidylinositol-3-kinase (PI3K) p85, PI3Kp110, as well as the downstream target protein kinase B/Akt (at Ser473). Besides, D-pinitol inhibited the expression of glycogen synthesis kinase-3β (GSK-3β) protein and regulated the expression of glycogen synthesis (GS) protein and then accelerated the glycogen synthesis. Above all, D-pinitol played a positive role in regulating insulin-mediated glucose uptake in the liver through translocation and activation of the PI3K/Akt signaling pathway in T2DM rats.

  5. A Novel mTORC1-Dependent, Akt-Independent Pathway Differentiates the Gut Tropism of Regulatory and Conventional CD4 T Cells.

    Science.gov (United States)

    Chen, Leo C; Nicholson, Yawah T; Rosborough, Brian R; Thomson, Angus W; Raimondi, Giorgio

    2016-08-15

    The vitamin A metabolite all-trans retinoic acid (ATRA) induces a gut-homing phenotype in activated CD4(+) conventional T cells (Tconv) by upregulating the integrin α4β7 and the chemokine receptor CCR9. We report that, in contrast to mouse Tconv, only ∼50% of regulatory T cells (Treg) upregulate CCR9 when stimulated by physiological levels of ATRA, even though Tconv and Treg express similar levels of the retinoic acid receptor (RAR). The resulting bimodal CCR9 expression is not associated with differences in the extent of their proliferation, level of Foxp3 expression, or affiliation with naturally occurring Treg or induced Treg in the circulating Treg pool. Furthermore, we find that exposure of Treg to the mechanistic target of rapamycin (mTOR) inhibitor rapamycin suppresses upregulation of both CCR9 and α4β7, an effect that is not evident with Tconv. This suggests that in Treg, ATRA-induced upregulation of CCR9 and α4β7 is dependent on activation of a mTOR signaling pathway. The involvement of mTOR is independent of Akt activity, because specific inhibition of Akt, pyruvate dehydrogenase kinase-1, or its downstream target glycogen synthase kinase-3 did not prevent CCR9 expression. Additionally, Rictor (mTOR complex [mTORC]2)-deficient Treg showed unaltered ability to express CCR9, whereas Raptor (mTORC1)-deficient Treg were unable to upregulate CCR9, suggesting the selective participation of mTORC1. These findings reveal a novel difference between ATRA signaling and chemokine receptor induction in Treg versus Tconv and provide a framework via which the migratory behavior of Treg versus Tconv might be regulated differentially for therapeutic purposes. PMID:27402696

  6. Co-targeting ALK and EGFR parallel signaling in oral squamous cell carcinoma.

    Science.gov (United States)

    Gonzales, Cara B; De La Chapa, Jorge J; Saikumar, Pothana; Singha, Prajjal K; Dybdal-Hargreaves, Nicholas F; Chavez, Jeffery; Horning, Aaron M; Parra, Jamie; Kirma, Nameer B

    2016-08-01

    Squamous cell carcinoma (SCC) comprises 90% of all head and neck cancers and has a poor survival rate due to late-stage disease that is refractive to traditional therapies. Epidermal growth factor receptor (EGFR) is over-expressed in greater than 80% of head and neck SCC (HNSCC). However, EGFR targeted therapies yielded little to no efficacy in clinical trials. This study investigated the efficacy of co-targeting EGFR and the anaplastic lymphoma kinase (ALK) whose promoter is hypomethylated in late-stage oral SCC (OSCC). We observed increased ALK activity in late-stage human OSCC tumors and invasive OSCC cell lines. We also found that while ALK inhibition alone had little effect on proliferation, co-targeting ALK and EGFR significantly reduced OSCC cell proliferation in vitro. Further analysis showed significant efficacy of combined treatment in HSC3-derived xenografts resulting in a 30% decrease in tumor volumes by 14days (panalysis showed that co-targeting ALK and EGFR significantly reduced EGFR phosphorylation (Y1148) in HSC3 cells but not Cal27 cells. ALK and EGFR downstream signaling interactions are also demonstrated by Western blot analysis in which lone EGFR and ALK inhibitors attenuated AKT activity whereas co-targeting ALK and EGFR completely abolished AKT activation. No effects were observed on ERK1/2 activation. STAT3 activity was significantly induced by lone ALK inhibition in HSC3 cells and to a lower extent in Cal27 cells. Together, these data illustrate that ALK inhibitors enhance anti-tumor activity of EGFR inhibitors in susceptible tumors that display increased ALK expression, most likely through abolition of AKT activation. PMID:27424178

  7. Matrine inhibits proliferation and induces apoptosis of human colon cancer LoVo cells by inactivating Akt pathway.

    Science.gov (United States)

    Zhang, Shujun; Cheng, Binglin; Li, Hali; Xu, Wei; Zhai, Bo; Pan, Shangha; Wang, Lei; Liu, Ming; Sun, Xueying

    2014-01-01

    The present study has investigated the anti-tumor activity and the underlying mechanisms of matrine on human colon cancer LoVo cells. Matrine inhibited the proliferation of the cells in dose- and time-dependent manners. The concentration required for 50 % inhibition (IC50) was 1.15, 0.738, and 0.414 mg/ml, when cell were incubated with matrine for 24, 48, and 72 h, respectively. Matrine induced cell cycle arrest at G1 phase by downregulating cyclin D1 and upregulating p27 and p21. Matrine induced cell apoptosis by reducing the ratio of Bcl-2/Bax and increasing the activation of caspase-9 in a dose-dependent manner. Matrine displayed its anti-tumor activity by inactivating Akt, the upstream factor of the above proteins. Matrine significantly reduced the protein levels of pAkt, and increased the protein levels of other downstream factors, pBad and pGSK-3β. Specific inhibition of pAkt induced cell apoptosis, and synergized with matrine to inhibit the proliferation of LoVo cells; whereas activation of Akt neutralized the inhibitory effect of matrine on cell proliferation. The present study has demonstrated that matrine inhibits proliferation and induces apoptosis of human colon cancer LoVo cells by inactivating Akt pathway, indicating matrine may be a potential anti-cancer agent for colon cancer.

  8. CNOOC Advancing into Downstream of Petroleum Industry

    Institute of Scientific and Technical Information of China (English)

    1997-01-01

    @@ "China National Offshore Oil Corporation is starting to expand its business into the downstream sector after making achievements continuously in exploration and development," Wang Yan, president of the corporation,told reporter in an interview, adding that the downstream sector is the third development stage for China National Offshore Oil Corporation (CNOOC).

  9. Inhibition of Akt signaling by exclusion from lipid rafts in normal and transformed epidermal keratinocytes

    DEFF Research Database (Denmark)

    Calay, Damien; Vind-Kezunovic, Dina; Frankart, Aurelie;

    2010-01-01

    Lipid rafts are cholesterol-rich plasma membrane domains that regulate signal transduction. Because our earlier work indicated that raft disruption inhibited proliferation and caused cell death, we investigated here the role of membrane cholesterol, the crucial raft constituent, in the regulation...... of the phosphatidylinositol-3 kinase (PI3K)/Akt pathway. Raft disruption was achieved in normal human keratinocytes and precancerous (HaCaT) or transformed (A431) keratinocytes by cholesterol extraction or inactivation with methyl-beta-cyclodextrin, filipin III, or 5-cholestene-5-beta-ol. Lipid raft disruption did not affect...... in deactivation of mammalian target of rapamycin, activation of FoxO3a, and increased sensitivity to apoptosis stimuli. Lipid raft disruption abrogated the binding of Akt and the major Akt kinase, phosphatidylinositol-dependent kinase 1, to the membrane by pleckstrin-homology domains. Thus, the integrity of lipid...

  10. Inhibition of Akt signaling by exclusion from lipid rafts in normal and transformed epidermal keratinocytes

    DEFF Research Database (Denmark)

    Calay, Damien; Vind-Kezunovic, Dina; Frankart, Aurelie;

    2010-01-01

    Lipid rafts are cholesterol-rich plasma membrane domains that regulate signal transduction. Because our earlier work indicated that raft disruption inhibited proliferation and caused cell death, we investigated here the role of membrane cholesterol, the crucial raft constituent, in the regulation...... in deactivation of mammalian target of rapamycin, activation of FoxO3a, and increased sensitivity to apoptosis stimuli. Lipid raft disruption abrogated the binding of Akt and the major Akt kinase, phosphatidylinositol-dependent kinase 1, to the membrane by pleckstrin-homology domains. Thus, the integrity of lipid...... of the phosphatidylinositol-3 kinase (PI3K)/Akt pathway. Raft disruption was achieved in normal human keratinocytes and precancerous (HaCaT) or transformed (A431) keratinocytes by cholesterol extraction or inactivation with methyl-beta-cyclodextrin, filipin III, or 5-cholestene-5-beta-ol. Lipid raft disruption did not affect...

  11. Mechanistic Analysis of AKT1 Regulation by the CBL-CIPK-PP2CA Interactions

    Institute of Scientific and Technical Information of China (English)

    Wen-Zhi Lan; Sung-Chul Lee; Yu-Fen Che; Yuan-Qing Jiang; Sheng Luan

    2011-01-01

    T Arabidopsis K+ transporter 1 (AKT1) participates in K+ uptake in roots, especially under low-K conditions. We recently identified a Ca2+ signaling pathway consisting of multiple calcineurin B-like calcium sensors (CBLs) and multiple target kinases (CBL-interacting protein kinases or CIPKs) that phosphorylate and activate AKT1, whereas a specific PP2C-type phosphatase inactivates CIPK-dependent AKT1 activity. In this study, we analyzed the interactions between PP2Cs and the CBL-CIPK pathway and found previously unsuspected mechanisms underlying the CBL-CIPK-PP2C signaling processes. The interaction between the CIPKs and PP2Cs involves the kinase domain of the CIPK component, in addition to the protein phosphatase interacting motif (PPI) in the regulatory domain. Furthermore, specific CBLs physically interact with and inactivate PP2C phosphatases to recover the CIPK-dependent AKT1 channel activity. These findings provide further insights into the signaling network consisting of CBL-CIPK-PP2C interactions in the activation of the AKT1 channel.

  12. WA7 - View from downstream

    CERN Multimedia

    1977-01-01

    With this set-up the Annecy (LAPP)-CERN-Copenhagen(Niels Bohr Institute)-Genova-Oslo-University College London Collaboration investigated large-angle elastic scattering of charged mesons and antiprotons on protons, first at 20 and 30 GeV/c, later at 50 GeV/c. The beam H1 (from top) passes two CEDAR counters (centre) before entering the one-metre long liquid hydrogen target, partly inside a magnet of 1.56 T, with a field volume of about 1.50x0.75x1.50 m3. In the foreground one sees on the left the hadron calorimeter of the 'slow' arm; on the right, are scintillation hodoscopes and behind them, a treshold Cerenkov counter of the 'fast' arm.

  13. Frequent alterations of the PI3K/AKT/mTOR pathways in hereditary nonpolyposis colorectal cancer

    DEFF Research Database (Denmark)

    Ekstrand, Anna Isinger; Jönsson, Mats; Lindblom, Annika;

    2010-01-01

    The phosphatidylinositol 3-kinases-AKT-mammalian target of rapamycin pathway (PI3K/AKT/mTOR) is central in colorectal tumors. Data on its role in hereditary cancers are, however, scarce and we therefore characterized mutations in PIK3CA and KRAS, and expression of PIK3CA, phosphorylated AKT...... and PTEN in 58 HNPCC-associated colorectal cancers. Derangements of at least one of the PI3K/AKT/mTOR components analyzed were found in 51/58 (88%) tumors. Mutations in PIK3CA and KRAS were identified in 14 and 31% of the tumors respectively. Overexpression of PIK3CA and phosphorylated AKT occurred in 59...... and 75% and were strongly associated (P = 0.005). Reduced/lost PTEN expression was found in 63% of the tumors. Though HNPCC-associated colorectal cancers show simple genetic profiles with few chromosomal alterations, we demonstrate frequent and repeated targeting of the PI3K/AKT/mTOR pathway, which...

  14. Tamoxifen-induced cytotoxicity in breast cancer cells is mediated by glucose-regulated protein 78 (GRP78) via AKT (Thr308) regulation.

    Science.gov (United States)

    Pujari, Radha; Jose, Jemy; Bhavnani, Varsha; Kumar, Natesh; Shastry, Padma; Pal, Jayanta K

    2016-08-01

    Glucose regulated protein 78 (GRP78) has recently been suggested to be associated with drug resistance in breast cancer patients. However, the precise role of GRP78 in drug resistance and the involved signaling pathways are not clearly understood. In the present study, we show that among a panel of drugs, namely Paclitaxel (TAX), Doxorubicin (DOX), 5-fluorouracil (5-FU), UCN-01 and Tamoxifen (TAM) used, TAM alone up-regulated the expression of GRP78 significantly and induced apoptosis in MCF-7 and MDA-MB-231 cells. Interestingly, inhibition of GRP78 by a specific pharmacological inhibitor, VER-155008 augmented TAM-induced apoptosis, and overexpression of GRP78 rendered the cells resistant to TAM-induced cell death suggesting a role for GRP78 in TAM-induced cytotoxicity. Mechanistically, the expression of phosphorylated AKT as determined by Western blot analyses revealed that TAM selectively upregulated phosphorylation of AKT at Thr308 but not at Ser473, and siRNA silencing of GRP78 resulted in inhibition of AKT phosphorylation at Thr308 but not at Ser473. Further, a GRP78 inhibitor, VER155008 inhibited TAM-induced phosphorylation of GSK3β, a downstream substrate of AKT. These results, thus suggests a role for GRP78 in TAM-induced AKT activation. Additionally, co-localization studies by immunofluorescence, and immunoprecipitation experiments demonstrated a complex formation of AKT and GRP78. Furthermore, in glucose-free medium, the cells were sensitized to TAM-induced cell death that was associated with reduced AKT phosphorylation at Thr308, thus strengthening the association of AKT regulation with drug response. Collectively, our findings identify a role of GRP78 in AKT regulation in response to TAM in breast cancer cells. PMID:27262235

  15. Insulin inhibits inflammation and promotes atherosclerotic plaque stability via PI3K-Akt pathway activation.

    Science.gov (United States)

    Yan, Hao; Ma, Ying; Li, Yan; Zheng, Xiaohui; Lv, Ping; Zhang, Yuan; Li, Jia; Ma, Meijuan; Zhang, Le; Li, Congye; Zhang, Rongqing; Gao, Feng; Wang, Haichang; Tao, Ling

    2016-02-01

    Toll-like receptor (TLR) 4 induced inflammation was reported to play an important role in atherosclerotic plaque stability. Recent studies indicated that insulin could inhibit inflammation by activating phosphatidylinositol 3-kinase-Akt-dependent (PI3K-Akt) signaling pathway. In the current study, we hypothesized that insulin would inhibit TLR4 induced inflammation via promoting PI3K-Akt activation, thus enhancing the stabilization of atherosclerotic plaques. In order to mimic the process of plaque formation, monocyte-macrophage lineage RAW264.7 were cultured and induced to form foam cells by oxidized LDL (ox-LDL). Oil red O staining results showed that insulin significantly restrained ox-LDL-induced foam cell formation. Analysis of inflammatory reaction during foam cell formation indicated that insulin significantly down-regulated the expression of tumor necrosis factor (TNF)-α, interleukin (IL)-6 levels, inhibited TLR4, myeloid differentiation primary response gene (MyD) 88 and nuclear factor (NF)-κB. Further mechanism analysis showed that pretreating with the PI3K blocker, wortmannin dramatically dampened the insulin-induced up-regulation of pAkt expression. Additionally, blockade of PI3K-Akt signaling also dampened the immunosuppression effect brought by insulin. Following the construction of a rodent atherosclerosis model, pretreatment of insulin resulted in an evident decrease in lipid deposition of the blood vessel wall, serum levels of TNF-α and IL-6, and numbers of infiltrated macrophages and foam cells. Taken together, these results suggested that insulin might inhibit inflammation and promote atherosclerotic plaque stability via the PI3K-Akt pathway by targeting TLR4-MyD88-NF-κB signaling. Our findings may provide a potential target for the prevention of cardiovascular disease. PMID:26681144

  16. Reactive oxygen species and PI3K/Akt signaling in cancer.

    Science.gov (United States)

    Jin, Seo Yeon; Lee, Hye Sun; Kim, Eun Kyoung; Ha, Jung Min; Kim, Young Whan; Bae, SunSik

    2014-10-01

    Reactive oxygen species (ROS) are chemically reactive molecules containing oxygen and associates with multiple cellular functions such as cell proliferation, differentiation, and apoptosis. In the present study, we showed that Insulin-like growth factor-1(IGF-1) modulates SKOV-3 ovarian cancer cell by regulation of generation of ROS. Akt mediates cellular signaling pathways in association with mammalian target of rapamycin complex (mTOR) and Rac small G protein. Insulin-like growth factor-1 (IGF-1)-induced generation of ROS was completely abolished by phosphatidylinositol 3-kinase (PI3K) (LY294002, 10?µM) or Akt inhibitors (SH-5, 50?µM), whereas inhibition of extracellular-regulated kinase by an ERK inhibitor (PD98059, 10?µM) or inhibition of mammalian target of rapamycin complex 1 (mTORC1) by an mTORC1 inhibitor (Rapamycin, 100?nM) did not affect IGF-1-induced generation of ROS. Inactivation of mTORC2 by silencing Rapamycin-insensitive companion of mTOR (Rictor), abolished IGF-1-induced SKOV-3 cell migration as well as activation of Akt. However, inactivation of mTORC1 by silencing of Raptor had no effect. Silencing of Akt1 but not Akt2 attenuated IGF-1-induced generation of ROS. Expression of PIP3-dependent Rac exchanger1 (P-Rex1), a Rac guanosine exchange factor and a component of the mTOR complex. Silencing of P-Rex1 abolished IGF-1-induced generation of ROS. Finally, inhibition of NADPH oxidase system completely blunted IGF-1-induced generation of ROS, whereas inhibition of xanthine oxiase,cyclooxygenase, and mitochondrial respiratory chain complex was not effective. Given these results, we suggest that IGF-1 induces ROS generation through the PI3K/Akt/ mTOR2/NADPH oxidase signaling axis. PMID:26461347

  17. Novel PI3K/Akt inhibitors screened by the cytoprotective function of human immunodeficiency virus type 1 Tat.

    Directory of Open Access Journals (Sweden)

    Yuri Kim

    Full Text Available The PI3K/Akt pathway regulates various stress-related cellular responses such as cell survival, cell proliferation, metabolism and protein synthesis. Many cancer cell types display the activation of this pathway, and compounds inhibiting this cell survival pathway have been extensively evaluated as anti-cancer agents. In addition to cancers, several human viruses, such as HTLV, HPV, HCV and HIV-1, also modulate this pathway, presumably in order to extend the life span of the infected target cells for productive viral replication. The expression of HIV-1 Tat protein exhibited the cytoprotective effect in macrophages and a human microglial cell line by inhibiting the negative regulator of this pathway, PTEN. This cytoprotective effect of HIV-1 appears to contribute to the long-term survival and persistent HIV-1 production in human macrophage reservoirs. In this study we exploited the PI3K/Akt dependent cytoprotective effect of Tat-expressing CHME5 cells. We screened a collection of compounds known to modulate inflammation, and identified three novel compounds: Lancemaside A, Compound K and Arctigenin that abolished the cytoprotective phenotype of Tat-expressing CHME5 cells. All three compounds antagonized the kinase activity of Akt. Further detailed signaling studies revealed that each of these three compounds targeted different steps of the PI3K/Akt pathway. Arctigenin regulates the upstream PI3K enzyme from converting PIP2 to PIP3. Lancemaside A1 inhibited the movement of Akt to the plasma membrane, a critical step for Akt activation. Compound K inhibited Akt phosphorylation. This study supports that Tat-expressing CHME5 cells are an effective model system for screening novel PI3K/Akt inhibitors.

  18. Angiotensin Ⅱ suppresses adriamycin-induced apoptosis through activation of phosphatidylinositol 3-kinase/Akt signaling in human breast cancer cells

    Institute of Scientific and Technical Information of China (English)

    Yanbin Zhao; Xuesong Chen; Li Cai; Yanmei Yang; Guangjie Sui; Jin Wu

    2008-01-01

    Angiotensin Ⅱ (Ang Ⅱ) stimulates tumor growth and angiogenesis in some solid cancer cells, but its anti-apoptosis role in breast cancer remains unclear. To address this issue, we investigated the effect of Ang Ⅱ on adriamycin-induced apoptosis in breast cancer MCF-7 cells. Treatment of human breast cancer MCF-7 cells with adriamycin, a DNA topoisomerase Hα inhibitor, caused apoptosis. However, cells pretreated with Ang Ⅱ were resistant to this apoptosis. Ang Ⅱ significantly reduced the ratio of apoptotic cells and stimulation of phospho-Akt-Thr308 and phospho-Akt-Ser473 in a dose-dependent and time-dependent manner. In addition, Ang Ⅱ significantly prevented apoptosis through inhibiting the cleavage of procaspase-9, a major downstream effector of Akt.The Ang Ⅱ type 1 receptor (AT1R) was responsible for these effects. Among the signaling molecules downstream of AT1R,we revealed that the phosphatidylinositol 3-kinase/Akt pathway plays a predominant role in the anti-apoptotic effect of Ang Ⅱ. Our data indicated that Ang Ⅱ plays a critical antiapoptotic role in breast cancer cells by a mechanism involving AT1R/phosphatidylinositol 3-kinase/Akt activation and the subsequent suppression of caspase-9 activation.

  19. mTORC2 phosphorylation of Akt1: a possible mechanism for hydrogen sulfide-induced cardioprotection.

    Directory of Open Access Journals (Sweden)

    Yue Zhou

    Full Text Available Hydrogen sulfide (H2S is known to have cardiac protective effects through Akt activation. Akt acts as a 'central sensor' for myocyte survival or death; its activity is regulated by multiple kinases including PI3K, mTORC2, PDK1 and phosphatases including PTEN, PP2A and PHLPPL. Based on the previous finding that PI3K inhibitor LY294002 abolishes H2S-induced Akt phosphorylation and cardioprotection, it is accepted that PI3K is the mediator of H2S-induced Akt phosphorylation. However, LY294002 inhibits both PI3K and mTOR, and PI3K only recruits Akt to the membrane where Akt is phosphorylated by Akt kinases. We undertook a series of experiments to further evaluate the role of mTORC2, PDK1, PTEN, PP2A and PHLPPL in H2S-induced Akt phosphorylation and cardioprotection, which, we believe, has not been investigated before. Hearts from adult Sprague-Dawley rats were isolated and subjected to (i normoxia, (ii global ischemia and (iii ischemia/reperfusion in the presence or absence of 50 µM of H2S donor NaHS. Cardiac mechanical function and lactate dehydrogenase (LDH release were assessed. All hearts also were Western analyzed at the end of perfusion for Akt and a panel of appropriate Akt regulators and targets. Hearts pretreated with 50 µM NaHS had improved function at the end of reperfusion (Rate pressure product; 19±4×10(3 vs. 10±3×10(3 mmHg/min, p<0.05 and reduced cell injury (LDH release 19±10 vs. 170±87 mU/ml p<0.05 compared to untreated hearts. NaHS significantly increased phospho-Akt, phospho-mTOR, phospho-Bim and Bcl-2 in reperfused hearts (P<0.05. Furthermore using H9c2 cells we demonstrate that NaHS pretreatment reduces apoptosis following hypoxia/re-oxygenation. Importantly, PP242, a specific mTOR inhibitor, abolished both cardioprotection and protein phosphorylation in isolated heart and reduced apoptotic effects in H9c2 cells. Treating hearts with NaHS only during reperfusion produced less cardioprotection through a similar mechanism

  20. Establishment of a luciferase assay-based screening system: Fumitremorgin C selectively inhibits cellular proliferation of immortalized astrocytes expressing an active form of AKT

    International Nuclear Information System (INIS)

    The AKT pathway is frequently activated in glioblastoma, and as such, inhibitors of this pathway could prove very useful as anti-glioblastoma therapies. Here we established immortalized astrocytes expressing Renilla luciferase as well as those expressing both an active form of AKT and firefly luciferase. Since both luciferase activities represent the numbers of corresponding cell lines, novel inhibitors of the AKT pathway can be identified by treating co-cultures containing the two types of luciferase-expressing cells with individual compounds. Indeed, such a screening system succeeded in identifying fumitremorgin C as an efficient inhibitor of the AKT pathway, which was further confirmed by the ability of fumitremorgin C to selectively inhibit the growth of immortalized astrocytes expressing an active form of AKT. The present study proposes a broadly applicable approach for identifying therapeutic agents that target the pathways and/or molecules responsible for cancer development

  1. Pyrvinium attenuates Hedgehog signaling downstream of smoothened.

    Science.gov (United States)

    Li, Bin; Fei, Dennis Liang; Flaveny, Colin A; Dahmane, Nadia; Baubet, Valérie; Wang, Zhiqiang; Bai, Feng; Pei, Xin-Hai; Rodriguez-Blanco, Jezabel; Hang, Brian; Orton, Darren; Han, Lu; Wang, Baolin; Capobianco, Anthony J; Lee, Ethan; Robbins, David J

    2014-09-01

    The Hedgehog (HH) signaling pathway represents an important class of emerging developmental signaling pathways that play critical roles in the genesis of a large number of human cancers. The pharmaceutical industry is currently focused on developing small molecules targeting Smoothened (Smo), a key signaling effector of the HH pathway that regulates the levels and activity of the Gli family of transcription factors. Although one of these compounds, vismodegib, is now FDA-approved for patients with advanced basal cell carcinoma, acquired mutations in Smo can result in rapid relapse. Furthermore, many cancers also exhibit a Smo-independent activation of Gli proteins, an observation that may underlie the limited efficacy of Smo inhibitors in clinical trials against other types of cancer. Thus, there remains a critical need for HH inhibitors with different mechanisms of action, particularly those that act downstream of Smo. Recently, we identified the FDA-approved anti-pinworm compound pyrvinium as a novel, potent (IC50, 10 nmol/L) casein kinase-1α (CK1α) agonist. We show here that pyrvinium is a potent inhibitor of HH signaling, which acts by reducing the stability of the Gli family of transcription factors. Consistent with CK1α agonists acting on these most distal components of the HH signaling pathway, pyrvinium is able to inhibit the activity of a clinically relevant, vismodegib -resistant Smo mutant, as well as the Gli activity resulting from loss of the negative regulator suppressor of fused. We go on to demonstrate the utility of this small molecule in vivo, against the HH-dependent cancer medulloblastoma, attenuating its growth and reducing the expression of HH biomarkers. PMID:24994715

  2. Akt2/PKBbeta-sensitive regulation of renal phosphate transport

    OpenAIRE

    Kempe, D S; Ackermann, T F; Boini, K M; Klaus, F; Umbach, A T; Dërmaku-Sopjani, M; Judenhofer, M S; Pichler, B J; Capuano, P.; Stange, G.; Wagner, C. A.; Birnbaum, M J; Pearce, D.; Föller, M; Lang, F.

    2010-01-01

    AIM: The protein kinase B (PKB)/Akt is known to stimulate the cellular uptake of glucose and amino acids. The kinase is expressed in proximal renal tubules. The present study explored the influence of Akt/PKB on renal tubular phosphate transport. METHODS: The renal phosphate transporter NaPi-IIa was expressed in Xenopus oocytes with or without PKB/Akt and Na(+) phosphate cotransport determined using dual electrode voltage clamp. Renal phosphate excretion was determined in Akt2/PKBbeta knockou...

  3. Akt is negatively regulated by the MULAN E3 ligase

    OpenAIRE

    Bae, Seunghee; Kim, Sun-Yong; Jung, Jin Hyuk; Yoon, Yeongmin; CHA, HWA JUN; Lee, Hyunjin; Kim, Karam; Kim, Jongran; An, In-Sook; Kim, Jongdoo; UM, HONG-DUCK; Park, In-Chul; Lee, Su-Jae; Nam, Seon Young; Jin, Young-Woo

    2012-01-01

    The serine/threonine kinase Akt functions in multiple cellular processes, including cell survival and tumor development. Studies of the mechanisms that negatively regulate Akt have focused on dephosphorylation-mediated inactivation. In this study, we identified a negative regulator of Akt, MULAN, which possesses both a RING finger domain and E3 ubiquitin ligase activity. Akt was found to directly interact with MULAN and to be ubiquitinated by MULAN in vitro and in vivo. Other molecular assays...

  4. Upstream-downstream cooperation approach in Guanting Reservoir watershed

    Institute of Scientific and Technical Information of China (English)

    YANG Zhi-feng; ZHANG Wen-guo

    2005-01-01

    A case study is introduced and discussed concerning water dispute of misuse and pollution between up- and down-stream parts.The relations between water usage and local industrial structures are analyzed. Results show it is important to change industrial structures of the target region along with controlling water pollution by technical and engineering methods. Three manners of upstream-downstream cooperation are presented and discussed based on the actual conditions of Guangting Reservoir watershed, Two typical scenarios are supposed and studied along with the local plan on water resources development. The best solution for this cooperation presents a good way to help the upstream developing in a new pattern of eco-economy.

  5. Adenosine triphosphate-sensitive potassium channel opener protects PC12 cells against hypoxia-induced apoptosis through PI3K/Akt and Bcl-2 signaling pathways

    Institute of Scientific and Technical Information of China (English)

    Hong Zhang; Chunhong Jia; Danyang Zhao; Yang Lu; Runling Wang; Jia Li

    2010-01-01

    Although previous studies have shown the neuroprotective effects of the adenosine triphosphate (ATP)-sensitive potassium (KATP) channel opener against ischemic neuronal damage, little is known about the mechanisms involved. Phosphatidylinositol-3 kinase (PI3K)/v-akt murine thy-moma viral oncogene homolog (Akt) and Bcl-2 are thought to be important factors that mediate neuroprotection. The present study investigated the effects of KATP openers on hypoxia-induced PC12 cell apoptosis, as well as mRNA and protein expression of Akt and Bcl-2. Results demon-strated that pretreatment of PC12 cells with pinacidil, a KATP opener, resulted in decreased PC12 cell apoptosis following hypoxia, as detected by Annexin-V fluorescein isothiocyanate/ propidium iodide double staining flow cytometry. In addition, mRNA and protein expression of phosphorylated Akt (p-Akt) and Bcl-2 increased, as detected by immunofluorescence, Western blot analysis, and reverse-transcription polymerase chain reaction. The protective effect of this preconditioning was attenuated by glipizide, a selective KATP blocker. These results demonstrate for the first time that the protective mechanisms of KATP openers on PC12 cell apoptosis following hypoxia could result from activation of the PI3K/Akt signaling pathway, which further activates expression of the downstream Bcl-2 gene.

  6. Methylmercury, an environmental electrophile capable of activation and disruption of the Akt/CREB/Bcl-2 signal transduction pathway in SH-SY5Y cells

    Science.gov (United States)

    Unoki, Takamitsu; Abiko, Yumi; Toyama, Takashi; Uehara, Takashi; Tsuboi, Koji; Nishida, Motohiro; Kaji, Toshiyuki; Kumagai, Yoshito

    2016-01-01

    Methylmercury (MeHg) modifies cellular proteins via their thiol groups in a process referred to as “S-mercuration”, potentially resulting in modulation of the cellular signal transduction pathway. We examined whether low-dose MeHg could affect Akt signaling involved in cell survival. Exposure of human neuroblastoma SH-SY5Y cells of up to 2 μM MeHg phosphorylated Akt and its downstream signal molecule CREB, presumably due to inactivation of PTEN through S-mercuration. As a result, the anti-apoptotic protein Bcl-2 was up-regulated by MeHg. The activation of Akt/CREB/Bcl-2 signaling mediated by MeHg was, at least in part, linked to cellular defence because either pretreatment with wortmannin to block PI3K/Akt signaling or knockdown of Bcl-2 enhanced MeHg-mediated cytotoxicity. In contrast, increasing concentrations of MeHg disrupted Akt/CREB/Bcl-2 signaling. This phenomenon was attributed to S-mercuration of CREB through Cys286 rather than Akt. These results suggest that although MeHg is an apoptosis-inducing toxicant, this environmental electrophile is able to activate the cell survival signal transduction pathway at lower concentrations prior to apoptotic cell death. PMID:27357941

  7. Differential regulation of mTOR signaling determines sensitivity to AKT inhibition in diffuse large B cell lymphoma

    OpenAIRE

    Ezell, Scott A.; Wang, Suping; Bihani, Teeru; Lai, Zhongwu; Grosskurth, Shaun E.; Tepsuporn, Suprawee; Davies, Barry R; Huszar, Dennis; Byth, Kate F.

    2016-01-01

    Agents that target components of the PI3K/AKT/mTOR pathway are under investigation for the treatment of diffuse large B cell lymphoma (DLBCL). Given the highly heterogeneous nature of DLBCL, it is not clear whether all subtypes of DLBCL will be susceptible to PI3K pathway inhibition, or which kinase within this pathway is the most favorable target. Pharmacological profiling of a panel of DLBCL cell lines revealed a subset of DLBCL that was resistant to AKT inhibition. Strikingly, sensitivity ...

  8. Akt Links Insulin Signaling to Albumin Endocytosis in Proximal Tubule Epithelial Cells.

    Science.gov (United States)

    Coffey, Sam; Costacou, Tina; Orchard, Trevor; Erkan, Elif

    2015-01-01

    Diabetes mellitus (DM) has become an epidemic, causing a significant decline in quality of life of individuals due to its multisystem involvement. Kidney is an important target organ in DM accounting for the majority of patients requiring renal replacement therapy at dialysis units. Microalbuminuria (MA) has been a valuable tool to predict end-organ damage in DM but its low sensitivity has driven research efforts to seek other alternatives. Albumin is taken up by albumin receptors, megalin and cubilin in the proximal tubule epithelial cells. We demonstrated that insulin at physiological concentrations induce albumin endocytosis through activation of protein kinase B (Akt) in proximal tubule epithelial cells. Inhibition of Akt by a phosphorylation deficient construct abrogated insulin induced albumin endocytosis suggesting a role for Akt in insulin-induced albumin endocytosis. Furthermore we demonstrated a novel interaction between Akt substrate 160kDa (AS160) and cytoplasmic tail of megalin. Mice with type 1 DM (T1D) displayed decreased Akt, megalin, cubilin and AS160 expression in their kidneys in association with urinary cubilin shedding preceding significant MA. Patients with T1D who have developed MA in the EDC (The Pittsburgh Epidemiology of Diabetes Complications) study demonstrated urinary cubilin shedding prior to development of MA. We hypothesize that perturbed insulin-Akt cascade in DM leads to alterations in trafficking of megalin and cubilin, which results in urinary cubilin shedding as a prelude to MA in early diabetic nephropathy. We propose that utilization of urinary cubilin shedding, as a urinary biomarker, will allow us to detect and intervene in diabetic nephropathy (DN) at an earlier stage. PMID:26465605

  9. Akt Links Insulin Signaling to Albumin Endocytosis in Proximal Tubule Epithelial Cells.

    Directory of Open Access Journals (Sweden)

    Sam Coffey

    Full Text Available Diabetes mellitus (DM has become an epidemic, causing a significant decline in quality of life of individuals due to its multisystem involvement. Kidney is an important target organ in DM accounting for the majority of patients requiring renal replacement therapy at dialysis units. Microalbuminuria (MA has been a valuable tool to predict end-organ damage in DM but its low sensitivity has driven research efforts to seek other alternatives. Albumin is taken up by albumin receptors, megalin and cubilin in the proximal tubule epithelial cells. We demonstrated that insulin at physiological concentrations induce albumin endocytosis through activation of protein kinase B (Akt in proximal tubule epithelial cells. Inhibition of Akt by a phosphorylation deficient construct abrogated insulin induced albumin endocytosis suggesting a role for Akt in insulin-induced albumin endocytosis. Furthermore we demonstrated a novel interaction between Akt substrate 160kDa (AS160 and cytoplasmic tail of megalin. Mice with type 1 DM (T1D displayed decreased Akt, megalin, cubilin and AS160 expression in their kidneys in association with urinary cubilin shedding preceding significant MA. Patients with T1D who have developed MA in the EDC (The Pittsburgh Epidemiology of Diabetes Complications study demonstrated urinary cubilin shedding prior to development of MA. We hypothesize that perturbed insulin-Akt cascade in DM leads to alterations in trafficking of megalin and cubilin, which results in urinary cubilin shedding as a prelude to MA in early diabetic nephropathy. We propose that utilization of urinary cubilin shedding, as a urinary biomarker, will allow us to detect and intervene in diabetic nephropathy (DN at an earlier stage.

  10. PARP1 inhibitors attenuate AKT phosphorylation via the upregulation of PHLPP1

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Shuai [State Key Laboratory of Medical Genomics, Shanghai Institute of Hematology, Rui Jin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200025 (China); Division of Hematology and Oncology, Comprehensive Cancer Center, University of Alabama at Birmingham, Birmingham, AL 35205 (United States); Wang, Huibo; Davis, Ben C. [Division of Hematology and Oncology, Comprehensive Cancer Center, University of Alabama at Birmingham, Birmingham, AL 35205 (United States); Liang, Jiyong [Department of Systems Biology, University of Texas MD Anderson Cancer Center, Houston, TX 77054 (United States); Cui, Rutao [Department of Dermatology, Boston University School of Medicine, Boston, MA 02118 (United States); Chen, Sai-Juan, E-mail: sjchen@stn.sh.cn [State Key Laboratory of Medical Genomics, Shanghai Institute of Hematology, Rui Jin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200025 (China); Xu, Zhi-Xiang, E-mail: zhi-xiang.xu@ccc.uab.edu [Division of Hematology and Oncology, Comprehensive Cancer Center, University of Alabama at Birmingham, Birmingham, AL 35205 (United States)

    2011-08-26

    Highlights: {yields} PARP1 inhibitors cause a cytotoxic effect independent of DNA repair impairment. {yields} PARP1 inhibitors attenuated AKT-FOXO3A signaling by activating PHLPP1. {yields} PHLPP1 regulates the sensitivity of cancer cells to PARP1 inhibitors. -- Abstract: Poly(ADP-ribose) polymerase-1 (PARP1) inhibitors are emerging as an important class of drugs for treating BRCA-deficient cancers. Recent discoveries have shown that PARP1 inhibitors may treat other cancer patients in addition to the relatively small proportion of patients carrying BRCA mutations. However, the additional targets by which PARP1 inhibitor-mediated tumor suppression remain poorly understood. In this study, we show that two PARP1 inhibitors, PJ-34 and 3-AB, attenuate AKT phosphorylation at serine 473 (S473) independent of DNA repair impairment. These inhibitors decrease the AKT-associated phosphorylation of FOXO3A, enhance the nuclear retention of FOXO3A, and activate its transcriptional activity. We further demonstrate that treatment with PJ-34 or 3-AB dramatically increases the level of PHLPP1. Overexpression of PHLPP1 enhances the PARP1 inhibitor-induced downregulation of AKT phosphorylation and increases tumor cell death. In contrast, knockdown of PHLPP1 abrogates the PARP1 inhibitor-mediated AKT inhibition and desensitizes cells to its treatment. Therefore, our findings not only show the robust role of PARP1 inhibitors in AKT inhibition but also develop a novel strategy to increase the effectiveness of cancer treatment via PARP1 inhibitor-induced PHLPP1 upregulation.

  11. Metformin and rapamycin have distinct effects on the AKT pathway and proliferation in breast cancer cells.

    Science.gov (United States)

    Zakikhani, Mahvash; Blouin, Marie-José; Piura, Esther; Pollak, Michael N

    2010-08-01

    Rapamycin and its analogues inhibit mTOR, which leads to decreased protein synthesis and decreased cancer cell proliferation in many experimental systems. Adenosine 5'- monophosphate-activated protein kinase (AMPK) activators such as metformin have similar actions, in keeping with the TSC2/1 pathway linking activation of AMPK to inhibition of mTOR. As mTOR inhibition by rapamycin is associated with attenuation of negative feedback to IRS-1, rapamycin is known to increase activation of AKT, which may reduce its anti-neoplastic activity. We observed that metformin exposure decreases AKT activation, an action opposite to that of rapamycin. We show that metformin (but not rapamycin) exposure leads to increased phosphorylation of IRS-1 at Ser(789), a site previously reported to inhibit downstream signaling and to be an AMPK substrate phosphorylated under conditions of cellular energy depletion. siRNA methods confirmed that reduction of AMPK levels attenuates both the IRS-1 Ser(789) phosphorylation and the inhibition of AKT activation associated with metformin exposure. Although both rapamycin and metformin inhibit mTOR (the former directly and the latter through AMPK signaling), our results demonstrate previously unrecognized differences between these agents. The data are consistent with the observation that maximal induction of apoptosis and inhibition of proliferation are greater for metformin than rapamycin. PMID:20135346

  12. Targeted disruption of fibrinogen like protein-1 accelerates hepatocellular carcinoma development

    Energy Technology Data Exchange (ETDEWEB)

    Nayeb-Hashemi, Hamed; Desai, Anal; Demchev, Valeriy [Division of Gastroenterology, Hepatology and Endoscopy, Department of Medicine. Brigham and Women' s Hospital and Harvard Medical School, Boston, MA 02115 (United States); Bronson, Roderick T. [Department of Microbiology and Immunology, Harvard Medical School, Boston, MA 02115 (United States); Hornick, Jason L. [Department of Pathology, Brigham and Women' s Hospital and Harvard Medical School, Boston, MA 02115 (United States); Cohen, David E. [Division of Gastroenterology, Hepatology and Endoscopy, Department of Medicine. Brigham and Women' s Hospital and Harvard Medical School, Boston, MA 02115 (United States); Ukomadu, Chinweike, E-mail: cukomadu@partners.org [Division of Gastroenterology, Hepatology and Endoscopy, Department of Medicine. Brigham and Women' s Hospital and Harvard Medical School, Boston, MA 02115 (United States)

    2015-09-18

    Fibrinogen like protein-1 (Fgl1) is a predominantly liver expressed protein that has been implicated as both a hepatoprotectant and a hepatocyte mitogen. Fgl1 expression is decreased in hepatocellular carcinoma (HCC) and its loss correlates with a poorly differentiated phenotype. To better elucidate the role of Fgl1 in hepatocarcinogenesis, we treated mice wild type or null for Fgl1 with diethyl nitrosamine and monitored for incidence of hepatocellular cancer. We find that mice lacking Fgl1 develop HCC at more than twice the rate of wild type mice. We show that hepatocellular cancers from Fgl1 null mice are molecularly distinct from those of the wild type mice. In tumors from Fgl1 null mice there is enhanced activation of Akt and downstream targets of the mammalian target of rapamycin (mTOR). In addition, there is paradoxical up regulation of putative hepatocellular cancer tumor suppressors; tripartite motif-containing protein 35 (Trim35) and tumor necrosis factor super family 10b (Tnfrsf10b). Taken together, these findings suggest that Fgl1 acts as a tumor suppressor in hepatocellular cancer through an Akt dependent mechanism and supports its role as a potential therapeutic target in HCC. - Highlights: • Fgl1 knockout mice (Fgl1KO) are more prone to carcinogen-induced liver cancer compared to wild type (WT) mates. • Tumors from the Fgl1KO are molecularly distinct with enhanced Akt and mTOR activity in comparison with Fgl1WT tumors. • Tumors from the Fgl1KO have enhanced expression of Trim35 and Tnfrsf10b, putative HCC tumor suppressors.

  13. Development of Drugs Targeting the PI3K Signalling Pathway in Leukaemias and Lymphomas

    Directory of Open Access Journals (Sweden)

    Alexandre Arcaro

    2015-03-01

    Full Text Available The phosphoinositide 3-kinase (PI3K family of signalling enzymes play a key role in the transduction of signals from activated cell surface receptors controlling cell growth and proliferation, survival, metabolism, and migration. The intracellular signalling pathway from activated receptors to PI3K and its downstream targets v-akt murine thymoma viral oncogene homolog (Akt and mechanistic target of rapamycin (mTOR is very frequently deregulated by genetic and epigenetic mechanisms in human cancer, including leukaemia and lymphoma. In the past decade, an arsenal of small molecule inhibitors of key enzymes in this pathway has been developed and evaluated in pre-clinical studies and clinical trials in cancer patients. These include pharmacological inhibitors of Akt, mTOR, and PI3K, some of which are approved for the treatment of leukaemia and lymphoma. The PI3K family comprises eight different catalytic isoforms in humans, which have been subdivided into three classes. Class I PI3K isoforms have been extensively studied in the context of human cancer, and the isoforms p110α and p110δ are validated drug targets. The recent approval of a p110δ-specific PI3K inhibitor (idelalisib/Zydelig® for the treatment of selected B cell malignancies represents the first success in developing these molecules into anti-cancer drugs. In addition to PI3K inhibitors, mTOR inhibitors are intensively studied in leukaemia and lymphoma, and temsirolimus (Torisel® is approved for the treatment of a type of lymphoma. Based on these promising results it is hoped that additional novel PI3K pathway inhibitors will in the near future be further developed into new drugs for leukaemia and lymphoma.

  14. Characterization of AKT independent effects of the synthetic AKT inhibitors SH-5 and SH-6 using an integrated approach combining transcriptomic profiling and signaling pathway perturbations

    International Nuclear Information System (INIS)

    confocal laser scanning microscopy and time lapse recording we identified a specific defect in the last step of the cytokinesis as responsible for the binucleation. The PIAs SH-5 and SH-6 impinge on additional cellular targets apart from AKT in colorectal cancer cells. The effects are mostly cell line specific and have an influence at the outcome of the treatment. In view of potential clinical trials it will be necessary to take these diverse effects into consideration to optimize patient treatment

  15. The Novel Small Molecule Inhibitor, OSU-T315, Suppresses Vestibular Schwannoma and Meningioma Growth by Inhibiting PDK2 Function in the AKT Pathway Activation

    Science.gov (United States)

    Mercado-Pimentel, ME; Igarashi, S; Dunn, AM; Behbahani, M; Miller, C; Read, CM; Jacob, A

    2016-01-01

    Activation of PKB/AKT signaling, which requires PDK1 and PDK2 function, drives Vestibular Schwannoma (VS) and meningioma growth. PDK2 function is defined as a molecule that phosphorylates AKT-Ser473. Integrin-Linked Kinase (ILK) functions as PDK2 in PKB/AKT activation in many cancers; therefore, we hypothesized that OSU-T315, a small molecule ILK inhibitor, will inhibit the ILK-PDK2 function in PKB/AKT signaling activation in VS and meningioma cell growth. OSU-T315 decreased cell viability at IC50 < 2μM in VS (HEI193) and meningioma (Ben-Men-1) cell lines, in primary cells at < 3.5μM, while in normal primary Schwann cells at 7.1μM. OSU-T315 inhibits AKT signaling by decreasing phosphorylation at AKT-Ser473, AKT-Thr308, ILK-Ser246 and ILK-Thr173. In addition, OSU-T315 affected the phosphorylation or expression levels of AKT downstream proliferation effectors as well as autophagy markers. Flow cytometry shows that OSU-T315 increased the percentage of cells arrested at G2/M for both, HEI193 (39.99%) and Ben-Men-1 (26.96%) cells, compared to controls (21.54%, 8.47%). Two hours of OSU-T315 treatment increased cell death in both cell lines (34.3%, 9.1%) versus untreated (12.1%, 8.1%). Though longer exposure increased cell death in Ben-Men-1, TUNEL assays showed that OSU-T315 does not induce apoptosis. OSU-T315 was primarily cytotoxic for HEI193 and Ben-Men-1 inducing a dysregulated autophagy. Our studies suggest that OSU-T315 has translational potential as a chemotherapeutic agent against VS and meningioma.

  16. ChAkt1 involvement in orchestrating the immune and heat shock responses in Crassostrea hongkongensis: Molecular cloning and functional characterization.

    Science.gov (United States)

    Wang, Fuxuan; Xiao, Shu; Zhang, Yang; Zhang, Yuehuan; Liu, Ying; Yan, Yan; Xiang, Zhiming; Yu, Ziniu

    2015-12-01

    G-protein-coupled receptors (GPCRs) are the largest class of cell-surface receptors and play crucial roles in virtually every organ system. As one of the major downstream effectors of GPCRs, Akt can acquire information from the receptors and coordinate intracellular responses for many signaling pathways, through which the serine/threonine kinase masters numerous aspects of biological processes, such as cell survival, growth, proliferation, migration, angiogenesis, and metabolism. In the present study, we have characterized the first Akt1 ortholog in mollusks using the Hong Kong oyster, Crassostrea hongkongensis (designed ChAkt1). The full-length cDNA is 2223 bp and encodes a putative protein of 493 amino acids that contains an amino-terminal pleckstin homology (PH) domain, a central catalytic domain, and a carboxy-terminal regulatory domain. Quantitative real-time PCR analysis showed that ChAkt1 mRNA is broadly expressed in various tissues and during different stages of the oyster's embryonic and larval development. Upon exposure to two stressors (microbial infection and heat shock), the expression level of ChAkt1 mRNA increases significantly. Furthermore, ChAkt1 is located in the cytoplasm in HEK293T cells, where the over-expression of ChAkt1 regulates the transcriptional activities of NF-κB and p53 reporter genes. Taken together, our results indicate that ChAkt1 most likely plays a central role in response to various stimuli in oysters and has a particular response to microbial pathogens and high temperature.

  17. Phosphoinositide-3-kinase/akt - dependent signaling is required for maintenance of [Ca2+]i,ICa, and Ca2+ transients in HL-1 cardiomyocytes

    Directory of Open Access Journals (Sweden)

    Graves Bridget M

    2012-06-01

    Full Text Available Abstract The phosphoinositide 3-kinases (PI3K/Akt dependent signaling pathway plays an important role in cardiac function, specifically cardiac contractility. We have reported that sepsis decreases myocardial Akt activation, which correlates with cardiac dysfunction in sepsis. We also reported that preventing sepsis induced changes in myocardial Akt activation ameliorates cardiovascular dysfunction. In this study we investigated the role of PI3K/Akt on cardiomyocyte function by examining the role of PI3K/Akt-dependent signaling on [Ca2+]i, Ca2+ transients and membrane Ca2+ current, ICa, in cultured murine HL-1 cardiomyocytes. LY294002 (1–20 μM, a specific PI3K inhibitor, dramatically decreased HL-1 [Ca2+]i, Ca2+ transients and ICa. We also examined the effect of PI3K isoform specific inhibitors, i.e. α (PI3-kinase α inhibitor 2; 2–8 nM; β (TGX-221; 100 nM and γ (AS-252424; 100 nM, to determine the contribution of specific isoforms to HL-1 [Ca2+]i regulation. Pharmacologic inhibition of each of the individual PI3K isoforms significantly decreased [Ca2+]i, and inhibited Ca2+ transients. Triciribine (1–20 μM, which inhibits AKT downstream of the PI3K pathway, also inhibited [Ca2+]i, and Ca2+ transients and ICa. We conclude that the PI3K/Akt pathway is required for normal maintenance of [Ca2+]i in HL-1 cardiomyocytes. Thus, myocardial PI3K/Akt-PKB signaling sustains [Ca2+]i required for excitation-contraction coupling in cardiomyoctyes.

  18. Biliverdin Reductase: a Target for Cancer Therapy?

    Directory of Open Access Journals (Sweden)

    Peter eGibbs

    2015-06-01

    Full Text Available Biliverdin reductase (BVR is a multifunctional protein that is the primary source of the potent antioxidant, bilirubin. BVR regulates activities/functions in the insulin/IGF-1/IRK/PI3K/MAPK pathways. Activation of certain kinases in these pathways is/are hallmark(s of cancerous cells. The protein is a scaffold/bridge and intracellular transporter of kinases that regulate growth and proliferation of cells, including PKCs, ERK and Akt, and their targets including NF-κB, Elk1, HO-1 and iNOS. The scaffold and transport functions enable activated BVR to relocate from the cytosol to the nucleus or to the plasma membrane, depending on the activating stimulus. This enables the reductase to function in diverse signaling pathways. And, its expression at the transcript and protein levels are increased in human tumors and the infiltrating T-cells, monocytes and circulating lymphocytes, as well as the circulating and infiltrating macrophages. These functions suggest that the cytoprotective role of BVR may be permissive for cancer/tumor growth. In this review, we summarize the recent developments that define the pro-growth activities of BVR, particularly with respect to its input into the MAPK signaling pathway and present evidence that BVR-based peptides inhibit activation of protein kinases, including MEK, PKCδ and ERK as well as downstream targets including Elk1 and iNOS, and thus offers a credible novel approach to reduce cancer cell proliferation.

  19. Lovastatin inhibits VEGFR and AKT activation: synergistic cytotoxicity in combination with VEGFR inhibitors.

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    Tong T Zhao

    Full Text Available BACKGROUND: In a recent study, we demonstrated the ability of lovastatin, a potent inhibitor of mevalonate synthesis, to inhibit the function of the epidermal growth factor receptor (EGFR. Lovastatin attenuated ligand-induced receptor activation and downstream signaling through the PI3K/AKT pathway. Combining lovastatin with gefitinib, a potent EGFR inhibitor, induced synergistic cytotoxicity in a variety of tumor derived cell lines. The vascular endothelial growth factor receptor (VEGFR and EGFR share similar activation, internalization and downstream signaling characteristics. METHODOLOGY/PRINCIPAL FINDINGS: The VEGFRs, particularly VEGFR-2 (KDR, Flt-1, play important roles in regulating tumor angiogenesis by promoting endothelial cell proliferation, survival and migration. Certain tumors, such as malignant mesothelioma (MM, also express both the VEGF ligand and VEGFRs that act in an autocrine loop to directly stimulate tumor cell growth and survival. In this study, we have shown that lovastatin inhibits ligand-induced VEGFR-2 activation through inhibition of receptor internalization and also inhibits VEGF activation of AKT in human umbilical vein endothelial cells (HUVEC and H28 MM cells employing immunofluorescence and Western blotting. Combinations of lovastatin and a VEGFR-2 inhibitor showed more robust AKT inhibition than either agent alone in the H28 MM cell line. Furthermore, combining 5 µM lovastatin treatment, a therapeutically relevant dose, with two different VEGFR-2 inhibitors in HUVEC and the H28 and H2052 mesothelioma derived cell lines demonstrated synergistic cytotoxicity as demonstrated by MTT cell viability and flow cytometric analyses. CONCLUSIONS/SIGNIFICANCE: These results highlight a novel mechanism by which lovastatin can regulate VEGFR-2 function and a potential therapeutic approach for MM through combining statins with VEGFR-2 inhibitors.

  20. A genome-wide RNAi screen identifies FOXO4 as a metastasis-suppressor through counteracting PI3K/AKT signal pathway in prostate cancer.

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    Bing Su

    Full Text Available Activation of the PI3K/AKT signal pathway is a known driving force for the progression to castration-recurrent prostate cancer (CR-CaP, which constitutes the major lethal phenotype of CaP. Here, we identify using a genomic shRNA screen the PI3K/AKT-inactivating downstream target, FOXO4, as a potential CaP metastasis suppressor. FOXO4 protein levels inversely correlate with the invasive potential of a panel of human CaP cell lines, with decreased mRNA levels correlating with increased incidence of clinical metastasis. Knockdown (KD of FOXO4 in human LNCaP cells causes increased invasion in vitro and lymph node (LN metastasis in vivo without affecting indices of proliferation or apoptosis. Increased Matrigel invasiveness was found by KD of FOXO1 but not FOXO3. Comparison of differentially expressed genes affected by FOXO4-KD in LNCaP cells in culture, in primary tumors and in LN metastases identified a panel of upregulated genes, including PIP, CAMK2N1, PLA2G16 and PGC, which, if knocked down by siRNA, could decrease the increased invasiveness associated with FOXO4 deficiency. Although only some of these genes encode FOXO promoter binding sites, they are all RUNX2-inducible, and RUNX2 binding to the PIP promoter is increased in FOXO4-KD cells. Indeed, the forced expression of FOXO4 reversed the increased invasiveness of LNCaP/shFOXO4 cells; the forced expression of FOXO4 did not alter RUNX2 protein levels, yet it decreased RUNX2 binding to the PIP promoter, resulting in PIP downregulation. Finally, there was a correlation between FOXO4, but not FOXO1 or FOXO3, downregulation and decreased metastasis-free survival in human CaP patients. Our data strongly suggest that increased PI3K/AKT-mediated metastatic invasiveness in CaP is associated with FOXO4 loss, and that mechanisms to induce FOXO4 re-expression might suppress CaP metastatic aggressiveness.

  1. Hybrid cells derived from breast epithelial cell/breast cancer cell fusion events show a differential RAF-AKT crosstalk

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    Özel Cem

    2012-04-01

    differential active RAF-AKT crosstalk. Because PI3K/AKT signalling has been chosen as a target for anti-cancer therapies our data might point to a possible severe side effect of AKT targeted cancer therapies. Inhibition of PI3K/AKT signalling in RAF-AKT crosstalk positive cancer (hybrid cells could result in a progression of these cells. Thus, not only the receptor (activation status, but also the activation of signal transduction molecules should be analysed thoroughly prior to therapy.

  2. MiR-223 suppresses cell proliferation by targeting IGF-1R.

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    Cheng You Jia

    Full Text Available To study the roles of microRNA-223 (miR-223 in regulation of cell growth, we established a miR-223 over-expression model in HeLa cells infected with miR-223 by Lentivirus pLL3.7 system. We observed in this model that miR-223 significantly suppressed the proliferation, growth rate, colony formation of HeLa cells in vitro, and in vivo tumorigenicity or tumor formation in nude mice. To investigate the mechanisms involved, we scanned and examined the potential and putative target molecules of miR-223 by informatics, quantitative PCR and Western blot, and found that insulin-like growth factor-1 receptor (IGF-1R was the functional target of miR-223 inhibition of cell proliferation. Targeting IGF-1R by miR-223 was not only seen in HeLa cells, but also in leukemia and hepatoma cells. The downstream pathway, Akt/mTOR/p70S6K, to which the signal was mediated by IGF-1R, was inhibited as well. The relative luciferase activity of the reporter containing wild-type 3'UTR(3'untranslated region of IGF-1R was significantly suppressed, but the mutant not. Silence of IGF-1R expression by vector-based short hairpin RNA resulted in the similar inhibition with miR-223. Contrarily, rescued IGF-1R expression in the cells that over-expressed miR-223, reversed the inhibition caused by miR-223 via introducing IGF-1R cDNA that didn't contain the 3'UTR. Meanwhile, we also noted that miR-223 targeted Rasa1, but the downstream molecules mediated by Rasa1 was neither targeted nor regulated. Therefore we believed that IGF-1R was the functional target for miR-223 suppression of cell proliferation and its downstream PI3K/Akt/mTOR/p70S6K pathway suppressed by miR-223 was by targeting IGF-1R.

  3. Evaluation of cancer stem cell markers CD133, CD44, CD24: association with AKT isoforms and radiation resistance in colon cancer cells.

    Directory of Open Access Journals (Sweden)

    Sara Häggblad Sahlberg

    Full Text Available The cell surface proteins CD133, CD24 and CD44 are putative markers for cancer stem cell populations in colon cancer, associated with aggressive cancer types and poor prognosis. It is important to understand how these markers may predict treatment outcomes, determined by factors such as radioresistance. The scope of this study was to assess the connection between EGFR, CD133, CD24, and CD44 (including isoforms expression levels and radiation sensitivity, and furthermore analyze the influence of AKT isoforms on the expression patterns of these markers, to better understand the underlying molecular mechanisms in the cell. Three colon cancer cell-lines were used, HT-29, DLD-1, and HCT116, together with DLD-1 isogenic AKT knock-out cell-lines. All three cell-lines (HT-29, HCT116 and DLD-1 expressed varying amounts of CD133, CD24 and CD44 and the top ten percent of CD133 and CD44 expressing cells (CD133high/CD44high were more resistant to gamma radiation than the ten percent with lowest expression (CD133low/CD44low. The AKT expression was lower in the fraction of cells with low CD133/CD44. Depletion of AKT1 or AKT2 using knock out cells showed for the first time that CD133 expression was associated with AKT1 but not AKT2, whereas the CD44 expression was influenced by the presence of either AKT1 or AKT2. There were several genes in the cell adhesion pathway which had significantly higher expression in the AKT2 KO cell-line compared to the AKT1 KO cell-line; however important genes in the epithelial to mesenchymal transition pathway (CDH1, VIM, TWIST1, SNAI1, SNAI2, ZEB1, ZEB2, FN1, FOXC2 and CDH2 did not differ. Our results demonstrate that CD133high/CD44high expressing colon cancer cells are associated with AKT and increased radiation resistance, and that different AKT isoforms have varying effects on the expression of cancer stem cell markers, which is an important consideration when targeting AKT in a clinical setting.

  4. Regulation of AKT phosphorylation at Ser473 and Thr308 by endoplasmic reticulum stress modulates substrate specificity in a severity dependent manner.

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    Hong Wa Yung

    Full Text Available Endoplasmic reticulum (ER stress is a common factor in the pathophysiology of diverse human diseases that are characterised by contrasting cellular behaviours, from proliferation in cancer to apoptosis in neurodegenerative disorders. Coincidently, dysregulation of AKT/PKB activity, which is the central regulator of cell growth, proliferation and survival, is often associated with the same diseases. Here, we demonstrate that ER stress modulates AKT substrate specificity in a severity-dependent manner, as shown by phospho-specific antibodies against known AKT targets. ER stress also reduces both total and phosphorylated AKT in a severity-dependent manner, without affecting activity of the upstream kinase PDK1. Normalisation to total AKT revealed that under ER stress phosphorylation of Thr308 is suppressed while that of Ser473 is increased. ER stress induces GRP78, and siRNA-mediated knock-down of GRP78 enhances phosphorylation at Ser473 by 3.6 fold, but not at Thr308. Substrate specificity is again altered. An in-situ proximity ligation assay revealed a physical interaction between GRP78 and AKT at the plasma membrane of cells following induction of ER stress. Staining was weak in cells with normal nuclear morphology but stronger in those displaying rounded, condensed nuclei. Co-immunoprecipitation of GRP78 and P-AKT(Ser473 confirmed the immuno-complex consists of non-phosphorylated AKT (Ser473 and Thr308. The interaction is likely specific as AKT did not bind to all molecular chaperones, and GRP78 did not bind to p70 S6 kinase. These findings provide one mechanistic explanation for how ER stress contributes to human pathologies demonstrating contrasting cell fates via modulation of AKT signalling.

  5. Inhibition of AKT promotes FOXO3a-dependent apoptosis in prostate cancer.

    Science.gov (United States)

    Das, T P; Suman, S; Alatassi, H; Ankem, M K; Damodaran, C

    2016-01-01

    Growth factor-induced activation of protein kinase-B (PKB), also known as AKT, induces pro-survival signaling and inhibits activation of pro-apoptotic signaling molecules including the Forkhead box O-3a (FOXO3a) transcription factor and caspase in transformed prostate cells in vitro. Earlier we reported that Withaferin-A (WA), a small herbal molecule, induces pro-apoptotic response-4 (Par-4) mediated apoptosis in castration-resistant prostate cancer (CRPC) cells. In the present study, we demonstrate that inhibition of AKT facilitates nuclear shuttling of FOXO3a where it regulates Par-4 transcription in CRPC cells. FOXO3a is upstream of Par-4 signaling, which is required for induction of apoptosis in CRPC cells. Promoter bashing studies and Ch-IP analysis confirm a direct interaction of FOXO3a and Par-4; a sequential deletion of FOXO3a-binding sites in the Par-4 promoter fails to induce Par-4 activation. To confirm these observations, we either overexpressed AKT or silenced FOXO3a activation in CRPC cells. Both methods inhibit Par-4 function and apoptosis is significantly compromised. In xenograft tumors derived from AKT-overexpressed CRPC cells, FOXO3a and Par-4 expression is downregulated, leading to aggressive tumor growth. Oral administration of WA to mice with xenograft tumors restores FOXO3a-mediated Par-4 functions and results in inhibited tumor growth. Finally, an inverse correlation of nuclear localization of AKT expression corresponds to cytoplasmic Par-4 localization in human prostate tissue array. Our studies suggest that Par-4 is one of the key transcriptional targets of FOXO3a, and Par-4 activation is required for induction of apoptosis in CRPC cells. Activation of FOXO3a appears to be an attractive target for the treatment of CRPC and molecules such as WA can be explored further for the treatment of CRPC. PMID:26913603

  6. Causal reasoning identifies mechanisms of sensitivity for a novel AKT kinase inhibitor, GSK690693

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    Macoritto Michael

    2010-07-01

    Full Text Available Abstract Background Inappropriate activation of AKT signaling is a relatively common occurrence in human tumors, and can be caused by activation of components of, or by loss or decreased activity of inhibitors of, this signaling pathway. A novel, pan AKT kinase inhibitor, GSK690693, was developed in order to interfere with the inappropriate AKT signaling seen in these human malignancies. Causal network modeling is a systematic computational analysis that identifies upstream changes in gene regulation that can serve as explanations for observed changes in gene expression. In this study, causal network modeling is employed to elucidate mechanisms of action of GSK690693 that contribute to its observed biological effects. The mechanism of action of GSK690693 was evaluated in multiple human tumor cell lines from different tissues in 2-D cultures and xenografts using RNA expression and phosphoproteomics data. Understanding the molecular mechanism of action of novel targeted agents can enhance our understanding of various biological processes regulated by the intended target and facilitate their clinical development. Results Causal network modeling on transcriptomic and proteomic data identified molecular networks that are comprised of activated or inhibited mechanisms that could explain observed changes in the sensitive cell lines treated with GSK690693. Four networks common to all cell lines and xenografts tested were identified linking GSK690693 inhibition of AKT kinase activity to decreased proliferation. These networks included increased RB1 activity, decreased MYC activity, decreased TFRC activity, and increased FOXO1/FOXO3 activity. Conclusion AKT is involved in regulating both cell proliferation and apoptotic pathways; however, the primary effect with GSK690693 appears to be anti-proliferative in the cell lines and xenografts evaluated. Furthermore, these results indicate that anti-proliferative responses to GSK690693 in either 2-D culture or

  7. AKT and ERK1/2 signaling in intrahepatic cholangiocarcinoma

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    Intrahepatic cholangiocarcinomas (ICC) are neoplasms that originate from cholangiocytes and can occur at any level of the biliary tree. Surgical resection is the current therapy of choice for this highly aggressive cancer. However, the 5-year survival still is poor, with high recurrence rates. Due to the intrahepatic growth a significant proportion of patients present with advanced disease and are not candidates for curative surgery or transplantation. The existing palliative options are of limited benefit and there is a great necessity for novel therapeutic options. In this article we review the role of the phosphoinositide 3-kinase(PI3K)/ AKT and extracellular regulated kinase (ERK) signaling pathways in ICC and present new data on the prognostic value of these protein kinases. Finally, we discuss future upcoming therapeutic options based on targeting these signaling pathways.

  8. Driving neural regeneration through the mammalian target of rapamycin

    Institute of Scientific and Technical Information of China (English)

    Kenneth Maiese

    2014-01-01

    Neurodegenerative disorders affect more than 30 million individuals throughout the world and lead to signiifcant disability as well as death. These statistics will increase almost exponentially as the lifespan and age of individuals increase globally and individuals become more susceptible to acute disorders such as stroke as well as chronic diseases that involve cognitive loss, Alzheimer’s disease, and Parkinson’s disease. Current therapies for such disorders are effective only for a small subset of individuals or provide symptomatic relief but do not alter disease progression. One exciting therapeutic approach that may turn the tide for addressing neurodegenerative disorders involves the mammalian target of rapamycin (mTOR). mTOR is a component of the protein complexes mTOR Complex 1 (mTORC1) and mTOR Complex 2 (mTORC2) that are ubiquitous throughout the body and control multiple functions such as gene transcription, metabolism, cell survival, and cell senescence. mTOR through its relationship with phosphoinositide 3-kinas e (PI 3-K) and protein kinase B (Akt) and multiple downstream signaling pathways such as p70 ribosomal S6 kinase (p70S6K) and proline rich Akt substrate 40 kDa (PRAS40) promotes neuro-nal cell regeneration through stem cell renewal and oversees critical pathways such as apoptosis, autophagy, and necroptosis to foster protection against neurodegenerative disorders. Targeting by mTOR of speciifc pathways that drive long-term potentiation, synaptic plasticity, andβ-amyl oid toxicity may offer new strategies for disorders such as stroke and Alzheimer’s disease. Overall, mTOR is an essential neuroprotective pathway but must be carefully targeted to maximize clini-cal efifcacy and eliminate any clinical toxic side effects.

  9. Molecular Characterization of Synovial Sarcoma in Children and Adolescents: Evidence of Akt Activation1

    Science.gov (United States)

    Bozzi, Fabio; Ferrari, Andrea; Negri, Tiziana; Conca, Elena; Luca, Da Riva; Losa, Marco; Casieri, Paola; Orsenigo, Marta; Lampis, Andrea; Meazza, Cristina; Casanova, Michela; Pierotti, Marco A; Tamborini, Elena; Pilotti, Silvana

    2008-01-01

    Synovial sarcoma (SS) is the most frequent nonrhabdomyosarcomatous soft tissue sarcoma encountered in adolescents and young adults, and despite advances in the treatment of local disease, metastases remain the main cause of death. The aim of this study was to characterize a single-center series of pediatric SS molecularly to seek any biomarkers or pathways that might make suitable targets for new agents. Seventeen cases of pediatric SS showing the SYT-SSX fusion transcript were screened immunohistochemically, biochemically, molecularly, and cytogenetically (depending on the available material) to investigate any expression/activation of epidermal growth factor receptor, platelet-derived growth factor receptor alpha (PDGFRα), PDGFRβ, Akt, and deregulated Wnt pathway. The most relevant outcome was the finding of activated epidermal growth factor receptor, PDGFRα, and PDGFRβ, which activated Akt in both the monophasic and biphasic histologic subtypes. Consistently, Akt activation was completely abolished in an SS cell line assay when stimulated by PDGF-AA and treated with the phosphatidylinositol 3-kinase inhibitor LY294002. Our results also showed the nuclear localization of β-catenin and cyclin D1 gene products in monophasic SS and the movement of β-catenin into the cytoplasm in the glandular component of the biphasic subtype. Although they need to be confirmed in larger series, these preliminary data suggest that therapeutic strategies including specific inhibitors of the phosphatidylinositol 3-kinase/Akt pathway might be exploited in SS. PMID:18633459

  10. AKT-mediated enhanced aerobic glycolysis causes acquired radioresistance by human tumor cells

    International Nuclear Information System (INIS)

    Background and purpose: Cellular radioresistance is a major impediment to effective radiotherapy. Here, we demonstrated that long-term exposure to fractionated radiation conferred acquired radioresistance to tumor cells due to AKT-mediated enhanced aerobic glycolysis. Material and methods: Two human tumor cell lines with acquired radioresistance were established by long-term exposure to fractionated radiation with 0.5 Gy of X-rays. Glucose uptake was inhibited using 2-deoxy-D-glucose, a non-metabolizable glucose analog. Aerobic glycolysis was assessed by measuring lactate concentrations. Cells were then used for assays of ROS generation, survival, and cell death as assessed by annexin V staining. Results: Enhanced aerobic glycolysis was shown by increased glucose transporter Glut1 expression and a high lactate production rate in acquired radioresistant cells compared with parental cells. Inhibiting the AKT pathway using the AKT inhibitor API-2 abrogated these phenomena. Moreover, we found that inhibiting glycolysis with 2-deoxy-D-glucose suppressed acquired tumor cell radioresistance. Conclusions: Long-term fractionated radiation confers acquired radioresistance to tumor cells by AKT-mediated alterations in their glucose metabolic pathway. Thus, tumor cell metabolic pathway is an attractive target to eliminate radioresistant cells and improve radiotherapy efficacy

  11. A dual-color fluorescence-based platform to identify selective inhibitors of Akt signaling.

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    Aranzazú Rosado

    Full Text Available BACKGROUND: Inhibition of Akt signaling is considered one of the most promising therapeutic strategies for many cancers. However, rational target-orientated approaches to cell based drug screens for anti-cancer agents have historically been compromised by the notorious absence of suitable control cells. METHODOLOGY/PRINCIPAL FINDINGS: In order to address this fundamental problem, we have developed BaFiso, a live-cell screening platform to identify specific inhibitors of this pathway. BaFiso relies on the co-culture of isogenic cell lines that have been engineered to sustain interleukin-3 independent survival of the parental Ba/F3 cells, and that are individually tagged with different fluorescent proteins. Whilst in the first of these two lines cell survival in the absence of IL-3 is dependent on the expression of activated Akt, the cells expressing constitutively-activated Stat5 signaling display IL-3 independent growth and survival in an Akt-independent manner. Small molecules can then be screened in these lines to identify inhibitors that rescue IL-3 dependence. CONCLUSIONS/SIGNIFICANCE: BaFiso measures differential cell survival using multiparametric live cell imaging and permits selective inhibitors of Akt signaling to be identified. BaFiso is a platform technology suitable for the identification of small molecule inhibitors of IL-3 mediated survival signaling.

  12. AKT1 genotype moderates the acute psychotomimetic effects of naturalistically smoked cannabis in young cannabis smokers.

    Science.gov (United States)

    Morgan, C J A; Freeman, T P; Powell, J; Curran, H V

    2016-01-01

    Smoking cannabis daily doubles an individual's risk of developing a psychotic disorder, yet indicators of specific vulnerability have proved largely elusive. Genetic variation is one potential risk modifier. Single-nucleotide polymorphisms in the AKT1 and catechol-O-methyltransferase (COMT) genes have been implicated in the interaction between cannabis, psychosis and cognition, but no studies have examined their impact on an individual's acute response to smoked cannabis. A total 442 healthy young cannabis users were tested while intoxicated with their own cannabis-which was analysed for delta-9-tetrahydrocannbinol (THC) and cannabidiol content-and also ± 7 days apart when drug-free. Psychotomimetic symptoms and working memory were assessed on both the sessions. Variation at the rs2494732 locus of the AKT1 gene predicted acute psychotic response to cannabis along with dependence on the drug and baseline schizotypal symptoms. Working memory following cannabis acutely was worse in females, with some suggestion of an impact of COMT polymorphism on working memory when drug-free. These findings are the first to demonstrate that AKT1 mediates the acute response to cannabis in otherwise healthy individuals and implicate the AKT1 pathway as a possible target for prevention and treatment of cannabis psychosis. PMID:26882038

  13. The downstream industry compared to market

    International Nuclear Information System (INIS)

    J.L. Schilansky introduces here the difficult question of the downstream industry compared to market in recalling the recent structural changes (behaviour of customers, behaviour of the USA- and China-governments), the increase of the European and French regulations, the climatic change and the conjectural impact of the crisis on the refining industry. (O.M.)

  14. The Akt-mTOR axis is a pivotal regulator of eccentric hypertrophy during volume overload

    OpenAIRE

    Masataka Ikeda; Tomomi Ide; Takeo Fujino; Yuka Matsuo; Shinobu Arai; Keita Saku; Takamori Kakino; Yasuhiro Oga; Akiko Nishizaki; Kenji Sunagawa

    2015-01-01

    The heart has two major modalities of hypertrophy in response to hemodynamic loads: concentric and eccentric hypertrophy caused by pressure and volume overload (VO), respectively. However, the molecular mechanism of eccentric hypertrophy remains poorly understood. Here we demonstrate that the Akt-mammalian target of rapamycin (mTOR) axis is a pivotal regulator of eccentric hypertrophy during VO. While mTOR in the heart was activated in a left ventricular end-diastolic pressure (LVEDP)-depende...

  15. MITIGATION OF SEDIMENTATION HAZARDS DOWNSTREAM FROM RESERVOIRS

    Institute of Scientific and Technical Information of China (English)

    Ellen WOHL; Sara RATHBURN

    2003-01-01

    Many reservoirs currently in operation trap most or all of the sediment entering the reservoir,creating sediment-depleted conditions downstream. This may cause channel adjustment in the form of bank erosion, bed erosion, substrate coarsening, and channel planform change. Channel adjustment may also result from episodic sediment releases during reservoir operation, or from sediment evacuation following dam removal. Channel adjustment to increased sediment influx depends on the magnitude, frequency, duration and grain-size distribution of the sediment releases, and on the downstream channel characteristics. Channel adjustment may occur as a change in substrate sizedistribution, filling of pools, general bed aggradation, lateral instability, change in channel planform,and/or floodplain aggradation. The increased sediment availability may alter aquatic and riparian habitat, reduce water quality, distribute adsorbed contaminants along the river corridor, and provide germination sites for exotic vegetation. Mitigation of these sedimentation hazards requires: (1)mapping grain-size distribution within the reservoir and estimating the grain-size distributions of sediment that will be mobilized through time; (2) mapping shear stress and sediment transport capacity as a function of discharge on the basis of channel units for the length of the river likely to be affected; (3) mapping potential depositional zones, and aquatic habitat and "acceptable losses," along the downstream channel, and comparing these volumes to the total sediment volume stored in the reservoir as a means of estimating total transport capacity required to mobilize reservoir sediment delivered to the channel; (4) designing discharge and sediment release regime (magnitude, frequency,duration) to minimize adverse downstream impacts; and (5) developing plans to remove, treat, contain,or track contaminants, and to restrict establishment of exotic vegetation. The North Fork Poudre River in Colorado is used to

  16. Mechanisms of antiprostate cancer by gum mastic: NF-κB signal as target

    Institute of Scientific and Technical Information of China (English)

    Mei-lan HE; Ang LI; Chun-su XU; Shun-li WANG; Meng-jie ZHANG; Hua GU; Yao-qin YANG; Hui-hong TAO

    2007-01-01

    Aim: To study the effect of gum mastic, a natural resin, on the proliferation of androgen-independent prostate cancer PC-3 cells, and further investigate the mechanisms involved in this regulatory system, taking nuclear factor kB (NF-kB) signal as the target. Methods: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and a flow cytometer were used to detect the effect of gum mastic on the proliferation of PC-3 cells. Then, reporter gene assay, RT-PCR, and Western blotting were carried out to study the effects of gum mastic on the NF-κB protein level and the NF-kB signal pathway. The expression of genes involved in the NF-kB signal pathway, including cyclin D1, inhibitors of kBs (IkBα), and phosphorylated Akt (p-AKT), were measured. In addition, transient transfection assays with the 5×NF-κB consensus sequence promoter was also used to test the effects of gum mastic. Results: Gum mastic inhibited PC-3 cell growth and blocked the PC-3 cell cycle in the G1 phase. Gum mastic also suppressed NF-κB activity in the PC-3 cells. The expression of cyclin D1, a crucial cell cycle regulator and an NF-kB downstream target gene, was reduced as well. Moreover, gum mastic decreased the p-AKT protein level and increased the IkBα protein level.Conclusion: Gum mastic inhibited the proliferation and blocked the cell cycle progression in PC-3 cells by suppressing NF-κB activity and the NF-κB signal pathway.

  17. Presence of both alterations in FGFR/FGF and PI3K/AKT/mTOR confer improved outcomes for patients with metastatic breast cancer treated with PI3K/AKT/mTOR inhibitors

    Science.gov (United States)

    Wheler, Jennifer J.; Atkins, Johnique T.; Janku, Filip; Moulder, Stacy L.; Stephens, Philip J.; Yelensky, Roman; Valero, Vicente; Miller, Vincent; Kurzrock, Razelle; Meric-Bernstam, Funda

    2016-01-01

    There is limited data on co-expression of FGFR/FGR amplifications and PI3K/ AKT/mTOR alterations in breast cancer. Tumors from patients with metastatic breast cancer referred to our Phase I Program were analyzed by next generation sequencing (NGS). Genomic libraries were selected for all exons of 236 (or 182) cancer-related genes sequenced to average depth of >500× in a CLIA laboratory (Foundation Medicine, Cambridge, MA, USA) and analyzed for all classes of genomic alterations. We report genomic profiles of 112 patients with metastatic breast cancer, median age 55 years (range, 27-78). Twenty-four patients (21%) had at least one amplified FGFR or FGF. Fifteen of the 24 patients (63%) also had an alteration in the PI3K/ AKT/mTOR pathway. There was no association between alterations in FGFR/FGF and PI3K/AKT/mTOR (P=0.49). Patients with simultaneous amplification in FGFR/FGF signaling and the PI3K/AKT/mTOR pathway had a higher rate of SD≥6 months/PR/ CR when treated with therapies targeting the PI3K/AKT/mTOR pathway than patients with only alterations in the PI3K/AKT/mTOR pathway (73% vs. 34%; P=0.0376) and remained on treatment longer (6.8 vs. 3.7 months; P=0.053). Higher response rates were seen in patients with simultaneous amplification in FGFR/FGF signaling and alterations in the PI3K/AKT/mTOR pathway who were treated with inhibitors of that pathway. PMID:27489863

  18. Activation of Akt is essential for the propagation of mitochondrial respiratory stress signaling and activation of the transcriptional coactivator heterogeneous ribonucleoprotein A2.

    Science.gov (United States)

    Guha, Manti; Fang, Ji-Kang; Monks, Robert; Birnbaum, Morris J; Avadhani, Narayan G

    2010-10-15

    Mitochondrial respiratory stress (also called mitochondrial retrograde signaling) activates a Ca(2+)/calcineurin-mediated signal that culminates in transcription activation/repression of a large number of nuclear genes. This signal is propagated through activation of the regulatory proteins NFκB c-Rel/p50, C/EBPδ, CREB, and NFAT. Additionally, the heterogeneous ribonucleoprotein A2 (hnRNPA2) functions as a coactivator in up-regulating the transcription of Cathepsin L, RyR1, and Glut-4, the target genes of stress signaling. Activation of IGF1R, which causes a metabolic switch to glycolysis, cell invasiveness, and resistance to apoptosis, is a phenotypic hallmark of C2C12 myoblasts subjected to mitochondrial stress. In this study, we report that mitochondrial stress leads to increased expression, activation, and nuclear localization of Akt1. Mitochondrial respiratory stress also activates Akt1-gene expression, which involves hnRNPA2 as a coactivator, indicating a complex interdependency of these two factors. Using Akt1(-/-) mouse embryonic fibroblasts and Akt1 mRNA-silenced C2C12 cells, we show that Akt1-mediated phosphorylation is crucial for the activation and recruitment of hnRNPA2 to the enhanceosome complex. Akt1 mRNA silencing in mtDNA-depleted cells resulted in reversal of the invasive phenotype, accompanied by sensitivity to apoptotic stimuli. These results show that Akt1 is an important regulator of the nuclear transcriptional response to mitochondrial stress.

  19. Research progress of protein kinase B/Akt inhibitors%蛋白激酶B/Akt抑制剂

    Institute of Scientific and Technical Information of China (English)

    郝茜; 钟嫄; 王朋; 赵桂森

    2011-01-01

    PI3K (phosphatidylinositol 3-kinase)/Akt (protein kinase B, PKB) signaling pathway plays a key role in cell growth and survival. Excessive activation of PI3K/Akt pathway has been found in many tumors. In more than 50% of human tumors, Akt or/and its upstream regulatory molecules (such as PTEN and PI3K) changes abnormally. So Akt has become a hot target for cancer prevention and therapy. Recently, many effective small-molecule Akt inhibitors with different mechanisms have been found. According to the binding site and/or the chemical structures of various Akt inhibitors, they are divided into ATP competitive inhibitor, Akt allosteric inhibitor and phosphatidylinositol analog inhibitor. This paper reviews the relationship between PI3K/Akt pathway and tumors, and the research progress of Akt inhibitor, which will contribute to the design of new anti-tumor drugs.%磷脂酰肌醇-3-激酶(phosphatidylinositol 3-kinase,PI3K)/蛋白激酶B(protein kinase B,PKB/Akt)信号通路在细胞生长与存活中起着关键作用,PI3K/Akt通路的过度激活在多种肿瘤中常见.Akt激酶本身以及Akt激酶上游调节分子,例如PTEN和PI3K,在超过50%的人类肿瘤中均有异常变化.因此Akt成为肿瘤预防和肿瘤靶向治疗的热点之一.许多小分子化合物通过不同机制抑制Akt活性,根据小分子抑制剂与激酶的结合部位和化学结构不同,主要分为ATP竞争性抑制剂、Akt变构抑制剂和磷脂酰肌醇类似物抑制剂.本文综述了PI3K/Akt通路与肿瘤的关系和Akt抑制剂的研究现状,为新型抗癌药物的设计研究提供参考.

  20. Evaluation of Intracellular Signaling Downstream Chimeric Antigen Receptors.

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    Hannah Karlsson

    Full Text Available CD19-targeting CAR T cells have shown potency in clinical trials targeting B cell leukemia. Although mainly second generation (2G CARs carrying CD28 or 4-1BB have been investigated in patients, preclinical studies suggest that third generation (3G CARs with both CD28 and 4-1BB have enhanced capacity. However, little is known about the intracellular signaling pathways downstream of CARs. In the present work, we have analyzed the signaling capacity post antigen stimulation in both 2G and 3G CARs. 3G CAR T cells expanded better than 2G CAR T cells upon repeated stimulation with IL-2 and autologous B cells. An antigen-driven accumulation of CAR+ cells was evident post antigen stimulation. The cytotoxicity of both 2G and 3G CAR T cells was maintained by repeated stimulation. The phosphorylation status of intracellular signaling proteins post antigen stimulation showed that 3G CAR T cells had a higher activation status than 2G. Several proteins involved in signaling downstream the TCR were activated, as were proteins involved in the cell cycle, cell adhesion and exocytosis. In conclusion, 3G CAR T cells had a higher degree of intracellular signaling activity than 2G CARs which may explain the increased proliferative capacity seen in 3G CAR T cells. The study also indicates that there may be other signaling pathways to consider when designing or evaluating new generations of CARs.

  1. Systemic bisperoxovanadium activates Akt/mTOR, reduces autophagy, and enhances recovery following cervical spinal cord injury.

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    Chandler L Walker

    Full Text Available Secondary damage following primary spinal cord injury extends pathology beyond the site of initial trauma, and effective management is imperative for maximizing anatomical and functional recovery. Bisperoxovanadium compounds have proven neuroprotective effects in several central nervous system injury/disease models, however, no mechanism has been linked to such neuroprotection from bisperoxovanadium treatment following spinal trauma. The goal of this study was to assess acute bisperoxovanadium treatment effects on neuroprotection and functional recovery following cervical unilateral contusive spinal cord injury, and investigate a potential mechanism of the compound's action. Two experimental groups of rats were established to 1 assess twice-daily 7 day treatment of the compound, potassium bisperoxo (picolinato vanadium, on long-term recovery of skilled forelimb activity using a novel food manipulation test, and neuroprotection 6 weeks following injury and 2 elucidate an acute mechanistic link for the action of the drug post-injury. Immunofluorescence and Western blotting were performed to assess cellular signaling 1 day following SCI, and histochemistry and forelimb functional analysis were utilized to assess neuroprotection and recovery 6 weeks after injury. Bisperoxovanadium promoted significant neuroprotection through reduced motorneuron death, increased tissue sparing, and minimized cavity formation in rats. Enhanced forelimb functional ability during a treat-eating assessment was also observed. Additionally, bisperoxovanadium significantly enhanced downstream Akt and mammalian target of rapamycin signaling and reduced autophagic activity, suggesting inhibition of the phosphatase and tensin homologue deleted on chromosome ten as a potential mechanism of bisperoxovanadium action following traumatic spinal cord injury. Overall, this study demonstrates the efficacy of a clinically applicable pharmacological therapy for rapid initiation of

  2. Upstream regulators and downstream effectors of NF-κB in Alzheimer's disease.

    Science.gov (United States)

    Shi, Zhe-Min; Han, Ya-Wei; Han, Xiao-Hui; Zhang, Kun; Chang, Ya-Nan; Hu, Zhi-Mei; Qi, Hai-Xia; Ting, Chen; Zhen, Zhang; Hong, Wei

    2016-07-15

    Since Alzheimer's disease (AD) is becoming the prevalent dementia in the whole world, more underlying mechanisms are emerging. Long time has the transcription factor NF-κB been identified to participate in AD pathogenesis, various studies have focused on the causes and effects of AD that are linked to NF-κB. In this review we discuss diverse environmental stimuli including oxidative stress, neuroinflammation and metabolism, involved signaling pathways such as PI3K/AKT, MAPK and AGE/RAGE/GSK-3 and newly found ncRNAs that mediate neuron toxicity or neuron protection through NF-κB activation and the following response associated with the same factors in AD. These may provide future orientation of investigation at transcription level and support efficient treatment to AD by a better understanding of the upstream regulators and downstream effectors of NF-κB. PMID:27288790

  3. Cytoplasmic Skp2 expression is associated with p-Akt1 and predicts poor prognosis in human breast carcinomas.

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    Jing Liu

    Full Text Available BACKGROUND: S-phase kinase protein 2 (Skp2, an oncogenic protein, is a key regulator in different cellular and molecular processes, through ubiquitin-proteasome degradation pathway. Increased levels of Skp2 are observed in various types of cancer and associated with poor prognosis. However, in human breast carcinomas, the underlying mechanism and prognostic significance of cytoplasmic Skp2 is still undefined. METHODS: To investigate the role of cytoplasmic Skp2 expression in human breast carcinomas, we immnohistochemically assessed cytoplasmic Skp2, p-Akt1, and p27 expression in 251 patients with invasive ductal carcinomas of the breast. Association of cytoplasmic Skp2 expression with p-Akt1 and p27 was analyzed as well as correspondence with other clinicopathological parameters. Disease-free survival and overall survival were determined based on the Kaplan-Meier method and Cox regression models. RESULTS: Cytoplasmic of Skp2 was detected in 165 out of 251 (65.7% patients. Cytoplasmic Skp2 expression was associated with larger tumor size, more advanced histological grade, and positive HER2 expression. Increased cytoplasmic Skp2 expression correlated with p-Akt1 expression, with 54.2% (51/94 of low p-Akt1-expressing breast carcinomas, but 72.6% (114/157 of high p-Akt1-expressing breast carcinomas exhibiting cytoplasmic Skp2 expression. Elevated cytoplasmic Skp2 expression with low p-Akt1 expression was associated with poor disease-free and overall survival (DFS and OS, and Cox regression models demonstrated that cytoplasmic Skp2 expression was an independent prognostic marker for invasive breast carcinomas. CONCLUSION: Cytoplasmic Skp2 expression is associated with aggressive prognostic factors, such as larger tumor size, and advanced histological grade of the breast cancers. Results demonstrate that combined cytoplasmic Skp2 and p-Akt1 expression may be prognostic for patients with invasive breast carcinomas, and cytoplasmic Skp2 may serve as a

  4. Stem cell factor (SCF) protects osteoblasts from oxidative stress through activating c-Kit-Akt signaling

    Energy Technology Data Exchange (ETDEWEB)

    Yang, Lei [Department of Orthopedics, Changzhou Wujin People’s Hospital-South Division, Affiliated Hospital of Jiangsu University, Changzhou (China); Wu, Zhong [Department of Orthopedics, Shanghai Tenth People’s Hospital, Tongji University School of Medicine, Shanghai (China); Yin, Gang; Liu, Haifeng; Guan, Xiaojun; Zhao, Xiaoqiang [Department of Orthopedics, Changzhou Wujin People’s Hospital-South Division, Affiliated Hospital of Jiangsu University, Changzhou (China); Wang, Jianguang, E-mail: jianguangwang@163.com [Department of Orthopedics, Shanghai Tenth People’s Hospital, Tongji University School of Medicine, Shanghai (China); Zhu, Jianguo, E-mail: gehujianguo68@163.com [Department of Orthopedics, Changzhou Wujin People’s Hospital-South Division, Affiliated Hospital of Jiangsu University, Changzhou (China)

    2014-12-12

    Highlights: • SCF receptor c-Kit is functionally expressed in primary and transformed osteoblasts. • SCF protects primary and transformed osteoblasts from H{sub 2}O{sub 2}. • SCF activation of c-Kit in osteoblasts, required for its cyto-protective effects. • c-Kit mediates SCF-induced Akt activation in cultured osteoblasts. • Akt activation is required for SCF-regulated cyto-protective effects in osteoblasts. - Abstract: Osteoblasts regulate bone formation and remodeling, and are main target cells of oxidative stress in the progression of osteonecrosis. The stem cell factor (SCF)-c-Kit pathway plays important roles in the proliferation, differentiation and survival in a range of cell types, but little is known about its functions in osteoblasts. In this study, we found that c-Kit is functionally expressed in both osteoblastic-like MC3T3-E1 cells and primary murine osteoblasts. Its ligand SCF exerted significant cyto-protective effects against hydrogen peroxide (H{sub 2}O{sub 2}). SCF activated its receptor c-Kit in osteoblasts, which was required for its cyto-protective effects against H{sub 2}O{sub 2}. Pharmacological inhibition (by Imatinib and Dasatinib) or shRNA-mediated knockdown of c-Kit thus inhibited SCF-mediated osteoblast protection. Further investigations showed that protection by SCF against H{sub 2}O{sub 2} was mediated via activation of c-Kit-dependent Akt pathway. Inhibition of Akt activation, through pharmacological or genetic means, suppressed SCF-mediated anti-H{sub 2}O{sub 2} activity in osteoblasts. In summary, we have identified a new SCF-c-Kit-Akt physiologic pathway that protects osteoblasts from H{sub 2}O{sub 2}-induced damages, and might minimize the risk of osteonecrosis caused by oxidative stress.

  5. Role of PI3-K/Akt pathway and its effect on glial cell line-derived neurotrophic factor in midbrain dopamine cells

    Institute of Scientific and Technical Information of China (English)

    Hong-jun WANG; Jun-ping CAO; Jing-kao YU; Dian-shuai GAO

    2007-01-01

    Aim: To explore the intracellular mechanisms underlying the survival/differentia-don effect of the glial cell line-derived neurotrophic factor (GDNF) on dopamine(DA) cells. Methods: Midbrain slice culture and primary cell culture were established, and the cultures were divided into 3 groups: control group, GDNF group, and the phosphatidylinositol 3-kinase/Akt (PI3-K/Akt) pathway-inhibited group. Then the expression of tyrosine hydroxylase (TH) was detected by immunostaining as well as Western blotting. Results: GDNF treatment induced an increase in the number of TH-immunoreactive (ir) cells and the neurite number of TH-ir cells, as well as in the level of TH expression in cultures (Number of TH-ir cells in the slice culture: control group, 8.76±0.75; GDNF group, 18.63±0.95.Number of TH-ir cells and neurite number of TH-ir cells in cell culture: controlgroup, 3.65±0.88 and 2.49±0.42; GDNF group, 6.01±0.43 and 4.89±0.46). Meanwhile, the stimulation of cultured cells with GDNF increased the phosphorylation of Akt, which is a downstream effector of PI3-K/Akt. The effects of GDNF were specifically blocked by the inhibitor of the PI3-K/Akt pathway, wortmannin (Number of TH-ir cells in slice culture: PI3-K/Akt pathway-inhibited group, 6.98±0.58. Num-ber of TH-ir cells and neurite number of TH-ir cells in cell culture: PI3-K/Aktpathway-inhibited group, 3.79±0.62 and 2.50±0.25, respectively). Conclusion: The PI3-K/Akt pathway mediates the survival/differentiation effect of GDNF on DA cells.8±0.58.

  6. Targeting TORC1/2 enhances sensitivity to EGFR inhibitors in head and neck cancer preclinical models.

    Science.gov (United States)

    Cassell, Andre; Freilino, Maria L; Lee, Jessica; Barr, Sharon; Wang, Lin; Panahandeh, Mary C; Thomas, Sufi M; Grandis, Jennifer R

    2012-11-01

    Head and neck squamous cell carcinoma (HNSCC) is characterized by overexpression of the epidermal growth factor receptor (EGFR) where treatments targeting EGFR have met with limited clinical success. Elucidation of the key downstream-pathways that remain activated in the setting of EGFR blockade may reveal new therapeutic targets. The present study was undertaken to test the hypothesis that inhibition of the mammalian target of rapamycin (mTOR) complex would enhance the effects of EGFR blockade in HNSCC preclinical models. Treatment of HNSCC cell lines with the newly developed TORC1/TORC2 inhibitor OSI-027/ASP4876 resulted in dose-dependent inhibition of proliferation with abrogation of phosphorylation of known downstream targets including phospho-AKT (Ser473), phospho-4E-BP1, phospho-p70s6K, and phospho-PRAS40. Furthermore, combined treatment with OSI-027 and erlotinib resulted in enhanced biochemical effects and synergistic growth inhibition in vitro. Treatment of mice bearing HNSCC xenografts with a combination of the Food and Drug Administration (FDA)-approved EGFR inhibitor cetuximab and OSI-027 demonstrated a significant reduction of tumor volumes compared with either treatment alone. These findings suggest that TORC1/TORC2 inhibition in conjunction with EGFR blockade represents a plausible therapeutic strategy for HNSCC. PMID:23226094

  7. Targeting TORC1/2 Enhances Sensitivity to EGFR Inhibitors in Head and Neck Cancer Preclinical Models

    Directory of Open Access Journals (Sweden)

    Andre Cassell

    2012-11-01

    Full Text Available Head and neck squamous cell carcinoma (HNSCC is characterized by overexpression of the epidermal growth factor receptor (EGFR where treatments targeting EGFR have met with limited clinical success. Elucidation of the key downstream-pathways that remain activated in the setting of EGFR blockade may reveal new therapeutic targets. The present study was undertaken to test the hypothesis that inhibition of the mammalian target of rapamycin (mTOR complex would enhance the effects of EGFR blockade in HNSCC preclinical models. Treatment of HNSCC cell lines with the newly developed TORC1/TORC2 inhibitor OSI-027/ASP4876 resulted in dose-dependent inhibition of proliferation with abrogation of phosphorylation of known downstream targets including phospho-AKT (Ser473, phospho-4E-BP1, phospho-p70s6K, and phospho-PRAS40. Furthermore, combined treatment with OSI-027 and erlotinib resulted in enhanced biochemical effects and synergistic growth inhibition in vitro. Treatment of mice bearing HNSCC xenografts with a combination of the Food and Drug Administration (FDA-approved EGFR inhibitor cetuximab and OSI-027 demonstrated a significant reduction of tumor volumes compared with either treatment alone. These findings suggest that TORC1/TORC2 inhibition in conjunction with EGFR blockade represents a plausible therapeutic strategy for HNSCC.

  8. Twist induces epithelial-mesenchymal transition and cell motility in breast cancer via ITGB1-FAK/ILK signaling axis and its associated downstream network.

    Science.gov (United States)

    Yang, Jiajia; Hou, Yixuan; Zhou, Mingli; Wen, Siyang; Zhou, Jian; Xu, Liyun; Tang, Xi; Du, Yan-e; Hu, Ping; Liu, Manran

    2016-02-01

    Twist, a highly conserved basic Helix-Loop-Helix transcription factor, functions as a major regulator of epithelial-mesenchymal transition (EMT) and tumor metastasis. In different cell models, signaling pathways such as TGF-β, MAPK/ERK, WNT, AKT, JAK/STAT, Notch, and P53 have also been shown to play key roles in the EMT process, yet little is known about the signaling pathways regulated by Twist in tumor cells. Using iTRAQ-labeling combined with 2D LC-MS/MS analysis, we identified 194 proteins with significant changes of expression in MCF10A-Twist cells. These proteins reportedly play roles in EMT, cell junction organization, cell adhesion, and cell migration and invasion. ECM-receptor interaction, MAPK, PI3K/AKT, P53 and WNT signaling were found to be aberrantly activated in MCF10A-Twist cells. Ingenuity Pathways Analysis showed that integrin β1 (ITGB1) acts as a core regulator in linking integrin-linked kinase (ILK), Focal-adhesion kinase (FAK), MAPK/ERK, PI3K/AKT, and WNT signaling. Increased Twist and ITGB1 are associated with breast tumor progression. Twist transcriptionally regulates ITGB1 expression. Over-expression of ITGB1 or Twist in MCF10A led to EMT, activation of FAK/ILK, MAPK/ERK, PI3K/AKT, and WNT signaling. Knockdown of Twist or ITGB1 in BT549 and Hs578T cells decreased activity of FAK, ILK, and their downstream signaling, thus specifically impeding EMT and cell invasion. Knocking down ILK or inhibiting FAK, MAPK/ERK, or PI3K/AKT signaling also suppressed Twist-driven EMT and cell invasion. Thus, the Twist-ITGB1-FAK/ILK pathway and their downstream signaling network dictate the Twist-induced EMT process in human mammary epithelial cells and breast cancer cells. PMID:26693891

  9. Caveolin-1 is critical in the proliferative effect of leptin on osteoblasts through the activation of Akt.

    Science.gov (United States)

    Zou, Lin; Zhang, Guichun; Liu, Lifeng; Chen, Chen; Cao, Xuecheng; Cai, Jinfang

    2016-09-01

    Osteoblasts are critical in bone remodeling and the repair of bone fractures. Leptin is involved in bone metabolism and osteoblast survival through the downstream signaling pathway, however, the exact mechanism of the effect of leptin on osteoblasts remains to be fully elucidated. In the present study, hFOB 1.19 cells were used to observe the effects of leptin on cell proliferation and apoptosis, and to investigate the underlying mechanism. The results confirmed that treatment of hFOB 1.19 cells with leptin significantly induced cell proliferation. Western blot analysis showed that the expression of caveolin‑1 and the activation of Akt in the cells treated with leptin were significantly increased, compared with the control cells. Additionally, inhibiting Akt activation eliminated the effects on cell proliferation induced by leptin. The rates of cell apoptosis and cell cycle distribution were examined using flow cytometry, which revealed a decrease in the apoptotic rate and an increase in the proportion of cells in the S phase. This indicated that leptin was capable of inducing cell proliferation by inhibiting apoptosis and stimulating cell progression to the S phase. Transfection of the cells with caveolin‑1 small interfering RNA showed that the activation of Akt induced by leptin was significantly inhibited. Furthermore, caveolin‑1 knockdown and inhibiting Akt activation eliminated the increased proliferation, increased proportion of cells in the S phase and increased anti‑apoptotic effects induced by leptin. Taken together, the data obtained in the present study demonstrated that caveolin‑1 was critical in the proliferative effect of leptin on osteoblasts via the activation of Akt. PMID:27430651

  10. Upstream versus Downstream Implementation of Climate Policy

    OpenAIRE

    Erin T. Mansur

    2010-01-01

    This chapter examines the tradeoffs of regulating upstream (e.g., coal, natural gas, and refined petroleum product producers) versus regulating downstream (e.g., direct sources of greenhouse gases (GHG)). In general, regulating at the source provides polluters with incentives to choose among more opportunities to abate pollution. This chapter develops a simple theoretical model that shows why this added flexibility achieves the lowest overall costs. I broaden the theory to incorporate several...

  11. Mechanical downstream processing of Single Cell Oils

    OpenAIRE

    De Coninck, Maarten; Van Hecke, Renaat; Deprez, Koen; De Baerdemaeker, Josse

    2011-01-01

    During the last years, the third generation of bio fuels has been arousing more and more interest. Under certain conditions some micro organisms: yeasts, algae, fungi and bacteria, can accumulate up to 50% oil (based on dry weight). These so-called ‘Single cell oils’ (SCO) are well known in this context. Nowadays, harvesting and recovery of interesting products from microalgae is one of the most problematic areas of algal biofuel production technology. The traditional downstream process,...

  12. India's Downstream Petroleum Sector

    Energy Technology Data Exchange (ETDEWEB)

    NONE

    2010-07-01

    This study provides a holistic examination of pricing and investment dynamics in India's downstream petroleum sector. It analyses the current pricing practices, highlights the tremendous fiscal cost of current pricing and regulatory arrangements, and examines the sectoral investment dynamics. It also looks at potential paths towards market-based reform along which the Indian government may move, while at the same time protecting energy market access for India's large poor population.

  13. Clinicopathological Research and Expression of PTEN/PI3K/Akt Signaling Pathway in Non-small Cell Lung Cancer

    Directory of Open Access Journals (Sweden)

    Hong SHU

    2009-08-01

    Full Text Available Background and objective It has been known that abnormality of PTEN/PI3K/Akt signal pathway played an important role in initiation of some malignant tumors. The aim of this study is to examine the expression and clinicopathological significance of PTEN, PI3K and Akt in non-small cell lung cancer (NSCLC. Methods Expression levels of PTEN, PI3K and Akt protein were determined using immunohistochemistry S-P in 61 specimens of NSCLC with follow-up. Results ①The levels of PTEN protein was higher than that of control group, and levels of PI3K and Akt protein were lower than that of control group; ②Expression of PTEN and PI3K were related to histotype, clinical stage, lymphonode metastasis and survival rate; Expression of Akt was related to clinical stage, lymphonode metastasis and survival rate; ③The Cox Monovariable Analyses revealed that both smoking and negative expression of PTEN were the risking factors on the death of the NSCLC patients after surgery; ④The expression of PTEN protein was negatively correlated to that of PI3K and Akt respectively, while the expression of PI3K was positively correlated to that of Akt. Conclusion In NSCLC, the lack of PTEN induced up-regulation of PI3K and Akt, which demonstrated that PTEN/PI3K/Akt signaling pathway contributed to the tumorigenesis and development of NSCLC. They could be used as the indicators of prognosis and targets of therapy.

  14. Detection of pAkt protein in imprint cytology of invasive breast cancer: Correlation with HER2/neu, hormone receptors, and other clinicopathological variables

    Directory of Open Access Journals (Sweden)

    Olympia Vasou

    2015-01-01

    Full Text Available Purpose: Akt is a serine/threonine protein kinase and has emerged as a crucial regulator of widely divergent cellular processes, including apoptosis, proliferation, differentiation, and metabolism. Activation of Akt/protein kinase B has been positively associated with human epidermal growth-factor receptor 2 (HER2/neu overexpression in breast carcinoma and a worse outcome among endocrine treated patients. The Akt signaling pathway currently attracts considerable attention as a new target for effective therapeutic strategies. We therefore investigated the relationship between activation of Akt and clinicopathologic variables including hormone receptor and HER2/neu status. Methods: Archival tumor tissues from 100 patients with invasive breast carcinoma were analyzed by immunocytochemistry. This study describes the results of immunocytochemical pAkt expression in breast carcinoma imprints, prepared from cut surfaces of freshly removed tumors . Both nuclear and cytoplasmic expressions were evaluated for pAkt. Results: Nuclear and cytoplasmic positive scores of 72% (72/100 and 42% (42/100, respectively, were found. Coexistence of nuclear and cytoplasmic staining was observed in 32 cases (32/100. Nuclear positive staining correlated with HER2/neu overexpression (P = 0.043 and was significantly associated with positive involvement of axillary lymph nodes (P = 0.013. No correlation was found between cytoplasmic pAkt rate and clinicopathological parameters, estrogen receptor, progesterone receptor or HER2/neu expression. Conclusions: pAkt expression can be evaluated in cytological material and may add valuable information to current prognostic models for breast cancer. pAkt overexpression appears to be linked with potentially aggressive tumor phenotype in invasive breast carcinoma.

  15. Heat Stress-Induced PI3K/mTORC2-Dependent AKT Signaling Is a Central Mediator of Hepatocellular Carcinoma Survival to Thermal Ablation Induced Heat Stress.

    Science.gov (United States)

    Thompson, Scott M; Callstrom, Matthew R; Jondal, Danielle E; Butters, Kim A; Knudsen, Bruce E; Anderson, Jill L; Lien, Karen R; Sutor, Shari L; Lee, Ju-Seog; Thorgeirsson, Snorri S; Grande, Joseph P; Roberts, Lewis R; Woodrum, David A

    2016-01-01

    Thermal ablative therapies are important treatment options in the multidisciplinary care of patients with hepatocellular carcinoma (HCC), but lesions larger than 2-3 cm are plagued with high local recurrence rates and overall survival of these patients remains poor. Currently no adjuvant therapies exist to prevent local HCC recurrence in patients undergoing thermal ablation. The molecular mechanisms mediating HCC resistance to thermal ablation induced heat stress and local recurrence remain unclear. Here we demonstrate that the HCC cells with a poor prognostic hepatic stem cell subtype (Subtype HS) are more resistant to heat stress than HCC cells with a better prognostic hepatocyte subtype (Subtype HC). Moreover, sublethal heat stress rapidly induces phosphoinositide 3-kinase (PI3K)/mammalian target of rapamycin (mTOR) dependent-protein kinase B (AKT) survival signaling in HCC cells in vitro and at the tumor ablation margin in vivo. Conversely, inhibition of PI3K/mTOR complex 2 (mTORC2)-dependent AKT phosphorylation or direct inhibition of AKT function both enhance HCC cell killing and decrease HCC cell survival to sublethal heat stress in both poor and better prognostic HCC subtypes while mTOR complex 1 (mTORC1)-inhibition has no impact. Finally, we showed that AKT isoforms 1, 2 and 3 are differentially upregulated in primary human HCCs and that overexpression of AKT correlates with worse tumor biology and pathologic features (AKT3) and prognosis (AKT1). Together these findings define a novel molecular mechanism whereby heat stress induces PI3K/mTORC2-dependent AKT survival signaling in HCC cells and provide a mechanistic rationale for adjuvant AKT inhibition in combination with thermal ablation as a strategy to enhance HCC cell killing and prevent local recurrence, particularly at the ablation margin. PMID:27611696

  16. Downstream mode of competition with upstream market power

    OpenAIRE

    Manasakis, Constantine; Vlassis, Minas

    2013-01-01

    In a two-tier oligopoly, where the downstream firms are locked in pair-wise exclusive relationships with their upstream input suppliers, the equilibrium mode of competition in the downstream market is endogenously determined as a renegotiation-proof contract signed between each downstream firm and its exclusive upstream input supplier. We find that the upstream-downstream exclusive relationships credibly sustain the Cournot (Bertrand) mode of competition in the downstream market, when the goo...

  17. Vertical Integration and Market Foreclosure with Convex Downstream Costs

    OpenAIRE

    Baake, Pio; Kamecke, Ulrich; Normann, Hans-Theo

    2001-01-01

    In a framework with an upstream monopoly and a downstream duopoly, we analyze the impact of convex costs on the downstream level. In contrast to the case of constant marginal costs, vertical integration does not imply complete market foreclosure. While the non-integrated downstream firm receives a strictly positive amount of the intermediate good, the downstream allocation is inefficient. However, a parametrized example indicates that competition at the downstream level may increase aggregate...

  18. Overcoming Akt Induced Therapeutic Resistance in Breast Cancer through siRNA and Thymoquinone Encapsulated Multilamellar Gold Niosomes.

    Science.gov (United States)

    Rajput, Shashi; Puvvada, Nagaprasad; Kumar, B N Prashanth; Sarkar, Siddik; Konar, Suraj; Bharti, Rashmi; Dey, Goutam; Mazumdar, Abhijit; Pathak, Amita; Fisher, Paul B; Mandal, Mahitosh

    2015-12-01

    Akt overexpression in cancer causes resistance to traditional chemotherapeutics. Silencing Akt through siRNA provides new therapeutic options; however, poor in vivo siRNA pharmacokinetics impede translation. We demonstrate that acidic milieu-sensitive multilamellar gold niosomes (Nio-Au) permit targeted delivery of both Akt-siRNA and thymoquinone (TQ) in tamoxifen-resistant and Akt-overexpressing MCF7 breast cancer cells. Octadecylamine groups of functionalized gold nanoparticles impart cationic attribute to niosomes, stabilized through polyethylene glycol. TQ's aqueous insolubility renders its encapsulation within hydrophobic core, and negatively charged siRNA binds in hydrophilic region of cationic niosomes. These niosomes were exploited to effectively knockdown Akt, thereby sensitizing cells to TQ. Immunoblot studies revealed enhanced apoptosis by inducing p53 and inhibiting MDM2 expression, which was consistent with in vivo xenograft studies. This innovative strategy, using Nio-Au to simultaneously deliver siRNA (devoid of any chemical modification) and therapeutic drug, provides an efficacious approach for treating therapy-resistant cancers with significant translational potential. PMID:26505213

  19. Intestinal trefoil factor activates the PI3K/Akt signaling pathway to protect gastric mucosal epithelium from damage.

    Science.gov (United States)

    Sun, Zhaorui; Liu, Hongmei; Yang, Zhizhou; Shao, Danbing; Zhang, Wei; Ren, Yi; Sun, Baodi; Lin, Jinfeng; Xu, Min; Nie, Shinan

    2014-09-01

    Intestinal trefoil factor (ITF, also named as trefoil factor 3, TFF3) is a member of the TFF-domain peptide family, which plays an essential role in the regulation of cell survival, cell migration and maintains mucosal epithelial integrity in the gastrointestinal tract. However, the underlying mechanisms and associated molecules remain unclear. The aim of this study was to explore the protective effects of ITF on gastric mucosal epithelium injury and its possible molecular mechanisms of action. In the present study, we show that ITF was able to promote the proliferation and migration of GES-1 cells via a mechanism that involves the PI3K/Akt signaling pathway. Western blot results indicated that ITF induced a dose- and time-dependent increase in the Akt signaling pathway. ITF also plays an essential role in the restitution of GES-1 cell damage induced by lipopolysaccharide (LPS). LPS induced the apoptosis of GES-1 cells, decreased cell viability significantly (Pinhibition of the PI3K/Akt pathway. Taken together, our results demonstrate that ITF promotes the proliferation and migration of gastric mucosal epithelial cells and preserves gastric mucosal epithelial integrity after damage is mediated by activation of the PI3K/Akt signaling pathway. This study suggested that the PI3K/Akt pathway could act as a key intracellular pathway in the gastric mucosal epithelium that may serve as a therapeutic target to preserve epithelial integrity during injury.

  20. COX-2 activation is associated with Akt phosphorylation and poor survival in ER-negative, HER2-positive breast cancer

    Directory of Open Access Journals (Sweden)

    Goodman Julie E

    2010-11-01

    Full Text Available Abstract Background Inducible cyclooxgenase-2 (COX-2 is commonly overexpressed in breast tumors and is a target for cancer therapy. Here, we studied the association of COX-2 with breast cancer survival and how this association is influenced by tumor estrogen and HER2 receptor status and Akt pathway activation. Methods Tumor COX-2, HER2 and estrogen receptor α (ER expression and phosphorylation of Akt, BAD, and caspase-9 were analyzed immunohistochemically in 248 cases of breast cancer. Spearman's correlation and multivariable logistic regression analyses were used to examine the relationship between COX-2 and tumor characteristics. Kaplan-Meier survival and multivariable Cox proportional hazards regression analyses were used to examine the relationship between COX-2 and disease-specific survival. Results COX-2 was significantly associated with breast cancer outcome in ER-negative [Hazard ratio (HR = 2.72; 95% confidence interval (CI, 1.36-5.41; comparing high versus low COX-2] and HER2 overexpressing breast cancer (HR = 2.84; 95% CI, 1.07-7.52. However, the hazard of poor survival associated with increased COX-2 was highest among patients who were both ER-negative and HER2-positive (HR = 5.95; 95% CI, 1.01-34.9. Notably, COX-2 expression in the ER-negative and HER2-positive tumors correlated significantly with increased phosphorylation of Akt and of the two Akt targets, BAD at Ser136 and caspase-9 at Ser196. Conclusions Up-regulation of COX-2 in ER-negative and HER2-positive breast tumors is associated with Akt pathway activation and is a marker of poor outcome. The findings suggest that COX-2-specific inhibitors and inhibitors of the Akt pathway may act synergistically as anticancer drugs in the ER-negative and HER2-positive breast cancer subtype.

  1. Effects of MicroRNA-153 on the Expression of Its Target Gene Downstream Signaling Molecule GSK-3β and on the Cellular Anti-Injury Ability%MicroRNA-153对靶基因下游信号分子GSK-3β表达水平及细胞抗损伤能力的影响

    Institute of Scientific and Technical Information of China (English)

    梁春联; 朱华; 黄澜; 许艳峰; 邓巍; 马春梅; 刘颖; 秦川

    2011-01-01

    Objective Mir-153 can negatively regulate the expression of APP and APLP2 protein, the crucial Alzheimer' s disease related genes, and consequently lower the level of their intracellular degradation fragment (intracellular domains, ICDs). Considering the transcriptional activity and pro-apoptotic role of ICDs, the aim of this study was to explore the effect of mir-153 on the expression of GSK-3β, the downstream signaling molecule of the two target genes, and on the ability of cells against damage stress to further identify the role of mir-153 in Alzheimer' s disease.Methods A stably transfected cell line over-expressing mir-153 was developed and mir-153 transgenic mice were generated. Western blot was used to detect the expression of phosphorylated GSK-3β, Tau and their total protein in the cells and mice. The mir-153 stably transfected cells were treated with Aβ42peptide and H202. respectively, to determine the changes of cell viability by MTS and analyze the cell apoptosis by flow cytometry. Results The expression of phosphorylated GSK-3β and it's total protein were decreased and the phosphorylation of Tau was reduced in the mir-153 stably transfected cells. The expression of phosphorylated GSK-3β and it' s total protein were down-regulated and the level of phosphorylated Tau and its total protein were not significantly changed in the brain of mir-153 transgenic mice. Under the treatment of Aβ42 peptide and H2O2, the viability of mir-153 stably transfected cells were clearly decreased and the apoptosis level of the cells was increased. Conclusion Mir-153 can negatively regulate the expression of GSK-3β, the downstream signaling molecule of its target genes. Over-expressed mir-153 lowers the cellular anti-injury ability.%目的 mir-153可负调控阿尔茨海默病(Alzheimer'S disease,AD)主要致病基因APP及APLP2的蛋白表达,降低其胞内降解片段(intracellular domains,ICDs)的生成.因ICDs具有转录活化及促凋亡

  2. Stabilization of LKB1 and Akt by neddylation regulates energy metabolism in liver cancer

    Science.gov (United States)

    Barbier-Torres, Lucía; Delgado, Teresa C.; García-Rodríguez, Juan L.; Zubiete-Franco, Imanol; Fernández-Ramos, David; Buqué, Xabier; Cano, Ainara; Juan, Virginia Gutiérrez-de; Fernández-Domínguez, Itziar; Lopitz-Otsoa, Fernando; Fernández-Tussy, Pablo; Boix, Loreto; Bruix, Jordi; Villa, Erica; Castro, Azucena; Lu, Shelly C.; Aspichueta, Patricia; Xirodimas, Dimitris; Varela-Rey, Marta; Mato, José M.; Beraza, Naiara; Martínez-Chantar, María L.

    2015-01-01

    The current view of cancer progression highlights that cancer cells must undergo through a post-translational regulation and metabolic reprogramming to progress in an unfriendly environment. In here, the importance of neddylation modification in liver cancer was investigated. We found that hepatic neddylation was specifically enriched in liver cancer patients with bad prognosis. In addition, the treatment with the neddylation inhibitor MLN4924 in Phb1-KO mice, an animal model of hepatocellular carcinoma showing elevated neddylation, reverted the malignant phenotype. Tumor cell death in vivo translating into liver tumor regression was associated with augmented phosphatidylcholine synthesis by the PEMT pathway, known as a liver-specific tumor suppressor, and restored mitochondrial function and TCA cycle flux. Otherwise, in protumoral hepatocytes, neddylation inhibition resulted in metabolic reprogramming rendering a decrease in oxidative phosphorylation and concomitant tumor cell apoptosis. Moreover, Akt and LKB1, hallmarks of proliferative metabolism, were altered in liver cancer being new targets of neddylation. Importantly, we show that neddylation-induced metabolic reprogramming and apoptosis were dependent on LKB1 and Akt stabilization. Overall, our results implicate neddylation/signaling/metabolism, partly mediated by LKB1 and Akt, in the development of liver cancer, paving the way for novel therapeutic approaches targeting neddylation in hepatocellular carcinoma. PMID:25650664

  3. MicroRNA-520a attenuates proliferation of Raji cells through inhibition of AKT1/NF-κB and PERK/eIF2α signaling pathway.

    Science.gov (United States)

    Wang, Xiaojuan; Wang, Pei; Zhu, Yan; Zhang, Zhi; Zhang, Jinqian; Wang, Hongwei

    2016-09-01

    Burkitt's lymphoma (BL) is a fast growing cancer of the human lymphatic system, and an extremely invasive B-cell non-Hodgkin's lymphoma. We explored the mechanism of apoptosis in Raji cells associated with the post-transcriptional regulation factors. To confirm that the predicted microRNA-520a (miR-520a) is matched with AKT1, 3' untranslated region (UTR) luciferase activity of AKT1 was used in the assessment. In the presence of the mimics or inhibitors of miR-520a, cell function of Raji, such as proliferation, growth and apoptosis were analyzed. The expression of endoplasmic reticulum (ER) stress‑related proteins were examined. Luciferase reporter analysis showed that miR‑520a leads to decreased activity of luciferase gene fused with AKT1 3'UTR. Therefore, AKT1 is a direct target of miR‑520a. Our data indicated that the mimics of miR‑520a inhibited growth, proliferation of Raji cells and promoted its apoptosis, which was related to downregulation of AKT1, NF‑κB and ER stress response mediated by PERK/eIF2α pathway. On the contrary, the inhibitors of miR‑520a promoted growth, proliferation of Raji cells and inhibited its apoptosis, which was related to AKT1/NF‑κB and PERK/eIF2α pathway. We identified miR‑520a, which specifically binds to AKT1 mRNA 3'UTR. miR‑520a is a crucial mediator for proliferation and ER stress in Raji cells through regulating the AKT1/NF‑κB or PERK/eIF2α signaling pathway. Our findings suggest that targeting miR‑520a is a promising therapeutic strategy in BL. PMID:27461820

  4. Make Yourself at Home: Viral Hijacking of the PI3K/Akt Signaling Pathway

    Directory of Open Access Journals (Sweden)

    Nora Diehl

    2013-12-01

    Full Text Available As viruses do not possess genes encoding for proteins required for translation, energy metabolism or membrane biosynthesis, they are classified as obligatory intracellular parasites that depend on a host cell to replicate. This genome limitation forces them to gain control over cellular processes to ensure their successful propagation. A diverse spectrum of virally encoded proteins tackling a broad spectrum of cellular pathways during most steps of the viral life cycle ranging from the host cell entry to viral protein translation has evolved. Since the host cell PI3K/Akt signaling pathway plays a critical regulatory role in many cellular processes including RNA processing, translation, autophagy and apoptosis, many viruses, in widely varying ways, target it. This review focuses on a number of remarkable examples of viral strategies, which exploit the PI3K/Akt signaling pathway for effective viral replication.

  5. Combined inhibition of p38 and Akt signaling pathways abrogates cyclosporine A-mediated pathogenesis of aggressive skin SCCs

    Energy Technology Data Exchange (ETDEWEB)

    Arumugam, Aadithya; Walsh, Stephanie B.; Xu, Jianmin; Afaq, Farrukh [Department of Dermatology, University of Alabama at Birmingham, Birmingham, AL 35294-0019 (United States); Elmets, Craig A. [Department of Dermatology, University of Alabama at Birmingham, Birmingham, AL 35294-0019 (United States); Skin Diseases Research Center, University of Alabama at Birmingham, Birmingham, AL 35294 (United States); Athar, Mohammad, E-mail: mathar@uab.edu [Department of Dermatology, University of Alabama at Birmingham, Birmingham, AL 35294-0019 (United States); Skin Diseases Research Center, University of Alabama at Birmingham, Birmingham, AL 35294 (United States)

    2012-08-24

    Highlights: Black-Right-Pointing-Pointer p38 and Akt are the crucial molecular targets in the pathogenesis of SCCs in OTRs. Black-Right-Pointing-Pointer Combined inhibition of these targets diminished tumor growth by 90%. Black-Right-Pointing-Pointer Inhibition of these targets act through downregulating mTOR signaling pathway. -- Abstract: Non-melanoma skin cancers (NMSCs) are the most common neoplasm in organ transplant recipients (OTRs). These cancers are more invasive and metastatic as compared to those developed in normal cohorts. Previously, we have shown that immunosuppressive drug, cyclosporine A (CsA) directly alters tumor phenotype of cutaneous squamous cell carcinomas (SCCs) by activating TGF-{beta} and TAK1/TAB1 signaling pathways. Here, we identified novel molecular targets for the therapeutic intervention of these SCCs. We observed that combined blockade of Akt and p38 kinases-dependent signaling pathways in CsA-promoted human epidermoid carcinoma A431 xenograft tumors abrogated their growth by more than 90%. This diminution in tumor growth was accompanied by a significant decrease in proliferation and an increase in apoptosis. The residual tumors following the combined treatment with Akt inhibitor triciribine and p38 inhibitors SB-203580 showed significantly diminished expression of phosphorylated Akt and p38 and these tumors were less invasive and highly differentiated. Diminished tumor invasiveness was associated with the reduced epithelial-mesenchymal transition as ascertained by the enhanced E-cadherin and reduced vimentin and N-cadherin expression. Consistently, these tumors also manifested reduced MMP-2/9. The decreased p-Akt expression was accompanied by a significant reduction in p-mTOR. These data provide first important combinatorial pharmacological approach to block the pathogenesis of CsA-induced highly aggressive cutaneous neoplasm in OTRs.

  6. Combined inhibition of p38 and Akt signaling pathways abrogates cyclosporine A-mediated pathogenesis of aggressive skin SCCs

    International Nuclear Information System (INIS)

    Highlights: ► p38 and Akt are the crucial molecular targets in the pathogenesis of SCCs in OTRs. ► Combined inhibition of these targets diminished tumor growth by 90%. ► Inhibition of these targets act through downregulating mTOR signaling pathway. -- Abstract: Non-melanoma skin cancers (NMSCs) are the most common neoplasm in organ transplant recipients (OTRs). These cancers are more invasive and metastatic as compared to those developed in normal cohorts. Previously, we have shown that immunosuppressive drug, cyclosporine A (CsA) directly alters tumor phenotype of cutaneous squamous cell carcinomas (SCCs) by activating TGF-β and TAK1/TAB1 signaling pathways. Here, we identified novel molecular targets for the therapeutic intervention of these SCCs. We observed that combined blockade of Akt and p38 kinases-dependent signaling pathways in CsA-promoted human epidermoid carcinoma A431 xenograft tumors abrogated their growth by more than 90%. This diminution in tumor growth was accompanied by a significant decrease in proliferation and an increase in apoptosis. The residual tumors following the combined treatment with Akt inhibitor triciribine and p38 inhibitors SB-203580 showed significantly diminished expression of phosphorylated Akt and p38 and these tumors were less invasive and highly differentiated. Diminished tumor invasiveness was associated with the reduced epithelial–mesenchymal transition as ascertained by the enhanced E-cadherin and reduced vimentin and N-cadherin expression. Consistently, these tumors also manifested reduced MMP-2/9. The decreased p-Akt expression was accompanied by a significant reduction in p-mTOR. These data provide first important combinatorial pharmacological approach to block the pathogenesis of CsA-induced highly aggressive cutaneous neoplasm in OTRs.

  7. The novel Akt inhibitor Palomid 529 (P529) enhances the effect of radiotherapy in prostate cancer

    Science.gov (United States)

    Diaz, R; Nguewa, P A; Diaz-Gonzalez, J A; Hamel, E; Gonzalez-Moreno, O; Catena, R; Serrano, D; Redrado, M; Sherris, D; Calvo, A

    2009-01-01

    Radiotherapy (RT) is a common treatment for localised prostate cancer, but can cause important side effects. The therapeutic efficacy of RT can be enhanced by pharmacological compounds that target specific pathways involved in cell survival. This would elicit a similar therapeutic response using lower doses of RT and, in turn, reducing side effects. This study describes the antitumour activity of the novel Akt inhibitor 8-(1-Hydroxy-ethyl)-2-methoxy-3-(4-methoxy-benzyloxy)-benzo[c]chromen-6-one (Palomid 529 or P529) as well as its ability to decrease radiation-activated phospho-Akt (p-Akt) signalling in a prostate cancer model. P529 showed a potent antiproliferative activity in the NCI-60 cell lines panel, with growth inhibitory 50 (GI50) <35 μM. In addition, P529 significantly enhanced the antiproliferative effect of radiation in prostate cancer cells (PC-3). Analysis of signalling pathways targeted by P529 exhibited a decrease in p-Akt, VEGF, MMP-2, MMP-9, and Id-1 levels after radiation treatment. Moreover, the Bcl-2/Bax ratio was also reduced. Treatment of PC-3 tumour-bearing mice with 20 mg kg−1 P529 or 6 Gy radiation dose decreased tumour size by 42.9 and 53%, respectively. Combination of both treatments resulted in 77.4% tumour shrinkage. Decreased tumour growth was due to reduced proliferation and increased apoptosis (as assessed by PCNA and caspase-3 immunostaining). Our results show the antitumour efficacy of P529 alone, and as a radiosensitiser, and suggest that this compound could be used in the future to treat human prostate cancer. PMID:19240717

  8. AKT2-knockdown suppressed viability with enhanced apoptosis, and attenuated chemoresistance to temozolomide of human glioblastoma cells in vitro and in vivo

    Directory of Open Access Journals (Sweden)

    Cui Y

    2015-07-01

    Full Text Available Yong Cui,1,* Jing Lin,1,* Jianling Zuo,2 Lei Zhang,1 Yan Dong,1 Guohan Hu,1 Chun Luo,1 Juxiang Chen,1 Yicheng Lu1 1Department of Neurosurgery, Changzheng Hospital, Second Military Medical University, Shanghai, People’s Republic of China; 2Department of Neurosurgery, First Affiliated Hospital of Soochow University, Suzhou, People’s Republic of China *These authors contributed equally to this work Abstract: The AKT2 kinase (protein kinase Bβ is overexpressed in high-grade gliomas. Upregulation of the AKT2 gene has been previously observed in glioblastoma patients suffering from chemotherapy failure and tumor progress. In this study, we aimed to evaluate the effect of AKT2 on viability and chemoresistance in the human glioblastoma cell line U251. The U251 cell line was stably transfected with short hairpin RNA (shRNA targeting AKT2. U251 cells underexpressing AKT2 were then examined for viability with temozolomide (TMZ treatment, and tested for cell apoptosis both in vitro and in tumor-implanted mice. Next, expressions of several chemoresistance-related molecules were measured by quantitative reverse-transcription polymerase chain reaction (qRT-PCR and western blot analysis. The results showed that the 50% inhibitory concentration (IC50 of AKT2 shRNA-transfected cells was significantly lower compared with Lenti-GFP-transfected and nontransfected controls and that the tumor growth of the AKT2-shRNA and TMZ combined-treated mice was obviously suppressed in either mass or volume. Concomitantly, the apoptosis of TMZ-treated tumor cells was significantly enhanced after knockdown of AKT2, as measured by flow cytometry and in situ terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL analysis. Furthermore, AKT2-inhibition in TMZ-treated glioblastoma U251 cells upregulated apoptotic effector caspase-3, whereas it downregulated antiapoptotic protein Bcl-2, DNA repairing protein MGMT, and drug efflux pump protein MRP1. Our study

  9. Propolis augments apoptosis induced by butyrate via targeting cell survival pathways.

    Directory of Open Access Journals (Sweden)

    Eric Drago

    Full Text Available Diet is one of the major lifestyle factors affecting incidence of colorectal cancer (CC, and despite accumulating evidence that numerous diet-derived compounds modulate CC incidence, definitive dietary recommendations are not available. We propose a strategy that could facilitate the design of dietary supplements with CC-preventive properties. Thus, nutrient combinations that are a source of apoptosis-inducers and inhibitors of compensatory cell proliferation pathways (e.g., AKT signaling may produce high levels of programmed death in CC cells. Here we report the combined effect of butyrate, an apoptosis inducer that is produced through fermentation of fiber in the colon, and propolis, a honeybee product, on CC cells. We established that propolis increases the apoptosis of CC cells exposed to butyrate through suppression of cell survival pathways such as the AKT signaling. The programmed death of CC cells by combined exposure to butyrate and propolis is further augmented by inhibition of the JNK signaling pathway. Analyses on the contribution of the downstream targets of JNK signaling, c-JUN and JAK/STAT, to the apoptosis of butyrate/propolis-treated CC cells ascertained that JAK/STAT signaling has an anti-apoptotic role; whereas, the role of cJUN might be dependent upon regulatory cell factors. Thus, our studies ascertained that propolis augments apoptosis of butyrate-sensitive CC cells and re-sensitizes butyrate-resistant CC cells to apoptosis by suppressing AKT signaling and downregulating the JAK/STAT pathway. Future in vivo studies should evaluate the CC-preventive potential of a dietary supplement that produces high levels of colonic butyrate, propolis, and diet-derived JAK/STAT inhibitors.

  10. Role of beta-arrestin 2 downstream of dopamine receptors in the basal ganglia

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    Thomas eDel'Guidice

    2011-09-01

    Full Text Available Multifunctional scaffolding protein beta-arrestins (βArr and the G protein receptor kinases (GRK are involved in the desensitization of several G protein coupled-receptors (GPCR. However, arrestins can also contribute to GPCR signaling independently from G proteins. In this review, we focus on the role of βArr in the regulation of dopamine receptor functions in the striatum. First, we present in vivo evidence supporting a role for these proteins in the regulation of dopamine receptor desensitization. Second, we provide an overview of the roles of βArr-2 in the regulation of ERK/MAPkinases and Akt/GSK3 signaling pathways downstream of the D1 and D2 dopamine receptors. Thereafter, we examine the possible involvement of βArr-mediated signaling in the action of dopaminergic drugs used for the treatment of mental disorders. Finally, we focus on different potential cellular proteins regulated by βArr-mediated signaling which could contribute to the regulation of behavioral responses to dopamine. Overall, the identification of a cell signaling function for βArr downstream of dopamine receptors underscores the intricate complexity of the intertwined mechanisms regulating and mediating cell signaling in the basal ganglia. Understanding these mechanisms may lead to a better comprehension of the several roles played by these structures in the regulation of mood and to the development of new psychoactive drugs having better therapeutic efficacy.

  11. PLZF mediates the PTEN/AKT/FOXO3a signaling in suppression of prostate tumorigenesis.

    Directory of Open Access Journals (Sweden)

    JingPing Cao

    Full Text Available Promyelocytic leukemia zinc finger (PLZF protein expression is closely related to the progression of human cancers, including prostate cancer (PCa. However, the according context of a signaling pathway for PLZF to suppress prostate tumorigenesis remains greatly unknown. Here we report that PLZF is a downstream mediator of the PTEN signaling pathway in PCa. We found that PLZF expression is closely correlated with PTEN expression in a cohort of prostate cancer specimens. Interestingly, both PTEN rescue and phosphoinositide 3-kinase (PI3K inhibitor LY294002 treatment increase the PLZF expression in prostate cancer cell lines. Further, luciferase reporter assay and chromatin immunoprecipitation assay demonstrate that FOXO3a, a transcriptional factor phosphorylated by PI3K/AKT, could directly bind to the promoter of PLZF gene. These results indicate that PTEN regulates PLZF expression by AKT/FOXO3a. Moreover, our animal experiments also demonstrate that PLZF is capable of inhibiting prostate tumorigenesis in vivo. Taken together, our study defines a PTEN/PLZF pathway and would shed new lights for developing therapeutic strategy of prostate cancer.

  12. Subsequent to Suppression: Downstream Comprehension Consequences of Noun/Verb Ambiguity in Natural Reading

    Science.gov (United States)

    Stites, Mallory C.; Federmeier, Kara D.

    2015-01-01

    We used eye tracking to investigate the downstream processing consequences of encountering noun/verb (NV) homographs (i.e., park) in semantically neutral but syntactically constraining contexts. Target words were followed by a prepositional phrase containing a noun that was plausible for only 1 meaning of the homograph. Replicating previous work,…

  13. Plasma waves downstream of weak collisionless shocks

    Science.gov (United States)

    Coroniti, F. V.; Greenstadt, E. W.; Moses, S. L.; Smith, E. J.; Tsurutani, B. T.

    1993-01-01

    In September 1983 the International Sun Earth Explorer 3 (ISEE 3) International Cometary Explorer (ICE) spacecraft made a long traversal of the distant dawnside flank region of the Earth's magnetosphere and had many encounters with the low Mach number bow shock. These weak shocks excite plasma wave electric field turbulence with amplitudes comparable to those detected in the much stronger bow shock near the nose region. Downstream of quasi-perpendicular (quasi-parallel) shocks, the E field spectra exhibit a strong peak (plateau) at midfrequencies (1 - 3 kHz); the plateau shape is produced by a low-frequency (100 - 300 Hz) emission which is more intense behind downstream of two quasi-perpendicular shocks show that the low frequency signals are polarized parallel to the magnetic field, whereas the midfrequency emissions are unpolarized or only weakly polarized. A new high frequency (10 - 30 kHz) emission which is above the maximum Doppler shift exhibit a distinct peak at high frequencies; this peak is often blurred by the large amplitude fluctuations of the midfrequency waves. The high-frequency component is strongly polarized along the magnetic field and varies independently of the lower-frequency waves.

  14. Clinical prognostic significance and pro-metastatic activity of RANK/RANKL via the AKT pathway in endometrial cancer.

    Science.gov (United States)

    Wang, Jing; Liu, Yao; Wang, Lihua; Sun, Xiao; Wang, Yudong

    2016-02-01

    RANK/RANKL plays a key role in metastasis of certain malignant tumors, which makes it a promising target for developing novel therapeutic strategies for cancer. However, the prognostic value and pro-metastatic activity of RANK in endometrial cancer (EC) remain to be determined. Thus, the present study investigated the effect of RANK on the prognosis of EC patients, as well as the pro-metastatic activity of EC cells. The results indicated that those with high expression of RANK showed decreased overall survival and progression-free survival. Statistical analysis revealed the positive correlations between RANK/RANKL expression and metastasis-related factors. Additionally, RANK/RANKL significantly promoted cell migration/invasion via activating AKT/β-catenin/Snail pathway in vitro. However, RANK/RANKL-induced AKT activation could be suppressed after osteoprotegerin (OPG) treatment. Furthermore, the combination of medroxyprogesterone acetate (MPA) and RANKL could in turn attenuate the effect of RANKL alone. Similarly, MPA could partially inhibit the RANK-induced metastasis in an orthotopic mouse model via suppressing AKT/β-catenin/Snail pathway. Therefore, therapeutic inhibition of MPA in RANK/RANKL-induced metastasis was mediated by AKT/β-catenin/Snail pathway both in vitro and in vivo, suggesting a potential target of RANK for gene-based therapy for EC. PMID:26734994

  15. SG2NA enhances cancer cell survival by stabilizing DJ-1 and thus activating Akt

    Energy Technology Data Exchange (ETDEWEB)

    Tanti, Goutam Kumar, E-mail: goutamjnu@hotmail.com; Pandey, Shweta; Goswami, Shyamal K.

    2015-08-07

    SG2NA in association with striatin and zinedin forms a striatin family of WD-40 repeat proteins. This family of proteins functions as scaffold in different signal transduction pathways. They also act as a regulatory subunit of protein phosphatase 2A. We have shown that SG2NA which evolved first in the metazoan evolution among the striatin family members expresses different isoforms generated out of alternative splicing. We have also shown that SG2NA protects cells from oxidative stress by recruiting DJ-1 and Akt to mitochondria and membrane in the post-mitotic neuronal cells. DJ-1 is both cancer and Parkinson's disease related protein. In the present study we have shown that SG2NA protects DJ-1 from proteasomal degradation in cancer cells. Hence, downregulation of SG2NA reduces DJ-1/Akt colocalization in cancer cells resulting in the reduction of anchorage dependent and independent growth. Thus SG2NA enhances cancer cell survival. Reactive oxygen species enhances SG2NA, DJ-1 and Akt trimerization. Removal of the reactive oxygen species by N-acetyl-cysteine thus reduces cancer cell growth. - Highlights: • Reactive oxygen species (ROS) play potential role in cancer cell proliferation. • It enhances the association between DJ-1 and Akt mediated by SG2NA. • In cancer cells SG2NA stabilizes DJ-1 by inhibiting it from proteosomal degradation. • DJ-1 then activates Akt and cancer cells get their property of enhanced proliferation by sustained activation of Akt. • Further study on this field could lead to new target for cancer therapy.

  16. PI3K-AKT-mTOR signaling in prostate cancer progression and androgen deprivation therapy resistance

    Institute of Scientific and Technical Information of China (English)

    Merritt P Edlind; Andrew C Hsieh

    2014-01-01

    Prostate cancer (PCa) is the second most common malignancy among men in the world. Castration-resistant prostate cancer (CRPC) is the lethal form of the disease, which develops upon resistance to ifrst line androgen deprivation therapy (ADT). Emerging evidence demonstrates a key role for the PI3K-AKT-mTOR signaling axis in the development and maintenance of CRPC. This pathway, which is deregulated in the majority of advanced PCas, serves as a critical nexus for the integration of growth signals with downstream cellular processes such as protein synthesis, proliferation, survival, metabolism and differentiation, thus providing mechanisms for cancer cells to overcome the stress associated with androgen deprivation. Furthermore, preclinical studies have elucidated a direct connection between the PI3K-AKT-mTOR and androgen receptor (AR) signaling axes, revealing a dynamic interplay between these pathways during the development of ADT resistance. Thus, there is a clear rationale for the continued clinical development of a number of novel inhibitors of the PI3K pathway, which offer the potential of blocking CRPC growth and survival. In this review, we will explore the relevance of the PI3K-AKT-mTOR pathway in PCa progression and castration resistance in order to inform the clinical development of speciifc pathway inhibitors in advanced PCa. In addition, we will highlight current deifciencies in our clinical knowledge, most notably the need for biomarkers that can accurately predict for response to PI3K pathway inhibitors.

  17. Inhibition of oxidative stress-elicited AKT activation facilitates PPARγ agonist-mediated inhibition of stem cell character and tumor growth of liver cancer cells.

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    Lanlan Liu

    Full Text Available Emerging evidence suggests that tumor-initiating cells (TICs are the most malignant cell subpopulation in tumors because of their resistance to chemotherapy or radiation treatment. Targeting TICs may be a key innovation for cancer treatment. In this study, we found that PPARγ agonists inhibited the cancer stem cell-like phenotype and attenuated tumor growth of human hepatocellular carcinoma (HCC cells. Reactive oxygen species (ROS initiated by NOX2 upregulation were partially responsible for the inhibitory effects mediated by PPARγ agonists. However, PPARγ agonist-mediated ROS production significantly activated AKT, which in turn promoted TIC survival by limiting ROS generation. Inhibition of AKT, by either pharmacological inhibitors or AKT siRNA, significantly enhanced PPARγ agonist-mediated inhibition of cell proliferation and stem cell-like properties in HCC cells. Importantly, in nude mice inoculated with HCC Huh7 cells, we demonstrated a synergistic inhibitory effect of the PPARγ agonist rosiglitazone and the AKT inhibitor triciribine on tumor growth. In conclusion, we observed a negative feedback loop between oxidative stress and AKT hyperactivation in PPARγ agonist-mediated suppressive effects on HCCs. Combinatory application of an AKT inhibitor and a PPARγ agonist may provide a new strategy for inhibition of stem cell-like properties in HCCs and treatment of liver cancer.

  18. Hydrogen sulfide promotes cell proliferation of oral cancer through activation of the COX2/AKT/ERK1/2 axis.

    Science.gov (United States)

    Zhang, Shuai; Bian, Huan; Li, Xiaoxu; Wu, Huanhuan; Bi, Qingwei; Yan, Yingbin; Wang, Yixiang

    2016-05-01

    Hydrogen sulfide, the third gaseous transmitter, is one of the main causes of halitosis in the oral cavity. It is generally considered as playing a deleterious role in many oral diseases including oral cancer. However, the regulatory mechanisms involved in the effects of hydrogen sulfide on oral cancer growth remain largely unknown. In the present study, we investigated the underlying mechanisms through CCK-8 assay, EdU incorporation, real-time PCR, western blot and pathway blockade assays. Our results showed that hydrogen sulfide promoted oral cancer cell proliferation through activation of the COX2, AKT and ERK1/2 pathways in a dose-dependent manner. Blocking any of the three above pathways inhibited hydrogen sulfide-induced oral cancer cell proliferation. Meanwhile, blockade of COX2 by niflumic acid downregulated NaHS-induced p-ERK and p-AKT expression. Inactivation of the AKT pathway by GSK690693 significantly decreased NaHS‑induced p-ERK1/2 expression, and inhibition of the ERK1/2 pathway by U0126 markedly increased NaHS-induced p-AKT expression. Either the AKT or ERK1/2 inhibitor did not significantly alter the COX2 expression level. Our data revealed, for the first time, that hydrogen sulfide promotes oral cancer cell proliferation through activation of the COX2/AKT/ERK1/2 axis, suggesting new potential targets to eliminate the effect of hydrogen sulfide on the development of oral cancer. PMID:26987083

  19. Valproic acid and butyrate induce apoptosis in human cancer cells through inhibition of gene expression of Akt/protein kinase B

    Directory of Open Access Journals (Sweden)

    Li Qiao

    2006-12-01

    Full Text Available Abstract Background In eukaryotic cells, the genomic DNA is packed with histones to form the nucleosome and chromatin structure. Reversible acetylation of the histone tails plays an important role in the control of specific gene expression. Mounting evidence has established that histone deacetylase inhibitors selectively induce cellular differentiation, growth arrest and apoptosis in variety of cancer cells, making them a promising class of anticancer drugs. However, the molecular mechanisms of the anti-cancer effects of these inhibitors have yet to be understood. Results Here, we report that a key determinant for the susceptibility of cancer cells to histone deacetylase inhibitors is their ability to maintain cellular Akt activity in response to the treatment. Also known as protein kinase B, Akt is an essential pro-survival factor in cell proliferation and is often deregulated during tumorigenesis. We show that histone deacetylase inhibitors, such as valproic acid and butyrate, impede Akt1 and Akt2 expression, which leads to Akt deactivation and apoptotic cell death. In addition, valproic acid and butyrate induce apoptosis through the caspase-dependent pathway. The activity of caspase-9 is robustly activated upon valproic acid or butyrate treatment. Constitutively active Akt is able to block the caspase activation and rescues cells from butyrate-induced apoptotic cell death. Conclusion Our study demonstrates that although the primary target of histone deacetylase inhibitors is transcription, it is the capacity of cells to maintain cellular survival networks that determines their fate of survival.

  20. Arsenic trioxide overcomes rapamycin-induced feedback activation of AKT and ERK signaling to enhance the anti-tumor effects in breast cancer.

    Directory of Open Access Journals (Sweden)

    Cynthia Guilbert

    Full Text Available Inhibitors of the mammalian target of rapamycin (mTORi have clinical activity; however, the benefits of mTOR inhibition by rapamycin and rapamycin-derivatives (rapalogs may be limited by a feedback mechanism that results in AKT activation. Increased AKT activity resulting from mTOR inhibition can be a result of increased signaling via the mTOR complex, TORC2. Previously, we published that arsenic trioxide (ATO inhibits AKT activity and in some cases, decreases AKT protein expression. Therefore, we propose that combining ATO and rapamycin may circumvent the AKT feedback loop and increase the anti-tumor effects. Using a panel of breast cancer cell lines, we find that ATO, at clinically-achievable doses, can enhance the inhibitory activity of the mTORi temsirolimus. In all cell lines, temsirolimus treatment resulted in AKT activation, which was decreased by concomitant ATO treatment only in those cell lines where ATO enhanced growth inhibition. Treatment with rapalog also results in activated ERK signaling, which is decreased with ATO co-treatment in all cell lines tested. We next tested the toxicity and efficacy of rapamycin plus ATO combination therapy in a MDA-MB-468 breast cancer xenograft model. The drug combination was well-tolerated, and rapamycin did not increase ATO-induced liver enzyme levels. In addition, combination of these drugs was significantly more effective at inhibiting tumor growth compared to individual drug treatments, which corresponded with diminished phospho-Akt and phospho-ERK levels when compared with rapamycin-treated tumors. Therefore, we propose that combining ATO and mTORi may overcome the feedback loop by decreasing activation of the MAPK and AKT signaling pathways.

  1. 40 CFR 80.69 - Requirements for downstream oxygenate blending.

    Science.gov (United States)

    2010-07-01

    ... 40 Protection of Environment 16 2010-07-01 2010-07-01 false Requirements for downstream oxygenate... downstream oxygenate blending. The requirements of this section apply to all reformulated gasoline blendstock... annual compliance period; (D) A process for notifying oxygenate blenders and other downstream parties...

  2. Dormancy of cancer cells with suppression of AKT activity contributes to survival in chronic hypoxia.

    Directory of Open Access Journals (Sweden)

    Hiroko Endo

    Full Text Available A hypoxic microenvironment in tumors has been recognized as a cause of malignancy or resistance to various cancer therapies. In contrast to recent progress in understanding the acute response of cancer cells to hypoxia, the characteristics of tumor cells in chronic hypoxia remain elusive. We have identified a pancreatic cancer cell line, AsPC-1, that is exceptionally able to survive for weeks under 1% oxygen conditions while most tested cancer cell lines die after only some days under these conditions. In chronic hypoxia, AsPC-1 cells entered a state of dormancy characterized by no proliferation, no death, and metabolic suppression. They reversibly switched to active status after being placed again in optimal culture conditions. ATP turnover, an indicator of energy demand, was markedly decreased and accompanied by reduced AKT phosphorylation. Forced activation of AKT resulted in increased ATP turnover and massive cell death in vitro and a decreased number of dormant cells in vivo. In contrast to most cancer cell lines, primary-cultured colorectal cancer cells easily entered the dormant status with AKT suppression under hypoxia combined with growth factor-depleted conditions. Primary colorectal cancer cells in dormancy were resistant to chemotherapy. Thus, the ability to survive in a deteriorated microenvironment by entering into dormancy under chronic hypoxia might be a common property among cancer cells. Targeting the regulatory mechanism inducing this dormant status could provide a new strategy for treating cancer.

  3. Channel changes downstream from a dam

    Science.gov (United States)

    Hadley, R.F.; Emmett, W.W.

    1998-01-01

    A flood-control dam was completed during 1979 on Bear Creek, a small tributary stream to the South Platte River in the Denver, Colorado, area. Before and after dam closure, repetitive surveys between 1977 and 1992 at five cross sections downstream of the dam documented changes in channel morphology. During this 15-year period, channel width increased slightly, but channel depth increased by more than 40 percent. Within the study reach, stream gradient decreased and median bed material sizes coarsened from sand in the pools and fine gravel on the riffle to a median coarse gravel throughout the reach. The most striking visual change was from a sparse growth of streamside grasses to a dense growth of riparian woody vegetation.

  4. Experimental Study on Scours Downstream of Floodgates

    Institute of Scientific and Technical Information of China (English)

    张玮; 陈锡林; 徐金环; 李国臣; 王志谦

    2000-01-01

    The river reach downstream of a floodgate at the estuary of the Xinyihe River is about 1.3km long, and the riverbed is composed of clotty clay. In the experiment, soil samples are taken from the construction site, and the incipient velocity is determined in a laboratory flume, and it is used to design the scour model and to select model sand material. The experimental results show that scours below the floodgate is unavoidable due to large discharge and low tidal level. Scours is caused by two factors: the rapid flow passing though the floodgate and the water drop near the river mouth during low tide, and the scour below the floodgate is more critical to the structural design. It is suggested that anti-scour walls should be used instead of riprap. The ideas and methods adopted in the experiment can be used as reference in the study on river scour under similar conditions.

  5. Downstream process options for the ABE fermentation.

    Science.gov (United States)

    Friedl, Anton

    2016-05-01

    Butanol is a very interesting substance both for the chemical industry and as a biofuel. The classical distillation process for the removal of butanol is far too energy demanding, at a factor of 220% of the energy content of butanol. Alternative separation processes studied are hybrid processes of gas-stripping, liquid-liquid extraction and pervaporation with distillation and a novel adsorption/drying/desorption hybrid process. Compared with the energy content of butanol, the resulting energy demand for butanol separation and concentration of optimized hybrid processes is 11%-22% for pervaporation/distillation and 11%-17% for liquid-liquid extraction/distillation. For a novel adsorption/drying/desorption process, the energy demand is 9.4%. But all downstream process options need further proof of industrial applicability. PMID:27020411

  6. Modeling of channel erosion downstream spillway dams

    Directory of Open Access Journals (Sweden)

    M.A. Mikhalev

    2013-03-01

    Full Text Available The channel erosion downstream spillway dams in non-cohesive materials has been analyzed from the viewpoint of methods of similarity and dimension theory. The obtained criterion equation connects the maximum depth of the local erosion with its determining parameters: length of concrete lining of bed in the down water of the spillway dam; Froude number at the contracted cross section; Archimedes and Reynolds criterions; submergence factor of hydraulic jump. The problem may be formulated as follows: the geometric size of the structure, kinematics and dynamics of the flows in the model are similar to that in the prototype. Conditions under which the characteristic depth of the local erosion in the model would be recomputed into the prototype, like any geometric size, are being discussed.

  7. The jet membrane-experiment: downstream sampling

    International Nuclear Information System (INIS)

    The invasion separation effect of the free jet structure was found in 1966 at Saclay. In the Downstream Sampling Configuration patended by Campargue (1967), the light fraction is withdrawn from the supersonic central core, by skimming the separating free jet. From experimental and theoretical results obtained for gas and isotopic mixtures, the following points linked to operation and equipment costs, are considered: system description; influence of mass ratio, expansion ratio, nature of separating gas, ratio of upflow to separating jet flow, rarefaction. Fron an uninteresting aspect of Jet Membrane (elimination of background penetration), a new principle has been discovered to produce nozzle beams which may be of great interest for other separation processes involving free jets and/or molecular beams

  8. Ammonia downstream from HH 80 North

    Science.gov (United States)

    Girart, Jose M.; Rodriguez, Luis F.; Anglada, Guillem; Estalella, Robert; Torrelles, Jose, M.; Marti, Josep; Pena, Miriam; Ayala, Sandra; Curiel, Salvador; Noriega-Crespo, Alberto

    1994-01-01

    HH 80-81 are two optically visible Herbig-Haro (HH) objects located about 5 minutes south of their exciting source IRAS 18162-2048. Displaced symmetrically to the north of this luminous IRAS source, a possible HH counterpart was recently detected as a radio continuum source with the very large array (VLA). This radio source, HH 80 North, has been proposed to be a member of the Herbig-Haro class since its centimeter flux density, angular size, spectral index, and morphology are all similar to those of HH 80. However, no object has been detected at optical wavelengths at the position of HH 80 North, possibly because of high extinction, and the confirmation of the radio continuum source as an HH object has not been possible. In the prototypical Herbig-Haro objects HH 1 and 2, ammonia emission has been detected downstream of the flow in both objects. This detection has been intepreted as a result of an enhancement in the ammonia emission produced by the radiation field of the shock associated with the HH object. In this Letter we report the detection of the (1,1) and (2,2) inversion transitions of ammonia downstream HH 80 North. This detection gives strong suppport to the interpretation of HH 80 North as a heavily obscured HH object. In addition, we suggest that ammonia emission may be a tracer of embedded Herbig-Haro objects in other regions of star formation. A 60 micrometer IRAS source could be associated with HH 80 North and with the ammonia condensation. A tentative explanation for the far-infrared emission as arising in dust heated by their optical and UV radiation of the HH object is presented.

  9. CCN1 acutely increases nitric oxide production via integrin αvβ3-Akt-S6K-phosphorylation of endothelial nitric oxide synthase at the serine 1177 signaling axis.

    Science.gov (United States)

    Hwang, Soojin; Lee, Hyeon-Ju; Kim, Gyungah; Won, Kyung-Jong; Park, Yoon Shin; Jo, Inho

    2015-12-01

    Although CCN1 (also known as cysteine-rich, angiogenic inducer 61, CYR61) has been reported to promote angiogenesis and neovascularization in endothelial cells (ECs), its effects on endothelial nitric oxide (NO) production have never been studied. Using human umbilical vein ECs, we investigated whether and how CCN1 regulates NO production. CCN1 acutely increased NO production in a time- and dose-dependent manner, which was accompanied by increased phosphorylation of endothelial NO synthase (eNOS) at serine 1177 (eNOS-Ser(1177)), but not that of eNOS-Thr(495) or eNOS-Ser(114). The level of total eNOS expression was unaltered. Treatment with either LY294002, a selective inhibitor of phosphoinositide 3-kinase known as an upstream kinase of Akt, or H-89, an inhibitor of protein kinase A, mitogen- and stress-activated protein kinase 1, Rho-associated protein kinase 2, and ribosomal protein S6 kinase (S6K), inhibited CCN1-stimulated eNOS-Ser(1177) phosphorylation and subsequent NO production. Ectopic expression of small interfering RNA against Akt and S6K significantly inhibited the effects of CCN1. Consistently, CCN1 increased the phosphorylation of Akt-Ser(473) and S6K-Thr(389). However, CCN1 did not alter the expression or secretion of VEGF, a known downstream factor of CCN1 and a potential upstream factor of Akt-mediated eNOS-Ser(1177) phosphorylation. Furthermore, neutralization of integrin αvβ3 with corresponding antibody completely reversed all of the observed effects of CCN1. Moreover, CCN1 increased acetylcholine-induced relaxation in the rat aortas. Finally, we also found that CCN1-stimulated eNOS-Ser(1177) phosphorylation and NO production are true for other types of EC tested. In conclusion, CCN1 acutely increases NO production via activation of a signaling axis in integrin αvβ3-Akt-S6K-eNOS-Ser(1177) phosphorylation, suggesting an important role for CCN1 in vasodilation.

  10. Functional Role of mTORC2 versus Integrin-Linked Kinase in Mediating Ser473-Akt Phosphorylation in PTEN-Negative Prostate and Breast Cancer Cell Lines.

    Science.gov (United States)

    Lee, Su-Lin; Chou, Chih-Chien; Chuang, Hsiao-Ching; Hsu, En-Chi; Chiu, Po-Chen; Kulp, Samuel K; Byrd, John C; Chen, Ching-Shih

    2013-01-01

    Although the rictor-mTOR complex (mTORC2) has been shown to act as phosphoinositide-dependent kinase (PDK)2 in many cell types, other kinases have also been implicated in mediating Ser473-Akt phosphorylation. Here, we demonstrated the cell line specificity of integrin-linked kinase (ILK) versus mTORC2 as PDK2 in LNCaP and PC-3 prostate and MDA-MB-468 breast cancer cells, of which the PTEN-negative status allowed the study of Ser473-Akt phosphorylation independent of external stimulation. PC-3 and MDA-MB-468 cells showed upregulated ILK expression relative to LNCaP cells, which expressed a high abundance of mTOR. Exposure to Ku-0063794, a second-generation mTOR inhibitor, decreased Ser473-Akt phosphorylation in LNCaP cells, but not in PC-3 or MDA-MB-468 cells. In contrast, treatment with T315, a novel ILK inhibitor, reduced the phosphorylation of Ser473-Akt in PC-3 and MDA-MB-468 cells without affecting that in LNCaP cells. This cell line specificity was verified by comparing Ser473-Akt phosphorylation status after genetic knockdown of rictor, ILK, and other putative Ser-473-Akt kinases. Genetic knockdown of rictor, but not ILK or the other kinases examined, inhibited Ser473-Akt phosphorylation in LNCaP cells. Conversely, PC-3 and MDA-MB-468 cells were susceptible to the effect of ILK silencing on Ser473-Akt phosphorylation, while knockdown of rictor or any of the other target kinases had no appreciable effect. Co-immunoprecipitation analysis demonstrated the physical interaction between ILK and Akt in PC-3 cells, and T315 blocked ILK-mediated Ser473 phosphorylation of bacterially expressed Akt. ILK also formed complexes with rictor in PC-3 and MDA-MB-468 cells that were disrupted by T315, but such complexes were not observed in LNCaP cells. In the PTEN-functional MDA-MB-231 cell line, both T315 and Ku-0063794 suppressed EGF-induced Ser473-Akt phosphorylation. Inhibition of ILK by T315 or siRNA-mediated knockdown suppressed epithelial-mesenchymal transition in MDA

  11. Functional Role of mTORC2 versus Integrin-Linked Kinase in Mediating Ser473-Akt Phosphorylation in PTEN-Negative Prostate and Breast Cancer Cell Lines.

    Directory of Open Access Journals (Sweden)

    Su-Lin Lee

    Full Text Available Although the rictor-mTOR complex (mTORC2 has been shown to act as phosphoinositide-dependent kinase (PDK2 in many cell types, other kinases have also been implicated in mediating Ser473-Akt phosphorylation. Here, we demonstrated the cell line specificity of integrin-linked kinase (ILK versus mTORC2 as PDK2 in LNCaP and PC-3 prostate and MDA-MB-468 breast cancer cells, of which the PTEN-negative status allowed the study of Ser473-Akt phosphorylation independent of external stimulation. PC-3 and MDA-MB-468 cells showed upregulated ILK expression relative to LNCaP cells, which expressed a high abundance of mTOR. Exposure to Ku-0063794, a second-generation mTOR inhibitor, decreased Ser473-Akt phosphorylation in LNCaP cells, but not in PC-3 or MDA-MB-468 cells. In contrast, treatment with T315, a novel ILK inhibitor, reduced the phosphorylation of Ser473-Akt in PC-3 and MDA-MB-468 cells without affecting that in LNCaP cells. This cell line specificity was verified by comparing Ser473-Akt phosphorylation status after genetic knockdown of rictor, ILK, and other putative Ser-473-Akt kinases. Genetic knockdown of rictor, but not ILK or the other kinases examined, inhibited Ser473-Akt phosphorylation in LNCaP cells. Conversely, PC-3 and MDA-MB-468 cells were susceptible to the effect of ILK silencing on Ser473-Akt phosphorylation, while knockdown of rictor or any of the other target kinases had no appreciable effect. Co-immunoprecipitation analysis demonstrated the physical interaction between ILK and Akt in PC-3 cells, and T315 blocked ILK-mediated Ser473 phosphorylation of bacterially expressed Akt. ILK also formed complexes with rictor in PC-3 and MDA-MB-468 cells that were disrupted by T315, but such complexes were not observed in LNCaP cells. In the PTEN-functional MDA-MB-231 cell line, both T315 and Ku-0063794 suppressed EGF-induced Ser473-Akt phosphorylation. Inhibition of ILK by T315 or siRNA-mediated knockdown suppressed epithelial

  12. Enhanced targeted anticancer effects and inhibition of tumor metastasis by the TMTP1 compound peptide TMTP1-TAT-NBD.

    Science.gov (United States)

    Liu, Ronghua; Xi, Ling; Luo, Danfeng; Ma, Xiangyi; Yang, Wanhua; Xi, Yandong; Wang, Hongyan; Qian, Ming; Fan, Liangsheng; Xia, Xi; Li, Kezheng; Wang, Daowen; Zhou, Jianfeng; Meng, Li; Wang, Shixuan; Ma, Ding

    2012-08-10

    Micromolecular agents that block tumor development and metastasis hold great promise as cancer-targeted therapies. Tumor molecular targeted peptide 1 (TMTP1) was previously shown to target primary tumors and metastatic foci specifically. In this study, a group of composite peptides were incorporated to TMPT1. The NF-κB essential modulator-binding domain (NBD), and the trans-activator of transcription (TAT) peptide, were synthesized to enhance the targeted anti-tumor effects of TMTP1. TMTP1-NBD did not exhibit strong affinity to tumor cells as we had expected. Conjugating TAT with TMTP1-NBD ameliorated the poor hydrophilicity and negative charge of TMTP1-NBD. Therefore TMTP1-TAT-NBD displayed strong affinity and anti-tumor effects as we expected in vivo and in vitro. Interestingly cytoplasmic glycogen accumulation as well as apoptosis was observed in TMTP1-TAT-NBD treated PC-3M-1E8 cells. The downstream signaling pathways including AKT, GSK-3β, IκBα and NF-κB activity were verified to decrease by TMTP1-TAT-NBD. The pharmacokinetics and distribution of TMTP1-TAT-NBD in MDA-MB-231 tumor-bearing mice model provided some evidence for safety of the composite peptide, which showed the fluorescence of the peptide peaked in the tumor 6h after injection, with little fluorescence detected in normal organs except for very weak fluorescence in kidney. In conclusion, TMTP1-TAT-NBD may be a promising targeted anti-tumor agent for primary tumor and metastatic foci, which enhances the anticancer effects through inhibiting the AKT/GSK-3β/NF-κB pathway comparing with TMTP1. PMID:22580115

  13. The inhibition of cell proliferation and induction of apoptosis in pancreatic ductal adenocarcinoma cells by verrucarin A, a macrocyclic trichothecene, is associated with the inhibition of Akt/NF-кB/mTOR prosurvival signaling.

    Science.gov (United States)

    Deeb, Dorrah; Gao, Xiaohua; Liu, Yongbo; Zhang, Yiguan; Shaw, Jiajiu; Valeriote, Frederick A; Gautam, Subhash C

    2016-09-01

    Pancreatic ductal adenocarcinoma (PDA) remains one of the most difficult to treat of all malignancies. Multimodality regimens provide only short-term symptomatic improvement with minor impact on survival, underscoring the urgent need for novel therapeutics and treatment strategies for PDA. Trichothecenes are powerful mycotoxins that inhibit protein synthesis and induce ribotoxic stress response in mammalian cells. Verrucarin A (VC-A) is a Type D macrocyclic mycotoxin which inhibited cell proliferation and induced apoptosis in breast cancer cells. However, the antitumor activity of VC-A for PDA cells has not been investigated. Here we show potent antitumor activity and the mechanism of action of VC-A in PDA cell lines. VC-A strongly inhibited the proliferation and arrested cells in the S phase of the cell cycle. The blocking of cell cycle progression by VC-A was associated with the inhibition of cell cycle regulatory proteins cyclin D1, cyclin E, cyclin-dependent kinases (cdks) cdk2, cdk4 and cdk inhibitor WAF1/21. VC-A induced apoptosis in PDA cells as indicated by the increased Annexin V FITC-binding, cleavage of poly(ADP-ribose) polymerase‑1 (PARP-1) and procaspases-3, -8 and -9. VC-A also induced mitochondrial depolarization and release of cytochrome c and it inhibited Bcl-2 family proteins that regulate apoptosis (Bcl-2, Bcl-xL, Bax and Bad). In addition, VC-A reduced the levels of inhibitors of apoptosis survivin and c-IAP-2. Finally, VC-A downregulated the expression of prosurvival phospho-Akt (p-Akt), nuclear factor κB (NF-κB) (p65) and mammalian target of rapamycin (p-mTOR) signaling proteins and their downstream mediators. Together, these results demonstrated strong antiproliferative and apoptosis-inducing activity of verrucarin A for PDA cells through cell cycle arrest and inhibition of the prosurvival (antiapoptotic) AKT/NF-κB/mTOR signaling. PMID:27573873

  14. Amyloid precursor protein cooperates with c-KIT mutation/overexpression to regulate cell apoptosis in AML1-ETO-positive leukemia via the PI3K/AKT signaling pathway.

    Science.gov (United States)

    Yu, Guopan; Yin, Changxin; Jiang, Ling; Zheng, Zhongxin; Wang, Zhixiang; Wang, Chunli; Zhou, Hongsheng; Jiang, Xuejie; Liu, Qifa; Meng, Fanyi

    2016-09-01

    It has been reported that amyloid precursor protein (APP) promotes cell proliferation and metastasis in various types of solid cancers. In our previous study, we showed that APP is highly expressed and regulates leukemia cell migration in AML1‑ETO-positive (AE) leukemia. Whether APP is involved in the regulation of AE leukemia cell proliferation or apoptosis is unclear. In the present study we focused on the correlation of APP with c-KIT mutation/overexpression and cell proliferation and apoptosis in AE leukemia. APP and c-KIT expression detected by quantitative real-time (qPCR) method, and c-KIT mutations screened using PCR in bone marrow cells from 65 patients with AE leukemia before their first chemotherapy, were simultaneously assessed. Furthermore, the Kasumi-1 cell line was chosen as the cell model, and the APP gene was knocked down using siRNA technology. The correlation of cell cycle distribution and apoptosis and c-Kit expression with APP expression levels, as well as the regulation of the PI3K/AKT signaling pathway by APP were analyzed in the Kasumi-1 cell line. The results showed that peripheral white blood cell counts (P=0.008) and bone marrow cellularity (P=0.031), but not bone marrow blasts, were correlated with APP expression. Moreover, the patients with APP high expression had a significantly higher incidence of c-KIT mutations (Ptype and negative control cells, cell apoptosis, both early (P0.05). Moreover, the knockdown of APP markedly decreased c-KIT expression at both the transcription (as evidenced by qPCR analysis) and translation (as confirmed by CD117 assay and western blot analysis) levels, as well as p-AKT and its downstream targets including NF-κB, p53 and Bcl-2. In conclusion, APP may cooperate with c-KIT mutation/overexpression in the regulation of cell apoptosis but not proliferation in AE leukemia via the PI3K/AKT signaling pathway. PMID:27460334

  15. Effects of exercise on AMPK signaling and downstream components to PI3K in rat with type 2 diabetes.

    Directory of Open Access Journals (Sweden)

    Shicheng Cao

    Full Text Available Exercise can increase skeletal muscle sensitivity to insulin, improve insulin resistance and regulate glucose homeostasis in rat models of type 2 diabetes. However, the potential mechanism remains poorly understood. In this study, we established a male Sprague-Dawley rat model of type 2 diabetes, with insulin resistance and β cell dysfunction, which was induced by a high-fat diet and low-dose streptozotocin to replicate the pathogenesis and metabolic characteristics of type 2 diabetes in humans. We also investigated the possible mechanism by which chronic and acute exercise improves metabolism, and the phosphorylation and expression of components of AMP-activated protein kinase (AMPK and downstream components of phosphatidylinositol 3-kinase (PI3K signaling pathways in the soleus. As a result, blood glucose, triglyceride, total cholesterol, and free fatty acid were significantly increased, whereas insulin level progressively declined in diabetic rats. Interestingly, chronic and acute exercise reduced blood glucose, increased phosphorylation and expression of AMPKα1/2 and the isoforms AMPKα1 and AMPKα2, and decreased phosphorylation and expression of AMPK substrate, acetyl CoA carboxylase (ACC. Chronic exercise upregulated phosphorylation and expression of AMPK upstream kinase, LKB1. But acute exercise only increased LKB1 expression. In particular, exercise reversed the changes in protein kinase C (PKCζ/λ phosphorylation, and PKCζ phosphorylation and expression. Additionally, exercise also increased protein kinase B (PKB/Akt1, Akt2 and GLUT4 expression, but AS160 protein expression was unchanged. Chronic exercise elevated Akt (Thr(308 and (Ser(473 and AS160 phosphorylation. Finally, we found that exercise increased peroxisome proliferator-activated receptor-γ coactivator 1 (PGC1 mRNA expression in the soleus of diabetic rats. These results indicate that both chronic and acute exercise influence the phosphorylation and expression of

  16. Zinc promotes proliferation and activation of myogenic cells via the PI3K/Akt and ERK signaling cascade

    Energy Technology Data Exchange (ETDEWEB)

    Ohashi, Kazuya, E-mail: asuno10k@yahoo.co.jp [Department of Life Sciences, Graduate School of Arts and Sciences, The University of Tokyo (Japan); Nagata, Yosuke, E-mail: cynagata@mail.ecc.u-tokyo.ac.jp [Department of Life Sciences, Graduate School of Arts and Sciences, The University of Tokyo (Japan); Wada, Eiji, E-mail: gacchu1@yahoo.co.jp [Department of Life Sciences, Graduate School of Arts and Sciences, The University of Tokyo (Japan); Zammit, Peter S., E-mail: peter.zammit@kcl.ac.uk [Randall Division of Cell and Molecular Biophysics, King' s College London, London SE1 1UL (United Kingdom); Shiozuka, Masataka, E-mail: cmuscle@mail.ecc.u-tokyo.ac.jp [Department of Life Sciences, Graduate School of Arts and Sciences, The University of Tokyo (Japan); Matsuda, Ryoichi, E-mail: cmatsuda@mail.ecc.u-tokyo.ac.jp [Department of Life Sciences, Graduate School of Arts and Sciences, The University of Tokyo (Japan)

    2015-05-01

    Skeletal muscle stem cells named muscle satellite cells are normally quiescent but are activated in response to various stimuli, such as injury and overload. Activated satellite cells enter the cell cycle and proliferate to produce a large number of myogenic progenitor cells, and these cells then differentiate and fuse to form myofibers. Zinc is one of the essential elements in the human body, and has multiple roles, including cell growth and DNA synthesis. However, the role of zinc in myogenic cells is not well understood, and is the focus of this study. We first examined the effects of zinc on differentiation of murine C2C12 myoblasts and found that zinc promoted proliferation, with an increased number of cells incorporating EdU, but inhibited differentiation with reduced myogenin expression and myotube formation. Furthermore, we used the C2C12 reserve cell model of myogenic quiescence to investigate the role of zinc on activation of myogenic cells. The number of reserve cells incorporating BrdU was increased by zinc in a dose dependent manner, with the number dramatically further increased using a combination of zinc and insulin. Akt and extracellular signal-regulated kinase (ERK) are downstream of insulin signaling, and both were phosphorylated after zinc treatment. The zinc/insulin combination-induced activation involved the phosphoinositide 3-kinase (PI3K)/Akt and ERK cascade. We conclude that zinc promotes activation and proliferation of myogenic cells, and this activation requires phosphorylation of PI3K/Akt and ERK as part of the signaling cascade. - Highlights: • Zinc has roles for promoting proliferation and inhibition differentiation of C2C12. • Zinc promotes activation of reserve cells. • Insulin and zinc synergize activation of reserve cells. • PI3K/Akt and ERK cascade affect zinc/insulin-mediated activation of reserve cells.

  17. Zinc promotes proliferation and activation of myogenic cells via the PI3K/Akt and ERK signaling cascade

    International Nuclear Information System (INIS)

    Skeletal muscle stem cells named muscle satellite cells are normally quiescent but are activated in response to various stimuli, such as injury and overload. Activated satellite cells enter the cell cycle and proliferate to produce a large number of myogenic progenitor cells, and these cells then differentiate and fuse to form myofibers. Zinc is one of the essential elements in the human body, and has multiple roles, including cell growth and DNA synthesis. However, the role of zinc in myogenic cells is not well understood, and is the focus of this study. We first examined the effects of zinc on differentiation of murine C2C12 myoblasts and found that zinc promoted proliferation, with an increased number of cells incorporating EdU, but inhibited differentiation with reduced myogenin expression and myotube formation. Furthermore, we used the C2C12 reserve cell model of myogenic quiescence to investigate the role of zinc on activation of myogenic cells. The number of reserve cells incorporating BrdU was increased by zinc in a dose dependent manner, with the number dramatically further increased using a combination of zinc and insulin. Akt and extracellular signal-regulated kinase (ERK) are downstream of insulin signaling, and both were phosphorylated after zinc treatment. The zinc/insulin combination-induced activation involved the phosphoinositide 3-kinase (PI3K)/Akt and ERK cascade. We conclude that zinc promotes activation and proliferation of myogenic cells, and this activation requires phosphorylation of PI3K/Akt and ERK as part of the signaling cascade. - Highlights: • Zinc has roles for promoting proliferation and inhibition differentiation of C2C12. • Zinc promotes activation of reserve cells. • Insulin and zinc synergize activation of reserve cells. • PI3K/Akt and ERK cascade affect zinc/insulin-mediated activation of reserve cells

  18. Regulatory network of inflammation downstream of proteinase-activated receptors

    Directory of Open Access Journals (Sweden)

    Hurst Robert E

    2007-03-01

    Full Text Available Abstract Background Protease-activated receptors (PAR are present in the urinary bladder, and their expression is altered in response to inflammation. PARs are a unique class of G protein-coupled that carry their own ligands, which remain cryptic until unmasked by proteolytic cleavage. Although the canonical signal transduction pathway downstream of PAR activation and coupling with various G proteins is known and leads to the rapid transcription of genes involved in inflammation, the effect of PAR activation on the downstream transcriptome is unknown. We have shown that intravesical administration of PAR-activating peptides leads to an inflammatory reaction characterized by edema and granulocyte infiltration. Moreover, the inflammatory response to intravesical instillation of known pro-inflammatory stimuli such as E. coli lipopolysaccharide (LPS, substance P (SP, and antigen was strongly attenuated by PAR1- and to a lesser extent by PAR2-deficiency. Results Here, cDNA array experiments determined inflammatory genes whose expression is dependent on PAR1 activation. For this purpose, we compared the alteration in gene expression in wild type and PAR1-/- mice induced by classical pro-inflammatory stimuli (LPS, SP, and antigen. 75 transcripts were considered to be dependent on PAR-1 activation and further annotated in silico by Ingenuity Pathways Analysis (IPA and gene ontology (GO. Selected transcripts were target validated by quantitative PCR (Q-PCR. Among PAR1-dependent transcripts, the following have been implicated in the inflammatory process: b2m, ccl7, cd200, cd63, cdbpd, cfl1, dusp1, fkbp1a, fth1, hspb1, marcksl1, mmp2, myo5a, nfkbia, pax1, plaur, ppia, ptpn1, ptprcap, s100a10, sim2, and tnfaip2. However, a balanced response to signals of injury requires a transient cellular activation of a panel of genes together with inhibitory systems that temper the overwhelming inflammation. In this context, the activation of genes such as dusp1 and

  19. EGFR inhibition evokes innate drug resistance in lung cancer cells by preventing Akt activity and thus inactivating Ets-1 function.

    Science.gov (United States)

    Phuchareon, Janyaporn; McCormick, Frank; Eisele, David W; Tetsu, Osamu

    2015-07-21

    Nonsmall cell lung cancer (NSCLC) is the leading cause of cancer death worldwide. About 14% of NSCLCs harbor mutations in epidermal growth factor receptor (EGFR). Despite remarkable progress in treatment with tyrosine kinase inhibitors (TKIs), only 5% of patients achieve tumor reduction >90%. The limited primary responses are attributed partly to drug resistance inherent in the tumor cells before therapy begins. Recent reports showed that activation of receptor tyrosine kinases (RTKs) is an important determinant of this innate drug resistance. In contrast, we demonstrate that EGFR inhibition promotes innate drug resistance despite blockade of RTK activity in NSCLC cells. EGFR TKIs decrease both the mitogen-activated protein kinase (MAPK) and Akt protein kinase pathways for a short time, after which the Ras/MAPK pathway becomes reactivated. Akt inhibition selectively blocks the transcriptional activation of Ets-1, which inhibits its target gene, dual specificity phosphatase 6 (DUSP6), a negative regulator specific for ERK1/2. As a result, ERK1/2 is activated. Furthermore, elevated c-Src stimulates Ras GTP-loading and activates Raf and MEK kinases. These observations suggest that not only ERK1/2 but also Akt activity is essential to maintain Ets-1 in an active state. Therefore, despite high levels of ERK1/2, Ets-1 target genes including DUSP6 and cyclins D1, D3, and E2 remain suppressed by Akt inhibition. Reduction of DUSP6 in combination with elevated c-Src renews activation of the Ras/MAPK pathway, which enhances cell survival by accelerating Bim protein turnover. Thus, EGFR TKIs evoke innate drug resistance by preventing Akt activity and inactivating Ets-1 function in NSCLC cells.

  20. CCL21 Facilitates Chemoresistance and Cancer Stem Cell-Like Properties of Colorectal Cancer Cells through AKT/GSK-3β/Snail Signals

    Directory of Open Access Journals (Sweden)

    Lin-Lin Lu

    2016-01-01

    Full Text Available Some evidence indicated that chemoresistance associates with the acquisition of cancer stem-like properties. Recent studies suggested that chemokines can promote the chemoresistance and stem cell properties in various cancer cells, while the underling mechanism is still not completely illustrated. In our study, we found that CCL21 can upregulate the expression of P-glycoprotein (P-gp and stem cell property markers such as Bmi-1, Nanog, and OCT-4 in colorectal cancer (CRC HCT116 cells and then improve the cell survival rate and mammosphere formation. Our results suggested that Snail was crucial for CCL21-mediated chemoresistance and cancer stem cell property in CRC cells. Further, we observed that CCL21 treatment increased the protein but not mRNA levels of Snail, which suggested that CCL21 upregulates Snail via posttranscriptional ways. The downstream signals AKT/GSK-3β mediated CCL21 induced the upregulation of Snail due to the fact that CCL21 treatment can obviously phosphorylate both AKT and GSK-3β. The inhibitor of PI3K/Akt, LY294002 significantly abolished CCL21 induced chemoresistance and mammosphere formation of HCT116 cells. Collectively, our results in the present study revealed that CCL21 can facilitate chemoresistance and stem cell property of CRC cells via the upregulation of P-gp, Bmi-1, Nanog, and OCT-4 through AKT/GSK-3β/Snail signals, which suggested a potential therapeutic approach to CRC patients.

  1. CCL21 Facilitates Chemoresistance and Cancer Stem Cell-Like Properties of Colorectal Cancer Cells through AKT/GSK-3β/Snail Signals.

    Science.gov (United States)

    Lu, Lin-Lin; Chen, Xiao-Hui; Zhang, Ge; Liu, Zong-Cai; Wu, Nong; Wang, Hao; Qi, Yi-Fei; Wang, Hong-Sheng; Cai, Shao Hui; Du, Jun

    2016-01-01

    Some evidence indicated that chemoresistance associates with the acquisition of cancer stem-like properties. Recent studies suggested that chemokines can promote the chemoresistance and stem cell properties in various cancer cells, while the underling mechanism is still not completely illustrated. In our study, we found that CCL21 can upregulate the expression of P-glycoprotein (P-gp) and stem cell property markers such as Bmi-1, Nanog, and OCT-4 in colorectal cancer (CRC) HCT116 cells and then improve the cell survival rate and mammosphere formation. Our results suggested that Snail was crucial for CCL21-mediated chemoresistance and cancer stem cell property in CRC cells. Further, we observed that CCL21 treatment increased the protein but not mRNA levels of Snail, which suggested that CCL21 upregulates Snail via posttranscriptional ways. The downstream signals AKT/GSK-3β mediated CCL21 induced the upregulation of Snail due to the fact that CCL21 treatment can obviously phosphorylate both AKT and GSK-3β. The inhibitor of PI3K/Akt, LY294002 significantly abolished CCL21 induced chemoresistance and mammosphere formation of HCT116 cells. Collectively, our results in the present study revealed that CCL21 can facilitate chemoresistance and stem cell property of CRC cells via the upregulation of P-gp, Bmi-1, Nanog, and OCT-4 through AKT/GSK-3β/Snail signals, which suggested a potential therapeutic approach to CRC patients. PMID:27057280

  2. Detection of the transforming AKT1 mutation E17K in non-small cell lung cancer by high resolution melting

    Directory of Open Access Journals (Sweden)

    Fox Stephen B

    2008-05-01

    Full Text Available Abstract Background A recurrent somatic mutation, E17K, in the pleckstrin homology domain of the AKT1 gene, has been recently described in breast, colorectal, and ovarian cancers. AKT1 is a pivotal mediator of signalling pathways involved in cell survival, proliferation and growth. The E17K mutation stimulates downstream signalling and exhibits transforming activity in vitro and in vivo. Findings We developed a sensitive high resolution melting (HRM assay to detect the E17K mutation from formalin-fixed paraffin-embedded tumours. We screened 219 non-small cell lung cancer biopsies for the mutation using HRM analysis. Four samples were identified as HRM positive. Subsequent sequencing of those samples confirmed the E17K mutation in one of the cases. A rare single nucleotide polymorphism was detected in each of the remaining three samples. The E17K was found in one of the 14 squamous cell carcinomas. No mutations were found in 141 adenocarcinomas and 39 large cell carcinomas. Conclusion The AKT1 E17K mutation is very rare in lung cancer and might be associated with tumorigenesis in squamous cell carcinoma. HRM represents a rapid cost-effective and robust screening of low frequency mutations such as AKT1 mutations in clinical samples.

  3. Neuroprotection of Insulin against Oxidative Stress-induced Apoptosis in Cultured Retinal Neurons: Involvement of Phosphoinositide 3-kinase/Akt Signal Pathway

    Institute of Scientific and Technical Information of China (English)

    Xiao-Rui YU; Guo-Rong JIA; Guang-Dao GAO; Shu-Hong WANG; Yan HAN; Wei CAO

    2006-01-01

    In order to investigate the neuroprotection of insulin in retinal neurons, we used retinal neuronal culture as a model system to study the protective effects of insulin against H2O2-induced cytotoxicity and apoptotic death. Primary retinal neuronal cultures were grown from retinas of 0-2-day old Sprague-Dawley rats. Cell viability was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide assay.Apoptotic cell death was evaluated by the TdT-mediated digoxigenin-dUTP nick-end labeling assay, and by DNA laddering analysis. Phosphoinositide 3-kinase (PI3K) activity was measured using phosphoinositide 4,5-bisphophate and [γ-32p]ATP as substrate. Western blot analysis with anti-phospho-Akt (pS473) antibody was performed to examine the level of phosphorylated Akt. We observed that treatment with 100 μM H2O2 for 24 h significantly decreased cell viability and induced apoptotic death of retinal neurons, and that pretreatment with 10 nM insulin significantly inhibited or attenuated H2O2-induced cytotoxicity and apoptosis. Pretreatment with LY294002, a specific PI3K inhibitor, abolished the cytoprotective effect of insulin. Insulin also strongly activated both PI3K and the downstream effector Akt. These results suggest that insulin protects retinal neurons from oxidative stress-induced apoptosis and that the PI3K/Akt signal pathway is involved in insulinmediated retinal neuroprotection.

  4. Targeted therapy in lymphoma

    Directory of Open Access Journals (Sweden)

    Cavalli Franco

    2010-11-01

    Full Text Available Abstract Discovery of new treatments for lymphoma that prolong survival and are less toxic than currently available agents represents an urgent unmet need. We now have a better understanding of the molecular pathogenesis of lymphoma, such as aberrant signal transduction pathways, which have led to the discovery and development of targeted therapeutics. The ubiquitin-proteasome and the Akt/mammalian target of rapamycin (mTOR pathways are examples of pathological mechanisms that are being targeted in drug development efforts. Bortezomib (a small molecule protease inhibitor and the mTOR inhibitors temsirolimus, everolimus, and ridaforolimus are some of the targeted therapies currently being studied in the treatment of aggressive, relapsed/refractory lymphoma. This review will discuss the rationale for and summarize the reported findings of initial and ongoing investigations of mTOR inhibitors and other small molecule targeted therapies in the treatment of lymphoma.

  5. Reducing the weight of cancer: mechanistic targets for breaking the obesity-carcinogenesis link.

    Science.gov (United States)

    Hursting, Stephen D; Lashinger, Laura M; Wheatley, Karrie W; Rogers, Connie J; Colbert, Lisa H; Nunez, Nomeli P; Perkins, Susan N

    2008-08-01

    The prevalence of obesity, an established epidemiologic risk factor for many cancers, has risen steadily for the past several decades in the US. The increasing rates of obesity among children are especially alarming and suggest continuing increases in the rates of obesity-related cancers for many years to come. Unfortunately, the mechanisms underlying the association between obesity and cancer are not well understood. In particular, the effects on the carcinogenesis process and mechanistic targets of interventions that modulate energy balance, such as reduced-calorie diets and physical activity, have not been well characterized. The purpose of this review is to provide a strong foundation for the translation of mechanism-based research in this area by describing key animal and human studies of energy balance modulations involving diet or physical activity and by focusing on the interrelated pathways affected by alterations in energy balance. Particular attention is placed on signaling through the insulin and insulin-like growth factor-1 receptors, including components of the Akt and mammalian target of rapamycin (mTOR) signaling pathways downstream of these growth factor receptors. These pathways have emerged as potential targets for disrupting the obesity-cancer link. The ultimate goal of this work is to provide the missing mechanistic information necessary to identify targets for the prevention and control of cancers related to or caused by excess body weight.

  6. Synthesis of new pyrrolo[1,2-a]quinoxaline derivatives as potential inhibitors of Akt kinase.

    Science.gov (United States)

    Desplat, Vanessa; Geneste, Ambre; Begorre, Marc-Antoine; Fabre, Solene Belisle; Brajot, Stephane; Massip, Stephane; Thiolat, Denis; Mossalayi, Djavad; Jarry, Christian; Guillon, Jean

    2008-10-01

    Akt kinases are attractive targets for small molecule drug discovery because of their key role in tumor cell survival/proliferation and their overexpression/activation in many human cancers. Recent efforts in the development and biological evaluation of small molecule inhibitors of Akt have led to the identification of novel Akt kinase inhibitors, based on a quinoxaline or pyrazinone scaffold. A series of new substituted pyrrolo[1,2-a]quinoxaline derivatives, structural analogues of these active quinoxaline or pyrazinone pharmacophores, was synthesized from various substituted 2-nitroanilines or 1,2-phenylenediamine via multistep heterocyclization process. These new compounds were tested for their in vitro ability to inhibit the proliferation of the human leukemic cell lines K562, U937 and HL60, and the breast cancer cell line MCF7. Three of these human cell lines (K562, U937 and MCF7) exhibited an active phosphorylated Akt form. The most promising active pyrroloquinoxalines were found to be 1a that inhibited K562 cell line proliferation with an IC(50) of 4.5 microM, and 1h that inhibited U937 and MCF7 cell lines with IC(50) of 5 and 8 microM, respectively. These two candidates exhibited more potent activities than the reference inhibitor A6730.

  7. B Cell Receptor Activation Predominantly Regulates AKT-mTORC1/2 Substrates Functionally Related to RNA Processing

    Science.gov (United States)

    Mohammad, Dara K.; Ali, Raja H.; Turunen, Janne J.; Nore, Beston F.; Smith, C. I. Edvard

    2016-01-01

    Protein kinase B (AKT) phosphorylates numerous substrates on the consensus motif RXRXXpS/T, a docking site for 14-3-3 interactions. To identify novel AKT-induced phosphorylation events following B cell receptor (BCR) activation, we performed proteomics, biochemical and bioinformatics analyses. Phosphorylated consensus motif-specific antibody enrichment, followed by tandem mass spectrometry, identified 446 proteins, containing 186 novel phosphorylation events. Moreover, we found 85 proteins with up regulated phosphorylation, while in 277 it was down regulated following stimulation. Up regulation was mainly in proteins involved in ribosomal and translational regulation, DNA binding and transcription regulation. Conversely, down regulation was preferentially in RNA binding, mRNA splicing and mRNP export proteins. Immunoblotting of two identified RNA regulatory proteins, RBM25 and MEF-2D, confirmed the proteomics data. Consistent with these findings, the AKT-inhibitor (MK-2206) dramatically reduced, while the mTORC-inhibitor PP242 totally blocked phosphorylation on the RXRXXpS/T motif. This demonstrates that this motif, previously suggested as an AKT target sequence, also is a substrate for mTORC1/2. Proteins with PDZ, PH and/or SH3 domains contained the consensus motif, whereas in those with an HMG-box, H15 domains and/or NF-X1-zinc-fingers, the motif was absent. Proteins carrying the consensus motif were found in all eukaryotic clades indicating that they regulate a phylogenetically conserved set of proteins. PMID:27487157

  8. MicroRNA-150 negatively regulates the function of CD4(+) T cells through AKT3/Bim signaling pathway.

    Science.gov (United States)

    Sang, Wei; Sun, Cai; Zhang, Cong; Zhang, Dianzheng; Wang, Ying; Xu, Linyan; Zhang, Zhe; Wei, Xiangyu; Pan, Bin; Yan, Dongmei; Zhu, Feng; Yan, Zhiling; Cao, Jiang; Loughran, Thomas P; Xu, Kailin

    2016-01-01

    Donor-derived CD4(+) T lymphocytes are the major effector cells directly involved in the development of graft-versus-host disease (GVHD). As a negative regulator of immune cell differentiation and development, microRNA-150 (miR-150) induces immunological tolerance in CD4(+) T cells after transplantation. However, the specific mechanisms have not been fully elucidated. In this study, we demonstrated that miR-150 is capable of not only inhibiting proliferation and activation of CD4(+) T cells but also promoting apoptosis. Mechanistically, miR-150 targets v-akt murine thymoma viral oncogene homolog 3 (AKT3), and subsequently downregulates B-cell lymphoma 2 (Bcl-2) interacting mediator of cell death (BIM). We have also demonstrated that re-expression of AKT3 reversed miR-150-mediated inhibition of CD4(+) T lymphocyte development. Therefore, we conclude that miR-150 negatively regulates CD4(+) T cell function by inhibiting the AKT3/BIM signaling pathway. These findings also suggest that manipulating the levels of miRNA-150 could be a valuable strategy in prevention and/or treatment of acute graft-versus-host disease. PMID:27329362

  9. RASSF4 promotes EV71 replication to accelerate the inhibition of the phosphorylation of AKT.

    Science.gov (United States)

    Zhang, Fengfeng; Liu, Yongjuan; Chen, Xiong; Dong, Lanlan; Zhou, Bingfei; Cheng, Qingqing; Han, Song; Liu, Zhongchun; Peng, Biwen; He, Xiaohua; Liu, Wanhong

    2015-03-20

    Enterovirus 71 (EV71) is a neurotropic virus that causes hand, foot and mouth disease (HFMD), occasionally leading to death. As a member of the RAS association domain family (RASSFs), RASSF4 plays important roles in cell death, tumor development and signal transduction. However, little is known about the relationship between RASSF4 and EV71. Our study reveals for the first time that RASSF4 promotes EV71 replication and then accelerates AKT phosphorylation inhibition in EV71-infected 293T cells, suggesting that RASSF4 may be a potential new target for designing therapeutic measures to prevent and control EV71 infection.

  10. Microbial production of scleroglucan and downstream processing

    Directory of Open Access Journals (Sweden)

    Natalia Alejandra Castillo

    2015-10-01

    Full Text Available Synthetic petroleum-based polymers and natural plant polymers have the disadvantage of restricted sources, in addition to the non-biodegradability of the former ones. In contrast, eco-sustainable microbial polysaccharides, of low-cost and standardized production, represent an alternative to address this situation. With a strong global market, they attracted worldwide attention because of their novel and unique physico-chemical properties as well as varied industrial applications, and many of them are promptly becoming economically competitive. Scleroglucan, a beta-1,3-beta-1,6-glucan secreted by Sclerotium fungi, exhibits high potential for commercialization and may show different branching frequency, side-chain length and/or molecular weight depending on the producing strain or culture conditions. Water-solubility, viscosifying ability and wide stability over temperature, pH and salinity make scleroglucan useful for different biotechnological (enhanced oil recovery, food additives, drug delivery, cosmetic and pharmaceutical products, biocompatible materials, etc., and biomedical (immunoceutical, antitumor, etc. applications. It can be copiously produced at bioreactor scale under standardized conditions, where a high EPS concentration normally governs the process optimization. Operative and nutritional conditions, as well as the incidence of scleroglucan downstream processing will be discussed in this chapter. The relevance of using standardized inocula from selected strains and experiences concerning the intricate scleroglucan scaling-up will be also herein outlined.

  11. Microbial production of scleroglucan and downstream processing

    Science.gov (United States)

    Castillo, Natalia A.; Valdez, Alejandra L.; Fariña, Julia I.

    2015-01-01

    Synthetic petroleum-based polymers and natural plant polymers have the disadvantage of restricted sources, in addition to the non-biodegradability of the former ones. In contrast, eco-sustainable microbial polysaccharides, of low-cost and standardized production, represent an alternative to address this situation. With a strong global market, they attracted worldwide attention because of their novel and unique physico-chemical properties as well as varied industrial applications, and many of them are promptly becoming economically competitive. Scleroglucan, a β-1,3-β-1,6-glucan secreted by Sclerotium fungi, exhibits high potential for commercialization and may show different branching frequency, side-chain length, and/or molecular weight depending on the producing strain or culture conditions. Water-solubility, viscosifying ability and wide stability over temperature, pH and salinity make scleroglucan useful for different biotechnological (enhanced oil recovery, food additives, drug delivery, cosmetic and pharmaceutical products, biocompatible materials, etc.), and biomedical (immunoceutical, antitumor, etc.) applications. It can be copiously produced at bioreactor scale under standardized conditions, where a high exopolysaccharide concentration normally governs the process optimization. Operative and nutritional conditions, as well as the incidence of scleroglucan downstream processing will be discussed in this chapter. The relevance of using standardized inocula from selected strains and experiences concerning the intricate scleroglucan scaling-up will be also herein outlined. PMID:26528259

  12. New challenges in downstream retail markets

    International Nuclear Information System (INIS)

    The role and purposes of deregulation in the natural gas industry and the way in which downstream markets operate today were reviewed. The question of what form deregulation will take in the future, and how it will affect market participants was also addressed. Load imbalance has been identified as the most significant system inefficiency. It was argued that as long as LCDs hold control over the imbalances, end-users will be in a position where they will have no control over a variable which can significantly affect their cost. It is this lack of control that can be viewed as a system inefficiency. It was suggested that in the future deregulation will focus on load balancing, and will support the continued development of a retail market for products and services which exist independently of the wholesale market. Security of supply and control of the transportation and storage assets were identified as the principal issues associated with end-user customers gaining more control over their gas accounts

  13. Akt deficiency attenuates muscle size and function but not the response to ActRIIB inhibition.

    Directory of Open Access Journals (Sweden)

    Marcus D Goncalves

    Full Text Available BACKGROUND: Akt is a critical mediator of developmental skeletal muscle growth. Treatment with a soluble ActRIIB fusion protein (ActRIIB-mFc increases skeletal muscle mass and strength by inhibiting myostatin and related peptides. Recent in vitro studies have suggested that Akt signaling is necessary for the ability of ActRIIB inhibition to induce muscle hypertrophy. Thus, we hypothesized that mice deficient in either Akt1 or Akt2 would not respond to in vivo inhibition of ActRIIB with ActRIIB-mFc treatment. METHODOLOGY AND PRINCIPAL FINDINGS: We analyzed body composition and muscle parameters in wild-type C57BL/6J and Akt1 and Akt2 knockout mice, and compared the responses to blockade of ActRIIB signaling via ActRIIB-mFc treatment. Mice lacking Akt1 or Akt2 had reduced muscle mass, grip strength and contractile force. However, deficiency of Akt1 or Akt2 did not prevent the ability of ActRIIB-mFc treatment to induce muscle hypertrophy, or increase grip strength and contractile force. Akt1 and Akt2 deficient mice responded similarly as wild type mice to ActRIIB-mFc treatment by increasing fiber size. CONCLUSIONS AND SIGNIFICANCE: Akt1 and Akt2 are important for the regulation of skeletal muscle mass and function. However, these Akt isoforms are not essential for the ability of ActRIIB inhibition to regulate muscle size, fiber type, strength or contractile force.

  14. Multiple host kinases contribute to Akt activation during Salmonella infection.

    Directory of Open Access Journals (Sweden)

    Bernhard Roppenser

    Full Text Available SopB is a type 3 secreted effector with phosphatase activity that Salmonella employs to manipulate host cellular processes, allowing the bacteria to establish their intracellular niche. One important function of SopB is activation of the pro-survival kinase Akt/protein kinase B in the infected host cell. Here, we examine the mechanism of Akt activation by SopB during Salmonella infection. We show that SopB-mediated Akt activation is only partially sensitive to PI3-kinase inhibitors LY294002 and wortmannin in HeLa cells, suggesting that Class I PI3-kinases play only a minor role in this process. However, depletion of PI(3,4 P2/PI(3-5 P3 by expression of the phosphoinositide 3-phosphatase PTEN inhibits Akt activation during Salmonella invasion. Therefore, production of PI(3,4 P2/PI(3-5 P3 appears to be a necessary event for Akt activation by SopB and suggests that non-canonical kinases mediate production of these phosphoinositides during Salmonella infection. We report that Class II PI3-kinase beta isoform, IPMK and other kinases identified from a kinase screen all contribute to Akt activation during Salmonella infection. In addition, the kinases required for SopB-mediated activation of Akt vary depending on the type of infected host cell. Together, our data suggest that Salmonella has evolved to use a single effector, SopB, to manipulate a remarkably large repertoire of host kinases to activate Akt for the purpose of optimizing bacterial replication in its host.

  15. Dependence of mountain moisture retention on downstream saturation

    CERN Document Server

    November, L J

    2007-01-01

    We derive 1D steady equilibria for partially saturated downslope flow as a solution to the reposed vadose-zone problem. The equilibria are determined by two free parameters: a mean flow and a downstream saturation. Where there is no downstream saturation, a uniform outflow of constant saturation is obtained, but if an isolated zone of excess downstream saturation exists, the upstream flow speed is reduced and saturation increased. The saturated zone produces a capillary head jump that blocks the downward percolation. With a fully saturated downstream zone in medium clay mixtures, flow backs up more than 20 km projected vertically, but with 90% downstream saturation, the back up is reduced to less than a km. The drying of downstream saturated zones with the increased rate of mountain moisture outflow gives a viable quantitative explanation for the mysterious 56% unaccounted drop seen in the output of the southern NM La Luz / Fresnal Watershed at Alamogordo's upstream spring-box diversions.

  16. Coprinus comatus cap inhibits adipocyte differentiation via regulation of PPARγ and Akt signaling pathway.

    Directory of Open Access Journals (Sweden)

    Hyoung Joon Park

    Full Text Available This study assessed the effects of Coprinus comatus cap (CCC on adipogenesis in 3T3-L1 adipocytes and the effects of CCC on the development of diet-induced obesity in rats. Here, we showed that the CCC has an inhibitory effect on the adipocyte differentiation of 3T3-L1 cells, resulting in a significant decrease in lipid accumulation through the downregulation of several adipocyte specific-transcription factors, including CCAAT/enhancer binding protein β, C/EBPδ, and peroxisome proliferator-activated receptor gamma (PPARγ. Moreover, treatment with CCC during adipocyte differentiation induced a significant down-regulation of PPARγ and adipogenic target genes, including adipocyte protein 2, lipoprotein lipase, and adiponectin. Interestingly, the CCC treatment of the 3T3-L1 adipocytes suppressed the insulin-stimulated Akt and GSK3β phosphorylation, and these effects were stronger in the presence of an inhibitor of Akt phosphorylation, LY294002, suggesting that CCC inhibited adipocyte differentiation through the down-regulation of Akt signaling. In the animal study, CCC administration significantly reduced the body weight and adipose tissue weight of rats fed a high fat diet (HFD and attenuated lipid accumulation in the adipose tissues of the HFD-induced obese rats. The size of the adipocyte in the epididymal fat of the CCC fed rats was significantly smaller than in the HFD rats. CCC treatment significantly reduced the total cholesterol and triglyceride levels in the serum of HFD rats. These results strongly indicated that the CCC-mediated decrease in body weight was due to a reduction in adipose tissue mass. The expression level of PPARγ and phospho-Akt was significantly lower in the CCC-treated HFD rats than that in the HFD obesity rats. These results suggested that CCC inhibited adipocyte differentiation by the down-regulation of major transcription factor involved in the adipogenesis pathway including PPARγ through the regulation of the

  17. Cutaneous Human Papillomaviruses Down-regulate AKT1, whereas AKT2 Up-regulation and Activation Associates with Tumors

    OpenAIRE

    O'Shaughnessy, Ryan F L; Akgũl, Baki; Storey, Alan; Pfister, Herbert; Harwood, Catherine A; Byrne, Carolyn

    2007-01-01

    Epithelial tumorigenesis has been linked to AKT up-regulation. Human papillomaviruses (HPV) cause anogenital cancers and anogenital HPV infection up-regulates AKT activity. Mounting evidence points to a role for cutaneous HPVs as etiologic factors in skin tumorigenesis. High-risk cutaneous β HPVs have been linked to carcinogenesis in immunosuppressed patients, and high-risk cutaneous HPV8 genes enhance tumorigenesis in transgenic mice. We find that, in contrast to anogenital HPVs, cutaneous H...

  18. PAC exhibits potent anti-colon cancer properties through targeting cyclin D1 and suppressing epithelial-to-mesenchymal transition.

    Science.gov (United States)

    Al-Qasem, Abeer; Al-Howail, Huda A; Al-Swailem, Mashael; Al-Mazrou, Amer; Al-Otaibi, Basem; Al-Jammaz, Ibrahim; Al-Khalaf, Huda H; Aboussekhra, Abdelilah

    2016-03-01

    Colorectal cancer (CRC) is a major cause of cancer morbidity and mortality worldwide. Although response rates and overall survival have been improved in recent years, resistance to multiple drug combinations is inevitable. Therefore, the development of more efficient drugs, with fewer side effects is urgently needed. To this end, we have investigated in the present report the effect of PAC, a novel cucumin analogue, on CRC cells both in vitro and in vivo. We have shown that PAC induces apoptosis, mainly via the internal mitochondrial route, and inhibits cell proliferation through delaying the cell cycle at G2/M phase. Interestingly, the pro-apoptotic effect was mediated through STAT3-dependent down-regulation of cyclin D1 and its downstream target survivin. Indeed, change in the expression level of cyclin D1 modulated the expression of survivin and the response of CRC cells to PAC. Furthermore, using the ChIP assay, we have shown PAC-dependent reduction in the binding of STAT3 to the cyclin D1 promoter in vivo. Additionally, PAC suppressed the epithelial-to-mesenchymal process through down-regulating the mesenchymal markers (N-cadherin, vimentin and Twist1) and inhibiting the invasion/migration abilities of the CRC cells via repressing the pro-migration/invasion protein kinases AKT and ERK1/2. In addition, PAC inhibited tumor growth and repressed the JAK2/STAT3, AKT/mTOR and MEK/ERK pathways as well as their common downstream effectors cyclin D1 and survivin in humanized CRC xenografts. Collectively, these results indicate that PAC has potent anti-CRC effects, and therefore could constitute an effective alternative chemotherapeutic agent, which may consolidate the adjuvant treatment of colon cancer.

  19. Genomically amplified Akt3 activates DNA repair pathway and promotes glioma progression.

    Science.gov (United States)

    Turner, Kristen M; Sun, Youting; Ji, Ping; Granberg, Kirsi J; Bernard, Brady; Hu, Limei; Cogdell, David E; Zhou, Xinhui; Yli-Harja, Olli; Nykter, Matti; Shmulevich, Ilya; Yung, W K Alfred; Fuller, Gregory N; Zhang, Wei

    2015-03-17

    Akt is a robust oncogene that plays key roles in the development and progression of many cancers, including glioma. We evaluated the differential propensities of the Akt isoforms toward progression in the well-characterized RCAS/Ntv-a mouse model of PDGFB-driven low grade glioma. A constitutively active myristoylated form of Akt1 did not induce high-grade glioma (HGG). In stark contrast, Akt2 and Akt3 showed strong progression potential with 78% and 97% of tumors diagnosed as HGG, respectively. We further revealed that significant variations in polarity and hydropathy values among the Akt isoforms in both the pleckstrin homology domain (P domain) and regulatory domain (R domain) were critical in mediating glioma progression. Gene expression profiles from representative Akt-derived tumors indicated dominant and distinct roles for Akt3, consisting primarily of DNA repair pathways. TCGA data from human GBM closely reflected the DNA repair function, as Akt3 was significantly correlated with a 76-gene signature DNA repair panel. Consistently, compared with Akt1 and Akt2 overexpression models, Akt3-expressing human GBM cells had enhanced activation of DNA repair proteins, leading to increased DNA repair and subsequent resistance to radiation and temozolomide. Given the wide range of Akt3-amplified cancers, Akt3 may represent a key resistance factor.

  20. Cadmium Activates Reactive Oxygen Species-dependent AKT/mTOR and Mitochondrial Apoptotic Pathways in Neuronal Cells

    Institute of Scientific and Technical Information of China (English)

    YUAN Yan; BIAN Jian Chun; LIU Zong Ping; WANG Yi; HU Fei Fei; JIANG Chen Yang; ZHANG Ya Jing; YANG Jin Long; ZHAO Shi Wen; GU Jian Hong; LIU Xue Zhong

    2016-01-01

    ObjectiveTo examine the role of Cd-induced reactive oxygen species (ROS) generation in the apoptosis of neuronal cells. MethodsNeuronal cells (primary rat cerebral cortical neurons and PC12cells) were incubated with or without Cd post-pretreatment with rapamycin (Rap) or N-acetyl-L-cysteine (NAC). Cell viability was determined by MTT assay, apoptosis was examined using flow cytometry and fluorescence microscopy, and the activation of phosphoinositide 3'-kinase/protein kinase B (Akt)/mammalian target of rapamycin (mTOR) and mitochondrial apoptotic pathways were measured by western blotting or immunofluorescence assays. ResultsCd-induced activation of Akt/mTOR signaling, including Akt, mTOR,p70 S6 kinase (p70 S6K), and eukaryotic initiation factor 4E binding protein 1(4E-BP1). Rap, an mTOR inhibitor and NAC, a ROS scavenger, blocked Cd-induced activation of Akt/mTOR signaling and apoptosis of neuronal cells. Furthermore, NAC blocked the decrease of B-cell lymphoma 2/Bcl-2 associated X protein (Bcl-2/Bax) ratio, release of cytochrome c, cleavage of caspase-3 and poly(ADP-ribose) polymerase (PARP), and nuclear translocation of apoptosis-inducing factor(AIF)and endonuclease G (Endo G). ConclusionCd-induced ROS generation activates Akt/mTOR and mitochondrial pathways, leading to apoptosis ofneuronal cells. Our findings suggest that mTOR inhibitors or antioxidants have potential for preventing Cd-induced neurodegenerative diseases.

  1. Pyrvinium attenuates Hedgehog signaling downstream of Smoothened

    OpenAIRE

    Li, Bin; Fei, Dennis Liang; Flaveny, Colin A.; Dahmane, Nadia; Baubet, Valérie; Wang, Zhiqiang; Bai, Feng; Pei, Xin-Hai; Rodriguez-Blanco, Jezabel; Hang, Brian; Orton, Darren; Han, Lu; Wang, Baolin; Capobianco, Anthony J.; Lee, Ethan

    2014-01-01

    The Hedgehog (HH) signaling pathway represents an important class of emerging developmental signaling pathways that play critical roles in the genesis of a large number of human cancers. The pharmaceutical industry is currently focused on developing small molecules targeting Smoothened (Smo), a key signaling effector of the HH pathway that regulates the levels and activity of the Gli family of transcription factors. Although one of these compounds vismodegib is now FDA-approved for advanced b...

  2. Hydroeconomic optimization of reservoir management under downstream water quality constraints

    Science.gov (United States)

    Davidsen, Claus; Liu, Suxia; Mo, Xingguo; Holm, Peter E.; Trapp, Stefan; Rosbjerg, Dan; Bauer-Gottwein, Peter

    2015-10-01

    A hydroeconomic optimization approach is used to guide water management in a Chinese river basin with the objectives of meeting water quantity and water quality constraints, in line with the China 2011 No. 1 Policy Document and 2015 Ten-point Water Plan. The proposed modeling framework couples water quantity and water quality management and minimizes the total costs over a planning period assuming stochastic future runoff. The outcome includes cost-optimal reservoir releases, groundwater pumping, water allocation, wastewater treatments and water curtailments. The optimization model uses a variant of stochastic dynamic programming known as the water value method. Nonlinearity arising from the water quality constraints is handled with an effective hybrid method combining genetic algorithms and linear programming. Untreated pollutant loads are represented by biochemical oxygen demand (BOD), and the resulting minimum dissolved oxygen (DO) concentration is computed with the Streeter-Phelps equation and constrained to match Chinese water quality targets. The baseline water scarcity and operational costs are estimated to 15.6 billion CNY/year. Compliance to water quality grade III causes a relatively low increase to 16.4 billion CNY/year. Dilution plays an important role and increases the share of surface water allocations to users situated furthest downstream in the system. The modeling framework generates decision rules that result in the economically efficient strategy for complying with both water quantity and water quality constraints.

  3. DOWNSTREAM ECOCIDE FROM UPSTREAM WATER PIRACY

    Directory of Open Access Journals (Sweden)

    Miah Muhammad Adel

    2012-01-01

    Full Text Available Upstream India and downstream Bangladesh share more than 50 international rivers. India has set up water diversion constructions in more than 50% of these rivers, the largest one being on the Bangladesh’s northwest upon the Ganges River, puts Bangladesh’s Gangetic ecosystem at stake. In some border rivers, India has set up groins on her side of river banks. Also, Indian side pumps Bangladesh river water stealthily from border-rivers. Further, India is constructing another dam and reservoir upstream on the Barak River on the northeast of Bangladesh. Furthermore, India has chalked out a grand plan for river networking. Exploration has been made to assess the degree of the ecosystem degradation both inland and on the coast due to all water diversion constructions around the border, except for the Tipaimukh Dam in which case estimation of projected ecosystem degradation has been mentioned. Finally, Indian grand plan of river networking plan has been briefly touched upon. Site visitations, observations, surveys, measurements and interviews of professionals were made in the project country. Relevant literatures on this issue were reviewed in electronic and print databases. Related published articles in electronic and print media were systematically searched following the key words for the case. Finally, both electronic and print news media have been closely followed to know the latest developments on this issue. The reduced flow of the Ganges in Bangladesh has caused scarcity of fresh water, species endangerment and extinction, obstruction to livestock raising, loss of livelihoods, people’s displacement, changes in crop production, reduction in navigable routes, extreme weather, increased flood occurrences, scarcity of potable water, groundwater contamination, reduction in coastal sediment deposition, deterioration of the Ganges water quality and inland intrusion of saline water front. Water diversion constructions in other rivers have

  4. PI3K-AKT Signaling via Nrf2 Protects against Hyperoxia-Induced Acute Lung Injury, but Promotes Inflammation Post-Injury Independent of Nrf2 in Mice.

    Directory of Open Access Journals (Sweden)

    Narsa M Reddy

    Full Text Available Lung epithelial and endothelial cell death accompanied by inflammation contributes to hyperoxia-induced acute lung injury (ALI. Impaired resolution of ALI can promote and/or perpetuate lung pathogenesis, including fibrosis. Previously, we have shown that the transcription factor Nrf2 induces cytoprotective gene expression and confers protection against hyperoxic lung injury, and that Nrf2-mediated signaling is also crucial for the restoration of lung homeostasis post-injury. Although we have reported that PI3K/AKT signaling is required for Nrf2 activation in lung epithelial cells, significance of the PI3K/AKT-Nrf2 crosstalk during hyperoxic lung injury and repair remains unclear. Thus, we evaluated this aspect using Nrf2 knockout (Nrf2(-/- and wild-type (Nrf2(+/+ mouse models. Here, we show that pharmacologic inhibition of PI3K/AKT signaling increased lung inflammation and alveolar permeability in Nrf2(+/+ mice, accompanied by decreased expression of Nrf2-target genes such as Nqo1 and Hmox1. PI3K/AKT inhibition dampened hyperoxia-stimulated Nqo1 and Hmox1 expression in lung epithelial cells and alveolar macrophages. Contrasting with its protective effects, PI3K/AKT inhibition suppressed lung inflammation in Nrf2(+/+ mice during post-injury. In Nrf2(-/- mice exposed to room-air, PI3K/AKT inhibition caused lung injury and inflammation, but it did not exaggerate hyperoxia-induced ALI. During post-injury, PI3K/AKT inhibition did not augment, but rather attenuated, lung inflammation in Nrf2(-/- mice. These results suggest that PI3K/AKT-Nrf2 signaling is required to dampen hyperoxia-induced lung injury and inflammation. Paradoxically, the PI3K/AKT pathway promotes lung inflammation, independent of Nrf2, during post-injury.

  5. Targeted Inhibition of Multiple Receptor Tyrosine Kinases in Mesothelioma

    Directory of Open Access Journals (Sweden)

    Wen-Bin Ou

    2011-01-01

    Full Text Available The receptor tyrosine kinases (RTKs epidermal growth factor receptor (EGFR and MET are activated in subsets of mesothelioma, suggesting that these kinases might represent novel therapeutic targets in this notoriously chemotherapy-resistant cancer. However, clinical trials have shown little activity for EGFR inhibitors in mesothelioma. Despite the evidence for RTK activation in mesothelioma pathogenesis, it is unclear whether transforming activity is dependent on an individual kinase oncoprotein or the coordinated activity of multiple kinases. Using phospho-RTK and immunoblot assays, we herein demonstrate activation of multiple RTKs (EGFR, MET, AXL, and ERBB3 in individual mesothelioma cell lines but not in normal mesothelioma cells. Inhibition of mesothelioma multi-RTK signaling was accomplished using combinations of RTK direct inhibitors or by inhibition of the RTK chaperone, heat shock protein 90 (HSP90. Multi-RTK inhibition by the HSP90 inhibitor 17-allyloamino-17demethoxygeldanamycin (17-AAG had a substantially greater effect on mesothelioma proliferation and survival compared with inhibition of individual activated RTKs. HSP90 inhibition also suppressed phosphorylation of down-stream signaling intermediates (AKT, mitogen-activated protein kinase, and S6; upregulated the p53, p21, and p27 cell cycle checkpoints; induced G2 phase arrest; induced caspase 3/7 activity; and led to an increase in the sub-G1 apoptotic population. These compelling proapoptotic and antiproliferative responses indicate that HSP90 inhibition warrants clinical evaluation as a novel therapeutic strategy in mesothelioma.

  6. Isorhamnetin inhibits cell proliferation and induces apoptosis in breast cancer via Akt and mitogen‑activated protein kinase kinase signaling pathways.

    Science.gov (United States)

    Hu, Shan; Huang, Liming; Meng, Liwei; Sun, He; Zhang, Wei; Xu, Yingchun

    2015-11-01

    Breast cancer is the most common cause of female cancer-associated mortality. Although treatment options, including chemotherapy, radiotherapy and surgery have led to a decline in the mortality rates associated with breast cancer, drug resistance remains one of the predominant causes for poor prognosis and high recurrence rates. The present study investigated the potential effects of the natural product, isorhamnetin on breast cancer, and examined the effects of isorhamnetin on the Akt/mammalian target of rapamycin (mTOR) and the mitogen-activated protein kinase (MAPK)/MAPK kinase (MEK) signaling cascades, which are two important signaling pathways for endocrine therapy resistance in breast cancer. The results of the present study indicate that isorhamnetin inhibits cell proliferation and induces cell apoptosis. In addition, isorhamnetin was observed to inhibit the Akt/mTOR and the MEK/extracellular signal-regulated kinase phosphorylation cascades. The inhibition of these two signaling pathways was attenuated by the two Akt and MEK1 inhibitors, but not by the nuclear factor-κB inhibitor. Furthermore, epidermal growth factor inhibited the effects of isorhamnetin via activation of the Akt and MEK signaling pathways. These results indicate that isorhamnetin exhibits antitumor effects in breast cancer, which are mediated by the Akt and MEK signaling pathways. PMID:26502751

  7. Downstream Evolution of Longitudinal Embedded Vortices with Helical Structure

    DEFF Research Database (Denmark)

    Velte, Clara Marika; Okulov, Valery; Hansen, Martin Otto Laver

    2009-01-01

    In the present work the downstream development of device induced vortices with helical symmetry embedded in wall bounded flow on a bump is studied with the aid of Stereoscopic Particle Image Velocimetry (SPIV). The downstream evolution of characteristic parameters of helical vortices is studied...

  8. Metformin inhibits age-related centrosome amplification in Drosophila midgut stem cells through AKT/TOR pathway.

    Science.gov (United States)

    Na, Hyun-Jin; Park, Joung-Sun; Pyo, Jung-Hoon; Jeon, Ho-Jun; Kim, Young-Shin; Arking, Robert; Yoo, Mi-Ae

    2015-07-01

    We delineated the mechanism regulating the inhibition of centrosome amplification by metformin in Drosophila intestinal stem cells (ISCs). Age-related changes in tissue-resident stem cells may be closely associated with tissue aging and age-related diseases, such as cancer. Centrosome amplification is a hallmark of cancers. Our recent work showed that Drosophila ISCs are an excellent model for stem cell studies evaluating age-related increase in centrosome amplification. Here, we showed that metformin, a recognized anti-cancer drug, inhibits age- and oxidative stress-induced centrosome amplification in ISCs. Furthermore, we revealed that this effect is mediated via down-regulation of AKT/target of rapamycin (TOR) activity, suggesting that metformin prevents centrosome amplification by inhibiting the TOR signaling pathway. Additionally, AKT/TOR signaling hyperactivation and metformin treatment indicated a strong correlation between DNA damage accumulation and centrosome amplification in ISCs, suggesting that DNA damage might mediate centrosome amplification. Our study reveals the beneficial and protective effects of metformin on centrosome amplification via AKT/TOR signaling modulation. We identified a new target for the inhibition of age- and oxidative stress-induced centrosome amplification. We propose that the Drosophila ISCs may be an excellent model system for in vivo studies evaluating the effects of anti-cancer drugs on tissue-resident stem cell aging.

  9. A novel piggyBac transposon inducible expression system identifies a role for AKT signalling in primordial germ cell migration.

    Directory of Open Access Journals (Sweden)

    James D Glover

    Full Text Available In this work, we describe a single piggyBac transposon system containing both a tet-activator and a doxycycline-inducible expression cassette. We demonstrate that a gene product can be conditionally expressed from the integrated transposon and a second gene can be simultaneously targeted by a short hairpin RNA contained within the transposon, both in vivo and in mammalian and avian cell lines. We applied this system to stably modify chicken primordial germ cell (PGC lines in vitro and induce a reporter gene at specific developmental stages after injection of the transposon-modified germ cells into chicken embryos. We used this vector to express a constitutively-active AKT molecule during PGC migration to the forming gonad. We found that PGC migration was retarded and cells could not colonise the forming gonad. Correct levels of AKT activation are thus essential for germ cell migration during early embryonic development.

  10. Galectin-1 induces hepatocellular carcinoma EMT and sorafenib resistance by activating FAK/PI3K/AKT signaling.

    Science.gov (United States)

    Zhang, P-F; Li, K-S; Shen, Y-H; Gao, P-T; Dong, Z-R; Cai, J-B; Zhang, C; Huang, X-Y; Tian, M-X; Hu, Z-Q; Gao, D-M; Fan, J; Ke, A-W; Shi, G-M

    2016-01-01

    Galectin-1 (Gal-1) is involved in several pathological activities associated with tumor progression and chemoresistance, however, the role and molecular mechanism of Gal-1 activity in hepatocellular carcinoma (HCC) epithelial-mesenchymal transition (EMT) and sorafenib resistance remain enigmatic. In the present study, forced Gal-1 expression promoted HCC progression and sorafenib resistance. Gal-1 elevated αvβ3-integrin expression, leading to AKT activation. Moreover, Gal-1 overexpression induced HCC cell EMT via PI3K/AKT cascade activation. Clinically, our data revealed that Gal-1 overexpression is correlated with poor HCC survival outcomes and sorafenib response. These data suggest that Gal-1 may be a potential therapeutic target for HCC and a biomarker for predicting response to sorafenib treatment. PMID:27100895

  11. DNA–PKcs–SIN1 complexation mediates low-dose X-ray irradiation (LDI)-induced Akt activation and osteoblast differentiation

    Energy Technology Data Exchange (ETDEWEB)

    Xu, Yong; Fang, Shi-ji [The Department of Orthopedics, The Second Affiliated Hospital of Soochow University, Suzhou 215000 (China); Zhu, Li-juan [Jiangsu Key Laboratory of Translational Research and Therapy for Neuro-Psycho-Diseases and Institute of Neuroscience, Soochow University, Suzhou, Jiangsu 215021 (China); Zhu, Lun-qing, E-mail: xiaodongwangsz@163.com [The Center of Diagnosis and Treatment for Children’s Bone Diseases, The Children’s Hospital Affiliated to Soochow University, Suzhou, Jiangsu 215000 (China); Zhou, Xiao-zhong, E-mail: zhouxz@suda.edu.cn [The Department of Orthopedics, The Second Affiliated Hospital of Soochow University, Suzhou 215000 (China)

    2014-10-24

    Highlights: • LDI increases ALP activity, promotes type I collagen (Col I)/Runx2 mRNA expression. • LDI induces DNA–PKcs activation, which is required for osteoblast differentiation. • Akt activation mediates LDI-induced ALP activity and Col I/Runx2 mRNA increase. • DNA–PKcs–SIN1 complexation mediates LDI-induced Akt Ser-473 phosphorylation. • DNA–PKcs–SIN1 complexation is important for osteoblast differentiation. - Abstract: Low-dose irradiation (LDI) induces osteoblast differentiation, however the underlying mechanisms are not fully understood. In this study, we explored the potential role of DNA-dependent protein kinase catalytic subunit (DNA–PKcs)–Akt signaling in LDI-induced osteoblast differentiation. We confirmed that LDI promoted mouse calvarial osteoblast differentiation, which was detected by increased alkaline phosphatase (ALP) activity as well as mRNA expression of type I collagen (Col I) and runt-related transcription factor 2 (Runx2). In mouse osteoblasts, LDI (1 Gy) induced phosphorylation of DNA–PKcs and Akt (mainly at Ser-473). The kinase inhibitors against DNA–PKcs (NU-7026 and NU-7441) or Akt (LY294002, perifosine and MK-2206), as well as partial depletion of DNA–PKcs or Akt1 by targeted-shRNA, dramatically inhibited LDI-induced Akt activation and mouse osteoblast differentiation. Further, siRNA-knockdown of SIN1, a key component of mTOR complex 2 (mTORC2), also inhibited LDI-induced Akt Ser-473 phosphorylation as well as ALP activity increase and Col I/Runx2 expression in mouse osteoblasts. Co-immunoprecipitation (Co-IP) assay results demonstrated that LDI-induced DNA–PKcs–SIN1 complexation, which was inhibited by NU-7441 or SIN1 siRNA-knockdown in mouse osteoblasts. In summary, our data suggest that DNA–PKcs–SIN1 complexation-mediated Akt activation (Ser-473 phosphorylation) is required for mouse osteoblast differentiation.

  12. MET and AKT genetic influence on facial emotion perception.

    Directory of Open Access Journals (Sweden)

    Ming-Teng Lin

    Full Text Available BACKGROUND: Facial emotion perception is a major social skill, but its molecular signal pathway remains unclear. The MET/AKT cascade affects neurodevelopment in general populations and face recognition in patients with autism. This study explores the possible role of MET/AKT cascade in facial emotion perception. METHODS: One hundred and eighty two unrelated healthy volunteers (82 men and 100 women were recruited. Four single nucleotide polymorphisms (SNP of MET (rs2237717, rs41735, rs42336, and rs1858830 and AKT rs1130233 were genotyped and tested for their effects on facial emotion perception. Facial emotion perception was assessed by the face task of Mayer-Salovey-Caruso Emotional Intelligence Test (MSCEIT. Thorough neurocognitive functions were also assessed. RESULTS: Regarding MET rs2237717, individuals with the CT genotype performed better in facial emotion perception than those with TT (p = 0.016 by ANOVA, 0.018 by general linear regression model [GLM] to control for age, gender, and education duration, and showed no difference with those with CC. Carriers with the most common MET CGA haplotype (frequency = 50.5% performed better than non-carriers of CGA in facial emotion perception (p = 0.018, df = 1, F = 5.69, p = 0.009 by GLM. In MET rs2237717/AKT rs1130233 interaction, the C carrier/G carrier group showed better facial emotion perception than those with the TT/AA genotype (p = 0.035 by ANOVA, 0.015 by GLM, even when neurocognitive functions were controlled (p = 0.046 by GLM. CONCLUSIONS: To our knowledge, this is the first study to suggest that genetic factors can affect performance of facial emotion perception. The findings indicate that MET variances and MET/AKT interaction may affect facial emotion perception, implicating that the MET/AKT cascade plays a significant role in facial emotion perception. Further replication studies are needed.

  13. Dioscin inhibits colon tumor growth and tumor angiogenesis through regulating VEGFR2 and AKT/MAPK signaling pathways

    Energy Technology Data Exchange (ETDEWEB)

    Tong, Qingyi [Regenerative Medicine Research Center, State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, Sichuan University, Chengdu, Sichuan 610041 (China); Qing, Yong, E-mail: qingyongxy@yahoo.co.jp [Department of Pharmacology, West China School of Pharmacy, Sichuan University, Chengdu, Sichuan 610041 (China); Wu, Yang [State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, Sichuan University, Chengdu, Sichuan 610041 (China); Hu, Xiaojuan; Jiang, Lei [Department of Pharmacology, West China School of Pharmacy, Sichuan University, Chengdu, Sichuan 610041 (China); Wu, Xiaohua, E-mail: wuxh@scu.edu.cn [Regenerative Medicine Research Center, State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, Sichuan University, Chengdu, Sichuan 610041 (China)

    2014-12-01

    Dioscin has shown cytotoxicity against cancer cells, but its in vivo effects and the mechanisms have not elucidated yet. The purpose of the current study was to assess the antitumor effects and the molecular mechanisms of dioscin. We showed that dioscin could inhibit tumor growth in vivo and has no toxicity at the test condition. The growth suppression was accompanied by obvious blood vessel decrease within solid tumors. We also found dioscin treatment inhibited the proliferation of cancer and endothelial cell lines, and most sensitive to primary cultured human umbilical vein endothelial cells (HUVECs). What's more, analysis of HUVECs migration, invasion, and tube formation exhibited that dioscin has significantly inhibitive effects to these actions. Further analysis of blood vessel formation in the matrigel plugs indicated that dioscin could inhibit VEGF-induced blood vessel formation in vivo. We also identified that dioscin could suppress the downstream protein kinases of VEGFR2, including Src, FAK, AKT and Erk1/2, accompanied by the increase of phosphorylated P38MAPK. The results potently suggest that dioscin may be a potential anticancer drug, which efficiently inhibits angiogenesis induced by VEGFR2 signaling pathway as well as AKT/MAPK pathways. - Highlights: • Dioscin inhibits tumor growth in vivo and does not exhibit any toxicity. • Dioscin inhibits angiogenesis within solid tumors. • Dioscin inhibits the proliferation, migration, invasion, and tube formation of HUVECs. • Dioscin inhibits VEGF–induced blood vessel formation in vivo. • Dioscin inhibits VEGFR2 signaling pathway as well as AKT/MAPK pathway.

  14. SMAD-PI3K-Akt-mTOR pathway mediates BMP-7 polarization of monocytes into M2 macrophages.

    Directory of Open Access Journals (Sweden)

    Crystal Rocher

    Full Text Available Previously we demonstrated that bone morphogenetic protein-7 (BMP-7 treatment polarizes monocytes into M2 macrophages and increases the expression of anti-inflammatory cytokines. Despite these findings, the mechanisms for the observed BMP-7 induced monocyte polarization into M2 macrophages are completely unknown. In this study, we demonstrate the mechanisms involved in the polarization of monocytes into M2 macrophages. Apoptotic conditioned media (ACM was generated to mimic the stressed conditions, inducing monocyte polarization. Monocytes were treated with ACM along with BMP-7 and/or its inhibitor, follistatin, for 48 hours. Furthermore, an inhibitor of the PI3K pathway, LY-294002, was also studied. Our data show that BMP-7 induces polarization of monocytes into M2 macrophages while significantly increasing the expression of anti-inflammatory markers, arginase-1 and IL-10, and significantly (p<0.05 decreasing the expression of pro-inflammatory markers iNOS, IL-6, TNF-α and MCP-1; (p<0.05. Moreover, addition of the PI3K inhibitor, LY-294002, significantly (p<0.05 decreases upregulation of IL-10 and arginase-1, suggesting involvement of the PI3K pathway in M2 macrophage polarization. Next, following BMP-7 treatment, a significant (p<0.05 increase in p-SMAD1/5/8 and p-PI3K expression resulting in downstream activation of p-Akt and p-mTOR was observed. Furthermore, expression of p-PTEN, an inhibitor of the PI3K pathway, was significantly (p<0.05 increased in the ACM group. However, BMP-7 treatment inhibited its expression, suggesting involvement of the PI3K-Akt-mTOR pathway. In conclusion, we demonstrate that BMP-7 polarizes monocytes into M2 macrophages and enhances anti-inflammatory cytokine expression which is mediated by the activated SMAD-PI3K-Akt-mTOR pathway.

  15. Role of a novel PH-kinase domain interface in PKB/Akt regulation: structural mechanism for allosteric inhibition.

    Directory of Open Access Journals (Sweden)

    Véronique Calleja

    2009-01-01

    Full Text Available Protein kinase B (PKB/Akt belongs to the AGC superfamily of related serine/threonine protein kinases. It is a key regulator downstream of various growth factors and hormones and is involved in malignant transformation and chemo-resistance. Full-length PKB protein has not been crystallised, thus studying the molecular mechanisms that are involved in its regulation in relation to its structure have not been simple. Recently, the dynamics between the inactive and active conformer at the molecular level have been described. The maintenance of PKB's inactive state via the interaction of the PH and kinase domains prevents its activation loop to be phosphorylated by its upstream activator, phosphoinositide-dependent protein kinase-1 (PDK1. By using a multidisciplinary approach including molecular modelling, classical biochemical assays, and Förster resonance energy transfer (FRET/two-photon fluorescence lifetime imaging microscopy (FLIM, a detailed model depicting the interaction between the different domains of PKB in its inactive conformation was demonstrated. These findings in turn clarified the molecular mechanism of PKB inhibition by AKT inhibitor VIII (a specific allosteric inhibitor and illustrated at the molecular level its selectivity towards different PKB isoforms. Furthermore, these findings allude to the possible function of the C-terminus in sustaining the inactive conformer of PKB. This study presents essential insights into the quaternary structure of PKB in its inactive conformation. An understanding of PKB structure in relation to its function is critical for elucidating its mode of activation and discovering how to modulate its activity. The molecular mechanism of inhibition of PKB activation by the specific drug AKT inhibitor VIII has critical implications for determining the mechanism of inhibition of other allosteric inhibitors and for opening up opportunities for the design of new generations of modulator drugs.

  16. Terpinolene, a component of herbal sage, downregulates AKT1 expression in K562 cells

    OpenAIRE

    Okumura, Naoko; Yoshida, Hitomi; Nishimura, Yuri; Kitagishi, Yasuko; Matsuda, Satoru

    2011-01-01

    Protein kinase AKT mediates cell proliferation and survival signals, and also contributes to cancer progression. Increased expression and/or activation of AKT is involved in a variety of human cancers. In cells treated with sage or rosemary extract, mRNA and protein expression levels of AKT1 were reduced compared with those of the control cells 48 h after the herbal treatments. We found that terpinolene, a common component of sage and rosemary, markedly reduced the protein expression of AKT1 ...

  17. Akt activation induces hypertrophy without contractile phenotypic maturation in airway smooth muscle

    OpenAIRE

    Ma, Lan; Brown, Melanie; Kogut, Paul; Serban, Karina; Li, Xiaojing; McConville, John; Chen, Bohao; Bentley, J. Kelley; Hershenson, Marc B.; Dulin, Nickolai; Solway, Julian; Camoretti-Mercado, Blanca

    2011-01-01

    Airway smooth muscle (ASM) hypertrophy is a cardinal feature of severe asthma, but the underlying molecular mechanisms remain uncertain. Forced protein kinase B/Akt 1 activation is known to induce myocyte hypertrophy in other muscle types, and, since a number of mediators present in asthmatic airways can activate Akt signaling, we hypothesized that Akt activation could contribute to ASM hypertrophy in asthma. To test this hypothesis, we evaluated whether Akt activation occurs naturally within...

  18. Impact of mTORC1 inhibition on keratinocyte proliferation during skin tumor promotion in wild-type and BK5.AktWT mice.

    Science.gov (United States)

    Rho, Okkyung; Kiguchi, Kaoru; Jiang, Guiyu; DiGiovanni, John

    2014-11-01

    In this study, we examined the impact of rapamycin on mTORC1 signaling during 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced keratinocyte proliferation and skin tumor promotion in both wild-type (FVB/N) and BK5.Akt(WT) mice. TPA activated mTORC1 signaling in a time-dependent manner in cultured primary mouse keratinocytes and a mouse keratinocyte cell line. Early activation (15-30 min) of mTORC1 signaling induced by TPA was mediated in part by PKC activation, whereas later activation (2-4 h) was mediated by activation of EGFR and Akt. BK5.Akt(WT) transgenic mice, where Akt1 is overexpressed in basal epidermis, are highly sensitive to TPA-induced epidermal proliferation and two-stage skin carcinogenesis. Targeting mTORC1 with rapamycin effectively inhibited TPA-induced epidermal hyperplasia and hyperproliferation as well as tumor promotion in a dose-dependent manner in both wild-type and BK5.Akt(WT) mice. A significant expansion (∼threefold) of the label retaining cell (LRC) population per hair follicle was observed in BK5.Akt(WT) mice compared to FVB/N mice. There was also a significant increase in K15 expressing cells in the hair follicle of transgenic mice that coincided with expression of phospho-Akt, phospho-S6K, and phospho-PRAS40, suggesting an important role of mTORC1 signaling in bulge-region keratinocyte stem cell (KSC) homeostasis. After 2 weeks of TPA treatment, LRCs had moved upward into the interfollicular epidermis from the bulge region of both wild-type and BK5.Akt(WT) mice. TPA-mediated LRC proliferation and migration was significantly inhibited by rapamycin. Collectively, the current data indicate that signaling through mTORC1 contributes significantly to the process of skin tumor promotion through effects on proliferation of the target cells for tumor development.

  19. Impact of mTORC1 inhibition on keratinocyte proliferation during skin tumor promotion in wild-type and BK5.AktWT mice.

    Science.gov (United States)

    Rho, Okkyung; Kiguchi, Kaoru; Jiang, Guiyu; DiGiovanni, John

    2014-11-01

    In this study, we examined the impact of rapamycin on mTORC1 signaling during 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced keratinocyte proliferation and skin tumor promotion in both wild-type (FVB/N) and BK5.Akt(WT) mice. TPA activated mTORC1 signaling in a time-dependent manner in cultured primary mouse keratinocytes and a mouse keratinocyte cell line. Early activation (15-30 min) of mTORC1 signaling induced by TPA was mediated in part by PKC activation, whereas later activation (2-4 h) was mediated by activation of EGFR and Akt. BK5.Akt(WT) transgenic mice, where Akt1 is overexpressed in basal epidermis, are highly sensitive to TPA-induced epidermal proliferation and two-stage skin carcinogenesis. Targeting mTORC1 with rapamycin effectively inhibited TPA-induced epidermal hyperplasia and hyperproliferation as well as tumor promotion in a dose-dependent manner in both wild-type and BK5.Akt(WT) mice. A significant expansion (∼threefold) of the label retaining cell (LRC) population per hair follicle was observed in BK5.Akt(WT) mice compared to FVB/N mice. There was also a significant increase in K15 expressing cells in the hair follicle of transgenic mice that coincided with expression of phospho-Akt, phospho-S6K, and phospho-PRAS40, suggesting an important role of mTORC1 signaling in bulge-region keratinocyte stem cell (KSC) homeostasis. After 2 weeks of TPA treatment, LRCs had moved upward into the interfollicular epidermis from the bulge region of both wild-type and BK5.Akt(WT) mice. TPA-mediated LRC proliferation and migration was significantly inhibited by rapamycin. Collectively, the current data indicate that signaling through mTORC1 contributes significantly to the process of skin tumor promotion through effects on proliferation of the target cells for tumor development. PMID:24114993

  20. Arctigenin Increases Hemeoxygenase-1 Gene Expression by Modulating PI3K/AKT Signaling Pathway in Rat Primary Astrocytes.

    Science.gov (United States)

    Jeong, Yeon-Hui; Park, Jin-Sun; Kim, Dong-Hyun; Kim, Hee-Sun

    2014-11-01

    In the present study, we found that the natural compound arctigenin inhibited hydrogen peroxide-induced reactive oxygen species (ROS) production in rat primary astrocytes. Since hemeoxygenase-1 (HO-1) plays a critical role as an antioxidant defense factor in the brain, we examined the effect of arctigenin on HO-1 expression in rat primary astrocytes. We found that arctigenin increased HO-1 mRNA and protein levels. Arctigenin also increases the nuclear translocation and DNA binding of Nrf2/c-Jun to the antioxidant response element (ARE) on HO-1 promoter. In addition, arctigenin increased ARE-mediated transcriptional activities in rat primary astrocytes. Further mechanistic studies revealed that arctigenin increased the phosphorylation of AKT, a downstream substrate of phosphatidylinositol 3-kinase (PI3K). Treatment of cells with a PI3K-specific inhibitor, LY294002, suppressed the HO-1 expression, Nrf2 DNA binding and ARE-mediated transcriptional activities in arctigenin-treated astrocyte cells. The results collectively suggest that PI3K/AKT signaling pathway is at least partly involved in HO-1 expression by arctigenin via modulation of Nrf2/ARE axis in rat primary astrocytes. PMID:25489416

  1. Both mitogen-activated protein kinase (MAPK)/extracellular-signal-regulated kinases (ERK) 1/2 and phosphatidylinositide-3-OH kinase (PI3K)/Akt pathways regulate activation of E-twenty-six (ETS)-like transcription factor 1 (Elk-1) in U138 glioblastoma cells.

    Science.gov (United States)

    Mut, Melike; Lule, Sevda; Demir, Ozlem; Kurnaz, Isil Aksan; Vural, Imran

    2012-02-01

    Epidermal growth factor (EGF) and its receptor (EGFR) have been shown to play a significant role in the pathogenesis of glioblastoma. In our study, the EGFR was stimulated with EGF in human U138 glioblastoma cells. We show that the activated mitogen-activated protein kinase (MAPK)/extracellular-signal-regulated kinases (ERK) 1/2 pathway phosphorylated the E twenty-six (ETS)-like transcription factor 1 (Elk-1) mainly at serine 383 residue. Mitogen-activated protein kinase kinase (MEK) 1/2 inhibitor, UO126 and ERK inhibitor II, FR180204 blocked the Elk-1 phosphorylation and activation. The phosphatidylinositide-3-OH kinase (PI3K)/Akt pathway was also involved in the Elk-1 activation. Activation of the Elk-1 led to an increased survival and a proliferative response with the EGF stimulation in the U138 glioblastoma cells. Knocking-down the Elk-1 using an RNA interference technique caused a decrease in survival of the unstimulated U138 glioblastoma cells and also decreased the proliferative response to the EGF stimulation. The Elk-1 transcription factor was important for the survival and proliferation of U138 glioblastoma cells upon the stimulation of EGFR with EGF. The MAPK/ERK1/2 and PI3K/Akt pathways regulated this response via activation of the Elk-1 transcription factor. The Elk-1 may be one of the convergence points for pathways located downstream of EGFR in glioblastoma cells. Utilization of the Elk-1 as a therapeutic target may lead to a novel strategy in treatment of glioblastoma.

  2. Nanobiopolymer for direct targeting and inhibition of EGFR expression in triple negative breast cancer.

    Directory of Open Access Journals (Sweden)

    Satoshi Inoue

    Full Text Available Treatment options for triple negative breast cancer (TNBC are generally limited to cytotoxic chemotherapy. Recently, anti-epidermal growth factor receptor (EGFR therapy has been introduced for TNBC patients. We engineered a novel nanobioconjugate based on a poly(β-L-malic acid (PMLA nanoplatform for TNBC treatment. The nanobioconjugate carries anti-tumor nucleosome-specific monoclonal antibody (mAb 2C5 to target breast cancer cells, anti-mouse transferrin receptor (TfR antibody for drug delivery through the host endothelial system, and Morpholino antisense oligonucleotide (AON to inhibit EGFR synthesis. The nanobioconjugates variants were: (1 P (BioPolymer with AON, 2C5 and anti-TfR for tumor endothelial and cancer cell targeting, and EGFR suppression (P/AON/2C5/TfR, and (2 P with AON and 2C5 (P/AON/2C5. Controls included (3 P with 2C5 but without AON (P/2C5, (4 PBS, and (5 P with PEG and leucine ester (LOEt for endosomal escape (P/mPEG/LOEt. Drugs were injected intravenously to MDA-MB-468 TNBC bearing mice. Tissue accumulation of injected nanobioconjugates labeled with Alexa Fluor 680 was examined by Xenogen IVIS 200 (live imaging and confocal microscopy of tissue sections. Levels of EGFR, phosphorylated and total Akt in tumor samples were detected by western blotting. In vitro western blot showed that the leading nanobioconjugate P/AON/2C5/TfR inhibited EGFR synthesis significantly better than naked AON. In vivo imaging revealed that 2C5 increased drug-tumor accumulation. Significant tumor growth inhibition was observed in mice treated with the lead nanobioconjugate (1 [P = 0.03 vs. controls; P<0.05 vs. nanobioconjugate variant (2]. Lead nanobioconjugate (1 also showed stronger inhibition of EGFR expression and Akt phosphorylation than other treatments. Treatment of TNBC with the new nanobioconjugate results in tumor growth arrest by inhibiting EGFR and its downstream signaling intermediate, phosphorylated Akt. The nanobioconjugate

  3. Intracellular reactive oxygen species are essential for PI3K/Akt/mTOR-dependent IL-7-mediated viability of T-cell acute lymphoblastic leukemia cells.

    Science.gov (United States)

    Silva, A; Gírio, A; Cebola, I; Santos, C I; Antunes, F; Barata, J T

    2011-06-01

    Interleukin-7 (IL-7) activates phosphoinositide 3-kinase/Akt/mammalian target of rapamycin (PI3K/Akt/mTOR) pathway, thereby mediating viability, proliferation and growth of T-cell acute lymphoblastic leukemia (T-ALL) cells. Reactive oxygen species (ROS) can be upregulated by growth factors and are known to regulate proliferation and viability. Here, we show that IL-7 upregulates ROS in T-ALL cells in a manner that is dependent on PI3K/Akt/mTOR pathway activity and that relies on both NADPH oxidase and mitochondrial respiratory chain. Conversely, IL-7-induced activation of PI3K signaling pathway requires mitochondrial respiration and ROS. We have previously shown that IL-7-mediated activation of PI3K pathway drives the upregulation of the glucose transporter Glut1, promoting glucose uptake in T-ALL cells. Using phloretin to inhibit Glut function, we demonstrate that glucose uptake is mandatory for ROS upregulation in IL-7-treated T-ALL cells, suggesting that IL-7 stimulation leads to increased ROS via PI3K pathway activation and consequent upregulation of Glut1 and glucose uptake. Overall, our data reveal the existence of a critical crosstalk between PI3K/Akt signaling pathway and ROS that is essential for IL-7-mediated T-ALL cell survival, and that may constitute a novel target for therapeutic intervention. PMID:21455214

  4. Extraction and downstream processing of plant-derived recombinant proteins.

    Science.gov (United States)

    Buyel, J F; Twyman, R M; Fischer, R

    2015-11-01

    Plants offer the tantalizing prospect of low-cost automated manufacturing processes for biopharmaceutical proteins, but several challenges must be addressed before such goals are realized and the most significant hurdles are found during downstream processing (DSP). In contrast to the standardized microbial and mammalian cell platforms embraced by the biopharmaceutical industry, there are many different plant-based expression systems vying for attention, and those with the greatest potential to provide inexpensive biopharmaceuticals are also the ones with the most significant drawbacks in terms of DSP. This is because the most scalable plant systems are based on the expression of intracellular proteins in whole plants. The plant tissue must therefore be disrupted to extract the product, challenging the initial DSP steps with an unusually high load of both particulate and soluble contaminants. DSP platform technologies can accelerate and simplify process development, including centrifugation, filtration, flocculation, and integrated methods that combine solid-liquid separation, purification and concentration, such as aqueous two-phase separation systems. Protein tags can also facilitate these DSP steps, but they are difficult to transfer to a commercial environment and more generic, flexible and scalable strategies to separate target and host cell proteins are preferable, such as membrane technologies and heat/pH precipitation. In this context, clarified plant extracts behave similarly to the feed stream from microbes or mammalian cells and the corresponding purification methods can be applied, as long as they are adapted for plant-specific soluble contaminants such as the superabundant protein RuBisCO. Plant-derived pharmaceutical proteins cannot yet compete directly with established platforms but they are beginning to penetrate niche markets that allow the beneficial properties of plants to be exploited, such as the ability to produce 'biobetters' with tailored

  5. Chronic modulation of AMP-Kinase, Akt and mTOR pathways by ionizing radiation in human lung cancer xenografts

    International Nuclear Information System (INIS)

    Earlier, we showed that in cancer cells, AMP-activated kinase (AMPK) participates in a signal transduction pathway involving ATM-AMPK-p53/p21cip1 which is activated by ionizing radiation (IR) to mediate G2-M arrest and enhanced cytotoxicity. We also observed that AMPK modulates ATM expression and activity and the IR response of the Akt-mTOR pathway. Since the ATM, AMPK and Akt pathways are key targets of novel radio-sensitizing therapeutics, we examined the chronic modultion of expression and activity of those pathways by IR alone in xenograft models of lung cancer. Immuno-compromised mice were grafted with human lung A549 and H1299 cells, were treated with a single fraction of 0 or 10 Gy, and left to grow for 8 weeks. Extracted tumors were subjected to lysis and immunoblotting or fixation and immunohistochemical analysis. IR inhibited significantly xenograft growth and was associated with increased expression of Ataxia Telengiectasia Mutated (ATM) and enhanced phosphorylation of two ATM targets, H2Ax and checkpoint kinase Chk2. Irradiated tumours showed increased total AMPK levels and phosphorylation of AMPK and its substrate Acetyl-CoA Carboxylase (ACC). IR led to enhanced expression and phosphorylation of p53 and cyclin dependent kinase inhibitors p21cip1 and p27kip1. However, irradiated tumours had reduced phosphorylation of Akt, mTOR and it‘s target translation initiation inhibitor 4EBP1. Irradiated xenografts showed reduced microvessel density, reduced expression of CD31 but increased expression of hypoxia-induced factor 1A (HIF1a) compared to controls. IR inhibits epithelial cancer tumour growth and results in sustained expression and activation of ATM-Chk2, and AMPK-p53/p21cip1/p27kip1 but partial inhibition of the Akt-mTOR signaling pathways. Future studies should examine causality between those events and explore whether further modulation of the AMPK and Akt-mTOR pathways by novel therapeutics can sensitize lung tumours to radiation

  6. MATHEMATICAL MODELLING OF DEGRADATION AND FLUVIAL PROCESS DOWNSTREAM RESERVOIRS

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    To research into the problem of degradation and fluvial process downstream reservoirs and its influence on flood control and navigation, a 1-D mathematical model of degradation and fluvial process downstream the reservoir was established in this paper. The non-equilibrium transport of non-uniform suspended load, the non-uniform bedload transport and bed material sorting were considered in the model. Some techniques were suggested for some problems in calculation, such as the effective suspended load carrying capacity of the different reaches of bed materials, the coefficient of suspended load carrying capacity, the recovering coefficient of carrying capacity, the mixed layer thickness, the bedload transport width, bifurcation and confluence of main and branch channel, and the distribution of deposition and erosion along the cross section, etc. The model was tested by the data of degradation downstream the Danjiangkou reservoir on the Hanjiang River and the data of degradation downstream the Gezhouba Project on the Yangtze River.

  7. Corrosion rate and downstream effect in liquid metal systems

    International Nuclear Information System (INIS)

    Results of experiments performed, especially of runs 1.10 and 1.12 have been evaluated with respect to the downstream effect in isothermal test sections in sodium at 1000 K. It is evident that there is not dependence of the selective Ni loss on position. This loss will be reduced considerably with high Ni concentration in sodium. The downstream function is influenced by different causes for short and extended exposure periods. Up to 400h Cr, together with impurities in sodium dominates the downstream effect. After longer exposure periods the steel surface will become equilibrated with respect to sodium conditions and the downstream effect will be dominated by elemental dissolution, essentially of Fe and saturation of dissolved elements in sodium. (author)

  8. Fucoidan/FGF-2 induces angiogenesis through JNK- and p38-mediated activation of AKT/MMP-2 signalling

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Beom Su [Wonkwang Bone Regeneration Research Institute, Wonkwang University, Iksan, Jeonbuk 570-749 (Korea, Republic of); Bonecell Biotech Inc., 77, Dunsan-dong, Seo-gu, Daejeon 302-830 (Korea, Republic of); Park, Ji-Yun [Bonecell Biotech Inc., 77, Dunsan-dong, Seo-gu, Daejeon 302-830 (Korea, Republic of); Kang, Hyo-Jin [Wonkwang Bone Regeneration Research Institute, Wonkwang University, Iksan, Jeonbuk 570-749 (Korea, Republic of); Kim, Hyung-Jin [Department of Microbiology, School of Medicine, Wonkwang University, Iksan, Jeonbuk 570-749 (Korea, Republic of); Lee, Jun, E-mail: omslee@wku.ac.kr [Wonkwang Bone Regeneration Research Institute, Wonkwang University, Iksan, Jeonbuk 570-749 (Korea, Republic of); Bonecell Biotech Inc., 77, Dunsan-dong, Seo-gu, Daejeon 302-830 (Korea, Republic of)

    2014-08-08

    Graphical abstract: Schematic diagram of the angiogenic activity mechanism by FGF-2/fucoidan treatment in HUVECs. Fucoidan enhances the FGF-2-induced phosphorylation of p38, JNK, and ERK MAPKs. However, p38 and JNK were involved in AKT phosphorylation and MMP-2 activation and resulted in enhanced angiogenic activity, such as tube formation and migration, in HUVECs. - Highlights: • The angiogenic activity of fucoidan in HUVECs was explored. • Fucoidan enhanced HUVEC proliferation, migration, and tube formation. • Fucoidan enhanced angiogenesis through p38 and JNK but not ERK in HUVECs. • Fucoidan targeted angiogenesis-mediated AKT/MMP-2 signalling in HUVECs. - Abstract: Angiogenesis is an important biological process in tissue development and repair. Fucoidan has previously been shown to potentiate in vitro tube formation in the presence of basic fibroblast growth factor (FGF-2). However, the underlying molecular mechanism remains largely unknown. This study was designed to investigate the action of fucoidan in angiogenesis in human umbilical vein endothelial cells (HUVECs) and to explore fucoidan-signalling pathways. First, we evaluated the effect of fucoidan on cell proliferation. Matrigel-based tube formation and wound healing assays were performed to investigate angiogenesis. Matrix metalloproteinase-2 (MMP-2) mRNA expression and activity levels were analysed by reverse transcription polymerase chain reaction (RT-PCR) and zymography, respectively. Additionally, phosphorylation of mitogen-activated protein kinases (MAPKs) and protein kinase B (AKT) was detected by Western blot. The results indicate that fucoidan treatment significantly increased cell proliferation in the presence of FGF-2. Moreover, compared to the effect of FGF-2 alone, fucoidan and FGF-2 had a greater effect on tube formation and cell migration, and this effect was found to be synergistic. Furthermore, fucoidan enhanced the phosphorylation of extracellular signal-regulated kinase (ERK

  9. MiR-20a Induces Cell Radioresistance by Activating the PTEN/PI3K/Akt Signaling Pathway in Hepatocellular Carcinoma

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Yuqin [Department of Radiation Oncology, Nanfang Hospital, Southern Medical University, Guangzhou, Guangdong Province (China); Zheng, Lin [Department of Pathology, School of Basic Medical Sciences, Southern Medical University, Guangzhou, Guangdong Province (China); Department of Pathology, Nanfang Hospital, Southern Medical University, Guangzhou, Guangdong Province (China); Ding, Yi [Department of Radiation Oncology, Nanfang Hospital, Southern Medical University, Guangzhou, Guangdong Province (China); Li, Qi [Department of Gastroenterology, Nanfang Hospital, Southern Medical University, Guangzhou, Guangdong Province (China); Wang, Rong [Department of Radiation Oncology, Nanfang Hospital, Southern Medical University, Guangzhou, Guangdong Province (China); Liu, Tongxin; Sun, Quanquan [Department of Radiation Oncology, Cancer Hospital, Hangzhou, Zhejiang Province (China); Yang, Hua [Department of Radiation Oncology, Nanhai Hospital, Southern Medical University, Guangzhou, Guangdong Province (China); Peng, Shunli [Department of Radiation Oncology, Nanfang Hospital, Southern Medical University, Guangzhou, Guangdong Province (China); Wang, Wei, E-mail: wangwei9500@hotmail.com [Department of Radiation Oncology, Nanfang Hospital, Southern Medical University, Guangzhou, Guangdong Province (China); Chen, Longhua, E-mail: chenlhsmu@126.com [Department of Radiation Oncology, Nanfang Hospital, Southern Medical University, Guangzhou, Guangdong Province (China)

    2015-08-01

    Purpose: To investigate the role of miR-20a in hepatocellular carcinoma (HCC) cell radioresistance, which may reveal potential strategies to improve treatment. Methods and Materials: The expression of miR-20a and PTEN were detected in HCC cell lines and paired primary tissues by quantitative real-time polymerase chain reaction. Cell radiation combined with colony formation assays was administrated to discover the effect of miR-20a on radiosensitivity. Bioinformatics prediction and luciferase assay were used to identify the target of miR-20a. The phosphatidylinositol 3-kinase inhibitor LY294002 was used to inhibit phosphorylation of Akt, to verify whether miR-20a affects HCC cell radioresistance through activating the PTEN/PI3K/Akt pathway. Results: MiR-20a levels were increased in HCC cell lines and tissues, whereas PTEN was inversely correlated with it. Overexpression of miR-20a in Bel-7402 and SMMC-7721 cells enhances their resistance to the effect of ionizing radiation, and the inhibition of miR-20a in HCCLM3 and QGY-7701 cells sensitizes them to it. PTEN was identified as a direct functional target of miR-20a for the induction of radioresistance. Overexpression of miR-20a activated the PTEN/PI3K/Akt signaling pathway. Additionally, the kinase inhibitor LY294002 could reverse the effect of miR-20a–induced radioresistance. Conclusion: MiR-20a induces HCC cell radioresistance by activating the PTEN/PI3K/Akt pathway, which suggests that miR-20a/PTEN/PI3K/Akt might represent a target of investigation for developing effective therapeutic strategies against HCC.

  10. PI3K/Akt/mTOR inhibitors in breast cancer

    Institute of Scientific and Technical Information of China (English)

    Joycelyn JX Lee; Kiley Loh; Yoon-Sim Yap

    2015-01-01

    Activation of the phosphoinositide 3 kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) pathway is common in breast cancer. hTere is preclinical data to support inhibition of the pathway, and phase I to III trials involving inhibitors of the pathway have been or are being conducted in solid tumors and breast cancer. Everolimus, an mTOR inhibitor, is currently approved for the treatment of hormone receptor (HR)-positive, human epidermal growth factor receptor 2 (HER2)-negative breast cancer. In this review, we summarise the efficacy and toxicity findings from the randomised clinical trials, with simpliifed guidelines on the management of potential adverse effects. Education of healthcare professionals and patients is critical for safety and compliance. While there is some clinical evidence of activity of mTOR inhibition in HR-positive and HER2-positive breast cancers, the benefits may be more pronounced in selected subsets rather than in the overall population. Further development of predictive biomarkers will be useful in the selection of patients who will beneift from inhibition of the PI3K/Akt/mTOR (PAM) pathway.

  11. Insulin Attenuates Beta-Amyloid-Associated Insulin/Akt/EAAT Signaling Perturbations in Human Astrocytes.

    Science.gov (United States)

    Han, Xiaojuan; Yang, Liling; Du, Heng; Sun, Qinjian; Wang, Xiang; Cong, Lin; Liu, Xiaohui; Yin, Ling; Li, Shan; Du, Yifeng

    2016-08-01

    The excitatory amino acid transporters 1 and 2 (EAAT1 and EAAT2), mostly located on astrocytes, are the main mediators for glutamate clearance in humans. Malfunctions of these transporters may lead to excessive glutamate accumulation and subsequent excitotoxicity to neurons, which has been implicated in many kinds of neurodegenerative disorders including Alzheimer's disease (AD). Yet, the specific mechanism of the glutamate system dysregulation remains vague. To explore whether the insulin/protein kinase B (Akt)/EAAT signaling in human astrocytes could be disturbed by beta-amyloid protein (Aβ) and be protected by insulin, we incubated HA-1800 cells with varying concentrations of Aβ1-42 oligomers and insulin. Then the alterations of several key substrates in this signal transduction pathway were determined. Our results showed that expressions of insulin receptor, phospho-insulin receptor, phospho-protein kinase B, phospho-mammalian target of rapamycin, and EAAT1 and EAAT2 were decreased by the Aβ1-42 oligomers in a dose-dependent manner (p  0.05), and the mRNA levels of EAAT1 and EAAT2 were also unchanged (p > 0.05). Taken together, this study indicates that Aβ1-42 oligomers could cause disturbances in insulin/Akt/EAAT signaling in astrocytes, which might be responsible for AD onset and progression. Additionally, insulin can exert protective functions to the brain by modulating protein modifications or expressions. PMID:26358886

  12. Vasopressin activates Akt/mTOR pathway in smooth muscle cells cultured in high glucose concentration

    Energy Technology Data Exchange (ETDEWEB)

    Montes, Daniela K.; Brenet, Marianne; Muñoz, Vanessa C.; Burgos, Patricia V.; Villanueva, Carolina I. [Department of Physiology, Universidad Austral de Chile, Valdivia 509-9200 (Chile); Figueroa, Carlos D. [Department of Anatomy, Histology and Pathology, Universidad Austral de Chile, Valdivia 509-9200 (Chile); González, Carlos B., E-mail: cbgonzal@uach.cl [Department of Physiology, Universidad Austral de Chile, Valdivia 509-9200 (Chile); Department of Neuroscience and Cell Biology, University of Texas Medical Branch, Galveston, TX 77555 (United States)

    2013-11-29

    Highlights: •AVP induces mTOR phosphorylation in A-10 cells cultured in high glucose concentration. •The mTOR phosphorylation is mediated by the PI3K/Akt pathway activation. •The AVP-induced mTOR phosphorylation inhibited autophagy and stimulated cell proliferation. -- Abstract: Mammalian target of rapamycin (mTOR) complex is a key regulator of autophagy, cell growth and proliferation. Here, we studied the effects of arginine vasopressin (AVP) on mTOR activation in vascular smooth muscle cells cultured in high glucose concentration. AVP induced the mTOR phosphorylation in A-10 cells grown in high glucose, in contrast to cells cultured in normal glucose; wherein, only basal phosphorylation was observed. The AVP-induced mTOR phosphorylation was inhibited by a PI3K inhibitor. Moreover, the AVP-induced mTOR activation inhibited autophagy and increased thymidine incorporation in cells grown in high glucose. This increase was abolished by rapamycin which inhibits the mTORC1 complex formation. Our results suggest that AVP stimulates mTOR phosphorylation by activating the PI3K/Akt signaling pathway and, subsequently, inhibits autophagy and raises cell proliferation in A-10 cells maintained in high glucose concentration.

  13. Downstream influence scaling of turbulent flow past expansion corners

    Science.gov (United States)

    Lu, Frank K.; Chung, Kung-Ming

    1992-01-01

    Previous studies of the high-speed viscous inviscid interaction between a turbulent boundary layer and an expansion at a convex corner have noted that surface pressure decreases toward the downstream inviscid value yielded by a Prandtl-Meyer expansion. A downstream influence on the corner is presently identified which is based on the mean surface pressure distribution; a scaling law is proposed for this distance.

  14. Modelling Downstream Effects in the Presence of Technological Change

    OpenAIRE

    Duncan Mortimer

    2008-01-01

    Downstream effects are typically evaluated given current technology and current practice patterns rather than for technology and practice patterns that will be available at the time when downstream effects accrue. Where a relatively short time horizon can be expected to capture all relevant costs and effects, the current approach is unlikely to introduce substantial error into estimates of the costs and benefits attributed to an intervention; the estimates will remain valid so long as the con...

  15. MATHEMATICAL MODEL OF RIVER BED CHANGE DOWNSTREAM OF XIAOLANGDI RESERVOIR

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    A mathematical model of river bed change downstream of the Xiaolangdi Reservoir was developed based on the most recent achievement of sediment theory in the Yellow River. The model was verified by the comparison of computed results and measured data from 1986 to 1996. Numerical prediction of the erosion and deposition downstream of the Xiaolangdi Reservoir in its first operation year was carried out, and a series of suggestions were given for reservoir operation mode in its early operation period.

  16. Downstream processing of Isochrysis galbana: a step towards microalgal biorefinery

    OpenAIRE

    Gilbert-López, Bienvenida; Mendiola, José A.; Fontecha, Javier; SIJTSMA Lolke; Cifuentes, Alejandro; Herrero, Miguel; Ibáñez, Elena; van den Broek, Lambertus A. M.

    2015-01-01

    An algae-based biorefinery relies on the efficient use of algae biomass through its fractionation of several valuable/bioactive compounds that can be used in industry. If this biorefinery includes green platforms as downstream processing technologies able to fulfill the requirements of green chemistry, it will end-up with sustainable processes. In the present study, a downstream processing platform has been developed to extract bioactive compounds from the microalga Isochrysis galbana using v...

  17. HER3/ErbB3, an emerging cancer therapeutic target.

    Science.gov (United States)

    Zhang, Ningyan; Chang, Yujun; Rios, Adan; An, Zhiqiang

    2016-01-01

    HER3 is a member of the HER (EGFR/ErbB) receptor family consisting of four closely related type 1 transmembrane receptors (EGFR, HER2, HER3, and HER4). HER receptors are part of a complex signaling network intertwined with the Ras/Raf/MAPK, PI3K/AKT, JAK/STAT, and PKC signaling pathways. Aberrant activation of the HER receptors and downstream signaling molecules tips the balance on cellular events, leading to various types of cancers. Monoclonal antibodies (mAbs) and small molecule inhibitors targeting EGFR and HER2 tyrosine kinase activities exhibit clinical benefits in the treatment of several types of cancers, but their clinical efficacy is limited by the occurrence of drug resistance. HER3 is the preferred dimerization partner of HER2 and it is well established that HER3 plays an important role in drug resistance to EGFR- and HER2-targeting therapies. Since HER3 has limited kinase activity, mAbs are being explored to target HER3 for cancer therapy. Currently, approximately a dozen of anti-HER3 mAbs are at different stages of clinical development. However, the lack of established biomarkers has made it more challenging to stratify cancer patients to whom HER3-targeting therapies can be more effective. In this review, we focus on the validation of HER3 as a cancer drug target, the recent development in biomarker discovery for anti-HER3 therapies, and the progress made in the clinical development of HER3-targeting mAbs. PMID:26496898

  18. Modeling Neutral Densities Downstream of a Gridded Ion Thruster

    Science.gov (United States)

    Soulas, George C.

    2010-01-01

    The details of a model for determining the neutral density downstream of a gridded ion thruster are presented. An investigation of the possible sources of neutrals emanating from and surrounding a NEXT ion thruster determined that the most significant contributors to the downstream neutral density include discharge chamber neutrals escaping through the perforated grids, neutrals escaping from the neutralizer, and vacuum facility background neutrals. For the neutral flux through the grids, near- and far-field equations are presented for rigorously determining the neutral density downstream of a cylindrical aperture. These equations are integrated into a spherically-domed convex grid geometry with a hexagonal array of apertures for determining neutral densities downstream of the ion thruster grids. The neutrals escaping from an off-center neutralizer are also modeled assuming diffuse neutral emission from the neutralizer keeper orifice. Finally, the effect of the surrounding vacuum facility neutrals is included and assumed to be constant. The model is used to predict the neutral density downstream of a NEXT ion thruster with and without neutralizer flow and a vacuum facility background pressure. The impacts of past simplifying assumptions for predicting downstream neutral densities are also examined for a NEXT ion thruster.

  19. Downstream-based Scheduling for Energy Conservation in Green EPONs

    KAUST Repository

    Chen, Shen

    2012-05-01

    Maximizing the optical network unit’s (ONU) sleep time is an effective approach for achieving maximum energy conservation in green Ethernet passive optical networks (EPONs). While overlapping downstream and upstream ONU transmissions can maximize the ONU sleep time, it jeopardizes the quality of service (QoS) performance of the network, especially for downstream traffic in case the overlapping is based on the upstream time slot. In this paper, we study the downstream traffic performance in green EPONs under the limited service discipline and the upstream-based overlapped time window. Specifically, we first derive the expected mean packet delay, and then present a closed-form expression of the ONU sleep time, setting identical upstream/downstream transmission cycle times based on a maximum downstream traffic delay re-quirement. With the proposed system model, we present a novel downstream bandwidth allocation scheme for energy conservation in green EPONs. Simulation results verify the proposed model and highlight the advantages of our scheme over conventional approaches.

  20. Piperlongumine, an alkaloid causes inhibition of PI3 K/Akt/mTOR signaling axis to induce caspase-dependent apoptosis in human triple-negative breast cancer cells.

    Science.gov (United States)

    Shrivastava, Shweta; Kulkarni, Prasad; Thummuri, Dinesh; Jeengar, Manish Kumar; Naidu, V G M; Alvala, Mallika; Redddy, G Bhanuprakash; Ramakrishna, Sistla

    2014-07-01

    The phosphatidylinositol 3-kinase (PI3 K)/Akt/mammalian target of rapamycin (mTOR) signaling axis plays a central role in cell proliferation, growth and survival under physiological conditions. However, aberrant PI3 K/Akt/mTOR signaling has been implicated in many human cancers, including human triple negative breast cancer. Therefore, dual inhibitors of PI3 K/Akt and mTOR signaling could be valuable agents for treating breast cancer. The objective of this study was to investigate the effect of piperlongumine (PPLGM), a natural alkaloid on PI3 K/Akt/mTOR signaling, Akt mediated regulation of NF-kB and apoptosis evasion in human breast cancer cells. Using molecular docking studies, we found that PPLGM physically interacts with the conserved domain of PI3 K and mTOR kinases and the results were comparable with standard dual inhibitor PF04691502. Our results demonstrated that treatment of different human triple-negative breast cancer cells with PPLGM resulted in concentration- and time-dependent growth inhibition. The inhibition of cancer cell growth was associated with G1-phase cell cycle arrest and down-regulation of the NF-kB pathway leads to activation of the mitochondrial apoptotic pathway. It was also found that PPLGM significantly decreased the expression of p-Akt, p70S6K1, 4E-BP1, cyclin D1, Bcl-2, p53 and increased expression of Bax, cytochrome c in human triple-negative breast cancer cells. Although insulin treatment increased the phosphorylation of Akt (Ser473), p70S6K1, 4E-BP1, PPLGM abolished the insulin mediated phosphorylation, it clearly indicates that PPLGM acts through PI3 k/Akt/mTOR axis. Our results suggest that PPLGM may be an effective therapeutic agent for the treatment of human triple negative breast cancer. PMID:24729100

  1. Propentofylline inhibits glioblastoma cell invasion and survival by targeting the TROY signaling pathway.

    Science.gov (United States)

    Dhruv, Harshil D; Roos, Alison; Tomboc, Patrick J; Tuncali, Serdar; Chavez, Ashley; Mathews, Ian; Berens, Michael E; Loftus, Joseph C; Tran, Nhan L

    2016-02-01

    Glioblastoma (GBM) is the most common primary tumor of the CNS and carries a dismal prognosis. The aggressive invasion of GBM cells into the surrounding normal brain makes complete resection impossible, significantly increases resistance to the standard therapy regimen, and virtually assures tumor recurrence. Median survival for newly diagnosed GBM is 14.6 months and declines to 8 months for patients with recurrent GBM. New therapeutic strategies that target the molecular drivers of invasion are required for improved clinical outcome. We have demonstrated that TROY (TNFRSF19), a member of the TNFR super-family, plays an important role in GBM invasion and resistance. Knockdown of TROY expression inhibits GBM cell invasion, increases sensitivity to temozolomide, and prolongs survival in an intracranial xenograft model. Propentofylline (PPF), an atypical synthetic methylxanthine compound, has been extensively studied in Phase II and Phase III clinical trials for Alzheimer's disease and vascular dementia where it has demonstrated blood-brain permeability and minimal adverse side effects. Here we showed that PPF decreased GBM cell expression of TROY, inhibited glioma cell invasion, and sensitized GBM cells to TMZ. Mechanistically, PPF decreased glioma cell invasion by modulating TROY expression and downstream signaling, including AKT, NF-κB, and Rac1 activation. Thus, PPF may provide a pharmacologic approach to target TROY, inhibit cell invasion, and reduce therapeutic resistance in GBM. PMID:26559543

  2. Sphingosylphosphorylcholine protects cardiomyocytes against ischemic apoptosis via lipid raft/PTEN/Akt1/mTOR mediated autophagy.

    Science.gov (United States)

    Yue, Hong-Wei; Liu, Jing; Liu, Ping-Ping; Li, Wen-Jing; Chang, Fen; Miao, Jun-Ying; Zhao, Jing

    2015-09-01

    Autophagy, evoked by diverse stresses including myocardial ischemia/reperfusion (I/R), profoundly affects the development of heart failure. However, the specific molecular basis of autophagy remains to be elucidated. Here we report that sphingosylphosphorylcholine (SPC), a bioactive sphingolipid, significantly suppressed apoptosis and induced autophagy in cardiomyocytes. Blocking this SPC evoked autophagy by 3-methyladenine (3MA)-sensitized cardiomyocytes to serum deprivation-induced apoptosis. Subsequent studies revealed that SPC downregulated the phosphorylation of p70S6K and 4EBP1 (two substrates of mTOR) but enhanced that of JNK when inducing autophagy. We identified SPC as a switch for the activity of Akt1, a supposed upstream modulator of both mTOR and JNK. Furthermore, β-cyclodextrin, which destroys membrane cholesterol, abolished the SPC-reduced phosphorylation of both Akt and PTEN, thus inhibiting SPC-induced autophagy. In conclusion, SPC is a novel molecule protecting cardiomyocytes against apoptosis by promoting autophagy. The lipid raft/PTEN/Akt1/mTOR signal pathway is the underlying mechanism and might provide novel targets for cardiac failure therapy.

  3. Titration of signalling output: insights into clinical combinations of MEK and AKT inhibitors

    OpenAIRE

    STEWART, A.; Thavasu, P.; de Bono, J S; Banerji, U

    2015-01-01

    Background We aimed to understand the relative contributions of inhibiting MEK and AKT on cell growth to guide combinations of these agents. Materials and methods A panel of 20 cell lines was exposed to either the MEK inhibitor, PD0325901, or AKT inhibitor, AKT 1/2 inhibitor. p-ERK and p-S6 ELISAs were used to define degrees of MEK and AKT inhibition, respectively. Growth inhibition to different degrees of MEK and AKT inhibition, either singly or in combination using 96-h sulphorhodamine assa...

  4. Protein Kinase B/Akt Binds and Phosphorylates PED/PEA-15, Stabilizing Its Antiapoptotic Action

    OpenAIRE

    Trencia, Alessandra; Perfetti, Anna; Cassese, Angela; Vigliotta, Giovanni; Miele, Claudia; Oriente, Francesco; Santopietro, Stefania; Giacco, Ferdinando; Condorelli, Gerolama; Formisano, Pietro; Beguinot, Francesco

    2003-01-01

    The antiapoptotic protein PED/PEA-15 features an Akt phosphorylation motif upstream from Ser116. In vitro, recombinant PED/PEA-15 was phosphorylated by Akt with a stoichiometry close to 1. Based on Western blotting with specific phospho-Ser116 PED/PEA-15 antibodies, Akt phosphorylation of PED/PEA-15 occurred mainly at Ser116. In addition, a mutant of PED/PEA-15 featuring the substitution of Ser116→Gly (PEDS116→G) showed 10-fold-decreased phosphorylation by Akt. In intact 293 cells, Akt also i...

  5. The insulin-like growth factor 1 pathway is a potential therapeutic target for low-grade serous ovarian carcinoma

    Science.gov (United States)

    King, Erin R.; Zu, Zhifei; Tsang, Yvonne T.M.; Deavers, Michael T.; Malpica, Anais; Mok, Samuel C.; Gershenson, David M.; Wong, Kwong-Kwok

    2011-01-01

    Objective To validate the overexpression of insulin-like growth factor 1 (IGF-1) and its receptor (IGF-1R) in low-grade serous ovarian carcinoma (SOC), and to investigate whether the IGF-1 pathway is a potential therapeutic target for low-grade SOC. Methods Gene expression profiling was performed on serous borderline ovarian tumors (SBOTs) and low-grade SOC, and overexpression of IGF-1 in low-grade SOC was validated by RT-PCR and immunohistochemistry. The effect of exogenous IGF-1 on cell proliferation was determined in cell lines by cell proliferation assays, cell migration assays, and Western blot. Signaling pathways downstream of IGF-1 and the effects of the AKT inhibitor MK-2206 were investigated by Western blot analysis and by generating IGF-1R short hairpin RNA stable knockdown cell lines. Low- and high-grade cell lines were treated with the dual IGF-1R- and insulin receptor-directed tyrosine kinase inhibitor OSI-906, and cellular proliferation was measured. Results mRNA analysis and immunostaining revealed significantly higher IGF-1 expression in low-grade SOCs than in SBOTs or high-grade SOCs. In response to exogenous treatment with IGF-1, low-grade cell lines exhibited more intense upregulation of phosphorylated AKT than did high-grade cell lines, an effect that was diminished with IGF-1R knockdown and MK-2206 treatment. Low-grade SOC cell lines were more sensitive to growth inhibition with OSI-906 than were high-grade cell lines. Conclusions IGF-1 is overexpressed in low-grade SOCs compared with SBOTs and high-grade SOCs. Additionally, low-grade SOC cell lines were more responsive to IGF-1 stimulation and IGF-1R inhibition than were high-grade lines. The IGF-1 pathway is therefore a potential therapeutic target in low-grade SOC. PMID:21726895

  6. Combined Inhibition of Cyclin-Dependent Kinases (Dinaciclib) and AKT (MK-2206) Blocks Pancreatic Tumor Growth and Metastases in Patient-Derived Xenograft Models.

    Science.gov (United States)

    Hu, Chaoxin; Dadon, Tikva; Chenna, Venugopal; Yabuuchi, Shinichi; Bannerji, Rajat; Booher, Robert; Strack, Peter; Azad, Nilofer; Nelkin, Barry D; Maitra, Anirban

    2015-07-01

    KRAS is activated by mutation in the vast majority of cases of pancreatic cancer; unfortunately, therapeutic attempts to inhibit KRAS directly have been unsuccessful. Our previous studies showed that inhibition of cyclin-dependent kinase 5 (CDK5) reduces pancreatic cancer growth and progression, through blockage of the centrally important RAL effector pathway, downstream of KRAS. In the current study, the therapeutic effects of combining the CDK inhibitor dinaciclib (SCH727965; MK-7965) with the pan-AKT inhibitor MK-2206 were evaluated using orthotopic and subcutaneous patient-derived human pancreatic cancer xenograft models. The combination of dinaciclib (20 mg/kg, i.p., three times a week) and MK-2206 (60 mg/kg, orally, three times a week) dramatically blocked tumor growth and metastasis in all eight pancreatic cancer models examined. Remarkably, several complete responses were induced by the combination treatment of dinaciclib and MK-2206. The striking results obtained in these models demonstrate that the combination of dinaciclib with the pan-AKT inhibitor MK-2206 is promising for therapeutic evaluation in pancreatic cancer, and strongly suggest that blocking RAL in combination with other effector pathways downstream from KRAS may provide increased efficacy in pancreatic cancer. Based on these data, an NCI-CTEP-approved multicenter phase I clinical trial for pancreatic cancer of the combination of dinaciclib and MK-2206 (NCT01783171) has now been opened. PMID:25931518

  7. Bakuchiol suppresses proliferation of skin cancer cells by directly targeting Hck, Blk, and p38 MAP kinase.

    Science.gov (United States)

    Kim, Jong-Eun; Kim, Jae Hwan; Lee, Younghyun; Yang, Hee; Heo, Yong-Seok; Bode, Ann M; Lee, Ki Won; Dong, Zigang

    2016-03-22

    Bakuchiol is a meroterpene present in the medicinal plant Psoralea corylifolia, which has been traditionally used in China, India, Japan and Korea for the treatment of premature ejaculation, knee pain, alopecia spermatorrhea, enuresis, backache, pollakiuria, vitiligo, callus, and psoriasis. Here, we report the chemopreventive properties of bakuchiol, which acts by inhibiting epidermal growth factor (EGF)-induced neoplastic cell transformation. Bakuchiol also decreased viability and inhibited anchorage-independent growth of A431 human epithelial carcinoma cells. Bakuchiol reduced A431 xenograft tumor growth in an in vivo mouse model. Using kinase profiling, we identified Hck, Blk and p38 mitogen activated protein kinase (MAPK) as targets of bakuchiol, which directly bound to each kinase in an ATP-competitive manner. Bakuchiol also inhibited EGF-induced signaling pathways downstream of Hck, Blk and p38 MAPK, including the MEK/ERKs, p38 MAPK/MSK1 and AKT/p70S6K pathways. This report is the first mechanistic study identifying molecular targets for the anticancer activity of bakuchiol and our findings indicate that bakuchiol exhibits potent anticancer activity by targeting Hck, Blk and p38 MAPK. PMID:26910280

  8. miR-200c and phospho-AKT as prognostic factors and mediators of osteosarcoma progression and lung metastasis.

    Science.gov (United States)

    Berlanga, Pablo; Muñoz, Lisandra; Piqueras, Marta; Sirerol, J Antoni; Sánchez-Izquierdo, María Dolors; Hervás, David; Hernández, Miguel; Llavador, Margarita; Machado, Isidro; Llombart-Bosch, Antonio; Cañete, Adela; Castel, Victoria; Font de Mora, Jaime

    2016-08-01

    Lung metastasis is the major cause of death in osteosarcoma patients. However, molecular mechanisms underlying this metastasis remain poorly understood. To identify key molecules related with pulmonary metastasis of pediatric osteosarcomas, we analyzed high-throughput miRNA expression in a cohort of 11 primary tumors and 15 lung metastases. Results were further validated with an independent cohort of 10 primary tumors and 6 metastases. In parallel, we performed immunohistochemical analysis of activated signaling pathways in 36 primary osteosarcomas. Only phospho-AKT associated with lower overall survival in primary tumors, supporting its role in osteosarcoma progression. CTNNB1 expression also associated with lower overall survival but was not strong enough to be considered an independent variable. Interestingly, miR-200c was overexpressed in lung metastases, implicating an inhibitory feed-back loop to PI3K-AKT. Moreover, transfection of miR200c-mimic in U2-OS cells reduced phospho-AKT levels but increased cellular migration and proliferation. Notably, miR-200c expression strongly correlated with miR-141 and with the osteogenic inhibitor miR-375, all implicated in epithelial to mesenchymal transition. These findings contrast epithelial tumors where reduced miR-200c expression promotes metastasis. Indeed, we noted that osteosarcoma cells in the lung also expressed the epithelial marker CDH1, revealing a change in their mesenchymal phenotype. We propose that miR-200c upregulation occurs late in osteosarcoma progression to provide cells with an epithelial phenotype that facilitates their integration in the metastatic lung niche. Thus, our findings identify phospho-AKT in the primary tumor and miR-200c later during tumor progression as prognostic molecules and potential therapeutic targets to prevent progression and metastasis of pediatric osteosarcomas. PMID:27155790

  9. GLP1 protects cardiomyocytes from palmitate-induced apoptosis via Akt/GSK3b/b-catenin pathway.

    Science.gov (United States)

    Ying, Ying; Zhu, Huazhang; Liang, Zhen; Ma, Xiaosong; Li, Shiwei

    2015-12-01

    Activation of apoptosis in cardiomyocytes by saturated palmitic acids contributes to cardiac dysfunction in diabetic cardiomyopathy. Beta-catenin (b-catenin) is a transcriptional regulator of several genes involved in survival/anti-apoptosis. However, its role in palmitate-induced cardiomyocyte apoptosis remains unclear. Glucagon-like peptide 1 (GLP1) has been shown to exhibit potential cardioprotective properties. This study was designed to evaluate the role of b-catenin signalling in palmitate-induced cardiomyocyte apoptosis and the molecular mechanism underlying the protective effects of GLP1 on palmitate-stressed cardiomyocytes. Exposure of neonatal rat cardiomyocytes to palmitate increased the fatty acid transporter CD36-mediated intracellular lipid accumulation and cardiomyocyte apoptosis, decreased accumulation and nuclear translocation of active b-catenin, and reduced expression of b-catenin target protein survivin and BCL2. These detrimental effects of palmitate were significantly attenuated by GLP1 co-treatment. However, the anti-apoptotic effects of GLP1 were markedly abolished when b-catenin was silenced with a specific short hairpin RNA. Furthermore, analysis of the upstream molecules and mechanisms responsible for GLP1-associated cardiac protection revealed that GLP1 restored the decreased phosphorylation of protein kinase B (Akt) and glycogen synthase kinase-3b (GSK3b) in palmitate-stimulated cardiomyocytes. In contrast, inhibition of Akt with an Akt-specific inhibitor MK2206 or blockade of GLP1 receptor (GLP1R) with a competitive antagonist exendin-(9-39) significantly abrogated the GLP1-mediated activation of GSK3b/b-catenin signalling, leading to increased apoptosis in palmitate-stressed cardiomyocytes. Collectively, our results demonstrated for the first time that the attenuated b-catenin signalling may contribute to palmitate-induced cardiomyocyte apoptosis, while GLP1 can protect cardiomyocytes from palmitate-induced apoptosis through

  10. WISP1 overexpression promotes proliferation and migration of human vascular smooth muscle cells via AKT signaling pathway.

    Science.gov (United States)

    Lu, Shun; Liu, Hao; Lu, Lihe; Wan, Heng; Lin, Zhiqi; Qian, Kai; Yao, Xingxing; Chen, Qing; Liu, Wenjun; Yan, Jianyun; Liu, Zhengjun

    2016-10-01

    Proliferation and migration of vascular smooth muscle cells (VSMCs) play crucial roles in the development of vascular restenosis. Our previous study showed that CCN4, namely Wnt1 inducible signaling pathway protein 1 (WISP1), significantly promotes proliferation and migration of rat VSMCs, but its mechanism remains unclear. This study aims to investigate whether and how WISP1 stimulates proliferation and migration of human VSMCs. Western blot analysis showed that FBS treatment increased WISP1 protein levels in human VSMCs in a dose-dependent manner. Overexpression of WISP1 using adenovirus encoding WISP1 (AD-WISP1) significantly increased proliferation rate of human VSMCs by 2.98-fold compared with empty virus (EV)-transfected cells, shown by EdU incorporation assay. Additionally, Scratch-induced wound healing assay revealed that adenovirus-mediated overexpression of WISP1 significantly increased cell migration compared with EV-transfected cells from 6h (4.56±1.14% vs. 11.23±2.25%, PMigration Assay confirmed that WISP1 overexpression significantly promoted human VSMC migration by 2.25-fold compared with EV. Furthermore, WISP1 overexpression stimulated Akt signaling activation in human VSMCs. Blockage of Akt signaling by Akt inhibitor AZD5363 or PI3K inhibitor LY294002, led to an inhibitory effect of WISP1-induced proliferation and migration in human VSMCs. Moreover, we found that WISP1 overexpression stimulated GSK3α/β phosphorylation, and increased expression of cyclin D1 and MMP9 in human VSMCs, and this effect was abolished by AZD5363. Collectively, we demonstrated that Akt signaling pathway mediates WISP1-induced migration and proliferation of human VSMCs, suggesting that WISP1 may act as a novel potential therapeutic target for vascular restenosis.

  11. Akt1 intramitochondrial cycling is a crucial step in the redox modulation of cell cycle progression.

    Directory of Open Access Journals (Sweden)

    Valeria Gabriela Antico Arciuch

    Full Text Available Akt is a serine/threonine kinase involved in cell proliferation, apoptosis, and glucose metabolism. Akt is differentially activated by growth factors and oxidative stress by sequential phosphorylation of Ser(473 by mTORC2 and Thr(308 by PDK1. On these bases, we investigated the mechanistic connection of H(2O(2 yield, mitochondrial activation of Akt1 and cell cycle progression in NIH/3T3 cell line with confocal microscopy, in vivo imaging, and directed mutagenesis. We demonstrate that modulation by H(2O(2 entails the entrance of cytosolic P-Akt1 Ser(473 to mitochondria, where it is further phosphorylated at Thr(308 by constitutive PDK1. Phosphorylation of Thr(308 in mitochondria determines Akt1 passage to nuclei and triggers genomic post-translational mechanisms for cell proliferation. At high H(2O(2, Akt1-PDK1 association is disrupted and P-Akt1 Ser(473 accumulates in mitochondria in detriment to nuclear translocation; accordingly, Akt1 T308A is retained in mitochondria. Low Akt1 activity increases cytochrome c release to cytosol leading to apoptosis. As assessed by mass spectra, differential H(2O(2 effects on Akt1-PDK interaction depend on the selective oxidation of Cys(310 to sulfenic or cysteic acids. These results indicate that Akt1 intramitochondrial-cycling is central for redox modulation of cell fate.

  12. Assessing the impacts of Three Gorges Dam on lake inundation areas across the downstream Yangtze floodplain

    Science.gov (United States)

    Wang, J.; Sheng, Y.; Wada, Y.

    2013-12-01

    China's Three Gorges Dam (TGD) has received worldwide attention due to its profound impacts on the downstream hydraulic, morphological, and ecological systems. The TGD operation results in direct alternation of the discharge to the middle and lower Yangtze reach, manifested as regulated flow and reduced sediment load. TGD's flow regulation, typically described as water storage in fall while release in winter and spring, interferes with the natural seasonality of downstream Yangtze River levels which are essentially important to the inundation dynamics of surrounding lakes/wetlands in the Yangtze floodplain. Concurrent decrease of sediment load has caused chronic downstream channel erosion which lowers Yangtze level in relation to flow and further affects the sustainability of riparian lakes and the related ecosystems. By integrating satellite observations, in situ measurement, and hydrologic simulations, this study presents a systematic assessment of the TGD impacts on the inundation areas of six major freshwater lakes across the entire Yangtze basin downstream of the TGD, during the time period from TGD's initial impoundment in June 2003 to early 2012. Despite the small number, the six targeted lakes cover a total area of ~5,000 km2 accounting for ~25% of the freshwater lake area in China, and were identified as the only natural lakes that remain in open connection to the Yangtze River across the downstream floodplain. Using daily MODIS imagery from 2000 to 2012, we revealed a significant year-round decline in the aggregated inundation area of the studied lakes by an average of ~580 km2 or 17.7% from the pre-dam to post-dam period (i.e., before and after June, 2003). To diagnose TGD's contribution to such lake area decline, we followed a two-step procedure by first quantifying the TGD impacts on the seasonal level regime along the complete longitudinal range of the Yangtze River downstream from TGD to the estuary [Wang et al., 2013], and then estimating the

  13. PI3K/Akt contributes to increased expression of Toll-like receptor 4 in macrophages exposed to hypoxic stress

    Energy Technology Data Exchange (ETDEWEB)

    Kim, So Young; Jeong, Eunshil; Joung, Sun Myung [School of Life Sciences, Gwangju Institute of Science and Technology, Gwangju 500-712 (Korea, Republic of); Lee, Joo Young, E-mail: joolee@catholic.ac.kr [School of Life Sciences, Gwangju Institute of Science and Technology, Gwangju 500-712 (Korea, Republic of); College of Pharmacy, The Catholic University of Korea, Bucheon 420-743 (Korea, Republic of)

    2012-03-16

    Highlights: Black-Right-Pointing-Pointer Hypoxic stress-induced TLR4 expression is mediated by PI3K/Akt in macrophages. Black-Right-Pointing-Pointer PI3K/Akt regulated HIF-1 activation leading to TLR4 expression. Black-Right-Pointing-Pointer p38 mitogen-activated protein kinase was not involved in TLR4 expression by hypoxic stress. Black-Right-Pointing-Pointer Sulforaphane suppressed hypoxia-mediated TLR4 expression by inhibiting PI3K/Akt. -- Abstract: Toll-like receptors (TLRs) play critical roles in triggering immune and inflammatory responses by detecting invading microbial pathogens and endogenous danger signals. Increased expression of TLR4 is implicated in aggravated inflammatory symptoms in ischemic tissue injury and chronic diseases. Results from our previous study showed that TLR4 expression was upregulated by hypoxic stress mediated by hypoxia-inducible factor-1 (HIF-1) at a transcriptional level in macrophages. In this study, we further investigated the upstream signaling pathway that contributed to the increase of TLR4 expression by hypoxic stress. Either treatment with pharmacological inhibitors of PI3K and Akt or knockdown of Akt expression by siRNA blocked the increase of TLR4 mRNA and protein levels in macrophages exposed to hypoxia and CoCl{sub 2}. Phosphorylation of Akt by hypoxic stress preceded nuclear accumulation of HIF-1{alpha}. A PI3K inhibitor (LY294002) attenuated CoCl{sub 2}-induced nuclear accumulation and transcriptional activation of HIF-1{alpha}. In addition, HIF-1{alpha}-mediated upregulation of TLR4 expression was blocked by LY294002. Furthermore, sulforaphane suppressed hypoxia- and CoCl{sub 2}-induced upregulation of TLR4 mRNA and protein by inhibiting PI3K/Akt activation and the subsequent nuclear accumulation and transcriptional activation of HIF-1{alpha}. However, p38 was not involved in HIF-1{alpha} activation and TLR4 expression induced by hypoxic stress in macrophages. Collectively, our results demonstrate that PI3K/Akt

  14. Modulation of Akt and ERK1/2 pathways by resveratrol in chronic myelogenous leukemia (CML cells results in the downregulation of Hsp70.

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    Soumyajit Banerjee Mustafi

    Full Text Available BACKGROUND: Resveratrol is known to downregulate the high endogenous level of Heat shock protein 70 (Hsp70 in Chronic Myelogenous Leukemia (CML K562 cells and induce apoptosis. Since Heat Shock Factor 1 (HSF1 controls transcription of Hsp70, we wanted to probe the signaling pathways responsible for transcriptional activation of HSF1. METHODOLOGY/PRINCIPAL FINDINGS: Cells exposed to 40microM Resveratrol rapidly abolished serine473 phosphorylation of Akt and significantly reduced its kinase activity. Inactivation of Akt pathway by Resveratrol subsequently blocked serine9 phosphorylation of Gsk3beta. Active non-phosphorylated Gsk3beta rendered HSF1 transcriptionally inactive and reduced Hsp70 production. Blocking PI3K/Akt activity also demonstrated similar effects on Hsp70 comparable to Resveratrol. Inactivation of Gsk3beta activity by inhibitors SB261763 or LiCl upregulated Hsp70. Resveratrol significantly modulated ERK1/2 activity as evident from hyper phosphorylation at T302/Y304 residues and simultaneous upregulation in kinase activity. Blocking ERK1/2 activation resulted in induction of Hsp70. Therefore, increase in ERK1/2 activity by Resveratrol provided another negative influence on Hsp70 levels through negative regulation of HSF1 activity. 17-allylamino-17-demethoxygeldanamycin (17AAG, a drug that inhibits Hsp90 chaperone and degrades its client protein Akt concomitantly elevated Hsp70 levels by promoting nuclear translocation of HSF1 from the cytosol. This effect is predominantly due to inhibition of both Akt and ERK1/2 activation by 17AAG. Simultaneously treating K562 with Resveratrol and 17AAG maintained phosho-ERK1/2 levels close to untreated controls demonstrating their opposite effects on ERK1/2 pathway. Resveratrol was found not to interfere with Bcr-Abl activation in K562 cells. CONCLUSION/SIGNIFICANCE: Thus our study comprehensively illustrates that Resveratrol acts downstream of Bcr-Abl and inhibits Akt activity but stimulates ERK

  15. Anti-fibrotic actions of interleukin-10 against hypertrophic scarring by activation of PI3K/AKT and STAT3 signaling pathways in scar-forming fibroblasts.

    Directory of Open Access Journals (Sweden)

    Jihong Shi

    inhibits fibrosis by activating AKT and STAT3 phosphorylation downstream of the IL-10 receptor, and by facilitating crosstalk between the PI3K/AKT and STAT3 signal transduction pathways.

  16. Abberent expression of oncogenic and tumor-suppressive microRNAs and their target genes in human adenocarcinoma alveolar basal epithelial cells

    Directory of Open Access Journals (Sweden)

    Elham Tafsiri

    2016-01-01

    Conclusion: The significant differential expression level of these miRNAs made them as candidate biomarkers in NSCLC tumor tissues of patients. Perhaps Bcl-2 down-regulation and Akt-3 up-regulation can be linked with survival signals in A549 cell line. We can conclude that Bcl-2 and Akt-3 might be therapeutic targets to inhibit cell proliferation in NSCLC.

  17. Ellagic acid protects endothelial cells from oxidized low-density lipoprotein-induced apoptosis by modulating the PI3K/Akt/eNOS pathway

    International Nuclear Information System (INIS)

    Endothelial apoptosis is a driving force in atherosclerosis development. Oxidized low-density lipoprotein (oxLDL) promotes inflammatory and thrombotic processes and is highly atherogenic, as it stimulates macrophage cholesterol accumulation and foam cell formation. Previous studies have shown that the phosphatidylinositol 3-kinase/Akt/endothelial nitric oxide synthase/nitric oxide (PI3K/Akt/eNOS/NO) pathway is involved in oxLDL-induced endothelial apoptosis. Ellagic acid, a natural polyphenol found in berries and nuts, has in recent years been the subject of intense research within the fields of cancer and inflammation. However, its protective effects against oxLDL-induced injury in vascular endothelial cells have not been clarified. In the present study, we investigated the anti-apoptotic effect of ellagic acid in human umbilical vein endothelial cells (HUVECs) exposed to oxLDL and explored the possible mechanisms. Our results showed that pretreatment with ellagic acid (5-20 μM) significantly attenuated oxLDL-induced cytotoxicity, apoptotic features, and generation of reactive oxygen species (ROS). In addition, the anti-apoptotic effect of ellagic acid was partially inhibited by a PI3K inhibitor (wortmannin) and a specific eNOS inhibitor (cavtratin) but not by an ERK inhibitor (PD98059). In exploring the underlying mechanisms of ellagic acid action, we found that oxLDL decreased Akt and eNOS phosphorylation, which in turn activated NF-κB and downstream pro-apoptotic signaling events including calcium accumulation, destabilization of mitochondrial permeability, and disruption of the balance between pro- and anti-apoptotic Bcl-2 proteins. Those alterations induced by oxLDL, however, were attenuated by pretreatment with ellagic acid. The inhibition of oxLDL-induced endothelial apoptosis by ellagic acid is due at least in part to its anti-oxidant activity and its ability to modulate the PI3K/Akt/eNOS signaling pathway.

  18. Protective Effect of DHT on Apoptosis Induced by U18666A via PI3K/Akt Signaling Pathway in C6 Glial Cell Lines.

    Science.gov (United States)

    Yao, Kai; Wu, Junfeng; Zhang, Jianfeng; Bo, Jimei; Hong, Zhen; Zu, Hengbing

    2016-07-01

    Various useful animal models, such as Alzheimer's disease and Niemann-Pick disease, were provided by U18666A. However, the pathogenesis of U18666A-induced diseases, including U18666A-mediated apoptosis, remains incompletely elucidated, and therapeutic strategies are still limited. Dihydrotestosterone (DHT) has been reported to contribute to the prevention and treatment of neurodegenerative disorders. Our study investigated the neuroprotective activity of DHT in U18666A-related diseases. Apoptosis of C6 cells was detected by Hoechst 33258 fluorescent staining and flow cytometry with annexin V-FITC/PI dual staining. Cell viability was assessed using Cell Counting Kit-8. Expression of apoptosis-related proteins, such as Akt, seladin-1, Bcl-2 family proteins, and caspase-3, was determined using Western blot. Our results demonstrated that the apoptotic rate of C6 cells significantly increased after U18666A addition, but was remarkably reduced after DHT treatment. Pretreatment with DHT attenuated U18666A-induced cell viability loss. PI3K inhibitor LY294002 could suppress DHT anti-apoptotic effect. Furthermore, we discovered that U18666A could significantly downregulate seladin-1 expression in a dose-dependent manner, but no significant change was observed in Bcl-xL, Bax, and P-Akt protein expressions. Compared with U18666A-treated group, the expression of P-Akt, seladin-1, and Bcl-xL significantly increased, and the expression of Bax and caspase-3 remarkably reduced after DHT treatment. However, in the presence of LY294002, the effect of DHT was reversed. In conclusion, we found that seladin-1 may take part in U18666A-induced apoptosis. DHT may inhibit U18666A-induced apoptosis by regulating downstream apoptosis-related proteins including seladin-1, caspase-3, Bcl-xL, and Bax through activation of the PI3K/Akt signal pathway. PMID:26340949

  19. Modeling downstream fining in sand-bed rivers. I: Formulation

    Science.gov (United States)

    Wright, S.; Parker, G.

    2005-01-01

    In this paper a numerical modeling formulation is presented for simulation of the development of the longitudinal profile and bed sediment distribution in sand-bed rivers. The objective of the model application, which is presented in the companion paper (Wright and Parker, 2005), is to study the development of two characteristics of large, low-slope, sand-bed rivers: (1) a downstream decrease in bed slope (i.e. concave upward longitudinal profile) and (2) a downstream decrease in characteristic bed sediment diameter (e.g. the median bed surface size D50). Three mechanisms that lead to an upward concave profile and downstream fining are included in the modeling formulation: (1) a delta prograding into standing water at the downstream boundary, (2) sea-level rise, and (3) tectonic subsidence. In the companion paper (Wright and Parker, 2005) the model is applied to simulate the development of the longitudinal profile and downstream fining in sand-bed rivers flowing into the ocean during the past 5000 years of relatively slow sea-level rise. ?? 2005 International Association of Hydraulic Engineering and Research.

  20. Mortality of zebra mussel, Dreissena polymorpha, veligers during downstream transport

    Science.gov (United States)

    Horvath, T.G.; Lamberti, G.A.

    1999-01-01

    1. Streams flowing from lakes which contain zebra mussels, Dreissena polymorpha, provide apparently suitable habitats for mussel colonization and downstream range expansion, yet most such streams contain few adult mussels. We postulated that mussel veligers experience high mortality during dispersal via downstream transport. They tested this hypothesis in Christiana Creek, a lake-outlet stream in south-western Michigan, U.S.A., in which adult mussel density declined exponentially with distance downstream. 2. A staining technique using neutral red was developed and tested to distinguish quickly live and dead veligers. Live and dead veligers were distinguishable after an exposure of fresh samples to 13.3 mg L-1 of neutral red for 3 h. 3. Neutral red was used to determine the proportion of live veligers in samples taken longitudinally along Christiana Creek. The proportion of live veligers (mean ?? SE) declined from 90 ?? 3% at the lake outlet to 40 ?? 8% 18 km downstream. 4. Veligers appear to be highly susceptible to damage by physical forces (e.g. shear), and therefore, mortality in turbulent streams could be an important mechanism limiting zebra mussel dispersal to downstream reaches. Predictions of zebra mussel spread and population growth should consider lake-stream linkages and high mortality in running waters.

  1. Downstream Yangtze River levels impacted by Three Gorges Dam

    Science.gov (United States)

    Wang, Jida; Sheng, Yongwei; Gleason, Colin J.; Wada, Yoshihide

    2013-12-01

    Changes in the Yangtze River level induced by large-scale human water regulation have profound implications on the inundation dynamics of surrounding lakes/wetlands and the integrity of related ecosystems. Using in situ measurements and hydrological simulation, this study reveals an altered Yangtze level regime downstream from the Three Gorges Dam (TGD) to the Yangtze estuary in the East China Sea as a combined result of (i) TGD’s flow regulation and (ii) Yangtze channel erosion due to reduced sediment load. During the average annual cycle of TGD’s regular flow control in 2009-2012, downstream Yangtze level variations were estimated to have been reduced by 3.9-13.5% at 15 studied gauging stations, manifested as evident level decrease in fall and increase in winter and spring. The impacts on Yangtze levels generally diminished in a longitudinal direction from the TGD to the estuary, with a total time lag of ˜9-12 days. Chronic Yangtze channel erosion since the TGD closure has lowered water levels in relation to flows at most downstream stations, which in turn counteracts the anticipated level increase by nearly or over 50% in winter and spring while reinforcing the anticipated level decrease by over 20% in fall. Continuous downstream channel erosion in the near future may further counteract the benefit of increased Yangtze levels during TGD’s water supplement in winter and accelerate the receding of inundation areas/levels of downstream lakes in fall.

  2. CHIP promotes thyroid cancer proliferation via activation of the MAPK and AKT pathways.

    Science.gov (United States)

    Zhang, Li; Liu, Lianyong; He, Xiaohua; Shen, Yunling; Liu, Xuerong; Wei, Jing; Yu, Fang; Tian, Jianqing

    2016-08-26

    The carboxyl terminus of Hsp70-interacting protein (CHIP) is a U box-type ubiquitin ligase that plays crucial roles in various biological processes, including tumor progression. To date, the functional mechanism of CHIP in thyroid cancer remains unknown. Here, we obtained evidence of upregulation of CHIP in thyroid cancer tissues and cell lines. CHIP overexpression markedly enhanced thyroid cancer cell viability and colony formation in vitro and accelerated tumor growth in vivo. Conversely, CHIP knockdown impaired cell proliferation and tumor growth. Notably, CHIP promoted cell growth through activation of MAPK and AKT pathways, subsequently decreasing p27 and increasing cyclin D1 and p-FOXO3a expression. Our findings collectively indicate that CHIP functions as an oncogene in thyroid cancer, and is therefore a potential therapeutic target for this disease. PMID:27342662

  3. Francisella subverts innate immune signaling: Focus on PI3K/Akt

    Directory of Open Access Journals (Sweden)

    Thomas John Cremer

    2011-02-01

    Full Text Available Intracellular bacterial pathogens exploit host cells as a part of their lifecycle, and they do so by manipulating host cell signaling events. Many such bacteria are known to produce effector proteins that promote cell invasion, alter membrane trafficking and disrupt signaling cascades. This review highlights recent advances in our understanding of signaling pathways involved in host cell responses to Francisella tularensis, a facultative Gram-negative intracellular pathogen that causes tularemia. We highlight several key pathways that are targeted by Francisella, with a focus on the PI3K/Akt pathway. Lastly, we discuss the emerging role of microRNAs, specifically miR-155, as a key regulator of host signaling and defense.

  4. Knockdown of Pokemon protein expression inhibits hepatocellular carcinoma cell proliferation by suppression of AKT activity.

    Science.gov (United States)

    Zhu, Xiaosan; Dai, Yichen; Chen, Zhangxin; Xie, Junpei; Zeng, Wei; Lin, Yuanyuan

    2013-01-01

    Overexpression of Pokemon, which is an erythroid myeloid ontogenic factor protein, occurs in different cancers, including hepatocellular carcinoma (HCC). Pokemon is also reported to have an oncogenic activity in various human cancers. This study investigated the effect of Pokemon knockdown on the regulation of HCC growth. POK shRNA suppressed the expression of Pokemon protein in HepG2 cells compared to the negative control vector-transfected HCC cells. Pokemon knockdown also reduced HCC cell viability and enhanced cisplatin-induced apoptosis in HCC cells. AKT activation and the expression of various cell cycle-related genes were inhibited following Pokemon knockdown. These data demonstrate that Pokemon may play a role in HCC progression, suggesting that inhibition of Pokemon expression using Pokemon shRNA should be further evaluated as a novel target for the control of HCC. PMID:23924858

  5. CHIP promotes thyroid cancer proliferation via activation of the MAPK and AKT pathways.

    Science.gov (United States)

    Zhang, Li; Liu, Lianyong; He, Xiaohua; Shen, Yunling; Liu, Xuerong; Wei, Jing; Yu, Fang; Tian, Jianqing

    2016-08-26

    The carboxyl terminus of Hsp70-interacting protein (CHIP) is a U box-type ubiquitin ligase that plays crucial roles in various biological processes, including tumor progression. To date, the functional mechanism of CHIP in thyroid cancer remains unknown. Here, we obtained evidence of upregulation of CHIP in thyroid cancer tissues and cell lines. CHIP overexpression markedly enhanced thyroid cancer cell viability and colony formation in vitro and accelerated tumor growth in vivo. Conversely, CHIP knockdown impaired cell proliferation and tumor growth. Notably, CHIP promoted cell growth through activation of MAPK and AKT pathways, subsequently decreasing p27 and increasing cyclin D1 and p-FOXO3a expression. Our findings collectively indicate that CHIP functions as an oncogene in thyroid cancer, and is therefore a potential therapeutic target for this disease.

  6. The influence of the stem cell marker ALDH and the EGFR-PI3 kinase act signaling pathway on the radiation resistance of human tumor cell lines; Der Einfluss des Stammzellmarkers ALDH und des EGFR-PI3 Kinase-Akt Signalwegs auf die Strahlenresistenz humaner Tumorzelllinien

    Energy Technology Data Exchange (ETDEWEB)

    Mihatsch, Julia

    2014-07-14

    Cancer is the second leading cause of death in industriated nations. Besides surgery and chemotherapy, radiotherapy (RT) is an important approach by which about 60% of patients are treated. The response of these patients to RT is very heterogenous. On the one hand, there are patients with tumors which are radiosensitive and can be cured, but on the other hand patients bear tumors which are quite resistant to radiotherapy. A Radioresistant phenotype of tumor cells causes treatment failure consequently leading to a limited response to radiotherapy. It is proposed, that radiotherapy outcome mainly depends on the potential of radiation on controlling growth, proliferation and survival of a specific population of tumor cells called cancer stem cells (CSCs) or tumor-initiating cells. Based on experimental studies so far reported it is assumed that the population of CSC varies in tumors from different entities and is relatively low compared to the tumor bulk cells in general. According to the CSC hypothesis, it might be concluded that the differential response of tumors to radiotherapy depends on CSC populations, since these supposedly slow replicating cells are able to initiate a tumor, to self renew indefinitely and to generate the differentiated progeny of a tumor. Besides the role of cancer stem cells in radiotherapy response, ionizing radiation (IR) activates the epidermal growth factor receptor (EGFR) and its downstream signaling pathways such as phosphoinositide 3-kinase (PI3K)/Akt, mitogen-activated protein kinase (MAPK) and Janus kinase/Signal Transducer and Activator of Transcription (JAK/STAT) pathways. Among these pathways, PI3K/Akt is one of the most important pathways involved in post-irradiation survival: Activation of Akt results in activation of DNA-dependent protein kinase, catalytic subunit (DNA-PKcs). DNA-PKcs is a core enzyme involved in repair of IR-induced DNA-double strand breaks (DNA-DSB) through non-homologous end joining (NHEJ). The aim of the

  7. Suppression of inflammatory responses during MOG-induced experimental autoimmune encephalomyelitis is regulated by AKT3 signaling

    OpenAIRE

    Tsiperson, Vladislav; Gruber, Ross C; Goldberg, Michael; Jordan, Ayana; Weinger, Jason G.; Macian, Fernando; Shafit-Zagardo, Bridget

    2013-01-01

    AKT3, a member of the serine/threonine kinase AKT family, is involved in a variety of biological processes. AKT3 is expressed in immune cells, and is the major AKT isoform in the CNS representing 30% of the total AKT expressed in spinal cord, and 50% in the brain. Myelin-oligodendrocyte glycoprotein (MOG)-induced experimental autoimmune encephalomyelitis (EAE) is a mouse model in which lymphocytes and monocytes enter the CNS, resulting in inflammation, demyelination, and axonal injury. We hyp...

  8. DNA Source Selection for Downstream Applications Based on DNA Quality Indicators Analysis.

    Science.gov (United States)

    Lucena-Aguilar, Gema; Sánchez-López, Ana María; Barberán-Aceituno, Cristina; Carrillo-Ávila, José Antonio; López-Guerrero, José Antonio; Aguilar-Quesada, Rocío

    2016-08-01

    High-quality human DNA samples and associated information of individuals are necessary for biomedical research. Biobanks act as a support infrastructure for the scientific community by providing a large number of high-quality biological samples for specific downstream applications. For this purpose, biobank methods for sample preparation must ensure the usefulness and long-term functionality of the products obtained. Quality indicators are the tool to measure these parameters, the purity and integrity determination being those specifically used for DNA. This study analyzes the quality indicators in DNA samples derived from 118 frozen human tissues in optimal cutting temperature (OCT) reactive, 68 formalin-fixed paraffin-embedded (FFPE) tissues, 119 frozen blood samples, and 26 saliva samples. The results obtained for DNA quality are discussed in association with the usefulness for downstream applications and availability of the DNA source in the target study. In brief, if any material is valid, blood is the most approachable option of prospective collection of samples providing high-quality DNA. However, if diseased tissue is a requisite or samples are available, the recommended source of DNA would be frozen tissue. These conclusions will determine the best source of DNA, according to the planned downstream application. Furthermore our results support the conclusion that a complete procedure of DNA quantification and qualification is necessary to guarantee the appropriate management of the samples, avoiding low confidence results, high costs, and a waste of samples. PMID:27158753

  9. Critical effects of downstream boundary conditions on vortex breakdown

    Science.gov (United States)

    Kandil, Osama; Kandil, Hamdy A.; Liu, C. H.

    1992-01-01

    The unsteady, compressible, full Navier-Stokes (NS) equations are used to study the critical effects of the downstream boundary conditions on the supersonic vortex breakdown. The present study is applied to two supersonic vortex breakdown cases. In the first case, quasi-axisymmetric supersonic swirling flow is considered in a configured circular duct, and in the second case, quasi-axisymmetric supersonic swirling jet, that is issued from a nozzle into a supersonic jet of lower Mach number, is considered. For the configured duct flow, four different types of downstream boundary conditions are used, and for the swirling jet flow from the nozzle, two types of downstream boundary conditions are used. The solutions are time accurate which are obtained using an implicit, upwind, flux-difference splitting, finite-volume scheme.

  10. Weight Loss Upregulates the Small GTPase DIRAS3 in Human White Adipose Progenitor Cells, Which Negatively Regulates Adipogenesis and Activates Autophagy via Akt-mTOR Inhibition.

    Science.gov (United States)

    Ejaz, Asim; Mitterberger, Maria C; Lu, Zhen; Mattesich, Monika; Zwierzina, Marit E; Hörl, Susanne; Kaiser, Andreas; Viertler, Hans-Peter; Rostek, Ursula; Meryk, Andreas; Khalid, Sana; Pierer, Gerhard; Bast, Robert C; Zwerschke, Werner

    2016-04-01

    Long-term weight-loss (WL) interventions reduce insulin serum levels, protect from obesity, and postpone age-associated diseases. The impact of long-term WL on adipose-derived stromal/progenitor cells (ASCs) is unknown. We identified DIRAS3 and IGF-1 as long-term WL target genes up-regulated in ASCs in subcutaneous white adipose tissue of formerly obese donors (WLDs). We show that DIRAS3 negatively regulates Akt, mTOR and ERK1/2 signaling in ASCs undergoing adipogenesis and acts as a negative regulator of this pathway and an activator of autophagy. Studying the IGF-1-DIRAS3 interaction in ASCs of WLDs, we demonstrate that IGF-1, although strongly up-regulated in these cells, hardly activates Akt, while ERK1/2 and S6K1 phosphorylation is activated by IGF-1. Overexpression of DIRAS3 in WLD ASCs completely inhibits Akt phosphorylation also in the presence of IGF-1. Phosphorylation of ERK1/2 and S6K1 is lesser reduced under these conditions. In conclusion, our key findings are that DIRAS3 down-regulates Akt-mTOR signaling in ASCs of WLDs. Moreover, DIRAS3 inhibits adipogenesis and activates autophagy in these cells. PMID:27211557

  11. Association study of polymorphisms in FOXO3, AKT1 and IGF-2R genes with human longevity in a Han Chinese population.

    Science.gov (United States)

    Li, Ning; Luo, Huaichao; Liu, Xiaoqi; Ma, Shi; Lin, He; Chen, Rong; Hao, Fang; Zhang, Dingding

    2016-01-01

    FOXO3, AKT1 and IGF-2R are critical members of the insulin/IGF-1 signaling pathway. Previous studies showed that polymorphisms (SNPs) in FOXO3, AKT1 and IGF-2R were associated with human longevity in Caucasian population. However, the association of these SNPs in different ethnic groups is often inconsistent. Here, we investigated the association of genetic variants in three genes with human longevity in Han Chinese population. Twelve SNPs from FOXO3, AKT1 and IGF-2R were selected and genotyped in 1202 long-lived individuals (nonagenarians and centenarians) and younger individuals. Rs9486902 of FOXO3 was found to be associated with human longevity in both genders combined in this study (allelic P = 0.002, corrected P = 0.024). The other eleven SNPs were not significantly associated with human longevity in Han Chinese population. The haplotypes TTCTT, CCTTC and CTCCT of FOXO3 as well as GGTCGG and GGTCAG of AKT1 were shown to have a significant difference between case and control (P =0.006, 2.78×10-5, 4.68×10-6, 0.003,0.005, respectively). The estimated prevalence of diabetes and prediabetes in long-lived individuals was significantly lower than in common adult populations (P = 0.001, 2.3×10-26) .Therefore, the search for longevity-associated genes provides the identification of new potential targets beneficial for the treatment of diabetes. PMID:26683100

  12. Increased Phosphorylation of PI3K/Akt/mTOR in the Obstructed Kidney of Rats with Unilateral Ureteral Obstruction

    OpenAIRE

    Ma, Seong Kwon; Joo, Soo Yeon; Kim, Chang Seong; Choi, Joon Seok; Bae, Eun Hui; Lee, JongUn; Kim, Soo Wan

    2013-01-01

    The present study aimed to investigate changes in the mammalian target of rapamycin (mTOR) signaling pathway in the obstructed kidney of rats with unilateral ureteral obstruction (UUO). Male Sprague-Dawley rats were unilaterally obstructed by ligation of the left proximal ureter for 7 days. Control rats were treated in the same way except that no ligature was made. The expression levels of phosphorylated phosphatidylinositol 3-kinase (PI3K), Akt, and mTOR were determined in the kidney by semi...

  13. Akt2- and ETS1-Dependent IP3 Receptor 2 Expression in Dendritic Cell Migration

    Directory of Open Access Journals (Sweden)

    Wenting Yang

    2014-01-01

    Full Text Available Background/Aims: The protein kinase Akt2/PKBβ is a known regulator of macrophage and dendritic cell (DC migration. The mechanisms linking Akt2 activity to migration remained, however, elusive. DC migration is governed by Ca2+ signaling. We thus explored whether Akt2 regulates DC Ca2+ signaling. Methods: DCs were derived from bone marrow of Akt2-deficient mice (akt2-/- and their wild type littermates (akt2+/+. DC maturation was induced by lipopolysaccharides (LPS and evaluated by flow cytometry. Cytosolic Ca2+ concentration was determined by Fura-2 fluorescence, channel activity by whole cell recording, transcript levels by RT-PCR, migration utilizing transwells. Results: Upon maturation, chemokine CCL21 stimulated migration of akt2+/+ but not akt2-/- DCs. CCL21-induced increase in cytosolic Ca2+ concentration, thapsigargin-induced release of Ca2+ from intracellular stores with subsequent store-operated Ca2+ entry (SOCE, ATP-induced inositol 1,4,5-trisphosphate (IP3-dependent Ca2+ release as well as Ca2+ release-activated Ca2+ (CRAC channel activity were all significantly lower in mature akt2-/- than in mature akt2+/+ DCs. Transcript levels of IP3 receptor IP3R2 and of IP3R2 regulating transcription factor ETS1 were significantly higher in akt2+/+ than in akt2-/- DCs prior to maturation and were upregulated by LPS stimulation (1h in akt2+/+ and to a lower extent in akt2-/- DCs. Following maturation, protein abundance of IP3R2 and ETS1 were similarly higher in akt2+/+ than in akt2-/- DCs. The IP3R inhibitor Xestospongin C significantly decreased CCL21-induced migration of akt2+/+DCs and abrogated the differences between genotypes. Finally, knock-down of ETS1 with siRNA decreased IP3R2 mRNA abundance, thapsigargin- and ATP-induced Ca2+ release, SOCE and CRAC channel activation, as well as DC migration. Conclusion: Akt2 upregulates DC migration at least in part by ETS1-dependent stimulation of IP3R2 transcription.

  14. Transition duct with divided upstream and downstream portions

    Science.gov (United States)

    McMahan, Kevin Weston; LeBegue, Jeffrey Scott; Maldonado, Jaime Javier; Dillard, Daniel Jackson; Flanagan, James Scott

    2015-07-14

    Turbine systems are provided. In one embodiment, a turbine system includes a transition duct comprising an inlet, an outlet, and a duct passage extending between the inlet and the outlet and defining a longitudinal axis, a radial axis, and a tangential axis. The outlet of the transition duct is offset from the inlet along the longitudinal axis and the tangential axis. The duct passage includes an upstream portion extending from the inlet and a downstream portion extending from the outlet. The turbine system further includes a rib extending from an outer surface of the duct passage, the rib dividing the upstream portion and the downstream portion.

  15. Interaction of fan rotor flow with downstream struts

    Science.gov (United States)

    Obrien, W. F., Jr.; Reimers, S. L.; Richardson, S. W.

    1983-01-01

    The detailed unsteady pressure field produced on the rotor blades of an axial-flow fan by interaction with downstream struts was investigated. The experimental arrangement was similar to that found in the fan casings of turbofan aircraft engines. Acoustically significant pressure fluctuations were measured on both thy suction and pressure sides of the rotor blades for several positions of the downstream struts. The level of the observed interaction decreased with increased spacing of the struts behind the rotor. An inviscid flow analysis for the disturbance level is compared with trends of the measured results.

  16. Elucidation of a downstream boundary of the 3' IgH regulatory region.

    Science.gov (United States)

    Manis, John P; Michaelson, Jennifer S; Birshtein, Barbara K; Alt, Frederick W

    2003-01-01

    Class switch recombination (CSR) changes the immunoglobulin heavy chain (IgH) constant region gene (C(H)) in B cells from IgM to IgG, IgA, or IgE, without modifying the variable region gene segment. This process requires transcription through switch (S) regions located upstream of the C(H) genes targeted for CSR, a process that relies on the activity of an uncharacterized regulatory region at the 3' end of the C(H) locus (3' IgH RR) that has been implicated via the effects of pgk-neo cassettes inserted into the locus. The 30kb region just downstream of the most 3' C(H) gene (Ca) contains four known enhancer elements including HS3a, HS1,2, HS3b, and HS4. Replacement of either of the proximal two enhancer elements (HS3a or HS1,2) with a pgk-neo gene cassette disrupted germline transcription of and CSR to most C(H) genes. However, replacement of either of the enhancers with a loxP sequence had no effect on CSR indicating that these elements are not critical for CSR. Insertion of a pgk-neo cassette at various sites within the C(H) locus inhibited CSR to upstream, but not downstream C(H) genes, supporting the notion that the pgk-neo cassette insertion into the locus short-circuits the ability of the 3' RR to facilitate CSR of dependent C(H) genes upstream of the insertion. These analyses also indicated that the key elements of the 3' IgH RR were downstream from HS1,2. In this study, we have sought to localize the 3' IgH RR by defining its 3' boundary. For this purpose, a pgk-neo gene cassette was targeted 2kb downstream of the HS4 element in ES cells that had normal ability to undergo CSR. We then employed Rag-2 deficient blastocyst complementation to generate chimeric mice that harbored B cells homozygous for this mutation. Such chimeras exhibited normal reconstitution of the splenic compartment and had normal serum immunoglobulin levels. Upon in vitro activation, transcription from the pgk-neo cassette was induced in B cells, however, CSR to all measured IgH isotypes

  17. Polyunsaturated fatty acids affect the localization and signaling of PIP3/AKT in prostate cancer cells.

    Science.gov (United States)

    Gu, Zhennan; Wu, Jiansheng; Wang, Shihua; Suburu, Janel; Chen, Haiqin; Thomas, Michael J; Shi, Lihong; Edwards, Iris J; Berquin, Isabelle M; Chen, Yong Q

    2013-09-01

    AKT is a serine-threonine protein kinase that plays important roles in cell growth, proliferation and apoptosis. It is activated after binding to phosphatidylinositol phosphates (PIPs) with phosphate groups at positions 3,4 and 3,4,5 on the inositol ring. In spite of extensive research on AKT, one aspect has been largely overlooked, namely the role of the fatty acid chains on PIPs. PIPs are phospholipids composed of a glycerol backbone with fatty acids at the sn-1 and sn-2 position and inositol at the sn-3 position. Here, we show that polyunsaturated fatty acids (PUFAs) modify phospholipid content. Docosahexaenoic acid (DHA), an ω3 PUFA, can replace the fatty acid at the sn-2 position of the glycerol backbone, thereby changing the species of phospholipids. DHA also inhibits AKT(T308) but not AKT(S473) phosphorylation, alters PI(3,4,5)P3 (PIP3) and phospho-AKT(S473) protein localization, decreases pPDPK1(S241)-AKT and AKT-BAD interaction and suppresses prostate tumor growth. Our study highlights a potential novel mechanism of cancer inhibition by ω3 PUFA through alteration of PIP3 and AKT localization and affecting the AKT signaling pathway.

  18. Akt2 Deficiency is Associated with Anxiety and Depressive Behavior in Mice

    Directory of Open Access Journals (Sweden)

    Christina Leibrock

    2013-09-01

    Full Text Available Background: The economic burden associated with major depressive disorder and anxiety disorders render both disorders the most common and debilitating psychiatric illnesses. To date, the exact cellular and molecular mechanisms underlying the pathophysiology, successful treatment and prevention of these highly associated disorders have not been identified. Akt2 is a key protein in the phosphatidylinositide-3 (PI3K / glycogen synthase 3 kinase (GSK3 signaling pathway, which in turn is involved in brain-derived neurotrophic factor (BDNF effects on fear memory, mood stabilisation and action of several antidepressant drugs. The present study thus explored the impact of Akt2 on behaviour of mice. Methods: Behavioural studies (Open-Field, Light-Dark box, O-Maze, Forced Swimming Test, Emergence Test, Object Exploration Test, Morris Water Maze, Radial Maze have been performed with Akt2 knockout mice (akt-/- and corresponding wild type mice (akt+/+. Results: Anxiety and depressive behavior was significantly higher in akt-/- than in akt+/+ mice. The akt-/- mice were cognitively unimpaired but displayed increased anxiety in several behavioral tests (O-Maze test, Light-Dark box, Open Field test. Moreover, akt-/- mice spent more time floating in the Forced Swimming test, which is a classical feature of experimental depression. Conclusion: Akt2 might be a key factor in the pathophysiology of depression and anxiety.

  19. Pragmalinguistische Analyse der illokutionären Akte im Dramendialog

    Directory of Open Access Journals (Sweden)

    Noha Desouky

    2012-07-01

    Full Text Available Die Sprechakttheorie ist ein Beitrag zur Klärung der Frage: „Was tut man, indem man handelt?“. Sagt ein Sprecher z. B.: „Morgen komme ich.“, dann äußert der Sprecher einen bestimmten Sprechakt, der sprachlich bedeutungsvoll ist. Mit dieser Äußerung kann der Sprecher verschiedene Dinge tun: Er kann dem Hörer etwas mitteilen oder ihm versprechen, morgen zu kommen, oder ihn vor seinem Besuch warnen. Alle diese vollzogenen Akte werden illokutionäre Akte genannt. Der illokutionäre Teilakt wird - im Vergleich zu anderen lokutionären und perlokutionären Teilakten der Sprechakttheorie - als ein zentrales Forschungsgebiet der Pragmalinguistik beschrieben.

  20. Regulation of Akt/FOXO3a/GSK-3β/AR Signaling Network by Isoflavone in Prostate Cancer Cells*

    OpenAIRE

    Li, Yiwei; Wang, Zhiwei; Kong, Dejuan; Li, Ran; Sarkar, Sanila H.; Sarkar, Fazlul H

    2008-01-01

    We have previously shown that genistein could inhibit Akt activation and down-regulate AR (androgen receptor) and PSA (prostate-specific antigen) expression in prostate cancer (PCa) cells. However, pure genistein showed increased lymph node metastasis in an animal model, but such an adverse effect was not seen with isoflavone, suggesting that further mechanistic studies are needed for elucidating the role of isoflavone in PCa. It is known that FOXO3a and GSK-3β, target...

  1. Forskningsnettverk for aktørorientert tjenesteinnovasjon i velferdssektoren

    OpenAIRE

    Keitsch, Martina; Nakrem, Sigrid; Kiran, Asle H.; Sigurjonsson, Johannes; Pettersen, Ida Nilstad; Solbjør, Marit; Ljunggren, Birgitte; Grønning, Kjersti

    2013-01-01

    Forskningsnettverk for aktørorientert tjenesteinnovasjon i velferdssektoren gjennomførte 17.- 18. juni 2013 en workshop på Ørlandet Kysthotell. Workshop’en er en del av forprosjektet “Sustainable services and technology for home medication – A pre-project developed by the Research network for actor-oriented service innovation in the welfare sector” som er finansiert av SAK-midler (Kunnskapsdepartementet), tildelt gjennom Midt-Norsk Nettverk, Helse og Sosialfag (MNHS) for 2013.

  2. Bimatoprost protects retinal neuronal damage via Akt pathway.

    Science.gov (United States)

    Takano, Norihito; Tsuruma, Kazuhiro; Ohno, Yuta; Shimazawa, Masamitsu; Hara, Hideaki

    2013-02-28

    Worldwide, prostaglandin analogs, such as bimatoprost, have become the major therapeutic class for medical treatment of glaucoma because of their efficacy and generally well tolerated systemic safety profile. However, the detailed mechanism of the direct action of bimatoprost on retinal ganglion cells (RGC) has rarely been understood. Thus, in this study, we elucidated the mechanism of the protective effects of bimatoprost on RGC against oxidative stress. To examine the protective effects of bimatoprost, cultured RGC with various concentrations of bimatoprost (in both free acid and amide form) were exposed to l-buthionin-(S,R)-sulfoximine (BSO) plus glutamate or serum depletion in vitro and intravitreal injection of N-methyl-D-aspartate (NMDA) was used to induce retinal damage in vivo. To elucidate the protective mechanism of bimatoprost, we used western blot analysis to investigate the phosphorylation of Akt and extracellular signal-regulated kinase (ERK). Bimatoprost significantly reduced BSO plus glutamate- and serum deprivation-induced death in concentration-dependent manners. Bimatoprost induced activation of Akt and ERK, and a phosphatidylinositol 3-kinase inhibitor, LY294002, attenuated the protective effect of bimatoprost. On the other hand, a mitogen-activated protein kinase kinase inhibitor, U0126, exhibited protective effect unexpectedly. Moreover, ERK was more phosphorylated by attenuation of Akt activity in cultured RGC. In an in vivo study, bimatoprost reduced NMDA-induced RGC death. Taken together, these findings indicate that bimatoprost has protective effects on in vitro and in vivo retinal damage, suggesting that the mechanism underlying may be via the Akt pathway, which may modulate the ERK pathway.

  3. RLIP76-dependent suppression of PI3K/AKT/Bcl-2 pathway by miR-101 induces apoptosis in prostate cancer

    Energy Technology Data Exchange (ETDEWEB)

    Yang, Jing; Song, Qi; Cai, Yi; Wang, Peng; Wang, Min; Zhang, Dong, E-mail: zhangd1117@yahoo.com

    2015-08-07

    MicroRNA-101 (miR-101) participates in carcinogenesis and tumor progression in various cancers. However, its biological functions in prostate cancer are still unclear. Here, we demonstrate that miR-101 represents a critical role in regulating cell apoptosis in prostate cancer cells. We first demonstrated that miR-101 treatment promoted apoptosis in DU145 and PC3 cells by using flow cytometric analysis and transmission electron microscopy (TEM). To verify the mechanisms, we identified a novel miR-101 target, Ral binding protein 1 (RLIP76). We found miR-101 transfection significantly suppresses RLIP76 expression, which can transactivate phosphorylation of PI3K-Akt signaling, and resulted in an amplification of Bcl2-induced apoptosis. Furthermore, we demonstrated that RLIP76 overexpression could reverse the anti-tumor effects of miR-101 in DU145 and PC3 cells by using flow cytometry assay and MTT assay. Taken together, our results revealed that the effect of miR-101 on prostate cancer cell apoptosis was due to RLIP76 regulation of the PI3K/Akt/Bcl-2 signaling pathway. - Highlights: • miR-101 inhibited prostate cancer cell proliferation and enhanced apoptosis. • miR-101 directly targeted and regulated RLIP76 expression. • miR-101 suppressed PI3K/Akt/Bcl-2 signaling pathway by targeting RLIP76.

  4. RLIP76-dependent suppression of PI3K/AKT/Bcl-2 pathway by miR-101 induces apoptosis in prostate cancer

    International Nuclear Information System (INIS)

    MicroRNA-101 (miR-101) participates in carcinogenesis and tumor progression in various cancers. However, its biological functions in prostate cancer are still unclear. Here, we demonstrate that miR-101 represents a critical role in regulating cell apoptosis in prostate cancer cells. We first demonstrated that miR-101 treatment promoted apoptosis in DU145 and PC3 cells by using flow cytometric analysis and transmission electron microscopy (TEM). To verify the mechanisms, we identified a novel miR-101 target, Ral binding protein 1 (RLIP76). We found miR-101 transfection significantly suppresses RLIP76 expression, which can transactivate phosphorylation of PI3K-Akt signaling, and resulted in an amplification of Bcl2-induced apoptosis. Furthermore, we demonstrated that RLIP76 overexpression could reverse the anti-tumor effects of miR-101 in DU145 and PC3 cells by using flow cytometry assay and MTT assay. Taken together, our results revealed that the effect of miR-101 on prostate cancer cell apoptosis was due to RLIP76 regulation of the PI3K/Akt/Bcl-2 signaling pathway. - Highlights: • miR-101 inhibited prostate cancer cell proliferation and enhanced apoptosis. • miR-101 directly targeted and regulated RLIP76 expression. • miR-101 suppressed PI3K/Akt/Bcl-2 signaling pathway by targeting RLIP76

  5. Downstream processing of Isochrysis galbana: a step towards microalgal biorefinery

    NARCIS (Netherlands)

    Gilbert-López, B.; Mendiola, J.A.; Fontecha, J.; Broek, van den L.A.M.; Sijtsma, L.; Cifuentes, A.; Herrero, M.; Ibáñez, E.

    2015-01-01

    An algae-based biorefinery relies on the efficient use of algae biomass through its fractionation of several valuable/bioactive compounds that can be used in industry. If this biorefinery includes green platforms as downstream processing technologies able to fulfill the requirements of green chemist

  6. Modeling downstream fining in sand-bed rivers. II: Application

    Science.gov (United States)

    Wright, S.; Parker, G.

    2005-01-01

    In this paper the model presented in the companion paper, Wright and Parker (2005) is applied to a generic river reach typical of a large, sand-bed river flowing into the ocean in order to investigate the mechanisms controlling longitudinal profile development and downstream fining. Three mechanisms which drive downstream fining are studied: a delta prograding into standing water, sea-level rise, and tectonic subsidence. Various rates of sea-level rise (typical of the late Holocene) and tectonic subsidence are modeled in order to quantify their effects on the degree of profile concavity and downstream fining. Also, several other physical mechanisms which may affect fining are studied, including the relative importance of the suspended versus bed load, the effect of the loss of sediment overbank, and the influence of the delta bottom slope. Finally, sensitivity analysis is used to show that the grain-size distribution at the interface between the active layer and substrate has a significant effect on downstream fining. ?? 2005 International Association of Hydraulic Engineering and Research.

  7. Patents and Downstream Innovation Suppression - Facts or Fiction?

    DEFF Research Database (Denmark)

    Howells, John

    Merges and Nelson have proposed that pioneer patents have enabled their owners to 'block' or 'hold-up' downstream innovation in cases as important as the car, radio, aircraft and electric lighting (Merges and Nelson 1990, ; Merges and Nelson 1994). Merges and Nelson use their work to question...

  8. Peru's Downstream Natural Gas Sector : A Preliminary Assessment

    OpenAIRE

    Reinstein, David; Benítez, Daniel A. Benítez; Todd M. Johnson

    2011-01-01

    This study assesses the natural gas market in Peru. In the process of evaluating the downstream market, the study identifies opportunities for meeting the Government s aspirational goals with respect to energy and natural gas development, including the efficient use of natural gas in the power and other sectors, strengthening and coordinating national energy planning for the gas sector, in...

  9. Simulating potential structural and operational changes for Detroit Dam on the North Santiam River, Oregon, for downstream temperature management

    Science.gov (United States)

    Buccola, Norman L.; Rounds, Stewart A.; Sullivan, Annett B.; Risley, John C.

    2012-01-01

    structural options were explored with the model scenarios. Multiple downstream temperature targets were used along with three sets of environmental forcing conditions representing cool/wet, normal, and hot/dry conditions. Five structural options at Detroit Dam were modeled, including the use of existing outlets, one hypothetical variable-elevation outlet such as a sliding gate, a hypothetical combination of a floating outlet and a fixed-elevation outlet, and a hypothetical combination of a floating outlet and a sliding gate. Finally, 14 sets of operational guidelines for Detroit Dam were explored to gain an understanding of the effects of imposing different downstream minimum streamflows, imposing minimum outflow rules to specific outlets, and managing the level of the lake with different timelines through the year. Selected subsets of these combinations of operational and structural scenarios were run through the downstream models of Big Cliff Reservoir and the North Santiam and Santiam Rivers to explore how hypothetical changes at Detroit Dam might provide improved temperatures for endangered salmonids downstream of the Detroit-Big Cliff Dam complex. Conclusions that can be drawn from these model scenarios include: *The water-temperature targets set by the U.S. Army Corps of Engineers for releases from Detroit Dam can be met through a combination of new dam outlets or a delayed drawdown of the lake in autumn. *Spring and summer dam operations greatly affect the available release temperatures and operational flexibility later in the autumn. Releasing warm water during midsummer tends to keep more cool water available for release in autumn. *The ability to meet downstream temperature targets during spring depends on the characteristics of the available outlets. Under existing conditions, although warm water sometimes is present at the lake surface in spring and early summer, such water may not be available for release if the lake level is either well below or well above the

  10. Methylation of Hg downstream from the Bonanza Hg mine, Oregon

    Science.gov (United States)

    Gray, John E.; Hines, Mark E.; Krabbenhoft, David P.; Thoms, Bryn

    2012-01-01

    Speciation of Hg and conversion to methyl-Hg were evaluated in stream sediment, stream water, and aquatic snails collected downstream from the Bonanza Hg mine, Oregon. Total production from the Bonanza mine was >1360t of Hg, during mining from the late 1800s to 1960, ranking it as an intermediate sized Hg mine on an international scale. The primary objective of this study was to evaluate the distribution, transport, and methylation of Hg downstream from a Hg mine in a coastal temperate climatic zone. Data shown here for m