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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. Akt Regulates TNFα synthesis downstream of RIP1 kinase activation during necroptosis.

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    Colleen R McNamara

    Full Text Available Necroptosis is a regulated form of necrotic cell death that has been implicated in the pathogenesis of various diseases including intestinal inflammation and systemic inflammatory response syndrome (SIRS. In this work, we investigated the signaling mechanisms controlled by the necroptosis mediator receptor interacting protein-1 (RIP1 kinase. We show that Akt kinase activity is critical for necroptosis in L929 cells and plays a key role in TNFα production. During necroptosis, Akt is activated in a RIP1 dependent fashion through its phosphorylation on Thr308. In L929 cells, this activation requires independent signaling inputs from both growth factors and RIP1. Akt controls necroptosis through downstream targeting of mammalian Target of Rapamycin complex 1 (mTORC1. Akt activity, mediated in part through mTORC1, links RIP1 to JNK activation and autocrine production of TNFα. In other cell types, such as mouse lung fibroblasts and macrophages, Akt exhibited control over necroptosis-associated TNFα production without contributing to cell death. Overall, our results provide new insights into the mechanism of necroptosis and the role of Akt kinase in both cell death and inflammatory regulation.

  3. Recent Development of Anticancer Therapeutics Targeting Akt

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    Morrow, John K.; Du-Cuny, Lei; Chen, Lu; Meuillet, Emmanuelle J.; Eugene A Mash; Powis, Garth; Zhang, Shuxing

    2011-01-01

    The serine/threonine kinase Akt has proven to be a significant signaling target, involved in various biological functions. Because of its cardinal role in numerous cellular responses, Akt has been implicated in many human diseases, particularly cancer. It has been established that Akt is a viable and feasible target for anticancer therapeutics. Analysis of all Akt kinases reveals conserved homology for an N-terminal regulatory domain, which contains a pleckstrin-homology (PH) domain for cellu...

  4. Recent development of anticancer therapeutics targeting Akt.

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    Morrow, John K; Du-Cuny, Lei; Chen, Lu; Meuillet, Emmanuelle J; Mash, Eugene A; Powis, Garth; Zhang, Shuxing

    2011-01-01

    The serine/threonine kinase Akt has proven to be a significant signaling target, involved in various biological functions. Because of its cardinal role in numerous cellular responses, Akt has been implicated in many human diseases, particularly cancer. It has been established that Akt is a viable and feasible target for anticancer therapeutics. Analysis of all Akt kinases reveals conserved homology for an N-terminal regulatory domain, which contains a pleckstrin-homology (PH) domain for cellular translocation, a kinase domain with serine/threonine specificity, and a C-terminal extension domain. These well defined regions have been targeted, and various approaches, including in silico methods, have been implemented to develop Akt inhibitors. In spite of unique techniques and a prolific body of knowledge surrounding Akt, no targeted Akt therapeutics have reached the market yet. Here we will highlight successes and challenges to date on the development of anticancer agents modulating the Akt pathway in recent patents as well as discuss the methods employed for this task. Special attention will be given to patents with focus on those discoveries using computer-aided drug design approaches.

  5. 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.

  6. 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....

  7. AS101 prevents diabetic nephropathy progression and mesangial cell dysfunction: regulation of the AKT downstream pathway.

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    Itay Israel Shemesh

    Full Text Available Diabetic nephropathy (DN is characterized by proliferation of mesangial cells, mesangial expansion, hypertrophy and extracellular matrix accumulation. Previous data have cross-linked PKB (AKT to TGFβ induced matrix modulation. The non-toxic compound AS101 has been previously shown to favorably affect renal pathology in various animal models and inhibits AKT activity in leukemic cells. Here, we studied the pharmacological properties of AS101 against the progression of rat DN and high glucose-induced mesangial dysfunction. In-vivo administration of AS101 to Streptozotocin injected rats didn't decreased blood glucose levels but ameliorated kidney hypotrophy, proteinuria and albuminuria and downregulated cortical kidney phosphorylation of AKT, GSK3β and SMAD3. AS101 treatment of primary rat glomerular mesangial cells treated with high glucose significantly reduced their elevated proliferative ability, as assessed by XTT assay and cell cycle analysis. This reduction was associated with decreased levels of p-AKT, increased levels of PTEN and decreased p-GSK3β and p-FoxO3a expression. Pharmacological inhibition of PI3K, mTORC1 and SMAD3 decreased HG-induced collagen accumulation, while inhibition of GSK3β did not affect its elevated levels. AS101 also prevented HG-induced cell growth correlated to mTOR and (rpS6 de-phosphorylation. Thus, pharmacological inhibition of the AKT downstream pathway by AS101 has clinical potential in alleviating the progression of diabetic nephropathy.

  8. 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

  9. 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.

  10. 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.

  11. 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.

  12. Activation of RAF/MEK/ERK and PI3K/AKT/mTOR pathways in pituitary adenomas and their effects on downstream effectors.

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    Dworakowska, D; Wlodek, E; Leontiou, C A; Igreja, S; Cakir, M; Teng, M; Prodromou, N; Góth, M I; Grozinsky-Glasberg, S; Gueorguiev, M; Kola, B; Korbonits, M; Grossman, A B

    2009-12-01

    Raf/MEK/ERK and phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) cascades are key signalling pathways interacting with each other to regulate cell growth and tumourigenesis. We have previously shown B-Raf and Akt overexpression and/or overactivation in pituitary adenomas. The aim of this study is to assess the expression of their downstream components (MEK1/2, ERK1/2, mTOR, TSC2, p70S6K) and effectors (c-MYC and CYCLIN D1). We studied tissue from 16 non-functioning pituitary adenomas (NFPAs), six GH-omas, six prolactinomas and six ACTH-omas, all collected at transsphenoidal surgery; 16 normal autopsy pituitaries were used as controls. The expression of phospho and total protein was assessed with western immunoblotting, and the mRNA expression with quantitative RT-PCR. The expression of pSer217/221 MEK1/2 and pThr183 ERK1/2 (but not total MEK1/2 or ERK1/2) was significantly higher in all tumour subtypes in comparison to normal pituitaries. There was no difference in the expression of phosphorylated/total mTOR, TSC2 or p70S6K between pituitary adenomas and controls. Neither c-MYC phosphorylation at Ser 62 nor total c-MYC was changed in the tumours. However, c-MYC phosphorylation at Thr58/Ser62 (a response target for Akt) was decreased in all tumour types. CYCLIN D1 expression was higher only in NFPAs. The mRNA expression of MEK1, MEK2, ERK1, ERK2, c-MYC and CCND1 was similar in all groups. Our data indicate that in pituitary adenomas both the Raf/MEK/ERK and PI3K/Akt/mTOR pathways are upregulated in their initial cascade, implicating a pro-proliferative signal derangement upstream to their point of convergence. However, we speculate that other processes, such as senescence, attenuate the changes downstream in these benign tumours.

  13. In vitro and in vivo activity of novel small-molecule inhibitors targeting the pleckstrin homology domain of protein kinase B/AKT.

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    Moses, Sylvestor A; Ali, M Ahad; Zuohe, Song; Du-Cuny, Lei; Zhou, Li Li; Lemos, Robert; Ihle, Nathan; Skillman, A Geoffrey; Zhang, Shuxing; Mash, Eugene A; Powis, Garth; Meuillet, Emmanuelle J

    2009-06-15

    The phosphatidylinositol 3-kinase/AKT signaling pathway plays a critical role in activating survival and antiapoptotic pathways within cancer cells. Several studies have shown that this pathway is constitutively activated in many different cancer types. The goal of this study was to discover novel compounds that bind to the pleckstrin homology (PH) domain of AKT, thereby inhibiting AKT activation. Using proprietary docking software, 22 potential PH domain inhibitors were identified. Surface plasmon resonance spectroscopy was used to measure the binding of the compounds to the expressed PH domain of AKT followed by an in vitro activity screen in Panc-1 and MiaPaCa-2 pancreatic cancer cell lines. We identified a novel chemical scaffold in several of the compounds that binds selectively to the PH domain of AKT, inducing a decrease in AKT activation and causing apoptosis at low micromolar concentrations. Structural modifications of the scaffold led to compounds with enhanced inhibitory activity in cells. One compound, 4-dodecyl-N-(1,3,4-thiadiazol-2-yl)benzenesulfonamide, inhibited AKT and its downstream targets in cells as well as in pancreatic cancer cell xenografts in immunocompromised mice; it also exhibited good antitumor activity. In summary, a pharmacophore for PH domain inhibitors targeting AKT function was developed. Computer-aided modeling, synthesis, and testing produced novel AKT PH domain inhibitors that exhibit promising preclinical properties.

  14. 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.

  15. 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

  16. Targeting the PI3K/Akt pathway in prostate cancer: challenges and opportunities (review).

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    Toren, Paul; Zoubeidi, Amina

    2014-11-01

    The PI3K/Akt pathway is an actively pursued therapeutic target in oncology. In prostate cancer, the activation of this pathway appears to be characteristic of many aggressive prostate cancers. Further, activation of the PI3K/Akt pathway is more frequently observed as prostate cancer progresses toward a resistant, metastatic disease. Signalling from this pathway activates numerous survival, growth, metabolic and metastatic functions characteristic of aggressive cancer. Biomarkers of this pathway have correlated activation of this pathway to high grade disease and higher risk of disease progression. Therefore there is significant interest in developing effective strategies to target this pathway in prostate cancer. In this review, we discuss the pre-clinical and clinical data relevant to targeting of the PI3K/Akt pathway in prostate cancer. In particular, we review the rationale and relevance of co-targeting approaches against the PI3K/Akt pathway. It is anticipated that through an improved understanding of the biology of the PI3K/Akt pathway in prostate cancer, relevant biomarkers and rationale combination therapies will optimize targeting of this pathway to improve outcomes among patients with aggressive prostate cancer.

  17. 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.

  18. 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.

  19. Skeletal muscle insulin signaling defects downstream of phosphatidylinositol 3-kinase at the level of akt are associated with impaired nonoxidative glucose disposal in HIV lipodystrophy

    DEFF Research Database (Denmark)

    Haugaard, Steen B.; Andersen, Ove; Madsbad, Sten

    2005-01-01

    More than 40% of HIV-infected patients on highly active antiretroviral therapy (HAART) experience fat redistribution (lipodystrophy), a syndrome associated with insulin resistance primarily affecting insulin-stimulated nonoxidative glucose metabolism (NOGM(ins)). Skeletal muscle biopsies, obtaine...... defects were downstream of PI 3-kinase at the level of Akt. These results suggest mechanisms for the insulin resistance greatly enhancing the risk of type 2 diabetes in HIV lipodystrophy....... from 18 lipodystrophic nondiabetic patients (LIPO) and 18 nondiabetic patients without lipodystrophy (NONLIPO) before and during hyperinsulinemic (40 mU.m(-2).min(-1))-euglycemic clamps, were analyzed for insulin signaling effectors. All patients were on HAART. Both LIPO and NONLIPO patients were...... normoglycemic (4.9 +/- 0.1 and 4.8 +/- 0.1 mmol/l, respectively); however, NOGM(ins) was reduced by 49% in LIPO patients (P correlated positively with insulin-stimulated glycogen synthase activity (I-form, P correlated inversely...

  20. 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.

  1. Molecular targets for cancer therapy in the PI3K/AKT/mTOR pathway.

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    Polivka, Jiri; Janku, Filip

    2014-05-01

    Aberrations in various cellular signaling pathways are instrumental in regulating cellular metabolism, tumor development, growth, proliferation, metastasis and cytoskeletal reorganization. The fundamental cellular signaling cascade involved in these processes, the phosphatidylinositol 3-kinase/protein kinase-B/mammalian target of rapamycin (PI3K/AKT/mTOR), closely related to the mitogen-activated protein kinase (MAPK) pathway, is a crucial and intensively explored intracellular signaling pathway in tumorigenesis. Various activating mutations in oncogenes together with the inactivation of tumor suppressor genes are found in diverse malignancies across almost all members of the pathway. Substantial progress in uncovering PI3K/AKT/mTOR alterations and their roles in tumorigenesis has enabled the development of novel targeted molecules with potential for developing efficacious anticancer treatment. Two approved anticancer drugs, everolimus and temsirolimus, exemplify targeted inhibition of PI3K/AKT/mTOR in the clinic and many others are in preclinical development as well as being tested in early clinical trials for many different types of cancer. This review focuses on targeted PI3K/AKT/mTOR signaling from the perspective of novel molecular targets for cancer therapy found in key pathway members and their corresponding experimental therapeutic agents. Various aberrant prognostic and predictive biomarkers are also discussed and examples are given. Novel approaches to PI3K/AKT/mTOR pathway inhibition together with a better understanding of prognostic and predictive markers have the potential to significantly improve the future care of cancer patients in the current era of personalized cancer medicine.

  2. 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

    Science.gov (United States)

    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…

  3. Inositol hexaphosphate suppresses growth and induces apoptosis in prostate carcinoma cells in culture and nude mouse xenograft: PI3K-Akt pathway as potential target.

    Science.gov (United States)

    Gu, Mallikarjuna; Roy, Srirupa; Raina, Komal; Agarwal, Chapla; Agarwal, Rajesh

    2009-12-15

    Constitutive activation of phosphoinositide 3-kinase (PI3K)-Akt pathway transmits growth-regulatory signals that play a central role in promoting survival, proliferation, and angiogenesis in human prostate cancer cells. Here, we assessed the efficacy of inositol hexaphosphate (IP6) against invasive human prostate cancer PC-3 and C4-2B cells and regulation of PI3K-Akt pathway. IP6 treatment of cells suppressed proliferation, induced apoptosis along with caspase-3 and poly(ADP-ribose) polymerase (PARP) cleavage, and inhibited constitutive activation of Akt and its upstream regulators PI3K, phosphoinositide-dependent kinase-1 and integrin-linked kinase-1 (ILK1). Downstream of Akt, IP6 inhibited the phosphorylation of glycogen synthase kinase-3alpha/beta at Ser(21/9) and consequently reduced cyclin D1 expression. Efficacy studies employing PC-3 tumor xenograft growth in nude mice showed that 2% (w/v) IP6 feeding in drinking water inhibits tumor growth and weight by 52% to 59% (P IP6 significantly reduces the expression of molecules associated with cell survival/proliferation (ILK1, phosphorylated Akt, cyclin D1, and proliferating cell nuclear antigen) and angiogenesis (platelet endothelial cell adhesion molecule-1 or CD31, vascular endothelial growth factor, endothelial nitric oxide synthase, and hypoxia-inducible factor-1alpha) together with an increase in apoptotic markers (cleaved caspase-3 and PARP). These findings suggest that, by targeting the PI3K-ILK1-Akt pathway, IP6 suppresses cell survival, proliferation, and angiogenesis but induces death in prostate cancer cells, which might have translational potential in preventing and controlling the growth of advanced and aggressive prostate cancer for which conventional chemotherapy is not effective.

  4. Energy deposition in a thin copper target downstream and off-axis of a proton-radiography target

    CERN Document Server

    Greene, G A; Snead, C L; Hanson, A L; Murray, M M

    2002-01-01

    A series of proton energy-deposition experiments was conducted to measure the energy deposited in a copper target located downstream and off-axis of a high-energy proton-radiography target. The proton/target interactions involved low-intensity bunches of protons at 24 GeV/c onto a spherical target consisting of concentric shells of tungsten and copper. The energy-deposition target was placed at five locations downstream of the proton-radiography target, off-axis of the primary beam transport, and was either unshielded or shielded by 5 or 10 cm of lead. Maximum temperature rises measured in the energy-deposition target due to single bunches of 5x10 sup 1 sup 0 protons on the proton-radiography target were approximately 20 mK per bunch. The data indicated that the scattered radiation was concentrated close to the primary transport axis of the beam line. The energy deposited in the energy-deposition target was reduced by moving the target radially away from the primary transport axis. Placing lead shielding in f...

  5. 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.

  6. 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.

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

    Science.gov (United States)

    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.

  8. Proteomics analysis of cellular imatinib targets and their candidate downstream effectors.

    Science.gov (United States)

    Breitkopf, Susanne B; Oppermann, Felix S; Keri, Gyorgy; Grammel, Markus; Daub, Henrik

    2010-11-05

    Inhibition of deregulated protein kinases by small molecule drugs has evolved into a major therapeutic strategy for the treatment of human malignancies. Knowledge about direct cellular targets of kinase-selective drugs and the identification of druggable downstream mediators of oncogenic signaling are relevant for both initial therapy selection and the nomination of alternative targets in case molecular resistance emerges. To address these issues, we performed a proof-of-concept proteomics study designed to monitor drug effects on the pharmacologically tractable subproteome isolated by affinity purification with immobilized, nonselective kinase inhibitors. We applied this strategy to chronic myeloid leukemia cells that express the transforming Bcr-Abl fusion kinase. We used SILAC to measure how cellular treatment with the Bcr-Abl inhibitor imatinib affects protein binding to a generic kinase inhibitor resin and further quantified site-specific phosphorylations on resin-retained proteins. Our integrated approach indicated additional imatinib target candidates, such as flavine adenine dinucleotide synthetase, as well as repressed phosphorylation events on downstream effectors not yet implicated in imatinib-regulated signaling. These included activity-regulating phosphorylations on the kinases Btk, Fer, and focal adhesion kinase, which may qualify them as alternative target candidates in Bcr-Abl-driven oncogenesis. Our approach is rather generic and may have various applications in kinase drug discovery.

  9. 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...

  10. MMP14 as a novel downstream target of VEGFR2 in migratory glioma-tropic neural stem cells

    Directory of Open Access Journals (Sweden)

    Nikita G. Alexiades

    2015-11-01

    Full Text Available Neural stem cell (NSC-based carriers have been presented as promising therapeutic tools for the treatment of infiltrative brain tumors due to their intrinsic tumor homing property. They have demonstrated the ability to migrate towards distant tumor microsatellites and effectively deliver the therapeutic payload, thus significantly improving survival in experimental animal models for brain tumor. Despite such optimistic results, the efficacy of NSC-based anti-cancer therapy has been limited due to the restricted tumor homing ability of NSCs. To examine this issue, we investigated the mechanisms of tumor-tropic migration of an FDA-approved NSC line, HB1.F3.CD, by performing a gene expression analysis. We identified vascular endothelial growth factor-A (VEGFA and membrane-bound matrix metalloproteinase (MMP14 as molecules whose expression are significantly elevated in migratory NSCs. We observed increased expression of VEGF receptor 2 (VEGFR2 in the focal adhesion complexes of migratory NSCs, with downstream activation of VEGFR2-dependent kinases such as p-PLCγ, p-FAK, and p-Akt, a signaling cascade reported to be required for cellular migration. In an in vivo orthotopic glioma xenograft model, analysis of the migratory trail showed that NSCs maintained expression of VEGFR2 and preferentially migrated within the perivascular space. Knockdown of VEGFR2 via shRNAs led to significant downregulation of MMP14 expression, which resulted in inhibited tumor-tropic migration. Overall, our results suggest, the involvement of VEGFR2-regulated MMP14 in the tumor-tropic migratory behavior of NSCs. Our data warrant investigation of MMP14 as a target for enhancing the migratory properties of NSC carriers and optimizing the delivery of therapeutic payloads to disseminated tumor burdens.

  11. 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.

  12. TRAF5 is a downstream target of MAVS in antiviral innate immune signaling.

    Directory of Open Access Journals (Sweden)

    Eric D Tang

    Full Text Available The recognition of nucleic acids by the innate immune system during viral infection results in the production of type I interferons and the activation of antiviral immune responses. The RNA helicases RIG-I and MDA-5 recognize distinct types of cytosolic RNA species and signal through the mitochondrial protein MAVS to stimulate the phosphorylation and activation of the transcription factors IRF3 and IRF7, thereby inducing type I interferon expression. Alternatively, the activation of NF-kappaB leads to proinflammatory cytokine production. The function of MAVS is dependent on both its C-terminal transmembrane (TM domain and N-terminal caspase recruitment domain (CARD. The TM domain mediates MAVS dimerization in response to viral RNA, allowing the CARD to bind to and activate the downstream effector TRAF3. Notably, dimerization of the MAVS CARD alone is sufficient to activate IRF3, IRF7, and NF-kappaB. However, TRAF3-deficient cells display only a partial reduction in interferon production in response to RNA virus infection and are not defective in NF-kappaB activation. Here we find that the related ubiquitin ligase TRAF5 is a downstream target of MAVS that mediates both IRF3 and NF-kappaB activation. The TM domain of MAVS allows it to dimerize and thereby associate with TRAF5 and induce its ubiquitination in a CARD-dependent manner. Also, NEMO is recruited to the dimerized MAVS CARD domain in a TRAF3 and TRAF5-dependent manner. Thus, our findings reveal a possible function for TRAF5 in mediating the activation of IRF3 and NF-kappaB downstream of MAVS through the recruitment of NEMO. TRAF5 may be a key molecule in the innate response against viral infection.

  13. Identifying master regulators of cancer and their downstream targets by integrating genomic and epigenomic features.

    Science.gov (United States)

    Gevaert, Olivier; Plevritis, Sylvia

    2013-01-01

    Vast amounts of molecular data characterizing the genome, epigenome and transcriptome are becoming available for a variety of cancers. The current challenge is to integrate these diverse layers of molecular biology information to create a more comprehensive view of key biological processes underlying cancer. We developed a biocomputational algorithm that integrates copy number, DNA methylation, and gene expression data to study master regulators of cancer and identify their targets. Our algorithm starts by generating a list of candidate driver genes based on the rationale that genes that are driven by multiple genomic events in a subset of samples are unlikely to be randomly deregulated. We then select the master regulators from the candidate driver and identify their targets by inferring the underlying regulatory network of gene expression. We applied our biocomputational algorithm to identify master regulators and their targets in glioblastoma multiforme (GBM) and serous ovarian cancer. Our results suggest that the expression of candidate drivers is more likely to be influenced by copy number variations than DNA methylation. Next, we selected the master regulators and identified their downstream targets using module networks analysis. As a proof-of-concept, we show that the GBM and ovarian cancer module networks recapitulate known processes in these cancers. In addition, we identify master regulators that have not been previously reported and suggest their likely role. In summary, focusing on genes whose expression can be explained by their genomic and epigenomic aberrations is a promising strategy to identify master regulators of cancer.

  14. Activation of phosphatidylinositol 3-kinase/Akt-mammalian target of Rapamycin signaling pathway in the hippocampus is essential for the acquisition of morphine-induced place preference in rats.

    Science.gov (United States)

    Cui, Yue; Zhang, X Q; Cui, Y; Xin, W J; Jing, J; Liu, X G

    2010-11-24

    Hippocampus is a critical structure for the acquisition of morphine-induced conditioned place preference (CPP), which is a usual learning paradigm for assessing drug reward. However, the precise mechanisms remain largely unknown. Phosphatidylinositol 3-kinase (PI3K) and its downstream targets, including Akt, mammalian target of Rapamycin (mTOR) and 70-kDa ribosomal S6 kinase (p70S6K), are critical molecules implicated in learning and memory. Here, we tested the role of PI3K/Akt-mTOR-p70S6K signaling pathway in morphine-induced CPP in the hippocampus. Our results showed that the acquisition of morphine CPP increased phosphorylation of Akt in the hippocampal CA3, but not in the nucleus accumbens (NAc), the ventral tegmental area (VTA) or the CA1. Moreover, the phosphorylated Akt exclusively expressed in the CA3 neurons. Likewise, levels of phosphorylated mTOR and p70S6K were significantly enhanced in the CA3 following morphine CPP. The alterations of these phosphorylated proteins are positively correlated with the acquisition of morphine CPP. More importantly, microinjection of PI3K inhibitor (LY294002) or mTOR inhibitor (Rapamycin) into the CA3 prevented the acquisition of CPP and inhibited the activation of PI3K-Akt signaling pathway. In addition, pre-infusion of β-FNA (β-funaltrexamine hydrochloride), a selective irreversible μ opioid receptor antagonist, into CA3 significantly prevented the acquisition of CPP and impaired Akt phosphorylation. All these results strongly implied that the PI3K-Akt signaling pathway activated by μ opioid receptor in hippocampal CA3 plays an important role in acquisition of morphine-induced CPP.

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

    Directory of Open Access Journals (Sweden)

    Catríona M. Dowling

    2015-07-01

    Full Text Available 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.

  16. 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.

  17. 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.

  18. Downstream targets of GATA3 in the vestibular sensory organs of the inner ear.

    Science.gov (United States)

    Alvarado, David M; Veile, Rose; Speck, Judith; Warchol, Mark; Lovett, Michael

    2009-12-01

    Haploinsufficiency for the transcription factor GATA3 leads to hearing loss in humans. It is expressed throughout the auditory sensory epithelium (SE). In the vestibular organs, GATA3 is limited to the striola reversal zone of the utricle. Stereocilia orientation shifts 180 degrees at this region, which contains morphologically distinct type-I hair cells. The striola is conserved in all amniotes, its function is unknown, and GATA3 is the only known marker of the reversal zone. To identify downstream targets of GATA3 that might point to striolar function, we measured gene expression differences between striolar and extra-striolar SE. These were compared with profiles after GATA3 RNAi and GATA3 over-expression. We identified four genes (BMP2, FKHL18, LMO4, and MBNL2) that consistently varied with GATA3. Two of these (LMO4 and MBNL2) were shown to be direct targets of GATA3 by ChIP. Our results suggest that GATA3 impacts WNT signaling in this region of the sensory macula.

  19. Target regulation of PI3K/Akt/mTOR pathway by cannabidiol in treatment of experimental multiple sclerosis.

    Science.gov (United States)

    Giacoppo, Sabrina; Pollastro, Federica; Grassi, Gianpaolo; Bramanti, Placido; Mazzon, Emanuela

    2017-01-01

    This study was aimed to investigate whether treatment with purified cannabidiol (CBD) may counteract the development of experimental multiple sclerosis (MS), by targeting the PI3K/Akt/mTOR pathway. Although the PI3K/Akt/mTOR pathway was found to be activated by cannabinoids in several immune and non-immune cells, currently, there is no data about the effects of CBD in the PI3K/Akt/mTOR activity in MS. Experimental Autoimmune Encephalomyelitis (EAE), the most common model of MS, was induced in C57BL/6 mice by immunization with myelin oligodendroglial glycoprotein peptide (MOG)35-55. After EAE onset, which occurs approximately 14days after disease induction, mice were daily intraperitoneally treated with CBD (10mg/kg mouse) and observed for clinical signs of EAE. At 28days from EAE-induction, mice were euthanized and spinal cord tissues were sampled to perform immunohistochemical evaluations and western blot analysis. Our results showed a clear downregulation of the PI3K/Akt/mTOR pathway following EAE induction. CBD treatment was able to restore it, increasing significantly the phosphorylation of PI3K, Akt and mTOR. Also, an increased level of BNDF in CBD-treated mice seems to be involved in the activation of PI3K/Akt/mTOR pathway. In addition, our data demonstrated that therapeutic efficacy of CBD treatment is due to reduction of pro-inflammatory cytokines, like IFN-γ and IL-17 together with an up-regulation of PPARγ. Finally, CBD was found to promote neuronal survival by inhibiting JNK and p38 MAP kinases. These results provide an interesting discovery about the regulation of the PI3K/Akt/mTOR pathway by cannabidiol administration, that could be a new potential therapeutic target for MS management.

  20. Synthesis and evaluation of indazole based analog sensitive Akt inhibitors.

    Science.gov (United States)

    Okuzumi, Tatsuya; Ducker, Gregory S; Zhang, Chao; Aizenstein, Brian; Hoffman, Randy; Shokat, Kevan M

    2010-08-01

    The kinase Akt is a key signaling node in regulating cellular growth and survival. It is implicated in cancer by mutation and its role in the downstream transmission of aberrant PI3K signaling. For these reasons, Akt has become an increasingly important target of drug development efforts and several inhibitors are now reaching clinical trials. Paradoxically it has been observed that active site kinase inhibitors of Akt lead to hyperphosphorylation of Akt itself. To investigate this phenomenon we here describe the application of a chemical genetics strategy that replaces native Akt with a mutant version containing an active site substitution that allows for the binding of an engineered inhibitor. This analog sensitive strategy allows for the selective inhibition of a single kinase. In order to create the inhibitor selective for the analog sensitive kinase, a diversity of synthetic approaches was required, finally resulting in the compound PrINZ, a 7-substituted version of the Abbott Labs Akt inhibitor A-443654.

  1. Cereblon and its downstream substrates as molecular targets of immunomodulatory drugs.

    Science.gov (United States)

    Ito, Takumi; Handa, Hiroshi

    2016-09-01

    Thalidomide was first developed as a sedative around 60 years ago, but exhibited teratogenicity, leading to serious defects such as limb deformities. Nevertheless, thalidomide is now recognized as a therapeutic drug for the treatment of Hansen's disease and myeloma. Immunomodulatory drugs (IMiDs), a new class of anti-cancer drug derived from thalidomide, have also been developed and exert potent anti-cancer effects. Although the molecular mechanism of thalidomide and IMiDs remained unclear for a long time, cereblon, a substrate receptor of the CRL4 E3 ubiquitin ligase was identified as a primary direct target by a new affinity technique. A growing body of evidence suggests that the effect of IMiDs on myeloma and other cancer cells is mediated by CRBN. Each IMiD binds to CRBN and alters the substrate specificity of the CRBN E3 ubiquitin ligase complex, resulting in breakdown of intrinsic downstream proteins such as Ikaros and Aiolos. Here we give an overview of the current understanding of mechanism of action of IMiDs via CRBN and prospects for the development of new drugs that degrade protein of interest.

  2. 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.

  3. Mechanisms contributing to differential regulation of PAX3 downstream target genes in normal human epidermal melanocytes versus melanoma cells.

    Science.gov (United States)

    Bartlett, Danielle; Boyle, Glen M; Ziman, Mel; Medic, Sandra

    2015-01-01

    Melanoma is a highly aggressive and drug resistant form of skin cancer. It arises from melanocytes, the pigment producing cells of the skin. The formation of these melanocytes is driven by the transcription factor PAX3 early during embryonic development. As a result of alternative splicing, the PAX3 gene gives rise to eight different transcripts which encode isoforms that have different structures and activate different downstream target genes involved in pathways of cell proliferation, migration, differentiation and survival. Furthermore, post-translational modifications have also been shown to alter the functions of PAX3. We previously identified PAX3 downstream target genes in melanocytes and melanoma cells. Here we assessed the effects of PAX3 down-regulation on this panel of target genes in primary melanocytes versus melanoma cells. We show that PAX3 differentially regulates various downstream target genes involved in cell proliferation in melanoma cells compared to melanocytes. To determine mechanisms behind this differential downstream target gene regulation, we performed immunoprecipitation to assess post-translational modifications of the PAX3 protein as well as RNAseq to determine PAX3 transcript expression profiles in melanocytes compared to melanoma cells. Although PAX3 was found to be post-translationally modified, there was no qualitative difference in phosphorylation and ubiquitination between melanocytes and melanoma cells, while acetylation of PAX3 was reduced in melanoma cells. Additionally, there were differences in PAX3 transcript expression profiles between melanocytes and melanoma cells. In particular the PAX3E transcript, responsible for reducing melanocyte proliferation and increasing apoptosis, was found to be down-regulated in melanoma cells compared to melanocytes. These results suggest that alternate transcript expression profiles activate different downstream target genes leading to the melanoma phenotype.

  4. Effect of high fat diet on pulmonary expression of parathyroid hormone-related protein and its downstream targets

    Directory of Open Access Journals (Sweden)

    Learta Oruqaj

    2016-10-01

    Significance: This study established that physiological regulation of leptin plasma levels by high fat diet affects the pulmonary PTHrP expression and of PTHrP downstream targets. Modification of pulmonary expression of PTH-1 receptors by high fat diet after myocardial infarction suggests that the identified interaction may participate in the obesity paradox.

  5. Expression of hypoxia-inducible factor 1 alpha and its downstream targets in fibroepithelial tumors of the breast

    NARCIS (Netherlands)

    Kuijper, Arno; Groep, P. van der; Wall, E. van der; Diest, P.J. van

    2005-01-01

    INTRODUCTION Hypoxia-inducible factor 1 (HIF-1) alpha and its downstream targets carbonic anhydrase IX (CAIX) and vascular endothelial growth factor (VEGF) are key factors in the survival of proliferating tumor cells in a hypoxic microenvironment. We studied the expression and prognostic relevance o

  6. Targeting hyperactivation of the AKT survival pathway to overcome therapy resistance of melanoma brain metastases.

    Science.gov (United States)

    Niessner, Heike; Forschner, Andrea; Klumpp, Bernhard; Honegger, Jürgen B; Witte, Maria; Bornemann, Antje; Dummer, Reinhard; Adam, Annemarie; Bauer, Jürgen; Tabatabai, Ghazaleh; Flaherty, Keith; Sinnberg, Tobias; Beck, Daniela; Leiter, Ulrike; Mauch, Cornelia; Roesch, Alexander; Weide, Benjamin; Eigentler, Thomas; Schadendorf, Dirk; Garbe, Claus; Kulms, Dagmar; Quintanilla-Martinez, Leticia; Meier, Friedegund

    2013-02-01

    Brain metastases are the most common cause of death in patients with metastatic melanoma, and the RAF-MEK-ERK and PI3K-AKT signaling pathways are key players in melanoma progression and drug resistance. The BRAF inhibitor vemurafenib significantly improved overall survival. However, brain metastases still limit the effectiveness of this therapy. In a series of patients, we observed that treatment with vemurafenib resulted in substantial regression of extracerebral metastases, but brain metastases developed. This study aimed to identify factors that contribute to treatment resistance in brain metastases. Matched brain and extracerebral metastases from melanoma patients had identical ERK, p-ERK, and AKT immunohistochemistry staining patterns, but there was hyperactivation of AKT (p-AKT) and loss of PTEN expression in the brain metastases. Mutation analysis revealed no differences in BRAF, NRAS, or KIT mutation status in matched brain and extracerebral metastases. In contrast, AKT, p-AKT, and PTEN expression was identical in monolayer cultures derived from melanoma brain and extracerebral metastases. Furthermore, melanoma cells stimulated by astrocyte-conditioned medium showed higher AKT activation and invasiveness than melanoma cells stimulated by fibroblast-conditioned medium. Inhibition of PI3K-AKT signaling resensitized melanoma cells isolated from a vemurafenib-resistant brain metastasis to vemurafenib. Brain-derived factors appear to induce hyperactivation of the AKT survival pathway and to promote the survival and drug resistance of melanoma cells in the brain. Thus, inhibition of PI3K-AKT signaling shows potential for enhancing and/or prolonging the antitumor effect of BRAF inhibitors or other anticancer agents in melanoma brain metastases.

  7. 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.

  8. 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.

  9. 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.

  10. Suppression of Glioma-Cell Survival by Antisense and Dominant-Negative AKT2 RNA

    Institute of Scientific and Technical Information of China (English)

    Peiyu Pu; Chunsheng Kang; Jie Li; Guangxiu Wang

    2005-01-01

    OBJECTIVE Overexpression of growth factors and their receptors such as PDGF, FGF, VEGF, IGF, EGF, TGFα etc. Play a critical role in the development and progression of malignant gliomas. AKT, one of the most potent downstream signaling effectors of these growth factor receptors is usually overactivated in malignant gliomas. The present study was undertaken to investigate the effects of antisense and dominant negative AKT2 RNA on the survival of glioma cells with overexpression of AKT2.METHODS Antisense and dominant negative AKT2 constructs (AS-AKT2,DN-AKT2) were transfected into human glioblastoma cell line TJ905 with overexpression of AKT2. Using Western blotting, MTT assay, Ki67 labeling index (Ki67 LI), flow cytometry and the TUNEL method, the expression of AKT2 and GFAP, the proliferation rate and apoptosis of glioma cells transfected with AS-AKT2 or DN-AKT2 were compared to those characteristics of parental and glioma cells transfected with an empty vector.RESULTS Cell proliferation was inhibited in glioma cells transfected with ASAKT2 and DN-AKT2 RNA, while GFAP expression and apoptosis were markedly increased in those cells.CONCLUSION AKT is an important mediator in the growth signaling pathway of malignant gliomas and is a potential promising therapeutic target for malignant gliomas.

  11. 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...

  12. Downstream Regulatory Element Antagonist Modulator (DREAM), a target for anti-thrombotic agents.

    Science.gov (United States)

    Cho, Jaehyung

    2017-03-01

    Circulating platelets participate in the process of numerous diseases including thrombosis, inflammation, and cancer. Thus, it is of great importance to understand the underlying mechanisms mediating platelet activation under disease conditions. Emerging evidence indicates that despite the lack of a nucleus, platelets possess molecules that are involved in gene transcription in nucleated cells. This review will summarize downstream regulatory element antagonist modulator (DREAM), a transcriptional repressor, and highlight recent findings suggesting its novel non-transcriptional role in hemostasis and thrombosis.

  13. ABCB2 (TAP1) as the downstream target of SHH signaling enhances pancreatic ductal adenocarcinoma drug resistance.

    Science.gov (United States)

    Xu, Min; Li, Lei; Liu, Zhiqiang; Jiao, Zhijun; Xu, Ping; Kong, Xiangyu; Huang, Haojie; Zhang, Youli

    2013-06-10

    Hedgehog signaling plays critical roles in drug resistance of PDAC. We demonstrate that SHH is highly expressed in PDAC patients and cell lines. SHH signaling protects PDAC cells against gemcitabine induced apoptosis, because either over-expression or knockdown of SHH in PDAC cells affects the sensitivity to gemcitabine. Mechanistic studies show that ABCB2 serves as the downstream target of SHH signaling, leading to the drug resistance of PDAC cells. Combinational treatments with gemcitabine and cyclopamine yield synergistic antitumor effects in vitro and in vivo. Our study suggests that inhibiting SHH signaling or targeting ABCB2 gene improves the efficacy of chemotherapy in patients with PDAC.

  14. 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.

  15. MicroRNA-21 accelerates hepatocyte proliferation in vitro via PI3K/Akt signaling by targeting PTEN

    Energy Technology Data Exchange (ETDEWEB)

    Yan-nan, Bai [Department of Hepato-Biliary-Pancreatic Surgery, West China Hospital, Sichuan University, Chengdu 610041, Sichuan Province (China); Department of Hepatobiliary Surgery, Fujian Provincial Hospital, Provincial Clinical College of Fujian Medical University, Fuzhou 350001, Fujian Province (China); Zhao-yan, Yu; Li-xi, Luo; Jiang, Yi [Department of Hepato-Biliary-Pancreatic Surgery, West China Hospital, Sichuan University, Chengdu 610041, Sichuan Province (China); Qing-jie, Xia [Translational Neuroscience Center, West China Hospital/West China Medical School of Sichuan University, Chengdu 610041, Sichuan Province (China); Yong, Zeng, E-mail: yongzengmd@gmail.com [Department of Hepato-Biliary-Pancreatic Surgery, West China Hospital, Sichuan University, Chengdu 610041, Sichuan Province (China)

    2014-01-17

    Highlights: •miRNAs-expression patterns of primary hepatocytes under proliferative status. •miR-21 expression level peaked at 12 h after stimulated by EGF. •miR-21 drive rapid S phase entry of primary hepatocytes. •PI3K/Akt signaling was modulated via targeting PTEN by miR-21. -- Abstract: MicroRNAs (miRNAs) are involved in controlling hepatocyte proliferation during liver regeneration. In this study, we established the miRNAs-expression patterns of primary hepatocytes in vitro under stimulation of epidermal growth factor (EGF), and found that microRNA-21 (miR-21) was appreciably up-regulated and peaked at 12 h. In addition, we further presented evidences indicating that miR-21 promotes primary hepatocyte proliferation through in vitro transfecting with miR-21 mimics or inhibitor. We further demonstrated that phosphatidylinositol 3′-OH kinase (PI3K)/Akt signaling was altered accordingly, it is, by targeting phosphatase and tensin homologue deleted on chromosome 10, PI3K/Akt signaling is activated by miR-21 to accelerate hepatocyte rapid S-phase entry and proliferation in vitro.

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

    Science.gov (United States)

    Zhu, Houbao; Xu, Wangyang; Zhang, Hongxin; Liu, Jianbing; Xu, Haimin; Lu, Shunyuan; Dang, Suying; Kuang, Ying; Jin, Xiaolong; Wang, Zhugang

    2013-08-16

    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.

  17. Essential role of AKT in tumor cells addicted to FGFR.

    Science.gov (United States)

    Hu, Yi; Lu, Huiru; Zhang, Jinchao; Chen, Jun; Chai, Zhifang; Zhang, Jingxin

    2014-02-01

    Tumor cells with genetic amplifications or mutations in the fibroblast growth factor receptor (FGFR) family are often addicted to FGFR and heavily dependent on its signaling to survive. Although it is critical to understand which signaling pathway downstream of FGFR plays an essential role to guide the research and development of FGFR inhibitors, it has remained unclear partly because the tool compounds used in the literature also hit many other kinases, making the results difficult to interpret. With the development of a potent FGFR-specific inhibitor, BGJ398, we are now able to dissect various pathways with low drug concentrations to minimize multiple-target effects. Importantly, here, we show that inhibition of FGFR signaling by BGJ398 leads to only transient inhibition of ERK1/2 phosphorylation, whereas the inhibitory effect on AKT phosphorylation is sustainable, indicating that AKT, not ERK as commonly believed, serves as an appropriate pharmacodynamic biomarker for BGJ398. Although AKT inhibition by a pan-PI3K inhibitor alone has almost no effect on cell growth, heterologous expression of myr-AKT, an active form of AKT, rescues BGJ398-mediated suppression of tumor cell proliferation. These results indicate that AKT is an essential component downstream of FGFR. Finally, combination of the FGFR inhibitor BGJ398 with rapamycin significantly inhibits AKT phosphorylation and enhances their antiproliferative effects in FGFR-addicted cells, suggesting an effective combination strategy for clinical development of FGFR inhibitors.

  18. 4-Hydroxy-3-Methoxybenzoic Acid Methyl Ester: A Curcumin Derivative Targets Akt/NFκB Cell Survival Signaling Pathway: Potential for Prostate Cancer Management

    Directory of Open Access Journals (Sweden)

    Addanki P. Kumar

    2003-05-01

    Full Text Available Transcription factor NFKB and the serine/threonine kinase Akt play critical roles in mammalian cell survival signaling and have been shown to be activated in various malignancies including prostate cancer (PCA. We have developed an analogue of curcumin called 4hydroxy-3-methoxybenzoic acid methyl ester (HMBME that targets the Akt/NFκB signaling pathway. Here, we demonstrate the ability of this novel compound to inhibit the proliferation of human and mouse PCA cells. HMBME-induced apoptosis in these cells was tested by using multiple biochemical approaches, in addition to morphological analysis. Overexpression of constitutively active Akt reversed the HMBME-induced growth inhibition and apoptosis, illustrating the direct role of Akt signaling in HMBME-mediated growth inhibition and apoptosis. Further, investigation of the molecular events associated with its action in LNCaP cells shows that: 1 HMBME reduces the level of activated form of Akt (phosphorylated Akt; and 2 inhibits the Akt kinase activity. Further, the transcriptional activity of NFκB, the DNA-binding activity of NFκB, and levels of p65 were all significantly reduced following treatment with HMBME. Overexpression of constitutively active Akt, but not the kinase dead mutant of Akt, activated the basal NFκB transcriptional activity. HMBME treatment had no influence on this constitutively active Aktaugmented NFκB transcriptional activity. These data indicate that HMBME-mediated inhibition of Akt kinase activity may have a potential in suppressing/decreasing the activity of major survival/antiapoptotic pathways. The potential use of HMBME as an agent that targets survival mechanisms in PCA cells is discussed.

  19. Enhanced Cardiac Akt/Protein Kinase B Signaling Contributes to Pathological Cardiac Hypertrophy in Part by Impairing Mitochondrial Function via Transcriptional Repression of Mitochondrion-Targeted Nuclear Genes

    Science.gov (United States)

    Wende, Adam R.; O'Neill, Brian T.; Bugger, Heiko; Riehle, Christian; Tuinei, Joseph; Buchanan, Jonathan; Tsushima, Kensuke; Wang, Li; Caro, Pilar; Guo, Aili; Sloan, Crystal; Kim, Bum Jun; Wang, Xiaohui; Pereira, Renata O.; McCrory, Mark A.; Nye, Brenna G.; Benavides, Gloria A.; Darley-Usmar, Victor M.; Shioi, Tetsuo; Weimer, Bart C.

    2014-01-01

    Sustained Akt activation induces cardiac hypertrophy (LVH), which may lead to heart failure. This study tested the hypothesis that Akt activation contributes to mitochondrial dysfunction in pathological LVH. Akt activation induced LVH and progressive repression of mitochondrial fatty acid oxidation (FAO) pathways. Preventing LVH by inhibiting mTOR failed to prevent the decline in mitochondrial function, but glucose utilization was maintained. Akt activation represses expression of mitochondrial regulatory, FAO, and oxidative phosphorylation genes in vivo that correlate with the duration of Akt activation in part by reducing FOXO-mediated transcriptional activation of mitochondrion-targeted nuclear genes in concert with reduced signaling via peroxisome proliferator-activated receptor α (PPARα)/PGC-1α and other transcriptional regulators. In cultured myocytes, Akt activation disrupted mitochondrial bioenergetics, which could be partially reversed by maintaining nuclear FOXO but not by increasing PGC-1α. Thus, although short-term Akt activation may be cardioprotective during ischemia by reducing mitochondrial metabolism and increasing glycolysis, long-term Akt activation in the adult heart contributes to pathological LVH in part by reducing mitochondrial oxidative capacity. PMID:25535334

  20. 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

  1. 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

  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. 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.

  4. 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.

  5. Activation of Akt is increased in the dysplasia-carcinoma sequence in Barrett's oesophagus and contributes to increased proliferation and inhibition of apoptosis: a histopathological and functional study

    Directory of Open Access Journals (Sweden)

    El-Amin Khalid

    2007-06-01

    stimulate Akt phosphorylation. Downstream targets of Akt include Bad and Forkhead transcription factors. Activation of Akt in obesity and by reflux of gastric acid may be important in the pathogenesis of Barrett's adenocarcinoma

  6. 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.

  7. Anti-myeloma activity of Akt inhibition is linked to the activation status of PI3K/Akt and MEK/ERK pathway.

    Directory of Open Access Journals (Sweden)

    Vijay Ramakrishnan

    Full Text Available The PI3K/Akt/mTOR signal transduction pathway plays a central role in multiple myeloma (MM disease progression and development of therapeutic resistance. mTORC1 inhibitors have shown limited efficacy in the clinic, largely attributed to the reactivation of Akt due to rapamycin induced mTORC2 activity. Here, we present promising anti-myeloma activity of MK-2206, a novel allosteric pan-Akt inhibitor, in MM cell lines and patient cells. MK-2206 was able to induce cytotoxicity and inhibit proliferation in all MM cell lines tested, albeit with significant heterogeneity that was highly dependent on basal pAkt levels. MK-2206 was able to inhibit proliferation of MM cells even when cultured with marrow stromal cells or tumor promoting cytokines. The induction of cytotoxicity was due to apoptosis, which at least partially was mediated by caspases. MK-2206 inhibited pAkt and its down-stream targets and up-regulated pErk in MM cells. Using MK-2206 in combination with rapamycin (mTORC1 inhibitor, LY294002 (PI3K inhibitor, or U0126 (MEK1/2 inhibitor, we show that Erk- mediated downstream activation of PI3K/Akt pathway results in resistance to Akt inhibition. These provide the basis for clinical evaluation of MK-2206 alone or in combination in MM and potential use of baseline pAkt and pErk as biomarkers for patient selection.

  8. Anti-myeloma activity of Akt inhibition is linked to the activation status of PI3K/Akt and MEK/ERK pathway.

    Science.gov (United States)

    Ramakrishnan, Vijay; Kimlinger, Teresa; Haug, Jessica; Painuly, Utkarsh; Wellik, Linda; Halling, Timothy; Rajkumar, S Vincent; Kumar, Shaji

    2012-01-01

    The PI3K/Akt/mTOR signal transduction pathway plays a central role in multiple myeloma (MM) disease progression and development of therapeutic resistance. mTORC1 inhibitors have shown limited efficacy in the clinic, largely attributed to the reactivation of Akt due to rapamycin induced mTORC2 activity. Here, we present promising anti-myeloma activity of MK-2206, a novel allosteric pan-Akt inhibitor, in MM cell lines and patient cells. MK-2206 was able to induce cytotoxicity and inhibit proliferation in all MM cell lines tested, albeit with significant heterogeneity that was highly dependent on basal pAkt levels. MK-2206 was able to inhibit proliferation of MM cells even when cultured with marrow stromal cells or tumor promoting cytokines. The induction of cytotoxicity was due to apoptosis, which at least partially was mediated by caspases. MK-2206 inhibited pAkt and its down-stream targets and up-regulated pErk in MM cells. Using MK-2206 in combination with rapamycin (mTORC1 inhibitor), LY294002 (PI3K inhibitor), or U0126 (MEK1/2 inhibitor), we show that Erk- mediated downstream activation of PI3K/Akt pathway results in resistance to Akt inhibition. These provide the basis for clinical evaluation of MK-2206 alone or in combination in MM and potential use of baseline pAkt and pErk as biomarkers for patient selection.

  9. miR-24 represses metastasis of human osteosarcoma cells by targeting Ack1 via AKT/MMPs pathway.

    Science.gov (United States)

    Liu, Zhendong; Liu, Zhitao; Zhang, Yuanjun; Li, Yan; Liu, Bo; Zhang, Kexiang

    2017-02-08

    The expression levels of the protein tyrosine kinase Ack1 has been reported to be dysregulated in various cancers and involve in oncogenesis and progression. However, the expression and role of Ack1 in osteosarcoma remains unknown. In this study, we found that Ack1 were evidently upregulated in human osteosarcoma tissues and cell lines. In addition, the clinical data showed that high expression level of Ack1 is closely associated with clinical stage and positive distant metastasis, and negatively correlated with overall survival. Then, bioinformatics prediction and luciferase reporter assay indicated Ack1 as a direct target of miR-24, and Ack1 could be downregulated by miR-24 at both the mRNA and protein expression levels. Moreover, Ack1 expression levels were inversely correlated with that of miR-24 in osteosarcoma tissues. Furthermore, functional assay showed that miR-24 significantly suppressed osteosarcoma progression partially mediated by inhibiting Ack1 expression. Finally, western bolt assay revealed that miR-24 regulate AKT/MMPs pathway via Ack1 in osteosarcoma cells. In conclusion, our study demonstrated the suppression of miR-24 on osteosarcoma metastasis by targeting Ack1 via AKT/MMPs pathways, providing a novel strategy for the diagnosis and treatment of osteosarcoma patients.

  10. 3-phosphoinositide-dependent protein kinase-1/Akt signaling represents a major cyclooxygenase-2-independent target for celecoxib in prostate cancer cells.

    Science.gov (United States)

    Kulp, Samuel K; Yang, Ya-Ting; Hung, Chin-Chun; Chen, Kuen-Feng; Lai, Ju-Ping; Tseng, Ping-Hui; Fowble, Joseph W; Ward, Patrick J; Chen, Ching-Shih

    2004-02-15

    Regarding the involvement of cyclooxygenase-2 (COX-2)-independent pathways in celecoxib-mediated antineoplastic effects, the following two issues remain outstanding: identity of the non-COX-2 targets and relative contributions of COX-2-dependent versus -independent mechanisms. We use a close celecoxib analog deficient in COX-2-inhibitory activity, DMC (4-[5-(2,5-dimethylphenyl)-3(trifluoromethyl)-1H-pyrazol-1-yl]benzene-sulfonamide), to examine the premise that Akt signaling represents a major non-COX-2 target. Celecoxib and DMC block Akt activation in PC-3 cells through the inhibition of phosphoinositide-dependent kinase-1 (PDK-1) with IC(50) of 48 and 38 micro M, respectively. The consequent effect on Akt activation is more pronounced (IC(50) values of 28 and 20 micro M, respectively), which might be attributed to the concomitant dephosphorylation by protein phosphatase 2A. In serum-supplemented medium, celecoxib and DMC cause G(1) arrest, and at higher concentrations, they induce apoptosis with relative potency comparable with that in blocking Akt activation. Moreover, the effect of daily oral celecoxib and DMC at 100 and 200 mg/kg on established PC-3 xenograft tumors is assessed. Celecoxib at both doses and DMC at 100 mg/kg had marginal impacts. However, a correlation exists between the in vitro potency of DMC and its ability at 200 mg/kg to inhibit xenograft tumor growth through the inhibition of Akt activation. Analysis of the tumor samples indicates that a differential reduction in the phospho-Akt/Akt ratio was noted in celecoxib- and DMC-treated groups vis-à-vis the control group. Together, these data underscore the role of 3-phosphoinositide-dependent protein kinase-1/Akt signaling in celecoxib-mediated in vitro antiproliferative effects in prostate cancer cells.

  11. 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

  12. Genomic analyses of musashi1 downstream targets show a strong association with cancer-related processes.

    Science.gov (United States)

    de Sousa Abreu, Raquel; Sanchez-Diaz, Patricia C; Vogel, Christine; Burns, Suzanne C; Ko, Daijin; Burton, Tarea L; Vo, Dat T; Chennasamudaram, Soudhamini; Le, Shu-Yun; Shapiro, Bruce A; Penalva, Luiz O F

    2009-05-01

    Musashi1 (Msi1) is a highly conserved RNA-binding protein with pivotal functions in stem cell maintenance, nervous system development, and tumorigenesis. Despite its importance, only three direct mRNA targets have been characterized so far: m-numb, CDKN1A, and c-mos. Msi1 has been shown to affect their translation by binding to short elements located in the 3'-untranslated region. To better understand Msi1 functions, we initially performed an RIP-Chip analysis in HEK293T cells; this method consists of isolation of specific RNA-protein complexes followed by identification of the RNA component via microarrays. A group of 64 mRNAs was found to be enriched in the Msi1-associated population compared with controls. These genes belong to two main functional categories pertinent to tumorigenesis: 1) cell cycle, cell proliferation, cell differentiation, and apoptosis and 2) protein modification (including ubiquitination and ubiquitin cycle). To corroborate our findings, we examined the impact of Msi1 expression on both mRNA (transcriptomic) and protein (proteomic) expression levels. Genes whose mRNA levels were affected by Msi1 expression have a Gene Ontology distribution similar to RIP-Chip results, reinforcing Msi1 participation in cancer-related processes. The proteomics study revealed that Msi1 can have either positive or negative effects on gene expression of its direct targets. In summary, our results indicate that Msi1 affects a network of genes and could function as a master regulator during development and tumor formation.

  13. 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

  14. 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.

  15. Activator protein 1 promotes gemcitabine-induced apoptosis in pancreatic cancer by upregulating its downstream target Bim.

    Science.gov (United States)

    Ren, Xiaoxia; Zhao, Wenjing; Du, Yongxing; Zhang, Taiping; You, Lei; Zhao, Yupei

    2016-12-01

    Gemcitabine is a commonly used chemotherapy drug in pancreatic cancer. The function of activator protein 1 (AP-1) is cell-specific, and its function depends on the expression of other complex members. In the present study, we added gemcitabine to the media of Panc-1 and SW1990 cells at clinically achieved concentrations (10 µM). Compared with constitutive c-Fos expression, c-Jun expression increased in a dose-dependent manner upon gemcitabine treatment. c-Jun overexpression increased gemcitabine-induced apoptosis through Bim activation, while cell apoptosis and Bim expression decreased following c-Jun knockdown. Furthermore, gemcitabine-induced apoptosis and Bim levels decreased when c-Jun phosphorylation was blocked by SP600125. Our findings suggest that c-Jun, which is a member of the AP-1 complex, functions in gemcitabine-induced apoptosis by regulating its downstream target Bim in pancreatic cancer cells.

  16. Abnormal Protein Glycosylation and Activated PI3K/Akt/mTOR Pathway: Role in Bladder Cancer Prognosis and Targeted Therapeutics

    Science.gov (United States)

    Lima, Luís; Peixoto, Andreia; Fernandes, Elisabete; Neves, Diogo; Neves, Manuel; Gaiteiro, Cristiana; Tavares, Ana; Gil da Costa, Rui M.; Cruz, Ricardo; Amaro, Teresina; Oliveira, Paula A.; Ferreira, José Alexandre; Santos, Lúcio L.

    2015-01-01

    ). This decreased the number of invasive lesions and, concomitantly, the expression of STn and also pS6, the downstream effector of the PI3K/Akt/mTOR pathway. In conclusion, STn was found to be marker of poor prognosis in bladder cancer and, in combination with PI3K/Akt/mTOR pathway evaluation, holds potential to improve the stratification of stage disease. Animal experiments suggest that mTOR pathway inhibition could be a potential therapeutic approach for this specific subtype of MIBC. PMID:26569621

  17. Abnormal Protein Glycosylation and Activated PI3K/Akt/mTOR Pathway: Role in Bladder Cancer Prognosis and Targeted Therapeutics.

    Science.gov (United States)

    Costa, Céu; Pereira, Sofia; Lima, Luís; Peixoto, Andreia; Fernandes, Elisabete; Neves, Diogo; Neves, Manuel; Gaiteiro, Cristiana; Tavares, Ana; Gil da Costa, Rui M; Cruz, Ricardo; Amaro, Teresina; Oliveira, Paula A; Ferreira, José Alexandre; Santos, Lúcio L

    2015-01-01

    ). This decreased the number of invasive lesions and, concomitantly, the expression of STn and also pS6, the downstream effector of the PI3K/Akt/mTOR pathway. In conclusion, STn was found to be marker of poor prognosis in bladder cancer and, in combination with PI3K/Akt/mTOR pathway evaluation, holds potential to improve the stratification of stage disease. Animal experiments suggest that mTOR pathway inhibition could be a potential therapeutic approach for this specific subtype of MIBC.

  18. 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.

  19. 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.

  20. Mefloquine effectively targets gastric cancer cells through phosphatase-dependent inhibition of PI3K/Akt/mTOR signaling pathway.

    Science.gov (United States)

    Liu, Yanwei; Chen, Sen; Xue, Rui; Zhao, Juan; Di, Maojun

    2016-02-05

    Deregulation of PI3K/Akt/mTOR pathway has been recently identified to play a crucial role in the progress of human gastric cancer. In this study, we show that mefloquine, a FDA-approved anti-malarial drug, effectively targets human gastric cancer cells. Mefloquine potently inhibits proliferation and induces apoptosis of a panel of human gastric cancer cell lines, with EC50 ∼ 0.5-0.7 μM. In two independent gastric cancer xenograft mouse models, mefloquine significantly inhibits growth of both tumors. The combination of mefloquine with paclitaxel enhances the activity of either drug alone in in vitro and in vivo. In addition, mefloquine potently decreased phosphorylation of PI3K, Akt, mTOR and rS6. Overexpression of constitutively active Akt significantly restored mefloquine-mediated inhibition of mTOR phosphorylation and growth, and induction of apoptosis, suggesting that mefloquine acts on gastric cancer cells via suppressing PI3K/Akt/mTOR pathway. We further show that mefloquine-mediated inhibition of Akt/mTOR singaling is phosphatase-dependent as pretreatment with calyculin A does-dependently reversed mefloquine-mediated inhibition of Akt/mTOR phosphorylation. Since mefloquine is already available for clinic use, these results suggest that it is a useful addition to the treatment armamentarium for gastric cancer.

  1. miR-218 inhibits the invasion and migration of colon cancer cells by targeting the PI3K/Akt/mTOR signaling pathway.

    Science.gov (United States)

    Zhang, Xiangliang; Shi, Huijuan; Tang, Hongsheng; Fang, Zhiyuan; Wang, Jiping; Cui, Shuzhong

    2015-05-01

    Colon cancer is one of the most common and lethal malignancies worldwide. Despite major advances in the treatment of colon cancer, the prognosis remains very poor. Thus, novel and effective therapies for colon cancer are urgently needed. In the present study, the expression status of miR-218 and the role of the phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt)/mammalian target of rapamycin (mTOR) pathway were investigated in colon cancer samples. Firstly, we observed that miR-218 expression was significantly reduced, while PI3K/Akt/mTOR pathway activity was enhanced. The overexpression of miR-218 suppressed the proliferation, migration and invasion of LoVo colon cancer cells, whereas the inhibition of miR-218 promoted these processes. Furthermore, the PI3K/Akt/mTOR signaling pathway was identified as a direct target of miR-218. The upregulation of miR-218 inhibited the activation of the PI3K/Akt/mTOR signaling pathway, as well as the expression of matrix metalloproteinase (MMP)9. The downregulation of miR-218 activated the PI3K/Akt/mTOR signaling pathway and promoted MMP9 expression. Taken together, our results demonstrate that miR-218 suppresses the proliferation, migration and invasion of LoVo colon cancer cells by targeting the PI3K/Akt/mTOR signaling pathway and MMP9. Our data indicate that miR-218 is a potential target in the treatment of colon cancer.

  2. 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.

  3. Grainyhead-like 2 downstream targets act to suppress epithelial-to-mesenchymal transition during neural tube closure.

    Science.gov (United States)

    Ray, Heather J; Niswander, Lee A

    2016-04-01

    The transcription factor grainyhead-like 2 (GRHL2) is expressed in non-neural ectoderm (NNE) and Grhl2 loss results in fully penetrant cranial neural tube defects (NTDs) in mice. GRHL2 activates expression of several epithelial genes; however, additional molecular targets and functional processes regulated by GRHL2 in the NNE remain to be determined, as well as the underlying cause of the NTDs in Grhl2 mutants. Here, we find that Grhl2 loss results in abnormal mesenchymal phenotypes in the NNE, including aberrant vimentin expression and increased cellular dynamics that affects the NNE and neural crest cells. The resulting loss of NNE integrity contributes to an inability of the cranial neural folds to move toward the midline and results in NTD. Further, we identified Esrp1, Sostdc1, Fermt1, Tmprss2 and Lamc2 as novel NNE-expressed genes that are downregulated in Grhl2 mutants. Our in vitro assays show that they act as suppressors of the epithelial-to-mesenchymal transition (EMT). Thus, GRHL2 promotes the epithelial nature of the NNE during the dynamic events of neural tube formation by both activating key epithelial genes and actively suppressing EMT through novel downstream EMT suppressors.

  4. Nuclear cereblon modulates transcriptional activity of Ikaros and regulates its downstream target, enkephalin, in human neuroblastoma cells.

    Science.gov (United States)

    Wada, Takeyoshi; Asahi, Toru; Sawamura, Naoya

    2016-08-26

    The gene coding cereblon (CRBN) was originally identified in genetic linkage analysis of mild autosomal recessive nonsyndromic intellectual disability. CRBN has broad localization in both the cytoplasm and nucleus. However, the significance of nuclear CRBN remains unknown. In the present study, we aimed to elucidate the role of CRBN in the nucleus. First, we generated a series of CRBN deletion mutants and determined the regions responsible for the nuclear localization. Only CRBN protein lacking the N-terminal region was localized outside of the nucleus, suggesting that the N-terminal region is important for its nuclear localization. CRBN was also identified as a thalidomide-binding protein and component of the cullin-4-containing E3 ubiquitin ligase complex. Thalidomide has been reported to be involved in the regulation of the transcription factor Ikaros by CRBN-mediated degradation. To investigate the nuclear functions of CRBN, we performed co-immunoprecipitation experiments and evaluated the binding of CRBN to Ikaros. As a result, we found that CRBN was associated with Ikaros protein, and the N-terminal region of CRBN was required for Ikaros binding. In luciferase reporter gene experiments, CRBN modulated transcriptional activity of Ikaros. Furthermore, we found that CRBN modulated Ikaros-mediated transcriptional repression of the proenkephalin gene by binding to its promoter region. These results suggest that CRBN binds to Ikaros via its N-terminal region and regulates transcriptional activities of Ikaros and its downstream target, enkephalin.

  5. 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.

  6. N-Cadherin in Prostate Cancer: Downstream Pathways and Their Translational Application for Castrate-Resistant Prostate Cancer

    Science.gov (United States)

    2012-09-01

    inhibition of downstream signaling pathways especially NF-κB and the PI3K/AKT pathways may serve as complementary therapies to N-cadherin targeting...N-cadherin–positive cells from LAPC9-CR tumors are also more tum- origenic than N-cadherin–negative cells (E.K. and R.E.R., unpublished data). A

  7. 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.

  8. 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.

  9. 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.

  10. Targeting of cell metabolism in human acute myeloid leukemia--more than targeting of isocitrate dehydrogenase mutations and PI3K/AKT/mTOR signaling?

    Science.gov (United States)

    Hauge, Michelle; Bruserud, Øystein; Hatfield, Kimberley Joanne

    2016-03-01

    Targeting of cellular metabolism has emerged as a possible strategy in the treatment of human malignancies, and several experimental studies suggest that this therapeutic approach should also be considered in acute myeloid leukemia (AML). Clinical studies of metabolic intervention in AML patients with isocitrate dehydrogenase mutations have shown promising results. Moreover, metabolic targeting of the PI3K/AKT/mTOR signaling pathway as an anticancer strategy has been extensively studied. In this review, we focus on other emerging therapeutic alternatives for metabolic inhibition in human AML, in particular targeting of glycolysis and the AMP kinase signaling pathway. Pharmacological drugs for these metabolic interventions are already available and they seem to have an acceptable toxicity, even when used in combination with conventional chemotherapy. Future clinical studies of these therapeutic strategies should focus on the following: (i) heterogeneity of patients and the possibility that this treatment is most effective only for certain subsets of patients, (ii) toxic effects in AML patients with an existing disease-induced bone marrow failure prior to treatment, and (iii) whether this strategy should be used as part of a potentially curative treatment and/or as disease-stabilizing treatment to prolong survival in elderly or unfit patients.

  11. 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.

  12. Dual-Targeting of AR and Akt Pathways by Berberine in Castration-Resistant Prostate Cancer

    Science.gov (United States)

    2015-08-01

    maintained by serial passaging in castrated SCID mice. The tumors were cut into 25 mm3 pieces and implanted subcutaneously into castrated SCID mice...transactivate target genes. Cancer Research 2015 Jun 9, Epub ahead of print . Abstracts: Xichun Liu, Elisa Ledet, Yanfeng Qi, Yan Don, Oliver Sartor...expressed at a much lower level than AR-FL (Fig. S5). Moreover, AR-V7 and ARv567es can 230 heterodimerize (Fig. 3D ). Mutating D-box, but not the FxxLF

  13. The Phosphoinositide 3-OH Kinase/AKT2 Pathway as a Critical Target for Farnesyltransferase Inhibitor-Induced Apoptosis

    OpenAIRE

    Jiang, Kun; Coppola, Domenico; Crespo, Nichole C.; Nicosia, Santo V.; Hamilton, Andrew D.; Sebti, Said M.; Cheng, Jin Q.

    2000-01-01

    Farnesyltransferase inhibitors (FTIs) represent a novel class of anticancer drugs that exhibit a remarkable ability to inhibit malignant transformation without toxicity to normal cells. However, the mechanism by which FTIs inhibit tumor growth is not well understood. Here, we demonstrate that FTI-277 inhibits phosphatidylinositol 3-OH kinase (PI 3-kinase)/AKT2-mediated growth factor- and adhesion-dependent survival pathways and induces apoptosis in human cancer cells that overexpress AKT2. Fu...

  14. 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

  15. 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.

  16. Reversing melanoma cross-resistance to BRAF and MEK inhibitors by co-targeting the AKT/mTOR pathway.

    Directory of Open Access Journals (Sweden)

    Mohammad Atefi

    Full Text Available BACKGROUND: The sustained clinical activity of the BRAF inhibitor vemurafenib (PLX4032/RG7204 in patients with BRAF(V600 mutant melanoma is limited primarily by the development of acquired resistance leading to tumor progression. Clinical trials are in progress using MEK inhibitors following disease progression in patients receiving BRAF inhibitors. However, the PI3K/AKT pathway can also induce resistance to the inhibitors of MAPK pathway. METHODOLOGY/PRINCIPAL FINDINGS: The sensitivity to vemurafenib or the MEK inhibitor AZD6244 was tested in sensitive and resistant human melanoma cell lines exploring differences in activation-associated phosphorylation levels of major signaling molecules, leading to the testing of co-inhibition of the AKT/mTOR pathway genetically and pharmacologically. There was a high degree of cross-resistance to vemurafenib and AZD6244, except in two vemurafenib-resistant cell lines that acquired a secondary mutation in NRAS. In other cell lines, acquired resistance to both drugs was associated with persistence or increase in activity of AKT pathway. siRNA-mediated gene silencing and combination therapy with an AKT inhibitor or rapamycin partially or completely reversed the resistance. CONCLUSIONS/SIGNIFICANCE: Primary and acquired resistance to vemurafenib in these in vitro models results in frequent cross resistance to MEK inhibitors, except when the resistance is the result of a secondary NRAS mutation. Resistance to BRAF or MEK inhibitors is associated with the induction or persistence of activity within the AKT pathway in the presence of these drugs. This resistance can be potentially reversed by the combination of a RAF or MEK inhibitor with an AKT or mTOR inhibitor. These combinations should be available for clinical testing in patients progressing on BRAF inhibitors.

  17. 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

  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. 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.

  20. Inhibition of hydrogen peroxide signaling by 4-hydroxynonenal due to differential regulation of Akt1 and Akt2 contributes to decreases in cell survival and proliferation in hepatocellular carcinoma cells.

    Science.gov (United States)

    Shearn, Colin T; Reigan, Philip; Petersen, Dennis R

    2012-07-01

    Dysregulation of cell signaling by electrophiles such as 4-hydroxynonenal (4-HNE) is a key component in the pathogenesis of chronic inflammatory liver disease. Another consequence of inflammation is the perpetuation of oxidative damage by the production of reactive oxidative species such as hydrogen peroxide. Previously, we have demonstrated Akt2 as a direct target of 4-HNE in hepatocellular carcinoma cells. In the present study, we used the hepatocellular carcinoma cell line HepG2 as model to understand the combinatorial effects of 4-HNE and hydrogen peroxide. We demonstrate that 4-HNE inhibits hydrogen peroxide-mediated phosphorylation of Akt1 but not Akt2. Pretreatment of HepG2 cells with 4-HNE prevented hydrogen peroxide stimulation of Akt-dependent phosphorylation of downstream targets and intracellular Akt activity compared with untreated control cells. Using biotin hydrazide capture, it was confirmed that 4-HNE treatment resulted in carbonylation of Akt1, which was not observed in untreated control cells. Using a synthetic GSK3α/β peptide as a substrate, treatment of recombinant human myristoylated Akt1 (rAkt1) with 20 or 40 μΜ 4-HNE inhibited rAkt1 activity by 29 and 60%, respectively. We further demonstrate that 4-HNE activates Erk via a PI3 kinase and PP2A-dependent mechanism leading to increased Jnk phosphorylation. At higher concentrations, 4-HNE decreased both cell survival and proliferation as evidenced by MTT assays and EdU incorporation as well as decreased expression of cyclin D1 and β-catenin, an effect only moderately increased by the addition of hydrogen peroxide. The ability of 4-HNE to exert combinatorial effects on Erk, Jnk, and Akt-dependent cell survival pathways provides additional insight into the mechanisms of cellular damage associated with chronic inflammation.

  1. MicroRNA-130b targets PTEN to mediate drug resistance and proliferation of breast cancer cells via the PI3K/Akt signaling pathway

    Science.gov (United States)

    Miao, Yuan; Zheng, Wei; Li, Nana; Su, Zhen; Zhao, Lifen; Zhou, Huimin; Jia, Li

    2017-01-01

    Multidrug resistance (MDR) correlates with treatment failure and poor prognosis among breast cancer patients. This study was aimed to investigate the possible mechanism by which microRNA-130b-3p (miR-130b) mediates the chemoresistance and proliferation of breast cancer. MiR-130b was found to be up-regulated in tumor tissues versus adjacent tissues of breast cancer, as well as in adriamycin (ADR) resistant breast cancer cell line (MCF-7/ADR) versus its parental line (MCF-7) and the non-malignant breast epithelial cell line (MCF-10A), demonstrating its crucial relevance for breast cancer biology. We identified that PTEN was a direct target of miR-130b and inversely correlated with miR-130b expression in breast cancer. Moreover, over-expression of miR-130b promoted drug resistance, proliferation and decreased apoptosis of MCF-7 cells, while suppression of miR-130b enhanced drug cytotoxicity and apoptosis, as well as reduced proliferation of MCF-7/ADR cells in vitro and in vivo. Particularly, miR-130b mediated the activity of phosphoinositide-3 kinase (PI3K)/Akt signaling pathway as well as the chemoresistance and proliferation of breast cancer cell lines, which was partially blocked following knockdown of PTEN. Altogether, miR-130b targets PTEN to induce MDR, proliferation, and apoptosis via PI3K/Akt signaling pathway. This provides a novel promising candidate for breast cancer therapy. PMID:28165066

  2. Targeting of PI3K/AKT/mTOR pathway to inhibit T cell activation and prevent graft-versus-host disease development

    Directory of Open Access Journals (Sweden)

    Mª Carmen Herrero-Sánchez

    2016-10-01

    Full Text Available Abstract Background Graft-versus-host disease (GvHD remains the major obstacle to successful allogeneic hematopoietic stem cell transplantation, despite of the immunosuppressive regimens administered to control T cell alloreactivity. PI3K/AKT/mTOR pathway is crucial in T cell activation and function and, therefore, represents an attractive therapeutic target to prevent GvHD development. Recently, numerous PI3K inhibitors have been developed for cancer therapy. However, few studies have explored their immunosuppressive effect. Methods The effects of a selective PI3K inhibitor (BKM120 and a dual PI3K/mTOR inhibitor (BEZ235 on human T cell proliferation, expression of activation-related molecules, and phosphorylation of PI3K/AKT/mTOR pathway proteins were analyzed. Besides, the ability of BEZ235 to prevent GvHD development in mice was evaluated. Results Simultaneous inhibition of PI3K and mTOR was efficient at lower concentrations than PI3K specific targeting. Importantly, BEZ235 prevented naïve T cell activation and induced tolerance of alloreactive T cells, while maintaining an adequate response against cytomegalovirus, more efficiently than BKM120. Finally, BEZ235 treatment significantly improved the survival and decreased the GvHD development in mice. Conclusions These results support the use of PI3K inhibitors to control T cell responses and show the potential utility of the dual PI3K/mTOR inhibitor BEZ235 in GvHD prophylaxis.

  3. 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.

  4. Antagonism of miR-21 reverses epithelial-mesenchymal transition and cancer stem cell phenotype through AKT/ERK1/2 inactivation by targeting PTEN.

    Directory of Open Access Journals (Sweden)

    Mingli Han

    Full Text Available BACKGROUND: Accumulating evidence suggested that epithelial-mesenchymal transition (EMT and cancer stem cell (CSC characteristics, both of which contribute to tumor invasion and metastasis, are interrelated with miR-21. MiR-21 is one of the important microRNAs associated with tumor progression and metastasis, but the molecular mechanisms underlying EMT and CSC phenotype during miR-21 contributes to migration and invasion of breast cancer cells remain to be elucidated. METHODOLOGY/PRINCIPAL FINDINGS: In this study, MDA-MB-231/anti-miR-21 cells were established by transfected hsa-miR-21 antagomir into breast cancer MDA-MB-231 cells. EMT was evaluated by the changes of mesenchymal cell markers (N-cadherin, Vimentin, and alpha-SMA, epithelial cell marker (E-cadherin, as well as capacities of cell migration and invasion; CSC phenotype was measured using the changes of CSC surface markers (ALDH1 and CD44, and the capacity of sphereforming (mammospheres. We found that antagonism of miR-21 reversed EMT and CSC phenotype, accompanied with PTEN up-regulation and AKT/ERK1/2 inactivation. Interestingly, down-regulation of PTEN by siPTEN suppressed the effects of miR-21 antagomir on EMT and CSC phenotype, confirming that PTEN is a target of miR-21 in reversing EMT and CSC phenotype. The inhibitors of PI3K-AKT and ERK1/2 pathways, LY294002 and U0126, both significantly suppressed EMT and CSC phenotype, indicating that AKT and ERK1/2 pathways are required for miR-21 mediating EMT and CSC phenotype. CONCLUSIONS/SIGNIFICANCE: In conclusion, our results demonstrated that antagonism of miR-21 reverses EMT and CSC phenotype through targeting PTEN, via inactivation of AKT and ERK1/2 pathways, and showed a novel mechanism of which might relieve the malignant biological behaviors of breast cancer.

  5. 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.

  6. Characterization of TCF21 Downstream Target Regions Identifies a Transcriptional Network Linking Multiple Independent Coronary Artery Disease Loci.

    Science.gov (United States)

    Sazonova, Olga; Zhao, Yuqi; Nürnberg, Sylvia; Miller, Clint; Pjanic, Milos; Castano, Victor G; Kim, Juyong B; Salfati, Elias L; Kundaje, Anshul B; Bejerano, Gill; Assimes, Themistocles; Yang, Xia; Quertermous, Thomas

    2015-05-01

    To functionally link coronary artery disease (CAD) causal genes identified by genome wide association studies (GWAS), and to investigate the cellular and molecular mechanisms of atherosclerosis, we have used chromatin immunoprecipitation sequencing (ChIP-Seq) with the CAD associated transcription factor TCF21 in human coronary artery smooth muscle cells (HCASMC). Analysis of identified TCF21 target genes for enrichment of molecular and cellular annotation terms identified processes relevant to CAD pathophysiology, including "growth factor binding," "matrix interaction," and "smooth muscle contraction." We characterized the canonical binding sequence for TCF21 as CAGCTG, identified AP-1 binding sites in TCF21 peaks, and by conducting ChIP-Seq for JUN and JUND in HCASMC confirmed that there is significant overlap between TCF21 and AP-1 binding loci in this cell type. Expression quantitative trait variation mapped to target genes of TCF21 was significantly enriched among variants with low P-values in the GWAS analyses, suggesting a possible functional interaction between TCF21 binding and causal variants in other CAD disease loci. Separate enrichment analyses found over-representation of TCF21 target genes among CAD associated genes, and linkage disequilibrium between TCF21 peak variation and that found in GWAS loci, consistent with the hypothesis that TCF21 may affect disease risk through interaction with other disease associated loci. Interestingly, enrichment for TCF21 target genes was also found among other genome wide association phenotypes, including height and inflammatory bowel disease, suggesting a functional profile important for basic cellular processes in non-vascular tissues. Thus, data and analyses presented here suggest that study of GWAS transcription factors may be a highly useful approach to identifying disease gene interactions and thus pathways that may be relevant to complex disease etiology.

  7. Characterization of TCF21 Downstream Target Regions Identifies a Transcriptional Network Linking Multiple Independent Coronary Artery Disease Loci.

    Directory of Open Access Journals (Sweden)

    Olga Sazonova

    2015-05-01

    Full Text Available To functionally link coronary artery disease (CAD causal genes identified by genome wide association studies (GWAS, and to investigate the cellular and molecular mechanisms of atherosclerosis, we have used chromatin immunoprecipitation sequencing (ChIP-Seq with the CAD associated transcription factor TCF21 in human coronary artery smooth muscle cells (HCASMC. Analysis of identified TCF21 target genes for enrichment of molecular and cellular annotation terms identified processes relevant to CAD pathophysiology, including "growth factor binding," "matrix interaction," and "smooth muscle contraction." We characterized the canonical binding sequence for TCF21 as CAGCTG, identified AP-1 binding sites in TCF21 peaks, and by conducting ChIP-Seq for JUN and JUND in HCASMC confirmed that there is significant overlap between TCF21 and AP-1 binding loci in this cell type. Expression quantitative trait variation mapped to target genes of TCF21 was significantly enriched among variants with low P-values in the GWAS analyses, suggesting a possible functional interaction between TCF21 binding and causal variants in other CAD disease loci. Separate enrichment analyses found over-representation of TCF21 target genes among CAD associated genes, and linkage disequilibrium between TCF21 peak variation and that found in GWAS loci, consistent with the hypothesis that TCF21 may affect disease risk through interaction with other disease associated loci. Interestingly, enrichment for TCF21 target genes was also found among other genome wide association phenotypes, including height and inflammatory bowel disease, suggesting a functional profile important for basic cellular processes in non-vascular tissues. Thus, data and analyses presented here suggest that study of GWAS transcription factors may be a highly useful approach to identifying disease gene interactions and thus pathways that may be relevant to complex disease etiology.

  8. Genomic Analyses of Musashi1 Downstream Targets Show a Strong Association with Cancer-related Processes*S⃞

    OpenAIRE

    2009-01-01

    Musashi1 (Msi1) is a highly conserved RNA-binding protein with pivotal functions in stem cell maintenance, nervous system development, and tumorigenesis. Despite its importance, only three direct mRNA targets have been characterized so far: m-numb, CDKN1A, and c-mos. Msi1 has been shown to affect their translation by binding to short elements located in the 3′-untranslated region. To better understand Msi1 functions, we initially performed an RIP-Chip analysis in H...

  9. 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.

  10. 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

  11. Akt1 signaling coordinates BMP signaling and β-catenin activity to regulate second heart field progenitor development.

    Science.gov (United States)

    Luo, Wen; Zhao, Xia; Jin, Hengwei; Tao, Lichan; Zhu, Jingai; Wang, Huijuan; Hemmings, Brian A; Yang, Zhongzhou

    2015-02-15

    Second heart field (SHF) progenitors exhibit continued proliferation and delayed differentiation, which are modulated by FGF4/8/10, BMP and canonical Wnt/β-catenin signaling. PTEN-Akt signaling regulates the stem cell/progenitor cell homeostasis in several systems, such as hematopoietic stem cells, intestinal stem cells and neural progenitor cells. To address whether PTEN-Akt signaling is involved in regulating cardiac progenitors, we deleted Pten in SHF progenitors. Deletion of Pten caused SHF expansion and increased the size of the SHF derivatives, the right ventricle and the outflow tract. Cell proliferation of cardiac progenitors was enhanced, whereas cardiac differentiation was unaffected by Pten deletion. Removal of Akt1 rescued the phenotype and early lethality of Pten deletion mice, suggesting that Akt1 was the key downstream target that was negatively regulated by PTEN in cardiac progenitors. Furthermore, we found that inhibition of FOXO by Akt1 suppressed the expression of the gene encoding the BMP ligand (BMP7), leading to dampened BMP signaling in the hearts of Pten deletion mice. Cardiac activation of Akt also increased the Ser552 phosphorylation of β-catenin, thus enhancing its activity. Reducing β-catenin levels could partially rescue heart defects of Pten deletion mice. We conclude that Akt signaling regulates the cell proliferation of SHF progenitors through coordination of BMP signaling and β-catenin activity.

  12. 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

  13. 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

  14. 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.

  15. MicroRNA-320 inhibits invasion and induces apoptosis by targeting CRKL and inhibiting ERK and AKT signaling in gastric cancer cells

    Science.gov (United States)

    Zhao, Yue; Dong, Qianze; Wang, Enhua

    2017-01-01

    MicroRNA-320 (miR-320) downregulation has been reported in several human cancers. Until now, its expression pattern and biological roles in human cancer remain unknown. This study aims to clarify its clinical expression pattern and biological function in gastric cancers. We found miR-320 level was downregulated in gastric cancer tissues. miR-320 mimic was transfected in SGC-7901 cells with low endogenous expression. miR-320 inhibitor was used in BGC-823 cells with high endogenous expression. We found that miR-320 inhibited SGC-7901 proliferation and invasion, with decreased expression of cyclin D1 and MMP9 at both mRNA and protein levels. We also found that miR-320 mimic downregulated chemoresistance and cell survival of gastric cancer cells when treated with 5-fluorouracil. miR-320 inhibitor displayed the opposite effects in BGC-823 cell line. In addition, we discovered that miR-320 mimic could inhibit AKT and ERK activity. By using luciferase reporter assay, we found that CRKL serves as the target of miR-320. miR-320 mimic downregulated CRKL expression, whereas miR-320 inhibitor upregulated CRKL expression. miR-320 suppressed CRKL-3′-untranslated region reporter intensity in SGC-7901 cells. Furthermore, CRKL depletion abrogated the effects of miR-320. In gastric cancer tissues, we observed a negative correlation between CRKL and miR-320. In conclusion, our study demonstrated that downregulation of miR-320 was closely related with malignant progression of gastric cancer. miR-320 inhibits proliferation, invasion, and chemoresistance through ERK and AKT signaling by targeting CRKL. PMID:28255248

  16. 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.

  17. Regulation of PI3-kinase/Akt signaling by muscle-enriched microRNA-486

    Science.gov (United States)

    Small, Eric M.; O’Rourke, Jason R.; Moresi, Viviana; Sutherland, Lillian B.; McAnally, John; Gerard, Robert D.; Richardson, James A.; Olson, Eric N.

    2010-01-01

    microRNAs (miRNAs) play key roles in modulating a variety of cellular processes through repression of mRNA targets. In a screen for miRNAs regulated by myocardin-related transcription factor-A (MRTF-A), a coactivator of serum response factor (SRF), we discovered a muscle-enriched miRNA, miR-486, controlled by an alternative promoter within intron 40 of the Ankyrin-1 gene. Transcription of miR-486 is directly controlled by SRF and MRTF-A, as well as by MyoD. Among the most strongly predicted targets of miR-486 are phosphatase and tensin homolog (PTEN) and Foxo1a, which negatively affect phosphoinositide-3-kinase (PI3K)/Akt signaling. Accordingly, PTEN and Foxo1a protein levels are reduced by miR-486 overexpression, which, in turn, enhances PI3K/Akt signaling. Similarly, we show that MRTF-A promotes PI3K/Akt signaling by up-regulating miR-486 expression. Conversely, inhibition of miR-486 expression enhances the expression of PTEN and Foxo1a and dampens signaling through the PI3K/Akt-signaling pathway. Our findings implicate miR-486 as a downstream mediator of the actions of SRF/MRTF-A and MyoD in muscle cells and as a potential modulator of PI3K/Akt signaling. PMID:20142475

  18. Infection of Female BWF1 Lupus Mice with Malaria Parasite Attenuates B Cell Autoreactivity by Modulating the CXCL12/CXCR4 Axis and Its Downstream Signals PI3K/AKT, NFκB and ERK.

    Science.gov (United States)

    Badr, Gamal; Sayed, Ayat; Abdel-Maksoud, Mostafa A; Mohamed, Amany O; El-Amir, Azza; Abdel-Ghaffar, Fathy A; Al-Quraishy, Saleh; Mahmoud, Mohamed H

    2015-01-01

    Systemic lupus erythematosus (SLE) is a prototypic autoimmune disease characterized by abnormal autoreactivity in B cells. Lymphocytes and their soluble mediators contribute to the disease pathogenesis. We recently demonstrated that infecting lupus mice with malaria confers protection against lupus nephritis by attenuating oxidative stress in both liver and kidney tissues. In the current study, we further investigated B cell autoreactivity in female BWF1 lupus mice after infection with either live or gamma-irradiated malaria, using ELISA, flow cytometry and Western blot analysis. The lupus mice exhibited a significant elevation in plasma levels of IL-4, IL-6, IL-7, IL-12, IL-17, IFN-α, IFN-γ, TGF-β, BAFF and APRIL and a marked elevation of IgG2a, IgG3 and ant-dsDNA autoantibodies compared with normal healthy mice. Infecting lupus mice with live but not gamma-irradiated malaria parasite partially and significantly restored the levels of the soluble mediators that contribute to the progression of lupus. Furthermore, the B cells of lupus mice exhibited an increased proliferative capacity; aberrant overexpression of the chemokine receptor CXCR4; and a marked elevation in responsiveness to their cognate ligand (CXCL12) via aberrant activation of the PI3K/AKT, NFκB and ERK signaling pathways. Interestingly, infecting lupus mice with live but not gamma-irradiated malaria parasite restored a normal proliferative capacity, surface expression of CXCR4 and B cell response to CXCL-12. Taken together, our data present interesting findings that clarify, for the first time, the molecular mechanisms of how infection of lupus mice with malaria parasite controls B cell autoreactivity and thus confers protection against lupus severity.

  19. Arsenic-induced mitochondrial oxidative damage is mediated by decreased PGC-1α expression and its downstream targets in rat brain.

    Science.gov (United States)

    Prakash, Chandra; Kumar, Vijay

    2016-08-25

    The present study was carried out to investigate the molecular mechanism of arsenic-induced mitochondrial oxidative damage and its relation to biogenesis in rat brain. Chronic sodium arsenite (25 ppm, orally) administration for 12 weeks decreased mitochondrial complexes activities and mRNA expression of selective complexes subunits. The expression of mitochondrial biogenesis regulator PGC-1α, and its downstream targets NRF-1, NRF-2 and Tfam were decreased significantly both at mRNA and protein levels suggesting impaired biogenesis following chronic arsenic-exposure. In addition to this, protein expression analysis also revealed activation of Bax and caspase-3, leading to translocation of cytochrome c from mitochondria to cytosol suggesting induction of apoptotic pathway under oxidative stress. This was further confirmed by electron microscopy study which depicted morphological changes in mitochondria in terms of altered nuclear and mitochondrial shape and chromatin condensation in arsenic-treated rats. The immunohistochemical studies showed both nuclear and cytosolic localization of NRF-1 and NRF-2 in arsenic-exposed rat brain further suggesting regulatory role of these transcription factors under arsenic neurotoxicity. The results of present study indicate that arsenic-induced mitochondrial oxidative damage is associated with decreased mitochondrial biogenesis in rat brain that may present as important target to reveal the mechanism for arsenic-induced neurotoxicity.

  20. miR-2861 acts as a tumor suppressor via targeting EGFR/AKT2/CCND1 pathway in cervical cancer induced by human papillomavirus virus 16 E6.

    Science.gov (United States)

    Xu, Junfen; Wan, Xiaoyun; Chen, Xiaojing; Fang, Yifeng; Cheng, Xiaodong; Xie, Xing; Lu, Weiguo

    2016-07-01

    Persistent infection with oncogenic human papillomavirus viruses (HPVs) is a casual factor for cervical cancer and its precursors, and the abnormal constitutive expression of viral oncoprotein E6 is a key event during the malignant transformation. Here, we performed miRNA microarray to identify changes of miRNAs following ectopic HPV16 E6 overexpression in HEK293T cells and found miR-2861 was greatly decreased in both HEK293T and HaCaT cells expressing HPV16 E6 compared to vector control. Further, we demonstrated a biological link among HPV16 E6, miR-2861, EGFR, AKT2, and CCND1 in cervical cancer cells. We showed that miR-2861 was downregulated in cervical cancer tissues and negatively correlated with advanced tumor stage and lymph node metastasis. Overexpression of miR-2861 suppressed cervical cancer cell proliferation and invasion and enhanced apoptosis. Subsequent investigation revealed that EGFR, AKT2, and CCND1 were all the direct targets of miR-2861. Importantly, silencing EGFR, AKT2, and/or CCND1 recapitulated the cellular effects seen upon miR-2861 overexpression. Restoration of EGFR, AKT2, and/or CCND1 counteracted the effects of miR-2861 expression. Thus, we identified a new pathway employing miR-2861, EGFR, AKT2, and CCND1 that may mediate HPV16 E6 induced initiation and progression of cervical cancer.

  1. Analysis of miRNA profiles identified miR-196a as a crucial mediator of aberrant PI3K/AKT signaling in lung cancer cells.

    Science.gov (United States)

    Guerriero, Ilaria; D'Angelo, Daniela; Pallante, Pierlorenzo; Santos, Mafalda; Scrima, Marianna; Malanga, Donatella; De Marco, Carmela; Ravo, Maria; Weisz, Alessandro; Laudanna, Carmelo; Ceccarelli, Michele; Falco, Geppino; Rizzuto, Antonia; Viglietto, Giuseppe

    2016-11-17

    Hyperactivation of the PI3K/AKT pathway is observed in most human cancer including lung carcinomas. Here we have investigated the role of miRNAs as downstream targets of activated PI3K/AKT signaling in Non Small Cell Lung Cancer (NSCLC). To this aim, miRNA profiling was performed in human lung epithelial cells (BEAS-2B) expressing active AKT1 (BEAS-AKT1-E17K), active PI3KCA (BEAS-PIK3CA-E545K) or with silenced PTEN (BEAS-shPTEN).Twenty-four differentially expressed miRNAs common to BEAS-AKT1-E17K, BEAS-PIK3CA-E545K and BEAS-shPTEN cells were identified through this analysis, with miR-196a being the most consistently up-regulated miRNA. Interestingly, miR-196a was significantly overexpressed also in human NSCLC-derived cell lines (n=11) and primary lung cancer samples (n=28).By manipulating the expression of miR-196a in BEAS-2B and NCI-H460 cells, we obtained compelling evidence that this miRNA acts downstream the PI3K/AKT pathway, mediating some of the proliferative, pro-migratory and tumorigenic activity that this pathway exerts in lung epithelial cells, possibly through the regulation of FoxO1, CDKN1B (hereafter p27) and HOXA9.

  2. 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.

  3. 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

  4. Development of a new model system to dissect isoform specific Akt signalling in adipocytes

    Science.gov (United States)

    Kajno, Esi; McGraw, Timothy E.; Gonzalez, Eva

    2015-01-01

    Protein kinase B (Akt) kinases are critical signal transducers mediating insulin action. Genetic studies revealed that Akt1 and Akt2 signalling differentially contribute to sustain lipid and glucose homoeostasis; however Akt isoform-specific effectors remain elusive due to the lack of a suitable model system to mechanistically interrogate Akt isoform-specific signalling. To overcome those technical limitations we developed a novel model system that provides acute and specific control of signalling by Akt isoforms. We generated mutants of Akt1 and Akt2 resistant to the allosteric Akt inhibitor MK-2206. We then developed adipocyte cell lines, in which endogenous Akt1 or Akt2 has been replaced by their corresponding drug-resistant Akt mutant. Treatment of those cells with MK-2206 allowed for acute and specific control of either Akt1 or Akt2 function. Our data showed that Akt1W80A and Akt2W80A mutants are resistant to MK-2206, dynamically regulated by insulin and able to signal to Akt downstream effectors. Analyses of insulin action in this cellular system showed that Akt1 and Akt2 are both able to mediate insulin regulation of the transcription factor forkhead box O1 (FoxO1) and the glucose transporter 4 (GLUT4), revealing a redundant role for these Akt kinases in the control of glucose transport into fat cells. In contrast, Akt1 signalling is uniquely required for adipogenesis, by controlling the mitotic clonal expansion (MCE) of pre-adipocytes that precedes white adipose cell differentiation. Our data provide new insights into the role of Akt kinases in glucose transport and adipogenesis and support our model system as a valuable tool for the biochemical characterization of signalling by specific Akt isoforms. PMID:25856301

  5. Fucoidan inhibits the migration and proliferation of HT-29 human colon cancer cells via the phosphoinositide-3 kinase/Akt/mechanistic target of rapamycin pathways.

    Science.gov (United States)

    Han, Yong-Seok; Lee, Jun Hee; Lee, Sang Hun

    2015-09-01

    Fucoidan, a sulfated polysaccharide, has a variety of biological activities, including anti-cancer, anti-angiogenic and anti-inflammatory effects. However, the underlying mechanisms of fucoidan as an anti‑cancer agent remain to be elucidated. The present study examined the anti‑cancer effect of fucoidan on HT‑29 human colon cancer cells. The cell growth of HT29 cells was significantly decreased following treatment with fucoidan (200 µg/ml). In addition, fucoidan inhibited the migration of HT‑29 cells by decreasing the expression levels of matrix metalloproteinase‑2 in a dose‑dependent manner (0‑200 µg/ml). The underlying mechanism of these inhibitory effects included the downregulation of phosphoinositide 3‑kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) by treatment with fucoidan. Furthermore, fucoidan increased the expression of cleaved caspase‑3 and decreased cancer sphere formation. The present study suggested that fucoidan exerts an anti‑cancer effect on HT‑29 human colon cancer cells by downregulating the PI3K‑Akt‑mTOR signaling pathway. Therefore, fucoidan may be a potential therapeutic reagent against the growth of human colon cancer cells.

  6. 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.

  7. 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.

  8. 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

  9. MiR-181a Targets PHLPP2 to Augment AKT Signaling and Regulate Proliferation and Apoptosis in Human Keloid Fibroblasts

    Directory of Open Access Journals (Sweden)

    Zhen Rang

    2016-12-01

    Full Text Available Background/Aims: Keloids are fibrous overgrowths induced by cutaneous injury. MicroRNAs (miRNAs have recently emerged as post-transcriptional gene repressors and participants in a diverse array of pathophysiological processes leading to skin disease. The purpose of the current study was to explore the precise functions of miR-181a in human keloid development and the underlying mechanisms. Methods: A miRNA microarray analysis was performed to compare expression profiles between keloid and normal skin tissues. Quantitative real-time PCR was conducted to estimate miR-181a expression. Cell proliferation was determined using the cell counting kit-8 (CCK-8 and 5-ethynyl-2-deoxyuridine (EdU assays, and cell cycle and apoptosis were detected with flow cytometry. Direct targets of miR-181a were identified using the luciferase reporter assay. Results: miR-181a was significantly upregulated in human keloid tissues and fibroblasts, compared with their control counterparts. Overexpression of miR-181a enhanced keloid fibroblast DNA synthesis and proliferation and inhibited apoptosis, whereas miR-181a suppression triggered the opposite effects. Moreover, miR-181a suppressed the expression of PH domain leucine-rich repeat protein phosphatase 2 (PHLPP2 through direct interactions with its 3′UTR region and subsequently enhanced AKT activation. Overexpression of PHLPP2 without its 3′UTR attenuated the effects of miR-181a on cell proliferation and apoptosis in keloid fibroblast cells. Furthermore, miR-181a mimics increased normal skin fibroblast proliferation. Conclusions: Our results highlight a novel pathway mediated by miR-181a, which may be effectively used as a therapeutic target for treatment of keloids.

  10. 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...

  11. Constitutively nuclear FOXO3a localization predicts poor survival and promotes Akt phosphorylation in breast cancer.

    Directory of Open Access Journals (Sweden)

    Jie Chen

    Full Text Available BACKGROUND: The PI3K-Akt signal pathway plays a key role in tumorigenesis and the development of drug-resistance. Cytotoxic chemotherapy resistance is linked to limited therapeutic options and poor prognosis. METHODOLOGY/PRINCIPAL FINDINGS: Examination of FOXO3a and phosphorylated-Akt (P-Akt expression in breast cancer tissue microarrays showed nuclear FOXO3a was associated with lymph node positivity (p = 0.052, poor prognosis (p = 0.014, and P-Akt expression in invasive ductal carcinoma. Using tamoxifen and doxorubicin-sensitive and -resistant breast cancer cell lines as models, we found that doxorubicin- but not tamoxifen-resistance is associated with nuclear accumulation of FOXO3a, consistent with the finding that sustained nuclear FOXO3a is associated with poor prognosis. We also established that doxorubicin treatment induces proliferation arrest and FOXO3a nuclear relocation in sensitive breast cancer cells. Induction of FOXO3a activity in doxorubicin-sensitive MCF-7 cells was sufficient to promote Akt phosphorylation and arrest cell proliferation. Conversely, knockdown of endogenous FOXO3a expression reduced PI3K/Akt activity. Using MDA-MB-231 cells, in which FOXO3a activity can be induced by 4-hydroxytamoxifen, we showed that FOXO3a induction up-regulates PI3K-Akt activity and enhanced doxorubicin resistance. However FOXO3a induction has little effect on cell proliferation, indicating that FOXO3a or its downstream activity is deregulated in the cytotoxic drug resistant breast cancer cells. Thus, our results suggest that sustained FOXO3a activation can enhance hyperactivation of the PI3K/Akt pathway. CONCLUSIONS/SIGNIFICANCE: Together these data suggest that lymph node metastasis and poor survival in invasive ductal breast carcinoma are linked to an uncoupling of the Akt-FOXO3a signaling axis. In these breast cancers activated Akt fails to inactivate and re-localize FOXO3a to the cytoplasm, and nuclear-targeted FOXO3a does not induce cell

  12. 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.

  13. 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.

  14. PI3K/Akt/mTOR signaling pathway and targeting therapy of skin cancer%PI3K/Akt/mTOR信号通路与皮肤肿瘤靶向治疗

    Institute of Scientific and Technical Information of China (English)

    李金超; 许爱娥

    2009-01-01

    P13K/AkdmTOR信号转导通路是促存活通路,在很多肿瘤中组成性激活.该通路激活的机制是肿瘤抑制基PTEN功能缺失、P13K扩增或突变、Akt扩增或突变.近年研究发现,该通路失常可促进肿瘤细胞的存活和生长,持续活化在皮肤肿瘤发病中起着重要的作用,已经发现抑制该通路中的信号分子可以治疗多种肿瘤,目前,针对该通路的抑制药物也在研究中,主要集中于mTOR抑制剂.%The PI3K/Akt/mTOR pathway is a prototypic survival pathway that is constitutively activated in various types of cancer. This pathway may be activated by function loss of tumor suppressor gene PTEN, amplification or mutation of PI3K and Akt genes. Recent studies have shown that the abnormality of PI3K/Akt/mTOR pathway may promote the survival and development of tumor cells, and continuous activation of this pathway is associated with the development of cutaneous tumors. It has been found that many tumors can be treated by inhibition of this pathway. Multiple inhibitors of this pathway have been developed and are being evaluated in laboratory and clinical trials, with much attention focused on mTOR inhibition.

  15. 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.

  16. 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.

  17. 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.

  18. 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.

  19. 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

  20. Endomembrane PtdIns(3,4,5)P3 activates the PI3K-Akt pathway.

    Science.gov (United States)

    Jethwa, Nirmal; Chung, Gary H C; Lete, Marta G; Alonso, Alicia; Byrne, Richard D; Calleja, Véronique; Larijani, Banafshé

    2015-09-15

    PKB/Akt activation is a common step in tumour growth, proliferation and survival. Akt activation is understood to occur at the plasma membrane of cells in response to growth factor stimulation and local production of the phosphoinositide lipid phosphatidylinositol (3,4,5)-trisphosphate [PtdIns(3,4,5)P3] following phosphoinositide 3-kinase (PI3K) activation. The metabolism and turnover of phosphoinositides is complex--they act as signalling molecules as well as structural components of biological membranes. The localisation and significance of internal pools of PtdIns(3,4,5)P3 has long been speculated upon. By using transfected and recombinant protein probes for PtdIns(3,4,5)P3, we show that PtdIns(3,4,5)P3 is enriched in the nuclear envelope and early endosomes. By exploiting an inducible dimerisation device to recruit Akt to these compartments, we demonstrate that Akt can be locally activated in a PtdIns(3,4,5)P3-dependent manner and has the potential to phosphorylate compartmentally localised downstream substrates. This could be an important mechanism to regulate Akt isoform substrate specificity or influence the timing and duration of PI3K pathway signalling. Defects in phosphoinositide metabolism and localisation are known to contribute to cancer, suggesting that interactions at subcellular compartments might be worthwhile targets for therapeutic intervention.

  1. 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.

  2. Regulation of sonic hedgehog-GLI1 downstream target genes PTCH1, Cyclin D2, Plakoglobin, PAX6 and NKX2.2 and their epigenetic status in medulloblastoma and astrocytoma

    Directory of Open Access Journals (Sweden)

    Eberhart Charles G

    2010-11-01

    Full Text Available Abstract Background The Sonic hedgehog (Shh signaling pathway is critical for cell growth and differentiation. Impairment of this pathway can result in both birth defects and cancer. Despite its importance in cancer development, the Shh pathway has not been thoroughly investigated in tumorigenesis of brain tumors. In this study, we sought to understand the regulatory roles of GLI1, the immediate downstream activator of the Shh signaling pathway on its downstream target genes PTCH1, Cyclin D2, Plakoglobin, NKX2.2 and PAX6 in medulloblastoma and astrocytic tumors. Methods We silenced GLI1 expression in medulloblastoma and astrocytic cell lines by transfection of siRNA against GLI1. Subsequently, we performed RT-PCR and quantitative real time RT-PCR (qRT-PCR to assay the expression of downstream target genes PTCH1, Cyclin D2, Plakoglobin, NKX2.2 and PAX6. We also attempted to correlate the pattern of expression of GLI1 and its regulated genes in 14 cell lines and 41 primary medulloblastoma and astrocytoma tumor samples. We also assessed the methylation status of the Cyclin D2 and PTCH1 promoters in these 14 cell lines and 58 primary tumor samples. Results Silencing expression of GLI1 resulted up-regulation of all target genes in the medulloblastoma cell line, while only PTCH1 was up-regulated in astrocytoma. We also observed methylation of the cyclin D2 promoter in a significant number of astrocytoma cell lines (63% and primary astrocytoma tumor samples (32%, but not at all in any medulloblastoma samples. PTCH1 promoter methylation was less frequently observed than Cyclin D2 promoter methylation in astrocytomas, and not at all in medulloblastomas. Conclusions Our results demonstrate different regulatory mechanisms of Shh-GLI1 signaling. These differences vary according to the downstream target gene affected, the origin of the tissue, as well as epigenetic regulation of some of these genes.

  3. 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.

  4. Cellular and Molecular Roles of the Akt Protein Kinase in Breast Carcinomas

    Science.gov (United States)

    1999-06-01

    are in progress. Identification of Akt interacting proteins We proposed to identify targets of Akt using a yeast two-hybrid screen (1). We have...studies in Task 2. Key Research Accomplishments "* Identified Akt interacting proteins using a yeast two-hybrid screen "* Provided secondary evidence...human breast cancer lines (5). Therefore, our studies in the future will also focus on the regulation of Oct3 by Akt. Identification of AKT Interacting

  5. 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.

  6. MiR-126 regulates proliferation and invasion in the bladder cancer BLS cell line by targeting the PIK3R2-mediated PI3K/Akt signaling pathway

    Directory of Open Access Journals (Sweden)

    Xiao J

    2016-08-01

    Full Text Available Jun Xiao,1 Huan-Yi Lin,2 Yuan-Yuan Zhu,3 Yu-Ping Zhu,1 Ling-Wu Chen2 1Department of Urology, Anhui Provincial Hospital, Anhui Medical University, Hefei, 2Department of Urology, First Affiliated Hospital of Sun Yat-Sen University, Guangzhou, 3Clinical Laboratory, Anhui Provincial Hospital, Anhui Medical University, Hefei, People’s Republic of China Objective: To assess whether microRNA-126 (miR-126 targets phosphatidylinositol 3-kinase regulatory subunit beta (PIK3R2 and to determine the potential roles of miR-126 in regulating proliferation and invasion via the PIK3R2-mediated phosphatidylinositol 3 kinase (PI3K-protein kinase B (Akt signaling pathway in the human bladder BLS cell line. Materials and methods: A recombinant lentivirus (Lv vector expressing miR-216 (Lv-miR-126 was successfully constructed, and Lv-miR-126 and Lv vector were transfected into the BLS cell line. A direct regulatory relationship between miR-126 and the PIK3R2 gene was demonstrated by luciferase reporter assays. To determine whether PIK3R2 directly participates in the miR-126-induced effects in BLS cells, anti-miR-126 and a PIK3R2 small interfering RNA (siRNA were transfected into the BLS cells. Quantitative real-time polymerase chain reaction was used to measure miR-126 and PIK3R2 expressions. 5-Ethynyl-2'-deoxyuridine and colony formation assays to assess cell proliferation, flow cytometry for cell apoptosis and cell cycle analysis, Transwell assays for cell migration and invasion, and Western blots for PIK3R2, PI3K, phosphorylated PI3K (p-PI3K, Akt, and phosphorylated Akt (p-Akt protein expressions were performed. Results: Lv-miR-126 significantly enhanced the relative expression of miR-126 in the BLS cells after infection (P<0.0001. MiR-126 overexpression inhibited the proliferation, cloning, migration, and invasion of BLS cells, promoted cell apoptosis, and induced S phase arrest (all P<0.05. PIK3R2, p-PI3K, and p-Akt protein expressions were significantly

  7. Inhibitory effect of adenovirus-mediated short hairpin RNA targeting P85 and Akt1 on growth of human gastric adenocarcinoma cell%腺病毒介导的靶向P85和Akt1短发夹RNA对人胃腺癌细胞生长抑制作用的研究

    Institute of Scientific and Technical Information of China (English)

    张靖; 付彦超; 康春生; 张庆瑜; 王涛; 张洁

    2009-01-01

    目的 构建靶向P85和蛋白激酶B1(PKB1/Akt1)的短发夹RNA(shRNA)腺病毒载体,研究其对人胃腺癌细胞SGC-7901生长的抑制效果.方法 构建腺病毒载体rAd5-P+A,体外转染SGC-7901细胞后,以实时定量PCR和Western blot分别检测P85和Akt1的mRNA和蛋白质的表达.以噻唑蓝比色分析法(MTT法)和流式细胞法评价转染后胃癌细胞的增殖活性.构建裸鼠皮下荷瘤模型进一步观察rAd5-P+A对SGC-7901细胞生长的抑制效果,并应用原位末端标记技术(TUNEL法)检测肿瘤细胞的凋亡情况.结果 成功构建的rAd5-P+A重组腺病毒载体转染SGC-7901细胞后可显著抑制p85和Akt1的mRNA表达,而P85和Aktl蛋白表达量在转染48 h、72 h后分别下调57.5%、63.7%和67.8%、75.6%,与空白对照组和通用腺病毒对照(rAd5-HK)组相比,差异具有统计学意义(P=0.005,P=0.003).与空白对照组和rAd5-HK组相比,SGC-7901细胞的增殖活性在rAd5-P+A转染后第2天明显下降(P<0.001),且rAd5-P+A转染组进入S期的细胞数减少了5.9%~7.1%,而进入G0/G1期的细胞增加了12.1%~13.7%.裸鼠皮下荷瘤模型治疗实验也显示,rAd5-P+A可抑制胃癌细胞的生长,诱导细胞的凋亡.结论 腺病毒介导的靶向P85和Akt1的shRNA可抑制人胃腺癌细胞的生长,这可能为胃腺癌靶向性联合基因治疗提供新的策略.%Objective To construct a short hairpin RNA (shRNA) adenovirus vector targeting P85 and protein kinase B1 (PKB1/Akt1) and study its effects on the growth of SGC-7901 human gastric adenocareinoma cells. Methods P85 and Aktl shRNA expression frames were subcloned to pGSadeno adenovirus vector with homologous recombination technology to construct pGSadeno-P85 + Akt1 (rAd5-P + A) vector. After screening and amplification, the recombinant adenovirus vector was digested with PacI and transfected into SGC-7901 cells and then its titer and transfection efficiency were detected with fluorescent microscope. P85 and Akt1 mRNA protein

  8. 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.

  9. Compound library screening identified Akt/PKB kinase pathway inhibitors as potential key molecules for the development of new chemotherapeutics against schistosomiasis.

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    Morel, Marion; Vanderstraete, Mathieu; Cailliau, Katia; Lescuyer, Arlette; Lancelot, Julien; Dissous, Colette

    2014-12-01

    Protein kinases (PKs) are one of the largest protein families in most eukaryotic organisms. These enzymes are involved in the control of cell proliferation, differentiation and metabolism and a large number of the anticancer drugs currently used are directed against PKs. The structure and function of PKs are well conserved throughout evolution. In schistosome parasites, PKs were shown to be involved in essential functions at every stage of the parasite life cycle, making these enzymes promising anti-parasite drug targets. In this study, we tested a panel of commercial inhibitors for various PKs and analyzed their effects on pairing and egg production by schistosomes as well as their toxicity towards schistosomula larvae. Results obtained confirmed the deleterious effect of PK targeting on Schistosoma mansoni physiology and the important function of different tyrosine and serine/threonine kinases in the biology and reproduction of this parasite. They also indicated for the first time that the Protein kinase B (also called Akt) which is a major downstream target of many receptor tyrosine kinases and a central player at the crossroads of signal transduction pathways activated in response to growth factors and insulin, can constitute a novel target for anti-schistosome chemotherapy. Structural and functional studies have shown that SmAkt is a conserved kinase and that its activity can be inhibited by commercially available Akt inhibitors. In treated adult worms, Akt/PKB kinase pathway inhibitors induced profound alterations in pairing and egg laying and they also greatly affected the viability of schistosomula larvae.

  10. Salvianolic acid A reverses paclitaxel resistance in human breast cancer MCF-7 cells via targeting the expression of transgelin 2 and attenuating PI3 K/Akt pathway.

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    Cai, Jiangxia; Chen, Siying; Zhang, Weipeng; Zheng, Xiaowei; Hu, Sasa; Pang, Chengsen; Lu, Jun; Xing, Jianfeng; Dong, Yalin

    2014-10-15

    Chemotherapy resistance represents a major problem for the treatment of patients with breast cancer and greatly restricts the use of first-line chemotherapeutics paclitaxel. The purpose of this study was to investigate the role of transgelin 2 in human breast cancer paclitaxel resistance cell line (MCF-7/PTX) and the reversal mechanism of salvianolic acid A (SAA), a phenolic active compound extracted from Salvia miltiorrhiza. Western blotting and real-time quantitative polymerase chain reaction (qRT-PCR) indicated that transgelin 2 may mediate paclitaxel resistance by activating the phosphatidylinositol 3-kinase (PI3 K)/Akt signaling pathway to suppress MCF-7/PTX cells apoptosis. The reversal ability of SAA was confirmed by MTT assay and flow cytometry, with a superior 9.1-fold reversal index and enhancement of the apoptotic cytotoxicity induced by paclitaxel. In addition, SAA effectively prevented transgelin 2 and adenosine-triphosphate binding cassette transporter (ABC transporter) including P-glycoprotein (P-gp), multidrug resistance associated protein 1 (MRP1), and breast cancer resistance protein (BCRP) up-regulation and exhibited inhibitory effect on PI3 K/Akt signaling pathway in MCF-7/PTX cells. Taken together, SAA can reverse paclitaxel resistance through suppressing transgelin 2 expression by mechanisms involving attenuation of PI3 K/Akt pathway activation and ABC transporter up-regulation. These results not only provide insight into the potential application of SAA in reversing paclitaxel resistance, thus facilitating the sensitivity of breast cancer chemotherapy, but also highlight a potential role of transgelin 2 in the development of paclitaxel resistance in breast cancer.

  11. Prostate specific membrane antigen knockdown impairs the tumorigenicity of LNCaP prostate cancer cells by inhibiting the phosphatidylinositol 3-kinase/Akt signaling pathway

    Institute of Scientific and Technical Information of China (English)

    Guo Zhenghui; Lai Yiming; Du Tao; Zhang Yiming; Chen Jieqing; Bi Liangkuan; Lin Tianxin

    2014-01-01

    Background Prostate specific membrane antigen (PSMA) can facilitate the growth,migration,and invasion of the LNCaP prostate cancer cell lines,but the underlying molecular mechanisms have not yet been clearly defined.Here,we investigated whether PSMA serves as a novel regulator of the phosphatidylinositol 3-kinase (PI3K)/Akt signaling by employing PSMA knockdown model and PI3K pharmacological inhibitor (LY294002) in LNCaP prostate cancer cells.Methods PSMA knockdown had been stably established by transfecting with lentivirus-mediated siRNA in our previous study.Then,LNCaP cells were divided into interference,non-interference,and blank groups.We first testified the efficacy of PSMA knockdown in our LNCaP cell line.Then,we compared the expression of PSMA and total/activated Akt by Westem blotting in the above three groups with or without LY294002 treatment.Furthermore,immunocytochemistry was performed to confirm the changes of activated Akt (p-Akt,Ser473) in groups.Besides,cell proliferation,migration,and cell cycle were measured by CCK-8 assay,Transwell analysis,and Flow cytometry respectively.Results After PSMA knockdown,the level of p-Akt (Ser473) but not of total-Akt (Akt1/2) was significantly decreased when compared with the non-interference and blank groups.However,LY294002 administration significantly reduced the expression of p-Akt (Ser473) in all the three groups.The results of immunocytochemistry further confirmed that PSMA knockdown or LY294002 treatment was associated with p-Akt (Ser473) down-regulation.Decrease of cell proliferation,migration,and survival were also observed upon PSMA knockdown and LY294002 treatment.Conclusions Taken together,our results reveal that PI3K/Akt signaling pathway inhibition may serve as a novel molecular mechanism in LNCaP prostate cancer cells of PSMA knockdown and suggest that Akt (Ser473) may play a critical role as a downstream signaling target effector of PSMA in this cellular model.

  12. Effect of eccentric exercise velocity on akt/mtor/p70(s6k) signaling in human skeletal muscle.

    Science.gov (United States)

    Roschel, Hamilton; Ugrinowistch, Carlos; Barroso, Renato; Batista, Mauro A B; Souza, Eduardo O; Aoki, Marcelo S; Siqueira-Filho, Mario A; Zanuto, Ricardo; Carvalho, Carla R O; Neves, Manoel; Mello, Marco T; Tricoli, Valmor

    2011-04-01

    It has been suggested that muscle tension plays a major role in the activation of intracellular pathways for skeletal muscle hypertrophy via an increase in mechano growth factor (MGF) and other downstream targets. Eccentric exercise (EE) imposes a greater amount of tension on the active muscle. In particular, high-speed EE seems to exert an additional effect on muscle tension and, thus, on muscle hypertrophy. However, little is known about the effect of EE velocity on hypertrophy signaling. This study investigated the effect of acute EE-velocity manipulation on the Akt/mTORCI/p70(S6K) hypertrophy pathway. Twenty subjects were assigned to either a slow (20°·s(-1); ES) or fast EE (210°·s(-1); EF) group. Biopsies were taken from vastus lateralis at baseline (B), immediately after (T1), and 2 h after (T2) the completion of 5 sets of 8 repetitions of eccentric knee extensions. Akt, mTOR, and p70(S6K) total protein were similar between groups, and did not change postintervention. Further, Akt and p70(S6K) protein phosphorylation were higher at T2 than at B for ES and EF. MGF messenger RNA was similar between groups, and only significantly higher at T2 than at B in ES. The acute manipulation of EE velocity does not seem to differently influence intracellular hypertrophy signaling through the Akt/mTORCI/p70S6K pathway.

  13. Dual Inhibition of PI3K/AKT and MEK/ERK Pathways Induces Synergistic Antitumor Effects in Diffuse Intrinsic Pontine Glioma Cells

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    Y. Linda Wu

    2017-04-01

    Full Text Available Diffuse intrinsic pontine glioma (DIPG is a devastating disease with an extremely poor prognosis. Recent studies have shown that platelet-derived growth factor receptor (PDGFR and its downstream effector pathway, PI3K/AKT/mTOR, are frequently amplified in DIPG, and potential therapies targeting this pathway have emerged. However, the addition of targeted single agents has not been found to improve clinical outcomes in DIPG, and targeting this pathway alone has produced insufficient clinical responses in multiple malignancies investigated, including lung, endometrial, and bladder cancers. Acquired resistance also seems inevitable. Activation of the Ras/Raf/MEK/ERK pathway, which shares many nodes of cross talk with the PI3K/AKT pathway, has been implicated in the development of resistance. In the present study, perifosine, a PI3K/AKT pathway inhibitor, and trametinib, a MEK inhibitor, were combined, and their therapeutic efficacy on DIPG cells was assessed. Growth delay assays were performed with each drug individually or in combination. Here, we show that dual inhibition of PI3K/AKT and MEK/ERK pathways synergistically reduced cell viability. We also reveal that trametinib induced AKT phosphorylation in DIPG cells that could not be effectively attenuated by the addition of perifosine, likely due to the activation of other compensatory mechanisms. The synergistic reduction in cell viability was through the pronounced induction of apoptosis, with some effect from cell cycle arrest. We conclude that the concurrent inhibition of the PI3K/AKT and MEK/ERK pathways may be a potential therapeutic strategy for DIPG.

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

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    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.

  15. 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.

  16. Cyclooxygenase 2-dependent and independent activation of Akt through casein kinase 2α contributes to human bladder cancer cell survival

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    Fujimoto Kiyohide

    2011-05-01

    Full Text Available Abstract Background Survival rate for patients presenting muscle invasive bladder cancer is very low, and useful therapeutic target has not been identified yet. In the present study, new COX2 downstream signals involved in urothelial carcinoma cell survival were investigated in vitro and in vivo. Methods COX2 gene was silenced by siRNA transfection. Orthotopic implantation animal model and transurethral instillation of siRNA with atelocollagen was constructed to examine the effects of COX2 knockdown in vivo. Cell cycle was examined by flowcytoketry. Surgical specimens derived from patients with urinary bladder cancer (all were initially diagnosed cases were used for immunohistochemical analysis of the indicated protein expression in urothelial carcinoma cells. Results Treatment with the COX2 inhibitor or knockdown of COX2 reduced expression of casein kinase (CK 2 α, a phophorylated Akt and urokinase type plasminogen activator (uPA, resulting in p27 induction, cell cycle arrest at G1 phase and cell growth suppression in human urothelial carcinoma cell lines expressing COX2. Silencing of CK2α exhibited the similar effects. Even in UMUC3 cells lacking the COX2 gene, COX2 inhibition also inhibited cell growth through down-regulation of the CK2α-Akt/uPA axis. The mouse orthotropic bladder cancer model demonstrated that the COX2 inhibitor, meloxicam significantly reduced CK2α, phosphorylated Akt and uPA expression, whereas induced p27 by which growth and invasiveness of bladder cancer cells were strongly inhibited. Immunohistochemically, high expression of COX2, CK2α and phosphorylated form of Akt was found in high-grade, invasive carcinomas as well as carcinoma in situ, but not in low-grade and noninvasive phenotypes. Conclusions COX2-dependent and independent activation of CK2α-Akt/uPA signal is mainly involved in urothelial carcinoma cell survival, moreover, not only COX2 but also CK2α could be direct targets of COX2 inhibitors.

  17. 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

  18. Reduced signaling of PI3K-Akt and RAS-MAPK pathways is the key target for weight-loss-induced cancer prevention by dietary calorie restriction and/or physical activity.

    Science.gov (United States)

    Standard, Joseph; Jiang, Yu; Yu, Miao; Su, Xiaoyu; Zhao, Zhihui; Xu, Jianteng; Chen, Jie; King, Brenee; Lu, Lizhi; Tomich, John; Baybutt, Richard; Wang, Weiqun

    2014-12-01

    Weight control through either dietary calorie restriction (DCR) or exercise has been associated with cancer prevention in animal models. However, the underlying mechanisms are not fully defined. Bioinformatics using genomics, proteomics and lipidomics was employed to elucidate the molecular targets of weight control in a mouse skin cancer model. SENCAR mice were randomly assigned into four groups for 10 weeks: ad-libitum-fed sedentary control, ad-libitum-fed exercise (AE), exercise but pair-fed isocaloric amount of control (PE) and 20% DCR. Two hours after topical TPA treatment, skin epidermis was analyzed by Affymetrix for gene expression, DIGE for proteomics and lipidomics for phospholipids. Body weights were significantly reduced in both DCR and PE but not AE mice versus the control. Among 39,000 transcripts, 411, 67 and 110 genes were significantly changed in DCR, PE and AE, respectively. The expression of genes relevant to PI3K-Akt and Ras-MAPK signaling was effectively reduced by DCR and PE but not AE as measured through GenMAPP software. Proteomics analysis identified ~120 proteins, with 27 proteins significantly changed by DCR, including up-regulated apolipoprotein A-1, a key antioxidant protein that decreases Ras-MAPK activity. Of the total 338 phospholipids analyzed by lipidomics, 57 decreased by PE including 5 phophatidylinositol species that serve as PI3K substrates. Although a full impact has not been determined yet, it appears that the reduction of both Ras-MAPK and PI3K-Akt signaling pathways is a cancer preventive target that has been consistently demonstrated by three bioinformatics approaches.

  19. 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

    2016-08-08

    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.

  20. DARPP-32 and Akt regulation in ethanol-preferring AA and ethanol-avoiding ANA rats.

    Science.gov (United States)

    Nuutinen, Saara; Kiianmaa, Kalervo; Panula, Pertti

    2011-09-26

    Ethanol and other addictive drugs affect many intracellular phosphorylation and dephosphorylation cascades. These cascades are thought to be highly important in the regulation of neuronal activity. The present experiments characterized the regulation of three key signaling molecules, DARPP-32 (dopamine and cAMP regulated phosphoprotein, 32kDa), Akt kinase and ERK1/2 (extracellular signal-regulated kinase 1 and 2) in ethanol-preferring AA (Alko, alcohol) and ethanol-avoiding ANA (Alko, non-alcohol) rat lines. Radioactive in situ hybridization was used in drug naïve animals and Western blotting after acute ethanol administration in striatum, hippocampus and prefrontal cortex. The mRNA levels of DARPP-32 in striatal areas were higher in ANA rats than in AA rats. There was no difference in the striatal enriched phosphatase (STEP61), the downstream target of DARPP-32 expression between the rat lines. Ethanol (1.5g/kg) increased phosphorylation of DARPP-32 at threonine 34 in both AA and in ANA rats indicating that acute ethanol activates DARPP-32 similarly in these rat lines. The expression of Akt kinase was higher in the CA1 of hippocampus in ANA than in AA rats and acute ethanol activated Akt in hippocampus in ANA but not in AA rats. No significant alterations in the regulation of ERK1/2 were found in either rat line. Our findings suggest that DARPP-32 and Akt are regulated by ethanol and differences in the regulation of these molecules might contribute to the dramatically different ethanol drinking patterns seen in AA and ANA rats.

  1. 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.

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

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    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.

  3. 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

    Science.gov (United States)

    Sharov, Alexei A; Masui, Shinji; Sharova, Lioudmila V; Piao, Yulan; Aiba, Kazuhiro; Matoba, Ryo; Xin, Li; Niwa, Hitoshi; Ko, Minoru SH

    2008-01-01

    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 < 0.2) to a compendium of published and new microarray data (3, 6, 12, and 24 hr after 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. PMID:18522731

  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. miR-92a-3p Exerts Various Effects in Glioma and Glioma Stem-Like Cells Specifically Targeting CDH1/β-Catenin and Notch-1/Akt Signaling Pathways

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    Hang Song

    2016-10-01

    Full Text Available MicroRNAs (miRNAs are implicated in the regulation of tumor progression and stemness of cancer stem-like cells. Recently, miR-92a-3p was reported to be up-regulated in human glioma samples. Nevertheless, the precise role of miR-92a-3p in glioma cells and glioma stem-like cells (GSCs has not been fully elucidated. It is necessary to clarify the function of miR-92a-3p in glioma and GSCs to develop novel therapeutic approaches for glioma patients. In the present study, we applied methyl-thiazolyl-tetrazolium (MTT assay and Transwell assay to measure the proliferation rate and metastatic potential of glioma cells. Meanwhile, the self-renewal ability of GSCs was detected by tumor sphere formation assay. The results revealed that down-regulation of miR-92a-3p suppressed the glioma cell malignancy in vitro. Moreover, knockdown of miR-92a-3p led to a reduction of tumorgenesis in vivo. Interestingly, we also observed that up-regulation of miR-92a-3p could inhibit the stemness of GSCs. Subsequent mechanistic investigation indicated that cadherin 1 (CDH1/β-catenin signaling and Notch-1/Akt signaling were the downstream pathways of miR-92a-3p in glioma cells and GSCs, respectively. These results suggest that miR-92a-3p plays different roles in glioma cells and GSCs through regulating different signaling pathways.

  6. Characterization of VPS34-IN1, a selective inhibitor of Vps34, reveals that the phosphatidylinositol 3-phosphate-binding SGK3 protein kinase is a downstream target of class III phosphoinositide 3-kinase.

    Science.gov (United States)

    Bago, Ruzica; Malik, Nazma; Munson, Michael J; Prescott, Alan R; Davies, Paul; Sommer, Eeva; Shpiro, Natalia; Ward, Richard; Cross, Darren; Ganley, Ian G; Alessi, Dario R

    2014-11-01

    The Vps34 (vacuolar protein sorting 34) class III PI3K (phosphoinositide 3-kinase) phosphorylates PtdIns (phosphatidylinositol) at endosomal membranes to generate PtdIns(3)P that regulates membrane trafficking processes via its ability to recruit a subset of proteins possessing PtdIns(3)P-binding PX (phox homology) and FYVE domains. In the present study, we describe a highly selective and potent inhibitor of Vps34, termed VPS34-IN1, that inhibits Vps34 with 25 nM IC50 in vitro, but does not significantly inhibit the activity of 340 protein kinases or 25 lipid kinases tested that include all isoforms of class I as well as class II PI3Ks. Administration of VPS34-IN1 to cells induces a rapid dose-dependent dispersal of a specific PtdIns(3)P-binding probe from endosome membranes, within 1 min, without affecting the ability of class I PI3K to regulate Akt. Moreover, we explored whether SGK3 (serum- and glucocorticoid-regulated kinase-3), the only protein kinase known to interact specifically with PtdIns(3)P via its N-terminal PX domain, might be controlled by Vps34. Mutations disrupting PtdIns(3)P binding ablated SGK3 kinase activity by suppressing phosphorylation of the T-loop [PDK1 (phosphoinositide-dependent kinase 1) site] and hydrophobic motif (mammalian target of rapamycin site) residues. VPS34-IN1 induced a rapid ~50-60% loss of SGK3 phosphorylation within 1 min. VPS34-IN1 did not inhibit activity of the SGK2 isoform that does not possess a PtdIns(3)P-binding PX domain. Furthermore, class I PI3K inhibitors (GDC-0941 and BKM120) that do not inhibit Vps34 suppressed SGK3 activity by ~40%. Combining VPS34-IN1 and GDC-0941 reduced SGK3 activity ~80-90%. These data suggest SGK3 phosphorylation and hence activity is controlled by two pools of PtdIns(3)P. The first is produced through phosphorylation of PtdIns by Vps34 at the endosome. The second is due to the conversion of class I PI3K product, PtdIns(3,4,5)P3 into PtdIns(3)P, via the sequential actions of the Ptd

  7. Chronic low-dose radiation protects cells from high-dose radiation via increase of AKT expression by NF-{sub k}B

    Energy Technology Data Exchange (ETDEWEB)

    Park, Hyung Sun; Seong, Ki Moon; Kim, Ji Young; Kim, Cha Soon; Yang, Kwang Hee; Nam, Seon Young [Radiation Effect Research Team, Radiation Health Research Institute, Korea Hydro and Nuclear Power Co., LTD., Gyeongju (Korea, Republic of)

    2013-04-15

    Exposure to low-dose and low-dose rate of ionizing radiation is an important issue in radiation protection. Low-dose ionizing radiation has been observed to elicit distinctly different responses compared to high-dose radiation, in various biological systems including the reproductive, immune, and hematopoietic systems (Liu et al. 2006). Some data were reported that low-dose radiation could initiate beneficial effects by stimulating cell growth, DNA repair, activation of transcription factors and gene expression (Calabrese et al., 2004). Cells exposed to low-dose radiation can develop adaptive resistance to subsequent high-dose radiation induced DNA damage, gene mutation, and cell death. We previously reported that low-dose of ionizing radiation induced cell survival through the activation of AKT (protein kinase B, PKB) pathway (Park et al., 2009). AKT has been shown to be potently activated in response to a wide variety of growth factors and ionizing radiation. Cell survival against ionizing radiation seems to be associated with the activation of AKT pathway via phosphorylation of its downstream nuclear target molecules. In the present study, we examined the effects of chronic low-dose irradiation in human lung fibroblast cells. The aim was to explore the possibility of a low-dose radiation-induced adaptive cellular response against subsequent challenging high-dose irradiation. In the present study, we examined the regulatory mechanism responsible for cellular response induced by chronic low-dose of ionizing radiation in normal human cells. We found that the level of AKT protein was closely associated with cell survival. In addition, NF-{sub k}B activation by chronic low-dose radiation regulates AKT activation via gene expression and acinus expression. In conclusion, our data demonstrate that chronic low-dose radiation could inhibit the cell death induced by cytotoxic high-dose radiation through the modulation of the level of AKT and acinus proteins via NF-{sub k

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

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    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.

  9. 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

  10. 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

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

    DEFF Research Database (Denmark)

    Jönsson, Mats; Ekstrand, Anna Isinger; Jönsson, Mats;

    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...

  12. Theobromine, the primary methylxanthine found in Theobroma cacao, prevents malignant glioblastoma proliferation by negatively regulating phosphodiesterase-4, extracellular signal-regulated kinase, Akt/mammalian target of rapamycin kinase, and nuclear factor-kappa B.

    Science.gov (United States)

    Sugimoto, Naotoshi; Miwa, Shinji; Hitomi, Yoshiaki; Nakamura, Hiroyuki; Tsuchiya, Hiroyuki; Yachie, Akihiro

    2014-01-01

    Theobromine, a caffeine derivative, is the primary methylxanthine produced by Theobroma cacao. We previously showed that methylxanthines, including caffeine and theophylline, have antitumor and antiinflammatory effects, which are in part mediated by their inhibition of phosphodiesterase (PDE). A member of the PDE family, PDE4, is widely expressed in and promotes the growth of glioblastoma, the most common type of brain tumor. The purpose of this study was to determine whether theobromine could exert growth inhibitory effects on U87-MG, a cell line derived from human malignant glioma. We show that theobromine treatment elevates intracellular cAMP levels and increases the activity of p38 mitogen-activated protein kinase and c-Jun N-terminal kinase, whereas it attenuates p44/42 extracellular signal-regulated kinase activity and the Akt/mammalian target of rapamycin kinase and nuclear factor-kappa B signal pathways. It also inhibits cell proliferation. These results suggest that foods and beverages containing cocoa bean extracts, including theobromine, might be extremely effective in preventing human glioblastoma.

  13. Oncogenic AKT1(E17K) mutation induces mammary hyperplasia but prevents HER2-driven tumorigenesis.

    Science.gov (United States)

    Mancini, Maria L; Lien, Evan C; Toker, Alex

    2016-04-05

    One of the most frequently deregulated signaling pathways in breast cancer is the PI 3-K/Akt cascade. Genetic lesions are commonly found in PIK3CA, PTEN, and AKT, which lead to excessive and constitutive activation of Akt and downstream signaling that results in uncontrolled proliferation and increased cellular survival. One such genetic lesion is the somatic AKT1(E17K) mutation, which has been identified in 4-8% of breast cancer patients. To determine how this mutation contributes to mammary tumorigenesis, we constructed a genetically engineered mouse model that conditionally expresses human AKT1(E17K) in the mammary epithelium. Although AKT1(E17K) is only weakly constitutively active and does not promote proliferation in vitro, it is capable of escaping negative feedback inhibition to exhibit sustained signaling dynamics in vitro. Consistently, both virgin and multiparous AKT1(E17K) mice develop mammary gland hyperplasia that do not progress to carcinoma. This hyperplasia is accompanied by increased estrogen receptor expression, although exposure of the mice to estrogen does not promote tumor development. Moreover, AKT1(E17K) prevents HER2-driven mammary tumor formation, in part through negative feedback inhibition of RTK signaling. Analysis of TCGA breast cancer data revealed that the mRNA expression, total protein levels, and phosphorylation of various RTKs are decreased in human tumors harboring AKT1(E17K).

  14. PI3K-Akt signaling pathway upregulates hepatitis C virus RNA translation through the activation of SREBPs.

    Science.gov (United States)

    Shi, Qing; Hoffman, Brett; Liu, Qiang

    2016-03-01

    Hepatitis C virus (HCV) activates PI3K-Akt signaling to enhance entry and replication. Here, we found that this pathway also increased HCV translation. Knocking down the three Akt isoforms significantly decreased, whereas ectopic expression increased HCV translation. HCV translation upregulation by Akt required their kinase activities because Akt kinase-dead mutants downregulated HCV translation; and was dependent on PI3K activity since it was sensitive to PI3K inhibitor wortmannin. The viral 3'UTR was not involved in translation upregulation by Akt. HCV NS5A increased Akt phosphorylation/activity and HCV translation in the absence of the viral 3'UTR. Sterol regulatory element-binding proteins (SREBPs) were the downstream effectors of the PI3K-Akt pathway in regulating HCV translation because Akt1 and Akt2 activated both SREBP-1 and SREBP-2, whereas Akt3 upregulated SREBP-1. Knocking down SREBPs significantly decreased, while ectopic expression of SREBPs increased HCV translation. Taken together, we showed that the PI3K-Akt signaling pathway positively regulates HCV translation through SREBPs.

  15. 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.

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

    Science.gov (United States)

    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.

  17. Compound library screening identified Akt/PKB kinase pathway inhibitors as potential key molecules for the development of new chemotherapeutics against schistosomiasis

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    Marion Morel

    2014-12-01

    Full Text Available Protein kinases (PKs are one of the largest protein families in most eukaryotic organisms. These enzymes are involved in the control of cell proliferation, differentiation and metabolism and a large number of the anticancer drugs currently used are directed against PKs. The structure and function of PKs are well conserved throughout evolution. In schistosome parasites, PKs were shown to be involved in essential functions at every stage of the parasite life cycle, making these enzymes promising anti-parasite drug targets. In this study, we tested a panel of commercial inhibitors for various PKs and analyzed their effects on pairing and egg production by schistosomes as well as their toxicity towards schistosomula larvae. Results obtained confirmed the deleterious effect of PK targeting on Schistosoma mansoni physiology and the important function of different tyrosine and serine/threonine kinases in the biology and reproduction of this parasite. They also indicated for the first time that the Protein kinase B (also called Akt which is a major downstream target of many receptor tyrosine kinases and a central player at the crossroads of signal transduction pathways activated in response to growth factors and insulin, can constitute a novel target for anti-schistosome chemotherapy. Structural and functional studies have shown that SmAkt is a conserved kinase and that its activity can be inhibited by commercially available Akt inhibitors. In treated adult worms, Akt/PKB kinase pathway inhibitors induced profound alterations in pairing and egg laying and they also greatly affected the viability of schistosomula larvae.

  18. Activating Akt1 mutations alter DNA double strand break repair and radiosensitivity

    Science.gov (United States)

    Oeck, S.; Al-Refae, K.; Riffkin, H.; Wiel, G.; Handrick, R.; Klein, D.; Iliakis, G.; Jendrossek, V.

    2017-01-01

    The survival kinase Akt has clinical relevance to radioresistance. However, its contributions to the DNA damage response, DNA double strand break (DSB) repair and apoptosis remain poorly defined and often contradictory. We used a genetic approach to explore the consequences of genetic alterations of Akt1 for the cellular radiation response. While two activation-associated mutants with prominent nuclear access, the phospho-mimicking Akt1-TDSD and the clinically relevant PH-domain mutation Akt1-E17K, accelerated DSB repair and improved survival of irradiated Tramp-C1 murine prostate cancer cells and Akt1-knockout murine embryonic fibroblasts in vitro, the classical constitutively active membrane-targeted myrAkt1 mutant had the opposite effects. Interestingly, DNA-PKcs directly phosphorylated Akt1 at S473 in an in vitro kinase assay but not vice-versa. Pharmacological inhibition of DNA-PKcs or Akt restored radiosensitivity in tumour cells expressing Akt1-E17K or Akt1-TDSD. In conclusion, Akt1-mediated radioresistance depends on its activation state and nuclear localization and is accessible to pharmacologic inhibition. PMID:28209968

  19. K-ras/PI3K-Akt signaling is essential for zebrafish hematopoiesis and angiogenesis.

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    Lihui Liu

    Full Text Available The RAS small GTPases orchestrate multiple cellular processes. Studies on knock-out mice showed the essential and sufficient role of K-RAS, but not N-RAS and H-RAS in embryonic development. However, many physiological functions of K-RAS in vivo remain unclear. Using wild-type and fli1:GFP transgenic zebrafish, we showed that K-ras-knockdown resulted in specific hematopoietic and angiogenic defects, including the impaired expression of erythroid-specific gene gata1 and sse3-hemoglobin, reduced blood circulation and disorganized blood vessels. Expression of either K-rasC40 that links to phosphoinositide 3-kinase (PI3K activation, or Akt2 that acts downstream of PI3K, could rescue both hematopoietic and angiogenic defects in the K-ras knockdown. Consistently, the functional rescue by k-ras mRNA was significantly suppressed by wortmannin, a PI3K-specific inhibitor. Our results provide direct evidence that PI3K-Akt plays a crucial role in mediating K-ras signaling during hematopoiesis and angiogenesis in vivo, thus offering new targets and alternative vertebrate model for studying these processes and their related diseases.

  20. 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

  1. Phospho-Akt overexpression is prognostic and can be used to tailor the synergistic interaction of Akt inhibitors with gemcitabine in pancreatic cancer

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    Daniela Massihnia

    2017-01-01

    decreased cell migration and invasion, which was additionally reduced by the combination with gemcitabine. This combination significantly increased apoptosis, associated with induction of caspase-3/6/8/9, PARP and BAD, and inhibition of Bcl-2 and NF-kB in LPC028, but not in LPC006 cells. However, targeting the key glucose transporter Glut1 resulted in similar apoptosis induction in LPC006 cells. Conclusions These data support the analysis of phospho-Akt expression as both a prognostic and a predictive biomarker, for the rational development of new combination therapies targeting the Akt pathway in PDAC. Finally, inhibition of Glut1 might overcome resistance to these therapies and warrants further studies.

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

    Science.gov (United States)

    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.

  3. Sann-Joong-Kuey-Jian-Tang induces autophagy in HepG2 cells via regulation of the phosphoinositide-3 kinase/Akt/mammalian target of rapamycin and p38 mitogen-activated protein kinase pathways.

    Science.gov (United States)

    Chuang, Wan-Ling; Su, Chin-Cheng; Lin, Ping-Yi; Lin, Chi-Chen; Chen, Yao-Li

    2015-08-01

    Sann-Joong-Kuey-Jian-Tang (SJKJT), a traditional Chinese medicine, was previously reported to induce autophagy and inhibit the proliferation of the human HepG2 hepatocellular carcinoma cell line via an extrinsic pathway. In the present study, the effects of SJKJT-induced autophagy and the cytotoxic mechanisms mediating these effects were investigated in HepG2 cells. The cytotoxicity of SJKJT in the HepG2 cells was evaluated using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. The results demonstrated that the half-maximal inhibitory concentration of SJKJT was 2.91 mg/ml at 24 h, 1.64 mg/ml at 48 h and 1.26 mg/ml at 72 h. The results of confocal fluorescence microscopy indicated that SJKJT resulted in the accumulation of green fluorescent protein-LC3 and vacuolation of the cytoplasm. Flow cytometric analysis revealed the accumulation of acidic vesicular organelles. Furthermore, western blot analysis, used to determine the expression levels of autophagy-associated proteins, demonstrated that the HepG2 cells treated with SJKJT exhibited LC3B-I/LC3B-II conversion, increased expression levels of Beclin, Atg-3 and Atg-5 and reduced expression levels of p62 and decreased signaling of the phosphoinositide-3 kinase/Akt/mammalian target of rapamycin and the p38 mitogen-activated protein kinase pathways. Taken together, these findings may assist in the development of novel chemotherapeutic agents for the treatment of malignant types of liver cancer.

  4. 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

    Directory of Open Access Journals (Sweden)

    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.

  5. 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.

  6. Regulation of mTORC1 Signaling by Src Kinase Activity Is Akt1-Independent in RSV-Transformed Cells

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    Martina Vojtěchová

    2008-02-01

    Full Text Available Increased activity of the Src tyrosine protein kinase that has been observed in a large number of human malignancies appears to be a promising target for drug therapy. In the present study, a critical role of the Src activity in the deregulation of mTOR signaling pathway in Rous sarcoma virus (RSV-transformed hamster fibroblasts, H19 cells, was shown using these cells treated with the Src-specific inhibitor, SU6656, and clones of fibroblasts expressing either the active Src or the dominant-negative Src kinase-dead mutant. Disruption of the Src kinase activity results in substantial reduction of the phosphorylation and activity of the Akt/protein kinase B (PKB, phosphorylation of tuberin (TSC2, mammalian target of rapamycin (mTOR, S6K1, ribosomal protein S6, and eukaryotic initiation factor 4E-binding protein 4E-BP1. The ectopic, active Akt1 that was expressed in Src-deficient cells significantly enhanced phosphorylation of TSC2 in these cells, but it failed to activate the inhibited components of the mTOR pathway that are downstream of TSC2. The data indicate that the Src kinase activity is essential for the activity of mTOR-dependent signaling pathway and suggest that mTOR targets may be controlled by Src independently of Akt1/TSC2 cascade in cells expressing hyperactive Src protein. These observations might have an implication in drug resistance to mTOR inhibitor-based cancer therapy in certain cell types.

  7. BMX acts downstream of PI3K to promote colorectal cancer cell survival and pathway inhibition sensitizes to the BH3 mimetic ABT-737.

    Science.gov (United States)

    Potter, Danielle S; Kelly, Paul; Denneny, Olive; Juvin, Veronique; Stephens, Len R; Dive, Caroline; Morrow, Christopher J

    2014-02-01

    Evasion of apoptosis is a hallmark of cancer, and reversing this process by inhibition of survival signaling pathways is a potential therapeutic strategy. Phosphoinositide 3-kinase (PI3K) signaling can promote cell survival and is upregulated in solid tumor types, including colorectal cancer (CRC), although these effects are context dependent. The role of PI3K in tumorigenesis combined with their amenability to specific inhibition makes them attractive drug targets. However, we observed that inhibition of PI3K in HCT116, DLD-1, and SW620 CRC cells did not induce apoptotic cell death. Moreover, these cells were relatively resistant to the Bcl-2 homology domain 3 (BH3) mimetic ABT-737, which directly targets the Bcl-2 family of apoptosis regulators. To test the hypothesis that PI3K inhibition lowers the apoptotic threshold without causing apoptosis per se, PI3K inhibitors were combined with ABT-737. PI3K inhibition enhanced ABT-737-induced apoptosis by 2.3- to 4.5-fold and reduced expression levels of MCL-1, the resistance biomarker for ABT-737. PI3K inhibition enhanced ABT-737-induced apoptosis a further 1.4- to 2.4-fold in CRC cells with small interfering RNA-depleted MCL-1, indicative of additional sensitizing mechanisms. The observation that ABT-737-induced apoptosis was unaffected by inhibition of PI3K downstream effectors AKT and mTOR, implicated a novel PI3K-dependant pathway. To elucidate this, an RNA interference (RNAi) screen of potential downstream effectors of PI3K signaling was conducted, which demonstrated that knockdown of the TEC kinase BMX sensitized to ABT-737. This suggests that BMX is an antiapoptotic downstream effector of PI3K, independent of AKT.

  8. BMX Acts Downstream of PI3K to Promote Colorectal Cancer Cell Survival and Pathway Inhibition Sensitizes to the BH3 Mimetic ABT-737

    Directory of Open Access Journals (Sweden)

    Danielle S. Potter

    2014-02-01

    Full Text Available Evasion of apoptosis is a hallmark of cancer, and reversing this process by inhibition of survival signaling pathways is a potential therapeutic strategy. Phosphoinositide 3-kinase (PI3K signaling can promote cell survival and is upregulated in solid tumor types, including colorectal cancer (CRC, although these effects are context dependent. The role of PI3K in tumorigenesis combined with their amenability to specific inhibition makes them attractive drug targets. However, we observed that inhibition of PI3K in HCT116, DLD-1, and SW620 CRC cells did not induce apoptotic cell death. Moreover, these cells were relatively resistant to the Bcl-2 homology domain 3 (BH3 mimetic ABT-737, which directly targets the Bcl-2 family of apoptosis regulators. To test the hypothesis that PI3K inhibition lowers the apoptotic threshold without causing apoptosis per se, PI3K inhibitors were combined with ABT-737. PI3K inhibition enhanced ABT-737-induced apoptosis by 2.3- to 4.5-fold and reduced expression levels of MCL-1, the resistance biomarker for ABT-737. PI3K inhibition enhanced ABT-737-induced apoptosis a further 1.4- to 2.4-fold in CRC cells with small interfering RNA-depleted MCL-1, indicative of additional sensitizing mechanisms. The observation that ABT-737-induced apoptosis was unaffected by inhibition of PI3K downstream effectors AKT and mTOR, implicated a novel PI3K-dependant pathway. To elucidate this, an RNA interference (RNAi screen of potential downstream effectors of PI3K signaling was conducted, which demonstrated that knockdown of the TEC kinase BMX sensitized to ABT-737. This suggests that BMX is an antiapoptotic downstream effector of PI3K, independent of AKT.

  9. REDD1 integrates hypoxia-mediated survival signaling downstream of phosphatidylinositol 3-kinase.

    Science.gov (United States)

    Schwarzer, Rolf; Tondera, Daniel; Arnold, Wolfgang; Giese, Klaus; Klippel, Anke; Kaufmann, Jörg

    2005-02-10

    Cancer cells frequently evade apoptosis during tumorigenesis by acquiring mutations in apoptotic regulators. Chronic activation of the PI 3-kinase-Akt pathway through loss of the tumor suppressor PTEN is one mechanism by which these cells can gain increased protection against apoptosis. We report here that REDD1 (RTP801) can act as a transcriptional downstream target of PI 3-kinase signaling in human prostate cancer cells (PC-3). REDD1 expression is markedly reduced in PC-3 cells treated with LY294002 (LY) or Rapamycin and strongly induced under hypoxic conditions in a hypoxia-inducible factor-1 (HIF-1)-dependent manner. Loss of function studies employing antisense molecules or RNA interference indicate that REDD1 is essential for invasive growth of prostate cancer cells in vitro and in vivo. Reduced REDD1 levels can sensitize cells towards apoptosis, whereas elevated levels of REDD1 induced by hypoxia or overexpression desensitize cells to apoptotic stimuli. Taken together our data designate REDD1 as a novel target for therapeutic intervention in prostate cancer.

  10. 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.

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

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    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.

  12. ApoA-IV improves insulin sensitivity and glucose uptake in mouse adipocytes via PI3K-Akt Signaling

    Science.gov (United States)

    Li, Xiaoming; Wang, Fei; Xu, Min; Howles, Philip; Tso, Patrick

    2017-01-01

    Insulin resistance is a risk factor for type 2 diabetes mellitus. We investigated the effect of ApoA-IV on glucose uptake in the adipose and muscle tissues of mice and cultured 3T3-L1 adipocytes. We found that treatment with ApoA-IV lowered fasting blood glucose in both WT and diabetic KKAy mice by increasing glucose uptake in cardiac muscle, white adipose tissue, and brown adipose tissue through a mechanism that was partially insulin independent. Cell culture experiments showed that ApoA-IV improved glucose uptake in adipocytes in the absence of insulin by upregulating GLUT4 translocation by PI3K mediated activation of Akt signaling pathways. Considering our previous finding that ApoA-IV treatment enhanced pancreatic insulin secretion, these results suggests that ApoA-IV acts directly upon adipose tissue to improve glucose uptake and indirectly via insulin signaling. Our findings warrant future studies to identify the receptor for ApoA-IV and the downstream targets of PI3K-Akt signaling that regulate glucose uptake in adipocytes as potential therapeutic targets for treating insulin resistance. PMID:28117404

  13. 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.

  14. 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

    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...

  15. A critical role of the small GTPase Rac1 in Akt2-mediated GLUT4 translocation in mouse skeletal muscle.

    Science.gov (United States)

    Takenaka, Nobuyuki; Izawa, Rumi; Wu, Junyuan; Kitagawa, Kaho; Nihata, Yuma; Hosooka, Tetsuya; Noguchi, Tetsuya; Ogawa, Wataru; Aiba, Atsu; Satoh, Takaya

    2014-03-01

    Insulin promotes glucose uptake in skeletal muscle by inducing the translocation of the glucose transporter GLUT4 to the plasma membrane. The serine/threonine kinase Akt2 has been implicated as a key regulator of this insulin action. However, the mechanisms whereby Akt2 regulates multiple steps of GLUT4 translocation remain incompletely understood. Recently, the small GTPase Rac1 has been identified as a skeletal muscle-specific regulator of insulin-stimulated glucose uptake. Here, we show that Rac1 is a critical downstream component of the Akt2 pathway in mouse skeletal muscle as well as cultured myocytes. GLUT4 translocation induced by constitutively activated Akt2 was totally dependent on the expression of Rac1 in L6 myocytes. Moreover, we observed the activation of Rac1 when constitutively activated Akt2 was ectopically expressed. Constitutively activated Akt2-triggered Rac1 activation was diminished by knockdown of FLJ00068, a guanine nucleotide exchange factor for Rac1. Knockdown of Akt2, on the other hand, markedly reduced Rac1 activation by a constitutively activated mutant of phosphoinositide 3-kinase. In mouse skeletal muscle, constitutively activated mutants of Akt2 and phosphoinositide 3-kinase, when ectopically expressed, induced GLUT4 translocation. Muscle-specific rac1 knockout markedly diminished Akt2- or phosphoinositide 3-kinase-induced GLUT4 translocation, highlighting a crucial role of Rac1 downstream of Akt2. Taken together, these results strongly suggest a novel regulatory link between Akt2 and Rac1 in insulin-dependent signal transduction leading to glucose uptake in skeletal muscle.

  16. 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,

  17. 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

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    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.

  18. 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.)

  19. Increased Akt signaling in the mosquito fat body increases adult survivorship.

    Science.gov (United States)

    Arik, Anam J; Hun, Lewis V; Quicke, Kendra; Piatt, Michael; Ziegler, Rolf; Scaraffia, Patricia Y; Badgandi, Hemant; Riehle, Michael A

    2015-04-01

    Akt signaling regulates diverse physiologies in a wide range of organisms. We examine the impact of increased Akt signaling in the fat body of 2 mosquito species, the Asian malaria mosquito Anopheles stephensi and the yellow fever mosquito Aedes aegypti. Overexpression of a myristoylated and active form of A. stephensi and Ae. aegypti Akt in the fat body of transgenic mosquitoes led to activation of the downstream signaling molecules forkhead box O (FOXO) and p70 S6 kinase in a tissue and blood meal-specific manner. In both species, increased Akt signaling in the fat body after blood feeding significantly increased adult survivorship relative to nontransgenic sibling controls. In A. stephensi, survivorship was increased by 15% to 45%, while in Ae. aegypti, it increased 14% to 47%. Transgenic mosquitoes fed only sugar, and thus not expressing active Akt, had no significant difference in survivorship relative to nontransgenic siblings. Expression of active Akt also increased expression of fat body vitellogenin, but the number of viable eggs did not differ significantly between transgenic and nontransgenic controls. This work demonstrates a novel mechanism of enhanced survivorship through increased Akt signaling in the fat bodies of multiple mosquito genera and provides new tools to unlock the molecular underpinnings of aging in eukaryotic organisms.

  20. A Positive Feedback Loop between Akt and mTORC2 via SIN1 Phosphorylation

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    Guang Yang

    2015-08-01

    Full Text Available The mechanistic target of rapamycin complex 2 (mTORC2 regulates cell survival and cytoskeletal organization by phosphorylating its AGC kinase substrates; however, little is known about the regulation of mTORC2 itself. It was previously reported that Akt phosphorylates the mTORC2 subunit SIN1 at T86, activating mTORC2 through a positive feedback loop, though another study reported that S6K phosphorylates SIN1 at the same site, inhibiting mTORC2 activity. We performed extensive analysis of SIN1 phosphorylation upon inhibition of Akt, S6K, and mTOR under diverse cellular contexts, and we found that, in all cell lines and conditions studied, Akt is the major kinase responsible for SIN1 phosphorylation. These findings refine the activation mechanism of the Akt-mTORC2 signaling branch as follows: PDK1 phosphorylates Akt at T308, increasing Akt kinase activity. Akt phosphorylates SIN1 at T86, enhancing mTORC2 kinase activity, which leads to phosphorylation of Akt S473 by mTORC2, thereby catalyzing full activation of Akt.

  1. A Positive Feedback Loop between Akt and mTORC2 via SIN1 Phosphorylation.

    Science.gov (United States)

    Yang, Guang; Murashige, Danielle S; Humphrey, Sean J; James, David E

    2015-08-11

    The mechanistic target of rapamycin complex 2 (mTORC2) regulates cell survival and cytoskeletal organization by phosphorylating its AGC kinase substrates; however, little is known about the regulation of mTORC2 itself. It was previously reported that Akt phosphorylates the mTORC2 subunit SIN1 at T86, activating mTORC2 through a positive feedback loop, though another study reported that S6K phosphorylates SIN1 at the same site, inhibiting mTORC2 activity. We performed extensive analysis of SIN1 phosphorylation upon inhibition of Akt, S6K, and mTOR under diverse cellular contexts, and we found that, in all cell lines and conditions studied, Akt is the major kinase responsible for SIN1 phosphorylation. These findings refine the activation mechanism of the Akt-mTORC2 signaling branch as follows: PDK1 phosphorylates Akt at T308, increasing Akt kinase activity. Akt phosphorylates SIN1 at T86, enhancing mTORC2 kinase activity, which leads to phosphorylation of Akt S473 by mTORC2, thereby catalyzing full activation of Akt.

  2. 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.

  3. Drosophila tribbles antagonizes insulin signaling-mediated growth and metabolism via interactions with Akt kinase.

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    Rahul Das

    Full Text Available Drosophila Tribbles (Trbl is the founding member of the Trib family of kinase-like docking proteins that modulate cell signaling during proliferation, migration and growth. In a wing misexpression screen for Trbl interacting proteins, we identified the Ser/Thr protein kinase Akt1. Given the central role of Akt1 in insulin signaling, we tested the function of Trbl in larval fat body, a tissue where rapid increases in size are exquisitely sensitive to insulin/insulin-like growth factor levels. Consistent with a role in antagonizing insulin-mediated growth, trbl RNAi knockdown in the fat body increased cell size, advanced the timing of pupation and increased levels of circulating triglyceride. Complementarily, overexpression of Trbl reduced fat body cell size, decreased overall larval size, delayed maturation and lowered levels of triglycerides, while circulating glucose levels increased. The conserved Trbl kinase domain is required for function in vivo and for interaction with Akt in a yeast two-hybrid assay. Consistent with direct regulation of Akt, overexpression of Trbl in the fat body decreased levels of activated Akt (pSer505-Akt while misexpression of trbl RNAi increased phospho-Akt levels, and neither treatment affected total Akt levels. Trbl misexpression effectively suppressed Akt-mediated wing and muscle cell size increases and reduced phosphorylation of the Akt target FoxO (pSer256-FoxO. Taken together, these data show that Drosophila Trbl has a conserved role to bind Akt and block Akt-mediated insulin signaling, and implicate Trib proteins as novel sites of signaling pathway integration that link nutrient availability with cell growth and proliferation.

  4. SDF-1α/CXCR4 Signaling in Lipid Rafts Induces Platelet Aggregation via PI3 Kinase-Dependent Akt Phosphorylation

    Science.gov (United States)

    Hayashi, Moyuru; Kaneda, Mizuho; Iida, Kazuko; Shimonaka, Motoyuki; Hara, Takahiko; Arai, Morio; Koike, Yuichi; Yamamoto, Naomasa; Kasahara, Kohji

    2017-01-01

    Stromal cell-derived factor-1α (SDF-1α)-induced platelet aggregation is mediated through its G protein-coupled receptor CXCR4 and phosphatidylinositol 3 kinase (PI3K). Here, we demonstrate that SDF-1α induces phosphorylation of Akt at Thr308 and Ser473 in human platelets. SDF-1α-induced platelet aggregation and Akt phosphorylation are inhibited by pretreatment with the CXCR4 antagonist AMD3100 or the PI3K inhibitor LY294002. SDF-1α also induces the phosphorylation of PDK1 at Ser241 (an upstream activator of Akt), GSK3β at Ser9 (a downstream substrate of Akt), and myosin light chain at Ser19 (a downstream element of the Akt signaling pathway). SDF-1α-induced platelet aggregation is inhibited by pretreatment with the Akt inhibitor MK-2206 in a dose-dependent manner. Furthermore, SDF-1α-induced platelet aggregation and Akt phosphorylation are inhibited by pretreatment with the raft-disrupting agent methyl-β-cyclodextrin. Sucrose density gradient analysis shows that 35% of CXCR4, 93% of the heterotrimeric G proteins Gαi-1, 91% of Gαi-2, 50% of Gβ and 4.0% of PI3Kβ, and 4.5% of Akt2 are localized in the detergent-resistant membrane raft fraction. These findings suggest that SDF-1α/CXCR4 signaling in lipid rafts induces platelet aggregation via PI3K-dependent Akt phosphorylation. PMID:28072855

  5. SDF-1α/CXCR4 Signaling in Lipid Rafts Induces Platelet Aggregation via PI3 Kinase-Dependent Akt Phosphorylation.

    Science.gov (United States)

    Ohtsuka, Hiroko; Iguchi, Tomohiro; Hayashi, Moyuru; Kaneda, Mizuho; Iida, Kazuko; Shimonaka, Motoyuki; Hara, Takahiko; Arai, Morio; Koike, Yuichi; Yamamoto, Naomasa; Kasahara, Kohji

    2017-01-01

    Stromal cell-derived factor-1α (SDF-1α)-induced platelet aggregation is mediated through its G protein-coupled receptor CXCR4 and phosphatidylinositol 3 kinase (PI3K). Here, we demonstrate that SDF-1α induces phosphorylation of Akt at Thr308 and Ser473 in human platelets. SDF-1α-induced platelet aggregation and Akt phosphorylation are inhibited by pretreatment with the CXCR4 antagonist AMD3100 or the PI3K inhibitor LY294002. SDF-1α also induces the phosphorylation of PDK1 at Ser241 (an upstream activator of Akt), GSK3β at Ser9 (a downstream substrate of Akt), and myosin light chain at Ser19 (a downstream element of the Akt signaling pathway). SDF-1α-induced platelet aggregation is inhibited by pretreatment with the Akt inhibitor MK-2206 in a dose-dependent manner. Furthermore, SDF-1α-induced platelet aggregation and Akt phosphorylation are inhibited by pretreatment with the raft-disrupting agent methyl-β-cyclodextrin. Sucrose density gradient analysis shows that 35% of CXCR4, 93% of the heterotrimeric G proteins Gαi-1, 91% of Gαi-2, 50% of Gβ and 4.0% of PI3Kβ, and 4.5% of Akt2 are localized in the detergent-resistant membrane raft fraction. These findings suggest that SDF-1α/CXCR4 signaling in lipid rafts induces platelet aggregation via PI3K-dependent Akt phosphorylation.

  6. 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.

  7. Resveratrol chemosensitizes HER-2-overexpressing breast cancer cells to docetaxel chemoresistance by inhibiting docetaxel-mediated activation of HER-2-Akt axis.

    Science.gov (United States)

    Vinod, B S; Nair, H H; Vijayakurup, V; Shabna, A; Shah, S; Krishna, A; Pillai, K S; Thankachan, S; Anto, R J

    2015-01-01

    As breast cancer cells often develop chemoresistance, better therapeutic options are in search to circumvent it. Here we demonstrate that human epidermal growth factor receptor-2 (HER-2)-overexpressing breast cancer cells resist docetaxel-induced cytotoxicity by upregulating HER-2 and its activity downstream, through Akt and mitogen-activated protein kinase (MAPK) pathways. We observed that introducing resveratrol as a chemosensitizer in docetaxel chemotherapy blocks upregulation and activation of HER-2 in addition to blocking downstream signaling pathways such as Akt. Resveratrol and docetaxel combination results in the synergistic induction of cell death in HER-2-overexpressing SK-BR-3 cells, whereas introduction of wild-type HER-2 in MDA-MD-231 cells increased the resistance to docetaxel. Dominant-negative HER-2 sensitizes SK-BR-3 cells to docetaxel. Our study identified a new synergistic therapeutic combination that targets HER-2-induced breast cancer resistance and might help to overcome therapeutic resistance during breast cancer therapy. The synergism of docetaxel and resveratrol was maximum in SK-BR-3, which is unique among the cell lines studied, due to its high expression status of HER-2, a receptor known to dictate the signaling environment of breast cancer cells. Docetaxel could further induce HER-2 activity in these cells, which was downregulated on resveratrol treatment. Transfection of DN-HER-2 in SK-BR-3 cells inhibits the synergism as the transfection itself sensitizes these cells to docetaxel, leaving no role for resveratrol, whereas ectopic expression of HER-2 introduces the synergism in MDA-MB-231, the triple-negative cell line, in which the synergism was minimum, attesting the crucial role of HER-2 in suppressing the sensitivity to docetaxel. Single-agent docetaxel induced HER-2-mediated resistance to cell death, which was blocked by resveratrol. Resveratrol also downregulated docetaxel-induced activation of MAPK and Akt, survival signaling

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

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    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.

  9. Reversing hypomyelination in BACE1-null mice with Akt-DD overexpression.

    Science.gov (United States)

    Hu, Xiangyou; Schlanger, Rita; He, Wanxia; Macklin, Wendy B; Yan, Riqiang

    2013-05-01

    β-Site amyloid precursor protein convertase enzyme 1 (BACE1), a type I transmembrane aspartyl protease required to cleave amyloid precursor protein for releasing a toxic amyloid peptide, also cleaves type I and type III neuregulin-1 (Nrg-1). BACE1 deficiency in mice causes hypomyelination during development and impairs remyelination if injured. In BACE1-null mice, the abolished cleavage of neuregulin-1 by BACE1 is speculated to cause reduced myelin sheath thickness in both the central nervous system and peripheral nervous system because reduced cleavage of Nrg-1 correlates with reduced Akt phosphorylation, a downstream signaling molecule of the Nrg-1/ErbB pathway. Here we tested specifically whether increasing Akt activity alone in oligodendrocytes would be sufficient to reverse the hypomyelination phenotype in BACE1-null mice. BACE1-null mice were bred with transgenic mice expressing constitutively active Akt (Akt-DD; mutations with D(308)T and D(473)S) in oligodendrocytes. Relative to littermate BACE1-null controls, BACE1(-/-)/Akt-DD mice exhibited enhanced expression of myelin basic protein and promoter of proteolipid protein. The elevated expression of myelin proteins correlated with a thicker myelin sheath in optic nerves; comparison of quantified g ratios with statistic significance was used to confirm this reversion. However, it appeared that myelin sheath thickness in the sciatic nerves was not increased in BACE1(-/-)/Akt-DD mice, as the g ratio was not significantly different from the control. Hence, increased Akt activity in BACE1-null myelinating cells only compensates for the loss of BACE1 activity in the central nervous system, which is consistent with the observation that overexpression of Akt-DD in Schwann cells did not induce hypermyelination. Our results suggest that signaling activity other than Akt may also contribute to proper myelination in peripheral nerves.

  10. Association of MTOR and AKT Gene Polymorphisms with Susceptibility and Survival of Gastric Cancer.

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    Ying Piao

    Full Text Available The phosphoinositide 3-kinase (PI3K/protein kinase B (PKB, AKT/mammalian target of rapamycin (mTOR signaling pathway plays a critical role in angiogenesis and cell growth, proliferation, metabolism, migration, differentiation, and apoptosis. Genetic diversity in key factors of this pathway may influence protein function and signal transduction, contributing to disease initiation and progression. Studies suggest that MTOR rs1064261 and AKT rs1130233 polymorphisms are associated with risk and/or prognosis of multiple cancer types. However, this relationship with gastric cancer (GC remains unclear. The aim of this study was to investigate the role of MTOR and AKT polymorphisms in the risk and prognosis of GC.The Sequenom MassARRAY platform was used to genotype 1842 individuals for MTOR rs1064261 T→C and AKT rs1130233 G→A polymorphisms. ELISA was used to detect Helicobacter pylori antibodies in serum. Immunohistochemical analysis was used to detect total and phosphorylated MTOR and AKT proteins.The MTOR rs1064261 (TC+CC genotype and the AKT rs1130233 (GA+AA genotype were associated with increased risk of GC in men (P = 0.049, P = 0.030. In H. pylori-negative individuals, the AKT rs1130233 GA and (GA+AA genotypes were related to increased risk of atrophic gastritis (AG; P = 0.012, P = 0.024. Notably, the AKT rs1130233 (GA+AA genotype demonstrated significant interactions with H. pylori in disease progression from healthy controls (CON to AG (P = 0.013 and from AG to GC (P = 0.049. Additionally, for individuals with the AKT rs1130233 variant, those in the H. pylori-positive group had higher levels of phosphorylated AKT (p-AKT expression. The AKT rs1130233 genotype was found to be associated with clinicopathological parameters including lymph node metastasis and alcohol drinking (P<0.05.MTOR rs1064261and AKT rs1130233 polymorphisms were associated with increased GC risk in males and increased AG risk in H. pylori-negative individuals. A significant

  11. Computational Insights into the Inhibitory Mechanism of Human AKT1 by an Orally Active Inhibitor, MK-2206

    OpenAIRE

    Mohd Rehan; Beg, Mohd A.; Shadma Parveen; Ghazi A Damanhouri; Galila F Zaher

    2014-01-01

    The AKT signaling pathway has been identified as an important target for cancer therapy. Among small-molecule inhibitors of AKT that have shown tremendous potential in inhibiting cancer, MK-2206 is a highly potent, selective and orally active allosteric inhibitor. Promising preclinical anticancer results have led to entry of MK-2206 into Phase I/II clinical trials. Despite such importance, the exact binding mechanism and the molecular interactions of MK-2206 with human AKT are not available. ...

  12. Recurrent BCAM-AKT2 fusion gene leads to a constitutively activated AKT2 fusion kinase in high-grade serous ovarian carcinoma

    Science.gov (United States)

    Kannan, Kalpana; Coarfa, Cristian; Chao, Pei-Wen; Luo, Liming; Wang, Yan; Brinegar, Amy E.; Hawkins, Shannon M.; Milosavljevic, Aleksandar; Matzuk, Martin M.; Yen, Laising

    2015-01-01

    High-grade serous ovarian cancer (HGSC) is among the most lethal forms of cancer in women. Excessive genomic rearrangements, which are expected to create fusion oncogenes, are the hallmark of this cancer. Here we report a cancer-specific gene fusion between BCAM, a membrane adhesion molecule, and AKT2, a key kinase in the PI3K signaling pathway. This fusion is present in 7% of the 60 patient cancers tested, a significant frequency considering the highly heterogeneous nature of this malignancy. Further, we provide direct evidence that BCAM-AKT2 is translated into an in-frame fusion protein in the patient’s tumor. The resulting AKT2 fusion kinase is membrane-associated, constitutively phosphorylated, and activated as a functional kinase in cells. Unlike endogenous AKT2, whose activity is tightly regulated by external stimuli, BCAM-AKT2 escapes the regulation from external stimuli. Moreover, a BCAM-AKT2 fusion gene generated via chromosomal translocation using the CRISPR/Cas9 system leads to focus formation in both OVCAR8 and HEK-293T cell lines, suggesting that BCAM-AKT2 is oncogenic. Together, the results indicate that BCAM-AKT2 expression is a new mechanism of AKT2 kinase activation in HGSC. BCAM-AKT2 is the only fusion gene in HGSC that is proven to translate an aberrant yet functional kinase fusion protein with oncogenic properties. This recurrent genomic alteration is a potential therapeutic target and marker of a clinically relevant subtype for tailored therapy of HGSC. PMID:25733895

  13. 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.

  14. 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

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

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    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.

  16. 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).

  17. 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.

  18. Breast tumor cells with PI3K mutation or HER2 amplification are selectively addicted to Akt signaling.

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    Qing-Bai She

    Full Text Available BACKGROUND: Dysregulated PI3K/Akt signaling occurs commonly in breast cancers and is due to HER2 amplification, PI3K mutation or PTEN inactivation. The objective of this study was to determine the role of Akt activation in breast cancer as a function of mechanism of activation and whether inhibition of Akt signaling is a feasible approach to therapy. METHODOLOGY/PRINCIPAL FINDINGS: A selective allosteric inhibitor of Akt kinase was used to interrogate a panel of breast cancer cell lines characterized for genetic lesions that activate PI3K/Akt signaling: HER2 amplification or PI3K or PTEN mutations in order to determine the biochemical and biologic consequences of inhibition of this pathway. A variety of molecular techniques and tissue culture and in vivo xenograft models revealed that tumors with mutational activation of Akt signaling were selectively dependent on the pathway. In sensitive cells, pathway inhibition resulted in D-cyclin loss, G1 arrest and induction of apoptosis, whereas cells without pathway activation were unaffected. Most importantly, the drug effectively inhibited Akt kinase and its downstream effectors in vivo and caused complete suppression of the growth of breast cancer xenografts with PI3K mutation or HER2 amplification, including models of the latter selected for resistance to Herceptin. Furthermore, chronic administration of the drug was well-tolerated, causing only transient hyperglycemia without gross toxicity to the host despite the pleiotropic normal functions of Akt. CONCLUSIONS/SIGNIFICANCE: These data demonstrate that breast cancers with PI3K mutation or HER2 amplification are selectively dependent on Akt signaling, and that effective inhibition of Akt in tumors is feasible and effective in vivo. These findings suggest that direct inhibition of Akt may represent a therapeutic strategy for breast and other cancers that are addicted to the pathway including tumors with resistant to Herceptin.

  19. 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 < 0.05). In conclusion, our data indicate that aberrant Akt signalling contributes to maintaining stemness in lung cancer TICs through a NF-kB/IL-6/STAT3 pathway and provide novel potential therapeutic targets for eliminating these malignant cells in NSCLC.

  20. 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

    2015-01-01

    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 < 0.05). In conclusion, our data indicate that aberrant Akt signalling contributes to maintaining stemness in lung cancer TICs through a NF-kB/IL-6/STAT3 pathway and provide novel potential therapeutic targets for eliminating these malignant cells in NSCLC. PMID:26486080

  1. Statins exhibit anticancer effects through modifications of the pAkt signaling pathway.

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    Miraglia, Erica; Högberg, Johan; Stenius, Ulla

    2012-03-01

    Statins are cholesterol lowering drugs that exhibit antitumor effects in several in vitro and in vivo models, and epidemiological studies indicate that statins prevent cancer. However, the molecular mechanism underlying the effects of statins still needs to be elucidated. We previously demonstrated that single doses of different statins rapidly affect Akt signaling via the purinergic receptor P2X7. In particular, statins down-regulated nuclear pAkt. Here, we report that long-term treatment of A549 cells with high concentrations of statins (15-75 µM) selects cell sub-populations exhibiting altered P2X receptor expression, signs of increased PTEN activity, enhanced PHLPP2, decreased PI3K p110β and inhibited downstream pAkt signaling. Furthermore, the nuclear accumulation of pAkt in response to insulin was inhibited in selected cells. Statin-selected cells displayed reduced proliferation rate and were more vulnerable to etoposide- and 5-fluorouracil-elicited cytotoxic effects. The stability of a selected phenotype (50 µM) was tested for three weeks in the absence of statins. This resulted in a reversal of some, but not all alterations. Importantly, the truncated nuclear insulin response was retained. We conclude that long-term treatment with high doses of statins selects cells exhibiting stable alterations in insulin-Akt signaling and which are vulnerable to DNA damage. Our studies strengthen the hypothesis that an altered Akt signaling has a role in chemopreventive effects of statins.

  2. 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.

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

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    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)

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

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    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.

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

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    Kuo Selena Z

    2012-11-01

    Full Text Available Abstract Background 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. Methods 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. Results 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. Conclusions These results demonstrate that in HNSCC cancer stem cells, salinomycin can cause cell death and

  6. A novel PKB/Akt inhibitor, MK-2206, effectively inhibits insulin-stimulated glucose metabolism and protein synthesis in isolated rat skeletal muscle.

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    Lai, Yu-Chiang; Liu, Yang; Jacobs, Roxane; Rider, Mark H

    2012-10-01

    PKB (protein kinase B), also known as Akt, is a key component of insulin signalling. Defects in PKB activation lead to insulin resistance and metabolic disorders, whereas PKB overactivation has been linked to tumour growth. Small-molecule PKB inhibitors have thus been developed for cancer treatment, but also represent useful tools to probe the roles of PKB in insulin action. In the present study, we examined the acute effects of two allosteric PKB inhibitors, MK-2206 and Akti 1/2 (Akti) on PKB signalling in incubated rat soleus muscles. We also assessed the effects of the compounds on insulin-stimulated glucose uptake, glycogen and protein synthesis. MK-2206 dose-dependently inhibited insulin-stimulated PKB phosphorylation, PKBβ activity and phosphorylation of PKB downstream targets (including glycogen synthase kinase-3α/β, proline-rich Akt substrate of 40 kDa and Akt substrate of 160 kDa). Insulin-stimulated glucose uptake, glycogen synthesis and glycogen synthase activity were also decreased by MK-2206 in a dose-dependent manner. Incubation with high doses of MK-2206 (10 μM) inhibited insulin-induced p70 ribosomal protein S6 kinase and 4E-BP1 (eukaryotic initiation factor 4E-binding protein-1) phosphorylation associated with increased eEF2 (eukaryotic elongation factor 2) phosphorylation. In contrast, Akti only modestly inhibited insulin-induced PKB and mTOR (mammalian target of rapamycin) signalling, with little or no effect on glucose uptake and protein synthesis. MK-2206, rather than Akti, would thus be the tool of choice for studying the role of PKB in insulin action in skeletal muscle. The results point to a key role for PKB in mediating insulin-stimulated glucose uptake, glycogen synthesis and protein synthesis in skeletal muscle.

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

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    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.

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

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    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.

  9. The Role of PI3K/Akt/mTOR Signaling in Gastric Carcinoma

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    Matsuoka, Tasuku [Department of Surgical Oncology, Osaka City University Graduate School of Medicine, 1-4-3 Asahi-machi, Abeno-ku, Osaka 545-8585 (Japan); Yashiro, Masakazu, E-mail: m9312510@med.osaka-cu.ac.jp [Department of Surgical Oncology, Osaka City University Graduate School of Medicine, 1-4-3 Asahi-machi, Abeno-ku, Osaka 545-8585 (Japan); Oncology Institute of Geriatrics and Medical Science, Osaka City University Graduate School of Medicine, 1-4-3 Asahi-machi, Abeno-ku, Osaka 545-8585 (Japan)

    2014-07-07

    The phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) pathway is one of the key signaling pathways induced by various receptor-tyrosine kinases. Accumulating evidence shows that this pathway is an important promoter of cell growth, metabolism, survival, metastasis, and resistance to chemotherapy. Genetic alterations in the PI3K/Akt/mTOR pathway in gastric carcinoma have often been demonstrated. Many kinds of molecular targeting therapies are currently undergoing clinical testing in patients with solid tumors. However, with the exception of the ErbB2-targeting antibody, targeting agents, including PI3K/Akt/mTOR inhibitors, have not been approved for treatment of patients with gastric carcinoma. This review summarizes the current knowledge on PI3K/Akt/mTOR signaling in the pathogenesis of gastric carcinoma and the possible therapeutic targets for gastric carcinoma. Improved knowledge of the PI3K/Akt/mTOR pathway in gastric carcinoma will be useful in understanding the mechanisms of tumor development and for identifying ideal targets of anticancer therapy for gastric carcinoma.

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

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    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

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

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    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.

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

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    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

  13. Wnt1 Neuroprotection Translates into Improved Neurological Function during Oxidant Stress and Cerebral Ischemia Through AKT1 and Mitochondrial Apoptotic Pathways

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    Zhao Zhong Chong

    2010-01-01

    Full Text Available Although essential for the development of the nervous system, Wnt1 also has been associated with neurodegenerative disease and cognitive loss during periods of oxidative stress. Here we show that endogenous expression of Wnt1 is suppressed during oxidative stress in both in vitro and in vivo experimental models. Loss of endogenous Wnt1 signaling directly correlates with neuronal demise and increased functional deficit, illustrating that endogenous neuronal Wnt1 offers a vital level of intrinsic cellular protection against oxidative stress. Furthermore, transient overexpression of Wnt1 or application of exogenous Wnt1 recombinant protein is necessary to preserve neurological function and rescue neurons from apoptotic membrane phosphatidylserine externalization and genomic DNA degradation, since blockade of Wnt1 signaling with a Wnt1 antibody or dickkopf related protein 1 abrogates neuronal protection by Wnt1. Wnt1 ultimately relies upon the activation of Akt1, the modulation of mitochondrial membrane permeability, and the release of cytochrome c to control the apoptotic cascade, since inhibition of Wnt1 signaling, the phosphatidylinositol 3-kinase pathway, or Akt1 activity abrogates the ability of Wnt1 to block these apoptotic components. Our work identifies Wnt1 and its downstream signaling as cellular targets with high clinical potential for novel treatment strategies for multiple disorders precipitated by oxidative stress.

  14. Wnt1 neuroprotection translates into improved neurological function during oxidant stress and cerebral ischemia through AKT1 and mitochondrial apoptotic pathways.

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    Chong, Zhao Zhong; Shang, Yan Chen; Hou, Jinling; Maiese, Kenneth

    2010-01-01

    Although essential for the development of the nervous system, Wnt1 also has been associated with neurodegenerative disease and cognitive loss during periods of oxidative stress. Here we show that endogenous expression of Wnt1 is suppressed during oxidative stress in both in vitro and in vivo experimental models. Loss of endogenous Wnt1 signaling directly correlates with neuronal demise and increased functional deficit, illustrating that endogenous neuronal Wnt1 offers a vital level of intrinsic cellular protection against oxidative stress. Furthermore, transient overexpression of Wnt1 or application of exogenous Wnt1 recombinant protein is necessary to preserve neurological function and rescue neurons from apoptotic membrane phosphatidylserine externalization and genomic DNA degradation, since blockade of Wnt1 signaling with a Wnt1 antibody or dickkopf related protein 1 abrogates neuronal protection by Wnt1. Wnt1 ultimately relies upon the activation of Akt1, the modulation of mitochondrial membrane permeability, and the release of cytochrome c to control the apoptotic cascade, since inhibition of Wnt1 signaling, the phosphatidylinositol 3-kinase pathway, or Akt1 activity abrogates the ability of Wnt1 to block these apoptotic components. Our work identifies Wnt1 and its downstream signaling as cellular targets with high clinical potential for novel treatment strategies for multiple disorders precipitated by oxidative stress.

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

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    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.

  16. mTORC2 controls dendritic arbor development through Akt-mTORC1-p70S6K pathway

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    Malgorzata Barbara Urbanska

    2014-03-01

    Full Text Available Mammalian target of rapamycin (mTOR is a serine/threonine protein kinase that was identified as one of the important regulators of dendritogenesis. mTOR forms two functionally separate complexes (mTORC in mammalian cells. The complexes are defined by unique mTOR binding partners, Raptor for the mTORC1, and Rictor for the mTORC2. These two complexes control different cellular processes and their roles seem non-overlapping. mTORC1 was initially characterized as a protein translation regulator while mTORC2 was shown to affect cytoskeleton. Both processes are known to be essential for neuron development, but until now the role of particular mTORCs in dendritogenesis was not studied. Therefore, in our study we precisely characterized mTOR involvement in dendrite development. Using RNA interference in developing rat hippocampal neurons in culture we targeted either Raptor or Rictor to separate activities of mTORC1 and mTORC2, respectively. Dendritic arbors of neurons, depleted of either one of these proteins were reduced. However, no obvious differences were observed in context of dendrite number, length, dendritic arbor complexity and dynamics of dendritic growth and retraction between cells with either Raptor or Rictor knockdown. Yet on the molecular level we were able to pin-point differences between cells lacking Rictor and Raptor. For example, Rictor, but not Raptor knockdown phenotype could be rescued by overexpression of a constitutively active mutant of Akt (myr-Akt, a kinase known to be mTORC2 effector. Besides of being mTORC2 effector, Akt is the most important positive regulator of mTORC1. Since, Rictor and Raptor knockdown have similar and not additive dendritic phenotypes, we hypothesized that mTORC1 acts downstream of mTORC2 during dendritic growth. Therefore, we tested the effects of Rictor knockdown in neurons on phosphorylation of S6 (Ser-235/236 and eIF4B (Ser-422, canonical targets for mTORC1. Indeed, downregulation of Rictor

  17. The Prdm13 histone methyltransferase encoding gene is a Ptf1a-Rbpj downstream target that suppresses glutamatergic and promotes GABAergic neuronal fate in the dorsal neural tube.

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    Hanotel, Julie; Bessodes, Nathalie; Thélie, Aurore; Hedderich, Marie; Parain, Karine; Van Driessche, Benoit; Brandão, Karina De Oliveira; Kricha, Sadia; Jorgensen, Mette C; Grapin-Botton, Anne; Serup, Palle; Van Lint, Carine; Perron, Muriel; Pieler, Tomas; Henningfeld, Kristine A; Bellefroid, Eric J

    2014-02-15

    The basic helix-loop-helix (bHLH) transcriptional activator Ptf1a determines inhibitory GABAergic over excitatory glutamatergic neuronal cell fate in progenitors of the vertebrate dorsal spinal cord, cerebellum and retina. In an in situ hybridization expression survey of PR domain containing genes encoding putative chromatin-remodeling zinc finger transcription factors in Xenopus embryos, we identified Prdm13 as a histone methyltransferase belonging to the Ptf1a synexpression group. Gain and loss of Ptf1a function analyses in both frog and mice indicates that Prdm13 is positively regulated by Ptf1a and likely constitutes a direct transcriptional target. We also showed that this regulation requires the formation of the Ptf1a-Rbp-j complex. Prdm13 knockdown in Xenopus embryos and in Ptf1a overexpressing ectodermal explants lead to an upregulation of Tlx3/Hox11L2, which specifies a glutamatergic lineage and a reduction of the GABAergic neuronal marker Pax2. It also leads to an upregulation of Prdm13 transcription, suggesting an autonegative regulation. Conversely, in animal caps, Prdm13 blocks the ability of the bHLH factor Neurog2 to activate Tlx3. Additional gain of function experiments in the chick neural tube confirm that Prdm13 suppresses Tlx3(+)/glutamatergic and induces Pax2(+)/GABAergic neuronal fate. Thus, Prdm13 is a novel crucial component of the Ptf1a regulatory pathway that, by modulating the transcriptional activity of bHLH factors such as Neurog2, controls the balance between GABAergic and glutamatergic neuronal fate in the dorsal and caudal part of the vertebrate neural tube.

  18. Tomosyn is a novel Akt substrate mediating insulin-dependent GLUT4 exocytosis.

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    Nagano, Koki; Takeuchi, Hiroshi; Gao, Jing; Mori, Yoshihide; Otani, Takahito; Wang, DaGuang; Hirata, Masato

    2015-05-01

    Insulin triggers glucose uptake into skeletal muscle and adipose tissues by gaining the available number of glucose transporter 4 (GLUT4) on the cell surface. GLUT4-loaded vesicles are targeted to plasma membrane from the intracellular reservoir through multiple trafficking and fusion processes that are mainly regulated by Akt. However, it is still largely unknown how GLUT4 expression in the cell surface is promoted by insulin. In the present study, we identified tomosyn at Ser-783 as a possible Akt-substrate motif and examined whether the phosphorylation at Ser-783 is involved in the regulation of GLUT4 expression. Both Akt1 and Akt2 phosphorylated the wild-type tomosyn, but not the mutant tomosyn in which Ser-783 was replaced with Ala. Phosphorylation of tomosyn at Ser-783 was also observed in the intact cells by insulin stimulation, which was blocked by PI3K inhibitor, LY294002. In vitro pull-down assay showed that phosphorylation of tomosyn at Ser-783 by Akt inhibited the interaction with syntaxin 4. Insulin stimulation increased GLUT4 in the cell surface of CHO-K1 cells to promote glucose uptake, however exogenous expression of the mutant tomosyn attenuated the increase by insulin. These results suggest that Ser-783 of tomosyn is a target of Akt and is implicated in the interaction with syntaxin 4.

  19. Transforming growth factor-beta1 upregulation triggers pulmonary artery smooth muscle cell proliferation and apoptosis imbalance in rats with hypoxic pulmonary hypertension via the PTEN/AKT pathways.

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    Liu, Yun; Cao, Yonggang; Sun, Shuyang; Zhu, Jinquan; Gao, Shan; Pang, Jie; Zhu, Daling; Sun, Zengxian

    2016-08-01

    Transforming growth factor-beta1 (TGFβ1) and Phosphatase and Tensin homolog deleted on chromosome ten (PTEN) are involved in the regulation of proliferation, differentiation, migration and apoptosis of various cell types. In previous studies, we have shown that TGFβ1 and PTEN play an important role in the progression of pulmonary vascular remodeling induced by pulmonary artery smooth muscle cells (PASMCs). However, the mechanisms involved in the activation of PASMCs between TGFβ1 and PTEN pathways remain unknown. We found that pulmonary vascular walls in hypoxic pulmonary arterial hypertension (PAH) rats were thicker than the vessels from normal rats in vivo. Substantially higher levels of TGFβ1 and significant loss of PTEN expression were observed in the lungs of PAH rats when compared with normoxia. Meanwhile, AKT, a downstream proliferative signaling protein of the PTEN antagonist PI3K, was markedly activated in the lungs of PAH rats. In vitro studies using PASMCs showed that TGFβ1 increased cell proliferation in PTEN-dependent manner. Moreover, we found that TGFβ1 enhanced cell survival, up-regulated the expression of Bcl-2 and procaspase-3, decreased the number of TUNEL-positive cells and caspase-3 expression in PASMCs under serum-deprived (SD) condition via PI3K/AKT pathway. The results further establish that TGFβ1 promoted PAH by decreasing PTEN expression and increasing PI3K/AKT activation in the lung. In conclusion, TGFβ1 mediated PTEN inactivation and resistance to apoptosis seems to be key mediators of lung vascular remodeling associated with PAH. These findings further clarify molecular mechanisms that support targeting PTEN/AKT signaling pathway to attenuate pathogenic derangements in PAH.

  20. Catalase expression in MCF-7 breast cancer cells is mainly controlled by PI3K/Akt/mTor signaling pathway.

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    Glorieux, Christophe; Auquier, Julien; Dejeans, Nicolas; Sid, Brice; Demoulin, Jean-Baptiste; Bertrand, Luc; Verrax, Julien; Calderon, Pedro Buc

    2014-05-15

    Catalase is an antioxidant enzyme that catalyzes mainly the transformation of hydrogen peroxide into water and oxygen. Although catalase is frequently down-regulated in tumors the underlying mechanism remains unclear. Few transcription factors have been reported to directly bind the human catalase promoter. Among them FoxO3a has been proposed as a positive regulator of catalase expression. Therefore, we decided to study the role of the transcription factor FoxO3a and the phosphatidylinositol-3 kinase (PI3K) signaling pathway, which regulates FoxO3a, in the expression of catalase. To this end, we developed an experimental model of mammary breast MCF-7 cancer cells that acquire resistance to oxidative stress, the so-called Resox cells, in which catalase is overexpressed as compared with MCF-7 parental cell line. In Resox cells, Akt expression is decreased but its phosphorylation is enhanced when compared with MCF-7 cells. A similar profile is observed for FoxO3a, with less total protein but more phosphorylated FoxO3a in Resox cells, correlating with its higher Akt activity. The modulation of FoxO3a expression by knockdown and overexpression strategies did not affect catalase expression, neither in MCF-7 nor in Resox cells. Inhibition of PI3K and mTOR by LY295002 and rapamycin, respectively, decreases the phosphorylation of downstream targets (i.e. GSK3β and p70S6K) and leads to an increase of catalase expression only in MCF-7 but not in Resox cells. In conclusion, FoxO3a does not appear to play a critical role in the regulation of catalase expression in both cancer cells. Only MCF-7 cells are sensitive and dependent on PI3K/Akt/mTOR signaling.

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

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    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.

  2. Essential role of caveolae in interleukin-6- and insulin-like growth factor I-triggered Akt-1-mediated survival of multiple myeloma cells.

    Science.gov (United States)

    Podar, Klaus; Tai, Yu-Tzu; Cole, Craig E; Hideshima, Teru; Sattler, Martin; Hamblin, Angela; Mitsiades, Nicholas; Schlossman, Robert L; Davies, Faith E; Morgan, Gareth J; Munshi, Nikhil C; Chauhan, Dharminder; Anderson, Kenneth C

    2003-02-21

    Caveolae, specialized flask-shaped lipid rafts on the cell surface, are composed of cholesterol, sphingolipids, and structural proteins termed caveolins; functionally, these plasma membrane microdomains have been implicated in signal transduction and transmembrane transport. In the present study, we examined the role of caveolin-1 in multiple myeloma cells. We show for the first time that caveolin-1, which is usually absent in blood cells, is expressed in multiple myeloma cells. Analysis of myeloma cell-derived plasma membrane fractions shows that caveolin-1 is co-localized with interleukin-6 receptor signal transducing chain gp130 and with insulin-like growth factor-I receptor. Cholesterol depletion by beta-cyclodextrin results in the loss of caveola structure in myeloma cells, as shown by transmission electron microscopy, and loss of caveolin-1 function. Interleukin-6 and insulin-like growth factor-I, growth and survival factors in multiple myeloma, induce caveolin-1 phosphorylation, which is abrogated by pre-treatment with beta-cyclodextrin. Importantly, inhibition of caveolin-1 phosphorylation blocks both interleukin-6-induced protein complex formation with caveolin-1 and downstream activation of the phosphatidylinositol 3-kinase/Akt-1 pathway. beta-Cyclodextrin also blocks insulin-like growth factor-I-induced tyrosine phosphorylation of insulin-responsive substrate-1 and downstream activation of the phosphatidylinositol 3-kinase/Akt-1 pathway. Therefore, cholesterol depletion by beta-cyclodextrin abrogates both interleukin-6- and insulin-like growth factor-I-triggered multiple myeloma cell survival via negative regulation of caveolin-1. Taken together, this study identifies caveolin-1 and other structural membrane components as potential new therapeutic targets in multiple myeloma.

  3. Extracellular acidosis induces neutrophil activation by a mechanism dependent on activation of phosphatidylinositol 3-kinase/Akt and ERK pathways.

    Science.gov (United States)

    Martínez, Diego; Vermeulen, Mónica; Trevani, Analía; Ceballos, Ana; Sabatté, Juan; Gamberale, Romina; Alvarez, María Eugenia; Salamone, Gabriela; Tanos, Tamara; Coso, Omar A; Geffner, Jorge

    2006-01-15

    Inflammation in peripheral tissues is usually associated with the development of local acidosis; however, there are few studies aimed at analyzing the influence of acidosis on immune cells. We have shown previously that extracellular acidosis triggers human neutrophil activation, inducing a transient increase in intracellular Ca2+ concentration, a shape change response, the up-regulation of CD18 expression, and a delay of apoptosis. In this study, we analyzed the signaling pathways responsible for neutrophil activation. We found that acidosis triggers the phosphorylation of Akt (the main downstream target of PI3K) and ERK MAPK, but not that of p38 and JNK MAPK. No degradation of IkappaB was observed, supporting the hypothesis that NF-kappaB is not activated under acidosis. Inhibition of PI3K by wortmannin or LY294002 markedly decreased the shape change response and the induction of Ca2+ transients triggered by acidosis, whereas the inhibition of MEK by PD98059 or U0126 significantly inhibited the shape change response without affecting the induction of Ca2+ transients. We also found that acidosis not only induces a shape change response and the induction of Ca2+ transients in human neutrophils but also stimulates the endocytosis of FITC-OVA and FITC-dextran. Stimulation of endocytosis was partially prevented by inhibitors of PI3K and MEK. Together, our results support the notion that the stimulation of human neutrophils by extracellular acidosis is dependent on the activation of PI3K/Akt and ERK pathways. Of note, using mouse peritoneal neutrophils we observed that the enhancement of endocytosis induced by acidosis was associated with an improved ability to present extracellular Ags through a MHC class I-restricted pathway.

  4. Upregulation of Akt/NF-κB-regulated inflammation and Akt/Bad-related apoptosis signaling pathway involved in hepatic carcinoma process: suppression by carnosic acid nanoparticle.

    Science.gov (United States)

    Tang, Bo; Tang, Fang; Wang, Zhenran; Qi, Guangying; Liang, Xingsi; Li, Bo; Yuan, Shengguang; Liu, Jie; Yu, Shuiping; He, Songqing

    Primary liver cancer is globally the sixth most frequent cancer, and the second leading cause of cancer death and its incidence is increasing in many countries, becoming a serious threat to human health. Many researches focused on the treatment and prevention of liver cancer. However, due to the underlying molecular mechanism of liver cancer still not fully understood, the studies and development of treatments were forced to be delayed. Akt has been suggested to play an essential role in the progression of inflammation response and apoptosis. Hence, in this study, Akt-knockout mice and cells of liver cancer were used as a model to investigate the molecular mechanism of Akt-associated inflammatory and apoptotic signaling pathway linked with NF-κB and Bcl-2-associated death promoter (Bad) for the progression of liver cancer. Carnosic acid (CA), as a phenolic diterpene with anticancer, antibacterial, antidiabetic, as well as neuroprotective properties, is produced by many species from Lamiaceae family. Administration of CA nanoparticles was sufficient to lead to considerable inhibition of liver cancer progression. The results indicated that, compared to the normal liver cells, the expression of Akt was significantly higher in liver cancer cell lines. Also, we found that Akt-knockout cancer cell lines modulated inflammation response and apoptosis via inhibiting NF-κB activation and inducing apoptotic reaction. Our results indicated that the downstream signals, including cytokines regulated by NF-κB and caspase-3-activated apoptosis affected by Bad, were re-modulated for knockout of Akt. And CA nanoparticles, acting as Akt-knockout, could inhibit inflammation and accelerate apoptosis in liver cancer by altering NF-κB activation and activating caspase-3 through Bad pathway. These findings demonstrated that the nanoparticulate drug CA performed its effective role owing to its ability to reduce inflammatory action and enhance apoptosis for the overexpression of NF

  5. 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.

  6. 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).

  7. 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.

  8. 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.

  9. 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.

  10. IL-13 induces YY1 through the AKT pathway in lung fibroblasts.

    Science.gov (United States)

    Guo, Jia; Yao, Hongwei; Lin, Xin; Xu, Haodong; Dean, David; Zhu, Zhou; Liu, Gang; Sime, Patricia

    2015-01-01

    A key feature of lung fibrosis is the accumulation of myofibroblasts. Interleukin 13 (IL-13) is a pro-fibrotic mediator that directly and indirectly influences the activation of myofibroblasts. Transforming growth factor beta (TGF-β) promotes the differentiation of fibroblasts into myofibroblasts, and can be regulated by IL-13. However, IL-13's downstream signaling pathways are not completely understood. We previously reported that the transcription factor Yin Yang 1 (YY1) is upregulated in fibroblasts treated with TGF-β and in the lungs of mice and patients with pulmonary fibrosis. Moreover, YY1 directly regulates collagen and alpha smooth muscle actin (α-SMA) expression in fibroblasts. However, it is not known if IL-13 regulates fibroblast activation through YY1 expression. We hypothesize that IL-13 up-regulates YY1 expression through regulation of AKT activation, leading to fibroblast activation. In this study we found that YY1 was upregulated by IL-13 in lung fibroblasts in a dose- and time-dependent manner, resulting in increased α-SMA. Conversely, knockdown of YY1 blocked IL-13-induced α-SMA expression in fibroblasts. Furthermore, AKT phosphorylation was increased in fibroblasts treated with IL-13, and AKT overexpression upregulated YY1, whereas blockade of AKT phosphorylation suppressed the induction of YY1 by IL-13 in vitro. In vivo YY1 was upregulated in fibrotic lungs from CC10-IL-13 transgenic mice compared to that from wild-type littermates, which was associated with increased AKT phosphorylation. Taken together, these findings demonstrate that IL-13 is a potent stimulator and activator of fibroblasts, at least in part, through AKT-mediated YY1 activation.

  11. 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.

  12. 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.

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

    Directory of Open Access Journals (Sweden)

    Anthony J. Robertson

    2013-03-01

    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. 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.

  17. 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.

  18. Metastable primordial germ cell-like state induced from mouse embryonic stem cells by Akt activation

    Energy Technology Data Exchange (ETDEWEB)

    Yamano, Noriko [Graduate School of Frontier Biosciences, Osaka University, 2-2 Yamada-oka, Suita, Osaka 565-0871 (Japan); Kimura, Tohru, E-mail: tkimura@patho.med.osaka-u.ac.jp [Department of Pathology, Medical School, Osaka University, 2-2 Yamada-oka, Suita, Osaka 565-0871 (Japan); Watanabe-Kushima, Shoko [Graduate School of Frontier Biosciences, Osaka University, 2-2 Yamada-oka, Suita, Osaka 565-0871 (Japan); Shinohara, Takashi [Department of Molecular Genetics, Graduate School of Medicine, Kyoto University, Kyoto 606-8501 (Japan); Nakano, Toru, E-mail: tnakano@patho.med.osaka-u.ac.jp [Graduate School of Frontier Biosciences, Osaka University, 2-2 Yamada-oka, Suita, Osaka 565-0871 (Japan); Department of Pathology, Medical School, Osaka University, 2-2 Yamada-oka, Suita, Osaka 565-0871 (Japan)

    2010-02-12

    Specification to primordial germ cells (PGCs) is mediated by mesoderm-induction signals during gastrulation. We found that Akt activation during in vitro mesodermal differentiation of embryonic stem cells (ESCs) generated self-renewing spheres with differentiation states between those of ESCs and PGCs. Essential regulators for PGC specification and their downstream germ cell-specific genes were expressed in the spheres, indicating that the sphere cells had commenced differentiation to the germ lineage. However, the spheres did not proceed to spermatogenesis after transplantation into testes. Sphere cell transfer to the original feeder-free ESC cultures resulted in chaotic differentiation. In contrast, when the spheres were cultured on mouse embryonic fibroblasts or in the presence of ERK-cascade and GSK3 inhibitors, reversion to the ESC-like state was observed. These results indicate that Akt signaling promotes a novel metastable and pluripotent state that is intermediate to those of ESCs and PGCs.

  19. 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

    2015-07-08

    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.

  20. 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

  1. Perifosine as a potential novel anti-cancer agent inhibits EGFR/MET-AKT axis in malignant pleural mesothelioma.

    Directory of Open Access Journals (Sweden)

    Giulia Pinton

    Full Text Available BACKGROUND: PI3K/AKT signalling pathway is aberrantly active and plays a critical role for cell cycle progression of human malignant pleural mesothelioma (MMe cells. AKT is one of the important cellular targets of perifosine, a novel bio-available alkylphospholipid that has displayed significant anti-proliferative activity in vitro and in vivo in several human tumour model systems and is currently being tested in clinical trials. METHODS: We tested Perifosine activity on human mesothelial cells and different mesothelioma cell lines, in order to provide evidence of its efficacy as single agent and combined therapy. RESULTS: We demonstrate here that perifosine, currently being evaluated as an anti-cancer agent in phase 1 and 2 clinical trials, caused a dose-dependent reduction of AKT activation, at concentrations causing MMe cell growth arrest. In this study we firstly describe that MMe cells express aside from AKT1 also AKT3 and that either the myristoylated, constitutively active, forms of the two proteins, abrogated perifosine-mediated cell growth inhibition. Moreover, we describe here a novel mechanism of perifosine that interferes, upstream of AKT, affecting EGFR and MET phosphorylation. Finally, we demonstrate a significant increase in cell toxicity when MMe cells were treated with perifosine in combination with cisplatin. CONCLUSIONS: This study provides a novel mechanism of action of perifosine, directly inhibiting EGFR/MET-AKT1/3 axis, providing a rationale for a novel translational approach to the treatment of MMe.

  2. Hepatitis C Virus RNA-Dependent RNA Polymerase Interacts with the Akt/PKB Kinase and Induces Its Subcellular Relocalization.

    Science.gov (United States)

    Valero, María Llanos; Sabariegos, Rosario; Cimas, Francisco J; Perales, Celia; Domingo, Esteban; Sánchez-Prieto, Ricardo; Mas, Antonio

    2016-06-01

    Hepatitis C virus (HCV) interacts with cellular components and modulates their activities for its own benefit. These interactions have been postulated as a target for antiviral treatment, and some candidate molecules are currently in clinical trials. The multifunctional cellular kinase Akt/protein kinase B (PKB) must be activated to increase the efficacy of HCV entry but is rapidly inactivated as the viral replication cycle progresses. Viral components have been postulated to be responsible for Akt/PKB inactivation, but the underlying mechanism remained elusive. In this study, we show that HCV polymerase NS5B interacts with Akt/PKB. In the presence of transiently expressed NS5B or in replicon- or virus-infected cells, NS5B changes the cellular localization of Akt/PKB from the cytoplasm to the perinuclear region. Sequestration of Akt/PKB by NS5B could explain its exclusion from its participation in early Akt/PKB inactivation. The NS5B-Akt/PKB interaction represents a new regulatory step in the HCV infection cycle, opening possibilities for new therapeutic options.

  3. Combination treatment of prostate cancer with FGF receptor and AKT kinase inhibitors

    Science.gov (United States)

    Feng, Shu; Shao, Longjiang; Castro, Patricia; Coleman, Ilsa; Nelson, Peter S; Smith, Paul D; Davies, Barry R; Ittmann, Michael

    2017-01-01

    Activation of the PI3K/AKT pathway occurs in the vast majority of advanced prostate cancers (PCas). Activation of fibroblast growth factor receptor (FGFR) signaling occurs in a wide variety of malignancies, including PCa. RNA-Seq of castration resistant PCa revealed expression of multiple FGFR signaling components compatible with FGFR signaling in all cases, with multiple FGF ligands expressed in 90% of cases. Immunohistochemistry confirmed FGFR signaling in the majority of xenografts and advanced PCas. AZD5363, an AKT kinase inhibitor and AZD4547, a FGFR kinase inhibitor are under active clinical development. We therefore sought to determine if these two drugs have additive effects in PCa models. The effect of both agents, singly and in combination was evaluated in a variety of PCa cell lines in vitro and in vivo. All cell lines tested responded to both drugs with decreased invasion, soft agar colony formation and growth in vivo, with additive effects seen with combination treatment. Activation of the FGFR, AKT, ERK and STAT3 pathways was examined in treated cells. AZD5363 inhibited AKT signaling and increased FGFR1 signaling, which partially compensated for decreased AKT kinase activity. While AZD4547 could effectively block the ERK pathway, combination treatment was needed to completely block STAT3 activation. Thus combination treatment with AKT and FGFR kinase inhibitors have additive effects on malignant phenotypes in vitro and in vivo by inhibiting multiple signaling pathways and mitigating the compensatory upregulation of FGFR signaling induced by AKT kinase inhibition. Our studies suggest that co-targeting these pathways may be efficacious in advanced PCa. PMID:28008155

  4. 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.

  5. AKT inhibitor suppresses hyperthermia-induced Ndrg2 phosphorylation in gastric cancer cells

    Energy Technology Data Exchange (ETDEWEB)

    Tao, Yurong; Guo, Yan; Liu, Wenchao [Department of Oncology, State Key Discipline of Cell Biology, Xijing Hospital, The Fourth Military Medical University, Shaanxi, Xi' an (China); Zhang, Jian; Li, Xia; Shen, Lan; Ru, Yi [State Key Laboratory of Cancer Biology, Department of Biochemistry and Molecular Biology, The Fourth Military Medical University, Shaanxi, Xi' an (China); Xue, Yan [Department of Oncology, State Key Discipline of Cell Biology, Xijing Hospital, The Fourth Military Medical University, Shaanxi, Xi' an (China); Zheng, Jin [Department of Traditional Chinese and Western Medicine of Oncology, Tangdu Hospital, The Fourth Military Medical University, Shaanxi, Xi' an (China); Liu, Xinping; Zhang, Jing; Yao, Libo [State Key Laboratory of Cancer Biology, Department of Biochemistry and Molecular Biology, The Fourth Military Medical University, Shaanxi, Xi' an (China)

    2013-04-05

    Hyperthermia is one of the most effective adjuvant treatments for various cancers with few side effects. However, the underlying molecular mechanisms still are not known. N-myc downstream-regulated gene 2 (NDRG2), a tumor suppressor, has been shown to be involved in diverse cellular stresses including hypoxia, lipotoxicity, etc. In addition, Ndrg2 has been reported to be related to progression of gastric cancer. In the current study, our data showed that the apoptosis rate of MKN28 cells increased relatively rapidly to 13.4% by 24 h after treatment with hyperthermia (42°C for 1 h) compared to 5.1% in control cells (P < 0.05). Nevertheless, there was no obvious change in the expression level of total Ndrg2 during this process. Further investigation demonstrated that the relative phosphorylation levels of Ndrg2 at Ser332, Thr348 increased up to 3.2- and 1.9-fold (hyperthermia group vs control group) at 3 h in MKN28 cells, respectively (P < 0.05). We also found that heat treatment significantly increased AKT phosphorylation. AKT inhibitor VIII (10 µM) decreased the phosphorylation level of Ndrg2 induced by hyperthermia. Accordingly, the apoptosis rate rose significantly in MKN28 cells (16.4%) treated with a combination of AKT inhibitor VIII and hyperthermia compared to that (6.8%) of cells treated with hyperthermia alone (P < 0.05). Taken together, these data demonstrated that Ndrg2 phosphorylation could be induced by hyperthermia in an AKT-dependent manner in gastric cancer cells. Furthermore, AKT inhibitor VIII suppressed Ndrg2 phosphorylation and rendered gastric cancer cells susceptible to apoptosis induced by hyperthermia.

  6. 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.

  7. Akt-dependent enhanced migratory capacity of Th17 cells from children with lupus nephritis.

    Science.gov (United States)

    Kshirsagar, Sudhir; Riedl, Magdalena; Billing, Heiko; Tönshoff, Burkhard; Thangavadivel, Shanmugapriya; Steuber, Christian; Staude, Hagen; Wechselberger, Gottfried; Edelbauer, Monika

    2014-11-15

    Th17 cells infiltrate the kidneys of patients with lupus nephritis (LN) and are critical for the pathogenesis of this disease. In this study, we show that enhanced activity of Stat3 in CD4(+)CD45RA(-)Foxp3(-) and Foxp3(low) effector T cells from children with LN correlates with increased frequencies of IL-17-producing cells within these T cell populations. The levels of retinoic acid-related orphan receptor c and IL-17 mRNA are significantly higher in PBMCs from children with LN than in those from controls. Mammalian target of rapamycin inhibition by rapamycin reduces both Stat3 activation in effector T cells and the frequency of IL-17-producing T cells in lupus patients. Complement factor C5a slightly increases the expression of IL-17 and induces activation of Akt in anti-CD3-activated lupus effector T cells. Th17 cells from children with LN exhibit high Akt activity and enhanced migratory capacity. Inhibition of the Akt signaling pathway significantly decreases Th17 cell migration. These findings indicate that the Akt signaling pathway plays a significant role in the migratory activity of Th17 cells from children with LN and suggest that therapeutic modulation of the Akt activity may inhibit Th17 cell trafficking to sites of inflammation and thus suppress chronic inflammatory processes in children with LN.

  8. 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...

  9. Novel agents and associated toxicities of inhibitors of the pi3k/Akt/mtor pathway for the treatment of breast cancer

    OpenAIRE

    Chia, S.; Gandhi, S.; Joy, A.A.; Edwards, S.; Gorr, M.; Hopkins, S; Kondejewski, J.; Ayoub, J.P.; Califaretti, N.; Rayson, D.; Dent, S.F.

    2015-01-01

    The pi3k/Akt/mtor (phosphatidylinositol 3 kinase/ Akt/mammalian target of rapamycin) signalling pathway is an established driver of oncogenic activity in human malignancies. Therapeutic targeting of this pathway holds significant promise as a treatment strategy. Everolimus, an mtor inhibitor, is the first of this class of agents approved for the treatment of hormone receptor–positive, human epidermal growth factor receptor 2–negative advanced breast cancer. Everolimus has been associated with...

  10. Tubb3 regulation by the Erk and Akt signaling pathways: a mechanism involved in the effect of arginine ADP-ribosyltransferase 1 (Art1) on apoptosis of colon carcinoma CT26 cells.

    Science.gov (United States)

    Xiao, Ming; Tang, Yi; Chen, Wen-Wen; Wang, Ya-Lan; Yang, Lian; Li, Xian; Song, Guang-Lin; Kuang, Jing

    2016-02-01

    The influence of the most important classical mono-ADP-ribosyltransferase, arginine ADP-ribosyltransferase 1 (Art1), on survival and apoptosis of colon carcinoma cells and the potential mechanisms have been partly discussed in our previous study but still need to be further studied. In this present study, Art1 of colon carcinoma CT26 cells was silenced with lentiviral vector-mediated short hairpin RNA (shRNA) or overexpressed with lentiviral vector-mediated complementary DNA (cDNA) and allograft transplant tumors are established in Balb/c mice. We verified Art1 knockdown increases apoptosis of CT26 cells transplant tumor; Art1 overexpression acts oppositely. Accordingly, growth of transplant tumors is inhibited in Art1 knockdown transplant tumors and increases in Art1 overexpression transplant tumors. Furthermore, activity of Akt and Erk cell signal pathways and expression of an apoptosis biomarker, βIII-tubulin (Tubb3), decrease when Art1 was silenced and increase when Art1 was overexpressed. Inhibiting Akt pathway or Erk pathway both downregulates expression of Tubb3 on protein and messenger RNA (mRNA) level, indicating that Tubb3 could be regulated by both Akt and Erk pathways, and plays a role in the influence of Art1 on apoptosis of Balb/c mice allograft transplant tumor. We also demonstrated that Bcl-2 family is not the responsible downstream factor of the Erk pathway in colon carcinoma cells which is undergoing apoptosis. These findings enrich the molecular mechanism for the function of Art1 in colon carcinoma and provide a complementary support for Art1 to be a potential therapeutic target of the treatment of this kind of malignant tumor.

  11. Activating E17K mutation in the gene encoding the protein kinase AKT1 in a subset of squamous cell carcinoma of the lung.

    Science.gov (United States)

    Malanga, Donatella; Scrima, Marianna; De Marco, Carmela; Fabiani, Fernanda; De Rosa, Nicla; De Gisi, Silvia; Malara, Natalia; Savino, Rocco; Rocco, Gaetano; Chiappetta, Gennaro; Franco, Renato; Tirino, Virginia; Pirozzi, Giuseppe; Viglietto, Giuseppe

    2008-03-01

    Somatic mutation (E17K) that constitutively activates the protein kinase AKT1 has been found in human cancer patients. We determined the role of the E17K mutation of AKT1 in lung cancer, through sequencing of AKT1 exon 4 in 105 resected, clinically annotated non-small cell lung cancer specimens. We detected a missense mutations G-->A transition at nucleotide 49 (that results in the E17K substitution) in two squamous cell carcinoma (2/36) but not in adenocarcinoma (0/53). The activity of the endogenous kinase carrying the E17K mutation immunoprecipitated by tumour tissue was significantly higher compared with the wild-type kinase immunoprecipitated by the adjacent normal tissue as determined both by in vitro kinase assay using a consensus peptide as substrate and by in vivo analysis of the phosphorylation status of AKT1 itself (pT308, pS473) or of known downstream substrates such as GSK3 (pS9/S22) and p27 (T198). Immunostaining or immunoblot analysis on membrane-enriched extracts indicated that the enhanced membrane localization exhibited by the endogenous E17K-AKT1 may account for the observed increased activity of mutant E17K kinase in comparison with the wild-type AKT1 from adjacent normal tissue. In conclusion, this is the first report of AKT1 mutation in lung cancer. Our data provide evidence that, although AKT1 mutations are apparently rare in lung cancer (1.9%), the oncogenic properties of E17K-AKT1 may contribute to the development of a fraction of lung carcinoma with squamous histotype (5.5%).

  12. 1α,25(OH)2D3-dependent modulation of Akt in proliferating and differentiating C2C12 skeletal muscle cells.

    Science.gov (United States)

    Buitrago, Claudia G; Arango, Nadia S; Boland, Ricardo L

    2012-04-01

    We previously reported that 1α,25-dihydroxy-vitamin D(3) [1α,25(OH)(2)D(3)] induces non-transcriptional rapid responses through activation of Src and MAPKs in the skeletal muscle cell line C2C12. In the present study we investigated the modulation of Akt by the secosteroid hormone in C2C12 cells at proliferative stage (myoblasts) and at early differentiation stage. In proliferating cells, 1α,25(OH)(2)D(3) activates Akt by phosphorylation in Ser473 in a time-dependent manner (5-60 min). When these cells were pretreated with methyl-beta-cyclodextrin to disrupt caveolae microdomains, hormone-induced activation of Akt was suppressed. Similar results were obtained by siRNA silencing of caveolin-1 expression, further indicating that hormone effects on cell membrane caveolae are required for downstream signaling. PI3K and p38 MAPK, but not ERK1/2, participate in 1α,25(OH)(2)D(3) activation of Akt in myoblasts. The involvement of p38 MAPK in Akt phosphorylation by the hormone probably occurs through MAPK-activated protein kinase 2 (MK2), which is activated by the steroid. In addition, the participation of Src in Akt phosphorylation by 1α,25(OH)(2)D(3) was demonstrated using the inhibitor PP2 and antisense oligodeoxynucleotides that suppress Src expression. We also observed that PI3K participates in hormone-induced proliferation. During the early phase of C2C12 cell differentiation 1α,25(OH)(2)D(3) also increases Akt phosphorylation and activates Src. Of relevance, Src and PI3K are involved in Akt activation and in MHC and myogenin increased expression by 1α,25(OH)(2)D(3). Altogether, these data suggest that 1α,25(OH)(2)D(3) upregulates Akt through Src, PI(3)K, and p38 MAPK to stimulate myogenesis in C2C12 cells.

  13. Downstream bioprocess characterisation within microfluidic devices

    DEFF Research Database (Denmark)

    Marques, Marco; Krühne, Ulrich; Szita, Nicolas

    2016-01-01

    Miniaturising bioprocess unit operation steps is a well-established approach to find novel routes for process intensification and improved process economics. While a number of microbioreactors have been presented over the last 15 years, miniaturised downstream unit operations (mDUO) are less deve...

  14. 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.

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

    Science.gov (United States)

    Nayeb-Hashemi, Hamed; Desai, Anal; Demchev, Valeriy; Bronson, Roderick T.; Hornick, Jason L.; Cohen, David E.; Ukomadu, Chinweike

    2015-01-01

    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. PMID:26225745

  16. 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.

  17. 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

  18. Upregulation of Akt/NF-κB-regulated inflammation and Akt/Bad-related apoptosis signaling pathway involved in hepatic carcinoma process: suppression by carnosic acid nanoparticle

    Directory of Open Access Journals (Sweden)

    Tang B

    2016-11-01

    Full Text Available Bo Tang,1,2,* Fang Tang,1,2,* Zhenran Wang,1,2,* Guangying Qi,3 Xingsi Liang,1,2 Bo Li,1,2 Shengguang Yuan,1,2 Jie Liu,1,2 Shuiping Yu,1,2 Songqing He1,2 1Department of Hepatobiliary Surgery, Guilin Medical University Affiliated Hospital, 2Laboratory of Liver Injury and Repair Molecular Medicine, Guilin Medical University, 3Department of Pathology and Physiopathology, Guilin Medical University, Guilin, Guangxi, People’s Republic of China *These authors contributed equally to this work Abstract: Primary liver cancer is globally the sixth most frequent cancer, and the second leading cause of cancer death and its incidence is increasing in many countries, becoming a serious threat to human health. Many researches focused on the treatment and prevention of liver cancer. However, due to the underlying molecular mechanism of liver cancer still not fully understood, the studies and development of treatments were forced to be delayed. Akt has been suggested to play an essential role in the progression of inflammation response and apoptosis. Hence, in this study, Akt-knockout mice and cells of liver cancer were used as a model to investigate the molecular mechanism of Akt-associated inflammatory and apoptotic signaling pathway linked with NF-κB and Bcl-2-associated death promoter (Bad for the progression of liver cancer. Carnosic acid (CA, as a phenolic diterpene with anticancer, antibacterial, antidiabetic, as well as neuroprotective properties, is produced by many species from Lamiaceae family. Administration of CA nanoparticles was sufficient to lead to considerable inhibition of liver cancer progression. The results indicated that, compared to the normal liver cells, the expression of Akt was significantly higher in liver cancer cell lines. Also, we found that Akt-knockout cancer cell lines modulated inflammation response and apoptosis via inhibiting NF-κB activation and inducing apoptotic reaction. Our results indicated that the downstream

  19. Rac1 Activation Caused by Membrane Translocation of a Guanine Nucleotide Exchange Factor in Akt2-Mediated Insulin Signaling in Mouse Skeletal Muscle.

    Directory of Open Access Journals (Sweden)

    Nobuyuki Takenaka

    Full Text Available Insulin-stimulated glucose uptake in skeletal muscle is mediated by the glucose transporter GLUT4, which is translocated to the plasma membrane following insulin stimulation. Several lines of evidence suggested that the protein kinase Akt2 plays a key role in this insulin action. The small GTPase Rac1 has also been implicated as a regulator of insulin-stimulated GLUT4 translocation, acting downstream of Akt2. However, the mechanisms whereby Akt2 regulates Rac1 activity remain obscure. The guanine nucleotide exchange factor FLJ00068 has been identified as a direct regulator of Rac1 in Akt2-mediated signaling, but its characterization was performed mostly in cultured myoblasts. Here, we provide in vivo evidence that FLJ00068 indeed acts downstream of Akt2 as a Rac1 regulator by using mouse skeletal muscle. Small interfering RNA knockdown of FLJ00068 markedly diminished GLUT4 translocation to the sarcolemma following insulin administration or ectopic expression of a constitutively activated mutant of either phosphoinositide 3-kinase or Akt2. Additionally, insulin and these constitutively activated mutants caused the activation of Rac1 as shown by immunofluorescent microscopy using a polypeptide probe specific to activated Rac1 in isolated gastrocnemius muscle fibers and frozen sections of gastrocnemius muscle. This Rac1 activation was also abrogated by FLJ00068 knockdown. Furthermore, we observed translocation of FLJ00068 to the cell periphery following insulin stimulation in cultured myoblasts. Localization of FLJ00068 in the plasma membrane in insulin-stimulated, but not unstimulated, myoblasts and mouse gastrocnemius muscle was further affirmed by subcellular fractionation and subsequent immunoblotting. Collectively, these results strongly support a critical role of FLJ00068 in Akt2-mediated Rac1 activation in mouse skeletal muscle insulin signaling.

  20. Repression of BIM mediates survival signaling by MYC and AKT in high-risk T-cell acute lymphoblastic leukemia.

    Science.gov (United States)

    Reynolds, C; Roderick, J E; LaBelle, J L; Bird, G; Mathieu, R; Bodaar, K; Colon, D; Pyati, U; Stevenson, K E; Qi, J; Harris, M; Silverman, L B; Sallan, S E; Bradner, J E; Neuberg, D S; Look, A T; Walensky, L D; Kelliher, M A; Gutierrez, A

    2014-09-01

    Treatment resistance in T-cell acute lymphoblastic leukemia (T-ALL) is associated with phosphatase and tensin homolog (PTEN) deletions and resultant phosphatidylinositol 3'-kinase (PI3K)-AKT pathway activation, as well as MYC overexpression, and these pathways repress mitochondrial apoptosis in established T-lymphoblasts through poorly defined mechanisms. Normal T-cell progenitors are hypersensitive to mitochondrial apoptosis, a phenotype that is dependent on the expression of proapoptotic BIM. In a conditional zebrafish model, MYC downregulation induced BIM expression in T-lymphoblasts, an effect that was blunted by expression of constitutively active AKT. In human T-ALL cell lines and treatment-resistant patient samples, treatment with MYC or PI3K-AKT pathway inhibitors each induced BIM upregulation and apoptosis, indicating that BIM is repressed downstream of MYC and PI3K-AKT in high-risk T-ALL. Restoring BIM function in human T-ALL cells using a stapled peptide mimetic of the BIM BH3 domain had therapeutic activity, indicating that BIM repression is required for T-ALL viability. In the zebrafish model, where MYC downregulation induces T-ALL regression via mitochondrial apoptosis, T-ALL persisted despite MYC downregulation in 10% of bim wild-type zebrafish, 18% of bim heterozygotes and in 33% of bim homozygous mutants (P=0.017). We conclude that downregulation of BIM represents a key survival signal downstream of oncogenic MYC and PI3K-AKT signaling in treatment-resistant T-ALL.

  1. 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.

  2. 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

  3. The phosphatidylinositol 3-kinase/Akt and c-Jun N-terminal kinase signaling in cancer: Alliance or contradiction? (Review).

    Science.gov (United States)

    Zhao, Hua-Fu; Wang, Jing; Tony To, Shing-Shun

    2015-08-01

    The phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway and c-Jun N-terminal kinase (JNK) pathway are responsible for regulating a variety of cellular processes including cell growth, migration, invasion and apoptosis. These two pathways are essential to the development and progression of tumors. The dual roles of JNK signaling in apoptosis and tumor development determine the different interactions between the PI3K/Akt and JNK pathways. Activation of PI3K/Akt signaling can inhibit stress- and cytokine-induced JNK activation through Akt antagonizing and the formation of the JIP1-JNK module, as well as the activities of upstream kinases ASK1, MKK4/7 and MLK. On the other hand, hyperactivation of Akt and JNK is also found in cancers that harbor EGFR overexpression or loss of PTEN. Understanding the activation mechanism of PI3K/Akt and JNK pathways, as well as the interplays between these two pathways in cancer may contribute to the identification of novel therapeutic targets. In the present report, we summarized the current understanding of the PI3K/Akt and JNK signaling networks, as well as their biological roles in cancers. In addition, the interactions and regulatory network between PI3K/Akt and JNK pathways in cancer were discussed.

  4. Regulation of mammary stem/progenitor cells by PTEN/Akt/beta-catenin signaling.

    Directory of Open Access Journals (Sweden)

    Hasan Korkaya

    2009-06-01

    Full Text Available Recent evidence suggests that many malignancies, including breast cancer, are driven by a cellular subcomponent that displays stem cell-like properties. The protein phosphatase and tensin homolog (PTEN is inactivated in a wide range of human cancers, an alteration that is associated with a poor prognosis. Because PTEN has been reported to play a role in the maintenance of embryonic and tissue-specific stem cells, we investigated the role of the PTEN/Akt pathway in the regulation of normal and malignant mammary stem/progenitor cell populations. We demonstrate that activation of this pathway, via PTEN knockdown, enriches for normal and malignant human mammary stem/progenitor cells in vitro and in vivo. Knockdown of PTEN in normal human mammary epithelial cells enriches for the stem/progenitor cell compartment, generating atypical hyperplastic lesions in humanized NOD/SCID mice. Akt-driven stem/progenitor cell enrichment is mediated by activation of the Wnt/beta-catenin pathway through the phosphorylation of GSK3-beta. In contrast to chemotherapy, the Akt inhibitor perifosine is able to target the tumorigenic cell population in breast tumor xenografts. These studies demonstrate an important role for the PTEN/PI3-K/Akt/beta-catenin pathway in the regulation of normal and malignant stem/progenitor cell populations and suggest that agents that inhibit this pathway are able to effectively target tumorigenic breast cancer cells.

  5. 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.

  6. 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.

  7. Polycystin-1 Induces Resistance to Apoptosis through the Phosphatidylinositol 3-Kinase/Akt Signaling Pathway

    Science.gov (United States)

    Boca, Manila; Distefano, Gianfranco; Boletta, Alessandra; Qian, Feng; Bhunia, Anil K.; Germino, Gregory G.

    2006-01-01

    Polycystin-1 (PC-1), the PKD1 gene product, is a large receptor whose expression in renal epithelial cells results in resistance to apoptosis and tubulogenesis, a model consistent with the phenotype observed in patients. This study links PC-1 expression to a signaling pathway that is known to be both antiapoptotic and important for normal tubulogenesis. This study found that PC-1 expression results in phosphorylation of Akt and downstream effectors and that phosphatidylinositol 3-kinase (PI3-K) inhibitors prevent this process. In addition, it is shown that dominant negative Akt can revert PC-1-induced protection from apoptosis. Furthermore, it was observed that increased PI3-K β activity in PC-1- expressing MDCK cells seems to be dependent on both tyrosine-kinase activity and heterotrimeric G proteins. It also was found that PC-1-induced tubulogenesis is inhibited by PI3-K inhibitors. Taken together, these data suggest that the PI3-K/Akt cascade may be a central modulator of PC-1 function and that its deregulation might be important in autosomal dominant polycystic kidney disease. PMID:16452497

  8. Fish reproductive guilds downstream of dams.

    Science.gov (United States)

    Vasconcelos, L P; Alves, D C; Gomes, L C

    2014-11-01

    Fish reproductive guilds were used to evaluate the responses of species with different reproductive strategies during two different periods of post-dam construction. The data used for the comparisons were collected in the upper Paraná River floodplain (Brazil), downstream of the Porto Primavera dam, 2 and 10 years after impoundment. The abundance (catch per unit effort, CPUE), species richness, evenness and structure of communities, all within reproductive guilds, were used to test the hypothesis that these metrics vary spatially and temporally. The influence of damming on species structure and the diversity of fish reproductive guilds varied spatiotemporally, and species with opportunistic reproductive strategies tended to be less affected. Conversely, long-distance migratory species responded more markedly to spatiotemporal variations, indicating that the ecosystem dynamics exert greater effects on populations of these species. Thus, the effects of a dam, even if attenuated, may extend over several years, especially downstream. This finding emphasizes the importance of maintaining large undammed tributaries downstream of reservoirs.

  9. 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.

  10. 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.

  11. 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.

  12. 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.

  13. Astaxanthin Alleviates Early Brain Injury Following Subarachnoid Hemorrhage in Rats: Possible Involvement of Akt/Bad Signaling

    Directory of Open Access Journals (Sweden)

    Xiang-Sheng Zhang

    2014-07-01

    Full Text Available Apoptosis has been proven to play a crucial role in early brain injury pathogenesis and to represent a target for the treatment of subarachnoid hemorrhage (SAH. Previously, we demonstrated that astaxanthin (ATX administration markedly reduced neuronal apoptosis in the early period after SAH. However, the underlying molecular mechanisms remain obscure. In the present study, we tried to investigate whether ATX administration is associated with the phosphatidylinositol 3-kinase-Akt (PI3K/Akt pathway, which can play an important role in the signaling of apoptosis. Our results showed that post-SAH treatment with ATX could cause a significant increase of phosphorylated Akt and Bad levels, along with a significant decrease of cleaved caspase-3 levels in the cortex after SAH. In addition to the reduced neuronal apoptosis, treatment with ATX could also significantly reduce secondary brain injury characterized by neurological dysfunction, cerebral edema and blood-brain barrier disruption. In contrast, the PI3K/Akt inhibitor, LY294002, could partially reverse the neuroprotection of ATX in the early period after SAH by downregulating ATX-induced activation of Akt/Bad and upregulating cleaved caspase-3 levels. These results provided the evidence that ATX could attenuate apoptosis in a rat SAH model, potentially, in part, through modulating the Akt/Bad pathway.

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

    Directory of Open Access Journals (Sweden)

    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.

  15. The fibrotic role of phosphatidylinositol-3-kinase/Akt pathway in injured skeletal muscle after acute contusion.

    Science.gov (United States)

    Li, H-Y; Zhang, Q-G; Chen, J-W; Chen, S-Q; Chen, S-Y

    2013-09-01

    Transforming growth factor β (TGF-β) is a multifunctional cytokine with fibrogenic properties. Previous studies demonstrated that Phosphatidylinositol 3-Kinase (PI3K)/Akt/ mammalian target of Ramycin (mTOR), a non-Smad TGF-β pathway, plays an important role in the fibrotic pathogenesis of different organs such as the lung, kidney, skin and liver. However, the role of PI3k-Akt pathway in fibrosis in injured skeletal muscle is still unclear. In this study, we determined the fibrotic role of PI3K-Akt pathway in injured skeletal muscle. We established a mouse model for acute muscle contusion. Western blotting analysis showed that TGF-β, phosphorylated Akt and phosphorylated mTOR were increased in muscles after acute contusion, which indicated that the PI3K-Akt- mTOR pathway was activated in skeletal muscle after acute contusion. The pathway was inhibited by a PI3K inhibitor, LY294002. Moreover, the expression of fibrosis markers vimentin, α SMA and collagen I and the area of scar decreased in injured skeletal muscle after PI3K pathway was blocked. The muscle function improved in terms of both fast-twitch and tetanic strength after PI3K/Akt pathway was inhibited in injured skeletal muscle. In conclusion, activation of PI3K-Akt-mTOR pathway might promote collagen production and scar formation in the acute contused skeletal muscle. Blocking of PI3K-Akt-mTOR pathway could improve the function of injured skeletal muscle.

  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. AKT1 Activation is Obligatory for Spontaneous BCC Tumor Growth in a Murine Model that Mimics Some Features of Basal Cell Nevus Syndrome.

    Science.gov (United States)

    Kim, Arianna L; Back, Jung Ho; Zhu, Yucui; Tang, Xiuwei; Yardley, Nathan P; Kim, Katherine J; Athar, Mohammad; Bickers, David R

    2016-10-01

    Patients with basal cell nevus syndrome (BCNS), also known as Gorlin syndrome, develop numerous basal cell carcinomas (BCC) due to germline mutations in the tumor suppressor PTCH1 and aberrant activation of Hedgehog (Hh) signaling. Therapies targeted at components of the Hh pathway, including the smoothened (SMO) inhibitor vismodegib, can ablate these tumors clinically, but tumors recur upon drug discontinuation. Using SKH1-Ptch1(+/-) as a model that closely mimics the spontaneous and accelerated growth pattern of BCCs in patients with BCNS, we show that AKT1, a serine/threonine protein kinase, is intrinsically activated in keratinocytes derived from the skin of newborn Ptch1(+/-) mice in the absence of carcinogenic stimuli. Introducing Akt1 haplodeficiency in Ptch1(+/-) mice (Akt1(+/-) Ptch1(+/-)) significantly abrogated BCC growth. Similarly, pharmacological inhibition of AKT with perifosine, an alkyl phospholipid AKT inhibitor, diminished the growth of spontaneous and UV-induced BCCs. Our data demonstrate an obligatory role for AKT1 in BCC growth, and targeting AKT may help reduce BCC tumor burden in BCNS patients. Cancer Prev Res; 9(10); 794-802. ©2016 AACR.

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

    Directory of Open Access Journals (Sweden)

    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.

    Directory of Open Access Journals (Sweden)

    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. microRNA-181b promotes migration and invasion of osteosarcoma cells by targeting N-myc downstream regulated gene 2%miR-181b通过靶向调控N-myc下游调节基因2影响骨肉瘤细胞的迁移和侵袭

    Institute of Scientific and Technical Information of China (English)

    邵建立; 李志忠; 王亮; 焦根龙; 周志刚; 孙国栋

    2016-01-01

    Objective To investigate the effects of miR-181b on the migration and invasion of osteosarcoma cells. Methods Three cultured osteosarcoma cell lines and MG-63 cells transfected with miR-181b inhibitor were examined for miR-181b expression using qRT-PCR analysis. The cell migration and invasion of the transfected cells were assessed with Transwell assay. The targets of miR-181b were predicted using a miRNA target prediction software and the results were verified with luciferase reporter assay. The target protein expression in osteosarcoma cells lines was determined by Western blotting, and the cell migration and invasion changes following inhibition of miR-181b or its target protein were assessed using Transwell assay. Results All the 3 osteosarcoma cells lines showed significantly up-regulated miR-181b expression. Inhibition of miR-181b expression obviously suppressed the migration and invasion of MG-63 cells. Based on luciferase reporter assay, N-myc downstream regulated gene 2 (NDRG2) was identified as the direct target gene of miR-181b, and inhibition of NDRG2 expression significantly reversed the effect of miR-181b on cell migration and invasion in MG-63 cells. Conclusion miR-181b is over-expressed in osteosarcoma cells, and inhibition of miR-181b, which directly targets NDRG2, can suppress the migration and invasion of osteosarcoma cells.%目的:探讨miR-181b对骨肉瘤细胞迁移和侵袭的影响。方法培养骨肉瘤细胞,实时荧光定量PCR检测miR-181b在骨肉瘤细胞中的表达。抑制miR-181b的表达,Transwell检测对骨肉瘤迁移和侵袭的影响;生物信息学分析miR-181b靶基因并采用荧光素酶报告基因分析miR-181b和靶基因在骨肉瘤中是否直接作用,同时采用Western Blotting检测靶基因在骨肉瘤细胞中的表达以及Transwell检测靶基因对骨肉瘤迁移和侵袭的影响。结果实时荧光定量PCR结果表明miR-181b在骨肉瘤细胞中高表达。抑制miR-181b的表达

  2. 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.

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

    Directory of Open Access Journals (Sweden)

    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.

  4. 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

  5. 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.

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

    Directory of Open Access Journals (Sweden)

    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.

  7. Ion Effects in the DARHT-II Downstream Transport

    CERN Document Server

    Chan, Kwok-Chi D; Ekdahl, Carl; Genoni, Thomas C; Hughes, Thomas P; Schulze, Martin E

    2005-01-01

    The DARHT-II accelerator produces an 18-MeV, 2-kA, 2-μs electron beam pulse. After the accelerator, the pulse is delivered to the final focus on an x-ray producing target via a beam transport section called the Downstream Transport. Ions produced due to beam ionization of residual gases in the Downstream Transport can affect the beam dynamics. Ions generated by the head of the pulse will cause modification of space-charge forces at the tail of the pulse so that the beam head and tail will have different beam envelopes. They may also induce ion-hose instability at the tail of the pulse. If these effects are significant, the focusing requirements of beam head and tail at the final focus will become very different. The focusing of the complete beam pulse will be time dependent and difficult to achieve, leading to less efficient x-ray production. In this paper, we will describe the results of our calculations of these ion effects at different residual-gas pressure levels. Our goal is to determine the ma...

  8. Downstream processing of biopharmaceutical proteins produced in plants

    Science.gov (United States)

    Buyel, Johannes Felix; Fischer, Rainer

    2014-01-01

    All biological platforms for the manufacture of biopharmaceutical proteins produce an initially turbid extract that must be clarified to avoid fouling sensitive media such as chromatography resins. Clarification is more challenging if the feed stream contains large amounts of dispersed particles, because these rapidly clog the filter media typically used to remove suspended solids. Charged polymers (flocculants) can increase the apparent size of the dispersed particles by aggregation, facilitating the separation of solids and liquids, and thus reducing process costs. However, many different factors can affect the behavior of flocculants, including the pH and conductivity of the medium, the size and charge distribution of the particulates, and the charge density and molecular mass of the polymer. Importantly, these properties can also affect the recovery of the target protein and the overall safety profile of the process. We therefore used a design of experiments approach to establish reliable predictive models that characterize the impact of flocculants during the downstream processing of biopharmaceutical proteins. We highlight strategies for the selection of flocculants during process optimization. These strategies will contribute to the quality by design aspects of process development and facilitate the development of safe and efficient downstream processes for plant-derived pharmaceutical proteins. PMID:24637706

  9. Upstream and Downstream Influence in STBLI Instability

    Science.gov (United States)

    Martin, Pino; Priebe, Stephan; Helm, Clara

    2016-11-01

    Priebe and Martín (JFM, 2012) show that the low-frequency unsteadiness in shockwave and turbulent boundary layer interactions (STBLI) is governed by an inviscid instability. Priebe, Tu, Martín and Rowley (JFM, 2016) show that the instability is an inviscid centrifugal one, i.e Görtlerlike vortices. Previous works had given differing conclusions as to whether the low-frequency unsteadiness in STBLI is caused by an upstream or downstream mechanism. In this paper, we reconcile these opposite views and show that upstream and downstream correlations co-exist in the context of the nature of Görtler vortices. We find that the instability is similar to that in separated subsonic and laminar flows. Since the turbulence is modulated but passive to the global mode, the turbulent separated flows are amenable to linear global analysis. As such, the characteristic length and time scales, and the receptivity of the global mode might be determined, and low-order models that represent the low-frequency dynamics in STBLI might be developed. The centrifugal instability persists even under hypersonic conditions. This work is funded by the AFOSR Grant Number AF9550-15-1-0284 with Dr. Ivett Leyva.

  10. Induction of Apoptosis by Luteolin Involving Akt Inactivation in Human 786-O Renal Cell Carcinoma Cells

    Directory of Open Access Journals (Sweden)

    Yen-Chuan Ou

    2013-01-01

    Full Text Available There is a growing interest in the health-promoting effects of natural substances obtained from plants. Although luteolin has been identified as a potential therapeutic and preventive agent for cancer because of its potent cancer cell-killing activity, the molecular mechanisms have not been well elucidated. This study provides evidence of an alternative target for luteolin and sheds light on the mechanism of its physiological benefits. Treatment of 786-O renal cell carcinoma (RCC cells (as well as A498 and ACHN with luteolin caused cell apoptosis and death. This cytotoxicity was caused by the downregulation of Akt and resultant upregulation of apoptosis signal-regulating kinase-1 (Ask1, p38, and c-Jun N-terminal kinase (JNK activities, probably via protein phosphatase 2A (PP2A activation. In addition to being a concurrent substrate of caspases and event of cell death, heat shock protein-90 (HSP90 cleavage might also play a role in driving further cellular alterations and cell death, at least in part, involving an Akt-related mechanism. Due to the high expression of HSP90 and Akt-related molecules in RCC and other cancer cells, our findings suggest that PP2A activation might work in concert with HSP90 cleavage to inactivate Akt and lead to a vicious caspase-dependent apoptotic cycle in luteolin-treated 786-O cells.

  11. 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.

  12. Testosterone induces cardiomyocyte hypertrophy through mammalian target of rapamycin complex 1 pathway.

    Science.gov (United States)

    Altamirano, Francisco; Oyarce, César; Silva, Patricio; Toyos, Marcela; Wilson, Carlos; Lavandero, Sergio; Uhlén, Per; Estrada, Manuel

    2009-08-01

    Elevated testosterone concentrations induce cardiac hypertrophy but the molecular mechanisms are poorly understood. Anabolic properties of testosterone involve an increase in protein synthesis. The mammalian target of rapamycin complex 1 (mTORC1) pathway is a major regulator of cell growth, but the relationship between testosterone action and mTORC1 in cardiac cells remains unknown. Here, we investigated whether the hypertrophic effects of testosterone are mediated by mTORC1 signaling in cultured cardiomyocytes. Testosterone increases the phosphorylation of mTOR and its downstream targets 40S ribosomal protein S6 kinase 1 (S6K1; also known as RPS6KB1) and eukaryotic initiation factor 4E-binding protein 1 (4E-BP1). The S6K1 phosphorylation induced by testosterone was blocked by rapamycin and small interfering RNA to mTOR. Moreover, the hormone increased both extracellular-regulated kinase (ERK1/2) and protein kinase B (Akt) phosphorylation. ERK1/2 inhibitor PD98059 blocked the testosterone-induced S6K1 phosphorylation, whereas Akt inhibition (Akt-inhibitor-X) had no effect. Testosterone-induced ERK1/2 and S6K1 phosphorylation increases were blocked by either 1,2-bis(2-aminophenoxy)ethane-N,N,N,N-tetraacetic acid-acetoxymethylester or by inhibitors of inositol 1,4,5-trisphosphate (IP(3)) pathway: U-73122 and 2-aminoethyl diphenylborate. Finally, cardiomyocyte hypertrophy was evaluated by, the expression of beta-myosin heavy chain, alpha-skeletal actin, cell size, and amino acid incorporation. Testosterone increased all four parameters and the increase being blocked by mTOR inhibition. Our findings suggest that testosterone activates the mTORC1/S6K1 axis through IP(3)/Ca(2+) and MEK/ERK1/2 to induce cardiomyocyte hypertrophy.

  13. Methylglyoxal activates the target of rapamycin complex 2-protein kinase C signaling pathway in Saccharomyces cerevisiae.

    Science.gov (United States)

    Nomura, Wataru; Inoue, Yoshiharu

    2015-04-01

    Methylglyoxal is a typical 2-oxoaldehyde derived from glycolysis. We show here that methylglyoxal activates the Pkc1-Mpk1 mitogen-activated protein (MAP) kinase cascade in a target of rapamycin complex 2 (TORC2)-dependent manner in the budding yeast Saccharomyces cerevisiae. We demonstrate that TORC2 phosphorylates Pkc1 at Thr(1125) and Ser(1143). Methylglyoxal enhanced the phosphorylation of Pkc1 at Ser(1143), which transmitted the signal to the downstream Mpk1 MAP kinase cascade. We found that the phosphorylation status of Pkc1(T1125) affected the phosphorylation of Pkc1 at Ser(1143), in addition to its protein levels. Methylglyoxal activated mammalian TORC2 signaling, which, in turn, phosphorylated Akt at Ser(473). Our results suggest that methylglyoxal is a conserved initiator of TORC2 signaling among eukaryotes.

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

    Directory of Open Access Journals (Sweden)

    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

  16. 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.

  17. [Regulative mechanisms of mammalian target of rapamycin signaling pathway in glomerular hypertrophy in diabetic nephropathy and interventional effects of Chinese herbal medicine].

    Science.gov (United States)

    Yang, Jing-Jing; Huang, Yan-ru; Wan, Yi-gang; Shen, Shan-mei; Mao, Zhi-min; Wu, Wei; Yao, Jian

    2015-08-01

    Glomerular hypertrophy is the main pathological characteristic in the early stage of diabetic nephropathy (DN), and its regulatory mechanism is closely related to mammalian target of rapamycin (mTOR) signaling pathway activity. mTOR includes mTOR complex 1 (mTORC1) and mTOR complex 2(mTORC2), in which, the upstream pathway of mTORC1 is phosphatidylinositol-3-kinase (PI3K)/serine-threonine kinase(Akt)/adenosine monophosphate activated protein kinase(AMPK), and the representative signaling molecules in the downstream pathway of mTORC1 are 4E-binding proteins(4EBP) and phosphoprotein 70 S6Kinase(p70S6K). Some Chinese herbal extracts could improve cell proliferation via intervening the expressions of the key molecules in the upstream or downstream of PIK/Akt/mTOR signaling pathway in vivo. As for glomerular mesangial cells(MC) and podocyte, mTOR plays an important role in regulating glomerular inherent cells, including adjusting cell cycle, energy metabolism and matrix protein synthesis. Rapamycin, the inhibitor of mTOR, could suppress glomerular inherent cell hypertrophy, cell proliferation, glomerular basement membrane (GBM) thickening and mesangial matrix deposition in model rats with DN. Some Chinese herbal extracts could alleviate glomerular lesions by intervening mTOR signaling pathway activity in renal tissue of DN animal models or in renal inherent cells in vivo and in vitro.

  18. 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.

  19. Downstream Thermal Evolution of Vortex Cores

    Science.gov (United States)

    Gómez-Barea, A.; Herrada, M. A.; Pérez-Saborid, M.; Barrero, A.

    1999-11-01

    The downstream evolution of the total temperature field in a quasi-incompressible axisymmetric vortex core has been computed. Starting at an initial station (z=0) with velocity profiles of the Burgers type and given temperature distributions, the numerical results of the evolution show that, according to experimental results, the total temperature in the near-axis region decreases substantially due to the work done by pressure and viscous forces together with the effect of both convection and conduction of heat. Depending on the values of the parameters characterizing the initial profiles and on the value of the Prandtl number, the vortex either breaks down or eventually reaches a self-similar regime. The results obtained shed light on the basic physics involved in the thermal separation phenomenon which appears inside Ranque-Hilsch vortex tubes.

  20. 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.

  1. 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.

  2. 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.

  3. 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.

  4. 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

  5. Acute mechanical stretch promotes eNOS activation in venous endothelial cells mainly via PKA and Akt pathways.

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    Zhenqian Hu

    Full Text Available In the vasculature, physiological levels of nitric oxide (NO protect against various stressors, including mechanical stretch. While endothelial NO production in response to various stimuli has been studied extensively, the precise mechanism underlying stretch-induced NO production in venous endothelial cells remains incompletely understood. Using a model of continuous cellular stretch, we found that stretch promoted phosphorylation of endothelial NO synthase (eNOS at Ser¹¹⁷⁷, Ser⁶³³ and Ser⁶¹⁵ and NO production in human umbilical vein endothelial cells. Although stretch activated the kinases AMPKα, PKA, Akt, and ERK1/2, stretch-induced eNOS activation was only inhibited by kinase-specific inhibitors of PKA and PI3K/Akt, but not of AMPKα and Erk1/2. Similar results were obtained with knockdown by shRNAs targeting the PKA and Akt genes. Furthermore, inhibition of PKA preferentially attenuated eNOS activation in the early phase, while inhibition of the PI3K/Akt pathway reduced eNOS activation in the late phase, suggesting that the PKA and PI3K/Akt pathways play distinct roles in a time-dependent manner. Finally, we investigated the role of these pathways in stretch-induced endothelial exocytosis and leukocyte adhesion. Interestingly, we found that inhibition of the PI3K/Akt pathway increased stretch-induced Weibel-Palade body exocytosis and leukocyte adhesion, while inhibition of the PKA pathway had the opposite effects, suggesting that the exocytosis-promoting effect of PKA overwhelms the inhibitory effect of PKA-mediated NO production. Taken together, the results suggest that PKA and Akt are important regulators of eNOS activation in venous endothelial cells under mechanical stretch, while playing different roles in the regulation of stretch-induced endothelial exocytosis and leukocyte adhesion.

  6. Role of the guanine nucleotide exchange factor in Akt2-mediated plasma membrane translocation of GLUT4 in insulin-stimulated skeletal muscle.

    Science.gov (United States)

    Takenaka, Nobuyuki; Yasuda, Naoto; Nihata, Yuma; Hosooka, Tetsuya; Noguchi, Tetsuya; Aiba, Atsu; Satoh, Takaya

    2014-11-01

    The small GTPase Rac1 plays a key role in insulin-promoted glucose uptake mediated by the GLUT4 glucose transporter in skeletal muscle. Our recent studies have demonstrated that the serine/threonine protein kinase Akt2 is critically involved in insulin-dependent Rac1 activation. The purpose of this study is to clarify the role of the guanine nucleotide exchange factor FLJ00068 in Akt2-mediated Rac1 activation and GLUT4 translocation in mouse skeletal muscle and cultured myocytes. Constitutively activated FLJ00068 induced GLUT4 translocation in a Rac1-dependent and Akt2-independent manner in L6 myocytes. On the other hand, knockdown of FLJ00068 significantly reduced constitutively activated Akt2-triggered GLUT4 translocation. Furthermore, Rac1 activation and GLUT4 translocation induced by constitutively activated phosphoinositide 3-kinase were inhibited by knockdown of FLJ00068. In mouse gastrocnemius muscle, constitutively activated FLJ00068 actually induced GLUT4 translocation to the sarcolemma. GLUT4 translocation by constitutively activated FLJ00068 was totally abolished in rac1 knockout mouse gastrocnemius muscle. Additionally, we were successful in detecting the activation of Rac1 following the expression of constitutively activated FLJ00068 in gastrocnemius muscle by immunofluorescence microscopy using an activation-specific probe. Collectively, these results strongly support the notion that FLJ00068 regulates Rac1 downstream of Akt2, leading to the stimulation of glucose uptake in skeletal muscle.

  7. 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.

  8. Survival advantages conferred to colon cancer cells by E-selectin-induced activation of the PI3K-NFκB survival axis downstream of Death receptor-3

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    Paquet Éric R

    2011-07-01

    Full Text Available Abstract Background Extravasation of circulating cancer cells is a key event of metastatic dissemination that is initiated by the adhesion of cancer cells to endothelial cells. It requires interactions between adhesion receptors on endothelial cells and their counter-receptors on cancer cells. Notably, E-selectin, a major endothelial adhesion receptor, interacts with Death receptor-3 present on metastatic colon carcinoma cells. This interaction confers metastatic properties to colon cancer cells by promoting the adhesion of cancer cells to endothelial cells and triggering the activation of the pro-migratory p38 and pro-survival ERK pathways in the cancer cells. In the present study, we investigated further the mechanisms by which the E-selectin-activated pathways downstream of DR3 confer a survival advantage to colon cancer cells. Methods Cell survival has been ascertained by using the WST-1 assay and by evaluating the activation of the PI3 kinase/NFκB survival axis. Apoptosis has been assayed by determining DNA fragmentation by Hoechst staining and by measuring cleavage of caspases-8 and -3. DR3 isoforms have been identified by PCR. For more precise quantification, targeted PCR reactions were carried out, and the amplified products were analyzed by automated chip-based microcapillary electrophoresis on an Agilent 2100 Bioanalyzer instrument. Results Interaction between DR3-expressing HT29 colon carcinoma cells and E-selectin induces the activation of the PI3K/Akt pathway. Moreover, p65/RelA, the anti-apoptotic subunit of NFκB, is rapidly translocated to the nucleus in response to E-selectin. This translocation is impaired by the PI3K inhibitor LY294002. Furthermore, inhibition of the PI3K/Akt pathway increases the cleavage of caspase 8 in colon cancer cells treated with E-selectin and this effect is still further increased when both ERK and PI3K pathways are concomitantly inhibited. Intriguingly, metastatic colon cancer cell lines such as HT

  9. 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.

  10. Computational drugs repositioning identifies inhibitors of oncogenic PI3K/AKT/P70S6K-dependent pathways among FDA-approved compounds

    Science.gov (United States)

    Carrella, Diego; Manni, Isabella; Tumaini, Barbara; Dattilo, Rosanna; Papaccio, Federica; Mutarelli, Margherita; Sirci, Francesco; Amoreo, Carla A.; Mottolese, Marcella; Iezzi, Manuela; Ciolli, Laura; Aria, Valentina; Bosotti, Roberta; Isacchi, Antonella; Loreni, Fabrizio; Bardelli, Alberto; Avvedimento, Vittorio E.; di Bernardo, Diego; Cardone, Luca

    2016-01-01

    The discovery of inhibitors for oncogenic signalling pathways remains a key focus in modern oncology, based on personalized and targeted therapeutics. Computational drug repurposing via the analysis of FDA-approved drug network is becoming a very effective approach to identify therapeutic opportunities in cancer and other human diseases. Given that gene expression signatures can be associated with specific oncogenic mutations, we tested whether a “reverse” oncogene-specific signature might assist in the computational repositioning of inhibitors of oncogenic pathways. As a proof of principle, we focused on oncogenic PI3K-dependent signalling, a molecular pathway frequently driving cancer progression as well as raising resistance to anticancer-targeted therapies. We show that implementation of “reverse” oncogenic PI3K-dependent transcriptional signatures combined with interrogation of drug networks identified inhibitors of PI3K-dependent signalling among FDA-approved compounds. This led to repositioning of Niclosamide (Niclo) and Pyrvinium Pamoate (PP), two anthelmintic drugs, as inhibitors of oncogenic PI3K-dependent signalling. Niclo inhibited phosphorylation of P70S6K, while PP inhibited phosphorylation of AKT and P70S6K, which are downstream targets of PI3K. Anthelmintics inhibited oncogenic PI3K-dependent gene expression and showed a cytostatic effect in vitro and in mouse mammary gland. Lastly, PP inhibited the growth of breast cancer cells harbouring PI3K mutations. Our data indicate that drug repositioning by network analysis of oncogene-specific transcriptional signatures is an efficient strategy for identifying oncogenic pathway inhibitors among FDA-approved compounds. We propose that PP and Niclo should be further investigated as potential therapeutics for the treatment of tumors or diseases carrying the constitutive activation of the PI3K/P70S6K signalling axis. PMID:27542212

  11. Targeting ILK and {beta}4 integrin abrogates the invasive potential of ovarian cancer

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    Choi, Yoon Pyo; Kim, Baek Gil [BK21 Project for Medical Science, Yonsei University College of Medicine, Seoul (Korea, Republic of); Department of Pathology, Yonsei University College of Medicine, Seoul (Korea, Republic of); Gao, Ming-Qing; Kang, Suki [Department of Pathology, Yonsei University College of Medicine, Seoul (Korea, Republic of); Cho, Nam Hoon, E-mail: cho1988@yuhs.ac [BK21 Project for Medical Science, Yonsei University College of Medicine, Seoul (Korea, Republic of); Department of Pathology, Yonsei University College of Medicine, Seoul (Korea, Republic of)

    2012-10-26

    Highlights: Black-Right-Pointing-Pointer The potential of targeting ILK and integrins for highly aggressive ovarian cancer. Black-Right-Pointing-Pointer Unanticipated synergistic effect for the combination of ILK/{beta}4 integrin. Black-Right-Pointing-Pointer Combination of ILK/{beta}4 integrin effectively inhibited the PI3K/Akt/Rac1 cascade. Black-Right-Pointing-Pointer Targeting of {beta}4 integrin/ILK had potent inhibitory effects in ovarian cancer. -- Abstract: Integrins and integrin-linked kinase (ILK) are essential to cancerous invasion because they mediate physical interactions with the extracellular matrix, and regulate oncogenic signaling pathways. The purpose of our study is to determine whether deletion of {beta}1 and {beta}4 integrin and ILK, alone or in combination, has antitumoral effects in ovarian cancer. Expression of {beta}1 and {beta}4 integrin and ILK was analyzed by immunohistochemistry in 196 ovarian cancer tissue samples. We assessed the effects of depleting these molecules with shRNAs in ovarian cancer cells by Western blot, conventional RT-PCR, cell proliferation, migration, invasion, and in vitro Rac1 activity assays, and in vivo xenograft formation assays. Overexpression of {beta}4 integrin and ILK in human ovarian cancer specimens was found to correlate with tumor aggressiveness. Depletion of these targets efficiently suppresses ovarian cancer cell proliferation, migration, and invasion in vitro and xenograft tumor formation in vivo. We also demonstrated that single depletion of ILK or combination depletion of {beta}4 integrin/ILK inhibits phosphorylation of downstream signaling targets, p-Ser 473 Akt and p-Thr202/Tyr204 Erk1/2, and activation of Rac1, as well as reduce expression of MMP-2 and MMP-9 and increase expression of caspase-3 in vitro. In conclusion, targeting {beta}4 integrin combined with ILK can instigate the latent tumorigenic potential and abrogate the invasive potential in ovarian cancer.

  12. Wortmannin, PI3K/Akt signaling pathway inhibitor, attenuates thyroid injury associated with severe acute pancreatitis in rats.

    Science.gov (United States)

    Abliz, Ablikim; Deng, Wenhong; Sun, Rongze; Guo, Wenyi; Zhao, Liang; Wang, Weixing

    2015-01-01

    Increasing evidences suggest that PI3K/AKT pathway plays an important role in the pathogenesis of inflammatory diseases such as acute pancreatitis. However, the exact effect of PI3K/AKT on thyroid injury associated with acute pancreatitis has not been investigated. This study aimed to investigate the protective effects of wortmannin, PI3K/AKT inhibitor, on thyroid injury in a rat model of severe acute pancreatitis (SAP). Sixty male SD rats were randomly divided into four groups: sham operating group (SO), SAP group, wortmannin treatment (WOR) group and drug control (WOR-CON) group. Serum amylase (AMY), lipase (LIP) and thyroid hormone levels were evaluated. The morphological change of thyroid tissue was analyzed under the light and transmission electron microscopy. AKT, P38MAPK and NF-κB expression in the thyroid tissue was evaluated by immunohistochemical staining. Oxidative stress and inflammatory cytokines were detected. Results showed that wortmannin attenuated the following: (1) serum AMY, LIP and thyroid hormone (2) pancreatic and thyroid pathological injuries (3) thyroid MDA, (4) thyroid ultrastructural change, (5) serum TNF-α, IL-6 and IL-1β (6) AKT, MAPKP38 and NF-κB expression in thyroid tissues. These results suggested that wortmannin attenuates thyroid injury in SAP rats, presumably because of its role on prevent ROS generation and inhibits the activation of P38MAPK, NF-κB pathway. Our findings provide new therapeutic targets for thyroid injury associated with SAP.

  13. 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.

  14. Inhibition of constitutively activated phosphoinositide 3-kinase/AKT pathway enhances antitumor activity of chemotherapeutic agents in breast cancer susceptibility gene 1-defective breast cancer cells.

    Science.gov (United States)

    Yi, Yong Weon; Kang, Hyo Jin; Kim, Hee Jeong; Hwang, Jae Seok; Wang, Antai; Bae, Insoo

    2013-09-01

    Loss or decrease of wild type BRCA1 function, by either mutation or reduced expression, has a role in hereditary and sporadic human breast and ovarian cancers. We report here that the PI3K/AKT pathway is constitutively active in BRCA1-defective human breast cancer cells. Levels of phospho-AKT are sustained even after serum starvation in breast cancer cells carrying deleterious BRCA1 mutations. Knockdown of BRCA1 in MCF7 cells increases the amount of phospho-AKT and sensitizes cells to small molecule protein kinase inhibitors (PKIs) targeting the PI3K/AKT pathway. Restoration of wild type BRCA1 inhibits the activated PI3K/AKT pathway and de-sensitizes cells to PKIs targeting this pathway in BRCA1 mutant breast cancer cells, regardless of PTEN mutations. In addition, clinical PI3K/mTOR inhibitors, PI-103, and BEZ235, showed anti-proliferative effects on BRCA1 mutant breast cancer cell lines and synergism in combination with chemotherapeutic drugs, cisplatin, doxorubicin, topotecan, and gemcitabine. BEZ235 synergizes with the anti-proliferative effects of gemcitabine by enhancing caspase-3/7 activity. Our results suggest that the PI3K/AKT pathway can be an important signaling pathway for the survival of BRCA1-defective breast cancer cells and pharmacological inhibition of this pathway is a plausible treatment for a subset of breast cancers.

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

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    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.

  16. 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

  17. 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.

  18. 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

  19. AKTivation of the PI3K/AKT/mTOR signaling pathway by KSHV

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    Aadra P Bhatt

    2013-01-01

    Full Text Available As an obligate intracellular parasite, the Kaposi sarcoma-associated herpesvirus (KSHV relies on host cell machinery to meet its needs for survival, viral replication, production, and dissemination of progeny virions. KSHV is a ɣ-herpesvirus that is associated with three different malignancies: Kaposi sarcoma (KS, and two B cell lymphoproliferative disorders, primary effusion lymphoma (PEL and multicentric Castleman disease (MCD. KSHV viral proteins modulate cellular phosphatidylinositol-3-kinase (PI3K/AKT/mammalian target of rapamycin (mTOR signaling pathway, which is a ubiquitous pathway that also controls B lymphocyte proliferation and development. We review the mechanisms by which KSHV manipulates the PI3K/AKT/mTOR pathway, with a specific focus on B cells.

  20. 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.

  1. PI3K/AKT/mTOR: role in breast cancer progression, drug resistance, and treatment.

    Science.gov (United States)

    Guerrero-Zotano, Angel; Mayer, Ingrid A; Arteaga, Carlos L

    2016-12-01

    Anti-cancer cancer-targeted therapies are designed to exploit a particular vulnerability in the tumor, which in most cases results from its dependence on an oncogene and/or loss of a tumor suppressor. Mutations in the phosphoinositide 3-kinase (PI3K)/AKT/mTOR pathway are freqcuently found in breast cancers and associated with cellular transformation, tumorigenesis, cancer progression, and drug resistance. Several drugs targeting PI3K/ATK/mTOR are currently in clinical trials, mainly in combination with endocrine therapy and anti-HER2 therapy. These drugs are the focus of this review.

  2. 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.

  3. 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.

  4. Ischemic post-conditioning facilitates brain recovery after stroke by promoting Akt/mTOR activity in nude rats.

    Science.gov (United States)

    Xie, Rong; Wang, Peng; Ji, Xunming; Zhao, Heng

    2013-12-01

    While pre-conditioning is induced before stroke onset, ischemic post-conditioning (IPostC) is performed after reperfusion, which typically refers to a series of mechanical interruption of blood reperfusion after stroke. IPostC is known to reduce infarction in wild-type animals. We investigated if IPostC protects against brain injury induced by focal ischemia in Tcell-deficient nude rats and to examine its effects on Akt and the mammalian target of rapamycin (mTOR) pathway. Although IPostC reduced infarct size at 2 days post-stroke in wild-type rats, it did not attenuate infarction in nude rats. Despite the unaltered infarct size in nude rats, IPostC increased levels of phosphorylated Akt (p-Akt) and Akt isoforms (Akt1, Akt2, Akt3), and p-mTOR, p-S6K and p-4EBP1 in the mTOR pathway, as well as growth associated Protein 43 (GAP43), both in the peri-infarct area and core, 24 h after stroke. IPostC improved neurological function in nude rats 1-30 days after stroke and reduced the extent of brain damage 30 days after stroke. The mTOR inhibitor rapamycin abolished the long-term protective effects of IPostC. We determined that IPostC did not inhibit acute infarction in nude rats but did provide long-term protection by enhancing Akt and mTOR activity during the acute post-stroke phase. Post-conditioning did not attenuate infarction in nude rats measured 2 days post-stroke, but improved neurological function in nude rats and reduced brain damage 30 days after stroke. It resulted in increased-activities of Akt and mTOR, S6K and p-4EBP1. The mTOR inhibitor rapamycin abolished the long-term protective effects of IPostC.

  5. 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

  6. Hepatic stellate cell is activated by microRNA-181b via PTEN/Akt pathway.

    Science.gov (United States)

    Zheng, Jianjian; Wu, Cunzao; Xu, Ziqiang; Xia, Peng; Dong, Peihong; Chen, Bicheng; Yu, Fujun

    2015-01-01

    Activation of hepatic stellate cells (HSCs) is an essential event in the initiation and progression of liver fibrosis. MicroRNAs have been shown to play a pivotal role in regulating HSC functions such as cell proliferation, differentiation, and apoptosis. Recently, miR-181b has been reported to promote HSCs proliferation by targeting p27. But whether alpha-smooth muscle actin (α-SMA) or collagens could be promoted by miR-181b in activated HSCs is still not clear. Therefore, the understanding of the role of miR-181b in liver fibrosis remains limited. Our results showed that miR-181b expression was increased much higher than miR-181a expression in vitro in transforming growth factor-β1-induced HSC activation as well as in vivo in carbon tetrachloride-induced rat liver fibrosis. Of note, overexpression of miR-181b significantly increased the expressions level of α-SMA and type I collagen, and further promoted HSCs proliferation. Furthermore, phosphatase and tensin homologs deleted on chromosome 10 (PTEN), a negative regulator of PI3K/Akt pathway, were confirmed as a direct target of miR-181b. We demonstrated that miR-181b could suppress PTEN expression and increase Akt phosphorylation in HSCs. Interestingly, the effects of miR-181b on the activation of HSCs were blocked down by Akt inhibitor LY294002. Our results revealed a profibrotic role of miR-181b in HSC activation and demonstrated that miR-181b could activate HSCs, at least in part, via PTEN/Akt pathway.

  7. 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

  8. DOWNSTREAM ECOCIDE FROM UPSTREAM WATER PIRACY

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    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

  9. 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.

  10. 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.

  11. KU004 induces G1 cell cycle arrest in human breast cancer SKBR-3 cells by modulating PI3K/Akt pathway.

    Science.gov (United States)

    Fu, Jing; Tian, Chongchong; Xing, Mengtao; Wang, Xinzhi; Guo, Hongli; Sun, Lixin; Sun, Lan; Jiang, Zhenzhou; Zhang, Luyong

    2014-06-01

    KU004 is a newly synthesized compound which has been demonstrated possessing potent anti-cancer activities through targeting the highly-expressed protein HER2 on the surface of the cells. In this study, we investigated the potential roles of KU004 in the induced-cell cycle arrest in human breast cancer SK-BR-3 cells. KU004 could not only inhibit the proliferation of SK-BR-3 in a concentration-dependent manner but also induce G1 phase arrest in SK-BR-3 cells. The western blot results showed KU004 decreased the expression of cyclin D, CDK-4, p-Rb708/780, and up-regulated the p21. In order to verify whether KU004 takes the anti-tumor effect thought the regulation of PI3K/Akt pathway, we used western blot to detect the expression of protein Akt, Her2, p-Akt and p-Her2. Our results shown that after KU004 treatment, the amount of p-Akt and p-Her2 decreased but the total amount of Akt and Her2 remained unchanged. In conclusion, these results provide a framework for further exploration of KU004 as a novel chemotherapeutic for human breast tumors by modulating PI3K/Akt pathway.

  12. 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.

  13. 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.

  14. 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.

  15. Apigenin suppresses GLUT-1 and p-AKT expression to enhance the chemosensitivity to cisplatin of laryngeal carcinoma Hep-2 cells: an in vitro study.

    Science.gov (United States)

    Xu, Ying-Ying; Wu, Ting-Ting; Zhou, Shui-Hong; Bao, Yang-Yang; Wang, Qin-Ying; Fan, Jun; Huang, Ya-Ping

    2014-01-01

    Glucose transporter-1 (GLUT-1) and PI3K/Akt are known to be closely involved in resistance to chemotherapy. Co-targeted therapy reducing GLUT-1 expression and PI3K/Akt pathway activity may overcome the chemoresistance of human cancers. Apigenin may inhibit the expression of GLUT-1 and the PI3K/Akt pathway. We hypothesized that over-expression of GLUT-1 and p-Akt was associated with the resistance to cisplatin of laryngeal carcinoma Hep-2 cells. We explored whether apigenin inhibited GLUT-1 and p-Akt, resulting in sensitization of laryngeal carcinoma Hep-2 cells to cisplatin. Real-time RT-PCR and Western blotting confirmed the presence of GLUT-1 mRNA, and GLUT-1 and p-Akt proteins in Hep-2 cells. We found that resistance or insensitivity of Hep-2 cells to cisplatin might be associated with such expression. Apigenin markedly enhanced the cisplatin-induced suppression of Hep-2 cell growth. This effect was concentration- and time-dependent. Thus apigenin may significantly reduce the levels of GLUT-1 mRNA, and GLUT-1 and p-Akt proteins, in cisplatin-treated Hep-2 cells, in a concentration- and time-dependent manner. To conclude, overexpression of GLUT-1 mRNA may be associated with the resistance to cisplatin of laryngeal carcinoma Hep-2 cells. Apigenin may enhance the sensitivity to cisplatin of laryngeal carcinoma cells via inhibition of GLUT-1 and p-Akt expression.

  16. Isorhamnetin protects against cardiac hypertrophy through blocking PI3K-AKT pathway.

    Science.gov (United States)

    Gao, Lu; Yao, Rui; Liu, Yuzhou; Wang, Zheng; Huang, Zhen; Du, Binbin; Zhang, Dianhong; Wu, Leiming; Xiao, Lili; Zhang, Yanzhou

    2017-02-07

    Isorhamnetin, a flavonoid compound extracted from the Chinese herb Hippophae rhamnoides L., is well known for its anti-inflammatory, anti-oxidative, anti-adipogenic, anti-proliferative, and anti-tumor activities. However, the role of isorhamnetin in cardiac hypertrophy has not been reported. The aims of the present study were to find whether isorhamnetin could alleviate cardiac hypertrophy and to define the underlying molecular mechanisms. Here, we investigated the effects of isorhamnetin (100 mg/kg/day) on cardiac hypertrophy induced by aortic banding in mice. Cardiac hypertrophy was evaluated by echocardiographic, hemodynamic, pathological, and molecular analyses. Our data demonstrated that isorhamnetin could inhibit cardiac hypertrophy and fibrosis 8 weeks after aortic banding. The results further revealed that the effect of isorhamnetin on cardiac hypertrophy was mediated by blocking the activation of phosphatidylinositol 3-kinase-AKT signaling pathway. In vitro studies performed in neonatal rat cardiomyocytes confirmed that isorhamnetin could attenuate cardiomyocyte hypertrophy induced by angiotensin II, which was associated with phosphatidylinositol 3-kinase-AKT signaling pathway. In conclusion, these data indicate for the first time that isorhamnetin has protective potential for targeting cardiac hypertrophy by blocking the phosphatidylinositol 3-kinase-AKT signaling pathway. Thus, our study suggests that isorhamnetin may represent a potential therapeutic strategy for the treatment of cardiac hypertrophy and heart failure.

  17. Dynamic Akt/mTOR Signaling in Children with Autism Spectrum Disorder

    Science.gov (United States)

    Onore, Charity; Yang, Houa; Van de Water, Judy; Ashwood, Paul

    2017-01-01

    Autism spectrum disorder (ASD) is a behaviorally defined disorder affecting 1 in 68 children. Currently, there is no known cause for the majority of ASD cases nor are there physiological diagnostic tools or biomarkers to aid behavioral diagnosis. Whole-genome linkage studies, genome-wide association studies, copy number variation screening, and SNP analyses have identified several ASD candidate genes, but which vary greatly among individuals and family clusters, suggesting that a variety of genetic mutations may result in a common pathology or alter a common mechanistic pathway. The Akt/mammalian target of rapamycin (mTOR) pathway is involved in many cellular processes including synaptic plasticity and immune function that can alter neurodevelopment. In this study, we examined the activity of the Akt/mTOR pathway in cells isolated from children with ASD and typically developing controls. We observed higher activity of mTOR, extracellular receptor kinase, and p70S6 kinase and lower activity of glycogen synthase kinase 3 (GSK3)α and tuberin (TSC2) in cells from children with ASD. These data suggest a phosphorylation pattern indicative of higher activity in the Akt/mTOR pathway in children with general/idiopathic ASD and may suggest a common pathological pathway of interest for ASD.

  18. The PI3K/Akt pathway contributes to arenavirus budding.

    Science.gov (United States)

    Urata, Shuzo; Ngo, Nhi; de la Torre, Juan Carlos

    2012-04-01

    Several arenaviruses, chiefly Lassa virus (LASV), cause hemorrhagic fever (HF) disease in humans and pose a significant public health concern in regions where they are endemic. On the other hand, evidence indicates that the globally distributed prototypic arenavirus lymphocytic choriomeningitis virus (LCMV) is a neglected human pathogen. The phosphatidylinositol 3-kinase (PI3K)/Akt pathway participates in many cellular processes, including cell survival and differentiation, and also has been shown to play important roles in different steps of the life cycles of a variety of viruses. Here we report that the inhibition of the PI3K/Akt pathway inhibited budding and to a lesser extent RNA synthesis, but not cell entry, of LCMV. Accordingly, BEZ-235, a PI3K inhibitor currently in cancer clinical trials, inhibited LCMV multiplication in cultured cells. These findings, together with those previously reported for Junin virus (JUNV), indicate that targeting the PI3K/Akt pathway could represent a novel antiviral strategy to combat human-pathogenic arenaviruses.

  19. 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.

  20. 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...

  1. Up-regulation of the mammalian target of rapamycin complex 1 subunit Raptor by aldosterone induces abnormal pulmonary artery smooth muscle cell survival patterns to promote pulmonary arterial hypertension.

    Science.gov (United States)

    Aghamohammadzadeh, Reza; Zhang, Ying-Yi; Stephens, Thomas E; Arons, Elena; Zaman, Paula; Polach, Kevin J; Matar, Majed; Yung, Lai-Ming; Yu, Paul B; Bowman, Frederick P; Opotowsky, Alexander R; Waxman, Aaron B; Loscalzo, Joseph; Leopold, Jane A; Maron, Bradley A

    2016-07-01

    Activation of the mammalian target of rapamycin complex 1 (mTORC1) subunit Raptor induces cell growth and is a downstream target of Akt. Elevated levels of aldosterone activate Akt, and, in pulmonary arterial hypertension (PAH), correlate with pulmonary arteriole thickening, which suggests that mTORC1 regulation by aldosterone may mediate adverse pulmonary vascular remodeling. We hypothesized that aldosterone-Raptor signaling induces abnormal pulmonary artery smooth muscle cell (PASMC) survival patterns to promote PAH. Remodeled pulmonary arterioles from SU-5416/hypoxia-PAH rats and monocrotaline-PAH rats with hyperaldosteronism expressed increased levels of the Raptor target, p70S6K, which provided a basis for investigating aldosterone-Raptor signaling in human PASMCs. Aldosterone (10(-9) to 10(-7) M) increased Akt/mTOR/Raptor to activate p70S6K and increase proliferation, viability, and apoptosis resistance in PASMCs. In PASMCs transfected with Raptor-small interfering RNA or treated with spironolactone/eplerenone, aldosterone or pulmonary arterial plasma from patients with PAH failed to increase p70S6K activation or to induce cell survival in vitro Optimal inhibition of pulmonary arteriole Raptor was achieved by treatment with Staramine-monomethoxy polyethylene glycol that was formulated with Raptor-small interfering RNA plus spironolactone in vivo, which decreased arteriole muscularization and pulmonary hypertension in 2 experimental animal models of PAH in vivo Up-regulation of mTORC1 by aldosterone is a critical pathobiologic mechanism that controls PASMC survival to promote hypertrophic vascular remodeling and PAH.-Aghamohammadzadeh, R., Zhang, Y.-Y., Stephens, T. E., Arons, E., Zaman, P., Polach, K. J., Matar, M., Yung, L.-M., Yu, P. B., Bowman, F. P., Opotowsky, A. R., Waxman, A. B., Loscalzo, J., Leopold, J. A., Maron, B. A. Up-regulation of the mammalian target of rapamycin complex 1 subunit Raptor by aldosterone induces abnormal pulmonary artery smooth

  2. KIF14 Promotes AKT Phosphorylation and Contributes to Chemoresistance in Triple-Negative Breast Cancer

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    Stina M. Singel

    2014-03-01

    Full Text Available Despite evidence that kinesin family member 14 (KIF14 can serve as a prognostic biomarker in various solid tumors, how it contributes to tumorigenesis remains unclear. We observed that experimental decrease in KIF14 expression increases docetaxel chemosensitivity in estrogen receptor–negative/progesterone receptor–negative/human epidermal growth factor receptor 2-negative, “triple-negative” breast cancers (TNBC. To investigate the oncogenic role of KIF14, we used noncancerous human mammary epithelial cells and ectopically expressed KIF14 and found increased proliferative capacity, increased anchorage-independent grown in vitro, and increased resistance to docetaxel but not to doxorubicin, carboplatin, or gemcitabine. Seventeen benign breast biopsies of BRCA1 or BRCA2 mutation carriers showed increased KIF14 mRNA expression by fluorescence in situ hybridization compared to controls with no known mutations in BRCA1 or BRCA2, suggesting increased KIF14 expression as a biomarker of high-risk breast tissue. Evaluation of 34 cases of locally advanced TNBC showed that KIF14 expression significantly correlates with chemotherapy-resistant breast cancer. KIF14 knockdown also correlates with decreased AKT phosphorylation and activity. Live-cell imaging confirmed an insulin-induced temporal colocalization of KIF14 and AKT at the plasma membrane, suggesting a potential role of KIF14 in promoting activation of AKT. An experimental small-molecule inhibitor of KIF14 was then used to evaluate the potential anticancer benefits of downregulating KIF14 activity. Inhibition of KIF14 shows a chemosensitizing effect and correlates with decreasing activation of AKT. Together, these findings show an early and critical role for KIF14 in the tumorigenic potential of TNBC, and therapeutic targeting of KIF14 is feasible and effective for TNBC.

  3. Functional Role of mTORC2 versus Integrin-Linked Kinase in Mediating Ser473-Akt Phosphorylation in PTEN-Negative Prostate and Breast Cancer Cell Lines.

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    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

  4. 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.

  5. Prostaglandin E2 reduces radiation-induced epithelial apoptosis through a mechanism involving AKT activation and bax translocation.

    Science.gov (United States)

    Tessner, Teresa G; Muhale, Filipe; Riehl, Terrence E; Anant, Shrikant; Stenson, William F

    2004-12-01

    Prostaglandin E2 (PGE2) synthesis modulates the response to radiation injury in the mouse intestinal epithelium through effects on crypt survival and apoptosis; however, the downstream signaling events have not been elucidated. WT mice receiving 16,16-dimethyl PGE2 (dmPGE2) had fewer apoptotic cells per crypt than untreated mice. Apoptosis in Bax(-/-) mice receiving 12 Gy was approximately 50% less than in WT mice, and the ability of dmPGE2 to attenuate apoptosis was lost in Bax(-/-) mice. Positional analysis revealed that apoptosis in the Bax(-/-) mice was diminished only in the bax-expressing cells of the lower crypts and that in WT mice, dmPGE2 decreased apoptosis only in the bax-expressing cells. The HCT-116 intestinal cell line and Bax(-/-) HCT-116 recapitulated the apoptotic response of the mouse small intestine with regard to irradiation and dmPGE2. Irradiation of HCT-116 cells resulted in phosphorylation of AKT that was enhanced by dmPGE2 through transactivation of the EGFR. Inhibition of AKT phosphorylation prevented the reduction of apoptosis by dmPGE2 following radiation. Transfection of HCT-116 cells with a constitutively active AKT reduced apoptosis in irradiated cells to the same extent as in nontransfected cells treated with dmPGE2. Treatment with dmPGE2 did not alter bax or bcl-x expression but suppressed bax translocation to the mitochondrial membrane. Our in vivo studies indicate that there are bax-dependent and bax-independent radiation-induced apoptosis in the intestine but that only the bax-dependent apoptosis is reduced by dmPGE2. The in vitro studies indicate that dmPGE2, most likely by signaling through the E prostaglandin receptor EP2, reduces radiation-induced apoptosis through transactivation of the EGFR and enhanced activation of AKT and that this results in reduced bax translocation to the mitochondria.

  6. 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.

  7. Overcoming resistance of targeted EGFR monotherapy by inhibition of STAT3 escape pathway in soft tissue sarcoma

    Science.gov (United States)

    Wang, Xiaochun; Goldstein, David; Crowe, Philip J.; Yang, Mark; Garrett, Kerryn; Zeps, Nikolajs; Yang, Jia-Lin

    2016-01-01

    Although epidermal growth factor receptor (EGFR) is often over-expressed in soft tissue sarcoma (STS), a phase II trial using an EGFR inhibitor gefitinib showed a low response rate. This study identified a new secondary resistance mechanism of gefitinib in STS, and developed new strategies to improve the effectiveness of EGFR inhibition particularly by blocking the STAT3 pathway. We demonstrated that seven STS cell lines of diverse histological origin showed resistance to gefitinib despite blockade of phosphorylated EGFR (pEGFR) and downstream signal transducers (pAKT and pERK) in PI3K/AKT and RAS/ERK pathways. Gefitinib exposure was not associated with decrease in the ratio of pSTAT3/pSTAT1. The relative STAT3 abundance and activation may be responsible for the drug resistance. We therefore hypothesized that the addition of a STAT3 inhibitor could overcome the STAT3 escape pathway. We found that the addition of STAT3 inhibitor S3I-201 to gefitinib achieved synergistic anti-proliferative and pro-apoptotic effects in all three STS cell lines examined. This was confirmed in a fibrosarcoma xenografted mouse model, where the tumours from the combination group (418mm3) were significantly smaller than those from untreated (1032mm3) or single drug (912 and 798mm3) groups. Our findings may have clinical implications for optimising EGFR-targeted therapy in STS. PMID:26909593

  8. Detection of the transforming AKT1 mutation E17K in non-small cell lung cancer by high resolution melting

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    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.

  9. Migration of Th1 lymphocytes is regulated by CD152 (CTLA-4-mediated signaling via PI3 kinase-dependent Akt activation.

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    Karin Knieke

    Full Text Available Efficient adaptive immune responses require the localization of T lymphocytes in secondary lymphoid organs and inflamed tissues. To achieve correct localization of T lymphocytes, the migration of these cells is initiated and directed by adhesion molecules and chemokines. It has recently been shown that the inhibitory surface molecule CD152 (CTLA-4 initiates Th cell migration, but the molecular mechanism underlying this effect remains to be elucidated. Using CD4 T lymphocytes derived from OVA-specific TCR transgenic CD152-deficient and CD152-competent mice, we demonstrate that chemokine-triggered signal transduction is differentially regulated by CD152 via phosphoinositide 3-kinase (PI3K-dependent activation of protein kinase B (PKB/Akt. In the presence of CD152 signaling, the chemoattractant CCL4 selectively induces the full activation of Akt via phosphorylation at threonine 308 and serine 473 in pro-inflammatory Th lymphocytes expressing the cognate chemokine receptor CCR5. Akt signals lead to cytoskeleton rearrangements, which are indispensable for migration. Therefore, this novel Akt-modulating function of CD152 signals affecting T cell migration demonstrates that boosting CD152 or its down-stream signal transduction could aid therapies aimed at sensitizing T lymphocytes for optimal migration, thus contributing to a precise and effective immune response.

  10. 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.

  11. Adrenoceptors promote glucose uptake into adipocytes and muscle by an insulin-independent signaling pathway involving mechanistic target of rapamycin complex 2.

    Science.gov (United States)

    Mukaida, Saori; Evans, Bronwyn A; Bengtsson, Tore; Hutchinson, Dana S; Sato, Masaaki

    2017-02-01

    Uptake of glucose into skeletal muscle and adipose tissue plays a vital role in metabolism and energy balance. Insulin released from β-islet cells of the pancreas promotes glucose uptake in these target tissues by stimulating translocation of GLUT4 transporters to the cell surface. This process is complex, involving signaling proteins including the mechanistic (or mammalian) target of rapamycin (mTOR) and Akt that intersect with multiple pathways controlling cell survival, growth and proliferation. mTOR exists in two forms, mTOR complex 1 (mTORC1), and mTOR complex 2 (mTORC2). mTORC1 has been intensively studied, acting as a key regulator of protein and lipid synthesis that integrates cellular nutrient availability and energy balance. Studies on mTORC2 have focused largely on its capacity to activate Akt by phosphorylation at Ser473, however recent findings demonstrate a novel role for mTORC2 in cellular glucose uptake. For example, agonists acting at β2-adrenoceptors (ARs) in skeletal muscle or β3-ARs in brown adipose tissue increase glucose uptake in vitro and in vivo via mechanisms dependent on mTORC2 but not Akt. In this review, we will focus on the signaling pathways downstream of β-ARs that promote glucose uptake in skeletal muscle and brown adipocytes, and will highlight how the insulin and adrenergic pathways converge and interact in these cells. The identification of insulin-independent mechanisms that promote glucose uptake should facilitate novel treatment strategies for metabolic disease.

  12. 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.

  13. A Positive Feedback Loop between Akt and mTORC2 via SIN1 Phosphorylation

    OpenAIRE

    Guang Yang; Danielle S. Murashige; Sean J. Humphrey; David E. James

    2015-01-01

    The mechanistic target of rapamycin complex 2 (mTORC2) regulates cell survival and cytoskeletal organization by phosphorylating its AGC kinase substrates; however, little is known about the regulation of mTORC2 itself. It was previously reported that Akt phosphorylates the mTORC2 subunit SIN1 at T86, activating mTORC2 through a positive feedback loop, though another study reported that S6K phosphorylates SIN1 at the same site, inhibiting mTORC2 activity. We performed extensive analysis of SIN...

  14. 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.

  15. miR-222 induces Adriamycin resistance in breast cancer through PTEN/Akt/p27(kip1) pathway.

    Science.gov (United States)

    Wang, Dan-Dan; Yang, Su-Jin; Chen, Xiu; Shen, Hong-Yu; Luo, Long-Ji; Zhang, Xiao-Hui; Zhong, Shan-Liang; Zhao, Jian-Hua; Tang, Jin-Hai

    2016-11-01

    The high resistant rate of Adriamycin (Adr) is associated with a poor prognosis of breast cancer in women worldwide. Since miR-222 might contribute to chemoresistance in many cancer types, in this study, we aimed to investigate its efficacy in breast cancer through PTEN/Akt/p27 (kip1) pathway. Firstly, in vivo, we verified that miR-222 was upregulated in chemoresistant tissues after surgery compared with the paired preneoadjuvant samples of 21 breast cancer patients. Then, human breast cancer Adr-resistant cell line (MCF-7/Adr) was constructed to validate the pathway from the parental sensitive cell line (MCF-7/S). MCF-7/Adr and MCF-7/S were transfected with miR-222 mimics, miR-222 inhibitors, or their negative controls, respectively. The results showed that inhibition of miR-222 in MCF-7/Adr significantly increased the expressions of PTEN and p27 (kip1) and decreased phospho-Akt (p-Akt) both in mRNA and protein levels (p cancer cells to Adr through PTEN/Akt/p27 (kip1) signaling pathway, which provided a potential target to increase the sensitivity to Adr in breast cancer treatment and further improved the prognosis of breast cancer patients.

  16. Expression of p-AKT characterizes adenoid cystic carcinomas of head and neck with a higher risk for tumor relapses

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    Müller-Hermelink Hans-Konrad

    2009-06-01

    Full Text Available Abstract Background Adenoid cystic carcinomas are rare tumors with an indolent clinical course, but frequent local relapses. The identification of tumors with a higher relapse risk seems to be interesting. Hence we investigated parameters of glucose metabolism, which were found associated with poor prognosis in other malignancies. Methods Specimen of 29 patients were investigated immunohistochemically with antibodies against p-AKT, TKTL-1 (transketolase-like 1, M2PK (M2 pyruvate kinase, and GLUT-1. Proliferation was investigated by staining with Ki67. The tumors were located at the major or minor salivary glands. Only the typical cribriform subtype was investigated. The initial tumor stage was pT1 or pT2. Results Expression of p-AKT was significantly (P = 0.036 associated with a higher relapse risk in multivariate analysis. Low expression of M2PK was non-significantly (P = 0.065 predictive for a higher risk. TKTL-1 and GLUT-1 were expressed in the majority of cases, albeit not associated with relapse risk. Conclusion Adenoid cystic carcinomas positive for p-AKT show a higher relapse risk. However, other parameters of glucose metabolism investigated here or proliferation (Ki67 were not predictive in this entity. Our findings demonstrate a possible background for therapeutic approaches targeting the inhibition of PI3K/AKT pathway.

  17. Multiple host kinases contribute to Akt activation during Salmonella infection.

    Science.gov (United States)

    Roppenser, Bernhard; Kwon, Hyunwoo; Canadien, Veronica; Xu, Risheng; Devreotes, Peter N; Grinstein, Sergio; Brumell, John H

    2013-01-01

    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.

  18. 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.

  19. Akt mediates an angiogenic switch in transformed keratinocytes.

    Science.gov (United States)

    Segrelles, Carmen; Ruiz, Sergio; Santos, Mirentxu; Martínez-Palacio, Jesús; Lara, M Fernanda; Paramio, Jesús M

    2004-07-01

    Akt signaling is involved in tumorigenesis via a number of different mechanisms that result in increased proliferation and decreased apoptosis. Previous data have demonstrated that Akt-mediated signaling is functionally involved in keratinocyte transformation. This work investigates the involvement of angiogenesis as a mediator of tumorigenesis in Akt-transformed keratinocytes. Tumors produced by subcutaneous injection of the latter showed increased angiogenic profiles associated with increased vascular endothelial growth factor (VEGF) protein levels. However, in contrast to v-ras(Ha)-transformed keratinocytes, VEGF mRNA levels were not increased. The induction of VEGF protein by Akt is associated with increased phosphorylation and thus activation of p70S6K and eIF4E-binding protein 1, leading to increased VEGF translation. In addition, we observed increased metaloproteinases 2 and 9 expression, but not thrombospondin 1, in tumors derived from Akt-transformed keratinocytes. Collectively, these results demonstrate that Akt is an important mediator of angiogenesis in malignant keratinocytes through a post-transcriptional mechanism.

  20. AKT1 Activation Promotes Development of Melanoma Metastases

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    Joseph H. Cho

    2015-11-01

    Full Text Available Metastases are the major cause of melanoma-related mortality. Previous studies implicating aberrant AKT signaling in human melanoma metastases led us to evaluate the effect of activated AKT1 expression in non-metastatic BRAFV600E/Cdkn2aNull mouse melanomas in vivo. Expression of activated AKT1 resulted in highly metastatic melanomas with lung and brain metastases in 67% and 17% of our mice, respectively. Silencing of PTEN in BRAFV600E/Cdkn2aNull melanomas cooperated with activated AKT1, resulting in decreased tumor latency and the development of lung and brain metastases in nearly 80% of tumor-bearing mice. These data demonstrate that AKT1 activation is sufficient to elicit lung and brain metastases in this context and reveal that activation of AKT1 is distinct from PTEN silencing in metastatic melanoma progression. These findings advance our knowledge of the mechanisms driving melanoma metastasis and may provide valuable insights for clinical management of this disease.

  1. 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.

  2. 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

  3. 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.

  4. Early molecular and behavioral response to lipopolysaccharide in the WAG/Rij rat model of absence epilepsy and depressive-like behavior, involves interplay between AMPK, AKT/mTOR pathways and neuroinflammatory cytokine release.

    Science.gov (United States)

    Russo, Emilio; Andreozzi, Francesco; Iuliano, Rodolfo; Dattilo, Vincenzo; Procopio, Teresa; Fiume, Giuseppe; Mimmi, Selena; Perrotti, Nicola; Citraro, Rita; Sesti, Giorgio; Constanti, Andrew; De Sarro, Giovambattista

    2014-11-01

    The mammalian target of rapamycin (mTOR) pathway has been recently indicated as a suitable drug target for the prevention of epileptogenesis. The mTOR pathway is known for its involvement in the control of the immune system. Since neuroinflammation is recognized as a major contributor to epileptogenesis, we wished to examine whether the neuroprotective effects of mTOR modulation could involve a suppression of the neuroinflammatory process in epileptic brain. We have investigated the early molecular mechanisms involved in the effects of intracerebral administration of the lipopolysaccharide (LPS) in the WAG/Rij rat model of absence epilepsy, in relation to seizure generation and depressive-like behavior; we also tested whether the effects of LPS could be modulated by treatment with rapamycin (RAP), a specific mTOR inhibitor. We determined, in specific rat brain areas, levels of p-mTOR/p-p70S6K and also p-AKT/p-AMPK as downstream or upstream indicators of mTOR activity and tested the effects of LPS and RAP co-administration. Changes in the brain levels of pro-inflammatory cytokines IL-1β and TNF-α and their relative mRNA expression levels were measured, and the involvement of nuclear factor-κB (NF-κB) was also examined in vitro. We confirmed that RAP inhibits the aggravation of absence seizures and depressive-like/sickness behavior induced by LPS in the WAG/Rij rats through the activation of mTOR and show that this effect is correlated with the ability of RAP to dampen and delay LPS increases in neuroinflammatory cytokines IL-1β and TNF-α, most likely through inhibition of the activation of NF-κB. Our results suggest that such a mechanism could contribute to the antiseizure, antiepileptogenic and behavioral effects of RAP and further highlight the potential therapeutic usefulness of mTOR inhibition in the management of human epilepsy and other neurological disorders. Furthermore, we show that LPS-dependent neuroinflammatory effects are also mediated by a

  5. Mechanical stimulation and IGF-1 enhance mRNA translation rate in osteoblasts via activation of the AKT-mTOR pathway

    NARCIS (Netherlands)

    Bakker, A.D.; Gakes, T.; Hogervorst, J.M.A.; de Wit, G.M.J.; Klein-Nulend, J.; Jaspers, R.T.

    2016-01-01

    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 th

  6. Detection, Validation, and Downstream Analysis of Allelic Variation in Gene Expression

    Science.gov (United States)

    Ciobanu, Daniel C.; Lu, Lu; Mozhui, Khyobeni; Wang, Xusheng; Jagalur, Manjunatha; Morris, John A.; Taylor, William L.; Dietz, Klaus; Simon, Perikles; Williams, Robert W.

    2010-01-01

    Common sequence variants within a gene often generate important differences in expression of corresponding mRNAs. This high level of local (allelic) control—or cis modulation—rivals that produced by gene targeting, but expression is titrated finely over a range of levels. We are interested in exploiting this allelic variation to study gene function and downstream consequences of differences in expression dosage. We have used several bioinformatics and molecular approaches to estimate error rates in the discovery of cis modulation and to analyze some of the biological and technical confounds that contribute to the variation in gene expression profiling. Our analysis of SNPs and alternative transcripts, combined with eQTL maps and selective gene resequencing, revealed that between 17 and 25% of apparent cis modulation is caused by SNPs that overlap probes rather than by genuine quantitative differences in mRNA levels. This estimate climbs to 40–50% when qualitative differences between isoform variants are included. We have developed an analytical approach to filter differences in expression and improve the yield of genuine cis-modulated transcripts to ∼80%. This improvement is important because the resulting variation can be successfully used to study downstream consequences of altered expression on higher-order phenotypes. Using a systems genetics approach we show that two validated cis-modulated genes, Stk25 and Rasd2, are likely to control expression of downstream targets and affect disease susceptibility. PMID:19884314

  7. 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.

  8. 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.

  9. 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.

  10. Defective Connective Tissue Remodeling in Smad3 Mice Leads to Accelerated Aneurysmal Growth Through Disturbed Downstream TGF-β Signaling

    Directory of Open Access Journals (Sweden)

    I. van der Pluijm, PhD

    2016-10-01

    Smad3 deficiency leads to imbalanced activation of downstream genes, no activation of MMPs in VSMCs, and immune responses resulting in rapid aortic wall dilatation and rupture. Our findings uncover new possibilities for treatment of SMAD3 patients; instead of targeting TGF-β signaling, immune suppression may be more beneficial.

  11. The greedy nature of mutant RAS: a boon for drug discovery targeting cancer metabolism?

    Science.gov (United States)

    Lv, Jing; Wang, Jieqiong; Chang, Siyu; Liu, Mingyao; Pang, Xiufeng

    2016-01-01

    RAS oncogene mutations are frequently detected in human cancers. Among RAS-mediated tumorigenesis, KRAS-driven cancers are the most frequently diagnosed and resistant to current therapies. Despite more than three decades of intensive efforts, there are still no specific therapies for mutant RAS proteins. While trying to block those well-established downstream pathways, such as the RAF-MAPK pathway and the PI3K-AKT pathway, attentions have been paid to potential effects of RAS on metabolic pathways and the feasibility for targeting these pathways. Recent studies have proved that RAS not only promotes aerobic glycolysis and glutamine metabolism reprograming to provide energy, but it also facilitates branched metabolism pathways, autophagy, and macropinocytosis. These alterations generate building blocks for tumor growth and strengthen antioxidant defense in tumor cells. All of these metabolic changes meet different demands of RAS-driven cancers, making them distinct from normal cells. Indeed, some achievements have been made to inhibit tumor growth through targeting specific metabolism rewiring in preclinical models. Although there is still a long way to elucidate the landscape of altered metabolism, we believe that specific metabolic enzymes or pathways could be therapeutically targeted for selective inhibition of RAS-driven cancers.

  12. Causal Network Models for Predicting Compound Targets and Driving Pathways in Cancer.

    Science.gov (United States)

    Jaeger, Savina; Min, Junxia; Nigsch, Florian; Camargo, Miguel; Hutz, Janna; Cornett, Allen; Cleaver, Stephen; Buckler, Alan; Jenkins, Jeremy L

    2014-06-01

    Gene-expression data are often used to infer pathways regulating transcriptional responses. For example, differentially expressed genes (DEGs) induced by compound treatment can help characterize hits from phenotypic screens, either by correlation with known drug signatures or by pathway enrichment. Pathway enrichment is, however, typically computed with DEGs rather than "upstream" nodes that are potentially causal of "downstream" changes. Here, we present graph-based models to predict causal targets from compound-microarray data. We test several approaches to traversing network topology, and show that a consensus minimum-rank score (SigNet) beat individual methods and could highly rank compound targets among all network nodes. In addition, larger, less canonical networks outperformed linear canonical interactions. Importantly, pathway enrichment using causal nodes rather than DEGs recovers relevant pathways more often. To further validate our approach, we used integrated data sets from the Cancer Genome Atlas to identify driving pathways in triple-negative breast cancer. Critical pathways were uncovered, including the epidermal growth factor receptor 2-phosphatidylinositide 3-kinase-AKT-MAPK growth pathway andATR-p53-BRCA DNA damage pathway, in addition to unexpected pathways, such as TGF-WNT cytoskeleton remodeling, IL12-induced interferon gamma production, and TNFR-IAP (inhibitor of apoptosis) apoptosis; the latter was validated by pooled small hairpin RNA profiling in cancer cells. Overall, our approach can bridge transcriptional profiles to compound targets and driving pathways in cancer.

  13. Knockdown of AKT3 (PKBγ and PI3KCA Suppresses Cell Viability and Proliferation and Induces the Apoptosis of Glioblastoma Multiforme T98G Cells

    Directory of Open Access Journals (Sweden)

    Monika Paul-Samojedny

    2014-01-01

    Full Text Available Glioblastoma multiforme (GBM is the most malignant and invasive human brain tumor that is difficult to treat and has a very poor prognosis. Thus, new therapeutic strategies that target GBM are urgently needed. The PI3K/AKT/PTEN signaling pathway is frequently deregulated in a wide range of cancers. The present study was designed to examine the inhibitory effect of AKT3 or PI3KCA siRNAs on GBM cell growth, viability, and proliferation.T98G cells were transfected with AKT3 and/or PI3KCA siRNAs. AKT3 and PI3KCA protein-positive cells were identified using FC and Western blotting. The influence of specific siRNAs on T98G cell viability, proliferation, cell cycle, and apoptosis was evaluated as well using FC. Alterations in the mRNA expression of AKT3, PI3KCA, and apoptosis-related genes were analyzed using QRT-PCR. Knockdown of AKT3 and/or PI3KCA genes in T98G cells led to a significant reduction in cell viability, the accumulation of subG1-phase cells and, a reduced fraction of cells in the S and G2/M phases. Additionally, statistically significant differences in the BAX/BCL-2 ratio and an increased percentage of apoptotic cells were found. The siRNA-induced AKT3 and PI3KCA mRNA knockdown may offer a novel therapeutic strategy to control the growth of human GBM cells.

  14. 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.

  15. Control of fibroblast fibronectin expression and alternative splicing via the PI3K/Akt/mTOR pathway

    Energy Technology Data Exchange (ETDEWEB)

    White, Eric S., E-mail: docew@umich.edu [Division of Pulmonary and Critical Care Medicine, Department of Internal Medicine, University of Michigan Medical School, Ann Arbor, MI (United States); Sagana, Rommel L.; Booth, Adam J.; Yan, Mei; Cornett, Ashley M.; Bloomheart, Christopher A.; Tsui, Jessica L.; Wilke, Carol A.; Moore, Bethany B. [Division of Pulmonary and Critical Care Medicine, Department of Internal Medicine, University of Michigan Medical School, Ann Arbor, MI (United States); Ritzenthaler, Jeffrey D.; Roman, Jesse [Department of Medicine, University of Louisville School of Medicine, Louisville, KY (United States); Muro, Andres F. [International Centre for Genetic Engineering and Biotechnology, Trieste (Italy)

    2010-10-01

    Fibronectin (FN), a ubiquitous glycoprotein that plays critical roles in physiologic and pathologic conditions, undergoes alternative splicing which distinguishes plasma FN (pFN) from cellular FN (cFN). Although both pFN and cFN can be incorporated into the extracellular matrix, a distinguishing feature of cFN is the inclusion of an alternatively spliced exon termed EDA (for extra type III domain A). The molecular steps involved in EDA splicing are well-characterized, but pathways influencing EDA splicing are less clear. We have previously found an obligate role for inhibition of the tumor suppressor phosphatase and tensin homologue on chromosome 10 (PTEN), the primary regulator of the PI3K/Akt pathway, in fibroblast activation. Here we show TGF-{beta}, a potent inducer of both EDA splicing and fibroblast activation, inhibits PTEN expression and activity in mesenchymal cells, corresponding with enhanced PI3K/Akt signaling. In pten{sup -/-} fibroblasts, which resemble activated fibroblasts, inhibition of Akt attenuated FN production and decreased EDA alternative splicing. Moreover, inhibition of mammalian target of rapamycin (mTOR) in pten{sup -/-} cells also blocked FN production and EDA splicing. This effect was due to inhibition of Akt-mediated phosphorylation of the primary EDA splicing regulatory protein SF2/ASF. Importantly, FN silencing in pten{sup -/-} cells resulted in attenuated proliferation and migration. Thus, our results demonstrate that the PI3K/Akt/mTOR axis is instrumental in FN transcription and alternative splicing, which regulates cell behavior.

  16. 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.

  17. Inhibition of Aβ(25-35)-induced cell apoptosis by low-power-laser-irradiation (LPLI) through promoting Akt-dependent YAP cytoplasmic translocation.

    Science.gov (United States)

    Zhang, Heng; Wu, Shengnan; Xing, Da

    2012-01-01

    Deposition of amyloid-β-peptide (Aβ) in the brain is considered a pathological hallmark of Alzheimer's disease (AD). Our previous studies show that Yes-associated protein (YAP) is involved in the regulation of apoptosis induced by Aβ(25-35) through YAP nuclear translocation and its pro-apoptotic function is mediated by its interaction with p73. In the present study, we first found that Low-power laser irradiation (LPLI) promoted YAP cytoplasmic translocation and inhibited Aβ(25-35)-induced YAP nuclear translocation. Moreover, the cytoplasmic translocation was in an Akt-dependent manner. Activated Akt by LPLI phosphorylated YAP on ser127 (S127) and resulted in decreasing the interaction between YAP and p73, and in suppressing the proapoptotic gene bax expression following Aβ(25-35) treatment. Inhibition of Akt expression by siRNA significantly abolished the effect of LPLI. More importantly, LPLI could inhibit Aβ(25-35)-induced cell apoptosis through activation of Akt/YAP/p73 signaling pathway. Therefore, our findings first suggest that YAP may be a therapeutic target and these results directly point to a potential therapeutic strategy for the treatment of AD through Akt/YAP/p73 signaling pathway with LPLI.

  18. Apocynin inhibits Toll-like receptor-4-mediated activation of NF-κB by suppressing the Akt and mTOR pathways.

    Science.gov (United States)

    Nam, Yoon Jeong; Kim, Arum; Sohn, Dong Suep; Lee, Chung Soo

    2016-12-01

    Microbial product lipopolysaccharide has been shown to be involved in the pathogenesis of inflammatory skin diseases. Apocynin has demonstrated to have an anti-inflammatory effect. However, the effect of apocynin on the Toll-like receptor-4-dependent activation of Akt, mammalian target of rapamycin (mTOR), and nuclear factor (NF)-κB pathway, which is involved in productions of inflammatory mediators in keratinocytes, has not been studied. Using human keratinocytes, we investigated the effect of apocynin on the inflammatory mediator production in relation to the Toll-like receptor-4-mediated-Akt/mTOR and NF-κB pathways, which regulates the transcription genes involved in immune and inflammatory responses. Apocynin, Akt inhibitor SH-5, Bay 11-7085 and N-acetylcysteine each attenuated the lipopolysaccharide-induced production of cytokines, PGE2, and chemokines, changes in the levels of Toll-like receptor-4, p-Akt, mTOR, and NF-κB, and production of reactive oxygen species in keratinocytes. The results show that apocynin appears to attenuate the lipopolysaccharide-stimulated production of inflammatory mediators in keratinocytes by suppressing the Toll-like receptor-4-mediated activation of the Akt, mTOR, and NF-κB pathways. The effect of apocynin appears to be attributed to its inhibitory effect on the production of reactive oxygen species. Apocynin appears to attenuate the microbial product-mediated inflammatory skin diseases.

  19. 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.

  20. Complex regulation of PKCβ2 and PDK-1/AKT by ROCK2 in diabetic heart.

    Directory of Open Access Journals (Sweden)

    Guorong Lin

    Full Text Available OBJECTIVES: The RhoA/ROCK pathway contributes to diabetic cardiomyopathy in part by promoting the sustained activation of PKCβ2 but the details of their interaction are unclear. The purpose of this study was to investigate if over-activation of ROCK in the diabetic heart leads to direct phosphorylation and activation of PKCβ2, and to determine if their interaction affects PDK-1/Akt signaling. METHODS: Regulation by ROCK of PKCβ2 and related kinases was investigated by Western blotting and co-immunoprecipitation in whole hearts and isolated cardiomyocytes from 12 to 14-week diabetic rats. Direct ROCK2 phosphorylation of PKCβ2 was examined in vitro. siRNA silencing was used to confirm role of ROCK2 in PKCβ2 phosphorylation in vascular smooth muscle cells cultured in high glucose. Furthermore, the effect of ROCK inhibition on GLUT4 translocation was determined in isolated cardiomyocytes by confocal microscopy. RESULTS: Expression of ROCK2 and expression and phosphorylation of PKCβ2 were increased in diabetic hearts. A physical interaction between the two kinases was demonstrated by reciprocal immunoprecipitation, while ROCK2 directly phosphorylated PKCβ2 at T641 in vitro. ROCK2 siRNA in vascular smooth muscle cells or inhibition of ROCK in diabetic hearts reduced PKCβ2 T641 phosphorylation, and this was associated with attenuation of PKCβ2 activity. PKCβ2 also formed a complex with PDK-1 and its target AKT, and ROCK inhibition resulted in upregulation of the phosphorylation of PDK-1 and AKT, and increased translocation of glucose transporter 4 (GLUT4 to the plasma membrane in diabetic hearts. CONCLUSION: This study demonstrates that over-activation of ROCK2 contributes to diabetic cardiomyopathy by multiple mechanisms, including direct phosphorylation and activation of PKCβ2 and interference with the PDK-1-mediated phosphorylation and activation of AKT and translocation of GLUT4. This suggests that ROCK2 is a critical node in the

  1. PREX1 Protein Function Is Negatively Regulated Downstream of Receptor Tyrosine Kinase Activation by p21-activated Kinases (PAKs).

    Science.gov (United States)

    Barrows, Douglas; He, John Z; Parsons, Ramon

    2016-09-16

    Downstream of receptor tyrosine kinase and G protein-coupled receptor (GPCR) stimulation, the phosphatidylinositol 3,4,5-trisphosphate (PIP3)-dependent Rac exchange factor (PREX) family of guanine nucleotide exchange factors (GEFs) activates Rho GTPases, leading to important roles for PREX proteins in numerous cellular processes and diseases, including cancer. PREX1 and PREX2 GEF activity is activated by the second messengers PIP3 and Gβγ, and further regulation of PREX GEF activity occurs by phosphorylation. Stimulation of receptor tyrosine kinases by neuregulin and insulin-like growth factor 1 (IGF1) leads to the phosphorylation of PREX1; however, the kinases that phosphorylate PREX1 downstream of these ligands are not known. We recently reported that the p21-activated kinases (PAKs), which are activated by GTP-bound Ras-related C3 botulinum toxin substrate 1 (Rac1), mediate the phosphorylation of PREX2 after insulin receptor activation. Here we show that certain phosphorylation events on PREX1 after insulin, neuregulin, and IGF1 treatment are PAK-dependent and lead to a reduction in PREX1 binding to PIP3 Like PREX2, PAK-mediated phosphorylation also negatively regulates PREX1 GEF activity. Furthermore, the onset of PREX1 phosphorylation was delayed compared with the phosphorylation of AKT, supporting a model of negative feedback downstream of PREX1 activation. We also found that the phosphorylation of PREX1 after isoproterenol and prostaglandin E2-mediated GPCR activation is partially PAK-dependent and likely also involves protein kinase A, which is known to reduce PREX1 function. Our data point to multiple mechanisms of PREX1 negative regulation by PAKs within receptor tyrosine kinase and GPCR-stimulated signaling pathways that have important roles in diseases such as diabetes and cancer.

  2. 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.

  3. Cytoplasmic localization of wild-type survivin is associated with constitutive activation of the PI3K/Akt signaling pathway and represents a favorable prognostic factor in patients with acute myeloid leukemia.

    Science.gov (United States)

    Serrano-López, Juana; Serrano, Josefina; Figueroa, Vianihuini; Torres-Gomez, Antonio; Tabares, Salvador; Casaño, Javier; Fernandez-Escalada, Noemi; Sánchez-Garcia, Joaquín

    2013-12-01

    Survivin is over-expressed in most hematologic malignancies but the prognostic significance of the subcompartmental distribution of wild-type or splicing variants in acute myeloid leukemia has not been addressed yet. Using western blotting, we assessed the expression of wild-type survivin and survivin splice variants 2B and Delta-Ex3 in nuclear and cytoplasmic protein extracts in samples taken from 105 patients at the time of their diagnosis of acute myeloid leukemia. Given that survivin is a downstream effector of the PI3K/Akt signaling pathway, survivin expression was also correlated with pSer473-Akt. Wild-type survivin and the 2B splice variant were positive in 76.3% and 78.0% of samples in the nucleus, cytoplasm or both, whereas the Delta-Ex3 isoform was only positive in the nucleus in 37.7% of samples. Cytoplasmic localization of wild-type survivin was significantly associated with the presence of high levels of pSer473-Akt (P<0.001). Inhibition of the PI3K/Akt pathway with wortmannin and Ly294002 caused a significant reduction in the expression of cytoplasmic wild-type survivin. The presence of cytoplasmic wild-type survivin and pSer473-Akt was associated with a lower fraction of quiescent leukemia stem cells (P=0.02). The presence of cytoplasmic wild-type survivin and pSer473-Akt were favorable independent prognostic factors. Moreover, the activation of the PI3K/Akt pathway with expression of cytoplasmic wild-type survivin identified a subgroup of acute myeloid leukemia patients with an excellent outcome (overall survival rate of 60.0±21.9% and relapse-free survival of 63.0±13.5%). Our findings suggest that cytoplasmic wild-type survivin is a critical downstream effector of the PI3K/Akt pathway leading to more chemosensitive cells and a more favorable outcome in acute myeloid leukemia.

  4. 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.

  5. Experimental investigation of unsteady fan flow interaction with downstream struts

    Science.gov (United States)

    Ng, W. F.; Obrien, W. F.; Olsen, T. L.

    1986-07-01

    In the present study of the unsteady pressure field produced on fan rotor blades by interaction with downstream struts, a single stage, low speed axial-flow fan was instrumented with blade-mounted high frequency pressure transducers. In addition, stationary pressure problems were used to map out the flowfield. Fluctuating pressure measurements are presented for blade midspan and 85-percent span on both the suction and pressure surfaces of the rotor blades at several positions of the downstream struts, and for two different flow coefficients. The strut is found to produce an effect on the unsteady pressure field on the rotor blades; this effect exceeds that due to the stator at design rotor-stator-strut spacing, but it rapidly declines as the struts are moved downstream.

  6. Transition duct with divided upstream and downstream portions

    Energy Technology Data Exchange (ETDEWEB)

    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.

  7. 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.

  8. Immunoexpression status and prognostic value of mammalian target of rapamycin and hypoxia-induced pathway members in papillary cell renal cell carcinomas.

    Science.gov (United States)

    Chaux, Alcides; Schultz, Luciana; Albadine, Roula; Hicks, Jessica; Kim, Jenny J; Allaf, Mohamad E; Carducci, Michael A; Rodriguez, Ronald; Hammers, Hans-Joerg; Argani, Pedram; Reuter, Victor E; Netto, George J

    2012-12-01

    Dysregulation of the mammalian target of rapamycin and hypoxia-induced pathways has been consistently identified in clear cell renal cell carcinomas. However, experience with non-clear cell renal cell carcinoma subtypes is scant. In this study, we evaluated the immunohistochemical expression of upstream (PTEN and phosphorylated AKT) and downstream (phosphorylated S6 and 4EBP1) effectors of the mammalian target of rapamycin pathway, as well as related cell-cycle proteins (p27 and c-MYC), and a member of the hypoxia-induced pathway (HIF-1α) in 54 patients with papillary renal cell carcinoma treated by nephrectomy. PTEN was lower in tumor than in normal kidney, and loss of PTEN expression was found in 48% of the patients. In tumor tissues, phosphorylated S6, 4EBP1, and HIF-1α were higher than in normal kidney. Conversely, scores of p27 were lower in tumor than in normal kidney. Finally, scores of c-MYC and phosphorylated AKT were similar in tumor and in normal kidney. Overall mortality and cancer-specific mortality were 24% and 11%, respectively. Tumor progression was observed in 17% of the patients. None of the tested biomarkers predicted cancer-specific mortality or tumor progression. As expected, patients with high T-stage tumors had higher hazard ratios for cancer-specific mortality (hazard ratio, 6.9) and tumor progression (hazard ratio, 6.7). Patients with higher Fuhrman grades also had higher risks for cancer-specific mortality (hazard ratio, 11.4) and tumor progression (hazard ratio, 4.5). In summary, our study provides evidence of dysregulation of the mammalian target of rapamycin and hypoxia-induced pathways in papillary renal cell carcinoma. Immunohistochemistry for members of the mammalian target of rapamycin pathway and for HIF-1α lacked prognostic significance in our cohort.

  9. 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.

  10. 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.

  11. AKT signaling as a novel factor associated with in vitro resistance of human AML to gemtuzumab ozogamicin.

    Directory of Open Access Journals (Sweden)

    David B Rosen

    Full Text Available Gemtuzumab ozogamicin (GO, an immunoconjugate between an anti-CD33 antibody and a calicheamicin-γ(1 derivative, induces remissions and improves survival in a subset of patients with acute myeloid leukemia (AML. As the mechanisms underlying GO and calicheamicin-γ(1 resistance are incompletely understood, we herein used flow cytometry-based single cell network profiling (SCNP assays to study cellular responses of primary human AML cells to GO. Our data indicate that the extent of DNA damage is quantitatively impacted by CD33 expression and drug efflux activity. However, although DNA damage is required for GO-induced cytotoxicity, it is not sufficient for effective cell kill, suggesting that downstream anti-apoptotic pathways may function as relevant resistance mechanisms. Supporting this notion, we found activated PI3K/AKT signaling to be associated with GO resistance in vitro in primary AML cells. Consistently, the investigational AKT inhibitor MK-2206 significantly sensitized various human AML cells to GO or free calicheamicin-γ(1 with particularly pronounced effects in otherwise GO or free calicheamicin-γ(1-resistant cells. Likewise, MK-2206 also sensitized primary AML cells to calicheamicin-γ(1. Together, our findings illustrate the capacity of SCNP assays to discover chemotherapy-related biological pathways and signaling networks relevant to GO-induced genotoxic stress. The identification of AKT signaling as being associated with GO resistance in vitro may provide a novel approach to improve the in vivo efficacy of GO/calicheamicin-γ(1 and, by extrapolation, other DNA damage-based therapeutics.

  12. The PI3K/Akt pathway is involved in procyanidin-mediated suppression of human colorectal cancer cell growth.

    Science.gov (United States)

    Choy, Ying Yng; Fraga, Magdalena; Mackenzie, Gerardo G; Waterhouse, Andrew L; Cremonini, Eleonora; Oteiza, Patricia I

    2016-12-01

    Colorectal cancer (CRC) has the third highest incidence worldwide. Epidemiological studies showed that the consumption of fruit and vegetables containing procyanidins (PCA), polymers of flavan-3-ols, is associated with lower CRC risk. However, the molecular mechanisms supporting this positive association are unclear. This study investigated the capacity of PCA with different degrees of polymerization to reduce CRC cell growth, characterizing the underlying mechanisms. Compared to the monomer ((-)-epicatechin) and the trimer, the hexamer (Hex) was the most active at reducing CRC cell viability. Hex caused a concentration- (2.5-50 μM) and time- (24-72 h) dependent decrease in the viability of six human CRC cell lines in culture. Hex caused CRC apoptotic Caco-2 cell death within 24 h, as evidenced by caspase 3 and caspase 9 activation, DNA fragmentation, and changes in nuclear morphology/staining. Hex-induced apoptosis occurs through the mitochondrial pathway, as evidenced by an increased Bad mitochondrial translocation, and cytochrome c release from the mitochondria to the cytosol. Hex also arrested the Caco-2 cell cycle at G2 /M phase and upregulated genes involved in autophagy. Mechanistically, in Caco-2 cells Hex inhibited the PI3K/Akt signaling pathway, causing the downstream downregulation of proteins involved in the regulation of cell survival (Bad, GSK-3β). Accordingly, the Akt inhibitor MKK-2206 decreased Bad and GSK-3β phosphorylation. MKK-2206 decreased cell growth, having an additive effect with Hex. In conclusion, our results show that large PCA can inhibit CRC cell growth via the Akt kinase pathway, demonstrating a mechanism to explain the epidemiological evidence linking PCA-rich diets with lower CRC risk. © 2016 Wiley Periodicals, Inc.

  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. 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.

  15. 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

  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. 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.

  18. 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.

  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.

  20. 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

  1. 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...

  2. 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.

  3. Extreme wave phenomena in down-stream running modulated waves

    NARCIS (Netherlands)

    Andonowati,; Karjanto, N.; Groesen, van E.

    2006-01-01

    Modulational, Benjamin-Feir, instability is studied for the down-stream evolution of surface gravity waves. An explicit solution, the soliton on finite background, of the NLS equation in physical space is used to study various phenomena in detail. It is shown that for sufficiently long modulation le

  4. DNS and RANS Simulation of Dispersion Downstream of an Obstacle

    Science.gov (United States)

    2008-12-01

    DNS AND RANS SIMULATION OF DISPERSION DOWNSTREAM OF AN OBSTACLE Riccardo Rossi*, Gianluca Iaccarino** * Laboratorio di Termofluidodinamica...ORGANIZATION NAME(S) AND ADDRESS(ES) Laboratorio di Termofluidodinamica Computazionale, Seconda Facolt‘a di Ingegneria di Forl‘ý Universit‘a di Bologna

  5. Flow diagnostics downstream of a tribladed rotor model

    DEFF Research Database (Denmark)

    Naumov, I. V.; Rahmanov, V. V.; Okulov, Valery

    2012-01-01

    This paper presents results of a study of vortex wake structures and measurements of instantaneous 3D velocity fields downstream of a triblade turbine model. Two operation modes of flow around the rotor with different tip speed ratios were tested. Initially the wake structures were visualized and...

  6. Signaling intermediates (MAPK and PI3K) as therapeutic targets in NSCLC.

    Science.gov (United States)

    Ciuffreda, Ludovica; Incani, Ursula Cesta; Steelman, Linda S; Abrams, Stephen L; Falcone, Italia; Curatolo, Anais Del; Chappell, William H; Franklin, Richard A; Vari, Sabrina; Cognetti, Francesco; McCubrey, James A; Milella, Michele

    2014-01-01

    The RAS/RAF/MEK/ ERK and the PI3K/AKT/mTOR pathways govern fundamental physiological processes, such as cell proliferation, differentiation, metabolism, cytoskeleton reorganization and cell death and survival. Constitutive activation of these signal transduction pathways is a required hallmark of cancer and dysregulation, on either genetic or epigenetic grounds, of these pathways has been implicated in the initiation, progression and metastastic spread of lung cances. Targeting components of the MAPK and PI3K cascades is thus an attractive strategy in the development of novel therapeutic approaches to treat lung cancer, although the use of single pathway inhibitors has met with limited clinical success so far. Indeed, the presence of intra- and inter-pathway compensatory loops that re-activate the very same cascade, either upstream or downstream the point of pharmacological blockade, or activate the alternate pathway following the blockade of one signaling cascade has been demonstrated, potentially driving preclinical (and possibly clinical) resistance. Therefore, the blockade of both pathways with combinations of signaling inhibitors might result in a more efficient anti-tumor effect, and thus potentially overcome and/or delay clinical resistance, as compared with single agent. The current review aims at summarizing the current status of preclinical and clinical research with regard to pathway crosstalks between the MAPK and PI3K cascades in NSCLC and the rationale for combined therapeutic pathway targeting.

  7. Downstream-migrating fluvial point bars in the rock record

    Science.gov (United States)

    Ghinassi, Massimiliano; Ielpi, Alessandro; Aldinucci, Mauro; Fustic, Milovan

    2016-04-01

    Classical models developed for ancient fluvial point bars are based on the assumption that meander bends invariably increase their radius as meander-bend apices migrate in a direction transverse to the channel-belt axis (i.e., meander bend expansion). However, many modern meandering rivers are also characterized by down-valley migration of the bend apex, a mechanism that takes place without a significant change in meander radius and wavelength. Downstream-migrating fluvial point bars (DMFPB) are the dominant architectural element of these types of meander belts. Yet they are poorly known from ancient fluvial-channel belts, since their disambiguation from expansional point bars often requires fully-3D perspectives. This study aims to review DMFPB deposits spanning in age from Devonian to Holocene, and to discuss their main architectural and sedimentological features from published outcrop, borehole and 3D-seismic datasets. Fluvial successions hosting DMFPB mainly accumulated in low accommodation conditions, where channel belts were affected by different degrees of morphological (e.g., valleys) or tectonic (e.g., axial drainage of