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Sample records for rapamycin mtor regulates

  1. PTEN and rapamycin inhibiting the growth of K562 cells through regulating mTOR signaling pathway

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    Chen Hao

    2008-12-01

    Full Text Available Abstract Objective To investigate, in vitro, the regulatory effects of tumor-suppressing gene PTEN on mTOR (mammalian target of rapamycin signaling pathway, the effects of transfected PTEN and rapamycin on the growth inhibition, and apoptosis induction for human leukemia cell line K562 cells. Methods K562 cells were transfected with recombined adenovirus-PTEN vector containing green fluorescent protein (Ad-PTEN-GFP, followed by the treatment of the cells with or without rapamycin. The proliferation inhibition rate and apoptotic rate of these transfected and/or rapamycin treated K562 cells were measured by MTT assay and flow cytometry (FCM, the expression levels of PTEN-, mTOR-, cyclinD1- and P27kip1- mRNA were measured by real-time fluorescent relative-quantification reverse transcriptional PCR (FQ-PCR, the protein expression levels of PTEN, Akt, p-Akt were detected by western blotting. Results The proliferation of K562 cells was inhibited by PTEN gene transfection with/without the treatment of rapamycin. The expression levels of PTEN- and P27kip1- mRNA were up-regulated, and the mTOR- and cyclinD1- mRNA were down-regulated in K562 cells after the cells transfected with wild type PTEN gene and treated with rapamycin. Conclusion PTEN and rapamycin inhibited mTOR expression by acting as an upstream regulator of mTOR. Low dose rapamycin in combination with over-expressed PTEN might have synergistic effects on inhibiting the proliferation and promoting apoptosis of K562 cells.

  2. Synergism between the mTOR inhibitor rapamycin and FAK down-regulation in the treatment of acute lymphoblastic leukemia

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    Pei-Jie Shi

    2016-02-01

    Full Text Available Abstract Background Acute lymphoblastic leukemia (ALL is an aggressive malignant disorder of lymphoid progenitor cells in both children and adults. Although improvements in contemporary therapy and development of new treatment strategies have led to dramatic increases in the cure rate in children with ALL, the relapse rate remains high and the prognosis of relapsed childhood ALL is poor. Molecularly targeted therapies have emerged as the leading treatments in cancer therapy. Multi-cytotoxic drug regimens have achieved success, yet many studies addressing targeted therapies have focused on only one single agent. In this study, we attempted to investigate whether the effect of the mammalian target of rapamycin (mTOR inhibitor rapamycin is synergistic with the effect of focal adhesion kinase (FAK down-regulation in the treatment of ALL. Methods The effect of rapamycin combined with FAK down-regulation on cell proliferation, the cell cycle, and apoptosis was investigated in the human precursor B acute lymphoblastic leukemia cells REH and on survival time and leukemia progression in a non-obese diabetic/severe combined immunodeficiency (NOD/SCID mouse model. Results When combined with FAK down-regulation, rapamycin-induced suppression of cell proliferation, G0/G1 cell cycle arrest, and apoptosis were significantly enhanced. In addition, REH cell-injected NOD/SCID mice treated with rapamycin and a short-hairpin RNA (shRNA to down-regulate FAK had significantly longer survival times and slower leukemia progression compared with mice injected with REH-empty vector cells and treated with rapamycin. Moreover, the B-cell CLL/lymphoma-2 (BCL-2 gene family was shown to be involved in the enhancement, by combined treatment, of REH cell apoptosis. Conclusions FAK down-regulation enhanced the in vitro and in vivo inhibitory effects of rapamycin on REH cell growth, indicating that the simultaneous targeting of mTOR- and FAK-related pathways might offer a novel

  3. Rapamycin regulates biochemical metabolites

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    Tucci, Paola; Porta, Giovanni; Agostini, Massimiliano; Antonov, Alexey; Garabadgiu, Alexander Vasilievich; Melino, Gerry; Willis, Anne E

    2013-01-01

    The mammalian target of rapamycin (mTOR) kinase is a master regulator of protein synthesis that couples nutrient sensing to cell growth, and deregulation of this pathway is associated with tumorigenesis. p53, and its less investigated family member p73, have been shown to interact closely with mTOR pathways through the transcriptional regulation of different target genes. To investigate the metabolic changes that occur upon inhibition of the mTOR pathway and the role of p73 in this response p...

  4. Two mTOR inhibitors, rapamycin and Torin 1, differentially regulate iron-induced generation of mitochondrial ROS.

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    Huang, Hui; Chen, Jun; Lu, Huiru; Zhou, Mengxue; Chai, Zhifang; Hu, Yi

    2017-12-01

    It is generally believed that gene-environment interaction may contribute to neurodegeneration. Of particular note is that iron overload may be one of the risk factors for neurodegeneration. However, the mechanisms underlying iron-associated neurotoxicity are not fully understood. Here we explored the effects of mechanistic target of rapamycin (mTOR) inhibition in iron-stressed human neuroblastoma cells. Two mTOR inhibitors, rapamycin and Torin 1, had similar effects in cells exposed to a relatively low concentration of iron. At a higher concentration of iron, Torin 1, instead of rapamycin, could further aggravate iron-induced cytotoxicity, and mitochondrial ROS levels were significantly higher in Torin 1-treated cells. These results suggest that mTOR inhibition may not be able to alleviate iron-induced neurotoxicity.

  5. Rapamycin

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    Srivastava, Rupesh K.; Utley, Adam; Shrikant, Protul A.

    2012-01-01

    Vaccines that generate Ag-specific CD8+ T-cell responses of appropriate quality, magnitude and duration are highly desirable. The ability of mTOR to regulate CD8+ T-cell functional differentiation must be exploited for clinical benefit. In a recent paper, we report that varying the regimen of rapamycin administration regulates viral vaccine-induced CD8+ T-cell responses for tumor immunity. These observations validate the use of rapamycin in vaccination strategies and demonstrate the efficacy ...

  6. The Role of Mammalian Target of Rapamycin (mTOR) in Insulin Signaling.

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    Yoon, Mee-Sup

    2017-10-27

    The mammalian target of rapamycin (mTOR) is a serine/threonine kinase that controls a wide spectrum of cellular processes, including cell growth, differentiation, and metabolism. mTOR forms two distinct multiprotein complexes known as mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2), which are characterized by the presence of raptor and rictor, respectively. mTOR controls insulin signaling by regulating several downstream components such as growth factor receptor-bound protein 10 (Grb10), insulin receptor substrate (IRS-1), F-box/WD repeat-containing protein 8 (Fbw8), and insulin like growth factor 1 receptor/insulin receptor (IGF-IR/IR). In addition, mTORC1 and mTORC2 regulate each other through a feedback loop to control cell growth. This review outlines the current understanding of mTOR regulation in insulin signaling in the context of whole body metabolism.

  7. mTOR Ser-2481 autophosphorylation monitors mTORC-specific catalytic activity and clarifies rapamycin mechanism of action.

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    Soliman, Ghada A; Acosta-Jaquez, Hugo A; Dunlop, Elaine A; Ekim, Bilgen; Maj, Nicole E; Tee, Andrew R; Fingar, Diane C

    2010-03-12

    The mammalian target of rapamycin (mTOR) Ser/Thr kinase signals in at least two multiprotein complexes distinguished by their different partners and sensitivities to rapamycin. Acute rapamycin inhibits signaling by mTOR complex 1 (mTORC1) but not mTOR complex 2 (mTORC2), which both promote cell growth, proliferation, and survival. Although mTORC2 regulation remains poorly defined, diverse cellular mitogens activate mTORC1 signaling in a manner that requires sufficient levels of amino acids and cellular energy. Before the identification of distinct mTOR complexes, mTOR was reported to autophosphorylate on Ser-2481 in vivo in a rapamycin- and amino acid-insensitive manner. These results suggested that modulation of mTOR intrinsic catalytic activity does not universally underlie mTOR regulation. Here we re-examine the regulation of mTOR Ser-2481 autophosphorylation (Ser(P)-2481) in vivo by studying mTORC-specific Ser(P)-2481 in mTORC1 and mTORC2, with a primary focus on mTORC1. In contrast to previous work, we find that acute rapamycin and amino acid withdrawal markedly attenuate mTORC1-associated mTOR Ser(P)-2481 in cycling cells. Although insulin stimulates both mTORC1- and mTORC2-associated mTOR Ser(P)-2481 in a phosphatidylinositol 3-kinase-dependent manner, rapamycin acutely inhibits insulin-stimulated mTOR Ser(P)-2481 in mTORC1 but not mTORC2. By interrogating diverse mTORC1 regulatory input, we find that without exception mTORC1-activating signals promote, whereas mTORC1-inhibitory signals decrease mTORC1-associated mTOR Ser(P)-2481. These data suggest that mTORC1- and likely mTORC2-associated mTOR Ser-2481 autophosphorylation directly monitors intrinsic mTORC-specific catalytic activity and reveal that rapamycin inhibits mTORC1 signaling in vivo by reducing mTORC1 catalytic activity.

  8. Mammalian target of rapamycin (mTor) mediates tau protein dyshomeostasis: implication for Alzheimer disease.

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    Tang, Zhi; Bereczki, Erika; Zhang, Haiyan; Wang, Shan; Li, Chunxia; Ji, Xinying; Branca, Rui M; Lehtiö, Janne; Guan, Zhizhong; Filipcik, Peter; Xu, Shaohua; Winblad, Bengt; Pei, Jin-Jing

    2013-05-31

    Previous evidence from post-mortem Alzheimer disease (AD) brains and drug (especially rapamycin)-oriented in vitro and in vivo models implicated an aberrant accumulation of the mammalian target of rapamycin (mTor) in tangle-bearing neurons in AD brains and its role in the formation of abnormally hyperphosphorylated tau. Compelling evidence indicated that the sequential molecular events such as the synthesis and phosphorylation of tau can be regulated through p70 S6 kinase, the well characterized immediate downstream target of mTor. In the present study, we further identified that the active form of mTor per se accumulates in tangle-bearing neurons, particularly those at early stages in AD brains. By using mass spectrometry and Western blotting, we identified three phosphoepitopes of tau directly phosphorylated by mTor. We have developed a variety of stable cell lines with genetic modification of mTor activity using SH-SY5Y neuroblastoma cells as background. In these cellular systems, we not only confirmed the tau phosphorylation sites found in vitro but also found that mTor mediates the synthesis and aggregation of tau, resulting in compromised microtubule stability. Changes of mTor activity cause fluctuation of the level of a battery of tau kinases such as protein kinase A, v-Akt murine thymoma viral oncogene homolog-1, glycogen synthase kinase 3β, cyclin-dependent kinase 5, and tau protein phosphatase 2A. These results implicate mTor in promoting an imbalance of tau homeostasis, a condition required for neurons to maintain physiological function.

  9. The Role of Mammalian Target of Rapamycin (mTOR in Insulin Signaling

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    Mee-Sup Yoon

    2017-10-01

    Full Text Available The mammalian target of rapamycin (mTOR is a serine/threonine kinase that controls a wide spectrum of cellular processes, including cell growth, differentiation, and metabolism. mTOR forms two distinct multiprotein complexes known as mTOR complex 1 (mTORC1 and mTOR complex 2 (mTORC2, which are characterized by the presence of raptor and rictor, respectively. mTOR controls insulin signaling by regulating several downstream components such as growth factor receptor-bound protein 10 (Grb10, insulin receptor substrate (IRS-1, F-box/WD repeat-containing protein 8 (Fbw8, and insulin like growth factor 1 receptor/insulin receptor (IGF-IR/IR. In addition, mTORC1 and mTORC2 regulate each other through a feedback loop to control cell growth. This review outlines the current understanding of mTOR regulation in insulin signaling in the context of whole body metabolism.

  10. [Effects of mTOR Inhibitor Rapamycin on Burkitt's Lymphoma Cells].

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    Zhou, Lun-Huan; Zhu, Xiong-Peng; Xiao, Hui-Fang; Xin, Peng-Liang; Li, Chun-Tuan

    2017-10-01

    To explore the effects of mTOR inhibitor rapamycin on proliferation, cell cycle and apoptosis of Burkitt's lymphoma cell line Raji and CA46 cells and its mechanism, so as to provide the experimental evidence for a therapeutic target of Burkitt's lymphoma. 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide(MTT) assay was performed to assess the inhibitory effect of rapamycin on proliferation of Burkitt's lymphoma cell line Raji and CA46 cells. The cell cycle distribution of Raji and CA46 cells was analyzed by flow cytometry with propidium iodide(PI) single staining. The cell apoptosis of Raji and CA46 cells was analyzed by flow cytometry with FITC Annexin V+PI double staining. The expressions of RPS6, p-RPS6, survivin and caspase-3 proteins were detected by Western blot after treating with rapamycin. Rapamycin markedly inhibited the proliferation of both Raji and CA46 cells in a time- and concentration-dependent manners, showing good biological activity, the cell proliferation inhibition rate reached about 20% after treatment with 1 nmol/L rapamycin. After treatment with different concentrations of rapamycin for 24 and 48 hours, the proportion of both cells in G1/G0 phase in the treated groups was significantly increased in a time- and concentration-dependent manners in comparison with the solvent control group. With regard to the cells in S and G2/M phase, the decreased population was accompanied by the increase of G1/G0 phase cells. After treatment with 100 nmol/L rapamycin for 48 hours, both Raji and CA46 cells demonstrated an apparent apoptosis,especially late apoptosis by flow cytometry with Annexin V+PI staining. After treatment with rapamycin, the expression of p-RPS6 and survivin of Raji and CA46 cells was obviously down-regulated, the expression of caspase-3 was obviously up-regulated in a time- and dose-dependent manners. However, rapamycin did not obviously affect the expression of RPS6. The rapamycin can effectively inhibit cell proliferation

  11. Targeting mTOR with rapamycin: One dose does not fit all

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    Foster, David A.; Toschi, Alfredo

    2009-01-01

    A puzzling aspect of rapamycin-based therapeutic strategies is the wide disparity in the doses needed to suppress mTOR under different circumstances. A recent study revealing mechanistically how rapamycin suppresses mTOR provides two explanations for the differential sensitivities to rapamycin. First, mTOR exists as two functionally distinct complexes (mTORC1 and mTORC2), and while rapamycin suppresses both, it does so at very different concentrations. Whereas mTORC1 is suppressed by concentrations of rapamycin in the low nM range, mTORC2 generally requires low μM concentrations. Second, the efficacy of rapamycin is dependent on the level of phosphatidic acid (PA), which is required for the assembly of both mTORC1 and mTORC2 complexes. Rapamycin interacts with mTOR in a manner that is competitive with PA. Therefore, elevated levels of PA, which is common in cancer cells, increases the level of rapamycin needed to suppress both mTORC1 and mTORC2. A practical outcome of the recent study is that if PA levels are suppressed, mTORC2 becomes sensitive to concentrations of rapamycin that can be achieved clinically. Since mTORC2 is likely more critical for survival signals in cancer cells, the recent findings suggest new strategies for enhancing the efficacy of rapamycin-based therapeutic approaches in cancer cells. PMID:19270529

  12. The mTOR inhibitor rapamycin has limited acute anticonvulsant effects in mice.

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    Adam L Hartman

    Full Text Available The mammalian target of rapamycin (mTOR pathway integrates signals from different nutrient sources, including amino acids and glucose. Compounds that inhibit mTOR kinase activity such as rapamycin and everolimus can suppress seizures in some chronic animal models and in patients with tuberous sclerosis. However, it is not known whether mTOR inhibitors exert acute anticonvulsant effects in addition to their longer term antiepileptogenic effects. To gain insights into how rapamycin suppresses seizures, we investigated the anticonvulsant activity of rapamycin using acute seizure tests in mice.Following intraperitoneal injection of rapamycin, normal four-week-old male NIH Swiss mice were evaluated for susceptibility to a battery of acute seizure tests similar to those currently used to screen potential therapeutics by the US NIH Anticonvulsant Screening Program. To assess the short term effects of rapamycin, mice were seizure tested in ≤ 6 hours of a single dose of rapamycin, and for longer term effects of rapamycin, mice were tested after 3 or more daily doses of rapamycin.The only seizure test where short-term rapamycin treatment protected mice was against tonic hindlimb extension in the MES threshold test, though this protection waned with longer rapamycin treatment. Longer term rapamycin treatment protected against kainic acid-induced seizure activity, but only at late times after seizure onset. Rapamycin was not protective in the 6 Hz or PTZ seizure tests after short or longer rapamycin treatment times. In contrast to other metabolism-based therapies that protect in acute seizure tests, rapamycin has limited acute anticonvulsant effects in normal mice.The efficacy of rapamycin as an acute anticonvulsant agent may be limited. Furthermore, the combined pattern of acute seizure test results places rapamycin in a third category distinct from both fasting and the ketogenic diet, and which is more similar to drugs acting on sodium channels.

  13. Blocking mTOR Signalling with Rapamycin Ameliorates Imiquimod-induced Psoriasis in Mice.

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    Bürger, Claudia; Shirsath, Nitesh; Lang, Victoria; Diehl, Sandra; Kaufmann, Roland; Weigert, Andreas; Han, Ying-Ying; Ringel, Christian; Wolf, Peter

    2017-10-02

    The mTOR (mechanistic target of rapamycin) inhibitor rapamycin has long been known for its immune suppressive properties, but it has shown limited therapeutic success when given systemically to patients with psoriasis. Recent data have shown that the mTOR pathway is hyperactivated in lesional psoriatic skin, which probably contributes to the disease by interfering with maturation of keratinocytes. This study investigated the effect of topical rapamycin treatment in an imiquimod-induced psoriatic mouse model. The disease was less severe if the mice had received rapamycin treatment. Immunohistological analysis revealed that rapamycin not only prevented the activation of mTOR signalling (P-mTOR and P-S6 levels), but almost normalized the expression of epidermal differentiation markers. In addition, the influx of innate immune cells into the draining lymph nodes was partially reduced by rapamycin treatment. These data emphasize the role of mTOR signalling in the pathogenesis of psoriasis, and support the investigation of topical mTOR inhibition as a novel anti-psoriatic strategy.

  14. Akt-dependent and independent mechanisms of mTOR regulation in cancer

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    Memmott, Regan M.; Dennis, Phillip A.

    2009-01-01

    The protein kinase mTOR (mammalian target of rapamycin) is a critical regulator of cellular metabolism, growth, and proliferation. These processes contribute to tumor formation, and many cancers are characterized by aberrant activation of mTOR. Although activating mutations in mTOR itself have not been identified, deregulation of upstream components that regulate mTOR are prevalent in cancer. The prototypic mechanism of mTOR regulation in cells is through activation of the PI3K/Akt pathway, b...

  15. Akt-dependent and independent mechanisms of mTOR regulation in cancer

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    Memmott, Regan M.; Dennis, Phillip A.

    2009-01-01

    The protein kinase mTOR (mammalian target of rapamycin) is a critical regulator of cellular metabolism, growth, and proliferation. These processes contribute to tumor formation, and many cancers are characterized by aberrant activation of mTOR. Although activating mutations in mTOR itself have not been identified, deregulation of upstream components that regulate mTOR are prevalent in cancer. The prototypic mechanism of mTOR regulation in cells is through activation of the PI3K/Akt pathway, but mTOR receives input from multiple signaling pathways. This review will discuss Akt-dependent and independent mechanisms of mTOR regulation in response to mitogenic signals, as well as its regulation in response to energy and nutrient-sensing pathways. Preclinical and clinical studies have demonstrated that tumors bearing genetic alterations that activate mTOR are sensitive to pharmacologic inhibition of mTOR. Elucidation of novel pathways that regulate mTOR may help identify predictive factors for sensitivity to mTOR inhibitors and could provide new therapeutic targets for inhibiting the mTOR pathway in cancer. This review will also highlight pharmacologic approaches that inhibit mTOR via activation of the AMP-activated protein kinase (AMPK), an important inhibitor of the mTOR pathway and an emerging target in cancer. PMID:19166931

  16. The nuclear import of ribosomal proteins is regulated by mTOR

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    Kazyken, Dubek; Kaz, Yelimbek; Kiyan, Vladimir; Zhylkibayev, Assylbek A.; Chen, Chien-Hung; Agarwal, Nitin K.; Sarbassov, Dos D.

    2014-01-01

    Mechanistic target of rapamycin (mTOR) is a central component of the essential signaling pathway that regulates cell growth and proliferation by controlling anabolic processes in cells. mTOR exists in two distinct mTOR complexes known as mTORC1 and mTORC2 that reside mostly in cytoplasm. In our study, the biochemical characterization of mTOR led to discovery of its novel localization on nuclear envelope where it associates with a critical regulator of nuclear import Ran Binding Protein 2 (RanBP2). We show that association of mTOR with RanBP2 is dependent on the mTOR kinase activity that regulates the nuclear import of ribosomal proteins. The mTOR kinase inhibitors within thirty minutes caused a substantial decrease of ribosomal proteins in the nuclear but not cytoplasmic fraction. Detection of a nuclear accumulation of the GFP-tagged ribosomal protein rpL7a also indicated its dependence on the mTOR kinase activity. The nuclear abundance of ribosomal proteins was not affected by inhibition of mTOR Complex 1 (mTORC1) by rapamycin or deficiency of mTORC2, suggesting a distinctive role of the nuclear envelope mTOR complex in the nuclear import. Thus, we identified that mTOR in association with RanBP2 mediates the active nuclear import of ribosomal proteins. PMID:25294810

  17. Rapamycin targeting mTOR and hedgehog signaling pathways blocks human rhabdomyosarcoma growth in xenograft murine model

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    Kaylani, Samer Z. [Division of Hematology and Oncology, Department of Pediatrics, University of Alabama at Birmingham, 1600 7th Avenue South, ACC 414, Birmingham, AL 35233 (United States); Xu, Jianmin; Srivastava, Ritesh K. [Department of Dermatology and Skin Diseases Research Center, University of Alabama at Birmingham, 1530 3rd Avenue South, VH 509, Birmingham, AL 35294-0019 (United States); Kopelovich, Levy [Division of Cancer Prevention, National Cancer Institute, Bethesda (United States); Pressey, Joseph G. [Division of Hematology and Oncology, Department of Pediatrics, University of Alabama at Birmingham, 1600 7th Avenue South, ACC 414, Birmingham, AL 35233 (United States); Athar, Mohammad, E-mail: mathar@uab.edu [Department of Dermatology and Skin Diseases Research Center, University of Alabama at Birmingham, 1530 3rd Avenue South, VH 509, Birmingham, AL 35294-0019 (United States)

    2013-06-14

    Graphical abstract: Intervention of poorly differentiated RMS by rapamycin: In poorly differentiated RMS, rapamycin blocks mTOR and Hh signaling pathways concomitantly. This leads to dampening in cell cycle regulation and induction of apoptosis. This study provides a rationale for the therapeutic intervention of poorly differentiated RMS by treating patients with rapamycin alone or in combination with other chemotherapeutic agents. -- Highlights: •Rapamycin abrogates RMS tumor growth by modulating proliferation and apoptosis. •Co-targeting mTOR/Hh pathways underlie the molecular basis of effectiveness. •Reduction in mTOR/Hh pathways diminish EMT leading to reduced invasiveness. -- Abstract: Rhabdomyosarcomas (RMS) represent the most common childhood soft-tissue sarcoma. Over the past few decades outcomes for low and intermediate risk RMS patients have slowly improved while patients with metastatic or relapsed RMS still face a grim prognosis. New chemotherapeutic agents or combinations of chemotherapies have largely failed to improve the outcome. Based on the identification of novel molecular targets, potential therapeutic approaches in RMS may offer a decreased reliance on conventional chemotherapy. Thus, identification of effective therapeutic agents that specifically target relevant pathways may be particularly beneficial for patients with metastatic and refractory RMS. The PI3K/AKT/mTOR pathway has been found to be a potentially attractive target in RMS therapy. In this study, we provide evidence that rapamycin (sirolimus) abrogates growth of RMS development in a RMS xenograft mouse model. As compared to a vehicle-treated control group, more than 95% inhibition in tumor growth was observed in mice receiving parenteral administration of rapamycin. The residual tumors in rapamycin-treated group showed significant reduction in the expression of biomarkers indicative of proliferation and tumor invasiveness. These tumors also showed enhanced apoptosis

  18. Mechanism of Metformin-dependent Inhibition of Mammalian Target of Rapamycin (mTOR) and Ras Activity in Pancreatic Cancer

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    Nair, Vijayalekshmi; Sreevalsan, Sandeep; Basha, Riyaz; Abdelrahim, Maen; Abudayyeh, Ala; Rodrigues Hoffman, Aline; Safe, Stephen

    2014-01-01

    The antidiabetic drug metformin exhibits both chemopreventive and chemotherapeutic activity for multiple cancers including pancreatic cancer; however, the underlying mechanism of action of metformin is unclear. A recent study showed that metformin down-regulated specificity protein (Sp) transcription factors (TFs) Sp1, Sp3, and Sp4 in pancreatic cancer cells and tumors, and this was accompanied by down-regulation of several pro-oncogenic Sp-regulated genes. Treatment with metformin or down-regulation of Sp TFs by RNAi also inhibits two major pro-oncogenic pathways in pancreatic cancer cells, namely mammalian target of rapamycin (mTOR) signaling and epidermal growth factor (EGFR)-dependent activation of Ras. Metformin and Sp knockdown by RNAi decreased expression of the insulin-like growth factor-1 receptor (IGF-1R), resulting in inhibition of mTOR signaling. Ras activity was also decreased by metformin and Sp knockdown of EGFR, another Sp-regulated gene. Thus, the antineoplastic activities of metformin in pancreatic cancer are due, in part, to down-regulation of Sp TFs and Sp-regulated IGF-1R and EGFR, which in turn results in inhibition of mTOR and Ras signaling, respectively. PMID:25143389

  19. Hypothalamic roles of mTOR complex I: Integration of nutrient and hormone signals to regulate energy homeostasis

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    Mammalian or mechanistic target of rapamycin (mTOR) senses nutrient, energy, and hormone signals to regulate metabolism and energy homeostasis. mTOR activity in the hypothalamus, which is associated with changes in energy status, plays a critical role in the regulation of food intake and body weight...

  20. Rapamycin up-regulates triglycerides in hepatocytes by down-regulating Prox1.

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    Kwon, Sora; Jeon, Ji-Sook; Kim, Su Bin; Hong, Young-Kwon; Ahn, Curie; Sung, Jung-Suk; Choi, Inho

    2016-02-27

    Although the prolonged use of rapamycin may cause unwanted side effects such as hyperlipidemia, the underlying mechanism remains unknown. Prox1 is a transcription factor responsible for the development of several tissues including lymphatics and liver. There is growing evidences that Prox1 participates in metabolism in addition to embryogenesis. However, whether Prox1 is directly related to lipid metabolism is currently unknown. HepG2 human hepatoma cells were treated with rapamycin and total lipids were analyzed by thin layer chromatography. The effect of rapamycin on the expression of Prox1 was determined by western blotting. To investigate the role of Prox1 in triglycerides regulation, siRNA and overexpression system were employed. Rapamycin was injected into mice for 2 weeks and total lipids and proteins in liver were measured by thin layer chromatography and western blot analysis, respectively. Rapamycin up-regulated the amount of triglyceride and down-regulated the expression of Prox1 in HepG2 cells by reducing protein half-life but did not affect its transcript. The loss-of-function of Prox1 was coincident with the increase of triglycerides in HepG2 cells treated with rapamycin. The up-regulation of triglycerides by rapamycin in HepG2 cells reverted to normal levels by the compensation of Prox1 using the overexpression system. Rapamycin also down-regulated Prox1 expression but increased triglycerides in mouse liver. This study suggests that rapamycin can increase the amount of triglycerides by down-regulating Prox1 expression in hepatocytes, which means that the mammalian target of rapamycin (mTOR) signaling is important for the regulation of triglycerides by maintaining Prox1 expression.

  1. Inhibition of the mammalian target of rapamycin (mTOR in advanced pancreatic cancer: results of two phase II studies

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    Zhang Yujian

    2010-07-01

    Full Text Available Abstract Background The phosphoinositide 3-kinase (PI3K/Akt pathway is constitutively activated in pancreatic cancer and the mammalian target of rapamycin (mTOR kinase is an important mediator for its signaling. Our recent in vitro studies suggest that prolonged exposure of pancreatic cancer cells to mTOR inhibitors can promote insulin receptor substrate-PI3K interactions and paradoxically increase Akt phosphorylation and cyclin D1 expression in pancreatic cancer cells (negative feedback loop. The addition of erlotinib to rapamycin can down-regulate rapamycin-stimulated Akt and results in synergistic antitumor activity with erlotinib in preclinical tumor models. Methods Two studies prospectively enrolled adult patients with advanced pancreatic cancer, Eastern Cooperative Oncology Group performance status 0-1, adequate hematologic, hepatic and renal parameters and measurable disease. In Study A, temsirolimus was administered intravenously at 25 mg weekly. In Study B, everolimus was administered orally at 30 mg weekly and erlotinib was administered at 150 mg daily. The primary endpoint in both studies was overall survival at 6 months. Secondary endpoints included time to progression, progression-free survival, overall survival, response rate, safety and toxicity. Pretreatment tumor biopsies were analyzed by immunofluorescence and laser scanning cytometry for the expression of pmTOR/mTOR, pAkt/Akt, pErk/Erk, pS6, p4EBP-1 and PTEN. Results Five patients enrolled in Study A; Two patients died within a month (rapid disease progression and hemorrhagic stroke, respectively. One patient developed dehydration and another developed asthenia. Sixteen patients enrolled in Study B.: 12 males, all ECOG PS = 1. Median cycles = 1 (range 1-2. Grade 4 toxicity: hyponatremia (n = 1, Grade 3: diarrhea (n = 1, cholangitis (n = 3, hyperglycemia (n = 1, fatigue (n = 1. Grade 2: pneumonia (n = 2, dehydration (n = 2, nausea (n = 2, neutropenia (n = 1, mucositis (n = 2

  2. p21WAF1/CIP1 Expression is Differentially Regulated by Metformin and Rapamycin

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    Zoltan Molnar

    2014-01-01

    Full Text Available The mammalian target of rapamycin (mTOR pathway plays an important role in the development of diabetic nephropathy and other age-related diseases. One of the features of DN is the elevated expression of p21WAF1/CIP1. However, the importance of the mTOR signalling pathway in p21 regulation is poorly understood. Here we investigated the effect of metformin and rapamycin on mTOR-related phenotypes in cell lines of epithelial origin. This study reports that metformin inhibits high glucose-induced p21 expression. High glucose opposed metformin in regulating cell size, proliferation, and protein synthesis. These effects were associated with reduced AMPK activation, affecting downstream mTOR signalling. However, the inhibition of the mTOR pathway by rapamycin did not have a negative effect on p21 expression, suggesting that metformin regulates p21 upstream of mTOR. These findings provide support for the hypothesis that AMPK activation may regulate p21 expression, which may have implications for diabetic nephropathy and other age-related pathologies.

  3. Molecular mechanisms of mTOR regulation by stress.

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    Heberle, Alexander Martin; Prentzell, Mirja Tamara; van Eunen, Karen; Bakker, Barbara Marleen; Grellscheid, Sushma Nagaraja; Thedieck, Kathrin

    2015-01-01

    Tumors are prime examples of cell growth in unfavorable environments that elicit cellular stress. The high metabolic demand and insufficient vascularization of tumors cause a deficiency of oxygen and nutrients. Oncogenic mutations map to signaling events via mammalian target of rapamycin (mTOR), metabolic pathways, and mitochondrial function. These alterations have been linked with cellular stresses, in particular endoplasmic reticulum (ER) stress, hypoxia, and oxidative stress. Yet tumors survive these challenges and acquire highly energy-demanding traits, such as overgrowth and invasiveness. In this review we focus on stresses that occur in cancer cells and discuss them in the context of mTOR signaling. Of note, many tumor traits require mTOR complex 1 (mTORC1) activity, but mTORC1 hyperactivation eventually sensitizes cells to apoptosis. Thus, mTORC1 activity needs to be balanced in cancer cells. We provide an overview of the mechanisms contributing to mTOR regulation by stress and suggest a model wherein stress granules function as guardians of mTORC1 signaling, allowing cancer cells to escape stress-induced cell death.

  4. Rapamycin: one drug, many effects

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    Li, Jing; Kim, Sang Gyun; Blenis, John

    2014-01-01

    The mammalian target of rapamycin (mTOR) signaling pathway is a master regulator of cell growth and metabolism. Deregulation of the mTOR pathway has been implicated in a number of human diseases such as cancer, diabetes, obesity, neurological diseases and genetic disorders. Rapamycin, a specific inhibitor of mTOR, has been shown to be useful in the treatment of certain diseases. Here we discuss its mechanism of action and highlight recent findings regarding the effects and limitations of rapamycin monotherapy and the potential utility of combination therapy with rapamycin. PMID:24508508

  5. Rapamycin: One Drug, Many Effects

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    Li, Jing; Kim, Sang Gyun; Blenis, John

    2014-01-01

    The mammalian target of rapamycin (mTOR) signaling pathway is a master regulator of cell growth and metabolism. Deregulation of the mTOR pathway has been implicated in a number of human diseases such as cancer, diabetes, obesity, neurological diseases and genetic disorders. Rapamycin, a specific inhibitor of mTOR, has been shown to be useful in the treatment of certain diseases. Here we discuss its mechanism of action and highlight recent findings regarding the effects and limitations of rapa...

  6. Inhibition of mTOR by Rapamycin Results in Auditory Hair Cell Damage and Decreased Spiral Ganglion Neuron Outgrowth and Neurite Formation In Vitro

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    Katharina Leitmeyer

    2015-01-01

    Full Text Available Rapamycin is an antifungal agent with immunosuppressive properties. Rapamycin inhibits the mammalian target of rapamycin (mTOR by blocking the mTOR complex 1 (mTORC1. mTOR is an atypical serine/threonine protein kinase, which controls cell growth, cell proliferation, and cell metabolism. However, less is known about the mTOR pathway in the inner ear. First, we evaluated whether or not the two mTOR complexes (mTORC1 and mTORC2, resp. are present in the mammalian cochlea. Next, tissue explants of 5-day-old rats were treated with increasing concentrations of rapamycin to explore the effects of rapamycin on auditory hair cells and spiral ganglion neurons. Auditory hair cell survival, spiral ganglion neuron number, length of neurites, and neuronal survival were analyzed in vitro. Our data indicates that both mTOR complexes are expressed in the mammalian cochlea. We observed that inhibition of mTOR by rapamycin results in a dose dependent damage of auditory hair cells. Moreover, spiral ganglion neurite number and length of neurites were significantly decreased in all concentrations used compared to control in a dose dependent manner. Our data indicate that the mTOR may play a role in the survival of hair cells and modulates spiral ganglion neuronal outgrowth and neurite formation.

  7. mTOR regulates peripheral nerve response to tensile strain.

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    Love, James M; Bober, Brian G; Orozco, Elisabeth; White, Amanda T; Bremner, Shannon N; Lovering, Richard M; Schenk, Simon; Shah, Sameer B

    2017-05-01

    While excessive tensile strain can be detrimental to nerve function, strain can be a positive regulator of neuronal outgrowth. We used an in vivo rat model of sciatic nerve strain to investigate signaling mechanisms underlying peripheral nerve response to deformation. Nerves were deformed by 11% and did not demonstrate deficits in compound action potential latency or amplitude during or after 6 h of strain. As revealed by Western blotting, application of strain resulted in significant upregulation of mammalian target of rapamycin (mTOR) and S6 signaling in nerves, increased myelin basic protein (MBP) and β-actin levels, and increased phosphorylation of neurofilament subunit H (NF-H) compared with unstrained (sham) contralateral nerves (P nerve tubulin levels compared with unstrained controls. Systemic rapamycin treatment, thought to selectively target mTOR complex 1 (mTORC1), suppressed mTOR/S6 signaling, reduced levels of MBP and overall tubulin, and decreased NF-H phosphorylation in nerves strained for 6 h, revealing a role for mTOR in increasing MBP expression and NF-H phosphorylation, and maintaining tubulin levels. Consistent with stretch-induced increases in MBP, immunolabeling revealed increased S6 signaling in Schwann cells of stretched nerves compared with unstretched nerves. In addition, application of strain to cultured adult dorsal root ganglion neurons showed an increase in axonal protein synthesis based on a puromycin incorporation assay, suggesting that neuronal translational pathways also respond to strain. This work has important implications for understanding mechanisms underlying nerve response to strain during development and regeneration.NEW & NOTEWORTHY Peripheral nerves experience tensile strain (stretch) during development and movement. Excessive strain impairs neuronal function, but moderate strains are accommodated by nerves and can promote neuronal growth; mechanisms underlying these phenomena are not well understood. We demonstrated

  8. Down-regulation of mTOR leads to up-regulation of osteoprotegerin in bone marrow cells

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    Mogi, Makio, E-mail: makio@dpc.aichi-gakuin.ac.jp [Department of Medicinal Biochemistry, School of Pharmacy, Aichi-Gakuin University, 1-100 Kusumoto, Chikusa, Nagoya, Aichi 464-8650 (Japan); Kondo, Ayami [Department of Medicinal Biochemistry, School of Pharmacy, Aichi-Gakuin University, 1-100 Kusumoto, Chikusa, Nagoya, Aichi 464-8650 (Japan)

    2009-06-19

    Osteoprotegerin (OPG)/osteoclastogenesis inhibitory factor regulates bone mass by inhibiting osteoclastic bone resorption. mTOR, which is the mammalian target of rapamycin, is a kinase and central regulator of cell growth, proliferation, and survival. By using Rapamycin, we studied whether mTOR pathway is associated with OPG protein production in the mouse bone marrow-derived stromal cell line ST2. Rapamycin markedly increased the level of soluble OPG in ST2 cells. This antibiotic treatment resulted in the suppression of phosphorylation of mTOR. Rapamycin had no effects on the proliferation, differentiation, or apoptosis of the cells. Treatment with bone morphogenetic protein-4, which can induce OPG protein in ST2 cells, also resulted in a decrease in the density of the phospho-mTOR-band, suggesting that the suppression of the phospho-mTOR pathway is necessary for OPG production in ST2 cells. Thus, suitable suppression of mTOR phosphorylation is a necessary requirement for OPG production in bone marrow stromal cells.

  9. mTOR signaling and its involvement in the regulation of cell movements through remodeling the cytoskeleton architecture

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    Kosach V. R.

    2015-02-01

    Full Text Available mTOR kinase is one of the basic links at the crossroad of several signal transduction pathways. De­re­gulated mTOR kinase signaling accompanies the progress of cancer, diabetes, neurodegenerative disorders and aging. Implication of mTOR inhibitor rapamycin decreases migration and invasion of malignant cells, and metastasis formation. However, a precise mechanism of the regulation of cellular locomotion by mTOR kinase is not fully understood. This article focuses on the recent findings that demonstrated a possible role of mTOR kinase in the regulation of cytoskeleton remodeling and cell migration properties. Detailed studies on this non-canonical mTOR function will extend our knowledge about cell migration and metastasis formation and might improve anti-cancer therapeutic approaches.

  10. The role of diacylglycerol kinase ζ and phosphatidic acid in the mechanical activation of mammalian target of rapamycin (mTOR) signaling and skeletal muscle hypertrophy.

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    You, Jae-Sung; Lincoln, Hannah C; Kim, Chan-Ran; Frey, John W; Goodman, Craig A; Zhong, Xiao-Ping; Hornberger, Troy A

    2014-01-17

    The activation of mTOR signaling is essential for mechanically induced changes in skeletal muscle mass, and previous studies have suggested that mechanical stimuli activate mTOR (mammalian target of rapamycin) signaling through a phospholipase D (PLD)-dependent increase in the concentration of phosphatidic acid (PA). Consistent with this conclusion, we obtained evidence which further suggests that mechanical stimuli utilize PA as a direct upstream activator of mTOR signaling. Unexpectedly though, we found that the activation of PLD is not necessary for the mechanically induced increases in PA or mTOR signaling. Motivated by this observation, we performed experiments that were aimed at identifying the enzyme(s) that promotes the increase in PA. These experiments revealed that mechanical stimulation increases the concentration of diacylglycerol (DAG) and the activity of DAG kinases (DGKs) in membranous structures. Furthermore, using knock-out mice, we determined that the ζ isoform of DGK (DGKζ) is necessary for the mechanically induced increase in PA. We also determined that DGKζ significantly contributes to the mechanical activation of mTOR signaling, and this is likely driven by an enhanced binding of PA to mTOR. Last, we found that the overexpression of DGKζ is sufficient to induce muscle fiber hypertrophy through an mTOR-dependent mechanism, and this event requires DGKζ kinase activity (i.e. the synthesis of PA). Combined, these results indicate that DGKζ, but not PLD, plays an important role in mechanically induced increases in PA and mTOR signaling. Furthermore, this study suggests that DGKζ could be a fundamental component of the mechanism(s) through which mechanical stimuli regulate skeletal muscle mass.

  11. The Role of Diacylglycerol Kinase ζ and Phosphatidic Acid in the Mechanical Activation of Mammalian Target of Rapamycin (mTOR) Signaling and Skeletal Muscle Hypertrophy*

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    You, Jae-Sung; Lincoln, Hannah C.; Kim, Chan-Ran; Frey, John W.; Goodman, Craig A.; Zhong, Xiao-Ping; Hornberger, Troy A.

    2014-01-01

    The activation of mTOR signaling is essential for mechanically induced changes in skeletal muscle mass, and previous studies have suggested that mechanical stimuli activate mTOR (mammalian target of rapamycin) signaling through a phospholipase D (PLD)-dependent increase in the concentration of phosphatidic acid (PA). Consistent with this conclusion, we obtained evidence which further suggests that mechanical stimuli utilize PA as a direct upstream activator of mTOR signaling. Unexpectedly though, we found that the activation of PLD is not necessary for the mechanically induced increases in PA or mTOR signaling. Motivated by this observation, we performed experiments that were aimed at identifying the enzyme(s) that promotes the increase in PA. These experiments revealed that mechanical stimulation increases the concentration of diacylglycerol (DAG) and the activity of DAG kinases (DGKs) in membranous structures. Furthermore, using knock-out mice, we determined that the ζ isoform of DGK (DGKζ) is necessary for the mechanically induced increase in PA. We also determined that DGKζ significantly contributes to the mechanical activation of mTOR signaling, and this is likely driven by an enhanced binding of PA to mTOR. Last, we found that the overexpression of DGKζ is sufficient to induce muscle fiber hypertrophy through an mTOR-dependent mechanism, and this event requires DGKζ kinase activity (i.e. the synthesis of PA). Combined, these results indicate that DGKζ, but not PLD, plays an important role in mechanically induced increases in PA and mTOR signaling. Furthermore, this study suggests that DGKζ could be a fundamental component of the mechanism(s) through which mechanical stimuli regulate skeletal muscle mass. PMID:24302719

  12. Mechanism of metformin-dependent inhibition of mammalian target of rapamycin (mTOR) and Ras activity in pancreatic cancer: role of specificity protein (Sp) transcription factors.

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    Nair, Vijayalekshmi; Sreevalsan, Sandeep; Basha, Riyaz; Abdelrahim, Maen; Abudayyeh, Ala; Rodrigues Hoffman, Aline; Safe, Stephen

    2014-10-03

    The antidiabetic drug metformin exhibits both chemopreventive and chemotherapeutic activity for multiple cancers including pancreatic cancer; however, the underlying mechanism of action of metformin is unclear. A recent study showed that metformin down-regulated specificity protein (Sp) transcription factors (TFs) Sp1, Sp3, and Sp4 in pancreatic cancer cells and tumors, and this was accompanied by down-regulation of several pro-oncogenic Sp-regulated genes. Treatment with metformin or down-regulation of Sp TFs by RNAi also inhibits two major pro-oncogenic pathways in pancreatic cancer cells, namely mammalian target of rapamycin (mTOR) signaling and epidermal growth factor (EGFR)-dependent activation of Ras. Metformin and Sp knockdown by RNAi decreased expression of the insulin-like growth factor-1 receptor (IGF-1R), resulting in inhibition of mTOR signaling. Ras activity was also decreased by metformin and Sp knockdown of EGFR, another Sp-regulated gene. Thus, the antineoplastic activities of metformin in pancreatic cancer are due, in part, to down-regulation of Sp TFs and Sp-regulated IGF-1R and EGFR, which in turn results in inhibition of mTOR and Ras signaling, respectively. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  13. BMAL1-dependent regulation of the mTOR signaling pathway delays aging.

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    Khapre, Rohini V; Kondratova, Anna A; Patel, Sonal; Dubrovsky, Yuliya; Wrobel, Michelle; Antoch, Marina P; Kondratov, Roman V

    2014-01-01

    The circadian clock, an internal time-keeping system, has been linked with control of aging, but molecular mechanisms of regulation are not known. BMAL1 is a transcriptional factor and core component of the circadian clock; BMAL1 deficiency is associated with premature aging and reduced lifespan. Here we report that activity of mammalian Target of Rapamycin Complex 1 (mTORC1) is increased upon BMAL1 deficiency both in vivo and in cell culture. Increased mTOR signaling is associated with accelerated aging; in accordance with that, treatment with the mTORC1 inhibitor rapamycin increased lifespan of Bmal1-/- mice by 50%. Our data suggest that BMAL1 is a negative regulator of mTORC1 signaling. We propose that the circadian clock controls the activity of the mTOR pathway through BMAL1-dependent mechanisms and this regulation is important for control of aging and metabolism.

  14. The rapamycin-regulated gene expression signature determines prognosis for breast cancer

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    Tsavachidis Spiridon

    2009-09-01

    Full Text Available Abstract Background Mammalian target of rapamycin (mTOR is a serine/threonine kinase involved in multiple intracellular signaling pathways promoting tumor growth. mTOR is aberrantly activated in a significant portion of breast cancers and is a promising target for treatment. Rapamycin and its analogues are in clinical trials for breast cancer treatment. Patterns of gene expression (metagenes may also be used to simulate a biologic process or effects of a drug treatment. In this study, we tested the hypothesis that the gene-expression signature regulated by rapamycin could predict disease outcome for patients with breast cancer. Results Colony formation and sulforhodamine B (IC50 in vitro and in vivo gene expression data identified a signature, termed rapamycin metagene index (RMI, of 31 genes upregulated by rapamycin treatment in vitro as well as in vivo (false discovery rate of 10%. In the Miller dataset, RMI did not correlate with tumor size or lymph node status. High (>75th percentile RMI was significantly associated with longer survival (P = 0.015. On multivariate analysis, RMI (P = 0.029, tumor size (P = 0.015 and lymph node status (P = 0.001 were prognostic. In van 't Veer study, RMI was not associated with the time to develop distant metastasis (P = 0.41. In the Wang dataset, RMI predicted time to disease relapse (P = 0.009. Conclusion Rapamycin-regulated gene expression signature predicts clinical outcome in breast cancer. This supports the central role of mTOR signaling in breast cancer biology and provides further impetus to pursue mTOR-targeted therapies for breast cancer treatment.

  15. mTOR as a Key Regulator in Maintaining Skeletal Muscle Mass

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    Mee-Sup Yoon

    2017-10-01

    Full Text Available Maintenance of skeletal muscle mass is regulated by the balance between anabolic and catabolic processes. Mammalian target of rapamycin (mTOR is an evolutionarily conserved serine/threonine kinase, and is known to play vital roles in protein synthesis. Recent findings have continued to refine our understanding of the function of mTOR in maintaining skeletal muscle mass. mTOR controls the anabolic and catabolic signaling of skeletal muscle mass, resulting in the modulation of muscle hypertrophy and muscle wastage. This review will highlight the fundamental role of mTOR in skeletal muscle growth by summarizing the phenotype of skeletal-specific mTOR deficiency. In addition, the evidence that mTOR is a dual regulator of anabolism and catabolism in skeletal muscle mass will be discussed. A full understanding of mTOR signaling in the maintenance of skeletal muscle mass could help to develop mTOR-targeted therapeutics to prevent muscle wasting.

  16. Distinct amino acid-sensing mTOR pathways regulate skeletal myogenesis.

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    Yoon, Mee-Sup; Chen, Jie

    2013-12-01

    Signaling through the mammalian target of rapamycin (mTOR) in response to amino acid availability controls many cellular and developmental processes. mTOR is a master regulator of myogenic differentiation, but the pathways mediating amino acid signals in this process are not known. Here we examine the Rag GTPases and the class III phosphoinositide 3-kinase (PI3K) Vps34, two mediators of amino acid signals upstream of mTOR complex 1 (mTORC1) in cell growth regulation, for their potential involvement in myogenesis. We find that, although both Rag and Vps34 mediate amino acid activation of mTORC1 in C2C12 myoblasts, they have opposing functions in myogenic differentiation. Knockdown of RagA/B enhances, whereas overexpression of active RagB/C mutants impairs, differentiation, and this inhibitory function of Rag is mediated by mTORC1 suppression of the IRS1-PI3K-Akt pathway. On the other hand, Vps34 is required for myogenic differentiation. Amino acids activate a Vps34-phospholipase D1 (PLD1) pathway that controls the production of insulin-like growth factor II, an autocrine inducer of differentiation, through the Igf2 muscle enhancer. The product of PLD, phosphatidic acid, activates the enhancer in a rapamycin-sensitive but mTOR kinase-independent manner. Our results uncover amino acid-sensing mechanisms controlling the homeostasis of myogenesis and underline the versatility and context dependence of mTOR signaling.

  17. mTOR (Mechanistic Target of Rapamycin) Inhibition Decreases Mechanosignaling, Collagen Accumulation, and Stiffening of the Thoracic Aorta in Elastin-Deficient Mice.

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    Jiao, Yang; Li, Guangxin; Li, Qingle; Ali, Rahmat; Qin, Lingfeng; Li, Wei; Qyang, Yibing; Greif, Daniel M; Geirsson, Arnar; Humphrey, Jay D; Tellides, George

    2017-09-01

    Elastin deficiency because of heterozygous loss of an ELN allele in Williams syndrome causes obstructive aortopathy characterized by medial thickening and fibrosis and consequent aortic stiffening. Previous work in Eln-null mice with a severe arterial phenotype showed that inhibition of mTOR (mechanistic target of rapamycin), a key regulator of cell growth, lessened the aortic obstruction but did not prevent early postnatal death. We investigated the effects of mTOR inhibition in Eln-null mice partially rescued by human ELN that manifest a less severe arterial phenotype and survive long term. Thoracic aortas of neonatal and juvenile mice with graded elastin deficiency exhibited increased signaling through both mTOR complex 1 and 2. Despite lower predicted wall stress, there was increased phosphorylation of focal adhesion kinase, suggestive of greater integrin activation, and increased transforming growth factor-β-signaling mediators, associated with increased collagen expression. Pharmacological blockade of mTOR by rapalogs did not improve luminal stenosis but reduced mechanosignaling (in delayed fashion after mTOR complex 1 inhibition), medial collagen accumulation, and stiffening of the aorta. Rapalog administration also retarded somatic growth, however, and precipitated neonatal deaths. Complementary, less-toxic strategies to inhibit mTOR via altered growth factor and nutrient responses were not effective. In addition to previously demonstrated therapeutic benefits of rapalogs decreasing smooth muscle cell proliferation in the absence of elastin, we find that rapalogs also prevent aortic fibrosis and stiffening attributable to partial elastin deficiency. Our findings suggest that mTOR-sensitive perturbation of smooth muscle cell mechanosensing contributes to elastin aortopathy. © 2017 American Heart Association, Inc.

  18. mTOR regulates tau phosphorylation and degradation: implications for Alzheimer's disease and other tauopathies.

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    Caccamo, Antonella; Magrì, Andrea; Medina, David X; Wisely, Elena V; López-Aranda, Manuel F; Silva, Alcino J; Oddo, Salvatore

    2013-06-01

    Accumulation of tau is a critical event in several neurodegenerative disorders, collectively known as tauopathies, which include Alzheimer's disease and frontotemporal dementia. Pathological tau is hyperphosphorylated and aggregates to form neurofibrillary tangles. The molecular mechanisms leading to tau accumulation remain unclear and more needs to be done to elucidate them. Age is a major risk factor for all tauopathies, suggesting that molecular changes contributing to the aging process may facilitate tau accumulation and represent common mechanisms across different tauopathies. Here, we use multiple animal models and complementary genetic and pharmacological approaches to show that the mammalian target of rapamycin (mTOR) regulates tau phosphorylation and degradation. Specifically, we show that genetically increasing mTOR activity elevates endogenous mouse tau levels and phosphorylation. Complementary to it, we further demonstrate that pharmacologically reducing mTOR signaling with rapamycin ameliorates tau pathology and the associated behavioral deficits in a mouse model overexpressing mutant human tau. Mechanistically, we provide compelling evidence that the association between mTOR and tau is linked to GSK3β and autophagy function. In summary, we show that increasing mTOR signaling facilitates tau pathology, while reducing mTOR signaling ameliorates tau pathology. Given the overwhelming evidence that reducing mTOR signaling increases lifespan and healthspan, the data presented here have profound clinical implications for aging and tauopathies and provide the molecular basis for how aging may contribute to tau pathology. Additionally, these results provide preclinical data indicating that reducing mTOR signaling may be a valid therapeutic approach for tauopathies. © 2013 John Wiley & Sons Ltd and the Anatomical Society.

  19. Effects of ketamine administration on mTOR and reticulum stress signaling pathways in the brain after the infusion of rapamycin into prefrontal cortex.

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    Abelaira, Helena M; Réus, Gislaine Z; Ignácio, Zuleide M; Dos Santos, Maria Augusta B; de Moura, Airam B; Matos, Danyela; Demo, Júlia P; da Silva, Júlia B I; Michels, Monique; Abatti, Mariane; Sonai, Beatriz; Dal Pizzol, Felipe; Carvalho, André F; Quevedo, João

    2017-04-01

    Recent studies show that activation of the mTOR signaling pathway is required for the rapid antidepressant actions of glutamate N-methyl-D-aspartate (NMDA) receptor antagonists. A relationship between mTOR kinase and the endoplasmic reticulum (ER) stress pathway, also known as the unfolded protein response (UPR) has been shown. We evaluate the effects of ketamine administration on the mTOR signaling pathway and proteins of UPR in the prefrontal cortex (PFC), hippocampus, amygdala and nucleus accumbens, after the inhibiton of mTOR signaling in the PFC. Male adult Wistar rats received pharmacological mTOR inhibitor, rapamycin (0.2 nmol), or vehicle into the PFC and then a single dose of ketamine (15 mg/kg, i.p.). The immunocontent of mTOR, eukaryotic initiation factor 4E-binding protein 1 (4E-BP1), eukaryotic elongation factor 2 kinase (eEF2K) homologous protein (CHOP), PKR-like ER kinase (PERK) and inositol-requiring enzyme 1 (IRE1) - alpha were determined in the brain. The mTOR levels were reduced in the rapamycin group treated with saline and ketamine in the PFC; p4EBP1 levels were reduced in the rapamycin group treated with ketamine in the PFC and nucleus accumbens; the levels of peEF2K were increased in the PFC in the vehicle group treated with ketamine and reduced in the rapamycin group treated with ketamine. The PERK and IRE1-alpha levels were decreased in the PFC in the rapamycin group treated with ketamine. Our results suggest that mTOR signaling inhibition by rapamycin could be involved, at least in part, with the mechanism of action of ketamine; and the ketamine antidepressant on ER stress pathway could be also mediated by mTOR signaling pathway in certain brain structures. Copyright © 2016 Elsevier Ltd. All rights reserved.

  20. Regulation of mTOR by mechanically induced signaling events in skeletal muscle.

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    Hornberger, Troy Alan; Sukhija, Kunal Balu; Chien, Shu

    2006-07-01

    Mechanical stimuli play a major role in the regulation of skeletal muscle mass, and the maintenance of muscle mass contributes significantly to disease prevention and the quality of life. Although a link between mechanical stimuli and the regulation of muscle mass has been recognized for decades, the mechanisms involved in converting mechanical information into the molecular events that control this process have not been defined. Nevertheless, significant advancements are being made in this field, and it has recently been established that signaling through a rapamycin-sensitive pathway is necessary for mechanically induced growth of skeletal muscle. Since rapamycin is a highly specific inhibitor of a protein kinase called the mammalian target of rapamycin (mTOR), many investigators have concluded that mTOR signaling is necessary for the mechanically induced growth of skeletal muscle. In this review, we have summarized the current knowledge regarding how mechanical stimuli activate mTOR signaling, discussed the newly discovered role of phospholipase D (PLD) and phosphatidic acid (PA) in this pathway, and considered the potential roles of PLD and PA in the mechanical regulation of skeletal muscle mass.

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

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

    2016-01-01

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

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

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    Amilcar Rivera Rivera

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

  3. Glucose Regulation of Load‐Induced mTOR Signaling and ER Stress in Mammalian Heart

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    Sen, Shiraj; Kundu, Bijoy K.; Wu, Henry Cheng‐Ju; Hashmi, S. Shahrukh; Guthrie, Patrick; Locke, Landon W.; Roy, R. Jack; Matherne, G. Paul; Berr, Stuart S.; Terwelp, Matthew; Scott, Brian; Carranza, Sylvia; Frazier, O. Howard; Glover, David K.; Dillmann, Wolfgang H.; Gambello, Michael J.; Entman, Mark L.; Taegtmeyer, Heinrich

    2013-01-01

    Background Changes in energy substrate metabolism are first responders to hemodynamic stress in the heart. We have previously shown that hexose‐6‐phosphate levels regulate mammalian target of rapamycin (mTOR) activation in response to insulin. We now tested the hypothesis that inotropic stimulation and increased afterload also regulate mTOR activation via glucose 6‐phosphate (G6P) accumulation. Methods and Results We subjected the working rat heart ex vivo to a high workload in the presence of different energy‐providing substrates including glucose, glucose analogues, and noncarbohydrate substrates. We observed an association between G6P accumulation, mTOR activation, endoplasmic reticulum (ER) stress, and impaired contractile function, all of which were prevented by pretreating animals with rapamycin (mTOR inhibition) or metformin (AMPK activation). The histone deacetylase inhibitor 4‐phenylbutyrate, which relieves ER stress, also improved contractile function. In contrast, adding the glucose analogue 2‐deoxy‐d‐glucose, which is phosphorylated but not further metabolized, to the perfusate resulted in mTOR activation and contractile dysfunction. Next we tested our hypothesis in vivo by transverse aortic constriction in mice. Using a micro‐PET system, we observed enhanced glucose tracer analog uptake and contractile dysfunction preceding dilatation of the left ventricle. In contrast, in hearts overexpressing SERCA2a, ER stress was reduced and contractile function was preserved with hypertrophy. Finally, we examined failing human hearts and found that mechanical unloading decreased G6P levels and ER stress markers. Conclusions We propose that glucose metabolic changes precede and regulate functional (and possibly also structural) remodeling of the heart. We implicate a critical role for G6P in load‐induced mTOR activation and ER stress. PMID:23686371

  4. Fluoxetine regulates mTOR signalling in a region-dependent manner in depression-like mice.

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    Liu, Xiao-Long; Luo, Liu; Mu, Rong-Hao; Liu, Bin-Bin; Geng, Di; Liu, Qing; Yi, Li-Tao

    2015-11-02

    Previous studies have demonstrated that the mammalian target of rapamycin (mTOR) signaling pathway has an important role in ketamine-induced, rapid antidepressant effects despite the acute administration of fluoxetine not affecting mTOR phosphorylation in the brain. However, the effects of long-term fluoxetine treatment on mTOR modulation have not been assessed to date. In the present study, we examined whether fluoxetine, a type of commonly used antidepressant agent, alters mTOR signaling following chronic administration in different brain regions, including the frontal cortex, hippocampus, amygdala and hypothalamus. We also investigated whether fluoxetine enhanced synaptic protein levels in these regions via the activation of the mTOR signaling pathway and its downstream regulators, p70S6K and 4E-BP-1. The results indicated that chronic fluoxetine treatment attenuated the chronic, unpredictable, mild stress (CUMS)-induced mTOR phosphorylation reduction in the hippocampus and amygdala of mice but not in the frontal cortex or the hypothalamus. Moreover, the CUMS-decreased PSD-95 and synapsin I levels were reversed by fluoxetine, and these effects were blocked by rapamycin only in the hippocampus. In conclusion, our findings suggest that chronic treatment with fluoxetine can induce synaptic protein expression by activating the mTOR signaling pathway in a region-dependent manner and mainly in the hippocampus.

  5. Different patterns of Akt and ERK feedback activation in response to rapamycin, active-site mTOR inhibitors and metformin in pancreatic cancer cells.

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    Soares, Heloisa P; Ni, Yang; Kisfalvi, Krisztina; Sinnett-Smith, James; Rozengurt, Enrique

    2013-01-01

    The mTOR pathway is aberrantly stimulated in many cancer cells, including pancreatic ductal adenocarcinoma (PDAC), and thus it is a potential target for therapy. However, the mTORC1/S6K axis also mediates negative feedback loops that attenuate signaling via insulin/IGF receptor and other tyrosine kinase receptors. Suppression of these feed-back loops unleashes over-activation of upstream pathways that potentially counterbalance the antiproliferative effects of mTOR inhibitors. Here, we demonstrate that treatment of PANC-1 or MiaPaCa-2 pancreatic cancer cells with either rapamycin or active-site mTOR inhibitors suppressed S6K and S6 phosphorylation induced by insulin and the GPCR agonist neurotensin. Rapamycin caused a striking increase in Akt phosphorylation at Ser(473) while the active-site inhibitors of mTOR (KU63794 and PP242) completely abrogated Akt phosphorylation at this site. Conversely, active-site inhibitors of mTOR cause a marked increase in ERK activation whereas rapamycin did not have any stimulatory effect on ERK activation. The results imply that first and second generation of mTOR inhibitors promote over-activation of different pro-oncogenic pathways in PDAC cells, suggesting that suppression of feed-back loops should be a major consideration in the use of these inhibitors for PDAC therapy. In contrast, metformin abolished mTORC1 activation without over-stimulating Akt phosphorylation on Ser(473) and prevented mitogen-stimulated ERK activation in PDAC cells. Metformin induced a more pronounced inhibition of proliferation than either KU63794 or rapamycin while, the active-site mTOR inhibitor was more effective than rapamycin. Thus, the effects of metformin on Akt and ERK activation are strikingly different from allosteric or active-site mTOR inhibitors in PDAC cells, though all these agents potently inhibited the mTORC1/S6K axis.

  6. Longitudinal imaging studies of tumor microenvironment in mice treated with the mTOR inhibitor rapamycin.

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    Keita Saito

    Full Text Available Rapamycin is an allosteric inhibitor of mammalian target of rapamycin, and inhibits tumor growth and angiogenesis. Recent studies suggested a possibility that rapamycin renormalizes aberrant tumor vasculature and improves tumor oxygenation. The longitudinal effects of rapamycin on angiogenesis and tumor oxygenation were evaluated in murine squamous cell carcinoma (SCCVII by electron paramagnetic resonance imaging (EPRI and magnetic resonance imaging (MRI to identify an optimal time after rapamycin treatment for enhanced tumor radioresponse. Rapamycin treatment was initiated on SCCVII solid tumors 8 days after implantation (500-750 mm(3 and measurements of tumor pO(2 and blood volume were conducted from day 8 to 14 by EPRI/MRI. Microvessel density was evaluated over the same time period by immunohistochemical analysis. Tumor blood volume as measured by MRI significantly decreased 2 days after rapamycin treatment. Tumor pO(2 levels modestly but significantly increased 2 days after rapamycin treatment; whereas, it decreased in non-treated control tumors. Furthermore, the fraction of hypoxic area (pixels with pO(2<10 mm Hg in the tumor region decreased 2 days after rapamycin treatments. Immunohistochemical analysis of tumor microvessel density and pericyte coverage revealed that microvessel density decreased 2 days after rapamycin treatment, but pericyte coverage did not change, similar to what was seen with anti-angiogenic agents such as sunitinib which cause vascular renormalization. Collectively, EPRI/MRI co-imaging can provide non-invasive evidence of rapamycin-induced vascular renormalization and resultant transient increase in tumor oxygenation. Improved oxygenation by rapamycin treatment provides a temporal window for anti-cancer therapies to realize enhanced response to radiotherapy.

  7. Impairment of object recognition memory by rapamycin inhibition of mTOR in the amygdala or hippocampus around the time of learning or reactivation.

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    Jobim, Paulo F C; Pedroso, Thiago R; Werenicz, Aline; Christoff, Raissa R; Maurmann, Natasha; Reolon, Gustavo K; Schröder, Nadja; Roesler, Rafael

    2012-03-01

    The role of the basolateral complex of the amygdala (BLA) in recognition memory remains poorly understood. The mammalian target of rapamycin (mTOR) in the BLA and other brain areas has been implicated in synaptic plasticity and memory. We have recently shown that mTOR signaling in both the BLA and the dorsal hippocampus (DH) is required for formation and reconsolidation of inhibitory avoidance, a fear-motivated memory task. Here we examined the effects of infusions of the mTOR inhibitor rapamycin into the BLA before or after either training or reactivation on retention of novel object recognition (NOR) memory in rats, and compared the effects with those obtained using intra-DH infusions. Male Wistar rats received bilateral infusions of vehicle or rapamycin into the BLA or DH before or after NOR training or reactivation. Rapamycin impaired NOR retention tested 24h after training when given either before or immediately after training into the BLA or DH. Rapamycin also impaired retention measured 24h after reactivation when infused before reactivation into the BLA or DH, or immediately after reactivation into the BLA, but not when given 6h after reactivation into either the BLA or DH. The results suggest that mTOR signaling in the BLA and DH is involved in NOR memory formation and stabilization. Copyright © 2011 Elsevier B.V. All rights reserved.

  8. Critical analysis of the potential for therapeutic targeting of mammalian target of rapamycin (mTOR in gastric cancer

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    Inokuchi M

    2014-04-01

    Full Text Available Mikito Inokuchi,1 Keiji Kato,1 Kazuyuki Kojima,2 Kenichi Sugihara1 1Department of Surgical Oncology, 2Department of Minimally Invasive Surgery, Tokyo Medical and Dental University, Tokyo, Japan Abstract: Multidisciplinary treatment including chemotherapy has become the global standard of care for patients with metastatic gastric cancer (mGC; nonetheless, survival remains poor. Although many molecular-targeted therapies have been developed for various cancers, only anti-HER2 treatment has produced promising results in patients with mGC. Mammalian target of rapamycin (mTOR plays a key role in cell proliferation, antiapoptosis, and metastasis in signaling pathways from the tyrosine kinase receptor, and its activation has been demonstrated in gastric cancer (GC cells. This review discusses the clinical relevance of mTOR in GC and examines its potential as a therapeutic target in patients with mGC. Preclinical studies in animal models suggest that suppression of the mTOR pathway inhibits the proliferation of GC cells and delays tumor progression. The mTOR inhibitor everolimus has been evaluated as second- or third-line treatment in clinical trials. Adverse events were well tolerated although the effectiveness of everolimus alone was limited. Everolimus is now being evaluated in combination with chemotherapy in Phase III clinical studies in this subgroup of patients. Two Phase III studies include exploratory biomarker research designed to evaluate the predictive value of the expression or mutation of molecules related to the Akt/mTOR signaling pathway. These biomarker studies may lead to the realization of targeted therapy for selected patients with mGC in the future. Keywords: gastric cancer, mTOR, everolimus

  9. Up-Regulation of the Excitatory Amino Acid Transporters EAAT1 and EAAT2 by Mammalian Target of Rapamycin

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    Abeer Abousaab

    2016-11-01

    Full Text Available Background: The excitatory amino-acid transporters EAAT1 and EAAT2 clear glutamate from the synaptic cleft and thus terminate neuronal excitation. The carriers are subject to regulation by various kinases. The EAAT3 isoform is regulated by mammalian target of rapamycin (mTOR. The present study thus explored whether mTOR influences transport by EAAT1 and/or EAAT2. Methods: cRNA encoding wild type EAAT1 (SLC1A3 or EAAT2 (SLC1A2 was injected into Xenopus oocytes without or with additional injection of cRNA encoding mTOR. Dual electrode voltage clamp was performed in order to determine electrogenic glutamate transport (IEAAT. EAAT2 protein abundance was determined utilizing chemiluminescence. Results: Appreciable IEAAT was observed in EAAT1 or EAAT2 expressing but not in water injected oocytes. IEAAT was significantly increased by coexpression of mTOR. Coexpression of mTOR increased significantly the maximal IEAAT in EAAT1 or EAAT2 expressing oocytes, without significantly modifying affinity of the carriers. Moreover, coexpression of mTOR increased significantly EAAT2 protein abundance in the cell membrane. Conclusions: The kinase mTOR up-regulates the excitatory amino acid transporters EAAT1 and EAAT2.

  10. mTOR: A Link from the Extracellular Milieu to Transcriptional Regulation of Oligodendrocyte Development

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    Teresa L. Wood

    2013-02-01

    Full Text Available Oligodendrocyte development is controlled by numerous extracellular signals that regulate a series of transcription factors that promote the differentiation of oligodendrocyte progenitor cells to myelinating cells in the central nervous system. A major element of this regulatory system that has only recently been studied is the intracellular signalling from surface receptors to transcription factors to down-regulate inhibitors and up-regulate inducers of oligodendrocyte differentiation and myelination. The current review focuses on one such pathway: the mTOR (mammalian target of rapamycin pathway, which integrates signals in many cell systems and induces cell responses including cell proliferation and cell differentiation. This review describes the known functions of mTOR as they relate to oligodendrocyte development, and its recently discovered impact on oligodendrocyte differentiation and myelination. A potential model for its role in oligodendrocyte development is proposed.

  11. mTOR mediates human trophoblast invasion through regulation of matrix-remodeling enzymes and is associated with serine phosphorylation of STAT3

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    Busch, Susann [Placenta-Lab, Department of Obstetrics, Friedrich-Schiller-University Jena, Bachstrasse 18, 07740 Jena (Germany); Renaud, Stephen J. [Department of Anatomy and Cell Biology, Queen' s University, Kingston, Ontario, Canada K7L3N6 (Canada); Schleussner, Ekkehard [Placenta-Lab, Department of Obstetrics, Friedrich-Schiller-University Jena, Bachstrasse 18, 07740 Jena (Germany); Graham, Charles H. [Department of Anatomy and Cell Biology, Queen' s University, Kingston, Ontario, Canada K7L3N6 (Canada); Markert, Udo R., E-mail: markert@med.uni-jena.de [Placenta-Lab, Department of Obstetrics, Friedrich-Schiller-University Jena, Bachstrasse 18, 07740 Jena (Germany)

    2009-06-10

    The intracellular signaling molecule mammalian target of rapamycin (mTOR) is essential for cell growth and proliferation. It is involved in mouse embryogenesis, murine trophoblast outgrowth and linked to tumor cell invasiveness. In order to assess the role of mTOR in human trophoblast invasion we analyzed the in vitro invasiveness of HTR-8/SVneo immortalized first-trimester trophoblast cells in conjunction with enzyme secretion upon mTOR inhibition and knockdown of mTOR protein expression. Additionally, we also tested the capability of mTOR to trigger signal transducer and activator of transcription (STAT)-3 by its phosphorylation status. Rapamycin inhibited mTOR kinase activity as demonstrated with a lower phosphorylation level of the mTOR substrate p70 S6 kinase (S6K). With the use of rapamycin and siRNA-mediated mTOR knockdown we could show that cell proliferation, invasion and secretion of matrix-metalloproteinases (MMP)-2 and -9, urokinase-like plasminogen activator (uPA) and its major physiological uPA inhibitor (PAI)-1 were inhibited. While tyrosine phosphorylation of STAT3 was unaffected by mTOR inhibition and knockdown, serine phosphorylation was diminished. We conclude that mTOR signaling is one major mechanism in a tightly regulated network of intracellular signal pathways including the JAK/STAT system to regulate invasion in human trophoblast cells by secretion of enzymes that remodel the extra-cellular matrix (ECM) such as MMP-2, -9, uPA and PAI-1. Dysregulation of mTOR may contribute to pregnancy-related pathologies caused through impaired trophoblast invasion.

  12. Rapamycin Loaded Solid Lipid Nanoparticles as a New Tool to Deliver mTOR Inhibitors: Formulation and in Vitro Characterization.

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    Polchi, Alice; Magini, Alessandro; Mazuryk, Jarosław; Tancini, Brunella; Gapiński, Jacek; Patkowski, Adam; Giovagnoli, Stefano; Emiliani, Carla

    2016-05-09

    Recently, the use of mammalian target of rapamycin (mTOR) inhibitors, in particular rapamycin (Rp), has been suggested to improve the treatment of neurodegenerative diseases. However, as Rp is a strong immunosuppressant, specific delivery to the brain has been postulated to avoid systemic exposure. In this work, we fabricated new Rp loaded solid lipid nanoparticles (Rp-SLN) stabilized with polysorbate 80 (PS80), comparing two different methods and lipids. The formulations were characterized by differential scanning calorimetry (DSC), nuclear magnetic resonance (NMR), wide angle X-ray scattering (WAXS), cryo-transmission electron microscopy (cryo-TEM), dynamic light scattering (DLS) and particle tracking. In vitro release and short-term stability were assessed. Biological behavior of Rp-SLN was tested in SH-SY5Y neuroblastoma cells. The inhibition of mTOR complex 1 (mTORC1) was evaluated over time by a pulse-chase study compared to free Rp and Rp nanocrystals. Compritol Rp-SLN resulted more stable and possessing proper size and surface properties with respect to cetyl palmitate Rp-SLN. Rapamycin was entrapped in an amorphous form in the solid lipid matrix that showed partial crystallinity with stable Lβ, sub-Lα and Lβ' arrangements. PS80 was stably anchored on particle surface. No drug release was observed over 24 h and Rp-SLN had a higher cell uptake and a more sustained effect over a week. The mTORC1 inhibition was higher with Rp-SLN. Overall, compritol Rp-SLN show suitable characteristics and stability to be considered for further investigation as Rp brain delivery system.

  13. Rapamycin Loaded Solid Lipid Nanoparticles as a New Tool to Deliver mTOR Inhibitors: Formulation and in Vitro Characterization

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    Alice Polchi

    2016-05-01

    Full Text Available Recently, the use of mammalian target of rapamycin (mTOR inhibitors, in particular rapamycin (Rp, has been suggested to improve the treatment of neurodegenerative diseases. However, as Rp is a strong immunosuppressant, specific delivery to the brain has been postulated to avoid systemic exposure. In this work, we fabricated new Rp loaded solid lipid nanoparticles (Rp-SLN stabilized with polysorbate 80 (PS80, comparing two different methods and lipids. The formulations were characterized by differential scanning calorimetry (DSC, nuclear magnetic resonance (NMR, wide angle X-ray scattering (WAXS, cryo-transmission electron microscopy (cryo-TEM, dynamic light scattering (DLS and particle tracking. In vitro release and short-term stability were assessed. Biological behavior of Rp-SLN was tested in SH-SY5Y neuroblastoma cells. The inhibition of mTOR complex 1 (mTORC1 was evaluated over time by a pulse-chase study compared to free Rp and Rp nanocrystals. Compritol Rp-SLN resulted more stable and possessing proper size and surface properties with respect to cetyl palmitate Rp-SLN. Rapamycin was entrapped in an amorphous form in the solid lipid matrix that showed partial crystallinity with stable Lβ, sub-Lα and Lβ′ arrangements. PS80 was stably anchored on particle surface. No drug release was observed over 24 h and Rp-SLN had a higher cell uptake and a more sustained effect over a week. The mTORC1 inhibition was higher with Rp-SLN. Overall, compritol Rp-SLN show suitable characteristics and stability to be considered for further investigation as Rp brain delivery system.

  14. Cross-talk between AMPK and mTOR in regulating energy balance.

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    Xu, Jia; Ji, Jian; Yan, Xiang-Hua

    2012-01-01

    Energy balance is maintained by a complex homeostatic system involving some signaling pathways and "nutrient sensors" in multiple tissues and organs. Any defect associated with the pathways can lead to metabolic disorders including obesity, type 2 diabetes, and the metabolic syndrome. The 5'-adenosine monophosphate-activated protein kinase (AMPK) and mammalian target of rapamycin (mTOR) appear to play a significant role in the intermediary metabolism of these diseases. AMPK is involved in the fundamental regulation of energy balance at the whole body level by responding to hormonal and nutrient signals in the central nervous system and peripheral tissues that modulate food intake and energy expenditure. Mammalian target of rapamycin (mTOR),is one of the downstream targets of AMPK functions as an intracellular nutrient sensor to control protein synthesis, cell growth, and metabolism. Recent research demonstrated the possible interplay between mTOR and AMPK signaling pathways. In this review, we will present current knowledge of AMPK and mTOR pathways in regulating energy balance and demonstrate the convergence between these two pathways.

  15. Translational regulation of GPx-1 and GPx-4 by the mTOR pathway.

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    Emily N Reinke

    Full Text Available Glutathione peroxidase activity was previously determined to be elevated in lymphocytes obtained from patients treated with the Bcr-Abl kinase inhibitor imatinib mesylate. In order to expand upon this observation, the established chronic myelogenous leukemia cell lines KU812 and MEG-01 were treated with imatinib and the effect on several anti-oxidant proteins was determined. The levels of GPx-1 were significantly increased following treatment with imatinib. This increase was not due to altered steady-state mRNA levels, and appeared to be dependent on the expression of Bcr-Abl, as no increases were observed following imatinib treatment of cells that did not express the fusion protein. The nutrient-sensing signaling protein, mammalian target of rapamycin (mTOR, can be activated by Bcr-Abl and its activity regulates the translation of many different proteins. Treatment of those same cells used in the imatinib studies with rapamycin, an inhibitor of mTOR, resulted in elevated GPx-1 and GPx-4 protein levels independent of Bcr-Abl expression. These proteins all belong to the selenoprotein family of peptides that contain the UGA-encoded amino acid selenocysteine. Collectively, these data provide evidence of a novel means of regulating anti-oxidants of the selenoprotein family via the mTOR pathway.

  16. mTOR inhibitor rapamycin induce polymorphonuclear myeloid-derived suppressor cells mobilization and function in protecting against acute graft-versus-host disease after bone marrow transplantation.

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    Lin, Yu; Wang, Binsheng; Shan, Wei; Tan, Yamin; Feng, Jingjing; Xu, Lin; Wang, Limengmeng; Han, Biqing; Zhang, Mingming; Yu, Jian; Yu, Xiaohong; Huang, He

    2017-11-10

    The mammalian target of rapamycin (mTOR) inhibitor rapamycin (RAPA) has been shown to be an effective immunosuppressor in the management of acute graft-versus-host disease (aGVHD) after bone marrow transplantation. Myeloid-derived suppressor cells (MDSCs) also have a protective effect in aGVHD regulation. However, the relationship between RAPA and MDSCs in aGVHD models is unclear. Meanwhile, the effect of RAPA on different subgroups of MDSCs is also less well described. In this study, we demonstrate that in vivo administration of RAPA results in the expansion and functional enhancement of polymorphonuclear MDSCs (PMN-MDSCs) in a murine model of aGVHD. RAPA treatment can enhance the suppressive function of PMN-MDSCs via up-regulation of arginase1 (Arg1) and induced nitric oxide synthase (iNOS) at later time points. Moreover, RAPA can also induce a strong immunosuppressive function in PMN-MDSCs from murine bone marrow in vitro, but has a contrary effect on monocytic MDSCs (M-MDSCs). We found that RAPA-treated PMN-MDSCs can restrain the differentiation of Th1/Th2 cells and promote induction of regulatory T cells in in vitro studies. Copyright © 2017 Elsevier Inc. All rights reserved.

  17. BDNF activates mTOR to regulate GluR1 expression required for memory formation.

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    Leandro Slipczuk

    Full Text Available BACKGROUND: The mammalian target of Rapamycin (mTOR kinase plays a key role in translational control of a subset of mRNAs through regulation of its initiation step. In neurons, mTOR is present at the synaptic region, where it modulates the activity-dependent expression of locally-translated proteins independently of mRNA synthesis. Indeed, mTOR is necessary for different forms of synaptic plasticity and long-term memory (LTM formation. However, little is known about the time course of mTOR activation and the extracellular signals governing this process or the identity of the proteins whose translation is regulated by this kinase, during mnemonic processing. METHODOLOGY/PRINCIPAL FINDINGS: Here we show that consolidation of inhibitory avoidance (IA LTM entails mTOR activation in the dorsal hippocampus at the moment of and 3 h after training and is associated with a rapid and rapamycin-sensitive increase in AMPA receptor GluR1 subunit expression, which was also blocked by intra-hippocampal delivery of GluR1 antisense oligonucleotides (ASO. In addition, we found that pre- or post-training administration of function-blocking anti-BDNF antibodies into dorsal CA1 hampered IA LTM retention, abolished the learning-induced biphasic activation of mTOR and its readout, p70S6K and blocked GluR1 expression, indicating that BDNF is an upstream factor controlling mTOR signaling during fear-memory consolidation. Interestingly, BDNF ASO hindered LTM retention only when given into dorsal CA1 1 h after but not 2 h before training, suggesting that BDNF controls the biphasic requirement of mTOR during LTM consolidation through different mechanisms: an early one involving BDNF already available at the moment of training, and a late one, happening around 3 h post-training that needs de novo synthesis of this neurotrophin. CONCLUSIONS/SIGNIFICANCE: IN CONCLUSION, OUR FINDINGS DEMONSTRATE THAT: 1 mTOR-mediated mRNA translation is required for memory consolidation during

  18. PREDICTION OF THE COURSE OF OSTEOARTHROSIS FROM mTOR (MAMMALIAN TARGET OF RAPAMYCIN GENE EXPRESSION

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    E V Chetina

    2012-01-01

    Results. Analysis of gene expression in the outpatients with OA identified two subgroups: in one subgroup (n = 13 mTOR expression was considerably much less than that in the control group; the expression of ATG1 and p21 did not differ greatly from the control and that of caspase 3 and TNF-α was significantly higher. The other outpatients (n = 20 and all the examined patients needing endoprosthetic replacement were ascertained to have a higher gene expression of mTOR, ATG1, p21, caspase 3, and TNF-α than in the control group. Before endoprosthetic replacement, severe joint destruction in patients with OA was associated with enhanced gene expression of mTOR, ATG1, p21, and caspase 3. Conclusion. In early-stage disease, increased mTOR gene expression may serve as a prognostic marker of the severity of the disease and articular cartilage destruction.

  19. The interaction between early life epilepsy and autistic-like behavioral consequences: a role for the mammalian target of rapamycin (mTOR pathway.

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    Delia M Talos

    Full Text Available Early life seizures can result in chronic epilepsy, cognitive deficits and behavioral changes such as autism, and conversely epilepsy is common in autistic children. We hypothesized that during early brain development, seizures could alter regulators of synaptic development and underlie the interaction between epilepsy and autism. The mammalian Target of Rapamycin (mTOR modulates protein translation and is dysregulated in Tuberous Sclerosis Complex, a disorder characterized by epilepsy and autism. We used a rodent model of acute hypoxia-induced neonatal seizures that results in long term increases in neuronal excitability, seizure susceptibility, and spontaneous seizures, to determine how seizures alter mTOR Complex 1 (mTORC1 signaling. We hypothesized that seizures occurring at a developmental stage coinciding with a critical period of synaptogenesis will activate mTORC1, contributing to epileptic networks and autistic-like behavior in later life. Here we show that in the rat, baseline mTORC1 activation peaks during the first three postnatal weeks, and induction of seizures at postnatal day 10 results in further transient activation of its downstream targets phospho-4E-BP1 (Thr37/46, phospho-p70S6K (Thr389 and phospho-S6 (Ser235/236, as well as rapid induction of activity-dependent upstream signaling molecules, including BDNF, phospho-Akt (Thr308 and phospho-ERK (Thr202/Tyr204. Furthermore, treatment with the mTORC1 inhibitor rapamycin immediately before and after seizures reversed early increases in glutamatergic neurotransmission and seizure susceptibility and attenuated later life epilepsy and autistic-like behavior. Together, these findings suggest that in the developing brain the mTORC1 signaling pathway is involved in epileptogenesis and altered social behavior, and that it may be a target for development of novel therapies that eliminate the progressive effects of neonatal seizures.

  20. mTOR REGULATES TAU PHOSPHORYLATION AND DEGRADATION: IMPLICATIONS FOR ALZHEIMER’S DISEASE AND OTHER TAUOPATHIES

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    Caccamo, Antonella; Magrì, Andrea; Medina, David X.; Wisely, Elena V.; López-Aranda, Manuel F.; Silva, Alcino J.; Oddo, Salvatore

    2013-01-01

    SUMMARY Accumulation of tau is a critical event in several neurodegenerative disorders, collectively known as tauopathies, which include Alzheimer’s disease and frontotemporal dementia. Pathological tau is hyperphosphorylated and aggregates to form neurofibrillary tangles. The molecular mechanisms leading to tau accumulation remain unclear and more needs to be done to elucidate them. Age is a major risk factor for all tauopathies, suggesting that molecular changes contributing to the aging process may facilitate tau accumulation and represent common mechanisms across different tauopathies. Here, we use multiple animal models and complementary genetic and pharmacological approaches to show that the mammalian target of rapamycin (mTOR) regulates tau phosphorylation and degradation. Specifically, we show that genetically increasing mTOR activity elevates endogenous mouse tau levels and phosphorylation. Complementary to it, we further demonstrate that pharmacologically reducing mTOR signaling with rapamycin ameliorates tau pathology and the associated behavioral deficits in a mouse model overexpressing mutant human tau. Mechanistically, we provide compelling evidence that the association between mTOR and tau is linked to GSK3β and autophagy function. In summary, we show that increasing mTOR signaling facilitates tau pathology while reducing mTOR signaling ameliorates tau pathology. Given the overwhelming evidence showing that reducing mTOR signaling increases lifespan and health span, the data presented here have profound clinical implications for aging and tauopathies and provide the molecular basis for how aging may contribute to tau pathology. Additionally, these results provide pre-clinical data indicating that reducing mTOR signaling may be a valid therapeutic approach for tauopathies. PMID:23425014

  1. Lysophosphatidic acid acyltransferase beta regulates mTOR signaling.

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    Michelle A Blaskovich

    Full Text Available Lysophosphatidic acid acyltransferase (LPAAT-β is a phosphatidic acid (PA generating enzyme that plays an essential role in triglyceride synthesis. However, LPAAT-β is now being studied as an important regulator of cell growth and differentiation and as a potential therapeutic target in cancer since PA is necessary for the activity of key proteins such as Raf, PKC-ζ and mTOR. In this report we determine the effect of LPAAT-β silencing with siRNA in pancreatic adenocarcinoma cell lines. We show for the first time that LPAAT-β knockdown inhibits proliferation and anchorage-independent growth of pancreatic cancer cells. This is associated with inhibition of signaling by mTOR as determined by levels of mTORC1- and mTORC2-specific phosphorylation sites on 4E-BP1, S6K and Akt. Since PA regulates the activity of mTOR by modulating its binding to FKBP38, we explored the possibility that LPAAT-β might regulate mTOR by affecting its association with FKBP38. Coimmunoprecipitation studies of FKBP38 with mTOR show increased levels of FKBP38 associated with mTOR when LPAAT-β protein levels are knocked down. Furthermore, depletion of LPAAT-β results in increased Lipin 1 nuclear localization which is associated with increased nuclear eccentricity, a nuclear shape change that is dependent on mTOR, further confirming the ability of LPAAT-β to regulate mTOR function. Our results provide support for the hypothesis that PA generated by LPAAT-β regulates mTOR signaling. We discuss the implications of these findings for using LPAAT-β as a therapeutic target.

  2. Regulation of mTOR activity in Snell dwarf and GH receptor gene-disrupted mice.

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    Dominick, Graham; Berryman, Darlene E; List, Edward O; Kopchick, John J; Li, Xinna; Miller, Richard A; Garcia, Gonzalo G

    2015-02-01

    The involvement of mammalian target of rapamycin (mTOR) in lifespan control in invertebrates, calorie-restricted rodents, and extension of mouse lifespan by rapamycin have prompted speculation that diminished mTOR function may contribute to mammalian longevity in several settings. We show here that mTOR complex-1 (mTORC1) activity is indeed lower in liver, muscle, heart, and kidney tissue of Snell dwarf and global GH receptor (GHR) gene-disrupted mice (GHR-/-), consistent with previous studies. Surprisingly, activity of mTORC2 is higher in fasted Snell and GHR-/- than in littermate controls in all 4 tissues tested. Resupply of food enhanced mTORC1 activity in both controls and long-lived mutant mice but diminished mTORC2 activity only in the long-lived mice. Mice in which GHR has been disrupted only in the liver do not show extended lifespan and also fail to show the decline in mTORC1 and increase in mTORC2 seen in mice with global loss of GHR. The data suggest that the antiaging effects in the Snell dwarf and GHR-/- mice are accompanied by both a decline in mTORC1 in multiple organs and an increase in fasting levels of mTORC2. Neither the lifespan nor mTOR effects appear to be mediated by direct GH effects on liver or by the decline in plasma IGF-I, a shared trait in both global and liver-specific GHR-/- mice. Our data suggest that a more complex pattern of hormonal effects and intertissue interactions may be responsible for regulating both lifespan and mTORC2 function in these mouse models of delayed aging.

  3. ATP-Competitive Inhibitors of the Mammalian Target of Rapamycin: Design and Synthesis of Highly Potent and Selective Pyrazolopyrimidines

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    Zask, Arie; Verheijen, Jeroen C.; Curran, Kevin; Kaplan, Joshua; Richard, David J.; Nowak, Pawel; Malwitz, David J.; Brooijmans, Natasja; Bard, Joel; Svenson, Kristine; Lucas, Judy; Toral-Barza, Lourdes; Zhang, Wei-Guo; Hollander, Irwin; Gibbons, James J.; Abraham, Robert T.; Ayral-Kaloustian, Semiramis; Mansour, Tarek S.; Yu, Ker; (Wyeth)

    2009-09-18

    The mammalian target of rapamycin (mTOR), a central regulator of growth, survival, and metabolism, is a validated target for cancer therapy. Rapamycin and its analogues, allosteric inhibitors of mTOR, only partially inhibit one mTOR protein complex. ATP-competitive, global inhibitors of mTOR that have the potential for enhanced anticancer efficacy are described. Structural features leading to potency and selectivity were identified and refined leading to compounds with in vivo efficacy in tumor xenograft models.

  4. Clinical significance of chemokine receptor CXCR4 and mammalian target of rapamycin (mTOR) expression in patients with diffuse large B-cell lymphoma.

    Science.gov (United States)

    Xu, Zi-Zhen; Shen, Jian-Kang; Zhao, Shu-Qing; Li, Jun-Min

    2017-09-27

    To assess the relevance of C-X-C chemokine receptor type 4 (CXCR4) and mammalian target of rapamycin (mTOR) to large-B-cell lymphoma (DLBCL), levels of protein expression were measured in 56 DLBCL patients who had received rituximab-based therapy. Of these, 34 were positive for CXCR4 expression (60.7%) and 31 for mTOR (55.4%). CXCR4 expression was positively correlated with mTOR expression (r = 0.602; p = .000). CXCR4 expression was significantly associated with high lactate dehydrogenase (LDH) level (p = .009), high IPI score (p = .030) and non-GCB subtype (p = .006). Furthermore, the expression levels of CXCR4 and mTOR were negatively correlated with the chance of remission (p < .05). Kaplan-Meier analysis indicated significantly shorter progression-free survival (PFS) and overall survival (OS) in patients positive for CXCR4 and mTOR expression. The combination therapy with CXCR4 inhibitor WZ811 and mTOR inhibitor everolimus showed syncergistic effect in DLBCL cell lines. These results suggest that the expression of CXCR4 and mTOR may be suitable as biomarkers of the prognosis of DLBCL and for development of new therapeutic strategies.

  5. The role of mTOR signaling in the regulation of protein synthesis and muscle mass during immobilization in mice.

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    You, Jae-Sung; Anderson, Garrett B; Dooley, Matthew S; Hornberger, Troy A

    2015-09-01

    The maintenance of skeletal muscle mass contributes substantially to health and to issues associated with the quality of life. It has been well recognized that skeletal muscle mass is regulated by mechanically induced changes in protein synthesis, and that signaling by mTOR is necessary for an increase in protein synthesis and the hypertrophy that occurs in response to increased mechanical loading. However, the role of mTOR signaling in the regulation of protein synthesis and muscle mass during decreased mechanical loading remains largely undefined. In order to define the role of mTOR signaling, we employed a mouse model of hindlimb immobilization along with pharmacological, mechanical and genetic means to modulate mTOR signaling. The results first showed that immobilization induced a decrease in the global rates of protein synthesis and muscle mass. Interestingly, immobilization also induced an increase in mTOR signaling, eIF4F complex formation and cap-dependent translation. Blocking mTOR signaling during immobilization with rapamycin not only impaired the increase in eIF4F complex formation, but also augmented the decreases in global protein synthesis and muscle mass. On the other hand, stimulating immobilized muscles with isometric contractions enhanced mTOR signaling and rescued the immobilization-induced decrease in global protein synthesis through a rapamycin-sensitive mechanism that was independent of ribosome biogenesis. Unexpectedly, the effects of isometric contractions were also independent of eIF4F complex formation. Similar to isometric contractions, overexpression of Rheb in immobilized muscles enhanced mTOR signaling, cap-dependent translation and global protein synthesis, and prevented the reduction in fiber size. Therefore, we conclude that the activation of mTOR signaling is both necessary and sufficient to alleviate the decreases in protein synthesis and muscle mass that occur during immobilization. Furthermore, these results indicate that the

  6. Carboxy terminal tail of polycystin-1 regulates localization of TSC2 to repress mTOR.

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    Ruhee Dere

    2010-02-01

    Full Text Available Autosomal dominant polycystic kidney disease (ADPKD is a commonly inherited renal disorder caused by defects in the PKD1 or PKD2 genes. ADPKD is associated with significant morbidity, and is a major underlying cause of end-stage renal failure (ESRF. Commonly, treatment options are limited to the management of hypertension, cardiovascular risk factors, dialysis, and transplantation when ESRF develops, although several new pharmacotherapies, including rapamycin, have shown early promise in animal and human studies. Evidence implicates polycystin-1 (PC-1, the gene product of the PKD1 gene, in regulation of the mTOR pathway. Here we demonstrate a mechanism by which the intracellular, carboxy-terminal tail of polycystin-1 (CP1 regulates mTOR signaling by altering the subcellular localization of the tuberous sclerosis complex 2 (TSC2 tumor suppressor, a gatekeeper for mTOR activity. Phosphorylation of TSC2 at S939 by AKT causes partitioning of TSC2 away from the membrane, its GAP target Rheb, and its activating partner TSC1 to the cytosol via 14-3-3 protein binding. We found that TSC2 and a C-terminal polycystin-1 peptide (CP1 directly interact and that a membrane-tethered CP1 protects TSC2 from AKT phosphorylation at S939, retaining TSC2 at the membrane to inhibit the mTOR pathway. CP1 decreased binding of 14-3-3 proteins to TSC2 and increased the interaction between TSC2 and its activating partner TSC1. Interestingly, while membrane tethering of CP1 was required to activate TSC2 and repress mTOR, the ability of CP1 to inhibit mTOR signaling did not require primary cilia and was independent of AMPK activation. These data identify a unique mechanism for modulation of TSC2 repression of mTOR signaling via membrane retention of this tumor suppressor, and identify PC-1 as a regulator of this downstream component of the PI3K signaling cascade.

  7. Synergistic inhibition of human melanoma proliferation by combination treatment with B-Raf inhibitor BAY43-9006 and mTOR inhibitor Rapamycin

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    Slingluff Craig L

    2005-10-01

    Full Text Available Abstract Background Targeted inhibition of protein kinases is now acknowledged as an effective approach for cancer therapy. However, targeted therapies probably have limited success because cancer cells have alternate pathways for survival and proliferation thereby avoiding inhibition. We tested the hypothesis that combination of targeted agents would be more effective than single agents in arresting melanoma cell proliferation. Methods We evaluated whether BAY43-9006, an inhibitor of the B-Raf kinase, and rapamycin, an inhibitor of the mTOR kinase, would inhibit serum-stimulated proliferation of human melanoma cell lines, either alone or in combination. Proliferation was measured by quantitating melanoma cell numbers with a luciferase for ATP. Phosphorylation of proteins downstream of targeted kinase(s was assayed by immunoblots. Statistical significance was determined with the Student-T test. Isobologram analysis was performed to distinguish additive versus synergistic effects of combinations of drugs. Results Serum-stimulated proliferation of multiple human melanoma cell lines was inhibited by BAY43-9006 and by rapamycin. Melanoma cells containing the B-Raf mutation V599E were more sensitive than cells with wild-type B-raf to 10 nM doses of both BAY43-9006 and rapamycin. Regardless of B-Raf mutational status, the combination of low dose rapamycin and BAY43-9006 synergistically inhibited melanoma cell proliferation. As expected, rapamycin inhibited the phosphorylation of mTOR substrates, p70S6K and 4EBP1, and BAY43-9006 inhibited phosphorylation of ERK, which is dependent on B-Raf activity. We also observed unexpected rapamycin inhibition of the phosphorylation of ERK, as well as BAY43-9006 inhibition of the phosphorylation of mTOR substrates, p70S6K and 4EBP1. Conclusion There was synergistic inhibition of melanoma cell proliferation by the combination of rapamycin and BAY 43-9006, and unexpected inhibition of two signaling pathways by agents

  8. eIF4E-Overexpression imparts perillyl alcohol and rapamycin-mediated regulation of telomerase reverse transcriptase.

    Science.gov (United States)

    Sundin, Tabetha; Peffley, Dennis; Hentosh, Patricia

    2013-08-01

    Translation is mediated partly by regulation of free eukaryotic initiation factor 4E (eIF4E) levels through PI3K-Akt-mTOR signaling. Cancer cells treated with the plant-derived perillyl alcohol (POH) or the mechanistic target of rapamycin (mTOR) inhibitor rapamycin dephosphorylate eIF4E-binding protein (4E-BP1) and attenuate cap-dependent translation. We previously showed in cancer cell lines with elevated eIF4E that POH and rapamycin regulate telomerase activity through this pathway. Here, immortalized Chinese hamster ovary (CHO) control cells and CHO cells with forced eIF4E expression (rb4E) were used to elucidate eIF4E's role in telomerase regulation by POH and rapamycin. Despite 5-fold higher eIF4E amounts in rb4E, telomerase activity, telomerase reverse transcriptase (TERT) mRNA, and TERT protein were nearly equivalent in control and rb4E cells. In control cells, telomerase activity, TERT mRNA and protein levels were unaffected by either compound. In contrast, telomerase activity and TERT protein were both attenuated by either agent in rb4E cells, but without corresponding TERT mRNA decreases indicating a translational/post-translational process. S6K, Akt, and 4E-BP1 were modulated by mTOR mediators only in the presence of increased eIF4E. Thus, eIF4E-overexpression in rb4E cells enables inhibitory effects of POH and rapamycin on telomerase and TERT protein. Importantly, eIF4E-overexpression modifies cellular protein synthetic processes and gene regulation. Copyright © 2013 Elsevier Inc. All rights reserved.

  9. Rapamycin inhibits IGF-1-mediated up-regulation of MDM2 and sensitizes cancer cells to chemotherapy.

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    Wei Du

    Full Text Available The Murine Double Minute 2 (MDM2 protein is a key regulator of cell proliferation and apoptosis that acts primarily by inhibiting the p53 tumor suppressor. Similarly, the PI3-Kinase (PI3K/AKT pathway is critical for growth factor-mediated cell survival. Additionally, it has been reported that AKT can directly phosphorylate and activate MDM2. In this study, we show that IGF-1 up-regulates MDM2 protein levels in a PI3K/AKT-dependent manner. Inhibition of mTOR by rapamycin or expression of a dominant negative eukaryotic initiation factor 4E binding protein 1 (4EBP1 mutant protein, as well as ablation of eukaryotic initiation factor 4E (eIF4E, efficiently abolishes IGF-1-mediated up-regulation of MDM2. In addition, we show that rapamycin effectively inhibits MDM2 expression and sensitizes cancer cells to chemotherapy. Taken together, this study reveals a novel mechanism by which IGF-1 activates MDM2 via the mTOR pathway, and that pharmacologic inhibition of mTOR combined with chemotherapy may be more effective in treatment of a subset of cancers harboring increased MDM2 activation.

  10. Bone Size and Quality Regulation: Concerted Actions of mTOR in Mesenchymal Stromal Cells and Osteoclasts

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    Hongguang Wu

    2017-06-01

    Full Text Available The bone size and quality, acquired during adolescent growth under the influence of anabolic hormones, growth factors, and nutrients, determine the height and bone stability and forecast osteoporosis risks in late life. Yet bone size and quality control mechanisms remain enigmatic. To study the roles of mammalian target of rapamycin (mTOR signaling, sensor of growth factors and nutrients, in bone size and quality regulation, we ablated Tsc1, a suppressor of mTOR, in mesenchymal stromal cells (MSCs, monocytes, or their progenies osteoblasts and osteoclasts. mTOR activation in MSCs, but much less in osteoblasts, increased bone width and mass due to MSC hyperproliferation, but decreased bone length and mineral contents due to defective MSC differentiation. mTOR activation promotes bone mineral accretion by inhibiting osteoclast differentiation and activity directly or via coupling with MSCs. Tuberous sclerosis complex patient studies confirmed these findings. Thus, mTOR regulates bone size via MSCs and bone quality by suppressing catabolic activities of osteoclasts.

  11. [Role of the mTOR pathway in the central regulation of energy balance].

    Science.gov (United States)

    Haissaguerre, Magalie; Cota, Daniela

    2015-01-01

    The pathway of the mammalian (or mechanistic) target of rapamycin kinase (mTOR) responds to different signals such as nutrients and hormones and regulates many cellular functions as the synthesis of proteins and lipids, mitochondrial activity and the organization of the cytoskeleton. At the cellular level, mTOR forms two distinct complexes: mTORC1 and mTORC2. This review intends to summarize the various recent advances on the role of these two protein complexes in the central regulation of energy balance. mTORC1 activity modulates energy balance and metabolic responses by regulating the activity of neuronal populations, such as those located in the arcuate nucleus of the hypothalamus. Recent studies have shown that activity of the hypothalamic mTORC1 pathway varies according to cell and stimulus types, and that this signaling cascade regulates food intake and body weight in response to nutrients, such as leucine, and hormones like leptin, ghrelin and triiodothyronine. On the other hand, mTORC2 seems to be involved in the regulation of neuronal morphology and synaptic activity. However, its function in the central regulation of the energy balance is less known. Dysregulation of mTORC1 and mTORC2 is described in obesity and type 2 diabetes. Therefore, a better understanding of the molecular mechanisms involved in the regulation of energy balance by mTOR may lead to the identification of new therapeutic targets for the treatment of these metabolic pathologies. © Société de Biologie, 2016.

  12. Dysregulation of Mammalian Target of Rapamycin Signaling in Mouse Models of Autism

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    Huber, Kimberly M.; Klann, Eric; Costa-Mattioli, Mauro; Zukin, R. Suzanne

    2015-01-01

    The mammalian target of rapamycin (mTOR) is a central regulator of a diverse array of cellular processes, including cell growth, proliferation, autophagy, translation, and actin polymerization. Components of the mTOR cascade are present at synapses and influence synaptic plasticity and spine morphogenesis. A prevailing view is that the study of mTOR and its role in autism spectrum disorders (ASDs) will elucidate the molecular mechanisms by which mTOR regulates neuronal function under physiolo...

  13. Regulation of autophagy by amino acids and MTOR-dependent signal transduction

    NARCIS (Netherlands)

    Meijer, Alfred J.; Lorin, Séverine; Blommaart, Edward F.; Codogno, Patrice

    2015-01-01

    Amino acids not only participate in intermediary metabolism but also stimulate insulin-mechanistic target of rapamycin (MTOR)-mediated signal transduction which controls the major metabolic pathways. Among these is the pathway of autophagy which takes care of the degradation of long-lived proteins

  14. PRKCI negatively regulates autophagy via PIK3CA/AKT–MTOR signaling

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    Qu, Liujing; Li, Ge; Xia, Dan; Hongdu, Beiqi; Xu, Chentong; Lin, Xin [Key Laboratory of Medical Immunology, Ministry of Health, Peking University Health Sciences Center, Beijing (China); Peking University Center for Human Disease Genomics, Peking University, Beijing (China); Chen, Yingyu, E-mail: yingyu_chen@bjmu.edu.cn [Key Laboratory of Medical Immunology, Ministry of Health, Peking University Health Sciences Center, Beijing (China); Peking University Center for Human Disease Genomics, Peking University, Beijing (China)

    2016-02-05

    The atypical protein kinase C isoform PRKC iota (PRKCI) plays a key role in cell proliferation, differentiation, and carcinogenesis, and it has been shown to be a human oncogene. Here, we show that PRKCI overexpression in U2OS cells impaired functional autophagy in normal or cell stress conditions, as characterized by decreased levels of light chain 3B-II protein (LC3B-II) and weakened degradation of endogenous and exogenous autophagic substrates. Conversely, PRKCI knockdown by small interference RNA resulted in opposite effects. Additionally, we identified two novel PRKCI mutants, PRKCI{sup L485M} and PRKCI{sup P560R}, which induced autophagy and exhibited dominant negative effects. Further studies indicated that PRKCI knockdown–mediated autophagy was associated with the inactivation of phosphatidylinositol 3-kinase alpha/AKT–mammalian target of rapamycin (PIK3CA/AKT–MTOR) signaling. These data underscore the importance of PRKCI in the regulation of autophagy. Moreover, the finding may be useful in treating PRKCI-overexpressing carcinomas that are characterized by increased levels of autophagy. - Highlights: • The atypical protein kinase C iota isoform (PRKCI) is a human oncogene. • PRKCI overexpression impairs functional autophagy in U2OS cells. • It reduces LC3B-II levels and weakens SQSTM1 and polyQ80 aggregate degradation. • PRKCI knockdown has the opposite effect. • The effect of PRKCI knockdown is related to PIK3CA/AKT–MTOR signaling inactivation.

  15. Platelet-derived growth factor regulates vascular smooth muscle phenotype via mammalian target of rapamycin complex 1

    Energy Technology Data Exchange (ETDEWEB)

    Ha, Jung Min; Yun, Sung Ji; Kim, Young Whan; Jin, Seo Yeon; Lee, Hye Sun [Medical Research Institute, Department of Pharmacology, Pusan National University School of Medicine, Yangsan (Korea, Republic of); Song, Sang Heon [Department of Internal Medicine, Pusan National University Hospital, Busan (Korea, Republic of); Shin, Hwa Kyoung [Department of Anatomy, Pusan National University School of Korean Medicine, Yangsan (Korea, Republic of); Bae, Sun Sik, E-mail: sunsik@pusan.ac.kr [Medical Research Institute, Department of Pharmacology, Pusan National University School of Medicine, Yangsan (Korea, Republic of)

    2015-08-14

    Mammalian target of rapamycin complex (mTORC) regulates various cellular processes including proliferation, growth, migration and differentiation. In this study, we showed that mTORC1 regulates platelet-derived growth factor (PDGF)-induced phenotypic conversion of vascular smooth muscle cells (VSMCs). Stimulation of contractile VSMCs with PDGF significantly reduced the expression of contractile marker proteins in a time- and dose-dependent manner. In addition, angiotensin II (AngII)-induced contraction of VSMCs was completely blocked by the stimulation of VSMCs with PDGF. PDGF-dependent suppression of VSMC marker gene expression was significantly blocked by inhibition of phosphatidylinositol 3-kinase (PI3K), extracellular signal-regulated kinase (ERK), and mTOR whereas inhibition of p38 MAPK had no effect. In particular, inhibition of mTORC1 by rapamycin or by silencing of Raptor significantly blocked the PDGF-dependent phenotypic change of VSMCs whereas silencing of Rictor had no effect. In addition, loss of AngII-dependent contraction by PDGF was significantly retained by silencing of Raptor. Inhibition of mTORC1 by rapamycin or by silencing of Raptor significantly blocked PDGF-induced proliferation of VSMCs. Taken together, we suggest that mTORC1 plays an essential role in PDGF-dependent phenotypic changes of VSMCs. - Graphical abstract: Regulation of VSMC phenotype by PDGF-dependent activation of mTORC1. - Highlights: • The expression of contractile marker proteins was reduced by PDGF stimulation. • PDGF-dependent phenotypic conversion of VSMCs was blocked by inhibition of mTOR. • PDGF-induced proliferation of VSMCs was attenuated by inhibition of mTORC1. • mTORC1 plays a critical role in PDGF-dependent phenotypic conversion of VSMCs.

  16. Coupled down-regulation of mTOR and telomerase activity during fluorouracil-induced apoptosis of hepatocarcinoma Cells

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    Wu Mengchao

    2007-11-01

    Full Text Available Abstract Background Hepatocellular carcinoma (HCC is the most invasive and frequently diagnosed malignancy and the second leading cause of cancer death in many regions of Asia. The PI3K/Akt/mTOR signal pathway is involved in multiple cellular functions including proliferation, differentiation, tumorigenesis, and apoptosis. Up-regulation of telomerase activity is thought to be a critical step leading to cell transformation. Methods This study investigated changes in mTOR pathway and telomerase activity in hepatocarcinoma cell line SMMC-7721 treated with chemotherapeutic agent 5-fluorouracil (5-Fu. We detected apoptosis of hepatocarcinoma cells by TUNEL assay. Telomerase activity, hTERT transcription level and p- p70 S6k was demonstrated by the telomeric repeat amplification protocol and silver staining assay, Dual-Luciferase Reporter Assay and Western blot analysis respectively. Results Treating SMMC-7721 cells with 5-Fu leads to apoptosis of the cells, and reduction in telomerase activity, as well as a dramatic reduction in the activated form of p70 S6 kinase, a mTOR substrate. The 5-Fu treatment nearly abolishes transcription of hTERT (the major component of telomerase mRNA. Treating SMMC-7721 cells with Rapamycin, a specific mTOR inhibitor, significantly reduce hTERT protein level but did not affect hTERT transcription. 5-Fu and rapamycin were synergistic in regards to down-regulation of telomerase activity in hepatocarcinoma cells. Conclusion These results suggest that chemotherapeutic agent 5-Fu may down-regulate telomerase activity at both transcriptional level and PI3K/Akt/mTOR pathway-dependent post-transcriptional level to facilitate hepatocellular carcinoma cell apoptosis.

  17. Finding a better drug for epilepsy: the mTOR pathway as an antiepileptogenic target.

    NARCIS (Netherlands)

    Galanopoulou, A.S.; Gorter, J.A.; Cepeda, C.

    2012-01-01

    The mammalian target of rapamycin (mTOR) signaling pathway regulates cell growth, differentiation, proliferation, and metabolism. Loss-of-function mutations in upstream regulators of mTOR have been highly associated with dysplasias, epilepsy, and neurodevelopmental disorders. These include tuberous

  18. mTOR regulates phagosome and entotic vacuole fission.

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    Krajcovic, Matej; Krishna, Shefali; Akkari, Leila; Joyce, Johanna A; Overholtzer, Michael

    2013-12-01

    Macroendocytic vacuoles formed by phagocytosis, or the live-cell engulfment program entosis, undergo sequential steps of maturation, leading to the fusion of lysosomes that digest internalized cargo. After cargo digestion, nutrients must be exported to the cytosol, and vacuole membranes must be processed by mechanisms that remain poorly defined. Here we find that phagosomes and entotic vacuoles undergo a late maturation step characterized by fission, which redistributes vacuolar contents into lysosomal networks. Vacuole fission is regulated by the serine/threonine protein kinase mammalian target of rapamycin complex 1 (mTORC1), which localizes to vacuole membranes surrounding engulfed cells. Degrading engulfed cells supply engulfing cells with amino acids that are used in translation, and rescue cell survival and mTORC1 activity in starved macrophages and tumor cells. These data identify a late stage of phagocytosis and entosis that involves processing of large vacuoles by mTOR-regulated membrane fission.

  19. Regulation of autophagy by amino acids and MTOR-dependent signal transduction.

    Science.gov (United States)

    Meijer, Alfred J; Lorin, Séverine; Blommaart, Edward F; Codogno, Patrice

    2015-10-01

    Amino acids not only participate in intermediary metabolism but also stimulate insulin-mechanistic target of rapamycin (MTOR)-mediated signal transduction which controls the major metabolic pathways. Among these is the pathway of autophagy which takes care of the degradation of long-lived proteins and of the elimination of damaged or functionally redundant organelles. Proper functioning of this process is essential for cell survival. Dysregulation of autophagy has been implicated in the etiology of several pathologies. The history of the studies on the interrelationship between amino acids, MTOR signaling and autophagy is the subject of this review. The mechanisms responsible for the stimulation of MTOR-mediated signaling, and the inhibition of autophagy, by amino acids have been studied intensively in the past but are still not completely clarified. Recent developments in this field are discussed.

  20. The emerging role of m-TOR up-regulation in brain Astrocytoma.

    Science.gov (United States)

    Ryskalin, Larisa; Limanaqi, Fiona; Biagioni, Francesca; Frati, Alessandro; Esposito, Vincenzo; Calierno, Maria Teresa; Lenzi, Paola; Fornai, Francesco

    2017-05-01

    The present manuscript is an overview of various effects of mTOR up-regulation in astrocytoma with an emphasis on its deleterious effects on the proliferation of Glioblastoma Multiforme. The manuscript reports consistent evidence indicating the occurrence of mTOR up-regulation both in experimental and human astrocytoma. The grading of human astrocytoma is discussed in relationship with mTOR up-regulation. In the second part of the manuscript, the biochemical pathways under the influence of mTOR are translated to cell phenotypes which are generated by mTOR up-regulation and reverted by its inhibition. A special section is dedicated to the prominent role of autophagy in mediating the effects of mTOR in glioblastoma. In detail, autophagy inhibition produced by mTOR up-regulation determines the fate of cancer stem cells. On the other hand, biochemical findings disclose the remarkable effects of autophagy activators as powerful inducers of cell differentiation with a strong prevalence towards neuronal phenotypes. Thus, mTOR modulation acts on the neurobiology of glioblastoma just like it operates in vivo at the level of brain stem cell niches by altering autophagy-dependent cell differentiation. In the light of such a critical role of autophagy we analyzed the ubiquitin proteasome system. The merging between autophagy and proteasome generates a novel organelle, named autophagoproteasome which is strongly induced by mTOR inhibitors in glioblastoma cells. Remarkably, when mTOR is maximally inhibited the proteasome component selectively moves within autophagy vacuoles, thus making the proteasome activity dependent on the entry within autophagy compartment.

  1. Cell size and fat content of dietary-restricted Caenorhabditis elegans are regulated by ATX-2, an mTOR repressor.

    Science.gov (United States)

    Bar, Daniel Z; Charar, Chayki; Dorfman, Jehudith; Yadid, Tam; Tafforeau, Lionel; Lafontaine, Denis L J; Gruenbaum, Yosef

    2016-08-09

    Dietary restriction (DR) is a metabolic intervention that extends the lifespan of multiple species, including yeast, flies, nematodes, rodents, and, arguably, rhesus monkeys and humans. Hallmarks of lifelong DR are reductions in body size, fecundity, and fat accumulation, as well as slower development. We have identified atx-2, the Caenorhabditis elegans homolog of the human ATXN2L and ATXN2 genes, as the regulator of these multiple DR phenotypes. Down-regulation of atx-2 increases the body size, cell size, and fat content of dietary-restricted animals and speeds animal development, whereas overexpression of atx-2 is sufficient to reduce the body size and brood size of wild-type animals. atx-2 regulates the mechanistic target of rapamycin (mTOR) pathway, downstream of AMP-activated protein kinase (AMPK) and upstream of ribosomal protein S6 kinase and mTOR complex 1 (TORC1), by its direct association with Rab GDP dissociation inhibitor β, which likely regulates RHEB shuttling between GDP-bound and GTP-bound forms. Taken together, this work identifies a previously unknown mechanism regulating multiple aspects of DR, as well as unknown regulators of the mTOR pathway. They also extend our understanding of diet-dependent growth retardation, and offers a potential mechanism to treat obesity.

  2. Six1 induces protein synthesis signaling expression in duck myoblasts mainly via up-regulation of mTOR

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    Haohan Wang

    2016-03-01

    Full Text Available Abstract As a critical transcription factor, Six1 plays an important role in the regulation of myogenesis and muscle development. However, little is known about its regulatory mechanism associated with muscular protein synthesis. The objective of this study was to investigate the effects of overexpression ofSix1 on the expression of key protein metabolism-related genes in duck myoblasts. Through an experimental model where duck myoblasts were transfected with a pEGFP-duSix1 construct, we found that overexpression of duckSix1 could enhance cell proliferation activity and increase mRNA expression levels of key genes involved in the PI3K/Akt/mTOR signaling pathway, while the expression of FOXO1, MuRF1and MAFbx was not significantly altered, indicating thatSix1 could promote protein synthesis in myoblasts through up-regulating the expression of several related genes. Additionally, in duck myoblasts treated with LY294002 and rapamycin, the specific inhibitors ofPI3K and mTOR, respectively, the overexpression of Six1 could significantly ameliorate inhibitive effects of these inhibitors on protein synthesis. Especially, the mRNA expression levels of mTOR and S6K1 were observed to undergo a visible change, and a significant increase in protein expression of S6K1 was seen. These data suggested that Six1plays an important role in protein synthesis, which may be mainly due to activation of the mTOR signaling pathway.

  3. Rapamycin-insensitive up-regulation of adipocyte phospholipase A2 in tuberous sclerosis and lymphangioleiomyomatosis.

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    Chenggang Li

    Full Text Available Tuberous sclerosis syndrome (TSC is an autosomal dominant tumor suppressor gene syndrome affecting multiple organs, including renal angiomyolipomas and pulmonary lymphangioleiomyomatosis (LAM. LAM is a female-predominant interstitial lung disease characterized by the progressive cyst formation and respiratory failure, which is also seen in sporadic patients without TSC. Mutations in TSC1 or TSC2 cause TSC, result in hyperactivation of mammalian target of rapamycin (mTOR, and are also seen in LAM cells in sporadic LAM. We recently reported that prostaglandin biosynthesis and cyclooxygenase-2 were deregulated in TSC and LAM. Phospholipase A2 (PLA2 is the rate-limiting enzyme that catalyzes the conversion of plasma membrane phospholipids into prostaglandins. In this study, we identified upregulation of adipocyte AdPLA2 (PLA2G16 in LAM nodule cells using publicly available expression data. We showed that the levels of AdPLA2 transcript and protein were higher in LAM lungs compared with control lungs. We then showed that TSC2 negatively regulates the expression of AdPLA2, and loss of TSC2 is associated with elevated production of prostaglandin E2 (PGE2 and prostacyclin (PGI2 in cell culture models. Mouse model studies also showed increased expression of AdPLA2 in xenograft tumors, estrogen-induced lung metastatic lesions of Tsc2 null leiomyoma-derived cells, and spontaneous renal cystadenomas from Tsc2+/- mice. Importantly, rapamycin treatment did not affect the expression of AdPLA2 and the production of PGE2 by TSC2-deficient mouse embryonic fibroblast (Tsc2-/-MEFs, rat uterine leiomyoma-derived ELT3 cells, and LAM patient-associated renal angiomyolipoma-derived "mesenchymal" cells. Furthermore, methyl arachidonyl fluorophosphate (MAFP, a potent irreversible PLA2 inhibitor, selectively suppressed the growth and induced apoptosis of TSC2-deficient LAM patient-derived cells relative to TSC2-addback cells. Our findings suggest that AdPLA2 plays an

  4. High glucose up-regulates Semaphorin 3A expression via the mTOR signaling pathway in keratinocytes: A potential mechanism and therapeutic target for diabetic small fiber neuropathy.

    Science.gov (United States)

    Wu, Liang-Yan; Li, Mei; Qu, Min-Li; Li, Xin; Pi, Lin-Hua; Chen, Zi; Zhou, Shan-Lei; Yi, Xiao-Qing; Shi, Xia-Jie; Wu, Jing; Wang, Shan

    2017-12-05

    Small fiber neuropathy (SFN) is a common complication in diabetes, and is characterized by decreased intraepidermal nerve fiber density (IENFD). Semaphorin 3A (Sema3A), which is produced by keratinocytes, has a chemorepulsive effect on intraepidermal nerve fibers. mTOR signaling can mediate local protein synthesis that is critical for growth of axons and dendrites. Therefore, this study aimed to investigate whether Sema3A is up-regulated in diabetic keratinocytes via the mTOR-mediated p70 S6K and 4E-BP1 signaling pathways, and furthermore whether it is involved in the pathogenesis of diabetic SFN. IENFD, expression of Sema3A, and mTOR signaling, were evaluated in the skin of diabetic patients with SFN as well as control subjects. Sema3A and mTOR signaling were also assessed in HaCaT cells which had been treated with high glucose (HG) or recombinant Sema3A (rSema3A) in the presence or absence of rapamycin. Small fiber dysfunction was evaluated by examining IENFD and using behavioral tests in control and streptozotocin-induced diabetic rats treated with or without rapamycin. We found that higher Sema3A expression and over-activation of mTOR signaling, was accompanied by reduced IENFD in the skin of diabetic patients compared with control subjects. The expression of Sema3A, and mTOR signaling were up-regulated in HaCaT cells incubated with HG or rSema3A, and this could be attenuated by rapamycin. Hyperalgesia, reduced IENFD, and up-regulated Sema3A and mTOR signaling were also detected in diabetic rats. These effects were ameliorated by rapamycin treatment. Our data indicate that HG up-regulates Sema3A expression by activating mTOR signaling in diabetic keratinocytes. This pathway may therefore play a critical role in diabetic SFN. Copyright © 2017 Elsevier B.V. All rights reserved.

  5. Signaling through the Phosphatidylinositol 3-Kinase (PI3K)/Mammalian Target of Rapamycin (mTOR) Axis Is Responsible for Aerobic Glycolysis mediated by Glucose Transporter in Epidermal Growth Factor Receptor (EGFR)-mutated Lung Adenocarcinoma.

    Science.gov (United States)

    Makinoshima, Hideki; Takita, Masahiro; Saruwatari, Koichi; Umemura, Shigeki; Obata, Yuuki; Ishii, Genichiro; Matsumoto, Shingo; Sugiyama, Eri; Ochiai, Atsushi; Abe, Ryo; Goto, Koichi; Esumi, Hiroyasu; Tsuchihara, Katsuya

    2015-07-10

    Oncogenic epidermal growth factor receptor (EGFR) signaling plays an important role in regulating global metabolic pathways, including aerobic glycolysis, the pentose phosphate pathway (PPP), and pyrimidine biosynthesis. However, the molecular mechanism by which EGFR signaling regulates cancer cell metabolism is still unclear. To elucidate how EGFR signaling is linked to metabolic activity, we investigated the involvement of the RAS/MEK/ERK and PI3K/AKT/mammalian target of rapamycin (mTOR) pathways on metabolic alteration in lung adenocarcinoma (LAD) cell lines with activating EGFR mutations. Although MEK inhibition did not alter lactate production and the extracellular acidification rate, PI3K/mTOR inhibitors significantly suppressed glycolysis in EGFR-mutant LAD cells. Moreover, a comprehensive metabolomics analysis revealed that the levels of glucose 6-phosphate and 6-phosphogluconate as early metabolites in glycolysis and PPP were decreased after inhibition of the PI3K/AKT/mTOR pathway, suggesting a link between PI3K signaling and the proper function of glucose transporters or hexokinases in glycolysis. Indeed, PI3K/mTOR inhibition effectively suppressed membrane localization of facilitative glucose transporter 1 (GLUT1), which, instead, accumulated in the cytoplasm. Finally, aerobic glycolysis and cell proliferation were down-regulated when GLUT1 gene expression was suppressed by RNAi. Taken together, these results suggest that PI3K/AKT/mTOR signaling is indispensable for the regulation of aerobic glycolysis in EGFR-mutated LAD cells. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  6. Response to mTOR inhibition: activity of eIF4E predicts sensitivity in cell lines and acquired changes in eIF4E regulation in breast cancer

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    Bartlett John MS

    2011-02-01

    Full Text Available Abstract Background Inhibitors of the kinase mTOR, such as rapamycin and everolimus, have been used as cancer therapeutics with limited success since some tumours are resistant. Efforts to establish predictive markers to allow selection of patients with tumours likely to respond have centred on determining phosphorylation states of mTOR or its targets 4E-BP1 and S6K in cancer cells. In an alternative approach we estimated eIF4E activity, a key effector of mTOR function, and tested the hypothesis that eIF4E activity predicts sensitivity to mTOR inhibition in cell lines and in breast tumours. Results We found a greater than three fold difference in sensitivity of representative colon, lung and breast cell lines to rapamycin. Using an assay to quantify influences of eIF4E on the translational efficiency specified by structured 5'UTRs, we showed that this estimate of eIF4E activity was a significant predictor of rapamycin sensitivity, with higher eIF4E activities indicative of enhanced sensitivity. Surprisingly, non-transformed cell lines were not less sensitive to rapamycin and did not have lower eIF4E activities than cancer lines, suggesting the mTOR/4E-BP1/eIF4E axis is deregulated in these non-transformed cells. In the context of clinical breast cancers, we estimated eIF4E activity by analysing expression of eIF4E and its functional regulators within tumour cells and combining these scores to reflect inhibitory and activating influences on eIF4E. Estimates of eIF4E activity in cancer biopsies taken at diagnosis did not predict sensitivity to 11-14 days of pre-operative everolimus treatment, as assessed by change in tumour cell proliferation from diagnosis to surgical excision. However, higher pre-treatment eIF4E activity was significantly associated with dramatic post-treatment changes in expression of eIF4E and 4E-binding proteins, suggesting that eIF4E is further deregulated in these tumours in response to mTOR inhibition. Conclusions

  7. Regulation of the Target of Rapamycin and Other Phosphatidylinositol 3-Kinase-Related Kinases by Membrane Targeting

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    Maristella De Cicco

    2015-09-01

    Full Text Available Phosphatidylinositol 3-kinase-related kinases (PIKKs play vital roles in the regulation of cell growth, proliferation, survival, and consequently metabolism, as well as in the cellular response to stresses such as ionizing radiation or redox changes. In humans six family members are known to date, namely mammalian/mechanistic target of rapamycin (mTOR, ataxia-telangiectasia mutated (ATM, ataxia- and Rad3-related (ATR, DNA-dependent protein kinase catalytic subunit (DNA-PKcs, suppressor of morphogenesis in genitalia-1 (SMG-1, and transformation/transcription domain-associated protein (TRRAP. All fulfill rather diverse functions and most of them have been detected in different cellular compartments including various cellular membranes. It has been suggested that the regulation of the localization of signaling proteins allows for generating a locally specific output. Moreover, spatial partitioning is expected to improve the reliability of biochemical signaling. Since these assumptions may also be true for the regulation of PIKK function, the current knowledge about the regulation of the localization of PIKKs at different cellular (membrane compartments by a network of interactions is reviewed. Membrane targeting can involve direct lipid-/membrane interactions as well as interactions with membrane-anchored regulatory proteins, such as, for example, small GTPases, or a combination of both.

  8. Divergent tissue and sex effects of rapamycin on the proteasome-chaperone network of old mice

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    Karl Andrew Rodriguez

    2014-11-01

    Full Text Available Rapamycin, an allosteric inhibitor of the mTOR kinase, increases longevity in mice in a sex-specific manner. In contrast to the widely accepted theory that a loss of proteasome activity is detrimental to both life- and healthspan, biochemical studies in vitro reveal that rapamycin inhibits 20S proteasome peptidase activity. We tested if this unexpected finding is also evident after chronic rapamycin treatment in vivo by measuring peptidase activities for both the 26S and 20S proteasome in liver, fat, and brain tissues of old, male and female mice fed encapsulated chow containing 2.24mg/kg (14 ppm rapamycin for 6 months. Further we assessed if rapamycin altered expression of the chaperone proteins known to interact with the proteasome-mediated degradation system (PMDS, heat shock factor 1 (HSF1, and the levels of key mTOR pathway proteins. Rapamycin had little effect on liver proteasome activity in either gender, but increased proteasome activity in female brain lysates and lowered its activity in female fat tissue. Rapamycin-induced changes in molecular chaperone levels were also more substantial in tissues from female animals. Furthermore, mTOR pathway proteins showed more significant changes in female tissues compared to those from males. These data show collectively that there are divergent tissue and sex effects of rapamycin on the proteasome-chaperone network and that these may be linked to the disparate effects of rapamycin on males and females. Further our findings suggest that rapamycin induces indirect regulation of the PMDS/heat-shock response through its modulation of the mTOR pathway rather than via direct interactions between rapamycin and the proteasome.

  9. Inhibition of MDM2 Re-Sensitizes Rapamycin Resistant Renal Cancer Cells via the Activation of p53.

    Science.gov (United States)

    Tian, Xin; Dai, Shundong; Sun, Jing; Jiang, Shenyi; Sui, Chengguang; Meng, Fandong; Li, Yan; Fu, Liye; Jiang, Tao; Wang, Yang; Su, Jia; Jiang, Youhong

    2016-01-01

    Rapamycin is a potential anti-cancer agent, which modulates the activity of mTOR, a key regulator of cell growth and proliferation. However, several types of cancer cells are resistant to the anti-proliferative effects of rapamycin. In this study, we report a MDM2/p53-mediated rapamycin resistance in human renal cancer cells. Trypan blue exclusion tests were used to determine the cell viability. Changes in mRNA and protein expression were measured using real-time PCR and western blot, respectively. Xenograft models were established to evaluate the in vivo effects of rapamycin combined with a MDM2 inhibitor. Rapamycin treatment suppresses the expression of MDM2 and exogenous overexpression of MDM2 in A498 cells contributes to rapamycin resistance. By establishing a rapamycin resistant cell line, we observed that MDM2 was significantly upregulated in rapamycin resistant cells than that in rapamycin sensitive cells. Importantly, the rapamycin resistant cells demonstrated attenuated accumulation of p53 in the nucleus in response to rapamycin treatment. Moreover, the inhibition of MDM2 by siMDM2 sensitizes A498 cells to rapamycin through the activation of p53. In both in vitro and in vivo models, the combination of rapamycin with the MDM2 inhibitor, MI-319, demonstrated a synergistic inhibitory effect on rapamycin resistant cells. Our study reports a novel mechanism for rapamycin resistance in human renal cancer and provides a new perspective for the development of anti-cancer drugs. © 2016 S. Karger AG, Basel.

  10. Regulation and function of ribosomal protein S6 kinase (S6K) within mTOR signalling networks.

    Science.gov (United States)

    Magnuson, Brian; Ekim, Bilgen; Fingar, Diane C

    2012-01-01

    The ribosomal protein S6K (S6 kinase) represents an extensively studied effector of the TORC1 [TOR (target of rapamycin) complex 1], which possesses important yet incompletely defined roles in cellular and organismal physiology. TORC1 functions as an environmental sensor by integrating signals derived from diverse environmental cues to promote anabolic and inhibit catabolic cellular functions. mTORC1 (mammalian TORC1) phosphorylates and activates S6K1 and S6K2, whose first identified substrate was rpS6 (ribosomal protein S6), a component of the 40S ribosome. Studies over the past decade have uncovered a number of additional S6K1 substrates, revealing multiple levels at which the mTORC1-S6K1 axis regulates cell physiology. The results thus far indicate that the mTORC1-S6K1 axis controls fundamental cellular processes, including transcription, translation, protein and lipid synthesis, cell growth/size and cell metabolism. In the present review we summarize the regulation of S6Ks, their cellular substrates and functions, and their integration within rapidly expanding mTOR (mammalian TOR) signalling networks. Although our understanding of the role of mTORC1-S6K1 signalling in physiology remains in its infancy, evidence indicates that this signalling axis controls, at least in part, glucose homoeostasis, insulin sensitivity, adipocyte metabolism, body mass and energy balance, tissue and organ size, learning, memory and aging. As dysregulation of this signalling axis contributes to diverse disease states, improved understanding of S6K regulation and function within mTOR signalling networks may enable the development of novel therapeutics.

  11. Developmental differences in megakaryocytopoiesis are associated with up-regulated TPO signaling through mTOR and elevated GATA-1 levels in neonatal megakaryocytes

    Science.gov (United States)

    Liu, Zhi-Jian; Italiano, Joseph; Ferrer-Marin, Francisca; Gutti, Ravi; Bailey, Matthew; Poterjoy, Brandon; Rimsza, Lisa

    2011-01-01

    Multiple observations support the existence of developmental differences in megakaryocytopoiesis. We have previously shown that neonatal megakaryocyte (MK) progenitors are hyperproliferative and give rise to MKs smaller and of lower ploidy than adult MKs. Based on these characteristics, neonatal MKs have been considered immature. The molecular mechanisms underlying these differences are unclear, but contribute to the pathogenesis of disorders of neonatal megakaryocytopoiesis. In the present study, we demonstrate that low-ploidy neonatal MKs, contrary to traditional belief, are more mature than adult low-ploidy MKs. These mature MKs are generated at a 10-fold higher rate than adult MKs, and result from a developmental uncoupling of proliferation, polyploidization, and terminal differentiation. This pattern is associated with up-regulated thrombopoietin (TPO) signaling through mammalian target of rapamycin (mTOR) and elevated levels of full-length GATA-1 and its targets. Blocking of mTOR with rapamycin suppressed the maturation of neonatal MKs without affecting ploidy, in contrast to the synchronous inhibition of polyploidization and cytoplasmic maturation in adult MKs. We propose that these mechanisms allow fetuses/neonates to populate their rapidly expanding bone marrow and intravascular spaces while maintaining normal platelet counts, but also set the stage for disorders restricted to fetal/neonatal MK progenitors, including the Down syndrome–transient myeloproliferative disorder and the thrombocytopenia absent radius syndrome. PMID:21304100

  12. mTOR Signaling in Protein Translation Regulation: Implications in Cancer Genesis and Therapeutic Interventions

    Directory of Open Access Journals (Sweden)

    Mehvish Showkat

    2014-01-01

    Full Text Available mTOR is a central nutrient sensor that signals a cell to grow and proliferate. Through distinct protein complexes it regulates different levels of available cellular energy substrates required for cell growth. One of the important functions of the complex is to maintain available amino acid pool by regulating protein translation. Dysregulation of mTOR pathway leads to aberrant protein translation which manifests into various pathological states. Our review focuses on the role mTOR signaling plays in protein translation and its physiological role. It also throws some light on available data that show translation dysregulation as a cause of pathological complexities like cancer and the available drugs that target the pathway for cancer treatment.

  13. The mTOR Signalling Pathway in Human Cancer

    Directory of Open Access Journals (Sweden)

    Paula Soares

    2012-02-01

    Full Text Available The conserved serine/threonine kinase mTOR (the mammalian target of rapamycin, a downstream effector of the PI3K/AKT pathway, forms two distinct multiprotein complexes: mTORC1 and mTORC2. mTORC1 is sensitive to rapamycin, activates S6K1 and 4EBP1, which are involved in mRNA translation. It is activated by diverse stimuli, such as growth factors, nutrients, energy and stress signals, and essential signalling pathways, such as PI3K, MAPK and AMPK, in order to control cell growth, proliferation and survival. mTORC2 is considered resistant to rapamycin and is generally insensitive to nutrients and energy signals. It activates PKC-α and AKT and regulates the actin cytoskeleton. Deregulation of multiple elements of the mTOR pathway (PI3K amplification/mutation, PTEN loss of function, AKT overexpression, and S6K1, 4EBP1 and eIF4E overexpression has been reported in many types of cancers, particularly in melanoma, where alterations in major components of the mTOR pathway were reported to have significant effects on tumour progression. Therefore, mTOR is an appealing therapeutic target and mTOR inhibitors, including the rapamycin analogues deforolimus, everolimus and temsirolimus, are submitted to clinical trials for treating multiple cancers, alone or in combination with inhibitors of other pathways. Importantly, temsirolimus and everolimus were recently approved by the FDA for the treatment of renal cell carcinoma, PNET and giant cell astrocytoma. Small molecules that inhibit mTOR kinase activity and dual PI3K-mTOR inhibitors are also being developed. In this review, we aim to survey relevant research, the molecular mechanisms of signalling, including upstream activation and downstream effectors, and the role of mTOR in cancer, mainly in melanoma.

  14. Longevity, aging and rapamycin

    OpenAIRE

    Ehninger, Dan; Neff, Frauke; Xie, Kan

    2014-01-01

    The federal drug administration (FDA)-approved compound rapamycin was the first pharmacological agent shown to extend maximal lifespan in both genders in a mammalian species. A major question then is whether the drug slows mammalian aging or if it has isolated effects on longevity by suppressing cancers, the main cause of death in many mouse strains. Here, we review what is currently known about the effects that pharmacological or genetic mammalian target of rapamycin (mTOR) inhibition have o...

  15. The role of phosphoinositide 3-kinase and phosphatidic acid in the regulation of mammalian target of rapamycin following eccentric contractions.

    Science.gov (United States)

    O'Neil, T K; Duffy, L R; Frey, J W; Hornberger, T A

    2009-07-15

    Resistance exercise induces a hypertrophic response in skeletal muscle and recent studies have begun to shed light on the molecular mechanisms involved in this process. For example, several studies indicate that signalling by the mammalian target of rapamycin (mTOR) is necessary for a hypertrophic response. Furthermore, resistance exercise has been proposed to activate mTOR signalling through an upstream pathway involving the phosphoinositide 3-kinase (PI3K) and protein kinase B (PKB); however, this hypothesis has not been thoroughly tested. To test this hypothesis, we first evaluated the temporal pattern of signalling through PI3K-PKB and mTOR following a bout of resistance exercise with eccentric contractions (EC). Our results indicated that the activation of signalling through PI3K-PKB is a transient event (12 h). Furthermore, inhibition of PI3K-PKB activity did not prevent the activation of mTOR signalling by ECs, indicating that PI3K-PKB is not part of the upstream regulatory pathway. These observations led us to investigate an alternative pathway for the activation of mTOR signalling involving the synthesis of phosphatidic acid (PA) by phospholipase D (PLD). Our results demonstrate that ECs induce a sustained elevation in [PA] and inhibiting the synthesis of PA by PLD prevented the activation of mTOR. Furthermore, we determined that similar to ECs, PA activates mTOR signalling through a PI3K-PKB-independent mechanism. Combined, the results of this study indicate that the activation of mTOR following eccentric contractions occurs through a PI3K-PKB-independent mechanism that requires PLD and PA.

  16. Decreasing Eukaryotic Initiation Factor 3C (EIF3C) Suppresses Proliferation and Stimulates Apoptosis in Breast Cancer Cell Lines Through Mammalian Target of Rapamycin (mTOR) Pathway.

    Science.gov (United States)

    Zhao, Weipeng; Li, Xichuan; Wang, Jun; Wang, Chen; Jia, Yongsheng; Yuan, Shunzong; Huang, Yong; Shi, Yehui; Tong, Zhongsheng

    2017-08-30

    BACKGROUND Translation initiation is the rate limiting step of protein synthesis and is highly regulated. Eukaryotic initiation factor 3C (EIF3C), an oncogene overexpressed in several human cancers, plays an important role in tumorigenesis and cell proliferation. MATERIAL AND METHODS Immunohistochemistry was used to determine the expression of EIF3C in breast cancer tissues from 42 patients. We investigated whether EIF3C silencing decreases breast cancer cell proliferation as assessed by colony formation assay, and whether EIF3C gene knockdown induces apoptosis as assessed by flow cytometry analysis. We utilized the stress and apoptosis signaling antibody array kit, while p-ERK1/2, p-Akt, p-Smad2, p-p38 MAPK, cleaved caspase-3, and cleaved caspase-7 were explored between EIF3C-siRNA and controls. Furthermore, the effects of EIF3C gene knockdown in mTOR pathway were analyzed by western blotting for different cell lines. RESULTS In EIF3C-positive tumors, 32 out of 42 showed significantly higher frequencies of high grade group by immunoreactivity (p=0.0016). BrdU incorporation after four days of cell plating was significantly suppressed in MDA-MB-231 cells by EIF3C knockdown compared with controls, with average changes of 7.8-fold (p<0.01). Clone number was significantly suppressed in MDA-MB-231 cells by EIF3C knockdown compared with controls (p<0.05). Cell apoptosis was significantly increased in the EIF3C-siRNA group when compared with the cells that were transfected with scrambled siRNA (3.51±0.0842 versus 13.24±0.2307, p<0.01). The mTOR signaling pathway was involved in decreasing EIF3C translational efficiency. CONCLUSIONS Unveiling the mechanisms of EIF3 action in tumorigenesis may help identify attractive targets for cancer therapy.

  17. Inhibition of mammalian target of rapamycin decreases intrarenal oxygen availability and alters glomerular permeability.

    Science.gov (United States)

    Sivertsson, Ebba; Friederich-Persson, Malou; Öberg, Carl M; Fasching, Angelica; Hansell, Peter; Rippe, Bengt; Palm, Fredrik

    2017-09-27

    Increased kidney oxygen consumption causing tissue hypoxia is suggested as a common pathway to chronic kidney disease. Mammalian target of rapamycin (mTOR) regulates cell proliferation and mitochondrial function. mTOR inhibitors, e.g. rapamycin, are used clinically to prevent graft rejection. mTOR has been identified as a key player in diabetes, which has stimulated the use of mTOR inhibitors to counter diabetic nephropathy. However, the effect of mTOR inhibition on kidney oxygen consumption is unknown. We therefore investigated the effects of mTOR inhibition on in vivo kidney function, oxygen homeostasis and glomerular permeability. Control and streptozotocin-induced diabetic rats were chronically treated with rapamycin and the functional consequences studied fourteen days thereafter. In both groups, mTOR inhibition induced mitochondrial uncoupling resulting in increased total kidney oxygen consumption and decreased intrarenal oxygen availability. Concomitantly, mTOR inhibition induced tubular injury, as estimated from urinary excretion of kidney injury molecule-1 (KIM-1), and reduced urinary protein excretion. The latter corresponded to reduced sieving coefficient for large molecules. In conclusion, mTOR inhibition induces mitochondrial dysfunction leading to decreased oxygen availability in normal and diabetic kidneys, which translates to increased KIM-1 in the urine. Reduced proteinuria after mTOR inhibition is an effect of reduced glomerular permeability for large molecules. Since hypoxia has been suggested as a common pathway to development of chronic kidney disease, mTOR inhibition to patients with pre-existing nephropathy should be used with caution since it may accelerate the progression of disease. Copyright © 2017, American Journal of Physiology-Renal Physiology.

  18. Alternative rapamycin treatment regimens mitigate the impact of rapamycin on glucose homeostasis and the immune system

    OpenAIRE

    Arriola Apelo, Sebastian I.; Neuman, Joshua C.; Baar, Emma L.; Syed, Faizan A.; Cummings, Nicole E.; Brar, Harpreet K.; Pumper, Cassidy P.; Kimple, Michelle E.; Lamming, Dudley W.

    2015-01-01

    Summary Inhibition of the mechanistic target of rapamycin (mTOR) signaling pathway by the FDA?approved drug rapamycin has been shown to promote lifespan and delay age?related diseases in model organisms including mice. Unfortunately, rapamycin has potentially serious side effects in humans, including glucose intolerance and immunosuppression, which may preclude the long?term prophylactic use of rapamycin as a therapy for age?related diseases. While the beneficial effects of rapamycin are larg...

  19. Evaluation of an AAV2-based rapamycin-regulated glial cell line-derived neurotrophic factor (GDNF expression vector system.

    Directory of Open Access Journals (Sweden)

    Piotr Hadaczek

    Full Text Available Effective regulation of transgene product in anatomically circumscribed brain tissue is dependent on the pharmacokinetics of the regulating agent, the kinetics of transcriptional activation and degradation of the transgene product. We evaluated rapamycin-regulated AAV2-GDNF expression in the rat brain (striatum. Regulated (a dual-component system: AAV2-FBZhGDNF + AAV2-TF1Nc and constitutive (CMV-driven expression vectors were compared. Constitutively active AAV2-GDNF directed stable GDNF expression in a dose-dependent manner and it increased for the first month, thereafter reaching a plateau that was maintained over a further 3 months. For the AAV2-regGDNF, rapamycin was administered in a 3-days on/4-days off cycle. Intraperitoneal, oral, and direct brain delivery (CED of rapamycin were evaluated. Two cycles of rapamycin at an intraperitoneal dose of 10 mg/kg gave the highest GDNF level (2.75±0.01 ng/mg protein. Six cycles at 3 mg/kg resulted in lower GDNF values (1.36±0.3 ng/mg protein. Interestingly, CED of rapamycin into the brain at a very low dose (50 ng induced GDNF levels comparable to a 6-week intraperitoneal rapamycin cycle. This study demonstrates the effectiveness of rapamycin regulation in the CNS. However, the kinetics of the transgene in brain tissue, the regulator dosing amount and schedule are critical parameters that influence the kinetics of accumulation and zenith of the encoded transgene product.

  20. Genetic and pharmacologic evidence that mTOR targeting outweighs mTORC1 inhibition as an antimyeloma strategy.

    Science.gov (United States)

    Chen, Xi; Díaz-Rodríguez, Elena; Ocio, Enrique M; Paiva, Bruno; Mortensen, Deborah S; Lopez-Girona, Antonia; Chopra, Rajesh; Miguel, Jesús San; Pandiella, Atanasio

    2014-02-01

    The mammalian target of rapamycin (mTOR) is a serine/threonine kinase that regulates cell growth, proliferation, metabolism, and cell survival, and plays those roles by forming two functionally distinct multiprotein complexes: mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2). Deregulation of the mTOR pathway has been found in different cancers, including multiple myeloma. Agents acting on mTORC1, such as rapamycin and derivatives, are being explored as antitumoral strategies. However, whether targeting mTOR would be a more effective antimyeloma strategy than exclusively acting on the mTORC1 branch remains to be established. In this report, we explored the activation status of mTOR routes in malignant plasma cells, and analyzed the contribution of mTOR and its two signaling branches to the proliferation of myeloma cells. Gene expression profiling demonstrated deregulation of mTOR pathway-related genes in myeloma plasma cells from patients. Activation of the mTOR pathway in myelomatous plasma cells was corroborated by flow cytometric analyses. RNA interference (RNAi) experiments indicated that mTORC1 predominated over mTORC2 in the control of myeloma cell proliferation. However, mTOR knockdown had a superior antiproliferative effect than acting only on mTORC1 or mTORC2. Pharmacologic studies corroborated that the neutralization of mTOR has a stronger antimyeloma effect than the individual inhibition of mTORC1 or mTORC2. Together, our data support the clinical development of agents that widely target mTOR, instead of agents, such as rapamycin or its derivatives, that solely act on mTORC1.

  1. Rapamycin inhibits poly(ADP-ribosyl)ation in intact cells

    Energy Technology Data Exchange (ETDEWEB)

    Fahrer, Joerg, E-mail: joerg.fahrer@uni-ulm.de [Molecular Toxicology Group, Department of Biology, University of Konstanz (Germany); Wagner, Silvia [Clinic of General, Visceral- and Transplantation Surgery, ZMF, University Hospital Tuebingen (Germany); Buerkle, Alexander [Molecular Toxicology Group, Department of Biology, University of Konstanz (Germany); Koenigsrainer, Alfred [Clinic of General, Visceral- and Transplantation Surgery, ZMF, University Hospital Tuebingen (Germany)

    2009-08-14

    Rapamycin is an immunosuppressive drug, which inhibits the mammalian target of rapamycin (mTOR) kinase activity inducing changes in cell proliferation. Synthesis of poly(ADP-ribose) (PAR) is an immediate cellular response to genotoxic stress catalyzed mostly by poly(ADP-ribose) polymerase 1 (PARP-1), which is also controlled by signaling pathways. Therefore, we investigated whether rapamycin affects PAR production. Strikingly, rapamycin inhibited PAR synthesis in living fibroblasts in a dose-dependent manner as monitored by immunofluorescence. PARP-1 activity was then assayed in vitro, revealing that down-regulation of cellular PAR production by rapamycin was apparently not due to competitive PARP-1 inhibition. Further studies showed that rapamycin did not influence the cellular NAD pool and the activation of PARP-1 in extracts of pretreated fibroblasts. Collectively, our data suggest that inhibition of cellular PAR synthesis by rapamycin is mediated by formation of a detergent-sensitive complex in living cells, and that rapamycin may have a potential as therapeutic PARP inhibitor.

  2. Mammalian target of rapamycin inhibitors for treatment in tuberous sclerosis

    Directory of Open Access Journals (Sweden)

    Won Seop Kim

    2011-06-01

    Full Text Available Tuberous sclerosis complex (TSC is a genetic multisystem disorder that results from mutations in the TSC1 or TSC2 genes, and is associated with hamartomas in several organs, including subependymal giant cell tumors. The neurological manifestations of TSC are particularly challenging and include infantile spasms, intractable epilepsy, cognitive disabilities, and autism. The TSC1- and TSC2-encoded proteins modulate cell function via the mammalian target of rapamycin (mTOR signaling cascade, and are key factors in the regulation of cell growth and proliferation. The mTOR pathway provides an intersection for an intricate network of protein cascades that respond to cellular nutrition, energy levels, and growth factor stimulation. In the brain, TSC1 and TSC2 have been implicated in cell body size, dendritic arborization, axonal outgrowth and targeting, neuronal migration, cortical lamination, and spine formation. The mTOR pathway represents a logical candidate for drug targeting, because mTOR regulates multiple cellular functions that may contribute to epileptogenesis, including protein synthesis, cell growth and proliferation, and synaptic plasticity. Antagonism of the mTOR pathway with rapamycin and related compounds may provide new therapeutic options for TSC patients.

  3. Muscle inactivation of mTOR causes metabolic and dystrophin defects leading to severe myopathy

    Science.gov (United States)

    Risson, Valérie; Mazelin, Laetitia; Roceri, Mila; Sanchez, Hervé; Moncollin, Vincent; Corneloup, Claudine; Richard-Bulteau, Hélène; Vignaud, Alban; Baas, Dominique; Defour, Aurélia; Freyssenet, Damien; Tanti, Jean-François; Le-Marchand-Brustel, Yannick; Ferrier, Bernard; Conjard-Duplany, Agnès; Romanino, Klaas; Bauché, Stéphanie; Hantaï, Daniel; Mueller, Matthias; Kozma, Sara C.; Thomas, George; Rüegg, Markus A.; Ferry, Arnaud; Pende, Mario; Bigard, Xavier; Koulmann, Nathalie

    2009-01-01

    Mammalian target of rapamycin (mTOR) is a key regulator of cell growth that associates with raptor and rictor to form the mTOR complex 1 (mTORC1) and mTORC2, respectively. Raptor is required for oxidative muscle integrity, whereas rictor is dispensable. In this study, we show that muscle-specific inactivation of mTOR leads to severe myopathy, resulting in premature death. mTOR-deficient muscles display metabolic changes similar to those observed in muscles lacking raptor, including impaired oxidative metabolism, altered mitochondrial regulation, and glycogen accumulation associated with protein kinase B/Akt hyperactivation. In addition, mTOR-deficient muscles exhibit increased basal glucose uptake, whereas whole body glucose homeostasis is essentially maintained. Importantly, loss of mTOR exacerbates the myopathic features in both slow oxidative and fast glycolytic muscles. Moreover, mTOR but not raptor and rictor deficiency leads to reduced muscle dystrophin content. We provide evidence that mTOR controls dystrophin transcription in a cell-autonomous, rapamycin-resistant, and kinase-independent manner. Collectively, our results demonstrate that mTOR acts mainly via mTORC1, whereas regulation of dystrophin is raptor and rictor independent. PMID:20008564

  4. Brain Injury-Induced Synaptic Reorganization in Hilar Inhibitory Neurons Is Differentially Suppressed by Rapamycin.

    Science.gov (United States)

    Butler, Corwin R; Boychuk, Jeffery A; Smith, Bret N

    2017-01-01

    Following traumatic brain injury (TBI), treatment with rapamycin suppresses mammalian (mechanistic) target of rapamycin (mTOR) activity and specific components of hippocampal synaptic reorganization associated with altered cortical excitability and seizure susceptibility. Reemergence of seizures after cessation of rapamycin treatment suggests, however, an incomplete suppression of epileptogenesis. Hilar inhibitory interneurons regulate dentate granule cell (DGC) activity, and de novo synaptic input from both DGCs and CA3 pyramidal cells after TBI increases their excitability but effects of rapamycin treatment on the injury-induced plasticity of interneurons is only partially described. Using transgenic mice in which enhanced green fluorescent protein (eGFP) is expressed in the somatostatinergic subset of hilar inhibitory interneurons, we tested the effect of daily systemic rapamycin treatment (3 mg/kg) on the excitability of hilar inhibitory interneurons after controlled cortical impact (CCI)-induced focal brain injury. Rapamycin treatment reduced, but did not normalize, the injury-induced increase in excitability of surviving eGFP+ hilar interneurons. The injury-induced increase in response to selective glutamate photostimulation of DGCs was reduced to normal levels after mTOR inhibition, but the postinjury increase in synaptic excitation arising from CA3 pyramidal cell activity was unaffected by rapamycin treatment. The incomplete suppression of synaptic reorganization in inhibitory circuits after brain injury could contribute to hippocampal hyperexcitability and the eventual reemergence of the epileptogenic process upon cessation of mTOR inhibition. Further, the cell-selective effect of mTOR inhibition on synaptic reorganization after CCI suggests possible mechanisms by which rapamycin treatment modifies epileptogenesis in some models but not others.

  5. Leucyl-tRNA Synthetase Regulates Lactation and Cell Proliferation via mTOR Signaling in Dairy Cow Mammary Epithelial Cells

    Science.gov (United States)

    Wang, Lina; Lin, Ye; Bian, Yanjie; Liu, Lili; Shao, Li; Lin, Lin; Qu, Bo; Zhao, Feng; Gao, Xuejun; Li, Qingzhang

    2014-01-01

    The role of LeuRS, an aminoacyl-tRNA synthetase, as an intracellular l-leucine sensor for the mTORC1 pathway has been the subject of much research recently. Despite this, the association between LeuRS and lactation in dairy cow mammary epithelial cells (DCMECs) remains unknown. In this study, we found that LeuRS expression in mammary gland tissue was significantly higher during lactation than pregnancy. Moreover, our data demonstrates that LeuRS is localized in the cytoplasm. Treatment with leucine increased DCMECs viability and proliferation, as well as mammalian target of rapamycin (mTOR), p-mTOR, ribosomal protein S6 kinase 1 (S6K1), p-S6K1, β-Casein, sterol regulatory element binding protein 1c (SREBP-1c), glucose transporter 1 (GLUT1), and Cyclin D1 mRNA and protein expression. Secretion of lactose and triglyceride were also increased. siRNA-mediated knockdown of LeuRS led to reduction in all of these processes. Based on these data, LeuRS up-regulates the mTOR pathway to promote proliferation and lactation of DCMECs in response to changes in the intracellular leucine concentration. PMID:24722568

  6. The mitochondrial uncoupler DNP triggers brain cell mTOR signaling network reprogramming and CREB pathway up-regulation.

    Science.gov (United States)

    Liu, Dong; Zhang, Yongqing; Gharavi, Robert; Park, Hee Ra; Lee, Jaewon; Siddiqui, Sana; Telljohann, Richard; Nassar, Matthew R; Cutler, Roy G; Becker, Kevin G; Mattson, Mark P

    2015-08-01

    Mitochondrial metabolism is highly responsive to nutrient availability and ongoing activity in neuronal circuits. The molecular mechanisms by which brain cells respond to an increase in cellular energy expenditure are largely unknown. Mild mitochondrial uncoupling enhances cellular energy expenditure in mitochondria and can be induced with 2,4-dinitrophenol (DNP), a proton ionophore previously used for weight loss. We found that DNP treatment reduces mitochondrial membrane potential, increases intracellular Ca(2+) levels and reduces oxidative stress in cerebral cortical neurons. Gene expression profiling of the cerebral cortex of DNP-treated mice revealed reprogramming of signaling cascades that included suppression of the mammalian target of rapamycin (mTOR) and insulin--PI3K - MAPK pathways, and up-regulation of tuberous sclerosis complex 2, a negative regulator of mTOR. Genes encoding proteins involved in autophagy processes were up-regulated in response to DNP. CREB (cAMP-response element-binding protein) signaling, Arc and brain-derived neurotrophic factor, which play important roles in synaptic plasticity and adaptive cellular stress responses, were up-regulated in response to DNP, and DNP-treated mice exhibited improved performance in a test of learning and memory. Immunoblot analysis verified that key DNP-induced changes in gene expression resulted in corresponding changes at the protein level. Our findings suggest that mild mitochondrial uncoupling triggers an integrated signaling response in brain cells characterized by reprogramming of mTOR and insulin signaling, and up-regulation of pathways involved in adaptive stress responses, molecular waste disposal, and synaptic plasticity. Physiological bioenergetic challenges such as exercise and fasting can enhance neuroplasticity and protect neurons against injury and neurodegeneration. Here, we show that the mitochondrial uncoupling agent 2,4-dinitrophenol (DNP) elicits adaptive signaling responses in the

  7. Rapamycin causes growth arrest and inhibition of invasion in human chondrosarcoma cells.

    Science.gov (United States)

    Song, Jian; Wang, Xiaobo; Zhu, Jiaxue; Liu, Jun

    2016-01-01

    Chondrosarcoma is a highly malignant tumor that is characterized by a potent capacity to invade locally and cause distant metastasis and notable for its lack of response to conventional chemotherapy or radiotherapy. Rapamycin, the inhibitor of mammalian target of rapamycin (mTOR), is a valuable drug with diverse clinical applications and regulates many cellular processes. However, the effects of rapamycin on cell growth and invasion of human chondrosarcoma cells are not well known. We determined the effect of rapamycin on cell proliferation, cell cycle arrest and invasion by using MTS, flow cytometry and invasion assays in two human chondrosarcoma cell lines, SW1353 and JJ012. Cell cycle regulatory and invasion-related genes' expression analysis was performed by quantitative RT-PCR (qRT-PCR). We also evaluated the effect of rapamycin on tumor growth by using mice xenograph models. Rapamycin significantly inhibited the cell proliferation, induced cell cycle arrest and decreased the invasion ability of human chondrosarcoma cells. Meanwhile, rapamycin modulated the cell cycle regulatory and invasion-related genes' expression. Furthermore, the tumor growth of mice xenograph models with human chondrosarcoma cells was significantly inhibited by rapamycin. These results provided further insight into the role of rapamycin in chondrosarcoma. Therefore, rapamycin targeted therapy may be a potential treatment strategy for chondrosarcoma.

  8. Identification of S664 TSC2 phosphorylation as a marker for extracellular signal-regulated kinase mediated mTOR activation in tuberous sclerosis and human cancer.

    Science.gov (United States)

    Ma, Li; Teruya-Feldstein, Julie; Bonner, Pauline; Bernardi, Rosa; Franz, David Neal; Witte, David; Cordon-Cardo, Carlos; Pandolfi, Pier Paolo

    2007-08-01

    Constitutive activation of extracellular signal-regulated kinases (Erk1/2) is frequently implicated in human cancers. Recently, aberrantly activated Erk was also found in brain lesions associated with tuberous sclerosis (TSC). We reported previously that Erk might contribute to tumorigenesis by phosphorylating TSC2 at specific residues, particularly S664. In our present study, 25 TSC-related cortical tubers or subependymal giant cell astrocytomas, as well as tissue microarrays of six types of human cancers, were analyzed for the expression of phospho-Erk (pErk) 1/2, S664-phospho-TSC2 (pTSC2), and phospho-S6 (pS6) by immunohistochemistry. We found that Erk-mediated TSC2 phosphorylation occurred at a high incidence and positively correlated with mitogen-activated protein kinase (MAPK) and mammalian target of rapamycin (mTOR) activation in TSC-associated brain lesions as well as in various cancers. Interestingly, in certain types of cancers (e.g., breast carcinoma and colon carcinoma), S664-pTSC2 seemed to be a more sensitive marker than pErk. Furthermore, most of the pTSC2-positive samples ( approximately 75%) were positive for pS6, but only 40% to 55% of the pS6-positive tumors exhibited TSC2 phosphorylation. Our results show that S664 TSC2 phosphorylation is a marker for Erk-mediated (as opposed to Akt-mediated) mTOR activation in TSC and human cancer. On the basis of these findings, TSC2 phosphorylation at S664 can be used to identify patients that may benefit from antitumor therapy with MAPK and mTOR inhibitors. Importantly, our results indicate that Erk-mediated phosphorylation and inactivation of TSC2 can be critical in development of hamartomatous lesions in TSC and cancer pathogenesis.

  9. mTOR is necessary for proper satellite cell activity and skeletal muscle regeneration

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Pengpeng [Key Laboratory of Swine Genetics and Breeding of Agricultural Ministry & Key Laboratory of Agricultural Animal Genetics, Breeding and Reproduction of Ministry of Education, College of Animal Science and Technology, Huazhong Agricultural University, Wuhan 430070 (China); Department of Animal Sciences, Purdue University, West Lafayette, IN 47907 (United States); Liang, Xinrong; Shan, Tizhong [Department of Animal Sciences, Purdue University, West Lafayette, IN 47907 (United States); Jiang, Qinyang [Department of Animal Sciences, Purdue University, West Lafayette, IN 47907 (United States); College of Animal Science and Technology, Guangxi University, Nanning 530004 (China); Deng, Changyan [Key Laboratory of Swine Genetics and Breeding of Agricultural Ministry & Key Laboratory of Agricultural Animal Genetics, Breeding and Reproduction of Ministry of Education, College of Animal Science and Technology, Huazhong Agricultural University, Wuhan 430070 (China); Zheng, Rong, E-mail: zhengrong@mail.hzau.edu.cn [Key Laboratory of Swine Genetics and Breeding of Agricultural Ministry & Key Laboratory of Agricultural Animal Genetics, Breeding and Reproduction of Ministry of Education, College of Animal Science and Technology, Huazhong Agricultural University, Wuhan 430070 (China); Kuang, Shihuan, E-mail: skuang@purdue.edu [Department of Animal Sciences, Purdue University, West Lafayette, IN 47907 (United States)

    2015-07-17

    The serine/threonine kinase mammalian target of rapamycin (mTOR) is a key regulator of protein synthesis, cell proliferation and energy metabolism. As constitutive deletion of Mtor gene results in embryonic lethality, the function of mTOR in muscle stem cells (satellite cells) and skeletal muscle regeneration remains to be determined. In this study, we established a satellite cell specific Mtor conditional knockout (cKO) mouse model by crossing Pax7{sup CreER} and Mtor{sup flox/flox} mice. Skeletal muscle regeneration after injury was severely compromised in the absence of Mtor, indicated by increased number of necrotic myofibers infiltrated by Evans blue dye, and reduced number and size of regenerated myofibers in the Mtor cKO mice compared to wild type (WT) littermates. To dissect the cellular mechanism, we analyzed satellite cell-derived primary myoblasts grown on single myofibers or adhered to culture plates. The Mtor cKO myoblasts exhibited defective proliferation and differentiation kinetics when compared to myoblasts derived from WT littermates. At the mRNA and protein levels, the Mtor cKO myoblasts expressed lower levels of key myogenic determinant genes Pax7, Myf5, Myod, Myog than did the WT myoblasts. These results suggest that mTOR is essential for satellite cell function and skeletal muscle regeneration through controlling the expression of myogenic genes. - Highlights: • Pax7{sup CreER} was used to delete Mtor gene in satellite cells. • Satellite cell specific deletion of Mtor impairs muscle regeneration. • mTOR is necessary for satellite cell proliferation and differentiation. • Deletion of Mtor leads to reduced expression of key myogenic genes.

  10. mTOR Inhibition in Epilepsy: Rationale and Clinical Perspectives

    OpenAIRE

    Ostendorf, Adam P.; Wong, Michael

    2015-01-01

    Despite a large number of available medical options, many individuals with epilepsy are refractory to existing therapies that mainly target neurotransmitter or ion channel activity. A growing body of preclinical data has uncovered a molecular pathway that appears crucial in many genetic and acquired epilepsy syndromes. The mammalian target of rapamycin (mTOR) pathway regulates a number of cellular processes required in the growth, metabolism, structure and cell-cell interactions of neurons an...

  11. Finding a better drug for epilepsy: the mTOR pathway as an antiepileptogenic target.

    Science.gov (United States)

    Galanopoulou, Aristea S; Gorter, Jan A; Cepeda, Carlos

    2012-07-01

    The mammalian target of rapamycin (mTOR) signaling pathway regulates cell growth, differentiation, proliferation, and metabolism. Loss-of-function mutations in upstream regulators of mTOR have been highly associated with dysplasias, epilepsy, and neurodevelopmental disorders. These include tuberous sclerosis, which is due to mutations in TSC1 or TSC2 genes; mutations in phosphatase and tensin homolog (PTEN) as in Cowden syndrome, polyhydramnios, megalencephaly, symptomatic epilepsy syndrome (PMSE) due to mutations in the STE20-related kinase adaptor alpha (STRADalpha); and neurofibromatosis type 1 attributed to neurofibromin 1 mutations. Inhibition of the mTOR pathway with rapamycin may prevent epilepsy and improve the underlying pathology in mouse models with disrupted mTOR signaling, due to PTEN or TSC mutations. However the timing and duration of its administration appear critical in defining the seizure and pathology-related outcomes. Rapamycin application in human cortical slices from patients with cortical dysplasias reduces the 4-aminopyridine-induced oscillations. In the multiple-hit model of infantile spasms, pulse high-dose rapamycin administration can reduce the cortical overactivation of the mTOR pathway, suppresses spasms, and has disease-modifying effects by partially improving cognitive deficits. In post-status epilepticus models of temporal lobe epilepsy, rapamycin may ameliorate the development of epilepsy-related pathology and reduce the expression of spontaneous seizures, but its effects depend on the timing and duration of administration, and possibly the model used. The observed recurrence of seizures and epilepsy-related pathology after rapamycin discontinuation suggests the need for continuous administration to maintain the benefit. However, the use of pulse administration protocols may be useful in certain age-specific epilepsy syndromes, like infantile spasms, whereas repetitive-pulse rapamycin protocols may suffice to sustain a long

  12. ARD1 Stabilization of TSC2 Suppresses Tumorigenesis Through the mTOR Signaling Pathway

    Science.gov (United States)

    Kuo, Hsu-Ping; Lee, Dung-Fang; Chen, Chun-Te; Liu, Mo; Chou, Chao-Kai; Lee, Hong-Jen; Du, Yi; Xie, Xiaoming; Wei, Yongkun; Xia, Weiya; Weihua, Zhang; Yang, Jer-Yen; Yen, Chia-Jui; Huang, Tzu-Hsuan; Tan, Minjia; Xing, Gang; Zhao, Yingming; Lin, Chien-Hsing; Tsai, Shih-Feng; Fidler, Isaiah J.; Hung, Mien-Chie

    2010-01-01

    Mammalian target of rapamycin (mTOR) regulates various cellular functions, including tumorigenesis, and is inhibited by the tuberous sclerosis 1 (TSC1)–TSC2 complex. Here, we demonstrate that arrest-defective protein 1 (ARD1) physically interacts with, acetylates, and stabilizes TSC2, thereby repressing mTOR activity. The inhibition of mTOR by ARD1 inhibits cell proliferation and increases autophagy, thereby inhibiting tumorigenicity. Correlation between ARD1 and TSC2 abundance was apparent in multiple tumor types. Moreover, evaluation of loss of heterozygosity at Xq28 revealed allelic loss in 31% of tested breast cancer cell lines and tumor samples. Together, our findings suggest that ARD1 functions as an inhibitor of the mTOR pathway and that dysregulation of the ARD1-TSC2-mTOR axis may contribute to cancer development. PMID:20145209

  13. mTOR Inhibition in Epilepsy: Rationale and Clinical Perspectives

    Science.gov (United States)

    Ostendorf, Adam P.; Wong, Michael

    2015-01-01

    Despite a large number of available medical options, many individuals with epilepsy are refractory to existing therapies that mainly target neurotransmitter or ion channel activity. A growing body of preclinical data has uncovered a molecular pathway that appears crucial in many genetic and acquired epilepsy syndromes. The mammalian target of rapamycin (mTOR) pathway regulates a number of cellular processes required in the growth, metabolism, structure and cell-cell interactions of neurons and glia. Rapamycin and similar compounds inhibit mTOR complex 1 (mTORC1) and decrease seizures, delay seizure development or prevent epileptogenesis in many animal models of mTOR hyperactivation. However, the exact mechanisms by which mTOR inhibition drives decreased seizure activity have not been completely determined. Nonetheless, these preclinical data have led to limited use in humans with epilepsy due to tuberous sclerosis complex (TSC) and polyhydramnios, megalencephaly and symptomatic epilepsy (PMSE) with promising results. Currently, larger controlled studies are underway using mTOR inhibitors in individuals with TSC and intractable epilepsy. PMID:25633849

  14. mTOR inhibition in epilepsy: rationale and clinical perspectives.

    Science.gov (United States)

    Ostendorf, Adam P; Wong, Michael

    2015-02-01

    Despite a large number of available medical options, many individuals with epilepsy are refractory to existing therapies that mainly target neurotransmitter or ion channel activity. A growing body of preclinical data has uncovered a molecular pathway that appears crucial in many genetic and acquired epilepsy syndromes. The mammalian target of rapamycin (mTOR) pathway regulates a number of cellular processes required in the growth, metabolism, structure, and cell-cell interactions of neurons and glia. Rapamycin and similar compounds inhibit mTOR complex 1 and decrease seizures, delay seizure development, or prevent epileptogenesis in many animal models of mTOR hyperactivation. However, the exact mechanisms by which mTOR inhibition drives decreased seizure activity have not been completely determined. Nonetheless, these preclinical data have led to limited use in humans with epilepsy due to tuberous sclerosis complex and polyhydramnios, megalencephaly, and symptomatic epilepsy with promising results. Currently, larger controlled studies are underway using mTOR inhibitors in individuals with tuberous sclerosis complex and intractable epilepsy.

  15. Mammalian target of rapamycin/eukaryotic initiation factor 4F pathway regulates follicle growth and development of theca cells in mice.

    Science.gov (United States)

    Zhang, Chao; Liu, Xiao-Ran; Cao, Yong-Chun; Tian, Jin-Ling; Zhen, Di; Luo, Xiao-Fei; Wang, Xin-Mei; Tian, Jian-Hui; Gao, Jian-Ming

    2017-04-01

    The aim of the present study was to clarify the roles of the mammalian target of rapamycin (mTOR) signalling pathway in follicular growth and development of thecal cells. Using in vivo-grown and in vitro-cultured ovaries, histological changes were evaluated using haematoxylin and eosin (HE) staining. Differentially expressed genes (DEGs) from 0 day post partum (d.p.p.) to 8 d.p.p. ovaries were screened by microarray and verified by quantitative real-time polymerase chain reaction. Forty-two DEGs related to cell proliferation and differentiation were screened out, with most DEGs being related to the to mTOR signalling pathway. Then, 3 d.p.p. ovaries were retrieved and used to verify the role of mTOR signalling in follicle and thecal cell development using its activators (Ras homologue enriched in brain (Rheb) and GTP) and inhibitor (rapamycin). The development of follicles and thecal cells was significantly impaired in ovaries cultured in vitro Day 3 to Day 8. In in vitro-cultured ovaries, Rheb and GTP (is 100ngmL-1 Rheb and 500ngmL-1 GTP for 48h) significantly increased follicle diameter, the percentage of primary and secondary follicles and the umber of thecal cells, and upregulated expression of mTOR, phosphorylated eukaryotic translation initiation factor 4E-binding protein 1 (4EBP1), eukaryotic initiation factor (eIF) 4F and cytochrome P450, family 17, subfamily A, polypeptide 1 (CYP17A1). Rapamycin (10nM rapamycin for 24h) had opposite effects to those of Rheb and GTP, and partly abrogated (significant) the effects of Rheb and GTP when added to the culture in combination with these drugs. Thus, mTOR signalling plays an important role in follicle growth and thecal cell development.

  16. Stimulation of the creatine transporter SLC6A8 by the protein kinase mTOR.

    Science.gov (United States)

    Shojaiefard, Manzar; Christie, David L; Lang, Florian

    2006-03-24

    Cellular accumulation of creatine is accomplished by the Na(+), Cl(-), and creatine transporter CreaT (SLC6A8). The mammalian target of rapamycin (mTOR) is a kinase stimulating cellular nutrient uptake. The present experiments explored whether SLC6A8 is regulated by mTOR. In Xenopus oocytes expressing SLC6A8 but not in water injected oocytes, creatine-induced a current which was significantly enhanced by coexpression of mTOR. Kinetic analysis revealed that mTOR enhanced maximal current without significantly altering affinity. Preincubation of the oocytes for 32 h with rapamycin (50 nM) decreased the creatine-induced current and abrogated its stimulation by mTOR. The effect of mTOR on CreaT was blunted by additional coexpression of the inactive mutant of the serum and glucocorticoid-inducible kinase (K119N)SGK1 and mimicked by coexpression of wild type SGK1. In conclusion, mTOR stimulates the creatine transporter SLC6A8 through mechanisms at least partially shared by the serum and glucocorticoid-inducible kinase SGK1.

  17. Discovery and optimization of 2-(4-substituted-pyrrolo[2,3-b]pyridin-3-yl)methylene-4-hydroxybenzofuran-3(2H)-ones as potent and selective ATP-competitive inhibitors of the mammalian target of rapamycin (mTOR)

    Energy Technology Data Exchange (ETDEWEB)

    Tsoua, Hwei-Ru; MacEwana, Gloria; Birnberga, Gary; Grosua, George; Bursavicha, Matthew G.; Barda, Joel; Brooijmansa, Natasja; Toral-Barzab, Lourdes; Hollanderb, Irwin; Mansoura, Tarek S.; Ayral-Kaloustiana, Semiramis; Yub, Ker (Wyeth)

    2010-07-19

    We discovered 2-(4-substituted-pyrrolo[2,3-b]pyridin-3-yl)methylene-4-hydroxybenzofuran-3(2H)-ones as potent and selective ATP-competitive inhibitors of the mammalian target of rapamycin (mTOR). Since phenolic OH groups pose metabolic liability, one of the two hydroxyl groups was selectively removed. The SAR data showed the structural features necessary for subnanomolar inhibitory activity against mTOR kinase as well as selectivity over PI3K?. An X-ray co-crystal structure of one inhibitor with the mTOR-related PI3K? revealed the key hydrogen bonding interactions.

  18. Active Mechanistic Target of Rapamycin plays an ancillary rather than essential role in Zebrafish CNS axon regeneration

    Directory of Open Access Journals (Sweden)

    Heike eDiekmann

    2015-07-01

    Full Text Available The developmental decrease of the intrinsic regenerative ability of the mammalian central nervous system (CNS is associated with reduced activity of mechanistic target of rapamycin (mTOR in mature neurons such as retinal ganglion cells (RGCs. While mTOR activity is further decreased upon axonal injury, maintenance of its pre-injury level, for instance by genetic deletion of the phosphatase and tensin homolog (PTEN, markedly promotes axon regeneration in mammals. The current study now addressed the question whether active mTOR might generally play a central role in axon regeneration by analyzing its requirement in regeneration-competent zebrafish. Remarkably, regulation of mTOR activity after optic nerve injury in zebrafish is fundamentally different compared to mammals. Hardly any activity was detected in naïve RGCs, whereas it was markedly increased upon axotomy in vivo as well as in dissociated cell cultures. After a short burst, mTOR activity was quickly attenuated, which is contrary to the requirements for axon regeneration in mammals. Surprisingly, mTOR activity was not essential for axonal growth per se, but correlated with cytokine- and PTEN inhibitor-induced neurite extension in vitro. Moreover, inhibition of mTOR using rapamycin significantly reduced axonal regeneration in vivo and delayed functional recovery after optic nerve injury. Therefore, axotomy-induced mTOR activity is involved in CNS axon regeneration in zebrafish similar to mammals, although it plays an ancillary rather than essential role in this regeneration-competent species.

  19. Effects of chronic overload on muscle hypertrophy and mTOR signaling in adult and aged rats

    Science.gov (United States)

    We examined the effect of 28 days of overload on mammalian target of rapamycin (mTOR) and extracellular signal-regulated kinase (ERK) signaling in young adult (Y; 6 mo old) and aged (O; 30 mo old) Fischer 344 x Brown Norway rats subjected to bilateral synergist ablation (SA) of two-thirds of the gas...

  20. Estradiol-Induced Object Recognition Memory Consolidation Is Dependent on Activation of mTOR Signaling in the Dorsal Hippocampus

    Science.gov (United States)

    Fortress, Ashley M.; Fan, Lu; Orr, Patrick T.; Zhao, Zaorui; Frick, Karyn M.

    2013-01-01

    The mammalian target of rapamycin (mTOR) signaling pathway is an important regulator of protein synthesis and is essential for various forms of hippocampal memory. Here, we asked whether the enhancement of object recognition memory consolidation produced by dorsal hippocampal infusion of 17[Beta]-estradiol (E[subscript 2]) is dependent on mTOR…

  1. Differential Effects of Rapamycin and Dexamethasone in Mouse Models of Established Allergic Asthma

    Science.gov (United States)

    Mushaben, Elizabeth M.; Brandt, Eric B.; Hershey, Gurjit K. Khurana; Le Cras, Timothy D.

    2013-01-01

    The mammalian target of rapamycin (mTOR) plays an important role in cell growth/differentiation, integrating environmental cues, and regulating immune responses. Our lab previously demonstrated that inhibition of mTOR with rapamycin prevented house dust mite (HDM)-induced allergic asthma in mice. Here, we utilized two treatment protocols to investigate whether rapamycin, compared to the steroid, dexamethasone, could inhibit allergic responses during the later stages of the disease process, namely allergen re-exposure and/or during progression of chronic allergic disease. In protocol 1, BALB/c mice were sensitized to HDM (three i.p. injections) and administered two intranasal HDM exposures. After 6 weeks of rest/recovery, mice were re-exposed to HDM while being treated with rapamycin or dexamethasone. In protocol 2, mice were exposed to HDM for 3 or 6 weeks and treated with rapamycin or dexamethasone during weeks 4–6. Characteristic features of allergic asthma, including IgE, goblet cells, airway hyperreactivity (AHR), inflammatory cells, cytokines/chemokines, and T cell responses were assessed. In protocol 1, both rapamycin and dexamethasone suppressed goblet cells and total CD4+ T cells including activated, effector, and regulatory T cells in the lung tissue, with no effect on AHR or total inflammatory cell numbers in the bronchoalveolar lavage fluid. Rapamycin also suppressed IgE, although IL-4 and eotaxin 1 levels were augmented. In protocol 2, both drugs suppressed total CD4+ T cells, including activated, effector, and regulatory T cells and IgE levels. IL-4, eotaxin, and inflammatory cell numbers were increased after rapamycin and no effect on AHR was observed. Dexamethasone suppressed inflammatory cell numbers, especially eosinophils, but had limited effects on AHR. We conclude that while mTOR signaling is critical during the early phases of allergic asthma, its role is much more limited once disease is established. PMID:23349887

  2. Finding a better drug for epilepsy: The mTOR pathway as an antiepileptogenic target

    Science.gov (United States)

    Galanopoulou, Aristea S.; Gorter, Jan A.; Cepeda, Carlos

    2012-01-01

    Summary The mTOR signaling pathway regulates cell growth, differentiation, proliferation and metabolism. Loss of function mutations in upstream regulators of mTOR have been highly associated with dysplasias, epilepsy and neurodevelopmental disorders. These include tuberous sclerosis, which is due to mutations in TSC1 or TSC2 genes, mutations in phosphatase and tensin homolog (PTEN) as in Cowden syndrome, polyhydramnios, megalencephaly, symptomatic epilepsy syndrome (PMSE) due to mutations in the STE20-related kinase adaptor alpha (STRADalpha), and neurofibromatosis type 1 attributed to neurofibromin 1 mutations. Inhibition of the mTOR pathway with rapamycin may prevent epilepsy and improve the underlying pathology in mouse models with disrupted mTOR signaling, due to PTEN or TSC mutations. However the timing and duration of its administration appear critical in defining the seizure and pathology-related outcomes. Rapamycin application in human cortical slices from patients with cortical dysplasias reduces the 4-aminopyridine induced oscillations. In the multiple-hit model of infantile spasms, pulse high dose rapamycin administration can reduce the cortical overactivation of the mTOR pathway, suppresses spasms and has disease-modifying effects by partially improving cognitive deficits. In post-status epilepticus models of temporal lobe epilepsy, rapamycin may ameliorate the development of epilepsy-related pathology and reduce the expression of spontaneous seizures, but its effects depend on the timing and duration of administration, and possibly the model used. The observed recurrence of seizures and epilepsy-related pathology after rapamycin discontinuation suggests the need for continuous administration to maintain the benefit. However, the use of pulse administration protocols may be useful in certain age-specific epilepsy syndromes, like infantile spasms, whereas repetitive pulse rapamycin protocols may suffice to sustain a long-term benefit in genetic disorders

  3. Immunohistochemical expression of mTOR in germinal center and nongerminal center group of diffuse large B-cell lymphoma: a clinicopathological study.

    Science.gov (United States)

    Vajpayee, Neerja; Burack, Richard; Wang, Dongliang; Hutchison, Robert E; Gajra, Ajeet

    2015-03-01

    The mammalian target of rapamycin (mTOR) pathway regulates many major cellular processes and is implicated in an increasing number of neoplasms, including lymphoma. We correlated immunohistochemical expression of mTOR with germinal center and nongerminal center phenotype, B cell lymphoma-2 (bcl-2) and cellular homolog of the retroviral v-myconcogene (c-myc) expression, and International Prognostic Index (IPI) score in 31 patients with diffuse large B-cell lymphoma (DLBCL). Virtually all patients in our study with high mTOR scores had a germinal center phenotype. Furthermore within the germinal center subgroup, patients with high mTOR scores were associated with higher IPI scores (P germinal center phenotype of DLBCL, mTOR expression might help identify a subset of patients with potentially more aggressive tumors who might benefit from use of targeted therapy using mTOR inhibitors. Copyright © 2015 Elsevier Inc. All rights reserved.

  4. mTOR Inhibition: From Aging to Autism and Beyond

    Directory of Open Access Journals (Sweden)

    Matt Kaeberlein

    2013-01-01

    Full Text Available The mechanistic target of rapamycin (mTOR is a highly conserved protein that regulates growth and proliferation in response to environmental and hormonal cues. Broadly speaking, organisms are constantly faced with the challenge of interpreting their environment and making a decision between “grow or do not grow.” mTOR is a major component of the network that makes this decision at the cellular level and, to some extent, the tissue and organismal level as well. Although overly simplistic, this framework can be useful when considering the myriad functions ascribed to mTOR and the pleiotropic phenotypes associated with genetic or pharmacological modulation of mTOR signaling. In this review, I will consider mTOR function in this context and attempt to summarize and interpret the growing body of literature demonstrating interesting and varied effects of mTOR inhibitors. These include robust effects on a multitude of age-related parameters and pathologies, as well as several other processes not obviously linked to aging or age-related disease.

  5. Rapamycin sensitizes T-ALL cells to dexamethasone-induced apoptosis

    Directory of Open Access Journals (Sweden)

    Mu Dezhi

    2010-11-01

    Full Text Available Abstract Background Glucocorticoid (GC resistance is frequently seen in acute lymphoblastic leukemia of T-cell lineage (T-ALL. In this study we investigate the potential and mechanism of using rapamycin to restore the sensitivity of GC-resistant T-ALL cells to dexamethasone (Dex treatment. Methods Cell proliferation was detected by 3-(4,5-dimethylthiazol-2-yl- 2,5-diphenyltetrazolium bromide (MTT assay. Fluorescence-activated cell sorting (FACS analysis was used to analyze apoptosis and cell cycles. Western blot analysis was performed to test the expression of the downstream effector proteins of mammalian target of rapamycin (mTOR, the cell cycle regulatory proteins, and apoptosis associated proteins. Results 10 nM rapamycin markedly increased GC sensitivity in GC-resistant T-ALL cells and this effect was mediated, at least in part, by inhibition of mTOR signaling pathway. Cell cycle arrest was associated with modulation of G1-S phase regulators. Both rapamycin and Dex can induce up-regulation of cyclin-dependent kinase (CDK inhibitors of p21 and p27 and co-treatment of rapamycin with Dex resulted in a synergistic induction of their expressions. Rapamycin did not obviously affect the expression of cyclin A, whereas Dex induced cyclin A expression. Rapamycin prevented Dex-induced expression of cyclin A. Rapamycin had a stronger inhibition of cyclin D1 expression than Dex. Rapamycin enhanced GC-induced apoptosis and this was not achieved by modulation of glucocorticoid receptor (GR expression, but synergistically up-regulation of pro-apoptotic proteins like caspase-3, Bax, and Bim, and down-regulation of anti-apoptotic protein of Mcl-1. Conclusion Our data suggests that rapamycin can effectively reverse GC resistance in T-ALL and this effect is achieved by inducing cell cycles arrested at G0/G1 phase and activating the intrinsic apoptotic program. Therefore, combination of mTOR inhibitor rapamycin with GC containing protocol might be an attracting

  6. Mechanistic target of rapamycin (mTOR: a point of convergence in the action of insulin/IGF-1 and G protein-coupled receptor agonists in pancreatic cancer cells

    Directory of Open Access Journals (Sweden)

    Enrique eRozengurt

    2014-09-01

    Full Text Available Pancreatic ductal adenocarcinoma (PDAC, the most common form of pancreatic cancer, is one of the most lethal human diseases. PDAC is now the fourth leading cause of cancer mortality in both men and women and deaths due to PDAC are projected to increase dramatically. Novel targets and agents for chemoprevention are urgently needed and will most likely arise from a more detailed understanding of the signaling mechanisms that stimulate the promotion and progression of sub-malignant cells into pancreatic cancer cells and from the identification of modifiable risk factors for PDAC. Many epidemiological studies have linked obesity and long-standing type 2 diabetes mellitus (T2DM with increased risk and worse clinical outcomes for developing PDAC. These diet-related metabolic disorders are multifaceted but characterized by peripheral insulin resistance, compensatory overproduction of insulin and increased bioavailability of IGF-1. Mounting evidence indicates that the insulin/IGF-1R system plays a critical role in PDAC development and multiple studies support the notion that crosstalk between the insulin receptor and heptahelical G protein-coupled receptor (GPCR signaling systems is an important element in the biological responses elicited by these signaling systems, including cell proliferation. This article highlights the central role of the mechanistic target of rapamycin (mTOR in mediating crosstalk between insulin/IGF-1 and G protein-coupled receptor (GPCR signaling in pancreatic cancer cells and proposes strategies, including the use of metformin, to target this signaling system in PDAC cells.

  7. Rapamycin reversal of VEGF-C-driven lymphatic anomalies in the respiratory tract.

    Science.gov (United States)

    Baluk, Peter; Yao, Li-Chin; Flores, Julio C; Choi, Dongwon; Hong, Young-Kwon; McDonald, Donald M

    2017-08-17

    Lymphatic malformations are serious but poorly understood conditions that present therapeutic challenges. The goal of this study was to compare strategies for inducing regression of abnormal lymphatics and explore underlying mechanisms. CCSP-rtTA/tetO-VEGF-C mice, in which doxycycline regulates VEGF-C expression in the airway epithelium, were used as a model of pulmonary lymphangiectasia. After doxycycline was stopped, VEGF-C expression returned to normal, but lymphangiectasia persisted for at least 9 months. Inhibition of VEGFR-2/VEGFR-3 signaling, Notch, β-adrenergic receptors, or autophagy and antiinflammatory steroids had no noticeable effect on the amount or severity of lymphangiectasia. However, rapamycin inhibition of mTOR reduced lymphangiectasia by 76% within 7 days without affecting normal lymphatics. Efficacy of rapamycin was not increased by coadministration with the other agents. In prevention trials, rapamycin suppressed VEGF-C-driven mTOR phosphorylation and lymphatic endothelial cell sprouting and proliferation. However, in reversal trials, no lymphatic endothelial cell proliferation was present to block in established lymphangiectasia, and rapamycin did not increase caspase-dependent apoptosis. However, rapamycin potently suppressed Prox1 and VEGFR-3. These experiments revealed that lymphangiectasia is remarkably resistant to regression but is responsive to rapamycin, which rapidly reduces and normalizes the abnormal lymphatics without affecting normal lymphatics.

  8. Rapamycin Influences the Efficiency of Fertilization and Development in the Mouse: A Role for Autophagic Activation

    Directory of Open Access Journals (Sweden)

    Geun-Kyung Lee

    2016-08-01

    Full Text Available The mammalian target of rapamycin (mTOR regulates cellular processes such as cell growth, metabolism, transcription, translation, and autophagy. Rapamycin is a selective inhibitor of mTOR, and induces autophagy in various systems. Autophagy contributes to clearance and recycling of macromolecules and organelles in response to stress. We previously reported that vitrified-warmed mouse oocytes show acute increases in autophagy during warming, and suggested that it is a natural response to cold stress. In this follow-up study, we examined whether the modulation of autophagy influences survival, fertilization, and developmental rates of vitrified-warmed mouse oocytes. We used rapamycin to enhance autophagy in metaphase II (MII oocytes before and after vitrification. The oocytes were then subjected to in vitro fertilization (IVF. The fertilization and developmental rates of vitrified-warmed oocytes after rapamycin treatment were significantly lower than those for control groups. Modulation of autophagy with rapamycin treatment shows that rapamycin-induced autophagy exerts a negative influence on fertilization and development of vitrified-warmed oocytes.

  9. Adapting the Stress Response: Viral Subversion of the mTOR Signaling Pathway

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    Valerie Le Sage

    2016-05-01

    Full Text Available The mammalian target of rapamycin (mTOR is a central regulator of gene expression, translation and various metabolic processes. Multiple extracellular (growth factors and intracellular (energy status molecular signals as well as a variety of stressors are integrated into the mTOR pathway. Viral infection is a significant stress that can activate, reduce or even suppress the mTOR signaling pathway. Consequently, viruses have evolved a plethora of different mechanisms to attack and co-opt the mTOR pathway in order to make the host cell a hospitable environment for replication. A more comprehensive knowledge of different viral interactions may provide fruitful targets for new antiviral drugs.

  10. Next-generation mTOR inhibitors in clinical oncology: how pathway complexity informs therapeutic strategy.

    LENUS (Irish Health Repository)

    Wander, Seth A

    2011-04-01

    Mammalian target of rapamycin (mTOR) is a PI3K-related kinase that regulates cell growth, proliferation, and survival via mTOR complex 1 (mTORC1) and mTORC2. The mTOR pathway is often aberrantly activated in cancers. While hypoxia, nutrient deprivation, and DNA damage restrain mTORC1 activity, multiple genetic events constitutively activate mTOR in cancers. Here we provide a brief overview of the signaling pathways up- and downstream of mTORC1 and -2, and discuss the insights into therapeutic anticancer targets - both those that have been tried in the clinic with limited success and those currently under clinical development - that knowledge of these pathways gives us.

  11. mTOR, a Potential Target to Treat Autism Spectrum Disorder.

    Science.gov (United States)

    Sato, Atsushi

    2016-01-01

    Mammalian target of rapamycin (mTOR) is a key regulator in various cellular processes, including cell growth, gene expression, and synaptic functions. Autism spectrum disorder (ASD) is frequently accompanied by monogenic disorders, such as tuberous sclerosis complex, phosphatase and tensin homolog tumor hamartoma syndrome, neurofibromatosis 1, and fragile X syndrome, in which mTOR is hyperactive. Mutations in the genes involved in the mTOR-mediated signaling pathway have been identified in some cases of syndromic ASD. Evidences indicate a pathogenic role for hyperactive mTOR-mediated signaling in ASD associated with these monogenic disorders, and mTOR inhibitors are a potential pharmacotherapy for ASD. Abnormal synaptic transmission through metabotropic glutamate receptor 5 may underlie in a part of ASD associated with hyperactive mTOR-mediated signaling. In this review, the relationship between mTOR and ASD is discussed.

  12. GADD34 Keeps the mTOR Pathway Inactivated in Endoplasmic Reticulum Stress Related Autophagy.

    Science.gov (United States)

    Holczer, Marianna; Bánhegyi, Gábor; Kapuy, Orsolya

    2016-01-01

    The balance of protein synthesis and proteolysis (i.e. proteostasis) is maintained by a complex regulatory network in which mTOR (mechanistic target of rapamycin serine/threonine kinase) pathway and unfolded protein response are prominent positive and negative actors. The interplay between the two systems has been revealed; however the mechanistic details of this crosstalk are largely unknown. The aim of the present study was to investigate the elements of crosstalk during endoplasmic reticulum stress and to verify the key role of GADD34 in the connection with the mTOR pathway. Here, we demonstrate that a transient activation of autophagy is present in endoplasmic reticulum stress provoked by thapsigargin or tunicamycin, which is turned into apoptotic cell death. The transient phase can be characterized by the elevation of the autophagic marker LC3II/I, by mTOR inactivation, AMP-activated protein kinase activation and increased GADD34 level. The switch from autophagy to apoptosis is accompanied with the appearance of apoptotic markers, mTOR reactivation, AMP-activated protein kinase inactivation and a decrease in GADD34. Inhibition of autophagy by 3-methyladenine shortens the transient phase, while inhibition of mTOR by rapamycin or resveratrol prolongs it. Inhibition of GADD34 by guanabenz or transfection of the cells with siGADD34 results in down-regulation of autophagy-dependent survival and a quick activation of mTOR, followed by apoptotic cell death. The negative effect of GADD34 inhibition is diminished when guanabenz or siGADD34 treatment is combined with rapamycin or resveratrol addition. These data confirm that GADD34 constitutes a mechanistic link between endoplasmic reticulum stress and mTOR inactivation, therefore promotes cell survival during endoplasmic reticulum stress.

  13. Rapamycin-insensitive mTORC1 activity controls eIF4E:4E-BP1 binding [v1; ref status: indexed, http://f1000r.es/NM6hpo

    Directory of Open Access Journals (Sweden)

    Mark Livingstone

    2012-07-01

    Full Text Available The recent development of mammalian target of rapamycin (mTOR kinase domain inhibitors and genetic dissection of rapamycin-sensitive and -insensitive mTOR protein complexes (mTORC1 and mTORC2 have revealed that phosphorylation of the mTOR substrate 4E-BP1 on amino acids Thr37 and/or Thr46 represents a rapamycin-insensitive activity of mTORC1. Despite numerous previous reports utilizing serine (Ser-to-alanine (Ala and threonine (Thr-to-Ala phosphorylation site mutants of 4E-BP1 to assess which post-translational modification(s directly regulate binding to eIF4E, an ambiguous understanding persists. This manuscript demonstrates that the initial, rapamycin-insensitive phosphorylation event at Thr46 is sufficient to prevent eIF4E:4E-BP1 binding. This finding is relevant, particularly as mTOR kinase domain inhibitors continue to be assessed for clinical efficacy, since it clarifies a difference between the action of these second-generation mTOR inhibitors and those of rapamycin analogues.

  14. Mammalian target of rapamycin complex 2 (mTORC2) controls glycolytic gene expression by regulating Histone H3 Lysine 56 acetylation.

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    Vadla, Raghavendra; Haldar, Devyani

    2017-11-16

    Metabolic reprogramming is a hallmark of cancer cells, but the mechanisms are not well understood. The mammalian target of rapamycin complex 2 (mTORC2) controls cell growth and proliferation and plays a critical role in metabolic reprogramming in glioma. mTORC2 regulates cellular processes such as cell survival, metabolism, and proliferation by phosphorylation of AGC kinases. Components of mTORC2 are shown to localize to the nucleus, but whether mTORC2 modulates epigenetic modifications to regulate gene expression is not known. Here, we identified histone H3 lysine 56 acetylation (H3K56Ac) is regulated by mTORC2 and show that global H3K56Ac levels were downregulated on mTORC2 knockdown but not on mTORC1 knockdown. mTORC2 promotes H3K56Ac in a tuberous sclerosis complex 1/2 (TSC1/2) mediated signaling pathway. We show that knockdown of sirtuin6 (SIRT6) prevented H3K56 deacetylation in mTORC2 depleted cells. Using glioma model consisting of U87EGFRvIII cells, we established that mTORC2 promotes H3K56Ac in glioma. Finally, we show that mTORC2 regulates the expression of glycolytic genes by regulating H3K56Ac levels at the promoters of these genes in glioma cells and depletion of mTOR leads to increased recruitment of SIRT6 to these promoters. Collectively, these results identify mTORC2 signaling pathway positively promotes H3K56Ac through which it may mediate metabolic reprogramming in glioma.

  15. Blood-brain barrier leakage after status epilepticus in rapamycin-treated rats I : Magnetic resonance imaging

    NARCIS (Netherlands)

    van Vliet, Erwin A; Otte, Wim M; Wadman, Wytse J; Aronica, Eleonora; Kooij, Gijs; de Vries, Helga E; Dijkhuizen, Rick M; Gorter, Jan A

    OBJECTIVE: The mammalian target of rapamycin (mTOR) pathway has received increasing attention as a potential antiepileptogenic target. Treatment with the mTOR inhibitor rapamycin after status epilepticus reduces the development of epilepsy in a rat model. To study whether rapamycin mediates this

  16. A combination of eicosapentaenoic acid-free fatty acid, epigallocatechin-3-gallate and proanthocyanidins has a strong effect on mTOR signaling in colorectal cancer cells

    NARCIS (Netherlands)

    Angelo, D' L.; Piazzi, G.; Pacilli, A.; Prossomariti, A.; Fazio, C.; Montanaro, L.; Graziani, G.; Fogliano, V.; Munarini, A.; Bianchi, F.; Belluzzi, A.; Bazzoli, F.; Ricciardiello, L.

    2014-01-01

    Colorectal cancer (CRC) is one of the major causes of cancer death worldwide. The development of novel anti-CRC agents able to overcome drug resistance and/or off-target toxicity is of pivotal importance. The mammalian target of rapamycin (mTOR) plays a critical role in CRC, regulating protein

  17. Cap-independent translation ensures mTOR expression and function upon protein synthesis inhibition.

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    Marques-Ramos, Ana; Candeias, Marco M; Menezes, Juliane; Lacerda, Rafaela; Willcocks, Margaret; Teixeira, Alexandre; Locker, Nicolas; Romão, Luísa

    2017-11-01

    The mechanistic/mammalian target of rapamycin (mTOR) is a conserved serine/threonine kinase that integrates cellular signals from the nutrient and energy status to act, namely, on the protein synthesis machinery. While major advances have emerged regarding the regulators and effects of the mTOR signaling pathway, little is known about the regulation of mTOR gene expression. Here, we show that the human mTOR transcript can be translated in a cap-independent manner, and that its 5' untranslated region (UTR) is a highly folded RNA scaffold capable of binding directly to the 40S ribosomal subunit. We further demonstrate that mTOR is able to bypass the cap requirement for translation both in normal and hypoxic conditions. Moreover, our data reveal that the cap-independent translation of mTOR is necessary for its ability to induce cell-cycle progression into S phase. These results suggest a novel regulatory mechanism for mTOR gene expression that integrates the global protein synthesis changes induced by translational inhibitory conditions. © 2017 Marques-Ramos et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

  18. mTOR activation is a biomarker and a central pathway to autoimmune disorders, cancer, obesity, and aging.

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    Perl, Andras

    2015-06-01

    The mechanistic target of rapamycin (mTOR) is a ubiquitous serine/threonine kinase, which plays pivotal roles in integrating growth signals on a cellular level. To support proliferation and survival under stress, two interacting complexes that harbor mTOR, mTORC1 and mTORC2, promote the transcription of genes involved in carbohydrate metabolism and lipogenesis, enhance protein translation, and inhibit autophagy. Although rapamycin was originally developed as an inhibitor of T cell proliferation for preventing organ transplant rejection, its molecular target, mTOR, has been subsequently identified as a central regulator of metabolic cues that drive lineage specification in the immune system. Owing to oxidative stress, the activation of mTORC1 has emerged as a central pathway for the pathogenesis of systemic lupus erythematosus and other autoimmune diseases. Paradoxically, mTORC1 has also been identified as a mediator of the Warburg effect that allows cell survival under hypoxia. Rapamycin and new classes of mTOR inhibitors are being developed to block not only transplant rejection and autoimmunity but also to treat obesity and various forms of cancer. Through preventing these diseases, personalized mTOR blockade holds promise to extend life span. © 2015 New York Academy of Sciences.

  19. Whey Protein Concentrate Renders MDA-MB-231 Cells Sensitive to Rapamycin by Altering Cellular Redox State and Activating GSK3β/mTOR Signaling.

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    Cheng, Shih-Hsuan; Tseng, Yang-Ming; Wu, Szu-Hsien; Tsai, Shih-Meng; Tsai, Li-Yu

    2017-11-21

    Whey protein concentrate (WPC) is an amino acid-rich supplement that has been shown to increase cellular antioxidant capacity. Mammalian target of rapamycin (mTOR) is a crucial regulator of signaling in mammalian cells, and serves as a therapeutic target for triple-negative breast cancer (TNBC). This study was designed to investigate the effect of combining WPC with rapamycin on MDA-MB-231 human breast cancer cells. These cells were found to be insensitive to rapamycin and exhibited higher glutathione (GSH) and reactive oxygen species levels than non-tumorigenic MCF-10A cells. However, for MDA-MB-231 cells, the half maximal inhibitory concentration of rapamycin was lower when this drug was administered in combination with WPC than when used alone. Furthermore, combining WPC with rapamycin depleted GSH levels and reduced Nrf2 nuclear accumulation. In addition, WPC activated GSK3β/mTOR signaling, and GSK3β appeared to be involved in the WPC-mediated Nrf2 reduction and mTOR activation. In conclusion, WPC induced rapamycin sensitivity in MDA-MB-231 cells by altering their redox state and activating GSK3β/mTOR signaling. These results not only suggest a novel therapeutic approach for breast cancer treatment, but also provide insight into the critical pathways affecting the resistance to mTOR inhibition observed in a subgroup of TNBC patients.

  20. Rapamycin regulates autophagy and cell adhesion in induced pluripotent stem cells

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    Areechun Sotthibundhu

    2016-11-01

    Full Text Available Abstract Background Cellular reprogramming is a stressful process, which requires cells to engulf somatic features and produce and maintain stemness machineries. Autophagy is a process to degrade unwanted proteins and is required for the derivation of induced pluripotent stem cells (iPSCs. However, the role of autophagy during iPSC maintenance remains undefined. Methods Human iPSCs were investigated by microscopy, immunofluorescence, and immunoblotting to detect autophagy machinery. Cells were treated with rapamycin to activate autophagy and with bafilomycin to block autophagy during iPSC maintenance. High concentrations of rapamycin treatment unexpectedly resulted in spontaneous formation of round floating spheres of uniform size, which were analyzed for differentiation into three germ layers. Mass spectrometry was deployed to reveal altered protein expression and pathways associated with rapamycin treatment. Results We demonstrate that human iPSCs express high basal levels of autophagy, including key components of APMKα, ULK1/2, BECLIN-1, ATG13, ATG101, ATG12, ATG3, ATG5, and LC3B. Block of autophagy by bafilomycin induces iPSC death and rapamycin attenuates the bafilomycin effect. Rapamycin treatment upregulates autophagy in iPSCs in a dose/time-dependent manner. High concentration of rapamycin reduces NANOG expression and induces spontaneous formation of round and uniformly sized embryoid bodies (EBs with accelerated differentiation into three germ layers. Mass spectrometry analysis identifies actin cytoskeleton and adherens junctions as the major targets of rapamycin in mediating iPSC detachment and differentiation. Conclusions High levels of basal autophagy activity are present during iPSC derivation and maintenance. Rapamycin alters expression of actin cytoskeleton and adherens junctions, induces uniform EB formation, and accelerates differentiation. IPSCs are sensitive to enzyme dissociation and require a lengthy differentiation time

  1. Effects of mTOR on neurological deficits after transient global ischemia

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    Xing Jihong

    2017-04-01

    Full Text Available Mammalian target of rapamycin (mTOR is a serine/threonine protein kinase and activation of its signal pathway plays an important role in regulating protein growth and synthesis as well as cell proliferation and survival. In the present study, we examined the contribution of mTOR and its downstream products to brain injuries and neurological deficiencies after cardiac arrest (CA induced-transient global ischemia. CA was induced by asphyxia followed by cardiopulmonary resuscitation (CPR in rats. Our results showed that expression of p-mTOR, mTOR-mediated phosphorylation of 4E-binding protein 4 (4E-BP1 and p70 ribosomal S6 protein kinase 1 (S6K1 pathways were amplified in CA rats compared to their controls. Blocking mTOR using rapamycin attenuated upregulation of pro-inflammatory cytokines (namely IL-1β, IL-6 and TNF-α, and Caspase-3, indicating cell apoptosis and also promoting the levels of vascular endothelial growth factor (VEGF and its subtype receptor VEGFR-2 in the hippocampus. Moreover, the effects of rapamycin were linked to improvement of neurological deficits and increased brain water content observed in CA rats. In conclusion, activation of mTOR signal is engaged in pathophysiological process during CA-induced transient global ischemia and blocking mTOR pathway plays a beneficial role in regulating injured neuronal tissues and neurological deficits via PIC, apoptotic Caspase-3 and VEGF mechanisms. Targeting one or more of these specific mTOR pathways and its downstream signaling molecules may present new opportunities for neural dysfunction and vulnerability related to transient global ischemia.

  2. Overnutrition, mTOR signaling, and cardiovascular diseases

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    Jia, Guanghong; Aroor, Annayya R.; Martinez-Lemus, Luis A.

    2014-01-01

    The prevalence of obesity and associated medical disorders has increased dramatically in the United States and throughout much of the world in the past decade. Obesity, induced by excess intake of carbohydrates and fats, is a major cause of Type 2 diabetes, hypertension, and the cardiorenal metabolic syndrome. There is emerging evidence that excessive nutrient intake promotes signaling through the mammalian target of rapamycin (mTOR), which, in turn, may lead to alterations of cellular metabolic signaling leading to insulin resistance and obesity-related diseases, such as diabetes, cardiovascular and kidney disease, as well as cancer. While the pivotal role of mTOR signaling in regulating metabolic stress, autophagy, and adaptive immune responses has received increasing attention, there remain many gaps in our knowledge regarding this important nutrient sensor. For example, the precise cellular signaling mechanisms linking excessive nutrient intake and enhanced mTOR signaling with increased cardiovascular and kidney disease, as well as cancer, are not well understood. In this review, we focus on the effects that the interaction between excess intake of nutrients and enhanced mTOR signaling have on the promotion of obesity-associated diseases and potential therapeutic strategies involving targeting mTOR signaling. PMID:25253086

  3. A central role for the mammalian target of rapamycin in LPS-induced anorexia in mice.

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    Yue, Yunshuang; Wang, Yi; Li, Dan; Song, Zhigang; Jiao, Hongchao; Lin, Hai

    2015-01-01

    Bacterial lipopolysaccharide (LPS), also known as endotoxin, induces profound anorexia. However, the LPS-provoked pro-inflammatory signaling cascades and the neural mechanisms underlying the development of anorexia are not clear. Mammalian target of rapamycin (mTOR) is a key regulator of metabolism, cell growth, and protein synthesis. This study aimed to determine whether the mTOR pathway is involved in LPS-induced anorexia. Effects of LPS on hypothalamic gene/protein expression in mice were measured by RT-PCR or western blotting analysis. To determine whether inhibition of mTOR signaling could attenuate LPS-induced anorexia, we administered an i.c.v. injection of rapamycin, an mTOR inhibitor, on LPS-treated male mice. In this study, we showed that LPS stimulates the mTOR signaling pathway through the enhanced phosphorylation of mTOR(Ser2448) and p70S6K(Thr389). We also showed that LPS administration increased the phosphorylation of FOXO1(Ser256), the p65 subunit of nuclear factor kappa B (Panorexia by decreasing the phosphorylation of p70S6K(Thr389), FOXO1(Ser256), and FOXO1/3a(Thr) (24) (/) (32). These results suggest promising approaches for the prevention and treatment of LPS-induced anorexia. © 2015 Society for Endocrinology.

  4. Mechanistic Target of Rapamycin-Independent Antidepressant Effects of (R)-Ketamine in a Social Defeat Stress Model.

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    Yang, Chun; Ren, Qian; Qu, Youge; Zhang, Ji-Chun; Ma, Min; Dong, Chao; Hashimoto, Kenji

    2018-01-01

    The role of the mechanistic target of rapamycin (mTOR) signaling in the antidepressant effects of ketamine is controversial. In addition to mTOR, extracellular signal-regulated kinase (ERK) is a key signaling molecule in prominent pathways that regulate protein synthesis. (R)-Ketamine has a greater potency and longer-lasting antidepressant effects than (S)-ketamine. Here we investigated whether mTOR signaling and ERK signaling play a role in the antidepressant effects of two enantiomers. The effects of mTOR inhibitors (rapamycin and AZD8055) and an ERK inhibitor (SL327) on the antidepressant effects of ketamine enantiomers in the chronic social defeat stress (CSDS) model (n = 7 or 8) and on those of ketamine enantiomers in these signaling pathways in mouse brain regions were examined. The intracerebroventricular infusion of rapamycin or AZD8055 blocked the antidepressant effects of (S)-ketamine, but not (R)-ketamine, in the CSDS model. Furthermore, (S)-ketamine, but not (R)-ketamine, significantly attenuated the decreased phosphorylation of mTOR and its downstream effector, ribosomal protein S6 kinase, in the prefrontal cortex of susceptible mice after CSDS. Pretreatment with SL327 blocked the antidepressant effects of (R)-ketamine but not (S)-ketamine. Moreover, (R)-ketamine, but not (S)-ketamine, significantly attenuated the decreased phosphorylation of ERK and its upstream effector, mitogen-activated protein kinase/ERK kinase, in the prefrontal cortex and hippocampal dentate gyrus of susceptible mice after CSDS. This study suggests that mTOR plays a role in the antidepressant effects of (S)-ketamine, but not (R)-ketamine, and that ERK plays a role in (R)-ketamine's antidepressant effects. Thus, it is unlikely that the activation of mTOR signaling is necessary for antidepressant actions of (R)-ketamine. Copyright © 2017 Society of Biological Psychiatry. Published by Elsevier Inc. All rights reserved.

  5. Rapamycin improves sociability in the BTBR T(+)Itpr3(tf)/J mouse model of autism spectrum disorders.

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    Burket, Jessica A; Benson, Andrew D; Tang, Amy H; Deutsch, Stephen I

    2014-01-01

    Overactivation of the mammalian target of rapamycin (mTOR) has been implicated in the pathogenesis of syndromic forms of autism spectrum disorders (ASDs), such as tuberous sclerosis complex, neurofibromatosis 1, and fragile X syndrome. Administration of mTORC1 (mTOR complex 1) inhibitors (e.g. rapamycin) in syndromic mouse models of ASDs improved behavior, cognition, and neuropathology. However, since only a minority of ASDs are due to the effects of single genes (∼10%), there is a need to explore inhibition of mTOR activity in mouse models that may be more relevant to the majority of nonsyndromic presentations, such as the genetically inbred BTBR T(+)Itpr3(tf)/J (BTBR) mouse model of ASDs. BTBR mice have social impairment and exhibit increased stereotypic behavior. In prior work, d-cycloserine, a partial glycineB site agonist that targets the N-methyl-d-aspartate (NMDA) receptor, was shown to improve sociability in both Balb/c and BTBR mouse models of ASDs. Importantly, NMDA receptor activation regulates mTOR signaling activity. The current study investigated the ability of rapamycin (10mg/kg, i.p.×four days), an mTORC1 inhibitor, to improve sociability and stereotypic behavior in BTBR mice. Using a standard paradigm to assess mouse social behavior, rapamycin improved several measures of sociability in the BTBR mouse, suggesting that mTOR overactivation represents a therapeutic target that mediates or contributes to impaired sociability in the BTBR mouse model of ASDs. Interestingly, there was no effect of rapamycin on stereotypic behaviors in this mouse model. Copyright © 2013 Elsevier Inc. All rights reserved.

  6. Mechanical stimulation induces mTOR signaling via an ERK-independent mechanism: implications for a direct activation of mTOR by phosphatidic acid.

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    Jae Sung You

    Full Text Available Signaling by mTOR is a well-recognized component of the pathway through which mechanical signals regulate protein synthesis and muscle mass. However, the mechanisms involved in the mechanical regulation of mTOR signaling have not been defined. Nevertheless, recent studies suggest that a mechanically-induced increase in phosphatidic acid (PA may be involved. There is also evidence which suggests that mechanical stimuli, and PA, utilize ERK to induce mTOR signaling. Hence, we reasoned that a mechanically-induced increase in PA might promote mTOR signaling via an ERK-dependent mechanism. To test this, we subjected mouse skeletal muscles to mechanical stimulation in the presence or absence of a MEK/ERK inhibitor, and then measured several commonly used markers of mTOR signaling. Transgenic mice expressing a rapamycin-resistant mutant of mTOR were also used to confirm the validity of these markers. The results demonstrated that mechanically-induced increases in p70(s6k T389 and 4E-BP1 S64 phosphorylation, and unexpectedly, a loss in total 4E-BP1, were fully mTOR-dependent signaling events. Furthermore, we determined that mechanical stimulation induced these mTOR-dependent events, and protein synthesis, through an ERK-independent mechanism. Similar to mechanical stimulation, exogenous PA also induced mTOR-dependent signaling via an ERK-independent mechanism. Moreover, PA was able to directly activate mTOR signaling in vitro. Combined, these results demonstrate that mechanical stimulation induces mTOR signaling, and protein synthesis, via an ERK-independent mechanism that potentially involves a direct interaction of PA with mTOR. Furthermore, it appears that a decrease in total 4E-BP1 may be part of the mTOR-dependent mechanism through which mechanical stimuli activate protein synthesis.

  7. Mechanical stimulation induces mTOR signaling via an ERK-independent mechanism: implications for a direct activation of mTOR by phosphatidic acid.

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    You, Jae Sung; Frey, John W; Hornberger, Troy A

    2012-01-01

    Signaling by mTOR is a well-recognized component of the pathway through which mechanical signals regulate protein synthesis and muscle mass. However, the mechanisms involved in the mechanical regulation of mTOR signaling have not been defined. Nevertheless, recent studies suggest that a mechanically-induced increase in phosphatidic acid (PA) may be involved. There is also evidence which suggests that mechanical stimuli, and PA, utilize ERK to induce mTOR signaling. Hence, we reasoned that a mechanically-induced increase in PA might promote mTOR signaling via an ERK-dependent mechanism. To test this, we subjected mouse skeletal muscles to mechanical stimulation in the presence or absence of a MEK/ERK inhibitor, and then measured several commonly used markers of mTOR signaling. Transgenic mice expressing a rapamycin-resistant mutant of mTOR were also used to confirm the validity of these markers. The results demonstrated that mechanically-induced increases in p70(s6k) T389 and 4E-BP1 S64 phosphorylation, and unexpectedly, a loss in total 4E-BP1, were fully mTOR-dependent signaling events. Furthermore, we determined that mechanical stimulation induced these mTOR-dependent events, and protein synthesis, through an ERK-independent mechanism. Similar to mechanical stimulation, exogenous PA also induced mTOR-dependent signaling via an ERK-independent mechanism. Moreover, PA was able to directly activate mTOR signaling in vitro. Combined, these results demonstrate that mechanical stimulation induces mTOR signaling, and protein synthesis, via an ERK-independent mechanism that potentially involves a direct interaction of PA with mTOR. Furthermore, it appears that a decrease in total 4E-BP1 may be part of the mTOR-dependent mechanism through which mechanical stimuli activate protein synthesis.

  8. Increased expression of (immuno)proteasome subunits during epileptogenesis is attenuated by inhibition of the mammalian target of rapamycin pathway.

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    Broekaart, Diede W M; van Scheppingen, Jackelien; Geijtenbeek, Karlijne W; Zuidberg, Mark R J; Anink, Jasper J; Baayen, Johannes C; Mühlebner, Angelika; Aronica, Eleonora; Gorter, Jan A; van Vliet, Erwin A

    2017-08-01

    Inhibition of the mammalian target of rapamycin (mTOR) pathway reduces epileptogenesis in various epilepsy models, possibly by inhibition of inflammatory processes, which may include the proteasome system. To study the role of mTOR inhibition in the regulation of the proteasome system, we investigated (immuno)proteasome expression during epileptogenesis, as well as the effects of the mTOR inhibitor rapamycin. The expression of constitutive (β1, β5) and immunoproteasome (β1i, β5i) subunits was investigated during epileptogenesis using immunohistochemistry in the electrical post-status epilepticus (SE) rat model for temporal lobe epilepsy (TLE). The effect of rapamycin was studied on (immuno)proteasome subunit expression in post-SE rats that were treated for 6 weeks. (Immuno)proteasome expression was validated in the brain tissue of patients who had SE or drug-resistant TLE and the effect of rapamycin was studied in primary human astrocyte cultures. In post-SE rats, increased (immuno)proteasome expression was detected throughout epileptogenesis in neurons and astrocytes within the hippocampus and piriform cortex and was most evident in rats that developed a progressive form of epilepsy. Rapamycin-treated post-SE rats had reduced (immuno)proteasome protein expression and a lower number of spontaneous seizures compared to vehicle-treated rats. (Immuno)proteasome expression was also increased in neurons and astrocytes within the human hippocampus after SE and in patients with drug-resistant TLE. In vitro studies using cultured human astrocytes showed that interleukin (IL)-1β-induced (immuno)proteasome gene expression could be attenuated by rapamycin. Because dysregulation of the (immuno)proteasome system is observed before the occurrence of spontaneous seizures in rats, is associated with progression of epilepsy, and can be modulated via the mTOR pathway, it may represent an interesting novel target for drug treatment in epilepsy. Wiley Periodicals, Inc. © 2017

  9. Rapamycin Regulates Bleomycin-Induced Lung Damage in SP-C-Deficient Mice

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    Satish K. Madala

    2011-01-01

    Full Text Available Injury to the distal respiratory epithelium has been implicated as an underlying cause of idiopathic lung diseases. Mutations that result in SP-C deficiencies are linked to a small subset of spontaneous and familial cases of interstitial lung disease (ILD and interstitial pulmonary fibrosis (IPF. Gene-targeted mice that lack SP-C (−/− develop an irregular ILD-like disease with age and are a model of the human SP-C related disease. In the current study, we investigated whether rapamycin could ameliorate bleomycin-induced fibrosis in the lungs of −/− mice. +/+ and −/− mice were exposed to bleomycin with either preventative administration of rapamycin or therapeutic administration beginning eight days after the bleomycin injury. Rapamycin-treatment increased weight loss and decreased survival of bleomycin-treated +/+ and −/− mice. Rapamycin did not reduce the fibrotic disease in the prophylactic or rescue experiments of either genotype of mice. Further, rapamycin treatment augmented airway resistance and reduced lung compliance of bleomycin-treated −/− mice. Rapamycin treatment was associated with an increased expression of profibrotic Th2 cytokines and reduced expression of INF-γ. These findings indicate that novel therapeutics will be required to treat individuals with SP-C deficient ILD/IPF.

  10. Mammalian Target of Rapamycin: Hitting the Bull's-Eye for Neurological Disorders

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    Zhao Zhong Chong

    2010-01-01

    Full Text Available The mammalian target of rapamycin (mTOR and its associated cell signaling pathways have garnered significant attention for their roles in cell biology and oncology. Interestingly,the explosion of information in this field has linked mTOR to neurological diseases with promising initial studies. mTOR, a 289 kDa serine/threonine protein kinase, plays an important role in cell growth and proliferation and is activated through phosphorylation in response to growth factors, mitogens and hormones. Growth factors, amino acids, cellular nutrients and oxygen deficiency can downregulate mTOR activity. The function of mTOR signaling is mediated primarily through two mTOR complexes: mTORC1 and mTORC2. mTORC1 initiates cap-dependent protein translation, a rate-limiting step of protein synthesis, through the phosphorylation of the targets eukaryotic initiation factor 4E-binding protein 1 (4EBP1 and p70 ribosomal S6 kinase (p70S6K. In contrast, mTORC2 regulates development of the cytoskeleton and also controls cell survival. Although closely tied to tumorigenesis, mTOR and the downstream signaling pathways are significantly involved in the central nervous system (CNS with synaptic plasticity, memory retention, neuroendocrine regulation associated with food intake and puberty and modulation of neuronal repair following injury. The signaling pathways of mTOR also are believed to be a significant component in a number of neurological diseases, such as Alzheimer disease, Parkinson disease and Huntington disease, tuberous sclerosis, neurofibromatosis, fragile X syndrome, epilepsy, traumatic brain injury and ischemic stroke. Here we describe the role of mTOR in the CNS and illustrate the potential for new strategies directed against neurological disorders.

  11. Dysregulation of Mammalian Target of Rapamycin Signaling in Mouse Models of Autism.

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    Huber, Kimberly M; Klann, Eric; Costa-Mattioli, Mauro; Zukin, R Suzanne

    2015-10-14

    The mammalian target of rapamycin (mTOR) is a central regulator of a diverse array of cellular processes, including cell growth, proliferation, autophagy, translation, and actin polymerization. Components of the mTOR cascade are present at synapses and influence synaptic plasticity and spine morphogenesis. A prevailing view is that the study of mTOR and its role in autism spectrum disorders (ASDs) will elucidate the molecular mechanisms by which mTOR regulates neuronal function under physiological and pathological conditions. Although many ASDs arise as a result of mutations in genes with multiple molecular functions, they appear to converge on common biological pathways that give rise to autism-relevant behaviors. Dysregulation of mTOR signaling has been identified as a phenotypic feature common to fragile X syndrome, tuberous sclerosis complex 1 and 2, neurofibromatosis 1, phosphatase and tensin homolog, and potentially Rett syndrome. Below are a summary of topics covered in a symposium that presents dysregulation of mTOR as a unifying theme in a subset of ASDs. Copyright © 2015 the authors 0270-6474/15/3513836-07$15.00/0.

  12. Phospholipase D regulates the size of skeletal muscle cells through the activation of mTOR signaling.

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    Jaafar, Rami; De Larichaudy, Joffrey; Chanon, Stéphanie; Euthine, Vanessa; Durand, Christine; Naro, Fabio; Bertolino, Philippe; Vidal, Hubert; Lefai, Etienne; Némoz, Georges

    2013-08-02

    mTOR is a major actor of skeletal muscle mass regulation in situations of atrophy or hypertrophy. It is established that Phospholipase D (PLD) activates mTOR signaling, through the binding of its product phosphatidic acid (PA) to mTOR protein. An influence of PLD on muscle cell size could thus be suspected. We explored the consequences of altered expression and activity of PLD isoforms in differentiated L6 myotubes. Inhibition or down-regulation of the PLD1 isoform markedly decreased myotube size and muscle specific protein content. Conversely, PLD1 overexpression induced muscle cell hypertrophy, both in vitro in myotubes and in vivo in mouse gastrocnemius. In the presence of atrophy-promoting dexamethasone, PLD1 overexpression or addition of exogenous PA protected myotubes against atrophy. Similarly, exogenous PA protected myotubes against TNFα-induced atrophy. Moreover, the modulation of PLD expression or activity in myotubes showed that PLD1 negatively regulates the expression of factors involved in muscle protein degradation, such as the E3-ubiquitin ligases Murf1 and Atrogin-1, and the Foxo3 transcription factor. Inhibition of mTOR by PP242 abolished the positive effects of PLD1 on myotubes, whereas modulating PLD influenced the phosphorylation of both S6K1 and Akt, which are respectively substrates of mTORC1 and mTORC2 complexes. These observations suggest that PLD1 acts through the activation of both mTORC1 and mTORC2 to induce positive trophic effects on muscle cells. This pathway may offer interesting therapeutic potentialities in the treatment of muscle wasting.

  13. Regulation of cardiac miR-208a, an inducer of obesity, by rapamycin and nebivolol.

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    Gul, Rukhsana; Mahmood, Abuzar; Luck, Christian; Lum-Naihe, Kelly; Alfadda, Assim A; Speth, Robert C; Pulakat, Lakshmi

    2015-11-01

    Resistance to obesity is observed in rodents and humans treated with rapamycin (Rap) or nebivolol (Neb). Because cardiac miR-208a promotes obesity, this study tested whether the modes of actions of Rap and Neb involve inhibition of miR-208a. Mouse cardiomyocyte HL-1 cells and Zucker obese (ZO) rats were used to investigate regulation of cardiac miR-208a. Angiotensin II (Ang II) increased miR-208a expression in HL-1 cells. Pretreatment with an AT1 receptor (AT1R) antagonist, losartan (1 μM), antagonized this effect, whereas a phospholipase C inhibitor, U73122 (10 μM), and an NADPH oxidase inhibitor, apocynin (0.5 mM), did not. Ang II-induced increase in miR-208a was suppressed by Rap (10 nM), an inhibitor of nutrient sensor kinase mTORC1, and Neb (1 μM), a 3rd generation β-blocker that suppressed bioavailable AT1R binding of (125) I-Ang II. Thus, suppression of AT1R expression by Neb, inhibition of AT1R activation by losartan, and inhibition of AT1R-induced activation of mTORC1 by Rap attenuated the Ang II-induced increase in miR-208a. In ZO rats, Rap treatment (750 μg kg(-1)  day(-1) ; 12 weeks) reduced obesity despite similar food intake, suppressed cardiac miR-208a, and increased cardiac MED13, a suppresser of obesity. Rap and Neb suppressed cardiac miR-208a. Suppression of miR-208a and increase in MED13 correlated with attenuated weight gain despite leptin resistance. © 2015 The Obesity Society.

  14. mTOR direct interactions with Rheb-GTPase and raptor: sub-cellular localization using fluorescence lifetime imaging

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    Yadav Rahul B

    2013-01-01

    Full Text Available Abstract Background The mammalian target of rapamycin (mTOR signalling pathway has a key role in cellular regulation and several diseases. While it is thought that Rheb GTPase regulates mTOR, acting immediately upstream, while raptor is immediately downstream of mTOR, direct interactions have yet to be verified in living cells, furthermore the localisation of Rheb has been reported to have only a cytoplasmic cellular localization. Results In this study a cytoplasmic as well as a significant sub-cellular nuclear mTOR localization was shown , utilizing green and red fluorescent protein (GFP and DsRed fusion and highly sensitive single photon counting fluorescence lifetime imaging microscopy (FLIM of live cells. The interaction of the mTORC1 components Rheb, mTOR and raptor, tagged with EGFP/DsRed was determined using fluorescence energy transfer-FLIM. The excited-state lifetime of EGFP-mTOR of ~2400 ps was reduced by energy transfer to ~2200 ps in the cytoplasm and to 2000 ps in the nucleus when co-expressed with DsRed-Rheb, similar results being obtained for co-expressed EGFP-mTOR and DsRed-raptor. The localization and distribution of mTOR was modified by amino acid withdrawal and re-addition but not by rapamycin. Conclusions The results illustrate the power of GFP-technology combined with FRET-FLIM imaging in the study of the interaction of signalling components in living cells, here providing evidence for a direct physical interaction between mTOR and Rheb and between mTOR and raptor in living cells for the first time.

  15. mTOR is essential for the proteotoxic stress response, HSF1 activation and heat shock protein synthesis.

    Directory of Open Access Journals (Sweden)

    Shiuh-Dih Chou

    Full Text Available The target of rapamycin (TOR is a high molecular weight protein kinase that regulates many processes in cells in response to mitogens and variations in nutrient availability. Here we have shown that mTOR in human tissue culture cells plays a key role in responses to proteotoxic stress and that reduction in mTOR levels by RNA interference leads to increase sensitivity to heat shock. This effect was accompanied by a drastic reduction in ability to synthesize heat shock proteins (HSP, including Hsp70, Hsp90 and Hsp110. As HSP transcription is regulated by heat shock transcription factor 1 (HSF1, we examined whether mTOR could directly phosphorylate this factor. Indeed, we determined that mTOR could directly phosphorylate HSF1 on serine 326, a key residue in transcriptional activation. HSF1 was phosphorylated on S326 immediately after heat shock and was triggered by other cell stressors including proteasome inhibitors and sodium arsenite. Null mutation of S326 to alanine led to loss of ability to activate an HSF1-regulated promoter-reporter construct, indicating a direct role for mTOR and S326 in transcriptional regulation of HSP genes during stress. As mTOR is known to exist in at least two intracellular complexes, mTORC1 and mTOR2 we examined which complex might interact with HSF1. Indeed mTORC1 inhibitor rapamycin prevented HSF1-S326 phosphorylation, suggesting that this complex is involved in HSF1 regulation in stress. Our experiments therefore suggest a key role for mTORC1 in transcriptional responses to proteotoxic stress.

  16. Co-Localization of Insulin-Like Growth Factor Binding Protein-1, Casein Kinase-2β, and Mechanistic Target of Rapamycin in Human Hepatocellular Carcinoma Cells as Demonstrated by Dual Immunofluorescence and in Situ Proximity Ligation Assay.

    Science.gov (United States)

    Singal, Sahil S; Nygard, Karen; Dhruv, Manthan R; Biggar, Kyle; Abu Shehab, Majida; Shun-Cheng Li, Shawn; Jansson, Thomas; Gupta, Madhulika B

    2017-10-14

    Insulin-like growth factor binding protein (IGFBP)-1 influences fetal growth by modifying insulin-like growth factor-I (IGF-I) bioavailability. IGFBP-1 phosphorylation, which markedly increases its affinity for IGF-I, is regulated by mechanistic target of rapamycin (mTOR) and casein kinase (CSNK)-2. However, the underlying molecular mechanisms remain unknown. We examined the cellular localization and potential interactions of IGFBP-1, CSNK-2β, and mTOR as a prerequisite for protein-protein interaction. Analysis of dual immunofluorescence images indicated a potential perinuclear co-localization between IGFBP-1 and CSNK-2β and a nuclear co-localization between CSNK-2β and mTOR. Proximity ligation assay (PLA) indicated proximity between IGFBP-1 and CSNK-2β as well as mTOR and CSNK-2β but not between mTOR and IGFBP-1. Three-dimensional rendering of the PLA images validated that IGFBP-1 and CSNK-2β interactions were in the perinuclear region and mTOR and CSNK-2β interactions were predominantly perinuclear rather than nuclear as indicated by mTOR and CSNK-2β co-localization. Compared with control, hypoxia and rapamycin treatment showed markedly amplified PLA signals for IGFBP-1 and CSNK-2β (approximately 18-fold, P = 0.0002). Stable isotope labeling with multiple reaction monitoring-mass spectrometry demonstrated that hypoxia and rapamycin treatment increased IGFBP-1 phosphorylation at Ser98/Ser101/Ser119/Ser174 but most considerably (106-fold) at Ser169. We report interactions between CSNK-2β and IGFBP-1 as well as mTOR and CSNK-2β, providing strong evidence of a mechanistic link between mTOR and IGF-I signaling, two critical regulators of cell growth via CSNK-2. Copyright © 2018 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

  17. Mammalian target of rapamycin inhibitor-associated stomatitis

    NARCIS (Netherlands)

    Boers-Doets, Christine B.; Raber-Durlacher, Judith E.; Treister, Nathaniel S.; Epstein, Joel B.; Arends, Anniek B. P.; Wiersma, Diede R.; Lalla, Rajesh V.; Logan, Richard M.; van Erp, Nielka P.; Gelderblom, Hans

    2013-01-01

    With the recent introduction of inhibitors of mammalian target of rapamycin (mTOR) in oncology, distinct cutaneous and oral adverse events have been identified. In fact, stomatitis and rash are documented as the most frequent and potentially dose-limiting side effects. Clinically, mTOR

  18. Enoyl-CoA hydratase-1 regulates mTOR signaling and apoptosis by sensing nutrients.

    Science.gov (United States)

    Zhang, Ya-Kun; Qu, Yuan-Yuan; Lin, Yan; Wu, Xiao-Hui; Chen, Hou-Zao; Wang, Xu; Zhou, Kai-Qiang; Wei, Yun; Guo, Fushen; Yao, Cui-Fang; He, Xia-Di; Liu, Li-Xia; Yang, Chen; Guan, Zong-Yuan; Wang, Shi-Dong; Zhao, Jianyuan; Liu, De-Pei; Zhao, Shi-Min; Xu, Wei

    2017-09-06

    The oncogenic mechanisms of overnutrition, a confirmed independent cancer risk factor, remain poorly understood. Herein, we report that enoyl-CoA hydratase-1 (ECHS1), the enzyme involved in the oxidation of fatty acids (FAs) and branched-chain amino acids (BCAAs), senses nutrients and promotes mTOR activation and apoptotic resistance. Nutrients-promoted acetylation of lys(101) of ECHS1 impedes ECHS1 activity by impairing enoyl-CoA binding, promoting ECHS1 degradation and blocking its mitochondrial translocation through inducing ubiquitination. As a result, nutrients induce the accumulation of BCAAs and FAs that activate mTOR signaling and stimulate apoptosis, respectively. The latter was overcome by selection of BCL-2 overexpressing cells under overnutrition conditions. The oncogenic effects of nutrients were reversed by SIRT3, which deacetylates lys(101) acetylation. Severely decreased ECHS1, accumulation of BCAAs and FAs, activation of mTOR and overexpression of BCL-2 were observed in cancer tissues from metabolic organs. Our results identified ECHS1, a nutrients-sensing protein that transforms nutrient signals into oncogenic signals.Overnutrition has been linked to increased risk of cancer. Here, the authors show that exceeding nutrients suppress Enoyl-CoA hydratase-1 (ECHS1) activity by inducing its acetylation resulting in accumulation of fatty acids and branched-chain amino acids and oncogenic mTOR activation.

  19. 14-3-3γ Regulates Lipopolysaccharide-Induced Inflammatory Responses and Lactation in Dairy Cow Mammary Epithelial Cells by Inhibiting NF-κB and MAPKs and Up-Regulating mTOR Signaling

    Science.gov (United States)

    Liu, Lixin; Lin, Ye; Liu, Lili; Bian, Yanjie; Zhang, Li; Gao, Xuejun; Li, Qingzhang

    2015-01-01

    As a protective factor for lipopolysaccharide (LPS)-induced injury, 14-3-3γ has been the subject of recent research. Nevertheless, whether 14-3-3γ can regulate lactation in dairy cow mammary epithelial cells (DCMECs) induced by LPS remains unknown. Here, the anti-inflammatory effect and lactation regulating ability of 14-3-3γ in LPS-induced DCMECs are investigated for the first time, and the molecular mechanisms responsible for their effects are explored. The results of qRT-PCR showed that 14-3-3γ overexpression significantly inhibited the mRNA expression of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), interleukin-1β (IL-1β) and inducible nitric oxide synthase (iNOS). Enzyme-linked immunosorbent assay (ELISA) analysis revealed that 14-3-3γ overexpression also suppressed the production of TNF-α and IL-6 in cell culture supernatants. Meanwhile, CASY-TT Analyser System showed that 14-3-3γ overexpression clearly increased the viability and proliferation of cells. The results of kit methods and western blot analysis showed that 14-3-3γ overexpression promoted the secretion of triglycerides and lactose and the synthesis of β-casein. Furthermore, the expression of genes relevant to nuclear factor-κB (NF-κB) and mitogen-activated protein kinase (MAPKs) and lactation-associated proteins were assessed by western blot, and the results suggested that 14-3-3γ overexpression inactivated the NF-κB and MAPK signaling pathways by down-regulating extracellular signal regulated protein kinase (ERK), p38 mitogen-activated protein kinase (p38MAPK) and inhibitor of NF-κB (IκB) phosphorylation levels, as well as by inhibiting NF-κB translocation. Meanwhile, 14-3-3γ overexpression enhanced the expression levels of β-casein, mammalian target of rapamycin (mTOR), ribosomal protein S6 kinase 1 (S6K1), serine/threonine protein kinase Akt 1 (AKT1), sterol regulatory element binding protein 1 (SREBP1) and peroxisome proliferator-activated receptor gamma (PPAR

  20. 14-3-3γ Regulates Lipopolysaccharide-Induced Inflammatory Responses and Lactation in Dairy Cow Mammary Epithelial Cells by Inhibiting NF-κB and MAPKs and Up-Regulating mTOR Signaling

    Directory of Open Access Journals (Sweden)

    Lixin Liu

    2015-07-01

    Full Text Available As a protective factor for lipopolysaccharide (LPS-induced injury, 14-3-3γ has been the subject of recent research. Nevertheless, whether 14-3-3γ can regulate lactation in dairy cow mammary epithelial cells (DCMECs induced by LPS remains unknown. Here, the anti-inflammatory effect and lactation regulating ability of 14-3-3γ in LPS-induced DCMECs are investigated for the first time, and the molecular mechanisms responsible for their effects are explored. The results of qRT-PCR showed that 14-3-3γ overexpression significantly inhibited the mRNA expression of tumor necrosis factor-α (TNF-α, interleukin-6 (IL-6, interleukin-1β (IL-1β and inducible nitric oxide synthase (iNOS. Enzyme-linked immunosorbent assay (ELISA analysis revealed that 14-3-3γ overexpression also suppressed the production of TNF-α and IL-6 in cell culture supernatants. Meanwhile, CASY-TT Analyser System showed that 14-3-3γ overexpression clearly increased the viability and proliferation of cells. The results of kit methods and western blot analysis showed that 14-3-3γ overexpression promoted the secretion of triglycerides and lactose and the synthesis of β-casein. Furthermore, the expression of genes relevant to nuclear factor-κB (NF-κB and mitogen-activated protein kinase (MAPKs and lactation-associated proteins were assessed by western blot, and the results suggested that 14-3-3γ overexpression inactivated the NF-κB and MAPK signaling pathways by down-regulating extracellular signal regulated protein kinase (ERK, p38 mitogen-activated protein kinase (p38MAPK and inhibitor of NF-κB (IκB phosphorylation levels, as well as by inhibiting NF-κB translocation. Meanwhile, 14-3-3γ overexpression enhanced the expression levels of β-casein, mammalian target of rapamycin (mTOR, ribosomal protein S6 kinase 1 (S6K1, serine/threonine protein kinase Akt 1 (AKT1, sterol regulatory element binding protein 1 (SREBP1 and peroxisome proliferator-activated receptor gamma

  1. Differing Effects of Systemically Administered Rapamycin on Consolidation and Reconsolidation of Context vs. Cued Fear Memories

    Science.gov (United States)

    Glover, Ebony M.; Ressler, Kerry J.; Davis, Michael

    2010-01-01

    Rapamycin, an inhibitor of the mammalian target of rapamycin (mTOR) kinase, has attracted interest as a possible prophylactic for post-traumatic stress disorder (PTSD)-associated fear memories. We report here that although rapamycin (40 mg/kg, i.p.) disrupted the consolidation and reconsolidation of fear-potentiated startle paradigm to a…

  2. Oncolytic virotherapy synergism with signaling inhibitors: Rapamycin increases myxoma virus tropism for human tumor cells.

    Science.gov (United States)

    Stanford, Marianne M; Barrett, John W; Nazarian, Steven H; Werden, Steven; McFadden, Grant

    2007-02-01

    Myxoma virus is a rabbit-specific poxvirus pathogen that also exhibits a unique tropism for human tumor cells and is dramatically oncolytic for human cancer xenografts. Most tumor cell lines tested are permissive for myxoma infection in a fashion intimately tied to the activation state of Akt kinase. A host range factor of myxoma virus, M-T5, directly interacts with Akt and mediates myxoma virus tumor cell tropism. mTOR is a regulator of cell growth and metabolism downstream of Akt and is specifically inhibited by rapamycin. We report that treatment of nonpermissive human tumor cell lines, which normally restrict myxoma virus replication, with rapamycin dramatically increased virus tropism and spread in vitro. This increased myxoma replication is concomitant with global effects on mTOR signaling, specifically, an increase in Akt kinase. In contrast to the effects on human cancer cells, rapamycin does not increase myxoma virus replication in rabbit cell lines or permissive human tumor cell lines with constitutively active Akt. This indicates that rapamycin increases the oncolytic capacity of myxoma virus for human cancer cells by reconfiguring the internal cell signaling environment to one that is optimal for productive virus replication and suggests the possibility of a potentially therapeutic synergism between kinase signaling inhibitors and oncolytic poxviruses for cancer treatment.

  3. Target-of-rapamycin complex 1 (Torc1) signaling modulates cilia size and function through protein synthesis regulation

    Science.gov (United States)

    Yuan, Shiaulou; Li, Jade; Diener, Dennis R.; Choma, Michael A.; Rosenbaum, Joel L.; Sun, Zhaoxia

    2012-01-01

    The cilium serves as a cellular antenna by coordinating upstream environmental cues with numerous downstream signaling processes that are indispensable for the function of the cell. This role is supported by the revelation that defects of the cilium underlie an emerging class of human disorders, termed “ciliopathies.” Although mounting interest in the cilium has demonstrated the essential role that the organelle plays in vertebrate development, homeostasis, and disease pathogenesis, the mechanisms regulating cilia morphology and function remain unclear. Here, we show that the target-of-rapamycin (TOR) growth pathway modulates cilia size and function during zebrafish development. Knockdown of tuberous sclerosis complex 1a (tsc1a), which encodes an upstream inhibitor of TOR complex 1 (Torc1), increases cilia length. In contrast, treatment of embryos with rapamycin, an inhibitor of Torc1, shortens cilia length. Overexpression of ribosomal protein S6 kinase 1 (S6k1), which encodes a downstream substrate of Torc1, lengthens cilia. Furthermore, we provide evidence that TOR-mediated cilia assembly is evolutionarily conserved and that protein synthesis is essential for this regulation. Finally, we demonstrate that TOR signaling and cilia length are pivotal for a variety of downstream ciliary functions, such as cilia motility, fluid flow generation, and the establishment of left-right body asymmetry. Our findings reveal a unique role for the TOR pathway in regulating cilia size through protein synthesis and suggest that appropriate and defined lengths are necessary for proper function of the cilium. PMID:22308353

  4. Central exercise action increases the AMPK and mTOR response to leptin.

    Directory of Open Access Journals (Sweden)

    Eduardo R Ropelle

    Full Text Available AMP-activated protein kinase (AMPK and mammalian Target of Rapamycin (mTOR are key regulators of cellular energy balance and of the effects of leptin on food intake. Acute exercise is associated with increased sensitivity to the effects of leptin on food intake in an IL-6-dependent manner. To determine whether exercise ameliorates the AMPK and mTOR response to leptin in the hypothalamus in an IL-6-dependent manner, rats performed two 3-h exercise bouts, separated by one 45-min rest period. Intracerebroventricular IL-6 infusion reduced food intake and pretreatment with AMPK activators and mTOR inhibitor prevented IL-6-induced anorexia. Activators of AMPK and fasting increased food intake in control rats to a greater extent than that observed in exercised ones, whereas inhibitor of AMPK had the opposite effect. Furthermore, the reduction of AMPK and ACC phosphorylation and increase in phosphorylation of proteins involved in mTOR signal transduction, observed in the hypothalamus after leptin infusion, were more pronounced in both lean and diet-induced obesity rats after acute exercise. Treatment with leptin reduced food intake in exercised rats that were pretreated with vehicle, although no increase in responsiveness to leptin-induced anorexia after pretreatment with anti-IL6 antibody, AICAR or Rapamycin was detected. Thus, the effects of leptin on the AMPK/mTOR pathway, potentiated by acute exercise, may contribute to appetite suppressive actions in the hypothalamus.

  5. Mammalian target of rapamycin is essential for cardiomyocyte survival and heart development in mice

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Pengpeng [Key Laboratory of Swine Genetics and Breeding, Ministry of Agriculture, College of Animal Science and Technology, Huazhong Agricultural University, Wuhan 430070 (China); Key Laboratory of Agricultural Animal Genetics, Breeding and Reproduction, Ministry of Education, College of Animal Science and Technology, Huazhong Agricultural University, Wuhan 430070 (China); Department of Animal Sciences, Purdue University, West Lafayette, IN 47907 (United States); Shan, Tizhong; Liang, Xinrong [Department of Animal Sciences, Purdue University, West Lafayette, IN 47907 (United States); Deng, Changyan [Key Laboratory of Swine Genetics and Breeding, Ministry of Agriculture, College of Animal Science and Technology, Huazhong Agricultural University, Wuhan 430070 (China); Key Laboratory of Agricultural Animal Genetics, Breeding and Reproduction, Ministry of Education, College of Animal Science and Technology, Huazhong Agricultural University, Wuhan 430070 (China); Kuang, Shihuan, E-mail: skuang@purdue.edu [Department of Animal Sciences, Purdue University, West Lafayette, IN 47907 (United States)

    2014-09-12

    Highlights: • mTOR is a critical regulator of many biological processes yet its function in heart is not well understood. • MCK-Cre/Mtor{sup flox/flox} mice were established to delete Mtor in cardiomyocytes. • The mTOR-mKO mice developed normally but die prematurely within 5 weeks after birth due to heart disease. • The mTOR-mKO mice had dilated myocardium and increased cell death. • mTOR-mKO hearts had reduced expression of metabolic genes and activation of mTOR target proteins. - Abstract: Mammalian target of rapamycin (mTOR) is a critical regulator of protein synthesis, cell proliferation and energy metabolism. As constitutive knockout of Mtor leads to embryonic lethality, the in vivo function of mTOR in perinatal development and postnatal growth of heart is not well defined. In this study, we established a muscle-specific mTOR conditional knockout mouse model (mTOR-mKO) by crossing MCK-Cre and Mtor{sup flox/flox} mice. Although the mTOR-mKO mice survived embryonic and perinatal development, they exhibited severe postnatal growth retardation, cardiac muscle pathology and premature death. At the cellular level, the cardiac muscle of mTOR-mKO mice had fewer cardiomyocytes due to apoptosis and necrosis, leading to dilated cardiomyopathy. At the molecular level, the cardiac muscle of mTOR-mKO mice expressed lower levels of fatty acid oxidation and glycolysis related genes compared to the WT littermates. In addition, the mTOR-mKO cardiac muscle had reduced Myh6 but elevated Myh7 expression, indicating cardiac muscle degeneration. Furthermore, deletion of Mtor dramatically decreased the phosphorylation of S6 and AKT, two key targets downstream of mTORC1 and mTORC2 mediating the normal function of mTOR. These results demonstrate that mTOR is essential for cardiomyocyte survival and cardiac muscle function.

  6. Inhibition of mammalian target of rapamycin by rapamycin increases the radiosensitivity of esophageal carcinoma Eca109 cells

    OpenAIRE

    ZHANG, DEJUN; XIANG, JIE; GU, YUMING; XU, WEI; XU, HAO; ZU, MAOHENG; PEI, DONGSHENG; ZHENG, JUNNIAN

    2014-01-01

    The aim of the present study was to investigate whether radiation induces the mammalian target of rapamycin (Rap) (mTOR) signaling pathway in esophageal carcinoma Eca109 cells, and whether mTOR inhibition by rapamycin increases Eca109 cell radiosensitivity. Changes in the levels of mTOR signaling pathway and DNA damage-repair proteins in Eca109 cells prior to and following radiation were determined. The Eca109 cells were treated with Rap (0, 100, 200 and 400 nmol/l) in combination with radiat...

  7. Discovery of GSK2126458, a Highly Potent Inhibitor of PI3K and the Mammalian Target of Rapamycin

    Energy Technology Data Exchange (ETDEWEB)

    Knight, Steven D.; Adams, Nicholas D.; Burgess, Joelle L.; Chaudhari, Amita M.; Darcy, Michael G.; Donatelli, Carla A.; Luengo, Juan I.; Newlander, Ken A.; Parrish, Cynthia A.; Ridgers, Lance H.; Sarpong, Martha A.; Schmidt, Stanley J.; Aller, Glenn S.Van; Carson, Jeffrey D.; Diamond, Melody A.; Elkins, Patricia A.; Gardiner, Christine M.; Garver, Eric; Gilbert, Seth A.; Gontarek, Richard R.; Jackson, Jeffrey R.; Kershner, Kevin L.; Luo, Lusong; Raha, Kaushik; Sherk, Christian S.; Sung, Chiu-Mei; Sutton, David; Tummino, Peter J.; Wegrzyn, Ronald J.; Auger, Kurt R.; Dhanak, Dashyant (GSKPA)

    2010-09-30

    Phosphoinositide 3-kinase {alpha} (PI3K{alpha}) is a critical regulator of cell growth and transformation, and its signaling pathway is the most commonly mutated pathway in human cancers. The mammalian target of rapamycin (mTOR), a class IV PI3K protein kinase, is also a central regulator of cell growth, and mTOR inhibitors are believed to augment the antiproliferative efficacy of PI3K/AKT pathway inhibition. 2,4-Difluoro-N-{l_brace}2-(methyloxy)-5-[4-(4-pyridazinyl)-6-quinolinyl]-3-pyridinyl{r_brace}benzenesulfonamide (GSK2126458, 1) has been identified as a highly potent, orally bioavailable inhibitor of PI3K{alpha} and mTOR with in vivo activity in both pharmacodynamic and tumor growth efficacy models. Compound 1 is currently being evaluated in human clinical trials for the treatment of cancer.

  8. Protective Effects of the Mechanistic Target of Rapamycin against Excess Iron and Ferroptosis in Cardiomyocytes.

    Science.gov (United States)

    Baba, Yuichi; Higa, Jason K; Shimada, Briana K; Horiuchi, Kate M; Suhara, Tomohiro; Kobayashi, Motoi; Woo, Jonathan D; Aoyagi, Hiroko; Marh, Karra S; Kitaoka, Hiroaki; Matsui, Takashi

    2017-11-10

    Clinical studies suggest that myocardial iron is a risk factor for left ventricular remodeling in patients after myocardial infarction (MI). Ferroptosis was recently reported as a mechanism of iron-dependent non-apoptotic cell death. However, ferroptosis in the heart is not well understood. The mechanistic target of rapamycin (mTOR) protects the heart against pathological stimuli such as ischemia. To define the role of cardiac mTOR on cell survival in iron-mediated cell death, we examined cardiomyocyte (CM) cell viability under excess iron and ferroptosis conditions. Adult mouse CMs were isolated from cardiac-specific mTOR transgenic (mTOR-Tg), cardiac-specific mTOR knockout (mTOR-KO), or control mice. CMs were treated with ferric iron [Fe (III)]-citrate, erastin, a class 1 ferroptosis inducer, or Ras Selective Lethal 3 (RSL3), a class 2 ferroptosis inducer. Live/Dead Cell Viability Assays revealed that Fe (III)-citrate, erastin, and RSL3 induced cell death. Co-treatment with ferrostatin-1, a ferroptosis inhibitor, inhibited cell death in all conditions. mTOR overexpression suppressed Fe (III)-citrate, erastin, and RSL3-induced cell death, while mTOR deletion exaggerated cell death in these conditions. H2DCFDA (2',7'-dichlorodihydrofluorescein diacetate) measurement of reactive oxygen species (ROS) production showed that erastin-induced ROS production was significantly lower in mTOR-Tg versus control CMs. These findings suggest that ferroptosis is a significant type of cell death in CMs, and that mTOR plays an important role in protecting CMs against excess iron and ferroptosis, at least in part by regulating ROS production. Understanding the effects of mTOR in preventing iron-mediated cell death will provide a new therapy for patients with MI. Copyright © 2017, American Journal of Physiology-Heart and Circulatory Physiology.

  9. Rapamycin inhibits IGF-1 stimulated cell motility through PP2A pathway.

    Directory of Open Access Journals (Sweden)

    Lei Liu

    2010-05-01

    Full Text Available Serine/threonine (Ser/Thr protein phosphatase 2A (PP2A has been implicated as a novel component of the mammalian target of rapamycin (mTOR signaling pathway. Recently we have demonstrated that mTOR regulates cell motility in part through p70 S6 kinase 1 (S6K1 and eukaryotic initiation factor 4E (eIF4E binding protein 1 (4E-BP1 pathways. Little is known about the role of PP2A in the mTOR-mediated cell motility. Here we show that rapamycin inhibited the basal or insulin-like growth factor 1 (IGF-1-induced motility of human Ewing sarcoma (Rh1 and rhabdomyosarcoma (Rh30 cells. Treatment of the cells with rapamycin activated PP2A activity, and concurrently inhibited IGF-1 stimulated phosphorylation of Erk1/2. Inhibition of Erk1/2 with PD98059 did not significantly affect the basal mobility of the cells, but dramatically inhibited IGF-1-induced cell motility. Furthermore, inhibition of PP2A with okadaic acid significantly attenuated the inhibitory effect of rapamycin on IGF-1-stimulated phosphorylation of Erk1/2 as well as cell motility. Consistently, expression of dominant negative PP2A conferred resistance to IGF-1-stimulated phosphorylation of Erk1/2 and cell motility. Expression of constitutively active MKK1 also attenuated rapamycin inhibition of IGF-1-stimulated phosphorylation of Erk1/2 and cell motility. The results suggest that rapamycin inhibits cell motility, in part by targeting PP2A-Erk1/2 pathway.

  10. mTOR signaling disruption from myeloid-derived suppressive cells protects against immune-mediated hepatic injury through the HIF1α-dependent glycolytic pathway.

    Science.gov (United States)

    Chen, Xi; Zhang, Zhengguo; Bi, Yujing; Fu, Zan; Gong, Pingsheng; Li, Yan; Yu, Qing; Jia, Anna; Wang, Jian; Xue, Lixiang; Yang, Hui; Liu, Guangwei

    2016-12-01

    The mechanistic target of rapamycin (mTOR) pathway integrates diverse environmental inputs, including immune signals and metabolic cues, to direct innate and adaptive immune responses. Myeloid-derived suppressive cells (MDSCs) are a heterogeneous cell population that plays a crucial regulatory effect in immune-related diseases. However, whether mTOR signaling affects the functions of MDSCs remains largely unexplored. Here, we show that mTOR signaling is a pivotal, negative determinant of MDSC function in immune-mediated hepatic injury (IMH) diseases. In the context of IMH, the blocking of mTOR with rapamycin or mTOR-deficient CD11b+Gr1+ MDSCs mediates the protection against IMH; mTOR with rapamycin and mTOR-deficient CD11b+Gr1+ MDSCs are suppressive immune modulators that result in less IFN-γ-producing TH1 cells and more Foxp3+ Tregs Mechanistically, mTOR activity down-regulation in MDSCs induced iNOS expressions and NO productions. Pharmacologic inhibitions of iNOS completely eliminate MDSC-suppressive function and lose their inducible effects on T cell differentiation. Importantly, HIF1α-dependent glycolytic activity is responsible for mTOR-deficient, increased MDSC functional changes in IMH inflammation. Thus, these data demonstrate that mTOR acts as a fundamental "rheostat" in MDSCs to link immunologic signals to glycolytic pathways and functional fitness and highlights a central role of metabolic programming of MDSC-suppressive activity in protecting against immune hepatic injuries. © Society for Leukocyte Biology.

  11. Correlation between telomerase and mTOR pathway in cancer stem cells.

    Science.gov (United States)

    Dogan, Fatma; Biray Avci, Cigir

    2018-01-30

    Cancer stem cells (CSCs), which are defined as a subset of tumor cells, are able to self-renew, proliferate, differentiate similar to normal stem cells. Therefore, targeting CSCs has been considered as a new approach in cancer therapy. The mammalian target of rapamycin (mTOR) is a receptor tyrosine kinase which plays an important role in regulating cell proliferation, differentiation, cell growth, self-renewal in CSCs. On the other hand, hTERT overactivation provides replicative feature and immortality to CSCs, so the stemness and replicative properties of CSCs depend on telomerase activity. Therefore hTERT/telomerase activity may become a universal biomarker for anticancer therapy and it is an attractive therapeutic target for CSCs. It is known that mTOR regulates telomerase activity at the translational and post-translational level. Researchers show that mTOR inhibitor rapamycin reduces telomerase activity without changing hTERT mRNA activity. Correlation between mTOR and hTERT is important for survival and immortality of cancer cells. In addition, the PI3K/AKT/mTOR signaling pathway and hTERT up-regulation are related with cancer stemness features and drug resistance. mTOR inhibitor and TERT inhibitor combination may construct a novel strategy in cancer stem cells and it can make a double effect on telomerase enzyme. Consequently, inhibition of PI3K/AKT/mTOR signaling pathway components and hTERT activation may prohibit CSC self-renewal and surpass CSC-mediated resistance in order to develop new cancer therapeutics. Copyright © 2017 Elsevier B.V. All rights reserved.

  12. 14-3-3γ affects mTOR pathway and regulates lactogenesis in dairy cow mammary epithelial cells.

    Science.gov (United States)

    Khudhair, Nagam; Luo, Chaochao; Khalid, Ahmed; Zhang, Li; Zhang, Shuang; Ao, Jinxia; Li, Qingzhang; Gao, Xuejun

    2015-08-01

    14-3-3 proteins are an acidic protein family that is highly conserved and widely distributed in eukaryotic cells. Recent studies have found that 14-3-3 proteins play critical roles in cell signal transductions, cell growth and differentiation, and protein synthesis. 14-3-3γ is an important member of 14-3-3 protein family. In our previous study, we found that 14-3-3γ was upregulated by estrogen in dairy cow mammary epithelial cell (DCMEC), but the function and mechanism of 14-3-3γ is not known. In this experiment, we first cultured and purified the primary DCMEC and found 14-3-3γ located both in the cytoplasm and nucleus by using immunofluorescence assay. Methionine, lysine, estrogen, and prolactin could upregulate the expression of 14-3-3γ, stimulate the secretion of β-casein and triglyceride, and raise the cell viability of DCMEC. We constructed a stable 14-3-3γ overexpression cell line of DCMEC and found that the expressions of mTOR and p-mTOR, the secretion of triglyceride and β-casein (CSN2), and the cell viability of DCMEC were all upregulated. We also observed the effects of 14-3-3γ gene silencing and gained consistent results with 14-3-3γ overexpression. These findings reveal that 14-3-3γ affects the mTOR pathway and regulates lactogenesis in DCMECs.

  13. Mammalian target of rapamycin complex 1 signalling is essential for germinal centre reaction.

    Science.gov (United States)

    Li, Bingshou; Li, Zhirong; Wang, Pengcheng; Huang, Qizhao; Xu, Lifan; He, Ran; Ye, Lilin; Bai, Qiang

    2017-10-01

    The mammalian target of rapamycin (mTOR) is a serine-threonine kinase that has been shown to be essential for the differentiation and function of various immune cells. Earlier in vitro studies showed that mTOR signalling regulates B-cell biology by supporting their activation and proliferation. However, how mTOR signalling temporally regulates in vivo germinal centre B (GCB) cell development and differentiation into short-lived plasma cells, long-lived plasma cells and memory cells is still not well understood. In this study, we used a combined conditional/inducible knock-out system to investigate the temporal regulation of mTOR complex 1 (mTORC1) in the GCB cell response to acute lymphocytic choriomeningitis virus infection by deleting Raptor, a main component of mTORC1, specifically in B cells in pre- and late GC phase. Early Raptor deficiency strongly inhibited GCB cell proliferation and differentiation and plasma cell differentiation. Nevertheless, late GC Raptor deficiency caused only decreases in the size of memory B cells and long-lived plasma cells through poor maintenance of GCB cells, but it did not change their differentiation. Collectively, our data revealed that mTORC1 signalling supports GCB cell responses at both early and late GC phases during viral infection but does not regulate GCB cell differentiation into memory B cells and plasma cells at the late GC stage. © 2017 John Wiley & Sons Ltd.

  14. Moderate mammalian target of rapamycin inhibition induces autophagy in HTR8/SVneo cells via O-linked β-N-acetylglucosamine signaling.

    Science.gov (United States)

    Zhang, Qiuxia; Na, Quan; Song, Weiwei

    2017-10-01

    Autophagy, a highly regulated process with a dual role (pro-survival or pro-death), has been implicated in adverse pregnancy outcomes. The aim of this study was to explore the mechanism whereby mammalian target of rapamycin (mTOR) signaling regulates autophagy by modulating protein O-GlcNAcylation in human trophoblasts. HTR8/SVneo cells were incubated in serum-free medium for different time intervals or treated with varying doses of Torin1. Protein expression and cell apoptosis were detected by immunoblotting and flow cytometry, respectively. Short-term serum starvation or slight suppression of mTOR signaling promoted autophagy and decreased apoptosis in HTR8/SVneo cells. Conversely, prolonged serum starvation or excessive inhibition of mTOR reduced autophagy and enhanced cell apoptosis. Both serum starvation and mTOR signaling suppression reduced protein O-GlcNAcylation. Upregulation and downregulation of O-linked β-N-acetylglucosamine (O-GlcNAc) levels attenuated and augmented autophagy, respectively. Moderate mTOR inhibition-induced autophagy was blocked by upregulation of protein O-GlcNAcylation. Furthermore, immunoprecipitation studies revealed that Beclin1 and synaptosome associated protein 29 (SNAP29) could be O-GlcNAcylated, and that slight mTOR inhibition resulted in decreased O-GlcNAc modification of Beclin1 and SNAP29. Notably, we observed an inverse correlation between phosphorylation (Ser15) and O-GlcNAcylation of Beclin1. mTOR signaling inhibition played dual roles in regulating autophagy and apoptosis in HTR8/SVneo cells. Moderate mTOR suppression might induce autophagy via modulating O-GlcNAcylation of Beclin1 and SNAP29. Moreover, the negative interplay between Beclin1 O-GlcNAcylation and phosphorylation (Ser15) may be involved in autophagy regulation by mTOR signaling. © 2017 Japan Society of Obstetrics and Gynecology.

  15. Involvement of mTOR signaling in sphingosylphosphorylcholine-induced hypopigmentation effects

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    Jeong Hyo-Soon

    2011-08-01

    Full Text Available Abstract Background Sphingosylphosphorylcholine (SPC acts as a potent lipid mediator and signaling molecule in various cell types. In the present study, we investigated the effects of SPC on melanogenesis and SPC-modulated signaling pathways related to melanin synthesis. Methods Melanin production was measured in Mel-Ab cells. A luciferase assay was used to detect transcriptional activity of the MITF promoter. Western blot analysis was performed to examine SPC-induced signaling pathways. Results SPC produced significant hypopigmentation effects in a dose-dependent manner. It was found that SPC induced not only activation of Akt but also stimulation of mTOR, a downstream mediator of the Akt signaling pathway. Moreover, SPC decreased the levels of LC3 II, which is known to be regulated by mTOR. Treatment with the mTOR inhibitor rapamycin eliminated decreases in melanin and LC3 II levels by SPC. Furthermore, we found that the Akt inhibitor LY294002 restored SPC-mediated downregulation of LC3 II and inhibited the activation of mTOR by SPC. Conclusions Our data suggest that the mTOR signaling pathway is involved in SPC-modulated melanin synthesis.

  16. Inhibiting the Mammalian target of rapamycin blocks the development of experimental cerebral malaria.

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    Gordon, Emile B; Hart, Geoffrey T; Tran, Tuan M; Waisberg, Michael; Akkaya, Munir; Skinner, Jeff; Zinöcker, Severin; Pena, Mirna; Yazew, Takele; Qi, Chen-Feng; Miller, Louis H; Pierce, Susan K

    2015-06-02

    Malaria is an infectious disease caused by parasites of several Plasmodium spp. Cerebral malaria (CM) is a common form of severe malaria resulting in nearly 700,000 deaths each year in Africa alone. At present, there is no adjunctive therapy for CM. Although the mechanisms underlying the pathogenesis of CM are incompletely understood, it is likely that both intrinsic features of the parasite and the human host's immune response contribute to disease. The kinase mammalian target of rapamycin (mTOR) is a central regulator of immune responses, and drugs that inhibit the mTOR pathway have been shown to be antiparasitic. In a mouse model of CM, experimental CM (ECM), we show that the mTOR inhibitor rapamycin protects against ECM when administered within the first 4 days of infection. Treatment with rapamycin increased survival, blocked breakdown of the blood-brain barrier and brain hemorrhaging, decreased the influx of both CD4(+) and CD8(+) T cells into the brain and the accumulation of parasitized red blood cells in the brain. Rapamycin induced marked transcriptional changes in the brains of infected mice, and analysis of transcription profiles predicted that rapamycin blocked leukocyte trafficking to and proliferation in the brain. Remarkably, animals were protected against ECM even though rapamycin treatment significantly increased the inflammatory response induced by infection in both the brain and spleen. These results open a new avenue for the development of highly selective adjunctive therapies for CM by targeting pathways that regulate host and parasite metabolism. Malaria is a highly prevalent infectious disease caused by parasites of several Plasmodium spp. Malaria is usually uncomplicated and resolves with time; however, in about 1% of cases, almost exclusively among young children, malaria becomes severe and life threatening, resulting in nearly 700,000 deaths each year in Africa alone. Among the most severe complications of Plasmodium falciparum infection

  17. Mammalian target of rapamycin hyperactivity mediates the detrimental effects of a high sucrose diet on Alzheimer's disease pathology.

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    Orr, Miranda E; Salinas, Angelica; Buffenstein, Rochelle; Oddo, Salvatore

    2014-06-01

    High sugar consumption and diabetes increase the risk of developing Alzheimer's disease (AD) by unknown mechanisms. Using an animal model of AD, here we show that high sucrose intake induces obesity with changes in central and peripheral insulin signaling. These pre-diabetic changes are associated with an increase in amyloid-β production and deposition. Moreover, high sucrose ingestion exacerbates tau phosphorylation by increasing Cdk5 activity. Mechanistically, the sucrose-mediated increase in AD-like pathology results from hyperactive mammalian target of rapamycin (mTOR), a key nutrient sensor important in regulating energy homeostasis. Specifically, we show that rapamycin, an mTOR inhibitor, prevents the detrimental effects of sucrose in the brain without altering changes in peripheral insulin resistance. Overall, our data suggest that high sucrose intake and dysregulated insulin signaling, which are known to contribute to the occurrence of diabetes, increase the risk of developing AD by upregulating brain mTOR signaling. Therefore, early interventions to modulate mTOR activity in individuals at high risk of developing diabetes may decrease their AD susceptibility. Copyright © 2014 Elsevier Inc. All rights reserved.

  18. Pasteurella multocida toxin- induced osteoclastogenesis requires mTOR activation.

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    Kloos, Bianca; Chakraborty, Sushmita; Lindner, Sonja G; Noack, Katrin; Harre, Ulrike; Schett, Georg; Krämer, Oliver H; Kubatzky, Katharina F

    2015-09-14

    Pasteurella multocida toxin (PMT) is a potent inducer of osteoclast formation. Pigs suffering from an infection with toxigenic Pasteurella multocida strains develop atrophic rhinitis characterised by a loss of turbinate bones and conchae. However, on the molecular level the process of bone loss remains largely uncharacterised. Recently it was found that PMT activates the serine/threonine kinase mammalian target of rapamycin (mTOR) in fibroblasts. Using RAW264.7 macrophages, we investigated the role of the mTOR complex 1 (mTORC1) in PMT-mediated osteoclast formation. PMT induces the differentiation of RAW264.7 macrophages into multinucleated, tartrate resistant acid phosphatase (TRAP) positive osteoclasts that are capable to resorb bone. In the presence of the mTORC1 inhibitor rapamycin, PMT was significantly less able to induce the formation of TRAP-positive osteoclasts. Accordingly, the resulting resorption of bone was strongly reduced. A major target of mTOR is the 70 kDa ribosomal protein S6 kinase 1 (p70 S6K1). Activated p70 S6K1 decreases the expression of programmed cell death protein 4 (PDCD4), a negative transcriptional regulator of osteoclastogenesis, at the protein and gene level. Ultimately this results in the activation of c-Jun, a component of the activator protein 1 (AP-1) complex, which is a major transcription factor for the induction of osteoclast-specific genes. We now demonstrate that c-Jun and its downstream target, the osteoclast-specific bone degrading protease cathepsin K, are upregulated upon PMT treatment in an mTOR-dependent manner. Activation of mTOR signalling plays a central role in the formation of osteoclasts through the bacterial toxin PMT. On the molecular level, PMT-induced activation of mTOR leads to down regulation of PDCD4, a known repressor of AP-1 complex, culminating in the activation of c-Jun, an essential transcription factor for triggering osteoclastogenesis.

  19. Mammalian Target of Rapamycin Signaling Pathway Changes with Intestinal Epithelial Cells Renewal Along Crypt-Villus Axis

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    Huansheng Yang

    2016-07-01

    Full Text Available Background/Aims: Understanding the mechanism that involves in regulating epithelial cells renewal is the fundamental of regulating intestinal mucosa development and functions and related diseases. The mechanistic target of rapamycin (mTOR signaling pathway involves in controlling various major processes by integrating intracellular and extracellular cues. The present experiment was conducted to test the correlation between the mTOR signaling pathway and intestinal epithelial cells renewal along crypt-villus axis (CVA. Methods: Intestinal epithelial cells were sequentially isolated from the jejunum of piglets along CVA, and the amount or phosphorylation level of proteins involved in cell cycle, mTOR signaling pathway, gene expression, and the antioxidant capacity in the isolated cells were measured. Results: The results showed that the amount of proteins involved in cell cycle decreased from crypt to villus tip. The amount or phosphorylation level of proteins related to mTOR signaling pathway in intestinal epithelial cells mainly decreased during maturation along CVA. The amount of proteins involved in gene expression and the antioxidant capacity also decreased from crypt to the top of villi. Conclusions: These results indicate that the mTOR signaling pathway may be involved in regulating the intestinal epithelial cells renewal along CVA and it may partly through affecting the antioxidant capacity and gene expression of intestinal epithelial cells. Further histological verification is needed to confirm the results of the present experiments.

  20. Inhibition of mammalian target of rapamycin reduces epileptogenesis and blood-brain barrier leakage but not microglia activation.

    NARCIS (Netherlands)

    van Vliet, E.A.; Forte, G.; Holtman, L.; den Burger, J.C.G.; Sinjewel, A.; de Vries, H.E.; Aronica, E.; Gorter, J.A.

    2012-01-01

    Purpose: Previous studies have shown that inhibition of the mammalian target of rapamycin (mTOR) pathway with rapamycin prevents epileptogenesis after pharmacologically induced status epilepticus (SE) in rat models of temporal lobe epilepsy. Because rapamycin is also known for its immunosuppressant

  1. The mechanical activation of mTOR signaling: an emerging role for late endosome/lysosomal targeting.

    Science.gov (United States)

    Jacobs, Brittany L; Goodman, Craig A; Hornberger, Troy A

    2014-02-01

    It is well recognized that mechanical signals play a critical role in the regulation of skeletal muscle mass, and the maintenance of muscle mass is essential for mobility, disease prevention and quality of life. Furthermore, over the last 15 years it has become established that signaling through a protein kinase called the mammalian (or mechanistic) target of rapamycin (mTOR) is essential for mechanically-induced changes in protein synthesis and muscle mass, however, the mechanism(s) via which mechanical stimuli regulate mTOR signaling have not been defined. Nonetheless, advancements are being made, and an emerging body of evidence suggests that the late endosome/lysosomal (LEL) system might play a key role in this process. Therefore, the purpose of this review is to summarize this body of evidence. Specifically, we will first explain why the Ras homologue enriched in brain (Rheb) and phosphatidic acid (PA) are considered to be direct activators of mTOR signaling. We will then describe the process of endocytosis and its involvement in the formation of LEL structures, as well as the evidence which indicates that mTOR and its direct activators (Rheb and PA) are all enriched at the LEL. Finally, we will summarize the evidence that has implicated the LEL in the regulation of mTOR by various growth regulatory inputs such as amino acids, growth factors and mechanical stimuli.

  2. ESAT6 inhibits autophagy flux and promotes BCG proliferation through MTOR

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    Dong, Hu, E-mail: austhudong@126.com [Department of Medical Immunology, Medical School, Anhui University of Science and Technology (China); Medical Inspection Center, Anhui University of Science and Technology, Huainan (China); Jing, Wu, E-mail: wujing8008@126.com [Department of Medical Immunology, Medical School, Anhui University of Science and Technology (China); Medical Inspection Center, Anhui University of Science and Technology, Huainan (China); Runpeng, Zhao; Xuewei, Xu; Min, Mu; Ru, Cai [Department of Medical Immunology, Medical School, Anhui University of Science and Technology (China); Yingru, Xing; Shengfa, Ni [Affiliated Cancer Hospital, Anhui University of Science and Technology (China); Rongbo, Zhang [Department of Medical Immunology, Medical School, Anhui University of Science and Technology (China); Medical Inspection Center, Anhui University of Science and Technology, Huainan (China)

    2016-08-19

    In recent years, increasing studies have found that pathogenic Mycobacterium tuberculosis (Mtb) inhibits autophagy, which mediates the anti-mycobacterial response, but the mechanism is not clear. We previously reported that secretory acid phosphatase (SapM) of Mtb can negatively regulate autophagy flux. Recently, another virulence factor of Mtb, early secretory antigenic target 6 (ESAT6), has been found to be involved in inhibiting autophagy, but the mechanism remains unclear. In this study, we show that ESAT6 hampers autophagy flux to boost bacillus Calmette-Guerin (BCG) proliferation and reveals a mechanism by which ESAT6 blocks autophagosome-lysosome fusion in a mammalian target of rapamycin (MTOR)-dependent manner. In both Raw264.7 cells and primary macrophages derived from the murine abdominal cavity (ACM), ESAT6 repressed autophagy flux by interfering with the autophagosome-lysosome fusion, which resulted in an increased load of BCG. Impaired degradation of LC3Ⅱ and SQSTM1 by ESAT6 was related to the upregulated activity of MTOR. Contrarily, inhibiting MTOR with Torin1 removed the ESAT6-induced autophagy block and lysosome dysfunction. Furthermore, in both Raw264.7 and ACM cells, MTOR inhibition significantly suppressed the survival of BCG. In conclusion, our study highlights how ESAT6 blocks autophagy and promotes BCG survival in a way that activates MTOR. - Highlights: • A mechanism for disruping autophagy flux induced by ESAT6. • ESAT6-inhibited autophagy is MTOR-dependent. • ESAT6-boosted BCG is MTOR-dependent.

  3. Novel nervous and multi-system regenerative therapeutic strategies for diabetes mellitus with mTOR

    Directory of Open Access Journals (Sweden)

    Kenneth Maiese

    2016-01-01

    Full Text Available Throughout the globe, diabetes mellitus (DM is increasing in incidence with limited therapies presently available to prevent or resolve the significant complications of this disorder. DM impacts multiple organs and affects all components of the central and peripheral nervous systems that can range from dementia to diabetic neuropathy. The mechanistic target of rapamycin (mTOR is a promising agent for the development of novel regenerative strategies for the treatment of DM. mTOR and its related signaling pathways impact multiple metabolic parameters that include cellular metabolic homeostasis, insulin resistance, insulin secretion, stem cell proliferation and differentiation, pancreatic β-cell function, and programmed cell death with apoptosis and autophagy. mTOR is central element for the protein complexes mTOR Complex 1 (mTORC1 and mTOR Complex 2 (mTORC2 and is a critical component for a number of signaling pathways that involve phosphoinositide 3-kinase (PI 3-K, protein kinase B (Akt, AMP activated protein kinase (AMPK, silent mating type information regulation 2 homolog 1 (Saccharomyces cerevisiae (SIRT1, Wnt1 inducible signaling pathway protein 1 (WISP1, and growth factors. As a result, mTOR represents an exciting target to offer new clinical avenues for the treatment of DM and the complications of this disease. Future studies directed to elucidate the delicate balance mTOR holds over cellular metabolism and the impact of its broad signaling pathways should foster the translation of these targets into effective clinical regimens for DM.

  4. Rapamycin-Sensitive Late-LTP is Enhanced in the Hippocampus of IL-6 Transgenic Mice.

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    Olde Engberink, Anneke; Hernandez, Ruben; de Graan, Pierre; Gruol, Donna L

    2017-11-10

    The neuroimmune factor IL-6 has been shown to regulate hippocampal long-term potentiation (LTP), an activity-dependent enhancement of synaptic transmission that plays a central role in memory and learning. This IL-6 action was demonstrated with relatively short IL-6 exposure, and may reflect physiological actions of IL-6. IL-6 is also expressed chronically at elevated levels in the central nervous system (CNS) under pathological conditions such as neurological disorders. Little is known about the effects IL-6 on LTP under such conditions, an issue that we are addressing by electrophysiological recordings from CA1 pyramidal neurons of hippocampal slices from transgenic mice that persistently express elevated levels of IL-6 in the CNS (IL-6 tg). The current studies examined the long-lasting phase of LTP (late LTP; L-LTP) and the potential involvement mammalian target of rapamycin (mTOR), a known regulator of L-LTP and a downstream partner of IL-6 signal transduction pathways. Results show that basal synaptic transmission and L-LTP were increased in hippocampal slices from IL-6 tg mice compared to slices from non-transgenic (non-tg) control mice. An inhibitor of mTOR, rapamycin, reduced L-LTP in slices from both genotypes, and eliminated the difference in magnitude of L-LTP between IL-6 and non-tg hippocampus. There were no genotypic effect of rapamycin on basal synaptic transmission, but synaptic responses during the LTP induction protocol were reduced in IL-6 tg slices, an effect that could contribute to the reduction of L-LTP in the IL-6 tg slices. These results indicate that persistently increased levels of IL-6 can lead to alterations in mTOR regulation of L-LTP, possibly affecting learning and memory. Copyright © 2017 IBRO. Published by Elsevier Ltd. All rights reserved.

  5. A possible link between BDNF and mTOR in control of food intake

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    Nobuyuki eTakei

    2014-09-01

    Full Text Available Food intake is intricately regulated by glucose, amino acids, hormones, neuropeptides, and trophic factors through a neural circuit in the hypothalamus. Brain-derived neurotrophic factor (BDNF, the most prominent neurotrophic factor in the brain, regulates differentiation, maturation, and synaptic plasticity throughout life. Among its many roles, BDNF exerts an anorexigenic function in the brain. However, the intracellular signaling induced by BDNF to control food intake is not fully understood. One candidate for the molecule involved in transducing the anorexigenic activity of BDNF is the mammalian target of rapamycin (mTOR. mTOR senses extracellular amino acids, glucose, growth factors, and neurotransmitters, and regulates anabolic reactions response to these signals. Activated mTOR increases protein and lipid synthesis and inhibits protein degradation. In the hypothalamus, mTOR activation is thought to reduce food intake. Here we summarize recent findings regarding BDNF- and mTOR-mediated feeding control, and propose a link between these molecules in eating behavior.

  6. FK506 Binding Protein Mediates Glioma Cell Growth and Sensitivity to Rapamycin Treatment by Regulating NF-κB Signaling Pathway

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    Wei Jiang

    2008-03-01

    Full Text Available FK506 binding protein 5 (FKBP5 belongs to a family of immunophilins named for their ability to bind immunosuppressive drugs, also known as peptidyl-prolyl cis-trans isomerases, and also with chaperones to help protein folding. Using glioma cDNA microarray analysis, we found that FKBP5 was overexpressed in glioma tumors. This finding was further validated by real-time reverse transcription-polymerase chain reaction and Western blot analysis. The roles of FKBP5 in glioma cells were then examined. We found that cell growth was suppressed after FKBP5 expression was inhibited by short interfering RNA transfection and enhanced by FKBP5 overexpression. Electrophoretic mobility shift assay showed that nuclear factor-kappa B (NF-κB and DNA binding was enhanced by FKBP5 overexpression. The expression level of I-kappa B alpha and phosphorylated NF-κB was regulated by the expression of FKBP5. These data suggest that FKBP5 is involved in NF-κB pathway activation in glioma cells. In addition, FKBP5 overexpression in rapamycin-sensitive U87 cells blocked the cells' response to rapamycin treatment, whereas rapamycin-resistant glioma cells, both PTEN-positive and -negative, were synergistically sensitive to rapamycin after FKBP5 was knocked down, suggesting that the FKBP5 regulates glioma cell response to rapamycin treatment. In conclusion, our study demonstrates that FKBP5 plays an important role in glioma growth and chemoresistance through regulating signal transduction of the NF-κB pathway.

  7. mTOR inhibition increases cell viability via autophagy induction during endoplasmic reticulum stress – An experimental and modeling study

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    Orsolya Kapuy

    2014-01-01

    Full Text Available Unfolded or misfolded proteins in the endoplasmic reticulum (ER trigger an adaptive ER stress response known as unfolded protein response (UPR. Depending on the severity of ER stress, either autophagy-controlled survival or apoptotic cell death can be induced. The molecular mechanisms by which UPR controls multiple fate decisions have started to emerge. One such molecular mechanism involves a master regulator of cell growth, mammalian target of rapamycin (mTOR, which paradoxically is shown to have pro-apoptotic role by mutually interacting with ER stress response. How the interconnections between UPR and mTOR influence the dynamics of autophagy and apoptosis activation is still unclear. Here we make an attempt to explore this problem by using experiments and mathematical modeling. The effect of perturbed mTOR activity in ER stressed cells was studied on autophagy and cell viability by using agents causing mTOR pathway inhibition (such as rapamycin or metyrapone. We observed that mTOR inhibition led to an increase in cell viability and was accompanied by an increase in autophagic activity. It was also shown that autophagy was activated under conditions of severe ER stress but that in the latter phase of stress it was inhibited at the time of apoptosis activation. Our mathematical model shows that both the activation threshold and temporal dynamics of autophagy and apoptosis inducers are sensitive to variation in mTOR activity. These results confirm that autophagy has cytoprotective role and is activated in mutually exclusive manner with respect to ER stress levels.

  8. FXR blocks the growth of liver cancer cells through inhibiting mTOR-s6K pathway

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    Huang, Xiongfei, E-mail: xiongfeihuang@hotmail.com [Department of Pathology and Institute of Oncology, Preclinical School, Fujian Medical University, Fuzhou 350108, Fujian (China); Key Laboratory of Ministry of Education for Gastrointestinal Cancer, Fujian Medical University, Fuzhou 350108, Fujian (China); Zeng, Yeting [Department of Pathology and Institute of Oncology, Preclinical School, Fujian Medical University, Fuzhou 350108, Fujian (China); Wang, Xinrui [Department of Biochemistry and Molecular Biology, Fujian Medical University, Fuzhou 350108, Fujian (China); Ma, Xiaoxiao [Department of Diabetes Complications and Metabolism, Diabetes & Metabolism Research Institute, Beckman Research Institute, City of Hope, CA 91010 (United States); Li, Qianqian; Li, Ningbo; Su, Hongying [Department of Pathology and Institute of Oncology, Preclinical School, Fujian Medical University, Fuzhou 350108, Fujian (China); Huang, Wendong [Department of Diabetes Complications and Metabolism, Diabetes & Metabolism Research Institute, Beckman Research Institute, City of Hope, CA 91010 (United States)

    2016-05-27

    The nuclear receptor Farnesoid X Receptor (FXR) is likely a tumor suppressor in liver tissue but its molecular mechanism of suppression is not well understood. In this study, the gene expression profile of human liver cancer cells was investigated by microarray. Bioinformatics analysis of these data revealed that FXR might regulate the mTOR/S6K signaling pathway. This was confirmed by altering the expression level of FXR in liver cancer cells. Overexpression of FXR prevented the growth of cells and induced cell cycle arrest, which was enhanced by the mTOR/S6K inhibitor rapamycin. FXR upregulation also intensified the inhibition of cell growth by rapamycin. Downregulation of FXR produced the opposite effect. Finally, we found that ectopic expression of FXR in SK-Hep-1 xenografts inhibits tumor growth and reduces expression of the phosphorylated protein S6K. Taken together, our data provide the first evidence that FXR suppresses proliferation of human liver cancer cells via the inhibition of the mTOR/S6K signaling pathway. FXR expression can be used as a biomarker of personalized mTOR inhibitor treatment assessment for liver cancer patients. -- Highlights: •FXR inhibits the proliferation of liver cancer cells by prolonging G0/G1 phase. •Microarray results indicate that mTOR-S6k signaling is involved in cellular processes in which FXR plays an important role. •FXR blocks the growth of liver cancer cells via the inhibition of the mTOR/S6K signaling pathway in vitro and in vivo.

  9. Alcohol consumption mitigates apoptosis and mammalian target of rapamycin signaling in myocardium.

    Science.gov (United States)

    Elmadhun, Nassrene Y; Sabe, Ashraf A; Lassaletta, Antonio D; Sellke, Frank W

    2014-06-01

    Epidemiologic studies have shown that individuals who consume low to moderate alcohol have a lower risk of cardiovascular disease developing compared with abstainers. Although experimental studies confirmed this observation, the effect of alcohol on ischemic myocardium is still unclear. We developed a clinically relevant animal model of chronic myocardial ischemia to investigate the effects of moderate alcohol consumption on the myocardium. Fourteen Yorkshire swine underwent placement of an ameroid constrictor to induce chronic myocardial ischemia. Postoperatively, one group was supplemented with 90 mL 50% EtOH daily (n = 7) and one group was supplemented with 80 g sucrose daily to normalize caloric intake between groups (n = 7). After 7 weeks, all animals underwent sternotomy, and harvest of the chronically ischemic myocardium and nonischemic myocardium. Tissues were analyzed for protein expression and stained for apoptosis quantification. In the ischemic myocardium, alcohol down-regulated the following proapoptotic proteins: tumor necrosis factor-α, forkhead box protein 03, BCL2-associated death promoter, and cysteine aspartic acid-specific protease 9; up-regulated the following prosurvival proteins: 5'adenosine monophosphate-activated protein kinase, phosphorylated 5'adenosine monophosphate-activated protein kinase, and phosphorylated forkhead box protein 03; and down-regulated mammalian target of rapamycin (MTOR) signaling by down-regulating MTOR, phosphorylated MTOR, and up-regulating Deptor. In the nonischemic myocardium, alcohol up-regulated prosurvival proteins: protein kinase B, phosphorylated protein kinase B, phosphorylated B-cell CLL/lymphoma 2, 5'adenosine monophosphate-activated protein kinase, phosphorylated BCL2-associated death promoter, phosphorylated forkhead box protein 03, and down-regulated MTOR signaling by down-regulating phosphorylated MTOR and up-regulating Deptor. Alcohol also decreased cell death as measured by terminal

  10. mTOR regulates the expression of DNA damage response enzymes in long-lived Snell dwarf, GHRKO, and PAPPA-KO mice.

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    Dominick, Graham; Bowman, Jacqueline; Li, Xinna; Miller, Richard A; Garcia, Gonzalo G

    2017-02-01

    Studies of the mTOR pathway have prompted speculation that diminished mTOR complex-1 (mTORC1) function may be involved in controlling the aging process. Our previous studies have shown diminished mTORC1 activity in tissues of three long-lived mutant mice: Snell dwarf mice, growth hormone receptor gene disrupted mice (GHRKO), and in this article, mice deficient in the pregnancy-associated protein-A (PAPPA-KO). The ways in which lower mTOR signals slow aging and age-related diseases are, however, not well characterized. Here, we show that Snell, GHKRO, and PAPPA-KO mice express high levels of two proteins involved in DNA repair, O-6-methylguanine-DNA methyltransferase (MGMT) and N-myc downstream-regulated gene 1 (NDRG1). Furthermore, we report that lowering mTOR enhances MGMT and NDRG1 protein expression via post-transcriptional mechanisms. We show that the CCR4-NOT complex, a post-transcriptional regulator of gene expression, is downstream of the mTORC1 pathway and may be responsible for the upregulation of MGMT and NDRG1 in all three varieties of long-lived mice. Our data thus suggest a novel link between DNA repair and mTOR signaling via post-transcriptional regulation involving specific alteration in the CCR4-NOT complex, whose modulation could control multiple aspects of the aging process. © 2016 The Authors. Aging Cell published by the Anatomical Society and John Wiley & Sons Ltd.

  11. mTOR Kinase: A Possible Pharmacological Target in the Management of Chronic Pain

    Directory of Open Access Journals (Sweden)

    Lucia Lisi

    2015-01-01

    Full Text Available Chronic pain represents a major public health problem worldwide. Current pharmacological treatments for chronic pain syndromes, including neuropathic pain, are only partially effective, with significant pain relief achieved in 40–60% of patients. Recent studies suggest that the mammalian target of rapamycin (mTOR kinase and downstream effectors may be implicated in the development of chronic inflammatory, neuropathic, and cancer pain. The expression and activity of mTOR have been detected in peripheral and central regions involved in pain transmission. mTOR immunoreactivity was found in primary sensory axons, in dorsal root ganglia (DRG, and in dorsal horn neurons. This kinase is a master regulator of protein synthesis, and it is critically involved in the regulation of several neuronal functions, including the synaptic plasticity that is a major mechanism leading to the development of chronic pain. Enhanced activation of this pathway is present in different experimental models of chronic pain. Consistently, pharmacological inhibition of the kinase activity turned out to have significant antinociceptive effects in several experimental models of inflammatory and neuropathic pain. We will review the main evidence from animal and human studies supporting the hypothesis that mTOR may be a novel pharmacological target for the management of chronic pain.

  12. Biphasic Rapamycin Effects in Lymphoma and Carcinoma Treatment.

    Science.gov (United States)

    Liu, Yang; Pandeswara, Srilakshmi; Dao, Vinh; Padrón, Álvaro; Drerup, Justin M; Lao, Shunhua; Liu, Aijie; Hurez, Vincent; Curiel, Tyler J

    2017-01-15

    mTOR drives tumor growth but also supports T-cell function, rendering the applications of mTOR inhibitors complex especially in T-cell malignancies. Here, we studied the effects of the mTOR inhibitor rapamycin in mouse EL4 T-cell lymphoma. Typical pharmacologic rapamycin (1-8 mg/kg) significantly reduced tumor burden via direct suppression of tumor cell proliferation and improved survival in EL4 challenge independent of antitumor immunity. Denileukin diftitox (DD)-mediated depletion of regulatory T cells significantly slowed EL4 growth in vivo in a T-cell-dependent fashion. However, typical rapamycin inhibited T-cell activation and tumor infiltration in vivo and failed to boost DD treatment effects. Low-dose (LD) rapamycin (75 μg/kg) increased potentially beneficial CD44hiCD62L(+) CD8(+) central memory T cells in EL4 challenge, but without clinical benefit. LD rapamycin significantly enhanced DD treatment efficacy, but DD plus LD rapamycin treatment effects were independent of antitumor immunity. Instead, rapamycin upregulated EL4 IL2 receptor in vitro and in vivo, facilitating direct DD tumor cell killing. LD rapamycin augmented DD efficacy against B16 melanoma and a human B-cell lymphoma, but not against human Jurkat T-cell lymphoma or ID8agg ovarian cancer cells. Treatment effects correlated with IL2R expression, but mechanisms in some tumors were not fully defined. Overall, our data define a distinct, biphasic mechanisms of action of mTOR inhibition at doses that are clinically exploitable, including in T-cell lymphomas. Cancer Res; 77(2); 520-31. ©2016 AACR. ©2016 American Association for Cancer Research.

  13. Resveratrol Inhibits Cancer Cell Metabolism by Down Regulating Pyruvate Kinase M2 via Inhibition of Mammalian Target of Rapamycin

    Science.gov (United States)

    Iqbal, Mohd Askandar; Bamezai, Rameshwar N. K.

    2012-01-01

    Metabolism of cancer cells with pyruvate kinase M2 (PKM2) at its centre stage has assumed a prime significance in cancer research in recent times. Cancer cell metabolism, characterized by enhanced glucose uptake, production of lactate and anabolism is considered an ideal target for therapeutic interventions. Expression of PKM2 switches metabolism in favor of cancer cells, therefore, the present study was designed to investigate the hitherto unknown effect of resveratrol, a phytoalexin, on PKM2 expression and resultant implications on cancer metabolism. We observed that resveratrol down-regulated PKM2 expression by inhibiting mTOR signaling and suppressed cancer metabolism, adjudged by decreased glucose uptake, lactate production (aerobic glycolysis) and reduced anabolism (macromolecule synthesis) in various cancer cell lines. A contingent decrease in intracellular levels of ribose-5-phosphate (R5P), a critical intermediate of pentose phosphate pathway, accounted for a reduced anabolism. Consequently, the state of suppressed cancer metabolism resulted in decreased cellular proliferation. Interestingly, shRNA-mediated silencing of PKM2 inhibited glucose uptake and lactate production, providing evidence for the critical role of PKM2 and its mediation in the observed effects of resveratrol on cancer metabolism. Further, an over-expression of PKM2 abolished the observed effects of resveratrol, signifying the role of PKM2 downregulation as a critical function of resveratrol. The study reports a novel PKM2-mediated effect of resveratrol on cancer metabolism and provides a new dimension to its therapeutic potential. PMID:22574221

  14. hNAG-1 increases lifespan by regulating energy metabolism and insulin/IGF-1/mTOR signaling.

    Science.gov (United States)

    Wang, Xingya; Chrysovergis, Kali; Kosak, Justin; Kissling, Grace; Streicker, Mike; Moser, Glenda; Li, Ruifang; Eling, Thomas E

    2014-08-01

    Nonsteroidal anti-inflammatory drug-activated gene (NAG-1) or GDF15 is a divergent member of the transforming growth factor beta (TGF-β) superfamily and mice expressing hNAG-1/hGDF15 have been shown to be resistant to HFD-induced obesity and inflammation. This study investigated if hNAG-1 increases lifespan in mice and its potential mechanisms. Here we report that female hNAG-1 mice had significantly increased both mean and median life spans in two transgenic lines, with a larger difference in life spans in mice on a HFD than on low fat diet. hNAG-1 mice displayed significantly reduced body and adipose tissue weight, lowered serum IGF-1, insulin and glucose levels, improved insulin sensitivity, and increased oxygen utilization, oxidative metabolism and energy expenditure. Gene expression analysis revealed significant differences in conserved gene pathways that are important regulators of longevity, including IGF-1, p70S6K, and PI3K/Akt signaling cascades. Phosphorylation of major components of IGF-1/mTOR signaling pathway was significantly lower in hNAG-1mice. Collectively, hNAG-1 is an important regulator of mammalian longevity and may act as a survival factor. Our study suggests that hNAG-1 has potential therapeutic uses in obesity-related diseases where life span is frequently shorter.

  15. Caveat mTOR: aberrant signaling disrupts corticogenesis

    OpenAIRE

    Osborne, Lucy R.

    2010-01-01

    The mammalian target of rapamycin (mTOR) signaling pathway is activated in several disorders associated with benign tumors and malformations of the cerebral cortex. In this issue of the JCI, Orlova et al. have now definitively added another disorder to this group by demonstrating that activation of mTOR signaling is associated with polyhydramnios, megalencephaly, and symptomatic epilepsy syndrome (PMSE), which is characterized by severe intractable epilepsy and megalencephaly. PMSE is caused ...

  16. Gemcitabine resistance in breast cancer cells regulated by PI3K/AKT-mediated cellular proliferation exerts negative feedback via the MEK/MAPK and mTOR pathways

    Directory of Open Access Journals (Sweden)

    Yang XL

    2014-06-01

    Full Text Available Xiao Li Yang, Feng Juan Lin, Ya Jie Guo, Zhi Min Shao, Zhou Luo Ou Key Laboratory of Breast Cancer in Shanghai, Breast Cancer Institute, Cancer Hospital, Department of Oncology, Shanghai Medical College, Fudan University, Shanghai, People's Republic of China Abstract: Chemoresistance is a major cause of cancer treatment failure and leads to a reduction in the survival rate of cancer patients. Phosphatidylinositol 3-kinase/protein kinase B/mammalian target of rapamycin (PI3K/AKT/mTOR and mitogen-activated protein kinase (MAPK pathways are aberrantly activated in many malignant tumors, including breast cancer, which may indicate an association with breast cancer chemoresistance. In this study, we generated a chemoresistant human breast cancer cell line, MDA-MB-231/gemcitabine (simplified hereafter as “231/Gem”, from MDA-MB-231 human breast cancer cells. Flow cytometry studies revealed that with the same treatment concentration of gemcitabine, 231/Gem cells displayed more robust resistance to gemcitabine, which was reflected by fewer apoptotic cells and enhanced percentage of S-phase cells. Through the use of inverted microscopy, Cell Counting Kit-8, and Transwell assays, we found that compared with parental 231 cells, 231/Gem cells displayed more morphologic projections, enhanced cell proliferative ability, and improved cell migration and invasion. Mechanistic studies revealed that the PI3K/AKT/mTOR and mitogen-activated protein kinase kinase (MEK/MAPK signaling pathways were activated through elevated expression of phosphorylated (p-extracellular signal-regulated kinase (ERK, p-AKT, mTOR, p-mTOR, p-P70S6K, and reduced expression of p-P38 and LC3-II (the marker of autophagy in 231/Gem in comparison to control cells. However, there was no change in the expression of Cyclin D1 and p-adenosine monophosphate-activated protein kinase (AMPK. In culture, inhibitors of PI3K/AKT and mTOR, but not of MEK/MAPK, could reverse the enhanced proliferative

  17. Fluoride-Induced Autophagy via the Regulation of Phosphorylation of Mammalian Targets of Rapamycin in Mice Leydig Cells.

    Science.gov (United States)

    Zhang, Jianhai; Zhu, Yuchen; Shi, Yan; Han, Yongli; Liang, Chen; Feng, Zhiyuan; Zheng, Heping; Eng, Michelle; Wang, Jundong

    2017-10-11

    Fluoride is known to impair testicular function and decrease testosterone levels, yet the underlying mechanisms remain inconclusive. The objective of this study is to investigate the roles of autophagy in fluoride-induced male reproductive toxicity using both in vivo and in vitro Leydig cell models. Using transmission electron microscopy and monodansylcadaverine staining, we observed increasing numbers of autophagosomes in testicular tissue, especially in Leydig cells of fluoride-exposed mice. Further study revealed that fluoride increased the levels of mRNA and protein expression of autophagy markers LC3, Beclin1, and Atg 5 in primary Leydig cells. Furthermore, fluoride inhibited the phosphorylation of mammalian targets of rapamycin and 4EBP1, which in turn resulted in a decrease in the levels of AKT and PI3K mRNA expression, as well as an elevation of the level of AMPK expression in both testes and primary Leydig cells. Additionally, fluoride exposure significantly changed the mRNA expression of the PDK1, TSC, and Atg13 regulator genes in primary Leydig cells but not in testicular cells. Taken together, our findings highlight the roles of autophagy in fluoride-induced testicular and Leydig cell damage and contribute to the elucidation of the underlying mechanisms of fluoride-induced male reproductive toxicity.

  18. Mammalian target of rapamycin complex 2 regulates muscle glucose uptake during exercise in mice

    DEFF Research Database (Denmark)

    Kleinert, Maximilian; Parker, Benjamin L; Fritzen, Andreas Mæchel

    2017-01-01

    Exercise increases glucose uptake into insulin-resistant muscle. Thus, elucidating the exercise signalling network in muscle may uncover new therapeutic targets. mTORC2, a regulator of insulin-controlled glucose uptake, has been reported to interact with Rac1, which plays a role in exercise......-induced glucose uptake in muscle. Therefore, we tested the hypothesis that mTORC2 activity is necessary for muscle glucose uptake during treadmill exercise. We used mice that specifically lack mTORC2 signalling in muscle, by deletion of the obligatory mTORC2 component, Rictor (Ric mKO). Running capacity...... potential exercise-dependent mTORC2 substrates, including contractile proteins, kinases, transcriptional regulators, actin cytoskeleton regulators and ion-transport proteins. Our study suggests that mTORC2 is a component of the exercise signalling network that regulates muscle glucose uptake and we provide...

  19. Towards natural mimetics of metformin and rapamycin.

    Science.gov (United States)

    Aliper, Alexander; Jellen, Leslie; Cortese, Franco; Artemov, Artem; Karpinsky-Semper, Darla; Moskalev, Alexey; Swick, Andrew G; Zhavoronkov, Alex

    2017-11-15

    Aging is now at the forefront of major challenges faced globally, creating an immediate need for safe, widescale interventions to reduce the burden of chronic disease and extend human healthspan. Metformin and rapamycin are two FDA-approved mTOR inhibitors proposed for this purpose, exhibiting significant anti-cancer and anti-aging properties beyond their current clinical applications. However, each faces issues with approval for off-label, prophylactic use due to adverse effects. Here, we initiate an effort to identify nutraceuticals-safer, naturally-occurring compounds-that mimic the anti-aging effects of metformin and rapamycin without adverse effects. We applied several bioinformatic approaches and deep learning methods to the Library of Integrated Network-based Cellular Signatures (LINCS) dataset to map the gene- and pathway-level signatures of metformin and rapamycin and screen for matches among over 800 natural compounds. We then predicted the safety of each compound with an ensemble of deep neural network classifiers. The analysis revealed many novel candidate metformin and rapamycin mimetics, including allantoin and ginsenoside (metformin), epigallocatechin gallate and isoliquiritigenin (rapamycin), and withaferin A (both). Four relatively unexplored compounds also scored well with rapamycin. This work revealed promising candidates for future experimental validation while demonstrating the applications of powerful screening methods for this and similar endeavors.

  20. Blood-brain barrier leakage after status epilepticus in rapamycin-treated rats II : Potential mechanisms

    NARCIS (Netherlands)

    van Vliet, Erwin A; Otte, Wim M; Wadman, Wytse J; Aronica, Eleonora; Kooij, Gijs; de Vries, Helga E; Dijkhuizen, Rick M; Gorter, Jan A

    OBJECTIVE: Blood-brain barrier (BBB) leakage may play a pro-epileptogenic role after status epilepticus. In the accompanying contrast-enhanced magnetic resonance imaging (CE-MRI) study we showed that the mammalian target of rapamycin (mTOR) inhibitor rapamycin reduced BBB leakage and seizure

  1. Mammalian Target of Rapamycin Inhibition in Trypanosoma cruzi-Infected Macrophages Leads to an Intracellular Profile That Is Detrimental for Infection

    Directory of Open Access Journals (Sweden)

    Jorge David Rojas Márquez

    2018-02-01

    Full Text Available The causative agent of Chagas’ disease, Trypanosoma cruzi, affects approximately 10 million people living mainly in Latin America, with macrophages being one of the first cellular actors confronting the invasion during T. cruzi infection and their function depending on their proper activation and polarization into distinct M1 and M2 subtypes. Macrophage polarization is thought to be regulated not only by cytokines and growth factors but also by environmental signals. The metabolic checkpoint kinase mammalian target of rapamycin (mTOR-mediated sensing of environmental and metabolic cues influences macrophage polarization in a complex and as of yet incompletely understood manner. Here, we studied the role of the mTOR pathway in macrophages during T. cruzi infection. We demonstrated that the parasite activated mTOR, which was beneficial for its replication since inhibition of mTOR in macrophages by different inhibitors decreased parasite replication. Moreover, in rapamycin pretreated and infected macrophages, we observed a decreased arginase activity and expression, reduced IL-10 and increased interleukin-12 production, compared to control infected macrophages treated with DMSO. Surprisingly, we also found a reduced iNOS activity and expression in these macrophages. Therefore, we investigated possible alternative mechanisms involved in controlling parasite replication in rapamycin pretreated and infected macrophages. Although, cytoplasmic ROS and the enzyme indoleamine 2, 3-dioxygenase (IDO were not involved, we observed a significant increase in IL-6, TNF-α, and IL-1β production. Taking into account that IL-1β is produced by activation of the cytoplasmic receptor NLRP3, which is one of the main components of the inflammasome, we evaluated NLRP3 expression during mTOR inhibition and T. cruzi infection. We observed that rapamycin-pretreated and infected macrophages showed a significant increase in NLRP3 expression and produced higher levels of

  2. FUNCTIONS OF THE mTOR SIGNALING PATHWAY IN NORMAL ARTICULAR CARTILAGE CHONDROCYTES AND IN OSTEOARTHRITIS

    Directory of Open Access Journals (Sweden)

    E. V. Chetina

    2016-01-01

    Full Text Available Osteoarthritis  (OA is a chronic disease associated with pain, stiffness, limited mobility and joint inflammation, as well as articular cartilage destruction.  Recent studies have shown the importance  of chondrocyte  differentiation (hypertrophy as one of the mechanisms  of cartilage degradation in OA. This suggests that chondrocyte  metabolism undergoes the profound changes during cartilage resorption,  which are due to dysregulation of cell function. One of the major cellular metabolic regulators is the protein mTOR (mechanistic target of rapamycin that controls cell growth, proliferation, protein biosynthesis and integrates extracellular signals from growth factors and hormones with amino acid availability and intracellular energy status. The importance  of mTOR activity for articular cartilage destruction  in OAis confirmed by significant changes in the work of mTOR regulatory network that involves multiple intracellular (growth factors, adenosine triphosphate, oxygen availability, and autophagy and extracellular (glucose, amino acids, lipids, and hexosamine signals. Moreover, the altered expression of the mTOR gene in the blood of patients with OA is associated with either increased pain or synovitis, which indicates that there is a strong metabolic heterogeneity in patients with OA and a need for a differentiated therapeutic  approach. The above problems are discussed in this review.

  3. The Importance of the mTOR Regulatory Network in Chondrocyte Biology and Osteoarthritis

    Directory of Open Access Journals (Sweden)

    Elena V. Tchetina

    2014-07-01

    Full Text Available Osteoarthritis (OA is a chronic disorder associated mainly with pain, limited range of motion, stiffness, joint inflammation, and articular cartilage (AC destruction. Recent studies demonstrated the involvement of chondrocyte differentiation (hypertrophy as one of the mechanisms of cartilage degradation in OA. This indicates the involvement of profound alterations in chondrocyte metabolism in the course of cartilage resorption orchestrated by principal changes in the regulation of cellular function. Mammalian target of rapamycin (mTOR controls critical cellular processes such as growth, proliferation, and protein synthesis, and integrates extracellular signals from growth factors and hormones with amino acid availability and intracellular energy status. The importance of mTOR activity during AC destruction in OA is supported by considerable alterations in the mTOR regulatory network, involving multiple intracellular (availability of growth factors, adenosine triphosphate [ATP], and oxygen as well as autophagy and extracellular (glucose, amino acid, lipid, and hexosamine signals. Moreover, variable mTOR gene expression in the peripheral blood of OA patients is associated with increases in pain or synovitis, and indicates a profound metabolic dissimilarity among patients that might require differential approaches to treatment. These issues are discussed in the present review article.

  4. Trichomonas vaginalis metalloproteinase induces mTOR cleavage of SiHa cells.

    Science.gov (United States)

    Quan, Juan-Hua; Choi, In-Wook; Yang, Jung-Bo; Zhou, Wei; Cha, Guang-Ho; Zhou, Yu; Ryu, Jae-Sook; Lee, Young-Ha

    2014-12-01

    Trichomonas vaginalis secretes a number of proteases which are suspected to be the cause of pathogenesis; however, little is understood how they manipulate host cells. The mammalian target of rapamycin (mTOR) regulates cell growth, cell proliferation, cell motility, cell survival, protein synthesis, and transcription. We detected various types of metalloproteinases including GP63 protein from T. vaginalis trophozoites, and T. vaginalis GP63 metalloproteinase was confirmed by sequencing and western blot. When SiHa cells were stimulated with live T. vaginalis, T. vaginalis excretory-secretory products (ESP) or T. vaginalis lysate, live T. vaginalis and T. vaginalis ESP induced the mTOR cleavage in both time- and parasite load-dependent manner, but T. vaginalis lysate did not. Pretreatment of T. vaginalis with a metalloproteinase inhibitor, 1,10-phenanthroline, completely disappeared the mTOR cleavage in SiHa cells. Collectively, T. vaginalis metallopeptidase induces host cell mTOR cleavage, which may be related to survival of the parasite.

  5. Dihydroartemisinin potentiates the anticancer effect of cisplatin via mTOR inhibition in cisplatin-resistant ovarian cancer cells: involvement of apoptosis and autophagy

    Energy Technology Data Exchange (ETDEWEB)

    Feng, Xue [Department of Gynecology and Obstetrics, The First Affiliated Hospital of Harbin Medical University, Harbin 150001 (China); Li, Ling [Department of Brain Cognition Computing Lab, University of Kent, Kent CT2 7NZ (United Kingdom); Jiang, Hong; Jiang, Keping; Jin, Ye [Department of Gynecology and Obstetrics, The First Affiliated Hospital of Harbin Medical University, Harbin 150001 (China); Zheng, Jianhua, E-mail: zhengjianhua1115@126.com [Department of Gynecology and Obstetrics, The First Affiliated Hospital of Harbin Medical University, Harbin 150001 (China)

    2014-02-14

    Highlights: • Phosphorylation of mTOR is abnormal activation in SKOV3/DDP ovarian cancer cells. • Downregulation of mTOR by DHA helps to sensitize the SKOV3/DDP cells to chemotherapy. • DHA has the potential of induce autophagy in cancer cells. - Abstract: Dihydroartemisinin (DHA) exhibits anticancer activity in tumor cells but its mechanism of action is unclear. Cisplatin (DDP) is currently the best known chemotherapeutic available for ovarian cancer. However, tumors return de novo with acquired resistance over time. Mammalian target of rapamycin (mTOR) is an important kinase that regulates cell apoptosis and autophagy, and its dysregulation has been observed in chemoresistant human cancers. Here, we show that compared with control ovarian cancer cells (SKOV3), mTOR phosphorylation was abnormally activated in cisplatin-resistant ovarian cancer cells (SKOV3/DDP) following cisplatin monotherapy. Treatment with cisplatin combined with DHA could enhance cisplatin-induced proliferation inhibition in SKOV3/DDP cells. This mechanism is at least partially due to DHA deactivation of mTOR kinase and promotion of apoptosis. Although autophagy was also induced by DHA, the reduced cell death was not found by suppressing autophagic flux by Bafilomycin A1 (BAF). Taken together, we conclude that inhibition of cisplatin-induced mTOR activation is one of the main mechanisms by which DHA dramatically promotes its anticancer effect in cisplatin-resistant ovarian cancer cells.

  6. Dietary interventions that reduce mTOR activity rescue autistic-like behavioral deficits in mice

    NARCIS (Netherlands)

    Wu, Jiangbo; de Theije, Caroline G M; Lopes da Silva, Sofia; Abbring, Suzanne; van der Horst, Hilma; Broersen, Laus M; Willemsen, Linette; Kas, Martien J; Garssen, Johan; Kraneveld, Aletta D

    Enhanced mammalian target of rapamycin (mTOR) signaling in the brain has been implicated in the pathogenesis of autism spectrum disorder (ASD). Inhibition of the mTOR pathway improves behavior and neuropathology in mouse models of ASD containing mTOR-associated single gene mutations. The current

  7. Dietary interventions that reduce mTOR activity rescue autistic-like behavioral deficits in mice

    NARCIS (Netherlands)

    Wu, Jiangbo; de Theije, Caroline G M; da Silva, Sofia Lopes; Abbring, Suzanne; van der Horst, Hilma; Broersen, Laus M; Willemsen, Linette; Kas, Martien; Garssen, Johan; Kraneveld, Aletta D

    2016-01-01

    Enhanced mammalian target of rapamycin (mTOR) signaling in the brain has been implicated in the pathogenesis of autism spectrum disorder (ASD). Inhibition of the mTOR pathway improves behavior and neuropathology in mouse models of ASD containing mTOR-associated single gene mutations. The current

  8. Rapamycin extends life- and health span because it slows aging.

    Science.gov (United States)

    Blagosklonny, Mikhail V

    2013-08-01

    Making headlines, a thought-provocative paper by Neff, Ehninger and coworkers claims that rapamycin extends life span but has limited effects on aging. How is that possibly possible? And what is aging if not an increase of the probability of death with age. I discuss that the JCI paper actually shows that rapamycin slows aging and also extends lifespan regardless of its direct anti-cancer activities. Aging is, in part, MTOR-driven: a purposeless continuation of developmental growth. Rapamycin affects the same processes in young and old animals: young animals' traits and phenotypes, which continuations become hyperfunctional, harmful and lethal later in life.

  9. The Role of mTOR Signaling in the Regulation of RAG Expression and Genomic Stability During B Lymphocyte Development

    Science.gov (United States)

    2014-07-01

    Mammalian TORC2 is resistant to acute rapamycin inhibition. However, chronic rapamycin exposure also inhibits mTORC2 in vitro and in vivo (Facchinetti... gentamicin , and 50M -mercaptoethanol at 37°C in a 5% CO2 incubator. Cells were monitored daily and fresh medium were exchanged when needed...is found frequently in blood cancers, resulting in an increased resistance of tumor cells to cytotoxic chemotherapies. Therefore, the inhibition of

  10. Stoichiometry and assembly of mTOR complexes revealed by single-molecule pulldown.

    Science.gov (United States)

    Jain, Ankur; Arauz, Edwin; Aggarwal, Vasudha; Ikon, Nikita; Chen, Jie; Ha, Taekjip

    2014-12-16

    The mammalian target of rapamycin (mTOR) kinase is a master regulator of cellular, developmental, and metabolic processes. Deregulation of mTOR signaling is implicated in numerous human diseases including cancer and diabetes. mTOR functions as part of either of the two multisubunit complexes, mTORC1 and mTORC2, but molecular details about the assembly and oligomerization of mTORCs are currently lacking. We use the single-molecule pulldown (SiMPull) assay that combines principles of conventional pulldown assays with single-molecule fluorescence microscopy to investigate the stoichiometry and assembly of mTORCs. After validating our approach with mTORC1, confirming a dimeric assembly as previously reported, we show that all major components of mTORC2 exist in two copies per complex, indicating that mTORC2 assembles as a homodimer. Interestingly, each mTORC component, when free from the complexes, is present as a monomer and no single subunit serves as the dimerizing component. Instead, our data suggest that dimerization of mTORCs is the result of multiple subunits forming a composite surface. SiMPull also allowed us to distinguish complex disassembly from stoichiometry changes. Physiological conditions that abrogate mTOR signaling such as nutrient deprivation or energy stress did not alter the stoichiometry of mTORCs. On the other hand, rapamycin treatment leads to transient appearance of monomeric mTORC1 before complete disruption of the mTOR-raptor interaction, whereas mTORC2 stoichiometry is unaffected. These insights into assembly of mTORCs may guide future mechanistic studies and exploration of therapeutic potential.

  11. Inflammatory stress increases hepatic CD36 translational efficiency via activation of the mTOR signalling pathway.

    Directory of Open Access Journals (Sweden)

    Chuan Wang

    Full Text Available Inflammatory stress is an independent risk factor for the development of non-alcoholic fatty liver disease (NAFLD. Although CD36 is known to facilitate long-chain fatty acid uptake and contributes to NAFLD progression, the mechanisms that link inflammatory stress to hepatic CD36 expression and steatosis remain unclear. As the mammalian target of rapamycin (mTOR signalling pathway is involved in CD36 translational activation, this study was undertaken to investigate whether inflammatory stress enhances hepatic CD36 expression via mTOR signalling pathway and the underlying mechanisms. To induce inflammatory stress, we used tumour necrosis factor alpha (TNF-α and interleukin-6 (IL-6 stimulation of the human hepatoblastoma HepG2 cells in vitro and casein injection in C57BL/6J mice in vivo. The data showed that inflammatory stress increased hepatic CD36 protein levels but had no effect on mRNA expression. A protein degradation assay revealed that CD36 protein stability was not different between HepG2 cells treated with or without TNF-α or IL-6. A polysomal analysis indicated that CD36 translational efficiency was significantly increased by inflammatory stress. Additionally, inflammatory stress enhanced the phosphorylation of mTOR and its downstream translational regulators including p70S6K, 4E-BP1 and eIF4E. Rapamycin, an mTOR-specific inhibitor, reduced the phosphorylation of mTOR signalling pathway and decreased the CD36 translational efficiency and protein level even under inflammatory stress resulting in the alleviation of inflammatory stress-induced hepatic lipid accumulation. This study demonstrates that the activation of the mTOR signalling pathway increases hepatic CD36 translational efficiency, resulting in increased CD36 protein expression under inflammatory stress.

  12. The actions of exogenous leucine on mTOR signalling and amino acid transporters in human myotubes

    Directory of Open Access Journals (Sweden)

    Cameron-Smith David

    2011-06-01

    Full Text Available Abstract Background The branched-chain amino acid (BCAA leucine has been identified to be a key regulator of skeletal muscle anabolism. Activation of anabolic signalling occurs via the mammalian target of rapamycin (mTOR through an undefined mechanism. System A and L solute carriers transport essential amino acids across plasma membranes; however it remains unknown whether an exogenous supply of leucine regulates their gene expression. The aim of the present study was to investigate the effects of acute and chronic leucine stimulation of anabolic signalling and specific amino acid transporters, using cultured primary human skeletal muscle cells. Results Human myotubes were treated with leucine, insulin or co-treated with leucine and insulin for 30 min, 3 h or 24 h. Activation of mTOR signalling kinases were examined, together with putative nutrient sensor human vacuolar protein sorting 34 (hVps34 and gene expression of selected amino acid transporters. Phosphorylation of mTOR and p70S6K was transiently increased following leucine exposure, independently to insulin. hVps34 protein expression was also significantly increased. However, genes encoding amino acid transporters were differentially regulated by insulin and not leucine. Conclusions mTOR signalling is transiently activated by leucine within human myotubes independently of insulin stimulation. While this occurred in the absence of changes in gene expression of amino acid transporters, protein expression of hVps34 increased.

  13. Transcriptional Profiling of Rapamycin-Treated Fibroblasts From Hypertrophic and Keloid Scars

    Science.gov (United States)

    Wong, Victor W.; You, Fanglei; Januszyk, Michael; Gurtner, Geoffrey C.; Kuang, Anna A.

    2016-01-01

    Excess scar formation after cutaneous injury can result in hypertrophic scar (HTS) or keloid formation. Modern strategies to treat pathologic scarring represent nontargeted approaches that produce suboptimal results. Mammalian target of rapamycin (mTOR), a central mediator of inflammation, has been proposed as a novel target to block fibroproliferation. To examine its mechanism of action, we performed genomewide microarray on human fibroblasts (from normal skin, HTS, and keloid scars) treated with the mTOR inhibitor, rapamycin. Hypertrophic scar and keloid fibroblasts demonstrated overexpression of collagen I and III that was effectively abrogated with rapamycin. Blockade of mTOR specifically impaired fibroblast expression of the collagen biosynthesis genes PLOD, PCOLCE, and P4HA, targets significantly overexpressed in HTS and keloid scars. These data suggest that pathologic scarring can be abrogated via modulation of mTOR pathways in procollagen and collagen processing. PMID:24835866

  14. Autophagy inhibits high glucose induced cardiac microvascular endothelial cells apoptosis by mTOR signal pathway.

    Science.gov (United States)

    Zhang, Zheng; Zhang, Shenwei; Wang, Yong; Yang, Ming; Zhang, Ning; Jin, Zhitao; Ding, Liping; Jiang, Wei; Yang, Junke; Sun, Zhimin; Qiu, Chunguang; Hu, Taohong

    2017-08-20

    Cardiac microvascular endothelial cells (CMECs) dysfunction is an important pathophysiological event in the cardiovascular complications induced by diabetes. However, the underlying mechanism is not fully clarified. Autophagy is involved in programmed cell death. Here we investigated the potential role of autophagy on the CMECs injury induced by high glucose. CMECs were cultured in normal or high glucose medium for 6, 12 and 24 h respectively. The autophagy of CMECs was measured by green fluorescence protein (GFP)-LC3 plasmid transfection. Moreover, the apoptosis of CMEC was determined by flow cytometry. Furthermore, 3-Methyladenine (3MA), ATG7 siRNA and rapamycin were administrated to regulate the autophagy state. Moreover, Western blotting assay was performed to measure the expressions of Akt, mTOR, LC3 and p62. High glucose stress decreased the autophagy, whereas increased the apoptosis in CMECs time dependently. Meanwhile, high glucose stress activated the Akt/mTOR signal pathway. Furthermore, autophagy inhibitor, 3-MA and ATG7 siRNA impaired the autophagy and increased the apoptosis in CMECs induced by high glucose stress. Conversely, rapamycin up-regulated the autophagy and decreased the apoptosis in CMECs under high glucose condition. Our data provide evidence that high glucose directly inhibits autophagy, as a beneficial adaptive response to protect CMECs against apoptosis. Furthermore, the autophagy was mediated, at least in part, by mTOR signaling.

  15. Regulation of the mTOR Pathway by a Novel Rheb Binding Protein BNIP3

    Science.gov (United States)

    2008-05-01

    vivo. Much progress has been made regarding the mechanisms of TORC1 regulation. Tuberous sclerosis complex (TSC) is a genetic disease characterized...and TORC2, TOR complex 1 and 2, respectively; TSC, tuberous sclerosis complex; TM, transmembrane; BH3, Bcl-2 homol- ogy 3; CD, highly conserved domain...cancers (50–52). In addi- tion, knockdown Bnip3 enables breast cancer metastases in the lung, liver, and bone (53). In this study, we identified Bnip3 as a

  16. Grape polyphenols inhibit Akt/mammalian target of rapamycin signaling and potentiate the effects of gefitinib in breast cancer.

    Science.gov (United States)

    Castillo-Pichardo, Linette; Dharmawardhane, Suranganie F

    2012-01-01

    We recently reported that a combination of dietary grape polyphenols resveratrol, quercetin, and catechin (RQC), at low concentrations, was effective at inhibiting metastatic cancer progression. Herein, we investigate the molecular mechanisms of RQC in breast cancer and explore the potential of RQC as a potentiation agent for the epidermal growth factor receptor (EGFR) therapeutic gefitinib. Our in vitro experiments showed RQC induced apoptosis in gefitinib-resistant breast cancer cells via regulation of a myriad of proapoptotic proteins. Because the Akt/mammalian target of rapamycin (mTOR) signaling pathway is often elevated during development of anti-EGFR therapy resistance, the effect of RQC on the mTOR upstream effector Akt and the negative regulator AMP kinase (AMPK) was investigated. RQC was found to reduce Akt activity, induce the activation of AMPK, and inhibit mTOR signaling in breast cancer cells. Combined RQC and gefitinib decreased gefitinib resistant breast cancer cell viability to a greater extent than RQC or gefitinib alone. Moreover, RQC inhibited Akt and mTOR and activated AMPK even in the presence of gefitinib. Our in vivo experiments showed combined RQC and gefitinib was more effective than the individual treatments at inhibiting mammary tumor growth and metastasis in nude mice. Therefore, RQC treatment inhibits breast cancer progression and may potentiate anti-EGFR therapy by inhibition of Akt/mTOR signaling.

  17. mTOR Regulates Endocytosis and Nutrient Transport in Proximal Tubular Cells.

    Science.gov (United States)

    Grahammer, Florian; Ramakrishnan, Suresh K; Rinschen, Markus M; Larionov, Alexey A; Syed, Maryam; Khatib, Hazim; Roerden, Malte; Sass, Jörn Oliver; Helmstaedter, Martin; Osenberg, Dorothea; Kühne, Lucas; Kretz, Oliver; Wanner, Nicola; Jouret, Francois; Benzing, Thomas; Artunc, Ferruh; Huber, Tobias B; Theilig, Franziska

    2017-01-01

    Renal proximal tubular cells constantly recycle nutrients to ensure minimal loss of vital substrates into the urine. Although most of the transport mechanisms have been discovered at the molecular level, little is known about the factors regulating these processes. Here, we show that mTORC1 and mTORC2 specifically and synergistically regulate PTC endocytosis and transport processes. Using a conditional mouse genetic approach to disable nonredundant subunits of mTORC1, mTORC2, or both, we showed that mice lacking mTORC1 or mTORC1/mTORC2 but not mTORC2 alone develop a Fanconi-like syndrome of glucosuria, phosphaturia, aminoaciduria, low molecular weight proteinuria, and albuminuria. Interestingly, proteomics and phosphoproteomics of freshly isolated kidney cortex identified either reduced expression or loss of phosphorylation at critical residues of different classes of specific transport proteins. Functionally, this resulted in reduced nutrient transport and a profound perturbation of the endocytic machinery, despite preserved absolute expression of the main scavenger receptors, MEGALIN and CUBILIN. Our findings highlight a novel mTOR-dependent regulatory network for nutrient transport in renal proximal tubular cells. Copyright © 2016 by the American Society of Nephrology.

  18. Current Models of Mammalian Target of Rapamycin Complex 1 (mTORC1) Activation by Growth Factors and Amino Acids

    Science.gov (United States)

    Zheng, Xu; Liang, Yan; He, Qiburi; Yao, Ruiyuan; Bao, Wenlei; Bao, Lili; Wang, Yanfeng; Wang, Zhigang

    2014-01-01

    Mammalian target of rapamycin (mTOR), which is now referred to as mechanistic target of rapamycin, integrates many signals, including those from growth factors, energy status, stress, and amino acids, to regulate cell growth and proliferation, protein synthesis, protein degradation, and other physiological and biochemical processes. The mTOR-Rheb-TSC-TBC complex co-localizes to the lysosome and the phosphorylation of TSC-TBC effects the dissociation of the complex from the lysosome and activates Rheb. GTP-bound Rheb potentiates the catalytic activity of mTORC1. Under conditions with growth factors and amino acids, v-ATPase, Ragulator, Rag GTPase, Rheb, hVps34, PLD1, and PA have important but disparate effects on mTORC1 activation. In this review, we introduce five models of mTORC1 activation by growth factors and amino acids to provide a comprehensive theoretical foundation for future research. PMID:25402640

  19. Current Models of Mammalian Target of Rapamycin Complex 1 (mTORC1 Activation by Growth Factors and Amino Acids

    Directory of Open Access Journals (Sweden)

    Xu Zheng

    2014-11-01

    Full Text Available Mammalian target of rapamycin (mTOR, which is now referred to as mechanistic target of rapamycin, integrates many signals, including those from growth factors, energy status, stress, and amino acids, to regulate cell growth and proliferation, protein synthesis, protein degradation, and other physiological and biochemical processes. The mTOR-Rheb-TSC-TBC complex co-localizes to the lysosome and the phosphorylation of TSC-TBC effects the dissociation of the complex from the lysosome and activates Rheb. GTP-bound Rheb potentiates the catalytic activity of mTORC1. Under conditions with growth factors and amino acids, v-ATPase, Ragulator, Rag GTPase, Rheb, hVps34, PLD1, and PA have important but disparate effects on mTORC1 activation. In this review, we introduce five models of mTORC1 activation by growth factors and amino acids to provide a comprehensive theoretical foundation for future research.

  20. The Effect of Different Dosing Schedules of Intravitreal Sirolimus, a Mammalian Target of Rapamycin (mTOR) Inhibitor, in the Treatment of Non-Infectious Uveitis (An American Ophthalmological Society Thesis).

    Science.gov (United States)

    Nguyen, Quan Dong; Sadiq, Mohammad Ali; Soliman, Mohamed Kamel; Agarwal, Aniruddha; Do, Diana V; Sepah, Yasir J

    2016-08-01

    To determine if two different doses of intravitreal sirolimus, an mTOR inhibitor, can decrease inflammation and is safe in eyes with non-infectious posterior, intermediate, or panuveitis in the Sirolimus as a Therapeutic Approach UVEitis: Protocol-2 (SAVE-2) Study. SAVE-2 is a prospective randomized, phase II, open-label interventional clinical trial conducted at 4 clinical centers in the United States. Eligible subjects were randomized into one of two treatments. Group 1 received 440µg of intravitreal sirolimus in study eyes on days 0, 30, 60, 90, 120, and 150; group 2 received 880µg of intravitreal sirolimus on days 0, 60, and 120. Fellow eyes were also eligible to receive sirolimus (of opposite dose to that of study eye). Primary endpoint of the study was at month 6 (M6). 24 subjects have been randomized in SAVE-2 and are included in the analysis. Vitreous haze decreased by ≥2 steps in 63.6% and 50% of patients in groups 1 and 2, respectively at M6 (p=0.695). Mean change in best-corrected visual acuity for subjects was +3.66 and -2.91 ETDRS letters in group 1 and 2, respectively. Among subjects with macular edema at baseline (n=13), the mean change in foveal thickness was -89.42µm in group 1 and +81.5µm in group 2 at M6. Both low and high doses of intravitreal sirolimus were found to decrease vitreous haze in eyes with non-infectious uveitis. Low dose (440µg) sirolimus administered monthly may be more efficacious in reducing uveitic macular edema than high dose (880µg) administered every 2 months.

  1. Chondroitin sulfate proteoglycan-4 does not protect melanoma cells during inhibition of PI3K and mTOR pathways.

    Science.gov (United States)

    Javaid, Sehrish; Terai, Kaoru; Dudek, Arkadiusz Z

    2015-03-01

    Chondroitin sulfate proteoglycan-4 (CSPG4) is commonly expressed in melanoma cells and induces melanoma cell proliferation and migration by enhancement of activation of the extracellular signal-regulated kinase 1, 2 (ERK1,2) pathway. The phosphoinositide 3-kinase (PI3K) -protein kinase B (AKT) and mammalian target of rapamycin (mTOR) pathways are also frequently de-regulated in melanoma. We hypothesized that CSPG4, by sustained activation of PI3K, may reduce the effect of the dual inhibition of PI3K-AKT and mTOR pathways. CSPG4-negative melanoma cell line WM1552C was transfected with CSPG4 and CSPG4 lacking cytoplasmic domain (melanoma-associated chondroitin sulfate proteoglycan (MCSP)ΔCD). To assess the effect of CSPG4 on the mTOR pathway, PF-5212384, a dual PI3K/mTOR inhibitor was used. Cell proliferation and downstream signaling from mTOR was assayed in the presence of CSPG4. Forced CSPG4 expression did not provide any protection to melanoma cells from the pharmacological inhibition of mTOR pathway in vitro. In addition, we demonstrated that inhibition of signaling molecules downstream of AKT and mTOR was not diminished in the presence of CSPG4 when the cells were treated with the PI3K/mTOR inhibitor. CSPG4 expression does not have any impact on survival and signaling activity of melanoma cells during PI3K/mTOR inhibition. Copyright© 2015 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

  2. Long-term mTOR inhibitors administration evokes altered calcium homeostasis and platelet dysfunction in kidney transplant patients

    OpenAIRE

    López, Esther; Berna-Erro, Alejandro; Bermejo, Nuria; Brull, José María; Martinez, Rocío; Garcia Pino, Guadalupe; Alvarado, Raul; Salido, Ginés María; Rosado, Juan Antonio; Cubero, Juan José; Redondo, Pedro Cosme

    2013-01-01

    The use of the mammal target of rapamycin (mTOR) inhibitors has been consolidated as the therapy of election for preventing graft rejection in kidney transplant patients, despite their immunosuppressive activity is less strong than anti-calcineurin agents like tacrolimus and cyclosporine A. Furthermore, as mTOR is widely expressed, rapamycin (a macrolide antibiotic produced by Streptomyces hygroscopicus) is recommended in patients presenting neoplasia due to its antiproliferative actions. Hen...

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

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    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.

  4. Rapamycin reverses status epilepticus-induced memory deficits and dendritic damage.

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    Amy L Brewster

    Full Text Available Cognitive impairments are prominent sequelae of prolonged continuous seizures (status epilepticus; SE in humans and animal models. While often associated with dendritic injury, the underlying mechanisms remain elusive. The mammalian target of rapamycin complex 1 (mTORC1 pathway is hyperactivated following SE. This pathway modulates learning and memory and is associated with regulation of neuronal, dendritic, and glial properties. Thus, in the present study we tested the hypothesis that SE-induced mTORC1 hyperactivation is a candidate mechanism underlying cognitive deficits and dendritic pathology seen following SE. We examined the effects of rapamycin, an mTORC1 inhibitor, on the early hippocampal-dependent spatial learning and memory deficits associated with an episode of pilocarpine-induced SE. Rapamycin-treated SE rats performed significantly better than the vehicle-treated rats in two spatial memory tasks, the Morris water maze and the novel object recognition test. At the molecular level, we found that the SE-induced increase in mTORC1 signaling was localized in neurons and microglia. Rapamycin decreased the SE-induced mTOR activation and attenuated microgliosis which was mostly localized within the CA1 area. These findings paralleled a reversal of the SE-induced decreases in dendritic Map2 and ion channels levels as well as improved dendritic branching and spine density in area CA1 following rapamycin treatment. Taken together, these findings suggest that mTORC1 hyperactivity contributes to early hippocampal-dependent spatial learning and memory deficits and dendritic dysregulation associated with SE.

  5. Role of mTOR Inhibitors in Kidney Disease

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    Moto Kajiwara

    2016-06-01

    Full Text Available The first compound that inhibited the mammalian target of rapamycin (mTOR, sirolimus (rapamycin was discovered in the 1970s as a soil bacterium metabolite collected on Easter Island (Rapa Nui. Because sirolimus showed antiproliferative activity, researchers investigated its molecular target and identified the TOR1 and TOR2. The mTOR consists of mTOR complex 1 (mTORC1 and mTORC2. Rapalogues including sirolimus, everolimus, and temsirolimus exert their effect mainly on mTORC1, whereas their inhibitory effect on mTORC2 is mild. To obtain compounds with more potent antiproliferative effects, ATP-competitive inhibitors of mTOR targeting both mTORC1 and mTORC2 have been developed and tested in clinical trials as anticancer drugs. Currently, mTOR inhibitors are used as anticancer drugs against several solid tumors, and immunosuppressive agents for transplantation of various organs. This review discusses the role of mTOR inhibitors in renal disease with a particular focus on renal cancer, diabetic nephropathy, and kidney transplantation.

  6. Rapamycin extends murine lifespan but has limited effects on aging.

    Science.gov (United States)

    Neff, Frauke; Flores-Dominguez, Diana; Ryan, Devon P; Horsch, Marion; Schröder, Susanne; Adler, Thure; Afonso, Luciana Caminha; Aguilar-Pimentel, Juan Antonio; Becker, Lore; Garrett, Lillian; Hans, Wolfgang; Hettich, Moritz M; Holtmeier, Richard; Hölter, Sabine M; Moreth, Kristin; Prehn, Cornelia; Puk, Oliver; Rácz, Ildikó; Rathkolb, Birgit; Rozman, Jan; Naton, Beatrix; Ordemann, Rainer; Adamski, Jerzy; Beckers, Johannes; Bekeredjian, Raffi; Busch, Dirk H; Ehninger, Gerhard; Graw, Jochen; Höfler, Heinz; Klingenspor, Martin; Klopstock, Thomas; Ollert, Markus; Stypmann, Jörg; Wolf, Eckhard; Wurst, Wolfgang; Zimmer, Andreas; Fuchs, Helmut; Gailus-Durner, Valérie; Hrabe de Angelis, Martin; Ehninger, Dan

    2013-08-01

    Aging is a major risk factor for a large number of disorders and functional impairments. Therapeutic targeting of the aging process may therefore represent an innovative strategy in the quest for novel and broadly effective treatments against age-related diseases. The recent report of lifespan extension in mice treated with the FDA-approved mTOR inhibitor rapamycin represented the first demonstration of pharmacological extension of maximal lifespan in mammals. Longevity effects of rapamycin may, however, be due to rapamycin's effects on specific life-limiting pathologies, such as cancers, and it remains unclear if this compound actually slows the rate of aging in mammals. Here, we present results from a comprehensive, large-scale assessment of a wide range of structural and functional aging phenotypes, which we performed to determine whether rapamycin slows the rate of aging in male C57BL/6J mice. While rapamycin did extend lifespan, it ameliorated few studied aging phenotypes. A subset of aging traits appeared to be rescued by rapamycin. Rapamycin, however, had similar effects on many of these traits in young animals, indicating that these effects were not due to a modulation of aging, but rather related to aging-independent drug effects. Therefore, our data largely dissociate rapamycin's longevity effects from effects on aging itself.

  7. Phosphorylation of Ribosomal Protein S6 Mediates Mammalian Target of Rapamycin Complex 1-Induced Parathyroid Cell Proliferation in Secondary Hyperparathyroidism.

    Science.gov (United States)

    Volovelsky, Oded; Cohen, Gili; Kenig, Ariel; Wasserman, Gilad; Dreazen, Avigail; Meyuhas, Oded; Silver, Justin; Naveh-Many, Tally

    2016-04-01

    Secondary hyperparathyroidism is characterized by increased serum parathyroid hormone (PTH) level and parathyroid cell proliferation. However, the molecular pathways mediating the increased parathyroid cell proliferation remain undefined. Here, we found that the mTOR pathway was activated in the parathyroid of rats with secondary hyperparathyroidism induced by either chronic hypocalcemia or uremia, which was measured by increased phosphorylation of ribosomal protein S6 (rpS6), a downstream target of the mTOR pathway. This activation correlated with increased parathyroid cell proliferation. Inhibition of mTOR complex 1 by rapamycin decreased or prevented parathyroid cell proliferation in secondary hyperparathyroidism rats and in vitro in uremic rat parathyroid glands in organ culture. Knockin rpS6(p-/-) mice, in which rpS6 cannot be phosphorylated because of substitution of all five phosphorylatable serines with alanines, had impaired PTH secretion after experimental uremia- or folic acid-induced AKI. Uremic rpS6(p-/-) mice had no increase in parathyroid cell proliferation compared with a marked increase in uremic wild-type mice. These results underscore the importance of mTOR activation and rpS6 phosphorylation for the pathogenesis of secondary hyperparathyroidism and indicate that mTORC1 is a significant regulator of parathyroid cell proliferation through rpS6. Copyright © 2016 by the American Society of Nephrology.

  8. Rapalogs and mTOR inhibitors as anti-aging therapeutics.

    Science.gov (United States)

    Lamming, Dudley W; Ye, Lan; Sabatini, David M; Baur, Joseph A

    2013-03-01

    Rapamycin, an inhibitor of mechanistic target of rapamycin (mTOR), has the strongest experimental support to date as a potential anti-aging therapeutic in mammals. Unlike many other compounds that have been claimed to influence longevity, rapamycin has been repeatedly tested in long-lived, genetically heterogeneous mice, in which it extends both mean and maximum life spans. However, the mechanism that accounts for these effects is far from clear, and a growing list of side effects make it doubtful that rapamycin would ultimately be beneficial in humans. This Review discusses the prospects for developing newer, safer anti-aging therapies based on analogs of rapamycin (termed rapalogs) or other approaches targeting mTOR signaling.

  9. Effects of rapamycin and curcumin treatment on the development of epilepsy after electrically induced status epilepticus in rats.

    NARCIS (Netherlands)

    Drion, C.M.; Borm, L.E.; Kooijman, L.; Aronica, E.; Wadman, W.J.; Hartog, A.F.; van Vliet, E.A.; Gorter, J.A.

    2016-01-01

    OBJECTIVE: Inhibition of the mammalian target of rapamycin (mTOR) pathway has been suggested as a possible antiepileptogenic strategy in temporal lobe epilepsy (TLE). Here we aim to elucidate whether mTOR inhibition has antiepileptogenic and/or antiseizure effects using different treatment

  10. AMPK and mTOR: sensors and regulators of immunometabolic changes during Salmonella infection in the chicken.

    Science.gov (United States)

    Kogut, Michael H; Genovese, Kenneth J; He, Haiqi; Arsenault, Ryan J

    2016-02-01

    Non-typhoidal Salmonella enterica induce an early pro-inflammatory response in chickens, but the response is short-lived, asymptomatic of clinical disease, results in a persistent colonization of the gastrointestinal (GI) tract, and can transmit infections to naïve hosts via fecal shedding of bacteria. The underlying mechanisms that facilitate this persistent colonization of the ceca of chickens by Salmonella are unknown. We have begun to concentrate on the convergence of metabolism and immune function as playing a major role in regulating the host responsiveness to infection. It is now recognized that the immune system monitors the metabolic state of tissues and responds by modulating metabolic function. The aim in this review is to summarize the literature that has defined a series of genotypic and phenotypic alterations in the regulatory host immune-metabolic signaling pathways in the local cecal microenvironment during the first 4 d following infection with Salmonella enterica serovar Enteritidis. Using chicken-specific kinomic immune-metabolism peptide arrays and quantitative real-time-PCR of cecal tissue during the early (4 to 48 h) and late stages (4 to 17 d) of a Salmonella infection in young broiler chickens, the local immunometabolic microenvironment has been ascertained. Distinct immune and metabolic pathways are altered between 2 to 4 d post-infection that dramatically changed the local immunometabolic environment. Thus, the tissue immunometabolic phenotype of the cecum plays a major role in the ability of the bacterium to establish a persistent cecal colonization. In general, our findings show that AMPK and mTOR are key players linking specific extracellular milieu and intracellular metabolism. Phenotypically, the early response (4 to 48 h) to Salmonella infection is pro-inflammatory, fueled by glycolysis and mTOR-mediated protein synthesis, whereas by the later phase (4 to 5 d), the local environment has undergone an immune-metabolic reprogramming to

  11. mTOR hyperactivity mediates the detrimental effects of a high sucrose diet on Alzheimer’s disease pathology

    Science.gov (United States)

    Orr, Miranda E.; Salinas, Angelica; Buffenstein, Rochelle; Oddo, Salvatore

    2014-01-01

    High sugar consumption and diabetes increase the risk of developing Alzheimer’s disease (AD) by unknown mechanisms. Using an animal model of AD, here we show that high sucrose intake induces obesity with changes in central and peripheral insulin signaling. These pre-diabetic changes are associated with an increase in Aβ production and deposition. Moreover, high sucrose ingestion exacerbates tau phosphorylation by increasing Cdk5 activity. Mechanistically, the sucrose-mediated increase in AD-like pathology is due to hyperactive mTOR, a key nutrient sensor important in regulating energy homeostasis. Specifically, we show that rapamycin, an mTOR inhibitor, prevents the detrimental effects of sucrose in the brain without altering changes in peripheral insulin resistance. Overall, our data suggest that high sucrose intake and dysregulated insulin signaling, which are known to contribute to the occurrence of diabetes, increase the risk of developing AD by upregulating mTOR signaling. Therefore, early interventions to modulate mTOR activity in individuals at high risk of developing diabetes may decrease their AD susceptibility. PMID:24411482

  12. Caloric restriction protects against electrical kindling of the amygdala by inhibiting the mTOR signaling pathway

    Directory of Open Access Journals (Sweden)

    Bryan Victor Phillips-Farfan

    2015-03-01

    Full Text Available Caloric restriction (CR has been shown to possess antiepileptic properties; however its mechanism of action is poorly understood. CR might inhibit the activity of the mammalian or mechanistic target of rapamycin (mTOR signaling cascade, which seems to participate crucially in the generation of epilepsy. Thus, we investigated the effect of CR on the mTOR pathway and whether CR modified epilepsy generation due to electrical amygdala kindling. The former was studied by analyzing the phosphorylation of adenosine monophosphate-activated protein kinase, protein kinase B and the ribosomal protein S6. The mTOR cascade is regulated by energy and by insulin levels, both of which may be changed by CR; thus we investigated if CR altered the levels of energy substrates in the blood or the level of insulin in plasma. Finally, we studied if CR modified the expression of genes that encode proteins participating in the mTOR pathway. CR increased the after-discharge threshold and tended to reduce the after-discharge duration, indicating an anti-convulsive action. CR diminished the phosphorylation of protein kinase B and ribosomal protein S6, suggesting an inhibition of the mTOR cascade. However, CR did not change glucose, β-hydroxybutyrate or insulin levels; thus the effects of CR were independent from them. Interestingly, CR also did not modify the expression of any investigated gene. The results suggest that the anti-epileptic effect of CR may be partly due to inhibition of the mTOR pathway.

  13. Inhibition of hemangioma growth using polymer-lipid hybrid nanoparticles for delivery of rapamycin.

    Science.gov (United States)

    Li, Haitao; Teng, Yunfei; Sun, Jin; Liu, Jianyong

    2017-11-01

    Although infantile hemangiomas is benign, its rapid growth may induce serious complications. However, only one drug Hemangeol™ has been approved by US Food and Drug Administration (FDA) to treat infantile hemangiomas. Thus it is necessary to develop novel alternative drugs to treat infantile hemangiomas. Rapamycin is a well-know potent antiangiogenic agent, whereas the daily oral administration of rapamycin exerts undesired metabolic effects due to its inhibition of mechanistic target of rapamycin (mTOR) which is critical in cell metabolism. We hereby developed rapamycin-loaded polymer-lipid hybrid nanoparticles (Rapamycin-PLNPs) as a local controlled release system to realize local and sustained release of rapamycin, aiming to reduce the side effects and frequency of administration of rapamycin. Rapamycin-PLNPs are of a small size (129.1nm), desired drug encapsulation efficiency (63.7%), and sustained drug release for 5 days. Rapamycin-PLNPs were shown to be able to effectively bind to hemangioma endothelia cells (HemECs), induce significant proliferation inhibition and reduce expression of angiogenesis factors in HemECs. The therapeutic effect of Rapamycin-PLNPs against infantile hemangioma in vivo was superior to rapamycin, as reflected by reduced hemangioma volume, weight and microvessel density. Taken together, Rapamycin-PLNPs represent a very promising local approach in the treatment of infantile hemangiomas. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

  14. Targeting of mTORC1/2 by the mTOR kinase inhibitor PP242 induces apoptosis in AML cells under conditions mimicking the bone marrow microenvironment

    Science.gov (United States)

    Zeng, Zhihong; Shi, Yue Xi; Tsao, Twee; Qiu, YiHua; Kornblau, Steven M.; Baggerly, Keith A.; Liu, Wenbin; Jessen, Katti; Liu, Yi; Kantarjian, Hagop; Rommel, Christian; Fruman, David A.; Andreeff, Michael

    2012-01-01

    The interactions between the bone marrow (BM) microenvironment and acute myeloid leukemia (AML) is known to promote survival of AML cells. In this study, we used reverse phase-protein array (RPPA) technology to measure changes in multiple proteins induced by stroma in leukemic cells. We then investigated the potential of an mTOR kinase inhibitor, PP242, to disrupt leukemia/stroma interactions, and examined the effects of PP242 in vivo using a mouse model. Using RPPA, we confirmed that multiple survival signaling pathways, including the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT)/mammalian target of rapamycin (mTOR), were up-regulated in primary AML cells cocultured with stroma. PP242 effectively induced apoptosis in primary samples cultured with or without stroma. Mechanistically, PP242 attenuated the activities of mTORC1 and mTORC2, sequentially inhibited phosphorylated AKT, S6K, and 4EBP1, and concurrently suppressed chemokine receptor CXCR4 expression in primary leukemic cells and in stromal cells cultured alone or cocultured with leukemic cells. In the in vivo leukemia mouse model, PP242 inhibited mTOR signaling in leukemic cells and demonstrated a greater antileukemia effect than rapamycin. Our findings indicate that disrupting mTOR/AKT signaling with a selective mTOR kinase inhibitor can effectively target leukemic cells within the BM microenvironment. PMID:22826565

  15. Dose-dependent Effects of mTOR Inhibition on Weight and Mitochondrial Disease in Mice

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    Simon C Johnson

    2015-07-01

    Full Text Available Rapamycin extends lifespan and attenuates age-related pathologies in mice when administered through diet at 14 parts per million (PPM. Recently, we reported that daily intraperitoneal injection of rapamycin at 8 mg/kg attenuates mitochondrial disease symptoms and progression in the Ndufs4 knockout mouse model of Leigh Syndrome. Although rapamycin is a widely used pharmaceutical agent dosage has not been rigorously examined and no dose-response profile has been established. Given these observations we sought to determine if increased doses of oral rapamycin would result in more robust impact on mTOR driven parameters. To test this hypothesis, we compared the effects of dietary rapamycin at doses ranging from 14 to 378 PPM on growth in control and Ndufs4 knockout mice and on health and survival in the Ndufs4 knockout model. High dose rapamycin was well tolerated, dramatically reduced growth, and overcame gender differences. The highest oral dose, approximately 27-times the dose shown to extend murine lifespan, increased survival in Ndufs4 knockout mice similarly to daily rapamycin injection without observable adverse effects. These findings have broad implications for the effective use of rapamycin in murine studies and for the translational potential of rapamycin in the treatment of mitochondrial disease. This data, further supported by a comparison of available literature, suggests that 14 PPM dietary rapamycin is a sub-optimal dose for targeting mTOR systemically in mice. Our findings suggest that the role of mTOR in mammalian biology may be broadly underestimated when determined through treatment with rapamycin at commonly used doses.

  16. Exercise increases mTOR signaling in brain regions involved in cognition and emotional behavior.

    Science.gov (United States)

    Lloyd, Brian A; Hake, Holly S; Ishiwata, Takayuki; Farmer, Caroline E; Loetz, Esteban C; Fleshner, Monika; Bland, Sondra T; Greenwood, Benjamin N

    2017-04-14

    Exercise can enhance learning and memory and produce resistance against stress-related psychiatric disorders such as depression and anxiety. In rats, these beneficial effects of exercise occur regardless of exercise controllability: both voluntary and forced wheel running produce stress-protective effects. The mechanisms underlying these beneficial effects of exercise remain unknown. The mammalian target of rapamycin (mTOR) is a translation regulator important for cell growth, proliferation, and survival. mTOR has been implicated in enhancing learning and memory as well as antidepressant effects. Moreover, mTOR is sensitive to exercise signals such as metabolic factors. The effects of exercise on mTOR signaling, however, remain unknown. The goal of the present study was to test the hypothesis that exercise, regardless of controllability, increases levels of phosphorylated mTOR (p-mTOR) in brain regions important for learning and emotional behavior. Rats were exposed to 6 weeks of either sedentary (locked wheel), voluntary, or forced wheel running conditions. At 6 weeks, rats were sacrificed during peak running and levels of p-mTOR were measured using immunohistochemistry. Overall, both voluntary and forced exercise increased p-mTOR-positive neurons in the medial prefrontal cortex, striatum, hippocampus, hypothalamus, and amygdala compared to locked wheel controls. Exercise, regardless of controllability, also increased numbers of p-mTOR-positive glia in the striatum, hippocampus, and amygdala. For both neurons and glia, the largest increase in p-mTOR positive cells was observed after voluntary running, with forced exercise causing a more modest increase. Interestingly, voluntary exercise preferentially increased p-mTOR in astrocytes (GFAP+), while forced running increased p-mTOR in microglia (CD11+) in the inferior dentate gyrus. Results suggest that mTOR signaling is sensitive to exercise, but subtle differences exist depending on exercise controllability

  17. Biliverdin modulates the expression of C5aR in response to endotoxin in part via mTOR signaling.

    Science.gov (United States)

    Bisht, Kavita; Wegiel, Barbara; Tampe, Jens; Neubauer, Oliver; Wagner, Karl-Heinz; Otterbein, Leo E; Bulmer, Andrew C

    2014-06-20

    Macrophages play a crucial role in the maintenance and resolution of inflammation and express a number of pro- and anti-inflammatory molecules in response to stressors. Among them, the complement receptor 5a (C5aR) plays an integral role in the development of inflammatory disorders. Biliverdin and bilirubin, products of heme catabolism, exert anti-inflammatory effects and inhibit complement activation. Here, we define the effects of biliverdin on C5aR expression in macrophages and the roles of Akt and mammalian target of rapamycin (mTOR) in these responses. Biliverdin administration inhibited lipopolysaccharide (LPS)-induced C5aR expression (without altering basal expression), an effect partially blocked by rapamycin, an inhibitor of mTOR signaling. Biliverdin also reduced LPS-dependent expression of the pro-inflammatory cytokines TNF-α and IL-6. Collectively, these data indicate that biliverdin regulates LPS-mediated expression of C5aR via the mTOR pathway, revealing an additional mechanism underlying biliverdin's anti-inflammatory effects. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.

  18. Putrescine stimulates the mTOR signaling pathway and protein synthesis in porcine trophectoderm cells.

    Science.gov (United States)

    Kong, Xiangfeng; Wang, Xiaoqiu; Yin, Yulong; Li, Xilong; Gao, Haijun; Bazer, Fuller W; Wu, Guoyao

    2014-11-01

    Insufficient placental growth is a major factor contributing to intrauterine growth retardation in mammals. There is growing evidence that putrescine produced from arginine (Arg) and proline via ornithine decarboxylase is a key regulator of angiogenesis, embryogenesis, as well as placental and fetal growth. However, the underlying mechanisms are largely unknown. The present study tested the hypothesis that putrescine stimulates protein synthesis by activating the mechanistic target of rapamycin (mTOR) signaling pathway in porcine trophectoderm cell line 2 cells. The cells were cultured for 2 to 4 days in customized Arg-free Dulbecco modified Eagle Ham medium containing 0, 10, 25, or 50 μM putrescine or 100 μM Arg. Cell proliferation, protein synthesis, and degradation, as well as the abundance of total and phosphorylated mTOR, ribosomal protein S6 kinase 1, and eukaryotic initiation factor 4E-binding protein-1 (4EBP1), were determined. Our results indicate that putrescine promotes cell proliferation and protein synthesis in a dose- and time-dependent manner, which was inhibited by difluoro-methylornithine (an inhibitor of ornithine decarboxylase). Moreover, supplementation of culture medium with putrescine increased the abundance of phosphorylated mTOR and its downstream targets, 4EBP1 and p70 S6K1 proteins. Collectively, these findings reveal a novel and important role for putrescine in regulating the mTOR signaling pathway in porcine placental cells. We suggest that dietary supplementation with or intravenous administration of putrescine may provide a new and effective strategy to improve survival and growth of embryos/fetuses in mammals. © 2014 by the Society for the Study of Reproduction, Inc.

  19. Fasting Increases Human Skeletal Muscle Net Phenylalanine Release and This Is Associated with Decreased mTOR Signaling

    Science.gov (United States)

    Vendelbo, Mikkel Holm; Møller, Andreas Buch; Christensen, Britt; Nellemann, Birgitte; Clasen, Berthil Frederik Forrest; Nair, K. Sreekumaran; Jørgensen, Jens Otto Lunde; Jessen, Niels; Møller, Niels

    2014-01-01

    Aim Fasting is characterised by profound changes in energy metabolism including progressive loss of body proteins. The underlying mechanisms are however unknown and we therefore determined the effects of a 72-hour-fast on human skeletal muscle protein metabolism and activation of mammalian target of rapamycin (mTOR), a key regulator of cell growth. Methods Eight healthy male volunteers were studied twice: in the postabsorptive state and following 72 hours of fasting. Regional muscle amino acid kinetics was measured in the forearm using amino acid tracers. Signaling to protein synthesis and breakdown were assessed in skeletal muscle biopsies obtained during non-insulin and insulin stimulated conditions on both examination days. Results Fasting significantly increased forearm net phenylalanine release and tended to decrease phenylalanine rate of disappearance. mTOR phosphorylation was decreased by ∼50% following fasting, together with reduced downstream phosphorylation of 4EBP1, ULK1 and rpS6. In addition, the insulin stimulated increase in mTOR and rpS6 phosphorylation was significantly reduced after fasting indicating insulin resistance in this part of the signaling pathway. Autophagy initiation is in part regulated by mTOR through ULK1 and fasting increased expression of the autophagic marker LC3B-II by ∼30%. p62 is degraded during autophagy but was increased by ∼10% during fasting making interpretation of autophagic flux problematic. MAFbx and MURF1 ubiquitin ligases remained unaltered after fasting indicating no change in protesomal protein degradation. Conclusions Our results show that during fasting increased net phenylalanine release in skeletal muscle is associated to reduced mTOR activation and concomitant decreased downstream signaling to cell growth. PMID:25020061

  20. Fasting increases human skeletal muscle net phenylalanine release and this is associated with decreased mTOR signaling.

    Directory of Open Access Journals (Sweden)

    Mikkel Holm Vendelbo

    Full Text Available Fasting is characterised by profound changes in energy metabolism including progressive loss of body proteins. The underlying mechanisms are however unknown and we therefore determined the effects of a 72-hour-fast on human skeletal muscle protein metabolism and activation of mammalian target of rapamycin (mTOR, a key regulator of cell growth.Eight healthy male volunteers were studied twice: in the postabsorptive state and following 72 hours of fasting. Regional muscle amino acid kinetics was measured in the forearm using amino acid tracers. Signaling to protein synthesis and breakdown were assessed in skeletal muscle biopsies obtained during non-insulin and insulin stimulated conditions on both examination days.Fasting significantly increased forearm net phenylalanine release and tended to decrease phenylalanine rate of disappearance. mTOR phosphorylation was decreased by ∼50% following fasting, together with reduced downstream phosphorylation of 4EBP1, ULK1 and rpS6. In addition, the insulin stimulated increase in mTOR and rpS6 phosphorylation was significantly reduced after fasting indicating insulin resistance in this part of the signaling pathway. Autophagy initiation is in part regulated by mTOR through ULK1 and fasting increased expression of the autophagic marker LC3B-II by ∼30%. p62 is degraded during autophagy but was increased by ∼10% during fasting making interpretation of autophagic flux problematic. MAFbx and MURF1 ubiquitin ligases remained unaltered after fasting indicating no change in protesomal protein degradation.Our results show that during fasting increased net phenylalanine release in skeletal muscle is associated to reduced mTOR activation and concomitant decreased downstream signaling to cell growth.

  1. Mammalian target of rapamycin activity is required for expansion of CD34(+) hematopoietic progenitor cells

    NARCIS (Netherlands)

    Geest, Christian R.; Zwartkruis, Fried J.; Vellenga, Edo; Coffer, Paul J.; Buitenhuis, Miranda

    Background The mammalian target of rapamycin is a conserved protein kinase known to regulate protein synthesis, cell size and proliferation. Aberrant regulation of mammalian target of rapamycin activity has been observed in hematopoietic malignancies, including acute leukemias and myelodysplastic

  2. Mammalian target of rapamycin activity is required for expansion of CD34+ hematopoietic progenitor cells

    NARCIS (Netherlands)

    Geest, C.R.; Zwartkruis, G.J.T.; Vellenga, E.; Coffer, P.J.; Buitenhuis, M.

    2009-01-01

    Background The mammalian target of rapamycin is a conserved protein kinase known to regulate protein synthesis, cell size and proliferation. Aberrant regulation of mammalian target of rapamycin activity has been observed in hematopoietic malignancies, including acute leukemias and

  3. Effects of rapamycin treatment after controlled cortical impact injury on neurogenesis and synaptic reorganization in the mouse dentate gyrus

    Directory of Open Access Journals (Sweden)

    Corwin R Butler

    2015-11-01

    Full Text Available Post-traumatic epilepsy (PTE is one consequence of traumatic brain injury (TBI. A prominent cell signaling pathway activated in animal models of both TBI and epilepsy is the mammalian target of rapamycin (mTOR. Inhibition of mTOR with rapamycin has shown promise as a potential modulator of epileptogenesis in several animal models of epilepsy, but cellular mechanisms linking mTOR expression and epileptogenesis are unclear. In this study, the role of mTOR in modifying functional hippocampal circuit reorganization after focal TBI induced by controlled cortical impact was investigated. Rapamycin (3 or 10 mg/kg, an inhibitor of mTOR signaling, was administered by intraperitoneal injection beginning on the day of injury and continued daily until tissue collection. Relative to controls, rapamycin treatment reduced dentate granule cell area in the hemisphere ipsilateral to the injury two weeks post-injury. Brain injury resulted in a significant increase in doublecortin immunolabeling in the dentate gyrus ipsilateral to the injury, indicating increased neurogenesis shortly after TBI. Rapamycin treatment prevented the increase in doublecortin labeling, with no overall effect on Fluoro-Jade B staining in the ipsilateral hemisphere, suggesting that rapamycin treatment reduced posttraumatic neurogenesis but did not prevent cell loss after injury. At later times post-injury (8-13 weeks, evidence of mossy fiber sprouting and increased recurrent excitation of dentate granule cells was detected, which were attenuated by rapamycin treatment. Rapamycin treatment also diminished seizure prevalence relative to vehicle-treated controls after TBI. Collectively, these results support a role for adult neurogenesis in PTE development and suggest that suppression of epileptogenesis by mTOR inhibition includes effects on post-injury neurogenesis.

  4. Rapamycin drives selection against a pathogenic heteroplasmic mitochondrial DNA mutation.

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    Dai, Ying; Zheng, Kangni; Clark, Joanne; Swerdlow, Russell H; Pulst, Stefan M; Sutton, James P; Shinobu, Leslie A; Simon, David K

    2014-02-01

    Mitochondrial DNA (mtDNA) mutations cause a variety of mitochondrial disorders for which effective treatments are lacking. Emerging data indicate that selective mitochondrial degradation through autophagy (mitophagy) plays a critical role in mitochondrial quality control. Inhibition of mammalian target of rapamycin (mTOR) kinase activity can activate mitophagy. To test the hypothesis that enhancing mitophagy would drive selection against dysfunctional mitochondria harboring higher levels of mutations, thereby decreasing mutation levels over time, we examined the impact of rapamycin on mutation levels in a human cytoplasmic hybrid (cybrid) cell line expressing a heteroplasmic mtDNA G11778A mutation, the most common cause of Leber's hereditary optic neuropathy. Inhibition of mTORC1/S6 kinase signaling by rapamycin induced colocalization of mitochondria with autophagosomes, and resulted in a striking progressive decrease in levels of the G11778A mutation and partial restoration of ATP levels. Rapamycin-induced upregulation of mitophagy was confirmed by electron microscopic evidence of increased autophagic vacuoles containing mitochondria-like organelles. The decreased mutational burden was not due to rapamycin-induced cell death or mtDNA depletion, as there was no significant difference in cytotoxicity/apoptosis or mtDNA copy number between rapamycin and vehicle-treated cells. These data demonstrate the potential for pharmacological inhibition of mTOR kinase activity to activate mitophagy as a strategy to drive selection against a heteroplasmic mtDNA G11778A mutation and raise the exciting possibility that rapamycin may have therapeutic potential for the treatment of mitochondrial disorders associated with heteroplasmic mtDNA mutations, although further studies are needed to determine if a similar strategy will be effective for other mutations and other cell types.

  5. Assessment of Response of Kidney Tumors to Rapamycin and Atorvastatin in Tsc1+/- Mice.

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    Shen, Ming Hong; Samsel, Paulina; Shen, Louise L; Narov, Kalin; Yang, Jian; Sampson, Julian R

    2017-10-01

    Atorvastatin is widely used to lower blood cholesterol and to reduce risk of cardiovascular disease-associated complications. Epidemiological investigations and preclinical studies suggest that statins such as atorvastatin have antitumor activity for various types of cancer. Tuberous sclerosis (TSC) is a tumor syndrome caused by TSC1 or TSC2 mutations that lead to aberrant activation of mTOR and tumor formation in multiple organs. Previous studies have demonstrated that atorvastatin selectively suppressed growth and proliferation of mouse Tsc2 null embryonic fibroblasts through inhibition of mTOR. However, atorvastatin alone did not reduce tumor burden in the liver and kidneys of Tsc2+/- mice as assessed by histological analysis, and no combination therapy of rapamycin and atorvastatin has been tried. In this study, we used T2-weighted magnetic resonance imaging to track changes in tumor number and size in the kidneys of a Tsc1+/- mouse model and to assess the efficacy of rapamycin and atorvastatin alone and as a combination therapy. We found that rapamycin alone or rapamycin combined with atorvastatin significantly reduced tumor burden, while atorvastatin alone did not. Combined therapy with rapamycin and atorvastatin appeared to be more effective for treating renal tumors than rapamycin alone, but the difference was not statistically significant. We conclude that combined therapy with rapamycin and atorvastatin is unlikely to provide additional benefit over rapamycin as a single agent in the treatment of Tsc-associated renal tumors. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

  6. Metabolic effects of acute thiamine depletion are reversed by rapamycin in breast and leukemia cells.

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    Shuqian Liu

    reversed by rapamycin. Thus, these studies demonstrate that acute intracellular thiamine depletion by recombinant thiaminase results in metabolic changes in thiamine-dependent metabolism, and demonstrate a previously unrecognized role of mTOR signaling in the regulation of thiamine-dependent metabolism.

  7. mTOR Overactivation and Compromised Autophagy in the Pathogenesis of Pulmonary Fibrosis.

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    Yao-Song Gui

    Full Text Available The mammalian target of rapamycin (mTOR signaling pathway in pulmonary fibrosis was investigated in cell and animal models. mTOR overactivation in alveolar epithelial cells (AECs was achieved in the conditional and inducible Tsc1 knock-down mice SPC-rtTA/TetO-Cre/Tsc1(fx/+ (STT. Doxycycline caused Tsc1 knock-down and consequently mTOR activation in AECs for the STT mice. Mice treated with bleomycin exhibited increased mortality and pulmonary fibrosis compared with control mice. In wild-type C57BL/6J mice, pretreatment with rapamycin attenuated the bleomycin-mediated mortality and fibrosis. Rapamycin-mediated mouse survival benefit was inhibited by chloroquine, an autophagy inhibitor. Autophagosomes were decreased in the lungs after bleomycin exposure. Rapamycin induced the production of autophagosomes and diminished p62. We concluded that mTOR overactivation in AECs and compromised autophagy in the lungs are involved in the pathogenesis of pulmonary fibrosis. The suppression of mTOR and enhancement of autophagy may be used for treatment of pulmonary fibrosis.

  8. Imbalanced mechanistic target of rapamycin C1 and C2 activity in the cerebellum of Angelman syndrome mice impairs motor function.

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    Sun, Jiandong; Liu, Yan; Moreno, Stephanie; Baudry, Michel; Bi, Xiaoning

    2015-03-18

    Angelman syndrome (AS) is a neurogenetic disorder caused by deficiency of maternally expressed ubiquitin-protein ligase E3A (UBE3A), an E3 ligase that targets specific proteins for proteasomal degradation. Although motor function impairment occurs in all patients with AS, very little research has been done to understand and treat it. The present study focuses on Ube3A deficiency-induced alterations in signaling through the mechanistic target of rapamycin (mTOR) pathway in the cerebellum of the AS mouse model and on potential therapeutic applications of rapamycin. Levels of tuberous sclerosis complex 2 (TSC2), a negative regulator of mTOR, were increased in AS mice compared with wild-type mice; however, TSC2 inhibitory phosphorylation was also increased. Correspondingly, levels of phosphorylated/active mTOR were increased. Phosphorylation of the mTORC1 substrates S6 kinase 1 (S6K1) and S6 was elevated, whereas that of the mTORC2 substrates AKT and N-myc downstream regulated 1 was decreased, suggesting enhanced mTORC1 but inhibited mTORC2 signaling. Semi-chronic treatment of AS mice with rapamycin not only improved their motor performance but also normalized mTORC1 and mTORC2 signaling. Furthermore, inhibitory phosphorylation of rictor, a key regulatory/structural subunit of the mTORC2 complex, was increased in AS mice and decreased after rapamycin treatment. These results indicate that Ube3A deficiency leads to overactivation of the mTORC1-S6K1 pathway, which in turn inhibits rictor, resulting in decreased mTORC2 signaling in Purkinje neurons of AS mice. Finally, rapamycin treatment also improved dendritic spine morphology in AS mice, through inhibiting mTORC1 and possibly enhancing mTORC2-mediated regulation of synaptic cytoskeletal elements. Collectively, our results indicate that the imbalance between mTORC1 and mTORC2 activity may contribute to synaptic pathology and motor impairment in AS. Copyright © 2015 the authors 0270-6474/15/354706-13$15.00/0.

  9. Rapamycin suppresses brain aging in senescence-accelerated OXYS rats.

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    Kolosova, Nataliya G; Vitovtov, Anton O; Muraleva, Natalia A; Akulov, Andrey E; Stefanova, Natalia A; Blagosklonny, Mikhail V

    2013-06-01

    Cellular and organismal aging are driven in part by the MTOR (mechanistic target of rapamycin) pathway and rapamycin extends life span inC elegans, Drosophila and mice. Herein, we investigated effects of rapamycin on brain aging in OXYS rats. Previously we found, in OXYS rats, an early development of age-associated pathological phenotypes similar to several geriatric disorders in humans, including cerebral dysfunctions. Behavioral alterations as well as learning and memory deficits develop by 3 months. Here we show that rapamycin treatment (0.1 or 0.5 mg/kg as a food mixture daily from the age of 1.5 to 3.5 months) decreased anxiety and improved locomotor and exploratory behavior in OXYS rats. In untreated OXYS rats, MRI revealed an increase of the area of hippocampus, substantial hydrocephalus and 2-fold increased area of the lateral ventricles. Rapamycin treatment prevented these abnormalities, erasing the difference between OXYS and Wister rats (used as control). All untreated OXYS rats showed signs of neurodegeneration, manifested by loci of demyelination. Rapamycin decreased the percentage of animals with demyelination and the number of loci. Levels of Tau and phospho-Tau (T181) were increased in OXYS rats (compared with Wistar). Rapamycin significantly decreased Tau and inhibited its phosphorylation in the hippocampus of OXYS and Wistar rats. Importantly, rapamycin treatment caused a compensatory increase in levels of S6 and correspondingly levels of phospo-S6 in the frontal cortex, indicating that some downstream events were compensatory preserved, explaining the lack of toxicity. We conclude that rapamycin in low chronic doses can suppress brain aging.

  10. Combinatorial Antitumor Effect of Rapamycin and β-Elemene in Follicular Thyroid Cancer Cells

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    Jun Zhou

    2016-01-01

    Full Text Available Background. mTOR signaling would be a promising target for thyroid cancer therapy. However, in clinical trials, objective response rate with mTOR inhibitor monotherapy in most cancer types was modest. A new focus on development of combinatorial strategies with rapalogs is increasing. Objective. Investigating the combinatorial antitumor effect of rapamycin and β-elemene in follicular thyroid cancer cells. Methods. MTT assay was used to determine the FTC-133 cell proliferation after culturing with rapamycin and/or β-elemene. To analyze their combinatorial effect, immunoblotting was performed to analyze the activation status of AKT. Moreover, β-elemene attenuated rapamycin-induced immunosuppression was tested in mice. Results. Combination of rapamycin and β-elemene exerted significant synergistic antiproliferative effects in FTC-133 cell lines in vitro, based on inhibiting the AKT feedback activation induced by rapamycin. In vivo, the β-elemene could attenuate rapamycin-induced immunosuppression via reversing imbalance of Treg/Th17, with the underlying mechanism needed to be declared. Conclusions. We demonstrate that the novel combination of mTOR inhibitor with β-elemene synergistically attenuates tumor cell growth in follicular thyroid cancer, which requires additional preclinical validation.

  11. Rapamycin inhibits spermatogenesis by changing the autophagy status through suppressing mechanistic target of rapamycin-p70S6 kinase in male rats.

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    Liu, Shangjing; Huang, Longxian; Geng, Yanqing; He, Junlin; Chen, Xuemei; Xu, Hao; Li, Rong; Wang, Yingxiong; Ding, Yubin; Liu, Xueqing

    2017-10-01

    Rapamycin (sirolimus) is an antiproliferative drug that has been widely used in the clinic as an immunosuppressant and a potential anticancer agent. Certain reports have indicated that rapamycin may induce male infertility through impairing sperm quality. The present study investigated the mechanism of male infertility caused by rapamycin and examined whether withdrawal of rapamycin could recover the number of sperm in rats. Male Sprague‑Dawley rats (n=100) were divided randomly into 5 groups: 3 rapamycin‑treated groups (2, 4 and 6 mg/kg) and 2 control groups [Blank and dimethyl sulfoxide (DMSO)]. Organ coefficients of the testes, number of sperm and hematoxylin‑eosin staining analyses demonstrated that rapamycin treatment markedly damaged the structure of the seminiferous tubule and reduced the number of sperm. Immunohistochemistry of mechanistic target of rapamycin (mTOR) and Ki67 in testes tissue, and western blotting of phosphorylated‑p70S6K and p70S6K, supported the hypothesis that rapamycin causes sperm reduction through inhibiting proliferation of spermatogonia. Unfortunately, 24 weeks after cessation of rapamycin treatment, only the number of sperm in 2 mg/kg group was restored back to the normal level. In addition, to the best of our knowledge, the present study was the first to demonstrate that low doses rapamycin leads to activation of autophagy in rat testes. This may be a self‑protective mechanism of the cell in response to external stress. Thus, spermatogenesis can be recovered in the testes from rats in the low dose group. High doses of rapamycin resulted in excessive consumption of autophagy proteins, and the damage could not be compensated. In addition, it was revealed that cell apoptosis increased after treatment with rapamycin. In conclusion, the present study demonstrated that rapamycin inhibits spermatogenesis through suppressing phosphorylation of p70S6K and changing the autophagy status, ultimately reducing the number of sperm

  12. The role of phospholipase D and phosphatidic acid in the mechanical activation of mTOR signaling in skeletal muscle.

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    Hornberger, T A; Chu, W K; Mak, Y W; Hsiung, J W; Huang, S A; Chien, S

    2006-03-21

    Signaling by the mammalian target of rapamycin (mTOR) has been reported to be necessary for mechanical load-induced growth of skeletal muscle. The mechanisms involved in the mechanical activation of mTOR signaling are not known, but several studies indicate that a unique [phosphotidylinositol-3-kinase (PI3K)- and nutrient-independent] mechanism is involved. In this study, we have demonstrated that a regulatory pathway for mTOR signaling that involves phospholipase D (PLD) and the lipid second messenger phosphatidic acid (PA) plays a critical role in the mechanical activation of mTOR signaling. First, an elevation in PA concentration was sufficient for the activation of mTOR signaling. Second, the isozymes of PLD (PLD1 and PLD2) are localized to the z-band in skeletal muscle (a critical site of mechanical force transmission). Third, mechanical stimulation of skeletal muscle with intermittent passive stretch ex vivo induced PLD activation, PA accumulation, and mTOR signaling. Finally, pharmacological inhibition of PLD blocked the mechanically induced increase in PA and the activation of mTOR signaling. Combined, these results indicate that mechanical stimuli activate mTOR signaling through a PLD-dependent increase in PA. Furthermore, we showed that mTOR signaling was partially resistant to rapamycin in muscles subjected to mechanical stimulation. Because rapamycin and PA compete for binding to the FRB domain on mTOR, these results suggest that mechanical stimuli activate mTOR signaling through an enhanced binding of PA to the FRB domain on mTOR.

  13. Caveat mTOR: aberrant signaling disrupts corticogenesis.

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    Osborne, Lucy R

    2010-05-01

    The mammalian target of rapamycin (mTOR) signaling pathway is activated in several disorders associated with benign tumors and malformations of the cerebral cortex. In this issue of the JCI, Orlova et al. have now definitively added another disorder to this group by demonstrating that activation of mTOR signaling is associated with polyhydramnios, megalencephaly, and symptomatic epilepsy syndrome (PMSE), which is characterized by severe intractable epilepsy and megalencephaly. PMSE is caused by lack of the pseudokinase STE20-related kinase adaptor alpha (STRADalpha), and Orlova et al. show that reduction of STRADalpha levels during corticogenesis in the mouse results in a cellular phenotype and neuronal migration defects similar to those observed in patients with PMSE, clearly demonstrating a pivotal role for STRADalpha in cell polarity and growth. This study helps pave the way for possible therapeutic intervention with rapamycin to control the epilepsy and learning disabilities associated with this disorder.

  14. Synergistic Effects between mTOR Complex 1/2 and Glycolysis Inhibitors in Non-Small-Cell Lung Carcinoma Cells.

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    Suhua Jiang

    Full Text Available Cancer metabolism has greatly interested researchers. Mammalian target of rapamycin (mTOR is dysregulated in a variety of cancers and considered to be an appealing therapeutic target. It has been proven that growth factor signal, mediated by mTOR complex 1 (mTORC1, drives cancer metabolism by regulating key enzymes in metabolic pathways. However, the role of mTORC2 in cancer metabolism has not been thoroughly investigated. In this study, by employing automated spectrophotometry, we found the level of glucose uptake was decreased in non-small-cell lung carcinoma (NSCLC A549, PC-9 and SK-MES-1 cells treated with rapamycin or siRNA against Raptor, indicating that the inhibition of mTORC1 attenuated glycolytic metabolism in NSCLC cells. Moreover, the inhibition of AKT reduced glucose uptake in the cells as well, suggesting the involvement of AKT pathway in mTORC1 mediated glycolytic metabolism. Furthermore, our results showed a significant decrease in glucose uptake in rictor down-regulated NSCLC cells, implying a critical role of mTORC2 in NSCLC cell glycolysis. In addition, the experiments for MTT, ATP, and clonogenic assays demonstrated a reduction in cell proliferation, cell viability, and colony forming ability in mTOR inhibiting NSCLC cells. Interestingly, the combined application of mTORC1/2 inhibitors and glycolysis inhibitor not only suppressed the cell proliferation and colony formation, but also induced cell apoptosis, and such an effect of the combined application was stronger than that caused by mTORC1/2 inhibitors alone. In conclusion, this study reports a novel effect of mTORC2 on NSCLC cell metabolism, and reveals the synergistic effects between mTOR complex 1/2 and glycolysis inhibitors, suggesting that the combined application of mTORC1/2 and glycolysis inhibitors may be a new promising approach to treat NSCLC.

  15. Pivotal Role of mTOR Signaling in Hepatocellular Carcinoma

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    Villanueva, Augusto; Chiang, Derek Y.; Newell, Pippa; Peix, Judit; Thung, Swan; Alsinet, Clara; Tovar, Victoria; Roayaie, Sasan; Minguez, Beatriz; Sole, Manel; Battiston, Carlo; van Laarhoven, Stijn; Fiel, Maria I; Di Feo, Analisa; Hoshida, Yujin; Yea, Steven; Toffanin, Sara; Ramos, Alex; Martignetti, John A.; Mazzaferro, Vincenzo; Bruix, Jordi; Waxman, Samuel; Schwartz, Myron; Meyerson, Matthew; Friedman, Scott L.; Llovet, Josep M.

    2008-01-01

    BACKGROUND The advent of targeted therapies in hepatocellular carcinoma (HCC) has underscored the importance of pathway characterization to identify novel molecular targets for treatment. Based on its role in cell growth and differentiation, we evaluated mTOR signaling activation in human HCC, as well as the anti-tumoral effect of a dual-level blockade of the mTOR pathway. METHODS The mTOR pathway was assessed using integrated data from mutation analysis (direct sequencing), DNA copy number changes (SNP-array), mRNA levels (qRT-PCR and gene expression microarray), and protein activation (immunostaining) in 351 human samples, including HCC (n=314), and non-tumoral tissue (n=37). Effects of dual blockade of mTOR signaling using a rapamycin analog (everolimus) and an EGFR/VEGFR inhibitor (AEE788) were evaluated in liver cancer cell lines, and in a tumor xenograft model. RESULTS Aberrant mTOR signaling (phosphorylated-RPS6) was present in half of the cases, associated with IGF pathway activation, EGF upregulation, and PTEN dysregulation. PTEN and PI3KCA-B mutations were rare events. Chromosomal gains in RICTOR (25% of patients) and positive pRPS6 staining correlated with recurrence. RICTOR-specific siRNA downregulation reduced tumor cell viability in vitro. Blockage of mTOR signaling with everolimus in vitro and in a xenograft model decelerated tumor growth and increased survival. This effect was enhanced in vivo after EGFR blockade. CONCLUSIONS MTOR signaling has a critical role in the pathogenesis of HCC, with evidence for the role of RICTOR in tumor oncogenesis. MTOR blockade with everolimus is effective in vivo. These findings establish a rationale for targeting mTOR pathway in clinical trials in HCC. PMID:18929564

  16. Phosphorylation of Ribosomal Protein S6 Mediates Mammalian Target of Rapamycin Complex 1–Induced Parathyroid Cell Proliferation in Secondary Hyperparathyroidism

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    Volovelsky, Oded; Cohen, Gili; Kenig, Ariel; Wasserman, Gilad; Dreazen, Avigail; Meyuhas, Oded; Silver, Justin

    2016-01-01

    Secondary hyperparathyroidism is characterized by increased serum parathyroid hormone (PTH) level and parathyroid cell proliferation. However, the molecular pathways mediating the increased parathyroid cell proliferation remain undefined. Here, we found that the mTOR pathway was activated in the parathyroid of rats with secondary hyperparathyroidism induced by either chronic hypocalcemia or uremia, which was measured by increased phosphorylation of ribosomal protein S6 (rpS6), a downstream target of the mTOR pathway. This activation correlated with increased parathyroid cell proliferation. Inhibition of mTOR complex 1 by rapamycin decreased or prevented parathyroid cell proliferation in secondary hyperparathyroidism rats and in vitro in uremic rat parathyroid glands in organ culture. Knockin rpS6p−/− mice, in which rpS6 cannot be phosphorylated because of substitution of all five phosphorylatable serines with alanines, had impaired PTH secretion after experimental uremia- or folic acid–induced AKI. Uremic rpS6p−/− mice had no increase in parathyroid cell proliferation compared with a marked increase in uremic wild–type mice. These results underscore the importance of mTOR activation and rpS6 phosphorylation for the pathogenesis of secondary hyperparathyroidism and indicate that mTORC1 is a significant regulator of parathyroid cell proliferation through rpS6. PMID:26283674

  17. mTOR signaling in proteostasis and its relevance to autism spectrum disorders

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    Judit Faus-Garriga

    2017-01-01

    Full Text Available Proteins are extremely labile cellular components, especially at physiological temperatures. The appropriate regulation of protein levels, or proteostasis, is essential for all cells. In the case of highly polarized cells like neurons, proteostasis is also crucial at synapses, where quick confined changes in protein composition occur to support synaptic activity and plasticity. The accurate regulation of those cellular processes controlling protein synthesis and degradation is necessary for proteostasis, and its deregulation has deleterious consequences in brain function. Alterations in those cellular mechanisms supporting synaptic protein homeostasis have been pinpointed in autism spectrum disorders such as tuberous sclerosis, neurofibromatosis 1, PTEN-related disorders, fragile X syndrome, MECP2 disorders and Angelman syndrome. Proteostasis alterations in these disorders share the alterations in mechanistic/mammalian target of rapamycin (mTOR signaling pathway, an intracellular pathway with key synaptic roles. The aim of the present review is to describe the recent literature on the major cellular mechanisms involved in proteostasis regulation in the synaptic context, and its association with mTOR signaling deregulations in various autism spectrum disorders. Altogether, the cellular and molecular mechanisms in synaptic proteostasis could be the foundation for novel shared therapeutic strategies that would take advantage of targeting common disorder mechanisms.

  18. Transient mTOR inhibition facilitates continuous growth of liver tumors by modulating the maintenance of CD133+ cell populations.

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    Zhaojuan Yang

    Full Text Available The mammalian target of the rapamycin (mTOR pathway, which drives cell proliferation, is frequently hyperactivated in a variety of malignancies. Therefore, the inhibition of the mTOR pathway has been considered as an appropriate approach for cancer therapy. In this study, we examined the roles of mTOR in the maintenance and differentiation of cancer stem-like cells (CSCs, the conversion of conventional cancer cells to CSCs and continuous tumor growth in vivo. In H-Ras-transformed mouse liver tumor cells, we found that pharmacological inhibition of mTOR with rapamycin greatly increased not only the CD133+ populations both in vitro and in vivo but also the expression of stem cell-like genes. Enhancing mTOR activity by over-expressing Rheb significantly decreased CD133 expression, whereas knockdown of the mTOR yielded an opposite effect. In addition, mTOR inhibition severely blocked the differentiation of CD133+ to CD133- liver tumor cells. Strikingly, single-cell culture experiments revealed that CD133- liver tumor cells were capable of converting to CD133+ cells and the inhibition of mTOR signaling substantially promoted this conversion. In serial implantation of tumor xenografts in nude BALB/c mice, the residual tumor cells that were exposed to rapamycin in vivo displayed higher CD133 expression and had increased secondary tumorigenicity compared with the control group. Moreover, rapamycin treatment also enhanced the level of stem cell-associated genes and CD133 expression in certain human liver tumor cell lines, such as Huh7, PLC/PRC/7 and Hep3B. The mTOR pathway is significantly involved in the generation and the differentiation of tumorigenic liver CSCs. These results may be valuable for the design of more rational strategies to control clinical malignant HCC using mTOR inhibitors.

  19. Rapamycin extends murine lifespan but has limited effects on aging

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    Neff, Frauke; Flores-Dominguez, Diana; Ryan, Devon P.; Horsch, Marion; Schröder, Susanne; Adler, Thure; Afonso, Luciana Caminha; Aguilar-Pimentel, Juan Antonio; Becker, Lore; Garrett, Lillian; Hans, Wolfgang; Hettich, Moritz M.; Holtmeier, Richard; Hölter, Sabine M.; Moreth, Kristin; Prehn, Cornelia; Puk, Oliver; Rácz, Ildikó; Rathkolb, Birgit; Rozman, Jan; Naton, Beatrix; Ordemann, Rainer; Adamski, Jerzy; Beckers, Johannes; Bekeredjian, Raffi; Busch, Dirk H.; Ehninger, Gerhard; Graw, Jochen; Höfler, Heinz; Klingenspor, Martin; Klopstock, Thomas; Ollert, Markus; Stypmann, Jörg; Wolf, Eckhard; Wurst, Wolfgang; Zimmer, Andreas; Fuchs, Helmut; Gailus-Durner, Valérie; Hrabe de Angelis, Martin; Ehninger, Dan

    2013-01-01

    Aging is a major risk factor for a large number of disorders and functional impairments. Therapeutic targeting of the aging process may therefore represent an innovative strategy in the quest for novel and broadly effective treatments against age-related diseases. The recent report of lifespan extension in mice treated with the FDA-approved mTOR inhibitor rapamycin represented the first demonstration of pharmacological extension of maximal lifespan in mammals. Longevity effects of rapamycin may, however, be due to rapamycin’s effects on specific life-limiting pathologies, such as cancers, and it remains unclear if this compound actually slows the rate of aging in mammals. Here, we present results from a comprehensive, large-scale assessment of a wide range of structural and functional aging phenotypes, which we performed to determine whether rapamycin slows the rate of aging in male C57BL/6J mice. While rapamycin did extend lifespan, it ameliorated few studied aging phenotypes. A subset of aging traits appeared to be rescued by rapamycin. Rapamycin, however, had similar effects on many of these traits in young animals, indicating that these effects were not due to a modulation of aging, but rather related to aging-independent drug effects. Therefore, our data largely dissociate rapamycin’s longevity effects from effects on aging itself. PMID:23863708

  20. Negative regulation of HLA-DR expression on endothelial cells by anti-blood group A/B antibody ligation and mTOR inhibition.

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    Iwasaki, Kenta; Miwa, Yuko; Uchida, Kazuharu; Kodera, Yasuhiro; Kobayashi, Takaaki

    2017-02-01

    Donor-specific antibody (DSA), particularly against HLA class II, is a major cause of chronic antibody-mediated rejection (CAMR) after transplantation, although ABO-incompatible kidney transplantation has recently demonstrated favorable graft outcomes. The condition of no injury even in the presence of anti-donor antibody has been referred to as "accommodation", which would be one of the key factors for successful long-term graft survival. The purpose of this study was to analyze the beneficial effect of anti-blood group A/B antibody ligation on endothelial cells against HLA-DR antibody-mediated, complement-dependent cytotoxicity (CDC). Blood group A/B-expressing endothelial cells EA.hy926 or Human Umbilical Vein Endothelia Cells (HUVEC) were incubated with IFNγ in the presence or absence of anti-blood group A/B antibody or mTOR inhibitor (mTOR-i) for 48h. The effects on signaling pathway, HLA expression, complement regulatory factors, and CDC were investigated. Expression of HLA-DR on EA.hy926 or HUVEC were successfully elicited by IFNγ treatment, although little or no expression was observed in quiescent cells. Pre-incubation with anti-blood group A/B antibody had resistance to HLA-DR antibody-mediated CDC against IFNγ-treated cells in a concentration-dependent manner. This finding was ascribed to decreased expression of HLA-DR by post-translational regulation and increased expression of CD55/59, which was related to ERK and mTOR pathway inhibition. mTOR-i also inhibited HLA-DR expression by itself. Furthermore, the combination of mTOR-I and anti-blood group A/B ligation had an additive effect in preventing HLA-DR antibody-mediated CDC. Anti-blood group A/B antibody might play a preventive role in CAMR. Inhibition of the ERK and mTOR pathways may contribute to the development of a novel treatment in the maintenance period after transplantation. Copyright © 2016 Elsevier B.V. All rights reserved.

  1. Reperfusion Therapy with Rapamycin Attenuates Myocardial Infarction through Activation of AKT and ERK

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    Scott M. Filippone

    2017-01-01

    Full Text Available Prompt coronary reperfusion is the gold standard for minimizing injury following acute myocardial infarction. Rapamycin, mammalian target of Rapamycin (mTOR inhibitor, exerts preconditioning-like cardioprotective effects against ischemia/reperfusion (I/R injury. We hypothesized that Rapamycin, given at the onset of reperfusion, reduces myocardial infarct size through modulation of mTOR complexes. Adult C57 male mice were subjected to 30 min of myocardial ischemia followed by reperfusion for 1 hour/24 hours. Rapamycin (0.25 mg/kg or DMSO (7.5% was injected intracardially at the onset of reperfusion. Post-I/R survival (87% and cardiac function (fractional shortening, FS: 28.63±3.01% were improved in Rapamycin-treated mice compared to DMSO (survival: 63%, FS: 17.4±2.6%. Rapamycin caused significant reduction in myocardial infarct size (IS: 26.2±2.2% and apoptosis (2.87±0.64% as compared to DMSO-treated mice (IS: 47.0±2.3%; apoptosis: 7.39±0.81%. Rapamycin induced phosphorylation of AKT S473 (target of mTORC2 but abolished ribosomal protein S6 phosphorylation (target of mTORC1 after I/R. Rapamycin induced phosphorylation of ERK1/2 but inhibited p38 phosphorylation. Infarct-limiting effect of Rapamycin was abolished with ERK inhibitor, PD98059. Rapamycin also attenuated Bax and increased Bcl-2/Bax ratio. These results suggest that reperfusion therapy with Rapamycin protects the heart against I/R injury by selective activation of mTORC2 and ERK with concurrent inhibition of mTORC1 and p38.

  2. Pre-clinical study of drug combinations that reduce breast cancer burden due to aberrant mTOR and metabolism promoted by LKB1 loss

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    Andrade-Vieira, Rafaela; Goguen, Donna; Bentley, Heidi A.; Bowen, Chris V.; Marignani, Paola A.

    2014-01-01

    Cancer therapies that simultaneously target activated mammalian target of rapamycin (mTOR) and cell metabolism are urgently needed. The goal of our study was to identify therapies that effectively inhibited both mTOR activity and cancer cell metabolism in primary tumors in vivo. Using our mouse model of spontaneous breast cancer promoted by loss of LKB1 expression in an ErbB2 activated model; referred to as LKB1−/−NIC mice, we evaluated the effect of novel therapies in vivo on primary tumors. Treatment of LKB1−/−NIC mice with AZD8055 and 2-DG mono-therapies significantly reduced mammary gland tumorigenesis by inhibiting mTOR pathways and glycolytic metabolism; however simultaneous inhibition of these pathways with AZD8055/2-DG combination was significantly more effective at reducing tumor volume and burden. At the molecular level, combination treatment inhibited mTORC1/mTORC2 activity, selectively inhibited mitochondria function and blocked MAPK pro-survival signaling responsible for the ERK-p90RSK feedback loop. Our findings suggest that loss of LKB1 expression be considered a marker for metabolic dysfunction given its role in regulating AMPK and mTOR function. Finally, the outcome of our pre-clinical study confirms therapies that simultaneously target mTORC1/mTORC2 and glycolytic metabolism in cancer produce the best therapeutic outcome for the treatment of patients harboring metabolically active HER2 positive breast cancers. PMID:25436981

  3. MicroRNA-145 inhibits the activation of the mTOR signaling pathway to suppress the proliferation and invasion of invasive pituitary adenoma cells by targeting AKT3 in vivo and in vitro

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    Zhou K

    2017-03-01

    Full Text Available Kai Zhou, Yan-Dong Fan, Peng-Fei Wu, Serick Duysenbi, Zhao-Hai Feng, Guo-Jia Du, Ting-Rong Zhang Department of Neurosurgery, The First Affiliated Hospital of Xinjiang Medical University, Urumqi, People’s Republic of China Purpose: This study was designed to explore how miR-145 regulates the mTOR signaling pathway in invasive pituitary adenoma (IPA by targeting AKT3.Methods: A total of 71 cases of IPA tissues and 66 cases of non-IPA tissues were obtained in this study. In vitro, the IPA cells were assigned into blank control, empty plasmid, miR-145 mimic, miR-145 inhibitor, miR-145 mimic + rapamycin, miR-145 inhibitor + rapamycin and rapamycin groups. Quantitative real-time polymerase chain reaction (qRT-PCR and Western blotting were performed to detect the protein expressions of PI3K, AKT3, mTOR mRNA and the mRNA expression of miR-145 both in vivo and in vitro. Additionally, the S6K and RPS6 mRNA and protein expressions as well as the relative phosphorylation levels were determined in vitro. MTT assay, flow cytometry and transwell assay were used to testify the cell proliferation, apoptosis and invasion ability, respectively.Results: The IPA tissues exhibited significantly lower expression of miR-145 but higher PI3K, AKT3 and mTOR mRNA and protein expressions when compared with the non-IPA tissues. Compared with the blank control and empty plasmid groups, the miR-145 mimic group showed significantly decreased PI3K, AKT3, mTOR, S6K and RPS6 mRNA and protein expressions as well as phosphorylation levels; besides, the IPA cell proliferation, migration and invasion ability were strongly inhibited, accompanied with the increased number of apoptotic cells. In the miR-145 inhibitor group, the PI3K, AKT3, mTOR, S6K and RPS6 mRNA and protein expressions as well as the phosphorylation levels were significantly increased; cell proliferation, migration and invasion ability were remarkably elevated, accompanied with reduced apoptotic cell number

  4. Antitumor activity of rapamycin in a Phase I trial for patients with recurrent PTEN-deficient glioblastoma.

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    Tim F Cloughesy

    2008-01-01

    Full Text Available There is much discussion in the cancer drug development community about how to incorporate molecular tools into early-stage clinical trials to assess target modulation, measure anti-tumor activity, and enrich the clinical trial population for patients who are more likely to benefit. Small, molecularly focused clinical studies offer the promise of the early definition of optimal biologic dose and patient population.Based on preclinical evidence that phosphatase and tensin homolog deleted on Chromosome 10 (PTEN loss sensitizes tumors to the inhibition of mammalian target of rapamycin (mTOR, we conducted a proof-of-concept Phase I neoadjuvant trial of rapamycin in patients with recurrent glioblastoma, whose tumors lacked expression of the tumor suppressor PTEN. We aimed to assess the safety profile of daily rapamycin in patients with glioma, define the dose of rapamycin required for mTOR inhibition in tumor tissue, and evaluate the antiproliferative activity of rapamycin in PTEN-deficient glioblastoma. Although intratumoral rapamycin concentrations that were sufficient to inhibit mTOR in vitro were achieved in all patients, the magnitude of mTOR inhibition in tumor cells (measured by reduced ribosomal S6 protein phosphorylation varied substantially. Tumor cell proliferation (measured by Ki-67 staining was dramatically reduced in seven of 14 patients after 1 wk of rapamycin treatment and was associated with the magnitude of mTOR inhibition (p = 0.0047, Fisher exact test but not the intratumoral rapamycin concentration. Tumor cells harvested from the Ki-67 nonresponders retained sensitivity to rapamycin ex vivo, indicating that clinical resistance to biochemical mTOR inhibition was not cell-intrinsic. Rapamycin treatment led to Akt activation in seven patients, presumably due to loss of negative feedback, and this activation was associated with shorter time-to-progression during post-surgical maintenance rapamycin therapy (p < 0.05, Logrank test.Rapamycin

  5. Rapamycin and mTORC1 Inhibition in the Mouse: Skin Cancer Prevention

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    Athar, Mohammad; Kopelovich, Levy

    2011-01-01

    Therapeutic and preventive effects of rapamycin include reduced risk of non-melanoma skin cancer (NMSC). In this issue of the journal (beginning on page XXX), Checkley et al. report that rapamycin inhibits mammalian target of rapamycin (mTOR) complex 1 in murine epidermis, thereby inhibiting tumor promotion mediated by tetradecanoyl phorbol-13 acetate (TPA) in association with a strong anti-inflammatory effect. Rapamycin is an immunosuppressive drug for preventing graft rejection in organ transplant recipients and reduces the risk of NMSC and Kaposi’s sarcoma in this population, albeit by mechanisms distinct from immunosuppression. Important future directions include identifying molecular predictors of rapamycin/rapalog sensitivity or resistance (potentially, for example, PI3K pathway alterations and KRAS mutations) and combined non-rapalog, mTOR-targeting approaches, all of which should increase efficacy and minimize toxicity. PMID:21733819

  6. GSK-3/Rb12 Pathway as a Novel Target of Rapamycin in Prostate Cancer

    Science.gov (United States)

    2005-11-01

    that also have a potent tumor suppressor effect. These drugs are currently being evaluated in clinical trials to treat human cancers including...tumors to inhibition of FRAP /mTOR." Proc Natl Acad Sci U S A 98(18): 10314-9. Noh, W. C., W. H. Mondesire, et al. (2004). "Determinants of rapamycin

  7. Growth differentiation factor 15 stimulates rapamycin-sensitive ovarian cancer cell growth and invasion.

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    Griner, Samantha E; Joshi, Jayashree P; Nahta, Rita

    2013-01-01

    Identification of novel molecular markers and therapeutic targets may improve survival rates for patients with ovarian cancer. In the current study, immunohistochemical (IHC) analysis of two human ovarian tumor tissue arrays showed high staining for GDF15 in a majority of tissues. Exogenous stimulation of ovarian cancer cell lines with recombinant human GDF15 (rhGDF15) or stable over-expression of a GDF15 expression plasmid promoted anchorage-independent growth, increased invasion, and up-regulation of matrix metalloproteinases (MMPs) and vascular endothelial growth factor (VEGF). MMP inhibition suppressed GDF15-mediated invasion. In addition, IHC analysis of human ovarian tumor tissue arrays indicated that GDF15 expression correlated significantly with high MMP2 and MMP9 expression. Exogenous and endogenous GDF15 over-expression stimulated phosphorylation of p38, Erk1/2, and Akt. Pharmacologic inhibition of p38, MEK, or PI3K suppressed GDF15-stimulated growth. Further, proliferation, growth, and invasion of GDF15 stable clones were blocked by rapamycin. IHC analysis demonstrated significant correlation between GDF15 expression and phosphorylation of mTOR. Finally, knockdown of endogenous GDF15 or neutralization of secreted GDF15 suppressed invasion and growth of a GDF15-over-expressing ovarian cancer cell line. These data indicate that GDF15 over-expression, which occurred in a majority of human ovarian cancers, promoted rapamycin-sensitive invasion and growth of ovarian cancer cells. Inhibition of mTOR may be an effective therapeutic strategy for ovarian cancers that over-express GDF15. Future studies should examine GDF15 as a novel molecular target for blocking ovarian cancer progression. Copyright © 2012 Elsevier Inc. All rights reserved.

  8. Rapamycin Rescues the Poor Developmental Capacity of Aged Porcine Oocytes

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    Seung Eun Lee

    2014-05-01

    Full Text Available Unfertilized oocytes age inevitably after ovulation, which limits their fertilizable life span and embryonic development. Rapamycin affects mammalian target of rapamycin (mTOR expression and cytoskeleton reorganization during oocyte meiotic maturation. The goal of this study was to examine the effects of rapamycin treatment on aged porcine oocytes and their in vitro development. Rapamycin treatment of aged oocytes for 24 h (68 h in vitro maturation [IVM]; 44 h+10 μM rapamycin/24 h, 47.52±5.68 or control oocytes (44 h IVM; 42.14±4.40 significantly increased the development rate and total cell number compared with untreated aged oocytes (68 h IVM, 22.04±5.68 (p<0.05. Rapamycin treatment of aged IVM oocytes for 24 h also rescued aberrant spindle organization and chromosomal misalignment, blocked the decrease in the level of phosphorylated-p44/42 mitogen-activated protein kinase (MAPK, and increased the mRNA expression of cytoplasmic maturation factor genes (MOS, BMP15, GDF9, and CCNB1 compared with untreated, 24 h-aged IVM oocytes (p<0.05. Furthermore, rapamycin treatment of aged oocytes decreased reactive oxygen species (ROS activity and DNA fragmentation (p<0.05, and downregulated the mRNA expression of mTOR compared with control or untreated aged oocytes. By contrast, rapamycin treatment of aged oocytes increased mitochondrial localization (p<0.05 and upregulated the mRNA expression of autophagy (BECN1, ATG7, MAP1LC3B, ATG12, GABARAP, and GABARAPL1, anti-apoptosis (BCL2L1 and BIRC5; p<0.05, and development (NANOG and SOX2; p<0.05 genes, but it did not affect the mRNA expression of pro-apoptosis genes (FAS and CASP3 compared with the control. This study demonstrates that rapamycin treatment can rescue the poor developmental capacity of aged porcine oocytes.

  9. Rapamycin prevents drug seeking via disrupting reconsolidation of reward memory in rats.

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    Lin, Jue; Liu, Lingqi; Wen, Quan; Zheng, Chunming; Gao, Yang; Peng, Shuxian; Tan, Yalun; Li, Yanqin

    2014-01-01

    The maladaptive drug memory developed between the drug-rewarding effect and environmental cues contributes to difficulty in preventing drug relapse. Established reward memories can be disrupted by pharmacologic interventions following their reactivation. Rapamycin, an inhibitor of mammalian target of rapamycin (mTOR) kinase, has been proved to be involved in various memory consolidation. However, it is less well characterized in drug memory reconsolidation. Using a conditioned place preference (CPP) procedure, we examined the effects of systemically administered rapamycin on reconsolidation of drug memory in rats. We found that systemically administered rapamycin (0.1 or 10 mg/kg, i.p.) after re-exposure to drug-paired environment, dose dependently decreased the expression of CPP 1 d later, and the effect lasted for up to 14 d and could not be reversed by a priming injection of morphine. The effect of rapamycin on morphine-associated memory was specific to drug-paired context, and rapamycin had no effect on subsequent CPP expression when rats were exposed to saline-paired context or homecage. These results indicated that systemic administration of rapamycin after memory reactivation can persistently inhibit the drug seeking behaviour via disruption of morphine memory reconsolidation in rats. Additionally, the effect of rapamycin on memory reconsolidation was reproduced in cocaine CPP and alcohol CPP. Furthermore, rapamycin did not induce conditioned place aversion and had no effect on locomotor activity and anxiety behaviour. These findings suggest that rapamycin could erase the acquired drug CPP in rats, and that mTOR activity plays an important role in drug reconsolidation and is required for drug relapse.

  10. Rapamycin pharmacokinetic and pharmacodynamic relationships in osteosarcoma: a comparative oncology study in dogs.

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    Melissa C Paoloni

    2010-06-01

    Full Text Available Signaling through the mTOR pathway contributes to growth, progression and chemoresistance of several cancers. Accordingly, inhibitors have been developed as potentially valuable therapeutics. Their optimal development requires consideration of dose, regimen, biomarkers and a rationale for their use in combination with other agents. Using the infrastructure of the Comparative Oncology Trials Consortium many of these complex questions were asked within a relevant population of dogs with osteosarcoma to inform the development of mTOR inhibitors for future use in pediatric osteosarcoma patients.This prospective dose escalation study of a parenteral formulation of rapamycin sought to define a safe, pharmacokinetically relevant, and pharmacodynamically active dose of rapamycin in dogs with appendicular osteosarcoma. Dogs entered into dose cohorts consisting of 3 dogs/cohort. Dogs underwent a pre-treatment tumor biopsy and collection of baseline PBMC. Dogs received a single intramuscular dose of rapamycin and underwent 48-hour whole blood pharmacokinetic sampling. Additionally, daily intramuscular doses of rapamycin were administered for 7 days with blood rapamycin trough levels collected on Day 8, 9 and 15. At Day 8 post-treatment collection of tumor and PBMC were obtained. No maximally tolerated dose of rapamycin was attained through escalation to the maximal planned dose of 0.08 mg/kg (2.5 mg/30 kg dog. Pharmacokinetic analysis revealed a dose-dependent exposure. In all cohorts modulation of the mTOR pathway in tumor and PBMC (pS6RP/S6RP was demonstrated. No change in pAKT/AKT was seen in tumor samples following rapamycin therapy.Rapamycin may be safely administered to dogs and can yield therapeutic exposures. Modulation pS6RP/S6RP in tumor tissue and PBMCs was not dependent on dose. Results from this study confirm that the dog may be included in the translational development of rapamycin and potentially other mTOR inhibitors. Ongoing studies of

  11. Influence of mTOR in energy and metabolic homeostasis.

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    Haissaguerre, Magalie; Saucisse, Nicolas; Cota, Daniela

    2014-11-01

    The mechanistic (or mammalian) target of rapamycin couples a variety of different environmental signals, including nutrients and hormones, with the regulation of several energy-demanding cellular functions, spanning from protein and lipid synthesis to mitochondrial activity and cytoskeleton dynamics. mTOR forms two distinct protein complexes in cells, mTORC1 and mTORC2. This review focuses on recent advances made in understanding the roles played by these two complexes in the regulation of whole body metabolic homeostasis. Studies carried out in the past few years have shown that mTORC1 activity in the hypothalamus varies by cell and stimulus type, and that this complex is critically implicated in the regulation of food intake and body weight and in the central actions of both nutrients and hormones, such as leptin, ghrelin and triiodothyronine. As a regulator of cellular anabolic processes, mTORC1 activity in the periphery favors adipogenesis, lipogenesis, glucose uptake and beta-cell mass expansion. Much less is known about the function of mTORC2 in the hypothalamus, while in peripheral organs this second complex exerts roles strikingly similar to those described for mTORC1. Deregulation of mTORC1 and mTORC2 is associated with obesity, type 2 diabetes, cancer and neurodegenerative disorders. Insights on the exact relationship between mTORC1 and mTORC2 in the context of the regulation of metabolic homeostasis and on the specific molecular mechanisms engaged by these two complexes in such regulation may provide new avenues for therapy. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

  12. Activation of the Mammalian Target of Rapamycin in the Rostral Ventromedial Medulla Contributes to the Maintenance of Nerve Injury-Induced Neuropathic Pain in Rat

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    Jian Wang

    2015-01-01

    Full Text Available The mammalian target of rapamycin (mTOR, a serine-threonine protein kinase, integrates extracellular signals, thereby modulating several physiological and pathological processes, including pain. Previous studies have suggested that rapamycin (an mTOR inhibitor can attenuate nociceptive behaviors in many pain models, most likely at the spinal cord level. However, the mechanisms of mTOR at the supraspinal level, particularly at the level of the rostral ventromedial medulla (RVM, remain unclear. Thus, the aim of this study was to elucidate the role of mTOR in the RVM, a key relay region for the descending pain control pathway, under neuropathic pain conditions. Phosphorylated mTOR was mainly expressed in serotonergic spinally projecting neurons and was significantly increased in the RVM after spared nerve injury- (SNI- induced neuropathic pain. Moreover, in SNI rat brain slices, rapamycin infusion both decreased the amplitude instead of the frequency of spontaneous excitatory postsynaptic currents and reduced the numbers of action potentials in serotonergic neurons. Finally, intra-RVM microinjection of rapamycin effectively alleviated established mechanical allodynia but failed to affect the development of neuropathic pain. In conclusion, our data provide strong evidence for the role of mTOR in the RVM in nerve injury-induced neuropathic pain, indicating a novel mechanism of mTOR inhibitor-induced analgesia.

  13. A systems study reveals concurrent activation of AMPK and mTOR by amino acids

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    Pezze, Piero Dalle; Ruf, Stefanie; Sonntag, Annika G; Langelaar-Makkinje, Miriam; Hall, Philip; Heberle, Alexander M; Navas, Patricia Razquin; van Eunen, Karen; Tölle, Regine C; Schwarz, Jennifer J; Wiese, Heike; Warscheid, Bettina; Deitersen, Jana; Stork, Björn; Fäßler, Erik; Schäuble, Sascha; Hahn, Udo; Horvatovich, Peter; Shanley, Daryl P; Thedieck, Kathrin

    2016-01-01

    Amino acids (aa) are not only building blocks for proteins, but also signalling molecules, with the mammalian target of rapamycin complex 1 (mTORC1) acting as a key mediator. However, little is known about whether aa, independently of mTORC1, activate other kinases of the mTOR signalling network. To

  14. Target of rapamycin signaling orchestrates growth-defense trade-offs in plants.

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    De Vleesschauwer, David; Filipe, Osvaldo; Hoffman, Gena; Seifi, Hamed Soren; Haeck, Ashley; Canlas, Patrick; Van Bockhaven, Jonas; De Waele, Evelien; Demeestere, Kristof; Ronald, Pamela; Hofte, Monica

    2018-01-01

    Plant defense to microbial pathogens is often accompanied by significant growth inhibition. How plants merge immune system function with normal growth and development is still poorly understood. Here, we investigated the role of target of rapamycin (TOR), an evolutionary conserved serine/threonine kinase, in the plant defense response. We used rice as a model system and applied a combination of chemical, genetic, genomic and cell-based analyses. We demonstrate that ectopic expression of TOR and Raptor (regulatory-associated protein of mTOR), a protein previously demonstrated to interact with TOR in Arabidopsis, positively regulates growth and development in rice. Transcriptome analysis of rice cells treated with the TOR-specific inhibitor rapamycin revealed that TOR not only dictates transcriptional reprogramming of extensive gene sets involved in central and secondary metabolism, cell cycle and transcription, but also suppresses many defense-related genes. TOR overexpression lines displayed increased susceptibility to both bacterial and fungal pathogens, whereas plants with reduced TOR signaling displayed enhanced resistance. Finally, we found that TOR antagonizes the action of the classic defense hormones salicylic acid and jasmonic acid. Together, these results indicate that TOR acts as a molecular switch for the activation of cell proliferation and plant growth at the expense of cellular immunity. © 2017 The Authors. New Phytologist © 2017 New Phytologist Trust.

  15. The effects of melatonin on oxidative stress and prevention of primordial follicle loss via activation of mTOR pathway in the rat ovary.

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    Behram Kandemir, Y; Aydin, C; Gorgisen, G

    2017-02-28

    Mammalian Target of Rapamycin (mTOR) signaling pathway has important roles in the regulation of puberty onset, gonadotropin secretion, follicular development and ovulation. Melatonin (N-acetyl-5-methoxytryptamine) is a lipophilic hormone has multiple functions in regulating the fertility. Recent studies have shown that melatonin affected the number or maturation of follicles in the ovary. The aim of this study was to investigate the effects of melatonin on mTOR expression and quantity of follicle in rat ovary. In the present study, a total of 45 female rats were randomly divided into three groups. Group 1; Control (C), Group 2: Vehicle (V) and Group 3; Melatonin (M). Melatonin was administered intraperitoneally at a dose of 50 mg/kg/day for 30 days in Melatonin group. The effects of Melatonin on the expression of mTOR and downstream components  were determined by Western Blot and Reverse Transcriptase PCR analysis. Upon Western Blot and RT-PCR evaluations, we detected higher expression and activation of mTOR, P70S6K, PKCalpha, PCNA and higher numbers of primordial  follicles in melatonin group compared with V and C group. In addition to this results, melatonin decreased oxidative stress markers, such as MDA, on the contrary, levels of antioxidative markers, such as CAT and GPx, were increased by melatonin in rat ovary. This study indicated that melatonin may have a significant protective effect on primordial follicles and increase the expression of mTOR and downstream components in rat ovary. Melatonin treatment may have a beneficial effect on fertility.

  16. Loss of Tuberous Sclerosis Complex 2 (TSC2 as a Predictive Biomarker of Response to mTOR Inhibitor Treatment in Patients with Hepatocellular Carcinoma

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    Jinhyun Cho

    2016-10-01

    Full Text Available BACKGROUND: Hepatocellular carcinoma (HCC is a leading cause of cancer-related death globally. Mechanistic target of rapamycin (mTOR is frequently up-regulated in HCC and plays an important role in HCC tumorigenesis. Tumors with loss of tuberous sclerosis complex 2 (TSC2, a negative regulator of mTOR signaling, tend to respond well to mTOR inhibitors. We analyzed TSC2 expression status in Korean patients with HCC and evaluated the correlation between TSC2 loss and response to the mTOR inhibitor, everolimus. METHODS: We retrospectively assessed 36 patients with advanced HCC who had received sorafenib at a single center in Korea between 2008 and 2014, and for whom tumor specimens were available for TSC2 immunohistochemical analysis (IHC. Three patient-derived tumor cell lines (PDCs were analyzed by western blotting to determine TSC2 expression and drug sensitivity to mTOR. RESULTS: Twelve of 36 patients (33.3% showed low to undetectable levels of TSC2 expression. No significant differences were observed in progression-free survival (PFS or overall survival with sorafenib treatment based on TSC2 expression status. Two patients were treated with everolimus after sorafenib failure; one patient, with moderate TSC2 expression, experienced stable disease with a PFS of 5.8 months; the other, with high TSC2 expression, experienced rapid progression. PDC models demonstrated that the TSC2-low HCC PDC line was significantly more sensitive to everolimus than the TSC2-high HCC PDC lines. CONCLUSION: Loss of TSC2 may predict improved response to everolimus in HCC patients, but further studies are needed to confirm the predictive role of TSC2 expression for everolimus treatment.

  17. Drosophila insulin and target of rapamycin (TOR pathways regulate GSK3 beta activity to control Myc stability and determine Myc expression in vivo

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    Parisi Federica

    2011-09-01

    Full Text Available Abstract Background Genetic studies in Drosophila melanogaster reveal an important role for Myc in controlling growth. Similar studies have also shown how components of the insulin and target of rapamycin (TOR pathways are key regulators of growth. Despite a few suggestions that Myc transcriptional activity lies downstream of these pathways, a molecular mechanism linking these signaling pathways to Myc has not been clearly described. Using biochemical and genetic approaches we tried to identify novel mechanisms that control Myc activity upon activation of insulin and TOR signaling pathways. Results Our biochemical studies show that insulin induces Myc protein accumulation in Drosophila S2 cells, which correlates with a decrease in the activity of glycogen synthase kinase 3-beta (GSK3β a kinase that is responsible for Myc protein degradation. Induction of Myc by insulin is inhibited by the presence of the TOR inhibitor rapamycin, suggesting that insulin-induced Myc protein accumulation depends on the activation of TOR complex 1. Treatment with amino acids that directly activate the TOR pathway results in Myc protein accumulation, which also depends on the ability of S6K kinase to inhibit GSK3β activity. Myc upregulation by insulin and TOR pathways is a mechanism conserved in cells from the wing imaginal disc, where expression of Dp110 and Rheb also induces Myc protein accumulation, while inhibition of insulin and TOR pathways result in the opposite effect. Our functional analysis, aimed at quantifying the relative contribution of Myc to ommatidial growth downstream of insulin and TOR pathways, revealed that Myc activity is necessary to sustain the proliferation of cells from the ommatidia upon Dp110 expression, while its contribution downstream of TOR is significant to control the size of the ommatidia. Conclusions Our study presents novel evidence that Myc activity acts downstream of insulin and TOR pathways to control growth in Drosophila. At

  18. Phosphatidic acid regulates systemic inflammatory responses by modulating the Akt-mammalian target of rapamycin-p70 S6 kinase 1 pathway.

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    Lim, Hyung-Kyu; Choi, Young-Ae; Park, Wan; Lee, Taehoon; Ryu, Sung Ho; Kim, Seong-Yong; Kim, Jae-Ryong; Kim, Jung-Hye; Baek, Suk-Hwan

    2003-11-14

    Macrophages are pivotal effector cells in the innate immune system. When microbial products bind to pathogen recognition receptors, macrophages are activated and release a broad array of mediators, such as cytokines, that orchestrate the inflammatory responses of the host. Phosphatidic acid (PA) has been implicated as an important metabolite of phospholipid biosynthesis and in membrane remodeling and has been further suggested to be a crucial second messenger in various cellular signaling events. Here we show that PA is an essential regulator of inflammatory response. Deleterious effects of PA are associated with the secretion of proinflammatory cytokines, such as tumor necrosis factor-alpha, interleukin-1beta, interleukin-6, and the production of nitric oxide, prostaglandin E2, which are predominantly released by macrophage Raw264.7 cells. Furthermore, the administration of PA to mice increased the serum cytokine level. Moreover, direct or lipopolysaccharide-induced PA accumulation by macrophages led to the Akt-dependent activation of the mammalian target of rapamycin-p70 S6 kinase 1, a process required for the induction of inflammatory mediators. These findings demonstrate the importance of the role of PA in systemic inflammatory responses, and provide a potential usefulness as specific targets for the development of therapies.

  19. A Recollection of mTOR Signaling in Learning and Memory

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    Graber, Tyson E.; McCamphill, Patrick K.; Sossin, Wayne S.

    2013-01-01

    Mechanistic target of rapamcyin (mTOR) is a central player in cell growth throughout the organism. However, mTOR takes on an additional, more specialized role in the developed neuron, where it regulates the protein synthesis-dependent, plastic changes underlying learning and memory. mTOR is sequestered in two multiprotein complexes (mTORC1 and…

  20. The Neuroprotective Effect of Rapamycin as a Modulator of the mTOR-NF-κB Axis during Retinal Inflammation.

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    Tomohiro Okamoto

    Full Text Available The determination of the molecular mechanism underlying retinal pathogenesis and visual dysfunction during innate inflammation, and the treatment effect of rapamycin thereon.The endotoxin-induced uveitis and retinitis mouse model was established by injecting lipopolysaccharide. The mice were subsequently treated with rapamycin, a mammalian target of rapamycin (mTOR inhibitor. The rhodopsin mRNA and protein expression level in the retina and the photoreceptor outer segment (OS length in immunohistochemical stainings were measured, and visual function was recorded by electroretinography. Inflammatory cytokines, their related molecules, mTOR, and LC3 levels were measured by real-time PCR and/or immunoblotting. Leukocyte adhesion during inflammation was analyzed using concanavalin A lectin.The post-transcriptional reduction in the visual pigment of rod photoreceptor cells, rhodopsin, OS shortening, and rod photoreceptor cell dysfunction during inflammation were suppressed by rapamycin. Activation of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB and induction of inflammatory cytokines, such as interleukin-6 (IL-6 and monocyte chemoattractant protein-1 (MCP-1, and the activation of the downstream signaling protein, signal transducer and activator of transcription 3 (STAT3, which reduces rhodopsin in the retina during inflammation, were attenuated by rapamycin. Increased leukocyte adhesion was also attenuated by rapamycin. Interestingly, although mTOR activation was observed after NF-κB activation, mTOR inhibition suppressed NF-κB activation at the early phase, indicating that the basal level of activated mTOR was sufficient to activate NF-κB in the retina. In addition, the inhibition of NF-κB suppressed mTOR activation, suggesting a positive feedback loop of mTOR and NF-κB during inflammation. The ratio of LC3II to LC3I, which reflects autophagy induction, was not changed by inflammation but was increased by rapamycin

  1. Genetic variability of the mTOR pathway and prostate cancer risk in the European Prospective Investigation on Cancer (EPIC.

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    Daniele Campa

    2011-02-01

    Full Text Available The mTOR (mammalian target of rapamycin signal transduction pathway integrates various signals, regulating ribosome biogenesis and protein synthesis as a function of available energy and amino acids, and assuring an appropriate coupling of cellular proliferation with increases in cell size. In addition, recent evidence has pointed to an interplay between the mTOR and p53 pathways. We investigated the genetic variability of 67 key genes in the mTOR pathway and in genes of the p53 pathway which interact with mTOR. We tested the association of 1,084 tagging SNPs with prostate cancer risk in a study of 815 prostate cancer cases and 1,266 controls nested within the European Prospective Investigation into Cancer and Nutrition (EPIC. We chose the SNPs (n = 11 with the strongest association with risk (p<0.01 and sought to replicate their association in an additional series of 838 prostate cancer cases and 943 controls from EPIC. In the joint analysis of first and second phase two SNPs of the PRKCI gene showed an association with risk of prostate cancer (OR(allele = 0.85, 95% CI 0.78-0.94, p = 1.3 x 10⁻³ for rs546950 and OR(allele = 0.84, 95% CI 0.76-0.93, p = 5.6 x 10⁻⁴ for rs4955720. We confirmed this in a meta-analysis using as replication set the data from the second phase of our study jointly with the first phase of the Cancer Genetic Markers of Susceptibility (CGEMS project. In conclusion, we found an association with prostate cancer risk for two SNPs belonging to PRKCI, a gene which is frequently overexpressed in various neoplasms, including prostate cancer.

  2. Inhibition of akt enhances the chemopreventive effects of topical rapamycin in mouse skin

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    Dickinson, Sally E; Janda, Jaroslav; Criswell, Jane; Blohm-Mangone, Karen; Olson, Erik R.; Liu, Zhonglin; Barber, Christie; Rusche, Jadrian J.; Petricoin, Emmanuel; Calvert, Valerie; Einspahr, Janine G.; Dickinson, Jesse; Stratton, Steven P.; Curiel-Lewandrowski, Clara; Saboda, Kathylynn; Hu, Chengcheng; Bode, Ann M.; Dong, Zigang; Alberts, David S.; Bowden, G. Timothy

    2016-01-01

    The PI3Kinase/Akt/mTOR pathway has important roles in cancer development for multiple tumor types, including UV-induced non-melanoma skin cancer. Immunosuppressed populations are at increased risk of aggressive cutaneous squamous cell carcinoma (SCC). Individuals who are treated with rapamycin, (sirolimus, a classical mTOR inhibitor) have significantly decreased rates of developing new cutaneous SCCs compared to those that receive traditional immunosuppression. However, systemic rapamycin use can lead to significant adverse events. Here we explored the use of topical rapamycin as a chemopreventive agent in the context of solar simulated light (SSL)-induced skin carcinogenesis. In SKH-1 mice, topical rapamycin treatment decreased tumor yields when applied after completion of 15 weeks of SSL exposure compared to controls. However, applying rapamycin during SSL exposure for 15 weeks, and continuing for 10 weeks after UV treatment, increased tumor yields. We also examined whether a combinatorial approach might result in more significant tumor suppression by rapamycin. We validated that rapamycin causes increased Akt (S473) phosphorylation in the epidermis after SSL, and show for the first time that this dysregulation can be inhibited in vivo by a selective PDK1/Akt inhibitor, PHT-427. Combining rapamycin with PHT-427 on tumor prone skin additively caused a significant reduction of tumor multiplicity compared to vehicle controls. Our findings indicate that patients taking rapamycin should avoid sun exposure, and that combining topical mTOR inhibitors and Akt inhibitors may be a viable chemoprevention option for individuals at high risk for cutaneous SCC.

  3. Branched-chain amino acids enhance premature senescence through mammalian target of rapamycin complex I-mediated upregulation of p21 protein.

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    Masayuki Nakano

    Full Text Available Branched-chain amino acids (BCAAs have been applied as an oral supplementation to patients with liver cirrhosis. BCAAs not only improve nutritional status of patients but also decrease the incidence of liver cancer. Mammalian target of rapamycin (mTOR links cellular metabolism with growth and proliferation in response to nutrients, energy, and growth factors. BCAAs, especially leucine, have been shown to regulate protein synthesis through mTOR activities. On the other hand, cellular senescence is suggested to function as tumor suppressor mechanisms, and induced by a variety of stimuli including DNA damage-inducing drugs. However, it is not clear how BCAA supplementation prevents the incidence of liver cancer in patients with cirrhosis. Here we showed that human cancer cells, HepG2 and U2OS, cultured in medium containing BCAAs with Fischer's ratio about 3, which was shown to have highest activities to synthesize and secrete of albumin, had higher activities to induce premature senescence and elevate mTORC1 activities. Furthermore, BCAAs themselves enhanced the execution of premature senescence induced by DNA damage-inducing drugs, which was effectively prevented by rapamycin. These results strongly suggested the contribution of the mTORC1 pathway to the regulation of premature senescence. Interestingly, the protein levels of p21, a p53 target and well-known gene essential for the execution of cellular senescence, were upregulated in the presence of BCAAs. These results suggested that BCAAs possibly contribute to tumor suppression by enhancing cellular senescence mediated through the mTOR signalling pathway.

  4. The Role of mTOR Inhibitors for the Treatment of B-Cell Lymphomas

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    Pinelopi Argyriou

    2012-01-01

    Full Text Available Despite the fact that the majority of lymphomas initially respond to treatment, many patients relapse and die from disease that is refractory to current regimens. The need for new treatment strategies in lymphomas has led to the investigation and evaluation of novel agents that target cellular pathways. The mammalian target of rapamycin (mTOR is a representative pathway that may be implicated in lymphomagenesis. Rapamycin and especially its derivatives (temsirolimus, everolimus, and deforolimus represent the first described mTOR inhibitors. These agents have shown promising results in the treatment of lymphoid malignancies. On the other hand, new ATP-competitive mTOR inhibitors that provoke a broader inhibition of mTOR activity are in early stages of clinical development. The purpose of this paper is to summarize the existing knowledge about mTOR inhibitors and their use in the treatment of B-cell lymphomas. Relevant issues regarding mTOR biology in general as well as in B-cell lymphoid neoplasms are also discussed in short.

  5. Topical application of rapamycin ointment ameliorates Dermatophagoides farina body extract-induced atopic dermatitis in NC/Nga mice.

    Science.gov (United States)

    Yang, Fei; Tanaka, Mari; Wataya-Kaneda, Mari; Yang, Lingli; Nakamura, Ayumi; Matsumoto, Shoji; Attia, Mostafa; Murota, Hiroyuki; Katayama, Ichiro

    2014-08-01

    Atopic dermatitis (AD), a chronic inflammatory skin disease characterized by relapsing eczema and intense prurigo, requires effective and safe pharmacological therapy. Recently, rapamycin, an mTOR (mammalian target of rapamycin) inhibitor, has been reported to play a critical role in immune responses and has emerged as an effective immunosuppressive drug. In this study, we assessed whether inhibition of mTOR signalling could suppress dermatitis in mice. Rapamycin was topically applied to inflamed skin in a murine AD model that was developed by repeated topical application of Dermatophagoides farina body (Dfb) extract antigen twice weekly for 7 weeks in NC/Nga mice. The efficacy of topical rapamycin treatment was evaluated immunologically and serologically. Topical application of rapamycin reduced inflammatory cell infiltration in the dermis, alleviated the increase of serum IgE levels and resulted in a significant reduction in clinical skin condition score and marked improvement of histological findings. In addition, increased mTOR phosphorylation in the lesional skin was observed in our murine AD model. Topical application of rapamycin ointment inhibited Dfb antigen-induced dermatitis in NC/Nga mice, promising a new therapy for atopic dermatitis. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  6. AKT (v-akt murine thymoma viral oncogene homolog 1) and N-Ras (neuroblastoma ras viral oncogene homolog) coactivation in the mouse liver promotes rapid carcinogenesis by way of mTOR (mammalian target of rapamycin complex 1), FOXM1 (forkhead box M1)/SKP2, and c-Myc pathways.

    Science.gov (United States)

    Ho, Coral; Wang, Chunmei; Mattu, Sandra; Destefanis, Giulia; Ladu, Sara; Delogu, Salvatore; Armbruster, Julia; Fan, Lingling; Lee, Susie A; Jiang, Lijie; Dombrowski, Frank; Evert, Matthias; Chen, Xin; Calvisi, Diego F

    2012-03-01

    Activation of v-akt murine thymoma viral oncogene homolog (AKT) and Ras pathways is often implicated in carcinogenesis. However, the oncogenic cooperation between these two cascades in relationship to hepatocellular carcinoma (HCC) development remains undetermined. To investigate this issue, we generated a mouse model characterized by combined overexpression of activated forms of AKT and neuroblastoma Ras viral oncogene homolog (N-Ras) protooncogenes in the liver by way of hydrodynamic gene transfer. The molecular mechanisms underlying crosstalk between AKT and N-Ras were assessed in the mouse model and further evaluated in human and murine HCC cell lines. We found that coexpression of AKT and N-Ras resulted in a dramatic acceleration of liver tumor development when compared with mice overexpressing AKT alone, whereas N-Ras alone did not lead to tumor formation. At the cellular level, concomitant up-regulation of AKT and N-Ras resulted in increased proliferation and microvascularization when compared with AKT-injected mice. Mechanistic studies suggested that accelerated hepatocarcinogenesis driven by AKT and N-Ras resulted from a strong activation of mammalian target of rapamycin complex 1 (mTORC1). Furthermore, elevated expression of FOXM1/SKP2 and c-Myc also contributed to rapid tumor growth in AKT/Ras mice, yet by way of mTORC1-independent mechanisms. The biological effects of coactivation of AKT and N-Ras were then recapitulated in vitro using HCC cell lines, which supports the functional significance of mTORC1, FOXM1/SKP2, and c-Myc signaling cascades in mediating AKT and N-Ras-induced liver tumor development. Our data demonstrate the in vivo crosstalk between the AKT and Ras pathways in promoting liver tumor development, and the pivotal role of mTORC1-dependent and independent pathways in mediating AKT and Ras induced hepatocarcinogenesis. Copyright © 2011 American Association for the Study of Liver Diseases.

  7. Salidroside protects against hydrogen peroxide-induced injury in HUVECs via the regulation of REDD1 and mTOR activation.

    Science.gov (United States)

    Xu, Mao-Chun; Shi, Hai-Ming; Wang, Hao; Gao, Xiu-Fang

    2013-07-01

    Antioxidative therapy is considered an effective strategy for treating oxidative stress-induced apoptosis in cardiovascular diseases. Salidroside has been used as an antioxidative therapy for oxidative injury in cardiac diseases. However, the mechanism underlying its antioxidant effect is poorly understood. The present study aimed to investigate the pharmacological effects of salidroside on cultured human umbilical vein endothelial cells (HUVECs) under conditions of oxidative injury induced by hydrogen peroxide (H2O2) and the underlying mechanisms in vitro. HUVECs pretreated with or without salidroside for 24 h were exposed to H2O2-induced oxidative stress conditions for 6 h and then cell viability, apoptosis, HIF-1α, regulated in development and DNA damage responses-1 (REDD1) and the PI3K/Akt/mTOR pathway were investigated. The results demonstrated that salidroside effectively attenuated H2O2-impaired cell viability and the production of reactive oxygen species (ROS) in a concentration-dependent manner. Reduced H2O2-induced apoptosis and activation of the cellular PI3K/Akt/mTOR pathway were demonstrated in HUVECs pretreated with salidroside. Furthermore, the level of REDD1, a direct regulator of mitochondrial metabolism, significantly increased in parallel with the level of HIF-1α following pretreatment with salidroside. The antioxidative effect of salidroside was abrogated in REDD1 knockdown cells. However, LY294002, a PI3K inhibitor, attenuated the anti-apoptotic effect of salidroside and blocked the increase of Akt and mTOR; however, did not affect the antioxidative effect of salidroside. These findings suggested that salidroside was capable of protecting HUVECs against H2O2-induced apoptosis by activating the PI3K/Akt/mTOR-dependent pathway and inhibiting ROS production by activating REDD1.

  8. Health Effects of Long-Term Rapamycin Treatment: The Impact on Mouse Health of Enteric Rapamycin Treatment from Four Months of Age throughout Life.

    Directory of Open Access Journals (Sweden)

    Kathleen E Fischer

    Full Text Available Rapamycin, an mTOR inhibitor, has been shown to extend lifespan in a range of model organisms. It has been reported to extend lifespan in multiple strains of mice, administered chronically or acutely early or late in life. The ability of rapamycin to extend health (healthspan as opposed to life is less well documented. To assess the effects chronic rapamycin treatment on healthspan, enteric rapamycin was given to male and female C57BL/6J mice starting at 4 months of age and continued throughout life. Repeated, longitudinal assessments of health in individual animals were made starting at 16 months of age (=12 months of treatment until death. A number of health parameters were improved (female grip strength, female body mass and reduced sleep fragmentation in both sexes, others showed no significant difference, while at least one (male rotarod performance was negatively affected. Rapamycin treatment affected many measures of health in a highly sex-specific manner. While sex-specific phenotypic effects of rapamycin treatment have been widely reported, in this study we document sex differences in the direction of phenotypic change. Rapamycin-fed males and females were both significantly different from controls; however the differences were in the opposite direction in measures of body mass, percent fat and resting metabolic rate, a pattern not previously reported.

  9. Rapamycin carbonate esters

    OpenAIRE

    Rhodes, A; Sandhu, S S; Onis, J. E; McKendrick, John

    2009-01-01

    Certain embodiments include carbonate esters of rapamycin at position 42 that are synthesized by a lipase catalyzed regio-specific process. These compounds or a pharmaceutically acceptable salt thereof are useful in the treatment of organ and tissue transplant rejection, autoimmune disease, proliferative disorder, restenosis, cancer, or microbial infection.

  10. Docosahexaenoic Acid Induces Cell Death in Human Non-Small Cell Lung Cancer Cells by Repressing mTOR via AMPK Activation and PI3K/Akt Inhibition

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    Nayeong Kim

    2015-01-01

    Full Text Available The anticancer properties and mechanism of action of omega-3 polyunsaturated fatty acids (ω3-PUFAs have been demonstrated in several cancers; however, the mechanism in lung cancer remains unclear. Here, we show that docosahexaenoic acid (DHA, a ω3-PUFA, induced apoptosis and autophagy in non-small cell lung cancer (NSCLC cells. DHA-induced cell death was accompanied by AMP-activated protein kinase (AMPK activation and inactivated phosphatidylinositol 3-kinase (PI3K/Akt/mammalian target of rapamycin (mTOR signaling. Knocking down AMPK and overexpressing Akt increased mTOR activity and attenuated DHA-induced cell death, suggesting that DHA induces cell death via AMPK- and Akt-regulated mTOR inactivation. This was confirmed in Fat-1 transgenic mice, which produce ω3-PUFAs. Lewis lung cancer (LLC tumor cells implanted into Fat-1 mice showed slower growth, lower phospho-Akt levels, and higher levels of apoptosis and autophagy than cells implanted into wild-type mice. Taken together, these data suggest that DHA-induced apoptosis and autophagy in NSCLC cells are associated with AMPK activation and PI3K/Akt inhibition, which in turn lead to suppression of mTOR; thus ω3-PUFAs may be utilized as potential therapeutic agents for NSCLC treatment.

  11. Effects of mTOR inhibition on cardiac and adipose tissue pathology and glucose metabolism in rats with metabolic syndrome.

    Science.gov (United States)

    Uchinaka, Ayako; Yoneda, Mamoru; Yamada, Yuichiro; Murohara, Toyoaki; Nagata, Kohzo

    2017-08-01

    The mammalian target of rapamycin (mTOR) is a regulator of metabolism and is implicated in pathological conditions such as obesity and diabetes. We aimed to investigate the role of mTOR in obesity. A new animal model of metabolic syndrome (MetS), named DahlS.Z-Lepr(fa) /Lepr(fa) (DS/obese) rats was established previously in our laboratory. In this study, we used this model to evaluate the effects of mTOR inhibition on cardiac and adipose tissue pathology and glucose metabolism. DS/obese rats were treated with the mTOR inhibitor, everolimus, (0.83 mg/kg per day, per os) for 4 weeks at 9 weeks of age. Age-matched homozygous lean (DahlS.Z-Lepr(+) /Lepr(+) or DS/lean) littermates of DS/obese rats were used as controls. Treatment with everolimus ameliorated hypertension, left ventricular (LV) hypertrophy and fibrosis, and LV diastolic dysfunction, and attenuated cardiac oxidative stress and inflammation in DS/obese rats, but had no effect on these parameters in DS/lean rats. Treatment with everolimus reduced Akt Thr308 phosphorylation in the heart of DS/obese rats. It also alleviated obesity, hyperphagia, adipocyte hypertrophy, and adipose tissue inflammation in DS/obese rats. Everolimus treatment exacerbated glucose intolerance, but did not affect Akt phosphorylation levels in the fat or liver in these rats. Pancreatic β-cell mass was increased in DS/obese rats compared with that in DS/lean rats and this effect was attenuated by everolimus. Activation of mTOR signaling contributes to the pathophysiology of MetS and its associated complications. And mTOR inhibition with everolimus ameliorated obesity as well as cardiac and adipose tissue pathology, but exacerbated glucose metabolism in rats with MetS. © 2017 The Authors. Pharmacology Research & Perspectives published by John Wiley & Sons Ltd, British Pharmacological Society and American Society for Pharmacology and Experimental Therapeutics.

  12. Rapamycin Inhibits ALDH Activity, Resistance to Oxidative Stress, and Metastatic Potential in Murine Osteosarcoma Cells

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    Xiaodong Mu

    2013-01-01

    Full Text Available Osteosarcoma (OS is the most common primary malignancy of bone. Mortality is determined by the presence of metastatic disease, but little is known regarding the biochemical events that drive metastases. Two murine OS cell lines, K7M2 and K12, are related but differ significantly in their metastatic potentials: K7M2 is highly metastatic whereas K12 displays much less metastatic potential. Using this experimental system, the mammalian target of rapamycin (mTOR pathway has been implicated in OS metastasis. We also discovered that aldehyde dehydrogenase (ALDH, a stem cell marker activity is higher in K7M2 cells than K12 cells. Rapamycin treatment reduces the expression and enzymatic activity of ALDH in K7M2 cells. ALDH inhibition renders these cells more susceptible to apoptotic death when exposed to oxidative stress. Furthermore, rapamycin treatment reduces bone morphogenetic protein-2 (BMP2 and vascular endothelial growth factor (VEGF gene expression and inhibits K7M2 proliferation, migration, and invasion in vitro. Inhibition of ALDH with disulfiram correlated with decreased mTOR expression and activity. In conclusion, we provide evidence for interaction between mTOR activity, ALDH activity, and metastatic potential in murine OS cells. Our work suggests that mTOR and ALDH are therapeutic targets for the treatment and prevention of OS metastasis.

  13. Activated mammalian target of rapamycin is associated with T regulatory cell insufficiency in nasal polyps

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    Shi Jianbo

    2009-02-01

    Full Text Available Abstract Background Decreased infiltration of Foxp3+ T regulatory cell (Treg is considered to be critical for the Th1/Th2 dysregulation of nasal polyps, while the cellular mechanism underlying Foxp3+ Treg insufficiency is currently not well defined. Methods We attempted to investigate the tissue expression of phosphorylated mammalian target of rapamycin (pmTOR and infiltration of Foxp3+ Tregs in 28 nasal polyps and 16 controls by histological staining. We also evaluated the effects of blocking the mTOR signaling pathway with rapamycin on T cell phenotype selection and Foxp3+CD4+ Tregs expansion in a tissue culture system. Results Significantly increased infiltration of pmTOR+ inflammatory cells and decreased infiltration of Foxp3+CD4+ Tregs into nasal polyps was observed, with an inverse association. In the tissue culture system, we detected significantly elevated Foxp3 expression and IL-10 production, as well as an increased percentage of Foxp3+ Tregs in nasal polyps after blocking the mTOR signaling pathway with rapamycin. Conclusion Here we demonstrate for the first time that the mTOR signaling pathway is associated with Foxp3+ Tregs insufficiency in nasal polyps. Inhibition of the mTOR signaling pathway may be helpful for enhancement of Foxp3+ Treg expansion, as well as modulation of T cell phenotype imbalances in nasal polyps.

  14. Improved Clearance during Treatment of HPV-Positive Head and Neck Cancer through mTOR Inhibition

    Directory of Open Access Journals (Sweden)

    Joseph D. Coppock

    2013-06-01

    Full Text Available Human papillomavirus (HPV-related head and neck squamous cell carcinoma (HNSCC incidence is increasing at a near epidemic rate. We investigated whether the mammalian (or mechanistic target of rapamycin (mTOR inhibitor, rapamycin, can be used as a concurrent agent to standard-of-care cisplatin/radiation therapy (CRT to attenuate tumor lactate production, thus enhancing CRT-induced immune-mediated clearance of this antigenic tumor type. A C57Bl/ 6-derived mouse oropharyngeal epithelial cell line retrovirally transduced with HPV type 16 E6/E7 and human squamous cell carcinoma cell lines were evaluated for their response to rapamycin in vitro with proliferation assays, Western blots, and lactate assays. Clonogenic assays and a preclinical mouse model were used to assess rapamycin as a concurrent agent to CRT. The potential of rapamycin to enhance immune response through lactate attenuation was assessed using quantitative tumor lactate bioluminescence and assessment of cell-mediated immunity using E6/E7-vaccinated mouse splenocytes. Rapamycin alone inhibited mTOR signaling of all cancer cell lines tested in vitro and in vivo. Furthermore, rapamycin administered alone significantly prolonged survival in vivo but did not result in any long-term cures. Given concurrently, CRT/rapamycin significantly enhanced direct cell killing in clonogenic assays and prolonged survival in immunocompromised mice. However, in immunocompetent mice, concurrent CRT/rapamycin increased long-term cures by 21%. Preliminary findings suggest that improved survival involves increased cell killing and enhanced immune-mediated clearance in part due to decreased lactate production. The results may provide rationale for the clinical evaluation of mTOR inhibitors concurrent with standard-of-care CRT for treatment of HPV-positive HNSCC.

  15. mTOR as a multifunctional therapeutic target in HIV infection

    DEFF Research Database (Denmark)

    Nicoletti, Ferdinando; Fagone, Paolo; Meroni, PierLuigi

    2011-01-01

    Patients undergoing long-term highly active antiretroviral therapy treatment are probably at a higher risk of various HIV-related complications. Hyperactivation of The mammalian target of rapamycin (mTOR) has been found to contribute to dysregulated apoptosis and autophagy which determine CD4(+)-T......-cell loss, impaired function of innate immunity and development of neurocognitive disorders. Dysregulated mTOR activation has also been shown to play a key part in the development of nephropathy and in the pathogenesis of HIV-associated malignancies. These studies strongly support a multifunctional key role...... for mTOR in the pathogenesis of HIV-related disorders and suggest that specific mTOR inhibitors could represent a novel approach for the prevention and treatment of these pathologies....

  16. Campylobacter jejuni induces colitis through activation of mammalian target of rapamycin signaling.

    Science.gov (United States)

    Sun, Xiaolun; Threadgill, Deborah; Jobin, Christian

    2012-01-01

    Campylobacter jejuni is the worldwide leading cause of bacterial-induced enteritis. The molecular and cellular events that lead to campylobacteriosis are poorly understood. We identify mammalian target of rapamycin (mTOR) as a signaling pathway that leads to C jejuni-induced intestinal inflammation. Germ-free (control) or conventionally derived Il10(-/-) mice that express enhanced green fluorescent protein (EGFP) under the control of nuclear factor κB (Il10(-/-); NF-κB(EGFP) mice) were infected with C jejuni (10(9) colony-forming units/mouse) for 12 days; their responses were determined using histologic, semiquantitative reverse-transcription polymerase chain reaction, fluorescence in situ hybridization, transmission electron microscopy, and tissue culture analyses. mTOR signaling was blocked by daily intraperitoneal injections of the pharmacologic inhibitor rapamycin (1.5 mg/kg). CD4(+) T cells were depleted by intraperitoneal injections of antibodies against CD4 (0.5 mg/mouse every 3 days). Bacterial survival in splenocytes was measured using a gentamycin killing assay. C jejuni induced intestinal inflammation, which correlated with activation of mTOR signaling and neutrophil infiltration. The inflamed intestines of these mice had increased levels of interleukin-1β, Cxcl2, interleukin-17a, and EGFP; C jejuni localized to colons and extraintestinal tissues of infected Il10(-/-); NF-κB(EGFP) mice compared with controls. Rapamycin, administered before or after introduction of C jejuni, blocked C jejuni-induced intestinal inflammation and bacterial accumulation. LC3II processing and killing of C jejuni were increased in splenocytes incubated with rapamycin compared with controls. mTOR signaling mediates C jejuni-induced colitis in Il10(-/-) mice, independently of T-cell activation. Factors involved in mTOR signaling might be therapeutic targets for campylobacteriosis. Copyright © 2012 AGA Institute. Published by Elsevier Inc. All rights reserved.

  17. Caffeine affects the biological responses of human hematopoietic cells of myeloid lineage via downregulation of the mTOR pathway and xanthine oxidase activity.

    Science.gov (United States)

    Gibbs, Bernhard F; Gonçalves Silva, Isabel; Prokhorov, Alexandr; Abooali, Maryam; Yasinska, Inna M; Casely-Hayford, Maxwell A; Berger, Steffen M; Fasler-Kan, Elizaveta; Sumbayev, Vadim V

    2015-10-06

    Correction of human myeloid cell function is crucial for the prevention of inflammatory and allergic reactions as well as leukaemia progression. Caffeine, a naturally occurring food component, is known to display anti-inflammatory effects which have previously been ascribed largely to its inhibitory actions on phosphodiesterase. However, more recent studies suggest an additional role in affecting the activity of the mammalian target of rapamycin (mTOR), a master regulator of myeloid cell translational pathways, although detailed molecular events underlying its mode of action have not been elucidated. Here, we report the cellular uptake of caffeine, without metabolisation, by healthy and malignant hematopoietic myeloid cells including monocytes, basophils and primary acute myeloid leukaemia mononuclear blasts. Unmodified caffeine downregulated mTOR signalling, which affected glycolysis and the release of pro-inflammatory/pro-angiogenic cytokines as well as other inflammatory mediators. In monocytes, the effects of caffeine were potentiated by its ability to inhibit xanthine oxidase, an enzyme which plays a central role in human purine catabolism by generating uric acid. In basophils, caffeine also increased intracellular cyclic adenosine monophosphate (cAMP) levels which further enhanced its inhibitory action on mTOR. These results demonstrate an important mode of pharmacological action of caffeine with potentially wide-ranging therapeutic impact for treating non-infectious disorders of the human immune system, where it could be applied directly to inflammatory cells.

  18. Caffeine affects the biological responses of human hematopoietic cells of myeloid lineage via downregulation of the mTOR pathway and xanthine oxidase activity

    Science.gov (United States)

    Abooali, Maryam; Yasinska, Inna M.; Casely-Hayford, Maxwell A.; Berger, Steffen M.; Fasler-Kan, Elizaveta; Sumbayev, Vadim V.

    2015-01-01

    Correction of human myeloid cell function is crucial for the prevention of inflammatory and allergic reactions as well as leukaemia progression. Caffeine, a naturally occurring food component, is known to display anti-inflammatory effects which have previously been ascribed largely to its inhibitory actions on phosphodiesterase. However, more recent studies suggest an additional role in affecting the activity of the mammalian target of rapamycin (mTOR), a master regulator of myeloid cell translational pathways, although detailed molecular events underlying its mode of action have not been elucidated. Here, we report the cellular uptake of caffeine, without metabolisation, by healthy and malignant hematopoietic myeloid cells including monocytes, basophils and primary acute myeloid leukaemia mononuclear blasts. Unmodified caffeine downregulated mTOR signalling, which affected glycolysis and the release of pro-inflammatory/pro-angiogenic cytokines as well as other inflammatory mediators. In monocytes, the effects of caffeine were potentiated by its ability to inhibit xanthine oxidase, an enzyme which plays a central role in human purine catabolism by generating uric acid. In basophils, caffeine also increased intracellular cyclic adenosine monophosphate (cAMP) levels which further enhanced its inhibitory action on mTOR. These results demonstrate an important mode of pharmacological action of caffeine with potentially wide-ranging therapeutic impact for treating non-infectious disorders of the human immune system, where it could be applied directly to inflammatory cells. PMID:26384306

  19. Rapamycin increases fetal hemoglobin and ameliorates the nociception phenotype in sickle cell mice.

    Science.gov (United States)

    Khaibullina, Alfia; Almeida, Luis E F; Wang, Li; Kamimura, Sayuri; Wong, Edward C C; Nouraie, Mehdi; Maric, Irina; Albani, Sarah; Finkel, Julia; Quezado, Zenaide M N

    2015-12-01

    Fetal hemoglobin-inducing therapies are disease-modifying and ameliorate the pain phenotype in sickle cell disease (SCD). Rapamycin, a mammalian target of rapamycin (mTOR) inhibitor, increases HbF in erythroid precursor cells in vitro. We hypothesized that rapamycin would increase HbF levels and improve nociception phenotype in SCD mice. We used sine-wave electrical stimulation to examine nocifensive phenotype and evaluate myelinated [2000Hz (Aβ-fiber) and 250Hz (Aδ-fiber)] and unmyelinated (5Hz C-fibers)] sensory fiber function. Rapamycin significantly increased γ-globin mRNA and HbF levels [+2.3% (0.7, 3.9), mean increase (95% confidence interval, CI), p=0.006]. In homozygous (sickling) mice, long- (16 weeks), but not short-term (6 weeks), rapamycin treatment increased 2000Hz and 250Hz current thresholds in a pattern that varied according to sex. In male, but not female mice, rapamycin (compared with vehicle) was associated with increases in 2000Hz [21Units (7, 35), mean difference (95% CI), p=0.009 for sex∗treatment interaction] and 250Hz [9Units (1, 16), p=0.01] current thresholds. In rapamycin-treated homozygotes, HbF levels directly correlated with myelinated [2000Hz(Aβ-fiber, r=0.58, p=0.01) and 250Hz(Aδ-fiber, r=0.6, p=0.01)] but not unmyelinated sensory fiber current thresholds. These findings suggest that in SCD mice, rapamycin increases HbF and modulates current thresholds of myelinated fibers. Therefore, mTOR signaling might be implicated in the pathobiology of SCD. Copyright © 2015 Elsevier Inc. All rights reserved.

  20. The mTOR inhibitor sirolimus suppresses renal, hepatic, and cardiac tissue cellular respiration.

    Science.gov (United States)

    Albawardi, Alia; Almarzooqi, Saeeda; Saraswathiamma, Dhanya; Abdul-Kader, Hidaya Mohammed; Souid, Abdul-Kader; Alfazari, Ali S

    2015-01-01

    The purpose of this in vitro study was to develop a useful biomarker (e.g., cellular respiration, or mitochondrial O2 consumption) for measuring activities of mTOR inhibitors. It measured the effects of commonly used immunosuppressants (sirolimus-rapamycin, tacrolimus, and cyclosporine) on cellular respiration in target tissues (kidney, liver, and heart) from C57BL/6 mice. The mammalian target of rapamycin (mTOR), a serine/ threonine kinase that supports nutrient-dependent cell growth and survival, is known to control energy conversion processes within the mitochondria. Consistently, inhibitors of mTOR (e.g., rapamycin, also known as sirolimus or Rapamune®) have been shown to impair mitochondrial function. Inhibitors of the calcium-dependent serine/threonine phosphatase calcineurin (e.g., tacrolimus and cyclosporine), on the other hand, strictly prevent lymphokine production leading to a reduced T-cell function. Sirolimus (10 μM) inhibited renal (22%, P=0.002), hepatic (39%, Pcellular respiration. Tacrolimus and cyclosporine had no or minimum effects on cellular respiration in these tissues. Thus, these results clearly demonstrate that impaired cellular respiration (bioenergetics) is a sensitive biomarker of the immunosuppressants that target mTOR.

  1. Role of adenosine 5'-monophosphate-activated protein kinase subunits in skeletal muscle mammalian target of rapamycin signaling

    DEFF Research Database (Denmark)

    Deshmukh, Atul S.; Treebak, Jonas Thue; Long, Yun Chau

    2008-01-01

    AMP-activated protein kinase (AMPK) is an important energy-sensing protein in skeletal muscle. Mammalian target of rapamycin (mTOR) mediates translation initiation and protein synthesis through ribosomal S6 kinase 1 (S6K1) and eukaryotic initiation factor 4E-binding protein 1 (4E-BP1). AMPK activ...

  2. Long-term mTOR inhibitors administration evokes altered calcium homeostasis and platelet dysfunction in kidney transplant patients

    Science.gov (United States)

    López, Esther; Berna-Erro, Alejandro; Bermejo, Nuria; Brull, José María; Martinez, Rocío; Garcia Pino, Guadalupe; Alvarado, Raul; Salido, Ginés María; Rosado, Juan Antonio; Cubero, Juan José; Redondo, Pedro Cosme

    2013-01-01

    The use of the mammal target of rapamycin (mTOR) inhibitors has been consolidated as the therapy of election for preventing graft rejection in kidney transplant patients, despite their immunosuppressive activity is less strong than anti-calcineurin agents like tacrolimus and cyclosporine A. Furthermore, as mTOR is widely expressed, rapamycin (a macrolide antibiotic produced by Streptomyces hygroscopicus) is recommended in patients presenting neoplasia due to its antiproliferative actions. Hence, we have investigated whether rapamycin presents side effects in the physiology of other cell types different from leucocytes, such as platelets. Blood samples were drawn from healthy volunteers and kidney transplant patients long-term medicated with rapamycin: sirolimus and everolimus. Platelets were either loaded with fura-2 or directly stimulated, and immunoassayed or fixed with Laemmli's buffer to perform the subsequent analysis of platelet physiology. Our results indicate that rapamycin evokes a biphasic time-dependent alteration in calcium homeostasis and function in platelets from kidney transplant patients under rapamycin regime, as demonstrated by the reduction in granule secretion observed and subsequent impairment of platelet aggregation in these patients compared with healthy volunteers. Platelet count was also reduced in these patients, thus 41% of patients presented thrombocytopenia. All together our results show that long-term administration of rapamycin to kidney transplant patients evokes alteration in platelet function. PMID:23577651

  3. The role of mTOR signaling in Alzheimer disease.

    Science.gov (United States)

    Oddo, Salvatore

    2012-01-01

    The buildup of Abeta and tau is believed to directly cause or contribute to the progressive cognitive deficits characteristic of Alzheimer disease. However, the molecular pathways linking Abeta and tau accumulation to learning and memory deficits remain elusive. There is growing evidence that soluble forms of Abeta and tau can obstruct learning and memory by interfering with several signaling cascades. In this review, I will present data showing that the mammalian target of rapamycin (mTOR) may play a role in Abeta and tau induced neurodegeneration.

  4. Disruption of Parallel and Converging Signaling Pathways Contributes to the Synergistic Antitumor Effects of Simultaneous mTOR and EGFR Inhibition in GBM Cells

    Directory of Open Access Journals (Sweden)

    Ravi D. Rao

    2005-10-01

    Full Text Available Elevated epidermal growth factor receptor (EGFR and mammalian target of rapamycin (mTOR signaling are known to contribute to the malignant properties of glioblastoma multiforme (GBM, which include uncontrolled cell proliferation and evasion of apoptosis. Small molecule inhibitors that target these protein kinases have been evaluated in multiple clinical trials for cancer patients, including those with GBM. Here we have examined the cellular and molecular effects of a combined kinase inhibition of mTOR (rapamycin and EGFR (EKI-785 in U87 and U251 GBM cells. Simultaneous treatment with rapamycin and EKI-785 results in synergistic antiproliferative as well as proapoptotic effects. At a molecular level, rapamycin alone significantly decreases S6 phosphorylation, whereas EKI-785 alone promotes substantially reduced signal transducer and activator of transcription (STAT3 phosphorylation. Treatment with rapamycin alone also increases Akt phosphorylation on Ser-473, but this effect is blocked by a simultaneous administration of EKI-785. Individually, EKI-785 diminishes while rapamycin promotes the binding of the translation inhibitor eukaryotic initiation factor 4E binding protein (4EBP1 to the eukaryotic translation initiation factor 4E (eIF4E. In spite of these opposing effects, the highest level of 4EBP1-eIF4E binding occurs with the combination of the two inhibitors. These results indicate that the inhibition of EGFR and mTOR has distinct as well as common signaling consequences and provides a molecular rationale forthe synergistic antitumor effects of EKI-785 and rapamycin administration.

  5. Differential mTOR pathway profiles in bladder cancer cell line subtypes to predict sensitivity to mTOR inhibition.

    Science.gov (United States)

    Hau, Andrew M; Nakasaki, Manando; Nakashima, Kazufumi; Krish, Goutam; Hansel, Donna E

    2017-10-01

    Molecular classification of bladder cancer has been increasingly proposed as a potential tool to predict clinical outcomes and responses to chemotherapy. Here we focused on mechanistic target of rapamycin (mTOR) inhibition as a chemotherapeutic strategy and characterized the expression profile of mTOR signaling targets in representative bladder cancer cell lines from basal, luminal, and either basal/luminal ("non-type") molecular subtypes. Protein and mRNA expression of mTOR signaling components from representative luminal (RT4 and RT112), basal (SCaBER and 5637), and nontype (T24 and J82) bladder cancer cell line subtypes were determined by Western blot and database mining analysis of the Cancer Cell Line Encyclopedia. Cell viability following treatment with either, Torin-2 or KU-0063794, 2 dual mTOR complex 1/2 inhibitors, was determined by MTT assay. Immunoblot analysis of cells treated with Torin-2 or KU-0063794 was performed to determine the effects of mTOR inhibition on expression and phosphorylation status of mTOR signaling components, Akt, 4E-BP1, and ribosomal protein S6. Molecular subtypes of bladder cancer cell lines each exhibited a distinct pattern of expression of mTOR-associated genes and baseline phosphorylation level of Akt and 4E-BP1. Cells with low levels of Akt Ser-473 phosphorylation were more resistant to the cytotoxic effects of mTOR inhibition with Torin-2, but not KU-0063794. Exposure to Torin-2 and KU-0063794 both potently and rapidly inhibited phosphorylation of Akt Ser-473 and Thr-308, and 4E-BP1 T37/46 in cell lines that included basal and nontype subtypes. Differential gene expression and protein activity associated with mTOR signaling is observed among bladder cancer cell lines stratified into basal, luminal, and nontype subtypes. Urothelial carcinomas characterized by high baseline Akt Ser-473 phosphorylation may be best suited for targeted mTOR therapies. Copyright © 2017 Elsevier Inc. All rights reserved.

  6. Inhibition of mTOR promotes hyperthermia sensitivity in SMMC-7721 human hepatocellular carcinoma cell line

    Science.gov (United States)

    WANG, QING-LIANG; LIU, BO; LI, XIAO-JIE; HU, KUN-PENG; ZHAO, KUN; YE, XIAO-MING

    2016-01-01

    The mammalian target of rapamycin (mTOR) is a critical mediator of the phosphoinositide 3-kinase/protein kinase B/mTOR signaling pathway, and mTOR activity is induced following heat shock. Thermotherapy is used to treat hepatocellular carcinoma (HCC). However, the role of mTOR in modulating thermosensitivity in HCC has yet to be elucidated. In the present study, the antisense plasmid pEGFP-C1-mTOR was transfected into SMMC-7721 cells, and the expression levels of mTOR were analyzed by reverse transcription-polymerase chain reaction and western blot analysis. The thermal responses of the transfected cells were also examined. The results revealed that SMMC-7721 cells were sensitive to heat treatment, and cell viability was significantly inhibited following hyperthermia treatment (P<0.01). The mRNA and protein expression levels of mTOR decreased post-transfection. Cell proliferation, colony-forming ability and motility were all significantly decreased following hyperthermia treatment in the transfected cells. Flow cytometry analysis demonstrated that apoptosis was significantly increased following treatment (P<0.01). The number of cells in S phase was increased, and the cell cycle was arrested in S phase. In conclusion, inhibition of mTOR increased the thermosensitivity of SMMC-7721 cells by increasing cellular apoptosis and inducing S phase arrest. PMID:26998020

  7. Nuclear PIM1 confers resistance to rapamycin-impaired endothelial proliferation

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    Walpen, Thomas; Kalus, Ina [Research Unit, Division Internal Medicine, University Hospital Zuerich, 8091 Zuerich (Switzerland); Schwaller, Juerg [Department of Biomedicine, University of Basel, 4031 Basel (Switzerland); Peier, Martin A. [Research Unit, Division Internal Medicine, University Hospital Zuerich, 8091 Zuerich (Switzerland); Battegay, Edouard J. [Research Unit, Division Internal Medicine, University Hospital Zuerich, 8091 Zuerich (Switzerland); Zurich Center for Integrative Human Physiology (ZIHP), 8057 Zuerich (Switzerland); Humar, Rok, E-mail: Rok.Humar@usz.ch [Research Unit, Division Internal Medicine, University Hospital Zuerich, 8091 Zuerich (Switzerland); Zurich Center for Integrative Human Physiology (ZIHP), 8057 Zuerich (Switzerland)

    2012-12-07

    Highlights: Black-Right-Pointing-Pointer Pim1{sup -/-} endothelial cell proliferation displays increased sensitivity to rapamycin. Black-Right-Pointing-Pointer mTOR inhibition by rapamycin enhances PIM1 cytosolic and nuclear protein levels. Black-Right-Pointing-Pointer Truncation of Pim1 beyond serine 276 results in nuclear localization of the kinase. Black-Right-Pointing-Pointer Nuclear PIM1 increases endothelial proliferation independent of rapamycin. -- Abstract: The PIM serine/threonine kinases and the mTOR/AKT pathway integrate growth factor signaling and promote cell proliferation and survival. They both share phosphorylation targets and have overlapping functions, which can partially substitute for each other. In cancer cells PIM kinases have been reported to produce resistance to mTOR inhibition by rapamycin. Tumor growth depends highly on blood vessel infiltration into the malignant tissue and therefore on endothelial cell proliferation. We therefore investigated how the PIM1 kinase modulates growth inhibitory effects of rapamycin in mouse aortic endothelial cells (MAEC). We found that proliferation of MAEC lacking Pim1 was significantly more sensitive to rapamycin inhibition, compared to wildtype cells. Inhibition of mTOR and AKT in normal MAEC resulted in significantly elevated PIM1 protein levels in the cytosol and in the nucleus. We observed that truncation of the C-terminal part of Pim1 beyond Ser 276 resulted in almost exclusive nuclear localization of the protein. Re-expression of this Pim1 deletion mutant significantly increased the proliferation of Pim1{sup -/-} cells when compared to expression of the wildtype Pim1 cDNA. Finally, overexpression of the nuclear localization mutant and the wildtype Pim1 resulted in complete resistance to growth inhibition by rapamycin. Thus, mTOR inhibition-induced nuclear accumulation of PIM1 or expression of a nuclear C-terminal PIM1 truncation mutant is sufficient to increase endothelial cell proliferation

  8. Synergic antiproliferative and antiangiogenic effects of EGFR and mTor inhibitors on pancreatic cancer cells.

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    Azzariti, Amalia; Porcelli, Letizia; Gatti, Giuliana; Nicolin, Angelo; Paradiso, Angelo

    2008-03-01

    The in vitro efficacy of both EGFR inhibitor gefitinib and mTor inhibitor rapamycin, either administrated alone or in different combination schedules, was analysed in four pancreas cancer cell lines. Both drugs were found to induce cell growth inhibition, apoptosis as well as a slight but stable accumulation of cells in the G0/G1 phase. In all cell lines, neither gefitinib nor rapamycin affected EGFR and the expression of its downstream effectors. By contrast, gefitinib inhibited in a fast and completely way p-EGFR and partially p-Akt while a 3 days-rapamycin exposure resulted in the inhibition of the expression of both mTor and p70S6K. Moreover, after early stimulation, the mTor inhibitor produced a progressive, and almost complete inhibition of p-Akt. The analysis of combined gefitinib and rapamycin administration showed a clear schedule-dependent activity which turned out to be synergic only when gefitinib was given before rapamycin. This synergism seemed to depend on increase of both p-Akt and p70S6K inhibition, the greater the induction of apoptosis, the higher the decrease in cell cycle rate. Moreover, the antiangiogenic activity of the two drugs given in combination was demonstrated by a strong reduction of VEGF release which turned out to be more pronounced in the synergic schedule, and HIF-1alpha inhibition-independent. Our results suggest that the schedule of gefitinib followed by rapamycin, acting at different levels of the EGFR cellular pathway, could induce antitumor and antiangiogenic effects of clinical interest in the pancreas cancer model.

  9. MicroRNA-101 mediates the suppressive effect of laminar shear stress on mTOR expression in vascular endothelial cells

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    Chen, Kui; Fan, Wendong; Wang, Xing; Ke, Xiao [Division of Cardiology, First Affiliated Hospital of Sun Yat-sen University, Guangzhou 510080 (China); Wu, Guifu, E-mail: eecpchina@yahoo.com.cn [Key Laboratory on Assisted Circulation, Ministry of Health, Guangzhou 510080 (China); Hu, Chengheng, E-mail: huchenghengpci@yahoo.com.cn [Division of Cardiology, First Affiliated Hospital of Sun Yat-sen University, Guangzhou 510080 (China)

    2012-10-12

    Highlights: Black-Right-Pointing-Pointer Laminar shear stress upregulates miR-101 expression in vascular endothelial cells. Black-Right-Pointing-Pointer miR-101 represses mTOR expression through a specific 3 Prime UTR binding site. Black-Right-Pointing-Pointer Overexpression of miR-101 inhibits G1/S transition and endothelial cell proliferation. Black-Right-Pointing-Pointer Blockade of miR-101 attenuates the suppressive effect of laminar flow on mTOR expression. -- Abstract: Shear stress associated with blood flow plays an important role in regulating gene expression and cell function in endothelial cells (ECs). MicroRNAs (miRNAs) are highly conserved, small non-coding RNAs that negatively regulate the expression of target genes by binding to the mRNA 3 Prime -untranslated region (3 Prime UTR) at the posttranscriptional level involved in diverse cellular processes. This study demonstrates that microRNA-101 in response to laminar shear stress (LSS) is involved in the flow regulation of gene expression in ECs. qRT-PCR analysis showed that miR-101 expression was significantly upregulated in human umbilical vein endothelial cells (HUVECs) exposed to 12 dyn/cm{sup 2} laminar shear stress for 12 h. We found that transfection of miR-101 significantly decreased the luciferase activity of plasmid reporter containing the 3 Prime UTR of mammalian target of rapamycin (mTOR) gene. Western analysis revealed that the protein level of mTOR was significantly reduced in ECs transfected with miR-101. Furthermore, miR-101 overexpression induced cell cycle arrest at the G1/S transition and suppressed endothelial cell proliferation. Finally, transfection of miR-101 inhibitors attenuated the suppressive effects of LSS on mTOR expression, which identified the efficacy of loss-of-function of miR-101 in laminar flow-treated ECs. In conclusion, we have demonstrated that upregulation of miR-101 in response to LSS contributes to the suppressive effects of LSS on mTOR expression and EC

  10. MicroRNA-99a inhibits insulin-induced proliferation, migration, dedifferentiation, and rapamycin resistance of vascular smooth muscle cells by inhibiting insulin-like growth factor-1 receptor and mammalian target of rapamycin.

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    Zhang, Zi-Wei; Guo, Rui-Wei; Lv, Jin-Lin; Wang, Xian-Mei; Ye, Jin-Shan; Lu, Ni-Hong; Liang, Xing; Yang, Li-Xia

    2017-04-29

    Patients with type 2 diabetes mellitus (T2DM) are characterized by insulin resistance and are subsequently at high risk for atherosclerosis. Hyperinsulinemia has been associated with proliferation, migration, and dedifferentiation of vascular smooth muscle cells (VSMCs) during the pathogenesis of atherosclerosis. Moreover, insulin-like growth factor-1 receptor (IGF-1R) and mammalian target of rapamycin (mTOR) have been demonstrated to be the underlying signaling pathways. Recently, microRNA-99a (miR-99a) has been suggested to regulate the phenotypic changes of VSMCs in cancer cells. However, whether it is involved in insulin-induced changes of VSCMs has not been determined. In this study, we found that insulin induced proliferation, migration, and dedifferentiation of mouse VSMCs in a dose-dependent manner. Furthermore, the stimulating effects of high-dose insulin on proliferation, migration, and dedifferentiation of mouse VSMCs were found to be associated with the attenuation of the inhibitory effects of miR-99a on IGF-1R and mTOR signaling activities. Finally, we found that the inducing effect of high-dose insulin on proliferation, migration, and dedifferentiation of VSMCs was partially inhibited by an active mimic of miR-99a. Taken together, these results suggest that miR-99a plays a key regulatory role in the pathogenesis of insulin-induced proliferation, migration, and phenotype conversion of VSMCs at least partly via inhibition of IGF-1R and mTOR signaling. Our results provide evidence that miR-99a may be a novel target for the treatment of hyperinsulinemia-induced atherosclerosis. Copyright © 2017 Elsevier Inc. All rights reserved.

  11. Bone growth during rapamycin therapy in young rats

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    He Yu-Zhu

    2009-01-01

    Full Text Available Abstract Background Rapamycin is an effective immunosuppressant widely used to maintain the renal allograft in pediatric patients. Linear growth may be adversely affected in young children since rapamycin has potent anti-proliferative and anti-angiogenic properties. Methods Weanling three week old rats were given rapamycin at 2.5 mg/kg daily by gavage for 2 or 4 weeks and compared to a Control group given equivalent amount of saline. Morphometric measurements and biochemical determinations for serum calcium, phosphate, iPTH, urea nitrogen, creatinine and insulin-growth factor I (IGF-I were obtained. Histomorphometric analysis of the growth plate cartilage, in-situ hybridization experiments and immunohistochemical studies for various proteins were performed to evaluate for chondrocyte proliferation, chondrocyte differentiation and chondro/osteoclastic resorption. Results At the end of the 2 weeks, body and tibia length measurements were shorter after rapamycin therapy associated with an enlargement of the hypertrophic zone in the growth plate cartilage. There was a decrease in chondrocyte proliferation assessed by histone-4 and mammalian target of rapamycin (mTOR expression. A reduction in parathyroid hormone/parathyroid hormone related peptide (PTH/PTHrP and an increase in Indian hedgehog (Ihh expression may explain in part, the increase number of hypertrophic chondrocytes. The number of TRAP positive multinucleated chondro/osteoclasts declined in the chondro-osseous junction with a decrease in the receptor activator of nuclear factor kappa β ligand (RANKL and vascular endothelial growth factor (VEGF expression. Although body and tibial length remained short after 4 weeks of rapamycin, changes in the expression of chondrocyte proliferation, chondrocyte differentiation and chondro/osteoclastic resorption which were significant after 2 weeks of rapamycin improved at the end of 4 weeks. Conclusion When given to young rats, 2 weeks of rapamycin

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

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    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. Copyright © 2016 Elsevier Ltd. All rights reserved.

  13. THE EFFECT OF RAPAMYCIN ON SECRETORY ACTIVITY OF THE RABBIT OVARIAN FRAGMENTS

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    Sushmita Nath

    2012-02-01

    Full Text Available The aim of our study was to examine the effect of rapamycin on secretory activity of the rabbit ovarian fragments. The secretion of steroid (progesterone, testosterone, estradiol and peptide (prolactine hormones by ovarian fragments after rapamycin addition at the doses 0, 1, 10, 100 μg.ml-1 was determined. Fragments were incubated with rapamycin for 48 hours. Hormones were determinated by RIA. The experimental data showed that, addition of rapamycin did not affect progesterone and prolactine release (at all doses. Estradiol secretion was inhibited by rapamycin at the doses of 1, 10 and 100 μg.ml-1. Testosterone was inhibited by the rapamycin at the doses of 1 and 10 μg.ml-1 but not at 100 μg.ml-1. In conclusion, our results suggest a direct effect of rapamycin on ovarian functionsand a possible involvement in the regulation of steroidogenesis.

  14. Predicting mTOR inhibitors with a classifier using recursive partitioning and Naïve Bayesian approaches.

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    Ling Wang

    Full Text Available BACKGROUND: Mammalian target of rapamycin (mTOR is a central controller of cell growth, proliferation, metabolism, and angiogenesis. Thus, there is a great deal of interest in developing clinical drugs based on mTOR. In this paper, in silico models based on multi-scaffolds were developed to predict mTOR inhibitors or non-inhibitors. METHODS: First 1,264 diverse compounds were collected and categorized as mTOR inhibitors and non-inhibitors. Two methods, recursive partitioning (RP and naïve Bayesian (NB, were used to build combinatorial classification models of mTOR inhibitors versus non-inhibitors using physicochemical descriptors, fingerprints, and atom center fragments (ACFs. RESULTS: A total of 253 models were constructed and the overall predictive accuracies of the best models were more than 90% for both the training set of 964 and the external test set of 300 diverse compounds. The scaffold hopping abilities of the best models were successfully evaluated through predicting 37 new recently published mTOR inhibitors. Compared with the best RP and Bayesian models, the classifier based on ACFs and Bayesian shows comparable or slightly better in performance and scaffold hopping abilities. A web server was developed based on the ACFs and Bayesian method (http://rcdd.sysu.edu.cn/mtor/. This web server can be used to predict whether a compound is an mTOR inhibitor or non-inhibitor online. CONCLUSION: In silico models were constructed to predict mTOR inhibitors using recursive partitioning and naïve Bayesian methods, and a web server (mTOR Predictor was also developed based on the best model results. Compound prediction or virtual screening can be carried out through our web server. Moreover, the favorable and unfavorable fragments for mTOR inhibitors obtained from Bayesian classifiers will be helpful for lead optimization or the design of new mTOR inhibitors.

  15. miR-199a-3p is involved in estrogen-mediated autophagy through the IGF-1/mTOR pathway in osteocyte-like MLO-Y4 cells.

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    Fu, Jiayao; Hao, Lingyu; Tian, Yawen; Liu, Yang; Gu, Yijing; Wu, Junhua

    2018-03-01

    To date, evidence indicates that estrogen partially modulates cellular processes through microRNAs. Autophagy is a catabolic process that is regulated by multiple factors and is associated with skeletal diseases. However, whether estrogen regulates osteocyte autophagy via microRNAs is largely unknown. In this study, we observed the up-regulation of microRNA-199a-3p, a post-transcriptional regulatory factor, in osteocytic areas in ovariectomized (OVX) mice. The mature forms of miR-199a-3p and pri-miR-199a were produced in response to estrogen signaling in osteocyte-like MLO-Y4 cells. Western blotting, autophagic flux detection, mRFP-GFP-LC3 fluorescence, and electron microscopy confirmed that miR-199a-3p induced autophagy in MLO-Y4 cells, although cellular apoptosis was not affected. Additionally, we documented the ability of estrogen to mediate osteocyte autophagy. Based on our in vivo data, estrogen deficiency induced autophagy in osteocytes. Treatment of starved MLO-Y4 cells with 17β-estradiol suppressed the excess autophagy induced by starvation via activation of mammalian target of rapamycin (mTOR)-related signaling cascades, while administration of rapamycin reversed the effects of 17β-estradiol. Meanwhile, miR-199a-3p overexpression reversed 17β-estradiol-mediated regulation of autophagy in MLO-Y4 cells. According to mechanistic studies, miR-199a-3p inhibited the mTOR pathway by directly binding to the 3'-untranslated regions of insulin growth factor-1 (IGF-1) and mTOR. However, overexpression of miR-199a-3p inhibited IGF-1 phosphorylation and mTOR-related pathways. Knockdown of mTOR and IGF-1 abolished estrogen signaling and restored LC3-II expression through mTOR re-activation, respectively. Thus, miR-199a-3p appears to be involved in the estrogen regulatory networks that mediate bone cell autophagy, potentially by targeting IGF-1 and mTOR. © 2017 Wiley Periodicals, Inc.

  16. Effects of rapamycin and curcumin treatment on the development of epilepsy after electrically induced status epilepticus in rats.

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    Drion, Cato M; Borm, Lars E; Kooijman, Lieneke; Aronica, Eleonora; Wadman, Wytse J; Hartog, Aloysius F; van Vliet, Erwin A; Gorter, Jan A

    2016-05-01

    Inhibition of the mammalian target of rapamycin (mTOR) pathway has been suggested as a possible antiepileptogenic strategy in temporal lobe epilepsy (TLE). Here we aim to elucidate whether mTOR inhibition has antiepileptogenic and/or antiseizure effects using different treatment strategies in the electrogenic post-status epilepticus (SE) rat model. Effects of mTOR inhibitor rapamycin were tested using the following three treatment protocols: (1) "stop-treatment"-post-SE treatment (6 mg/kg/day) was discontinued after 3 weeks; rats were monitored for 5 more weeks thereafter, (2) "pretreatment"-rapamycin (3 mg/kg/day) was applied during 3 days preceding SE; and (3) "chronic phase-treatment"-5 days rapamycin treatment (3 mg/kg/day) in the chronic phase. We also tested curcumin, an alternative mTOR inhibitor with antiinflammatory and antioxidant effects, using chronic phase treatment. Seizures were continuously monitored using video-electroencephalography (EEG) recordings; mossy fiber sprouting, cell death, and inflammation were studied using immunohistochemistry. Blood was withdrawn regularly to assess rapamycin and curcumin levels with high performance liquid chromatography (HPLC). Stop-treatment led to a strong reduction of seizures during the 3-week treatment and a gradual reappearance of seizures during the following 5 weeks. Three days pretreatment did not prevent seizure development, whereas 5-day rapamycin treatment in the chronic phase reduced seizure frequency. Washout of rapamycin was slow and associated with a gradual reappearance of seizures. Rapamycin treatment (both 3 and 6 mg/kg) led to body growth reduction. Curcumin treatment did not reduce seizure frequency or lead to a decrease in body weight. The present study indicates that rapamycin cannot prevent epilepsy in the electrical stimulation post-SE rat model but has seizure-suppressing properties as long as rapamycin blood levels are sufficiently high. Oral curcumin treatment had no effect on chronic

  17. Protective Macroautophagy Is Involved in Vitamin E Succinate Effects on Human Gastric Carcinoma Cell Line SGC-7901 by Inhibiting mTOR Axis Phosphorylation.

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    Liying Hou

    Full Text Available Vitamin E succinate (VES, a potential cancer therapeutic agent, potently induces apoptosis and inhibits the growth of various cancer cells. Autophagy has been supposed to promote cancer cell survival or trigger cell death, depending on particular cancer types and tumor microenvironments. The role of autophagy in the growth suppressive effect of VES on gastric cancer cell is basically unknown. We aimed to determine whether and how autophagy affected the VES-induced inhibition of SGC-7901 human gastric carcinoma cell growth. SGC-7901 cells were treated with VES or pre-treated with autophagy inhibitor, chloroquine (CQ and 3-methyladenine (3-MA. Electron microscopy, fluorescence microscopy and Western blot were used to study whether VES induced autophagy reaction in SGC-7901 cells. Western blot evaluated the activities of the mammalian target of rapamycin (mTOR axis. Then we used 3-(4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide (MTT and flow cytometry to detect the level of cell viability and apoptosis. Collectively, our data indeed strongly support our hypothesis that VES treatment produced cytological variations that depict autophagy, increased the amount of intracellular green fluorescent protein-microtubule associated protein 1 light chain 3 (GFP-LC3 punctate fluorescence and the number of autophagic vacuoles. It altered the expression of endogenous autophagy marker LC3. VES activated the suppression of mTOR through inhibiting upstream regulators p38 MAPK and Akt. mTOR suppression consequently inhibited the activation of mTOR downstream targets p70S6K and 4E-BP-1. The activation of the upstream mTOR inhibitor AMPK had been up-regulated by VES. The results showed that pre-treatment SGC-7901 with autophagy inhibitors before VES treatment could increase the capacity of VES to reduce cell viability and to provoke apoptosis. In conclusion, VES-induced autophagy participates in SGC-7901 cell protection by inhibiting mTOR axis

  18. Both mTORC1 and mTORC2 are involved in the regulation of cell adhesion

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    Chen, Long; Xu, Baoshan; Liu, Lei; Liu, Chunxiao; Luo, Yan; Chen, Xin; Barzegar, Mansoureh; Chung, Jun; Huang, Shile

    2015-01-01

    mTOR is a central controller for cell growth/proliferation and survival. Recent studies have shown that mTOR also regulates cell adhesion, yet the underlying mechanism is not known. Here we found that inhibition of mTOR by rapamycin reduced the basal or type I insulin-like growth factor (IGF-1)-stimulated adhesion of cancer cells. Further research revealed that both mTORC1 and mTORC2 were involved in the regulation of cell adhesion, as silencing expression of raptor or rictor inhibited cell adhesion. Also, PP242, an mTORC1/2 kinase inhibitor, inhibited cell adhesion more potently than rapamycin (mTORC1 inhibitor). Of interest, ectopic expression of constitutively active and rapamycin-resistant mutant of p70 kinase 1 (S6K1) or downregulation of eukaryotic initiation factor 4E (eIF4E)-binding protein 1 (4E-BP1) conferred resistance to rapamycin inhibition of cell adhesion, whereas expression of constitutively hypophosphorylated 4E-BP1 (4EBP1-5A) or downregulation of S6K1 suppressed cell adhesion. In contrast, neither genetic manipulation of Akt activity nor pharmacological inhibition of Akt affected cell adhesion. The results suggest that both mTORC1 and mTORC2 are involved in the regulation of cell adhesion; and mTORC1 regulates cell adhesion through S6K1 and 4E-BP1 pathways, but mTORC2 regulates cell adhesion via Akt-independent mechanism. PMID:25762619

  19. mTOR complex-2 stimulates acetyl-CoA and de novo lipogenesis through ATP citrate lyase in HER2/PIK3CA-hyperactive breast cancer.

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    Chen, Yaqing; Qian, Jianchang; He, Qun; Zhao, Hui; Toral-Barza, Lourdes; Shi, Celine; Zhang, Xuesai; Wu, Jiang; Yu, Ker

    2016-05-03

    The mechanistic target of rapamycin (mTOR) is a major regulator of cell growth and is frequently dysregulated in cancer. While mTOR complex-1 (mTORC1) is a validated cancer target, the role of mTOR complex-2 (mTORC2) remains less defined. Here, we reveal mTORC2 as a critical regulator of breast cancer metabolism. We showed that hyperphosphorylation in ATP citrate lyase (ACL) occurs frequently in human breast tumors and correlates well with HER2+ and/or PIK3CA-mutant (HER2+/PIK3CAmut) status in breast tumor cell lines. In HER2+/PIK3CAmut cells, mTORC2 controls Ser-455 phosphorylation of ACL thereby promoting acetyl-CoA production, de novo lipogenesis and mitochondrial physiology, all of which were inhibited by an mTORC1/mTORC2 kinase inhibitor (mTOR-KI) or cellular depletion of mTORC2 or ACL. mTOR-KI but not rapamycin blocked the IGF-1-induced ACL phosphorylation and glucose to lipid conversion. Depletion of mTORC2 but not mTORC1 specifically inhibited the ACL-dependent acetyl-CoA production. In the HER2+/PIK3CAmut MDA361, MDA453, BT-474 and T47D cells, depletion of mTORC2 or ACL led to growth inhibition and mitochondrial hyperpolarization, which were partially rescued by an alternate source of acetyl-CoA. These same changes were not apparent in mTORC2- or ACL-depleted HER2-/PIK3CAwt MDA231 and HCC1806 cells, highlighting a differential dependence of mTORC2-ACL for survival in these two cell types. Moreover, ACL Ser-455 mutants S455E (phosphomimetic) and S455A (non-phosphorylatable) each increased or decreased, respectively, the acetyl-CoA production, mitochondrial homeostasis and survival in ACL-depleted MDA453 cells. These studies define a new and rapamycin-resistant mechanism of mTORC2-ACL in lipogenesis and acetyl-CoA biology and provide a rationale for targeting of mTORC1 and mTORC2 in HER2+/PIK3CAmut breast cancer.

  20. Mammalian target of rapamycin and the kidney. II. Pathophysiology and therapeutic implications.

    Science.gov (United States)

    Lieberthal, Wilfred; Levine, Jerrold S

    2012-07-15

    The mTOR pathway plays an important role in a number of common renal diseases, including acute kidney injury (AKI), diabetic nephropathy (DN), and polycystic kidney diseases (PKD). The activity of mTOR complex 1 (mTORC1) is necessary for renal regeneration and repair after AKI, and inhibition of mTORC1 by rapamycin has been shown to delay recovery from ischemic AKI in animal studies, and to prolong delayed graft function in humans who have received a kidney transplant. For this reason, administration of rapamycin should be delayed or discontinued in patients with AKI until full recovery of renal function has occurred. On the other hand, inappropriately high mTORC1 activity contributes to the progression of the metabolic syndrome, the development of type 2 diabetes, and the pathogenesis of DN. In addition, chronic hyperactivity of mTORC1, and possibly also mTORC2, contributes to cyst formation and enlargement in a number of forms of PKD. Inhibition of mTOR, using either rapamycin (which inhibits predominantly mTORC1) or "catalytic" inhibitors (which effectively inhibit both mTORC1 and mTORC2), provide exciting possibilities for novel forms of treatment of DN and PKD. In this second part of the review, we will examine the role of mTOR in the pathophysiology of DN and PKD, as well as the potential utility of currently available and newly developed inhibitors of mTOR to slow the progression of DN and/or PKD.

  1. Beneficial metabolic effects of rapamycin are associated with enhanced regulatory cells in diet-induced obese mice.

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    Makki, Kassem; Taront, Solenne; Molendi-Coste, Olivier; Bouchaert, Emmanuel; Neve, Bernadette; Eury, Elodie; Lobbens, Stéphane; Labalette, Myriam; Duez, Hélène; Staels, Bart; Dombrowicz, David; Froguel, Philippe; Wolowczuk, Isabelle

    2014-01-01

    The "mechanistic target of rapamycin" (mTOR) is a central controller of growth, proliferation and/or motility of various cell-types ranging from adipocytes to immune cells, thereby linking metabolism and immunity. mTOR signaling is overactivated in obesity, promoting inflammation and insulin resistance. Therefore, great interest exists in the development of mTOR inhibitors as therapeutic drugs for obesity or diabetes. However, despite a plethora of studies characterizing the metabolic consequences of mTOR inhibition in rodent models, its impact on immune changes associated with the obese condition has never been questioned so far. To address this, we used a mouse model of high-fat diet (HFD)-fed mice with and without pharmacologic mTOR inhibition by rapamycin. Rapamycin was weekly administrated to HFD-fed C57BL/6 mice for 22 weeks. Metabolic effects were determined by glucose and insulin tolerance tests and by indirect calorimetry measures of energy expenditure. Inflammatory response and immune cell populations were characterized in blood, adipose tissue and liver. In parallel, the activities of both mTOR complexes (e. g. mTORC1 and mTORC2) were determined in adipose tissue, muscle and liver. We show that rapamycin-treated mice are leaner, have enhanced energy expenditure and are protected against insulin resistance. These beneficial metabolic effects of rapamycin were associated to significant changes of the inflammatory profiles of both adipose tissue and liver. Importantly, immune cells with regulatory functions such as regulatory T-cells (Tregs) and myeloid-derived suppressor cells (MDSCs) were increased in adipose tissue. These rapamycin-triggered metabolic and immune effects resulted from mTORC1 inhibition whilst mTORC2 activity was intact. Taken together, our results reinforce the notion that controlling immune regulatory cells in metabolic tissues is crucial to maintain a proper metabolic status and, more generally, comfort the need to search for novel

  2. 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.

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    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.

  3. Synergistic antitumor activity of rapamycin and EF24 via increasing ROS for the treatment of gastric cancer

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    Weiqian Chen

    2016-12-01

    Full Text Available Mechanistic/mammalian target of rapamycin (mTOR has emerged as a new potential therapeutic target for gastric cancer. Rapamycin and rapamycin analogs are undergoing clinical trials and have produced clinical responses in a subgroup of cancer patients. However, monotherapy with rapamycin at safe dosage fails to induce cell apoptosis and tumor regression which has hampered its clinical application. This has led to the exploration of more effective combinatorial regimens to enhance the effectiveness of rapamycin. In our present study, we have investigated the combination of rapamycin and a reactive oxygen species (ROS inducer EF24 in gastric cancer. We show that rapamycin increases intracellular ROS levels and displays selective synergistic antitumor activity with EF24 in gastric cancer cells. This activity was mediated through the activation of c-Jun N terminal kinase and endoplasmic reticulum stress (ER pathways in cancer cells. We also show that inhibiting ROS accumulation reverses ER stress and prevents apoptosis induced by the combination of rapamycin and EF24. These mechanisms were confirmed using human gastric cancer xenografts in immunodeficient mice. Taken together, our work provides a novel therapeutic strategy for the treatment of gastric cancer. The work reveals that ROS generation could be an important target for the development of new combination therapies for cancer treatment.

  4. Combination of rapamycin, CI-1040, and 17-AAG inhibits metastatic capacity of prostate cancer via Slug inhibition.

    Directory of Open Access Journals (Sweden)

    Guanxiong Ding

    Full Text Available Though prostate cancer (PCa has slow progression, the hormone refractory (HRCP and metastatic entities are substantially lethal and lack effective treatments. Transcription factor Slug is critical in regulating metastases of various tumors including PCa. Here we studied targeted therapy against Slug using combination of 3 drugs targeting 3 pathways respectively converging via Slug and further regulating PCa metastasis. Using in vitro assays we confirmed that Slug up-regulation incurred inhibition of E-cadherin that was anti-metastatic, and inhibited Bim-regulated cell apoptosis in PCa. Upstream PTEN/Akt, mTOR, Erk, and AR/Hsp90 pathways were responsible for Slug up-regulation and each of these could be targeted by rapamycin, CI-1040, and 17-AAG respectively. In 4 PCa cell lines with different traits in terms of PTEN loss and androgen sensitivity we tested the efficacy of mono- and combined therapy with the drugs. We found that metastatic capacity of the cells was maximally inhibited only when all 3 drugs were combined, due to the crosstalk between the pathways. 17-AAG decreases Slug expression via blockade of HSP90-dependent AR stability. Combination of rapamycin and CI-1040 diminishes invasiveness more potently in PCa cells that are androgen insensitive and with PTEN loss. Slug inhibited Bim-mediated apoptosis that could be rescued by mTOR/Erk/HSP90 inhibitors. Using mouse models for circulating PCa DNA quantification, we found that combination of mTOR/Erk/HSP90 inhibitors reduced circulating PCa cells in vivo significantly more potently than combination of 2 or monotherapy. Conclusively, combination of mTOR/Erk/Hsp90 inhibits metastatic capacity of prostate cancer via Slug inhibition.

  5. The mammalian target of rapamycin at the crossroad between cognitive aging and Alzheimer’s disease

    Science.gov (United States)

    Talboom, Joshua S; Velazquez, Ramon; Oddo, Salvatore

    2015-01-01

    Age-dependent cognitive decline is a major debilitating event affecting even individuals who are otherwise healthy. Understanding the molecular basis underlying these changes may increase the healthspan of the elderly population. It may also reveal insights into the pathogenesis of numerous neurodegenerative disorders characterized by cognitive deficits, as aging is the major risk factor for most of these disorders. Alzheimer’s disease (AD), the most common neurodegenerative disorder, first manifests itself as deficits in encoding new memories. As AD progresses, these deficits spread to other cognitive domains that further debilitate the person before contributing to their demise. Suppression of the mammalian target of rapamycin (mTOR) increases healthspan and lifespan in several organisms. Numerous reports have linked alterations in mTOR signaling to age-dependent cognitive decline and the pathogenesis of AD. This review will discuss recent work highlighting the complex role of mTOR in cognitive aging and in the pathogenesis of AD. PMID:28721257

  6. The mammalian target of rapamycin at the crossroad between cognitive aging and Alzheimer's disease.

    Science.gov (United States)

    Talboom, Joshua S; Velazquez, Ramon; Oddo, Salvatore

    2015-01-01

    Age-dependent cognitive decline is a major debilitating event affecting even individuals who are otherwise healthy. Understanding the molecular basis underlying these changes may increase the healthspan of the elderly population. It may also reveal insights into the pathogenesis of numerous neurodegenerative disorders characterized by cognitive deficits, as aging is the major risk factor for most of these disorders. Alzheimer's disease (AD), the most common neurodegenerative disorder, first manifests itself as deficits in encoding new memories. As AD progresses, these deficits spread to other cognitive domains that further debilitate the person before contributing to their demise. Suppression of the mammalian target of rapamycin (mTOR) increases healthspan and lifespan in several organisms. Numerous reports have linked alterations in mTOR signaling to age-dependent cognitive decline and the pathogenesis of AD. This review will discuss recent work highlighting the complex role of mTOR in cognitive aging and in the pathogenesis of AD.

  7. Rapamycin Inhibits Proliferation of Hemangioma Endothelial Cells by Reducing HIF-1-Dependent Expression of VEGF

    Science.gov (United States)

    Medici, Damian; Olsen, Bjorn R.

    2012-01-01

    Hemangiomas are tumors formed by hyper-proliferation of vascular endothelial cells. This is caused by elevated vascular endothelial growth factor (VEGF) signaling through VEGF receptor 2 (VEGFR2). Here we show that elevated VEGF levels produced by hemangioma endothelial cells are reduced by the mTOR inhibitor rapamycin. mTOR activates p70S6K, which controls translation of mRNA to generate proteins such as hypoxia inducible factor-1 (HIF-1). VEGF is a known HIF-1 target gene, and our data show that VEGF levels in hemangioma endothelial cells are reduced by HIF-1α siRNA. Over-expression of HIF-1α increases VEGF levels and endothelial cell proliferation. Furthermore, both rapamycin and HIF-1α siRNA reduce proliferation of hemangioma endothelial cells. These data suggest that mTOR and HIF-1 contribute to hemangioma endothelial cell proliferation by stimulating an autocrine loop of VEGF signaling. Furthermore, mTOR and HIF-1 may be therapeutic targets for the treatment of hemangiomas. PMID:22900063

  8. Rapamycin inhibits proliferation of hemangioma endothelial cells by reducing HIF-1-dependent expression of VEGF.

    Directory of Open Access Journals (Sweden)

    Damian Medici

    Full Text Available Hemangiomas are tumors formed by hyper-proliferation of vascular endothelial cells. This is caused by elevated vascular endothelial growth factor (VEGF signaling through VEGF receptor 2 (VEGFR2. Here we show that elevated VEGF levels produced by hemangioma endothelial cells are reduced by the mTOR inhibitor rapamycin. mTOR activates p70S6K, which controls translation of mRNA to generate proteins such as hypoxia inducible factor-1 (HIF-1. VEGF is a known HIF-1 target gene, and our data show that VEGF levels in hemangioma endothelial cells are reduced by HIF-1α siRNA. Over-expression of HIF-1α increases VEGF levels and endothelial cell proliferation. Furthermore, both rapamycin and HIF-1α siRNA reduce proliferation of hemangioma endothelial cells. These data suggest that mTOR and HIF-1 contribute to hemangioma endothelial cell proliferation by stimulating an autocrine loop of VEGF signaling. Furthermore, mTOR and HIF-1 may be therapeutic targets for the treatment of hemangiomas.

  9. Inhibition of mTOR is required for optimal antitumor effect of HER2 inhibitors against HER2-overexpressing cancer cells

    Science.gov (United States)

    Miller, Todd W.; Forbes, James T.; Shah, Chirayu; Wyatt, Shelby K.; Manning, H. Charles; Olivares, Maria G.; Sanchez, Violeta; Dugger, Teresa C.; Granja, Nara de Matos; Narasanna, Archana; Cook, Rebecca S.; Kennedy, J. Phillip; Lindsley, Craig W.; Arteaga, Carlos L.

    2009-01-01

    Purpose A significant fraction of HER2-overexpressing breast cancers exhibit resistance to the HER2 antibody trastuzumab. Hyperactivity of the phosphatidylinositol-3 kinase (PI3K)/AKT pathway confers trastuzumab resistance, and mTOR is a major downstream effector of PI3K/AKT. Therefore, we examined whether mTOR inhibitors synergize with trastuzumab. Experimental Design Immunocompetent mice bearing HER2-positive mammary tumors were treated with trastuzumab, the mTOR inhibitor rapamycin, or the combination. Mice were imaged for tumor cell death using an optical Annexin-V probe and with [18F]FDG-PET. The signaling and growth effects of the mTOR inhibitor RAD001 on HER2+ cells treated with trastuzumab or lapatinib were evaluated. Results Treatment of mice with trastuzumab plus rapamycin was more effective than single-agent treatments, inducing complete regression of 26/26 tumors. The combination induced tumor cell death (Annexin-V binding) and inhibited FDG uptake. Rapamycin inhibited mTOR and tumor cell proliferation as determined by phospho-S6 and Ki67 immunohistochemistry, respectively. In culture, the combination of RAD001 plus trastuzumab inhibited cell growth more effectively than either drug alone. Trastuzumab partially decreased PI3K but not mTOR activity. Knockdown of TSC2 resulted in HER2-independent activation of mTOR and dampened the response to trastuzumab and lapatinib. Treatment with the HER2 inhibitor lapatinib decreased phospho-S6 and growth in TSC2-expressing but not in TSC2-knockdown cells. Conclusions Inhibition of PI3K and mTOR are required for the growth inhibitory effect of HER2 antagonists. These findings collectively support the combined use of trastuzumab and mTOR inhibitors for the treatment of HER2+ breast cancer. PMID:19934303

  10. Discovery and Development of ATP-Competitive mTOR Inhibitors Using Computational Approaches.

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    Luo, Yao; Wang, Ling

    2017-11-16

    The mammalian target of rapamycin (mTOR) is a central controller of cell growth, proliferation, metabolism, and angiogenesis. This protein is an attractive target for new anticancer drug development. Significant progress has been made in hit discovery, lead optimization, drug candidate development and determination of the three-dimensional (3D) structure of mTOR. Computational methods have been applied to accelerate the discovery and development of mTOR inhibitors helping to model the structure of mTOR, screen compound databases, uncover structure-activity relationship (SAR) and optimize the hits, mine the privileged fragments and design focused libraries. Besides, computational approaches were also applied to study protein-ligand interactions mechanisms and in natural product-driven drug discovery. Herein, we survey the most recent progress on the application of computational approaches to advance the discovery and development of compounds targeting mTOR. Future directions in the discovery of new mTOR inhibitors using computational methods are also discussed. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  11. Targeting tumorigenesis: development and use of mTOR inhibitors in cancer therapy

    Directory of Open Access Journals (Sweden)

    Kay Andrea

    2009-10-01

    Full Text Available Abstract The mammalian target of rapamycin (mTOR is an intracellular serine/threonine protein kinase positioned at a central point in a variety of cellular signaling cascades. The established involvement of mTOR activity in the cellular processes that contribute to the development and progression of cancer has identified mTOR as a major link in tumorigenesis. Consequently, inhibitors of mTOR, including temsirolimus, everolimus, and ridaforolimus (formerly deforolimus have been developed and assessed for their safety and efficacy in patients with cancer. Temsirolimus is an intravenously administered agent approved by the US Food and Drug Administration (FDA and the European Medicines Agency (EMEA for the treatment of advanced renal cell carcinoma (RCC. Everolimus is an oral agent that has recently obtained US FDA and EMEA approval for the treatment of advanced RCC after failure of treatment with sunitinib or sorafenib. Ridaforolimus is not yet approved for any indication. The use of mTOR inhibitors, either alone or in combination with other anticancer agents, has the potential to provide anticancer activity in numerous tumor types. Cancer types in which these agents are under evaluation include neuroendocrine tumors, breast cancer, leukemia, lymphoma, hepatocellular carcinoma, gastric cancer, pancreatic cancer, sarcoma, endometrial cancer, and non-small-cell lung cancer. The results of ongoing clinical trials with mTOR inhibitors, as single agents and in combination regimens, will better define their activity in cancer.

  12. Mammalian Target of Rapamycin Inhibitor Induced Complete Remission of a Recurrent Subependymal Giant Cell Astrocytoma in a Patient Without Features of Tuberous Sclerosis Complex.

    Science.gov (United States)

    Appalla, Deepika; Depalma, Andres; Calderwood, Stanley

    2016-07-01

    The majority of patients with subependymal giant cell astrocytoma (SEGA) have tuberous sclerosis complex (TSC). In such patients, the mammalian target of rapamycin (mTOR) inhibitor everolimus has been shown to induce responses. Isolated SEGA have been reported in patients without clinical or genetic features of TSC. The treatment of these patients with everolimus has not previously been reported. We treated a patient with a recurrent isolated SEGA with an mTOR inhibitor. The patient tolerated therapy well and had a sustained complete remission. MTOR inhibitors may be useful for the treatment of isolated SEGA. Further study is warranted. © 2016 Wiley Periodicals, Inc.

  13. Rapamycin potentiates cytotoxicity by docetaxel possibly through downregulation of Survivin in lung cancer cells

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    Li Hui

    2011-03-01

    Full Text Available Abstract Background To elucidate whether rapamycin, the inhibitor of mTOR (mammalian target of rapamycin, can potentiate the cytotoxic effect of docetaxel in lung cancer cells and to probe the mechanism underlying such enhancement. Methods Lung cancer cells were treated with docetaxel and rapamycin. The effect on the proliferation of lung cancer cells was evaluated using the MTT method, and cell apoptosis was measured by flow cytometry. Protein expression and level of phosphorylation were assayed using Western Blot method. Results Co-treatment of rapamycin and docetaxel was found to favorably enhance the cytotoxic effect of docetaxel in four lung cancer cell lines. This tumoricidal boost is associated with a reduction in the expression and phosphorylation levels of Survivin and ERK1/2, respectively. Conclusion The combined application of mTOR inhibitor and docetaxel led to a greater degree of cancer cell killing than that by either compound used alone. Therefore, this combination warrants further investigation in its suitability of serving as a novel therapeutic scheme for treating advanced and recurrent lung cancer patients.

  14. mTOR pathway inhibition prevents neuroinflammation and neuronal death in a mouse model of cerebral palsy.

    Science.gov (United States)

    Srivastava, Isha N; Shperdheja, Jona; Baybis, Marianna; Ferguson, Tanya; Crino, Peter B

    2016-01-01

    Mammalian target of rapamycin (mTOR) pathway signaling governs cellular responses to hypoxia and inflammation including induction of autophagy and cell survival. Cerebral palsy (CP) is a neurodevelopmental disorder linked to hypoxic and inflammatory brain injury however, a role for mTOR modulation in CP has not been investigated. We hypothesized that mTOR pathway inhibition would diminish inflammation and prevent neuronal death in a mouse model of CP. Mouse pups (P6) were subjected to hypoxia-ischemia and lipopolysaccharide-induced inflammation (HIL), a model of CP causing neuronal injury within the hippocampus, periventricular white matter, and neocortex. mTOR pathway inhibition was achieved with rapamycin (an mTOR inhibitor; 5mg/kg) or PF-4708671 (an inhibitor of the downstream p70S6kinase, S6K, 75 mg/kg) immediately following HIL, and then for 3 subsequent days. Phospho-activation of the mTOR effectors p70S6kinase and ribosomal S6 protein and expression of hypoxia inducible factor 1 (HIF-1α) were assayed. Neuronal cell death was defined with Fluoro-Jade C (FJC) and autophagy was measured using Beclin-1 and LC3II expression. Iba-1 labeled, activated microglia were quantified. Neuronal death, enhanced HIF-1α expression, and numerous Iba-1 labeled, activated microglia were evident at 24 and 48 h following HIL. Basal mTOR signaling, as evidenced by phosphorylated-S6 and -S6K levels, was unchanged by HIL. Rapamycin or PF-4,708,671 treatment significantly reduced mTOR signaling, neuronal death, HIF-1α expression, and microglial activation, coincident with enhanced expression of Beclin-1 and LC3II, markers of autophagy induction. mTOR pathway inhibition prevented neuronal death and diminished neuroinflammation in this model of CP. Persistent mTOR signaling following HIL suggests a failure of autophagy induction, which may contribute to neuronal death in CP. These results suggest that mTOR signaling may be a novel therapeutic target to reduce neuronal cell death in

  15. Inhibition of mTOR improves the impairment of acidification in autophagic vesicles caused by hepatic steatosis

    Energy Technology Data Exchange (ETDEWEB)

    Nakadera, Eisuke [Department of Gastroenterology, Juntendo University School of Medicine, Hongo 2-1-1, Bunkyo-ku, Tokyo 113-8421 (Japan); Yamashina, Shunhei, E-mail: syamashi@juntendo.ac.jp [Department of Gastroenterology, Juntendo University School of Medicine, Hongo 2-1-1, Bunkyo-ku, Tokyo 113-8421 (Japan); Izumi, Kousuke; Inami, Yoshihiro; Sato, Toshifumi; Fukushima, Hirofumi; Kon, Kazuyoshi; Ikejima, Kenichi [Department of Gastroenterology, Juntendo University School of Medicine, Hongo 2-1-1, Bunkyo-ku, Tokyo 113-8421 (Japan); Ueno, Takashi [Division of Proteomics and Biomolecular Science, Juntendo University, School of Medicine, Hongo 2-1-1, Bunkyo-ku, Tokyo 113-8421 (Japan); Watanabe, Sumio [Department of Gastroenterology, Juntendo University School of Medicine, Hongo 2-1-1, Bunkyo-ku, Tokyo 113-8421 (Japan)

    2016-01-22

    Recent investigations revealed that dysfunction of autophagy involved in the progression of chronic liver diseases such as alcoholic and nonalcoholic steatohepatitis and hepatocellular neoplasia. Previously, it was reported that hepatic steatosis disturbs autophagic proteolysis via suppression of both autophagic induction and lysosomal function. Here, we demonstrate that autophagic acidification was altered by a decrease in lysosomal proton pump vacuolar-ATPase (V-ATPase) in steatohepatitis. The number of autophagic vesicles was increased in hepatocytes from obese KKAy mice as compared to control. Similarly, autophagic membrane protein LC3-II and lysosomal protein LAMP-2 expression were enhanced in KKAy mice liver. Nevertheless, both phospho-mTOR and p62 expression were augmented in KKAy mice liver. More than 70% of autophagosomes were stained by LysoTracker Red (LTR) in hepatocytes from control mice; however, the percentage of acidic autolysosomes was decreased in hepatocytes from KKAy mice significantly (40.1 ± 3.48%). Both protein and RNA level of V-ATPase subunits ATP6v1a, ATP6v1b, ATP6v1d in isolated lysosomes were suppressed in KKAy mice as compared to control. Interestingly, incubation with mTOR inhibitor rapamycin increased in the rate of LTR-positive autolysosomes in hepatocytes from KKAy mice and suppressed p62 accumulation in the liver from KKAy mice which correlated to an increase in the V-ATPase subunits expression. These results indicate that down-regulation of V-ATPase due to hepatic steatosis causes autophagic dysfunction via disruption of lysosomal and autophagic acidification. Moreover, activation of mTOR plays a pivotal role on dysregulation of lysosomal and autophagic acidification by modulation of V-ATPase expression and could therefore be a useful therapeutic target to ameliorate dysfunction of autophagy in NAFLD. - Highlights: • Hepatic steatosis causes accumulation of autophagic vesicles in hepatocytes. • Hepatic steatosis disturbs

  16. mTOR and the health benefits of exercise.

    Science.gov (United States)

    Watson, Kurt; Baar, Keith

    2014-12-01

    Exercise is the greatest physiological stress that our bodies experience. For example, during maximal endurance exercise in elite athlete's cardiac output can increase up to 8-fold and the working muscles receive 21-times more blood each minute than at rest. Given the physiological stress associated with exercise and the adaptations that occur to handle this stress, it is not surprising that exercise training is known to prevent or effectively treat a multitude of degenerative conditions including cardiovascular disease, cancer, diabetes, depression, Alzheimer's disease, Parkinson's disease, and many others. Many of the health benefits of exercise are mediated by the mammalian/mechanistic target of rapamycin (mTOR), either in complex 1 or 2, not only within the working muscle, but also in distant tissues such as fat, liver, and brain. This review will discuss how exercise activates mTOR in diverse tissues and the ways that mTOR is important in the adaptive response that makes us bigger, stronger, and healthier as a result of exercise. Copyright © 2014 Elsevier Ltd. All rights reserved.

  17. Ketamine Exhibits Different Neuroanatomical Profile After Mammalian Target of Rapamycin Inhibition in the Prefrontal Cortex: the Role of Inflammation and Oxidative Stress.

    Science.gov (United States)

    Abelaira, Helena M; Réus, Gislaine Z; Ignácio, Zuleide M; Dos Santos, Maria Augusta B; de Moura, Airam B; Matos, Danyela; Demo, Júlia P; da Silva, Júlia B I; Danielski, Lucineia G; Petronilho, Fabricia; Carvalho, André F; Quevedo, João

    2017-09-01

    Studies indicated that mammalian target of rapamycin (mTOR), oxidative stress, and inflammation are involved in the pathophysiology of major depressive disorder (MDD). Ketamine, an N-methyl-D-aspartate (NMDA) receptor antagonist, has been identified as a novel MDD therapy; however, the antidepressant mechanism is not fully understood. In addition, the effects of ketamine after mTOR inhibition have not been fully investigated. In the present study, we examined the behavioral and biochemical effects of ketamine in the prefrontal cortex (PFC), hippocampus, amygdala, and nucleus accumbens after inhibition of mTOR signaling in the PFC. Male adult Wistar rats received pharmacological mTOR inhibitor, rapamycin (0.2 nmol) or vehicle into the PFC and then a single dose of ketamine (15 mg/kg, i.p.). Immobility was assessed in forced swimming tests, and then oxidative stress parameters and inflammatory markers were evaluated in the brain and periphery. mTOR activation in the PFC was essential to ketamine's antidepressant-like effects. Ketamine increased lipid damage in the PFC, hippocampus, and amygdala. Protein carbonyl was elevated in the PFC, amygdala, and NAc after ketamine administration. Ketamine also increased nitrite/nitrate in the PFC, hippocampus, amygdala, and NAc. Myeloperoxidase activity increased in the hippocampus and NAc after ketamine administration. The activities of superoxide dismutase and catalase were reduced after ketamine administration in all brain areas studied. Inhibition of mTOR signaling pathways by rapamycin in the PFC was required to protect against oxidative stress by reducing damage and increasing antioxidant enzymes. Finally, the TNF-α level was increased in serum by ketamine; however, the rapamycin plus treatment group was not able to block this increase. Activation of mTOR in the PFC is involved in the antidepressant-like effects of ketamine; however, the inhibition of this pathway was able to protect certain brain areas against

  18. Clustered, Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9-coupled Affinity Purification/Mass Spectrometry Analysis Revealed a Novel Role of Neurofibromin in mTOR Signaling.

    Science.gov (United States)

    Li, Xu; Gao, Min; Choi, Jong Min; Kim, Beom-Jun; Zhou, Mao-Tian; Chen, Zhen; Jain, Antrix N; Jung, Sung Yun; Yuan, Jingsong; Wang, Wenqi; Wang, Yi; Chen, Junjie

    2017-04-01

    Neurofibromin (NF1) is a well known tumor suppressor that is commonly mutated in cancer patients. It physically interacts with RAS and negatively regulates RAS GTPase activity. Despite the importance of NF1 in cancer, a high quality endogenous NF1 interactome has yet to be established. In this study, we combined c lustered, r egularly i nterspaced s hort p alindromic r epeats (CRISPR)/Cas9-mediated gene knock-out technology with affinity purification using antibodies against endogenous proteins, followed by mass spectrometry analysis, to sensitively and accurately detect NF1 protein-protein interactions in unaltered in vivo settings. Using this system, we analyzed endogenous NF1-associated protein complexes and identified 49 high-confidence candidate interaction proteins, including RAS and other functionally relevant proteins. Through functional validation, we found that NF1 negatively regulates mechanistic target of rapamycin signaling (mTOR) in a LAMTOR1-dependent manner. In addition, the cell growth and survival of NF1-deficient cells have become dependent on hyperactivation of the mTOR pathway, and the tumorigenic properties of these cells have become dependent on LAMTOR1. Taken together, our findings may provide novel insights into therapeutic approaches targeting NF1-deficient tumors. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  19. Low-dose rapamycin reduces kidney volume angiomyolipomas and prevents the loss of renal function in a patient with tuberous sclerosis complex.

    Science.gov (United States)

    Peces, Ramón; Peces, Carlos; Cuesta-López, Emilio; Pérez-Dueñas, Virginia; Vega-Cabrera, Cristina; Azorín, Sebastián; Selgas, Rafael

    2010-11-01

    Tuberous sclerosis complex (TSC) is caused by constitutively activated mammalian target of rapamycin (mTOR) resulting in non-malignant tumours of several organs including renal angiomyolipomas (AMLs). AMLs may originate renal failure, hypertension and spontaneous life-threatening bleeding. Recent reports suggest a possible beneficial role of the mTOR inhibitor rapamycin for TSC. However, safety and efficiency of rapamycin in TSC patients as an anti-proliferative agent are still undefined. A 40-year-old man with sporadic TSC and a history of spontaneous bleeding from his left kidney AMLs received low-dose rapamycin for 12 months, and this was associated with a reduction in bilateral kidney AML volume, stabilization and even improvement of renal function. There was also a reduction of facial angiofibromas, improvement of blood pressure control and absence of AML bleeding over this time period. Brain lesion images remained stable, and no significant rapamycin-associated side effects were noted. To the best of our knowledge, this is the first report of a case of reduction in renal AML volume together with preservation of renal function in a patient with TSC receiving low-dose rapamycin. These data suggest that it could be the result of the anti-angiogenic, anti-fibrotic and anti-proliferative effects of rapamycin.

  20. The integrated role of ACh, ERK and mTOR in the mechanisms of hippocampal inhibitory avoidance memory.

    Science.gov (United States)

    Giovannini, Maria Grazia; Lana, Daniele; Pepeu, Giancarlo

    2015-03-01

    The purpose of this review is to summarize the present knowledge on the interplay among the cholinergic system, Extracellular signal-Regulated Kinase (ERK) and Mammalian Target of Rapamycin (mTOR) pathways in the development of short and long term memories during the acquisition and recall of the step-down inhibitory avoidance in the hippocampus. The step-down inhibitory avoidance is a form of associative learning that is acquired in a relatively simple one-trial test through several sensorial inputs. Inhibitory avoidance depends on the integrated activity of hippocampal CA1 and other brain areas. Recall can be performed at different times after acquisition, thus allowing for the study of both short and long term memory. Among the many neurotransmitter systems involved, the cholinergic neurons that originate in the basal forebrain and project to the hippocampus are of crucial importance in inhibitory avoidance processes. Acetylcholine released from cholinergic fibers during acquisition and/or recall of behavioural tasks activates muscarinic and nicotinic acetylcholine receptors and brings about a long-lasting potentiation of the postsynaptic membrane followed by downstream activation of intracellular pathway (ERK, among others) that create conditions favourable for neuronal plasticity. ERK appears to be salient not only in long term memory, but also in the molecular mechanisms underlying short term memory formation in the hippocampus. Since ERK can function as a biochemical coincidence detector in response to extracellular signals in neurons, the activation of ERK-dependent downstream effectors is determined, in part, by the duration of ERK phosphorylation itself. Long term memories require protein synthesis, that in the synapto-dendritic compartment represents a direct mechanism that can produce rapid changes in protein content in response to synaptic activity. mTOR in the brain regulates protein translation in response to neuronal activity, thereby modulating

  1. Rictor/mammalian target of rapamycin complex 2 promotes macrophage activation and kidney fibrosis.

    Science.gov (United States)

    Ren, Jiafa; Li, Jianzhong; Feng, Ye; Shu, Bingyan; Gui, Yuan; Wei, Wei; He, Weichun; Yang, Junwei; Dai, Chunsun

    2017-08-01

    Mammalian target of rapamycin (mTOR) signalling controls many essential cellular functions. However, the role of Rictor/mTOR complex 2 (mTORC2) in regulating macrophage activation and kidney fibrosis remains largely unknown. We report here that Rictor/mTORC2 was activated in macrophages from the fibrotic kidneys of mice. Ablation of Rictor in macrophages reduced kidney fibrosis, inflammatory cell accumulation, macrophage proliferation and polarization after unilateral ureter obstruction or ischaemia/reperfusion injury. In bone marrow-derived macrophages (BMMs), deletion of Rictor or blockade of protein kinase Cα inhibited cell migration. Additionally, deletion of Rictor or blockade of Akt abolished interleukin-4-stimulated or transforming growth factor (TGF)-β1-stimulated macrophage M2 polarization. Furthermore, deletion of Rictor downregulated TGF-β1-stimulated upregulation of multiple profibrotic cytokines, including platelet-derived growth factor, vascular endothelial growth factor and connective tissue growth factor, in BMMs. Conditioned medium from TGF-β1-pretreated Rictor-/- macrophages stimulated fibroblast activation less efficiently than that from TGF-β1-pretreated Rictor+/+ macrophages. These results demonstrate that Rictor/mTORC2 signalling can promote macrophage activation and kidney fibrosis. Targeting this signalling pathway in macrophages may shine light on ways to protect against kidney fibrosis in patients with chronic kidney diseases. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

  2. OP16, a novel ent-kaurene diterpenoid, potentiates the antitumor effect of rapamycin by inhibiting rapamycin-induced feedback activation of Akt signaling in esophageal squamous cell carcinoma.

    Science.gov (United States)

    Peng, Ke-Zheng; Ke, Yu; Zhao, Qi; Tian, Fei; Liu, Hong-Min; Hou, Guiqin; Lu, Zhaoming

    2017-09-15

    Hyperactivation of mTOR signaling pathway has been viewed as a significant molecular pathogenesis of cancer. However, inhibition of mTOR by rapamycin and its analogs could induce numerous negative feedback loops to attenuate their therapeutic efficacy. As a traditional Chinese herbal medicine, Rabdosia rubescens has been used to treat esophageal squamous cell carcinoma (ESCC) for hundreds of years, and its major effective component is oridonin. Here we reported that OP16, a novel analog of oridonin, showed potent inhibition of cell proliferation and Akt phosphorylation in ESCC cells. The combination of OP16 and rapamycin possesses synergistic anti-proliferative and pro-apoptotic effects both in ESCC cells and ESCC xenografts, and no obvious adverse effect was observed in vivo. Mechanistic analysis revealed that OP16 could inhibit rapamycin-induced Akt activation through the p70S6K-mediated negative feedback loops, and the combination of OP16 and rapamycin was more effective in activating caspase-dependent apoptotic signaling cascade. This study supports the combined use of OP16 with rapamycin as a feasible and effective therapeutic approach for future treatment of ESCC. Copyright © 2017 Elsevier Inc. All rights reserved.

  3. The Mammalian Target of Rapamycin and DNA methyltransferase 1 axis mediates vascular endothelial dysfunction in response to disturbed flow.

    Science.gov (United States)

    Zhang, Yun-Peng; Huang, Yi-Tao; Huang, Tse-Shun; Pang, Wei; Zhu, Juan-Juan; Liu, Yue-Feng; Tang, Run-Ze; Zhao, Chuan-Rong; Yao, Wei-Juan; Li, Yi-Shuan; Chien, Shu; Zhou, Jing

    2017-11-08

    The earliest atherosclerotic lesions preferentially develop in arterial regions experienced disturbed blood flow, which induces endothelial expression of pro-atherogenic genes and the subsequent endothelial dysfunction. Our previous study has demonstrated an up-regulation of DNA methyltransferase 1 (DNMT1) and a global hypermethylation in vascular endothelium subjected to disturbed flow. Here, we determined that DNMT1-specific inhibition in arterial wall ameliorates the disturbed flow-induced atherosclerosis through, at least in part, targeting cell cycle regulator cyclin A and connective tissue growth factor (CTGF). We identified the signaling pathways mediating the flow-induction of DNMT1. Inhibition of the mammalian target of rapamycin (mTOR) suppressed the DNMT1 up-regulation both in vitro and in vivo. Together, our results demonstrate that disturbed flow influences endothelial function and induces atherosclerosis in an mTOR/DNMT1-dependent manner. The conclusions obtained from this study might facilitate further evaluation of the epigenetic regulation of endothelial function during the pathological development of atherosclerosis and offer novel prevention and therapeutic targets of this disease.

  4. Tomato FK506 Binding Protein 12KD (FKBP12 mediates the interaction between rapamycin and Target of Rapamycin (TOR

    Directory of Open Access Journals (Sweden)

    Fangjie Xiong

    2016-11-01

    Full Text Available Target of Rapamycin (TOR signaling is an important regulator in multiple organisms including yeast, plants and animals. However, the TOR signaling in plants is much less understood as compared to that in yeast and animals. TOR kinase can be efficiently suppressed by rapamycin in the presence of functional FK506 Binding Protein 12KD (FKBP12 in yeast and animals. In most examined higher plants rapamycin fails to inhibit TOR kinase due to the non-functional FKBP12. Here we find that tomato plants showed obvious growth inhibition when treated with rapamycin and the inhibitory phenotype is similar to suppression of TOR causing by active-site TOR inhibitors (asTORis such as KU63794, AZD8055 and Torin1. The chemical genetic assays using TOR inhibitors and heterologous expressing SlFKBP12 in Arabidopsis indicated that the TOR signaling is functional in tomato. The protein gel shifting and TOR inhibitors combination assays showed that SlFKBP12 can mediate the interaction between rapamycin and TOR. Furthermore, comparative expression profiling analysis between treatments with rapamycin and KU63794 identified highly overlapped Differentially Expressed Genes (DEGs which are involved in many anabolic and catabolic processes, such as photosynthesis, cell wall restructuring, and senescence in tomato. These observations suggest that SlFFBP12 is functional in tomato. The results provided basic information of TOR signaling in tomato, and also some new insights into how TOR controls plant growth and development through reprogramming the transcription profiles

  5. Analysis list: Mtor [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Mtor + mm9 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/target/Mtor.1.tsv http://dbarchive.bioscience...dbc.jp/kyushu-u/mm9/target/Mtor.5.tsv http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/tar...get/Mtor.10.tsv http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/colo/Mtor..tsv http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/colo/.gml ...

  6. Sustained activation of the mammalian target of rapamycin nutrient sensing pathway is associated with hepatic insulin resistance, but not with steatosis, in mice

    NARCIS (Netherlands)

    Korsheninnikova, E.; van der Zon, G. C. M.; Voshol, P. J.; Janssen, G. M.; Havekes, L. M.; Grefhorst, A.; Kuipers, F.; Reijngoud, D. -J.; Romijn, J. A.; Ouwens, D. M.; Maassen, J. A.

    2006-01-01

    Aims/hypothesis Activation of nutrient sensing through mammalian target of rapamycin (mTOR) has been linked to the pathogenesis of insulin resistance. We examined activation of mTOR-signalling in relation to insulin resistance and hepatic steatosis in mice. Materials and methods Chronic hepatic

  7. Topical rapamycin as a treatment for fibrofolliculomas in Birt-Hogg-Dubé syndrome: a double-blind placebo-controlled randomized split-face trial.

    Directory of Open Access Journals (Sweden)

    Lieke M C Gijezen

    Full Text Available Birt-Hogg-Dubé syndrome (BHD is a rare autosomal dominant disorder characterised by the occurrence of benign, mostly facial, skin tumours called fibrofolliculomas, multiple lung cysts, spontaneous pneumothorax and an increased renal cancer risk. Current treatments for fibrofolliculomas have high rates of recurrence and carry a risk of complications. It would be desirable to have a treatment that could prevent fibrofolliculomas from growing. Animal models of BHD have previously shown deregulation of mammalian target of rapamycin (mTOR. Topical use of the mTOR inhibitor rapamycin is an effective treatment for the skin tumours (angiofibromas in tuberous sclerosis complex, which is also characterised by mTOR deregulation. In this study we aimed to determine if topical rapamycin is also an effective treatment for fibrofolliculomas in BHD.We performed a double blinded, randomised, facial left-right controlled trial of topical rapamycin 0.1% versus placebo in 19 BHD patients. Trial duration was 6 months. The primary outcome was cosmetic improvement as measured by doctors and patients. Changes in fibrofolliculoma number and size were also measured, as was occurrence of side effects.No change in cosmetic status of fibrofolliculomas was reported in the majority of cases for the rapamycin treated (79% by doctors, 53% by patients as well as the placebo treated facial sides (both 74%. No significant differences between rapamycin and placebo treated facial halves were observed (p = 1.000 for doctors opinion, p = 0.344 for patients opinion. No significant difference in fibrofolliculoma number or change in size of the fibrofolliculomas was seen after 6 months. Side effects occurred more often after rapamycin treatment (68% of patients than after placebo (58% of patients; p = 0.625. A burning sensation, erythema, itching and dryness were most frequently reported.This study provides no evidence that treatment of fibrofolliculomas with topical

  8. Salidroside alleviates cachexia symptoms in mouse models of cancer cachexia via activating mTOR signalling.

    Science.gov (United States)

    Chen, Xiangzheng; Wu, Yangping; Yang, Tinghan; Wei, Mingtian; Wang, Yuxi; Deng, Xiangbing; Shen, Congcong; Li, Wenting; Zhang, Hang; Xu, Weiyong; Gou, Lantu; Zeng, Yong; Zhang, Yonghui; Wang, Ziqiang; Yang, Jinliang

    2016-05-01

    Cachexia has a devastating impact on survival and quality of life for many cancer patients and contributes to nearly one-third of all cancer deaths; also, it is associated with poor responses to chemotherapy and survival. A better understanding of the underlying mechanisms of cancer-associated cachexia (CAC), coupled with effective therapeutic approaches, will improve management of progressive functional impairment in cancer patients. Salidroside, a phenylpropanoid glycoside in Rhodiola rosea L, has been reported to possess potential anti-fatigue, anti-ageing, and anti-Alzheimer's disease properties. It is widely consumed as a nutritional supplement, but its effects on CAC and the possible mechanism remain a mystery. In the murine models of cachexia induced by CT-26 and Lewis lung carcinoma (LLC) tumour, respectively, main features of CAC were determined after treatment of salidroside or chemotherapy. In vitro experiments were performed using murine C2C12 myotubes, which were treated by tumour necrosis factor-α. Levels of several critical muscle-related signal proteins such as mammalian target of rapamycin (mTOR), p-mTOR, and myosin heavy chain (MyHC) were examined using western blot both in vitro and in vivo. In the present study, we showed the exciting effect of salidroside on the treatment of CAC. In CT-26 and LLC models, respectively, salidroside treatment could effectively preserve the tumour-free body weight, decrease loss of adipose and gastrocnemius muscles, alleviate tumour burden, and prolong their survival time. Additionally, in combined chemotherapy, salidroside could synergistically enhance the anti-tumour activity of cisplatin, especially decreased or eliminated chemotherapy-induced cachexia. Further analysis demonstrated that salidroside could significantly increase expression of mTOR, p-mTOR, and MyHC in gastrocnemius muscle. Also, results in vitro showed that salidroside could not only obviously increase mTOR, p-mTOR, and MyHC expression in C2C12

  9. mTOR complex 1: a key player in neuroadaptations induced by drugs of abuse.

    Science.gov (United States)

    Neasta, Jeremie; Barak, Segev; Hamida, Sami Ben; Ron, Dorit

    2014-07-01

    The mammalian (or mechanistic) target of rapamycin (mTOR) complex 1 (mTORC1) is a serine and threonine kinase that regulates cell growth, survival, and proliferation. mTORC1 is a master controller of the translation of a subset of mRNAs. In the central nervous system mTORC1 plays a crucial role in mechanisms underlying learning and memory by controlling synaptic protein synthesis. Here, we review recent evidence suggesting that the mTORC1 signaling pathway promotes neuroadaptations following exposure to a diverse group of drugs of abuse including stimulants, cannabinoids, opiates, and alcohol. We further describe potential molecular mechanisms by which drug-induced mTORC1 activation may alter brain functions. Finally, we propose that mTORC1 is a focal point shared by drugs of abuse to mediate drug-related behaviors such as reward seeking and excessive drug intake, and offer future directions to decipher the contribution of the kinase to mechanisms underlying addiction. Recent studies suggesting that exposure to diverse classes of drugs of abuse as well as exposure to drug-associated memories lead to mTORC1 kinase activation in the limbic system. In turn, mTORC1 controls the onset and the maintenance of pathological neuroadaptions that underlie several features of drug addiction such as drug seeking and relapse. Therefore, we propose that targeting mTORC1 and its effectors is a promising strategy to treat drug disorders. © 2014 International Society for Neurochemistry.

  10. Retraction: "Down-regulation of Notch-1 and Jagged-1 inhibits prostate cancer cell growth, migration and invasion, and induces apoptosis via inactivation of Akt, mTOR, and NF-κB signaling pathways" by Wang et al.

    Science.gov (United States)

    2016-08-01

    The above article, published online on January 5, 2010 in Wiley Online Library (wileyonlinelibrary.com), has been retracted by agreement between the journal Editor in Chief, Gary S. Stein, and Wiley Periodicals, Inc. The retraction has been agreed following an investigation from Wayne State University involving the first author and the corresponding author that found Figure 5A to be inappropriately manipulated. REFERENCE Wang Z, Li Y, Banerjee S, Kong D, Ahmad A, Nogueira V, Hay N, Sarkar FH. 2010. Down-regulation of Notch-1 and Jagged-1 inhibits prostate cancer cell growth, migration and invasion, and induces apoptosis via inactivation of Akt, mTOR, and NF-κB signaling pathways. J Cell Biochem 109:726-736; doi: 10.1002/jcb.22451. © 2016 Wiley Periodicals, Inc.

  11. Cord Blood Cells Responses to IL2, IL7 and IL15 Cytokines for mTOR Expression.

    Science.gov (United States)

    Mohammadian, Anahita; Naderali, Elahe; Mohammadi, Seyedeh Momeneh; Movasaghpour, Aliakbar; Valipour, Behnaz; Nouri, Mohammad; Nozad Charoudeh, Hojjatollah

    2017-04-01

    Purpose: Mammalian target of rapamycin (mTOR)is important in hematopoiesis and affect cell growth,differentiation and survival. Although previous studies were identified the effect of cytokines on the mononuclear cells development however the cytokines effect on mTOR in cord blood mononuclear cells was unclear. The aim of this study was to evaluate mTOR expression in cord blood mononuclear and cord blood stem cells (CD34+ cells) in culture conditions for lymphoid cell development. Methods: Isolation of The mononuclear cells (MNCs) from umbilical cord blood were done with use of Ficollpaque density gradient. We evaluated cultured cord blood mononuclear and CD34+ cells in presece of IL2, IL7 and IL15 at distinct time points during 21 days by using flow cytometry. In this study, we presented the role of IL2, IL7 and IL15 on the expression of mTOR in cord blood cells. Results: mTOR expression were increased in peresence of IL2, IL7 and IL15 in day 14 and afterword reduced. However in persence of IL2 and IL15 expression of mTOR significantly reduced. mTOR expression in CD34+ cells decreased significantly from day7 to day 21 in culture. Conclusion: cytokines play important role in mTOR expression during hematopoiesis and development of cord blood mononuclear cells.

  12. Cord Blood Cells Responses to IL2, IL7 and IL15 Cytokines for mTOR Expression

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    Anahita Mohammadian

    2017-04-01

    Full Text Available Purpose: Mammalian target of rapamycin (mTORis important in hematopoiesis and affect cell growth,differentiation and survival. Although previous studies were identified the effect of cytokines on the mononuclear cells development however the cytokines effect on mTOR in cord blood mononuclear cells was unclear. The aim of this study was to evaluate mTOR expression in cord blood mononuclear and cord blood stem cells (CD34+ cells in culture conditions for lymphoid cell development. Methods: Isolation of The mononuclear cells (MNCs from umbilical cord blood were done with use of Ficollpaque density gradient. We evaluated cultured cord blood mononuclear and CD34+ cells in presece of IL2, IL7 and IL15 at distinct time points during 21 days by using flow cytometry. In this study, we presented the role of IL2, IL7 and IL15 on the expression of mTOR in cord blood cells. Results: mTOR expression were increased in peresence of IL2, IL7 and IL15 in day 14 and afterword reduced. However in persence of IL2 and IL15 expression of mTOR significantly reduced. mTOR expression in CD34+ cells decreased significantly from day7 to day 21 in culture. Conclusion: cytokines play important role in mTOR expression during hematopoiesis and development of cord blood mononuclear cells.

  13. Rapamycin inhibits the growth and muscle-sparing effects of clenbuterol.

    Science.gov (United States)

    Kline, William O; Panaro, Frank J; Yang, Hayung; Bodine, Sue C

    2007-02-01

    Clenbuterol and other beta2-adrenergic agonists are effective at inducing muscle growth and attenuating muscle atrophy through unknown mechanisms. This study tested the hypothesis that clenbuterol-induced growth and muscle sparing is mediated through the activation of Akt and mammalian target of rapamycin (mTOR) signaling pathways. Clenbuterol was administered to normal weight-bearing adult rats to examine the growth-inducing effects and to adult rats undergoing muscle atrophy as the result of hindlimb suspension or denervation to examine the muscle-sparing effects. The pharmacological inhibitor rapamycin was administered in combination with clenbuterol in vivo to determine whether activation of mTOR was involved in mediating the effects of clenbuterol. Clenbuterol administration increased the phosphorylation status of PKB/Akt, S6 kinase 1/p70(s6k), and eukaryotic initiation factor 4E binding protein 1/PHAS-1. Clenbuterol treatment induced growth by 27-41% in normal rats and attenuated muscle loss during hindlimb suspension by 10-20%. Rapamycin treatment resulted in a 37-97% suppression of clenbuterol-induced growth and a 100% reduction of the muscle-sparing effect. In contrast, rapamycin was unable to block the muscle-sparing effects of clenbuterol after denervation. Clenbuterol was also shown to suppress the expression of the MuRF1 and MAFbx transcripts in muscles from normal, denervated, and hindlimb-suspended rats. These results demonstrate that the effects of clenbuterol are mediated, in part, through the activation of Akt and mTOR signaling pathways.

  14. Differential Role of Rapamycin and Torin/KU63794 in Inflammatory Response of 264.7 RAW Macrophages Stimulated by CA-MRSA

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    Rebekah K. H. Shappley

    2014-01-01

    Full Text Available Background. Rapamycin suppresses the RAW264.7 macrophage mediated inflammatory response but in lower doses induces it. In the present study, we tested the suppression of the inflammatory response in the presence of mTOR 1 and 2 inhibitors, Torin and KU63794. Methods. RAW264.7 cells were stimulated for 18 hrs with 106 to 107 CFU/mL inocula of community-acquired- (CA- MRSA isolate, USA400 strain MW2, in the presence of Vancomycin. Then, in sequential experiments, we added Torin, KU63794, and Rapamycin alone and in various combinations. Supernatants were collected and assayed for TNF, IL-1, IL-6, INF, and NO. Results. Rapamycin induces 10–20% of the inflammatory cascade at dose of 0.1 ng/mL and suppresses it by 60% at dose of 10 ng/mL. The induction is abolished in the presence of Torin KU63794. Torin and KU63794 are consistently suppressing cytokine production 50–60%. Conclusions. There is a differential response between Rapamycin (mTOR-1 inhibitor and Torin KU63794 (mTOR 1 and 2 inhibitors. Torin and KU63794 exhibit a dose related suppression. Rapamycin exhibits a significant induction-suppression biphasic response. Knowledge of such response may allow manipulation of the septic inflammatory cascade for clinical advantages.

  15. Sepsis and mechnaical ventilation restrain translation initiation in skeletal muscle by inducing AMPK-associated TSC[2] restriction of mTOR signaling in pigs

    Science.gov (United States)

    In skeletal muscle, AMP-activated protein kinase (AMPK) acts as a cellular energy sensor of AMP: ATP and modulates translation by repressing mammalian target of rapamycin (mTOR) activation. Endotoxin (LPS)-induced sepsis reduces muscle protein synthesis by blunting translation initiation. We hypothe...

  16. Differential effects of chronic overload-induced muscle hypertrophy on mTOR and MAPK signaling pathways in adult and aged rats

    Science.gov (United States)

    We examined activation of the mammalian target of rapamycin (mTOR) and mitogen-activated protein kinase (MAPK) signaling pathways in adult (Y; 6 mo old; n = 16) and aged (O; 30 mo old; n = 16) male rats (Fischer 344 x Brown Norway) subjected to chronic overload-induced muscle hypertrophy of the plan...

  17. Leptin-induced mTOR activation defines a specific molecular and transcriptional signature controlling CD4+ effector T cell responses

    DEFF Research Database (Denmark)

    Procaccini, Claudio; De Rosa, Veronica; Galgani, Mario

    2012-01-01

    The sensing by T cells of metabolic and energetic changes in the microenvironment can determine the differentiation, maturation, and activation of these cells. Although it is known that mammalian target of rapamycin (mTOR) gauges nutritonal and energetic signals in the extracellular milieu, it is...

  18. Recent Advances in Mammalian Target of Rapamycin Inhibitor Use in Heart and Lung Transplantation.

    Science.gov (United States)

    Fine, Nowell M; Kushwaha, Sudhir S

    2016-12-01

    The mammalian target of rapamycin (mTOR) inhibitors sirolimus and everolimus are increasingly used in cardiothoracic transplantation. Several recent clinical trials have demonstrated their efficacy in combination with reduced cyclosporine dosing in de novo heart transplant recipients, in particular with everolimus. A number of other studies have demonstrated their efficacy for improving renal function and reducing calcineurin inhibitor use, attenuating cardiac allograft vasculopathy progression and reducing cytomegalovirus infections in maintenance heart transplant populations. A growing body of literature, including a small number of clinical trials, now describes the use mTOR inhibitors in lung transplant recipients. The benefits in this population include improved lung and renal function in limited studies. Considerably less evidence is available in pediatric heart transplantation, though similar indications in the maintenance therapy population have been described. The benefits of mTOR inhibitors must be weighed against the increased risk of adverse events and drug intolerance compared with other primary immunosuppressants, and discontinuation rates are particularly high in lung transplant recipients. The risks of surgical wound healing complications in transplant recipients receiving mTOR inhibitors previously or actively supported by mechanical circulatory support devices remains poorly described in the current literature. The current role and recent evidence for mTOR inhibitor use in heart and lung transplantation is examined in this review.

  19. The ever-evolving role of mTOR in translation.

    Science.gov (United States)

    Fonseca, Bruno D; Smith, Ewan M; Yelle, Nicolas; Alain, Tommy; Bushell, Martin; Pause, Arnim

    2014-12-01

    Control of translation allows for the production of stoichiometric levels of each protein in the cell. Attaining such a level of fine-tuned regulation of protein production requires the coordinated temporal and spatial control of numerous cellular signalling cascades impinging on the various components of the translational machinery. Foremost among these is the mTOR signalling pathway. The mTOR pathway regulates both the initiation and elongation steps of protein synthesis through the phosphorylation of numerous translation factors, while simultaneously ensuring adequate folding of nascent polypeptides through co-translational degradation of misfolded proteins. Perhaps most remarkably, mTOR is also a key regulator of the synthesis of ribosomal proteins and translation factors themselves. Two seminal studies have recently shown in translatome analysis that the mTOR pathway preferentially regulates the translation of mRNAs encoding ribosomal proteins and translation factors. Therefore, the role of the mTOR pathway in the control of protein synthesis extends far beyond immediate translational control. By controlling ribosome production (and ultimately ribosome availability), mTOR is a master long-term controller of protein synthesis. Herein, we review the literature spanning the early discoveries of mTOR on translation to the latest advances in our understanding of how the mTOR pathway controls the synthesis of ribosomal proteins. Crown Copyright © 2014. Published by Elsevier Ltd. All rights reserved.

  20. Negative Effects of Chronic Rapamycin Treatment on Behavior in a Mouse Model of Fragile X Syndrome

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    Rachel M. Saré

    2018-01-01

    Full Text Available Fragile X syndrome (FXS, the most common form of inherited intellectual disability, is also highly associated with autism spectrum disorders (ASD. It is caused by expansion of a CGG repeat sequence on the X chromosome resulting in silencing of the FMR1 gene. This is modeled in the mouse by deletion of Fmr1 (Fmr1 KO. Fmr1 KO mice recapitulate many of the behavioral features of the disorder including seizure susceptibility, hyperactivity, impaired social behavior, sleep problems, and learning and memory deficits. The mammalian target of rapamycin pathway (mTORC1 is upregulated in Fmr1 KO mice and is thought to be important for the pathogenesis of this disorder. We treated Fmr1 KO mice chronically with an mTORC1 inhibitor, rapamycin, to determine if rapamycin treatment could reverse behavioral phenotypes. We performed open field, zero maze, social behavior, sleep, passive avoidance, and audiogenic seizure testing. We found that pS6 was upregulated in Fmr1 KO mice and normalized by rapamycin treatment, but, except for an anxiogenic effect, it did not reverse any of the behavioral phenotypes examined. In fact, rapamycin treatment had an adverse effect on sleep and social behavior in both control and Fmr1 KO mice. These results suggest that targeting the mTOR pathway in FXS is not a good treatment strategy and that other pathways should be considered.

  1. E-selectin targeted immunoliposomes for rapamycin delivery to activated endothelial cells.

    Science.gov (United States)

    Gholizadeh, Shima; Visweswaran, Ganesh Ram R; Storm, Gert; Hennink, Wim E; Kamps, Jan A A M; Kok, Robbert J

    2017-10-13

    Activated endothelial cells play a pivotal role in the pathology of inflammatory disorders and thus present a target for therapeutic intervention by drugs that intervene in inflammatory signaling cascades, such as rapamycin (mammalian target of rapamycin (mTOR) inhibitor). In this study we developed anti-E-selectin immunoliposomes for targeted delivery to E-selectin over-expressing tumor necrosis factor-α (TNF-α) activated endothelial cells. Liposomes composed of 1,2-dipalmitoyl-sn-glycero-3.;hosphocholine (DPPC), Cholesterol, and 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)-2000]-maleimide (DSPE-PEG-Mal) were loaded with rapamycin via lipid film hydration, after which they were further functionalized by coupling N-succinimidyl-S-acetylthioacetate (SATA)-modified mouse anti human E-selectin antibodies to the distal ends of the maleimidyl (Mal)-PEG groups. In cell binding assays, these immunoliposomes bound specifically to TNF-α activated endothelial cells. Upon internalization, rapamycin loaded immunoliposomes inhibited proliferation and migration of endothelial cells, as well as expression of inflammatory mediators. Our findings demonstrate that rapamycin-loaded immunoliposomes can specifically inhibit inflammatory responses in inflamed endothelial cells. Copyright © 2017. Published by Elsevier B.V.

  2. Hyperactive mTOR signals in the proopiomelanocortin-expressing hippocampal neurons cause age-dependent epilepsy and premature death in mice.

    Science.gov (United States)

    Matsushita, Yuki; Sakai, Yasunari; Shimmura, Mitsunori; Shigeto, Hiroshi; Nishio, Miki; Akamine, Satoshi; Sanefuji, Masafumi; Ishizaki, Yoshito; Torisu, Hiroyuki; Nakabeppu, Yusaku; Suzuki, Akira; Takada, Hidetoshi; Hara, Toshiro

    2016-03-10

    Epilepsy is a frequent comorbidity in patients with focal cortical dysplasia (FCD). Recent studies utilizing massive sequencing data identified subsets of genes that are associated with epilepsy and FCD. AKT and mTOR-related signals have been recently implicated in the pathogenic processes of epilepsy and FCD. To clarify the functional roles of the AKT-mTOR pathway in the hippocampal neurons, we generated conditional knockout mice harboring the deletion of Pten (Pten-cKO) in Proopiomelanocortin-expressing neurons. The Pten-cKO mice developed normally until 8 weeks of age, then presented generalized seizures at 8-10 weeks of age. Video-monitored electroencephalograms detected paroxysmal discharges emerging from the cerebral cortex and hippocampus. These mice showed progressive hypertrophy of the dentate gyrus (DG) with increased expressions of excitatory synaptic markers (Psd95, Shank3 and Homer). In contrast, the expression of inhibitory neurons (Gad67) was decreased at 6-8 weeks of age. Immunofluorescence studies revealed the abnormal sprouting of mossy fibers in the DG of the Pten-cKO mice prior to the onset of seizures. The treatment of these mice with an mTOR inhibitor rapamycin successfully prevented the development of seizures and reversed these molecular phenotypes. These data indicate that the mTOR pathway regulates hippocampal excitability in the postnatal brain.

  3. Inhibition of p70S6K does not mimic the enhancement of Akt phosphorylation by rapamycin.

    Science.gov (United States)

    Wang, Xuerong; Yue, Ping; Tao, Hui; Sun, Shi-Yong

    2017-08-01

    It has been suggested that the mTOR complex 1 (mTORC1)/p70S6K axis represses upstream PI3K/Akt signaling through phosphorylation of IRS-1 and its subsequent degradation. One potential and current model that explains Akt activation induced by the mTOR inhibitor rapamycin is the relief of mTORC1/p70S6K-mediated feedback inhibition of IRS-1/PI3K/Akt signaling, although this has not been experimentally proven. In this study, we found that chemical inhibition of p70S6K did not increase Akt phosphorylation. Surprisingly, knockdown of p70S6K even substantially inhibited Akt phosphorylation. Hence, p70S6K inhibition clearly does not mimic the activation of Akt by rapamycin. Inhibition or enforced activation of p70S6K did not affect the ability of rapamycin to increase Akt phosphorylation. Moreover, inhibition of mTORC1 with either rapamycin or raptor knockdown did not elevate IRS-1 levels, despite potently increasing Akt phosphorylation. Critically, knockdown or knockout of IRS-1 or IRS-2 failed to abolish the ability of rapamycin to increase Akt phosphorylation. Therefore, IRS-1 and IRS-2 are not essential for mediating rapamycin-induced Akt activation. Collectively, our findings suggest that Akt activation by rapamycin or mTORC1 inhibition is unlikely due to relief of p70S6K-mediated feedback inhibition of IRS-1/PI3K/Akt signaling.

  4. mTORC1 inhibitors rapamycin and metformin affect cardiovascular markers differentially in ZDF rats.

    Science.gov (United States)

    Nistala, Ravi; Raja, Ahmad; Pulakat, Lakshmi

    2017-03-01

    Mammalian target for rapamycin complex 1 (mTORC1) is a common target for the action of immunosuppressant macrolide rapamycin and glucose-lowering metformin. Inhibition of mTORC1 can exert both beneficial and detrimental effects in different pathologies. Here, we investigated the differential effects of rapamycin (1.2 mg/kg per day delivered subcutaneously for 6 weeks) and metformin (300 mg/kg per day delivered orally for 11 weeks) treatments on male Zucker diabetic fatty (ZDF) rats that mimic the cardiorenal pathology of type 2 diabetic patients and progress to insulin insufficiency. Rapamycin and metformin improved proteinuria, and rapamycin also reduced urinary gamma glutamyl transferase (GGT) indicating improvement of tubular health. Metformin reduced food and water intake, and urinary sodium and potassium, whereas rapamycin increased urinary sodium. Metformin reduced plasma alkaline phosphatase, but induced transaminitis as evidenced by significant increases in plasma AST and ALT. Metformin also induced hyperinsulinemia, but did not suppress fasting plasma glucose after ZDF rats reached 17 weeks of age, and worsened lipid profile. Rapamycin also induced mild transaminitis. Additionally, both rapamycin and metformin increased plasma uric acid and creatinine, biomarkers for cardiovascular and renal disease. These observations define how rapamycin and metformin differentially modulate metabolic profiles that regulate cardiorenal pathology in conditions of severe type 2 diabetes.

  5. Anorexigenic and Orexigenic Hormone Modulation of Mammalian Target of Rapamycin Complex 1 Activity and the Regulation of Hypothalamic Agouti-Related Protein mRNA Expression

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    Kenneth R. Watterson

    2012-03-01

    Full Text Available Activation of mammalian target of rapamycin 1 (mTORC1 by nutrients, insulin and leptin leads to appetite suppression (anorexia. Contrastingly, increased AMP-activated protein kinase (AMPK activity by ghrelin promotes appetite (orexia. However, the interplay between these mechanisms remains poorly defined. The relationship between the anorexigenic hormones, insulin and leptin, and the orexigenic hormone, ghrelin, on mTORC1 signalling was examined using S6 kinase phosphorylation as a marker for changes in mTORC1 activity in mouse hypothalamic GT1-7 cells. Additionally, the contribution of AMPK and mTORC1 signalling in relation to insulin-, leptin- and ghrelin-driven alterations to mouse hypothalamic agouti-related protein (AgRP mRNA levels was examined. Insulin and leptin increase mTORC1 activity in a phosphoinositide-3-kinase (PI3K- and protein kinase B (PKB-dependent manner, compared to vehicle controls, whereas increasing AMPK activity inhibits mTORC1 activity and blocks the actions of the anorexigenic hormones. Ghrelin mediates an AMPK-dependent decrease in mTORC1 activity and increases hypothalamic AgRP mRNA levels, the latter effect being prevented by insulin in an mTORC1-dependent manner. In conclusion, mTORC1 acts as an integration node in hypothalamic neurons for hormone-derived PI3K and AMPK signalling and mediates at least part of the assimilated output of anorexigenic and orexigenic hormone actions in the hypothalamus.

  6. Dissociation of the Pharmacological Effects of THC by mTOR Blockade

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    Puighermanal, Emma; Busquets-Garcia, Arnau; Gomis-González, Maria; Marsicano, Giovanni; Maldonado, Rafael; Ozaita, Andrés

    2013-01-01

    The potential therapeutic benefits of cannabinoid compounds have raised interest in understanding the molecular mechanisms that underlie cannabinoid-mediated effects. We previously showed that the acute amnesic-like effects of delta9-tetrahydrocannabinol (THC) were prevented by the subchronic inhibition of the mammalian target of rapamycin (mTOR) pathway. In the present study, we assess the relevance of the mTOR pathway in other acute and chronic pharmacological effects of THC. The rapamycin derivative temsirolimus, an inhibitor of the mTOR pathway approved by the Food and Drug Administration, prevents both the anxiogenic- and the amnesic-like effects produced by acute THC. In contrast, THC-induced anxiolysis, hypothermia, hypolocomotion, and antinociception are not sensitive to the mTOR inhibition. In addition, a clear tolerance to THC-induced anxiolysis, hypothermia, hypolocomotion, and antinociception was observed after chronic treatment, but not to its anxiogenic- and amnesic-like effects. Temsirolimus pre-treatment prevented the amnesic-like effects of chronic THC without affecting the downregulation of CB1 receptors (CB1R) induced by this chronic treatment. Instead, temsirolimus blockade after chronic THC cessation did not prevent the residual cognitive deficit produced by chronic THC. Using conditional knockout mice lacking CB1R in GABAergic or glutamatergic neurons, we found that GABAergic CB1Rs are mainly downregulated under chronic THC treatment conditions, and CB1–GABA–KO mice did not develop cognitive deficits after chronic THC exposure. Therefore, mTOR inhibition by temsirolimus allows the segregation of the potentially beneficial effects of cannabinoid agonists, such as the anxiolytic and antinociceptive effects, from the negative effects, such as anxiogenic- and amnesic-like responses. Altogether, these results provide new insights for targeting the endocannabinoid system in order to prevent possible side effects. PMID:23358238

  7. Allomyrina Dichotoma Larvae Regulate Food Intake and Body Weight in High Fat Diet-Induced Obese Mice Through mTOR and Mapk Signaling Pathways

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    Jongwan Kim

    2016-02-01

    Full Text Available Recent evidence has suggested that the Korean horn beetle (Allomyrina dichotoma has anti-hepatofibrotic, anti-neoplastic, and antibiotic effects and is recognized as a traditional medicine. In our previous works, Allomyrina dichotoma larvae (ADL inhibited differentiation of adipocytes both in vitro and in vivo. However, the anorexigenic and endoplasmic reticulum(ER stress-reducing effects of ADL in obesity has not been examined. In this study, we investigated the anorexigenic and ER stress-reducing effects of ADL in the hypothalamus of diet-induced obese (DIO mice. Intracerebroventricular (ICV administration of ethanol extract of ADL (ADE suggested that an antagonizing effect on ghrelin-induced feeding behavior through the mTOR and MAPK signaling pathways. Especially, ADE resulted in strong reduction of ER stress both in vitro and in vivo. These findings strongly suggest that ADE and its constituent bioactive compounds are available and valuable to use for treatment of various diseases driven by prolonged ER stress.

  8. Rapamycin has a biphasic effect on insulin sensitivity in C2C12 myotubes due to sequential disruption of mTORC1 and mTORC2

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    Lan eYe

    2012-09-01

    Full Text Available Rapamycin, an inhibitor of mTOR complex 1 (mTORC1, improves insulin sensitivity in acute studies in vitro and in vivo by disrupting a negative feedback loop mediated by S6 kinase. We find that rapamycin has a clear biphasic effect on insulin sensitivity in C2C12 myotubes, with enhanced responsiveness during the first hour that declines to almost complete insulin resistance by 24-48 hours. We and others have recently observed that chronic rapamycin treatment induces insulin resistance in rodents, at least in part due to disruption of mTORC2, an mTOR-containing complex that is not acutely sensitive to the drug. Chronic rapamycin treatment may also impair insulin action via the inhibition of mTORC1-dependent mitochondrial biogenesis and activity, which could result in a buildup of lipid intermediates that are known to trigger insulin resistance. We confirmed that rapamycin inhibits expression of PGC-1α, a key mitochondrial transcription factor, and acutely reduces respiration rate in myotubes. However, rapamycin did not stimulate phosphorylation of PKCθ, a central mediator of lipid-induced insulin resistance. Instead, we found dramatic disruption of mTORC2, which coincided with the onset of insulin resistance. Selective inhibition of mTORC1 or mTORC2 by shRNA-mediated knockdown of specific components (Raptor and Rictor, respectively confirmed that mitochondrial effects of rapamycin are mTORC1-dependent, whereas insulin resistance was recapitulated only by knockdown of mTORC2. Thus, mTORC2 disruption, rather than inhibition of mitochondria, causes insulin resistance in rapamycin-treated myotubes, and this system may serve as a useful model to understand the effects of rapamycin on mTOR signaling in vivo.

  9. Activation of the unfolded protein response in sarcoma cells treated with rapamycin or temsirolimus.

    Science.gov (United States)

    Briggs, Joseph W; Ren, Ling; Chakrabarti, Kristi R; Tsai, Yien Che; Weissman, Allan M; Hansen, Ryan J; Gustafson, Daniel L; Khan, Yousuf A; Dinman, Jonathan D; Khanna, Chand

    2017-01-01

    Activation of the unfolded protein response (UPR) in eukaryotic cells represents an evolutionarily conserved response to physiological stress. Here, we report that the mTOR inhibitors rapamycin (sirolimus) and structurally related temsirolimus are capable of inducing UPR in sarcoma cells. However, this effect appears to be distinct from the classical role for these drugs as mTOR inhibitors. Instead, we detected these compounds to be associated with ribosomes isolated from treated cells. Specifically, temsirolimus treatment resulted in protection from chemical modification of several rRNA residues previously shown to bind rapamycin in prokaryotic cells. As an application for these findings, we demonstrate maximum tumor cell growth inhibition occurring only at doses which induce UPR and which have been shown to be safely achieved in human patients. These results are significant because they challenge the paradigm for the use of these drugs as anticancer agents and reveal a connection to UPR, a conserved biological response that has been implicated in tumor growth and response to therapy. As a result, eIF2 alpha phosphorylation and Xbp-1 splicing may serve as useful biomarkers of treatment response in future clinical trials using rapamycin and rapalogs.

  10. Activation of the unfolded protein response in sarcoma cells treated with rapamycin or temsirolimus.

    Directory of Open Access Journals (Sweden)

    Joseph W Briggs

    Full Text Available Activation of the unfolded protein response (UPR in eukaryotic cells represents an evolutionarily conserved response to physiological stress. Here, we report that the mTOR inhibitors rapamycin (sirolimus and structurally related temsirolimus are capable of inducing UPR in sarcoma cells. However, this effect appears to be distinct from the classical role for these drugs as mTOR inhibitors. Instead, we detected these compounds to be associated with ribosomes isolated from treated cells. Specifically, temsirolimus treatment resulted in protection from chemical modification of several rRNA residues previously shown to bind rapamycin in prokaryotic cells. As an application for these findings, we demonstrate maximum tumor cell growth inhibition occurring only at doses which induce UPR and which have been shown to be safely achieved in human patients. These results are significant because they challenge the paradigm for the use of these drugs as anticancer agents and reveal a connection to UPR, a conserved biological response that has been implicated in tumor growth and response to therapy. As a result, eIF2 alpha phosphorylation and Xbp-1 splicing may serve as useful biomarkers of treatment response in future clinical trials using rapamycin and rapalogs.

  11. The role of 18F-FDG PET/CT imaging in patient with malignant PEComa treated with mTOR inhibitor

    Directory of Open Access Journals (Sweden)

    Sun L

    2015-07-01

    Full Text Available Lu Sun,1 Xiaorong Sun,2 Yuhui Li,3 Ligang Xing4 1School of Medicine and Life Sciences, University of Jinan-Shandong Academy of Medical Sciences, 2PET/CT Center, Department of Radiology, 3Department of Pathology, 4Department of Radiation Oncology, Shandong Cancer Hospital and Institute, Jinan, Shandong, People’s Republic of China Abstract: Malignant perivascular epithelioid cell tumor (malignant PEComa is a rare disease for which the diagnostic criteria and treatment options have not been established. Since PEComa is associated with upregulation of mammalian target of rapamycin (mTOR pathway which controls Glut-1 (glucose transporter function, increased 18F-fluorodeoxyglucose (18F-FDG uptake may indicate the over activation of mTOR pathway and may guide selectively inhibiting mTOR pathway treatment. We report a malignant PEComa patient who presented for 18F-FDG positron emission tomography/computed tomography (PET/CT restaging. The tumor had shown significant avidity on PET/CT as well as an evident response to sirolimus (rapamycin, Rapamune™ that supports the utility of mTOR inhibitors as an effective treatment for malignant PEComa. Therefore, 18F-FDG PET/CT is helpful in restaging and guiding treatment for malignant PEComa with mTOR inhibitors. Keywords: malignant perivascular epithelioid cell tumor, PEComa, mTOR inhibitor, FDG, PET/CT 

  12. 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.

    2016-01-01

    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

  13. Long-Term Alcohol-Induced Activation of Mammalian Target of Rapamycin is a Key Risk Factor of Epilepsy.

    Science.gov (United States)

    Fu, Xiaoling; Guo, Zhe; Gao, Chang; Chu, Qinying; Li, Jianhua; Ma, Hongying; Shu, Gangming

    2016-10-25

    BACKGROUND The aim of this study was to determine whether activation of mammalian target of rapamycin (mTOR) is a key epileptogenic mechanism in the development of alcohol-related seizure. MATERIAL AND METHODS C57BL/6 mice were administered 10% ethanol in drinking water for 9 weeks. Video-electroencephalography (EEG) monitoring was then used to assess seizure frequency after alcohol and rapamycin treatment. In addition, mouse neuroblastoma NG108-15 cells were treated ethanol for 3 days and subsequently treated with AKT inhibitor LY294002 for 2-12 h. The in vitro kinase assay was performed for determining mTOR activity. Western blot analysis was used to determine the expression of P-AKT, P-S6K, and P-S6. RESULTS Long-term ethanol treatment markedly increased the seizure frequency of C57/BL6 mice over time. Moreover, ethanol treatment increased the expression level of P-S6 over time. Ethanol-induced seizure can be reversed by rapamycin. In addition, the in vitro kinase assay showed mTOR activity was activated by ethanol. Compared with NG108-15 cells treated without both ethanol and LY294002, ethanol increased the expression level of P-AKT, P-S6K, and P-S6, whereas LY294002 had opposite effects on expression levels of these proteins. CONCLUSIONS Our findings indicate that long-term alcohol intake increases the risk of epilepsy via activation of mTOR signaling. Moreover, ethanol-induced mTOR activation may be dependent on the AKT-mTOR signaling pathway. The key molecules involved in AKT-mTOR signaling pathway may serve as potential targets in the treatment of epilepsy.

  14. Genome-wide association study for biomarker identification of Rapamycin and Everolimus using a lymphoblastoid cell line system

    Directory of Open Access Journals (Sweden)

    Jing eJiang

    2013-08-01

    Full Text Available The mammalian target of rapamycin (mTOR inhibitors, a set of promising potential anti-cancer agents, has shown response variability among individuals. This study aimed to identify novel biomarkers and mechanisms that might influence the response to Rapamycin and Everolimus. Genome-wide association (GWA analyses involving single nucleotide polymorphisms (SNPs, mRNA and microRNAs microarray data were assessed for association with area under the cytotoxicity dose response curve (AUC of two mTOR inhibitors in 272 human lymphoblastoid cell lines (LCLs. Integrated analysis among SNPs, expression data, microRNA data and AUC values were also performed to help select candidate genes for further functional characterization. Functional validation of candidate genes using siRNA screening in multiple cell lines followed by MTS assays for the two mTOR inhibitors were performed. We found that 16 expression probe sets (genes that overlapped between the two drugs were associated with AUC values of two mTOR inhibitors. 127 and 100 SNPs had P<10-4, while 8 and 10 SNPs had P<10-5 with Rapamycin and Everolimus AUC, respectively. Functional studies indicated that 13 genes significantly altered cell sensitivity to either one or both drugs in at least one cell line. Additionally, one microRNA, miR-10a, was significantly associated with AUC values for both drugs and was shown to repress expression of genes that were associated with AUC and desensitize cells to both drugs. In summary, this study identified genes and a microRNA that might contribute to response to mTOR inhibitors.

  15. Rapamycin inhibits CaCl2-induced thoracic aortic aneurysm formation in rats through mTOR-mediated suppression of proinflammatory mediators.

    Science.gov (United States)

    Cao, Jiumei; Wu, Qihong; Geng, Liang; Chen, Xiaonan; Shen, Weifeng; Wu, Fang; Chen, Ying

    2017-08-01

    The aim of the present study was to investigate the effect of the mammalian target of rapamycin (mTOR) signaling pathway on thoracic aortic aneurysm (TAA) development. The study used a calcium chloride (CaCl2)‑induced rat TAA model to explore the potential role of mTOR signaling pathway in the disease development. Adult male Sprague‑Dawley rats underwent the periarterial exposure of thoracic aorta to either 0.5 M CaCl2 or normal saline, and a subgroup of CaCl2‑treated rats received rapamycin 1 day prior to surgery. Without pre‑administering rapamycin, significantly enhanced phosphorylation of mTOR and expression of proinflammatory cytokines [i.e., tumor necrosis factor α (TNF‑α), interleukin 6 (IL‑6), and interleukin (IL)‑1β] were observed in the CaCl2‑treated aortic segments 2 days post‑treatment compared with the NaCl‑treated segments. At 2 weeks post‑treatment, hematoxylin and eosin and Verhoeff‑Van Gieson staining revealed aneurysmal alteration and disappearance of normal wavy elastic structures in the aortic segments exposed to CaCl2. In contrast, the CaCl2‑induced TAA formation was inhibited by pre‑administering rapamycin to CaCl2‑treated rats, which demonstrated attenuated mTOR phosphorylation and downregulation of the proinflammatory mediators (i.e., TNF‑α, IL‑6, IL‑1β, matrix metallopeptidases 2 and 9) to the control level. Further in vitro cell culture experiments using aortic smooth muscle cell (SMC) suggested that the inhibition of the mTOR signaling pathway by rapamycin could promote the differentiation of SMCs, as reflected by the reduced expression of S100A4 and osteopontin. The present study indicated that the early enhanced mTOR signaling pathway in the TAA development and mTOR inhibitor rapamycin may inhibit CaCl2‑induced TAA formation.

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

    Science.gov (United States)

    Guilbert, Cynthia; Annis, Matthew G; Dong, Zhifeng; Siegel, Peter M; Miller, Wilson H; Mann, Koren K

    2013-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Cynthia Guilbert

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

  18. Paradigmenwechsel in der Anti-Aging-Medizin: Hormesis, Target-of-Rapamycin-Komplex und erste Anti-Aging-Pillen // Paradigm Shift in Anti-Aging Medicine: Hormesis, Target of Rapamycin Complex and First Human Anti-Aging-Pills

    Directory of Open Access Journals (Sweden)

    Römmler A

    2016-01-01

    Full Text Available Studies in model organisms have shown that some drugs and lifestyle practices (calorie-restricted diets, regular exercise, e.g. can extend life and health span and protect against the onset of age-related chronic diseases by targeting physiological pathways.brA common mode of action was found via mTOR (mechanistic Target Of Rapamycin pathway signalling. This intracellular protein kinase complex plays a key role in stimulating anabolic and cell growth promoting processes, while inhibiting autophagy. On the other hand, downregulation results in antiproliferative, anticancer and intensive cell-repairing effects leading to life and health span extension and stress resistance. The mTOR complex regulates such basic cell activities and integrates signals from nutrition sensing, energy metabolism, insulin and growth factors, stress and hypoxia.brImportantly, mTOR can be inhibited by some molecules and their analogs (rapamycin, resveratrol, metformin, e.g., which are released naturally from plants, yeast or bacteria to protect against natural enemies. Its dosage resembles an adaptive hormetic response relationship, as high concentrations are toxic and mild doses are associated with anticancer and antiaging effects. This opens up new avenues for their use as „anti-aging pills“ in humans.brRecent human data suggest that metformin, rapamycin and other mTOR-inhibitors could delay heart disease, cancer, cognitive decline and improve survival time in people with diabetes mellitus. In addition, response to influenca vaccine was enhanced by rapamycin in adults with immunosenescence, indicating beneficial anti-aging effects in the elderly.br“Treat aging” is an actual call to recognize aging as an indication appropriate for clinical trials and treatments, as it was recently approved by the Federal Drug Administration (FDA USA. p bKurzfassung/b: Die ansteigende Morbidität und Invalidität in alternden Industrienationen stößt an die Grenzen der Ressourcen

  19. Scalable and Anonymous Group Communication with MTor

    National Research Council Canada - National Science Library

    Dong Lin; Micah Sherr; Boon Thau Loo

    2016-01-01

    This paper presents MTor, a low-latency anonymous group communication system. We construct MTor as an extension to Tor, allowing the construction of multi-source multicast trees on top of the existing Tor infrastructure...

  20. Rapamycin-Induced Apoptosis in HGF-Stimulated Lens Epithelial Cells by AKT/mTOR, ERK and JAK2/STAT3 Pathways

    Directory of Open Access Journals (Sweden)

    Fang Tian

    2014-08-01

    Full Text Available Hepatocyte growth factor (HGF induced the proliferation of lens epithelial cells (LECs and may be a major cause of posterior capsule opacification (PCO, which is the most frequent postoperative complication of cataract surgery. To date, several agents that can block LECs proliferation have been studied, but none have been used in clinic. Recently, accumulating evidence has suggested rapamycin, the inhibitor of mTOR (mammalian target of Rapamycin, was associated with the induction of apoptosis in LECs. The purpose of our study was to investigate the potential effects of rapamycin on HGF-induced LECs and the underlying mechanisms by which rapamycin exerted its actions. Using cell proliferation, cell viability and flow cytometric apoptosis assays, we found that rapamycin potently not only suppressed proliferation but also induced the apoptosis of LECs in a dose-dependent manner under HGF administration. Further investigation of the underlying mechanism using siRNA transfection revealed that rapamycin could promote apoptosis of LECs via inhibiting HGF-induced phosphorylation of AKT/mTOR, ERK and JAK2/STAT3 signaling molecules. Moreover, the forced expression of AKT, ERK and STAT3 could induce a significant suppression of apoptosis in these cells after treatment of rapamycin. Together, these findings suggested that rapamycin-induced apoptosis in HGF-stimulated LECs is accompanied by inhibition of AKT/mTOR, ERK and JAK2/STAT3 pathways, which supports its use to inhibit PCO in preclinical studies and provides theoretical foundation for future possible practice.

  1. Addition of rapamycin to anti-CD3 antibody improves long-term glycaemia control in diabetic NOD mice.

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    Shira Perl

    Full Text Available Non-Fc-binding Anti CD3 antibody has proven successful in reverting diabetes in the non-obese diabetes mouse model of type 1 diabetes and limited efficacy has been observed in human clinical trials. We hypothesized that addition of rapamycin, an mTOR inhibitor capable of inducing operational tolerance in allogeneic bone marrow transplantation, would result in improved diabetes reversal rates and overall glycemia.Seventy hyperglycemic non-obese diabetic mice were randomized to either a single injection of anti CD3 alone or a single injection of anti CD3 followed by 14 days of intra-peritoneal rapamycin. Mice were monitored for hyperglycemia and metabolic control.Mice treated with the combination of anti CD3 and rapamycin had similar rates of diabetes reversal compared to anti CD3 alone (25/35 vs. 22/35. Mice treated with anti CD3 plus rapamycin had a significant improvement in glycemia control as exhibited by lower blood glucose levels in response to an intra-peritoneal glucose challenge; average peak blood glucose levels 30 min post intra-peritoneal injection of 2 gr/kg glucose were 6.9 mmol/L in the anti CD3 plus rapamycin group vs. 10 mmo/L in the anti CD3 alone (P<0.05.The addition of rapamycin to anti CD3 results in significant improvement in glycaemia control in diabetic NOD mice.

  2. l-Tryptophan-mediated Enhancement of Susceptibility to Nonalcoholic Fatty Liver Disease Is Dependent on the Mammalian Target of Rapamycin*

    Science.gov (United States)

    Osawa, Yosuke; Kanamori, Hiromitsu; Seki, Ekihiro; Hoshi, Masato; Ohtaki, Hirofumi; Yasuda, Yoichi; Ito, Hiroyasu; Suetsugu, Atsushi; Nagaki, Masahito; Moriwaki, Hisataka; Saito, Kuniaki; Seishima, Mitsuru

    2011-01-01

    Nonalcoholic fatty liver disease is one of the most common liver diseases. l-Tryptophan and its metabolite serotonin are involved in hepatic lipid metabolism and inflammation. However, it is unclear whether l-tryptophan promotes hepatic steatosis. To explore this issue, we examined the role of l-tryptophan in mouse hepatic steatosis by using a high fat and high fructose diet (HFHFD) model. l-Tryptophan treatment in combination with an HFHFD exacerbated hepatic steatosis, expression of HNE-modified proteins, hydroxyproline content, and serum alanine aminotransaminase levels, whereas l-tryptophan alone did not result in these effects. We also found that l-tryptophan treatment increases serum serotonin levels. The introduction of adenoviral aromatic amino acid decarboxylase, which stimulates the serotonin synthesis from l-tryptophan, aggravated hepatic steatosis induced by the HFHFD. The fatty acid-induced accumulation of lipid was further increased by serotonin treatment in cultured hepatocytes. These results suggest that l-tryptophan increases the sensitivity to hepatic steatosis through serotonin production. Furthermore, l-tryptophan treatment, adenoviral AADC introduction, and serotonin treatment induced phosphorylation of the mammalian target of rapamycin (mTOR), and a potent mTOR inhibitor rapamycin attenuated hepatocyte lipid accumulation induced by fatty acid with serotonin. These results suggest the importance of mTOR activation for the exacerbation of hepatic steatosis. In conclusion, l-tryptophan exacerbates hepatic steatosis induced by HFHFD through serotonin-mediated activation of mTOR. PMID:21841000

  3. The TSC-mTOR pathway regulates macrophage polarization

    Science.gov (United States)

    Byles, Vanessa; Covarrubias, Anthony J.; Ben-Sahra, Issam; Lamming, Dudley W.; Sabatini, David M.; Manning, Brendan D.; Horng, Tiffany

    2013-01-01

    Macrophages are able to polarize to proinflammatory M1 or alternative M2 states with distinct phenotypes and physiological functions. How metabolic status regulates macrophage polarization remains not well understood, and here we examine the role of mTOR (Mechanistic Target of Rapamycin), a central metabolic pathway that couples nutrient sensing to regulation of metabolic processes. Using a mouse model in which myeloid lineage specific deletion of Tsc1 (Tsc1Δ/Δ) leads to constitutive mTOR Complex 1 (mTORC1) activation, we find that Tsc1Δ/Δ macrophages are refractory to IL-4 induced M2 polarization, but produce increased inflammatory responses to proinflammatory stimuli. Moreover, mTORC1-mediated downregulation of Akt signaling critically contributes to defective polarization. These findings highlight a key role for the mTOR pathway in regulating macrophage polarization, and suggest how nutrient sensing and metabolic status could be “hard-wired” to control of macrophage function, with broad implications for regulation of Type 2 immunity, inflammation, and allergy. PMID:24280772

  4. Preclinical evaluation of WYE-687, a mTOR kinase inhibitor, as a potential anti-acute myeloid leukemia agent

    Energy Technology Data Exchange (ETDEWEB)

    Cheng, Feng; Wang, Lingling; Shen, Yunfeng; Xia, Jun; Chen, Heng; Jiang, Yuanqiang, E-mail: jiangyuanqiangwuxi@163.com; Lu, Mize, E-mail: lumizewuxi9@sina.com

    2016-02-05

    Mammalian target of rapamycin (mTOR) as a potential drug target for treatment of acute myeloid leukemia (AML). Here, we investigated the potential anti-leukemic activity by WYE-687, a potent mTOR kinase inhibitor. We demonstrated that WYE-687 potently inhibited survival and proliferation of established (HL-60, U937, AML-193 and THP-1 lines) and human AML progenitor cells. Yet, same WYE-687 treatment was non-cytotoxic to the primary peripheral blood mononuclear leukocytes (PBMCs) isolated from healthy donors. WYE-687 induced caspase-dependent apoptotic death in above AML cells/progenitor cells. On the other hand, the pan-caspase inhibitor (Z-VAD-FMK), the caspase-3 specific inhibitor (Z-DEVD-FMK) or the caspase-9 specific inhibitor (z-LEHD-fmk) attenuated WYE-687-induced cytotoxicity. At the molecular level, WYE-687 concurrently inhibited activation of mTORC1 (p70S6K1 and S6 phosphorylations) and mTORC2 (AKT Ser-473 and FoxO1/3a phosphorylations), whiling downregulating mTORC1/2-regulated genes (Bcl-xL and hypoxia-inducible factor 1/2α) in both HL-60/U937 cells and human AML progenitor cells. In vivo, oral administration of WYE-687 potently inhibited U937 leukemic xenograft tumor growth in severe combined immunodeficient (SCID) mice, without causing significant toxicities. In summary, our results demonstrate that targeting mTORC1/2 by WYE-687 leads to potent antitumor activity in preclinical models of AML. - Highlights: • WYE-687 inhibits survival and proliferation of human AML cells/progenitor cells. • WYE-687 induces apoptotic death of human AML cells/progenitor cells. • WYE-687 inhibits mTORC1/2 activation in human AML cells/progenitor cells. • WYE-687 inhibits U937 xenograft growth in SCID mice.

  5. Effect of rapamycin on spleen size in longstanding renal transplant recipients.

    Science.gov (United States)

    Araújo, N C; Sampaio Gonçalves de Lucena, S B; da Silveira Rioja, S

    2014-06-01

    Based on evidence available in the literature, rapamycin, a mammalian target of rapamycin (mTOR) inhibitor, but not calcineurin inhibitors (CNIs), has been shown to decrease spleen size. Small spleen, in some instances, is associated with hyposplenism, a condition recently reported in patients with longstanding renal transplant. Accordingly, the effect of immunosuppressive drugs on spleen size was evaluated. Renal transplant recipients (35 taking mTOR and 68 CNI) were included, in whom a standardized investigation of the kidney allograft and spleen with the use of color Doppler ultrasound was performed and a peripheral smear were reviewed for the presence of Howell-Jolly bodies (HJBs). We enrolled 103 patients (64 men; 66 from a deceased donor). The mean age was 47.7 years (range, 23.0-74.0 y). Mean transplant duration was 1,899 days (range, 181-6,883 d). According to the presence of HJBs, the prevalence of hyposplenism was 47.6% for the entire cohort. The differences between the mTOR and CNI groups regarding sex and the presence of HJBs were not statistically significant (P > .05). Age, creatinine, hemoglobin, leukocytes, platelets, and Doppler parameters in spleen and kidney were similar in both groups (P > .05). mTOR patients had a decreased spleen length size (90.09 ± 13.02 mm vs 111.95 ± 18.66 mm; P < .001), a longer transplant duration (3,576 ± 1,594 d vs 1,036 ± 1,369 d; P < .001) and higher serum cholesterol (227.50 ± 38.75 mg/dL vs 182.67 ± 37.74 mg/dL; P < .001) and triglycerides (194.23 ± 79.88 mg/dL vs 148.70 ± 55.54 mg/dL; P = .003) levels compared with the CNI group. A multivariate analysis showed mTOR inhibitor to be the most important predictor of spleen size. In both the mTOR and CNI groups, the comparison between the subgroups of present and absent HJBs did not show any difference. The findings of this study suggest that small spleens in transplant recipients may be linked to treatment with an mTOR inhibitor, although this apparently does

  6. Deficiency of cardiac Acyl-CoA synthetase-1 induces diastolic dysfunction, but pathologic hypertrophy is reversed by rapamycin

    DEFF Research Database (Denmark)

    Paul, David S; Grevengoed, Trisha J; Pascual, Florencia

    2014-01-01

    In mice with temporally-induced cardiac-specific deficiency of acyl-CoA synthetase-1 (Acsl1(H-/-)), the heart is unable to oxidize long-chain fatty acids and relies primarily on glucose for energy. These metabolic changes result in the development of both a spontaneous cardiac hypertrophy...... of sarco/endoplasmic reticulum calcium ATPase and phospholamban showed no difference between genotypes. To determine the role of mTOR in the development of cardiac hypertrophy, we treated Acsl1(H-/-) mice with rapamycin. Six to eight week old Acsl1(H-/-) mice and their littermate controls were given i.......p. tamoxifen to eliminate cardiac Acsl1, then concomitantly treated for 10weeks with i.p. rapamycin or vehicle alone. Rapamycin completely blocked the enhanced ventricular S6K phosphorylation and cardiac hypertrophy and attenuated the expression of hypertrophy-associated fetal genes, including α-skeletal actin...

  7. Recent Advances and Challenges of mTOR Inhibitors Use in the Treatment of Patients with Tuberous Sclerosis Complex

    Directory of Open Access Journals (Sweden)

    Filipe Palavra

    2017-01-01

    Full Text Available Tuberous sclerosis complex (TSC is a genetic condition characterized by the presence of benign, noninvasive, and tumor-like lesions called hamartomas that can affect multiple organ systems and are responsible for the clinical features of the disease. In the majority of cases, TSC results from mutations in the TSC1 and TSC2 genes, leading to the overactivation of the mammalian target of rapamycin (mTOR signalling pathway, which controls several cell functions, including cell growth, proliferation, and survival. The establishment of a connection between TSC and mTOR led to the clinical use of drugs known as mTOR inhibitors (like rapamycin, also known as sirolimus and everolimus, which are becoming an increasingly interesting tool in the management of TSC-associated features, such as subependymal giant cell astrocytomas, renal angiomyolipomas, and also epilepsy. However, the intrinsic characteristics of these drugs and their systemic effects in such a heterogeneous condition pose many challenges in clinical practice, so that some questions remain unanswered. This article provides an overview of the pharmacological aspects of mTOR inhibitors about the clinical trials leading to their approval in TSC-related conditions and exposes current challenges and future directions associated with this promising therapeutic line.

  8. Phosphoproteomic profiling of in vivo signaling in liver by the mammalian target of rapamycin complex 1 (mTORC1.

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    Gokhan Demirkan

    Full Text Available Our understanding of signal transduction networks in the physiological context of an organism remains limited, partly due to the technical challenge of identifying serine/threonine phosphorylated peptides from complex tissue samples. In the present study, we focused on signaling through the mammalian target of rapamycin (mTOR complex 1 (mTORC1, which is at the center of a nutrient- and growth factor-responsive cell signaling network. Though studied extensively, the mechanisms involved in many mTORC1 biological functions remain poorly understood.We developed a phosphoproteomic strategy to purify, enrich and identify phosphopeptides from rat liver homogenates. Using the anticancer drug rapamycin, the only known target of which is mTORC1, we characterized signaling in liver from rats in which the complex was maximally activated by refeeding following 48 hr of starvation. Using protein and peptide fractionation methods, TiO(2 affinity purification of phosphopeptides and mass spectrometry, we reproducibly identified and quantified over four thousand phosphopeptides. Along with 5 known rapamycin-sensitive phosphorylation events, we identified 62 new rapamycin-responsive candidate phosphorylation sites. Among these were PRAS40, gephyrin, and AMP kinase 2. We observed similar proportions of increased and reduced phosphorylation in response to rapamycin. Gene ontology analysis revealed over-representation of mTOR pathway components among rapamycin-sensitive phosphopeptide candidates.In addition to identifying potential new mTORC1-mediated phosphorylation events, and providing information relevant to the biology of this signaling network, our experimental and analytical approaches indicate the feasibility of large-scale phosphoproteomic profiling of tissue samples to study physiological signaling events in vivo.

  9. A brain proteomic investigation of rapamycin effects in the Tsc1+/- mouse model.

    Science.gov (United States)

    Wesseling, Hendrik; Elgersma, Ype; Bahn, Sabine

    2017-01-01

    Tuberous sclerosis complex (TSC) is a rare monogenic disorder characterized by benign tumors in multiple organs as well as a high prevalence of epilepsy, intellectual disability and autism. TSC is caused by inactivating mutations in the TSC1 or TSC2 genes. Heterozygocity induces hyperactivation of mTOR which can be inhibited by mTOR inhibitors, such as rapamycin, which have proven efficacy in the treatment of TSC-associated symptoms. The aim of the present study was (1) to identify molecular changes associated with social and cognitive deficits in the brain tissue of Tsc1+/- mice and (2) to investigate the molecular effects of rapamycin treatment, which has been shown to ameliorate genotype-related behavioural deficits. Molecular alterations in the frontal cortex and hippocampus of Tsc1+/- and control mice, with or without rapamycin treatment, were investigated. A quantitative mass spectrometry-based shotgun proteomic approach (LC-MSE) was employed as an unbiased method to detect changes in protein levels. Changes identified in the initial profiling stage were validated using selected reaction monitoring (SRM). Protein Set Enrichment Analysis was employed to identify dysregulated pathways. LC-MSE analysis of Tsc1+/- mice and controls (n = 30) identified 51 proteins changed in frontal cortex and 108 in the hippocampus. Bioinformatic analysis combined with targeted proteomic validation revealed several dysregulated molecular pathways. Using targeted assays, proteomic alterations in the hippocampus validated the pathways "myelination", "dendrite," and "oxidative stress", an upregulation of ribosomal proteins and the mTOR kinase. LC-MSE analysis was also employed on Tsc1+/- and wildtype mice (n = 34) treated with rapamycin or vehicle. Rapamycin treatment exerted a stronger proteomic effect in Tsc1+/- mice with significant changes (mainly decreased expression) in 231 and 106 proteins, respectively. The cellular pathways "oxidative stress" and "apoptosis" were found

  10. Down regulation of Peroxiredoxin-3 in 3T3-L1 adipocytes leads to oxidation of Rictor in the mammalian-target of rapamycin complex 2 (mTORC2).

    Science.gov (United States)

    Olson, Dalay H; Burrill, Joel S; Kuzmicic, Jovan; Hahn, Wendy S; Park, Ji-Man; Kim, Do-Hyung; Bernlohr, David A

    2017-11-25

    Mitochondrially-derived oxidative stress has been implicated in the development of obesity-induced insulin resistance and is correlated with down regulation of Peroxiredoxin-3 (Prdx3). Prdx3 knockout mice exhibit whole-body insulin resistance, while Prdx3 transgenic animals remain insulin sensitive when placed on a high fat diet. To define the molecular events linking mitochondrial oxidative stress to insulin action, Prdx3 was silenced in 3T3-L1 adipocytes (Prdx3 KD) and the resultant cells evaluated for mitochondrial function, endoplasmic reticulum stress (ER stress), mitochondrial unfolded protein response (mtUPR) and insulin signaling. Prdx3 KD cells exhibit a two-fold increase in H2O2, reduced insulin-stimulated glucose transport and attenuated S473 phosphorylation of the mTORC2 substrate, Akt. Importantly, the decrease in glucose uptake can be rescued by pre-treatment with the antioxidant N-acetyl-cysteine (NAC). The changes in insulin sensitivity occur independently from activation of the ER stress or mtUPR pathways. Analysis of mTORC2, the complex responsible for phosphorylating Akt at S473, reveals increased cysteine oxidation of Rictor in Prdx3 KD cells that can be rescued with NAC. Taken together, these data suggest mitochondrial dysfunction in adipocytes may attenuate insulin signaling via oxidation of the mammalian-target of rapamycin complex 2 (mTORC2). Copyright © 2017. Published by Elsevier Inc.

  11. Finding a better drug for epilepsy: The mTOR pathway as an antiepileptogenic target

    OpenAIRE

    Galanopoulou, Aristea S.; Gorter, Jan A.; Cepeda, Carlos

    2012-01-01

    The mTOR signaling pathway regulates cell growth, differentiation, proliferation and metabolism. Loss of function mutations in upstream regulators of mTOR have been highly associated with dysplasias, epilepsy and neurodevelopmental disorders. These include tuberous sclerosis, which is due to mutations in TSC1 or TSC2 genes, mutations in phosphatase and tensin homolog (PTEN) as in Cowden syndrome, polyhydramnios, megalencephaly, symptomatic epilepsy syndrome (PMSE) due to mutations in the STE2...

  12. α1A-Adrenoceptors activate mTOR signalling and glucose uptake in cardiomyocytes.

    Science.gov (United States)

    Sato, Masaaki; Evans, Bronwyn A; Sandström, Anna L; Chia, Ling Yeong; Mukaida, Saori; Thai, Bui San; Nguyen, Anh; Lim, Linzi; Tan, Christina Y R; Baltos, Jo-Anne; White, Paul J; May, Lauren T; Hutchinson, Dana S; Summers, Roger J; Bengtsson, Tore

    2018-02-01

    The capacity of G protein-coupled receptors to modulate mechanistic target of rapamycin (mTOR) activity is a newly emerging paradigm with the potential to link cell surface receptors with cell survival. Cardiomyocyte viability is linked to signalling pathways involving Akt and mTOR, as well as increased glucose uptake and utilization. Our aim was to determine whether the α 1A -adrenoceptor (AR) couples to these protective pathways, and increased glucose uptake. We characterised α 1A -AR signalling in CHO-K1 cells co-expressing the human α 1A -AR and GLUT4 (CHOα 1A GLUT4myc) and in neonatal rat ventricular cardiomyocytes (NRVM), and measured glucose uptake, intracellular Ca 2+ mobilization, and phosphorylation of mTOR, Akt, 5' adenosine monophosphate-activated kinase (AMPK) and S6 ribosomal protein (S6rp). In both systems, noradrenaline and the α 1A -AR selective agonist A61603 stimulated glucose uptake by parallel pathways involving mTOR and AMPK, whereas another α 1 -AR agonist oxymetazoline increased glucose uptake predominantly by mTOR. All agonists promoted phosphorylation of mTOR at Ser2448 and Ser2481, indicating activation of both mTORC1 and mTORC2, but did not increase Akt phosphorylation. In CHOα 1A GLUT4myc cells, siRNA directed against rictor but not raptor suppressed α 1A -AR mediated glucose uptake. We have thus identified mTORC2 as a key component in glucose uptake stimulated by α 1A -AR agonists. Our findings identify a novel link between the α 1A -AR, mTORC2 and glucose uptake, that have been implicated separately in cardiomyocyte survival. Our studies provide an improved framework for examining the utility of α 1A -AR selective agonists as tools in the treatment of cardiac dysfunction. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

  13. mTOR inhibition sensitizes human hepatocellular carcinoma cells to resminostat

    Energy Technology Data Exchange (ETDEWEB)

    Peng, Xingang, E-mail: pengxinggang26@sina.com [Department of Emergency General Surgery, The Affiliated Hospital of Qingdao University, Qingdao (China); Zhang, Donghui, E-mail: zhangdonghuiyx@sina.com [Department of Infectious Disease, Linyi People’s Hospital, Linyi (China); Li, Zhengling, E-mail: lizhenglingzz@sina.com [Department of Nursing, Tengzhou Central People’s Hospital, Tengzhou (China); Fu, Meili, E-mail: fumeilidrlinyi@tom.com [Department of Infectious Disease, Linyi People’s Hospital, Linyi (China); Liu, Haiyan, E-mail: liuhaiyanlinyi5@sina.com [Department of Nursing, Linyi People’s Hospital, Linyi (China)

    2016-09-02

    Histone deacetylases (HDACs) hyper-activity in hepatocellular carcinoma (HCC) is often associated with patients’ poor prognosis. Our previous study has shown that resminostat, a novel HDAC inhibitor (HDACi), activated mitochondrial permeability transition pore (mPTP)-dependent apoptosis pathway in HCC cells. Here we explored the potential resminostat resistance factor by focusing on mammalian target of rapamycin (mTOR). We showed that AZD-2014, a novel mTOR kinase inhibitor, potentiated resminostat-induced cytotoxicity and proliferation inhibition in HCC cells. Molecularly, AZD-2014 enhanced resminostat-induced mPTP apoptosis pathway activation in HCC cells. Inhibition of this apoptosis pathway, by the caspase-9 specific inhibitor Ac-LEHD-CHO, the mPTP blockers (sanglifehrin A/cyclosporine A), or by shRNA-mediated knockdown of mPTP component cyclophilin-D (Cyp-D), significantly attenuated resminostat plus AZD-2014-induced cytotoxicity and apoptosis in HCC cells. Significantly, mTOR shRNA knockdown or kinase-dead mutation (Asp-2338-Ala) also sensitized HCC cells to resminostat, causing profound cytotoxicity and apoptosis induction. Together, these results suggest that mTOR could be a primary resistance factor of resminostat. Targeted inhibition of mTOR may thus significantly sensitize HCC cells to resminostat. - Highlights: • AZD-2014 potentiates resminostat’s cytotoxicity against HCC cells. • AZD-2014 facilitates resminostat-induced HCC cell apoptosis. • AZD-2014 augments resminostat-induced mitochondrial apoptosis pathway activation. • mTOR shRNA or kinase-dead mutation significantly sensitizes HCC cells to resminostat.

  14. The role of mTOR during cisplatin treatment in an in vitro and ex vivo model of cervical cancer.

    Science.gov (United States)

    Leisching, G R; Loos, B; Botha, M H; Engelbrecht, A-M

    2015-09-01

    Cisplatin is used as a cytotoxic agent for the management of cervical cancer. However, the severity of the side-effects limits the use of this drug, particularly at high doses. Resistance to cisplatin is often attributed to a disruption in the normal apoptotic response via aberrant activation of pathways such as the mTOR pathway. Here we assess the role of mTOR and its effect on cell death sensitization and autophagy in response to a low concentration of cisplatin in cervical cancer cells. Additionally we measured the expression profile of mTOR in normal, low- and high-grade squamous intraepithelial (LSIL and HSIL) lesions and cancerous tissue. An in vitro model of cervical cancer was established using HeLa and CaSki cells. mTOR protein expression as well as autophagy-related proteins were evaluated through Western blotting. Inhibition of mTOR was achieved with the use of rapamycin and RNA silencing. A low concentration of cisplatin administered as a single agent induces autophagy, but not apoptosis. Cisplatin cytotoxicity was greatly enhanced in cancer cells when mTOR had been inhibited prior to cisplatin treatment which was likely due to autophagy being increased above cisplatin-induced levels, thereby inducing apoptosis. Cervical tissue samples revealed an increase in mTOR protein expression in LSIL and carcinoma tissue which suggests a change in autophagy control. Our data suggest that utilising a lower dose of cisplatin combined with mTOR inhibition is a viable treatment option and addresses the challenge of cisplatin dose-dependent toxicity, however future studies are required to confirm this in a clinical setting. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  15. Attenuation of pentylenetrazole-induced acute status epilepticus in rats by adenosine involves inhibition of the mammalian target of rapamycin pathway.

    Science.gov (United States)

    Wang, Yuliang; Liu, Xuewu; Wang, Yuan; Chen, Jinbo; Han, Tao; Su, Lei; Zang, Kejun

    2017-10-18

    Adenosine (ADO) has been characterized as an endogenous anticonvulsant and alternative therapeutic drug, but its mechanism is not entirely clear. This study aimed to examine the relationship of ADO with the mammalian target of rapamycin (mTOR) in a Wistar rat model of pentylenetetrazole (PTZ)-induced acute status epilepticus. ADO (200 mg/kg) was administered intraperitoneally 30 min before PTZ (55-65 mg/kg) treatment, and Western blot assays and immunohistochemistry were performed 3 h after the onset of acute status epilepticus to detect phospho-TOR and the downstream target of mTOR, phospho-S6. The expression of these phosphoproteins in the hippocampus was significantly increased in PTZ-treated rats, but this increase was attenuated by the addition of ADO. To further verify a role for ADO in attenuating mTOR activity, we also evaluated its ability to suppress mTOR activity in normal rats that were not treated with PTZ. Our results suggest that ADO suppresses mTOR and S6 phosphorylation in normal rats and that this suppression can be reversed by the application of Compound C, an inhibitor of AMP-activated protein kinase, which functions as an upstream suppressor of the mTOR pathway. Thus, our results provide a novel antiepileptic mechanism for ADO in suppressing mTOR pathway activation upon PTZ-induced acute status epilepticus.

  16. Genetic reduction of mammalian target of rapamycin ameliorates Alzheimer's disease-like cognitive and pathological deficits by restoring hippocampal gene expression signature.

    Science.gov (United States)

    Caccamo, Antonella; De Pinto, Vito; Messina, Angela; Branca, Caterina; Oddo, Salvatore

    2014-06-04

    Elevated mammalian target of rapamycin (mTOR) signaling has been found in Alzheimer's disease (AD) patients and is linked to diabetes and aging, two known risk factors for AD. However, whether hyperactive mTOR plays a role in the cognitive deficits associated with AD remains elusive. Here, we genetically reduced mTOR signaling in the brains of Tg2576 mice, a widely used animal model of AD. We found that suppression of mTOR signaling reduced amyloid-β deposits and rescued memory deficits. Mechanistically, the reduction in mTOR signaling led to an increase in autophagy induction and restored the hippocampal gene expression signature of the Tg2576 mice to wild-type levels. Our results implicate hyperactive mTOR signaling as a previous unidentified signaling pathway underlying gene-expression dysregulation and cognitive deficits in AD. Furthermore, hyperactive mTOR signaling may represent a molecular pathway by which aging contributes to the development of AD. Copyright © 2014 the authors 0270-6474/14/347988-11$15.00/0.

  17. Rapamycin promotes osteogenesis under inflammatory conditions.

    Science.gov (United States)

    Li, Xing; Chang, Bei; Wang, Banchao; Bu, Wenhuan; Zhao, Liang; Liu, Jie; Meng, Lin; Wang, Lu; Xin, Ying; Wang, Dandan; Tang, Qi; Zheng, Changyu; Sun, Hongchen

    2017-12-01

    Chronic periodontitis, a common oral disease, usually results in irreversible bone resorption. Bone regeneration is a complex process between bone‑forming activity of osteoblasts and bone‑resorbing activity of osteoclasts, and still remains a challenge for physicians clinically. A previous study demonstrated that the mechanistic target of rapamycin signaling pathway is involved in osteogenic differentiation of mesenchymal stromal cells. Herein, whether rapamycin could be used to induce osteogenic differentiation of primary bone marrow‑derived mesenchymal stem cells (BMSCs) in vitro and promote new bone formation in vivo were evaluated. The results demonstrated that rapamycin alone was not enough to fully induce osteoblast differentiation in vitro and enhanced bone regeneration in vivo. Interestingly, rapamycin in rapamycin plus lipopolysaccharide (LPS)‑treated BMSCs significantly increased the gene expression levels of Sp7 transcription factor, runt related transcription factor 2, alkaline phosphatase (ALP) and collagen I (Col I), ALP activity, and calcium nodule at different time points in vitro, indicating that osteoblast differentiation occurs by rapamycin when BMSCs are exposed to LPS simultaneously. It was also demonstrated that rapamycin in rapamycin plus LPS‑treated rats promoted bone regeneration in vivo. These results suggest that rapamycin may influence osteoblast differentiation and new bone formation after LPS induces an inflammatory environment. Rapamycin may be used to treat periodontitis associated with bone loss in future clinical practice.

  18. PRAS40 is an integral regulatory component of erythropoietin mTOR signaling and cytoprotection.

    Directory of Open Access Journals (Sweden)

    Zhao Zhong Chong

    Full Text Available Emerging strategies that center upon the mammalian target of rapamycin (mTOR signaling for neurodegenerative disorders may bring effective treatment for a number of difficult disease entities. Here we show that erythropoietin (EPO, a novel agent for nervous system disorders, prevents apoptotic SH-SY5Y cell injury in an oxidative stress model of oxygen-glucose deprivation through phosphatidylinositol-3-kinase (PI 3-K/protein kinase B (Akt dependent activation of mTOR signaling and phosphorylation of the downstream pathways of p70 ribosomal S6 kinase (p70S6K, eukaryotic initiation factor 4E-binding protein 1 (4EBP1, and proline rich Akt substrate 40 kDa (PRAS40. PRAS40 is an important regulatory component either alone or in conjunction with EPO signal transduction that can determine cell survival through apoptotic caspase 3 activation. EPO and the PI 3-K/Akt pathways control cell survival and mTOR activity through the inhibitory post-translational phosphorylation of PRAS40 that leads to subcellular binding of PRAS40 to the cytoplasmic docking protein 14-3-3. However, modulation and phosphorylation of PRAS40 is independent of other protective pathways of EPO that involve extracellular signal related kinase (ERK 1/2 and signal transducer and activator of transcription (STAT5. Our studies highlight EPO and PRAS40 signaling in the mTOR pathway as potential therapeutic strategies for development against degenerative disorders that lead to cell demise.

  19. The Role of mTOR Inhibitors in Liver Transplantation: Reviewing the Evidence

    Science.gov (United States)

    Klintmalm, Goran B.; Nashan, Björn

    2014-01-01

    Despite the success of liver transplantation, long-term complications remain, including de novo malignancies, metabolic syndrome, and the recurrence of hepatitis C virus (HCV) and hepatocellular carcinoma (HCC). The current mainstay of treatment, calcineurin inhibitors (CNIs), can also worsen posttransplant renal dysfunction, neurotoxicity, and diabetes. Clearly there is a need for better immunosuppressive agents that maintain similar rates of efficacy and renal function whilst minimizing adverse effects. The mammalian target of rapamycin (mTOR) inhibitors with a mechanism of action that is different from other immunosuppressive agents has the potential to address some of these issues. In this review we surveyed the literature for reports of the use of mTOR inhibitors in adult liver transplantation with respect to renal function, efficacy, safety, neurological symptoms, de novo tumors, and the recurrence of HCC and HCV. The results of our review indicate that mTOR inhibitors are associated with efficacy comparable to CNIs while having benefits on renal function in liver transplantation. We also consider newer dosing schedules that may limit side effects. Finally, we discuss evidence that mTOR inhibitors may have benefits in the oncology setting and in relation to HCV-related allograft fibrosis, metabolic syndrome, and neurotoxicity. PMID:24719752

  20. IL-7 Restores T Lymphocyte Immunometabolic Failure in Septic Shock Patients through mTOR Activation.

    Science.gov (United States)

    Venet, Fabienne; Demaret, Julie; Blaise, Benjamin J; Rouget, Christelle; Girardot, Thibaut; Idealisoa, Estellie; Rimmelé, Thomas; Mallet, François; Lepape, Alain; Textoris, Julien; Monneret, Guillaume

    2017-09-01

    T lymphocyte alterations are central to sepsis pathophysiology, whereas related mechanisms remain poorly understood. We hypothesized that metabolic alterations could play a role in sepsis-induced T lymphocyte dysfunction. Samples from septic shock patients were obtained at day 3 and compared with those from healthy donors. T cell metabolic status was evaluated in the basal condition and after T cell stimulation. We observed that basal metabolic content measured in lymphocytes by nuclear magnetic resonance spectroscopy was altered in septic patients. Basal ATP concentration, oxidative phosphorylation (OXPHOS), and glycolysis pathways in T cells were decreased as well. After stimulation, T lymphocytes from patients failed to induce glycolysis, OXPHOS, ATP production, GLUT1 expression, glucose entry, and proliferation to similar levels as controls. This was associated with significantly altered mTOR, but not Akt or HIF-1α, activation and only minor AMPKα phosphorylation dysfunction. IL-7 treatment improved mTOR activation, GLUT1 expression, and glucose entry in septic patients' T lymphocytes, leading to their enhanced proliferation. mTOR activation was central to this process, because rapamycin systematically inhibited the beneficial effect of recombinant human IL-7. We demonstrate the central role of immunometabolism and, in particular, mTOR alterations in the pathophysiology of sepsis-induced T cell alterations. Our results support the rationale for targeting metabolism in sepsis with recombinant human IL-7 as a treatment option. Copyright © 2017 by The American Association of Immunologists, Inc.

  1. Acute mTOR inhibition induces insulin resistance and alters substrate utilization in vivo

    DEFF Research Database (Denmark)

    Kleinert, Maximilian; Sylow, Lykke; Fazakerley, Daniel J

    2014-01-01

    , but not rapamycin reduced insulin-stimulated glucose uptake into incubated muscles, despite normal GLUT4 translocation in muscle cells. AZD8055 inhibited glycolysis in MEF cells. Abrogation of mTORC2 activity by SIN1 deletion impaired glycolysis and AZD8055 had no effect in SIN1 KO MEFs. Re-expression of wildtype...... SIN1 rescued glycolysis. Glucose intolerance following AZD8055 administration was absent in mice lacking the mTORC2 subunit Rictor in muscle, and in vivo glucose uptake into Rictor-deficient muscle was reduced despite normal Akt activity. Taken together, acute mTOR inhibition is detrimental to glucose...

  2. DPP in the matrix activates AKT and mTOR signaling pathway to promote preodontoblast survival and differentiation

    Directory of Open Access Journals (Sweden)

    ANNE eGEORGE

    2015-08-01

    Full Text Available Dentin phosphophoryn (DPP is an extracellular matrix protein synthesized by odontoblasts. It is highly acidic and the phosphorylated protein possesses a strong affinity for calcium ions. Therefore, DPP in the extracellular matrix can promote hydroxyapatite nucleation and can regulate the size of the growing crystal. Besides its calcium binding property, DPP can initiate signaling functions from the ECM (Extracellular matrix. The signals that promote the cytodifferentiation of preodontoblasts to fully functional odontoblasts are not known. In this study, we demonstrate that preodontoblasts on a DPP matrix, generates mechanical and biochemical signals. This is initiated by the ligation of the integrins with the RGD containing DPP. The downstream biochemical response observed is the activation of the AKT( protein kinase B and mTOR (mammalian target of rapamycin signaling pathways leading to the activation of the transcription factor NF- κB (Nuclear factor κB . Terminal differentiation of the preodontoblasts was assessed by identifying phosphate and calcium deposits in the matrix using von Kossa and Alizarin red staining respectively. Identifying the signaling pathways initiated by DPP in the dentin matrix would help in devising strategies for dentin tissue engineering.

  3. Potential use of rapamycin in HIV infection

    DEFF Research Database (Denmark)

    Donia, Marco; McCubrey, James A; Bendtzen, Klaus

    2010-01-01

    The strong need for the development of alternative anti-HIV agents is primarily due to the emergence of strain-resistant viruses, the need for sustained adherence to complex treatment regimens and the toxicity of currently used antiviral drugs. This review analyzes proof of concept studies...... indicating that the immunomodulatory drug rapamycin (RAPA) possesses anti-HIV properties both in vitro and in vivo that qualifies it as a potential new anti-HIV drug. It represents a literature review of published studies that evaluated the in vitro and in vivo activity of RAPA in HIV. RAPA represses HIV-1...... replication in vitro through different mechanisms including, but not limited, to down regulation of CCR5. In addition RAPA synergistically enhances the anti-HIV activity of entry inhibitors such as vicriviroc, aplaviroc and enfuvirtide in vitro. RAPA also inhibits HIV-1 infection in human peripheral blood...

  4. Vibrio vulnificus induces mTOR activation and inflammatory responses in macrophages.

    Directory of Open Access Journals (Sweden)

    Dan-Li Xie

    Full Text Available Vibrio vulnificus (V. vulnificus, a Gram-negative marine bacterium, can cause life-threatening primary septicemia, especially in patients with liver diseases. How V. vulnificus affects the liver and how it acts on macrophages are not well understood. In this report, we demonstrated that V. vulnificus infection causes a strong inflammatory response, marked expansion of liver-resident macrophages, and liver damage in mice. We demonstrated further that V. vulnificus activates mTOR in macrophages and inhibition of mTOR differentially regulates V. vulnificus induced inflammatory responses, suggesting the possibility of targeting mTOR as a strategy to modulate V. vulnificus induced inflammatory responses.

  5. Gingipain-dependent degradation of mammalian target of rapamycin pathway proteins by the periodontal pathogen Porphyromonas gingivalis during invasion.

    Science.gov (United States)

    Stafford, P; Higham, J; Pinnock, A; Murdoch, C; Douglas, C W I; Stafford, G P; Lambert, D W

    2013-10-01

    Porphyromonas gingivalis and Tannerella forsythia are gram-negative pathogens strongly associated with periodontitis. Their abilities to interact, invade and persist within host cells are considered crucial to their pathogenicity, but the mechanisms by which they subvert host defences are not well understood. In this study, we set out to investigate whether P. gingivalis and T. forsythia directly target key signalling molecules that may modulate the host cell phenotype to favour invasion and persistence. Our data identify, for the first time, that P. gingivalis, but not T. forsythia, reduces levels of intracellular mammalian target of rapamycin (mTOR) in oral epithelial cells following invasion over a 4-h time course, via the action of gingipains. The ability of cytochalasin D to abrogate P. gingivalis-mediated mTOR degradation suggests that this effect is dependent upon cellular invasion. We also show that levels of several other proteins in the mTOR signalling pathway are modulated by gingipains, either directly or as a consequence of mTOR degradation including p-4E-BP1. Taken together, our data suggest that P. gingivalis manipulates the mTOR pathway, providing evidence for a potentially novel mechanism by which P. gingivalis mediates its effects on host cell responses to infection. 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd

  6. Life-long rapamycin administration ameliorates age-dependent cognitive deficits by reducing IL-1β and enhancing NMDA signaling

    Science.gov (United States)

    Majumder, Smita; Caccamo, Antonella; Medina, David X.; Benavides, Adriana D.; Javors, Martin A.; Kraig, Ellen; Strong, Randy; Richardson, Arlan; Oddo, Salvatore

    2012-01-01

    Summary Understanding the factors that contribute to age-related cognitive decline is imperative, particularly as age is the major risk factor for several neurodegenerative disorders. Levels of several cytokines increase in the brain during aging, including IL-1β, whose levels positively correlate with cognitive deficits. Previous reports show that reducing the activity of the mammalian target of rapamycin (mTOR) extends lifespan in yeast, nematodes, Drosophila, and mice. It remains to be established, however, whether extending lifespan with rapamycin is accompanied by an improvement in cognitive function. In this study, we show that 18-month-old mice treated with rapamycin starting at two months of age perform significantly better on a task measuring spatial learning and memory compared to age-matched mice on the control diet. In contrast, rapamycin does not improve cognition when given to 15-month-old mice with pre-existing, age-dependent learning and memory deficits. We further show that the rapamycin-mediated improvement in learning and memory is associated with a decrease in IL-1β levels and an increase in NMDA signaling. This is the first evidence to show that a small molecule known to increase lifespan also ameliorates age-dependent learning and memory deficits. PMID:22212527

  7. Programmed Death-1 Inhibition of Phosphatidylinositol 3-Kinase/AKT/Mechanistic Target of Rapamycin Signaling Impairs Sarcoidosis CD4+ T Cell Proliferation.

    Science.gov (United States)

    Celada, Lindsay J; Rotsinger, Joseph E; Young, Anjuli; Shaginurova, Guzel; Shelton, Debresha; Hawkins, Charlene; Drake, Wonder P

    2017-01-01

    Patients with progressive sarcoidosis exhibit increased expression of programmed death-1 (PD-1) receptor on their CD4+ T cells. Up-regulation of this marker of T cell exhaustion is associated with a reduction in the proliferative response to T cell receptor (TCR) stimulation, a defect that is reversed by PD-1 pathway blockade. Genome-wide association studies and microarray analyses have correlated signaling downstream from the TCR with sarcoidosis disease severity, but the mechanism is not yet known. Reduced phosphatidylinositol 3-kinase (PI3K)/AKT expression inhibits proliferation by inhibiting cell cycle progression. To test the hypothesis that PD-1 expression attenuates TCR-dependent activation of PI3K/AKT activity in progressive systemic sarcoidosis, we analyzed PI3K/AKT/mechanistic target of rapamycin (mTOR) expression at baseline and after PD-1 pathway blockade in CD4+ T cells isolated from patients with sarcoidosis and healthy control subjects. We confirmed an increased percentage of PD-1+ CD4+ T cells and reduced proliferative capacity in patients with sarcoidosis compared with healthy control subjects (P sarcoidosis CD4+ T cell proliferative capacity is secondary to altered expression of key mediators of cell cycle progression, including the PI3K/AKT/mTOR pathway, via PD-1 up-regulation. This supports the concept that PD-1 up-regulation drives the immunologic deficits associated with sarcoidosis severity by inducing signaling aberrancies in key mediators of cell cycle progression.

  8. mTOR Hyperactivation by Ablation of Tuberous Sclerosis Complex 2 in the Mouse Heart Induces Cardiac Dysfunction with the Increased Number of Small Mitochondria Mediated through the Down-Regulation of Autophagy.

    Directory of Open Access Journals (Sweden)

    Manabu Taneike

    Full Text Available Mammalian target of rapamycin complex 1 (mTORC1 is a key regulator of cell growth, proliferation and metabolism. mTORC1 regulates protein synthesis positively and autophagy negatively. Autophagy is a major system to manage bulk degradation and recycling of cytoplasmic components and organelles. Tuberous sclerosis complex (TSC 1 and 2 form a heterodimeric complex and inactivate Ras homolog enriched in brain, resulting in inhibition of mTORC1. Here, we investigated the effects of hyperactivation of mTORC1 on cardiac function and structure using cardiac-specific TSC2-deficient (TSC2-/- mice. TSC2-/- mice were born normally at the expected Mendelian ratio. However, the median life span of TSC2-/- mice was approximately 10 months and significantly shorter than that of control mice. TSC2-/- mice showed cardiac dysfunction and cardiomyocyte hypertrophy without considerable fibrosis, cell infiltration or apoptotic cardiomyocyte death. Ultrastructural analysis of TSC2-/- hearts revealed misalignment, aggregation and a decrease in the size and an increase in the number of mitochondria, but the mitochondrial function was maintained. Autophagic flux was inhibited, while the phosphorylation level of S6 or eukaryotic initiation factor 4E -binding protein 1, downstream of mTORC1, was increased. The upregulation of autophagic flux by trehalose treatment attenuated the cardiac phenotypes such as cardiac dysfunction and structural abnormalities of mitochondria in TSC2-/- hearts. The results suggest that autophagy via the TSC2-mTORC1 signaling pathway plays an important role in maintenance of cardiac function and mitochondrial quantity and size in the heart and could be a therapeutic target to maintain mitochondrial homeostasis in failing hearts.

  9. Rapamycin enhances docetaxel-induced cytotoxicity in a androgen-independent prostate cancer xenograft model by survivin downregulation

    Energy Technology Data Exchange (ETDEWEB)

    Morikawa, Yasuyuki, E-mail: yasu-m@med.gunma-u.ac.jp [Department of Urology, Gunma University Graduate School of Medicine, 3-39-22 Showa-machi, Maeabshi, Gunma 3718511 (Japan); Koike, Hidekazu; Sekine, Yoshitaka; Matsui, Hiroshi; Shibata, Yasuhiro; Ito, Kazuto; Suzuki, Kazuhiro [Department of Urology, Gunma University Graduate School of Medicine, 3-39-22 Showa-machi, Maeabshi, Gunma 3718511 (Japan)

    2012-03-16

    Highlights: Black-Right-Pointing-Pointer Rapamycin (RPM) enhances the susceptibility of PC3 cells to docetaxel. Black-Right-Pointing-Pointer Low-dosage of docetaxel (DTX) did not reduce survivin expression levels in PC3 cells. Black-Right-Pointing-Pointer Combination treatment of RPM with DTX suppressed the expression of surviving. Black-Right-Pointing-Pointer SiRNA against survivin enhanced the susceptibility of PC3 cells to DTX. Black-Right-Pointing-Pointer RPM and DTX cotreatment inhibited PC3 cell growth and decreased surviving in vivo. -- Abstract: Background: Docetaxel is a first-line treatment choice in castration-resistant prostate cancer (CRPC). However, the management of CRPC remains an important challenge in oncology. There have been many reports on the effects of rapamycin, which is an inhibitor of the mammalian target of rapamycin (mTOR), in the treatment of carcinogenesis. We assessed the cytotoxic effects of the combination treatment of docetaxel and rapamycin in prostate cancer cells. Furthermore, we examined the relationship between these treatments and survivin, which is a member of the inhibitory apoptosis family. Methods: Prostate cancer cells were cultured and treated with docetaxel and rapamycin. The effects on proliferation were evaluated with the MTS assay. In addition, we evaluated the effect on proliferation of the combination treatment induced knockdown of survivin expression by small interfering RNA transfection and docetaxel. Protein expression levels were assayed using western blotting. PC3 cells and xenograft growth in nude mice were used to evaluate the in vivo efficacy of docetaxel and its combination with rapamycin. Results: In vitro and in vivo, the combination of rapamycin with docetaxel resulted in a greater inhibition of proliferation than treatment with rapamycin or docetaxel alone. In addition, in vitro and in vivo, rapamycin decreased basal surviving levels, and cotreatment with docetaxel further decreased these levels

  10. Comparative Analysis of Muscle Hypertrophy Models Reveals Divergent Gene Transcription Profiles and Points to Translational Regulation of Muscle Growth through Increased mTOR Signaling

    Directory of Open Access Journals (Sweden)

    Marcelo G. Pereira

    2017-12-01

    Full Text Available Skeletal muscle mass is a result of the balance between protein breakdown and protein synthesis. It has been shown that multiple conditions of muscle atrophy are characterized by the common regulation of a specific set of genes, termed atrogenes. It is not known whether various models of muscle hypertrophy are similarly regulated by a common transcriptional program. Here, we characterized gene expression changes in three different conditions of muscle growth, examining each condition during acute and chronic phases. Specifically, we compared the transcriptome of Extensor Digitorum Longus (EDL muscles collected (1 during the rapid phase of postnatal growth at 2 and 4 weeks of age, (2 24 h or 3 weeks after constitutive activation of AKT, and (3 24 h or 3 weeks after overload hypertrophy caused by tenotomy of the Tibialis Anterior muscle. We observed an important overlap between significantly regulated genes when comparing each single condition at the two different timepoints. Furthermore, examining the transcriptional changes occurring 24 h after a hypertrophic stimulus, we identify an important role for genes linked to a stress response, despite the absence of muscle damage in the AKT model. However, when we compared all different growth conditions, we did not find a common transcriptional fingerprint. On the other hand, all conditions showed a marked increase in mTORC1 signaling and increased ribosome biogenesis, suggesting that muscle growth is characterized more by translational, than transcriptional regulation.

  11. BRAF gene alterations and enhanced mammalian target of rapamycin signaling in gangliogliomas.

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    Kakkar, Aanchal; Majumdar, Atreye; Pathak, Pankaj; Kumar, Anupam; Kumari, Kalpana; Tripathi, Manjari; Sharma, Mehar C; Suri, Vaishali; Tandon, Vivek; Chandra, Sarat P; Sarkar, Chitra

    2017-01-01

    Gangliogliomas (GGs) are slow-growing glioneuronal tumors seen in children and young adults. They are associated with intractable epilepsy, and have recently been found to harbor BRAF (B- rapidly accelerated fibrosarcoma) gene mutations. However, the mammalian target of rapamycin (mTOR) signaling pathway, downstream of BRAF, has not been evaluated extensively in GGs. GG cases were retrieved, clinical data obtained, and histopathological features reviewed. Sequencing for BRAF V600E mutation, analysis of BRAF copy number by quantitative real-time polymerase chain reaction, and immunohistochemistry for mTOR pathway markers p-S6 and p-4EBP1 were performed. Sixty-four cases of GG were identified (0.9% of central nervous system tumors). Of these, 28 had sufficient tumor tissue for further evaluation. Mixed glial and neuronal morphology was the commonest (64%) type. Focal cortical dysplasia was identified in the adjacent cortex (6 cases). BRAF V600E mutation was identified in 30% of GGs; BRAF copy number gain was observed in 50% of them. p-S6 and p-4EBP1 immunopositivity was seen in 57% cases each. Thus, mTOR pathway activation was seen in 81% cases, and was independent of BRAF alterations. 87% patients had Engel grade I outcome, while 13% had Engel grade II outcome. Both the Engel grade II cases analyzed showed BRAF V600E mutation. BRAF V600E mutation is frequent in GGs, as is BRAF gain; the former may serve as a target for personalized therapy in patients with residual tumors, necessitating its assessment in routine pathology reporting of these tumors. Evidence of mTOR pathway activation highlights similarities in the pathogenetic mechanisms underlying GG and focal cortical dysplasia, and suggests that mTOR inhibitors may be of utility in GG patients with persistent seizures after surgery.

  12. The effect of Bortezomib and Rapamycin on Telomerase Activity in Mantle Cell Lymphoma

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    Orit Uziel

    2014-12-01

    In the light of the crucial role of telomerase in cancer cells, it was important to characterize the possible relations between telomerase and bortezomib and to distinguish the biochemical mechanisms of its regulation and its interactions with other signal transduction inhibitors such as rapamycin. The results of this work encourage the in vivo examination of the therapeutic potential of the combination of bortezomib and rapamycin in Mantle Cell Lymphoma patients.

  13. Cardiac mTOR rescues the detrimental effects of diet-induced obesity in the heart after ischemia-reperfusion.

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    Aoyagi, Toshinori; Higa, Jason K; Aoyagi, Hiroko; Yorichika, Naaiko; Shimada, Briana K; Matsui, Takashi

    2015-06-15

    Diet-induced obesity deteriorates the recovery of cardiac function after ischemia-reperfusion (I/R) injury. While mechanistic target of rapamycin (mTOR) is a key mediator of energy metabolism, the effects of cardiac mTOR in ischemic injury under metabolic syndrome remains undefined. Using cardiac-specific transgenic mice overexpressing mTOR (mTOR-Tg mice), we studied the effect of mTOR on cardiac function in both ex vivo and in vivo models of I/R injury in high-fat diet (HFD)-induced obese mice. mTOR-Tg and wild-type (WT) mice were fed a HFD (60% fat by calories) for 12 wk. Glucose intolerance and insulin resistance induced by the HFD were comparable between WT HFD-fed and mTOR-Tg HFD-fed mice. Functional recovery after I/R in the ex vivo Langendorff perfusion model was significantly lower in HFD-fed mice than normal chow diet-fed mice. mTOR-Tg mice demonstrated better cardiac function recovery and had less of the necrotic markers creatine kinase and lactate dehydrogenase in both feeding conditions. Additionally, mTOR overexpression suppressed expression of proinflammatory cytokines, including IL-6 and TNF-α, in both feeding conditions after I/R injury. In vivo I/R models showed that at 1 wk after I/R, HFD-fed mice exhibited worse cardiac function and larger myocardial scarring along myofibers compared with normal chow diet-fed mice. In both feeding conditions, mTOR overexpression preserved cardiac function and prevented myocardial scarring. These findings suggest that cardiac mTOR overexpression is sufficient to prevent the detrimental effects of diet-induced obesity on the heart after I/R, by reducing cardiac dysfunction and myocardial scarring. Copyright © 2015 the American Physiological Society.

  14. Regulation of Ketone Body Metabolism and the Role of PPARα

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    Maja Grabacka

    2016-12-01

    Full Text Available Ketogenesis and ketolysis are central metabolic processes activated during the response to fasting. Ketogenesis is regulated in multiple stages, and a nuclear receptor peroxisome proliferator activated receptor α (PPARα is one of the key transcription factors taking part in this regulation. PPARα is an important element in the metabolic network, where it participates in signaling driven by the main nutrient sensors, such as AMP-activated protein kinase (AMPK, PPARγ coactivator 1α (PGC-1α, and mammalian (mechanistic target of rapamycin (mTOR and induces hormonal mediators, such as fibroblast growth factor 21 (FGF21. This work describes the regulation of ketogenesis and ketolysis in normal and malignant cells and briefly summarizes the positive effects of ketone bodies in various neuropathologic conditions.

  15. Suppression of mTOR signaling pathway promotes bone marrow mesenchymal stem cells differentiation into osteoblast in degenerative scoliosis: in vivo and in vitro.

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    Wang, Yu; Yi, Xiao-Dong; Li, Chun-De

    2017-02-01

    To investigate the role of mTOR signaling pathway in bone marrow mesenchymal stem cells (BMSCs) differentiation into osteoblast in degenerative scoliosis (DS). The rat model of DS was established. Thirty-two Sprague-Dawley (SD) rats were selected and divided into the normal control group, the positive control group (normal rats injected with rapamycin), the negative control group (DS rats injected with PBS) and the experiment group (DS rats injected with rapamycin). H&E staining was performed to observe the osteogenesis of scoliosis. The BMSCs were obtained and assigned into seven groups: the normal control group, the positive control group, the negative control group and 1.0/10.0/100.0/1000.0 nmol/L experiment groups. Flow cytometry was conducted to testify cell cycle. The mRNA and protein expressions of mTOR and osteoblastic differentiation markers were measured by qRT-PCR and western blotting. In vivo, compared with the negative control group, bone trabecular area and the number of differentiated bone cells were significantly increased in the experiment groups. In vitro, at 24 and 48 h after rapamycin treatment, compared with the negative control group, BMSCs at G0/G1 stage increased, but BMSCs at S stage decreased in the 1.0/10.0/100.0/1000.0 nmol/L experiment groups; the expressions of mTOR and p70-S6K1 proteins were reduced in the 1.0/10.0/100.0/1000.0 nmol/L experiment groups, while ALP activity, OC levels, calcium deposition, Co1-I protein expression and the mRNA expressions of OC and Co1-I were significantly increased. Suppression of mTOR signaling pathway by rapamycin could promote BMSCs differentiation into osteoblast in DS.

  16. Presurgical Administration of mTOR Inhibitors in Patients with Large Subependymal Giant Cell Astrocytoma Associated with Tuberous Sclerosis Complex.

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    Jiang, Tao; Du, Jiang; Raynald; Wang, Junmei; Li, Chunde

    2017-11-01

    Direct surgical resection remains the standard treatment for patients with tuberous sclerosis complex (TSC) with a large subependymal giant cell astrocytoma (SEGA). Rapamycin or everolimus is seldom used in these patients because of the risk of increased intracranial pressure and possibility of sudden death. Three patients with TSC and a large intracranial SEGA received oral rapamycin (0.5 mg/day) or everolimus (2.5 mg/day) before surgery for tumor resection. After mTOR inhibitor therapy, computed tomography scans and magnetic resonance imaging revealed tumor reduction. Tumor bleeding was easy to control during surgery, and the border between tumor and surrounding brain tissue was clearly differentiated. Analysis of postsurgical tumor specimens showed low blood density and focal necrosis. Preoperative mTOR inhibitors could be a potentially novel treatment modality in large TSC-SEGA with hydrocephalus. In this series, mTOR inhibitors were not only safe and well tolerated, but also beneficial for tumor resection. Copyright © 2017. Published by Elsevier Inc.

  17. Decreased mTOR signalling reduces mitochondrial ROS in brain via accumulation of the telomerase protein TERT within mitochondria.

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    Miwa, Satomi; Czapiewski, Rafal; Wan, Tengfei; Bell, Amy; Hill, Kirsten N; von Zglinicki, Thomas; Saretzki, Gabriele

    2016-10-22

    Telomerase in its canonical function maintains telomeres in dividing cells. In addition, the telomerase protein TERT has non-telomeric functions such as shuttling to mitochondria resulting in a decreased oxidative stress, DNA damage and apoptosis. TERT protein persists in adult neurons and can co-localise to mitochondria under various stress conditions. We show here that TERT expression decreased in mouse brain during aging while release of reactive oxygen species (ROS) from the mitochondrial electron transport chain increased. Dietary restriction (DR) caused accumulation of TERT protein in mouse brain mitochondria correlating to decreased ROS release and improved learning and spatial short-term memory. Decreased mTOR signalling is a mediator of DR. Accordingly, feeding mice with rapamycin increased brain mitochondrial TERT and reduced ROS release. Importantly, the beneficial effects of rapamycin on mitochondrial function were absent in brains and fibroblasts from first generation TERT -/- mice, and when TERT shuttling was inhibited by the Src kinase inhibitor bosutinib. Taken together, our data suggests that the mTOR signalling pathway impinges on the mitochondrial localisation of TERT protein, which might in turn contribute to the protection of the brain by DR or rapamycin against age-associated mitochondrial ROS increase and cognitive decline.

  18. Nanoparticle Albumin-Bound Rapamycin in Treating Patients With Advanced Cancer With mTOR Mutations

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    2017-09-19

    Advanced Malignant Neoplasm; Cervical Squamous Cell Carcinoma; Endometrial Carcinoma; Malignant Uterine Neoplasm; Recurrent Bladder Carcinoma; Recurrent Breast Carcinoma; Recurrent Cervical Carcinoma; Recurrent Head and Neck Carcinoma; Recurrent Malignant Neoplasm; Recurrent Ovarian Carcinoma; Recurrent Prostate Carcinoma; Recurrent Renal Cell Carcinoma; Solid Neoplasm; Stage III Bladder Cancer; Stage III Prostate Cancer; Stage III Renal Cell Cancer; Stage IIIA Breast Cancer; Stage IIIA Cervical Cancer; Stage IIIA Ovarian Cancer; Stage IIIB Breast Cancer; Stage IIIB Cervical Cancer; Stage IIIB Ovarian Cancer; Stage IIIC Breast Cancer; Stage IIIC Ovarian Cancer; Stage IV Breast Cancer; Stage IV Ovarian Cancer; Stage IV Prostate Cancer; Stage IV Renal Cell Cancer; Stage IVA Bladder Cancer; Stage IVA Cervical Cancer; Stage IVB Bladder Cancer; Stage IVB Cervical Cancer

  19. Rapamycin bypasses vesicle-mediated signaling events to activate Gln3 in Saccharomyces cerevisiae

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    Puria, Rekha

    2008-01-01

    Growth of Saccharomyces cerevisiae in poor nitrogen sources or exposure to the Tor inhibitor rapamycin results in expression of the nitrogen catabolite repressed (NCR) genes whose products are involved in scavenging and metabolizing nitrogen. The NCR genes are regulated by the GATA-like transactivators Gln3 and Gat1, which are thought to be under control of the rapamycin-sensitive Tor complex 1 (TORC1). We have recently shown that Gln3 nuclear translocation in response to nitrogen source quality but not in response to rapamycin requires Golgi to endosome trafficking. These and previous findings that several TORC1 components localize to low density endomembranes are discussed in a model that underscores a prominent role for the vesicular trafficking system in facilitating molecular interactions in response to nitrogen source. In addition, these findings have important implications for Tor signaling and rapamycin mechanism of action, both in yeast and in metazoans. PMID:19430540

  20. Mammalian target of rapamycin inhibition in polycystic kidney disease: From bench to bedside

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    Hyun-Jung Kim

    2012-09-01

    Full Text Available Autosomal dominant polycystic kidney disease (ADPKD is the most common life-threatening hereditary disease in the USA resulting in chronic kidney disease and the need for dialysis and transplantation. Approximately 85% of cases of ADPKD are caused by a mutation in the Pkd1 gene that encodes polycystin-1, a large membrane receptor. The Pkd1 gene mutation results in abnormal proliferation in tubular epithelial cells, which plays a crucial role in cyst development and/or growth in PKD. Activation of the proliferative mammalian target of rapamycin (mTOR signaling pathway has been demonstrated in polycystic kidneys from rodents and humans. mTOR inhibition with sirolimus or everolimus decreases cysts in most animal models of PKD including Pkd1 and Pkd2 gene deficient orthologous models of human disease. On the basis of animal studies, human studies were undertaken. Two large randomized clinical trials published in the New England Journal of Medicine of everolimus or sirolimus in ADPKD patients were very unimpressive and associated with a high side-effect profile. Possible reasons for the unimpressive nature of the human studies include their short duration, the high drop-out rate, suboptimal dosing, lack of randomization of “fast” and “slow progressors” and the lack of correlation between kidney size and kidney function in ADPKD. The future of mTOR inhibition in ADPKD is discussed.

  1. Rapamycin nanoparticles localize in diseased lung vasculature and prevent pulmonary arterial hypertension.

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    Segura-Ibarra, Victor; Amione-Guerra, Javier; Cruz-Solbes, Ana S; Cara, Francisca E; Iruegas-Nunez, David A; Wu, Suhong; Youker, Keith A; Bhimaraj, Arvind; Torre-Amione, Guillermo; Ferrari, Mauro; Karmouty-Quintana, Harry; Guha, Ashrith; Blanco, Elvin

    2017-05-30

    Vascular remodeling resulting from pulmonary arterial hypertension (PAH) leads to endothelial fenestrations. This feature can be exploited by nanoparticles (NP), allowing them to extravasate from circulation and accumulate in remodeled pulmonary vessels. Hyperactivation of the mTOR pathway in PAH drives pulmonary arterial smooth muscle cell proliferation. We hypothesized that rapamycin (RAP)-loaded NPs, an mTOR inhibitor, would accumulate in diseased lungs, selectively targeting vascular mTOR and preventing PAH progression. RAP poly(ethylene glycol)-block-poly(ε-caprolactone) (PEG-PCL) NPs were fabricated. NP accumulation and efficacy were examined in a rat monocrotaline model of PAH. Following intravenous (IV) administration, NP accumulation in diseased lungs was verified via LC/MS analysis and confocal imaging. Pulmonary arteriole thickness, right ventricular systolic pressures, and ventricular remodeling were determined to assess the therapeutic potential of RAP NPs. Monocrotaline-exposed rats showed increased NP accumulation within lungs compared to healthy controls, with NPs present to a high extent within pulmonary perivascular regions. RAP, in both free and NP form, attenuated PAH development, with histological analysis revealing minimal changes in pulmonary arteriole thickness and no ventricular remodeling. Importantly, NP-treated rats showed reduced systemic side effects compared to free RAP. This study demonstrates the potential for nanoparticles to significantly impact PAH through site-specific delivery of therapeutics. Copyright © 2017 Elsevier B.V. All rights reserved.

  2. mTOR Inhibition Attenuates Dextran Sulfate Sodium-Induced Colitis by Suppressing T Cell Proliferation and Balancing TH1/TH17/Treg Profile.

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    Shurong Hu

    Full Text Available It has been established that mammalian target of Rapamycin (mTOR inhibitors have anti-inflammatory effects in models of experimental colitis. However, the underlying mechanism is largely unknown. In this research, we investigate the anti-inflammatory effects of AZD8055, a potent mTOR inhibitor, on T cell response in dextran sulfate sodium (DSS-induced colitis in mice, a commonly used animal model of inflammatory bowel diseases (IBD. Severity of colitis is evaluated by changing of body weight, bloody stool, fecal consistency, histology evaluation and cytokine expression. We find that AZD8055 treatment attenuates DSS-induced body weight loss, colon length shortening and pathological damage of the colon. And AZD8055 treatment decreases colonic expression of genes encoding the pro-inflammatory cytokines interferon-γ, interleukin (IL-17A, IL-1β,IL-6 and tumor necrosis factor(TNF-a and increases colonic expression of anti-inflammatory cytokines IL-10. We show that AZD8055 treatment decreases the percentages of CD4+ T cells and CD8+ T cells in spleen, lymph nodes and peripheral blood of mice. We also find that AZD8055 treatment significantly reduces the number of T helper 1(TH1 cells and TH17 cells and increases regulatory T (Treg cells in the lamina propria and mesenteric lymph nodes. Furthermore, we demonstrates that AZD8055 suppresses the proliferation of CD4+ and CD8+ T cells and the differentiation of TH1/TH17 cells and expands Treg cells in vitro. The results suggest that, in experimental colitis, AZD8055 exerts anti-inflammatory effect by regulating T helper cell polarization and proliferation.

  3. A modelling-experimental approach reveals insulin receptor substrate (IRS)-dependent regulation of adenosine monosphosphate-dependent kinase (AMPK) by insulin

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    Sonntag, Annika G; Dalle Pezze, Piero; Shanley, Daryl P; Thedieck, Kathrin

    Mammalian target of rapamycin (mTOR) kinase responds to growth factors, nutrients and cellular energy status and is a central controller of cellular growth. mTOR exists in two multiprotein complexes that are embedded into a complex signalling network. Adenosine monophosphate-dependent kinase (AMPK)

  4. 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

  5. Differential Reponses of Hematopoietic Stem and Progenitor Cells to mTOR Inhibition

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    Aimin Yang

    2015-01-01

    Full Text Available Abnormal activation of the mammalian target of rapamycin (mTOR signaling pathway has been observed in a variety of human cancers. Therefore, targeting of the mTOR pathway is an attractive strategy for cancer treatment and several mTOR inhibitors, including AZD8055 (AZD, a novel dual mTORC1/2 inhibitor, are currently in clinical trials. Although bone marrow (BM suppression is one of the primary side effects of anticancer drugs, it is not known if pharmacological inhibition of dual mTORC1/2 affects BM hematopoietic stem and progenitor cells (HSPCs function and plasticity. Here we report that dual inhibition of mTORC1/2 by AZD or its analogue (KU-63794 depletes mouse BM Lin−Sca-1+c-Kit+ cells in cultures via the induction of apoptotic cell death. Subsequent colony-forming unit (CFU assays revealed that inhibition of mTORC1/2 suppresses the clonogenic function of hematopoietic progenitor cells (HPCs in a dose-dependent manner. Surprisingly, we found that dual inhibition of mTORC1/2 markedly inhibits the growth of day-14 cobblestone area-forming cells (CAFCs but enhances the generation of day-35 CAFCs. Given the fact that day-14 and day-35 CAFCs are functional surrogates of HPCs and hematopoietic stem cells (HSCs, respectively, these results suggest that dual inhibition of mTORC1/2 may have distinct effects on HPCs versus HSCs.

  6. Cryptotanshinone inhibition of mammalian target of rapamycin pathway is dependent on oestrogen receptor alpha in breast cancer.

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    Pan, Yanhong; Shi, Junfeng; Ni, Wenting; Liu, Yuping; Wang, Siliang; Wang, Xu; Wei, Zhonghong; Wang, Aiyun; Chen, Wenxing; Lu, Yin

    2017-09-01

    Cryptotanshinone (CPT) has been demonstrated to inhibit proliferation and mammalian target of rapamycin (mTOR) pathway in MCF-7 breast cancer cells. However, the same results are unable to be repeated in MDA-MB-231 cells. Given the main difference of oestrogen receptor α (ERα) between two types of breast cancer cells, It is possibly suggested that CPT inhibits mTOR pathway dependent on ERα in breast cancer. CPT could significantly inhibit cell proliferation of ERα-positive cancer cells, whereas ERα-negative cancer cells are insensitive to CPT. The molecular docking results indicated that CPT has a high affinity with ERα, and the oestrogen receptor element luciferase reporter verified CPT distinct anti-oestrogen effect. Furthermore, CPT inhibits mTOR signalling in MCF-7 cells, but not in MDA-MB-231 cells, which is independent on binding to the FKBP12 and disrupting the mTOR complex. Meanwhile, increased expression of phosphorylation AKT and insulin receptor substrate (IRS1) induced by insulin-like growth factor 1 (IGF-1) was antagonized by CPT, but other molecules of IGF-1/AKT/mTOR signalling pathway such as phosphatase and tensin homolog (PTEN) and phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K) were negatively affected. Finally, the MCF-7 cells transfected with shERα for silencing ERα show resistant to CPT, and p-AKT, phosphorylation of p70 S6 kinase 1 (p-S6K1) and eukaryotic initiation factor 4E binding protein 1 (4E-BP1) were partially recovered, suggesting ERα is required for CPT inhibition of mTOR signalling. Overall, CPT inhibition of mTOR is dependent on ERα in breast cancer and should be a potential anti-oestrogen agent and a natural adjuvant for application in endocrine resistance therapy. © 2017 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.

  7. Branched Chain Amino Acid Suppresses Hepatocellular Cancer Stem Cells through the Activation of Mammalian Target of Rapamycin

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    Nishitani, Shinobu; Horie, Mayumi; Ishizaki, Sonoko; Yano, Hirohisa

    2013-01-01

    Differentiation of cancer stem cells (CSCs) into cancer cells causes increased sensitivity to chemotherapeutic agents. Although inhibition of mammalian target of rapamycin (mTOR) leads to CSC survival, the effect of branched chain amino acids (BCAAs), an mTOR complex 1 (mTORC1) activator remains unknown. In this study, we examined the effects of BCAA on hepatocellular carcinoma (HCC) cells expressing a hepatic CSC marker, EpCAM. We examined the effects of BCAA and/or 5-fluorouracil (FU) on expression of EpCAM and other CSC-related markers, as well as cell proliferation in HCC cells and in a xenograft mouse model. We also characterized CSC-related and mTOR signal-related molecule expression and tumorigenicity in HCC cells with knockdown of Rictor or Raptor, or overexpression of constitutively active rheb (caRheb). mTOR signal-related molecule expression was also examined in BCAA-treated HCC cells. In-vitro BCAA reduced the frequency of EpCAM-positive cells and improved sensitivity to the anti-proliferative effect of 5-FU. Combined 5-FU and BCAA provided better antitumor efficacy than 5-FU alone in the xenograft model. Stimulation with high doses of BCAA activated mTORC1. Knockdown and overexpression experiments revealed that inhibition of mTOR complex 2 (mTORC2) or activation of mTORC1 led to decreased EpCAM expression and little or no tumorigenicity. BCAA may enhance the sensitivity to chemotherapy by reducing the population of cscs via the mTOR pathway. This result suggests the utility of BCAA in liver cancer therapy. PMID:24312415

  8. Branched chain amino acid suppresses hepatocellular cancer stem cells through the activation of mammalian target of rapamycin.

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    Shinobu Nishitani

    Full Text Available Differentiation of cancer stem cells (CSCs into cancer cells causes increased sensitivity to chemotherapeutic agents. Although inhibition of mammalian target of rapamycin (mTOR leads to CSC survival, the effect of branched chain amino acids (BCAAs, an mTOR complex 1 (mTORC1 activator remains unknown. In this study, we examined the effects of BCAA on hepatocellular carcinoma (HCC cells expressing a hepatic CSC marker, EpCAM. We examined the effects of BCAA and/or 5-fluorouracil (FU on expression of EpCAM and other CSC-related markers, as well as cell proliferation in HCC cells and in a xenograft mouse model. We also characterized CSC-related and mTOR signal-related molecule expression and tumorigenicity in HCC cells with knockdown of Rictor or Raptor, or overexpression of constitutively active rheb (caRheb. mTOR signal-related molecule expression was also examined in BCAA-treated HCC cells. In-vitro BCAA reduced the frequency of EpCAM-positive cells and improved sensitivity to the anti-proliferative effect of 5-FU. Combined 5-FU and BCAA provided better antitumor efficacy than 5-FU alone in the xenograft model. Stimulation with high doses of BCAA activated mTORC1. Knockdown and overexpression experiments revealed that inhibition of mTOR complex 2 (mTORC2 or activation of mTORC1 led to decreased EpCAM expression and little or no tumorigenicity. BCAA may enhance the sensitivity to chemotherapy by reducing the population of cscs via the mTOR pathway. This result suggests the utility of BCAA in liver cancer therapy.

  9. Mathematical Modelling of Metabolic Regulation in Aging

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    Mark T. Mc Auley

    2015-04-01

    Full Text Available The underlying cellular mechanisms that characterize aging are complex and multifaceted. However, it is emerging that aging could be regulated by two distinct metabolic hubs. These hubs are the pathway defined by the mammalian target of rapamycin (mTOR and that defined by the NAD+-dependent deacetylase enzyme, SIRT1. Recent experimental evidence suggests that there is crosstalk between these two important pathways; however, the mechanisms underpinning their interaction(s remains poorly understood. In this review, we propose using computational modelling in tandem with experimentation to delineate the mechanism(s. We briefly discuss the main modelling frameworks that could be used to disentangle this relationship and present a reduced reaction pathway that could be modelled. We conclude by outlining the limitations of computational modelling and by discussing opportunities for future progress in this area.