Adeno-Associated Viral Vector-Mediated mTOR Inhibition by Short Hairpin RNA Suppresses Laser-Induced Choroidal Neovascularization

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Tae Kwann Park

2017-09-01

Full Text Available Choroidal neovascularization (CNV is the defining characteristic feature of the wet subtype of age-related macular degeneration (AMD and may result in irreversible blindness. Based on anti-vascular endothelial growth factor (anti-VEGF, the current therapeutic approaches to CNV are fraught with difficulties, and mammalian target of rapamycin (mTOR has recently been proposed as a possible therapeutic target, although few studies have been conducted. Here, we show that a recombinant adeno-associated virus-delivered mTOR-inhibiting short hairpin RNA (rAAV-mTOR shRNA, which blocks the activity of both mTOR complex 1 and 2, represents a promising therapeutic approach for the treatment of CNV. Eight-week-old male C57/B6 mice were treated with the short hairpin RNA (shRNA after generating CNV lesions in the eyes via laser photocoagulation. The recombinant adeno-associated virus (rAAV delivery vehicle was able to effectively transduce cells in the inner retina, and significantly fewer inflammatory cells and less extensive CNV were observed in the animals treated with rAAV-mTOR shRNA when compared with control- and rAAV-scrambled shRNA-treated groups. Presumably related to the reduction of CNV, increased autophagy was detected in CNV lesions treated with rAAV-mTOR shRNA, whereas significantly fewer apoptotic cells detected in the outer nuclear layer around the CNV indicate that mTOR inhibition may also have neuroprotective effects. Taken together, these results demonstrate the therapeutic potential of mTOR inhibition, resulting from rAAV-mTOR shRNA activity, in the treatment of AMD-related CNV. Keywords: retinal neovascularization, choroidal neovascularization, adeno-associated virus, mTOR, RNA interference, mTOR shRNA, autophagy

Silencing Nrf2 impairs glioma cell proliferation via AMPK-activated mTOR inhibition

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Jia, Yue [Department of Neurosurgery, Jinling Hospital, School of Medicine, Nanjing University, 305 East Zhongshan Road, Nanjing 210002, Jiangsu Province (China); Wang, Handong, E-mail: njhdwang@hotmail.com [Department of Neurosurgery, Jinling Hospital, School of Medicine, Nanjing University, 305 East Zhongshan Road, Nanjing 210002, Jiangsu Province (China); Wang, Qiang [Department of Neurosurgery, Jinling Hospital, School of Medicine, Nanjing University, 305 East Zhongshan Road, Nanjing 210002, Jiangsu Province (China); Ding, Hui [Department of Neurosurgery, Jinling Hospital, School of Medicine, Southern Medical University (Guangzhou), 305 East Zhongshan Road, Nanjing 210002, Jiangsu Province (China); Wu, Heming [Department of Neurosurgery, Nanjing Jingdu Hospital, No. 34, Biao 34, Yanggongjing Road, Nanjing 210002, Jiangsu Province (China); Pan, Hao [Department of Neurosurgery, Jinling Hospital, School of Medicine, Nanjing University, 305 East Zhongshan Road, Nanjing 210002, Jiangsu Province (China)

2016-01-15

Gliomas are the leading cause of death among adults with primary brain malignancies. Treatment for malignant gliomas remains limited, and targeted therapies have been incompletely explored. Nuclear factor erythroid 2-related factor 2 (Nrf2), a key transcription regulator for antioxidant and detoxification enzymes, is abundantly expressed in cancer cells. In this study, the role and mechanism of Nrf2 in cancer cell proliferation was investigated in multiple glioma cell lines. We first evaluated the expression patterns of Nrf2 in four glioma cell lines and found all four cell lines expressed Nrf2, but the highest level was observed in U251 cells. We further evaluated the biological functions of Nrf2 in U251 glioma cell proliferation by specific inhibition of Nrf2 using short hairpin RNA (shRNA). We found that Nrf2 depletion inhibited glioma cell proliferation. Nrf2 depletion also decreased colony formation in U251 cells stably expressing Nrf2 shRNA compared to scrambled control shRNA. Moreover, suppression of Nrf2 expression could lead to ATP depletion (with concomitant rise in AMP/ATP ratio) and consequently to AMPK-activated mTOR inhibition. Finally, activation of adenosine monophosphate–activated protein kinase (AMPK) by treated with phenformin, an AMPK agonist, can mimic the inhibitory effect of Nrf2 knockdown in U251 cells. In conclusion, our findings will shed light to the role and mechanism of Nrf2 in regulating glioma proliferation via ATP-depletion-induced AMPK activation and consequent mTOR inhibition, a novel insight into our understanding the role and mechanism of Nrf2 in glioma pathoetiology. To our knowledge, this is also the first report to provide a rationale for the implication of cross-linking between Nrf2 and mTOR signaling.

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.

Inhibition of radiation-induced lipid peroxidation by means of gallic polydisulphide

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Losev, Yu.P.; Amadyan, M.G.; Oganesyan, N.M.; Fedulov, A.S.; Abramyan, A.K.; Shagoyan, A.G.; Khachkavanktsyan, A.S.

1999-01-01

Inhibition of radiation-induced lipid peroxidation by means of gallic polydisulphade has been studied. Rats were exposed to X-rays in doses 4,8 and 5,25 Gy. Lipid peroxidation was analysed in blood plasma, membranes of erythrocytes and homogenates of liver and spleen tissues of rats. Polydisulphide of gallic acid was used as inhibitor of lipid peroxidation because of its effective antioxidant properties as have been reported previously. It has been demonstrated that gallic disulphide exhibited high inhibition efficiency in conditions of radiation-induced lipid peroxidation due to the effect of intra-molecular synergism

Simvastatin and metformin inhibit cell growth in hepatitis C virus infected cells via mTOR increasing PTEN and autophagy.

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José A Del Campo

Full Text Available Hepatitis C virus (HCV infection has been related to increased risk of development of hepatocellular carcinoma (HCC while metformin (M and statins treatment seemed to protect against HCC development. In this work, we aim to identify the mechanisms by which metformin and simvastatin (S could protect from liver cancer. Huh7.5 cells were infected with HCV particles and treated with M+S. Human primary hepatocytes were treated with M+S. Treatment with both drugs inhibited Huh7.5 cell growth and HCV infection. In non-infected cells S increased translational controlled tumor protein (TCTP and phosphatase and tensin homolog (PTEN proteins while M inhibited mammalian target of rapamycin (mTOR and TCTP. Simvastatin and metformin co-administered down-regulated mTOR and TCTP, while PTEN was increased. In cells infected by HCV, mTOR, TCTP, p62 and light chain 3B II (LC3BII were increased and PTEN was decreased. S+M treatment increased PTEN, p62 and LC3BII in Huh7.5 cells. In human primary hepatocytes, metformin treatment inhibited mTOR and PTEN, but up-regulated p62, LC3BII and Caspase 3. In conclusion, simvastatin and metformin inhibited cell growth and HCV infection in vitro. In human hepatocytes, metformin increased cell-death markers. These findings suggest that M+S treatment could be useful in therapeutic prevention of HCV-related hepatocellular carcinoma.

mTOR inhibition in macrophages of asymptomatic HIV+ persons reverses the decrease in TLR4-mediated TNFα release through prolongation of MAPK pathway activation1

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Li, Xin; Han, Xinbing; Llano, Juliana; Bole, Medhavi; Zhou, Xiuqin; Swan, Katharine; Anandaiah, Asha; Nelson, Benjamin; Patel, Naimish R.; Reinach, Peter S.; Koziel, Henry; Tachado, Souvenir D.

2011-01-01

Toll-like receptor 4 (TLR4) mediated signaling is significantly impaired in macrophages from HIV+ persons predominantly due to altered MyD88-dependent pathway signaling caused in part by constitutive activation of PI3K. Here we assessed in these macrophages if the blunted increase in TLR4-mediated TNFα release induced by lipid A are associated with PI3K-induced upregulation of mammalian target of rapamycin (mTOR) activity. mTOR inhibition with rapamycin enhanced TLR4-mediated TNFα release, but instead suppressed anti-inflammatory IL-10 release. Targeted gene silencing of mTOR in macrophages resulted in lipid A-induced TNFα and IL-10 release patterns similar to those induced by rapamycin. Rapamycin restored MyD88-IRAK interaction in a dose-dependent manner. Targeted gene silencing of MyD88 (shRNA) and mTOR (RNAi) inhibition resulted in TLR4-mediated p70s6K activation and enhanced TNFα release, whereas IL-10 release was inhibited in both silenced and non-silenced HIV+ macrophages. Furthermore, mTOR inhibition augmented lipid A-induced TNFα release through enhanced and prolonged phosphorylation of ERK1/2 and JNK1/2 MAP kinases, which was associated with time-dependent MKP-1 destabilization. Taken together, impaired TLR4-mediated TNFα release in HIV+ macrophages is attributable in part to mTOR activation by constitutive PI3K expression in a MyD88-dependent signaling pathway. These changes result in MKP-1 stabilization, which shortens and blunts MAP kinase activation. mTOR inhibition may serve as a potential therapeutic target to upregulate macrophage innate immune host defense responsiveness in HIV+ persons. PMID:22025552

DUAL INHIBITION OF PI3K/AKT AND mTOR SIGNALING IN HUMAN NON-SMALL CELL LUNG CANCER CELLS BY A DIETARY FLAVONOID FISETIN

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Khan, Naghma; Afaq, Farrukh; Khusro, Fatima H.; Adhami, Vaqar Mustafa; Suh, Yewseok; Mukhtar, Hasan

2011-01-01

Lung cancer is one of the most commonly occurring malignancies. It has been reported that mTOR is phosphorylated in lung cancer and its activation was more frequent in tumors with over-expression of PI3K/Akt. Therefore, dual inhibitors of PI3K/Akt and mTOR signaling could be valuable agents for treating lung cancer. In the present study, we show that fisetin, a dietary tetrahydroxyflavone inhibits cell-growth with the concomitant suppression of PI3K/Akt and mTOR signaling in human non-small cell lung cancer (NSCLC) cells. Using autodock 4, we found that fisetin physically interacts with the mTOR complex at two sites. Fisetin treatment was also found to reduce the formation of A549 cell colonies in a dose-dependent manner. Treatment of cells with fisetin caused decrease in the protein expression of PI3K (p85 and p110), inhibition of phosphorylation of Akt, mTOR, p70S6K1, eIF-4E and 4E-BP1. Fisetin-treated cells also exhibited dose-dependent inhibition of the constituents of mTOR signaling complex like Rictor, Raptor, GβL and PRAS40. There was increase in the phosphorylation of AMPKα and decrease in the phosphorylation of TSC2 on treatment of cells with fisetin. We also found that treatment of cells with mTOR inhibitor rapamycin and mTOR-siRNA caused decrease in phosphorylation of mTOR and its target proteins which were further downregulated on treatment with fisetin, suggesting that these effects are mediated in part, through mTOR signaling. Our results show that fisetin suppressed PI3K/Akt and mTOR signaling in NSCLC cells and thus, could be developed as a chemotherapeutic agent against human lung cancer. PMID:21618507

Hormone resistance in two MCF-7 breast cancer cell lines is associated with reduced mTOR signaling, decreased glycolysis and increased sensitivity to cytotoxic drugs

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Euphemia Yee Leung

2014-09-01

Full Text Available The mTOR pathway is a key regulator of multiple cellular signaling pathways and is a potential target for therapy. We have previously developed two hormone-resistant sub-lines of the MCF-7 human breast cancer line, designated TamC3 and TamR3, which were characterized by reduced mTOR signaling, reduced cell volume and resistance to mTOR inhibition. Here we show that these lines exhibit increased sensitivity to carboplatin, oxaliplatin, 5-fluorouracil, camptothecin, doxorubicin, paclitaxel, docetaxel and hydrogen peroxide. The mechanisms underlying these changes have not yet been characterized but may include a shift from glycolysis to mitochondrial respiration. If this phenotype is found in clinical hormone-resistant breast cancers, conventional cytotoxic therapy may be a preferred option for treatment.

Denervation atrophy is independent from Akt and mTOR activation and is not rescued by myostatin inhibition

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Elizabeth M. MacDonald

2014-04-01

Full Text Available The purpose of our study was to compare two acquired muscle atrophies and the use of myostatin inhibition for their treatment. Myostatin naturally inhibits skeletal muscle growth by binding to ActRIIB, a receptor on the cell surface of myofibers. Because blocking myostatin in an adult wild-type mouse induces profound muscle hypertrophy, we applied a soluble ActRIIB receptor to models of disuse (limb immobilization and denervation (sciatic nerve resection atrophy. We found that treatment of immobilized mice with ActRIIB prevented the loss of muscle mass observed in placebo-treated mice. Our results suggest that this protection from disuse atrophy is regulated by serum and glucocorticoid-induced kinase (SGK rather than by Akt. Denervation atrophy, however, was not protected by ActRIIB treatment, yet resulted in an upregulation of the pro-growth factors Akt, SGK and components of the mTOR pathway. We then treated the denervated mice with the mTOR inhibitor rapamycin and found that, despite a reduction in mTOR activation, there is no alteration of the atrophy phenotype. Additionally, rapamycin prevented the denervation-induced upregulation of the mTORC2 substrates Akt and SGK. Thus, our studies show that denervation atrophy is not only independent from Akt, SGK and mTOR activation but also has a different underlying pathophysiological mechanism than disuse atrophy.

Changes in mitochondrial function by lipid peroxidation and their inhibition by biscoclaurin alkaloid

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Aono, K.; Shiraishi, N.; Arita, T.; Inouye, B.; Nakazawa, T.; Utsumi, K.

1981-01-01

During in vitro investigation of changes in mitochondrial function accompanying lipid peroxidation, it was found that cepharanthine, a biscoclaurin alkaloid, protects against such change. Results obtained were as follows: (1) Fe2+ induces lipid peroxidation of isolated mitochondria, resulting in diminished oxidative phosphorylation. (2) This diminishment largely depends on deterioration of ion compartmentation of the membrane and an increase in latent ATPase activity. (3) The Fe2+-induced deterioration in ion compartmentation is inhibited by cepharanthine. (4) Cepharanthine inhibits the mitochondrial lipid peroxidation induced by Fe2+. (5) Cepharanthine inhibits the lipid peroxidation of soybean lecithin liposomes by 60Co-irradiation

Eicosapentaenoic acid abolishes inhibition of insulin-induced mTOR phosphorylation by LPS via PTP1B downregulation in skeletal muscle.

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Wei, Hong-Kui; Deng, Zhao; Jiang, Shu-Zhong; Song, Tong-Xing; Zhou, Yuan-Fei; Peng, Jian; Tao, Ya-Xiong

2017-01-05

Dietary n-3 polyunsaturated fatty acids (n-3 PUFAs) increase insulin signaling in skeletal muscle. In the current study, we investigated the effect of eicosapentaenoic acid (EPA) on insulin-induced mammalian target of rapamycin (mTOR) phosphorylation in myotubes. We showed that EPA did not affect basal and insulin-induced mTOR phosphorylation in myotubes. However, EPA abolished lipopolysaccharide (LPS) -induced deficiency in insulin signaling (P  0.05). In myotubes, LPS stimulated PTP1B expression via NF-κB and activation protein-1 (AP1). Pre-incubation of 50 μM EPA prevented the LPS-induced activation of AP1 and NF-κΒ as well as PTP1B expression (P < 0.05). Interestingly, incubation of peroxisome proliferator-activated receptor γ (PPARγ) antagonist (GW9662) prior to EPA treatment, the effect of EPA on insulin-induced mTOR phosphorylation was blocked. Accordingly, EPA did not inhibit the LPS-induced activation of AP1 or NF-κΒ as well as PTP1B expression when incubation of GW9662 prior to EPA treatment. The in vivo study showed that EPA prevented LPS-induced PTPT1B expression and a decrease in insulin-induced mTOR phosphorylation in muscle of mice. In summary, EPA abolished LPS inhibition of insulin-induced mTOR phosphorylation in myotubes, and one of the key mechanisms was to inhibit AP1 and NF-κB activation and PTP1B transcription. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

TBK1 Regulates Prostate Cancer Dormancy through mTOR Inhibition

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Jin Koo Kim

2013-09-01

Full Text Available The mechanisms that regulate hematopoietic stem cell (HSC dormancy and self-renewal are well established and are largely dependent on signals emanating from the HSC niche. Recently, we found that prostate cancer (PCa cells target the HSC niche in mouse bone marrow (BM during metastasis. Little is known, however, as to how the HSC niche may regulate dormancy in cancer cells. In this study, we investigated the effects of TANK binding kinase 1 (TBK1 on PCa dormancy in the BM niche. We found that binding with niche osteoblasts induces the expression of TBK1 in PCa cells PC3 and C4-2B. Interestingly, TBK1 interacts with mammalian target of rapamycin (mTOR and inhibits its function. Rapamycin, an mTOR inhibitor, induces cell cycle arrest of PCa cells and enhances chemotherapeutic resistance of PCa cells. As a result, the knockdown of TBK1 decreases PCa stem-like cells and drug resistance in vitro and in vivo. Taken together, these results strongly indicate that TBK1 plays an important role in the dormancy and drug resistance of PCa.

mTOR Inhibition and Clinical Transplantation: Pancreas and Islet.

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Berney, Thierry; Andres, Axel; Toso, Christian; Majno, Pietro; Squifflet, Jean-Paul

2018-02-01

This brief overview discusses the beneficial and deleterious effects of mammalian target of rapamycin (mTOR) inhibitors on β cells, and how sirolimus- and everolimus-based immunosuppression have impacted on practices and outcomes of pancreas and islet transplantation. Sirolimus was the cornerstone of immunosuppressive regimens in islet transplantation at the turn of the millenium, but utilization of mTOR inhibitors has progressively decreased from greater than 80% to less than 50% of islet transplant recipients in more recent years. For whole pancreas transplantation, mTOR inhibitors were used in approximately 20% of patients in the early 2000s, but this dropped over the years to less than 10% currently. This decrease is arguably due to less well-tolerated side effects without the advantage of better outcomes. Nonetheless, mTOR inhibitors remain extremely valuable as second-line immunosuppressants in pancreas and islet transplantation.

Inhibition of DNA nanotube-conjugated mTOR siRNA on the growth of pulmonary arterial smooth muscle cells

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Zaichun You

2015-12-01

Full Text Available Here we provide raw and processed data and methods behind mTOR siRNA loaded DNA nanotubes (siRNA-DNA-NTs in the growth of pulmonary arterial smooth muscle cells (PASMCs under both normoxic and hypoxic condition, and also related to (You et al., Biomaterials, 2015, 67:137–150, [1]. The MTT analysis, Semi-quantitative RT-PCR data presented here were used to probe cytotoxicity of mTOR siRNA-DNA-NT complex in its TAE-Mg2+ buffer. siRNA-DNA-NTs have a lower cytotoxicity and higher transfection efficiency and can, based on inhibition of mTOR expression, decrease PASMCs growth both hypoxic and normal condition.

The Involvement of Mutual Inhibition of ERK and mTOR in PLCγ1-Mediated MMP-13 Expression in Human Osteoarthritis Chondrocytes

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Zejun Liu

2015-08-01

Full Text Available The issue of whether ERK activation determines matrix synthesis or degradation in osteoarthritis (OA pathogenesis currently remains controversial. Our previous study shows that PLCγ1 and mTOR are involved in the matrix metabolism of OA cartilage. Investigating the interplays of PLCγ1, mTOR and ERK in matrix degradation of OA will facilitate future attempts to manipulate ERK in OA prevention and therapy. Here, cultured human normal chondrocytes and OA chondrocytes were treated with different inhibitors or transfected with expression vectors, respectively. The levels of ERK, p-ERK, PLCγ1, p-PLCγ1, mTOR, p-mTOR and MMP-13 were then evaluated by Western blotting analysis. The results manifested that the expression level of ERK in human OA chondrocytes was lower than that in human normal articular chondrocytes, and the up-regulation of ERK could promote matrix synthesis, including the decrease in MMP-13 level and the increase in Aggrecan level in human OA chondrocytes. Furthermore, the PLCγ1/ERK axis and a mutual inhibition of mTOR and ERK were observed in human OA chondrocytes. Interestingly, activated ERK had no inhibitory effect on MMP-13 expression in PLCγ1-transformed OA chondrocytes. Combined with our previous study, the non-effective state of ERK activation by PLCγ1 on MMP-13 may be partly attributed to the inhibition of the PLCγ1/mTOR axis on the PLCγ1/ERK axis. Therefore, the study indicates that the mutual inhibition of ERK and mTOR is involved in PLCγ1-mediated MMP-13 expression in human OA chondrocytes, with important implication for the understanding of OA pathogenesis as well as for its prevention and therapy.

Hypothalamic mTOR signaling regulates food intake.

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Cota, Daniela; Proulx, Karine; Smith, Kathi A Blake; Kozma, Sara C; Thomas, George; Woods, Stephen C; Seeley, Randy J

2006-05-12

The mammalian Target of Rapamycin (mTOR) protein is a serine-threonine kinase that regulates cell-cycle progression and growth by sensing changes in energy status. We demonstrated that mTOR signaling plays a role in the brain mechanisms that respond to nutrient availability, regulating energy balance. In the rat, mTOR signaling is controlled by energy status in specific regions of the hypothalamus and colocalizes with neuropeptide Y and proopiomelanocortin neurons in the arcuate nucleus. Central administration of leucine increases hypothalamic mTOR signaling and decreases food intake and body weight. The hormone leptin increases hypothalamic mTOR activity, and the inhibition of mTOR signaling blunts leptin's anorectic effect. Thus, mTOR is a cellular fuel sensor whose hypothalamic activity is directly tied to the regulation of energy intake.

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

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

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Feng, Xue; Li, Ling; Jiang, Hong; Jiang, Keping; Jin, Ye; Zheng, Jianhua

2014-01-01

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

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

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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.

[Interleukin-37 induces apoptosis and autophagy of SMMC-7721 cells by inhibiting phosphorylation of mTOR].

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Li, Tingting; Zhu, Di; Mou, Tong; Guo, Zhen; Pu, Junliang; Wu, Zhongjun

2017-04-01

Objective To investigate the underlying mechanism by which interleukin-37 (IL-37) induces the apoptosis and autophagy in SMMC-7721 cells. Methods SMMC-7721 cells were incubated in vitro and divided into two groups, IL-37 treated group and control group. The cells were treated with (50, 100, 200) ng/mL of recombinant human interleukin-37 (rhIL-37). CCK-8 assay was used to detect the cell proliferation of SMMC-7721 cells. Cell apoptosis was measured by flow cytometry. Western blot analysis was performed to examine the expressions of apoptosis-related proteins, Bax, Bcl-2, and autophagy related proteins, microtubule-associated proteins 1 light chain 3 (LC3), beclin 1 and mammalian target of rapamycin (mTOR). Transmission electron microscopy (TEM) was used to observe the ultrastructures of autophagosomes. Results The rhIL-37 inhibited the proliferation of hepatocellular carcinoma SMMC-7721 cells. It induced the apoptosis and autophagy in SMMC-7721 cells. In the IL-37 treated group, the levels of Bax, LC3 and beclin 1 increased but Bcl-2 decreased. The phosphorylation of mTOR was inhibited in the IL-37 treated group. Autophagosome was obvious in the IL-37 treated group. Conclusion IL-37 induces the apoptosis and autophagy in SMMC-7721 cells, which may be related to the phosphorylation of mTOR.

Anthelminthic drug niclosamide sensitizes the responsiveness of cervical cancer cells to paclitaxel via oxidative stress-mediated mTOR inhibition

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Chen, Liping; Wang, Li; Shen, Haibin; Lin, Hui; Li, Dan

2017-01-01

Drug repurposing represents an alternative therapeutic strategy to cancer treatment. The potent anti-cancer activities of a FDA-approved anthelminthic drug niclosamide have been demonstrated in various cancers. However, whether niclosamide is active against cervical cancer is unknown. In this study, we investigated the effects of niclosamide alone and its combination with paclitaxel in cervical cancer in vitro and in vivo. We found that niclosamide significantly inhibited proliferation and induced apoptosis of a panel of cervical cancer cell lines, regardless of their cellular origin and genetic pattern. Niclosamide also inhibited tumor growth in cervical cancer xenograft mouse model. Importantly, niclosamide significantly enhanced the responsiveness of cervical cancer cell to paclitaxel. We further found that niclosamide induced mitochondrial dysfunctions via inhibiting mitochondrial respiration, complex I activity and ATP generation, which led to oxidative stress. ROS scavenge agent N-acetyl-L-cysteine (NAC) completely reversed the effects of niclosamide in increasing cellular ROS, inhibiting proliferation and inducing apoptosis, suggesting that oxidative stress induction is the mechanism of action of niclosamide in cervical cancer cells. In addition, niclosamide significantly inhibited mammalian target of rapamycin (mTOR) signaling pathway in cervical cancer cells and its inhibitory effect on mTOR is modulated by oxidative stress. Our work suggests that niclosamide is a useful addition to the treatment armamentarium for cervical cancer and induction of oxidative stress may be a potential therapeutic strategy in cervical cancer. - Highlights: • Niclosamide is active against cervical cancer cells in vitro and in vivo. • Niclosamide sensitizes cervical cancer cell response to paclitaxel. • Niclosamide induces mitochondrial dysfunction and oxidative damage. • Niclosamide inhibits mTOR signaling in an oxidative stress-dependent manner.

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...

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

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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.

Huaier Extract Induces Autophagic Cell Death by Inhibiting the mTOR/S6K Pathway in Breast Cancer Cells.

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Xiaolong Wang

Full Text Available Huaier extract is attracting increased attention due to its biological activities, including antitumor, anti-parasite and immunomodulatory effects. Here, we investigated the role of autophagy in Huaier-induced cytotoxicity in MDA-MB-231, MDA-MB-468 and MCF7 breast cancer cells. Huaier treatment inhibited cell viability in all three cell lines and induced various large membranous vacuoles in the cytoplasm. In addition, electron microscopy, MDC staining, accumulated expression of autophagy markers and flow cytometry revealed that Huaier extract triggered autophagy. Inhibition of autophagy attenuated Huaier-induced cell death. Furthermore, Huaier extract inhibited the mammalian target of the rapamycin (mTOR/S6K pathway in breast cancer cells. After implanting MDA-MB-231 cells subcutaneously into the right flank of BALB/c nu/nu mice, Huaier extract induced autophagy and effectively inhibited xenograft tumor growth. This study is the first to show that Huaier-induced cytotoxicity is partially mediated through autophagic cell death in breast cancer cells through suppression of the mTOR/S6K pathway.

mTOR Inhibition and Tuberous Sclerosis Prevention

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J Gordon Millichap

2013-09-01

Full Text Available Investigators at Children's Memorial Health Institute, Warsaw, Poland, report monozygotic twin sisters with tuberous sclerosis complex (TSC, one treated with the mTOR inhibitor everolimus since age 4 years.

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.

Targeting both IGF-1R and mTOR synergistically inhibits growth of renal cell carcinoma in vitro

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Cardillo, Thomas M; Trisal, Preeti; Arrojo, Roberto; Goldenberg, David M; Chang, Chien-Hsing

2013-01-01

Advanced or metastatic renal cell carcinoma (RCC) has a poor prognosis, because it is relatively resistant to conventional chemotherapy or radiotherapy. Treatments with human interferon-α2b alone or in combination with mammalian target of rapamycin (mTOR) inhibitors have led to only a modest improvement in clinical outcome. One observation made with mTOR inhibitors is that carcinomas can overcome these inhibitory effects by activating the insulin-like growth factor-I (IGF-I) signaling pathway. Clinically, there is an association of IGF-I receptor (IGF-IR) expression in RCC and poor long-term patient survival. We have developed a humanized anti-IGF-IR monoclonal antibody, hR1, which binds to RCC, resulting in effective down-regulation of IGF-IR and moderate inhibition of cell proliferation in vitro. In this work, we evaluate the anti-tumor activity of two novel IGF-1R-targeting agents against renal cell carcinoma given alone or in combination with an mTOR inhibitor. hR1 was linked by the DOCK-AND-LOCK™ (DNL™) method to four Fabs of hR1, generating Hex-hR1, or to four molecules of interferon-α2b, generating 1R-2b. Eight human RCC cell lines were screened for IGF-1R expression and sensitivity to treatment with hR1 in vitro. Synergy with an mTOR inhibitor, temsirolimus, was tested in a cell line (ACHN) with low sensitivity to hR1. Hex-hR1 induced the down-regulation of IGF-IR at 10-fold lower concentrations compared to the parental hR1. Sensitivity to growth inhibition mediated by hR1 and Hex-hR1 treatments correlated with IGF-1R expression (higher expression was more sensitive). The potency of 1R-2b to inhibit the in vitro growth of RCC was also demonstrated in two human cell lines, ACHN and 786-O, with EC 50 –values of 63 and 48 pM, respectively. When combined with temsirolimus, a synergistic growth-inhibition with hR1, Hex-hR1, and 1R-2b was observed in ACHN cells at concentrations as low as 10 nM for hR1, 1 nM for Hex-hR1, and 2.6 nM for 1R-2b. Both Hex-hR1

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...

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.

Inhibition of Lipid Peroxidation by Enzymatic Hydrolysates from Wheat Bran

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Yanping Cao

2011-01-01

Full Text Available Wheat bran, an important by-product of the cereal industry, is rich in potentially health-promoting phenolic compounds. The phenolics are mainly esterified to the cell wall polysaccharides. In our previous paper, wheat bran was destarched and deproteinated by α-amylase, protease and amyloglucosidase successively and further hydrolyzed using Bacillus subtilis xylanases, and the enzymatic hydrolysates from wheat bran (EHWB showed good scavenging activity in vitro. The aim of this study is to further characterize the antioxidant potential of EHWB against various systems, both ex vivo and in vivo, namely, rat liver microsomal lipid peroxidation systems induced by Fe2+/H2O2 and Fe3+-adenosine diphosphate (ADP/dihydronicotinamide adenine dinucleotide phosphate (NADPH, copper- and 2,2’-azo-bis(2-amidinopropane dihydrochloride (AAPH-induced human low-density lipoprotein (LDL oxidation systems, and alloxan-induced in vivo lipid peroxidation in mice. EHWB inhibited lipid peroxidation in rat liver microsomes induced by Fe2+/H2O2 and Fe3+-ADP/NADPH in a concentration-dependent manner with 90.3 and 87 % inhibition of lipid peroxidation at 50 mg/L, respectively, which were similar to that of butylated hydroxytoluene (BHT at 20 mg/L. The antioxidant potential of EHWB at a concentration ranging from 10 to 20 mg/L in the nonenzymatic system was more effective than in the enzymatic system. EHWB strongly inhibited in vitro copper- and AAPH-mediated oxidation of LDL in a concentration- and time-dependent manner with 52.41 and 63.03 % inhibition at 20 mg/L, respectively, which were similar to that of ascorbate at 10 mg/L. EHWB significantly decreased the level of thiobarbituric acid reactive substances (TBARS and increased the activities of glutathione peroxidase (GSH-Px, catalase (CAT and superoxide dismutase (SOD in serum and liver of alloxan-treated mice compared with the control. These results demonstrated that EHWB might be efficient in the protection of

PirB Overexpression Exacerbates Neuronal Apoptosis by Inhibiting TrkB and mTOR Phosphorylation After Oxygen and Glucose Deprivation Injury.

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Zhao, Zhao-Hua; Deng, Bin; Xu, Hao; Zhang, Jun-Feng; Mi, Ya-Jing; Meng, Xiang-Zhong; Gou, Xing-Chun; Xu, Li-Xian

2017-05-01

Previous studies have proven that paired immunoglobulin-like receptor B (PirB) plays a crucial suppressant role in neurite outgrowth and neuronal plasticity after central nervous system injury. However, the role of PirB in neuronal survival after cerebral ischemic injury and its mechanisms remains unclear. In the present study, the role of PirB is investigated in the survival and apoptosis of cerebral cortical neurons in cultured primary after oxygen and glucose deprivation (OGD)-induced injury. The results have shown that rebarbative PirB exacerbates early neuron apoptosis and survival. PirB gene silencing remarkably decreases early apoptosis and promotes neuronal survival after OGD. The expression of bcl-2 markedly increased and the expression of bax significantly decreased in PirB RNAi-treated neurons, as compared with the control- and control RNAi-treated ones. Further, phosphorylated TrkB and mTOR levels are significantly downregulated in the damaged neurons. However, the PirB silencing markedly upregulates phosphorylated TrkB and mTOR levels in the neurons after the OGD. Taken together, the overexpression of PirB inhibits the neuronal survival through increased neuron apoptosis. Importantly, the inhibition of the phosphorylation of TrkB and mTOR may be one of its mechanisms.

Device-based local delivery of siRNA against mammalian target of rapamycin (mTOR) in a murine subcutaneous implant model to inhibit fibrous encapsulation

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Takahashi, Hironobu; Wang, Yuwei; Grainger, David W.

2010-01-01

Fibrous encapsulation of surgically implant devices is associated with elevated proliferation and activation of fibroblasts in tissues surrounding these implants, frequently causing foreign body complications. Here we test the hypothesis that inhibition of the expression of mammalian target of rapamycin (mTOR) in fibroblasts can mitigate the soft tissue implant foreign body response by suppressing fibrotic responses around implants. In this study, mTOR was knocked down using small interfering...

Device-based local delivery of siRNA against mammalian target of rapamycin (mTOR) in a murine subcutaneous implant model to inhibit fibrous encapsulation.

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Takahashi, Hironobu; Wang, Yuwei; Grainger, David W

2010-11-01

Fibrous encapsulation of surgically implanted devices is associated with elevated proliferation and activation of fibroblasts in tissues surrounding these implants, frequently causing foreign body complications. Here we test the hypothesis that inhibition of the expression of mammalian target of rapamycin (mTOR) in fibroblasts can mitigate the soft tissue implant foreign body response by suppressing fibrotic responses around implants. In this study, mTOR was knocked down using small interfering RNA (siRNA) conjugated with branched polyethylenimine (bPEI) in fibroblastic lineage cells in serum-based cell culture as shown by both gene and protein analysis. This mTOR knock-down led to an inhibition in fibroblast proliferation by 70% and simultaneous down-regulation in the expression of type I collagen in fibroblasts in vitro. These siRNA/bPEI complexes were released from poly(ethylene glycol) (PEG)-based hydrogel coatings surrounding model polymer implants in a subcutaneous rodent model in vivo. No significant reduction in fibrous capsule thickness and mTOR expression in the foreign body capsules were observed. The siRNA inefficacy in this in vivo implant model was attributed to siRNA dosing limitations in the gel delivery system, and lack of targeting ability of the siRNA complex specifically to fibroblasts. While in vitro data supported mTOR knock-down in fibroblast cultures, in vivo siRNA delivery must be further improved to produce clinically relevant effects on fibrotic encapsulation around implants. Copyright © 2010 Elsevier B.V. All rights reserved.

The mTOR signalling pathway in cancer and the potential mTOR inhibitory activities of natural phytochemicals.

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Tan, Heng Kean; Moad, Ahmed Ismail Hassan; Tan, Mei Lan

2014-01-01

The mammalian target of rapamycin (mTOR) kinase plays an important role in regulating cell growth and cell cycle progression in response to cellular signals. It is a key regulator of cell proliferation and many upstream activators and downstream effectors of mTOR are known to be deregulated in various types of cancers. Since the mTOR signalling pathway is commonly activated in human cancers, many researchers are actively developing inhibitors that target key components in the pathway and some of these drugs are already on the market. Numerous preclinical investigations have also suggested that some herbs and natural phytochemicals, such as curcumin, resveratrol, timosaponin III, gallic acid, diosgenin, pomegranate, epigallocatechin gallate (EGCC), genistein and 3,3'-diindolylmethane inhibit the mTOR pathway either directly or indirectly. Some of these natural compounds are also in the clinical trial stage. In this review, the potential anti-cancer and chemopreventive activities and the current status of clinical trials of these phytochemicals are discussed.

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.

Fisetin inhibits human melanoma cell growth through direct binding to p70S6K and mTOR: findings from 3-D melanoma skin equivalents and computational modeling.

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Syed, Deeba N; Chamcheu, Jean-Christopher; Khan, Mohammad Imran; Sechi, Mario; Lall, Rahul K; Adhami, Vaqar M; Mukhtar, Hasan

2014-06-01

The incidence of melanoma continues to rise. Inspite of treatment advances, the prognosis remains grim once the disease has metastasized, emphasizing the need to explore additional therapeutic strategies. One such approach is through the use of mechanism-based dietary intervention. We previously showed that the flavonoid fisetin inhibits melanoma cell proliferation, in vitro and in vivo. Here, we studied fisetin-mediated regulation of kinases involved in melanoma growth and progression. Time-course analysis in 3-D melanoma constructs that transitioned from radial to vertical growth showed that fisetin treatment resulted in significant decrease in melanocytic lesions in contrast to untreated controls that showed large tumor nests and invading disseminated cells. Further studies in melanoma cultures and mouse xenografts showed that fisetin-mediated growth inhibition was associated with dephosphorylation of AKT, mTOR and p70S6K proteins. In silico modeling indicated direct interaction of fisetin with mTOR and p70S6K with favorable free energy values. These findings were validated by cell-free competition assays that established binding of fisetin to p70S6K and mTOR while little affinity was detected with AKT. Kinase activity studies reflected similar trend with % inhibition observed for p70S6K and mTOR at lower doses than AKT. Our studies characterized, for the first time, the differential interactions of any botanical agent with kinases involved in melanoma growth and demonstrate that fisetin inhibits mTOR and p70S6K through direct binding while the observed inhibitory effect of fisetin on AKT is mediated indirectly, through targeting interrelated pathways. Published by Elsevier Inc.

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

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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

2017-01-01

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 study demonstrated that the amino acids histidine, lysine, threonine inhibited mTOR signaling and IgE-mediated mast cell activation, while the amino acids leucine, isoleucine, valine had no effect on mTOR signaling in BMMCs. Based on these results, we designed an mTOR-targeting amino acid diet (Active 1 diet) and assessed the effects of dietary interventions with the amino acid diet or a multi-nutrient supplementation diet (Active 2 diet) on autistic-like behavior and mTOR signaling in food allergic mice and in inbred BTBR T+Itpr3tf/J mice. Cow's milk allergic (CMA) or BTBR male mice were fed a Control, Active 1, or Active 2 diet for 7 consecutive weeks. CMA mice showed reduced social interaction and increased self-grooming behavior. Both diets reversed behavioral impairments and inhibited the mTOR activity in the prefrontal cortex and amygdala of CMA mice. In BTBR mice, only Active 1 diet reduced repetitive self-grooming behavior and attenuated the mTOR activity in the prefrontal and somatosensory cortices. The current results suggest that activated mTOR signaling pathway in the brain may be a convergent pathway in the pathogenesis of ASD bridging genetic background and environmental triggers (food allergy) and that mTOR over-activation could serve as a potential therapeutic target for the treatment of ASD. Copyright © 2016. Published by Elsevier Inc.

The Inhibition of microRNA-128 on IGF-1-Activating mTOR Signaling Involves in Temozolomide-Induced Glioma Cell Apoptotic Death.

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Peng-Hsu Chen

Full Text Available Temozolomide (TMZ, an alkylating agent of the imidazotetrazine series, is a first-line chemotherapeutic drug used in the clinical therapy of glioblastoma multiforme, the most common and high-grade primary glioma in adults. Micro (miRNAs, which are small noncoding RNAs, post-transcriptionally regulate gene expressions and are involved in gliomagenesis. However, no studies have reported relationships between TMZ and miRNA gene regulation. We investigated TMZ-mediated miRNA profiles and its molecular mechanisms underlying the induction of glioma cell death. By performing miRNA microarray and bioinformatics analyses, we observed that expression of 248 miRNAs was altered, including five significantly upregulated and 17 significantly downregulated miRNAs, in TMZ-treated U87MG cells. miR-128 expression levels were lower in different glioma cells and strongly associated with poor survival. TMZ treatment significantly upregulated miR-128 expression. TMZ significantly enhanced miR-128-1 promoter activity and transcriptionally regulated miR-128 levels through c-Jun N-terminal kinase 2/c-Jun pathways. The overexpression and knockdown of miR-128 expression significantly affected TMZ-mediated cell viability and apoptosis-related protein expression. Furthermore, the overexpression of miR-128 alone enhanced apoptotic death of glioma cells through caspase-3/9 activation, poly(ADP ribose polymerase degradation, reactive oxygen species generation, mitochondrial membrane potential loss, and non-protective autophagy formation. Finally, we identified that key members in mammalian target of rapamycin (mTOR signaling including mTOR, rapamycin-insensitive companion of mTOR, insulin-like growth factor 1, and PIK3R1, but not PDK1, were direct target genes of miR-128. TMZ inhibited mTOR signaling through miR-128 regulation. These results indicate that miR-128-inhibited mTOR signaling is involved in TMZ-mediated cytotoxicity. Our findings may provide a better understanding

Mammalian target of rapamycin (mTOR): a central regulator of male fertility?

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Jesus, Tito T; Oliveira, Pedro F; Sousa, Mário; Cheng, C Yan; Alves, Marco G

2017-06-01

Mammalian target of rapamycin (mTOR) is a central regulator of cellular metabolic phenotype and is involved in virtually all aspects of cellular function. It integrates not only nutrient and energy-sensing pathways but also actin cytoskeleton organization, in response to environmental cues including growth factors and cellular energy levels. These events are pivotal for spermatogenesis and determine the reproductive potential of males. Yet, the molecular mechanisms by which mTOR signaling acts in male reproductive system remain a matter of debate. Here, we review the current knowledge on physiological and molecular events mediated by mTOR in testis and testicular cells. In recent years, mTOR inhibition has been explored as a prime strategy to develop novel therapeutic approaches to treat cancer, cardiovascular disease, autoimmunity, and metabolic disorders. However, the physiological consequences of mTOR dysregulation and inhibition to male reproductive potential are still not fully understood. Compelling evidence suggests that mTOR is an arising regulator of male fertility and better understanding of this atypical protein kinase coordinated action in testis will provide insightful information concerning its biological significance in other tissues/organs. We also discuss why a new generation of mTOR inhibitors aiming to be used in clinical practice may also need to include an integrative view on the effects in male reproductive system.

Down-Regulation of Homer1b/c Protects Against Chemically Induced Seizures Through Inhibition of mTOR Signaling

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Lei Cao

2015-03-01

Full Text Available Background: Homer is a family of post synaptic density proteins functionally and physically attached to target proteins at proline-rich sequences. Reducing Homer1b/c expression has been shown in previous studies to be protective against excitotoxic insults, implicating Homer1b/c in the physiological regulation of aberrant neuronal excitability. Methods: To test the efficacy of a Homer1b/c reducing therapy for disorders with a detrimental hyperexcitability profile in mice, we used small interfere RNA (siRNA to decrease endogenous Homer1b/c expression in mouse hippocampus. The baseline motor and cognitive behavior was measured by sensorimotor tests, Morris water maze and elevated plus maze tasks. The anti-epileptic effects of Homer1b/c knockdown were determined in two chemically induced seizure models induced by Picrotoxin (PTX or pentylenetetrazole (PTZ administration. Results: The results of sensorimotor tests, Morris water maze and elevated plus maze tasks showed that Homer1b/c reduction had no effect on baseline motor or cognitive behavior. In two chemically induced seizure models, mice with reduced Homerb/c protein had less severe seizures than control mice. Total Homer1b/c protein levels and seizure severity were highly correlated, such that those mice with the most severe seizures also had the highest levels of Homer1b/c. In addition, the phosphorylation of mammalian target of rapamycin (mTOR and its target protein S6 was significantly inhibited in Homer1b/c down-regulated mice. Homer1b/c knockdown-induced inhibition of mTOR pathway was partially ablated by the metabotropic glutamate receptor 5 (mGluR5 agonist CHPG. Conclusion: Our results demonstrate that endogenous Homer1b/c is integral for regulating neuronal hyperexcitability in adult animals and suggest that reduction of Homer1b/c could protect against chemically induced seizures through inhibition mTOR pathway.

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

Lipid peroxidation inhibition and antiradical activities of some leaf fractions of Mangifera indica.

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Badmus, Jelili A; Adedosu, Temitope O; Fatoki, John O; Adegbite, Victor A; Adaramoye, Oluwatosin A; Odunola, Oyeronke A

2011-01-01

This study was undertaken to assess in vitro lipid peroxidation inhibitions and anti-radical activities of methanolic, chloroform, ethyl acetate and water fractions of Mangifera indica leaf. Inhibition of Fe(2+)-induced lipid peroxidation (LPO) in egg, brain, and liver homogenates, 1,1-diphenyl-2-picrylhydrazyl (DPPH) and hydroxyl (OH-) radical scavenging activities were evaluated. Total phenol was assessed in all fractions, and the reducing power of methanolic fraction was compared to gallic acid and ascorbic acid. The results showed that Fe2+ induced significant lipid peroxidation (LPO) in all the homogenates. Ethyl acetate fraction showed the highest percentage inhibition of LPO in both egg yolk (68.3%) and brain (66.3%), while the aqueous fraction exerted the highest inhibition in liver homogenate (89.1%) at a concentration of 10 microg/mL. These observed inhibitions of LPO by these fractions were higher than that of ascorbic acid used as a standard. The DPPH radical scavenging ability exhibited by ethyl acetate fraction was found to be the highest with IC50 value of 1.5 microg/mL. The ethyl acetate and methanolic fractions had the highest OH- radical scavenging ability with the same IC50 value of 5 microg/mL. The total phenol content of ethyl acetate fraction was the highest with 0.127 microg/mg gallic acid equivalent (GAE). The reductive potential of methanolic fraction showed a concentration-dependent increase. This study showed that inhibition of LPO and the DPPH and OH- radicals scavenging abilities of Mangifera indica leaf could be related to the presence of phenolic compounds. Therefore, the ethyl acetate fraction of the leaf may be a good source of natural antioxidative agent.

Enhancement of Autophagy by Simvastatin through Inhibition of Rac1-mTOR Signaling Pathway in Coronary Arterial Myocytes

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Yu-Miao Wei

2013-06-01

Full Text Available Background/Aims: In addition to their action of lowering blood cholesterol levels, statins modulate biological characteristics and functions of arterial myocytes such as viability, proliferation, apoptosis, survival and contraction. The present study tested whether simvastatin, as a prototype statin, enhances autophagy in coronary arterial myocytes (CAMs to thereby exert their beneficial effects in atherosclerosis. Methods and Results: Using flow cytometry, we demonstrated that simvastatin significantly increased the autophagsome formation in CAMs. Western blot analysis confirmed that simvastatin significantly increased protein expression of typical autophagy markers LC3B and Beclin1 in these CAMs. Confocal microscopy further demonstrated that simvastatin increased fusion of autophagosomes with lysosomes, which was blocked by autophagy inhibitor 3-methyladenine or silencing of Atg7 genes. Simvastatin reduced mammalian target of rapamycin (mTOR activity, which was reversed by Rac1-GTPase overexpression and the mTOR agonist phosphatidic acid. Moreover, both Rac1-GTPase overexpression and activation of mTOR by phosphatidic acid drastically blocked simvastatin-induced autophagosome formation in CAMs. Interestingly, simvastatin increased protein expression of a contractile phenotype marker calponin in CAMs, which was blocked by autophagy inhibitor 3-methyladenine. Simvastatin markedly reduced proliferation of CAMs under both control and proatherogenic stimulation. However, this inhibitory effect of simvastatin on CAM proliferation was blocked by by autophagy inhibitor 3-methyladenine or silencing of Atg7 genes. Lastly, animal experiments demonstrated that simvastatin increased protein expression of LC3B and calponin in mouse coronary arteries. Conclusion: Our results indicate that simvastatin inhibits the Rac1-mTOR pathway and thereby increases autophagy in CAMs which may stabilize CAMs in the contractile phenotype to prevent proliferation and growth

Inhibition of rat microsomal lipid peroxidation by the oral administration of D002

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Menéndez R.

2000-01-01

Full Text Available The effect of D002, a defined mixture of higher primary alcohols purified from bee wax, on in vivo and in vitro lipid peroxidation was studied. The extent of lipid peroxidation was measured on the basis of the levels of thiobarbituric acid reactive substances (TBARS. When D002 (5-100 mg/kg body weight was administered orally to rats for two weeks, a partial inhibition of the in vitro enzymatic and non-enzymatic lipid peroxidation was observed in liver and brain microsomes. Maximal protection (46% occurred at a dose of 25 mg/kg. D002 behaved differently depending on both the presence of NADPH and the integrity of liver microsomes, which suggests that under conditions where microsomal metabolism was favored the protective effect of D002 was increased. D002 (25 mg/kg also completely inhibited carbon tetrachloride- and toluene-induced in vivo lipid peroxidation in liver and brain. Also, D002 significantly lowered in a dose-dependent manner the basal level of TBARS in liver (19-40% and brain (28-44% microsomes. We conclude that the oral administration of D002 (5, 25 and 100 mg/kg for two weeks protected rat liver and brain microsomes against microsomal lipid peroxidation in vitro and in vivo. Thus, D002 could be useful as a dietary natural antioxidant supplement. More studies are required before these data can be extrapolated to the recommendation for the use of D002 as a dietary antioxidant supplement for humans.

SAR405, a PIK3C3/Vps34 inhibitor that prevents autophagy and synergizes with MTOR inhibition in tumor cells.

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Pasquier, Benoit

2015-04-03

Autophagy plays an important role in cancer and it has been suggested that it functions not only as a tumor suppressor pathway to prevent tumor initiation, but also as a prosurvival pathway that helps tumor cells endure metabolic stress and resist death triggered by chemotherapeutic agents. We recently described the discovery of inhibitors of PIK3C3/Vps34 (phosphatidylinositol 3-kinase, catalytic subunit type 3), the lipid kinase component of the class III phosphatidylinositol 3-kinase (PtdIns3K). This PtdIns3K isoform has attracted significant attention in recent years because of its role in autophagy. Following chemical optimization we identified SAR405, a low molecular mass kinase inhibitor of PIK3C3, highly potent and selective with regard to other lipid and protein kinases. We demonstrated that inhibiting the catalytic activity of PIK3C3 disrupts vesicle trafficking from late endosomes to lysosomes. SAR405 treatment also inhibits autophagy induced either by starvation or by MTOR (mechanistic target of rapamycin) inhibition. Finally our results show that combining SAR405 with everolimus, the FDA-approved MTOR inhibitor, results in a significant synergy on the reduction of cell proliferation using renal tumor cells. This result indicates a potential therapeutic application for PIK3C3 inhibitors in cancer.

mTOR signaling promotes foam cell formation and inhibits foam cell egress through suppressing the SIRT1 signaling pathway.

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Zheng, Haixiang; Fu, Yucai; Huang, Yusheng; Zheng, Xinde; Yu, Wei; Wang, Wei

2017-09-01

Atherosclerosis (AS) is a chronic immuno‑inflammatory disease accompanied by dyslipidemia. The authors previously demonstrated that sirtuin 1 (SIRT1) may prevent atherogenesis through influencing the liver X receptor/C‑C chemokine receptor type 7/nuclear factor‑κB (LXR‑CCR7/NF‑κB) signaling pathway. Previous studies have suggested a role for mammalian target of rapamycin (mTOR) signaling in the pathogenesis of cardiovascular diseases. The present study investigated the potential association between mTOR signaling and SIRT1‑LXR‑CCR7/NF‑κB signaling (SIRT1 signaling) in AS pathogenesis. To induce foam cell formation, U937 cells were differentiated into macrophages by exposure to phorbol 12‑myristate 13‑acetate (PMA) for 24 h, followed by treatment with palmitate and oxidized low density lipoprotein for a further 24 h. Oil red O staining revealed a large accumulation of lipid droplets present in foam cells. Western blot analysis demonstrated increased protein levels of phosphorylated (p)‑mTOR and its downstream factor p‑ribosomal protein S6 kinase (p70S6K). Reverse transcription‑quantitative polymerase chain reaction and western blot analyses additionally revealed decreased expression of SIRT1, LXRα and CCR7 and increased expression of NF‑κB and its downstream factor tumor necrosis factor‑α (TNF‑α) in an atherogenetic condition induced by lysophosphatidic acid (LPA). In addition, abundant lipid droplets accumulated in U937‑LPA‑treated foam cells. Rapamycin, an mTOR inhibitor, suppressed the expression and activity of mTOR and p70S6K, however enhanced expression of SIRT1, LXRα, and CCR7. Conversely, rapamycin deceased TNF‑α and NF‑κB activity, the latter of which was further confirmed by immunofluorescence analysis demonstrating increased levels of NF‑κB present in the cytoplasm compared with the nucleus. The findings of the present study suggest that mTOR signaling promotes foam cell formation and inhibits foam

Antidepressant action of ketamine via mTOR is mediated by inhibition of nitrergic Rheb degradation.

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Harraz, M M; Tyagi, R; Cortés, P; Snyder, S H

2016-03-01

As traditional antidepressants act only after weeks/months, the discovery that ketamine, an antagonist of glutamate/N-methyl-D-aspartate (NMDA) receptors, elicits antidepressant actions in hours has been transformative. Its mechanism of action has been elusive, though enhanced mammalian target of rapamycin (mTOR) signaling is a major feature. We report a novel signaling pathway wherein NMDA receptor activation stimulates generation of nitric oxide (NO), which S-nitrosylates glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Nitrosylated GAPDH complexes with the ubiquitin-E3-ligase Siah1 and Rheb, a small G protein that activates mTOR. Siah1 degrades Rheb leading to reduced mTOR signaling, while ketamine, conversely, stabilizes Rheb that enhances mTOR signaling. Drugs selectively targeting components of this pathway may offer novel approaches to the treatment of depression.

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

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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

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

Poly(neutral red) based hydrogen peroxide biosensor for chromium determination by inhibition measurements.

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Attar, Aisha; Emilia Ghica, M; Amine, Aziz; Brett, Christopher M A

2014-08-30

Amperometric hydrogen peroxide enzyme inhibition biosensors based on horseradish peroxidase (HRP) immobilised on electropolymerised neutral red (NR) or directly on the surface of carbon film electrodes (CFE) have been successfully applied to the determination of toxic Cr(III) and Cr(VI). Parameters influencing the performance of the biosensor including the enzyme immobilisation method, the amount of hydrogen peroxide, applied potential and electrolyte pH were optimised. The inhibition of horseradish peroxidase by the chromium species was studied under the optimised conditions. Results from the quantitative analysis of chromium ions are discussed in terms of detection limit, linear range and sensitivity. The HRP kinetic interactions reveal mixed binding of Cr(III) with I50=3.8μM and inhibition binding constant Ki=11.3μM at HRP/PNR/CFE biosensors and uncompetitive binding of Cr(VI) with I50=3.9μM and Ki=0.78μM at HRP/CFE biosensors in the presence of H2O2 substrate. Interferences from other heavy metal ions were studied and the inhibition show very good selectivity towards Cr(III) and Cr(VI). Copyright © 2014 Elsevier B.V. All rights reserved.

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.

Inhibition of rat liver microsomal lipid peroxidation by N-acyldehydroalanines: An in vitro comparative study

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Buc-Calderon, P.; Roberfroid, M. (Universite Catholique de Louvain, Brussels (Belgium))

1989-09-01

Captodative substituted olefins are radical scavengers which react with free radicals to form stabilized radical adducts. One of those compounds, N-(paramethoxyphenylacetyl)dehydroalanine (AD-5), may react and scavenge both superoxide anion (O-2) and alk-oxyl radicals (RO.), and in this way prevent the appearance of their mediated biological effects. Nitrofurantoin and tert-butyl hydroperoxide were used as model compounds to stimulate free radical production and their mediated lipid peroxidation in rat liver microsomes. In addition, lipid peroxidation was also initiated by exposure of rat liver microsomal suspensions to ionizing radiation (gamma rays). The microsomal lipid peroxidation induced by these chemicals and physical agents was inhibited by the addition of AD-5. These effects were dose-dependent in a millimolar range of concentration. In addition, AD-5 has no effect on microsomal electron transport, showing that NADPH-cytochrome P450 reductase activity was not modified. These data, together with the comparisons of the effects of AD-5 and some antioxidant molecules such as superoxide dismutase, uric acid, and mannitol, support the conclusion that inhibition of lipid peroxidation by AD-5 is the result of its free radical scavenger activity. In addition, the inhibitory effect of AD-5 on microsomal lipid peroxidation was dependent of the nature of the free radical species involved in the initiation of the process, suggesting that O-2 is scavenged more efficiently than RO.

mTOR inhibitors in urinary bladder cancer.

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Pinto-Leite, R; Arantes-Rodrigues, R; Sousa, Nuno; Oliveira, P A; Santos, L

2016-09-01

Despite the great scientific advances that have been made in cancer treatment, there is still much to do, particularly with regard to urinary bladder cancer. Some of the drugs used in urinary bladder cancer treatment have been in use for more than 30 years and show reduced effectiveness and high recurrence rates. There have been several attempts to find new and more effective drugs, to be used alone or in combination with the drugs already in use, in order to overcome this situation.The biologically important mammalian target of rapamycin (mTOR) pathway is altered in cancer and mTOR inhibitors have raised many expectations as potentially important anticancer drugs. In this article, the authors will review the mTOR pathway and present their experiences of the use of some mTOR inhibitors, sirolimus, everolimus and temsirolimus, in isolation and in conjunction with non-mTOR inhibitors cisplatin and gemcitabine, on urinary bladder tumour cell lines. The non-muscle-invasive cell line, 5637, is the only one that exhibits a small alteration in the mTOR and AKT phosphorylation after rapalogs exposure. Also, there was a small inhibition of cell proliferation. With gemcitabine plus everolimus or temsirolimus, the results were encouraging as a more effective response was noticed with both combinations, especially in the 5637 and T24 cell lines. Cisplatin associated with everolimus or temsirolimus also gave promising results, as an antiproliferative effect was observed when the drugs were associated, in particular on the 5637 and HT1376 cell lines. Everolimus or temsirolimus in conjunction with gemcitabine or cisplatin could have an important role to play in urinary bladder cancer treatment, depending on the tumour grading.

MTOR-Driven Metabolic Reprogramming Regulates Legionella pneumophila Intracellular Niche Homeostasis

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Abshire, Camille F.; Roy, Craig R.

2016-01-01

Vacuolar bacterial pathogens are sheltered within unique membrane-bound organelles that expand over time to support bacterial replication. These compartments sequester bacterial molecules away from host cytosolic immunosurveillance pathways that induce antimicrobial responses. The mechanisms by which the human pulmonary pathogen Legionella pneumophila maintains niche homeostasis are poorly understood. We uncovered that the Legionella-containing vacuole (LCV) required a sustained supply of host lipids during expansion. Lipids shortage resulted in LCV rupture and initiation of a host cell death response, whereas excess of host lipids increased LCVs size and housing capacity. We found that lipids uptake from serum and de novo lipogenesis are distinct redundant supply mechanisms for membrane biogenesis in Legionella-infected macrophages. During infection, the metabolic checkpoint kinase Mechanistic Target of Rapamycin (MTOR) controlled lipogenesis through the Serum Response Element Binding Protein 1 and 2 (SREBP1/2) transcription factors. In Legionella-infected macrophages a host-driven response that required the Toll-like receptors (TLRs) adaptor protein Myeloid differentiation primary response gene 88 (Myd88) dampened MTOR signaling which in turn destabilized LCVs under serum starvation. Inactivation of the host MTOR-suppression pathway revealed that L. pneumophila sustained MTOR signaling throughout its intracellular infection cycle by a process that required the upstream regulator Phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K) and one or more Dot/Icm effector proteins. Legionella-sustained MTOR signaling facilitated LCV expansion and inhibition of the PI3K-MTOR-SREPB1/2 axis through pharmacological or genetic interference or by activation of the host MTOR-suppression response destabilized expanding LCVs, which in turn triggered cell death of infected macrophages. Our work identified a host metabolic requirement for LCV homeostasis and demonstrated that L

Gβγ interacts with mTOR and promotes its activation

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Robles-Molina, Evelyn [Department of Pharmacology, Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional (CINVESTAV-IPN), Apartado postal 14-740, México, D.F. 07360 (Mexico); Dionisio-Vicuña, Misael [Department of Cell Biology, Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional (CINVESTAV-IPN), Apartado postal 14-740, México, D.F. 07360 (Mexico); Guzmán-Hernández, María Luisa [Department of Pharmacology, Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional (CINVESTAV-IPN), Apartado postal 14-740, México, D.F. 07360 (Mexico); Reyes-Cruz, Guadalupe [Department of Cell Biology, Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional (CINVESTAV-IPN), Apartado postal 14-740, México, D.F. 07360 (Mexico); Vázquez-Prado, José, E-mail: jvazquez@cinvestav.mx [Department of Pharmacology, Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional (CINVESTAV-IPN), Apartado postal 14-740, México, D.F. 07360 (Mexico)

2014-02-07

Highlights: • Gβγ interacts with mTOR kinase domain via a mechanism sensitive to chronic treatment with rapamycin. • Gβγ interacts with mTORC1 and mTORC2 which correlates with its ability to promote mTORC1 and mTORC2 signaling. • Gβγ heterodimers containing different Gβ subunits, except Gβ{sub 4}, interact with mTOR. - Abstract: Diverse G protein-coupled receptors depend on Gβγ heterodimers to promote cell polarization and survival via direct activation of PI3Kγ and potentially other effectors. These events involve full activation of AKT via its phosphorylation at Ser473, suggesting that mTORC2, the kinase that phosphorylates AKT at Ser473, is activated downstream of Gβγ. Thus, we tested the hypothesis that Gβγ directly contributes to mTOR signaling. Here, we demonstrate that endogenous mTOR interacts with Gβγ. Cell stimulation with serum modulates Gβγ interaction with mTOR. The carboxyl terminal region of mTOR, expressed as a GST-fusion protein, including the serine/threonine kinase domain, binds Gβγ heterodimers containing different Gβ subunits, except Gβ{sub 4}. Both, mTORC1 and mTORC2 complexes interact with Gβ{sub 1}γ{sub 2} which promotes phosphorylation of their respective substrates, p70S6K and AKT. In addition, chronic treatment with rapamycin, a condition known to interfere with assembly of mTORC2, reduces the interaction between Gβγ and mTOR and the phosphorylation of AKT; whereas overexpression of Gαi interfered with the effect of Gβγ as promoter of p70S6K and AKT phosphorylation. Altogether, our results suggest that Gβγ positively regulates mTOR signaling via direct interactions and provide further support to emerging strategies based on the therapeutical potential of inhibiting different Gβγ signaling interfaces.

mTOR inhibitors alone and in combination with JAK2 inhibitors effectively inhibit cells of myeloproliferative neoplasms.

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Costanza Bogani

Full Text Available BACKGROUND: Dysregulated signaling of the JAK/STAT pathway is a common feature of chronic myeloproliferative neoplasms (MPN, usually associated with JAK2V617F mutation. Recent clinical trials with JAK2 inhibitors showed significant improvements in splenomegaly and constitutional symptoms in patients with myelofibrosis but meaningful molecular responses were not documented. Accordingly, there remains a need for exploring new treatment strategies of MPN. A potential additional target for treatment is represented by the PI3K/AKT/mammalian target of rapamycin (mTOR pathway that has been found constitutively activated in MPN cells; proof-of-evidence of efficacy of the mTOR inhibitor RAD001 has been obtained recently in a Phase I/II trial in patients with myelofibrosis. The aim of the study was to characterize the effects in vitro of mTOR inhibitors, used alone and in combination with JAK2 inhibitors, against MPN cells. FINDINGS: Mouse and human JAK2V617F mutated cell lines and primary hematopoietic progenitors from MPN patients were challenged with an allosteric (RAD001 and an ATP-competitive (PP242 mTOR inhibitor and two JAK2 inhibitors (AZD1480 and ruxolitinib. mTOR inhibitors effectively reduced proliferation and colony formation of cell lines through a slowed cell division mediated by changes in cell cycle transition to the S-phase. mTOR inhibitors also impaired the proliferation and prevented colony formation from MPN hematopoietic progenitors at doses significantly lower than healthy controls. JAK2 inhibitors produced similar antiproliferative effects in MPN cell lines and primary cells but were more potent inducers of apoptosis, as also supported by differential effects on cyclinD1, PIM1 and BcLxL expression levels. Co-treatment of mTOR inhibitor with JAK2 inhibitor resulted in synergistic activity against the proliferation of JAK2V617F mutated cell lines and significantly reduced erythropoietin-independent colony growth in patients with

Repression of protein translation and mTOR signaling by proteasome inhibitor in colon cancer cells

International Nuclear Information System (INIS)

Wu, William Ka Kei; Volta, Viviana; Cho, Chi Hin; Wu, Ya Chun; Li, Hai Tao; Yu, Le; Li, Zhi Jie; Sung, Joseph Jao Yiu

2009-01-01

Protein homeostasis relies on a balance between protein synthesis and protein degradation. The ubiquitin-proteasome system is a major catabolic pathway for protein degradation. In this respect, proteasome inhibition has been used therapeutically for the treatment of cancer. Whether inhibition of protein degradation by proteasome inhibitor can repress protein translation via a negative feedback mechanism, however, is unknown. In this study, proteasome inhibitor MG-132 lowered the proliferation of colon cancer cells HT-29 and SW1116. In this connection, MG-132 reduced the phosphorylation of mammalian target of rapamycin (mTOR) at Ser2448 and Ser2481 and the phosphorylation of its downstream targets 4E-BP1 and p70/p85 S6 kinases. Further analysis revealed that MG-132 inhibited protein translation as evidenced by the reductions of 35 S-methionine incorporation and polysomes/80S ratio. Knockdown of raptor, a structural component of mTOR complex 1, mimicked the anti-proliferative effect of MG-132. To conclude, we demonstrate that the inhibition of protein degradation by proteasome inhibitor represses mTOR signaling and protein translation in colon cancer cells.

Discovery and optimization of potent and selective imidazopyridine and imidazopyridazine mTOR inhibitors.

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Peterson, Emily A; Boezio, Alessandro A; Andrews, Paul S; Boezio, Christiane M; Bush, Tammy L; Cheng, Alan C; Choquette, Deborah; Coats, James R; Colletti, Adria E; Copeland, Katrina W; DuPont, Michelle; Graceffa, Russell; Grubinska, Barbara; Kim, Joseph L; Lewis, Richard T; Liu, Jingzhou; Mullady, Erin L; Potashman, Michele H; Romero, Karina; Shaffer, Paul L; Stanton, Mary K; Stellwagen, John C; Teffera, Yohannes; Yi, Shuyan; Cai, Ti; La, Daniel S

2012-08-01

mTOR is a critical regulator of cellular signaling downstream of multiple growth factors. The mTOR/PI3K/AKT pathway is frequently mutated in human cancers and is thus an important oncology target. Herein we report the evolution of our program to discover ATP-competitive mTOR inhibitors that demonstrate improved pharmacokinetic properties and selectivity compared to our previous leads. Through targeted SAR and structure-guided design, new imidazopyridine and imidazopyridazine scaffolds were identified that demonstrated superior inhibition of mTOR in cellular assays, selectivity over the closely related PIKK family and improved in vivo clearance over our previously reported benzimidazole series. Copyright © 2012. Published by Elsevier Ltd.

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.

A chemical genetic screen for mTOR pathway inhibitors based on 4E-BP-dependent nuclear accumulation of eIF4E.

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Livingstone, Mark; Larsson, Ola; Sukarieh, Rami; Pelletier, Jerry; Sonenberg, Nahum

2009-12-24

The signal transduction pathway wherein mTOR regulates cellular growth and proliferation is an active target for drug discovery. The search for new mTOR inhibitors has recently yielded a handful of promising compounds that hold therapeutic potential. This search has been limited by the lack of a high-throughput assay to monitor the phosphorylation of a direct rapamycin-sensitive mTOR substrate in cells. Here we describe a novel cell-based chemical genetic screen useful for efficiently monitoring mTOR signaling to 4E-BPs in response to stimuli. The screen is based on the nuclear accumulation of eIF4E, which occurs in a 4E-BP-dependent manner specifically upon inhibition of mTOR signaling. Using this assay in a small-scale screen, we have identified several compounds not previously known to inhibit mTOR signaling, demonstrating that this method can be adapted to larger screens. Copyright 2009 Elsevier Ltd. All rights reserved.

TIS21/(BTG2) negatively regulates estradiol-stimulated expansion of hematopoietic stem cells by derepressing Akt phosphorylation and inhibiting mTOR signal transduction.

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Kim, Bong Cho; Ryu, Min Sook; Oh, S Paul; Lim, In Kyoung

2008-09-01

It has been known that 12-O-tetradecanoyl phorbol-13-acetate-inducible sequence 21 (TIS21), ortholog of human B-cell translocation gene 2, regulates expansions of stage-specific thymocytes and hematopoietic progenitors. In the present study, lineage-negative (Lin(-))/stem cell antigen-1-positive (Sca-1+)/c-Kit+ (LSK) cell content was significantly elevated in bone marrow (BM) of TIS21-knockout (TIS21(-/-)) female mice, suggesting 17beta-estradiol (E(2))-regulated progenitor expansion. E(2) induced DNA synthesis and cell proliferation of mouse embryonic fibroblasts (MEFs) isolated from TIS21(-/-) mice, but not wild type (WT). In contrast to WT, E(2) failed to activate protein kinase B (Akt) in the TIS21(-/-) MEFs, independent of extracellular signal-regulated kinase 1/2 (Erk1/2) activation. Despite attenuation of Akt activation, mammalian target of rapamycin (mTOR) was constitutively activated in the TIS21(-/-) MEFs. Furthermore, mitogen-activated protein kinase 1/2 inhibitor or knockdown of Erk1 could restore activation of Akt and downregulate mTOR. Immunoprecipitation showed Akt preferentially bound to phosphorylated Erk1/2 (p-Erk1/2) in TIS21(-/-) cells, but reconstitution of TIS21 inhibited their interaction. E(2)-injected TIS21(-/-) male mice also increased LSK cells in BM. Taken together, expansion of hematopoietic progenitors in TIS21(-/-) female mice might be through inhibition of Akt activation, and constitutive activation of mTOR via preferential binding of TIS21 to E(2)-induced p-Erk1/2, compared with that of Akt. Our results suggest that TIS21 plays a pivotal role in maintaining the hematopoietic stem cell compartment and hematopoiesis.

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

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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.

Skeletal myocyte hypertrophy requires mTOR kinase activity and S6K1

International Nuclear Information System (INIS)

Park, In-Hyun; Erbay, Ebru; Nuzzi, Paul; Chen Jie

2005-01-01

The protein kinase mammalian target of rapamycin (mTOR) is a central regulator of cell proliferation and growth, with the ribosomal subunit S6 kinase 1 (S6K1) as one of the key downstream signaling effectors. A critical role of mTOR signaling in skeletal muscle differentiation has been identified recently, and an unusual regulatory mechanism independent of mTOR kinase activity and S6K1 is revealed. An mTOR pathway has also been reported to regulate skeletal muscle hypertrophy, but the regulatory mechanism is not completely understood. Here, we report the investigation of mTOR's function in insulin growth factor I (IGF-I)-induced C2C12 myotube hypertrophy. Added at a later stage when rapamycin no longer had any effect on normal myocyte differentiation, rapamycin completely blocked myocyte hypertrophy as measured by myotube diameter. Importantly, a concerted increase of average myonuclei per myotube was observed in IGF-I-stimulated myotubes, which was also inhibited by rapamycin added at a time when it no longer affected normal differentiation. The mTOR protein level, its catalytic activity, its phosphorylation on Ser2448, and the activity of S6K1 were all found increased in IGF-I-stimulated myotubes compared to unstimulated myotubes. Using C2C12 cells stably expressing rapamycin-resistant forms of mTOR and S6K1, we provide genetic evidence for the requirement of mTOR and its downstream effector S6K1 in the regulation of myotube hypertrophy. Our results suggest distinct mTOR signaling mechanisms in different stages of skeletal muscle development: While mTOR regulates the initial myoblast differentiation in a kinase-independent and S6K1-independent manner, the hypertrophic function of mTOR requires its kinase activity and employs S6K1 as a downstream effector

mTOR signaling promotes stem cell activation via counterbalancing BMP-mediated suppression during hair regeneration.

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Deng, Zhili; Lei, Xiaohua; Zhang, Xudong; Zhang, Huishan; Liu, Shuang; Chen, Qi; Hu, Huimin; Wang, Xinyue; Ning, Lina; Cao, Yujing; Zhao, Tongbiao; Zhou, Jiaxi; Chen, Ting; Duan, Enkui

2015-02-01

Hair follicles (HFs) undergo cycles of degeneration (catagen), rest (telogen), and regeneration (anagen) phases. Anagen begins when the hair follicle stem cells (HFSCs) obtain sufficient activation cues to overcome suppressive signals, mainly the BMP pathway, from their niche cells. Here, we unveil that mTOR complex 1 (mTORC1) signaling is activated in HFSCs, which coincides with the HFSC activation at the telogen-to-anagen transition. By using both an inducible conditional gene targeting strategy and a pharmacological inhibition method to ablate or inhibit mTOR signaling in adult skin epithelium before anagen initiation, we demonstrate that HFs that cannot respond to mTOR signaling display significantly delayed HFSC activation and extended telogen. Unexpectedly, BMP signaling activity is dramatically prolonged in mTOR signaling-deficient HFs. Through both gain- and loss-of-function studies in vitro, we show that mTORC1 signaling negatively affects BMP signaling, which serves as a main mechanism whereby mTORC1 signaling facilitates HFSC activation. Indeed, in vivo suppression of BMP by its antagonist Noggin rescues the HFSC activation defect in mTORC1-null skin. Our findings reveal a critical role for mTOR signaling in regulating stem cell activation through counterbalancing BMP-mediated repression during hair regeneration. © The Author (2015). Published by Oxford University Press on behalf of Journal of Molecular Cell Biology, IBCB, SIBS, CAS. All rights reserved.

Photochemistry of peroxoborates: borate inhibition of the photodecomposition of hydrogen peroxide.

Science.gov (United States)

Rey, Sébastien; Davies, D Martin

2006-12-13

The UV absorbance and photochemical decomposition kinetics of hydrogen peroxide in borate/boric acid buffers were investigated as a function of pH, total peroxide concentration, and total boron concentration. At higher pH borate/boric acid inhibits the photodecomposition of hydrogen peroxide (molar absorptivity and quantum yield of H(2)O(2) and HO(2) (-), (19.0+/-0.3) M(-1) cm(-1) and 1, and (237+/-7) M(-1) cm(-1) and 0.8+/-0.1, respectively). The results are consistent with the equilibrium formation of the anions monoperoxoborate, K(BOOH)=[H(+)][HOOB(OH)(3) (-)]/([B(OH)(3)][H(2)O(2)]), 2.0 x 10(-8), R. Pizer, C. Tihal, Inorg. Chem. 1987, 26, 3639-3642, and monoperoxodiborate, K(BOOB)=[BOOB(2-)]/([B(OH)(4) (-)][HOOB(OH)(3) (-)]), 1.0+/-0.3 or 4.3+/-0.9, depending upon the conditions, with molar absorptivity, (19+/-1) M(-1) cm(-1) and (86+/-15) M(-1) cm(-1), respectively, and respective quantum yields, 1.1+/-0.1 and 0.04+/-0.04. The low quantum yield of monoperoxodiborate is discussed in terms of the slower diffusion apart of incipient (.)OB(OH)(3) (-) radicals than may be possible for (.)OH radicals, or a possible oxygen-bridged cyclic structure of the monoperoxodiborate.

The Protective Effects of Κ-Opioid Receptor Stimulation in Hypoxic Pulmonary Hypertension Involve Inhibition of Autophagy Through the AMPK-MTOR Pathway

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Yaguang Zhou

2017-12-01

Full Text Available Background/Aims: In a previous study, we showed that κ-opioid receptor stimulation with the selective agonist U50,488H ameliorated hypoxic pulmonary hypertension (HPH. However, the roles that pulmonary arterial smooth muscle cell (PASMC proliferation, apoptosis, and autophagy play in κ-opioid receptor-mediated protection against HPH are still unknown. The goal of the present study was to investigate the role of autophagy in U50,488H-induced HPH protection and the underlying mechanisms. Methods: Rats were exposed to 10% oxygen for three weeks to induce HPH. After hypoxia, the mean pulmonary arterial pressure (mPAP and the right ventricular pressure (RVP were measured. Cell viability was monitored using the Cell Counting Kit-8 (CCK-8 assay. Cell apoptosis was detected by flow cytometry and Western blot. Autophagy was assessed by means of the mRFP-GFP-LC3 adenovirus transfection assay and by Western blot. Results: Inhibition of autophagy by the administration of chloroquine prevented the development of HPH in the rat model, as evidenced by significantly reduced mPAP and RVP, as well as decreased autophagy. U50,488H mimicked the effects of chloroquine, and the effects of U50,488H were blocked by nor-BNI, a selective κ-opioid receptor antagonist. In vitro experiments showed that the inhibition of autophagy by chloroquine was associated with decreased proliferation and increased apoptosis of PASMCs. Under hypoxia, U50,488H also significantly inhibited autophagy, reduced proliferation and increased apoptosis of PASMCs. These effects of U50,488H were blocked by nor-BNI. Moreover, exposure to hypoxic conditions significantly increased AMPK phosphorylation and reduced mTOR phosphorylation, and these effects were abrogated by U50,488H. The effects of U50,488H on PASMC autophagy were inhibited by AICAR, a selective AMPK agonist, or by rapamycin, a selective mTOR inhibitor. Conclusion: Our data provide evidence for the first time that κ-opioid receptor

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 ...

AMP-Activated Protein Kinase Alleviates Extracellular Matrix Accumulation in High Glucose-Induced Renal Fibroblasts through mTOR Signaling Pathway

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Xia Luo

2015-01-01

Full Text Available Background/Aims: Extracellular matrix accumulation contributes significantly to the pathogenesis of diabetic nephropathy. Although AMP-activated protein kinase (AMPK has been found to inhibit extracellular matrix synthesis by experiments in vivo and vitro, its role in alleviating the deposition of extracellular matrix in renal interstitial fibroblasts has not been well defined. Methods: Currently, we conducted this study to investigate the effects of AMPK on high glucose-induced extracellular matrix synthesis and involved intracellular signaling pathway by using western blot in the kidney fibroblast cell line (NRK-49f. Results: Collagen IV protein levels were significantly increased by high glucose in a time-dependent manner. This was associated with a decrease in Thr72 phosphorylation of AMPK and an increase in phosphorylation of mTOR on Ser2448. High glucose-induced extracellular matrix accumulation and mTOR activation were significantly inhibited by the co-treatment of rAAV-AMPKα1312 (encoding constitutively active AMPKα1 whereas activated by r-AAV-AMPKα1D157A (encoding dominant negative AMPKα1. In cultured renal fibroblasts, overexpression of AMPKα1D157A upregulated mTOR signaling and matrix synthesis, which were ameliorated by co-treatment with the inhibitor of mTOR, rapamycin. Conclusion: Collectively, these findings indicate that AMPK exerts renoprotective effects by inhibiting the accumulation of extracellular matrix through mTOR signaling pathway.

Digestibility of Quinoa (Chenopodium quinoa Willd.) Protein Concentrate and Its Potential to Inhibit Lipid Peroxidation in the Zebrafish Larvae Model.

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Vilcacundo, R; Barrio, D; Carpio, C; García-Ruiz, A; Rúales, J; Hernández-Ledesma, B; Carrillo, W

2017-09-01

Quinoa protein concentrate (QPC) was extracted and digested under in vitro gastrointestinal conditions. The protein content of QPC was in the range between 52.40 and 65.01% depending on the assay used. Quinoa proteins were almost completely hydrolyzed by pepsin at pH of 1.2, 2.0, and 3.2. At high pH, only partial hydrolysis was observed. During the duodenal phase, no intact proteins were visible, indicating their susceptibility to the in vitro simulated digestive conditions. Zebrafish larvae model was used to evaluate the in vivo ability of gastrointestinal digests to inhibit lipid peroxidation. Gastric digestion at pH 1.2 showed the highest lipid peroxidation inhibition percentage (75.15%). The lipid peroxidation activity increased after the duodenal phase. The digest obtained at the end of the digestive process showed an inhibition percentage of 82.10%, comparable to that showed when using BHT as positive control (87.13%).

Systemic inhibition of mTOR kinase via rapamycin disrupts consolidation and reconsolidation of auditory fear memory.

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Mac Callum, Phillip E; Hebert, Mark; Adamec, Robert E; Blundell, Jacqueline

2014-07-01

The mammalian target of rapamycin (mTOR) kinase is a critical regulator of mRNA translation and is known to be involved in various long lasting forms of synaptic and behavioural plasticity. However, information concerning the temporal pattern of mTOR activation and susceptibility to pharmacological intervention during both consolidation and reconsolidation of long-term memory (LTM) remains scant. Male C57BL/6 mice were injected systemically with rapamycin at various time points following conditioning or retrieval in an auditory fear conditioning paradigm, and compared to vehicle (and/or anisomycin) controls for subsequent memory recall. Systemic blockade of mTOR with rapamycin immediately or 12h after training or reactivation impairs both consolidation and reconsolidation of an auditory fear memory. Further behavioural analysis revealed that the enduring effects of rapamycin on reconsolidation are dependent upon reactivation of the memory trace. Rapamycin, however, has no effect on short-term memory or the ability to retrieve an established fear memory. Collectively, our data suggest that biphasic mTOR signalling is essential for both consolidation and reconsolidation-like activities that contribute to the formation, re-stabilization, and persistence of long term auditory-fear memories, while not influencing other aspects of the memory trace. These findings also provide evidence for a cogent treatment model for reducing the emotional strength of established, traumatic memories analogous to those observed in acquired anxiety disorders such as posttraumatic stress disorder (PTSD) and specific phobias, through pharmacologic blockade of mTOR using systemic rapamycin following reactivation. Copyright © 2013 Elsevier Inc. All rights reserved.

mTOR signaling plays a critical role in the defects observed in muscle-derived stem/progenitor cells isolated from a murine model of accelerated aging.

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Takayama, Koji; Kawakami, Yohei; Lavasani, Mitra; Mu, Xiaodong; Cummins, James H; Yurube, Takashi; Kuroda, Ryosuke; Kurosaka, Masahiro; Fu, Freddie H; Robbins, Paul D; Niedernhofer, Laura J; Huard, Johnny

2017-07-01

Mice expressing reduced levels of ERCC1-XPF (Ercc1 -/Δ mice) demonstrate premature onset of age-related changes due to decreased repair of DNA damage. Muscle-derived stem/progenitor cells (MDSPCs) isolated from Ercc1 -/Δ mice have an impaired capacity for cell differentiation. The mammalian target of rapamycin (mTOR) is a critical regulator of cell growth in response to nutrient, hormone, and oxygen levels. Inhibition of the mTOR pathway extends the lifespan of several species. Here, we examined the role of mTOR in regulating the MDSPC dysfunction that occurs with accelerated aging. We show that mTOR signaling pathways are activated in Ercc1 -/Δ MDSPCs compared with wild-type (WT) MDSPCs. Additionally, inhibiting mTOR with rapamycin promoted autophagy and improved the myogenic differentiation capacity of the Ercc1 -/Δ MDSPCs. The percent of apoptotic and senescent cells in Ercc1 -/Δ MDSPC cultures was decreased upon mTOR inhibition. These results establish that mTOR signaling contributes to stem cell dysfunction and cell fate decisions in response to endogenous DNA damage. Therefore, mTOR represents a potential therapeutic target for improving defective, aged stem cells. © 2016 The Authors. Journal of Orthopaedic Research Published by Wiley Periodicals, Inc. on behalf of Orthopaedic Research Society. J Orthop Res 35:1375-1382, 2017. © 2016 The Authors. Journal of Orthopaedic Research Published by Wiley Periodicals, Inc. on behalf of Orthopaedic Research Society.

Inhibition of hydrogen peroxide induced injuring on human skin fibroblast by Ulva prolifera polysaccharide.

Science.gov (United States)

Cai, Chuner; Guo, Ziye; Yang, Yayun; Geng, Zhonglei; Tang, Langlang; Zhao, Minglin; Qiu, Yuyan; Chen, Yifan; He, Peimin

2016-10-01

Ulva prolifera can protect human skin fibroblast from being injured by hydrogen peroxide. This work studied the composition of Ulva prolifera polysaccharide and identified its physicochemical properties. The results showed that the cell proliferation of 0.5mg/mL crude polysaccharide was 154.4% of that in negative control group. Moreover, ROS detection indices, including DCFH-DA, GSH-PX, MDA and CAT, indicated that crude polysaccharide could improve cellular ability to scavenge free radical and decrease the injury on human skin fibroblast by hydrogen peroxide. In purified polysaccharide, the activity of fraction P1-1 was the highest, with 174.6% of that in negative control group. The average molecular weight of P1-1 was 137kD with 18.0% of sulfate content. This work showed the inhibition of hydrogen peroxide induced injuries on human skin fibroblast by Ulva prolifera polysaccharide, which may further evaluate the application of U. prolifera on cosmetics. Copyright © 2016 Elsevier B.V. All rights reserved.

Arctigenin inhibits the activation of the mTOR pathway, resulting in autophagic cell death and decreased ER expression in ER-positive human breast cancer cells.

Science.gov (United States)

Maxwell, Thressi; Lee, Kyu Shik; Kim, Soyoung; Nam, Kyung-Soo

2018-04-01

Arctigenin, a member of the Asteraceae family, is a biologically active lignan that is consumed worldwide due to its several health benefits. However, its use may pose a problem for patients with estrogen receptor (ER)α-positive breast cancer, since studies have shown that arctigenin is a phytoestrogen that exerts a proliferative effect by binding to the ER. Thus, in this study, we examined the effect of arctigenin on ERα-positive MCF-7 human breast cancer cells to determine whether the consumption of arctigenin is safe for patients with breast cancer. First, we found that arctigenin inhibited the viability of the MCF-7 cells, and colony formation assay confirmed that this effect was cytotoxic rather than cytostatic. The cytotoxic effects were not mediated by cell cycle arrest, apoptosis, or necroptosis, despite DNA damage, as indicated by poly(ADP-ribose) polymerase (PARP) cleavage and phosphorylated H2A.X. An increase in lipidated LC3, a marker of autophagosome formation, was observed, indicating that autophagy was induced by arctigenin, which was found to be triggered by the inhibition of the mechanistic target of rapamycin (mTOR) pathway. We then examined the effects of arctigenin on ERα expression and determined whether it affects the sensitivity of the cells to tamoxifen, as tamoxifen is commonly used against hormone-responsive cancers and is known to act via the ERα. We found that treatment with arctigenin effectively downregulated ERα expression, which was found to be a consequence of the inhibition of the mTOR pathway. However, treatment with arctigenin in combination with tamoxifen did not affect the sensitivity of the cells to tamoxifen, but instead, exerted a synergistic effect. On the whole, our data indicate that the phytoestrogen, arctigenin, mainly targeted the mTOR pathway in ERα-positive MCF-7 human breast cancer cells, leading to autophagy-induced cell death and the downregulation of ERα expression. Furthermore, the synergistic effects

Resveratrol engages AMPK to attenuate ERK and mTOR signaling in sensory neurons and inhibits incision-induced acute and chronic pain

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Tillu Dipti V

2012-01-01

Full Text Available Abstract Background Despite advances in our understanding of basic mechanisms driving post-surgical pain, treating incision-induced pain remains a major clinical challenge. Moreover, surgery has been implicated as a major cause of chronic pain conditions. Hence, more efficacious treatments are needed to inhibit incision-induced pain and prevent the transition to chronic pain following surgery. We reasoned that activators of AMP-activated protein kinase (AMPK may represent a novel treatment avenue for the local treatment of incision-induced pain because AMPK activators inhibit ERK and mTOR signaling, two important pathways involved in the sensitization of peripheral nociceptors. Results To test this hypothesis we used a potent and efficacious activator of AMPK, resveratrol. Our results demonstrate that resveratrol profoundly inhibits ERK and mTOR signaling in sensory neurons in a time- and concentration-dependent fashion and that these effects are mediated by AMPK activation and independent of sirtuin activity. Interleukin-6 (IL-6 is thought to play an important role in incision-induced pain and resveratrol potently inhibited IL-6-mediated signaling to ERK in sensory neurons and blocked IL-6-mediated allodynia in vivo through a local mechanism of action. Using a model of incision-induced allodynia in mice, we further demonstrate that local injection of resveratrol around the surgical wound strongly attenuates incision-induced allodynia. Intraplantar IL-6 injection and plantar incision induces persistent nociceptive sensitization to PGE2 injection into the affected paw after the resolution of allodynia to the initial stimulus. We further show that resveratrol treatment at the time of IL-6 injection or plantar incision completely blocks the development of persistent nociceptive sensitization consistent with the blockade of a transition to a chronic pain state by resveratrol treatment. Conclusions These results highlight the importance of signaling

mTOR drives cerebral blood flow and memory deficits in LDLR-/- mice modeling atherosclerosis and vascular cognitive impairment.

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Jahrling, Jordan B; Lin, Ai-Ling; DeRosa, Nicholas; Hussong, Stacy A; Van Skike, Candice E; Girotti, Milena; Javors, Martin; Zhao, Qingwei; Maslin, Leigh Ann; Asmis, Reto; Galvan, Veronica

2018-01-01

We recently showed that mTOR attenuation blocks progression and abrogates established cognitive deficits in Alzheimer's disease (AD) mouse models. These outcomes were associated with the restoration of cerebral blood flow (CBF) and brain vascular density (BVD) resulting from relief of mTOR inhibition of NO release. Recent reports suggested a role of mTOR in atherosclerosis. Because mTOR drives aging and vascular dysfunction is a universal feature of aging, we hypothesized that mTOR may contribute to brain vascular and cognitive dysfunction associated with atherosclerosis. We measured CBF, BVD, cognitive function, markers of inflammation, and parameters of cardiovascular disease in LDLR -/- mice fed maintenance or high-fat diet ± rapamycin. Cardiovascular pathologies were proportional to severity of brain vascular dysfunction. Aortic atheromas were reduced, CBF and BVD were restored, and cognitive dysfunction was attenuated potentially through reduction in systemic and brain inflammation following chronic mTOR attenuation. Our studies suggest that mTOR regulates vascular integrity and function and that mTOR attenuation may restore neurovascular function and cardiovascular health. Together with our previous studies in AD models, our data suggest mTOR-driven vascular damage may be a mechanism shared by age-associated neurological diseases. Therefore, mTOR attenuation may have promise for treatment of cognitive impairment in atherosclerosis.

Metabolic Symbiosis Enables Adaptive Resistance to Anti-angiogenic Therapy that Is Dependent on mTOR Signaling

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Elizabeth Allen

2016-05-01

Full Text Available Therapeutic targeting of tumor angiogenesis with VEGF inhibitors results in demonstrable, but transitory efficacy in certain human tumors and mouse models of cancer, limited by unconventional forms of adaptive/evasive resistance. In one such mouse model, potent angiogenesis inhibitors elicit compartmental reorganization of cancer cells around remaining blood vessels. The glucose and lactate transporters GLUT1 and MCT4 are induced in distal hypoxic cells in a HIF1α-dependent fashion, indicative of glycolysis. Tumor cells proximal to blood vessels instead express the lactate transporter MCT1, and p-S6, the latter reflecting mTOR signaling. Normoxic cancer cells import and metabolize lactate, resulting in upregulation of mTOR signaling via glutamine metabolism enhanced by lactate catabolism. Thus, metabolic symbiosis is established in the face of angiogenesis inhibition, whereby hypoxic cancer cells import glucose and export lactate, while normoxic cells import and catabolize lactate. mTOR signaling inhibition disrupts this metabolic symbiosis, associated with upregulation of the glucose transporter GLUT2.

Essential oil from lemon peels inhibit key enzymes linked to neurodegenerative conditions and pro-oxidant induced lipid peroxidation.

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Oboh, Ganiyu; Olasehinde, Tosin A; Ademosun, Ayokunle O

2014-01-01

This study sought to investigate the effects of essential oil from lemon (Citrus limoni) peels on acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) activities in vitro. The essential oil was extracted by hydrodistillation, dried with anhydrous Na2SO4 and characterized using gas chromatography. Antioxidant properties of the oil and inhibition of pro-oxidant-induced lipid peroxidation in rats brain homogenate were also assessed. The essential oil inhibited AChE and BChE activities in a concentration-dependent manner. GC analysis revealed the presence of sabinene, limonene, α-pinene, β-pinene, neral, geranial, 1,8-cineole, linalool, borneol, α-terpineol, terpinen-4-ol, linalyl acetate and β-caryophyllene. Furthermore, the essential oil exhibited antioxidant activities as typified by ferric reducing property, Fe(2+)-chelation and radicals [DPPH, ABTS, OH, NO] scavenging abilities. The inhibition of AChE and BChE activities, inhibition of pro-oxidant induced lipid peroxidation and antioxidant activities could be possible mechanisms for the use of the essential oil in the management and prevention of oxidative stress-induced neurodegeneration.

Hypothalamic mTOR pathway mediates thyroid hormone-induced hyperphagia in hyperthyroidism.

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Varela, Luis; Martínez-Sánchez, Noelia; Gallego, Rosalía; Vázquez, María J; Roa, Juan; Gándara, Marina; Schoenmakers, Erik; Nogueiras, Rubén; Chatterjee, Krishna; Tena-Sempere, Manuel; Diéguez, Carlos; López, Miguel

2012-06-01

Hyperthyroidism is characterized in rats by increased energy expenditure and marked hyperphagia. Alterations of thermogenesis linked to hyperthyroidism are associated with dysregulation of hypothalamic AMPK and fatty acid metabolism; however, the central mechanisms mediating hyperthyroidism-induced hyperphagia remain largely unclear. Here, we demonstrate that hyperthyroid rats exhibit marked up-regulation of the hypothalamic mammalian target of rapamycin (mTOR) signalling pathway associated with increased mRNA levels of agouti-related protein (AgRP) and neuropeptide Y (NPY), and decreased mRNA levels of pro-opiomelanocortin (POMC) in the arcuate nucleus of the hypothalamus (ARC), an area where mTOR co-localizes with thyroid hormone receptor-α (TRα). Central administration of thyroid hormone (T3) or genetic activation of thyroid hormone signalling in the ARC recapitulated hyperthyroidism effects on feeding and the mTOR pathway. In turn, central inhibition of mTOR signalling with rapamycin in hyperthyroid rats reversed hyperphagia and normalized the expression of ARC-derived neuropeptides, resulting in substantial body weight loss. The data indicate that in the hyperthyroid state, increased feeding is associated with thyroid hormone-induced up-regulation of mTOR signalling. Furthermore, our findings that different neuronal modulations influence food intake and energy expenditure in hyperthyroidism pave the way for a more rational design of specific and selective therapeutic compounds aimed at reversing the metabolic consequences of this disease. Copyright © 2012 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Follistatin-mediated skeletal muscle hypertrophy is regulated by Smad3 and mTOR independently of myostatin

Science.gov (United States)

Winbanks, Catherine E.; Weeks, Kate L.; Thomson, Rachel E.; Sepulveda, Patricio V.; Beyer, Claudia; Qian, Hongwei; Chen, Justin L.; Allen, James M.; Lancaster, Graeme I.; Febbraio, Mark A.; Harrison, Craig A.; McMullen, Julie R.; Chamberlain, Jeffrey S.

2012-01-01

Follistatin is essential for skeletal muscle development and growth, but the intracellular signaling networks that regulate follistatin-mediated effects are not well defined. We show here that the administration of an adeno-associated viral vector expressing follistatin-288aa (rAAV6:Fst-288) markedly increased muscle mass and force-producing capacity concomitant with increased protein synthesis and mammalian target of rapamycin (mTOR) activation. These effects were attenuated by inhibition of mTOR or deletion of S6K1/2. Furthermore, we identify Smad3 as the critical intracellular link that mediates the effects of follistatin on mTOR signaling. Expression of constitutively active Smad3 not only markedly prevented skeletal muscle growth induced by follistatin but also potently suppressed follistatin-induced Akt/mTOR/S6K signaling. Importantly, the regulation of Smad3- and mTOR-dependent events by follistatin occurred independently of overexpression or knockout of myostatin, a key repressor of muscle development that can regulate Smad3 and mTOR signaling and that is itself inhibited by follistatin. These findings identify a critical role of Smad3/Akt/mTOR/S6K/S6RP signaling in follistatin-mediated muscle growth that operates independently of myostatin-driven mechanisms. PMID:22711699

Outside-in HLA class I signaling regulates ICAM-1 clustering and endothelial cell-monocyte interactions via mTOR in transplant antibody-mediated rejection.

Science.gov (United States)

Salehi, Sahar; Sosa, Rebecca A; Jin, Yi-Ping; Kageyama, Shoichi; Fishbein, Michael C; Rozengurt, Enrique; Kupiec-Weglinski, Jerzy W; Reed, Elaine F

2018-05-01

Antibody-mediated rejection (AMR) resulting in transplant allograft vasculopathy (TAV) is the major obstacle for long-term survival of solid organ transplants. AMR is caused by donor-specific antibodies to HLA, which contribute to TAV by initiating outside-in signaling transduction pathways that elicit monocyte recruitment to activated endothelium. Mechanistic target of rapamycin (mTOR) inhibitors can attenuate TAV; therefore, we sought to understand the mechanistic underpinnings of mTOR signaling in HLA class I Ab-mediated endothelial cell activation and monocyte recruitment. We used an in vitro model to assess monocyte binding to HLA I Ab-activated endothelial cells and found mTOR inhibition reduced ezrin/radixin/moesin (ERM) phosphorylation, intercellular adhesion molecule 1 (ICAM-1) clustering, and monocyte firm adhesion to HLA I Ab-activated endothelium. Further, in a mouse model of AMR, in which C57BL/6. RAG1 -/- recipients of BALB/c cardiac allografts were passively transferred with donor-specific MHC I antibodies, mTOR inhibition significantly reduced vascular injury, ERM phosphorylation, and macrophage infiltration of the allograft. Taken together, these studies indicate mTOR inhibition suppresses ERM phosphorylation in endothelial cells, which impedes ICAM-1 clustering in response to HLA class I Ab and prevents macrophage infiltration into cardiac allografts. These findings indicate a novel therapeutic application for mTOR inhibitors to disrupt endothelial cell-monocyte interactions during AMR. © 2017 The American Society of Transplantation and the American Society of Transplant Surgeons.

Evaluation of antioxidant activity of Ruta graveolens L. extract on inhibition of lipid peroxidation and DPPH radicals and the effects of some external factors on plant extract's potency.

Directory of Open Access Journals (Sweden)

S. Mohammadi- Motamed

2014-01-01

Full Text Available The antioxidant properties of Ruta graveolens L. were evaluated by two different methods; free radical scavenging using DPPH and inhibition of lipid peroxidation by the ferric thiocyanate method. The IC50 value of the methanol extract in DPPH inhibition was 200.5 μg/mL which was acceptable in comparison with BHT (41.8 μg/mL. In thiocyanate method, the plant extract demonstrated activity as much as BHT in prevention of lipid peroxidation. Increasing the temperature during extraction, significantly decreased the extract power in inhibition of DPPH radicals. The storage time and temperature had no effect on lipid peroxidation inhibition.

The anti-hepatocellular carcinoma cell activity by a novel mTOR kinase inhibitor CZ415

International Nuclear Information System (INIS)

Zhang, Wei; Chen, Bingyu; Zhang, Yu; Li, Kaiqiang; Hao, Ke; Jiang, Luxi; Wang, Ying; Mou, Xiaozhou; Xu, Xiaodong; Wang, Zhen

2017-01-01

Dysregulation of mammalian target of rapamycin (mTOR) in hepatocellular carcinoma (HCC) represents a valuable treatment target. Recent studies have developed a highly-selective and potent mTOR kinase inhibitor, CZ415. Here, we showed that nM concentrations of CZ415 efficiently inhibited survival and induced apoptosis in HCC cell lines (HepG2 and Huh-7) and primary-cultured human HCC cells. Meanwhile, CZ415 inhibited proliferation of HCC cells, more potently than mTORC1 inhibitors (rapamycin and RAD001). CZ415 was yet non-cytotoxic to the L02 human hepatocytes. Mechanistic studies showed that CZ415 disrupted assembly of mTOR complex 1 (mTORC1) and mTORC2 in HepG2 cells. Meanwhile, activation of mTORC1 (p-S6K1) and mTORC2 (p-AKT, Ser-473) was almost blocked by CZ415. In vivo studies revealed that oral administration of CZ415 significantly suppressed HepG2 xenograft tumor growth in severe combined immuno-deficient (SCID) mice. Activation of mTORC1/2 was also largely inhibited in CZ415-treated HepG2 tumor tissue. Together, these results show that CZ415 blocks mTORC1/2 activation and efficiently inhibits HCC cell growth in vitro and in vivo. - Highlights: • CZ415 is anti-survival and pro-apoptotic to hepatocellular carcinoma (HCC) cells. • CZ415 inhibits HCC cell proliferation, more efficiently than mTORC1 inhibitors. • CZ415 blocks assembly and activation of both mTORC1 and mTORC2 in HCC cells. • CZ415 oral administration inhibits HepG2 tumor growth in SCID mice. • mTORC1/2 activation in HepG2 tumor is inhibited with CZ415 administration.

GFR meets mTOR: value of different methods to measure and estimate GFR & (side) effects of mTOR inhibition in renal transplantation

NARCIS (Netherlands)

Baas, M.C.

2011-01-01

The subject of this thesis is twofold: where GFR and mTOR meet. Precise measurement of kidney function is difficult and cumbersome and many, simpler alternatives have been developed to determine GFR. Determination of GFR remains an approximation since the GFR itself is not a static phenomenon. This

Role of the Phosphorylation of mTOR in the Differentiation of AML Cells Triggered with CD44 Antigen

KAUST Repository

Darwish, Manar M

2013-05-01

Acute myeloid leukemia (AML) is a hematological disorder characterized by blockage of differentiation of myeloblasts. To date, the main therapy for AML is chemotherapy. Yet, studies are seeking a better treatment to enhance the survival rate of patients and minimize the relapsing of the disease. Since the major problem in these cells is that they are arrested in cellular differentiation, drugs that could induce their differentiation have proven to be efficient and of major interest for AML therapy. CD44 triggering appeared as a promising target for AML therapy as it has been shown that specific monoclonal antibodies, such as A3D8 and H90, reversed the blockage of differentiation, inhibited the proliferation of all AML subtypes, and in some cases, induced cell apoptosis. Studies conducted in our laboratory have added strength to these antibodies as potential treatment for AML. Indeed, our laboratory found that treating HL60 cells with A3D8 shows a decrease in the phosphorylation of the mammalian target of Rapamycin (mTOR) kinase correlated with the inhibition of proliferation/induction of differentiation of AML cells.The relationship between the induction of differentiation and the inhibition of proliferation and the decrease of mTOR phosphorylation remains to be clarified. To study the importance of the de-phosphorylation of mTOR and the observed effect of CD44 triggering on differentiation and/or proliferation, we sought to prepare phospho-mimic mutants of the mTOR kinase that will code for a constitutively phosphorylated form of mTOR and used two main methods to express this mutant in HL60 cells: lentiviral and simple transfection (cationic-liposomal transfection).

Alcohol Inhibits Odontogenic Differentiation of Human Dental Pulp Cells by Activating mTOR Signaling

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Wei Qin

2017-01-01

Full Text Available Long-term heavy alcohol consumption could result in a range of health, social, and behavioral problems. People who abuse alcohol are at high risks of seriously having osteopenia, periodontal disease, and compromised oral health. However, the role of ethanol (EtOH in the biological functions of human dental pulp cells (DPCs is unknown. Whether EtOH affects the odontoblastic differentiation of DPCs through the mechanistic target of rapamycin (mTOR remains unexplored. The objective of this study was to investigate the effects of EtOH on DPC differentiation and mineralization. DPCs were isolated and purified from human dental pulps. The proliferation and odontoblastic differentiation of DPCs treated with EtOH were subsequently investigated. Different doses of EtOH were shown to be cytocompatible with DPCs. EtOH significantly activated the mTOR pathway in a dose-dependent manner. In addition, EtOH downregulated the alkaline phosphatase activity, attenuated the mineralized nodule formation, and suppressed the expression of odontoblastic markers including ALP, DSPP, DMP-1, Runx2, and OCN. Moreover, the pretreatment with rapamycin, a specific mTOR inhibitor, markedly reversed the EtOH-induced odontoblastic differentiation and cell mineralization. Our findings show for the first time that EtOH can suppress DPC differentiation and mineralization in a mTOR-dependent manner, indicating that EtOH may be involved in negatively regulating the dental pulp repair.

Alcohol Inhibits Odontogenic Differentiation of Human Dental Pulp Cells by Activating mTOR Signaling.

Science.gov (United States)

Qin, Wei; Huang, Qi-Ting; Weir, Michael D; Song, Zhi; Fouad, Ashraf F; Lin, Zheng-Mei; Zhao, Liang; Xu, Hockin H K

2017-01-01

Long-term heavy alcohol consumption could result in a range of health, social, and behavioral problems. People who abuse alcohol are at high risks of seriously having osteopenia, periodontal disease, and compromised oral health. However, the role of ethanol (EtOH) in the biological functions of human dental pulp cells (DPCs) is unknown. Whether EtOH affects the odontoblastic differentiation of DPCs through the mechanistic target of rapamycin (mTOR) remains unexplored. The objective of this study was to investigate the effects of EtOH on DPC differentiation and mineralization. DPCs were isolated and purified from human dental pulps. The proliferation and odontoblastic differentiation of DPCs treated with EtOH were subsequently investigated. Different doses of EtOH were shown to be cytocompatible with DPCs. EtOH significantly activated the mTOR pathway in a dose-dependent manner. In addition, EtOH downregulated the alkaline phosphatase activity, attenuated the mineralized nodule formation, and suppressed the expression of odontoblastic markers including ALP, DSPP, DMP-1, Runx2, and OCN. Moreover, the pretreatment with rapamycin, a specific mTOR inhibitor, markedly reversed the EtOH-induced odontoblastic differentiation and cell mineralization. Our findings show for the first time that EtOH can suppress DPC differentiation and mineralization in a mTOR-dependent manner, indicating that EtOH may be involved in negatively regulating the dental pulp repair.

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

International Nuclear Information System (INIS)

Mogi, Makio; Kondo, Ayami

2009-01-01

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.

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

National Research Council Canada - National Science Library

Guan, Kun-Liang

2008-01-01

.... We demonstrate that BNIP3 plays a critical role in hypoxia-induced mTOR inhibition. Furthermore we found that BNIP3 itself has a growth inhibitory activity and inactivation of BNIP3 promotes cell growth...

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.

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%, Prespiration. 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.

Antioxidant effect of bisphosphonates and simvastatin on chondrocyte lipid peroxidation

International Nuclear Information System (INIS)

Dombrecht, E.J.; De Tollenaere, C.B.; Aerts, K.; Cos, P.; Schuerwegh, A.J.; Bridts, C.H.; Van Offel, J.F.; Ebo, D.G.; Stevens, W.J.; De Clerck, L.S.

2006-01-01

The objective of this study was to evaluate the effect of bisphosphonates (BPs) and simvastatin on chondrocyte lipid peroxidation. For this purpose, a flow cytometrical method using C11-BODIPY 581/591 was developed to detect hydroperoxide-induced lipid peroxidation in chondrocytes. Tertiary butylhydroperoxide (t-BHP) induced a time and concentration dependent increase in chondrocyte lipid peroxidation. Addition of a Fe 2+ /EDTA complex to t-BHP or hydrogen peroxide (H 2 O 2 ) clearly enhanced lipid peroxidation. The lipophilic simvastatin demonstrated a small inhibition in the chondrocyte lipid peroxidation. None of three tested BPs (clodronate, pamidronate, and risedronate) had an effect on chondrocyte lipid peroxidation induced by t-BHP. However, when Fe 2+ /EDTA complex was added to t-BHP or H 2 O 2 , BPs inhibited the lipid peroxidation process varying from 25% to 58%. This study demonstrates that BPs have antioxidant properties as iron chelators, thereby inhibiting the chondrocyte lipid peroxidation. These findings add evidence to the therapeutic potential of bisphosphonates and statins in rheumatoid arthritis

Combination of mTOR and MAPK Inhibitors—A Potential Way to Treat Renal Cell Carcinoma

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Ashutosh Chauhan

2016-10-01

Full Text Available Renal cell carcinoma (RCC is the most common neoplasm that occurs in the kidney and is marked by a unique biology, with a long history of poor response to conventional cancer treatments. In the past few years, there have been significant advancements to understand the biology of RCC. This has led to the introduction of novel targeted therapies in the management of patients with metastatic disease. Patients treated with targeted therapies for RCC had shown positive impact on overall survival, however, no cure is possible and patients need to undergo treatment for long periods of time, which raises challenges to manage the associated adverse events. Moreover, many patients may not respond to it and even response may not last long enough in the responders. Many inhibitors of the Mammalian target of Rapamycin (mTOR signaling pathway are currently being used in treatment of advanced RCC. Studies showed that inhibitions of mTOR pathways induce Mitogen-Activated Protein Kinase (MAPK escape cell death and cells become resistant to mTOR inhibitors. Because of this, there is a need to inhibit both pathways with their inhibitors comparatively for a better outcome and treatment of patients with RCC.

Quantitative Structure-Activity Relationships Predicting the Antioxidant Potency of 17β-Estradiol-Related Polycyclic Phenols to Inhibit Lipid Peroxidation

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Katalin Prokai-Tatrai

2013-01-01

Full Text Available The antioxidant potency of 17β-estradiol and related polycyclic phenols has been well established. This property is an important component of the complex events by which these types of agents are capable to protect neurons against the detrimental consequences of oxidative stress. In order to relate their molecular structure and properties with their capacity to inhibit lipid peroxidation, a marker of oxidative stress, quantitative structure-activity relationship (QSAR studies were conducted. The inhibition of Fe3+-induced lipid peroxidation in rat brain homogenate, measured through an assay detecting thiobarbituric acid reactive substances for about seventy compounds were correlated with various molecular descriptors. We found that lipophilicity (modeled by the logarithm of the n-octanol/water partition coefficient, logP was the property that influenced most profoundly the potency of these compounds to inhibit lipid peroxidation in the biological medium studied. Additionally, the important contribution of the bond dissociation enthalpy of the phenolic O-H group, a shape index, the solvent-accessible surface area and the energy required to remove an electron from the highest occupied molecular orbital were also confirmed. Several QSAR equations were validated as potentially useful exploratory tools for identifying or designing novel phenolic antioxidants incorporating the structural backbone of 17β-estradiol to assist therapy development against oxidative stress-associated neurodegeneration.

Ammonia Induces Autophagy through Dopamine Receptor D3 and MTOR

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Li, Zhiyuan; Ji, Xinmiao; Wang, Wenchao; Liu, Juanjuan; Liang, Xiaofei; Wu, Hong; Liu, Jing; Eggert, Ulrike S.; Liu, Qingsong

2016-01-01

Hyperammonemia is frequently seen in tumor microenvironments as well as in liver diseases where it can lead to severe brain damage or death. Ammonia induces autophagy, a mechanism that tumor cells may use to protect themselves from external stresses. However, how cells sense ammonia has been unclear. Here we show that culture medium alone containing Glutamine can generate milimolar of ammonia at 37 degrees in the absence of cells. In addition, we reveal that ammonia acts through the G protein-coupled receptor DRD3 (Dopamine receptor D3) to induce autophagy. At the same time, ammonia induces DRD3 degradation, which involves PIK3C3/VPS34-dependent pathways. Ammonia inhibits MTOR (mechanistic target of Rapamycin) activity and localization in cells, which is mediated by DRD3. Therefore, ammonia has dual roles in autophagy: one to induce autophagy through DRD3 and MTOR, the other to increase autophagosomal pH to inhibit autophagic flux. Our study not only adds a new sensing and output pathway for DRD3 that bridges ammonia sensing and autophagy induction, but also provides potential mechanisms for the clinical consequences of hyperammonemia in brain damage, neurodegenerative diseases and tumors. PMID:27077655

D-Glucosamine inhibits proliferation of human cancer cells through inhibition of p70S6K

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Oh, Hyun-Ji; Lee, Jason S.; Song, Dae-Kyu; Shin, Dong-Hoon; Jang, Byeong-Churl; Suh, Seong-Il; Park, Jong-Wook; Suh, Min-Ho; Baek, Won-Ki

2007-01-01

Although D-glucosamine has been reported as an inhibitor of tumor growth both in vivo and in vitro, the mechanism for the anticancer effect of D-glucosamine is still unclear. Since there are several reports suggesting D-glucosamine inhibits protein synthesis, we examined whether D-glucosamine affects p70S6 K activity, an important signaling molecule involved in protein translation. In the present study, we found D-glucosamine inhibited the activity of p70S6K and the proliferation of DU145 prostate cancer cells and MDA-MB-231 breast cancer cells. D-Glucosamine decreased phosphorylation of p70S6K, and its downstream substrates RPS6, and eIF-4B, but not mTOR and 4EBP1 in DU145 cells, suggesting that D-glucosamine induced inhibition of p70S6K is not through the inhibition of mTOR. In addition, D-glucosamine enhanced the growth inhibitory effects of rapamycin, a specific inhibitor of mTOR. These findings suggest that D-glucosamine can inhibit growth of cancer cells through dephosphorylation of p70S6K

Selective regulation of YB-1 mRNA translation by the mTOR signaling pathway is not mediated by 4E-binding protein.

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Lyabin, D N; Ovchinnikov, L P

2016-03-02

The Y-box binding protein 1 (YB-1) is a key regulator of gene expression at the level of both translation and transcription. The mode of its action on cellular events depends on its subcellular distribution and the amount in the cell. So far, the regulatory mechanisms of YB-1 synthesis have not been adequately studied. Our previous finding was that selective inhibition of YB-1 mRNA translation was caused by suppression of activity of the mTOR signaling pathway. It was suggested that this event may be mediated by phosphorylation of the 4E-binding protein (4E-BP). Here, we report that 4E-BP alone can only slightly inhibit YB-1 synthesis both in the cell and in vitro, although it essentially decreases binding of the 4F-group translation initiation factors to mRNA. With inhibited mTOR kinase, the level of mRNA binding to the eIF4F-group factors was decreased, while that to 4E-BP1 was increased, as was observed for both mTOR kinase-sensitive mRNAs and those showing low sensitivity. This suggests that selective inhibition of translation of YB-1 mRNA, and probably some other mRNAs as well, by mTOR kinase inhibitors is not mediated by the action of the 4E-binding protein upon functions of the 4F-group translation initiation factors.

Restoration of Corticosteroid Sensitivity in Chronic Obstructive Pulmonary Disease by Inhibition of Mammalian Target of Rapamycin.

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Mitani, Akihisa; Ito, Kazuhiro; Vuppusetty, Chaitanya; Barnes, Peter J; Mercado, Nicolas

2016-01-15

Corticosteroid resistance is a major barrier to the effective treatment of chronic obstructive pulmonary disease (COPD). Several molecular mechanisms have been proposed, such as activations of the phosphoinositide-3-kinase/Akt pathway and p38 mitogen-activated protein kinase. However, the mechanism for corticosteroid resistance is still not fully elucidated. To investigate the role of mammalian target of rapamycin (mTOR) in corticosteroid sensitivity in COPD. The corticosteroid sensitivity of peripheral blood mononuclear cells collected from patients with COPD, smokers, and nonsmoking control subjects, or of human monocytic U937 cells exposed to cigarette smoke extract (CSE), was quantified as the dexamethasone concentration required to achieve 30% inhibition of tumor necrosis factor-α-induced CXCL8 production in the presence or absence of the mTOR inhibitor rapamycin. mTOR activity was determined as the phosphorylation of p70 S6 kinase, using Western blotting. mTOR activity was increased in peripheral blood mononuclear cells from patients with COPD, and treatment with rapamycin inhibited this as well as restoring corticosteroid sensitivity. In U937 cells, CSE stimulated mTOR activity and c-Jun expression, but pretreatment with rapamycin inhibited both and also reversed CSE-induced corticosteroid insensitivity. mTOR inhibition by rapamycin restores corticosteroid sensitivity via inhibition of c-Jun expression, and thus mTOR is a potential novel therapeutic target for COPD.

Identification of Palmitoleic Acid Controlled by mTOR Signaling as a Biomarker of Polymyositis

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Geng Yin

2017-01-01

Full Text Available Polymyositis (PM is a chronic disease characterized by muscle pain, weakness, and increase in muscle-related enzymes, accompanied with inflammations in lymphocytes. However, it is not well understood how the molecular alternations in lymphocytes contribute to the development of polymyositis. The mechanistic target of rapamycin (mTOR signaling is the central regulator of metabolism and inflammation in mammalian cells. Based on previous studies, we proposed that mTOR signaling may control inflammatory reactions via lipid metabolism. In this study, we aim to figure out the role of mTOR signaling in the development of polymyositis and identify novel biomarkers for the detection and therapy of polymyositis. After screening and validation, we found that palmitoleic acid, a monounsaturated fatty acid, is highly regulated by mTOR signaling. Inhibition of mTORC1 activity decreases palmitoleic acid level. Moreover, mTORC1 regulates the level of palmitoleic acid by controlling its de novo synthesis. Importantly, increased palmitoleic acid has been proven to be a marker of polymyositis. Our work identifies palmitoleic acid in peripheral blood mononuclear cells (PBMC as a biomarker of polymyositis and offers new targets to the clinical therapy.

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

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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

Inhibitory effect of mTOR activator MHY1485 on autophagy: suppression of lysosomal fusion.

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Yeon Ja Choi

Full Text Available Autophagy is a major degradative process responsible for the disposal of cytoplasmic proteins and dysfunctional organelles via the lysosomal pathway. During the autophagic process, cells form double-membraned vesicles called autophagosomes that sequester disposable materials in the cytoplasm and finally fuse with lysosomes. In the present study, we investigated the inhibition of autophagy by a synthesized compound, MHY1485, in a culture system by using Ac2F rat hepatocytes. Autophagic flux was measured to evaluate the autophagic activity. Autophagosomes were visualized in Ac2F cells transfected with AdGFP-LC3 by live-cell confocal microscopy. In addition, activity of mTOR, a major regulatory protein of autophagy, was assessed by western blot and docking simulation using AutoDock 4.2. In the result, treatment with MHY1485 suppressed the basal autophagic flux, and this inhibitory effect was clearly confirmed in cells under starvation, a strong physiological inducer of autophagy. The levels of p62 and beclin-1 did not show significant change after treatment with MHY1485. Decreased co-localization of autophagosomes and lysosomes in confocal microscopic images revealed the inhibitory effect of MHY1485 on lysosomal fusion during starvation-induced autophagy. These effects of MHY1485 led to the accumulation of LC3II and enlargement of the autophagosomes in a dose- and time-dependent manner. Furthermore, MHY1485 induced mTOR activation and correspondingly showed a higher docking score than PP242, a well-known ATP-competitive mTOR inhibitor, in docking simulation. In conclusion, MHY1485 has an inhibitory effect on the autophagic process by inhibition of fusion between autophagosomes and lysosomes leading to the accumulation of LC3II protein and enlarged autophagosomes. MHY1485 also induces mTOR activity, providing a possibility for another regulatory mechanism of autophagy by the MHY compound. The significance of this study is the finding of a novel

Study on mechanism of decreased lipid peroxide by low dose radiation

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Yamaoka, Kiyonori; Okazoe, Yoko; Akimaru, Kunihiro; Sato, E.F.; Utsumi, Kozo.

1991-01-01

We examined the effect of SOD on lipid peroxidation in biomembrane from V.E-deficient rats, in order to study the mechanism of increased SOD activities and decreased lipid peroxide by low dose irradiation. The following results were obtained. i. Active oxygen (O 2 - ) strongly enhances lipid peroxidations in biomembrane with the Fe 3+ as catalyst. ii. SOD evidently inhibits lipid peroxidations under above conditions. iii. We suggested that the effect of SOD enhanced by low dose irradiation results in inhibition of lipid peroxidation. (author)

Hydrogen peroxide induce modifications of human extracellular superoxide dismutase that results in enzyme inhibition

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Randi H. Gottfredsen

2013-01-01

Full Text Available Superoxide dismutase (EC-SOD controls the level of superoxide in the extracellular space by catalyzing the dismutation of superoxide into hydrogen peroxide and molecular oxygen. In addition, the enzyme reacts with hydrogen peroxide in a peroxidase reaction which is known to disrupt enzymatic activity. Here, we show that the peroxidase reaction supports a site-specific bond cleavage. Analyses by peptide mapping and mass spectrometry shows that oxidation of Pro112 supports the cleavage of the Pro112–His113 peptide bond. Substitution of Ala for Pro112 did not inhibit fragmentation, indicating that the oxidative fragmentation at this position is dictated by spatial organization and not by side-chain specificity. The major part of EC-SOD inhibited by the peroxidase reaction was not fragmented but found to encompass oxidations of histidine residues involved in the coordination of copper (His98 and His163. These oxidations are likely to support the dissociation of copper from the active site and thus loss of enzymatic activity. Homologous modifications have also been described for the intracellular isozyme, Cu/Zn-SOD, reflecting the almost identical structures of the active site within these enzymes. We speculate that the inactivation of EC-SOD by peroxidase activity plays a role in regulating SOD activity in vivo, as even low levels of superoxide will allow for the peroxidase reaction to occur.

Deregulation of mTOR signaling is involved in thymic lymphoma development in Atm-/- mice

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Kuang, Xianghong; Shen, Jianjun; Wong, Paul K.Y.; Yan, Mingshan

2009-01-01

Abnormal thymocyte development with thymic lymphomagenesis inevitably occurs in Atm-/- mice, indicating that ATM plays a pivotal role in regulating postnatal thymocyte development and preventing thymic lymphomagenesis. The mechanism for ATM controls these processes is unclear. We have shown previously that c-Myc, an oncoprotein regulated by the mammalian target of rapamycin (mTOR), is overexpressed in Atm-/- thymocytes. Here, we show that inhibition of mTOR signaling with its specific inhibitor, rapamycin, suppresses normal thymocyte DNA synthesis by downregulating 4EBP1, but not S6K, and that 4EBP1 phosphorylation and cyclin D1 expression are coordinately increased in Atm-/- thymocytes. Administration of rapamycin to Atm-/- mice attenuates elevated phospho-4EBP1, c-Myc and cyclin D1 in their thymocytes, and delays thymic lymphoma development. These results indicate that mTOR downstream effector 4EBP1 is essential for normal thymocyte proliferation, but deregulation of 4EBP1 in Atm deficiency is a major factor driving thymic lymphomagenesis in the animals.

CXCR4/CXCL12/CXCR7 axis is functional in neuroendocrine tumors and signals on mTOR.

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Circelli, Luisa; Sciammarella, Concetta; Guadagno, Elia; Tafuto, Salvatore; del Basso de Caro, Marialaura; Botti, Giovanni; Pezzullo, Luciano; Aria, Massimo; Ramundo, Valeria; Tatangelo, Fabiana; Losito, Nunzia Simona; Ieranò, Caterina; D'Alterio, Crescenzo; Izzo, Francesco; Ciliberto, Gennaro; Colao, Annamaria; Faggiano, Antongiulio; Scala, Stefania

2016-04-05

To evaluate the possible crosstalk between C-X-C chemokine receptor 4 (CXCR4)/C-X-C motif chemokine 12 (CXCL12)/C-X-C chemokine receptor 7 (CXCR7) axis with the mammalian target of rapamycin (mTOR) pathway in neuroendocrine tumors (NETs). Sixty-one human NETs were included into the study. CXCR4/CXCL12/CXCR7 axis and mTOR pathway were assessed by qRT-PCR and immunohistochemistry (IHC). The effect of mTOR inhibitor, RAD001, was evaluated on CXCR4 pathway through proliferation and p-Erk and p-AKT induction. CXCR4/CXCL12/CXCR7 axis and p-mTOR were found to be active and correlated with grading, Ki67 index and tumor stage. mTOR pathway activation significantly correlated with poor prognosis. In human NET cells, CXCL12 induced mTOR signalling while AMD3100 (CXCR4-antagonist) impaired it. The mTOR-antagonist, RAD001, impaired the CXCL12-dependent induction of CXCR4 downstream effectors. Combination of AMD3100 and RAD001 potentiate cell growth inhibition. CXCR4/CXCL12/CXCR7 axis is active in NETs and signals on mTOR. CXCR4 might be considered a prognostic factor in NETs. Combined treatment with AMD3100 and RAD001 may provide clinical benefits in NET patients with drug-resistant.

CDK2 and mTOR are direct molecular targets of isoangustone A in the suppression of human prostate cancer cell growth

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Lee, Eunjung; Son, Joe Eun; Byun, Sanguine; Lee, Seung Joon; Kim, Yeong A [WCU Biomodulation Major, Department of Agricultural Biotechnology and Center for Food and Bioconvergence, Seoul National University, Seoul 151-921 (Korea, Republic of); Liu, Kangdong [The Hormel Institute, University of Minnesota, 801 16th Avenue NE, Austin, MN 55912 (United States); Kim, Jiyoung [WCU Biomodulation Major, Department of Agricultural Biotechnology and Center for Food and Bioconvergence, Seoul National University, Seoul 151-921 (Korea, Republic of); Lim, Soon Sung; Park, Jung Han Yoon [Department of Food Science and Nutrition, College of Natural Science, Hallym University, Chuncheon, 200-702 (Korea, Republic of); Dong, Zigang [The Hormel Institute, University of Minnesota, 801 16th Avenue NE, Austin, MN 55912 (United States); Lee, Ki Won, E-mail: kiwon@snu.ac.kr [WCU Biomodulation Major, Department of Agricultural Biotechnology and Center for Food and Bioconvergence, Seoul National University, Seoul 151-921 (Korea, Republic of); Advanced Institutes of Convergence Technology, Seoul National University, Suwon 443-270 (Korea, Republic of); Lee, Hyong Joo, E-mail: leehyjo@snu.ac.kr [WCU Biomodulation Major, Department of Agricultural Biotechnology and Center for Food and Bioconvergence, Seoul National University, Seoul 151-921 (Korea, Republic of); Advanced Institutes of Convergence Technology, Seoul National University, Suwon 443-270 (Korea, Republic of)

2013-10-01

Licorice extract which is used as a natural sweetener has been shown to possess inhibitory effects against prostate cancer, but the mechanisms responsible are poorly understood. Here, we report a compound, isoangustone A (IAA) in licorice that potently suppresses the growth of aggressive prostate cancer and sought to clarify its mechanism of action. We analyzed its inhibitory effects on the growth of PTEN-deleted human prostate cancer cells, in vitro and in vivo. Administration of IAA significantly attenuated the growth of prostate cancer cell cultures and xenograft tumors. These effects were found to be attributable to inhibition of the G1/S phase cell cycle transition and the accumulation of p27{sup kip1}. The elevated p27{sup kip1} expression levels were concurrent with the decrease of its phosphorylation at threonine 187 through suppression of CDK2 kinase activity and the reduced phosphorylation of Akt at Serine 473 by diminishing the kinase activity of the mammalian target of rapamycin (mTOR). Further analysis using recombinant proteins and immunoprecipitated cell lysates determined that IAA exerts suppressive effects against CDK2 and mTOR kinase activity by direct binding with both proteins. These findings suggested that the licorice compound IAA is a potent molecular inhibitor of CDK2 and mTOR, with strong implications for the treatment of prostate cancer. Thus, licorice-derived extracts with high IAA content warrant further clinical investigation for nutritional sources for prostate cancer patients. - Highlights: • Isoangustone A suppresses growth of PC3 and LNCaP prostate cancer cells. • Administration of isoangustone A inhibits tumor growth in mice. • Treatment of isoangustone A induces cell cycle arrest and accumulation of p27{sup kip1}. • Isoangustone A inhibits CDK2 and mTOR activity. • Isoangustone A directly binds with CDK2 and mTOR complex in prostate cancer cells.

Water extractable phytochemicals from Capsicum pubescens (tree pepper) inhibit lipid peroxidation induced by different pro-oxidant agents in brain

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Oboh, G [Biochemistry Department, Federal University of Technology, Akure, Ondo State (Nigeria); [Departamento de Quimica, Universidade Federal de Santa Maria (UFSM), Campus Universitario - Camobi, Santa Maria RS (Brazil); [Abdus Salam International Centre for Theoretical Physics, Trieste (Italy)]. E-mail: goboh2001@yahoo.com; Rocha, J B.T. [Campus Universitario - Camobi, Santa Maria RS (Brazil)

2006-03-15

Reactive oxygen species (ROS) is the cause of neurodegenerative disorders such as Lou Gehrig's disease, Parkinson's disease and Huntington's disease; one practical way to prevent and manage neurodegenerative diseases is through the eating of food rich in antioxidants (dietary means). In this study, the antioxidant and neuroprotective properties of aqueous extract of ripe and unripe Capsicum pubescens (popularly known as tree pepper) on different pro-oxidant induced lipid peroxidation in Rat's brain (in vitro) is been investigated. Aqueous extract of freshly harvested pepper was prepared, and the total phenol content, vitamin C, ferric reducing antioxidant property (FRAP) and Fe (II) chelating ability was determined. In addition, the ability of the extracts to protect the Rat's brain against some pro-oxidant FeSO{sub 4}, Sodium nitroprusside and Quinolinic acid - induced oxidative stress was also determined. The results of the study revealed that ripe Capsicum pubescens had a significantly higher (P<0.05) total phenol [ripe (113.7mg/100g), unripe (70.5mg/100g)] content and ferric reducing antioxidant property than the unripe pepper. However, there was no significant difference in the vitamin C [ripe (231.5{mu}g/g), unripe (224.4{mu}g/g)] content and Fe (II) chelating ability. Furthermore, the pepper extracts caused a significant decrease (P<0.05) in 25{mu}M Fe(II), 7{mu}M Sodium Nitroprusside and 1mM Quinolinic acid induced lipid peroxidation in the Rat's brain in a dose-dependent manner. However, the ripe pepper inhibited MDA (Malondialdehyhide) production in the Rat's brain than the unripe pepper. Conversely, both extract did not significantly inhibit Fe (II)/H{sub 2}O{sub 2} induced decomposition of deoxyribose. Therefore, ripe and unripe Capsicum pubescens would inhibit lipid peroxidation in vitro. However, the ripe potent was a more potent inhibitor of lipid peroxidation, which is probably due to its higher vitamin C and phenol content, reducing power and Fe

Requirement for the eIF4E binding proteins for the synergistic down-regulation of protein synthesis by hypertonic conditions and mTOR inhibition.

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Clemens, Michael J; Elia, Androulla; Morley, Simon J

2013-01-01

The protein kinase mammalian target of rapamycin (mTOR) regulates the phosphorylation and activity of several proteins that have the potential to control translation, including p70S6 kinase and the eIF4E binding proteins 4E-BP1 and 4E-BP2. In spite of this, in exponentially growing cells overall protein synthesis is often resistant to mTOR inhibitors. We report here that sensitivity of wild-type mouse embryonic fibroblasts (MEFs) to mTOR inhibitors can be greatly increased when the cells are subjected to the physiological stress imposed by hypertonic conditions. In contrast, protein synthesis in MEFs with a double knockout of 4E-BP1 and 4E-BP2 remains resistant to mTOR inhibitors under these conditions. Phosphorylation of p70S6 kinase and protein kinase B (Akt) is blocked by the mTOR inhibitor Ku0063794 equally well in both wild-type and 4E-BP knockout cells, under both normal and hypertonic conditions. The response of protein synthesis to hypertonic stress itself does not require the 4E-BPs. These data suggest that under certain stress conditions: (i) translation has a greater requirement for mTOR activity and (ii) there is an absolute requirement for the 4E-BPs for regulation by mTOR. Importantly, dephosphorylation of p70S6 kinase and Akt is not sufficient to affect protein synthesis acutely.

A kinase-dead knock-in mutation in mTOR leads to early embryonic lethality and is dispensable for the immune system in heterozygous mice

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Cavender Druie

2009-05-01

Full Text Available Abstract Background The mammalian target of rapamycin protein (mTOR is an evolutionarily conserved kinase that regulates protein synthesis, cell cycle progression and proliferation in response to various environmental cues. As a critical downstream mediator of PI3K signaling, mTOR is important for lymphocyte development and function of mature T and B-cells. Most studies of mTOR in immune responses have relied on the use of pharmacological inhibitors, such as rapamycin. Rapamycin-FKBP12 complex exerts its immunosuppressive and anti-proliferative effect by binding outside the kinase domain of mTOR, and subsequently inhibiting downstream mTOR signaling. Results To determine the requirement for mTOR kinase activity in the immune system function, we generated knock-in mice carrying a mutation (D2338 in the catalytic domain of mTOR. While homozygous mTOR kd/kd embryos died before embryonic day 6.5, heterozygous mTOR+/kd mice appeared entirely normal and are fertile. mTOR +/kd mice exhibited normal T and B cell development and unaltered proliferative responses of splenocytes to IL-2 and TCR/CD28. In addition, heterozygousity for the mTOR kinase-dead allele did not sensitize T cells to rapamycin in a CD3-mediated proliferation assay. Unexpectedly, mTOR kinase activity towards its substrate 4E-BP1 was not decreased in hearts and livers from heterozygous animals. Conclusion Altogether, our findings indicate that mTOR kinase activity is indispensable for the early development of mouse embryos. Moreover, a single wild type mTOR allele is sufficient to maintain normal postnatal growth and lymphocyte development and proliferation.

Characteristic mTOR activity in Hodgkin-lymphomas offers a potential therapeutic target in high risk disease – a combined tissue microarray, in vitro and in vivo study

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Márk, Ágnes; Kopper, László; Sebestyén, Anna; Hajdu, Melinda; Váradi, Zsófia; Sticz, Tamás Béla; Nagy, Noémi; Csomor, Judit; Berczi, Lajos; Varga, Viktória; Csóka, Monika

2013-01-01

Targeting signaling pathways is an attractive approach in many malignancies. The PI3K/Akt/mTOR pathway is activated in a number of human neoplasms, accompanied by lower overall and/or disease free survival. mTOR kinase inhibitors have been introduced in the therapy of renal cell carcinoma and mantle cell lymphoma, and several trials are currently underway. However, the pathological characterization of mTOR activity in lymphomas is still incomplete. mTOR activity and the elements of mTOR complexes were investigated by immunohistochemistry on tissue microarrays representing different human non-Hodgkin-lymphomas (81 cases) and Hodgkin-lymphomas (87 cases). The expression of phospho-mTOR, phospho-4EBP1, phospho-p70S6K, phospho-S6, Rictor, Raptor and Bcl-2, Bcl-xL, Survivin and NF-kappaB-p50 were evaluated, and mTOR activity was statistically analyzed along with 5-year survival data. The in vitro and in vivo effect of the mTOR inhibitor rapamycin was also examined in human Hodgkin-lymphoma cell lines. The majority (>50%) of mantle cell lymphoma, Burkitt lymphoma, diffuse large B-cell lymphoma, anaplastic large-cell lymphoma and Hodgkin-lymphoma cases showed higher mTOR activity compared to normal lymphoid tissues. Hodgkin-lymphoma was characterized by high mTOR activity in 93% of the cases, and Bcl-xL and NF-kappaB expression correlated with this mTOR activity. High mTOR activity was observed in the case of both favorable and unfavorable clinical response. Low mTOR activity was accompanied by complete remission and at least 5-year disease free survival in Hodgkin-lymphoma patients. However, statistical analysis did not identify correlation beetween mTOR activity and different clinical data of HL patients, such as survival. We also found that Rictor (mTORC2) was not overexpressed in Hodgkin-lymphoma biopsies and cell lines. Rapamycin inhibited proliferation and induced apoptosis in Hodgkin-lymphoma cells both in vitro and in vivo, moreover, it increased the apoptotic

Wnt signaling inhibits CTL memory programming.

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Xiao, Zhengguo; Sun, Zhifeng; Smyth, Kendra; Li, Lei

2013-12-01

Induction of functional CTLs is one of the major goals for vaccine development and cancer therapy. Inflammatory cytokines are critical for memory CTL generation. Wnt signaling is important for CTL priming and memory formation, but its role in cytokine-driven memory CTL programming is unclear. We found that wnt signaling inhibited IL-12-driven CTL activation and memory programming. This impaired memory CTL programming was attributed to up-regulation of eomes and down-regulation of T-bet. Wnt signaling suppressed the mTOR pathway during CTL activation, which was different to its effects on other cell types. Interestingly, the impaired memory CTL programming by wnt was partially rescued by mTOR inhibitor rapamycin. In conclusion, we found that crosstalk between wnt and the IL-12 signaling inhibits T-bet and mTOR pathways and impairs memory programming which can be recovered in part by rapamycin. In addition, direct inhibition of wnt signaling during CTL activation does not affect CTL memory programming. Therefore, wnt signaling may serve as a new tool for CTL manipulation in autoimmune diseases and immune therapy for certain cancers. Copyright © 2013 Elsevier Ltd. All rights reserved.

Smad3 induces atrogin-1, inhibits mTOR and protein synthesis, and promotes muscle atrophy in vivo.

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Goodman, Craig A; McNally, Rachel M; Hoffmann, F Michael; Hornberger, Troy A

2013-11-01

Myostatin, a member of the TGF superfamily, is sufficient to induce skeletal muscle atrophy. Myostatin-induced atrophy is associated with increases in E3-ligase atrogin-1 expression and protein degradation and decreases in Akt/mechanistic target of rapamycin (mTOR) signaling and protein synthesis. Myostatin signaling activates the transcription factor Smad3 (Small Mothers Against Decapentaplegic), which has been shown to be necessary for myostatin-induced atrogin-1 expression and atrophy; however, it is not known whether Smad3 is sufficient to induce these events or whether Smad3 simply plays a permissive role. Thus, the aim of this study was to address these questions with an in vivo model. To accomplish this goal, in vivo transfection of plasmid DNA was used to create transient transgenic mouse skeletal muscles, and our results show for the first time that Smad3 expression is sufficient to stimulate atrogin-1 promoter activity, inhibit Akt/mTOR signaling and protein synthesis, and induce muscle fiber atrophy. Moreover, we propose that Akt/mTOR signaling is inhibited by a Smad3-induced decrease in microRNA-29 (miR-29) expression and a subsequent increase in the translation of phosphatase and tensin homolog (PTEN) mRNA. Smad3 is also sufficient to inhibit peroxisome proliferator-activated receptor-γ coactivator-1α (PGC1α) promoter activity and to increase FoxO (Forkhead Box Protein, Subclass O)-mediated signaling and the promoter activity of plasminogen activator inhibitor 1 (PAI-1). Combined, this study provides the first evidence that Smad3 is sufficient to regulate many of the events associated with myostatin-induced atrophy and therefore suggests that Smad3 signaling may be a viable target for therapies aimed at preventing myostatin-induced muscle atrophy.

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.

Inhibition of lipid peroxidation induced by γ- radiation and AAPH in rat liver and brain mitochondria by mushrooms

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Lakshmi, B.; Janardhanan, K.K.; Tilak, J.C.; Devasagayam, T.P.A.; Adhikari, S.

2005-01-01

Exposure to radiation or 2.2' Azobis(2-amidopropane) dihydrochloride (AAPH) induces generation of reactive oxygen species (ROS) especially hydroxyl radical ( . OH) and peroxyl radical (ROO . ), which are capable of inducing lipid peroxidation. Our earlier studies have demonstrated that extracts of the medicinal and edible mushrooms Ganoderma lucidum, Pleurotus florida, Pleurotus sajor-caju and Phellinus rimosus possessed significant antioxidant activity, measured as radical scavenging. In the present study, we examined the protective effect of these mushroom extracts against radiation- and AAPH-induced lipid peroxidation using rat liver and brain mitochondria as model systems. The results obtained showed that the investigated mushroom extracts significantly inhibited the formation of lipid hydroperoxide and thiobarbituric acid reactive substances, indicating membrane protective effects. The finding suggests the profound protective effect of the extracts of the fruiting bodies of G. lucidum, P. florida, P. sajor-caju and P. rimosus against lipid peroxidation by two major forms of ROS capable of inducing this type of damage in a major organelle, the mitochondria from both rat liver and brain. This observation can possibly explain the health benefits of these mushrooms. (author)

Combined MTOR and autophagy inhibition: phase I trial of hydroxychloroquine and temsirolimus in patients with advanced solid tumors and melanoma.

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Rangwala, Reshma; Chang, Yunyoung C; Hu, Janice; Algazy, Kenneth M; Evans, Tracey L; Fecher, Leslie A; Schuchter, Lynn M; Torigian, Drew A; Panosian, Jeffrey T; Troxel, Andrea B; Tan, Kay-See; Heitjan, Daniel F; DeMichele, Angela M; Vaughn, David J; Redlinger, Maryann; Alavi, Abass; Kaiser, Jonathon; Pontiggia, Laura; Davis, Lisa E; O'Dwyer, Peter J; Amaravadi, Ravi K

2014-08-01

The combination of temsirolimus (TEM), an MTOR inhibitor, and hydroxychloroquine (HCQ), an autophagy inhibitor, augments cell death in preclinical models. This phase 1 dose-escalation study evaluated the maximum tolerated dose (MTD), safety, preliminary activity, pharmacokinetics, and pharmacodynamics of HCQ in combination with TEM in cancer patients. In the dose escalation portion, 27 patients with advanced solid malignancies were enrolled, followed by a cohort expansion at the top dose level in 12 patients with metastatic melanoma. The combination of HCQ and TEM was well tolerated, and grade 3 or 4 toxicity was limited to anorexia (7%), fatigue (7%), and nausea (7%). An MTD was not reached for HCQ, and the recommended phase II dose was HCQ 600 mg twice daily in combination with TEM 25 mg weekly. Other common grade 1 or 2 toxicities included fatigue, anorexia, nausea, stomatitis, rash, and weight loss. No responses were observed; however, 14/21 (67%) patients in the dose escalation and 14/19 (74%) patients with melanoma achieved stable disease. The median progression-free survival in 13 melanoma patients treated with HCQ 1200mg/d in combination with TEM was 3.5 mo. Novel 18-fluorodeoxyglucose positron emission tomography (FDG-PET) measurements predicted clinical outcome and provided further evidence that the addition of HCQ to TEM produced metabolic stress on tumors in patients that experienced clinical benefit. Pharmacodynamic evidence of autophagy inhibition was evident in serial PBMC and tumor biopsies only in patients treated with 1200 mg daily HCQ. This study indicates that TEM and HCQ is safe and tolerable, modulates autophagy in patients, and has significant antitumor activity. Further studies combining MTOR and autophagy inhibitors in cancer patients are warranted.

Inhibition of key enzymes linked to type 2 diabetes and sodium nitroprusside-induced lipid peroxidation in rat pancreas by water extractable phytochemicals from some tropical spices.

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Adefegha, Stephen Adeniyi; Oboh, Ganiyu

2012-07-01

Spices have been used as food adjuncts and in folklore for ages. Inhibition of key enzymes (α-amylase and α-glucosidase) involved in the digestion of starch and protection against free radicals and lipid peroxidation in pancreas could be part of the therapeutic approach towards the management of hyperglycemia and dietary phenolics have shown promising potentials. This study investigated and compared the inhibitory properties of aqueous extracts of some tropical spices: Xylopia aethiopica [Dun.] A. Rich (Annonaceae), Monodora myristica (Gaertn.) Dunal (Annonaceae), Syzygium aromaticum [L.] Merr. et Perry (Myrtaceae), Piper guineense Schumach. et Thonn (Piperaceae), Aframomum danielli K. Schum (Zingiberaceae) and Aframomum melegueta (Rosc.) K. Schum (Zingiberaceae) against α-amylase, α-glucosidase, 1,1-diphenyl-2-picrylhydrazyl (DPPH) radicals and sodium nitroprusside (SNP)-induced lipid peroxidation in rat pancreas--in vitro using different spectrophotometric method. Aqueous extract of the spices was prepared and the ability of the spice extracts to inhibit α-amylase, α-glucosidase, DPPH radicals and SNP-induced lipid peroxidation in rat pancreas--in vitro was investigated using various spectrophotometric methods. All the spice extracts inhibited α-amylase (IC(50) = 2.81-4.83 mg/mL), α-glucosidase (IC(50) = 2.02-3.52 mg/mL), DPPH radicals (EC(50) = 15.47-17.38 mg/mL) and SNP-induced lipid peroxidation (14.17-94.38%), with the highest α-amylase & α-glucosidase inhibitory actions and DPPH radical scavenging ability exhibited by X. aethiopica, A. danielli and S. aromaticum, respectively. Also, the spices possess high total phenol (0.88-1.3 mg/mL) and flavonoid (0.24-0.52 mg/mL) contents with A. melegueta having the highest total phenolic and flavonoid contents. The inhibitory effects of the spice extracts on α-amylase, α-glucosidase, DPPH radicals and SNP-induced lipid peroxidation in pancreas (in vitro) could be attributed to the presence of biologically

The proliferation of amplifying neural progenitor cells is impaired in the aging brain and restored by the mTOR pathway activation.

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Romine, Jennifer; Gao, Xiang; Xu, Xiao-Ming; So, Kwok Fai; Chen, Jinhui

2015-04-01

A decrease in neurogenesis in the aged brain has been correlated with cognitive decline. The molecular signaling that regulates age-related decline in neurogenesis is still not fully understood. We found that different subtypes of neural stem cells (NSCs) in the hippocampus were differentially impaired by aging. The quiescent NSCs decreased slowly, although the active NSCs exhibited a sharp and dramatic decline from the ages of 6-9 months and became more quiescent at an early stage during the aging process. The activity of the mammalian target of rapamycin (mTOR) signal pathway is compromised in the NSCs of the aged brain. Activating the mTOR signaling pathway increased NSC proliferation and promoted neurogenesis in aged mice. In contrast, inhibiting the mTOR signaling pathway decreased NSCs proliferation. These results indicate that an age-associated decline in neurogenesis is mainly because of the reduction in proliferation of active NSCs, at least partially because of the compromise in the mTOR signaling activity. Stimulating the mTOR signaling revitalizes the NSCs, restores their proliferation, and enhances neurogenesis in the hippocampus of the aged brain. Copyright © 2015 Elsevier Inc. All rights reserved.

Scalable and Anonymous Group Communication with MTor

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Lin Dong

2016-04-01

Full Text Available 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. MTor does not depend on an external service to broker the group communication, and avoids central points of failure and trust. MTor’s substantial bandwidth savings and graceful scalability enable new classes of anonymous applications that are currently too bandwidth-intensive to be viable through traditional unicast Tor communication-e.g., group file transfer, collaborative editing, streaming video, and real-time audio conferencing.

Autophagy in muscle of glucose-infusion hyperglycemia rats and streptozotocin-induced hyperglycemia rats via selective activation of m-TOR or FoxO3.

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Pengfei Lv

Full Text Available Autophagy is a conserved process in eukaryotes required for metabolism and is involved in diverse diseases. To investigate autophagy in skeletal muscle under hyperglycemia status, we established two hyperglycemia-rat models that differ in their circulating insulin levels, by glucose infusion and singe high-dose streptozotocin injection. We then detected expression of autophagy related genes with real-time PCR and western blot. We found that under hyperglycemia status induced by glucose-infusion, autophagy was inhibited in rat skeletal muscle, whereas under streptozotocin-induced hyperglycemia status autophagy was enhanced. Meanwhile, hyperglycemic gastrocnemius muscle was more prone to autophagy than soleus muscle. Furthermore, inhibition of autophagy in skeletal muscle in glucose-infusion hyperglycemia rats was mediated by the m-TOR pathway while m-TOR and FoxO3 both contributed to enhancement of autophagy in gastrocnemius muscle in streptozotocin-induced hyperglycemia rats. These data shows that insulin plays a relatively more important role than hyperglycemia in regulating autophagy in hyperglycemia rat muscle through selectively activating the m-TOR or FoxO3 pathway in a fiber-selective manner.

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

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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.

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.

Reproducing Phenomenology of Peroxidation Kinetics via Model Optimization

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Ruslanov, Anatole D.; Bashylau, Anton V.

2010-06-01

We studied mathematical modeling of lipid peroxidation using a biochemical model system of iron (II)-ascorbate-dependent lipid peroxidation of rat hepatocyte mitochondrial fractions. We found that antioxidants extracted from plants demonstrate a high intensity of peroxidation inhibition. We simplified the system of differential equations that describes the kinetics of the mathematical model to a first order equation, which can be solved analytically. Moreover, we endeavor to algorithmically and heuristically recreate the processes and construct an environment that closely resembles the corresponding natural system. Our results demonstrate that it is possible to theoretically predict both the kinetics of oxidation and the intensity of inhibition without resorting to analytical and biochemical research, which is important for cost-effective discovery and development of medical agents with antioxidant action from the medicinal plants.

Blocking mammalian target of rapamycin (mTOR) improves neuropathic pain evoked by spinal cord injury.

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Wang, Xiaoping; Li, Xiaojia; Huang, Bin; Ma, Shuai

2016-01-01

Spinal cord injury (SCI) is an extremely serious type of physical trauma observed in clinics. Neuropathic pain resulting from SCI has a lasting and significant impact on most aspects of daily life. Thus, a better understanding of the molecular pathways responsible for the cause of neuropathic pain observed in SCI is important to develop effective therapeutic agents and treatment strategies. Mammalian target of rapamycin (mTOR) is a serine/threonine protein kinase that is well known for its critical roles in regulating protein synthesis and growth. Furthermore, compelling evidence supports the notion that widespread dysregulation of mTOR and its downstream pathways are involved in neuropathic pain. Thus, in this study we specifically examined the underlying mechanisms by which mTOR and its signaling pathways are involved in SCI-evoked neuropathic pain in a rat model. Overall, we demonstrated that SCI increased the protein expression of p-mTOR, and mTORmediated- phosphorylation of 4E-binding protein 4 (4E-BP1) and p70 ribosomal S6 protein kinase 1 (S6K1) in the superficial dorsal horn of the spinal cord. Also, we showed that blocking spinal mTOR by intrathecal injection of rapamycin significantly inhibited pain responses induced by mechanical and thermal stimulation. In addition, blocking spinal phosphatidylinositide 3-kinase (p-PI3K) pathway significantly attenuated activities of p-mTOR pathways as well as mechanical and thermal hyperalgesia in SCI rats. Moreover, blocking mTOR and PI3K decreased the enhanced levels of substance P and calcitonin gene-related peptide (CGRP) in the dorsal horn of SCI rats. We revealed specific signaling pathways leading to SCI-evoked neuropathic pain, including the activation of PI3K, mTOR and its downstream signaling pathways. Targeting one or more of these signaling molecules may present new opportunities for treatment and management of neuropathic pain often observed in patients with SCI.

Blocking mammalian target of rapamycin (mTOR improves neuropathic pain evoked by spinal cord injury

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Wang Xiaoping

2016-01-01

Full Text Available Spinal cord injury (SCI is an extremely serious type of physical trauma observed in clinics. Neuropathic pain resulting from SCI has a lasting and significant impact on most aspects of daily life. Thus, a better understanding of the molecular pathways responsible for the cause of neuropathic pain observed in SCI is important to develop effective therapeutic agents and treatment strategies. Mammalian target of rapamycin (mTOR is a serine/threonine protein kinase that is well known for its critical roles in regulating protein synthesis and growth. Furthermore, compelling evidence supports the notion that widespread dysregulation of mTOR and its downstream pathways are involved in neuropathic pain. Thus, in this study we specifically examined the underlying mechanisms by which mTOR and its signaling pathways are involved in SCI-evoked neuropathic pain in a rat model. Overall, we demonstrated that SCI increased the protein expression of p-mTOR, and mTORmediated- phosphorylation of 4E–binding protein 4 (4E-BP1 and p70 ribosomal S6 protein kinase 1 (S6K1 in the superficial dorsal horn of the spinal cord. Also, we showed that blocking spinal mTOR by intrathecal injection of rapamycin significantly inhibited pain responses induced by mechanical and thermal stimulation. In addition, blocking spinal phosphatidylinositide 3-kinase (p-PI3K pathway significantly attenuated activities of p-mTOR pathways as well as mechanical and thermal hyperalgesia in SCI rats. Moreover, blocking mTOR and PI3K decreased the enhanced levels of substance P and calcitonin gene-related peptide (CGRP in the dorsal horn of SCI rats. We revealed specific signaling pathways leading to SCI-evoked neuropathic pain, including the activation of PI3K, mTOR and its downstream signaling pathways. Targeting one or more of these signaling molecules may present new opportunities for treatment and management of neuropathic pain often observed in patients with SCI.

mTOR pathway is activated in endothelial cells from patients with Takayasu arteritis and is modulated by serum immunoglobulin G.

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Hadjadj, Jérôme; Canaud, Guillaume; Mirault, Tristan; Samson, Maxime; Bruneval, Patrick; Régent, Alexis; Goulvestre, Claire; Witko-Sarsat, Véronique; Costedoat-Chalumeau, Nathalie; Guillevin, Loïc; Mouthon, Luc; Terrier, Benjamin

2018-06-01

Takayasu arteritis (TA) and GCA are large-vessel vasculitides characterized by vascular remodelling involving endothelial cells (ECs) and vascular smooth muscle cells. Mammalian target of rapamycin (mTOR) pathway has been involved in vascular remodelling. We hypothesized that the mTOR pathway was involved in the pathogenesis of large-vessel vasculitis. We used IF analysis on aortic and temporal artery biopsies from patients with TA and GCA to assess the involvement of the mTOR pathway and searched for antibodies targeting ECs in serum by IIF and cellular ELISA. We evaluated in vitro the effect of purified IgG from patients on mTOR pathway activation and cell proliferation. IF analyses on tissues revealed that both mTORC1 and mTORC2 are activated specifically in ECs from TA patients but not in ECs from GCA patients and healthy controls (HCs). Using IIF and ELISA, we observed higher levels of antibodies binding to ECs in TA patients compared with GCA patients and HCs. Using western blot, we demonstrated that purified IgG from TA patients caused mTORC1 activation in ECs, whereas this effect was not observed with purified IgG from GCA patients or HCs. Purified IgG from TA patients induced a significant EC proliferation compared with to GCA and HC IgG, and this effect was decreased after EC exposure with sirolimus, a specific mTOR inhibitor and PI3K inhibitor. Our results suggest that antibodies targeting ECs drive endothelial remodelling in TA through activation of the mTOR pathway, but not in GCA. Inhibition of the mTOR pathway could represent a therapeutic option in TA.

Biphasic activation of the mTOR pathway in the gustatory cortex is correlated with and necessary for taste learning.

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Belelovsky, Katya; Kaphzan, Hanoch; Elkobi, Alina; Rosenblum, Kobi

2009-06-10

Different forms of memories and synaptic plasticity require synthesis of new proteins at the time of acquisition or immediately after. We are interested in the role of translation regulation in the cortex, the brain structure assumed to store long-term memories. The mammalian target of rapamycin, mTOR (also known as FRAP and RAFT-1), is part of a key signal transduction mechanism known to regulate translation of specific subset of mRNAs and to affect learning and synaptic plasticity. We report here that novel taste learning induces two waves of mTOR activation in the gustatory cortex. Interestingly, the first wave can be identified both in synaptoneurosomal and cellular fractions, whereas the second wave is detected in the cellular fraction but not in the synaptic one. Inhibition of mTOR, specifically in the gustatory cortex, has two effects. First, biochemically, it modulates several known downstream proteins that control translation and reduces the expression of postsynaptic density-95 in vivo. Second, behaviorally, it attenuates long-term taste memory. The results suggest that the mTOR pathway in the cortex modulates both translation factor activity and protein expression, to enable normal taste memory consolidation.

Inhibitory heterotrimeric GTP-binding proteins inhibit hydrogen peroxide-induced apoptosis by up-regulation of Bcl-2 via NF-κB in H1299 human lung cancer cells

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Seo, Mi Ran; Nam, Hyo-Jung; Kim, So-Young; Juhnn, Yong-Sung

2009-01-01

Inhibitory heterotrimeric GTP-binding proteins (Gi proteins) mediate a variety of signaling pathways by coupling receptors and effectors to regulate cellular proliferation, differentiation, and apoptosis. However, the role of Gi proteins in the modulation of hydrogen peroxide-induced apoptosis is not clearly understood. Thus, we investigated the effect of Gi proteins on hydrogen peroxide-induced apoptosis and the underlying mechanisms in H1299 human lung cancer cells. The stable expression of constitutively active alpha subunits of Gi1 (Gαi1QL), Gi2, or Gi3 inhibited hydrogen peroxide-induced apoptosis. The expression of Gαi1QL up-regulated Bcl-2 expression, and the knockdown of Bcl-2 with siRNA abolished the anti-apoptotic effect of Gαi1QL. Gαi1 induced the transcription of Bcl-2 by activation of NF-κB, which resulted from an increase in NF-κB p50 protein. We conclude that Gαi1 inhibits hydrogen peroxide-induced apoptosis of H1299 lung cancer cells by up-regulating the transcription of Bcl-2 through a p50-mediated NF-κB activation.

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

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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.

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

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Zhang, Pengpeng; Liang, Xinrong; Shan, Tizhong; Jiang, Qinyang; Deng, Changyan; Zheng, Rong; Kuang, Shihuan

2015-01-01

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 CreER and Mtor 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 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

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…

Advanced Research of mTOR and Lung Carcinoid Tumors

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Zixuan ZHANG

2013-01-01

Full Text Available Mammalian target of rapamycin (mTOR, a main protein kinase in the phosphoinositide 3-kinase (PI3K/AKT/mTOR signaling pathway, is an important intracellular mediator involved in multiple celluar functions including proliferation, differentiation, apoptosis, tumorigenesis, and angiogenesis. Recently, the high expression of mTOR and mTOR-related kinase have been found in neuroendocrin tumors. Therefore, mTOR pathway represents an attractive target for new anticancer therapies except surgery.

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.

mTOR signaling and its roles in normal and abnormal brain development.

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Nobuyuki eTakei

2014-04-01

Full Text Available Target of rapamycin (TOR was first identified in yeast as a target molecule of rapamycin, an anti-fugal and immunosuppressant macrolide compound. In mammals, its orthologue is called mTOR (mammalian TOR. mTOR is a serine/threonine kinase that converges different extracellular stimuli, such as nutrients and growth factors, and diverges into several biochemical reactions, including translation, autophagy, transcription, and lipid synthesis among others. These biochemical reactions govern cell growth and cause cells to attain an anabolic state. Thus, the disruption of mTOR signaling is implicated in a wide array of diseases such as cancer, diabetes, and obesity. In the central nervous system (CNS, the mTOR signaling cascade is activated by nutrients, neurotrophic factors, and neurotransmitters that enhances protein (and possibly lipid synthesis and suppresses autophagy. These processes contribute to normal neuronal growth by promoting their differentiation, neurite elongation and branching, and synaptic formation during development. Therefore, disruption of mTOR signaling may cause neuronal degeneration and abnormal neural development. While reduced mTOR signaling is associated with neurodegeneration, excess activation of mTOR signaling causes abnormal development of neurons and glia, leading to brain malformation. In this review, we first introduce the current state of molecular knowledge of mTOR complexes and signaling in general. We then describe mTOR activation in neurons, which leads to translational enhancement, and finally discuss the link between mTOR and normal/abnormal neuronal growth during development.

mTOR dysregulation and tuberous sclerosis-related epilepsy

NARCIS (Netherlands)

Curatolo, Paolo; Moavero, Romina; van Scheppingen, Jackelien; Aronica, Eleonora

2018-01-01

The mammalian target of rapamycin (mTOR) pathway has emerged as a key player for proper neural network development, and it is involved in epileptogenesis triggered by both genetic or acquired factors. Areas covered. The robust mTOR signaling deregulation observed in a large spectrum of epileptogenic

Long non-coding RNA BCAR4 promotes chondrosarcoma cell proliferation and migration through activation of mTOR signaling pathway.

Science.gov (United States)

Shui, Xiaolong; Zhou, Chengwei; Lin, Wei; Yu, Yang; Feng, Yongzeng; Kong, Jianzhong

2017-05-01

Chondrosarcoma is one of the common malignant histologic tumors, very difficult to treat, but the concrete cause and mechanism have not yet been elucidated. The present study aimed to investigate the functional involvement of BCAR4 in chondrosarcoma and its potentially underlying mechanism. QRT-PCR and western blot were used to determine the expression of BCAR4 and mTOR signaling pathway proteins both in chondrosarcoma tissues and cells. Chondrosarcoma cell proliferation and migration were assessed by MTT assay and transwell migration assay, respectively. The expression vectors were constructed and used to modulate the expression of BCAR4 and mTOR. Chondrosarcoma xenograft mouse model was established by subcutaneous injection with chondrosarcoma cell lines. The tumor volume was monitored to evaluate the effect of BCAR4 on chondrosarcoma cell tumorigenicity. The expressions of BCAR4, p-mTOR and p-P70S6K were up-regulated in chondrosarcoma tissues and cell lines. Moreover, BCAR4 overexpression had significant promoting effect on cell proliferation and migration in chondrosarcoma cells. Furthermore, mTOR signaling pathway was epigenetically activated by BCAR4-induced hyperacetylation of histone H3. We also found that mTOR overexpression abolished the decrease of chondrosarcoma cell proliferation and migration induced by BCAR4 knockdown. In vivo experiments confirmed that BCAR4 overexpression significantly accelerated tumor growth, while the knockdown of BCAR4 significantly inhibited tumor growth. BCAR4 promoted chondrosarcoma cell proliferation and migration through activation of mTOR signaling pathway, and thus contributed to chondrosarcoma progression. Impact statement LncRNA BCAR4 promoted chondrosarcoma cell proliferation and migration through activation of mTOR signaling pathway, and thus contributed to chondrosarcoma progression.

Ascorbic acid improves the antioxidant activity of European grape juices by improving the juices' ability to inhibit lipid peroxidation of human LDL in vitro

DEFF Research Database (Denmark)

Landbo, Anne-Katrine Regel; Meyer, Anne Boye Strunge

2001-01-01

. Red grape juice concentrate inhibited lipid peroxidation of LDL by prolonging the lag phase by 2.7 times relative to a control when evaluated at a total phenolic concentration of 10 muM gallic acid equivalents (GAE). Both red grape juices tested blocked lipid peroxidation of LDL at 20 muM GAE. White.......96, P acid alone did not exert antioxidant activity towards LDL, but combinations of 5 muM ascorbic acid with 5 muM GAE juice phenols eliminated the prooxidant activity of white grape juice, and significantly...

Inhibition of Oxidative Stress and Lipid Peroxidation by Anthocyanins from Defatted Canarium odontophyllum Pericarp and Peel Using In Vitro Bioassays

Science.gov (United States)

Khoo, Hock Eng; Azlan, Azrina; Ismail, Amin; Abas, Faridah; Hamid, Muhajir

2014-01-01

Canarium odontophyllum, also known as CO, is a highly nutritious fruit. Defatted parts of CO fruit are potent sources of nutraceutical. This study aimed to determine oxidative stress and lipid peroxidation effects of defatted CO pericarp and peel extracts using in vitro bioassays. Cell cytotoxic effect of the CO pericarp and peel extracts were also evaluated using HUVEC and Chang liver cell lines. The crude extracts of defatted CO peel and pericarp showed cytoprotective effects in t-BHP and 40% methanol-induced cell death. The crude extracts also showed no toxic effect to Chang liver cell line. Using CD36 ELISA, NAD+ and LDL inhibition assays, inhibition of oxidative stress were found higher in the crude extract of defatted CO peel compared to the pericarp extract. Hemoglobin and LDL oxidation assays revealed both crude extracts had significantly reduced lipid peroxidation as compared to control. TBARS values among defatted CO pericarp, peel, and cyanidin-3-glucoside showed no significant differences for hemoglobin and LDL oxidation assays. The protective effects of defatted CO parts, especially its peel is related to the presence of high anthocyanin that potentially offers as a pharmaceutical ingredient for cardioprotection. PMID:24416130

Inhibition of oxidative stress and lipid peroxidation by anthocyanins from defatted Canarium odontophyllum pericarp and peel using in vitro bioassays.

Directory of Open Access Journals (Sweden)

Hock Eng Khoo

Full Text Available Canarium odontophyllum, also known as CO, is a highly nutritious fruit. Defatted parts of CO fruit are potent sources of nutraceutical. This study aimed to determine oxidative stress and lipid peroxidation effects of defatted CO pericarp and peel extracts using in vitro bioassays. Cell cytotoxic effect of the CO pericarp and peel extracts were also evaluated using HUVEC and Chang liver cell lines. The crude extracts of defatted CO peel and pericarp showed cytoprotective effects in t-BHP and 40% methanol-induced cell death. The crude extracts also showed no toxic effect to Chang liver cell line. Using CD36 ELISA, NAD(+ and LDL inhibition assays, inhibition of oxidative stress were found higher in the crude extract of defatted CO peel compared to the pericarp extract. Hemoglobin and LDL oxidation assays revealed both crude extracts had significantly reduced lipid peroxidation as compared to control. TBARS values among defatted CO pericarp, peel, and cyanidin-3-glucoside showed no significant differences for hemoglobin and LDL oxidation assays. The protective effects of defatted CO parts, especially its peel is related to the presence of high anthocyanin that potentially offers as a pharmaceutical ingredient for cardioprotection.

Addition of Everolimus Post VEGFR Inhibition Treatment Failure in Advanced Sarcoma Patients Who Previously Benefited from VEGFR Inhibition: A Case Series

OpenAIRE

ElNaggar, Adam C.; Hays, John L.; Chen, James L.

2016-01-01

Background Patients with metastatic sarcoma who progress on vascular endothelial growth factor receptor inhibitors (VEGFRi) have limited treatment options. Upregulation of the mTOR pathway has been demonstrated to be a means of resistance to targeted VEGFRi in metastatic sarcoma. Patients and methods Retrospective cohort study to evaluate the clinical benefit at four months of combining mTOR inhibition (mTORi) via everolimus with VEGFRi in patients who have derived benefit from single-agent V...

EGFR targeting monoclonal antibody combines with an mTOR inhibitor and potentiates tumor inhibition by acting on complementary signaling hubs

International Nuclear Information System (INIS)

James, Roshan; Vishwakarma, Siddharth; Chivukula, Indira V; Basavaraj, Chetana; Melarkode, Ramakrishnan; Montero, Enrique; Nair, Pradip

2012-01-01

Nimotuzumab, an anti-epidermal growth factor receptor (anti-EGFR) monoclonal antibody, has been used extensively in many solid tumors and confers significant survival advantage. The antibody has limited skin toxicity and is generally well tolerated. Similar to other anti-EGFR therapies, patients may relapse a few months after treatment. In this study we show for the first time, the use of Nimotuzumab along with Sirolimus has synergistic effect on tumor inhibition as compared with the drugs used individually, in Nimotuzumab responsive and nonresponsive cell lines. In vitro studies prove that while Sirolimus (25 nmol/L) affects the signal downstream to mammalian target of rapamycin (mTOR), Nimotuzumab (83 nmol/L) downregulates pTYR, pMAPK and pSTAT3 by 40%, 20% and 30%, respectively. The combination, targeting these two different signaling hubs, may be associated with the synergistic inhibition observed. In vivo, the use of half human therapeutic equivalent doses for both the drugs substantially reduces tumors established in nude as well as severe combined immunodeficiency (SCID) mice by EGFR overexpressing A-431 cells. The drug combination reduces cell proliferation and the expression of signal transduction molecules. Treated tumors are better differentiated as compared with those established in the control mice. Tumor microarray demonstrates that Nimotuzumab and the combination groups segregate independently to the Sirolimus and the control treatment. The combination uniquely downregulated 55% of the altered tumor genes, extending beyond the typical pathways associated with Nimotuzumab and Sirolimus downstream pathways inhibition. These results would suggest that this nontoxic drug combination improves therapeutic benefit even in patients with low-EGFR expression and severely immunocompromised because of their current medication

Inactivation of possible micromycete food contaminants using the low-temperature plasma and hydrogen peroxide

International Nuclear Information System (INIS)

Čeřovský, M.; Khun, J.; Rusová, K.; Scholtz, V.; Soušková, H.

2013-01-01

The inhibition effect of hydrogen peroxide aerosol, low-temperature plasma and their combinations has been studied on several micromycetes spores. The low-temperature plasma was generated in corona discharges in the open air apparatus with hydrogen peroxide aerosol. Micromycete spores were inoculated on the surface of agar plates, exposed solely to the hydrogen peroxide aerosol, corona discharge or their combination. After incubation the diameter of inhibition zone was measured. The solely positive corona discharge exhibits no inactivation effect, the solely negative corona discharge and solely hydrogen peroxide aerosol exhibit the inactivation effect, however their combinations exhibit to be much more effective. Low-temperature plasma and hydrogen peroxide aerosol present a possible alternative method of microbial decontamination of food, food packages or other thermolabile materials

Inactivation of possible micromycete food contaminants using the low-temperature plasma and hydrogen peroxide

Energy Technology Data Exchange (ETDEWEB)

Čeřovský, M., E-mail: scholtz@aldebaran.cz [Institute of Chemical Technology in Prague, Department of Food Preservation, Faculty of Food and Biochemical Technology (Czech Republic); Khun, J. [Institute of Chemical Technology in Prague, Department of Physics and Measurements, Faculty of Chemical Engineering (Czech Republic); Rusová, K. [Institute of Chemical Technology in Prague, Department of Food Preservation, Faculty of Food and Biochemical Technology (Czech Republic); Scholtz, V. [Institute of Chemical Technology in Prague, Department of Physics and Measurements, Faculty of Chemical Engineering (Czech Republic); Soušková, H. [Institute of Chemical Technology in Prague, Department of Computing and Control Engineering, Faculty of Chemical Engineering (Czech Republic)

2013-09-15

The inhibition effect of hydrogen peroxide aerosol, low-temperature plasma and their combinations has been studied on several micromycetes spores. The low-temperature plasma was generated in corona discharges in the open air apparatus with hydrogen peroxide aerosol. Micromycete spores were inoculated on the surface of agar plates, exposed solely to the hydrogen peroxide aerosol, corona discharge or their combination. After incubation the diameter of inhibition zone was measured. The solely positive corona discharge exhibits no inactivation effect, the solely negative corona discharge and solely hydrogen peroxide aerosol exhibit the inactivation effect, however their combinations exhibit to be much more effective. Low-temperature plasma and hydrogen peroxide aerosol present a possible alternative method of microbial decontamination of food, food packages or other thermolabile materials.

Microsomal lipid peroxidation as a mechanism of cellular damage. [Dissertation

Energy Technology Data Exchange (ETDEWEB)

Kornbrust, D.J.

1979-01-01

The NADPH/iron-dependent peroxidation of lipids in rat liver microsomes was found to be dependent on the presence of free ferrous ion and maintains iron in the reduced Fe/sup 2 +/ state. Chelation of iron by EDTA inhibited peroxidation. Addition of iron, after preincubation of microsomes in the absence of iron, did not enhance the rate of peroxidation suggesting that iron acts by initiating peroxidative decomposition of membrane lipids rather than by catalyzing the breakdown of pre-formed hydroperoxides. Liposomes also underwent peroxidation in the presence of ferrous iron at a rate comparable to intact microsomes and was stimulated by ascorbate. Carbon tetrachloride initiated lipid peroxidation in the absence of free metal ions. Rates of in vitro lipid peroxidation of microsomes and homogenates were found to vary widely between different tissues and species. The effects of paraquat on lipid peroxidation was also studied. (DC)

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....

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

Directory of Open Access Journals (Sweden)

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

Water extractable phytochemicals from Capsicum pubescens (tree pepper) inhibit lipid peroxidation induced by different pro-oxidant agents in brain

International Nuclear Information System (INIS)

Oboh, G.; Rocha, J.B.T.

2006-03-01

Reactive oxygen species (ROS) is the cause of neurodegenerative disorders such as Lou Gehrig's disease, Parkinson's disease and Huntington's disease; one practical way to prevent and manage neurodegenerative diseases is through the eating of food rich in antioxidants (dietary means). In this study, the antioxidant and neuroprotective properties of aqueous extract of ripe and unripe Capsicum pubescens (popularly known as tree pepper) on different pro-oxidant induced lipid peroxidation in Rat's brain (in vitro) is been investigated. Aqueous extract of freshly harvested pepper was prepared, and the total phenol content, vitamin C, ferric reducing antioxidant property (FRAP) and Fe (II) chelating ability was determined. In addition, the ability of the extracts to protect the Rat's brain against some pro-oxidant FeSO 4 , Sodium nitroprusside and Quinolinic acid) - induced oxidative stress was also determined. The results of the study revealed that ripe Capsicum pubescens had a significantly higher (P 2 O 2 induced decomposition of deoxyribose. Therefore, ripe and unripe Capsicum pubescens would inhibit lipid peroxidation in vitro. However, the ripe potent was a more potent inhibitor of lipid peroxidation, which is probably due to its higher vitamin C and phenol content, reducing power and Fe (II) chelating ability. (author)

The Role of the Mammalian Target of Rapamycin (mTOR) in Pulmonary Fibrosis

Science.gov (United States)

Nho, Richard

2018-01-01

The phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT)/mammalian target of rapamycin (mTOR)-dependent pathway is one of the most integral pathways linked to cell metabolism, proliferation, differentiation, and survival. This pathway is dysregulated in a variety of diseases, including neoplasia, immune-mediated diseases, and fibroproliferative diseases such as pulmonary fibrosis. The mTOR kinase is frequently referred to as the master regulator of this pathway. Alterations in mTOR signaling are closely associated with dysregulation of autophagy, inflammation, and cell growth and survival, leading to the development of lung fibrosis. Inhibitors of mTOR have been widely studied in cancer therapy, as they may sensitize cancer cells to radiation therapy. Studies also suggest that mTOR inhibitors are promising modulators of fibroproliferative diseases such as idiopathic pulmonary fibrosis (IPF) and radiation-induced pulmonary fibrosis (RIPF). Therefore, mTOR represents an attractive and unique therapeutic target in pulmonary fibrosis. In this review, we discuss the pathological role of mTOR kinase in pulmonary fibrosis and examine how mTOR inhibitors may mitigate fibrotic progression. PMID:29518028

Enhancing mTOR-targeted cancer therapy by preventing mTOR/raptor inhibition-initiated, mTOR/rictor-independent Akt activation

OpenAIRE

Wang, Xuerong; Yue, Ping; Kim, Young Ae; Fu, Haian; Khuri, Fadlo R.; Sun, Shi-Yong

2008-01-01

It has been shown that mTOR inhibitors activate Akt while inhibiting mTOR signaling. However, the underlying mechanisms and the impact of the Akt activation on mTOR-targeted cancer therapy are unclear. The present work focused on addressing the role of mTOR/rictor in mTOR inhibitor-induced Akt activation and the impact of sustained Akt activation on mTOR-targeted cancer therapy. Thus, we have demonstrated that mTOR inhibitors increase Akt phosphorylation through a mechanism independent of mTO...

Cross regulation between mTOR signaling and O-GlcNAcylation.

Science.gov (United States)

Very, Ninon; Steenackers, Agata; Dubuquoy, Caroline; Vermuse, Jeanne; Dubuquoy, Laurent; Lefebvre, Tony; El Yazidi-Belkoura, Ikram

2018-06-01

The hexosamine biosynthetic pathway (HBP) integrates glucose, amino acids, fatty acids and nucleotides metabolisms for uridine diphosphate N-acetylglucosamine (UDP-GlcNAc) synthesis. UDP-GlcNAc is the nucleotide sugar donor for O-linked β-N-acetylglucosaminylation (O-GlcNAcylation) processes. O-GlcNAc transferase (OGT) is the enzyme which transfers the N-acetylglucosamine (O-GlcNAc) residue onto target proteins. Several studies previously showed that glucose metabolism dysregulations associated with obesity, diabetes or cancer correlated with an increase of OGT expression and global O-GlcNAcylation levels. Moreover, these diseases present an increased activation of the nutrient sensing mammalian target of rapamycin (mTOR) pathway. Other works demonstrate that mTOR regulates protein O-GlcNAcylation in cancer cells through stabilization of OGT. In this context, we studied the cross-talk between these two metabolic sensors in vivo in obese mice predisposed to diabetes and in vitro in normal and colon cancer cells. We report that levels of OGT and O-GlcNAcylation are increased in obese mice colon tissues and colon cancer cells and are associated with a higher activation of mTOR signaling. In parallel, treatments with mTOR regulators modulate OGT and O-GlcNAcylation levels in both normal and colon cancer cells. However, deregulation of O-GlcNAcylation affects mTOR signaling activation only in cancer cells. Thus, a crosstalk exists between O-GlcNAcylation and mTOR signaling in contexts of metabolism dysregulation associated to obesity or cancer.

Determination and validation of mTOR kinase-domain 3D structure by homology modeling

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Lakhlili W

2015-07-01

Full Text Available Wiame Lakhlili,1 Gwénaël Chevé,2 Abdelaziz Yasri,2 Azeddine Ibrahimi1 1Laboratoire de Biotechnologie (MedBiotech, Faculté de Médecine et de Pharmacie de Rabat, Université Mohammed V de Rabat, Rabat, Morroco; 2OriBase Pharma, Cap Gamma, Parc Euromédecine, Montpellier, France Abstract: The AKT/mammalian target of rapamycin (mTOR pathway is considered as one of the commonly activated and deregulated signaling pathways in human cancer. mTOR is associated with other proteins in two molecular complexes: mTOR complex 1/Raptor and the mTOR complex 2/Rictor. Using the crystal structure of the related lipid kinase PI3Kγ, we built a model of the catalytic region of mTOR. The modeling of the three-dimensional (3D structure of the mTOR was performed by homology modeling program SWISS-MODEL. The quality and validation of the obtained model were performed using PROCHECK and PROVE softwares. The overall stereochemical property of the protein was assessed by the Ramachandran plot. The model validation was also done by docking of known inhibitors. In this paper, we describe and validate a 3D model for the mTOR catalytic site.Keywords: mTOR, homology modeling, mTOR kinase-domain, docking

Discovery and Development of ATP-Competitive mTOR Inhibitors Using Computational Approaches.

Science.gov (United States)

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.

Influence of Growth Medium on Hydrogen Peroxide and Bacteriocin Production of Lactobacillus Strains

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Edina Németh

2005-01-01

Full Text Available This study was conducted to investigate the inhibitory effect of bacteriocin and the production of hydrogen peroxide by four non-starter lactic acid bacteria, Lactobacillus plantarum 2142, Lactobacillus curvatus 2770, Lactobacillus curvatus 2775, Lactobacillus casei subsp. pseudoplantarum 2750 and the probiotic strain Lactobacillus casei Shirota, propagated in de Man Rogosa Sharpe (MRS and tomato juice (TJ broth. The methods were a commonly used agar diffusion technique and a microtiter assay method. The best peroxide-producing Lactobacillus strain was selected for screening the inhibitory activity against Listeria monocytogenes, Bacillus cereus, Escherichia coli and the activity of bacteriocins against Lactobacillus sakei and Candida glabrata. All of the investigated lactic acid bacteria (LAB strains grown in MRS broth produced the highest concentration of hydrogen peroxide ranging from 2–6 g/mL after 72 h of storage. L. plantarum 2142 produced enough hydrogen peroxide already after 24 h at 5 °C in phosphate buffer to inhibit the growth of L. monocytogenes and B. cereus. Crude bacteriocin suspension from the investigated LAB inhibited only slightly the growth of L. sakei, however, the same suspension from MRS completely inhibited the 6-fold diluted yeast suspension. The concentrated bacteriocin suspensions from the both broths inhibited the growth of L. sakei completely. Among the strains, L. plantarum 2142 seemed to be the best peroxide and bacteriocin producer, and the antimicrobial metabolite production was better in MRS than in TJ broth.

Regulation of androgen receptor transactivity and mTOR-S6 kinase pathway by Rheb in prostate cancer cell proliferation.

Science.gov (United States)

Kobayashi, Takashi; Shimizu, Yosuke; Terada, Naoki; Yamasaki, Toshinari; Nakamura, Eijiro; Toda, Yoshinobu; Nishiyama, Hiroyuki; Kamoto, Toshiyuki; Ogawa, Osamu; Inoue, Takahiro

2010-06-01

Ras homolog-enriched in brain (Rheb), a small GTP-binding protein, is associated with prostate carcinogenesis through activating mammalian target of rapamycin (mTOR) signaling pathway. This study aimed to elucidate whether Rheb promotes proliferation of prostate cancer cells and can act as a potent therapeutic target in prostate cancer. Prostate cancer cell lines and human prostatic tissues were examined for the expression of Rheb. The effects of forced expression or knockdown of Rheb on cell proliferation were also examined. Semi-quantitative and quantitative RT-PCR were performed to evaluate mRNA expression. Western blotting was used to examine protein expression. Cell count and WST-1 assay were used to measure cell proliferation. Fluorescence-activated cell sorting was used to assess the cell cycle. Rheb mRNA and protein expression was higher in more aggressive, androgen-independent prostate cancer cell lines PC3, DU145, and C4-2, compared with the less aggressive LNCaP. Rheb expression was higher in cancer tissues than in benign prostatic epithelia. Forced expression of Rheb in LNCaP cells accelerated proliferation without enhancing androgen receptor transactivity. Attenuation of Rheb expression or treatment with the mTOR inhibitor rapamycin decreased proliferation of PC3 and DU145 cells, with a decrease in the activated form of p70S6 kinase, one of the main targets of mTOR. Rheb potentiates proliferation of prostate cancer cells and inhibition of Rheb or mTOR can lead to suppressed proliferation of aggressive prostate cancer cell lines in vitro. Rheb and the mTOR pathway are therefore probable targets for suppressing prostate cancer.

Inhibitors of mTOR

NARCIS (Netherlands)

Klümpen, Heinz-Josef; Beijnen, Jos H.; Gurney, Howard; Schellens, Jan H. M.

2010-01-01

Inhibitors of mammalian target of rapamycin (mTOR) have been approved for the treatment of renal cell carcinoma and appear to have a role in the treatment of other malignancies. The primary objective of this drug review is to provide pharmacokinetic and dynamic properties of the commonly used drugs

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

Membrane lipid peroxidation by UV-A: Mechanism and implications

International Nuclear Information System (INIS)

Bose, B.; Agarwal, S.; Chatterjee, S.N.

1990-01-01

UV-A produced a dose-dependent linear increase of lipid peroxidation in liposomal membrane, as detected by the assay of (i) conjugated dienes, (ii) lipid hydroperoxides, (iii) malondialdehydes (MDA), and (iv) the fluorescent adducts formed by the reaction of MDA with glycine and also a linear dose-dependent increase of [ 14 C]glucose efflux from the liposomes. UV-A-induced MDA production could not be inhibited by any significant degree by sodium formate, dimethyl sulfoxide, EDTA, or superoxide dismutase but was very significantly inhibited by butylated hydroxytoluene, alpha-tocopherol, sodium azide, L-histidine, dimethylfuran, and beta-carotene. MDA formation increased with an increase in the D 2 O content in water, leading to a maximal amount of nearly 50% enhancement of lipid peroxidation in 100% D 2 O vis-a-vis water used as dispersion medium. The experimental findings indicate the involvement of singlet oxygen as the initiator of the UV-A-induced lipid peroxidation

Targeting mTOR in HIV-Negative Classic Kaposi's Sarcoma

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Ofer Merimsky

2008-01-01

Full Text Available A 66-year old female with HIV-negative classic Kaposi's sarcoma responded to mTOR targeting by rapamycin. The response was well documented by PET-CT. This case provides supporting evidence that the mTOR pathway may be important in the tumorigenesis of KS and that rapamycin may have activity in this disease.

Kinetic modelling of in vitro data of PI3K, mTOR1, PTEN enzymes and on-target inhibitors Rapamycin, BEZ235, and LY294002.

Science.gov (United States)

Goltsov, Alexey; Tashkandi, Ghassan; Langdon, Simon P; Harrison, David J; Bown, James L

2017-01-15

The phosphatidylinositide 3-kinases (PI3K) and mammalian target of rapamycin-1 (mTOR1) are two key targets for anti-cancer therapy. Predicting the response of the PI3K/AKT/mTOR1 signalling pathway to targeted therapy is made difficult because of network complexities. Systems biology models can help explore those complexities but the value of such models is dependent on accurate parameterisation. Motivated by a need to increase accuracy in kinetic parameter estimation, and therefore the predictive power of the model, we present a framework to integrate kinetic data from enzyme assays into a unified enzyme kinetic model. We present exemplar kinetic models of PI3K and mTOR1, calibrated on in vitro enzyme data and founded on Michaelis-Menten (MM) approximation. We describe the effects of an allosteric mTOR1 inhibitor (Rapamycin) and ATP-competitive inhibitors (BEZ235 and LY294002) that show dual inhibition of mTOR1 and PI3K. We also model the kinetics of phosphatase and tensin homolog (PTEN), which modulates sensitivity of the PI3K/AKT/mTOR1 pathway to these drugs. Model validation with independent data sets allows investigation of enzyme function and drug dose dependencies in a wide range of experimental conditions. Modelling of the mTOR1 kinetics showed that Rapamycin has an IC 50 independent of ATP concentration and that it is a selective inhibitor of mTOR1 substrates S6K1 and 4EBP1: it retains 40% of mTOR1 activity relative to 4EBP1 phosphorylation and inhibits completely S6K1 activity. For the dual ATP-competitive inhibitors of mTOR1 and PI3K, LY294002 and BEZ235, we derived the dependence of the IC 50 on ATP concentration that allows prediction of the IC 50 at different ATP concentrations in enzyme and cellular assays. Comparison of drug effectiveness in enzyme and cellular assays showed that some features of these drugs arise from signalling modulation beyond the on-target action and MM approximation and require a systems-level consideration of the whole PI3K

Preclinical evaluation of WYE-687, a mTOR kinase inhibitor, as a potential anti-acute myeloid leukemia agent

International Nuclear Information System (INIS)

Cheng, Feng; Wang, Lingling; Shen, Yunfeng; Xia, Jun; Chen, Heng; Jiang, Yuanqiang; Lu, Mize

2016-01-01

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.

Preclinical evaluation of WYE-687, a mTOR kinase inhibitor, as a potential anti-acute myeloid leukemia agent

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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.

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

PRAS40 and PRR5-like protein are new mTOR interactors that regulate apoptosis.

Directory of Open Access Journals (Sweden)

Kathrin Thedieck

Full Text Available TOR (Target of Rapamycin is a highly conserved protein kinase and a central controller of cell growth. TOR is found in two functionally and structurally distinct multiprotein complexes termed TOR complex 1 (TORC1 and TOR complex 2 (TORC2. In the present study, we developed a two-dimensional liquid chromatography tandem mass spectrometry (2D LC-MS/MS based proteomic strategy to identify new mammalian TOR (mTOR binding proteins. We report the identification of Proline-rich Akt substrate (PRAS40 and the hypothetical protein Q6MZQ0/FLJ14213/CAE45978 as new mTOR binding proteins. PRAS40 binds mTORC1 via Raptor, and is an mTOR phosphorylation substrate. PRAS40 inhibits mTORC1 autophosphorylation and mTORC1 kinase activity toward eIF-4E binding protein (4E-BP and PRAS40 itself. HeLa cells in which PRAS40 was knocked down were protected against induction of apoptosis by TNFalpha and cycloheximide. Rapamycin failed to mimic the pro-apoptotic effect of PRAS40, suggesting that PRAS40 mediates apoptosis independently of its inhibitory effect on mTORC1. Q6MZQ0 is structurally similar to proline rich protein 5 (PRR5 and was therefore named PRR5-Like (PRR5L. PRR5L binds specifically to mTORC2, via Rictor and/or SIN1. Unlike other mTORC2 members, PRR5L is not required for mTORC2 integrity or kinase activity, but dissociates from mTORC2 upon knock down of tuberous sclerosis complex 1 (TSC1 and TSC2. Hyperactivation of mTOR by TSC1/2 knock down enhanced apoptosis whereas PRR5L knock down reduced apoptosis. PRR5L knock down reduced apoptosis also in mTORC2 deficient cells. The above suggests that mTORC2-dissociated PRR5L may promote apoptosis when mTOR is hyperactive. Thus, PRAS40 and PRR5L are novel mTOR-associated proteins that control the balance between cell growth and cell death.

mTOR is involved in 17β-estradiol-induced, cultured immature boar Sertoli cell proliferation via regulating the expression of SKP2, CCND1, and CCNE1.

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Yang, Wei-Rong; Wang, Yong; Wang, Yi; Zhang, Jiao-Jiao; Zhang, Jia-Hua; Lu, Cheng; Wang, Xian-Zhong

2015-04-01

Mammalian target of rapamycin (mTOR) is known to be involved in mammalian cell proliferation, while S-phase kinase-associated protein 2 (SKP2) plays a vital role in the cell cycle. Within the testis, estrogen also plays an important role in Sertoli cell proliferation, although it is not clear how. The present study asked if mTOR is involved in 17β-estradiol-dependent Sertoli cell proliferation. We specifically assessed if extracellular signal-regulated kinase 1/2 (ERK1/2) and/or phosphoinositide 3-kinase/protein kinase B (PI3K/AKT) exert convergent effects toward the activation of mTOR signaling, and if this signaling regulates the expression of SKP2 through retinoblastoma (RB) and early mitotic inhibitor 1 (EMI1) protein and on CCNE1 and CCND1 mRNA levels. Treatment with 17β-estradiol for 15-90 min activated mTOR, with mTOR phosphorylation peaking after 30 min. U0126 (5 μM), a specific inhibitor of (MEK1/2), and 10-DEBC (2 μM), a selective inhibitor of AKT, both significantly reduced 17β-estradiol-induced phosphorylation of mTOR. Rapamycin suppressed 17β-estradiol-induced Sertoli cell proliferation, appearing to act by reducing the abundance of SKP2, CCND1, and CCNE1 mRNA as well as RB and EMI1 protein. These data indicated that 17β-estradiol enhances Sertoli cell proliferation via mTOR activation, which involves both ERK1/2 and PI3K/AKT signaling. Activated mTOR subsequently increases SKP2 mRNA and protein expression by enhancing the expression of CCND1 and CCNE1, and inhibits SKP2 protein degradation by increasing EMI1 abundance. © 2015 Wiley Periodicals, Inc.

mTOR Signaling Confers Resistance to Targeted Cancer Drugs.

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Guri, Yakir; Hall, Michael N

2016-11-01

Cancer is a complex disease and a leading cause of death worldwide. Extensive research over decades has led to the development of therapies that target cancer-specific signaling pathways. However, the clinical benefits of such drugs are at best transient due to tumors displaying intrinsic or adaptive resistance. The underlying compensatory pathways that allow cancer cells to circumvent a drug blockade are poorly understood. We review here recent studies suggesting that mammalian TOR (mTOR) signaling is a major compensatory pathway conferring resistance to many cancer drugs. mTOR-mediated resistance can be cell-autonomous or non-cell-autonomous. These findings suggest that mTOR signaling should be monitored routinely in tumors and that an mTOR inhibitor should be considered as a co-therapy. Copyright © 2016 Elsevier Inc. All rights reserved.

The role of mTOR in ovarian cancer, polycystic ovary syndrome and ovarian aging.

Science.gov (United States)

Liu, Jin; Wu, Dai-Chao; Qu, Li-Hua; Liao, Hong-Qing; Li, Mei-Xiang

2018-05-12

The mammalian target of rapamycin, mTOR, is a serine-threonine protein kinase downstream of the phosphatidylinositol 3-kinase (PI3K)-AKT axis. The pathway can regulate cell growth, proliferation, and survival by activating ribosomal kinases. Recent studies have implicated the mTOR signaling pathway in ovarian neoplasms, polycystic ovary syndrome (PCOS) and premature ovarian failure (POF). Preclinical investigations have demonstrated that the PI3K/AKT/mTOR pathway is frequently activated in the control of various ovarian functions. mTOR allows cancer cells to escape the normal biochemical system and regulates the balance between apoptosis and survival. Some recent studies have suggested that involvement of the mTOR signaling system is an important pathophysiological basis of PCOS. Overexpression of the mTOR pathway can impair the interaction of cumulus cells, lead to insulin resistance, and affect the growth of follicles directly. The roles of mTOR signaling in follicular development have been extensively studied in recent years; abnormalities in this process lead to a series of pathologies such as POF and infertility. To improve understanding of the role of the mTOR signaling pathway in the pathogenesis and development of ovarian diseases, here we review the roles of mTOR signaling in such diseases and discuss the corresponding therapeutic strategies that target this pathway. This article is protected by copyright. All rights reserved. © 2018 Wiley Periodicals, Inc.

Promotion of ovarian follicle growth following mTOR activation: synergistic effects of AKT stimulators.

Directory of Open Access Journals (Sweden)

Yuan Cheng

Full Text Available Mammalian target of rapamycin (mTOR is a serine/threonine kinase and mTOR signaling is important in regulating cell growth and proliferation. Recent studies using oocyte- and granulosa cell-specific deletion of mTOR inhibitor genes TSC1 or TSC2 demonstrated the important role of mTOR signaling in the promotion of ovarian follicle development. We now report that treatment of ovaries from juvenile mice with an mTOR activator MHY1485 stimulated mTOR, S6K1 and rpS6 phosphorylation. Culturing ovaries for 4 days with MHY1485 increased ovarian explant weights and follicle development. In vivo studies further demonstrated that pre-incubation of these ovaries with MHY1485 for 2 days, followed by allo-grafting into kidney capsules of adult ovariectomized hosts for 5 days, led to marked increases in graft weights and promotion of follicle development. Mature oocytes derived from MHY1485-activated ovarian grafts could be successfully fertilized, leading the delivery of healthy pups. We further treated ovaries with the mTOR activator together with AKT activators (PTEN inhibitor and phosphoinositol-3-kinase stimulator before grafting and found additive enhancement of follicle growth. Our studies demonstrate the ability of an mTOR activator in promoting follicle growth, leading to a potential strategy to stimulate preantral follicle growth in infertile patients.

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.

Germline and somatic mutations in the MTOR gene in focal cortical dysplasia and epilepsy

DEFF Research Database (Denmark)

Møller, Rikke S; Weckhuysen, Sarah; Chipaux, Mathilde

2016-01-01

OBJECTIVE: To assess the prevalence of somatic MTOR mutations in focal cortical dysplasia (FCD) and of germline MTOR mutations in a broad range of epilepsies. METHODS: We collected 20 blood-brain paired samples from patients with FCD and searched for somatic variants using deep-targeted gene panel...... sequencing. Germline mutations in MTOR were assessed in a French research cohort of 93 probands with focal epilepsies and in a diagnostic Danish cohort of 245 patients with a broad range of epilepsies. Data sharing among collaborators allowed us to ascertain additional germline variants in MTOR. RESULTS: We...... detected recurrent somatic variants (p.Ser2215Phe, p.Ser2215Tyr, and p.Leu1460Pro) in the MTOR gene in 37% of participants with FCD II and showed histologic evidence for activation of the mTORC1 signaling cascade in brain tissue. We further identified 5 novel de novo germline missense MTOR variants in 6...

PED/PEA-15 inhibits hydrogen peroxide-induced apoptosis in Ins-1E pancreatic beta-cells via PLD-1.

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Francesca Fiory

Full Text Available The small scaffold protein PED/PEA-15 is involved in several different physiologic and pathologic processes, such as cell proliferation and survival, diabetes and cancer. PED/PEA-15 exerts an anti-apoptotic function due to its ability to interfere with both extrinsic and intrinsic apoptotic pathways in different cell types. Recent evidence shows that mice overexpressing PED/PEA-15 present larger pancreatic islets and increased beta-cells mass. In the present work we investigated PED/PEA-15 role in hydrogen peroxide-induced apoptosis in Ins-1E beta-cells. In pancreatic islets isolated from Tg(PED/PEA-15 mice hydrogen peroxide-induced DNA fragmentation was lower compared to WT islets. TUNEL analysis showed that PED/PEA-15 overexpression increases the viability of Ins-1E beta-cells and enhances their resistance to apoptosis induced by hydrogen peroxide exposure. The activity of caspase-3 and the cleavage of PARP-1 were markedly reduced in Ins-1E cells overexpressing PED/PEA-15 (Ins-1E(PED/PEA-15. In parallel, we observed a decrease of the mRNA levels of pro-apoptotic genes Bcl-xS and Bad. In contrast, the expression of the anti-apoptotic gene Bcl-xL was enhanced. Accordingly, DNA fragmentation was higher in control cells compared to Ins-1E(PED/PEA-15 cells. Interestingly, the preincubation with propranolol, an inhibitor of the pathway of PLD-1, a known interactor of PED/PEA-15, responsible for its deleterious effects on glucose tolerance, abolishes the antiapoptotic effects of PED/PEA-15 overexpression in Ins-1E beta-cells. The same results have been obtained by inhibiting PED/PEA-15 interaction with PLD-1 in Ins-1E(PED/PEA-15. These results show that PED/PEA-15 overexpression is sufficient to block hydrogen peroxide-induced apoptosis in Ins-1E cells through a PLD-1 mediated mechanism.

Nesfatin-1 inhibits ovarian epithelial carcinoma cell proliferation in vitro

International Nuclear Information System (INIS)

Xu, Yang; Pang, Xiaoyan; Dong, Mei; Wen, Fang; Zhang, Yi

2013-01-01

Highlights: •Nesfatin-1 inhibits the proliferation and growth of HO-8910 cells by G1 phase arrest. •Nesfatin-1 enhances HO-8910 cell apoptosis. •Nesfatin-1 inhibits HO-8910 cell proliferation via mTOR and RhoA/ROCK signaling pathway. •The first report of nesfatin-1-mediated proliferation in ovarian epithelial carcinoma. -- Abstract: Nesfatin-1, an 82-amino-acid peptide derived from a 396-amino-acid precursor protein nucleobindin 2 (NUCB2), was originally identified in hypothalamic nuclei involved in the regulation of food intake. It was recently reported that nesfatin-1 is a novel depot specific adipokine preferentially produced by subcutaneous tissue, with obesity- and food deprivation-regulated expression. Although a relation between ovarian cancer mortality and obesity has been previously established, a role of nesfatin-1 in ovarian epithelial carcinoma remains unknown. The aim of the present study is to examine the effect of nesfatin-1 on ovary carcinoma cells proliferation. We found that nesfatin-1 inhibits the proliferation and growth of HO-8910 cells by G1 phase arrest, this inhibition could be abolished by nesfatin-1 neutralizing antibody. Nesfatin-1 enhances HO-8910 cell apoptosis, activation of mammalian target of rapamycin (mTOR) and RhoA/ROCK signaling pathway block the effects of nesfatin-1-induced apoptosis, therefore reverses the inhibition of HO-8910 cell proliferation by nesfatin-1. In conclusion, the present study demonstrated that nesfatin-1 can inhibit the proliferation in human ovarian epithelial carcinoma cell line HO-8910 cells through inducing apoptosis via mTOR and RhoA/ROCK signaling pathway. This study provides a novel regulatory signaling pathway of nesfatin-1-regulated ovarian epithelial carcinoma growth and may contribute to ovarian cancer prevention and therapy, especially in obese patients

Nesfatin-1 inhibits ovarian epithelial carcinoma cell proliferation in vitro

Energy Technology Data Exchange (ETDEWEB)

Xu, Yang; Pang, Xiaoyan; Dong, Mei; Wen, Fang, E-mail: wenfang64@hotmail.com; Zhang, Yi, E-mail: syzi960@yahoo.com

2013-11-01

Highlights: •Nesfatin-1 inhibits the proliferation and growth of HO-8910 cells by G1 phase arrest. •Nesfatin-1 enhances HO-8910 cell apoptosis. •Nesfatin-1 inhibits HO-8910 cell proliferation via mTOR and RhoA/ROCK signaling pathway. •The first report of nesfatin-1-mediated proliferation in ovarian epithelial carcinoma. -- Abstract: Nesfatin-1, an 82-amino-acid peptide derived from a 396-amino-acid precursor protein nucleobindin 2 (NUCB2), was originally identified in hypothalamic nuclei involved in the regulation of food intake. It was recently reported that nesfatin-1 is a novel depot specific adipokine preferentially produced by subcutaneous tissue, with obesity- and food deprivation-regulated expression. Although a relation between ovarian cancer mortality and obesity has been previously established, a role of nesfatin-1 in ovarian epithelial carcinoma remains unknown. The aim of the present study is to examine the effect of nesfatin-1 on ovary carcinoma cells proliferation. We found that nesfatin-1 inhibits the proliferation and growth of HO-8910 cells by G1 phase arrest, this inhibition could be abolished by nesfatin-1 neutralizing antibody. Nesfatin-1 enhances HO-8910 cell apoptosis, activation of mammalian target of rapamycin (mTOR) and RhoA/ROCK signaling pathway block the effects of nesfatin-1-induced apoptosis, therefore reverses the inhibition of HO-8910 cell proliferation by nesfatin-1. In conclusion, the present study demonstrated that nesfatin-1 can inhibit the proliferation in human ovarian epithelial carcinoma cell line HO-8910 cells through inducing apoptosis via mTOR and RhoA/ROCK signaling pathway. This study provides a novel regulatory signaling pathway of nesfatin-1-regulated ovarian epithelial carcinoma growth and may contribute to ovarian cancer prevention and therapy, especially in obese patients.

Hydrogen peroxide as a fungicide for fish culture

Science.gov (United States)

Dawson, V.K.; Rach, J.J.; Schreier, Theresa M.

1994-01-01

Antifungal agents are needed to maintain healthy stocks of fish in the intensive culture systems currently employed in fish hatcheries. Malachite green has been the most widely used antifungal agent; however, its potential for producing teratology in animals and fish precludes further use in fish culture. Preliminary studies at the National Fisheries Research Center, La Crosse, WI, USA (La Crosse Center) indicate that hydrogen peroxide is effective for control of Saprolegnia sp. fungus on incubating eggs of rainbow trout. It is also effective against a wide variety of other organisms such as bacteria, yeasts, viruses, and spores, and has been proposed as a treatment for sea lice on salmon. Hydrogen peroxide and its primary decomposition products, oxygen and water, are not systemic poisons and are considered environmentally compatible. In response to a petition from the La Crosse Center, the U.S. Food and Drug Administration (FDA) recently classified hydrogen peroxide as a 'low regulatory priority' when used for control of fungus on fish and fish eggs. Preliminary tests conducted at the La Crosse Center suggest that prophylactic treatments of 250 to 500 ppm (based on 100% active ingredient) for 15 minutes every other day will inhibit fungal infections on healthy rainbow trout (Oncorhynchus mykiss) eggs. This treatment regime also seems to inhibit fungal development and increase hatching success among infected eggs. Efficacy and safety of hydrogen peroxide as a fungicide for fish are currently being evaluated.

Transient activation of mTOR following forced treadmill exercise in rats

DEFF Research Database (Denmark)

Elfving, Betina; Christensen, Tina; Ratner, Cecilia

2013-01-01

, while the induction of neurogenesis requires signaling through the VEGF receptor, Flk-1 (VEGFR-2). VEGF expression is believed to be regulated by two distinct mTOR (mammalian Target of Rapamycin)-containing multiprotein complexes mTORC1 and mTORC2, respectively. This study was initiated to investigate...... of mTOR was regulated after a single bout of exercise. In conclusion, the effect of treadmill exercise on the VEGF system is acute rather than chronic and there is a transient activation of mTOR. More studies are needed to understand whether this could be beneficial in the treatment of neuropsychiatric...

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

2014-01-01

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. PMID:25548410

Thiol peroxidases mediate specific genome-wide regulation of gene expression in response to hydrogen peroxide

OpenAIRE

Fomenko, Dmitri E.; Koc, Ahmet; Agisheva, Natalia; Jacobsen, Michael; Kaya, Alaattin; Malinouski, Mikalai; Rutherford, Julian C.; Siu, Kam-Leung; Jin, Dong-Yan; Winge, Dennis R.; Gladyshev, Vadim N.

2011-01-01

Hydrogen peroxide is thought to regulate cellular processes by direct oxidation of numerous cellular proteins, whereas antioxidants, most notably thiol peroxidases, are thought to reduce peroxides and inhibit H2O2 response. However, thiol peroxidases have also been implicated in activation of transcription factors and signaling. It remains unclear if these enzymes stimulate or inhibit redox regulation and whether this regulation is widespread or limited to a few cellular components. Herein, w...

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.

Targeting tumorigenesis: development and use of mTOR inhibitors in cancer therapy

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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.

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

Science.gov (United States)

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

2015-01-01

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. PMID:26522512

Agmatine produces antidepressant-like effects by activating AMPA receptors and mTOR signaling.

Science.gov (United States)

Neis, Vivian Binder; Moretti, Morgana; Bettio, Luis Eduardo B; Ribeiro, Camille M; Rosa, Priscila Batista; Gonçalves, Filipe Marques; Lopes, Mark William; Leal, Rodrigo Bainy; Rodrigues, Ana Lúcia S

2016-06-01

The activation of AMPA receptors and mTOR signaling has been reported as mechanisms underlying the antidepressant effects of fast-acting agents, specially the NMDA receptor antagonist ketamine. In the present study, oral administration of agmatine (0.1mg/kg), a neuromodulator that has been reported to modulate NMDA receptors, caused a significant reduction in the immobility time of mice submitted to the tail suspension test (TST), an effect prevented by the administration of DNQX (AMPA receptor antagonist, 2.5μg/site, i.c.v.), BDNF antibody (1μg/site, i.c.v.), K-252a (TrkB receptor antagonist, 1μg/site, i.c.v.), LY294002 (PI3K inhibitor, 10nmol/site, i.c.v.) or rapamycin (selective mTOR inhibitor, 0.2nmol/site, i.c.v.). Moreover, the administration of lithium chloride (non-selective GSK-3β inhibitor, 10mg/kg, p.o.) or AR-A014418 (selective GSK-3β inhibitor, 0.01μg/site, i.c.v.) in combination with a sub-effective dose of agmatine (0.0001mg/kg, p.o.) reduced the immobility time in the TST when compared with either drug alone. Furthermore, increased immunocontents of BDNF, PSD-95 and GluA1 were found in the prefrontal cortex of mice just 1h after agmatine administration. These results indicate that the antidepressant-like effect of agmatine in the TST may be dependent on the activation of AMPA and TrkB receptors, PI3K and mTOR signaling as well as inhibition of GSK-3β, and increase in synaptic proteins. The results contribute to elucidate the complex signaling pathways involved in the antidepressant effect of agmatine and reinforce the pivotal role of these molecular targets for antidepressant responses. Copyright © 2016 Elsevier B.V. and ECNP. All rights reserved.

Antioxidant, lipid peroxidation inhibition and free radical scavenging efficacy of a diterpenoid compound sugiol isolated from Metasequoia glyptostroboides.

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Bajpai, Vivek K; Sharma, Ajay; Kang, Sun Chul; Baek, Kwang-Hyun

2014-01-01

To investigate the antioxidant efficacy of a biologically active diterpenoid compound sugiol isolated from Metasequoia glyptostroboides (M. glyptostroboides) in various antioxidant models. An abietane type diterpenoid sugiol, isolated from ethyl acetate extract of M. glyptostroboides cones, was analyzed for its antioxidant efficacy as reducing power ability and lipid peroxidation inhibition as well as its ability to scavenge free radicals such as 1,1-diphenyl-2-picryl hydrazyl, nitric oxide, superoxide and hydroxyl radicals. The sugiol showed significant and concentration-dependent antioxidant and free radical scavenging activities. Consequently, the sugiol exerted lipid peroxidation inhibitory effect by 76.5% as compared to α-tocopherol (80.13%) and butylated hydroxyanisole (76.59%). In addition, the sugiol had significant scavenging activities of 1,1-diphenyl-2-picryl hydrazyl, nitric oxide, superoxide and hydroxyl free radicals in a concentration-dependent manner by 78.83%, 72.42%, 72.99% and 85.04%, when compared to the standard compound ascorbic acid (81.69%, 74.62%, 73.00% and 73.79%) and α-tocopherol/butylated hydroxyanisole (84.09%, 78.61%, 74.45% and 70.02%), respectively. These findings justify the biological and traditional uses of M. glyptostroboides or its secondary metabolites as confirmed by its promising antioxidant efficacy. Copyright © 2014 Hainan Medical College. Published by Elsevier B.V. All rights reserved.

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

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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.

Evaluation of the antioxidant activity of root extract of pepper fruit (Dennetia tripetala), and it's potential for the inhibition of lipid peroxidation.

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Okolie, Ngozi Paulinus; Falodun, Abiodun; Davids, Oluseyi

2014-01-01

The antioxidant properties of ethanolic root extract of pepper fruit (Donnetia tripetala), and its effect on lipid peroxidation of some fresh beef tissues during frozen storage were investigated. The antioxidant parameters were assessed using standard methods, while malondialdehyde levels of different fresh beef tissue sections treated with the extract prior to freezing, were estimated in a colorimetric reaction with thiobarbituric acid. The H2O2-scavenging ability of the extract was similar to that of ascorbic acid, with a maximum scavenging power of 55.61 ±4.98%, and an IC50 value of 86µg/ml. The extract exhibited a concentration-dependent ferric ion-reducing power, although this was significantly lower relative to that of the ascorbic acid (p capacity of the extract to inhibit lipid peroxidation in frozen heart muscle slices was significantly higher than that of vitamin C (p extract of D. tripetala is rich in antioxidants which can be applied to meat preservation during refrigerated storage.

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.

Comment on "A dynamic network model of mTOR signaling reveals TSC-independent mTORC2 regulation": building a model of the mTOR signaling network with a potentially faulty tool.

Science.gov (United States)

Manning, Brendan D

2012-07-10

In their study published in Science Signaling (Research Article, 27 March 2012, DOI: 10.1126/scisignal.2002469), Dalle Pezze et al. tackle the dynamic and complex wiring of the signaling network involving the protein kinase mTOR, which exists within two distinct protein complexes (mTORC1 and mTORC2) that differ in their regulation and function. The authors use a combination of immunoblotting for specific phosphorylation events and computational modeling. The primary experimental tool employed is to monitor the autophosphorylation of mTOR on Ser(2481) in cell lysates as a surrogate for mTOR activity, which the authors conclude is a specific readout for mTORC2. However, Ser(2481) phosphorylation occurs on both mTORC1 and mTORC2 and will dynamically change as the network through which these two complexes are connected is manipulated. Therefore, models of mTOR network regulation built using this tool are inherently imperfect and open to alternative explanations. Specific issues with the main conclusion made in this study, involving the TSC1-TSC2 (tuberous sclerosis complex 1 and 2) complex and its potential regulation of mTORC2, are discussed here. A broader goal of this Letter is to clarify to other investigators the caveats of using mTOR Ser(2481) phosphorylation in cell lysates as a specific readout for either of the two mTOR complexes.

Labor Inhibits Placental Mechanistic Target of Rapamycin Complex 1 Signaling

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LAGER, Susanne; AYE, Irving L.M.H.; GACCIOLI, Francesca; RAMIREZ, Vanessa I.; JANSSON, Thomas; POWELL, Theresa L.

2014-01-01

Introduction Labor induces a myriad of changes in placental gene expression. These changes may represent a physiological adaptation inhibiting placental cellular processes associated with a high demand for oxygen and energy (e.g., protein synthesis and active transport) thereby promoting oxygen and glucose transfer to the fetus. We hypothesized that mechanistic target of rapamycin complex 1 (mTORC1) signaling, a positive regulator of trophoblast protein synthesis and amino acid transport, is inhibited by labor. Methods Placental tissue was collected from healthy, term pregnancies (n=15 no-labor; n=12 labor). Activation of Caspase-1, IRS1/Akt, STAT, mTOR, and inflammatory signaling pathways was determined by Western blot. NFκB p65 and PPARγ DNA binding activity was measured in isolated nuclei. Results Labor increased Caspase-1 activation and mTOR complex 2 signaling, as measured by phosphorylation of Akt (S473). However, mTORC1 signaling was inhibited in response to labor as evidenced by decreased phosphorylation of mTOR (S2448) and 4EBP1 (T37/46 and T70). Labor also decreased NFκB and PPARγ DNA binding activity, while having no effect on IRS1 or STAT signaling pathway. Discussion and conclusion Several placental signaling pathways are affected by labor, which has implications for experimental design in studies of placental signaling. Inhibition of placental mTORC1 signaling in response to labor may serve to down-regulate protein synthesis and amino acid transport, processes that account for a large share of placental oxygen and glucose consumption. We speculate that this response preserves glucose and oxygen for transfer to the fetus during the stressful events of labor. PMID:25454472

Phase I study of the mTOR inhibitor ridaforolimus and the HDAC inhibitor vorinostat in advanced renal cell carcinoma and other solid tumors.

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Zibelman, Matthew; Wong, Yu-Ning; Devarajan, Karthik; Malizzia, Lois; Corrigan, Alycia; Olszanski, Anthony J; Denlinger, Crystal S; Roethke, Susan K; Tetzlaff, Colleen H; Plimack, Elizabeth R

2015-10-01

Drugs inhibiting the mammalian target of rapamycin (mTOR) are approved in the treatment of renal cell carcinoma (RCC), but resistance inevitably emerges. Proposed escape pathways include increased phosphorylation of Akt, which can be down regulated by histone deacetylase (HDAC) inhibitors. We hypothesized that co-treatment with the mTOR inhibitor ridaforolimus and the HDAC inhibitor vorinostat may abrogate resistance in RCC. This phase 1 study evaluated the co-administration of ridaforolimus and vorinostat in patients with advanced solid tumors. The primary objective was to determine the maximum tolerated dose (MTD) in RCC patients. Although all solid tumors were allowed, prior cytotoxic chemotherapy was limited to 1 regimen. Using a modified 3 + 3 dose escalation design, various dose combinations were tested concurrently in separate cohorts. Efficacy was a secondary endpoint. Fifteen patients were treated at one of three dose levels, thirteen with RCC (10 clear cell, 3 papillary). Dosing was limited by thrombocytopenia. The MTD was determined to be ridaforolimus 20 mg daily days 1-5 with vorinostat 100 mg BID days 1-3 weekly, however late onset thrombocytopenia led to a lower recommended phase II dose: ridaforolimus 20 mg daily days 1-5 with vorinostat 100 mg daily days 1-3 weekly. Two patients, both with papillary RCC, maintained disease control for 54 and 80 weeks, respectively. The combination of ridaforolimus and vorinostat was tolerable at the recommended phase II dose. Two patients with papillary RCC experienced prolonged disease stabilization, thus further study of combined HDAC and mTOR inhibition in this population is warranted.

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.

Anti-lymphangiogenic properties of mTOR inhibitors in head and neck squamous cell carcinoma experimental models

International Nuclear Information System (INIS)

Ekshyyan, Oleksandr; Moore-Medlin, Tara N; Raley, Matthew C; Sonavane, Kunal; Rong, Xiaohua; Brodt, Michael A; Abreo, Fleurette; Alexander, Jonathan Steven; Nathan, Cherie-Ann O

2013-01-01

Tumor dissemination to cervical lymph nodes via lymphatics represents the first step in the metastasis of head and neck squamous cell carcinoma (HNSCC) and is the most significant predictor of tumor recurrence decreasing survival by 50%. The lymphatic suppressing properties of mTOR inhibitors are not yet well understood. Lymphatic inhibiting effects of rapamycin were evaluated in vitro using two lymphatic endothelial cell (LEC) lines. An orthotopic mouse model of HNSCC (OSC-19 cells) was used to evaluate anti-lymphangiogenic effects of rapamycin in vivo. The incidence of cervical lymph node metastases, numbers of tumor-free lymphatic vessels and those invaded by tumor cells in mouse lingual tissue, and expression of pro-lymphangiogenic markers were assessed. Rapamycin significantly decreased lymphatic vascular density (p = 0.027), reduced the fraction of lymphatic vessels invaded by tumor cells in tongue tissue (p = 0.013) and decreased metastasis-positive lymph nodes (p = 0.04). Rapamycin also significantly attenuated the extent of metastatic tumor cell spread within lymph nodes (p < 0.0001). We found that rapamycin significantly reduced LEC proliferation and was correlated with decreased VEGFR-3 expression in both LEC, and in some HNSCC cell lines. The results of this study demonstrate anti-lymphangiogenic properties of mTOR inhibitors in HNSCC. mTOR inhibitors suppress autocrine and paracrine growth stimulation of tumor and lymphatic endothelial cells by impairing VEGF-C/VEGFR-3 axis and release of soluble VEGFR-2. In a murine HNSCC orthotopic model rapamycin significantly suppressed lymphovascular invasion, decreased cervical lymph node metastasis and delayed the spread of metastatic tumor cells within the lymph nodes

ERK inhibition sensitizes CZ415-induced anti-osteosarcoma activity in vitro and in vivo.

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Yin, Gang; Fan, Jin; Zhou, Wei; Ding, Qingfeng; Zhang, Jun; Wu, Xuan; Tang, Pengyu; Zhou, Hao; Wan, Bowen; Yin, Guoyong

2017-10-10

mTOR is a valuable oncotarget for osteosarcoma. The anti-osteosarcoma activity by a novel mTOR kinase inhibitor, CZ415, was evaluated. We demonstrated that CZ415 potently inhibited survival and proliferation of known osteosarcoma cell lines (U2OS, MG-63 and SaOs2), and primary human osteosarcoma cells. Further, CZ415 provoked apoptosis and disrupted cell cycle progression in osteosarcoma cells. CZ415 treatment in osteosarcoma cells concurrently blocked mTORC1 and mTORC2 activation. Intriguingly, ERK-MAPK activation could be a major resistance factor of CZ415. ERK inhibition (by MEK162/U0126) or knockdown (by targeted ERK1/2 shRNAs) dramatically sensitized CZ415-induced osteosarcoma cell apoptosis. In vivo , CZ415 oral administration efficiently inhibited U2OS tumor growth in mice. Its activity was further potentiated with co-administration of MEK162. Collectively, we demonstrate that ERK inhibition sensitizes CZ415-induced anti-osteosarcoma activity in vitro and in vivo . CZ415 could be further tested as a promising anti-osteosarcoma agent, alone or in combination of ERK inhibition.

β-Amyloid promotes accumulation of lipid peroxides by inhibiting CD36-mediated clearance of oxidized lipoproteins

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Khan Tayeba

2004-11-01

Full Text Available Abstract Background Recent studies suggest that hypercholesterolemia, an established risk factor for atherosclerosis, is also a risk factor for Alzheimer's disease. The myeloid scavenger receptor CD36 binds oxidized lipoproteins that accumulate with hypercholesterolemia and mediates their clearance from the circulation and peripheral tissues. Recently, we demonstrated that CD36 also binds fibrillar β-amyloid and initiates a signaling cascade that regulates microglial recruitment and activation. As increased lipoprotein oxidation and accumulation of lipid peroxidation products have been reported in Alzheimer's disease, we investigated whether β-amyloid altered oxidized lipoprotein clearance via CD36. Methods The availability of mice genetically deficient in class A (SRAI & II and class B (CD36 scavenger receptors has facilitated studies to discriminate their individual actions. Using primary microglia and macrophages, we assessed the impact of Aβ on: (a cholesterol ester accumulation by GC-MS and neutral lipid staining, (b binding, uptake and degradation of 125I-labeled oxidized lipoproteins via CD36, SR-A and CD36/SR-A-independent pathways, (c expression of SR-A and CD36. In addition, using mice with targeted deletions in essential kinases in the CD36-signaling cascade, we investigated whether Aβ-CD36 signaling altered metabolism of oxidized lipoproteins. Results In primary microglia and macrophages, Aβ inhibited binding, uptake and degradation of oxidized low density lipoprotein (oxLDL in a dose-dependent manner. While untreated cells accumulated abundant cholesterol ester in the presence of oxLDL, cells treated with Aβ were devoid of cholesterol ester. Pretreatment of cells with Aβ did not affect subsequent degradation of oxidized lipoproteins, indicating that lysosomal accumulation of Aβ did not disrupt this degradation pathway. Using mice with targeted deletions of the scavenger receptors, we demonstrated that Aβ inhibited oxidized

Lactic acid in tumor microenvironments causes dysfunction of NKT cells by interfering with mTOR signaling.

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Xie, Di; Zhu, Shasha; Bai, Li

2016-12-01

Cellular metabolism has been shown to regulate differentiation and function of immune cells. Tumor associated immune cells undergo phenotypic and functional alterations due to the change of cellular metabolism in tumor microenvironments. NKT cells are good candidates for immunotherapies against tumors and have been used in several clinical trials. However, the influences of tumor microenvironments on NKT cell functions remain unclear. In our studies, lactic acid in tumor microenvironments inhibited IFNγ and IL4 productions from NKT cells, and more profound influence on IFNγ was observed. By adjusting the pH of culture medium we further showed that, dysfunction of NKT cells could simply be induced by low extracellular pH. Moreover, low extracellular pH inhibited NKT cell functions by inhibiting mammalian target of rapamycin (mTOR) signaling and nuclear translocation of promyelocytic leukemia zinc-finger (PLZF). Together, our results suggest that tumor acidic microenvironments could interfere with NKT cell functions through metabolic controls.

Learning from the Cardiologists and Developing Eluting Stents Targeting the Mtor Pathway for Pulmonary Application; A Future Concept for Tracheal Stenosis

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Zarogoulidis, Paul; Darwiche, Kaid; Tsakiridis, Kosmas; Teschler, Helmut; Yarmus, Lonny; Zarogoulidis, Konstantinos; Freitag, Lutz

2014-01-01

Tracheal stenosis due to either benign or malignant disease is a situation that the pulmonary physicians and thoracic surgeons have to cope in their everyday clinical practice. In the case where tracheal stenosis is caused due to malignancy mini-interventional interventions with laser, apc, cryoprobe, balloon dilation or with combination of more than one equipment and technique can be used. On the other hand, in the case of a benign disease such as; tracheomalacia the clinician can immediately upon diagnosis proceed to the stent placement. In both situations however; it has been observed that the stents induce formation of granuloma tissue in both or one end of the stent. Therefore a frequent evaluation of the patient is necessary, taking also into account the nature of the primary disease. Evaluation methodologies identifying different types and extent of the trachea stenosis have been previously published. However; we still do not have an effective adjuvant therapy to prevent granuloma tissue formation or prolong already treated granuloma lesions. There have been proposed many mechanisms which induce the abnormal growth of the local tissue, such as; local pressure, local stress, inflammation and vascular endothelial growth factor overexpression. Immunomodulatory agents inhibiting the mTOR pathway are capable of inhibiting the inflammatory cascade locally. In the current mini-review we will try to present the current knowledge of drug eluting stents inhibiting the mTOR pathway and propose a future application of these stents as a local anti-proliferative treatment. PMID:24454525

Learning from the Cardiologists and Developing Eluting Stents Targeting the Mtor Pathway for Pulmonary Application; A Future Concept for Tracheal Stenosis.

Science.gov (United States)

Zarogoulidis, Paul; Darwiche, Kaid; Tsakiridis, Kosmas; Teschler, Helmut; Yarmus, Lonny; Zarogoulidis, Konstantinos; Freitag, Lutz

2013-08-26

Tracheal stenosis due to either benign or malignant disease is a situation that the pulmonary physicians and thoracic surgeons have to cope in their everyday clinical practice. In the case where tracheal stenosis is caused due to malignancy mini-interventional interventions with laser, apc, cryoprobe, balloon dilation or with combination of more than one equipment and technique can be used. On the other hand, in the case of a benign disease such as; tracheomalacia the clinician can immediately upon diagnosis proceed to the stent placement. In both situations however; it has been observed that the stents induce formation of granuloma tissue in both or one end of the stent. Therefore a frequent evaluation of the patient is necessary, taking also into account the nature of the primary disease. Evaluation methodologies identifying different types and extent of the trachea stenosis have been previously published. However; we still do not have an effective adjuvant therapy to prevent granuloma tissue formation or prolong already treated granuloma lesions. There have been proposed many mechanisms which induce the abnormal growth of the local tissue, such as; local pressure, local stress, inflammation and vascular endothelial growth factor overexpression. Immunomodulatory agents inhibiting the mTOR pathway are capable of inhibiting the inflammatory cascade locally. In the current mini-review we will try to present the current knowledge of drug eluting stents inhibiting the mTOR pathway and propose a future application of these stents as a local anti-proliferative treatment.

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

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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

Wnt1 inhibits hydrogen peroxide-induced apoptosis in mouse cardiac stem cells.

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Jingjin Liu

Full Text Available BACKGROUND: Because of their regenerative and paracrine abilities, cardiac stem cells (CSCs are the most appropriate, optimal and promising candidates for the development of cardiac regenerative medicine strategies. However, native and exogenous CSCs in ischemic hearts are exposed to various pro-apoptotic or cytotoxic factors preventing their regenerative and paracrine abilities. METHODS AND RESULTS: We examined the effects of H2O2 on mouse CSCs (mCSCs, and observed that hydrogen peroxide (H2O2 treatment induces mCSCs apoptosis via the caspase 3 pathway, in a dose-dependent manner. We then examined the effects of Wnt1 over-expression on H2O2-induced apoptosis in mCSCs and observed that Wnt1 significantly decreased H2O2-induced apoptosis in mCSCs. On the other hand, inhibition of the canonical Wnt pathway by the secreted frizzled related protein 2 (SFRP2 or knockdown of β-catenin in mCSCs reduced cells resistance to H2O2-induced apoptosis, suggesting that Wnt1 predominantly prevents H2O2-induced apoptosis through the canonical Wnt pathway. CONCLUSIONS: Our results provide the first evidences that Wnt1 plays an important role in CSCs' defenses against H2O2-induced apoptosis through the canonical Wnt1/GSK3β/β-catenin signaling pathway.

Lipid peroxidation in liver homogenates. Effects of membrane lipid composition and irradiation

International Nuclear Information System (INIS)

Vaca, C.; Ringdahl, M.H.

1984-01-01

The rate of lipid peroxidation has been followed in whole liver homogenates from mice using the TBA-method. Liver homogenates with different membrane fatty acid composition were obtained from mice fed diets containing different sources of fat i.e. sunflower seed oil (S), coconut oil (C) and hydrogenated lard (L). The yields of the TBA-chromophore (TBA-c) were 4 times higher in the liver homogenates S compared to C and L after 4 hour incubation at 37 0 C. Irradiation of the liver homogenates before incubation inhibited the formation of lipid peroxidation products in a dose dependent way. The catalytic capacity of the homogenates was investigated, followed as the autooxidation of cysteamine or modified by addition of the metal chelator EDTA. The rate of autooxidation of cysteamine, which is dependent on the presence of metal ions (Fe/sup 2+/ or Cu/sup 2+/), was decreased with increasing dose, thus indicating an alteration in the availability of metal catalysts in the system. The addition of Fe/sup 2+/ to the system restored the lipid peroxidation yields in the irradiated systems and the presence of EDTA inhibited the formation of lipid peroxidation products in all three dietary groups. It is suggested that irradiation alters the catalytic activity needed in the autooxidation processes of polyunsaturated fatty acids

mTOR in breast cancer: differential expression in triple-negative and non-triple-negative tumors.

LENUS (Irish Health Repository)

Walsh, S

2012-04-01

Triple-negative breast cancer (TNBC) is defined by the absence of estrogen receptors (ER), progesterone receptors (PR) and overexpression of HER2. Targeted therapy is currently unavailable for this subgroup of breast cancer patients. mTOR controls cancer cell growth, survival and invasion and is thus a potential target for the treatment of patients with TNBC. Using immunohistochemistry, mTOR and p-mTOR were measured in 89 TNBCs and 99 non-TNBCs. While mTOR expression was confined to tumor cell cytoplasm, p-mTOR staining was located in the nucleus, perinuclear area and in the cytoplasm. Potentially important, was our finding that nuclear p-mTOR was found more frequently in triple-negative than non triple-negative cancers (p < 0.001). These results suggest that mTOR may play a more important role in the progression of TNBC compared to non-TNBC. Based on these findings, we conclude that mTOR may be a new target for the treatment of triple-negative breast cancer.

Translation initiation complex eIF4F is a therapeutic target for dual mTOR kinase inhibitors in non-Hodgkin lymphoma

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Stenson, Mary J.; Maurer, Matthew J.; Wellik, Linda E.; Link, Brian; Hege, Kristen; Dogan, Ahmet; Sotomayor, Eduardo; Witzig, Thomas; Gupta, Mamta

2015-01-01

Deregulated mRNA translation has been implicated in disease development and in part is controlled by a eukaryotic initiation complex eIF4F (composed of eIF4E, eIF4G and eIF4A). We demonstrate here that the cap bound fraction from lymphoma cells was enriched with eIF4G and eIF4E indicating that lymphoma cells exist in an activated translational state. Moreover, 77% (110/142) of diffuse large B cell lymphoma tumors expressed eIF4E and this was associated with an inferior event free survival. Over-expression of wild-type eIF4E (eIF4EWT) but not cap-mutant eIF4E (eIF4Ecap mutant) increased the activation of the eIF4F complex. Treatment with the active-site dual mTOR inhibitor CC214-1 reduced the level of the eIF4F complex by decreasing the cap bound fraction of eIF4G and increasing the levels of 4E-BP1. CC214-1 inhibited both the cap dependent and global protein translation. CC214-1 inhibited c-Myc, and cyclin D3 translation by decreasing polysomal fractions from lymphoma cells. Inhibition of eIF4E with shRNA further decreased the CC214-1 induced inhibition of the eIF4F complex, c-Myc, cyclin D3 translation, and colony formation. These studies demonstrate that the eIF4F complex is deregulated in aggressive lymphoma and that dual mTOR therapy has therapeutic potential in these patients. PMID:25839159

Differential control of ageing and lifespan by isoforms and splice variants across the mTOR network.

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Razquin Navas, Patricia; Thedieck, Kathrin

2017-07-15

Ageing can be defined as the gradual deterioration of physiological functions, increasing the incidence of age-related disorders and the probability of death. Therefore, the term ageing not only reflects the lifespan of an organism but also refers to progressive functional impairment and disease. The nutrient-sensing kinase mTOR (mammalian target of rapamycin) is a major determinant of ageing. mTOR promotes cell growth and controls central metabolic pathways including protein biosynthesis, autophagy and glucose and lipid homoeostasis. The concept that mTOR has a crucial role in ageing is supported by numerous reports on the lifespan-prolonging effects of the mTOR inhibitor rapamycin in invertebrate and vertebrate model organisms. Dietary restriction increases lifespan and delays ageing phenotypes as well and mTOR has been assigned a major role in this process. This may suggest a causal relationship between the lifespan of an organism and its metabolic phenotype. More than 25 years after mTOR's discovery, a wealth of metabolic and ageing-related effects have been reported. In this review, we cover the current view on the contribution of the different elements of the mTOR signalling network to lifespan and age-related metabolic impairment. We specifically focus on distinct roles of isoforms and splice variants across the mTOR network. The comprehensive analysis of mouse knockout studies targeting these variants does not support a tight correlation between lifespan prolongation and improved metabolic phenotypes and questions the strict causal relationship between them. © 2017 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.

DEPTOR-related mTOR suppression is involved in metformin's anti-cancer action in human liver cancer cells

Energy Technology Data Exchange (ETDEWEB)

Obara, Akio; Fujita, Yoshihito; Abudukadier, Abulizi; Fukushima, Toru; Oguri, Yasuo; Ogura, Masahito; Harashima, Shin-ichi; Hosokawa, Masaya; Inagaki, Nobuya, E-mail: inagaki@metab.kuhp.kyoto-u.ac.jp

2015-05-15

Metformin, one of the most commonly used drugs for patients with type 2 diabetes, recently has received much attention regarding its anti-cancer action. It is thought that the suppression of mTOR signaling is involved in metformin's anti-cancer action. Although liver cancer is one of the most responsive types of cancer for reduction of incidence by metformin, the molecular mechanism of the suppression of mTOR in liver remains unknown. In this study, we investigated the mechanism of the suppressing effect of metformin on mTOR signaling and cell proliferation using human liver cancer cells. Metformin suppressed phosphorylation of p70-S6 kinase, and ribosome protein S6, downstream targets of mTOR, and suppressed cell proliferation. We found that DEPTOR, an endogenous substrate of mTOR suppression, is involved in the suppressing effect of metformin on mTOR signaling and cell proliferation in human liver cancer cells. Metformin increases the protein levels of DEPTOR, intensifies binding to mTOR, and exerts a suppressing effect on mTOR signaling. This increasing effect of DEPTOR by metformin is regulated by the proteasome degradation system; the suppressing effect of metformin on mTOR signaling and cell proliferation is in a DEPTOR-dependent manner. Furthermore, metformin exerts a suppressing effect on proteasome activity, DEPTOR-related mTOR signaling, and cell proliferation in an AMPK-dependent manner. We conclude that DEPTOR-related mTOR suppression is involved in metformin's anti-cancer action in liver, and could be a novel target for anti-cancer therapy. - Highlights: • We elucidated a novel pathway of metformin's anti-cancer action in HCC cells. • DEPTOR is involved in the suppressing effect of metformin on mTOR signaling. • Metformin increases DEPTOR protein levels via suppression of proteasome activity. • DEPTOR-related mTOR suppression is involved in metformin's anti-cancer action.

DEPTOR-related mTOR suppression is involved in metformin's anti-cancer action in human liver cancer cells

International Nuclear Information System (INIS)

Obara, Akio; Fujita, Yoshihito; Abudukadier, Abulizi; Fukushima, Toru; Oguri, Yasuo; Ogura, Masahito; Harashima, Shin-ichi; Hosokawa, Masaya; Inagaki, Nobuya

2015-01-01

Metformin, one of the most commonly used drugs for patients with type 2 diabetes, recently has received much attention regarding its anti-cancer action. It is thought that the suppression of mTOR signaling is involved in metformin's anti-cancer action. Although liver cancer is one of the most responsive types of cancer for reduction of incidence by metformin, the molecular mechanism of the suppression of mTOR in liver remains unknown. In this study, we investigated the mechanism of the suppressing effect of metformin on mTOR signaling and cell proliferation using human liver cancer cells. Metformin suppressed phosphorylation of p70-S6 kinase, and ribosome protein S6, downstream targets of mTOR, and suppressed cell proliferation. We found that DEPTOR, an endogenous substrate of mTOR suppression, is involved in the suppressing effect of metformin on mTOR signaling and cell proliferation in human liver cancer cells. Metformin increases the protein levels of DEPTOR, intensifies binding to mTOR, and exerts a suppressing effect on mTOR signaling. This increasing effect of DEPTOR by metformin is regulated by the proteasome degradation system; the suppressing effect of metformin on mTOR signaling and cell proliferation is in a DEPTOR-dependent manner. Furthermore, metformin exerts a suppressing effect on proteasome activity, DEPTOR-related mTOR signaling, and cell proliferation in an AMPK-dependent manner. We conclude that DEPTOR-related mTOR suppression is involved in metformin's anti-cancer action in liver, and could be a novel target for anti-cancer therapy. - Highlights: • We elucidated a novel pathway of metformin's anti-cancer action in HCC cells. • DEPTOR is involved in the suppressing effect of metformin on mTOR signaling. • Metformin increases DEPTOR protein levels via suppression of proteasome activity. • DEPTOR-related mTOR suppression is involved in metformin's anti-cancer action

Hyperosmotic stress inhibits insulin receptor substrate-1 function by distinct mechanisms in 3T3-L1 adipocytes

DEFF Research Database (Denmark)

Gual, Philippe; Gonzalez, Teresa; Grémeaux, Thierry

2003-01-01

. Furthermore, the mammalian target of rapamycin (mTOR) inhibitor rapamycin prevented the osmotic shock-induced phosphorylation of IRS-1 on Ser307. The inhibition of mTOR completely reversed the inhibitory effect of hyperosmotic stress on insulin-induced IRS-1 tyrosine phosphorylation and PI 3-kinase activation......In 3T3-L1 adipocytes, hyperosmotic stress was found to inhibit insulin signaling, leading to an insulin-resistant state. We show here that, despite normal activation of insulin receptor, hyperosmotic stress inhibits both tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1) and IRS-1....... In addition, prolonged osmotic stress enhanced the degradation of IRS proteins through a rapamycin-insensitive pathway and a proteasome-independent process. These data support evidence of new mechanisms involved in osmotic stress-induced cellular insulin resistance. Short-term osmotic stress induces...

Vorinostat, an HDAC inhibitor attenuates epidermoid squamous cell carcinoma growth by dampening mTOR signaling pathway in a human xenograft murine model

Energy Technology Data Exchange (ETDEWEB)

Kurundkar, Deepali; Srivastava, Ritesh K.; Chaudhary, Sandeep C. [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); Ballestas, Mary E. [Department of Pediatrics Infectious Disease, Children' s of Alabama, School of Medicine, University of Alabama at Birmingham, AL (United States); Kopelovich, Levy [Division of Cancer Prevention, National Cancer Institute, 6130 Executive Blvd., Suite 2114, Bethesda, MD 20892 (United States); Elmets, Craig A. [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); 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)

2013-01-15

Histone deacetylase (HDAC) inhibitors are potent anticancer agents and show efficacy against various human neoplasms. Vorinostat is a potent HDAC inhibitor and has shown potential to inhibit growth of human xenograft tumors. However, its effect on the growth of skin neoplasm remains undefined. In this study, we show that vorinostat (2 μM) reduced expression of HDAC1, 2, 3, and 7 in epidermoid carcinoma A431 cells. Consistently, it increased acetylation of histone H3 and p53. Vorinostat (100 mg/kg body weight, IP) treatment reduced human xenograft tumor growth in highly immunosuppressed nu/nu mice. Histologically, the vorinostat-treated tumor showed features of well-differentiation with large necrotic areas. Based on proliferating cell nuclear antigen (PCNA) staining and expression of cyclins D1, D2, E, and A, vorinostat seems to impair proliferation by down-regulating the expression of these proteins. However, it also induced apoptosis. The mechanism by which vorinostat blocks proliferation and makes tumor cells prone to apoptosis, involved inhibition of mTOR signaling which was accompanied by reduction in cell survival AKT and extracellular-signal regulated kinase (ERK) signaling pathways. Our data provide a novel mechanism-based therapeutic intervention for cutaneous squamous cell carcinoma (SCC). Vorinostat may be utilized to cure skin neoplasms in organ transplant recipient (OTR). These patients have high morbidity and surgical removal of these lesions which frequently develop in these patients, is difficult. -- Highlights: ► Vorinostat reduces SCC growth in a xenograft murine model. ► Vorinostat dampens proliferation and induces apoptosis in tumor cells. ► Diminution in mTOR, Akt and ERK signaling underlies inhibition in proliferation. ► Vorinostat by inhibiting HDACs inhibits epithelial–mesenchymal transition.

Vorinostat, an HDAC inhibitor attenuates epidermoid squamous cell carcinoma growth by dampening mTOR signaling pathway in a human xenograft murine model

International Nuclear Information System (INIS)

Kurundkar, Deepali; Srivastava, Ritesh K.; Chaudhary, Sandeep C.; Ballestas, Mary E.; Kopelovich, Levy; Elmets, Craig A.; Athar, Mohammad

2013-01-01

Histone deacetylase (HDAC) inhibitors are potent anticancer agents and show efficacy against various human neoplasms. Vorinostat is a potent HDAC inhibitor and has shown potential to inhibit growth of human xenograft tumors. However, its effect on the growth of skin neoplasm remains undefined. In this study, we show that vorinostat (2 μM) reduced expression of HDAC1, 2, 3, and 7 in epidermoid carcinoma A431 cells. Consistently, it increased acetylation of histone H3 and p53. Vorinostat (100 mg/kg body weight, IP) treatment reduced human xenograft tumor growth in highly immunosuppressed nu/nu mice. Histologically, the vorinostat-treated tumor showed features of well-differentiation with large necrotic areas. Based on proliferating cell nuclear antigen (PCNA) staining and expression of cyclins D1, D2, E, and A, vorinostat seems to impair proliferation by down-regulating the expression of these proteins. However, it also induced apoptosis. The mechanism by which vorinostat blocks proliferation and makes tumor cells prone to apoptosis, involved inhibition of mTOR signaling which was accompanied by reduction in cell survival AKT and extracellular-signal regulated kinase (ERK) signaling pathways. Our data provide a novel mechanism-based therapeutic intervention for cutaneous squamous cell carcinoma (SCC). Vorinostat may be utilized to cure skin neoplasms in organ transplant recipient (OTR). These patients have high morbidity and surgical removal of these lesions which frequently develop in these patients, is difficult. -- Highlights: ► Vorinostat reduces SCC growth in a xenograft murine model. ► Vorinostat dampens proliferation and induces apoptosis in tumor cells. ► Diminution in mTOR, Akt and ERK signaling underlies inhibition in proliferation. ► Vorinostat by inhibiting HDACs inhibits epithelial–mesenchymal transition.

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

Blood lipid metabolites and meat lipid peroxidation responses of ...

African Journals Online (AJOL)

Esnart Mukumbo

2017-06-19

Jun 19, 2017 ... Fat and protein contents of thigh muscle and abdominal fat weight were measured and reported. Chickens fed LPO had greater serum triacylglycerol and very low ... favour lipid peroxidation, inhibit synthesis of higher homologous of ... The ambient temperature was gradually decreased from 33 °C at first.

Architecture of human mTOR complex 1.

Science.gov (United States)

Aylett, Christopher H S; Sauer, Evelyn; Imseng, Stefan; Boehringer, Daniel; Hall, Michael N; Ban, Nenad; Maier, Timm

2016-01-01

Target of rapamycin (TOR), a conserved protein kinase and central controller of cell growth, functions in two structurally and functionally distinct complexes: TORC1 and TORC2. Dysregulation of mammalian TOR (mTOR) signaling is implicated in pathologies that include diabetes, cancer, and neurodegeneration. We resolved the architecture of human mTORC1 (mTOR with subunits Raptor and mLST8) bound to FK506 binding protein (FKBP)-rapamycin, by combining cryo-electron microscopy at 5.9 angstrom resolution with crystallographic studies of Chaetomium thermophilum Raptor at 4.3 angstrom resolution. The structure explains how FKBP-rapamycin and architectural elements of mTORC1 limit access to the recessed active site. Consistent with a role in substrate recognition and delivery, the conserved amino-terminal domain of Raptor is juxtaposed to the kinase active site. Copyright © 2016, American Association for the Advancement of Science.

Identification of a Non-Gatekeeper Hot Spot for Drug-Resistant Mutations in mTOR Kinase.

Science.gov (United States)

Wu, Tzung-Ju; Wang, Xiaowen; Zhang, Yanjie; Meng, Linghua; Kerrigan, John E; Burley, Stephen K; Zheng, X F Steven

2015-04-21

Protein kinases are therapeutic targets for human cancer. However, "gatekeeper" mutations in tyrosine kinases cause acquired clinical resistance, limiting long-term treatment benefits. mTOR is a key cancer driver and drug target. Numerous small-molecule mTOR kinase inhibitors have been developed, with some already in human clinical trials. Given our clinical experience with targeted therapeutics, acquired drug resistance in mTOR is thought likely, but not yet documented. Herein, we describe identification of a hot spot (L2185) for drug-resistant mutations, which is distinct from the gatekeeper site, and a chemical scaffold refractory to drug-resistant mutations. We also provide new insights into mTOR kinase structure and function. The hot spot mutations are potentially useful as surrogate biomarkers for acquired drug resistance in ongoing clinical trials and future treatments and for the design of the next generation of mTOR-targeted drugs. Our study provides a foundation for further research into mTOR kinase function and targeting. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

Inhibition of mTOR improves the impairment of acidification in autophagic vesicles caused by hepatic steatosis

International Nuclear Information System (INIS)

Nakadera, Eisuke; Yamashina, Shunhei; Izumi, Kousuke; Inami, Yoshihiro; Sato, Toshifumi; Fukushima, Hirofumi; Kon, Kazuyoshi; Ikejima, Kenichi; Ueno, Takashi; Watanabe, Sumio

2016-01-01

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

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

mTOR at the Transmitting and Receiving Ends in Tumor Immunity.

Science.gov (United States)

Guri, Yakir; Nordmann, Thierry M; Roszik, Jason

2018-01-01

Cancer is a complex disease and a leading cause of death worldwide. Immunity is critical for cancer control. Cancer cells exhibit high mutational rates and therefore altered self or neo-antigens, eliciting an immune response to promote tumor eradication. Failure to mount a proper immune response leads to cancer progression. mTOR signaling controls cellular metabolism, immune cell differentiation, and effector function. Deregulated mTOR signaling in cancer cells modulates the tumor microenvironment, thereby affecting tumor immunity and possibly promoting carcinogenesis.

Cord Blood Cells Responses to IL2, IL7 and IL15 Cytokines for mTOR Expression

Directory of Open Access Journals (Sweden)

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.

Role of nutrients and mTOR signaling in the regulation of pancreatic progenitors development

Directory of Open Access Journals (Sweden)

Lynda Elghazi

2017-06-01

Full Text Available Objective: Poor fetal nutrition increases the risk of type 2 diabetes in the offspring at least in part by reduced embryonic β-cell growth and impaired function. However, it is not entirely clear how fetal nutrients and growth factors impact β-cells during development to alter glucose homeostasis and metabolism later in life. The current experiments aimed to test the impact of fetal nutrients and growth factors on endocrine development and how these signals acting on mTOR signaling regulate β-cell mass and glucose homeostasis. Method: Pancreatic rudiments in culture were used to study the role of glucose, growth factors, and amino acids on β-cell development. The number and proliferation of pancreatic and endocrine progenitor were assessed in the presence or absence of rapamycin. The impact of mTOR signaling in vivo on pancreas development and glucose homeostasis was assessed in models deficient for mTOR or Raptor in Pdx1 expressing pancreatic progenitors. Results: We found that amino acid concentrations, and leucine in particular, enhance the number of pancreatic and endocrine progenitors and are essential for growth factor induced proliferation. Rapamycin, an mTORC1 complex inhibitor, reduced the number and proliferation of pancreatic and endocrine progenitors. Mice lacking mTOR in pancreatic progenitors exhibited hyperglycemia in neonates, hypoinsulinemia and pancreatic agenesis/hypoplasia with pancreas rudiments containing ductal structures lacking differentiated acinar and endocrine cells. In addition, loss of mTORC1 by deletion of raptor in pancreatic progenitors reduced pancreas size with reduced number of β-cells. Conclusion: Together, these results suggest that amino acids concentrations and in particular leucine modulates growth responses of pancreatic and endocrine progenitors and that mTOR signaling is critical for these responses. Inactivation of mTOR and raptor in pancreatic progenitors suggested that alterations in some of

Delayed recovery of skeletal muscle mass following hindlimb immobilization in mTOR heterozygous mice.

Directory of Open Access Journals (Sweden)

Susan M Lang

Full Text Available The present study addressed the hypothesis that reducing mTOR, as seen in mTOR heterozygous (+/- mice, would exaggerate the changes in protein synthesis and degradation observed during hindlimb immobilization as well as impair normal muscle regrowth during the recovery period. Atrophy was produced by unilateral hindlimb immobilization and data compared to the contralateral gastrocnemius. In wild-type (WT mice, the gradual loss of muscle mass plateaued by day 7. This response was associated with a reduction in basal protein synthesis and development of leucine resistance. Proteasome activity was consistently elevated, but atrogin-1 and MuRF1 mRNAs were only transiently increased returning to basal values by day 7. When assessed 7 days after immobilization, the decreased muscle mass and protein synthesis and increased proteasome activity did not differ between WT and mTOR(+/- mice. Moreover, the muscle inflammatory cytokine response did not differ between groups. After 10 days of recovery, WT mice showed no decrement in muscle mass, and this accretion resulted from a sustained increase in protein synthesis and a normalization of proteasome activity. In contrast, mTOR(+/- mice failed to fully replete muscle mass at this time, a defect caused by the lack of a compensatory increase in protein synthesis. The delayed muscle regrowth of the previously immobilized muscle in the mTOR(+/- mice was associated with a decreased raptor•4EBP1 and increased raptor•Deptor binding. Slowed regrowth was also associated with a sustained inflammatory response (e.g., increased TNFα and CD45 mRNA during the recovery period and a failure of IGF-I to increase as in WT mice. These data suggest mTOR is relatively more important in regulating the accretion of muscle mass during recovery than the loss of muscle during the atrophy phase, and that protein synthesis is more sensitive than degradation to the reduction in mTOR during muscle regrowth.

Delayed recovery of skeletal muscle mass following hindlimb immobilization in mTOR heterozygous mice.

Science.gov (United States)

Lang, Susan M; Kazi, Abid A; Hong-Brown, Ly; Lang, Charles H

2012-01-01

The present study addressed the hypothesis that reducing mTOR, as seen in mTOR heterozygous (+/-) mice, would exaggerate the changes in protein synthesis and degradation observed during hindlimb immobilization as well as impair normal muscle regrowth during the recovery period. Atrophy was produced by unilateral hindlimb immobilization and data compared to the contralateral gastrocnemius. In wild-type (WT) mice, the gradual loss of muscle mass plateaued by day 7. This response was associated with a reduction in basal protein synthesis and development of leucine resistance. Proteasome activity was consistently elevated, but atrogin-1 and MuRF1 mRNAs were only transiently increased returning to basal values by day 7. When assessed 7 days after immobilization, the decreased muscle mass and protein synthesis and increased proteasome activity did not differ between WT and mTOR(+/-) mice. Moreover, the muscle inflammatory cytokine response did not differ between groups. After 10 days of recovery, WT mice showed no decrement in muscle mass, and this accretion resulted from a sustained increase in protein synthesis and a normalization of proteasome activity. In contrast, mTOR(+/-) mice failed to fully replete muscle mass at this time, a defect caused by the lack of a compensatory increase in protein synthesis. The delayed muscle regrowth of the previously immobilized muscle in the mTOR(+/-) mice was associated with a decreased raptor•4EBP1 and increased raptor•Deptor binding. Slowed regrowth was also associated with a sustained inflammatory response (e.g., increased TNFα and CD45 mRNA) during the recovery period and a failure of IGF-I to increase as in WT mice. These data suggest mTOR is relatively more important in regulating the accretion of muscle mass during recovery than the loss of muscle during the atrophy phase, and that protein synthesis is more sensitive than degradation to the reduction in mTOR during muscle regrowth.

mTOR up-regulation of PFKFB3 is essential for acute myeloid leukemia cell survival

International Nuclear Information System (INIS)

Feng, Yonghuai; Wu, Liusong

2017-01-01

Although mTOR (mammalian target of rapamycin) activation is frequently observed in acute myeloid leukemia (AML) patients, the precise function and the downstream targets of mTOR are poorly understood. Here we revealed that PFKFB3, but not PFKFB1, PFKFB2 nor PFKFB4 was a novel downstream substrate of mTOR signaling pathway as PFKFB3 level was augmented after knocking down TSC2 in THP1 and OCI-AML3 cells. Importantly, PFKFB3 silencing suppressed glycolysis and cell proliferation of TSC2 silencing OCI-AML3 cells and activated apoptosis pathway. These results suggested that mTOR up-regulation of PFKFB3 was essential for AML cells survival. Mechanistically, Rapamycin treatment or Raptor knockdown reduced the expression of PFKFB3 in TSC2 knockdown cells, while Rictor silencing did not have such effect. Furthermore, we also revealed that mTORC1 up-regulated PFKFB3 was dependent on hypoxia-inducible factor 1α (HIF1α), a positive regulator of glycolysis. Moreover, PFKFB3 inhibitor PFK15 and rapamycin synergistically blunted the AML cell proliferation. Taken together, PFKFB3 was a promising drug target in AML patients harboring mTOR hyper-activation.

mTOR at the Transmitting and Receiving Ends in Tumor Immunity

Directory of Open Access Journals (Sweden)

Yakir Guri

2018-03-01

Full Text Available Cancer is a complex disease and a leading cause of death worldwide. Immunity is critical for cancer control. Cancer cells exhibit high mutational rates and therefore altered self or neo-antigens, eliciting an immune response to promote tumor eradication. Failure to mount a proper immune response leads to cancer progression. mTOR signaling controls cellular metabolism, immune cell differentiation, and effector function. Deregulated mTOR signaling in cancer cells modulates the tumor microenvironment, thereby affecting tumor immunity and possibly promoting carcinogenesis.

Phosphatidylinositol-3-OH kinase and nutrient-sensing mTOR pathways control T lymphocyte trafficking

NARCIS (Netherlands)

Sinclair, Linda V.; Finlay, David; Feijoo, Carmen; Cornish, Georgina H.; Gray, Alex; Ager, Ann; Okkenhaug, Klaus; Hagenbeek, Thijs J.; Spits, Hergen; Cantrell, Doreen A.

2008-01-01

Phosphatidylinositol-3-OH kinase (PI(3)K) and the nutrient sensor mTOR are evolutionarily conserved regulators of cell metabolism. Here we show that PI(3)K and mTOR determined the repertoire of adhesion and chemokine receptors expressed by T lymphocytes. The key lymph node-homing receptors CD62L

Artemisinic acid exhibits antitumor activity in MCF-7 breast cancer cells through the inhibition of angiogenesis, VEGF, m-TOR and AKT signalling pathways

Directory of Open Access Journals (Sweden)

Yan Cui

2016-09-01

Full Text Available The aim of the present study was to evaluate the antitumor and anti-angiogenic effects of artemisinic acid in MCF-7 human breast cancer cells. Various cell signalling pathways (VEGF, m-TOR and AKT signalling pathways and MTT assay were used. The in vivo antitumor activity of artemisinic acid was evaluated by means of tumor xenograft mouse model. Transwell cell migration assay was used to examine the chemotactic motility of the human umbilical vascular endothelial cells (HUVECs, while as endothelial cell capillary-like tube formation assay was used to evaluate the effect of artemisinic acid on the tube formation in HUVECs. We found that artemisinic acid considerably reduced both the volume and weight of concrete tumors and reduced angiogenesis in a xenograft mouse tumor model in vivo. Further, artemisinic acid suppressed the VEGF-induced cell migration and capillary-like tube formation of HUVECs in a dose-dependent manner. Artemisinic acid was found to suppress the VEGF-induced phosphorylation of VEGFR2 and also the activity of AKT and m-TOR.

A pilot trial of the mTOR (mammalian target of rapamycin) inhibitor RAD001 in patients with advanced B-CLL.

Science.gov (United States)

Decker, Thomas; Sandherr, Michael; Goetze, Katharina; Oelsner, Madlen; Ringshausen, Ingo; Peschel, Christian

2009-03-01

Although B-cell chronic lymphocytic leukemia (CLL) is treatable, it remains an incurable disease and most patients inevitably suffer relapse. Many therapeutic options exist for those requiring therapy, including monoclonal antibodies and stem cell transplantation, but remissions tend to last shorter in the course of the disease. Targeting the cell cycle has recently been realized to be an attractive therapeutic approach in solid and hematological malignancies, and the proliferative nature of B-CLL is increasingly accepted. Here, we report data on a phase II pilot trial with the oral mammalian target of rapamycin (mTOR) inhibitor RAD001 5 mg/daily in patients with advanced B-CLL who had progressive disease after at least two lines of treatment. After treatment of seven patients, this trial was stopped because of toxicity concerns, although some degree of activity was observed (one partial remission, three patients with stable disease). Interestingly, cyclin E expression decreased in responding patients. Further strategies of mTOR inhibition by RAD001 in B-CLL should focus on different treatment schedules, adequate anti-infectious prophylaxis, or combinations with cytotoxic drugs.

Inhibition of alloxan diabetes by low dose γ-irradiation before alloxan administration

International Nuclear Information System (INIS)

Yamaoka, Kiyonori; Takehara, Yoshiki; Yoshioka, Tamotsu; Utsumi, Kozo.

1994-01-01

We evaluated the inhibitory effects of whole body 60 Co-γ irradiation at a single low dose on alloxan-induced hyperglycemia in rats. (1) In rats that received alloxan, SOD activity in pancreas significantly decreased, but the decrease was inhibited by irradiation at a dose of 0.5 Gy. (2) Similarly, plasma peroxide, pancreatic peroxide, and blood glucose increased. However, the increase in pancreatic peroxide was inhibited by irradiation at a dose of 0.5 or 1.0 Gy and the increase in blood glucose by irradiation at 0.5 Gy. (3) After alloxan administration, degranulation was observed in cells, but this was inhibited by irradiation at 0.5 Gy. These results suggest that alloxan diabetes was inhibited by the increase of SOD activity in pancreas after low dose irradiation at 0.5 Gy. (author)

Radioprotection by dipyridamole in the aging mouse. Effects on lipid peroxidation in mouse liver, spleen and brain after whole-body X-ray irradiation

International Nuclear Information System (INIS)

Seino, Noritaka

1995-01-01

To investigate the radioprotective effect of dipyridamole in the aging mouse, the lipid peroxide content in aging mouse liver, spleen and brain irradiated by X-ray were measured both before and after injection of dipyridamole. The lipid peroxide content increased with aging from 2 months old to 16 months old in the mouse liver, spleen and brain. The content of lipid peroxide in the liver and spleen of the aging mouse was significantly increased in 7 days after whole-body irradiation with 8 Gy, but was unchanged in the brain. Dipyridamole, given before irradiation, significantly inhibited the increase of lipid peroxide after irradiation. These results suggest that dipyridamole may have radioprotective effects on aging mouse liver and spleen as well as on young mouse, and that inhibition of lipid peroxidation is a possible factor in the radioprotective effect of dipyridamole. (author)

A trypsin inhibitor from Tecoma stans leaves inhibits growth and promotes ATP depletion and lipid peroxidation in Candida albicans and Candida krusei

Directory of Open Access Journals (Sweden)

Leydianne Leite de Siqueira Patriota

2016-04-01

Full Text Available Tecoma stans (yellow elder has shown medicinal properties and antimicrobial activity. Previous reports on antifungal activity of T. stans preparations and presence of trypsin inhibitor activity from T. stans leaves stimulated the investigation reported here. In this work, we proceeded to the purification and characterization of a trypsin inhibitor (TesTI, which was investigated for anti-Candida activity. Finally, in order to determine the potential of TesTI as a new natural chemotherapeutic product, its cytotoxicity to human peripheral blood mononuclear cells (PBMCs was evaluated. TesTI was isolated from saline extract by ammonium sulphate fractionation followed by ion exchange and gel filtration chromatographies. Antifungal activity was evaluated by determining the minimal inhibitory (MIC and fungicide (MFC concentrations using fungal cultures containing only yeast form or both yeast and hyphal forms. Candida cells treated with TesTI were evaluated for intracellular ATP levels and lipid peroxidation. Cytotoxicity of TesTI to PBMCs was evaluated by MTT assay. TesTI (39.8 kDa, pI 3.41, Ki 43 nM inhibited similarly the growth of both C. albicans and C. krusei culture types at MIC of 100 µg/mL. The MFCs were 200 µg/mL for C. albicans and C. krusei. Time-response curves revealed that TesTI (at MIC was more effective at inhibiting the replication of C. albicans cells. At MIC, TesTI promoted reduction of ATP levels and lipid peroxidation in the Candida cells, being not cytotoxic to PBMCs. In conclusion, TesTI is an antifungal agent against C. albicans and C. krusei, without toxicity to human cells.

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.

mTOR in squamous cell carcinoma of the oesophagus: a potential target for molecular therapy?

NARCIS (Netherlands)

Boone, J.; ten Kate, F. J. W.; Offerhaus, G. J. A.; van Diest, P. J.; Borel Rinkes, I. H. M.; van Hillegersberg, R.

2008-01-01

AIMS: The mammalian target of rapamycin (mTOR), an important regulator of protein translation and cell proliferation, is activated in various malignancies. In a randomised controlled trial of advanced renal cell carcinoma patients, targeted therapy to mTOR by means of rapamycin analogues has been

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

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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

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

Mechanistic target of rapamycin (MTOR) protein expression in the tumor and its microenvironment correlates with more aggressive pathology at cystectomy

NARCIS (Netherlands)

Winters, B.R. (Brian R.); Vakar-Lopez, F. (Funda); Brown, L. (Lisha); Montgomery, B. (Bruce); Seiler, R. (Roland); P.C. Black (Peter C.); J.L. Boormans (Joost); Dall′Era, M. (Marc); Davincioni, E. (Elai); Douglas, J. (James); Gibb, E.A. (Ewan A.); B.W. van Rhijn (Bas); M.S. van der Heijden (Michiel); Hsieh, A.C. (Andrew C.); Wright, J.L. (Jonathan L.); Lam, H.-M. (Hung-Ming)

2018-01-01

textabstractBackground: The mechanistic target of rapamycin (mTOR) has been implicated in driving tumor biology in multiple malignancies, including urothelial carcinoma (UC). We investigate how mTOR and phosphorylated mTOR (pmTOR) protein expression correlate with chemoresponsiveness in the tumor

Germline activating MTOR mutation arising through gonadal mosaicism in two brothers with megalencephaly and neurodevelopmental abnormalities.

Science.gov (United States)

Mroske, Cameron; Rasmussen, Kristen; Shinde, Deepali N; Huether, Robert; Powis, Zoe; Lu, Hsiao-Mei; Baxter, Ruth M; McPherson, Elizabeth; Tang, Sha

2015-11-05

In humans, Mammalian Target of Rapamycin (MTOR) encodes a 300 kDa serine/ threonine protein kinase that is ubiquitously expressed, particularly at high levels in brain. MTOR functions as an integrator of multiple cellular processes, and in so doing either directly or indirectly regulates the phosphorylation of at least 800 proteins. While somatic MTOR mutations have been recognized in tumors for many years, and more recently in hemimegalencephaly, germline MTOR mutations have rarely been described. We report the successful application of family-trio Diagnostic Exome Sequencing (DES) to identify the underlying molecular etiology in two brothers with multiple neurological and developmental lesions, and for whom previous testing was non-diagnostic. The affected brothers, who were 6 and 23 years of age at the time of DES, presented symptoms including but not limited to mild Autism Spectrum Disorder (ASD), megalencephaly, gross motor skill delay, cryptorchidism and bilateral iris coloboma. Importantly, we determined that each affected brother harbored the MTOR missense alteration p.E1799K (c.5395G>A). This exact variant has been previously identified in multiple independent human somatic cancer samples and has been shown to result in increased MTOR activation. Further, recent independent reports describe two unrelated families in whom p.E1799K co-segregated with megalencephaly and intellectual disability (ID); in both cases, p.E1799K was shown to have originated due to germline mosaicism. In the case of the family reported herein, the absence of p.E1799K in genomic DNA extracted from the blood of either parent suggests that this alteration most likely arose due to gonadal mosaicism. Further, the p.E1799K variant exerts its effect by a gain-of-function (GOF), autosomal dominant mechanism. Herein, we describe the use of DES to uncover an activating MTOR missense alteration of gonadal mosaic origin that is likely to be the causative mutation in two brothers who present

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.

2015-01-01

ABSTRACT 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

Inhibition of alloxan diabetes by low dose {gamma}-irradiation before alloxan administration

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Yamaoka, Kiyonori [Central Research Inst. of Electric Power Industry, Komae, Tokyo (Japan). Komae Research Lab.; Takehara, Yoshiki; Yoshioka, Tamotsu; Utsumi, Kozo

1994-10-01

We evaluated the inhibitory effects of whole body {sup 60}Co-{gamma} irradiation at a single low dose on alloxan-induced hyperglycemia in rats. (1) In rats that received alloxan, SOD activity in pancreas significantly decreased, but the decrease was inhibited by irradiation at a dose of 0.5 Gy. (2) Similarly, plasma peroxide, pancreatic peroxide, and blood glucose increased. However, the increase in pancreatic peroxide was inhibited by irradiation at a dose of 0.5 or 1.0 Gy and the increase in blood glucose by irradiation at 0.5 Gy. (3) After alloxan administration, degranulation was observed in cells, but this was inhibited by irradiation at 0.5 Gy. These results suggest that alloxan diabetes was inhibited by the increase of SOD activity in pancreas after low dose irradiation at 0.5 Gy. (author).

Curcumin significantly enhances dual PI3K/Akt and mTOR inhibitor NVP-BEZ235-induced apoptosis in human renal carcinoma Caki cells through down-regulation of p53-dependent Bcl-2 expression and inhibition of Mcl-1 protein stability.

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Bo Ram Seo

Full Text Available The PI3K/Akt and mTOR signaling pathways are important for cell survival and growth, and they are highly activated in cancer cells compared with normal cells. Therefore, these signaling pathways are targets for inducing cancer cell death. The dual PI3K/Akt and mTOR inhibitor NVP-BEZ235 completely inhibited both signaling pathways. However, NVP-BEZ235 had no effect on cell death in human renal carcinoma Caki cells. We tested whether combined treatment with natural compounds and NVP-BEZ235 could induce cell death. Among several chemopreventive agents, curcumin, a natural biologically active compound that is extracted from the rhizomes of Curcuma species, markedly induced apoptosis in NVP-BEZ235-treated cells. Co-treatment with curcumin and NVP-BEZ235 led to the down-regulation of Mcl-1 protein expression but not mRNA expression. Ectopic expression of Mcl-1 completely inhibited curcumin plus NVP-NEZ235-induced apoptosis. Furthermore, the down-regulation of Bcl-2 was involved in curcumin plus NVP-BEZ235-induced apoptosis. Curcumin or NVP-BEZ235 alone did not change Bcl-2 mRNA or protein expression, but co-treatment reduced Bcl-2 mRNA and protein expression. Combined treatment with NVP-BEZ235 and curcumin reduced Bcl-2 expression in wild-type p53 HCT116 human colon carcinoma cells but not p53-null HCT116 cells. Moreover, Bcl-2 expression was completely reversed by treatment with pifithrin-α, a p53-specific inhibitor. Ectopic expression of Bcl-2 also inhibited apoptosis in NVP-BE235 plus curcumin-treated cells. In contrast, NVP-BEZ235 combined with curcumin did not have a synergistic effect on normal human skin fibroblasts and normal human mesangial cells. Taken together, combined treatment with NVP-BEZ235 and curcumin induces apoptosis through p53-dependent Bcl-2 mRNA down-regulation at the transcriptional level and Mcl-1 protein down-regulation at the post-transcriptional level.

Peroxidized mineral oil increases the oxidant status of culture media and inhibits in vitro porcine embryo development.

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Martinez, C A; Nohalez, A; Ceron, J J; Rubio, C P; Roca, J; Cuello, C; Rodriguez-Martinez, H; Martinez, E A; Gil, M A

2017-11-01

The use of oils with undetected alterations is a long-recognized problem for in vitro embryo production systems. Since peroxides in oils have been associated with reduced embryo production outcomes, our goals were (1) to evaluate the effects of a batch of mineral oil (MO) that was suspected to be altered on the in vitro production of pig embryos and (2) to determine oil peroxide values throughout culture and the transfer of oxidant agents from oil to culture media. Sunflower oil, which has a completely different chemical composition than MO but a higher oxidative status, and unaltered MO were used as controls. Oocyte maturation, fertilization and embryo development were affected differently depending on the oil overlay used. While the suspected MO was not able to sustain in vitro maturation and fertilization, the oocytes incubated in the presence of sunflower oil were matured and fertilized similarly to those of the unaltered MO group. Moreover, the cleavage rate of presumed zygotes cultured under the suspected MO was severely reduced compared with those cultured under the other oils, and none of the cleaved embryos developed to the blastocyst stage. Although the cleavage rates in the sunflower oil and unaltered MO groups were similar, embryos cultured under sunflower oil also failed to develop to the blastocyst stage. Our results revealed that the suspected MO and sunflower oil had similar levels of peroxides and that these levels were much higher than those of the unaltered MO. The total oxidant status was higher in media incubated under peroxidized oils than in fresh media or media incubated without an oil overlay or under unaltered MO, indicating that oxidant agents were transferred to the incubation media. However, unlike the sunflower oil group, the culture media incubated under the suspected MO had high levels of total oxidant status and low levels of hydrogen peroxide and reactive oxygen species, suggesting the presence of other unknown oxidant agents in

Reduced AMPK-ACC and mTOR signaling in muscle from older men, and effect of resistance exercise.

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Li, Mengyao; Verdijk, Lex B; Sakamoto, Kei; Ely, Brian; van Loon, Luc J C; Musi, Nicolas

2012-01-01

AMP-activated protein kinase (AMPK) is a key energy-sensitive enzyme that controls numerous metabolic and cellular processes. Mammalian target of rapamycin (mTOR) is another energy/nutrient-sensitive kinase that controls protein synthesis and cell growth. In this study we determined whether older versus younger men have alterations in the AMPK and mTOR pathways in skeletal muscle, and examined the effect of a long term resistance type exercise training program on these signaling intermediaries. Older men had decreased AMPKα2 activity and lower phosphorylation of AMPK and its downstream signaling substrate acetyl-CoA carboxylase (ACC). mTOR phosphylation also was reduced in muscle from older men. Exercise training increased AMPKα1 activity in older men, however, AMPKα2 activity, and the phosphorylation of AMPK, ACC and mTOR, were not affected. In conclusion, older men have alterations in the AMPK-ACC and mTOR pathways in muscle. In addition, prolonged resistance type exercise training induces an isoform-selective up regulation of AMPK activity. Published by Elsevier Ireland Ltd.

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.

Protective role of edible clam Paphia malabarica (Chemnitz) against lipid peroxidation and free radicals

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Pawar, R.T.; Nagvenkar, S.S.; Jagtap, T.G.

In vitro inhibition of lipid peroxidation and free radical scavenging properties of a seafood Paphia malabarica (Chemnitz) as a natural source of antioxidants was observed. Antioxidant activities of Paphia malabarica extracts were tested...

Nutrient sensing via mTOR in T cells maintains a tolerogenic microenvironment

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Duncan eHowie

2014-08-01

Full Text Available We have proposed that tolerance can be maintained by the induction, by Treg cells, of a tolerogenic microenvironment within tolerated tissues that inhibits effector cell activity but which supports the generation of further Treg cells by infectious tolerance. Two important components of this tolerogenic microenvironment depend on metabolism and nutrient sensing. The first is due to the up-regulation of multiple enzymes that consume essential amino acids (EAAs, which are sensed in naive T cells primarily via inhibition of the mTOR pathway, which in turn encourages their further differentiation into foxp3+ Treg cells. The second mechanism is the metabolism of extracellular ATP to adenosine by the ectoenzymes CD39 and CD73. These two enzymes are constitutively co-expressed on Treg cells, but can also be induced on a wide variety of cell types by TGFbeta and the adenosine generated can be shown to be a potent inhibitor of T cell proliferation. This review will focus on mechanisms of nutrient sensing in T cells, how these are integrated with TCR and cytokine signals via the mnTOR pathway, and what impact this has on intracellular metabolism and subsequently the control of differentiation into different effector or regulatory T cell subsets.

Glutathione protects liver and kidney tissue from cadmium- and lead-provoked lipid peroxidation

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Jovanović Jasmina M.

2013-01-01

Full Text Available Cd and Pb represent a serious ecological problem due to their soluble nature, their mobility and ability to accumulate in the soil. The exposure to these heavy metals can originate from different sources (drinking water, food, air, and they can make their way into the human body through the respiratory and digestive system. We investigated the effects of glutathione on Cd and Pb accumulation and lipid peroxidation effects in the liver and kidneys of heavy metal intoxicated rats. The content of the marker of lipid peroxidation - malondialdehyde was increased several fold the in tissues of exposed animals, the effects being more pronounced in liver. The treatment of intoxicated animals with glutathione drastically suppressed lipid peroxidation. Our results imply that the application of glutathione may have protective role in heavy metal intoxication by inhibiting lipid peroxidation. However, precaution should be made when it comes to Cd, since it seems that glutathione promoted Cd accumulation in the liver.

AKT inhibitors promote cell death in cervical cancer through disruption of mTOR signaling and glucose uptake.

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Ramachandran Rashmi

Full Text Available PI3K/AKT pathway alterations are associated with incomplete response to chemoradiation in human cervical cancer. This study was performed to test for mutations in the PI3K pathway and to evaluate the effects of AKT inhibitors on glucose uptake and cell viability.Mutational analysis of DNA from 140 pretreatment tumor biopsies and 8 human cervical cancer cell lines was performed. C33A cells (PIK3CAR88Q and PTENR233* were treated with increasing concentrations of two allosteric AKT inhibitors (SC-66 and MK-2206 with or without the glucose analogue 2-deoxyglucose (2-DG. Cell viability and activation status of the AKT/mTOR pathway were determined in response to the treatment. Glucose uptake was evaluated by incubation with 18F-fluorodeoxyglucose (FDG. Cell migration was assessed by scratch assay.Activating PIK3CA (E545K, E542K and inactivating PTEN (R233* mutations were identified in human cervical cancer. SC-66 effectively inhibited AKT, mTOR and mTOR substrates in C33A cells. SC-66 inhibited glucose uptake via reduced delivery of Glut1 and Glut4 to the cell membrane. SC-66 (1 µg/ml-56% and MK-2206 (30 µM-49% treatment decreased cell viability through a non-apoptotic mechanism. Decreases in cell viability were enhanced when AKT inhibitors were combined with 2-DG. The scratch assay showed a substantial reduction in cell migration upon SC-66 treatment.The mutational spectrum of the PI3K/AKT pathway in cervical cancer is complex. AKT inhibitors effectively block mTORC1/2, decrease glucose uptake, glycolysis, and decrease cell viability in vitro. These results suggest that AKT inhibitors may improve response to chemoradiation in cervical cancer.

MenTORing Immunity: mTOR Signaling in the Development and Function of Tissue-Resident Immune Cells.

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Jones, Russell G; Pearce, Edward J

2017-05-16

Tissue-resident immune cells must balance survival in peripheral tissues with the capacity to respond rapidly upon infection or tissue damage, and in turn couple these responses with intrinsic metabolic control and conditions in the tissue microenvironment. The serine/threonine kinase mammalian/mechanistic target of rapamycin (mTOR) is a central integrator of extracellular and intracellular growth signals and cellular metabolism and plays important roles in both innate and adaptive immune responses. This review discusses the function of mTOR signaling in the differentiation and function of tissue-resident immune cells, with focus on the role of mTOR as a metabolic sensor and its impact on metabolic regulation in innate and adaptive immune cells. We also discuss the impact of metabolic constraints in tissues on immune homeostasis and disease, and how manipulating mTOR activity with drugs such as rapamycin can modulate immunity in these contexts. Copyright © 2017. Published by Elsevier Inc.

Production of uranium peroxide

International Nuclear Information System (INIS)

Caropreso, F.E.; Kreuz, D.F.

1977-01-01

A process is claimed of recovering uranium values as uranium peroxide from an aqueous uranyl solution containing dissolved vanadium and sodium impurities by treating the uranyl solution with hydrogen peroxide in an amount sufficient to have an excess of at least 0.5 parts H 2 O 2 per part of vanadium (V 2 O 5 ) above the stoichiometric amount required to form the uranium peroxide, the hydrogen peroxide treatment is carried out in three sequential phases consisting of I, a precipitation phase in which the hydrogen peroxide is added to the uranyl solution to precipitate the uranium peroxide and the pH of the reaction medium maintained in the range of 2.5 to 5.5 for a period of from about 1 to 60 minutes after the hydrogen peroxide addition; II, a digestion phase in which the pH of the reaction medium is maintained in the range of 3.0 to 7.0 for a period of about 5 to 180 minutes and III, a final phase in which the pH of the reaction medium is maintained in the range of 4.0 to 7.0 for a period of about 1 to 60 minutes during which time the uranium peroxide is separated from the reaction solution containing the dissolved vanadium and sodium impurities. The excess hydrogen peroxide is maintained during the entire treatment up until the uranium peroxide is separated from the reaction medium

Early LC3 lipidation induced by d-limonene does not rely on mTOR inhibition, ERK activation and ROS production and it is associated with reduced clonogenic capacity of SH-SY5Y neuroblastoma cells.

Science.gov (United States)

Berliocchi, Laura; Chiappini, Carlotta; Adornetto, Annagrazia; Gentile, Debora; Cerri, Silvia; Russo, Rossella; Bagetta, Giacinto; Corasaniti, Maria Tiziana

2018-02-01

d-Limonene is a natural monoterpene abundant in Citrus essential oils. It is endowed with several biological activities, including inhibition of carcinogenesis and promotion of tumour regression. Recently, d-limonene has been shown to modulate autophagic markers in vitro at concentrations found in vivo, in clinical pharmacokinetic studies. Autophagy is an intracellular catabolic process serving as both an adaptive metabolic response and a quality control mechanism. Because autophagy defects have been linked to a wide range of human pathologies, including neurodegeneration and cancer, there is a need for new pharmacological tools to control deregulated autophagy. To better understand the effects of d-limonene on autophagy, to identify the molecular mechanisms through which this monoterpene rapidly triggers LC3 lipidation and to evaluate the role for autophagy in long-term effects of d-limonene. Human SH-SY5Y neuroblastoma, HepG2 hepatocellular carcinoma and MCF7 breast cancer cells were used. Endogenous LC3-II levels were evaluated by western blotting. Autophagic flux assay was performed using bafilomycin A1 and chloroquine. Intracellular distribution of LC3 protein was studied by confocal microscopy analysis of LC3B-GFP transduced cells. Expression of lysosomal-membrane protein LAMP-1 was assessed by immunofluorescence analysis. Phosphorylated levels of downstream substrates of mTOR kinase (p70S6 kinase, 4E-BP1, and ULK1) and ERK were analyzed by western blotting. Production of reactive oxygen species (ROS) was assessed by live confocal microscopy of cells loaded with CellROX ® Green Reagent. Clonogenic assay was used to evaluate the ability of treated cells to proliferate and form colonies. LC3 lipidation promoted by d-limonene correlates with autophagosome formation and stimulation of basal autophagy. LC3 lipidation does not rely on inhibition of mTOR kinase, which instead appears to be transiently activated. In addition, d-limonene rapidly activates ERK and

Reactive oxygen species and lipid peroxidation product-scavenging ability of yogurt organisms.

Science.gov (United States)

Lin, M Y; Yen, C L

1999-08-01

The antioxidative activity of the intracellular extracts of yogurt organisms was investigated. All 11 strains tested, including five strains of Streptococcus thermophilus and six strains of Lactobacillus delbrueckii ssp. bulgaricus, demonstrated an antioxidative effect on the inhibition of linoleic acid peroxidation. The antioxidative effect of intracellular extracts of 10(8) cells of yogurt organisms was equivalent to 25 to 96 ppm butylated hydroxytoluene, which indicated that all strains demonstrated excellent antioxidative activity. The scavenging of reactive oxygen species, hydroxyl radical, and hydrogen peroxide was studied for intracellular extracts of yogurt organisms. All strains showed reactive oxygen species-scavenging ability. Lactobacillus delbrueckii ssp. bulgaricus Lb demonstrated the highest hydroxyl radical-scavenging ability at 234 microM. Streptococcus thermophilus MC and 821 and L. delbrueckii ssp. bulgaricus 448 and 449 scavenged the most hydrogen peroxide at approximately 50 microM. The scavenging ability of lipid peroxidation products, t-butylhydroperoxide and malondialdehyde, was also evaluated. Results showed that the extracts were not able to scavenge the t-butylhydroperoxide. Nevertheless, malondialdehyde was scavenged well by most strains.

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

Effect of americium-241 on luminous bacteria. Role of peroxides

Energy Technology Data Exchange (ETDEWEB)

Alexandrova, M., E-mail: maka-alexandrova@rambler.r [Siberian Federal University, Svobodny 79, 660041 Krasnoyarsk (Russian Federation); Rozhko, T. [Siberian Federal University, Svobodny 79, 660041 Krasnoyarsk (Russian Federation); Vydryakova, G. [Institute of Biophysics SB RAS, Akademgorodok 50, 660036 Krasnoyarsk (Russian Federation); Kudryasheva, N. [Siberian Federal University, Svobodny 79, 660041 Krasnoyarsk (Russian Federation); Institute of Biophysics SB RAS, Akademgorodok 50, 660036 Krasnoyarsk (Russian Federation)

2011-04-15

The effect of americium-241 ({sup 241}Am), an alpha-emitting radionuclide of high specific activity, on luminous bacteria Photobacterium phosphoreum was studied. Traces of {sup 241}Am in nutrient media (0.16-6.67 kBq/L) suppressed the growth of bacteria, but enhanced luminescence intensity and quantum yield at room temperature. Lower temperature (4 {sup o}C) increased the time of bacterial luminescence and revealed a stage of bioluminescence inhibition after 150 h of bioluminescence registration start. The role of conditions of exposure the bacterial cells to the {sup 241}Am is discussed. The effect of {sup 241}Am on luminous bacteria was attributed to peroxide compounds generated in water solutions as secondary products of radioactive decay. Increase of peroxide concentration in {sup 241}Am solutions was demonstrated; and the similarity of {sup 241}Am and hydrogen peroxide effects on bacterial luminescence was revealed. The study provides a scientific basis for elaboration of bioluminescence-based assay to monitor radiotoxicity of alpha-emitting radionuclides in aquatic solutions. - Highlights: {yields} Am-241 in water solutions (A = 0.16-6.7 kBq/L) suppresses bacterial growth.{yields} Am-241 (A = 0.16-6.7 kBq/L) stimulate bacterial luminescence. {yields} Peroxides, secondary radiolysis products, cause increase of bacterial luminescence.

Hydrogen peroxide safety issues

International Nuclear Information System (INIS)

Conner, W.V.

1993-01-01

A literature survey was conducted to review the safety issues involved in handling hydrogen peroxide solutions. Most of the information found in the literature is not directly applicable to conditions at the Rocky Flats Plant, but one report describes experimental work conducted previously at Rocky Flats to determine decomposition reaction-rate constants for hydrogen peroxide solutions. Data from this report were used to calculate decomposition half-life times for hydrogen peroxide in solutions containing several decomposition catalysts. The information developed from this survey indicates that hydrogen peroxide will undergo both homogeneous and heterogeneous decomposition. The rate of decomposition is affected by temperature and the presence of catalytic agents. Decomposition of hydrogen peroxide is catalyzed by alkalies, strong acids, platinum group and transition metals, and dissolved salts of transition metals. Depending upon conditions, the consequence of a hydrogen peroxide decomposition can range from slow evolution of oxygen gas to a vapor, phase detonation of hydrogen peroxide vapors

The Rapamycin-Binding Domain of the Protein Kinase mTOR is a Destabilizing Domain*

Science.gov (United States)

Edwards, Sarah R.; Wandless, Thomas J.

2013-01-01

Rapamycin is an immunosuppressive drug that binds simultaneously to the 12-kDa FK506- and rapamycin-binding protein (FKBP12, or FKBP) and the FKBP-rapamycin binding domain (FRB) of the mammalian target of rapamycin (mTOR) kinase. The resulting ternary complex has been used to conditionally perturb protein function, and one such method involves perturbation of a protein of interest through its mislocalization. We synthesized two rapamycin derivatives that possess large substituents at the C16 position within the FRB-binding interface, and these derivatives were screened against a library of FRB mutants using a three-hybrid assay in Saccharomyces cerevisiae. Several FRB mutants responded to one of the rapamycin derivatives, and twenty of these mutants were further characterized in mammalian cells. The mutants most responsive to the ligand were fused to yellow fluorescent protein, and fluorescence levels in the presence and absence of the ligand were measured to determine stability of the fusion proteins. Wild-type and mutant FRB domains were expressed at low levels in the absence of the rapamycin derivative, and expression levels rose up to ten-fold upon treatment with ligand. The synthetic rapamycin derivatives were further analyzed using quantitative mass spectrometry, and one of the compounds was found to contain contaminating rapamycin. Furthermore, uncontaminated analogs retain the ability to inhibit mTOR, albeit with diminished potency relative to rapamycin. The ligand-dependent stability displayed by wildtype FRB and FRB mutants as well as the inhibitory potential and purity of the rapamycin derivatives should be considered as potentially confounding experimental variables when using these systems. PMID:17350953

Discrepancy between low levels of mTOR activity and high levels of p-S6 in primary central nervous system lymphoma may be explained by PAS domain-containing serine/threonine-protein kinase-mediated phosphorylation

DEFF Research Database (Denmark)

Marosvari, Dora; Nagy, Noemi; Kriston, Csilla

2018-01-01

The primary aim of this study was to determine mTOR-pathway activity in primary central nervous system lymphoma (PCNSL), which could be a potential target for therapy. After demonstrating that p-S6 positivity largely exceeded mTOR activity, we aimed to identify other pathways that may lead to S6...... phosphorylation. We measured mTOR activity with immunohistochemistry for p-mTOR and its downstream effectors p(T389)-p70S6K1, p-S6, and p-4EBP1 in 31 cases of PCNSL and 51 cases of systemic diffuse large B-cell lymphoma (DLBCL) and evaluated alternative S6 phosphorylation pathways with p-RSK, p(T229)-p70S6K1...... responsible for S6 phosphorylation, PASK proved to be positive in all cases of PCNSL and DLBCL. Inhibition of PASK resulted in reduced expression of p-S6 in BHD1-cells. This is the first study demonstrating an mTOR independent p-S6 activity in PCNSL and that PASK may contribute to the phosphorylation of S6...

Peroxidation of the dried thin film of lipid by high-energy alpha particles from a cyclotron

International Nuclear Information System (INIS)

Agarwal, S.; Chatterjee, S.N.

1984-01-01

High-energy α particles produced a dose-dependent linear increase in different lipid peroxidation products (e.g., malondialdehyde (MDA), conjugated dienes, and hydroperoxides) in the dried thin film state. An inverse dose-rate effect was observed when the dose rate was varied by changing either the α-particle fluence rate or the α-particle energy. The antioxidants α-tocopherol and butylated hydroxytoluene (BHT) suppressed the α-particle-induced lipid peroxidation in the dried thin film state, and in this respect α-tocopherol was found superior to BHT. It was found that α-tocopherol was equally efficient in inhibiting lipid peroxidations by α particles and ultraviolet light

Elimination of hydrogen peroxide by Haemophilus somnus, a catalase-negative pathogen of cattle.

OpenAIRE

Sample, A K; Czuprynski, C J

1991-01-01

Haemophilus somnus is a catalase-negative, gram-negative pathogen of cattle which is refractory to killing by bovine neutrophils. In this report, we showed that H. somnus rapidly inhibited Luminol-dependent chemiluminescence of bovine neutrophils costimulated with opsonized zymosan or phorbol myristate acetate. We have postulated that this inhibition resulted in part from H. somnus preventing the accumulation of hydrogen peroxide (H2O2) during the oxidative burst. In support of this hypothesi...

Delayed Recovery of Skeletal Muscle Mass following Hindlimb Immobilization in mTOR Heterozygous Mice

OpenAIRE

Lang, Susan M.; Kazi, Abid A.; Hong-Brown, Ly; Lang, Charles H.

2012-01-01

The present study addressed the hypothesis that reducing mTOR, as seen in mTOR heterozygous (+/-) mice, would exaggerate the changes in protein synthesis and degradation observed during hindlimb immobilization as well as impair normal muscle regrowth during the recovery period. Atrophy was produced by unilateral hindlimb immobilization and data compared to the contralateral gastrocnemius. In wild-type (WT) mice, the gradual loss of muscle mass plateaued by day 7. This response was associated ...

Association of CAD, a multifunctional protein involved in pyrimidine synthesis, with mLST8, a component of the mTOR complexes

Science.gov (United States)

2013-01-01

Background mTOR is a genetically conserved serine/threonine protein kinase, which controls cell growth, proliferation, and survival. A multifunctional protein CAD, catalyzing the initial three steps in de novo pyrimidine synthesis, is regulated by the phosphorylation reaction with different protein kinases, but the relationship with mTOR protein kinase has not been known. Results CAD was recovered as a binding protein with mLST8, a component of the mTOR complexes, from HEK293 cells transfected with the FLAG-mLST8 vector. Association of these two proteins was confirmed by the co-immuoprecipitaiton followed by immunoblot analysis of transfected myc-CAD and FLAG-mLST8 as well as that of the endogenous proteins in the cells. Analysis using mutant constructs suggested that CAD has more than one region for the binding with mLST8, and that mLST8 recognizes CAD and mTOR in distinct ways. The CAD enzymatic activity decreased in the cells depleted of amino acids and serum, in which the mTOR activity is suppressed. Conclusion The results obtained indicate that mLST8 bridges between CAD and mTOR, and plays a role in the signaling mechanism where CAD is regulated in the mTOR pathway through the association with mLST8. PMID:23594158

Inhibition of protein kinase C delta attenuates allergic airway inflammation through suppression of PI3K/Akt/mTOR/HIF-1 alpha/VEGF pathway.

Directory of Open Access Journals (Sweden)

Yun Ho Choi

Full Text Available Vascular endothelial growth factor (VEGF is supposed to contribute to the pathogenesis of allergic airway disease. VEGF expression is regulated by a variety of stimuli such as nitric oxide, growth factors, and hypoxia-inducible factor-1 alpha (HIF-1α. Recently, inhibition of the mammalian target of rapamycin (mTOR has been shown to alleviate cardinal asthmatic features, including airway hyperresponsiveness, eosinophilic inflammation, and increased vascular permeability in asthma models. Based on these observations, we have investigated whether mTOR is associated with HIF-1α-mediated VEGF expression in allergic asthma. In studies with the mTOR inhibitor rapamycin, we have elucidated the stimulatory role of a mTOR-HIF-1α-VEGF axis in allergic response. Next, the mechanisms by which mTOR is activated to modulate this response have been evaluated. mTOR is known to be regulated by phosphoinositide 3-kinase (PI3K/Akt or protein kinase C-delta (PKC δ in various cell types. Consistent with these, our results have revealed that suppression of PKC δ by rottlerin leads to the inhibition of PI3K/Akt activity and the subsequent blockade of a mTOR-HIF-1α-VEGF module, thereby attenuating typical asthmatic attack in a murine model. Thus, the present data indicate that PKC δ is necessary for the modulation of the PI3K/Akt/mTOR signaling cascade, resulting in a tight regulation of HIF-1α activity and VEGF expression. In conclusion, PKC δ may represent a valuable target for innovative therapeutic treatment of allergic airway disease.

miR-199a Links MeCP2 with mTOR Signaling and Its Dysregulation Leads to Rett Syndrome Phenotypes

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Keita Tsujimura

2015-09-01

Full Text Available Rett syndrome (RTT is a neurodevelopmental disorder caused by MECP2 mutations. Although emerging evidence suggests that MeCP2 deficiency is associated with dysregulation of mechanistic target of rapamycin (mTOR, which functions as a hub for various signaling pathways, the mechanism underlying this association and the molecular pathophysiology of RTT remain elusive. We show here that MeCP2 promotes the posttranscriptional processing of particular microRNAs (miRNAs as a component of the microprocessor Drosha complex. Among the MeCP2-regulated miRNAs, we found that miR-199a positively controls mTOR signaling by targeting inhibitors for mTOR signaling. miR-199a and its targets have opposite effects on mTOR activity, ameliorating and inducing RTT neuronal phenotypes, respectively. Furthermore, genetic deletion of miR-199a-2 led to a reduction of mTOR activity in the brain and recapitulated numerous RTT phenotypes in mice. Together, these findings establish miR-199a as a critical downstream target of MeCP2 in RTT pathogenesis by linking MeCP2 with mTOR signaling.

Impact of mTOR Inhibitors on Cancer Development in Kidney Transplantation Recipients: A Population-Based Study.

Science.gov (United States)

Kao, C-C; Liu, J-S; Lin, M-H; Hsu, C-Y; Chang, F-C; Lin, Y-C; Chen, H-H; Chen, T-W; Hsu, C-C; Wu, M-S

2016-04-01

The mammalian target of rapamycin (mTOR) inhibitor is an immunosuppressive drug used in kidney transplantation. Whether the mTOR inhibitor is associated with reduced risk of cancer development and mortality after kidney transplantation is controversial. We conducted a nationwide population-based study. Patients who did not have malignancy history and received kidney transplantation between 2010 and 2013 were enrolled. Recipients who had mTOR inhibitors (n = 430) for more than 30 days comprised the study group; 1720 recipients who did not have mTOR inhibitors comprised the control group. The primary outcome is the development of cancer after kidney transplantation. These patients were followed until the first-time admission with diagnosis of cancer, death, or the end of 2014. A Cox proportional-hazard model was used to determine the risk of cancer development and all-cause mortality. During the 35-month median duration of observation, there were 16 and 61 patients with cancer development in the study group and the control group, respectively. The cancer incidence was 12.8 and 12.4 per 1000 person-years. There were 10 and 135 mortality cases, with the incidence rate of 7.8 and 26.9 per 1000 person-years. After multivariable adjustment, the mTOR inhibitors users were not associated with reduced risk of new cancer development as compared with control (hazard ratio [HR], 0.86; 95% confidence interval [CI], 0.46-1.60; P = .63), nor risk of all-cause mortality (HR, 0.70; 95% CI, 0.33-1.46; P = .34). The use of mTOR inhibitors was not associated with a reduction in the risk of cancer development and all-cause mortality in kidney transplantation recipients. Copyright © 2016 Elsevier Inc. All rights reserved.

Rapamycin inhibits BMP-7-induced osteogenic and lipogenic marker expressions in fetal rat calvarial cells.

Science.gov (United States)

Yeh, Lee-Chuan C; Ma, Xiuye; Ford, Jeffery J; Adamo, Martin L; Lee, John C

2013-08-01

Bone morphogenetic proteins (BMPs) promote osteoblast differentiation and bone formation in vitro and in vivo. BMPs canonically signal through Smad transcription factors, but BMPs may activate signaling pathways traditionally stimulated by growth factor tyrosine kinase receptors. Of these, the mTOR pathway has received considerable attention because BMPs activate P70S6K, a downstream effector of mTOR, suggesting that BMP-induced osteogenesis is mediated by mTOR activation. However, contradictory effects of the mTOR inhibitor rapamycin (RAPA) on bone formation have been reported. Since bone formation is thought to be inversely related to lipid accumulation and mTOR is also important for lipid synthesis, we postulated that BMP-7 may stimulate lipogenic enzyme expression in a RAPA-sensitive mechanism. To test this hypothesis, we determined the effects of RAPA on BMP-7-stimulated expression of osteogenic and lipogenic markers in cultured fetal rat calvarial cells. Our study showed that BMP-7 promoted the expression of osteogenic and lipogenic markers. The effect of BMP-7 on osteogenic markers was greater in magnitude than on lipogenic markers and was temporally more sustained. RAPA inhibited basal and BMP-7-stimulated osteogenic and lipogenic marker expression and bone nodule mineralization. The acetyl CoA carboxylase inhibitor TOFA stimulated the expression of osteoblast differentiation markers, whereas palmitate suppressed their expression. We speculate that the BMP-7-stimulated adipogenesis is part of the normal anabolic response to BMPs, but that inappropriate activation of the lipid biosynthetic pathway by mTOR could have deleterious effects on bone formation and could explain paradoxical effects of RAPA to promote bone formation. Copyright © 2013 Wiley Periodicals, Inc.

High levels of hydrogen peroxide in overnight tooth-whitening formulas: effects on enamel and pulp.

Science.gov (United States)

Pugh, George; Zaidel, Lynette; Lin, Nora; Stranick, Michael; Bagley, Daniel

2005-01-01

Limited data are available to assess the safety of high levels of hydrogen peroxide in overnight tooth-whitening formulas. The purpose of this study was to assess the effects of hydrogen peroxide on enamel microhardness, pulp penetration, and enamel morphology. Colgate Platinum Professional Overnight Whitening System (Colgate Oral Pharmaceuticals, Inc., Canton, MA, USA) (10% carbamide peroxide, equivalent to 3.5% hydrogen peroxide) was compared with two prototype formulations containing either 7.0% or 12.0% hydrogen peroxide. In the pulp chamber studies, human extracted teeth were exposed to 3.5%, 7.0%, or 12.0% hydrogen peroxide for 30 minutes, 4 hours, or 7 hours. Microhardness, electron spectroscopy for chemical analysis, and atomic force microscopy evaluations were made from enamel blocks cut from human extracted molars. The enamel blocks were evaluated following 14 7-hour treatments (98 h total). At 7 hours' post-treatment, hydrogen peroxide penetrated the pulp chamber at 23.12 +/- 10.09, 24.58 +/- 6.90, and 26.39 +/- 5.43 microg for 3.5%, 7.0%, and 12.0% hydrogen peroxide, respectively. With regard to enamel morphology, pulp penetration, microhardness, and elemental composition, no statistically significant differences were observed between treatment groups following 98 hours of treatment. Hydrogen peroxide does not adversely affect enamel morphology or microhardness. The levels recovered in pulp indicate that hydrogen peroxide is not expected to inhibit pulpal enzymes. Overnight tray products containing levels of hydrogen peroxide of 3.5%, 7.0%, and 12.0% are not expected to adversely affect the enamel or pulpal enzymes. Additional safety studies are needed to assess the potential for tooth sensitivity and gingival irritation.

mTOR inhibition elicits a dramatic response in PI3K-dependent colon cancers.

Directory of Open Access Journals (Sweden)

Dustin A Deming

Full Text Available The phosphatidylinositide-3-kinase (PI3K signaling pathway is critical for multiple cellular functions including metabolism, proliferation, angiogenesis, and apoptosis, and is the most commonly altered pathway in human cancers. Recently, we developed a novel mouse model of colon cancer in which tumors are initiated by a dominant active PI3K (FC PIK3ca. The cancers in these mice are moderately differentiated invasive mucinous adenocarcinomas of the proximal colon that develop by 50 days of age. Interestingly, these cancers form without a benign intermediary or aberrant WNT signaling, indicating a non-canonical mechanism of tumorigenesis. Since these tumors are dependent upon the PI3K pathway, we investigated the potential for tumor response by the targeting of this pathway with rapamycin, an mTOR inhibitor. A cohort of FC PIK3ca mice were treated with rapamycin at a dose of 6 mg/kg/day or placebo for 14 days. FDG dual hybrid PET/CT imaging demonstrated a dramatic tumor response in the rapamycin arm and this was confirmed on necropsy. The tumor tissue remaining after treatment with rapamycin demonstrated increased pERK1/2 or persistent phosphorylated ribosomal protein S6 (pS6, indicating potential resistance mechanisms. This unique model will further our understanding of human disease and facilitate the development of therapeutics through pharmacologic screening and biomarker identification.

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

Science.gov (United States)

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...

The antidepressant sertraline inhibits translation initiation by curtailing mammalian target of rapamycin signaling.

Science.gov (United States)

Lin, Chen-Ju; Robert, Francis; Sukarieh, Rami; Michnick, Stephen; Pelletier, Jerry

2010-04-15

Sertraline, a selective serotonin reuptake inhibitor, is a widely used antidepressant agent. Here, we show that sertraline also exhibits antiproliferative activity. Exposure to sertraline leads to a concentration-dependent decrease in protein synthesis. Moreover, polysome profile analysis of sertraline-treated cells shows a reduction in polysome content and a concomitant increase in 80S ribosomes. The inhibition in translation caused by sertraline is associated with decreased levels of the eukaryotic initiation factor (eIF) 4F complex, altered localization of eIF4E, and increased eIF2alpha phosphorylation. The latter event leads to increased REDD1 expression, which in turn impinges on the mammalian target of rapamycin (mTOR) pathway by affecting TSC1/2 signaling. Sertraline also independently targets the mTOR signaling pathway downstream of Rheb. In the Emu-myc murine lymphoma model where carcinogenesis is driven by phosphatase and tensin homologue (PTEN) inactivation, sertraline is able to enhance chemosensitivity to doxorubicin. Our results indicate that sertraline exerts antiproliferative activity by targeting the mTOR signaling pathway in a REDD1-dependent manner. (c) 2010 AACR.

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

Science.gov (United States)

Hu, Fang; Xu, Yong; Liu, Feng

2016-06-01

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. mTOR integrates signals from a variety of "energy balancing" hormones such as leptin, insulin, and ghrelin, although its action varies in response to these distinct hormonal stimuli as well as across different neuronal populations. In this review, we summarize and highlight recent findings regarding the functional roles of mTOR complex 1 (mTORC1) in the hypothalamus specifically in its regulation of body weight, energy expenditure, and glucose/lipid homeostasis. Understanding the role and underlying mechanisms behind mTOR-related signaling in the brain will undoubtedly pave new avenues for future therapeutics and interventions that can combat obesity, insulin resistance, and diabetes. Copyright © 2016 the American Physiological Society.

Turmeric and black pepper spices decrease lipid peroxidation in meat patties during cooking

Science.gov (United States)

Zhang, Yanjun; Henning, Susanne M.; Lee, Ru-Po; Huang, Jianjun; Zerlin, Alona; Li, Zhaoping; Heber, David

2015-01-01

Abstract Spices are rich in natural antioxidants and have been shown to be potent inhibitors of lipid peroxidation during cooking of meat. Turmeric contains unique conjugated curcuminoids with strong antioxidant activity. Piperine, one of the main constituents of black pepper, is known to increase the bioavailability of curcuminoids in mouse and human studies when consumed with turmeric. We investigated whether adding black pepper to turmeric powder may further inhibit lipid peroxidation when added to meat patties prior to cooking. The addition of black pepper to turmeric significantly decreased the lipid peroxidation in hamburger meat. When investigating the antioxidant activity of the main chemical markers, we determined that piperine did not exhibit any antioxidant activity. Therefore, we conclude that other black pepper ingredients are responsible for the increased antioxidant activity of combining black pepper with turmeric powder. PMID:25582173

Intrathecal administration of rapamycin inhibits the phosphorylation of DRG Nav1.8 and attenuates STZ-induced painful diabetic neuropathy in rats.

Science.gov (United States)

He, Wan-You; Zhang, Bin; Xiong, Qing-Ming; Yang, Cheng-Xiang; Zhao, Wei-Cheng; He, Jian; Zhou, Jun; Wang, Han-Bing

2016-04-21

The mammalian target of rapamycin (mTOR) is a key regulator of mRNA translation and protein synthesis, and it is specifically inhibited by rapamycin. In chronic pain conditions, mTOR-mediated local protein synthesis is crucial for neuronal hyperexcitability and synaptic plasticity. The tetrodotoxin-resistant (TTX-R) sodium channel Nav1.8 plays a major role in action potential initiation and propagation and cellular excitability in DRG (dorsal root ganglion) neurons. In this study, we investigated if mTOR modulates the phosphorylation of Nav1.8 that is associated with neuronal hyperexcitability and behavioral hypersensitivity in STZ-induced diabetic rats. Painful diabetic neuropathy (PDN) was induced in Sprague-Dawley rats by intraperitoneal injection with streptozotocin (STZ) at 60mg/kg. After the onset of PDN, the rats received daily intrathecal administrations of rapamycin (1μg, 3μg, or 10μg/day) for 7 days; other diabetic rats received the same volumes of dimethyl sulfoxide (DMSO). Herein, we demonstrate a marked increase in protein expression of total mTOR and phospho-mTOR (p-mTOR) together with the up-regulation of phosphor-Nav1.8 (p-Nav1.8) prior to the mechanical withdrawal threshold reaching a significant reduction in dorsal root ganglions (DRGs). Furthermore, the intrathecal administration of rapamycin, inhibiting the activity of mTOR, suppressed the phosphorylation of DRG Nav1.8, reduced the TTX-R current density, heightened the voltage threshold for activation and lowered the voltage threshold for inactivation and relieved mechanical hypersensitivity in diabetic rats. An intrathecal injection (i.t.) of rapamycin inhibited the phosphorylation and enhanced the functional availability of DRG Nav1.8 attenuated STZ-induced hyperalgesia. These results suggest that rapamycin is a potential therapeutic intervention for clinical PDN. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Effect of tea catechins on the structure of lipid membrane and beta-ray induced lipid peroxidation

International Nuclear Information System (INIS)

Kubota, M.; Haga, H.; Takeuchi, Y.; Okuno, K.; Yoshioka, H.; Yoshioka, H.

2007-01-01

Inhibiting effect of four tea catechins, (-)-epicatechin (EC), (-)-epicatechin gallate (ECG), (-)-epigallocatechin (EGC), (-)-epigallocatechin gallate (EGCG), on the lipid peroxidation induced by β-ray in tritiated water was examined using a spin probe method. 16-Doxylstearic acid (16NS) was incorporated into the liposome prepared from egg yolk phosphatidylcholine and the rate of the decrease of ESR intensity of 16NS was used as a measure of the inhibiting effect. In the low concentration region below 10 -5 M, catechins showed their inhibitions on the lipid peroxidation according to the order of ECG>EGCG>EC>EGC. This result was explained by a model that the initiator of the peroxidation is the hydroxyl radical (·OH) and the catechins adsorbed on the lipid membrane surface acting as scavengers of ·OH. In the high concentration range, however, the effect was diverse and it decreased with the increase of it in the case of EGCG. EGCG in this range was considered to enter into the interior of the membrane and break the structure, which causes the decrease of 16NS. Observation with transmission electron microscope (TEM) revealed that the size of the liposome became larger with the increasing concentration of EGCG and finally it was broken into fragments, showing that EGCG broadened the area of the liposome as expected from the result of ESR. (author)

Novel small molecule drugs inhibit tumor cell metabolism and show potent anti-tumorigenic potential

DEFF Research Database (Denmark)

Trojel-Hansen, Christina; Erichsen, Kamille Dumong; Christensen, Mette Knak

2011-01-01

oxyphenisatine analogs TOP001 and TOP216 exert their anti-cancer effect by affecting tumor cell metabolism and inducing intracellular amino acid deprivation, leading to a block of cell proliferation. GCN2-mediated phosphorylation of eIF2a as well as mTOR pathway inhibition supports the above notion. In addition...

Novel small molecule drugs inhibit tumor cell metabolism and show potent anti-tumorigenic potential

DEFF Research Database (Denmark)

Trojel-Hansen, Christina; Erichsen, Kamille Dumong; Christensen, Mette Knak

2011-01-01

oxyphenisatine analogs TOP001 and TOP216 exert their anti-cancer effect by affecting tumor cell metabolism and inducing intracellular amino acid deprivation, leading to a block of cell proliferation. GCN2-mediated phosphorylation of eIF2α as well as mTOR pathway inhibition supports the above notion. In addition...

Simultaneous inhibition of aberrant cancer kinome using rationally designed polymer-protein core-shell nanomedicine.

Science.gov (United States)

Chandran, Parwathy; Gupta, Neha; Retnakumari, Archana Payickattu; Malarvizhi, Giridharan Loghanathan; Keechilat, Pavithran; Nair, Shantikumar; Koyakutty, Manzoor

2013-11-01

Simultaneous inhibition of deregulated cancer kinome using rationally designed nanomedicine is an advanced therapeutic approach. Herein, we have developed a polymer-protein core-shell nanomedicine to inhibit critically aberrant pro-survival kinases (mTOR, MAPK and STAT5) in primitive (CD34(+)/CD38(-)) Acute Myeloid Leukemia (AML) cells. The nanomedicine consists of poly-lactide-co-glycolide core (~250 nm) loaded with mTOR inhibitor, everolimus, and albumin shell (~25 nm thick) loaded with MAPK/STAT5 inhibitor, sorafenib and the whole construct was surface conjugated with monoclonal antibody against CD33 receptor overexpressed in AML. Electron microscopy confirmed formation of core-shell nanostructure (~290 nm) and flow cytometry and confocal studies showed enhanced cellular uptake of targeted nanomedicine. Simultaneous inhibition of critical kinases causing synergistic lethality against leukemic cells, without affecting healthy blood cells, was demonstrated using immunoblotting, cytotoxicity and apoptosis assays. This cell receptor plus multi-kinase targeted core-shell nanomedicine was found better specific and tolerable compared to current clinical regime of cytarabine and daunorubicin. These authors demonstrate simultaneous inhibition of critical kinases causing synergistic lethality against leukemic cells, without affecting healthy blood cells by using rationally designed polymer-protein core-shell nanomedicine, provoding an advanced method to eliminate cancer cells, with the hope of future therapeutic use. Copyright © 2013 Elsevier Inc. All rights reserved.

Peroxide organometallic compounds and their transformations

International Nuclear Information System (INIS)

Razuvaev, G.A.; Brilkina, T.G.

1976-01-01

A survey is given experimental works on synthesis and reactions of peroxide organometallic compounds. Reactions have been considered of organometallic compounds with oxygen and organic peroxides which result in formation of both peroxide and non-peroxide products. Possible routes and mechanisms of chemical transformations of peroxide organometallic compounds have been discussed. Reactions of organometallic compounds with oxygen and peroxides have been considered

Invasive pulmonary aspergillosis mimicking organizing pneumonia after mTOR inhibitor therapy: A case report

Directory of Open Access Journals (Sweden)

Yuki Iijima

2018-04-01

Full Text Available A 67-year-old man presented to the hospital with complaints of fever and cough. He had a past medical history of renal cell carcinoma and had just started treatment with temsirolimus, a mammalian target of rapamycin (mTOR inhibitor. A 1-week course of antibiotics did not have any effect on his symptoms. A chest computed tomography (CT scan showed the reversed halo sign (RHS. Organizing pneumonia induced by mTOR inhibitor treatment was initially considered. However, transbronchial biopsy revealed clusters of fungal organisms, suggesting infection with Aspergillus spp. Within just 2 weeks, a CT scan showed drastic enlargement of the cavitary lesion, with multiple newly formed consolidations. The patient was diagnosed with invasive pulmonary aspergillosis. Concomitant treatment with voriconazole and micafungin was started. Two weeks after the initiation of treatment, he became afebrile with gradual regression of the cavitary lesion and consolidations. Keywords: mTOR inhibitor, Organizing pneumonia, Reversed halo sign, Invasive pulmonary aspergillosis, Immunocompromise

An in vitro model to test relative antioxidant potential: Ultraviolet-induced lipid peroxidation in liposomes

International Nuclear Information System (INIS)

Pelle, E.; Maes, D.; Padulo, G.A.; Kim, E.K.; Smith, W.P.

1990-01-01

Since antioxidants have been shown to play a major role in preventing some of the effects of aging and photoaging in skin, it is important to study this phenomenon in a controlled manner. This was accomplished by developing a simple and reliable in vitro technique to assay antioxidant efficacy. Inhibition of peroxidation by antioxidants was used as a measure of relative antioxidant potential. Liposomes, high in polyunsaturated fatty acids (PUFA), were dispersed in buffer and irradiated with ultraviolet (UV) light. Irradiated liposomes exhibited a significantly higher amount of hydroperoxides than liposomes containing antioxidants in a dose- and concentration-dependent manner. Lipid peroxidation was determined spectrophotometrically by an increase in thiobarbituric acid reacting substances. To further substantiate the production of lipid peroxides, gas chromatography was used to measure a decrease in PUFA substrate. In order of decreasing antioxidant effectiveness, the following results were found among lipophilic antioxidants: BHA greater than catechin greater than BHT greater than alpha-tocopherol greater than chlorogenic acid. Among hydrophilic antioxidants, ascorbic acid and dithiothreitol were effective while glutathione was ineffective. In addition, ascorbic acid was observed to act synergistically with alpha-tocopherol, which is in agreement with other published reports on the interaction of these two antioxidants. Although peroxyl radical scavengers seem to be at a selective advantage in this liposomal/UV system, these results demonstrate the validity of this technique as an assay for measuring an antioxidant's potential to inhibit UV-induced peroxidation

Hydrogen peroxide stimulates cell motile activity through LPA receptor-3 in liver epithelial WB-F344 cells

Energy Technology Data Exchange (ETDEWEB)

Shibata, Ayano; Tanabe, Eriko; Inoue, Serina; Kitayoshi, Misaho; Okimoto, Souta; Hirane, Miku; Araki, Mutsumi [Division of Cancer Biology and Bioinformatics, Department of Life Science, Faculty of Science and Engineering, Kinki University, 3-4-1, Kowakae, Higashiosaka, Osaka 577-8502 (Japan); Fukushima, Nobuyuki [Division of Molecular Neurobiology, Department of Life Science, Faculty of Science and Engineering, Kinki University, 3-4-1, Kowakae, Higashiosaka, Osaka 577-8502 (Japan); Tsujiuchi, Toshifumi, E-mail: ttujiuch@life.kindai.ac.jp [Division of Cancer Biology and Bioinformatics, Department of Life Science, Faculty of Science and Engineering, Kinki University, 3-4-1, Kowakae, Higashiosaka, Osaka 577-8502 (Japan)

2013-04-12

Highlights: •Hydrogen peroxide stimulates cell motility of WB-F344 cells. •LPA{sub 3} is induced by hydrogen peroxide in WB-F344 cells. •Cell motility by hydrogen peroxide is inhibited in LPA{sub 3} knockdown cells. •LPA signaling is involved in cell migration by hydrogen peroxide. -- Abstract: Hydrogen peroxide which is one of reactive oxygen species (ROS) mediates a variety of biological responses, including cell proliferation and migration. In the present study, we investigated whether lysophosphatidic acid (LPA) signaling is involved in cell motile activity stimulated by hydrogen peroxide. The rat liver epithelial WB-F344 cells were treated with hydrogen peroxide at 0.1 or 1 μM for 48 h. In cell motility assays, hydrogen peroxide treated cells showed significantly high cell motile activity, compared with untreated cells. To measure the expression levels of LPA receptor genes, quantitative real time RT-PCR analysis was performed. The expressions of LPA receptor-3 (Lpar3) in hydrogen peroxide treated cells were significantly higher than those in control cells, but not Lpar1 and Lpar2 genes. Next, to assess the effect of LPA{sub 3} on cell motile activity, the Lpar3 knockdown cells from WB-F344 cells were also treated with hydrogen peroxide. The cell motile activity of the knockdown cells was not stimulated by hydrogen peroxide. Moreover, in liver cancer cells, hydrogen peroxide significantly activated cell motility of Lpar3-expressing cells, but not Lpar3-unexpressing cells. These results suggest that LPA signaling via LPA{sub 3} may be mainly involved in cell motile activity of WB-F344 cells stimulated by hydrogen peroxide.

Safe handling of potential peroxide forming compounds and their corresponding peroxide yielded derivatives.

Energy Technology Data Exchange (ETDEWEB)

Sears, Jeremiah Matthew; Boyle, Timothy J.; Dean, Christopher J.

2013-06-01

This report addresses recent developments concerning the identification and handling of potential peroxide forming (PPF) and peroxide yielded derivative (PYD) chemicals. PPF chemicals are described in terms of labeling, shelf lives, and safe handling requirements as required at SNL. The general peroxide chemistry concerning formation, prevention, and identification is cursorily presented to give some perspective to the generation of peroxides. The procedure for determining peroxide concentrations and the proper disposal methods established by the Hazardous Waste Handling Facility are also provided. Techniques such as neutralization and dilution are provided for the safe handling of any PYD chemicals to allow for safe handling. The appendices are a collection of all available SNL documentation pertaining to PPF/PYD chemicals to serve as a single reference.

A novel mTOR activating protein protects dopamine neurons against oxidative stress by repressing autophagy related cell death.

Science.gov (United States)

Choi, Kyou-Chan; Kim, Shin-Hee; Ha, Ji-Young; Kim, Sang-Tae; Son, Jin H

2010-01-01

Our previous microarray analysis identified a neuroprotective protein Oxi-alpha, that was down-regulated during oxidative stress (OS)-induced cell death in dopamine neurons [Neurochem. Res. (2004) vol. 29, pp. 1223]. Here we find that the phylogenetically conserved Oxi-alpha protects against OS by a novel mechanism: activation of the mammalian target of rapamycin (mTOR) kinase and subsequent repression of autophagic vacuole accumulation and cell death. To the best of our knowledge, Oxi-alpha is the first molecule discovered in dopamine neurons, which activates mTOR kinase. Indeed, the down-regulation of Oxi-alpha by OS suppresses the activation of mTOR kinase. The pathogenic effect of down-regulated Oxi-alpha was confirmed by gene-specific knockdown experiment, which resulted in not only the repression of mTOR kinase and the subsequent phosphorylation of p70 S6 kinase and 4E-BP1, but also enhanced susceptibility to OS. In accordance with these observations, treatment with rapamycin, an mTOR inhibitor and autophagy inducer, potentiated OS-induced cell death, while similar treatment with an autophagy inhibitor, 3-methyladenine protected the dopamine cells. Our findings present evidence for the presence of a novel class of molecule involved in autophagic cell death triggered by OS in dopamine neurons.

21 CFR 172.802 - Acetone peroxides.

Science.gov (United States)

2010-04-01

... 21 Food and Drugs 3 2010-04-01 2009-04-01 true Acetone peroxides. 172.802 Section 172.802 Food and... Multipurpose Additives § 172.802 Acetone peroxides. The food additive acetone peroxides may be safely used in... acetone peroxide, with minor proportions of higher polymers, manufactured by reaction of hydrogen peroxide...

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.

Vasopressin activates Akt/mTOR pathway in smooth muscle cells cultured in high glucose concentration

Energy Technology Data Exchange (ETDEWEB)

Montes, Daniela K.; Brenet, Marianne; Muñoz, Vanessa C.; Burgos, Patricia V.; Villanueva, Carolina I. [Department of Physiology, Universidad Austral de Chile, Valdivia 509-9200 (Chile); Figueroa, Carlos D. [Department of Anatomy, Histology and Pathology, Universidad Austral de Chile, Valdivia 509-9200 (Chile); González, Carlos B., E-mail: cbgonzal@uach.cl [Department of Physiology, Universidad Austral de Chile, Valdivia 509-9200 (Chile); Department of Neuroscience and Cell Biology, University of Texas Medical Branch, Galveston, TX 77555 (United States)

2013-11-29

Highlights: •AVP induces mTOR phosphorylation in A-10 cells cultured in high glucose concentration. •The mTOR phosphorylation is mediated by the PI3K/Akt pathway activation. •The AVP-induced mTOR phosphorylation inhibited autophagy and stimulated cell proliferation. -- Abstract: Mammalian target of rapamycin (mTOR) complex is a key regulator of autophagy, cell growth and proliferation. Here, we studied the effects of arginine vasopressin (AVP) on mTOR activation in vascular smooth muscle cells cultured in high glucose concentration. AVP induced the mTOR phosphorylation in A-10 cells grown in high glucose, in contrast to cells cultured in normal glucose; wherein, only basal phosphorylation was observed. The AVP-induced mTOR phosphorylation was inhibited by a PI3K inhibitor. Moreover, the AVP-induced mTOR activation inhibited autophagy and increased thymidine incorporation in cells grown in high glucose. This increase was abolished by rapamycin which inhibits the mTORC1 complex formation. Our results suggest that AVP stimulates mTOR phosphorylation by activating the PI3K/Akt signaling pathway and, subsequently, inhibits autophagy and raises cell proliferation in A-10 cells maintained in high glucose concentration.

mTORC2 promotes type I insulin-like growth factor receptor and insulin receptor activation through the tyrosine kinase activity of mTOR.

Science.gov (United States)

Yin, Yancun; Hua, Hui; Li, Minjing; Liu, Shu; Kong, Qingbin; Shao, Ting; Wang, Jiao; Luo, Yuanming; Wang, Qian; Luo, Ting; Jiang, Yangfu

2016-01-01

Mammalian target of rapamycin (mTOR) is a core component of raptor-mTOR (mTORC1) and rictor-mTOR (mTORC2) complexes that control diverse cellular processes. Both mTORC1 and mTORC2 regulate several elements downstream of type I insulin-like growth factor receptor (IGF-IR) and insulin receptor (InsR). However, it is unknown whether and how mTOR regulates IGF-IR and InsR themselves. Here we show that mTOR possesses unexpected tyrosine kinase activity and activates IGF-IR/InsR. Rapamycin induces the tyrosine phosphorylation and activation of IGF-IR/InsR, which is largely dependent on rictor and mTOR. Moreover, mTORC2 promotes ligand-induced activation of IGF-IR/InsR. IGF- and insulin-induced IGF-IR/InsR phosphorylation is significantly compromised in rictor-null cells. Insulin receptor substrate (IRS) directly interacts with SIN1 thereby recruiting mTORC2 to IGF-IR/InsR and promoting rapamycin- or ligand-induced phosphorylation of IGF-IR/InsR. mTOR exhibits tyrosine kinase activity towards the general tyrosine kinase substrate poly(Glu-Tyr) and IGF-IR/InsR. Both recombinant mTOR and immunoprecipitated mTORC2 phosphorylate IGF-IR and InsR on Tyr1131/1136 and Tyr1146/1151, respectively. These effects are independent of the intrinsic kinase activity of IGF-IR/InsR, as determined by assays on kinase-dead IGF-IR/InsR mutants. While both rictor and mTOR immunoprecitates from rictor(+/+) MCF-10A cells exhibit tyrosine kinase activity towards IGF-IR and InsR, mTOR immunoprecipitates from rictor(-/-) MCF-10A cells do not induce IGF-IR and InsR phosphorylation. Phosphorylation-deficient mutation of residue Tyr1131 in IGF-IR or Tyr1146 in InsR abrogates the activation of IGF-IR/InsR by mTOR. Finally, overexpression of rictor promotes IGF-induced cell proliferation. Our work identifies mTOR as a dual-specificity kinase and clarifies how mTORC2 promotes IGF-IR/InsR activation.

Hydrogen peroxide kinetics in water radiolysis

Science.gov (United States)

Iwamatsu, Kazuhiro; Sundin, Sara; LaVerne, Jay A.

2018-04-01

The kinetics of the formation and reaction of hydrogen peroxide in the long time γ- radiolysis of water is examined using a combination of experiment with model calculations. Escape yields of hydrogen peroxide on the microsecond time scale are easily measured with added radical scavengers even with substantial amounts of initial added hydrogen peroxide. The γ-radiolysis of aqueous hydrogen peroxide solutions without added radical scavengers reach a steady state limiting concentration of hydrogen peroxide with increasing dose, and that limit is directly proportional to the initial concentration of added hydrogen peroxide. The dose necessary to reach that limiting hydrogen peroxide concentration is also proportional to the initial concentration, but dose rate has a very small effect. The addition of molecular hydrogen to aqueous solutions of hydrogen peroxide leads to a decrease in the high dose limiting hydrogen peroxide concentration that is linear with the initial hydrogen concentration, but the amount of decrease is not stoichiometric. Proton irradiations of solutions with added hydrogen peroxide and hydrogen are more difficult to predict because of the decreased yields of radicals; however, with a substantial increase in dose rate there is a sufficient decrease in radical yields that hydrogen addition has little effect on hydrogen peroxide decay.

Stabilization of hydrogen peroxide using tartaric acids in Fenton and fenton-like oxidation

Energy Technology Data Exchange (ETDEWEB)

Oh, Hyung Suk; Kim, Jeong-Jin; Kim, Young-Hun [Andong National University, Andong (Korea, Republic of)

2016-03-15

The stabilization of hydrogen peroxide is a key factor in the efficiency of a Fenton reaction. The stability of hydrogen peroxide was evaluated in a Fenton reaction and Fenton-like reactions in the presence of tartaric acid as a stabilizer. The interactions between ferrous or ferric iron and tartaric acid were observed through spectroscopic monitoring at variable pH around pKa{sub 1} and pKa{sub 2} of the stabilizer. Ferric iron had a strong interaction with the stabilizer, and the strong interaction was dominant above pKa{sub 2}. At a low pH, below pKa{sub 1}, the stabilizing effect was at its maximum and the prolonged life-time of hydrogen peroxide gave a higher efficiency to the oxidative degradation of nitrobenzene. In Fenton-like reactions with hematite, the acidic conditions caused dissolution of iron from an iron oxide, and an increase in iron species was the result. Tartaric acid showed a stabilizing effect on hydrogen peroxide in the Fentonlike system. The stabilization by tartaric acid might be due to an inhibition of catalytic activity of dissolved iron, and the stabilization strongly depends on the ionization state of the stabilizer.

Stabilization of hydrogen peroxide using tartaric acids in Fenton and fenton-like oxidation

International Nuclear Information System (INIS)

Oh, Hyung Suk; Kim, Jeong-Jin; Kim, Young-Hun

2016-01-01

The stabilization of hydrogen peroxide is a key factor in the efficiency of a Fenton reaction. The stability of hydrogen peroxide was evaluated in a Fenton reaction and Fenton-like reactions in the presence of tartaric acid as a stabilizer. The interactions between ferrous or ferric iron and tartaric acid were observed through spectroscopic monitoring at variable pH around pKa 1 and pKa 2 of the stabilizer. Ferric iron had a strong interaction with the stabilizer, and the strong interaction was dominant above pKa 2 . At a low pH, below pKa 1 , the stabilizing effect was at its maximum and the prolonged life-time of hydrogen peroxide gave a higher efficiency to the oxidative degradation of nitrobenzene. In Fenton-like reactions with hematite, the acidic conditions caused dissolution of iron from an iron oxide, and an increase in iron species was the result. Tartaric acid showed a stabilizing effect on hydrogen peroxide in the Fentonlike system. The stabilization by tartaric acid might be due to an inhibition of catalytic activity of dissolved iron, and the stabilization strongly depends on the ionization state of the stabilizer.

Inhibition by acrolein of light-induced stomatal opening through inhibition of inward-rectifying potassium channels in Arabidopsis thaliana.

Science.gov (United States)

Islam, Md Moshiul; Ye, Wenxiu; Matsushima, Daiki; Khokon, Md Atiqur Rahman; Munemasa, Shintaro; Nakamura, Yoshimasa; Murata, Yoshiyuki

2015-01-01

Acrolein is a reactive α,β-unsaturated aldehyde derived from lipid peroxides, which are produced in plants under a variety of stress. We investigated effects of acrolein on light-induced stomatal opening using Arabidopsis thaliana. Acrolein inhibited light-induced stomatal opening in a dose-dependent manner. Acrolein at 100 μM inhibited plasma membrane inward-rectifying potassium (Kin) channels in guard cells. Acrolein at 100 μM inhibited Kin channel KAT1 expressed in a heterologous system using Xenopus leaves oocytes. These results suggest that acrolein inhibits light-induced stomatal opening through inhibition of Kin channels in guard cells.

Free radical scavenging activity and lipid peroxidation inhibition of Hypericum helianthemoides (spach Boiss

Directory of Open Access Journals (Sweden)

Soheila Moein

2015-06-01

Full Text Available Antioxidants are compounds that obstruct the oxidation of macromolecules in the body. In general, there are two categories of antioxidants, natural and synthetic. Recently, interest has been increased considerably for obtaining new natural antioxidants. In this study, the scavenging of free radicals such as DPPH, NO and OH by Hypericum helianthemoides extract was evaluated. Also, the antioxidant properties of this extract were evaluated by FRAP, FTC methods and determination phenolic compounds. The plant was collected from north of Fars Province and plant extraction was obtained using ethanol. In DPPH radical scavenging, different concentrations of the Hypericum extract were added to DPPH radical. In hydroxyl radical scavenging, Fenton reaction mixture, TCA and TBA were mixed with Hypericum extract. In nitric radical scavenging, nitropruside was mixed with Hypericum extract and then sulphanilic acid, naphthylene diamine were added. In determination of phenolic compounds, Folin-ciocalteu and sodium carbonate were added to Hypericum extract. In DPPH radical scavenging, the IC50 of Hypericum extract (309.35±6.5μg/ml was higher than the antioxidant standards, BHT (IC50=81.9±2.6 μg/ml and quercetin (IC50=60.04±6.48 μg/ml. The highest scavenging of hydroxyl radicals was observed in Hypericum extract (70.3±0.8%, 125 μg/ml. In gallic acid it was (73.8±3.3%. In 200 μg/ml of Hypericum extract scavenged NO radical (85.2±2.7%. In FRAP method, the IC50 of this extract was 109.7±10.5 μg/ml. In FTC method, the inhibition of lipid peroxidation by Hypericum extract, BHT and ascorbic acid were 59.2±2.2, 66.9±0.15, 64.06±0.02 respectively. Total phenol of the plant extract was 3±0.4 mg/g.

Effects of the single and combined treatment with dopamine agonist, somatostatin analog and mTOR inhibitors in a human lung carcinoid cell line: an in vitro study.

Science.gov (United States)

Pivonello, Claudia; Rousaki, Panagoula; Negri, Mariarosaria; Sarnataro, Maddalena; Napolitano, Maria; Marino, Federica Zito; Patalano, Roberta; De Martino, Maria Cristina; Sciammarella, Concetta; Faggiano, Antongiulio; Rocco, Gaetano; Franco, Renato; Kaltsas, Gregory A; Colao, Annamaria; Pivonello, Rosario

2017-06-01

Somatostatin analogues and mTOR inhibitors have been used as medical therapy in lung carcinoids with variable results. No data are available on dopamine agonists as treatment for lung carcinoids. The main aim of the current study was to evaluate the effect of the combined treatment of somatostatin analogue octreotide and the dopamine agonist cabergoline with mTOR inhibitors in an in vitro model of typical lung carcinoids: the NCI-H727 cell line. In NCI-H727 cell line, reverse transcriptase-quantitative polymerase chain reaction and immunofluorescence were assessed to characterize the expression of the somatostatin receptor 2 and 5, dopamine receptor 2 and mTOR pathway components. Fifteen typical lung carcinoids tissue samples have been used for somatostatin receptor 2, dopamine receptor 2, and the main mTOR pathway component p70S6K expression and localization by immunohistochemistry. Cell viability, fluorescence-activated cell sorting analysis and western blot have been assessed to test the pharmacological effects of octreotide, cabergoline and mTOR inhibitors, and to evaluate the activation of specific cell signaling pathways in NCI-H727 cell line. NCI-H727 cell line expressed somatostatin receptor 2, somatostatin receptor 5 and dopamine receptor 2 and all mTOR pathway components at messenger and protein levels. Somatostatin receptor 2, dopamine receptor 2, and p70S6K (non phosphorylated and phosphorylated) proteins were expressed in most typical lung carcinoids tissue samples. Octreotide and cabergoline did not reduce cell viability as single agents but, when combined with mTOR inhibitors, they potentiate mTOR inhibitors effect after long-term exposure, reducing Akt and ERK phosphorylation, mTOR escape mechanisms, and increasing the expression DNA-damage-inducible transcript 4, an mTOR suppressor. In conclusion, the single use of octreotide and cabergoline is not sufficient to block cell viability but the combined approach of these agents with mTOR inhibitors

PI3K-Akt signaling activates mTOR-mediated epileptogenesis in organotypic hippocampal culture model of post-traumatic epilepsy.

Science.gov (United States)

Berdichevsky, Yevgeny; Dryer, Alexandra M; Saponjian, Yero; Mahoney, Mark M; Pimentel, Corrin A; Lucini, Corrina A; Usenovic, Marija; Staley, Kevin J

2013-05-22

mTOR is activated in epilepsy, but the mechanisms of mTOR activation in post-traumatic epileptogenesis are unknown. It is also not clear whether mTOR inhibition has an anti-epileptogenic, or merely anticonvulsive effect. The rat hippocampal organotypic culture model of post-traumatic epilepsy was used to study the effects of long-term (four weeks) inhibition of signaling pathways that interact with mTOR. Ictal activity was quantified by measurement of lactate production and electrical recordings, and cell death was quantified with lactate dehydrogenase (LDH) release measurements and Nissl-stained neuron counts. Lactate and LDH measurements were well correlated with electrographic activity and neuron counts, respectively. Inhibition of PI3K and Akt prevented activation of mTOR, and was as effective as inhibition of mTOR in reducing ictal activity and cell death. A dual inhibitor of PI3K and mTOR, NVP-BEZ235, was also effective. Inhibition of mTOR with rapamycin reduced axon sprouting. Late start of rapamycin treatment was effective in reducing epileptic activity and cell death, while early termination of rapamycin treatment did not result in increased epileptic activity or cell death. The conclusions of the study are as follows: (1) the organotypic hippocampal culture model of post-traumatic epilepsy comprises a rapid assay of anti-epileptogenic and neuroprotective activities and, in this model (2) mTOR activation depends on PI3K-Akt signaling, and (3) transient inhibition of mTOR has sustained effects on epilepsy.

PI3K-Akt signaling activates mTOR-mediated epileptogenesis in organotypic hippocampal culture model of posttraumatic epilepsy

Science.gov (United States)

Berdichevsky, Yevgeny; Dryer, Alexandra M.; Saponjian, Yero; Mahoney, Mark M.; Pimentel, Corrin A.; Lucini, Corrina A.; Usenovic, Marija; Staley, Kevin J.

2013-01-01

mTOR is activated in epilepsy, but the mechanisms of mTOR activation in post-traumatic epileptogenesis are unknown. It is also not clear whether mTOR inhibition has an antiepileptogenic, or merely anti-convulsive effect. The rat hippocampal organotypic culture model of post-traumatic epilepsy was used to study the effects of long term (four weeks) inhibition of signaling pathways that interact with mTOR. Ictal activity was quantified by measurement of lactate production and electrical recordings, and cell death was quantified with LDH release measurements and Nissl-stained neuron counts. Lactate and LDH measurements were well-correlated with electrographic activity and neuron counts, respectively. Inhibition of PI3K and Akt prevented activation of mTOR, and was as effective as inhibition of mTOR in reducing ictal activity and cell death. A dual inhibitor of PI3K and mTOR, NVP-BEZ235, was also effective. Inhibition of mTOR with rapamycin reduced axon sprouting. Late start of rapamycin treatment was effective in reducing epileptic activity and cell death, while early termination of rapamycin treatment did not result in increased epileptic activity or cell death. The conclusions of the study are: (1), the organotypic hippocampal culture model of posttraumatic epilepsy comprises a rapid assay of antiepileptogenic and neuroprotective activities and, in this model (2), mTOR activation depends on PI3K-Akt signaling, and (3) transient inhibition of mTOR has sustained effects on epilepsy. PMID:23699517

Treatment of Advanced Malignant Uterine Perivascular Epithelioid Cell Tumor with mTOR Inhibitors: Single-institution Experience and Review of the Literature.

Science.gov (United States)

Starbuck, Kristen D; Drake, Richard D; Budd, G Thomas; Rose, Peter G

2016-11-01

Uterine perivascular epithelioid cell tumors (PEComas) are rare mesenchymal tumors. Many have malignant behavior, and no successful treatment strategy has been established. Identification of mutations in the tuberous sclerosis 1 (TSC1) and TSC2 genes producing constitutive activation of the mammalian target of rapamycin (mTOR) pathway presents an opportunity for targeted therapy. Patients with advanced malignant uterine PEComa treated with mTOR inhibitors were identified and records were retrospectively reviewed for treatment response based on radiographic assessment. Three patients with advanced uterine PEComas underwent debulking surgery followed by mTOR inhibitor therapy; two had a complete response to therapy and disease in one patient progressed. Given the absence of effective therapies for malignant uterine PEComas, targeting the mTOR pathway is a logical strategy to pursue given the known pathobiology involving the Tuberous Sclerosis complex. Treatment of malignant uterine PEComas with mTOR inhibitors was effective in two out of three patients after surgical resection, with durable response. Copyright© 2016 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

PKI-587 and sorafenib targeting PI3K/AKT/mTOR and Ras/Raf/MAPK pathways synergistically inhibit HCC cell proliferation.

Science.gov (United States)

Gedaly, Roberto; Angulo, Paul; Hundley, Jonathan; Daily, Michael F; Chen, Changguo; Evers, B Mark

2012-08-01

Deregulated Ras/Raf/MAPK and PI3K/AKT/mTOR signaling pathways are found in hepatocellular carcinoma (HCC). This study aimed to test the inhibitory effects of PKI-587 and sorafenib as single agents or in combination on HCC (Huh7 cell line) proliferation. (3)H-thymidine incorporation and MTT assay were used to assess Huh7 cell proliferation. Phosphorylation of the key enzymes in the Ras/Raf/MAPK and PI3K/AKT/mTOR pathways was detected by Western blot. We found that PKI-587 is a more potent PI3K/mTOR inhibitor than PI-103. Combination of PKI-587 and sorafenib was a more effective inhibitor of Huh7 proliferation than the combination of PI-103 and sorafenib. Combination of PKI-587 and sorafenib synergistically inhibited epidermal growth factor (EGF)-stimulated Huh7 proliferation compared with monodrug therapy. EGF increased phosphorylation of Ras/Raf downstream signaling proteins MEK and ERK; EGF-stimulated activation was inhibited by sorafenib. However, sorafenib, as a single agent, increased AKT (Ser473) phosphorylation. EGF-stimulated AKT (ser473) activation was inhibited by PKI-587. PKI-587 is a potent inhibitor of AKT (Ser473), mTOR (Ser2448), and S6K (Thr389) phosphorylation; in contrast, rapamycin stimulated mTOR complex 2 substrate AKT(Ser473) phosphorylation although it inhibited mTOR complex 1 substrate S6K phosphorylation. PKI-587, as a single agent, stimulated MEK and ERK phosphorylation. However, when PKI-587 and sorafenib were used in combination, they inhibited all the tested kinases in the Ras/Raf /MAPK and PI3K/AKT/mTOR pathways. The combination of PKI-587 and sorafenib has the advantage over monodrug therapy on inhibition of HCC cell proliferation by blocking both PI3K/AKT/mTOR and Ras/Raf/MAPK signaling pathways. Copyright © 2012 Elsevier Inc. All rights reserved.

mTOR Hyperactivation in down syndrome hippocampus appears early during development

NARCIS (Netherlands)

Iyer, Anand M.; van Scheppingen, Jackelien; Milenkovic, Ivan; Anink, Jasper J.; Adle-Biassette, Homa; Kovacs, Gabor G.; Aronica, Eleonora

2014-01-01

The mammalian target of rapamycin (mTOR) signaling pathway is a key developmental pathway involved in mechanisms underlying cellular aging and neurodegeneration. We hypothesized that its deregulation may occur during early brain development in patients with Down syndrome (DS). The expression

MEK-ERK inhibition potentiates WAY-600-induced anti-cancer efficiency in preclinical hepatocellular carcinoma (HCC) models

Energy Technology Data Exchange (ETDEWEB)

Wang, Kaifeng, E-mail: kaifeng_wangdr@sina.com [Cancer center, the Affiliated Hospital of Hangzhou Normal University, Hangzhou (China); Fan, Yaohua [Oncology Department, No. 1 Hospital of Jiaxing, Zhejiang Province, Jiaxing (China); Chen, Gongying [Oncology Department, The Affiliated Hospital Hangzhou Normal University, Hangzhou (China); Wang, Zhengrong [Taizhou Hospital, Zhejiang Province, Taizhou (China); Kong, Dexin; Zhang, Peng [Oncology Department, Sir Run Run Shaw Hospital, Medical School, Zhejiang University, Hangzhou (China)

2016-05-27

The search for novel anti-hepatocellular carcinoma (HCC) agents is important. Mammalian target of rapamycin (mTOR) hyper-activation plays a pivotal role in promoting HCC tumorigenesis and chemoresistance. The current preclinical study evaluated the potential anti-HCC activity by a potent mTOR kinase inhibitor, WAY-600. We showed that WAY-600 inhibited survival and proliferation of HCC cell lines (HepG2 and Huh7) and primary human HCC cells. Caspase-dependent apoptosis was activated by WAY-600 in above HCC cells. Reversely, caspase inhibitors largely attenuated WAY-600's lethality against HCC cells. At the signaling level, WAY-600 blocked mTOR complex 1/2 (mTORC1/2) assemble and activation, yet activated MEK-ERK pathway in HCC cells. MEK-ERK inhibitors, PD-98059 and MEK-162, or MEK1/2 shRNA significantly potentiated WAY-600's cytotoxicity in HCC cells. Further studies showed that WAY-600 intraperitoneal (i.p.) administration in nude mice inhibited p-AKT Ser-473 and displayed significant anti-cancer activity against HepG2 xenografts. Remarkably, co-administration of MEK-162 further potentiated WAY-600's anti-HCC activity in vivo. These preclinical results demonstrate the potent anti-HCC activity by WAY-600, either alone or with MEK-ERK inhibitors. -- Highlights: •WAY-600 inhibits HCC cell survival and proliferation in vitro. •WAY-600 activates caspase-dependent apoptosis in HCC cells. •WAY-600 blocks mTORC1/2 activation, but activates MEK-ERK in HCC cells. •MEK-ERK inhibitors or MEK1/2 shRNA enhances WAY-600's cytotoxicity against HCC cells. •MEK-162 co-administration potentiates WAY-600-induced the anti-HepG2 tumor efficacy.

Hydrogen peroxide as a sustainable energy carrier: Electrocatalytic production of hydrogen peroxide and the fuel cell

International Nuclear Information System (INIS)

Fukuzumi, Shunichi; Yamada, Yusuke; Karlin, Kenneth D.

2012-01-01

This review describes homogeneous and heterogeneous catalytic reduction of dioxygen with metal complexes focusing on the catalytic two-electron reduction of dioxygen to produce hydrogen peroxide. Whether two-electron reduction of dioxygen to produce hydrogen peroxide or four-electron O 2 -reduction to produce water occurs depends on the types of metals and ligands that are utilized. Those factors controlling the two processes are discussed in terms of metal–oxygen intermediates involved in the catalysis. Metal complexes acting as catalysts for selective two-electron reduction of oxygen can be utilized as metal complex-modified electrodes in the electrocatalytic reduction to produce hydrogen peroxide. Hydrogen peroxide thus produced can be used as a fuel in a hydrogen peroxide fuel cell. A hydrogen peroxide fuel cell can be operated with a one-compartment structure without a membrane, which is certainly more promising for the development of low-cost fuel cells as compared with two compartment hydrogen fuel cells that require membranes. Hydrogen peroxide is regarded as an environmentally benign energy carrier because it can be produced by the electrocatalytic two-electron reduction of O 2 , which is abundant in air, using solar cells; the hydrogen peroxide thus produced could then be readily stored and then used as needed to generate electricity through the use of hydrogen peroxide fuel cells.

Hydrogen Peroxide as a Sustainable Energy Carrier: Electrocatalytic Production of Hydrogen Peroxide and the Fuel Cell.

Science.gov (United States)

Fukuzumi, Shunichi; Yamada, Yusuke; Karlin, Kenneth D

2012-11-01

This review describes homogeneous and heterogeneous catalytic reduction of dioxygen with metal complexes focusing on the catalytic two-electron reduction of dioxygen to produce hydrogen peroxide. Whether two-electron reduction of dioxygen to produce hydrogen peroxide or four-electron O 2 -reduction to produce water occurs depends on the types of metals and ligands that are utilized. Those factors controlling the two processes are discussed in terms of metal-oxygen intermediates involved in the catalysis. Metal complexes acting as catalysts for selective two-electron reduction of oxygen can be utilized as metal complex-modified electrodes in the electrocatalytic reduction to produce hydrogen peroxide. Hydrogen peroxide thus produced can be used as a fuel in a hydrogen peroxide fuel cell. A hydrogen peroxide fuel cell can be operated with a one-compartment structure without a membrane, which is certainly more promising for the development of low-cost fuel cells as compared with two compartment hydrogen fuel cells that require membranes. Hydrogen peroxide is regarded as an environmentally benign energy carrier because it can be produced by the electrocatalytic two-electron reduction of O 2 , which is abundant in air, using solar cells; the hydrogen peroxide thus produced could then be readily stored and then used as needed to generate electricity through the use of hydrogen peroxide fuel cells.

mTOR Inhibition Attenuates Dextran Sulfate Sodium-Induced Colitis by Suppressing T Cell Proliferation and Balancing TH1/TH17/Treg Profile.

Directory of Open Access Journals (Sweden)

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.

Systemic and Nonrenal Adverse Effects Occurring in Renal Transplant Patients Treated with mTOR Inhibitors

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Gianluigi Zaza

2013-01-01

Full Text Available The mammalian target of rapamycin inhibitors (mTOR-I, sirolimus and everolimus, are immunosuppressive drugs largely used in renal transplantation. The main mechanism of action of these drugs is the inhibition of the mammalian target of rapamycin (mTOR, a regulatory protein kinase involved in lymphocyte proliferation. Additionally, the inhibition of the crosstalk among mTORC1, mTORC2, and PI3K confers the antineoplastic activities of these drugs. Because of their specific pharmacological characteristics and their relative lack of nephrotoxicity, these inhibitors are valid option to calcineurine inhibitors (CNIs for maintenance immunosuppression in renal transplant recipients with chronic allograft nephropathy. However, as other immunosuppressive drugs, mTOR-I may induce the development of several adverse effects that need to be early recognized and treated to avoid severe illness in renal transplant patients. In particular, mTOR-I may induce systemic nonnephrological side effects including pulmonary toxicity, hematological disorders, dysmetabolism, lymphedema, stomatitis, cutaneous adverse effects, and fertility/gonadic toxicity. Although most of the adverse effects are dose related, it is extremely important for clinicians to early recognize them in order to reduce dosage or discontinue mTOR-I treatment avoiding the onset and development of severe clinical complications.

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

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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.

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

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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

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

International Nuclear Information System (INIS)

Fahrer, Joerg; Wagner, Silvia; Buerkle, Alexander; Koenigsrainer, Alfred

2009-01-01

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.

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.

Influence of pH on inhibition of Streptococcus mutans by Streptococcus oligofermentans.

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Liu, Ying; Chu, Lei; Wu, Fei; Guo, Lili; Li, Mengci; Wang, Yinghui; Wu, Ligeng

2014-02-01

Streptococcus oligofermentans is a novel strain of oral streptococcus that can specifically inhibit the growth of Streptococcus mutans. The aims of this study were to assess the growth of S. oligofermentans and the ability of S. oligofermentans to inhibit growth of Streptococcus mutans at different pH values. Growth inhibition was investigated in vitro using an interspecies competition assay. The 4-aminoantipyine method was used to measure the initial production rate and the total yield of hydrogen peroxide in S. oligofermentans. S. oligofermentans grew best at pH 7.0 and showed the most pronounced inhibitory effect when it was inoculated earlier than S. mutans. In terms of the total yield and the initial production rate of hydrogen peroxide by S. oligofermentans, the effects of the different culture pH values were as follows: pH 7.0 > 6.5 > 6.0 > 7.5 > 5.5 = 8.0 (i.e. there was no significant difference between pH 5.5 and pH 8.0). Environmental pH and the sequence of inoculation significantly affected the ability of S. oligofermentans to inhibit the growth of S. mutans. The degree of inhibition may be attributed to the amount of hydrogen peroxide produced. © 2013 Eur J Oral Sci.

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

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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

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

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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.

Androgen interacts with exercise through the mTOR pathway to induce skeletal muscle hypertrophy.

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Zeng, Fanxing; Zhao, Hua; Liao, Jingwen

2017-12-01

This study was designed to investigate the effects of exogenous androgen and resistance exercise on skeletal muscle hypertrophy and the role of the mammalian target of rapamycin (mTOR) signalling during the process. A total of 24 male Sprague-Dawley rats were randomly assigned to sham operation and dihydrotestosterone (DHT) implantation groups with subgroups subjected to sedentary conditions or resistance exercise (SHAM+SED, SHAM+EX, DHT+SED, and DHT+EX). The experimental procedure lasted for 10 days. The mRNA expression of androgen receptor (AR) and insulin-like growth factor I (IGF-I), the expression of myosin heavy chain (MHC), as well as the phosphorylation statuses of AR, mTOR, p70 ribosomal S6 kinase (p70 S6K ), and eukaryotic translation initiation factor 4E-binding protein 1 (4EBP1) were determined in the white gastrocnemius muscle. The cross sectional area and wet mass of the muscle were also measured. The cross sectional area and MHC expression were significantly higher in SHAM+EX, DHT+SED, and DHT+EX than in SHAM+SED. There was no significant difference among groups in muscle mass. The mRNA expression of AR and IGF-I and the phosphorylation of mTOR, p70 S6K , and 4EBP1 were significantly increased in DHT+SED and SHAM+EX and were significantly enhanced in DHT+EX compared with either DHT or exercise alone. These data show that DHT causes hypertrophy in skeletal muscle and that exercise has a synergistic effect on DHT-induced hypertrophy. Exercise enhances androgen-induced rapid anabolic action, which involves activation of the mTOR pathway.

Lipid peroxidation regulates podocyte migration and cytoskeletal structure through redox sensitive RhoA signaling

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Claudia Kruger

2018-06-01

Full Text Available Early podocyte loss is characteristic of chronic kidney diseases (CKD in obesity and diabetes. Since treatments for hyperglycemia and hypertension do not prevent podocyte loss, there must be additional factors causing podocyte depletion. The role of oxidative stress has been implicated in CKD but it is not known how exactly free radicals affect podocyte physiology. To assess this relationship, we investigated the effects of lipid radicals on podocytes, as lipid peroxidation is a major form of oxidative stress in diabetes. We found that lipid radicals govern changes in podocyte homeostasis through redox sensitive RhoA signaling: lipid radicals inhibit migration and cause loss of F-actin fibers. These effects were prevented by mutating the redox sensitive cysteines of RhoA. We therefore suggest that in diseases associated with increased lipid peroxidation, lipid radicals can determine podocyte function with potentially pathogenic consequences for kidney physiology. Keywords: Lipid peroxidation, Reactive lipids, Podocyte, RhoA, Cysteine, Chronic kidney disease

Enhanced Peroxide Resistance of In Vitro Mutagenized Fluorideresistant Klebsiella pneumoniae Ureases for Catalytic Buffering of Agent Decontamination Reactions

National Research Council Canada - National Science Library

Fry, Ilona J; DeFrank, Joseph J

2004-01-01

.... Fluoride-resistant (FR) ureases developed previously in our laboratory were >95% inhibited by 1% hydrogen peroxide. To overcome this problem, the FR mutant urease structural genes of Klebsiella pneumoniae were mutagenized in vitro in an E...

A study of the relative importance of the peroxiredoxin-, catalase-, and glutathione-dependent systems in neural peroxide metabolism.

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Mitozo, Péricles Arruda; de Souza, Luiz Felipe; Loch-Neckel, Gecioni; Flesch, Samira; Maris, Angelica Francesca; Figueiredo, Cláudia Pinto; Dos Santos, Adair Roberto Soares; Farina, Marcelo; Dafre, Alcir Luiz

2011-07-01

Cells are endowed with several overlapping peroxide-degrading systems whose relative importance is a matter of debate. In this study, three different sources of neural cells (rat hippocampal slices, rat C6 glioma cells, and mouse N2a neuroblastoma cells) were used as models to understand the relative contributions of individual peroxide-degrading systems. After a pretreatment (30 min) with specific inhibitors, each system was challenged with either H₂O₂ or cumene hydroperoxide (CuOOH), both at 100 μM. Hippocampal slices, C6 cells, and N2a cells showed a decrease in the H₂O₂ decomposition rate (23-28%) by a pretreatment with the catalase inhibitor aminotriazole. The inhibition of glutathione reductase (GR) by BCNU (1,3-bis(2-chloroethyl)-1-nitrosourea) significantly decreased H₂O₂ and CuOOH decomposition rates (31-77%). Inhibition of catalase was not as effective as BCNU at decreasing cell viability (MTT assay) and cell permeability or at increasing DNA damage (comet test). Impairing the thioredoxin (Trx)-dependent peroxiredoxin (Prx) recycling by thioredoxin reductase (TrxR) inhibition with auranofin neither potentiated peroxide toxicity nor decreased the peroxide-decomposition rate. The results indicate that neural peroxidatic systems depending on Trx/TrxR for recycling are not as important as those depending on GSH/GR. Dimer formation, which leads to Prx2 inactivation, was observed in hippocampal slices and N2a cells treated with H₂O₂, but not in C6 cells. However, Prx-SO₃ formation, another form of Prx inactivation, was observed in all neural cell types tested, indicating that redox-mediated signaling pathways can be modulated in neural cells. These differences in Prx2 dimerization suggest specific redox regulation mechanisms in glia-derived (C6) compared to neuron-derived (N2a) cells and hippocampal slices. Copyright © 2011 Elsevier Inc. All rights reserved.

Fisetin Suppresses Lipid Accumulation in Mouse Adipocytic 3T3-L1 Cells by Repressing GLUT4-Mediated Glucose Uptake through Inhibition of mTOR-C/EBPα Signaling.

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Watanabe, Marina; Hisatake, Mitsuhiro; Fujimori, Ko

2015-05-27

3,7,3',4'-Tetrahydroxyflavone (fisetin) is a flavonoid found in vegetables and fruits having broad biological activities. Here the effects of fisetin on adipogenesis and its regulatory mechanism in mouse adipocytic 3T3-L1 cells are studied. Fisetin inhibited the accumulation of intracellular lipids and lowered the expression of adipogenic genes such as peroxisome proliferator-activated receptor γ and CCAAT/enhancer-binding protein (C/EBP) α and fatty acid-binding protein 4 (aP2) during adipogenesis. Moreover, the mRNA levels of genes such as acetyl-CoA carboxylase, fatty acid synthase, and stearoyl-CoA desaturase involved in the fatty acid biosynthesis (lipogenesis) were reduced by the treatment with fisetin. The expression level of the glucose transporter 4 (GLUT4) gene was also decreased by fisetin, resulting in down-regulation of glucose uptake. Furthermore, fisetin inhibited the phosphorylation of the mammalian target of rapamycin (mTOR) and that of p70 ribosomal S6 kinase, a target of the mTOR complex, the inhibition of which was followed by a decreased mRNA level of the C/EBPα gene. The results obtained from a chromatin immunoprecipitation assay demonstrated that the ability of C/EBPα to bind to the GLUT4 gene promoter was reduced by the treatment with fisetin, which agreed well with those obtained when 3T3-L1 cells were allowed to differentiate into adipocytes in medium in the presence of rapamycin, an inhibitor for mTOR. These results indicate that fisetin suppressed the accumulation of intracellular lipids by inhibiting GLUT4-mediated glucose uptake through inhibition of the mTOR-C/EBPα signaling in 3T3-L1 cells.

[Inhibition of Linseed Oil Autooxidation by Essential Oils and Extracts from Spice Plants].

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Misharina, T A; Alinkina, E S; Terenina, M B; Krikunova, N I; Kiseleva, V I; Medvedeva, I B; Semenova, M G

2015-01-01

Clove bud essential oil, extracts from ginger, pimento and black pepper, or ascorbyl palmytate were studied as natural antioxidants for the inhibition of autooxidation of polyunsaturated fatty acids in linseed oil. Different methods were used to estimate antioxidant efficiency. These methods are based on the following parameters: peroxide values; peroxide concentration; content of degradation products of unsaturated fatty acid peroxides, which acted with thiobarbituric acid; diene conjugate content; the content of volatile compounds that formed as products of unsaturated fatty acid peroxide degradation; and the composition of methyl esters of fatty acids in samples of oxidized linseed oil.

Detection of hydrogen peroxide with graphyne

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Majidi, R.; Karami, A. R.

2013-12-01

The effect of hydrogen peroxide on the electronic properties of graphyne has been investigated to explore the possibility of using graphyne based biosensor. We have used density functional theory to study the electronic properties of γ-graphyne in the presence of different number of hydrogen peroxide. The optimal adsorption position, orientation, and distance of hydrogen peroxide adsorbed on the graphyne sheet have been determined by calculating adsorption energy. It is found that γ-graphyne which is an intrinsic semiconductor becomes an n-type semiconductor due to the presence of hydrogen peroxide. The energy band gap of γ-graphyne is decreased by increasing the number of hydrogen peroxide. The results demonstrate that γ-graphyne is a promising candidate for biosensor application because of its electrical sensitivity to hydrogen peroxide.

The Effects of Krill Oil on mTOR Signaling and Resistance Exercise: A Pilot Study

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John Georges

2018-01-01

Full Text Available Introduction. Krill oil supplementation has been shown to improve postexercise immune function; however, its effect on muscle hypertrophy is currently unknown. Therefore, the aim of present study was to investigate the ability of krill oil to stimulate mTOR signaling and its ability to augment resistance training-induced changes in body composition and performance. Methods. C2C12 myoblasts cells were stimulated with krill oil or soy-derived phosphatidylcholine (S-PC, and then, the ratio of P-p70-389 to total p70 was used as readout for mTOR signaling. In double-blind,placebo-controlled study, resistance trained subjects consumed either 3 g krill oil daily or placebo, and each took part in an 8-week periodized resistance training program. Body composition, maximal strength, peak power, and rate of perceived recovery were assessed collectively at the end of weeks 0 and 8. In addition, safety parameters (comprehensive metabolic panel (CMP, complete blood count (CBC, and urine analysis (UA and cognitive performance were measured pre- and posttesting. Results. Krill oil significantly stimulated mTOR signaling in comparison to S-PC and control. No differences for markers on the CMP, CBC, or UA were observed. Krill oil significantly increased lean body mass from baseline (p=0.021, 1.4 kg, +2.1%; however, there were no statistically significant differences between groups for any measures taken. Conclusion. Krill oil activates mTOR signaling. Krill oil supplementation in athletes is safe, and its effect on resistance exercise deserves further research.

Evaluation of the toxicity and efficacy of hydrogen peroxide treatments on eggs of warm and cool water fishes

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Rach, J.J.; Gaikowski, M.P.; Howe, G.E.; Schreier, Theresa M.

1998-01-01

The use of hydrogen peroxide in aquaculture is growing and there is a need to develop fundamental guidelines to effectively treat diseased fish. The safety (toxicity) of hydrogen peroxide treatments was determined on eggs of representative warm- and coolwater fish species. Eggs of northern pike (Esox lucius), walleye (Stizostedion vitreum), yellow perch (Pel ca flavescens), white sucker (Catostomus commersoni), lake sturgeon (Acipenser fulvescens), paddlefish (Polyodon spathula), common carp (Cyprinus carpio), and channel catfish (Ictalurus punctatus) were cultured in egg jars or aquaria. Treatments were initiated with non-eyed eggs and continued until all viable eggs had hatched. Eggs were treated daily for 15 min Monday through Friday with either 0, 500, 1000, 3000, or 6000 mu l l(-1) of hydrogen peroxide. For all species, the mean percent hatch was greater in eggs treated with 1000 mu l l(-1) hydrogen peroxide for 15 min than in the untreated controls. Common carp, lake sturgeon, and paddlefish were the least sensitive to hydrogen peroxide with percent hatch ranging from 40 to 48% in the 6000 mu l l(-1) hydrogen peroxide treatment. Fungal infections reduced or eliminated the hatch in most controls whereas nearly all treated eggs remained free of infection; hydrogen peroxide inhibited fungal infections on fish eggs. (C) 1998 Elsevier Science B.V. All rights reserved.

Metformin combined with aspirin significantly inhibit pancreatic cancer cell growth in vitro and in vivo by suppressing anti-apoptotic proteins Mcl-1 and Bcl-2

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Yue, Wen; Zheng, Xi; Lin, Yong; Yang, Chung S.; Xu, Qing; Carpizo, Darren; Huang, Huarong; DiPaola, Robert S.; Tan, Xiang-Lin

2015-01-01

Metformin and aspirin have been studied extensively as cancer preventive or therapeutic agents. However, the effects of their combination on pancreatic cancer cells have not been investigated. Herein, we evaluated the effects of metformin and aspirin, alone or in combination, on cell viability, migration, and apoptosis as well as the molecular changes in mTOR, STAT3 and apoptotic signaling pathways in PANC-1 and BxPC3 cells. Metformin and aspirin, at relatively low concentrations, demonstrated synergistically inhibitory effects on cell viability. Compared to the untreated control or individual drug, the combination of metformin and aspirin significantly inhibited cell migration and colony formation of both PANC-1 and BxPC-3 cells. Metformin combined with aspirin significantly inhibited the phosphorylation of mTOR and STAT3, and induced apoptosis as measured by caspase-3 and PARP cleavage. Remarkably, metformin combined with aspirin significantly downregulated the anti-apoptotic proteins Mcl-1 and Bcl-2, and upregulated the pro-apoptotic proteins Bim and Puma, as well as interrupted their interactions. The downregulation of Mcl-1 and Bcl-2 was independent of AMPK or STAT3 pathway but partially through mTOR signaling and proteasome degradation. In a PANC-1 xenograft mouse model, we demonstrated that the combination of metformin and aspirin significantly inhibited tumor growth and downregulated the protein expression of Mcl-1 and Bcl-2 in tumors. Taken together, the combination of metformin and aspirin significantly inhibited pancreatic cancer cell growth in vitro and in vivo by regulating the pro- and anti-apoptotic Bcl-2 family members, supporting the continued investigation of this two drug combination as chemopreventive or chemotherapeutic agents for pancreatic cancer. PMID:26056043

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

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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.

Protein S-glutathionylation lowers superoxide/hydrogen peroxide release from skeletal muscle mitochondria through modification of complex I and inhibition of pyruvate uptake.

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Robert M Gill

Full Text Available Protein S-glutathionylation is a reversible redox modification that regulates mitochondrial metabolism and reactive oxygen species (ROS production in liver and cardiac tissue. However, whether or not it controls ROS release from skeletal muscle mitochondria has not been explored. In the present study, we examined if chemically-induced protein S-glutathionylation could alter superoxide (O2●-/hydrogen peroxide (H2O2 release from isolated muscle mitochondria. Disulfiram, a powerful chemical S-glutathionylation catalyst, was used to S-glutathionylate mitochondrial proteins and ascertain if it can alter ROS production. It was found that O2●-/H2O2 release rates from permeabilized muscle mitochondria decreased with increasing doses of disulfiram (100-500 μM. This effect was highest in mitochondria oxidizing succinate or palmitoyl-carnitine, where a ~80-90% decrease in the rate of ROS release was observed. Similar effects were detected in intact mitochondria respiring under state 4 conditions. Incubation of disulfiram-treated mitochondria with DTT (2 mM restored ROS release confirming that these effects were associated with protein S-glutathionylation. Disulfiram treatment also inhibited phosphorylating and proton leak-dependent respiration. Radiolabelled substrate uptake experiments demonstrated that disulfiram inhibited pyruvate import but had no effect on carnitine uptake. Immunoblot analysis of complex I revealed that it contained several protein S-glutathionylation targets including NDUSF1, a subunit required for NADH oxidation. Taken together, these results demonstrate that O2●-/H2O2 release from muscle mitochondria can be altered by protein S-glutathionylation. We attribute these changes to the protein S-glutathionylation complex I and inhibition of mitochondrial pyruvate carrier.

Role of hydrogen peroxide during the interaction between the hemibiotrophic fungal pathogen Septoria tritici and wheat

NARCIS (Netherlands)

Shetty, N.P.; Mehrabi, R.; Lütken, H.; Haldrup, A.; Kema, G.H.J.

2007-01-01

Hydrogen peroxide (H2O2) is reported to inhibit biotrophic but benefit necrotrophic pathogens. Infection by necrotrophs can result in a massive accumulation of H2O2 in hosts. Little is known of how pathogens with both growth types are affected (hemibiotrophs). The hemibiotroph, Septoria tritici,

Inositol Hexaphosphate Inhibits Proliferation and Induces Apoptosis of Colon Cancer Cells by Suppressing the AKT/mTOR Signaling Pathway

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Małgorzata Kapral

2017-10-01

Full Text Available Abstract: AKT, a serine/threonine protein kinase and mammalian target of rapamycin (mTOR plays a critical role in the proliferation and resistance to apoptosis that are essential to the development and progression of colon cancer. Therefore, AKT/mTOR signaling pathway has been recognized as an attractive target for anticancer therapy. Inositol hexaphosphate (InsP6, a natural occurring phytochemical, has been shown to have both preventive and therapeutic effects against various cancers, however, its exact molecular mechanisms of action are not fully understood. The aim of the in vitro study was to investigate the anticancer activity of InsP6 on colon cancer with the focus on inhibiting the AKT1 kinase and p70S6K1 as mTOR effector, in relation to proliferation and apoptosis of cells. The colon cancer Caco-2 cells were cultured using standard techniques and exposed to InsP6 at different concentrations (1 mM, 2.5 mM and 5 mM. Cellular proliferative activity was monitored by 5-bromo-2′-deoxyuridine (BrdU incorporation into cellular DNA. Flow cytometric analysis was performed for cell cycle progression and apoptosis studies. Real-time RT-qPCR was used to validate mRNA levels of CDNK1A, CDNK1B, CASP3, CASP9, AKT1 and S6K1 genes. The concentration of p21 protein as well as the activities of caspase 3, AKT1 and p70S6K1 were determined by the ELISA method. The results revealed that IP6 inhibited proliferation and stimulated apoptosis of colon cancer cells. This effect was mediated by an increase in the expression of genes encoding p21, p27, caspase 3, caspase 9 as well a decrease in transcription of AKT1 and S6K1. InsP6 suppressed phosphorylation of AKT1 and p70S6K1, downstream effector of mTOR. Based on these studies it may be concluded that InsP6 can reduce proliferation and induce apoptosis through inhibition of the AKT/mTOR pathway and mTOR effector followed by modulation of the expression and activity of several key components of these pathways in

Antioxidant effects of crude extracts from Baccharis species: inhibition of myeloperoxidase activity, protection against lipid peroxidation, and action as oxidative species scavenger

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Tiago O. Vieira

2011-05-01

Full Text Available The objective of this study was to show a comparison of the antioxidant properties of aqueous and ethanolic extracts obtained from Baccharis articulata (Lam. Pers., Baccharis trimera (Less. DC., Baccharis spicata (Lam. Baill. and Baccharis usterii Heering, Asteraceae, by several techniques covering a range of oxidant species and of biotargets. We have investigated the ability of the plant extracts to scavenge DPPH (1,1-diphenyl-2-picryl-hydrazyl free radical, action against lipid peroxidation of membranes including rat liver microsomes and soy bean phosphatidylcholine liposomes by ascorbyl radical and peroxynitrite. Hydroxyl radical scavenger activity was measured monitoring the deoxyribose oxidation. The hypochlorous acid scavenger activity was also evaluated by the prevention of protein carbonylation and finally the myeloperoxidase (MPO activity inhibition. The results obtained suggest that the Baccharis extracts studied present a significant antioxidant activity scavenging free radicals and protecting biomolecules from the oxidation. We can suggest that the supposed therapeutic efficacy of this plant could be due, in part, to these properties.

Problem of the lithium peroxide thermal stability

International Nuclear Information System (INIS)

Nefedov, R A; Ferapontov, Yu A; Kozlova, N P

2016-01-01

The behavior of lithium peroxide and lithium peroxide monohydrate samples under heating in atmospheric air was studied by the method of thermogravimetric analysis (TGA) and differential thermal analysis (DTA). It was found that in the temperature range of 32°C to 82°C the interaction of lithium peroxides and steam with the formation of lithium peroxide monohydrate occurs, which was confirmed chemically and by X-ray Single-qualitative analysis. It was experimentally found that lithium peroxide starts to decompose into the lithium oxide and oxygen in the temperature range of 340 ÷ 348°C. It was established that the resulting thermal decomposition of lithium oxide, lithium peroxide at the temperature of 422°C melts with lithium carbonate eutecticly. The manifestation of polymorphism was not marked(seen or noticed) under the heating of studied samples of lithium peroxide and lithium peroxide monohydrate in the temperature range of 25°C ÷ 34°C. (paper)

FBXW7 mutations in patients with advanced cancers: clinical and molecular characteristics and outcomes with mTOR inhibitors.

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Denis L Jardim

Full Text Available FBXW7 is a tumor suppressor gene responsible for the degradation of several proto-oncogenes. Preclinical data suggest that FBXW7 mutations sensitize cells to mTOR inhibitors. Clinicopathologic characteristics of cancer patients with FBXW7 mutations and their responses to mTOR inhibitors remain unknown.Using multiplex gene panels we evaluated how the FBXW7 mutation affected the cancer phenotype of patients referred to a phase I clinic starting January 2012. Whenever possible patients positive for FBXW7 mutation were treated with regimens containing an mTOR inhibitors and their outcomes were reviewed.FBXW7 mutations were detected in 17 of 418 patients (4.0%. Among tumor types with more than 10 patients tested, FBXW7 mutations occurred in colorectal cancer (7/49; 14.3%, squamous cell cancer of head and neck (2/18; 11.1%, liver (1/13; 7.7%, and ovarian cancers (1/40; 2.5%. No one clinical, pathological or demographic feature was characteristic of the FBXW7-mutated patient population. The mutation occurred in isolation in only 2/17 (12% patients, and KRAS was frequently found as a concomitant mutation, especially in patients with colorectal cancer (6/7; 86%. Ten patients were treated on a protocol containing an mTOR inhibitor, with a median time to treatment failure of 2.8 months (range, 1.3-6.8. One patient with liver cancer (fibrolamellar subtype continues to have a prolonged stable disease for 6.8+ months.In patients with advanced cancers, somatic mutations in FBXW7 usually occur with other simultaneous molecular aberrations, which can contribute to limited therapeutic efficacy of mTOR inhibitors.

The dual mTORC1 and mTORC2 inhibitor AZD8055 inhibits head and neck squamous cell carcinoma cell growth in vivo and in vitro

Energy Technology Data Exchange (ETDEWEB)

Li, Qiang; Song, Xin-mao; Ji, Yang-yang; Jiang, Hui; Xu, Lin-gen, E-mail: drlingenxu@126.com

2013-11-01

Highlights: •AZD8055 induces significant cytotoxic effects in cultured HNSCC cells. •AZD8055 blocks mTORC1 and mTORC2 activation in cultured HNSCC cells. •JNK activation is required for AZD8055-induced HNSCC cell death. •AZD8055 inhibits Hep-2 cell growth in vivo, and was more efficient than rapamycin. -- Abstract: The serine/threonine kinase mammalian target of rapamycin (mTOR) promotes cell survival and proliferation, and is constitutively activated in head and neck squamous cell carcinoma (HNSCC). Thus mTOR is an important target for drug development in this disease. Here we tested the anti-tumor ability of AZD8055, the novel mTOR inhibitor, in HNSCC cells. AZD8055 induced dramatic cell death of HNSCC lines (Hep-2 and SCC-9) through autophagy. AZD8055 blocked both mTOR complex (mTORC) 1 and mTORC2 activation without affecting Erk in cultured HNSCC cells. Meanwhile, AZD8055 induced significant c-Jun N-terminal kinase (JNK) activation, which was also required for cancer cell death. JNK inhibition by its inhibitors (SP 600125 and JNK-IN-8), or by RNA interference (RNAi) alleviated AZD8055-induced cell death. Finally, AZD8055 markedly increased the survival of Hep-2 transplanted mice through a significant reduction of tumor growth, without apparent toxicity, and its anti-tumor ability was more potent than rapamycin. Meanwhile, AZD8055 administration activated JNK while blocking mTORC1/2 in Hep-2 tumor engrafts. Our current results strongly suggest that AZD8055 may be further investigated for HNSCC treatment in clinical trials.

Ameliorating effects of genestein: Study on mice liver glutathione and lipid peroxidation after irradiation

International Nuclear Information System (INIS)

Gaur, A.

2010-01-01

Genistein is a soya isoflavone, which is found naturally in legumes. such as soybeans and chickpeas. Radiation-induced free radicals in turn impair the antioxidative defense mechanism, leading to an increased membrane lipid peroxidation that results in damage of the membrane bound enzyme and may lead to damage or death of cell. Hence, the lipid peroxidation is a good biomarker of damage occurs due to radiation and the inhibition of lipid peroxidation is suggestive of radioprotective action. Glutathione has been shown to protect cells against oxidative stress by reacting with peroxides and hydroperoxides and determines the inherent radiosensitivity of cells. Materials and Methods: For experimentation, healthy Swiss Albino male mice of 6-8 weeks old were selected from inbred colony. Genistein was dissolved in dimethyl sulfoxide and then prepared different concentration solutions so that the volume administered intraperitoneally was 0.5 ml. Lipid peroxidation was estimated by the method of Ohkawa and GSH was estimated by the method of Moron. Results: The intraperitoneal administration of optimum dose (200 mg/kg body weight) of Genistein before 24 hours and 15 minutes of irradiation (8 Gy at a dose rate of 1.02 Gy/min)reverted the increase in lipid peroxidation (by 18.01% ± 3.05) and decrease of Glutathione (by 62.05%±21.58) caused by irradiation in liver of Swiss albino mice. Statistically analyzed survival data produced a dose reduction factor = 1.24. Conclusion: The results indicate that Genistein against radiation effect may pave way to the formulation of medicine in radiotherapy for normal tissue and possible against radiomimetic drug induced toxicity.

Antitumor and antimicrobial activities and inhibition of in-vitro lipid ...

African Journals Online (AJOL)

The antitumor activity was measured in DLA cell line induced mice. Inhibition of in vitro lipid peroxidation activity of the D. nobile in both liver homogenate and RBC ghosts was also carried out. The aqueous extracts of stem and flower of D. nobile showed better zone of bacterial inhibition than that of ethanol and chloroform

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.

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

International Nuclear Information System (INIS)

Zhang, Zi-wei; Guo, Rui-wei; Lv, Jin-lin; Wang, Xian-mei; Ye, Jin-shan; Lu, Ni-hong; Liang, Xing; Yang, Li-xia

2017-01-01

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. - Highlights: • Suggesting a new mechanism of insulin-triggered VSMC functions. • Providing a new therapeutic strategies that target atherosclerosis in T2DM patients. • Providing a new strategies that target in-stent restenosis in T2DM patients.

Quality control of photosystem II: lipid peroxidation accelerates photoinhibition under excessive illumination.

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Tiffanie Chan

Full Text Available Environmental stresses lower the efficiency of photosynthesis and sometimes cause irreversible damage to plant functions. When spinach thylakoids and Photosystem II membranes were illuminated with excessive visible light (100-1,000 µmol photons m(-1 s(-1 for 10 min at either 20°C or 30°C, the optimum quantum yield of Photosystem II decreased as the light intensity and temperature increased. Reactive oxygen species and endogenous cationic radicals produced through a photochemical reaction at and/or near the reaction center have been implicated in the damage to the D1 protein. Here we present evidence that lipid peroxidation induced by the illumination is involved in the damage to the D1 protein and the subunits of the light-harvesting complex of Photosystem II. This is reasoned from the results that considerable lipid peroxidation occurred in the thylakoids in the light, and that lipoxygenase externally added in the dark induced inhibition of Photosystem II activity in the thylakoids, production of singlet oxygen, which was monitored by electron paramagnetic resonance spin trapping, and damage to the D1 protein, in parallel with lipid peroxidation. Modification of the subunits of the light-harvesting complex of Photosystem II by malondialdehyde as well as oxidation of the subunits was also observed. We suggest that mainly singlet oxygen formed through lipid peroxidation under light stress participates in damaging the Photosystem II subunits.

Thiol peroxidases mediate specific genome-wide regulation of gene expression in response to hydrogen peroxide

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Fomenko, Dmitri E.; Koc, Ahmet; Agisheva, Natalia; Jacobsen, Michael; Kaya, Alaattin; Malinouski, Mikalai; Rutherford, Julian C.; Siu, Kam-Leung; Jin, Dong-Yan; Winge, Dennis R.; Gladyshev, Vadim N.

2011-01-01

Hydrogen peroxide is thought to regulate cellular processes by direct oxidation of numerous cellular proteins, whereas antioxidants, most notably thiol peroxidases, are thought to reduce peroxides and inhibit H2O2 response. However, thiol peroxidases have also been implicated in activation of transcription factors and signaling. It remains unclear if these enzymes stimulate or inhibit redox regulation and whether this regulation is widespread or limited to a few cellular components. Herein, we found that Saccharomyces cerevisiae cells lacking all eight thiol peroxidases were viable and withstood redox stresses. They transcriptionally responded to various redox treatments, but were unable to activate and repress gene expression in response to H2O2. Further studies involving redox transcription factors suggested that thiol peroxidases are major regulators of global gene expression in response to H2O2. The data suggest that thiol peroxidases sense and transfer oxidative signals to the signaling proteins and regulate transcription, whereas a direct interaction between H2O2 and other cellular proteins plays a secondary role. PMID:21282621

Plasticity and mTOR: Towards Restoration of Impaired Synaptic Plasticity in mTOR-Related Neurogenetic Disorders

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Tanjala T. Gipson

2012-01-01

Full Text Available Objective. To review the recent literature on the clinical features, genetic mutations, neurobiology associated with dysregulation of mTOR (mammalian target of rapamycin, and clinical trials for tuberous sclerosis complex (TSC, neurofibromatosis-1 (NF1 and fragile X syndrome (FXS, and phosphatase and tensin homolog hamartoma syndromes (PTHS, which are neurogenetic disorders associated with abnormalities in synaptic plasticity and mTOR signaling. Methods. Pubmed and Clinicaltrials.gov were searched using specific search strategies. Results/Conclusions. Although traditionally thought of as irreversible disorders, significant scientific progress has been made in both humans and preclinical models to understand how pathologic features of these neurogenetic disorders can be reduced or reversed. This paper revealed significant similarities among the conditions. Not only do they share features of impaired synaptic plasticity and dysregulation of mTOR, but they also share clinical features—autism, intellectual disability, cutaneous lesions, and tumors. Although scientific advances towards discovery of effective treatment in some disorders have outpaced others, progress in understanding the signaling pathways that connect the entire group indicates that the lesser known disorders will become treatable as well.

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.

Spectroscopic studies of europium-tetracyclines complexes and their applications in detection of hydrogen peroxide and urea peroxide

International Nuclear Information System (INIS)

Grasso, Andrea Nastri

2010-01-01

In this work were studied the spectroscopic properties of trivalent europium ion complexed with components of tetracycline family, chlorotetracycline, oxytetracycline and metacycline, in the presence of hydrogen peroxide and urea peroxide. Optical parameters were obtained such as absorption, emission, lifetime and calibration curves were constructed for luminescence spectra. Experiments were carried out with both inorganic compounds and europium-tetracyclines complexes in order to verify possible interferences. Studies for glucose determination were also described using europium-tetracyclines complexes as biosensors. Results show that europium tetracyclines complexes emit a narrow band in the visible region and, in the presence of hydrogen peroxide or urea peroxide there is a greater enhancement in their luminescence and lifetime. Thus, europium-tetracyclines complexes studied can be used as biosensors for hydrogen and urea peroxides determination as a low cost and room temperature method. An indirect method for glucose determination was studied by adding glucose oxidase enzyme in europium-tetracyclines complex in the presence of glucose promoting as product hydrogen peroxide. (author)

Myostatin inhibits eEF2K-eEF2 by regulating AMPK to suppress protein synthesis.

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Deng, Zhao; Luo, Pei; Lai, Wen; Song, Tongxing; Peng, Jian; Wei, Hong-Kui

2017-12-09

Growth of skeletal muscle is dependent on the protein synthesis, and the rate of protein synthesis is mainly regulated in the stage of translation initiation and elongation. Myostatin, a member of the transforming growth factor-β (TGF-β) superfamily, is a negative regulator of protein synthesis. C2C12 myotubes was incubated with 0, 0.01, 0.1, 1, 2, 3 μg/mL myostatin recombinant protein, and then we detected the rates of protein synthesis by the method of SUnSET. We found that high concentrations of myostatin (2 and 3 μg/mL) inhibited protein synthesis by blocking mTOR and eEF2K-eEF2 pathway, while low concentration of myostatin (0.01, 0.1 and 1 μg/mL) regulated eEF2K-eEF2 pathway activity to block protein synthesis without affected mTOR pathway, and myostatin inhibited eEF2K-eEF2 pathway through regulating AMPK pathway to suppress protein synthesis. It provided a new mechanism for myostatin regulating protein synthesis and treating muscle atrophy. Copyright © 2017. Published by Elsevier Inc.

21 CFR 529.1150 - Hydrogen peroxide.

Science.gov (United States)

2010-04-01

... 21 Food and Drugs 6 2010-04-01 2010-04-01 false Hydrogen peroxide. 529.1150 Section 529.1150 Food... DRUGS, FEEDS, AND RELATED PRODUCTS CERTAIN OTHER DOSAGE FORM NEW ANIMAL DRUGS § 529.1150 Hydrogen peroxide. (a) Specifications. Each milliliter of solution contains 396.1 milligrams (mg) hydrogen peroxide...

On the Mechanism of Cytoprotection by Ferrostatin-1 and Liproxstatin-1 and the Role of Lipid Peroxidation in Ferroptotic Cell Death.

Science.gov (United States)

Zilka, Omkar; Shah, Ron; Li, Bo; Friedmann Angeli, José Pedro; Griesser, Markus; Conrad, Marcus; Pratt, Derek A

2017-03-22

Ferroptosis is a form of regulated necrosis associated with the iron-dependent accumulation of lipid hydroperoxides that may play a key role in the pathogenesis of degenerative diseases in which lipid peroxidation has been implicated. High-throughput screening efforts have identified ferrostatin-1 (Fer-1) and liproxstatin-1 (Lip-1) as potent inhibitors of ferroptosis - an activity that has been ascribed to their ability to slow the accumulation of lipid hydroperoxides. Herein we demonstrate that this activity likely derives from their reactivity as radical-trapping antioxidants (RTAs) rather than their potency as inhibitors of lipoxygenases. Although inhibited autoxidations of styrene revealed that Fer-1 and Lip-1 react roughly 10-fold more slowly with peroxyl radicals than reactions of α-tocopherol (α-TOH), they were significantly more reactive than α-TOH in phosphatidylcholine lipid bilayers - consistent with the greater potency of Fer-1 and Lip-1 relative to α-TOH as inhibitors of ferroptosis. None of Fer-1, Lip-1, and α-TOH inhibited human 15-lipoxygenase-1 (15-LOX-1) overexpressed in HEK-293 cells when assayed at concentrations where they inhibited ferroptosis. These results stand in stark contrast to those obtained with a known 15-LOX-1 inhibitor (PD146176), which was able to inhibit the enzyme at concentrations where it was effective in inhibiting ferroptosis. Given the likelihood that Fer-1 and Lip-1 subvert ferroptosis by inhibiting lipid peroxidation as RTAs, we evaluated the antiferroptotic potential of 1,8-tetrahydronaphthyridinols (hereafter THNs): rationally designed radical-trapping antioxidants of unparalleled reactivity. We show for the first time that the inherent reactivity of the THNs translates to cell culture, where lipophilic THNs were similarly effective to Fer-1 and Lip-1 at subverting ferroptosis induced by either pharmacological or genetic inhibition of the hydroperoxide-detoxifying enzyme Gpx4 in mouse fibroblasts, and glutamate

Lycopene protects human SH-SY5Y neuroblastoma cells against hydrogen peroxide-induced death via inhibition of oxidative stress and mitochondria-associated apoptotic pathways

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FENG, CHUNSHENG; LUO, TIANFEI; ZHANG, SHUYAN; LIU, KAI; ZHANG, YANHONG; LUO, YINAN; GE, PENGFEI

2016-01-01

Oxidative stress, which is characterized by excessive production of reactive oxygen species (ROS), is a common pathway that results in neuronal injury or death due to various types of pathological stress. Although lycopene has been identified as a potent antioxidant, its effect on hydrogen peroxide (H2O2)-induced neuronal damage remains unclear. In the present study, pretreatment with lycopene was observed to protect SH-SY5Y neuroblastoma cells against H2O2-induced death via inhibition of apoptosis resulting from activation of caspase-3 and translocation of apoptosis inducing factor (AIF) to the nucleus. Furthermore, the over-produced ROS, as well as the reduced activities of anti-oxidative enzymes, superoxide dismutase and catalase, were demonstrated to be alleviated by lycopene. Additionally, lycopene counteracted H2O2-induced mitochondrial dysfunction, which was evidenced by suppression of mitochondrial permeability transition pore opening, attenuation of the decline of the mitochondrial membrane potential, and inhibition of the increase of Bax and decrease of Bcl-2 levels within the mitochondria. The release of cytochrome c and AIF from the mitochondria was also reduced. These results indicate that lycopene is a potent neuroprotectant against apoptosis, oxidative stress and mitochondrial dysfunction, and could be administered to prevent neuronal injury or death. PMID:27035331

Glutathione delays varies as-tocopherol oxidation and subsequent lipid peroxidation in rat liver microsomes

International Nuclear Information System (INIS)

Robey, S.; Mavis, R.

1986-01-01

A method has been developed for in vitro trace radiolabeling of rat liver microsomes with 3 H-α-tocopherol (αT*) which allows virtually complete oxidation of the αT* under oxidizing conditions. The supernatant of a 16,000 xg centrifugation of homogenized rat liver, containing the cytosolic rat liver vitamin E (VE) transfer protein, was incubated with an ethanolic solution of αT* for 10 minutes at 37 0 C. Labeled microsomes were collected in the washed 100,000 xg pellet. Microsomes were then incubated with 30 μM Fe 2+ in an NADPH-generating system, and both production of malondialdehyde (MDA) (a product of lipid peroxidation) and oxidation of αT* were monitored over a time course in the presence and absence of glutathione (GSH). The results indicate virtually complete oxidation of αT* precedes significant membrane lipid peroxidation, and that addition of 5 mM GSH delays both αT* oxidation and subsequent MDA production. This suggests that the previously observed VE-dependent heat labile inhibition of microsomal lipid peroxidation by GSH involves maintaining membrane levels of α-tocopherol

mTOR activity in AIDS-related diffuse large B-cell lymphoma.

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Sara H Browne

Full Text Available Patients infected with HIV have a significantly increased risk of developing non-Hodgkin lymphomas despite the widespread use of HAART. To investigate mTOR pathway activity in acquired immunodeficiency syndrome (AIDS related diffuse large B-cell lymphoma AR-DLBCL, we used immunohistochemistry to examine the presence of the phosphorylated 70 ribosomal S6 protein-kinase (p70S6K, an extensively studied effector of mTOR Complex 1 (mTORC1 and the phosphorylated phosphatase and tensin homolog (pPTEN, a negative regulator of mTORC1 pathway.We evaluated tissue samples from 126 patients with AR-DLBCL. Among them, 98 samples were from tissue microarrays (TMAs supplied by the Aids and Cancer Specimen Resource (ACSR, the remaining 28 samples were from cases diagnosed and treated at the University of California, San Diego (UCSD. The presence of p70S6K was evaluated with two antibodies directed against the combined epitopes Ser235/236 and Ser240/244, respectively; and additional monoclonal anti-bodies were used to identify pPTEN and phosphorylated proline-rich Akt substrate of 40kDa (pPRAS40. The degree of intensity and percentage of cells positive for p70S6K and pPTEN were assessed in all the samples. In addition, a subgroup of 28 patients from UCSD was studied to assess the presence of pPRAS40, an insulin-regulated activator of the mTORC1. The expression of each of these markers was correlated with clinical and histopathologic features.The majority of the patients evaluated were males (88%; only two cases (1.6% were older than 65 years of age. We found high levels of both p70S6K-paired epitopes studied, 48% positivity against Ser235/236 (44% in ACSR and 64% in UCSD group, and 86% positivity against Ser240/244 (82% in ACSR and 100% in UCSD group. We observed more positive cells and stronger intensity with epitope Ser240/244 in comparison to Ser235/236 (p<0.0001. The degree of intensity and percentage of cells positive for pPTEN was positively correlated with

Bromate Formation Characteristics of UV Irradiation, Hydrogen Peroxide Addition, Ozonation, and Their Combination Processes

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Naoyuki Kishimoto

2012-01-01

Full Text Available Bromate formation characteristics of six-physicochemical oxidation processes, UV irradiation, single addition of hydrogen peroxide, ozonation, UV irradiation with hydrogen peroxide addition (UV/H2O2, ozonation with hydrogen peroxide addition (O3/H2O2, and ozonation with UV irradiation (O3/UV were investigated using 1.88 μM of potassium bromide solution with or without 6.4 μM of 4-chlorobenzoic acid. Bromate was not detected during UV irradiation, single addition of H2O2, and UV/H2O2, whereas ozone-based treatments produced . Hydroxyl radicals played more important role in bromate formation than molecular ozone. Acidification and addition of radical scavengers such as 4-chlorobenzoic acid were effective in inhibiting bromate formation during the ozone-based treatments because of inhibition of hydroxyl radical generation and consumption of hydroxyl radicals, respectively. The H2O2 addition was unable to decompose 4-chlorobenzoic acid, though O3/UV and O3/H2O2 showed the rapid degradation, and UV irradiation and UV/H2O2 showed the slow degradation. Consequently, if the concentration of organic contaminants is low, the UV irradiation and/or UV/H2O2 are applicable to organic contaminants removal without bromate formation. However, if the concentration of organic contaminants is high, O3/H2O2 and O3/UV should be discussed as advanced oxidation processes because of their high organic removal efficiency and low bromate formation potential at the optimum condition.

Acetaminophen and aspirin inhibit superoxide anion generation and lipid peroxidation, and protect against 1-methyl-4-phenyl pyridinium-induced dopaminergic neurotoxicity in rats.

Science.gov (United States)

Maharaj, D S; Saravanan, K S; Maharaj, H; Mohanakumar, K P; Daya, S

2004-04-01

We assessed the antioxidant activity of non-narcotic analgesics, acetaminophen and aspirin in rat brain homogenates and neuroprotective effects in vivo in rats intranigrally treated with 1-methyl-4-phenyl pyridinium (MPP+). Both drugs inhibited cyanide-induced superoxide anion generation, as well as lipid peroxidation in rat brain homogenates, the combination of the agents resulting in a potentiation of this effect. Acetaminophen or aspirin when administered alone or in combination, did not alter dopamine (DA) levels in the forebrain or in the striatum. Intranigral infusion of MPP+ in rats caused severe depletion of striatal DA levels in the ipsilateral striatum in rats by the third day. Systemic post-treatment of acetaminophen afforded partial protection, whereas similar treatment of aspirin resulted in complete blockade of MPP+-induced striatal DA depletion. While these findings suggest usefulness of non-narcotic analgesics in neuroprotective therapy in neurodegenerative diseases, aspirin appears to be a potential candidate in prophylactic as well as in adjuvant therapy in Parkinson's disease.

PARP-1 modulation of mTOR signaling in response to a DNA alkylating agent.

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Chantal Ethier

Full Text Available Poly(ADP-ribose polymerase-1 (PARP-1 is widely involved in cell death responses. Depending on the degree of injury and on cell type, PARP activation may lead to autophagy, apoptosis or necrosis. In HEK293 cells exposed to the alkylating agent N-methyl-N'-nitro-N'-nitrosoguanine (MNNG, we show that PARP-1 activation triggers a necrotic cell death response. The massive poly(ADP-ribose (PAR synthesis following PARP-1 activation leads to the modulation of mTORC1 pathway. Shortly after MNNG exposure, NAD⁺ and ATP levels decrease, while AMP levels drastically increase. We characterized at the molecular level the consequences of these altered nucleotide levels. First, AMP-activated protein kinase (AMPK is activated and the mTORC1 pathway is inhibited by the phosphorylation of Raptor, in an attempt to preserve cellular energy. Phosphorylation of the mTORC1 target S6 is decreased as well as the phosphorylation of the mTORC2 component Rictor on Thr1135. Finally, Akt phosphorylation on Ser473 is lost and then, cell death by necrosis occurs. Inhibition of PARP-1 with the potent PARP inhibitor AG14361 prevents all of these events. Moreover, the antioxidant N-acetyl-L-cysteine (NAC can also abrogate all the signaling events caused by MNNG exposure suggesting that reactive oxygen species (ROS production is involved in PARP-1 activation and modulation of mTOR signaling. In this study, we show that PARP-1 activation and PAR synthesis affect the energetic status of cells, inhibit the mTORC1 signaling pathway and possibly modulate the mTORC2 complex affecting cell fate. These results provide new evidence that cell death by necrosis is orchestrated by the balance between several signaling pathways, and that PARP-1 and PAR take part in these events.

Rheb Inhibits Protein Synthesis by Activating the PERK-eIF2α Signaling Cascade

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Richa Tyagi

2015-02-01

Full Text Available Rheb, a ubiquitous small GTPase, is well known to bind and activate mTOR, which augments protein synthesis. Inhibition of protein synthesis is also physiologically regulated. Thus, with cell stress, the unfolded protein response system leads to phosphorylation of the initiation factor eIF2α and arrest of protein synthesis. We now demonstrate a major role for Rheb in inhibiting protein synthesis by enhancing the phosphorylation of eIF2α by protein kinase-like ER kinase (PERK. Interplay between the stimulatory and inhibitory roles of Rheb may enable cells to modulate protein synthesis in response to varying environmental stresses.

Dexamethasone and BCAA Failed to Modulate Muscle Mass and mTOR Signaling in GH-Deficient Rats.

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Nishida, Hikaru; Ikegami, Ayaka; Kaneko, Chiaki; Kakuma, Hitomi; Nishi, Hisano; Tanaka, Noriko; Aoyama, Michiko; Usami, Makoto; Okimura, Yasuhiko

2015-01-01

Branched-chain amino acids (BCAAs) and IGF-I, the secretion of which is stimulated by growth hormone (GH), prevent muscle atrophy. mTOR plays a pivotal role in the protective actions of BCAA and IGF-1. The pathway by which BCAA activates mTOR is different from that of IGF-1, which suggests that BCAA and GH work independently. We tried to examine whether BCAA exerts a protective effect against dexamethasone (Dex)-induced muscle atrophy independently of GH using GH-deficient spontaneous dwarf rats (SDRs). Unexpectedly, Dex did not induce muscle atrophy assessed by the measurement of cross-sectional area (CSA) of the muscle fibers and did not increase atrogin-1, MuRF1 and REDD1 expressions, which are activated during protein degradation. Glucocorticoid (GR) mRNA levels were higher in SDRs compared to GH-treated SDRs, indicating that the low expression of GR is not the reason of the defect of Dex's action in SDRs. BCAA did not stimulate the phosphorylation of p70S6K or 4E-BP1, which stimulate protein synthesis. BCAA did not decrease the mRNA level of atrogin-1 or MuRF1. These findings suggested that Dex failed to modulate muscle mass and that BCAA was unable to activate mTOR in SDRs because these phosphorylations of p70S6K and 4E-BP1 and the reductions of these mRNAs are regulated by mTOR. In contrast, after GH supplementation, these responses to Dex were normalized and muscle fiber CSA was decreased by Dex. BCAA prevented the Dex-induced decrease in CSA. BCAA increased the phosphorylation of p70S6K and decreased the Dex-induced elevations of atrogin-1 and Bnip3 mRNAs. However, the amount of mTORC1 components including mTOR was not decreased in the SDRs compared to the normal rats. These findings suggest that GH increases mTORC1 activity but not its content to recover the action of BCAA in SDRs and that GH is required for actions of Dex and BCAA in muscles.

Dexamethasone and BCAA Failed to Modulate Muscle Mass and mTOR Signaling in GH-Deficient Rats.

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Hikaru Nishida

Full Text Available Branched-chain amino acids (BCAAs and IGF-I, the secretion of which is stimulated by growth hormone (GH, prevent muscle atrophy. mTOR plays a pivotal role in the protective actions of BCAA and IGF-1. The pathway by which BCAA activates mTOR is different from that of IGF-1, which suggests that BCAA and GH work independently. We tried to examine whether BCAA exerts a protective effect against dexamethasone (Dex-induced muscle atrophy independently of GH using GH-deficient spontaneous dwarf rats (SDRs. Unexpectedly, Dex did not induce muscle atrophy assessed by the measurement of cross-sectional area (CSA of the muscle fibers and did not increase atrogin-1, MuRF1 and REDD1 expressions, which are activated during protein degradation. Glucocorticoid (GR mRNA levels were higher in SDRs compared to GH-treated SDRs, indicating that the low expression of GR is not the reason of the defect of Dex's action in SDRs. BCAA did not stimulate the phosphorylation of p70S6K or 4E-BP1, which stimulate protein synthesis. BCAA did not decrease the mRNA level of atrogin-1 or MuRF1. These findings suggested that Dex failed to modulate muscle mass and that BCAA was unable to activate mTOR in SDRs because these phosphorylations of p70S6K and 4E-BP1 and the reductions of these mRNAs are regulated by mTOR. In contrast, after GH supplementation, these responses to Dex were normalized and muscle fiber CSA was decreased by Dex. BCAA prevented the Dex-induced decrease in CSA. BCAA increased the phosphorylation of p70S6K and decreased the Dex-induced elevations of atrogin-1 and Bnip3 mRNAs. However, the amount of mTORC1 components including mTOR was not decreased in the SDRs compared to the normal rats. These findings suggest that GH increases mTORC1 activity but not its content to recover the action of BCAA in SDRs and that GH is required for actions of Dex and BCAA in muscles.

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

Energy Technology Data Exchange (ETDEWEB)

Liu, Yanwei [Department of General Surgery, Shiyan Taihe Hospital, Hubei University of Medicine, Shiyan, Hubei Province (China); Chen, Sen [Department of Academic Affairs, Hubei University of Medicine, Shiyan, Hubei Province (China); Xue, Rui [Department of Anesthesiology, Renmin Hospital, Hubei University of Medicine, Shiyan, Hubei Province (China); Zhao, Juan [Department of Oncology, Xiangyang Central Hospital, Shiyan, Hubei Province (China); Di, Maojun, E-mail: maoojun_di@163.com [Department of General Surgery, Shiyan Taihe Hospital, Hubei University of Medicine, Shiyan, Hubei Province (China)

2016-02-05

Deregulation of PI3K/Akt/mTOR pathway has been recently identified to play a crucial role in the progress of human gastric cancer. In this study, we show that mefloquine, a FDA-approved anti-malarial drug, effectively targets human gastric cancer cells. Mefloquine potently inhibits proliferation and induces apoptosis of a panel of human gastric cancer cell lines, with EC{sub 50} ∼0.5–0.7 μM. In two independent gastric cancer xenograft mouse models, mefloquine significantly inhibits growth of both tumors. The combination of mefloquine with paclitaxel enhances the activity of either drug alone in in vitro and in vivo. In addition, mefloquine potently decreased phosphorylation of PI3K, Akt, mTOR and rS6. Overexpression of constitutively active Akt significantly restored mefloquine-mediated inhibition of mTOR phosphorylation and growth, and induction of apoptosis, suggesting that mefloquine acts on gastric cancer cells via suppressing PI3K/Akt/mTOR pathway. We further show that mefloquine-mediated inhibition of Akt/mTOR singaling is phosphatase-dependent as pretreatment with calyculin A does-dependently reversed mefloquine-mediated inhibition of Akt/mTOR phosphorylation. Since mefloquine is already available for clinic use, these results suggest that it is a useful addition to the treatment armamentarium for gastric cancer. - Highlights: • Mefloquine targets a panel of gastric cancer cell lines in vitro and in vivo. • Combination of mefloquine and paclitaxel is synergistic. • Mefloquine acts on gastric cancer via inhibition of PI3K/Akt/mTOR pathway. • Mefloquine can be repurposed for gastric cancer treatment.

L-Arginine Enhances Protein Synthesis by Phosphorylating mTOR (Thr 2446 in a Nitric Oxide-Dependent Manner in C2C12 Cells

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Ruxia Wang

2018-01-01

Full Text Available Muscle atrophy may arise from many factors such as inactivity, malnutrition, and inflammation. In the present study, we investigated the stimulatory effect of nitric oxide (NO on muscle protein synthesis. Primarily, C2C12 cells were supplied with extra L-arginine (L-Arg in the culture media. L-Arg supplementation increased the activity of inducible nitric oxide synthase (iNOS, the rate of protein synthesis, and the phosphorylation of mTOR (Thr 2446 and p70S6K (Thr 389. L-NAME, an NOS inhibitor, decreased NO concentrations within cells and abolished the stimulatory effect of L-Arg on protein synthesis and the phosphorylation of mTOR and p70S6K. In contrast, SNP (sodium nitroprusside, an NO donor, increased NO concentrations, enhanced protein synthesis, and upregulated mTOR and p70S6K phosphorylation, regardless of L-NAME treatment. Blocking mTOR with rapamycin abolished the stimulatory effect of both L-Arg and SNP on protein synthesis and p70S6K phosphorylation. These results indicate that L-Arg stimulates protein synthesis via the activation of the mTOR (Thr 2446/p70S6K signaling pathway in an NO-dependent manner.

Hydrogen peroxide stabilization in one-dimensional flow columns

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Schmidt, Jeremy T.; Ahmad, Mushtaque; Teel, Amy L.; Watts, Richard J.

2011-09-01

Rapid hydrogen peroxide decomposition is the primary limitation of catalyzed H 2O 2 propagations in situ chemical oxidation (CHP ISCO) remediation of the subsurface. Two stabilizers of hydrogen peroxide, citrate and phytate, were investigated for their effectiveness in one-dimensional columns of iron oxide-coated and manganese oxide-coated sand. Hydrogen peroxide (5%) with and without 25 mM citrate or phytate was applied to the columns and samples were collected at 8 ports spaced 13 cm apart. Citrate was not an effective stabilizer for hydrogen peroxide in iron-coated sand; however, phytate was highly effective, increasing hydrogen peroxide residuals two orders of magnitude over unstabilized hydrogen peroxide. Both citrate and phytate were effective stabilizers for manganese-coated sand, increasing hydrogen peroxide residuals by four-fold over unstabilized hydrogen peroxide. Phytate and citrate did not degrade and were not retarded in the sand columns; furthermore, the addition of the stabilizers increased column flow rates relative to unstabilized columns. These results demonstrate that citrate and phytate are effective stabilizers of hydrogen peroxide under the dynamic conditions of one-dimensional columns, and suggest that citrate and phytate can be added to hydrogen peroxide before injection to the subsurface as an effective means for increasing the radius of influence of CHP ISCO.

PI3K-Akt signaling activates mTOR-mediated epileptogenesis in organotypic hippocampal culture model of posttraumatic epilepsy

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Berdichevsky, Yevgeny; Dryer, Alexandra M.; Saponjian, Yero; Mahoney, Mark M.; Pimentel, Corrin A.; Lucini, Corrina A.; Usenovic, Marija; Staley, Kevin J.

2013-01-01

mTOR is activated in epilepsy, but the mechanisms of mTOR activation in post-traumatic epileptogenesis are unknown. It is also not clear whether mTOR inhibition has an antiepileptogenic, or merely anti-convulsive effect. The rat hippocampal organotypic culture model of post-traumatic epilepsy was used to study the effects of long term (four weeks) inhibition of signaling pathways that interact with mTOR. Ictal activity was quantified by measurement of lactate production and electrical recordi...

Fisetin inhibits TNF-α-induced inflammatory action and hydrogen peroxide-induced oxidative damage in human keratinocyte HaCaT cells through PI3K/AKT/Nrf-2-mediated heme oxygenase-1 expression.

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Seo, Seung-Hee; Jeong, Gil-Saeng

2015-12-01

Oxidative skin damage and skin inflammation play key roles in the pathogenesis of skin-related diseases. Fisetin is a naturally occurring flavonoid abundantly found in several vegetables and fruits. Fisetin has been shown to exert various positive biological effects, such as anti-cancer, anti-proliferative, neuroprotective and anti-oxidative effects. In this study, we investigate the skin protective effects and anti-inflammatory properties of fisetin in hydrogen peroxide- and TNF-α-challenged human keratinocyte HaCaT cells. When HaCaT cells were treated with non-cytotoxic concentrations of fisetin (1-20μM), heme oxygenase (HO)-1 mRNA and protein expression increased in a dose-dependent manner. Furthermore, fisetin dose-dependently increased cell viability and reduced ROS production in hydrogen peroxide-treated HaCaT cells. Fisetin also inhibited the production of NO, PGE2 IL-1β, IL-6, expression of iNOS and COX-2, and activation of NF-κB in HaCaT cells treated with TNF-α. Fisetin induced Nrf2 translocation to the nuclei. HO-1 siRNA transient transfection reversed the effects of fisetin on cytoprotection, ROS reduction, NO, PGE2, IL-1β, IL-6, and TNF-α production, and NF-κB DNA-binding activity. Moreover, fisetin increased Akt phosphorylation and a PI3K pathway inhibitor (LY294002) abolished fisetin-induced cytoprotection and NO inhibition. Taken together, these results provide evidence for a beneficial role of fisetin in skin therapy. Copyright © 2015. Published by Elsevier B.V.

20(S-Protopanaxadiol-Induced Apoptosis in MCF-7 Breast Cancer Cell Line through the Inhibition of PI3K/AKT/mTOR Signaling Pathway

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Hong Zhang

2018-04-01

Full Text Available 20(S-Protopanaxadiol (PPD is one of the major active metabolites of ginseng. It has been reported that 20(S-PPD shows a broad spectrum of antitumor effects. Our research study aims were to investigate whether apoptosis of human breast cancer MCF-7 cells could be induced by 20(S-PPD by targeting the Phosphatidylinositol 3-kinase/Protein kinase B/Mammalian target of rapamycin (PI3K/AKT/mTOR signal pathway in vitro and in vivo. Cell cycle analysis was performed by Propidium Iodide (PI staining. To overexpress and knock down the expression of mTOR, pcDNA3.1-mTOR and mTOR small interfering RNA (siRNA transient transfection assays were used, respectively. Cell viability and apoptosis were evaluated by 3-(4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide (MTT-test and Annexin V /PI double-staining after transfection. The antitumor effect in vivo was determined by the nude mice xenograft assay. After 24 h of incubation, treatment with 20(S-PPD could upregulate phosphorylated-Phosphatase and tensin homologue deleted on chromosome 10 (p-PTEN expression and downregulate PI3K/AKT/mTOR-pathway protein expression. Moreover, G0/G1 cell cycle arrest in MCF-7 cells could be induced by 20(S-PPD treatment at high concentrations. Furthermore, overexpression or knockdown of mTOR could inhibit or promote the apoptotic effects of 20(S-PPD. In addition, tumor volumes were partially reduced by 20(S-PPD at 100 mg/kg in a MCF-7 xenograft model. Immunohistochemical staining indicated a close relationship between the inhibition of tumor growth and the PI3K/AKT/mTOR signal pathway. PI3K/AKT/mTOR pathway-mediated apoptosis may be one of the potential mechanisms of 20(S-PPD treatment.

Rearrangements of organic peroxides and related processes

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Ivan A. Yaremenko

2016-08-01

Full Text Available This review is the first to collate and summarize main data on named and unnamed rearrangement reactions of peroxides. It should be noted, that in the chemistry of peroxides two types of processes are considered under the term rearrangements. These are conventional rearrangements occurring with the retention of the molecular weight and transformations of one of the peroxide moieties after O–O-bond cleavage. Detailed information about the Baeyer−Villiger, Criegee, Hock, Kornblum−DeLaMare, Dakin, Elbs, Schenck, Smith, Wieland, and Story reactions is given. Unnamed rearrangements of organic peroxides and related processes are also analyzed. The rearrangements and related processes of important natural and synthetic peroxides are discussed separately.

Glycolysis in Panc-1 human pancreatic cancer cells is inhibited by everolimus.

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Liu, Ling; Gong, Liansheng; Zhang, Yangde; Li, Nianfeng

2013-01-01

The aim of this study was to evaluate the effects and molecular mechanisms of everolimus on Panc-1 human pancreatic cancer cells. Panc-1 human pancreatic cancer cells were treated with everolimus (10 μg/ml) at selected time points (6, 12 and 24 h). Cell proliferation and apoptosis were evaluated by MTT and flow cytometric analyses. The glycolytic activity was determined by measuring the activity of the key enzyme lactate dehydrogenase (LDH) and lactate production. The activity of mammalian target of rapamycin (mTOR) signaling was measured by western blotting. The expression of genes, including hexokinase 2 (HK2) and microRNA-143 (miR-143), was evaluated by real-time polymerase chain reaction (PCR). The administration of everolimus time-dependently inhibited proliferation and glycolysis and induced apoptosis in the Panc-1 human pancreatic cancer cells. As the time of treatment with everolimus increased, the mTOR signaling activity decreased, indicated by lower phosphorylation levels of S6 kinase; however, the phosphorylation levels of mTOR barely changed. Moreover, our data showed an everolimus-induced increase in miR-143 and decrease in HK2 in Panc-1 cells in a time-dependent manner. In conclusion, the current study indicates a novel role of everolimus in its antitumor effect as an inhibitor of glycolysis in Panc-1 human pancreatic cancer cells. Furthermore, our data highlights the significance of exploring the mechanisms of everolimus and miR-143 in malignant tumors.

Protein oxidation and peroxidation

DEFF Research Database (Denmark)

Davies, Michael Jonathan

2016-01-01

Proteins are major targets for radicals and two-electron oxidants in biological systems due to their abundance and high rate constants for reaction. With highly reactive radicals damage occurs at multiple side-chain and backbone sites. Less reactive species show greater selectivity with regard...... to the residues targeted and their spatial location. Modification can result in increased side-chain hydrophilicity, side-chain and backbone fragmentation, aggregation via covalent cross-linking or hydrophobic interactions, protein unfolding and altered conformation, altered interactions with biological partners...... and modified turnover. In the presence of O2, high yields of peroxyl radicals and peroxides (protein peroxidation) are formed; the latter account for up to 70% of the initial oxidant flux. Protein peroxides can oxidize both proteins and other targets. One-electron reduction results in additional radicals...

Incidence and risk of treatment-related mortality with mTOR inhibitors everolimus and temsirolimus in cancer patients: a meta-analysis.

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Wei-Xiang Qi

Full Text Available BACKGROUND: Two novel mammalian targets of rapamycin (mTOR inhibitors everolimus and temsirolimus are now approved by regulatory agencies and have been widely investigated among various types of solid tumors, but the risk of fatal adverse events (FAEs with these drugs is not well defined. METHODS: We searched PubMed, EMBASE, and Cochrane library databases for relevant trials. Eligible studies included prospective phase II and III trials evaluating everolimus and temsirolimus in patients with all malignancies and data on FAEs were available. Statistical analyses were conducted to calculate the summary incidence, RRs and 95% confidence intervals (CIs by using either random effects or fixed effect models according to the heterogeneity of the included studies. RESULTS: A total of 3322 patients with various advanced solid tumors from 12 trials were included. The overall incidence of mTOR inhibitors associated FAEs was 1.8% (95%CI: 1.3-2.5%, and the incidences of everolimus related FAEs were comparable to that of temsirolimus (1.7% versus 1.8%. Compared with the controls, the use of mTOR inhibitors was associated with an increased risk of FAEs, with a RR of 3.24 (95%CI: 1.21-8.67, p = 0.019. On subgroup analysis, a non-statistically significant increase in the risk of FAEs was found according to different mTOR inhibitors, tumor types or controlled therapy. No evidence of publication bias was observed. CONCLUSION: With the present evidence, the use of mTOR inhibitors seems to increase the risk of FAEs in patients with advanced solid tumors. More high quality trials are still needed to investigate this association.

Glucose Induces Mouse β-Cell Proliferation via IRS2, MTOR, and Cyclin D2 but Not the Insulin Receptor

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Stamateris, Rachel E.; Sharma, Rohit B.; Kong, Yahui; Ebrahimpour, Pantea; Panday, Deepika; Ranganath, Pavana; Zou, Baobo; Levitt, Helena; Parambil, Nisha Abraham; O’Donnell, Christopher P.; García-Ocaña, Adolfo

2016-01-01

An important goal in diabetes research is to understand the processes that trigger endogenous β-cell proliferation. Hyperglycemia induces β-cell replication, but the mechanism remains debated. A prime candidate is insulin, which acts locally through the insulin receptor. Having previously developed an in vivo mouse hyperglycemia model, we tested whether glucose induces β-cell proliferation through insulin signaling. By using mice lacking insulin signaling intermediate insulin receptor substrate 2 (IRS2), we confirmed that hyperglycemia-induced β-cell proliferation requires IRS2 both in vivo and ex vivo. Of note, insulin receptor activation was not required for glucose-induced proliferation, and insulin itself was not sufficient to drive replication. Glucose and insulin caused similar acute signaling in mouse islets, but chronic signaling differed markedly, with mammalian target of rapamycin (MTOR) and extracellular signal–related kinase (ERK) activation by glucose and AKT activation by insulin. MTOR but not ERK activation was required for glucose-induced proliferation. Cyclin D2 was necessary for glucose-induced β-cell proliferation. Cyclin D2 expression was reduced when either IRS2 or MTOR signaling was lost, and restoring cyclin D2 expression rescued the proliferation defect. Human islets shared many of these regulatory pathways. Taken together, these results support a model in which IRS2, MTOR, and cyclin D2, but not the insulin receptor, mediate glucose-induced proliferation. PMID:26740601

Curcumin inhibited HGF-induced EMT and angiogenesis through regulating c-Met dependent PI3K/Akt/mTOR signaling pathways in lung cancer

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Demin Jiao

2016-01-01

Full Text Available The epithelial-mesenchymal transition (EMT and angiogenesis have emerged as two pivotal events in cancer progression. Curcumin has been extensively studied in preclinical models and clinical trials of cancer prevention due to its favorable toxicity profile. However, the possible involvement of curcumin in the EMT and angiogenesis in lung cancer remains unclear. This study found that curcumin inhibited hepatocyte growth factor (HGF-induced migration and EMT-related morphological changes in A549 and PC-9 cells. Moreover, pretreatment with curcumin blocked HGF-induced c-Met phosphorylation and downstream activation of Akt, mTOR, and S6. These effects mimicked that of c-Met inhibitor SU11274 or PI3 kinase inhibitor LY294002 or mTOR inhibitor rapamycin treatment. c-Met gene overexpression analysis further demonstrated that curcumin suppressed lung cancer cell EMT by inhibiting c-Met/Akt/mTOR signaling pathways. In human umbilical vein endothelial cells (HUVECs, we found that curcumin also significantly inhibited PI3K/Akt/mTOR signaling and induced apoptosis and reduced migration and tube formation of HGF-treated HUVEC. Finally, in the experimental mouse model, we showed that curcumin inhibited HGF-stimulated tumor growth and induced an increase in E-cadherin expression and a decrease in vimentin, CD34, and vascular endothelial growth factor (VEGF expression. Collectively, these findings indicated that curcumin could inhibit HGF-promoted EMT and angiogenesis by targeting c-Met and blocking PI3K/Akt/mTOR pathways.

Strong synergism between small molecule inhibitors of HER2, PI3K, mTOR and Bcl-2 in human breast cancer cells.

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Hamunyela, Roswita H; Serafin, Antonio M; Akudugu, John M

2017-02-01

Targeting pro-survival cell signaling components has been promising in cancer therapy, but the benefit of targeting with single agents is limited. For malignancies such as triple-negative breast cancer, there is a paucity of targets that are amenable to existing interventions as they are devoid of the human epidermal growth factor receptor 2 (HER2), progesterone receptor (PR), and estrogen receptor (ER). Concurrent targeting of cell signaling entities other than HER2, PR and ER with multiple agents may be more effective. Evaluating modes of interaction between agents can inform efficient selection of agents when used in cocktails. Using clonogenic cell survival, interaction between inhibitors of HER2 (TAK-165), phosphoinositide 3-kinase (PI3K) and mammalian target of rapamycin (mTOR) (NVP-BEZ235), and the pro-survival gene (Bcl-2) (ABT-263) in three human breast cell lines (MDA-MB-231, MCF-7 and MCF-12A) ranged from strong to very strong synergism. The strongest synergy was demonstrated in PR and ER negative cells. Inhibition of PI3K, mTOR and Bcl-2 could potentially be effective in the treatment of triple-negative cancers. The very strong synergy observed even at lowest concentrations of inhibitors indicates that these cocktails might be able to be used at a minimised risk of systemic toxicity. Concurrent use of multiple inhibitors can potentiate conventional interventions like radiotherapy and chemotherapy. Copyright © 2016 Elsevier B.V. All rights reserved.

Influence of supplemental ultraviolet-B radiation on lipid peroxidation of Chinese cabbage

International Nuclear Information System (INIS)

Huang Shaobai; Zhang Jingjuan; Liu Xiaozhong

1998-01-01

Chinese cabbage cultivar Aijiaohuang was grown in an indoor experiment treated by 0.0,130 (simulating 20% ozone depletion)kJm~(-2)day~(-1) of ultraviolet-B(UV-B) for 4 and 7 days to study the effect of supplemental UV-B radiation on flavoniods and lipid peroxidation in the leaves of Chinese cabbage. Accumulation of UV-ABSORBING flavonoids in the leaves of Chinese cabbage was induced by UV-B radiation. Enhanced UV-B radiation reduced ascorbic acid content in the leaves of Chinese cabbage. It was also found that 13.0kJm~(2)day~(-1) UV-B inhibited catalase and superoxide dismutase activities and increased malondiadehyde content in the leaves of Chinese cabbage. These effects induced by UV-B radiation was enhanced with the time course of treatment. The results above suggested that supplemental UV-B radiation enhanced lipid peroxidation of Chinese cabbage, and the accumulation of UV-absorbing flavonoid could not alleviate the damage of UV-B radiation

LY294002 inhibits glucocorticoid-induced COX-2 gene expression in cardiomyocytes through a phosphatidylinositol 3 kinase-independent mechanism

International Nuclear Information System (INIS)

Sun Haipeng; Xu Beibei; Sheveleva, Elena; Chen, Qin M.

2008-01-01

Glucocorticoids induce COX-2 expression in rat cardiomyocytes. While investigating whether phosphatidylinositol 3 kinase (PI3K) plays a role in corticosterone (CT)-induced COX-2, we found that LY294002 (LY29) but not wortmannin (WM) attenuates CT from inducing COX-2 gene expression. Expression of a dominant-negative mutant of p85 subunit of PI3K failed to inhibit CT from inducing COX-2 expression. CT did not activate PI3K/AKT signaling pathway whereas LY29 and WM decreased the activity of PI3K. LY303511 (LY30), a structural analogue and a negative control for PI3K inhibitory activity of LY29, also suppressed COX-2 induction. These data suggest PI3K-independent mechanisms in regulating CT-induced COX-2 expression. LY29 and LY30 do not inhibit glucocorticoid receptor transactivity. Both compounds have been reported to inhibit Casein Kinase 2 activity and modulate potassium and calcium levels independent of PI3K, while LY29 has been reported to inhibit mammalian Target of Rapamycin (mTOR), and DNA-dependent Protein Kinase (DNA-PK). Inhibitor of Casein Kinase 2 (CK2), mTOR or DNA-PK failed to prevent CT from inducing COX-2 expression. Tetraethylammonium (TEA), a potassium channel blocker, and nimodipine, a calcium channel blocker, both attenuated CT from inducing COX-2 gene expression. CT was found to increase intracellular Ca 2+ concentration, which can be inhibited by LY29, TEA or nimodipine. These data suggest a possible role of calcium instead of PI3K in CT-induced COX-2 expression in cardiomyocytes

Inhibition of mTORC1 Enhances the Translation of Chikungunya Proteins via the Activation of the MnK/eIF4E Pathway

Science.gov (United States)

Joubert, Pierre-Emmanuel; Stapleford, Kenneth; Guivel-Benhassine, Florence; Vignuzzi, Marco; Schwartz, Olivier; Albert, Matthew L.

2015-01-01

Chikungunya virus (CHIKV), the causative agent of a major epidemic spanning five continents, is a positive stranded mRNA virus that replicates using the cell’s cap-dependent translation machinery. Despite viral infection inhibiting mTOR, a metabolic sensor controls cap-dependent translation, viral proteins are efficiently translated. Rapalog treatment, silencing of mtor or raptor genes, but not rictor, further enhanced CHIKV infection in culture cells. Using biochemical assays and real time imaging, we demonstrate that this effect is independent of autophagy or type I interferon production. Providing in vivo evidence for the relevance of our findings, mice treated with mTORC1 inhibitors exhibited increased lethality and showed a higher sensitivity to CHIKV. A systematic evaluation of the viral life cycle indicated that inhibition of mTORC1 has a specific positive effect on viral proteins, enhancing viral replication by increasing the translation of both structural and nonstructural proteins. Molecular analysis defined a role for phosphatidylinositol-3 kinase (PI3K) and MAP kinase-activated protein kinase (MnKs) activation, leading to the hyper-phosphorylation of eIF4E. Finally, we demonstrated that in the context of CHIKV inhibition of mTORC1, viral replication is prioritized over host translation via a similar mechanism. Our study reveals an unexpected bypass pathway by which CHIKV protein translation overcomes viral induced mTORC1 inhibition. PMID:26317997

Inhibition of mTORC1 Enhances the Translation of Chikungunya Proteins via the Activation of the MnK/eIF4E Pathway.

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Pierre-Emmanuel Joubert

2015-08-01

Full Text Available Chikungunya virus (CHIKV, the causative agent of a major epidemic spanning five continents, is a positive stranded mRNA virus that replicates using the cell's cap-dependent translation machinery. Despite viral infection inhibiting mTOR, a metabolic sensor controls cap-dependent translation, viral proteins are efficiently translated. Rapalog treatment, silencing of mtor or raptor genes, but not rictor, further enhanced CHIKV infection in culture cells. Using biochemical assays and real time imaging, we demonstrate that this effect is independent of autophagy or type I interferon production. Providing in vivo evidence for the relevance of our findings, mice treated with mTORC1 inhibitors exhibited increased lethality and showed a higher sensitivity to CHIKV. A systematic evaluation of the viral life cycle indicated that inhibition of mTORC1 has a specific positive effect on viral proteins, enhancing viral replication by increasing the translation of both structural and nonstructural proteins. Molecular analysis defined a role for phosphatidylinositol-3 kinase (PI3K and MAP kinase-activated protein kinase (MnKs activation, leading to the hyper-phosphorylation of eIF4E. Finally, we demonstrated that in the context of CHIKV inhibition of mTORC1, viral replication is prioritized over host translation via a similar mechanism. Our study reveals an unexpected bypass pathway by which CHIKV protein translation overcomes viral induced mTORC1 inhibition.

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.

Endocrine responses and acute mTOR pathway phosphorylation to resistance exercise with leucine and whey

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MT Lane

2017-02-01

Full Text Available Leucine ingestion reportedly activates the mTOR pathway in skeletal muscle, contributing to a hypertrophy response. The purpose of the study was to compare the post-resistance exercise effects of leucine and whey protein supplementation on endocrine responses and muscle mTOR pathway phosphorylation. On visit 1, subjects (X±SD; n=20; age=27.8±2.8yrs provided baseline blood samples for analysis of cortisol, glucose and insulin; a muscle biopsy of the vastus lateralis muscle to assess mTOR signaling pathway phosphorylation; and were tested for maximum strength on the leg press and leg extension exercises. For visits 2 and 3, subjects were randomized in a double-blind crossover design to ingest either leucine and whey protein (10g+10g; supplement or a non-caloric placebo. During these visits, 5 sets of 10 repetitions were performed on both exercises, immediately followed by ingestion of the supplement or placebo. Blood was sampled 30 min post-, and a muscle biopsy 45 min post-exercise. Western blots quantified total and phosphorylated proteins. Insulin increased (α<.05 with supplementation with no change in glucose compared to placebo. Relative phosphorylation of AKT and rpS6 were greater with leucine and whey supplementation compared to placebo. Supplementation of leucine and whey protein immediately after heavy resistance exercise increases anabolic signaling in human skeletal muscle.

Nutrient-Deprived Retinal Progenitors Proliferate in Response to Hypoxia: Interaction of the HIF-1 and mTOR Pathway

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Helena Khaliullina

2016-05-01

Full Text Available At a cellular level, nutrients are sensed by the mechanistic Target of Rapamycin (mTOR. The response of cells to hypoxia is regulated via action of the oxygen sensor Hypoxia-Inducible Factor 1 (HIF-1. During development, injury and disease, tissues might face conditions of both low nutrient supply and low oxygen, yet it is not clear how cells adapt to both nutrient restriction and hypoxia, or how mTOR and HIF-1 interact in such conditions. Here we explore this question in vivo with respect to cell proliferation using the ciliary marginal zone (CMZ of Xenopus. We found that both nutrient-deprivation and hypoxia cause retinal progenitors to decrease their proliferation, yet when nutrient-deprived progenitors are exposed to hypoxia there is an unexpected rise in cell proliferation. This increase, mediated by HIF-1 signalling, is dependent on glutaminolysis and reactivation of the mTOR pathway. We discuss how these findings in non-transformed tissue may also shed light on the ability of cancer cells in poorly vascularised solid tumours to proliferate.

Kinetics of Platinum-Catalyzed Decomposition of Hydrogen Peroxide

Science.gov (United States)

Vetter, Tiffany A.; Colombo, D. Philip, Jr.

2003-07-01

CIBA Vision Corporation markets a contact lens cleaning system that consists of an AOSEPT disinfectant solution and an AOSEPT lens cup. The disinfectant is a buffered 3.0% m/v hydrogen peroxide solution and the cup includes a platinum-coated AOSEPT disc. The hydrogen peroxide disinfects by killing bacteria, fungi, and viruses found on the contact lenses. Because the concentration of hydrogen peroxide needed to disinfect is irritating to eyes, the hydrogen peroxide needs to be neutralized, or decomposed, before the contact lenses can be used again. A general chemistry experiment is described where the kinetics of the catalyzed decomposition of the hydrogen peroxide are studied by measuring the amount of oxygen generated as a function of time. The order of the reaction with respect to the hydrogen peroxide, the rate constant, and the energy of activation are determined. The integrated rate law is used to determine the time required to decompose the hydrogen peroxide to a concentration that is safe for eyes.

Hydrogen Peroxide Probes Directed to Different Cellular Compartments

Science.gov (United States)

Malinouski, Mikalai; Zhou, You; Belousov, Vsevolod V.; Hatfield, Dolph L.; Gladyshev, Vadim N.

2011-01-01

Background Controlled generation and removal of hydrogen peroxide play important roles in cellular redox homeostasis and signaling. We used a hydrogen peroxide biosensor HyPer, targeted to different compartments, to examine these processes in mammalian cells. Principal Findings Reversible responses were observed to various redox perturbations and signaling events. HyPer expressed in HEK 293 cells was found to sense low micromolar levels of hydrogen peroxide. When targeted to various cellular compartments, HyPer occurred in the reduced state in the nucleus, cytosol, peroxisomes, mitochondrial intermembrane space and mitochondrial matrix, but low levels of the oxidized form of the biosensor were also observed in each of these compartments, consistent with a low peroxide tone in mammalian cells. In contrast, HyPer was mostly oxidized in the endoplasmic reticulum. Using this system, we characterized control of hydrogen peroxide in various cell systems, such as cells deficient in thioredoxin reductase, sulfhydryl oxidases or subjected to selenium deficiency. Generation of hydrogen peroxide could also be monitored in various compartments following signaling events. Conclusions We found that HyPer can be used as a valuable tool to monitor hydrogen peroxide generated in different cellular compartments. The data also show that hydrogen peroxide generated in one compartment could translocate to other compartments. Our data provide information on compartmentalization, dynamics and homeostatic control of hydrogen peroxide in mammalian cells. PMID:21283738

Hydrogen peroxide probes directed to different cellular compartments.

Directory of Open Access Journals (Sweden)

Mikalai Malinouski

2011-01-01

Full Text Available Controlled generation and removal of hydrogen peroxide play important roles in cellular redox homeostasis and signaling. We used a hydrogen peroxide biosensor HyPer, targeted to different compartments, to examine these processes in mammalian cells.Reversible responses were observed to various redox perturbations and signaling events. HyPer expressed in HEK 293 cells was found to sense low micromolar levels of hydrogen peroxide. When targeted to various cellular compartments, HyPer occurred in the reduced state in the nucleus, cytosol, peroxisomes, mitochondrial intermembrane space and mitochondrial matrix, but low levels of the oxidized form of the biosensor were also observed in each of these compartments, consistent with a low peroxide tone in mammalian cells. In contrast, HyPer was mostly oxidized in the endoplasmic reticulum. Using this system, we characterized control of hydrogen peroxide in various cell systems, such as cells deficient in thioredoxin reductase, sulfhydryl oxidases or subjected to selenium deficiency. Generation of hydrogen peroxide could also be monitored in various compartments following signaling events.We found that HyPer can be used as a valuable tool to monitor hydrogen peroxide generated in different cellular compartments. The data also show that hydrogen peroxide generated in one compartment could translocate to other compartments. Our data provide information on compartmentalization, dynamics and homeostatic control of hydrogen peroxide in mammalian cells.

Mechanisms of amino acid sensing in mTOR signaling pathway

OpenAIRE

Kim, Eunjung

2009-01-01

Amino acids are fundamental nutrients for protein synthesis and cell growth (increase in cell size). Recently, many compelling evidences have shown that the level of amino acids is sensed by extra- or intra-cellular amino acids sensor(s) and regulates protein synthesis/degradation. Mammalian target of rapamycin complex 1 (mTORC1) is placed in a central position in cell growth regulation and dysregulation of mTOR signaling pathway has been implicated in many serious human diseases including ca...

Fisetin inhibits laryngeal carcinoma through regulation of AKT/NF-κB/mTOR and ERK1/2 signaling pathways.

Science.gov (United States)

Zhang, Xi-Jun; Jia, Shen-Shan

2016-10-01

Targeting cancer cells is crucial for improving the efficiency of laryngeal cancer treatment. However, the signaling pathway and therapeutic strategy, related to the tumor, still need further research. Dietary flavonoid fisetin (3,3',4',7-tetrahydroxyflavone) found in many fruits and vegetables has been shown in preclinical studies to inhibit cancer growth through regulating cell cycle, apoptosis, angiogenesis, invasion and metastasis without causing any toxicity to normal cells. PI3K/AKT and ERK1/2 have been known as essential signaling pathways to modulate cell proliferation, apoptosis as well as autophagy via mTOR, Caspase-3 and NF-κB signals. In our study, flow cytometry and western blot assays suggested that apoptosis was induced by fisetin administration, promoting Caspase-3 expressions by regulating PI3K/AKT/NF-κB. Additionally, fisetin suppressed TU212 cells proliferation, which was linked with ERK1/2 inactivation. Further, the activation of PI3K/AKT-regulated mTOR was inhibited by fisetin, leading to transcription suppression and proliferation inhibition of TU212 cells. In vivo studies also showed that the tumor volume and weight of nude mice were reduced for fisetin use with KI-67 decrease and LC3II increase in tumor tissue samples. Together, our data indicated that fisetin had a potential role in controlling human laryngeal cancer through inhibiting tumor cell proliferation, inducing apoptosis and autophagy regulated by ERK1/2 and AKT/NF-κB/mTOR signaling pathways, which might provide a therapeutic strategy for laryngeal cancer inhibition in future. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

Amyloid-beta binds catalase with high affinity and inhibits hydrogen peroxide breakdown.

OpenAIRE

Milton, N G

1999-01-01

Amyloid-beta (Abeta) specifically bound purified catalase with high affinity and inhibited catalase breakdown of H(2)O(2). The Abeta-induced catalase inhibition involved formation of the inactive catalase Compound II and was reversible. CatalaseAbeta interactions provide rapid functional assays for the cytotoxic domain of Abeta and suggest a mechanism for some of the observed actions of Abeta plus catalase in vitro.

Leucine minimizes denervation-induced skeletal muscle atrophy of rats through akt/mtor signaling pathways

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Carolina Barbosa Ribeiro

2015-03-01

Full Text Available The aim of the present study was to evaluate the effect of leucine treatment (0.30 mM on muscle weight and signaling of myoproteins related to synthesis and degradation pathways of soleus muscle following seven days of complete sciatic nerve lesion.Wistar rats (n=24 of 3 to 4 months of age (192 ± 23 g were used. The animals were randomly distributed into four experimental groups (n=6/group: control, treated with leucine (L, denervated (D and denervated treated with leucine (DL.Dependent measures were proteins levels of AKT, AMPK, mTOR, and ACC performed by Western blot. Leucine induced a reduction in the phosphorylation of AMPK (p<0.05 by 16% in the L and by 68% in the DL groups as compared with control group. Denervation increased AMPK by 24% in the D group as compared with the control group (p<0.05. AKT was also modulated by denervation and leucine treatment, highlighted by the elevation of AKT phosphorylation in the D (65%, L (98% and DL (146% groups as compared with the control group (p<0.05. AKT phosphorylation was 49% higher in the D group as compared with the DL group.Furthermore, denervation decreased mTOR phosphorylation by 29% in the D group as compared with the control group. However, leucine treatment induced an increase of 49% in the phosphorylation of mTOR in the L group as compared with the control group, and an increase of 154% in the DL as compared with the D group ( p<0.05. ACC phosphorylation was 20% greater in the D group than the control group. Furthermore, ACC in the soleus was 22% lower in the in the L group and 50% lower in the DL group than the respective control group (p<0.05.In conclusion, leucine treatment minimized the deleterious effects of denervation on rat soleus muscle by increasing anabolic (AKT and mTOR and decreasing catabolic (AMPK pathways. These results may be interesting for muscle recovery following acute denervation, which may contribute to musculoskeletal rehabilitation after denervation.

Hydrogen peroxide treatment of TCE contaminated soil

International Nuclear Information System (INIS)

Hurst, D.H.; Robinson, K.G.; Siegrist, R.L.

1993-01-01

Solvent contaminated soils are ubiquitous in the industrial world and represent a significant environmental hazard due to their persistence and potentially negative impacts on human health and the environment. Environmental regulations favor treatment of soils with options which reduce the volume and toxicity of contaminants in place. One such treatment option is the in-situ application of hydrogen peroxide to soils contaminated with chlorinated solvents such as trichloroethylene (TCE). This study investigated hydrogen peroxide mass loading rates on removal of TCE from soils of varying organic matter content. Batch experiments conducted on contaminated loam samples using GC headspace analysis showed up to 80% TCE removal upon peroxide treatment. Column experiments conducted on sandy loam soils with high organic matter content showed only 25% TCE removal, even at hydrogen peroxide additions of 25 g peroxide per kg soil

Crosstalk of the mTOR network with stress granules and the TGF-beta pathway

NARCIS (Netherlands)

Prentzell, Mirja Tamara

2018-01-01

Alle organismen en cellen hebben voedingsstoffen nodig om te kunnen groeien en overleven. Het mTOR (mechanistic of mammalian target of rapamycin) kinase is een knooppunt in een complex signaaltransductie netwerk dat celgroei in reactie op voedingsstoffen faciliteert. Ontregeling van dit netwerk

Dietary protein sources differentially affect microbiota, mTOR activity and transcription of mTOR signaling pathways in the small intestine.

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Soumya K Kar

Full Text Available Dietary protein sources can have profound effects on host-microbe interactions in the gut that are critically important for immune resilience. However more knowledge is needed to assess the impact of different protein sources on gut and animal health. Thirty-six wildtype male C57BL/6J mice of 35 d age (n = 6/group; mean ± SEM body weight 21.9 ± 0.25 g were randomly assigned to groups fed for four weeks with semi synthetic diets prepared with one of the following protein sources containing (300 g/kg as fed basis: soybean meal (SBM, casein, partially delactosed whey powder, spray dried plasma protein, wheat gluten meal and yellow meal worm. At the end of the experiment, mice were sacrificed to collect ileal tissue to acquire gene expression data, and mammalian (mechanistic target of rapamycin (mTOR activity, ileal digesta to study changes in microbiota and serum to measure cytokines and chemokines. By genome-wide transcriptome analysis, we identified fourteen high level regulatory genes that are strongly affected in SBM-fed mice compared to the other experimental groups. They mostly related to the mTOR pathway. In addition, an increased (P < 0.05 concentration of granulocyte colony-stimulating factor was observed in serum of SBM-fed mice compared to other dietary groups. Moreover, by 16S rRNA sequencing, we observed that SBM-fed mice had higher (P < 0.05 abundances of Bacteroidales family S24-7, compared to the other dietary groups. We showed that measurements of genome-wide expression and microbiota composition in the mouse ileum reveal divergent responses to diets containing different protein sources, in particular for a diet based on SBM.

Ethanol extract of Tetrapleura tetraptera fruit peels: Chemical characterization, and antioxidant potentials against free radicals and lipid peroxidation in hepatic tissues

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Ochuko L. Erukainure

2017-11-01

Full Text Available The chemical and antioxidant properties of the ethanolic extract of Tetrapleura tetraptera fruit peels were investigated. Dried peels of T. tetraptera fruits were extracted with ethanol. The extract was subjected to preliminary phytochemical screening using standard procedures. GC–MS was used in identifying the secondary metabolites. The antioxidant properties of the extract were determined by its ferric reducing activity, 2,2′-diphenyl-1-picrylhydrazyl (DPPH and nitric oxide (NO radicals scavenging activities, and the inhibition of lipid peroxidation in hepatic tissues of albino male rats. Preliminary phytochemical screening revealed the presence of flavonoids, phenols, tannins, saponins, terpenoids and phlebotannin. GC–MS analysis revealed the presence of D-fructose, piperazine, octodrine, glycidol, glyceraldehydes, 6-octadecenoic acid and 9,12-octadecenoic acid, with D–fructose being the most predominant compound. The extract exhibited high antioxidant activities both in vitro and ex vivo, as indicated by its ability to scavenge DPPH and nitric oxide as well as inhibition of lipid peroxidation. This is further portrayed by its ferric reducing activity. These results suggest an antioxidant protective effect of the extract against oxidative hepatic damage and can be attributed to a synergetic action of the identified bioactive compounds. Keywords: Antioxidant, Lipid peroxidation, Phytochemicals, Secondary metabolites

Raptor, a positive regulatory subunit of mTOR complex 1, is a novel phosphoprotein of the rDNA transcription machinery in nucleoli and chromosomal nucleolus organizer regions (NORs).

Science.gov (United States)

Vazquez-Martin, Alejandro; Cufí, Sílvia; Oliveras-Ferraros, Cristina; Menendez, Javier A

2011-09-15

Raptor is the key scaffolding protein that recruits mTOR substrates to rapamycin-sensitive mTOR complex 1 (mTORC1), a molecular integrator of mitogenic and nutrient/energy environmental inputs into protein translation and cell growth. Although Raptor phosphorylation on various sites is pivotal in the regulation of mTORC1 activity, it remains to be elucidated whether site-specific phosphorylation differentially distributes Raptor to unique subcellular compartments. When exploring the spatiotemporal cell cycle dynamics of six different phospho (P)-Raptor isoforms (Thr ( 706) , Ser ( 722) , Ser ( 863) , Ser ( 792) and Ser ( 877) ), a number of remarkable events differentially defined a topological resetting of P-RaptorThr706 on interphasic and mitotic chromosomes. In interphase nuclei, P-Raptor (Thr706) co-localized with fibrillarin, a component of the nucleolar small nuclear ribonucleoprotein particle, as well as with RNA polymerase I, the enzyme that transcribes nucleolar rRNA. Upon Actinomycin D-induced nucleolar segregation and disaggregation, P-RaptorThr706 was excluded from the nucleolus to accumulate at discrete nucleoplasmic bodies. During mitosis, CDK1 inhibition-induced premature assembly of nucleoli relocated fibrillarin to the surrounding regions of chromosomal-associated P-Raptor (Thr706) , suggesting that a subpopulation of mitotic P-Raptor (Thr706) remained targeted at chromosomal loops of rDNA or nuclear organizer regions (NORs). At the end of mitosis and cytokinesis, when reassembly of incipient nucleoli begins upon NORs activation of rDNA transcription, fibrillarin spatially reorganized with P-Raptor (Thr706) to give rise to daughter nucleoli. Treatment with IGF1 exclusively hyperactivated nuclear P-Raptor (Ser706) and concomitantly promoted Ser ( 2481) autophosphorylation of mTOR, which monitors mTORC1-associated catalytic activity. Nucleolar- and NOR-associated P-Raptor (Ser706) may physically link mTORC1 signaling to ever-growing nucleolus

Effect of antioxidants and silicates on peroxides in povidone.

Science.gov (United States)

Narang, Ajit S; Rao, Venkatramana M; Desai, Divyakant S

2012-01-01

Reactive peroxides in povidone often lead to degradation of oxidation-labile drugs. To reduce peroxide concentration in povidone, the roles of storage conditions, antioxidants, and silicates were investigated. Povidone alone and its physical mixtures with ascorbic acid, propyl gallate, sodium sulfite, butylated hydroxyanisole (BHA), or butylated hydroxytoluene (BHT) were stored at 25 °C and 40 °C, at 11%, 32%, and 50% relative humidity. In addition, povidone solution in methanol was equilibrated with silicates (silica gel and molecular sieves), followed by solvent evaporation to recover povidone powder. Peroxide concentrations in povidone were measured. The concentration of peroxides in povidone increased under very-low-humidity storage conditions. Among the antioxidants, ascorbic acid, propyl gallate, and sodium sulfite reduced the peroxide concentration in povidone, whereas BHA and BHT did not. Water solubility appeared to determine the effectiveness of antioxidants. Also, some silicates significantly reduced peroxide concentration in povidone without affecting its functionality as a tablet binder. Porosity of silicates was critical to their ability to reduce the peroxide concentration in povidone. A combination of these approaches can reduce the initial peroxide concentration in povidone and minimize peroxide growth under routine storage conditions. Copyright © 2011 Wiley-Liss, Inc.

Possible involvement of membrane lipids peroxidation and oxidation of catalytically essential thiols of the cerebral transmembrane sodium pump as component mechanisms of iron-mediated oxidative stress-linked dysfunction of the pump's activity

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T.I. Omotayo

2015-04-01

Full Text Available The precise molecular events defining the complex role of oxidative stress in the inactivation of the cerebral sodium pump in radical-induced neurodegenerative diseases is yet to be fully clarified and thus still open. Herein we investigated the modulation of the activity of the cerebral transmembrane electrogenic enzyme in Fe2+-mediated in vitro oxidative stress model. The results show that Fe2+ inhibited the transmembrane enzyme in a concentration dependent manner and this effect was accompanied by a biphasic generation of aldehydic product of lipid peroxidation. While dithiothreitol prevented both Fe2+ inhibitory effect on the pump and lipid peroxidation, vitamin E prevented only lipid peroxidation but not inhibition of the pump. Besides, malondialdehyde (MDA inhibited the pump by a mechanism not related to oxidation of its critical thiols. Apparently, the low activity of the pump in degenerative diseases mediated by Fe2+ may involve complex multi-component mechanisms which may partly involve an initial oxidation of the critical thiols of the enzyme directly mediated by Fe2+ and during severe progression of such diseases; aldehydic products of lipid peroxidation such as MDA may further exacerbate this inhibitory effect by a mechanism that is likely not related to the oxidation of the catalytically essential thiols of the ouabain-sensitive cerebral electrogenic pump.

Determination of hydrogen peroxide in water by chemiluminescence detection, (1). Flow injection type hydrogen peroxide detection system

International Nuclear Information System (INIS)

Yamashiro, Naoya; Uchida, Shunsuke; Satoh, Yoshiyuki; Morishima, Yusuke; Yokoyama, Hiroaki; Satoh, Tomonori; Sugama, Junichi; Yamada, Rie

2004-01-01

A flow injection type hydrogen peroxide detection system with a sub-ppb detection limit has been developed to determine hydrogen peroxide concentration in water sampled from a high temperature, high pressure hydrogen peroxide water loop. The hydrogen peroxide detector is based on luminol chemiluminescence spectroscopy. A small amount of sample water (20 μl) is mixed with a reagent mixture, an aqueous solution of luminol and Co 2+ catalyst, in a mixing cell which is installed just upstream from the detection cell. The optimum values for pH and the concentrations of luminol and Co 2+ ion have been determined to ensure a lower detectable limit and a higher reproducibility. The photocurrent detected by the detection system is expressed by a linear function of the hydrogen peroxide concentration in the region of lower concentration ([H 2 O 2 ] 2 O 2 ] in the region of higher concentration ([H 2 O 2 ] > 10 ppb). The luminous intensity of luminol chemiluminescence is the highest when pH of the reagent mixture is 11.0. Optimization of the major parameters gives the lowest detectable limit of 0.3 ppb. (author)

Treatment of post-transplantation lymphoproliferative disorders after kidney transplant with rituximab and conversion to m-TOR inhibitor.

Science.gov (United States)

Nieto-Rios, John Fredy; Gómez de Los Ríos, Sandra Milena; Serna-Higuita, Lina María; Ocampo-Kohn, Catalina; Aristizabal-Alzate, Arbey; Gálvez-Cárdenas, Kenny Mauricio; Zuluaga-Valencia, Gustavo Adolfo

2016-12-30

Post-transplantation lymphoproliferative disorders are serious complications of organ transplantation which treatment is not yet standardized. To describe the clinical response, overall and graft survival of patients in our center with this complication after kidney transplantation, which received rituximab as part of their treatment as well as conversion to m-TOR. Retrospective study, which included patients, diagnosed with post-transplant lymphoproliferative disorders after kidney transplantation from January 2011 to July 2014. Eight cases were found with a wide spectrum of clinical presentations. Most had monomorphic histology, 85% were associated with Epstein-Barr virus, 25% of patients had tumor involvement of the renal graft, and 12.5% ​​had primary central nervous system lymphoma. All patients were managed with reduction of immunosuppression, conversion to m-TOR (except one who lost the graft at diagnosis) and rituximab-based therapy. The overall response rate was 87.5% (62.5% complete response, 25% partial response). Survival was 87.5% with a median follow-up of 34 months. An additional patient lost the graft, with chronic nephropathy already known. All the remaining patients had stable renal function. There are no standardized treatment regimens for lymphoproliferative disorders after kidney transplantation, but these patients can be managed successfully with reduction of immunosuppression, conversion to m-TOR and rituximab-based schemes.

Novel Eicosapentaenoic Acid-derived F3-isoprostanes as Biomarkers of Lipid Peroxidation*

Science.gov (United States)

Song, Wen-Liang; Paschos, Georgios; Fries, Susanne; Reilly, Muredach P.; Yu, Ying; Rokach, Joshua; Chang, Chih-Tsung; Patel, Pranav; Lawson, John A.; FitzGerald, Garret A.

2009-01-01

Isoprostanes (iPs) are prostaglandin (PG) isomers generated by free radical-catalyzed peroxidation of polyunsaturated fatty acids (PUFAs). Urinary F2-iPs, PGF2α isomers derived from arachidonic acid (AA) are used as indices of lipid peroxidation in vivo. We now report the characterization of two major F3-iPs, 5-epi-8,12-iso-iPF3α-VI and 8,12-iso-iPF3α-VI, derived from the ω-3 fatty acid, eicosapentaenoic acid (EPA). Although the potential therapeutic benefits of EPA receive much attention, a shift toward a diet rich in ω-3 PUFAs may also predispose to enhanced lipid peroxidation. Urinary 5-epi-8,12-iso-iPF3α-VI and 8,12-iso-iPF3α-VI are highly correlated and unaltered by cyclooxygenase inhibition in humans. Fish oil dose-dependently elevates urinary F3-iPs in mice and a shift in dietary ω-3/ω-6 PUFAs is reflected by an increasing slope [m] of the line relating urinary 8, 12-iso-iPF3α-VI and 8,12-iso-iPF2α-VI. Administration of bacterial lipopolysaccharide evokes a reversible increase in both urinary 8,12-iso-iPF3α-VI and 8,12-iso-iPF2α-VI in humans on an ad lib diet. However, while excretion of the iPs is highly correlated (R2 median = 0.8), [m] varies by an order of magnitude, reflecting marked inter-individual variability in the relative peroxidation of ω-3 versus ω-6 substrates. Clustered analysis of F2- and F3-iPs refines assessment of the oxidant stress response to an inflammatory stimulus in vivo by integrating variability in dietary intake of ω-3/ω-6 PUFAs. PMID:19520854

Radiation-induced peroxidation of egg lecithin liposomes

International Nuclear Information System (INIS)

Bisby, R.H.; Cundall, R.B.; Tomaszewski, K.E.; Coleman, M.H.; Gould, G.

1983-01-01

Peroxidation of multilamellar vesicles of egg lecithin was measured following γ-irradiation of oxygen saturated suspensions. The addition of hydroxyl radical scavengers and the enzymes superoxide dismutase and catalase was used to show that hydroxyl radicals were the major species initiating peroxidation. Superoxide radicals were found to be much less effective initiators of peroxidation. Trolox C, a water soluble analogue of vitamin E, was found to act as an efficient antioxidant in this system. (author)

7 CFR 58.431 - Hydrogen peroxide.

Science.gov (United States)

2010-01-01

... 7 Agriculture 3 2010-01-01 2010-01-01 false Hydrogen peroxide. 58.431 Section 58.431 Agriculture Regulations of the Department of Agriculture (Continued) AGRICULTURAL MARKETING SERVICE (Standards... Material § 58.431 Hydrogen peroxide. The solution shall comply with the specification of the U.S...

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.

PEROXIDE PROCESS FOR SEPARATION OF RADIOACTIVE MATERIALS

Science.gov (United States)

Seaborg, G.T.; Perlman, I.

1958-09-16

reduced state, from hexavalent uranium. It consists in treating an aqueous solution containing such uranium and plutonium ions with sulfate ions in order to form a soluble uranium sulfate complex and then treating the solution with a soluble thorium compound and a soluble peroxide compound in order to ferm a thorium peroxide carrier precipitate which carries down with it the plutonium peroxide present. During this treatment the pH of the solution must be maintained between 2 and 3.

Inorganic precursor peroxides for antifouling coatings

DEFF Research Database (Denmark)

Olsen, S.M.; Pedersen, L.T.; Hermann, M.H.

2009-01-01

Modern antifouling coatings are generally based on cuprous oxide (Cu2O) and organic biocides as active ingredients. Cu2O is prone to bioaccumulation, and should therefore be replaced by more environmentally benign compounds when technically possible. However, cuprous oxide does not only provide...... antifouling properties, it is also a vital ingredient for the antifouling coating to obtain its polishing and leaching mechanism. In this paper, peroxides of strontium, calcium, magnesium, and zinc are tested as pigments in antifouling coatings. The peroxides react with seawater to create hydrogen peroxide...... matrix provides antifouling properties exceeding those of a similar coating based entirely on zinc oxide....

Essential Oil from Clove Bud (Eugenia aromatica Kuntze) Inhibit Key Enzymes Relevant to the Management of Type-2 Diabetes and Some Pro-oxidant Induced Lipid Peroxidation in Rats Pancreas in vitro.

Science.gov (United States)

Oboh, Ganiyu; Akinbola, Ifeoluwa A; Ademosun, Ayokunle O; Sanni, David M; Odubanjo, Oluwatoyin V; Olasehinde, Tosin A; Oyeleye, Sunday I

2015-01-01

The inhibition of enzymes involved in the breakdown of carbohydrates is considered a therapeutic approach to the management of type-2 diabetes. This study sought to investigate the effects of essential oil from clove bud on α-amylase and α-glucosidase activities. Essential oil from clove bud was extracted by hydrodistillation, dried with anhydrous Na2SO4 and characterized using gas chromatography-mass spectrometry (GC-MS). The effects of the essential oil on α-amylase and α-glucosidase activities were investigated. The antioxidant properties of the oil and the inhibition of Fe(2+) and sodium nitroprusside-induced malondialdehyde (MDA) production in rats pancreas homogenate were also carried out. The essential oil inhibited α-amylase (EC50=88.9 μl/L) and α-glucosidase (EC50=71.94 μl/L) activities in a dose-dependent manner. Furthermore, the essential oil inhibited Fe(2+) and SNP-induced MDA production and exhibited antioxidant activities through their NO*, OH*, scavenging and Fe(2+)- chelating abilities. The total phenolic and flavonoid contents of the essential oil were 12.95 mg/g and 6.62 mg/g respectively. GC-MS analysis revealed the presence of α-pinene, β-pinene, neral, geranial, gamma terpinene, cis-ocimene, allo ocimene, 1,8-cineole, linalool, borneol, myrcene and pinene-2-ol in significant amounts. Furthermore, the essential oils exhibited antioxidant activities as typified by hydroxyl (OH) and nitric oxide (NO)] radicals scavenging and Fe(2+)-chelating abilities. The inhibition of α-amylase and α-glucosidase activities, inhibition of pro-oxidant induced lipid peroxidation in rat pancreas and antioxidant activities could be possible mechanisms for the use of the essential oil in the management and prevention of oxidative stress induced type-2 diabetes.

EFFLUENT TREATMENT FACILITY PEROXIDE DESTRUCTION CATALYST TESTING

International Nuclear Information System (INIS)

HALGREN DL

2008-01-01

The 200 Area Effluent Treatment Facility (ETF) main treatment train includes the peroxide destruction module (PDM) where the hydrogen peroxide residual from the upstream ultraviolet light/hydrogen peroxide oxidation unit is destroyed. Removal of the residual peroxide is necessary to protect downstream membranes from the strong oxidizer. The main component of the PDM is two reaction vessels utilizing granular activated carbon (GAC) as the reaction media. The PDM experienced a number of operability problems, including frequent plugging, and has not been utilized since the ETF changed to groundwater as the predominant feed. The unit seemed to be underperforming in regards to peroxide removal during the early periods of operation as well. It is anticipated that a functional PDM will be required for wastewater from the vitrification plant and other future streams. An alternate media or methodology needs to be identified to replace the GAC in the PDMs. This series of bench scale tests is to develop information to support an engineering study on the options for replacement of the existing GAC method for peroxide destruction at the ETF. A number of different catalysts will be compared as well as other potential methods such as strong reducing agents. The testing should lead to general conclusions on the viability of different catalysts and identify candidates for further study and evaluation

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

Mechanical activation of mammalian target of rapamycin pathway is required for cartilage development.

Science.gov (United States)

Guan, Yingjie; Yang, Xu; Yang, Wentian; Charbonneau, Cherie; Chen, Qian

2014-10-01

Mechanical stress regulates development by modulating cell signaling and gene expression. However, the cytoplasmic components mediating mechanotransduction remain unclear. In this study, elimination of muscle contraction during chicken embryonic development resulted in a reduction in the activity of mammalian target of rapamycin (mTOR) in the cartilaginous growth plate. Inhibition of mTOR activity led to significant inhibition of chondrocyte proliferation, cartilage tissue growth, and expression of chondrogenic genes, including Indian hedgehog (Ihh), a critical mediator of mechanotransduction. Conversely, cyclic loading (1 Hz, 5% matrix deformation) of embryonic chicken growth plate chondrocytes in 3-dimensional (3D) collagen scaffolding induced sustained activation of mTOR. Mechanical activation of mTOR occurred in serum-free medium, indicating that it is independent of growth factor or nutrients. Treatment of chondrocytes with Rapa abolished mechanical activation of cell proliferation and Ihh gene expression. Cyclic loading of chondroprogenitor cells deficient in SH2-containing protein tyrosine phosphatase 2 (Shp2) further enhanced mechanical activation of mTOR, cell proliferation, and chondrogenic gene expression. This result suggests that Shp2 is an antagonist of mechanotransduction through inhibition of mTOR activity. Our data demonstrate that mechanical activation of mTOR is necessary for cell proliferation, chondrogenesis, and cartilage growth during bone development, and that mTOR is an essential mechanotransduction component modulated by Shp2 in the cytoplasm. © FASEB.

Ionizing radiation and lipid peroxidation in human body

International Nuclear Information System (INIS)

Giubileo, Gianfranco

1997-07-01

Lipids are organic compounds constituting the living cells. Lipid molecules can be disassembled through peroxidative pathways and hydrocarbons can be bred as end-product of lipid peroxidation in vivo. Lipid peroxidation can be started by an indirect effect of ionizing radiation. So a radioinduced cellular damage in human body can be detected by monitoring the production of specific hydrocarbons

Branched Chain Amino Acids Cause Liver Injury in Obese/Diabetic Mice by Promoting Adipocyte Lipolysis and Inhibiting Hepatic Autophagy

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Fuyang Zhang

2016-11-01

Full Text Available The Western meat-rich diet is both high in protein and fat. Although the hazardous effect of a high fat diet (HFD upon liver structure and function is well recognized, whether the co-presence of high protein intake contributes to, or protects against, HF-induced hepatic injury remains unclear. Increased intake of branched chain amino acids (BCAA, essential amino acids compromising 20% of total protein intake reduces body weight. However, elevated circulating BCAA is associated with non-alcoholic fatty liver disease and injury. The mechanisms responsible for this quandary remain unknown; the role of BCAA in HF-induced liver injury is unclear. Utilizing HFD or HFD + BCAA models, we demonstrated BCAA supplementation attenuated HFD-induced weight gain, decreased fat mass, activated mammalian target of rapamycin (mTOR, inhibited hepatic lipogenic enzymes, and reduced hepatic triglyceride content. However, BCAA caused significant hepatic damage in HFD mice, evidenced by exacerbated hepatic oxidative stress, increased hepatic apoptosis, and elevated circulation hepatic enzymes. Compared to solely HFD-fed animals, plasma levels of free fatty acids (FFA in the HFD + BCAA group are significantly further increased, due largely to AMPKα2-mediated adipocyte lipolysis. Lipolysis inhibition normalized plasma FFA levels, and improved insulin sensitivity. Surprisingly, blocking lipolysis failed to abolish BCAA-induced liver injury. Mechanistically, hepatic mTOR activation by BCAA inhibited lipid-induced hepatic autophagy, increased hepatic apoptosis, blocked hepatic FFA/triglyceride conversion, and increased hepatocyte susceptibility to FFA-mediated lipotoxicity. These data demonstrated that BCAA reduces HFD-induced body weight, at the expense of abnormal lipolysis and hyperlipidemia, causing hepatic lipotoxicity. Furthermore, BCAA directly exacerbate hepatic lipotoxicity by reducing lipogenesis and inhibiting autophagy in the hepatocyte.

INTERACTION OF ALDEHYDES DERIVED FROM LIPID PEROXIDATION AND MEMBRANE PROTEINS.

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Stefania ePizzimenti

2013-09-01

Full Text Available A great variety of compounds are formed during lipid peroxidation of polyunsaturated fatty acids of membrane phospholipids. Among them, bioactive aldehydes, such as 4-hydroxyalkenals, malondialdehyde (MDA and acrolein, have received particular attention since they have been considered as toxic messengers that can propagate and amplify oxidative injury. In the 4-hydroxyalkenal class, 4-hydroxy-2-nonenal (HNE is the most intensively studied aldehyde, in relation not only to its toxic function, but also to its physiological role. Indeed, HNE can be found at low concentrations in human tissues and plasma and participates in the control of biological processes, such as signal transduction, cell proliferation and differentiation. Moreover, at low doses, HNE exerts an anti-cancer effect, by inhibiting cell proliferation, angiogenesis, cell adhesion and by inducing differentiation and/or apoptosis in various tumor cell lines. It is very likely that a substantial fraction of the effects observed in cellular responses, induced by HNE and related aldehydes, be mediated by their interaction with proteins, resulting in the formation of covalent adducts or in the modulation of their expression and/or activity. In this review we focus on membrane proteins affected by lipid peroxidation-derived aldehydes, under physiological and pathological conditions.

Antibiotic drug tigecycline inhibited cell proliferation and induced autophagy in gastric cancer cells

International Nuclear Information System (INIS)

Tang, Chunling; Yang, Liqun; Jiang, Xiaolan; Xu, Chuan; Wang, Mei; Wang, Qinrui; Zhou, Zhansong; Xiang, Zhonghuai; Cui, Hongjuan

2014-01-01

Highlights: • Tigecycline inhibited cell growth and proliferation in human gastric cancer cells. • Tigecycline induced autophagy not apoptosis in human gastric cancer cells. • AMPK/mTOR/p70S6K pathway was activated after tigecycline treatment. • Tigecycline inhibited tumor growth in xenograft model of human gastric cancer cells. - Abstract: Tigecycline acts as a glycylcycline class bacteriostatic agent, and actively resists a series of bacteria, specifically drug fast bacteria. However, accumulating evidence showed that tetracycline and their derivatives such as doxycycline and minocycline have anti-cancer properties, which are out of their broader antimicrobial activity. We found that tigecycline dramatically inhibited gastric cancer cell proliferation and provided an evidence that tigecycline induced autophagy but not apoptosis in human gastric cancer cells. Further experiments demonstrated that AMPK pathway was activated accompanied with the suppression of its downstream targets including mTOR and p70S6K, and ultimately induced cell autophagy and inhibited cell growth. So our data suggested that tigecycline might act as a candidate agent for pre-clinical evaluation in treatment of patients suffering from gastric cancer

Antibiotic drug tigecycline inhibited cell proliferation and induced autophagy in gastric cancer cells

Energy Technology Data Exchange (ETDEWEB)

Tang, Chunling; Yang, Liqun; Jiang, Xiaolan [State Key Laboratory of Silkworm Genome Biology, Southwest University, Chongqing 400716 (China); Xu, Chuan [Division of Scientific Research and Training, General Hospital of PLA Chengdu Military Area Command, Chengdu, Sichuan 610083 (China); Wang, Mei; Wang, Qinrui [State Key Laboratory of Silkworm Genome Biology, Southwest University, Chongqing 400716 (China); Zhou, Zhansong, E-mail: zhouzhans@sina.com [Institute of Urinary Surgery, Southwest Hospital, Third Military Medical University, Chongqing 400038 (China); Xiang, Zhonghuai [State Key Laboratory of Silkworm Genome Biology, Southwest University, Chongqing 400716 (China); Cui, Hongjuan, E-mail: hcui@swu.edu.cn [State Key Laboratory of Silkworm Genome Biology, Southwest University, Chongqing 400716 (China)

2014-03-28

Highlights: • Tigecycline inhibited cell growth and proliferation in human gastric cancer cells. • Tigecycline induced autophagy not apoptosis in human gastric cancer cells. • AMPK/mTOR/p70S6K pathway was activated after tigecycline treatment. • Tigecycline inhibited tumor growth in xenograft model of human gastric cancer cells. - Abstract: Tigecycline acts as a glycylcycline class bacteriostatic agent, and actively resists a series of bacteria, specifically drug fast bacteria. However, accumulating evidence showed that tetracycline and their derivatives such as doxycycline and minocycline have anti-cancer properties, which are out of their broader antimicrobial activity. We found that tigecycline dramatically inhibited gastric cancer cell proliferation and provided an evidence that tigecycline induced autophagy but not apoptosis in human gastric cancer cells. Further experiments demonstrated that AMPK pathway was activated accompanied with the suppression of its downstream targets including mTOR and p70S6K, and ultimately induced cell autophagy and inhibited cell growth. So our data suggested that tigecycline might act as a candidate agent for pre-clinical evaluation in treatment of patients suffering from gastric cancer.

PI3K inhibition enhances doxorubicin-induced apoptosis in sarcoma cells.

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Diana Marklein

Full Text Available We searched for a drug capable of sensitization of sarcoma cells to doxorubicin (DOX. We report that the dual PI3K/mTOR inhibitor PI103 enhances the efficacy of DOX in several sarcoma cell lines and interacts with DOX in the induction of apoptosis. PI103 decreased the expression of MDR1 and MRP1, which resulted in DOX accumulation. However, the enhancement of DOX-induced apoptosis was unrelated to DOX accumulation. Neither did it involve inhibition of mTOR. Instead, the combination treatment of DOX plus PI103 activated Bax, the mitochondrial apoptosis pathway, and caspase 3. Caspase 3 activation was also observed in xenografts of sarcoma cells in nude mice upon combination of DOX with the specific PI3K inhibitor GDC-0941. Although the increase in apoptosis did not further impact on tumor growth when compared to the efficient growth inhibition by GDC-0941 alone, these findings suggest that inhibition of PI3K may improve DOX-induced proapoptotic effects in sarcoma. Taken together with similar recent studies of neuroblastoma- and glioblastoma-derived cells, PI3K inhibition seems to be a more general option to sensitize tumor cells to anthracyclines.

PROCESS OF ELIMINATING HYDROGEN PEROXIDE IN SOLUTIONS CONTAINING PLUTONIUM VALUES

Science.gov (United States)

Barrick, J.G.; Fries, B.A.

1960-09-27

A procedure is given for peroxide precipitation processes for separating and recovering plutonium values contained in an aqueous solution. When plutonium peroxide is precipitated from an aqueous solution, the supernatant contains appreciable quantities of plutonium and peroxide. It is desirable to process this solution further to recover plutonium contained therein, but the presence of the peroxide introduces difficulties; residual hydrogen peroxide contained in the supernatant solution is eliminated by adding a nitrite or a sulfite to this solution.

Localization of ανβ6 integrin-TGF-β1/Smad3, mTOR and PPARγ in experimental colorectal fibrosis

Directory of Open Access Journals (Sweden)

G. Latella

2013-12-01

Full Text Available A simultaneous action of several pro-fibrotic mediators appears relevant in the development of fibrosis. There are evidences that transforming growth factor-β (TGF-β/Smad3 pathway forms with αvβ6 integrin, mammalian target of Rapamycin (mTOR and peroxisome proliferator-activated receptor-γ (PPARγ a complex signalling network with extensive crosstalk and strong effects on fibrosis development. The present study evaluated the expression of TGFβ, Smad3, αvβ6 integrin, mTOR and PPARγ in 2, 4, 6-trinitrobenzenesulphonic acid (TNBS-induced colorectal fibrosis in Smad3 wild-type (WT and null mice. Smad3 WT mice treated with TNBS developed a marked colorectal fibrosis and showed a concomitant up-regulation of TGFβ, Smad3, αvβ6 and mTOR and a reduction of PPARγ expression. On the other hand, Smad3 Null mice similarly treated with TNBS did not develop fibrosis and showed a very low or even absent expression of TGFβ, Smad3, αvβ6 and mTOR and a marked over-expression of PPARγ. At the same time the expression of α-smooth muscle actin (a marker of activated myofibroblasts, collagen I-III and connective tissue growth factor (a downstream effector of TGFβ/Smad3-induced extracellular matrix proteins were up-regulated in Smad3 WT mice treated with TNBS compared to Null TNBS-treated mice. These preliminary results suggest a possible interaction between these pro-fibrotic molecules in the development of intestinal fibrosis.

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

NARCIS (Netherlands)

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

2016-01-01

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

Efficacy of formalin, hydrogen-peroxide, and sodium-chloride on fungal-infected rainbow-trout eggs

Science.gov (United States)

Schreier, Theresa M.; Rach, J.J.; Howe, G.E.

1996-01-01

Antifungal agents are essential for the maintenance of healthy stocks of fish and their eggs in intensive aquaculture operations. In the usa, formalin is the only fungicide approved for use in fish culture, however, hydrogen peroxide and sodium chloride have been granted low regulatory priority drug status by the united states food and drug administration (fda) and their use is allowed. We evaluated the efficacy of these fungicides for controlling fungal infections on rainbow trout eggs. A pilot study was conducted to determine the minimum water flow rate required to administer test chemicals accurately in heath incubators. A minimum water flow rate of 7.6 1 min(-1) was necessary to maintain treatment concentrations during flow-through chemical exposures, the antifungal activity of formalin, hydrogen peroxide, and sodium chloride was evaluated by treating uninfected and 10% fungal-infected (saprolegnia parasitica) rainbow trout eggs (oncorhynchus mykiss) for 15 min every other day until hatch. There were no significant differences among treatments in percent hatch or final infection for uninfected eggs receiving prophylactic chemical treatments, eggs of the negative control group (uninfected and untreated) had a mean hatch exceeding 86%, all chemical treatments conducted on the infected egg groups controlled the spread of fungus and improved hatching success compared with the positive control groups (infected and untreated), formalin treatments of 1000 and 1500 mu l 1(-1) and hydrogen peroxide treatments of 500 and 1000 mu l 1(-1) were the most effective. Sodium chloride treatments of 30000 mg 1(-1) improved fry hatch, but the compound was less effective at inhibiting fungal growths compared with hydrogen peroxide and formalin treatments.

Radiation effect on lipid peroxide content of spices

International Nuclear Information System (INIS)

Kaneko, Nobutada; Ito, Hitoshi; Ishigaki, Isao

1990-01-01

To evaluate the radiation-induced deterioration of lipid in spices, peroxide value, iodine value and acid value were measured after extraction by chloroform. Peroxide values of black pepper and white pepper were not increased by gamma-irradiation with doses below 30 kGy and gradually increased at higher dose up to 80 kGy in this study. On contrary, peroxide values of clove and rosemary increased rather quickly below 20 kGy of gamma-irradiation, and they became stationary at higher dose. Iodine values and acid values had relationship with peroxide values on each kind of spices. On the storage study of irradiated spices, peroxide values decreased quickly during 20 days storage as same as nonirradiated spices, and it became stationary after 20 to 50 days storage at 30degC. Enhancement of oxidized deterioration were not observed even higher irradiation doses up to 80 kGy in this study. (author)

Antioxidative effects of fermented sesame sauce against hydrogen peroxide-induced oxidative damage in LLC-PK1 porcine renal tubule cells

Science.gov (United States)

Song, Jia-Le; Choi, Jung-Ho; Seo, Jae-Hoon; Kil, Jeung-Ha

2014-01-01

BACKGROUND/OBJECTIVES This study was performed to investigate the in vitro antioxidant and cytoprotective effects of fermented sesame sauce (FSeS) against hydrogen peroxide (H2O2)-induced oxidative damage in renal proximal tubule LLC-PK1 cells. MATERIALS/METHODS 1,1-diphenyl-2-picrylhydrazyl (DPPH), hydroxyl radical (•OH), and H2O2 scavenging assay was used to evaluate the in vitro antioxidant activity of FSeS. To investigate the cytoprotective effect of FSeS against H2O2-induced oxidative damage in LLC-PK1 cells, the cellular levels of reactive oxygen species (ROS), lipid peroxidation, and endogenous antioxidant enzymes including catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GSH-px) were measured. RESULTS The ability of FSeS to scavenge DPPH, •OH and H2O2 was greater than that of FSS and AHSS. FSeS also significantly inhibited H2O2-induced (500 µM) oxidative damage in the LLC-PK1 cells compared to FSS and AHSS (P sauces, FSeS also significantly increased cellular CAT, SOD, and GSH-px activities and mRNA expression (P < 0.05). CONCULUSIONS These results from the present study suggest that FSeS is an effective radical scavenger and protects against H2O2-induced oxidative damage in LLC-PK1 cells by reducing ROS levels, inhibiting lipid peroxidation, and stimulating antioxidant enzyme activity. PMID:24741396

The effects of β-elemene on the expression of mTOR, HIF-1α ...

African Journals Online (AJOL)

The purpose of this manuscript was to study the regulation effects of â-elemene combined with radiotherapy on three different gene expressions in lung adenocarcinoma A549 cell. mTOR gene, HIF-1á gene, Survivin gene were included in the gene group. Cell culture and RT-PCR were applied to finish this research.

Radioprotective effect of Panax ginseng on the phosphatases and lipid peroxidation level in testes of Swiss albino mice

Energy Technology Data Exchange (ETDEWEB)

Kumar M.; Sharma M.K.; Saxena P.S.; Kumar A. [Rajasthan Univ., Jaipur (India)

2003-03-01

The Panax ginseng has been used as traditional medicine for past several years among oriental people. The present investigation has been made to assess the radioprotective efficacy of ginseng root extract in the testicular enzymes of Swiss albino mice. The Swiss albino mice were divided into different groups. Ginseng treated group: The animals were administered 10 mg/kg body weight ginseng root extract intraperitoneal (i.p.). Radiation treated group: The animals were exposed to 8 Gy gamma radiation at the dose rate of 1.69 Gy/min at the distance of 80 cm. Combination group: Animals were administered ginseng extract continuously for 4 d and on 4th day they were irradiated to 8 Gy gamma radiation after 30 min of extract administration. The animals from above groups were autopsied on day 1, 3, 7, 14 and 30. Biochemical estimations of acid and alkaline phosphatases and Lipid peroxidation (LPO) in testes were done. In ginseng treated group acid and alkaline phosphatases activity and LPO level did not show any significant alteration. In irradiated animals there was a significant increase in acid phosphatase activity and LPO level. However, significant decline in alkaline phosphatase activity was observed. The treatment of ginseng before irradiation causes significant decrease in acid phosphatase and LPO level and significant increase in alkaline phosphatase activity. One of the cause of radiation damage is lipid peroxidation. Due to lipid peroxidation, lysosomal membrane permeability alters and thus results in release of hydrolytic enzymes. So, an increase in acid phosphatase was noticed after radiation treatment. The alkaline phosphatase activity is associated with membrane permeability and different stages of spermatogenesis. Due to membrane damage and depletion of germ cells of testes after irradiation the enzyme activity was decreased. Ginseng markedly inhibits lipid peroxidation. It acts in indirect fashion to protect radical processes by inhibition of initiation of

Radioprotective effect of Panax ginseng on the phosphatases and lipid peroxidation level in testes of Swiss albino mice

International Nuclear Information System (INIS)

Kumar, M.; Sharma, M.K.; Saxena, P.S.; Kumar, A.

2003-01-01

The Panax ginseng has been used as traditional medicine for past several years among oriental people. The present investigation has been made to assess the radioprotective efficacy of ginseng root extract in the testicular enzymes of Swiss albino mice. The Swiss albino mice were divided into different groups. Ginseng treated group: The animals were administered 10 mg/kg body weight ginseng root extract intraperitoneal (i.p.). Radiation treated group: The animals were exposed to 8 Gy gamma radiation at the dose rate of 1.69 Gy/min at the distance of 80 cm. Combination group: Animals were administered ginseng extract continuously for 4 d and on 4th day they were irradiated to 8 Gy gamma radiation after 30 min of extract administration. The animals from above groups were autopsied on day 1, 3, 7, 14 and 30. Biochemical estimations of acid and alkaline phosphatases and Lipid peroxidation (LPO) in testes were done. In ginseng treated group acid and alkaline phosphatases activity and LPO level did not show any significant alteration. In irradiated animals there was a significant increase in acid phosphatase activity and LPO level. However, significant decline in alkaline phosphatase activity was observed. The treatment of ginseng before irradiation causes significant decrease in acid phosphatase and LPO level and significant increase in alkaline phosphatase activity. One of the cause of radiation damage is lipid peroxidation. Due to lipid peroxidation, lysosomal membrane permeability alters and thus results in release of hydrolytic enzymes. So, an increase in acid phosphatase was noticed after radiation treatment. The alkaline phosphatase activity is associated with membrane permeability and different stages of spermatogenesis. Due to membrane damage and depletion of germ cells of testes after irradiation the enzyme activity was decreased. Ginseng markedly inhibits lipid peroxidation. It acts in indirect fashion to protect radical processes by inhibition of initiation of

Linoleic Acid-Induced Ultra-Weak Photon Emission from Chlamydomonas reinhardtii as a Tool for Monitoring of Lipid Peroxidation in the Cell Membranes

Science.gov (United States)

Prasad, Ankush; Pospíšil, Pavel

2011-01-01

Reactive oxygen species formed as a response to various abiotic and biotic stresses cause an oxidative damage of cellular component such are lipids, proteins and nucleic acids. Lipid peroxidation is considered as one of the major processes responsible for the oxidative damage of the polyunsaturated fatty acid in the cell membranes. Various methods such as a loss of polyunsaturated fatty acids, amount of the primary and the secondary products are used to monitor the level of lipid peroxidation. To investigate the use of ultra-weak photon emission as a non-invasive tool for monitoring of lipid peroxidation, the involvement of lipid peroxidation in ultra-weak photon emission was studied in the unicellular green alga Chlamydomonas reinhardtii. Lipid peroxidation initiated by addition of exogenous linoleic acid to the cells was monitored by ultra-weak photon emission measured with the employment of highly sensitive charged couple device camera and photomultiplier tube. It was found that the addition of linoleic acid to the cells significantly increased the ultra-weak photon emission that correlates with the accumulation of lipid peroxidation product as measured using thiobarbituric acid assay. Scavenging of hydroxyl radical by mannitol, inhibition of intrinsic lipoxygenase by catechol and removal of molecular oxygen considerably suppressed ultra-weak photon emission measured after the addition of linoleic acid. The photon emission dominated at the red region of the spectrum with emission maximum at 680 nm. These observations reveal that the oxidation of linoleic acid by hydroxyl radical and intrinsic lipoxygenase results in the ultra-weak photon emission. Electronically excited species such as excited triplet carbonyls are the likely candidates for the primary excited species formed during the lipid peroxidation, whereas chlorophylls are the final emitters of photons. We propose here that the ultra-weak photon emission can be used as a non-invasive tool for the

Hydrogen peroxide probes directed to different cellular compartments.

OpenAIRE

Mikalai Malinouski; You Zhou; Vsevolod V Belousov; Dolph L Hatfield; Vadim N Gladyshev

2011-01-01

Background Controlled generation and removal of hydrogen peroxide play important roles in cellular redox homeostasis and signaling. We used a hydrogen peroxide biosensor HyPer, targeted to different compartments, to examine these processes in mammalian cells. Principal Findings Reversible responses were observed to various redox perturbations and signaling events. HyPer expressed in HEK 293 cells was found to sense low micromolar levels of hydrogen peroxide. When targeted to various cellular ...

Effective Therapeutic Intervention and Comprehensive Genetic Analysis of mTOR Signaling in PEComa : A Case Report

NARCIS (Netherlands)

Weeber, Fleur; Koudijs, Marco J; Hoogstraat, Marlous; Besselink, Nicolle J M; VAN Lieshout, Stef; Nijman, Isaac J; Cuppen, Edwin; Offerhaus, G Johan; Voest, Emile E

BACKGROUND/AIM: Perivascular epithelioid cell tumors (PEComas) are rare mesenchymal neoplasms. The exact genetic alterations underlying the pathophysiology of PEComas are largely unknown, although it has been shown that activation of the Mammalian target of rapamycin (mTOR) signaling pathway plays a

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

Directory of Open Access Journals (Sweden)

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.

Deregulation of miR-100, miR-99a and miR-199b in tissues and plasma coexists with increased expression of mTOR kinase in endometrioid endometrial carcinoma

International Nuclear Information System (INIS)

Torres, Anna; Torres, Kamil; Pesci, Anna; Ceccaroni, Marcello; Paszkowski, Tomasz; Cassandrini, Paola; Zamboni, Giuseppe; Maciejewski, Ryszard

2012-01-01

Alterations of mTOR gene expression have been implicated in the pathogenesis of endometrioid endometrial cancer however only few studies explored the cause of increased mTOR activation in this malignancy. miRNAs are small, noncoding RNAs, which were proven to regulated gene expression at the posttranscriptional level. The study aimed to explore deregulation of miRNAs targeting mTOR kinase (miR-99a, miR-100 and miR-199b) as a possible cause of its altered expression in EEC tissues. In addition expression of the three miRNAs was investigated in plasma of EEC patients and was assessed in terms of diagnostic and prognostic utility. We investigated expression of mTOR kinase transcripts in 46 fresh tissue samples. Expression of miR-99a, miR-100 and miR-199b was investigated in the same group of fresh samples, and in additional 58 FFPE sections as well as in 48 plasma samples using qPCR. Relative quantification was performed using experimentally validated endogenous controls. mTOR kinase expression was increased in EEC tissues and was accompanied by decreased expression of all three miRNAs. Down-regulation of the investigated miRNAs was discovered in plasma of EEC patients and miRNA signatures classified EEC tissues (miR-99a/miR-100/miR-199b) and plasma (miR-99a/miR-199b) samples with higher accuracy in comparison to single miRNAs. We also revealed that miR-100 was an independent prognostic marker of overall survival. We conclude that increased expression of mTOR kinase coexists with down-regulation of its targeting miRNAs, which could suggest a new mechanism of mTOR pathway alterations in EEC. In addition, our findings implicate that miRNA signatures can be considered promising biomarkers for early detection and prognosis of endometrioid endometrial carcinoma

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

Energy Technology Data Exchange (ETDEWEB)

Bu, Xinxin; Jia, Fengqi; Wang, Weifeng; Guo, Xianling; Wu, Mengchao; Wei, Lixin [Tumor Immunology and Gene Therapy Center, Eastern Hepatobiliary Hospital, Second Military Medical Universisty, 225 Changhai Road, Shanghai 200438 (China)

2007-11-12

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. 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. 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. 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.

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

International Nuclear Information System (INIS)

Bu, Xinxin; Jia, Fengqi; Wang, Weifeng; Guo, Xianling; Wu, Mengchao; Wei, Lixin

2007-01-01

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. 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. 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. 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

Anticancer effects of kaempferol in A375 human malignant melanoma cells are mediated via induction of apoptosis, cell cycle arrest, inhibition of cell migration and downregulation of m-TOR/PI3K/AKT pathway.

Science.gov (United States)

Yang, Jia; Xiao, Peng; Sun, Jiaming; Guo, Liang

2018-01-01

Melanoma is an aggressive form of human cancer with limited treatment options currently available. The present study was aimed to evaluate the anticancer activity of kaempferol (KAM) against the human malignant melanoma A375 cell line along with evaluation of its effects on apoptosis, cell cycle, cell migration and m-TOR/PI3K/AKT pathway. Effects on cell viability were assessed by MTT assay while clonogenic assay measured the effects of KAM on colony formation. Annexin V assay evaluated the apoptotic effects of KAM in these cells using flow cytometry. Effects on cell cycle were determined by using flow cytometry with propidium iodide (PI) as probe. The effects of KAM on m-TOR/ PI3K/AKT signalling pathway were evaluated by western blot assay. MTT assay indicated that KAM exhibits a significant anticancer activity against A375 cells with an IC50 of 20 μM. These antiproliferative effects of KAM were also supported by the colony formation assay wherein KAM reduced the colony formation in a dose-dependent manner. The anticancer effect of KAM was found to be due to the initiation of apoptosis in human malignant melanoma A375 cells. Additionally, KAM also exhibited the capacity to trigger G2/M cell cycle arrest and to inhibit the cell migratory potential of A375 cells. KAM caused significant downregulation of m-TOR, phosphorylated (p) m-TOR, PI3K, p-PI3K and Akt protein levels in A375 malignantmelanoma cells. KAM exerts potent anticancer effects via induction of apoptosis, G2/M cell cycle arrest, cell migration inhibition and downregulation of m-TOR, pm-TOR, PI3K, p-PI3K and Akt protein levels.

Effects of dietary poly-β-hydroxybutyrate (PHB) on microbiota composition and the mTOR signaling pathway in the intestines of litopenaeus vannamei.

Science.gov (United States)

Duan, Yafei; Zhang, Yue; Dong, Hongbiao; Wang, Yun; Zhang, Jiasong

2017-12-01

Poly-β-hydroxybutyrate (PHB) is a natural polymer of the short chain fatty acid β-hydroxybutyrate, which acts as a microbial control agent. The mammalian target of the rapamycin (mTOR) signaling pathway plays a crucial role in intestine inflammation and epithelial morphogenesis. In this study, we examined the composition of intestine microbiota, and mTOR signaling-related gene expression in Pacific white shrimp Litopenaeus vannamei fed diets containing different levels of PHB: 0% (Control), 1% (PHB1), 3% (PHB3), and 5% (PHB5) (w/w) for 35 days. High-throughput sequencing analysis revealed that dietary PHB altered the composition and diversity of intestine microbiota, and that the microbiota diversity decreased with the increasing doses of PHB. Specifically, dietary PHB increased the relative abundance of Proteobacteria and Tenericutes in the PHB1 and PHB5 groups, respectively, and increased that of Gammaproteobacteria in the three PHB groups. Alternatively, PHB decreased Alphaproteobacteria in the PHB3 and PHB5 groups. At the genus level, dietary PHB increased the abundance of beneficial bacteria, such as Bacillus, Lactobacillus, Lactococcus, Clostridium, and Bdellovibrio. The relative mRNA expression levels of the mTOR signaling-related genes TOR, 4E-BP, eIF4E1α, and eIF4E2 all increased in the three PHB treatment groups. These results revealed that dietary PHB supplementation had a beneficial effect on intestine health of L. vannamei by modulating the composition of intestine microbiota and activating mTOR signaling.

Active oxygen participation in chlorophyll destruction and lipid peroxidation in SO/sub 2/-fumigated leaves of spinach

Energy Technology Data Exchange (ETDEWEB)

Shimazaki, K; Sakaki, T; Sugahara, K

1980-01-01

Chlorophyll a and carotenoids of spinach plants began to be destroyed in 2 to 3 hr after the initiation of fumigation with 2.0 ppM sulfur dioxide (SO/sub 2/) in light, whereas chlorophyll b was apparently undamaged during 8 hr of exposure to SO/sub 2/. The content of pheophytin a, chromatographically determined, was not changed by SO/sub 2/ fumigation. When leaf disks (phi = 10 mm), excised from the leaves fumigated with SO/sub 2/ at 2.0 ppM for 2 hr, were illuminated, chlorophyll a and carotenoids were broken down, but they were not destroyed in darkness. The destruction of chlorophyll a and carotenoids was suppressed under a stream of nitrogen. Chlorophyll a destruction was inhibited by free radical scavengers, 1,2-dihydroxbenzene-3,5-disulfonate (tiron), hydroquinone and ascorbate. The singlet oxygen scavengers, 1,4-diazabicyclo-(2,2,2)-octane (DABCO), methionine and histidine, and hydroxyl radical scavengers, benzoate and formate were without effect on the destruction of chlorophyll a. Chlorophyll a destruction was inhibited by the addition of superoxide dismutase (SOD) to the homogenate of SO/sub 2/-fumigated leaves. SO/sub 2/ fumigation for 2 hr reduced the activity of superoxide dismutase to 40% without producing the significant loss of chlorophyll. From these results we concluded that chlorophyll a destruction by SO/sub 2/ was due to superoxide radicals. Moreover, malondialdehyde (MDA), an indicator of lipid peroxidation, was accumulated in SO/sub 2/-fumigated leaves in light. MDA formation was inhibited by tiron and hydroquinone, and by DABCO but was not inhibited by benzoate and formate. MDA formation was increased by D/sub 2/O. From these results it was concluded that /sup 1/O/sub 2/ was involved in lipid peroxidation in SO/sub 2/-fumigated leaves.

PRKCI negatively regulates autophagy via PIK3CA/AKT–MTOR signaling

Energy Technology Data Exchange (ETDEWEB)

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.

High Glucose Inhibits Neural Stem Cell Differentiation Through Oxidative Stress and Endoplasmic Reticulum Stress.

Science.gov (United States)

Chen, Xi; Shen, Wei-Bin; Yang, Penghua; Dong, Daoyin; Sun, Winny; Yang, Peixin

2018-06-01

Maternal diabetes induces neural tube defects by suppressing neurogenesis in the developing neuroepithelium. Our recent study further revealed that high glucose inhibited embryonic stem cell differentiation into neural lineage cells. However, the mechanism whereby high glucose suppresses neural differentiation is unclear. To investigate whether high glucose-induced oxidative stress and endoplasmic reticulum (ER) stress lead to the inhibition of neural differentiation, the effect of high glucose on neural stem cell (the C17.2 cell line) differentiation was examined. Neural stem cells were cultured in normal glucose (5 mM) or high glucose (25 mM) differentiation medium for 3, 5, and 7 days. High glucose suppressed neural stem cell differentiation by significantly decreasing the expression of the neuron marker Tuj1 and the glial cell marker GFAP and the numbers of Tuj1 + and GFAP + cells. The antioxidant enzyme superoxide dismutase mimetic Tempol reversed high glucose-decreased Tuj1 and GFAP expression and restored the numbers of neurons and glial cells differentiated from neural stem cells. Hydrogen peroxide treatment imitated the inhibitory effect of high glucose on neural stem cell differentiation. Both high glucose and hydrogen peroxide triggered ER stress, whereas Tempol blocked high glucose-induced ER stress. The ER stress inhibitor, 4-phenylbutyrate, abolished the inhibition of high glucose or hydrogen peroxide on neural stem cell differentiation. Thus, oxidative stress and its resultant ER stress mediate the inhibitory effect of high glucose on neural stem cell differentiation.

Branched Chain Amino Acids Cause Liver Injury in Obese/Diabetic Mice by Promoting Adipocyte Lipolysis and Inhibiting Hepatic Autophagy.

Science.gov (United States)

Zhang, Fuyang; Zhao, Shihao; Yan, Wenjun; Xia, Yunlong; Chen, Xiyao; Wang, Wei; Zhang, Jinglong; Gao, Chao; Peng, Cheng; Yan, Feng; Zhao, Huishou; Lian, Kun; Lee, Yan; Zhang, Ling; Lau, Wayne Bond; Ma, Xinliang; Tao, Ling

2016-11-01

The Western meat-rich diet is both high in protein and fat. Although the hazardous effect of a high fat diet (HFD) upon liver structure and function is well recognized, whether the co-presence of high protein intake contributes to, or protects against, HF-induced hepatic injury remains unclear. Increased intake of branched chain amino acids (BCAA, essential amino acids compromising 20% of total protein intake) reduces body weight. However, elevated circulating BCAA is associated with non-alcoholic fatty liver disease and injury. The mechanisms responsible for this quandary remain unknown; the role of BCAA in HF-induced liver injury is unclear. Utilizing HFD or HFD+BCAA models, we demonstrated BCAA supplementation attenuated HFD-induced weight gain, decreased fat mass, activated mammalian target of rapamycin (mTOR), inhibited hepatic lipogenic enzymes, and reduced hepatic triglyceride content. However, BCAA caused significant hepatic damage in HFD mice, evidenced by exacerbated hepatic oxidative stress, increased hepatic apoptosis, and elevated circulation hepatic enzymes. Compared to solely HFD-fed animals, plasma levels of free fatty acids (FFA) in the HFD+BCAA group are significantly further increased, due largely to AMPKα2-mediated adipocyte lipolysis. Lipolysis inhibition normalized plasma FFA levels, and improved insulin sensitivity. Surprisingly, blocking lipolysis failed to abolish BCAA-induced liver injury. Mechanistically, hepatic mTOR activation by BCAA inhibited lipid-induced hepatic autophagy, increased hepatic apoptosis, blocked hepatic FFA/triglyceride conversion, and increased hepatocyte susceptibility to FFA-mediated lipotoxicity. These data demonstrated that BCAA reduces HFD-induced body weight, at the expense of abnormal lipolysis and hyperlipidemia, causing hepatic lipotoxicity. Furthermore, BCAA directly exacerbate hepatic lipotoxicity by reducing lipogenesis and inhibiting autophagy in the hepatocyte. Copyright © 2016. Published by Elsevier

A chemiluminescent method for determination of lipid peroxidation

International Nuclear Information System (INIS)

Liang Xiaofeng; Hu Tianxi; Fan Xiaobing

2003-01-01

We established a chemiluminescent system for determination of lipid peroxidation and screening anti-oxidants. The lipid containing unsaturated fatly acids was injected into a galls tube. Luminol solution and the deionized water were added into it too. The glass tube was put into a preincubation box to incubate it for 0.5 h at 37 degree C. AAPH solution was injected into the tube for immediate measurement in a biochemiluminometer at 38-39 degree C. The pulses /6s(CP6s) were determined with T-2 program. Chemiluminescent dynamic and lipid peroxidation changes were observed continuously. Once the CL intensity of lipid peroxidation got peak, the antioxidant which has different concentration was added immediately in situ. A certain CL intensity (CP6s) was chosen as evaluation index to compare the activity of antioxidants. A luminol chemiluminescent system for determination of lipid peroxidation has been made. It was found that Vit. C, teapolyphenol, and glutathione have effects on scavenging lipid free radicals. The new method is quick, sensitive, and simple for determination of lipid peroxidation and screening antioxidants

A dynamic network model of mTOR signaling reveals TSC-independent mTORC2 regulation

NARCIS (Netherlands)

Dalle Pezze, Piero; Sonntag, Annika G; Thien, Antje; Prentzell, Mirja T; Gödel, Markus; Fischer, Sven; Neumann-Haefelin, Elke; Huber, Tobias B; Baumeister, Ralf; Shanley, Daryl P; Thedieck, Kathrin

2012-01-01

The kinase mammalian target of rapamycin (mTOR) exists in two multiprotein complexes (mTORC1 and mTORC2) and is a central regulator of growth and metabolism. Insulin activation of mTORC1, mediated by phosphoinositide 3-kinase (PI3K), Akt, and the inhibitory tuberous sclerosis complex 1/2

Dual mTORC1/C2 inhibitors suppress cellular geroconversion (a senescence program).

Science.gov (United States)

Leontieva, Olga V; Demidenko, Zoya N; Blagosklonny, Mikhail V

2015-09-15

In proliferating cells, mTOR is active and promotes cell growth. When the cell cycle is arrested, then mTOR converts reversible arrest to senescence (geroconversion). Rapamycin and other rapalogs suppress geroconversion, maintaining quiescence instead. Here we showed that ATP-competitive kinase inhibitors (Torin1 and PP242), which inhibit both mTORC1 and TORC2, also suppressed geroconversion. Despite inhibition of proliferation (in proliferating cells), mTOR inhibitors preserved re-proliferative potential (RP) in arrested cells. In p21-arrested cells, Torin 1 and PP242 detectably suppressed geroconversion at concentrations as low as 1-3 nM and 10-30 nM, reaching maximal gerosuppression at 30 nM and 300 nM, respectively. Near-maximal gerosuppression coincided with inhibition of p-S6K(T389) and p-S6(S235/236). Dual mTOR inhibitors prevented senescent morphology and hypertrophy. Our study warrants investigation into whether low doses of dual mTOR inhibitors will prolong animal life span and delay age-related diseases. A new class of potential anti-aging drugs can be envisioned.

CMV and BKPyV Infections in Renal Transplant Recipients Receiving an mTOR Inhibitor-Based Regimen Versus a CNI-Based Regimen: A Systematic Review and Meta-Analysis of Randomized, Controlled Trials.

Science.gov (United States)

Mallat, Samir G; Tanios, Bassem Y; Itani, Houssam S; Lotfi, Tamara; McMullan, Ciaran; Gabardi, Steven; Akl, Elie A; Azzi, Jamil R

2017-08-07

The objective of this meta-analysis is to compare the incidences of cytomegalovirus and BK polyoma virus infections in renal transplant recipients receiving a mammalian target of rapamycin inhibitor (mTOR)-based regimen compared with a calcineurin inhibitor-based regimen. We conducted a comprehensive search for randomized, controlled trials up to January of 2016 addressing our objective. Other outcomes included acute rejection, graft loss, serious adverse events, proteinuria, wound-healing complications, and eGFR. Two review authors selected eligible studies, abstracted data, and assessed risk of bias. We assessed quality of evidence using the Grading of Recommendations Assessment, Development and Evaluation methodology. We included 28 randomized, controlled trials with 6211 participants classified into comparison 1: mTOR inhibitor versus calcineurin inhibitor and comparison 2: mTOR inhibitor plus reduced dose of calcineurin inhibitor versus regular dose of calcineurin inhibitor. Results showed decreased incidence of cytomegalovirus infection in mTOR inhibitor-based group in both comparison 1 (risk ratio, 0.54; 95% confidence interval, 0.41 to 0.72), with high quality of evidence, and comparison 2 (risk ratio, 0.43; 95% confidence interval, 0.24 to 0.80), with moderate quality of evidence. The available evidence neither confirmed nor ruled out a reduction of BK polyoma virus infection in mTOR inhibitor-based group in both comparisons. Secondary outcomes revealed more serious adverse events and acute rejections in mTOR inhibitor-based group in comparison 1 and no difference in comparison 2. There was no difference in graft loss in both comparisons. eGFR was higher in the mTOR inhibitor-based group in comparison 1 (mean difference =4.07 ml/min per 1.73 m 2 ; 95% confidence interval, 1.34 to 6.80) and similar to the calcineurin inhibitor-based group in comparison 2. More proteinuria and wound-healing complications occurred in the mTOR inhibitor-based groups. We found

Synthesis and thermal properties of strontium and calcium peroxides

Science.gov (United States)

Philipp, Warren H.; Kraft, Patricia A.

1989-01-01

A practical synthesis and a discussion of some chemical properties of pure strontium peroxide and calcium peroxide are presented. The general synthesis of these peroxides involves precipitation of their octahydrates by addition of H2O2 to aqueous ammoniacal Sr(NO3)2 or CaCl2. The octahydrates are converted to the anhydrous peroxides by various dehydration techniques. A new x-ray diffraction powder pattern for CaO2 x 8H2O is given from which lattice parameters a=6.212830 and c=11.0090 were calculated on the basis of the tetragonal crystal system.

Tangeretin and its metabolite 4'-hydroxytetramethoxyflavone attenuate EGF-stimulated cell cycle progression in hepatocytes; role of inhibition at the level of mTOR/p70S6K.

Science.gov (United States)

Cheng, Z; Surichan, S; Ruparelia, K; Arroo, R; Boarder, M R

2011-04-01

The mechanisms by which the dietary compound tangeretin has anticancer effects may include acting as a prodrug, forming an antiproliferative product in cancer cells. Here we show that tangeretin also inhibits cell cycle progression in hepatocytes and investigate the role of its primary metabolite 4'-hydroxy-5,6,7,8-tetramethoxyflavone (4'-OH-TMF) in this effect. We used epidermal growth factor (EGF)-stimulated rat hepatocytes, with [(3)H]-thymidine incorporation into DNA as an index of progression to S-phase of the cell cycle, and Western blots for phospho-proteins involved in the cell signalling cascade. Incubation of tangeretin with microsomes expressing CYP1A, or with hepatocytes, generated a primary product we identified as 4'-OH-TMF. Low micromolar concentrations of tangeretin or 4'-OH-TMF gave a concentration-dependent inhibition of EGF-stimulated progression to S-phase while having little effect on cell viability. To determine whether time for conversion of tangeretin to an active metabolite would enhance the inhibitory effect we used long pre-incubations; this reduced the inhibitory effect, in parallel with a reduction in the concentration of tangeretin. The EGF-stimulation of hepatocyte cell cycle progression requires signalling through Akt/mTOR/p70S6K kinase cascades. The tangeretin metabolite 4'-OH-TMF selectively inhibited S6K phosphorylation in the absence of significant inhibition of upstream Akt activity, suggesting an effect at the level of mTOR. Tangeretin and 4'-OH-TMF both inhibit cell cycle progression in primary hepatocytes. The inhibition of p70S6K phosphorylation by 4'-OH-TMF raises the possibility that inhibition of the mTOR pathway may contribute to the anticancer influence of a flavonoid-rich diet. © 2011 The Authors. British Journal of Pharmacology © 2011 The British Pharmacological Society.

Determination of active oxygen content in rare earth peroxides

International Nuclear Information System (INIS)

Queiroz, Carlos A.S.; Abrao, Alcidio

1993-01-01

The content of active oxygen in rare earth peroxides have been determined after the dissolution of the samples with hydrocloridic acid in the presence of potassium iodide. The free generated iodine is titrated with sodium thiosulfate using starch as indicator. The oxidation of iodide to the free iodine indicates the presence of a higher valence state rare earth oxide, until now specifically recognized for the oxides of cerium (Ce O 2 ), praseodymium (Pr 6 O 1 1) and terbium (TB 4 O 7 ). recently the authors synthesized a new series of rare earth compounds, the peroxides. These new compounds were prepared by precipitating the rare earth elements complexed with carbonate ion by addition of hydrogen peroxide. the authors demonstrated that all rare earth elements, once solubilized by complexing with carbonate ion, are quantitatively precipitated as peroxide by addition of hydrogen peroxide. (author)

The problem of peroxidation in radiolis logy

International Nuclear Information System (INIS)

Baraboj, V.A.; Chebotarev, E.E.

1986-01-01

A hypothesis is validated concerning the products of freeradical oxidation of lipids and the phenol compounds as a mediator of the stress-syndrome. The data are reviewed on activation of peroxidation under the effect of radiation, cytochemical agents, etc., secondarily stimulating the neurohumoral system function of homeostasis regulation. With the emotional-algesic and cold-stresses, the regulatory system stimulation is of primary, reflex, nature, but it secondarily promotes the peroxidation activation. The radiotoxins (of the quinoid and lipid nature) appearing in tissues under the effect of ionizing radiation, are smilar in structure and mechanism of action to peroxidation activation products formed under the effect of other stress-agents

Induction of mitochondrial biogenesis and respiration is associated with mTOR regulation in hepatocytes of rats treated with the pan-PPAR activator tetradecylthioacetic acid (TTA)

Energy Technology Data Exchange (ETDEWEB)

Hagland, Hanne R.; Nilsson, Linn I.H. [Department of Biomedicine, University of Bergen (Norway); Burri, Lena [Institute of Medicine, University of Bergen, Haukeland University Hospital (Norway); Nikolaisen, Julie [Department of Biomedicine, University of Bergen (Norway); Berge, Rolf K. [Institute of Medicine, University of Bergen, Haukeland University Hospital (Norway); Department of Heart Disease, Haukeland University Hospital (Norway); Tronstad, Karl J., E-mail: karl.tronstad@biomed.uib.no [Department of Biomedicine, University of Bergen (Norway)

2013-01-11

Highlights: Black-Right-Pointing-Pointer We investigated mechanisms of mitochondrial regulation in rat hepatocytes. Black-Right-Pointing-Pointer Tetradecylthioacetic acid (TTA) was employed to activate mitochondrial oxidation. Black-Right-Pointing-Pointer Mitochondrial biogenesis and respiration were induced. Black-Right-Pointing-Pointer It was confirmed that PPAR target genes were induced. Black-Right-Pointing-Pointer The mechanism involved activation mTOR. -- Abstract: The hypolipidemic effect of peroxisome proliferator-activated receptor (PPAR) activators has been explained by increasing mitochondrial fatty acid oxidation, as observed in livers of rats treated with the pan-PPAR activator tetradecylthioacetic acid (TTA). PPAR-activation does, however, not fully explain the metabolic adaptations observed in hepatocytes after treatment with TTA. We therefore characterized the mitochondrial effects, and linked this to signalling by the metabolic sensor, the mammalian target of rapamycin (mTOR). In hepatocytes isolated from TTA-treated rats, the changes in cellular content and morphology were consistent with hypertrophy. This was associated with induction of multiple mitochondrial biomarkers, including mitochondrial DNA, citrate synthase and mRNAs of mitochondrial proteins. Transcription analysis further confirmed activation of PPAR{alpha}-associated genes, in addition to genes related to mitochondrial biogenesis and function. Analysis of mitochondrial respiration revealed that the capacity of both electron transport and oxidative phosphorylation were increased. These effects coincided with activation of the stress related factor, ERK1/2, and mTOR. The protein level and phosphorylation of the downstream mTOR actors eIF4G and 4E-BP1 were induced. In summary, TTA increases mitochondrial respiration by inducing hypertrophy and mitochondrial biogenesis in rat hepatocytes, via adaptive regulation of PPARs as well as mTOR.

Effect of cadmium chloride on hepatic lipid peroxidation in mice