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Sample records for akt mammalian target

  1. Plumbagin induces G2-M arrest and autophagy by inhibiting the AKT/mammalian target of rapamycin pathway in breast cancer cells.

    PubMed

    Kuo, Po-Lin; Hsu, Ya-Ling; Cho, Chien-Yu

    2006-12-01

    This study is the first to investigate the anticancer effect of plumbagin in human breast cancer cells. Plumbagin exhibited cell proliferation inhibition by inducing cells to undergo G2-M arrest and autophagic cell death. Blockade of the cell cycle was associated with increased p21/WAF1 expression and Chk2 activation, and reduced amounts of cyclin B1, cyclin A, Cdc2, and Cdc25C. Plumbagin also reduced Cdc2 function by increasing the association of p21/WAF1/Cdc2 complex and the levels of inactivated phospho-Cdc2 and phospho-Cdc25C by Chk2 activation. Plumbagin triggered autophagic cell death but not predominantly apoptosis. Pretreatment of cells with autophagy inhibitor bafilomycin suppressed plumbagin-mediated cell death. We also found that plumbagin inhibited survival signaling through the phosphatidylinositol 3-kinase/AKT signaling pathway by blocking the activation of AKT and downstream targets, including the mammalian target of rapamycin, forkhead transcription factors, and glycogen synthase kinase 3beta. Phosphorylation of both of mammalian target of rapamycin downstream targets, p70 ribosomal protein S6 kinase and 4E-BP1, was also diminished. Overexpression of AKT by AKT cDNA transfection decreased plumbagin-mediated autophagic cell death, whereas reduction of AKT expression by small interfering RNA potentiated the effect of plumbagin, supporting the inhibition of AKT being beneficial to autophagy. Furthermore, suppression of AKT by plumbagin enhanced the activation of Chk2, resulting in increased inactive phosphorylation of Cdc25C and Cdc2. Further investigation revealed that plumbagin inhibition of cell growth was also evident in a nude mouse model. Taken together, these results imply a critical role for AKT inhibition in plumbagin-induced G2-M arrest and autophagy of human breast cancer cells. PMID:17172425

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

    PubMed

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

    2009-09-01

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

  3. Protection Against Epithelial Damage During Candida albicans Infection Is Mediated by PI3K/Akt and Mammalian Target of Rapamycin Signaling

    PubMed Central

    Moyes, David L.; Shen, Chengguo; Murciano, Celia; Runglall, Manohursingh; Richardson, Jonathan P.; Arno, Matthew; Aldecoa-Otalora, Estibaliz; Naglik, Julian R.

    2014-01-01

    Background. The ability of epithelial cells (ECs) to discriminate between commensal and pathogenic microbes is essential for healthy living. Key to these interactions are mucosal epithelial responses to pathogen-induced damage. Methods. Using reconstituted oral epithelium, we assessed epithelial gene transcriptional responses to Candida albicans infection by microarray. Signal pathway activation was monitored by Western blotting and transcription factor enzyme-linked immunosorbent assay, and the role of these pathways in C. albicans–induced damage protection was determined using chemical inhibitors. Results. Transcript profiling demonstrated early upregulation of epithelial genes involved in immune responses. Many of these genes constituted components of signaling pathways, but only NF-κB, MAPK, and PI3K/Akt pathways were functionally activated. We demonstrate that PI3K/Akt signaling is independent of NF-κB and MAPK signaling and plays a key role in epithelial immune activation and damage protection via mammalian target of rapamycin (mTOR) activation. Conclusions. PI3K/Akt/mTOR signaling may play a critical role in protecting epithelial cells from damage during mucosal fungal infections independent of NF-κB or MAPK signaling. PMID:24357630

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2016-01-01

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

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

    ERIC Educational Resources Information Center

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

    2008-01-01

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

  7. Inhibition of insulin-like growth factor receptor/AKT/mammalian target of rapamycin axis targets colorectal cancer stem cells by attenuating mevalonate-isoprenoid pathway in vitro and in vivo

    PubMed Central

    Sharon, Chetna; Baranwal, Somesh; Patel, Nirmita J.; Rodriguez-Agudo, Daniel; Pandak, William M.; Majumdar, Adhip PN; Krystal, Geoffrey; Patel, Bhaumik B.

    2015-01-01

    We observed a co-upregulation of the insulin-like growth factor receptor (IGF-1R)/AKT/mammalian target of rapamycin (mTOR) [InAT] axis and the mevalonate-isoprenoid biosynthesis (MIB) pathways in colorectal cancer stem cells (CSCs) in an unbiased approach. Hence, we hypothesized that the InAT axis might regulate the MIB pathway to govern colorectal CSCs growth. Stimulation (IGF-1) or inhibition (IGF-1R depletion and pharmacological inhibition of IGF-1R/mTOR) of the InAT axis produced induction or attenuation of CSC growth as well as expression of CSC markers and self-renewal factors respectively. Intriguingly, activation of the InAT axis (IGF-1) caused significant upregulation of the MIB pathway genes (both mRNA and protein); while its inhibition produced the opposite effects in colonospheres. More importantly, supplementation with dimethylallyl- and farnesyl-PP, MIB metabolites downstream of isopentenyl-diphosphate delta isomerase (IDI), but not mevalonate and isopentenyl-pp that are upstream of IDI, resulted in a near-complete reversal of the suppressive effect of the InAT axis inhibitors on CSCs growth. The latter findings suggest a specific regulation of the MIB pathway by the InAT axis distal to the target of statins that inhibit 3-hydroxy-3-methyl-glutaryl-CoA reductase (HMGCR). Effects of IGF-1R inhibition on colonic CSCs proliferation and the MIB pathway were confirmed in an ‘in vivo’ HCT-116 xenograft model. These observations establish a novel mechanistic link between the InAT axis that is commonly deregulated in colorectal cancer and the MIB pathway in regulation of colonic CSCs growth. Hence, the InAT-MIB corridor is a novel target for developing paradigm shifting optimum anti-CSCs therapies for colorectal cancer. PMID:25895029

  8. All Akt Isoforms (Akt1, Akt2, Akt3) Are Involved in Normal Hearing, but Only Akt2 and Akt3 Are Involved in Auditory Hair Cell Survival in the Mammalian Inner Ear

    PubMed Central

    Brand, Yves; Levano, Soledad; Radojevic, Vesna; Naldi, Arianne Monge; Setz, Cristian; Ryan, Allen F.; Pak, Kwang; Hemmings, Brian A.; Bodmer, Daniel

    2015-01-01

    The kinase Akt is a key downstream mediator of the phosphoinositide-3-kinase signaling pathway and participates in a variety of cellular processes. Akt comprises three isoforms each encoded by a separate gene. There is evidence to indicate that Akt is involved in the survival and protection of auditory hair cells in vitro. However, little is known about the physiological role of Akt in the inner ear—especially in the intact animal. To elucidate this issue, we first analyzed the mRNA expression of the three Akt isoforms in the inner ear of C57/BL6 mice by real-time PCR. Next, we tested the susceptibility to gentamicin-induced auditory hair cell loss in isoform-specific Akt knockout mice compared to wild-types (C57/BL6) in vitro. To analyze the effect of gene deletion in vivo, hearing and cochlear microanatomy were evaluated in Akt isoform knockout animals. In this study, we found that all three Akt isoforms are expressed in the cochlea. Our results further indicate that Akt2 and Akt3 enhance hair cell resistance to ototoxicity, while Akt1 does not. Finally, we determined that untreated Akt1 and Akt2/Akt3 double knockout mice display significant hearing loss, indicating a role for these isoforms in normal hearing. Taken together, our results indicate that each of the Akt isoforms plays a distinct role in the mammalian inner ear. PMID:25811375

  9. Effects of inhibitors of vascular endothelial growth factor receptor 2 and downstream pathways of receptor tyrosine kinases involving phosphatidylinositol 3-kinase/Akt/mammalian target of rapamycin or mitogen-activated protein kinase in canine hemangiosarcoma cell lines.

    PubMed

    Adachi, Mami; Hoshino, Yuki; Izumi, Yusuke; Sakai, Hiroki; Takagi, Satoshi

    2016-07-01

    Canine hemangiosarcoma (HSA) is a progressive malignant neoplasm with no current effective treatment. Previous studies showed that receptor tyrosine kinases and molecules within their downstream pathways involving phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin (m-TOR) or mitogen-activated protein kinase (MAPK) were overexpressed in canine, human, and murine tumors, including HSA. The present study investigated the effects of inhibitors of these pathways in canine splenic and hepatic HSA cell lines using assays of cell viability and apoptosis. Inhibitors of the MAPK pathway did not affect canine HSA cell viability. However, cell viability was significantly reduced by exposure to inhibitors of vascular endothelial growth factor receptor 2 and the PI3K/Akt/m-TOR pathway; these inhibitors also induced apoptosis in these cell lines. These results suggest that these inhibitors reduce the proliferation of canine HSA cells by inducing apoptosis. Further study of these inhibitors, using xenograft mouse models of canine HSA, are warranted to explore their potential for clinical application. PMID:27408334

  10. A hexane fraction of guava Leaves (Psidium guajava L.) induces anticancer activity by suppressing AKT/mammalian target of rapamycin/ribosomal p70 S6 kinase in human prostate cancer cells.

    PubMed

    Ryu, Nae Hyung; Park, Kyung-Ran; Kim, Sung-Moo; Yun, Hyung-Mun; Nam, Dongwoo; Lee, Seok-Geun; Jang, Hyeung-Jin; Ahn, Kyoo Seok; Kim, Sung-Hoon; Shim, Bum Sang; Choi, Seung-Hoon; Mosaddik, Ashik; Cho, Somi K; Ahn, Kwang Seok

    2012-03-01

    This study was carried out to evaluate the anticancer effects of guava leaf extracts and its fractions. The chemical compositions of the active extracts were also determined. In the present study, we set out to determine whether the anticancer effects of guava leaves are linked with their ability to suppress constitutive AKT/mammalian target of rapamycin (mTOR)/ribosomal p70 S6 kinase (S6K1) and mitogen-activated protein kinase (MAPK) activation pathways in human prostate cancer cells. We found that guava leaf hexane fraction (GHF) was the most potent inducer of cytotoxic and apoptotic effects in PC-3 cells. The molecular mechanism or mechanisms of GHF apoptotic potential were correlated with the suppression of AKT/mTOR/S6K1 and MAPK signaling pathways. This effect of GHF correlated with down-regulation of various proteins that mediate cell proliferation, cell survival, metastasis, and angiogenesis. Analysis of GHF by gas chromatography and gas chromatography-mass spectrometry tentatively identified 60 compounds, including β-eudesmol (11.98%), α-copaene (7.97%), phytol (7.95%), α-patchoulene (3.76%), β-caryophyllene oxide (CPO) (3.63%), caryophylla-3(15),7(14)-dien-6-ol (2.68%), (E)-methyl isoeugenol (1.90%), α-terpineol (1.76%), and octadecane (1.23%). Besides GHF, CPO, but not phytol, also inhibited the AKT/mTOR/S6K1 signaling pathway and induced apoptosis in prostate cancer cells. Overall, these findings suggest that guava leaves can interfere with multiple signaling cascades linked with tumorigenesis and provide a source of potential therapeutic compounds for both the prevention and treatment of cancer. PMID:22280146

  11. A Hexane Fraction of Guava Leaves (Psidium guajava L.) Induces Anticancer Activity by Suppressing AKT/Mammalian Target of Rapamycin/Ribosomal p70 S6 Kinase in Human Prostate Cancer Cells

    PubMed Central

    Ryu, Nae Hyung; Park, Kyung-Ran; Kim, Sung-Moo; Yun, Hyung-Mun; Nam, Dongwoo; Lee, Seok-Geun; Jang, Hyeung-Jin; Ahn, Kyoo Seok; Kim, Sung-Hoon; Shim, Bum Sang; Choi, Seung-Hoon; Mosaddik, Ashik

    2012-01-01

    Abstract This study was carried out to evaluate the anticancer effects of guava leaf extracts and its fractions. The chemical compositions of the active extracts were also determined. In the present study, we set out to determine whether the anticancer effects of guava leaves are linked with their ability to suppress constitutive AKT/mammalian target of rapamycin (mTOR)/ribosomal p70 S6 kinase (S6K1) and mitogen-activated protein kinase (MAPK) activation pathways in human prostate cancer cells. We found that guava leaf hexane fraction (GHF) was the most potent inducer of cytotoxic and apoptotic effects in PC-3 cells. The molecular mechanism or mechanisms of GHF apoptotic potential were correlated with the suppression of AKT/mTOR/S6K1 and MAPK signaling pathways. This effect of GHF correlated with down-regulation of various proteins that mediate cell proliferation, cell survival, metastasis, and angiogenesis. Analysis of GHF by gas chromatography and gas chromatography–mass spectrometry tentatively identified 60 compounds, including β-eudesmol (11.98%), α-copaene (7.97%), phytol (7.95%), α-patchoulene (3.76%), β-caryophyllene oxide (CPO) (3.63%), caryophylla-3(15),7(14)-dien-6-ol (2.68%), (E)-methyl isoeugenol (1.90%), α-terpineol (1.76%), and octadecane (1.23%). Besides GHF, CPO, but not phytol, also inhibited the AKT/mTOR/S6K1 signaling pathway and induced apoptosis in prostate cancer cells. Overall, these findings suggest that guava leaves can interfere with multiple signaling cascades linked with tumorigenesis and provide a source of potential therapeutic compounds for both the prevention and treatment of cancer. PMID:22280146

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed Central

    CHUANG, WAN-LING; SU, CHIN-CHENG; LIN, PING-YI; LIN, CHI-CHEN; CHEN, YAO-LI

    2015-01-01

    Sann-Joong-Kuey-Jian-Tang (SJKJT), a traditional Chinese medicine, was previously reported to induce autophagy and inhibit the proliferation of the human HepG2 hepatocellular carcinoma cell line via an extrinsic pathway. In the present study, the effects of SJKJT-induced autophagy and the cytotoxic mechanisms mediating these effects were investigated in HepG2 cells. The cytotoxicity of SJKJT in the HepG2 cells was evaluated using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. The results demonstrated that the half-maximal inhibitory concentration of SJKJT was 2.91 mg/ml at 24 h, 1.64 mg/ml at 48 h and 1.26 mg/ml at 72 h. The results of confocal fluorescence microscopy indicated that SJKJT resulted in the accumulation of green fluorescent protein-LC3 and vacuolation of the cytoplasm. Flow cytometric analysis revealed the accumulation of acidic vesicular organelles. Furthermore, western blot analysis, used to determine the expression levels of autophagy-associated proteins, demonstrated that the HepG2 cells treated with SJKJT exhibited LC3B-I/LC3B-II conversion, increased expression levels of Beclin, Atg-3 and Atg-5 and reduced expression levels of p62 and decreased signaling of the phosphoinositide-3 kinase/Akt/mammalian target of rapamycin and the p38 mitogen-activated protein kinase pathways. Taken together, these findings may assist in the development of novel chemotherapeutic agents for the treatment of malignant types of liver cancer. PMID:25847489

  14. Follicle-stimulating Hormone Activation of Hypoxia-inducible Factor-1 by the Phosphatidylinositol 3-Kinase/AKT/Ras Homolog Enriched in Brain (Rheb)/Mammalian Target of Rapamycin (mTOR) Pathway Is Necessary for Induction of Select Protein Markers of Follicular Differentiation*

    PubMed Central

    Alam, Hena; Maizels, Evelyn T.; Park, Youngkyu; Ghaey, Shail; Feiger, Zachary J.; Chandel, Navdeep S.; Hunzicker-Dunn, Mary

    2006-01-01

    We sought to elucidate the role of AKT in follicle-stimulating hormone (FSH)-mediated granulosa cell (GC) differentiation. Our results define a signaling pathway in GCs whereby the inactivating phosphorylation of tuberin downstream of phosphatidylinositol (PI) 3-kinase/AKT activity leads to Rheb (Ras homolog enriched in brain) and subsequent mTOR (mammalian target of rapamycin) activation. mTOR then stimulates translation by phosphorylating p70 S6 kinase and, consequently, the 40 S ribosomal protein S6. Activation of this pathway is required for FSH-mediated induction of several follicular differentiation markers, including luteinizing-hormone receptor (LHR), inhibin-α, microtubule-associated protein 2D, and the PKA type IIβ regulatory subunit. FSH also promotes activation of the transcription factor hypoxia-inducible factor-1 (HIF-1). FSH-stimulated HIF-1 activity is inhibited by the PI 3-kinase inhibitor LY294002, the Rheb inhibitor FTI-277 (farne-syltransferase inhibitor-277), and the mTOR inhibitor rapamycin. Finally, we find that the FSH-mediated up-regulation of reporter activities for LHR, inhibin-α, and vascular endothelial growth factor is dependent upon HIF-1 activity, because a dominant negative form of HIF-1α interferes with the up-regulation of these genes. These results show that FSH enhances HIF-1 activity downstream of the PI 3-kinase/AKT/Rheb/mTOR pathway in GCs and that HIF-1 activity is necessary for FSH to induce multiple follicular differentiation markers. PMID:14982927

  15. Rapamycin interacts synergistically with idarubicin to induce T-leukemia cell apoptosis in vitro and in a mesenchymal stem cell simulated drug-resistant microenvironment via Akt/mammalian target of rapamycin and extracellular signal-related kinase signaling pathways.

    PubMed

    Wu, Kang-Ni; Zhao, Yan-Min; He, Ying; Wang, Bin-Sheng; Du, Kai-Li; Fu, Shan; Hu, Kai-Min; Zhang, Li-Fei; Liu, Li-Zhen; Hu, Yong-Xian; Wang, Ying-Jia; Huang, He

    2014-03-01

    T-cell acute lymphoblastic leukemias (T-ALLs) are clonal lymphoid malignancies with a poor prognosis, and still a lack of effective treatment. Here we examined the interactions between the mammalian target of rapamycin (mTOR) inhibitor rapamycin and idarubicin (IDA) in a series of human T-ALL cell lines Molt-4, Jurkat, CCRF-CEM and CEM/C1. Co-exposure of cells to rapamycin and IDA synergistically induced T-ALL cell growth inhibition and apoptosis mediated by caspase activation via the intrinsic mitochondrial pathway and extrinsic pathway. Combined treatment with rapamycin and IDA down-regulated Bcl-2 and Mcl-1, and inhibited the activation of phosphoinositide 3-kinase (PI3K)/mTOR and extracellular signal-related kinase (ERK). They also played synergistic pro-apoptotic roles in the drug-resistant microenvironment simulated by mesenchymal stem cells (MSCs) as a feeder layer. In addition, MSCs protected T-ALL cells from IDA cytotoxicity by up-regulating ERK phosphorylation, while rapamycin efficiently reversed this protective effect. Taken together, we confirm the synergistic antitumor effects of rapamycin and IDA, and provide an insight into the potential future clinical applications of combined rapamycin-IDA regimens for treating T-cell malignancies. PMID:23741975

  16. [mTOR, the mammalian target of rapamycin].

    PubMed

    Julien, Louis-André; Roux, Philippe P

    2010-12-01

    The discovery of rapamycin from a soil sample on Easter Island in the mid 60's marked the beginning of an exciting field of research in cell biology and medicine. While it was first used as an antifungal and as an immunosuppressive drug, more recent studies confirmed rapamycin's antiproliferative properties over a variety of solid tumors. Research aimed at identifying its mechanism of action uncovered mTOR (mammalian target of rapamycin), a protein kinase that regulates mRNA translation and protein synthesis, an essential step in cell division and proliferation. Recent evidence suggests a more complex role for mTOR in the regulation of several growth factor-stimulated protein kinases, including the proto-oncogene Akt. This article reviews mTOR function and regulation, and briefly details the future challenges for anti-cancer therapies based on mTOR inhibition. PMID:21187044

  17. Mammalian Tribbles Homologs at the Crossroads of Endoplasmic Reticulum Stress and Mammalian Target of Rapamycin Pathways

    PubMed Central

    Cunard, Robyn

    2013-01-01

    In 2000, investigators discovered Tribbles, a Drosophila protein that coordinates morphogenesis by inhibiting mitosis. Further work has delineated Xenopus (Xtrb2), Nematode (Nipi-3), and mammalian homologs of Drosophila tribbles, which include TRB1, TRB2, and TRB3. The sequences of tribbles homologs are highly conserved, and despite their protein kinase structure, to date they have not been shown to have kinase activity. TRB family members play a role in the differentiation of macrophages, lymphocytes, muscle cells, adipocytes, and osteoblasts. TRB isoforms also coordinate a number of critical cellular processes including glucose and lipid metabolism, inflammation, cellular stress, survival, apoptosis, and tumorigenesis. TRB family members modulate multiple complex signaling networks including mitogen activated protein kinase cascades, protein kinase B/AKT signaling, mammalian target of rapamycin, and inflammatory pathways. The following review will discuss metazoan homologs of Drosophila tribbles, their structure, expression patterns, and functions. In particular, we will focus on TRB3 function in the kidney in podocytes. This review will also discuss the key signaling pathways with which tribbles proteins interact and provide a rationale for developing novel therapeutics that exploit these interactions to provide better treatment options for both acute and chronic kidney disease. PMID:24490110

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

    PubMed Central

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

    2016-01-01

    Cellular resistance in tumour cells to different therapeutic approaches has been a limiting factor in the curative treatment of cancer. Resistance to therapeutic radiation is a common phenomenon which significantly reduces treatment options and impacts survival. One of the mechanisms of acquiring resistance to ionizing radiation is the overexpression or activation of various oncogenes like the EGFR (epidermal growth factor receptor), RAS (rat sarcoma) oncogene or loss of PTEN (phosphatase and tensin homologue) which in turn activates the phosphatidyl inositol 3-kinase/protein kinase B (PI3-K)/AKT pathway responsible for radiation resistance in various tumours. Blocking the pathway enhances the radiation response both in vitro and in vivo. Due to the differential activation of this pathway (constitutively activated in tumour cells and not in the normal host cells), it is an excellent candidate target for molecular targeted therapy to enhance radiation sensitivity. In this regard, HIV protease inhibitors (HPIs) known to interfere with PI3-K/AKT signaling in tumour cells, have been shown to sensitize various tumour cells to radiation both in vitro and in vivo. As a result, HPIs are now being investigated as possible radiosensitizers along with various chemotherapeutic drugs. This review describes the mechanisms by which PI3-K/AKT pathway causes radioresistance and the role of HIV protease inhibitors especially nelfinavir as a potential candidate drug to target the AKT pathway for overcoming radioresistance and its use in various clinical trials for different malignancies. PMID:27121513

  19. Driving neural regeneration through the mammalian target of rapamycin.

    PubMed

    Maiese, Kenneth

    2014-08-01

    Neurodegenerative disorders affect more than 30 million individuals throughout the world and lead to significant disability as well as death. These statistics will increase almost exponentially as the lifespan and age of individuals increase globally and individuals become more susceptible to acute disorders such as stroke as well as chronic diseases that involve cognitive loss, Alzheimer's disease, and Parkinson's disease. Current therapies for such disorders are effective only for a small subset of individuals or provide symptomatic relief but do not alter disease progression. One exciting therapeutic approach that may turn the tide for addressing neurodegenerative disorders involves the mammalian target of rapamycin (mTOR). mTOR is a component of the protein complexes mTOR Complex 1 (mTORC1) and mTOR Complex 2 (mTORC2) that are ubiquitous throughout the body and control multiple functions such as gene transcription, metabolism, cell survival, and cell senescence. mTOR through its relationship with phosphoinositide 3-kinase (PI 3-K) and protein kinase B (Akt) and multiple downstream signaling pathways such as p70 ribosomal S6 kinase (p70S6K) and proline rich Akt substrate 40 kDa (PRAS40) promotes neuronal cell regeneration through stem cell renewal and oversees critical pathways such as apoptosis, autophagy, and necroptosis to foster protection against neurodegenerative disorders. Targeting by mTOR of specific pathways that drive long-term potentiation, synaptic plasticity, and β-amyloid toxicity may offer new strategies for disorders such as stroke and Alzheimer's disease. Overall, mTOR is an essential neuroprotective pathway but must be carefully targeted to maximize clinical efficacy and eliminate any clinical toxic side effects. PMID:25317149

  20. Targeting AKT1-E17K and the PI3K/AKT Pathway with an Allosteric AKT Inhibitor, ARQ 092

    PubMed Central

    Yu, Yi; Savage, Ronald E.; Eathiraj, Sudharshan; Meade, Justin; Wick, Michael J.; Hall, Terence; Abbadessa, Giovanni; Schwartz, Brian

    2015-01-01

    As a critical component in the PI3K/AKT/mTOR pathway, AKT has become an attractive target for therapeutic intervention. ARQ 092 and a next generation AKT inhibitor, ARQ 751 are selective, allosteric, pan-AKT and AKT1-E17K mutant inhibitors that potently inhibit phosphorylation of AKT. Biochemical and cellular analysis showed that ARQ 092 and ARQ 751 inhibited AKT activation not only by dephosphorylating the membrane-associated active form, but also by preventing the inactive form from localizing into plasma membrane. In endometrial PDX models harboring mutant AKT1-E17K and other tumor models with an activated AKT pathway, both compounds exhibited strong anti-tumor activity. Combination studies conducted in in vivo breast tumor models demonstrated that ARQ 092 enhanced tumor inhibition of a common chemotherapeutic agent (paclitaxel). In a large panel of diverse cancer cell lines, ARQ 092 and ARQ 751 inhibited proliferation across multiple tumor types but were most potent in leukemia, breast, endometrial, and colorectal cancer cell lines. Moreover, inhibition by ARQ 092 and ARQ 751 was more prevalent in cancer cell lines containing PIK3CA/PIK3R1 mutations compared to those with wt-PIK3CA/PIK3R1 or PTEN mutations. For both ARQ 092 and ARQ 751, PIK3CA/PIK3R1 and AKT1-E17K mutations can potentially be used as predictive biomarkers for patient selection in clinical studies. PMID:26469692

  1. Dual Targeting of Akt and mTORC1 Impairs Repair of DNA Double-Strand Breaks and Increases Radiation Sensitivity of Human Tumor Cells

    PubMed Central

    Holler, Marina; Grottke, Astrid; Mueck, Katharina; Manes, Julia; Jücker, Manfred

    2016-01-01

    Inhibition of mammalian target of rapamycin-complex 1 (mTORC1) induces activation of Akt. Because Akt activity mediates the repair of ionizing radiation-induced DNA double-strand breaks (DNA-DSBs) and consequently the radioresistance of solid tumors, we investigated whether dual targeting of mTORC1 and Akt impairs DNA-DSB repair and induces radiosensitization. Combining mTORC1 inhibitor rapamycin with ionizing radiation in human non-small cell lung cancer (NSCLC) cells (H661, H460, SK-MES-1, HTB-182, A549) and in the breast cancer cell line MDA-MB-231 resulted in radiosensitization of H661 and H460 cells (responders), whereas only a very slight effect was observed in A549 cells, and no effect was observed in SK-MES-1, HTB-182 or MDA-MB-231 cells (non-responders). In responder cells, rapamycin treatment did not activate Akt1 phosphorylation, whereas in non-responders, rapamycin mediated PI3K-dependent Akt activity. Molecular targeting of Akt by Akt inhibitor MK2206 or knockdown of Akt1 led to a rapamycin-induced radiosensitization of non-responder cells. Compared to the single targeting of Akt, the dual targeting of mTORC1 and Akt1 markedly enhanced the frequency of residual DNA-DSBs by inhibiting the non-homologous end joining repair pathway and increased radiation sensitivity. Together, lack of radiosensitization induced by rapamycin was associated with rapamycin-mediated Akt1 activation. Thus, dual targeting of mTORC1 and Akt1 inhibits repair of DNA-DSB leading to radiosensitization of solid tumor cells. PMID:27137757

  2. Targeting the PI3K/AKT/mTOR Signaling Axis in Children with Hematologic Malignancies

    PubMed Central

    Barrett, David; Brown, Valerie I.; Grupp, Stephan A.; Teachey, David T.

    2014-01-01

    The phosphatidylinositiol 3-kinase (PI3K), AKT, mammalian target of rapamycin (mTOR) signaling pathway (PI3K/AKT/mTOR) is frequently dysregulated in disorders of cell growth and survival, including a number of pediatric hematologic malignancies. The pathway can be abnormally activated in childhood acute lymphoblastic leukemia (ALL), acute myelogenous leukemia (AML), and chronic myelogenous leukemia (CML), as well as in some pediatric lymphomas and lymphoproliferative disorders. Most commonly, this abnormal activation occurs as a consequence of constitutive activation of AKT, providing a compelling rationale to target this pathway in many of these conditions. A variety of agents, beginning with the rapamycin analogue (rapalog) sirolimus, have been used successfully to target this pathway in a number of pediatric hematologic malignancies. Rapalogs demonstrate significant preclinical activity against ALL, which has led to a number of clinical trials. Moreover, rapalogs can synergize with a number of conventional cytotoxic agents and overcome pathways of chemotherapeutic resistance for drugs commonly used in ALL treatment, including methotrexate and corticosteroids. Based on preclinical data, rapalogs are also being studied in AML, CML, and non-Hodgkin’s lymphoma. Recently, significant progress has been made using rapalogs to treat pre-malignant lymphoproliferative disorders, including the autoimmune lymphoproliferative syndrome (ALPS); complete remissions in children with otherwise therapy-resistant disease have been seen. Rapalogs only block one component of the pathway (mTORC1), and newer agents are under preclinical and clinical development that can target different and often multiple protein kinases in the PI3K/AKT/mTOR pathway. Most of these agents have been tolerated in early-phase clinical trials. A number of PI3K inhibitors are under investigation. Of note, most of these also target other protein kinases. Newer agents are under development that target both m

  3. Will targeting PI3K/Akt/mTOR signaling work in hematopoietic malignancies?

    PubMed Central

    Gao, Yanan; Yuan, Chase Y.

    2016-01-01

    The constitutive activation of phosphatidylinositol 3-kinase/protein kinase B/mammalian target of rapamycin (PI3K/Akt/mTOR) signaling pathway has been demonstrated to be critical in clinical cancer patients as well as in laboratory cancer models including hematological malignancies. Great efforts have been made to develop inhibitors targeting this pathway in hematological malignancies but so far the efficacies of these inhibitors were not as good as expected. By analyzing existing literatures and datasets available, we found that mutations of genes in the pathway only constitute a very small subset of hematological malignancies. Deep understanding of the function of gene, the pathway and/or its regulators, and the cellular response to inhibitors, may help us design better drugs targeting the hematological malignancies. PMID:27583254

  4. Rationale for targeting the PI3K/Akt/mTOR pathway in myeloproliferative neoplasms.

    PubMed

    Bartalucci, Niccolò; Guglielmelli, Paola; Vannucchi, Alessandro M

    2013-09-01

    The chronic myeloproliferative neoplasms (MPNs), are characterized by a Janus Kinase (JAK)-2 V617F point mutation but this molecular abnormality does not explain by itself the pathogenesis of these disorders, or the phenotypic diversity associated with essential thrombocythemia, polycythemia vera (PV), and myelofibrosis. Beyond the JAK/signal transducer and activator of transcription network, a wide number of molecular alterations were described in MPN including the fosfatidilinositolo-3-chinasi (PI3K)/protein kinase B (Akt)/mammalian target of rapamycin (mTOR) pathway constitutive activation. Several pathway inhibitors were developed, including everolimus, up to the latest class of catalytic inhibitors such as BKM120 and BEZ235. In this review, we present some clinical and experimental evidence showing that the PI3K/Akt/mTOR pathway could represent a therapeutic target in MPNs. In in vitro studies, everolimus has been shown to inhibit cell proliferation and clonogenic potential in human and murine JAK2 V617F mutated cell lines. Patients with PV and primary myelofibrosis hematopoietic progenitors were significantly more sensitive to everolimus compared with healthy control subjects. Of much interest, a combination of everolimus and the JAK1/2 inhibitor, ruxolitinib, showed strong synergism in inducing cell cycle arrest and blockade of cell proliferation. Similar data were obtained using a dual PI3K/mTOR inhibitor, BEZ235, with activity that was also shown in preclinical murine models. A multicenter phase I/II trial with everolimus in myelofibrosis documented a well tolerated clinical efficiency in terms of spleen size reduction and resolution of systemic symptoms and pruritus. These observations indicate that the PI3K/Akt/mTOR pathway might represent a novel target for treatment in MPN. The synergism demonstrated in vitro with JAK2 inhibitors could open additional therapeutic possibilities based on concurrent targeting of different pathways that might optimize

  5. Targeting the Raft-Associated Akt Signaling in Hepatocellular Carcinoma

    PubMed Central

    Liu, Yuan; Lv, Ji-Yun; Shi, Jian-Fei; Yang, Mei; Liu, Shu-Hong; Li, Zhi-Wei; Wang, Hong-Bo; Zhang, Shao-Geng; Liu, Zhen-Wen; Ding, Jin-Biao; Xu, Dong-Ping; Zhao, Jing-Min

    2014-01-01

    Caveolin-1 and flotillin-1 are considered as markers of lipid rafts which can be regarded as sorting platforms for targeted transport of transmembrane proteins and are involved in fundamental cellular events such as signal transduction, cell adhesion, lipid/protein sorting, and human cancer. We addressed caveolin-1 and flotillin-1 expression in 90 human hepatocellular carcinoma (HCC) and adjacent noncancerous tissues (ANT) samples by SDS-PAGE and immunoblotting with specific antibodies. Significant caveolin-1 and flotillin-1 overexpression was found in HCC tissues compared to ANT and was confirmed by immunohistochemistry. Raft-associated Akt signaling pathway components involved in the regulation of cell survival were altered by western blotting in HCC microdomain-enriched subcellular fractions purified from paired HCC and ANT samples. Our results demonstrated that the activity of raft-associated but not total membrane Akt determines its cellular functions. Lipid rafts differ in different types of tissues, which allows for the possibility of tissue-type-specific targeting for cell survival. PMID:25243186

  6. Novel Targeting of Phoshatidylinositol-3 Kinase and Mammalian Target of Rapamycin (mTOR) in Renal Cell Carcinoma

    PubMed Central

    Cho, Daniel

    2013-01-01

    Allosteric inhibitors of the kinase mammalian target of rapamycin (mTOR) have demonstrated significant clinical activity in patients with advanced renal cell carcinoma (RCC). Unfortunately, substantial clinical responses to these rapalogues are only seen in a subset of patients with advanced RCC. Preclinical studies have identified multiple theoretical shortcomings of the rapalogues and numerous novel agents directed against the PI3-K/Akt/mTOR pathway which address many of these shortcomings are in active clinical development. In this review, we will discuss the preclinical and clinical experience with the rapalogues in RCC, potential mechanisms of resistance to the rapalogues, and the progress in the clinical development of novel agents directed against the PI3-K/Akt/mTOR Pathway. PMID:23867512

  7. The ketogenic diet inhibits the mammalian target of rapamycin (mTOR) pathway

    PubMed Central

    McDaniel, Sharon S.; Rensing, Nicholas R.; Thio, Liu Lin; Yamada, Kelvin A.; Wong, Michael

    2011-01-01

    Summary The ketogenic diet (KD) is an effective treatment for epilepsy, but its mechanisms of action are poorly understood. We investigated the hypothesis that KD inhibits mammalian target of rapamycin (mTOR) pathway signaling. The expression of pS6 and pAkt, markers of mTOR pathway activation, was reduced in hippocampus and liver of rats fed KD. In the kainate model of epilepsy, KD blocked the hippocampal pS6 elevation that occurs after status epilepticus. As mTOR signaling has been implicated in epileptogenesis, these results suggest that the KD may have anticonvulsant or antiepileptogenic actions via mTOR pathway inhibition. PMID:21371020

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

    PubMed

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

    2016-08-15

    The serine-threonine protein kinase Akt, also known as protein kinase B, is a key component of the phosphoinositide 3-kinase (PI3K)-Akt-mTOR axis. Deregulated activation of this pathway is frequent in human tumors and Akt-dependent signaling appears to be critical in cell survival. PI3K activation generates 3-phosphorylated phosphatidylinositols that bind Akt pleckstrin homology (PH) domain. The blockage of Akt PH domain/phosphoinositides interaction represents a promising approach to interfere with the oncogenic potential of over-activated Akt. In the present study, phosphatidyl inositol mimics based on a β-glucoside scaffold have been synthesized as Akt inhibitors. The compounds possessed one or two lipophilic moieties of different length at the anomeric position of glucose, and an acidic or basic group at C-6. Docking studies, ELISA Akt inhibition assays, and cellular assays on different cell models highlighted 1-O-octadecanoyl-2-O-β-d-sulfoquinovopyranosyl-sn-glycerol as the best Akt inhibitor among the synthesized compounds, which could be considered as a lead for further optimization in the design of Akt inhibitors. PMID:27316541

  9. The cytotoxic T cell proteome and its shaping by mammalian Target of Rapamycin

    PubMed Central

    Hukelmann, Jens L.; Anderson, Karen E.; Sinclair, Linda V.; Grzes, Katarzyna M.; Murillo, Alejandro Brenes; Hawkins, Phillip T.; Stephens, Len R.; Lamond, Angus I.; Cantrell, Doreen A.

    2015-01-01

    High-resolution mass spectrometry maps the cytotoxic T lymphocyte (CTL) proteome and the impact of mammalian target of rapamycin complex 1 (mTORC1) on CTLs. The CTL proteome was dominated by metabolic regulators and granzymes and mTORC1 selectively repressed and promoted expression of subset of CTL proteins (~10%). These included key CTL effector molecules, signaling proteins and a subset of metabolic enzymes. Proteomic data highlighted the potential for mTORC1 negative control of phosphatidylinositol (3,4,5)-trisphosphate (PtdIns(3,4,5)P3) production in CTL. mTORC1 was shown to repress PtdIns(3,4,5)P3 production and to determine the mTORC2 requirement for activation of the kinase Akt. Unbiased proteomic analysis thus provides a comprehensive understanding of CTL identity and mTORC1 control of CTL function. PMID:26551880

  10. Targeting the PI3K/AKT/mTOR pathway: potential for lung cancer treatment

    PubMed Central

    Cheng, Haiying; Shcherba, Marina; Pendurti, Gopichand; Liang, Yuanxin; Piperdi, Bilal; Perez-Soler, Roman

    2014-01-01

    SUMMARY The PI3K/AKT/mTOR pathway is commonly activated in non-small-cell lung cancer. It plays important roles in promoting oncogenesis in lung cancer and mediating resistance to EGF receptor tyrosine kinase inhibitors. Targeted agents against the components of this pathway are currently in development and their clinical benefits remain to be defined. This review provides an overview of the pathway dysregulation and novel agents targeting the pathway in lung cancer. In addition, potential predictive biomarkers guiding patient selection for targeted PI3K/AKT/mTOR inhibition is also discussed. PMID:25342981

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

    PubMed Central

    Pal, Ipsita; Mandal, Mahitosh

    2012-01-01

    The PI3K-Akt pathway is a vital regulator of cell proliferation and survival. Alterations in the PIK3CA gene that lead to enhanced PI3K kinase activity have been reported in many human cancer types, including cancers of the colon, breast, brain, liver, stomach and lung. Deregulation of PI3K causes aberrant Akt activity. Therefore targeting this pathway could have implications for cancer treatment. The first generation PI3K-Akt inhibitors were proven to be highly effective with a low IC50, but later, they were shown to have toxic side effects and poor pharmacological properties and selectivity. Thus, these inhibitors were only effective in preclinical models. However, derivatives of these first generation inhibitors are much more selective and are quite effective in targeting the PI3K-Akt pathway, either alone or in combination. These second-generation inhibitors are essentially a specific chemical moiety that helps to form a strong hydrogen bond interaction with the PI3K/Akt molecule. The goal of this review is to delineate the current efforts that have been undertaken to inhibit the various components of the PI3K and Akt pathway in different types of cancer both in vitro and in vivo. Our focus here is on these novel therapies and their inhibitory effects that depend upon their chemical nature, as well as their development towards clinical trials. PMID:22983389

  12. Taking aim at Alzheimer’s disease through the mammalian target of rapamycin

    PubMed Central

    Maiese, Kenneth

    2014-01-01

    A significant portion of the world’s population suffers from sporadic Alzheimer’s disease (AD) with available present therapies limited to symptomatic care that does not alter disease progression. Over the next decade, advancing age of the global population will dramatically increase the incidence of AD and severely impact health care resources, necessitating novel, safe, and efficacious strategies for AD. The mammalian target of rapamycin (mTOR) and its protein complexes mTOR Complex 1 (mTORC1) and mTOR Complex 2 (mTORC2) offer exciting and unique avenues of intervention for AD through the oversight of programmed cell death pathways of apoptosis, autophagy, and necroptosis. mTOR modulates multi-faceted signal transduction pathways that involve phosphoinositide 3-kinase (PI 3-K), protein kinase B (Akt), hamartin (tuberous sclerosis 1)/tuberin (tuberous sclerosis 2) (TSC1/TSC2) complex, proline-rich Akt substrate 40 kDa (PRAS40), and p70 ribosomal S6 kinase (p70S6K) and can interface with the neuroprotective pathways of growth factors, sirtuins, wingless, fork-head transcription factors, and glycogen synthase kinase-3β. With the ability of mTOR to broadly impact cellular function, clinical strategies for AD that implement mTOR must achieve parallel objectives of protecting neuronal, vascular, and immune cell survival in conjunction with preserving networks that determine memory and cognitive function. PMID:25105207

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

    PubMed

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

    2014-02-01

    Preclinical data and tumor specimen studies report that AKT kinases are related to many human cancers. Therefore, identification and development of small molecule inhibitors targeting AKT and its signaling pathway can be therapeutic in treatment of cancer. Numerous studies report inhibitors that target the ATP-binding pocket in the kinase domains, but the similarity of this site, within the kinase family makes selectivity a major problem. The sequence identity amongst PH domains is significantly lower than that in kinase domains and developing more selective inhibitors is possible if PH domain is targeted. This in silico screening study is the first time report toward the identification of potential allosteric inhibitors expected to bind the cavity between kinase and PH domains of Akt1. Structural information of Akt1 was used to develop structure-based pharmacophore models comprising hydrophobic, acceptor, donor and ring features. The 3D structural information of previously identified allosteric Akt inhibitors obtained from literature was employed to develop a ligand-based pharmacophore model. Database was generated with drug like subset of ZINC and screening was performed based on 3D similarity to the selected pharmacophore hypotheses. Binding modes and affinities of the ligands were predicted by Glide software. Top scoring hits were further analyzed considering 2D similarity between the compounds, interactions with Akt1, fitness to pharmacophore models, ADME, druglikeness criteria and Induced-Fit docking. Using virtual screening methodologies, derivatives of 3-methyl-xanthine, quinoline-4-carboxamide and 2-[4-(cyclohexa-1,3-dien-1-yl)-1H-pyrazol-3-yl]phenol were proposed as potential leads for allosteric inhibition of Akt1. PMID:24291487

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

    PubMed

    Iskandar, Kartini; Rezlan, Majidah; Pervaiz, Shazib

    2014-10-01

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

  15. Efficacy of targeted AKT inhibition in genetically engineered mouse models of PTEN-deficient prostate cancer

    PubMed Central

    De Velasco, Marco A.; Kura, Yurie; Yoshikawa, Kazuhiro; Nishio, Kazuto; Davies, Barry R.; Uemura, Hirotsugu

    2016-01-01

    The PI3K/AKT pathway is frequently altered in advanced human prostate cancer mainly through the loss of functional PTEN, and presents as potential target for personalized therapy. Our aim was to determine the therapeutic potential of the pan-AKT inhibitor, AZD5363, in PTEN-deficient prostate cancer. Here we used a genetically engineered mouse (GEM) model of PTEN-deficient prostate cancer to evaluate the in vivo pharmacodynamic and antitumor activity of AZD5363 in castration-naïve and castration-resistant prostate cancer. An additional GEM model, based on the concomitant inactivation of PTEN and Trp53 (P53), was established as an aggressive model of advanced prostate cancer and was used to further evaluate clinically relevant endpoints after treatment with AZD5363. In vivo pharmacodynamic studies demonstrated that AZD5363 effectively inhibited downstream targets of AKT. AZD5363 monotherapy significantly reduced growth of tumors in castration-naïve and castration-resistant models of PTEN-deficient prostate cancer. More importantly, AZD5363 significantly delayed tumor growth and improved overall survival and progression-free survival in PTEN/P53 double knockout mice. Our findings demonstrate that AZD5363 is effective against GEM models of PTEN-deficient prostate cancer and provide lines of evidence to support further investigation into the development of treatment strategies targeting AKT for the treatment of PTEN-deficient prostate cancer. PMID:26910118

  16. miR-382 targeting PTEN-Akt axis promotes liver regeneration

    PubMed Central

    Wang, Fei; Dimitrova-Shumkovska, Jasmina; Xiang, Yang; Zhao, Yingying; Liu, Jingqi; Xiao, Junjie; Yang, Changqing

    2016-01-01

    Liver regeneration is a highly orchestrated process which can be regulated by microRNAs (miRNAs, miRs), though the mechanisms are largely unclear. This study was aimed to identify miRNAs responsible for hepatocyte proliferation during liver regeneration. Here we detected a marked elevation of miR-382 in the mouse liver at 48 hrs after partial hepatectomy (PH-48h) using microarray analysis and qRT-PCRs. miR-382 overexpression accelerated the proliferation and the G1 to S phase transition of the cell cycle both in mouse NCTC1469 and human HL7702 normal liver cells, while miR-382 downregulation had inverse effects. Moreover, miR-382 negatively regulated PTEN expression and increased Akt phosphorylation both in vitro and in vivo. Using PTEN siRNA and Akt activator/inhibitor, we further found that PTEN inhibition and Akt phosphorylation were essential for mediating the promotive effect of miR-382 in the proliferation and cell growth of hepatocytes. Collectively, our findings identify miR-382 as a promoter for hepatocyte proliferation and cell growth via targeting PTEN-Akt axis which might be a novel therapeutic target to enhance liver regeneration capability. PMID:26636539

  17. Novel Kinase Inhibitors Targeting the PH Domain of AKT for Preventing and Treating Cancer | NCI Technology Transfer Center | TTC

    Cancer.gov

    The National Cancer Institute's Medical Oncology Branch is seeking statements of capability or interest from parties interested in licensing and co-development collaborative research to further develop, evaluate, or commercialize novel kinase inhibitors targeting the PH domain of AKT.

  18. Concurrent targeting Akt and sphingosine kinase 1 by A-674563 in acute myeloid leukemia cells.

    PubMed

    Xu, Lin; Zhang, Yanan; Gao, Meng; Wang, Guangping; Fu, Yunfeng

    2016-04-15

    Akt signaling plays a pivotal role in acute myeloid leukemia (AML) development and progression. In the present study, we evaluated the potential anti-AML activity by a novel Akt kinase inhibitor A-674563. Our results showed that A-674563 dose-dependently inhibited survival and proliferation of U937 AML cells and six lines of human AML progenitor cells, yet sparing human peripheral blood mononuclear leukocytes (PBMCs). A-674563 activated caspase-3/9 and apoptosis in the AML cells. Reversely, the pan-caspase inhibitor z-VAD-CHO dramatically alleviated A-674563-induced AML cell apoptosis and cytotoxicity. For the molecular study, we showed that A-674563 blocked Akt activation in U937 cells and human AML progenitor cells. Further, A-674563 decreased sphingosine kinase 1 (SphK1) activity in above AML cells to deplete pro-survival sphingosine-1-phosphate (S1P) and boost pro-apoptotic ceramide production. Such an effect on SphK1 signaling by A-674563 appeared independent of Akt blockage. Significantly, K6PC-5, a novel SphK1 activator, or supplement with S1P attenuated A-674563-induced ceramide production, and subsequent U937 cell death and apoptosis. Importantly, intraperitoneal injection of A-674563 at well-tolerated doses suppressed U937 leukemic xenograft tumor growth in nude mice, whiling significantly improving the animal survival. The results of the current study demonstrate that A-674563 exerts potent anti-leukemic activity in vitro and in vivo, possibly via concurrent targeting Akt and SphK1 signalings. PMID:26920060

  19. Targeted Apoptotic Effects of Thymoquinone and Tamoxifen on XIAP Mediated Akt Regulation in Breast Cancer

    PubMed Central

    Rajput, Shashi; Kumar, B. N. Prashanth; Sarkar, Siddik; Das, Subhasis; Azab, Belal; Santhekadur, Prasanna K.; Das, Swadesh K.; Emdad, Luni; Sarkar, Devanand; Fisher, Paul B.; Mandal, Mahitosh

    2013-01-01

    X-linked inhibitor of apoptosis protein (XIAP) is constitutively expressed endogenous inhibitor of apoptosis, exhibit its antiapoptotic effect by inactivating key caspases such as caspase-3, caspase-7 and caspase-9 and also play pivotal role in rendering cancer chemoresistance. Our studies showed the coadministration of TQ and TAM resulting in a substantial increase in breast cancer cell apoptosis and marked inhibition of cell growth both in vitro and in vivo. Anti-angiogenic and anti-invasive potential of TQ and TAM was assessed through in vitro studies. This novel combinatorial regimen leads to regulation of multiple cell signaling targets including inactivation of Akt and XIAP degradation. At molecular level, TQ and TAM synergistically lowers XIAP expression resulting in binding and activation of caspase-9 in apoptotic cascade, and interfere with cell survival through PI3-K/Akt pathway by inhibiting Akt phosphorylation. Cleaved caspase-9 further processes other intracellular death substrates such as PARP thereby shifting the balance from survival to apoptosis, indicated by rise in the sub-G1 cell population. This combination also downregulates the expression of Akt-regulated downstream effectors such as Bcl-xL, Bcl-2 and induce expression of Bax, AIF, cytochrome C and p-27. Consistent with these results, overexpression studies further confirmed the involvement of XIAP and its regulatory action on Akt phosphorylation along with procaspase-9 and PARP cleavage in TQ-TAM coadministrated induced apoptosis. The ability of TQ and TAM in inhibiting XIAP was confirmed through siRNA-XIAP cotransfection studies. This novel modality may be a promising tool in breast cancer treatment. PMID:23613836

  20. Antineoplastic effects of mammalian target of rapamycine inhibitors.

    PubMed

    Salvadori, Maurizio

    2012-10-24

    Cancer after transplantation is the third cause of death and one of the more relevant comorbidities. Aim of this review is to verify the role of different pathogenetic mechanisms in cancer development in transplant patients and in general population as well. In particular has been outlined the different role exerted by two different families of drug as calcineurin inhibitor and mammalian target of rapamycin (mTOR) inhibitor. The role of mTOR pathways in cell homeostasis is complex but enough clear. As a consequence the mTOR pathway deregulation is involved in the genesis of several cancers. Hence the relevant role of mTOR inhibitors. The authors review the complex mechanism of action of mTOR inhibitors, not only for what concerns the immune system but also other cells as endothelial, smooth muscle and epithelial cells. The mechanism of action is still now not completely defined and understood. It implies the inhibition of mTOR pathway at different levels, but mainly at level of the phosphorylation of several intracellular kinases that contribute to activate mTOR complex. Many prospective and retrospective studies in transplant patients document the antineoplastic role of mTOR inhibition. More recently mTOR inhibitors proven to be effective in the treatment of some cancers also in general population. Kidney cancers, neuroendocrine tumors and liver cancers seem to be the most sensitive to these drugs. Best results are obtained with a combination treatment, targeting the mTOR pathway at different levels. PMID:24175199

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

    SciTech Connect

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

    2010-09-30

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

  2. Alisertib induces apoptosis and autophagy through targeting the AKT/mTOR/AMPK/p38 pathway in leukemic cells.

    PubMed

    Fu, Yunfeng; Zhang, Yanan; Gao, Meng; Quan, Lingli; Gui, Rong; Liu, Jing

    2016-07-01

    Alisertib, a potent and selective Aurora kinase A inhibitor, has been demonstrated to exert potent anti-cancer effects in pre-clinical and clinical studies. However, mechanisms of action of alisertib, including the molecular pathways involved in alisertib-induced apoptosis and autophagy of leukemic cells, have remained elusive. The aim of the present study was to investigate the effects of alisertib on cell growth, apoptosis and autophagy and to delineate the possible molecular mechanisms in leukemic cells. Acid phosphatase, MTT and Annexin V/propidium iodide staining assays as well as immunostaining for light chain 3B showed that treatment of the REH leukemia cell line with alisertib exerted potent growth inhibitory effects, and induced apoptosis and autophagy in a dose‑dependent manner. Western blot analysis indicated that these effects may be attributed to the suppression of the activity of the Akt/mammalian target of rapamycin/5'-AMP-dependent kinase/p38 mitogen-activated protein kinase signaling pathways in REH cells. The present study confirmed that alisertib may represent a promising autophagy-inducing drug for the treatment of leukemia and shed light on its molecular mechanism of action. PMID:27177156

  3. Irradiation promotes Akt-targeting therapeutic gene delivery to the tumor vasculature

    SciTech Connect

    Sonveaux, Pierre; Frerart, Francoise; Bouzin, Caroline; Brouet, Agnes; Wever, Julie de; Jordan, Benedicte F.; Gallez, Bernard; Feron, Olivier . E-mail: feron@mint.ucl.ac.be

    2007-03-15

    Purpose: To determine whether radiation-induced increases in nitric oxide (NO) production can influence tumor blood flow and improve delivery of Akt-targeting therapeutic DNA lipocomplexes to the tumor. Methods and Materials: The contribution of NO to the endothelial response to radiation was identified using NO synthase (NOS) inhibitors and endothelial NOS (eNOS)-deficient mice. Reporter-encoding plasmids complexed with cationic lipids were used to document the tumor vascular specificity and the efficacy of in vivo lipofection after irradiation. A dominant-negative Akt gene construct was used to evaluate the facilitating effects of radiotherapy on the therapeutic transgene delivery. Results: The abundance of eNOS protein was increased in both irradiated tumor microvessels and endothelial cells, leading to a stimulation of NO release and an associated increase in tumor blood flow. Transgene expression was subsequently improved in the irradiated vs. nonirradiated tumor vasculature. This effect was not apparent in eNOS-deficient mice and could not be reproduced in irradiated cultured endothelial cells. Finally, we combined low-dose radiotherapy with a dominant-negative Akt gene construct and documented synergistic antitumor effects. Conclusions: This study offers a new rationale to combine radiotherapy with gene therapy, by directly exploiting the stimulatory effects of radiation on NO production by tumor endothelial cells. The preferential expression of the transgene in the tumor microvasculature underscores the potential of such an adjuvant strategy to limit the angiogenic response of irradiated tumors.

  4. Colocalized delivery of rapamycin and paclitaxel to tumors enhances synergistic targeting of the PI3K/Akt/mTOR pathway.

    PubMed

    Blanco, Elvin; Sangai, Takafumi; Wu, Suhong; Hsiao, Angela; Ruiz-Esparza, Guillermo U; Gonzalez-Delgado, Carlos A; Cara, Francisca E; Granados-Principal, Sergio; Evans, Kurt W; Akcakanat, Argun; Wang, Ying; Do, Kim-Anh; Meric-Bernstam, Funda; Ferrari, Mauro

    2014-07-01

    Ongoing clinical trials target the aberrant PI3K/Akt/mammalian target of rapamycin (mTOR) pathway in breast cancer through administration of rapamycin, an allosteric mTOR inhibitor, in combination with paclitaxel. However, synergy may not be fully exploited clinically because of distinct pharmacokinetic parameters of drugs. This study explores the synergistic potential of site-specific, colocalized delivery of rapamycin and paclitaxel through nanoparticle incorporation. Nanoparticle drug loading was accurately controlled, and synergistic drug ratios established in vitro. Precise drug ratios were maintained in tumors 48 hours after nanoparticle administration to mice, at levels twofold greater than liver and spleen, yielding superior antitumor activity compared to controls. Simultaneous and preferential in vivo delivery of rapamycin and paclitaxel to tumors yielded mechanistic insights into synergy involving suppression of feedback loop Akt phosphorylation and its downstream targets. Findings demonstrate that a same time, same place, and specific amount approach to combination chemotherapy by means of nanoparticle delivery has the potential to successfully translate in vitro synergistic findings in vivo. Predictive in vitro models can be used to determine optimum drug ratios for antitumor efficacy, while nanoparticle delivery of combination chemotherapies in preclinical animal models may lead to enhanced understanding of mechanisms of synergy, ultimately opening several avenues for personalized therapy. PMID:24569835

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

    SciTech Connect

    Wang, Yelin; Hu, Chen; Cheng, Jun; Chen, Binquan; Ke, Qinghong; Lv, Zhen; Wu, Jian; Zhou, Yanfeng

    2014-04-18

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

  6. Suppression of esophageal tumor growth and chemoresistance by directly targeting the PI3K/AKT pathway.

    PubMed

    Li, Bin; Li, Jin; Xu, Wen Wen; Guan, Xin Yuan; Qin, Yan Ru; Zhang, Li Yi; Law, Simon; Tsao, Sai Wah; Cheung, Annie L M

    2014-11-30

    Esophageal cancer is the sixth most common cause of cancer-related deaths worldwide. Novel therapeutic intervention is urgently needed for this deadly disease. The functional role of PI3K/AKT pathway in esophageal cancer is little known. In this study, our results from 49 pairs of human esophageal tumor and normal specimens demonstrated that AKT was constitutively active in the majority (75.5%) of esophageal tumors compared with corresponding normal tissues. Inhibition of the PI3K/AKT pathway with specific inhibitors, wortmannin and LY294002, significantly reduced Bcl-xL expression, induced caspase-3-dependent apoptosis, and repressed cell proliferation and tumor growth in vitro and in vivo without obvious toxic effects. Moreover, significantly higher expression level of p-AKT was observed in fluorouracil (5-FU)-resistant esophageal cancer cells. Inactivation of PI3K/AKT pathway markedly increased the sensitivity and even reversed acquired resistance of esophageal cancer cells to chemotherapeutic drugs in vitro. More importantly, the resistance of tumor xenografts derived from esophageal cancer cells with acquired 5-FU resistance to chemotherapeutic drugs was significantly abrogated by wortmannin treatment in animals. In summary, our data support PI3K/AKT as a valid therapeutic target and strongly suggest that PI3K/AKT inhibitors used in conjunction with conventional chemotherapy may be a potentially useful therapeutic strategy in treating esophageal cancer patients. PMID:25344912

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

    SciTech Connect

    Hehlgans, Stephanie; Department of Radiotherapy and Oncology, University of Frankfurt, Frankfurt am Main; Institute of Radiopharmacy, Helmholtz Center Dresden-Rossendorf, Dresden ; Eke, Iris; Cordes, Nils; Institute of Radiopharmacy, Helmholtz Center Dresden-Rossendorf, Dresden; Department of Radiation Oncology, University Hospital and Medical Faculty Carl Gustav Carus, Dresden University of Technology, Dresden

    2012-08-01

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

  8. Role of Calcineurin, hnRNPA2 and Akt in Mitochondrial Respiratory Stress-Mediated Transcription Activation of Nuclear Gene Targets

    PubMed Central

    Guha, Manti; Tang, Weigang; Sondheimer, Neal; Avadhani, Narayan G.

    2010-01-01

    Pathophysiological conditions causing mitochondrial dysfunction and altered transmembrane potential (Δψm) initiate a mitochondrial respiratory stress response, also known as mitochondrial retrograde response, in a variety of mammalian cells. An increase in the cytosolic Ca2+ [Ca2+]c as part of this signaling cascade activates Ca2+ responsive phosphatase, Calcineurin (Cn). Activation of IGF1R accompanied by increased glycolysis, invasiveness, and resistance to apoptosis are phenotypic hallmarks of C2C12 rhabdomyoblast cells subjected to this stress. The signaling is associated with activation and increased nuclear translocation of a number of transcription factors including a novel NFκB (cRel: p50) pathway, NFAT, CREB and C/EBPδ. This culminates in the upregulation of a number of nuclear genes including Cathepsin L, RyR1, Glut4 and Akt1. We observed that stress regulated transcription activation of nuclear genes involves a cooperative interplay between NFκB (cRel:p50), C/EBPδ, CREB, NFAT. Our results show that the functional synergy of these factors requires the stress-activated heterogeneous nuclear ribonucleoprotein, hnRNPA2 as a transcriptional co-activator. We report here that mitochondrial stress leads to induced expression and activation of serine threonine kinase Akt1. Interestingly, we observe that Akt1 phosphorylates hnRNPA2 under mitochondrial stress conditions, which is a crucial step for the recruitment of this coactivator to the stress target promoters and culmination in mitochondrial stress-mediated transcription activation of target genes. We propose that mitochondrial stress plays an important role in tumor progression and emergence of invasive phenotypes. PMID:20153290

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

    SciTech Connect

    Zhang, Pengpeng; Shan, Tizhong; Liang, Xinrong; Deng, Changyan; Kuang, Shihuan

    2014-09-12

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

  10. [Significance of mTOR (mammalian target of rapamycin) activity in human lymphomas].

    PubMed

    Márk, Ágnes

    2014-06-01

    Neoplastic processes, tumor growth, and tumor cell proliferation and survival are often due to the altered activation of different signaling pathways. The increased activity of PI3K/AKT/mTOR signaling has been shown to be an important regulator of tumor growth in several solid tumors and in mantle cell lymphomas. The active form of mTOR kinase (mammalian target of rapamycin) is a key signaling molecule, and it exists in two different complexes, mTORC1 and mTORC2. In the present work, mTOR activity was investigated in different lymphoma types, in parallel with clinical data. We also examined in Hodgkin lymphomas (HL) the role of mTOR activity in survival mechanisms such as antiapoptotic protein expression and alterations in the microenvironment. We determined which lymphoma types display characteristic high mTOR activity in our TMA (tissue microarray) study. We observed that mTOR activity is increased in mitotic lymphoid cells compared to interphasic cells. The number of diffuse large B cell lymphoma (DLBCL) and HL cases was extended in a further set of TMA. We observed significantly higher mTOR activity in the non-centrum germinativum derived subtype of DLBCL than in the centrum germinativum derived subtype, which was a prognostic marker; 63% of mTOR active cases showed Rictor overexpression, indicating mTORC2 activity. High mTOR activity was also established in 92% of HL cases, which was linked to mTORC1. This finding was not a prognostic marker, however, it can be useful in targeted therapy. We observed the overexpression of the antiapoptotic protein BCL-xL and NFκB-p50 in the majority of mTOR active HLs. HLs showed high numbers of regulatory T cells in the microenvironment and high expression of galectin-1 in tumor cells and in the extracellular matrix, when compared to reactive lymph nodes. We confirmed that mTOR inhibition had significant antiproliferative and antiapoptotic effects in lymphoma cell lines and in lymphoma xenografts (HL, DLBCL, Burkitt lymphoma

  11. Targeting the glucose-regulated protein-78 abrogates Pten-null driven AKT activation and endometrioid tumorigenesis.

    PubMed

    Lin, Y G; Shen, J; Yoo, E; Liu, R; Yen, H-Y; Mehta, A; Rajaei, A; Yang, W; Mhawech-Fauceglia, P; DeMayo, F J; Lydon, J; Gill, P; Lee, A S

    2015-10-01

    Rates of the most common gynecologic cancer, endometrioid adenocarcinoma (EAC), continue to rise, mirroring the global epidemic of obesity, a well-known EAC risk factor. Thus, identifying novel molecular targets to prevent and/or mitigate EAC is imperative. The prevalent Type 1 EAC commonly harbors loss of the tumor suppressor, Pten, leading to AKT activation. The major endoplasmic reticulum (ER) chaperone, GRP78, is a potent pro-survival protein to maintain ER homeostasis, and as a cell surface protein, is known to regulate the phosphatidylinositol 3-kinase (PI3K)/AKT pathway. To determine whether targeting GRP78 could suppress EAC development, we created a conditional knockout mouse model using progesterone receptor-Cre-recombinase to achieve Pten and Grp78 (cPten(f/f)Grp78(f/f)) deletion in the endometrial epithelium. Mice with a single Pten (cPten(f/f)) deletion developed well-differentiated EAC by 4 weeks. In contrast, no cPten(f/f)Grp78(f/f) mice developed EAC, even after more than 8 months of observation. Histologic examination of uteri from cPten(f/f)Grp78(f/f) mice also revealed no complex atypical hyperplasia, a well-established EAC precursor. These histologic observations among the cPten(f/f)Grp78(f/f) murine uteri also corresponded to abrogation of AKT activation within the endometrium. We further observed that GRP78 co-localized with activated AKT on the surface of EAC, thus providing an opportunity for therapeutic targeting. Consistent with previous findings that cell surface GRP78 is an upstream regulator of PI3K/AKT signaling, we show here that in vivo short-term systemic treatment with a highly specific monoclonal antibody against GRP78 suppressed AKT activation and increased apoptosis in the cPten(f/f) tumors. Collectively, these findings present GRP78-targeting therapy as an efficacious therapeutic option for EAC. PMID:25684138

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

    PubMed Central

    HAN, YONG-SEOK; LEE, JUN HEE; LEE, SANG HUN

    2015-01-01

    Fucoidan, a sulfated polysaccharide, has a variety of biological activities, including anti-cancer, anti-angiogenic and anti-inflammatory effects. However, the underlying mechanisms of fucoidan as an anti-cancer agent remain to be elucidated. The present study examined the anti-cancer effect of fucoidan on HT-29 human colon cancer cells. The cell growth of HT29 cells was significantly decreased following treatment with fucoidan (200 μg/ml). In addition, fucoidan inhibited the migration of HT-29 cells by decreasing the expression levels of matrix metalloproteinase-2 in a dose-dependent manner (0–200 μg/ml). The underlying mechanism of these inhibitory effects included the downregulation of phosphoinositide 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) by treatment with fucoidan. Furthermore, fucoidan increased the expression of cleaved caspase-3 and decreased cancer sphere formation. The present study suggested that fucoidan exerts an anti-cancer effect on HT-29 human colon cancer cells by downregulating the PI3K-Akt-mTOR signaling pathway. Therefore, fucoidan may be a potential therapeutic reagent against the growth of human colon cancer cells. PMID:25998232

  13. PI3K/Akt/mTOR: A promising therapeutic target for non-medullary thyroid carcinoma.

    PubMed

    Petrulea, Mirela S; Plantinga, Theo S; Smit, Jan W; Georgescu, Carmen E; Netea-Maier, Romana T

    2015-09-01

    Thyroid carcinoma (TC) is the most common endocrine malignancy. The pathogenesis of TC is complex and involves multiple genetic events that lead to activation of oncogenic pathways such as the MAP kinase (MAPK) pathway and the PI3K/Akt/mTOR pathway. The PI3K/Akt pathway has emerged as an important player in the pathogenesis of TC, particularly in follicular and advanced anaplastic or poorly differentiated TC. Because these patients have a poor prognosis, particularly when their tumors become resistant to the conventional treatment with radioactive iodine, efforts have been made to identify possible targets for therapy within these pathways. Orally available drugs targeting the PI3K/Akt/mTOR pathway are being used with success in treatment of several types of malignant tumors. There is an increasing amount of preclinical and clinical data supporting that this pathway may represent a promising target for systemic therapy in TC. The present review focuses on the most recent developments on the role of the PI3K/Akt pathway in the pathogenesis of non-medullary TC and will provide insight into how this pathway can be targeted either alone or in the context of multimodal therapeutic strategies for treatment of advanced TC. PMID:26138515

  14. Guggulsterone Targets Smokeless Tobacco Induced PI3K/Akt Pathway in Head and Neck Cancer Cells

    PubMed Central

    Macha, Muzafar A.; Matta, Ajay; Chauhan, Shyam Singh; Siu, K. W. Michael; Ralhan, Ranju

    2011-01-01

    Background Epidemiological association of head and neck cancer with smokeless tobacco (ST) emphasizes the need to unravel the molecular mechanisms implicated in cancer development, and identify pharmacologically safe agents for early intervention and prevention of disease recurrence. Guggulsterone (GS), a biosafe nutraceutical, inhibits the PI3K/Akt pathway that plays a critical role in HNSCC development. However, the potential of GS to suppress ST and nicotine (major component of ST) induced HNSCC remains unexplored. We hypothesized GS can abrogate the effects of ST and nicotine on apoptosis in HNSCC cells, in part by activation of PI3K/Akt pathway and its downstream targets, Bax and Bad. Methods and Results Our results showed ST and nicotine treatment resulted in activation of PI3K, PDK1, Akt, and its downstream proteins - Raf, GSK3β and pS6 while GS induced a time dependent decrease in activation of PI3K/Akt pathway. ST and nicotine treatment also resulted in induction of Bad and Bax phosphorylation, increased the association of Bad with 14-3-3ζresulting in its sequestration in the cytoplasm of head and neck cancer cells, thus blocking its pro-apoptotic function. Notably, GS pre-treatment inhibited ST/nicotine induced activation of PI3K/Akt pathway, and inhibited the Akt mediated phosphorylation of Bax and Bad. Conclusions In conclusion, GS treatment not only inhibited proliferation, but also induced apoptosis by abrogating the effects of ST / nicotine on PI3K/Akt pathway in head and neck cancer cells. These findings provide a rationale for designing future studies to evaluate the chemopreventive potential of GS in ST / nicotine associated head and neck cancer. PMID:21383988

  15. Targeting cytosolic phospholipase A2 α in colorectal cancer cells inhibits constitutively activated protein kinase B (AKT) and cell proliferation

    PubMed Central

    Xie, Chanlu; Hua, Sheng; Li, Jianfang; Wang, Tingfeng; Yao, Mu; Vignarajan, Soma; Teng, Ying; Hejazi, Leila; Liu, Bingya; Dong, Qihan

    2014-01-01

    A constitutive activation of protein kinase B (AKT) in a hyper-phosphorylated status at Ser473 is one of the hallmarks of anti-EGFR therapy-resistant colorectal cancer (CRC). The aim of this study was to examine the role of cytosolic phospholipase A2α (cPLA2α) on AKT phosphorylation at Ser473 and cell proliferation in CRC cells with mutation in phosphoinositide 3-kinase (PI3K). AKT phosphorylation at Ser473 was resistant to EGF stimulation in CRC cell lines of DLD-1 (PIK3CAE545K mutation) and HT-29 (PIK3CAP499T mutation). Over-expression of cPLA2α by stable transfection increased basal and EGF-stimulated AKT phosphorylation and proliferation in DLD-1 cells. In contrast, silencing of cPLA2α with siRNA or inhibition with Efipladib decreased basal and EGF-stimulated AKT phosphorylation and proliferation in HT-29. Treating animals transplanted with DLD-1 with Efipladib (10 mg/kg, i.p. daily) over 14 days reduced xenograft growth by >90% with a concomitant decrease in AKT phosphorylation. In human CRC tissue, cPLA2α expression and phosphorylation were increased in 63% (77/120) compared with adjacent normal mucosa determined by immunohistochemistry. We conclude that cPLA2α is required for sustaining AKT phosphorylation at Ser473 and cell proliferation in CRC cells with PI3K mutation, and may serve as a potential therapeutic target for treatment of CRC resistant to anti-EGFR therapy. PMID:25365190

  16. MiR-20b targets AKT3 and modulates vascular endothelial growth factor-mediated changes in diabetic retinopathy.

    PubMed

    Qin, Bo; Liu, Jinwen; Liu, Shenwen; Li, Baijun; Ren, Jing

    2016-08-01

    Diabetic retinopathy (DR) is the leading cause of new-onset blindness. The roles of microRNAs in diabetic retinopathy are largely unknown. The aim of this study is to investigate the role of miR-20b in DR. Transfection of miR-20b mimic in high glucose (HG)-treated human retinal endothelial cells (HRECs) increased miR-20b expression and decreased the expression level of VEGF mRNA, while transfection of miR-20b inhibitor in control HRECs reduced the miR-20b expression with a corresponding increase of VEGF mRNA. In vitro functional assay showed that transfection of miR-20b mimic prevented HG-induced increase in transendothelial permeability and tube formation in HRECs. Transfection of miR-20b inhibitor or treatment of VEGF increased transendothelial permeability and tube formation in control HRECs. Luciferase reported assay showed that AKT3 is a target of miR-20b. Transfection of miR-20b mimic prevented the up-regulation of AKT3 induced by HG without changing the protein levels of other isoforms of AKT, and silencing of AKT3 caused decrease of VEGF mRNA and protein levels as well as prevented HG-induced increase in transendothelial permeability and tube formation. Finally, we showed that miR-20b was down-regulated in the retina and retinal endothelial cells in diabetic rats, with a correlated up-regulation of VEGF and AKT3. Intravitreal injection of miR-20b mimic in the diabetic rat significantly increased the miR-20b expression and decreased the expression levels of AKT3 and VEGF in the retina tissues, and intravitreal delivery of AKT3 siRNA in the diabetic rat significantly decreased the expressions of AKT3 and VEGF. Collectively, miR-20b is important for the regulation of VEGF-mediated changes in HRECs and rat retinal tissues under hyperglycemic conditions possibly via targeting AKT3. PMID:27421659

  17. miR-214 promotes osteoclastogenesis by targeting Pten/PI3k/Akt pathway.

    PubMed

    Zhao, Chenyang; Sun, Weijia; Zhang, Pengfei; Ling, Shukuan; Li, Yuheng; Zhao, Dingsheng; Peng, Jiang; Wang, Aiyuan; Li, Qi; Song, Jinping; Wang, Cheng; Xu, Xiaolong; Xu, Zi; Zhong, Guohui; Han, Bingxing; Chang, Yan-Zhong; Li, Yingxian

    2015-01-01

    microRNA is necessary for osteoclast differentiation, function and survival. It has been reported that miR-199/214 cluster plays important roles in vertebrate skeletal development and miR-214 inhibits osteoblast function by targeting ATF4. Here, we show that miR-214 is up-regulated during osteoclastogenesis from bone marrow monocytes (BMMs) with macrophage colony stimulating factor (M-CSF) and receptor activator of nuclear factor-κB ligand (RANKL) induction, which indicates that miR-214 plays a critical role in osteoclast differentiation. Overexpression of miR-214 in BMMs promotes osteoclastogenesis, whereas inhibition of miR-214 attenuates it. We further find that miR-214 functions through PI3K/Akt pathway by targeting phosphatase and tensin homolog (Pten). In vivo, osteoclast specific miR-214 transgenic mice (OC-TG214) exhibit down-regulated Pten levels, increased osteoclast activity, and reduced bone mineral density. These results reveal a crucial role of miR-214 in the differentiation of osteoclasts, which will provide a potential therapeutic target for osteoporosis. PMID:25826666

  18. Fangchinoline targets PI3K and suppresses PI3K/AKT signaling pathway in SGC7901 cells.

    PubMed

    Tian, Feng; Ding, Ding; Li, Dandan

    2015-01-01

    Fangchinoline, an important compound in Stephania tetrandra S. Moore, as a novel antitumor agent, has been implicated in several types of cancers cells except gastric cancer. To investigate whether fangchinoline affects gastric cancer cells, we detected the signaling pathway by which fangchinoline plays a role in different human gastric cancer cells lines. We found that fangchinoline effectively suppressed proliferation and invasion of SGC7901 cell lines, but not MKN45 cell lines by inhibiting the expression of PI3K and its downstream pathway. All of the Akt/MMP2/MMP9 pathway, Akt/Bad pathway, and Akt/Gsk3β/CDK2 pathway could be inhibited by fangchinoline through inhibition of PI3K. Taken together, these results suggest that fangchinoline targets PI3K in tumor cells that express PI3K abundantly and inhibits the growth and invasive ability of the tumor cells. PMID:25872479

  19. Fangchinoline targets PI3K and suppresses PI3K/AKT signaling pathway in SGC7901 cells

    PubMed Central

    TIAN, FENG; DING, DING; LI, DANDAN

    2015-01-01

    Fangchinoline, an important compound in Stephania tetrandra S. Moore, as a novel antitumor agent, has been implicated in several types of cancers cells except gastric cancer. To investigate whether fangchinoline affects gastric cancer cells, we detected the signaling pathway by which fangchinoline plays a role in different human gastric cancer cells lines. We found that fangchinoline effectively suppressed proliferation and invasion of SGC7901 cell lines, but not MKN45 cell lines by inhibiting the expression of PI3K and its downstream pathway. All of the Akt/MMP2/MMP9 pathway, Akt/Bad pathway, and Akt/Gsk3β/CDK2 pathway could be inhibited by fangchinoline through inhibition of PI3K. Taken together, these results suggest that fangchinoline targets PI3K in tumor cells that express PI3K abundantly and inhibits the growth and invasive ability of the tumor cells. PMID:25872479

  20. Distinct roles for mammalian target of rapamycin complexes in the fibroblast response to transforming growth factor-beta.

    PubMed

    Rahimi, Rod A; Andrianifahanana, Mahefatiana; Wilkes, Mark C; Edens, Maryanne; Kottom, Theodore J; Blenis, John; Leof, Edward B

    2009-01-01

    Transforming growth factor-beta (TGF-beta) promotes a multitude of diverse biological processes, including growth arrest of epithelial cells and proliferation of fibroblasts. Although the TGF-beta signaling pathways that promote inhibition of epithelial cell growth are well characterized, less is known about the mechanisms mediating the positive response to this growth factor. Given that TGF-beta has been shown to promote fibrotic diseases and desmoplasia, identifying the fibroblast-specific TGF-beta signaling pathways is critical. Here, we investigate the role of mammalian target of rapamycin (mTOR), a known effector of phosphatidylinositol 3-kinase (PI3K) and promoter of cell growth, in the fibroblast response to TGF-beta. We show that TGF-beta activates mTOR complex 1 (mTORC1) in fibroblasts but not epithelial cells via a PI3K-Akt-TSC2-dependent pathway. Rapamycin, the pharmacologic inhibitor of mTOR, prevents TGF-beta-mediated anchorage-independent growth without affecting TGF-beta transcriptional responses or extracellular matrix protein induction. In addition to mTORC1, we also examined the role of mTORC2 in TGF-beta action. mTORC2 promotes TGF-beta-induced morphologic transformation and is required for TGF-beta-induced Akt S473 phosphorylation but not mTORC1 activation. Interestingly, both mTOR complexes are necessary for TGF-beta-mediated growth in soft agar. These results define distinct and overlapping roles for mTORC1 and mTORC2 in the fibroblast response to TGF-beta and suggest that inhibitors of mTOR signaling may be useful in treating fibrotic processes, such as desmoplasia. PMID:19117990

  1. MicroRNA-542-3p Suppresses Tumor Cell Invasion via Targeting AKT Pathway in Human Astrocytoma.

    PubMed

    Cai, Junchao; Zhao, JingJing; Zhang, Nu; Xu, Xiaonan; Li, Rong; Yi, Yang; Fang, Lishan; Zhang, Le; Li, Mengfeng; Wu, Jueheng; Zhang, Heng

    2015-10-01

    The molecular mechanism underlying constitutive activation of AKT signaling, which plays essential roles in astrocytoma progression, is not fully characterized. Increasing numbers of studies have reported that microRNAs are involved in the malignant behavior of astrocytoma cells via directly targeting multiple oncogenes or tumor suppressors. Here, we found that microRNA (miR)-542-3p expression was decreased in glioblastoma cell lines and astrocytoma tissues, and reduced levels of miR-542-3p expression correlated with high histopathological grades and poor prognosis of astrocytoma patients. Exogenous miR-542-3p suppressed glioblastoma cell invasion through not only targeting AKT1 itself but also directly down-regulating its two important upstream regulators, namely, integrin-linked kinase and PIK3R1. Notably, overexpressing miR-542-3p decreased AKT1 phosphorylation and directly and indirectly repressed nuclear translocation and transactivation activity of β-catenin to exert its anti-invasive effect. Furthermore, the miR-542-3p expression level negatively correlated with AKT activity as well as levels of integrin-linked kinase and PIK3R1 in human astrocytoma specimens. These findings suggest that miR-542-3p acts as a negative regulator in astrocytoma progression and that miR-542-3p down-regulation contributes to aberrant activation of AKT signaling, leaving open the possibility that miR-542-3p may be a potential therapeutic target for high grade astrocytoma. PMID:26286747

  2. The Marine Fungal Metabolite, AD0157, Inhibits Angiogenesis by Targeting the Akt Signaling Pathway

    PubMed Central

    García-Caballero, Melissa; Cañedo, Librada; Fernández-Medarde, Antonio; Medina, Miguel Ángel; Quesada, Ana R.

    2014-01-01

    In the course of a screening program for the inhibitors of angiogenesis from marine sources, AD0157, a pyrrolidinedione fungal metabolite, was selected for its angiosupressive properties. AD0157 inhibited the growth of endothelial and tumor cells in culture in the micromolar range. Our results show that subtoxic doses of this compound inhibit certain functions of endothelial cells, namely, differentiation, migration and proteolytic capability. Inhibition of the mentioned essential steps of in vitro angiogenesis is in agreement with the observed antiangiogenic activity, substantiated by using two in vivo angiogenesis models, the chorioallantoic membrane and the zebrafish embryo neovascularization assays, and by the ex vivo mouse aortic ring assay. Our data indicate that AD0157 induces apoptosis in endothelial cells through chromatin condensation, DNA fragmentation, increases in the subG1 peak and caspase activation. The data shown here altogether indicate for the first time that AD0157 displays antiangiogenic effects, both in vitro and in vivo, that are exerted partly by targeting the Akt signaling pathway in activated endothelial cells. The fact that these effects are carried out at lower concentrations than those required for other inhibitors of angiogenesis makes AD0157 a new promising drug candidate for further evaluation in the treatment of cancer and other angiogenesis-related pathologies. PMID:24441613

  3. The novel miR-9500 regulates the proliferation and migration of human lung cancer cells by targeting Akt1

    PubMed Central

    Yoo, J K; Jung, H Y; Lee, J M; Yi, H; Oh, S-H; Ko, H Y; Yoo, H; Kim, H-R; Song, H; Kim, S; Kim, J K

    2014-01-01

    MicroRNAs have crucial roles in lung cancer cell development. They regulate cell growth, proliferation and migration by mediating the expression of tumor suppressor genes and oncogenes. We identified and characterized the novel miR-9500 in human lung cancer cells. The miR-9500 forms a stem-loop structure and is conserved in other mammals. The expression levels of miR-9500 were reduced in lung cancer cells and lung cancer tissues compared with normal tissues, as verified by TaqMan miRNA assays. It was confirmed that the putative target gene, Akt1, was directly suppressed by miR-9500, as demonstrated by a luciferase reporter assay. The miR-9500 significantly repressed the protein expression levels of Akt1, as demonstrated via western blot, but did not affect the corresponding mRNA levels. Akt1 has an important role in lung carcinogenesis, and depletion of Akt1 has been shown to have antiproliferative and anti-migratory effects in previous studies. In the current study, the overexpression of miR-9500 inhibited cell proliferation and the expression of cell cycle-related proteins. Likewise, the overexpression of miR-9500 impeded cell migration in human lung cancer cells. In an in vivo assay, miR-9500 significantly suppressed Fluc expression compared with NC and ASO-miR-9500, suggesting that cell proliferation was inhibited in nude mice. Likewise, miR-9500 repressed tumorigenesis and metastasis by targeting Akt1. These data indicate that miR-9500 might be applicable for lung cancer therapy. PMID:24658401

  4. Strategies to Detect Endogenous Ubiquitination of a Target Mammalian Protein.

    PubMed

    Sigismund, Sara; Polo, Simona

    2016-01-01

    Different biochemical techniques are well established to investigate target's ubiquitination in mammals without overexpressing a tagged version of ubiquitin (Ub). The simplest and more direct approach is to immunoprecipitate (IP) your target protein from cell lysate (stimulated and/or properly treated), followed by western blot analysis utilizing specific antibodies against Ub (see Subheading 3.1). This approach requires a good antibody against the target working in IP; alternatively, one could express a tagged version of the protein, possibly at the endogenous level. Another approach consists in IP ubiquitinated proteins from total cell lysate followed by detection with the antibody against the protein of interest. This second method relies on the availability of specific and very efficient antibodies against Ub (see Subheading 3.2). A more quantitative approach is the DELFIA assay (Perkin Elmer), an ELISA-based assay, which allows comparing more samples and conditions (see Subheading 3.3). Cross-validation with more than one approach is usually recommended in order to prove that your protein is modified by ubiquitin.Here we will use the EGFR as model system but protocols can be easily modified according to the protein of interest. PMID:27613032

  5. Akt kinase C-terminal modifications control activation loop dephosphorylation and enhance insulin response

    PubMed Central

    Chan, Tung O.; Zhang, Jin; Tiegs, Brian C.; Blumhof, Brian; Yan, Linda; Keny, Nikhil; Penny, Morgan; Li, Xue; Pascal, John M.; Armen, Roger S.; Rodeck, Ulrich; Penn, Raymond B.

    2015-01-01

    The Akt protein kinase, also known as protein kinase B, plays key roles in insulin receptor signalling and regulates cell growth, survival and metabolism. Recently, we described a mechanism to enhance Akt phosphorylation that restricts access of cellular phosphatases to the Akt activation loop (Thr308 in Akt1 or protein kinase B isoform alpha) in an ATP-dependent manner. In the present paper, we describe a distinct mechanism to control Thr308 dephosphorylation and thus Akt deactivation that depends on intramolecular interactions of Akt C-terminal sequences with its kinase domain. Modifications of amino acids surrounding the Akt1 C-terminal mTORC2 (mammalian target of rapamycin complex 2) phosphorylation site (Ser473) increased phosphatase resistance of the phosphorylated activation loop (pThr308) and amplified Akt phosphorylation. Furthermore, the phosphatase-resistant Akt was refractory to ceramide-dependent dephosphorylation and amplified insulin-dependent Thr308 phosphorylation in a regulated fashion. Collectively, these results suggest that the Akt C-terminal hydrophobic groove is a target for the development of agents that enhance Akt phosphorylation by insulin. PMID:26201515

  6. Dual inhibition of phosphatidylinositol 3'-kinase and mammalian target of rapamycin using NVP-BEZ235 as a novel therapeutic approach for mucinous adenocarcinoma of the ovary.

    PubMed

    Kudoh, Akiko; Oishi, Tetsuro; Itamochi, Hiroaki; Sato, Seiya; Naniwa, Jun; Sato, Shinya; Shimada, Muneaki; Kigawa, Junzo; Harada, Tasuku

    2014-03-01

    Ovarian mucinous adenocarcinoma (MAC) resists standard chemotherapy and is associated with poor prognosis. A more effective treatment is needed urgently. The present study assessed the possibility of molecular-targeted therapy with a novel dual inhibitor of phosphatidylinositol 3'-kinase (PI3K) and mammalian target of rapamycin (mTOR), NVP-BEZ235 (BEZ235) to treat of MAC. Seven human MAC cell lines were used in this study. The sensitivity of the cells to BEZ235, temsirolimus, and anticancer agents was determined with the WST-8 assay. Cell cycle distribution was assessed by flow cytometry, and the expression of proteins in apoptotic pathways and molecules of the PI3K/Akt/mTOR signaling pathways was determined by Western blot analysis. We also examined the effects of BEZ235 on tumor growth in nude mice xenograft models. The cell lines showed half-maximal inhibitory concentration values of BEZ235 from 13 to 328 nmol/L. Low half-maximal inhibitory concentration values to BEZ235 were observed in MCAS and OMC-1 cells; these 2 lines have an activating mutation in the PIK3CA gene. NVP-BEZ235 down-regulated the protein expression of phosphorylated (p-) Akt, p-p70S6K, and p-4E-BP1, suppressed cell cycle progression, up-regulated the expression of cleaved PARP and cleaved caspase 9, and increased apoptotic cells. Synergistic effects were observed on more than 5 cell lines when BEZ235 was combined with paclitaxel or cisplatin. The treatment of mice bearing OMC-1 or RMUG-S with BEZ235 significantly suppressed tumor growth in MAC xenograft models without severe weight loss. We conclude that the PI3K/Akt/mTOR pathway is a potential therapeutic target and that BEZ235 should be explored as a therapeutic agent for MAC. PMID:24552895

  7. Targeting mammalian organelles with internalizing phage (iPhage) libraries

    PubMed Central

    Rangel, Roberto; Dobroff, Andrey S.; Guzman-Rojas, Liliana; Salmeron, Carolina C.; Gelovani, Juri G.; Sidman, Richard L.; Pasqualini, Renata; Arap, Wadih

    2015-01-01

    Techniques largely used for protein interaction studies and discovery of intracellular receptors, such as affinity capture complex purification and yeast two-hybrid, may produce inaccurate datasets due to protein insolubility, transient or weak protein interactions, or irrelevant intracellular context. A versatile tool to overcome these limitations as well as to potentially create vaccines and engineer peptides and antibodies as targeted diagnostic and therapeutic agents, is the phage display technique. We have recently developed a new technology for screening internalizing phage (iPhage) vectors and libraries utilizing a ligand/receptor-independent mechanism to penetrate eukaryotic cells. iPhage particles provide a unique discovery platform for combinatorial intracellular targeting of organelle ligands along with their corresponding receptors and to fingerprint functional protein domains in living cells. Here we explain the design, cloning, construction, and production of iPhage-based vectors and libraries, along with basic ligand-receptor identification and validation methodologies for organelle receptors. An iPhage library screening can be performed in ~8 weeks. PMID:24030441

  8. Antimyeloma activity of the orally bioavailable dual phosphatidylinositol 3-kinase/mammalian target of rapamycin inhibitor NVP-BEZ235.

    PubMed

    McMillin, Douglas W; Ooi, Melissa; Delmore, Jake; Negri, Joseph; Hayden, Patrick; Mitsiades, Nicolas; Jakubikova, Jana; Maira, Sauveur-Michel; Garcia-Echeverria, Carlos; Schlossman, Robert; Munshi, Nikhil C; Richardson, Paul G; Anderson, Kenneth C; Mitsiades, Constantine S

    2009-07-15

    The phosphatidylinositol 3-kinase (PI3K)-Akt-mammalian target of rapamycin (mTOR) pathway mediates proliferation, survival, and drug resistance in multiple myeloma (MM) cells. Here, we tested the anti-MM activity of NVP-BEZ235 (BEZ235), which inhibits PI3K/Akt/mTOR signaling at the levels of PI3K and mTOR. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide colorimetric survival assays showed that MM cell lines exhibited dose- and time-dependent decreased viability after exposure to BEZ235 (IC(50), 25-800 nmol/L for 48 hours). MM cells highly sensitive (IC(50), <25 nmol/L) to BEZ235 (e.g., MM.1S, MM.1R, Dox40, and KMS-12-PE) included both lines sensitive and resistant to conventional (dexamethasone, cytotoxic chemotherapeutics) agents. Pharmacologically relevant BEZ235 concentrations (25-400 nmol/L) induced rapid commitment to and induction of MM.1S and OPM-2 cell death. Furthermore, normal donor peripheral blood mononuclear cells were less sensitive (IC(50), >800 nmol/L) than the majority of MM cell lines tested, suggesting a favorable therapeutic index. In addition, BEZ235 was able to target MM cells in the presence of exogenous interleukin-6, insulin-like growth factor-1, stromal cells, or osteoclasts, which are known to protect against various anti-MM agents. Molecular profiling revealed that BEZ235 treatment decreased the amplitude of transcriptional signatures previously associated with myc, ribosome, and proteasome function, as well as high-risk MM and undifferentiated human embryonic stem cells. In vivo xenograft studies revealed significant reduction in tumor burden (P = 0.011) and survival (P = 0.028) in BEZ235-treated human MM tumor-bearing mice. Combinations of BEZ235 with conventional (e.g., dexamethasone and doxorubicin) or novel (e.g., bortezomib) anti-MM agents showed lack of antagonism. These results indicate that BEZ235 merits clinical testing, alone and in combination with other agents, in MM. PMID:19584292

  9. Discovery of a novel class of highly potent, selective, ATP-competitive, and orally bioavailable inhibitors of the mammalian target of rapamycin (mTOR).

    PubMed

    Takeuchi, Craig S; Kim, Byung Gyu; Blazey, Charles M; Ma, Sunghoon; Johnson, Henry W B; Anand, Neel K; Arcalas, Arlyn; Baik, Tae Gon; Buhr, Chris A; Cannoy, Jonah; Epshteyn, Sergey; Joshi, Anagha; Lara, Katherine; Lee, Matthew S; Wang, Longcheng; Leahy, James W; Nuss, John M; Aay, Naing; Aoyama, Ron; Foster, Paul; Lee, Jae; Lehoux, Isabelle; Munagala, Narsimha; Plonowski, Arthur; Rajan, Sharmila; Woolfrey, John; Yamaguchi, Kyoko; Lamb, Peter; Miller, Nicole

    2013-03-28

    A series of novel, highly potent, selective, and ATP-competitive mammalian target of rapamycin (mTOR) inhibitors based on a benzoxazepine scaffold have been identified. Lead optimization resulted in the discovery of inhibitors with low nanomolar activity and greater than 1000-fold selectivity over the closely related PI3K kinases. Compound 28 (XL388) inhibited cellular phosphorylation of mTOR complex 1 (p-p70S6K, pS6, and p-4E-BP1) and mTOR complex 2 (pAKT (S473)) substrates. Furthermore, this compound displayed good pharmacokinetics and oral exposure in multiple species with moderate bioavailability. Oral administration of compound 28 to athymic nude mice implanted with human tumor xenografts afforded significant and dose-dependent antitumor activity. PMID:23394126

  10. Optimization of a Series of Triazole Containing Mammalian Target of Rapamycin (mTOR) Kinase Inhibitors and the Discovery of CC-115.

    PubMed

    Mortensen, Deborah S; Perrin-Ninkovic, Sophie M; Shevlin, Graziella; Elsner, Jan; Zhao, Jingjing; Whitefield, Brandon; Tehrani, Lida; Sapienza, John; Riggs, Jennifer R; Parnes, Jason S; Papa, Patrick; Packard, Garrick; Lee, Branden G S; Harris, Roy; Correa, Matthew; Bahmanyar, Sogole; Richardson, Samantha J; Peng, Sophie X; Leisten, Jim; Khambatta, Godrej; Hickman, Matt; Gamez, James C; Bisonette, René R; Apuy, Julius; Cathers, Brian E; Canan, Stacie S; Moghaddam, Mehran F; Raymon, Heather K; Worland, Peter; Narla, Rama Krishna; Fultz, Kimberly E; Sankar, Sabita

    2015-07-23

    We report here the synthesis and structure-activity relationship (SAR) of a novel series of triazole containing mammalian target of rapamycin (mTOR) kinase inhibitors. SAR studies examining the potency, selectivity, and PK parameters for a series of triazole containing 4,6- or 1,7-disubstituted-3,4-dihydropyrazino[2,3-b]pyrazine-2(1H)-ones resulted in the identification of triazole containing mTOR kinase inhibitors with improved PK properties. Potent compounds from this series were found to block both mTORC1(pS6) and mTORC2(pAktS473) signaling in PC-3 cancer cells, in vitro and in vivo. When assessed in efficacy models, analogs exhibited dose-dependent efficacy in tumor xenograft models. This work resulted in the selection of CC-115 for clinical development. PMID:26102506

  11. Recombination induced by triple-helix-targeted DNA damage in mammalian cells.

    PubMed Central

    Faruqi, A F; Seidman, M M; Segal, D J; Carroll, D; Glazer, P M

    1996-01-01

    Gene therapy has been hindered by the low frequency of homologous recombination in mammalian cells. To stimulate recombination, we investigated the use of triple-helix-forming oligonucleotides (TFOs) to target DNA damage to a selected site within cells. By treating cells with TFOs linked to psoralen, recombination was induced within a simian virus 40 vector carrying two mutant copies of the supF tRNA reporter gene. Gene conversion events, as well as mutations at the target site, were also observed. The variety of products suggests that multiple cellular pathways can act on the targeted damage, and data showing that the triple helix can influence these pathways are presented. The ability to specifically induce recombination or gene conversion within mammalian cells by using TFOs may provide a new research tool and may eventually lead to novel applications in gene therapy. PMID:8943337

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

    SciTech Connect

    Yan-nan, Bai; Zhao-yan, Yu; Li-xi, Luo; Jiang, Yi; Qing-jie, Xia

    2014-01-17

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

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

    PubMed

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

    2013-06-01

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

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

    PubMed Central

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

    2013-01-01

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

  15. Regulation of insulin receptor substrate-1 by mTORC2 (mammalian target of rapamycin complex 2)

    PubMed Central

    DeStefano, Michael A.; Jacinto, Estela

    2016-01-01

    mTOR (mammalian target of rapamycin) responds to the presence of nutrients, energy and growth factors to link cellular metabolism, growth and proliferation. The rapamycin-sensitive mTORC (mTOR complex) 1 activates the translational regulator S6K (S6 kinase), leading to increased protein synthesis in the presence of nutrients. On the other hand, the rapamycin-insensitive mTORC2 responds to the presence of growth factors such as insulin by phosphorylating Akt to promote its maturation and allosteric activation. We recently found that mTORC2 can also regulate insulin signalling at the level of IRS-1 (insulin receptor substrate-1). Whereas mTORC1 promotes IRS-1 serine phosphorylation that is linked to IRS-1 down-regulation, we uncovered that mTORC2 mediates its degradation. In mTORC2-disrupted cells, inactive IRS-1 accumulated despite undergoing phosphorylation at the mTORC1-mediated serine sites. Defective IRS-1 degradation was due to attenuated expression of the CUL7 (Cullin 7) ubiquitin ligase substrate-targeting subunit Fbw8. mTORC2 and Fbw8 co-localize at the membrane where mTORC2 phosphorylates Ser86 to stabilize Fbw8 and promotes its cytosolic localization upon insulin stimulation. Under conditions of chronic insulin exposure, inactive serine-phosphorylated IRS-1 and Fbw8 co-localize to the cytosol where the former becomes ubiquitylated via CUL7/Fbw8. Thus mTORC2 negatively feeds back to IRS-1 via control of Fbw8 stability and localization. Our findings reveal that, in addition to persistent mTORC1 signalling, increased mTORC2 signals can promote insulin resistance due to mTORC2-mediated degradation of IRS-1. PMID:23863152

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

    PubMed Central

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

    2014-01-01

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

  17. Targeting of a histone acetyltransferase domain to a promoter enhances protein expression levels in mammalian cells.

    PubMed

    Kwaks, T H J; Sewalt, R G A B; van Blokland, R; Siersma, T J; Kasiem, M; Kelder, A; Otte, A P

    2005-01-12

    Silencing of transfected genes in mammalian cells is a fundamental problem that probably involves the (in)accessibility status of chromatin. A potential solution to this problem is to provide a cell with protein factors that make the chromatin of a promoter more open or accessible for transcription. We tested this by targeting such proteins to different promoters. We found that targeting the p300 histone acetyltransferase (HAT) domain to strong viral or cellular promoters is sufficient to result in higher expression levels of a reporter protein. In contrast, targeting the chromatin-remodeling factor Brahma does not result in stable, higher protein expression levels. The long-term effects of the targeted p300HAT domain on protein expression levels are positively reinforced, when also anti-repressor elements are applied to flank the reporter construct. These elements were previously shown to be potent blockers of chromatin-associated repressors. The simultaneous application of the targeted p300HAT domain and anti-repressor elements conveys long-term stability to protein expression. Whereas no copy number dependency is achieved by targeting of the p300HAT domain alone, copy number dependency is improved when anti-repressor elements are included. We conclude that targeting of protein domains such as HAT domains helps to facilitate expression of transfected genes in mammalian cells. However, the simultaneous application of other genomic elements such as the anti-repressor elements prevents silencing more efficiently. PMID:15607223

  18. Familial cortical dysplasia caused by mutation in the mammalian target of rapamycin regulator NPRL3.

    PubMed

    Sim, Joe C; Scerri, Thomas; Fanjul-Fernández, Miriam; Riseley, Jessica R; Gillies, Greta; Pope, Kate; van Roozendaal, Hanna; Heng, Julian I; Mandelstam, Simone A; McGillivray, George; MacGregor, Duncan; Kannan, Lakshminarayanan; Maixner, Wirginia; Harvey, A Simon; Amor, David J; Delatycki, Martin B; Crino, Peter B; Bahlo, Melanie; Lockhart, Paul J; Leventer, Richard J

    2016-01-01

    We describe first cousin sibling pairs with focal epilepsy, one of each pair having focal cortical dysplasia (FCD) IIa. Linkage analysis and whole-exome sequencing identified a heterozygous germline frameshift mutation in the gene encoding nitrogen permease regulator-like 3 (NPRL3). NPRL3 is a component of GAP Activity Towards Rags 1, a negative regulator of the mammalian target of rapamycin complex 1 signaling pathway. Immunostaining of resected brain tissue demonstrated mammalian target of rapamycin activation. Screening of 52 unrelated individuals with FCD identified 2 additional patients with FCDIIa and germline NPRL3 mutations. Similar to DEPDC5, NPRL3 mutations may be considered as causal variants in patients with FCD or magnetic resonance imaging-negative focal epilepsy. PMID:26285051

  19. Resveratrol Inhibits Inflammatory Responses via the Mammalian Target of Rapamycin Signaling Pathway in Cultured LPS-Stimulated Microglial Cells

    PubMed Central

    Guo, Jia-Zhi; Zhang, Wei; He, Ying; Song, Rui; Wang, Wen-Min; Xiao, Chun-Jie; Lu, Di

    2012-01-01

    Background Resveratrol have been known to possess many pharmacological properties including antioxidant, cardioprotective and anticancer effects. Although current studies indicate that resveratrol produces neuroprotection against neurological disorders, the precise mechanisms for its beneficial effects are still not fully understood. We investigate the effect of anti-inflammatory and mechamisms of resveratrol by using lipopolysaccharide (LPS)-stimulated murine microglial BV-2 cells. Methodology/Principal Findings BV-2 cells were treated with resveratrol (25, 50, and 100 µM) and/or LPS (1 µg/ml). Nitric oxide (NO) and prostaglandin E2 (PGE2) were measured by Griess reagent and ELISA. The mRNA and protein levels of proinflammatory proteins and cytokines were analysed by RT-PCR and double immunofluorescence labeling, respectively. Phosphorylation levels of PTEN (phosphatase and tensin homolog deleted on chromosome 10), Akt, mammalian target of rapamycin (mTOR), mitogen-activated protein kinases (MAPKs) cascades, inhibitor κB-α (IκB-α) and cyclic AMP-responsive element-binding protein (CREB) were measured by western blot. Resveratrol significantly attenuated the LPS-induced expression of NO, PGE2, inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and nuclear factor-κB (NF-κB) in BV-2 cells. Resveratrol increased PTEN, Akt and mTOR phosphorylation in a dose-dependent manner or a time-dependent manner. Rapamycin (10 nM), a specific mTOR inhibitor, blocked the effects of resveratrol on LPS-induced microglial activation. In addition, mTOR inhibition partially abolished the inhibitory effect of resveratrol on the phosphorylation of IκB-α, CREB, extracellular signal-regulated kinase 1/2 (ERK1/2), c-Jun N-terminal protein kinase (JNK), and p38 mitogen-activated protein kinase (p38 MAPK). Conclusion and Implications This study indicates that resveratrol inhibited LPS-induced proinflammatory

  20. Myopathy caused by mammalian target of rapamycin complex 1 (mTORC1) inactivation is not reversed by restoring mitochondrial function

    PubMed Central

    Romanino, Klaas; Mazelin, Laetitia; Albert, Verena; Conjard-Duplany, Agnès; Lin, Shuo; Bentzinger, C. Florian; Handschin, Christoph; Puigserver, Pere; Zorzato, Francesco; Schaeffer, Laurent; Gangloff, Yann-Gaël; Rüegg, Markus A.

    2011-01-01

    Mammalian target of rapamycin complex 1 (mTORC1) is central to the control of cell, organ, and body size. Skeletal muscle-specific inactivation of mTORC1 in mice results in smaller muscle fibers, fewer mitochondria, increased glycogen stores, and a progressive myopathy that causes premature death. In mTORC1-deficient muscles, peroxisome proliferator-activated receptor gamma coactivator 1-α (PGC-1α), which regulates mitochondrial biogenesis and glucose homeostasis, is strongly down-regulated. Here we tested whether induction of mitochondrial biogenesis pharmacologically or by the overexpression of PGC-1α is sufficient to reverse the phenotype of mice deficient for mTORC1. We show that both approaches normalize mitochondrial function, such as oxidative capacity and expression of mitochondrial genes. However, they do not prevent or delay the progressive myopathy. In addition, we find that mTORC1 has a much stronger effect than PGC-1α on the glycogen content in muscle. This effect is based on the strong activation of PKB/Akt in mTORC1-deficient mice. We also show that activation of PKB/Akt not only affects glycogen synthesis but also diminishes glycogen degradation. Thus, our work provides strong functional evidence that mitochondrial dysfunction in mice with inactivated mTORC1 signaling is caused by the down-regulation of PGC-1α. However, our data also show that the impairment of mitochondria does not lead directly to the lethal myopathy. PMID:22143799

  1. Myopathy caused by mammalian target of rapamycin complex 1 (mTORC1) inactivation is not reversed by restoring mitochondrial function.

    PubMed

    Romanino, Klaas; Mazelin, Laetitia; Albert, Verena; Conjard-Duplany, Agnès; Lin, Shuo; Bentzinger, C Florian; Handschin, Christoph; Puigserver, Pere; Zorzato, Francesco; Schaeffer, Laurent; Gangloff, Yann-Gaël; Rüegg, Markus A

    2011-12-20

    Mammalian target of rapamycin complex 1 (mTORC1) is central to the control of cell, organ, and body size. Skeletal muscle-specific inactivation of mTORC1 in mice results in smaller muscle fibers, fewer mitochondria, increased glycogen stores, and a progressive myopathy that causes premature death. In mTORC1-deficient muscles, peroxisome proliferator-activated receptor gamma coactivator 1-α (PGC-1α), which regulates mitochondrial biogenesis and glucose homeostasis, is strongly down-regulated. Here we tested whether induction of mitochondrial biogenesis pharmacologically or by the overexpression of PGC-1α is sufficient to reverse the phenotype of mice deficient for mTORC1. We show that both approaches normalize mitochondrial function, such as oxidative capacity and expression of mitochondrial genes. However, they do not prevent or delay the progressive myopathy. In addition, we find that mTORC1 has a much stronger effect than PGC-1α on the glycogen content in muscle. This effect is based on the strong activation of PKB/Akt in mTORC1-deficient mice. We also show that activation of PKB/Akt not only affects glycogen synthesis but also diminishes glycogen degradation. Thus, our work provides strong functional evidence that mitochondrial dysfunction in mice with inactivated mTORC1 signaling is caused by the down-regulation of PGC-1α. However, our data also show that the impairment of mitochondria does not lead directly to the lethal myopathy. PMID:22143799

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

    PubMed

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

    2015-08-01

    Glomerular hypertrophy is the main pathological characteristic in the early stage of diabetic nephropathy (DN), and its regulatory mechanism is closely related to mammalian target of rapamycin (mTOR) signaling pathway activity. mTOR includes mTOR complex 1 (mTORC1) and mTOR complex 2(mTORC2), in which, the upstream pathway of mTORC1 is phosphatidylinositol-3-kinase (PI3K)/serine-threonine kinase(Akt)/adenosine monophosphate activated protein kinase(AMPK), and the representative signaling molecules in the downstream pathway of mTORC1 are 4E-binding proteins(4EBP) and phosphoprotein 70 S6Kinase(p70S6K). Some Chinese herbal extracts could improve cell proliferation via intervening the expressions of the key molecules in the upstream or downstream of PIK/Akt/mTOR signaling pathway in vivo. As for glomerular mesangial cells(MC) and podocyte, mTOR plays an important role in regulating glomerular inherent cells, including adjusting cell cycle, energy metabolism and matrix protein synthesis. Rapamycin, the inhibitor of mTOR, could suppress glomerular inherent cell hypertrophy, cell proliferation, glomerular basement membrane (GBM) thickening and mesangial matrix deposition in model rats with DN. Some Chinese herbal extracts could alleviate glomerular lesions by intervening mTOR signaling pathway activity in renal tissue of DN animal models or in renal inherent cells in vivo and in vitro. PMID:26790279

  3. miR-9 and miR-124 synergistically affect regulation of dendritic branching via the AKT/GSK3β pathway by targeting Rap2a

    PubMed Central

    Xue, Qian; Yu, Caiyong; Wang, Yan; Liu, Ling; Zhang, Kun; Fang, Chao; Liu, Fangfang; Bian, Ganlan; Song, Bing; Yang, Angang; Ju, Gong; Wang, Jian

    2016-01-01

    A single microRNA (miRNA) can regulate expression of multiple proteins, and expression of an individual protein may be controlled by numerous miRNAs. This regulatory pattern strongly suggests that synergistic effects of miRNAs play critical roles in regulating biological processes. miR-9 and miR-124, two of the most abundant miRNAs in the mammalian nervous system, have important functions in neuronal development. In this study, we identified the small GTP-binding protein Rap2a as a common target of both miR-9 and miR-124. miR-9 and miR-124 together, but neither miRNA alone, strongly suppressed Rap2a, thereby promoting neuronal differentiation of neural stem cells (NSCs) and dendritic branching of differentiated neurons. Rap2a also diminished the dendritic complexity of mature neurons by decreasing the levels of pAKT and pGSK3β. Our results reveal a novel pathway in which miR-9 and miR-124 synergistically repress expression of Rap2a to sustain homeostatic dendritic complexity during neuronal development and maturation. PMID:27221778

  4. Targeting the PI3K/Akt signaling pathway in gastric carcinoma: A reality for personalized medicine?

    PubMed Central

    Singh, Shikha Satendra; Yap, Wei Ney; Arfuso, Frank; Kar, Shreya; Wang, Chao; Cai, Wanpei; Dharmarajan, Arunasalam M; Sethi, Gautam; Kumar, Alan Prem

    2015-01-01

    Frequent activation of phosphatidylinositol-3 kinases (PI3K)/Akt/mTOR signaling pathway in gastric cancer (GC) is gaining immense popularity with identification of mutations and/or amplifications of PIK3CA gene or loss of function of PTEN, a tumor suppressor protein, to name a few; both playing a crucial role in regulating this pathway. These aberrations result in dysregulation of this pathway eventually leading to gastric oncogenesis, hence, there is a need for targeted therapy for more effective anticancer treatment. Several inhibitors are currently in either preclinical or clinical stages for treatment of solid tumors like GC. With so many inhibitors under development, further studies on predictive biomarkers are needed to measure the specificity of any therapeutic intervention. Herein, we review the common dysregulation of PI3K/Akt/mTOR pathway in GC and the various types of single or dual pathway inhibitors under development that might have a superior role in GC treatment. We also summarize the recent developments in identification of predictive biomarkers and propose use of predictive biomarkers to facilitate more personalized cancer therapy with effective PI3K/Akt/mTOR pathway inhibition. PMID:26604635

  5. Sorafenib inhibits lymphoma xenografts by targeting MAPK/ERK and AKT pathways in tumor and vascular cells.

    PubMed

    Carlo-Stella, Carmelo; Locatelli, Silvia L; Giacomini, Arianna; Cleris, Loredana; Saba, Elena; Righi, Marco; Guidetti, Anna; Gianni, Alessandro M

    2013-01-01

    The anti-lymphoma activity and mechanism(s) of action of the multikinase inhibitor sorafenib were investigated using a panel of lymphoma cell lines, including SU-DHL-4V, Granta-519, HD-MyZ, and KMS-11 cell lines. In vitro, sorafenib significantly decreased cell proliferation and phosphorylation levels of MAPK and PI3K/Akt pathways while increased apoptotic cell death. In vivo, sorafenib treatment resulted in a cytostatic rather than cytotoxic effect on tumor cell growth associated with a limited inhibition of tumor volumes. However, sorafenib induced an average 50% reduction of tumor vessel density and a 2-fold increase of necrotic areas. Upon sorafenib treatment, endothelial and tumor cells from SU-DHL-4V, Granta-519, and KMS-11 nodules showed a potent inhibition of either phospho-ERK or phospho-AKT, whereas a concomitant inhibition of phospho-ERK and phospho-AKT was only observed in HD-MyZ nodules. In conclusion, sorafenib affects the growth of lymphoid cell lines by triggering antiangiogenic mechanism(s) and directly targeting tumor cells. PMID:23620775

  6. Sorafenib Inhibits Lymphoma Xenografts by Targeting MAPK/ERK and AKT Pathways in Tumor and Vascular Cells

    PubMed Central

    Carlo-Stella, Carmelo; Locatelli, Silvia L.; Giacomini, Arianna; Cleris, Loredana; Saba, Elena; Righi, Marco; Guidetti, Anna; Gianni, Alessandro M.

    2013-01-01

    The anti-lymphoma activity and mechanism(s) of action of the multikinase inhibitor sorafenib were investigated using a panel of lymphoma cell lines, including SU-DHL-4V, Granta-519, HD-MyZ, and KMS-11 cell lines. In vitro, sorafenib significantly decreased cell proliferation and phosphorylation levels of MAPK and PI3K/Akt pathways while increased apoptotic cell death. In vivo, sorafenib treatment resulted in a cytostatic rather than cytotoxic effect on tumor cell growth associated with a limited inhibition of tumor volumes. However, sorafenib induced an average 50% reduction of tumor vessel density and a 2-fold increase of necrotic areas. Upon sorafenib treatment, endothelial and tumor cells from SU-DHL-4V, Granta-519, and KMS-11 nodules showed a potent inhibition of either phospho-ERK or phospho-AKT, whereas a concomitant inhibition of phospho-ERK and phospho-AKT was only observed in HD-MyZ nodules. In conclusion, sorafenib affects the growth of lymphoid cell lines by triggering antiangiogenic mechanism(s) and directly targeting tumor cells. PMID:23620775

  7. TRIB3 suppresses tumorigenesis by controlling mTORC2/AKT/FOXO signaling

    PubMed Central

    Salazar, María; Lorente, Mar; García-Taboada, Elena; Gómez, Eduardo Pérez; Dávila, David; Zúñiga-García, Patricia; Flores, Juana M; Rodríguez, Antonio; Hegedus, Zoltan; Mosén-Ansorena, David; Aransay, Ana M; Hernández-Tiedra, Sonia; López-Valero, Israel; Quintanilla, Miguel; Sánchez, Cristina; Iovanna, Juan L; Dusetti, Nelson; Guzmán, Manuel; Francis, Sheila E; Carracedo, Arkaitz; Kiss-Toth, Endre; Velasco, Guillermo

    2015-01-01

    In a recent article, we found that Tribbles pseudokinase 3 (TRIB3) plays a tumor suppressor role and that this effect relies on the dysregulation of the phosphorylation of v-akt murine thymoma viral oncogene homolog (AKT) by the mammalian target of rapamycin complex 2 (mTORC2 complex), and the subsequent hyperphosphorylation and inactivation of the transcription factor Forkhead box O3 (FOXO3). PMID:27308456

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

    PubMed

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

    2015-10-27

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

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

    SciTech Connect

    Oommen, Deepu; Prise, Kevin M.

    2012-05-11

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

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

    PubMed

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

    2013-09-12

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

  11. Targeted mutagenesis in mammalian cells mediated by intracellular triple helix formation.

    PubMed Central

    Wang, G; Levy, D D; Seidman, M M; Glazer, P M

    1995-01-01

    As an alternative to standard gene transfer techniques for genetic manipulation, we have investigated the use of triple helix-forming oligonucleotides to target mutations to selected genes within mammalian cells. By treating monkey COS cells with oligonucleotides linked to psoralen, we have generated targeted mutations in a simian virus 40 (SV40) vector contained within the cells via intracellular triple helix formation. Oligonucleotide entry into the cells and sequence-specific triplex formation within the SV40 DNA deliver the psoralen to the targeted site. Photoactivation of the psoralen by long-wavelength UV light yields adducts and thereby mutations at that site. We engineered into the SV40 vector novel supF mutation reporter genes containing modified polypurine sites amenable to triplex formation. By comparing the abilities of a series of oligonucleotides to target these new sites, we show that targeted mutagenesis in vivo depends on the strength and specificity of the third-strand binding. Oligonucleotides with weak target site binding affinity or with only partial target site homology were ineffective at inducing mutations in the SV40 vectors within the COS cells. We also show that the targeted mutagenesis is dependent on the oligonucleotide concentration and is influenced by the timing of the oligonucleotide treatment and of the UV irradiation of the cells. Frequencies of intracellular targeted mutagenesis in the range of 1 to 2% were observed, depending upon the conditions of the experiment. DNA sequence analysis revealed that most of the mutations were T.A-to-A.T transversions precisely at the targeted psoralen intercalation site. Several deletions encompassing that site were also seen. The ability to target mutations to selected sites within mammalian cells by using modified triplex-forming oligonucleotides may provide a new research tool and may eventually lead to therapeutic applications. PMID:7862165

  12. Targeted gene knockout in mammalian cells by using engineered zinc-finger nucleases

    PubMed Central

    Santiago, Yolanda; Chan, Edmond; Liu, Pei-Qi; Orlando, Salvatore; Zhang, Lin; Urnov, Fyodor D.; Holmes, Michael C.; Guschin, Dmitry; Waite, Adam; Miller, Jeffrey C.; Rebar, Edward J.; Gregory, Philip D.; Klug, Aaron; Collingwood, Trevor N.

    2008-01-01

    Gene knockout is the most powerful tool for determining gene function or permanently modifying the phenotypic characteristics of a cell. Existing methods for gene disruption are limited by their efficiency, time to completion, and/or the potential for confounding off-target effects. Here, we demonstrate a rapid single-step approach to targeted gene knockout in mammalian cells, using engineered zinc-finger nucleases (ZFNs). ZFNs can be designed to target a chosen locus with high specificity. Upon transient expression of these nucleases the target gene is first cleaved by the ZFNs and then repaired by a natural—but imperfect—DNA repair process, nonhomologous end joining. This often results in the generation of mutant (null) alleles. As proof of concept for this approach we designed ZFNs to target the dihydrofolate reductase (DHFR) gene in a Chinese hamster ovary (CHO) cell line. We observed biallelic gene disruption at frequencies >1%, thus obviating the need for selection markers. Three new genetically distinct DHFR−/− cell lines were generated. Each new line exhibited growth and functional properties consistent with the specific knockout of the DHFR gene. Importantly, target gene disruption is complete within 2–3 days of transient ZFN delivery, thus enabling the isolation of the resultant DHFR−/− cell lines within 1 month. These data demonstrate further the utility of ZFNs for rapid mammalian cell line engineering and establish a new method for gene knockout with application to reverse genetics, functional genomics, drug discovery, and therapeutic recombinant protein production. PMID:18359850

  13. Novel roles of Akt and mTOR in suppressing TGF-β/ALK5-mediated Smad3 activation

    PubMed Central

    Song, Kyung; Wang, Hui; Krebs, Tracy L; Danielpour, David

    2006-01-01

    Insulin-like growth factor-I inhibits transforming growth factor-β (TGF-β) signaling by blocking activation of Smad3 (S3), via a phosphatidylinositol 3-kinase (PI3K)/Akt-dependent pathway. Here we provide the first report that the kinase activity of Akt is necessary for its ability to suppress many TGF-β responses, including S3 activation and induction of apoptosis. Wild-type and myristoylated Akts (AktWT and AktMyr) suppress TGF-β-induced phospho-activation of S3 but not Smad2 (S2), whereas kinase-dead Akt1 (Akt1K179M) or dominant-negative PI3K enhances TGF-β-induced phospho-activation of both S2 and S3. Using siRNA, rapamycin (Rap), and adenoviral expression for FKBP12-resistant and constitutively active TGF-β type I receptor (ALK5), we demonstrate that mammalian target of Rap (mTOR) mediates Akt1 suppression of phospho-activation of S3. These and further data on Akt1-S3 binding do not support a recently proposed model that Akt blocks S3 activation through physical interaction and sequestration of S3 from TGF-β receptors. We propose a novel model whereby Akt suppresses activation of S3 in an Akt kinase-dependent manner through mTOR, a likely route for loss of tumor suppression by TGF-β in cancers. PMID:16362038

  14. Trisubstituted-Imidazoles Induce Apoptosis in Human Breast Cancer Cells by Targeting the Oncogenic PI3K/Akt/mTOR Signaling Pathway.

    PubMed

    Mohan, Chakrabhavi Dhananjaya; Srinivasa, V; Rangappa, Shobith; Mervin, Lewis; Mohan, Surender; Paricharak, Shardul; Baday, Sefer; Li, Feng; Shanmugam, Muthu K; Chinnathambi, Arunachalam; Zayed, M E; Alharbi, Sulaiman Ali; Bender, Andreas; Sethi, Gautam; Basappa; Rangappa, Kanchugarakoppal S

    2016-01-01

    Overactivation of PI3K/Akt/mTOR is linked with carcinogenesis and serves a potential molecular therapeutic target in treatment of various cancers. Herein, we report the synthesis of trisubstituted-imidazoles and identified 2-chloro-3-(4, 5-diphenyl-1H-imidazol-2-yl) pyridine (CIP) as lead cytotoxic agent. Naïve Base classifier model of in silico target prediction revealed that CIP targets RAC-beta serine/threonine-protein kinase which comprises the Akt. Furthermore, CIP downregulated the phosphorylation of Akt, PDK and mTOR proteins and decreased expression of cyclin D1, Bcl-2, survivin, VEGF, procaspase-3 and increased cleavage of PARP. In addition, CIP significantly downregulated the CXCL12 induced motility of breast cancer cells and molecular docking calculations revealed that all compounds bind to Akt2 kinase with high docking scores compared to the library of previously reported Akt2 inhibitors. In summary, we report the synthesis and biological evaluation of imidazoles that induce apoptosis in breast cancer cells by negatively regulating PI3K/Akt/mTOR signaling pathway. PMID:27097161

  15. Trisubstituted-Imidazoles Induce Apoptosis in Human Breast Cancer Cells by Targeting the Oncogenic PI3K/Akt/mTOR Signaling Pathway

    PubMed Central

    Mervin, Lewis; Mohan, Surender; Paricharak, Shardul; Baday, Sefer; Li, Feng; Shanmugam, Muthu K.; Chinnathambi, Arunachalam; Zayed, M. E.; Alharbi, Sulaiman Ali; Bender, Andreas; Sethi, Gautam; Basappa; Rangappa, Kanchugarakoppal S.

    2016-01-01

    Overactivation of PI3K/Akt/mTOR is linked with carcinogenesis and serves a potential molecular therapeutic target in treatment of various cancers. Herein, we report the synthesis of trisubstituted-imidazoles and identified 2-chloro-3-(4, 5-diphenyl-1H-imidazol-2-yl) pyridine (CIP) as lead cytotoxic agent. Naïve Base classifier model of in silico target prediction revealed that CIP targets RAC-beta serine/threonine-protein kinase which comprises the Akt. Furthermore, CIP downregulated the phosphorylation of Akt, PDK and mTOR proteins and decreased expression of cyclin D1, Bcl-2, survivin, VEGF, procaspase-3 and increased cleavage of PARP. In addition, CIP significantly downregulated the CXCL12 induced motility of breast cancer cells and molecular docking calculations revealed that all compounds bind to Akt2 kinase with high docking scores compared to the library of previously reported Akt2 inhibitors. In summary, we report the synthesis and biological evaluation of imidazoles that induce apoptosis in breast cancer cells by negatively regulating PI3K/Akt/mTOR signaling pathway. PMID:27097161

  16. Selective inhibition of regulatory T cells by targeting the PI3K-Akt pathway.

    PubMed

    Abu-Eid, Rasha; Samara, Raed N; Ozbun, Laurent; Abdalla, Maher Y; Berzofsky, Jay A; Friedman, Kevin M; Mkrtichyan, Mikayel; Khleif, Samir N

    2014-11-01

    Despite the strides that immunotherapy has made in mediating tumor regression, the clinical effects are often transient, and therefore more durable responses are still needed. The temporary nature of the therapy-induced immune response can be attributed to tumor immune evasion mechanisms, mainly the effect of suppressive immune cells and, in particular, regulatory T cells (Treg). Although the depletion of Tregs has been shown to be effective in enhancing immune responses, selective depletion of these suppressive cells without affecting other immune cells has not been very successful, and new agents are sought. We found that PI3K-Akt pathway inhibitors selectively inhibit Tregs with minimal effect on conventional T cells (Tconv). Our results clearly show selective in vitro inhibition of activation (as represented by a decrease in downstream signaling) and proliferation of Tregs in comparison with Tconvs when treated with different Akt and PI3K inhibitors. This effect has been observed in both human and murine CD4 T cells. In vivo treatment with these inhibitors resulted in a significant and selective reduction in Tregs in both naïve and tumor-bearing mice. Furthermore, these PI3K-Akt inhibitors led to a significant therapeutic antitumor effect, which was shown to be Treg dependent. Here, we report the use of PI3K-Akt pathway inhibitors as potent agents for the selective depletion of suppressive Tregs. We show that these inhibitors are able to enhance the antitumor immune response and are therefore promising clinical reagents for Treg depletion. PMID:25080445

  17. Selective inhibition of regulatory T cells by targeting PI3K-Akt pathway

    PubMed Central

    Abu-Eid, R; Samara, RN; Ozbun, L; Abdalla, MY; Berzofsky, JA; Friedman, KM; Mkrtichyan, M; Khleif, SN

    2014-01-01

    Despite the strides that immunotherapy has made in mediating tumor regression, the clinical effects are often transient, and therefore more durable responses still are needed. The temporary nature of the therapy-induced immune response can be attributed to tumor immune evasion mechanisms, mainly the effect of suppressive immune cells and, in particular, T regulatory cells (Treg). Although the depletion of Treg has been shown to be effective in enhancing immune responses, selective depletion of these suppressive cells without affecting other immune cells has not been very successful, and new agents are sought. We found that PI3K-Akt pathway inhibitors selectively inhibit Treg with minimal effect on conventional T cells (Tconv). Our results clearly show selective in vitro inhibition of activation (as represented by a decrease in downstream signaling) and proliferation of Treg in comparison to Tconv when treated with different Akt and PI3K inhibitors. This effect has been observed in both human and murine CD4 T cells. In vivo treatment with these inhibitors resulted in a significant and selective reduction in Treg both in naïve and tumor-bearing mice. Furthermore, these PI3K-Akt inhibitors led to a significant therapeutic antitumor effect, which was shown to be Treg-dependent. Here, we report the use of PI3K-Akt pathway inhibitors as potent agents for the selective depletion of suppressive Treg. We show that these inhibitors are able to enhance the antitumor immune response and are therefore promising clinical reagents for Treg-depletion. PMID:25080445

  18. MicroRNA-92a promotes metastasis of nasopharyngeal carcinoma by targeting the PTEN/AKT pathway

    PubMed Central

    Zhang, Haixiong; Cao, Hui; Xu, Dadao; Zhu, Kang

    2016-01-01

    MicroRNAs have been confirmed to be a group of important regulators during the pathogenesis of nasopharyngeal carcinoma (NPC). This study confirmed that the expression of microRNA-92a (miR-92a) was significantly upregulated in NPC as compared to noncancerous nasopharyngeal epithelial tissues. Furthermore, high expression of miR-92a was observed in all NPC cell lines, especially in high metastatic cell lines. Clinical analysis indicated that high expression of miR-92a was associated with adverse clinicopathological features including the advanced tumor-node-metastasis stage and distant metastasis, and conferred poor prognosis of patients. In vitro assays showed that miR-92a overexpression potentiated the migration and invasion of 6-10B cells, and miR-92a silencing reduced the number of migrated and invaded 5-8F cells. Phosphatase and tensin homolog (PTEN) was confirmed as a direct downstream target of miR-92a in NPC cells. Otherwise, alteration of miR-92a expression regulated PTEN/AKT pathway in NPC cells. Mechanistically, miR-92a exerted its promoting effects on the metastatic behaviors of NPC cells through suppressing PTEN/AKT pathway. Taken together, this study demonstrates that miR-92a is a promising prognostic biomarker for patients with NPC, and may be a potential therapeutic target to prevent the metastasis of NPC. PMID:27366095

  19. Hepatic FOXO1 Target Genes Are Co-regulated by Thyroid Hormone via RICTOR Protein Deacetylation and MTORC2-AKT Protein Inhibition.

    PubMed

    Singh, Brijesh K; Sinha, Rohit A; Zhou, Jin; Tripathi, Madhulika; Ohba, Kenji; Wang, Mu-En; Astapova, Inna; Ghosh, Sujoy; Hollenberg, Anthony N; Gauthier, Karine; Yen, Paul M

    2016-01-01

    MTORC2-AKT is a key regulator of carbohydrate metabolism and insulin signaling due to its effects on FOXO1 phosphorylation. Interestingly, both FOXO1 and thyroid hormone (TH) have similar effects on carbohydrate and energy metabolism as well as overlapping transcriptional regulation of many target genes. Currently, little is known about the regulation of MTORC2-AKT or FOXO1 by TH. Accordingly, we performed hepatic transcriptome profiling in mice after FOXO1 knockdown in the absence or presence of TH, and we compared these results with hepatic FOXO1 and THRB1 (TRβ1) ChIP-Seq data. We identified a subset of TH-stimulated FOXO1 target genes that required co-regulation by FOXO1 and TH. TH activation of FOXO1 was directly linked to an increase in SIRT1-MTORC2 interaction and RICTOR deacetylation. This, in turn, led to decreased AKT and FOXO1 phosphorylation. Moreover, TH increased FOXO1 nuclear localization, DNA binding, and target gene transcription by reducing AKT-dependent FOXO1 phosphorylation in a THRB1-dependent manner. These events were associated with TH-mediated oxidative phosphorylation and NAD(+) production and suggested that downstream metabolic effects by TH can post-translationally activate other transcription factors. Our results showed that RICTOR/MTORC2-AKT can integrate convergent hormonal and metabolic signals to provide coordinated and sensitive regulation of hepatic FOXO1-target gene expression. PMID:26453307

  20. Akt phosphorylation and regulation of transketolase is a nodal point for amino acid control of purine synthesis.

    PubMed

    Saha, Arindam; Connelly, Stephen; Jiang, Jingjing; Zhuang, Shunhui; Amador, Deron T; Phan, Tony; Pilz, Renate B; Boss, Gerry R

    2014-07-17

    The phosphatidylinositol 3-kinase (PI3K)/Akt pathway integrates environmental clues to regulate cell growth and survival. We showed previously that depriving cells of a single essential amino acid rapidly and reversibly arrests purine synthesis. Here we demonstrate that amino acids via mammalian target of rapamycin 2 and IκB kinase regulate Akt activity and Akt association and phosphorylation of transketolase (TKT), a key enzyme of the nonoxidative pentose phosphate pathway (PPP). Akt phosphorylates TKT on Thr382, markedly enhancing enzyme activity and increasing carbon flow through the nonoxidative PPP, thereby increasing purine synthesis. Mice fed a lysine-deficient diet for 2 days show decreased Akt activity, TKT activity, and purine synthesis in multiple organs. These results provide a mechanism whereby Akt coordinates amino acid availability with glucose utilization, purine synthesis, and RNA and DNA synthesis. PMID:24981175

  1. Recent advances in developing molecular tools for targeted genome engineering of mammalian cells.

    PubMed

    Lim, Kwang-il

    2015-01-01

    Various biological molecules naturally existing in diversified species including fungi, bacteria, and bacteriophage have functionalities for DNA binding and processing. The biological molecules have been recently actively engineered for use in customized genome editing of mammalian cells as the molecule-encoding DNA sequence information and the underlying mechanisms how the molecules work are unveiled. Excitingly, multiple novel methods based on the newly constructed artificial molecular tools have enabled modifications of specific endogenous genetic elements in the genome context at efficiencies that are much higher than that of the conventional homologous recombination based methods. This minireview introduces the most recently spotlighted molecular genome engineering tools with their key features and ongoing modifications for better performance. Such ongoing efforts have mainly focused on the removal of the inherent DNA sequence recognition rigidity from the original molecular platforms, the addition of newly tailored targeting functions into the engineered molecules, and the enhancement of their targeting specificity. Effective targeted genome engineering of mammalian cells will enable not only sophisticated genetic studies in the context of the genome, but also widely-applicable universal therapeutics based on the pinpointing and correction of the disease-causing genetic elements within the genome in the near future. PMID:25104401

  2. AMPK reverses the mesenchymal phenotype of cancer cells by targeting the Akt-MDM2-Foxo3a signaling axis.

    PubMed

    Chou, Chih-Chien; Lee, Kuen-Haur; Lai, I-Lu; Wang, Dasheng; Mo, Xiaokui; Kulp, Samuel K; Shapiro, Charles L; Chen, Ching-Shih

    2014-09-01

    In cancer cells, the epithelial-mesenchymal transition (EMT) confers the ability to invade basement membranes and metastasize to distant sites, establishing it as an appealing target for therapeutic intervention. Here, we report a novel function of the master metabolic kinase AMPK in suppressing EMT by modulating the Akt-MDM2-Foxo3 signaling axis. This mechanistic link was supported by the effects of siRNA-mediated knockdown and pharmacologic activation of AMPK on epithelial and mesenchymal markers in established breast and prostate cancer cells. Exposure of cells to OSU-53, a novel allosteric AMPK activator, as well as metformin and AICAR, was sufficient to reverse their mesenchymal phenotype. These effects were abrogated by AMPK silencing. Phenotypic changes were mediated by Foxo3a activation, insofar as silencing or overexpressing Foxo3a mimicked the effects of AMPK silencing or OSU-53 treatment on EMT, respectively. Mechanistically, Foxo3a activation led to the transactivation of the E-cadherin gene and repression of genes encoding EMT-inducing transcription factors. OSU-53 activated Foxo3a through two Akt-dependent pathways, one at the level of nuclear localization by blocking Akt- and IKKβ-mediated phosphorylation, and a second at the level of protein stabilization via cytoplasmic sequestration of MDM2, an E3 ligase responsible for Foxo3a degradation. The suppressive effects of OSU-53 on EMT had therapeutic implications illustrated by its ability to block invasive phenotypes in vitro and metastatic properties in vivo. Overall, our work illuminates a mechanism of EMT regulation in cancer cells mediated by AMPK, along with preclinical evidence supporting a tractable therapeutic strategy to reverse mesenchymal phenotypes associated with invasion and metastasis. PMID:24994714

  3. Discovery of mammalian target of rapamycin (mTOR) kinase inhibitor CC-223.

    PubMed

    Mortensen, Deborah S; Perrin-Ninkovic, Sophie M; Shevlin, Graziella; Zhao, Jingjing; Packard, Garrick; Bahmanyar, Sogole; Correa, Matthew; Elsner, Jan; Harris, Roy; Lee, Branden G S; Papa, Patrick; Parnes, Jason S; Riggs, Jennifer R; Sapienza, John; Tehrani, Lida; Whitefield, Brandon; Apuy, Julius; Bisonette, René R; Gamez, James C; Hickman, Matt; Khambatta, Godrej; Leisten, Jim; Peng, Sophie X; Richardson, Samantha J; Cathers, Brian E; Canan, Stacie S; Moghaddam, Mehran F; Raymon, Heather K; Worland, Peter; Narla, Rama Krishna; Fultz, Kimberly E; Sankar, Sabita

    2015-07-01

    We report here the synthesis and structure-activity relationship (SAR) of a novel series of mammalian target of rapamycin (mTOR) kinase inhibitors. A series of 4,6- or 1,7-disubstituted-3,4-dihydropyrazino[2,3-b]pyrazine-2(1H)-ones were optimized for in vivo efficacy. These efforts resulted in the identification of compounds with excellent mTOR kinase inhibitory potency, with exquisite kinase selectivity over the related lipid kinase PI3K. The improved PK properties of this series allowed for exploration of in vivo efficacy and ultimately the selection of CC-223 for clinical development. PMID:26083478

  4. High-affinity triplex-forming oligonucleotide target sequences in mammalian genomes.

    PubMed

    Wu, Qi; Gaddis, Sara S; MacLeod, Michael C; Walborg, Earl F; Thames, Howard D; DiGiovanni, John; Vasquez, Karen M

    2007-01-01

    Site-specific recognition of duplex DNA by triplex-forming oligonucleotides (TFOs) provides a promising approach to manipulate mammalian genomes. A prerequisite for successful gene targeting using this approach is that the targeted gene must contain specific, high-affinity TFO target sequences (TTS). To date, TTS have been identified and characterized in only approximately 37 human or rodent genes, limiting the application of triplex-directed gene targeting. We searched the complete human and mouse genomes using an algorithm designed to identify high-affinity TTS. The resulting data set contains 1.9 million potential TTS for each species. We found that 97.8% of known human and 95.2% of known mouse genes have at least one potential high-affinity TTS in the promoter and/or transcribed gene regions. Importantly, 86.5% of known human and 83% of the known mouse genes have at least one TTS that is unique to that gene. Thus, it is possible to target the majority of human and mouse genes with specific TFOs. We found substantially more potential TTS in the promoter sequences than in the transcribed gene sequences or intergenic sequences in both genomes. We selected 12 mouse genes and 2 human genes critical for cell signaling, proliferation, and/or carcinogenesis, identified potential TTS in each, and determined TFO binding affinities to these sites in vitro. We identified at least one high-affinity, specific TFO binding site within each of these genes. Using this information, many genes involved in mammalian cell proliferation and carcinogenesis can now be targeted. PMID:17013831

  5. miR-612 negatively regulates colorectal cancer growth and metastasis by targeting AKT2

    PubMed Central

    Sheng, L; He, P; Yang, X; Zhou, M; Feng, Q

    2015-01-01

    Colorectal cancer (CRC) is one of the most common cancers worldwide, with a particularly high incidence in developed countries. Distant metastasis and recurrence are the main causes of CRC-related deaths. MicroRNAs (miRNAs) in the serum make them potential biomarkers for cancers, as reported in serum or tumor tissues from CRC patients. In this study, we found that miR-612 expression was significantly lower in CRC tissues or cells compared with peritumor tissues or normal cells, and lower in metastatic CRC specimens compared with non-metastatic specimens, whereas AKT2 exhibited opposite trend. Gain-of-function and loss-of-function assays showed that miR-612 inhibited CRC cell proliferation and migration in vitro by Cell Counting Kit-8 and transwell assays. Further analysis revealed that miR-612 directly suppressed AKT2, which in turn inhibited the downstream epithelial–mesenchymal transition-related signaling pathway. These results were additionally validated in vivo by tumorigenesis and liver metastasis experiments. The results of this study suggested a critical role of miR-612 in the development of CRC. PMID:26158514

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

    PubMed

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

    2016-02-01

    Deregulation of PI3K/Akt/mTOR pathway has been recently identified to play a crucial role in the progress of human gastric cancer. In this study, we show that mefloquine, a FDA-approved anti-malarial drug, effectively targets human gastric cancer cells. Mefloquine potently inhibits proliferation and induces apoptosis of a panel of human gastric cancer cell lines, with EC50 ∼ 0.5-0.7 μM. In two independent gastric cancer xenograft mouse models, mefloquine significantly inhibits growth of both tumors. The combination of mefloquine with paclitaxel enhances the activity of either drug alone in in vitro and in vivo. In addition, mefloquine potently decreased phosphorylation of PI3K, Akt, mTOR and rS6. Overexpression of constitutively active Akt significantly restored mefloquine-mediated inhibition of mTOR phosphorylation and growth, and induction of apoptosis, suggesting that mefloquine acts on gastric cancer cells via suppressing PI3K/Akt/mTOR pathway. We further show that mefloquine-mediated inhibition of Akt/mTOR singaling is phosphatase-dependent as pretreatment with calyculin A does-dependently reversed mefloquine-mediated inhibition of Akt/mTOR phosphorylation. Since mefloquine is already available for clinic use, these results suggest that it is a useful addition to the treatment armamentarium for gastric cancer. PMID:26780727

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

    SciTech Connect

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

    2013-08-16

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

  8. Targeted gene conversion induced by triplex-directed psoralen interstrand crosslinks in mammalian cells.

    PubMed

    Liu, Yaobin; Nairn, Rodney S; Vasquez, Karen M

    2009-10-01

    Correction of a defective gene is a promising approach for both basic research and clinical gene therapy. However, the absence of site-specific targeting and the low efficiency of homologous recombination in human cells present barriers to successful gene targeting. In an effort to overcome these barriers, we utilized triplex-forming oligonucleotides (TFOs) conjugated to a DNA interstrand crosslinking (ICL) agent, psoralen (pTFO-ICLs), to improve the gene targeting efficiency at a specific site in DNA. Gene targeting events were monitored by the correction of a deletion on a recipient plasmid with the homologous sequence from a donor plasmid in human cells. The mechanism underlying this event is stimulation of homologous recombination by the pTFO-ICL. We found that pTFO-ICLs are efficient in inducing targeted gene conversion (GC) events in human cells. The deletion size in the recipient plasmid influenced both the recombination frequency and spectrum of recombinants; i.e. plasmids with smaller deletions had a higher frequency and proportion of GC events. The polarity of the pTFO-ICL also had a prominent effect on recombination. Our results suggest that pTFO-ICL induced intermolecular recombination provides an efficient method for targeted gene correction in mammalian cells. PMID:19726585

  9. Transcriptional regulation of gene expression during osmotic stress responses by the mammalian target of rapamycin.

    PubMed

    Ortells, M Carmen; Morancho, Beatriz; Drews-Elger, Katherine; Viollet, Benoit; Laderoute, Keith R; López-Rodríguez, Cristina; Aramburu, Jose

    2012-05-01

    Although stress can suppress growth and proliferation, cells can induce adaptive responses that allow them to maintain these functions under stress. While numerous studies have focused on the inhibitory effects of stress on cell growth, less is known on how growth-promoting pathways influence stress responses. We have approached this question by analyzing the effect of mammalian target of rapamycin (mTOR), a central growth controller, on the osmotic stress response. Our results showed that mammalian cells exposed to moderate hypertonicity maintained active mTOR, which was required to sustain their cell size and proliferative capacity. Moreover, mTOR regulated the induction of diverse osmostress response genes, including targets of the tonicity-responsive transcription factor NFAT5 as well as NFAT5-independent genes. Genes sensitive to mTOR-included regulators of stress responses, growth and proliferation. Among them, we identified REDD1 and REDD2, which had been previously characterized as mTOR inhibitors in other stress contexts. We observed that mTOR facilitated transcription-permissive conditions for several osmoresponsive genes by enhancing histone H4 acetylation and the recruitment of RNA polymerase II. Altogether, these results reveal a previously unappreciated role of mTOR in regulating transcriptional mechanisms that control gene expression during cellular stress responses. PMID:22287635

  10. Pharmacological Targeting of the Mammalian Clock Regulates Sleep Architecture and Emotional Behavior

    PubMed Central

    Banerjee, Subhashis; Wang, Yongjun; Solt, Laura A.; Griffett, Kristine; Kazantzis, Melissa; Amador, Ariadna; El-Gendy, Bahaa M.; Huitron-Resendiz, Salvador; Roberts, Amanda J.; Shin, Youseung; Kamenecka, Theodore M.; Burris, Thomas P.

    2014-01-01

    Synthetic drug-like molecules that directly modulate the activity of key clock proteins offer the potential to directly modulate the endogenous circadian rhythm and treat diseases associated with clock dysfunction. Here, we demonstrate that synthetic ligands targeting a key component of the mammalian clock, the nuclear receptors REV-ERBα and β, regulate sleep architecture and emotional behavior in mice. REV-ERB agonists induce wakefulness and reduce REM and slow-wave sleep. Interestingly, REV-ERB agonists also reduce anxiety-like behavior. These data are consistent with increased anxiety-like behavior of REV-ERBβ null mice, in which REV-ERB agonists have no effect Also consistent with these effects being mediated by REV-ERB, the effect of the agonist on sleep and anxiety was suppressed by lithium treatment. These results indicate that pharmacological targeting of REVERB may lead to the development of novel therapeutics to treat sleep disorders and anxiety. PMID:25536025

  11. Most mammalian mRNAs are conserved targets of microRNAs

    PubMed Central

    Friedman, Robin C.; Farh, Kyle Kai-How; Burge, Christopher B.; Bartel, David P.

    2009-01-01

    MicroRNAs (miRNAs) are small endogenous RNAs that pair to sites in mRNAs to direct post-transcriptional repression. Many sites that match the miRNA seed (nucleotides 2–7), particularly those in 3′ untranslated regions (3′UTRs), are preferentially conserved. Here, we overhauled our tool for finding preferential conservation of sequence motifs and applied it to the analysis of human 3′UTRs, increasing by nearly threefold the detected number of preferentially conserved miRNA target sites. The new tool more efficiently incorporates new genomes and more completely controls for background conservation by accounting for mutational biases, dinucleotide conservation rates, and the conservation rates of individual UTRs. The improved background model enabled preferential conservation of a new site type, the “offset 6mer,” to be detected. In total, >45,000 miRNA target sites within human 3′UTRs are conserved above background levels, and >60% of human protein-coding genes have been under selective pressure to maintain pairing to miRNAs. Mammalian-specific miRNAs have far fewer conserved targets than do the more broadly conserved miRNAs, even when considering only more recently emerged targets. Although pairing to the 3′ end of miRNAs can compensate for seed mismatches, this class of sites constitutes less than 2% of all preferentially conserved sites detected. The new tool enables statistically powerful analysis of individual miRNA target sites, with the probability of preferentially conserved targeting (PCT) correlating with experimental measurements of repression. Our expanded set of target predictions (including conserved 3′-compensatory sites), are available at the TargetScan website, which displays the PCT for each site and each predicted target. PMID:18955434

  12. miR-185 inhibits hepatocellular carcinoma growth by targeting the DNMT1/PTEN/Akt pathway.

    PubMed

    Qadir, Ximena V; Han, Chang; Lu, Dongdong; Zhang, Jinqiang; Wu, Tong

    2014-08-01

    miRNAs have recently been implicated in hepatocarcinogenesis, although the actions and mechanisms of individual miRNAs remain incompletely understood. We examined the biological functions and molecular mechanisms of miR-185 in hepatocellular carcinoma (HCC). The expression of miR-185 is decreased in human HCC tissues compared with the nonneoplastic liver parenchyma. Quantitative RT-PCR showed a reduction of miR-185 in human HCC cells compared with primary hepatocytes. miR-185 overexpression in human HCC cells inhibited cell proliferation and invasion in vitro and prevented tumor growth in SCID mice. miR-185 overexpression inhibited DNMT1 3' untranslated region luciferase reporter activity in HCC cells; this effect was abolished when the miR-185 binding site was mutated. miR-185 mimic or overexpression decreased the level of DNMT1 protein in HCC cells. These findings establish DNMT1 as a bona fide target of miR-185 in HCC cells. The role of DNMT1 in miR-185-induced inhibition of HCC growth was further supported by the fact that DNMT1 overexpression prevented miR-185-induced inhibition of HCC cell proliferation/invasion. miR-185 mimic or overexpression reduced PTEN promoter DNA methylation and enhanced PTEN expression, leading to the inhibition of Akt phosphorylation; these effects were partially reversed by DNMT1 overexpression. These results provide novel evidence that miR-185 inhibits HCC cell growth by targeting DNMT1, leading to PTEN induction and Akt inhibition. Thus, reactivation or induction of miR-185 may represent a novel therapeutic strategy for HCC treatment. PMID:24911372

  13. miR-185 Inhibits Hepatocellular Carcinoma Growth by Targeting the DNMT1/PTEN/Akt Pathway

    PubMed Central

    Qadir, Ximena V.; Han, Chang; Lu, Dongdong; Zhang, Jinqiang; Wu, Tong

    2015-01-01

    miRNAs have recently been implicated in hepatocarcinogenesis, although the actions and mechanisms of individual miRNAs remain incompletely understood. We examined the biological functions and molecular mechanisms of miR-185 in hepatocellular carcinoma (HCC). The expression of miR-185 is decreased in human HCC tissues compared with the nonneoplastic liver parenchyma. Quantitative RT-PCR showed a reduction of miR-185 in human HCC cells compared with primary hepatocytes. miR-185 overexpression in human HCC cells inhibited cell proliferation and invasion in vitro and prevented tumor growth in SCID mice. miR-185 overexpression inhibited DNMT1 3′ untranslated region luciferase reporter activity in HCC cells; this effect was abolished when the miR-185 binding site was mutated. miR-185 mimic or overexpression decreased the level of DNMT1 protein in HCC cells. These findings establish DNMT1 as a bona fide target of miR-185 in HCC cells. The role of DNMT1 in miR-185–induced inhibition of HCC growth was further supported by the fact that DNMT1 overexpression prevented miR-185–induced inhibition of HCC cell proliferation/invasion. miR-185 mimic or overexpression reduced PTEN promoter DNA methylation and enhanced PTEN expression, leading to the inhibition of Akt phosphorylation; these effects were partially reversed by DNMT1 overexpression. These results provide novel evidence that miR-185 inhibits HCC cell growth by targeting DNMT1, leading to PTEN induction and Akt inhibition. Thus, reactivation or induction of miR-185 may represent a novel therapeutic strategy for HCC treatment. PMID:24911372

  14. Novel role of the small GTPase Rheb: its implication in endocytic pathway independent of the activation of mammalian target of rapamycin.

    PubMed

    Saito, Kota; Araki, Yasuhiro; Kontani, Kenji; Nishina, Hiroshi; Katada, Toshiaki

    2005-03-01

    The Ras-homologous GTPase Rheb that is conserved from yeast to human appears to be involved not only in cell growth but also in nutrient uptake. Recent biochemical analysis revealed that tuberous sclerosis complex (TSC), a GTPase-activating protein (GAP), deactivates Rheb and that phosphatidylinositol 3'-kinase (PI3k)-Akt/PKB kinase pathway activates Rheb through inhibition of the GAP-mediated deactivation. Although mammalian target of rapamycin (mTOR) kinase is implicated in the downstream target of Rheb, the direct effector(s) and exact functions of Rheb have not been fully elucidated. Here we identified that Rheb expression in cultured cells induces the formation of large cytoplasmic vacuoles, which are characterized as late endocytic (late endosome- and lysosome-like) components. The vacuole formation required the GTP form of Rheb, but not the activation of the downstream mTOR kinase. These results suggest that Rheb regulates endocytic trafficking pathway independent of the previously identified mTOR pathway. The physiological roles of the two Rheb-dependent signaling pathways are discussed in terms of nutrient uptake and cell growth or cell cycle progression. PMID:15809346

  15. Transforming Growth Factor-β Is an Upstream Regulator of Mammalian Target of Rapamycin Complex 2-Dependent Bladder Cancer Cell Migration and Invasion.

    PubMed

    Gupta, Sounak; Hau, Andrew M; Al-Ahmadie, Hikmat A; Harwalkar, Jyoti; Shoskes, Aaron C; Elson, Paul; Beach, Jordan R; Hussey, George S; Schiemann, William P; Egelhoff, Thomas T; Howe, Philip H; Hansel, Donna E

    2016-05-01

    Our prior work identified the mammalian target of rapamycin complex 2 (mTORC2) as a key regulator of bladder cancer cell migration and invasion, although upstream growth factor mediators of this pathway in bladder cancer have not been well delineated. We tested whether transforming growth factor (TGF)-β, which can function as a promotility factor in bladder cancer cells, could regulate mTORC2-dependent bladder cancer cell motility and invasion. In human bladder cancers, the highest levels of phosphorylated SMAD2, a TGF-β signaling intermediate, were present in high-grade invasive bladder cancers and associated with more frequent recurrence and decreased disease-specific survival. Increased expression of TGF-β isoforms, receptors, and signaling components was detected in invasive high-grade bladder cancer cells that expressed Vimentin and lacked E-cadherin. Application of TGF-β induced phosphorylation of the Ser473 residue of AKT, a selective target of mTORC2, in a SMAD2- and SMAD4-independent manner and increased bladder cancer cell migration in a modified scratch wound assay and invasion through Matrigel. Inhibition of TGF-β receptor I using SB431542 ablated TGF-β-induced migration and invasion. A similar effect was seen when Rictor, a key mTORC2 component, was selectively silenced. Our results suggest that TGF-β can induce bladder cancer cell invasion via mTORC2 signaling, which may be applicable in most bladder cancers. PMID:26988652

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

    PubMed Central

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

    2015-01-01

    Oncogenic epidermal growth factor receptor (EGFR) signaling plays an important role in regulating global metabolic pathways, including aerobic glycolysis, the pentose phosphate pathway (PPP), and pyrimidine biosynthesis. However, the molecular mechanism by which EGFR signaling regulates cancer cell metabolism is still unclear. To elucidate how EGFR signaling is linked to metabolic activity, we investigated the involvement of the RAS/MEK/ERK and PI3K/AKT/mammalian target of rapamycin (mTOR) pathways on metabolic alteration in lung adenocarcinoma (LAD) cell lines with activating EGFR mutations. Although MEK inhibition did not alter lactate production and the extracellular acidification rate, PI3K/mTOR inhibitors significantly suppressed glycolysis in EGFR-mutant LAD cells. Moreover, a comprehensive metabolomics analysis revealed that the levels of glucose 6-phosphate and 6-phosphogluconate as early metabolites in glycolysis and PPP were decreased after inhibition of the PI3K/AKT/mTOR pathway, suggesting a link between PI3K signaling and the proper function of glucose transporters or hexokinases in glycolysis. Indeed, PI3K/mTOR inhibition effectively suppressed membrane localization of facilitative glucose transporter 1 (GLUT1), which, instead, accumulated in the cytoplasm. Finally, aerobic glycolysis and cell proliferation were down-regulated when GLUT1 gene expression was suppressed by RNAi. Taken together, these results suggest that PI3K/AKT/mTOR signaling is indispensable for the regulation of aerobic glycolysis in EGFR-mutated LAD cells. PMID:26023239

  17. Targeting the AKT/GSK3{beta}/Cyclin D1/Cdk4 Survival Signaling Pathway for Eradication of Tumor Radioresistance Acquired by Fractionated Radiotherapy

    SciTech Connect

    Shimura, Tsutomu; Kakuda, Satoshi; Ochiai, Yasushi; Kuwahara, Yoshikazu; Takai, Yoshihiro; Fukumoto, Manabu

    2011-06-01

    Purpose: Radioresistance is a major cause of treatment failure of radiotherapy (RT) in human cancer. We have recently revealed that acquired radioresistance of tumor cells induced by fractionated radiation is attributable to cyclin D1 overexpression as a consequence of the downregulation of GSK3{beta}-dependent cyclin D1 proteolysis mediated by a constitutively activated serine-threonine kinase, AKT. This prompted us to hypothesize that targeting the AKT/GSK3{beta}/cyclin D1 pathway may improve fractionated RT by suppressing acquired radioresistance of tumor cells. Methods and Materials: Two human tumor cell lines with acquired radioresistance were exposed to X-rays after incubation with either an AKT inhibitor, AKT/PKB signaling inhibitor-2 (API-2), or a Cdk4 inhibitor (Cdk4-I). Cells were then subjected to immunoblotting, clonogenic survival assay, cell growth analysis, and cell death analysis with TUNEL and annexin V staining. In vivo radiosensitivity was assessed by growth of human tumors xenografted into nude mice. Results: Treatment with API-2 resulted in downregulation of cyclin D1 expression in cells with acquired radioresistance. Cellular radioresistance disappeared completely both in vitro and in vivo with accompanying apoptosis when treated with API-2. Furthermore, inhibition of cyclin D1/Cdk4 by Cdk4-I was sufficient for abolishing radioresistance. Treatment with either API-2 or Cdk4-I was also effective in suppressing resistance to cis-platinum (II)-diamine-dichloride in the cells with acquired radioresistance. Interestingly, the radiosensitizing effect of API-2 was canceled by overexpression of cyclin D1 whereas Cdk4-I was still able to sensitize cells with cyclin D1 overexpression. Conclusion: Cyclin D1/Cdk4 is a critical target of the AKT survival signaling pathway responsible for tumor radioresistance. Targeting the AKT/GSK3{beta}/cyclin D1/Cdk4 pathway would provide a novel approach to improve fractionated RT and would have an impact on tumor

  18. Post-conversion targeted capture of modified cytosines in mammalian and plant genomes

    PubMed Central

    Li, Qing; Suzuki, Masako; Wendt, Jennifer; Patterson, Nicole; Eichten, Steven R.; Hermanson, Peter J.; Green, Dawn; Jeddeloh, Jeffrey; Richmond, Todd; Rosenbaum, Heidi; Burgess, Daniel; Springer, Nathan M.; Greally, John M.

    2015-01-01

    We present a capture-based approach for bisulfite-converted DNA that allows interrogation of pre-defined genomic locations, allowing quantitative and qualitative assessments of 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) at CG dinucleotides and in non-CG contexts (CHG, CHH) in mammalian and plant genomes. We show the technique works robustly and reproducibly using as little as 500 ng of starting DNA, with results correlating well with whole genome bisulfite sequencing data, and demonstrate that human DNA can be tested in samples contaminated with microbial DNA. This targeting approach will allow cell type-specific designs to maximize the value of 5mC and 5hmC sequencing. PMID:25813045

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2016-03-15

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

  1. ALTERATION OF AKT ACTIVITY INCREASES CHEMOTHERAPEUTIC DRUG AND HORMONAL RESISTANCE IN BREAST CANCER YET CONFERS AN ACHILLES HEEL BY SENSITIZATION TO TARGETED THERAPY

    PubMed Central

    Sokolosky, Melissa L.; Lehmann, Brian D.; Taylor, Jackson R.; Navolanic, Patrick M.; Chappell, William H.; Abrams, Stephen L.; Stadelman, Kristin M.; Wong, Ellis WT; Misaghian, Negin; Horn, Stefan; Bäsecke, Jörg; Libra, Massimo; Stivala, Franca; Ligresti, Giovanni; Tafuri, Agostino; Milella, Michele; Zarzycki, Marek; Dzugaj, Andrzej; Chiarini, Francesca; Evangelisti, Camilla; Martelli, Alberto M.; Terrian, David M.; Franklin, Richard A.; Steelman, Linda S.

    2008-01-01

    to potential therapeutic targets, mTOR and MEK. These studies indicate that activation of the Akt kinase or disruption of the normal activity of the PTEN phosphatase can have dramatic effects on activity of p70S6K and other downstream substrates and thereby altering the therapeutic sensitivity of breast cancer cells. The effects of doxorubicin and tamoxifen on induction of the Raf/MEK/ERK and PI3K/Akt survival pathways were examined in unmodified MCF-7 breast cells. Doxorubicin was a potent inducer of activated ERK and to a lesser extent Akt. Tamoxifen also induced ERK. Thus a consequence of doxorubicin and tamoxifen therapy of breast cancer is the induction of a pro-survival pathway which may contribute to the development of drug resistance. Unmodified MCF-7 cells were also sensitive to MEK and mTOR inhibitors which synergized with both tamoxifen and doxorubicin to induce death. In summary, our results point to the key interactions between the PI3K/PTEN/Akt/mTOR and Raf/MEK/ERK pathways in regulating chemotherapeutic drug resistance/sensitivity in breast cancer and indicate that targeting these pathways may prevent drug and hormonal resistance. PMID:18423407

  2. Development of a model describing regulation of casein synthesis by the mammalian target of rapamycin (mTOR) signaling pathway in response to insulin, amino acids, and acetate.

    PubMed

    Castro, J J; Arriola Apelo, S I; Appuhamy, J A D R N; Hanigan, M D

    2016-08-01

    To improve dietary protein use efficiency in lactating cows, mammary protein synthesis responses to AA, energy substrates, and hormones must be better understood. These entities exert their effects through stimulation of mRNA translation via control of initiation and elongation rates at the cellular level. A central protein kinase of this phenomenon is the mammalian target of rapamycin (mTOR), which transfers the nutritional and hormonal stimuli onto a series of proteins downstream through a cascade of phosphorylation reactions that ultimately affect protein synthesis. The objective of this work was to further develop an existing mechanistic model of mTOR phosphorylation responses to insulin and total essential AA to include the effects of specific essential AA and acetate mediated by signaling proteins including protein kinase B (Akt), adenosine monophosphate activated protein kinase (AMPK), and mTOR and to add a representation of milk protein synthesis. Data from 6 experiments in MAC-T cells and mammary tissue slices previously conducted in our laboratory were assembled and used to parameterize the dynamic system of differential equations representing Akt, AMPK, and mTOR in their phosphorylated and dephosphorylated states and the resulting regulation of milk protein synthesis. The model predicted phosphorylated Akt, mTOR, AMPK, and casein synthesis rates with root mean square prediction errors of 16.8, 28.4, 33.0, and 54.9%, respectively. All other dependent variables were free of mean and slope bias, indicating an adequate representation of the data. Whereas mTOR was not very sensitive to changes in insulin or acetate levels, it was highly sensitive to leucine and isoleucine, and this signal appeared to be effectively transduced to casein synthesis. Although prior work had observed a relationship with additional essential AA, and data supporting those conclusions were present in the data set, we were unable to derive significant relationships with any essential

  3. Down-Regulation of MicroRNA-223 Promotes Degranulation via the PI3K/Akt Pathway by Targeting IGF-1R in Mast Cells

    PubMed Central

    Xu, Hong; Zhou, Hui; Yang, Qian-Yuan; Liu, Feng; Zhou, Guo-Ping

    2015-01-01

    Background Mast cells play a central role in allergic and inflammatory disorders by inducing degranulation and inflammatory mediator release. Recent reports have shown that miRNAs play an important role in inflammatory response regulation. Therefore, the role of miR-223 in mast cells was investigated. Methods The expression of miR-223 was quantified by quantitative real-time polymerase chain reaction (qRT-PCR) in immunoglobulin E (IgE)-mediated mast cells. After successful miR-223 inhibition by transfection, degranulation was detected in IgE-mediated mast cells. The phosphorylation of IκB-α and Akt were examined using western blotting. NF-κB was tested using electrophoretic mobility shift assay. PI3K-inhibitor (LY294002) was used to investigate whether the PI3K/Akt pathway was essential for mast cell activation. The TargetScan database and a luciferase reporter system were used to identify whether insulin-like growth factor 1 receptor (IGF-1R) is a direct target of miR-223. Results MiR-223 expression was up-regulated in IgE-mediated mast cells, whereas its down-regulation promoted mast cell degranulation. Levels of IκB-α and Akt phosphorylation as well as NF-κB were increased in miR-223 inhibitor cells. LY294002 could block the PI3K/Akt signaling pathway and rescue the promotion caused by suppressing miR-223 in mast cells. IGF-1R was identified as a direct target of miR-223. Conclusions These findings suggest that down-regulation of miR-223 promotes degranulation via the PI3K/Akt pathway by targeting IGF-1R in mast cells. PMID:25875646

  4. Targeting the mammalian target of Rapamycin to inhibit VEGF and cytokines for the treatment of primary effusion lymphoma

    PubMed Central

    Gasperini, Paola; Tosato, Giovanna

    2009-01-01

    Primary effusion lymphoma (PEL) is a fatal malignancy, which typically presents as a lymphomatous effusion that later disseminates. Rapamycin (Rapa), which targets mTOR (mammalian target of Rapa), is currently evaluated as a treatment for PEL, but the recent development of PEL in Rapa-treated post-transplant recipients questions the drug's use in PEL. Here, we used a murine model of PEL effusion that mimics the human disease to investigate the anti-PEL activity of Rapa. We found that Rapa reduces ascites accumulation and extends mouse survival. Initially, Rapa reduced PEL load compared to control mice, but most mice rapidly showed PEL progression. Levels of VEGF, which promotes vascular permeability contributing to effusion formation, were significantly reduced in ascites of Rapa-treated mice compared to controls. Expression of IL-10, the principal autocrine growth factor for PEL, was initially reduced in PEL from Rapa-treated mice but rapidly increased despite treatment. We found that the hypoxic environment of ascites and Rapa cooperate in stimulating IL-10 expression in PEL, which likely contributes to the emergence of drug resistance. These results identify Rapa an effective drug to reduce PEL effusions but illustrate the rapid development of drug resistance, which likely limits the efficacy of Rapa in PEL. PMID:19554030

  5. MicroRNA-10b promotes nucleus pulposus cell proliferation through RhoC-Akt pathway by targeting HOXD10 in intervetebral disc degeneration.

    PubMed

    Yu, Xin; Li, Zheng; Shen, Jianxiong; Wu, William K K; Liang, Jinqian; Weng, Xisheng; Qiu, Guixing

    2013-01-01

    Aberrant proliferation of nucleus pulposus cell is implicated in the pathogenesis of intervertebral disc degeneration. Recent findings revealed that microRNAs, a class of small noncoding RNAs, could regulate cell proliferation in many pathological conditions. Here, we showed that miR-10b was dramatically upregulated in degenerative nucleus pulposus tissues when compared with nucleus pulposus tissues isolated from patients with idiopathic scoliosis. Moreover, miR-10b levels were associated with disc degeneration grade and downregulation of HOXD10. In cultured nucleus pulposus cells, miR-10b overexpression stimulated cell proliferation with concomitant translational inhibition of HOXD10 whereas restored expression of HOXD10 reversed the mitogenic effect of miR-10b. MiR-10b-mediated downregulation of HOXD10 led to increased RhoC expression and Akt phosphorylation. Either knockdown of RhoC or inhibition of Akt abolished the effect of miR-10b on nucleus pulposus cell proliferation. Taken together, aberrant miR-10b upregulation in intervertebral disc degeneration could contribute to abnormal nucleus pulposus cell proliferation through derepressing the RhoC-Akt pathway by targeting HOXD10. Our study also underscores the potential of miR-10b and the RhoC-Akt pathway as novel therapeutic targets in intervertebral disc degeneration. PMID:24376640

  6. MicroRNA-10b Promotes Nucleus Pulposus Cell Proliferation through RhoC-Akt Pathway by Targeting HOXD10 in Intervetebral Disc Degeneration

    PubMed Central

    Shen, Jianxiong; Wu, William K. K.; Liang, Jinqian; Weng, Xisheng; Qiu, Guixing

    2013-01-01

    Aberrant proliferation of nucleus pulposus cell is implicated in the pathogenesis of intervertebral disc degeneration. Recent findings revealed that microRNAs, a class of small noncoding RNAs, could regulate cell proliferation in many pathological conditions. Here, we showed that miR-10b was dramatically upregulated in degenerative nucleus pulposus tissues when compared with nucleus pulposus tissues isolated from patients with idiopathic scoliosis. Moreover, miR-10b levels were associated with disc degeneration grade and downregulation of HOXD10. In cultured nucleus pulposus cells, miR-10b overexpression stimulated cell proliferation with concomitant translational inhibition of HOXD10 whereas restored expression of HOXD10 reversed the mitogenic effect of miR-10b. MiR-10b-mediated downregulation of HOXD10 led to increased RhoC expression and Akt phosphorylation. Either knockdown of RhoC or inhibition of Akt abolished the effect of miR-10b on nucleus pulposus cell proliferation. Taken together, aberrant miR-10b upregulation in intervertebral disc degeneration could contribute to abnormal nucleus pulposus cell proliferation through derepressing the RhoC-Akt pathway by targeting HOXD10. Our study also underscores the potential of miR-10b and the RhoC-Akt pathway as novel therapeutic targets in intervertebral disc degeneration. PMID:24376640

  7. Mammalian target of rapamycin signaling modulates photic entrainment of the suprachiasmatic circadian clock

    PubMed Central

    Cao, Ruifeng; Li, Aiqing; Cho, Hee-yeon; Lee, Boyoung; Obrietan, Karl

    2010-01-01

    Inducible gene expression appears to be an essential event that couples light to entrainment of the master mammalian circadian clock located in the suprachiasmatic nucleus (SCN) of the hypothalamus. Recently, we reported that light triggers phase-dependent activation of the mammalian target of rapamycin (mTOR) signaling pathway, a major regulator of protein synthesis, in the SCN, thus raising the possibility that mTOR-evoked mRNA translation contributes to clock entrainment. Here, we employed a combination of cellular, molecular and behavioral assays to address this question. To this end, we show that the in vivo infusion of the mTOR inhibitor rapamycin led to a significant attenuation of the phase-delaying effect of early night light. Conversely, disruption of mTOR during the late night augmented the phase-advancing effect of light. To assess the role of mTOR signaling within the context of molecular entrainment, the effects of rapamycin on light-induced expression of PERIOD1 and PERIOD2 were examined. At both the early and late night time points, abrogation of mTOR signaling led to a significant attenuation of light-evoked PERIOD protein expression. Our results also reveal that light-induced mTOR activation leads to translation of mRNAs with a 5′-terminal oligopyrimidine tract such as eukaryotic elongation factor 1 A (eEF1A) and the immediate early gene JunB. Together, these data indicate that the mTOR pathway functions as potent and selective regulator of light-evoked protein translation and SCN clock entrainment. PMID:20445056

  8. Mechanisms of double-strand-break repair during gene targeting in mammalian cells.

    PubMed Central

    Ng, P; Baker, M D

    1999-01-01

    In the present study, the mechanism of double-strand-break (DSB) repair during gene targeting at the chromosomal immunoglobulin mu-locus in a murine hybridoma was examined. The gene-targeting assay utilized specially designed insertion vectors genetically marked in the region of homology to the chromosomal mu-locus by six diagnostic restriction enzyme site markers. The restriction enzyme markers permitted the contribution of vector-borne and chromosomal mu-sequences in the recombinant product to be determined. The use of the insertion vectors in conjunction with a plating procedure in which individual integrative homologous recombination events were retained for analysis revealed several important features about the mammalian DSB repair process:The presence of the markers within the region of shared homology did not affect the efficiency of gene targeting.In the majority of recombinants, the vector-borne marker proximal to the DSB was absent, being replaced with the corresponding chromosomal restriction enzyme site. This result is consistent with either formation and repair of a vector-borne gap or an "end" bias in mismatch repair of heteroduplex DNA (hDNA) that favored the chromosomal sequence. Formation of hDNA was frequently associated with gene targeting and, in most cases, began approximately 645 bp from the DSB and could encompass a distance of at least 1469 bp.The hDNA was efficiently repaired prior to DNA replication.The repair of adjacent mismatches in hDNA occurred predominantly on the same strand, suggesting the involvement of a long-patch repair mechanism. PMID:10049929

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

    PubMed

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

    2016-07-01

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

  10. Dazl is a target RNA suppressed by mammalian NANOS2 in sexually differentiating male germ cells

    PubMed Central

    Kato, Yuzuru; Katsuki, Takeo; Kokubo, Hiroki; Masuda, Aki; Saga, Yumiko

    2016-01-01

    Evolutionally conserved Nanos RNA-binding proteins play crucial roles in germ cell development. While a mammalian Nanos family protein, NANOS2, is required for sexual differentiation of male (XY) germ cells in mice, the underlying mechanisms and the identities of its target RNAs in vivo remain elusive. Using comprehensive microarray analysis and a bacterial artificial chromosome transgenic system, here we identify Dazl, a germ cell-specific gene encoding an RNA-binding protein implicated in translation, as a crucial target of NANOS2. Importantly, removal of the Dazl 3′-untranslated region in XY germ cells stabilizes the Dazl mRNA, resulting in elevated meiotic gene expression, abnormal resumption of the cell cycle and impaired processing-body formation, reminiscent of Nanos2-knockout phenotypes. Furthermore, our data suggest that NANOS2 acts as an antagonist of the DAZL protein. We propose a dual system of NANOS2-mediated suppression of Dazl expression as a pivotal molecular mechanism promoting sexual differentiation of XY germ cells. PMID:27072294

  11. Mammalian Target of Rapamycin Inhibitors and Life-Threatening Conditions in Tuberous Sclerosis Complex.

    PubMed

    Moavero, Romina; Romagnoli, Gloria; Graziola, Federica; Curatolo, Paolo

    2015-12-01

    Tuberous sclerosis complex (TSC) is a multisystem disease associated with an overall reduction in life expectancy due to the possible occurrence of different life-threatening conditions. Subjects affected by TSC are, in fact, at risk of hydrocephalus secondary to the growth of subependymal giant cell astrocytomas, or of sudden unexpected death in epilepsy. Other nonneurological life-threatening conditions include abdominal bleeding owing to renal angiomyolipomas rupture, renal insufficiency due to progressive parenchymal destruction by multiple cysts, pulmonary complications due to lymphangioleiomyomatosis, and cardiac failure or arrhythmias secondary to rhabdomyomas. In the last decades, there has been a great progress in understanding the pathophysiology of TSC-related manifestations, which are mainly linked to the hyperactivation of the so-called mammalian target of rapamycin (mTOR) pathway, as a consequence of the mutation in 1 of the 2 genes TSC1 or TSC2. This led to the development of new treatment strategies for this disease. In fact, it is now available as a biologically targeted therapy with everolimus, a selective mTOR inhibitor, which has been licensed in Europe and USA for the treatment of subependymal giant cell astrocytomas and angiomyolipomas in subjects with TSC. This drug also proved to benefit other TSC-related manifestations, including pulmonary lymphangioleiomyomatosis, cardiac rhabdomyomas, and presumably epileptic seizures. mTOR inhibitors are thus proving to be a systemic therapy able to simultaneously address different and potentially life-threatening complications, giving the hope of improving life expectation in individuals with TSC. PMID:26706015

  12. Combinatorial targeting and discovery of ligand-receptors in organelles of mammalian cells

    PubMed Central

    Rangel, Roberto; Guzman-Rojas, Liliana; le Roux, Lucia G.; Staquicini, Fernanda I.; Hosoya, Hitomi; Barbu, E. Magda; Ozawa, Michael G.; Nie, Jing; Jr, Kenneth Dunner; Langley, Robert R.; Sage, E. Helene; Koivunen, Erkki; Gelovani, Juri G.; Lobb, Roy R.; Sidman, Richard L.; Pasqualini, Renata; Arap, Wadih

    2012-01-01

    Phage display screening allows the study of functional protein–protein interactions at the cell surface, but investigating intracellular organelles remains a challenge. Here we introduce internalizing-phage libraries to identify clones that enter mammalian cells through a receptor-independent mechanism and target-specific organelles as a tool to select ligand peptides and identify their intracellular receptors. We demonstrate that penetratin, an antennapedia-derived peptide, can be displayed on the phage envelope and mediate receptor-independent uptake of internalizing phage into cells. We also show that an internalizing-phage construct displaying an established mitochondria-specific localization signal targets mitochondria, and that an internalizing-phage random peptide library selects for peptide motifs that localize to different intracellular compartments. As a proof-of-concept, we demonstrate that one such peptide, if chemically fused to penetratin, is internalized receptor-independently, localizes to mitochondria, and promotes cell death. This combinatorial platform technology has potential applications in cell biology and drug development. PMID:22510693

  13. Mammalian Target of Rapamycin Mediates Kidney Injury Molecule 1-Dependent Tubule Injury in a Surrogate Model.

    PubMed

    Yin, Wenqing; Naini, Said Movahedi; Chen, Guochun; Hentschel, Dirk M; Humphreys, Benjamin D; Bonventre, Joseph V

    2016-07-01

    Kidney injury molecule 1 (KIM-1), an epithelial phagocytic receptor, is markedly upregulated in the proximal tubule in various forms of acute and chronic kidney injury in humans and many other species. Whereas acute expression of KIM-1 has adaptive anti-inflammatory effects, chronic expression may be maladaptive in mice. Here, we characterized the zebrafish Kim family, consisting of Kim-1, Kim-3, and Kim-4. Kim-1 was markedly upregulated in kidney after gentamicin-induced injury and had conserved phagocytic activity in zebrafish. Both constitutive and tamoxifen-induced expression of Kim-1 in zebrafish kidney tubules resulted in loss of the tubule brush border, reduced GFR, pericardial edema, and increased mortality. Kim-1-induced kidney injury was associated with reduction of growth of adult fish. Kim-1 expression led to activation of the mammalian target of rapamycin (mTOR) pathway, and inhibition of this pathway with rapamycin increased survival. mTOR pathway inhibition in KIM-1-overexpressing transgenic mice also significantly ameliorated serum creatinine level, proteinuria, tubular injury, and kidney inflammation. In conclusion, persistent Kim-1 expression results in chronic kidney damage in zebrafish through a mechanism involving mTOR. This observation predicted the role of the mTOR pathway and the therapeutic efficacy of mTOR-targeted agents in KIM-1-mediated kidney injury and fibrosis in mice, demonstrating the utility of the Kim-1 renal tubule zebrafish models. PMID:26538632

  14. Dazl is a target RNA suppressed by mammalian NANOS2 in sexually differentiating male germ cells.

    PubMed

    Kato, Yuzuru; Katsuki, Takeo; Kokubo, Hiroki; Masuda, Aki; Saga, Yumiko

    2016-01-01

    Evolutionally conserved Nanos RNA-binding proteins play crucial roles in germ cell development. While a mammalian Nanos family protein, NANOS2, is required for sexual differentiation of male (XY) germ cells in mice, the underlying mechanisms and the identities of its target RNAs in vivo remain elusive. Using comprehensive microarray analysis and a bacterial artificial chromosome transgenic system, here we identify Dazl, a germ cell-specific gene encoding an RNA-binding protein implicated in translation, as a crucial target of NANOS2. Importantly, removal of the Dazl 3'-untranslated region in XY germ cells stabilizes the Dazl mRNA, resulting in elevated meiotic gene expression, abnormal resumption of the cell cycle and impaired processing-body formation, reminiscent of Nanos2-knockout phenotypes. Furthermore, our data suggest that NANOS2 acts as an antagonist of the DAZL protein. We propose a dual system of NANOS2-mediated suppression of Dazl expression as a pivotal molecular mechanism promoting sexual differentiation of XY germ cells. PMID:27072294

  15. Mammalian Target of Rapamycin Complex 1 and Cyclooxygenase 2 Pathways Cooperatively Exacerbate Endometrial Cancer

    PubMed Central

    Daikoku, Takiko; Terakawa, Jumpei; Hossain, Md M.; Yoshie, Mikihiro; Cappelletti, Monica; Yang, Peiying; Ellenson, Lora H.; Dey, Sudhansu K.

    2015-01-01

    The underlying causes of endometrial cancer (EMC) are poorly understood, and treatment options for patients with advanced stages of the disease are limited. Mutations in the phosphatase and tensin homologue gene are frequently detected in EMC. Cyclooxygenase 2 (Cox2) and mammalian target of rapamycin complex 1 (mTORC1) are known downstream targets of the phosphatase and tensin homologue protein, and their activities are up-regulated in EMC. However, it is not clear whether Cox2 and mTORC1 are crucial players in cancer progression or whether they work in parallel or cooperatively. In this study, we used a Cox2 inhibitor, celecoxib, and an mTORC1 inhibitor, rapamycin, in mouse models of EMC and in human EMC cell lines to explore the interactive roles of Cox2 and mTORC1 signaling. We found that a combined treatment with celecoxib and rapamycin markedly reduces EMC progression. We also observed that rapamycin reduces Cox2 expression, whereas celecoxib reduces mTORC1 activity. These results suggest that Cox2 and mTORC1 signaling is cross-regulated and cooperatively exacerbate EMC. PMID:25058027

  16. SHEDDING NEW LIGHT ON NEURODEGENERATIVE DISEASES THROUGH THE MAMMALIAN TARGET OF RAPAMYCIN

    PubMed Central

    Chong, Zhao Zhong; Shang, Yan Chen; Wang, Shaohui; Maiese, Kenneth

    2012-01-01

    Neurodegenerative disorders affect a significant portion of the world's population leading to either disability or death for almost 30 million individuals worldwide. One novel therapeutic target that may offer promise for multiple disease entities that involve Alzheimer's disease, Parkinson's disease, epilepsy, trauma, stroke, and tumors of the nervous system is the mammalian target of rapamycin (mTOR). mTOR signaling is dependent upon the mTORC1 and mTORC2 complexes that are composed of mTOR and several regulatory proteins including the tuberous sclerosis complex (TSC1, hamartin/ TSC2, tuberin). Through a number of integrated cell signaling pathways that involve those of mTORC1 and mTORC2 as well as more novel signaling tied to cytokines, Wnt, and forkhead, mTOR can foster stem cellular proliferation, tissue repair and longevity, and synaptic growth by modulating mechanisms that foster both apoptosis and autophagy. Yet, mTOR through its proliferative capacity may sometimes be detrimental to central nervous system recovery and even promote tumorigenesis. Further knowledge of mTOR and the critical pathways governed by this serine/threonine protein kinase can bring new light for neurodegeneration and other related diseases that currently require new and robust treatments. PMID:22980037

  17. Mammalian target of rapamycin signaling inhibition ameliorates vascular calcification via Klotho upregulation.

    PubMed

    Zhao, Yang; Zhao, Ming-Ming; Cai, Yan; Zheng, Ming-Fei; Sun, Wei-Liang; Zhang, Song-Yang; Kong, Wei; Gu, Jun; Wang, Xian; Xu, Ming-Jiang

    2015-10-01

    Vascular calcification (VC) is a major risk factor for cardiovascular mortality in chronic renal failure (CRF) patients, but the pathogenesis remains partially unknown and effective therapeutic targets should be urgently explored. Here we pursued the therapeutic role of rapamycin in CRF-related VC. Mammalian target of rapamycin (mTOR) signal was activated in the aortic wall of CRF rats. As expected, oral rapamycin administration significantly reduced VC by inhibiting mTOR in rats with CRF. Further in vitro results showed that activation of mTOR by both pharmacological agent and genetic method promoted, while inhibition of mTOR reduced, inorganic phosphate-induced vascular smooth muscle cell (VSMC) calcification and chondrogenic/osteogenic gene expression, which were independent of autophagy and apoptosis. Interestingly, the expression of Klotho, an antiaging gene that suppresses VC, was reduced in calcified vasculature, whereas rapamycin reversed membrane and secreted Klotho decline through mTOR inhibition. When mTOR signaling was enhanced by either mTOR overexpression or deletion of tuberous sclerosis 1, Klotho mRNA was further decreased in phosphate-treated VSMCs, suggesting a vital association between mTOR signaling and Klotho expression. More importantly, rapamycin failed to reduce VC in the absence of Klotho by using either siRNA knockdown of Klotho or Klotho knockout mice. Thus, Klotho has a critical role in mediating the observed decrease in calcification by rapamycin in vitro and in vivo. PMID:26061549

  18. Activation of mammalian target of rapamycin (mTOR) in triple negative feline mammary carcinomas

    PubMed Central

    2013-01-01

    Background Triple negative breast cancer (TNBC) in humans is defined by the absence of oestrogen receptor (ER), progesterone receptor (PR) and HER2 overexpression. Mammalian target of rapamycin (mTOR) is overexpressed in TNBC and it represents a potential target for the treatment of this aggressive tumour. Feline mammary carcinoma (FMC) is considered to be a model for hormone-independent human breast cancer. This study investigated mTOR and p-mTOR expression in FMC in relation to triple negative (TN) phenotype. Results The expression of mTOR, p-mTOR, ERα, PR and HER2 was evaluated in 58 FMCs by immunohistochemistry and in six FMC cell lines by Western blot analysis. 53.5% of FMC analyzed were ER, PR, HER2 negative (TN-FMC) while 56.9% and 55.2% of cases expressed mTOR and p-mTOR respectively. In this study we found that m-TOR and p-mTOR were more frequently detected in TN-FMC and in HER2 negative samples. Conclusions In this study, we demonstrate that there is also a FMC subset defined as TN FMC, which is characterised by a statistically significant association with m-TOR and p-mTOR expression as demonstrated in human breast cancer. PMID:23587222

  19. Inhibition of lung tumor growth by targeting EGFR/VEGFR-Akt/NF-κB pathways with novel theanine derivatives

    PubMed Central

    Zhang, Guoying; Ye, Xinshan; Wu, Erxi; Wang, Fengfei; Wu, Fei; Tian, Huihui; Liu, Xin; Chen, Linlin; Liu, Kun; Wang, Yishan; Liu, Hanchen; Zhang, Wenhua; Guan, Yukun; Wang, Qinwen; Zhao, Xiaohang; Wan, Xiaochun

    2014-01-01

    The molecularly targeted agents, including anti-VEGF or anti-EGFR monoclonal antibody and some inhibitors of EGFR tyrosine kinase, are effective in the treatment of non-small-cell lung cancer (NSCLC) to a certain extent, but the benefit for a proportion of patients is still limited. Hence, it is necessary and urgent to develop more selective and effective molecular targeted agents against lung cancer. Here, we have synthesized novel theanine derivatives, methyl coumarin-3-carboxylyl L-theanine (TMC), ethyl coumarin-3-carboxylyl L-theanine (TEC), ethyl 6-fluorocoumarin- 3-carboxylyl L-theanine (TFC), and ethyl 6-nitrocoumarin-3-carboxylyl L-theanine (TNC), which are fluorescent small molecules, based on their parental compound theanine and studied their anticancer activities in vitro, ex vivo and in vivo models of human and mouse cancers. Our results show that the four theanine derivatives significantly inhibit the lung cancer cell migration and the growth of lung cancer and leukemia cell lines. TFC and TNC display enhanced effects with anticancer drugs cytarabine, vincristine, and methotrexate on inhibition of lung cancer cell growth and no toxicity to the normal human embryonic lung fibroblast and peripheral blood lymphocytes. TFC and TNC exhibit strong suppression of the highly metastatic Lewis lung cancer (LLC) and A549 tumor growth in tumor-bearing mice without toxicity to mice. TFC and TNC can effectively suppress the growth of lung cancer cells in vitro, ex vivo and in vivo by targeting EGFR/VEGFR-Akt/NF-κB pathways. Our study has suggested that TFC and TNC may have the therapeutic and/or adjuvant therapeutic applications in the treatment of lung cancers and other cancer. PMID:25138052

  20. Inhibition of lung tumor growth by targeting EGFR/VEGFR-Akt/NF-κB pathways with novel theanine derivatives.

    PubMed

    Zhang, Guoying; Ye, Xinshan; Ji, Dexin; Zhang, Huarong; Sun, Fujia; Shang, Chunqing; Zhang, Ying; Wu, Erxi; Wang, Fengfei; Wu, Fei; Tian, Huihui; Liu, Xin; Chen, Linlin; Liu, Kun; Wang, Yishan; Liu, Hanchen; Zhang, Wenhua; Guan, Yukun; Wang, Qinwen; Zhao, Xiaohang; Wan, Xiaochun

    2014-09-30

    The molecularly targeted agents, including anti-VEGF or anti-EGFR monoclonal antibody and some inhibitors of EGFR tyrosine kinase, are effective in the treatment of non-small-cell lung cancer (NSCLC) to a certain extent, but the benefit for a proportion of patients is still limited. Hence, it is necessary and urgent to develop more selective and effective molecular targeted agents against lung cancer. Here, we have synthesized novel theanine derivatives, methyl coumarin-3-carboxylyl L-theanine (TMC), ethyl coumarin-3-carboxylyl L-theanine (TEC), ethyl 6-fluorocoumarin- 3-carboxylyl L-theanine (TFC), and ethyl 6-nitrocoumarin-3-carboxylyl L-theanine (TNC), which are fluorescent small molecules, based on their parental compound theanine and studied their anticancer activities in vitro, ex vivo and in vivo models of human and mouse cancers. Our results show that the four theanine derivatives significantly inhibit the lung cancer cell migration and the growth of lung cancer and leukemia cell lines. TFC and TNC display enhanced effects with anticancer drugs cytarabine vincristine, andmethotrexate on inhibition of lung cancer cell growth and no toxicity to the normal human embryonic lung fibroblast and peripheral blood lymphocytes. TFC and TNC exhibit strong suppression of the highly metastatic Lewis lung cancer (LLC) and A549 tumor growth in tumor-bearing mice without toxicity to mice. TFC and TNC can effectively suppress the growth of lung cancer cells in vitro, ex vivo and in vivo by targeting EGFR/VEGFR-Akt/NF-κB pathways. Our study has suggested that TFC and TNC may have the therapeutic and/or adjuvant therapeutic applications in the treatment of lung cancers and other cancer. PMID:25138052

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

    SciTech Connect

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

    2009-09-18

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

  2. The Upregulation of PI3K/Akt and MAP Kinase Pathways is Associated with Resistance of Microtubule-Targeting Drugs in Prostate Cancer.

    PubMed

    Liu, Zhi; Zhu, Guangjing; Getzenberg, Robert H; Veltri, Robert W

    2015-07-01

    Resistance is a significant limitation to the effectiveness of cancer therapies. The PI3K/Akt and MAP kinase pathways play important roles in a variety of normal cellular processes and tumorigenesis. This study is designed to explore the relationship of these signaling pathways with multidrug resistance in prostate cancer (PCa). The PI3K/Akt and MAP kinase pathways were investigated utilizing paclitaxel resistant DU145-TxR PCa cells and their parental non-resistant DU145 cells to determine their relationship with resistance to paclitaxel and other anticancer drugs. Our results demonstrate that the PI3K/Akt and MAP kinase pathways are upregulated in DU145-TxR cells compared to the DU145 cells. Inactivating these pathways using the PI3K/Akt pathway inhibitor LY294002 or the MAP kinase pathway inhibitor PD98059 renders the DU145-TxR cells more sensitive to paclitaxel. We investigated the effects of these inhibitors on other anticancer drugs including docetaxel, vinblastine, doxorubicin, 10-Hydroxycamptothecin (10-HCPT) and cisplatin and find that both inhibitors induces DU145-TxR cells to be more sensitive only to the microtubule-targeting drugs (paclitaxel, docetaxel and vinblastine). Furthermore, the treatment with these inhibitors induces cleaved-PARP production in DU145-TxR cells, suggesting that apoptosis induction might be one of the mechanisms for the reversal of drug resistance. In conclusion, the PI3K/Akt and MAP kinase pathways are associated with resistance to multiple chemotherapeutic drugs. Inactivating these pathways renders these PCa cells more sensitive to microtubule-targeting drugs such as paclitaxel, docetaxel and vinblastine. Combination therapies with novel inhibitors of these two signaling pathways potentially represents a more effective treatment for drug resistant PCa. PMID:25640606

  3. Efficacy and Safety of Mammalian Target of Rapamycin Inhibitors in Vascular Anomalies: A Systematic Review.

    PubMed

    Nadal, Marion; Giraudeau, Bruno; Tavernier, Elsa; Jonville-Bera, Annie-Pierre; Lorette, Gerárd; Maruani, Annabel

    2016-05-01

    Mammalian target of rapamycin (mTOR) inhibitors are a promising new treatment in vascular anomalies, but no published randomized controlled trials are available. The aim of this systematic review of all reported cases was to assess the efficacy and safety of mTOR inhibitors in all vascular anomalies, except cancers, in children and adults. In November 2014 MEDLINE, CENTRAL, LILACS and EMBASE were searched for studies of mTOR inhibitors in any vascular condition, except for malignant lesions, in humans. Fourteen publications and 9 posters, with data on 25 and 59 patients, respectively, all < 18 years old were included. Of these patients, 35.7% (n = 30) had vascular tumours, and 64.3% (n = 54) had malformations. Sirolimus was the most frequent mTOR inhibitor used (98.8%, n = 83). It was efficient in all cases, at a median time of 2 weeks (95% confidence interval 1-10 weeks). Sirolimus was well tolerated, the main side-effect being mouth sores, which led to treatment withdrawal in one case. The dosage of sirolimus was heterogeneous, the most common being 1.6 mg/m2/day. PMID:26607948

  4. Activation of Mammalian target of rapamycin in canine mammary carcinomas: an immunohistochemical study.

    PubMed

    Delgado, L; Gärtner, F; Dias Pereira, P

    2015-01-01

    Mammalian target of rapamycin (mTOR) is a serine-threonine kinase involved in cell growth, proliferation and survival. Activation of mTOR has been reported in various tumour types, including human breast cancer; however, the expression of mTOR in canine mammary tumours has not been examined. In the present study, expression of the activated form of mTOR (phospho-mTOR [p-mTOR]) was examined immunohistochemically in five normal canine mammary glands, 45 canine mammary carcinomas and their corresponding metastatic lesions (n = 15). Phospho-mTOR was not expressed in normal canine mammary tissue, but cytoplasmic labelling was observed in 78% of canine mammary carcinomas. Two carcinomas had both cytoplasmic and nuclear labelling. No significant relationship was found between p-mTOR cytoplasmic expression and histological type or grading of carcinomas, degree of tubular formation, anisokaryosis, mitotic activity or lymph node metastasis. In all except one case, the expression pattern of p-mTOR in lymph node metastases was similar or decreased when compared with the primary lesion. The findings suggest that p-mTOR is involved in mammary carcinogenesis in dogs. However, p-mTOR cytoplasmic expression does not appear to be a prognostic indicator in canine mammary carcinomas, which may be related to its subcellular location in the neoplastic cells. Canine mammary tumours may provide a model for the development of innovative medical strategies involving mTOR inhibitors in human breast cancer. PMID:25670666

  5. RBFox2 Binds Nascent RNA to Globally Regulate Polycomb Complex 2 Targeting in Mammalian Genomes.

    PubMed

    Wei, Chaoliang; Xiao, Rui; Chen, Liang; Cui, Hanwei; Zhou, Yu; Xue, Yuanchao; Hu, Jing; Zhou, Bing; Tsutsui, Taiki; Qiu, Jinsong; Li, Hairi; Tang, Liling; Fu, Xiang-Dong

    2016-06-16

    Increasing evidence suggests that diverse RNA binding proteins (RBPs) interact with regulatory RNAs to regulate transcription. RBFox2 is a well-characterized pre-mRNA splicing regulator, but we now encounter an unexpected paradigm where depletion of this RBP induces widespread increase in nascent RNA production in diverse cell types. Chromatin immunoprecipitation sequencing (ChIP-seq) reveals extensive interaction of RBFox2 with chromatin in a nascent RNA-dependent manner. Bayesian network analysis connects RBFox2 to Polycomb complex 2 (PRC2) and H3K27me3, and biochemical experiments demonstrate the ability of RBFox2 to directly interact with PRC2. Strikingly, RBFox2 inactivation eradicates PRC2 targeting on the majority of bivalent gene promoters and leads to transcriptional de-repression. Together, these findings uncover a mechanism underlying the enigmatic association of PRC2 with numerous active genes, highlight the importance of gene body sequences to gauge transcriptional output, and suggest nascent RNAs as critical signals for transcriptional feedback control to maintain homeostatic gene expression in mammalian genomes. PMID:27211866

  6. The Rapamycin-Sensitive Complex of Mammalian Target of Rapamycin Is Essential to Maintain Male Fertility.

    PubMed

    Schell, Christoph; Kretz, Oliver; Liang, Wei; Kiefer, Betina; Schneider, Simon; Sellung, Dominik; Bork, Tillmann; Leiber, Christian; Rüegg, Markus A; Mallidis, Con; Schlatt, Stefan; Mayerhofer, Artur; Huber, Tobias B; Grahammer, Florian

    2016-02-01

    The mammalian target of rapamycin complex 1 (mTORC1) inhibitor rapamycin and its analogs are being increasingly used in solid-organ transplantation. A commonly reported side effect is male subfertility to infertility, yet the precise mechanisms of mTOR interference with male fertility remain obscure. With the use of a conditional mouse genetic approach we demonstrate that deficiency of mTORC1 in the epithelial derivatives of the Wolffian duct is sufficient to cause male infertility. Analysis of spermatozoa from Raptor fl/fl*KspCre mice revealed an overall decreased motility pattern. Both epididymis and seminal vesicles displayed extensive organ regression with increasing age. Histologic and ultrastructural analyses demonstrated increased amounts of destroyed and absorbed spermatozoa in different segments of the epididymis. Mechanistically, genetic and pharmacologic mTORC1 inhibition was associated with an impaired cellular metabolism and a disturbed protein secretion of epididymal epithelial cells. Collectively, our data highlight the role of mTORC1 to preserve the function of the epididymis, ductus deferens, and the seminal vesicles. We thus reveal unexpected new insights into the frequently observed mTORC1 inhibitor side effect of male infertility in transplant recipients. PMID:26683665

  7. Mammalian target of rapamycin inhibition in polycystic kidney disease: From bench to bedside.

    PubMed

    Kim, Hyun-Jung; Edelstein, Charles L

    2012-09-01

    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. PMID:26894018

  8. A comprehensive review of everolimus clinical reports: a new mammalian target of rapamycin inhibitor.

    PubMed

    Gurk-Turner, Cheryle; Manitpisitkul, Wana; Cooper, Matthew

    2012-10-15

    As new immunosuppressive agents are introduced to the market, clinicians are faced with the daunting task of sifting through the published literature to decide the value that the agent will add to their own practice. We often must extrapolate information provided through study in other solid-organ transplantation populations than our specific area of interest as we interpret the results and outcomes. With these challenges in mind, this compilation of published work for the newest mammalian target of rapamycin inhibitor everolimus (Certican; Novartis Pharmaceuticals, Hanover, NJ) (Zortress; Novartis Pharmaceuticals, Basel, Switzerland) is intended to provide a concise but thorough presentation of available literature so that the reader who may be unfamiliar with the agent can make their own judgment. Both Ovid and PubMed search engines were queried with a particular focus on high-impact articles noted in the Web of Science or Citation Index. Work described solely in abstract or case report form was excluded, as well as meta-analyses or those that were editorial or commentary in nature. Included were publications presented using the English language that described adult human subjects who received a heart, lung, kidney, or liver allograft. The goal of this strategy was to allow for the inclusion of pertinent literature in an unbiased fashion. Tables are provided that outline trial specific information, leaving a discussion of major outcomes to the text of the review. PMID:22986894

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

    PubMed

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

    2016-07-01

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

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

    PubMed

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

    2016-05-28

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

  11. Roles of PI3K/AKT/PTEN Pathway as a Target for Pharmaceutical Therapy.

    PubMed

    Matsuda, Satoru; Nakanishi, Atsuko; Wada, Yoko; Kitagishi, Yasuko

    2013-01-01

    Multiple enzymes participate in the phosphorylation of a group of phosphoinositide lipids. Because of their important role in signal transduction, the dysregulated metabolism of phosphoinositides represents a key step in many disease settings. Loss of their function has been demonstrated to occur as an early event a wide variety of carcinogenesis and has therefore been suggested as a biomarker for the premalignant disease. In addition, genetic alterations at multiple nodes in the pathway have been implicated in several other diseases. Accordingly, given this pervasive involvement in many diseases, the development of molecules that modulates this pathway has been initiated in studies. They have been the focus of extensive research and drug discovery activities. A better understanding of the molecular connections could uncover new targets for drug development. PMID:24222802

  12. The novel orally bioavailable inhibitor of phosphoinositol-3-kinase and mammalian target of rapamycin, NVP-BEZ235, inhibits growth and proliferation in multiple myeloma

    SciTech Connect

    Baumann, Philipp Mandl-Weber, Sonja; Oduncu, Fuat; Schmidmaier, Ralf

    2009-02-01

    NVP-BEZ235 is a new inhibitor of phosphoinositol-3-kinase (PI3 kinase) and mammalian target of rapamycin (mTOR) whose efficacy in advanced solid tumours is currently being evaluated in a phase I/II clinical trial. Here we show that NVP-BEZ235 inhibits growth in common myeloma cell lines as well as primary myeloma cells at nanomolar concentrations in a time and dose dependent fashion. Further experiments revealed induction of apoptosis in three of four cell lines. Inhibition of cell growth was mainly due to inhibition of myeloma cell proliferation, as shown by the BrdU assay. Cell cycle analysis revealed induction of cell cycle arrest in the G1 phase, which was due to downregulation of cyclin D1, pRb and cdc25a. NVP-BEZ235 inhibited phosphorylation of protein kinase B (Akt), P70S6k and 4E-BP-1. Furthermore we show that the stimulatory effect of CD40-ligand (CD40L), insulin-like growth factor 1 (IGF-1), interleukin-6 (IL-6) and conditioned medium of HS-5 stromal cells on myeloma cell growth is completely abrogated by NVP-BEZ235. In addition, synergism studies revealed synergistic and additive activity of NVP-BEZ235 together with melphalan, doxorubicin and bortezomib. Taken together, inhibition of PI3 kinase/mTOR by NVP-BEZ235 is highly effective and NVP-BEZ235 represents a potential new candidate for targeted therapy in multiple myeloma.

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

    PubMed Central

    Matsuoka, Tasuku; Yashiro, Masakazu

    2014-01-01

    The phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) pathway is one of the key signaling pathways induced by various receptor-tyrosine kinases. Accumulating evidence shows that this pathway is an important promoter of cell growth, metabolism, survival, metastasis, and resistance to chemotherapy. Genetic alterations in the PI3K/Akt/mTOR pathway in gastric carcinoma have often been demonstrated. Many kinds of molecular targeting therapies are currently undergoing clinical testing in patients with solid tumors. However, with the exception of the ErbB2-targeting antibody, targeting agents, including PI3K/Akt/mTOR inhibitors, have not been approved for treatment of patients with gastric carcinoma. This review summarizes the current knowledge on PI3K/Akt/mTOR signaling in the pathogenesis of gastric carcinoma and the possible therapeutic targets for gastric carcinoma. Improved knowledge of the PI3K/Akt/mTOR pathway in gastric carcinoma will be useful in understanding the mechanisms of tumor development and for identifying ideal targets of anticancer therapy for gastric carcinoma. PMID:25003395

  14. Design and synthesis of 2-oxindole based multi-targeted inhibitors of PDK1/Akt signaling pathway for the treatment of glioblastoma multiforme.

    PubMed

    Sestito, Simona; Nesi, Giulia; Daniele, Simona; Martelli, Alma; Digiacomo, Maria; Borghini, Alice; Pietra, Daniele; Calderone, Vincenzo; Lapucci, Annalina; Falasca, Marco; Parrella, Paola; Notarangelo, Angelantonio; Breschi, Maria C; Macchia, Marco; Martini, Claudia; Rapposelli, Simona

    2015-11-13

    Aggressive behavior and diffuse infiltrative growth are the main features of Glioblastoma multiforme (GBM), together with the high degree of resistance and recurrence. Evidence indicate that GBM-derived stem cells (GSCs), endowed with unlimited proliferative potential, play a critical role in tumor development and maintenance. Among the many signaling pathways involved in maintaining GSC stemness, tumorigenic potential, and anti-apoptotic properties, the PDK1/Akt pathway is a challenging target to develop new potential agents able to affect GBM resistance to chemotherapy. In an effort to find new PDK1/Akt inhibitors, we rationally designed and synthesized a small family of 2-oxindole derivatives. Among them, compound 3 inhibited PDK1 kinase and downstream effectors such as CHK1, GS3Kα and GS3Kβ, which contribute to GCS survival. Compound 3 appeared to be a good tool for studying the role of the PDK1/Akt pathway in GCS self-renewal and tumorigenicity, and might represent the starting point for the development of more potent and focused multi-target therapies for GBM. PMID:26498573

  15. siDirect: highly effective, target-specific siRNA design software for mammalian RNA interference

    PubMed Central

    Naito, Yuki; Yamada, Tomoyuki; Ui-Tei, Kumiko; Morishita, Shinichi; Saigo, Kaoru

    2004-01-01

    siDirect (http://design.RNAi.jp/) is a web-based online software system for computing highly effective small interfering RNA (siRNA) sequences with maximum target-specificity for mammalian RNA interference (RNAi). Highly effective siRNA sequences are selected using novel guidelines that were established through an extensive study of the relationship between siRNA sequences and RNAi activity. Our efficient software avoids off-target gene silencing to enumerate potential cross-hybridization candidates that the widely used BLAST search may overlook. The website accepts an arbitrary sequence as input and quickly returns siRNA candidates, providing a wide scope of applications in mammalian RNAi, including systematic functional genomics and therapeutic gene silencing. PMID:15215364

  16. Congenital segmental lymphedema in tuberous sclerosis complex with associated subependymal giant cell astrocytomas treated with Mammalian target of rapamycin inhibitors.

    PubMed

    Prato, Giulia; Mancardi, Maria Margherita; Baglietto, Maria Giuseppina; Janis, Sara; Vercellino, Nadia; Rossi, Andrea; Consales, Alessandro; Raso, Alessandro; Garrè, Maria Luisa

    2014-09-01

    Tuberous sclerosis complex is a genetic, multisystemic disorder characterized by circumscribed benign lesions (hamartomas) in several organs, including brain. This is the result of defects in the TSC1 and/or TSC2 tumor suppressor genes, encoding the hamartin-tuberin complex that inhibits the mammalian target of rapamycin pathway. Specific inhibitors of this pathway have been shown to reduce the volume of subependymal giant cell astrocytomas associated with tuberous sclerosis. Congenital lymphedema is rarely seen in association with tuberous sclerosis, with only a few reported cases. Although this association can be coincidental, the dysgenetic lymphatic system can represent a hamartia as a consequence of gene mutation. We describe a child with congenital lymphedema in tuberous sclerosis and associated subependymal giant cell astrocytoma who experienced lymphangitis under treatment with mammalian target of rapamycin inhibitors. Because our patient did not show worsening of lymphedema, congenital lymphedema does not seem to be a contraindication for this therapy. PMID:24056156

  17. Metformin inhibits mammalian target of rapamycin-dependent translation initiation in breast cancer cells.

    PubMed

    Dowling, Ryan J O; Zakikhani, Mahvash; Fantus, I George; Pollak, Michael; Sonenberg, Nahum

    2007-11-15

    Metformin is used for the treatment of type 2 diabetes because of its ability to lower blood glucose. The effects of metformin are explained by the activation of AMP-activated protein kinase (AMPK), which regulates cellular energy metabolism. Recently, we showed that metformin inhibits the growth of breast cancer cells through the activation of AMPK. Here, we show that metformin inhibits translation initiation. In MCF-7 breast cancer cells, metformin treatment led to a 30% decrease in global protein synthesis. Metformin caused a dose-dependent specific decrease in cap-dependent translation, with a maximal inhibition of 40%. Polysome profile analysis showed an inhibition of translation initiation as metformin treatment of MCF-7 cells led to a shift of mRNAs from heavy to light polysomes and a concomitant increase in the amount of 80S ribosomes. The decrease in translation caused by metformin was associated with mammalian target of rapamycin (mTOR) inhibition, and a decrease in the phosphorylation of S6 kinase, ribosomal protein S6, and eIF4E-binding protein 1. The effects of metformin on translation were mediated by AMPK, as treatment of cells with the AMPK inhibitor compound C prevented the inhibition of translation. Furthermore, translation in MDA-MB-231 cells, which lack the AMPK kinase LKB1, and in tuberous sclerosis complex 2 null (TSC2(-/-)) mouse embryonic fibroblasts was unaffected by metformin, indicating that LKB1 and TSC2 are involved in the mechanism of action of metformin. These results show that metformin-mediated AMPK activation leads to inhibition of mTOR and a reduction in translation initiation, thus providing a possible mechanism of action of metformin in the inhibition of cancer cell growth. PMID:18006825

  18. Contribution of mammalian target of rapamycin in the pathophysiology of cirrhotic cardiomyopathy

    PubMed Central

    Saeedi Saravi, Seyed Soheil; Ghazi-Khansari, Mahmoud; Ejtemaei Mehr, Shahram; Nobakht, Maliheh; Mousavi, Seyyedeh Elaheh; Dehpour, Ahmad Reza

    2016-01-01

    AIM: To explore the role of mammalian target of rapamycin (mTOR) in the pathogenesis of cirrhotic cardiomyopathy and the potential of rapamycin to improve this pathologic condition. METHODS: Male albino Wistar rats weighing 100-120 g were treated with tetrachloride carbon (CCl4) for 8 wk to induce cirrhosis. Subsequently, animals were administered rapamycin (2 mg/kg per day). The QTc intervals were calculated in a 5-min electrocardiogram. Then, the left ventricular papillary muscles were isolated to examine inotropic responsiveness to β-adrenergic stimulation using a standard organ bath equipped by Powerlab system. Phosphorylated-mTOR localization in left ventricles was immunohistochemically assessed, and ventricular tumor necrosis factor (TNF)-α was measured. Western blot was used to measure levels of ventricular phosphorylated-mTOR protein. RESULTS: Cirrhosis was confirmed by hematoxylin and eosin staining of liver tissues, visual observation of lethargy, weight loss, jaundice, brown urine, ascites, liver stiffness, and a significant increase of spleen weight (P < 0.001). A significant prolongation in QTc intervals occurred in cirrhotic rats exposed to CCl4 (P < 0.001), while this prolongation was decreased with rapamycin treatment (P < 0.01). CCl4-induced cirrhosis caused a significant decrease of contractile responsiveness to isoproterenol stimulation and a significant increase in cardiac TNF-α. These findings were correlated with data from western blot and immunohistochemical studies on phosphorylated-mTOR expression in left ventricles. Phosphorylated-mTOR was significantly enhanced in cirrhotic rats, especially in the endothelium, compared to controls. Rapamycin treatment significantly increased contractile force and myocardial localization of phosphorylated-mTOR and decreased cardiac TNF-α concentration compared to cirrhotic rats with no treatment. CONCLUSION: In this study, we demonstrated a potential role for cardiac mTOR in the pathophysiology of

  19. Akt1 Mediates α-Smooth Muscle Actin Expression and Myofibroblast Differentiation via Myocardin and Serum Response Factor*

    PubMed Central

    Abdalla, Maha; Goc, Anna; Segar, Lakshman; Somanath, Payaningal R.

    2013-01-01

    Myofibroblast (MF) differentiation, marked by the de novo expression of smooth muscle α-actin (αSMA) stress fibers, plays a central role in wound healing and its persistence is a hallmark of fibrotic diseases. We have previously shown that Akt1 is necessary for wound healing through matrix regulation. However, the role of Akt1 in regulating MF differentiation with implications in fibrosis remains poorly defined. Here, we show that sustained activation of Akt1 was associated with a 6-fold increase in αSMA expression and assembly; an effect that is blunted in cells expressing inactive Akt1 despite TGFβ stimulation. Mechanistically, Akt1 mediated TGFβ-induced αSMA synthesis through the contractile gene transcription factors myocardin and serum response factor (SRF), independent of mammalian target of rapamycin in mouse embryonic fibroblasts and fibroblasts overexpressing active Akt1. Akt1 deficiency was associated with decreased myocardin, SRF, and αSMA expressions in vivo. Furthermore, sustained Akt1-induced αSMA synthesis markedly decreased upon RNA silencing of SRF and myocardin. In addition to its integral role in αSMA synthesis, we also show that Akt1 mediates fibronectin splice variant expression, which is required for MF differentiation, as well as total fibronectin, which generates the contractile force that promotes MF differentiation. In summary, our results constitute evidence that sustained Akt1 activation is crucial for TGFβ-induced MF formation and persistent differentiation. These findings highlight Akt1 as a novel potential therapeutic target for fibrotic diseases. PMID:24106278

  20. Rictor Phosphorylation on the THR-1135 Site Does Not Require Mammalian Target of Rapamycin Complex 2

    PubMed Central

    Boulbes, Delphine; Chen, Chien-Hung; Shaikenov, Tattym; Agarwal, Nitin K.; Peterson, Timothy R.; Addona, Terri A.; Keshishian, Hasmik; Carr, Steven A.; Magnuson, Mark A.; Sabatini, David M.; Sarbassov, Dos D.

    2010-01-01

    In animal cells growth factors coordinate cell proliferation and survival by regulating the PI3K/Akt signaling pathway. Deregulation of this signaling pathway is common in a variety of human cancers. The PI3K dependent signaling kinase complex defined as mTORC2 functions as a regulatory Ser-473 kinase of Akt. We find that activation of mTORC2 by growth factor signaling is linked to the specific phosphorylation of its component rictor on Thr-1135. The phosphorylation of this site is induced by the growth factor stimulation and expression of the oncogenic forms of ras or PI3K. Rictor phosphorylation is sensitive to inhibition of PI3K, mTOR, or expression of ILK. The substitution of wild-type rictor with its specific phospho-mutants in rictor null mouse embryonic fibroblasts did not alter the growth factor-dependent phosphorylation of Akt indicating that the rictor Thr-1135 phosphorylation is not critical in regulation of the mTORC2 kinase activity. We found that this rictor phosphorylation takes place in the mTORC2-deficient cells suggesting that this modification might play a role in regulation not only mTORC2 but also the mTORC2-independent function of rictor. PMID:20501647

  1. The Effects of Glucagon-like Peptide-2 on the Tight Junction and Barrier Function in IPEC-J2 Cells through Phosphatidylinositol 3-kinase–Protein Kinase B–Mammalian Target of Rapamycin Signaling Pathway

    PubMed Central

    Yu, Changsong; Jia, Gang; Deng, Qiuhong; Zhao, Hua; Chen, Xiaoling; Liu, Guangmang; Wang, Kangning

    2016-01-01

    Glucagon-like peptide-2 (GLP-2) is important for intestinal barrier function and regulation of tight junction (TJ) proteins, but the intracellular mechanisms of action remain undefined. The purpose of this research was to determine the protective effect of GLP-2 mediated TJ and transepithelial electrical resistance (TER) in lipopolysaccharide (LPS) stressed IPEC-J2 cells and to test the hypothesis that GLP-2 regulate TJ and TER through the phosphatidylinositol 3-kinase (PI3K)-protein kinase B (Akt)-mammalian target of rapamycin (mTOR) signaling pathway in IPEC-J2 cells. Wortmannin and LY294002 are specific inhibitors of PI3K. The results showed that 100 μg/mL LPS stress decreased TER and TJ proteins occludin, claudin-1 and zonula occludens protein 1 (ZO-1) mRNA, proteins expressions (p<0.01) respectively. GLP-2 (100 nmol/L) promote TER and TJ proteins occludin, claudin-1, and zo-1 mRNA, proteins expressions in LPS stressed and normal IPEC-J2 cells (p<0.01) respectively. In normal cells, both wortmannin and LY294002, PI3K inhibitors, prevented the mRNA and protein expressions of Akt and mTOR increase induced by GLP-2 (p<0.01) following with the significant decreasing of occludin, claudin-1, ZO-1 mRNA and proteins expressions and TER (p<0.01). In conclusion, these results indicated that GLP-2 can promote TJ’s expression and TER in LPS stressed and normal IPEC-J2 cells and GLP-2 could regulate TJ and TER through the PI3K/Akt/mTOR pathway. PMID:26954146

  2. miR-103 promotes 3T3-L1 cell adipogenesis through AKT/mTOR signal pathway with its target being MEF2D.

    PubMed

    Li, Meihang; Liu, Zhenjiang; Zhang, Zhenzhen; Liu, Guannv; Sun, Shiduo; Sun, Chao

    2015-03-01

    MicroRNAs are small non-coding RNAs that partially bind to the 3' untranslated (3'UTR) regions of target genes in animals and regulate protein production of the target transcripts. MiR-103 has been confirmed to play a critical role in lipid metabolism, however, the target genes and signaling pathway regulated by miR-103 is still unclear. In our experiment, we observed a positive function of miR-103 on the adipogenic differentiation of 3T3-L1 pre-adipocyte. Furthermore, we proved that this function of miR-103 worked through activating AKT/mTOR signal pathway and impairing target gene MEF2D. By inhibiting and over-expressing the MEF2D gene, we found that MEF2D had a negative role in regulating adipocyte key genes, and this function of MEF2D could be impaired by miR-103. In conclusion, we found that miR-103 can promote 3T3-L1 cells differentiation by targeting MEF2D and activating AKT/mTOR signal pathway. These results will shed a light on further study of microRNAs. PMID:25400071

  3. Structure-Activity Analysis of Niclosamide Reveals Potential Role for Cytoplasmic pH in Control of Mammalian Target of Rapamycin Complex 1 (mTORC1) Signaling*

    PubMed Central

    Fonseca, Bruno D.; Diering, Graham H.; Bidinosti, Michael A.; Dalal, Kush; Alain, Tommy; Balgi, Aruna D.; Forestieri, Roberto; Nodwell, Matt; Rajadurai, Charles V.; Gunaratnam, Cynthia; Tee, Andrew R.; Duong, Franck; Andersen, Raymond J.; Orlowski, John; Numata, Masayuki; Sonenberg, Nahum; Roberge, Michel

    2012-01-01

    Mammalian target of rapamycin complex 1 (mTORC1) signaling is frequently dysregulated in cancer. Inhibition of mTORC1 is thus regarded as a promising strategy in the treatment of tumors with elevated mTORC1 activity. We have recently identified niclosamide (a Food and Drug Administration-approved antihelminthic drug) as an inhibitor of mTORC1 signaling. In the present study, we explored possible mechanisms by which niclosamide may inhibit mTORC1 signaling. We tested whether niclosamide interferes with signaling cascades upstream of mTORC1, the catalytic activity of mTOR, or mTORC1 assembly. We found that niclosamide does not impair PI3K/Akt signaling, nor does it inhibit mTORC1 kinase activity. We also found that niclosamide does not interfere with mTORC1 assembly. Previous studies in helminths suggest that niclosamide disrupts pH homeostasis of the parasite. This prompted us to investigate whether niclosamide affects the pH balance of cancer cells. Experiments in both breast cancer cells and cell-free systems demonstrated that niclosamide possesses protonophoric activity in cells and in vitro. In cells, niclosamide dissipated protons (down their concentration gradient) from lysosomes to the cytosol, effectively lowering cytoplasmic pH. Notably, analysis of five niclosamide analogs revealed that the structural features of niclosamide required for protonophoric activity are also essential for mTORC1 inhibition. Furthermore, lowering cytoplasmic pH by means other than niclosamide treatment (e.g. incubation with propionic acid or bicarbonate withdrawal) recapitulated the inhibitory effects of niclosamide on mTORC1 signaling, lending support to a possible role for cytoplasmic pH in the control of mTORC1. Our data illustrate a potential mechanism for chemical inhibition of mTORC1 signaling involving modulation of cytoplasmic pH. PMID:22474287

  4. Structure-activity analysis of niclosamide reveals potential role for cytoplasmic pH in control of mammalian target of rapamycin complex 1 (mTORC1) signaling.

    PubMed

    Fonseca, Bruno D; Diering, Graham H; Bidinosti, Michael A; Dalal, Kush; Alain, Tommy; Balgi, Aruna D; Forestieri, Roberto; Nodwell, Matt; Rajadurai, Charles V; Gunaratnam, Cynthia; Tee, Andrew R; Duong, Franck; Andersen, Raymond J; Orlowski, John; Numata, Masayuki; Sonenberg, Nahum; Roberge, Michel

    2012-05-18

    Mammalian target of rapamycin complex 1 (mTORC1) signaling is frequently dysregulated in cancer. Inhibition of mTORC1 is thus regarded as a promising strategy in the treatment of tumors with elevated mTORC1 activity. We have recently identified niclosamide (a Food and Drug Administration-approved antihelminthic drug) as an inhibitor of mTORC1 signaling. In the present study, we explored possible mechanisms by which niclosamide may inhibit mTORC1 signaling. We tested whether niclosamide interferes with signaling cascades upstream of mTORC1, the catalytic activity of mTOR, or mTORC1 assembly. We found that niclosamide does not impair PI3K/Akt signaling, nor does it inhibit mTORC1 kinase activity. We also found that niclosamide does not interfere with mTORC1 assembly. Previous studies in helminths suggest that niclosamide disrupts pH homeostasis of the parasite. This prompted us to investigate whether niclosamide affects the pH balance of cancer cells. Experiments in both breast cancer cells and cell-free systems demonstrated that niclosamide possesses protonophoric activity in cells and in vitro. In cells, niclosamide dissipated protons (down their concentration gradient) from lysosomes to the cytosol, effectively lowering cytoplasmic pH. Notably, analysis of five niclosamide analogs revealed that the structural features of niclosamide required for protonophoric activity are also essential for mTORC1 inhibition. Furthermore, lowering cytoplasmic pH by means other than niclosamide treatment (e.g. incubation with propionic acid or bicarbonate withdrawal) recapitulated the inhibitory effects of niclosamide on mTORC1 signaling, lending support to a possible role for cytoplasmic pH in the control of mTORC1. Our data illustrate a potential mechanism for chemical inhibition of mTORC1 signaling involving modulation of cytoplasmic pH. PMID:22474287

  5. Unique roles of Akt1 and Akt2 in IGF-IR mediated lung tumorigenesis

    PubMed Central

    Franks, S. Elizabeth; Briah, Ritesh; Jones, Robert A.; Moorehead, Roger A.

    2016-01-01

    AKT is a serine-threonine kinase that becomes hyperactivated in a number of cancers including lung cancer. Based on AKT's association with malignancy, molecules targeting AKT have entered clinical trials for solid tumors including lung cancer. However, the AKT inhibitors being evaluated in clinical trials indiscriminately inhibit all three AKT isoforms (AKT1–3) and it remains unclear whether AKT isoforms have overlapping or divergent functions. Using a transgenic mouse model where IGF-IR overexpression drives lung tumorigenesis, we found that loss of Akt1 inhibited while loss of Akt2 enhanced lung tumor development. Lung tumors that developed in the absence of Akt2 were less likely to appear as discrete nodules and more frequently displayed a dispersed growth pattern. RNA sequencing revealed a number of genes differentially expressed in lung tumors lacking Akt2 and five of these genes, Actc1, Bpifa1, Mmp2, Ntrk2, and Scgb3a2 have been implicated in human lung cancer. Using 2 human lung cancer cell lines, we observed that a selective AKT1 inhibitor, A-674563, was a more potent regulator of cell survival than the pan-AKT inhibitor, MK-2206. This study suggests that compounds selectively targeting AKT1 may prove more effective than compounds that inhibit all three AKT isoforms at least in the treatment of lung adenocarcinoma. PMID:26654940

  6. The Akt switch model: Is location sufficient?

    PubMed

    Gray, Catheryn W; Coster, Adelle C F

    2016-06-01

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

  7. Developing a de novo targeted knock-in method based on in utero electroporation into the mammalian brain.

    PubMed

    Tsunekawa, Yuji; Terhune, Raymond Kunikane; Fujita, Ikumi; Shitamukai, Atsunori; Suetsugu, Taeko; Matsuzaki, Fumio

    2016-09-01

    Genome-editing technology has revolutionized the field of biology. Here, we report a novel de novo gene-targeting method mediated by in utero electroporation into the developing mammalian brain. Electroporation of donor DNA with the CRISPR/Cas9 system vectors successfully leads to knock-in of the donor sequence, such as EGFP, to the target site via the homology-directed repair mechanism. We developed a targeting vector system optimized to prevent anomalous leaky expression of the donor gene from the plasmid, which otherwise often occurs depending on the donor sequence. The knock-in efficiency of the electroporated progenitors reached up to 40% in the early stage and 20% in the late stage of the developing mouse brain. Furthermore, we inserted different fluorescent markers into the target gene in each homologous chromosome, successfully distinguishing homozygous knock-in cells by color. We also applied this de novo gene targeting to the ferret model for the study of complex mammalian brains. Our results demonstrate that this technique is widely applicable for monitoring gene expression, visualizing protein localization, lineage analysis and gene knockout, all at the single-cell level, in developmental tissues. PMID:27578183

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

    PubMed Central

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

    2016-01-01

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

  9. Protein-targeting strategy used to develop a selective inhibitor of the E17K point mutation in the PH Domain of Akt1

    PubMed Central

    Deyle, Kaycie M.; Farrow, Blake; Hee, Ying Qiao; Work, Jeremy; Wong, Michelle; Lai, Bert; Umeda, Aiko; Millward, Steven W.; Nag, Arundhati; Das, Samir; Heath, James R.

    2015-01-01

    Ligands that can selectively bind to proteins with single amino acid point mutations offer the potential to detect or treat an abnormal protein in the presence of the wildtype. However, it is difficult to develop a selective ligand if the point mutation is not associated with an addressable location, such as a binding pocket. Here we report an all-chemical, synthetic epitope-targeting strategy which we used to discover a 5-mer peptide with selectivity for the E17K transforming point mutation in the Pleckstrin Homology Domain of the Akt1 oncoprotein. A fragment of Akt1 containing the E17K mutation and a I19[Propargylglycine] substitution was synthesized to form an addressable synthetic epitope. Azide-presenting peptides that covalently clicked onto this alkyne-presenting epitope were selected from a library using in situ screening. One peptide exhibits a 10:1 in vitro selectivity for the oncoprotein relative to wildtype, with a similar selectivity in cells. This 5-mer peptide was expanded into a larger ligand that selectively blocks the E17K Akt1 interaction with its PIP3 substrate. PMID:25901825

  10. A protein-targeting strategy used to develop a selective inhibitor of the E17K point mutation in the PH domain of Akt1

    NASA Astrophysics Data System (ADS)

    Deyle, Kaycie M.; Farrow, Blake; Qiao Hee, Ying; Work, Jeremy; Wong, Michelle; Lai, Bert; Umeda, Aiko; Millward, Steven W.; Nag, Arundhati; Das, Samir; Heath, James R.

    2015-05-01

    Ligands that can bind selectively to proteins with single amino-acid point mutations offer the potential to detect or treat an abnormal protein in the presence of the wild type (WT). However, it is difficult to develop a selective ligand if the point mutation is not associated with an addressable location, such as a binding pocket. Here we report an all-chemical synthetic epitope-targeting strategy that we used to discover a 5-mer peptide with selectivity for the E17K-transforming point mutation in the pleckstrin homology domain of the Akt1 oncoprotein. A fragment of Akt1 that contained the E17K mutation and an I19[propargylglycine] substitution was synthesized to form an addressable synthetic epitope. Azide-presenting peptides that clicked covalently onto this alkyne-presenting epitope were selected from a library using in situ screening. One peptide exhibits a 10:1 in vitro selectivity for the oncoprotein relative to the WT, with a similar selectivity in cells. This 5-mer peptide was expanded into a larger ligand that selectively blocks the E17K Akt1 interaction with its PIP3 (phosphatidylinositol (3,4,5)-trisphosphate) substrate.

  11. Identification of the tuberous sclerosis complex-2 tumor suppressor gene product tuberin as a target of the phosphoinositide 3-kinase/akt pathway.

    PubMed

    Manning, Brendan D; Tee, Andrew R; Logsdon, M Nicole; Blenis, John; Cantley, Lewis C

    2002-07-01

    The S/T-protein kinases activated by phosphoinositide 3-kinase (PI3K) regulate a myriad of cellular processes. Here, we show that an approach using a combination of biochemistry and bioinformatics can identify substrates of these kinases. This approach identifies the tuberous sclerosis complex-2 gene product, tuberin, as a potential target of Akt/PKB. We demonstrate that, upon activation of PI3K, tuberin is phosphorylated on consensus recognition sites for PI3K-dependent S/T kinases. Moreover, Akt/PKB can phosphorylate tuberin in vitro and in vivo. We also show that S939 and T1462 of tuberin are PI3K-regulated phosphorylation sites and that T1462 is constitutively phosphorylated in PTEN(-/-) tumor-derived cell lines. Finally, we find that a tuberin mutant lacking the major PI3K-dependent phosphorylation sites can block the activation of S6K1, suggesting a means by which the PI3K-Akt pathway regulates S6K1 activity. PMID:12150915

  12. Angiographic and volumetric effects of mammalian target of rapamycin inhibitors on angiomyolipomas in tuberous sclerosis

    PubMed Central

    Sheth, Rahul A; Feldman, Adam S; Paul, Elahna; Thiele, Elizabeth A; Walker, T Gregory

    2016-01-01

    AIM: To investigate the angiographic and volumetric effects of mammalian target of rapamycin (mTOR) inhibitors on angiomyolipomas (AMLs) in a case series of patients with tuberous sclerosis complex. METHODS: All patients who underwent catheter angiography prior to and following mTOR inhibitor therapy (n = 3) were evaluated. All cross-sectional imaging studies were analyzed with three-dimensional volumetrics, and tumor volume curves for all three tissue compartments (soft tissue, vascular, and fat) were generated. Segmentation analysis tools were used to automatically create a region of interest (ROI) circumscribing the AML. On magnetic resonance images, the “fat only” map calculated from the in- and opposed-phase gradient recalled echo sequences was used to quantify fat volume within tumors. Tumor vascularity was measured by applying a thresholding tool within the ROI on post-contrast subtraction images. On computed tomography images, volume histogram analysis of Hounsfield unit was performed to quantify tumor tissue composition. The angiography procedures were also reviewed, and tumor vascularity based on pre-embolization angiography was characterized in a semi-quantitative manner. RESULTS: Patient 1 presented at the age of 15 with a 6.8 cm right lower pole AML and a 4.0 cm right upper pole AML. Embolization was performed of both tumors, and after a few years of size control, the tumors began to grow, and the patient was initiated on mTOR inhibitor therapy. There was an immediate reduction in the size of both lesions. The patient then underwent repeat embolization and discontinuation of mTOR inhibition, after which point there was a substantial regrowth in both tumors across all tissue compartments. Patient 2 presented at the age of 18 with a right renal AML. Following a brief period of tumor reduction after embolization, she was initiated on mTOR inhibitor therapy, with successful reduction in tumor size across all tissue compartments. As with patient 1

  13. Spontaneous Hepatocellular Carcinoma after the Combined Deletion of Akt Isoforms.

    PubMed

    Wang, Qi; Yu, Wan-Ni; Chen, Xinyu; Peng, Xiao-Ding; Jeon, Sang-Min; Birnbaum, Morris J; Guzman, Grace; Hay, Nissim

    2016-04-11

    Akt is frequently hyperactivated in human cancers and is targeted for cancer therapy. However, the physiological consequences of systemic Akt isoform inhibition were not fully explored. We showed that while combined Akt1 and Akt3 deletion in adult mice is tolerated, combined Akt1 and Akt2 deletion induced rapid mortality. Akt2(-/-) mice survived hepatic Akt1 deletion but all developed spontaneous hepatocellular carcinoma (HCC), which is associated with FoxO-dependent liver injury and inflammation. The gene expression signature of HCC-bearing livers is similar to aggressive human HCC. Consistently, neither Akt1(-/-) nor Akt2(-/-) mice are resistant to diethylnitrosamine-induced hepatocarcinogenesis, and Akt2(-/-) mice display a high incidence of lung metastasis. Thus, in contrast to other cancers, hepatic Akt inhibition induces liver injury that could promote HCC. PMID:26996309

  14. Long-term effects of rapamycin treatment on insulin mediated phosphorylation of Akt/PKB and glycogen synthase activity

    SciTech Connect

    Varma, Shailly; Shrivastav, Anuraag; Changela, Sheena; Khandelwal, Ramji L.

    2008-04-01

    Protein kinase B (Akt/PKB) is a Ser/Thr kinase that is involved in the regulation of cell proliferation/survival through mammalian target of rapamycin (mTOR) and the regulation of glycogen metabolism through glycogen synthase kinase 3{beta} (GSK-3{beta}) and glycogen synthase (GS). Rapamycin is an inhibitor of mTOR. The objective of this study was to investigate the effects of rapamycin pretreatment on the insulin mediated phosphorylation of Akt/PKB phosphorylation and GS activity in parental HepG2 and HepG2 cells with overexpression of constitutively active Akt1/PKB-{alpha} (HepG2-CA-Akt/PKB). Rapamycin pretreatment resulted in a decrease (20-30%) in the insulin mediated phosphorylation of Akt1 (Ser 473) in parental HepG2 cells but showed an upregulation of phosphorylation in HepG2-CA-Akt/PKB cells. Rictor levels were decreased (20-50%) in parental HepG2 cells but were not significantly altered in the HepG2-CA-Akt/PKB cells. Furthermore, rictor knockdown decreased the phosphorylation of Akt (Ser 473) by 40-60% upon rapamycin pretreatment. GS activity followed similar trends as that of phosphorylated Akt and so with rictor levels in these cells pretreated with rapamycin; parental HepG2 cells showed a decrease in GS activity, whereas as HepG2-CA-Akt/PKB cells showed an increase in GS activity. The changes in the levels of phosphorylated Akt/PKB (Ser 473) correlated with GS and protein phoshatase-1 activity.

  15. Sestrin2 integrates Akt and mTOR signaling to protect cells against energetic stress-induced death.

    PubMed

    Ben-Sahra, I; Dirat, B; Laurent, K; Puissant, A; Auberger, P; Budanov, A; Tanti, J-F; Bost, F

    2013-04-01

    The phosphoinositide-3 kinase/Akt (PI3K/Akt) pathway has a central role in cancer cell metabolism and proliferation. More importantly, it is one of the cardinal pro-survival pathways mediating resistance to apoptosis. The role of Akt in response to an energetic stress is presently unclear. Here, we show that Sestrin2 (Sesn2), also known as Hi95, a p53 target gene that protects cells against oxidative and genotoxic stresses, participates in the protective role of Akt in response to an energetic stress induced by 2-deoxyglucose (2-DG). Sesn2 is upregulated in response to an energetic stress such as 2-DG and metformin, and mediates the inhibition of mammalian target of rapamycin (mTOR), the major cellular regulator of energy metabolism. The increase of Sesn2 is independent of p53 but requires the anti-apoptotic pathway, PI3K/Akt. Inhibition of Akt, as well as loss of Sesn2, sensitizes cells to 2-DG-induced apoptosis. In addition, the rescue of Sesn2 partially reverses the pro-apoptotic effects of 2-DG. In conclusion, we identify Sesn2 as a new energetic stress sensor, which appears to be protective against energetic stress-induced apoptosis that integrates the pro-survival function of Akt and the negative regulation of mTOR. PMID:23238567

  16. Constitutively active Akt1 expression in mouse pancreas requires S6 kinase 1 for insulinoma formation

    PubMed Central

    Alliouachene, Samira; Tuttle, Robyn L.; Boumard, Stephanie; Lapointe, Thomas; Berissi, Sophie; Germain, Stephane; Jaubert, Francis; Tosh, David; Birnbaum, Morris J.; Pende, Mario

    2008-01-01

    Factors that promote pancreatic β cell growth and function are potential therapeutic targets for diabetes mellitus. In mice, genetic experiments suggest that signaling cascades initiated by insulin and IGFs positively regulate β cell mass and insulin secretion. Akt and S6 kinase (S6K) family members are activated as part of these signaling cascades, but how the interplay between these proteins controls β cell growth and function has not been determined. Here, we found that although transgenic mice overexpressing the constitutively active form of Akt1 under the rat insulin promoter (RIP-MyrAkt1 mice) had enlarged β cells and high plasma insulin levels, leading to improved glucose tolerance, a substantial proportion of the mice developed insulinomas later in life, which caused decreased viability. This oncogenic transformation tightly correlated with nuclear exclusion of the tumor suppressor PTEN. To address the role of the mammalian target of rapamycin (mTOR) substrate S6K1 in the MyrAkt1-mediated phenotype, we crossed RIP-MyrAkt1 and S6K1-deficient mice. The resulting mice displayed reduced insulinemia and glycemia compared with RIP-MyrAkt1 mice due to a combined effect of improved insulin secretion and insulin sensitivity. Importantly, although the increase in β cell size in RIP-MyrAkt1 mice was not affected by S6K1 deficiency, the hyperplastic transformation required S6K1. Our results therefore identify S6K1 as a critical element for MyrAkt1-induced tumor formation and suggest that it may represent a useful target for anticancer therapy downstream of mTOR. PMID:18846252

  17. Akt2 and Akt3 play a pivotal role in malignant gliomas

    PubMed Central

    Mure, Hideo; Matsuzaki, Kazuhito; Kitazato, Keiko T.; Mizobuchi, Yoshifumi; Kuwayama, Kazuyuki; Kageji, Teruyoshi; Nagahiro, Shinji

    2010-01-01

    Akt, one of the major downstream effectors of phosphatidylinositol 3-kinase, is hyper-expressed and activated in a variety of cancers including glioblastoma. However, the expression profiles of the Akt isoforms Akt1/PKBα, Akt2/PKBβ, and Akt3/PKBγ and their functional roles in malignant glioma are not well understood. Therefore, we examined the protein and mRNA expression patterns of Akt isoforms in tissues from human astrocytomas, glioblastomas, and non-neoplastic regions. We also explored the biological role of each Akt isoform in malignant glioma cells using RNA interference-mediated knock-down and the over-expression of plasmid DNA of each isoform. The expression of Akt1 protein and mRNA was similar in glioma and normal control tissues. Although the protein and mRNA level of Akt2 increased with the pathological grade of malignancy, the expression of Akt3 mRNA and protein decreased as the malignancy grade increased. In U87MG, T98G, and TGB cells, the down-regulation of Akt2 or Akt3 by RNA interference reduced the expression of the phosphorylated form of Bad, resulting in the induction of caspase-dependent apoptosis. Akt1 knock-down did not affect cell growth or survival. We first demonstrate that the over-expression of Akt2 or Akt3 down-regulated the expression of the other protein and that endogenous Akt3 protein showed high kinase activity in U87MG cells. Our data suggest that Akt2 and Akt3 play an important role in the viability of human malignant glioma cells. Targeting Akt2 and Akt3 may hold promise for the treatment of patients with gliomas. PMID:20167810

  18. ROS-mediated EB1 phosphorylation through Akt/GSK3β pathway: implication in cancer cell response to microtubule-targeting agents

    PubMed Central

    Grand, Marion Le; Rovini, Amandine; Bourgarel-Rey, Veronique; Honore, Stephane; Bastonero, Sonia; Braguer, Diane; Carre, Manon

    2014-01-01

    Microtubule-targeting agents (MTAs) are largely administered in adults and children cancers. Better deciphering their mechanism of action is of prime importance to develop more convenient therapy strategies. Here, we addressed the question of how reactive oxygen species (ROS) generation by mitochondria can be necessary for MTA efficacy. We showed for the first time that EB1 associates with microtubules in a phosphorylation-dependent manner, under control of ROS. By using phospho-defective mutants, we further characterized the Serine 155 residue as critical for EB1 accumulation at microtubule plus-ends, and both cancer cell migration and proliferation. Phosphorylation of EB1 on the Threonine 166 residue triggered opposite effects, and was identified as a requisite molecular switch in MTA activities. We then showed that GSK3β activation was responsible for MTA-triggered EB1 phosphorylation, resulting from ROS-mediated inhibition of upstream Akt. We thus disclosed here a novel pathway by which generation of mitochondrial ROS modulates microtubule dynamics through phosphorylation of EB1, improving our fundamental knowledge about this oncogenic protein, and pointing out the need to re-examine the current dogma of microtubule targeting by MTAs. The present work also provides a strong mechanistic rational to the promising therapeutic strategies that currently combine MTAs with anti-Akt targeted therapies. PMID:24930764

  19. Rap1 promotes multiple pancreatic islet cell functions and signals through mammalian target of rapamycin complex 1 to enhance proliferation.

    PubMed

    Kelly, Patrick; Bailey, Candice L; Fueger, Patrick T; Newgard, Christopher B; Casey, Patrick J; Kimple, Michelle E

    2010-05-21

    Recent studies have implicated Epac2, a guanine-nucleotide exchange factor for the Rap subfamily of monomeric G proteins, as an important regulator of insulin secretion from pancreatic beta-cells. Although the Epac proteins were originally identified as cAMP-responsive activators of Rap1 GTPases, the role of Rap1 in beta-cell biology has not yet been defined. In this study, we examined the direct effects of Rap1 signaling on beta-cell biology. Using the Ins-1 rat insulinoma line, we demonstrate that activated Rap1A, but not related monomeric G proteins, promotes ribosomal protein S6 phosphorylation. Using isolated rat islets, we show that this signaling event is rapamycin-sensitive, indicating that it is mediated by the mammalian target of rapamycin complex 1-p70 S6 kinase pathway, a known growth regulatory pathway. This newly defined beta-cell signaling pathway acts downstream of cAMP, in parallel with the stimulation of cAMP-dependent protein kinase, to drive ribosomal protein S6 phosphorylation. Activated Rap1A promotes glucose-stimulated insulin secretion, islet cell hypertrophy, and islet cell proliferation, the latter exclusively through mammalian target of rapamycin complex 1, suggesting that Rap1 is an important regulator of beta-cell function. This newly defined signaling pathway may yield unique targets for the treatment of beta-cell dysfunction in diabetes. PMID:20339002

  20. C-H activation generates period-shortening molecules that target cryptochrome in the mammalian circadian clock.

    PubMed

    Oshima, Tsuyoshi; Yamanaka, Iori; Kumar, Anupriya; Yamaguchi, Junichiro; Nishiwaki-Ohkawa, Taeko; Muto, Kei; Kawamura, Rika; Hirota, Tsuyoshi; Yagita, Kazuhiro; Irle, Stephan; Kay, Steve A; Yoshimura, Takashi; Itami, Kenichiro

    2015-06-01

    The synthesis and functional analysis of KL001 derivatives, which are modulators of the mammalian circadian clock, are described. By using cutting-edge C-H activation chemistry, a focused library of KL001 derivatives was rapidly constructed, which enabled the identification of the critical sites on KL001 derivatives that induce a rhythm-changing activity along with the components that trigger opposite modes of action. The first period-shortening molecules that target the cryptochrome (CRY) were thus discovered. Detailed studies on the effects of these compounds on CRY stability implicate the existence of an as yet undiscovered regulatory mechanism. PMID:25960183

  1. Akt activation enhances ribosomal RNA synthesis through casein kinase II and TIF-IA

    PubMed Central

    Nguyen, Le Xuan Truong; Mitchell, Beverly S.

    2013-01-01

    Transcription initiation factor I (TIF-IA) plays an essential role in regulating ribosomal RNA (rRNA) synthesis by tethering RNA polymerase I (Pol I) to the rDNA promoter. We have found that activated Akt enhances rRNA synthesis through the phosphorylation of casein kinase IIα (CK2α) on a threonine residue near its N terminus. CK2 in turn phosphorylates TIF-IA, thereby increasing rDNA transcription. Activated Akt also stabilizes TIF-IA, induces its translocation to the nucleolus, and enhances its interaction with Pol I. Treatment with AZD8055, an inhibitor of both Akt and mammalian target of rapamycin phosphorylation, but not with rapamycin, disrupts Akt-mediated TIF-IA stability, translocation, and activity. These data support a model in which activated Akt enhances rRNA synthesis both by preventing TIF-IA degradation and phosphorylating CK2α, which in turn phosphorylates TIF-IA. This model provides an explanation for the ability of activated Akt to promote cell proliferation and, potentially, transformation. PMID:24297901

  2. MiR-148a Functions as a Tumor Suppressor by Targeting CCK-BR via Inactivating STAT3 and Akt in Human Gastric Cancer

    PubMed Central

    Su, Liping; Li, Jianfang; Yu, Yingyan; Gu, Qinlong; Yan, Min; Zhu, Zhenggang; Liu, Bingya

    2016-01-01

    MicroRNAs (miRNAs) have been widely accepted as a class of gene expression regulators which post-translationally regulate protein expression. These small noncoding RNAs have been proved closely involved in the modulation of various pathobiological processes in cancer. In this research, we demonstrated that miR-148a expression was significantly down-regulated in gastric cancer tissues in comparison with the matched normal mucosal tissues, and its expression was statistically associated with lymph node metastasis. Ectopic expression of miR-148a inhibited tumor cell proliferation and migration in vitro, and inhibited tumor formation in vivo. Subsequently, we identified cholecystokinin B receptor (CCK-BR) as a direct target of miR-148a using western blot and luciferase activity assay. More importantly, siRNA-induced knockdown of CCK-BR elicited similar anti-oncogenic effects (decreased proliferation and migration) as those induced by enforced miR-148a expression. We also found that miR-148a-mediated anti-cancer effects are dependent on the inhibition of STAT3 and Akt activation, which subsequently regulates the pathways involved in cell proliferation and migration. Taken together, our results suggest that miR-148a serves as a tumor suppressor in human gastric carcinogenesis by targeting CCK-BR via inactivating STAT3 and Akt. PMID:27518872

  3. Immunohistochemical analysis of the Akt/mTOR/4E-BP1 signalling pathway in canine haemangiomas and haemangiosarcomas.

    PubMed

    Murai, A; Abou Asa, S; Kodama, A; Sakai, H; Hirata, A; Yanai, T

    2012-11-01

    The specific signalling pathways that are deregulated in canine endothelial tumours have not yet fully elucidated. Therefore, the aim of the present study was to examine activation of the Akt/mammalian target of rapamycin (mTOR)/eukaryotic initiation factor 4E-binding protein 1 (4E-BP1) signalling pathway in spontaneously arising canine haemangiomas (HAs) and haemangiosarcomas (HSAs) in order to identify novel molecular targets for treatment. Surgically-resected samples of HA (n = 27), HSA (n = 37), granulation tissue (n = 4) and normal skin (n = 4) were investigated by immunohistochemistry. Approximately 80% of the HSA samples had moderate to intense expression of phosphorylated Akt at Ser473 (p-Akt Ser473), p-Akt Thr308, p-4E-BP1 Thr37/46 and eukaryotic initiation factor 4E, which was significantly higher than in the HAs and was similar to the expression in activated endothelial cells (ECs). Although p-mTOR complex1 (p-mTORC1) Ser2448 was expressed by most of the activated ECs, only 35% of the HSA samples had weak to moderate expression. Because mTORC2 and phosphorylates Akt Ser473 was activated in HSA samples, the present findings suggest that the mTORC2/Akt/4E-BP1 pathway, regulated independently of mTORC1, may be important for targeting therapy in canine HSAs. PMID:22789858

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

    SciTech Connect

    Ma, Yanfei; Qin, Huadong; Cui, Yunfu

    2013-11-29

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

  5. CREB is a regulatory target for the protein kinase Akt/PKB in the differentiation of pancreatic ductal cells into islet {beta}-cells mediated by hepatocyte growth factor

    SciTech Connect

    Li, Xin-Yu; Zhan, Xiao-Rong; Liu, Xiao-Min; Wang, Xiao-Chen

    2011-01-14

    Research highlights: {yields} CREB is a regulatory target for the protein kinase Akt/PKB in pancreatic duct cells. {yields} Activation of the PI3K/AKT/CREB pathway plays a critical role in the HGF-mediated differentiation of pancreatic duct cells in vivo. {yields} CREB was causally linked to the expression of transcription factors during PDEC differentiation induced by HGF. -- Abstract: We have previously reported that the PI3K/Akt signaling pathway is involved in hepatocyte growth factor (HGF)-induced differentiation of adult rat pancreatic ductal epithelial cells (PDECs) into islet {beta}-cells in vitro. The transcription factor CREB is one of the downstream key effectors of the PI3K/Akt signaling pathway. Recent studies showing that CREB is required for the survival of certain cell types prompted us to examine whether CREB is a nuclear target for activation via the HGF-dependent Ser/Thr kinase Akt/PKB in the differentiation of pancreatic duct cell into islet {beta}-cells. In this study, we first attempted to examine whether HGF modulates the Akt-dependent activation of target gene CREB and then investigated whether CREB activity affects the differentiation of HGF-induced PDECs. Finally, we studied the role of CREB in modulating the expression of transcription factors in PDECs during the differentiation of HGF-induced PDECs. Our results demonstrated that CREB is a regulatory target for the protein kinase Akt/PKB in the differentiation of pancreatic ductal cells into islet {beta}-cells mediated by HGF.

  6. Probing the target search of DNA-binding proteins in mammalian cells using TetR as model searcher

    NASA Astrophysics Data System (ADS)

    Normanno, Davide; Boudarène, Lydia; Dugast-Darzacq, Claire; Chen, Jiji; Richter, Christian; Proux, Florence; Bénichou, Olivier; Voituriez, Raphaël; Darzacq, Xavier; Dahan, Maxime

    2015-07-01

    Many cellular functions rely on DNA-binding proteins finding and associating to specific sites in the genome. Yet the mechanisms underlying the target search remain poorly understood, especially in the case of the highly organized mammalian cell nucleus. Using as a model Tet repressors (TetRs) searching for a multi-array locus, we quantitatively analyse the search process in human cells with single-molecule tracking and single-cell protein-DNA association measurements. We find that TetRs explore the nucleus and reach their target by 3D diffusion interspersed with transient interactions with non-cognate sites, consistent with the facilitated diffusion model. Remarkably, nonspecific binding times are broadly distributed, underlining a lack of clear delimitation between specific and nonspecific interactions. However, the search kinetics is not determined by diffusive transport but by the low association rate to nonspecific sites. Altogether, our results provide a comprehensive view of the recruitment dynamics of proteins at specific loci in mammalian cells.

  7. Probing the target search of DNA-binding proteins in mammalian cells using TetR as model searcher.

    PubMed

    Normanno, Davide; Boudarène, Lydia; Dugast-Darzacq, Claire; Chen, Jiji; Richter, Christian; Proux, Florence; Bénichou, Olivier; Voituriez, Raphaël; Darzacq, Xavier; Dahan, Maxime

    2015-01-01

    Many cellular functions rely on DNA-binding proteins finding and associating to specific sites in the genome. Yet the mechanisms underlying the target search remain poorly understood, especially in the case of the highly organized mammalian cell nucleus. Using as a model Tet repressors (TetRs) searching for a multi-array locus, we quantitatively analyse the search process in human cells with single-molecule tracking and single-cell protein-DNA association measurements. We find that TetRs explore the nucleus and reach their target by 3D diffusion interspersed with transient interactions with non-cognate sites, consistent with the facilitated diffusion model. Remarkably, nonspecific binding times are broadly distributed, underlining a lack of clear delimitation between specific and nonspecific interactions. However, the search kinetics is not determined by diffusive transport but by the low association rate to nonspecific sites. Altogether, our results provide a comprehensive view of the recruitment dynamics of proteins at specific loci in mammalian cells. PMID:26151127

  8. Probing the target search of DNA-binding proteins in mammalian cells using TetR as model searcher

    PubMed Central

    Normanno, Davide; Boudarène, Lydia; Dugast-Darzacq, Claire; Chen, Jiji; Richter, Christian; Proux, Florence; Bénichou, Olivier; Voituriez, Raphaël; Darzacq, Xavier; Dahan, Maxime

    2015-01-01

    Many cellular functions rely on DNA-binding proteins finding and associating to specific sites in the genome. Yet the mechanisms underlying the target search remain poorly understood, especially in the case of the highly organized mammalian cell nucleus. Using as a model Tet repressors (TetRs) searching for a multi-array locus, we quantitatively analyse the search process in human cells with single-molecule tracking and single-cell protein–DNA association measurements. We find that TetRs explore the nucleus and reach their target by 3D diffusion interspersed with transient interactions with non-cognate sites, consistent with the facilitated diffusion model. Remarkably, nonspecific binding times are broadly distributed, underlining a lack of clear delimitation between specific and nonspecific interactions. However, the search kinetics is not determined by diffusive transport but by the low association rate to nonspecific sites. Altogether, our results provide a comprehensive view of the recruitment dynamics of proteins at specific loci in mammalian cells. PMID:26151127

  9. Involvement of PI3K/Akt Signaling Pathway and Its Downstream Intracellular Targets in the Antidepressant-Like Effect of Creatine.

    PubMed

    Cunha, Mauricio P; Budni, Josiane; Ludka, Fabiana K; Pazini, Francis L; Rosa, Julia Macedo; Oliveira, Ágatha; Lopes, Mark W; Tasca, Carla I; Leal, Rodrigo B; Rodrigues, Ana Lúcia S

    2016-07-01

    Creatine has been proposed to exert beneficial effects in the management of depression, but the cell signaling pathways implicated in its antidepressant effects are not well established. This study investigated the involvement of PI3K/Akt signaling pathway and its downstream intracellular targets in the antidepressant-like effect of creatine. The acute treatment of mice with creatine (1 mg/kg, po) increased the Akt and P70S6K phosphorylation, and HO-1, GPx and PSD95 immunocontents. The pretreatment of mice with LY294002 (10 nmol/mouse, icv, PI3K inhibitor), wortmannin (0.1 μg/mouse, icv, PI3K inhibitor), ZnPP (10 μg/mouse, icv, HO-1 inhibitor), or rapamycin (0.2 nmol/mouse, icv, mTOR inhibitor) prevented the antidepressant-like effect of creatine (1 mg/kg, po) in the TST. In addition, the administration of subeffective dose of either the selective GSK3 inhibitor AR-A014418 (0.01 μg/mouse, icv), the nonselective GSK3 inhibitor lithium chloride (10 mg/kg, po), or the HO-1 inductor CoPP (0.01 μg/mouse, icv), in combination with a subeffective dose of creatine (0.01 mg/kg, po) reduced the immobility time in the TST as compared with either drug alone. No treatment caused significant changes in the locomotor activity of mice. These results indicate that the antidepressant-like effect of creatine in the TST depends on the activation of Akt, Nrf2/HO-1, GPx, and mTOR, and GSK3 inhibition. PMID:25943184

  10. microRNA-29b prevents liver fibrosis by attenuating hepatic stellate cell activation and inducing apoptosis through targeting PI3K/AKT pathway

    PubMed Central

    Wang, Jia; Chu, Eagle S.H.; Chen, Hai-Yong; Man, Kwan; Go, Minnie Y.Y.; Huang, Xiao Ru; Lan, Hui Yao; Sung, Joseph J.Y.; Yu, Jun

    2015-01-01

    microRNA-29b (miR-29b) is known to be associated with TGF-β-mediated fibrosis, but the mechanistic action of miR-29b in liver fibrosis remains unclear and is warranted for investigation. We found that miR-29b was significantly downregulated in human and mice fibrotic liver tissues and in primary activated HSCs. miR-29b downregulation was directly mediated by Smad3 through binding to the promoter of miR-29b in hepatic stellate cell (HSC) line LX1, whilst miR-29b could in turn suppress Smad3 expression. miR-29b transduction in the liver of mice prevented CCl4 induced-fibrogenesis, concomitant with decreased expression of α-SMA, collagen I and TIMP-1. Ectopic expression of miR-29b in activated HSCs (LX-1, HSC-T6) inhibited cell viability and colony formation, and caused cell cycle arrest in G1 phase by downregulating cyclin D1 and p21cip1. Further, miR-29b induced apoptosis in HSCs mediated by caspase-9 and PARP. miR-29b inhibited its downstream effectors of PIK3R1 and AKT3 through direct targeting their 3′UTR regions. Moreover, knockdown of PIK3R1 or AKT3 suppressed α-SMA and collagen I and induced apoptosis in both HSCs and in mice. In conclusion, miR-29b prevents liver fibrogenesis by inhibiting HSC activation and inducing HSC apoptosis through inhibiting PI3K/AKT pathway. These results provide novel mechanistic insights for the anti-fibrotic effect of miR-29b. PMID:25356754

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

    PubMed

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

    2014-10-01

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

  12. Effective Targeted Gene Knockdown in Mammalian Cells Using the piggyBac Transposase-based Delivery System

    PubMed Central

    Owens, Jesse B; Mathews, Juanita; Davy, Philip; Stoytchev, Ilko; Moisyadi, Stefan; Allsopp, Rich

    2013-01-01

    Nonviral gene delivery systems are rapidly becoming a desirable and applicable method to overexpress genes in various types of cells. We have recently developed a piggyBac transposase-based, helper-independent and self-inactivating delivery system (pmGENIE-3) capable of high-efficiency transfection of mammalian cells including human cells. In the following study, we have assessed the potential of this delivery system to drive the expression of short hairpin RNAs to knock down genes in human cells. Two independent pmGENIE-3 vectors were developed to specifically target knockdown of an endogenous gene, telomerase reverse transcriptase (TERT), in telomerase-positive human immortalized cell lines. As compared with a transposase-deficient vector, pmGENIE-3 showed significantly improved short-term transfection efficiency (~4-fold enhancement, 48 hours posttransfection) and long-term integration efficiency (~5-fold enhancement) following antibiotic selection. We detected a significant reduction of both TERT expression and telomerase activity in both HEK293 and MCF-7 breast carcinoma cells transfected with two pmGENIE-3 construct targeting distinct regions of TERT. Importantly, this knockdown of expression was sufficient to abrogate telomerase function since telomeres were significantly shortened (3–4 Kb, P < 0.001) in both TERT-targeted cell lines following antibiotic selection of stable integrants. Together, these data show the capacity of the piggyBac nonviral delivery system to stably knockdown gene expression in mammalian cells and indicate the potential to develop novel tumor-targeting therapies. PMID:24326734

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

    PubMed

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

    2016-08-28

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

  14. Decreased Phosphorylated Protein Kinase B (Akt) in Individuals with Autism Associated with High Epidermal Growth Factor Receptor (EGFR) and Low Gamma-Aminobutyric Acid (GABA)

    PubMed Central

    Russo, Anthony J

    2015-01-01

    Dysregulation of the PI3K/AKT/mammalian target of rapamycin (mTOR) pathway could contribute to the pathogenesis of autism spectrum disorders. In this study, phosphorylated Akt concentration was measured in 37 autistic children and 12, gender and age similar neurotypical, controls using an enzyme-linked immunosorbent assay. Akt levels were compared to biomarkers known to be associated with epidermal growth factor receptor (EGFR) and c-Met (hepatocyte growth factor (HGF) receptor) pathways and severity levels of 19 autism-related symptoms. We found phosphorylated Akt levels significantly lower in autistic children and low Akt levels correlated with high EGFR and HGF and low gamma-aminobutyric acid, but not other biomarkers. Low Akt levels also correlated significantly with increased severity of receptive language, conversational language, hypotonia, rocking and pacing, and stimming, These results suggest a relationship between decreased phosphorylated Akt and selected symptom severity in autistic children and support the suggestion that the AKT pathways may be associated with the etiology of autism. PMID:26508828

  15. Hirsutenone in Alnus extract inhibits akt activity and suppresses prostate cancer cell proliferation.

    PubMed

    Kang, Soouk; Kim, Jong-Eun; Li, Yan; Jung, Sung Keun; Song, Nu Ry; Thimmegowda, N R; Kim, Bo Yeon; Lee, Hyong Joo; Bode, Ann M; Dong, Zigang; Lee, Ki Won

    2015-11-01

    Although specific compounds found in some East Asian traditional medicines have been shown to exhibit bioactive properties, their molecular mechanisms of action remain elusive. The bark of the Alnus species has been used for the treatment of various pathological conditions including hemorrhage, alcoholism, fever, diarrhea, skin diseases, inflammation, and cancer in East Asia for centuries. In this study, we show that hirsutenone, a bioactive compound in Alnus japonica, exhibits anti-cancer effects against prostate cancer through a direct physical inhibition of Akt1/2. Hirsutenone suppressed anchorage-dependent and independent cell growth of PC3 and LNCaP human prostate cancer cells. Annexin V and Propidium iodide (PI) staining results demonstrated that hirsutenone strongly induces apoptotic cell death in both PC3 and LNCaP cells. Furthermore, treatment of hirsutenone attenuated phosphorylation of mammalian target of rapamycin (mTOR), a downstream substrate of Akt, without affecting Akt phosphorylation. Kinase and pull-down assay results clearly show that hirsutenone inhibits Akt1 and 2 by direct binding in an adenosine triphosphate (ATP)-noncompetitive manner in vitro and ex vivo. Our results show that hirsutenone suppresses human prostate cancer by targeting Akt1 and 2 as a key component to explain for anti-cancer activity of Alnus species. PMID:25213146

  16. Reversal of the glycolytic phenotype of primary effusion lymphoma cells by combined targeting of cellular metabolism and PI3K/Akt/ mTOR signaling

    PubMed Central

    Bertacchini, Jessika; Frasson, Chiara; Bosco, Raffaella; Accordi, Benedetta; Basso, Giuseppe; Bonora, Massimo; Calabrò, Maria Luisa; Mattiolo, Adriana; Sgarbi, Gianluca; Baracca, Alessandra; Pinton, Paolo; Riva, Giovanni; Rampazzo, Enrico; Petrizza, Luca; Prodi, Luca; Milani, Daniela; Luppi, Mario; Potenza, Leonardo; De Pol, Anto; Cocco, Lucio; Capitani, Silvano; Marmiroli, Sandra

    2016-01-01

    PEL is a B-cell non-Hodgkin lymphoma, occurring predominantly as a lymphomatous effusion in body cavities, characterized by aggressive clinical course, with no standard therapy. Based on previous reports that PEL cells display a Warburg phenotype, we hypothesized that the highly hypoxic environment in which they grow in vivo makes them more reliant on glycolysis, and more vulnerable to drugs targeting this pathway. We established here that indeed PEL cells in hypoxia are more sensitive to glycolysis inhibition. Furthermore, since PI3K/Akt/mTOR has been proposed as a drug target in PEL, we ascertained that pathway-specific inhibitors, namely the dual PI3K and mTOR inhibitor, PF-04691502, and the Akt inhibitor, Akti 1/2, display improved cytotoxicity to PEL cells in hypoxic conditions. Unexpectedly, we found that these drugs reduce lactate production/extracellular acidification rate, and, in combination with the glycolysis inhibitor 2-deoxyglucose (2-DG), they shift PEL cells metabolism from aerobic glycolysis towards oxidative respiration. Moreover, the associations possess strong synergistic cytotoxicity towards PEL cells, and thus may reduce adverse reaction in vivo, while displaying very low toxicity to normal lymphocytes. Finally, we showed that the association of 2-DG and PF-04691502 maintains its cytotoxic and proapoptotic effect also in PEL cells co-cultured with human primary mesothelial cells, a condition known to mimic the in vivo environment and to exert a protective and pro-survival action. All together, these results provide a compelling rationale for the clinical development of new therapies for the treatment of PEL, based on combined targeting of glycolytic metabolism and constitutively activated signaling pathways. PMID:26575168

  17. Inhibition of Autophagy as a Strategy to Augment Radiosensitization by the Dual Phosphatidylinositol 3-Kinase/Mammalian Target of Rapamycin Inhibitor NVP-BEZ235S⃞

    PubMed Central

    Cerniglia, George J.; Karar, Jayashree; Tyagi, Sonia; Christofidou-Solomidou, Melpo; Rengan, Ramesh; Koumenis, Constantinos

    2012-01-01

    We investigated the effect of 2-methyl-2-{4-[3-methyl-2-oxo-8-(quinolin-3-yl)-2,3-dihydro-1H-imidazo[4,5-c]quinolin-1-yl]phenyl} propanenitrile (NVP-BEZ235) (Novartis, Basel Switzerland), a dual phosphatidylinositol 3-kinase (PI3K)/mammalian target of rapamycin (mTOR) inhibitor currently being tested in phase I clinical trials, in radiosensitization. NVP-BEZ235 radiosensitized a variety of cancer cell lines, including SQ20B head and neck carcinoma cells and U251 glioblastoma cells. NVP-BEZ235 also increased in vivo radiation response in SQ20B xenografts. Knockdown of Akt1, p110α, or mTOR resulted in radiosensitization, but not to the same degree as with NVP-BEZ235. NVP-BEZ235 interfered with DNA damage repair after radiation as measured by the CometAssay and resolution of phosphorylated H2A histone family member X foci. NVP-BEZ235 abrogated the radiation-induced phosphorylation of both DNA-dependent protein kinase catalytic subunit (DNA-PKcs) and ataxia telangiectasia mutated. Knockdown of either p110α or mTOR failed to decrease the phosphorylation of DNA-PKcs, suggesting that the effect of the drug was direct rather than mediated via p110α or mTOR. The treatment of cells with NVP-BEZ235 also promoted autophagy. To assess the importance of this process in radiosensitization, we used the autophagy inhibitors 3-methyladenine and chloroquine and found that either drug increased cell killing after NVP-BEZ235 treatment and radiation. Knocking down the essential autophagy proteins autophagy related 5 (ATG5) and beclin1 increased NVP-BEZ235-mediated radiosensitization. Furthermore, NVP-BEZ235 radiosensitized autophagy-deficient ATG5(−/−) fibroblasts to a greater extent than ATG5(+/+) cells. We conclude that NVP-BEZ235 radiosensitizes cells and induces autophagy by apparently distinct mechanisms. Inhibiting autophagy via pharmacologic or genetic means increases radiation killing after NVP-BEZ235 treatment; hence, autophagy seems to be cytoprotective in this

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

    PubMed

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

    2015-04-01

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

  19. Mammalian Target of Rapamycin: Its Role in Early Neural Development and in Adult and Aged Brain Function.

    PubMed

    Garza-Lombó, Carla; Gonsebatt, María E

    2016-01-01

    The kinase mammalian target of rapamycin (mTOR) integrates signals triggered by energy, stress, oxygen levels, and growth factors. It regulates ribosome biogenesis, mRNA translation, nutrient metabolism, and autophagy. mTOR participates in various functions of the brain, such as synaptic plasticity, adult neurogenesis, memory, and learning. mTOR is present during early neural development and participates in axon and dendrite development, neuron differentiation, and gliogenesis, among other processes. Furthermore, mTOR has been shown to modulate lifespan in multiple organisms. This protein is an important energy sensor that is present throughout our lifetime its role must be precisely described in order to develop therapeutic strategies and prevent diseases of the central nervous system. The aim of this review is to present our current understanding of the functions of mTOR in neural development, the adult brain and aging. PMID:27378854

  20. Mammalian Target of Rapamycin: Its Role in Early Neural Development and in Adult and Aged Brain Function

    PubMed Central

    Garza-Lombó, Carla; Gonsebatt, María E.

    2016-01-01

    The kinase mammalian target of rapamycin (mTOR) integrates signals triggered by energy, stress, oxygen levels, and growth factors. It regulates ribosome biogenesis, mRNA translation, nutrient metabolism, and autophagy. mTOR participates in various functions of the brain, such as synaptic plasticity, adult neurogenesis, memory, and learning. mTOR is present during early neural development and participates in axon and dendrite development, neuron differentiation, and gliogenesis, among other processes. Furthermore, mTOR has been shown to modulate lifespan in multiple organisms. This protein is an important energy sensor that is present throughout our lifetime its role must be precisely described in order to develop therapeutic strategies and prevent diseases of the central nervous system. The aim of this review is to present our current understanding of the functions of mTOR in neural development, the adult brain and aging. PMID:27378854

  1. Conophylline protects cells in cellular models of neurodegenerative diseases by inducing mammalian target of rapamycin (mTOR)-independent autophagy.

    PubMed

    Sasazawa, Yukiko; Sato, Natsumi; Umezawa, Kazuo; Simizu, Siro

    2015-03-01

    Macroautophagy is a cellular response that leads to the bulk, nonspecific degradation of cytosolic components, including organelles. In recent years, it has been recognized that autophagy is essential for prevention of neurodegenerative diseases, including Parkinson disease (PD) and Huntington disease (HD). Here, we show that conophylline (CNP), a vinca alkaloid, induces autophagy in an mammalian target of rapamycin-independent manner. Using a cellular model of PD, CNP suppressed protein aggregation and protected cells from cell death caused by treatment with 1-methyl-4-phenylpyridinium, a neurotoxin, by inducing autophagy. Moreover, in the HD model, CNP also eliminated mutant huntingtin aggregates. Our findings demonstrate the possible use of CNP as a therapeutic drug for neurodegenerative disorders, including PD and HD. PMID:25596530

  2. Conophylline Protects Cells in Cellular Models of Neurodegenerative Diseases by Inducing Mammalian Target of Rapamycin (mTOR)-independent Autophagy*

    PubMed Central

    Sasazawa, Yukiko; Sato, Natsumi; Umezawa, Kazuo; Simizu, Siro

    2015-01-01

    Macroautophagy is a cellular response that leads to the bulk, nonspecific degradation of cytosolic components, including organelles. In recent years, it has been recognized that autophagy is essential for prevention of neurodegenerative diseases, including Parkinson disease (PD) and Huntington disease (HD). Here, we show that conophylline (CNP), a vinca alkaloid, induces autophagy in an mammalian target of rapamycin-independent manner. Using a cellular model of PD, CNP suppressed protein aggregation and protected cells from cell death caused by treatment with 1-methyl-4-phenylpyridinium, a neurotoxin, by inducing autophagy. Moreover, in the HD model, CNP also eliminated mutant huntingtin aggregates. Our findings demonstrate the possible use of CNP as a therapeutic drug for neurodegenerative disorders, including PD and HD. PMID:25596530

  3. The molecular basis of multiple vector insertion by gene targeting in mammalian cells.

    PubMed Central

    Ng, P; Baker, M D

    1999-01-01

    Gene targeting using sequence insertion vectors generally results in integration of one copy of the targeting vector generating a tandem duplication of the cognate chromosomal region of homology. However, occasionally the target locus is found to contain >1 copy of the integrated vector. The mechanism by which the latter recombinants arise is not known. In the present study, we investigated the molecular basis by which multiple vectors become integrated at the chromosomal immunoglobulin mu locus in a murine hybridoma. To accomplish this, specially designed insertion vectors were constructed that included six diagnostic restriction enzyme markers in the Cmu region of homology to the target chromosomal mu locus. This enabled contributions by the vector-borne and chromosomal Cmu sequences at the recombinant locus to be ascertained. Targeted recombinants were isolated and analyzed to determine the number of vector copies integrated at the chromosomal immunoglobulin mu locus. Targeted recombinants identified as bearing >1 copy of the integrated vector resulted from a Cmu triplication formed by two vector copies in tandem. Examination of the fate of the Cmu region markers suggested that this class of recombinant was generated predominantly, if not exclusively, by two targeted vector integration events, each involving insertion of a single copy of the vector. Both vector insertion events into the chromosomal mu locus were consistent with the double-strand-break repair mechanism of homologous recombination. We interpret our results, taken together, to mean that a proportion of recipient cells is in a predetermined state that is amenable to targeted but not random vector integration. PMID:10049930

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

    PubMed

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

    2016-05-01

    Post-traumatic hypertrophic scar (HS) is a fibrotic disease with excessive extracellular matrix (ECM) production, which is a response to tissue injury by fibroblasts. Although emerging evidence has indicated that miRNA contributes to hypertrophic scarring, the role of miRNA in HS formation remains unclear. In this study, we found that miR-143-3p was markedly downregulated in HS tissues and fibroblasts (HSFs) using qRT-PCR. The expression of connective tissue growth factor (CTGF/CCN2) was upregulated both in HS tissues and HSFs, which is proposed to play a key role in ECM deposition in HS. The protein expression of collagen I (Col I), collagen III (Col III), and α-smooth muscle actin (α-SMA) was obviously inhibited after treatment with miR-143-3p in HSFs. The CCK-8 assay showed that miR-143-3p transfection reduced the proliferation ability of HSFs, and flow cytometry showed that either early or late apoptosis of HSFs was upregulated by miR-143-3p. In addition, the activity of caspase 3 and caspase 9 was increased after miR-143-3p transfection. On the contrary, the miR-143-3p inhibitor was demonstrated to increase cell proliferation and inhibit apoptosis of HSFs. Moreover, miR-143-3p targeted the 3'-UTR of CTGF and caused a significant decrease of CTGF. Western blot demonstrated that Akt/mTOR phosphorylation and the expression of CTGF, Col I, Col III, and α-SMA were inhibited by miR-143-3p, but increased by CTGF overexpression. In conclusion, we found that miR-143-3p inhibits hypertrophic scarring by regulating the proliferation and apoptosis of human HSFs, inhibiting ECM production-associated protein expression by targeting CTGF, and restraining the Akt/mTOR pathway. PMID:27075467

  5. Targeted toxin-based selectable drug-free enrichment of Mammalian cells with high transgene expression.

    PubMed

    Sato, Masahiro; Akasaka, Eri; Saitoh, Issei; Ohtsuka, Masato; Nakamura, Shingo; Sakurai, Takayuki; Watanabe, Satoshi

    2013-01-01

    Almost all transfection protocols for mammalian cells use a drug resistance gene for the selection of transfected cells. However, it always requires the characterization of each isolated clone regarding transgene expression, which is time-consuming and labor-intensive. In the current study, we developed a novel method to selectively isolate clones with high transgene expression without drug selection. Porcine embryonic fibroblasts were transfected with pCEIEnd, an expression vector that simultaneously expresses enhanced green fluorescent protein (EGFP) and endo-b-galactosidase C(EndoGalC; an enzyme capable of digesting cell surface a-Gal epitope) upon transfection. After transfection, the surviving cells were briefly treated with IB4SAP (a-Gal epitope-specific BS-I-B4 lectin conjugated with a toxin saporin). The treated cells were then allowed to grow in normal medium, during which only cells strongly expressing EndoGalC and EGFP would survive because of the absence of a-Gal epitopes on their cell surface. Almost all the surviving colonies after IB4SAP treatment were in fact negative for BS-I-B4 staining, and also strongly expressed EGFP. This system would be particularly valuable for researchers who wish to perform large-scale production of therapeutically important recombinant proteins. PMID:24832665

  6. Non-targeted Identification of Prions and Amyloid-forming Proteins from Yeast and Mammalian Cells*

    PubMed Central

    Kryndushkin, Dmitry; Pripuzova, Natalia; Burnett, Barrington G.; Shewmaker, Frank

    2013-01-01

    The formation of amyloid aggregates is implicated both as a primary cause of cellular degeneration in multiple human diseases and as a functional mechanism for providing extraordinary strength to large protein assemblies. The recent identification and characterization of several amyloid proteins from diverse organisms argues that the amyloid phenomenon is widespread in nature. Yet identifying new amyloid-forming proteins usually requires a priori knowledge of specific candidates. Amyloid fibers can resist heat, pressure, proteolysis, and denaturation by reagents such as urea or sodium dodecyl sulfate. Here we show that these properties can be exploited to identify naturally occurring amyloid-forming proteins directly from cell lysates. This proteomic-based approach utilizes a novel purification of amyloid aggregates followed by identification by mass spectrometry without the requirement for special genetic tools. We have validated this technique by blind identification of three amyloid-based yeast prions from laboratory and wild strains and disease-related polyglutamine proteins expressed in both yeast and mammalian cells. Furthermore, we found that polyglutamine aggregates specifically recruit some stress granule components, revealing a possible mechanism of toxicity. Therefore, core amyloid-forming proteins as well as strongly associated proteins can be identified directly from cells of diverse origin. PMID:23926098

  7. 17β-estradiol activates mTOR in chondrocytes by AKT-dependent and AKT-independent signaling pathways

    PubMed Central

    Tao, Yulei; Sun, Haibiao; Sun, Hongyan; Qiu, Xianxing; Xu, Changbo; Shi, Changxiu; Du, Jiahui

    2015-01-01

    To confirm whether 17β-estradiol (E2) activates mammalian target of rapamycin (mTOR) signaling pathway in chondrocytes and in what way activates mTOR. Human immortalized chondrocytes cell lines TC28a2 and C28/I2 were subjected to incubate with or without E2, LY294002 (the inhibitor of PI3K), rapamycin (the inhibitor of mTOR), or E2 in combination with LY294002 or rapamycin. Thereafter, protein levels of S6K1, p-S6K1, protein kinase B (AKT), and p-AKT were determined by Western blot analysis. Matrix metallopeptidase (MMP) 3 or MMP13 mRNA levels were evaluated by quantitative real-time PCR (qRT-PCR). Co-immunoprecipitation and Western blot analysis were performed to verify the interaction between ERα and mTOR. Both p-S6K1 and p-AKT protein levels in TC28a2 and C28/I2E2 cells were significantly increased by incubation with E2 (0.5 h and 1 h) (P < 0.05). Rapamycin did not affect the levels of p-AKT, but were significantly reduced by LY294002 or E2 in combination with LY294002. The levels of p-S6K1 were significantly decreased by incubation with LY294002, but the effect could be reversed by E2 in combination with LY294002. Rabbit anti-mTOR antibody was able to immunoprecipitate ERα after incubation with E2. Moreover, E2 inhibited the mRNA levels of MMP3 and MMP13 by mTOR pathway. E2 actives mTOR in chondrocytes through AKT-dependent and independent ways. PMID:26884863

  8. Calpain-2 activates Akt via TGF-β1-mTORC2 pathway in pulmonary artery smooth muscle cells.

    PubMed

    Abeyrathna, Prasanna; Kovacs, Laszlo; Han, Weihong; Su, Yunchao

    2016-07-01

    Calpain is a family of calcium-dependent nonlysosomal neutral cysteine endopeptidases. Akt is a serine/threonine kinase that belongs to AGC kinases and plays important roles in cell survival, growth, proliferation, angiogenesis, and cell metabolism. Both calpain and Akt are the downstream signaling molecules of platelet-derived growth factor (PDGF) and mediate PDGF-induced collagen synthesis and proliferation of pulmonary artery smooth muscle cells (PASMCs) in pulmonary vascular remodeling. We found that inhibitions of calpain-2 by using calpain inhibitor MDL28170 and calpain-2 small interfering RNA attenuated Akt phosphorylations at serine-473 (S473) and threonine-308 (T308), as well as collagen synthesis and cell proliferation of PASMCs induced by PDGF. Overexpression of calpain-2 in PASMCs induced dramatic increases in Akt phosphorylations at S473 and T308. Moreover, knockout of calpain attenuated Akt phosphorylations at S473 and T308 in smooth muscle of pulmonary arterioles of mice with chronic hypoxic pulmonary hypertension. The cell-permeable-specific transforming growth factor (TGF)-β receptor inhibitor SB431542 attenuated Akt phosphorylations at both S473 and T308 induced by PDGF and by overexpressed calpain-2 in PASMCs. Furthermore, SB-431452 and knocking down activin receptor-like kinase-5 significantly reduced PDGF-induced collagen synthesis and cell proliferation of PASMCs. Nevertheless, neutralizing extracellular TGF-β1 using a cell-impermeable TGF-β1 neutralizing antibody did not affect PDGF-induced Akt phosphorylations at S473 and T308. Furthermore, inhibition of mammalian target of rapamycin complex 2 (mTORC2) by knocking down its component protein Rictor prevented Akt phosphorylations at S473 and T308 induced by PDGF and by overexpressed calpain-2. These data provide first evidence supporting that calpain-2 upregulates PDGF-induced Akt phosphorylation in pulmonary vascular remodeling via an intracrine TGF-β1/mTORC2 mechanism. PMID:27099352

  9. Spatiotemporal regulation of GLI target genes in the mammalian limb bud.

    PubMed

    Lewandowski, Jordan P; Du, Fang; Zhang, Shilu; Powell, Marian B; Falkenstein, Kristin N; Ji, Hongkai; Vokes, Steven A

    2015-10-01

    GLI proteins convert Sonic hedgehog (Shh) signaling into a transcriptional output in a tissue-specific fashion. The Shh pathway has been extensively studied in the limb bud, where it helps regulate growth through a SHH-FGF feedback loop. However, the transcriptional response is still poorly understood. We addressed this by determining the gene expression patterns of approximately 200 candidate GLI-target genes and identified three discrete SHH-responsive expression domains. GLI-target genes expressed in the three domains are predominately regulated by derepression of GLI3 but have different temporal requirements for SHH. The GLI binding regions associated with these genes harbor both distinct and common DNA motifs. Given the potential for interaction between the SHH and FGF pathways, we also measured the response of GLI-target genes to inhibition of FGF signaling and found the majority were either unaffected or upregulated. These results provide the first characterization of the spatiotemporal response of a large group of GLI-target genes and lay the foundation for a systems-level understanding of the gene regulatory networks underlying SHH-mediated limb patterning. PMID:26238476

  10. Tunicamycins: translocase-I inhibitors that target bacterial cell wall and mammalian N-glycoproteins

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Tunicamycins, streptovirudins, and corynetoxins are natural products that target the biosynthesis of bacterial peptidoglycan and eukaryotic N-glycoproteins. The mechanism of action is known, with the tunicamycin-Mg**2+ complex established as a transition state analog for hexosamine-1-phosphate:pren...

  11. TARGETING SPHINGOSINE KINASE 1 INHIBITS AKT SIGNALING, INDUCES APOPTOSIS, AND SUPPRESSES GROWTH OF HUMAN GLIOBLASTOMA CELLS AND XENOGRAFTS

    PubMed Central

    Kapitonov, Dmitri; Allegood, Jeremy C.; Mitchell, Clint; Hait, Nitai C.; Almenara, Jorge A.; Adams, Jeffrey K.; Zipkin, Robert E.; Dent, Paul; Kordula, Tomasz; Milstien, Sheldon; Spiegel, Sarah

    2009-01-01

    Sphingosine-1-phosphate (S1P) is a potent sphingolipid mediator of diverse processes important for brain tumors, including cell growth, survival, migration, invasion, and angiogenesis. Sphingosine kinase 1 (SphK1), one of the two isoenzymes that produce S1P, is upregulated in glioblastoma and has been linked to poor prognosis in patients with glioblastoma multiforme (GBM). In the present study, we found that a potent isotype-specific SphK1 inhibitor, SK1-I, suppressed growth of LN229 and U373 glioblastoma cell lines and non-established human GBM6 cells. SK1-I also enhanced GBM cell death and inhibited their migration and invasion. SK1-I rapidly reduced phosphorylation of Akt but had no significant effect on activation of ERK1/2, another important survival pathway for GBM. Inhibition of the concomitant activation of the JNK pathway induced by SK1-I attenuated death of GBM cells. Importantly, SK1-I markedly reduced tumor growth rate of glioblastoma xenografts, inducing apoptosis and reducing tumor vascularization and enhanced the survival of mice harboring LN229 intracranial tumors. Our results support the notion that SphK1 may be an important factor in GBM and suggest that an isozyme-specific inhibitor of SphK1 deserves consideration as a new therapeutic agent for this disease. PMID:19723667

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

    PubMed

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

    2015-01-01

    The heart has two major modalities of hypertrophy in response to hemodynamic loads: concentric and eccentric hypertrophy caused by pressure and volume overload (VO), respectively. However, the molecular mechanism of eccentric hypertrophy remains poorly understood. Here we demonstrate that the Akt-mammalian target of rapamycin (mTOR) axis is a pivotal regulator of eccentric hypertrophy during VO. While mTOR in the heart was activated in a left ventricular end-diastolic pressure (LVEDP)-dependent manner, mTOR inhibition suppressed eccentric hypertrophy and induced cardiac atrophy even under VO. Notably, Akt was ubiquitinated and phosphorylated in response to VO, and blocking the recruitment of Akt to the membrane completely abolished mTOR activation. Various growth factors were upregulated during VO, suggesting that these might be involved in Akt-mTOR activation. Furthermore, the rate of eccentric hypertrophy progression was proportional to mTOR activity, which allowed accurate estimation of eccentric hypertrophy by time-integration of mTOR activity. These results suggested that the Akt-mTOR axis plays a pivotal role in eccentric hypertrophy, and mTOR activity quantitatively determines the rate of eccentric hypertrophy progression. As eccentric hypertrophy is an inherent system of the heart for regulating cardiac output and LVEDP, our findings provide a new mechanistic insight into the adaptive mechanism of the heart. PMID:26515499

  13. Pyruvate Kinase M2 Activates mTORC1 by Phosphorylating AKT1S1.

    PubMed

    He, Chang-Liang; Bian, Yang-Yang; Xue, Yu; Liu, Ze-Xian; Zhou, Kai-Qiang; Yao, Cui-Fang; Lin, Yan; Zou, Han-Fa; Luo, Fang-Xiu; Qu, Yuan-Yuan; Zhao, Jian-Yuan; Ye, Ming-Liang; Zhao, Shi-Min; Xu, Wei

    2016-01-01

    In cancer cells, the mammalian target of rapamycin complex 1 (mTORC1) that requires hormonal and nutrient signals for its activation, is constitutively activated. We found that overexpression of pyruvate kinase M2 (PKM2) activates mTORC1 signaling through phosphorylating mTORC1 inhibitor AKT1 substrate 1 (AKT1S1). An unbiased quantitative phosphoproteomic survey identified 974 PKM2 substrates, including serine202 and serine203 (S202/203) of AKT1S1, in the proteome of renal cell carcinoma (RCC). Phosphorylation of S202/203 of AKT1S1 by PKM2 released AKT1S1 from raptor and facilitated its binding to 14-3-3, resulted in hormonal- and nutrient-signals independent activation of mTORC1 signaling and led accelerated oncogenic growth and autophagy inhibition in cancer cells. Decreasing S202/203 phosphorylation by TEPP-46 treatment reversed these effects. In RCCs and breast cancers, PKM2 overexpression was correlated with elevated S202/203 phosphorylation, activated mTORC1 and inhibited autophagy. Our results provided the first phosphorylome of PKM2 and revealed a constitutive mTORC1 activating mechanism in cancer cells. PMID:26876154

  14. Pyruvate Kinase M2 Activates mTORC1 by Phosphorylating AKT1S1

    PubMed Central

    He, Chang-Liang; Bian, Yang-Yang; Xue, Yu; Liu, Ze-Xian; Zhou, Kai-Qiang; Yao, Cui-Fang; Lin, Yan; Zou, Han-Fa; Luo, Fang-Xiu; Qu, Yuan-Yuan; Zhao, Jian-Yuan; Ye, Ming-Liang; Zhao, Shi-Min; Xu, Wei

    2016-01-01

    In cancer cells, the mammalian target of rapamycin complex 1 (mTORC1) that requires hormonal and nutrient signals for its activation, is constitutively activated. We found that overexpression of pyruvate kinase M2 (PKM2) activates mTORC1 signaling through phosphorylating mTORC1 inhibitor AKT1 substrate 1 (AKT1S1). An unbiased quantitative phosphoproteomic survey identified 974 PKM2 substrates, including serine202 and serine203 (S202/203) of AKT1S1, in the proteome of renal cell carcinoma (RCC). Phosphorylation of S202/203 of AKT1S1 by PKM2 released AKT1S1 from raptor and facilitated its binding to 14-3-3, resulted in hormonal- and nutrient-signals independent activation of mTORC1 signaling and led accelerated oncogenic growth and autophagy inhibition in cancer cells. Decreasing S202/203 phosphorylation by TEPP-46 treatment reversed these effects. In RCCs and breast cancers, PKM2 overexpression was correlated with elevated S202/203 phosphorylation, activated mTORC1 and inhibited autophagy. Our results provided the first phosphorylome of PKM2 and revealed a constitutive mTORC1 activating mechanism in cancer cells. PMID:26876154

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

    PubMed Central

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

    2015-01-01

    The heart has two major modalities of hypertrophy in response to hemodynamic loads: concentric and eccentric hypertrophy caused by pressure and volume overload (VO), respectively. However, the molecular mechanism of eccentric hypertrophy remains poorly understood. Here we demonstrate that the Akt-mammalian target of rapamycin (mTOR) axis is a pivotal regulator of eccentric hypertrophy during VO. While mTOR in the heart was activated in a left ventricular end-diastolic pressure (LVEDP)-dependent manner, mTOR inhibition suppressed eccentric hypertrophy and induced cardiac atrophy even under VO. Notably, Akt was ubiquitinated and phosphorylated in response to VO, and blocking the recruitment of Akt to the membrane completely abolished mTOR activation. Various growth factors were upregulated during VO, suggesting that these might be involved in Akt-mTOR activation. Furthermore, the rate of eccentric hypertrophy progression was proportional to mTOR activity, which allowed accurate estimation of eccentric hypertrophy by time-integration of mTOR activity. These results suggested that the Akt-mTOR axis plays a pivotal role in eccentric hypertrophy, and mTOR activity quantitatively determines the rate of eccentric hypertrophy progression. As eccentric hypertrophy is an inherent system of the heart for regulating cardiac output and LVEDP, our findings provide a new mechanistic insight into the adaptive mechanism of the heart. PMID:26515499

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

    PubMed

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

    2016-08-01

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

  17. Overexpression of Akt1 Enhances Adipogenesis and Leads to Lipoma Formation in Zebrafish

    PubMed Central

    Rajendran, R. Samuel; Shen, Chia-Ning; Chen, Te-Hao; Yen, Chueh-Chuan; Chuang, Chih-Kuang; Lin, Dar-Shong; Hsiao, Chung-Der

    2012-01-01

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

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

    PubMed Central

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

    2016-01-01

    Persistent infection with oncogenic human papillomavirus viruses (HPVs) is a casual factor for cervical cancer and its precursors, and the abnormal constitutive expression of viral oncoprotein E6 is a key event during the malignant transformation. Here, we performed miRNA microarray to identify changes of miRNAs following ectopic HPV16 E6 overexpression in HEK293T cells and found miR-2861 was greatly decreased in both HEK293T and HaCaT cells expressing HPV16 E6 compared to vector control. Further, we demonstrated a biological link among HPV16 E6, miR-2861, EGFR, AKT2, and CCND1 in cervical cancer cells. We showed that miR-2861 was downregulated in cervical cancer tissues and negatively correlated with advanced tumor stage and lymph node metastasis. Overexpression of miR-2861 suppressed cervical cancer cell proliferation and invasion and enhanced apoptosis. Subsequent investigation revealed that EGFR, AKT2, and CCND1 were all the direct targets of miR-2861. Importantly, silencing EGFR, AKT2, and/or CCND1 recapitulated the cellular effects seen upon miR-2861 overexpression. Restoration of EGFR, AKT2, and/or CCND1 counteracted the effects of miR-2861 expression. Thus, we identified a new pathway employing miR-2861, EGFR, AKT2, and CCND1 that may mediate HPV16 E6 induced initiation and progression of cervical cancer. PMID:27364926

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

    PubMed Central

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

    2016-01-01

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

  20. 6″-Debromohamacanthin A, a Bis (Indole) Alkaloid, Inhibits Angiogenesis by Targeting the VEGFR2-Mediated PI3K/AKT/mTOR Signaling Pathways

    PubMed Central

    Kim, Gi Dae; Cheong, Oug Jae; Bae, Song Yi; Shin, Jongheon; Lee, Sang Kook

    2013-01-01

    Hamacanthins, bis (indole) alkaloids, are found in a few marine sponges, including Spongosorites sp. Hamacanthins have been shown to possess cytotoxic, antibacterial and antifungal activities. However, the precise mechanism for the biological activities of hamacanthins has not yet been elucidated. In the present study, the anti-angiogenic effects of 6″-debromohamacanthin A (DBHA), an active component of isolated hamacanthins, were evaluated in cultured human umbilical vascular endothelial cells (HUVEC) and endothelial-like cells differentiated from mouse embryonic stem (mES) cells. DBHA significantly inhibited vascular endothelial growth factor (VEGF)-induced cell proliferation, migration and tube formation in the HUVEC. DBHA also suppressed the capillary-like structure formation and the expression of platelet endothelial cell adhesion molecule (PECAM), an endothelial biomarker, in mES cell-derived endothelial-like cells. To further understand the precise molecular mechanism of action, VEGF-mediated signaling pathways were analyzed in HUVEC cells and mES cell-derived endothelial-like cells. DBHA suppressed the VEGF-induced expression of MAPKs (p38, ERK and SAPK/JNK) and the PI3K/AKT/mTOR signaling pathway. In addition, DBHA inhibited microvessel sprouting in mES/EB-derived embryoid bodies. In an ex vivo model, DBHA also suppressed the microvessel sprouting of mouse aortic rings. The findings suggest for the first time that DBHA inhibits angiogenesis by targeting the vascular endothelial growth factor receptor 2 (VEGFR2)-mediated PI3K/AKT/mTOR signaling pathway in endothelial cells. PMID:23549281

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

    PubMed

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

    2016-03-01

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

  2. End-targeting proteomics of isolated chromatin segments of a mammalian ribosomal RNA gene promoter

    PubMed Central

    Ide, Satoru; Dejardin, Jerome

    2015-01-01

    The unbiased identification of proteins associated with specific loci is crucial for understanding chromatin-based processes. The proteomics of isolated chromatin fragment (PICh) method has previously been developed to purify telomeres and identify associated proteins. This approach is based on the affinity capture of endogenous chromatin segments by hybridization with oligonucleotide containing locked nucleic acids. However, PICh is only efficient with highly abundant genomic targets, limiting its applicability. Here we develop an approach for identifying factors bound to the promoter region of the ribosomal RNA genes that we call end-targeting PICh (ePICh). Using ePICh, we could specifically enrich the RNA polymerase I pre-initiation complex, including the selectivity factor 1. The high purity of the ePICh material allowed the identification of ZFP106, a novel factor regulating transcription initiation by targeting RNA polymerase I to the promoter. Our results demonstrate that ePICh can uncover novel proteins controlling endogenous regulatory elements in mammals. PMID:25812914

  3. End-targeting proteomics of isolated chromatin segments of a mammalian ribosomal RNA gene promoter.

    PubMed

    Ide, Satoru; Dejardin, Jerome

    2015-01-01

    The unbiased identification of proteins associated with specific loci is crucial for understanding chromatin-based processes. The proteomics of isolated chromatin fragment (PICh) method has previously been developed to purify telomeres and identify associated proteins. This approach is based on the affinity capture of endogenous chromatin segments by hybridization with oligonucleotide containing locked nucleic acids. However, PICh is only efficient with highly abundant genomic targets, limiting its applicability. Here we develop an approach for identifying factors bound to the promoter region of the ribosomal RNA genes that we call end-targeting PICh (ePICh). Using ePICh, we could specifically enrich the RNA polymerase I pre-initiation complex, including the selectivity factor 1. The high purity of the ePICh material allowed the identification of ZFP106, a novel factor regulating transcription initiation by targeting RNA polymerase I to the promoter. Our results demonstrate that ePICh can uncover novel proteins controlling endogenous regulatory elements in mammals. PMID:25812914

  4. Targeted Cytoplasmic Irradiation with Alpha Particles Induces Mutations in Mammalian Cells

    NASA Astrophysics Data System (ADS)

    Wu, Li-Jun; Randers-Pehrson, Gerhard; Xu, An; Waldren, Charles A.; Geard, Charles R.; Yu, Zengliang; Hei, Tom K.

    1999-04-01

    Ever since x-rays were shown to induce mutation in Drosophila more than 70 years ago, prevailing dogma considered the genotoxic effects of ionizing radiation, such as mutations and carcinogenesis, as being due mostly to direct damage to the nucleus. Although there was indication that alpha particle traversal through cellular cytoplasm was innocuous, the full impact remained unknown. The availability of the microbeam at the Radiological Research Accelerator Facility of Columbia University made it possible to target and irradiate the cytoplasm of individual cells in a highly localized spatial region. By using dual fluorochrome dyes (Hoechst and Nile Red) to locate nucleus and cellular cytoplasm, respectively, thereby avoiding inadvertent traversal of nuclei, we show here that cytoplasmic irradiation is mutagenic at the CD59 (S1) locus of human-hamster hybrid (AL) cells, while inflicting minimal cytotoxicity. The principal class of mutations induced are similar to those of spontaneous origin and are entirely different from those of nuclear irradiation. Furthermore, experiments with radical scavenger and inhibitor of intracellular glutathione indicated that the mutagenicity of cytoplasmic irradiation depends on generation of reactive oxygen species. These findings suggest that cytoplasm is an important target for genotoxic effects of ionizing radiation, particularly radon, the second leading cause of lung cancer in the United States. In addition, cytoplasmic traversal by alpha particles may be more dangerous than nuclear traversal, because the mutagenicity is accomplished by little or no killing of the target cells.

  5. The antioxidant compound tert-butylhydroquinone activates Akt in myocardium, suppresses apoptosis and ameliorates pressure overload-induced cardiac dysfunction

    PubMed Central

    Zhang, Yongtao; Fang Liu, Fang; Bi, Xiaolei; Wang, Shuangxi; Wu, Xiao; Jiang, Fan

    2015-01-01

    Tert-butylhydroquinone (TBHQ) is an antioxidant compound which shows multiple cytoprotective actions. We evaluated the effects of TBHQ on pathological cardiac remodeling and dysfunction induced by chronic overload. Pressure overload was created by transverse aortic constriction (TAC) in male C57BL/6 mice. TBHQ was incorporated in the diet and administered for 4 weeks. TBHQ treatment prevented left ventricular dilatation and cardiac dysfunction induced by TAC, and decreased the prevalence of myocardial apoptosis. The beneficial effects of TBHQ were associated with an increase in Akt activation, but not related to activations of Nrf2 or AMP-activated protein kinase. TBHQ-induced Akt activation was accompanied by increased phosphorylation of Bad, glycogen synthase kinase-3β (GSK-3β) and mammalian target of rapamycin (mTOR). Mechanistically, we showed that in cultured H9c2 cells and primary cardiac myocytes, TBHQ stimulated Akt phosphorylation and suppressed oxidant-induced apoptosis; this effect was abolished by wortmannin or an Akt inhibitor. Blockade of the Akt pathway in vivo accelerated cardiac dysfunction, and abrogated the protective effects of TBHQ. TBHQ also reduced the reactive aldehyde production and protein carbonylation in stressed myocardium. We suggest that TBHQ treatment may represent a novel strategy for timely activation of the cytoprotective Akt pathway in stressed myocardium. PMID:26260024

  6. Tetrandrine suppresses metastatic phenotype of prostate cancer cells by regulating Akt/mTOR/MMP-9 signaling pathway.

    PubMed

    Kou, Bo; Liu, Wei; He, Wenbo; Zhang, Yuanyuan; Zheng, Jianjie; Yan, Yang; Zhang, Yongjian; Xu, Suochun; Wang, Haichen

    2016-05-01

    Tetrandrine (TET), a bisbenzylisoquinoline alkaloid found in traditional Chinese medicines, exerts anticancer activity in vitro and in vivo. However, its potential role in the prostate cancer metastatic process has not yet been elucidated. Thus, we investigated the inhibition effect of tetrandrine on prostate cancer migration and invasion and the corresponding molecular basis underlying its anticancer activity. Cell migration and invasion were determined using the Transwell chamber model. The protein expression of Akt, phosphorylated Akt, the mammalian target of rapamycin (mTOR), phosphorylated mTOR and matrix metalloproteinases 9 (MMP-9) was detected by western blot in the presence or absence of tetrandrine or in the group tetrandrine combination with LY294002 (inhibitor of Akt) and rapamycin (inhibitor of mTOR). Our studies showed that excluding the effect of tetrandrine on cell proliferation, tetrandrine significantly inhibited cell migration and invasion in prostate cancer DU145 and PC3 cells. Furthermore, tetrandrine decreased the protein levels of p-Akt, p-mTOR, and MMP-9. While the inhibition of Akt or mTOR by the respective inhibitors could potentiate this effect of tetrandrine on prostate cancer cells, the studies indicate that tetrandrine inhibits the metastasis process by negatively regulating the Akt/mTOR/MMP-9 signaling pathway. These results suggest that tetrandrine might serve as a potential metastasis suppressor to treat cancer cells that have escaped surgical removal or that have disseminated widely. PMID:26935264

  7. MiRNA-Target Interaction Reveals Cell-Specific Post-Transcriptional Regulation in Mammalian Cell Lines

    PubMed Central

    Kulkarni, Varun; Naqvi, Afsar Raza; Uttamani, Juhi Raju; Nares, Salvador

    2016-01-01

    MicroRNAs are 18–22 nucleotides long, non-coding RNAs that bind transcripts with complementary sequences leading to either mRNA degradation or translational suppression. However, the inherent differences in preferred mode of miRNA regulation among cells of different origin have not been examined. In our previous transcriptome profiling studies, we observed that post-transcriptional regulation can differ substantially depending on the cell in context. Here we examined mechanistic differences in the regulation of a let-7a targeted (wild type) or resistant (mutant) engineered renilla transcript across various mammalian cell lines of diverse origin. Dual luciferase assays show that compared to mutant (mut), the reporter gene containing wild type (wt) let-7a binding sites was efficiently suppressed upon transfection in various cell lines. Importantly, the strength of miRNA regulation varied across the cell lines. Total RNA analysis demonstrates that wt renilla mRNA was expressed to similar or higher levels compared to mut suggesting that translation repression is a predominant mode of miRNA regulation. Nonetheless, transcript degradation was observed in some cell lines. Ago-2 immunoprecipitation show that miRNA repressed renilla mRNA are associated with functional mi-RISC (miRNA-RNA induced silencing complex). Given the immense potential of miRNA as a therapeutic option, these findings highlight the necessity to thoroughly examine the mode of mRNA regulation in order to achieve the beneficial effects in targeting cells. PMID:26761000

  8. Blocking mammalian target of rapamycin alleviates bone cancer pain and morphine tolerance via µ-opioid receptor.

    PubMed

    Jiang, Zongming; Wu, Shaoyong; Wu, Xiujuan; Zhong, Junfeng; Lv, Anqing; Jiao, Jing; Chen, Zhonghua

    2016-04-15

    The current study was to examine the underlying mechanisms responsible for the role of mammalian target of rapamycin (mTOR) in regulating bone cancer-evoked pain and the tolerance of systemic morphine. Breast sarcocarcinoma Walker 256 cells were implanted into the tibia bone cavity of rats and this evoked significant mechanical and thermal hyperalgesia. Our results showed that the protein expression of p-mTOR, mTOR-mediated phosphorylation of 4E-binding protein 4 (4E-BP1), p70 ribosomal S6 protein kinase 1 (S6K1) as well as phosphatidylinositide 3-kinase (p-PI3K) pathways were amplified in the superficial dorsal horn of the spinal cord of bone cancer rats compared with control rats. Blocking spinal mTOR by using rapamycin significantly attenuated activities of PI3K signaling pathways as well as mechanical and thermal hyperalgesia. Additionally, rapamycin enhanced attenuations of protein kinase Cɛ (PKCɛ)/protein kinase A (PKA) induced by morphine and further extended analgesia of morphine via µ-opioid receptor (MOR). Our data for the first time revealed specific signaling pathways leading to bone cancer pain, including the activation of mTOR and PI3K and downstream PKCɛ/PKA, and resultant sensitization of MOR. Targeting one or more of these signaling molecules may present new opportunities for treatment and management of bone cancer pain often observed in clinics. PMID:26566757

  9. Mammalian Target of Rapamycin (mTOR) Inhibition as a Potential Antiepileptogenic Therapy: From Tuberous Sclerosis to Common Acquired Epilepsies

    PubMed Central

    Wong, Michael

    2011-01-01

    SUMMARY Most current treatments for epilepsy are symptomatic therapies that suppress seizures but do not affect the underlying course or prognosis of epilepsy. The need for disease-modifying or “antiepileptogenic” treatments for epilepsy is widely recognized, but no such preventative therapies have yet been established for clinical use. A rational strategy for preventing epilepsy is to target primary signaling pathways that initially trigger the numerous downstream mechanisms mediating epileptogenesis. The mammalian target of rapamycin (mTOR) pathway represents a logical candidate, because mTOR regulates multiple cellular functions that may contribute to epileptogenesis, including protein synthesis, cell growth and proliferation, and synaptic plasticity. The importance of the mTOR pathway in epileptogenesis is best illustrated by Tuberous Sclerosis Complex (TSC), one of the most common genetic causes of epilepsy. In mouse models of TSC, mTOR inhibitors prevent the development of epilepsy and underlying brain abnormalities associated with epileptogenesis. Accumulating evidence suggests that mTOR also participates in epileptogenesis due to a variety of other causes, including focal cortical dysplasia and acquired brain injuries, such as in animal models following status epilepticus or traumatic brain injury. Thus, mTOR inhibition may represent a potential antiepileptogenic therapy for diverse types of epilepsy, including both genetic and acquired epilepsies. PMID:19817806

  10. Mammalian Target of Rapamycin Complex 2 Controls CD8 T Cell Memory Differentiation in a Foxo1-Dependent Manner.

    PubMed

    Zhang, Lianjun; Tschumi, Benjamin O; Lopez-Mejia, Isabel C; Oberle, Susanne G; Meyer, Marten; Samson, Guerric; Rüegg, Markus A; Hall, Michael N; Fajas, Lluis; Zehn, Dietmar; Mach, Jean-Pierre; Donda, Alena; Romero, Pedro

    2016-02-01

    Upon infection, antigen-specific naive CD8 T cells are activated and differentiate into short-lived effector cells (SLECs) and memory precursor cells (MPECs). The underlying signaling pathways remain largely unresolved. We show that Rictor, the core component of mammalian target of rapamycin complex 2 (mTORC2), regulates SLEC and MPEC commitment. Rictor deficiency favors memory formation and increases IL-2 secretion capacity without dampening effector functions. Moreover, mTORC2-deficient memory T cells mount more potent recall responses. Enhanced memory formation in the absence of mTORC2 was associated with Eomes and Tcf-1 upregulation, repression of T-bet, enhanced mitochondrial spare respiratory capacity, and fatty acid oxidation. This transcriptional and metabolic reprogramming is mainly driven by nuclear stabilization of Foxo1. Silencing of Foxo1 reversed the increased MPEC differentiation and IL-2 production and led to an impaired recall response of Rictor KO memory T cells. Therefore, mTORC2 is a critical regulator of CD8 T cell differentiation and may be an important target for immunotherapy interventions. PMID:26804903

  11. Chemical Inhibitors and microRNAs (miRNA) Targeting the Mammalian Target of Rapamycin (mTOR) Pathway: Potential for Novel Anticancer Therapeutics

    PubMed Central

    AlQurashi, Naif; Hashimi, Saeed M.; Wei, Ming Q.

    2013-01-01

    The mammalian target of rapamycin (mTOR) is a critical regulator of many fundamental features in response to upstream cellular signals, such as growth factors, energy, stress and nutrients, controlling cell growth, proliferation and metabolism through two complexes, mTORC1 and mTORC2. Dysregulation of mTOR signalling often occurs in a variety of human malignant diseases making it a crucial and validated target in the treatment of cancer. Tumour cells have shown high susceptibility to mTOR inhibitors. Rapamycin and its derivatives (rapalogs) have been tested in clinical trials in several tumour types and found to be effective as anticancer agents in patients with advanced cancers. To block mTOR function, they form a complex with FKBP12 and then bind the FRB domain of mTOR. Furthermore, a new generation of mTOR inhibitors targeting ATP-binding in the catalytic site of mTOR showed potent and more selective inhibition. More recently, microRNAs (miRNA) have emerged as modulators of biological pathways that are essential in cancer initiation, development and progression. Evidence collected to date shows that miRNAs may function as tumour suppressors or oncogenes in several human neoplasms. The mTOR pathway is a promising target by miRNAs for anticancer therapy. Extensive studies have indicated that regulation of the mTOR pathway by miRNAs plays a major role in cancer progression, indicating a novel way to investigate the tumorigenesis and therapy of cancer. Here, we summarize current findings of the role of mTOR inhibitors and miRNAs in carcinogenesis through targeting mTOR signalling pathways and determine their potential as novel anti-cancer therapeutics. PMID:23434669

  12. An update on targeted gene repair in mammalian cells: methods and mechanisms.

    PubMed

    Jensen, Nanna M; Dalsgaard, Trine; Jakobsen, Maria; Nielsen, Roni R; Sørensen, Charlotte B; Bolund, Lars; Jensen, Thomas G

    2011-01-01

    Transfer of full-length genes including regulatory elements has been the preferred gene therapy strategy for clinical applications. However, with significant drawbacks emerging, targeted gene alteration (TGA) has recently become a promising alternative to this method. By means of TGA, endogenous DNA repair pathways of the cell are activated leading to specific genetic correction of single-base mutations in the genome. This strategy can be implemented using single-stranded oligodeoxyribonucleotides (ssODNs), small DNA fragments (SDFs), triplex-forming oligonucleotides (TFOs), adeno-associated virus vectors (AAVs) and zinc-finger nucleases (ZFNs). Despite difficulties in the use of TGA, including lack of knowledge on the repair mechanisms stimulated by the individual methods, the field holds great promise for the future. The objective of this review is to summarize and evaluate the different methods that exist within this particular area of human gene therapy research. PMID:21284895

  13. An update on targeted gene repair in mammalian cells: methods and mechanisms

    PubMed Central

    2011-01-01

    Transfer of full-length genes including regulatory elements has been the preferred gene therapy strategy for clinical applications. However, with significant drawbacks emerging, targeted gene alteration (TGA) has recently become a promising alternative to this method. By means of TGA, endogenous DNA repair pathways of the cell are activated leading to specific genetic correction of single-base mutations in the genome. This strategy can be implemented using single-stranded oligodeoxyribonucleotides (ssODNs), small DNA fragments (SDFs), triplex-forming oligonucleotides (TFOs), adeno-associated virus vectors (AAVs) and zinc-finger nucleases (ZFNs). Despite difficulties in the use of TGA, including lack of knowledge on the repair mechanisms stimulated by the individual methods, the field holds great promise for the future. The objective of this review is to summarize and evaluate the different methods that exist within this particular area of human gene therapy research. PMID:21284895

  14. The mammalian tachykinin ligand-receptor system: an emerging target for central neurological disorders

    PubMed Central

    Pantaleo, Nick; Chadwick, Wayne; Park, Sung-Soo; Wang, Liyun; Zhou, Yu; Martin, Bronwen; Maudsley, Stuart

    2010-01-01

    Our understanding of the complex signaling neurophysiology of the central nervous system has facilitated the exploration of potential novel receptor-ligand system targets for disorders of this most complex organ. In recent years, many relatively neglected receptor-ligand systems have been re-evaluated with respect to their ability to potently modulate discrete tracts in the central nervous system. One such system is the tachykinin (previously neurokinin) system. The multiple heptahelical G protein-coupled receptors and neuropeptide ligands that comprise this system may be significantly involved in more central nervous systems actions than previously thought, including sleep disorders, amyotrophic lateral sclerosis, Alzheimer’s and Machado-Joseph disease. The development of our understanding of the role of the tachykinin receptor-ligand system in higher order central functions is likely to allow the creation of more specific and selective tachykinin-related neurotherapeutics. PMID:20632965

  15. Akt signaling in platelets and thrombosis

    PubMed Central

    Woulfe, Donna S

    2010-01-01

    Akt is a Ser–Thr kinase with pleiotropic effects on cell survival, growth and metabolism. Recent evidence from gene-deletion studies in mice, and analysis of human platelets treated with Akt inhibitors, suggest that Akt regulates platelet activation, with potential consequences for thrombosis. Akt activation is regulated by the level of phosphoinositide 3-phosphates, and proteins that regulate concentrations of this lipid also regulate Akt activation and platelet function. Although the effectors through which Akt contributes to platelet activation are not definitively known, several candidates are discussed, including endothelial nitric oxide synthase, glycogen synthase kinase 3β, phosphodiesterase 3A and the integrin β3 tail. Selective inhibitors of Akt isoforms or of proteins that contribute to its activation, such as individual PI3K isoforms, may make attractive targets for antithrombotic therapy. This review summarizes the current literature describing Akt activity and its regulation in platelets, including speculation regarding the future of Akt or its regulatory pathways as targets for the development of antithrombotic therapies. PMID:20352060

  16. Small Molecule Membrane Transporters in the Mammalian Podocyte: A Pathogenic and Therapeutic Target

    PubMed Central

    Zennaro, Cristina; Artero, Mary; Di Maso, Vittorio; Carraro, Michele

    2014-01-01

    The intriguingly complex glomerular podocyte has been a recent object of intense study. Researchers have sought to understand its role in the pathogenesis of common proteinuric diseases such as minimal change disease and focal segmental glomerular sclerosis. In particular, considerable effort has been directed towards the anatomic and functional barrier to macromolecular filtration provided by the secondary foot processes, but little attention has been paid to the potential of podocytes to handle plasma proteins beyond the specialization of the slit diaphragm. Renal membrane transporters in the proximal tubule have been extensively studied for decades, particularly in relation to drug metabolism and elimination. Recently, uptake and efflux transporters for small organic molecules have also been found in the glomerular podocyte, and we and others have found that these transporters can engage not only common pharmaceuticals but also injurious endogenous and exogenous agents. We have also found that the activity of podocyte transporters can be manipulated to inhibit pathogen uptake and efflux. It is conceivable that podocyte transporters may play a role in disease pathogenesis and may be a target for future drug development. PMID:25411800

  17. Redox reactions in mammalian spermatogenesis and the potential targets of reactive oxygen species under oxidative stress

    PubMed Central

    Fujii, Junichi; Imai, Hirotaka

    2014-01-01

    Reduction-oxidation (Redox) reactions are ubiquitous mechanisms for vital activities in all organisms, and they play pivotal roles in the regulation of spermatogenesis as well. Here we focus on 3 redox-involved processes that have drawn much recent attention: the regulation of signal transduction by reactive oxygen species (ROS) such as hydrogen peroxide, oxidative protein folding in the endoplasmic reticulum (ER), and sulfoxidation of protamines during sperm chromatin condensation. The first 2 of these processes are emerging topics in cell biology and are applicable to most living cells, which includes spermatogenic cells. The roles of ROS in signal transduction have been elucidated in the last 2 decades and have received broad attention, most notably from the viewpoint of the proper control of mitotic signals. Redox processes in the ER are important because this is the organelle where secretory and membrane proteins are synthesized and proceed toward their functional structure, so that malfunction of the ER affects not only the involved cells but also the accepting cells of the secreted proteins in multicellular organisms. Sulfoxidation is the third of these processes, and the sulfoxidation of chromatin is a unique process in sperm maturation. During recent sulfoxidase research, GPX4 has emerged as a promising enzyme that plays essential roles in the production of fertile sperm, but the involvement of other redox proteins is also becoming evident. Because the molecules involved in the redox reactions are prone to oxidation, they can be sensitive to oxidative damage, which makes them potential targets for antioxidant therapy. PMID:26413390

  18. Isolation, characterization and targeted disruption of mouse ppia: cyclophilin A is not essential for mammalian cell viability.

    PubMed

    Colgan, J; Asmal, M; Luban, J

    2000-09-01

    Cyclophilins (CyPs) are a family of proteins found in organisms ranging from prokaryotes to humans. These molecules exhibit peptidyl-prolyl isomerase activity in vitro, suggesting that they influence the conformation of proteins in cells. CyPs also bind with varying affinities to the immunosuppressive drug cyclosporin A (CsA), a compound used clinically to prevent allograft rejection. The founding member of the family, cyclophilin A (CyPA), is an abundant, ubiquitously expressed protein of unknown function that binds with nanomolar affinity to CsA. Here, we describe the isolation and characterization of mouse Ppia (mPpia), the gene encoding CyPA. Ppia was isolated using a PCR screen that distinguishes the expressed gene from multiple pseudogenes present in the mouse genome. mPpia consists of 5 exons and 4 introns spanning roughly 4.5 kb and maps to chromosome 11 near the centromere. Sequence analysis of a 369-bp fragment from the proximal promoter region of mPpia revealed the presence of a TATA box and sites recognized by several transcriptional regulators, including Sp1, AP-2, GATA factors, c-Myb, and NF-IL-6. This region is sufficient to drive high-level reporter gene expression in transfected cells. Both copies of Ppia were disrupted in murine embryonic stem (ES) cells via gene targeting. Ppia(-/-) ES cells grow normally and differentiate into hematopoeitic precursor cells in vitro, indicating that CyPA is not essential for mammalian cell viability. PMID:10964515

  19. Syntaxin13 Expression Is Regulated by Mammalian Target of Rapamycin (mTOR) in Injured Neurons to Promote Axon Regeneration*

    PubMed Central

    Cho, Yongcheol; Di Liberto, Valentina; Carlin, Dan; Abe, Namiko; Li, Kathy H.; Burlingame, Alma L.; Guan, Shenheng; Michaelevski, Izhak; Cavalli, Valeria

    2014-01-01

    Injured peripheral neurons successfully activate intrinsic signaling pathways to enable axon regeneration. We have previously shown that dorsal root ganglia (DRG) neurons activate the mammalian target of rapamycin (mTOR) pathway following injury and that this activity enhances their axon growth capacity. mTOR plays a critical role in protein synthesis, but the mTOR-dependent proteins enhancing the regenerative capacity of DRG neurons remain unknown. To identify proteins whose expression is regulated by injury in an mTOR-dependent manner, we analyzed the protein composition of DRGs from mice in which we genetically activated mTOR and from mice with or without a prior nerve injury. Quantitative label-free mass spectrometry analyses revealed that the injury effects were correlated with mTOR activation. We identified a member of the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) family of proteins, syntaxin13, whose expression was increased by injury in an mTOR-dependent manner. Increased syntaxin13 levels in injured nerves resulted from local protein synthesis and not axonal transport. Finally, knockdown of syntaxin13 in cultured DRG neurons prevented axon growth and regeneration. Together, these data suggest that syntaxin13 translation is regulated by mTOR in injured neurons to promote axon regeneration. PMID:24737317

  20. Clustering of mammalian Hox genes with other H3K27me3 targets within an active nuclear domain.

    PubMed

    Vieux-Rochas, Maxence; Fabre, Pierre J; Leleu, Marion; Duboule, Denis; Noordermeer, Daan

    2015-04-14

    Embryogenesis requires the precise activation and repression of many transcriptional regulators. The Polycomb group proteins and the associated H3K27me3 histone mark are essential to maintain the inactive state of many of these genes. Mammalian Hox genes are targets of Polycomb proteins and form local 3D clusters centered on the H3K27me3 mark. More distal contacts have also been described, yet their selectivity, dynamics, and relation to other layers of chromatin organization remained elusive. We report that repressed Hox genes form mutual intra- and interchromosomal interactions with other genes located in strong domains labeled by H3K27me3. These interactions occur in a central and active nuclear environment that consists of the HiC compartment A, away from peripheral lamina-associated domains. Interactions are independent of nearby H3K27me3-marked loci and determined by chromosomal distance and cell-type-specific scaling factors, thus inducing a moderate reorganization during embryogenesis. These results provide a simplified view of nuclear organization whereby Polycomb proteins may have evolved to repress genes located in gene-dense regions whose position is restricted to central, active, nuclear environments. PMID:25825760

  1. Oral mucosal injury caused by mammalian target of rapamycin inhibitors: emerging perspectives on pathobiology and impact on clinical practice.

    PubMed

    Peterson, Douglas E; O'Shaughnessy, Joyce A; Rugo, Hope S; Elad, Sharon; Schubert, Mark M; Viet, Chi T; Campbell-Baird, Cynthia; Hronek, Jan; Seery, Virginia; Divers, Josephine; Glaspy, John; Schmidt, Brian L; Meiller, Timothy F

    2016-08-01

    In recent years oral mucosal injury has been increasingly recognized as an important toxicity associated with mammalian target of rapamycin (mTOR) inhibitors, including in patients with breast cancer who are receiving everolimus. This review addresses the state-of-the-science regarding mTOR inhibitor-associated stomatitis (mIAS), and delineates its clinical characteristics and management. Given the clinically impactful pain associated with mIAS, this review also specifically highlights new research focusing on the study of the molecular basis of pain. The incidence of mIAS varies widely (2-78%). As reported across multiple mTOR inhibitor clinical trials, grade 3/4 toxicity occurs in up to 9% of patients. Managing mTOR-associated oral lesions with topical oral, intralesional, and/or systemic steroids can be beneficial, in contrast to the lack of evidence supporting steroid treatment of oral mucositis caused by high-dose chemotherapy or radiation. However, steroid management is not uniformly efficacious in all patients receiving mTOR inhibitors. Furthermore, technology does not presently exist to permit clinicians to predict a priori which of their patients will develop these lesions. There thus remains a strategic need to define the pathobiology of mIAS, the molecular basis of pain, and risk prediction relative to development of the clinical lesion. This knowledge could lead to novel future interventions designed to more effectively prevent mIAS and improve pain management if clinically significant mIAS lesions develop. PMID:27334013

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

    PubMed

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

    2014-06-01

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

  3. Antisense Oligonucleotides Targeting Parasite Inositol 1,4,5-Trisphosphate Receptor Inhibits Mammalian Host Cell Invasion by Trypanosoma cruzi

    NASA Astrophysics Data System (ADS)

    Hashimoto, Muneaki; Nara, Takeshi; Hirawake, Hiroko; Morales, Jorge; Enomoto, Masahiro; Mikoshiba, Katsuhiko

    2014-02-01

    Chagas disease is caused by an intracellular parasitic protist, Trypanosoma cruzi. As there are no highly effective drugs against this agent that also demonstrate low toxicity, there is an urgent need for development of new drugs to treat Chagas disease. We have previously demonstrated that the parasite inositol 1,4,5-trisphosphate receptor (TcIP3R) is crucial for invasion of the mammalian host cell by T. cruzi. Here, we report that TcIP3R is a short-lived protein and that its expression is significantly suppressed in trypomastigotes. Treatment of trypomastigotes, an infective stage of T. cruzi, with antisense oligonucleotides specific to TcIP3R deceased TcIP3R protein levels and impaired trypomastigote invasion of host cells. Due to the resulting instability and very low expression level of TcIP3R in trypomastigotes indicates that TcIP3R is a promising target for antisense therapy in Chagas disease.

  4. Mis-regulation of Mammalian Target of Rapamycin (mTOR) Complexes Induced by Albuminuria in Proximal Tubules*

    PubMed Central

    Peruchetti, Diogo B.; Cheng, Jie; Caruso-Neves, Celso; Guggino, William B.

    2014-01-01

    High albumin concentrations in the proximal tubule of the kidney causes tubulointerstitial injury, but how this process occurs is not completely known. To address the signal transduction pathways mis-regulated in renal injury, we studied the modulation of mammalian target of rapamycin (mTOR) complexes by physiologic and pathophysiologic albumin concentrations in proximal tubule cells. Physiologic albumin concentrations activated the PI3K/mTORC2/PKB/mTORC1/S6 kinase (S6K) pathway, but pathophysiologically high albumin concentrations overactivated mTORC1 and inhibited mTORC2 activity. This control process involved the activation of ERK1/2, which promoted the inhibition of TSC2 and activation of S6K. Furthermore, S6K was crucial to promoting the over activation of mTORC1 and inhibition of mTORC2. Megalin expression at the luminal membrane is reduced by high concentrations of albumin. In addition, knockdown of megalin mimicked all the effects of pathophysiologic albumin concentrations, which disrupt normal signal transduction pathways and lead to an overactivation of mTORC1 and inhibition of mTORC2. These data provide new perspectives for understanding the molecular mechanisms behind the effects of albumin on the progression of renal disease. PMID:24790108

  5. Exogenous plant MIR168a specifically targets mammalian LDLRAP1: evidence of cross-kingdom regulation by microRNA

    PubMed Central

    Zhang, Lin; Hou, Dongxia; Chen, Xi; Li, Donghai; Zhu, Lingyun; Zhang, Yujing; Li, Jing; Bian, Zhen; Liang, Xiangying; Cai, Xing; Yin, Yuan; Wang, Cheng; Zhang, Tianfu; Zhu, Dihan; Zhang, Dianmu; Xu, Jie; Chen, Qun; Ba, Yi; Liu, Jing; Wang, Qiang; Chen, Jianqun; Wang, Jin; Wang, Meng; Zhang, Qipeng; Zhang, Junfeng; Zen, Ke; Zhang, Chen-Yu

    2012-01-01

    Our previous studies have demonstrated that stable microRNAs (miRNAs) in mammalian serum and plasma are actively secreted from tissues and cells and can serve as a novel class of biomarkers for diseases, and act as signaling molecules in intercellular communication. Here, we report the surprising finding that exogenous plant miRNAs are present in the sera and tissues of various animals and that these exogenous plant miRNAs are primarily acquired orally, through food intake. MIR168a is abundant in rice and is one of the most highly enriched exogenous plant miRNAs in the sera of Chinese subjects. Functional studies in vitro and in vivo demonstrated that MIR168a could bind to the human/mouse low-density lipoprotein receptor adapter protein 1 (LDLRAP1) mRNA, inhibit LDLRAP1 expression in liver, and consequently decrease LDL removal from mouse plasma. These findings demonstrate that exogenous plant miRNAs in food can regulate the expression of target genes in mammals. PMID:21931358

  6. N-terminal region of Mannheimia haemolytica leukotoxin serves as a mitochondrial targeting signal in mammalian cells.

    PubMed

    Kisiela, Dagmara I; Aulik, Nicole A; Atapattu, Dhammika N; Czuprynski, Charles J

    2010-07-01

    Mannheimia haemolytica leukotoxin (LktA) is a member of the RTX toxin family that specifically kills ruminant leukocytes. Previous studies have shown that LktA induces apoptosis in susceptible cells via a caspase-9-dependent pathway that involves binding of LktA to mitochondria. In this study, using the bioinformatics tool MitoProt II we identified an N-terminal amino acid sequence of LktA that represents a mitochondrial targeting signal (MTS). We show that expression of this sequence, as a GFP fusion protein within mammalian cells, directs GFP to mitochondria. By immunoprecipitation we demonstrate that LktA interacts with the Tom22 and Tom40 components of the translocase of the outer mitochondrial membrane (TOM), which suggests that import of this toxin into mitochondria involves a classical import pathway for endogenous proteins. We also analysed the amino acid sequences of other RTX toxins and found a MTS in the N-terminal region of Actinobacillus pleuropneumoniae ApxII and enterohaemorrhagic Escherichia coli EhxA, but not in A. pleuropneumoniae ApxI, ApxIII, Aggregatibacter actinomycetemcomitans LtxA or the haemolysin (HlyA) from uropathogenic strains of E. coli. These findings provide a new evidence for the importance of the N-terminal region in addressing certain RTX toxins to mitochondria. PMID:20109159

  7. Inhibition of mammalian target of rapamycin activation in the rostral anterior cingulate cortex attenuates pain-related aversion in rats.

    PubMed

    Lu, Bo; Jiang, Jingyan; Sun, Jianliang; Xiao, Chun; Meng, Bo; Zheng, Jinwei; Li, Xiaoyu; Wang, Ruichun; Wu, Guorong; Chen, Junping

    2016-09-01

    Pain is a complex experience that comprises both sensory and affective dimensions. Mammalian target of rapamycin (mTOR) plays an important role in the modulation of neuronal plasticity associated with the pathogenesis of pain sensation. However, the role of mTOR in pain affect is unclear. Using a formalin-induced conditioned place avoidance (F-CPA) test, the current study investigated the effects of the mTOR specific inhibitor rapamycin on noxious stimulation induced aversion in the rostral anterior cingulate cortex (rACC). Intraplantar injection of 5% formalin was associated with significant activation of mTOR, as well as p70 ribosomal S6 protein (p70S6K), its downstream effector, in the rACC. The inhibition of mTOR activation with rapamycin disrupted pain-related aversion; however, this inhibition did not affect formalin-induced spontaneous nociceptive behaviors in rats. These findings demonstrated for the first time that mTOR and its downstream pathway in the rACC contribute to the induction of pain-related negative emotion. PMID:27163752

  8. Phosphoproteomic Profiling of In Vivo Signaling in Liver by the Mammalian Target of Rapamycin Complex 1 (mTORC1)

    PubMed Central

    Demirkan, Gokhan; Yu, Kebing; Boylan, Joan M.; Salomon, Arthur R.; Gruppuso, Philip A.

    2011-01-01

    Background Our understanding of signal transduction networks in the physiological context of an organism remains limited, partly due to the technical challenge of identifying serine/threonine phosphorylated peptides from complex tissue samples. In the present study, we focused on signaling through the mammalian target of rapamycin (mTOR) complex 1 (mTORC1), which is at the center of a nutrient- and growth factor-responsive cell signaling network. Though studied extensively, the mechanisms involved in many mTORC1 biological functions remain poorly understood. Methodology/Principal Findings We developed a phosphoproteomic strategy to purify, enrich and identify phosphopeptides from rat liver homogenates. Using the anticancer drug rapamycin, the only known target of which is mTORC1, we characterized signaling in liver from rats in which the complex was maximally activated by refeeding following 48 hr of starvation. Using protein and peptide fractionation methods, TiO2 affinity purification of phosphopeptides and mass spectrometry, we reproducibly identified and quantified over four thousand phosphopeptides. Along with 5 known rapamycin-sensitive phosphorylation events, we identified 62 new rapamycin-responsive candidate phosphorylation sites. Among these were PRAS40, gephyrin, and AMP kinase 2. We observed similar proportions of increased and reduced phosphorylation in response to rapamycin. Gene ontology analysis revealed over-representation of mTOR pathway components among rapamycin-sensitive phosphopeptide candidates. Conclusions/Significance In addition to identifying potential new mTORC1-mediated phosphorylation events, and providing information relevant to the biology of this signaling network, our experimental and analytical approaches indicate the feasibility of large-scale phosphoproteomic profiling of tissue samples to study physiological signaling events in vivo. PMID:21738781

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

    PubMed

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

    2016-09-01

    T-cell immunoglobulin and mucin domain-con-taining protein-3 (TIM-3), a negative regulator of antitumor immune response, has been demonstrated to be involved in the onset and progression of several types of malignancies. The present study aimed to determine whether and how TIM‑3 plays such a role in esophageal squamous cell carcinoma (ESCC). TIM-3 expression was analyzed by immunohistochemistry and real‑time fluorescence quantitative PCR (qRT‑PCR) in ESCC and matched adjacent normal tissues. Functional experiments in vitro were performed to elucidate the effect of TIM‑3 knockdown on the proliferation, apoptosis, migration, invasion and epithelial-mesenchymal transition (EMT) in Eca109 and TE‑1 cell lines. Our data revealed that TIM‑3 expression was significantly elevated at both the mRNA and protein levels in ESCC tissues compared with the levels in the matched adjacent normal tissues (both P<0.001). TIM‑3 expression was significantly associated with lymph node metastasis (P=0.008), tumor‑node‑metastasis (TNM) stage (P=0.042) and depth of tumor invasion (P=0.042). In addition, we observed a strong correlation between high TIM‑3 expression and a worse overall survival of ESCC patients (P=0.001). Functional study demonstrated that TIM‑3 knockdown markedly inhibited proliferation, migration and invasion of ESCC cell lines without affecting apoptosis. In addition, TIM‑3 depletion was associated with downregulation of matrix metalloproteinase (MMP)-9 and upregulation of tissue inhibitor of metalloproteinase (TIMP)-1, and with reversion of EMT, as reflected by higher levels of the epithelial marker E‑cadherin and lower levels of the mesenchymal markers N‑cadherin and vimentin. Further study found that TIM‑3 depletion suppressed the signaling pathway involving p‑Akt, p‑GSK‑3β and Snail. Taken together, these results suggest that TIM‑3 is a novel therapeutic target and prognostic biomarker for ESCC and promotes metastasis of

  10. PTP4A3 is a target for inhibition of cell proliferatin, migration and invasion through Akt/mTOR signaling pathway in glioblastoma under the regulation of miR-137.

    PubMed

    Wang, Liling; Liu, Jianxun; Zhong, Zhiqiang; Gong, Xuhai; Liu, Wei; Shi, Lei; Li, Xuesong

    2016-09-01

    Glioblastoma multiforme (GBM) is one of the most common primary malignant adult brain tumors. It is characterized by aggressive progression and poor prognosis. There is significant need to understand the mechanism of GBM malignancy and develop improved therapeutic options for GBM patients. We systematically studied the function of PTP4A3 in the malignancy of GBM. We found that PTP4A3 was upregulated in GBM tissues and cells. Knockdown of PTP4A3 expression in GBM cells inhibited cell proliferation, migration, and invasion. PTP4A3 knockdown modulated the activity of the Akt/mTOR signaling pathway by inducing de-phosphorylation of Akt and mTOR. We identified PTP4A3 as a direct target of miR-137. MiR-137 has been reported as a tumor suppressor in GBM development. In this study, overexpression of miR-137 in GBM cells also inhibited cell proliferation, migration, and invasion. Finally, restoration of PTP4A3 expression in miR-137 overexpressing cells partially reversed the inhibition of GBM cell malignancy, and the de-phosphorylation of Akt and mTOR. We identified that PTP4A3 regulated GBM via miR-137-mediated Akt/mTOR signaling pathway. PMID:27328425

  11. Feedback loops blockade potentiates apoptosis induction and antitumor activity of a novel AKT inhibitor DC120 in human liver cancer.

    PubMed

    Yang, F; Deng, R; Qian, X-J; Chang, S-H; Wu, X-Q; Qin, J; Feng, G-K; Ding, K; Zhu, X-F

    2014-01-01

    The serine/threonine kinase AKT is generally accepted as a promising anticancer therapeutic target. However, the relief of feedback inhibition and enhancement of other survival pathways often attenuate the anticancer effects of AKT inhibitors. These compensatory mechanisms are very complicated and remain poorly understood. In the present study, we found a novel 2-pyrimidyl-5-amidothiazole compound, DC120, as an ATP competitive AKT kinase inhibitor that suppressed proliferation and induced apoptosis in liver cancer cells both in vitro and in vivo. DC120 blocked the phosphorylation of downstream molecules in the AKT signal pathway in dose- and time-dependent manners both in vitro and in vivo. However, unexpectedly, DC120 activated mammalian target of rapamycin complex 1 (mTORC1) pathway that was suggested by increased phosphorylation of 70KD ribosomal protein S6 kinase (P70S6K) and eukaryotic translation initiation factor 4E binding protein 1 (4E-BP1). The activated mTORC1 signal was because of increase of intracellular Ca(2+) via Ca(2+)/calmodulin (CaM)/ signaling to human vacuolar protein sorting 34 (hVps34) upon AKT inhibition. Meanwhile, DC120 attenuated the inhibitory effect of AKT on CRAF by decreasing phosphorylation of CRAF at Ser259 and thus activated the mitogen-activated protein kinase (MAPK) pathway. The activation of the mTORC1 and MAPK pathways by DC120 was not mutually dependent, and the combination of DC120 with mTORC1 inhibitor and/or MEK inhibitor induced significant apoptosis and growth inhibition both in vitro and in vivo. Taken together, the combination of AKT, mTORC1 and/or MEK inhibitors would be a promising therapeutic strategy for liver cancer treatment. PMID:24625973

  12. MicroRNA-21 Regulates PI3K/Akt/mTOR Signaling by Targeting TGFβI during Skeletal Muscle Development in Pigs

    PubMed Central

    Yang, Yalan; Hou, Xinhua; Wang, Zishuai; Zhu, Shiyun; Wang, Chuduan; Tang, Zhonglin; Li, Kui

    2015-01-01

    MicroRNAs (miRNAs), which are short (22–24 base pairs), non-coding RNAs, play critical roles in myogenesis. Using Solexa deep sequencing, we detected the expression levels of 229 and 209 miRNAs in swine skeletal muscle at 90 days post-coitus (E90) and 100 days postnatal (D100), respectively. A total of 138 miRNAs were up-regulated on E90, and 31 were up-regulated on D100. Of these, 9 miRNAs were selected for the validation of the small RNA libraries by quantitative RT-PCR (RT-qPCR). We found that miRNA-21 was down-regulated by 17-fold on D100 (P<0.001). Bioinformatics analysis suggested that the transforming growth factor beta-induced (TGFβI) gene was a potential target of miRNA-21. Both dual luciferase reporter assays and western blotting demonstrated that the TGFβI gene was regulated by miRNA-21. Co-expression analysis revealed that the mRNA expression levels of miRNA-21 and TGFβI were negatively correlated (r = -0.421, P = 0.026) in skeletal muscle during the 28 developmental stages. Our results revealed that more miRNAs are expressed in prenatal than in postnatal skeletal muscle. The miRNA-21 is a novel myogenic miRNA that is involved in skeletal muscle development and regulates PI3K/Akt/mTOR signaling by targeting the TGFβI gene. PMID:25950587

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

    PubMed

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

    2009-07-15

    Resistance exercise induces a hypertrophic response in skeletal muscle and recent studies have begun to shed light on the molecular mechanisms involved in this process. For example, several studies indicate that signalling by the mammalian target of rapamycin (mTOR) is necessary for a hypertrophic response. Furthermore, resistance exercise has been proposed to activate mTOR signalling through an upstream pathway involving the phosphoinositide 3-kinase (PI3K) and protein kinase B (PKB); however, this hypothesis has not been thoroughly tested. To test this hypothesis, we first evaluated the temporal pattern of signalling through PI3K-PKB and mTOR following a bout of resistance exercise with eccentric contractions (EC). Our results indicated that the activation of signalling through PI3K-PKB is a transient event (<15 min), while the activation of mTOR is sustained for a long duration (>12 h). Furthermore, inhibition of PI3K-PKB activity did not prevent the activation of mTOR signalling by ECs, indicating that PI3K-PKB is not part of the upstream regulatory pathway. These observations led us to investigate an alternative pathway for the activation of mTOR signalling involving the synthesis of phosphatidic acid (PA) by phospholipase D (PLD). Our results demonstrate that ECs induce a sustained elevation in [PA] and inhibiting the synthesis of PA by PLD prevented the activation of mTOR. Furthermore, we determined that similar to ECs, PA activates mTOR signalling through a PI3K-PKB-independent mechanism. Combined, the results of this study indicate that the activation of mTOR following eccentric contractions occurs through a PI3K-PKB-independent mechanism that requires PLD and PA. PMID:19470781

  14. Olanzapine Activates Hepatic Mammalian Target of Rapamycin: New Mechanistic Insight into Metabolic Dysregulation with Atypical Antipsychotic Drugs

    PubMed Central

    Schmidt, Robin H.; Jokinen, Jenny D.; Massey, Veronica L.; Falkner, K. Cameron; Shi, Xue; Yin, Xinmin; Zhang, Xiang; Beier, Juliane I.

    2013-01-01

    Olanzapine (OLZ), an effective treatment of schizophrenia and other disorders, causes weight gain and metabolic syndrome. Most studies to date have focused on the potential effects of OLZ on the central nervous system’s mediation of weight; however, peripheral changes in liver or other key metabolic organs may also play a role in the systemic effects of OLZ. Thus, the purpose of this study was to investigate the effects of OLZ on hepatic metabolism in a mouse model of OLZ exposure. Female C57Bl/6J mice were administered OLZ (8 mg/kg per day) or vehicle subcutaneously by osmotic minipumps for 28 days. Liver and plasma were taken at sacrifice for biochemical analyses and for comprehensive two-dimensional gas chromatography coupled to time-of-flight mass spectrometry metabolomics analysis. OLZ increased body weight, fat pad mass, and liver-to-body weight ratio without commensurate increase in food consumption, indicating that OLZ altered energy expenditure. Expression and biochemical analyses indicated that OLZ induced anaerobic glycolysis and caused a pseudo-fasted state, which depleted hepatic glycogen reserves. OLZ caused similar effects in cultured HepG2 cells, as determined by Seahorse analysis. Metabolomic analysis indicated that OLZ increased hepatic concentrations of amino acids that can alter metabolism via the mTOR pathway; indeed, hepatic mTOR signaling was robustly increased by OLZ. Interestingly, OLZ concomitantly activated AMP-activated protein kinase (AMPK) signaling. Taken together, these data suggest that disturbances in glucose and lipid metabolism caused by OLZ in liver may be mediated, at least in part, via simultaneous activation of both catabolic (AMPK) and anabolic (mammalian target of rapamycin) pathways, which yields new insight into the metabolic side effects of this drug. PMID:23926289

  15. An Emerging Role for the Mammalian Target of Rapamycin in “Pathological” Protein Translation: Relevance to Cocaine Addiction

    PubMed Central

    Dayas, Christopher V.; Smith, Doug W.; Dunkley, Peter R.

    2011-01-01

    Complex neuroadaptations within key nodes of the brain’s “reward circuitry” are thought to underpin long-term vulnerability to relapse. A more comprehensive understanding of the molecular and cellular signaling events that subserve relapse vulnerability may lead to pharmacological treatments that could improve treatment outcomes for psychostimulant-addicted individuals. Recent advances in this regard include findings that drug-induced perturbations to neurotrophin, metabotropic glutamate receptor, and dopamine receptor signaling pathways perpetuate plasticity impairments at excitatory glutamatergic synapses on ventral tegmental area and nucleus accumbens neurons. In the context of addiction, much previous work, in terms of downstream effectors to these receptor systems, has centered on the extracellular-regulated MAP kinase signaling pathway. The purpose of the present review is to highlight the evidence of an emerging role for another downstream effector of these addiction-relevant receptor systems – the mammalian target of rapamycin complex 1 (mTORC1). mTORC1 functions to regulate synaptic protein translation and is a potential critical link in our understanding of the neurobiological processes that drive addiction and relapse behavior. The precise cellular and molecular changes that are regulated by mTORC1 and contribute to relapse vulnerability are only just coming to light. Therefore, we aim to highlight evidence that mTORC1 signaling may be dysregulated by drug exposure and that these changes may contribute to aberrant translation of synaptic proteins that appear critical to increased relapse vulnerability, including AMPARs. The importance of understanding the role of this signaling pathway in the development of addiction vulnerability is underscored by the fact that the mTORC1 inhibitor rapamycin reduces drug-seeking in pre-clinical models and preliminary evidence indicating that rapamycin suppresses drug craving in humans. PMID:22347189

  16. Profiling the role of mammalian target of rapamycin in the vascular smooth muscle metabolome in pulmonary arterial hypertension

    PubMed Central

    Kudryashova, Tatiana V.; Goncharov, Dmitry A.; Pena, Andressa; Ihida-Stansbury, Kaori; DeLisser, Horace; Kawut, Steven M.

    2015-01-01

    Abstract Increased proliferation and resistance to apoptosis of pulmonary arterial vascular smooth muscle cells (PAVSMCs), coupled with metabolic reprogramming, are key components of pulmonary vascular remodeling, a major and currently irreversible pathophysiological feature of pulmonary arterial hypertension (PAH). We recently reported that activation of mammalian target of rapamycin (mTOR) plays a key role in increased energy generation and maintenance of the proliferative, apoptosis-resistant PAVSMC phenotype in human PAH, but the downstream effects of mTOR activation on PAH PAVSMC metabolism are not clear. Using liquid and gas chromatography–based mass spectrometry, we performed pilot metabolomic profiling of human microvascular PAVSMCs from idiopathic-PAH subjects before and after treatment with the selective adenosine triphosphate–competitive mTOR inhibitor PP242 and from nondiseased lungs. We have shown that PAH PAVSMCs have a distinct metabolomic signature of altered metabolites—components of fatty acid synthesis, deficiency of sugars, amino sugars, and nucleotide sugars—intermediates of protein and lipid glycosylation, and downregulation of key biochemicals involved in glutathione and nicotinamide adenine dinucleotide (NAD) metabolism. We also report that mTOR inhibition attenuated or reversed the majority of the PAH-specific abnormalities in lipogenesis, glycosylation, glutathione, and NAD metabolism without affecting altered polyunsaturated fatty acid metabolism. Collectively, our data demonstrate a critical role of mTOR in major PAH PAVSMC metabolic abnormalities and suggest the existence of de novo lipid synthesis in PAVSMCs in human PAH that may represent a new, important component of disease pathogenesis worthy of future investigation. PMID:26697174

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

    SciTech Connect

    Ha, Jung Min; Yun, Sung Ji; Kim, Young Whan; Jin, Seo Yeon; Lee, Hye Sun; Song, Sang Heon; Shin, Hwa Kyoung; Bae, Sun Sik

    2015-08-14

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

  18. Leptin induces macrophage lipid body formation by a phosphatidylinositol 3-kinase- and mammalian target of rapamycin-dependent mechanism.

    PubMed

    Maya-Monteiro, Clarissa M; Almeida, Patricia E; D'Avila, Heloisa; Martins, Aline S; Rezende, Ana Paula; Castro-Faria-Neto, Hugo; Bozza, Patricia T

    2008-01-25

    Leptin is an adipocyte-derived hormone/cytokine that links nutritional status with neuroendocrine and immune functions. Lipid bodies (lipid droplets) are emerging as dynamic organelles with roles in lipid metabolism and inflammation. Here we investigated the roles of leptin in signaling pathways involved in cytoplasmic lipid body biogenesis and leukotriene B(4) synthesis in macrophages. Our results demonstrated that leptin directly activated macrophages and induced the formation of adipose differentiation-related protein-enriched lipid bodies. Newly formed lipid bodies were sites of 5-lipoxygenase localization and correlated with an enhanced capacity of leukotriene B(4) production. We demonstrated that leptin-induced macrophage activation was dependent on phosphatidylinositol 3-kinase (PI3K) activity, since the lipid body formation was inhibited by LY294002 and was absent in the PI3K knock-out mice. Leptin induces phosphorylation of p70(S6K) and 4EBP1 key downstream signaling intermediates of the mammalian target of rapamycin (mTOR) pathway in a rapamycin-sensitive mechanism. The mTOR inhibitor, rapamycin, inhibited leptin-induced lipid body formation, both in vivo and in vitro. In addition, rapamycin inhibited leptin-induced adipose differentiation-related protein accumulation in macrophages and lipid body-dependent leukotriene synthesis, demonstrating a key role for mTOR in lipid body biogenesis and function. Our results establish PI3K/mTOR as an important signaling pathway for leptin-induced cytoplasmic lipid body biogenesis and adipose differentiation-related protein accumulation. Furthermore, we demonstrate a previously unrecognized link between intracellular (mTOR) and systemic (leptin) nutrient sensors in macrophage lipid metabolism. Leptin-induced increased formation of cytoplasmic lipid bodies and enhanced inflammatory mediator production in macrophages may have implications for obesity-related cardiovascular diseases. PMID:18039669

  19. Anticancer effect of celastrol on human triple negative breast cancer: possible involvement of oxidative stress, mitochondrial dysfunction, apoptosis and PI3K/Akt pathways.

    PubMed

    Shrivastava, Shweta; Jeengar, Manish Kumar; Reddy, V Sudhakar; Reddy, G Bhanuprakash; Naidu, V G M

    2015-06-01

    Signaling via the phosphatidylinositol-3 kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) is crucial for divergent physiological processes including transcription, translation, cell-cycle progression and apoptosis. The aim of work was to elucidate the anti-cancer effect of celastrol and the signal transduction pathways involved. Cytotoxic effect of celastrol was assessed by MTT assay on human triple negative breast cancer cells (TNBCs) and compared with that of MCF-7. Apoptosis induction was determined by AO/EtBr staining, mitochondrial membrane potential by JC-1, Annexin binding assays and modulation of apoptotic proteins and its effect on PI3K/Akt/mTOR pathway by western blotting. Celastrol induced apoptosis in TNBC cells, were supported by DNA fragmentation, caspase-3 activation and PARP cleavage. Meanwhile, celastrol triggered reactive oxygen species production with collapse of mitochondrial membrane potential, down-regulation of Bcl-2 and up-regulation of Bax expression. Celastrol effectively decreased PI3K 110α/85α enzyme activity, phosphorylation of Akt(ser473) and p70S6K1 and 4E-BP1. Although insulin treatment increased the phosphorylation of Akt(ser473), p70S6K1, 4E-BP1, celastrol abolished the insulin mediated phosphorylation. It clearly indicates that celastrol acts through PI3k/Akt/mTOR axis. We also found that celastrol inhibited the Akt/GSK3β and Akt/NFkB survival pathway. PI3K/Akt/mTOR inhibitor, PF-04691502 and mTOR inhibitor rapamycin enhanced the apoptosis-inducing effect of celastrol. These data demonstrated that celastrol induces apoptosis in TNBC cells and indicated that apoptosis might be mediated through mitochondrial dysfunction and PI3K/Akt signaling pathway. PMID:25818165

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

    PubMed

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

    2016-01-01

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

  1. Overexpression of microRNA-30a-5p inhibits liver cancer cell proliferation and induces apoptosis by targeting MTDH/PTEN/AKT pathway.

    PubMed

    Li, Wen-Fang; Dai, Hang; Ou, Qin; Zuo, Guo-Qing; Liu, Chang-An

    2016-05-01

    Increasing results suggest microRNAs (miRNAs) could function important roles in malignant tumor progression. miR-30a-5p is downregulated in variety of cancers and acts as a cancer suppressing gene. The functions and molecular mechanisms of miRNA-30a-5p in hepatocellular carcinoma (HCC) remain unclear. In the present study, quantitative real-time PCR (qRT-PCR) was used to detect miR-30a-5p expression in 16 pairs of HCC and their adjacent non-cancerous tissues and HCC cell lines. By overexpression of miRNA-30a-5p, CCK-8 and colon formation assay were used to evaluate cell growth and flow cytometry to evaluate cell apoptosis. Western blot was used to test protein expression. And potential mechanisms were analyzed with luciferase activity assay. In vivo HepG2 tumor growth was observed with nude mice. Our results showed that miR-30a-5p expression in HCC tissues was significantly lower compared to adjacent non-cancerous liver tissues, and lower miR-30a-5p expression was also observed in HCC cell lines compared to normal liver cell. Luciferase assay showed that metadherin (MTDH) mRNA was a direct target of miR-30a-5p. A significant reverse correlation between miR-30a-5p and MTDH in liver cancer tissues was observed. miR-30a-5p overexpression in HCC cells significantly inhibited cell proliferation, colon formation, and induced apoptosis while MTDH overexpression reversed growth inhibition and apoptosis induction of miRNA-30a-5p in HCC cells. miRNA-30a-5p upregulated phosphatase and tensin homolog (PTEN) protein expression and thus inhibited AKT activating by targeting MTDH. miRNA-30a-5p also significantly inhibited HepG2 tumor growth in vivo. Our results suggest that underexpression of miR-30a-5p might function as a tumor suppressing miRNA by directly targeting MTDH in HCC and is therefore a potential candidate biomarker for HCC targeting therapy. PMID:26589417

  2. A KSHV microRNA Directly Targets G Protein-Coupled Receptor Kinase 2 to Promote the Migration and Invasion of Endothelial Cells by Inducing CXCR2 and Activating AKT Signaling

    PubMed Central

    Bai, Zhiqiang; Qin, Di; Yan, Qin; Zhu, Jianzhong; Krueger, Brian J.; Renne, Rolf; Gao, Shou-Jiang; Lu, Chun

    2015-01-01

    Kaposi's sarcoma (KS) is a highly disseminated angiogenic tumor of endothelial cells linked to infection by Kaposi's sarcoma-associated herpesvirus (KSHV). KSHV encodes more than two dozens of miRNAs but their roles in KSHV-induced tumor dissemination and metastasis remain unknown. Here, we found that ectopic expression of miR-K12-3 (miR-K3) promoted endothelial cell migration and invasion. Bioinformatics and luciferase reporter analyses showed that miR-K3 directly targeted G protein-coupled receptor (GPCR) kinase 2 (GRK2, official gene symbol ADRBK1). Importantly, overexpression of GRK2 reversed miR-K3 induction of cell migration and invasion. Furthermore, the chemokine receptor CXCR2, which was negatively regulated by GRK2, was upregulated in miR-K3-transduced endothelial cells. Knock down of CXCR2 abolished miR-K3-induced cell migration and invasion. Moreover, miR-K3 downregulation of GRK2 relieved its direct inhibitory effect on AKT. Both CXCR2 induction and the release of AKT from GRK2 were required for miR-K3 maximum activation of AKT and induction of cell migration and invasion. Finally, deletion of miR-K3 from the KSHV genome abrogated its effect on the GRK2/CXCR2/AKT pathway and KSHV-induced migration and invasion. Our data provide the first-line evidence that, by repressing GRK2, miR-K3 facilitates cell migration and invasion via activation of CXCR2/AKT signaling, which likely contribute to the dissemination of KSHV-induced tumors. PMID:26402907

  3. Mechanistic target of rapamycin (mTOR) signaling genes in decapod crustaceans: cloning and tissue expression of mTOR, Akt, Rheb, and p70 S6 kinase in the green crab, Carcinus maenas, and blackback land crab, Gecarcinus lateralis.

    PubMed

    Abuhagr, Ali M; Maclea, Kyle S; Chang, Ernest S; Mykles, Donald L

    2014-02-01

    Mechanistic target of rapamycin (mTOR) controls global translation of mRNA into protein by phosphorylating p70 S6 kinase (S6K) and eIF4E-binding protein-1. Akt and Rheb, a GTP-binding protein, regulate mTOR protein kinase activity. Molting in crustaceans is regulated by ecdysteroids synthesized by a pair of molting glands, or Y-organs (YOs), located in the cephalothorax. During premolt, the YOs hypertrophy and increase production of ecdysteroids. Rapamycin (1μM) inhibited ecdysteroid secretion in Carcinus maenas and Gecarcinus lateralis YOs in vitro, indicating that ecdysteroidogenesis requires mTOR-dependent protein synthesis. The effects of molting on the expression of four key mTOR signaling genes (mTOR, Akt, Rheb, and S6K) in the YO was investigated. Partial cDNAs encoding green crab (C. maenas) mTOR (4031bp), Akt (855bp), and S6K (918bp) were obtained from expressed sequence tags. Identity/similarity of the deduced amino acid sequence of the C. maenas cDNAs to human orthologs were 72%/81% for Cm-mTOR, 58%/73% for Cm-Akt, and 77%/88% for Cm-S6K. mTOR, Akt, S6K, and elongation factor 2 (EF2) in C. maenas and blackback land crab (G. lateralis) were expressed in all tissues examined. The two species differed in the effects of molting on gene expression in the YO. In G. lateralis, Gl-mTOR, Gl-Akt, and Gl-EF2 mRNA levels were increased during premolt. By contrast, molting had no effect on the expression of Cm-mTOR, Cm-Akt, Cm-S6K, Cm-Rheb, and Cm-EF2. These data suggest that YO activation during premolt involves up regulation of mTOR signaling genes in G. lateralis, but is not required in C. maenas. PMID:24269559

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

    PubMed Central

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

    2016-01-01

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

  5. An AKT3-FOXG1-reelin network underlies defective migration in human focal malformations of cortical development.

    PubMed

    Baek, Seung Tae; Copeland, Brett; Yun, Eun-Jin; Kwon, Seok-Kyu; Guemez-Gamboa, Alicia; Schaffer, Ashleigh E; Kim, Sangwoo; Kang, Hoon-Chul; Song, Saera; Mathern, Gary W; Gleeson, Joseph G

    2015-12-01

    Focal malformations of cortical development (FMCDs) account for the majority of drug-resistant pediatric epilepsy. Postzygotic somatic mutations activating the phosphatidylinositol-4,5-bisphosphate-3-kinase (PI3K)-protein kinase B (AKT)-mammalian target of rapamycin (mTOR) pathway are found in a wide range of brain diseases, including FMCDs. It remains unclear how a mutation in a small fraction of cells disrupts the architecture of the entire hemisphere. Within human FMCD-affected brain, we found that cells showing activation of the PI3K-AKT-mTOR pathway were enriched for the AKT3(E17K) mutation. Introducing the FMCD-causing mutation into mouse brain resulted in electrographic seizures and impaired hemispheric architecture. Mutation-expressing neural progenitors showed misexpression of reelin, which led to a non-cell autonomous migration defect in neighboring cells, due at least in part to derepression of reelin transcription in a manner dependent on the forkhead box (FOX) transcription factor FOXG1. Treatments aimed at either blocking downstream AKT signaling or inactivating reelin restored migration. These findings suggest a central AKT-FOXG1-reelin signaling pathway in FMCD and support pathway inhibitors as potential treatments or therapies for some forms of focal epilepsy. PMID:26523971

  6. Hesperidin Inhibits Vascular Formation by Blocking the AKT/mTOR Signaling Pathways.

    PubMed

    Kim, Gi Dae

    2015-12-01

    Hesperidin has been shown to possess a potential inhibitory effect on vascular formation in endothelial cells. However, the fundamental mechanism for the anti-angiogenic activity of hesperidin is not fully understood. In the present study, we evaluated whether hesperidin has anti-angiogenic effects in mouse embryonic stem cell (mES)-derived endothelial-like cells, and human umbilical vascular endothelial cells (HUVECs), and evaluated their mechanism via the AKT/mammalian target of rapamycin (mTOR) signaling pathway. The endothelial cells were treated with several doses of hesperidin (12.5, 25, 50, and 100 μM) for 24 h. Cell viability and vascular formation were analyzed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and tube formation assay, respectively. Alteration of the AKT/mTOR signaling in vascular formation was analyzed by western blot. In addition, a mouse aortic ring assay was used to determine the effect of hesperidin on vascular formation. There were no differences between the viability of mES-derived endothelial-like cells and HUVECs after hesperidin treatment. However, hesperidin significantly inhibited cell migration and tube formation of HUVECs (P<0.05) and suppressed sprouting of microvessels in the mouse aortic ring assay. Moreover, hesperidin suppressed the expression of AKT and mTOR in HUVECs. Taken together, these findings suggest that hesperidin inhibits vascular formation by blocking the AKT/mTOR signaling pathways. PMID:26770908

  7. Sirt1 decreased adipose inflammation by interacting with Akt2 and inhibiting mTOR/S6K1 pathway in mice.

    PubMed

    Liu, Zhenjiang; Gan, Lu; Liu, Guannv; Chen, Yizhe; Wu, Tianjiao; Feng, Fei; Sun, Chao

    2016-08-01

    Sirtuin type 1 (Sirt1) and protein kinase B (Akt2) are associated with development of obesity and inflammation, but the molecular mechanisms of Sirt1 and Akt2 interaction on adipose inflammation remain unclear. To explore these mechanisms, a mouse model was used. Mice were fed with a high-fat diet (HFD) for 8 weeks, with interventions of resveratrol (RES) or nicotinamide (NAM) during the last 15 days. The HFD reduced Sirt1 mRNA in adipose tissue and elevated interleukin-6 (IL-6) expression. RES reduced the adipose tissue weight, increased the Sirt1 mRNA level, and reduced both mRNA and protein levels of IL-6, MCP-1, inducible nitric oxide synthase, and TNF-α by inhibiting phosphorylation of Akt2 in adipose tissue. Additionally, macrophage type I marker genes were reduced while macrophage type II marker genes were elevated by RES addition. Moreover, activation of Akt2 signal by using insulin significantly blunted the inhibitory effect of RES on adipose inflammation. Immunoprecipitation assay demonstrated that RES enhances the protein-protein interaction between Sirt1 and Akt2, but NAM inhibits this interaction. Furthermore, Sirt1 significantly reduced the levels of raptor and inactivated mammalian target of rapamycin (mTOR)C1 signal by interacting with Akt2, and confirmed that RES attenuated adipose inflammation by inhibiting the mTOR/S6K1 pathway via rapamycin. PMID:27317762

  8. GSK3β, But Not GSK3α, Inhibits the Neuronal Differentiation of Neural Progenitor Cells As a Downstream Target of Mammalian Target of Rapamycin Complex1

    PubMed Central

    Ahn, Jyhyun; Jang, Jiwon; Choi, Jinyong; Lee, Junsub; Oh, Seo-Ho; Lee, Junghun; Yoon, Keejung

    2014-01-01

    Glycogen synthase kinase 3 (GSK3) acts as an important regulator during the proliferation and differentiation of neural progenitor cells (NPCs), but the roles of the isoforms of this molecule (GSK3α and GSK3β) have not been clearly defined. In this study, we investigated the functions of GSK3α and GSK3β in the context of neuronal differentiation of murine NPCs. Treatment of primary NPCs with a GSK3 inhibitor (SB216763) resulted in an increase in the percentage of TuJ1-positive immature neurons, suggesting an inhibitory role of GSK3 in embryonic neurogenesis. Downregulation of GSK3β expression increased the percentage of TuJ1-positive cells, while knock-down of GSK3α seemed to have no effect. When primary NPCs were engineered to stably express either isoform of GSK3 using retroviral vectors, GSK3β, but not GSK3α, inhibited neuronal differentiation and helped the cells to maintain the characteristics of NPCs. Mutant GSK3β (Y216F) failed to suppress neuronal differentiation, indicating that the kinase activity of GSK3β is important for this regulatory function. Similar results were obtained in vivo when a retroviral vector expressing GSK3β was delivered to E9.5 mouse brains using the ultrasound image-guided gene delivery technique. In addition, SB216763 was found to block the rapamycin-mediated inhibition of neuronal differentiation of NPCs. Taken together, our results demonstrate that GSK3β, but not GSK3α, negatively controls the neuronal differentiation of progenitor cells and that GSK3β may act downstream of the mammalian target of rapamycin complex1 signaling pathway. PMID:24397546

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

    PubMed Central

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

    2015-01-01

    Muscle invasive bladder cancer (MIBC, stage ≥T2) is generally associated with poor prognosis, constituting the second most common cause of death among genitourinary tumours. Due to high molecular heterogeneity significant variations in the natural history and disease outcome have been observed. This has also delayed the introduction of personalized therapeutics, making advanced stage bladder cancer almost an orphan disease in terms of treatment. Altered protein glycosylation translated by the expression of the sialyl-Tn antigen (STn) and its precursor Tn as well as the activation of the PI3K/Akt/mTOR pathway are cancer-associated events that may hold potential for patient stratification and guided therapy. Therefore, a retrospective design, 96 bladder tumours of different stages (Ta, T1-T4) was screened for STn and phosphorylated forms of Akt (pAkt), mTOR (pmTOR), S6 (pS6) and PTEN, related with the activation of the PI3K/Akt/mTOR pathway. In our series the expression of Tn was residual and was not linked to stage or outcome, while STn was statically higher in MIBC when compared to non-muscle invasive tumours (p = 0.001) and associated decreased cancer-specific survival (log rank p = 0.024). Conversely, PI3K/Akt/mTOR pathway intermediates showed an equal distribution between non-muscle invasive bladder cancer (NMIBC) and MIBC and did not associate with cancer-specif survival (CSS) in any of these groups. However, the overexpression of pAKT, pmTOR and/or pS6 allowed discriminating STn-positive advanced stage bladder tumours facing worst CSS (p = 0.027). Furthermore, multivariate Cox regression analysis revealed that overexpression of PI3K/Akt/mTOR pathway proteins in STn+ MIBC was independently associated with approximately 6-fold risk of death by cancer (p = 0.039). Mice bearing advanced stage chemically-induced bladder tumours mimicking the histological and molecular nature of human tumours were then administrated with mTOR-pathway inhibitor sirolimus (rapamycin

  10. Synergistic targeting of PI3K/AKT pathway and androgen receptor axis significantly delays castration-resistant prostate cancer progression in vivo.

    PubMed

    Thomas, Christian; Lamoureux, Francois; Crafter, Claire; Davies, Barry R; Beraldi, Eliana; Fazli, Ladan; Kim, Soojin; Thaper, Daksh; Gleave, Martin E; Zoubeidi, Amina

    2013-11-01

    The progression to castration-resistant prostate cancer (CRPC) correlates with gain-of-function of the androgen receptor (AR) and activation of AKT. However, as single agents, AR or AKT inhibitors result in a reciprocal feedback loop. Therefore, we hypothesized that combination of an AKT inhibitor with an antiandrogen might result in a more profound, long-lasting remission of CRPC. Here, we report that the AKT inhibitor AZD5363 potently inhibits proliferation and induces apoptosis in prostate cancer cell lines expressing the AR and has anticancer activity in vivo in androgen-sensitive and castration-resistant phases of the LNCaP xenograft model. However, we found that the effect of castration-resistant tumor growth inhibition and prostate-specific antigen (PSA) stabilization is transient and resistance occurs with increasing PSA after approximately 30 days of treatment. Mechanistically, we found that single agent AZD5363 induces increase of AR binding to androgen response element, AR transcriptional activity, and AR-dependent genes such as PSA and NKX3.1 expression. These effects were overcome by the combination of AZD5363 with the antiandrogen bicalutamide, resulting in synergistic inhibition of cell proliferation and induction of apoptosis in vitro, and prolongation of tumor growth inhibition and PSA stabilization in CRPC in vivo. This study provides a preclinical proof-of-concept that combination of an AKT inhibitor with antiandrogen results in prolonged disease stabilization in a model of CRPC. PMID:23966621

  11. Proliferation, survival and metabolism: the role of PI3K/AKT/mTOR signalling in pluripotency and cell fate determination.

    PubMed

    Yu, Jason S L; Cui, Wei

    2016-09-01

    Phosphatidylinositide 3 kinases (PI3Ks) and their downstream mediators AKT and mammalian target of rapamycin (mTOR) constitute the core components of the PI3K/AKT/mTOR signalling cascade, regulating cell proliferation, survival and metabolism. Although these functions are well-defined in the context of tumorigenesis, recent studies - in particular those using pluripotent stem cells - have highlighted the importance of this pathway to development and cellular differentiation. Here, we review the recent in vitro and in vivo evidence for the role PI3K/AKT/mTOR signalling plays in the control of pluripotency and differentiation, with a particular focus on the molecular mechanisms underlying these functions. PMID:27578176

  12. Power of PTEN/AKT: Molecular switch between tumor suppressors and oncogenes

    PubMed Central

    XIE, YINGQIU; NAIZABEKOV, SANZHAR; CHEN, ZHANLIN; TOKAY, TURSONJAN

    2016-01-01

    An increasing amount of evidence has shown that tumor suppressors can become oncogenes, or vice versa, but the mechanism behind this is unclear. Recent findings have suggested that phosphatase and tensin homolog (PTEN) is one of the powerful switches for the conversion between tumor suppressors and oncogenes. PTEN regulates a number of cellular processes, including cell death and proliferation, through the phosphoinositide 3-kinase/protein kinase B/mammalian target of rapamycin (PI3K/AKT/mTOR) pathway. Furthermore, a number of studies have suggested that PTEN deletions may alter various functions of certain tumor suppressor and oncogenic proteins. The aim of the present review was to analyze specific cases driven by PTEN loss/AKT activation, including aberrant signaling pathways and novel drug targets for clinical application in personalized medicine. The findings illustrate how PTEN loss and/or AKT activation switches MDM2-dependent p53 downregulation, and induces conversion between oncogene and tumor suppressor in enhancer of zeste homolog 2, BTB domain-containing 7A, alternative reading frame 2, p27 and breast cancer 1, early onset, through multiple mechanisms. This review highlights the genetic basis of complex drug targets and provides insights into the rationale of precision cancer therapy. PMID:27347153

  13. Mammalian target of rapamycin (mTOR) inhibition reduces cerebral vasospasm following a subarachnoid hemorrhage injury in canines.

    PubMed

    Zhang, Weiguang; Khatibi, Nikan H; Yamaguchi-Okada, Mitsuo; Yan, Junhao; Chen, Chunhua; Hu, Qin; Meng, Haiwei; Han, Hongbin; Liu, Shuwei; Zhou, Changman

    2012-02-01

    Mammalian target of rapamycin (mTOR) pathway is a serine/threonine protein kinase that plays a vital role in regulating growth, proliferation, survival, and protein synthesis among cells. In the present study, we investigated the role of the mTOR pathway following subarachnoid hemorrhage brain injury--specifically investigating its ability to mediate the activation of cerebral vasospasm. Additionally, we investigated whether key signaling pathway molecules such as the mTOR, P70S6K1, and 4E-BP1 play a role in the process. Thirty dogs were randomly divided into 5 groups: sham, SAH (subarachnoid hemorrhage), SAH+DMSO (dimethyl sulfoxide), SAH+Rapamycin and SAH+AZD8055. An established canine double-hemorrhage model of SAH was used by injecting autologous arterial blood into the cisterna magna on days 0 and 2. Angiography was performed at days 0 and 7. Clinical behavior, histology, immunohistochemistry, and Western blot of mTOR, P70S6K1, 4E-BP1 and PCNA (proliferating cell nuclear antigen) in the basilar arteries were examined. In the SAH and SAH+DMSO groups, severe angiographic vasospasm was obtained (34.3±19.8%, 38.4±10.3) compared with that in Sham (93.9±5.0%) respectively. mTOR, P70S6K1, 4E-BP1 and PCNA increased in the sample of spastic basilar arteries (p<0.05). In the SAH+RAPA and SAH+AZD8055 groups, Rapamycin and AZD8055 attenuated angiographic vasospasm (62.3±15.9% and 65.2±10.3%) while improving appetite and activity scores (p<0.05) on days 5 through 7. Rapamycin and AZD8055 significantly reduced the level and expression of mTOR, P70S6K1, 4E-BP1 and PCNA (p<0.05). In conclusion, our study suggests that the mTOR molecular signaling pathway plays a significant role in cerebral vasospasm following SAH, and that inhibition of the mTOR pathway has the potential to become an attractive strategy to treat vasospasm following SAH. PMID:22177999

  14. Requirement of Mammalian Target of Rapamycin Complex 1 Downstream Effectors in Cued Fear Memory Reconsolidation and Its Persistence

    PubMed Central

    Huynh, Thu N.; Santini, Emanuela

    2014-01-01

    Memory retrieval, often termed reconsolidation, can render previously consolidated memories susceptible to manipulation that can lead to alterations in memory strength. Although it is known that reconsolidation requires mammalian target of rapamycin complex 1 (mTORC1)-dependent translation, the specific contributions of its downstream effectors in reconsolidation are unclear. Using auditory fear conditioning in mice, we investigated the role of eukaryotic translation initiation factor 4E (eIF4E)–eIF4G interactions and p70 S6 kinase polypeptide 1 (S6K1) in reconsolidation. We found that neither 4EGI-1 (2-[(4-(3,4-dichlorophenyl)-thiazol-2-ylhydrazono)-3-(2-nitrophenyl)]propionic acid), an inhibitor of eFI4E–eIF4G interactions, nor PF-4708671 [2-((4-(5-ethylpyrimidin-4-yl)piperazin-1-yl)methyl)-5-(trifluoromethyl)-1H-benzo[d]imidazole], an inhibitor of S6K1, alone blocked the reconsolidation of auditory fear memory. In contrast, using these drugs in concert to simultaneously block eIF4E–eIF4G interactions and S6K1 immediately after memory reactivation significantly attenuated fear memory reconsolidation. Moreover, the combination of 4EGI-1 and PF-4708671 further destabilized fear memory 10 d after memory reactivation, which was consistent with experiments using rapamycin, an mTORC1 inhibitor. Furthermore, inhibition of S6K1 immediately after retrieval resulted in memory destabilization 10 d after reactivation, whereas inhibition of eIF4E–eIF4G interactions did not. These results indicate that the reconsolidation of fear memory requires concomitant association of eIF4E to eIF4G as well as S6K1 activity and that the persistence of memory at longer intervals after memory reactivation also requires mTORC1-dependent processes that involve S6K1. These findings suggest a potential mechanism for how mTORC1-dependent translation is fine tuned to alter memory persistence. PMID:24990923

  15. Targeting of an interrupted polypurine:polypyrimidine sequence in mammalian cells by a triplex-forming oligonucleotide containing a novel base analogue.

    PubMed

    Semenyuk, A; Darian, E; Liu, J; Majumdar, A; Cuenoud, B; Miller, P S; Mackerell, A D; Seidman, M M

    2010-09-14

    The DNA triple helix consists of a third strand of nucleic acid lying in the major groove of an intact DNA duplex. The most stable triplexes form on polypurine:polypyrimidine sequences, and pyrimidine interruptions in the purine strand are destabilizing. Sequence stringency is imparted by specific Hoogsteen hydrogen bonds between third strand bases and the purine bases in the duplex. Appropriate base and sugar modifications of triple helix-forming oligonucleotides (TFOs) confer chromosome targeting activity in living cells. However, broad utilization of TFOs as gene targeting reagents in mammalian cells has been limited by the requirement for homopurine target sequences. Although there have been a number of base analogues described that appear to be promising as candidates for triplex target expansion, none has been examined in a biological system. We have employed a postsynthetic strategy to prepare a collection of TFOs with base analogues at a defined position. Following assessment of affinity for a triplex target with a single C:G inversion, TFOs with a second generation of analogues were synthesized. One of these, TFO-5a, with 2'-OMe-guanidinylethyl-5-methylcytosine at the position corresponding to the C:G interruption in the target sequence, was further modified to confer bioactivity. The activity of this TFO, linked to psoralen, was measured in a mammalian cell line that was engineered by directed sequence conversion to carry a triplex target with a single C:G interruption. TFO-5a was active against this target and inactive against the corresponding target with an uninterrupted polypurine:polypyrimidine sequence. PMID:20701359

  16. Targeting of an Interrupted Polypurine:Polypyrimidine Sequence in Mammalian Cells by a Triplex-Forming Oligonucleotide Containing a Novel Base Analogue†

    PubMed Central

    Semenyuk, A.; Darian, E.; Liu, J.; Majumdar, A.; Cuenoud, B.; Miller, P. S.; MacKerell, A. D.; Seidman, M. M.

    2010-01-01

    The DNA triple helix consists of a third strand of nucleic acid lying in the major groove of an intact DNA duplex. The most stable triplexes form on polypurine:polypyrimidine sequences, and pyrimidine interruptions in the purine strand are destabilizing. Sequence stringency is imparted by specific Hoogsteen hydrogen bonds between third strand bases and the purine bases in the duplex. Appropriate base and sugar modifications of triple helix-forming oligonucleotides (TFOs) confer chromosome targeting activity in living cells. However, broad utilization of TFOs as gene targeting reagents in mammalian cells has been limited by the requirement for homopurine target sequences. Although there have been a number of base analogues described that appear to be promising as candidates for triplex target expansion, none has been examined in a biological system. We have employed a postsynthetic strategy to prepare a collection of TFOs with base analogues at a defined position. Following assessment of affinity for a triplex target with a single C:G inversion, TFOs with a second generation of analogues were synthesized. One of these, TFO-5a, with 2′-OMeguanidinylethyl-5-methylcytosine at the position corresponding to the C:G interruption in the target sequence, was further modified to confer bioactivity. The activity of this TFO, linked to psoralen, was measured in a mammalian cell line that was engineered by directed sequence conversion to carry a triplex target with a single C:G interruption. TFO-5a was active against this target and inactive against the corresponding target with an uninterrupted polypurine:polypyrimidine sequence. PMID:20701359

  17. Combined inhibition of AKT/mTOR and MDM2 enhances Glioblastoma Multiforme cell apoptosis and differentiation of cancer stem cells

    PubMed Central

    Daniele, Simona; Costa, Barbara; Zappelli, Elisa; Da Pozzo, Eleonora; Sestito, Simona; Nesi, Giulia; Campiglia, Pietro; Marinelli, Luciana; Novellino, Ettore; Rapposelli, Simona; Martini, Claudia

    2015-01-01

    The poor prognosis of Glioblastoma Multiforme (GBM) is due to a high resistance to conventional treatments and to the presence of a subpopulation of glioma stem cells (GSCs). Combination therapies targeting survival/self-renewal signals of GBM and GSCs are emerging as useful tools to improve GBM treatment. In this context, the hyperactivated AKT/mammalian target of the rapamycin (AKT/mTOR) and the inhibited wild-type p53 appear to be good candidates. Herein, the interaction between these pathways was investigated, using the novel AKT/mTOR inhibitor FC85 and ISA27, which re-activates p53 functionality by blocking its endogenous inhibitor murine double minute 2 homologue (MDM2). In GBM cells, FC85 efficiently inhibited AKT/mTOR signalling and reactivated p53 functionality, triggering cellular apoptosis. The combined therapy with ISA27 produced a synergic effect on the inhibition of cell viability and on the reactivation of p53 pathway. Most importantly, FC85 and ISA27 blocked proliferation and promoted the differentiation of GSCs. The simultaneous use of these compounds significantly enhanced GSC differentiation/apoptosis. These findings suggest that FC85 actively enhances the downstream p53 signalling and that a combination strategy aimed at inhibiting the AKT/mTOR pathway and re-activating p53 signalling is potentially effective in GBM and in GSCs. PMID:25898313

  18. Vitamin E succinate induces apoptosis via the PI3K/AKT signaling pathways in EC109 esophageal cancer cells

    PubMed Central

    Yang, Peng; Zhao, Jiaying; Hou, Liying; Yang, Lei; Wu, Kun; Zhang, Linyou

    2016-01-01

    Esophageal cancer is the fourth most common gastrointestinal cancer, it generally has a poor prognosis and novel strategies are required for prevention and treatment. Vitamin E succinate (VES) is a potential chemical agent for cancer prevention and therapy as it exerts anti-tumor effects in a variety of cancers. However, the role of VES in tumorigenesis and progression of cancer remains to be elucidated. The present study aimed to determine the effects of VES in regulating the survival and apoptosis of human esophageal cancer cells. EC109 human esophageal cancer cells were used to investigate the anti-proliferative effects of VES. The MTT and Annexin V-fluorescein isothiocyanate/propidium iodide assays demonstrated that VES inhibited cell proliferation and induced apoptosis in esophageal cancer cells. Furthermore, VES downregulated constitutively active basal levels of phosphorylated (p)-serine-threonine kinase AKT (AKT) and p-mammalian target of rapamycin (mTOR), and decreased the phosphorylation of AKT substrates Bcl-2-associated death receptor and caspase-9, in addition to mTOR effectors, ribosomal protein S6 kinase β1 and eIF4E-binding protein 1. Phosphoinositide-3-kinase (PI3K) inhibitor, LY294002 suppressed p-AKT and p-mTOR, indicating PI3K is a common upstream mediator. The apoptosis induced by VES was increased by inhibition of AKT or mTOR with their respective inhibitor in esophageal cancer cells. The results of the present study suggested that VES targeted the PI3K/AKT signaling pathways and induced apoptosis in esophageal cancer cells. Furthermore, the current study suggests that VES may be useful in a combinational therapeutic strategy employing an mTOR inhibitor. PMID:27357907

  19. Sophoraflavanone G from Sophora alopecuroides inhibits lipopolysaccharide-induced inflammation in RAW264.7 cells by targeting PI3K/Akt, JAK/STAT and Nrf2/HO-1 pathways.

    PubMed

    Guo, Chao; Yang, Lei; Luo, Jun; Zhang, Chao; Xia, Yuanzheng; Ma, Ting; Kong, Lingyi

    2016-09-01

    Sophoraflavanone G (SG), a prenylated flavonoid from Sophora alopecuroides, has been reported to have many pharmacological activities including anti-inflammation. However, the molecular mechanisms of its anti-inflammatory activity remain largely unclear. In this study we investigated the effects and the underlying molecular mechanisms of SG on lipopolysaccharide (LPS)-induced inflammation in RAW264.7 cells. Pretreatment with SG inhibited LPS-induced production of nitric oxide (NO) and prostaglandin E2 (PGE2) through reducing the expressions of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2). SG also decreased the expressions of pro-inflammatory cytokines, tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and interleukin-1β (IL-1β), both in the protein and gene levels. Further experiments demonstrated that SG downregulated the LPS-induced upregulation of phosphorylated phosphoinositide-3-kinase and Akt (PI3K/Akt). SG also attenuated the expression of phosphorylated Janus kinase signal transducer and activator of transcription (JAK/STAT). In addition, SG upregulated heme oxygenase-1 (HO-1) expression via nuclear translocation of nuclear factor E2-related factor 2 (Nrf2). Taken together, SG may act as a natural agent to treat some inflammatory diseases by targeting PI3K/Akt, JAK/STAT and Nrf2/HO-1 pathways. PMID:27351825

  20. Genetic deletion and pharmacological inhibition of Akt1 isoform attenuates bladder cancer cell proliferation, motility and invasion.

    PubMed

    Sabbineni, Harika; Alwhaibi, Abdulrahman; Goc, Anna; Gao, Fei; Pruitt, Alanna; Somanath, Payaningal R

    2015-10-01

    Isoform specific expression, intracellular localization and function of Akt in bladder cancer are not known. In the current study, we identified Akt1, followed by Akt2 and Akt3 as the predominant Akt isoform in human T24 and UM-UC-3 metastatic bladder cancer cells. Whereas Akt1 is localized at the membrane, cytoplasm and nucleus, Akt2 is solely cytoplasmic and Akt3 is mostly localized in the nucleus in T24 cells. ShRNA-mediated Akt1 knockdown resulted in impaired T24 cell survival, proliferation, colony formation, migration and microinvasion. Whereas pharmacological inhibition of Akt1 resulted in impaired T24 and UM-UC-3 cell motility, viability and proliferation, effect of pharmacological inhibition by Akt2 inhibitor was limited to proliferation in T24, but not UM-UC-3 cells. Our data provide important clues on the therapeutic benefits of targeting Akt1 for bladder cancer therapy. PMID:26148825

  1. The role of amino acid-induced mammalian target of rapamycin complex 1(mTORC1) signaling in insulin resistance

    PubMed Central

    Yoon, Mee-Sup; Choi, Cheol Soo

    2016-01-01

    Mammalian target of rapamycin (mTOR) controls cell growth and metabolism in response to nutrients, energy, and growth factors. Recent findings have placed the lysosome at the core of mTOR complex 1 (mTORC1) regulation by amino acids. Two parallel pathways, Rag GTPase-Ragulator and Vps34-phospholipase D1 (PLD1), regulate mTOR activation on the lysosome. This review describes the recent advances in understanding amino acid-induced mTOR signaling with a particular focus on the role of mTOR in insulin resistance. PMID:27534530

  2. The role of amino acid-induced mammalian target of rapamycin complex 1(mTORC1) signaling in insulin resistance.

    PubMed

    Yoon, Mee-Sup; Choi, Cheol Soo

    2016-01-01

    Mammalian target of rapamycin (mTOR) controls cell growth and metabolism in response to nutrients, energy, and growth factors. Recent findings have placed the lysosome at the core of mTOR complex 1 (mTORC1) regulation by amino acids. Two parallel pathways, Rag GTPase-Ragulator and Vps34-phospholipase D1 (PLD1), regulate mTOR activation on the lysosome. This review describes the recent advances in understanding amino acid-induced mTOR signaling with a particular focus on the role of mTOR in insulin resistance. PMID:27534530

  3. Mammalian Target of Rapamycin Inhibitor Induced Complete Remission of a Recurrent Subependymal Giant Cell Astrocytoma in a Patient Without Features of Tuberous Sclerosis Complex.

    PubMed

    Appalla, Deepika; Depalma, Andres; Calderwood, Stanley

    2016-07-01

    The majority of patients with subependymal giant cell astrocytoma (SEGA) have tuberous sclerosis complex (TSC). In such patients, the mammalian target of rapamycin (mTOR) inhibitor everolimus has been shown to induce responses. Isolated SEGA have been reported in patients without clinical or genetic features of TSC. The treatment of these patients with everolimus has not previously been reported. We treated a patient with a recurrent isolated SEGA with an mTOR inhibitor. The patient tolerated therapy well and had a sustained complete remission. MTOR inhibitors may be useful for the treatment of isolated SEGA. Further study is warranted. PMID:26929034

  4. Akt phosphorylation is essential for nuclear translocation and retention in NGF-stimulated PC12 cells

    SciTech Connect

    Truong Le Xuan Nguyen; Choi, Joung Woo; Lee, Sang Bae; Ye, Keqiang; Woo, Soo-Dong; Lee, Kyung-Hoon; Ahn, Jee-Yin . E-mail: jyahn@med.skku.ac.kr

    2006-10-20

    Nerve growth factor (NGF) elicits Akt translocation into the nucleus, where it phosphorylates nuclear targets. Here, we describe that Akt phosphorylation can promote the nuclear translocation of Akt and is necessary for its nuclear retention. Overexpression of Akt-K179A, T308A, S473A-mutant failed to show either nuclear translocation or nuclear Akt phosphorylation, whereas expression of wild-type counterpart elicited profound Akt phosphorylation and induced nuclear translocation under NGF stimulation. Employing the PI3K inhibitor and a variety of mutants PI3K, we showed that nuclear translocation of Akt was mediated by activation of PI3K, and Akt phosphorylation status in the nucleus required PI3K activity. Thus the activity of PI3K might contribute to the nuclear translocation of Akt, and that Akt phosphorylation is essential for its nuclear retention under NGF stimulation conditions.

  5. Repression of AKT signaling by ARQ 092 in cells and tissues from patients with Proteus syndrome

    PubMed Central

    Lindhurst, Marjorie J.; Yourick, Miranda R.; Yu, Yi; Savage, Ronald E.; Ferrari, Dora; Biesecker, Leslie G.

    2015-01-01

    A somatic activating mutation in AKT1, c.49G>A, pGlu17Lys, that results in elevated AKT signaling in mutation-positive cells, is responsible for the mosaic overgrowth condition, Proteus syndrome. ARQ 092 is an allosteric pan-AKT inhibitor under development for treatment in cancer. We tested the efficacy of this drug for suppressing AKT signaling in cells and tissues from patients with Proteus syndrome. ARQ 092 reduced phosphorylation of AKT and downstream targets of AKT in a concentration-dependent manner in as little as two hours. While AKT signaling was suppressed with ARQ 092 treatment, cells retained their ability to respond to growth factor stimulation by increasing pAKT levels proportionally to untreated cells. At concentrations sufficient to decrease AKT signaling, little reduction in cell viability was seen. These results indicate that ARQ 092 can suppress AKT signaling and warrants further development as a therapeutic option for patients with Proteus syndrome. PMID:26657992

  6. p53-dependent inhibition of mammalian cell survival by a genetically selected peptide aptamer that targets the regulatory subunit of protein kinase CK2.

    PubMed

    Martel, V; Filhol, O; Colas, P; Cochet, C

    2006-11-30

    Based on the perturbation of its expression in human cancers and on its involvement in transformation and tumorigenesis, protein kinase CK2 has recently attracted attention as a potential therapeutic target. To assess the value of CK2 as a target for antiproliferative strategies, we have initiated a program aiming to develop inhibitors targeting specifically the regulatory CK2beta subunit. Here, we use a two-hybrid approach to isolate from combinatorial libraries, peptide aptamers that specifically interact with CK2beta. One of these (P1), which has significant sequence homology to the cytomegalovirus IE2 protein, binds with high affinity to the N-terminal domain of CK2beta without disrupting the formation of the CK2 holoenzyme. Expression of green fluorescent protein (GFP)-P1 in different mammalian cell lines activates p53 phosphorylation on serine 15, induces an upregulation of p21 and the release of the Cyt-C and apoptosis-inducing factor proapoptotic proteins triggering caspase-dependent and caspase-independent apoptosis. GFP-P1-induced apoptosis is associated with a p53-dependent pathway as cell death was abrogated in p53 knocked out cells. In summary, our data show that genetically selected peptide aptamers that specifically target CK2beta can induce apoptosis in mammalian cells through the recruitment of a p53-dependent apoptosis pathway. They also emphasize the critical role of CK2beta for cell survival and might allow the design of novel proapoptotic agents targeting this protein. PMID:16751801

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

    PubMed Central

    Xiao, Jun; Lin, Huan-Yi; Zhu, Yuan-Yuan; Zhu, Yu-Ping; Chen, Ling-Wu

    2016-01-01

    Objective To assess whether microRNA-126 (miR-126) targets phosphatidylinositol 3-kinase regulatory subunit beta (PIK3R2) and to determine the potential roles of miR-126 in regulating proliferation and invasion via the PIK3R2-mediated phosphatidylinositol 3 kinase (PI3K)-protein kinase B (Akt) signaling pathway in the human bladder BLS cell line. Materials and methods A recombinant lentivirus (Lv) vector expressing miR-216 (Lv-miR-126) was successfully constructed, and Lv-miR-126 and Lv vector were transfected into the BLS cell line. A direct regulatory relationship between miR-126 and the PIK3R2 gene was demonstrated by luciferase reporter assays. To determine whether PIK3R2 directly participates in the miR-126-induced effects in BLS cells, anti-miR-126 and a PIK3R2 small interfering RNA (siRNA) were transfected into the BLS cells. Quantitative real-time polymerase chain reaction was used to measure miR-126 and PIK3R2 expressions. 5-Ethynyl-2′-deoxyuridine and colony formation assays to assess cell proliferation, flow cytometry for cell apoptosis and cell cycle analysis, Transwell assays for cell migration and invasion, and Western blots for PIK3R2, PI3K, phosphorylated PI3K (p-PI3K), Akt, and phosphorylated Akt (p-Akt) protein expressions were performed. Results Lv-miR-126 significantly enhanced the relative expression of miR-126 in the BLS cells after infection (P<0.0001). MiR-126 overexpression inhibited the proliferation, cloning, migration, and invasion of BLS cells, promoted cell apoptosis, and induced S phase arrest (all P<0.05). PIK3R2, p-PI3K, and p-Akt protein expressions were significantly decreased in the BLS cells infected with Lv-miR-126. Luciferase assays showed that miR-126 significantly inhibited the PIK3R2 3′ untranslated region (3′UTR) luciferase reporter activity (P<0.05). The anti-miR-126 + PIK3R2 siRNA group had significantly decreased PIK3R2, p-PI3K, and p-Akt expressions compared with those of anti-miR-126 alone, as well as

  8. Mammalian target of rapamycin complex (mTOR) pathway modulates blood-testis barrier (BTB) function through F-actin organization and gap junction.

    PubMed

    Li, Nan; Cheng, C Yan

    2016-09-01

    mTOR (mammalian target of rapamycin) is one of the most important signaling molecules in mammalian cells which regulates an array of cellular events, ranging from cell metabolism to cell proliferation. Based on the association of mTOR with the core component proteins, such as Raptor or Rictor, mTOR can become the mTORC1 (mammalian target of rapamycin complex 1) or mTORC2, respectively. Studies have shown that during the epithelial cycle of spermatogenesis, mTORC1 promotes remodeling and restructuring of the blood-testis barrier (BTB) in vitro and in vivo, making the Sertoli cell tight junction (TJ)-permeability barrier "leaky"; whereas mTORC2 promotes BTB integrity, making the Sertoli cell TJ-barrier "tighter". These contrasting effects, coupled with the spatiotemporal expression of the core signaling proteins at the BTB that confer the respective functions of mTORC1 vs. mTORC2 thus provide a unique mechanism to modulate BTB dynamics, allowing or disallowing the transport of biomolecules and also preleptotene spermatocytes across the immunological barrier. More importantly, studies have shown that these changes to BTB dynamics conferred by mTORC1 and mTORC2 are mediated by changes in the organization of the actin microfilament networks at the BTB, and involve gap junction (GJ) intercellular communication. Since GJ has recently been shown to be crucial to reboot spermatogenesis and meiosis following toxicant-induced aspermatogenesis, these findings thus provide new insightful information regarding the integration of mTOR and GJ to regulate spermatogenesis. PMID:26957088

  9. A Novel Piggybac Transposon Inducible Expression System Identifies a Role for Akt Signalling in Primordial Germ Cell Migration

    PubMed Central

    Glover, James D.; Taylor, Lorna; Sherman, Adrian; Zeiger-Poli, Caroline; Sang, Helen M.; McGrew, Michael J.

    2013-01-01

    In this work, we describe a single piggyBac transposon system containing both a tet-activator and a doxycycline-inducible expression cassette. We demonstrate that a gene product can be conditionally expressed from the integrated transposon and a second gene can be simultaneously targeted by a short hairpin RNA contained within the transposon, both in vivo and in mammalian and avian cell lines. We applied this system to stably modify chicken primordial germ cell (PGC) lines in vitro and induce a reporter gene at specific developmental stages after injection of the transposon-modified germ cells into chicken embryos. We used this vector to express a constitutively-active AKT molecule during PGC migration to the forming gonad. We found that PGC migration was retarded and cells could not colonise the forming gonad. Correct levels of AKT activation are thus essential for germ cell migration during early embryonic development. PMID:24223709

  10. Benzo[b]furan derivatives induces apoptosis by targeting the PI3K/Akt/mTOR signaling pathway in human breast cancer cells.

    PubMed

    Kamal, Ahmed; Lakshma Nayak, V; Nagesh, Narayana; Vishnuvardhan, M V P S; Subba Reddy, N V

    2016-06-01

    The PI3K/Akt/mTOR signaling pathway plays a key regulatory function in cell survival, proliferation, migration, metabolism and apoptosis. Aberrant activation of the PI3K/Akt/mTOR pathway is found in many types of cancer and thus plays a major role in breast cancer cell proliferation. In our previous studies, benzo[b]furan derivatives were evaluated for their anticancer activity and the lead compounds identified were 26 and 36. These observations prompted us to investigate the molecular mechanism and apoptotic pathway of these lead molecules against breast cancer cells. Benzo[b]furan derivatives (26 and 36) were evaluated for their antiproliferative activity against human breast cancer cell lines MCF-7 and MDA MB-231. These compounds (26 and 36) have shown potent efficiency against breast cancer cells (MCF-7) with IC50 values 0.057 and 0.051μM respectively. Cell cycle analysis revealed that these compounds induced cell cycle arrest at G2/M phase in MCF-7 cells. Western blot analysis revealed that these compounds inhibit the PI3K/Akt/mTOR signaling pathway and induced mitochondrial mediated apoptosis in human breast cancer cells (MCF-7). PMID:27149364

  11. Targeted Regulation of PI3K/Akt/mTOR/NF-κB Signaling by Indole Compounds and their Derivatives: Mechanistic Details and Biological Implications for Cancer Therapy

    PubMed Central

    Ahmad, Aamir; Biersack, Bernhard; Li, Yiwei; Kong, Dejuan; Bao, Bin; Schobert, Rainer; Padhye, Subhash B.; Sarkar, Fazlul H.

    2014-01-01

    Indole compounds, found in cruciferous vegetables, are potent anti-cancer agents. Studies with indole-3-carbinol (I3C) and its dimeric product, 3,3’-diindolylmethane (DIM) suggest that these compounds have the ability to deregulate multiple cellular signaling pathways, including PI3K/Akt/mTOR signaling pathway. These natural compounds are also effective modulators of downstream transcription factor NF-κB signaling which might help explain their ability to inhibit invasion and angiogenesis, and the reversal of epithelial-to-mesenchymal transition (EMT) phenotype and drug resistance. Signaling through PI3K/Akt/mTOR and NF-κB pathway is increasingly being realized to play important role in EMT through the regulation of novel miRNAs which further validates the importance of this signaling network and its regulations by indole compounds. Here we will review the available literature on the modulation of PI3K/Akt/mTOR/NF-κB signaling by both parental I3C and DIM, as well as their analogs/derivatives, in an attempt to catalog their anticancer activity. PMID:23272910

  12. miR-125b inhibits keratinocyte proliferation and promotes keratinocyte apoptosis in oral lichen planus by targeting MMP-2 expression through PI3K/Akt/mTOR pathway.

    PubMed

    Wang, Jing; Luo, Hong; Xiao, Yan; Wang, Luyao

    2016-05-01

    Oral lichen planus (OLP) is a chronic inflammatory mucosal disease that involves the degeneration of keratinocytes. However, the etiology and mechanisms of OLP pathogenesis have not been fully elucidated. In this study, we used keratinocytes HaCaT stimulated with lipopolysaccharide (LPS) to mimic a local OLP immune environment, and investigated the regulatory role of miR-125b in keratinocyte proliferation and apoptosis under OLP conditions. Immunohistochemical analysis and quantitative real-time PCR (qRT-PCR) assay showed that MMP-2 expression was up-regulated and miR-125b expression was down-regulated in both OLP mucosa tissues and LPS-incubated HaCaT cells. Western blot analysis indicated that miR-125b overexpression suppressed LPS-induced MMP-2 expression in HaCaT cells. Molecularly, our results confirmed that MMP-2 is a target gene of miR-125b in HaCaT cells. The effect of miR-125b on cell proliferation was revealed by CCK-8 assay, BrdU assay and cell cycle analysis, which illustrated that miR-125b overexpression impeded LPS-induced HaCaT cell proliferation. Flow cytometry analysis further demonstrated that miR-125b overexpression promoted HaCaT cell apoptosis. Moreover, these effects were involved in PI3K/Akt/mTOR activation, as miR-125b overexpression inhibited LPS-enhanced expression of p-Akt and p-mTOR proteins. Taken together, these data confirm that miR-125b might inhibit keratinocyte proliferation and promote keratinocyte apoptosis in OLP pathogenesis by targeting MMP-2 through PI3K/Akt/mTOR pathway. PMID:27133077

  13. α-santalol inhibits the angiogenesis and growth of human prostate tumor growth by targeting vascular endothelial growth factor receptor 2-mediated AKT/mTOR/P70S6K signaling pathway

    PubMed Central

    2013-01-01

    Background VEGF receptor 2 (VEGFR2) inhibitors, as efficient antiangiogenesis agents, have been applied in the cancer treatment. However, recently, most of these anticancer drugs have some adverse effects. Discovery of novel VEGFR2 inhibitors as anticancer drug candidates is still needed. Methods We used α-santalol and analyzed its inhibitory effects on human umbilical vein endothelial cells (HUVECs) and Prostate tumor cells (PC-3 or LNCaP) in vitro. Tumor xenografts in nude mice were used to examine the in vivo activity of α-santalol. Results α-santalol significantly inhibits HUVEC proliferation, migration, invasion, and tube formation. Western blot analysis indicated that α-santalol inhibited VEGF-induced phosphorylation of VEGFR2 kinase and the downstream protein kinases including AKT, ERK, FAK, Src, mTOR, and pS6K in HUVEC, PC-3 and LNCaP cells. α-santalol treatment inhibited ex vivo and in vivo angiogenesis as evident by rat aortic and sponge implant angiogenesis assay. α-santalol significantly reduced the volume and the weight of solid tumors in prostate xenograft mouse model. The antiangiogenic effect by CD31 immunohistochemical staining indicated that α-santalol inhibited tumorigenesis by targeting angiogenesis. Furthermore, α-santalol reduced the cell viability and induced apoptosis in PC-3 cells, which were correlated with the downregulation of AKT, mTOR and P70S6K expressions. Molecular docking simulation indicated that α-santalol form hydrogen bonds and aromatic interactions within the ATP-binding region of the VEGFR2 kinase unit. Conclusion α-santalol inhibits angiogenesis by targeting VEGFR2 regulated AKT/mTOR/P70S6K signaling pathway, and could be used as a potential drug candidate for cancer therapy. PMID:24261856

  14. Loss of the repressor REST in uterine fibroids promotes aberrant G protein-coupled receptor 10 expression and activates mammalian target of rapamycin pathway

    PubMed Central

    Varghese, Binny V.; Koohestani, Faezeh; McWilliams, Michelle; Colvin, Arlene; Gunewardena, Sumedha; Kinsey, William H.; Nowak, Romana A.; Nothnick, Warren B.; Chennathukuzhi, Vargheese M.

    2013-01-01

    Uterine fibroids (leiomyomas) are the most common tumors of the female reproductive tract, occurring in up to 77% of reproductive-aged women, yet molecular pathogenesis remains poorly understood. A role for atypically activated mammalian target of rapamycin (mTOR) pathway in the pathogenesis of uterine fibroids has been suggested in several studies. We identified that G protein-coupled receptor 10 [GPR10, a putative signaling protein upstream of the phosphoinositide 3-kinase–protein kinase B/AKT–mammalian target of rapamycin (PI3K/AKT–mTOR) pathway] is aberrantly expressed in uterine fibroids. The activation of GPR10 by its cognate ligand, prolactin releasing peptide, promotes PI3K–AKT–mTOR pathways and cell proliferation specifically in cultured primary leiomyoma cells. Additionally, we report that RE1 suppressing transcription factor/neuron-restrictive silencing factor (REST/NRSF), a known tumor suppressor, transcriptionally represses GPR10 in the normal myometrium, and that the loss of REST in fibroids permits GPR10 expression. Importantly, mice overexpressing human GPR10 in the myometrium develop myometrial hyperplasia with excessive extracellular matrix deposition, a hallmark of uterine fibroids. We demonstrate previously unrecognized roles for GPR10 and its upstream regulator REST in the pathogenesis of uterine fibroids. Importantly, we report a unique genetically modified mouse model for a gene that is misexpressed in uterine fibroids. PMID:23284171

  15. Degradation of Akt Using Protein Catalyzed Capture Agents

    PubMed Central

    Das, Samir; Nag, Arundhati; Tang, Grace; Tang, Kevin; Sutherland, Alexander M.; Heath, James R.

    2016-01-01

    Abnormal signaling of the protein kinase Akt has been shown to contribute to human diseases such as diabetes and cancer, but Akt has proven to be a challenging target for drugging. Using iterative in situ click chemistry we recently developed multiple protein catalyzed capture (PCC) agents that allosterically modulate Akt enzymatic activity in a protein based assay. Here we utilize similar PCCs to exploit endogenous protein degradation pathways. We use the modularity of the anti-Akt PCCs to prepare Proteolysis Targeting Chimeric molecules (PROTACs) that are shown to promote the rapid degradation of Akt in live cancer cells. These novel PROTACs demonstrate that the epitope targeting selectivity of PCCs can be coupled with non-traditional drugging moieties to inhibit challenging targets. PMID:26880702

  16. The Akt-mTOR pathway in Down's syndrome: the potential use of rapamycin/rapalogs for treating cognitive deficits.

    PubMed

    Troca-Marín, Jose Antonio; Casañas, Juan José; Benito, Itziar; Montesinos, María Luz

    2014-02-01

    An increasing amount of evidence suggests that the dysregulation of the Akt-mTOR (Akt-mammalian Target Of Rapamycin) signaling network is associated with intellectual disabilities, such as fragile X, tuberous sclerosis and Rett's syndrome. The Akt-mTOR pathway is involved in dendrite morphogenesis and synaptic plasticity, and it has been shown to modulate both glutamatergic and GABAergic synaptic transmission. We have recently shown that the AktmTOR pathway is hyperactive in the hippocampus of Ts1Cje mice, a model of Down's syndrome, leading to increased local dendritic translation that could interfere with synaptic plasticity. Rapamycin and rapalogs are specific inhibitors of mTOR, and some of these inhibitors are Food and Drug Administration-approved drugs. In this review, we discuss the molecular basis and consequences of Akt-mTOR hyperactivation in Down's syndrome, paying close attention to alterations in the molecular mechanisms underlying synaptic plasticity. We also analyze the pros and cons of using rapamycin/rapalogs for the treatment of the cognitive impairments associated with this condition. PMID:24152334

  17. Mammalian target of rapamycin complex 1 (mTORC1) Is required for mouse spermatogonial differentiation in vivo.

    PubMed

    Busada, Jonathan T; Niedenberger, Bryan A; Velte, Ellen K; Keiper, Brett D; Geyer, Christopher B

    2015-11-01

    Spermatogonial stem cells (SSCs) must balance self-renewal with production of transit-amplifying progenitors that differentiate in response to retinoic acid (RA) before entering meiosis. This self-renewal vs. differentiation spermatogonial fate decision is critical for maintaining tissue homeostasis, as imbalances cause spermatogenesis defects that can lead to human testicular cancer or infertility. A great deal of effort has been exerted to understand how the SSC population is maintained. In contrast, little is known about the essential program of differentiation initiated by retinoic acid (RA) that precedes meiosis, and the pathways and proteins involved are poorly defined. We recently reported a novel role for RA in stimulating the PI3/AKT/mTOR kinase signaling pathway to activate translation of repressed mRNAs such as Kit. Here, we examined the requirement for mTOR complex 1 (mTORC1) in mediating the RA signal to direct spermatogonial differentiation in the neonatal testis. We found that in vivo inhibition of mTORC1 by rapamycin blocked spermatogonial differentiation, which led to an accumulation of undifferentiated spermatogonia. In addition, rapamycin also blocked the RA-induced translational activation of mRNAs encoding KIT, SOHLH1, and SOHLH2 without affecting expression of STRA8. These findings highlight dual roles for RA in germ cell development - transcriptional activation of genes, and kinase signaling to stimulate translation of repressed messages required for spermatogonial differentiation. PMID:26254600

  18. Inhibiting expression of specific genes in mammalian cells with 5′ end-mutated U1 small nuclear RNAs targeted to terminal exons of pre-mRNA

    PubMed Central

    Fortes, Puri; Cuevas, Yolanda; Guan, Fei; Liu, Peng; Pentlicky, Sara; Jung, Stephen P.; Martínez-Chantar, Maria L.; Prieto, Jesús; Rowe, David; Gunderson, Samuel I.

    2003-01-01

    Reducing or eliminating expression of a given gene is likely to require multiple methods to ensure coverage of all of the genes in a given mammalian cell. We and others [Furth, P. A., Choe, W. T., Rex, J. H., Byrne, J. C., and Baker, C. C. (1994) Mol. Cell. Biol. 14, 5278–5289] have previously shown that U1 small nuclear (sn) RNA, both natural or with 5′ end mutations, can specifically inhibit reporter gene expression in mammalian cells. This inhibition occurs when the U1 snRNA 5′ end base pairs near the polyadenylation signal of the reporter gene's pre-mRNA. This base pairing inhibits poly(A) tail addition, a key, nearly universal step in mRNA biosynthesis, resulting in degradation of the mRNA. Here we demonstrate that expression of endogenous mammalian genes can be efficiently inhibited by transiently or stably expressed 5′ end-mutated U1 snRNA. Also, we determine the inhibitory mechanism and establish a set of rules to use this technique and to improve the efficiency of inhibition. Two U1 snRNAs base paired to a single pre-mRNA act synergistically, resulting in up to 700-fold inhibition of the expression of specific reporter genes and 25-fold inhibition of endogenous genes. Surprisingly, distance from the U1 snRNA binding site to the poly(A) signal is not critical for inhibition, instead the U1 snRNA must be targeted to the terminal exon of the pre-mRNA. This could reflect a disruption by the 5′ end-mutated U1 snRNA of the definition of the terminal exon as described by the exon definition model. PMID:12826613

  19. Direct inhibition of the signaling functions of the mammalian target of rapamycin by the phosphoinositide 3-kinase inhibitors, wortmannin and LY294002.

    PubMed Central

    Brunn, G J; Williams, J; Sabers, C; Wiederrecht, G; Lawrence, J C; Abraham, R T

    1996-01-01

    The immunosuppressant, rapamycin, inhibits cell growth by interfering with the function of a novel kinase, termed mammalian target of rapamycin (mTOR). The putative catalytic domain of mTOR is similar to those of mammalian and yeast phosphatidylinositol (PI) 3-kinases. This study demonstrates that mTOR is a component of a cytokine-triggered protein kinase cascade leading to the phosphorylation of the eukaryotic initiation factor-4E (eIF-4E) binding protein, PHAS-1, in activated T lymphocytes. This event promotes G1 phase progression by stimulating eIF-4E-dependent translation initiation. A mutant YAC-1 T lymphoma cell line, which was selected for resistance to the growth-inhibitory action of rapamycin, was correspondingly resistant to the suppressive effect of this drug on PHAS-1 phosphorylation. In contrast, the PI 3-kinase inhibitor, wortmannin, reduced the phosphorylation of PHAS-1 in both rapamycin-sensitive and -resistant T cells. At similar drug concentrations (0.1-1 microM), wortmannin irreversibly inhibited the serine-specific autokinase activity of mTOR. The autokinase activity of mTOR was also sensitive to the structurally distinct PI 3-kinase inhibitor, LY294002, at concentrations (1-30 microM) nearly identical to those required for inhibition of the lipid kinase activity of the mammalian p85-p110 heterodimer. These studies indicate that the signaling functions of mTOR, and potentially those of other high molecular weight PI 3-kinase homologs, are directly affected by cellular treatment with wortmannin or LY294002. Images PMID:8895571

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

    PubMed

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

    2014-10-15

    Chemotherapy resistance represents a major problem for the treatment of patients with breast cancer and greatly restricts the use of first-line chemotherapeutics paclitaxel. The purpose of this study was to investigate the role of transgelin 2 in human breast cancer paclitaxel resistance cell line (MCF-7/PTX) and the reversal mechanism of salvianolic acid A (SAA), a phenolic active compound extracted from Salvia miltiorrhiza. Western blotting and real-time quantitative polymerase chain reaction (qRT-PCR) indicated that transgelin 2 may mediate paclitaxel resistance by activating the phosphatidylinositol 3-kinase (PI3 K)/Akt signaling pathway to suppress MCF-7/PTX cells apoptosis. The reversal ability of SAA was confirmed by MTT assay and flow cytometry, with a superior 9.1-fold reversal index and enhancement of the apoptotic cytotoxicity induced by paclitaxel. In addition, SAA effectively prevented transgelin 2 and adenosine-triphosphate binding cassette transporter (ABC transporter) including P-glycoprotein (P-gp), multidrug resistance associated protein 1 (MRP1), and breast cancer resistance protein (BCRP) up-regulation and exhibited inhibitory effect on PI3 K/Akt signaling pathway in MCF-7/PTX cells. Taken together, SAA can reverse paclitaxel resistance through suppressing transgelin 2 expression by mechanisms involving attenuation of PI3 K/Akt pathway activation and ABC transporter up-regulation. These results not only provide insight into the potential application of SAA in reversing paclitaxel resistance, thus facilitating the sensitivity of breast cancer chemotherapy, but also highlight a potential role of transgelin 2 in the development of paclitaxel resistance in breast cancer. PMID:25442283

  1. The Epithelial-Mesenchymal Transition (EMT) Regulatory Factor SLUG (SNAI2) Is a Downstream Target of SPARC and AKT in Promoting Melanoma Cell Invasion

    PubMed Central

    Fenouille, Nina; Tichet, Mélanie; Dufies, Maeva; Pottier, Anaïs; Mogha, Ariane; Soo, Julia K.; Rocchi, Stéphane; Mallavialle, Aude; Galibert, Marie-Dominique; Khammari, Amir; Lacour, Jean-Philippe; Ballotti, Robert; Deckert, Marcel; Tartare-Deckert, Sophie

    2012-01-01

    During progression of melanoma, malignant melanocytes can be reprogrammed into mesenchymal-like cells through a process similar to epithelial-mesenchymal transition (EMT), which is associated with downregulation of the junctional protein E-cadherin and acquisition of a migratory phenotype. Recent evidence supports a role for SLUG, a transcriptional repressor of E-cadherin, as a melanocyte lineage transcription factor that predisposes to melanoma metastasis. However, the signals responsible for SLUG expression in melanoma are unclear and its role in the invasive phenotype is not fully elucidated. Here, we report that SLUG expression and activation is driven by SPARC (also known as osteonectin), a secreted extracellular matrix-associated factor that promotes EMT-like changes. Ectopic expression or knockdown of SPARC resulted in increased or reduced expression of SLUG, respectively. SLUG increase occurred concomitantly with SPARC-mediated downregulation of E-cadherin and P-cadherin, and induction of mesenchymal traits in human melanocytes and melanoma cells. Pharmacological blockade of PI3 kinase/AKT signaling impeded SPARC-induced SLUG levels and cell migration, whereas adenoviral introduction of constitutively active AKT allowed rescue of SLUG and migratory capabilities of SPARC knockdown cells. We also observed that pharmacological inhibition of oncogenic BRAFV600E using PLX4720 did not influence SLUG expression in melanoma cells harboring BRAFV600E. Furthermore, SLUG is a bona fide transcriptional repressor of E-cadherin as well as a regulator of P-cadherin in melanoma cells and its knockdown attenuated invasive behavior and blocked SPARC-enhanced cell migration. Notably, inhibition of cell migration in SPARC-depleted cells was rescued by expression of a SLUG transgene. In freshly isolated metastatic melanoma cells, a positive association between SPARC and SLUG mRNA levels was also found. These findings reveal that autocrine SPARC maintains heightened SLUG

  2. Anti-autophagic and anti-apoptotic effects of memantine in a SH-SY5Y cell model of Alzheimer's disease via mammalian target of rapamycin-dependent and -independent pathways

    PubMed Central

    SONG, GUIJUN; LI, YU; LIN, LULU; CAO, YUNPENG

    2015-01-01

    Memantine non-competitively blocks the N-methyl-d-aspartate receptor in order to inhibit beta-amyloid (Aβ) secretion, and has been used to treat moderate-to-severe Alzheimer's disease (AD). However, the mechanisms underlying the role of memantine in the autophagy and apoptosis of neuronal cells in AD, as well as the association between neuronal autophagy and apoptosis have yet to be elucidated. The present study aimed to establish an AD cell model overexpressing the 695-amino-acid Swedish mutant of Aβ precursor protein (APP695swe) in order to observe the effects of memantine on the cell viability, autophagy and apoptosis of SH-SY5Y cells in the AD model, and to investigate the associated underlying mechanisms. A pcDNA3.1-APP695 plasmid was transfected into the SH-SY5Y cells. Reverse transcription-quantitative polymerase chain reaction and western blot analyses demonstrated that the AD cell model was successfully established. MTT assays demonstrated that memantine was able to upregulate neuronal cell survival, and acridine orange staining and flow cytometry demonstrated that memantine (5 µM) was able to inhibit neuronal autophagy and apoptosis. Following neuronal autophagy induction by rapamycin, cell apoptosis rates increased significantly. Further experiments revealed that memantine was able to upregulate the expression of signaling molecules phosphorylated (p)-phosphoinositide 3-kinase, p-Akt and p-mammalian target of rapamycin (mTOR), and also inhibited the phosphorylation of the B-cell lymphoma 2/Beclin-1 complex via mitogen-activated protein kinase 8. In conclusion, the results of the present study demonstrated that in the AD cell model, autophagy was able to promote apoptosis. Memantine exerted anti-autophagic and anti-apoptotic functions, and mTOR-dependent as well as-independent autophagic signaling pathways were involved in this process. Therefore, these results of the present study strongly supported the use of memantine as a potential therapeutic

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

    PubMed

    Chen, Jiezhong

    2012-12-12

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

  4. c-Src activation promotes nasopharyngeal carcinoma metastasis by inducing the epithelial-mesenchymal transition via PI3K/Akt signaling pathway: a new and promising target for NPC.

    PubMed

    Ke, Liangru; Xiang, Yanqun; Guo, Xiang; Lu, Jinping; Xia, Weixiong; Yu, Yahui; Peng, Yongjian; Wang, Li; Wang, Gang; Ye, Yanfang; Yang, Jing; Liang, Hu; Kang, Tiebang; Lv, Xing

    2016-05-10

    Aberrant activation of cellular Src (c-Src), a non-receptor tyrosine kinase, could promote cancer progression through activating its downstream signaling pathways. However, the roles of c-Src and phosphorylated-Src (p-Src) in nasopharyngeal carcinoma (NPC) progression are rarely investigated. Herein, we have identified high c-Src concentrations in the serum of NPC patients with distant metastasis using high-throughput protein microarrays. Levels of c-Src in serum and p-Src in human primary NPC samples were unfavorable independent prognostic factors for cancer-specific survival, disease-free survival, and distant metastasis-free survival. Depletion or inactivation of c-Src in NPC cells using sgRNA with CRISPR/Cas9 system or PP2 decreased cell viability, colony formation, migration and invasion in vitro and metastasis in vivo. In contrast, these malignancies could be up-regulated by overexpressed c-Src in a NPC cell line with low-metastasis potential. Furthermore, p-Src was involved in promoting NPC cell metastasis by inducing the epithelial-mesenchymal transition (EMT) process via activating the PI3K/Akt pathway and cytoskeleton remodeling. The p-Src-induced EMT process could be retarded by PP2, which mediated by down-regulating the PI3K/Akt pathway. In conclusion, elevated levels of c-Src in serum and p-Src in primary NPC tissue correlated with poor outcomes of NPC patients. And aberrant activation of c-Src facilitated NPC cells with malignant potential, especially metastasis ability, which mediated by the PI3K/Akt pathway activation and sequentially induced the EMT process. These findings unveiled a promising approach for targeted therapy of advanced NPC. PMID:27078847

  5. Targeting AKT with the Pro-apoptotic Peptide, TAT-CTMP: a Novel Strategy for the Treatment of Human Pancreatic Adenocarcinoma

    PubMed Central

    Simon, Peter O.; McDunn, Jonathan E.; Kashiwagi, Hiroyuki; Chang, Katherine; Goedegebuure, Peter S.; Hotchkiss, Richard S.; Hawkins, William G.

    2009-01-01

    Pancreatic adenocarcinoma carries an ominous prognosis and has little effective treatment. Several studies have demonstrated that the potently anti-apoptotic phosphatidyl inositol 3’-kinase (PI3K) - protein kinase B/AKT pathway is active in pancreas cancer. A recent study identified an endogenous AKT antagonist, carboxyl terminal modular protein (CTMP). CTMP inhibits the phosphorylation of AKT, preventing full activation of the kinase. We screened several cell permeable peptides from the N-terminal domain of CTMP (termed TAT-CTMP1 - 4) in vitro and found one that caused significant apoptosis in pancreatic adenocarcinoma cell lines. An inactive variant of this peptide was synthesized and used as a negative control. In all cell lines tested, TAT-CTMP4 induced a dose-dependent increase in apoptosis as detected by %-TUNEL positive cells and %-active caspase-3 (% active caspase-3 ranged from 31.2 to 61.9 at the highest dose tested (10µM)). A screening of various cell and tissue types revealed that the pro-apoptotic activity was highest in pancreatic adenocarcinoma. TAT-CTMP induced similar levels of active caspase-3 as several other known inducers of apoptosis: gemcitabine, radiation therapy, wortmannin, and recombinant tumor necrosis factor (TNF)-α. No apoptosis was observed in donor human peripheral blood mononuclear cells (PBMC, P<0.01). We further showed that TAT-CTMP4 could augment either gemcitabine chemotherapy or radiation therapy, standard therapies for pancreas cancer. Pancreatic adenocarcinoma xenografts, treated with a single dose of TAT-CTMP4 demonstrated a marked increase in caspase-3 positive tumor cells when compared to untreated controls. Additionally, pancreatic adenocarcinoma allografts treated with intratumoral TAT-CTMP and systemic gemcitabine displayed a significantly smaller tumor burden while undergoing treatment than mice in control groups (P<0.001). These data indicate that inhibiting AKT with CTMP may be of therapeutic benefit in the

  6. Controlled delivery of β-globin-targeting TALENs and CRISPR/Cas9 into mammalian cells for genome editing using microinjection

    PubMed Central

    Cottle, Renee N.; Lee, Ciaran M.; Archer, David; Bao, Gang

    2015-01-01

    Tal-effector nucleases (TALEN) and clustered regularly interspaced short palindromic repeats (CRISPR) with CRISPR-associated (Cas) proteins are genome editing tools with unprecedented potential. However, the ability to deliver optimal amounts of these nucleases into mammalian cells with minimal toxicity poses a major challenge. Common delivery approaches are transfection- and viral-based methods; each associated with significant drawbacks. An alternative method for directly delivering genome-editing reagents into single living cells with high efficiency and controlled volume is microinjection. Here, we characterize a glass microcapillary-based injection system and demonstrate controlled co-injection of TALENs or CRISPR/Cas9 together with donor template into single K562 cells for targeting the human β-globin gene. We quantified nuclease induced insertions and deletions (indels) and found that, with β-globin-targeting TALENs, similar levels of on- and off-target activity in cells could be achieved by microinjection compared with nucleofection. Furthermore, we observed 11% and 2% homology directed repair in single K562 cells co-injected with a donor template along with CRISPR/Cas9 and TALENs respectively. These results demonstrate that a high level of targeted gene modification can be achieved in human cells using glass-needle microinjection of genome editing reagents. PMID:26558999

  7. Controlled delivery of β-globin-targeting TALENs and CRISPR/Cas9 into mammalian cells for genome editing using microinjection.

    PubMed

    Cottle, Renee N; Lee, Ciaran M; Archer, David; Bao, Gang

    2015-01-01

    Tal-effector nucleases (TALEN) and clustered regularly interspaced short palindromic repeats (CRISPR) with CRISPR-associated (Cas) proteins are genome editing tools with unprecedented potential. However, the ability to deliver optimal amounts of these nucleases into mammalian cells with minimal toxicity poses a major challenge. Common delivery approaches are transfection- and viral-based methods; each associated with significant drawbacks. An alternative method for directly delivering genome-editing reagents into single living cells with high efficiency and controlled volume is microinjection. Here, we characterize a glass microcapillary-based injection system and demonstrate controlled co-injection of TALENs or CRISPR/Cas9 together with donor template into single K562 cells for targeting the human β-globin gene. We quantified nuclease induced insertions and deletions (indels) and found that, with β-globin-targeting TALENs, similar levels of on- and off-target activity in cells could be achieved by microinjection compared with nucleofection. Furthermore, we observed 11% and 2% homology directed repair in single K562 cells co-injected with a donor template along with CRISPR/Cas9 and TALENs respectively. These results demonstrate that a high level of targeted gene modification can be achieved in human cells using glass-needle microinjection of genome editing reagents. PMID:26558999

  8. Gefitinib induces lung cancer cell autophagy and apoptosis via blockade of the PI3K/AKT/mTOR pathway

    PubMed Central

    ZHAO, ZHONG-QUAN; YU, ZHONG-YANG; LI, JIE; OUYANG, XUE-NONG

    2016-01-01

    Gefitinib is a selective inhibitor of the tyrosine kinase epidermal growth factor receptor, which inhibits tumor pathogenesis, metastasis and angiogenesis, as well as promoting apoptosis. Therefore, gefitinib presents an effective drug for the targeted therapy of lung cancer. However, the underlying mechanisms by which gefitinib induces lung cancer cell death remain unclear. To investigate the effects of gefitinib on lung cancer cells and the mechanism of such, the present study analyzed the effect of gefitinib on the autophagy, apoptosis and proliferation of the A549 and A549-gefitinib-resistant (GR) cell lines GR. The regulation of the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT)/ mammalian target of rapamycin (mTOR) pathway was also investigated. Acridine orange staining revealed that gefitinib induced autophagy of A549 cells but not A549-GR cells. In addition, gefitinib promoted apoptosis and inhibited proliferation of A549 cells but not A549-GR cells. Furthermore, western blot analysis demonstrated that gefitinib treatment led to the downregulation of PI3K, AKT, pAKT, mTOR and phosphorylated-mTOR protein expression in A549 cells but not A549-GR cells. LY294002 blocked the PI3K/AKT/mTOR pathway and induced autophagy and apoptosis of A549 cells, however, no synergistic effect was observed following combined treatment with gefitinib and LY294002. In conclusion, the results of the present study indicate that gefitinib promotes autophagy and apoptosis of lung cancer cells via blockade of the PI3K/AKT/mTOR pathway, which leads to lung cancer cell death. PMID:27347100

  9. Upregulation of microRNA-31 targeting integrin α5 suppresses tumor cell invasion and metastasis by indirectly regulating PI3K/AKT pathway in human gastric cancer SGC7901 cells.

    PubMed

    Zhang, Xue-Bin; Song, Lei; Wen, Hong-Juan; Bai, Xiao-Xue; Li, Zhen-Juan; Ma, Lian-Jun

    2016-06-01

    To verify the hypothesis that upregulation of microRNA-31 (miR-31) targeting integrin α5 (ITGA5) suppresses tumor cell invasion and metastasis by indirectly regulating phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT) signaling pathway in human SGC7901 gastric cancer (GC) cells. The miRTarBase was used to predict whether ITGA5 is the target gene of miR-31, which was further confirmed by luciferase reporter gene assay. The SGC7901 GC cells were divided into five groups including the blank, miR-31 mimic, miR-31 mimic control, miR-31 inhibitor, and miR-31 inhibitor control groups. Reverse transcriptase-polymerase chain reaction (RT-PCR), western blotting, cell scratch test, and transwell assays were respectively performed in our study. TGA5 was found as the target gene of miR-31. The RT-PCR detection revealed that, compared with the blank group, ITGA5 messenger RNA (mRNA) expression decreased in the miR-31 mimic group, but increased in the miR-31 inhibitor group. The western blotting examination suggested that the expressions of ITGA5, PI3K, and AKT proteins reduced in the miR-31 mimic group, but enhanced in the miR-31 inhibitor group when compared to the blank group, respectively. The cell scratch and transwell assays indicated that the miR-31 expressions were negatively associated with GC cell migration and invasion. Besides, RT-PCR combined with western blotting demonstrated that the miR-31 expressions were higher in the normal tissues than those in the GC tissues, while the ITGA5 mRNA and protein showed lower expression in the normal tissues than they did in the GC tissues. Our study concluded that upregulation of miR-31 targeting ITGA5 may suppress tumor cell invasion and metastasis by indirectly regulating PI3K/AKT signaling pathway in human SGC7901 GC cells. PMID:26729197

  10. Bufalin suppresses hepatocellular carcinoma invasion and metastasis by targeting HIF-1α via the PI3K/AKT/mTOR pathway

    PubMed Central

    Xu, Litao; Zang, Kun; Ning, Zhouyu; Jiang, Feng; Chi, Huiying; Zhu, Xiaoyan; Meng, Zhiqiang

    2016-01-01

    It has been reported that there are multiple mechanisms by which bufalin could exert its antimetastatic effect. HIF-1α has been reported to be involved in tumor migration and invasion by regulating EMT. However, it is not known whether bufalin could exert the antimetastatic effect by modulating HIF-1α expression in hepatocellular carcinoma. In the present study, we aimed to evaluate the antimetastatic potential of bufalin in vivo and in vitro. Our results demonstrated that the liver/lung metastases were significantly reduced in bufalin-treated mice, as tested in the orthotopic transplanted and tail vein injection tumor models. Furthermore, the epithelial-to-mesenchymal transition (EMT) was inhibited in bufalin-treated tumors, as reflected the upregulation of E-cadherin, and downregulation of N-cadherin, vimentin, Snail. Similar results were observed in SMMC7721 cells treated with bufalin. Moreover, the transforming growth factor-β1 (TGF-β1)-induced EMT was also abrogated by bufalin. Mechanistically, our study demonstrated that hypoxia-inducible factor-1α (HIF-1α) played an important role in the antimetastatic effect of bufalin in hepatocellular carcinoma. Importantly, HIF-1α expression may be regulated through the inhibition of the PI3K/AKT/mTOR pathway. Taken together, our results suggest that bufalin suppresses hepatic tumor invasion and metastasis and that this process may be related to the PI3K/AKT/mTOR/ HIF-1α axis. PMID:26958938

  11. Silibinin ameliorates steatosis and insulin resistance during non-alcoholic fatty liver disease development partly through targeting IRS-1/PI3K/Akt pathway.

    PubMed

    Zhang, Yongxiang; Hai, Jie; Cao, Meng; Zhang, Yongli; Pei, Sujuan; Wang, Junbo; Zhang, Qinggui

    2013-11-01

    Silibinin (SIL) is a well-studied hepato-protective agent against a spectrum of liver diseases. However, the role of SIL in non-alcoholic fatty liver disease (NAFLD) induced insulin resistance and underlying signaling is not fully characterized. In this study, Sprague-Dawley (SD) rats were fed with high-fat diet to develop NAFLD with or without an SIL co-treatment. NAFLD rats showed typical NAFLD symptoms including histological changes, insulin resistance, and glucose metabolism dysfunction. SIL co-treatment significantly ameliorated these pathological features partly through restoring the IRS-1/PI3K/Akt pathway. In addition, BRL-3A and HepG2 cells were incubated with palmitic acid (PA) to induce steatosis. SIL co-treatment in cells also reduced lipid accumulation, recovered cell viability, and down-regulated the protein expression of resistin, the marker for insulin resistance. Specific blocker of PI3K abolished the ameliorative effects of SIL on cellular steatosis. In conclusion, SIL alleviated steatosis and insulin resistance both in vivo and in vitro partly through regulating the IRS-1/PI3K/Akt pathway. PMID:24036369

  12. Non-canonical GLI1/2 activation by PI3K/AKT signaling in renal cell carcinoma: A novel potential therapeutic target.

    PubMed

    Zhou, Jiancheng; Zhu, Guodong; Huang, Jun; Li, Lei; Du, Yuefeng; Gao, Yang; Wu, Dapeng; Wang, Xinyang; Hsieh, Jer-Tsong; He, Dalin; Wu, Kaijie

    2016-01-28

    Renal cell carcinoma (RCC) is the most lethal urologic malignancy; however, the molecular events supporting RCC carcinogenesis and progression remain poorly understood. In this study, based on the analysis of gene expression profile data from human clear cell RCC (ccRCC) and the corresponding normal tissues, we discovered that Hedgehog (HH) pathway component genes GLI1 and GLI2 were significantly elevated in ccRCC. Survival analysis of a large cohort of ccRCC samples demonstrated that the expression of GLI1 and GLI2 was negatively correlated with patient overall survival. Clinical sample-based VHL mutation and cell model-based VHL manipulation studies all indicated that the activation of GLI1 and GLI2 was not affected by VHL status. Further signaling pathway dissections demonstrated that GLI1 and GLI2 were activated by the phosphoinositide 3-kinase (PI3K)/AKT pathway, but not mediated by the canonical HH/SMO/GLI signaling. Up-regulation of GLI1 and GLI2 promoted RCC proliferation and clonogenic ability, whereas, a combination of GLIs inhibitor Gant61 and AKT inhibitor Perifosine synergistically suppressed RCC growth and induced apoptosis in vitro and in vivo. Therefore, this study identifies that GLI1 and GLI2 are critical for RCC carcinogenesis, and also provides an alternative therapeutic strategy for RCC. PMID:26577809

  13. Muscle Wasting in Fasting Requires Activation of NF-κB and Inhibition of AKT/Mechanistic Target of Rapamycin (mTOR) by the Protein Acetylase, GCN5.

    PubMed

    Lee, Donghoon; Goldberg, Alfred L

    2015-12-18

    NF-κB is best known for its pro-inflammatory and anti-apoptotic actions, but in skeletal muscle, NF-κB activation is important for atrophy upon denervation or cancer. Here, we show that also upon fasting, NF-κB becomes activated in muscle and is critical for the subsequent atrophy. Following food deprivation, the expression and acetylation of the p65 of NF-κB on lysine 310 increase markedly in muscles. NF-κB inhibition in mouse muscles by overexpression of the IκBα superrepressor (IκBα-SR) or of p65 mutated at Lys-310 prevented atrophy. Knockdown of GCN5 with shRNA or a dominant-negative GCN5 or overexpression of SIRT1 decreased p65K310 acetylation and muscle wasting upon starvation. In addition to reducing atrogene expression, surprisingly inhibiting NF-κB with IκBα-SR or by GCN5 knockdown in these muscles also enhanced AKT and mechanistic target of rapamycin (mTOR) activities, which also contributed to the reduction in atrophy. These new roles of NF-κB and GCN5 in regulating muscle proteolysis and AKT/mTOR signaling suggest novel approaches to combat muscle wasting. PMID:26515065

  14. Mammalian Protein Arginine Methyltransferase 7 (PRMT7) Specifically Targets RXR Sites in Lysine- and Arginine-rich Regions*

    PubMed Central

    Feng, You; Maity, Ranjan; Whitelegge, Julian P.; Hadjikyriacou, Andrea; Li, Ziwei; Zurita-Lopez, Cecilia; Al-Hadid, Qais; Clark, Amander T.; Bedford, Mark T.; Masson, Jean-Yves; Clarke, Steven G.

    2013-01-01

    The mammalian protein arginine methyltransferase 7 (PRMT7) has been implicated in roles of transcriptional regulation, DNA damage repair, RNA splicing, cell differentiation, and metastasis. However, the type of reaction that it catalyzes and its substrate specificity remain controversial. In this study, we purified a recombinant mouse PRMT7 expressed in insect cells that demonstrates a robust methyltransferase activity. Using a variety of substrates, we demonstrate that the enzyme only catalyzes the formation of ω-monomethylarginine residues, and we confirm its activity as the prototype type III protein arginine methyltransferase. This enzyme is active on all recombinant human core histones, but histone H2B is a highly preferred substrate. Analysis of the specific methylation sites within intact histone H2B and within H2B and H4 peptides revealed novel post-translational modification sites and a unique specificity of PRMT7 for methylating arginine residues in lysine- and arginine-rich regions. We demonstrate that a prominent substrate recognition motif consists of a pair of arginine residues separated by one residue (RXR motif). These findings will significantly accelerate substrate profile analysis, biological function study, and inhibitor discovery for PRMT7. PMID:24247247

  15. Intracellular reactive oxygen species are essential for PI3K/Akt/mTOR-dependent IL-7-mediated viability of T-cell acute lymphoblastic leukemia cells.

    PubMed

    Silva, A; Gírio, A; Cebola, I; Santos, C I; Antunes, F; Barata, J T

    2011-06-01

    Interleukin-7 (IL-7) activates phosphoinositide 3-kinase/Akt/mammalian target of rapamycin (PI3K/Akt/mTOR) pathway, thereby mediating viability, proliferation and growth of T-cell acute lymphoblastic leukemia (T-ALL) cells. Reactive oxygen species (ROS) can be upregulated by growth factors and are known to regulate proliferation and viability. Here, we show that IL-7 upregulates ROS in T-ALL cells in a manner that is dependent on PI3K/Akt/mTOR pathway activity and that relies on both NADPH oxidase and mitochondrial respiratory chain. Conversely, IL-7-induced activation of PI3K signaling pathway requires mitochondrial respiration and ROS. We have previously shown that IL-7-mediated activation of PI3K pathway drives the upregulation of the glucose transporter Glut1, promoting glucose uptake in T-ALL cells. Using phloretin to inhibit Glut function, we demonstrate that glucose uptake is mandatory for ROS upregulation in IL-7-treated T-ALL cells, suggesting that IL-7 stimulation leads to increased ROS via PI3K pathway activation and consequent upregulation of Glut1 and glucose uptake. Overall, our data reveal the existence of a critical crosstalk between PI3K/Akt signaling pathway and ROS that is essential for IL-7-mediated T-ALL cell survival, and that may constitute a novel target for therapeutic intervention. PMID:21455214

  16. Mango polyphenolics suppressed tumor growth in breast cancer xenografts in mice: role of the PI3K/AKT pathway and associated microRNAs.

    PubMed

    Banerjee, Nivedita; Kim, Hyemee; Krenek, Kimberly; Talcott, Stephen T; Mertens-Talcott, Susanne U

    2015-08-01

    The cytotoxic and anti-inflammatory properties of mango polyphenolics including gallic acid and gallotannins have been demonstrated in numerous types of cancers. We hypothesized that the phosphoinositide 3-kinase (PI3K)/AKT pathway and the expression of related miRNAs are involved in the chemotherapeutic activities of mango polyphenolics in a mouse xenograft model for breast cancer. The objectives of this research were to determine the tumor-cytotoxic activities of mango polyphenolics and the underlying molecular mechanisms involving posttranscriptional targets in BT474 breast cancer cells and xenografts in mice. In vitro findings showed cytotoxic effects of mango polyphenolics in BT474 breast cancer cells within a concentration range of 2.5 to 20 mg/L gallic acid equivalents. Mango polyphenolics suppressed the expression of PI3K, AKT, hypoxia inducible factor-1α, and vascular endothelial growth factor (VEGF) mRNA, and pAKT, AKT, pPI3K (p85), VEGF and nuclear factor-kappa B protein levels. The involvement of miR-126 was verified by using antagomiR for miR-126, where mango reversed the effect of the antagomiR of miR-126. In vivo, the intake of mango polyphenolics decreased the tumor volume by 73% in BT474 xenograft-bearing mice compared with the control group. In addition, mango reduced the expression of nuclear factor-kappa B (p65), pAKT, pPI3K, mammalian target of rapamycin, hypoxia inducible factor-1α, and VEGF protein in athymic nude mice. A screening for miRNA expression changes confirmed that mango polyphenolics modulated the expression of cancer-associated miRNAs including miR-126 in the xenografted tumors. In summary, mango polyphenolics have a chemotherapeutic potential against breast cancer that at least in part is mediated through the PI3K/AKT pathway and miR-126. PMID:26194618

  17. Functional Proteomics Identifies Acinus L as a Direct Insulin- and Amino Acid-Dependent Mammalian Target of Rapamycin Complex 1 (mTORC1) Substrate*

    PubMed Central

    Schwarz, Jennifer Jasmin; Wiese, Heike; Tölle, Regine Charlotte; Zarei, Mostafa; Dengjel, Jörn; Warscheid, Bettina; Thedieck, Kathrin

    2015-01-01

    The serine/threonine kinase mammalian target of rapamycin (mTOR) governs growth, metabolism, and aging in response to insulin and amino acids (aa), and is often activated in metabolic disorders and cancer. Much is known about the regulatory signaling network that encompasses mTOR, but surprisingly few direct mTOR substrates have been established to date. To tackle this gap in our knowledge, we took advantage of a combined quantitative phosphoproteomic and interactomic strategy. We analyzed the insulin- and aa-responsive phosphoproteome upon inhibition of the mTOR complex 1 (mTORC1) component raptor, and investigated in parallel the interactome of endogenous mTOR. By overlaying these two datasets, we identified acinus L as a potential novel mTORC1 target. We confirmed acinus L as a direct mTORC1 substrate by co-immunoprecipitation and MS-enhanced kinase assays. Our study delineates a triple proteomics strategy of combined phosphoproteomics, interactomics, and MS-enhanced kinase assays for the de novo-identification of mTOR network components, and provides a rich source of potential novel mTOR interactors and targets for future investigation. PMID:25907765

  18. Functional Proteomics Identifies Acinus L as a Direct Insulin- and Amino Acid-Dependent Mammalian Target of Rapamycin Complex 1 (mTORC1) Substrate.

    PubMed

    Schwarz, Jennifer Jasmin; Wiese, Heike; Tölle, Regine Charlotte; Zarei, Mostafa; Dengjel, Jörn; Warscheid, Bettina; Thedieck, Kathrin

    2015-08-01

    The serine/threonine kinase mammalian target of rapamycin (mTOR) governs growth, metabolism, and aging in response to insulin and amino acids (aa), and is often activated in metabolic disorders and cancer. Much is known about the regulatory signaling network that encompasses mTOR, but surprisingly few direct mTOR substrates have been established to date. To tackle this gap in our knowledge, we took advantage of a combined quantitative phosphoproteomic and interactomic strategy. We analyzed the insulin- and aa-responsive phosphoproteome upon inhibition of the mTOR complex 1 (mTORC1) component raptor, and investigated in parallel the interactome of endogenous mTOR. By overlaying these two datasets, we identified acinus L as a potential novel mTORC1 target. We confirmed acinus L as a direct mTORC1 substrate by co-immunoprecipitation and MS-enhanced kinase assays. Our study delineates a triple proteomics strategy of combined phosphoproteomics, interactomics, and MS-enhanced kinase assays for the de novo-identification of mTOR network components, and provides a rich source of potential novel mTOR interactors and targets for future investigation. PMID:25907765

  19. Combined antitumor gene therapy with herpes simplex virus-thymidine kinase and short hairpin RNA specific for mammalian target of rapamycin.

    PubMed

    Woo, Ha-Na; Lee, Won Il; Kim, Ji Hyun; Ahn, Jeonghyun; Han, Jeong Hee; Lim, Sue Yeon; Lee, Won Woo; Lee, Heuiran

    2015-12-01

    A proof-of-concept study is presented using dual gene therapy that employed a small hairpin RNA (shRNA) specific for mammalian target of rapamycin (mTOR) and a herpes simplex virus-thymidine kinase (HSV-TK) gene to inhibit the growth of tumors. Recombinant adeno-associated virus (rAAV) vectors containing a mutant TK gene (sc39TK) were transduced into HeLa cells, and the prodrug ganciclovir (GCV) was administered to establish a suicide gene-therapy strategy. Additionally, rAAV vectors expressing an mTOR-targeted shRNA were employed to suppress mTOR-dependent tumor growth. GCV selectively induced death in tumor cells expressing TK, and the mTOR-targeted shRNA altered the cell cycle to impair tumor growth. Combining the TK-GCV system with mTOR inhibition suppressed tumor growth to a greater extent than that achieved with either treatment alone. Furthermore, HSV-TK expression and mTOR inhibition did not mutually interfere with each other. In conclusion, gene therapy that combines the TK-GCV system and mTOR inhibition shows promise as a novel strategy for cancer therapy. PMID:26459571

  20. Specific and Nonspecific Membrane-binding Determinants Cooperate in Targeting Phosphatidylinositol Transfer Protein β-Isoform to the Mammalian Trans-Golgi Network

    PubMed Central

    Phillips, Scott E.; Ile, Kristina E.; Boukhelifa, Malika; Huijbregts, Richard P.H.; Bankaitis, Vytas A.

    2006-01-01

    Phosphatidylinositol transfer proteins (PITPs) regulate the interface between lipid metabolism and specific steps in membrane trafficking through the secretory pathway in eukaryotes. Herein, we describe the cis-acting information that controls PITPβ localization in mammalian cells. We demonstrate PITPβ localizes predominantly to the trans-Golgi network (TGN) and that this localization is independent of the phospholipid-bound state of PITPβ. Domain mapping analyses show the targeting information within PITPβ consists of three short C-terminal specificity elements and a nonspecific membrane-binding element defined by a small motif consisting of adjacent tryptophan residues (the W202W203 motif). Combination of the specificity elements with the W202W203 motif is necessary and sufficient to generate an efficient TGN-targeting module. Finally, we demonstrate that PITPβ association with the TGN is tolerant to a range of missense mutations at residue serine 262, we describe the TGN localization of a novel PITPβ isoform with a naturally occurring S262Q polymorphism, and we find no other genetic or pharmacological evidence to support the concept that PITPβ localization to the TGN is obligately regulated by conventional protein kinase C (PKC) or the Golgi-localized PKC isoforms δ or ε. These latter findings are at odds with a previous report that conventional PKC-mediated phosphorylation of residue Ser262 is required for PITPβ targeting to Golgi membranes. PMID:16540520

  1. Piperlongumine, an alkaloid causes inhibition of PI3 K/Akt/mTOR signaling axis to induce caspase-dependent apoptosis in human triple-negative breast cancer cells.

    PubMed

    Shrivastava, Shweta; Kulkarni, Prasad; Thummuri, Dinesh; Jeengar, Manish Kumar; Naidu, V G M; Alvala, Mallika; Redddy, G Bhanuprakash; Ramakrishna, Sistla

    2014-07-01

    The phosphatidylinositol 3-kinase (PI3 K)/Akt/mammalian target of rapamycin (mTOR) signaling axis plays a central role in cell proliferation, growth and survival under physiological conditions. However, aberrant PI3 K/Akt/mTOR signaling has been implicated in many human cancers, including human triple negative breast cancer. Therefore, dual inhibitors of PI3 K/Akt and mTOR signaling could be valuable agents for treating breast cancer. The objective of this study was to investigate the effect of piperlongumine (PPLGM), a natural alkaloid on PI3 K/Akt/mTOR signaling, Akt mediated regulation of NF-kB and apoptosis evasion in human breast cancer cells. Using molecular docking studies, we found that PPLGM physically interacts with the conserved domain of PI3 K and mTOR kinases and the results were comparable with standard dual inhibitor PF04691502. Our results demonstrated that treatment of different human triple-negative breast cancer cells with PPLGM resulted in concentration- and time-dependent growth inhibition. The inhibition of cancer cell growth was associated with G1-phase cell cycle arrest and down-regulation of the NF-kB pathway leads to activation of the mitochondrial apoptotic pathway. It was also found that PPLGM significantly decreased the expression of p-Akt, p70S6K1, 4E-BP1, cyclin D1, Bcl-2, p53 and increased expression of Bax, cytochrome c in human triple-negative breast cancer cells. Although insulin treatment increased the phosphorylation of Akt (Ser473), p70S6K1, 4E-BP1, PPLGM abolished the insulin mediated phosphorylation, it clearly indicates that PPLGM acts through PI3 k/Akt/mTOR axis. Our results suggest that PPLGM may be an effective therapeutic agent for the treatment of human triple negative breast cancer. PMID:24729100

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

    PubMed Central

    Memmott, Regan M.; Dennis, Phillip A.

    2009-01-01

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

  3. Berberis libanotica extract targets NF-κB/COX-2, PI3K/Akt and mitochondrial/caspase signalling to induce human erythroleukemia cell apoptosis.

    PubMed

    Diab, Saada; Fidanzi, Chloe; Léger, David Y; Ghezali, Lamia; Millot, Marion; Martin, Frédérique; Azar, Rania; Esseily, Fadi; Saab, Antoine; Sol, Vincent; Diab-Assaf, Mona; Liagre, Bertrand

    2015-07-01

    The aim of this study was to describe and understand the relationship between cyclooxygenase-2 (COX-2) expression and apoptosis rate in erythroleukemia cells after apoptosis induction by Berberis libanotica (Bl) extract. To achieve this goal we used erythroleukemia cell lines expressing COX‑2 (HEL cell line) or not (K562 cell line). Moreover, we made use of COX‑2 cDNA to overexpress COX‑2 in K562 cells. In light of the reported chemopreventive and chemosensitive effects of natural products on various tumor cells and animal models, we postulated that our Bl extract may mediate their effects through apoptosis induction with suppression of cell survival pathways. Our study is the first report on the specific examination of intrinsic apoptosis and Akt/NF-κB/COX‑2 pathways in human erythroleukemia cells upon Bl extract exposure. Even if Bl extract induced apoptosis of three human erythroleukemia cell lines, a dominant effect of Bl extract treatment on K562 cells was observed resulting in activation of the late markers of apoptosis with caspase-3 activation, PARP cleavage and DNA fragmentation. Whereas, we showed that Bl extract reduced significantly expression of COX‑2 by a dose-dependent manner in HEL and K562 (COX‑2+) cells. Furthermore, in regard to our results, it is clear that the simultaneous inhibition of Akt and NF-κB signalling can significantly contribute to the anticancer effects of Bl extract in human erythroleukemia cells. We observed that the Bl extract is clearly more active than the berberine alone on the induction of DNA fragmentation in human erythro-leukemia cells. PMID:25997834

  4. Tunicamycins: translocase-I inhibitors that target bacterial cell wall and mammalian N-glycoproteins. The potential for selective inhibitors

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Tunicamycins are a heterologous family of nucleoside antibiotics that target the biosynthesis of bacterial peptidoglycan and eukaryotic N-glycoproteins. The mechanism of action is known, with the tunicamycin-Mg2+ complex established as a transition state analog for hexosamine-1-phosphate: prenol pho...

  5. S9, a Novel Anticancer Agent, Exerts Its Anti-Proliferative Activity by Interfering with Both PI3K-Akt-mTOR Signaling and Microtubule Cytoskeleton

    PubMed Central

    Yang, Chun-hao; Ding, Hua-sheng; Luo, Cheng; Zhang, Yu; Wu, Mao-jiang; Zhang, Xiong-wen; Shen, Xu; Jiang, Hua-liang; Meng, Ling-hua; Ding, Jian

    2009-01-01

    Background Deregulation of the phosphatidylinositol 3-kinases (PI3K)/Akt/mammalian target of rapamycin (mTOR) pathway plays a central role in tumor formation and progression, providing validated targets for cancer therapy. S9, a hybrid of α-methylene-γ-lactone and 2-phenyl indole compound, possessed potent activity against this pathway. Methodology/Principal Findings Effects of S9 on PI3K-Akt-mTOR pathway were determined by Western blot, immunofluorescence staining and in vitro kinas assay. The interactions between tubulin and S9 were investigated by polymerization assay, CD, and SPR assay. The potential binding modes between S9 and PI3K, mTOR or tubulin were analyzed by molecular modeling. Anti-tumor activity of S9 was evaluated in tumor cells and in nude mice bearing human cancer xenografts. S9 abrogated EGF-activated PI3K-Akt-mTOR signaling cascade and Akt translocation to cellular membrane in human tumor cells. S9 possessed inhibitory activity against both PI3K and mTOR with little effect on other tested 30 kinases. S9 also completely impeded hyper-phosphorylation of Akt as a feedback of inhibition of mTOR by rapamycin. S9 unexpectedly arrested cells in M phase other than G1 phase, which was distinct from compounds targeting PI3K-Akt-mTOR pathway. Further study revealed that S9 inhibited tubulin polymerization via binding to colchicine-binding site of tubulin and resulted in microtubule disturbance. Molecular modeling indicated that S9 could potentially bind to the kinase domains of PI3K p110α subunit and mTOR, and shared similar hydrophobic interactions with colchicines in the complex with tubulin. Moreover, S9 induced rapid apoptosis in tumor cell, which might reflect a synergistic cooperation between blockade of both PI3-Akt-mTOR signaling and tubulin cytoskeleton. Finally, S9 displayed potent antiproliferative activity in a panel of tumor cells originated from different tissue types including drug-resistant cells and in nude mice bearing human tumor

  6. Akt1 binds focal adhesion kinase via the Akt1 kinase domain independently of the pleckstrin homology domain.

    PubMed

    Basson, M D; Zeng, B; Wang, S

    2015-10-01

    for developing novel inhibitors that target this specific Akt1/FAK interaction. PMID:26579576

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

    PubMed Central

    YAN, LI-XU; LIU, YAN-HUI; XIANG, JIAN-WEN; WU, QI-NIAN; XU, LEI-BO; LUO, XIN-LAN; ZHU, XIAO-LAN; LIU, CHAO; XU, FANG-PING; LUO, DONG-LAN; MEI, PING; XU, JIE; ZHANG, KE-PING; CHEN, JIE

    2016-01-01

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

  8. Mammalian DNA Repair. Final Report

    SciTech Connect

    2003-01-24

    The Gordon Research Conference (GRC) on Mammalian DNA Repair was held at Harbortown Resort, Ventura Beach, CA. Emphasis was placed on current unpublished research and discussion of the future target areas in this field.

  9. MicroRNA-141 and MicroRNA-146b-5p Inhibit the Prometastatic Mesenchymal Characteristics through the RNA-binding Protein AUF1 Targeting the Transcription Factor ZEB1 and the Protein Kinase AKT*

    PubMed Central

    Al-Khalaf, Huda H.; Aboussekhra, Abdelilah

    2014-01-01

    miR-141 and miR-146b-5p are two important tumor suppressor microRNAs, which control several cancer-related genes and processes. In the present report, we have shown that these microRNAs bind specific sites at the 3′-untranslated region (UTR) of the mRNA-binding protein AUF1, leading to its down-regulation. This inverse correlation between the levels of these microRNAs and AUF1 has been identified in various osteosarcoma cell lines. Additionally, we present clear evidence that AUF1 promotes mesenchymal features in osteosarcoma cells and that miR-141 and miR-146b-5p suppress this prometastatic process through AUF1 repression. Indeed, both microRNAs suppressed the invasion/migration and proliferation abilities of osteosarcoma cells through inhibiting the AKT protein kinase in an AUF1-dependent manner. We have also shown that AUF1 binds to and stabilizes the mRNA of the AKT activator phosphoinositide-dependent kinase-1 (PDK1). Furthermore, miR-141 and miR-146b-5p positively regulate the epithelial markers (E-cadherin and Epcam) and repress the mesenchymal markers (N-cadherin, Vimentin, Twist2, and ZEB1). These effects were mediated via the repression of the epithelial-to-mesenchymal inducer ZEB1 through targeting AUF1, which binds the 3′-UTR of the ZEB1 mRNA and reduces its turnover. These results indicate that at least some tumor suppressor functions of miR-141 and miR-146b-5p are mediated through the repression of the oncogenic potentials of AUF1. Therefore, these 3′-UTR-directed post-transcriptional gene expression regulators constitute promising new targets for diagnostic and/or therapeutic interventions. PMID:25261470

  10. Molecular cloning and functional characterization of a cell-permeable superoxide dismutase targeted to lung adenocarcinoma cells. Inhibition cell proliferation through the Akt/p27kip1 pathway.

    PubMed

    Lu, Min; Gong, Xingguo; Lu, Yuwen; Guo, Jianjun; Wang, Chenhui; Pan, Yuanjiang

    2006-05-12

    In clinical oncology, many trials with superoxide dismutase (SOD) have failed to demonstrate antitumor ability and in many cases even caused deleterious effects because of low tumor-targeting ability. In the current research, the Nostoc commune Fe-SOD coding sequence was amplified from genomic DNA. In addition, the single chain variable fragment (ScFv) was constructed from the cDNA of an LC-1 hybridoma cell line secreting anti-lung adenocarcinoma monoclonal antibody. After modification, the SOD and ScFv were fused and co-expressed, and the resulting fusion protein produced SOD and LC-1 antibody activity. Tracing SOD-ScFv by fluorescein isothiocyanate and superoxide anions (O2*-) in SPC-A-1 cells showed that the fusion protein could recognize and enter SPC-A-1 cells to eliminate O2*-. The lower oxidative stress resulting from the decrease in cellular O2*- delayed the cell cycle at G1 and significantly slowed SPC-A-1 cell growth in association with the dephosphorylation of the serine-threonine protein kinase Akt and expression of p27kip1. The tumor-targeting fusion protein resulting from this research overcomes two disadvantages of SODs previously used in the clinical setting, the inability to target tumor cells or permeate the cell membrane. These findings lay the groundwork for development of an efficient antitumor drug targeted by the ScFv. PMID:16551617

  11. Mitochondrial Akt Regulation of Hypoxic Tumor Reprogramming.

    PubMed

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

    2016-08-01

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

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

    SciTech Connect

    White, Eric S.; Sagana, Rommel L.; Booth, Adam J.; Yan, Mei; Cornett, Ashley M.; Bloomheart, Christopher A.; Tsui, Jessica L.; Wilke, Carol A.; Moore, Bethany B.; Ritzenthaler, Jeffrey D.; Roman, Jesse; Muro, Andres F.

    2010-10-01

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

  13. Valproic acid induces autophagy by suppressing the Akt/mTOR pathway in human prostate cancer cells

    PubMed Central

    Xia, Qinghua; Zheng, Yi; Jiang, Wei; Huang, Zhongxian; Wang, Muwen; Rodriguez, Ronald; Jin, Xunbo

    2016-01-01

    Previous studies have demonstrated that the chronic administration of valproic acid (VPA) suppresses angiogenesis in vivo; however, the mechanisms implicated in VPA-induced autophagy remain unclear. The current study aimed to assess VPA-induced autophagy in three prostate cancer cell lines (PC3, DU145 and LNCaP), in addition to analyzing the Akt/mammalian target of rapamycin (mTOR) signal pathway. Prostate cancer cell lines were cultured with various doses of VPA. Cell cycle was analyzed using flow cytometry, and autophagy markers [1A/1B-light chain 3 (LC3)-II and Beclin-1] were examined using transmission electron microscopy, fluorescent microscopy and western blotting. Activation of the Akt/mTOR signal pathway was also assessed by western blotting. The results demonstrated that VPA induced autophagosomes and suppressed the Akt/mTOR signal pathway. This was confirmed by detection of increased LC3-II and Beclin-1 in VPA-treated cells compared with untreated controls. Phosphorylated forms of Akt (PC3, P=0.048; DU145, P=0.045; LNCaP, P=0.039) and mTOR (PC3, P=0.012; DU145, P=0.41; LNCaP, P=0.35) were significantly reduced following VPA treatment. These results suggest that VPA may function as a histone deacetylase inhibitor, suppressing the growth of prostate cancer cells by modulating autophagy pathways, including inhibition of the Akt/mTOR pathway. Further experiments are required to determine the significance of all involved pathways regarding VPA-induced growth inhibition. PMID:27588130

  14. Drug-Related Pneumonitis During Mammalian Target of Rapamycin Inhibitor Therapy: Radiographic Pattern-Based Approach in Waldenström Macroglobulinemia as a Paradigm

    PubMed Central

    Boswell, Erica N.; Hatabu, Hiroto; Ghobrial, Irene M.; Ramaiya, Nikhil H.

    2015-01-01

    Background. This study determined the frequency of drug-related pneumonitis during mammalian target of rapamycin (mTOR) inhibitor therapy in Waldenström macroglobulinemia patients and investigated the imaging characteristics and radiographic patterns of pneumonitis. Materials and Methods. A total of 40 patients (23 men, 17 women; 43–84 years old) with Waldenström macroglobulinemia treated in 2 trials of the mTOR inhibitor everolimus were retrospectively studied. Chest computed tomography (CT) scans during therapy were reviewed for abnormalities suspicious for drug-related pneumonitis by the consensus of three radiologists, evaluating the extent, distributions, and specific findings. The radiographic patterns of pneumonitis were classified using the American Thoracic Society/European Respiratory Society classification of interstitial pneumonia. Results. Drug-related pneumonitis was noted in 23 patients (58%). The median time from the initiation of therapy to the onset of pneumonitis was 5.7 months. Lower lungs were involved in all 23 patients, with a higher extent than in the other zones (p < .001). The distribution was peripheral and lower in 11 patients (48%) and mixed and multifocal in 10 (44%). The findings were bilateral in 20 patients (87%). Ground glass opacities (GGOs) and reticular opacities were present in all 23 patients, with consolidation in 12, traction bronchiectasis in 2, and centrilobular nodularity in 1. The pattern of pneumonitis was classified as cryptogenic organizing pneumonia (COP) in 16 (70%) and nonspecific interstitial pneumonia (NSIP) in 7 (30%), with overlapping features of COP and NSIP in 7 patients. Conclusion. Drug-related pneumonitis was noted on CT in 58% of Waldenström macroglobulinemia patients treated with mTOR inhibitor therapy. Most common findings were bilateral GGOs and reticular opacities, with or without consolidation, in peripheral and lower lungs, demonstrating COP and NSIP patterns. Implications for Practice: The

  15. Pirarubicin induces an autophagic cytoprotective response through suppression of the mammalian target of rapamycin signaling pathway in human bladder cancer cells

    SciTech Connect

    Li, Kuiqing; Chen, Xu; Liu, Cheng; Gu, Peng; Li, Zhuohang; Wu, Shaoxu; Xu, Kewei; Lin, Tianxin; Huang, Jian

    2015-05-01

    Pirarubicin is widely used in intravesical chemotherapy for bladder cancer, but its efficacy is limited due to drug resistance; the mechanism has not been well studied. Emerging evidence shows that autophagy can be a novel target for cancer therapy. This study aimed to investigate the role of autophagy in pirarubicin-treated bladder cancer cells. Bladder cancer cells EJ and J82 were treated with pirarubicin, siRNA, 3-methyladenine or hydroxychloroquine. Cell proliferation and apoptosis were tested by cell survival assay and flow cytometric analysis, respectively. Autophagy was evaluated by immunoblotting before and after the treatments. The phosphorylated mammalian target of rapamycin, serine/threonine kinase p70 S6 kinase, and eukaryotic translation initiation factor 4E binding protein 1 were also investigated by immunoblotting. We found that pirarubicin could induce autophagy in bladder cancer cells. Inhibition of autophagy by 3-methyladenine, hydroxychloroquine or knockdown of autophagy related gene 3 significantly increased apoptosis in pirarubicin-treated bladder cancer cells. Pirarubicin-induced autophagy was mediated via the mTOR/p70S6K/4E-BP1 signaling pathway. In conclusion, autophagy induced by pirarubicin plays a cytoprotective role in bladder cancer cells, suggesting that inhibition of autophagy may improve efficacy over traditional pirarubicin chemotherapy in bladder cancer patients. - Highlights: • Pirarubicin induced autophagy in bladder cancer cells. • Inhibition of autophagy enhanced pirarubicin-induced apoptosis. • Pirarubicin induced autophagy through inhibition of mTOR signaling pathway.

  16. Pirarubicin induces an autophagic cytoprotective response through suppression of the mammalian target of rapamycin signaling pathway in human bladder cancer cells.

    PubMed

    Li, Kuiqing; Chen, Xu; Liu, Cheng; Gu, Peng; Li, Zhuohang; Wu, Shaoxu; Xu, Kewei; Lin, Tianxin; Huang, Jian

    2015-05-01

    Pirarubicin is widely used in intravesical chemotherapy for bladder cancer, but its efficacy is limited due to drug resistance; the mechanism has not been well studied. Emerging evidence shows that autophagy can be a novel target for cancer therapy. This study aimed to investigate the role of autophagy in pirarubicin-treated bladder cancer cells. Bladder cancer cells EJ and J82 were treated with pirarubicin, siRNA, 3-methyladenine or hydroxychloroquine. Cell proliferation and apoptosis were tested by cell survival assay and flow cytometric analysis, respectively. Autophagy was evaluated by immunoblotting before and after the treatments. The phosphorylated mammalian target of rapamycin, serine/threonine kinase p70 S6 kinase, and eukaryotic translation initiation factor 4E binding protein 1 were also investigated by immunoblotting. We found that pirarubicin could induce autophagy in bladder cancer cells. Inhibition of autophagy by 3-methyladenine, hydroxychloroquine or knockdown of autophagy related gene 3 significantly increased apoptosis in pirarubicin-treated bladder cancer cells. Pirarubicin-induced autophagy was mediated via the mTOR/p70S6K/4E-BP1 signaling pathway. In conclusion, autophagy induced by pirarubicin plays a cytoprotective role in bladder cancer cells, suggesting that inhibition of autophagy may improve efficacy over traditional pirarubicin chemotherapy in bladder cancer patients. PMID:25791481

  17. Quantitative Persulfide Site Identification (qPerS-SID) Reveals Protein Targets of H2S Releasing Donors in Mammalian Cells

    PubMed Central

    Longen, Sebastian; Richter, Florian; Köhler, Yvette; Wittig, Ilka; Beck, Karl-Friedrich; Pfeilschifter, Josef

    2016-01-01

    H2S is an important signalling molecule involved in diverse biological processes. It mediates the formation of cysteine persulfides (R-S-SH), which affect the activity of target proteins. Like thiols, persulfides show reactivity towards electrophiles and behave similarly to other cysteine modifications in a biotin switch assay. In this manuscript, we report on qPerS-SID a mass spectrometry-based method allowing the isolation of persulfide containing peptides in the mammalian proteome. With this method, we demonstrated that H2S donors differ in their efficacy to induce persulfides in HEK293 cells. Furthermore, data analysis revealed that persulfide formation affects all subcellular compartments and various cellular processes. Negatively charged amino acids appeared more frequently adjacent to cysteines forming persulfides. We confirmed our proteomic data using pyruvate kinase M2 as a model protein and showed that several cysteine residues are prone to persulfide formation finally leading to its inactivation. Taken together, the site-specific identification of persulfides on a proteome scale can help to identify target proteins involved in H2S signalling and enlightens the biology of H2S and its releasing agents. PMID:27411966

  18. Identification of the novel interacting partners of the mammalian target of rapamycin complex 1 in human CCRF-CEM and HEK293 cells.

    PubMed

    Rahman, Hazir; Qasim, Muhammad; Oellerich, Michael; Asif, Abdul R

    2014-01-01

    The present study was undertaken to identify proteins that interact with the mammalian target of rapamycin complex 1 (mTORC1) to enable it to carry out its crucial cell signaling functions. Endogenous and myc-tag mTORC1 was purified, in-gel tryptic digested and then identified by nano-LC ESI Q-TOF MS/MS analysis. A total of nine novel interacting proteins were identified in both endogenous and myc-tag mTORC1 purifications. These new mTORC1 interacting partners include heterogeneous nuclear ribonucleoproteins A2/B1, enhancer of mRNA decapping protein 4, 60S acidic ribosomal protein, P0, nucleolin, dynamin 2, glyceraldehyde 3 phosphate dehydrogenase, 2-oxoglutarate dehydrogenase, glycosyl transferase 25 family member 1 and prohibitin 2. Furthermore hnRNP A2/B1 and dynamin 2 interaction with mTORC1 was confirmed on immunoblotting. The present study has for the first time identified novel interacting partners of mTORC1 in human T lymphoblasts (CCRF-CEM) and human embryonic kidney (HEK293) cells. These new interacting proteins may offer new targets for therapeutic interventions in human diseases caused by perturbed mTORC1 signaling. PMID:24646917

  19. Quantitative Persulfide Site Identification (qPerS-SID) Reveals Protein Targets of H2S Releasing Donors in Mammalian Cells.

    PubMed

    Longen, Sebastian; Richter, Florian; Köhler, Yvette; Wittig, Ilka; Beck, Karl-Friedrich; Pfeilschifter, Josef

    2016-01-01

    H2S is an important signalling molecule involved in diverse biological processes. It mediates the formation of cysteine persulfides (R-S-SH), which affect the activity of target proteins. Like thiols, persulfides show reactivity towards electrophiles and behave similarly to other cysteine modifications in a biotin switch assay. In this manuscript, we report on qPerS-SID a mass spectrometry-based method allowing the isolation of persulfide containing peptides in the mammalian proteome. With this method, we demonstrated that H2S donors differ in their efficacy to induce persulfides in HEK293 cells. Furthermore, data analysis revealed that persulfide formation affects all subcellular compartments and various cellular processes. Negatively charged amino acids appeared more frequently adjacent to cysteines forming persulfides. We confirmed our proteomic data using pyruvate kinase M2 as a model protein and showed that several cysteine residues are prone to persulfide formation finally leading to its inactivation. Taken together, the site-specific identification of persulfides on a proteome scale can help to identify target proteins involved in H2S signalling and enlightens the biology of H2S and its releasing agents. PMID:27411966

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

    PubMed Central

    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 RNA conjugated with branched cationic polyethylenimine (bPEI) in fibroblastic lineage cells in serum-based cell culture as shown by both gene and protein analysis. This mTOR knockdown 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 was observed. Observed 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. PMID:20727922

  1. Tuberous sclerosis complex-1 and -2 gene products function together to inhibit mammalian target of rapamycin (mTOR)-mediated downstream signaling.

    PubMed

    Tee, Andrew R; Fingar, Diane C; Manning, Brendan D; Kwiatkowski, David J; Cantley, Lewis C; Blenis, John

    2002-10-15

    Tuberous sclerosis complex (TSC) is an autosomal dominant genetic disorder that occurs upon mutation of either the TSC1 or TSC2 genes, which encode the protein products hamartin and tuberin, respectively. Here, we show that hamartin and tuberin function together to inhibit mammalian target of rapamycin (mTOR)-mediated signaling to eukaryotic initiation factor 4E-binding protein 1 (4E-BP1) and ribosomal protein S6 kinase 1 (S6K1). First, coexpression of hamartin and tuberin repressed phosphorylation of 4E-BP1, resulting in increased association of 4E-BP1 with eIF4E; importantly, a mutant of TSC2 derived from TSC patients was defective in repressing phosphorylation of 4E-BP1. Second, the activity of S6K1 was repressed by coexpression of hamartin and tuberin, but the activity of rapamycin-resistant mutants of S6K1 were not affected, implicating mTOR in the TSC-mediated inhibitory effect on S6K1. Third, hamartin and tuberin blocked the ability of amino acids to activate S6K1 within nutrient-deprived cells, a process that is dependent on mTOR. These findings strongly implicate the tuberin-hamartin tumor suppressor complex as an inhibitor of mTOR and suggest that the formation of tumors within TSC patients may result from aberrantly high levels of mTOR-mediated signaling to downstream targets. PMID:12271141

  2. BCL-3 expression promotes colorectal tumorigenesis through activation of AKT signalling

    PubMed Central

    Urban, Bettina C; Collard, Tracey J; Eagle, Catherine J; Southern, Samantha L; Greenhough, Alexander; Hamdollah-Zadeh, Maryam; Ghosh, Anil; Paraskeva, Christos; Silver, Andrew; Williams, Ann C

    2016-01-01

    Objective Colorectal cancer remains the fourth most common cause of cancer-related mortality worldwide. Here we investigate the role of nuclear factor-κB (NF-κB) co-factor B-cell CLL/lymphoma 3 (BCL-3) in promoting colorectal tumour cell survival. Design Immunohistochemistry was carried out on 47 tumour samples and normal tissue from resection margins. The role of BCL-3/NF-κB complexes on cell growth was studied in vivo and in vitro using an siRNA approach and exogenous BCL-3 expression in colorectal adenoma and carcinoma cells. The question whether BCL-3 activated the AKT/protein kinase B (PKB) pathway in colorectal tumour cells was addressed by western blotting and confocal microscopy, and the ability of 5-aminosalicylic acid (5-ASA) to suppress BCL-3 expression was also investigated. Results We report increased BCL-3 expression in human colorectal cancers and demonstrate that BCL-3 expression promotes tumour cell survival in vitro and tumour growth in mouse xenografts in vivo, dependent on interaction with NF-κB p50 or p52 homodimers. We show that BCL-3 promotes cell survival under conditions relevant to the tumour microenvironment, protecting both colorectal adenoma and carcinoma cells from apoptosis via activation of the AKT survival pathway: AKT activation is mediated via both PI3K and mammalian target of rapamycin (mTOR) pathways, leading to phosphorylation of downstream targets GSK-3β and FoxO1/3a. Treatment with 5-ASA suppressed BCL-3 expression in colorectal cancer cells. Conclusions Our study helps to unravel the mechanism by which BCL-3 is linked to poor prognosis in colorectal cancer; we suggest that targeting BCL-3 activity represents an exciting therapeutic opportunity potentially increasing the sensitivity of tumour cells to conventional therapy. PMID:26033966

  3. Dissecting signalling by individual Akt/PKB isoforms, three steps at once.

    PubMed

    Osorio-Fuentealba, Cesar; Klip, Amira

    2015-09-01

    The serine/threonine kinase Akt/PKB (protein kinase B) is key for mammalian cell growth, survival, metabolism and oncogenic transformation. The diverse level and tissue expression of its three isoforms, Akt1/PKBα, Akt2/PKBβ and Akt3/PKBγ, make it daunting to identify isoform-specific actions in vivo and even in isolated tissues/cells. To date, isoform-specific knockout and knockdown have been the best strategies to dissect their individual overall functions. In a recent article in the Biochemical Journal, Kajno et al. reported a new strategy to study isoform selectivity in cell lines. Individual Akt/PKB isoforms in 3T3-L1 pre-adipocytes are first silenced via shRNA and stable cellular clones lacking one or the other isoform are selected. The stably silenced isoform is then replaced by a mutant engineered to be refractory to inhibition by MK-2206 (Akt1(W80A) or Akt2(W80A)). Akt1(W80A) or Akt2(W80A) are functional and effectively recruited to the plasma membrane in response to insulin. The system affords the opportunity to acutely control the activity of the endogenous non-silenced isoform through timely addition of MK-2206. Using this approach, it is confirmed that Akt1/PKBα is the preferred isoform sustaining adipocyte differentiation, but both Akt1/PKBα and Akt2/PKBβ can indistinctly support insulin-dependent FoxO1 (forkhead box O1) nuclear exclusion. Surprisingly, either isoform can also support insulin-dependent glucose transporter (GLUT) 4 translocation to the membrane, in contrast with the preferential role of Akt2/PKBβ assessed by knockdown studies. The new strategy should allow analysis of the plurality of Akt/PKB functions in other cells and in response to other stimuli. It should also be amenable to high-throughput studies to speed up advances in signal transmission by this pivotal kinase. PMID:26348913

  4. Inactivation of the tuberous sclerosis complex-1 and -2 gene products occurs by phosphoinositide 3-kinase/Akt-dependent and -independent phosphorylation of tuberin.

    PubMed

    Tee, Andrew R; Anjum, Rana; Blenis, John

    2003-09-26

    The tuberous sclerosis complex (TSC) is a genetic disorder that is caused through mutations in either one of the two tumor suppressor genes, TSC1 and TSC2, that encode hamartin and tuberin, respectively. Interaction of hamartin with tuberin forms a heterodimer that inhibits signaling by the mammalian target of rapamycin to its downstream targets: eukaryotic initiation factor 4E-binding protein 1 (4E-BP1) and ribosomal protein S6 kinase 1 (S6K1). During mitogenic sufficiency, the phosphoinositide 3-kinase (PI3K)/Akt pathway phosphorylates tuberin on Ser-939 and Thr-1462 that inhibits the tumor suppressor function of the TSC complex. Here we show that tuberin-hamartin heterodimers block protein kinase C (PKC)/MAPK- and phosphatidic acid-mediated signaling toward mammalian target of rapamycin-dependent targets. We also show that two TSC2 mutants derived from TSC patients are defective in repressing phorbol 12-myristate 13-acetate-induced 4E-BP1 phosphorylation. PKC/MAPK signaling leads to phosphorylation of tuberin at sites that overlap with and are distinct from Akt phosphorylation sites. Phosphorylation of tuberin by phorbol 12-myristate 13-acetate was reduced by treatment of cells with either bisindolylmaleimide I or UO126, inhibitors of PKC and MAPK/MEK (MAPK/ERK kinase), respectively, but not by wortmannin (an inhibitor of PI3K). This work reveals that both PI3K-independent and -dependent mechanisms modulate tuberin phosphorylation in vivo. PMID:12867426

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

    PubMed Central

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

    2015-01-01

    The pi3k/Akt/mtor (phosphatidylinositol 3 kinase/ Akt/mammalian target of rapamycin) signalling pathway is an established driver of oncogenic activity in human malignancies. Therapeutic targeting of this pathway holds significant promise as a treatment strategy. Everolimus, an mtor inhibitor, is the first of this class of agents approved for the treatment of hormone receptor–positive, human epidermal growth factor receptor 2–negative advanced breast cancer. Everolimus has been associated with significant improvements in progression-free survival; however, it is also associated with increased toxicity related to its specific mechanism of action. Methods A comprehensive review of the literature conducted using a focused medline search was combined with a search of current trials at http://ClinicalTrials.gov/. Summary tables of the toxicities of the various classes of pi3k/Akt/mtor inhibitors were created. A broad group of Canadian health care professionals was assembled to review the data and to produce expert opinion and summary recommendations for possible best practices in managing the adverse events associated with these pathway inhibitors. Results Differing toxicities are associated with the various classes of pi3k/Akt/mtor pathway inhibitors. The most common unique adverse events observed in everolimus clinical trials in breast cancer include stomatitis (all grades: approximately 60%), noninfectious pneumonitis (15%), rash (40%), hyperglycemia (15%), and immunosuppression (40%). To minimize grades 3 and 4 toxicities and to attempt to attain optimal outcomes, effective management of those adverse events is critical. Management should be interdisciplinary and should use approaches that include education, early recognition, active intervention, and potentially prophylactic strategies. Discussion Everolimus likely represents the first of many complex oral targeted therapies for the treatment of breast cancer. Using this agent as a template, it is essential to

  6. Mammalian Target of Rapamycin (mTOR) Activity Dependent Phospho-Protein Expression in Childhood Acute Lymphoblastic Leukemia (ALL)

    PubMed Central

    Márk, Ágnes; Hajdu, Melinda; Kenessey, István; Sticz, Tamás; Nagy, Eszter; Barna, Gábor; Váradi, Zsófia; Kovács, Gábor; Kopper, László; Csóka, Monika

    2013-01-01

    Modern treatment strategies have improved the prognosis of childhood ALL; however, treatment still fails in 25–30% of patients. Further improvement of treatment may depend on the development of targeted therapies. mTOR kinase, a central mediator of several signaling pathways, has recently attracted remarkable attention as a potential target in pediatric ALL. However, limited data exists about the activity of mTOR. In the present study, the amount of mTOR activity dependent phospho-proteins was characterized by ELISA in human leukemia cell lines and in lymphoblasts from childhood ALL patients (n = 49). Expression was measured before and during chemotherapy and at relapses. Leukemia cell lines exhibited increased mTOR activity, indicated by phospho-S6 ribosomal protein (p-S6) and phosphorylated eukaryotic initiation factor 4E binding protein (p-4EBP1). Elevated p-4EBP1 protein levels were detected in ALL samples at diagnosis; efficacy of chemotherapy was followed by the decrease of mTOR activity dependent protein phosphorylation. Optical density (OD) for p-4EBP1 (ELISA) was significantly higher in patients with poor prognosis at diagnosis, and in the samples of relapsed patients. Our results suggest that measuring mTOR activity related phospho-proteins such as p-4EBP1 by ELISA may help to identify patients with poor prognosis before treatment, and to detect early relapses. Determining mTOR activity in leukemic cells may also be a useful tool for selecting patients who may benefit from future mTOR inhibitor treatments. PMID:23573198

  7. Production of lentiviral vectors with enhanced efficiency to target dendritic cells by attenuating mannosidase activity of mammalian cells

    PubMed Central

    2011-01-01

    Background Dendritic cells (DCs) are antigen-presenting immune cells that interact with T cells and have been widely studied for vaccine applications. To achieve this, DCs can be manipulated by lentiviral vectors (LVs) to express antigens to stimulate the desired antigen-specific T cell response, which gives this approach great potential to fight diseases such as cancers, HIV, and autoimmune diseases. Previously we showed that LVs enveloped with an engineered Sindbis virus glycoprotein (SVGmu) could target DCs through a specific interaction with DC-SIGN, a surface molecule predominantly expressed by DCs. We hypothesized that SVGmu interacts with DC-SIGN in a mannose-dependent manner, and that an increase in high-mannose structures on the glycoprotein surface could result in higher targeting efficiencies of LVs towards DCs. It is known that 1-deoxymannojirimycin (DMJ) can inhibit mannosidase, which is an enzyme that removes high-mannose structures during the glycosylation process. Thus, we investigated the possibility of generating LVs with enhanced capability to modify DCs by supplying DMJ during vector production. Results Through western blot analysis and binding tests, we were able to infer that binding of SVGmu to DC-SIGN is directly related to amount of high-mannose structures on SVGmu. We also found that the titer for the LV (FUGW/SVGmu) produced with DMJ against 293T.DCSIGN, a human cell line expressing the human DC-SIGN atnibody, was over four times higher than that of vector produced without DMJ. In addition, transduction of a human DC cell line, MUTZ-3, yielded a higher transduction efficiency for the LV produced with DMJ. Conclusion We conclude that LVs produced under conditions with inhibited mannosidase activity can effectively modify cells displaying the DC-specific marker DC-SIGN. This study offers evidence to support the utilization of DMJ in producing LVs that are enhanced carriers for the development of DC-directed vaccines. PMID:21276219

  8. Autophagy inhibition enhances colorectal cancer apoptosis induced by dual phosphatidylinositol 3-kinase/mammalian target of rapamycin inhibitor NVP-BEZ235

    PubMed Central

    YANG, XIAOYU; NIU, BINGXUAN; WANG, LIBO; CHEN, MEILING; KANG, XIAOCHUN; WANG, LUONAN; JI, YINGHUA; ZHONG, JIATENG

    2016-01-01

    Phosphatidylinositol 3-kinase (PI3K)/mammalian target of rapamycin (mTOR) signaling pathway performs a central role in tumorigenesis and is constitutively activated in many malignancies. As a novel dual PI3K/mTOR inhibitor currently undergoing evaluation in a phase I/II clinical trial, NVP-BEZ235 indicates a significant antitumor efficacy in diverse solid tumors, including colorectal cancer (CRC). Autophagy is a catabolic process that maintains cellular homeostasis and reduces diverse stresses through lysosomal recycling of the unnecessary and damaged cell components. This process is also observed to antagonize the antitumor efficacy of PI3K/mTOR inhibitor agents such as NVP-BEZ235, via apoptosis inhibition. In the present study, we investigated anti-proliferative and apoptosis-inducing ability of NVP-BEZ235 in SW480 cells and the crosstalk between autophagy and apoptosis in SW480 cells treated with NVP-BEZ235 in combination with an autophagy inhibitor. The results revealed that, NVP-BEZ235 effectively inhibit the growth of SW480 cells by targeting the PI3K/mTOR signaling pathway and induced apoptosis. The inhibition of autophagy with 3-methyladenine or chloroquine inhibitors in combination with NVP-BEZ235 in SW480 cells enhanced the apoptotic rate as componets to NVP-BEZ235 alone. In conclusion, the findings provide a rationale for chemotherapy targeting the PI3K/mTOR signaling pathway presenting a potential therapeutic strategy to enhance the efficacy of dual PI3K/mTOR inhibitor NVP-BEZ235 in combination with an autophagy inhibitor in CRC treatment and treatment of other tumors. PMID:27347108

  9. The Prolyl Peptidases PRCP/PREP Regulate IRS-1 Stability Critical for Rapamycin-induced Feedback Activation of PI3K and AKT*

    PubMed Central

    Duan, Lei; Ying, Guoguang; Danzer, Brian; Perez, Ricardo E.; Shariat-Madar, Zia; Levenson, Victor V.; Maki, Carl G.

    2014-01-01

    The phosphatidylinositol 3-kinase (PI3K)/protein kinase B (PKB/AKT)/mammalian target of rapamycin (mTOR) pathway conveys signals from receptor tyrosine kinases (RTKs) to regulate cell metabolism, proliferation, survival, and motility. Previously we found that prolylcarboxypeptidase (PRCP) regulate proliferation and survival in breast cancer cells. In this study, we found that PRCP and the related family member prolylendopeptidase (PREP) are essential for proliferation and survival of pancreatic cancer cells. Depletion/inhibition of PRCP and PREP-induced serine phosphorylation and degradation of IRS-1, leading to inactivation of the cellular PI3K and AKT. Notably, depletion/inhibition of PRCP/PREP destabilized IRS-1 in the cells treated with rapamycin, blocking the feedback activation PI3K/AKT. Consequently, inhibition of PRCP/PREP enhanced rapamycin-induced cytotoxicity. Thus, we have identified PRCP and PREP as a stabilizer of IRS-1 which is critical for PI3K/AKT/mTOR signaling in pancreatic cancer cells. PMID:24936056

  10. Allosteric Small-Molecule Inhibitors of the AKT Kinase

    NASA Astrophysics Data System (ADS)

    Dalafave, D. S.

    This research addresses computational design of small druglike molecules for possible anticancer applications. AKT and SGK are kinases that control important cellular functions. They are highly homologous, having similar activators and targets. Cancers with increased SGK activity may develop resistance to AKT-specific inhibitors. Our goal was to design new molecules that would bind both AKT and SGK, thus preventing the development of drug resistance. Most kinase inhibitors target the kinase ATP-binding site. However, the high similarity in this site among kinases makes it difficult to target specifically. Furthermore, mutations in this site can cause resistance to ATP-competitive kinase inhibitors. We used existing AKT inhibitors as initial templates to design molecules that could potentially bind the allosteric sites of both AKT and SGK. Molecules with no implicit toxicities and optimal drug-like properties were used for docking studies. Binding energies of the stable complexes that the designed molecules formed with AKT and SGK were calculated. Possible applications of the designed putative inhibitors against cancers with overexpressed AKT/SGK is discussed.

  11. Protocatechuic acid inhibits cancer cell metastasis involving the down-regulation of Ras/Akt/NF-κB pathway and MMP-2 production by targeting RhoB activation

    PubMed Central

    Lin, Hui-Hsuan; Chen, Jing-Hsien; Chou, Fen-Pi; Wang, Chau-Jong

    2011-01-01

    BACKGROUND AND PURPOSE Protocatechuic acid (PCA) is plentiful in edible fruits and vegetables and is thus one anti-oxidative component of normal human diets. However, the molecular mechanisms involved in the chemopreventive activity of PCA are poorly understood. Here, we investigated the mechanism(s) underlying the anti-metastatic potential of PCA. EXPERIMENTAL APPROACH We used AGS cells in a wound healing model and Boyden chamber assays in vitro and injection of B16/F10 melanoma cells in mice (metastasis model in vivo) to analyse the effect of PCA on cancer cell invasion and metastasis. The activities and expression of molecular proteins were measured by zymographic assay, real-time RT-PCR and Western blotting. KEY RESULTS PCA inhibited cell migration and invasion at non-cytotoxic concentrations. Decreased expression of matrix metalloproteinase (MMP)-2 and a coincident increase in tissue inhibitor of MMP followed treatment with PCA. The PCA-inhibited MMP-2 activity and expression was accompanied by inactivation of NF-κB. All these effects of PCA could be mediated via the RhoB/ protein kinase Cε (PKCε) and Ras/Akt cascade pathways, as demonstrated by inhibition of PKCε and transfection of PKCε siRNA and ras overexpression vector. Finally, PCA inhibited metastasis of B16/F10 melanoma cells to the liver in mice. CONCLUSION AND IMPLICATIONS Our data imply that PCA down-regulated the Ras/Akt/NF-κB pathway by targeting RhoB activation, which in turn led to a reduction of MMP-mediated cellular events in cancer cells and provides a new mechanism for the anti-cancer activity of PCA. PMID:20840540

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

    PubMed

    Thamilselvan, Vijayalakshmi; Menon, Mani; Thamilselvan, Sivagnanam

    2016-10-01

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

  13. Targeting of the Akt-Nuclear Factor-κB Signaling Network by [1-(4-Chloro-3-nitrobenzenesulfonyl)-1H-indol-3-yl]-methanol (OSU-A9), a Novel Indole-3-Carbinol Derivative, in a Mouse Model of Hepatocellular Carcinoma

    PubMed Central

    Omar, Hany A.; Sargeant, Aaron M.; Weng, Jing-Ru; Wang, Dasheng; Kulp, Samuel K.; Patel, Tushar

    2009-01-01

    Constitutive activation of Akt and nuclear factor-κB (NF-κB) represents major cellular abnormalities associated with the development and progression of hepatocellular carcinoma (HCC). Based on the structure of indole-3-carbinol, a chemopreventive phytochemical, we developed a novel derivative, [1-(4-chloro-3-nitrobenzenesulfonyl)-1H-indol-3-yl]-methanol (OSU-A9), that exhibits higher potency in inducing apoptosis by targeting the Akt-NF-κB signaling network. This study was aimed at assessing the antitumor activity of OSU-A9 using both in vitro and in vivo models of HCC, a malignancy in which the Akt-NF-κB signaling network plays major roles in pathogenesis and therapeutic resistance. Our data show that OSU-A9 was 100 times more potent than indole-3-carbinol in suppressing the viability of Hep3B, Huh7, and PLC5 HCC cells with IC50 values ranging from 2.8 to 3.2 μM. OSU-A9 interfered with the interplay between Akt- and NF-κB-mediated oncogenic signaling, leading to changes in the functional status of diverse signaling effectors involved in cell cycle progression, apoptosis, angiogenesis, and metastasis. The in vivo efficacy of OSU-A9 was assessed in nude mice bearing luciferase-expressing Hep3B xenograft tumors. Daily oral treatments with OSU-A9 at 25 or 50 mg/kg for 56 days suppressed tumor growth by 67 and 80%, respectively, which was correlated with changes in intratumoral biomarkers pertinent to Akt-NF-κB signaling, and without apparent toxicity or evidence of hepatic biotransformation enzyme induction. Together, these findings indicate that OSU-A9 is a potent, orally bioavailable inhibitor of the Akt-NF-κB signaling network with a broad spectrum of antitumor activity that includes targets regulating multiple aspects of HCC pathogenesis and progression. PMID:19706731

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

    PubMed

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

    2015-01-01

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

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

    PubMed Central

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

    2015-01-01

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

  16. Heterophyllin B inhibits the adhesion and invasion of ECA-109 human esophageal carcinoma cells by targeting PI3K/AKT/β-catenin signaling

    PubMed Central

    TANTAI, JI-CHENG; ZHANG, YAO; ZHAO, HENG

    2016-01-01

    The present study aimed to measure the effect of heterophyllin B (HB) on the adhesion and invasion of ECA-109 human esophageal carcinoma cells, and examine the possible mechanism involved. A Cell Counting kit 8 assay was performed to determine the cell viability. Cell adhesion and invasion were determined following treatment of the ECA-109 cells with HB (0, 10, 25 and 50 µM) for 24 h. The levels of phosphorylated (p-)ATK and p-phosphoinositide 3-kinase (PI3K), and the protein levels of β-catenin were measured using western blot analysis. The mRNA and protein expression levels of E-cadherin, vimentin, snail, matrix metalloproteinase (MMP)2 and MMP9 were detected using reverse trancsription-quantitative polymerase chain reaction and western blot analyses, respectively. HB (10, 25 and 50 µM) significantly suppressed the adhesion and invasion of the ECA-109 human esophageal carcinoma cells in a dose-dependant manner. The expression levels of p-ATK, p-PI3K and β-catenin were markedly decreased. The expression of E-cadherin was promoted, whereas the expression levels of snail, vimentin, MMP 2 and MMP 9 were decreased significantly in the ECA-109 cells treated with HB. In addition, HB inhibited the adhesion and invasion induced by PI3K activating peptide in the ECA-109 cells, and the protein expression levels were also adjusted. These results suggested that HB effectively suppressed the adhesion and invasion of the human esophageal carcinoma cells by mediating the PI3K/AKT/β-catenin pathways and regulating the expression levels of adhesion- and invasion-associated genes. PMID:26647768

  17. Heterophyllin B inhibits the adhesion and invasion of ECA-109 human esophageal carcinoma cells by targeting PI3K/AKT/β-catenin signaling.

    PubMed

    Tantai, Ji-Cheng; Zhang, Yao; Zhao, Heng

    2016-02-01

    The present study aimed to measure the effect of heterophyllin B (HB) on the adhesion and invasion of ECA-109 human esophageal carcinoma cells, and examine the possible mechanism involved. A Cell Counting kit 8 assay was performed to determine the cell viability. Cell adhesion and invasion were determined following treatment of the ECA-109 cells with HB (0, 10, 25 and 50 µM) for 24 h. The levels of phosphorylated (p-)ATK and p-phosphoinositide 3-kinase (PI3K), and the protein levels of β-catenin were measured using western blot analysis. The mRNA and protein expression levels of E-cadherin, vimentin, snail, matrix metalloproteinase (MMP)2 and MMP9 were detected using reverse transcription-quantitative polymerase chain reaction and western blot analyses, respectively. HB (10, 25 and 50 µM) significantly suppressed the adhesion and invasion of the ECA-109 human esophageal carcinoma cells in a dose-dependant manner. The expression levels of p-ATK, p-PI3K and β-catenin were markedly decreased. The expression of E-cadherin was promoted, whereas the expression levels of snail, vimentin, MMP 2 and MMP 9 were decreased significantly in the ECA-109 cells treated with HB. In addition, HB inhibited the adhesion and invasion induced by PI3K activating peptide in the ECA-109 cells, and the protein expression levels were also adjusted. These results suggested that HB effectively suppressed the adhesion and invasion of the human esophageal carcinoma cells by mediating the PI3K/AKT/β-catenin pathways and regulating the expression levels of adhesion- and invasion-associated genes. PMID:26647768

  18. Akt-mediated regulation of NFκB and the essentialness of NFκB for the oncogenicity of PI3K and Akt

    PubMed Central

    Bai, Dong; Ueno, Lynn; Vogt, Peter K.

    2009-01-01

    The serine/threonine kinase Akt (cellular homolog of murine thymoma virus akt8 oncogene), also known as PKB (protein kinase B), is activated by lipid products of phosphatidylinositol 3-kinase (PI3K). Akt phosphorylates numerous protein targets that control cell survival, proliferation and motility. Previous studies suggest that Akt regulates transcriptional activity of the nuclear factor-κB (NFκB) by inducing phosphorylation and subsequent degradation of inhibitor of κB (IκB). We show here that NFκB-driven transcription increases in chicken embryonic fibroblasts (CEF) transformed by myristylated Akt (myrAkt). Accordingly, both a dominant negative mutant of Akt and Akt inhibitors repress NFκB-dependent transcription. The degradation of the IκB protein is strongly enhanced in Akt-transformed cells, and the loss of NFκB activity by introduction of a super-repressor of NFκB, IκBSR, interferes with PI3K- and Akt-induced oncogenic transformation of CEF. The phosphorylation of the p65 subunit of NFκB at serine 534 is also upregulated in Akt-transformed cells. Our data suggest that the stimulation of NFκB by Akt is dependent on the phosphorylation of p65 at S534, mediated by IKK (IκB kinase) α and β. Akt phosphorylates IKKα on T23, and this phosphorylation event is a prerequisite for the phosphorylation of p65 at S534 by IKKα and β. Our results demonstrate two separate functions of the IKK complex in NFκB activation in cells with constitutive Akt activity: the phosphorylation and consequent degradation of IκB and the phosphorylation of p65. The data further support the conclusion that NFκB activity is essential for PI3K- and Akt-induced oncogenic transformation. PMID:19609947

  19. Alternative Mammalian Target of Rapamycin (mTOR) Signal Activation in Sorafenib-resistant Hepatocellular Carcinoma Cells Revealed by Array-based Pathway Profiling*

    PubMed Central

    Masuda, Mari; Chen, Wei-Yu; Miyanaga, Akihiko; Nakamura, Yuka; Kawasaki, Kumiko; Sakuma, Tomohiro; Ono, Masaya; Chen, Chi-Long; Honda, Kazufumi; Yamada, Tesshi

    2014-01-01

    Sorafenib is a multi-kinase inhibitor that has been proven effective for the treatment of unresectable hepatocellular carcinoma (HCC). However, its precise mechanisms of action and resistance have not been well established. We have developed high-density fluorescence reverse-phase protein arrays and used them to determine the status of 180 phosphorylation sites of signaling molecules in the 120 pathways registered in the NCI-Nature curated database in 23 HCC cell lines. Among the 180 signaling nodes, we found that the level of ribosomal protein S6 phosphorylated at serine residue 235/236 (p-RPS6 S235/236) was most significantly correlated with the resistance of HCC cells to sorafenib. The high expression of p-RPS6 S235/236 was confirmed immunohistochemically in biopsy samples obtained from HCC patients who responded poorly to sorafenib. Sorafenib-resistant HCC cells showed constitutive activation of the mammalian target of rapamycin (mTOR) pathway, but whole-exon sequencing of kinase genes revealed no evident alteration in the pathway. p-RPS6 S235/236 is a potential biomarker that predicts unresponsiveness of HCC to sorafenib. The use of mTOR inhibitors may be considered for the treatment of such tumors. PMID:24643969

  20. Non-human primate fetal kidney transcriptome analysis indicates mammalian target of rapamycin (mTOR) is a central nutrient-responsive pathway

    PubMed Central

    Nijland, Mark J; Schlabritz-Loutsevitch, Natalia E; Hubbard, Gene B; Nathanielsz, Peter W; Cox, Laura A

    2007-01-01

    Developmental programming is defined as the process by which gene–environment interaction in the developing organism leads to permanent changes in phenotype and function. Numerous reports of maternal nutrient restriction during pregnancy demonstrate altered renal development. Typically this alteration manifests as a reduction in the total number of glomeruli in the mature kidney of the offspring, and suggests that predisposition to develop chronic renal disease may include an in utero origin. In a previous study, we defined the transcriptome in the kidney from fetuses of control (CON, fed ad libitum) and nutrient-restricted (NR, fed 70% of CON starting at 0.16 gestation (G)) pregnancies at half-way through gestation (0.5G), and established transcriptome and morphological changes in NR kidneys compared to CON. One goal of the present study was to use transcriptome data from fetal kidneys of CON and NR mothers at 0.5G with histological data to identify the molecular mechanisms that may regulate renal development. A second goal was to identify mechanisms by which NR elicits its affect on fetal baboon kidney. We have used an end-of-pathway gene expression analysis to prioritize and identify key pathways regulating the 0.5G kidney phenotype in response NR. From these data we have determined that the mammalian target of rapamycin (mTOR) signalling pathway is central to this phenotype. PMID:17185341

  1. Activation of mammalian target of rapamycin contributes to pain nociception induced in rats by BmK I, a sodium channel-specific modulator.

    PubMed

    Jiang, Feng; Hua, Li-Ming; Jiao, Yun-Lu; Ye, Pin; Fu, Jin; Cheng, Zhi-Jun; Ding, Gang; Ji, Yong-Hua

    2014-02-01

    The mammalian target of rapamycin (mTOR) pathway is essential for maintenance of the sensitivity of certain adult sensory neurons. Here, we investigated whether the mTOR cascade is involved in scorpion envenomation-induced pain hypersensitivity in rats. The results showed that intraplantar injection of a neurotoxin from Buthus martensii Karsch, BmK I (10 μg), induced the activation of mTOR, as well as its downstream molecules p70 ribosomal S6 protein kinase (p70 S6K) and eukaryotic initiation factor 4E-binding protein 1 (4E-BP1), in lumbar 5-6 dorsal root ganglia neurons on both sides in rats. The activation peaked at 2 h and recovered 1 day after injection. Compared with the control group, the ratios of p-mTOR/p-p70 S6K/p-4EBP1 in three types of neurons changed significantly. The cell typology of p-mTOR/p-p70 S6K/p-4E-BP1 immuno-reactive neurons also changed. Intrathecal administration of deforolimus, a specific inhibitor of mTOR, attenuated BmK I-induced pain responses (spontaneous flinching, paroxysmal pain-like behavior, and mechanical hypersensitivity). Together, these results imply that the mTOR signaling pathway is mobilized by and contributes to experimental scorpion sting-induced pain. PMID:24132796

  2. Phophatidylinositol-3 kinase/mammalian target of rapamycin/p70S6K regulates contractile protein accumulation in airway myocyte differentiation.

    PubMed

    Halayko, Andrew J; Kartha, Sreedharan; Stelmack, Gerald L; McConville, John; Tam, John; Camoretti-Mercado, Blanca; Forsythe, Sean M; Hershenson, Marc B; Solway, Julian

    2004-09-01

    Increased airway smooth muscle in airway remodeling results from myocyte proliferation and hypertrophy. Skeletal and vascular smooth muscle hypertrophy is induced by phosphatidylinositide-3 kinase (PI(3) kinase) via mammalian target of rapamycin (mTOR) and p70S6 kinase (p70S6K). We tested the hypothesis that this pathway regulates contractile protein accumulation in cultured canine airway myocytes acquiring an elongated contractile phenotype in serum-free culture. In vitro assays revealed a sustained activation of PI(3) kinase and p70S6K during serum deprivation up to 12 d, with concomitant accumulation of SM22 and smooth muscle myosin heavy chain (smMHC) proteins. Immunocytochemistry revealed that activation of PI3K/mTOR/p70S6K occurred almost exclusively in myocytes that acquire the contractile phenotype. Inhibition of PI(3) kinase or mTOR with LY294002 or rapamycin blocked p70S6K activation, prevented formation of large elongated contractile phenotype myocytes, and blocked accumulation of SM22 and smMHC. Inhibition of MEK had no effect. Steady-state mRNA abundance for SM22 and smMHC was unaffected by blocking p70S6K activation. These studies provide primary evidence that PI(3) kinase and mTOR activate p70S6K in airway myocytes leading to the accumulation of contractile apparatus proteins, differentiation, and growth of large, elongated contractile phenotype airway smooth muscle cells. PMID:15105162

  3. Different in vitro proliferation and cytokine-production inhibition of memory T-cell subsets after calcineurin and mammalian target of rapamycin inhibitors treatment.

    PubMed

    Merino, David; San Segundo, David; Medina, Juan M; Rodrigo, Emilio; Asensio, Esther; Irure, Juan; Fernández-Fresnedo, Gema; Arias, Manuel A; López-Hoyos, Marcos

    2016-06-01

    Calcineurin inhibitors (CNI) and mammalian target of rapamycin inhibitors (mTORi) are the main immunosuppressants used for long-term maintenance therapy in transplant recipients to avoid acute rejection episodes. Both groups of immunosuppressants have wide effects and are focused against the T cells, although different impacts on specific T-cell subsets, such as regulatory T cells, have been demonstrated. A greater knowledge of the impact of immunosuppression on the cellular components involved in allograft rejection could facilitate decisions for individualized immunosuppression when an acute rejection event is suspected. Memory T cells have recently gained focus because they might induce a more potent response compared with naive cells. The impact of immunosuppressants on different memory T-cell subsets remains unclear. In the present study, we have studied the specific impact of CNI (tacrolimus) and mTORi (rapamycin and everolimus) over memory and naive CD4(+) T cells. To do so, we have analysed the proliferation, phenotypic changes and cytokine synthesis in vitro in the presence of these immunosuppressants. The present work shows a more potent effect of CNI on proliferation and cytokine production in naive and memory T cells. However, the mTORi permit the differentiation of naive T cells to the memory phenotype and allow the production of interleukin-2. Taken together, our data show evidence to support the combined use of CNI and mTORi in transplant immunosuppression. PMID:26931075

  4. Spotting the enemy within: Targeted silencing of foreign DNA in mammalian genomes by the Krüppel-associated box zinc finger protein family.

    PubMed

    Wolf, Gernot; Greenberg, David; Macfarlan, Todd S

    2015-01-01

    Tandem C2H2-type zinc finger proteins (ZFPs) constitute the largest transcription factor family in animals. Tandem-ZFPs bind DNA in a sequence-specific manner through arrays of multiple zinc finger domains that allow high flexibility and specificity in target recognition. In tetrapods, a large proportion of tandem-ZFPs contain Krüppel-associated-box (KRAB) repression domains, which are able to induce epigenetic silencing through the KAP1 corepressor. The KRAB-ZFP family continuously amplified in tetrapods through segmental gene duplications, often accompanied by deletions, duplications, and mutations of the zinc finger domains. As a result, tetrapod genomes contain unique sets of KRAB-ZFP genes, consisting of ancient and recently evolved family members. Although several hundred human and mouse KRAB-ZFPs have been identified or predicted, the biological functions of most KRAB-ZFP family members have gone unexplored. Furthermore, the evolutionary forces driving the extraordinary KRAB-ZFP expansion and diversification have remained mysterious for decades. In this review, we highlight recent studies that associate KRAB-ZFPs with the repression of parasitic DNA elements in the mammalian germ line and discuss the hypothesis that the KRAB-ZFP family primarily evolved as an adaptive genomic surveillance system against foreign DNA. Finally, we comment on the computational, genetic, and biochemical challenges of studying KRAB-ZFPs and attempt to predict how these challenges may be soon overcome. PMID:26435754

  5. The mammalian target of rapamycin signaling pathway regulates myocyte enhancer factor-2C phosphorylation levels through integrin-linked kinase in goat skeletal muscle satellite cells.

    PubMed

    Wu, Haiqing; Ren, Yu; Pan, Wei; Dong, Zhenguo; Cang, Ming; Liu, Dongjun

    2015-11-01

    Mammalian target of rapamycin (mTOR) signaling pathway plays a key role in muscle development and is involved in multiple intracellular signaling pathways. Myocyte enhancer factor-2 (MEF2) regulates muscle cell proliferation and differentiation. However, how the mTOR signaling pathway regulates MEF2 activity remains unclear. We isolated goat skeletal muscle satellite cells (gSSCs) as model cells to explore mTOR signaling pathway regulation of MEF2C. We inhibited mTOR activity in gSSCs with PP242 and found that MEF2C phosphorylation was decreased and that muscle creatine kinase (MCK) expression was suppressed. Subsequently, we detected integrin-linked kinase (ILK) using MEF2C coimmunoprecipitation; ILK and MEF2C were colocalized in the gSSCs. We found that inhibiting mTOR activity increased ILK phosphorylation levels and that inhibiting ILK activity with Cpd 22 and knocking down ILK with small interfering RNA increased MEF2C phosphorylation and MCK expression. In the presence of Cpd 22, mTOR activity inhibition did not affect MEF2C phosphorylation. Moreover, ILK dephosphorylated MEF2C in vitro. These results suggest that the mTOR signaling pathway regulates MEF2C positively and regulates ILK negatively and that ILK regulates MEF2C negatively. It appears that the mTOR signaling pathway regulates MEF2C through ILK, further regulating the expression of muscle-related genes in gSSCs. PMID:26041412

  6. Fibrous Papule of the Face, Similar to Tuberous Sclerosis Complex-Associated Angiofibroma, Shows Activation of the Mammalian Target of Rapamycin Pathway: Evidence for a Novel Therapeutic Strategy?

    PubMed Central

    Chan, Jung-Yi Lisa; Wang, Kuo-Hsien; Fang, Chia-Lang; Chen, Wei-Yu

    2014-01-01

    Fibrous papules of the face are hamartomas characterized by stellate-shaped stromal cells, multinucleated giant cells, and proliferative blood vessels in the dermis. The pathogenesis of fibrous papules remains unclear. There is a striking microscopic resemblance between fibrous papules and tuberous sclerosis complex (TSC)-associated angiofibromas. A germline mutation of the TSC1 or TSC2 gene, leading to activation of the mammalian target of rapamycin (mTOR) pathway, accounts for the pathogenesis of TSC-associated angiofibromas. Activated mTOR subsequently activates p70 ribosomal protein S6 kinase (p70S6K) and ribosomal protein S6 (S6) by phosphorylation. Rapamycin, a mTOR inhibitor, is effective in treating TSC-associated angiofibromas. The aim of this study was to understand whether the mTOR pathway is activated in fibrous papules. We studied immunoexpressions of phosphorylated (p-) mTOR effectors in fibrous papules, TSC-associated angiofibromas, and normal skin controls. P-mTOR, p-p70S6K and p-S6 were highly expressed in dermal stromal cells and epidermal keratinocytes in fibrous papules and TSC-associated angiofibromas but not in fibroblasts and epidermal keratinocytes of normal skin controls (p<0.001). The results suggest topical rapamycin may be a novel treatment option for fibrous papules. PMID:24558502

  7. Inhibition of Mammalian Target of Rapamycin Complex 1 (mTORC1) Downregulates ELOVL1 Gene Expression and Fatty Acid Synthesis in Goat Fetal Fibroblasts

    PubMed Central

    Wang, Weipeng; He, Qiburi; Guo, Zhixin; Yang, Limin; Bao, Lili; Bao, Wenlei; Zheng, Xu; Wang, Yanfeng; Wang, Zhigang

    2015-01-01

    Elongation of very-long-chain fatty acids 1 (ELOVL1) is a ubiquitously expressed gene that belongs to the ELOVL family and regulates the synthesis of very-long-chain fatty acids (VLCFAs) and sphingolipids, from yeast to mammals. Mammalian target of rapamycin complex 1 (mTORC1) is a central regulator of cell metabolism and is associated with fatty acids synthesis. In this study, we cloned the cDNA that encodes Cashmere goat (Capra hircus) ELOVL1 (GenBank Accession number KF549985) and investigated its expression in 10 tissues. ELOVL1 cDNA was 840 bp, encoding a deduced protein of 279 amino acids, and ELOVL1 mRNA was expressed in a wide range of tissues. Inhibition of mTORC1 by rapamycin decreased ELOVL1 expression and fatty acids synthesis in Cashmere goat fetal fibroblasts. These data show that ELOVL1 expression is regulated by mTORC1 and that mTORC1 has significant function in fatty acids synthesis in Cashmere goat. PMID:26204830

  8. The Mammalian “Obesogen” Tributyltin Targets Hepatic Triglyceride Accumulation and the Transcriptional Regulation of Lipid Metabolism in the Liver and Brain of Zebrafish

    PubMed Central

    Lyssimachou, Angeliki; Santos, Joana G.; André, Ana; Soares, Joana; Lima, Daniela; Guimarães, Laura; Almeida, C. Marisa R.; Teixeira, Catarina; Castro, L. Filipe C.; Santos, Miguel M.

    2015-01-01

    Recent findings indicate that different Endocrine Disrupting Chemicals (EDCs) interfere with lipid metabolic pathways in mammals and promote fat accumulation, a previously unknown site of action for these compounds. The antifoulant and environmental pollutant tributyltin (TBT), which causes imposex in gastropod snails, induces an “obesogenic” phenotype in mammals, through the activation of the nuclear receptors retinoid X receptor (RXR) and peroxisome proliferator-activated receptor gamma (PPARγ). In teleosts, the effects of TBT on the lipid metabolism are poorly understood, particularly following exposure to low, environmental concentrations. In this context, the present work shows that exposure of zebrafish to 10 and 50 ng/L of TBT (as Sn) from pre-hatch to 9 months of age alters the body weight, condition factor, hepatosomatic index and hepatic triglycerides in a gender and dose related manner. Furthermore, TBT modulated the transcription of key lipid regulating factors and enzymes involved in adipogenesis, lipogenesis, glucocorticoid metabolism, growth and development in the brain and liver of exposed fish, revealing sexual dimorphic effects in the latter. Overall, the present study shows that the model mammalian obesogen TBT interferes with triglyceride accumulation and the transcriptional regulation of lipid metabolism in zebrafish and indentifies the brain lipogenic transcription profile of fish as a new target of this compound. PMID:26633012

  9. Screening of mammalian target of rapamycin inhibitors in natural product extracts by capillary electrophoresis in combination with high performance liquid chromatography-tandem mass spectrometry.

    PubMed

    Zhang, Yanmei; Li, Feng; Li, Mingxia; Kang, Jingwu

    2015-04-01

    In this study, capillary electrophoresis (CE) combined with HPLC-MS/MS were used as a powerful platform for screening of inhibitors of mammalian target of rapamycin (mTOR) in natural product extracts. The screening system has been established by using 5-carboxyfluorescein labeled substrate peptide F-4EBP1, a known mTOR inhibitor AZD8055, and a small chemical library consisted of 18 natural product extracts. Biochemical screening of natural product extracts was performed by CE with laser induced fluorescence detection. The CE separation allowed a quantitative measurement of the phosphorylated product, hence the quantitation of enzymatic inhibition as well as inhibition kinetics. The hits are readily identified as long as the peak area of the phosphorylated product is reduced in comparison with the negative control. Subsequent assay-guided isolation of the active natural product extract was performed with HPLC-MS/MS to track the particular active components. The structures of the identified active components were elucidated by the molecular ions and fragmentation information provided by MS/MS analysis. The CE-based assay method only requires minute pure compounds, which can be readily purified by HPLC. Therefore, the combination of CE and HPLC-MS/MS provides a high-throughput platform for screening bioactive compounds from the crude nature extracts. By taking the advantage of the screening system, salvianolic acid A and C in extract of Salvia miltiorrhiza were discovered as the new mTOR inhibitors. PMID:25725958

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

    PubMed Central

    Wang, Jian; Feng, Da-Yun; Li, Zhi-Hua; Feng, Ban; Zhang, Han; Zhang, Ting; Chen, Tao; Li, Yun-Qing

    2015-01-01

    The mammalian target of rapamycin (mTOR), a serine-threonine protein kinase, integrates extracellular signals, thereby modulating several physiological and pathological processes, including pain. Previous studies have suggested that rapamycin (an mTOR inhibitor) can attenuate nociceptive behaviors in many pain models, most likely at the spinal cord level. However, the mechanisms of mTOR at the supraspinal level, particularly at the level of the rostral ventromedial medulla (RVM), remain unclear. Thus, the aim of this study was to elucidate the role of mTOR in the RVM, a key relay region for the descending pain control pathway, under neuropathic pain conditions. Phosphorylated mTOR was mainly expressed in serotonergic spinally projecting neurons and was significantly increased in the RVM after spared nerve injury- (SNI-) induced neuropathic pain. Moreover, in SNI rat brain slices, rapamycin infusion both decreased the amplitude instead of the frequency of spontaneous excitatory postsynaptic currents and reduced the numbers of action potentials in serotonergic neurons. Finally, intra-RVM microinjection of rapamycin effectively alleviated established mechanical allodynia but failed to affect the development of neuropathic pain. In conclusion, our data provide strong evidence for the role of mTOR in the RVM in nerve injury-induced neuropathic pain, indicating a novel mechanism of mTOR inhibitor-induced analgesia. PMID:26770837

  11. Altered APP Processing in Insulin-Resistant Conditions Is Mediated by Autophagosome Accumulation via the Inhibition of Mammalian Target of Rapamycin Pathway

    PubMed Central

    Son, Sung Min; Song, Hyundong; Byun, Jayoung; Park, Kyong Soo; Jang, Hak Chul; Park, Young Joo; Mook-Jung, Inhee

    2012-01-01

    Insulin resistance, one of the major components of type 2 diabetes mellitus (T2DM), is a known risk factor for Alzheimer’s disease (AD), which is characterized by an abnormal accumulation of intra- and extracellular amyloid β peptide (Aβ). Insulin resistance is known to increase Aβ generation, but the underlying mechanism that links insulin resistance to increased Aβ generation is unknown. In this study, we examined the effect of high-fat diet–induced insulin resistance on amyloid precursor protein (APP) processing in mouse brains. We found that the induced insulin resistance promoted Aβ generation in the brain via altered insulin signal transduction, increased β- and γ-secretase activities, and accumulation of autophagosomes. These findings were confirmed in diabetic db/db mice brains. Furthermore, in vitro experiments in insulin-resistant SH-SY5Y cells and primary cortical neurons confirmed the alteration of APP processing by insulin resistance–induced autophagosome accumulation. Defects in insulin signal transduction affect autophagic flux by inhibiting the mammalian target of rapamycin pathway, resulting in altered APP processing in these cell culture systems. Thus, the insulin resistance that underlies the pathogenesis of T2DM might also trigger accumulation of autophagosomes, leading to increased Aβ generation, which might be involved in the pathogenesis of AD. PMID:22829447

  12. Mammalian pheromones.

    PubMed

    Liberles, Stephen D

    2014-01-01

    Mammalian pheromones control a myriad of innate social behaviors and acutely regulate hormone levels. Responses to pheromones are highly robust, reproducible, and stereotyped and likely involve developmentally predetermined neural circuits. Here, I review several facets of pheromone transduction in mammals, including (a) chemosensory receptors and signaling components of the main olfactory epithelium and vomeronasal organ involved in pheromone detection; (b) pheromone-activated neural circuits subject to sex-specific and state-dependent modulation; and (c) the striking chemical diversity of mammalian pheromones, which range from small, volatile molecules and sulfated steroids to large families of proteins. Finally, I review (d) molecular mechanisms underlying various behavioral and endocrine responses, including modulation of puberty and estrous; control of reproduction, aggression, suckling, and parental behaviors; individual recognition; and distinguishing of own species from predators, competitors, and prey. Deconstruction of pheromone transduction mechanisms provides a critical foundation for understanding how odor response pathways generate instinctive behaviors. PMID:23988175

  13. Mammalian Pheromones

    PubMed Central

    Liberles, Stephen D.

    2015-01-01

    Mammalian pheromones control a myriad of innate social behaviors and acutely regulate hormone levels. Responses to pheromones are highly robust, reproducible, and stereotyped and likely involve developmentally predetermined neural circuits. Here, I review several facets of pheromone transduction in mammals, including (a) chemosensory receptors and signaling components of the main olfactory epithelium and vomeronasal organ involved in pheromone detection; (b) pheromone-activated neural circuits subject to sex-specific and state-dependent modulation; and (c) the striking chemical diversity of mammalian pheromones, which range from small, volatile molecules and sulfated steroids to large families of proteins. Finally, I review (d ) molecular mechanisms underlying various behavioral and endocrine responses, including modulation of puberty and estrous; control of reproduction, aggression, suckling, and parental behaviors; individual recognition; and distinguishing of own species from predators, competitors, and prey. Deconstruction of pheromone transduction mechanisms provides a critical foundation for understanding how odor response pathways generate instinctive behaviors. PMID:23988175

  14. MicroRNA-195 targets VEGFR2 and has a tumor suppressive role in ACHN cells via PI3K/Akt and Raf/MEK/ERK signaling pathways.

    PubMed

    Sun, Pengcheng; Wang, Lu; Lu, Yunhan; Liu, Yuwei; Li, Lechen; Yin, Luyao; Zhang, Cheng; Zhao, Weiming; Shen, Baozhong; Xu, Wanhai

    2016-09-01

    Increasing evidence indicates that dysregulation of miR-195 may contribute to the occurrence and development of multiple types of human malignancies. However, the function and the mechanism of miR-195 in clear cell renal cell carcinoma (ccRCC) are still not fully understood. In the present study, we used qRT-PCR to detect the expression of miR-195 in ccRCC tissues and normal kidney tissues. MTT assay was performed to detect the cell viability of miR-195. Migration and invasion were evaluated by Transwell migration and Matrigel invasion assays, respectively. Additionally, apoptosis levels were evaluated using TUNEL assays, and signaling pathway changes were determined by western blot analysis. We observed that miR-195 was downregulated in clear cell renal cell carcinoma samples compared with normal renal samples. We identified that overexpression of miR-195 inhibited ACHN cell viability, migration, invasion, and it also induced cell apoptosis by targeting VEGFR2 via PI3K/Akt and Raf/MEK/ERK signaling pathways. These findings indicate that miR-195 has a tumor suppressive role in ACHN cells and miR-195 may be a promising candidate target for prevention and treatment of renal cell carcinoma. PMID:27572273

  15. Illuminating the phosphatidylinositol 3-kinase/Akt pathway

    NASA Astrophysics Data System (ADS)

    Ni, Qiang; Fosbrink, Matthew; Zhang, Jin

    2008-02-01

    Genetically encodable fluorescent biosensors based on fluorescence resonance energy transfer (FRET) are being developed for analyzing spatiotemporal dynamics of various signaling events in living cells, as these events are often dynamically regulated and spatially compartmentalized within specific signaling context. In particular, to investigate the phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway in the cellular context, we have developed a series of such biosensors that enable dynamic visualization of several key signaling events in this pathway, namely InPAkt for lipid second messenger dynamics, BAKR for Akt activity, and ReAktion for the action of Akt during its multi-step activation process. Discussed here are several studies that have been carried out with these novel biosensors. First, we examined nuclear phosphatidylinositol-3,4,5-triphosphate (PIP 3) in living cells using nucleus-targeted InPAkt. Second, we analyzed signal propagation from the plasma membrane to the nucleus by using plasma membrane-targeted InPAkt and nucleus-targeted BKAR to simultaneously monitor PIP 3 dynamics and Akt activity in the same cell. Of note, results from these co-imaging experiments suggest that active Akt can dissociate from the plasma membrane and translocate into the nucleus in the presence of high levels of PIP 3 at the plasma membrane. This finding has led to a further study of the action of Akt during its activation process, particularly focusing on how Akt dissociates from the membrane. In this regard, a live-cell molecular analysis using ReAktion reveals a conformational change in Akt that is critically dependent on the existence of a phosphorylatable T308 in the activation loop. Subsequently this has led to the discovery of new regulatory roles of this critical phosphorylation event of Akt for ensuring its proper activation and function.

  16. Radiation-induced Akt activation modulates radioresistance in human glioblastoma cells

    PubMed Central

    Li, Hui-Fang; Kim, Jung-Sik; Waldman, Todd

    2009-01-01

    Background Ionizing radiation (IR) therapy is a primary treatment for glioblastoma multiforme (GBM), a common and devastating brain tumor in humans. IR has been shown to induce PI3K-Akt activation in many cell types, and activation of the PI3K-Akt signaling pathway has been correlated with radioresistance. Methods Initially, the effects of IR on Akt activation were assessed in multiple human GBM cell lines. Next, to evaluate a potential causative role of IR-induced Akt activation on radiosensitivity, Akt activation was inhibited during IR with several complementary genetic and pharmacological approaches, and radiosensitivity measured using clonogenic survival assays. Results Three of the eight cell lines tested demonstrated IR-induced Akt activation. Further studies revealed that IR-induced Akt activation was dependent upon the presence of a serum factor, and could be inhibited by the EGFR inhibitor AG1478. Inhibition of PI3K activation with LY294002, or with inducible wild-type PTEN, inhibition of EGFR, as well as direct inhibition of Akt with two Akt inhibitors during irradiation increased the radiosensitivity of U87MG cells. Conclusion These results suggest that Akt may be a central player in a feedback loop whereby activation of Akt induced by IR increases radioresistance of GBM cells. Targeting the Akt signaling pathway may have important therapeutic implications when used in combination with IR in the treatment of a subset of brain tumor patients. PMID:19828040

  17. Cystinosin is a Component of the Vacuolar H+-ATPase-Ragulator-Rag Complex Controlling Mammalian Target of Rapamycin Complex 1 Signaling.

    PubMed

    Andrzejewska, Zuzanna; Nevo, Nathalie; Thomas, Lucie; Chhuon, Cerina; Bailleux, Anne; Chauvet, Véronique; Courtoy, Pierre J; Chol, Marie; Guerrera, Ida Chiara; Antignac, Corinne

    2016-06-01

    Cystinosis is a rare autosomal recessive storage disorder characterized by defective lysosomal efflux of cystine due to mutations in the CTNS gene encoding the lysosomal cystine transporter, cystinosin. Lysosomal cystine accumulation leads to crystal formation and functional impairment of multiple organs. Moreover, cystinosis is the most common inherited cause of renal Fanconi syndrome in children. Oral cysteamine therapy delays disease progression by reducing intracellular cystine levels. However, because cysteamine does not correct all complications of cystinosis, including Fanconi syndrome, we hypothesized that cystinosin could have novel roles in addition to transporting cystine out of the lysosome. By coimmunoprecipitation experiments and mass spectrometry, we found cystinosin interacts with almost all components of vacuolar H(+)-ATPase and the Ragulator complex and with the small GTPases Ras-related GTP-binding protein A (RagA) and RagC. Furthermore, the mammalian target of rapamycin complex 1 (mTORC1) pathway was downregulated in proximal tubular cell lines derived from Ctns(-/-) mice. Decrease of lysosomal cystine levels by cysteamine did not rescue mTORC1 activation in these cells, suggesting that the downregulation of mTORC1 is due to the absence of cystinosin rather than to the accumulation of cystine. Our results show a dual role for cystinosin as a cystine transporter and as a component of the mTORC1 pathway, and provide an explanation for the appearance of Fanconi syndrome in cystinosis. Furthermore, this study highlights the need to develop new treatments not dependent on lysosomal cystine depletion alone for this devastating disease. PMID:26449607

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

    PubMed

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

    2014-01-17

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

  19. Peroxisome proliferator-activated receptor-γ agonist inhibits the mammalian target of rapamycin signaling pathway and has a protective effect in a rat model of status epilepticus.

    PubMed

    San, Yong-Zhi; Liu, Yu; Zhang, Yu; Shi, Ping-Ping; Zhu, Yu-Lan

    2015-08-01

    Peroxisome proliferator-activated receptor γ (PPAR-γ) has a protective role in several neurological diseases. The present study investigated the effect of the PPAR-γ agonist, pioglitazone, on the mammalian target of rapamycin (mTOR) signaling pathway in a rat model of pentylenetetrazol (PTZ)-induced status epilepticus (SE). The investigation proceeded in two stages. First, the course of activation of the mTOR signaling pathway in PTZ-induced SE was examined to determine the time-point of peak activity, as reflected by phopshorylated (p)-mTOR/mTOR and p-S6/S6 ratios. Subsequently, pioglitazone was administrated intragastrically to investigate its effect on the mTOR signaling pathway, through western blot and immunochemical analyses. The levels of the interleukin (IL)-1β and IL-6 inflammatory cytokines were detected using ELISA, and neuronal loss was observed via Nissl staining. In the first stage of experimentation, the mTOR signaling pathway was activated, and the p-mTOR/mTOR and p-S6/S6 ratios peaked on the third day. Compared with the vehicle treated-SE group, pretreatment with pioglitazone was associated with the loss of fewer neurons, lower levels of IL-1β and IL-6, and inhibition of the activation of the mTOR signaling pathway. Therefore, the mTOR signaling pathway was activated in the PTZ-induced SE rat model, and the PPAR-γ agonist, pioglitazone, had a neuroprotective effect, by inhibiting activation of the mTOR pathway and preventing the increase in the levels of IL-1β and IL-6. PMID:25891824

  20. cAMP-dependent activation of mammalian target of rapamycin (mTOR) in thyroid cells. Implication in mitogenesis and activation of CDK4.

    PubMed

    Blancquaert, Sara; Wang, Lifu; Paternot, Sabine; Coulonval, Katia; Dumont, Jacques E; Harris, Thurl E; Roger, Pierre P

    2010-07-01

    How cAMP-dependent protein kinases [protein kinase A (PKA)] transduce the mitogenic stimulus elicited by TSH in thyroid cells to late activation of cyclin D3-cyclin-dependent kinase 4 (CDK4) remains enigmatic. Here we show in PC Cl3 rat thyroid cells that TSH/cAMP, like insulin, activates the mammalian target of rapamycin (mTOR)-raptor complex (mTORC1) leading to phosphorylation of S6K1 and 4E-BP1. mTORC1-dependent S6K1 phosphorylation in response to both insulin and cAMP required amino acids, whereas inhibition of AMP-activated protein kinase and glycogen synthase kinase 3 enhanced insulin but not cAMP effects. Unlike insulin, TSH/cAMP did not activate protein kinase B or induce tuberous sclerosis complex 2 phosphorylation at T1462 and Y1571. However, like insulin, TSH/cAMP produced a stable increase in mTORC1 kinase activity that was associated with augmented 4E-BP1 binding to raptor. This could be caused in part by T246 phosphorylation of PRAS40, which was found as an in vitro substrate of PKA. Both in PC Cl3 cells and primary dog thyrocytes, rapamycin inhibited DNA synthesis and retinoblastoma protein phosphorylation induced by TSH and insulin. Although rapamycin reduced cyclin D3 accumulation, the abundance of cyclin D3-CDK4 complexes was not affected. However, rapamycin inhibited the activity of these complexes by decreasing the TSH and insulin-mediated stimulation of activating T172 phosphorylation of CDK4. We propose that mTORC1 activation by TSH, at least in part through PKA-dependent phosphorylation of PRAS40, crucially contributes to mediate cAMP-dependent mitogenesis by regulating CDK4 T172-phosphorylation. PMID:20484410

  1. A treadmill exercise reactivates the signaling of the mammalian target of rapamycin (mTor) in the skeletal muscles of starved mice.

    PubMed

    Zheng, Dong-Mei; Bian, Zehua; Furuya, Norihiko; Oliva Trejo, Juan Alejandro; Takeda-Ezaki, Mitsue; Takahashi, Katsuyuki; Hiraoka, Yuka; Mineki, Reiko; Taka, Hikari; Ikeda, Shin-Ichi; Komatsu, Masaaki; Fujimura, Tsutomu; Ueno, Takashi; Ezaki, Junji

    2015-01-01

    It has been well established that a starvation-induced decrease in insulin/IGF-I and serum amino acids effectively suppresses the mammalian target of rapamycin (mTor) signaling to induce autophagy, which is a major degradative cellular pathway in skeletal muscles. In this study, we investigated the systematic effects of exercise on the mTor signaling of skeletal muscles. Wild type C57BL/6J mice were starved for 24h under synchronous autophagy induction conditions. Under these conditions, endogenous LC3-II increased, while both S6-kinse and S6 ribosomal protein were dephosphorylated in the skeletal muscles, which indicated mTor inactivation. Using GFP-LC3 transgenic mice, it was also confirmed that fluorescent GFP-LC3 dots in the skeletal muscles increased, including soleus, plantaris, and gastrocnemius, which clearly showed autophagosomal induction. These starved mice were then subjected to a single bout of running on a treadmill (12m/min, 2h, with a lean of 10 degrees). Surprisingly, biochemical analyses revealed that the exercise elicited a decrease in the LC3-II/LC3-I ratio as well as an inversion from the dephosphorylated state to the rephosphorylated state of S6-kinase and ribosomal S6 in these skeletal muscles. Consistently, the GFP-LC3 dots of the skeletal muscles were diminished immediately after the exercise. These results indicated that exercise suppressed starvation-induced autophagy through a reactivation of mTor signaling in the skeletal muscles of these starved mice. PMID:25485704

  2. Mammalian target of rapamycin/p70 ribosomal S6 protein kinase signaling is altered by sevoflurane and/or surgery in aged rats.

    PubMed

    Liu, Yongzhe; Ma, Li; Jiao, Linbo; Gao, Minglong; Guo, Wenzhi; Chen, Lin; Pan, Ningling; Ma, Yaqun

    2015-12-01

    The mammalian target of rapamycin (mTOR)/p70 ribosomal S6 protein kinase (p70S6k) pathway exerts anti‑apoptotic effects that may contribute to disease pathogenesis. The memory impairment in patients with Alzheimer's disease (AD) has been suggested to be contributed to by abnormal mTOR signaling. The aim of the current study was to investigate the association between sevoflurane and/or surgery and AD through the mTOR/p70S6K signaling pathway. Sprague‑Dawley rats were randomly assigned to the sevoflurane, surgery or control groups. The animals in the surgery group received a partial hepatectomy under sevoflurane anesthesia. The hippocampal levels of phosphorylated (p)‑mTOR, p‑p70S6K, caspase‑3 and p‑tau/total (t)‑tau were analyzed. The Morris water maze (MWM) was used to evaluate cognitive function following treatment. The levels of p‑mTOR and p‑p70S6K were reduced, whereas caspase‑3 levels were increased in the surgery group compared with the sevoflurane group. The p‑tau/t‑tau levels were increased, however, tau mRNA was unaffected by sevoflurane and/or surgery. The rats in the surgery group required a significantly longer time to locate the platform in the MWM test compared with the control and sevoflurane groups. Sevoflurane treatment and/or surgery reduced anti‑apoptotic activity, and the postoperative cognitive dysfunction following surgery may be due to mTOR signaling pathway inhibition in aged rats. Increased neuronal apoptosis and tau phosphorylation are suggested to be involved in the association between anesthesia and AD occurrence. PMID:26497858

  3. Redox Regulates Mammalian Target of Rapamycin Complex 1 (mTORC1) Activity by Modulating the TSC1/TSC2-Rheb GTPase Pathway*

    PubMed Central

    Yoshida, Sei; Hong, Sungki; Suzuki, Tsukasa; Nada, Shigeyuki; Mannan, Aristotle M.; Wang, Junying; Okada, Masato; Guan, Kun-Liang; Inoki, Ken

    2011-01-01

    Mammalian target of rapamycin (mTOR) is a kinase that plays a key role in a wide array of cellular processes and exists in two distinct functional complexes, mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2). Although mTORC2 is primarily activated by growth factors, mTORC1 is regulated by numerous extracellular and intracellular signals such as nutrients, growth factors, and cellular redox. Previous study has shown that cysteine oxidants sufficiently activate mTORC1 activity under amino acid-depleted conditions and that a reducing agent effectively suppresses amino acid-induced mTORC1 activity, thereby raising the possibility that redox-sensitive mechanisms underlie amino acid-dependent mTORC1 regulation. However, the molecular mechanism by which redox regulates mTORC1 activity is not well understood. In this study, we show that the redox-sensitive regulation of mTORC1 occurs via Rheb but not the Rag small GTPase. Enhancing cellular redox potential with cysteine oxidants significantly increases Rheb GTP levels. Importantly, modulation of the cellular redox potential with a cysteine oxidant or reducing agent failed to alter mTORC1 activity in TSC1−/− or TSC2−/− mouse embryonic fibroblast cells. Furthermore, a cysteine oxidant has little effect on mTOR localization but sufficiently activates mTORC1 activity in both p18−/− and control mouse embryonic fibroblast cells, suggesting that the redox-sensitive regulation of mTORC1 occurs independent of the Ragulator·Rag complex. Taken together, our results suggest that the TSC complex plays an important role in redox-sensitive mTORC1 regulation and argues for the activation of mTORC1 in places other than the lysosome upon inhibition of the TSC complex. PMID:21784859

  4. Activation of Autophagic Flux against Xenoestrogen Bisphenol-A-induced Hippocampal Neurodegeneration via AMP kinase (AMPK)/Mammalian Target of Rapamycin (mTOR) Pathways*

    PubMed Central

    Agarwal, Swati; Tiwari, Shashi Kant; Seth, Brashket; Yadav, Anuradha; Singh, Anshuman; Mudawal, Anubha; Chauhan, Lalit Kumar Singh; Gupta, Shailendra Kumar; Choubey, Vinay; Tripathi, Anurag; Kumar, Amit; Ray, Ratan Singh; Shukla, Shubha; Parmar, Devendra; Chaturvedi, Rajnish Kumar

    2015-01-01

    The human health hazards related to persisting use of bisphenol-A (BPA) are well documented. BPA-induced neurotoxicity occurs with the generation of oxidative stress, neurodegeneration, and cognitive dysfunctions. However, the cellular and molecular mechanism(s) of the effects of BPA on autophagy and association with oxidative stress and apoptosis are still elusive. We observed that BPA exposure during the early postnatal period enhanced the expression and the levels of autophagy genes/proteins. BPA treatment in the presence of bafilomycin A1 increased the levels of LC3-II and SQSTM1 and also potentiated GFP-LC3 puncta index in GFP-LC3-transfected hippocampal neural stem cell-derived neurons. BPA-induced generation of reactive oxygen species and apoptosis were mitigated by a pharmacological activator of autophagy (rapamycin). Pharmacological (wortmannin and bafilomycin A1) and genetic (beclin siRNA) inhibition of autophagy aggravated BPA neurotoxicity. Activation of autophagy against BPA resulted in intracellular energy sensor AMP kinase (AMPK) activation, increased phosphorylation of raptor and acetyl-CoA carboxylase, and decreased phosphorylation of ULK1 (Ser-757), and silencing of AMPK exacerbated BPA neurotoxicity. Conversely, BPA exposure down-regulated the mammalian target of rapamycin (mTOR) pathway by phosphorylation of raptor as a transient cell's compensatory mechanism to preserve cellular energy pool. Moreover, silencing of mTOR enhanced autophagy, which further alleviated BPA-induced reactive oxygen species generation and apoptosis. BPA-mediated neurotoxicity also resulted in mitochondrial loss, bioenergetic deficits, and increased PARKIN mitochondrial translocation, suggesting enhanced mitophagy. These results suggest implication of autophagy against BPA-mediated neurodegeneration through involvement of AMPK and mTOR pathways. Hence, autophagy, which arbitrates cell survival and demise during stress conditions, requires further assessment to be

  5. Characterization of a conserved C-terminal motif (RSPRR) in ribosomal protein S6 kinase 1 required for its mammalian target of rapamycin-dependent regulation.

    PubMed

    Schalm, Stefanie S; Tee, Andrew R; Blenis, John

    2005-03-25

    The mammalian target of rapamycin, mTOR, is a Ser/Thr kinase that promotes cell growth and proliferation by activating ribosomal protein S6 kinase 1 (S6K1). We previously identified a conserved TOR signaling (TOS) motif in the N terminus of S6K1 that is required for its mTOR-dependent activation. Furthermore, our data suggested that the TOS motif suppresses an inhibitory function associated with the C terminus of S6K1. Here, we have characterized the mTOR-regulated inhibitory region within the C terminus. We have identified a conserved C-terminal "RSPRR" sequence that is responsible for an mTOR-dependent suppression of S6K1 activation. Deletion or mutations within this RSPRR motif partially rescue the kinase activity of the S6K1 TOS motif mutant (S6K1-F5A), and this rescued activity is rapamycin resistant. Furthermore, we have shown that the RSPRR motif significantly suppresses S6K1 phosphorylation at two phosphorylation sites (Thr-389 and Thr-229) that are crucial for S6K1 activation. Importantly, introducing both the Thr-389 phosphomimetic and RSPRR motif mutations into the catalytically inactive S6K1 mutant S6K1-F5A completely rescues its activity and renders it fully rapamycin resistant. These data show that the N-terminal TOS motif suppresses an inhibitory function mediated by the C-terminal RSPRR motif. We propose that the RSPRR motif interacts with a negative regulator of S6K1 that is normally suppressed by mTOR. PMID:15659381

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

    SciTech Connect

    Montes, Daniela K.; Brenet, Marianne; Muñoz, Vanessa C.; Burgos, Patricia V.; Villanueva, Carolina I.; Figueroa, Carlos D.; González, Carlos B.

    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.

  7. Salmonella induce autophagy in melanoma by the downregulation of AKT/mTOR pathway.

    PubMed

    Lee, C-H; Lin, S-T; Liu, J-J; Chang, W-W; Hsieh, J-L; Wang, W-K

    2014-03-01

    Salmonella have been demonstrated to inhibit tumor growth. However, the mechanism of Salmonella-induced tumor cell death is less defined. Autophagy is a cellular process that mediates the degradation of long-lived proteins and unwanted organelles in the cytosol. Tumor cells frequently display lower levels of basal autophagic activity than their normal counterparts and fail to increase autophagic activity in response to stresses. Autophagy is involved in the cell defense elimination of bacteria. The signaling pathways leading to activation of Salmonella-induced autophagy in tumor cells remain to be elucidated. We used autophagy inhibitor (3-Methyladenine) and apoptosis inhibitor (Z-VAD-FMK) to demonstrate that Salmonella may induce cell death via apoptosis and autophagic pathway. Meanwhile, we suggested that Salmonella induce autophagy in a dose- and time-dependent manner. The autophagic markers were increased after tumor cell infected with Salmonella. In addition, the protein express levels of phosph-protein kinase B (P-AKT), phosph-mammalian targets of rapamycin (P-mTOR), phosph-p70 ribosomal s6 kinase (P-p70s6K) in tumor cells were decreased by western analysis after Salmonella infection. In conclusion, our results point out that Salmonella induce the autophagic signaling pathway via downregulation of AKT/mTOR pathway. Herein, our findings that Salmonella in controlling tumor growth may induce autophagic signal pathway. PMID:24451116

  8. PI3K/Akt/mTOR inhibitors in breast cancer

    PubMed Central

    Lee, Joycelyn JX; Loh, Kiley; Yap, Yoon-Sim

    2015-01-01

    Activation of the phosphoinositide 3 kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) pathway is common in breast cancer. There is preclinical data to support inhibition of the pathway, and phase I to III trials involving inhibitors of the pathway have been or are being conducted in solid tumors and breast cancer. Everolimus, an mTOR inhibitor, is currently approved for the treatment of hormone receptor (HR)-positive, human epidermal growth factor receptor 2 (HER2)-negative breast cancer. In this review, we summarise the efficacy and toxicity findings from the randomised clinical trials, with simplified guidelines on the management of potential adverse effects. Education of healthcare professionals and patients is critical for safety and compliance. While there is some clinical evidence of activity of mTOR inhibition in HR-positive and HER2-positive breast cancers, the benefits may be more pronounced in selected subsets rather than in the overall population. Further development of predictive biomarkers will be useful in the selection of patients who will benefit from inhibition of the PI3K/Akt/mTOR (PAM) pathway. PMID:26779371

  9. Multi-targeted neuroprotection by the HSV-2 gene ICP10PK includes robust bystander activity through PI3-K/Akt and/or MEK/ERK-dependent neuronal release of vascular endothelial growth factor and fractalkine

    PubMed Central

    Laing, Jennifer M.; Smith, Cynthia C.; Aurelian, Laure

    2013-01-01

    Hippocampal cultures infected with the ΔRR vector for the HSV-2 anti-apoptotic gene ICP10PK survive cell death triggered by a wide variety of insults. Survival includes robust protection of uninfected neurons, but the mechanism of this bystander activity is still unclear. Here we report that ICP10PK+ neurons release soluble factors that protect uninfected neurons from NMDA and MPP+-induced apoptosis. Release depends on ICP10PK-mediated activation of the Ras signaling pathways MEK/ERK and PI3-K/Akt, and it was not seen for cultures infected with the ICP10PK negative vector ΔPK. The released neuroprotective factors include vascular endothelial growth factor (VEGF) and fractalkine, the levels of which were significantly higher in conditioned media from hippocampal cultures infected with ΔRR (NCMΔRR) than ΔPK or phosphate-buffered saline (mock infection). VEGF neutralization inhibited the neuroprotective activity of NCMΔRR, indicating that the VEGF protective function is through neuron-neuron cross-talk. NCMΔRR also stimulated microglia to release increased levels of IL-10 and decreased levels of TNF-α that were protective for uninfected neurons. These release patterns were not seen for microglia given NCMΔRR in which fractalkine was neutralized, indicating that the fractalkine protective function is through bidirectional neuron-microglia communication. Collectively, the data indicate that ΔRR is a multiple target strategy to rescue neurons from excitotoxic injury. PMID:19891735

  10. Natural killer group 2D and CD28 receptors differentially activate mammalian/mechanistic target of rapamycin to alter murine effector CD8+ T-cell differentiation.

    PubMed

    McQueen, Bryan; Trace, Kelsey; Whitman, Emily; Bedsworth, Taylor; Barber, Amorette

    2016-03-01

    Memory CD8+ T cells are an essential component of anti-tumour and anti-viral immunity. Activation of the mammalian/mechanistic target of rapamycin (mTOR) pathway has been implicated in regulating the differentiation of effector and memory T cells. However, the mechanisms that control mTOR activity during immunity to tumours and infections are not well known. Activation of co-stimulatory receptors, including CD28 and natural killer group 2D (NKG2D), activate phosphatidylinositol-3 kinase and subsequently may activate the mTOR pathway in CD8+ T cells. This study compared the activation of the mTOR signalling pathway after co-stimulation through CD28 or NKG2D receptors in murine effector CD8+ T cells. Compared with CD28 co-stimulation, activation through CD3 and NKG2D receptors had weaker activation of mTORc1, as shown by decreased phosphorylation of mTORc1 targets S6K1, ribosomal protein S6 and eukaryotic initiation factor 4E binding protein 1. NKG2D co-stimulation also showed increased gene expression of tuberous sclerosis protein 2, a negative regulator of mTORc1, whereas CD28 co-stimulation increased gene expression of Ras homologue enriched in brain, an activator of mTORc1, and hypoxia-inducible factor-1α and vascular endothelial growth factor-α, pro-angiogenic factors downstream of mTORc1. Strong mTORc1 activation in CD28-co-stimulated cells also increased expression of transcription factors that support effector cell differentiation, namely T-bet, B lymphocyte-induced maturation protein (BLIMP-1), interferon regulatory factor 4, and inhibitor of DNA binding 2, whereas low levels of mTORc1 activation allowed for the expression of Eomes, B-cell lymphoma 6 (BCL6), and inhibitor of DNA binding 3 during NKG2D stimulation, and increased expression of memory markers CD62 ligand and CD127. These data show that compared with CD28, co-stimulation through the NKG2D receptor leads to the differential activation of the mTOR signalling pathway and potentially supports

  11. Phosphatidylinositol 3-kinase/Akt signaling as a key mediator of tumor cell responsiveness to radiation.

    PubMed

    Toulany, Mahmoud; Rodemann, H Peter

    2015-12-01

    The phosphatidylinositol 3-kinase (PI3K)/Akt pathway is a key cascade downstream of several protein kinases, especially membrane-bound receptor tyrosine kinases, including epidermal growth factor receptor (EGFR) family members. Hyperactivation of the PI3K/Akt pathway is correlated with tumor development, progression, poor prognosis, and resistance to cancer therapies, such as radiotherapy, in human solid tumors. Akt/PKB (Protein Kinase B) members are the major kinases that act downstream of PI3K, and these are involved in a variety of cellular functions, including growth, proliferation, glucose metabolism, invasion, metastasis, angiogenesis, and survival. Accumulating evidence indicates that activated Akt is one of the major predictive markers for solid tumor responsiveness to chemo/radiotherapy. DNA double-strand breaks (DNA-DSB), are the prime cause of cell death induced by ionizing radiation. Preclinical in vitro and in vivo studies have shown that constitutive activation of Akt and stress-induced activation of the PI3K/Akt pathway accelerate the repair of DNA-DSB and, consequently, lead to therapy resistance. Analyzing dysregulations of Akt, such as point mutations, gene amplification or overexpression, which results in the constitutive activation of Akt, might be of special importance in the context of radiotherapy outcomes. Such studies, as well as studies of the mechanism(s) by which activated Akt1 regulates repair of DNA-DSB, might help to identify combinations using the appropriate molecular targeting strategies with conventional radiotherapy to overcome radioresistance in solid tumors. In this review, we discuss the dysregulation of the components of upstream regulators of Akt as well as specific modifications of Akt isoforms that enhance Akt activity. Likewise, the mechanisms by which Akt interferes with repair of DNA after exposure to ionizing radiation, will be reviewed. Finally, the current status of Akt targeting in combination with radiotherapy will

  12. AKT Pathway Genes Define 5 Prognostic Subgroups in Glioblastoma

    PubMed Central

    Smirnov, Ivan; Reiser, Mark; Misra, Anjan; Shapiro, William R.; Mills, Gordon B.; Kim, Seungchan; Feuerstein, Burt G.

    2014-01-01

    Activity of GFR/PI3K/AKT pathway inhibitors in glioblastoma clinical trials has not been robust. We hypothesized variations in the pathway between tumors contribute to poor response. We clustered GBM based on AKT pathway genes and discovered new subtypes then characterized their clinical and molecular features. There are at least 5 GBM AKT subtypes having distinct DNA copy number alterations, enrichment in oncogenes and tumor suppressor genes and patterns of expression for PI3K/AKT/mTOR signaling components. Gene Ontology terms indicate a different cell of origin or dominant phenotype for each subgroup. Evidence suggests one subtype is very sensitive to BCNU or CCNU (median survival 5.8 vs. 1.5 years; BCNU/CCNU vs other treatments; respectively). AKT subtyping advances previous approaches by revealing additional subgroups with unique clinical and molecular features. Evidence indicates it is a predictive marker for response to BCNU or CCNU and PI3K/AKT/mTOR pathway inhibitors. We anticipate Akt subtyping may help stratify patients for clinical trials and augment discovery of class-specific therapeutic targets. PMID:24984002

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

    PubMed

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

    2014-02-01

    Tumor cells with genetic amplifications or mutations in the fibroblast growth factor receptor (FGFR) family are often addicted to FGFR and heavily dependent on its signaling to survive. Although it is critical to understand which signaling pathway downstream of FGFR plays an essential role to guide the research and development of FGFR inhibitors, it has remained unclear partly because the tool compounds used in the literature also hit many other kinases, making the results difficult to interpret. With the development of a potent FGFR-specific inhibitor, BGJ398, we are now able to dissect various pathways with low drug concentrations to minimize multiple-target effects. Importantly, here, we show that inhibition of FGFR signaling by BGJ398 leads to only transient inhibition of ERK1/2 phosphorylation, whereas the inhibitory effect on AKT phosphorylation is sustainable, indicating that AKT, not ERK as commonly believed, serves as an appropriate pharmacodynamic biomarker for BGJ398. Although AKT inhibition by a pan-PI3K inhibitor alone has almost no effect on cell growth, heterologous expression of myr-AKT, an active form of AKT, rescues BGJ398-mediated suppression of tumor cell proliferation. These results indicate that AKT is an essential component downstream of FGFR. Finally, combination of the FGFR inhibitor BGJ398 with rapamycin significantly inhibits AKT phosphorylation and enhances their antiproliferative effects in FGFR-addicted cells, suggesting an effective combination strategy for clinical development of FGFR inhibitors. PMID:24100276

  14. Insulin Attenuates Beta-Amyloid-Associated Insulin/Akt/EAAT Signaling Perturbations in Human Astrocytes.

    PubMed

    Han, Xiaojuan; Yang, Liling; Du, Heng; Sun, Qinjian; Wang, Xiang; Cong, Lin; Liu, Xiaohui; Yin, Ling; Li, Shan; Du, Yifeng

    2016-08-01

    The excitatory amino acid transporters 1 and 2 (EAAT1 and EAAT2), mostly located on astrocytes, are the main mediators for glutamate clearance in humans. Malfunctions of these transporters may lead to excessive glutamate accumulation and subsequent excitotoxicity to neurons, which has been implicated in many kinds of neurodegenerative disorders including Alzheimer's disease (AD). Yet, the specific mechanism of the glutamate system dysregulation remains vague. To explore whether the insulin/protein kinase B (Akt)/EAAT signaling in human astrocytes could be disturbed by beta-amyloid protein (Aβ) and be protected by insulin, we incubated HA-1800 cells with varying concentrations of Aβ1-42 oligomers and insulin. Then the alterations of several key substrates in this signal transduction pathway were determined. Our results showed that expressions of insulin receptor, phospho-insulin receptor, phospho-protein kinase B, phospho-mammalian target of rapamycin, and EAAT1 and EAAT2 were decreased by the Aβ1-42 oligomers in a dose-dependent manner (p < 0.05) and this trend could be recovered by insulin treatment (p < 0.05). However, the expressions of total Akt and mTOR were invariant (p > 0.05), and the mRNA levels of EAAT1 and EAAT2 were also unchanged (p > 0.05). Taken together, this study indicates that Aβ1-42 oligomers could cause disturbances in insulin/Akt/EAAT signaling in astrocytes, which might be responsible for AD onset and progression. Additionally, insulin can exert protective functions to the brain by modulating protein modifications or expressions. PMID:26358886

  15. PTEN-DEFICIENT TUMORS DEPEND ON AKT2 FOR MAINTENANCE AND SURVIVAL

    PubMed Central

    Chin, Y. Rebecca; Yuan, Xin; Balk, Steven P.; Toker, Alex

    2014-01-01

    Loss of PTEN is a common event in many cancers and leads to hyperactivation of the PI 3-K/Akt signaling pathway. The mechanisms by which Akt isoforms mediate signaling to phenotypes associated with PTEN-inactivation in cancer have not been defined. Here we show that Akt2 is exclusively required for PTEN-deficient prostate tumor spheroid maintenance whereas Akt1 is dispensable. shRNA silencing of Akt2 but not Akt1 promotes regression of prostate cancer xenografts. Mechanistically, we show that Akt2 silencing up-regulates p21 and the pro-apoptotic protein Bax and downregulates the insulin-like growth factor receptor-1. We also show that p21 is an effector of Akt2 in mediating prostate tumor maintenance. Moreover, Akt2 is also exclusively required for the maintenance and survival of other PTEN-deficient solid tumors, including breast cancer and glioblastoma. These findings identify a specific function for Akt2 in mediating survival of PTEN-deficient tumors and provide a rationale for developing therapeutics targeting Akt2. PMID:24838891

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

    PubMed

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

    2016-06-01

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

  17. Site Specific Activation of AKT Protects Cells from Death Induced by Glucose Deprivation

    PubMed Central

    Gao, Meng; Liang, Jiyong; Lu, Yiling; Guo, Huifang; German, Peter; Bai, Shanshan; Jonasch, Eric; Yang, Xingsheng; Mills, Gordon B.; Ding, Zhiyong

    2013-01-01

    The serine/threonine kinase AKT is a key mediator of cancer cell survival. We demonstrate that transient glucose deprivation modestly induces AKT phosphorylation at both Thr308 and Ser473. In contrast, prolonged glucose deprivation induces selective AKTThr308 phosphorylation and phosphorylation of a distinct subset of AKT downstream targets leading to cell survival under metabolic stress. Glucose deprivation-induced AKTThr308 phosphorylation is dependent on PDK1 and PI3K but not EGFR or IGF1R. Prolonged glucose deprivation induces the formation of a complex of AKT, PDK1, and the GRP78 chaperone protein, directing phosphorylation of AKTThr308 but AKTSer473. Our results reveal a novel mechanism of AKT activation under prolonged glucose deprivation that protects cells from metabolic stress. The selective activation of AKTThr308 phosphorylation that occurs during prolonged nutrient deprivation may provide an unexpected opportunity for the development and implementation of drugs targeting cell metabolism and aberrant AKT signaling. PMID:23396361

  18. Role of PI3K-AKT-mTOR and Wnt Signaling Pathways in Transition of G1-S Phase of Cell Cycle in Cancer Cells

    PubMed Central

    Vadlakonda, Lakshmipathi; Pasupuleti, Mukesh; Pallu, Reddanna

    2013-01-01

    The PI3K-Akt pathway together with one of its downstream targets, the mechanistic target of rapamycin (mTOR; also known as the mammalian target of rapamycin) is a highly deregulated pathway in cancers. mTOR exists in two complexes, mTORC1 and mTORC2. Akt phosphorylated at T308 inhibits TSC1/2 complex to activate mTORC1; mTORC2 is recognized as the kinase phosphorylating Akt at S473. Inhibition of autophagy by mTORC1 was shown to rescue disheveled (Dvl) leading to activation of Wnt pathway. Cyclin D1 and the c-Myc are activated by the Wnt signaling. Cyclin D1 is a key player in initiation of cell cycle. c-Myc triggers metabolic reprograming in G1 phase of cell cycle, which also activates the transcription factors like FoxO and p53 that play key roles in promoting the progression of cell cycle. While the role of p53 in cancer cell metabolism in arresting glycolysis and inhibition of pentose phosphate pathway has come to be recognized, there are confusions in the literature on the role of FoxO and that of rictor. FoxO was shown to be the transcription factor of rictor, in addition to the cell cycle inhibitors like p21. Rictor has dual roles; inhibition of c-Myc and constitution of mTORC2, both of which are key factors in the exit of G1-S phase and entry into G2 phase of cell cycle. A model is presented in this article, which suggests that the PI3K-Akt-mTOR and Wnt pathways converge and regulate the progression of cell cycle through G0-G1-S-phases and reprogram the metabolism in cancer cells. This model is different from the conventional method of looking at individual pathways triggering the cell cycle. PMID:23596569

  19. Hematopoietic Akt2 deficiency attenuates the progression of atherosclerosis

    PubMed Central

    Rotllan, Noemi; Chamorro-Jorganes, Aránzazu; Araldi, Elisa; Wanschel, Amarylis C.; Aryal, Binod; Aranda, Juan F.; Goedeke, Leigh; Salerno, Alessandro G.; Ramírez, Cristina M.; Sessa, William C.; Suárez, Yajaira; Fernández-Hernando, Carlos

    2015-01-01

    Atherosclerosis is the major cause of death and disability in diabetic and obese subjects with insulin resistance. Akt2, a phosphoinositide-dependent serine-threonine protein kinase, is highly express in insulin-responsive tissues; however, its role during the progression of atherosclerosis remains unknown. Thus, we aimed to investigate the contribution of Akt2 during the progression of atherosclerosis. We found that germ-line Akt2-deficient mice develop similar atherosclerotic plaques as wild-type mice despite higher plasma lipids and glucose levels. It is noteworthy that transplantation of bone marrow cells isolated from Akt2−/− mice to Ldlr−/− mice results in marked reduction of the progression of atherosclerosis compared with Ldlr−/− mice transplanted with wild-type bone marrow cells. In vitro studies indicate that Akt2 is required for macrophage migration in response to proatherogenic cytokines (monocyte chemotactic protein-1 and macrophage colony-stimulating factor). Moreover, Akt2−/− macrophages accumulate less cholesterol and have an alternative activated or M2-type phenotype when stimulated with proinflammatory cytokines. Together, these results provide evidence that macrophage Akt2 regulates migration, the inflammatory response and cholesterol metabolism and suggest that targeting Akt2 in macrophages might be beneficial for treating atherosclerosis.—Rotllan, N., Chamorro-Jorganes, A., Araldi, E., Wanschel, A. C., Aryal, B., Aranda, J. F., Goedeke, L., Salerno, A. G., Ramírez, C. M., Sessa,W. C., Suárez, Y., Fernández-Hernando, C. Hematopoietic Akt2 deficiency attenuates the progression of atherosclerosis. PMID:25392271

  20. Mammalian target of rapamycin (mTOR) inhibitors and combined chemotherapy in breast cancer: a meta-analysis of randomized controlled trials

    PubMed Central

    Qiao, Longwei; Liang, Yuting; Mira, Ranim R; Lu, Yaojuan; Gu, Junxia; Zheng, Qiping

    2014-01-01

    The mammalian target of rapamycin (mTOR) inhibitor, in combination with other chemotherapeutic drugs, has been used for treatment of breast cancer that develops resistance to endocrine therapy. However, the efficacy and safety need further evaluation. Here, we report a meta-analysis of randomized controlled trials (RCT) in breast cancer patients undergoing chemotherapy using steroid (exemestane) or nonsteroid (letrozole) aromatase inhibitors with or without mTOR inhibitors (everolimus). The overall response rate (ORR), progression-free survival (PFS), clinical benefi;t rate with 95% confidence interval (CI), and the major toxicities/adverse effects were analyzed. Data were extracted from twelve studies that meet the selection criteria. Among these, six studies that enrolled 3693 women received treatment of everolimus plus exemestane, or placebo with exemestane. The results showed that everolimus plus exemestane significantly increased the ORR relative risk (relative risk = 9.18, 95% CI = 5.21-16.15), PFS hazard ratio (hazard ratio = 0.44, 95% CI = 0.41-0.48), and clinical benefi;t rate (relative risk = 1.92, 95% CI 1.69-2.17) compared to placebo control, while the risks of stomatitis, rash, hyperglycemia, diarrhea, fatigue, anorexia and pneumonitis also increased. Three studies that enrolled 715 women who received everolimus as neoadjuvant therapy were analyzed. Compared to chemotherapy with placebo, chemotherapy plus everolimus did not increase the ORR relative risk (relative risk = 0.90, 95% CI = 0.77-1.05). Meanwhile, two other studies that enrolled 2104 women examined the efficacy of temsirolimus (or placebo control) plus letrozole. The results indicated that emsirolimus plus letrozole did not increase the ORR relative risk and clinical benefi;t rate (p > 0.05). Together, these data suggest that the combined mTOR inhibitor (everolimus) plus endocrine therapy (exemestane) is superior to endocrine therapy alone. As a neoadjuvant, everolimus did not increase the

  1. Mechanistic studies of semicarbazone triapine targeting human ribonucleotide reductase in vitro and in mammalian cells: tyrosyl radical quenching not involving reactive oxygen species.

    PubMed

    Aye, Yimon; Long, Marcus J C; Stubbe, JoAnne

    2012-10-12

    Triapine® (3-aminopyridine-2-carboxaldehyde thiosemicarbazone (3-AP)) is a drug in Phase II trials. One of its established cellular targets is the β(2) subunit of ribonucleotide reductase that requires a diferric-tyrosyl-radical [(Fe(III)(2)-Y·)(Fe(III)(2))] cofactor for de novo DNA biosynthesis. Several mechanisms for 3-AP inhibition of β(2) have been proposed; one involves direct iron chelation from β(2), whereas a second involves Y· destruction by reactive oxygen species formed in situ in the presence of O(2) and reductant by Fe(II)-(3-AP). Inactivation of β(2) can thus arise from cofactor destruction by loss of iron or Y·. In vitro kinetic data on the rates of (55)Fe and Y· loss from [((55)Fe(III)(2)-Y·)((55)Fe(III)(2))]-β(2) under aerobic and anaerobic conditions reveal that Y· loss alone is sufficient for rapid β(2) inactivation. Oxyblot(TM) and mass spectrometric analyses of trypsin-digested inhibited β(2), and lack of Y· loss from H(2)O(2) and O(2)(•) treatment together preclude reactive oxygen species involvement in Y· loss. Three mammalian cell lines treated with 5 μm 3-AP reveal Y· loss and β(2) inactivation within 30-min of 3-AP-exposure, analyzed by whole-cell EPR and lysate assays, respectively. Selective degradation of apo- over [(Fe(III)(2)-Y·)(Fe(III)(2))]-β(2) in lysates, similar iron-content in β(2) immunoprecipitated from 3-AP-treated and untreated [(55)Fe]-prelabeled cells, and prolonged (12 h) stability of the inhibited β(2) are most consistent with Y· loss being the predominant mode of inhibition, with β(2) remaining iron-loaded and stable. A model consistent with in vitro and cell-based biochemical studies is presented in which Fe(II)-(3-AP), which can be cycled with reductant, directly reduces Y· of the [(Fe(III)(2)-Y·)(Fe(III)(2))] cofactor of β(2). PMID:22915594

  2. BRAFV600E Negatively Regulates the AKT Pathway in Melanoma Cell Lines

    PubMed Central

    Chen, Brenden; Tardell, Christine; Higgins, Brian; Packman, Kathryn; Boylan, John F.; Niu, Huifeng

    2012-01-01

    Cross-feedback activation of MAPK and AKT pathways is implicated as a resistance mechanism for cancer therapeutic agents targeting either RAF/MEK or PI3K/AKT/mTOR. It is thus important to have a better understanding of the molecular resistance mechanisms to improve patient survival benefit from these agents. Here we show that BRAFV600E is a negative regulator of the AKT pathway. Expression of BRAFV600E in NIH3T3 cells significantly suppresses MEK inhibitor (RG7167) or mTORC1 inhibitor (rapamycin) induced AKT phosphorylation (pAKT) and downstream signal activation. Treatment-induced pAKT elevation is found in BRAF wild type melanoma cells but not in a subset of melanoma cell lines harboring BRAFV600E. Knock-down of BRAFV600E in these melanoma cells elevates basal pAKT and downstream signals, whereas knock-down of CRAF, MEK1/2 or ERK1/2 or treatment with a BRAF inhibitor have no impact on pAKT. Mechanistically, we show that BRAFV600E interacts with rictor complex (mTORC2) and regulates pAKT through mTORC2. BRAFV600E is identified in mTORC2 after immunoprecipitation of rictor. Knock-down of rictor abrogates BRAFV600E depletion induced pAKT. Knock-down of BRAFV600E enhances cellular enzyme activity of mTORC2. Aberrant activation of AKT pathway by PTEN loss appears to override the negative impact of BRAFV600E on pAKT. Taken together, our findings suggest that in a subset of BRAFV600E melanoma cells, BRAFV600E negatively regulates AKT pathway in a rictor-dependent, MEK/ERK and BRAF kinase-independent manner. Our study reveals a novel molecular mechanism underlying the regulation of feedback loops between the MAPK and AKT pathways. PMID:22880048

  3. BRAFV600E negatively regulates the AKT pathway in melanoma cell lines.

    PubMed

    Chen, Brenden; Tardell, Christine; Higgins, Brian; Packman, Kathryn; Boylan, John F; Niu, Huifeng

    2012-01-01

    Cross-feedback activation of MAPK and AKT pathways is implicated as a resistance mechanism for cancer therapeutic agents targeting either RAF/MEK or PI3K/AKT/mTOR. It is thus important to have a better understanding of the molecular resistance mechanisms to improve patient survival benefit from these agents. Here we show that BRAFV600E is a negative regulator of the AKT pathway. Expression of BRAFV600E in NIH3T3 cells significantly suppresses MEK inhibitor (RG7167) or mTORC1 inhibitor (rapamycin) induced AKT phosphorylation (pAKT) and downstream signal activation. Treatment-induced pAKT elevation is found in BRAF wild type melanoma cells but not in a subset of melanoma cell lines harboring BRAFV600E. Knock-down of BRAFV600E in these melanoma cells elevates basal pAKT and downstream signals, whereas knock-down of CRAF, MEK1/2 or ERK1/2 or treatment with a BRAF inhibitor have no impact on pAKT. Mechanistically, we show that BRAFV600E interacts with rictor complex (mTORC2) and regulates pAKT through mTORC2. BRAFV600E is identified in mTORC2 after immunoprecipitation of rictor. Knock-down of rictor abrogates BRAFV600E depletion induced pAKT. Knock-down of BRAFV600E enhances cellular enzyme activity of mTORC2. Aberrant activation of AKT pathway by PTEN loss appears to override the negative impact of BRAFV600E on pAKT. Taken together, our findings suggest that in a subset of BRAFV600E melanoma cells, BRAFV600E negatively regulates AKT pathway in a rictor-dependent, MEK/ERK and BRAF kinase-independent manner. Our study reveals a novel molecular mechanism underlying the regulation of feedback loops between the MAPK and AKT pathways. PMID:22880048

  4. Zinc stimulates glucose consumption by modulating the insulin signaling pathway in L6 myotubes: essential roles of Akt-GLUT4, GSK3β and mTOR-S6K1.

    PubMed

    Wu, Yuntang; Lu, Huizi; Yang, Huijun; Li, Chunlei; Sang, Qian; Liu, Xinyan; Liu, Yongzhe; Wang, Yongming; Sun, Zhong

    2016-08-01

    The present study was performed to evaluate the insulin-like effects of zinc in normal L6 myotubes as well as its ability to alleviate insulin resistance. Glucose consumption was measured in both normal and insulin-resistant L6 myotubes. Western blotting and immunofluorescence revealed that zinc exhibited insulin-like glucose transporting effects by activating key markers that are involved in the insulin signaling cascade (including Akt, GLUT4 and GSK3β), and downregulating members of the insulin signaling feedback cascade such as mammalian target of rapamycin (mTOR) and ribosomal protein S6 kinase (S6K1). In normal L6 myotubes, zinc enhanced glucose consumption via a mechanism that might involve the activation of Akt phosphorylation, glucose transporter 4 (GLUT4) translocation and GSK3β phosphorylation. In contrast, zinc exerted insulin-mimetic effects in insulin-resistant L6 myotubes by upregulating Akt phosphorylation, GLUT4 translocation and GSK3β phosphorylation, and downregulating the expression of mTOR and S6K1. In conclusion, zinc might enhance glucose consumption by modulating insulin signaling pathways including Akt-GLUT4, GSK3β, mTOR and S6K1. PMID:27295130

  5. Mammalian sleep

    NASA Astrophysics Data System (ADS)

    Staunton, Hugh

    2005-05-01

    This review examines the biological background to the development of ideas on rapid eye movement sleep (REM sleep), so-called paradoxical sleep (PS), and its relation to dreaming. Aspects of the phenomenon which are discussed include physiological changes and their anatomical location, the effects of total and selective sleep deprivation in the human and animal, and REM sleep behavior disorder, the latter with its clinical manifestations in the human. Although dreaming also occurs in other sleep phases (non-REM or NREM sleep), in the human, there is a contingent relation between REM sleep and dreaming. Thus, REM is taken as a marker for dreaming and as REM is distributed ubiquitously throughout the mammalian class, it is suggested that other mammals also dream. It is suggested that the overall function of REM sleep/dreaming is more important than the content of the individual dream; its function is to place the dreamer protagonist/observer on the topographical world. This has importance for the developing infant who needs to develop a sense of self and separateness from the world which it requires to navigate and from which it is separated for long periods in sleep. Dreaming may also serve to maintain a sense of ‘I’ness or “self” in the adult, in whom a fragility of this faculty is revealed in neurological disorders.

  6. Use of mammalian target of rapamycin inhibitors after failure of tyrosine kinase inhibitors in patients with metastatic renal cell carcinoma undergoing hemodialysis: A single-center experience with four cases.

    PubMed

    Omae, Kenji; Kondo, Tsunenori; Takagi, Toshio; Iizuka, Junpei; Kobayashi, Hirohito; Hashimoto, Yasunobu; Tanabe, Kazunari

    2016-07-01

    We retrospectively identified patients with end-stage renal disease undergoing hemodialysis treated with the mammalian target of rapamycin inhibitors as a second- and/or third-line targeted therapy after treatment failure with the tyrosine kinase inhibitors for metastatic renal cell carcinoma. Patient medical records were reviewed to evaluate the response to therapies and treatment-related toxicities. Four patients were identified. All patients had undergone nephrectomy, and one had received immunotherapy before targeted therapy. Two patients had clear cell histology, and the other two had papillary histology. All patients were classified into the intermediate risk group according to the Memorial Sloan-Kettering Cancer Center risk model. All patients were treated with everolimus as a second- or third-line therapy, and two patients were treated with temsirolimus as a second- or third-line therapy after treatment failure with sorafenib or sunitinib. The median duration of everolimus therapy was 6.7 months, whereas that of temsirolimus was 9.5 months. All patients had stable disease as the best response during each period of therapy. There were no severe adverse events. The use of mammalian target of rapamycin inhibitors in patients who previously failed to respond to tyrosine kinase inhibitors appears to be feasible in patients with end-stage renal disease requiring hemodialysis. PMID:26833674

  7. Oxidant Stress and Signal Transduction in the Nervous System with the PI 3-K, Akt, and mTOR Cascade

    PubMed Central

    Maiese, Kenneth; Chong, Zhao Zhong; Wang, Shaohui; Shang, Yan Chen

    2012-01-01

    Oxidative stress impacts multiple systems of the body and can lead to some of the most devastating consequences in the nervous system especially during aging. Both acute and chronic neurodegenerative disorders such as diabetes mellitus, cerebral ischemia, trauma, Alzheimer’s disease, Parkinson’s disease, Huntington’s disease, and tuberous sclerosis through programmed cell death pathways of apoptosis and autophagy can be the result of oxidant stress. Novel therapeutic avenues that focus upon the phosphoinositide 3-kinase (PI 3-K), Akt (protein kinase B), and the mammalian target of rapamycin (mTOR) cascade and related pathways offer exciting prospects to address the onset and potential reversal of neurodegenerative disorders. Effective clinical translation of these pathways into robust therapeutic strategies requires intimate knowledge of the complexity of these pathways and the ability of this cascade to influence biological outcome that can vary among disorders of the nervous system. PMID:23203037

  8. Mammalian aromatases.

    PubMed

    Conley, A; Hinshelwood, M

    2001-05-01

    Aromatase is the enzyme complex that catalyses the synthesis of oestrogens from androgens, and therefore it has unique potential to influence the physiological balance between the sex steroid hormones. Both aromatase cytochrome P450 (P450arom) and NADPH-cytochrome P450 reductase (reductase), the two essential components of the enzyme complex, are highly conserved among mammals and vertebrates. Aromatase expression occurs in the gonads and brain, and is essential for reproductive development and fertility. Of interest are the complex mechanisms involving alternative promoter utilization that have evolved to control tissue-specific expression in these tissues. In addition, in a number of species, including humans, expression of aromatase has a broader tissue distribution, including placenta, adipose and bone. The relevance of oestrogen synthesis and possibly androgen metabolism in these peripheral sites of expression is now becoming clear from studies in P450arom knockout (ArKO) mice and from genetic defects recognized recently in both men and women. Important species differences in the physiological roles of aromatase expression are also likely to emerge, despite the highly conserved nature of the enzyme system. The identification of functionally distinct, tissue-specific isozymes of P450arom in at least one mammal, pigs, and several species of fish indicates that there are additional subtle, but physiologically significant, species-specific roles for aromatase. Comparative studies of mammalian and other vertebrate aromatases will expand understanding of the role played by this ancient enzyme system in the evolution of reproduction and the adaptive influence of oestrogen synthesis on general health and well being. PMID:11427156

  9. Mouse hippocampal phosphorylation footprint induced by generalized seizures: Focus on ERK, mTORC1 and Akt/GSK-3 pathways.

    PubMed

    Gangarossa, Giuseppe; Sakkaki, Sophie; Lory, Philippe; Valjent, Emmanuel

    2015-12-17

    Exacerbated hippocampal activity has been associated to critical modifications of the intracellular signaling pathways. We have investigated rapid hippocampal adaptive responses induced by maximal electroshock seizure (MES). Here, we demonstrate that abnormal and exacerbated hippocampal activity induced by MES triggers specific and temporally distinct patterns of phosphorylation of extracellular signal-related kinase (ERK), mammalian target of rapamycin complex (mTORC) and Akt/glycogen synthase kinase-3 (Akt/GSK-3) pathways in the mouse hippocampus. While the ERK pathway is transiently activated, the mTORC1 cascade follows a rapid inhibition followed by a transient activation. This rebound of mTORC1 activity leads to the selective phosphorylation of p70S6K, which is accompanied by an enhanced phosphorylation of the ribosomal subunit S6. In contrast, the Akt/GSK-3 pathway is weakly altered. Finally, MES triggers a rapid upregulation of several plasticity-associated genes as a consequence exacerbated hippocampal activity. The results reported in the present study are reminiscent of the one observed in other models of generalized seizures, thus defining a common molecular footprint induced by intense and aberrant hippocampal activities. PMID:26545981

  10. Anticancer activity of pristimerin in ovarian carcinoma cells is mediated through the inhibition of prosurvival Akt/NF-κB/mTOR Signaling

    PubMed Central

    Gao, Xiaohua; Liu, Yongbo; Deeb, Dorrah; Arbab, Ali S.; Gautam, Subhash C.

    2014-01-01

    Pristimerin is a quinonemethide triterpenoid that has shown anticancer activity against some cancer types. However, the antitumor effects of pristimerin (PM) in ovarian cancer cells have not been adequately studied. The objective of the present study was to determine the anticancer activity and its mechanism of action in human ovarian carcinoma cell lines. PM strongly inhibited the proliferation of ovarian cancer cells by inducing apoptosis characterized by increased annexin V-binding, cleavage of poly (ADP-ribose) polymerase (PARP-1) and procaspases-3, -8 and -9. Furthermore, PM caused mitochondrial depolarization. Western blot analysis showed inhibition of prosurvival phospho-AKT (p-AKT), nuclear factor kappa B (NF-κB) (p65) and phospho-mammalian target of rapamycin (p-mTOR) signaling proteins in cells treated with PM. Treatment with PM also inhibited the expression of NF-κB-regulated antiapoptotic Bcl-2, Bcl-xL, c-IAP1 and survivin. Thus, our data showing potent antiproliferative and apoptosis-inducing activity of PM in ovarian carcinoma cells through the inhibition of AKT/NF-κB/mTOR signaling pathway warrant further investigation of PM for the management of ovarian cancer. PMID:25509983

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

    PubMed Central

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

    2016-01-01

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

  12. TCR-induced Akt serine 473 phosphorylation is regulated by protein kinase C-alpha

    SciTech Connect

    Yang, Lifen; Qiao, Guilin; Ying, Haiyan; Zhang, Jian; Yin, Fei

    2010-09-10

    Research highlights: {yields} Conventional PKC positively regulates TCR-induced phosphorylation of Akt. {yields} PKC-alpha is the PDK-2 responsible for phosphorylating Akt at Ser{sup 473} upon TCR stimulation. {yields} Knockdown of PKC-alpha decreases TCR-induced Akt phosphorylation. -- Abstract: Akt signaling plays a central role in T cell functions, such as proliferation, apoptosis, and regulatory T cell development. Phosphorylation at Ser{sup 473} in the hydrophobic motif, along with Thr{sup 308} in its activation loop, is considered necessary for Akt function. It is widely accepted that phosphoinositide-dependent kinase 1 (PDK-1) phosphorylates Akt at Thr{sup 308}, but the kinase(s) responsible for phosphorylating Akt at Ser{sup 473} (PDK-2) remains elusive. The existence of PDK-2 is considered to be specific to cell type and stimulus. PDK-2 in T cells in response to TCR stimulation has not been clearly defined. In this study, we found that conventional PKC positively regulated TCR-induced Akt Ser{sup 473} phosphorylation. PKC-alpha purified from T cells can phosphorylate Akt at Ser{sup 473} in vitro upon TCR stimulation. Knockdown of PKC-alpha in T-cell-line Jurkat cells reduced TCR-induced phosphorylation of Akt as well as its downstream targets. Thus our results suggest that PKC-alpha is a candidate for PDK-2 in T cells upon TCR stimulation.

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

    PubMed

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

    2016-03-22

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

  14. Molecular and functional interactions between AKT and SOX2 in breast carcinoma

    PubMed Central

    Mir, Perihan; Konantz, Martina; Pereboom, Tamara C.; Paczulla, Anna M.; Merz, Britta; Fehm, Tanja; Perner, Sven; Rothfuss, Oliver C.; Kanz, Lothar; Schulze-Osthoff, Klaus; Lengerke, Claudia

    2015-01-01

    The transcription factor SOX2 is a key regulator of pluripotency in embryonic stem cells and plays important roles in early organogenesis. Recently, SOX2 expression was documented in various cancers and suggested as a cancer stem cell (CSC) marker. Here we identify the Ser/Thr-kinase AKT as an upstream regulator of SOX2 protein turnover in breast carcinoma (BC). SOX2 and pAKT are co-expressed and co-regulated in breast CSCs and depletion of either reduces clonogenicity. Ectopic SOX2 expression restores clonogenicity and in vivo tumorigenicity of AKT-inhibited cells, suggesting that SOX2 acts as a functional downstream AKT target. Mechanistically, we show that AKT physically interacts with the SOX2 protein to modulate its subcellular distribution. AKT kinase inhibition results in enhanced cytoplasmic retention of SOX2, presumably via impaired nuclear import, and in successive cytoplasmic proteasomal degradation of the protein. In line, blockade of either nuclear transport or proteasomal degradation rescues SOX2 expression in AKT-inhibited BC cells. Finally, AKT inhibitors efficiently suppress the growth of SOX2-expressing putative cancer stem cells, whereas conventional chemotherapeutics select for this population. Together, our results suggest the AKT/SOX2 molecular axis as a regulator of BC clonogenicity and AKT inhibitors as promising drugs for the treatment of SOX2-positive BC. PMID:26498353

  15. Molecular and functional interactions between AKT and SOX2 in breast carcinoma.

    PubMed

    Schaefer, Thorsten; Wang, Hui; Mir, Perihan; Konantz, Martina; Pereboom, Tamara C; Paczulla, Anna M; Merz, Britta; Fehm, Tanja; Perner, Sven; Rothfuss, Oliver C; Kanz, Lothar; Schulze-Osthoff, Klaus; Lengerke, Claudia

    2015-12-22

    The transcription factor SOX2 is a key regulator of pluripotency in embryonic stem cells and plays important roles in early organogenesis. Recently, SOX2 expression was documented in various cancers and suggested as a cancer stem cell (CSC) marker. Here we identify the Ser/Thr-kinase AKT as an upstream regulator of SOX2 protein turnover in breast carcinoma (BC). SOX2 and pAKT are co-expressed and co-regulated in breast CSCs and depletion of either reduces clonogenicity. Ectopic SOX2 expression restores clonogenicity and in vivo tumorigenicity of AKT-inhibited cells, suggesting that SOX2 acts as a functional downstream AKT target. Mechanistically, we show that AKT physically interacts with the SOX2 protein to modulate its subcellular distribution. AKT kinase inhibition results in enhanced cytoplasmic retention of SOX2, presumably via impaired nuclear import, and in successive cytoplasmic proteasomal degradation of the protein. In line, blockade of either nuclear transport or proteasomal degradation rescues SOX2 expression in AKT-inhibited BC cells. Finally, AKT inhibitors efficiently suppress the growth of SOX2-expressing putative cancer stem cells, whereas conventional chemotherapeutics select for this population. Together, our results suggest the AKT/SOX2 molecular axis as a regulator of BC clonogenicity and AKT inhibitors as promising drugs for the treatment of SOX2-positive BC. PMID:26498353

  16. Kaposi sarcoma associated herpesvirus (KSHV) induces AKT hyperphosphorylation, bortezomib-resistance and GLUT-1 plasma membrane exposure in THP-1 monocytic cell line

    PubMed Central

    2013-01-01

    Background Phosphatidylinositol-3-kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) signaling pathway regulates multiple cellular processes such as cell proliferation, evasion from apoptosis, migration, glucose metabolism, protein synthesis and proper differentiation in immune cells. Kaposi sarcoma-associated herpesvirus (KSHV), an oncogenic virus associated with several human malignancies, expresses a variety of latent and lytic proteins able to activate PI3K/AKT pathway, promoting the growth of infected cells and a successful viral infection. Results We found that KSHV latent infection of THP-1 cells, a human monocytic cell line derived from an acute monocytic leukemia patient, resulted in an increase of AKT phoshorylation, not susceptible to bortezomib-induced dephosphorylation, compared to the mock-infected THP-1. Accordingly, THP-1-infected cells displayed increased resistance to the bortezomib cytotoxic effect in comparison to the uninfected cells, which was counteracted by pre-treatment with AKT-specific inhibitors. Finally, AKT hyperactivation by KSHV infection correlated with plasma membrane exposure of glucose transporter GLUT1, particularly evident during bortezomib treatment. GLUT1 membrane trafficking is a characteristic of malignant cells and underlies a change of glucose metabolism that ensures the survival to highly proliferating cells and render these cells highly dependent on glycolysis. GLUT1 membrane trafficking in KSHV-infected THP-1 cells indeed led to increased sensitivity to cell death induced by the glycolysis inhibitor 2-Deoxy-D-glucose (2DG), further potentiated by its combination with bortezomib. Conclusions KSHV confers to the THP-1 infected cells an oncogenic potential by altering the phosphorylation, expression and localization of key molecules that control cell survival and metabolism such as AKT and GLUT1. Such modifications in one hand lead to resistance to cell death induced by some chemotherapeutic drugs such as bortezomib

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

    NASA Astrophysics Data System (ADS)

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

    2016-04-01

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

  18. Phospholipase D2 Mediates Survival Signaling through Direct Regulation of Akt in Glioblastoma Cells*♦

    PubMed Central

    Bruntz, Ronald C.; Taylor, Harry E.; Lindsley, Craig W.; Brown, H. Alex

    2014-01-01

    The lack of innovative drug targets for glioblastoma multiforme (GBM) limits patient survival to approximately 1 year following diagnosis. The pro-survival kinase Akt provides an ideal target for the treatment of GBM as Akt signaling is frequently activated in this cancer type. However, the central role of Akt in physiological processes limits its potential as a therapeutic target. In this report, we show that the lipid-metabolizing enzyme phospholipase D (PLD) is a novel regulator of Akt in GBM. Studies using a combination of small molecule PLD inhibitors and siRNA knockdowns establish phosphatidic acid, the product of the PLD reaction, as an essential component for the membrane recruitment and activation of Akt. Inhibition of PLD enzymatic activity and subsequent Akt activation decreases GBM cell viability by specifically inhibiting autophagic flux. We propose a mechanism whereby phosphorylation of beclin1 by Akt prevents binding of Rubicon (RUN domain cysteine-rich domain containing beclin1-interacting protein), an interaction known to inhibit autophagic flux. These findings provide a novel framework through which Akt inhibition can be achieved without directly targeting the kinase. PMID:24257753

  19. AEG-1–AKT2: A novel complex controlling the aggressiveness of glioblastoma

    PubMed Central

    Emdad, Luni; Hu, Bin; Das, Swadesh K; Sarkar, Devanand; Fisher, Paul B

    2015-01-01

    Expression of AEG-1 (also known as MTDH or LYRIC) is elevated in many cancers including glioblastoma multiforme (GBM), in which it functions as an oncogene. AEG-1 activates AKT signaling and physically interacts with AKT2 in GBM. Disruption of this interaction reduces glioma cell survival and invasion, uncovering a novel potential target for development of an effective therapy against GBM.

  20. MYOCARDIAL AKT: THE OMNIPRESENT NEXUS

    PubMed Central

    Sussman, Mark A.; Völkers, Mirko; Fischer, Kimberlee; Bailey, Brandi; Cottage, Christopher T.; Din, Shabana; Gude, Natalie; Avitabile, Daniele; Alvarez, Roberto; Sundararaman, Balaji; Quijada, Pearl; Mason, Matt; Konstandin, Mathias H.; Malhowski, Amy; Cheng, Zhaokang; Khan, Mohsin; McGregor, Michael

    2013-01-01

    One of the greatest examples of integrated signal transduction is revealed by examination of effects mediated by AKT kinase in myocardial biology. Positioned at the intersection of multiple afferent and efferent signals, AKT exemplifies a molecular sensing node that coordinates dynamic responses of the cell in literally every aspect of biological responses. The balanced and nuanced nature of homeostatic signaling is particularly essential within the myocardial context, where regulation of survival, energy production, contractility, and response to pathological stress all flow through the nexus of AKT activation or repression. Equally important, the loss of regulated AKT activity is primarily the cause or consequence of pathological conditions leading to remodeling of the heart and eventual decompensation. This review presents an overview compendium of the complex world of myocardial AKT biology gleaned from more than a decade of research. Summarization of the widespread influence that AKT exerts upon myocardial responses leaves no doubt that the participation of AKT in molecular signaling will need to be reckoned with as a seemingly omnipresent regulator of myocardial molecular biological responses. PMID:21742795

  1. PI3K/Akt promotes feedforward mTORC2 activation through IKKα

    PubMed Central

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

    2016-01-01

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

  2. The targets of curcumin.

    PubMed

    Zhou, Hongyu; Beevers, Christopher S; Huang, Shile

    2011-03-01

    Curcumin (diferuloylmethane), an orange-yellow component of turmeric or curry powder, is a polyphenol natural product isolated from the rhizome of the plant Curcuma longa. For centuries, curcumin has been used in some medicinal preparation or used as a food-coloring agent. In recent years, extensive in vitro and in vivo studies suggested curcumin has anticancer, antiviral, antiarthritic, anti-amyloid, antioxidant, and anti-inflammatory properties. The underlying mechanisms of these effects are diverse and appear to involve the regulation of various molecular targets, including transcription factors (such as nuclear factor-kB), growth factors (such as vascular endothelial cell growth factor), inflammatory cytokines (such as tumor necrosis factor, interleukin 1 and interleukin 6), protein kinases (such as mammalian target of rapamycin, mitogen-activated protein kinases, and Akt) and other enzymes (such as cyclooxygenase 2 and 5 lipoxygenase). Thus, due to its efficacy and regulation of multiple targets, as well as its safety for human use, curcumin has received considerable interest as a potential therapeutic agent for the prevention and/or treatment of various malignant diseases, arthritis, allergies, Alzheimer's disease, and other inflammatory illnesses. This review summarizes various in vitro and in vivo pharmacological aspects of curcumin as well as the underlying action mechanisms. The recently identified molecular targets and signaling pathways modulated by curcumin are also discussed here. PMID:20955148

  3. Targets of curcumin

    PubMed Central

    Zhou, Hongyu; Beevers, Christopher S.; Huang, Shile

    2010-01-01

    Curcumin (diferuloylmethane), an orange-yellow component of turmeric or curry powder, is a polyphenol natural product isolated from the rhizome of the plant Curcuma longa. For centuries, curcumin has been used in some medicinal preparation or used as a food-coloring agent. In recent years, extensive in vitro and in vivo studies suggested curcumin has anticancer, antiviral, antiarthritic, anti-amyloid, antioxidant, and anti-inflammatory properties. The underlying mechanisms of these effects are diverse and appear to involve the regulation of various molecular targets, including transcription factors (such as nuclear factor-κB), growth factors (such as vascular endothelial cell growth factor), inflammatory cytokines (such as tumor necrosis factor, interleukin 1 and interleukin 6), protein kinases (such as mammalian target of rapamycin, mitogen-activated protein kinases, and Akt) and other enzymes (such as cyclooxygenase 2 and 5 lipoxygenase). Thus, due to its efficacy and regulation of multiple targets, as well as its safety for human use, curcumin has received considerable interest as a potential therapeutic agent for the prevention and/or treatment of various malignant diseases, arthritis, allergies, Alzheimer’s disease, and other inflammatory illnesses. This review summarizes various in vitro and in vivo pharmacological aspects of curcumin as well as the underlying action mechanisms. The recently identified molecular targets and signaling pathways modulated by curcumin are also discussed here. PMID:20955148

  4. Targeting isoprenylcysteine methylation ameliorates disease in a mouse model of progeria.

    PubMed

    Ibrahim, Mohamed X; Sayin, Volkan I; Akula, Murali K; Liu, Meng; Fong, Loren G; Young, Stephen G; Bergo, Martin O

    2013-06-14

    Several progeroid disorders, including Hutchinson-Gilford progeria syndrome (HGPS) and restrictive dermopathy (ZMPSTE24 deficiency), arise when a farnesylated and methylated form of prelamin A accumulates at the nuclear envelope. Here, we found that a hypomorphic allele of isoprenylcysteine carboxyl methyltransferase (ICMT) increased body weight, normalized grip strength, and prevented bone fractures and death in Zmpste24-deficient mice. The reduced ICMT activity caused prelamin A mislocalization within the nucleus and triggered prelamin A-dependent activation of AKT-mammalian target of rapamycin (mTOR) signaling, which abolished the premature senescence of Zmpste24-deficient fibroblasts. ICMT inhibition increased AKT-mTOR signaling and proliferation and delayed senescence in human HGPS fibroblasts but did not reduce the levels of misshapen nuclei in mouse and human cells. Thus, targeting ICMT might be useful for treating prelamin A-associated progeroid disorders. PMID:23686339

  5. The PI3K/Akt/mTOR pathway in ovarian cancer: therapeutic opportunities and challenges

    PubMed Central

    Cheaib, Bianca; Auguste, Aurélie; Leary, Alexandra

    2015-01-01

    The phosphatidylinositol 3 kinase (PI3K) pathway is frequently altered in cancer, including ovarian cancer (OC). Unfortunately, despite a sound biological rationale and encouraging activity in preclinical models, trials of first-generation inhibitors of mammalian target of rapamycin (mTOR) in OC have demonstrated negative results. The lack of patient selection as well as resistance to selective mTOR complex-1 (mTORC1) inhibitors could explain the disappointing results thus far. Nonetheless, a number of novel agents are being investigated, including dual mTORC1/mTORC2, Akt, and PI3K inhibitors. Although it is likely that inhibition of the PI3K/Akt/mTOR pathway may have little effect in unselected OC patients, certain histological types, such as clear cell or endometrioid OC with frequent phosphatidylinositol-4,5-biphosphate 3-kinase, catalytic subunit alpha (PIK3CA) and/or phosphatase and tensin homolog (PTEN) alterations, may be particularly suited to this approach. Given the complexity and redundancy of the PI3K signaling network, PI3K pathway inhibition may be most useful in combination with either chemotherapy or other targeted therapies, such as MEK inhibitors, anti-angiogenic therapy, and hormonal therapy, in appropriately selected OC patients. Here, we discuss the relevance of the PI3K pathway in OC and provide an up-to-date review of clinical trials of novel PI3K inhibitors alone or in combination with cytotoxics and novel therapies in OC. In addition, the challenges of drug resistance and predictive biomarkers are addressed. PMID:25556614

  6. Akt inhibitors in cancer treatment: The long journey from drug discovery to clinical use (Review)

    PubMed Central

    NITULESCU, GEORGE MIHAI; MARGINA, DENISA; JUZENAS, PETRAS; PENG, QIAN; OLARU, OCTAVIAN TUDOREL; SALOUSTROS, EMMANOUIL; FENGA, CONCETTINA; SPANDIDOS, DEMETRIOS A.; LIBRA, MASSIMO; TSATSAKIS, ARISTIDIS M.

    2016-01-01

    Targeted cancer therapies are used to inhibit the growth, progression, and metastasis of the tumor by interfering with specific molecular targets and are currently the focus of anticancer drug development. Protein kinase B, also known as Akt, plays a central role in many types of cancer and has been validated as a therapeutic target nearly two decades ago. This review summarizes the intracellular functions of Akt as a pivotal point of converging signaling pathways involved in cell growth, proliferation, apoptotis and neo-angiogenesis, and focuses on the drug design strategies to develop potent anticancer agents targeting Akt. The discovery process of Akt inhibitors has evolved from adenosine triphosphate (ATP)-competitive agents to alternative approaches employing allosteric sites in order to overcome the high degree of structural similarity between Akt isoforms in the catalytic domain, and considerable structural analogy to the AGC kinase family. This process has led to the discovery of inhibitors with greater specificity, reduced side-effects and lower toxicity. A second generation of Akt has inhibitors emerged by incorporating a chemically reactive Michael acceptor template to target the nucleophile cysteines in the catalytic activation loop. The review outlines the development of several promising drug candidates emphasizing the importance of each chemical scaffold. We explore the pipeline of Akt inhibitors and their preclinical and clinical examination status, presenting the potential clinical application of these agents as a monotherapy or in combination with ionizing radiation, other targeted therapies, or chemotherapy. PMID:26698230

  7. Activation of the PI3K/mTOR/AKT Pathway and Survival in Solid Tumors: Systematic Review and Meta-Analysis

    PubMed Central

    Ocana, Alberto; Vera-Badillo, Francisco; Al-Mubarak, Mustafa; Templeton, Arnoud J.; Corrales-Sanchez, Verónica; Diez-Gonzalez, Laura; Cuenca-Lopez, María D.; Seruga, Bostjan; Pandiella, Atanasio; Amir, Eitan

    2014-01-01

    Background Aberrations in the phosphatidylinositol 3-kinase (PI3K)/mammalian target of rapamycin (mTOR)/AKT pathway are common in solid tumors. Numerous drugs have been developed to target different components of this pathway. However the prognostic value of these aberrations is unclear. Methods PubMed was searched for studies evaluating the association between activation of the PI3K/mTOR/AKT pathway (defined as PI3K mutation [PIK3CA], lack of phosphatase and tensin homolog [PTEN] expression by immunohistochemistry or western-blot or increased expression/activation of downstream components of the pathway by immunohistochemistry) with overall survival (OS) in solid tumors. Published data were extracted and computed into odds ratios (OR) for death at 5 years. Data were pooled using the Mantel-Haenszel random-effect model. Results Analysis included 17 studies. Activation of the PI3K/mTOR/AKT pathway was associated with significantly worse 5-year survival (OR:2.12, 95% confidence intervals 1.42–3.16, p<0.001). Loss of PTEN expression and increased expression/activation of downstream components were associated with worse survival. No association between PIK3CA mutations and survival was observed. Differences between methods for assessing activation of the PI3K/mTOR/AKT pathway were statistically significant (p = 0.04). There was no difference in the effect of up-regulation of the pathway on survival between different cancer sites (p = 0.13). Conclusion Activation of the PI3K/AKT/mTOR pathway, especially if measured by loss of PTEN expression or increased expression/activation of downstream components is associated with poor survival. PIK3CA mutational status is not associated with adverse outcome, challenging its value as a biomarker of patient outcome or as a stratification factor for patients treated with agents acting on the PI3K/AKT/mTOR pathway. PMID:24777052

  8. AKT signaling in ERBB2-amplified breast cancer.

    PubMed

    Carmona, F Javier; Montemurro, Filippo; Kannan, Srinivasaraghavan; Rossi, Valentina; Verma, Chandra; Baselga, José; Scaltriti, Maurizio

    2016-02-01

    The PI3K/AKT pathway is the focus of several targeted therapeutic agents for a variety of malignancies. In ERBB2-amplified breast cancer, the hyperactivation of this signaling cascade is associated with resistance to ERBB2-targeted therapy. This can occur through gain-of-function alterations or compensatory mechanisms that enter into play upon pharmacological pressure. The strong rationale in combining anti-ERBB2 agents with PI3K/AKT inhibitors, together with the identification of genomic alterations conferring sensitivity to targeted inhibition, are guiding the design of clinical studies aimed at preventing the emergence of drug resistance and achieving more durable response. In the present review, we describe the involvement of this pathway in breast cancer pathogenesis, with an emphasis on AKT kinases, and provide insight into currently available targeted agents for the treatment of ERBB2-amplified breast cancer. Finally, we provide preliminary data on a novel AKT3 mutation detected in the context of resistance to anti-ERBB2 therapy as an example of genomics-based approaches towards uncovering novel actionable targets in this setting. PMID:26645663

  9. Electroporation into Cultured Mammalian Embryos

    NASA Astrophysics Data System (ADS)

    Nomura, Tadashi; Takahashi, Masanori; Osumi, Noriko

    Over the last century, mammalian embryos have been used extensively as a common animal model to investigate fundamental questions in the field of developmental biology. More recently, the establishment of transgenic and gene-targeting systems in laboratory mice has enabled researchers to unveil the genetic mechanisms under lying complex developmental processes (Mak, 2007). However, our understanding of cell—cell interactions and their molecular basis in the early stages of mammalian embryogenesis is still very fragmentary. One of the major problems is the difficulty of precise manipulation and limited accessibility to mammalian embryos via uterus wall. Unfortunately, existing tissue and organotypic culture systems per se do not fully recapitulate three-dimensional, dynamic processes of organogenesis observed in vivo. Although transgenic animal technology and virus-mediated gene delivery are useful to manipulate gene expression, these techniques take much time and financial costs, which limit their use.

  10. Nuclear Akt2 opposes limbal keratinocyte stem cell self-renewal by repressing a FOXO-mTORC1 signaling pathway.

    PubMed

    Saoncella, Stefania; Tassone, Beatrice; Deklic, Erika; Avolio, Fabio; Jon, Cristina; Tornillo, Giusy; De Luca, Elisa; Di Iorio, Enzo; Piva, Roberto; Cabodi, Sara; Turco, Emilia; Pandolfi, Pier Paolo; Calautti, Enzo

    2014-03-01

    Signals downstream of Akt can either favor or oppose stem cell (SC) maintenance, but how this dual role can be achieved is still undefined. Using human limbal keratinocyte stem cells (LKSCs), a SC type used in transplantation therapies for corneal regeneration, we show that Akt signaling is prominent in SC populations both in vivo and in vitro, and that Akt1 promotes while Akt2 opposes SC self-renewal. Noteworthy, loss of Akt2 signaling enhances LKSC maintenance ex vivo, whereas Akt1 depletion anticipates SC exhaustion. Mechanistically, the antagonistic functions of Akt1 and Akt2 in SC control are mainly dictated by their differential subcellular distribution, being nuclear Akt2 selectively implicated in FOXO inhibition. Akt2 downregulation favors LKSC maintenance as a result of a gain of FOXO functions, which attenuates the mechanistic target of rapamycin complex one signaling via tuberous sclerosis one gene induction, and promotes growth factor signaling through Akt1. Consistently, Akt2 deficiency also enhances limbal SCs in vivo. Thus, our findings reveal distinct roles for nuclear versus cytosolic Akt signaling in normal epithelial SC control and suggest that the selective Akt2 inhibition may provide novel pharmacological strategies for human LKSC expansion in therapeutic settings and mechanistic research. PMID:24123662

  11. Hinokitiol Inhibits Melanogenesis via AKT/mTOR Signaling in B16F10 Mouse Melanoma Cells.

    PubMed

    Tsao, Yu-Tzu; Huang, Yu-Fen; Kuo, Chun-Yu; Lin, Yu-Chiang; Chiang, Wei-Cheng; Wang, Wei-Kuang; Hsu, Chia-Wei; Lee, Che-Hsin

    2016-01-01

    H inokitiol purified from the heartwood of cupressaceous plants has had various biological functions of cell differentiation and growth. Hinokitiol has been demonstrated as having an important role in anti-inflammation and anti-bacteria effect, suggesting that it is potentially useful in therapies for hyperpigmentation. Previously, hinokitiol inhibited the production of melanin by inhibiting tyrosinase activity. The autophagic signaling pathway can induce hypopigmentation. This study is warranted to investigate the mechanism of hinokitiol-induced hypopigmentation through autophagy in B16F10 melanoma cells. The melanin contents and expression of microthphalmia associated transcription factor (MITF) and tyrosinase were inhibited by treatment with hinokitiol. Moreover, the phosphorylation of the protein express levels of phospho-protein kinase B (P-AKT) and phospho-mammalian targets of rapamycin (P-mTOR) were reduced after hinokitiol treatment. In addition, the microtubule associated protein 1 light chain 3 (LC3) -II and beclin 1 (autophagic markers) were increased after the B16F10 cell was treated with hinokitiol. Meanwhile, hinokitiol decreased cellular melanin contents in a dose-dependent manner. These findings establish that hinokitiol inhibited melanogenesis through the AKT/mTOR signaling pathway. PMID:26901194

  12. Akt is activated in chronic lymphocytic leukemia cells and delivers a pro-survival signal: the therapeutic potential of Akt inhibition

    PubMed Central

    Zhuang, Jianguo; Hawkins, Stephen F.; Glenn, Mark A.; Lin, Ke; Johnson, Gillian G.; Carter, Anthony; Cawley, John C.; Pettitt, Andrew R.

    2010-01-01

    Background The aims of the present study were to ascertain the activation status of Akt in the primary cells of chronic lymphocytic leukemia and to investigate the effects of specific Akt inhibition on chronic lymphocytic leukemia-cell survival. Design and Methods Anti-phospho-Akt (Ser473 or Thr308) antibodies and western blotting were used to establish the activation status of Akt. The effects of two different, specific small-molecule inhibitors (A-443654 or Akti-1/2) or small interfering RNA on cell survival and downstream targets of Akt were assessed. Apoptosis was determined by fluorescence-activated cell sorting analysis of phosphatidylserine exposure and by measurement of PARP cleavage. The phosphorylation status of GSK-3 and MDM2, two immediate downstream substrates of Akt, levels of the anti-apoptotic proteins BCL2 and MCL1, and expression of p53 and p21 were all measured by western blotting. Results Fully activated Akt was demonstrable in all chronic lymphocytic leukemia clones examined (n=26). These results were validated with extensive controls and it was shown that a harsh method of cell extraction is needed for detection of the active enzyme. Specific inhibition of Akt induced extensive apoptosis of chronic lymphocytic leukemia cells, which was associated with both a rapid loss of MCL1 through proteasomal degradation and increased expression of p53. Moreover, the Akt inhibitors, at concentrations that induced extensive apoptosis in chronic lymphocytic leukemia cells, had little or no effect on normal peripheral blood mononuclear cells. Conclusions Chronic lymphocytic leukemia clones consistently contain activated Akt which plays a pivotal role in maintaining cell survival. Inhibition of the Akt pathway may be of potential value as a novel therapeutic strategy in chronic lymphocytic leukemia. PMID:19713228

  13. Reactivation of AKT signaling following treatment of cancer cells with PI3K inhibitors attenuates their antitumor effects

    SciTech Connect

    Dufour, Marc; Dormond-Meuwly, Anne; Pythoud, Catherine; Demartines, Nicolas; Dormond, Olivier

    2013-08-16

    Highlights: •PI3K inhibitors inhibit AKT only transiently. •Re-activation of AKT limits the anti-cancer effect of PI3K inhibitors. •The results suggest to combine PI3K and AKT inhibitors in cancer therapy. -- Abstract: Targeting the phosphatidylinositol-3-kinase (PI3K) is a promising approach in cancer therapy. In particular, PI3K blockade leads to the inhibition of AKT, a major downstream effector responsible for the oncogenic activity of PI3K. However, we report here that small molecule inhibitors of PI3K only transiently block AKT signaling. Indeed, treatment of cancer cells with PI3K inhibitors results in a rapid inhibition of AKT phosphorylation and signaling which is followed by the reactivation of AKT signaling after 48 h as observed by Western blot. Reactivation of AKT signaling occurs despite effective inhibition of PI3K activity by PI3K inhibitors. In addition, wortmannin, a broad range PI3K inhibitor, did not block AKT reactivation suggesting that AKT signals independently of PI3K. In a therapeutical perspective, combining AKT and PI3K inhibitors exhibit stronger anti-proliferative and pro-apoptotic effects compared to AKT or PI3K inhibitors alone. Similarly, in a tumor xenograft mouse model, concomitant PI3K and AKT blockade results in stronger anti-cancer activity compared with either blockade alone. This study shows that PI3K inhibitors only transiently inhibit AKT which limits their antitumor activities. It also provides the proof of concept to combine PI3K inhibitors with AKT inhibitors in cancer therapy.

  14. Honokiol induces autophagic cell death in malignant glioma through reactive oxygen species-mediated regulation of the p53/PI3K/Akt/mTOR signaling pathway.

    PubMed

    Lin, Chien-Ju; Chen, Ta-Liang; Tseng, Yuan-Yun; Wu, Gong-Jhe; Hsieh, Ming-Hui; Lin, Yung-Wei; Chen, Ruei-Ming

    2016-08-01

    Honokiol, an active constituent extracted from the bark of Magnolia officinalis, possesses anticancer effects. Apoptosis is classified as type I programmed cell death, while autophagy is type II programmed cell death. We previously proved that honokiol induces cell cycle arrest and apoptosis of U87 MG glioma cells. Subsequently in this study, we evaluated the effect of honokiol on autophagy of glioma cells and examined the molecular mechanisms. Administration of honokiol to mice with an intracranial glioma increased expressions of cleaved caspase 3 and light chain 3 (LC3)-II. Exposure of U87 MG cells to honokiol also induced autophagy in concentration- and time-dependent manners. Results from the addition of 3-methyladenine, an autophagy inhibitor, and rapamycin, an autophagy inducer confirmed that honokiol-induced autophagy contributed to cell death. Honokiol decreased protein levels of PI3K, phosphorylated (p)-Akt, and p-mammalian target of rapamycin (mTOR) in vitro and in vivo. Pretreatment with a p53 inhibitor or transfection with p53 small interfering (si)RNA suppressed honokiol-induced autophagy by reversing downregulation of p-Akt and p-mTOR expressions. In addition, honokiol caused generation of reactive oxygen species (ROS), which was suppressed by the antioxidant, vitamin C. Vitamin C also inhibited honokiol-induced autophagic and apoptotic cell death. Concurrently, honokiol-induced alterations in levels of p-p53, p53, p-Akt, and p-mTOR were attenuated following vitamin C administration. Taken together, our data indicated that honokiol induced ROS-mediated autophagic cell death through regulating the p53/PI3K/Akt/mTOR signaling pathway. PMID:27236003

  15. Regulation of the cell cycle and PI3K/Akt/mTOR signaling pathway by tanshinone I in human breast cancer cell lines

    PubMed Central

    WANG, LI; WU, JIANZHONG; LU, JIANWEI; MA, RONG; SUN, DAWEI; TANG, JINHAI

    2015-01-01

    Breast cancer is the second leading cause of cancer-related mortality in females worldwide. Therefore, identifying alternative strategies to combat the disease mortality is important. The aim of the present study was to investigate the effect of tanshinone I (Tan I) on the tumorigenicity of estrogen-responsive MCF-7 and estrogen-independent MDA-MB-453 human breast cancer cells. The cytotoxicity of Tan I was evaluated using a Cell Counting Kit-8 assay, the apoptosis and cell cycle distribution were detected using flow cytometry and the cell morphology was observed using a fluorescence microscope. In addition, the cell cycle regulatory proteins and apoptosis-associated proteins involved in the phosphatidylinositide 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) signaling pathway were detected using western blot analysis using specific protein antibodies. The MCF-7 and MDA-MB-453 cells were equally sensitive to Tan I regardless of their responsiveness to estrogen. Tan I exerted similar antiproliferative activities and induction of apoptosis, resulting in S phase arrest accompanied by decreases in cyclin B and increases in cyclin E and cyclin A proteins, which may have been associated with the upregulation of cyclin-dependent kinase inhibitors p21Cip1 and p27Kip1. In addition, Tan I was found to downregulate anti-apoptotic and upregulate associated apoptotic components of the PI3K/Akt/mTOR signaling pathway. Notably, treatment with the PI3K inhibitor, LY294002, decreased the levels of phosphorylated (p)-PI3K, p-Akt and p-mTOR. These results clearly indicated that the mechanism of action of Tan I involved, at least partially, an effect on the PI3K/Akt/mTOR signaling pathway, providing new information for anticancer drug design and development. PMID:25355053

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

    PubMed

    Das, Rahul; Sebo, Zachary; Pence, Laramie; Dobens, Leonard L

    2014-01-01

    Drosophila Tribbles (Trbl) is the founding member of the Trib family of kinase-like docking proteins that modulate cell signaling during proliferation, migration and growth. In a wing misexpression screen for Trbl interacting proteins, we identified the Ser/Thr protein kinase Akt1. Given the central role of Akt1 in insulin signaling, we tested the function of Trbl in larval fat body, a tissue where rapid increases in size are exquisitely sensitive to insulin/insulin-like growth factor levels. Consistent with a role in antagonizing insulin-mediated growth, trbl RNAi knockdown in the fat body increased cell size, advanced the timing of pupation and increased levels of circulating triglyceride. Complementarily, overexpression of Trbl reduced fat body cell size, decreased overall larval size, delayed maturation and lowered levels of triglycerides, while circulating glucose levels increased. The conserved Trbl kinase domain is required for function in vivo and for interaction with Akt in a yeast two-hybrid assay. Consistent with direct regulation of Akt, overexpression of Trbl in the fat body decreased levels of activated Akt (pSer505-Akt) while misexpression of trbl RNAi increased phospho-Akt levels, and neither treatment affected total Akt levels. Trbl misexpression effectively suppressed Akt-mediated wing and muscle cell size increases and reduced phosphorylation of the Akt target FoxO (pSer256-FoxO). Taken together, these data show that Drosophila Trbl has a conserved role to bind Akt and block Akt-mediated insulin signaling, and implicate Trib proteins as novel sites of signaling pathway integration that link nutrient availability with cell growth and proliferation. PMID:25329475

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

    SciTech Connect

    Yang, Wonseok; Ju, Ji-hyun; Lee, Kyung-min; Nam, KeeSoo; Oh, Sunhwa; Shin, Incheol

    2013-02-01

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

  18. Drosophila Tribbles Antagonizes Insulin Signaling-Mediated Growth and Metabolism via Interactions with Akt Kinase

    PubMed Central

    Das, Rahul; Sebo, Zachary; Pence, Laramie; Dobens, Leonard L.

    2014-01-01

    Drosophila Tribbles (Trbl) is the founding member of the Trib family of kinase-like docking proteins that modulate cell signaling during proliferation, migration and growth. In a wing misexpression screen for Trbl interacting proteins, we identified the Ser/Thr protein kinase Akt1. Given the central role of Akt1 in insulin signaling, we tested the function of Trbl in larval fat body, a tissue where rapid increases in size are exquisitely sensitive to insulin/insulin-like growth factor levels. Consistent with a role in antagonizing insulin-mediated growth, trbl RNAi knockdown in the fat body increased cell size, advanced the timing of pupation and increased levels of circulating triglyceride. Complementarily, overexpression of Trbl reduced fat body cell size, decreased overall larval size, delayed maturation and lowered levels of triglycerides, while circulating glucose levels increased. The conserved Trbl kinase domain is required for function in vivo and for interaction with Akt in a yeast two-hybrid assay. Consistent with direct regulation of Akt, overexpression of Trbl in the fat body decreased levels of activated Akt (pSer505-Akt) while misexpression of trbl RNAi increased phospho-Akt levels, and neither treatment affected total Akt levels. Trbl misexpression effectively suppressed Akt-mediated wing and muscle cell size increases and reduced phosphorylation of the Akt target FoxO (pSer256-FoxO). Taken together, these data show that Drosophila Trbl has a conserved role to bind Akt and block Akt-mediated insulin signaling, and implicate Trib proteins as novel sites of signaling pathway integration that link nutrient availability with cell growth and proliferation. PMID:25329475

  19. Inhalation Toxicity of Particulate Matters Doped with Arsenic Induced Genotoxicity and Altered Akt Signaling Pathway in Lungs of Mice

    PubMed Central

    Park, Jin Hong; Kwon, Jung-Taek; Arassh, Minai-Teherani; Hwang, Soon-Kyung; Chang, Seung-Hee; Lim, Hwang Tae; Cho, Hyun-Seon

    2010-01-01

    In the workplace, the arsenic is used in the semiconductor production and the manufacturing of pigments, glass, pesticides and fungicides. Therefore, workers may be exposed to airborne arsenic during its use in manufacturing. The purpose of this study was to evaluate the potential toxicity of particulate matters (PMs) doped with arsenic (PMs-Arsenic) using a rodent model and to compare the genotoxicity in various concentrations and to examine the role of PMs-Arsenic in the induction of signaling pathway in the lung. Mice were exposed to PMs 124.4 ± 24.5 μg/m3 (low concentration) , 220.2 ± 34.5 μg/m3 (middle concentration) , 426.4 ± 40.3 μg/m3 (high concentration) doped with arsenic 1.4 μg/m3 (Low concentration) ,2.5 μg/m3 (middle concentration) , 5.7 μg/m3 (high concentration) for 4 wks (6 h/d, 5 d/wk) , respectively in the whole-body inhalation exposure chambers. To determine the level of genotoxicity, Chromosomal aberration (CA) assay in splenic lymphocytes and Supravital micronucleus (SMN) assay were performed. Then, signal pathway in the lung was analyzed. In the genotoxicity experiments, the increases of aberrant cells were concentration-dependent. Also, PMs-arsenic caused peripheral blood micronucleus frequency at high concentration. The inhalation of PMs-Arsenic increased an expression of phosphorylated Akt (p-Akt: protein kinase B) and phpsphorylated mammalian target of rapamycin (p-mTOR) at high concentration group. Taken together, inhaled PMs-Arsenic caused genotoxicity and altered Akt signaling pathway in the lung. Therefore, the inhalation of PMs-Arsenic needs for a careful risk assessment in the workplace. PMID:24278533

  20. Targeting mTOR signaling pathways and related negative feedback loops for the treatment of acute myeloid leukemia

    PubMed Central

    Carneiro, Benedito A; Kaplan, Jason B; Altman, Jessica K; Giles, Francis J; Platanias, Leonidas C

    2015-01-01

    An accumulating understanding of the complex pathogenesis of acute myeloid leukemia (AML) continues to lead to promising therapeutic approaches. Among the key aberrant intracellular signaling pathways involved in AML, the phosphatidylinositol 3-kinase/AKT/mammalian target of rapamycin (PI3K/AKT/mTOR) axis is of major interest. This axis modulates a wide array of critical cellular functions, including proliferation, metabolism, and survival. Pharmacologic inhibitors of components of this pathway have been developed over the past decade, but none has an established role in the treatment of AML. This review will discuss the preclinical data and clinical results driving ongoing attempts to exploit the PI3K/AKT/mTOR pathway in patients with AML and address issues related to negative feedback loops that account for leukemic cell survival. Targeting the PI3K/AKT/mTOR pathway is of high interest for the treatment of AML, but combination therapies with other targeted agents may be needed to block negative feedback loops in leukemia cells. PMID:25801978