Resistance of Akt kinases to dephosphorylation through ATP-dependent conformational plasticity.

PubMed

Chan, Tung O; Zhang, Jin; Rodeck, Ulrich; Pascal, John M; Armen, Roger S; Spring, Maureen; Dumitru, Calin D; Myers, Valerie; Li, Xue; Cheung, Joseph Y; Feldman, Arthur M

2011-11-15

Phosphorylation of a threonine residue (T308 in Akt1) in the activation loop of Akt kinases is a prerequisite for deregulated Akt activity frequently observed in neoplasia. Akt phosphorylation in vivo is balanced by the opposite activities of kinases and phosphatases. Here we describe that targeting Akt kinase to the cell membrane markedly reduced sensitivity of phosphorylated Akt to dephosphorylation by protein phosphatase 2A. This effect was amplified by occupancy of the ATP binding pocket by either ATP or ATP-competitive inhibitors. Mutational analysis revealed that R273 in Akt1 and the corresponding R274 in Akt2 are essential for shielding T308 in the activation loop against dephosphorylation. Thus, occupancy of the nucleotide binding pocket of Akt kinases enables intramolecular interactions that restrict phosphatase access and sustain Akt phosphorylation. This mechanism provides an explanation for the "paradoxical" Akt hyperphosphorylation induced by ATP-competitive inhibitor, A-443654. The lack of phosphatase resistance further contributes insight into the mechanism by which the human Akt2 R274H missense mutation may cause autosomal-dominant diabetes mellitus.

Critical role of PI3-kinase/Akt activation in the PARP inhibitor induced heart function recovery during ischemia-reperfusion.

PubMed

Kovacs, Krisztina; Toth, Ambrus; Deres, Peter; Kalai, Tamas; Hideg, Kalman; Gallyas, Ferenc; Sumegi, Balazs

2006-02-14

Poly(ADP-ribose) polymerase (PARP) inhibitors protect hearts from ischemia-reperfusion (IR)-induced damages by limiting nicotinamide adenine dinucleotide (NAD+) and ATP depletion, and by other, not yet elucidated mechanisms. Our preliminary data suggested that PARP catalyzed ADP-ribosylations may affect signaling pathways in cardiomyocytes. To clarify this possibility, we studied the effect of a well-characterized (4-hydroxyquinazoline) and a novel (carboxaminobenzimidazol-derivative) PARP inhibitor on the activation of phosphatidylinositol-3-kinase (PI3-kinase)/Akt pathway in Langendorff-perfused hearts. PARP inhibitors promoted the restoration of myocardial energy metabolism (assessed by 31P nuclear magnetic resonance spectroscopy) and cardiac function compared to untreated hearts. PARP inhibitors also attenuated the infarct size and reduced the IR-induced lipid peroxidation, protein oxidation and total peroxide concentration. Moreover, PARP inhibitors facilitated Akt phosphorylation and activation, as well as the phosphorylation of its downstream target glycogen synthase kinase-3beta (GSK-3beta) in normoxia and, more robustly, during IR. Blocking PI3-kinase by wortmannin or LY294002 reduced the PARP inhibitor-elicited robust Akt and GSK-3beta phosphorylation upon ischemia-reperfusion, and significantly diminished the recovery of ATP and creatine phosphate showing the importance of Akt activation in the recovery of energy metabolism. In addition, inhibition of PI3-kinase/Akt pathway decreased the protective effect of PARP inhibitors on infarct size and the recovery of heart functions. All these data suggest that contrary to the original view, which considered preservation of NAD+ and consequently ATP pools as the exclusive underlying mechanism for the cytoprotective effect of PARP inhibitors, the activation of PI3-kinase/Akt pathway and related processes are at least equally important in the cardioprotective effects of PARP inhibitors during ischemia-reperfusion.

Additive effect by combination of Akt inhibitor, MK-2206, and PDGFR inhibitor, tyrphostin AG 1296, in suppressing anaplastic thyroid carcinoma cell viability and motility.

PubMed

Che, Huan-Yong; Guo, Hang-Yuan; Si, Xu-Wei; You, Qiao-Ying; Lou, Wei-Ying

2014-01-01

The phosphatidylinositol-3-kinase/Akt pathway and receptor tyrosine kinases regulate many tumorigenesis related cellular processes including cell metabolism, cell survival, cell motility, and angiogenesis. Anaplastic thyroid carcinoma (ATC) is a rare type of thyroid cancer with no effective systemic therapy. It has been shown that Akt activation is associated with tumor progression in ATC. Here we observed the additive effect between an Akt inhibitor (MK-2206) and a novel platelet-derived growth factor receptor inhibitor (tyrphostin AG 1296) in ATC therapy. We found an additive effect between MK-2206 and tyrphostin AG 1296 in suppressing ATC cell viability. The combination of MK-2206 and tyrphostin AG 1296 induces additive apoptosis, additive suppression of the Akt signaling pathway, as well as additive inhibition of cell migration and invasion of ATC cells. Furthermore, the combination of MK-2206 and tyrphostin AG 1296 induced additive suppression of ATC tumor growth in vivo. In summary, our studies suggest that the combination of Akt and receptor tyrosine kinase inhibitors may be an efficient therapeutic strategy for ATC treatment, which might shed new light on ATC therapy.

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.

Sorafenib induces cathepsin B-mediated apoptosis of bladder cancer cells by regulating the Akt/PTEN pathway. The Akt inhibitor, perifosine, enhances the sorafenib-induced cytotoxicity against bladder cancer cells

PubMed Central

Amantini, Consuelo; Morelli, Maria Beatrice; Santoni, Matteo; Soriani, Alessandra; Cardinali, Claudio; Farfariello, Valerio; Eleuteri, Anna Maria; Bonfili, Laura; Mozzicafreddo, Matteo; Nabissi, Massimo; Cascinu, Stefano; Santoni, Giorgio

2015-01-01

Sorafenib, a tyrosine kinase inhibitor, has been demonstrated to exert anti-tumor effects. However, the molecular mechanisms underlying its effects on bladder cancer remain unknown. Here, we evaluated the mechanisms responsible for the sorafenib-induced anti-tumor effects on 5637 and T24 bladder cancer cells. We demonstrated that sorafenib reduces cell viability, stimulates lysosome permeabilization and induces apoptosis of bladder cancer cells. These effects are dependent by the activation of cathepsin B released from lysosomes. The sorafenib-increased cathepsin B activity induced the proteolysis of Bid into tBid that stimulates the intrinsic pathway of apoptosis characterized by mitochondrial membrane depolarization, oxygen radical generation and cytochrome c release. Moreover, we found that cathepsin B enzymatic activity, induced by sorafenib, is dependent on its dephosphorylation via PTEN activation and Akt inactivation. Pretreatment with orthovanadate rescued bladder cancer cells from apoptosis. In addition, the Akt inhibitor perifosine increased the sensitivity of bladder cancer cells to sorafenib-induced cytotoxicity. Overall, our results show that apoptotic cell death induced by sorafenib in bladder cancer cells is dependent on cathepsin B activity and involved PTEN and Akt signaling pathways. The Akt inhibitor perifosine increased the cytotoxic effects of sorafenib in bladder cancer cells. PMID:26097873

Recent Development of Anticancer Therapeutics Targeting Akt

PubMed Central

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

2013-01-01

The serine/threonine kinase Akt has proven to be a significant signaling target, involved in various biological functions. Because of its cardinal role in numerous cellular responses, Akt has been implicated in many human diseases, particularly cancer. It has been established that Akt is a viable and feasible target for anticancer therapeutics. Analysis of all Akt kinases reveals conserved homology for an N-terminal regulatory domain, which contains a pleckstrin-homology (PH) domain for cellular translocation, a kinase domain with serine/threonine specificity, and a C-terminal extension domain. These well defined regions have been targeted, and various approaches, including in silico methods, have been implemented to develop Akt inhibitors. In spite of unique techniques and a prolific body of knowledge surrounding Akt, no targeted Akt therapeutics have reached the market yet. Here we will highlight successes and challenges to date on the development of anticancer agents modulating the Akt pathway in recent patents as well as discuss the methods employed for this task. Special attention will be given to patents with focus on those discoveries using computer-aided drug design approaches. PMID:21110830

Discovery and preclinical pharmacology of a selective ATP-competitive Akt inhibitor (GDC-0068) for the treatment of human tumors.

PubMed

Blake, James F; Xu, Rui; Bencsik, Josef R; Xiao, Dengming; Kallan, Nicholas C; Schlachter, Stephen; Mitchell, Ian S; Spencer, Keith L; Banka, Anna L; Wallace, Eli M; Gloor, Susan L; Martinson, Matthew; Woessner, Richard D; Vigers, Guy P A; Brandhuber, Barbara J; Liang, Jun; Safina, Brian S; Li, Jun; Zhang, Birong; Chabot, Christine; Do, Steven; Lee, Leslie; Oeh, Jason; Sampath, Deepak; Lee, Brian B; Lin, Kui; Liederer, Bianca M; Skelton, Nicholas J

2012-09-27

The discovery and optimization of a series of 6,7-dihydro-5H-cyclopenta[d]pyrimidine compounds that are ATP-competitive, selective inhibitors of protein kinase B/Akt is reported. The initial design and optimization was guided by the use of X-ray structures of inhibitors in complex with Akt1 and the closely related protein kinase A. The resulting compounds demonstrate potent inhibition of all three Akt isoforms in biochemical assays and poor inhibition of other members of the cAMP-dependent protein kinase/protein kinase G/protein kinase C extended family and block the phosphorylation of multiple downstream targets of Akt in human cancer cell lines. Biological studies with one such compound, 28 (GDC-0068), demonstrate good oral exposure resulting in dose-dependent pharmacodynamic effects on downstream biomarkers and a robust antitumor response in xenograft models in which the phosphatidylinositol 3-kinase-Akt-mammalian target of rapamycin pathway is activated. 28 is currently being evaluated in human clinical trials for the treatment of cancer.

Selective Akt Inhibitors Synergize with Tyrosine Kinase Inhibitors and Effectively Override Stroma-Associated Cytoprotection of Mutant FLT3-Positive AML Cells

PubMed Central

Zhang, Xin; Nelson, Erik; Sattler, Martin; Liu, Feiyang; Nicolais, Maria; Zhang, Jianming; Mitsiades, Constantine; Smith, Robert W.; Stone, Richard; Galinsky, Ilene; Nonami, Atsushi; Griffin, James D.; Gray, Nathanael

2013-01-01

Objectives Tyrosine kinase inhibitor (TKI)-treated acute myeloid leukemia (AML) patients commonly show rapid and significant peripheral blood blast cell reduction, however a marginal decrease in bone marrow blasts. This suggests a protective environment and highlights the demand for a better understanding of stromal:leukemia cell communication. As a strategy to improve clinical efficacy, we searched for novel agents capable of potentiating the stroma-diminished effects of TKI treatment of mutant FLT3-expressing cells. Methods We designed a combinatorial high throughput drug screen using well-characterized kinase inhibitor-focused libraries to identify novel kinase inhibitors capable of overriding stromal-mediated resistance to TKIs, such as PKC412 and AC220. Standard liquid culture proliferation assays, cell cycle and apoptosis analysis, and immunoblotting were carried out with cell lines or primary AML to validate putative candidates from the screen and characterize the mechanism(s) underlying observed synergy. Results and Conclusions Our study led to the observation of synergy between selective Akt inhibitors and FLT3 inhibitors against mutant FLT3-positive AML in either the absence or presence of stroma. Our findings are consistent with evidence that Akt activation is characteristic of mutant FLT3-transformed cells, as well as observed residual Akt activity following FLT3 inhibitor treatment. In conclusion, our study highlights the potential importance of Akt as a signaling factor in leukemia survival, and supports the use of the co-culture chemical screen to identify agents able to potentiate TKI anti-leukemia activity in a cytoprotective microenvironment. PMID:23437141

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

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.

Inhibition of PTEN and activation of Akt by menadione.

PubMed

Yoshikawa, Kyoko; Nigorikawa, Kiyomi; Tsukamoto, Mariko; Tamura, Namiko; Hazeki, Kaoru; Hazeki, Osamu

2007-04-01

Menadione (vitamin K(3)) has been shown to activate Erk in several cell lines. This effect has been shown to be due to the activation of EGF receptors (EGFR) as a result of inhibition of some protein tyrosine phosphatases. In the present study, we examined the effects of menadione on Akt in Chinese hamster ovary cells. The phosphorylation of Akt by menadione was not inhibited by AG1478, an inhibitor of EGFR. Menadione inhibited the lipid phosphatase activity of PTEN in a cell-free system. In an intact cell system, menadione inhibited the effect of transfected PTEN on Akt. Thus, one mechanism of its action was considered the accelerated activation of Akt through inhibition of PTEN. This was not the sole mechanism responsible for the EGFR-independent activation of Akt, because menadione attenuated the rate of Akt dephosphorylation even in PTEN-null PC3 cells. The decelerated inactivation of Akt, probably through inhibition of some tyrosine phosphatases, was considered another mechanism of its action.

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

PubMed

Rudner, Justine; Ruiner, Carola-Ellen; Handrick, René; Eibl, Hans-Jörg; Belka, Claus; Jendrossek, Verena

2010-11-16

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

Dimethylarginine dimethylaminohydrolase 1 modulates endothelial cell growth through NO and Akt

PubMed Central

Zhang, Ping; Hu, Xinli; Xu, Xin; Chen, Yingjie; Bache, Robert J.

2011-01-01

Objective Dimethylarginine dimethylaminohydrolase 1 (DDAH1) modulates NO production by degrading the endogenous NO synthase (NOS) inhibitors ADMA and L-NMMA. This study examined whether, in addition to degrading ADMA, DDAH1 exerts ADMA independent effects that influence endothelial function. Methods and Results Using selective gene silencing of DDAH1 with small interfering RNA and overexpression of DDAH1 in HUVEC, we found that DDAH1 acts to promote endothelial cell proliferation, migration and tube formation both by Akt phosphorylation as well as through the traditional role of degrading ADMA. Incubation of HUVEC with the NOS inhibitors L-NAME or ADMA, the soluble guanylyl cyclase inhibitor ODQ, or the cGMP analog 8-pCPT-cGMP had no effect on p-AktSer473, indicating that the increase of p-AktSer473 produced by DDAH1 was independent of the NO-cGMP signaling pathway. DDAH1 formed a protein complex with Ras, and DDAH1 overexpression increased Ras activity. The Ras inhibitor manumycin-A or dominant-negative Ras significantly attenuated the DDAH1-induced increase of p-AktSer473. Furthermore, DDAH1 knockout impaired endothelial sprouting from cultured aortic rings, and overexpression of constitutively active Akt or DDAH1 rescued endothelial sprouting in the aortic rings from these mice. Conclusions DDAH1 exerts a unique role in activating Akt that affects endothelial function independent of degrading endogenous NOS inhibitors. PMID:21212404

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

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

Microsecond molecular dynamics simulations provide insight into the ATP-competitive inhibitor-induced allosteric protection of Akt kinase phosphorylation.

PubMed

Mou, Linkai; Cui, Tongwei; Liu, Weiguang; Zhang, Hong; Cai, Zhanxiu; Lu, Shaoyong; Gao, Guojun

2017-05-01

Akt is a serine/threonine protein kinase, a critical mediator of growth factor-induced survival in key cellular pathways. Allosteric signaling between protein intramolecular domains requires long-range communication mediated by hotspot residues, often triggered by ligand binding. Here, based on extensive 3 μs explicit solvent molecular dynamics (MD) simulations of Akt1 kinase domain in the unbound (apo) and ATP-competitive inhibitor, GDC-0068-bound states, we propose a molecular mechanism for allosteric regulation of Akt1 kinase phosphorylation by GDC-0068 binding to the ATP-binding site. MD simulations revealed that the apo Akt1 is flexible with two disengaged N- and C-lobes, equilibrated between the open and closed conformations. GDC-0068 occupancy of the ATP-binding site shifts the conformational equilibrium of Akt1 from the open conformation toward the closed conformation and stabilizes the closed state. This effect enables allosteric signal propagation from the GDC-0068 to the phosphorylated T308 (pT308) in the activation loop and restrains phosphatase access to pT308, thereby protecting the pT308 in the GDC-0068-bound Akt1. Importantly, functional hotspots involved in the allosteric communication from the GDC-0068 to the pT308 are identified. Our analysis of GDC-0068-induced allosteric protection of Akt kinase phosphorylation yields important new insights into the molecular mechanism of allosteric regulation of Akt kinase activity. © 2016 John Wiley & Sons A/S.

Efficacy of AKT Inhibitor ARQ 092 Compared with Sorafenib in a Cirrhotic Rat Model with Hepatocellular Carcinoma.

PubMed

Roth, Gaël S; Macek Jilkova, Zuzana; Zeybek Kuyucu, Ayca; Kurma, Keerthi; Ahmad Pour, Séyédéh Tayébéh; Abbadessa, Giovanni; Yu, Yi; Busser, Benoit; Marche, Patrice N; Leroy, Vincent; Decaens, Thomas

2017-10-01

Hepatocellular carcinoma (HCC) is the second most common cause of cancer-related mortality worldwide. The AKT pathway has been found activated in 50% of HCC cases, making it a promising target. Therefore, we assess efficacy of the allosteric AKT inhibitor ARQ 092 compared with untreated control and standard treatment, sorafenib, in vitro and in vivo ARQ 092 blocked phosphorylation of AKT in vitro and strongly inhibited cell growth with significantly higher potency than sorafenib. Similarly, apoptosis and cell migration were strongly reduced by ARQ 092 in vitro To mimic human advanced HCC, we used a diethylnitrosamine-induced cirrhotic rat model with fully developed HCC. MRI analyses showed that ARQ 092 significantly reduced overall tumor size. Furthermore, number of tumors was decreased by ARQ 092, which was associated with increased apoptosis and decreased proliferation. Tumor contrast enhancement was significantly decreased in the ARQ 092 group. Moreover, on tumor tissue sections, we observed a vascular normalization and a significant decrease in fibrosis in the surrounding liver of animals treated with ARQ 092. Finally, pAKT/AKT levels in ARQ 092-treated tumors were reduced, followed by downregulation of actors of AKT downstream signaling pathway: pmTOR, pPRAS40, pPLCγ1, and pS6K1. In conclusion, we demonstrated that ARQ 092 blocks AKT phosphorylation in vitro and in vivo In the HCC-rat model, ARQ 092 was well tolerated, showed antifibrotic effect, and had stronger antitumor effect than sorafenib. Our results confirm the importance of targeting AKT in HCC. Mol Cancer Ther; 16(10); 2157-65. ©2017 AACR . ©2017 American Association for Cancer Research.

Sensitivity to PI3K and AKT inhibitors is mediated by divergent molecular mechanisms in subtypes of DLBCL.

PubMed

Erdmann, Tabea; Klener, Pavel; Lynch, James T; Grau, Michael; Vočková, Petra; Molinsky, Jan; Tuskova, Diana; Hudson, Kevin; Polanska, Urszula M; Grondine, Michael; Mayo, Michele; Dai, Beiying; Pfeifer, Matthias; Erdmann, Kristian; Schwammbach, Daniela; Zapukhlyak, Myroslav; Staiger, Annette M; Ott, German; Berdel, Wolfgang E; Davies, Barry R; Cruzalegui, Francisco; Trneny, Marek; Lenz, Peter; Barry, Simon T; Lenz, Georg

2017-07-20

Activated B-cell-like (ABC) and germinal center B-cell-like diffuse large B-cell lymphoma (DLBCL) represent the 2 major molecular DLBCL subtypes. They are characterized by differences in clinical course and by divergent addiction to oncogenic pathways. To determine activity of novel compounds in these 2 subtypes, we conducted an unbiased pharmacologic in vitro screen. The phosphatidylinositol-3-kinase (PI3K) α/δ (PI3Kα/δ) inhibitor AZD8835 showed marked potency in ABC DLBCL models, whereas the protein kinase B (AKT) inhibitor AZD5363 induced apoptosis in PTEN-deficient DLBCLs irrespective of their molecular subtype. These in vitro results were confirmed in various cell line xenograft and patient-derived xenograft mouse models in vivo. Treatment with AZD8835 induced inhibition of nuclear factor κB signaling, prompting us to combine AZD8835 with the Bruton's tyrosine kinase inhibitor ibrutinib. This combination was synergistic and effective both in vitro and in vivo. In contrast, the AKT inhibitor AZD5363 was effective in PTEN-deficient DLBCLs through downregulation of the oncogenic transcription factor MYC. Collectively, our data suggest that patients should be stratified according to their oncogenic dependencies when treated with PI3K and AKT inhibitors. © 2017 by The American Society of Hematology.

Targeting of X-linked inhibitor of apoptosis protein and PI3-kinase/AKT signaling by embelin suppresses growth of leukemic cells

PubMed Central

Prabhu, Kirti S.; Siveen, Kodappully S.; Kuttikrishnan, Shilpa; Iskandarani, Ahmad; Tsakou, Magdalini; Achkar, Iman W.; Therachiyil, Lubna; Krishnankutty, Roopesh; Parray, Aijaz; Kulinski, Michal; Merhi, Maysaloun; Dermime, Said; Mohammad, Ramzi M.

2017-01-01

The X-linked inhibitor of apoptosis (XIAP) is a viable molecular target for anticancer drugs that overcome apoptosis-resistance of malignant cells. XIAP is an inhibitor of apoptosis, mediating through its association with BIR3 domain of caspase 9. Embelin, a quinone derivative isolated from the Embelia ribes plant, has been shown to exhibit chemopreventive, anti-inflammatory, and apoptotic activities via inhibiting XIAP activity. In this study, we found that embelin causes a dose-dependent suppression of proliferation in leukemic cell lines K562 and U937. Embelin mediated inhibition of proliferation correlates with induction of apoptosis. Furthermore, embelin treatment causes loss of mitochondrial membrane potential and release of cytochrome c, resulting in subsequent activation of caspase-3 followed by polyadenosin-5’-diphosphate-ribose polymerase (PARP) cleavage. In addition, embelin treatment of leukemic cells results in a decrease of constitutive phosphorylations/activation level of AKT and downregulation of XIAP. Gene silencing of XIAP and AKT expression showed a link between XIAP expression and activated AKT in leukemic cells. Interestingly, targeting of XIAP and PI3-kinase/AKT signaling augmented inhibition of proliferation and induction of apoptosis in leukemic cells. Altogether these findings raise the possibility that embelin alone or in combination with inhibitors of PI3-kinase/AKT pathway may have therapeutic usage in leukemia and possibly other malignancies with up-regulated XIAP pathway. PMID:28704451

Aberrant AKT activation drives well-differentiated liposarcoma

PubMed Central

Gutierrez, Alejandro; Snyder, Eric L.; Marino-Enriquez, Adrian; Zhang, Yi-Xiang; Sioletic, Stefano; Kozakewich, Elena; Grebliunaite, Ruta; Ou, Wen-bin; Sicinska, Ewa; Raut, Chandrajit P.; Demetri, George D.; Perez-Atayde, Antonio R.; Wagner, Andrew J.; Fletcher, Jonathan A.; Fletcher, Christopher D. M.; Look, A. Thomas

2011-01-01

Well-differentiated liposarcoma (WDLPS), one of the most common human sarcomas, is poorly responsive to radiation and chemotherapy, and the lack of animal models suitable for experimental analysis has seriously impeded functional investigation of its pathobiology and development of effective targeted therapies. Here, we show that zebrafish expressing constitutively active Akt2 in mesenchymal progenitors develop WDLPS that closely resembles the human disease. Tumor incidence rates were 8% in p53 wild-type zebrafish, 6% in p53 heterozygotes, and 29% in p53-homozygous mutant zebrafish (P = 0.013), indicating that aberrant Akt activation collaborates with p53 mutation in WDLPS pathogenesis. Analysis of primary clinical specimens of WDLPS, and of the closely related dedifferentiated liposarcoma (DDLPS) subtype, revealed immunohistochemical evidence of AKT activation in 27% of cases. Western blot analysis of a panel of cell lines derived from patients with WDLPS or DDLPS revealed robust AKT phosphorylation in all cell lines examined, even when these cells were cultured in serum-free media. Moreover, BEZ235, a small molecule inhibitor of PI3K and mammalian target of rapamycin that effectively inhibits AKT activation in these cells, impaired viability at nanomolar concentrations. Our findings are unique in providing an animal model to decipher the molecular pathogenesis of WDLPS, and implicate AKT as a previously unexplored therapeutic target in this chemoresistant sarcoma. PMID:21930930

Hsp27 regulates Akt activation and polymorphonuclear leukocyte apoptosis by scaffolding MK2 to Akt signal complex.

PubMed

Wu, Rui; Kausar, Hina; Johnson, Paul; Montoya-Durango, Diego E; Merchant, Michael; Rane, Madhavi J

2007-07-27

We have shown previously that Akt exists in a signal complex with p38 MAPK, MAPK-activated protein kinase-2 (MK2), and heat shock protein 27 (Hsp27) and MK2 phosphorylates Akt on Ser-473. Additionally, dissociation of Hsp27 from Akt, prior to Akt activation, induced polymorphonuclear leukocyte (PMN) apoptosis. However, the role of Hsp27 in regulating Akt activation was not examined. This study tested the hypothesis that Hsp27 regulates Akt activation and promotes cell survival by scaffolding MK2 to the Akt signal complex. Here we show that loss of Akt/Hsp27 interaction by anti-Hsp27 antibody treatment resulted in loss of Akt/MK2 interaction, loss of Akt-Ser-473 phosphorylation, and induced PMN apoptosis. Transfection of myristoylated Akt (AktCA) in HK-11 cells induced Akt-Ser-473 phosphorylation, activation, and Hsp27-Ser-82 phosphorylation. Cotransfection of AktCA with Hsp27 short interfering RNA, but not scrambled short interfering RNA, silenced Hsp27 expression, without altering Akt expression in HK-11 cells. Silencing Hsp27 expression inhibited Akt/MK2 interaction, inhibited Akt phosphorylation and Akt activation, and induced HK-11 cell death. Deletion mutagenesis studies identified acidic linker region (amino acids 117-128) on Akt as an Hsp27 binding region. Deletion of amino acids 117-128 on Akt resulted in loss of its interaction with Hsp27 and MK2 but not with Hsp90 as demonstrated by immunoprecipitation and glutathione S-transferase pulldown studies. Co-transfection studies demonstrated that constitutively active MK2 (MK2EE) phosphorylated Aktwt (wild type) on Ser-473 but failed to phosphorylate Akt(Delta117-128) mutant in transfixed cells. These studies collectively define a novel role of Hsp27 in regulating Akt activation and cellular apoptosis by mediating interaction between Akt and its upstream activator MK2.

Protein kinase C negatively regulates Akt activity and modifies UVC-induced apoptosis in mouse keratinocytes.

PubMed

Li, Luowei; Sampat, Keeran; Hu, Nancy; Zakari, Julia; Yuspa, Stuart H

2006-02-10

Skin keratinocytes are subject to frequent chemical and physical injury and have developed elaborate cell survival mechanisms to compensate. Among these, the Akt/protein kinase B (PKB) pathway protects keratinocytes from the toxic effects of ultraviolet light (UV). In contrast, the protein kinase C (PKC) family is involved in several keratinocyte death pathways. During an examination of potential interactions among these two pathways, we found that the insulin-like growth factor (IGF-1) activates both the PKC and the Akt signaling pathways in cultured primary mouse keratinocytes as indicated by increased phospho-PKC and phospho-Ser-473-Akt. IGF-1 also selectively induced translocation of PKCdelta and PKCepsilon from soluble to particulate fractions in mouse keratinocytes. Furthermore, the PKC-specific inhibitor, GF109203X, increased IGF-1-induced phospho-Ser-473-Akt and Akt kinase activity and enhanced IGF-1 protection from UVC-induced apoptosis. Selective activation of PKC by 12-O-tetradecanoylphorbol-13-acetate (TPA) reduced phospho-Ser-473-Akt, suggesting that activation of PKC inhibits Akt activity. TPA also attenuated IGF-1 and epidermal growth factor-induced phospho-Ser-473-Akt, reduced Akt kinase activity, and blocked IGF-1 protection from UVC-induced apoptosis. The inhibition of Akt activity by TPA was reduced by inhibitors of protein phosphatase 2A, and TPA stimulated the association of phosphatase 2A with Akt. Individual PKC isoforms were overexpressed in cultured keratinocytes by transduction with adenoviral vectors or inhibited with PKC-selective inhibitors. These studies indicated that PKCdelta and PKCepsilon were selectively potent at causing dephosphorylation of Akt and modifying cell survival, whereas PKCalpha enhanced phosphorylation of Akt on Ser-473. Our results suggested that activation of PKCdelta and PKCepsilon provide a negative regulation for Akt phosphorylation and kinase activity in mouse keratinocytes and serve as modulators of cell

AKT is a therapeutic target in myeloproliferative neoplasms

PubMed Central

Khan, Irum; Huang, Zan; Wen, Qiang; Stankiewicz, Monika J.; Gilles, Laure; Goldenson, Benjamin; Schultz, Rachael; Diebold, Lauren; Gurbuxani, Sandeep; Finke, Christy M.; Lasho, Terra L.; Koppikar, Priya; Pardanani, Animesh; Stein, Brady; Altman, Jessica K.; Levine, Ross L.; Tefferi, Ayalew; Crispino, John D.

2014-01-01

The majority of patients with BCR-ABL1-negative myeloproliferative neoplasms (MPN) harbor mutations in JAK2 or MPL, which lead to constitutive activation of the JAK/STAT, PI3K, and ERK signaling pathways. JAK inhibitors by themselves are inadequate in producing selective clonal suppression in MPN and are associated with hematopoietic toxicities. MK-2206 is a potent allosteric AKT inhibitor that was well tolerated, including no evidence of myelosuppression, in a phase I study of solid tumors. Herein, we show that inhibition of PI3K/AKT signaling by MK-2206 affected the growth of both JAK2V617F or MPLW515L-expressing cells via reduced phosphorylation of AKT and inhibition of its downstream signaling molecules. Moreover, we demonstrate that MK-2206 synergizes with Ruxolitinib in suppressing the growth of JAK2V617F mutant SET2 cells. Importantly MK-2206 suppressed colony formation from hematopoietic progenitor cells in patients with primary myelofibrosis (PMF) and alleviated hepatosplenomegaly and reduced megakaryocyte burden in the bone marrows, livers and spleens of mice with MPLW515L-induced MPN. Together, these findings establish AKT as a rational therapeutic target in the MPNs. PMID:23748344

AKT is a therapeutic target in myeloproliferative neoplasms.

PubMed

Khan, I; Huang, Z; Wen, Q; Stankiewicz, M J; Gilles, L; Goldenson, B; Schultz, R; Diebold, L; Gurbuxani, S; Finke, C M; Lasho, T L; Koppikar, P; Pardanani, A; Stein, B; Altman, J K; Levine, R L; Tefferi, A; Crispino, J D

2013-09-01

The majority of patients with BCR-ABL1-negative myeloproliferative neoplasms (MPN) harbor mutations in JAK2 or MPL, which lead to constitutive activation of the JAK/STAT, PI3K and ERK signaling pathways. JAK inhibitors by themselves are inadequate in producing selective clonal suppression in MPN and are associated with hematopoietic toxicities. MK-2206 is a potent allosteric AKT inhibitor that was well tolerated, including no evidence of myelosuppression, in a phase I study of solid tumors. Herein, we show that inhibition of PI3K/AKT signaling by MK-2206 affected the growth of both JAK2V617F- or MPLW515L-expressing cells via reduced phosphorylation of AKT and inhibition of its downstream signaling molecules. Moreover, we demonstrate that MK-2206 synergizes with ruxolitinib in suppressing the growth of JAK2V617F-mutant SET2 cells. Importantly, MK-2206 suppressed colony formation from hematopoietic progenitor cells in patients with primary myelofibrosis and alleviated hepatosplenomegaly and reduced megakaryocyte burden in the bone marrows, livers and spleens of mice with MPLW515L-induced MPN. Together, these findings establish AKT as a rational therapeutic target in the MPNs.

Calcineurin mediates AKT dephosphorylation in the ischemic rat retina.

PubMed

Park, Chang Hwan; Kim, Yoon Sook; Kim, Young Hee; Choi, Mee Young; Yoo, Ji Myong; Kang, Sang Soo; Choi, Wan Sung; Cho, Gyeong Jae

2008-10-09

Calcineurin (CaN) is a calcium/calmodulin-dependent protein phosphatase that has an important role in ischemia-induced apoptosis. The serine/threonine kinase, Akt, which is also known as protein kinase B, has an important role in the cell death/survival pathways. Akt is activated by its phosphorylation, which is positively regulated by phosphatidylinositol 3-kinase (PI3K) and negatively regulated by a class of protein phosphatases (PPs) in tissue. However, the relationship between CaN and Akt after transient ischemia remains unclear. In the present study, we investigated whether CaN is involved in neuronal cell apoptosis and Akt dephosphorylation that occur during ischemic injury. We examined the interdependence between CaN and Akt/protein kinase B (PKB) in the rat retina after transient ischemia. After ischemic damage, we detected changes in levels of CaN, Akt and Bad in rats in the presence or absence FK506, CaN inhibitor. Our results show that CaN cleavage reduced Akt phosphorylation at Thr308 and Ser473, and led to apoptosis via dephosphorylation of the proapoptotic Bcl-2 family member Bad. After treatment with FK506, Akt and Bad dephosphorylation was greatly reduced. The total number of TUNEL-positive neurons was reduced by intravitreal injection of FK506 after transient ischemia. These results indicate that CaN cleavage negatively regulates Akt phosphorylation and is involved in retinal cell apoptosis after transient ischemia.

Novel PI3K/Akt Inhibitors Screened by the Cytoprotective Function of Human Immunodeficiency Virus Type 1 Tat

PubMed Central

Kim, Dong-Hyun; Kim, Baek

2011-01-01

The PI3K/Akt pathway regulates various stress-related cellular responses such as cell survival, cell proliferation, metabolism and protein synthesis. Many cancer cell types display the activation of this pathway, and compounds inhibiting this cell survival pathway have been extensively evaluated as anti-cancer agents. In addition to cancers, several human viruses, such as HTLV, HPV, HCV and HIV-1, also modulate this pathway, presumably in order to extend the life span of the infected target cells for productive viral replication. The expression of HIV-1 Tat protein exhibited the cytoprotective effect in macrophages and a human microglial cell line by inhibiting the negative regulator of this pathway, PTEN. This cytoprotective effect of HIV-1 appears to contribute to the long-term survival and persistent HIV-1 production in human macrophage reservoirs. In this study we exploited the PI3K/Akt dependent cytoprotective effect of Tat-expressing CHME5 cells. We screened a collection of compounds known to modulate inflammation, and identified three novel compounds: Lancemaside A, Compound K and Arctigenin that abolished the cytoprotective phenotype of Tat-expressing CHME5 cells. All three compounds antagonized the kinase activity of Akt. Further detailed signaling studies revealed that each of these three compounds targeted different steps of the PI3K/Akt pathway. Arctigenin regulates the upstream PI3K enzyme from converting PIP2 to PIP3. Lancemaside A1 inhibited the movement of Akt to the plasma membrane, a critical step for Akt activation. Compound K inhibited Akt phosphorylation. This study supports that Tat-expressing CHME5 cells are an effective model system for screening novel PI3K/Akt inhibitors. PMID:21765914

Deficiency of Akt1, but not Akt2, attenuates the development of pulmonary hypertension

PubMed Central

Tang, Haiyang; Chen, Jiwang; Fraidenburg, Dustin R.; Song, Shanshan; Sysol, Justin R.; Drennan, Abigail R.; Offermanns, Stefan; Ye, Richard D.; Bonini, Marcelo G.; Minshall, Richard D.; Garcia, Joe G. N.; Machado, Roberto F.; Makino, Ayako

2014-01-01

Pulmonary vascular remodeling, mainly attributable to enhanced pulmonary arterial smooth muscle cell proliferation and migration, is a major cause for elevated pulmonary vascular resistance and pulmonary arterial pressure in patients with pulmonary hypertension. The signaling cascade through Akt, comprised of three isoforms (Akt1–3) with distinct but overlapping functions, is involved in regulating cell proliferation and migration. This study aims to investigate whether the Akt/mammalian target of rapamycin (mTOR) pathway, and particularly which Akt isoform, contributes to the development and progression of pulmonary vascular remodeling in hypoxia-induced pulmonary hypertension (HPH). Compared with the wild-type littermates, Akt1−/− mice were protected against the development and progression of chronic HPH, whereas Akt2−/− mice did not demonstrate any significant protection against the development of HPH. Furthermore, pulmonary vascular remodeling was significantly attenuated in the Akt1−/− mice, with no significant effect noted in the Akt2−/− mice after chronic exposure to normobaric hypoxia (10% O2). Overexpression of the upstream repressor of Akt signaling, phosphatase and tensin homolog deleted on chromosome 10 (PTEN), and conditional and inducible knockout of mTOR in smooth muscle cells were also shown to attenuate the rise in right ventricular systolic pressure and the development of right ventricular hypertrophy. In conclusion, Akt isoforms appear to have a unique function within the pulmonary vasculature, with the Akt1 isoform having a dominant role in pulmonary vascular remodeling associated with HPH. The PTEN/Akt1/mTOR signaling pathway will continue to be a critical area of study in the pathogenesis of pulmonary hypertension, and specific Akt isoforms may help specify therapeutic targets for the treatment of pulmonary hypertension. PMID:25416384

Dimethylarginine dimethylaminohydrolase 1 modulates endothelial cell growth through nitric oxide and Akt.

PubMed

Zhang, Ping; Hu, Xinli; Xu, Xin; Chen, Yingjie; Bache, Robert J

2011-04-01

Dimethylarginine dimethylaminohydrolase 1 (DDAH1) modulates NO production by degrading the endogenous nitric oxide (NO) synthase (NOS) inhibitors asymmetrical dimethylarginine (ADMA) and L-NG-monomethyl arginine (L-NMMA). This study examined whether, in addition to degrading ADMA, DDAH1 exerts ADMA-independent effects that influence endothelial function. Using selective gene silencing of DDAH1 with small interfering RNA and overexpression of DDAH1 in human umbilical vein endothelial cells, we found that DDAH1 acts to promote endothelial cell proliferation, migration, and tube formation by Akt phosphorylation, as well as through the traditional role of degrading ADMA. Incubation of human umbilical vein endothelial cells with the NOS inhibitors l-NG-nitro-arginine methyl ester (L-NAME) or ADMA, the soluble guanylyl cyclase inhibitor 1H-(1,2,4)oxadiazolo-(4,3-2)quinoxalin-1-one, or the cGMP analog 8-(4-Chlorophenylthio)-cGMP had no effect on phosphorylated (p)-Akt(Ser473), indicating that the increase in p-Akt(Ser473) produced by DDAH1 was independent of the NO-cGMP signaling pathway. DDAH1 formed a protein complex with Ras, and DDAH1 overexpression increased Ras activity. The Ras inhibitor manumycin-A or dominant-negative Ras significantly attenuated the DDAH1-induced increase in p-Akt(Ser473). Furthermore, DDAH1 knockout impaired endothelial sprouting from cultured aortic rings, and overexpression of constitutively active Akt or DDAH1 rescued endothelial sprouting in the aortic rings from these mice. DDAH1 exerts a unique role in activating Akt that affects endothelial function independently of degrading endogenous NOS inhibitors.

Inactivation of AKT Induces Cellular Senescence in Uterine Leiomyoma

PubMed Central

Xu, Xiaofei; Lu, Zhenxiao; Qiang, Wenan; Vidimar, Vania; Kong, Beihua

2014-01-01

Uterine leiomyomas (fibroids) are a major public health problem. Current medical treatments with GnRH analogs do not provide long-term benefit. Thus, permanent shrinkage or inhibition of fibroid growth via medical means remains a challenge. The AKT pathway is a major growth and survival pathway for fibroids. We propose that AKT inhibition results in a transient regulation of specific mechanisms that ultimately drive cells into cellular senescence or cell death. In this study, we investigated specific mechanisms of AKT inhibition that resulted in senescence. We observed that administration of MK-2206, an allosteric AKT inhibitor, increased levels of reactive oxygen species, up-regulated the microRNA miR-182 and several senescence-associated genes (including p16, p53, p21, and β-galactosidase), and drove leiomyoma cells into stress-induced premature senescence (SIPS). Moreover, induction of SIPS was mediated by HMGA2, which colocalized to senescence-associated heterochromatin foci. This study provides a conceivable molecular mechanism of SIPS by AKT inhibition in fibroids. PMID:24476133

Pan-Pim Kinase Inhibitor AZD1208 Suppresses Tumor Growth and Synergistically Interacts with Akt Inhibition in Gastric Cancer Cells.

PubMed

Lee, Miso; Lee, Kyung-Hun; Min, Ahrum; Kim, Jeongeun; Kim, Seongyeong; Jang, Hyemin; Lim, Jee Min; Kim, So Hyeon; Ha, Dong-Hyeon; Jeong, Won Jae; Suh, Koung Jin; Yang, Yae-Won; Kim, Tae Yong; Oh, Do-Youn; Bang, Yung-Jue; Im, Seock-Ah

2018-06-06

Pim kinases are highly conserved serine/threonine kinases, and different expression patterns of each isoform (Pim-1, Pim-2, and Pim-3) have been observed in various types of human cancers, including gastric cancer. AZD1208 is a potent and selective inhibitor that affects all three isoforms of Pim. We investigated the effects of AZD1208 as a single agent and in combination with an Akt inhibitor in gastric cancer cells. The antitumor activity of AZD1208 with/without an Akt inhibitor was evaluated in a large panel of gastric cancer cell lines through growth inhibition assays. The underlying mechanism was also examined by western blotting, immunofluorescence assay, and cell cycle analysis. AZD1208 treatment decreased gastric cancer cell proliferation rates and induced autophagy only in long-term culture systems. Light chain 3B (LC3B), a marker of autophagy, was increased in sensitive cells in a dose-dependent manner with AZD1208 treatment, which suggested that the growth inhibition effect of AZD1208 was achieved through autophagy, not apoptosis. Moreover, we found that cells damaged by Pim inhibition were repaired by activation of the DNA damage repair pathway, which promoted cell survival and led the cells to become resistant to AZD1208. We also confirmed that the combination of an Akt inhibitor with AZD1208 produced a highly synergistic effect in gastric cancer cell lines. Treatment with AZD1208 alone induced considerable cell death through autophagy in gastric cancer cells. Moreover, the combination of AZD1208 with an Akt inhibitor showed synergistic antitumor effects through regulation of the DNA damage repair pathway.

Inhibition of Akt with small molecules and biologics: historical perspective and current status of the patent landscape

PubMed Central

Mattmann, Margrith E; Stoops, Sydney L; Lindsley, Craig W

2014-01-01

Introduction Akt plays a pivotal role in cell survival and proliferation through a number of downstream effectors; unregulated activation of the PI3K/PTEN/Akt pathway is a prominent feature of many human cancers. Akt is considered an attractive target for cancer therapy by the inhibition of Akt alone or in combination with standard cancer chemotherapeutics. Both preclinical animal studies and clinical trials in humans have validated Akt as an important target of cancer drug discovery. Area covered A historical perspective of Akt inhibitors, including PI analogs, ATP-competitive and allosteric Akt inhibitors, along with other inhibitory mechanisms are reviewed in this paper with a focus on issued patents, patent applications and a summary of clinical trial updates since the last review in 2007. Expert opinion A vast diversity of inhibitors of Akt, both small molecule and biologic, have been developed in the past 5 years, with over a dozen in various phases of clinical development, and several displaying efficacy in humans. While it is not yet clear which mechanism of Akt inhibition will be optimal in humans, or which Akt isoforms to inhibit, or whether a small molecule or biologic agent will be best, data to all of these points will be available in the near future. PMID:21635152

Synemin promotes AKT-dependent glioblastoma cell proliferation by antagonizing PP2A.

PubMed

Pitre, Aaron; Davis, Nathan; Paul, Madhumita; Orr, A Wayne; Skalli, Omar

2012-04-01

The intermediate filament protein synemin is present in astrocyte progenitors and glioblastoma cells but not in mature astrocytes. Here we demonstrate a role for synemin in enhancing glioblastoma cell proliferation and clonogenic survival, as synemin RNA interference decreased both behaviors by inducing G1 arrest along with Rb hypophosphorylation and increased protein levels of the G1/S inhibitors p21(Cip1) and p27(Kip1). Akt involvement was demonstrated by decreased phosphorylation of its substrate, p21(Cip1), and reduced Akt catalytic activity and phosphorylation at essential activation sites. Synemin silencing, however, did not affect the activities of PDPK1 and mTOR complex 2, which directly phosphorylate Akt activation sites, but instead enhanced the activity of the major regulator of Akt dephosphorylation, protein phosphatase type 2A (PP2A). This was accompanied by changes in PP2A subcellular distribution resulting in increased physical interactions between PP2A and Akt, as shown by proximity ligation assays (PLAs). PLAs and immunoprecipitation experiments further revealed that synemin and PP2A form a protein complex. In addition, treatment of synemin-silenced cells with the PP2A inhibitor cantharidic acid resulted in proliferation and pAkt and pRb levels similar to those of controls. Collectively these results indicate that synemin positively regulates glioblastoma cell proliferation by helping sequester PP2A away from Akt, thereby favoring Akt activation.

The Akt DUBbed InAktive.

PubMed

Lin, Kui

2013-01-08

Akt is a central node in the phosphoinositide-3 kinase-Akt-mammalian target of rapamycin pathway and is activated by a multistep process in response to growth factor stimulation. An additional layer of posttranslational modification has emerged as a new paradigm in the regulation of Akt. The identification of an E3 ligase for Lys(63)-linked ubiquitination of Akt has now been complemented with the discovery of the tumor suppressor cylindromatosis as a deubiquitinating enzyme (DUB) for Akt. Thus, like phosphorylation and dephosphorylation, cycles of ubiquitination and deubiquitination provide additional on-off switches that keep Akt activity in balance, and disturbances in this balance have pathological consequences.

Substance P induces cardioprotection in ischemia-reperfusion via activation of AKT.

PubMed

Jubair, Shaiban; Li, Jianping; Dehlin, Heather M; Manteufel, Edward J; Goldspink, Paul H; Levick, Scott P; Janicki, Joseph S

2015-08-15

Accumulating evidence indicates that substance P is cardioprotective following ischemia-reperfusion primarily due to its potent coronary vasodilator actions. However, an anti-apoptotic effect of substance P has been observed in tenocytes following ischemia, which involved activation of the AKT pathway. This suggests the possibility that substance P also provides cardioprotection via direct actions to activate AKT in myocardial cells. The purpose of this study was to test the hypothesis that substance P attenuates ischemia-related cell death via direct effects on myocardial cells by activating cell survival pathways. Seven-week-old male Sprague-Dawley rats, anesthetized with intraperitoneal pentobarbital sodium (100 mg/kg), were used. The ability of substance P to prevent cellular damage was assessed following ischemia-reperfusion in an isolated heart preparation and in short-term hypoxia without reperfusion using a left ventricular tissue slice culture preparation. In addition, the NK-1 receptor and AKT involvement was assessed using the NK-1 receptor antagonist L732138 and the AKT inhibitor LY294002. The results indicate that substance P reduced the ischemia-related release of lactate dehydrogenase in both preparations and the degree of apoptosis and necrosis in the hypoxic left ventricular slices, indicating its ability to attenuate cell damage; and induced AKT phosphorylation, with both the AKT inhibitor and NK-1 receptor antagonist preventing the increased phosphorylation of AKT and the ability of substance P to attenuate hypoxic cellular damage. It is concluded that substance P reduces ischemia/hypoxia-induced myocardial cell death by acting directly on cardiac cells to initiate cell survival pathways via the NK-1 receptor and AKT. Copyright © 2015 the American Physiological Society.

Substance P induces cardioprotection in ischemia-reperfusion via activation of AKT

PubMed Central

Jubair, Shaiban; Li, Jianping; Dehlin, Heather M.; Manteufel, Edward J.; Goldspink, Paul H.; Levick, Scott P.

2015-01-01

Accumulating evidence indicates that substance P is cardioprotective following ischemia-reperfusion primarily due to its potent coronary vasodilator actions. However, an anti-apoptotic effect of substance P has been observed in tenocytes following ischemia, which involved activation of the AKT pathway. This suggests the possibility that substance P also provides cardioprotection via direct actions to activate AKT in myocardial cells. The purpose of this study was to test the hypothesis that substance P attenuates ischemia-related cell death via direct effects on myocardial cells by activating cell survival pathways. Seven-week-old male Sprague-Dawley rats, anesthetized with intraperitoneal pentobarbital sodium (100 mg/kg), were used. The ability of substance P to prevent cellular damage was assessed following ischemia-reperfusion in an isolated heart preparation and in short-term hypoxia without reperfusion using a left ventricular tissue slice culture preparation. In addition, the NK-1 receptor and AKT involvement was assessed using the NK-1 receptor antagonist L732138 and the AKT inhibitor LY294002. The results indicate that substance P reduced the ischemia-related release of lactate dehydrogenase in both preparations and the degree of apoptosis and necrosis in the hypoxic left ventricular slices, indicating its ability to attenuate cell damage; and induced AKT phosphorylation, with both the AKT inhibitor and NK-1 receptor antagonist preventing the increased phosphorylation of AKT and the ability of substance P to attenuate hypoxic cellular damage. It is concluded that substance P reduces ischemia/hypoxia-induced myocardial cell death by acting directly on cardiac cells to initiate cell survival pathways via the NK-1 receptor and AKT. PMID:26071541

AML sensitivity to YM155 is modulated through AKT and Mcl-1

PubMed Central

de Necochea-Campion, Rosalia; Diaz Osterman, Carlos J.; Hsu, Heng-Wei; Fan, Junjie; Mirshahidi, Saied; Wall, Nathan R.; Chen, Chien-Shing

2015-01-01

HL60 and U937 (acute myeloid leukemia (AML) cell lines) were assessed for sensitivity to YM155, and found to have distinct sensitive and resistant phenotypes, respectively. In HL60 cells, YM155 inhibition of growth proliferation was due to apoptosis which was measured by annexin V/PI staining. YM155 induced apoptosis through activation of intrinsic and extrinsic pathways that also culminated in caspase-3 activity and PARP cleavage. YM155 sensitivity was partially associated with this compound’s ability to downregulate survivin transcription since this was more pronounced in the HL60 cell line. However, marked differences were also observed in XIAP, Bcl-2, and Mcl-1L, and Mcl-1s. Furthermore, YM155 treatment completely inhibited production of total Akt protein in HL60, but not U937 cells. Importantly, Akt activity (pAkt-Ser473) levels were maintained in YM155 treated U937 cells which may help stabilize other anti-apoptotic proteins. Combination treatments with an Akt inhibitor, MK-2206, reduced levels of pAkt-Ser473 in U937 cells and synergistically sensitized them to YM155 cytotoxicity. Collectively our results indicate that Akt signaling may be an important factor mediating YM155 response in AML, and combinatorial therapies with Akt inhibitors could improve treatment efficacy in YM155-resistant cells. PMID:26118775

AKT capture by feline leukemia virus.

PubMed

Kawamura, Maki; Umehara, Daigo; Odahara, Yuka; Miyake, Ariko; Ngo, Minh Ha; Ohsato, Yoshiharu; Hisasue, Masaharu; Nakaya, Masa-Aki; Watanabe, Shinya; Nishigaki, Kazuo

2017-04-01

Oncogene-containing retroviruses are generated by recombination events between viral and cellular sequences, a phenomenon called "oncogene capture". The captured cellular genes, referred to as "v-onc" genes, then acquire new oncogenic properties. We report a novel feline leukemia virus (FeLV), designated "FeLV-AKT", that has captured feline c-AKT1 in feline lymphoma. FeLV-AKT contains a gag-AKT fusion gene that encodes the myristoylated Gag matrix protein and the kinase domain of feline c-AKT1, but not its pleckstrin homology domain. Therefore, it differs structurally from the v-Akt gene of murine retrovirus AKT8. AKT may be involved in the mechanisms underlying malignant diseases in cats.

Antagonism of EGFR and HER3 Enhances the Response to Inhibitors of the PI3K-Akt Pathway in Triple-Negative Breast Cancer

PubMed Central

Tao, Jessica J.; Castel, Pau; Radosevic-Robin, Nina; Elkabets, Moshe; Auricchio, Neil; Aceto, Nicola; Weitsman, Gregory; Barber, Paul; Vojnovic, Borivoj; Ellis, Haley; Morse, Natasha; Viola-Villegas, Nerissa Therese; Bosch, Ana; Juric, Dejan; Hazra, Saswati; Singh, Sharat; Kim, Phillip; Bergamaschi, Anna; Maheswaran, Shyamala; Ng, Tony; Penault-Llorca, Frédérique; Lewis, Jason S.; Carey, Lisa A.; Perou, Charles M.; Baselga, José; Scaltriti, Maurizio

2014-01-01

Both abundant epidermal growth factor receptor (EGFR or ErbB1) and high activity of the phosphatidyl-inositol 3-kinase (PI3K)–Akt pathway are common and therapeutically targeted in triple-negative breast cancer (TNBC). However, activation of another EGFR family member [human epidermal growth factor receptor 3 (HER3) (or ErbB3)] may limit the antitumor effects of these drugs. We found that TNBC cell lines cultured with the EGFR or HER3 ligand EGF or heregulin, respectively, and treated with either an Akt inhibitor (GDC-0068) or a PI3K inhibitor (GDC-0941) had increased abundance and phosphorylation of HER3. The phosphorylation of HER3 and EGFR in response to these treatments was reduced by the addition of a dual EGFR and HER3 inhibitor (MEHD7945A). MEHD7945A also decreased the phosphorylation (and activation) of EGFR and HER3 and the phosphorylation of downstream targets that occurred in response to the combination of EGFR ligands and PI3K-Akt pathway inhibitors. In culture, inhibition of the PI3K-Akt pathway combined with either MEHD7945A or knockdown of HER3 decreased cell proliferation compared with inhibition of the PI3K-Akt pathway alone. Combining either GDC-0068 or GDC-0941 with MEHD7945A inhibited the growth of xenografts derived from TNBC cell lines or from TNBC patient tumors, and this combination treatment was also more effective than combining either GDC-0068 or GDC-0941 with cetuximab, an EGFR-targeted antibody. After therapy with EGFR-targeted antibodies, some patients had residual tumors with increased HER3 abundance and EGFR/HER3 dimerization (an activating interaction). Thus, we propose that concomitant blockade of EGFR, HER3, and the PI3K-Akt pathway in TNBC should be investigated in the clinical setting. PMID:24667376

Antagonism of EGFR and HER3 enhances the response to inhibitors of the PI3K-Akt pathway in triple-negative breast cancer.

PubMed

Tao, Jessica J; Castel, Pau; Radosevic-Robin, Nina; Elkabets, Moshe; Auricchio, Neil; Aceto, Nicola; Weitsman, Gregory; Barber, Paul; Vojnovic, Borivoj; Ellis, Haley; Morse, Natasha; Viola-Villegas, Nerissa Therese; Bosch, Ana; Juric, Dejan; Hazra, Saswati; Singh, Sharat; Kim, Phillip; Bergamaschi, Anna; Maheswaran, Shyamala; Ng, Tony; Penault-Llorca, Frédérique; Lewis, Jason S; Carey, Lisa A; Perou, Charles M; Baselga, José; Scaltriti, Maurizio

2014-03-25

Both abundant epidermal growth factor receptor (EGFR or ErbB1) and high activity of the phosphatidylinositol 3-kinase (PI3K)-Akt pathway are common and therapeutically targeted in triple-negative breast cancer (TNBC). However, activation of another EGFR family member [human epidermal growth factor receptor 3 (HER3) (or ErbB3)] may limit the antitumor effects of these drugs. We found that TNBC cell lines cultured with the EGFR or HER3 ligand EGF or heregulin, respectively, and treated with either an Akt inhibitor (GDC-0068) or a PI3K inhibitor (GDC-0941) had increased abundance and phosphorylation of HER3. The phosphorylation of HER3 and EGFR in response to these treatments was reduced by the addition of a dual EGFR and HER3 inhibitor (MEHD7945A). MEHD7945A also decreased the phosphorylation (and activation) of EGFR and HER3 and the phosphorylation of downstream targets that occurred in response to the combination of EGFR ligands and PI3K-Akt pathway inhibitors. In culture, inhibition of the PI3K-Akt pathway combined with either MEHD7945A or knockdown of HER3 decreased cell proliferation compared with inhibition of the PI3K-Akt pathway alone. Combining either GDC-0068 or GDC-0941 with MEHD7945A inhibited the growth of xenografts derived from TNBC cell lines or from TNBC patient tumors, and this combination treatment was also more effective than combining either GDC-0068 or GDC-0941 with cetuximab, an EGFR-targeted antibody. After therapy with EGFR-targeted antibodies, some patients had residual tumors with increased HER3 abundance and EGFR/HER3 dimerization (an activating interaction). Thus, we propose that concomitant blockade of EGFR, HER3, and the PI3K-Akt pathway in TNBC should be investigated in the clinical setting.

The AKT-mTOR signalling pathway in kidney cancer tissues

NASA Astrophysics Data System (ADS)

Spirina, L. V.; Usynin, Y. A.; Kondakova, I. V.; Yurmazov, Z. A.; Slonimskaya, E. M.; Kolegova, E. S.

2015-11-01

An increased expression of phospho-AKT, m-TOR, glycogen regulator GSK-3-beta and transcription inhibitor 4E-BP1 was observed in kidney cancer tissues. Tumor size growth was associated with a high level of c-Raf and low content of phospho-m-TOR. Cancer metastasis development led to a decreased PTEN and phospho-AKT expression.

Akt SUMOylation regulates cell proliferation and tumorigenesis.

PubMed

Li, Rong; Wei, Jie; Jiang, Cong; Liu, Dongmei; Deng, Lu; Zhang, Kai; Wang, Ping

2013-09-15

Proto-oncogene Akt plays essential roles in cell proliferation and tumorigenesis. Full activation of Akt is regulated by phosphorylation, ubiquitination, and acetylation. Here we report that SUMOylation of Akt is a novel mechanism for its activation. Systematically analyzing the role of lysine residues in Akt activation revealed that K276, which is located in a SUMOylation consensus motif, is essential for Akt activation. Ectopic or endogenous Akt1 could be modified by SUMOylation. RNA interference-mediated silencing of UBC9 reduced Akt SUMOylation, which was promoted by SUMO E3 ligase PIAS1 and reversed by the SUMO-specific protease SENP1. Although multiple sites on Akt could be SUMOylated, K276 was identified as a major SUMO acceptor site. K276R or E278A mutation reduced SUMOylation of Akt but had little effect on its ubiquitination. Strikingly, these mutations also completely abolished Akt kinase activity. In support of these results, we found that expression of PIAS1 and SUMO1 increased Akt activity, whereas expression of SENP1 reduced Akt1 activity. Interestingly, the cancer-derived mutant E17K in Akt1 that occurs in various cancers was more efficiently SUMOylated than wild-type Akt. Moreover, SUMOylation loss dramatically reduced Akt1 E17K-mediated cell proliferation, cell migration, and tumorigenesis. Collectively, our findings establish that Akt SUMOylation provides a novel regulatory mechanism for activating Akt function. ©2013 AACR.

Brain Cancer Stem Cells Display Preferential Sensitivity to Akt Inhibition

PubMed Central

Eyler, Christine E.; Foo, Wen-Chi; LaFiura, Katherine M.; McLendon, Roger E.; Hjelmeland, Anita B.; Rich, Jeremy N.

2009-01-01

Malignant brain tumors are among the most lethal cancers, and conventional therapies are largely limited to palliation. Novel therapies targeted against specific molecular pathways may offer improved efficacy and reduced toxicity compared to conventional therapies, but initial clinical trials of molecular targeted agents in brain cancer therapy have been frequently disappointing. In brain tumors and other cancers, subpopulations of tumor cells have recently been characterized by their ability to self-renew and initiate tumors. Although these cancer stem cells, or tumor initiating cells, are often only present in small numbers in human tumors, mounting evidence suggests that cancer stem cells contribute to tumor maintenance and therapeutic resistance. Thus, the development of therapies that target cancer stem cell signal transduction and biologies may improve brain tumor patient survival. We now demonstrate that populations enriched for cancer stem cells are preferentially sensitive to an inhibitor of Akt, a prominent cell survival and invasion signaling node. Treatment with an Akt inhibitor more potently reduced the numbers of viable brain cancer stem cells relative to matched non-stem cancer cells associated with a preferential induction of apoptosis and a suppression of neurosphere formation. Akt inhibition also reduced the motility and invasiveness of all tumor cells but had a greater impact on cancer stem cell behaviors. Furthermore, inhibition of Akt activity in cancer stem cells increased survival of immunocompromised mice bearing human glioma xenografts in vivo. Together, these results suggest that Akt inhibitors may function as effective anti-cancer stem cell therapies. PMID:18802038

Celecoxib promotes c-FLIP degradation through Akt-independent inhibition of GSK3

PubMed Central

Chen, Shuzhen; Cao, Wei; Yue, Ping; Hao, Chunhai; Khuri, Fadlo R.; Sun, Shi-Yong

2011-01-01

Celecoxib is a COX2 inhibitor that reduces the risk of colon cancer. However, the basis for its cancer chemopreventive activity is not fully understood. In this study, we defined a mechanism of celecoxib action based on degradation of c-FLIP, a major regulator of the death receptor pathway of apoptosis. c-FLIP protein levels are regulated by ubiquitination and proteasome-mediated degradation. We found that celecoxib controlled c-FLIP ubiquitination through Akt-independent inhibition of GSK3 kinase, itself a candidate therapeutic target of interest in colon cancer. Celecoxib increased the levels of phosphorylated GSK3 (p-GSK3), including the α and β forms, even in cell lines where p-Akt levels were not increased. PI3K inhibitors abrogated Akt phosphorylation as expected but had no effect on celecoxib-induced GSK3 phosphorylation. In contrast, PKC inhibitors abolished celecoxib-induced GSK3 phosphorylation, implying that celecoxib influenced GSK3 phosphorylation through a mechanism relied upon PKC but not Akt. GSK3 blockade either by siRNA or kinase inhibitors was sufficient to attenuate c-FLIP levels. Combining celecoxib with GSK3 inhibition enhanced attenuation of c-FLIP and increased apoptosis. Proteasome inhibitor MG132 reversed the effects of GSK3 inhibition and increased c-FLIP ubiquitination, confirming that c-FLIP attenuation was mediated by proteasomal turnover as expected. Our findings reveal a novel mechanism through which the regulatory effects of c-FLIP on death receptor signaling are controlled by GSK3, which celecoxib acts at an upstream level to control independently of Akt. PMID:21868755

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

Glutaredoxin exerts an antiapoptotic effect by regulating the redox state of Akt.

PubMed

Murata, Hiroaki; Ihara, Yoshito; Nakamura, Hajime; Yodoi, Junji; Sumikawa, Koji; Kondo, Takahito

2003-12-12

Glutaredoxin (GRX) is a small dithiol protein involved in various cellular functions, including the redox regulation of certain enzyme activities. GRX functions via a disulfide exchange reaction by utilizing the active site Cys-Pro-Tyr-Cys. Here we demonstrated that overexpression of GRX protected cells from hydrogen peroxide (H2O2)-induced apoptosis by regulating the redox state of Akt. Akt was transiently phosphorylated, dephosphorylated, and then degraded in cardiac H9c2 cells undergoing H2O2-induced apoptosis. Under stress, Akt underwent disulfide bond formation between Cys-297 and Cys-311 and dephosphorylation in accordance with an increased association with protein phosphatase 2A. Overexpression of GRX protected Akt from H2O2-induced oxidation and suppressed recruitment of protein phosphatase 2A to Akt, resulting in a sustained phosphorylation of Akt and inhibition of apoptosis. This effect was reversed by cadmium, an inhibitor of GRX. Furthermore an in vitro assay revealed that GRX reduced oxidized Akt in concert with glutathione, NADPH, and glutathione-disulfide reductase. Thus, GRX plays an important role in protecting cells from apoptosis by regulating the redox state of Akt.

Activation of Akt by Advanced Glycation End Products (AGEs): Involvement of IGF-1 Receptor and Caveolin-1

PubMed Central

Yang, Su-Jung; Chen, Chen-Yu; Chang, Geen-Dong; Wen, Hui-Chin; Chen, Ching-Yu; Chang, Shi-Chuan; Liao, Jyh-Fei; Chang, Chung-Ho

2013-01-01

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

Hydrogen peroxide-induced Akt phosphorylation regulates Bax activation.

PubMed

Sadidi, Mahdieh; Lentz, Stephen I; Feldman, Eva L

2009-05-01

Reactive oxygen species such as hydrogen peroxide (H(2)O(2)) are involved in many cellular processes that positively and negatively regulate cell fate. H(2)O(2), acting as an intracellular messenger, activates phosphatidylinositol-3 kinase (PI3K) and its downstream target Akt, and promotes cell survival. The aim of the current study was to understand the mechanism by which PI3K/Akt signaling promotes survival in SH-SY5Y neuroblastoma cells. We demonstrate that PI3K/Akt mediates phosphorylation of the pro-apoptotic Bcl-2 family member Bax. This phosphorylation suppresses apoptosis and promotes cell survival. Increased survival in the presence of H(2)O(2) was blocked by LY294002, an inhibitor of PI3K activation. LY294002 prevented Bax phosphorylation and resulted in Bax translocation to the mitochondria, cytochrome c release, caspase-3 activation, and cell death. Collectively, these findings reveal a mechanism by which H(2)O(2)-induced activation of PI3K/Akt influences post-translational modification of Bax and inactivates a key component of the cell death machinery.

A novel AKT1 mutant amplifies an adaptive melanoma response to BRAF inhibition

PubMed Central

Shi, Hubing; Hong, Aayoung; Kong, Xiangju; Koya, Richard C.; Song, Chunying; Moriceau, Gatien; Hugo, Willy; Yu, Clarissa C.; Ng, Charles; Chodon, Thinle; Scolyer, Richard A.; Kefford, Richard F.; Ribas, Antoni; Long, Georgina V.; Lo, Roger S.

2013-01-01

BRAF inhibitor (BRAFi) therapy leads to remarkable anti-melanoma responses, but the initial tumor shrinkage is commonly incomplete, providing a nidus for subsequent disease progression. Adaptive signaling may underlie early BRAFi resistance and influence the selection pattern for genetic variants causing late, acquired resistance. We show here that BRAFi (or BRAFi+MEKi) therapy in patients frequently led to rebound p-AKT levels in their melanomas early on treatment. In cell lines, BRAFi treatment led to rebound levels of RTKs (including PDGFRβ), PIP3, pleckstrin homology domain (PHD) recruitment, and p-AKT. PTEN expression limited this BRAFi-elicited PI3K-AKT signaling, which could be rescued by introduction of a mutant AKT1 (Q79K) kown to confer acquired BRAFi resistance. Functionally, AKT1 Q79K conferred BRAFi resistance via amplifying BRAFi-elicited PI3K-AKT signaling. Additionally, MAPK pathway inhibition enhanced clonogenic growth dependency on PI3K or AKT. Thus, adaptive or genetic upregulation of AKT critically participates in melanoma survival during BRAFi therapy. PMID:24265152

Homocysteine enhances MMP-9 production in murine macrophages via ERK and Akt signaling pathways

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lee, Seung Jin; Lee, Yi Sle; Seo, Kyo Won

2012-04-01

Homocysteine (Hcy) at elevated levels is an independent risk factor of cardiovascular diseases, including atherosclerosis. In the present study, we investigated the effect of Hcy on the production of matrix metalloproteinases (MMP) in murine macrophages. Among the MMP known to regulate the activities of collagenase and gelatinase, Hcy exclusively increased the gelatinolytic activity of MMP-9 in J774A.1 cells as well as in mouse peritoneal macrophages. Furthermore, this activity was found to be correlated with Western blot findings in J774A.1 cells, which showed that MMP-9 expression was concentration- and time-dependently increased by Hcy. Inhibition of the ERK and Akt pathways ledmore » to a significant decrease in Hcy-induced MMP-9 expression, and combined treatment with inhibitors of the ERK and Akt pathways showed an additive effects. Activity assays for ERK and Akt showed that Hcy increased the phosphorylation of both, but these phosphorylation were not affected by inhibitors of the Akt and ERK pathways. In line with these findings, the molecular inhibition of ERK and Akt using siRNA did not affect the Hcy-induced phosphorylation of Akt and ERK, respectively. Taken together, these findings suggest that Hcy enhances MMP-9 production in murine macrophages by separately activating the ERK and Akt signaling pathways. -- Highlights: ► Homocysteine (Hcy) induced MMP-9 production in murine macrophages. ► Hcy induced MMP-9 production through ERK and Akt signaling pathways. ► ERK and Akt signaling pathways were activated by Hcy in murine macrophages. ► ERK and Akt pathways were additively act on Hcy-induced MMP-9 production. ► Hcy enhances MMP-9 production in macrophages via activation of ERK and Akt signaling pathways in an independent manner.« less

The role of the PTEN/AKT Pathway in NOTCH1-induced leukemia

PubMed Central

Palomero, Teresa; Dominguez, Maria; Ferrando, Adolfo A.

2008-01-01

Activating mutations in NOTCH1 are the most prominent genetic abnormality in T-cell acute Lymphoblastic Leukemia (T-ALL) and inhibition of NOTCH1 signaling with γ-secretase inhibitors (GSIs) has been proposed as targeted therapy in this disease. However, most T-ALL cell lines with mutations in NOTCH1 fail to respond to GSI therapy. Using gene expression profiling and mutation analysis we showed that mutational loss of PTEN is a common event in T-ALL and is associated with resistance to NOTCH inhibition. Furthermore, our studies revealed that NOTCH1 induces upregulation of the PI3K-AKT pathway via HES1, which negatively controls the expression of PTEN. This regulatory circuitry is evolutionary conserved from Drosophila to humans as demonstrated by the interaction of overexpression of Delta and Akt in a model of Notch-induced transformation in the fly eye. Loss of PTEN and constitutive activation of AKT in T-ALL induce increased glucose metabolism and bypass the requirement of NOTCH1 signaling to sustain cell growth. Importantly, PTEN-null/GSI resistant T-ALL cells switch their oncogene addiction from NOTCH1 to AKT and are highly sensitive to AKT inhibitors. These results should facilitate the development of molecular therapies targeting NOTCH1 and AKT for the treatment of T-ALL. PMID:18414037

Phosphorylated AKT preserves stallion sperm viability and motility by inhibiting caspases 3 and 7.

PubMed

Gallardo Bolaños, Juan M; Balao da Silva, Carolina M; Martín Muñoz, Patricia; Morillo Rodríguez, Antolín; Plaza Dávila, María; Rodríguez-Martínez, Heriberto; Aparicio, Inés M; Tapia, José A; Ortega Ferrusola, Cristina; Peña, Fernando J

2014-08-01

AKT, also referred to as protein kinase B (PKB or RAC), plays a critical role in controlling cell survival and apoptosis. To gain insights into the mechanisms regulating sperm survival after ejaculation, the role of AKT was investigated in stallion spermatozoa using a specific inhibitor and a phosphoflow approach. Stallion spermatozoa were washed and incubated in Biggers-Whitten-Whittingham medium, supplemented with 1% polyvinyl alcohol (PVA) in the presence of 0 (vehicle), 10, 20 or 30 μM SH5, an AKT inhibitor. SH5 treatment reduced the percentage of sperm displaying AKT phosphorylation, with inhibition reaching a maximum after 1 h of incubation. This decrease in phosphorylation was attributable to either dephosphorylation or suppression of the active phosphorylation pathway. Stallion spermatozoa spontaneously dephosphorylated during in vitro incubation, resulting in a lack of a difference in AKT phosphorylation between the SH5-treated sperm and the control after 4 h of incubation. AKT inhibition decreased the proportion of motile spermatozoa (total and progressive) and the sperm velocity. Similarly, AKT inhibition reduced membrane integrity, leading to increased membrane permeability and reduced the mitochondrial membrane potential concomitantly with activation of caspases 3 and 7. However, the percentage of spermatozoa exhibiting oxidative stress, the production of mitochondrial superoxide radicals, DNA oxidation and DNA fragmentation were not affected by AKT inhibition. It is concluded that AKT maintains the membrane integrity of ejaculated stallion spermatozoa, presumably by inhibiting caspases 3 and 7, which prevents the progression of spermatozoa to an incomplete form of apoptosis. © 2014 Society for Reproduction and Fertility.

Targeting the PI3K/Akt/mTOR pathway: effective combinations and clinical considerations

PubMed Central

LoPiccolo, Jaclyn; Blumenthal, Gideon M.; Bernstein, Wendy B.; Dennis, Phillip A.

2008-01-01

The PI3K/Akt/mTOR pathway is a prototypic survival pathway that is constitutively activated in many types of cancer. Mechanisms for pathway activation include loss of tumor suppressor PTEN function, amplification or mutation of PI3K, amplification or mutation of Akt, activation of growth factor receptors, and exposure to carcinogens. Once activated, signaling through Akt can be propagated to a diverse array of substrates, including mTOR, a key regulator of protein translation. This pathway is an attractive therapeutic target in cancer because it serves as a convergence point for many growth stimuli, and through its downstream substrates, controls cellular processes that contribute to the initiation and maintenance of cancer. Moreover, activation of the Akt/mTOR pathway confers resistance to many types of cancer therapy, and is a poor prognostic factor for many types of cancers. This review will provide an update on the clinical progress of various agents that target the pathway, such as the Akt inhibitors perifosine and PX-866 and mTOR inhibitors (rapamycin, CCI-779, RAD-001) and discuss strategies to combine these pathway inhibitors with conventional chemotherapy, radiotherapy, as well as newer targeted agents. We will also discuss how the complex regulation of the PI3K/Akt/mTOR pathway poses practical issues concerning the design of clinical trials, potential toxicities and criteria for patient selection. PMID:18166498

Celecoxib promotes c-FLIP degradation through Akt-independent inhibition of GSK3.

PubMed

Chen, Shuzhen; Cao, Wei; Yue, Ping; Hao, Chunhai; Khuri, Fadlo R; Sun, Shi-Yong

2011-10-01

Celecoxib is a COX-2 inhibitor that reduces the risk of colon cancer. However, the basis for its cancer chemopreventive activity is not fully understood. In this study, we defined a mechanism of celecoxib action based on degradation of cellular FLICE-inhibitory protein (c-FLIP), a major regulator of the death receptor pathway of apoptosis. c-FLIP protein levels are regulated by ubiquitination and proteasome-mediated degradation. We found that celecoxib controlled c-FLIP ubiquitination through Akt-independent inhibition of glycogen synthase kinase-3 (GSK3), itself a candidate therapeutic target of interest in colon cancer. Celecoxib increased the levels of phosphorylated GSK3, including the α and β forms, even in cell lines, where phosphorylated Akt levels were not increased. Phosphoinositide 3-kinase inhibitors abrogated Akt phosphorylation as expected but had no effect on celecoxib-induced GSK3 phosphorylation. In contrast, protein kinase C (PKC) inhibitors abolished celecoxib-induced GSK3 phosphorylation, implying that celecoxib influenced GSK3 phosphorylation through a mechanism that relied upon PKC and not Akt. GSK3 blockade either by siRNA or kinase inhibitors was sufficient to attenuate c-FLIP levels. Combining celecoxib with GSK3 inhibition enhanced attenuation of c-FLIP and increased apoptosis. Proteasome inhibitor MG132 reversed the effects of GSK3 inhibition and increased c-FLIP ubiquitination, confirming that c-FLIP attenuation was mediated by proteasomal turnover as expected. Our findings reveal a novel mechanism through which the regulatory effects of c-FLIP on death receptor signaling are controlled by GSK3, which celecoxib acts at an upstream level to control independently of Akt.

Discovery of 3-(3-(4-(1-Aminocyclobutyl)phenyl)-5-phenyl-3 H -imidazo[4,5- b ]pyridin-2-yl)pyridin-2-amine (ARQ 092): An Orally Bioavailable, Selective, and Potent Allosteric AKT Inhibitor

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lapierre, Jean-Marc; Eathiraj, Sudharshan; Vensel, David

The work in this paper describes the optimization of the 3-(3-phenyl-3H-imidazo[4,5-b]pyridin-2-yl)pyridin-2-amine chemical series as potent, selective allosteric inhibitors of AKT kinases, leading to the discovery of ARQ 092 (21a). The cocrystal structure of compound 21a bound to full-length AKT1 confirmed the allosteric mode of inhibition of this chemical class and the role of the cyclobutylamine moiety. Compound 21a demonstrated high enzymatic potency against AKT1, AKT2, and AKT3, as well as potent cellular inhibition of AKT activation and the phosphorylation of the downstream target PRAS40. Compound 21a also served as a potent inhibitor of the AKT1-E17K mutant protein and inhibited tumormore » growth in a human xenograft mouse model of endometrial adenocarcinoma.« less

Activation of Akt, not connexin 43 protein ubiquitination, regulates gap junction stability.

PubMed

Dunn, Clarence A; Su, Vivian; Lau, Alan F; Lampe, Paul D

2012-01-20

The pore-forming gap junctional protein connexin 43 (Cx43) has a short (1-3 h) half-life in cells in tissue culture and in whole tissues. Although critical for cellular function in all tissues, the process of gap junction turnover is not well understood because treatment of cells with a proteasomal inhibitor results in larger gap junctions but little change in total Cx43 protein whereas lysosomal inhibitors increase total, mostly nonjunctional Cx43. To better understand turnover and identify potential sites of Cx43 ubiquitination, we prepared constructs of Cx43 with different lysines converted to arginines. However, when transfected into cells, a mutant version of Cx43 with all lysines converted to arginines behaved similarly to wild type in the presence of proteasomal and lysosomal inhibitors, indicating that ubiquitination of Cx43 did not appear to be playing a role in gap junction stability. Through the use of inhibitors and dominant negative constructs, we found that Akt (protein kinase B) activity controlled gap junction stability and was necessary to form larger stable gap junctions. Akt activation was increased upon proteasomal inhibition and resulted in phosphorylation of Cx43 at Akt phosphorylation consensus sites. Thus, we conclude that Cx43 ubiquitination is not necessary for the regulation of Cx43 turnover; rather, Akt activity, probably through direct phosphorylation of Cx43, controls gap junction stability. This linkage of a kinase involved in controlling cell survival and growth to gap junction stability may mechanistically explain how gap junctions and Akt play similar regulatory roles.

Atorvastatin enhances neurite outgrowth in cortical neurons in vitro via up-regulating the Akt/mTOR and Akt/GSK-3β signaling pathways

PubMed Central

Jin, Ying; Sui, Hai-juan; Dong, Yan; Ding, Qi; Qu, Wen-hui; Yu, Sheng-xue; Jin, Ying-xin

2012-01-01

Aim: To investigate whether atorvastatin can promote formation of neurites in cultured cortical neurons and the signaling mechanisms responsible for this effect. Methods: Cultured rat cerebral cortical neurons were incubated with atorvastatin (0.05–10 μmol/L) for various lengths of time. For pharmacological experiments, inhibitors were added 30 min prior to addition of atorvastatin. Control cultures received a similar amount of DMSO. Following the treatment period, phase-contrast digital images were taken. Digital images of neurons were analyzed for total neurite branch length (TNBL), neurite number, terminal branch number, and soma area by SPOT Advanced Imaging software. After incubation with atorvastatin for 48 h, the levels of phosphorylated 3-phosphoinoside-dependent protein kinase-1 (PDK1), phospho-Akt, phosphorylated mammalian target of rapamycin (mTOR), phosphorylated 4E-binding protein 1 (4E-BP1), p70S6 kinase (p70S6K), and glycogen synthase kinase-3β (GSK-3β) in the cortical neurons were evaluated using Western blotting analyses. Results: Atorvastatin (0.05–10 μmol/L) resulted in dose-dependent increase in neurite number and length in these neurons. Pretreatment of the cortical neurons with phosphatidylinositol 3-kinase (PI3K) inhibitors LY294002 (30 μmol/L) and wortmannin (5 μmol/L), Akt inhibitor tricribine (1 μmol/L) or mTOR inhibitor rapamycin (100 nmol/L) blocked the atorvastatin-induced increase in neurite outgrowth, suggesting that atorvastatin promoted neurite outgrowth via activating the PI3K/Akt/mTOR signaling pathway. Atorvastatin (10 μmol/L) significantly increased the levels of phosphorylated PDK1, Akt and mTOR in the cortical neurons, which were prevented by LY294002 (30 μmol/L). Moreover, atorvastatin (10 μmol/L) stimulated the phosphorylation of 4E-BP1 and p70S6K, the substrates of mTOR, in the cortical neurons. In addition, atorvastatin (10 μmol/L) significantly increased the phosphorylated GSK-3β level in the cortical

A Novel Oncolytic Herpes Simplex Virus that Synergizes with Phosphatidylinositol 3-Kinase/Akt Pathway Inhibitors to Target Glioblastoma Stem Cells

PubMed Central

Kanai, Ryuichi; Wakimoto, Hiroaki; Martuza, Robert L.; Rabkin, Samuel D.

2011-01-01

Purpose To develop a new oncolytic herpes simplex virus (oHSV) for glioblastoma therapy that will be effective in glioblastoma stem cells (GSCs), an important and untargeted component of glioblastoma. One approach to enhance oHSV efficacy is by combination with other therapeutic modalities. Experimental design MG18L, containing a US3 deletion and an inactivating LacZ insertion in UL39, was constructed for the treatment of brain tumors. Safety was evaluated after intracerebral injection in HSV-susceptible mice. The efficacy of MG18L in human GSCs and glioma cell lines in vitro was compared to other oHSVs, alone or in combination with PI3K/Akt inhibitors (LY294002, triciribine, GDC-0941, BEZ235). Cytotoxic interactions between MG18L and PI3K/Akt inhibitors were determined using Chou-Talalay analysis. In vivo efficacy studies were performed using a clinically relevant mouse model of GSC-derived glioblastoma. Results MG18L was severely neuroattenuated in mice, replicated well in GSCs, and had anti-glioblastoma activity in vivo. PI3K/Akt inhibitors displayed significant but variable anti-proliferative activities in GSCs, while their combination with MG18L synergized in killing GSCs and glioma lines, but not human astrocytes, through enhanced induction of apoptosis. Importantly, synergy was independent of inhibitor sensitivity. In vivo, the combination of MG18L and LY294002 significantly prolonged survival of mice, as compared to either agent alone, achieving 50% long-term survival in glioblastoma-bearing mice. Conclusions This study establishes a novel therapeutic strategy: oHSV manipulation of critical oncogenic pathways to sensitize cancer cells to molecularly-targeted drugs. MG18L is a promising agent for the treatment of glioblastoma, being especially effective when combined with PI3K/Akt pathway-targeted agents. PMID:21505062

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

PubMed

Nguyen, Le Xuan Truong; Mitchell, Beverly S

2013-12-17

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.

Elongation Factor 1 alpha interacts with phospho-Akt in breast cancer cells and regulates their proliferation, survival and motility.

PubMed

Pecorari, Luisa; Marin, Oriano; Silvestri, Chiara; Candini, Olivia; Rossi, Elena; Guerzoni, Clara; Cattelani, Sara; Mariani, Samanta A; Corradini, Francesca; Ferrari-Amorotti, Giovanna; Cortesi, Laura; Bussolari, Rita; Raschellà, Giuseppe; Federico, Massimo R; Calabretta, Bruno

2009-08-03

Akt/PKB is a serine/threonine kinase that has attracted much attention because of its central role in regulating cell proliferation, survival, motility and angiogenesis. Activation of Akt in breast cancer portends aggressive tumour behaviour, resistance to hormone-, chemo-, and radiotherapy-induced apoptosis and it is correlated with decreased overall survival. Recent studies have identified novel tumor-specific substrates of Akt that may provide new diagnostic and prognostic markers and serve as therapeutic targets. This study was undertaken to identify pAkt-interacting proteins and to assess their biological roles in breast cancer cells. We confirmed that one of the pAkt interacting proteins is the Elongation Factor EF1alpha. EF1alpha contains a putative Akt phosphorylation site, but is not phosphorylated by pAkt1 or pAkt2, suggesting that it may function as a modulator of pAkt activity. Indeed, downregulation of EF1alpha expression by siRNAs led to markedly decreased expression of pAkt1 and to less extent of pAkt2 and was associated with reduced proliferation, survival and invasion of HCC1937 cells. Proliferation and survival was further reduced by combining EF1alpha siRNAs with specific pAkt inhibitors whereas EF1alpha downregulation slightly attenuated the decreased invasion induced by Akt inhibitors. We show here that EF1alpha is a pAkt-interacting protein which regulates pAkt levels. Since EF1alpha is often overexpressed in breast cancer, the consequences of EF1alpha increased levels for proliferation, survival and invasion will likely depend on the relative concentration of Akt1 and Akt2.

Ras/Raf/MEK/ERK and PI3K/PTEN/Akt/mTOR Cascade Inhibitors: How Mutations Can Result in Therapy Resistance and How to Overcome Resistance

PubMed Central

McCubrey, James A.; Steelman, Linda S.; Chappell, William H.; Abrams, Stephen L.; Franklin, Richard A.; Montalto, Giuseppe; Cervello, Melchiorre; Libra, Massimo; Candido, Saverio; Malaponte, Grazia; Mazzarino, Maria C.; Fagone, Paolo; Nicoletti, Ferdinando; Bäsecke, Jörg; Mijatovic, Sanja; Maksimovic-Ivanic, Danijela; Milella, Michele; Tafuri, Agostino; Chiarini, Francesca; Evangelisti, Camilla; Cocco, Lucio; Martelli, Alberto M.

2012-01-01

The Ras/Raf/MEK/ERK and PI3K/PTEN/Akt/mTOR cascades are often activated by genetic alterations in upstream signaling molecules such as receptor tyrosine kinases (RTK). Targeting these pathways is often complex and can result in pathway activation depending on the presence of upstream mutations (e.g., Raf inhibitors induce Raf activation in cells with wild type (WT) RAF in the presence of mutant, activated RAS) and rapamycin can induce Akt activation. Targeting with inhibitors directed at two constituents of the same pathway or two different signaling pathways may be a more effective approach. This review will first evaluate potential uses of Raf, MEK, PI3K, Akt and mTOR inhibitors that have been investigated in pre-clinical and clinical investigations and then discuss how cancers can become insensitive to various inhibitors and potential strategies to overcome this resistance. PMID:23085539

Ras/Raf/MEK/ERK and PI3K/PTEN/Akt/mTOR cascade inhibitors: how mutations can result in therapy resistance and how to overcome resistance.

PubMed

McCubrey, James A; Steelman, Linda S; Chappell, William H; Abrams, Stephen L; Franklin, Richard A; Montalto, Giuseppe; Cervello, Melchiorre; Libra, Massimo; Candido, Saverio; Malaponte, Grazia; Mazzarino, Maria C; Fagone, Paolo; Nicoletti, Ferdinando; Bäsecke, Jörg; Mijatovic, Sanja; Maksimovic-Ivanic, Danijela; Milella, Michele; Tafuri, Agostino; Chiarini, Francesca; Evangelisti, Camilla; Cocco, Lucio; Martelli, Alberto M

2012-10-01

The Ras/Raf/MEK/ERK and PI3K/PTEN/Akt/mTOR cascades are often activated by genetic alterations in upstream signaling molecules such as receptor tyrosine kinases (RTK). Targeting these pathways is often complex and can result in pathway activation depending on the presence of upstream mutations (e.g., Raf inhibitors induce Raf activation in cells with wild type (WT) RAF in the presence of mutant, activated RAS) and rapamycin can induce Akt activation. Targeting with inhibitors directed at two constituents of the same pathway or two different signaling pathways may be a more effective approach. This review will first evaluate potential uses of Raf, MEK, PI3K, Akt and mTOR inhibitors that have been investigated in pre-clinical and clinical investigations and then discuss how cancers can become insensitive to various inhibitors and potential strategies to overcome this resistance.

Disruption of PH–kinase domain interactions leads to oncogenic activation of AKT in human cancers

PubMed Central

Parikh, Chaitali; Janakiraman, Vasantharajan; Wu, Wen-I; Foo, Catherine K.; Kljavin, Noelyn M.; Chaudhuri, Subhra; Stawiski, Eric; Lee, Brian; Lin, Jie; Li, Hong; Lorenzo, Maria N.; Yuan, Wenlin; Guillory, Joseph; Jackson, Marlena; Rondon, Jesus; Franke, Yvonne; Bowman, Krista K.; Sagolla, Meredith; Stinson, Jeremy; Wu, Thomas D.; Wu, Jiansheng; Stokoe, David; Stern, Howard M.; Brandhuber, Barbara J.; Lin, Kui; Skelton, Nicholas J.; Seshagiri, Somasekar

2012-01-01

The protein kinase v-akt murine thymoma viral oncogene homolog (AKT), a key regulator of cell survival and proliferation, is frequently hyperactivated in human cancers. Intramolecular pleckstrin homology (PH) domain–kinase domain (KD) interactions are important in maintaining AKT in an inactive state. AKT activation proceeds after a conformational change that dislodges the PH from the KD. To understand these autoinhibitory interactions, we generated mutations at the PH–KD interface and found that most of them lead to constitutive activation of AKT. Such mutations are likely another mechanism by which activation may occur in human cancers and other diseases. In support of this likelihood, we found somatic mutations in AKT1 at the PH–KD interface that have not been previously described in human cancers. Furthermore, we show that the AKT1 somatic mutants are constitutively active, leading to oncogenic signaling. Additionally, our studies show that the AKT1 mutants are not effectively inhibited by allosteric AKT inhibitors, consistent with the requirement for an intact PH–KD interface for allosteric inhibition. These results have important implications for therapeutic intervention in patients with AKT mutations at the PH–KD interface. PMID:23134728

Cross regulation between cGMP-dependent protein kinase and Akt in vasodilatation of porcine pulmonary artery.

PubMed

Liu, Juan; Liu, Huixia; Li, Yanjing; Xu, Xiaojian; Chen, Zhengju; Liu, Limei; Yu, Xiaoxing; Gao, Yuansheng; Dou, Dou

2014-11-01

cGMP-dependent protein kinase (PKG) plays a crucial role in vasodilatation induced by cGMP-elevating agents. Akt has been demonstrated to be involved in modulating vasoreactivity. The present study was to determine the interaction between PKG and Akt and their influences on nitric oxide (NO)-induced vasodilatation. Isolated fourth-generation porcine pulmonary arteries were dissected from the lung and cut into rings in ice-cold modified Krebs-Ringer bicarbonate buffer. The relaxant responses of vessels were determined by organ chamber technique, cGMP was assayed by using enzyme-linked immunosorbent assay kit, the protein levels of phosphorylated Akt were examined by Western blotting, and the activity of phosphodiesterase type 5 (PDE5) was assayed by measuring the rate of cGMP degradation. Incubation with DETA NONOate (a stable NO donor) and 8-Br-cGMP (a cell membrane permeable analog of cGMP) attenuated Akt phosphorylation at Ser-473, which was prevented by Rp-8-Br-PET-cGMPS (a specific inhibitor of PKG) and calyculin A (an inhibitor of protein phosphatase 1 and 2A) but not by okadaic acid (a selective inhibitor of protein phosphatase 2A). Inhibition of Akt enhanced the relaxation and cGMP elevation of porcine pulmonary arteries induced by DETA NONOate or sodium nitroprusside, which was prevented by zaprinast, a specific inhibitor of PDE5. Incubation with LY294002 or Akt inhibitor reduced PDE5 activity in porcine pulmonary arteries. The present study indicates that PKG may attenuate Akt phosphorylation through protein phosphatase 1, which leads to an augmented cGMP elevation by inhibition of PDE5. The increased cGMP in turn activates PKG. Such a positive feedback may play an important role in NO-induced pulmonary vasodilatation.

Resveratrol Overcomes Cellular Resistance to Vemurafenib Through Dephosphorylation of AKT in BRAF-mutated Melanoma Cells.

PubMed

Luo, Hao; Umebayashi, Masayo; Doi, Keiko; Morisaki, Takashi; Shirasawa, Senji; Tsunoda, Toshiyuki

2016-07-01

The serine/threonine-protein kinase B-Raf (BRAF) V600E mutant (BRAF(V600E)) inhibitor vemurafenib, has improved clinical outcomes for patients with BRAF(V600E) melanoma, but acquired cellular resistance mediated by AKT serine/threonine kinase 1 (AKT) phosphorylation limits its efficacy. We examined the effect of resveratrol on vemurafenib-resistant melanoma cells. A vemurafenib-resistant human metastatic melanoma cell line positive for the BRAF V600E mutation was established. The anti-tumorigenic effects of vemurafenib and resveratrol, both alone and in combination, were examined through analysis of cell proliferation and protein expression. The level of phosphorylated AKT (p-AKT) was increased in the primary melanoma cells after treatment with vemurafenib, and the basal level of p-AKT was increased in vemurafenib-resistant melanoma cells. Notably, resveratrol both alone and in combination with vemurafenib effectively suppressed cell proliferation and AKT phosphorylation in both parental and vemurafenib-resistant melanoma cells. Vemurafenib resistance can be reversed by addition of resveratrol in patients undergoing treatment with BRAF inhibitors. Copyright© 2016 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

PKBγ/AKT3 loss-of-function causes learning and memory deficits and deregulation of AKT/mTORC2 signaling: Relevance for schizophrenia

PubMed Central

Floyd, Kirsten; Law, Amanda J.

2017-01-01

Psychiatric genetic studies have identified genome-wide significant loci for schizophrenia. The AKT3/1q44 locus is a principal risk region and gene-network analyses identify AKT3 polymorphisms as a constituent of several neurobiological pathways relevant to psychiatric risk; the neurobiological mechanisms remain unknown. AKT3 shows prenatal enrichment during human neocortical development and recurrent copy number variations involving the 1q43-44 locus are associated with cortical malformations and intellectual disability, implicating an essential role in early brain development. Here, we investigated the role of AKT3 as it relates to aspects of learning and memory and behavioral function, relevant to schizophrenia and cognitive disability, utilizing a novel murine model of Akt3 genetic deficiency. Akt3 heterozygous (Akt3-/+) or null mice (Akt3-/-) were assessed in a comprehensive test battery. Brain biochemical studies were conducted to assess the impact of Akt3 deficiency on cortical Akt/mTOR signaling. Akt3-/+ and Akt3-/- mice exhibited selective deficits of temporal order discrimination and spatial memory, tasks critically dependent on intact prefrontal-hippocampal circuitry, but showed normal prepulse inhibition, fear conditioned learning, memory for novel objects and social function. Akt3 loss-of-function, reduced brain size and dramatically impaired cortical Akt Ser473 activation in an allele-dose dependent manner. Such changes were observed in the absence of altered Akt1 or Akt2 protein expression. Concomitant reduction of the mTORC2 complex proteins, Rictor and Sin1 identifies a potential mechanism. Our findings provide novel insight into the neurodevelopmental role of Akt3, identify a non-redundant role for Akt3 in the development of prefrontal cortical-mediated cognitive function and show that Akt3 is potentially the dominant regulator of AKT/mTOR signaling in brain. PMID:28467426

Insulin induces drug resistance in melanoma through activation of the PI3K/Akt pathway

PubMed Central

Chi, Mengna; Ye, Yan; Zhang, Xu Dong; Chen, Jiezhong

2014-01-01

Introduction There is currently no curative treatment for melanoma once the disease spreads beyond the original site. Although activation of the PI3K/Akt pathway resulting from genetic mutations and epigenetic deregulation of its major regulators is known to cause resistance of melanoma to therapeutic agents, including the conventional chemotherapeutic drug dacarbazine and the Food and Drug Administration-approved mutant BRAF inhibitors vemurafenib and dabrafenib, the role of extracellular stimuli of the pathway, such as insulin, in drug resistance of melanoma remains less understood. Objective To investigate the effect of insulin on the response of melanoma cells to dacarbazine, and in particular, the effect of insulin on the response of melanoma cells carrying the BRAFV600E mutation to mutant BRAF inhibitors. An additional aim was to define the role of the PI3K/Akt pathway in the insulin-triggered drug resistance. Methods The effect of insulin on cytotoxicity induced by dacarbazine or the mutant BRAF inhibitor PLX4720 was tested by pre-incubation of melanoma cells with insulin. Cytotoxicity was determined by the MTS assay. The role of the PI3K/Akt pathway in the insulin-triggered drug resistance was examined using the PI3K inhibitor LY294002 and the PI3K and mammalian target of rapamycin dual inhibitor BEZ-235. Activation of the PI3K/Akt pathway was monitored by Western blot analysis of phosphorylated levels of Akt. Results Recombinant insulin attenuated dacarbazine-induced cytotoxicity in both wild-type BRAF and BRAFV600E melanoma cells, whereas it also reduced killing of BRAFV600E melanoma cells by PLX4720. Nevertheless, the protective effect of insulin was abolished by the PI3K and mTOR dual inhibitor BEZ-235 or the PI3K inhibitor LY294002. Conclusion Insulin attenuates the therapeutic efficacy of dacarbazine and PLX4720 in melanoma cells, which is mediated by activation of the PI3K/Akt pathway and can be overcome by PI3K inhibitors. PMID:24600206

Calmodulin-mediated activation of Akt regulates survival of c-Myc-overexpressing mouse mammary carcinoma cells.

PubMed

Deb, Tushar B; Coticchia, Christine M; Dickson, Robert B

2004-09-10

c-Myc-overexpressing mammary epithelial cells are proapoptotic; their survival is strongly promoted by epidermal growth factor (EGF). We now demonstrate that EGF-induced Akt activation and survival in transgenic mouse mammary tumor virus-c-Myc mouse mammary carcinoma cells are both calcium/calmodulin-dependent. Akt activation is abolished by the phospholipase C-gamma inhibitor U-73122, by the intracellular calcium chelator BAPTA-AM, and by the specific calmodulin antagonist W-7. These results implicate calcium/calmodulin in the activation of Akt in these cells. In addition, Akt activation by serum and insulin is also inhibited by W-7. EGF-induced and calcium/calmodulin-mediated Akt activation occurs in both tumorigenic and non-tumorigenic mouse and human mammary epithelial cells, independent of their overexpression of c-Myc. These results imply that calcium/calmodulin may be a common regulator of Akt activation, irrespective of upstream receptor activator, mammalian species, and transformation status in mammary epithelial cells. However, only c-Myc-overexpressing mouse mammary carcinoma cells (but not normal mouse mammary epithelial cells) undergo apoptosis in the presence of the calmodulin antagonist W-7, indicating the vital selective role of calmodulin for survival of these cells. Calcium/calmodulin-regulated Akt activation is mediated directly by neither calmodulin kinases nor phosphatidylinositol 3-kinase (PI-3 kinase). Pharmacological inhibitors of calmodulin kinase kinase and calmodulin kinases II and III do not inhibit EGF-induced Akt activation, and calmodulin antagonist W-7 does not inhibit phosphotyrosine-associated PI-3 kinase activation. Akt is, however, co-immunoprecipitated with calmodulin in an EGF-dependent manner, which is inhibited by calmodulin antagonist W-7. We conclude that calmodulin may serve a vital regulatory function to direct the localization of Akt to the plasma membrane for its activation by PI-3 kinase.

Efficacy of Histone Deacetylase and Estrogen Receptor Inhibition in Breast Cancer Cells Due to Concerted down Regulation of Akt

PubMed Central

Thomas, Scott; Thurn, K. Ted; Raha, Paromita; Chen, Stephanie; Munster, Pamela N.

2013-01-01

Hormonal therapy resistance remains a considerable barrier in the treatment of breast cancer. Activation of the Akt-PI3K-mTOR pathway plays an important role in hormonal therapy resistance. Our recent preclinical and clinical studies showed that the addition of a histone deacetylase inhibitor re-sensitized hormonal therapy resistant breast cancer to tamoxifen. As histone deacetylases are key regulators of Akt, we evaluated the effect of combined treatment with the histone deacetylase inhibitor PCI-24781 and tamoxifen on Akt in breast cancer cells. We demonstrate that while both histone deacetylase and estrogen receptor inhibition down regulate AKT mRNA and protein, their concerted effort results in down regulation of AKT activity with induction of cell death. Histone deacetylase inhibition exerts its effect on AKT mRNA through an estrogen receptor-dependent mechanism, primarily down regulating the most abundant isoform AKT1. Although siRNA depletion of AKT modestly induces cell death, when combined with an anti-estrogen, cytotoxicity is significantly enhanced. Thus, histone deacetylase regulation of AKT mRNA is a key mediator of this therapeutic combination and may represent a novel biomarker for predicting response to this regimen. PMID:23874830

Cervical spinal erythropoietin induces phrenic motor facilitation via ERK and Akt signaling

PubMed Central

Dale, Erica A.; Satriotomo, Irawan; Mitchell, Gordon S.

2012-01-01

Erythropoietin (EPO) is typically known for its role in erythropoiesis, but is also a potent neurotrophic/neuroprotective factor for spinal motor neurons. Another trophic factor regulated by Hypoxia-Inducible Factor-1, vascular endothelial growth factor (VEGF), signals via ERK and Akt activation to elicit long-lasting phrenic motor facilitation (pMF). Since EPO also signals via ERK and Akt activation, we tested the hypothesis that EPO elicits similar pMF. Using retrograde labeling and immunohistochemical techniques, we demonstrate in adult, male, Sprague-Dawley rats that EPO and its receptor, EPO-R, are expressed in identified phrenic motor neurons. Intrathecal EPO at C4 elicits long-lasting pMF; integrated phrenic nerve burst amplitude increased >90 min post-injection (63±12% baseline 90 min post-injection; p<0.001). EPO increased phosphorylation (and presumed activation) of ERK (1.6 fold vs controls; p<0.05) in phrenic motor neurons; EPO also increased pAkt (1.6 fold vs controls; p<0.05). EPO-induced pMF was abolished by the MEK/ERK inhibitor U0126 and the PI3 kinase/Akt inhibitor LY294002, demonstrating that ERK MAP kinases and Akt are both required for EPO-induced pMF. Pre-treatment with U0126 and LY294002 decreased both pERK and pAkt in phrenic motor neurons (p<0.05), indicating a complex interaction between these kinases. We conclude that EPO elicits spinal plasticity in respiratory motor control. Since EPO expression is hypoxia-sensitive, it may play a role in respiratory plasticity in conditions of prolonged or recurrent low oxygen. PMID:22539857

The significance of PTEN and AKT aberrations in pediatric T-cell acute lymphoblastic leukemia

PubMed Central

Zuurbier, Linda; Petricoin, Emanuel F.; Vuerhard, Maartje J.; Calvert, Valerie; Kooi, Clarissa; Buijs-Gladdines, Jessica G.C.A.M.; Smits, Willem K.; Sonneveld, Edwin; Veerman, Anjo J.P.; Kamps, Willem A.; Horstmann, Martin; Pieters, Rob; Meijerink, Jules P.P.

2012-01-01

Background PI3K/AKT pathway mutations are found in T-cell acute lymphoblastic leukemia, but their overall impact and associations with other genetic aberrations is unknown. PTEN mutations have been proposed as secondary mutations that follow NOTCH1-activating mutations and cause cellular resistance to γ-secretase inhibitors. Design and Methods The impact of PTEN, PI3K and AKT aberrations was studied in a genetically well-characterized pediatric T-cell leukemia patient cohort (n=146) treated on DCOG or COALL protocols. Results PTEN and AKT E17K aberrations were detected in 13% and 2% of patients, respectively. Defective PTEN-splicing was identified in incidental cases. Patients without PTEN protein but lacking exon-, splice-, promoter mutations or promoter hypermethylation were present. PTEN/AKT mutations were especially abundant in TAL- or LMO-rearranged leukemia but nearly absent in TLX3-rearranged patients (P=0.03), the opposite to that observed for NOTCH1-activating mutations. Most PTEN/AKT mutant patients either lacked NOTCH1-activating mutations (P=0.006) or had weak NOTCH1-activating mutations (P=0.011), and consequently expressed low intracellular NOTCH1, cMYC and MUSASHI levels. T-cell leukemia patients without PTEN/AKT and NOTCH1-activating mutations fared well, with a cumulative incidence of relapse of only 8% versus 35% for PTEN/AKT and/or NOTCH1-activated patients (P=0.005). Conclusions PI3K/AKT pathway aberrations are present in 18% of pediatric T-cell acute lymphoblastic leukemia patients. Absence of strong NOTCH1-activating mutations in these cases may explain cellular insensitivity to γ-secretase inhibitors. PMID:22491738

Role for pAKT in rat urinary bladder with cyclophosphamide (CYP)-induced cystitis

PubMed Central

Arms, Lauren

2011-01-01

AKT phosphorylation following peripheral nerve injury or inflammation may play a role in somatic pain processes and visceral inflammation. To examine such a role in micturition reflexes with bladder inflammation, we induced bladder inflammation in adult female Wistar rats (200–300 g) by injecting cyclophosphamide (CYP) intraperitoneally at acute (150 mg/kg; 4 h), intermediate (150 mg/kg; 48 h), and chronic (75 mg/kg; every third day for 10 days) time points. Western blot analyses of whole urinary bladders showed significant increases (P ≤ 0.01) in phosphorylated (p) AKT at all time points; however, the magnitude of AKT phosphorylation varied with duration of CYP treatment. Immunohistochemical analyses of pAKT immunoreactivity (pAKT-IR) in cryostat bladder sections demonstrated duration-dependent, significant (P ≤ 0.01) increases in pAKT-IR in both the urothelium and detrusor smooth muscle of CYP-inflamed bladders. Additionally, a suburothelial population of pAKT-IR macrophages (CD68-, MAC2-, and F4/80-positive) was present in chronic CYP-treated bladders. The functional role of pAKT in micturition was evaluated using open, conscious cystometry with continuous instillation of saline in conjunction with administration of an inhibitor of AKT phosphorylation, deguelin (1.0 μg/10 μl), or vehicle (1% DMSO in saline) in control (no inflammation) and CYP (48 h)-treated rats. Bladder capacity, void volume, and intercontraction void interval increased significantly (P ≤ 0.05) following intravesical instillation of deguelin in CYP (48 h)-treated rats. These results demonstrate increased AKT phosphorylation in the urinary bladder with urinary bladder inflammation and that blockade of AKT phosphorylation in the urothelium improves overall bladder function. PMID:21632956

Isoginkgetin inhibits tumor cell invasion by regulating phosphatidylinositol 3-kinase/Akt-dependent matrix metalloproteinase-9 expression.

PubMed

Yoon, Sang-Oh; Shin, Sejeong; Lee, Ho-Jae; Chun, Hyo-Kon; Chung, An-Sik

2006-11-01

Matrix metalloproteinase (MMP)-9 plays a key role in tumor invasion. Inhibitors of MMP-9 were screened from Metasequoia glyptostroboides (Dawn redwood) and one potent inhibitor, isoginkgetin, a biflavonoid, was identified. Noncytotoxic levels of isoginkgetin decreased MMP-9 production profoundly, but up-regulated the level of tissue inhibitor of metalloproteinase (TIMP)-1, an inhibitor of MMP-9, in HT1080 human fibrosarcoma cells. The major mechanism of Ras-dependent MMP-9 production in HT1080 cells was phosphatidylinositol 3-kinase (PI3K)/Akt/nuclear factor-kappaB (NF-kappaB) activation. Expression of dominant-active H-Ras and p85 (a subunit of PI3K) increased MMP-9 activity, whereas dominant-negative forms of these molecules decreased the level of MMP-9. H-Ras did not increase MMP-9 in the presence of a PI3K inhibitor, LY294002, and a NF-kappaB inhibitor, SN50. Further studies showed that isoginkgetin regulated MMP-9 production via PI3K/Akt/NF-kappaB pathway, as evidenced by the findings that isoginkgetin inhibited activities of both Akt and NF-kappaB. PI3K/Akt is a well-known key pathway for cell invasion, and isoginkgetin inhibited HT1080 tumor cell invasion substantially. Isoginkgetin was also quite effective in inhibiting the activities of Akt and MMP-9 in MDA-MB-231 breast carcinomas and B16F10 melanoma. Moreover, isoginkgetin treatment resulted in marked decrease in invasion of these cells. In summary, PI3K/Akt is a major pathway for MMP-9 expression and isoginkgetin markedly decreased MMP-9 expression and invasion through inhibition of this pathway. This suggests that isoginkgetin could be a potential candidate as a therapeutic agent against tumor invasion.

Low Phosphorylated AKT Expression in Laryngeal Cancer: Indications for a Higher Metastatic Risk

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nijkamp, Monique M.; Span, Paul N.; Stegeman, Hanneke

2013-10-01

Purpose: To validate the association of phosphorylated (p)AKT with lymph node metastasis in an independent, homogeneous cohort of patients with larynx cancer. Methods and Materials: Seventy-eight patients with laryngeal cancer were included. Epidermal growth factor receptor, pAKT, vimentin, E-cadherin, hypoxia, and blood vessels were visualized in biopsy material using immunohistochemistry. Positive tumor areas and spatial relationships between markers were assessed by automated image analysis. In 6 laryngeal cancer cell lines, E-cadherin and vimentin messenger RNA was quantified by real-time polymerase chain reaction and by immunohistochemistry before and after treatment with the pAKT inhibitor MK-2206. Results: A significant correlation was foundmore » between low pAKT in the primary tumor and positive lymph node status (P=.0005). Tumors with lymph node metastases had an approximately 10-fold lower median pAKT value compared with tumors without lymph node metastases, albeit with large intertumor variations, validating our previous results. After inhibition of pAKT in laryngeal cancer cells with MK-2206, up-regulation of vimentin and a downregulation of E-cadherin occurred, consistent with epithelial–mesenchymal transition. Conclusion: Low pAKT expression in larynx tumors is associated with lymph node metastases. Further, inhibition of pAKT in laryngeal cancer induces epithelial–mesenchymal transition, predisposing for an increased metastatic risk.« less

Cep55 regulates embryonic growth and development by promoting Akt stability in zebrafish.

PubMed

Jeffery, Jessie; Neyt, Christine; Moore, Wade; Paterson, Scott; Bower, Neil I; Chenevix-Trench, Georgia; Verkade, Heather; Hogan, Benjamin M; Khanna, Kum Kum

2015-05-01

CEP55 was initially described as a centrosome- and midbody-associated protein and a key mediator of cytokinesis. More recently, it has been implicated in PI3K/AKT pathway activation via an interaction with the catalytic subunit of PI3K. However, its role in embryonic development is unknown. Here we describe a cep55 nonsense mutant zebrafish with which we can study the in vivo physiologic role of Cep55. Homozygous mutants underwent extensive apoptosis by 24 hours postfertilization (hpf) concomitant with cell cycle defects, and heterozygous carriers were indistinguishable from their wild-type siblings. A similar phenotype was also observed in zebrafish injected with a cep55 morpholino, suggesting the mutant is a cep55 loss-of-function model. Further analysis revealed that Akt was destabilized in the homozygous mutants, which partially phenocopied Akt1 and Akt2 knockdown. Expression of either constitutively activated PIK3CA or AKT1 could partially rescue the homozygous mutants. Consistent with a role for Cep55 in regulation of Akt stability, treatment with proteasome inhibitor, MG132, partially rescued the homozygous mutants. Taken together, these results provide the first description of Cep55 in development and underline the importance of Cep55 in the regulation of Pi3k/Akt pathway and in particular Akt stability. © FASEB.

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.

Fluorescent indicators for Akt/protein kinase B and dynamics of Akt activity visualized in living cells.

PubMed

Sasaki, Kazuki; Sato, Moritoshi; Umezawa, Yoshio

2003-08-15

Akt/protein kinase B (PKB) is a serine/threonine kinase that regulates a variety of cellular responses. To provide information on the spatial and temporal dynamics of Akt/PKB activity, we have developed genetically encoded fluorescent indicators for Akt/PKB. The indicators contain two green fluorescent protein mutants, an Akt/PKB substrate domain, flexible linker sequence, and phosphorylation recognition domain. A phosphorylation of the substrate domain in the indicators caused change in the emission ratio based on fluorescent resonance energy transfer between the two green fluorescent protein mutants. To let the fluorescent indicators behave as endothelial nitric-oxide synthase and Bad, which are endogenous Akt/PKB substrates, they were fused with the Golgi target domain and mitochondria target domain, respectively. The indicators thus colocalized with the endogenous substrates conferred their susceptibilities to phosphorylation by Akt/PKB. We showed that the Golgi-localized indicator responded to the stimulation with 17beta-estradiol (E2) and insulin in endothelial cells. In addition, E2 elicited the phosphorylation of the mitochondria-localized indicator in the endothelial cells, but no phosphorylation was observed by E2 or by insulin of the diffusible indicator that has no targeting domain. The difference in the results with the three indicators suggests that the activated Akt/PKB is localized to subcellular compartments, including the Golgi apparatus and/or mitochondria, rather than diffusing in the cytosol, thereby efficiently phosphorylating its substrate proteins. E2 triggered the phosphorylation of the mitochondria-localized indicator, whereas insulin did not induce this phosphorylation, which suggests that the localization of the activated Akt/PKB to the mitochondria is directed differently between insulin and E2 via distinct mechanisms.

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

Role of Akt signaling in resistance to DNA-targeted therapy

PubMed Central

Avan, Abolfazl; Narayan, Ravi; Giovannetti, Elisa; Peters, Godefridus J

2016-01-01

The Akt signal transduction pathway controls most hallmarks of cancer. Activation of the Akt cascade promotes a malignant phenotype and is also widely implicated in drug resistance. Therefore, the modulation of Akt activity is regarded as an attractive strategy to enhance the efficacy of cancer therapy and irradiation. This pathway consists of phosphatidylinositol 3 kinase (PI3K), mammalian target of rapamycin, and the transforming serine-threonine kinase Akt protein isoforms, also known as protein kinase B. DNA-targeted agents, such as platinum agents, taxanes, and antimetabolites, as well as radiation have had a significant impact on cancer treatment by affecting DNA replication, which is aberrantly activated in malignancies. However, the caveat is that they may also trigger the activation of repairing mechanisms, such as upstream and downstream cascade of Akt survival pathway. Thus, each target can theoretically be inhibited in view of improving the potency of conventional treatment. Akt inhibitors, e.g., MK-2206 and perifosine, or PI3K modulators, e.g., LY294002 and Wortmannin, have shown some promising results in favor of sensitizing the cancer cells to the therapy in vitro and in vivo, which have provided the rationale for incorporation of these novel agents into multimodality treatment of different malignancies. Nevertheless, despite the acceptable safety profile of some of these agents in the clinical studies, with regard to the efficacy, the results are still too preliminary. Hence, we need to wait for the upcoming data from the ongoing trials before utilizing them into the standard care of cancer patients. PMID:27777878

Icariside II activates EGFR-Akt-Nrf2 signaling and protects osteoblasts from dexamethasone.

PubMed

Liu, Weidong; Mao, Li; Ji, Feng; Chen, Fengli; Wang, Shouguo; Xie, Yue

2017-01-10

The potential effect of icariside II on dexamethasone-induced osteoblast cell damages was evaluated here. In MC3T3-E1 osteoblastic cells and the primary murine osteoblasts, co-treatment with icariside II dramatically attenuated dexamethasone- induced cell death and apoptosis. Icariside II activated Akt signaling, which is required for its actions in osteoblasts. Akt inhibitors (LY294002, perifosine and MK-2206) almost abolished icariside II-induced osteoblast cytoprotection against dexamethasone. Further studies showed that icariside II activated Nrf2 signaling, downstream of Akt, to inhibit dexamethasone-induced reactive oxygen species (ROS) production in MC3T3-E1 cells and primary osteoblasts. On the other hand, Nrf2 shRNA knockdown inhibited icariside II-induced anti-dexamethasone cytoprotection in MC3T3-E1 cells. Finally, we showed that icariside II induced heparin-binding EGF (HB-EGF) production and EGFR trans-activation in MC3T3-E1 cells. EGFR inhibition, via anti-HB-EGF antibody, EGFR inhibitor AG1478 or EGFR shRNA knockdown, almost blocked icariside II-induced Akt-Nrf2 activation in MC3T3-E1 cells. Collectively, we conclude that icariside II activates EGFR-Akt-Nrf2 signaling and protects osteoblasts from dexamethasone. Icariside II might have translational value for the treatment of dexamethasone-associated osteoporosis/osteonecrosis.

Inhibitor of G protein-coupled receptor kinase 2 normalizes vascular endothelial function in type 2 diabetic mice by improving β-arrestin 2 translocation and ameliorating Akt/eNOS signal dysfunction.

PubMed

Taguchi, Kumiko; Matsumoto, Takayuki; Kamata, Katsuo; Kobayashi, Tsuneo

2012-07-01

In type 2 diabetes, although Akt/endothelial NO synthase (eNOS) activation is known to be negatively regulated by G protein-coupled receptor kinase 2 (GRK2), it is unclear whether the GRK2 inhibitor would have therapeutic effects. Here we examined the hypotensive effect of the GRK2 inhibitor and its efficacy agonist both vascular (aortic) endothelial dysfunction (focusing especially on the Akt/eNOS pathway) and glucose intolerance in two type 2 diabetic models (ob/ob mice and nicotinamide+streptozotocin-induced diabetic mice). Mice were treated with a single injection of the GRK2 inhibitor or vehicle, and the therapeutic effects were compared by examining vascular function and by Western blotting. The GRK2 inhibitor lowered blood pressure in both diabetic models but not in their age-matched controls. The GRK2 inhibitor significantly improved clonidine-induced relaxation only in diabetic (ob/ob and DM) mice, with accompanying attenuations of GRK2 activity and translocation to the plasma membrane. These protective effects of the GRK2 inhibitor may be attributable to the augmented Akt/eNOS pathway activation (as evidenced by increases in Akt phosphorylation at Ser(473) and at Thr(308), and eNOS phosphorylation at Ser(1177)) and to the prevention of the GRK2 translocation and promotion of β-arrestin 2 translocation to the membrane under clonidine stimulation. Moreover, the GRK2 inhibitor significantly improved the glucose intolerance seen in the ob/ob mice. Our work provides the first evidence that in diabetes, the GRK2 inhibitor ameliorates vascular endothelial dysfunction via the Akt/eNOS pathway by inhibiting GRK2 activity and enhancing β-arrestin 2 translocation under clonidine stimulation, thereby contributing to a blood pressure-lowering effect. We propose that the GRK2 inhibitor may be a promising therapeutic agent for cardiovascular complications in type 2 diabetes.

Akt2-Dependent Phosphorylation of Radixin in Regulation of Mrp-2 Trafficking in WIF-B Cells.

PubMed

Suda, Jo; Rockey, Don C; Karvar, Serhan

2016-02-01

The dominant ezrin/radixin/moesin protein in hepatocytes is radixin, which plays an important role in mediating the binding of F-actin to the plasma membrane after a conformational activation by phosphorylation at Thr564. Here we have investigated the importance of Akt-mediated radixin Thr564 phosphorylation on Mrp-2 distribution and function in WIF-B cells. Mrp-2 is an adenosine triphosphate (ATP)-binding cassette transporter that plays an important role in detoxification and chemoprotection by transporting a wide range of compounds, especially conjugates of lipophilic substances with glutathione, organic anions, and drug metabolites such as glucuronides. Akt1 and Akt2 expression were manipulated using dominant active and negative constructs as well as Akt1 and Akt2 siRNA. Cellular distribution of radixin and Mrp-2 was visualized by fluorescence microscopy. A 5-chloromethylfluorescein diacetate, which is a substrate of the Mrp-2 and is actively transported in canalicular lumina, was used to measure Mrp-2 function. Radixin phosphorylation was significantly increased in wild-type and dominant active Akt2 transfected cells. Furthermore, radixin and Mrp-2 were localized at the canalicular membrane, similar to control cells. In contrast, overexpression of dominant negative Akt2, siRNA knockdown of Akt2 and a specific Akt inhibitor prevented radixin phosphorylation and led to alteration of normal radixin and Mrp-2 localization; inhibition of Akt2, but not Akt1 function led to radixin localization to the cytoplasmic space. In addition, dominant negative and Akt2 knockdown led to a dramatically impaired hepatocyte secretory response, while wild-type and dominant active Akt2 transfected cells exhibited increased 5-chloromethylfluorescein diacetate excretion. In contrast to Akt2, Akt1 was not associated with radixin phosphorylation. These studies, therefore, identify Akt2 as a critical kinase that regulates radixin phosphorylation and leads to Mrp-2 translocation and

Temperature sensitivity of phospho-Ser{sup 473}-PKB/AKT

DOE Office of Scientific and Technical Information (OSTI.GOV)

Oehler-Jaenne, Christoph; Bueren, Andre O. von; Vuong, Van

2008-10-24

The phospho-PKB/Akt status is often used as surrogate marker to measure activation of the PI3K/Akt/mTOR signal transduction pathway. Though, inconsistencies of the p-Ser{sup 473}-PKB/Akt status have raised doubts in the validity of p-Ser{sup 473}-PKB/Akt phosphorylation as endpoint. Here, we determined that p-Ser{sup 473}-PKB/Akt but not p-Thr{sup 308}-PKB/Akt phosphorylation is highly temperature sensitive. p-Ser{sup 473}-PKB/Akt phosphorylation was rapidly reduced to levels below 50% on exposure to 20-25 deg. C in murine and human cell lines including cells expressing constitutively active PI3K or lacking PTEN. Down-regulation of p-Ser{sup 473}-PKB/Akt was reversible and re-exposure to physiological temperature resulted in increased p-Ser{sup 473}-PKB/Akt phosphorylationmore » levels. Phosphatase activity at low temperature was sustained at 75% baseline level and phosphatase inhibition prevented p-Ser{sup 473}-PKB/Akt dephosphorylation induced by the low temperature shift. Interestingly temperature-dependent deregulation of the p-Ser{sup 473}-PKB/Akt status was also observed in response to irradiation. Thus our data demonstrate that minimal additional stress factors deregulate the PI3K/Akt-survival pathway and the p-Ser{sup 473}-PKB/Akt status as experimental endpoint.« less

Shear stress stimulates phosphorylation of endothelial nitric-oxide synthase at Ser1179 by Akt-independent mechanisms: role of protein kinase A

NASA Technical Reports Server (NTRS)

Boo, Yong Chool; Sorescu, George; Boyd, Nolan; Shiojima, Ichiro; Walsh, Kenneth; Du, Jie; Jo, Hanjoong

2002-01-01

Recently, we have shown that shear stress stimulates NO(*) production by the protein kinase B/Akt (Akt)-dependent mechanisms in bovine aortic endothelial cells (BAEC) (Go, Y. M., Boo, Y. C., Park, H., Maland, M. C., Patel, R., Pritchard, K. A., Jr., Fujio, Y., Walsh, K., Darley-Usmar, V., and Jo, H. (2001) J. Appl. Physiol. 91, 1574-1581). Akt has been believed to regulate shear-dependent production of NO(*) by directly phosphorylating endothelial nitric-oxide synthase (eNOS) at the Ser(1179) residue (eNOS-S(1179)), but a critical evaluation using specific inhibitors or dominant negative mutants (Akt(AA) or Akt(AAA)) has not been reported. In addition, other kinases, including protein kinase A (PKA) and AMP kinase have also shown to phosphorylate eNOS-S(1179). Here, we show that shear-dependent phosphorylation of eNOS-S(1179) is mediated by an Akt-independent, but a PKA-dependent, mechanism. Expression of Akt(AA) or Akt(AAA) in BAEC by using recombinant adenoviral constructs inhibited phosphorylation of eNOS-S(1179) if cells were stimulated by vascular endothelial growth factor (VEGF), but not by shear stress. As shown before, expression of Akt(AA) inhibited shear-dependent NO(*) production, suggesting that Akt is still an important regulator in NO production. Further studies showed that a selective inhibitor of PKA, H89, inhibited shear-dependent phosphorylation of eNOS-S(1179) and NO(*) production. In contrast, H89 did not inhibit phosphorylation of eNOS-S(1179) induced by expressing a constitutively active Akt mutant (Akt(Myr)) in BAEC, showing that the inhibitor did not affect the Akt pathway. 8-Bromo-cAMP alone phosphorylated eNOS-S(1179) within 5 min without activating Akt, in an H89-sensitive manner. Collectively, these results demonstrate that shear stimulates phosphorylation of eNOS-S(1179) in a PKA-dependent, but Aktindependent manner, whereas the NO(*) production is regulated by the mechanisms dependent on both PKA and Akt. A coordinated interaction

Knockdown of Akt1 promotes Akt2 upregulation and resistance to oxidative-stress-induced apoptosis through control of multiple signaling pathways.

PubMed

Zhang, Lan; Sun, Shuming; Zhou, Jie; Liu, Jiao; Lv, Jia-Han; Yu, Xiang-Qiang; Li, Chi; Gong, Lili; Yan, Qin; Deng, Mi; Xiao, Ling; Ma, Haili; Liu, Jin-Ping; Peng, Yun-Lei; Wang, Dao; Liao, Gao-Peng; Zou, Li-Jun; Liu, Wen-Bin; Xiao, Ya-Mei; Li, David Wan-Cheng

2011-07-01

The Akt signaling pathway plays a key role in promoting the survival of various types of cells from stress-induced apoptosis, and different members of the Akt family display distinct physiological roles. Previous studies have shown that in response to UV irradiation, Akt2 is sensitized to counteract the induced apoptosis. However, in response to oxidative stress such as hydrogen peroxide, it remains to be elucidated what member of the Akt family would be activated to initiate the signaling cascades leading to resistance of the induced apoptosis. In the present study, we present the first evidence that knockdown of Akt1 enhances cell survival under exposure to 50 μM H(2)O(2). This survival is derived from selective upregulation and activation of Akt2 but not Akt3, which initiates 3 major signaling cascades. First, murine double minute 2 (MDM2) is hyperphosphorylated, which promotes p53 degradation and attenuates its Ser-15 phosphorylation, significantly attenuating Bcl-2 homologous antagonist killer (Bak) upregulation. Second, Akt2 activation inactivates glycogen synthase kinase 3 beta (GSK-3β) to promote stability of myeloid leukemia cell differentiation protein 1 (MCL-1). Finally, Akt2 activation promotes phosphorylation of FOXO3A toward cytosolic export and thus downregulates Bim expression. Overexpression of Bim enhances H(2)O(2)-induced apoptosis. Together, our results demonstrate that among the Akt family members, Akt2 is an essential kinase in counteracting oxidative-stress-induced apoptosis through multiple signaling pathways.

Preclinical rationale for PI3K/Akt/mTOR pathway inhibitors as therapy for epidermal growth factor receptor inhibitor-resistant non-small-cell lung cancer.

PubMed

Gadgeel, Shirish M; Wozniak, Antoinette

2013-07-01

Mutations in the epidermal growth factor receptor gene (EGFR) are frequently observed in non-small-cell lung cancer (NSCLC), occurring in about 40% to 60% of never-smokers and in about 17% of patients with adenocarcinomas. EGFR tyrosine kinase inhibitors (TKIs), such as gefitinib and erlotinib, have transformed therapy for patients with EGFR-mutant NSCLC and have proved superior to chemotherapy as first-line treatment for this patient group. Despite these benefits, there are currently 2 key challenges associated with EGFR inhibitor therapy for patients with NSCLC. First, only 85% to 90% of patients with the EGFR mutation derive clinical benefit from EGFR TKIs, with the remainder demonstrating innate resistance to therapy. Second, acquired resistance to EGFR TKIs inevitably occurs in patients who initially respond to therapy, with a median duration of response of about 10 months. Mutant EGFR activates various subcellular signaling cascades, including the phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) pathway, which demonstrates maintained activity in a variety of TKI-resistant cancers. Given the fundamental role of the PI3K/Akt/mTOR pathway in tumor oncogenesis, proliferation, and survival, PI3K pathway inhibitors have emerged as a possible solution to the problem of EGFR TKI resistance. However resistance to EGFR TKIs is associated with considerable heterogeneity and complexity. Preclinical experiments investigating these phenomena suggest that in some patients, PI3K inhibitors will have to be paired with other targeted agents if they are to be effective. This review discusses the preclinical data supporting PI3K/Akt/mTOR pathway inhibitor combinations in EGFR TKI-resistant NSCLC from the perspective of the various agents currently being investigated in clinical trials. Copyright © 2013 Elsevier Inc. All rights reserved.

The Akt signaling pathway

PubMed Central

Madhunapantula, SubbaRao V; Mosca, Paul J

2011-01-01

Studies using cultured melanoma cells and patient tumor biopsies have demonstrated deregulated PI3 kinase-Akt3 pathway activity in ∼70% of melanomas. Furthermore, targeting Akt3 and downstream PRAS40 has been shown to inhibit melanoma tumor development in mice. Although these preclinical studies and several other reports using small interfering RNAs and pharmacological agents targeting key members of this pathway have been shown to retard melanoma development, analysis of early Phase I and Phase II clinical trials using pharmacological agents to target this pathway demonstrate the need for (1) selection of patients whose tumors have PI3 kinase-Akt pathway deregulation, (2) further optimization of therapeutic agents for increased potency and reduced toxicity, (3) the identification of additional targets in the same pathway or in other signaling cascades that synergistically inhibit the growth and progression of melanoma, and (4) better methods for targeted delivery of pharmaceutical agents inhibiting this pathway. In this review we discuss key potential targets in PI3K-Akt3 signaling, the status of pharmacological agents targeting these proteins, drugs under clinical development, and strategies to improve the efficacy of therapeutic agents targeting this pathway. PMID:22157148

Methylglyoxal Mediates Adipocyte Proliferation by Increasing Phosphorylation of Akt1

PubMed Central

Jia, Xuming; Chang, Tuanjie; Wilson, Thomas W.; Wu, Lingyun

2012-01-01

Methylglyoxal (MG) is a highly reactive metabolite physiologically presented in all biological systems. The effects of MG on diabetes and hypertension have been long recognized. In the present study, we investigated the potential role of MG in obesity, one of the most important factors to cause metabolic syndrome. An increased MG accumulation was observed in the adipose tissue of obese Zucker rats. Cell proliferation assay showed that 5–20 µM of MG stimulated the proliferation of 3T3-L1 cells. Further study suggested that accumulated-MG stimulated the phosphorylation of Akt1 and its targets including p21 and p27. The activated Akt1 then increased the activity of CDK2 and accelerated the cell cycle progression of 3T3-L1 cells. The effects of MG were efficiently reversed by advanced glycation end product (AGE) breaker alagebrium and Akt inhibitor SH-6. In summary, our study revealed a previously unrecognized effect of MG in stimulating adipogenesis by up-regulation of Akt signaling pathway and this mechanism might offer a new approach to explain the development of obesity. PMID:22606274

SC79 protects retinal pigment epithelium cells from UV radiation via activating Akt-Nrf2 signaling

PubMed Central

Cao, Guo-fan; Cao, Cong; Jiang, Qin

2016-01-01

Excessive Ultra-violet (UV) radiation causes oxidative damages and apoptosis in retinal pigment epithelium (RPE) cells. Here we tested the potential activity of SC79, a novel small molecule activator of Akt, against the process. We showed that SC79 activated Akt in primary and established (ARPE-19 line) RPE cells. It protected RPE cells from UV damages possibly via inhibiting cell apoptosis. Akt inhibition, via an Akt specific inhibitor (MK-2206) or Akt1 shRNA silence, almost abolished SC79-induced RPE cytoprotection. Further studies showed that SC79 activated Akt-dependent NF-E2-related factor 2 (Nrf2) signaling and inhibited UV-induced oxidative stress in RPE cells. Reversely, Nrf2 shRNA knockdown or S40T mutation attenuated SC79-induced anti-UV activity. For the in vivo studies, we showed that intravitreal injection of SC79 significantly protected mouse retina from light damages. Based on these results, we suggest that SC79 protects RPE cells from UV damages possibly via activating Akt-Nrf2 signaling axis. PMID:27517753

Targeting the PI3K/Akt pathway in murine MDS/MPN driven by hyperactive Ras.

PubMed

Akutagawa, J; Huang, T Q; Epstein, I; Chang, T; Quirindongo-Crespo, M; Cottonham, C L; Dail, M; Slusher, B S; Friedman, L S; Sampath, D; Braun, B S

2016-06-01

Chronic and juvenile myelomonocytic leukemias (CMML and JMML) are myelodysplastic/myeloproliferative neoplasia (MDS/MPN) overlap syndromes that respond poorly to conventional treatments. Aberrant Ras activation because of NRAS, KRAS, PTPN11, CBL and NF1 mutations is common in CMML and JMML. However, no mechanism-based treatments currently exist for cancers with any of these mutations. An alternative therapeutic strategy involves targeting Ras-regulated effector pathways that are aberrantly activated in CMML and JMML, which include the Raf/MEK/ERK and phosphoinositide-3'-OH kinase (PI3K)/Akt cascades. Mx1-Cre, Kras(D12) and Mx1-Cre, Nf1(flox/)(-) mice accurately model many aspects of CMML and JMML. Treating Mx1-Cre, Kras(D12) mice with GDC-0941 (also referred to as pictilisib), an orally bioavailable inhibitor of class I PI3K isoforms, reduced leukocytosis, anemia and splenomegaly while extending survival. However, GDC-0941 treatment attenuated activation of both PI3K/Akt and Raf/MEK/ERK pathways in primary hematopoietic cells, suggesting it could be acting through suppression of Raf/MEK/ERK signals. To interrogate the importance of the PI3K/Akt pathway specifically, we treated mice with the allosteric Akt inhibitor MK-2206. This compound had no effect on Raf/MEK/ERK signaling, yet it also induced robust hematologic responses in Kras and Nf1 mice with MPN. These data support investigating PI3K/Akt pathway inhibitors as a therapeutic strategy in JMML and CMML patients.

Akt-Dependent Cytokine Production in Mast Cells

PubMed Central

Kitaura, Jiro; Asai, Koichi; Maeda-Yamamoto, Mari; Kawakami, Yuko; Kikkawa, Ushio; Kawakami, Toshiaki

2000-01-01

Cross-linking of FcεRI induces the activation of three protein tyrosine kinases, Lyn, Syk, and Bruton's tyrosine kinase (Btk), leading to the secretion of a panel of proinflammatory mediators from mast cells. This study showed phosphorylation at Ser-473 and enzymatic activation of Akt/protein kinase B, the crucial survival kinase, upon FcεRI stimulation in mouse mast cells. Phosphorylation of Akt is regulated positively by Btk and Syk and negatively by Lyn. Akt in turn can regulate positively the transcriptional activity of interleukin (IL)-2 and tumor necrosis factor (TNF)-α promoters. Transcription from the nuclear factor κB (NF-κB), nuclear factor of activated T cells (NF-AT), and activator protein 1 (AP-1) sites within these promoters is under the control of Akt activity. Accordingly, the signaling pathway involving IκB-α, a cytoplasmic protein that binds NF-κB and inhibits its nuclear translocation, appears to be regulated by Akt in mast cells. Catalytic activity of glycogen synthase kinase (GSK)-3β, a serine/threonine kinase that phosphorylates NF-AT and promotes its nuclear export, seems to be inhibited by Akt. Importantly, Akt regulates the production and secretion of IL-2 and TNF-α in FcεRI-stimulated mast cells. Altogether, these results revealed a novel function of Akt in transcriptional activation of cytokine genes via NF-κB, NF-AT, and AP-1 that contributes to the production of cytokines. PMID:10974038

Lithium ions attenuate serum-deprivation-induced apoptosis in PC12 cells through regulation of the Akt/FoxO1 signaling pathways.

PubMed

Zeng, Zhiwen; Wang, Haitao; Shang, Fu; Zhou, Lihua; Little, Peter J; Quirion, Remi; Zheng, Wenhua

2016-03-01

Lithium is currently used in the treatment of mental illness. We have previously reported that lithium stimulated the protein kinase B/Forkhead box O1 (Akt/FoxO1) pathway in rats. However, little information is available regarding its neuroprotective role of this pathway and underlying mechanisms. PC12 cells treated with serum deprivation were used as a toxicity model to study the protective effect of lithium and its underlying mechanisms. Cell viability was determined by methyl thiazolyl tetrazolium assay and Hoechst staining. FoxO1 subcellular location and its overexpression were used to study the underlying mechanisms. Various pathway inhibitors were used to investigate the possible pathways, while the phosphorylation of Akt and FoxO1 was analyzed by Western blot. Lithium pretreatment dose-dependently reduced PC12 cell apoptosis induced by serum starvation. The protective effect of lithium was abolished by LY294002, a PI3K-specific inhibitor, and Akt inhibitor Akt inhibitor VIII, whereas mitogen-activated protein kinase kinase (MEK kinase) inhibitor U0126 had no effect. Lithium induced the phosphorylation of Akt and FoxO1 in a time- and concentration-dependent manner. Lithium-induced phosphorylation of Akt and FoxO1 is mediated by the PI3K/Akt pathway. Serum deprivation caused nuclear translocation of FoxO1 while application of lithium reversed the effect of serum deprivation. Moreover, overexpression of FoxO1 enhanced cell apoptosis induced by serum withdrawal. Finally, lithium was found to reduce the exogenous and endogenous FoxO1 protein levels in PC12 cells in a concentration-dependent fashion. The protective effect of lithium against serum starvation cell death is mediated by the PI3K/Akt/FoxO1 pathway.

Vitamin E Facilitates the Inactivation of the Kinase Akt by the Phosphatase PHLPP1

PubMed Central

Huang, Po-Hsien; Chuang, Hsiao-Ching; Chou, Chih-Chien; Wang, Huiling; Lee, Su-Lin; Yang, Hsiao-Ching; Chiu, Hao-Chieh; Kapuriya, Naval; Wang, Dasheng; Kulp, Samuel K.; Chen, Ching-Shih

2014-01-01

rationale for the translational development of tocopherols into novel PH domain-targeted Akt inhibitors. PMID:23512990

Inhibition of Akt enhances the chemopreventive effects of topical rapamycin in mouse skin

USGS Publications Warehouse

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

2016-01-01

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

Inhibition of Akt Enhances the Chemopreventive Effects of Topical Rapamycin in Mouse Skin

PubMed Central

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

2016-01-01

The PI3Kinase/Akt/mTOR pathway has important roles in cancer development for multiple tumor types, including UV-induced non-melanoma skin cancer. Immunosuppressed populations are at increased risk of aggressive cutaneous squamous cell carcinoma (SCC). Individuals who are treated with rapamycin, (sirolimus, a classical mTOR inhibitor) have significantly decreased rates of developing new cutaneous SCCs compared to those that receive traditional immunosuppression. However, systemic rapamycin use can lead to significant adverse events. Here we explored the use of topical rapamycin as a chemopreventive agent in the context of solar simulated light (SSL)-induced skin carcinogenesis. In SKH-1 mice, topical rapamycin treatment decreased tumor yields when applied after completion of 15 weeks of SSL exposure compared to controls. However, applying rapamycin during SSL exposure for 15 weeks, and continuing for 10 weeks after UV treatment, increased tumor yields. We also examined whether a combinatorial approach might result in more significant tumor suppression by rapamycin. We validated that rapamycin causes increased Akt (S473) phosphorylation in the epidermis after SSL, and show for the first time that this dysregulation can be inhibited in vivo by a selective PDK1/Akt inhibitor, PHT-427. Combining rapamycin with PHT-427 on tumor prone skin additively caused a significant reduction of tumor multiplicity compared to vehicle controls. Our findings indicate that patients taking rapamycin should avoid sun exposure, and that combining topical mTOR inhibitors and Akt inhibitors may be a viable chemoprevention option for individuals at high risk for cutaneous SCC. PMID:26801880

Inhibition of Akt Enhances the Chemopreventive Effects of Topical Rapamycin in Mouse Skin.

PubMed

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

2016-03-01

The PI3Kinase/Akt/mTOR pathway has important roles in cancer development for multiple tumor types, including UV-induced nonmelanoma skin cancer. Immunosuppressed populations are at increased risk of aggressive cutaneous squamous cell carcinoma (SCC). Individuals who are treated with rapamycin (sirolimus, a classical mTOR inhibitor) have significantly decreased rates of developing new cutaneous SCCs compared with those that receive traditional immunosuppression. However, systemic rapamycin use can lead to significant adverse events. Here, we explored the use of topical rapamycin as a chemopreventive agent in the context of solar-simulated light (SSL)-induced skin carcinogenesis. In SKH-1 mice, topical rapamycin treatment decreased tumor yields when applied after completion of 15 weeks of SSL exposure compared with controls. However, applying rapamycin during SSL exposure for 15 weeks, and continuing for 10 weeks after UV treatment, increased tumor yields. We also examined whether a combinatorial approach might result in more significant tumor suppression by rapamycin. We validated that rapamycin causes increased Akt (S473) phosphorylation in the epidermis after SSL, and show for the first time that this dysregulation can be inhibited in vivo by a selective PDK1/Akt inhibitor, PHT-427. Combining rapamycin with PHT-427 on tumor prone skin additively caused a significant reduction of tumor multiplicity compared with vehicle controls. Our findings indicate that patients taking rapamycin should avoid sun exposure, and that combining topical mTOR inhibitors and Akt inhibitors may be a viable chemoprevention option for individuals at high risk for cutaneous SCC. ©2016 American Association for Cancer Research.

Transcriptional activation of peroxisome proliferator-activated receptor-{gamma} requires activation of both protein kinase A and Akt during adipocyte differentiation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kim, Sang-pil; Ha, Jung Min; Yun, Sung Ji

2010-08-13

Research highlights: {yields} Elevated cAMP activates both PKA and Epac. {yields} PKA activates CREB transcriptional factor and Epac activates PI3K/Akt pathway via Rap1. {yields} Akt modulates PPAR-{gamma} transcriptional activity in concert with CREB. -- Abstract: Peroxisome proliferator-activated receptor-{gamma} (PPAR-{gamma}) is required for the conversion of pre-adipocytes. However, the mechanism underlying activation of PPAR-{gamma} is unclear. Here we showed that cAMP-induced activation of protein kinase A (PKA) and Akt is essential for the transcriptional activation of PPAR-{gamma}. Hormonal induction of adipogenesis was blocked by a phosphatidylinositol 3-kinase (PI3K) inhibitor (LY294002), by a protein kinase A (PKA) inhibitor (H89), and by amore » Rap1 inhibitor (GGTI-298). Transcriptional activity of PPAR-{gamma} was markedly enhanced by 3-isobutyl-1-methylxanthine (IBMX), but not insulin and dexamethasone. In addition, IBMX-induced PPAR-{gamma} transcriptional activity was blocked by PI3K/Akt, PKA, or Rap1 inhibitors. 8-(4-Chlorophenylthio)-2'-O-methyl-cAMP (8-pCPT-2'-O-Me-cAMP) which is a specific agonist for exchanger protein directly activated by cAMP (Epac) significantly induced the activation of Akt. Furthermore, knock-down of Akt1 markedly attenuated PPAR-{gamma} transcriptional activity. These results indicate that both PKA and Akt signaling pathways are required for transcriptional activation of PPAR-{gamma}, suggesting post-translational activation of PPAR-{gamma} might be critical step for adipogenic gene expression.« less

Identification of Bombyx mori Akt and its phosphorylation by bombyxin stimulation.

PubMed

Nagata, Shinji; Hakuno, Fumihiko; Takahashi, Shin-Ichiro; Nagasawa, Hiromichi

2008-11-01

Akt, a Ser/Thr protein kinase involved in insulin signaling, was identified from the silkworm, Bombyx mori. Bombyx Akt (BomAkt) is composed of 493 amino acid residues including regions conserved in other Akts: the Pleckstrin homology and kinase domains, and a dual phosphorylation site essential for kinase activation. Commercially available antibodies against mammalian Akt and phosphoAkt were able to recognize BomAkt and phosphorylated BomAkt in HEK293 cells expressing BomAkt. Additionally, phosphorylation of BomAkt was detectable in insulin-like growth factor (IGF)-I stimulated-HEK293 cells expressing BomAkt. RT-PCR and immunoblotting analyses revealed that BomAkt is expressed ubiquitously in Bombyx larvae. Phosphorylation of BomAkt was observed both in the isolated fat body after exposure to bombyxin, an endogenous insulin-like peptide, and in the larval fat body by refeeding a diet after starvation. These results suggest that dietary intake may activate the insulin signaling pathway, including Akt, through bombyxin action in B. mori.

Delayed administration of parecoxib, a specific COX-2 inhibitor, attenuated postischemic neuronal apoptosis by phosphorylation Akt and GSK-3β.

PubMed

Ye, Zhi; Wang, Na; Xia, Pingping; Wang, E; Yuan, Yajing; Guo, Qulian

2012-02-01

Parecoxib is a recently described novel COX-2 inhibitor whose functional significance and neuroprotective mechanisms remain elusive. Therefore, in this study, we aimed to investigate whether delayed administration of parecoxib inhibited mitochondria-mediated neuronal apoptosis induced by ischemic reperfusion injury via phosphorylating Akt and its downstream target protein, glycogen synthase kinase 3β (GSK-3β). Adult male Sprague-Dawley rats were administered parecoxib (10 or 30 mg kg(-1), IP) or isotonic saline twice a day starting 24 h after middle cerebral artery occlusion (MCAO) for three consecutive days. Cerebral infarct volume, apoptotic neuron, caspase-3 immunoreactivity and the protein expression of p-Akt, p-GSK-3β and Cytochrome C in cerebral ischemic cortex were evaluated at 96 h after reperfusion. Parecoxib significantly diminished infarct volume and attenuated neuron apoptosis in a dose-independent manner, compared with MCAO group alone. Increased p-Akt and p-GSK-3β was observed in the ischemic penumbra of parecoxib group after stroke. Moreover, parecoxib also reduced the release of Cytochrome C from mitochondrial into cytosol and attenuated the caspase-3 immunoreactivity in the penumbra. Taken together, these results suggested that parecoxib ameliorated postischemic mitochondria-mediated neuronal apoptosis induced by focal cerebral ischemia in rats and this neuroprotective potential is involved in phosphorylation of Akt and GSK-3β.

Hepatic Proteomic Analysis Revealed Altered Metabolic Pathways in Insulin Resistant Akt1+/-/Akt2-/-Mice

PubMed Central

Pedersen, Brian A; Wang, Weiwen; Taylor, Jared F; Khattab, Omar S; Chen, Yu-Han; Edwards, Robert A; Yazdi, Puya G; Wang, Ping H

2015-01-01

Objective The aim of this study was to identify liver proteome changes in a mouse model of severe insulin resistance and markedly decreased leptin levels. Methods Two-dimensional differential gel electrophoresis was utilized to identify liver proteome changes in AKT1+/-/AKT2-/- mice. Proteins with altered levels were identified with tandem mass spectrometry. Ingenuity Pathway analysis was performed for the interpretation of the biological significance of the observed proteomic changes. Results 11 proteins were identified from 2 biological replicates to be differentially expressed by a ratio of at least 1.3 between age-matched insulin resistant (Akt1+/-/Akt2-/-) and wild type mice. Albumin and mitochondrial ornithine aminotransferase were detected from multiple spots, which suggest post-translational modifications. Enzymes of the urea cycle were common members of top regulated pathways. Conclusion Our results help to unveil the regulation of the liver proteome underlying altered metabolism in an animal model of severe insulin resistance. PMID:26455965

Imatinib-mediated inactivation of Akt regulates ABCG2 function in head and neck squamous cell carcinoma.

PubMed

Chu, Theresa S; Chen, Jocelyn S; Lopez, Jay Patrick; Pardo, Francisco S; Aguilera, Joseph; Ongkeko, Weg M

2008-09-01

To investigate whether the mechanism for the reversal of ABCG2 (also known as ABCP, MXR, and BCRP)-mediated drug resistance by imatinib mesylate (Gleevec, STI571; Novartis Pharmaceuticals Corp, East Hanover, New Jersey) is caused by the downregulation of Akt kinase. The adenosine triphosphatase-binding cassette protein ABCG2 has been suggested to be involved in the resistance against various anticancer drugs. Recent studies show that imatinib reverses ABCG2-mediated drug resistance to topotecan hydrochloride and SN-38. In addition, we have previously reported that imatinib downregulates Akt kinase activity, which is elevated in head and neck squamous cell carcinoma. Flow cytometric analysis was used to determine the levels of drug or dye extrusion from the cells. We used Akt kinase inhibitors, transfection with short interfering RNA (siRNA) Akt, and the tyrosine kinase inhibitor imatinib to show that these treatments decreased the side population by 50% to 70% in Hoechst 33342 extrusion studies. Doxorubicin hydrochloride extrusion experiments also demonstrated 20% to 26% decrease in doxorubicin efflux on cells treated with imatinib, 1L6-hydroxymethyl-chiro-inositol 2-(R)-2-O-methyl-3-O-octadecylcarbonate, and transfection with siRNA Akt. With Western blot and immunofluorescence experiments, our data suggest that ABCG2 translocation is the mechanism by which imatinib and Akt regulate drug resistance. Clonogenic survival assays performed with imatinib-treated cells resulted in a dose-dependent decrease in cell survival compared with the control population. Our findings demonstrate that imatinib confers greater doxorubicin retention, presumably via inhibition of Akt, which regulates ABCG2 function.

Class I PI3-kinase or Akt inhibition do not impair axonal polarization, but slow down axonal elongation.

PubMed

Diez, Héctor; Benitez, Ma José; Fernandez, Silvia; Torres-Aleman, Ignacio; Garrido, Juan José; Wandosell, Francisco

2016-11-01

PI3K proteins family have multiple and essential functions in most cellular events. This family is composed of class I, class II and class III PI3Ks, which upstream and downstream elements are not completely elucidated. Previous studies using the broad PI3K inhibitor, LY294002 allowed to propose that PI3 kinase>Akt pathway is a key element in the determination of axonal polarity in hippocampal neurons. Recently, new inhibitors with a higher selectivity for class I PI3K have been characterized. In the present study we have examined this widely accepted theory using a new class I PI3K inhibitor (GDC-0941), as well as Akt inhibitors, and PTEN phosphatase constructs to reduce PIP3 levels. Our present data show that both, class I PI3K inhibitor and Akt inhibitor did not alter axon specification in hippocampal neurons, but greatly reduced axon length. However, in the same experiments LY294002 effectively impeded axonal polarization, as previously reported. Our biochemical data show that both, class I PI3K and Akt inhibitors, effectively block downstream elements from Akt to S6K1 activity. Both inhibitors are stable in culture medium along the time period analysed, maintaining the inhibition better than LY294002. Besides, we found evidence that LY294002 directly inhibits mTORC1. However, further analysis using an mTORC1 inhibitor showed no change in neuron polarity. Same result was obtained using a general class III PI3K inhibitor. Interestingly, we found that either, wild-type PTEN, or a phosphatase-dead form of PTEN, disrupted axonal polarization, strongly suggesting that the role of PTEN in axonal polarity can be independent of PIP3. Copyright © 2016 Elsevier B.V. All rights reserved.

[Signaling pathways mTOR and AKT in epilepsy].

PubMed

Romero-Leguizamon, C R; Ramirez-Latorre, J A; Mora-Munoz, L; Guerrero-Naranjo, A

2016-07-01

The signaling pathway AKT/mTOR is a central axis in regulating cellular processes, particularly in neurological diseases. In the case of epilepsy, it has been observed alteration in the pathophysiological process of the same. However, they have not described all the mechanisms of these signaling pathways that could open the opportunity to new research and therapeutic strategies. To review existing partnerships between intracellular signaling pathways AKT and mTOR in the pathophysiology of epilepsy. Epilepsy is a disease with a high epidemiological impact globally, so it is widely investigated regarding the pathophysiological components thereof. In that search they have been involved different intracellular signaling pathways in neurons, as determinants epileptogenic. Advances in this field have even allowed the successful implementation of new therapeutic strategies and to open the way to new research in the field. Improving knowledge about the pathophysiological role of the signaling pathway mTOR/AKT in epilepsy can raise new investigations regarding therapeutic alternatives. The use of mTOR inhibitors, has emerged in recent years as effective in treating this disease entity alternative however is clear the necessity of continue the research for new drug therapies.

Cancer Associated E17K Mutation Causes Rapid Conformational Drift in AKT1 Pleckstrin Homology (PH) Domain

PubMed Central

Kumar, Ambuj; Purohit, Rituraj

2013-01-01

Background AKT1 (v-akt murine thymoma viral oncogene homologue 1) kinase is one of the most frequently activated proliferated and survival pathway of cancer. Recently it has been shown that E17K mutation in the Pleckstrin Homology (PH) domain of AKT1 protein leads to cancer by amplifying the phosphorylation and membrane localization of protein. The mutant has shown resistance to AKT1/2 inhibitor VIII drug molecule. In this study we have demonstrated the detailed structural and molecular consequences associated with the activity regulation of mutant protein. Methods The docking score exhibited significant loss in the interaction affinity to AKT1/2 inhibitor VIII drug molecule. Furthermore, the molecular dynamics simulation studies presented an evidence of rapid conformational drift observed in mutant structure. Results There was no stability loss in mutant as compared to native structure and the major cation–π interactions were also shown to be retained. Moreover, the active residues involved in membrane localization of protein exhibited significant rise in NHbonds formation in mutant. The rise in NHbond formation in active residues accounts for the 4-fold increase in the membrane localization potential of protein. Conclusion The overall result suggested that, although the mutation did not induce any stability loss in structure, the associated pathological consequences might have occurred due to the rapid conformational drifts observed in the mutant AKT1 PH domain. General Significance The methodology implemented and the results obtained in this work will facilitate in determining the core molecular mechanisms of cancer-associated mutations and in designing their potential drug inhibitors. PMID:23741320

TRPC3- and ETB receptor-mediated PI3K/AKT activation induces vasogenic edema formation following status epilepticus.

PubMed

Kim, Ji-Eun; Kang, Tae-Cheon

2017-10-01

Status epilepticus (SE, a prolonged seizure activity) is a high risk factor of developing vasogenic edema, which leads to secondary complications following SE. In the present study, we investigated whether transient receptor potential canonical channel-3 (TRPC3) may link vascular endothelial growth factor (VEGF) pathway to NFκB/ET B receptor axis in the rat piriform cortex during vasogenic edema formation. Following SE, TRPC3 and ET B receptor independently activated phosphatidylinositol 3 kinase (PI3K)/AKT/eNOS signaling pathway. SN50 (a NFκB inhibitor) attenuated the up-regulations of eNOS, TRPC3 and ET B receptor expressions following SE, accompanied by reductions in PI3K/AKT phosphorylations. Inhibition of SE-induced VEGF over-expression by leptomycin B also abrogated PI3K and AKT phosphorylations, but not TRPC3 expression. Wortmannin (a PI3K inhibitor) and 3CAI (an AKT inhibitor) effectively inhibited up-regulation of eNOS expressions and vasogenic edema lesion following SE. These findings indicate that PI3K/AKT may be common down-stream molecules for TRPC3- and ET B receptor signaling pathways during vasogenic edema formation. In addition, the present data demonstrate for the first time that TRPC3 may integrate VEGF- and NFκB-mediated vasogenic edema formation following SE. Thus, we suggest that PI3K/AKT signaling pathway may be one of considerable therapeutic targets for vasogenic edema. Copyright © 2017 Elsevier B.V. All rights reserved.

Intrinsic BET inhibitor resistance in SPOP-mutated prostate cancer is mediated by BET protein stabilization and AKT-mTORC1 activation.

PubMed

Zhang, Pingzhao; Wang, Dejie; Zhao, Yu; Ren, Shancheng; Gao, Kun; Ye, Zhenqing; Wang, Shangqian; Pan, Chun-Wu; Zhu, Yasheng; Yan, Yuqian; Yang, Yinhui; Wu, Di; He, Yundong; Zhang, Jun; Lu, Daru; Liu, Xiuping; Yu, Long; Zhao, Shimin; Li, Yao; Lin, Dong; Wang, Yuzhuo; Wang, Liguo; Chen, Yu; Sun, Yinghao; Wang, Chenji; Huang, Haojie

2017-09-01

Bromodomain and extraterminal domain (BET) protein inhibitors are emerging as promising anticancer therapies. The gene encoding the E3 ubiquitin ligase substrate-binding adaptor speckle-type POZ protein (SPOP) is the most frequently mutated in primary prostate cancer. Here we demonstrate that wild-type SPOP binds to and induces ubiquitination and proteasomal degradation of BET proteins (BRD2, BRD3 and BRD4) by recognizing a degron motif common among them. In contrast, prostate cancer-associated SPOP mutants show impaired binding to BET proteins, resulting in decreased proteasomal degradation and accumulation of these proteins in prostate cancer cell lines and patient specimens and causing resistance to BET inhibitors. Transcriptome and BRD4 cistrome analyses reveal enhanced expression of the GTPase RAC1 and cholesterol-biosynthesis-associated genes together with activation of AKT-mTORC1 signaling as a consequence of BRD4 stabilization. Our data show that resistance to BET inhibitors in SPOP-mutant prostate cancer can be overcome by combination with AKT inhibitors and further support the evaluation of SPOP mutations as biomarkers to guide BET-inhibitor-oriented therapy in patients with prostate cancer.

Acute Alcohol Modulates Cardiac Function as PI3K/Akt Regulates Oxidative Stress

PubMed Central

Umoh, Nsini A.; Walker, Robin K.; Al-Rubaiee, Mustafa; Jeffress, Miara A.; Haddad, Georges E.

2015-01-01

Background Clinical manifestations of alcohol abuse on the cardiac muscle include defective contractility with the development of heart failure. Interestingly, low alcohol consumption has been associated with reduced risk of cardiovascular disease. Although several hypotheses have been postulated for alcoholic cardiomyopathy and for the low-dose beneficial cardiovascular effects, the precise mechanisms and mediators remain largely undefined. We hypothesize that modulation of oxidative stress by PI3K/Akt plays a key role in the cardiac functional outcome to acute alcohol exposure. Methods Thus, acutely exposed rat cardiac tissue and cardiocytes to low (LA: 5 mM), moderate (MA: 25 mM), and high (HA: 100 mM) alcohol were assessed for markers of oxidative stress in the presence and absence of PI3K/Akt activators (IGF-1 0.1 μM or constitutively active PI3K: Ad.BD110 transfection) or inhibitor (LY294002 1 μMor Akt-negative construct Ad.Akt(K179M) transfection). Results Acute LA reduced Akt, superoxide dismutase (SOD-3) and NFκB, ERK1, and p38 MAPK gene expression. Acute HA only increased that of SOD-3 and NFκB. These effects were generally inhibited by Ad.Akt(K179M) and enhanced with Ad.BD110 transfection. In parallel, LA reduced but HA enhanced Akt activity, which was reversed by IGF-1 and inhibited by Ad.Akt(K179M), respectively. Also, LA reduced caspase 3/7 activity and oxidative stress, while HA increased both. The former was blocked, while the latter effect was enhanced by Ad.Akt(K179M). The reverse was true with PI3K/Akt activation. This translated into reduced viability with HA, with no effect with LA. On the functional level, acute LA improved cardiac output and ejection fraction, mainly through increased stroke volume. This was accompanied with enhanced end-systolic pressure–volume relationship and preload recruitable stroke work. Opposite effect was recorded for HA. LA and HA in vivo functional effects were alleviated by LY and enhanced by IGF-1 treatment

Cyclic mechanical strain maintains Nanog expression through PI3K/Akt signaling in mouse embryonic stem cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Horiuchi, Rie; Akimoto, Takayuki, E-mail: akimoto@m.u-tokyo.ac.jp; Institute for Biomedical Engineering, Consolidated Research Institute for Advanced Science and Medical Care, Waseda University, 513 Waseda-tsurumaki, Shinjuku, Tokyo 162-0041

2012-08-15

Mechanical strain has been reported to affect the proliferation/differentiation of many cell types; however, the effects of mechanotransduction on self-renewal as well as pluripotency of embryonic stem (ES) cells remains unknown. To investigate the effects of mechanical strain on mouse ES cell fate, we examined the expression of Nanog, which is an essential regulator of self-renewal and pluripotency as well as Nanog-associated intracellular signaling during uniaxial cyclic mechanical strain. The mouse ES cell line, CCE was plated onto elastic membranes, and we applied 10% strain at 0.17 Hz. The expression of Nanog was reduced during ES cell differentiation in responsemore » to the withdrawal of leukemia inhibitory factor (LIF); however, two days of cyclic mechanical strain attenuated this reduction of Nanog expression. On the other hand, the cyclic mechanical strain promoted PI3K-Akt signaling, which is reported as an upstream of Nanog transcription. The cyclic mechanical strain-induced Akt phosphorylation was blunted by the PI3K inhibitor wortmannin. Furthermore, cytochalasin D, an inhibitor of actin polymerization, also inhibited the mechanical strain-induced increase in phospho-Akt. These findings imply that mechanical force plays a role in regulating Nanog expression in ES cells through the actin cytoskeleton-PI3K-Akt signaling. -- Highlights: Black-Right-Pointing-Pointer The expression of Nanog, which is an essential regulator of 'stemness' was reduced during embryonic stem (ES) cell differentiation. Black-Right-Pointing-Pointer Cyclic mechanical strain attenuated the reduction of Nanog expression. Black-Right-Pointing-Pointer Cyclic mechanical strain promoted PI3K-Akt signaling and mechanical strain-induced Akt phosphorylation was blunted by the PI3K inhibitor and an inhibitor of actin polymerization.« less

Targeting the PI3K/Akt pathway in murine MDS/MPN driven by hyperactive Ras

PubMed Central

Akutagawa, Jon; Huang, Tannie Q.; Epstein, Inbal; Chang, Tiffany; Quirindongo-Crespo, Maricel; Cottonham, Charisa L.; Dail, Monique; Slusher, Barbara S.; Friedman, Lori S.; Sampath, Deepak; Braun, Benjamin S.

2016-01-01

Chronic and juvenile myelomonocytic leukemias (CMML and JMML) are myelodysplastic/myeloproliferative neoplasia (MDS/MPN) overlap syndromes that respond poorly to conventional treatments. Aberrant Ras activation due to NRAS, KRAS, PTPN11, CBL, and NF1 mutations is common in CMML and JMML. However, no mechanism-based treatments currently exist for cancers with any of these mutations. An alternative therapeutic strategy involves targeting Ras-regulated effector pathways that are aberrantly activated in CMML and JMML, which include the Raf/MEK/ERK and phosphoinositide-3´-OH kinase (PI3K)/Akt cascades. Mx1-Cre, KrasD12 and Mx1-Cre, Nf1flox/− mice accurately model many aspects of CMML and JMML. Treating Mx1-Cre, KrasD12 mice with GDC-0941 (also referred to as pictilisib), an orally bioavailable inhibitor of class I PI3K isoforms, reduced leukocytosis, anemia, and splenomegaly while extending survival. However, GDC-0941 treatment attenuated activation of both PI3K/Akt and Raf/MEK/ERK pathways in primary hematopoietic cells, suggesting it could be acting through suppression of Raf/MEK/ERK signals. To interrogate the importance of the PI3K/Akt pathway specifically, we treated mice with the allosteric Akt inhibitor MK-2206. This compound had no effect on Raf/MEK/ERK signaling, yet it also induced robust hematologic responses in Kras and Nf1 mice with MPN. These data support investigating PI3K/Akt pathway inhibitors as a therapeutic strategy in JMML and CMML patients. PMID:26965285

Functional characterization of lysosomal interaction of Akt with VRK2.

PubMed

Hirata, Noriyuki; Suizu, Futoshi; Matsuda-Lennikov, Mami; Tanaka, Tsutomu; Edamura, Tatsuma; Ishigaki, Satoko; Donia, Thoria; Lithanatudom, Pathrapol; Obuse, Chikashi; Iwanaga, Toshihiko; Noguchi, Masayuki

2018-06-05

Serine-threonine kinase Akt (also known as PKB, protein kinase B), a core intracellular mediator of cell survival, is involved in various human cancers and has been suggested to play an important role in the regulation of autophagy in mammalian cells. Nonetheless, the physiological function of Akt in the lysosomes is currently unknown. We have reported previously that PtdIns(3)P-dependent lysosomal accumulation of the Akt-Phafin2 complex is a critical step for autophagy induction. Here, to characterize the molecular function of activated Akt in the lysosomes in the process of autophagy, we searched for the molecules that interact with the Akt complex at the lysosomes after induction of autophagy. By time-of-flight-mass spectrometry (TOF/MS) analysis, kinases of the VRK family, a unique serine-threonine family of kinases in the human kinome, were identified. VRK2 interacts with Akt1 and Akt2, but not with Akt3; the C terminus of Akt and the N terminus of VRK2 facilitate the interaction of Akt and VRK2 in mammalian cells. The kinase-dead form of VRK2A (KD VRK2A) failed to interact with Akt in coimmunoprecipitation assays. Bimolecular fluorescence complementation (BiFC) experiments showed that, in the lysosomes, Akt interacted with VRK2A but not with VRK2B or KD VRK2A. Immunofluorescent assays revealed that VRK2 and phosphorylated Akt accumulated in the lysosomes after autophagy induction. WT VRK2A, but not KD VRK2A or VRK2B, facilitated accumulation of phosphorylated Akt in the lysosomes. Downregulation of VRK2 abrogated the lysosomal accumulation of phosphorylated Akt and impaired nuclear localization of TFEB; these events coincided to inhibition of autophagy induction. The VRK2-Akt complex is required for control of lysosomal size, acidification, bacterial degradation, and for viral replication. Moreover, lysosomal VRK2-Akt controls cellular proliferation and mitochondrial outer-membrane stabilization. Given the roles of autophagy in the pathogenesis of human

Exosomes promote cetuximab resistance via the PTEN/Akt pathway in colon cancer cells.

PubMed

Zhang, S; Zhang, Y; Qu, J; Che, X; Fan, Y; Hou, K; Guo, T; Deng, G; Song, N; Li, C; Wan, X; Qu, X; Liu, Y

2017-11-13

Cetuximab is widely used in patients with metastatic colon cancer expressing wildtype KRAS. However, acquired drug resistance limits its clinical efficacy. Exosomes are nanosized vesicles secreted by various cell types. Tumor cell-derived exosomes participate in many biological processes, including tumor invasion, metastasis, and drug resistance. In this study, exosomes derived from cetuximab-resistant RKO colon cancer cells induced cetuximab resistance in cetuximab-sensitive Caco-2 cells. Meanwhile, exosomes from RKO and Caco-2 cells showed different levels of phosphatase and tensin homolog (PTEN) and phosphor-Akt. Furthermore, reduced PTEN and increased phosphorylated Akt levels were found in Caco-2 cells after exposure to RKO cell-derived exosomes. Moreover, an Akt inhibitor prevented RKO cell-derived exosome-induced drug resistance in Caco-2 cells. These findings provide novel evidence that exosomes derived from cetuximab-resistant cells could induce cetuximab resistance in cetuximab-sensitive cells, by downregulating PTEN and increasing phosphorylated Akt levels.

PPARα induced NOS1 phosphorylation via PI3K/Akt in guinea pig antral mucous cells: NO-enhancement in Ca(2+)-regulated exocytosis.

PubMed

Tanaka, Saori; Hosogi, Shigekuni; Sawabe, Yukinori; Shimamoto, Chikao; Matsumura, Hitoshi; Inui, Toshio; Marunaka, Yoshinori; Nakahari, Takashi

2016-01-01

A PPARα (peroxisome proliferation activation receptor α) agonist (GW7647) activates nitric oxide synthase 1 (NOS1) to produce NO leading to cGMP accumulation in antral mucous cells. In this study, we examined how PPARα activates NOS1. The NO production stimulated by GW7647 was suppressed by inhibitors of PI3K (wortmannin) and Akt (AKT 1/2 Kinase Inhibitor, AKT-inh), although it was also suppressed by the inhibitors of PPARα (GW6471) and NOS1 (N-PLA). GW7647 enhanced the ACh (acetylcholine)-stimulated exocytosis (Ca(2+)-regulated exocytosis) mediated via NO, which was abolished by GW6471, N-PLA, wortmannin, and AKT-inh. The Western blotting revealed that GW7647 phosphorylates NOS1 via phosphorylation of PI3K/Akt in antral mucous cells. The immunofluorescence examinations demonstrated that PPARα existing with NOS1 co-localizes with PI3K and Akt in the cytoplasm of antral mucous cells. ACh alone and AACOCF3, an analogue of arachidonic acid (AA), induced the NOS1 phosphorylation via PI3K/Akt to produce NO, which was inhibited by GW6471. Since AA is a natural ligand for PPARα, ACh stimulates PPARα probably via AA. In conclusion, PPARα activates NOS1 via PI3K/Akt phosphorylation to produce NO in antral mucous cells during ACh stimulation.

LPS Increases 5-LO Expression on Monocytes via an Activation of Akt-Sp1/NF-κB Pathways.

PubMed

Lee, Seung Jin; Seo, Kyo Won; Kim, Chi Dae

2015-05-01

5-Lipoxygenase (5-LO) plays a pivotal role in the progression of atherosclerosis. Therefore, this study investigated the molecular mechanisms involved in 5-LO expression on monocytes induced by LPS. Stimulation of THP-1 monocytes with LPS (0~3 µg/ml) increased 5-LO promoter activity and 5-LO protein expression in a concentration-dependent manner. LPS-induced 5-LO expression was blocked by pharmacological inhibition of the Akt pathway, but not by inhibitors of MAPK pathways including the ERK, JNK, and p38 MAPK pathways. In line with these results, LPS increased the phosphorylation of Akt, suggesting a role for the Akt pathway in LPS-induced 5-LO expression. In a promoter activity assay conducted to identify transcription factors, both Sp1 and NF-κB were found to play central roles in 5-LO expression in LPS-treated monocytes. The LPS-enhanced activities of Sp1 and NF-κB were attenuated by an Akt inhibitor. Moreover, the LPS-enhanced phosphorylation of Akt was significantly attenuated in cells pretreated with an anti-TLR4 antibody. Taken together, 5-LO expression in LPS-stimulated monocytes is regulated at the transcriptional level via TLR4/Akt-mediated activations of Sp1 and NF-κB pathways in monocytes.

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wang Lei; Sasai, Ken; Akagi, Tsuyoshi

2008-08-29

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

Differential roles of ERRFI1 in EGFR and AKT pathway regulation affect cancer proliferation.

PubMed

Cairns, Junmei; Fridley, Brooke L; Jenkins, Gregory D; Zhuang, Yongxian; Yu, Jia; Wang, Liewei

2018-03-01

AKT signaling is modulated by a complex network of regulatory proteins and is commonly deregulated in cancer. Here, we present a dual mechanism of AKT regulation by the ERBB receptor feedback inhibitor 1 (ERRFI1). We show that in cells expressing high levels of EGFR, ERRF1 inhibits growth and enhances responses to chemotherapy. This is mediated in part through the negative regulation of AKT signaling by direct ERRFI1-dependent inhibition of EGFR In cells expressing low levels of EGFR, ERRFI1 positively modulates AKT signaling by interfering with the interaction of the inactivating phosphatase PHLPP with AKT, thereby promoting cell growth and chemotherapy desensitization. These observations broaden our understanding of chemotherapy response and have important implications for the selection of targeted therapies in a cell context-dependent manner. EGFR inhibition can only sensitize EGFR-high cells for chemotherapy, while AKT inhibition increases chemosensitivity in EGFR-low cells. By understanding these mechanisms, we can take advantage of the cellular context to individualize antineoplastic therapy. Finally, our data also suggest targeting of EFFRI1 in EGFR-low cancer as a promising therapeutic approach. © 2018 The Authors. Published under the terms of the CC BY NC ND 4.0 license.

FLT3-ITD induces expression of Pim kinases through STAT5 to confer resistance to the PI3K/Akt pathway inhibitors on leukemic cells by enhancing the mTORC1/Mcl-1 pathway.

PubMed

Okada, Keigo; Nogami, Ayako; Ishida, Shinya; Akiyama, Hiroki; Chen, Cheng; Umezawa, Yoshihiro; Miura, Osamu

2018-02-06

FLT3-ITD is the most frequent tyrosine kinase mutation in acute myeloid leukemia (AML) associated with poor prognosis. We previously reported that activation of STAT5 confers resistance to PI3K/Akt inhibitors on the FLT3-ITD-positive AML cell line MV4-11 and 32D cells driven by FLT3-ITD (32D/ITD) but not by FLT3 mutated in the tyrosine kinase domain (32D/TKD). Here, we report the involvement of Pim kinases expressed through STAT5 activation in acquisition of this resistance. The specific pan-Pim kinase inhibitor AZD1208 as well as PIM447 in combination with the PI3K inhibitor GDC-0941 or the Akt inhibitor MK-2206 cooperatively downregulated the mTORC1/4EBP1 pathway, formation of the eIF4E/eIF4G complex, and Mcl-1 expression leading to activation of Bak and Bax to induce caspase-dependent apoptosis synergistically in these cells. These cooperative effects were enhanced or inhibited by knock down of mTOR or expression of its activated mutant, respectively. Overexpression of Mcl-1 conferred the resistance on 32D/ITD cells to combined inhibition of the PI3K/Akt pathway and Pim kinases, while the Mcl-1-specific BH3 mimetic A-1210477 conquered the resistance of MV4-11 cells to GDC-0941. Furthermore, overexpression of Pim-1 in 32D/TKD enhanced the mTORC1/Mcl-1 pathway and partially protected it from the PI3K/Akt inhibitors or the FLT3 inhibitor gilteritinib to confer the resistance to PI3K/Akt inhibitors. Finally, AZD1208 and GDC-0941 cooperatively inhibited the mTORC1/Mcl-1 pathway and reduced viable cell numbers of primary AML cells from some FLT3-ITD positive cases. Thus, Pim kinases may protect the mTORC1/4EBP1/Mcl-1 pathway to confer the resistance to the PI3K/Akt inhibitors on FLT3-ITD cells and represent promising therapeutic targets.

Akt regulates the subcellular localization of the Rab27a-binding protein JFC1 by phosphorylation.

PubMed

Johnson, Jennifer L; Pacquelet, Sandrine; Lane, William S; Eam, Boreth; Catz, Sergio D

2005-08-01

Here, we show that the Rab27a-binding protein JFC1/Slp1 (synaptotagmin-like protein) is regulated by Akt-mediated phosphorylation. Using the phosphatase and tensin homolog-null LNCaP cells and the phosphatidylinositol 3-kinase inhibitor LY294002, we show that the phosphorylation of endogenous JFC1 is dependent on the phosphatidylinositol 3-kinase/Akt pathway. JFC1 was phosphorylated in cells expressing a constitutively active Akt, confirming that it is an Akt substrate in vivo. Direct phosphorylation of JFC1 by Akt was confirmed in vitro. Using microcapillary high-performance liquid chromatography tandem mass spectrometry, we identified five Akt-phosphorylation sites in JFC1. By mutagenesis analysis and subsequent immunoprecipitation (IP), we established that Akt phosphorylates JFC1 at serine 241. JFC1 and Rab27a colocalize in the proximity of the plasma membrane in LNCaP cells. The interaction was confirmed by IP analysis and was abolished by the point mutation W83S in JFC1. Phosphorylation did not alter the ability of JFC1 to bind to Rab27a. Instead, phosphorylation by Akt dramatically decreased when JFC1 was bound to Rab27a. Finally, we show that as a consequence of in vivo phosphorylation, JFC1 dissociates from the membrane, promoting JFC1 redistribution to the cytosol. Our results suggest that Akt regulates JFC1/Slp1 function by phosphorylation and may have implications on Rab27a-containing vesicle secretion.

Photoactivation of Akt1/GSK3β Isoform-Specific Signaling Axis Promotes Pancreatic β-Cell Regeneration.

PubMed

Huang, Lei; Jiang, Xiaoxiao; Gong, Longlong; Xing, Da

2015-08-01

Promotion of insulin-secreting β-cell regeneration in patients with diabetes is a promising approach for diabetes therapy, which can contribute to rescue the uncontrolled hyperglycemia. Low-power laser irradiation (LPLI) has been demonstrated to regulate multiple physiological processes both in vitro and in vivo through activation of various signaling pathways. In the present study, we showed that LPLI promoted β-cell replication and cell cycle progression through activation of Akt1/GSK3β isoform-specific signaling axis. Inhibition of PI3-K/Akt or GSK3 with specific inhibitors dramatically reduced or increased LPLI-induced β-cell replication, revealing Akt/GSK3 signaling axis was involved in β-cell replication and survival upon LPLI treatment. Furthermore, the results of shRNA-mediated knock down of Akt/GSK3 isoforms revealed that Akt1/GSK3β isoform-specific signaling axis regulated β-cell replication and survival in response to LPLI, but not Akt2/GSK3α. The mechanism by which LPLI promoted β-cell replication through Akt1/GSK3β signaling axis involved activation of β-catenin and down-regulation of p21. Taken together, these observations suggest that Akt1/GSK3β isoform signaling axis play a key role in β-cell replication and survival induced by LPLI. Moreover, our findings suggest that activation of Akt1/GSK3β isoform signaling axis by LPLI may provide guidance in practical applications for β-cell regenerative therapies. © 2015 Wiley Periodicals, Inc.

AKT signaling displays multifaceted functions in neural crest development.

PubMed

Sittewelle, Méghane; Monsoro-Burq, Anne H

2018-05-31

AKT signaling is an essential intracellular pathway controlling cell homeostasis, cell proliferation and survival, as well as cell migration and differentiation in adults. Alterations impacting the AKT pathway are involved in many pathological conditions in human disease. Similarly, during development, multiple transmembrane molecules, such as FGF receptors, PDGF receptors or integrins, activate AKT to control embryonic cell proliferation, migration, differentiation, and also cell fate decisions. While many studies in mouse embryos have clearly implicated AKT signaling in the differentiation of several neural crest derivatives, information on AKT functions during the earliest steps of neural crest development had remained relatively scarce until recently. However, recent studies on known and novel regulators of AKT signaling demonstrate that this pathway plays critical roles throughout the development of neural crest progenitors. Non-mammalian models such as fish and frog embryos have been instrumental to our understanding of AKT functions in neural crest development, both in neural crest progenitors and in the neighboring tissues. This review combines current knowledge acquired from all these different vertebrate animal models to describe the various roles of AKT signaling related to neural crest development in vivo. We first describe the importance of AKT signaling in patterning the tissues involved in neural crest induction, namely the dorsal mesoderm and the ectoderm. We then focus on AKT signaling functions in neural crest migration and differentiation. Copyright © 2018 Elsevier Inc. All rights reserved.

Suppression of transforming growth factor-beta-induced apoptosis through a phosphatidylinositol 3-kinase/Akt-dependent pathway.

PubMed

Chen, R H; Su, Y H; Chuang, R L; Chang, T Y

1998-10-15

Insulin and insulin receptor substrate 1 (IRS-1) are capable of protecting liver cells from apoptosis induced by transforming growth factor-beta1 (TGF-beta). The Ras/mitogen-activated protein kinase (MAP kinase) and the phosphatidylinositol 3-kinase (PI 3-kinase)/Akt pathways are both activated upon insulin stimulation and can protect against apoptosis under certain circumstances. We investigated which of these pathways is responsible for the protective effect of insulin on TGF-beta-induced apoptosis. An activated Ras, although elicited a strong mitogenic effect, could not protect Hep3B cells from TGF-beta-induced apoptosis. Furthermore, PD98059, a selective inhibitor of MEK, did not suppress the antiapoptotic effect of insulin. In contrast, the PI 3-kinase inhibitor, LY294002, efficiently blocked the effect of insulin. Protection against TGF-beta-induced apoptosis conferred by PI 3-kinase was further verified by stable transfection of an activated PI 3-kinase. Downstream targets of PI 3-kinase involved in this protection was further investigated. An activated Akt mimicked the antiapoptotic effect of insulin, whereas a dominant-negative Akt inhibited such effect. However, rapamycin, the p70S6 kinase inhibitor, had no effect on the protectivity of insulin against TGF-beta-induced apoptosis, suggesting that the antiapoptotic target of PI 3-kinase/Akt pathway is independent or lies upstream of the p70S6 kinase. The mechanism by which PI 3-kinase/Akt pathway interferes with the apoptotic signaling of TGF-beta was explored. Activation of PI 3-kinase did not lead to a suppression of Smad hetero-oligomerization or nuclear translocation but blocked TGF-beta-induced caspase-3-like activity. In summary, the PI 3-kinase/Akt pathway, but not the Ras/MAP kinase pathway, protects against TGF-beta-induced apoptosis by inhibiting a step downstream of Smad but upstream of caspase-3.

Analysis of the activation status of Akt, NFkappaB, and Stat3 in human diffuse gliomas.

PubMed

Wang, Huamin; Wang, Hua; Zhang, Wei; Huang, Helen J; Liao, Warren S L; Fuller, Gregory N

2004-08-01

Loss of phosphatase and tensin homolog (PTEN) and amplification of the epidermal growth factor receptor (EGFR) gene contribute to the progression of gliomas. As downstream targets of the PTEN and EGFR signaling pathways, Akt, NFkappaB, and signal transducer and activator of transcription-3 (Stat3) have been shown to play important roles in the control of cell proliferation, apoptosis, and oncogenesis. We examined the activation status of Akt, NFkappaB, and Stat3 in 259 diffuse gliomas using tissue microarrays and immunohistochemistry, and evaluated their association with glioma grade. We observed significant positive correlations between the activation status of Akt and NFkappaB and glioma grade. In contrast, only focal immunoreactivity for phospho-Stat3 was observed in < 9% of high-grade gliomas. In addition, we observed a significant correlation between the activation of Akt and NFkappaB. Functional correlation between Akt activation and the activation of NFkappaB was confirmed in U251MG GBM cells in which inhibition of Akt activation either by stable expression of PTEN or by the PI3-kinase inhibitors, wortmannin and LY294002, led to a concomitant decrease in NFkappaB-binding activity. Thus, our results demonstrate that constitutive activation of Akt and NFkappaB, but not Stat3, contributes significantly to the progression of diffuse gliomas, and activation of Akt may lead to NFkappaB activation in high-grade gliomas.

Apatinib Inhibits Angiogenesis Via Suppressing Akt/GSK3β/ANG Signaling Pathway in Anaplastic Thyroid Cancer.

PubMed

Jin, Zhijian; Cheng, Xi; Feng, Haoran; Kuang, Jie; Yang, Weiping; Peng, Chenghong; Shen, Baiyong; Qiu, Weihua

2017-01-01

Anaplastic thyroid carcinoma (ATC) is one of the most lethal human malignancies, and there is no efficient method to slow its process. Apatinib, a novel tyrosine kinase inhibitor (TKI), has been confirmed for its efficacy and safety in the treatment of advanced gastric carcinoma patients. However, the effects of Apatinib in ATC are still unknown. In this study, we explored the effects and mechanisms of Apatinib on tumor growth and angiogenesis in vitro and in vitro in ATC cells. Angiogenesis antibodies array was utilized to detect the expression of angiogenesis-related genes after Apatinib treatment in ATC cells. In addition, we used Akt activator, Akt inhibitor and GSK3β inhibitor to further study the mechanism for how Apatinib suppressed angiogenesis. Apatinib treatment could suppress the growth of ATC cells in a dose- and time-dependent manner via inducing apoptosis and blocking cell cycle progression at G0/G1 phase. Moreover, Apatinib treatment decreased the expression of angiogenin (ANG) and inhibited angiogenesis of ATC cells in vitro and in vitro. We further confirmed that recombinant human ANG (rhANG) significantly abrogated Apatinib-mediated anti-angiogenic ability in ATC cells. Additionally, Apatinib treatment decreased the level of p-Akt and p-GSK3β. Moreover, the Apatinib-mediated decrease of ANG and anti-angiogenic ability were partly reversed when an Akt activator, SC79, was administered. Furthermore, the anti-angiogenic ability of Apatinib can be enhanced in the presence of Akt inhibitor, and the inhibition of GSK3β attenuated the anti-angiogenic ability of Apatinib. Our results demonstrated that Apatinib treatment inhibited tumor growth, and Apatinib-induced suppression of Akt/GSK3β/ANG signaling pathway may play an important role in the inhibition of angiogenesis in ATC, supporting a potential therapeutic approach for using Apatinib in the treatment of ATC. © 2017 The Author(s). Published by S. Karger AG, Basel.

Dietary flavonoid fisetin: A novel dual inhibitor of PI3K/Akt and mTOR for prostate cancer management

PubMed Central

Adhami, Vaqar Mustafa; Syed, Deeba; Khan, Naghma; Mukhtar, Hasan

2013-01-01

Epidemiologic and case control population based studies over the past few decades have identified diet as an important determinant of cancer risk. This evidence has kindled an interest into research on bioactive food components and has till date resulted in the identification of many compounds with cancer preventive and therapeutic potential. Among such compounds has been fisetin (3,7,3’,4’-tetrahydroxyflavone), a flavonol and a member of the flavonoid polyphenols that also include quercetin, myricetin and kaempferol. Fisetin is commonly found in many fruits and vegetables such as apples, persimmons, grapes, kiwis, strawberries, onions and cucumbers. We evaluated the effects of fisetin against melanoma and cancers of the prostate, pancreas and the lungs. Using prostate and lung adenocarcinoma cells, we observed that fisetin acts as a dual inhibitor of the PI3K/Akt and the mTOR pathways. This is a significant finding considering the fact that mTOR is phosphorylated and its activation is more frequent in tumors with overexpression of PI3K/Akt. Dual inhibitors of PI3K/Akt and mTOR signaling have been suggested as valuable agents for treating such cancers. Here, we summarize our findings on the dietary flavonoid fisetin and its effects on cancer with particular focus on prostate cancer. Our observations and findings from other laboratories suggest that fisetin could be a useful chemotherapeutic agent that could be used either alone or as an adjuvant with conventional chemotherapeutic drugs for the management of prostate and other cancers. PMID:22842629

Akt/PKB activation in gastric carcinomas correlates with clinicopathologic variables and prognosis.

PubMed

Nam, Seon Young; Lee, Hye Seung; Jung, Gyung-Ah; Choi, Jimi; Cho, Sung Jin; Kim, Min Kyu; Kim, Woo Ho; Lee, Byung Lan

2003-12-01

Akt/protein kinase B (PKB) plays an important role in cell survival. However, the role of Akt in the biology of gastric cancer has not been well studied. We sought to investigate the expression of Akt or phosphorylated Akt (pAkt) in human gastric carcinomas and to analyze the relationship between Akt or pAkt and the clinicopathologic parameters. The expressions of Akt and pAkt were evaluated immunohistochemically in 311 gastric carcinomas using the tissue array method. Akt expression was detected in 74% of the tumors and pAkt expression in 78%. pAkt was highly expressed in the early stage of pTNM (p=0.011). We also found an inverse association between pAkt and lymphatic invasion (p=0.01) or lymph node metastasis (p=0.008). pAkt expression was significantly correlated with a higher survival in patients with stage I carcinomas (p=0.0003). Interestingly, combined evaluation revealed that the group with pAkt-positive and lymph node-negative carcinomas showed a better prognosis than the other groups (p<0.0001). In addition, pAkt was shown to correlate positively with APC (p=0.002) and Smad4 (p<0.0001) expression. These findings suggest that pAkt expression may help to predict the clinical outcome of gastric cancer patients.

Identification of a RAC/AKT-like gene in Leishmania parasites as a putative therapeutic target in leishmaniasis.

PubMed

Varela-M, Rubén E; Ochoa, Rodrigo; Muskus, Carlos E; Muro, Antonio; Mollinedo, Faustino

2017-10-10

Leishmaniasis is one of the world's most neglected diseases caused by at least 20 different species of the protozoan parasite Leishmania. Although new drugs have become recently available, current therapy for leishmaniasis is still unsatisfactory. A subgroup of serine/threonine protein kinases named as related to A and C protein kinases (RAC), or protein kinase B (PKB)/AKT, has been identified in several organisms including Trypanosoma cruzi parasites. PKB/AKT plays a critical role in mammalian cell signaling promoting cell survival and is a major drug target in cancer therapy. However, the role of protozoan parasitic PKB/AKT remains to be elucidated. We have found that anti-human AKT antibodies recognized a protein of about 57 kDa in Leishmania spp. parasites. Anti-human phospho-AKT(Thr308) antibodies identified a protein in extracts from Leishmania spp. that was upregulated following parasite exposure to stressful conditions, such as nutrient deprivation or heat shock. Incubation of AKT inhibitor X with Leishmania spp. promastigotes under stressful conditions or with Leishmania-infected macrophages led to parasite cell death. We have identified and cloned a novel gene from Leishmania donovani named Ld-RAC/AKT-like gene, encoding a 510-amino acid protein of approximately 57.6 kDa that shows a 26.5% identity with mammalian AKT1. Ld-RAC/AKT-like protein contains major mammalian PKB/AKT hallmarks, including the typical pleckstrin, protein kinase and AGC kinase domains. Unlike mammalian AKT that contains key phosphorylation sites at Thr308 and Ser473 in the activation loop and hydrophobic motif, respectively, Ld-RAC/AKT-like protein has a Thr residue in both motifs. By domain sequence comparison, we classified AKT proteins from different origins in four major subcategories that included different parasites. Our data suggest that Ld-RAC/AKT-like protein represents a Leishmania orthologue of mammalian AKT involved in parasite stress response and survival, and

Hyperforin inhibits Akt1 kinase activity and promotes caspase-mediated apoptosis involving Bad and Noxa activation in human myeloid tumor cells.

PubMed

Merhi, Faten; Tang, Ruoping; Piedfer, Marion; Mathieu, Julie; Bombarda, Isabelle; Zaher, Murhaf; Kolb, Jean-Pierre; Billard, Christian; Bauvois, Brigitte

2011-01-01

The natural phloroglucinol hyperforin HF displays anti-inflammatory and anti-tumoral properties of potential pharmacological interest. Acute myeloid leukemia (AML) cells abnormally proliferate and escape apoptosis. Herein, the effects and mechanisms of purified HF on AML cell dysfunction were investigated in AML cell lines defining distinct AML subfamilies and primary AML cells cultured ex vivo. HF inhibited in a time- and concentration-dependent manner the growth of AML cell lines (U937, OCI-AML3, NB4, HL-60) by inducing apoptosis as evidenced by accumulation of sub-G1 population, phosphatidylserine externalization and DNA fragmentation. HF also induced apoptosis in primary AML blasts, whereas normal blood cells were not affected. The apoptotic process in U937 cells was accompanied by downregulation of anti-apoptotic Bcl-2, upregulation of pro-apoptotic Noxa, mitochondrial membrane depolarization, activation of procaspases and cleavage of the caspase substrate PARP-1. The general caspase inhibitor Z-VAD-fmk and the caspase-9- and -3-specific inhibitors, but not caspase-8 inhibitor, significantly attenuated apoptosis. HF-mediated apoptosis was associated with dephosphorylation of active Akt1 (at Ser(473)) and Akt1 substrate Bad (at Ser(136)) which activates Bad pro-apoptotic function. HF supppressed the kinase activity of Akt1, and combined treatment with the allosteric Akt1 inhibitor Akt-I-VIII significantly enhanced apoptosis of U937 cells. Our data provide new evidence that HF's pro-apoptotic effect in AML cells involved inhibition of Akt1 signaling, mitochondria and Bcl-2 members dysfunctions, and activation of procaspases -9/-3. Combined interruption of mitochondrial and Akt1 pathways by HF may have implications for AML treatment.

Hyperforin Inhibits Akt1 Kinase Activity and Promotes Caspase-Mediated Apoptosis Involving Bad and Noxa Activation in Human Myeloid Tumor Cells

PubMed Central

Merhi, Faten; Tang, Ruoping; Piedfer, Marion; Mathieu, Julie; Bombarda, Isabelle; Zaher, Murhaf; Kolb, Jean-Pierre; Billard, Christian; Bauvois, Brigitte

2011-01-01

Background The natural phloroglucinol hyperforin HF displays anti-inflammatory and anti-tumoral properties of potential pharmacological interest. Acute myeloid leukemia (AML) cells abnormally proliferate and escape apoptosis. Herein, the effects and mechanisms of purified HF on AML cell dysfunction were investigated in AML cell lines defining distinct AML subfamilies and primary AML cells cultured ex vivo. Methodology and Results HF inhibited in a time- and concentration-dependent manner the growth of AML cell lines (U937, OCI-AML3, NB4, HL-60) by inducing apoptosis as evidenced by accumulation of sub-G1 population, phosphatidylserine externalization and DNA fragmentation. HF also induced apoptosis in primary AML blasts, whereas normal blood cells were not affected. The apoptotic process in U937 cells was accompanied by downregulation of anti-apoptotic Bcl-2, upregulation of pro-apoptotic Noxa, mitochondrial membrane depolarization, activation of procaspases and cleavage of the caspase substrate PARP-1. The general caspase inhibitor Z-VAD-fmk and the caspase-9- and -3-specific inhibitors, but not caspase-8 inhibitor, significantly attenuated apoptosis. HF-mediated apoptosis was associated with dephosphorylation of active Akt1 (at Ser473) and Akt1 substrate Bad (at Ser136) which activates Bad pro-apoptotic function. HF supppressed the kinase activity of Akt1, and combined treatment with the allosteric Akt1 inhibitor Akt-I-VIII significantly enhanced apoptosis of U937 cells. Significance Our data provide new evidence that HF's pro-apoptotic effect in AML cells involved inhibition of Akt1 signaling, mitochondria and Bcl-2 members dysfunctions, and activation of procaspases -9/-3. Combined interruption of mitochondrial and Akt1 pathways by HF may have implications for AML treatment. PMID:21998731

Effect of PPARG on AGEs-induced AKT/MTOR signaling-associated human chondrocytes autophagy.

PubMed

Wang, Zhao-Jun; Zhang, Hai-Bin; Chen, Cheng; Huang, Hao; Liang, Jian-Xia

2018-02-17

Accumulation of advanced glycation end products (AGEs) in articular cartilage is thought to represent a major risk factor for osteoarthritis development. In this study we aimed to probe the role of AGEs in human chondrocytes and to determine the impact of the peroxisome proliferator-activated receptor-γ (PPARG) on AGEs-induced cell autophagy. Cell viability was measured after human chondrocytes were treated with different concentrations of AGEs with or without the PPARG inhibitor, T0070907, or agonist, pioglitazone. Autophagy activation markers (MAP2LC3, BECN1 and SQSTM1/P62), expression of PPARG and the phosphorylation levels of Akt/MTOR were determined by Western blotting; autophagosome formation was analyzed by transmission electron microscopy (TEM); autophagic flux was detected with mRFP-GFP-LC3 tandem construct. Low doses of AGEs over a short amount of time stimulated chondrocyte proliferation and autophagy by limiting phosphorylation of Akt/MTOR signaling. The addition of PPARG inhibitor T0070907 lead to defective autophagy. High dose and long exposure to AGEs inhibited cell viability and autophagy by increasing phosphorylation levels of Akt/MTOR signaling. The agonist, pioglitazone, was shown to protect cell autophagy in a dose-dependent manner. Our findings suggest AGEs can downregulate PPARG and that PPARG maintains cell viability by activating the Akt/MTOR signaling pathway as well as inducing chondrocyte autophagy. © 2018 International Federation for Cell Biology.

Macrophage deficiency of Akt2 reduces atherosclerosis in Ldlr null mice[S

PubMed Central

Babaev, Vladimir R.; Hebron, Katie E.; Wiese, Carrie B.; Toth, Cynthia L.; Ding, Lei; Zhang, Youmin; May, James M.; Fazio, Sergio; Vickers, Kasey C.; Linton, MacRae F.

2014-01-01

Macrophages play crucial roles in the formation of atherosclerotic lesions. Akt, a serine/threonine protein kinase B, is vital for cell proliferation, migration, and survival. Macrophages express three Akt isoforms, Akt1, Akt2, and Akt3, but the roles of Akt1 and Akt2 in atherosclerosis in vivo remain unclear. To dissect the impact of macrophage Akt1 and Akt2 on early atherosclerosis, we generated mice with hematopoietic deficiency of Akt1 or Akt2. After 8 weeks on Western diet, Ldlr−/− mice reconstituted with Akt1−/− fetal liver cells (Akt1−/−→Ldlr−/−) had similar atherosclerotic lesion areas compared with control mice transplanted with WT cells (WT→Ldlr−/−). In contrast, Akt2−/−→Ldlr−/− mice had dramatically reduced atherosclerotic lesions compared with WT→Ldlr−/− mice of both genders. Similarly, in the setting of advanced atherosclerotic lesions, Akt2−/−→Ldlr−/− mice had smaller aortic lesions compared with WT→Ldlr−/− and Akt1−/−→Ldlr−/− mice. Importantly, Akt2−/−→Ldlr−/− mice had reduced numbers of proinflammatory blood monocytes expressing Ly-6Chi and chemokine C-C motif receptor 2. Peritoneal macrophages isolated from Akt2−/− mice were skewed toward an M2 phenotype and showed decreased expression of proinflammatory genes and reduced cell migration. Our data demonstrate that loss of Akt2 suppresses the ability of macrophages to undergo M1 polarization reducing both early and advanced atherosclerosis. PMID:25240046

Degradation of Akt using protein-catalyzed capture agents.

PubMed

Henning, Ryan K; Varghese, Joseph O; Das, Samir; Nag, Arundhati; Tang, Grace; Tang, Kevin; Sutherland, Alexander M; Heath, James R

2016-04-01

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 that are shown to promote the rapid degradation of Akt in live cancer cells. These novel proteolysis targeting chimeric molecules demonstrate that the epitope targeting selectivity of PCCs can be coupled with non-traditional drugging moieties to inhibit challenging targets. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.

Testosterone regulation of Akt/mTORC1/FoxO3a Signaling in Skeletal Muscle

PubMed Central

White, James P.; Gao, Song; Puppa, Melissa J.; Sato, Shuichi; Welle, Stephen L.; Carson, James A.

2012-01-01

Low endogenous testosterone production, known as hypogonadism is commonly associated with conditions inducing muscle wasting. Akt signaling can control skeletal muscle mass through mTOR regulation of protein synthesis and FoxO regulation of protein degradation, and this pathway has been previously identified as a target of androgen signaling. However, the testosterone sensitivity of Akt/mTOR signaling requires further understanding in order to grasp the significance of varied testosterone levels seen with wasting disease on muscle protein turnover regulation. Therefore, the purpose of this study is to determine the effect of androgen availability on muscle Akt/mTORC1/FoxO3a regulation in skeletal muscle and cultured C2C12 myotubes. C57BL/6 mice were either castrated for 42 days or castrated and treated with the nandrolone decanoate (ND) (6 mg/kg bw/wk). Testosterone loss (TL) significantly decreased volitional grip strength, body weight, and gastrocnemius (GAS) muscle mass, and ND reversed these changes. Related to muscle mass regulation, TL decreased muscle IGF-1 mRNA, the rate of myofibrillar protein synthesis, Akt phosphorylation, and the phosphorylation of Akt targets, GSK3β, PRAS40 and FoxO3a. TL induced expression of FoxO transcriptional targets, MuRF1, atrogin1 and REDD1. Muscle AMPK and raptor phosphorylation, mTOR inhibitors, were not altered by low testosterone. ND restored IGF-1 expression and Akt/mTORC1 signaling while repressing expression of FoxO transcriptional targets. Testosterone (T) sensitivity of Akt/mTORC1 signaling was examined in C2C12 myotubes, and mTOR phosphorylation was induced independent of Akt activation at low T concentrations, while a higher T concentration was required to activate Akt signaling. Interestingly, low concentration T was sufficient to amplify myotube mTOR and Akt signaling after 24h of T withdrawal, demonstrating the potential in cultured myotubes for a T initiated positive feedback mechanism to amplify Akt

Agmatine Protects Against 6-OHDA-Induced Apoptosis, and ERK and Akt/GSK Disruption in SH-SY5Y Cells.

PubMed

Amiri, Esmat; Ghasemi, Rasoul; Moosavi, Maryam

2016-08-01

6-Hydroxydopamine (6-OHDA), a metabolite of dopamine is known to induce dopaminergic cell toxicity which makes that a suitable agent inducing an experimental model of Parkinson's disease (PD). Agmatine has been shown to protect against some cellular and animal PD models. This study was aimed to assess whether agmatine prevents 6-OHDA-induced SH-SY5Y cell death and if yes, then how it affects Akt/glycogen synthesis kinase-3β (GSK-3β) and extracellular signal-regulated kinases (ERK) signals. The cells were treated with different drugs, and their viability was examined via MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) assay and morphological observation. Western blot studies were done to assess cleaved caspase-3, Akt/GSK-3β, and ERK proteins. 6-OHDA-induced cell death and caspase-3 cleavage, while agmatine prevented those changes. 6-OHDA also decreased the amount of phosphorylated Akt (pAkt)/Akt while increased GSK-3β activity which was prevented by agmatine. Additionally, this toxin increased pERK/ERK ratio which was averted again by agmatine. The PI3/Akt inhibitor, LY294002, impeded the changes induced by agmatine, while ERK inhibitor (PD98059) did not disturb the effects of agmatine, and by itself, it preserved the cells against 6-OHDA toxicity. This study revealed that agmatine is protective in 6-OHDA model of PD and affects Akt/GSK-3β and ERK pathways.

Propofol mediates signal transducer and activator of transcription 3 activation and crosstalk with phosphoinositide 3-kinase/AKT.

PubMed

Shravah, Jayant; Wang, Baohua; Pavlovic, Marijana; Kumar, Ujendra; Chen, David Dy; Luo, Honglin; Ansley, David M

2014-01-01

We previously demonstrated that propofol, an intravenous anesthetic with anti-oxidative properties, activated the phosphoinositide 3-kinase (PI3K)/AKT pathway to increase the expression of B cell lymphoma (Bcl)-2 and, therefore the anti-apoptotic potential on cardiomyocytes. Here, we wanted to determine if propofol can also activate the Janus kinase (JAK) 2/signal transducer and activator of transcription (STAT) 3 pathway, another branch of cardioprotective signaling. The cellular response of nuclear factor kappa B (NFκB) and STAT3 was also evaluated. Cardiac H9c2 cells were treated by propofol alone or in combination with pretreatment by inhibitors for JAK2/STAT3 or PI3K/AKT pathway. STAT3 and AKT phosphorylation, and STAT3 translocation were measured by western blotting and immunofluorescence staining, respectively. Propofol treatment significantly increased STAT3 phosphorylation at both tyrosine 705 and serine 727 residues. Sustained early phosphorylation of STAT3 was observed with 25~75 μM propofol at 10 and 30 min. Nuclear translocation of STAT3 was seen at 4 h after treatment with 50 μM propofol. In cultured H9c2 cells, we further demonstrated that propofol-induced STAT3 phosphorylation was reduced by pretreatment with PI3K/AKT pathway inhibitors wortmannin or API-2. Conversely, pretreatment with JAK2/STAT3 pathway inhibitor AG490 or stattic inhibited propofol-induced AKT phosphorylation. In addition, propofol induced NFκB p65 subunit perinuclear translocation. Inhibition or knockdown of STAT3 was associated with increased levels of the NFκB p65 subunit. Our results suggest that propofol induces an adaptive response by dual activation and crosstalk of cytoprotective PI3K/AKT and JAK2/STAT3 pathways. Rationale to apply propofol clinically as a preemptive cardioprotectant during cardiac surgery is supported by our findings.

Akt recruits Dab2 to albumin endocytosis in the proximal tubule.

PubMed

Koral, Kelly; Li, Hui; Ganesh, Nandita; Birnbaum, Morris J; Hallows, Kenneth R; Erkan, Elif

2014-12-15

Proximal tubule epithelial cells have a highly sophisticated endocytic machinery to retrieve the albumin in the glomerular filtrate. The megalin-cubilin complex and the endocytic adaptor disabled-2 (Dab2) play a pivotal role in albumin endocytosis. We previously demonstrated that protein kinase B (Akt) regulates albumin endocytosis in the proximal tubule through an interaction with Dab2. Here, we examined the nature of Akt-Dab2 interaction. The pleckstrin homology (PH) and catalytic domains (CD) of Akt interacted with the proline-rich domain (PRD) of Dab2 based on yeast-two hybrid (Y2H) experiments. Pull-down experiments utilizing the truncated constructs of Dab2 demonstrated that the initial 11 amino acids of Dab2-PRD were sufficient to mediate the interaction between Akt and Dab2. Endocytosis experiments utilizing Akt1- and Akt2-silencing RNA revealed that both Akt1 and Akt2 mediate albumin endocytosis in proximal tubule epithelial cells; therefore, Akt1 and Akt2 may play a compensatory role in albumin endocytosis. Furthermore, both Akt isoforms phosphorylated Dab2 at Ser residues 448 and 449. Ser-to-Ala mutations of these Dab2 residues inhibited albumin endocytosis and resulted in a shift in location of Dab2 from the peripheral to the perinuclear area, suggesting the physiological relevance of these phosphorylation sites in albumin endocytosis. We conclude that both Akt1 and Akt2 are involved in albumin endocytosis, and phosphorylation of Dab2 by Akt induces albumin endocytosis in proximal tubule epithelial cells. Further delineation of how Akt affects expression/phosphorylation of endocytic adaptors and receptors will enhance our understanding of the molecular network triggered by albumin overload in the proximal tubule. Copyright © 2014 the American Physiological Society.

New Insights into Protein Kinase B/Akt Signaling: Role of Localized Akt Activation and Compartment-Specific Target Proteins for the Cellular Radiation Response.

PubMed

Szymonowicz, Klaudia; Oeck, Sebastian; Malewicz, Nathalie M; Jendrossek, Verena

2018-03-18

Genetic alterations driving aberrant activation of the survival kinase Protein Kinase B (Akt) are observed with high frequency during malignant transformation and cancer progression. Oncogenic gene mutations coding for the upstream regulators or Akt, e.g., growth factor receptors, RAS and phosphatidylinositol-3-kinase (PI3K), or for one of the three Akt isoforms as well as loss of the tumor suppressor Phosphatase and Tensin Homolog on Chromosome Ten (PTEN) lead to constitutive activation of Akt. By activating Akt, these genetic alterations not only promote growth, proliferation and malignant behavior of cancer cells by phosphorylation of various downstream signaling molecules and signaling nodes but can also contribute to chemo- and radioresistance in many types of tumors. Here we review current knowledge on the mechanisms dictating Akt's activation and target selection including the involvement of miRNAs and with focus on compartmentalization of the signaling network. Moreover, we discuss recent advances in the cross-talk with DNA damage response highlighting nuclear Akt target proteins with potential involvement in the regulation of DNA double strand break repair.

Inhibition of ERK1/2 or AKT Activity Equally Enhances Radiation Sensitization in B16F10 Cells

PubMed Central

Kalal, Bhuvanesh Sukhlal; Fathima, Faraz; Pai, Vinitha Ramanath; Sanjeev, Ganesh; Krishna, Chilakapati Murali; Upadhya, Dinesh

2018-01-01

Background The aim of the study was to evaluate the radiation sensitizing ability of ERK1/2, PI3K-AKT and JNK inhibitors in highly radiation resistant and metastatic B16F10 cells which carry wild-type Ras and Braf. Methods Mouse melanoma cell line B16F10 was exposed to 1.0, 2.0 and 3.0 Gy of electron beam radiation. Phosphorylated ERK1/2, AKT and JNK levels were estimated by ELISA. Cells were exposed to 2.0 and 3.0 Gy of radiation with or without prior pharmacological inhibition of ERK1/2, AKT as well as JNK pathways. Cell death induced by radiation as well as upon inhibition of these pathways was measured by TUNEL assay using flow cytometry. Results Exposure of B16F10 cells to 1.0, 2.0 and 3.0 Gy of electron beam irradiation triggered an increase in all the three phosphorylated proteins compared to sham-treated and control groups. B16F10 cells pre-treated with either ERK1/2 or AKT inhibitors equally enhanced radiation-induced cell death at 2.0 as well as 3.0 Gy (P < 0.001), while inhibition of JNK pathway increased radiation-induced cell death to a lesser extent. Interestingly combined inhibition of ERK1/2 or AKT pathways did not show additional cell death compared to individual ERK1/2 or AKT inhibition. This indicates that ERK1/2 or AKT mediates radiation resistance through common downstream molecules in B16F10 cells. Conclusions Even without activating mutations in Ras or Braf genes, ERK1/2 and AKT play a critical role in B16F10 cell survival upon radiation exposure and possibly act through common downstream effector/s. PMID:29581812

Inhibition of ERK1/2 or AKT Activity Equally Enhances Radiation Sensitization in B16F10 Cells.

PubMed

Kalal, Bhuvanesh Sukhlal; Fathima, Faraz; Pai, Vinitha Ramanath; Sanjeev, Ganesh; Krishna, Chilakapati Murali; Upadhya, Dinesh

2018-02-01

The aim of the study was to evaluate the radiation sensitizing ability of ERK1/2, PI3K-AKT and JNK inhibitors in highly radiation resistant and metastatic B16F10 cells which carry wild-type Ras and Braf . Mouse melanoma cell line B16F10 was exposed to 1.0, 2.0 and 3.0 Gy of electron beam radiation. Phosphorylated ERK1/2, AKT and JNK levels were estimated by ELISA. Cells were exposed to 2.0 and 3.0 Gy of radiation with or without prior pharmacological inhibition of ERK1/2, AKT as well as JNK pathways. Cell death induced by radiation as well as upon inhibition of these pathways was measured by TUNEL assay using flow cytometry. Exposure of B16F10 cells to 1.0, 2.0 and 3.0 Gy of electron beam irradiation triggered an increase in all the three phosphorylated proteins compared to sham-treated and control groups. B16F10 cells pre-treated with either ERK1/2 or AKT inhibitors equally enhanced radiation-induced cell death at 2.0 as well as 3.0 Gy (P < 0.001), while inhibition of JNK pathway increased radiation-induced cell death to a lesser extent. Interestingly combined inhibition of ERK1/2 or AKT pathways did not show additional cell death compared to individual ERK1/2 or AKT inhibition. This indicates that ERK1/2 or AKT mediates radiation resistance through common downstream molecules in B16F10 cells. Even without activating mutations in Ras or Braf genes, ERK1/2 and AKT play a critical role in B16F10 cell survival upon radiation exposure and possibly act through common downstream effector/s.

Neuregulin-1β promotes glucose uptake via PI3K/Akt in neonatal rat cardiomyocytes.

PubMed

Pentassuglia, Laura; Heim, Philippe; Lebboukh, Sonia; Morandi, Christian; Xu, Lifen; Brink, Marijke

2016-05-01

Nrg1β is critically involved in cardiac development and also maintains function of the adult heart. Studies conducted in animal models showed that it improves cardiac performance under a range of pathological conditions, which led to its introduction in clinical trials to treat heart failure. Recent work also implicated Nrg1β in the regenerative potential of neonatal and adult hearts. The molecular mechanisms whereby Nrg1β acts in cardiac cells are still poorly understood. In the present study, we analyzed the effects of Nrg1β on glucose uptake in neonatal rat ventricular myocytes and investigated to what extent mTOR/Akt signaling pathways are implicated. We show that Nrg1β enhances glucose uptake in cardiomyocytes as efficiently as IGF-I and insulin. Nrg1β causes phosphorylation of ErbB2 and ErbB4 and rapidly induces the phosphorylation of FAK (Tyr(861)), Akt (Thr(308) and Ser(473)), and its effector AS160 (Thr(642)). Knockdown of ErbB2 or ErbB4 reduces Akt phosphorylation and blocks the glucose uptake. The Akt inhibitor VIII and the PI3K inhibitors LY-294002 and Byl-719 abolish Nrg1β-induced phosphorylation and glucose uptake. Finally, specific mTORC2 inactivation after knockdown of rictor blocks the Nrg1β-induced increases in Akt-p-Ser(473) but does not modify AS160-p-Thr(642) or the glucose uptake responses to Nrg1β. In conclusion, our study demonstrates that Nrg1β enhances glucose uptake in cardiomyocytes via ErbB2/ErbB4 heterodimers, PI3Kα, and Akt. Furthermore, although Nrg1β activates mTORC2, the resulting Akt-Ser(473) phosphorylation is not essential for glucose uptake induction. These new insights into pathways whereby Nrg1β regulates glucose uptake in cardiomyocytes may contribute to the understanding of its regenerative capacity and protective function in heart failure. Copyright © 2016 the American Physiological Society.

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Xu, Lin; Shaoyang Central Hospital, Hunan Province; Zhang, Yanan

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 AMLmore » 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. - Highlights: • A-674563 is cytotoxic and anti-proliferative in U937 and AML progenitor cells. • A-674563 activates caspase-3/9 and apoptosis in U937 and AML progenitor cells. • Whiling blocking Akt, A-674563 manipulates other signalings in AML cells. • A-674563 inhibits SphK1 activity in AML cells, independent of Akt blockage. • A-674563 injection inhibits U937 xenograft in vivo growth, and improves mice survival.« less

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. Copyright © 2015 Elsevier Masson SAS. All rights reserved.

Resveratrol Inhibits the Epidermal Growth Factor-Induced Migration of Osteoblasts: the Suppression of SAPK/JNK and Akt.

PubMed

Kawabata, Tetsu; Tokuda, Haruhiko; Fujita, Kazuhiko; Kainuma, Shingo; Sakai, Go; Matsushima-Nishiwaki, Rie; Kozawa, Osamu; Otsuka, Takanobu

2017-01-01

Resveratrol is a polyphenol enriched in the skins of grapes and berries, that shows various beneficial effects for human health. In the present study, we investigated the mechanism behind the epidermal growth factor (EGF)-induced migration of osteoblast-like MC3T3-E1 cells, and the effect of resveratrol on this cell migration. The cell migration was examined using Boyden chamber, and phosphorylation of each kinase was analyzed by Western blotting. The EGF-induced migration was suppressed by PD98059, an inhibitor of MEK1/2, as well as SB203580, an inhibitor of p38 MAP kinase, SP600125, an inhibitor of SAPK/JNK, and deguelin, an inhibitor of Akt. In contrast, rapamycin, an inhibitor of upstream kinase of p70 S6 kinase, and fasudil, an inhibitor of Rho-kinase, hardly affected the migration. Resveratrol significantly reduced the EGF-induced migration in a dose-dependent manner. SRT1720, an SIRT1 activator, suppressed the migration by EGF. In addition, resveratrol markedly attenuated the EGF-induced phosphorylation of SAPK/JNK and Akt without affecting the phosphorylation of p44/p42 MAP kinase or p38 MAP kinase. The phosphorylation of SAPK/JNK and Akt induced by EGF was down-regulated by SRT1720. Our results strongly suggest that resveratrol reduces the EGF-stimulated migration of osteoblasts via suppression of SAPK and Akt, and that the inhibitory effect of resveratrol is mediated in part via SIRT1. © 2017 The Author(s). Published by S. Karger AG, Basel.

Interaction of human biliverdin reductase with Akt/protein kinase B and phosphatidylinositol-dependent kinase 1 regulates glycogen synthase kinase 3 activity: a novel mechanism of Akt activation

PubMed Central

Miralem, Tihomir; Lerner-Marmarosh, Nicole; Gibbs, Peter E. M.; Jenkins, Jermaine L.; Heimiller, Chelsea; Maines, Mahin D.

2016-01-01

Biliverdin reductase A (BVR) and Akt isozymes have overlapping pleiotropic functions in the insulin/PI3K/MAPK pathway. Human BVR (hBVR) also reduces the hemeoxygenase activity product biliverdin to bilirubin and is directly activated by insulin receptor kinase (IRK). Akt isoenzymes (Akt1–3) are downstream of IRK and are activated by phosphatidylinositol-dependent kinase 1 (PDK1) phosphorylating T308 before S473 autophosphorylation. Akt (RxRxxSF) and PDK1 (RFxFPxFS) binding motifs are present in hBVR. Phosphorylation of glycogen synthase kinase 3 (GSK3) isoforms α/β by Akts inhibits their activity; nonphosphorylated GSK3β inhibits activation of various genes. We examined the role of hBVR in PDK1/Akt1/GSK3 signaling and Akt1 in hBVR phosphorylation. hBVR activates phosphorylation of Akt1 at S473 independent of hBVR’s kinase competency. hBVR and Akt1 coimmunoprecipitated, and in-cell Förster resonance energy transfer (FRET) and glutathione S-transferase pulldown analyses identified Akt1 pleckstrin homology domain as the interactive domain. hBVR activates phosphorylation of Akt1 at S473 independent of hBVR’s kinase competency. Site-directed mutagenesis, mass spectrometry, and kinetic analyses identified S230 in hBVR 225RNRYLSF sequence as the Akt1 target. Underlined amino acids are the essential residues of the signaling motifs. In cells, hBVR-activated Akt1 increased both GSK3α/β and forkhead box of the O class transcription class 3 (FoxO3) phosphorylation and inhibited total GSK3 activity; depletion of hBVR released inhibition and stimulated glucose uptake. Immunoprecipitation analysis showed that PDK1 and hBVR interact through hBVR’s PDK1 binding 161RFGFPAFS motif and formation of the PDK1/hBVR/Akt1 complex. sihBVR blocked complex formation. Findings identify hBVR as a previously unknown coactivator of Akt1 and as a key mediator of Akt1/GSK3 pathway, as well as define a key role for hBVR in Akt1 activation by PDK1.—Miralem, T., Lerner-Marmarosh, N

Interaction of human biliverdin reductase with Akt/protein kinase B and phosphatidylinositol-dependent kinase 1 regulates glycogen synthase kinase 3 activity: a novel mechanism of Akt activation.

PubMed

Miralem, Tihomir; Lerner-Marmarosh, Nicole; Gibbs, Peter E M; Jenkins, Jermaine L; Heimiller, Chelsea; Maines, Mahin D

2016-08-01

Biliverdin reductase A (BVR) and Akt isozymes have overlapping pleiotropic functions in the insulin/PI3K/MAPK pathway. Human BVR (hBVR) also reduces the hemeoxygenase activity product biliverdin to bilirubin and is directly activated by insulin receptor kinase (IRK). Akt isoenzymes (Akt1-3) are downstream of IRK and are activated by phosphatidylinositol-dependent kinase 1 (PDK1) phosphorylating T(308) before S(473) autophosphorylation. Akt (RxRxxSF) and PDK1 (RFxFPxFS) binding motifs are present in hBVR. Phosphorylation of glycogen synthase kinase 3 (GSK3) isoforms α/β by Akts inhibits their activity; nonphosphorylated GSK3β inhibits activation of various genes. We examined the role of hBVR in PDK1/Akt1/GSK3 signaling and Akt1 in hBVR phosphorylation. hBVR activates phosphorylation of Akt1 at S(473) independent of hBVR's kinase competency. hBVR and Akt1 coimmunoprecipitated, and in-cell Förster resonance energy transfer (FRET) and glutathione S-transferase pulldown analyses identified Akt1 pleckstrin homology domain as the interactive domain. hBVR activates phosphorylation of Akt1 at S(473) independent of hBVR's kinase competency. Site-directed mutagenesis, mass spectrometry, and kinetic analyses identified S(230) in hBVR (225)RNRYLSF sequence as the Akt1 target. Underlined amino acids are the essential residues of the signaling motifs. In cells, hBVR-activated Akt1 increased both GSK3α/β and forkhead box of the O class transcription class 3 (FoxO3) phosphorylation and inhibited total GSK3 activity; depletion of hBVR released inhibition and stimulated glucose uptake. Immunoprecipitation analysis showed that PDK1 and hBVR interact through hBVR's PDK1 binding (161)RFGFPAFS motif and formation of the PDK1/hBVR/Akt1 complex. sihBVR blocked complex formation. Findings identify hBVR as a previously unknown coactivator of Akt1 and as a key mediator of Akt1/GSK3 pathway, as well as define a key role for hBVR in Akt1 activation by PDK1.-Miralem, T., Lerner

Neuroprotective capabilities of TSA against cerebral ischemia/reperfusion injury via PI3K/Akt signaling pathway in rats.

PubMed

Ma, Xiao-Hui; Gao, Qiang; Jia, Zhen; Zhang, Ze-Wei

2015-02-01

Hundreds of previous studies demonstrated the cytoprotective effect of trichostatin-A (TSA), a kind of histone deacetylases inhibitors (HDACIs), against cerebral ischemia/reperfusion insult. Meanwhile, phosphatidylinositol-3 kinase/Akt (PI3K/Akt) is a well-known, important signaling pathway that mediates neuroprotection. However, it should be remains unclear whether the neuroprotective capabilities of TSA against cerebral ischemia/reperfusion is mediated by activation of the PI3K/Akt signaling pathway. Five groups rats (n = 12 each), with middle cerebral artery occlusion (MCAO) except sham group, were used to investigate the neuroprotective effect of certain concentration (0.05 mg/kg) of TSA, and whether the neuroprotective effect of TSA is associated with activation of the PI3K/Akt signaling pathway through using of wortmannin (0.25 mg/kg). TSA significantly increased the expression of p-Akt protein, reduced infarct volume, and attenuated neurological deficit in rats with transient MCAO, wortmannin weakened such effect of TSA dramatically. TSA could significantly decrease the neurological deficit scores and reduce the cerebral infarct volume during cerebral ischemia/reperfusion injury, which was achieved partly by activation of the PI3K/Akt signaling pathway via upgrading of p-Akt protein.

Akt regulates drug-induced cell death through Bcl-w downregulation.

PubMed

Garofalo, Michela; Quintavalle, Cristina; Zanca, Ciro; De Rienzo, Assunta; Romano, Giulia; Acunzo, Mario; Puca, Loredana; Incoronato, Mariarosaria; Croce, Carlo M; Condorelli, Gerolama

2008-01-01

Akt is a serine threonine kinase with a major role in transducing survival signals and regulating proteins involved in apoptosis. To find new interactors of Akt involved in cell survival, we performed a two-hybrid screening in yeast using human full-length Akt c-DNA as bait and a murine c-DNA library as prey. Among the 80 clones obtained, two were identified as Bcl-w. Bcl-w is a member of the Bcl-2 family that is essential for the regulation of cellular survival, and that is up-regulated in different human tumors, such as gastric and colorectal carcinomas. Direct interaction of Bcl-w with Akt was confirmed by immunoprecipitation assays. Subsequently, we addressed the function of this interaction: by interfering with the activity or amount of Akt, we have demonstrated that Akt modulates the amount of Bcl-w protein. We have found that inhibition of Akt activity may promote apoptosis through the downregulation of Bcl-w protein and the consequential reduction in interaction of Bcl-w with pro-apoptotic members of the Bcl-2 family. Our data provide evidence that Bcl-w is a new member of the Akt pathway and that Akt may induce anti-apoptotic signals at least in part through the regulation of the amount and activity of Bcl-w.

Development of highly sensitive cell-based AKT kinase ELISA for monitoring PI3K beta activity and compound efficacy.

PubMed

Yanamandra, Mahesh; Kole, Labanyamoy; Giri, Archana; Mitra, Sayan

2017-01-01

Phosphatidylinositol-3 kinase (PI3K) pathway regulates multiple cellular functions involving cell survival, growth, motility proliferation, apoptosis, and adhesion. These are deregulated in various diseases such as cancer, atherosclerosis, and inflammation. PI3Ks phosphorylate phosphatidylinositol 4,5-biphosphate (PIP2) yielding phosphatidylinositol 3, 4, 5 triphosphate (PIP3) which in turn activate AKT kinase (serine/threonine kinase), the central enzyme in regulation of metabolic functions. Due to their implications in disease pathophysiology, PI3K/AKT inhibitors became attractive targets for pharmaceutical industries. In order to assess the functional response generated by PI3K inhibitors, an appropriate cell-based screening system is essential in any screening cascade. Here we report the development of highly sensitive in-vitro cell-based kinase ELISA which quantifies the phosphorylated AKT kinase (serine 473) and total AKT kinase directly within the cells upon compound treatment. PI3Kβ overexpressing NIH3T3 cells stimulated by lysophosphatidic acid was used for PI3K/Akt pathway activation. Assay performance reliability and robustness were determined by percentage coefficient of variation (%CV) and Z factor which demonstrated an excellent agreement with assay guidelines. This 96-well plate medium throughput assay methodology was used to screen novel molecules and proved a commendable tool to study the mechanism of action property and target engagement of novel PI3K inhibitors in drug discovery.

delta opioid receptors stimulate Akt-dependent phosphorylation of c-jun in T cells.

PubMed

Shahabi, Nahid A; McAllen, Kathy; Sharp, Burt M

2006-02-01

Activation of naive T cells markedly up-regulates the expression of delta opioid receptors (DORs). These receptors are bound by DOR peptides released by T cells, modulating T cell functions such as interleukin-2 production, cellular proliferation, and chemotaxis. Previous studies have shown that DOR agonists [e.g., [D-Ala(2)-D-Leu(5)]-enkephalin (DADLE)] modulate T cell antigen receptor signaling through mitogen-activated protein kinases (MAPKs; i.e., extracellular signal-regulated kinases 1 and 2) and that DORs directly induce phosphorylation of activating transcription factor-2 (implicated in cytokine gene transcription) and its association with the MAPK c-jun1 NH(2)-terminal kinase (JNK). Such observations suggest that DORs may induce the phosphorylation of c-jun. These experiments were performed to test this hypothesis and determine the potential roles of phosphoinositide 3-kinase (PI3K) and Akt (protein kinase B). DADLE (10(-10) to 10(-6) M) dose-dependently induced c-jun phosphorylation. This was blocked by pertussis toxin and the DOR-specific antagonist naltindole. Fluorescence flow cytometry showed that DADLE significantly stimulated c-jun phosphorylation by T cells. DADLE stimulated phosphorylation of membrane-associated Akt; wortmannin and LY294002 ([2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one]), specific inhibitors of PI3K, abolished the DADLE-induced phosphorylation of c-jun. Finally, inhibitors of Akt and JNK blocked DADLE-induced phosphorylation of c-jun. Thus, activated DORs directly stimulate c-jun phosphorylation through a PI3K-dependent pathway in T cells, apparently involving Akt. This implies that DORs activate JNK through a novel pathway dependent on PI3K and Akt, thereby regulating the function of activator protein-1 transcription complexes containing c-jun and other transcription partners.

AKT Pathway Affects Bone Regeneration in Nonunion Treated with Umbilical Cord-Derived Mesenchymal Stem Cells.

PubMed

Qu, Zhiguo; Guo, Shengnan; Fang, Guojun; Cui, Zhenghong; Liu, Ying

2015-04-01

We have previously grafted human umbilical cord-derived mesenchymal stem cells (hUC-MSCs) with blood plasma to treat rat tibia nonunion. To further examine the biological characteristics of this process, we applied an established hUC-MSCs-treated rat nonunion model with the addition of an inhibitor of AKT. SD rats (80) were randomly divided into four groups: a fracture group (positive control); a nonunion group (negative control); a hUC-MSCs grafting with blood plasma group; and a hUC-MSCs grafting with blood plasma & AKT blocker group. The animals were sacrificed under deep anesthesia at 4 and 8 weeks post fracture for analysis. The fracture line became less defined at 4 weeks and disappeared at 8 weeks postoperatively in both the hUC-MSCs grafting with blood plasma and grafting with blood plasma & the AKT blocker, which is similar to the fracture group. Histological immunofluorescence studies showed that the numbers of hUC-MSCs in the calluses were significantly higher in the hUC-MSCs grafting with blood plasma than those in group with the AKT blocker. More bone morphogenetic protein 2 and bone sialoprotein expression and less osteoprotegerin and bone gla protein expression were observed in the AKT blocker group compared to the hUC-MSCs grafting with blood plasma. AKT gene expression in the AKT blocker group was decreased 50% compared to the hUC-MSCs with plasma group and decreased 70% compared to the fracture group, while the elastic modulus was decreased. In summary, our work demonstrates that AKT may play a role in modulating osteogenesis induced by hUC-MSCs.

ARQ 092, an orally-available, selective AKT inhibitor, attenuates neutrophil-platelet interactions in sickle cell disease

PubMed Central

Kim, Kyungho; Li, Jing; Barazia, Andrew; Tseng, Alan; Youn, Seock-Won; Abbadessa, Giovanni; Yu, Yi; Schwartz, Brian; Andrews, Robert K.; Gordeuk, Victor R.; Cho, Jaehyung

2017-01-01

Previous studies identified the Ser/Thr protein kinase, AKT, as a therapeutic target in thrombo-inflammatory diseases. Here we report that specific inhibition of AKT with ARQ 092, an orally-available AKT inhibitor currently in phase Ib clinical trials as an anti-cancer drug, attenuates the adhesive function of neutrophils and platelets from sickle cell disease patients in vitro and cell-cell interactions in a mouse model of sickle cell disease. Studies using neutrophils and platelets isolated from sickle cell disease patients revealed that treatment with 50–500 nM ARQ 092 significantly blocks αMβ2 integrin function in neutrophils and reduces P-selectin exposure and glycoprotein Ib/IX/V-mediated agglutination in platelets. Treatment of isolated platelets and neutrophils with ARQ 092 inhibited heterotypic cell-cell aggregation under shear conditions. Intravital microscopic studies demonstrated that short-term oral administration of ARQ 092 or hydroxyurea, a major therapy for sickle cell disease, diminishes heterotypic cell-cell interactions in venules of sickle cell disease mice challenged with tumor necrosis factor-α. Co-administration of hydroxyurea and ARQ 092 further reduced the adhesive function of neutrophils in venules and neutrophil transmigration into alveoli, inhibited expression of E-selectin and intercellular adhesion molecule-1 in cremaster vessels, and improved survival in these mice. Ex vivo studies in sickle cell disease mice suggested that co-administration of hydroxyurea and ARQ 092 efficiently blocks neutrophil and platelet activation and that the beneficial effect of hydroxyurea results from nitric oxide production. Our results provide important evidence that ARQ 092 could be a novel drug for the prevention and treatment of acute vaso-occlusive complications in patients with sickle cell disease. PMID:27758820

Protein Kinase C-δ Mediates Neuronal Apoptosis in the Retinas of Diabetic Rats via the Akt Signaling Pathway

PubMed Central

Kim, Young-Hee; Kim, Yoon-Sook; Park, Chang-Hwan; Chung, In-Yong; Yoo, Ji-Myong; Kim, Jae-Geun; Lee, Byung-Ju; Kang, Sang-Soo; Cho, Gyeong-Jae; Choi, Wan-Sung

2008-01-01

OBJECTIVE—Protein kinase C (PKC)-δ, an upstream regulator of the Akt survival pathway, contributes to cellular dysfunction in the pathogenesis of diabetes. Herein, we examined the role of PKC-δ in neuronal apoptosis through Akt in the retinas of diabetic rats. RESEARCH DESIGN AND METHODS—We used retinas from 24- and 35-week-old male Otsuka Long-Evans Tokushima fatty (OLETF) diabetic and Long-Evans Tokushima Otsuka (LETO) nondiabetic rats. To assess whether PKC-δ affects Akt signaling and cell death in OLETF rat retinas, we examined 1) PKC-δ activity and apoptosis; 2) protein levels of phosphatidylinositol 3-kinase (PI 3-kinase) p85, heat shock protein 90 (HSP90), and protein phosphatase 2A (PP2A); 3) Akt phosphorylation; and 4) Akt binding to HSP90 or PP2A in LETO and OLETF retinas in the presence or absence of rottlerin, a highly specific PKC-δ inhibitor, or small interfering RNAs (siRNAs) for PKC-δ and HSP90. RESULTS—In OLETF retinas from 35-week-old rats, ganglion cell death, PKC-δ and PP2A activity, and Akt-PP2A binding were significantly increased and Akt phosphorylation and Akt-HSP90 binding were decreased compared with retinas from 24-week-old OLETF and LETO rats. Rottlerin and PKC-δ siRNA abrogated these effects in OLETF retinas from 35-week-old rats. HSP90 siRNA significantly increased ganglion cell death and Akt-PP2A complexes and markedly decreased HSP90-Akt binding and Akt phosphorylation in LETO retinas from 35-week-old rats compared with those from nontreated LETO rats. CONCLUSIONS—PKC-δ activation contributes to neuro-retinal apoptosis in diabetic rats by inhibiting Akt-mediated signaling pathways. PMID:18443201

CCNG2 Overexpression Mediated by AKT Inhibits Tumor Cell Proliferation in Human Astrocytoma Cells.

PubMed

Zhang, Danfeng; Wang, Chunhui; Li, Zhenxing; Li, Yiming; Dai, Dawei; Han, Kaiwei; Lv, Liquan; Lu, Yicheng; Hou, Lijun; Wang, Junyu

2018-01-01

The cyclin family protein CCNG2 has an important inhibitory role in cancer initiation and progression, but the exact mechanism is still unknown. In this study, we examined the relationship between CCNG2 and the malignancy of astrocytomas and whether the AKT pathway, which is upregulated in astrocytomas, may inhibit CCNG2 expression. CCNG2 expression was found to be negatively associated with the pathological grade and proliferative activity of astrocytomas, as the highest expression was found in control brain tissue ( N  = 31), whereas the lowest expression was in high-grade glioma tissue ( N  = 31). Additionally, CCNG2 overexpression in glioma cell lines, T98G and U251 inhibited proliferation and arrested cells in the G0/G1 phase. Moreover, CCNG2 overexpression could increase glioma cells apoptosis. In contrast, AKT activity increased in glioma cells that had low CCNG2 expression. Expression of CCNG2 was higher in cells treated with the AKT kinase inhibitor MK-2206 indicating that the presence of phosphorylated AKT may inhibit the expression of CCNG2. Inhibition of AKT also led to decreased colony formation in T98G and U251 cells and knocked down of CCNG2 reversed the result. Finally, overexpression of CCNG2 in glioma cells reduced tumor volume in a murine model. To conclude, low expression of CCNG2 correlated with the severity astrocytoma and CCNG2 overexpression could induce apoptosis and inhibit proliferation. Inhibition of AKT activity increased the expression of CCNG2. The present study highlights the regulatory consequences of CCNG2 expression and AKT activity in astrocytoma tumorigenesis and the potential use of CCNG2 in anticancer treatment.

Lithium protects against methamphetamine-induced neurotoxicity in PC12 cells via Akt/GSK3β/mTOR pathway

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wu, Jintao; Zhu, Dexiao; Zhang, Jing

Methamphetamine (MA) is neurotoxic, especially in dopaminergic neurons. Long-lasting exposure to MA causes psychosis and increases the risk of Parkinson's disease. Lithium (Li) is a known mood stabilizer and has neuroprotective effects. Previous studies suggest that MA exposure decreases the phosphorylation of Akt/GSK3β pathway in vivo, whereas Li facilitates the phosphorylation of Akt/GSK3β pathway. Moreover, GSK3β and mTOR are implicated in the locomotor sensitization induced by psychostimulants and mTOR plays a critical role in MA induced toxicity. However, the effect of MA on Akt/GSK3β/mTOR pathway has not been fully investigated in vitro. Here, we found that MA exposure significantly dephosphorylated Akt/GSK3β/mTOR pathwaymore » in PC12 cells. In addition, Li remarkably attenuated the dephosphorylation effect of MA exposure on Akt/GSK3β/mTOR pathway. Furthermore, Li showed obvious protective effects against MA toxicity and LY294002 (Akt inhibitor) suppressed the protective effects of Li. Together, MA exposure dephosphorylates Akt/GSK3β/mTOR pathway in vitro, while lithium protects against MA-induced neurotoxicity via phosphorylation of Akt/GSK3β/mTOR pathway. - Highlights: • Lithium protects against methamphetamine-induced neurotoxicity in vitro. • Methamphetamine exposure dephosphorylates Akt/GSK3β/mTOR pathway. • Lithium attenuates methamphetamine-induced toxicity via phosphorylating Akt/GSK3β/mTOR pathway.« less

Activating Akt1 mutations alter DNA double strand break repair and radiosensitivity

PubMed Central

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

2017-01-01

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

Escin activates AKT-Nrf2 signaling to protect retinal pigment epithelium cells from oxidative stress

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wang, Kaijun; Zhejiang Provincial Key Lab of Ophthalmology, Hangzhou; Jiang, Yiqian

Here we explored the anti-oxidative and cytoprotective potentials of escin, a natural triterpene-saponin, against hydrogen peroxide (H{sub 2}O{sub 2}) in retinal pigment epithelium (RPE) cells. We showed that escin remarkably attenuated H{sub 2}O{sub 2}-induced death and apoptosis of established (ARPE-19) and primary murine RPE cells. Meanwhile, ROS production and lipid peroxidation by H{sub 2}O{sub 2} were remarkably inhibited by escin. Escin treatment in RPE cells resulted in NF-E2-related factor 2 (Nrf2) signaling activation, evidenced by transcription of anti-oxidant-responsive element (ARE)-regulated genes, including HO-1, NQO-1 and SRXN-1. Knockdown of Nrf2 through targeted shRNAs/siRNAs alleviated escin-mediated ARE gene transcription, and almost abolishedmore » escin-mediated anti-oxidant activity and RPE cytoprotection against H{sub 2}O{sub 2}. Reversely, escin was more potent against H{sub 2}O{sub 2} damages in Nrf2-over-expressed ARPE-19 cells. Further studies showed that escin-induced Nrf2 activation in RPE cells required AKT signaling. AKT inhibitors (LY294002 and perifosine) blocked escin-induced AKT activation, and dramatically inhibited Nrf2 phosphorylation, its cytosol accumulation and nuclear translocation in RPE cells. Escin-induced RPE cytoprotection against H{sub 2}O{sub 2} was also alleviated by the AKT inhibitors. Together, these results demonstrate that escin protects RPE cells from oxidative stress possibly through activating AKT-Nrf2 signaling.« less

Protooncogene TCL1b functions as an Akt kinase co-activator that exhibits oncogenic potency in vivo

PubMed Central

Hashimoto, M; Suizu, F; Tokuyama, W; Noguchi, H; Hirata, N; Matsuda-Lennikov, M; Edamura, T; Masuzawa, M; Gotoh, N; Tanaka, S; Noguchi, M

2013-01-01

Protooncogene T-cell leukemia 1 (TCL1), which is implicated in human T-cell prolymphocytic leukemia (T-PLL), interacts with Akt and enhances its kinase activity, functioning as an Akt kinase co-activator. Two major isoforms of TCL1 Protooncogenes (TCL1 and TCL1b) are present adjacent to each other on human chromosome 14q.32. In human T-PLL, both TCL1 and TCL1b are activated by chromosomal translocation. Moreover, TCL1b-transgenic mice have never been created. Therefore, it remains unclear whether TCL1b itself, independent of TCL1, exhibits oncogenicity. In co-immunoprecipitation assays, both ectopic and endogenous TCL1b interacted with Akt. In in vitro Akt kinase assays, TCL1b enhanced Akt kinase activity in dose- and time-dependent manners. Bioinformatics approaches utilizing multiregression analysis, cluster analysis, KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway mapping, Venn diagrams and Gene Ontology (GO) demonstrated that TCL1b showed highly homologous gene-induction signatures similar to Myr-Akt or TCL1. TCL1b exhibited oncogenicity in in vitro colony-transformation assay. Further, two independent lines of β-actin promoter-driven TCL1b-transgenic mice developed angiosarcoma on the intestinal tract. Angiosarcoma is a rare form of cancer in humans with poor prognosis. Using immunohistochemistry, 11 out of 13 human angiosarcoma samples were positively stained with both anti-TCL1b and anti-phospho-Akt antibodies. Consistently, in various cancer tissues, 69 out of 146 samples were positively stained with anti-TCL1b, out of which 46 were positively stained with anti-phospho-Akt antibodies. Moreover, TCL1b structure-based inhibitor ‘TCL1b-Akt-in' inhibited Akt kinase activity in in vitro kinase assays and PDGF (platelet-derived growth factor)-induced Akt kinase activities—in turn, ‘TCL1b-Akt-in' inhibited cellular proliferation of sarcoma. The current study disclosed TCL1b bears oncogenicity and hence serves as a novel therapeutic target for human

Halofuginone inhibits Smad3 phosphorylation via the PI3K/Akt and MAPK/ERK pathways in muscle cells: Effect on myotube fusion

DOE Office of Scientific and Technical Information (OSTI.GOV)

Roffe, Suzy; Hagai, Yosey; Institute of Animal Sciences, Volcani Center, Bet Dagan 50250

2010-04-01

Halofuginone, a novel inhibitor of Smad3 phosphorylation, has been shown to inhibit muscle fibrosis and to improve cardiac and skeletal muscle functions in the mdx mouse model of Duchenne muscular dystrophy. Here, we demonstrate that halofuginone promotes the phosphorylation of Akt and mitogen-activated protein kinase (MAPK) family members in a C2 muscle cell line and in primary myoblasts derived from wild-type and mdx mice diaphragms. Halofuginone enhanced the association of phosphorylated Akt and MAPK/extracellular signal-regulated protein kinase (ERK) with the non-phosphorylated form of Smad3, accompanied by a reduction in Smad3 phosphorylation levels. This reduction was reversed by inhibitors of themore » phosphoinositide 3'-kinase/Akt (PI3K/Akt) and MAPK/ERK pathways, suggesting their specific role in mediating halofuginone's inhibitory effect on Smad3 phosphorylation. Halofuginone enhanced Akt, MAPK/ERK and p38 MAPK phosphorylation and inhibited Smad3 phosphorylation in myotubes, all of which are crucial for myotube fusion. In addition, halofuginone increased the association Akt and MAPK/ERK with Smad3. As a consequence, halofuginone promoted myotube fusion, as reflected by an increased percentage of C2 and mdx myotubes containing high numbers of nuclei, and this was reversed by specific inhibitors of the PI3K and MAPK/ERK pathways. Together, the data suggest a role, either direct or via inhibition of Smad3 phosphorylation, for Akt or MAPK/ERK in halofuginone-enhanced myotube fusion, a feature which is crucial to improving muscle function in muscular dystrophies.« less

Phosphorylation of AKT induced by phosphorylated Hsp27 confers the apoptosis-resistance in t-AUCB-treated glioblastoma cells in vitro.

PubMed

Li, Rujun; Li, Junyang; Sang, Dongping; Lan, Qing

2015-01-01

The aim of this study is to determine whether phosphorylation of AKT could be effected by t-AUCB-induced p-Hsp27 and whether p-AKT inhibition sensitizes glioblastoma cells to t-AUCB, and to evaluate the effects of simultaneous inhibition of p-Hsp27 and p-AKT on t-AUCB treated glioblastoma cells. Cell growth was detected using CCK-8 assay; Caspase-3 activity assay kits and flow cytometry were used in apoptosis analysis; Western blot analysis was used to detect p-Hsp27 and p-AKT levels; RNA interference using the siRNA oligos of Hsp27 was performed to knockdown gene expression of Hsp27. All data were analyzed by the Student-Newman-Keul's test. We demonstrated that t-AUCB treatment induces AKT phosphorylation by activating Hsp27 in U251 and LN443 cell lines. Inhibition of AKT phosphorylation by AKT inhibitor IV sensitizes glioblastoma cells to t-AUCB, strengthens t-AUCB suppressing cell growth and inducing cell apoptosis. We also found inhibiting both p-Hsp27 and p-AKT synergistically strengthen t-AUCB suppressing cell growth. Thus, p-AKT induced by p-Hsp27 confers the apoptosis-resistance in t-AUCB-treated glioblastoma cells. Targeting p-Hsp27 and/or p-AKT may be a potential effective strategy for the treatment of glioblastoma.

The Atypical Antipsychotic Agent, Clozapine, Protects Against Corticosterone-Induced Death of PC12 Cells by Regulating the Akt/FoxO3a Signaling Pathway.

PubMed

Zeng, Zhiwen; Wang, Xue; Bhardwaj, Sanjeev K; Zhou, Xuanhe; Little, Peter J; Quirion, Remi; Srivastava, Lalit K; Zheng, Wenhua

2017-07-01

Schizophrenia is one of the most severe psychiatric disorders. Increasing evidence implicates that neurodegeneration is a component of schizophrenia pathology and some atypical antipsychotics are neuroprotective and successful in slowing the progressive morphological brain changes. As an antipsychotic agent, clozapine has superior and unique effects, but the intracellular signaling pathways that mediate clozapine action remain to be elucidated. The phosphatidylinositol-3-kinase/protein kinase B/Forkhead box O3 (PI3K/Akt/FoxO3a) pathway is crucial for neuronal survival. However, little information is available regarding this pathway with clozapine. In the present study, we investigated the protective effect of clozapine on the PC12 cells against corticosterone toxicity. Our results showed that corticosterone decreases the phosphorylation of Akt and FoxO3a, leading to the nuclear localization of FoxO3a and the apoptosis of PC12 cells, while clozapine concentration dependently protected PC12 cells against corticosterone insult. Pathway inhibitors studies displayed that the protective effect of clozapine was reversed by LY294002 and wortmannin, two PI3K inhibitors, or Akt inhibitor VIII although several other inhibitors had no effect. The shRNA knockdown results displayed that downregulated Akt1 or FoxO3a attenuated the protective effect of clozapine. Western blot analyses revealed that clozapine induced the phosphorylation of Akt and FoxO3a by the PI3K/Akt pathway and reversed the reduction of the phosphorylated Akt and FoxO3a and the nuclear translocation of FoxO3a evoked by corticosterone. Together, our data indicates that clozapine protects PC12 cells against corticosterone-induced cell death by modulating activity of the PI3K/Akt/FoxO3a pathway.

Extensive Crosstalk between O-GlcNAcylation and Phosphorylation Regulates Akt Signaling

PubMed Central

Sun, Danni; Xin, Xianliang; Pan, Qiuming; Peng, Shuying; Liang, Zhongjie; Luo, Cheng; Yang, Yiming; Jiang, Hualiang; Huang, Min; Chai, Wengang; Ding, Jian; Geng, Meiyu

2012-01-01

O-linked N-acetylglucosamine glycosylations (O-GlcNAc) and O-linked phosphorylations (O-phosphate), as two important types of post-translational modifications, often occur on the same protein and bear a reciprocal relationship. In addition to the well documented phosphorylations that control Akt activity, Akt also undergoes O-GlcNAcylation, but the interplay between these two modifications and the biological significance remain unclear, largely due to the technique challenges. Here, we applied a two-step analytic approach composed of the O-GlcNAc immunoenrichment and subsequent O-phosphate immunodetection. Such an easy method enabled us to visualize endogenous glycosylated and phosphorylated Akt subpopulations in parallel and observed the inhibitory effect of Akt O-GlcNAcylations on its phosphorylation. Further studies utilizing mass spectrometry and mutagenesis approaches showed that O-GlcNAcylations at Thr 305 and Thr 312 inhibited Akt phosphorylation at Thr 308 via disrupting the interaction between Akt and PDK1. The impaired Akt activation in turn resulted in the compromised biological functions of Akt, as evidenced by suppressed cell proliferation and migration capabilities. Together, this study revealed an extensive crosstalk between O-GlcNAcylations and phosphorylations of Akt and demonstrated O-GlcNAcylation as a new regulatory modification for Akt signaling. PMID:22629392

Antiangiogenic treatment diminishes renal injury and dysfunction via regulation of local AKT in early experimental diabetes.

PubMed

Bai, Xiaoyan; Li, Xiao; Tian, Jianwei; Zhou, Zhanmei

2014-01-01

In view of increased vascular endothelial growth factor-A (VEGF-A) expression and renal dysfunction in early diabetes, we designed a study to test whether VEGF-A inhibition can prevent early renal injury and dysfunction. We investigated the relationship and mechanism between VEGF-A and AKT regulation. In vitro, VEGF-A small interfering RNA (siRNA) and AKT inhibitor MK-2206 were employed to podocytes and NRK-52 cells cultured in high glucose (30 mM). In vivo, the antiangiogenic drug endostatin was administered in 12 week-old streptozotocin-induced male Sprague Dawley rats. The levels of VEGF-A, AKT, phosphorylated Ser⁴⁷³-AKT, phosphorylated Thr³⁰⁸-AKT, nephrin, angiotensin II (Ang II), angiotensin type II receptor 1 (ATR1) were examined using quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR), Western blot analysis and immunohistochemistry. Interactions between phosphorylated Thr³⁰⁸-AKT and either nephrin in podocytes or Ang II in renal tubules were studied, respectively, using confocal immunofluorescence microscopy and immunoprecipitation. Silencing VEGF-A in podocytes upregulated phosphorylated Thr³⁰⁸-AKT and nephrin. Silencing VEGF-A in NRK-52E cells upregulated phosphorylated Thr³⁰⁸-AKT while downregulated Ang II and ATR1. MK-2206 enhanced VEGF-A expression in both podocytes and NRK-52E cells by inhibiting AKT activities. In diabetic rat kidneys, VEGF-A was upregulated and phosphorylated Thr³⁰⁸-AKT colocalized with either nephrin in podocytes or Ang II in renal tubules. With the endostatin treatment, the level of VEGF-A decreased while phosphorylated Thr³⁰⁸-AKT increased in both glomeruli and renal tubules. Treatment with endostatin upregulated nephrin in podocytes while downregulated Ang II and AT1R in renal tubules. Glomerular mesangial expansion was attenuated by the endostatin treatment, however, differences did not reach statistical significance. Endostatin ameliorated the interstitial fibrosis

Antiangiogenic Treatment Diminishes Renal Injury and Dysfunction via Regulation of Local AKT in Early Experimental Diabetes

PubMed Central

Zhou, Zhanmei

2014-01-01

In view of increased vascular endothelial growth factor-A (VEGF-A) expression and renal dysfunction in early diabetes, we designed a study to test whether VEGF-A inhibition can prevent early renal injury and dysfunction. We investigated the relationship and mechanism between VEGF-A and AKT regulation. In vitro, VEGF-A small interfering RNA (siRNA) and AKT inhibitor MK-2206 were employed to podocytes and NRK-52 cells cultured in high glucose (30 mM). In vivo, the antiangiogenic drug endostatin was administered in 12 week-old streptozotocin-induced male Sprague Dawley rats. The levels of VEGF-A, AKT, phosphorylated Ser473-AKT, phosphorylated Thr308-AKT, nephrin, angiotensin II (Ang II), angiotensin type II receptor 1 (ATR1) were examined using quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR), Western blot analysis and immunohistochemistry. Interactions between phosphorylated Thr308-AKT and either nephrin in podocytes or Ang II in renal tubules were studied, respectively, using confocal immunofluorescence microscopy and immunoprecipitation. Silencing VEGF-A in podocytes upregulated phosphorylated Thr308-AKT and nephrin. Silencing VEGF-A in NRK-52E cells upregulated phosphorylated Thr308-AKT while downregulated Ang II and ATR1. MK-2206 enhanced VEGF-A expression in both podocytes and NRK-52E cells by inhibiting AKT activities. In diabetic rat kidneys, VEGF-A was upregulated and phosphorylated Thr308-AKT colocalized with either nephrin in podocytes or Ang II in renal tubules. With the endostatin treatment, the level of VEGF-A decreased while phosphorylated Thr308-AKT increased in both glomeruli and renal tubules. Treatment with endostatin upregulated nephrin in podocytes while downregulated Ang II and AT1R in renal tubules. Glomerular mesangial expansion was attenuated by the endostatin treatment, however, differences did not reach statistical significance. Endostatin ameliorated the interstitial fibrosis, urine albumin excretion rate

Constitutively expressed COX-2 in osteoblasts positively regulates Akt signal transduction via suppression of PTEN activity.

PubMed

Li, Ching-Ju; Chang, Je-Ken; Wang, Gwo-Jaw; Ho, Mei-Ling

2011-02-01

Cyclooxygenase-2 (COX-2) is thought to be an inducible enzyme, but increasing reports indicate that COX-2 is constitutively expressed in several organs. The status of COX-2 expression in bone and its physiological role remains undefined. Non-selective non-steroidal anti-inflammatory drugs (NSAIDs) and selective COX-2 inhibitors, which commonly suppress COX-2 activity, were reported to suppress osteoblast proliferation via Akt/FOXO3a/p27(Kip1) signaling, suggesting that COX-2 may be the key factor of the suppressive effects of NSAIDs on proliferation. Although Akt activation correlates with PTEN deficiency and cell viability, the role of COX-2 on PTEN/Akt regulation remains unclear. In this study, we hypothesized that COX-2 may be constitutively expressed in osteoblasts and regulate PTEN/Akt-related proliferation. We examined the localization and co-expression of COX-2 and p-Akt in normal mouse femurs and in cultured mouse (mOBs) and human osteoblasts (hOBs). Our results showed that osteoblasts adjacent to the trabeculae, periosteum and endosteum in mouse femurs constitutively expressed COX-2, while COX-2 co-expressed with p-Akt in osteoblasts sitting adjacent to trabeculae in vivo, and in mOBs and hOBs in vitro. We further used COX-2 siRNA to test the role of COX-2 in Akt signaling in hOBs; COX-2 silencing significantly inhibited PTEN phosphorylation, enhanced PTEN activity, and suppressed p-Akt level and proliferation. However, replenishment of the COX-2 enzymatic product, PGE2, failed to reverse COX-2-dependent Akt phosphorylation. Furthermore, transfection with recombinant human COX-2 (rhCOX-2) significantly reversed COX-2 siRNA-suppressed PTEN phosphorylation, but this effect was reduced when the enzymatic activity of rhCOX-2 was blocked. This finding indicated that the effect of COX-2 on PTEN/Akt signaling is not related to PGE2 but still dependent on COX-2 enzymatic activity. Conversely, COX-1 silencing did not affect PTEN/Akt signaling. Our findings provide

Phosphorylated Akt Protein at Ser473 Enables HeLa Cells to Tolerate Nutrient-Deprived Conditions

PubMed

Fathy, Moustafa; Awale, Suresh; Nikaido, Toshio

2017-12-29

Background: Despite angiogenesis, many tumours remain hypovascular and starved of nutrients while continuing to grow rapidly. The specific biochemical mechanisms associated with starvation resistance, austerity, may be new biological characters of cancer that are critical for cancer progression. Objective: This study aim was to investigate the effect of nutrient starvation on HeLa cells and the possible mechanism by which the cells are able to tolerate nutrient-deprived conditions. Methods: Nutrient starvation was achieved by culturing HeLa cells in nutrient-deprived medium (NDM) and cell survival was estimated by using cell counting kit-8. The effect of starvation on cell cycle distribution and the quantitative analysis of apoptotic cells were investigated by flow cytometry using propidium iodide staining. Western blotting was used to detect the expression levels of Akt and phosphorylated Akt at Ser473 (Ser473p-Akt) proteins. Results: HeLa cells displayed extremely long survival when cultured in NDM. The percentage of apoptotic HeLa cells was significantly increased by starvation in a time-dependent manner. A significant increase in the expression of Ser473p-Akt protein after starvation was also observed. Furthermore, it was found that Akt inhibitor III molecule inhibited the cells proliferation in a concentration- and time-dependent manner. Conclusion: Results of the present study provide evidence that Akt activation may be implicated in the tolerance of HeLa cells for nutrient starvation and may help to suggest new therapeutic strategies designed to prevent austerity of cervical cancer cells through inhibition of Akt activation. Creative Commons Attribution License

The Role of Akt Isoforms in Colorectal Cancer

DTIC Science & Technology

2015-09-01

AD_________________ Award Number: W81XWH-13-1-0198 TITLE: The Role of Akt Isoforms in Colorectal Cancer PRINCIPAL INVESTIGATOR: Jatin Roper...CONTRACT NUMBER The Role of Akt Isoforms in Colorectal Cancer 5b. GRANT NUMBER W81XWH-13-1-0198 5c. PROGRAM ELEMENT NUMBER 6. AUTHOR(S) 5d. PROJECT NUMBER...substantially reduces colorectal tumorigenesis in our genetically engineered mouse model. We also successfully ablated novel downstream targets of Akt in our

Membrane Heterogeneity in Akt Activation in Prostate Cancer

DTIC Science & Technology

2009-03-01

raft microdomains. The purpose of this project is to identify the mechanism of Akt1 recruitment to cholesterol-rich microdomains and to explore the...to identify the mecha- nism of Akt1 recruitment to cholesterol-rich microdomains and to explore the biological consequences for regulation of this...I was able to identify an im- portant function for serine 473 and lysine 179 in regulating the admission of Akt1 to the lipid raft signaling platform

Membrane Heterogeneity in Akt Activation in Prostate Cancer

DTIC Science & Technology

2009-11-01

promote oncogenesis by altering the nature of Akt1 signals that flow through lipid raft microdomains. The purpose of this project was to identify the...altering the nature of Akt1 signals that flow through lipid raft microdomains. The purpose of this project was to identify the mecha- nism of Akt1...predominantly in lipid rafts. Consequently, I was able to identify an important function for serine 473 and lysine 179 in regulating the admission

Akt activation by Ca2+/calmodulin-dependent protein kinase kinase 2 (CaMKK2) in ovarian cancer cells.

PubMed

Gocher, Angela M; Azabdaftari, Gissou; Euscher, Lindsey M; Dai, Shuhang; Karacosta, Loukia G; Franke, Thomas F; Edelman, Arthur M

2017-08-25

Hyperactivation of Akt is associated with oncogenic changes in the growth, survival, and chemoresistance of cancer cells. The PI3K/phosphoinositide-dependent kinase (PDK) 1 pathway represents the canonical mechanism for phosphorylation of Akt at its primary activation site, Thr-308. We observed that Ca 2+ /calmodulin (CaM)-dependent protein kinase kinase 2 (β) (CaMKK2) is highly expressed in high-grade serous ovarian cancer, and we investigated its role in Akt activation in ovarian cancer (OVCa) cell lines (OVCAR-3, SKOV-3, and Caov-3). Knockdown or pharmacological inhibition of CaMKK2 produced phenotypes expected of Akt inhibition, including reductions in cell growth and cell viability and in the regulation of Akt downstream targets involved in G 1 /S transition and apoptosis. CaMKK2 knockdown or inhibition decreased Akt phosphorylation at Thr-308 and Ser-473 to extents similar to those of PDK1 knockdown or PI3K inhibition. Combined CaMKK2 and PDK1 knockdown or CaMKK and PI3K inhibition, respectively, produced additive effects on p-Akt and cell growth, consistent with direct Akt phosphorylation by CaMKK2. This conclusion was supported by the absence of effects of CaMKK2 knockdown/inhibition on alternative means of activating Akt via p-Akt Thr-450, p-PDK1 Ser-241, or p-IRS1 Ser-636/639. Recombinant CaMKK2 directly activated recombinant Akt by phosphorylation at Thr-308 in a Ca 2+ /CaM-dependent manner. In OVCa cells, p-Akt Thr-308 was significantly inhibited by intracellular Ca 2+ i chelation or CaM inhibition. Ionomycin-induced Ca 2+ influx promoted p-Akt, an effect blocked by PDK1, and/or CaMKK2, siRNAs, and by PI3K and/or CaMKK inhibitors. CaMKK2 knockdown potentiated the effects of the chemotherapeutic drugs carboplatin and PX-866 to reduce proliferation and survival of OVCa cells. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

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. © 2015 Authors; published by Portland Press Limited.

Akt phosphorylation and NFkappaB activation are counterregulated under conditions of oxidative stress.

PubMed

Taylor, Juliet M; Crack, Peter J; Gould, Jodee A; Ali, Uğur; Hertzog, Paul J; Iannello, Rocco C

2004-11-01

This study was designed to elucidate the mechanisms involved in elevated cell death arising from an altered endogenous oxidant state. Increased levels of cell death were detected in cells lacking Gpx1 following the addition of exogenous H2O2. This increased apoptosis correlated with a down-regulation in the activation of the PI(3)K-Akt survival pathway. The importance of this pathway in protecting against H2O2-induced cell death was highlighted by the increased susceptibility of wild-type cells to apoptosis when treated with the PI(3)K inhibitor, LY294002. Activation of the oxidative stress sensitive transcription factor, NFkappaB, was elevated in the Gpx1-/- cells. Significantly, NFkappaB activation could be increased in wild-type cells through the addition of dominant-negative Akt. Therefore, our results suggest that the increased susceptibility of Gpx1-/- cells to H2O2-induced apoptosis can be attributed in part to diminished activation of Akt despite an up-regulation in the activation of the prosurvival NFkappaB. Thus, the PI(3)K-Akt and NFkappaB pathways can act independently of each other in an endogenous model of oxidative stress.

Complications of hyperglycaemia with PI3K-AKT-mTOR inhibitors in patients with advanced solid tumours on Phase I clinical trials.

PubMed

Geuna, E; Roda, D; Rafii, S; Jimenez, B; Capelan, M; Rihawi, K; Montemurro, F; Yap, T A; Kaye, S B; De Bono, J S; Molife, L R; Banerji, U

2015-12-01

PI3K-AKT-mTOR inhibitors (PAMi) are promising anticancer treatments. Hyperglycaemia is a mechanism-based toxicity of these agents and is becoming increasingly important with their use in larger numbers of patients. Retrospective case-control study comparing incidence and severity of hyperglycaemia (all grades) between a case group of 387 patients treated on 18 phase I clinical trials with PAMi (78 patients with PI3Ki, 138 with mTORi, 144 with AKTi and 27 with PI3K/mTORi) and a control group of 109 patients treated on 10 phase I clinical trials with agents not directly targeting the PAM pathway. Diabetic patients were excluded in both groups. The incidence of hyperglycaemia was not significantly different between cases and controls (86.6% vs 80.7%, respectively, P=0.129). However, high grade (grade 3-4) hyperglycaemia was more frequent in the PAMi group than in controls (6.7% vs 0%, respectively, P=0.005). The incidence of grade 3-4 hyperglycaemia was greater with AKT and multikinase inhibitors compared with other PAMi (P<0.001). All patients with high-grade hyperglycaemia received antihyperglycemic treatment and none developed severe metabolic complications (diabetic ketoacidosis or hyperosmolar hyperglycemic nonketotic state). High-grade hyperglycaemia was the cause of permanent PAMi discontinuation in nine patients. PI3K-AKT-mTOR inhibitors are associated with small (6.7%) but statistically significant increased risk of high-grade hyperglycaemia compared with non-PAM targeting agents. However, PAMi-induced hyperglycaemia was not found to be associated with severe metabolic complications in this non-diabetic population of patients with advanced cancers.

mTOR Complex 2 mediates Akt Phosphorylation that Requires PKCε in Adult Cardiac Muscle Cells

PubMed Central

Moschella, Phillip C.; McKillop, John; Pleasant, Dorea L.; Harston, Rebecca K.; Balasubramanian, Sundaravadivel; Kuppuswamy, Dhandapani

2013-01-01

Our earlier work showed that mammalian target of rapamycin (mTOR) is essential to the development of various hypertrophic responses, including cardiomyocyte survival. mTOR forms two independent complexes, mTORC1 and mTORC2, by associating with common and distinct cellular proteins. Both complexes are sensitive to a pharmacological inhibitor, torin1, although only mTORC1 is inhibited by rapamycin. Since mTORC2 is known to mediate the activation of a prosurvival kinase, Akt, we analyzed whether mTORC2 directly mediates Akt activation or whether it requires the participation of another prosurvival kinase, PKC ε (epsilon isoform of protein kinase-C). Our studies reveal that treatment of adult feline cardiomyocytes in vitro with insulin results in Akt phosphorylation at S473 for its activation which could be augmented with rapamycin but blocked by torin1. Silencing the expression of Rictor (rapamycin-insensitive companion of mTOR), an mTORC2 component, with a sh-RNA in cardiomyocytes lowers both insulin-stimulated Akt and PKC ε phosphorylation. Furthermore, phosphorylation of PKC ε and Akt at the critical S729 and S473 sites respectively was blocked by torin1 or Rictor knockdown but not by rapamycin, indicating that the phosphorylation at these specific sites occurs downstream of mTORC2. Additionally, expression of DN-PKC ε significantly lowered the insulin-stimulated Akt S473 phosphorylation, indicating an upstream role for PKC ε in the Akt activation. Biochemical analyses also revealed that PKC ε was part of Rictor but not Raptor (a binding partner and component of mTORC1). Together, these studies demonstrate that mTORC2 mediates prosurvival signaling in adult cardiomyocytes where PKC ε functions downstream of mTORC2 leading to Akt activation. PMID:23673367

mTOR complex 2 mediates Akt phosphorylation that requires PKCε in adult cardiac muscle cells.

PubMed

Moschella, Phillip C; McKillop, John; Pleasant, Dorea L; Harston, Rebecca K; Balasubramanian, Sundaravadivel; Kuppuswamy, Dhandapani

2013-09-01

Our earlier work showed that mammalian target of rapamycin (mTOR) is essential to the development of various hypertrophic responses, including cardiomyocyte survival. mTOR forms two independent complexes, mTORC1 and mTORC2, by associating with common and distinct cellular proteins. Both complexes are sensitive to a pharmacological inhibitor, torin1, although only mTORC1 is inhibited by rapamycin. Since mTORC2 is known to mediate the activation of a prosurvival kinase, Akt, we analyzed whether mTORC2 directly mediates Akt activation or whether it requires the participation of another prosurvival kinase, PKCε (epsilon isoform of protein kinase-C). Our studies reveal that treatment of adult feline cardiomyocytes in vitro with insulin results in Akt phosphorylation at S473 for its activation which could be augmented with rapamycin but blocked by torin1. Silencing the expression of Rictor (rapamycin-insensitive companion of mTOR), an mTORC2 component, with a sh-RNA in cardiomyocytes lowers both insulin-stimulated Akt and PKCε phosphorylation. Furthermore, phosphorylation of PKCε and Akt at the critical S729 and S473 sites respectively was blocked by torin1 or Rictor knockdown but not by rapamycin, indicating that the phosphorylation at these specific sites occurs downstream of mTORC2. Additionally, expression of DN-PKCε significantly lowered the insulin-stimulated Akt S473 phosphorylation, indicating an upstream role for PKCε in the Akt activation. Biochemical analyses also revealed that PKCε was part of Rictor but not Raptor (a binding partner and component of mTORC1). Together, these studies demonstrate that mTORC2 mediates prosurvival signaling in adult cardiomyocytes where PKCε functions downstream of mTORC2 leading to Akt activation. Copyright © 2013 Elsevier Inc. All rights reserved.

Akt1-Inhibitor of DNA binding2 is essential for growth cone formation and axon growth and promotes central nervous system axon regeneration

PubMed Central

Ko, Hyo Rim; Kwon, Il-Sun; Hwang, Inwoo; Jin, Eun-Ju; Shin, Joo-Ho; Brennan-Minnella, Angela M; Swanson, Raymond; Cho, Sung-Woo; Lee, Kyung-Hoon; Ahn, Jee-Yin

2016-01-01

Mechanistic studies of axon growth during development are beneficial to the search for neuron-intrinsic regulators of axon regeneration. Here, we discovered that, in the developing neuron from rat, Akt signaling regulates axon growth and growth cone formation through phosphorylation of serine 14 (S14) on Inhibitor of DNA binding 2 (Id2). This enhances Id2 protein stability by means of escape from proteasomal degradation, and steers its localization to the growth cone, where Id2 interacts with radixin that is critical for growth cone formation. Knockdown of Id2, or abrogation of Id2 phosphorylation at S14, greatly impairs axon growth and the architecture of growth cone. Intriguingly, reinstatement of Akt/Id2 signaling after injury in mouse hippocampal slices redeemed growth promoting ability, leading to obvious axon regeneration. Our results suggest that Akt/Id2 signaling is a key module for growth cone formation and axon growth, and its augmentation plays a potential role in CNS axonal regeneration. DOI: http://dx.doi.org/10.7554/eLife.20799.001 PMID:27938661

Akt, mTOR and NF-κB pathway activation in Treponema pallidum stimulates M1 macrophages.

PubMed

Lin, Li-Rong; Gao, Zheng-Xiang; Lin, Yong; Zhu, Xiao-Zhen; Liu, Wei; Liu, Dan; Gao, Kun; Tong, Man-Li; Zhang, Hui-Lin; Liu, Li-Li; Xiao, Yao; Niu, Jian-Jun; Liu, Fan; Yang, Tian-Ci

2018-06-01

The polarization of macrophages and the molecular mechanism involved during the early process of syphilis infection remain unknown. This study was conducted to explore the influence of Treponema pallidum (T. pallidum) treatment on macrophage polarization and the Akt-mTOR-NFκB signaling pathway mechanism involved in this process. M0 macrophages derived from the phorbol-12-myristate-13-acetate-induced human acute monocytic leukemia cell line THP-1 were cultured with T. pallidum. T. pallidum induced inflammatory cytokine (IL-1β and TNF-α) expression in a dose- and time-dependent manner. However IL-10 cytokine expression decreased at the mRNA and protein levels. Additionally, the expression of the M1 surface marker iNOS was up-regulated with incubation time, and the expression of the M2 surface marker CD206 was low (vs. PBS treated macrophages, P < 0.001) and did not fluctuate over 12 h. Further studies revealed that Akt-mTOR-NFκB pathway proteins, including p-Akt, p-mTOR, p-S6, p-p65, and p-IκBα, were significantly higher in the T. pallidum-treated macrophages than in the PBS-treated macrophages (P < 0.05). In addition, inflammatory cytokine expression was suppressed in T. pallidum-induced M1 macrophages pretreated with LY294002 (an Akt-specific inhibitor) or PDTC (an NF-κB inhibitor), while inflammatory cytokine levels increased in T. pallidum-induced M1 macrophages pretreated with rapamycin (an mTOR inhibitor). These findings revealed that T. pallidum promotes the macrophage transition to pro-inflammatory M1 macrophages in vitro. The present study also provides evidence that Akt, mTOR and NF-κB pathway activation in T. pallidum stimulates M1 macrophages. This study provides novel insights into the innate immune response to T. pallidum infection. Copyright © 2018. Published by Elsevier B.V.

Constitutively Active Akt Induces Ectodermal Defects and Impaired Bone Morphogenetic Protein Signaling

PubMed Central

Segrelles, Carmen; Moral, Marta; Lorz, Corina; Santos, Mirentxu; Lu, Jerry; Cascallana, José Luis; Lara, M. Fernanda; Carbajal, Steve; Martínez-Cruz, Ana Belén; García-Escudero, Ramón; Beltran, Linda; Segovia, José C.; Bravo, Ana

2008-01-01

Aberrant activation of the Akt pathway has been implicated in several human pathologies including cancer. However, current knowledge on the involvement of Akt signaling in development is limited. Previous data have suggested that Akt-mediated signaling may be an essential mediator of epidermal homeostasis through cell autonomous and noncell autonomous mechanisms. Here we report the developmental consequences of deregulated Akt activity in the basal layer of stratified epithelia, mediated by the expression of a constitutively active Akt1 (myrAkt) in transgenic mice. Contrary to mice overexpressing wild-type Akt1 (Aktwt), these myrAkt mice display, in a dose-dependent manner, altered development of ectodermally derived organs such as hair, teeth, nails, and epidermal glands. To identify the possible molecular mechanisms underlying these alterations, gene profiling approaches were used. We demonstrate that constitutive Akt activity disturbs the bone morphogenetic protein-dependent signaling pathway. In addition, these mice also display alterations in adult epidermal stem cells. Collectively, we show that epithelial tissue development and homeostasis is dependent on proper regulation of Akt expression and activity. PMID:17959825

Membrane Heterogeneity in Akt Activation in Prostate Cancer

DTIC Science & Technology

2009-07-01

oncogenesis by altering the nature of Akt1 signals that flow through lipid raft microdomains. The purpose of this project is to identify the mechanism of...nature of Akt1 signals that flow through lipid raft microdomains. The purpose of this project was to identify the mecha- nism of Akt1 recruitment to...predominantly in lipid rafts (for details see Annual Summary Report March 2008). Consequently, I was able to identify an im- portant function for

CD44 regulates cell migration in human colon cancer cells via Lyn kinase and AKT phosphorylation.

PubMed

Subramaniam, Venkateswaran; Vincent, Isabella R; Gardner, Helena; Chan, Emily; Dhamko, Helena; Jothy, Serge

2007-10-01

Colon cancer is among the leading causes of cancer death in North America. CD44, an adhesion and antiapoptotic molecule is overexpressed in colon cancer. Cofilin is involved in the directional motility of cells. In the present study, we looked at how CD44 might modulate cell migration in human colon cancer via cofilin. We used a human colon cancer cell line, HT29, which expresses CD44, HT29 where CD44 expression was knocked down by siRNA, SW620, a human colon cancer cell line which does not express CD44, stably transfected exons of CD44 in SW620 cells and the colon from CD44 knockout and wild-type mouse. Western blot analysis of siRNA CD44 lysates showed increased level of AKT phosphorylation and decreased level of cofilin expression. Similar results were also observed with SW620 cells and CD44 knockout mouse colon lysates. Experiments using the AKT phosphorylation inhibitor LY294002 indicate that AKT phosphorylation downregulates cofilin. Immunoprecipitation studies showed CD44 complex formation with Lyn, providing an essential link between CD44 and AKT phosphorylation. LY294002 also stabilized Lyn from phosphorylated AKT, suggesting an interaction between Lyn and AKT phosphorylation. Immunocytochemistry showed that cofilin and Lyn expression were downregulated in siRNA CD44 cells and CD44 knockout mouse colon. siRNA CD44 cells had significantly less migration compared to HT29 vector. Given the well-defined roles of CD44, phosphorylated AKT in apoptosis and cancer, these results indicate that CD44-induced cell migration is dependent on its complex formation with Lyn and its consequent regulation of AKT phosphorylation and cofilin expression.

[Exendin-4 promotes paracrine action of adipose-derived stem cells through PI3K/Akt signaling pathways].

PubMed

Zhou, Hao; Yang, Junjie; Wagn, Jing; Hu, Shunying; Chen, Guanghui; Chen, Yundai

2014-10-01

To investigate the mechanism by which exendin-4 promotes paracrine secretion of cytokines by adipose-derived stem cells (ADSCs). In vitro cultured SD rat ADSCs (fourth passage) with or without exendin-4 treatment underwent flow cytometry to characterize the surface markers. MTT assay was performed to assess the proliferation of the cells exposed to different concentrations (0-20 nm/L) of exendin-4, and the paracrine secretion of cytokines (bFGF, VEGF, HGF, and IGF-1) by the ADSCs was evaluated by qPCR. The changes in the expressions of p-Akt in the cells were analyzed by Western blotting and qPCR in response to exendin-4 (10 nm/L) with or without exposure to PI3K/Akt inhibitor LY-294002 (50 nm/L); bFGF, VEGF, HGF, and IGF-1 production in the cells were detected using ELISA kits. Treatment with exendin-4 for 12 h did not affect the surface marker profile of the ADSCs but promoted the cell proliferation (P<0.05). Exendin-4 significantly increased the mRNA expressions of VEGF, bFGF, HGF, and IGF-1 in a concentration-dependent manner, and 10 nm/L was the optimum concentration (P<0.05). Exendin-4 treatment resulted in significantly increased p-Akt expressions in the ADSCs, and PI3K/Akt inhibitor not only reversed such effects of exendin-4 on p-Akt but also diminished the exendin-4- mediated up-regulation of the paracrine cytokines. Exendin-4 can concentration-dependently promote the proliferative and paracrine capacities of ADSCs partially through the PI3K/Akt signaling pathway without affecting the surface marker profile of the cells.

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

NASA Technical Reports Server (NTRS)

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

2001-01-01

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

Id-1 activation of PI3K/Akt/NFkappaB signaling pathway and its significance in promoting survival of esophageal cancer cells.

PubMed

Li, Bin; Cheung, Pak Yan; Wang, Xianghong; Tsao, Sai Wah; Ling, Ming Tat; Wong, Yong Chuan; Cheung, Annie L M

2007-11-01

Inhibitor of differentiation or DNA binding (Id-1) is a helix-loop-helix protein that is over-expressed in many types of cancer including esophageal cancer. This study aims to investigate its effects on the phosphatidylinositol-3-kinase (PI3K)/Akt/ nuclear factor kappa B (NFkappaB) signaling pathway and the significance in protecting esophageal cancer cells against apoptosis. We found elevated expression of phosphorylated forms of Akt, glycogen synthase kinase 3beta and inhibitor of kappa B, as well as increased nuclear translocation of NFkappaB subunit p65 and NFkappaB DNA-binding activity, in esophageal cancer cells with stable ectopic Id-1 expression. Transient transfection of Id-1 into HEK293 cells confirmed activation of PI3K/Akt/NFkappaB signaling and the effects were counteracted by the PI3K inhibitor LY294002. Treatment with tumor necrosis factor-alpha (TNF-alpha) elicited a significantly weaker apoptotic response, following a marked and sustained activation of Akt and NFkappaB in the Id-1-over-expressing cells, compared with the vector control. The effects of Id-1 on the PI3K/Akt/NFkappaB signaling pathway and apoptosis were reversed in esophageal cancer cells transfected with siRNA against Id-1. In addition, inhibition of PI3K or NFkappaB signaling using the PI3K inhibitor LY294002 or the NFkappaB inhibitor Bay11-7082 increased the sensitivity of Id-1-over-expressing esophageal cancer cells to TNF-alpha-induced apoptosis. Our results provide the first evidence that Id-1 induces the activation of PI3K/Akt/NFkappaB signaling pathway, and protects esophageal cancer cells from TNF-alpha-induced apoptosis in vitro. Inactivation of Id-1 may provide us with a novel strategy to improve the treatment and survival of patients with esophageal cancer.

AKT Kinase Activity Is Required for Lithium to Modulate Mood-Related Behaviors in Mice

PubMed Central

Pan, Jen Q; Lewis, Michael C; Ketterman, Josh K; Clore, Elizabeth L; Riley, Misha; Richards, Keenan R; Berry-Scott, Erin; Liu, Xiulin; Wagner, Florence F; Holson, Edward B; Neve, Rachael L; Biechele, Travis L; Moon, Randall T; Scolnick, Edward M; Petryshen, Tracey L; Haggarty, Stephen J

2011-01-01

Bipolar disorder (BP) is a debilitating psychiatric disorder, affecting ∼2% of the worldwide population, for which the etiological basis, pathogenesis, and neurocircuitry remain poorly understood. Individuals with BP suffer from recurrent episodes of mania and depression, which are commonly treated with the mood stabilizer lithium. However, nearly half of BP patients do not respond adequately to lithium therapy and the clinically relevant mechanisms of lithium for mood stabilization remain elusive. Here, we modeled lithium responsiveness using cellular assays of glycogen synthase kinase 3 (GSK-3) signaling and mood-related behavioral assays in inbred strains of mice that differ in their response to lithium. We found that activating AKT through phosphosrylation of a key regulatory site (Thr308) was associated with lithium response—activation of signaling pathways downstream of GSK-3 in cells and attenuation of mood-related behaviors in mice—and this response was attenuated by selective and direct inhibition of AKT kinase activity. Conversely, the expression of constitutively active AKT1 in both the cellular and behavioral assays conferred lithium sensitivity. In contrast, selective and direct GSK-3 inhibition by the ATP-competitive inhibitor CHIR99021 bypassed the requirement for AKT activation and modulated behavior in both lithium-responsive and non-responsive mouse strains. These results distinguish the mechanism of action of lithium from direct GSK-3 inhibition both in vivo and in vitro, and highlight the therapeutic potential for selective GSK-3 inhibitors in BP treatment. PMID:21389981

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

PubMed

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

2012-08-01

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

oxLDL induces endothelial cell proliferation via Rho/ROCK/Akt/p27kip1 signaling: opposite effects of oxLDL and cholesterol loading.

PubMed

Zhang, Chongxu; Adamos, Crystal; Oh, Myung-Jin; Baruah, Jugajyoti; Ayee, Manuela A A; Mehta, Dolly; Wary, Kishore K; Levitan, Irena

2017-09-01

Oxidized modifications of LDL (oxLDL) play a key role in the development of endothelial dysfunction and atherosclerosis. However, the underlying mechanisms of oxLDL-mediated cellular behavior are not completely understood. Here, we compared the effects of two major types of oxLDL, copper-oxidized LDL (Cu 2+ -oxLDL) and lipoxygenase-oxidized LDL (LPO-oxLDL), on proliferation of human aortic endothelial cells (HAECs). Cu 2+ -oxLDL enhanced HAECs' proliferation in a dose- and degree of oxidation-dependent manner. Similarly, LPO-oxLDL also enhanced HAEC proliferation. Mechanistically, both Cu 2+ -oxLDL and LPO-oxLDL enhance HAEC proliferation via activation of Rho, Akt phosphorylation, and a decrease in the expression of cyclin-dependent kinase inhibitor 1B (p27 kip1 ). Both Cu 2+ -oxLDL or LPO-oxLDL significantly increased Akt phosphorylation, whereas an Akt inhibitor, MK2206, blocked oxLDL-induced increase in HAEC proliferation. Blocking Rho with C3 or its downstream target ROCK with Y27632 significantly inhibited oxLDL-induced Akt phosphorylation and proliferation mediated by both Cu 2+ - and LPO-oxLDL. Activation of RhoA was blocked by Rho-GDI-1, which also abrogated oxLDL-induced Akt phosphorylation and HAEC proliferation. In contrast, blocking Rac1 in these cells had no effect on oxLDL-induced Akt phosphorylation or cell proliferation. Moreover, oxLDL-induced Rho/Akt signaling downregulated cell cycle inhibitor p27 kip1 Preloading these cells with cholesterol, however, prevented oxLDL-induced Akt phosphorylation and HAEC proliferation. These findings provide a new understanding of the effects of oxLDL on endothelial proliferation, which is essential for developing new treatments against neovascularization and progression of atherosclerosis. Copyright © 2017 the American Physiological Society.

Phosphatidic acid induces decidualization by stimulating Akt-PP2A binding in human endometrial stromal cells.

PubMed

Lee, So Young; Lee, Yun Young; Choi, Joong Sub; Yoon, Mee-Sup; Han, Joong-Soo

2016-11-01

Decidualization of human endometrial stromal cells (hESCs) is crucial for successful uterine implantation and maintaining pregnancy. We previously reported that phospholipase D1 (PLD1) is required for cAMP-induced decidualization of hESCs. However, the mechanism by which phosphatidic acid (PA), the product of PLD1 action, might regulate decidualization is not known. We confirmed that PA induced decidualization of hESCs by observing morphological changes and measuring increased levels of decidualization markers such as IGFBP1 and prolactin transcripts (P < 0.05). Treatment with PA reduced phosphorylation of Akt and consequently that of FoxO1, which led to the increased IGFBP1 and prolactin mRNA levels (P < 0.05). Conversely, PLD1 knockdown rescued Akt phosphorylation. Binding of PP2A and Akt increased in response to cAMP or PA, suggesting that their binding is directly responsible for the inactivation of Akt during decidualization. Consistent with this observation, treatment with okadaic acid, a PP2A inhibitor, also inhibited cAMP-induced decidualization by blocking Akt dephosphorylation. © 2016 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.

Phosphorylated Akt/PKB controls cell growth and apoptosis in intraductal papillary-mucinous tumor and invasive ductal adenocarcinoma of the pancreas.

PubMed

Semba, Shuho; Moriya, Toshiyuki; Kimura, Wataru; Yamakawa, Mitsunori

2003-04-01

Akt/PKB promotes cell proliferation and rescues cells from apoptosis. To evaluate the role of Akt/PKB, a key molecule in phosphatidylinositol 3-OH kinase (PI3K) signaling, during the development of pancreatic duct neoplasias such as intraductal papillary-mucinous tumor (IPMT) and invasive ductal adenocarcinoma (IDAC) of the pancreas. In PK-45H pancreatic cancer cells, the growth-inhibitory and apoptosis-inducing effects of LY294002, a PI3K inhibitor, were detected in a concentration-dependent manner, followed by the reduction of phosphorylated Akt levels. Immunohistochemical analyses revealed that frequent overexpression of phosphorylated Akt (Ser473) was detected in 10 (63%) of 16 IPMTs and 14 (70%) of 20 IDACs. It is interesting that the incidence of Akt phosphorylation closely correlated with Ki-67 immunoreactivity and had an inverse association with the number of cases of apoptotic bodies in both IPMT and IDAC. Although there was no good correlation with other clinicopathologic parameters, the two patients with recurrent IPMT had high levels of phosphorylated Akt. Our findings suggest that activation of Akt plays an important role during the progression of these pancreatic duct neoplasias at the early stage. Furthermore, inhibition of the PI3K-Akt/PKB pathway may have therapeutic potential in the treatment of pancreatic duct tumors.

Akt1 Controls the Timing and Amplitude of Vascular Circadian Gene Expression

PubMed Central

Luciano, Amelia K.; Santana, Jeans M.; Velazquez, Heino; Sessa, William C.

2017-01-01

The AKT signaling pathway is important for circadian rhythms in mammals and flies (Drosophila). However, AKT signaling in mammals is more complicated since there are 3 isoforms of AKT, each performing slightly different functions. Here we study the most ubiquitous AKT isoform, Akt1, and its role at the organismal level in the central and vascular peripheral clocks. Akt1−/− mice exhibit relatively normal behavioral rhythms with only minor differences in circadian gene expression in the liver and heart. However, circadian gene expression in the Akt1−/− aorta, compared with control aorta, follows a distinct pattern. In the Akt1−/− aorta, positive regulators of circadian transcription have lower amplitude rhythms and peak earlier in the day, and negative circadian regulators are expressed at higher amplitudes and peak later in the day. In endothelial cells, negative circadian regulators exhibit an increased amplitude of expression, while the positive circadian regulators are arrhythmic with a decreased amplitude of expression. This indicates that Akt1 conditions the normal circadian rhythm in the vasculature more so than in other peripheral tissues where other AKT isoforms or kinases might be important for daily rhythms. PMID:28452287

Akt1 Controls the Timing and Amplitude of Vascular Circadian Gene Expression.

PubMed

Luciano, Amelia K; Santana, Jeans M; Velazquez, Heino; Sessa, William C

2017-06-01

The AKT signaling pathway is important for circadian rhythms in mammals and flies ( Drosophila). However, AKT signaling in mammals is more complicated since there are 3 isoforms of AKT, each performing slightly different functions. Here we study the most ubiquitous AKT isoform, Akt1, and its role at the organismal level in the central and vascular peripheral clocks. Akt1 -/- mice exhibit relatively normal behavioral rhythms with only minor differences in circadian gene expression in the liver and heart. However, circadian gene expression in the Akt1 -/- aorta, compared with control aorta, follows a distinct pattern. In the Akt1 -/- aorta, positive regulators of circadian transcription have lower amplitude rhythms and peak earlier in the day, and negative circadian regulators are expressed at higher amplitudes and peak later in the day. In endothelial cells, negative circadian regulators exhibit an increased amplitude of expression, while the positive circadian regulators are arrhythmic with a decreased amplitude of expression. This indicates that Akt1 conditions the normal circadian rhythm in the vasculature more so than in other peripheral tissues where other AKT isoforms or kinases might be important for daily rhythms.

A Phosphatidylinositol 3-kinase-regulated Akt-independent signaling promotes cigarette smoke-induced FRA-1 expression.

PubMed

Zhang, Qin; Adiseshaiah, Pavan; Kalvakolanu, Dhananjaya V; Reddy, Sekhar P

2006-04-14

The FRA-1 proto-oncogene is overexpressed in a variety of human tumors and is known to up-regulate the expression of genes involved in tumor progression and invasion. The phosphatidylinositol 3-kinase (PI3K)-Akt pathway is also known to regulate these cellular processes. More importantly, respiratory toxicants and carcinogens activate both the PI3K-Akt pathway and FRA-1 expression in human bronchial epithelial (HBE) cells. In this study we investigated a potential link between the PI3K-Akt pathway and the cigarette smoke (CS)-stimulated epidermal growth factor receptor-mediated FRA-1 induction in non-oncogenic HBE cells. Treatment of cells with LY294002, an inhibitor of the PI3K-Akt pathway, completely blocked CS-induced FRA-1 expression. Surprisingly pharmacological inhibition of Akt had no significant effect on CS-induced FRA-1 expression. Likewise the inhibition of protein kinase C zeta, which is a known downstream effector of PI3K, did not alter FRA-1 expression. We found that the PI3K through p21-activated kinase 1 regulates FRA-1 proto-oncogene induction by CS and the subsequent activation of the Elk1 and cAMP-response element-binding protein transcription factors that are bound to the promoter in HBE cells.

EZH2 promotes tumor progression via regulating VEGF-A/AKT signaling in non-small cell lung cancer.

PubMed

Geng, Jian; Li, Xiao; Zhou, Zhanmei; Wu, Chin-Lee; Dai, Meng; Bai, Xiaoyan

2015-04-10

Enhancer of Zeste Homologue 2 (EZH2) accounts for aggressiveness and unfavorable prognosis of tumor. We investigated the mechanisms and signaling pathways of EZH2 in non-small cell lung carcinoma (NSCLC) progression. Increased expression of EZH2, vascular endothelial growth factor-A (VEGF-A) and AKT phosphorylation correlated with differentiation, lymph node metastasis, size and TNM stage in NSCLC. There was a positive correlation between EZH2 and VEGF-A expression and high EZH2 expression, as an independent prognostic factor, predicted a shorter overall survival time for NSCLC patients. The expression of VEGF-A and phosphorylated Ser(473)-AKT, cell proliferation, migration and metastasis were enhanced in EZH2-overexpressing A549 cells, but inhibited in parental H2087 cells with EZH2 silencing or GSK126 treatment. AKT activity was enhanced by recombinant human VEGF-165 but suppressed by bevacizumab. An AKT inhibitor MK-2206 blocked VEGF-A expression and AKT phosphorylation in parental H2087 and EZH2-overexpressing A549 cells. EZH2 activity was not affected by either VEGF-A stimulation/depletion or MK-2206 inhibition. These results demonstrate that EZH2 promotes lung cancer progression via the VEGF-A/AKT signaling pathway. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

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

Apigenin inhibits cell proliferation, migration, and invasion by targeting Akt in the A549 human lung cancer cell line.

PubMed

Zhou, Zhongping; Tang, Miaomiao; Liu, Yi; Zhang, Zhuyi; Lu, Rongzhu; Lu, Jian

2017-04-01

Apigenin (APG), a widely distributed flavonoid in vegetables and fruits, with low toxicity, and a nonmutagenic characteristic, has been reported to have many targets. Evidence indicates that APG can inhibit the proliferation, migration, invasion, and metastasis of some tumor cells, but the mechanism, specifically in lung cancer, is unclear. The phosphoinositide 3-kinase (PI3K)/Akt signaling pathway regulates a diverse set of cellular functions relevant to the growth and progression of lung cancer, including proliferation, survival, migration, and invasion. Our results showed that APG exerted anti-proliferation, anti-migration, and anti-invasion effects in A549 human lung cancer cells by targeting the PI3K/Akt signaling pathway. 3-(4, 5-dimethylthiszol-2-yl)-2, 5-diphenytetrazolium bromide assay and colony formation assay showed that APG suppressed cell proliferation in a dose-dependent and time-dependent manner. Cell motility and invasiveness were assayed using a wound healing and Transwell assay, suggesting that APG inhibited the migration and invasion of A549 cells. Western blot analyses were carried out to examine the Akt signaling pathways. The results confirmed that APG decreased Akt expression and its activation. Then, cells were transfected with Akt-active and Akt-DN plasmids separately. The migration and invasion of A549 cells were significantly changed, constitutively activating Akt or knocking down Akt, indicating that APG can suppress the migration and invasion of lung cancer cells by modulating the PI3K/Akt signaling pathway. Furthermore, the results indicated that APG not only suppressed phosphorylation of Akt, thereby preventing its activation, but also inhibited its downstream gene expression of matrix metalloproteinases-9, glycogen synthase kinase-3β, and HEF1. Together, APG is a new inhibitor of Akt in lung cancer and a potential natural compound for cancer chemoprevention.

M-COPA suppresses endolysosomal Kit-Akt oncogenic signalling through inhibiting the secretory pathway in neoplastic mast cells.

PubMed

Hara, Yasushi; Obata, Yuuki; Horikawa, Keita; Tasaki, Yasutaka; Suzuki, Kyohei; Murata, Takatsugu; Shiina, Isamu; Abe, Ryo

2017-01-01

Gain-of-function mutations in Kit receptor tyrosine kinase result in the development of a variety of cancers, such as mast cell tumours, gastrointestinal stromal tumours (GISTs), acute myeloid leukemia, and melanomas. The drug imatinib, a selective inhibitor of Kit, is used for treatment of mutant Kit-positive cancers. However, mutations in the Kit kinase domain, which are frequently found in neoplastic mast cells, confer an imatinib resistance, and cancers expressing the mutants can proliferate in the presence of imatinib. Recently, we showed that in neoplastic mast cells that endogenously express an imatinib-resistant Kit mutant, Kit causes oncogenic activation of the phosphatidylinositol 3-kinase-Akt (PI3K-Akt) pathway and the signal transducer and activator of transcription 5 (STAT5) but only on endolysosomes and on the endoplasmic reticulum (ER), respectively. Here, we show a strategy for inhibition of the Kit-PI3K-Akt pathway in neoplastic mast cells by M-COPA (2-methylcoprophilinamide), an inhibitor of this secretory pathway. In M-COPA-treated cells, Kit localization in the ER is significantly increased, whereas endolysosomal Kit disappears, indicating that M-COPA blocks the biosynthetic transport of Kit from the ER. The drug greatly inhibits oncogenic Akt activation without affecting the association of Kit with PI3K, indicating that ER-localized Kit-PI3K complex is unable to activate Akt. Importantly, M-COPA but not imatinib suppresses neoplastic mast cell proliferation through inhibiting anti-apoptotic Akt activation. Results of our M-COPA treatment assay show that Kit can activate Erk not only on the ER but also on other compartments. Furthermore, Tyr568/570, Tyr703, Tyr721, and Tyr936 in Kit are phosphorylated on the ER, indicating that these five tyrosine residues are all phosphorylated before mutant Kit reaches the plasma membrane (PM). Our study provides evidence that Kit is tyrosine-phosphorylated soon after synthesis on the ER but is unable to

M-COPA suppresses endolysosomal Kit-Akt oncogenic signalling through inhibiting the secretory pathway in neoplastic mast cells

PubMed Central

Hara, Yasushi; Obata, Yuuki; Horikawa, Keita; Tasaki, Yasutaka; Suzuki, Kyohei; Murata, Takatsugu; Shiina, Isamu; Abe, Ryo

2017-01-01

Gain-of-function mutations in Kit receptor tyrosine kinase result in the development of a variety of cancers, such as mast cell tumours, gastrointestinal stromal tumours (GISTs), acute myeloid leukemia, and melanomas. The drug imatinib, a selective inhibitor of Kit, is used for treatment of mutant Kit-positive cancers. However, mutations in the Kit kinase domain, which are frequently found in neoplastic mast cells, confer an imatinib resistance, and cancers expressing the mutants can proliferate in the presence of imatinib. Recently, we showed that in neoplastic mast cells that endogenously express an imatinib-resistant Kit mutant, Kit causes oncogenic activation of the phosphatidylinositol 3-kinase-Akt (PI3K-Akt) pathway and the signal transducer and activator of transcription 5 (STAT5) but only on endolysosomes and on the endoplasmic reticulum (ER), respectively. Here, we show a strategy for inhibition of the Kit-PI3K-Akt pathway in neoplastic mast cells by M-COPA (2-methylcoprophilinamide), an inhibitor of this secretory pathway. In M-COPA-treated cells, Kit localization in the ER is significantly increased, whereas endolysosomal Kit disappears, indicating that M-COPA blocks the biosynthetic transport of Kit from the ER. The drug greatly inhibits oncogenic Akt activation without affecting the association of Kit with PI3K, indicating that ER-localized Kit-PI3K complex is unable to activate Akt. Importantly, M-COPA but not imatinib suppresses neoplastic mast cell proliferation through inhibiting anti-apoptotic Akt activation. Results of our M-COPA treatment assay show that Kit can activate Erk not only on the ER but also on other compartments. Furthermore, Tyr568/570, Tyr703, Tyr721, and Tyr936 in Kit are phosphorylated on the ER, indicating that these five tyrosine residues are all phosphorylated before mutant Kit reaches the plasma membrane (PM). Our study provides evidence that Kit is tyrosine-phosphorylated soon after synthesis on the ER but is unable to

Down-regulation of Akt by methanol extracts of Impatiens balsamina L. promotes apoptosis in human oral squamous cell carcinoma cell lines.

PubMed

Shin, Ji-Ae; Ryu, Mi Heon; Kwon, Ki-Han; Choi, BuYoung; Cho, Sung-Dae

2015-07-01

The apoptotic activity of methanol extracts of Impatiens balsamina L. (MEIB) and related mechanisms in human oral squamous cell carcinoma (OSCC) cells have been systematically investigated. The effects of MEIB on human OSCC cell lines were investigated using trypan blue exclusion assay, MTS assay, Western blot, 4'-6-diamidino-2-phenylindole (DAPI) staining, Live/Dead assay, Immunohistochemistry, reverse transcription-polymerase chain reaction, and promoter assay. MEIB decreased cell viability and induced apoptosis in HSC-4 cells. Higher levels of p-Akt expression were observed in OSCC than in normal oral mucosa (NOM), and it correlated with poor survival of the patients. MEIB dephosphorylated p-Akt and decreased Akt expression through proteasome-dependent degradation. LY294002 (PI3K inhibitor) decreased p-Akt and Akt, resulting in enhancing MEIB-induced apoptosis. MEIB down-regulated the expression level of survivin protein at the transcriptional level and YM155 (survivin inhibitor) decreased survivin, which facilitated MEIB-induced apoptosis. MEIB and LY294002 significantly increased Bax, thereby inducing the conformational change, mitochondrial translocation, and oligomerization. In addition, MEIB-induced growth inhibition and apoptosis in OSC-20, another human OSCC cells were mediated by regulating Akt and it downstream targets, survivin and Bax. These results suggest that MEIB may serve as a potential drug candidate for the treatment of human OSCC. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Forskolin increases angiogenesis through the coordinated cross-talk of PKA-dependent VEGF expression and Epac-mediated PI3K/Akt/eNOS signaling.

PubMed

Namkoong, Seung; Kim, Chun-Ki; Cho, Young-Lai; Kim, Ji-Hee; Lee, Hansoo; Ha, Kwon-Soo; Choe, Jongseon; Kim, Pyeung-Hyeun; Won, Moo-Ho; Kwon, Young-Geun; Shim, Eun Bo; Kim, Young-Myeong

2009-06-01

Forskolin, a potent activator of adenylyl cyclases, has been implicated in modulating angiogenesis, but the underlying mechanism has not been clearly elucidated. We investigated the signal mechanism by which forskolin regulates angiogenesis. Forskolin stimulated angiogenesis of human endothelial cells and in vivo neovascularization, which was accompanied by phosphorylation of CREB, ERK, Akt, and endothelial nitric oxide synthase (eNOS) as well as NO production and VEGF expression. Forskolin-induced CREB phosphorylation, VEGF promoter activity, and VEGF expression were blocked by the PKA inhibitor PKI.Moreover, phosphorylation of ERK by forskolin was inhibited by the MEK inhibitor PD98059, but not PKI. The forskolin-induced Akt/eNOS/NO pathway was completely inhibited by the phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002, but not significantly suppressed by PKI. These inhibitors and a NOS inhibitor partially inhibited forskolin-induced angiogenesis. The exchange protein directly activated by cAMP (Epac) activator, 8CPT-2Me-cAMP, promoted the Akt/eNOS/NO pathway and ERK phosphorylation,but did not induce CREB phosphorylation and VEGF expression. The angiogenic effect of the Epac activator was diminished by the inhibition of PI3K and MEK, but not by the PKA inhibitor. Small interfering RNA-mediated knockdown of Epac1 suppressed forskolin-induced angiogenesis and phosphorylation of ERK, Akt, and eNOS, but not CREB phosphorylation and VEGF expression. These results suggest that forskolin stimulates angiogenesis through coordinated cross-talk between two distinct pathways, PKA-dependent VEGF expression and Epac-dependent ERKactivation and PI3K/Akt/eNOS/NO signaling.

Icotinib, a potent and specific EGFR tyrosine kinase inhibitor, inhibits growth of squamous cell carcinoma cell line A431 through negatively regulating AKT signaling.

PubMed

Gao, Zhenzhen; Chen, Wei; Zhang, Xiaohua; Cai, Peifen; Fang, Xianying; Xu, Qiang; Sun, Yang; Gu, Yanhong

2013-06-01

Icotinib is a potent and specific epidermal growth factor receptor tyrosine kinase inhibitor. In this study, we reported that icotinib had the antitumor activity on human squamous cell carcinoma cell line A431 in vitro. Meanwhile, adhesion to fibronectin and expression of integrin α3 and β1 were significantly reduced in a dose-dependent manner after the treatment of icotinib. Moreover, icotinib induced cell cycle arrested and affected expression of various cell cycle related proteins in squamous cancer cell line A431, whereas it did not cause apoptosis. Furthermore, icotinib remarkably down-regulated phosphorylation of protein kinase B (AKT) though blocking the interaction between 3-phosphoinositide-dependent protein kinase-1 (PDK1) and AKT in A431 cells. Taken together, it is shown that the small molecular compound, icotinib, has an anti-squamous cell carcinoma activity in vitro and its antitumor mechanism is associated with the blockage of the interaction between PDK1 and AKT. These results provide a novel strategy for anti-squamous cell carcinoma therapy. Copyright © 2013 Elsevier Masson SAS. All rights reserved.

Critical role of endogenous Akt/IAPs and MEK1/ERK pathways in counteracting endoplasmic reticulum stress-induced cell death.

PubMed

Hu, Ping; Han, Zhang; Couvillon, Anthony D; Exton, John H

2004-11-19

Endoplasmic reticulum (ER) stress has been implicated in the pathogenesis of many diseases and in cancer therapy. Although the unfolded protein response is known to alleviate ER stress by reducing the accumulation of misfolded proteins, the exact survival elements and their downstream signaling pathways that directly counteract ER stress-stimulated apoptotic signaling remain elusive. Here, we have shown that endogenous Akt and ERK are rapidly activated and act as downstream effectors of phosphatidylinositol 3-kinase in thapsigargin- or tunicamycin-induced ER stress. Introduction of either dominant-negative Akt or MEK1 or the inhibitors LY294002 and U0126 sensitized cells to ER stress-induced cell death in different cell types. Reverse transcription-PCR analysis of gene expression during ER stress revealed that cIAP-2 and XIAP, members of the IAP family of potent caspase suppressors, were strongly induced. Transcription of cIAP-2 and XIAP was up-regulated by the phosphatidylinositol 3-kinase/Akt pathway as shown by its reversal by dominant-negative Akt or LY294002. Ablation of these IAPs by RNA interference sensitized cells to ER stress-induced death, which was reversed by the caspase inhibitor benzyloxycarbonyl-VAD-fluoromethyl ketone. The protective role of IAPs in ER stress coincided with Smac release from mitochondria to the cytosol. Furthermore, it was shown that mTOR was not required for Akt-mediated survival. These results represent the first demonstration that activation of endogenous Akt/IAPs and MEK/ERK plays a critical role in controlling cell survival by resisting ER stress-induced cell death signaling.

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

PubMed

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

2015-10-01

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 (Thr(308) in Akt1 or protein kinase B isoform alpha) in an ATP-dependent manner. In the present paper, we describe a distinct mechanism to control Thr(308) 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 (Ser(473)) increased phosphatase resistance of the phosphorylated activation loop (pThr(308)) and amplified Akt phosphorylation. Furthermore, the phosphatase-resistant Akt was refractory to ceramide-dependent dephosphorylation and amplified insulin-dependent Thr(308) 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. © 2015 Authors; published by Portland Press Limited.

Restoration of Akt activity by the bisperoxovanadium compound bpV(pic) attenuates hippocampal apoptosis in experimental neonatal pneumococcal meningitis

PubMed Central

Sury, Matthias D; Vorlet-Fawer, Lorianne; Agarinis, Claudia; Yousefi, Shida; Grandgirard, Denis; Leib, Stephen L; Christen, Stephan

2010-01-01

Pneumococcal meningitis causes apoptosis of developing neurons in the dentate gyrus of the hippocampus. The death of these cells is accompanied with long-term learning and memory deficits in meningitis survivors. Here, we studied the role of the PI3K/Akt (protein kinase B) survival pathway in hippocampal apoptosis in a well-characterized infant rat model of pneumococcal meningitis. Meningitis was accompanied by a significant decrease of the PI3K product phosphatidylinositol 3,4,5-triphosphate (PIP3) and of phosphorylated (i.e., activated) Akt in the hippocampus. At the cellular level, phosphorylated Akt was decreased in both the granular layer and the subgranular zone of the dentate gyrus, the region where the developing neurons undergo apoptosis. Protein levels and activity of PTEN, the major antagonist of PI3K, were unaltered by infection, suggesting that the observed decrease in PIP3 and Akt phosphorylation is a result of decreased PI3K signaling. Treatment with the PTEN inhibitor bpV(pic) restored Akt activity and significantly attenuated hippocampal apoptosis. Co-treatment with the specific PI3K inhibitor LY294002 reversed restoration of Akt activity and attenuation of hippocampal apoptosis, while it had no significant effect on these parameters on its own. These results indicate that the inhibitory effect of bpV(pic) on apoptosis was mediated by PI3K-dependent activation of Akt, strongly suggesting that bpV(pic) acted on PTEN. Treatment with bpV(pic) also partially inhibited the concentration of bacteria and cytokines in the CSF, but this effect was not reversed by LY294002, indicating that the effect of bpV(pic) on apoptosis was independent of its effect on CSF bacterial burden and cytokine levels. These results indicate that the PI3K/Akt pathway plays an important role in the death and survival of developing hippocampal neurons during the acute phase of pneumococcal meningitis. PMID:20875857

Neutral endopeptidase inhibits neuropeptide-mediated transactivation of the insulin-like growth factor receptor-Akt cell survival pathway.

PubMed

Sumitomo, M; Milowsky, M I; Shen, R; Navarro, D; Dai, J; Asano, T; Hayakawa, M; Nanus, D M

2001-04-15

G-protein coupled receptor (GPCR) agonists such as neuropeptides activate the insulin-like growth factor-1 receptor (IGF-IR) or the serine-threonine protein kinase Akt, suggesting that neuropeptides-GPCR signaling can cross-communicate with IGF-IR-Akt signaling pathways. Neutral endopeptidase 24.11 (NEP) is a cell-surface peptidase that cleaves and inactivates the neuropeptides endothelin-1 (ET-1) and bombesin, which are implicated in progression to androgen-independent prostate cancer (PC). We investigated the mechanisms of NEP regulation of neuropeptide-mediated cell survival in PC cells, including whether neuropeptide substrates of NEP induce phosphorylations of IGF-IR and Akt in PC cells. Western analyses revealed ET-1 and bombesin treatment induced phosphorylation of IGF-IRbeta and Akt independent of IGF-I in TSU-Pr1, DU145, and PC-3 PC cells, which lack NEP expression, but not in NEP-expressing LNCaP cells. Recombinant NEP and induced NEP expression in TSU-Pr1 cells using a tetracycline-repressive expression system inhibited ET-1-mediated phosphorylation of IGF-IRbeta and Akt, and blocked the protective effects of ET-1 against apoptosis induced by serum starvation. Incubation of TSU-Pr1 cells with specific kinase inhibitors together with ET-1 or bombesin showed that IGF-IR activation is required for neuropeptide-induced Akt phosphorylation, and that neuropeptide-induced Akt activation is predominantly mediated by Src and phosphatidylinositol 3-kinase but not by mitogen-activated protein kinase or protein kinase C. These data show that the neuropeptides ET-1 and bombesin stimulate ligand-independent activation of the IGF-IR, which results in Akt activation, and that this cross-communication between GPCR and IGF-IR signaling is inhibited by NEP.

Inhibition of Akt/mTOR Signaling by the Dietary Flavonoid Fisetin

PubMed Central

Syed, Deeba N.; Adhami, Vaqar M.; Khan, Mohammad Imran; Mukhtar, Hasan

2014-01-01

Plants have long been providing mankind with remedies of different ailments. Flavonoids, a family of naturally occurring polyphenolic compounds are ubiquitous in plants. Development of polyphenol-based drugs has not attracted much attention by researchers and drug companies. Therefore, despite extensive studies on polyphenols, this vast group of compounds is underrepresented in clinical medicine. Fisetin (3,7,3’,4’-tetrahydroxyflavone) belongs to the flavonol subgroup of flavonoids together with quercetin, myricetin and kaempferol and is found in several fruits and vegetables including strawberries, apples, persimmons and onions. Fisetin is showing promise as a useful natural agent against cancer and has been evaluated for its potential inhibitory role against cancer in several in vitro and in vivo studies. The Akt/mTOR pathway is known to play a central role in various cellular processes that contribute to the malignant phenotype. Accordingly, inhibition of this signaling cascade has been a focus of recent therapeutic studies. Novel inhibitors of PI3-K, Akt, and mTOR are now passing through early phase clinical trials. Herein, we review the effect of fisetin on the PI3-K/Akt/mTOR pathway as studied in different cancer cell models. PMID:23293889

Inhibition of Akt/mTOR signaling by the dietary flavonoid fisetin.

PubMed

Syed, Deeba N; Adhami, Vaqar M; Khan, Mohammad Imran; Mukhtar, Hasan

2013-09-01

Plants have long been providing mankind with remedies of different ailments. Flavonoids, a family of naturally occurring polyphenolic compounds are ubiquitous in plants. Development of polyphenol-based drugs has not attracted much attention by researchers and drug companies. Therefore, despite extensive studies on polyphenols, this vast group of compounds is underrepresented in clinical medicine. Fisetin (3,7,3',4'-tetrahydroxyflavone) belongs to the flavonol subgroup of flavonoids together with quercetin, myricetin and kaempferol and is found in several fruits and vegetables including strawberries, apples, persimmons and onions. Fisetin is showing promise as a useful natural agent against cancer and has been evaluated for its potential inhibitory role against cancer in several in vitro and in vivo studies. The Akt/mTOR pathway is known to play a central role in various cellular processes that contribute to the malignant phenotype. Accordingly, inhibition of this signaling cascade has been a focus of recent therapeutic studies. Novel inhibitors of PI3-K, Akt, and mTOR are now passing through early phase clinical trials. Herein, we review the effect of fisetin on the PI3- K/Akt/mTOR pathway as studied in different cancer cell models.

IKKβ and NFκB transcription govern lymphoma cell survival through AKT-induced plasma membrane trafficking of GLUT1

PubMed Central

Sommermann, Thomas; O’Neill, Kathleen; Plas, David R.; Cahir-McFarland, Ellen

2011-01-01

All cancer cells require increased nutrient uptake to support proliferation. Here we investigated the signals that govern glucose uptake in B-cell lymphomas and determined that the protein kinase IKKβ induced GLUT1 membrane trafficking in both viral and spontaneous B-cell lymphomas. IKKβ induced AKT activity, while IKKβ-driven NFκB transcription was required for GLUT1 surface localization downstream of AKT. Activated NFκB promoted AKT-mediated phosphorylation of the GLUT1 regulator, AKT Substrate 160kD (AS160), but was not required for AKT phosphorylation of the mammalian target of rapamycin (mTOR) regulator Tuberous Sclerosis 2 (TSC2). In Epstein Barr virus (EBV) transformed B-cells, NFκB inhibition repressed glucose uptake and induced caspase-independent cell death associated with autophagy. After NFκB inhibition, an alternate carbon source ameliorated both autophagy and cell death, whereas autophagy inhibitors specifically accelerated cell death. Taken together, the results suggest that NFκB signaling establishes a metabolic program supporting proliferation and apoptosis resistance by driving glucose import. PMID:21987722

Cellular context–mediated Akt dynamics regulates MAP kinase signaling thresholds during angiogenesis

PubMed Central

Hellesøy, Monica; Lorens, James B.

2015-01-01

The formation of new blood vessels by sprouting angiogenesis is tightly regulated by contextual cues that affect angiogeneic growth factor signaling. Both constitutive activation and loss of Akt kinase activity in endothelial cells impair angiogenesis, suggesting that Akt dynamics mediates contextual microenvironmental regulation. We explored the temporal regulation of Akt in endothelial cells during formation of capillary-like networks induced by cell–cell contact with vascular smooth muscle cells (vSMCs) and vSMC-associated VEGF. Expression of constitutively active Akt1 strongly inhibited network formation, whereas hemiphosphorylated Akt1 epi-alleles with reduced kinase activity had an intermediate inhibitory effect. Conversely, inhibition of Akt signaling did not affect endothelial cell migration or morphogenesis in vSMC cocultures that generate capillary-like structures. We found that endothelial Akt activity is transiently blocked by proteasomal degradation in the presence of SMCs during the initial phase of capillary-like structure formation. Suppressed Akt activity corresponded to the increased endothelial MAP kinase signaling that was required for angiogenic endothelial morphogenesis. These results reveal a regulatory principle by which cellular context regulates Akt protein dynamics, which determines MAP kinase signaling thresholds necessary drive a morphogenetic program during angiogenesis. PMID:26023089

Akt interaction with PLC(gamma) regulates the G(2)/M transition triggered by FGF receptors from MDA-MB-231 breast cancer cells.

PubMed

Browaeys-Poly, Edith; Perdereau, Dominique; Lescuyer, Arlette; Burnol, Anne-Françoise; Cailliau, Katia

2009-12-01

Estrogen-independent breast cancer cell growth is under the control of fibroblast growth factors receptors (FGFRs), but the role of phospholipase C gamma (PLC(gamma)) and Akt, the downstream effectors activated by FGFRs, in cell proliferation is still unresolved. FGFRs from highly invasive MDA-MB-231 cells were expressed in Xenopus oocyte, a powerful model system to assess the G(2)/M checkpoint regulation. Under FGF1 stimulation, an analysis of the progression in the M-phase of the cell cycle and of the Akt signaling cascades were performed using the phosphatidylinositol-3-kinase inhibitor, LY294002, and a mimetic peptide of the SH3 domain of PLC(gamma). Activated Akt binds and phosphorylates PLC(gamma) before Akt targets the tumor suppressor Chfr. Disruption of the Akt-PLC(gamma) interaction directs Akt binding to Chfr and accelerates the alleviation of the G(2)/M checkpoint. The PLC(gamma)-Akt interaction, triggered by FGF receptors from estrogen-independent breast cancer cells MDA-MB-231, regulates progression in the M-phase of the cell cycle.

Direct regulation of the Akt proto-oncogene product by phosphatidylinositol-3,4-bisphosphate.

PubMed

Franke, T F; Kaplan, D R; Cantley, L C; Toker, A

1997-01-31

The regulation of the serine-threonine kinase Akt by lipid products of phosphoinositide 3-kinase (PI 3-kinase) was investigated. Akt activity was found to correlate with the amount of phosphatidylinositol-3,4-bisphosphate (PtdIns-3,4-P2) in vivo, and synthetic PtdIns-3,4-P2 activated Akt both in vitro and in vivo. Binding of PtdIns-3,4-P2 occurred within the Akt pleckstrin homology (PH) domain and facilitated dimerization of Akt. Akt mutated in the PH domain was not activated by PI 3-kinase in vivo or by PtdIns-3, 4-P2 in vitro, and it was impaired in binding to PtdIns-3,4-P2. Examination of the binding to other phosphoinositides revealed that they bound to the Akt PH domain with much lower affinity than did PtdIns-3,4-P2 and failed to increase Akt activity. Thus, Akt is apparently regulated by the direct interaction of PtdIns-3,4-P2 with the Akt PH domain.

Epidermal growth factor–stimulated Akt phosphorylation requires clathrin or ErbB2 but not receptor endocytosis

PubMed Central

Garay, Camilo; Judge, Gurjeet; Lucarelli, Stefanie; Bautista, Stephen; Pandey, Rohan; Singh, Tanveer; Antonescu, Costin N.

2015-01-01

Epidermal growth factor (EGF) binding to its receptor (EGFR) activates several signaling intermediates, including Akt, leading to control of cell survival and metabolism. Concomitantly, ligand-bound EGFR is incorporated into clathrin-coated pits—membrane structures containing clathrin and other proteins—eventually leading to receptor internalization. Whether clathrin might regulate EGFR signaling at the plasma membrane before vesicle scission is poorly understood. We compared the effect of clathrin perturbation (preventing formation of, or receptor recruitment to, clathrin structures) to that of dynamin2 (allowing formation of clathrin structures but preventing EGFR internalization) under conditions in which EGFR endocytosis is clathrin dependent. Clathrin perturbation by siRNA gene silencing, with the clathrin inhibitor pitstop2, or knocksideways silencing inhibited EGF-simulated Gab1 and Akt phosphorylation in ARPE-19 cells. In contrast, perturbation of dynamin2 with inhibitors or by siRNA gene silencing did not affect EGF-stimulated Gab1 or Akt phosphorylation. EGF stimulation enriched Gab1 and phospho-Gab1 within clathrin structures. ARPE-19 cells have low ErbB2 expression, and overexpression and knockdown experiments revealed that robust ErbB2 expression bypassed the requirement for clathrin for EGF-stimulated Akt phosphorylation. Thus clathrin scaffolds may represent unique plasma membrane signaling microdomains required for signaling by certain receptors, a function that can be separated from vesicle formation. PMID:26246598

Akt2 deficiency is associated with anxiety and depressive behavior in mice.

PubMed

Leibrock, Christina; Ackermann, Teresa F; Hierlmeier, Michael; Lang, Florian; Borgwardt, Stefan; Lang, Undine E

2013-01-01

The economic burden associated with major depressive disorder and anxiety disorders render both disorders the most common and debilitating psychiatric illnesses. To date, the exact cellular and molecular mechanisms underlying the pathophysiology, successful treatment and prevention of these highly associated disorders have not been identified. Akt2 is a key protein in the phosphatidylinositide-3 (PI3K) / glycogen synthase 3 kinase (GSK3) signaling pathway, which in turn is involved in brain-derived neurotrophic factor (BDNF) effects on fear memory, mood stabilisation and action of several antidepressant drugs. The present study thus explored the impact of Akt2 on behaviour of mice. Behavioural studies (Open-Field, Light-Dark box, O-Maze, Forced Swimming Test, Emergence Test, Object Exploration Test, Morris Water Maze, Radial Maze) have been performed with Akt2 knockout mice (akt(-/-)) and corresponding wild type mice (akt(+/+)). Anxiety and depressive behavior was significantly higher in akt(-/-) than in akt(+/+) mice. The akt(-/-) mice were cognitively unimpaired but displayed increased anxiety in several behavioral tests (O-Maze test, Light-Dark box, Open Field test). Moreover, akt(-/-) mice spent more time floating in the Forced Swimming test, which is a classical feature of experimental depression. Akt2 might be a key factor in the pathophysiology of depression and anxiety. © 2013 S. Karger AG, Basel.

Cycles of Ubiquitination and Deubiquitination Critically Regulate Growth Factor-Mediated Activation of Akt Signaling

PubMed Central

Yang, Wei-Lei; Jin, Guoxiang; Li, Chien-Feng; Jeong, Yun Seong; Moten, Asad; Xu, Dazhi; Feng, Zizhen; Chen, Wei; Cai, Zhen; Darnay, Bryant; Gu, Wei; Lin, Hui-Kuan

2013-01-01

K63-linked ubiquitination of Akt is a posttranslational modification that plays a critical role in growth factor-mediated membrane recruitment and activation of Akt. Although E3 ligases involved in growth factor-induced Akt ubiquitination have been defined, the deubiquitinating enzyme (DUB) that triggers deubiquitination of Akt and the function of Akt deubiquitination remain largely unclear. Here, we showed that CYLD was a DUB for Akt and suppressed growth factor-mediated Akt ubiquitination and activation. CYLD directly removed ubiquitin moieties on Akt under serum-starved conditions. CYLD dissociated from Akt upon growth factor stimulation, thereby allowing E3 ligases to induce ubiquitination and activation of Akt. CYLD deficiency also promoted cancer cell proliferation, survival, glucose uptake and growth of prostate tumors. Our findings reveal the crucial role of cycles of ubiquitination and deubiquitination of Akt in its membrane recruitment and activation, and further identifies CYLD as a molecular switch for these processes. PMID:23300340

An essential role for the Id1/PI3K/Akt/NFkB/survivin signalling pathway in promoting the proliferation of endothelial progenitor cells in vitro.

PubMed

Li, Wei; Wang, Hang; Kuang, Chun-Yan; Zhu, Jin-Kun; Yu, Yang; Qin, Zhe-Xue; Liu, Jie; Huang, Lan

2012-04-01

The enhancement of re-endothelialisation is a critical therapeutic option for repairing injured blood vessels. Endothelial progenitor cells (EPCs) are the major source of cells that participate in endothelium repair and contribute to re-endothelialisation by reducing neointima formation after vascular injury. The over-expression of the inhibitor of differentiation or DNA binding 1 (Id1) significantly improved EPC proliferation. This study aimed to investigate the effects of Id1 on the phosphatidylinositol-3-kinase (PI3K)/Akt/nuclear factor kappa B (NFκB)/survivin signalling pathway and its significance in promoting EPC proliferation in vitro. Spleen-derived EPCs were cultured as previously described. Id1 was presented at low levels in EPCs, and was rapidly up-regulated by stimulation with vascular endothelial growth factor. We demonstrated that transient transfection of Id1 into EPCs activated the PI3K/Akt/NFκB/survivin signalling pathway and promoted EPC proliferation. The proliferation of EPCs was extensively inhibited by silencing of endogenous Id1, and knockdown of Id1 expression led to suppression of PI3K/Akt/NFκB/survivin signalling pathway in EPCs. In addition, blockade by the PI3K-specific inhibitor LY294002, Akt inhibitor, the NFκB inhibitor BAY 11-7082, the survivin inhibitor Curcumin, or the survivin inhibitor YM155 reduced the effects of Id1 transfection. These results suggest that the Id1/PI3K/Akt/NFκB/survivin signalling pathway plays a critical role in EPC proliferation. The Id1/PI3K/Akt/NFκB/survivin signalling pathway may represent a novel therapeutic target in the prevention of restenosis after vascular injury.

hnRNP L controls HPV16 RNA polyadenylation and splicing in an Akt kinase-dependent manner

PubMed Central

Kajitani, Naoko; Glahder, Jacob; Wu, Chengjun; Yu, Haoran; Nilsson, Kersti

2017-01-01

Abstract Inhibition of the Akt kinase activates HPV16 late gene expression by reducing HPV16 early polyadenylation and by activating HPV16 late L1 mRNA splicing. We identified ‘hot spots’ for RNA binding proteins at the early polyA signal and at splice sites on HPV16 late mRNAs. We observed that hnRNP L was associated with sequences at all HPV16 late splice sites and at the early polyA signal. Akt kinase inhibition resulted in hnRNP L dephosphorylation and reduced association of hnRNP L with HPV16 mRNAs. This was accompanied by an increased binding of U2AF65 and Sam68 to HPV16 mRNAs. Furthermore, siRNA knock-down of hnRNP L or Akt induced HPV16 gene expression. Treatment of HPV16 immortalized keratinocytes with Akt kinase inhibitor reduced hnRNP L binding to HPV16 mRNAs and induced HPV16 L1 mRNA production. Finally, deletion of the hnRNP L binding sites in HPV16 subgenomic expression plasmids resulted in activation of HPV16 late gene expression. In conclusion, the Akt kinase inhibits HPV16 late gene expression at the level of RNA processing by controlling the RNA-binding protein hnRNP L. We speculate that Akt kinase activity upholds an intracellular milieu that favours HPV16 early gene expression and suppresses HPV16 late gene expression. PMID:28934469

Targeting Phosphatidylinositide3-Kinase/Akt pathway by BKM120 for radiosensitization in hepatocellular carcinoma

PubMed Central

Liu, Wei-Lin; Gao, Ming; Tzen, Kai-Yuan; Tsai, Chiao-Ling; Hsu, Feng-Ming; Cheng, Ann-Lii; Cheng, Jason Chia-Hsien

2014-01-01

Tumor control of hepatocellular carcinoma by radiotherapy remains unsatisfactory. The phosphatidylinositol 3-kinase (PI3K)/Akt pathway plays a critical role in inhibiting cancer cell death. Elevated PI3K/Akt activity is associated with increased cellular resistance to irradiation. Our aim was to determine whether the inhibition of PI3K/Akt activity by a PI3K inhibitor, BKM120, contributes to the increased sensitivity of liver cancer cells to irradiation. The hepatocellular carcinoma cell lines (Huh7 and BNL) were used to evaluate the in vitro synergism between BKM120 and irradiation. Balb/c mice bearing ectopic BNL xenografts were treated with BKM120 and/or radiotherapy to assess the in vivo response. BKM120 increased cell killing by radiation, increased the expression of apoptotic markers, and suppressed the repair of radiation-induced DNA double-strand breaks. BKM120 pretreatment inhibited radiation-induced Akt phosphorylation and enhanced the tumor-suppressive effect and radiation-induced tumor cell apoptosis in ectopic xenografts. Inhibition of mTOR phosphorylation by rapamycin enhanced the radiosensitivity of BKM120-treated hepatocellular carcinoma cells. The synergism between BKM120 and irradiation likely inhibits the activation of Akt by radiation, leading to increased cell apoptosis and suppression of DNA-double-strand breaks repair in hepatocellular carcinoma cells. These data suggest that the BKM120/radiation combination may be a strategy worthy of clinical trials. PMID:25004403

Curcumin Suppresses Proliferation and Migration of MDA-MB-231 Breast Cancer Cells through Autophagy-Dependent Akt Degradation

PubMed Central

Zhang, Yemin; Zhou, Yu; Li, Mingxin; Wang, Changhua

2016-01-01

Previous studies have evidenced that the anticancer potential of curcumin (diferuloylmethane), a main yellow bioactive compound from plant turmeric was mediated by interfering with PI3K/Akt signaling. However, the underlying molecular mechanism is still poorly understood. This study experimentally revealed that curcumin treatment reduced Akt protein expression in a dose- and time-dependent manner in MDA-MB-231 breast cancer cells, along with an activation of autophagy and suppression of ubiquitin-proteasome system (UPS) function. The curcumin-reduced Akt expression, cell proliferation, and migration were prevented by genetic and pharmacological inhibition of autophagy but not by UPS inhibition. Additionally, inactivation of AMPK by its specific inhibitor compound C or by target shRNA-mediated silencing attenuated curcumin-activated autophagy. Thus, these results indicate that curcumin-stimulated AMPK activity induces activation of the autophagy-lysosomal protein degradation pathway leading to Akt degradation and the subsequent suppression of proliferation and migration in breast cancer cell. PMID:26752181

Caffeic acid phenethyl ester suppresses melanoma tumor growth by inhibiting PI3K/AKT/XIAP pathway.

PubMed

Pramanik, Kartick C; Kudugunti, Shashi K; Fofaria, Neel M; Moridani, Majid Y; Srivastava, Sanjay K

2013-09-01

Melanoma is highly metastatic and resistant to chemotherapeutic drugs. Our previous studies have demonstrated that caffeic acid phenethyl ester (CAPE) suppresses the growth of melanoma cells and induces reactive oxygen species generation. However, the exact mechanism of the growth suppressive effects of CAPE was not clear. Here, we determined the potential mechanism of CAPE against melanoma in vivo and in vitro. Administration of 10 mg/kg/day CAPE substantially suppressed the growth of B16F0 tumor xenografts in C57BL/6 mice. Tumors from CAPE-treated mice showed reduced phosphorylation of phosphoinositide 3-kinase, AKT, mammalian target of rapamycin and protein level of X-linked inhibitor of apoptosis protein (XIAP) and enhanced the cleavage of caspase-3 and poly (ADP ribose) polymerase. In order to confirm the in vivo observations, melanoma cells were treated with CAPE. CAPE treatment suppressed the activating phosphorylation of phosphoinositide 3-kinase at Tyr 458, phosphoinositide-dependent kinase-1 at Ser 241, mammalian target of rapamycin at Ser 2448 and AKT at Ser 473 in B16F0 and SK-MEL-28 cells in a concentration and time-dependent study. Furthermore, the expression of XIAP, survivin and BCL-2 was downregulated by CAPE treatment in both cell lines. Significant apoptosis was observed by CAPE treatment as indicated by cleavage of caspase-3 and poly (ADP ribose) polymerase. AKT kinase activity was inhibited by CAPE in a concentration-dependent manner. CAPE treatment increased the nuclear translocation of XIAP, indicating increased apoptosis in melanoma cells. To confirm the involvement of reactive oxygen species in the inhibition of AKT/XIAP pathway, cells were treated with antioxidant N-acetyl-cysteine (NAC) prior to CAPE treatment. Our results indicate that NAC blocked CAPE-mediated AKT/XIAP inhibition and protected the cells from apoptosis. Because AKT regulates XIAP, their interaction was examined by immunoprecipitation studies. Our results show that CAPE

Caffeic acid phenethyl ester suppresses melanoma tumor growth by inhibiting PI3K/AKT/XIAP pathway

PubMed Central

Srivastava, Sanjay K.

2013-01-01

Melanoma is highly metastatic and resistant to chemotherapeutic drugs. Our previous studies have demonstrated that caffeic acid phenethyl ester (CAPE) suppresses the growth of melanoma cells and induces reactive oxygen species generation. However, the exact mechanism of the growth suppressive effects of CAPE was not clear. Here, we determined the potential mechanism of CAPE against melanoma in vivo and in vitro. Administration of 10 mg/kg/day CAPE substantially suppressed the growth of B16F0 tumor xenografts in C57BL/6 mice. Tumors from CAPE-treated mice showed reduced phosphorylation of phosphoinositide 3-kinase, AKT, mammalian target of rapamycin and protein level of X-linked inhibitor of apoptosis protein (XIAP) and enhanced the cleavage of caspase-3 and poly (ADP ribose) polymerase. In order to confirm the in vivo observations, melanoma cells were treated with CAPE. CAPE treatment suppressed the activating phosphorylation of phosphoinositide 3-kinase at Tyr 458, phosphoinositide-dependent kinase-1 at Ser 241, mammalian target of rapamycin at Ser 2448 and AKT at Ser 473 in B16F0 and SK-MEL-28 cells in a concentration and time-dependent study. Furthermore, the expression of XIAP, survivin and BCL-2 was downregulated by CAPE treatment in both cell lines. Significant apoptosis was observed by CAPE treatment as indicated by cleavage of caspase-3 and poly (ADP ribose) polymerase. AKT kinase activity was inhibited by CAPE in a concentration-dependent manner. CAPE treatment increased the nuclear translocation of XIAP, indicating increased apoptosis in melanoma cells. To confirm the involvement of reactive oxygen species in the inhibition of AKT/XIAP pathway, cells were treated with antioxidant N-acetyl-cysteine (NAC) prior to CAPE treatment. Our results indicate that NAC blocked CAPE-mediated AKT/XIAP inhibition and protected the cells from apoptosis. Because AKT regulates XIAP, their interaction was examined by immunoprecipitation studies. Our results show that CAPE

Rhynchophylline Ameliorates Endothelial Dysfunction via Src-PI3K/Akt-eNOS Cascade in the Cultured Intrarenal Arteries of Spontaneous Hypertensive Rats

PubMed Central

Hao, Hui-Feng; Liu, Li-Mei; Pan, Chun-Shui; Wang, Chuan-She; Gao, Yuan-Sheng; Fan, Jing-Yu; Han, Jing-Yan

2017-01-01

Objectives: To examine the protective effect of Rhynchophylline (Rhy) on vascular endothelial function in spontaneous hypertensive rats (SHRs) and the underlying mechanism. Methods: Intrarenal arteries of SHRs and Wistar rats were suspended in myograph for force measurement. Expression and phosphorylation of endothelial nitric oxide (NO) synthase (eNOS), Akt, and Src kinase (Src) were examined by Western blotting. NO production was assayed by ELISA. Results: Rhy time- and concentration-dependently improved endothelium-dependent relaxation in the renal arteries from SHRs, but had no effect on endothelium-independent relaxation in SHR renal arteries. Wortmannin (an inhibitor of phosphatidylinositol 3-kinase) or PP2 (an inhibitor of Src) inhibited the improvement of relaxation in response to acetylcholine by 12 h-incubation with 300 μM Rhy. Western blot analysis revealed that Rhy elevated phosphorylations of eNOS, Akt, and Src in SHR renal arteries. Moreover, wortmannin reversed the increased phosphorylations of Akt and eNOS induced by Rhy, but did not affect the phosphorylation of Src. Furthermore, the enhanced phosphorylations of eNOS, Akt, and Src were blunted by PP2. Importantly, Rhy increased NO production and this effect was blocked by inhibition of Src or PI3K/Akt. Conclusion: The present study provides evidences for the first time that Rhy ameliorates endothelial dysfunction in SHRs through the activation of Src-PI3K/Akt-eNOS signaling pathway. PMID:29187825

Akt-Signal Integration Is Involved in the Differentiation of Embryonal Carcinoma Cells

PubMed Central

Chen, Bo; Xue, Zheng; Yang, Guanghui; Shi, Bingyang; Yang, Ben; Yan, Yuemin; Wang, Xue; Han, Daishu; Huang, Yue; Dong, Wenji

2013-01-01

The mechanism by which Akt modulates stem cell homeostasis is still incompletely defined. Here we demonstrate that Akt phosphorylates special AT-rich sequences binding protein 1 (SATB1) at serine 47 and protects SATB1 from apoptotic cleavage. Meanwhile, Akt phosphorylates Oct4 at threonine 228 and Klf4 at threonine 399, and accelerates their degradation. Moreover, PI3K/Akt signaling enhances the binding of SATB1 to Sox2, thereby probably impairing the formation of Oct4/Sox2 regulatory complexes. During retinoic acid (RA)-induced differentiation of mouse F9 embryonal carcinoma cells (ECCs), the Akt activation profile as well as its substrate spectrum is strikingly correlated with the down-regulation of Oct4, Klf4 and Nanog, which suggests Akt activation is coupled to the onset of differentiation. Accordingly, Akt-mediated phosphorylation is crucial for the capability of SATB1 to repress Nanog expression and to activate transcription of Bcl2 and Nestin genes. Taken together, we conclude that Akt is involved in the differentiation of ECCs through coordinated phosphorylations of pluripotency/differentiation factors. PMID:23762260

Aldosterone Induces Apoptosis in Rat Podocytes: Role of PI3-K/Akt and p38MAPK Signaling Pathways

PubMed Central

Chen, Cheng; Liang, Wei; Jia, Junya; van Goor, Harry; Singhal, Pravin C.; Ding, Guohua

2009-01-01

Background Podocytes play a critical role in the pathogenesis of glomerulosclerosis. Increasing evidence suggests that aldosterone (ALD) is involved in the initiation and progression of glomerular damage. It is, however, unknown whether there is a direct injurious effect of ALD on podocytes. Therefore, in the present study, we evaluated the effect of ALD on podocyte apoptosis and studied the role of phosphatidylinositol 3-kinase/Akt (PI3-K/Akt) and p38 mitogen-activated protein kinase (p38MAPK) signaling pathways in this process. Methods Podocytes were incubated in media containing either buffer or increasing concentrations of ALD (10–9∼10–5M) for variable time periods. The cells were also treated with either wortmannin (inhibitor of PI3-K, 100 nM), SB202190 (SB20, inhibitor of p38MAPK, 10 μM) or buffer. All treatments were performed with or without ALD (10–7M) for 24 h. At the end of the incubation period, apoptosis was evaluated by cell nucleus staining and flow cytometric analyses. Activation of PI3-K/Akt and p38MAPK phosphorylation of cultured rat podocytes was evaluated by performing Akt kinase assay and Western blot, respectively. Results Apoptosis of cultured rat podocytes was induced by ALD in a dose- and time-dependent manner. ALD inhibited the activity of PI3-K/Akt and increased the activation of p38MAPK. PI3-K/Akt activity was further inhibited by the addition of wortmannin to the cells in the presence of ALD. This was accompanied by a significant increase in apoptosis. ALD-induced p38MAPK phosphorylation and apoptosis were inhibited when the cells were pretreated with SB20. Furthermore, treatment with spironolactone not only attenuated the proapoptotic effect of ALD, but also significantly reversed its effects on PI3-K/Akt and p38MAPK signaling pathways. Conclusion ALD induces apoptosis in rat podocytes through inhibition of PI3-K/Akt and stimulation of p38 MAPK signaling pathways. Spironolactone attenuates ALD-induced podocyte apoptosis

DNA–PKcs–SIN1 complexation mediates low-dose X-ray irradiation (LDI)-induced Akt activation and osteoblast differentiation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Xu, Yong; Fang, Shi-ji; Zhu, Li-juan

Highlights: • LDI increases ALP activity, promotes type I collagen (Col I)/Runx2 mRNA expression. • LDI induces DNA–PKcs activation, which is required for osteoblast differentiation. • Akt activation mediates LDI-induced ALP activity and Col I/Runx2 mRNA increase. • DNA–PKcs–SIN1 complexation mediates LDI-induced Akt Ser-473 phosphorylation. • DNA–PKcs–SIN1 complexation is important for osteoblast differentiation. - Abstract: Low-dose irradiation (LDI) induces osteoblast differentiation, however the underlying mechanisms are not fully understood. In this study, we explored the potential role of DNA-dependent protein kinase catalytic subunit (DNA–PKcs)–Akt signaling in LDI-induced osteoblast differentiation. We confirmed that LDI promoted mouse calvarial osteoblast differentiation, which wasmore » detected by increased alkaline phosphatase (ALP) activity as well as mRNA expression of type I collagen (Col I) and runt-related transcription factor 2 (Runx2). In mouse osteoblasts, LDI (1 Gy) induced phosphorylation of DNA–PKcs and Akt (mainly at Ser-473). The kinase inhibitors against DNA–PKcs (NU-7026 and NU-7441) or Akt (LY294002, perifosine and MK-2206), as well as partial depletion of DNA–PKcs or Akt1 by targeted-shRNA, dramatically inhibited LDI-induced Akt activation and mouse osteoblast differentiation. Further, siRNA-knockdown of SIN1, a key component of mTOR complex 2 (mTORC2), also inhibited LDI-induced Akt Ser-473 phosphorylation as well as ALP activity increase and Col I/Runx2 expression in mouse osteoblasts. Co-immunoprecipitation (Co-IP) assay results demonstrated that LDI-induced DNA–PKcs–SIN1 complexation, which was inhibited by NU-7441 or SIN1 siRNA-knockdown in mouse osteoblasts. In summary, our data suggest that DNA–PKcs–SIN1 complexation-mediated Akt activation (Ser-473 phosphorylation) is required for mouse osteoblast differentiation.« less

PI3K/AKT signaling inhibits NOTCH1 lysosome-mediated degradation.

PubMed

Platonova, Natalia; Manzo, Teresa; Mirandola, Leonardo; Colombo, Michela; Calzavara, Elisabetta; Vigolo, Emilia; Cermisoni, Greta Chiara; De Simone, Daria; Garavelli, Silvia; Cecchinato, Valentina; Lazzari, Elisa; Neri, Antonino; Chiaramonte, Raffaella

2015-06-06

The pathways of NOTCH and PI3K/AKT are dysregulated in about 60% and 48% of T-cell acute lymphoblastic leukemia (T-ALL) patients, respectively. In this context, they interact and cooperate in controlling tumor cell biology. Here, we propose a novel mechanism by which the PI3K/AKT pathway regulates NOTCH1 in T-ALL, starting from the evidence that the inhibition of PI3K/AKT signaling induced by treatment with LY294002 or transient transfection with a dominant negative AKT mutant downregulates NOTCH1 protein levels and activity, without affecting NOTCH1 transcription. We showed that the withdrawal of PI3K/AKT signaling was associated to NOTCH1 phosphorylation in tyrosine residues and monoubiquitination of NOTCH1 detected by Ubiquitin capture assay. Co-immunoprecipitation assay and colocalization analysis further showed that the E3 ubiquitin ligase c-Cbl interacts and monoubiquitinates NOTCH1, activating its lysosomal degradation. These results suggest that the degradation of NOTCH1 could represent a mechanism of control by which NOTCH1 receptors are actively removed from the cell surface. This mechanism is finely regulated by the PI3K/AKT pathway in physiological conditions. In pathological conditions characterized by PI3K/AKT hyperactivation, such as T-ALL, the excessive AKT signaling could lead to NOTCH1 signaling dysregulation. Therefore, a therapeutic strategy directed to PI3K/AKT in T-ALL could contemporaneously inhibit the dysregulated NOTCH1 signaling. © 2015 Wiley Periodicals, Inc. © 2015 Wiley Periodicals, Inc.

TLR2 ligands induce cardioprotection against ischaemia/reperfusion injury through a PI3K/Akt-dependent mechanism

PubMed Central

Ha, Tuanzhu; Hu, Yulong; Liu, Li; Lu, Chen; McMullen, Julie R.; Kelley, Jim; Kao, Race L.; Williams, David L.; Gao, Xiang; Li, Chuanfu

2010-01-01

Aims Toll-like receptor (TLR)-mediated signalling pathways have been implicated in myocardial ischaemia/reperfusion (I/R) injury. Activation of the phosphoinositide 3-kinase (PI3K)/Akt pathway protects the myocardium from ischaemic injury. We hypothesized that the modulation of TLR2 would induce cardioprotection against I/R injury via activation of the PI3K/Akt signalling. Methods and results Mice were treated with TLR2 ligands, peptidoglycan (PGN) or Pam3CSK4, respectively, 1 h before the hearts were subjected to ischaemia (1 h), followed by reperfusion (4 h). Infarct size was determined by triphenyltetrazolium chloride staining. Cardiac function and haemodynamic performance were evaluated. Infarct size was significantly reduced in PGN- or Pam3CSK4-treated mice compared with untreated I/R mice. Administration of TLR2 ligands improved cardiac function following I/R. PGN treatment increased the levels of phospho-Akt and phospho-GSK-3β (glycogen synthase kinase-3β), compared with untreated I/R hearts. PGN stimulation increased TLR2 tyrosine phosphorylation and association of the p85 subunit of PI3K with TLR2. To investigate the role of PI3K/Akt signalling in PGN-induced cardioprotection, we administered the PI3K inhibitor, Wortmannin, to the mice 15 min before PGN treatment. We also administered PGN to kinase-deficient Akt (kdAkt) transgenic mice 1 h before myocardial I/R. Both PI3K inhibition and kdAkt mice abolished the cardioprotection induced by PGN. To examine the role of TLR2 in PGN-induced cardioprotection, we administrated PGN to TLR2 knockout mice 1 h before the hearts were subjected to I/R. PGN-induced cardioprotection was lost in TLR2-deficient mice. Conclusion These results demonstrate that TLR2 ligands induced cardioprotection, which is mediated through a TLR2/PI3K/Akt-dependent mechanism. PMID:20421349

Tectorigenin sensitizes paclitaxel-resistant human ovarian cancer cells through downregulation of the Akt and NFκB pathway.

PubMed

Yang, Yeong-In; Lee, Kyung-Tae; Park, Hee-Juhn; Kim, Tae Jin; Choi, Youn Seok; Shih, Ie-Ming; Choi, Jung-Hye

2012-12-01

Paclitaxel (Taxol) is currently used as the front-line chemotherapeutic agent for several cancers including ovarian carcinoma; however, the drug frequently induces drug resistance through multiple mechanisms. The new strategy of using natural compounds in combination therapies is highly attractive because those compounds may enhance the efficacy of chemotherapy. In this study, we found that tectorigenin, an isoflavonoid isolated from flower of Pueraria thunbergiana, enhanced the growth-inhibitory effect of paclitaxel in paclitaxel-resistant ovarian cancer cells (MPSC1(TR), A2780(TR) and SKOV3(TR)) as well as their naive counterparts. The combination of tectorigenin with paclitaxel resulted in a synergistic apoptosis compared with either agent alone through activation of caspases-3, -8 and -9. Treatment with tectorigenin inhibited the nuclear translocation of NFκB and the expression of NFκB-dependent genes such as FLIP, XIAP, Bcl-2, Bcl-xL and COX-2, which are known to be associated with chemoresistance. In addition, the tectorigenin-paclitaxel combination inhibited the phosphorylation of IκB and IKK and the activation of Akt in paclitaxel-resistant cancer cells. Moreover, tectorigenin-paclitaxel-induced cell growth inhibition was enhanced by pretreatment with the Akt inhibitor LY294002 or overexpression of the dominant negative Akt (Akt-DN), but reduced by overexpression of constitutively activated Akt (Akt-Myr). Furthermore, we found that Akt-Myr, at least in part, reversed tectorigenin-paclitaxel-induced nuclear translocation of NFκB and the phosphorylation of IκB and IKK. These data suggest that tectorigenin could sensitize paclitaxel-resistant human ovarian cancer cells through inactivation of the Akt/IKK/IκB/NFκB signaling pathway, and promise a new intervention to chemosensitize paclitaxel-induced cytotoxicity in ovarian cancer.

Over-activation of AKT signaling leading to 5-Fluorouracil resistance in SNU-C5/5-FU cells

PubMed Central

Kim, Eun-Ji; Kang, Gyeoung-Jin; Kang, Jung-Il; Boo, Hye-Jin; Hyun, Jin Won; Koh, Young Sang; Chang, Weon-Young; Kim, Young Ree; Kwon, Jung-Mi; Maeng, Young Hee; Yoo, Eun-Sook; Lee, Chang Hoon; Kang, Hee-Kyoung

2018-01-01

Here, we investigated whether over-activation of AKT pathway is important in the resistance to 5-fluorouracil (5-FU) in SNU-C5/5-FU cells, 5-FU-resistant human colon cancer cells. When compared to wild type SNU-C5 cells (WT), SNU-C5/5-FU cells showed over-activation of PI3K/AKT pathway, like increased phosphorylation of AKT, mTOR, and GSK-3β, nuclear localization of β-catenin, and decreased E-cadherin. Moreover, E-cadherin level was down-regulated in recurrent colon cancer tissues compared to primary colon cancer tissues. Gene silencing of AKT1 or treatment of LY294002 (PI3 kinase inhibitor) increased E-cadherin, whereas decreased phospho-GSK-3β. LY294002 also reduced protein level of β-catenin with no influence on mRNA level. PTEN level was higher in SNU-C5/WT than SNU-C5/5-FU cells, whereas the loss of PETN in SNU-C5/WT cells induced characteristics of SNU-C5/5-FU cells. In SNU-C5/5-FU cells, NF-κB signaling was activated, along with the overexpression of COX-2 and stabilization of survivin. However, increased COX-2 contributed to the stabilization of survivin, which directly interacts with cytoplasmic procaspase-3, while the inhibition of AKT reduced this cascade. We finally confirmed that combination treatment with 5-FU and LY294002 or Vioxx could induce apoptosis in SNU-C5/5-FU cells. These data suggest that inhibition of AKT activation may overcome 5-FU-resistance in SNU-C5/5-FU cells. These findings provide evidence that over-activation of AKT is crucial for the acquisition of resistance to anticancer drugs and AKT pathway could be a therapeutic target for cancer treatment. PMID:29731993

Gentamicin alters Akt-expression and its activation in the guinea pig cochlea.

PubMed

Heinrich, U-R; Strieth, S; Schmidtmann, I; Li, H; Helling, K

2015-12-17

Gentamicin treatment induces hair cell death or survival in the inner ear. Besides the well-known toxic effects, the phosphatidylinositol-3 kinase/Akt (PI3K/Akt) pathway was found to be involved in cell protection. After gentamicin application, the spatiotemporal expression patterns of Akt and its activated form (p-Akt) were determined in male guinea pigs. A single dose of 0.1 mL gentamicin (4 mg/ear/animal) was intratympanically injected. The auditory brainstem responses (ABRs) were recorded prior to application and 1, 2 and 7 days afterward. At these three time points the cochleae (n=10 in each case) were removed, transferred to fixative and embedded in paraffin. Seven ears were used as untreated controls. Gentamicin, Akt and p-Akt were identified immunohistochemically in various regions of the cochlea and their staining intensities were quantified on sections using digital image analysis. The application of gentamicin resulted in hearing loss with a concomitant up-regulation of Akt-expression in the organ of Corti and spiral ganglion cells and an additional activation in spiral ganglion cells. At the level of individual ears, clear intracellular correlations were found between Akt- and p-Akt-expression in the stria vascularis and interdental cells and, to a minor extent, in the spiral ligament and the organ of Corti. Furthermore, statistical evidence for the connection between gentamicin up-take and hearing loss was detected. The increase in Akt- and p-Akt-expression in the organ of Corti and spiral ganglion cells indicates a selected response of the cochlea against gentamicin toxicity. Copyright © 2015 IBRO. Published by Elsevier Ltd. All rights reserved.

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

PubMed

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

2016-05-18

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

Wogonin induces cross-regulation between autophagy and apoptosis via a variety of Akt pathway in human nasopharyngeal carcinoma cells.

PubMed

Chow, Shu-Er; Chen, Yu-Wen; Liang, Chi-Ang; Huang, Yao-Kuan; Wang, Jong-Shyan

2012-11-01

Autophagy as well as apoptosis is an emerging target for cancer therapy. Wogonin, a flavonoid compound derived from the traditional Chinese medicine of Huang-Qin, has anticancer activity in many cancer cells including human nasopharyngeal carcinoma (NPC). However, the involvement of autophagy in the wogonin-induced apoptosis of NPC cells was still uninvestigated. In this study, we found wogonin-induced autophagy had interference on the process of apoptosis. Wogonin-induced autophagy formation evidenced by LC3 I/II cleavage, acridine orange (AO)-stained vacuoles and the autophagosome/autolysosome images of TEM analysis. Activation of autophagy with rapamycin resulted in increased wogonin-mediated autophagy via inhibition of mTOR/P70S6K pathway. The functional relevance of autophagy in the antitumor activity was investigated by annexin V-positive stained cells and PARP cleavage. Induction of autophagy by rapamycin ameliorated the wogonin-mediated apoptosis, whereas inhibition of autophagy by 3-methyladenine (3-MA) or bafilomycin A1 increased the apoptotic effect. Interestingly, this study also found, in addition the mTOR/P70S6K pathway, wogonin also inhibited Raf/ERK pathway, a variety of Akt pathways. Inactivation of PI(3) K/Akt by their inhibitors significantly induced apoptosis and markedly sensitized the NPC cells to wogonin-induced apoptosis. This anticancer effect of Akt was further confirmed by SH6, a specific inhibitor of Akt. Importantly, inactivation of its downstream molecule ERK by PD98059, a MEK inhibitor, also induced apoptosis. This study indicated wogonin-induced both autophagy and apoptosis through a variety of Akt pathways and suggested modulation of autophagy might provide profoundly the potential therapeutic effect. Copyright © 2012 Wiley Periodicals, Inc.

Hepatoprotective Effects of Corilagin Following Hemorrhagic Shock are Through Akt-Dependent Pathway

PubMed Central

Liu, Fu-Chao; Chaudry, Irshad H.; Yu, Huang-Ping

2017-01-01

ABSTRACT Corilagin, a component of Phyllanthus urinaria extract, possesses antioxidant, thrombolytic, antiatherogenic, and hepatoprotective properties, but the mechanism underlying these effects remains unclear. Previous studies showed that the Akt (protein kinase B) signaling pathway exerts anti-inflammatory and organ protective effects. The aim of this study was to investigate the mechanism of action of corilagin and determine whether these effects are mediated through the Akt-dependent pathway in a trauma-hemorrhagic shock-induced liver injury rodent model. Hemorrhagic shock was induced in male Sprague–Dawley rats; mean blood pressure was maintained at 35 mm Hg to 40 mm Hg for 90 min, followed by fluid resuscitation. During resuscitation, three doses of corilagin alone (1 mg/kg, 5 mg/kg, or 10 mg/kg, intravenously) were administered. Furthermore, a single dose of corilagin (5 mg/kg) with and without Wortmannin (1 mg/kg, PI3K inhibitor), Wortmannin alone, or vehicle was administered. Twenty-four hours after resuscitation, plasma alanine aminotransferase and aspartate aminotransferase concentration and hepatic parameters were measured. One-way ANOVA was used for statistical analysis. Hepatic myeloperoxidase activity and the concentrations of plasma alanine aminotransferase and aspartate aminotransferase, interleukin-6, tumor necrosis factor-α, intercellular adhesion molecule-1, and cytokine-induced neutrophil chemoattractant-1 (CINC-1) and CINC-3 increased following hemorrhagic shock. These parameters were significantly attenuated in corilagin-treated rats following hemorrhagic shock. Hepatic phospho-Akt expression was also higher in corilagin-treated rats than in vehicle-treated rats. The elevation of phospho-Akt was abolished by combined treatment with Wortmannin and corilagin. Our results suggest that corilagin exerts its protective effects on hemorrhagic shock-induced liver injury, at least, via the Akt-dependent pathway. PMID:27559697

Urinary bladder organ hypertrophy is partially regulated by Akt1-mediated protein synthesis pathway.

PubMed

Qiao, Li-Ya; Xia, Chunmei; Shen, Shanwei; Lee, Seong Ho; Ratz, Paul H; Fraser, Matthew O; Miner, Amy; Speich, John E; Lysiak, Jeffrey J; Steers, William D

2018-05-15

The present study aims to investigate the role of Akt in the regulation of urinary bladder organ hypertrophy caused by partial bladder outlet obstruction (pBOO). Male rats were surgically induced for pBOO. Real-time PCR and western blot were used to examine the levels of mRNA and protein. A phosphoinositide 3-kinase (PI3K) inhibitor LY294002 was used to inhibit the activity of endogenous Akt. The urinary bladder developed hypertrophy at 2 weeks of pBOO. The protein but not mRNA levels of type I collagen and α-smooth muscle actin (αSMA) were increased in pBOO bladder when compared to sham control. The phosphorylation (activation) levels of Akt1 (p-Ser 473 ), mammalian target of rapamycin (mTOR), p70S6 kinase (p70S6K), and 4E-BP1 were also increased in pBOO bladder. LY294002 treatment reduced the phosphorylation levels of Akt1 and 4E-BP1, and the protein levels of type I collagen and αSMA in pBOO bladder. The mRNA and protein levels of proliferating cell nuclear antigen (PCNA) were increased in pBOO bladder, and PCNA up-regulation occurred in urothelial not muscular layer. LY294002 treatment had no effect on the mRNA and protein levels of PCNA in pBOO bladder. LY294002 treatment partially reduced the bladder weight caused by pBOO. pBOO-induced urinary bladder hypertrophy is attributable to fibrosis, smooth muscle cellular hypertrophy, and urothelium cell hyper-proliferation. Akt1-mediated protein synthesis in pBOO bladder contributes to type I collagen and αSMA but not PCNA up-regulation. Target of Akt1 is necessary but not sufficient in treatment of urinary bladder hypertrophy following pBOO. Copyright © 2018 Elsevier Inc. All rights reserved.

Prenatal retinoic acid upregulates pulmonary gene expression of PI3K and AKT in nitrofen-induced pulmonary hypoplasia.

PubMed

Doi, Takashi; Sugimoto, Kaoru; Ruttenstock, Elke; Dingemann, Jens; Puri, Prem

2010-10-01

The precise mechanism of pulmonary hypoplasia associated with congenital diaphragmatic hernia (CDH) still remains unclear. Recently, prenatal treatment with retinoic acid (RA) has been reported to stimulate alveologenesis in hypoplastic lungs in the nitrofen model of CDH. The serine/threonine protein kinase B (AKT) plays a key role in lung morphogenesis through epithelial-mesenchymal interaction in phosphatidylinositide 3-kinase (PI3K)-dependent manner. It has been reported that the lung morphogenesis in explants in mice is interfered by inhibitors of PI3K-AKT signaling pathway. Furthermore, we have recently shown that nitrofen inhibits PI3K-AKT signaling during mid-to-late lung morphogenesis in the nitrofen-induced hypoplastic lung. We hypothesized that prenatal administration of RA upregulates pulmonary gene expression of PI3K and AKT in the nitrofen-induced hypoplastic lung. Pregnant rats were exposed to either olive oil or nitrofen on day 9 of gestation (D9). 5 mg/kg of RA was given on D18, D19 and D20. The fetuses were harvested on D21, and fetal lungs were obtained and divided into four groups: control, control + RA, nitrofen, nitrofen + RA. The mRNA expression levels of PI3K and AKT were analyzed in each lung by real-time RT-PCR and statistically analyzed. Immunohistochemistry was also performed to evaluate protein expression of PI3K and AKT in the fetal lungs at D21. The pulmonary gene expression levels of PI3K and AKT were significantly upregulated in nitrofen + RA group compared to nitrofen group and control + RA group (p < 0.05), whereas there were no significant differences between controls and control + RA group. Immunoreactivity of PI3K and AKT was markedly increased in nitrofen + RA lungs compared to nitrofen-induced hypoplastic lungs. Upregulation of PI3K and AKT genes after prenatal treatment with RA in the nitrofen-induced hypoplastic lung suggests that RA may have a therapeutic potential in modulating lung alveologenesis by stimulating epithelial

Akt3 is a privileged first responder in isozyme-specific electrophile response.

PubMed

Long, Marcus J C; Parvez, Saba; Zhao, Yi; Surya, Sanjna L; Wang, Yiran; Zhang, Sheng; Aye, Yimon

2017-03-01

Isozyme-specific post-translational regulation fine tunes signaling events. However, redundancy in sequence or activity renders links between isozyme-specific modifications and downstream functions uncertain. Methods to study this phenomenon are underdeveloped. Here we use a redox-targeting screen to reveal that Akt3 is a first-responding isozyme sensing native electrophilic lipids. Electrophile modification of Akt3 modulated downstream pathway responses in cells and Danio rerio (zebrafish) and markedly differed from Akt2-specific oxidative regulation. Digest MS sequencing identified Akt3 C119 as the privileged cysteine that senses 4-hydroxynonenal. A C119S Akt3 mutant was hypomorphic for all downstream phenotypes shown by wild-type Akt3. This study documents isozyme-specific and chemical redox signal-personalized physiological responses.

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.

H2O2 treatment or serum deprivation induces autophagy and apoptosis in naked mole-rat skin fibroblasts by inhibiting the PI3K/Akt signaling pathway.

PubMed

Zhao, Shanmin; Li, Li; Wang, Shiyong; Yu, Chenlin; Xiao, Bang; Lin, Lifang; Cong, Wei; Cheng, Jishuai; Yang, Wenjing; Sun, Wei; Cui, Shufang

2016-12-20

Naked mole-rats (NMR; Heterocephalus glaber) display extreme longevity and resistance to cancer. Here, we examined whether autophagy contributes to the longevity of NMRs by assessing the effects of the PI3K/Akt pathway inhibitor LY294002 and the autophagy inhibitor chloroquine (CQ) on autophagy and apoptosis in NMR skin fibroblasts. Serum starvation, H2O2 treatment, and LY294002 treatment all increased the LC3-II/LC3-I ratio and numbers of double-membraned autophagosomes and autophagic vacuoles, and decreased levels of p70S6K, p-AktSer473, and p-AktThr308. By contrast, CQ treatment decreased p70S6K, AktSer473, and AktThr308 levels. The Bax/Bcl-2 ratio increased after 12 h of exposure to LY294002 or CQ. These data show that inhibiting the Akt pathway promotes autophagy and apoptosis in NMR skin fibroblasts. Furthermore, LY294002 or CQ treatment decreased caspase-3, p53, and HIF1-α levels, suggesting that serum starvation or H2O2 treatment increase autophagy and apoptosis in NMR skin fibroblasts by inhibiting the PI3K/Akt pathway. CQ-induced inhibition of late autophagy stages also prevented Akt activation and induced apoptosis. Finally, the HIF-1α and p53 pathways were involved in serum starvation- or H2O2-induced autophagy in NMR skin fibroblasts.

Genetic Deletion of Akt3 Induces an Endophenotype Reminiscent of Psychiatric Manifestations in Mice

PubMed Central

Bergeron, Yan; Bureau, Geneviève; Laurier-Laurin, Marie-Élaine; Asselin, Eric; Massicotte, Guy; Cyr, Michel

2017-01-01

The protein kinase B (PKB/Akt), found in three distinctive isoforms (PKBα/Akt1, PKBβ/Akt2, PKBγ/Akt3), is implicated in a variety of cellular processes such as cell development, growth and survival. Although Akt3 is the most expressed isoform in the brain, its role in cerebral functions is still unclear. In the present study, we investigated the behavioral, electrophysiological and biochemical consequences of Akt3 deletion in mice. Motor abilities, spatial navigation, recognition memory and LTP are intact in the Akt3 knockout (KO) mice. However, the prepulse inhibition, three-chamber social, forced swim, tail suspension, open field, elevated plus maze and light-dark transition tests revealed an endophenotype reminiscent of psychiatric manifestations such as schizophrenia, anxiety and depression. Biochemical investigations revealed that Akt3 deletion was associated with reduced levels of phosphorylated GSK3α/β at serine 21/9 in several brain regions, although Akt1 and Akt2 levels were unaffected. Notably, chronic administration of lithium, a mood stabilizer, restored the decreased phosphorylated GSK3α/β levels and rescued the depressive and anxiety-like behaviors in the Akt3 KO mice. Collectively, our data suggest that Akt3 might be a critical molecule underlying psychiatric-related behaviors in mice. PMID:28442992

I(2)(PP2A) regulates p53 and Akt correlatively and leads the neurons to abort apoptosis.

PubMed

Liu, Gong-Ping; Wei, Wei; Zhou, Xin; Zhang, Yao; Shi, Hai-Hong; Yin, Jun; Yao, Xiu-Qing; Peng, Cai-Xia; Hu, Juan; Wang, Qun; Li, Hong-Lian; Wang, Jian-Zhi

2012-02-01

A chronic neuron loss is the cardinal pathology in Alzheimer disease (AD), but it is still not understood why most neurons in AD brain do not accomplish apoptosis even though they are actually exposed to an environment with enriched proapoptotic factors. Protein phosphatase-2A inhibitor-2 (I(2)(PP2A)), an endogenous PP2A inhibitor, is significantly increased in AD brain, but the role of I(2)(PP2A) in AD-like neuron loss is elusive. Here, we show that I(2)(PP2A) regulates p53 and Akt correlatively. The mechanisms involve activated transcription and p38 MAPK activities. More importantly, we demonstrate that the simultaneous activation of Akt induced by I(2)(PP2A) counteracts the hyperactivated p53-induced cell apoptosis. Furthermore, I(2)(PP2A), p53 and Akt are all elevated in the brain of mouse model and AD patients. Our results suggest that the increased I(2)(PP2A) may trigger apoptosis by p53 upregulation, but due to simultaneous activation of Akt, the neurons are aborted from the apoptotic pathway. This finding contributes to the understanding of why most neurons in AD brain do not undergo apoptosis. Copyright © 2010. Published by Elsevier Inc.

EGFR trans-activation mediates pleiotrophin-induced activation of Akt and Erk in cultured osteoblasts.

PubMed

Fan, Jian-Bo; Liu, Wei; Yuan, Kun; Zhu, Xin-Hui; Xu, Da-Wei; Chen, Jia-Jia; Cui, Zhi-Ming

2014-05-09

Pleiotrophin (Ptn) plays an important role in bone growth through regulating osteoblasts' functions. The underlying signaling mechanisms are not fully understood. In the current study, we found that Ptn induced heparin-binding epidermal growth factor (HB-EGF) release to trans-activate EGF-receptor (EGFR) in both primary osteoblasts and osteoblast-like MC3T3-E1 cells. Meanwhile, Ptn activated Akt and Erk signalings in cultured osteoblasts. The EGFR inhibitor AG1478 as well as the monoclonal antibody against HB-EGF (anti-HB-EGF) significantly inhibited Ptn-induced EGFR activation and Akt and Erk phosphorylations in MC3T3-E1 cells and primary osteoblasts. Further, EGFR siRNA depletion or dominant negative mutation suppressed also Akt and Erk activation in MC3T3-E1 cells. Finally, we observed that Ptn increased alkaline phosphatase (ALP) activity and inhibited dexamethasone (Dex)-induced cell death in both MC3T3-E1 cells and primary osteoblasts, such effects were alleviated by AG1478 or anti-HB-EGF. Together, these results suggest that Ptn-induced Akt/Erk activation and some of its pleiotropic functions are mediated by EGFR trans-activation in cultured osteoblasts. Copyright © 2014 Elsevier Inc. All rights reserved.

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

PubMed

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

2014-09-01

Squamous cell lung carcinoma accounts for approximately 30% of all non-small cell lung cancers (NSCLCs). Despite progress in the understanding of the biology of cancer, cytotoxic chemotherapy remains the standard of care for patients with squamous cell lung carcinoma, but the prognosis is generally poor. The phosphatidylinositol 3-kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) pathway is one of the most commonly activated signaling pathways in cancer, leading to cell proliferation, survival, and differentiation. It has therefore become a major focus of clinical research. Various alterations in the PI3K/AKT/mTOR pathway have been identified in squamous cell lung carcinoma and a number of agents targeting these alterations are in clinical development for use as single agents and in combination with other targeted and conventional treatments. These include pan-PI3K inhibitors, isoform-specific PI3K inhibitors, AKT inhibitors, mTOR inhibitors, and dual PI3K/mTOR inhibitors. These agents have demonstrated antitumor activity in preclinical models of NSCLC and preliminary clinical evidence is also available for some agents. This review will discuss the role of the PI3K/AKT/mTOR pathway in cancer and how the discovery of genetic alterations in this pathway in patients with squamous cell lung carcinoma can inform the development of targeted therapies for this disease. An overview of ongoing clinical trials investigating PI3K/AKT/mTOR pathway inhibitors in squamous cell lung carcinoma will also be included. Copyright © 2014 Elsevier Ltd. All rights reserved.

Genetic Evidence Supports a Major Role for Akt1 in VSMCs During Atherogenesis

PubMed Central

Rotllan, Noemi; Wanschel, Amarylis C.; Fernandez-Hernando, Ana; Salerno, Alessandro G.; Offermanns, Stefan; Sessa, William C.; Fernández-Hernando, Carlos

2015-01-01

Rationale Coronary artery disease (CAD), the direct result of atherosclerosis, is the most common cause of death in Western societies. Vascular smooth muscle cell (VSMC) apoptosis occurs during the progression of atherosclerosis and in advanced lesions, promotes plaque necrosis, a common feature of high-risk/vulnerable atherosclerotic plaques. Akt1, a serine-threonine protein kinase, regulates several key endothelial cell (EC) and VSMC functions including cell growth, migration, survival and vascular tone. While global deficiency of Akt1 results in impaired angiogenesis and massive atherosclerosis, the specific contribution of VSMC Akt1 remains poorly characterized. Objective To investigate the contribution of VSMC Akt1 during atherogenesis and in established atherosclerotic plaques. Methods and Results We generated two mouse models in which Akt1 expression can be suppressed specifically in VSCMs before (Apoe−/−Akt1fl/flSm22αCRE) and after (Apoe−/−Akt1fl/flSM-MHC-CreERT2E) the formation of atherosclerotic plaques. This approach allows us to interrogate the role of Akt1 during the initial and late steps of atherogenesis. Absence of Akt1 in VSMCs during the progression of atherosclerosis results in larger atherosclerotic plaques characterized by bigger necrotic core areas, enhanced VSMC apoptosis and reduced fibrous cap and collagen content. In contrast, VSMC Akt1 inhibition in established atherosclerotic plaques does not influence lesion size but markedly reduces the relative fibrous cap area in plaques and increases VSMC apoptosis. Conclusions Akt1 expression in VSMCs influences early and late stages of atherosclerosis. Absence of Akt1 in VSMCs induces features of plaque vulnerability including fibrous cap thinning and extensive necrotic core areas. These observations suggest that interventions enhancing Akt1 expression specifically in VSMCs may lessen plaque progression. PMID:25868464

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

PubMed

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

2012-10-01

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

Osthole inhibits gastric cancer cell proliferation through regulation of PI3K/AKT

PubMed Central

Zhang, Yan

2018-01-01

Osthole is an active compound isolated from Chinese herb Cnidium monnieri (L.) Cusson, and had been reported to possess antitumor effect. However, the effect of osthole on the gastric cancer cells has not been investigated. In this study, the effects of osthole on the proliferation of human gastric cancer cells were tested. The data showed that osthole treatment significantly inhibited the proliferation of gastric cancer cells and resulted in the cell cycle arrest at G2/M phase in a dose-dependent manner. Western-blot study showed that the expression of cyclin B1 and cdc2 was markedly reduced by osthole. Moreover, expression of PI3K and pAKT was also significantly suppressed, and the results indicated that the inhibition of pAKT, cyclin B1, and cdc2 levels by osthole was notably enhanced by a PI3K inhibitor. These results demonstrate that osthole could inhibit gastric cancer cells proliferation via induction of cell cycle arrest at G2/M phase by the reduction of PI3K/AKT. PMID:29590128

Osthole inhibits gastric cancer cell proliferation through regulation of PI3K/AKT.

PubMed

Xu, Xiaojun; Liu, Xiaoyuan; Zhang, Yan

2018-01-01

Osthole is an active compound isolated from Chinese herb Cnidium monnieri (L.) Cusson, and had been reported to possess antitumor effect. However, the effect of osthole on the gastric cancer cells has not been investigated. In this study, the effects of osthole on the proliferation of human gastric cancer cells were tested. The data showed that osthole treatment significantly inhibited the proliferation of gastric cancer cells and resulted in the cell cycle arrest at G2/M phase in a dose-dependent manner. Western-blot study showed that the expression of cyclin B1 and cdc2 was markedly reduced by osthole. Moreover, expression of PI3K and pAKT was also significantly suppressed, and the results indicated that the inhibition of pAKT, cyclin B1, and cdc2 levels by osthole was notably enhanced by a PI3K inhibitor. These results demonstrate that osthole could inhibit gastric cancer cells proliferation via induction of cell cycle arrest at G2/M phase by the reduction of PI3K/AKT.

Aurora kinase A revives dormant laryngeal squamous cell carcinoma cells via FAK/PI3K/Akt pathway activation

PubMed Central

Yang, Li-yun; He, Chang-yu; Chen, Xue-hua; Su, Li-ping; Liu, Bing-ya; Zhang, Hao

2016-01-01

Revival of dormant tumor cells may be an important tumor metastasis mechanism. We hypothesized that aurora kinase A (AURKA), a cell cycle control kinase, promotes the transition of laryngeal squamous cell carcinoma (LSCC) cells from G0 phase to active division. We therefore investigated whether AURKA could revive dormant tumor cells to promote metastasis. Western blotting revealed that AURKA expression was persistently low in dormant laryngeal cancer Hep2 (D-Hep2) cells and high in non-dormant (T-Hep2) cells. Decreasing AURKA expression in T-Hep2 cells induced dormancy and reduced FAK/PI3K/Akt pathway activity. Increasing AURKA expression in D-Hep2 cells increased FAK/PI3K/Akt pathway activity and enhanced cellular proliferation, migration, invasion and metastasis. In addition, FAK/PI3K/Akt pathway inhibition caused dormancy-like behavior and reduced cellular mobility, migration and invasion. We conclude that AURKA may revive dormant tumor cells via FAK/PI3K/Akt pathway activation, thereby promoting migration and invasion in laryngeal cancer. AURKA/FAK/PI3K/Akt inhibitors may thus represent potential targets for clinical LSCC treatment. PMID:27356739

Activation of Akt rescues endoplasmic reticulum stress-impaired murine cardiac contractile function via glycogen synthase kinase-3β-mediated suppression of mitochondrial permeation pore opening.

PubMed

Zhang, Yingmei; Xia, Zhi; La Cour, Karissa H; Ren, Jun

2011-11-01

The present study was designed to examine the impact of chronic Akt activation on endoplasmic reticulum (ER) stress-induced cardiac mechanical anomalies, if any, and the underlying mechanism involved. Wild-type and transgenic mice with cardiac-specific overexpression of the active mutant of Akt (Myr-Akt) were subjected to the ER stress inducer tunicamycin (1 or 3 mg/kg). ER stress led to compromised echocardiographic (elevated left ventricular end-systolic diameter and reduced fractional shortening) and cardiomyocyte contractile function, intracellular Ca(2+) mishandling, and cell survival in wild-type mice associated with mitochondrial damage. In vitro ER stress induction in murine cardiomyocytes upregulated the ER stress proteins Gadd153, GRP78, and phospho-eIF2α, and promoted reactive oxygen species production, carbonyl formation, apoptosis, mitochondrial membrane potential loss, and mitochondrial permeation pore (mPTP) opening associated with overtly impaired cardiomyocyte contractile and intracellular Ca(2+) properties. Interestingly, these anomalies were mitigated by chronic Akt activation or the ER chaperon tauroursodeoxycholic acid (TUDCA). Treatment with tunicamycin also dephosphorylated Akt and its downstream signal glycogen synthase kinase 3β (GSK3β) (leading to activation of GSK3β), the effect of which was abrogated by Akt activation and TUDCA. The ER stress-induced cardiomyocyte contractile and mitochondrial anomalies were obliterated by the mPTP inhibitor cyclosporin A, GSK3β inhibitor SB216763, and ER stress inhibitor TUDCA. This research reported the direct relationship between ER stress and cardiomyocyte contractile and mitochondrial anomalies for the first time. Taken together, these data suggest that ER stress may compromise cardiac contractile and intracellular Ca(2+) properties, possibly through the Akt/GSK3β-dependent impairment of mitochondrial integrity.

Serine 1179 phosphorylation of endothelial nitric oxide synthase caused by 2,4,6-trinitrotoluene through PI3K/Akt signaling in endothelial cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sun Yang; Sumi, Daigo; Kumagai, Yoshito

2006-07-01

Although 2,4,6-trinitrotoluene (TNT) has been found to uncouple nitric oxide synthase (NOS), thereby leading to reactive oxygen species (ROS), cellular response against TNT still remains unclear. Exposure of bovine aortic endothelial cells (BAECs) to TNT (100 {mu}M) resulted in serine 1179 phosphorylation of endothelial NOS (eNOS). With specific inhibitors (wortmannin and LY294002), we found that PI3K/Akt signaling participated in the eNOS phosphorylation caused by TNT, whereas the ERK pathway did not. ROS were generated following exposure of BAECs to TNT. However, TNT-mediated phosphorylation of either eNOS or Akt was drastically blocked by NAC and PEG-CAT. Interestingly, pretreatment with apocynin, amore » specific inhibitor for NADPH oxidase, diminished the phosphorylation of eNOS and Akt. These results suggest that TNT affects NADPH oxidase, thereby generating hydrogen peroxide, which is capable of activating PI3K/Akt signaling associated with eNOS Ser 1179 phosphorylation.« less

Anti-inflammatory effects of fimasartan via Akt, ERK, and NFκB pathways on astrocytes stimulated by hemolysate.

PubMed

Yang, Xiu-Li; Kim, Chi Kyung; Kim, Tae Jung; Sun, Jing; Rim, Doeun; Kim, Young-Ju; Ko, Sang-Bae; Jang, Hyunduk; Yoon, Byung-Woo

2016-02-01

The aim of this study was to investigate whether fimasartan, a novel angiotensin II receptor blocker, modulates hemolysate-induced inflammation in astrocytes. We stimulated astrocytes with hemolysate to induce hemorrhagic inflammation in vitro. Astrocytes were pretreated with fimasartan and then incubated with hemolysate at different durations. Anti-inflammatory cell signaling molecules including Akt, extracellular signal regulated kinase (ERK), NFκB and cyclooxygenase-2 (COX-2) were assessed by western blotting. Pro-inflammatory mediators were evaluated by real-time RT-PCR and ELISA. The stimulation by hemolysate generated a robust activation of inflammatory signaling pathways in astrocytes. Hemolysate increased the phosphorylation of Akt at 1 h, and ERK1/2 at 20 min compared with the control group and promoted the degradation of IκBα. Pretreated fimasartan significantly decreased hemolysate-induced phosphorylation of Akt and ERK1/2. In addition, fimasartan also suppressed NFκB-related inflammatory pathways induced by hemolysate, including reduction of the gene expression of NFκB, and decreased nuclear translocation of NFκB and degradation of IκB. This reduction of inflammatory upstream pathways decreased the expression of inflammatory end-products: COX-2 and interleukin-1 (IL-1β). Furthermore, the expression of COX-2 was attenuated by both Akt inhibitor (LY294002) and ERK inhibitor (U0126), and IκBα degradation was suppressed by LY294002. These results demonstrate that pretreatment with fimasartan to astrocytes suppresses the inflammatory responses induced by hemolysate. Akt, ERK and NFκB were associated with hemolysate-induced COX-2 and IL-1β expression. Based on these mechanisms, fimasartan could be a candidate anti-inflammatory regulator for the treatment of intracerebral hemorrhage.

Characterization of the Akt2 Domain Essential for Binding Nuclear p21cip1 to Promote Cell Cycle Arrest during Myogenic Differentiation

PubMed Central

Heron-Milhavet, Lisa; Franckhauser, Celine; Fernandez, Anne; Lamb, Ned J.

2013-01-01

The binding of the cdk inhibitor p21cip1 to Akt2 in the nucleus is an essential component in determining the specific role of Akt2 in the cell cycle arrest that precedes myogenic differentiation. Here, through a combination of biochemical and cell biology approaches, we have addressed the molecular basis of this binding. Using amino-terminal truncation of Akt2, we show that p21cip1 binds at the carboxy terminal of Akt2 since deletion of the first 400 amino acids did not affect the interaction between Akt2 and p21cip1. Pull down using carboxy terminal-truncated Akt2 protein revealed the importance of the region between amino acids 400 and 445 for the binding to p21cip1. Since Akt2_400–445 and Akt2_420–445 peptides could both bind p21cip1, this refines the binding domain on Akt2 between amino acids 420 and 445. In order to confirm these data in living cells, we developed a protocol to synchronize myoblasts at the cell cycle exit point when p21cip1 expression is induced by MyoD before myogenic differentiation. When a synthetic Akt2 peptide spanning the region (410–437) was microinjected in p21-expressing myoblasts, p21cip1 no longer localized exclusively in the nucleus, instead being redistributed throughout the cell, thus showing that injected peptide 410–437 acts to compete with the binding of endogenous Akt2 to p21cip1. Taken together, our data suggest that this 27 amino acid sequence on Akt2 is necessary and sufficient to bind p21cip1 both in vitro and in living cells. PMID:24194853

DUOX enzyme activity promotes AKT signalling in prostate cancer cells.

PubMed

Pettigrew, Christopher A; Clerkin, John S; Cotter, Thomas G

2012-12-01

Reactive oxygen species (ROS) and oxidative stress are related to tumour progression, and high levels of ROS have been observed in prostate tumours compared to normal prostate. ROS can positively influence AKT signalling and thereby promote cell survival. The aim of this project was to establish whether the ROS generated in prostate cancer cells positively regulate AKT signalling and enable resistance to apoptotic stimuli. In PC3 cells, dual oxidase (DUOX) enzymes actively generate ROS, which inactivate phosphatases, thereby maintaining AKT phosphorylation. Inhibition of DUOX by diphenylene iodium (DPI), intracellular calcium chelation and small-interfering RNA (siRNA) resulted in lower ROS levels, lower AKT and glycogen synthase kinase 3β (GSK3β) phosphorylation, as well as reduced cell viability and increased susceptibility to apoptosis stimulating fragment (FAS) induced apoptosis. This report shows that ROS levels in PC3 cells are constitutively maintained by DUOX enzymes, and these ROS positively regulate AKT signalling through inactivating phosphatases, leading to increased resistance to apoptosis.

Regulation of ENaC-mediated alveolar fluid clearance by insulin via PI3K/Akt pathway in LPS-induced acute lung injury.

PubMed

Deng, Wang; Li, Chang-Yi; Tong, Jin; Zhang, Wei; Wang, Dao-Xin

2012-03-30

Stimulation of epithelial sodium channel (ENaC) increases Na(+) transport, a driving force of alveolar fluid clearance (AFC) to keep alveolar spaces free of edema fluid that is beneficial for acute lung injury (ALI). It is well recognized that regulation of ENaC by insulin via PI3K pathway, but the mechanism of this signaling pathway to regulate AFC and ENaC in ALI remains unclear. The aim of this study was to investigate the effect of insulin on AFC in ALI and clarify the pathway in which insulin regulates the expression of ENaC in vitro and in vivo. A model of ALI (LPS at a dose of 5.0 mg/kg) with non-hyperglycemia was established in Sprague-Dawley rats receiving continuous exogenous insulin by micro-osmotic pumps and wortmannin. The lungs were isolated for measurement of bronchoalveolar lavage fluid(BALF), total lung water content(TLW), and AFC after ALI for 8 hours. Alveolar epithelial type II cells were pre-incubated with LY294002, Akt inhibitor and SGK1 inhibitor 30 minutes before insulin treatment for 2 hours. The expressions of α-,β-, and γ-ENaC were detected by immunocytochemistry, reverse transcriptase polymerase chain reaction (RT-PCR) and western blotting. In vivo, insulin decreased TLW, enchanced AFC, increased the expressions of α-,β-, and γ-ENaC and the level of phosphorylated Akt, attenuated lung injury and improved the survival rate in LPS-induced ALI, the effects of which were blocked by wortmannin. Amiloride, a sodium channel inhibitor, significantly reduced insulin-induced increase in AFC. In vitro, insulin increased the expressions of α-,β-, and γ-ENaC as well as the level of phosphorylated Akt but LY294002 and Akt inhibitor significantly prevented insulin-induced increase in the expression of ENaC and the level of phosphorylated Akt respectively. Immunoprecipitation studies showed that levels of Nedd4-2 binding to ENaC were decreased by insulin via PI3K/Akt pathway. Our study demonstrated that insulin alleviated pulmonary edema and

PI3K/Akt contributes to increased expression of Toll-like receptor 4 in macrophages exposed to hypoxic stress

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kim, So Young; Jeong, Eunshil; Joung, Sun Myung

2012-03-16

Highlights: Black-Right-Pointing-Pointer Hypoxic stress-induced TLR4 expression is mediated by PI3K/Akt in macrophages. Black-Right-Pointing-Pointer PI3K/Akt regulated HIF-1 activation leading to TLR4 expression. Black-Right-Pointing-Pointer p38 mitogen-activated protein kinase was not involved in TLR4 expression by hypoxic stress. Black-Right-Pointing-Pointer Sulforaphane suppressed hypoxia-mediated TLR4 expression by inhibiting PI3K/Akt. -- Abstract: Toll-like receptors (TLRs) play critical roles in triggering immune and inflammatory responses by detecting invading microbial pathogens and endogenous danger signals. Increased expression of TLR4 is implicated in aggravated inflammatory symptoms in ischemic tissue injury and chronic diseases. Results from our previous study showed that TLR4 expression was upregulated by hypoxic stress mediated bymore » hypoxia-inducible factor-1 (HIF-1) at a transcriptional level in macrophages. In this study, we further investigated the upstream signaling pathway that contributed to the increase of TLR4 expression by hypoxic stress. Either treatment with pharmacological inhibitors of PI3K and Akt or knockdown of Akt expression by siRNA blocked the increase of TLR4 mRNA and protein levels in macrophages exposed to hypoxia and CoCl{sub 2}. Phosphorylation of Akt by hypoxic stress preceded nuclear accumulation of HIF-1{alpha}. A PI3K inhibitor (LY294002) attenuated CoCl{sub 2}-induced nuclear accumulation and transcriptional activation of HIF-1{alpha}. In addition, HIF-1{alpha}-mediated upregulation of TLR4 expression was blocked by LY294002. Furthermore, sulforaphane suppressed hypoxia- and CoCl{sub 2}-induced upregulation of TLR4 mRNA and protein by inhibiting PI3K/Akt activation and the subsequent nuclear accumulation and transcriptional activation of HIF-1{alpha}. However, p38 was not involved in HIF-1{alpha} activation and TLR4 expression induced by hypoxic stress in macrophages. Collectively, our results demonstrate that PI3K/Akt

The glycogen metabolism via Akt signaling is important for the secretion of enamel matrix in tooth development.

PubMed

Ida-Yonemochi, Hiroko; Otsu, Keishi; Ohshima, Hayato; Harada, Hidemitsu

2016-02-01

Cells alter their energy metabolism depending on the stage of differentiation or various environments. In the ameloblast differentiation of continuous growing mouse incisors, we found temporary glycogen storage in preameloblasts before the start of enamel matrix secretion and investigated the relationship between enamel matrix secretion and glycogen metabolism. Immunohistochemistry showed that in the transitional stage from preameloblasts to secretory ameloblasts, the glycogen synthase changed from the inactive form to the active form, the expression of glycogen phosphorylase increased, and further, the levels of IGF-1, IGF-1 receptor and activated Akt increased. These results suggested that the activation of Akt signaling via IGF is linked to the onset of both glycogen metabolism and enamel matrix deposition. In the experiments using organ culture and ameloblast cell line, the activation of Akt signaling by IGF-1 stimulated glycogen metabolism through the up-regulation of Glut-1,-4 and Gsk-3β and the dephosphorylation of glycogen synthase. Subsequently, they resulted in increased enamel matrix secretion. In contrast, some inhibitors of Akt signals and glycogen synthesis/degradation down-regulated enamel matrix secretion. Taking these findings together, glycogen metabolism via Akt signaling is an essential system for the secretion of enamel matrix in ameloblast differentiation. Copyright © 2016 Elsevier B.V. All rights reserved.

Hydrogen sulfide facilities production of nitric oxide via the Akt/endothelial nitric oxide synthases signaling pathway to protect human umbilical vein endothelial cells from injury by angiotensin II.

PubMed

Cui, Jiasen; Zhuang, Shunjiu; Qi, Shaohong; Li, Li; Zhou, Junwen; Zhang, Wan; Zhao, Yun; Qi, Ning; Yin, Yangjun; Huang, Lu

2017-11-01

Angiotensin II (Ang II) has been reported as key in inducing endothelial cell injury, and endothelial cells may produce nitric oxide (NO) to protect themselves. However, the underlying mechanism remains elusive. Human umbilical vein endothelial cells (HUVECs) were divided into five treatment groups as follows: Normal control, Ang II, Ang II + sodium hydrosulfide [NaHS; hydrogen sulfide (H2S) donor], Ang II + Akt inhibitors + NaHS, and Ang II + endothelial nitric oxide synthases (eNOS) inhibitors + NaHS. Subsequently, cell viability, apoptosis, migration, proliferation and adhesion ability were determined. In addition, tubular structure formation was observed, and the NO and phosphorylation levels of Akt and eNOS were evaluated. Compared with the normal control group, Ang II treatment reduced the viability of HUVECs and increased the level of cell apoptosis (P<0.05). Furthermore, Ang II treatment inhibited the phosphorylation level of eNOS and Akt, as well as the generation of NO (P<0.05). H2S reversed the above‑mentioned effects significantly and increased cell proliferation, adhesion ability and promoted tubular structure formation (P<0.05); however, H2S did not reverse the impact of eNOS and Akt phosphorylation levels after being processed with Akt and eNOS inhibitors, which indicates that H2S is capable of protecting HUVECs via the eNOS/Akt signaling pathway (P<0.05). Thus, H2S stimulates the production of NO and protects HUVECs via inducing the Akt/eNOS signaling pathway.

Apigenin in Combination with Akt Inhibition Significantly Enhances Thyrotropin-Stimulated Radioiodide Accumulation in Thyroid Cells

PubMed Central

Lakshmanan, Aparna; Doseff, Andrea I.; Ringel, Matthew D.; Saji, Motoyasu; Rousset, Bernard; Zhang, Xiaoli

2014-01-01

Background: Selectively increased radioiodine accumulation in thyroid cells by thyrotropin (TSH) allows targeted treatment of thyroid cancer. However, the extent of TSH-stimulated radioiodine accumulation in some thyroid tumors is not sufficient to confer therapeutic efficacy. Hence, it is of clinical importance to identify novel strategies to selectively further enhance TSH-stimulated thyroidal radioiodine accumulation. Methods: PCCl3 rat thyroid cells, PCCl3 cells overexpressing BRAFV600E, or primary cultured tumor cells from a thyroid cancer mouse model, under TSH stimulation were treated with various reagents for 24 hours. Cells were then subjected to radioactive iodide uptake, kinetics, efflux assays, and protein extraction followed by Western blotting against selected antibodies. Results: We previously reported that Akt inhibition increased radioiodine accumulation in thyroid cells under chronic TSH stimulation. Here, we identified Apigenin, a plant-derived flavonoid, as a reagent to further enhance the iodide influx rate increased by Akt inhibition in thyroid cells under acute TSH stimulation. Akt inhibition is permissive for Apigenin's action, as Apigenin alone had little effect. This action of Apigenin requires p38 MAPK activity but not PKC-δ. The increase in radioiodide accumulation by Apigenin with Akt inhibition was also observed in thyroid cells expressing BRAFV600E and in primary cultured thyroid tumor cells from TRβPV/PV mice. Conclusion: Taken together, Apigenin may serve as a dietary supplement in combination with Akt inhibitors to enhance therapeutic efficacy of radioiodine for thyroid cancer. PMID:24400871

GRAMD1B regulates cell migration in breast cancer cells through JAK/STAT and Akt signaling.

PubMed

Khanna, Puja; Lee, Joan Shuying; Sereemaspun, Amornpun; Lee, Haeryun; Baeg, Gyeong Hun

2018-06-22

Dysregulated JAK/STAT signaling has been implicated in breast cancer metastasis, which is associated with high relapse risks. However, mechanisms underlying JAK/STAT signaling-mediated breast tumorigenesis are poorly understood. Here, we showed that GRAMD1B expression is upregulated on IL-6 but downregulated upon treatment with the JAK2 inhibitor AG490 in the breast cancer MDA-MB-231 cells. Notably, Gramd1b knockdown caused morphological changes of the cells, characterized by the formation of membrane ruffling and protrusions, implicating its role in cell migration. Consistently, GRAMD1B inhibition significantly enhanced cell migration, with an increase in the levels of the Rho family of GTPases. We also found that Gramd1b knockdown-mediated pro-migratory phenotype is associated with JAK2/STAT3 and Akt activation, and that JAK2 or Akt inhibition efficiently suppresses the phenotype. Interestingly, AG490 dose-dependently increased p-Akt levels, and our epistasis analysis suggested that the effect of JAK/STAT inhibition on p-Akt is via the regulation of GRAMD1B expression. Taken together, our results suggest that GRAMD1B is a key signaling molecule that functions to inhibit cell migration in breast cancer by negating both JAK/STAT and Akt signaling, providing the foundation for its development as a novel biomarker in breast cancer.

Roles of methyltrienolone (R1881) in AKTs and AR expression patterns of cultured granulosa-lutein cells.

PubMed

Nekoonam, Saeid; Naji, Mohammad; Mortezaee, Keywan; Amidi, Fardin

2018-05-11

AR-mediated androgen signaling plays a key role in female reproductive system. Granulosa-lutein cells (GCs) are the main sites for expression of androgen receptor (AR). There is also a close relation between AKT signaling and AR. Here, we assayed the role for a synthetic AR ligand methyltrienolone (R1881) in expressions of AKTs and AR. Controlled ovarian hyperstimulation (COH) was performed in 20 normal women. Mural GCs were isolated by filtration method, cultured, and passaged. Then, the cells were starved for 48 h with 10% charcoal stripped FBS. The cells were then treated with R1881, bicalutamide (AR blocker), LY294002 (PI3K/AKT pathway blocker), and combination of them for 48 h. Finally, GCs were evaluated for quantitative real-time PCR analysis of AKT1, AKT2, AKT3, and AR, and also Western blot assessment of total AKT and phosphorylated AKT (p-AKT) [Ser473 and Thr308]. Addition of R1881 to the GCs culture showed high expressions of AKT1, AKT2, and AKT3 (P ≤ 0.05 vs LY294002 group and bicalutamide group). Expressions of AKT1 and AKT2 were decreased in the GCs under exposure to bicalutamide or LY294002 (P ≤ 0.05 vs R1881). AKT1, AKT2, and AKT3 showed decreased rates of expressions in the LY294002 + bicalutamide group (P ≤ 0.05 vs R1881). AR, total AKT and p-AKT showed no significant differences between groups. Our findings indicate that 46 h exposure with R1881 could affect AKTs expressions in the GCs of pre-ovulatory phase, but it cannot promote AR expression and AKTs activation. © 2018 Wiley Periodicals, Inc.

Kaposi sarcoma associated herpesvirus (KSHV) induces AKT hyperphosphorylation, bortezomib-resistance and GLUT-1 plasma membrane exposure in THP-1 monocytic cell line.

PubMed

Gonnella, Roberta; Santarelli, Roberta; Farina, Antonella; Granato, Marisa; D'Orazi, Gabriella; Faggioni, Alberto; Cirone, Mara

2013-10-23

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. 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. 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, but on the other hand, offer an

Carbachol induces p70S6K1 activation through an ERK-dependent but Akt-independent pathway in human colonic epithelial cells.

PubMed

Jiang, Xiaohua; Sinnett-Smith, James; Rozengurt, Enrique

2009-09-25

Stimulation of human colonic epithelial T84 cells with the muscarinic receptor agonist carbachol, a stable analog of acetylcholine, induced Akt, p70S6K1 and ERK activation. Treatment of T84 cells with the selective inhibitor of EGF receptor (EGFR) tyrosine kinase AG1478 abrogated Akt phosphorylation on Ser(473) induced by either carbachol or EGF, indicating that carbachol-induced Akt activation is mediated through EGFR transactivation. Surprisingly, AG1478 did not suppress p70S6K1 phosphorylation on Thr(389) in response to carbachol, indicating the G protein-coupled receptor (GPCR) stimulation induces p70S6K1 activation, at least in part, via an Akt-independent pathway. In contrast, treatment with the selective MEK inhibitor U0126 (but not with the inactive analog U0124) inhibited carbachol-induced p70S6K1 activation, indicating that the MEK/ERK/RSK pathway plays a critical role in p70S6K1 activation in GPCR-stimulated T84 cells. These findings imply that GPCR activation induces p70S6K1 via ERK rather than through the canonical PI 3-kinase/Akt/TSC/mTORC1 pathway in T84 colon carcinoma cells.

Carbachol induces p70S6K1 activation through an ERK-dependent but Akt-independent pathway in human colonic epithelial cells

PubMed Central

Jiang, Xiaohua; Sinnett-Smith, James; Rozengurt, Enrique

2009-01-01

Stimulation of human colonic epithelial T84 cells with the muscarinic receptor agonist carbachol, a stable analog of acetylcholine, induced Akt, p70S6K1 and ERK activation. Treatment of T84 cells with the selective inhibitor of EGF receptor (EGFR) tyrosine kinase AG1478 abrogated Akt phosphorylation on Ser473 induced by either carbachol or EGF, indicating that carbachol-induced Akt activation is mediated through EGFR transactivation. Surprisingly, AG1478 did not suppress p70S6K1 phosphorylation on Thr389 in response to carbachol, indicating the G protein-coupled receptor (GPCR) stimulation induces p70S6K1 activation, at least in part, via an Akt-independent pathway. In contrast, treatment with the selective MEK inhibitor U0126 (but not with the inactive analog U0124) inhibited carbachol-induced p70S6K1 activation, indicating that the MEK/ERK/RSK pathway plays a critical role in p70S6K1 activation in GPCR-stimulated T84 cells. These findings imply that GPCR activation induces p70S6K1 via ERK rather than through the canonical PI 3-kinase/Akt/TSC/mTORC1 pathway in T84 colon carcinoma cells. PMID:19615971

Polydatin inhibits cell proliferation and induces apoptosis in laryngeal cancer and HeLa cells via suppression of the PDGF/AKT signaling pathway.

PubMed

Li, Haixia; Shi, Baoyuan; Li, Yanyun; Yin, Fengfang

2017-07-01

Polydatin (PD), a stilbene compound extracted from Polygonum cuspidatum, is suggested to possess anti-cancer activities, including inhibition of cell proliferation, cell cycle arrest, and induction of apoptosis. The platelet-derived growth factor (PDGF)/AKT signaling pathway plays complex roles in tumor suppression. However, the effect of PD on the PDGF/AKT signaling pathway in laryngeal cancer and HeLa cells has not been explored. MTT assay and flow cytometry showed that PD inhibited cell proliferation and induced apoptosis in Hep-2 and AMC-HN-8 cells. Western blot analysis indicated that PD inhibited the expression levels of PDGF-B and phosphorylated AKT (p-AKT) in both cells. Treatment of PDGF-B siRNA or PDGFR inhibitor found that after the PDGF signaling was inactivated, p-AKT expression was significantly decreased in Hep-2 cells. Tumor xenograft experiment in nude mice indicated PD significantly inhibited the growth of Hep-2 cells in vivo. In conclusion, PD inhibited cell proliferation and induced apoptosis in laryngeal cancer and HeLa cells via inactivation of the PDGF/AKT signaling pathway. © 2017 Wiley Periodicals, Inc.

Akt1 deficiency diminishes skeletal muscle hypertrophy by reducing satellite cell proliferation.

PubMed

Moriya, Nobuki; Miyazaki, Mitsunori

2018-05-01

Skeletal muscle mass is determined by the net dynamic balance between protein synthesis and degradation. Although the Akt/mechanistic target of rapamycin (mTOR)-dependent pathway plays an important role in promoting protein synthesis and subsequent skeletal muscle hypertrophy, the precise molecular regulation of mTOR activity by the upstream protein kinase Akt is largely unknown. In addition, the activation of satellite cells has been indicated as a key regulator of muscle mass. However, the requirement of satellite cells for load-induced skeletal muscle hypertrophy is still under intense debate. In this study, female germline Akt1 knockout (KO) mice were used to examine whether Akt1 deficiency attenuates load-induced skeletal muscle hypertrophy through suppressing mTOR-dependent signaling and satellite cell proliferation. Akt1 KO mice showed a blunted hypertrophic response of skeletal muscle, with a diminished rate of satellite cell proliferation following mechanical overload. In contrast, Akt1 deficiency did not affect the load-induced activation of mTOR signaling and the subsequent enhanced rate of protein synthesis in skeletal muscle. These observations suggest that the load-induced activation of mTOR signaling occurs independently of Akt1 regulation and that Akt1 plays a critical role in regulating satellite cell proliferation during load-induced muscle hypertrophy.

Akt2/ZEB2 may be a biomarker for exfoliant cells in ascitic fluid in advanced grades of serous ovarian carcinoma.

PubMed

Liu, Changmei; Yang, Fangmei

2015-09-01

Ovarian cancers present a mild clinical course when diagnosed early but an aggressive pathway when diagnosed in the peri- and postmenopausal periods. However, the predictability of tumor progression is stochastic and is difficult to predict. In the present study, we hypothesized to examine the key pathways that are dysregulated to promote epithelial-mesenchymal transition in serous ovarian carcinoma. Examination of these steps would help to identify ascitic fluid with cells poised for metastasis or otherwise. We focused on examining the Akt2 expression, mainly because of its report as being overamplified in the aggressive variants of ovarian cancer, as well as TGFbeta-sensitivity of Akt2 that forms the key basis for metastasis initiation of most kinds of carcinoma. We obtained primary ovarian carcinoma samples as well as ascitic fluid and distantly metastatic ovarian carcinoma to examine the expression of Akt2. The results of the study demonstrated that in malignant exfoliated ovarian cancer cells, Smad4 expression was tremendously increased in the nuclei, suggesting nuclear translocation of Smad, which thereafter may have activated ZEB2, and thereafter genomically affected the expression of E-cadherin, myosin II, and vimentin, key components for initiating and sustaining metastasis. All of these may have been stimulated by increased cellular expression of Akt2 in metastatic variants of the serous ovarian carcinoma. The reliance on Akt2 and TGF beta signaling may also potentiate the case for Akt inhibitors or small molecule inhibitors of TGFbeta signaling like doxycycline as adjunct chemotherapy in serous ovarian carcinoma, especially the metastatic variants.

Acute Ethanol Inhibition of γ Oscillations Is Mediated by Akt and GSK3β

PubMed Central

Wang, JianGang; Zhao, JingXi; Liu, ZhiHua; Guo, FangLi; Wang, Yali; Wang, Xiaofang; Zhang, RuiLing; Vreugdenhil, Martin; Lu, Chengbiao

2016-01-01

Hippocampal network oscillations at gamma band frequency (γ, 30–80 Hz) are closely associated with higher brain functions such as learning and memory. Acute ethanol exposure at intoxicating concentrations (≥50 mM) impairs cognitive function. This study aimed to determine the effects and the mechanisms of acute ethanol exposure on γ oscillations in an in vitro model. Ethanol (25–100 mM) suppressed kainate-induced γ oscillations in CA3 area of the rat hippocampal slices, in a concentration-dependent, reversible manner. The ethanol-induced suppression was reduced by the D1R antagonist SCH23390 or the PKA inhibitor H89, was prevented by the Akt inhibitor triciribine or the GSk3β inhibitor SB415286, was enhanced by the NMDA receptor antagonist D-AP5, but was not affected by the MAPK inhibitor U0126 or PI3K inhibitor wortmanin. Our results indicate that the intracellular kinases Akt and GSk3β play a critical role in the ethanol-induced suppression of γ oscillations and reveal new cellular pathways involved in the ethanol-induced cognitive impairment. PMID:27582689

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

PubMed

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

2013-05-01

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

O-GlcNAcylation regulates ischemia-induced neuronal apoptosis through AKT signaling.

PubMed

Shi, Jianhua; Gu, Jin-hua; Dai, Chun-ling; Gu, Jianlan; Jin, Xiaoxia; Sun, Jianming; Iqbal, Khalid; Liu, Fei; Gong, Cheng-Xin

2015-09-28

Apoptosis plays an important role in neural development and neurological disorders. In this study, we found that O-GlcNAcylation, a unique protein posttranslational modification with O-linked β-N-acetylglucosamine (GlcNAc), promoted apoptosis through attenuating phosphorylation/activation of AKT and Bad. By using co-immunoprecipitation and mutagenesis techniques, we identified O-GlcNAc modification at both Thr308 and Ser473 of AKT. O-GlcNAcylation-induced apoptosis was attenuated by over-expression of AKT. We also found a dynamic elevation of protein O-GlcNAcylation during the first four hours of cerebral ischemia, followed by continuous decline after middle cerebral artery occlusion (MCAO) in the mouse brain. The elevation of O-GlcNAcylation coincided with activation of cell apoptosis. Finally, we found a negative correlation between AKT phosphorylation and O-GlcNAcylation in ischemic brain tissue. These results indicate that cerebral ischemia induces a rapid increase of O-GlcNAcylation that promotes apoptosis through down-regulation of AKT activity. These findings provide a novel mechanism through which O-GlcNAcylation regulates ischemia-induced neuronal apoptosis through AKT signaling.

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

PubMed Central

2010-01-01

Background Neuroblastoma is a paediatric cancer of the sympathetic nervous system. The single most important genetic indicator of poor clinical outcome is amplification of the MYCN transcription factor. One of many down-stream MYCN targets is miR-184, which is either directly or indirectly repressed by this transcription factor, possibly due to its pro-apoptotic effects when ectopically over-expressed in neuroblastoma cells. The purpose of this study was to elucidate the molecular mechanism by which miR-184 conveys pro-apoptotic effects. Results We demonstrate that the knock-down of endogenous miR-184 has the opposite effect of ectopic up-regulation, leading to enhanced neuroblastoma cell numbers. As a mechanism of how miR-184 causes apoptosis when over-expressed, and increased cell numbers when inhibited, we demonstrate direct targeting and degradation of AKT2, a major downstream effector of the phosphatidylinositol 3-kinase (PI3K) pathway, one of the most potent pro-survival pathways in cancer. The pro-apoptotic effects of miR-184 ectopic over-expression in neuroblastoma cell lines is reproduced by siRNA inhibition of AKT2, while a positive effect on cell numbers similar to that obtained by the knock-down of endogenous miR-184 can be achieved by ectopic up-regulation of AKT2. Moreover, co-transfection of miR-184 with an AKT2 expression vector lacking the miR-184 target site in the 3'UTR rescues cells from the pro-apoptotic effects of miR-184. Conclusions MYCN contributes to tumorigenesis, in part, by repressing miR-184, leading to increased levels of AKT2, a direct target of miR-184. Thus, two important genes with positive effects on cell growth and survival, MYCN and AKT2, can be linked into a common genetic pathway through the actions of miR-184. As an inhibitor of AKT2, miR-184 could be of potential benefit in miRNA mediated therapeutics of MYCN amplified neuroblastoma and other forms of cancer. PMID:20409325

Loss of Akt1 leads to severe atherosclerosis and occlusive coronary artery disease

PubMed Central

Fernández-Hernando, Carlos; Ackah, Eric; Yu, Jun; Suárez, Yajaira; Murata, Takahisa; Iwakiri, Yasuko; Prendergast, Jay; Miao, Robert Q.; Birnbaum, Morris J.; Sessa, William C.

2013-01-01

SUMMARY The Akt signaling pathway controls several cellular functions in the cardiovascular system; however, its role in atherogenesis is unknown. Here we show that the genetic ablation of Akt1 on an apolipoprotein E knockout background (ApoE−/−Akt1−/−) increases aortic lesion expansion and promotes coronary atherosclerosis. Mechanistically, lesion formation is due to enhanced expression of pro-inflammatory genes and endothelial cell and macrophage apoptosis. Bone marrow transfer experiments suggest that macrophages from ApoE−/−Akt1−/− donors were not sufficient to worsen atherogenesis when transferred to ApoE−/− recipients suggesting that lesion expansion in the ApoE−/− Akt1−/ strain may be of vascular origin. In the vessel wall, the loss of Akt1 increases inflammatory mediators and reduces eNOS phosphorylation suggesting that Akt1 exerts vascular protection against atherogenesis. The presence of coronary lesions in ApoE−/−/Akt1−/− mice provides a new model for studying the mechanisms of acute coronary syndrome in humans. PMID:18054314

Two Membrane-Associated Tyrosine Phosphatase Homologs Potentiate C. elegans AKT-1/PKB Signaling

PubMed Central

Hu, Patrick J; Xu, Jinling; Ruvkun, Gary

2006-01-01

Akt/protein kinase B (PKB) functions in conserved signaling cascades that regulate growth and metabolism. In humans, Akt/PKB is dysregulated in diabetes and cancer; in Caenorhabditis elegans, Akt/PKB functions in an insulin-like signaling pathway to regulate larval development. To identify molecules that modulate C. elegans Akt/PKB signaling, we performed a genetic screen for enhancers of the akt-1 mutant phenotype (eak). We report the analysis of three eak genes. eak-6 and eak-5/sdf-9 encode protein tyrosine phosphatase homologs; eak-4 encodes a novel protein with an N-myristoylation signal. All three genes are expressed primarily in the two endocrine XXX cells, and their predicted gene products localize to the plasma membrane. Genetic evidence indicates that these proteins function in parallel to AKT-1 to inhibit the FoxO transcription factor DAF-16. These results define two membrane-associated protein tyrosine phosphatase homologs that may potentiate C. elegans Akt/PKB signaling by cell autonomous and cell nonautonomous mechanisms. Similar molecules may modulate Akt/PKB signaling in human endocrine tissues. PMID:16839187

Interrogating two schedules of the AKT inhibitor MK-2206 in patients with advanced solid tumors incorporating novel pharmacodynamic and functional imaging biomarkers

PubMed Central

Yap, Timothy A.; Yan, Li; Patnaik, Amita; Tunariu, Nina; Biondo, Andrea; Fearen, Ivy; Papadopoulos, Kyriakos P.; Olmos, David; Baird, Richard; Delgado, Liliana; Tetteh, Ernestina; Beckman, Robert A.; Lupinacci, Lisa; Riisnaes, Ruth; Decordova, Shaun; Heaton, Simon P.; Swales, Karen; deSouza, Nandita M; Leach, Martin O.; Garrett, Michelle D.; Sullivan, Daniel M.; de Bono, Johann S.; Tolcher, Anthony W.

2014-01-01

Purpose Multiple cancers harbor genetic aberrations that impact AKT signaling. MK-2206 is a potent pan-AKT inhibitor with a maximum tolerated dose (MTD) previously established at 60mg on alternate days (QOD). Due to a long half-life (60-80h), a weekly (QW) MK-2206 schedule was pursued to compare intermittent QW and continuous QOD dosing. Experimental Design Patients with advanced cancers were enrolled onto a QW dose-escalation phase I study to investigate the safety and pharmacokinetic-pharmacodynamic profiles of tumor and platelet-rich plasma (PRP). The QOD MTD of MK-2206 was also assessed in patients with ovarian and castration-resistant prostate cancers, and patients with advanced cancers undergoing multiparametric functional magnetic resonance imaging (MRI) studies, including dynamic contrast-enhanced MRI, diffusion-weighted imaging, magnetic resonance spectroscopy and intrinsic susceptibility-weighted MRI. Results Seventy-one patients were enrolled; 38 patients had 60mg MK-2206 QOD, while 33 received MK-2206 at 90mg, 135mg, 150mg, 200mg, 250mg, and 300mg QW. The QW MK-2206 MTD was established at 200mg following dose-limiting rash at 250mg and 300mg. QW dosing appeared to be similarly tolerated to QOD, with toxicities including rash, gastrointestinal symptoms, fatigue, and hyperglycemia. Significant AKT pathway blockade was observed with both continuous QOD and intermittent QW dosing of MK-2206 in serially-obtained tumor and PRP specimens. The functional imaging studies demonstrated that complex multiparametric MRI protocols may be effectively implemented in a phase I trial. Conclusions MK-2206 safely results in significant AKT pathway blockade in QOD and QW schedules. The intermittent dose of 200mg QW is currently used in phase II MK-2206 monotherapy and combination studies. PMID:25239610

Interrogating two schedules of the AKT inhibitor MK-2206 in patients with advanced solid tumors incorporating novel pharmacodynamic and functional imaging biomarkers.

PubMed

Yap, Timothy A; Yan, Li; Patnaik, Amita; Tunariu, Nina; Biondo, Andrea; Fearen, Ivy; Papadopoulos, Kyriakos P; Olmos, David; Baird, Richard; Delgado, Liliana; Tetteh, Ernestina; Beckman, Robert A; Lupinacci, Lisa; Riisnaes, Ruth; Decordova, Shaun; Heaton, Simon P; Swales, Karen; deSouza, Nandita M; Leach, Martin O; Garrett, Michelle D; Sullivan, Daniel M; de Bono, Johann S; Tolcher, Anthony W

2014-11-15

Multiple cancers harbor genetic aberrations that impact AKT signaling. MK-2206 is a potent pan-AKT inhibitor with a maximum tolerated dose (MTD) previously established at 60 mg on alternate days (QOD). Due to a long half-life (60-80 hours), a weekly (QW) MK-2206 schedule was pursued to compare intermittent QW and continuous QOD dosing. Patients with advanced cancers were enrolled in a QW dose-escalation phase I study to investigate the safety and pharmacokinetic-pharmacodynamic profiles of tumor and platelet-rich plasma (PRP). The QOD MTD of MK-2206 was also assessed in patients with ovarian and castration-resistant prostate cancers and patients with advanced cancers undergoing multiparametric functional magnetic resonance imaging (MRI) studies, including dynamic contrast-enhanced MRI, diffusion-weighted imaging, magnetic resonance spectroscopy, and intrinsic susceptibility-weighted MRI. A total of 71 patients were enrolled; 38 patients had 60 mg MK-2206 QOD, whereas 33 received MK-2206 at 90, 135, 150, 200, 250, and 300 mg QW. The QW MK-2206 MTD was established at 200 mg following dose-limiting rash at 250 and 300 mg. QW dosing appeared to be similarly tolerated to QOD, with toxicities including rash, gastrointestinal symptoms, fatigue, and hyperglycemia. Significant AKT pathway blockade was observed with both continuous QOD and intermittent QW dosing of MK-2206 in serially obtained tumor and PRP specimens. The functional imaging studies demonstrated that complex multiparametric MRI protocols may be effectively implemented in a phase I trial. Treatment with MK-2206 safely results in significant AKT pathway blockade in QOD and QW schedules. The intermittent dose of 200 mg QW is currently used in phase II MK-2206 monotherapy and combination studies (NCT00670488). ©2014 American Association for Cancer Research.

The Akt signaling pathway is required for tissue maintenance and regeneration in planarians.

PubMed

Peiris, T Harshani; Ramirez, Daniel; Barghouth, Paul G; Oviedo, Néstor J

2016-04-11

Akt (PKB) is a serine threonine protein kinase downstream of the phosphoinositide 3-kinase (PI3K) pathway. In mammals, Akt is ubiquitously expressed and is associated with regulation of cellular proliferation, metabolism, cell growth and cell death. Akt has been widely studied for its central role in physiology and disease, in particular cancer where it has become an attractive pharmacological target. However, the mechanisms by which Akt signaling regulates stem cell behavior in the complexity of the whole body are poorly understood. Planarians are flatworms with large populations of stem cells capable of dividing to support adult tissue renewal and regeneration. The planarian ortholog Smed-Akt is molecularly conserved providing unique opportunities to analyze the function of Akt during cellular turnover and repair of adult tissues. Our findings abrogating Smed-Akt with RNA-interference in the planarian Schmidtea mediterranea led to a gradual decrease in stem cell (neoblasts) numbers. The reduced neoblast numbers largely affected the maintenance of adult tissues including the nervous and excretory systems and ciliated structures in the ventral epithelia, which impaired planarian locomotion. Downregulation of Smed-Akt function also resulted in an increase of cell death throughout the animal. However, in response to amputation, levels of cell death were decreased and failed to localize near the injury site. Interestingly, the neoblast mitotic response was increased around the amputation area but the regenerative blastema failed to form. We demonstrate Akt signaling is essential for organismal physiology and in late stages of the Akt phenotype the reduction in neoblast numbers may impair regeneration in planarians. Functional disruption of Smed-Akt alters the balance between cell proliferation and cell death leading to systemic impairment of adult tissue renewal. Our results also reveal novel roles for Akt signaling during regeneration, specifically for the timely

Arctigenin, a Natural Lignan Compound, Induces Apoptotic Death of Hepatocellular Carcinoma Cells via Suppression of PI3-K/Akt Signaling.

PubMed

Jiang, Xiaoxin; Zeng, Leping; Huang, Jufang; Zhou, Hui; Liu, Yubin

2015-04-28

In this study, we explored the cytotoxic effects of arctigenin, a natural lignan compound, on human hepatocellular carcinoma (HCC) cells and check the involvement of phosphatidylinositol 3-kinase (PI3-K)/Akt signaling. HCC cells were treated with different concentrations of arctigenin and cell viability and apoptosis were assessed. Manipulating Akt signaling was used to determine its role in the action of arctigenin. Arctigenin significantly inhibited the viability of HCC cells in a concentration-dependent manner. Arctigenin induced apoptosis and activation of caspase-9 and -3. Overexpression of a constitutively active Akt mutant blocked arctigenin-induced apoptosis. Combinational treatment with arctigenin and the PI3-K inhibitor LY294002 significantly enhanced apoptosis. Arctigenin reduced the expression of Bcl-xL, Mcl-1, and survivin and the phosphorylation of mTOR and S6K, which were significantly reversed by overexpression of constitutively active Akt. This is the first report about the anticancer activity of arctigenin in HCC cells, which is mediated by inactivation of PI3-K/Akt signaling. © 2015 Wiley Periodicals, Inc.

Identification of Akt Interaction Protein PHF20/TZP That Transcriptionally Regulates p53*

PubMed Central

Park, Sungman; Kim, Donghwa; Dan, Han C.; Chen, Huihua; Testa, Joseph R.; Cheng, Jin Q.

2012-01-01

Akt regulates a diverse array of cellular functions, including cell survival, proliferation, differentiation, and metabolism. Although a number of molecules have been identified as upstream regulators and downstream targets of Akt, the mechanisms by which Akt regulates these cellular processes remain elusive. Here, we demonstrate that a novel transcription factor, PHF20/TZP (referring to Tudor and zinc finger domain containing protein), binds to Akt and induces p53 expression at the transcription level. Knockdown of PHF20 significantly reduces p53. PHF20 inhibits cell growth, DNA synthesis, and cell survival. Akt phosphorylates PHF20 at Ser291 in vitro and in vivo, which results in its translocation from the nucleus to the cytoplasm and attenuation of PHF20 function. These data indicate that PHF20 is a substrate of Akt and plays a role in Akt cell survival/growth signaling. PMID:22334668

PI3K/Akt/mTOR Intracellular Pathway and Breast Cancer: Factors, Mechanism and Regulation.

PubMed

Sharma, Var Ruchi; Gupta, Girish Kumar; Sharma, A K; Batra, Navneet; Sharma, Daljit K; Joshi, Amit; Sharma, Anil K

2017-01-01

The most recurrent and considered second most frequent cause of cancer-related deaths worldwide in women is the breast cancer. The key to diagnosis is early prediction and a curable stage but still treatment remains a great clinical challenge. Origin of the Problem: A number of studies have been carried out for the treatment of breast cancer which includes the targeted therapies and increased survival rates in women. Essential PI3K/mTOR signaling pathway activation has been observed in most breast cancers. The cell growth and tumor development in such cases involve phosphoinositide 3 kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) complex intracellular pathway. Through preclinical and clinical trials, it has been observed that there are a number of other inhibitors of PI3K/Akt/mTOR pathway, which either alone or in combination with cytotoxic agents can be used for endocrine therapies. Structure and regulation/deregulation of mTOR provides a greater insight into the action mechanism. Also, through this review, one could easily scan first and second generation inhibitors for PI3K/Akt/mTOR pathway besides targeted therapies for breast cancer and the precise role of mTOR. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Amplification and Demultiplexing in Insulin-regulated Akt Protein Kinase Pathway in Adipocytes*

PubMed Central

Tan, Shi-Xiong; Ng, Yvonne; Meoli, Christopher C.; Kumar, Ansu; Khoo, Poh-Sim; Fazakerley, Daniel J.; Junutula, Jagath R.; Vali, Shireen; James, David E.; Stöckli, Jacqueline

2012-01-01

Akt plays a major role in insulin regulation of metabolism in muscle, fat, and liver. Here, we show that in 3T3-L1 adipocytes, Akt operates optimally over a limited dynamic range. This indicates that Akt is a highly sensitive amplification step in the pathway. With robust insulin stimulation, substantial changes in Akt phosphorylation using either pharmacologic or genetic manipulations had relatively little effect on Akt activity. By integrating these data we observed that half-maximal Akt activity was achieved at a threshold level of Akt phosphorylation corresponding to 5–22% of its full dynamic range. This behavior was also associated with lack of concordance or demultiplexing in the behavior of downstream components. Most notably, FoxO1 phosphorylation was more sensitive to insulin and did not exhibit a change in its rate of phosphorylation between 1 and 100 nm insulin compared with other substrates (AS160, TSC2, GSK3). Similar differences were observed between various insulin-regulated pathways such as GLUT4 translocation and protein synthesis. These data indicate that Akt itself is a major amplification switch in the insulin signaling pathway and that features of the pathway enable the insulin signal to be split or demultiplexed into discrete outputs. This has important implications for the role of this pathway in disease. PMID:22207758

AKT regulates BRCA1 stability in response to hormone signaling.

PubMed

Nelson, Andrew C; Lyons, Traci R; Young, Christian D; Hansen, Kirk C; Anderson, Steven M; Holt, Jeffrey T

2010-05-05

The observation that inherited mutations within BRCA1 result in breast and ovarian cancers suggests a functional relationship may exist between hormone signaling and BRCA1 function. We demonstrate that AKT activation promotes the expression of BRCA1 in response to estrogen and IGF-1 receptor signaling, and the rapid increase in BRCA1 protein levels appears to occur independently of new protein synthesis. Further, we identify a novel AKT phosphorylation site in BRCA1 at S694 which is responsive to activation of these signaling pathways. These data suggest AKT phosphorylation of BRCA1 increases total protein expression by preventing proteasomal degradation. AKT activation also appears to support nuclear localization of BRCA1, and co-expression of activated AKT with BRCA1 decreases radiation sensitivity, suggesting this interaction has functional consequences for BRCA1's role in DNA repair. Targets within this pathway could provide strategies for modulation of BRCA1 protein, which may prove therapeutically beneficial for breast and ovarian cancer treatment. Copyright 2010 Elsevier Ireland Ltd. All rights reserved.

Wortmannin Attenuates Seizure-Induced Hyperactive PI3K/Akt/mTOR Signaling, Impaired Memory, and Spine Dysmorphology in Rats

PubMed Central

Carter, Angela N.; Born, Heather A.; Levine, Amber T.; Dao, An T.; Zhao, Amanda J.; Lee, Wai L.

2017-01-01

Numerous studies have shown epilepsy-associated cognitive deficits, but less is known about the effects of one single generalized seizure. Recent studies demonstrate that a single, self-limited seizure can result in memory deficits and induces hyperactive phosphoinositide 3-kinase/Akt (protein kinase B)/mechanistic target of rapamycin (PI3K/Akt/mTOR) signaling. However, the effect of a single seizure on subcellular structures such as dendritic spines and the role of aberrant PI3K/Akt/mTOR signaling in these seizure-induced changes are unclear. Using the pentylenetetrazole (PTZ) model, we induced a single generalized seizure in rats and: (1) further characterized short- and long-term hippocampal and amygdala-dependent memory deficits, (2) evaluated whether there are changes in dendritic spines, and (3) determined whether inhibiting hyperactive PI3K/Akt/mTOR signaling rescued these alterations. Using the PI3K inhibitor wortmannin (Wort), we partially rescued short- and long-term memory deficits and altered spine morphology. These studies provide evidence that pathological PI3K/Akt/mTOR signaling plays a role in seizure-induced memory deficits as well as aberrant spine morphology. PMID:28612047

Akt-RSK-S6-kinase Signaling Networks Activated by Oncogenic Receptor Tyrosine Kinases

PubMed Central

Moritz, Albrecht; Li, Yu; Guo, Ailan; Villén, Judit; Wang, Yi; MacNeill, Joan; Kornhauser, Jon; Sprott, Kam; Zhou, Jing; Possemato, Anthony; Ren, Jian Min; Hornbeck, Peter; Cantley, Lewis C.; Gygi, Steven P.; Rush, John; Comb, Michael J.

2011-01-01

Receptor tyrosine kinases (RTKs) activate pathways mediated by serine/threonine (Ser/Thr) kinases such as the PI3K (phosphatidylinositol 3-kinase)-Akt pathway, the Ras-MAPK (mitogen-activated protein kinase)-RSK pathway, and the mTOR (mammalian target of rapamycin)-p70 S6 pathway that control important aspects of cell growth, proliferation, and survival. The Akt, RSK, and p70 S6 family of protein kinases transmit signals by phosphorylating substrates on a RxRxxS/T motif. Here, we developed a large-scale proteomic approach to identify over 200 substrates of this kinase family in cancer cell lines driven by the c-Met, epidermal growth factor receptor (EGFR), or platelet-derived growth factor receptor a (PDGFRα) RTKs. We identified a subset of proteins with RxRxxS/T sites for which phosphorylation was decreased by RTKIs as well as by inhibitors of the PI3K, mTOR, and MAPK pathways and determined the effects of siRNA directed against these substrates on cell viability. We found that phosphorylation of the protein chaperone SGTA (small glutamine-rich tetratricopeptide repeat-containing protein alpha) at Ser305 is essential for PDGFRα stabilization and cell survival in PDGFRα-dependent cancer cells. Our approach provides a new view of RTK and Akt-RSK-S6 kinase signaling, revealing many previously unidentified Akt-RSK-S6 kinase substrates that merit further consideration as targets for combination therapy with RTKIs. PMID:20736484

miR-21 inhibitor suppresses cell proliferation and colony formation through regulating the PTEN/AKT pathway and improves paclitaxel sensitivity in cervical cancer cells.

PubMed

Du, Guohui; Cao, Dongmei; Meng, Lingzheng

2017-05-01

The present study aimed to investigate the role and the molecular mechanisms underlying the effects of microRNA-21 (miR-21) on the proliferation, apoptosis and colony formation of cervical cancer cells, and to examine the role of miR-21 in mediating the sensitivity of cervical cancer cells to paclitaxel (PTX). Reverse transcription‑quantitative polymerase chain reaction was employed to determine the level of miR‑21 in various cervical cancer and normal cervical cells. The results revealed that the expression levels of miR-21 in cervical cancer cells were markedly higher when compared with normal cervical cells. Subsequently, a miR‑21 inhibitor or negative control (NC) was transfected into cervical cancer cells. Cell viability, colony formation and apoptosis were then analyzed using an MTT assay, crystal violet and Annexin V-fluorescein isothiocyanate/propidium iodide staining, respectively. The protein expression level of B-cell lymphoma‑2 (Bcl‑2), Bcl‑2‑associated X (Bax), programmed cell death 4 (PDCD4), survivin, c‑myc, phosphatase and tensin homolog (PTEN) and phosphorylated (p)‑AKT were determined by western blot analysis. The sensitivity of cervical cancer cells to PTX (25, 50 and 100 µg/ml) was characterized using an MTT assay. The results demonstrated that the miR-21 inhibitor promoted apoptosis of cervical cancer cells and suppressed their proliferation and colony formation when compared with the NC. In addition, the expression levels of Bcl‑2, survivin, c‑myc and p‑AKT were significantly downregulated in cells transfected with the miR‑21 inhibitor, whilst the expression levels of Bax, PDCD4 and PTEN were significantly upregulated. Furthermore, the miR‑21 inhibitor significantly enhanced the inhibition efficacy of PTX at a range of concentrations in cervical cancer cells. It was concluded that inhibition of miR‑21 suppressed cell proliferation and colony formation through regulating the PTEN/AKT pathway, and improved PTX

Gamma-glutamylcyclotransferase promotes the growth of human glioma cells by activating Notch-Akt signaling

DOE Office of Scientific and Technical Information (OSTI.GOV)

Shen, Shang-Hang; Yu, Ning; Liu, Xi-Yao

Glioma as an aggressive type tumor is rapidly growing and has become one of the leading cause of cancer-related death worldwide. γ-Glutamylcyclotransferase (GGCT) has been shown as a diagnostic marker in various cancers. To reveal whether there is a correlation between GGCT and human glioma, GGCT expression in human glioma tissues and cell lines was first determined. We found that GGCT expression was up-regulated in human glioma tissues and cell lines. Further, we demonstrate that GGCT knockdown inhibits glioma cell T98G and U251 proliferation and colony formation, whereas GGCT overexpression leads to oppose effects. GGCT overexpression promotes the expression ofmore » Notch receptors and activates Akt signaling in glioma cells, and Notch-Akt signaling is activated in glioma tissues with high expression of GGCT. Finally, we show that inhibition of Notch-Akt signaling with Notch inhibitor MK-0752 blocks the effects of GGCT on glioma proliferation and colony formation. In conclusion, GGCT plays a critical role in glioma cell proliferation and may be a potential cancer therapeutic target. - Highlights: • GGCT expression is up-regulated in human glioma tissues and cell lines. • GGCT promotes glioma cell growth and colony formation. • GGCT promotes the activation of Notch-Akt signaling in glioma cells and tissues. • Notch inhibition blocks the role of GGCT in human glioma cells.« less

The change tendency of PI3K/Akt pathway after spinal cord injury

PubMed Central

Zhang, Peixun; Zhang, Luping; Zhu, Lei; Chen, Fangmin; Zhou, Shuai; Tian, Ting; Zhang, Yuqiang; Jiang, Xiaorui; Li, Xuekun; Zhang, Chuansen; Xu, Lin; Huang, Fei

2015-01-01

Spinal cord injury (SCI) refers to the damage of spinal cord’s structure and function due to a variety of causes. At present, many scholars have confirmed that apoptosis is the main method of secondary injury in spinal cord injury. In view of understanding the function of PI3K/Akt pathway on spinal cord injury, this study observed the temporal variation of key molecules (PI3K, Akt, p-Akt) in the PI3K/Akt pathway after spinal cord injury by immunohistochemistry and Western-blot. The results showed that the expression of PI3K, Akt and p-Akt display a sharp increase one day after the spinal cord injury, and then it decreased gradually with the time passing by, but the absolute expression was certainly higher than the normal group. These results indicate that the PI3K/Akt signaling pathway is involved in the spinal cord injury and the mechanism may be related to apoptosis. PMID:26807170

Membrane Type 1–Matrix Metalloproteinase/Akt Signaling Axis Modulates TNF-α-Induced Procoagulant Activity and Apoptosis in Endothelial Cells

PubMed Central

Ohkawara, Hiroshi; Ishibashi, Toshiyuki; Sugimoto, Koichi; Ikeda, Kazuhiko; Ogawa, Kazuei; Takeishi, Yasuchika

2014-01-01

Membrane type 1–matrix metalloproteinase (MT1-MMP) functions as a signaling molecule in addition to a proteolytic enzyme. Our hypothesis was that MT1-MMP cooperates with protein kinase B (Akt) in tumor necrosis factor (TNF)-α-induced signaling pathways of vascular responses, including tissue factor (TF) procoagulant activity and endothelial apoptosis, in cultured human aortic endothelial cells (ECs). TNF-α (10 ng/mL) induced a decrease in Akt phosphorylation within 60 minutes in ECs. A chemical inhibitor of MMP, TIMP-2 and selective small interfering RNA (siRNA)-mediated suppression of MT1-MMP reversed TNF-α-triggered transient decrease of Akt phosphorylation within 60 minutes, suggesting that MT1-MMP may be a key regulator of Akt phosphorylation in TNF-α-stimulated ECs. In the downstream events, TNF-α increased TF antigen and activity, and suppressed the expression of thrombomodulin (TM) antigen. Inhibition of Akt markedly enhanced TNF-α-induced expression of TF antigen and activity, and further reduced the expression of TM antigen. Silencing of MT1-MMP by siRNA also reversed the changed expression of TF and TM induced by TNF-α. Moreover, TNF-α induced apoptosis of ECs through Akt- and forkhead box protein O1 (FoxO1)-dependent signaling pathway and nuclear factor-kB (NF-kB) activation. Knockdown of MT1-MMP by siRNA reversed apoptosis of ECs by inhibiting TNF-α-induced Akt-dependent regulation of FoxO1 in TNF-α-stimulated ECs. Immunoprecipitation demonstrated that TNF-α induced the changes in the associations between the cytoplasmic fraction of MT1-MMP and Akt in ECs. In conclusion, we show new evidence that MT1-MMP/Akt signaling axis is a key modifier for TNF-α-induced signaling pathways for modulation of procoagulant activity and apoptosis of ECs. PMID:25162582

B Cell Receptor Activation Predominantly Regulates AKT-mTORC1/2 Substrates Functionally Related to RNA Processing

PubMed Central

Mohammad, Dara K.; Ali, Raja H.; Turunen, Janne J.; Nore, Beston F.; Smith, C. I. Edvard

2016-01-01

Protein kinase B (AKT) phosphorylates numerous substrates on the consensus motif RXRXXpS/T, a docking site for 14-3-3 interactions. To identify novel AKT-induced phosphorylation events following B cell receptor (BCR) activation, we performed proteomics, biochemical and bioinformatics analyses. Phosphorylated consensus motif-specific antibody enrichment, followed by tandem mass spectrometry, identified 446 proteins, containing 186 novel phosphorylation events. Moreover, we found 85 proteins with up regulated phosphorylation, while in 277 it was down regulated following stimulation. Up regulation was mainly in proteins involved in ribosomal and translational regulation, DNA binding and transcription regulation. Conversely, down regulation was preferentially in RNA binding, mRNA splicing and mRNP export proteins. Immunoblotting of two identified RNA regulatory proteins, RBM25 and MEF-2D, confirmed the proteomics data. Consistent with these findings, the AKT-inhibitor (MK-2206) dramatically reduced, while the mTORC-inhibitor PP242 totally blocked phosphorylation on the RXRXXpS/T motif. This demonstrates that this motif, previously suggested as an AKT target sequence, also is a substrate for mTORC1/2. Proteins with PDZ, PH and/or SH3 domains contained the consensus motif, whereas in those with an HMG-box, H15 domains and/or NF-X1-zinc-fingers, the motif was absent. Proteins carrying the consensus motif were found in all eukaryotic clades indicating that they regulate a phylogenetically conserved set of proteins. PMID:27487157

Aspirin reduces the apoptotic effect of etoposide via Akt activation and up-regulation of p21(cip).

PubMed

Feng, Xiaocheng; Lu, Bin; Xu, Yingying; Li, Qin; Zhou, Wenbai; Yang, Zhihong; Yang, Zeng; Zhao, Weiwei; Shen, Zonghou; Hu, Renming

2011-10-01

Previous studies on the apoptotic effect of aspirin mainly focus on colorectal cancer and breast carcinoma. Few studies have been designed to explore the effect of aspirin on hepatocellular carcinoma. In the present study, we observed that aspirin caused G0/G1 phase cell cycle arrest and reduced etoposide induced caspase-3 activation in hepatocellular carcinoma G2 (HepG2) cells. Further investigation demonstrated that aspirin notably enhanced the activity of Akt and ERK1/2. Blocking the activation of Akt by the PI3-K-selective inhibitor wortmannin abrogated the anti-apoptotic effect of aspirin while the MEK inhibitor U0126 did not. p21(cip), an important substrate of Akt, is involved in the regulation of cell cycle arrest and apoptosis. Our data showed that the protein expression and ser146 phosphorylation levels of p21(cip) were significantly increased after treatment with aspirin, whereas p53 or p27 showed no change. The increase of p21(cip) protein levels was also scavenged by wortmannin but not by U0126. Moreover, reduction of caspase-3 activity induced by aspirin was attenuated by silencing p21(cip) expression. These results indicated that the anti-apoptotic effect of aspirin was dependent on activation of Akt which inhibited cell apoptosis by up-regulating p21(cip) and blocking caspase-3 activation. These findings could have clinical relevance in anticancer therapy and aspirin co-treatment of human malignancies.

Antitumor activity in RAS-driven tumors by blocking AKT and MEK.

PubMed

Tolcher, Anthony W; Khan, Khurum; Ong, Michael; Banerji, Udai; Papadimitrakopoulou, Vassiliki; Gandara, David R; Patnaik, Amita; Baird, Richard D; Olmos, David; Garrett, Christopher R; Skolnik, Jeffrey M; Rubin, Eric H; Smith, Paul D; Huang, Pearl; Learoyd, Maria; Shannon, Keith A; Morosky, Anne; Tetteh, Ernestina; Jou, Ying-Ming; Papadopoulos, Kyriakos P; Moreno, Victor; Kaiser, Brianne; Yap, Timothy A; Yan, Li; de Bono, Johann S

2015-02-15

KRAS is the most commonly mutated oncogene in human tumors. KRAS-mutant cells may exhibit resistance to the allosteric MEK1/2 inhibitor selumetinib (AZD6244; ARRY-142886) and allosteric AKT inhibitors (such as MK-2206), the combination of which may overcome resistance to both monotherapies. We conducted a dose/schedule-finding study evaluating MK-2206 and selumetinib in patients with advanced treatment-refractory solid tumors. Recommended dosing schedules were defined as MK-2206 at 135 mg weekly and selumetinib at 100 mg once daily. Grade 3 rash was the most common dose-limiting toxicity (DLT); other DLTs included grade 4 lipase increase, grade 3 stomatitis, diarrhea, and fatigue, and grade 3 and grade 2 retinal pigment epithelium detachment. There were no meaningful pharmacokinetic drug-drug interactions. Clinical antitumor activity included RECIST 1.0-confirmed partial responses in non-small cell lung cancer and low-grade ovarian carcinoma. Responses in KRAS-mutant cancers were generally durable. Clinical cotargeting of MEK and AKT signaling may be an important therapeutic strategy in KRAS-driven human malignancies (Trial NCT number NCT01021748). ©2014 American Association for Cancer Research.

Diallyl trisulfide exerts cardioprotection against myocardial ischemia-reperfusion injury in diabetic state, role of AMPK-mediated AKT/GSK-3β/HIF-1α activation

PubMed Central

Yu, Liming; Di, Wencheng; Dong, Xue; Li, Zhi; Xue, Xiaodong; Zhang, Jian; Wang, Qi; Xiao, Xiong; Han, Jinsong; Yang, Yang; Wang, Huishan

2017-01-01

Diallyl trisulfide (DATS), the major active ingredient in garlic, has been reported to confer cardioprotective effects. However, its effect on myocardial ischemia-reperfusion (MI/R) injury in diabetic state and the underlying mechanism are still unknown. We hypothesize that DATS reduces MI/R injury in diabetic state via AMPK-mediated AKT/GSK-3β/HIF-1α activation. Streptozotocin-induced diabetic rats received MI/R surgery with or without DATS (20mg/kg) treatment in the presence or absence of Compound C (Com.C, an AMPK inhibitor, 0.25mg/kg) or LY294002 (a PI3K inhibitor, 5mg/kg). We found that DATS significantly improved heart function and reduced myocardial apoptosis. Additionally, in cultured H9c2 cells, DATS (10μM) also attenuated simulated ischemia-reperfusion injury. We found that AMPK and AKT/GSK-3β/HIF-1α signaling were down-regulated under diabetic condition, while DATS markedly increased the phosphorylation of AMPK, ACC, AKT and GSK-3β as well as HIF-1α expression in MI/R-injured myocardium. However, these protective actions were all blunted by Com.C administration. Additionally, LY294002 abolished the stimulatory effect of DATS on AKT/GSK-3β/HIF-1α signaling without affecting AMPK signaling. While 2-methoxyestradiol (a HIF-1α inhibitor) reduced HIF-1α expression without affecting AKT/GSK-3β signaling. Taken together, these data showed that DATS protected against MI/R injury in diabetic state by attenuating cellular apoptosis via AMPK-mediated AKT/GSK-3β/HIF-1α signaling. Its cardioprotective effect deserves further study. PMID:29088824

Akt/p27kip1 Pathway Is Not Involved in Human Insulinoma Tumorigenesis

PubMed Central

de Lima, Andrea Paes; Garibaldi, Paula; Rubio, Maria de los Milagros; García, Florencia; Kral, Marta; Bruno, Oscar D.

2018-01-01

Insulinomas are pancreatic neuroendocrine tumors (pNET), usually benign. Akt/p27kip1 is an intracellular pathway overexpressed in many pNET. There are no data regarding its expression in human insulinomas. We aimed to investigate the expression of Akt and p27kip1 in 24 human insulinomas and to compare them to their expression in normal surrounding islets. Staining was performed on embedded paraffin tissue using polyclonal antibodies against total Akt, p-Akt, p27kip1, and pp27kip1. p-Akt was the predominant form in insulinomas; they presented lower Akt and p-Akt expression than normal islets in 83.3% and 87.5% of tumors, respectively. p27kip1 and pp27kip1 were mainly cytoplasmic in both insulinomas and normal tissue. Cytoplasmic pp27kip1 staining was higher in insulinomas and surprisingly nearly half of the insulinomas also presented nuclear p27kip1 (p = 0.029). No differences were observed in the subcellular localization of p27kip1 and activation of Akt between benign and malignant insulinomas. The low expression of Akt seen in insulinomas might explain the usual benign behavior of this type of pNET. Cytoplasmic p27kip1 in both insulinomas and normal islet cells could reflect the low rate of replication of beta cells, while nuclear p27kip1 would seem to indicate stabilization and nuclear anchoring of the cyclin D-Cdk4 complex. Our data seem to suggest that the Akt pathway is not involved in human insulinoma tumorigenesis. PMID:29853883

DNA-PKcs is important for Akt activation and gemcitabine resistance in PANC-1 pancreatic cancer cells.

PubMed

Hu, Hao; Gu, Yuanlong; Qian, Yi; Hu, Benshun; Zhu, Congyuan; Wang, Gaohe; Li, Jianping

2014-09-12

Pancreatic cancer is one of the most aggressive human malignancies with extremely poor prognosis. The moderate activity of the current standard gemcitabine and gemcitabine-based regimens was due to pre-existing or acquired chemo-resistance of pancreatic cancer cells. In this study, we explored the potential role of DNA-dependent protein kinase catalytic subunit (DNA-PKcs) in gemcitabine resistance, and studied the underlying mechanisms. We found that NU-7026 and NU-7441, two DNA-PKcs inhibitors, enhanced gemcitabine-induced cytotoxicity and apoptosis in PANC-1 pancreatic cancer cells. Meanwhile, PANC-1 cells with siRNA-knockdown of DNA-PKcs were more sensitive to gemcitabine than control PANC-1 cells. Through the co-immunoprecipitation (Co-IP) assay, we found that DNA-PKcs formed a complex with SIN1, the latter is an indispensable component of mammalian target of rapamycin (mTOR) complex 2 (mTORC2). DNA-PKcs-SIN1 complexation was required for Akt activation in PANC-1 cells, while inhibition of this complex by siRNA knockdown of DNA-PKcs/SIN1, or by DNA-PKcs inhibitors, prevented Akt phosphorylation in PANC-1 cells. Further, SIN1 siRNA-knockdown also facilitated gemcitabine-induced apoptosis in PANC-1 cells. Finally, DNA-PKcs and p-Akt expression was significantly higher in human pancreatic cancer tissues than surrounding normal tissues. Together, these results show that DNA-PKcs is important for Akt activation and gemcitabine resistance in PANC-1 pancreatic cancer cells. Copyright © 2014 Elsevier Inc. All rights reserved.

Endothelial Cell Autonomous Role of Akt1: Regulation of Vascular Tone and Ischemia-Induced Arteriogenesis.

PubMed

Lee, Monica Y; Gamez-Mendez, Ana; Zhang, Jiasheng; Zhuang, Zhenwu; Vinyard, David J; Kraehling, Jan; Velazquez, Heino; Brudvig, Gary W; Kyriakides, Themis R; Simons, Michael; Sessa, William C

2018-04-01

The importance of PI3K/Akt signaling in the vasculature has been demonstrated in several models, as global loss of Akt1 results in impaired postnatal ischemia- and VEGF-induced angiogenesis. The ubiquitous expression of Akt1, however, raises the possibility of cell-type-dependent Akt1-driven actions, thereby necessitating tissue-specific characterization. Herein, we used an inducible, endothelial-specific Akt1-deleted adult mouse model (Akt1iECKO) to characterize the endothelial cell autonomous functions of Akt1 in the vascular system. Endothelial-targeted ablation of Akt1 reduces eNOS (endothelial nitric oxide synthase) phosphorylation and promotes both increased vascular contractility in isolated vessels and elevated diastolic blood pressures throughout the diurnal cycle in vivo. Furthermore, Akt1iECKO mice subject to the hindlimb ischemia model display impaired blood flow and decreased arteriogenesis. Endothelial Akt1 signaling is necessary for ischemic resolution post-injury and likely reflects the consequence of NO insufficiency critical for vascular repair. © 2018 American Heart Association, Inc.

Alternative HER/PTEN/Akt Pathway Activation in HPV Positive and Negative Penile Carcinomas

PubMed Central

Stankiewicz, Elzbieta; Prowse, David M.; Ng, Mansum; Cuzick, Jack; Mesher, David; Hiscock, Frances; Lu, Yong-Jie; Watkin, Nicholas; Corbishley, Catherine; Lam, Wayne; Berney, Daniel M.

2011-01-01

Background The pathogenesis of penile squamous cell carcinoma (PSCC) is not well understood, though risk factors include human papillomavirus (HPV). Disruption of HER/PTEN/Akt pathway is present in many cancers; however there is little information on its function in PSCC. We investigated HER family receptors and phosphatase and tension homolog (PTEN) in HPV-positive and negative PSCC and its impact on Akt activation using immunohistochemistry and fluorescent in situ hybridisation (FISH). Methodology/Principal Findings 148 PSCCs were microarrayed and immunostained for phosphorylated EGFR (pEGFR), HER2, HER3, HER4, phosphorylated Akt (pAkt), Akt1 and PTEN proteins. EGFR and PTEN gene status were also evaluated using FISH. HPV presence was assessed by PCR. pEGFR expression was detected significantly less frequently in HPV-positive than HPV-negative tumours (p = 0.0143). Conversely, HER3 expression was significantly more common in HPV-positive cases (p = 0.0128). HER4, pAkt, Akt and PTEN protein expression were not related to HPV. HER3 (p = 0.0054) and HER4 (p = 0.0002) receptors significantly correlated with cytoplasmic Akt1 immunostaining. All three proteins positively correlated with tumour grade (HER3, p = 0.0029; HER4, p = 0.0118; Akt1, p = 0.0001). pEGFR expression correlated with pAkt but not with tumour grade or stage. There was no EGFR gene amplification. HER2 was not detected. PTEN protein expression was reduced or absent in 62% of tumours but PTEN gene copy loss was present only in 4% of PSCCs. Conclusions/Significance EGFR, HER3 and HER4 but not HER2 are associated with penile carcinogenesis. HPV-negative tumours tend to express significantly more pEGFR than HPV-positive cancers and this expression correlates with pAkt protein, indicating EGFR as an upstream regulator of Akt signalling in PSCC. Conversely, HER3 expression is significantly more common in HPV-positive cases and positively correlates with cytoplasmic Akt1 expression

Alternative HER/PTEN/Akt pathway activation in HPV positive and negative penile carcinomas.

PubMed

Stankiewicz, Elzbieta; Prowse, David M; Ng, Mansum; Cuzick, Jack; Mesher, David; Hiscock, Frances; Lu, Yong-Jie; Watkin, Nicholas; Corbishley, Catherine; Lam, Wayne; Berney, Daniel M

2011-03-02

The pathogenesis of penile squamous cell carcinoma (PSCC) is not well understood, though risk factors include human papillomavirus (HPV). Disruption of HER/PTEN/Akt pathway is present in many cancers; however there is little information on its function in PSCC. We investigated HER family receptors and phosphatase and tension homolog (PTEN) in HPV-positive and negative PSCC and its impact on Akt activation using immunohistochemistry and fluorescent in situ hybridisation (FISH). 148 PSCCs were microarrayed and immunostained for phosphorylated EGFR (pEGFR), HER2, HER3, HER4, phosphorylated Akt (pAkt), Akt1 and PTEN proteins. EGFR and PTEN gene status were also evaluated using FISH. HPV presence was assessed by PCR. pEGFR expression was detected significantly less frequently in HPV-positive than HPV-negative tumours (p = 0.0143). Conversely, HER3 expression was significantly more common in HPV-positive cases (p = 0.0128). HER4, pAkt, Akt and PTEN protein expression were not related to HPV. HER3 (p = 0.0054) and HER4 (p = 0.0002) receptors significantly correlated with cytoplasmic Akt1 immunostaining. All three proteins positively correlated with tumour grade (HER3, p = 0.0029; HER4, p = 0.0118; Akt1, p = 0.0001). pEGFR expression correlated with pAkt but not with tumour grade or stage. There was no EGFR gene amplification. HER2 was not detected. PTEN protein expression was reduced or absent in 62% of tumours but PTEN gene copy loss was present only in 4% of PSCCs. EGFR, HER3 and HER4 but not HER2 are associated with penile carcinogenesis. HPV-negative tumours tend to express significantly more pEGFR than HPV-positive cancers and this expression correlates with pAkt protein, indicating EGFR as an upstream regulator of Akt signalling in PSCC. Conversely, HER3 expression is significantly more common in HPV-positive cases and positively correlates with cytoplasmic Akt1 expression. HER4 and PTEN protein expression are not related to HPV infection

Chronic exposure of interleukin-13 suppress the induction of matrix metalloproteinase-1 by tumour necrosis factor α in normal and scleroderma dermal fibroblasts through protein kinase B/Akt.

PubMed

Brown Lobbins, M L; Shivakumar, B R; Postlethwaite, A E; Hasty, K A

2018-01-01

Peripheral blood mononuclear cells taken from patients with scleroderma express increased levels of interleukin (IL)-13. Moreover, the expression of matrix metalloproteinase-1 (MMP-1) from involved scleroderma skin fibroblasts is refractory to stimulation by tumour necrosis factor (TNF)-α. To elucidate the mechanism(s) involved, we examined the effect of IL-13 on TNF-α-induced MMP-1 expression in normal and scleroderma human dermal fibroblast lines and studied the involvement of serine/threonine kinase B/protein kinase B (Akt) in this response. Dermal fibroblast lines were stimulated with TNF-α in the presence of varying concentrations of IL-13. Total Akt and pAkt were quantitated using Western blot analyses. Fibroblasts were treated with or without Akt inhibitor VIII in the presence of IL-13 followed by TNF-α stimulation. MMP-1 expression was analysed by real-time polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA). Statistical analysis was performed using analysis of variance (anova) or Student's t-test. Upon TNF-α stimulation, normal dermal fibroblasts secrete more MMP-1 than systemic sclerosis (SSc) fibroblasts. This increase in MMP-1 is lost when fibroblasts are co-incubated with IL-13 and TNF-α. IL-13 induced a significant increase in levels of pAkt in dermal fibroblasts, while Akt inhibitor VIII reversed the suppressive effects of IL-13 on the response of cultured fibroblasts to TNF-α, increasing their expression of MMP-1. We show that IL-13 suppresses MMP-1 in TNF-α-stimulated normal and scleroderma dermal fibroblast. Akt inhibitor VIII is able to reverse the suppressive effect of IL-13 on MMP-1 expression and protein synthesis. Our data suggest that IL-13 regulates MMP-1 expression in response to TNF-α through an Akt-mediated pathway and may play a role in fibrotic diseases such as scleroderma. © 2017 British Society for Immunology.

3, 3′-Diindolylmethane Exhibits Antileukemic Activity In Vitro and In Vivo through a Akt-Dependent Process

PubMed Central

Gao, Ning; Cheng, Senping; Budhraja, Amit; Liu, E-Hu; Chen, Jieping; Chen, Deying; Yang, Zailin; Luo, Jia; Shi, Xianglin; Zhang, Zhuo

2012-01-01

3,3′-diindolylmethane (DIM), one of the active products derived from Brassica plants, is a promising antitumor agent. The present study indicated that DIM significantly induced apoptosis in U937 human leukemia cells in dose- and time-dependent manners. These events were also noted in other human leukemia cells (Jurkat and HL-60) and primary human leukemia cells (AML) but not in normal bone marrow mononuclear cells. We also found that DIM-induced lethality is associated with caspases activation, myeloid cell leukemia-1 (Mcl-1) down-regulation, p21cip1/waf1 up-regulation, and Akt inactivation accompanied by c-jun NH2-terminal kinase (JNK) activation. Enforced activation of Akt by a constitutively active Akt construct prevented DIM-mediated caspase activation, Mcl-1 down-regulation, JNK activation, and apoptosis. Conversely, DIM lethality was potentiated by the PI3K inhibitor LY294002. Interruption of the JNK pathway by pharmacologic or genetic approaches attenuated DIM-induced caspases activation, Mcl-1 down-regulation, and apoptosis. Lastly, DIM inhibits tumor growth of mouse U937 xenograft, which was related to induction of apoptosis and inactivation of Akt, as well as activation of JNK. Collectively, these findings suggest that DIM induces apoptosis in human leukemia cell lines and primary human leukemia cells, and exhibits antileukemic activity in vivo through Akt inactivation and JNK activation. PMID:22363731

MicroRNA-29b regulates TGF-β1-mediated epithelial–mesenchymal transition of retinal pigment epithelial cells by targeting AKT2

DOE Office of Scientific and Technical Information (OSTI.GOV)

Li, Min; Li, Hui; Liu, Xiaoqiang

2016-07-15

The role of microRNA (miRNA) in proliferative vitreoretinopathy (PVR) progression has not been studied extensively, especially in retinal pigment epithelial–mesenchymal transition (EMT) which is the main reason for formation of PVR. In this study, we first investigated the miRNA expression profile in transforming growth factor beta 1 (TGF-β1) mediated EMT of ARPE-19 cells. Among the five changed miRNAs, miR-29b showed the most significant downregulation. Enhanced expression of miR-29b could reverse TGF-β1 induced EMT through targeting Akt2. Akt2 downregulation could inhibit TGF-β1-induced EMT. Furthermore, inhibition of miR-29b in ARPE-19 cells directly triggered EMT process, which characterized by the phenotypic transition andmore » the upregulation of α-smooth muscle actin (α-SMA) and downregulation of E-cadherin and zona occludin-1 (ZO-1) with increased cell migration. Akt2-shRNA also inhibited miR-29 inhibitor-induced EMT process. These data indicate that miR-29b plays an important role in TGF-β1-mediated EMT in ARPE-19 cells by targeting Akt2. - Highlights: • MiR-29b expression is decreased in TGF-β1-induced EMT of ARPE-19 cells. • MiR-29b inhibits TGF-β1-induced EMT in ARPE-19 cells. • MiR-29b inhibitor induces EMT in ARPE-19 cells. • Akt2 is the target for miR-29b. • Downregulation of Akt2 prevents TGF-β1-induced EMT of ARPE-19 cells.« less

Prevention of TGF-beta-induced apoptosis by interlukin-4 through Akt activation and p70S6K survival signaling pathways.

PubMed

Lin, Sue-Jane; Chang, Chungming; Ng, Ah-Kau; Wang, Shu-Han; Li, Jia-Je; Hu, Cheng-po

2007-09-01

In this study, we demonstrate that interleukin-4 (IL-4) protects human hepatocellular carcinoma (HCC) cell line Hep3B from apoptosis induced by transforming growth factor-beta (TGF-beta). Further investigation of IL-4-transduced signaling pathways revealed that both insulin response substrate 1 and 2 (IRS-1/-2) and extracellular signal-regulated kinase (ERK) pathways were activated after IL-4 stimulation. The IRS-1/-2 activation was accompanied by the activation of phosphotidylinositol-3-kinase (PI3K), leading to Akt and p70 ribosomal protein S6 kinase (p70S6K). Interestingly, a protein kinase C (PKC) inhibitor, Gö6976, inhibited the phosphorylation of Akt, suggesting that the Akt activation was PKC-dependent. Using specific inhibitors for PI3K or ERK, we demonstrated that the PI3K pathway, but not the ERK pathway, was required for protection. The constitutively active form of PI3K almost completely rescued TGF-beta-induced apoptosis, further supporting the importance of the PI3K pathway in the protective effect of IL-4. Furthermore, a dominant negative Akt and/or Gö6976 only partially blocked the anti-apoptotic effect of IL-4. Similarly, rapamycin, which interrupted the activation of p70S6K, also only partially blocked the protective effect of IL-4. However, in the presence of both rapamycin and dominant negative Akt with or without Gö6976, IL-4 almost completely lost the anti-apoptotic effect, suggesting that both Akt and p70S6K pathways were required for the protective effect of IL-4 against TGF-beta-induced apoptosis.

Ex vivo Akt inhibition promotes the generation of potent CD19CAR T cells for adoptive immunotherapy.

PubMed

Urak, Ryan; Walter, Miriam; Lim, Laura; Wong, ChingLam W; Budde, Lihua E; Thomas, Sandra; Forman, Stephen J; Wang, Xiuli

2017-01-01

Insufficient persistence and effector function of chimeric antigen receptor (CAR)-redirected T cells have been challenging issues for adoptive T cell therapy. Generating potent CAR T cells is of increasing importance in the field. Studies have demonstrated the importance of the Akt pathway in the regulation of T cell differentiation and memory formation. We now investigate whether inhibition of Akt signaling during ex vivo expansion of CAR T cells can promote the generation of CAR T cells with enhanced antitumor activity following adoptive therapy in a murine leukemia xenograft model. Various T cell subsets including CD8+ T cells, bulk T cells, central memory T cells and naïve/memory T cells were isolated from PBMC of healthy donors, activated with CD3/CD28 beads, and transduced with a lentiviral vector encoding a second-generation CD19CAR containing a CD28 co-stimulatory domain. The transduced CD19CAR T cells were expanded in the presence of IL-2 (50U/mL) and Akt inhibitor (Akti) (1 μM) that were supplemented every other day. Proliferative/expansion potential, phenotypical characteristics and functionality of the propagated CD19CAR T cells were analyzed in vitro and in vivo after 17-21 day ex vivo expansion. Anti-tumor activity was evaluated after adoptive transfer of the CD19CAR T cells into CD19+ tumor-bearing immunodeficient mice. Tumor signals were monitored with biophotonic imaging, and survival rates were analyzed by the end of the experiments. We found that Akt inhibition did not compromise CD19CAR T cell proliferation and expansion in vitro, independent of the T cell subsets, as comparable CD19CAR T cell expansion was observed after culturing in the presence or absence of Akt inhibitor. Functionally, Akt inhibition did not dampen cell-mediated effector function, while Th1 cytokine production increased. With respect to phenotype, Akti-treated CD19CAR T cells expressed higher levels of CD62L and CD28 as compared to untreated CD19CAR T cells. Once

Inhibition of mTOR complexes protects cancer cells from glutamine starvation induced cell death by restoring Akt stability.

PubMed

Khan, Md Wasim; Layden, Brian T; Chakrabarti, Partha

2018-06-01

Glutamine, a well-established oncometabolite, anaplerotically fuels mitochondrial energy metabolism and modulates activity of mammalian/mechanistic target of rapamycin complexes (mTOR). Currently, mTOR inhibitors are in clinical use for certain types of cancer but with limited success. Since glutamine is essential for growth of many cancers, we reasoned that glutamine deprivation under conditions of mTOR inhibition should be more detrimental to cancer cell survival. However, our results show that when cells are deprived of glutamine concomitant with mTOR inhibition, hepatocarcinoma cells elicit an adaptive response which aids in their survival due to enhanced autophagic flux. Moreover, inhibition of mTOR promotes Akt ubiquitination and its proteasomal degradation however we show that Akt degradation is abrogated by increased autophagy following glutamine withdrawal. Under conditions of glutamine deficiency and mTOR inhibition, the enhanced stability of Akt protein may provide survival cues to cancer cells. Thus, our data uncovers a novel molecular link between glutamine metabolism, autophagy and stability of Akt with cancer cell survival. Copyright © 2018 Elsevier B.V. All rights reserved.

The CYP2E1 inhibitor DDC up-regulates MMP-1 expression in hepatic stellate cells via an ERK1/2- and Akt-dependent mechanism.

PubMed

Liu, Tianhui; Wang, Ping; Cong, Min; Xu, Youqing; Jia, Jidong; You, Hong

2013-06-05

DDC (diethyldithiocarbamate) could block collagen synthesis in HSC (hepatic stellate cells) through the inhibition of ROS (reactive oxygen species) derived from hepatocyte CYP2E1 (cytochrome P450 2E1). However, the effect of DDC on MMP-1 (matrix metalloproteinase-1), which is the main collagen degrading matrix metalloproteinase, has not been reported. In co-culture experiments, we found that DDC significantly enhanced MMP-1 expression in human HSC (LX-2) that were cultured with hepatocyte C3A cells either expressing or not expressing CYP2E1. The levels of both proenzyme and active MMP-1 enzyme were up-regulated in LX-2 cells, accompanied by elevated enzyme activity of MMP-1 and decreased collagen I, in both LX-2 cells and the culture medium. H2O2 treatment abrogated DDC-induced MMP-1 up-regulation and collagen I decrease, while catalase treatment slightly up-regulated MMP-1 expression. These data suggested that the decrease in ROS by DDC was partially responsible for the MMP-1 up-regulation. ERK1/2 (extracellular signal-regulated kinase 1/2), Akt (protein kinase B) and p38 were significantly activated by DDC. The ERK1/2 inhibitor (U0126) and Akt inhibitor (T3830) abrogated the DDC-induced MMP-1 up-regulation. In addition, a p38 inhibitor (SB203580) improved MMP-1 up-regulation through the stimulation of ERK1/2. Our data indicate that DDC significantly up-regulates the expression of MMP-1 in LX-2 cells which results in greater MMP-1 enzyme activity and decreased collagen I. The enhancement of MMP-1 expression by DDC was associated with H2O2 inhibition and coordinated regulation by the ERK1/2 and Akt pathways. These data provide some new insights into treatment strategies for hepatic fibrosis.

The CYP2E1 inhibitor DDC up-regulates MMP-1 expression in hepatic stellate cells via an ERK1/2- and Akt-dependent mechanism

PubMed Central

Liu, Tianhui; Wang, Ping; Cong, Min; Xu, Youqing; Jia, Jidong; You, Hong

2013-01-01

DDC (diethyldithiocarbamate) could block collagen synthesis in HSC (hepatic stellate cells) through the inhibition of ROS (reactive oxygen species) derived from hepatocyte CYP2E1 (cytochrome P450 2E1). However, the effect of DDC on MMP-1 (matrix metalloproteinase-1), which is the main collagen degrading matrix metalloproteinase, has not been reported. In co-culture experiments, we found that DDC significantly enhanced MMP-1 expression in human HSC (LX-2) that were cultured with hepatocyte C3A cells either expressing or not expressing CYP2E1. The levels of both proenzyme and active MMP-1 enzyme were up-regulated in LX-2 cells, accompanied by elevated enzyme activity of MMP-1 and decreased collagen I, in both LX-2 cells and the culture medium. H2O2 treatment abrogated DDC-induced MMP-1 up-regulation and collagen I decrease, while catalase treatment slightly up-regulated MMP-1 expression. These data suggested that the decrease in ROS by DDC was partially responsible for the MMP-1 up-regulation. ERK1/2 (extracellular signal-regulated kinase 1/2), Akt (protein kinase B) and p38 were significantly activated by DDC. The ERK1/2 inhibitor (U0126) and Akt inhibitor (T3830) abrogated the DDC-induced MMP-1 up-regulation. In addition, a p38 inhibitor (SB203580) improved MMP-1 up-regulation through the stimulation of ERK1/2. Our data indicate that DDC significantly up-regulates the expression of MMP-1 in LX-2 cells which results in greater MMP-1 enzyme activity and decreased collagen I. The enhancement of MMP-1 expression by DDC was associated with H2O2 inhibition and coordinated regulation by the ERK1/2 and Akt pathways. These data provide some new insights into treatment strategies for hepatic fibrosis. PMID:23577625

Immunohistochemical Evaluation of AKT Protein Activation in Canine Mast Cell Tumours

PubMed Central

Rodriguez, S.; Fadlalla, K.; Graham, T.; Tameru, B.; Fermin, C. D.; Samuel, T.

2011-01-01

Summary The pathogenesis of canine mast cell tumour (MCT) remains unknown. Moreover, therapeutic options are limited and resistance to targeted drugs and recurrences are common, necessitating the identification of additional cellular targets for therapy. In this study we investigated the expression of phosphorylated AKT protein in 25 archival canine MCT samples by immunohistochemistry and examined the correlation between the immunohistochemical scores and histopathological tumour grades. AKT protein was detected in all of the samples and 24 of the 25 samples expressed the phosphorylated form of the protein, albeit with variable intensity. However, when the immunohistochemical scores of weak, intermediate and strong labelling were compared with the histopathological grades of I to III, there was no strong correlation. This study suggests that canine MCT cells have activated AKT and indicates the need for further research on the role of the AKT protein and the possibility of targeting the AKT signalling pathway in MCTs. PMID:22289273

PI3K/Akt inhibitor LY294002 potentiates homoharringtonine antimyeloma activity in myeloma cells adhered to stromal cells and in SCID mouse xenograft.

PubMed

Chen, Ping; Wen, Xiaofang; Wang, Bin; Hou, Diyu; Zou, Hong; Yuan, Qin; Yang, Hui; Xie, Jieqiong; Huang, Huifang

2018-05-01

Homoharringtonine (HHT) is a known anti-leukemia drug that inhibits multiple myeloma (MM) cells both in vitro and in vivo. Our prior study demonstrated that the potency of HHT in MM cells was compromised significantly when myeloma cells were co-cultured with BM stromal cells. This study aimed to investigate whether PI3K/Akt inhibitor LY294002 could potentiate the antimyeloma activity of HHT against MM cells adhered to BM stromal cells and in vivo xenograft models. A co-culture system composed of MM cells and human stromal cells was employed to mimic MM cells in bone marrow niche. The inhibitory and pro-apoptotic effect of HHT and LY294002 was determined by CCK-8 assay or flow cytometry. Expression of PI3K/Akt signaling molecules and anti-apoptotic protein myeloid cell leukemia-1 (Mcl-1) was assessed by western blot analysis and/or reverse transcription real-time quantitative PCR (RT-qPCR). MM xenografts were used to evaluate antitumor effect of combined therapy with HHT and LY294002. Adhesion to BM stromal cells rendered MM cells resistant to HHT whereas silencing Mcl-1 partly reversed the resistance. LY294002 induced apoptosis in MM cells and potentiated the antimyeloma effects of HHT by inhibiting the PI3K/Akt signal pathway which was abnormally activated during adhesion. LY294002 also enhanced the antimyeloma effect of HHT in in vivo xenograft models. These findings suggest that activation of PI3K/Akt signal pathway was responsible for the resistance to HHT in MM cells adhered to stromal cells. LY294002 can potentiate the antimyeloma activity of HHT both in vitro and in vivo, which may represent a new clinical treatment in MM.

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

PubMed

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

2012-07-01

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

PI3K/Akt is involved in brown adipogenesis mediated by growth differentiation factor-5 in association with activation of the Smad pathway

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hinoi, Eiichi; Iezaki, Takashi; Fujita, Hiroyuki

2014-07-18

Highlights: • Akt is preferentially phosphorylated in BAT and sWAT of aP2-GDF5 mice. • PI3K/Akt signaling is involved in GDF5-induced brown adipogenesis. • PI3K/Akt signaling regulates GDF5-induced Smad5 phosphorylation. - Abstract: We have previously demonstrated promotion by growth differentiation factor-5 (GDF5) of brown adipogenesis for systemic energy expenditure through a mechanism relevant to activating the bone morphological protein (BMP) receptor/mothers against decapentaplegic homolog (Smad)/peroxisome proliferator-activated receptor gamma co-activator 1α (PGC-1α) pathway. Here, we show the involvement of the phosphatidylinositol 3-kinase (PI3K)/Akt pathway in brown adipogenesis mediated by GDF5. Overexpression of GDF5 in cells expressing adipocyte protein-2 markedly accelerated the phosphorylationmore » of Smad1/5/8 and Akt in white and brown adipose tissues. In brown adipose tissue from heterozygous GDF5{sup Rgsc451} mutant mice expressing a dominant-negative (DN) GDF5 under obesogenic conditions, the basal phosphorylation of Smad1/5/8 and Akt was significantly attenuated. Exposure to GDF5 not only promoted the phosphorylation of both Smad1/5/8 and Akt in cultured brown pre-adipocytes, but also up-regulated Pgc1a and uncoupling protein-1 expression in a manner sensitive to the PI3K/Akt inhibitor Ly294002 as well as retroviral infection with DN-Akt. GDF5 drastically promoted BMP-responsive luciferase reporter activity in a Ly294002-sensitive fashion. Both Ly294002 and DN-Akt markedly inhibited phosphorylation of Smad5 in the nuclei of brown pre-adipocytes. These results suggest that PI3K/Akt signals play a role in the GDF5-mediated brown adipogenesis through a mechanism related to activation of the Smad pathway.« less

Adenoviral gene transfer of Akt enhances myocardial contractility and intracellular calcium handling

PubMed Central

Cittadini, A; Monti, MG; Iaccarino, G; Di Rella, F; Tsichlis, PN; Di Gianni, A; Strömer, H; Sorriento, D; Peschle, C; Trimarco, B; Saccà, L; Condorelli, G

2010-01-01

The serine-threonine kinase Akt/PKB mediates stimuli from different classes of cardiomyocyte receptors, including the growth hormone/insulin like growth factor and the β-adrenergic receptors. Whereas the growth-promoting and antiapoptotic properties of Akt activation are well established, little is known about the effects of Akt on myocardial contractility, intracellular calcium (Ca2+) handling, oxygen consumption, and β-adrenergic pathway. To this aim, Sprague–Dawley rats were subjected to a wild-type Akt in vivo adenoviral gene transfer using a catheter-based technique combined with aortopulmonary crossclamping. Left ventricular (LV) contractility and intracellular Ca2+ handling were evaluated in an isolated isovolumic buffer-perfused, aequorin-loaded whole heart preparations 10 days after the surgery. The Ca2+–force relationship was obtained under steady-state conditions in tetanized muscles. No significant hypertrophy was detected in adenovirus with wild-type Akt (Ad.Akt) versus controls rats (LV-to-body weight ratio 2.6±0.2 versus 2.7±0.1 mg/g, controls versus Ad.Akt, P, NS). LV contractility, measured as developed pressure, increased by 41% in Ad.Akt. This was accounted for by both more systolic Ca2+ available to the contractile machinery (+19% versus controls) and by enhanced myofilament Ca2+ responsiveness, documented by an increased maximal Ca2+-activated pressure (+19% versus controls) and a shift to the left of the Ca2+–force relationship. Such increased contractility was paralleled by a slight increase of myocardial oxygen consumption (14%), while titrated dose of dobutamine providing similar inotropic effect augmented oxygen consumption by 39% (P<0.01). Phospholamban, calsequestrin, and ryanodine receptor LV mRNA and protein content were not different among the study groups, while sarcoplasmic reticulum Ca2+ ATPase protein levels were significantly increased in Ad.Akt rats. β-Adrenergic receptor density, affinity, kinase-1 levels, and

Curcuminoid EF24 enhances the anti-tumour activity of Akt inhibitor MK-2206 through ROS-mediated endoplasmic reticulum stress and mitochondrial dysfunction in gastric cancer.

PubMed

Chen, Xi; Dai, Xuanxuan; Zou, Peng; Chen, Weiqian; Rajamanickam, Vinothkumar; Feng, Chen; Zhuge, Weishan; Qiu, Chenyu; Ye, Qingqing; Zhang, Xiaohua; Liang, Guang

2017-05-01

Gastric cancer is one of the leading causes of morbidity and mortality worldwide. Akt is an anti-apoptotic kinase that plays a dynamic role in cell survival and is implicated in the pathogenesis of gastric cancer. MK-2206, the first allosteric inhibitor of Akt, is in clinical trials for a number of cancers. Although preclinical studies showed promise, clinical trials reported it had no effect when given alone at tolerated doses. The aim of our study was to delineate the effects of MK-2206 on gastric cancer cells and explore the ability of combination treatments to enhance the anti-tumour activity of MK-2206. SGC-7901, BGC-823 cells and immunodeficient mice were chosen as a model to study the treatment effects. Changes in cell viability, apoptosis and ROS, endoplasmic reticulum stress and mitochondrial dysfunction in the cells were analysed by MTT assays, ROS imaging and FACSCalibur, electron microscopy, JC-1 staining and western blotting. MK-2206 induced apoptotic cell death through the generation of ROS. We utilized ROS production to target gastric cancer cells by combining MK-2206 and an ROS inducer EF24. Our in vitro and in vivo xenograft studies showed that combined treatment with MK-2206 and EF24 synergistically induced apoptosis in gastric cancer cells and caused cell cycle arrest. These activities were mediated through ROS generation and the induction of endoplasmic reticulum stress and mitochondrial dysfunction. Targeting ROS generation by using a combination of an Akt inhibitor and EF24 could have potential as a therapy for gastric cancer. © 2017 The British Pharmacological Society.

Impaired Akt phosphorylation in B-cells of patients with common variable immunodeficiency.

PubMed

Yazdani, Reza; Ganjalikhani-Hakemi, Mazdak; Esmaeili, Mohammad; Abolhassani, Hassan; Vaeli, Shahram; Rezaei, Abbas; Sharifi, Zohre; Azizi, Gholamreza; Rezaei, Nima; Aghamohammadi, Asghar

2017-02-01

Common variable immunodeficiency (CVID) is a heterogeneous group of primary immunodeficiency characterized by recurrent infections. We evaluated whether defective PI3K/Akt/FoxO pathway could influence B-cell fate. Determination of B-cell subsets in CVD patients and healthy donors (HDs) were performed using flow cytometry. We evaluated mRNA and protein expression of PI3K, Akt and FoxO using real-time PCR and flow cytometry, respectively. Moreover, phosphorylated Akt (pAkt) expression in B-cells has been measured by flowcytometry. We identified a significant reduction in the percentage of marginal zone like B-cells, memory B-cells (total, switched and unswitched) and plasmablasts in patients, as these decreased B-cell subsets had a significant negative correlation with increased apoptosis in patients. Surprisingly, we identified decreased pAkt expression in B-cells of patients than HDs. We described for the first time impaired pAkt expression in B-cells of CVID patients that had a significant correlation with antibody response to the vaccine and worse clinical complications. Copyright © 2016 Elsevier Inc. All rights reserved.

Tocotrienols target PI3K/Akt signaling in anti-breast cancer therapy.

PubMed

Sylvester, Paul W; Ayoub, Nehad M

2013-09-01

The PI3K/Akt signaling pathway mediates mitogen-dependent growth and survival in various types of cancer cells, and inhibition of this pathway results in tumor cell growth arrest and apoptosis. Tocotrienols are natural forms of vitamin E that displays potent anticancer activity at treatment doses that had little or no effect on normal cell viability. Mechanistic studies revealed that the anticancer effects of γ-tocotrienol were associated with a suppression in PI3K/Akt signaling. Additional studies showed that cytotoxic LD50 doses of γ-tocotrienol were 3-5-fold higher than growth inhibitory IC50 treatment doses, suggesting that cytotoxic and antiproliferative effects of γ-tocotrienol might be mediated through different mechanisms. However, γ-tocotrienol-induced caspase activation and apoptosis in mammary tumor cells was also found to be associated with suppression in intracellular PI3K/Akt signaling and subsequent down-regulation of FLIP, an endogenous inhibitor of caspase processing and activation. Since breast cancer cells are significantly more sensitive to the inhibitory effects of γ-tocotrienol on PI3K/Akt signaling than normal cells, these findings suggest that γ-tocotrienol may provide significant health benefits in reducing the risk of breast cancer in women. Studies have also shown that combined treatment of γ-tocotrienol with other chemotherapeutic agents can result in a synergistic anticancer response. Combination therapy was most effective when the anticancer mechanism of action of γ-tocotrienol is complimentary to that of the other drug and can provide significant health benefits in the prevention and/or treatment of breast cancer, while at the same time avoiding tumor resistance or toxic effects that is commonly associated with high dose monotherapy.

MicroRNA-21 promotes bone mesenchymal stem cells migration in vitro by activating PI3K/Akt/MMPs pathway.

PubMed

Lv, Chen; Yang, Shengwu; Chen, Xin; Zhu, Xiongbai; Lin, Wenjun; Wang, Lu; Huang, Zhengxiang; Wang, Mingyue; Tu, Guanjun

2017-12-01

MicroRNA-21 (miR-21) contributes to anti-apoptosis in bone marrow mesenchymal stem cells (BMSC), but its role in the migration of BMSCs remains vague. The aim of this study was to determine the possible effect of miR-21 on regulating BMSCs directional migration and the expression of MMP-2/MMP-9 in BMSCs in vitro. BMSCs were successfully infected with miR-21-up lentivirus. Cell migration using Transwell assay indicated that upregulated expression of miR-21 could significantly promote BMSCs migration. Western blot analysis indicated that miR-21 significantly upregulated the expression of MMP-2 and MMP-9, which were related to metastasis-associated genes. GM6001, the specific MMPs inhibitor, abrogated the upregulated expression of MMP-2/MMP-9 and abolished the positive effect of miR-21 on promoting BMSCs migration. Meanwhile, miR-21 significantly enhanced Akt phosphorylation, as measured by Western blot analysis. LY294002, an inhibitor of Akt activation, abrogated the phosphorylation of Akt and abolished the positive effect of miR-21 on promoting BMSCs migration and upregulating MMP-2/MMP-9 expression. These results suggest that miR-21 contributes to BMSCs migration by upregulating MMP-2/MMP-9, potentially via the PI3K/Akt pathway. Copyright © 2017 Elsevier Ltd. All rights reserved.

The tumor suppressor gene TUSC2 (FUS1) sensitizes NSCLC to the AKT inhibitor MK2206 in LKB1-dependent manner.

PubMed

Meng, Jieru; Majidi, Mourad; Fang, Bingliang; Ji, Lin; Bekele, B Nebiyou; Minna, John D; Roth, Jack A

2013-01-01

TUSC2-defective gene expression is detected in the majority of lung cancers and is associated with worse overall survival. We analyzed the effects of TUSC2 re-expression on tumor cell sensitivity to the AKT inhibitor, MK2206, and explored their mutual signaling connections, in vitro and in vivo. TUSC2 transient expression in three LKB1-defective non-small cell lung cancer (NSCLC) cell lines combined with MK2206 treatment resulted in increased repression of cell viability and colony formation, and increased apoptotic activity. In contrast, TUSC2 did not affect the response to MK2206 treatment for two LKB1-wild type NSCLC cell lines. In vivo, TUSC2 systemic delivery, by nanoparticle gene transfer, combined with MK2206 treatment markedly inhibited growth of tumors in a human LKB1-defective H322 lung cancer xenograft mouse model. Biochemical analysis showed that TUSC2 transient expression in LKB1-defective NSCLC cells significantly stimulated AMP-activated protein kinase (AMPK) phosphorylation and enzymatic activity. More importantly, AMPK gene knockdown abrogated TUSC2-MK2206 cooperation, as evidenced by reduced sensitivity to the combined treatment. Together, TUSC2 re-expression and MK2206 treatment was more effective in inhibiting the phosphorylation and kinase activities of AKT and mTOR proteins than either single agent alone. In conclusion, these findings support the hypothesis that TUSC2 expression status is a biological variable that potentiates MK2206 sensitivity in LKB1-defective NSCLC cells, and identifies the AMPK/AKT/mTOR signaling axis as an important regulator of this activity.

A phase 1 study evaluating the combination of an allosteric AKT inhibitor (MK-2206) and trastuzumab in patients with HER2-positive solid tumors.

PubMed

Hudis, Clifford; Swanton, Charles; Janjigian, Yelena Y; Lee, Ray; Sutherland, Stephanie; Lehman, Robert; Chandarlapaty, Sarat; Hamilton, Nicola; Gajria, Devika; Knowles, James; Shah, Jigna; Shannon, Keith; Tetteh, Ernestina; Sullivan, Daniel M; Moreno, Carolina; Yan, Li; Han, Hyo Sook

2013-11-19

Trastuzumab is effective in human epidermal growth factor receptor 2 (HER2)-over-expressing breast and gastric cancers. However, patients may develop resistance through downstream signaling via the phosphatidylinositol 3-kinase (PI3K)/AKT pathway. This phase 1 trial was conducted to determine the safety and tolerability of the investigational AKT inhibitor MK-2206, to prepare for future studies to determine whether the combination with trastuzumab could inhibit compensatory signaling. Patients with HER2+ treatment-refractory breast and gastroesophageal cancer were enrolled. Treatment consisted of standard doses of intravenous trastuzumab and escalating dose levels of oral MK-2206 using either an every-other-day (45 mg and 60 mg QOD) or once-weekly (135 mg and 200 mg QW) schedule. A total of 34 patients with HER2+ disease were enrolled; 31 received study-drug. The maximum tolerated dose (MTD) for MK-2206 in combination with trastuzumab was 60 mg for the QOD schedule and 135 mg for the QW schedule, although a true MTD was not established due to early termination of the trial. The most common treatment-emergent toxicities included fatigue, hyperglycemia, and dermatologic rash, consistent with prior experience; one death unrelated to treatment was reported. There was one complete response in a patient with metastatic breast cancer, one patient achieved a partial response, and 5 patients had stable disease for at least 4 months, despite progression on multiple prior trastuzumab- and lapatinib-based therapies. Results also indicate that trastuzumab does not affect the pharmacokinetics of MK-2206. Results suggest the AKT inhibitor MK-2206 can be safely combined with trastuzumab, and is associated with clinical activity, supporting further investigation. ClinicalTrials.gov; identifier: NCT00963547.

Dual PI3K/mTOR inhibitor BEZ235 as a promising therapeutic strategy against paclitaxel-resistant gastric cancer via targeting PI3K/Akt/mTOR pathway.

PubMed

Chen, Dongshao; Lin, Xiaoting; Zhang, Cheng; Liu, Zhentao; Chen, Zuhua; Li, Zhongwu; Wang, Jingyuan; Li, Beifang; Hu, Yanting; Dong, Bin; Shen, Lin; Ji, Jiafu; Gao, Jing; Zhang, Xiaotian

2018-01-26

Paclitaxel (PTX) is widely used in the front-line chemotherapy for gastric cancer (GC), but resistance limits its use. Due to the lack of proper models, mechanisms underlying PTX resistance in GC were not well studied. Using established PTX-resistant GC cell sublines HGC-27R, we for the first time integrated biological traits and molecular mechanisms of PTX resistance in GC. Data revealed that PTX-resistant GC cells were characterized by microtubular disorders, an EMT phenotype, reduced responses to antimitotic drugs, and resistance to apoptosis (marked by upregulated β-tubulin III, vimentin, attenuated changes in G 2 /M molecules or pro-apoptotic factors in response to antimitotic drugs or apoptotic inducers, respectively). Activation of the phosphoinositide 3-kinase, the serine/threonine kinase Akt and mammalian target of rapamycin (PI3K/Akt/mTOR) and mitogen-activated protein kinase (MAPK) pathways were also observed, which might be the reason for above phenotypic alternations. In vitro data suggested that targeting these pathways were sufficient to elicit antitumor responses in PTX-resistant GC, in which the dual PI3K/mTOR inhibitor BEZ235 displayed higher therapeutic efficiency than the mTOR inhibitor everolimus or the MEK inhibitor AZD6244. Antitumor effects of BEZ235 were also confirmed in mice bearing HGC-27R tumors. Thus, these data suggest that PI3K/Akt/mTOR and MAPK pathway inhibition, especially PI3K/mTOR dual blockade, might be a promising therapeutic strategy against PTX-resistant GC.

Allosteric modulation of Ras and the PI3K/AKT/mTOR pathway: emerging therapeutic opportunities

PubMed Central

Hubbard, Paul A.; Moody, Colleen L.; Murali, Ramachandran

2014-01-01

GTPases and kinases are two predominant signaling modules that regulate cell fate. Dysregulation of Ras, a GTPase, and the three eponymous kinases that form key nodes of the associated phosphatidylinositol 4,5-bisphosphate 3-kinase (PI3K)/AKT/mTOR pathway have been implicated in many cancers, including pancreatic cancer, a disease noted for its current lack of effective therapeutics. The K-Ras isoform of Ras is mutated in over 90% of pancreatic ductal adenocarcinomas (PDAC) and there is growing evidence linking aberrant PI3K/AKT/mTOR pathway activity to PDAC. Although these observations suggest that targeting one of these nodes might lead to more effective treatment options for patients with pancreatic and other cancers, the complex regulatory mechanisms and the number of sequence-conserved isoforms of these proteins have been viewed as significant barriers in drug development. Emerging insights into the allosteric regulatory mechanisms of these proteins suggest novel opportunities for development of selective allosteric inhibitors with fragment-based drug discovery (FBDD) helping make significant inroads. The fact that allosteric inhibitors of Ras and AKT are currently in pre-clinical development lends support to this approach. In this article, we will focus on the recent advances and merits of developing allosteric drugs targeting these two inter-related signaling pathways. PMID:25566081

Cell survival under nutrient stress is dependent on metabolic conditions regulated by Akt and not by autophagic vacuoles.

PubMed

Bruno, P; Calastretti, A; Priulla, M; Asnaghi, L; Scarlatti, F; Nicolin, A; Canti, G

2007-10-01

Akt activation assists tumor cell survival and promotes resistance to chemotherapy. Here we show that constitutively active Akt (CA-Akt) cells are highly sensitized to cell death induced by nutrient and growth factor deprivation, whereas dominant-negative Akt (DN-Akt) cells have a high rate of survival. The content of autophagosomes in starved CA-Akt cells was high, while DN-Akt cells expressed autophagic vacuoles constitutively, independently of nutrition conditions. Thus Akt down-regulation and downstream events can induce autophagosomes which were not directly determinants of cell death. Biochemical analysis in Akt-mutated cells show that (i) Akt and mTOR proteins were degraded more rapidly than the housekeeping proteins, (ii) mTOR phosphorylation at position Thr(2446) was relatively high in DN-Akt and low in CA-Akt cells, induced by starvation in mock cells only, which suggests reduced autoregulation of these pathways in Akt-mutated cells, (iii) both protein synthesis and protein degradation were significantly higher in starved CA-Akt cells than in starved DN-Akt cells or mock cells. In conclusion, constitutively active Akt, unable to control synthesis and wasting of proteins, accelerates the death of starved cells.

The Akt1-eNOS axis illustrates the specificity of kinase-substrate relationships in vivo.

PubMed

Schleicher, Michael; Yu, Jun; Murata, Takahisa; Derakhshan, Berhad; Atochin, Dimitriy; Qian, Li; Kashiwagi, Satoshi; Di Lorenzo, Annarita; Harrison, Kenneth D; Huang, Paul L; Sessa, William C

2009-08-04

Akt1 is critical for many in vivo functions; however, the cell-specific substrates responsible remain to be defined. Here, we examine the importance of endothelial nitric oxide synthase (eNOS) as an Akt1 substrate by generating Akt1-deficient mice (Akt1(-/-) mice) carrying knock-in mutations (serine to aspartate or serine to alanine substitutions) of the critical Akt1 phosphorylation site on eNOS (serine 1176) that render the enzyme "constitutively active" or "less active." The eNOS mutations did not influence several phenotypes in Akt1(-/-) mice; however, the defective postnatal angiogenesis characteristic of Akt1(-/-) mice was rescued by crossing the Akt1(-/-) mice with mice carrying the constitutively active form of eNOS, but not by crossing with mice carrying the less active eNOS mutant. This genetic rescue resulted in the stabilization of hypoxia-inducible factor 1alpha (HIF-1alpha) and increased production of HIF-1alpha-responsive genes in vivo and in vitro. Thus, Akt1 regulates angiogenesis largely through phosphorylation of eNOS and NO-dependent signaling.

Mechanism of estrogen-mediated attenuation of hepatic injury following trauma-hemorrhage: Akt-dependent HO-1 up-regulation.

PubMed

Hsu, Jun-Te; Kan, Wen-Hong; Hsieh, Chi-Hsun; Choudhry, Mashkoor A; Schwacha, Martin G; Bland, Kirby I; Chaudry, Irshad H

2007-10-01

Protein kinase B (Akt) is known to be involved in proinflammatory and chemotactic events in response to injury. Akt activation also leads to the induction of heme oxygenase (HO)-1. Up-regulation of HO-1 mediates potent, anti-inflammatory effects and attenuates organ injury. Although studies have shown that 17beta-estradiol (E2) prevents organ damage following trauma-hemorrhage, it remains unknown whether Akt/HO-1 plays any role in E2-mediated attenuation of hepatic injury following trauma-hemorrhage. To study this, male rats underwent trauma-hemorrhage (mean blood pressure, approximately 40 mmHg for 90 min), followed by fluid resuscitation. At the onset of resuscitation, rats were treated with vehicle, E2 (1 mg/kg body weight), E2 plus the PI-3K inhibitor (Wortmannin), or the estrogen receptor (ER) antagonist (ICI 182,780). At 2 h after sham operation or trauma-hemorrhage, plasma alpha-GST and hepatic tissue myeloperoxidase (MPO) activity, IL-6, TNF-alpha, ICAM-1, cytokine-induced neutrophil chemoattractant-1, and MIP-2 levels were measured. Hepatic Akt and HO-1 protein levels were also determined. Trauma-hemorrhage increased hepatic injury markers (alpha-GST and MPO activity), cytokines, ICAM-1, and chemokine levels. These parameters were markedly improved in the E2-treated rats following trauma-hemorrhage. E2 treatment also increased hepatic Akt activation and HO-1 expression compared with vehicle-treated, trauma-hemorrhage rats, which were abolished by coadministration of Wortmannin or ICI 182,780. These results suggest that the salutary effects of E2 on hepatic injury following trauma-hemorrhage are in part mediated via an ER-related, Akt-dependent up-regulation of HO-1.

Resveratrol Reduces Prostate Cancer Growth and Metastasis by Inhibiting the Akt/MicroRNA-21 Pathway

PubMed Central

Sheth, Sandeep; Jajoo, Sarvesh; Kaur, Tejbeer; Mukherjea, Debashree; Sheehan, Kelly; Rybak, Leonard P.; Ramkumar, Vickram

2012-01-01

The consumption of foods containing resveratrol produces significant health benefits. Resveratrol inhibits cancer by reducing cell proliferation and metastasis and by inducing apoptosis. These actions could be explained by its ability to inhibit (ERK-1/2), Akt and suppressing the levels of estrogen and insulin growth factor -1 (IGF-1) receptor. How these processes are manifested into the antitumor actions of resveratrol is not clear. Using microarray studies, we show that resveratrol reduced the expression of various prostate-tumor associated microRNAs (miRs) including miR-21 in androgen-receptor negative and highly aggressive human prostate cancer cells, PC-3M-MM2. This effect of resveratrol was associated with reduced cell viability, migration and invasiveness. Additionally, resveratrol increased the expression of tumor suppressors, PDCD4 and maspin, which are negatively regulated by miR-21. Short interfering (si) RNA against PDCD4 attenuated resveratrol’s effect on prostate cancer cells, and similar effects were observed following over expression of miR-21 with pre-miR-21 oligonucleotides. PC-3M-MM2 cells also exhibited high levels of phospho-Akt (pAkt), which were reduced by both resveratrol and LY294002 (a PI3-kinase inhibitor). MiR-21 expression in these cells appeared to be dependent on Akt, as LY294002 reduced the levels of miR-21 along with a concurrent increase in PDCD4 expression. These in vitro findings were further corroborated in a severe combined immunodeficient (SCID) mouse xenograft model of prostate cancer. Oral administration of resveratrol not only inhibited the tumor growth but also decreased the incidence and number of metastatic lung lesions. These tumor- and metastatic-suppressive effects of resveratrol were associated with reduced miR-21 and pAkt, and elevated PDCD4 levels. Similar anti-tumor effects of resveratrol were observed in DU145 and LNCaP prostate cancer cells which were associated with suppression of Akt and PDCD4, but

Korean Red Ginseng inhibits apoptosis in neuroblastoma cells via estrogen receptor β-mediated phosphatidylinositol-3 kinase/Akt signaling

PubMed Central

Nguyen, Cuong Thach; Luong, Truc Thanh; Kim, Gyu-Lee; Pyo, Suhkneung; Rhee, Dong-Kwon

2014-01-01

Background Ginseng has been shown to exert antistress effects both in vitro and in vivo. However, the effects of ginseng on stress in brain cells are not well understood. This study investigated how Korean Red Ginseng (KRG) controls hydrogen peroxide-induced apoptosis via regulation of phosphatidylinositol-3 kinase (PI3K)/Akt and estrogen receptor (ER)-β signaling. Methods Human neuroblastoma SK-N-SH cells were pretreated with KRG and subsequently exposed to H2O2. The ability of KRG to inhibit oxidative stress-induced apoptosis was assessed in MTT cytotoxicity assays. Apoptotic protein expression was examined by Western blot analysis. The roles of ER-β, PI3K, and p-Akt signaling in KRG regulation of apoptosis were studied using small interfering RNAs and/or target antagonists. Results Pretreating SK-N-SH cells with KRG decreased expression of the proapoptotic proteins p-p53 and caspase-3, but increased expression of the antiapoptotic protein BCL2. KRG pretreatment was also associated with increased ER-β, PI3K, and p-Akt expression. Conversely, ER-β inhibition with small interfering RNA or inhibitor treatment increased p-p53 and caspase-3 levels, but decreased BCL2, PI3K, and p-Akt expression. Moreover, inhibition of PI3K/Akt signaling diminished p-p53 and caspase-3 levels, but increased BCL2 expression. Conclusion Collectively, the data indicate that KRG represses oxidative stress-induced apoptosis by enhancing PI3K/Akt signaling via upregulation of ER-β expression. PMID:25535479

PI3K/Akt signalling pathway and cancer.

PubMed

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

2004-04-01

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

CXCR6 promotes tumor cell proliferation and metastasis in osteosarcoma through the Akt pathway.

PubMed

Ma, Yunsheng; Xu, Xin; Luo, Mei

2017-01-01

Chemokine (C-X-C motif) receptor 6 (CXCR6) is up-regulated in many malignancies, indicating that CXCR6 plays an important role in tumor progression. However, the expression and function of CXCR6 in osteosarcoma (OS) remains unclear. This study aimed to explore the expression levels and function of CXCR6 in OS tissues and osteosarcoma cell lines MG-63, HOS and U2OS. The protein expression levels of CXCR6 in OS patient tissues and three osteosarcoma cell lines MG-63, HOS and U2OS were assessed. CXCR6-overexpression MG-63 cell lines were established and then the proliferation, invasion and the epithelial-mesenchymal transition (EMT) in those cells were assessed. CXCR6 mRNA levels in OS tissues were significantly higher than those in normal bone tissues. Consistently, both of the mRNA and protein levels of CXCR6 in OS cell lines MG-63, HOS and U2OS were higher than those in normal bone cells hFOB1.19. CXCR6 overexpression not only promoted cell proliferation, invasion and EMT, but also enhanced the phosphorylation of Akt in MG-63 cells. After inhibition of Akt-phosphorylation by Akt inhibitor, LY2940023, CXCR6-induced cell proliferation and invasion were dramatically attenuated. In conclusion, the present study demonstrated that CXCR6 enhances OS cell proliferation and invasion through the Akt pathway. Copyright © 2016 Elsevier Inc. All rights reserved.

Vorinostat Enhances Cytotoxicity of SN-38 and Temozolomide in Ewing Sarcoma Cells and Activates STAT3/AKT/MAPK Pathways

PubMed Central

Sampson, Valerie B.; Vetter, Nancy S.; Kamara, Davida F.; Collier, Anderson B.; Gresh, Renee C.; Kolb, E. Anders

2015-01-01

Histone deacetylase inhibitors (HDACi) have been evaluated in patients with Ewing sarcoma (EWS) but demonstrated limited activity. To better understand the potential for HDACi in EWS, we evaluated the combination of the HDACi vorinostat, with DNA damaging agents SN-38 (the active metabolite of irinotecan and topoisomerase 1 inhibitor) plus the alkylating agent temozolomide (ST). Drugs were evaluated in sequential and simultaneous combinations in two EWS cell lines. Results demonstrate that cell viability, DNA damage and reactive oxygen species (ROS) production are dependent on the sequence of drug administration. Enhanced cytotoxicity is exhibited in vitro in EWS cell lines treated with ST administered before vorinostat, which was modestly higher than concomitant treatment and superior to vorinostat administered before ST. Drug combinations downregulate cyclin D1 to induce G0/G1 arrest and promote apoptosis by cleavage of caspase-3 and PARP. When ST is administered before or concomitantly with vorinostat there is activation of STAT3, MAPK and the p53 pathway. In contrast, when vorinostat is administered before ST, there is DNA repair, increased AKT phosphorylation and reduced H2B acetylation. Inhibition of AKT using the small molecule inhibitor MK-2206 did not restore H2B acetylation. Combining ST with the dual ALK and IGF-1R inhibitor, AZD3463 simultaneously inhibited STAT3 and AKT to enhance the cytotoxic effects of ST and further reduce cell growth suggesting that STAT3 and AKT activation were in part mediated by ALK and IGF-1R signaling. In summary, potent antiproliferative and proapoptotic activity were demonstrated for ST induced DNA damage before or simultaneous with HDAC inhibition and cell death was mediated through the p53 pathway. These observations may aid in designing new protocols for treating pediatric patients with high-risk EWS. PMID:26571493

Whole-exome sequencing identifies recurrent AKT1 mutations in sclerosing hemangioma of lung

PubMed Central

Jung, Seung-Hyun; Kim, Min Sung; Lee, Sung-Hak; Park, Hyun-Chun; Choi, Hyun Joo; Maeng, Leeso; Min, Ki Ouk; Kim, Jeana; Park, Tae In; Shin, Ok Ran; Kim, Tae-Jung; Xu, Haidong; Lee, Kyo Young; Kim, Tae-Min; Song, Sang Yong; Lee, Charles; Chung, Yeun-Jun; Lee, Sug Hyung

2016-01-01

Pulmonary sclerosing hemangioma (PSH) is a benign tumor with two cell populations (epithelial and stromal cells), for which genomic profiles remain unknown. We conducted exome sequencing of 44 PSHs and identified recurrent somatic mutations of AKT1 (43.2%) and β-catenin (4.5%). We used a second subset of 24 PSHs to confirm the high frequency of AKT1 mutations (overall 31/68, 45.6%; p.E17K, 33.8%) and recurrent β-catenin mutations (overall 3 of 68, 4.4%). Of the PSHs without AKT1 mutations, two exhibited AKT1 copy gain. AKT1 mutations existed in both epithelial and stromal cells. In two separate PSHs from one patient, we observed two different AKT1 mutations, indicating they were not disseminated but independent arising tumors. Because the AKT1 mutations were not found to co-occur with β-catenin mutations (or any other known driver alterations) in any of the PSHs studied, we speculate that this may be the single-most common driver alteration to develop PSHs. Our study revealed genomic differences between PSHs and lung adenocarcinomas, including a high rate of AKT1 mutation in PSHs. These genomic features of PSH identified in the present study provide clues to understanding the biology of PSH and for differential genomic diagnosis of lung tumors. PMID:27601661

The inhibitory effect of alendronate, a nitrogen-containing bisphosphonate on the PI3K-Akt-NFkappaB pathway in osteosarcoma cells.

PubMed

Inoue, Ryosuke; Matsuki, Nori-aki; Jing, Gao; Kanematsu, Takashi; Abe, Kihachiro; Hirata, Masato

2005-11-01

1 Bisphosphonates are inhibitors of tumor cell growth as well as of bone resorption by inducing cell apoptosis. However, little is known regarding the mechanisms by which the drug induces cell apoptosis. The aim of the present study was to determine the effect of alendronate, one of the nitrogen-containing bisphosphonates on the phoshoinositide 3-kinase (PI3K)-Akt-NFkappaB pathway, the major cell survival pathway. 2 The PI3K-Akt-NFkappaB pathway was activated in the osteosarcoma cell line MG-63 treated with tumor necrosis factor-alpha or insulin. Saos-2 was also used in some experiments. This was assessed by the production of phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P(3)), increased PI3K activity, phosphorylation of Akt at serine 473 and threonine 308, increase in activity of the inhibitor of nuclear factor kappaB (IkappaB) kinase (IKK) and finally phosphorylation of IkappaB and its subsequent degradation. 3 Pretreatment with alendronate at 100 microM for 24 h prior to the stimulation with tumor necrosis factor-alpha or insulin partially inhibited the IkappaB phosphorylation and degradation. These events were more clearly observed in the presence of inhibitors of proteasomes, which are responsible for the degradation of IkappaB. The drug also partially inhibited the activity of IKK, but almost fully inhibited the phosphorylation of Akt and the production of PtdIns(3,4,5)P(3). 4 The inhibitory effect of alendronate on IkappaB phosphorylation and degradation was not attenuated by the exogenous addition of geranylgeraniol to replenish the cytosolic isoprenyl lipid substrate. 5 The present findings demonstrate that alendronate inhibited the PI3K-Akt-NFkappaB cell survival pathway at the point of PI3K activation, thus indicating the presence of new targets of alendronate.

Exercise activates the PI3K-AKT signal pathway by decreasing the expression of 5α-reductase type 1 in PCOS rats.

PubMed

Wu, Chuyan; Jiang, Feng; Wei, Ke; Jiang, Zhongli

2018-05-22

Hyperandrogenism and hyperinsulinemia are main clinical endocrine features of PCOS. Exercise can adjust the androgen level, as well as increase the sensitivity of insulin by activating PI3K-Akt insulin signaling pathways. 5αR1 has certain effects on insulin resistance and can synthesize dihydrotestosterone by metabolizing testosterone. So 5αR1 may be the target of androgen and insulin for exercise-induced regulation. To investigate the role of 5αR1 in the PI3K-Akt signaling pathway in skeletal muscle of PCOS rats activated by exercise, fifty-four female rats were randomly divided into the PCOS group (n = 42) and the control group(n = 12). After injection of testosterone propionate for 28 days, the remaining 36 rats in the PCOS group were randomly assigned to six groups: the sedentary group (PS, n = 6), sedentary and 5αRI (5α-reductase inhibitor) group (PS + RI, n = 6), sedentary and 5αR2I (5α-reductase type 2 selective inhibitor) group (PS + R2I, n = 6), exercise group (PE, n = 6), exercise and 5αRI group (PE + RI, n = 6), and exercise and 5αR2I group (PE + R2I, n = 6). The rats undergoing exercise were trained to swim for 14 days. Finasteride (5α-reductase type 2 selective inhibitor) and dutasteride (5α-reductase inhibitor) were administered once daily and were dosed based on weight. At the end, the expression of 5αR1 proteins, the phosphorylation level of PI3K and AKT, were determined by Western blot. The PCOS non-exercise group and the PE + RI group displayed significantly lower phosphorylation of Akt, PI3K p85 and GLUT4 expression, while in the PE + R2I group, the level of Akt phosphorylation and PI3K p85 expression was significantly higher than that of the PCOS non-exercise group and the PE + RI group. In summary, our study demonstrated that exercise can activate the PI3K/AKT signal pathway of PCOS rats by decreasing the expression of 5αR1.

Crosstalk between the IGF-1R/AKT/mTORC1 pathway and the tumor suppressors p53 and p27 determines cisplatin sensitivity and limits the effectiveness of an IGF-1R pathway inhibitor

PubMed Central

Davaadelger, Batzaya; Duan, Lei; Perez, Ricardo E.; Gitelis, Steven; Maki, Carl G.

2016-01-01

The insulin-like growth factor-1 receptor (IGF-1R) signaling pathway is aberrantly activated in multiple cancers and can promote proliferation and chemotherapy resistance. Multiple IGF-1R inhibitors have been developed as potential therapeutics. However, these inhibitors have failed to increase patient survival when given alone or in combination with chemotherapy agents. The reason(s) for the disappointing clinical effect of these inhibitors is not fully understood. Cisplatin (CP) activated the IGF-1R/AKT/mTORC1 pathway and stabilized p53 in osteosarcoma (OS) cells. p53 knockdown reduced IGF-1R/AKT/mTORC1 activation by CP, and IGF-1R inhibition reduced the accumulation of p53. These data demonstrate positive crosstalk between p53 and the IGF-1R/AKT/mTORC1 pathway in response to CP. Further studies showed the effect of IGF-1R inhibition on CP response is dependent on p53 status. In p53 wild-type cells treated with CP, IGF-1R inhibition increased p53s apoptotic function but reduced p53-dependent senescence, and had no effect on long term survival. In contrast, in p53-null/knockdown cells, IGF-1R inhibition reduced apoptosis in response to CP and increased long term survival. These effects were due to p27 since IGF-1R inhibition stabilized p27 in CP-treated cells, and p27 depletion restored apoptosis and reduced long term survival. Together, the results demonstrate 1) p53 expression determines the effect of IGF-1R inhibition on cancer cell CP response, and 2) crosstalk between the IGF-1R/AKT/mTORC1 pathway and p53 and p27 can reduce cancer cell responsiveness to chemotherapy and may ultimately limit the effectiveness of IGF-1R pathway inhibitors in the clinic. PMID:27050276

The death effector domain-containing DEDD forms a complex with Akt and Hsp90, and supports their stability

PubMed Central

Kurabe, Nobuya; Mori, Mayumi; Kurokawa, Jun; Taniguchi, Kaori; Aoyama, Hisatoshi; Atsuda, Kazuhiro; Nishijima, Akemi; Odawara, Nariaki; Harada, Saori; Nakashima, Katsuhiko; Arai, Satoko; Miyazaki, Toru

2010-01-01

Insulin secretion and glucose transport are the major mechanisms to balance glucose homeostasis. Recently, we found that the death effector domain-containing DEDD inhibits cyclin-dependent kinase 1 (Cdk1) function, thereby preventing Cdk1-dependent inhibitory phosphorylation of S6 kinase 1 (S6K1), downstream of phosphatidylinositol 3-kinase (PI3K), which overall results in maintenance of S6K1 activity. Here we newly show that DEDD forms a complex with Akt and heat-shock protein 90 (Hsp90), and supports the stability of both proteins. Hence, in DEDD−/− mice, Akt protein levels are diminished in skeletal muscles and adipose tissues, which interferes with the translocation of glucose transporter 4 (GLUT4) upon insulin stimulation, leading to inefficient incorporation of glucose in these organs. Interestingly, as for the activation of S6K1, suppression of Cdk1 is involved in the stabilization of Akt protein by DEDD, since diminishment of Cdk1 in DEDD−/− cells via siRNA expression or treatment with a Cdk1-inhibitor, increases both Akt and Hsp90 protein levels. Such multifaceted involvement of DEDD in glucose homeostasis by supporting both insulin secretion (via maintenance of S6K1 activity) and glucose uptake (via stabilizing Akt protein), may suggest an association of DEDD-deficiency with the pathogenesis of type 2 diabetes mellitus. PMID:20043882

Defibrotide Stimulates Angiogenesis and Protects Endothelial Cells from Calcineurin Inhibitor-Induced Apoptosis via Upregulation of AKT/Bcl-xL.

PubMed

Wang, Xiangmin; Pan, Bin; Hashimoto, Yuko; Ohkawara, Hiroshi; Xu, Kailin; Zeng, Lingyu; Ikezoe, Takayuki

2018-01-01

Sinusoidal obstruction syndrome is a life-threatening complication that can occur after haematopoietic stem cell transplantation. Defibrotide (DF) has been approved for the treatment of individuals with severe sinusoidal obstruction syndrome following haematopoietic stem cell transplantation in the European Union and the United States. However, the precise mechanisms by which DF protects endothelial cells remain to be elucidated. In this study, we found that DF stimulated angiogenesis in vitro and in vivo as assessed by vascular tube formation, scratch-wound repair and Matrigel plug assays. These effects were associated with an activation of pro-survival signalling pathways, including AKT (protein kinase B), ERK (extracellular signal-regulated kinases) and p38. More importantly, DF alleviated calcineurin inhibitor-induced growth inhibition and apoptosis of human umbilical vein endothelial cells and human hepatic sinusoidal endothelial cells in parallel with upregulation of anti-apoptotic protein B-cell lymphoma-extra-large (Bcl-xL), which was mediated by AKT (protein kinase B). Notably, these effects were abrogated when Bcl-xL was depleted by small interfering RNA (ribonucleic acid). In addition, DF counteracted calcineurin inhibitor-induced activation of nuclear factor-κB and Janus kinase 2 (JAK2)/Signal Transducer and Activator of Transcription 3 (STAT3) signalling and production of cytokines in vascular endothelial cell-derived EA.hy926 cells. Taken together, DF has pro-angiogenic, anti-apoptotic and anti-inflammatory effects on endothelial cells. DF is a potentially useful agent to prevent the development of, and treat individuals with, endothelial cell injury-related complications after haematopoietic stem cell transplantation. Schattauer GmbH Stuttgart.

Involvement of PI3K/AKT and MAPK Pathways for TNF-α Production in SiHa Cervical Mucosal Epithelial Cells Infected with Trichomonas vaginalis.

PubMed

Yang, Jung-Bo; Quan, Juan-Hua; Kim, Ye-Eun; Rhee, Yun-Ee; Kang, Byung-Hyun; Choi, In-Wook; Cha, Guang-Ho; Yuk, Jae-Min; Lee, Young-Ha

2015-08-01

Trichomonas vaginalis; induces proinflammation in cervicovaginal mucosal epithelium. To investigate the signaling pathways in TNF-α production in cervical mucosal epithelium after T. vaginalis infection, the phosphorylation of PI3K/AKT and MAPK pathways were evaluated in T. vaginalis-infected SiHa cells in the presence and absence of specific inhibitors. T. vaginalis increased TNF-α production in SiHa cells, in a parasite burden-dependent and incubation time-dependent manner. In T. vaginalis-infected SiHa cells, AKT, ERK1/2, p38 MAPK, and JNK were phosphorylated from 1 hr after infection; however, the phosphorylation patterns were different from each other. After pretreatment with inhibitors of the PI3K/AKT and MAPK pathways, TNF-α production was significantly decreased compared to the control; however, TNF-α reduction patterns were different depending on the type of PI3K/MAPK inhibitors. TNF-α production was reduced in a dose-dependent manner by treatment with wortmannin and PD98059, whereas it was increased by SP600125. These data suggested that PI3K/AKT and MAPK signaling pathways are important in regulation of TNF-α production in cervical mucosal epithelial SiHa cells. However, activation patterns of each pathway were different from the types of PI3K/MAPK pathways.

Estrogen levels regulate the subcellular distribution of phosphorylated Akt in hippocampal CA1 dendrites.

PubMed

Znamensky, Vladimir; Akama, Keith T; McEwen, Bruce S; Milner, Teresa A

2003-03-15

In addition to genomic pathways, estrogens may regulate gene expression by activating specific signal transduction pathways, such as that involving phosphatidylinositol 3-kinase (PI3-K) and the subsequent phosphorylation of Akt (protein kinase B). The Akt pathway regulates various cellular events, including the initiation of protein synthesis. Our previous studies showed that synaptogenesis in hippocampal CA1 pyramidal cell dendritic spines is highest when brain estrogen levels are highest. To address the role of Akt in this process, the subcellular distribution of phosphorylated Akt immunoreactivity (pAkt-I) in the hippocampus of female rats across the estrous cycle and male rats was analyzed by light microscopy (LM) and electron microscopy (EM). By LM, the density of pAkt-I in stratum radiatum of CA1 was significantly higher in proestrus rats (or in estrogen-supplemented ovariectomized females) compared with diestrus, estrus, or male rats. By EM, pAkt-I was found throughout the shafts and in select spines of stratum radiatum dendrites. Quantitative ultrastructural analysis identifying pAkt-I with immunogold particles revealed that proestrus rats compared with diestrus, estrus, and male rats contained significantly higher pAkt-I associated with (1) dendritic spines (both cytoplasm and plasmalemma), (2) spine apparati located within 0.1 microm of dendritic spine bases, (3) endoplasmic reticula and polyribosomes in the cytoplasm of dendritic shafts, and (4) the plasmalemma of dendritic shafts. These findings suggest that estrogens may regulate spine formation in CA1 pyramidal neurons via Akt-mediated signaling events.

The Akt1-eNOS Axis Illustrates the Specificity of Kinase-Substrate Relationships in Vivo

PubMed Central

Schleicher, Michael; Yu, Jun; Murata, Takahisa; Derakhshan, Berhad; Atochin, Dimitriy; Qian, Li; Kashiwagi, Satoshi; Lorenzo, Annarita Di; Harrison, Kenneth D.; Huang, Paul L.; Sessa, William C.

2016-01-01

Akt1 is critical for many in vivo functions; however, the cell-specific substrates responsible remain to be defined. Here, we examine the importance of endothelial nitric oxide synthase (eNOS) as an Akt1 substrate by generating Akt1-deficient mice (Akt1−/− mice) carrying knock-in mutations (serine to aspartate or serine to alanine substitutions) of the critical Akt1 phosphorylation site on eNOS (serine 1176) that render the enzyme “constitutively active” or “less active.” The eNOS mutations did not influence several phenotypes in Akt1−/− mice; however, the defective postnatal angiogenesis characteristic of Akt1−/− mice was rescued by crossing the Akt1−/− mice with mice carrying the constitutively active form of eNOS, but not by crossing with mice carrying the less active eNOS mutant. This genetic rescue resulted in the stabilization of hypoxia-inducible factor 1α (HIF-1α) and increased production of HIF-1α–responsive genes in vivo and in vitro. Thus, Akt1 regulates angiogenesis largely through phosphorylation of eNOS and NO-dependent signaling. PMID:19654415

Targeting HDAC3, a new partner protein of AKT in the reversal of chemoresistance in acute myeloid leukemia via DNA damage response.

PubMed

Long, J; Fang, W Y; Chang, L; Gao, W H; Shen, Y; Jia, M Y; Zhang, Y X; Wang, Y; Dou, H B; Zhang, W J; Zhu, J; Liang, A B; Li, J M; Hu, Jiong

2017-12-01

Resistance to cytotoxic chemotherapy drugs remains as the major cause of treatment failure in acute myeloid leukemia. Histone deacetylases (HDAC) are important regulators to maintain chromatin structure and control DNA damage; nevertheless, how each HDAC regulates genome stability remains unclear, especially under genome stress conditions. Here, we identified a mechanism by which HDAC3 regulates DNA damage repair and mediates resistance to chemotherapy drugs. In addition to inducing DNA damage, chemotherapy drugs trigger upregulation of HDAC3 expression in leukemia cells. Using genetic and pharmacological approaches, we show that HDAC3 contributes to chemotherapy resistance by regulating the activation of AKT, a well-documented factor in drug resistance development. HDAC3 binds to AKT and deacetylates it at the site Lys20, thereby promoting the phosphorylation of AKT. Chemotherapy drug exposure enhances the interaction between HDAC3 and AKT, resulting in decrease in AKT acetylation and increase in AKT phosphorylation. Whereas HDAC3 depletion or inhibition abrogates these responses and meanwhile sensitizes leukemia cells to chemotoxicity-induced apoptosis. Importantly, in vivo HDAC3 suppression reduces leukemia progression and sensitizes MLL-AF9 + leukemia to chemotherapy. Our findings suggest that combination therapy with HDAC3 inhibitor and genotoxic agents may constitute a successful strategy for overcoming chemotherapy resistance.

Isoorientin induces apoptosis through mitochondrial dysfunction and inhibition of PI3K/Akt signaling pathway in HepG2 cancer cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yuan, Li; Wang, Jing; Xiao, Haifang

Isoorientin (ISO) is a flavonoid compound that can be extracted from several plant species, such as Phyllostachys pubescens, Patrinia, and Drosophyllum lusitanicum; however, its biological activity remains poorly understood. The present study investigated the effects and putative mechanism of apoptosis induced by ISO in human hepatoblastoma cancer (HepG2) cells. The results showed that ISO induced cell death in a dose-dependent manner in HepG2 cells, but no toxicity in human liver cells (HL-7702) and buffalo rat liver cells (BRL-3A) treated with ISO at the indicated concentrations. ISO-induced cell death included apoptosis which characterized by the appearance of nuclear shrinkage, the cleavagemore » of poly (ADP-ribose) polymerase (PARP) and DNA fragmentation. ISO significantly (p < 0.01) increased the Bax/Bcl-2 ratio, disrupted the mitochondrial membrane potential (MMP), increased the release of cytochrome c, activated caspase-3, and enhanced intracellular levels of reactive oxygen species (ROS) and nitric oxide (NO). In addition, ISO effectively inhibited the phosphorylation of Akt and increased FoxO4 expression. The PI3K/Akt inhibitor LY294002 enhanced the apoptosis-inducing effect of ISO. However, LY294002 markedly quenched ROS and NO generation and diminished the protein expression of heme peroxidase enzyme (HO-1) and inducible nitric oxide synthase (iNOS). Furthermore, the addition of a ROS inhibitor (N-acetyl cysteine, NAC) or iNOS inhibitor (N-[3-(aminomethyl) benzyl] acetamidine, dihydrochloride, 1400W) significantly diminished the apoptosis induced by ISO and also blocked the phosphorylation of Akt. These results demonstrated for the first time that ISO induces apoptosis in HepG2 cells and indicate that this apoptosis might be mediated through mitochondrial dysfunction and PI3K/Akt signaling pathway, and has no toxicity in normal liver cells, suggesting that ISO may have good potential as a therapeutic and chemopreventive agent for liver cancer

Anti-tumour activity in RAS-driven tumours by blocking AKT and MEK

PubMed Central

Tolcher, Anthony W.; Khan, Khurum; Ong, Michael; Banerji, Udai; Papadimitrakopoulou, Vassiliki; Gandara, David R.; Patnaik, Amita; Baird, Richard D.; Olmos, David; Garrett, Christopher R.; Skolnik, Jeffrey M.; Rubin, Eric H.; Smith, Paul D.; Huang, Pearl; Learoyd, Maria; Shannon, Keith A.; Morosky, Anne; Tetteh, Ernestina; Jou, Ying-Ming; Papadopoulos, Kyriakos P.; Moreno, Victor; Kaiser, Brianne; Yap, Timothy A.; Yan, Li; de Bono, Johann S.

2014-01-01

Purpose KRAS is the most commonly mutated oncogene in human tumours. KRAS-mutant cells may exhibit resistance to the allosteric MEK1/2 inhibitor selumetinib (AZD6244; ARRY-142886) and allosteric AKT inhibitors (such as MK-2206), the combination of which may overcome resistance to both monotherapies. Experimental Design We conducted a dose/schedule-finding study evaluating MK-2206 and selumetinib in patients with advanced treatment-refractory solid tumours. Recommended dosing schedules were defined as MK-2206 135 mg weekly and selumetinib 100 mg once-daily. Results Grade 3 rash was the most common dose-limiting toxicity (DLT); other DLTs included grade 4 lipase increase, grade 3 stomatitis, diarrhoea, and fatigue, and grade 3 and grade 2 retinal pigment epithelium detachment. There were no meaningful pharmacokinetic drug-drug interactions. Clinical anti-tumour activity included RECIST 1.0-confirmed partial responses in non-small cell lung cancer and low-grade ovarian carcinoma. Conclusion Responses in KRAS-mutant cancers were generally durable. Clinical co-targeting of MEK and AKT signalling may be an important therapeutic strategy in KRAS-driven human malignancies (Trial NCT number NCT01021748). PMID:25516890

Akt Regulates TNFα Synthesis Downstream of RIP1 Kinase Activation during Necroptosis

PubMed Central

McNamara, Colleen R.; Ahuja, Ruchita; Osafo-Addo, Awo D.; Barrows, Douglas; Kettenbach, Arminja; Skidan, Igor; Teng, Xin; Cuny, Gregory D.; Gerber, Scott; Degterev, Alexei

2013-01-01

Necroptosis is a regulated form of necrotic cell death that has been implicated in the pathogenesis of various diseases including intestinal inflammation and systemic inflammatory response syndrome (SIRS). In this work, we investigated the signaling mechanisms controlled by the necroptosis mediator receptor interacting protein-1 (RIP1) kinase. We show that Akt kinase activity is critical for necroptosis in L929 cells and plays a key role in TNFα production. During necroptosis, Akt is activated in a RIP1 dependent fashion through its phosphorylation on Thr308. In L929 cells, this activation requires independent signaling inputs from both growth factors and RIP1. Akt controls necroptosis through downstream targeting of mammalian Target of Rapamycin complex 1 (mTORC1). Akt activity, mediated in part through mTORC1, links RIP1 to JNK activation and autocrine production of TNFα. In other cell types, such as mouse lung fibroblasts and macrophages, Akt exhibited control over necroptosis-associated TNFα production without contributing to cell death. Overall, our results provide new insights into the mechanism of necroptosis and the role of Akt kinase in both cell death and inflammatory regulation. PMID:23469174

The dopamine D2 receptor regulates Akt and GSK-3 via Dvl-3.

PubMed

Sutton, Laurie P; Rushlow, Walter J

2012-08-01

The dopamine D2 receptor (D2DR) regulates Akt and may also target the Wnt pathway, two signalling cascades that inhibit glycogen synthase kinase-3 (GSK-3). This study examined whether the Wnt pathway is regulated by D2DR and the role of Akt and dishevelled-3 (Dvl-3) in regulating GSK-3 and the transcription factor β-catenin in the rat brain. Western blotting showed that subchronic treatment of raclopride (D2DR antagonist) increase phosphorylated Akt, Dvl-3, GSK-3, phosphorylated GSK-3 and β-catenin, whereas subchronic treatment of quinpirole (D2DR agonist) induced the opposite response. Co-immunopreciptations revealed an association between GSK-3 and the D2DR complex that was altered following raclopride and quinpirole, albeit in opposite directions. SCH23390 (D1DR antagonist) and nafadotride (D3DR antagonist) were also used to determine if the response was specific to the D2DR. Neither subchronic treatment affected Dvl-3, GSK-3, Akt nor β-catenin protein levels, although nafadotride altered the phosphorylation state of Akt and GSK-3. In addition, in-vitro experiments were conducted to manipulate Akt and Dvl-3 activity in SH-SY5Y cells to elucidate how the pattern of change observed following manipulation of D2DR developed. Results indicate that Akt affects the phosphorylation state of GSK-3 but has no effect on β-catenin levels. However, altering Dvl-3 levels resulted in changes in Akt and the Wnt pathway similar to what was observed following raclopride or quinpirole treatment. Collectively, the data suggests that the D2DR very specifically regulates Wnt and Akt signalling via Dvl-3.

Harnessing the PI3K/Akt/mTOR pathway in T-cell acute lymphoblastic leukemia: Eliminating activity by targeting at different levels

PubMed Central

Ricci, Francesca; Tabellini, Giovanna; Chiarini, Francesca; Tazzari, Pier Luigi; Melchionda, Fraia; Buontempo, Francesca; Pagliaro, Pasqualepaolo; Pession, Andrea; McCubrey, James A.; Martelli, Alberto M.

2012-01-01

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive malignant hematological disorder arising in the thymus from T-cell progenitors. T-ALL mainly affects children and young adults, and remains fatal in 20% of adolescents and 50% of adults, despite progress in polychemotherapy protocols. Therefore, innovative targeted therapies are desperately needed for patients with a dismal prognosis. Aberrant activation of PI3K/Akt/mTOR signaling is a common event in T-ALL patients and portends a poor prognosis. Preclinical studies have highlighted that modulators of PI3K/Akt/mTOR signaling could have a therapeutic relevance in T-ALL. However, the best strategy for inhibiting this highly complex signal transduction pathway is still unclear, as the pharmaceutical companies have disclosed an impressive array of small molecules targeting this signaling network at different levels. Here, we demonstrate that a dual PI3K/PDK1 inhibitor, NVP-BAG956, displayed the most powerful cytotoxic effects against T-ALL cell lines and primary patients samples, when compared with a pan class I PI3K inhibitor (GDC-0941), an allosteric Akt inhibitor (MK-2206), an mTORC1 allosteric inhibitor (RAD-001), or an ATP-competitive mTORC1/mTORC2 inhibitor (KU-63794). Moreover, we also document that combinations of some of the aforementioned drugs strongly synergized against T-ALL cells at concentrations well below their respective IC50. This observation indicates that vertical inhibition at different levels of the PI3K/Akt/mTOR network could be considered as a future innovative strategy for treating T-ALL patients. PMID:22885370

Harnessing the PI3K/Akt/mTOR pathway in T-cell acute lymphoblastic leukemia: eliminating activity by targeting at different levels.

PubMed

Bressanin, Daniela; Evangelisti, Camilla; Ricci, Francesca; Tabellini, Giovanna; Chiarini, Francesca; Tazzari, Pier Luigi; Melchionda, Fraia; Buontempo, Francesca; Pagliaro, Pasqualepaolo; Pession, Andrea; McCubrey, James A; Martelli, Alberto M

2012-08-01

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive malignant hematological disorder arising in the thymus from T-cell progenitors. T-ALL mainly affects children and young adults, and remains fatal in 20% of adolescents and 50% of adults, despite progress in polychemotherapy protocols. Therefore, innovative targeted therapies are desperately needed for patients with a dismal prognosis. Aberrant activation of PI3K/Akt/mTOR signaling is a common event in T-ALL patients and portends a poor prognosis. Preclinical studies have highlighted that modulators of PI3K/Akt/mTOR signaling could have a therapeutic relevance in T-ALL. However, the best strategy for inhibiting this highly complex signal transduction pathway is still unclear, as the pharmaceutical companies have disclosed an impressive array of small molecules targeting this signaling network at different levels. Here, we demonstrate that a dual PI3K/PDK1 inhibitor, NVP-BAG956, displayed the most powerful cytotoxic affects against T-ALL cell lines and primary patients samples, when compared with a pan class I PI3K inhibitor (GDC-0941), an allosteric Akt inhibitor (MK-2206), an mTORC1 allosteric inhibitor (RAD-001), or an ATP-competitive mTORC1/mTORC2 inhibitor (KU63794). Moreover, we also document that combinations of some of the aforementioned drugs strongly synergized against T-ALL cells at concentrations well below their respective IC50. This observation indicates that vertical inhibition at different levels of the PI3K/Akt/mTOR network could be considered as a future innovative strategy for treating T-ALL patients.

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

Curcuminoid EF24 enhances the anti‐tumour activity of Akt inhibitor MK‐2206 through ROS‐mediated endoplasmic reticulum stress and mitochondrial dysfunction in gastric cancer

PubMed Central

Chen, Xi; Dai, Xuanxuan; Zou, Peng; Chen, Weiqian; Rajamanickam, Vinothkumar; Feng, Chen; Zhuge, Weishan; Qiu, Chenyu; Ye, Qingqing

2017-01-01

Background and Purpose Gastric cancer is one of the leading causes of morbidity and mortality worldwide. Akt is an anti‐apoptotic kinase that plays a dynamic role in cell survival and is implicated in the pathogenesis of gastric cancer. MK‐2206, the first allosteric inhibitor of Akt, is in clinical trials for a number of cancers. Although preclinical studies showed promise, clinical trials reported it had no effect when given alone at tolerated doses. The aim of our study was to delineate the effects of MK‐2206 on gastric cancer cells and explore the ability of combination treatments to enhance the anti‐tumour activity of MK‐2206. Experimental Approach SGC‐7901, BGC‐823 cells and immunodeficient mice were chosen as a model to study the treatment effects. Changes in cell viability, apoptosis and ROS, endoplasmic reticulum stress and mitochondrial dysfunction in the cells were analysed by MTT assays, ROS imaging and FACSCalibur, electron microscopy, JC‐1 staining and western blotting. Key Results MK‐2206 induced apoptotic cell death through the generation of ROS. We utilized ROS production to target gastric cancer cells by combining MK‐2206 and an ROS inducer EF24. Our in vitro and in vivo xenograft studies showed that combined treatment with MK‐2206 and EF24 synergistically induced apoptosis in gastric cancer cells and caused cell cycle arrest. These activities were mediated through ROS generation and the induction of endoplasmic reticulum stress and mitochondrial dysfunction. Conclusion and Implications Targeting ROS generation by using a combination of an Akt inhibitor and EF24 could have potential as a therapy for gastric cancer. PMID:28255993

Rictor and integrin-linked kinase interact and regulate Akt phosphorylation and cancer cell survival.

PubMed

McDonald, Paul C; Oloumi, Arusha; Mills, Julia; Dobreva, Iveta; Maidan, Mykola; Gray, Virginia; Wederell, Elizabeth D; Bally, Marcel B; Foster, Leonard J; Dedhar, Shoukat

2008-03-15

An unbiased proteomic screen to identify integrin-linked kinase (ILK) interactors revealed rictor as an ILK-binding protein. This finding was interesting because rictor, originally identified as a regulator of cytoskeletal dynamics, is also a component of mammalian target of rapamycin complex 2 (mTORC2), a complex implicated in Akt phosphorylation. These functions overlap with known ILK functions. Coimmunoprecipitation analyses confirmed this interaction, and ILK and rictor colocalized in membrane ruffles and leading edges of cancer cells. Yeast two-hybrid assays showed a direct interaction between the NH(2)- and COOH-terminal domains of rictor and the ILK kinase domain. Depletion of ILK and rictor in breast and prostate cancer cell lines resulted in inhibition of Akt Ser(473) phosphorylation and induction of apoptosis, whereas, in several cell lines, depletion of mTOR increased Akt phosphorylation. Akt and Ser(473)P-Akt were detected in ILK immunoprecipitates and small interfering RNA-mediated depletion of rictor, but not mTOR, inhibited the amount of Ser(473)P-Akt in the ILK complex. Expression of the NH(2)-terminal (1-398 amino acids) rictor domain also resulted in the inhibition of ILK-associated Akt Ser(473) phosphorylation. These data show that rictor regulates the ability of ILK to promote Akt phosphorylation and cancer cell survival.

PI3K/Akt signaling mediated Hexokinase-2 expression inhibits cell apoptosis and promotes tumor growth in pediatric osteosarcoma

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhuo, Baobiao; Li, Yuan; Li, Zhengwei

2015-08-21

Accumulating evidence has shown that PI3K/Akt pathway is frequently hyperactivated in osteosarcoma (OS) and contributes to tumor initiation and progression. Altered phenotype of glucose metabolism is a key hallmark of cancer cells including OS. However, the relationship between PI3K/Akt pathway and glucose metabolism in OS remains largely unexplored. In this study, we showed that elevated Hexokinase-2 (HK2) expression, which catalyzes the first essential step of glucose metabolism by conversion of glucose into glucose-6-phosphate, was induced by activated PI3K/Akt signaling. Immunohistochemical analysis showed that HK2 was overexpressed in 83.3% (25/30) specimens detected and was closely correlated with Ki67, a cell proliferationmore » index. Silencing of endogenous HK2 resulted in decreased aerobic glycolysis as demonstrated by reduced glucose consumption and lactate production. Inhibition of PI3K/Akt signaling also suppressed aerobic glycolysis and this effect can be reversed by reintroduction of HK2. Furthermore, knockdown of HK2 led to increased cell apoptosis and reduced ability of colony formation; meanwhile, these effects were blocked by 2-Deoxy-D-glucose (2-DG), a glycolysis inhibitor through its actions on hexokinase, indicating that HK2 functions in cell apoptosis and growth were mediated by altered aerobic glycolysis. Taken together, our study reveals a novel relationship between PI3K/Akt signaling and aerobic glycolysis and indicates that PI3K/Akt/HK2 might be potential therapeutic approaches for OS. - Highlights: • PI3K/Akt signaling contributes to elevated expression of HK2 in osteosarcoma. • HK2 inhibits cell apoptosis and promotes tumor growth through enhanced Warburg effect. • Inhibition of glycolysis blocks the oncogenic activity of HK2.« less

Regulation of Akt and Wnt signaling by the group II metabotropic glutamate receptor antagonist LY341495 and agonist LY379268.

PubMed

Sutton, Laurie P; Rushlow, Walter J

2011-06-01

Metabotropic glutamate receptors 2/3 (mGlu(2/3)) have been implicated in schizophrenia and as a novel treatment target for schizophrenia. The current study examined whether mGlu(2/3) regulates Akt (protein kinase B) and Wnt (Wingless/Int-1) signaling, two cascades associated with schizophrenia and modified by antipsychotics. Western blotting revealed increases in phosphorylated Akt (pAkt) and phosphorylated glycogen synthase kinase-3 (pGSK-3) following acute and repeated treatment of LY379268 (mGlu(2/3) agonist), whereas increases in dishevelled-2 (Dvl-2), dishevelled-3 (Dvl-3), GSK-3 and β-catenin were only observed following repeated treatment. LY341495 (mGlu(2/3) antagonist) induced the opposite response compared with LY379268. Co-immunoprecipitation experiments showed an association between the mGlu(2/3) complex and Dvl-2 providing a possible mechanism to explain how the mGlu(2/3) can mediate changes in Wnt signaling. However, there was no association between the mGlu(2/3) complex and Akt suggesting that changes in Akt signaling following LY341495 and LY379268 treatments may not be directly mediated by the mGlu(2/3) . Finally, an increase in locomotor activity induced by LY341495 treatment correlated with increased pAkt and pGSK-3 levels and was attenuated by the administration of the GSK-3 inhibitor, SB216763. Overall, the results suggest that mGlu(2/3) regulates Akt and Wnt signaling and LY379268 treatment has overlapping effects with D(2) dopamine receptor antagonists (antipsychotic drugs). © 2011 The Authors. Journal of Neurochemistry © 2011 International Society for Neurochemistry.

Heparanase induced by advanced glycation end products (AGEs) promotes macrophage migration involving RAGE and PI3K/AKT pathway

PubMed Central

2013-01-01

Background Advanced glycation end products (AGEs), inflammatory-associated macrophage migration and accumulation are crucial for initiation and progression of diabetic vascular complication. Enzymatic activity of heparanase (HPA) is implicated strongly in dissemination of metastatic tumor cells and cells of the immune system. In addition, HPA enhances the phosphorylation of selected signaling molecules including AKT pathway independent of enzymatic activity. However, virtually nothing is presently known the role of HPA during macrophage migration exposed to AGEs involving signal pathway. Methods These studies were carried out in Ana-1 macrophages. Macrophage viability was measured by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assays. HPA and AKT protein expression in macrophages are analysed by Western blotting and HPA mRNA expression by real time quantitative RT-PCR. Release of HPA was determined by ELISA. Macrophage migration was assessed by Transwell assays. Results HPA protein and mRNA were found to be increased significantly in AGEs-treated macrophages. Pretreatment with anti-HPA antibody which recognizes the nonenzymatic terminal of HPA prevented AGEs-induced AKT phosphorylation and macrophage migration. LY294002 (PI3k/AKT inhibitor) inhibited AGEs-induced macrophage migration. Furthermore, pretreatment with anti-receptor for advanced glycation end products (RAGE) antibody attenuated AGEs-induced HPA expression, AKT phosphorylation and macrophage migration. Conclusions These data indicate that AGEs-induced macrophage migration is dependent on HPA involving RAGE-HPA-PI3K/AKT pathway. The nonenzymatic activity of HPA may play a key role in AGEs-induced macrophage migration associated with inflammation in diabetic vascular complication. PMID:23442498

Phosphoinositide 3-kinase/Akt signalling is responsible for the differential susceptibility of myoblasts and myotubes to menadione-induced oxidative stress.

PubMed

Lim, Jeong A; Woo, Joo Hong; Kim, Hye Sun

2008-09-01

In this study, it was found that undifferentiated myoblasts were more vulnerable to menadione-induced oxidative stress than differentiated myotubes. Cell death occurred with a relatively low concentration of menadione in myoblasts compared to myotubes. With the same concentration of menadione, the Bcl-2/Bax ratio decreased and nuclei containing condensed chromatin were observed in myoblasts to a greater extent than in myotubes. However, myotubes became increasingly susceptible to menadione when phosphoinositide 3-kinase (PI3-K) was blocked by pre-incubation with LY294002, a PI3-K inhibitor. Actually, PI3-K activity was reduced by menadione in myoblasts but not in myotubes. In addition, the phosphorylation of Akt, a downstream effector of PI3-K, was inhibited in myoblasts by menadione but increased in myotubes. Both LY294002 and API-2, an Akt inhibitor, decreased the Bcl-2/Bax ratio in menadione-exposed myotubes. These results suggest that the differential activity of PI3-K/Akt signalling is responsible for the differential susceptibility of myoblasts and myotubes to menadione-induced oxidative stress.

Effective Identification of Akt Interacting Proteins by Two-Step Chemical Crosslinking, Co-Immunoprecipitation and Mass Spectrometry

PubMed Central

Huang, Bill X.; Kim, Hee-Yong

2013-01-01

Akt is a critical protein for cell survival and known to interact with various proteins. However, Akt binding partners that modulate or regulate Akt activation have not been fully elucidated. Identification of Akt-interacting proteins has been customarily achieved by co-immunoprecipitation combined with western blot and/or MS analysis. An intrinsic problem of the method is loss of interacting proteins during procedures to remove non-specific proteins. Moreover, antibody contamination often interferes with the detection of less abundant proteins. Here, we developed a novel two-step chemical crosslinking strategy to overcome these problems which resulted in a dramatic improvement in identifying Akt interacting partners. Akt antibody was first immobilized on protein A/G beads using disuccinimidyl suberate and allowed to bind to cellular Akt along with its interacting proteins. Subsequently, dithiobis[succinimidylpropionate], a cleavable crosslinker, was introduced to produce stable complexes between Akt and binding partners prior to the SDS-PAGE and nanoLC-MS/MS analysis. This approach enabled identification of ten Akt partners from cell lysates containing as low as 1.5 mg proteins, including two new potential Akt interacting partners. None of these but one protein was detectable without crosslinking procedures. The present method provides a sensitive and effective tool to probe Akt-interacting proteins. This strategy should also prove useful for other protein interactions, particularly those involving less abundant or weakly associating partners. PMID:23613850

Erk1/2 and Akt kinases are involved in the protective effect of aniracetam in astrocytes subjected to simulated ischemia in vitro.

PubMed

Gabryel, Bozena; Pudelko, Anna; Malecki, Andrzej

2004-06-28

The present study focused on the mechanism of cytoprotective effect of aniracetam on the primary rat astrocyte cultures exposed to simulated ischemia conditions in vitro. To study these mechanisms, the aniracetam-mediated modulation of the mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3-K)/Akt kinase pathways was determined. Simulated in vitro ischemia caused death of approximately 35% of astrocytes via apoptosis and decreased cell viability about 50% at 8 h. Exposure to aniracetam at concentrations of 0.1-10 microM in these conditions significantly decreased the number of apoptotic cells. Moreover, the intensification of 3-(4,5-dimethylthazol-2-yl)-2,5-diphenyltetrazolinum bromide (MTT) conversion and the decrease of lactate dehydrogenase (LDH) release after 1 and 10 microM aniracetam treatment were observed indicating a significant increase in cell viability. When cultured astrocytes were incubated during 8 h simulated ischemia with [1,4-diamino-2,3-dicyano-1,4-bis(2-aminophenylthio)butadiene] (U0126), an extracellular regulated kinase 1 and 2 (Erk1/2) inhibitor or wortmannin, a phosphatidylinositol 3-kinase (PI3 kinase)/Akt inhibitor, the cell apoptosis was accelerated. These effects of used kinase inhibitors (both U0126 and wortmannin) were antagonized by adding 1 and 10 microM aniracetam to the culture medium. In addition, aniracetam significantly stimulated of phospho-Erk1/2 kinase and phospho-Akt expression. Maximum levels of Erk1/2 and Akt activation were observed as a result of treatment with 10 microM aniracetam. U0126 and wortmannin markedly attenuated the effects of aniracetam on expression of activated kinases. Results of the present study indicate that both Erk1/2 and PI 3-K/Akt kinase pathways are vital for cytoprotective effect of aniracetam.

Computational Model of Gab1/2-Dependent VEGFR2 Pathway to Akt Activation

PubMed Central

Tan, Wan Hua; Popel, Aleksander S.; Mac Gabhann, Feilim

2013-01-01

Vascular endothelial growth factor (VEGF) signal transduction is central to angiogenesis in development and in pathological conditions such as cancer, retinopathy and ischemic diseases. However, no detailed mass-action models of VEGF receptor signaling have been developed. We constructed and validated the first computational model of VEGFR2 trafficking and signaling, to study the opposing roles of Gab1 and Gab2 in regulation of Akt phosphorylation in VEGF-stimulated endothelial cells. Trafficking parameters were optimized against 5 previously published in vitro experiments, and the model was validated against six independent published datasets. The model showed agreement at several key nodes, involving scaffolding proteins Gab1, Gab2 and their complexes with Shp2. VEGFR2 recruitment of Gab1 is greater in magnitude, slower, and more sustained than that of Gab2. As Gab2 binds VEGFR2 complexes more transiently than Gab1, VEGFR2 complexes can recycle and continue to participate in other signaling pathways. Correspondingly, the simulation results show a log-linear relationship between a decrease in Akt phosphorylation and Gab1 knockdown while a linear relationship was observed between an increase in Akt phosphorylation and Gab2 knockdown. Global sensitivity analysis demonstrated the importance of initial-concentration ratios of antagonistic molecular species (Gab1/Gab2 and PI3K/Shp2) in determining Akt phosphorylation profiles. It also showed that kinetic parameters responsible for transient Gab2 binding affect the system at specific nodes. This model can be expanded to study multiple signaling contexts and receptor crosstalk and can form a basis for investigation of therapeutic approaches, such as tyrosine kinase inhibitors (TKIs), overexpression of key signaling proteins or knockdown experiments. PMID:23805312

AKT in cancer: new molecular insights and advances in drug development

PubMed Central

Mundi, Prabhjot S.; Sachdev, Jasgit; McCourt, Carolyn

2016-01-01

The phosphatidylinositol‐3 kinase (PI3K)–AKT pathway is one of the most commonly dysregulated pathways in all of cancer, with somatic mutations, copy number alterations, aberrant epigenetic regulation and increased expression in a number of cancers. The carefully maintained homeostatic balance of cell division and growth on one hand, and programmed cell death on the other, is universally disturbed in tumorigenesis, and downstream effectors of the PI3K–AKT pathway play an important role in this disturbance. With a wide array of downstream effectors involved in cell survival and proliferation, the well‐characterized direct interactions of AKT make it a highly attractive yet elusive target for cancer therapy. Here, we review the salient features of this pathway, evidence of its role in promoting tumorigenesis and recent progress in the development of therapeutic agents that target AKT. PMID:27232857

The Role of Akt in Chronic Liver Disease and Liver Regeneration.

PubMed

Morales-Ruiz, Manuel; Santel, Ansgar; Ribera, Jordi; Jiménez, Wladimiro

2017-02-01

The liver is continuously exposed to diverse insults, which may culminate in pathological processes causing liver disease. An effective therapeutic strategy for chronic liver disease should control the causal factors of the disease and stimulate functional liver regeneration. Preclinical studies have shown that interventions aimed at maintaining Akt activity in a dysfunctional liver meet most of the criteria. Although the central function of Akt is cell survival, other cellular aspects such as glucose uptake, glycogen synthesis, cell-cycle progression, and lipid metabolism have been shown to be prominent functions of Akt in the context of hepatic physiology. In this review, the authors describe the benefits of the Akt signaling pathway, emphasizing its importance in coordinating proper cellular growth and differentiation during liver regeneration, hepatic function, and liver disease. Thieme Medical Publishers 333 Seventh Avenue, New York, NY 10001, USA.

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.

Pirfenidone Induces G1 Arrest in Human Tenon's Fibroblasts In Vitro Involving AKT and MAPK Signaling Pathways.

PubMed

Guo, Xiujuan; Yang, Yangfan; Liu, Liling; Liu, Xiaoan; Xu, Jiangang; Wu, Kaili; Yu, Minbin

2017-06-01

To investigate the underlying mechanism by which pirfenidone blocks the transition from the G1 to S phase in primary human Tenon's fibroblasts. Primary human Tenon's fibroblasts were characterized by immunocytofluorescence staining with vimentin, fibroblast surface protein, and cytokeratin. After treating Tenon's fibroblasts with pirfenidone under proliferation conditions (10% fetal bovine serum), cell proliferation was measured using a WST-1 assay. Progression through the cell cycle was analyzed by flow cytometry. The expression of CDK2, CDK6, cyclinD1, cyclinD3, and cyclinE and the phosphorylation of AKT, ERK1/2/MAPK, JNK/MAPK, and p38 MAPK were estimated using western blot analysis. Under proliferative conditions, pirfenidone inhibited Tenon's fibroblasts proliferation and arrested the cell cycle at the G1 phase; decreased the phosphorylation of AKT, GSK3β, ERK1/2/MAPK, and JNK/MAPK; increased the phosphorylation of p38 MAPK; and inhibited CDK2, CDK6, cyclin D1, cyclin D3, and cyclin E in a dose-dependent manner. Inhibitors of AKT (LY294002), ERK1/2 (U0126), and JNK (SP600125) arrested the G1/S transition, similar to the effect of pirfenidone. The p38 inhibitor (SB202190) decreased the G1-blocking effect of pirfenidone. The expression of CDK2, CDK6, cyclin D1, and cyclin D3 were inhibited by LY294002, U0126, and SP600125. SB202190 attenuated the pirfenidone-induced reduction of CDK2, CDK6, cyclin D1, cyclin D3, and cyclin E. Pirfenidone inhibited HTFs proliferation and induced G1 arrest by downregulating CDKs and cyclins involving the AKT/GSK3β and MAPK signaling pathways.

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

PubMed

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

2014-09-01

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

PKC{eta} is a negative regulator of AKT inhibiting the IGF-I induced proliferation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Shahaf, Galit; Rotem-Dai, Noa; Koifman, Gabriela

2012-04-15

The PI3K-AKT pathway is frequently activated in human cancers, including breast cancer, and its activation appears to be critical for tumor maintenance. Some malignant cells are dependent on activated AKT for their survival; tumors exhibiting elevated AKT activity show sensitivity to its inhibition, providing an Achilles heel for their treatment. Here we show that the PKC{eta} isoform is a negative regulator of the AKT signaling pathway. The IGF-I induced phosphorylation on Ser473 of AKT was inhibited by the PKC{eta}-induced expression in MCF-7 breast adenocarcinoma cancer cells. This was further confirmed in shRNA PKC{eta}-knocked-down MCF-7 cells, demonstrating elevated phosphorylation on AKTmore » Ser473. While PKC{eta} exhibited negative regulation on AKT phosphorylation it did not alter the IGF-I induced ERK phosphorylation. However, it enhanced ERK phosphorylation when stimulated by PDGF. Moreover, its effects on IGF-I/AKT and PDGF/ERK pathways were in correlation with cell proliferation. We further show that both PKC{eta} and IGF-I confer protection against UV-induced apoptosis and cell death having additive effects. Although the protective effect of IGF-I involved activation of AKT, it was not affected by PKC{eta} expression, suggesting that PKC{eta} acts through a different route to increase cell survival. Hence, our studies show that PKC{eta} provides negative control on AKT pathway leading to reduced cell proliferation, and further suggest that its presence/absence in breast cancer cells will affect cell death, which could be of therapeutic value.« less

Discovery of 1-(3-aryl-4-chlorophenyl)-3-(p-aryl)urea derivatives against breast cancer by inhibiting PI3K/Akt/mTOR and Hedgehog signalings.

PubMed

Li, Wenlu; Sun, Qinsheng; Song, Lu; Gao, Chunmei; Liu, Feng; Chen, Yuzong; Jiang, Yuyang

2017-12-01

PI3K/Akt/mTOR and hedgehog (Hh) signalings are two important pathways in breast cancer, which are usually connected with the drug resistance and cancer migration. Many studies indicated that PI3K/Akt/mTOR inhibitors and Hh inhibitors displayed synergistic effects, and the combination of the two signaling drugs could delay drug resistance and inhibit cancer migration in breast cancer. Therefore, the development of molecules simultaneously inhibiting these two pathways is urgent needed. Based on the structures of PI3K inhibitor buparlisib and Hh inhibitor vismodegib, a series of hybrid structures were designed and synthesized utilizing rational drug design and computer-based drug design. Several compounds displayed excellent antiproliferative activities against several breast cancer cell lines, including triple-negative breast cancer (TNBC) MDA-MB-231 cell. Further mechanistic studies demonstrated that the representative compound 9i could inhibit both PI3K/Akt/mTOR and hedgehog (Hh) signalings by inhibiting the phosphorylation of S6K and Akt as well as decreasing the SAG elevated expression of Gli1. Compound 9i could also induce apoptosis remarkably in T47D and MDA-MB-231 cells. In the transwell assay, 9i showed significant inhibition on the migration of MDA-MB-231. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

Evidence that Ser87 of BimEL is phosphorylated by Akt and regulates BimEL apoptotic function.

PubMed

Qi, Xiao-Jun; Wildey, Gary M; Howe, Philip H

2006-01-13

Bim, the Bcl-2 interacting mediator of cell death, is a member of the BH3-only family of pro-apoptotic proteins. Recent studies have demonstrated that the apoptotic activity of Bim can be regulated through a post-translational mechanism whereby ERK phosphorylation serves as a signal for Bim ubiquitination and proteasomal degradation. In this report, we investigated the signaling pathways leading to Bim phosphorylation in Ba/F3 cells, an interleukin-3 (IL-3)-dependent B-cell line. IL-3 stimulation induced phosphorylation of Bim(EL), one of the predominant isoforms of Bim expressed in cells, at multiple sites, as evidenced by the formation of at least three to four bands by Western blotting that were sensitive to phosphatase digestion. The appearance of multiple, phosphorylated species of Bim(EL) correlated with Akt, and not ERK, activation. The PI3K inhibitor, LY294002, blocked IL-3-stimulated Akt activity and partially blocked Bim(EL) phosphorylation. In vitro kinase assays showed that recombinant Akt could directly phosphorylate a GST-Bim(EL) fusion protein and identified the Akt phosphorylation site in the Bim(EL) domain as Ser(87). Further, we demonstrated that cytokine stimulation promotes Bim(EL) binding to 14-3-3 proteins. Finally, we show that mutation of Ser(87) dramatically increases the apoptotic potency of Bim(EL). We propose that Ser(87) of Bim(EL) is an important regulatory site that is targeted by Akt to attenuate the pro-apoptotic function of Bim(EL), thereby promoting cell survival.

Seasonal, tissue-specific regulation of Akt/protein kinase B and glycogen synthase in hibernators.

PubMed

Hoehn, Kyle L; Hudachek, Susan F; Summers, Scott A; Florant, Gregory L

2004-03-01

Yellow-bellied marmots (Marmota flaviventris) exhibit a circannual cycle of hyperphagia and nutrient storage in the summer followed by hibernation in the winter. This annual cycle of body mass gain and loss is primarily due to large-scale accumulation of lipid in the summer, which is then mobilized and oxidized for energy during winter. The rapid and predictable change in body mass makes these animals ideal for studies investigating the molecular basis for body weight regulation. In the study described herein, we monitored seasonal changes in the protein levels and activity of a central regulator of anabolic metabolism, the serine-threonine kinase Akt-protein kinase B (Akt/PKB), during the months accompanying maximal weight gain and entry into hibernation (June-November). Interestingly, under fasting conditions, Akt/PKB demonstrated a tissue-specific seasonal activation. Specifically, although Akt/PKB levels did not change, the activity of Akt/PKB (isoforms 1/alpha and 2/beta) in white adipose tissue (WAT) increased significantly in July. Moreover, glycogen synthase, which lies downstream of Akt/PKB on a linear pathway linking the enzyme to the stimulation of glycogen synthesis, demonstrated a similar pattern of seasonal activation. By contrast, Akt/PKB activity in skeletal muscle peaked much later (i.e., September). These data suggest the existence of a novel, tissue-specific mechanism regulating Akt/PKB activation during periods of marked anabolism.

Compound C induces protective autophagy in human cholangiocarcinoma cells via Akt/mTOR-independent pathway.

PubMed

Zhao, Xiaofang; Luo, Guosong; Cheng, Ying; Yu, Wenjing; Chen, Run; Xiao, Bin; Xiang, Yuancai; Feng, Chunhong; Fu, Wenguang; Duan, Chunyan; Yao, Fuli; Xia, Xianming; Tao, Qinghua; Wei, Mei; Dai, Rongyang

2018-07-01

Compound C, a well-known inhibitor of AMP-activated protein kinase (AMPK), has been reported to exert antitumor activities in some types of cells. Whether compound C can exert antitumor effects in human cholangiocarcinoma (CCA) remains unknown. Here, we demonstrated that compound C is a potent inducer of cell death and autophagy in human CCA cells. Autophagy inhibitors increased the cytotoxicity of compound C towards human CCA cells, as confirmed by increased LDH release, and PARP cleavage. It is notable that compound C treatment increased phosphorylated Akt, sustained high levels of phosphorylated p70S6K, and decreased mTOR regulated p-ULK1 (ser757). Based on the data that blocking PI3K/Akt or mTOR had no apparent influence on autophagic response, we suggest that compound C induces autophagy independent of Akt/mTOR signaling in human CCA cells. Further study demonstrated that compound C inhibited the phosphorylation of JNK and its target c-Jun. Blocking JNK by SP600125 or siRNA suppressed autophagy induction upon compound C treatment. Moreover, compound C induced p38 MAPK activation, and its inhibition promoted autophagy induction via JNK activation. In addition, compound C induced p53 expression, and its inhibition attenuated compound C-induced autophagic response. Thus, compound C triggers autophagy, at least in part, via the JNK and p53 pathways in human CCA cells. In conclusion, suppresses autophagy could increase compound C sensitivity in human CCA. © 2018 Wiley Periodicals, Inc.

Insulin regulates liver metabolism in vivo in the absence of hepatic Akt and Foxo1

PubMed Central

Lu, Mingjian; Wan, Min; Leavens, Karla F.; Chu, Qingwei; Monks, Bobby R.; Fernandez, Sully; Ahima, Rexford S.; Ueki, Kohjiro; Kahn, C. Ronald; Birnbaum, Morris J.

2012-01-01

Considerable data support the idea that Foxo1 drives the liver transcriptional program during fasting and is inhibited by Akt after feeding. Mice with hepatic deletion of Akt1 and Akt2 were glucose intolerant, insulin resistant, and defective in the transcriptional response to feeding in liver. These defects were normalized upon concomitant liver–specific deletion of Foxo1. Surprisingly, in the absence of both Akt and Foxo1, mice adapted appropriately to both the fasted and fed state, and insulin suppressed hepatic glucose production normally. Gene expression analysis revealed that deletion of Akt in liver led to constitutive activation of Foxo1–dependent gene expression, but once again concomitant ablation of Foxo1 restored postprandial regulation, preventing its inhibition of the metabolic response to nutrient intake. These results are inconsistent with the canonical model of hepatic metabolism in which Akt is an obligate intermediate for insulin’s actions. Rather they demonstrate that a major role of hepatic Akt is to restrain Foxo1 activity, and in the absence of Foxo1, Akt is largely dispensable for hepatic metabolic regulation in vivo. PMID:22344295

Protein Phosphatase 2A Isoforms Utilizing Aβ Scaffolds Regulate Differentiation through Control of Akt Protein*

PubMed Central

Hwang, Justin H.; Jiang, Tao; Kulkarni, Shreya; Faure, Nathalie; Schaffhausen, Brian S.

2013-01-01

Protein phosphatase 2A (PP2A) regulates almost all cell signaling pathways. It consists of a scaffolding A subunit to which a catalytic C subunit and one of many regulatory B subunits bind. Of the more than 80 PP2A isoforms, 10% use Aβ as a scaffold. This study demonstrates the isoform-specific function of the A scaffold subunits. Polyomaviruses have shown the importance of phosphotyrosine, PI3K, and p53 in transformation. Comparisons of polyoma and SV40 small T antigens implicate Aβ in the control of differentiation. Knockdown of Aβ enhanced differentiation. Akt signaling regulated differentiation; its activation or inhibition promoted or blocked it, respectively. Aβ bound Akt. Enhancement of PP2A Aβ/Akt interaction by polyoma small T antigen increased turnover of Akt Ser-473 phosphorylation. Conversely, knockdown of Aβ promoted Akt activity and reduced turnover of phosphate at Ser-473 of Akt. These data provide new insight into the regulation of Akt, a protein of extreme importance in cancer. Furthermore, our results suggest that the role for Aβ in differentiation and perhaps tumor suppression may lie partly in its ability to negatively regulate Akt. PMID:24052256

Edaravone protects rats and human pulmonary alveolar epithelial cells against hyperoxia injury: heme oxygenase-1 and PI3K/Akt pathway may be involved.

PubMed

Cao, Huifang; Feng, Ying; Ning, Yunye; Zhang, Zinan; Li, Weihao; Li, Qiang

2015-01-01

Hyperoxic acute lung injury (HALI) is a clinical syndrome as a result of prolonged supplement of high concentrations of oxygen. As yet, no specific treatment is available for HALI. The present study aims to investigate the effects of edaravone on hyperoxia-induced oxidative injury and the underlying mechanism. We treated rats and human pulmonary alveolar epithelial cells with hyperoxia and different concentration of edaravone, then examined the effects of edaravone on cell viability, cell injury and two oxidative products. The roles of heme oxygenase-1 (HO-1) and PI3K/Akt pathway were explored using Western blot and corresponding inhibitors. The results showed that edaravone reduced lung biochemical alterations induced by hyperoxia and mortality of rats, dose-dependently alleviated cell mortality, cell injury, and peroxidation of cellular lipid and DNA oxidative damage. It upregulated cellular HO-1 expression and activity, which was reversed by PI3K/Akt pathway inhibition. The administration of zinc protoporphyrin-IX, a HO-1 inhibitor, and LY249002, a PI3K/Akt pathway inhibitor, abolished the protective effects of edaravone in cells. This study indicates that edaravone protects rats and human pulmonary alveolar epithelial cells against hyperoxia-induced injury and the antioxidant effect may be related to upregulation of HO-1, which is regulated by PI3K/Akt pathway.

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

PubMed

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

2015-12-15

Here we report that the PI3K/Akt1/IL-6/STAT3 signalling pathway regulates generation and stem cell-like properties of Non-Small Cell Lung Cancer (NSCLC) tumor initiating cells (TICs). Mutant Akt1, mutant PIK3CA or PTEN loss enhances formation of lung cancer spheroids (LCS), self-renewal, expression of stemness markers and tumorigenic potential of human immortalized bronchial cells (BEAS-2B) whereas Akt inhibition suppresses these activities in established (NCI-H460) and primary NSCLC cells. Matched microarray analysis of Akt1-interfered cells and LCSs identified IL-6 as a critical target of Akt signalling in NSCLC TICs. Accordingly, suppression of Akt in NSCLC cells decreases IL-6 levels, phosphorylation of IkK and IkB, NF-kB transcriptional activity, phosphorylation and transcriptional activity of STAT3 whereas active Akt1 up-regulates them. Exposure of LCSs isolated from NSCLC cells to blocking anti-IL-6 mAbs, shRNA to IL-6 receptor or to STAT3 markedly reduces the capability to generate LCSs, to self-renew and to form tumors, whereas administration of IL-6 to Akt-interfered cells restores the capability to generate LCSs. Finally, immunohistochemical studies in NSCLC patients demonstrated a positive correlative trend between activated Akt, IL-6 expression and STAT3 phosphorylation (n = 94; p < 0.05). In conclusion, our data indicate that aberrant Akt signalling contributes to maintaining stemness in lung cancer TICs through a NF-kB/IL-6/STAT3 pathway and provide novel potential therapeutic targets for eliminating these malignant cells in NSCLC.

Sustained Activation of Akt Elicits Mitochondrial Dysfunction to Block Plasmodium falciparum Infection in the Mosquito Host

PubMed Central

Drexler, Anna L.; Antonova-Koch, Yevgeniya; Sakaguchi, Danielle; Napoli, Eleonora; Wong, Sarah; Price, Mark S.; Eigenheer, Richard; Phinney, Brett S.; Pakpour, Nazzy; Pietri, Jose E.; Cheung, Kong; Georgis, Martha; Riehle, Michael

2013-01-01

The overexpression of activated, myristoylated Akt in the midgut of female transgenic Anopheles stephensi results in resistance to infection with the human malaria parasite Plasmodium falciparum but also decreased lifespan. In the present study, the understanding of mitochondria-dependent midgut homeostasis has been expanded to explain this apparent paradox in an insect of major medical importance. Given that Akt signaling is essential for cell growth and survival, we hypothesized that sustained Akt activation in the mosquito midgut would alter the balance of critical pathways that control mitochondrial dynamics to enhance parasite killing at some cost to survivorship. Toxic reactive oxygen and nitrogen species (RNOS) rise to high levels in the midgut after blood feeding, due to a combination of high NO production and a decline in FOXO-dependent antioxidants. Despite an apparent increase in mitochondrial biogenesis in young females (3 d), energy deficiencies were apparent as decreased oxidative phosphorylation and increased [AMP]/[ATP] ratios. In addition, mitochondrial mass was lower and accompanied by the presence of stalled autophagosomes in the posterior midgut, a critical site for blood digestion and stem cell-mediated epithelial maintenance and repair, and by functional degradation of the epithelial barrier. By 18 d, the age at which An. stephensi would transmit P. falciparum to human hosts, mitochondrial dysfunction coupled to Akt-mediated repression of autophagy/mitophagy was more evident and midgut epithelial structure was markedly compromised. Inhibition of RNOS by co-feeding of the nitric-oxide synthase inhibitor L-NAME at infection abrogated Akt-dependent killing of P. falciparum that begins within 18 h of infection in 3–5 d old mosquitoes. Hence, Akt-induced changes in mitochondrial dynamics perturb midgut homeostasis to enhance parasite resistance and decrease mosquito infective lifespan. Further, quality control of mitochondrial function in the

Genetic alterations in the phosphatidylinositol-3 kinase/Akt pathway in thyroid cancer.

PubMed

Xing, Mingzhao

2010-07-01

Aberrant activation of the phosphatidylinositol-3 kinase (PI3K)/Akt pathway plays a fundamental role in thyroid tumorigenesis, particularly in follicular thyroid cancer (FTC) and aggressive thyroid cancer, such as anaplastic thyroid cancer (ATC). As the drivers of this process, many genetic alterations activating the PI3K/Akt pathway have been identified in thyroid cancer in recent years. This review summarizes the current knowledge on major genetic alterations in the PI3K/Akt pathway. These include PIK3CA mutations and genomic amplification/copy gain, Ras mutations, PTEN mutations, RET/PTC and PPARgamma/Pax8 rearrangements, as well as amplification/copy gain of PIK3CB, PDK1, Akt, and various receptor tyrosine kinase genes. Most of these genetic alterations are particularly common in FTC and many of them are even more common in ATC; they are generally less common in papillary thyroid cancer (PTC), in which the MAP kinase (MAPK) pathway activated by the BRAF mutation instead plays a major role. Methylation and, thus, epigenetic silencing of PTEN, a major negative regulator of the PI3K/Akt pathway, occurs in close association with activating genetic alterations of the PI3K/Akt pathway, constituting a unique self-enhancement mechanism for this pathway. Many of these genetic alterations are mutually exclusive in differentiated thyroid tumors, but with increasing concurrence from benign tumors to FTC to ATC. RET/PTC, Ras, and receptor tyrosine kinase could dually activate the PI3K/Akt and MAPK pathways. Most cases of ATC harbor genetic alterations in these genes or other genetic combinations that can activate both pathways. It is proposed that genetic alterations in the PI3K/Akt pathway promote thyroid cell transformation to FTC and that genetic alterations in the MAPK pathway promote cell transformation to PTC; accumulation of multiple genetic alterations that can activate both pathways promotes thyroid cancer aggressiveness and progression to ATC. Genetic alterations

The ectodomain of cadherin-11 binds to erbB2 and stimulates Akt phosphorylation to promote cranial neural crest cell migration

PubMed Central

Mathavan, Ketan; Khedgikar, Vikram; Bartolo, Vanessa

2017-01-01

During development, a multi-potent group of cells known as the cranial neural crest (CNC) migrate to form craniofacial structures. Proper migration of these cells requires proteolysis of cell adhesion molecules, such as cadherins. In Xenopus laevis, preventing extracellular cleavage of cadherin-11 impairs CNC migration. However, overexpression of the soluble cleavage product (EC1-3) is capable of rescuing this phenotype. The mechanism by which EC1-3 promotes CNC migration has not been investigated until now. Here we show that EC1-3 stimulates phosphorylation of Akt, a target of PI3K, in X.laevis CNC. Through immunoprecipitation experiments, we determined that EC1-3 interacts with all ErbB receptors, PDGFRα, and FGFR1. Of these receptors, only ErbB2 was able to produce an increase in Akt phosphorylation upon treatment with a recombinant EC1-3. This increase was abrogated by mubritinib, an inhibitor of ErbB2. We were able to recapitulate this decrease in Akt phosphorylation in vivo by knocking down ErbB2 in CNC cells. Knockdown of the receptor also significantly reduced CNC migration in vivo. We confirmed the importance of ErbB2 and ErbB receptor signaling in CNC migration using mubritinib and canertinib, respectively. Mubritinib and the PI3K inhibitor LY294002 significantly decreased cell migration while canertinib nearly prevented it altogether. These data show that ErbB2 and Akt are important for CNC migration and implicate other ErbB receptors and Akt-independent signaling pathways. Our findings provide the first example of a functional interaction between the extracellular domain of a type II classical cadherin and growth factor receptors. PMID:29190819

Clusterin protects H9c2 cardiomyocytes from oxidative stress-induced apoptosis via Akt/GSK-3β signaling pathway

PubMed Central

Jun, Hyoung-Oh; Kim, Dong-hun; Lee, Sae-Won; Lee, Hye Shin; Seo, Ji Hae; Kim, Jeong Hun; Kim, Jin Hyoung; Yu, Young Suk; Min, Bon Hong

2011-01-01

Clusterin is a secretory glycoprotein, which is highly up-regulated in a variety of normal and injury tissues undergoing apoptosis including infarct region of the myocardium. Here, we report that clusterin protects H9c2 cardiomyocytes from H2O2-induced apoptosis by triggering the activation of Akt and GSK-3β. Treatment with H2O2 induces apoptosis of H9c2 cells by promoting caspase cleavage and cytochrome c release from mitochondria. However, co-treatment with clusterin reverses the induction of apoptotic signaling by H2O2, thereby recovers cell viability. The protective effect of clusterin on H2O2-induced apoptosis is impaired by PI3K inhibitor LY294002, which effectively suppresses clusterin-induced activation of Akt and GSK-3β. In addition, the protective effect of clusterin is independednt on its receptor megalin, because inhibition of megalin has no effect on clusturin-mediated Akt/GSK-3β phosphoylation and H9c2 cell viability. Collectively, these results suggest that clusterin has a role protecting cardiomyocytes from oxidative stress and the Akt/GSK-3β signaling mediates anti-apoptotic effect of clusterin. PMID:21270507