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Sample records for active akt mutant

  1. The AKT inhibitor AZD5363 is selectively active in PI3KCA mutant gastric cancer, and sensitizes a patient-derived gastric cancer xenograft model with PTEN loss to Taxotere

    PubMed Central

    2013-01-01

    Introduction Activation of the PI3K/AKT pathway is a common phenomenon in cancer due to multiple mechanisms, including mutation of PI3KCA, loss or mutation of PTEN, or over-expression of receptor tyrosine kinases. We recently developed a novel AKT kinase inhibitor, AZD5363, and demonstrated that HGC27, a cell line harboring both PI3KCA mutation and PTEN loss, displayed the greatest sensitivity to this AKT inhibitor in vitro and in vivo. Case preparation To further elucidate the correlation between AZD5363 response and genetic alterations in gastric cancer (GC) and identify GC patients with both PI3KCA mutations and PTEN loss, we investigated the effects of pharmacological inhibition of AKT on a panel of 20 GC cell lines and genetic aberrations in tumor samples from a cohort of Chinese GC patients. We demonstrated that GC cells with PI3KCA mutations were selectively sensitive to AZD5363. Disease linkage studies showed that PI3KCA activating mutations or PTEN loss were found in 2.7% (4/150) and 23% (14/61) of Chinese GC patients respectively. To further dissect the role of PI3KCA mutation and PTEN loss in response to AKT inhibition, we tested the antitumor activity of AZD5363 in two patient-derived GC xenograft (PDGCX) models harboring either PI3KCA mutation or PTEN loss. Our data indicated that AZD5363 monotherapy treatment led to a moderate response in the PI3KCA mutant PDGCX model. Whilst monotherapy AZD5363 or Taxotere were ineffective in the PTEN negative PDGCX model, significant anti-tumor activity was observed when AZD5363 was combined with Taxotere. Conclusion Our results indicated that PI3KCA mutation is an important determinant of response to AKT inhibition in GC and combination with AZD5363 can overcome innate resistance to Taxotere in a PTEN loss PDGCX model. It is suggested that AKT inhibitor is an attractive option for treatment of a new segment of GC patients with aberrant PI3K/AKT signaling. PMID:24088382

  2. 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

  3. Activated AKT pathway promotes establishment of endometriosis.

    PubMed

    Kim, Tae Hoon; Yu, Yanni; Luo, Lily; Lydon, John P; Jeong, Jae-Wook; Kim, J Julie

    2014-05-01

    The pathogenesis of endometriosis remains unclear, and relatively little is known about the mechanisms that promote establishment and survival of the disease. Previously, we demonstrated that v-akt murine thymoma viral oncogene homolog (AKT) activity was increased in endometriosis tissues and cells from ovarian endometriomas and that this increase promoted cell survival as well as decreased levels of progesterone receptor. The objective of this study was to demonstrate a role for AKT in the establishment of ectopic lesions. First, a dose-dependent inhibition of AKT in stromal cells from human ovarian endometriomas (OSIS) as well as endometrial stromal cells from disease-free patients (ESC) with the allosteric AKT inhibitor MK-2206 was demonstrated by decreased levels of phosphorylated (p)(Ser473)-AKT. Levels of the AKT target protein, p(Ser256)-forkhead box O1 were increased in OSIS cells, which decreased with MK-2206 treatment, whereas levels of p(Ser9)-glycogen synthase kinase 3β did not change in response to MK-2206. Although MK-2206 decreased viability of both OSIS and ESC in a dose-dependent manner, proliferation of OSIS cells was differentially decreased significantly compared with ESC. Next, the role of hyperactive AKT in the establishment of ectopic lesions was studied using the bigenic, PR(cre/+)Pten(f/+) heterozygous mouse. Autologous implantation of uterine tissues was performed in these mice. After 4 weeks, an average of 4 ± 0.33 lesions per Pten(f/+) mouse and 7.5 ± 0.43 lesions in the PR(cre/+)Pten(f/+) mouse were found. Histological examination of the lesions showed endometrial tissue-like morphology, which was similar in both the Pten(f/+) and PR(cre/+)Pten(f/+) mice. Treatment of mice with MK-2206 resulted in a significantly decreased number of lesions established. Immunohistochemical staining of ectopic lesions revealed decreased p(Ser473)-AKT and the proliferation marker Ki67 from MK-2206-treated mice compared with vehicle-treated mice

  4. Mutant p53-R273H mediates cancer cell survival and anoikis resistance through AKT-dependent suppression of BCL2-modifying factor (BMF).

    PubMed

    Tan, B S; Tiong, K H; Choo, H L; Chung, F Fei-Lei; Hii, L-W; Tan, S H; Yap, I K S; Pani, S; Khor, N T W; Wong, S F; Rosli, R; Cheong, S-K; Leong, C-O

    2015-07-16

    p53 is the most frequently mutated tumor-suppressor gene in human cancers. Unlike other tumor-suppressor genes, p53 mutations mainly occur as missense mutations within the DNA-binding domain, leading to the expression of full-length mutant p53 protein. Mutant p53 proteins not only lose their tumor-suppressor function, but may also gain new oncogenic functions and promote tumorigenesis. Here, we showed that silencing of endogenous p53-R273H contact mutant, but not p53-R175H conformational mutant, reduced AKT phosphorylation, induced BCL2-modifying factor (BMF) expression, sensitized BIM dissociation from BCL-XL and induced mitochondria-dependent apoptosis in cancer cells. Importantly, cancer cells harboring endogenous p53-R273H mutant were also found to be inherently resistant to anoikis and lack BMF induction following culture in suspension. Underlying these activities is the ability of p53-R273H mutant to suppress BMF expression that is dependent on constitutively active PI3K/AKT signaling. Collectively, these findings suggest that p53-R273H can specifically drive AKT signaling and suppress BMF expression, resulting in enhanced cell survivability and anoikis resistance. These findings open the possibility that blocking of PI3K/AKT will have therapeutic benefit in mutant p53-R273H expressing cancers.

  5. Mutant p53-R273H mediates cancer cell survival and anoikis resistance through AKT-dependent suppression of BCL2-modifying factor (BMF)

    PubMed Central

    Tan, B S; Tiong, K H; Choo, H L; Fei-Lei Chung, F; Hii, L-W; Tan, S H; Yap, I KS; Pani, S; Khor, N TW; Wong, S F; Rosli, R; Cheong, S-K; Leong, C-O

    2015-01-01

    p53 is the most frequently mutated tumor-suppressor gene in human cancers. Unlike other tumor-suppressor genes, p53 mutations mainly occur as missense mutations within the DNA-binding domain, leading to the expression of full-length mutant p53 protein. Mutant p53 proteins not only lose their tumor-suppressor function, but may also gain new oncogenic functions and promote tumorigenesis. Here, we showed that silencing of endogenous p53-R273H contact mutant, but not p53-R175H conformational mutant, reduced AKT phosphorylation, induced BCL2-modifying factor (BMF) expression, sensitized BIM dissociation from BCL-XL and induced mitochondria-dependent apoptosis in cancer cells. Importantly, cancer cells harboring endogenous p53-R273H mutant were also found to be inherently resistant to anoikis and lack BMF induction following culture in suspension. Underlying these activities is the ability of p53-R273H mutant to suppress BMF expression that is dependent on constitutively active PI3K/AKT signaling. Collectively, these findings suggest that p53-R273H can specifically drive AKT signaling and suppress BMF expression, resulting in enhanced cell survivability and anoikis resistance. These findings open the possibility that blocking of PI3K/AKT will have therapeutic benefit in mutant p53-R273H expressing cancers. PMID:26181206

  6. Mutant p53-R273H mediates cancer cell survival and anoikis resistance through AKT-dependent suppression of BCL2-modifying factor (BMF).

    PubMed

    Tan, B S; Tiong, K H; Choo, H L; Chung, F Fei-Lei; Hii, L-W; Tan, S H; Yap, I K S; Pani, S; Khor, N T W; Wong, S F; Rosli, R; Cheong, S-K; Leong, C-O

    2015-01-01

    p53 is the most frequently mutated tumor-suppressor gene in human cancers. Unlike other tumor-suppressor genes, p53 mutations mainly occur as missense mutations within the DNA-binding domain, leading to the expression of full-length mutant p53 protein. Mutant p53 proteins not only lose their tumor-suppressor function, but may also gain new oncogenic functions and promote tumorigenesis. Here, we showed that silencing of endogenous p53-R273H contact mutant, but not p53-R175H conformational mutant, reduced AKT phosphorylation, induced BCL2-modifying factor (BMF) expression, sensitized BIM dissociation from BCL-XL and induced mitochondria-dependent apoptosis in cancer cells. Importantly, cancer cells harboring endogenous p53-R273H mutant were also found to be inherently resistant to anoikis and lack BMF induction following culture in suspension. Underlying these activities is the ability of p53-R273H mutant to suppress BMF expression that is dependent on constitutively active PI3K/AKT signaling. Collectively, these findings suggest that p53-R273H can specifically drive AKT signaling and suppress BMF expression, resulting in enhanced cell survivability and anoikis resistance. These findings open the possibility that blocking of PI3K/AKT will have therapeutic benefit in mutant p53-R273H expressing cancers. PMID:26181206

  7. Akt3 and Mutant V600EB-Raf Cooperate to Promote Early Melanoma Development

    PubMed Central

    Cheung, Mitchell; Sharma, Arati; Madhunapantula, SubbaRao V.; Robertson, Gavin P.

    2008-01-01

    B-Raf is the most mutated gene in melanoma; however, mechanism through which it promotes early melanomas remains uncertain. Most nevi contain activated V600EB-Raf but few develop into melanoma and expression in melanocytes is inhibitory with low protein levels present in surviving cells, suggesting unknown cooperative oncogenic events are necessary for melanoma development. Since many melanomas have V600EB-Raf and active Akt3, it is possible these proteins cooperatively facilitate melanocyte transformation. In this study, Akt3 is shown to phosphorylate V600EB-Raf to lower its activity as well as that of the downstream MAP kinase pathway to levels promoting early melanoma development. Expression of active Akt3 in early melanoma cells containing V600EB-Raf reduced MAP kinase signaling and promoted anchorage independent growth. Furthermore, expression of both V600EB-Raf and active Akt3 in melanocytes promoted a transformed phenotype. Mechanistically, aberrant Akt3 activity in early melanomas serves to phosphorylate serines 364 and 428 on V600EB-Raf in order to reduce activity of V600EB-Raf to levels that promote rather than inhibit proliferation, which aids melanocytic transformation. Inhibition of V600EB-Raf or Akt3 in advanced melanoma cells in which both pathways were active reduced anchorage independent growth and tumor development in a cooperatively acting manner. Inhibition of Akt3 alone in these cells led to increased MAP kinase signaling. In summary, these results suggest that activating B-Raf mutations initially promote nevi development but the resulting high, intense activation of the MAP kinase pathway inhibits further tumor progression requiring Akt3 activation to bypass this barrier and aid melanoma development. PMID:18451171

  8. Concurrent Targeting of KRAS and AKT by MiR-4689 Is a Novel Treatment Against Mutant KRAS Colorectal Cancer

    PubMed Central

    Hiraki, Masayuki; Nishimura, Junichi; Takahashi, Hidekazu; Wu, Xin; Takahashi, Yusuke; Miyo, Masaaki; Nishida, Naohiro; Uemura, Mamoru; Hata, Taishi; Takemasa, Ichiro; Mizushima, Tsunekazu; Soh, Jae-Won; Doki, Yuichiro; Mori, Masaki; Yamamoto, Hirofumi

    2015-01-01

    KRAS mutations are a major cause of drug resistance to molecular-targeted therapies. Aberrant epidermal growth factor receptor (EGFR) signaling may cause dysregulation of microRNA (miRNA) and gene regulatory networks, which leads to cancer initiation and progression. To address the functional relevance of miRNAs in mutant KRAS cancers, we transfected exogenous KRASG12V into human embryonic kidney 293 and MRC5 cells with wild-type KRAS and BRAF genes, and we comprehensively profiled the dysregulated miRNAs. The result showed that mature miRNA oligonucleotide (miR)-4689, one of the significantly down-regulated miRNAs in KRASG12V overexpressed cells, was found to exhibit a potent growth-inhibitory and proapoptotic effect both in vitro and in vivo. miR-4689 expression was significantly down-regulated in cancer tissues compared to normal mucosa, and it was particularly decreased in mutant KRAS CRC tissues. miR-4689 directly targets v-ki-ras2 kirsten rat sarcoma viral oncogene homolog (KRAS) and v-akt murine thymoma viral oncogene homolog 1(AKT1), key components of two major branches in EGFR pathway, suggesting KRAS overdrives this signaling pathway through inhibition of miR-4689. Overall, this study provided additional evidence that mutant KRAS functions as a broad regulator of the EGFR signaling cascade by inhibiting miR-4689, which negatively regulates both RAS/mitogen-activated protein kinase (MAPK) and phosphoinositide 3-kinase (PI3K)/AKT pathways. These activities indicated that miR-4689 may be a promising therapeutic agent in mutant KRAS CRC. PMID:25756961

  9. Mutant p53 promotes ovarian cancer cell adhesion to mesothelial cells via integrin β4 and Akt signals

    PubMed Central

    Lee, Jong-Gyu; Ahn, Ji-Hye; Jin Kim, Tae; Ho Lee, Jae; Choi, Jung-Hye

    2015-01-01

    Missense mutations in the TP53 gene resulting in the accumulation of mutant proteins are extremely common in advanced ovarian cancer, which is characterised by peritoneal metastasis. Attachment of cancer cells to the peritoneal mesothelium is regarded as an initial, key step for the metastatic spread of ovarian cancer. In the present study, we investigated the possible role of a p53 mutant in the mesothelial adhesion of ovarian cancer cells. We found that OVCAR-3 cells with the R248 TP53 mutation (p53R248) were more adhesive to mesothelial Met5A cells than were A2780 cells expressing wild-type p53. In addition, ectopic expression of p53R248 in p53-null SKOV-3 cells significantly increased adhesion to Met5A cells. Knockdown of mutant p53 significantly compromised p53R248-induced cell adhesion to Met5A cells. Microarray analysis revealed that several adhesion-related genes, including integrin β4, were markedly up-regulated, and certain signalling pathways, including PI3K/Akt, were activated in p53R248 transfectants of SKOV-3 cells. Inhibition of integrin β4 and Akt signalling using blocking antibody and the inhibitor LY294002, respectively, significantly attenuated p53R248-mediated ovarian cancer-mesothelial adhesion. These data suggest that the p53R248 mutant endows ovarian cancer cells with increased adhesiveness and that integrin β4 and Akt signalling are associated with the mutation-enhanced ovarian cancer-mesothelial cell adhesion. PMID:26223322

  10. Molecular dissection of AKT activation in lung cancer cell lines

    PubMed Central

    Guo, Yanan; Du, Jinyan; Kwiatkowski, David J

    2013-01-01

    AKT is a critical signaling node downstream of PI3K, which is often activated in cancer. We analyzed the state of activation of AKT in 80 human non-small cell lung cancer cell lines under serum starvation conditions. We identified 13 lines which showed persistent AKT activation in the absence of serum. In 12 of the 13 lines, AKT activation could be attributed to loss of PTEN, activating mutation in EGFR or PIK3CA, or amplification of ERBB2. HCC2429 was the only cell line that had no alterations in those genes, but had high phospho-AKT(Ser473) levels under serum starvation conditions. However, the activation of AKT in HCC2429 was PI3K- and mTORC2-dependent based upon use of specific inhibitors. Kinome tyrosine phosphorylation profiling showed that both Notch and SRC were highly activated in this cell line. Despite the activation of Notch, AKT activation and cell survival were not affected by Notch inhibitors DAPT or Compound E. In contrast, SRC inhibitors PP2 and dasatinib both significantly decreased pAKT(Ser473) levels and reduced cell survival by inducing apoptosis. Further, a combination of SRC and mTOR inhibition synergistically blocked activation of AKT and induced apoptosis. Over-expression of SRC has been identified previously in human lung cancers, and these results suggest that a combination of SRC and mTOR inhibitors may have unique therapeutic benefit for a subset of lung cancers with these molecular features. PMID:23319332

  11. Potassium uptake supporting plant growth in the absence of AKT1 channel activity: Inhibition by ammonium and stimulation by sodium

    NASA Technical Reports Server (NTRS)

    Spalding, E. P.; Hirsch, R. E.; Lewis, D. R.; Qi, Z.; Sussman, M. R.; Lewis, B. D.

    1999-01-01

    A transferred-DNA insertion mutant of Arabidopsis that lacks AKT1 inward-rectifying K+ channel activity in root cells was obtained previously by a reverse-genetic strategy, enabling a dissection of the K+-uptake apparatus of the root into AKT1 and non-AKT1 components. Membrane potential measurements in root cells demonstrated that the AKT1 component of the wild-type K+ permeability was between 55 and 63% when external [K+] was between 10 and 1,000 microM, and NH4+ was absent. NH4+ specifically inhibited the non-AKT1 component, apparently by competing for K+ binding sites on the transporter(s). This inhibition by NH4+ had significant consequences for akt1 plants: K+ permeability, 86Rb+ fluxes into roots, seed germination, and seedling growth rate of the mutant were each similarly inhibited by NH4+. Wild-type plants were much more resistant to NH4+. Thus, AKT1 channels conduct the K+ influx necessary for the growth of Arabidopsis embryos and seedlings in conditions that block the non-AKT1 mechanism. In contrast to the effects of NH4+, Na+ and H+ significantly stimulated the non-AKT1 portion of the K+ permeability. Stimulation of akt1 growth rate by Na+, a predicted consequence of the previous result, was observed when external [K+] was 10 microM. Collectively, these results indicate that the AKT1 channel is an important component of the K+ uptake apparatus supporting growth, even in the "high-affinity" range of K+ concentrations. In the absence of AKT1 channel activity, an NH4+-sensitive, Na+/H+-stimulated mechanism can suffice.

  12. Preclinical pharmacology, antitumor activity and development of pharmacodynamic markers for the novel, potent AKT inhibitor CCT128930

    PubMed Central

    Yap, Timothy A.; Walton, Mike I.; Hunter, Lisa-Jane K.; Valenti, Melanie; de Haven Brandon, Alexis; Eve, Paul D.; Ruddle, Ruth; Heaton, Simon P.; Henley, Alan; Pickard, Lisa; Vijayaraghavan, Gowri; Caldwell, John J.; Thompson, Neil T.; Aherne, Wynne; Raynaud, Florence I.; Eccles, Suzanne A.; Workman, Paul; Collins, Ian; Garrett, Michelle D.

    2016-01-01

    AKT is frequently deregulated in cancer, making it an attractive anticancer drug target. CCT128930 is a novel ATP-competitive AKT inhibitor discovered using fragment and structure-based approaches. It is a potent, advanced lead pyrrolopyrimidine compound exhibiting selectivity for AKT over PKA, achieved by targeting a single amino acid difference. CCT128930 exhibited marked antiproliferative activity and inhibited the phosphorylation of a range of AKT substrates in multiple tumor cell lines in vitro, consistent with AKT inhibition. CCT128930 caused a G1 arrest in PTEN-null U87MG human glioblastoma cells, consistent with AKT pathway blockade. Pharmacokinetic studies established that potentially active concentrations of CCT128930 could be achieved in human tumor xenografts. Furthermore, CCT128930 also blocked the phosphorylation of several downstream AKT biomarkers in U87MG tumor xenografts, indicating AKT inhibition in vivo. Antitumor activity was observed with CCT128930 in U87MG and HER2-positive, PIK3CA-mutant BT474 human breast cancer xenografts, consistent with its pharmacokinetic and pharmacodynamic properties. A quantitative immunofluorescence assay to measure the phosphorylation and total protein expression of the AKT substrate PRAS40 in hair follicles is presented. Significant decreases in pThr246 PRAS40 occurred in CCT128930-treated mouse whisker follicles in vivo and human hair follicles treated ex vivo, with minimal changes in total PRAS40. In conclusion, CCT128930 is a novel, selective and potent AKT inhibitor, which blocks AKT activity in vitro and in vivo and induces marked antitumor responses. We have also developed a novel biomarker assay for the inhibition of AKT in human hair follicles, which is currently being employed in clinical trials. PMID:21191045

  13. Preclinical pharmacology, antitumor activity, and development of pharmacodynamic markers for the novel, potent AKT inhibitor CCT128930.

    PubMed

    Yap, Timothy A; Walton, Mike I; Hunter, Lisa-Jane K; Valenti, Melanie; de Haven Brandon, Alexis; Eve, Paul D; Ruddle, Ruth; Heaton, Simon P; Henley, Alan; Pickard, Lisa; Vijayaraghavan, Gowri; Caldwell, John J; Thompson, Neil T; Aherne, Wynne; Raynaud, Florence I; Eccles, Suzanne A; Workman, Paul; Collins, Ian; Garrett, Michelle D

    2011-02-01

    AKT is frequently deregulated in cancer, making it an attractive anticancer drug target. CCT128930 is a novel ATP-competitive AKT inhibitor discovered using fragment- and structure-based approaches. It is a potent, advanced lead pyrrolopyrimidine compound exhibiting selectivity for AKT over PKA, achieved by targeting a single amino acid difference. CCT128930 exhibited marked antiproliferative activity and inhibited the phosphorylation of a range of AKT substrates in multiple tumor cell lines in vitro, consistent with AKT inhibition. CCT128930 caused a G(1) arrest in PTEN-null U87MG human glioblastoma cells, consistent with AKT pathway blockade. Pharmacokinetic studies established that potentially active concentrations of CCT128930 could be achieved in human tumor xenografts. Furthermore, CCT128930 also blocked the phosphorylation of several downstream AKT biomarkers in U87MG tumor xenografts, indicating AKT inhibition in vivo. Antitumor activity was observed with CCT128930 in U87MG and HER2-positive, PIK3CA-mutant BT474 human breast cancer xenografts, consistent with its pharmacokinetic and pharmacodynamic properties. A quantitative immunofluorescence assay to measure the phosphorylation and total protein expression of the AKT substrate PRAS40 in hair follicles is presented. Significant decreases in pThr246 PRAS40 occurred in CCT128930-treated mouse whisker follicles in vivo and human hair follicles treated ex vivo, with minimal changes in total PRAS40. In conclusion, CCT128930 is a novel, selective, and potent AKT inhibitor that blocks AKT activity in vitro and in vivo and induces marked antitumor responses. We have also developed a novel biomarker assay for the inhibition of AKT in human hair follicles, which is currently being used in clinical trials. PMID:21191045

  14. 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.

  15. Molecular mechanism underlying Akt activation in zinc-induced cardioprotection

    PubMed Central

    Lee, SungRyul; Chanoit, Guillaume; McIntosh, Rachel; Zvara, David A.; Xu, Zhelong

    2009-01-01

    Our previous study demonstrated that zinc prevents cardiac reperfusion injury by targeting the mitochondrial permeability transition pore (mPTP) via Akt and glycogen synthetase kinase 3β (GSK-3β). We aimed to address the mechanism by which zinc activates Akt. Treatment of H9c2 cells with ZnCl2 (10 μM) in the presence of the zinc ionophore pyrithione (4 μM) for 20 min enhanced Akt phosphorylation (Ser473), indicating that zinc can rapidly activate Akt. Zinc did not alter either phosphatase and tensin homolog deleted on chromosome 10 (PTEN) phosphorylation and total PTEN protein levels or PTEN oxidation, implying that PTEN may not play a role in the action of zinc. However, zinc-induced Akt phosphorylation was blocked by both the nonselective receptor tyrosine kinase (RTK) inhibitor genistein and the selective insulin-like growth factor-1 RTK (IGF-1RTK) inhibitor AG1024, indicating that zinc activates Akt via IGF-1RTK. Zinc-induced phosphorylation of protein tyrosine and Ser/Thr was also abolished by AG1024. In addition, zinc markedly enhanced phosphorylation of IGF-1 receptor (IGF-1R), which was again reversed by genistein and AG1024. A confocal imaging study revealed that AG1024 abolished the preventive effect of zinc on oxidant-induced mPTP opening, confirming that IGF-1RTK plays a role in zinc-induced cardioprotection. Furthermore, zinc decreased the activity of protein phosphatase 2A (PP2A), a major protein Ser/Thr phosphatase, implying that protein Ser/Thr phosphatases may also play a role in the action of zinc on Akt activity. Taken together, these findings demonstrate that exogenous zinc activates Akt via IGF-1RTK and prevents the mPTP opening in cardiac cells. Inactivation of Ser/Thr protein phosphatases may also contribute to zinc-induced Akt activation. PMID:19525380

  16. Expression, activation, and role of AKT isoforms in the uterus.

    PubMed

    Fabi, François; Asselin, Eric

    2014-11-01

    The three isoforms of AKT: AKT1, AKT2, and AKT3, are crucial regulators of both normal and pathological cellular processes. Each of these isoforms exhibits a high level of homology and functional redundancy with each other. However, while being highly similar and structurally homologous, a rising amount of evidence is showing that each isoform possesses specific targets as well as preferential subcellular localization. The role of AKT has been studied extensively in reproductive processes, but isoform-specific roles are yet to be fully understood. This review will focus on the role of AKT in the uterus and its function in processes related to cell death and proliferation such as embryo implantation, decidualization, endometriosis, and endometrial cancer in an isoform-centric manner. In this review, we will cover the activation of AKT in various settings, localization of isoforms in subcellular compartments, and the effect of isoform expression on cellular processes. To fully understand the dynamic molecular processes taking place in the uterus, it is crucial that we better understand the physiological role of AKT isoforms as well as their function in the emergence of diseases.

  17. Role for the adaptor protein Grb10 in the activation of Akt.

    PubMed

    Jahn, Thomas; Seipel, Petra; Urschel, Susanne; Peschel, Christian; Duyster, Justus

    2002-02-01

    Grb10 is a member of the Grb7 family of adapter proteins lacking intrinsic enzymatic function and encodes functional domains including a pleckstrin homology (PH) domain and an SH2 domain. The role of different Grb10 splice variants in signal transduction of growth factors like insulin or insulin-like growth factor has been described as inhibitory or stimulatory depending on the presence of a functional PH and/or SH2 domain. Performing a yeast two-hybrid screen with the c-kit cytoplasmic tail fused to LexA as a bait and a mouse embryo cDNA library as prey, we found that the Grb10 SH2 domain interacted with the c-kit receptor tyrosine kinase. In the course of SCF-mediated activation of c-kit, Grb10 is recruited to the c-kit receptor in an SH2 domain- and phosphotyrosine-dependent but PH domain-independent manner. We found that Akt and Grb10 form a constitutive complex, suggesting a role for Grb10 in the translocation of Akt to the cell membrane. Indeed, coexpression studies revealed that Grb10 and c-kit activate Akt in a synergistic manner. This dose-dependent effect of Grb10 is wortmannin sensitive and was also seen at a lower level in cells in which c-kit was not expressed. Expression of a Grb10 mutant lacking the SH2 domain as well as a mutant lacking the PH domain did not influence Akt activity. Grb10-induced Akt activation was observed without increased phosphatidylinositol 3-kinase (PI3-kinase) activity, suggesting that Grb10 is a positive regulator of Akt downstream of PI3-kinase. Significantly, deficient activation of Akt by a constitutively activated c-kit mutant lacking the binding site for PI3-kinase (c-kitD814V/Y719F) could be fully compensated by overexpression of Grb10. In Ba/F3 cells, the incapacity of c-kitD814V/Y719F to induce interleukin-3 (IL-3)-independent growth could be rescued by overexpression of Grb10. In contrast, expression of the SH2 deletion mutant of Grb10 together with c-kitD814V/Y719F did not render Ba/F3 cells independent of IL-3. In

  18. AKT mediated glycolytic shift regulates autophagy in classically activated macrophages.

    PubMed

    Matta, Sumit Kumar; Kumar, Dhiraj

    2015-09-01

    Autophagy is considered as an innate defense mechanism primarily due to its role in the targeting of intracellular pathogens for lysosomal degradation. Here we report inhibition of autophagy as an adaptive response in classically activated macrophages that helps achieve high cellular ROS production and cell death-another hallmark of innate mechanisms. We show prolonged classical activation of Raw 264.7 macrophages by treating them with IFN-γ and LPS inhibited autophagy. The inhibition of autophagy was dependent on nitric oxide (NO) production which activated the AKT-mTOR signaling, the known negative regulators of autophagy. Autophagy inhibition in these cells was accompanied with a shift to aerobic glycolysis along with a decline in the mitochondrial membrane potential (MOMP). The decline in MOMP coupled with autophagy inhibition led to increased mitochondrial content and considerably elevated cellular ROS, eventually causing cell death. Next, using specific siRNA mediated knockdowns we show AKT was responsible for the glycolytic shift and autophagy inhibition in activated macrophages. Surprisingly, AKT knockdown in activated macrophages also rescued them from cell death. Finally we show that AKT mediated autophagy inhibition in the activated macrophages correlated with the depletion of glucose from the extracellular medium, and glucose supplementation not only rescued autophagy levels and reversed other phenotypes of activated macrophages, but also inhibited cell death. Thus we report here a novel link between AKT mediated glycolytic metabolism and autophagy in the activated macrophages, and provide a possible mechanism for sustained macrophage activation in vivo.

  19. 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.

  20. Impaired translocation and activation of mitochondrial Akt1 mitigated mitochondrial oxidative phosphorylation Complex V activity in diabetic myocardium.

    PubMed

    Yang, Jia-Ying; Deng, Wu; Chen, Yumay; Fan, Weiwei; Baldwin, Kenneth M; Jope, Richard S; Wallace, Douglas C; Wang, Ping H

    2013-06-01

    Insulin can translocate Akt to mitochondria in cardiac muscle. The goals of this study were to define sub-mitochondrial localization of the translocated Akt, to dissect the effects of insulin on Akt isoform translocation, and to determine the direct effect of mitochondrial Akt activation on Complex V activity in normal and diabetic myocardium. The translocated Akt sequentially localized to the mitochondrial intermembrane space, inner membrane, and matrix. To confirm Akt translocation, in vitro import assay showed rapid entry of Akt into mitochondria. Akt isoforms were differentially regulated by insulin stimulation, only Akt1 translocated into mitochondria. In the insulin-resistant Type 2 diabetes model, Akt1 translocation was blunted. Mitochondrial activation of Akt1 increased Complex V activity by 24% in normal myocardium in vivo and restored Complex V activity in diabetic myocardium. Basal mitochondrial Complex V activity was lower by 22% in the Akt1(-/-) myocardium. Insulin-stimulated Complex V activity was not impaired in the Akt1(-/-) myocardium, due to compensatory translocation of Akt2 to mitochondria. Akt1 is the primary isoform that relayed insulin signaling to mitochondria and modulated mitochondrial Complex V activity. Activation of mitochondrial Akt1 enhanced ATP production and increased phosphocreatine in cardiac muscle cells. Dysregulation of this signal pathway might impair mitochondrial bioenergetics in diabetic myocardium.

  1. Ezrin-radixin-moesin-binding phosphoprotein-50 regulates EGF-induced AKT activation through interaction with EGFR and PTEN.

    PubMed

    Zheng, Junfang; Dai, Yuanping; Yang, Zhiyu; Yang, Longyan; Peng, Zhiqiang; Meng, Ran; Xiong, Ying; He, Junqi

    2016-01-01

    Dysregulated epidermal growth factor receptor (EGFR) signaling, especially EGFR/AKT signaling, plays important roles in tumorigenesis and progression, the study on intracellular regulation of this signaling pathway has great clinical significance. Phosphatase and tensin homolog deleted on chromosome ten (PTEN) is an important antagonist of AKT activity. Its regulation of AKT activity can be enhanced by ezrin-radixin-moesin-binding phosphoprotein-50 (EBP50)-mediated PTEN/EBP50/platelet-derived growth factor receptor (PDGFR) complex. EBP50 was reported to bind to EGFR, and that it may also mediate the formation of PTEN/EGFR complex to regulate EGFR/AKT signaling. In this study, experiments were performed to verify the hypothesis. Results showed that PTEN co-immunoprecipitated with EGFR, demonstrating PTEN/EGFR complex can form in tissue. Further studies showed that EBP50 knockdown decreased the amount of PTEN/EGFR complex by GST pull-down assay, and EBP50 overexpression increased the amount of PTEN/EGFR complex in a dose-dependent manner. While PTEN mutant (V403A), which can not bind with EBP50, only slightly mediated the formation of PTEN/EGFR complex, confirming that EBP50 specifically mediated the formation of the PTEN/EGFR complex. Both PTEN (V403A) and EGFR (L1043/1063F) mutants can not bind with EBP50. The expression of PTEN (V403A) or EGFR (L1043/1063F) mutant in cells resulted in higher AKT activation level than their respective wild-types by EGF stimulation, indicating that EBP50-mediated PTEN/EGFR complex can effectively inhibit EGF-induced AKT activation. EGF stimulation of siEBP50 cells induced higher AKT activation level compared with control cells, further confirming EBP50-mediated PTEN/EGFR complex can more effectively inhibit EGF-induced AKT activation. These results demonstrated the PTEN/EGFR complex formed under the mediation of EBP50, revealing a novel mechanism for negative regulation of EGF-induced AKT pathway, which may be an important molecular

  2. Akt2 Phosphorylates Ezrin to Trigger NHE3 Translocation and Activation*

    PubMed Central

    Shiue, Harn; Musch, Mark W.; Wang, Yingmin; Chang, Eugene B.; Turner, Jerrold R.

    2005-01-01

    Initiation of Na+-glucose cotransport in intestinal absorptive epithelia causes NHE3 to be translocated to the apical plasma membrane, leading to cytoplasmic alkalinization. We reported recently that this NHE3 translocation requires ezrin phosphorylation. However, the kinase that phosphorylates ezrin in this process has not been identified. Because Akt has also been implicated in NHE3 translocation, we investigated the hypothesis that Akt phosphorylates ezrin. After initiation of Na+-glucose cotransport, Akt is activated with kinetics that parallel those of ezrin phosphorylation. Inhibition of p38 MAP kinase, which blocks ezrin phosphorylation, also prevents Akt activation. Purified Akt directly phosphorylates recombinant ezrin at threonine 567 in vitro in an ATP-dependent manner. This in vitro phosphorylation can be prevented by Akt inhibitors. In intact cells, inhibition of either phosphoinositide 3-kinase, an upstream regulator of Akt, or inhibition of Akt itself using inhibitors validated in vitro prevents ezrin phosphorylation after initiation of Na+-glucose cotransport. Specific small interfering RNA knockdown of Akt2 prevented ezrin phosphorylation in intact cells. Pharmacological Akt inhibition or Akt2 knockdown also prevented NHE3 translocation and activation after initiation of Na+-glucose cotransport, confirming the functional role of Akt2. These studies therefore identify Akt2 as a critical kinase that regulates ezrin phosphorylation and activation. This Akt2-dependent ezrin phosphorylation leads to NHE3 translocation and activation. PMID:15531580

  3. Akt2 phosphorylates ezrin to trigger NHE3 translocation and activation.

    PubMed

    Shiue, Harn; Musch, Mark W; Wang, Yingmin; Chang, Eugene B; Turner, Jerrold R

    2005-01-14

    Initiation of Na(+)-glucose cotransport in intestinal absorptive epithelia causes NHE3 to be translocated to the apical plasma membrane, leading to cytoplasmic alkalinization. We reported recently that this NHE3 translocation requires ezrin phosphorylation. However, the kinase that phosphorylates ezrin in this process has not been identified. Because Akt has also been implicated in NHE3 translocation, we investigated the hypothesis that Akt phosphorylates ezrin. After initiation of Na(+)-glucose cotransport, Akt is activated with kinetics that parallel those of ezrin phosphorylation. Inhibition of p38 MAP kinase, which blocks ezrin phosphorylation, also prevents Akt activation. Purified Akt directly phosphorylates recombinant ezrin at threonine 567 in vitro in an ATP-dependent manner. This in vitro phosphorylation can be prevented by Akt inhibitors. In intact cells, inhibition of either phosphoinositide 3-kinase, an upstream regulator of Akt, or inhibition of Akt itself using inhibitors validated in vitro prevents ezrin phosphorylation after initiation of Na(+)-glucose cotransport. Specific small interfering RNA knockdown of Akt2 prevented ezrin phosphorylation in intact cells. Pharmacological Akt inhibition or Akt2 knockdown also prevented NHE3 translocation and activation after initiation of Na(+)-glucose cotransport, confirming the functional role of Akt2. These studies therefore identify Akt2 as a critical kinase that regulates ezrin phosphorylation and activation. This Akt2-dependent ezrin phosphorylation leads to NHE3 translocation and activation.

  4. Hydrogen Peroxide-Induced Akt Phosphorylation Regulates Bax Activation

    PubMed Central

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

    2009-01-01

    Reactive oxygen species such as hydrogen peroxide (H2O2) are involved in many cellular processes that positively and negatively regulate cell fate. H2O2, 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 H2O2 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 H2O2-induced activation of PI3K/Akt influences posttranslational modification of Bax and inactivate a key component of the cell death machinery. PMID:19278624

  5. Regulation of Akt/PKB activity by P21-activated kinase in cardiomyocytes.

    PubMed

    Mao, Kai; Kobayashi, Satoru; Jaffer, Zahara M; Huang, Yuan; Volden, Paul; Chernoff, Jonathan; Liang, Qiangrong

    2008-02-01

    Akt/PKB is a critical regulator of cardiac function and morphology, and its activity is governed by dual phosphorylation at active loop (Thr308) by phosphoinositide-dependent protein kinase-1 (PDK1) and at carboxyl-terminal hydrophobic motif (Ser473) by a putative PDK2. P21-activated kinase-1 (Pak1) is a serine/threonine protein kinase implicated in the regulation of cardiac hypertrophy and contractility and was shown previously to activate Akt through an undefined mechanism. Here we report Pak1 as a potential PDK2 that is essential for Akt activity in cardiomyocytes. Both Pak1 and Akt can be activated by multiple hypertrophic stimuli or growth factors in a phosphatidylinositol-3-kinase (PI3K)-dependent manner. Pak1 overexpression induces Akt phosphorylation at both Ser473 and Thr308 in cardiomyocytes. Conversely, silencing or inactivating Pak1 gene diminishes Akt phosphorylation in vitro and in vivo. Purified Pak1 can directly phosphorylate Akt only at Ser473, suggesting that Pak1 may be a relevant PDK2 responsible for AKT Ser473 phosphorylation in cardiomyocytes. In addition, Pak1 protects cardiomyocytes from cell death, which is blocked by Akt inhibition. Our results connect two important regulators of cellular physiological functions and provide a potential mechanism for Pak1 signaling in cardiomyocytes. PMID:18054038

  6. Genomically amplified Akt3 activates DNA repair pathway and promotes glioma progression.

    PubMed

    Turner, Kristen M; Sun, Youting; Ji, Ping; Granberg, Kirsi J; Bernard, Brady; Hu, Limei; Cogdell, David E; Zhou, Xinhui; Yli-Harja, Olli; Nykter, Matti; Shmulevich, Ilya; Yung, W K Alfred; Fuller, Gregory N; Zhang, Wei

    2015-03-17

    Akt is a robust oncogene that plays key roles in the development and progression of many cancers, including glioma. We evaluated the differential propensities of the Akt isoforms toward progression in the well-characterized RCAS/Ntv-a mouse model of PDGFB-driven low grade glioma. A constitutively active myristoylated form of Akt1 did not induce high-grade glioma (HGG). In stark contrast, Akt2 and Akt3 showed strong progression potential with 78% and 97% of tumors diagnosed as HGG, respectively. We further revealed that significant variations in polarity and hydropathy values among the Akt isoforms in both the pleckstrin homology domain (P domain) and regulatory domain (R domain) were critical in mediating glioma progression. Gene expression profiles from representative Akt-derived tumors indicated dominant and distinct roles for Akt3, consisting primarily of DNA repair pathways. TCGA data from human GBM closely reflected the DNA repair function, as Akt3 was significantly correlated with a 76-gene signature DNA repair panel. Consistently, compared with Akt1 and Akt2 overexpression models, Akt3-expressing human GBM cells had enhanced activation of DNA repair proteins, leading to increased DNA repair and subsequent resistance to radiation and temozolomide. Given the wide range of Akt3-amplified cancers, Akt3 may represent a key resistance factor.

  7. Genomically amplified Akt3 activates DNA repair pathway and promotes glioma progression

    PubMed Central

    Turner, Kristen M.; Sun, Youting; Ji, Ping; Granberg, Kirsi J.; Bernard, Brady; Hu, Limei; Cogdell, David E.; Zhou, Xinhui; Yli-Harja, Olli; Nykter, Matti; Shmulevich, Ilya; Yung, W. K. Alfred; Fuller, Gregory N.; Zhang, Wei

    2015-01-01

    Akt is a robust oncogene that plays key roles in the development and progression of many cancers, including glioma. We evaluated the differential propensities of the Akt isoforms toward progression in the well-characterized RCAS/Ntv-a mouse model of PDGFB-driven low grade glioma. A constitutively active myristoylated form of Akt1 did not induce high-grade glioma (HGG). In stark contrast, Akt2 and Akt3 showed strong progression potential with 78% and 97% of tumors diagnosed as HGG, respectively. We further revealed that significant variations in polarity and hydropathy values among the Akt isoforms in both the pleckstrin homology domain (P domain) and regulatory domain (R domain) were critical in mediating glioma progression. Gene expression profiles from representative Akt-derived tumors indicated dominant and distinct roles for Akt3, consisting primarily of DNA repair pathways. TCGA data from human GBM closely reflected the DNA repair function, as Akt3 was significantly correlated with a 76-gene signature DNA repair panel. Consistently, compared with Akt1 and Akt2 overexpression models, Akt3-expressing human GBM cells had enhanced activation of DNA repair proteins, leading to increased DNA repair and subsequent resistance to radiation and temozolomide. Given the wide range of Akt3-amplified cancers, Akt3 may represent a key resistance factor. PMID:25737557

  8. PI3K/Akt is involved in brown adipogenesis mediated by growth differentiation factor-5 in association with activation of the Smad pathway

    SciTech Connect

    Hinoi, Eiichi; Iezaki, Takashi; Fujita, Hiroyuki; Watanabe, Takumi; Odaka, Yoshiaki; Ozaki, Kakeru; Yoneda, Yukio

    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 phosphorylation 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.

  9. Juglanthraquinone C Induces Intracellular ROS Increase and Apoptosis by Activating the Akt/Foxo Signal Pathway in HCC Cells

    PubMed Central

    2016-01-01

    Juglanthraquinone C (JC), a naturally occurring anthraquinone extracted from Juglans mandshurica, could induce apoptosis of cancer cells. This study aims to investigate the detailed cytotoxicity mechanism of JC in HepG2 and BEL-7402 cells. The Affymetrix HG-U133 Plus 2.0 arrays were first used to analyze the mRNA expression exposed to JC or DMSO in HepG2 cells. Consistent with the previous results, the data indicated that JC could induce apoptosis and hyperactivated Akt. The Western blot analysis further revealed that Akt, a well-known survival protein, was strongly activated in HepG2 and BEL-7402 cells. Furthermore, an obvious inhibitory effect on JC-induced apoptosis was observed when the Akt levels were decreased, while the overexpression of constitutively active mutant Akt greatly accelerated JC-induced apoptosis. The subsequent results suggested that JC treatment suppressed nuclear localization and increased phosphorylated levels of Foxo3a, and the overexpression of Foxo3a abrogated JC-induced apoptosis. Most importantly, the inactivation of Foxo3a induced by JC further led to an increase of intracellular ROS levels by suppressing ROS scavenging enzymes, and the antioxidant N-acetyl-L-cysteine and catalase successfully decreased JC-induced apoptosis. Collectively, this study demonstrated that JC induced the apoptosis of hepatocellular carcinoma (HCC) cells by activating Akt/Foxo signaling pathway and increasing intracellular ROS levels. PMID:26682007

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

    SciTech Connect

    Truong Le Xuan Nguyen; Choi, Joung Woo; Lee, Sang Bae; Ye, Keqiang; Woo, Soo-Dong; Lee, Kyung-Hoon; Ahn, Jee-Yin . E-mail: jyahn@med.skku.ac.kr

    2006-10-20

    Nerve growth factor (NGF) elicits Akt translocation into the nucleus, where it phosphorylates nuclear targets. Here, we describe that Akt phosphorylation can promote the nuclear translocation of Akt and is necessary for its nuclear retention. Overexpression of Akt-K179A, T308A, S473A-mutant failed to show either nuclear translocation or nuclear Akt phosphorylation, whereas expression of wild-type counterpart elicited profound Akt phosphorylation and induced nuclear translocation under NGF stimulation. Employing the PI3K inhibitor and a variety of mutants PI3K, we showed that nuclear translocation of Akt was mediated by activation of PI3K, and Akt phosphorylation status in the nucleus required PI3K activity. Thus the activity of PI3K might contribute to the nuclear translocation of Akt, and that Akt phosphorylation is essential for its nuclear retention under NGF stimulation conditions.

  11. 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

  12. Molecular pharmacology and antitumor activity of PHT-427 a novel AKT/PDPK1 pleckstrin homology domain inhibitor

    PubMed Central

    Meuillet, Emmanuelle J.; Zuohe, Song; Lemos, Robert; Ihle, Nathan; Kingston, John; Watkins, Ryan; Moses, Sylvestor A.; Zhang, Shuxing; Cuny, Lei Du; Herbst, Roy; Jacoby, Jörg J.; Zhou, Li Li; Ahad, Ali M.; Mash, Eugene A; Kirkpatrick, D Lynn; Powis, Garth

    2010-01-01

    Phosphatidylinositol 3-kinase (PIK3)/ PtdIns dependent protein kinase-1(PDPK1)/Akt signaling plays a critical role in activating proliferation and survival pathways within cancer cells. We report the molecular pharmacology and antitumor activity of PHT-427 a compound designed to bind to the pleckstrin homology (PH) binding domain of signaling molecules important in cancer. Although originally designed to bind the PH domain of Akt, we now report that PHT-427 also binds to the PH domain of PDPK1. A series of PHT-427 analogs with variable C-4 to C-16 alkyl chain length were synthesized and tested. PHT-427 itself (C-12 chain) bound with the highest affinity to the PH domains of both PDPK1 and Akt. PHT-427 inhibited Akt and PDKP1 signaling and their downstream targets in sensitive but not resistant cells and tumor xenografts. When given orally PHT-427 inhibited the growth of human tumor xenografts in immunodeficient mice with up to 80% inhibition in the most sensitive tumors and showed greater activity than analogs with C4, C6 or C8 alkyl chains. Inhibition of PDKP1 was more closely correlated to antitumor activity than Akt inhibition. Tumors with PIK3CA mutation were the most sensitive and K-Ras mutant tumors the least sensitive. Combination studies showed that PHT-427 has greater than additive antitumor activity with paclitaxel in breast cancer, and with erlotinib in NSC lung cancer. When given over 5 days PHT-427 caused no weight loss or change in blood chemistry. Thus, we report a novel PH domain binding inhibitor of PDPK1/Akt signaling with significant in vivo antitumor activity and minimal toxicity. PMID:20197390

  13. Cell-cycle-regulated activation of Akt kinase by phosphorylation at its carboxyl terminus

    PubMed Central

    Liu, Pengda; Begley, Michael; Michowski, Wojciech; Inuzuka, Hiroyuki; Ginzberg, Miriam; Gao, Daming; Tsou, Peiling; Gan, Wenjian; Papa, Antonella; Kim, Byeong Mo; Wan, Lixin; Singh, Amrik; Zhai, Bo; Yuan, Min; Wang, Zhiwei; Gygi, Steven P.; Lee, Tae Ho; Lu, Kun-Ping; Toker, Alex; Pandolfi, Pier Paolo; Asara, John M.; Kirschner, Marc W.; Sicinski, Piotr; Cantley, Lewis; Wei, Wenyi

    2014-01-01

    Akt, also known as protein kinase B, plays key roles in cell proliferation, survival and metabolism. Akt hyperactivation contributes to many pathophysiological conditions, including human cancers1–3, and is closely associated with poor prognosis and chemo- or radio-therapeutic resistance4. Phosphorylation of Akt at S473 (ref. 5) and T308 (ref. 6) activates Akt. However, it remains unclear whether further mechanisms account for full Akt activation, and whether Akt hyperactivation is linked to misregulated cell cycle progression, another cancer hallmark7. Here we report that Akt activity fluctuates across the cell cycle, mirroring cyclin A expression. Mechanistically, phosphorylation of S477 and T479 at the Akt extreme carboxy terminus by cyclin-dependent kinase 2 (Cdk2)/cyclin A or mTORC2, under distinct physiological conditions, promotes Akt activation through facilitating, or functionally compensating for, S473 phosphorylation. Furthermore, deletion of the cyclin A2 allele in the mouse olfactory bulb leads to reduced S477/T479 phosphorylation and elevated cellular apoptosis. Notably, cyclin A2-deletion-induced cellular apoptosis in mouse embryonic stem cells is partly rescued by S477D/T479E-Akt1, supporting a physiological role for cyclin A2 in governing Akt activation. Together, the results of our study show Akt S477/T479 phosphorylation to be an essential layer of the Akt activation mechanism to regulate its physiological functions, thereby providing a new mechanistic link between aberrant cell cycle progression and Akt hyperactivation in cancer. PMID:24670654

  14. Activating PTEN by COX-2 inhibitors antagonizes radiation-induced AKT activation contributing to radiosensitization

    SciTech Connect

    Meng, Zhen; Gan, Ye-Hua

    2015-05-01

    Radiotherapy is still one of the most effective nonsurgical treatments for many tumors. However, radioresistance remains a major impediment to radiotherapy. Although COX-2 inhibitors can induce radiosensitization, the underlying mechanism is not fully understood. In this study, we showed that COX-2 selective inhibitor celecoxib enhanced the radiation-induced inhibition of cell proliferation and apoptosis in HeLa and SACC-83 cells. Treatment with celecoxib alone dephosphorylated phosphatase and tensin homolog deleted on chromosome ten (PTEN), promoted PTEN membrane translocation or activation, and correspondingly dephosphorylated or inactivated protein kinase B (AKT). By contrast, treatment with radiation alone increased PTEN phosphorylation, inhibited PTEN membrane translocation and correspondingly activated AKT in the two cell lines. However, treatment with celecoxib or another COX-2 selective inhibitor (valdecoxib) completely blocked radiation-induced increase of PTEN phosphorylation, rescued radiation-induced decrease in PTEN membrane translocation, and correspondingly inactivated AKT. Moreover, celecoxib could also upregulate PTEN protein expression by downregulating Sp1 expression, thereby leading to the activation of PTEN transcription. Our results suggested that COX-2 inhibitors could enhance radiosensitization at least partially by activating PTEN to antagonize radiation-induced AKT activation. - Highlights: • COX-2 inhibitor, celecoxib, could enhance radiosensitization. • Radiation induced PTEN inactivation (phosphorylation) and AKT activation. • COX-2 inhibitor induced PTEN expression and activation, and inactivated AKT. • COX-2 inhibitor enhanced radiosensitization through activating PTEN.

  15. 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

  16. 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.

  17. PI3K/Akt promotes feedforward mTORC2 activation through IKKα

    PubMed Central

    Dan, Han C.; Antonia, Ricardo J.; Baldwin, Albert S.

    2016-01-01

    The ser-thr Akt plays a critical role in the regulation of cell survival, cell growth and proliferation, as well as energy metabolism and is dysregulated in many cancers. The regulation of Akt activity depends on the phosphorylation at two sites: (i) Thr308 in the activation loop by phosphoinositide-dependent kinase-1 (PDK1) and (ii) Ser473 hydrophobic motif at the carboxyl terminus by a second activity termed PDK2, which is the mTORC2 complex composed of mTOR, rictor, and Sin1. Previously we demonstrated that IKKα, a component of the IKK complex that controls NF-κB activation, participates in the Akt-dependent regulation of mTORC1. Here we have explored a potential involvement of IKKα in controlling Akt activity and whether this may involve mTORC2. The experiments show that IKKα associates with mTORC2 in several cancer cells in a manner dependent on PI3K/Akt activity and that IKKα positively promotes Akt phosphorylation at Ser473 and at Thr308. Moreover, IKKα enhances mTORC2 kinase activity directed to Akt on Ser473 and Akt-mediated phosphorylation of FOXO3a and GSK3β, but not other Akt-associated targets such as TSC2 and PRAS40, indicating the existence of multiple mechanisms of Akt activation in cells. In addition, loss of IKKα suppresses growth factor-induced Akt activation associated with mTORC1 inhibition. These results indicate that IKKα serves as a feedforward regulator of mTORC2 and that IKKα could serve as a key therapeutic target to block mTORC2 and Akt activation in some cancers. PMID:27027448

  18. PI3K/Akt promotes feedforward mTORC2 activation through IKKα.

    PubMed

    Dan, Han C; Antonia, Ricardo J; Baldwin, Albert S

    2016-04-19

    The ser-thr Akt plays a critical role in the regulation of cell survival, cell growth and proliferation, as well as energy metabolism and is dysregulated in many cancers. The regulation of Akt activity depends on the phosphorylation at two sites: (i) Thr308 in the activation loop by phosphoinositide-dependent kinase-1 (PDK1) and (ii) Ser473 hydrophobic motif at the carboxyl terminus by a second activity termed PDK2, which is the mTORC2 complex composed of mTOR, rictor, and Sin1. Previously we demonstrated that IKKα, a component of the IKK complex that controls NF-κB activation, participates in the Akt-dependent regulation of mTORC1. Here we have explored a potential involvement of IKKα in controlling Akt activity and whether this may involve mTORC2. The experiments show that IKKα associates with mTORC2 in several cancer cells in a manner dependent on PI3K/Akt activity and that IKKα positively promotes Akt phosphorylation at Ser473 and at Thr308. Moreover, IKKα enhances mTORC2 kinase activity directed to Akt on Ser473 and Akt-mediated phosphorylation of FOXO3a and GSK3β, but not other Akt-associated targets such as TSC2 and PRAS40, indicating the existence of multiple mechanisms of Akt activation in cells. In addition, loss of IKKα suppresses growth factor-induced Akt activation associated with mTORC1 inhibition. These results indicate that IKKα serves as a feedforward regulator of mTORC2 and that IKKα could serve as a key therapeutic target to block mTORC2 and Akt activation in some cancers. PMID:27027448

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

    PubMed Central

    Takenaka, Nobuyuki; Nihata, Yuma; Satoh, Takaya

    2016-01-01

    Insulin-stimulated glucose uptake in skeletal muscle is mediated by the glucose transporter GLUT4, which is translocated to the plasma membrane following insulin stimulation. Several lines of evidence suggested that the protein kinase Akt2 plays a key role in this insulin action. The small GTPase Rac1 has also been implicated as a regulator of insulin-stimulated GLUT4 translocation, acting downstream of Akt2. However, the mechanisms whereby Akt2 regulates Rac1 activity remain obscure. The guanine nucleotide exchange factor FLJ00068 has been identified as a direct regulator of Rac1 in Akt2-mediated signaling, but its characterization was performed mostly in cultured myoblasts. Here, we provide in vivo evidence that FLJ00068 indeed acts downstream of Akt2 as a Rac1 regulator by using mouse skeletal muscle. Small interfering RNA knockdown of FLJ00068 markedly diminished GLUT4 translocation to the sarcolemma following insulin administration or ectopic expression of a constitutively activated mutant of either phosphoinositide 3-kinase or Akt2. Additionally, insulin and these constitutively activated mutants caused the activation of Rac1 as shown by immunofluorescent microscopy using a polypeptide probe specific to activated Rac1 in isolated gastrocnemius muscle fibers and frozen sections of gastrocnemius muscle. This Rac1 activation was also abrogated by FLJ00068 knockdown. Furthermore, we observed translocation of FLJ00068 to the cell periphery following insulin stimulation in cultured myoblasts. Localization of FLJ00068 in the plasma membrane in insulin-stimulated, but not unstimulated, myoblasts and mouse gastrocnemius muscle was further affirmed by subcellular fractionation and subsequent immunoblotting. Collectively, these results strongly support a critical role of FLJ00068 in Akt2-mediated Rac1 activation in mouse skeletal muscle insulin signaling. PMID:27163697

  20. Phanerochaete mutants with enhanced ligninolytic activity

    SciTech Connect

    Kakar, S.N.; Perez, A.; Gonzales, J.

    1993-06-01

    In addition to lignin, the white rot fungus Phanerochaete chrysosporium has the ability to degrade a wide spectrum of recalcitrant organopollutants in soils and aqueous media. Although some of the organic compounds are degraded under nonligninolytic conditions, most are degraded under ligninolytic conditions with the involvement of the extracellular enzymes, lignin peroxidases, and manganese-dependent peroxidases, which are produced as secondary metabolites triggered by conditions of nutrient starvation (e.g., nitrogen limitation). The fungus and its enzymes can thus provide alternative technologies for bioremediation, biopulping, biobleaching, and other industrial applications. The efficiency and effectiveness of the fungus can be enhanced by increasing production and secretion of the important enzymes in large quantities and as primary metabolites under enriched conditions. One way this can be achieved is through isolation of mutants that are deregulated or are hyperproducers or supersecretors of key enzymes under enriched conditions. Through ultraviolet-light and gamma-rays mutagenesis we have isolated a variety of mutants, some of which produce key enzymes of the ligninolytic system under high-nitrogen growth conditions. One of the mutants produced 272 units (U) of lignin peroxidases enzyme activity per liter after nine days under high nitrogen. The mutant and the parent strains produced up to 54 U/L and 62 U/L, respectively, of the enzyme activity under low-nitrogen growth conditions during this period. In some experiments the mutant showed 281 U/L of enzyme activity under high nitrogen after 17 days.

  1. Inositol polyphosphate multikinase is a physiologic PI3-kinase that activates Akt/PKB.

    PubMed

    Maag, David; Maxwell, Micah J; Hardesty, Douglas A; Boucher, Katie L; Choudhari, Namrata; Hanno, Adam G; Ma, Jenny F; Snowman, Adele S; Pietropaoli, Joseph W; Xu, Risheng; Storm, Phillip B; Saiardi, Adolfo; Snyder, Solomon H; Resnick, Adam C

    2011-01-25

    The second messenger phosphatidylinositol (3,4,5)-trisphosphate (PIP(3)), formed by the p110 family of PI3-kinases, promotes cellular growth, proliferation, and survival, in large part by activating the protein kinase Akt/PKB. We show that inositol polyphosphate multikinase (IPMK) physiologically generates PIP(3) as well as water soluble inositol phosphates. IPMK deletion reduces growth factor-elicited Akt signaling and cell proliferation caused uniquely by loss of its PI3-kinase activity. Inhibition of p110 PI3-kinases by wortmannin prevents IPMK phosphorylation and activation. Thus, growth factor stimulation of Akt signaling involves PIP(3) generation through the sequential activations of the p110 PI3-kinases and IPMK. As inositol phosphates inhibit Akt signaling, IPMK appears to act as a molecular switch, inhibiting or stimulating Akt via its inositol phosphate kinase or PI3-kinase activities, respectively. Drugs regulating IPMK may have therapeutic relevance in influencing cell proliferation.

  2. Nitric Oxide Synthase and Breast Cancer: Role of TIMP-1 in NO-mediated Akt Activation

    PubMed Central

    Ridnour, Lisa A.; Barasch, Kimberly M.; Windhausen, Alisha N.; Dorsey, Tiffany H.; Lizardo, Michael M.; Yfantis, Harris G.; Lee, Dong H.; Switzer, Christopher H.; Cheng, Robert Y. S.; Heinecke, Julie L.; Brueggemann, Ernst; Hines, Harry B.; Khanna, Chand; Glynn, Sharon A.; Ambs, Stefan; Wink, David A.

    2012-01-01

    Prediction of therapeutic response and cancer patient survival can be improved by the identification of molecular markers including tumor Akt status. A direct correlation between NOS2 expression and elevated Akt phosphorylation status has been observed in breast tumors. Tissue inhibitor matrix metalloproteinase-1 (TIMP-1) has been proposed to exert oncogenic properties through CD63 cell surface receptor pathway initiation of pro-survival PI3k/Akt signaling. We employed immunohistochemistry to examine the influence of TIMP-1 on the functional relationship between NOS2 and phosphorylated Akt in breast tumors and found that NOS2-associated Akt phosphorylation was significantly increased in tumors expressing high TIMP-1, indicating that TIMP-1 may further enhance NO-induced Akt pathway activation. Moreover, TIMP-1 silencing by antisense technology blocked NO-induced PI3k/Akt/BAD phosphorylation in cultured MDA-MB-231 human breast cancer cells. TIMP-1 protein nitration and TIMP-1/CD63 co-immunoprecipitation was observed at NO concentrations that induced PI3k/Akt/BAD pro-survival signaling. In the survival analysis, elevated tumor TIMP-1 predicted poor patient survival. This association appears to be mainly restricted to tumors with high NOS2 protein. In contrast, TIMP-1 did not predict poor survival in patient tumors with low NOS2 expression. In summary, our findings suggest that tumors with high TIMP-1 and NOS2 behave more aggressively by mechanisms that favor Akt pathway activation. PMID:22957045

  3. Low-power laser irradiation inhibits Aβ25-35-induced cell apoptosis through Akt activation

    NASA Astrophysics Data System (ADS)

    Zhang, Zhigang; Tang, Yonghong

    2009-08-01

    Low-power laser irradiation (LPLI) can modulate various cellular processes such as proliferation, differentiation and apoptosis. Recently, LPLI has been applied to moderate Alzheimer's disease (AD), but the underlying mechanism remains unknown. The protective role of LPLI against the amyloid beta peptide (Aβ), a major constituent of AD plaques, has not been studied. PI3K/Akt pathway is extremely important in protecting cells from apoptosis caused by diverse stress stimuli. However, whether LPLI can inhibit Aβ-induced apoptosis through Akt activation is still unclear. In current study, using FRET (fluorescence resonance energy transfer) technique, we investigated the activity of Akt in response to LPLI treatment. B kinase activity reporter (BKAR), a recombinant FRET probe of Akt, was utilized to dynamically detect the activation of Akt after LPLI treatment. The results show that LPLI promoted the activation of Akt. Moreover, LPLI inhibits apoptosis induced by Aβ25-35 and the apoptosis inhibition can be abolished by wortmannin, a specific inhibitor of PI3K/Akt. Taken together, these results suggest that LPLI can inhibit Aβ25-35-induced cell apoptosis through Akt activation.

  4. T Cells Expressing Constitutively Active Akt Resist Multiple Tumor-associated Inhibitory Mechanisms

    PubMed Central

    Sun, Jiali; Dotti, Gianpietro; Huye, Leslie E; Foster, Aaron E; Savoldo, Barbara; Gramatges, Maria M; Spencer, David M; Rooney, Cliona M

    2010-01-01

    Adoptive transfer of antigen-specific cytotoxic T lymphocytes has shown promise for the therapy of cancer. However, tumor-specific T cells are susceptible to diverse inhibitory signals from the tumor microenvironment. The Akt/protein kinase B plays a central role in T-cell proliferation, function, and survival and we hypothesized that expression of constitutively active Akt (caAkt) in T cells could provide resistance to many of these tumor-associated inhibitory mechanisms. caAkt expression in activated human T cells increased proliferation and cytokine production, a likely result of their sustained expression of nuclear factor-κB (NF-κB) and provided resistance to apoptosis by upregulating antiapoptotic molecules. caAkt expressing T cells (caAkt-T-cells) were also relatively resistant to suppression by and conversion into regulatory T cells (Tregs). These characteristics provided a survival advantage to T cells cocultured with tumor cells in vitro; CD3/28-stimulated T cells expressing a chimeric antigen receptor (CAR) specific for disialoganglioside (GD2) that redirected their activity to the immunosuppressive, GD2-expressing neuroblastoma cell line, LAN-1, resisted tumor-induced apoptosis when co-expressing transgenic caAkt. In conclusion, caAkt-transduced T cells showed resistance to several evasion strategies employed by tumors and may therefore enhance the antitumor activity of adoptively transferred T lymphocytes. PMID:20842106

  5. PP2A inhibition results in hepatic insulin resistance despite Akt2 activation.

    PubMed

    Galbo, Thomas; Perry, Rachel J; Nishimura, Erica; Samuel, Varman T; Quistorff, Bjørn; Shulman, Gerald I

    2013-10-01

    In the liver, insulin suppresses hepatic gluconeogenesis by activating Akt, which inactivates the key gluconeogenic transcription factor FoxO1 (Forkhead Box O1). Recent studies have implicated hyperactivity of the Akt phosphatase Protein Phosphatase 2A (PP2A) and impaired Akt signaling as a molecular defect underlying insulin resistance. We therefore hypothesized that PP2A inhibition would enhance insulin-stimulated Akt activity and decrease glucose production. PP2A inhibitors increased hepatic Akt phosphorylation and inhibited FoxO1in vitro and in vivo, and suppressed gluconeogenesis in hepatocytes. Paradoxically, PP2A inhibition exacerbated insulin resistance in vivo. This was explained by phosphorylation of both hepatic glycogen synthase (GS) (inactivation) and phosphorylase (activation) resulting in impairment of glycogen storage. Our findings underline the significance of GS and Phosphorylase as hepatic PP2A substrates and importance of glycogen metabolism in acute plasma glucose regulation. PMID:24150286

  6. Lithium potentiates GSK-3β activity by inhibiting phosphoinositide 3-kinase-mediated Akt phosphorylation

    SciTech Connect

    Tian, Nie; Kanno, Takeshi; Jin, Yu; Nishizaki, Tomoyuki

    2014-07-18

    Highlights: • Lithium suppresses Akt activity by reducing PI3K-mediated Akt phosphorylation. • Lithium enhances GSK-3β activity by reducing Akt-mediated GSK-3β phosphorylation. • Lithium suppresses GSK-3β activity through its direct inhibition. - Abstract: Accumulating evidence has pointed to the direct inhibitory action of lithium, an anti-depressant, on GSK-3β. The present study investigated further insight into lithium signaling pathways. In the cell-free assay Li{sub 2}CO{sub 3} significantly inhibited phosphoinositide 3-kinase (PI3K)-mediated phosphorylation of Akt1 at Ser473, but Li{sub 2}CO{sub 3} did not affect PI3K-mediated PI(3,4,5)P{sub 3} production and 3-phosphoinositide-dependent protein kinase 1 (PDK1)-mediated phosphorylation of Akt1 at Thr308. This indicates that lithium could enhance GSK-3β activity by suppressing Akt-mediated Ser9 phosphorylation of GSK-3β in association with inhibition of PI3K-mediated Akt activation. There was no direct effect of Li{sub 2}CO{sub 3} on Akt1-induced phosphorylation of GSK-3β at Ser9, but otherwise Li{sub 2}CO{sub 3} significantly reduced GSK-3β-mediated phosphorylation of β-catenin at Ser33/37 and Thr41. This indicates that lithium directly inhibits GSK-3β in an Akt-independent manner. In rat hippocampal slices Li{sub 2}CO{sub 3} significantly inhibited phosphorylation of Akt1/2 at Ser473/474, GSK-3β at Ser9, and β-catenin at Ser33/37 and Thr41. Taken together, these results indicate that lithium exerts its potentiating and inhibiting bidirectional actions on GSK-3β activity.

  7. Akt regulates basic helix-loop-helix transcription factor-coactivator complex formation and activity during neuronal differentiation.

    PubMed

    Vojtek, Anne B; Taylor, Jennifer; DeRuiter, Stacy L; Yu, Jenn-Yah; Figueroa, Claudia; Kwok, Roland P S; Turner, David L

    2003-07-01

    Neural basic helix-loop-helix (bHLH) transcription factors regulate neurogenesis in vertebrates. Signaling by peptide growth factors also plays critical roles in regulating neuronal differentiation and survival. Many peptide growth factors activate phosphatidylinositol 3-kinase (PI3K) and subsequently the Akt kinases, raising the possibility that Akt may impact bHLH protein function during neurogenesis. Here we demonstrate that reducing expression of endogenous Akt1 and Akt2 by RNA interference (RNAi) reduces neuron generation in P19 cells transfected with a neural bHLH expression vector. The reduction in neuron generation from decreased Akt expression is not solely due to decreased cell survival, since addition of the caspase inhibitor z-VAD-FMK rescues cell death associated with loss of Akt function but does not restore neuron formation. This result indicates that Akt1 and Akt2 have additional functions during neuronal differentiation that are separable from neuronal survival. We show that activated Akt1 enhances complex formation between bHLH proteins and the transcriptional coactivator p300. Activated Akt1 also significantly augments the transcriptional activity of the bHLH protein neurogenin 3 in complex with the coactivators p300 or CBP. In addition, inhibition of endogenous Akt activity by the PI3K/Akt inhibitor LY294002 abolishes transcriptional cooperativity between the bHLH proteins and p300. We propose that Akt regulates the assembly and activity of bHLH-coactivator complexes to promote neuronal differentiation.

  8. Unscheduled Akt-Triggered Activation of Cyclin-Dependent Kinase 2 as a Key Effector Mechanism of Apoptin's Anticancer Toxicity▿

    PubMed Central

    Maddika, Subbareddy; Panigrahi, Soumya; Wiechec, Emilia; Wesselborg, Sebastian; Fischer, Ute; Schulze-Osthoff, Klaus; Los, Marek

    2009-01-01

    Apoptin, a protein from the chicken anemia virus, has attracted attention because it specifically kills tumor cells while leaving normal cells unharmed. The reason for this tumor selectivity is unclear and depends on subcellular localization, as apoptin resides in the cytoplasm of normal cells but in the nuclei of transformed cells. It was shown that nuclear localization and tumor-specific killing crucially require apoptin's phosphorylation by an as yet unknown kinase. Here we elucidate the pathway of apoptin-induced apoptosis and show that it essentially depends on abnormal phosphatidylinositol 3-kinase (PI3-kinase)/Akt activation, resulting in the activation of the cyclin-dependent kinase CDK2. Inhibitors as well as dominant-negative mutants of PI3-kinase and Akt not only inhibited CDK2 activation but also protected cells from apoptin-induced cell death. Akt activated CDK2 by direct phosphorylation as well as by the phosphorylation-induced degradation of the inhibitor p27Kip1. Importantly, we also identified CDK2 as the principal kinase that phosphorylates apoptin and is crucially required for apoptin-induced cell death. Immortalized CDK2-deficient fibroblasts and CDK2 knockdown cells were markedly protected against apoptin. Thus, our results not only decipher the pathway of apoptin-induced cell death but also provide mechanistic insights for the selective killing of tumor cells. PMID:19103742

  9. 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.

  10. Fangchinoline suppresses the growth and invasion of human glioblastoma cells by inhibiting the kinase activity of Akt and Akt-mediated signaling cascades.

    PubMed

    Guo, Bingyu; Xie, Peng; Su, Jingyuan; Zhang, Tingting; Li, Xiaoming; Liang, Guobiao

    2016-02-01

    Glioblastoma multiforme (GBM) is one of the most palindromic and malignant central nervous system neoplasms, and the current treatment is not effectual for GBM. Research of specific medicine for GBM is significant. Fangchinoline possesses a wide range of pharmacological activities and attracts more attentions due to its anti-tumor effects. In this study, two WHO grade IV human GBM cell lines (U87 MG and U118 MG) were exposed to fangchinoline, and we found that fangchinoline specifically inhibits the kinase activity of Akt and markedly suppresses the phosphorylation of Thr308 and Ser473 of Akt in human GBM cells. We also observed that fangchinoline inhibits tumor cell proliferation and invasiveness and induces apoptosis through suppressing the Akt-mediated signaling cascades, including Akt/p21, Akt/Bad, and Akt/matrix metalloproteinases (MMPs). These data demonstrated that fangchinoline exerts its anti-tumor effects in human glioblastoma cells, at least partly by inhibiting the kinase activity of Akt and suppressing Akt-mediated signaling cascades. PMID:26408176

  11. Pemetrexed Induces S-Phase Arrest and Apoptosis via a Deregulated Activation of Akt Signaling Pathway

    PubMed Central

    Chen, Kun-Chieh; Yang, Tsung-Ying; Wu, Chun-Chi; Cheng, Chi-Chih; Hsu, Shih-Lan; Hung, Hsiao-Wen

    2014-01-01

    Pemetrexed is approved for first-line and maintenance treatment of patients with advanced or metastatic non-small-cell lung cancer (NSCLC). The protein kinase Akt/protein kinase B is a well-known regulator of cell survival which is activated by pemetrexed, but its role in pemetrexed-mediated cell death and its molecular mechanisms are unclear. This study showed that stimulation with pemetrexed induced S-phase arrest and cell apoptosis and a parallel increase in sustained Akt phosphorylation and nuclear accumulation in the NSCLC A549 cell line. Inhibition of Akt expression by Akt specific siRNA blocked S-phase arrest and protected cells from apoptosis, indicating an unexpected proapoptotic role of Akt in the pemetrexed-mediated toxicity. Treatment of A549 cells with pharmacological inhibitors of phosphatidylinositol 3-kinase (PI3K), wortmannin and Ly294002, similarly inhibited pemetrexed-induced S-phase arrest and apoptosis and Akt phosphorylation, indicating that PI3K is an upstream mediator of Akt and is involved in pemetrexed-mediated cell death. Previously, we identified cyclin A-associated cyclin-dependent kinase 2 (Cdk2) as the principal kinase that was required for pemetrexed-induced S-phase arrest and apoptosis. The current study showed that inhibition of Akt function and expression by pharmacological inhibitors as well as Akt siRNA drastically inhibited cyclin A/Cdk2 activation. These pemetrexed-mediated biological and molecular events were also observed in a H1299 cell line. Overall, our results indicate that, in contrast to its normal prosurvival role, the activated Akt plays a proapoptotic role in pemetrexed-mediated S-phase arrest and cell death through a mechanism that involves Cdk2/cyclin A activation. PMID:24847863

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

    PubMed Central

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

    2015-01-01

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

  13. Activating PTEN by COX-2 inhibitors antagonizes radiation-induced AKT activation contributing to radiosensitization.

    PubMed

    Meng, Zhen; Gan, Ye-Hua

    2015-05-01

    Radiotherapy is still one of the most effective nonsurgical treatments for many tumors. However, radioresistance remains a major impediment to radiotherapy. Although COX-2 inhibitors can induce radiosensitization, the underlying mechanism is not fully understood. In this study, we showed that COX-2 selective inhibitor celecoxib enhanced the radiation-induced inhibition of cell proliferation and apoptosis in HeLa and SACC-83 cells. Treatment with celecoxib alone dephosphorylated phosphatase and tensin homolog deleted on chromosome ten (PTEN), promoted PTEN membrane translocation or activation, and correspondingly dephosphorylated or inactivated protein kinase B (AKT). By contrast, treatment with radiation alone increased PTEN phosphorylation, inhibited PTEN membrane translocation and correspondingly activated AKT in the two cell lines. However, treatment with celecoxib or another COX-2 selective inhibitor (valdecoxib) completely blocked radiation-induced increase of PTEN phosphorylation, rescued radiation-induced decrease in PTEN membrane translocation, and correspondingly inactivated AKT. Moreover, celecoxib could also upregulate PTEN protein expression by downregulating Sp1 expression, thereby leading to the activation of PTEN transcription. Our results suggested that COX-2 inhibitors could enhance radiosensitization at least partially by activating PTEN to antagonize radiation-induced AKT activation.

  14. Activation of Akt protects alveoli from neonatal oxygen-induced lung injury.

    PubMed

    Alphonse, Rajesh S; Vadivel, Arul; Coltan, Lavinia; Eaton, Farah; Barr, Amy J; Dyck, Jason R B; Thébaud, Bernard

    2011-02-01

    Bronchopulmonary dysplasia (BPD) is the main complication of extreme prematurity, resulting in part from mechanical ventilation and oxygen therapy. Currently, no specific treatment exists for BPD. BPD is characterized by an arrest in alveolar development and increased apoptosis of alveolar epithelial cells (AECs). Type 2 AECs are putative distal lung progenitor cells, capable of regenerating alveolar homeostasis after injury. We hypothesized that the protection of AEC2 death via the activation of the prosurvival Akt pathway prevents arrested alveolar development in experimental BPD. We show that the pharmacologic inhibition of the prosurvival factor Akt pathway with wortmannin during the critical period of alveolar development impairs alveolar development in newborn rats, resulting in larger and fewer alveoli, reminiscent of BPD. Conversely, in an experimental model of BPD induced by oxygen exposure of newborn rats, alveolar simplification is associated with a decreased activation of lung Akt. In vitro studies with rat lung epithelial (RLE) cells cultured in hyperoxia (95% O(2)) showed decreased apoptosis and improved cell survival after the forced expression of active Akt by adenovirus-mediated gene transfer. In vivo, adenovirus-mediated Akt gene transfer preserves alveolar architecture in the newborn rat model of hyperoxia-induced BPD. We conclude that inhibition of the prosurvival factor Akt disrupts normal lung development, whereas the expression of active Akt in experimental BPD preserves alveolar development. We speculate that the modulation of apoptosis may have therapeutic potential in lung diseases characterized by alveolar damage. PMID:20348209

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

    PubMed Central

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

    2015-01-01

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

  16. Leishmania promastigotes activate PI3K/Akt signalling to confer host cell resistance to apoptosis.

    PubMed

    Ruhland, Aaron; Leal, Nicole; Kima, Peter E

    2007-01-01

    Previous reports have shown that cells infected with promastigotes of some Leishmania species are resistant to the induction of apoptosis. This would suggest that either parasites elaborate factors that block signalling from apoptosis inducers or that parasites engage endogenous host signalling pathways that block apoptosis. To investigate the latter scenario, we determined whether Leishmania infection results in the activation of signalling pathways that have been shown to mediate resistance to apoptosis in other infection models. First, we showed that infection with the promastigote form of Leishmania major, Leishmania pifanoi and Leishmania amazonensis activates signalling through p38 mitogen-activated protein kinase (MAPK), NFkappaB and PI3K/Akt. Then we found that inhibition of signalling through the PI3K/Akt pathway with LY294002 and Akt IV inhibitor reversed resistance of infected bone marrow-derived macrophages and RAW 264.7 macrophages to potent inducers of apoptosis. Moreover, reduction of Akt levels with small interfering RNAs to Akt resulted in the inability of infected macrophages to resist apoptosis. Further evidence of the role of PI3K/Akt signalling in the promotion of cell survival by infected cells was obtained with the finding that Bad, which is a substrate of Akt, becomes phosphorylated during the course of infection. In contrast to the observations with PI3K/Akt signalling, inhibition of p38 MAPK signalling with SB202190 or NFkappaB signalling with wedelolactone had limited effect on parasite-induced resistance to apoptosis. We conclude that Leishmania promastigotes engage PI3K/Akt signalling, which confers to the infected cell, the capacity to resist death from activators of apoptosis.

  17. Activation of PI3-kinase stimulates endocytosis of ROMK via Akt1/SGK1-dependent phosphorylation of WNK1.

    PubMed

    Cheng, Chih-Jen; Huang, Chou-Long

    2011-03-01

    WNK kinases stimulate endocytosis of ROMK channels to regulate renal K+ handling. Phosphatidylinositol 3-kinase (PI3K)-activating hormones, such as insulin and IGF 1, phosphorylate WNK1, but how this affects the regulation of ROMK abundance is unknown. Here, serum starvation of ROMK-transfected HEK cells led to an increase of ROMK current density; subsequent addition of insulin or IGF1 inhibited ROMK currents in a PI3K-dependent manner. Serum and insulin also increased phosphorylation of the downstream kinases Akt1 and SGK1 as well as WNK1. A biotinylation assay suggested that insulin and IGF1 inhibit ROMK by enhancing its endocytosis, a process that WNK1 may mediate. Knockdown of WNK1 with siRNA or expression of a phospho-deficient WNK1 mutant (T58A) both prevented insulin-induced inhibition of ROMK currents, suggesting that phosphorylation at Threonine-58 of WNK1 is important to mediate the inhibition of ROMK by PI3K-activating hormones or growth factors. In vitro and in vivo kinase assays supported the notion that Akt1 and SGK1 can phosphorylate WNK1 at this site, and we established that Akt1 and SGK1 synergistically inhibit ROMK through WNK1. We used dominant-negative intersectin and dynamin constructs to show that SGK1-mediated phosphorylation of WNK1 inhibits ROMK by promoting its endocytosis. Taken together, these results suggest that PI3K-activating hormones inhibit ROMK by enhancing its endocytosis via a mechanism that involves phosphorylation of WNK1 by Akt1 and SGK1. PMID:21355052

  18. Regulation of endothelial cell cycle by laminar versus oscillatory flow: distinct modes of interactions of AMP-activated protein kinase and Akt pathways.

    PubMed

    Guo, Deliang; Chien, Shu; Shyy, John Y-J

    2007-03-01

    Steady laminar flow in the straight parts of the arterial tree is atheroprotective, whereas disturbed flow with oscillation in branch points and the aortic root are athero-prone, in part, because of the distinct roles of the flow patterns in regulating the cell cycle of vascular endothelial cells (ECs). To elucidate the molecular basis underlying the endothelial cell cycle regulated by distinct flow patterns, we conducted flow-channel experiments to investigate the effects of laminar versus oscillatory flows on activation of AMP-activated protein kinase (AMPK) and Akt in ECs. Laminar flow caused a transient activation of both AMPK and Akt, but oscillatory flow activated only Akt, with AMPK being maintained at its basal level. Constitutively active and dominant-negative mutants of AMPK and Akt were used to elucidate further the positive effect of Akt and negative role of AMPK in mediating mTOR (mammalian target of rapamycin) and its target p70S6 kinase (S6K) in response to laminar and oscillatory flows. Measurements of phosphorylation of mTOR Ser2448 and S6K Thr389 showed that AMPK, by counteracting Akt under laminar flow, resulted in a transient activation of S6K. Under oscillatory flow, because of the lack of AMPK activation to effect negative regulation, S6K was activated in a sustained manner. As a functional consequence, AMPK activation attenuated cell cycle progression in response to both laminar and oscillatory flows. In contrast, AMPK inhibition promoted EC cycle progression by decreasing the cell population in the G(0)/G(1) phase and increasing it in the S+G(2)/M phase. In vivo, phosphorylation of the promitotic S6K in mouse thoracic aorta was much less than that in mouse aortic root. In contrast, AMPK phosphorylation was higher in the thoracic aorta. These results provide a molecular mechanism by which laminar versus oscillatory flow regulates the endothelial cell cycle.

  19. Osteopontin induces {beta}-catenin signaling through activation of Akt in prostate cancer cells

    SciTech Connect

    Robertson, Brian W.; Chellaiah, Meenakshi A.

    2010-01-01

    Secretion of osteopontin (OPN) by cancer cells is a known mediator of tumorigenesis and cancer progression in both experimental and clinical studies. Our work demonstrates that OPN can activate Akt, an important step in cancer progression. Both ILK and PI3K are integral proteins in the OPN/Akt pathway, as inhibition of either kinase leads to a loss of OPN-mediated Akt activation. Subsequent to OPN-induced Akt activation, we observe inactivation of GSK-3{beta}, a regulator of {beta}-catenin. Osteopontin stimulation leads to an overall increase in {beta}-catenin protein levels with a resultant transfer of {beta}-catenin to the nucleus. Through the nuclear import of {beta}-catenin, OPN increases both the transcription and protein levels of MMP-7 and CD44, which are known TCF/LEF transcription targets. This work describes an important aspect of cancer progression induced by OPN.

  20. Dual fluorescent molecular substrates selectively report the activation, sustainability and reversibility of cellular PKB/Akt activity

    NASA Astrophysics Data System (ADS)

    Shen, Duanwen; Bai, Mingfeng; Tang, Rui; Xu, Baogang; Ju, Xiaoming; Pestell, Richard G.; Achilefu, Samuel

    2013-04-01

    Using a newly developed near-infrared (NIR) dye that fluoresces at two different wavelengths (dichromic fluorescence, DCF), we discovered a new fluorescent substrate for Akt, also known as protein kinase B, and a method to quantitatively report this enzyme's activity in real time. Upon insulin activation of cellular Akt, the enzyme multi-phosphorylated a single serine residue of a diserine DCF substrate in a time-dependent manner, culminating in monophospho- to triphospho-serine products. The NIR DCF probe was highly selective for the Akt1 isoform, which was demonstrated using Akt1 knockout cells derived from MMTV-ErbB2 transgenic mice. The DCF mechanism provides unparalleled potential to assess the stimulation, sustainability, and reversibility of Akt activation longitudinally. Importantly, NIR fluorescence provides a pathway to translate findings from cells to living organisms, a condition that could eventually facilitate the use of these probes in humans.

  1. TEIF associated centrosome activity is regulated by EGF/PI3K/Akt signaling.

    PubMed

    Zhao, Jing; Zou, Yongxin; Liu, Haijing; Wang, Huali; Zhang, Hong; Hou, Wei; Li, Xin; Jia, Xinying; Zhang, Jing; Hou, Lin; Zhang, Bo

    2014-09-01

    Centrosome amplification, which is a characteristic of cancer cells, has been understood as a driving force of genetic instability in the development of cancer. In previous work, we demonstrated that TEIF (transcriptional element-interacting factor) distributes in the centrosomes and regulates centrosome status under both physiologic and pathologic conditions. Here we identify TEIF as a downstream effector in EGF/PI3K/Akt signaling. The addition of EGF or transfection of active Akt stimulates centrosome TEIF distribution, resulting in an increase of centrosome splitting and amplification, while inhibitors of either PI3K or Akt attenuate these changes in TEIF and the associated centrosome status. A consensus motif for Akt phosphorylation (RHRVLT) proved to be involved in centrosomal TEIF localization, and the 469-threonine of this motif may be phosphorylated by Akt both in vitro and in vivo. Elimination of this phosphorylated site on TEIF caused reduced centrosome distribution and centrosome splitting or amplification. Moreover, TEIF closely co-localized with C-NAP1 at the proximal ends of centrioles, and centriolar loading of TEIF stimulated by EGF/Akt could displace C-NAP1, resulting in centrosome splitting. These findings reveal linkage of the EGF/PI3K/Akt signaling pathway to regulation of centrosome status which may act as an oncogenic pathway and induce genetic instability in carcinogenesis. PMID:24769208

  2. Truncating PREX2 mutations activate its GEF activity and alter gene expression regulation in NRAS-mutant melanoma

    PubMed Central

    Lissanu Deribe, Yonathan; Shi, Yanxia; Rai, Kunal; Nezi, Luigi; Amin, Samir B.; Wu, Chia-Chin; Akdemir, Kadir C.; Mahdavi, Mozhdeh; Peng, Qian; Chang, Qing Edward; Hornigold, Kirsti; Arold, Stefan T.; Welch, Heidi C. E.; Garraway, Levi A.; Chin, Lynda

    2016-01-01

    PREX2 (phosphatidylinositol-3,4,5-triphosphate-dependent Rac-exchange factor 2) is a PTEN (phosphatase and tensin homolog deleted on chromosome 10) binding protein that is significantly mutated in cutaneous melanoma and pancreatic ductal adenocarcinoma. Here, genetic and biochemical analyses were conducted to elucidate the nature and mechanistic basis of PREX2 mutation in melanoma development. By generating an inducible transgenic mouse model we showed an oncogenic role for a truncating PREX2 mutation (PREX2E824*) in vivo in the context of mutant NRAS. Using integrative cross-species gene expression analysis, we identified deregulated cell cycle and cytoskeleton organization as significantly perturbed biological pathways in PREX2 mutant tumors. Mechanistically, truncation of PREX2 activated its Rac1 guanine nucleotide exchange factor activity, abolished binding to PTEN and activated the PI3K (phosphatidyl inositol 3 kinase)/Akt signaling pathway. We further showed that PREX2 truncating mutations or PTEN deletion induces down-regulation of the tumor suppressor and cell cycle regulator CDKN1C (also known as p57KIP2). This down-regulation occurs, at least partially, through DNA hypomethylation of a differentially methylated region in chromosome 11 that is a known regulatory region for expression of the CDKN1C gene. Together, these findings identify PREX2 as a mediator of NRAS-mutant melanoma development that acts through the PI3K/PTEN/Akt pathway to regulate gene expression of a cell cycle regulator. PMID:26884185

  3. ets-2 Is a Target for an Akt (Protein Kinase B)/Jun N-Terminal Kinase Signaling Pathway in Macrophages of motheaten-viable Mutant Mice

    PubMed Central

    Smith, James L.; Schaffner, Alicia E.; Hofmeister, Joseph K.; Hartman, Matthew; Wei, Guo; Forsthoefel, David; Hume, David A.; Ostrowski, Michael C.

    2000-01-01

    The transcription factor ets-2 was phosphorylated at residue threonine 72 in a colony-stimulating factor 1 (CSF-1)- and mitogen-activated protein kinase-independent manner in macrophages isolated from motheaten-viable (me-v) mice. The CSF-1 and ets-2 target genes coding for Bcl-x, urokinase plasminogen activator, and scavenger receptor were also expressed at high levels independent of CSF-1 addition to me-v cells. Akt (protein kinase B) was constitutively active in me-v macrophages, and an Akt immunoprecipitate catalyzed phosphorylation of ets-2 at threonine 72. The p54 isoform of c-jun N-terminal kinase–stress-activated kinase (JNK- SAPK) coimmunoprecipitated with Akt from me-v macrophages, and treatment of me-v cells with the specific phosphatidylinositol 3-kinase inhibitor LY294002 decreased cell survival, Akt and JNK kinase activities, ets-2 phosphorylation, and Bcl-x mRNA expression. Therefore, ets-2 is a target for phosphatidylinositol 3-kinase–Akt–JNK action, and the JNK p54 isoform is an ets-2 kinase in macrophages. Constitutive ets-2 activity may contribute to the pathology of me-v mice by increasing expression of genes like the Bcl-x gene that promote macrophage survival. PMID:11027273

  4. Triptolide has anticancer and chemosensitization effects by down-regulating Akt activation through the MDM2/REST pathway in human breast cancer

    PubMed Central

    Xiong, Jing; Su, Tiefen; Qu, Zhiling; Yang, Qin; Wang, Yu; Li, Jiansha; Zhou, Sheng

    2016-01-01

    Triptolide has been shown to exhibit anticancer activity. However, its mechanism of action is not clearly defined. Herein we report a novel signaling pathway, MDM2/Akt, is involved in the anticancer mechanism of triptolide. We observed that triptolide inhibits MDM2 expression in human breast cancer cells with either wild-type or mutant p53. This MDM2 inhibition resulted in decreased Akt activation. More specifically, triptolide interfered with the interaction between MDM2 and the transcription factor REST to increase expression of the regulatory subunit of PI3-kinase p85 and consequently inhibit Akt activation. We further showed that, regardless of p53 status, triptolide inhibited proliferation, induced apoptosis, and caused G1 phase cell cycle arrest. Triptolide also enhanced the cytotoxic effect of doxorubicin. MDM2 inhibition plays a causative role in these effects. The inhibitory effect of triptolide on MDM2-mediated Akt activation was eliminated with MDM2 overexpression. MDM2-overexpressing tumor cells, in turn, were less susceptible to the anticancer and chemosensitization effects of triptolide than control cells. Triptolide also exhibited anticancer and chemosensitization effects in nude mouse xenograft model. When it was administered to tumor-bearing nude mice, triptolide inhibited tumor growth and enhanced the antitumor effects of doxorubicin. In summary, triptolide has anticancer and chemosensitization effects by down-regulating Akt activation through the MDM2/REST pathway in human breast cancer. Our study helps to elucidate the p53-independent regulatory function of MDM2 in Akt signaling, offering a novel view of the mechanism by which triptolide functions as an anticancer agent. PMID:27004407

  5. Akt-dependent NF-kappaB activation is required for bile acids to rescue colon cancer cells from stress-induced apoptosis.

    PubMed

    Shant, Jasleen; Cheng, Kunrong; Marasa, Bernard S; Wang, Jian-Ying; Raufman, Jean-Pierre

    2009-02-01

    Conjugated secondary bile acids promote human colon cancer cell proliferation by activating EGF receptors (EGFR). We hypothesized that bile acid-induced EGFR activation also mediates cell survival by downstream Akt-regulated activation of NF-kappaB. Deoxycholyltaurine (DCT) treatment attenuated TNF-alpha-induced colon cancer cell apoptosis, and stimulated rapid and sustained NF-kappaB nuclear translocation and transcriptional activity (detected by NF-kappaB binding to an oligonucleotide consensus sequence and by activation of luciferase reporter gene constructs). Both DCT-induced NF-kappaB nuclear translocation and attenuation of TNF-alpha-stimulated apoptosis were dependent on EGFR activation. Inhibitors of nuclear translocation, proteosome activity, and IkappaBalpha kinase attenuated NF-kappaB transcriptional activity. Cell transfection with adenoviral vectors encoding a non-degradable IkappaBalpha 'super-repressor' blocked the actions of DCT on both NF-kappaB activation and TNF-alpha-induced apoptosis. Likewise, transfection with mutant akt and treatment with a chemical inhibitor of Akt attenuated effects of DCT on NF-kappaB transcriptional activity and TNF-alpha-induced apoptosis. Chemical inhibitors of Akt and NF-kappaB activation also attenuated DCT-induced rescue of H508 cells from ultraviolet radiation-induced apoptosis. Collectively, these observations indicate that, downstream of EGFR, bile acid-induced colon cancer cell survival is mediated by Akt-dependent NF-kappaB activation. These findings provide a mechanism whereby bile acids increase resistance of colon cancer to chemotherapy and radiation.

  6. Phosphatidylinositol 3-Kinase Couples Localised Calcium Influx to Activation of Akt in Central Nerve Terminals.

    PubMed

    Nicholson-Fish, Jessica C; Cousin, Michael A; Smillie, Karen J

    2016-03-01

    The efficient retrieval of synaptic vesicle membrane and cargo in central nerve terminals is dependent on the efficient recruitment of a series of endocytosis modes by different patterns of neuronal activity. During intense neuronal activity the dominant endocytosis mode is activity-dependent endocytosis (ADBE). Triggering of ADBE is linked to calcineurin-mediated dynamin I dephosphorylation since the same stimulation intensities trigger both. Dynamin I dephosphorylation is maximised by a simultaneous inhibition of its kinase glycogen synthase kinase 3 (GSK3) by the protein kinase Akt, however it is unknown how increased neuronal activity is transduced into Akt activation. To address this question we determined how the activity-dependent increases in intracellular free calcium ([Ca(2+)]i) control activation of Akt. This was achieved using either trains of high frequency action potentials to evoke localised [Ca(2+)]i increases at active zones, or a calcium ionophore to raise [Ca(2+)]i uniformly across the nerve terminal. Through the use of either non-specific calcium channel antagonists or intracellular calcium chelators we found that Akt phosphorylation (and subsequent GSK3 phosphorylation) was dependent on localised [Ca(2+)]i increases at the active zone. In an attempt to determine mechanism, we antagonised either phosphatidylinositol 3-kinase (PI3K) or calmodulin. Activity-dependent phosphorylation of both Akt and GSK3 was arrested on inhibition of PI3K, but not calmodulin. Thus localised calcium influx in central nerve terminals activates PI3K via an unknown calcium sensor to trigger the activity-dependent phosphorylation of Akt and GSK3.

  7. Hypoxia activates Akt and induces phosphorylation of GSK-3 in PC12 cells.

    PubMed

    Beitner-Johnson, D; Rust, R T; Hsieh, T C; Millhorn, D E

    2001-01-01

    Akt is a serine/threonine kinase that has been shown to play a central role in promoting cell survival and opposing apoptosis. We evaluated the effect of hypoxia on Akt in rat pheochromocytoma (PC12) cells. PC12 cells were exposed to varying levels of hypoxia, including 21%, 15%, 10%, 5%, and 1% O(2). Hypoxia dramatically increased phosphorylation of Akt (Ser(473)). This effect peaked after 6 h exposure to hypoxia, but persisted strongly for up to 24 h. Phosphorylation of Akt was paralleled with a progressive increase in phosphorylation of glycogen synthase kinase-3 (GSK-3), one of its downstream substrates. The effect of hypoxia on phosphorylation of Akt was completely blocked by pretreatment of the cells with wortmannin (100 nM), indicating that this effect is mediated by phosphatidylinositol 3-kinase (P13K). In contrast, whereas hypoxia also strongly induced phosphorylation of the transcription factors CREB and EPAS1, these effects persisted in the presence of wortmannin. Thus, hypoxia regulates both P13K-dependent and P13K-independent signaling pathways. Furthermore, activation of the P13K and Akt signaling pathways may be one mechanism by which cells adapt and survive under conditions of hypoxia. PMID:11257444

  8. Antiapoptotic effects of erythropoietin in differentiated neuroblastoma SH-SY5Y cells require activation of both the STAT5 and AKT signaling pathways.

    PubMed

    Um, Moonkyoung; Lodish, Harvey F

    2006-03-01

    The hematopoietic cytokine erythropoietin (Epo) prevents neuronal death during ischemic events in the brain and in neurodegenerative diseases, presumably through its antiapoptotic effects. To explore the role of different signaling pathways in Epo-mediated antiapoptotic effects in differentiated human neuroblastoma SH-SY5Y cells, we employed a prolactin receptor (PrlR)/erythropoietin receptor (EpoR) chimera system, in which binding of prolactin (Prl) to the extracellular domain activates EpoR signaling in the cytosol. On induction of apoptosis by staurosporine, Prl supports survival of the SH-SY5Y cells expressing the wild-type PrlR/EpoR chimera. In these cells Prl treatment strongly activates the STAT5, AKT, and MAPK signaling pathways and induces weak activation of the p65 NF-kappaB factor. Selective mutation of the eight tyrosine residues of the EpoR cytoplasmic domain results in impaired or absent activation of either STAT5 (mutation of Tyr(343)) or AKT (mutation of Tyr(479)) or both (mutation of all eight tyrosine residues). Most interestingly, Prl treatment does not prevent apoptosis in cells expressing mutant PrlR/EpoR chimeras in which either the STAT5 or the AKT signaling pathways are not activated. In contrast, ERK 1/2 is fully activated by all mutant PrlR/EpoR chimeras, comparable with the level seen with the wild-type PrlR/EpoR chimera, implying that activation of the MAPK signaling pathway per se is not sufficient for antiapoptotic activity. Therefore, the antiapoptotic effects of Epo in neuronal cells require the combinatorial activation of multiple signaling pathways, including STAT5, AKT, and potentially MAPK as well, in a manner similar to that observed in hematopoietic cells.

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

    PubMed

    Wang, Kaijun; Jiang, Yiqian; Wang, Wei; Ma, Jian; Chen, Min

    2015-12-25

    Here we explored the anti-oxidative and cytoprotective potentials of escin, a natural triterpene-saponin, against hydrogen peroxide (H2O2) in retinal pigment epithelium (RPE) cells. We showed that escin remarkably attenuated H2O2-induced death and apoptosis of established (ARPE-19) and primary murine RPE cells. Meanwhile, ROS production and lipid peroxidation by H2O2 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 abolished escin-mediated anti-oxidant activity and RPE cytoprotection against H2O2. Reversely, escin was more potent against H2O2 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 H2O2 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. PMID:26505797

  10. Cell Type-Specific Activation of AKT and ERK Signaling Pathways by Small Negatively-Charged Magnetic Nanoparticles

    NASA Astrophysics Data System (ADS)

    Rauch, Jens; Kolch, Walter; Mahmoudi, Morteza

    2012-11-01

    The interaction of nanoparticles (NPs) with living organisms has become a focus of public and scientific debate due to their potential wide applications in biomedicine, but also because of unwanted side effects. Here, we show that superparamagnetic iron oxide NPs (SPIONs) with different surface coatings can differentially affect signal transduction pathways. Using isogenic pairs of breast and colon derived cell lines we found that the stimulation of ERK and AKT signaling pathways by SPIONs is selectively dependent on the cell type and SPION type. In general, cells with Ras mutations respond better than their non-mutant counterparts. Small negatively charged SPIONs (snSPIONs) activated ERK to a similar extent as epidermal growth factor (EGF), and used the same upstream signaling components including activation of the EGF receptor. Importantly, snSPIONs stimulated the proliferation of Ras transformed breast epithelial cells as efficiently as EGF suggesting that NPs can mimic physiological growth factors.

  11. Antitumor Effects of Fucoidan on Human Colon Cancer Cells via Activation of Akt Signaling

    PubMed Central

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

    2015-01-01

    We identified a novel Akt signaling mechanism that mediates fucoidan-induced suppression of human colon cancer cell (HT29) proliferation and anticancer effects. Fucoidan treatment significantly inhibited growth, induced G1-phase-associated upregulation of p21WAF1 expression, and suppressed cyclin and cyclin-dependent kinase expression in HT29 colon cancer cells. Additionally, fucoidan treatment activated the Akt signaling pathway, which was inhibited by treatment with an Akt inhibitor. The inhibition of Akt activation reversed the fucoidan-induced decrease in cell proliferation, the induction of G1-phase-associated p21WAF1 expression, and the reduction in cell cycle regulatory protein expression. Intraperitoneal injection of fucoidan reduced tumor volume; this enhanced antitumor efficacy was associated with induction of apoptosis and decreased angiogenesis. These data suggest that the activation of Akt signaling is involved in the growth inhibition of colon cancer cells treated with fucoidan. Thus, fucoidan may serve as a potential therapeutic agent for colon cancer. PMID:25995820

  12. Antitumor Effects of Fucoidan on Human Colon Cancer Cells via Activation of Akt Signaling.

    PubMed

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

    2015-05-01

    We identified a novel Akt signaling mechanism that mediates fucoidan-induced suppression of human colon cancer cell (HT29) proliferation and anticancer effects. Fucoidan treatment significantly inhibited growth, induced G1-phase-associated upregulation of p21WAF1 expression, and suppressed cyclin and cyclin-dependent kinase expression in HT29 colon cancer cells. Additionally, fucoidan treatment activated the Akt signaling pathway, which was inhibited by treatment with an Akt inhibitor. The inhibition of Akt activation reversed the fucoidan-induced decrease in cell proliferation, the induction of G1-phase-associated p21WAF1 expression, and the reduction in cell cycle regulatory protein expression. Intraperitoneal injection of fucoidan reduced tumor volume; this enhanced antitumor efficacy was associated with induction of apoptosis and decreased angiogenesis. These data suggest that the activation of Akt signaling is involved in the growth inhibition of colon cancer cells treated with fucoidan. Thus, fucoidan may serve as a potential therapeutic agent for colon cancer.

  13. Phosphatidylinositol 3-kinase/Akt signaling enhances nuclear localization and transcriptional activity of BRCA1

    SciTech Connect

    Hinton, Cimona V.; Fitzgerald, Latricia D.; Thompson, Marilyn E. . E-mail: methompson@mmc.edu

    2007-05-15

    Signaling pathways involved in regulating nuclear-cytoplasmic distribution of BRCA1 have not been previously reported. Here, we provide evidence that heregulin {beta}1-induced activation of the Akt pathway increases the nuclear content of BRCA1. First, treatment of T47D breast cancer cells with heregulin {beta}1 results in a two-fold increase in nuclear BRCA1 as assessed by FACS analysis, immunoblotting and immunofluorescence. This heregulin-induced increase in nuclear BRCA1 is blocked by siRNA-mediated down-regulation of Akt. Second, mutation of threonine 509 in BRCA1, the site of Akt phosphorylation, to an alanine, attenuates the ability of heregulin to induce BRCA1 nuclear accumulation. These data suggest that Akt-catalyzed phosphorylation of BRCA1 is required for the heregulin-regulated nuclear concentration of BRCA1. Because most functions ascribed to BRCA1 occur within the nucleus, we postulated that phosphorylation-dependent nuclear accumulation of BRCA1 would result in enhanced nuclear activity, specifically transcriptional activity, of BRCA1. This postulate is affirmed by our observation that the ability of BRCA1 to transactivate GADD45 promoter constructs was enhanced in T47D cells treated with heregulin {beta}1. Furthermore, the heterologous expression of BRCA1 in HCC1937 human breast cancer cells, which have constitutively active Akt, also induces GADD45 promoter activity, whereas the expression of BRCA1 in which threonine 509 has been mutated to an alanine is able to only minimally induce promoter activity. These findings implicate Akt in upstream events leading to BRCA1 nuclear localization and function.

  14. Inhibition of Rb Phosphorylation Leads to mTORC2-Mediated Activation of Akt.

    PubMed

    Zhang, Jinfang; Xu, Kai; Liu, Pengda; Geng, Yan; Wang, Bin; Gan, Wenjian; Guo, Jianping; Wu, Fei; Chin, Y Rebecca; Berrios, Christian; Lien, Evan C; Toker, Alex; DeCaprio, James A; Sicinski, Piotr; Wei, Wenyi

    2016-06-16

    The retinoblastoma (Rb) protein exerts its tumor suppressor function primarily by inhibiting the E2F family of transcription factors that govern cell-cycle progression. However, it remains largely elusive whether the hyper-phosphorylated, non-E2F1-interacting form of Rb has any physiological role. Here we report that hyper-phosphorylated Rb directly binds to and suppresses the function of mTORC2 but not mTORC1. Mechanistically, Rb, but not p107 or p130, interacts with Sin1 and blocks the access of Akt to mTORC2, leading to attenuated Akt activation and increased sensitivity to chemotherapeutic drugs. As such, inhibition of Rb phosphorylation by depleting cyclin D or using CDK4/6 inhibitors releases Rb-mediated mTORC2 suppression. This, in turn, leads to elevated Akt activation to confer resistance to chemotherapeutic drugs in Rb-proficient cells, which can be attenuated with Akt inhibitors. Therefore, our work provides a molecular basis for the synergistic usage of CDK4/6 and Akt inhibitors in treating Rb-proficient cancer. PMID:27237051

  15. VAMP-associated protein B (VAPB) promotes breast tumor growth by modulation of Akt activity.

    PubMed

    Rao, Meghana; Song, Wenqiang; Jiang, Aixiang; Shyr, Yu; Lev, Sima; Greenstein, David; Brantley-Sieders, Dana; Chen, Jin

    2012-01-01

    VAPB (VAMP- associated protein B) is an ER protein that regulates multiple biological functions. Although aberrant expression of VAPB is associated with breast cancer, its function in tumor cells is poorly understood. In this report, we provide evidence that VAPB regulates breast tumor cell proliferation and AKT activation. VAPB protein expression is elevated in primary and metastatic tumor specimens, and VAPB mRNA expression levels correlated negatively with patient survival in two large breast tumor datasets. Overexpression of VAPB in mammary epithelial cells increased cell growth, whereas VAPB knockdown in tumor cells inhibited cell proliferation in vitro and suppressed tumor growth in orthotopic mammary gland allografts. The growth regulation of mammary tumor cells controlled by VAPB appears to be mediated, at least in part, by modulation of AKT activity. Overexpression of VAPB in MCF10A-HER2 cells enhances phosphorylation of AKT. In contrast, knockdown of VAPB in MMTV-Neu tumor cells inhibited pAKT levels. Pharmacological inhibition of AKT significantly reduced three-dimensional spheroid growth induced by VAPB. Collectively, the genetic, functional and mechanistic analyses suggest a role of VAPB in tumor promotion in human breast cancer.

  16. VAMP-Associated Protein B (VAPB) Promotes Breast Tumor Growth by Modulation of Akt Activity

    PubMed Central

    Rao, Meghana; Song, Wenqiang; Jiang, Aixiang; Shyr, Yu; Lev, Sima; Greenstein, David; Brantley-Sieders, Dana; Chen, Jin

    2012-01-01

    VAPB (VAMP- associated protein B) is an ER protein that regulates multiple biological functions. Although aberrant expression of VAPB is associated with breast cancer, its function in tumor cells is poorly understood. In this report, we provide evidence that VAPB regulates breast tumor cell proliferation and AKT activation. VAPB protein expression is elevated in primary and metastatic tumor specimens, and VAPB mRNA expression levels correlated negatively with patient survival in two large breast tumor datasets. Overexpression of VAPB in mammary epithelial cells increased cell growth, whereas VAPB knockdown in tumor cells inhibited cell proliferation in vitro and suppressed tumor growth in orthotopic mammary gland allografts. The growth regulation of mammary tumor cells controlled by VAPB appears to be mediated, at least in part, by modulation of AKT activity. Overexpression of VAPB in MCF10A-HER2 cells enhances phosphorylation of AKT. In contrast, knockdown of VAPB in MMTV-Neu tumor cells inhibited pAKT levels. Pharmacological inhibition of AKT significantly reduced three-dimensional spheroid growth induced by VAPB. Collectively, the genetic, functional and mechanistic analyses suggest a role of VAPB in tumor promotion in human breast cancer. PMID:23049696

  17. Physiological activation of Akt by PHLPP1 deletion protects against pathological hypertrophy

    PubMed Central

    Moc, Courtney; Taylor, Amy E.; Chesini, Gino P.; Zambrano, Cristina M.; Barlow, Melissa S.; Zhang, Xiaoxue; Gustafsson, Åsa B.; Purcell, Nicole H.

    2015-01-01

    Aims To examine the role of physiological Akt signalling in pathological hypertrophy through analysis of PHLPP1 (PH domain leucine-rich repeat protein phosphatase) knock-out (KO) mice. Methods and results To investigate the in vivo requirement for ‘physiological’ control of Akt activation in cardiac growth, we examined the effect of deleting the Akt phosphatase, PHLPP, on the induction of cardiac hypertrophy. Basal Akt phosphorylation increased nearly two-fold in the cardiomyocytes from PHLPP1 KO mice and physiological hypertrophy induced by swimming exercise was accentuated as assessed by increased heart size and myocyte cell area. In contrast, the development of pathophysiological hypertrophy induced by pressure overload and assessed by increases in heart size, myocyte cell area, and hypertrophic gene expression was attenuated. This attenuation coincided with decreased fibrosis and cell death in the KO mice. Cast moulding revealed increased capillary density basally in the KO hearts, which was further elevated relative to wild-type mouse hearts in response to pressure overload. In vitro studies with isolated myocytes in co-culture also demonstrated that PHLPP1 deletion in cardiomyocytes can enhance endothelial tube formation. Expression of the pro-angiogenic factor VEGF was also elevated basally and accentuated in response to transverse aortic constriction in hearts from KO mice. Conclusion Our data suggest that enhancing Akt activity by inhibiting its PHLPP1-mediated dephosphorylation promotes processes associated with physiological hypertrophy that may be beneficial in attenuating the development of pathological hypertrophy. PMID:25411382

  18. Heat stress activates AKT via focal adhesion kinase-mediated pathway in neonatal rat ventricular myocytes.

    PubMed

    Wei, Hongguang; Vander Heide, Richard S

    2008-08-01

    Heat stress (HS)-induced cardioprotection is associated with increased paxillin localization to the membrane fraction of neonatal rat ventricular myocytes (NRVM). The purpose of this study was 1) to examine the subcellular signaling pathways activated by HS; 2) to determine whether myocardial stress organizes and activates an integrated survival pathway; and 3) to investigate potential downstream cytoprotective proteins activated by HS. After HS, NRVM were subjected to chemical inhibitors (CI) designed to simulate ischemia by inhibiting both glycolysis and mitochondrial respiration. Protein kinase B (AKT) expression (wild type) was increased selectively with an adenoviral vector. Cell signaling was analyzed with Western blot analysis, while oncosis/apoptosis was assayed by measuring Trypan blue exclusion and/or terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL) staining, respectively. HS increased phosphorylation of focal adhesion kinase (FAK) at tyrosine 397 but did not adversely affect the viability of NRVM before CI. HS increased association between FAK and phosphatidylinositol 3-kinase as well as causing a significant increase in AKT activity. Increased expression of wild-type AKT protected myocytes from both oncotic and apoptotic cell death. Increased expression of a FAK inhibitor, FRNK, reduced AKT phosphorylation in response to HS both at time 0 and after 10 min of CI compared with myocytes expressing empty virus. We conclude that myocardial stress activates cytoskeleton-based signaling pathways that are associated with protection from lethal cell injury.

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

    SciTech Connect

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

    2008-04-01

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

  20. Sustained activation of Akt elicits mitochondrial dysfunction to block Plasmodium falciparum infection in the mosquito host.

    PubMed

    Luckhart, Shirley; Giulivi, Cecilia; 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

    2013-02-01

    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

  1. 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

  2. Insulin inhibits inflammation and promotes atherosclerotic plaque stability via PI3K-Akt pathway activation.

    PubMed

    Yan, Hao; Ma, Ying; Li, Yan; Zheng, Xiaohui; Lv, Ping; Zhang, Yuan; Li, Jia; Ma, Meijuan; Zhang, Le; Li, Congye; Zhang, Rongqing; Gao, Feng; Wang, Haichang; Tao, Ling

    2016-02-01

    Toll-like receptor (TLR) 4 induced inflammation was reported to play an important role in atherosclerotic plaque stability. Recent studies indicated that insulin could inhibit inflammation by activating phosphatidylinositol 3-kinase-Akt-dependent (PI3K-Akt) signaling pathway. In the current study, we hypothesized that insulin would inhibit TLR4 induced inflammation via promoting PI3K-Akt activation, thus enhancing the stabilization of atherosclerotic plaques. In order to mimic the process of plaque formation, monocyte-macrophage lineage RAW264.7 were cultured and induced to form foam cells by oxidized LDL (ox-LDL). Oil red O staining results showed that insulin significantly restrained ox-LDL-induced foam cell formation. Analysis of inflammatory reaction during foam cell formation indicated that insulin significantly down-regulated the expression of tumor necrosis factor (TNF)-α, interleukin (IL)-6 levels, inhibited TLR4, myeloid differentiation primary response gene (MyD) 88 and nuclear factor (NF)-κB. Further mechanism analysis showed that pretreating with the PI3K blocker, wortmannin dramatically dampened the insulin-induced up-regulation of pAkt expression. Additionally, blockade of PI3K-Akt signaling also dampened the immunosuppression effect brought by insulin. Following the construction of a rodent atherosclerosis model, pretreatment of insulin resulted in an evident decrease in lipid deposition of the blood vessel wall, serum levels of TNF-α and IL-6, and numbers of infiltrated macrophages and foam cells. Taken together, these results suggested that insulin might inhibit inflammation and promote atherosclerotic plaque stability via the PI3K-Akt pathway by targeting TLR4-MyD88-NF-κB signaling. Our findings may provide a potential target for the prevention of cardiovascular disease. PMID:26681144

  3. Stem cell factor (SCF) protects osteoblasts from oxidative stress through activating c-Kit-Akt signaling

    SciTech Connect

    Yang, Lei; Wu, Zhong; Yin, Gang; Liu, Haifeng; Guan, Xiaojun; Zhao, Xiaoqiang; Wang, Jianguang; Zhu, Jianguo

    2014-12-12

    Highlights: • SCF receptor c-Kit is functionally expressed in primary and transformed osteoblasts. • SCF protects primary and transformed osteoblasts from H{sub 2}O{sub 2}. • SCF activation of c-Kit in osteoblasts, required for its cyto-protective effects. • c-Kit mediates SCF-induced Akt activation in cultured osteoblasts. • Akt activation is required for SCF-regulated cyto-protective effects in osteoblasts. - Abstract: Osteoblasts regulate bone formation and remodeling, and are main target cells of oxidative stress in the progression of osteonecrosis. The stem cell factor (SCF)-c-Kit pathway plays important roles in the proliferation, differentiation and survival in a range of cell types, but little is known about its functions in osteoblasts. In this study, we found that c-Kit is functionally expressed in both osteoblastic-like MC3T3-E1 cells and primary murine osteoblasts. Its ligand SCF exerted significant cyto-protective effects against hydrogen peroxide (H{sub 2}O{sub 2}). SCF activated its receptor c-Kit in osteoblasts, which was required for its cyto-protective effects against H{sub 2}O{sub 2}. Pharmacological inhibition (by Imatinib and Dasatinib) or shRNA-mediated knockdown of c-Kit thus inhibited SCF-mediated osteoblast protection. Further investigations showed that protection by SCF against H{sub 2}O{sub 2} was mediated via activation of c-Kit-dependent Akt pathway. Inhibition of Akt activation, through pharmacological or genetic means, suppressed SCF-mediated anti-H{sub 2}O{sub 2} activity in osteoblasts. In summary, we have identified a new SCF-c-Kit-Akt physiologic pathway that protects osteoblasts from H{sub 2}O{sub 2}-induced damages, and might minimize the risk of osteonecrosis caused by oxidative stress.

  4. Ribonuclease 5 facilitates corneal endothelial wound healing via activation of PI3-kinase/Akt pathway

    PubMed Central

    Kim, Kyoung Woo; Park, Soo Hyun; Lee, Soo Jin; Kim, Jae Chan

    2016-01-01

    To maintain corneal transparency, corneal endothelial cells (CECs) exert a pump function against aqueous inflow. However, human CECs are arrested in the G1-phase and non-proliferative in vivo. Thus, treatment of corneal endothelial decompensation is limited to corneal transplantation, and grafts are vulnerable to immune rejection. Here, we show that ribonuclease (RNase) 5 is more highly expressed in normal human CECs compared to decompensated tissues. Furthermore, RNase 5 up-regulated survival of CECs and accelerated corneal endothelial wound healing in an in vitro wound of human CECs and an in vivo cryo-damaged rabbit model. RNase 5 treatment rapidly induced accumulation of cytoplasmic RNase 5 into the nucleus, and activated PI3-kinase/Akt pathway in human CECs. Moreover, inhibition of nuclear translocation of RNase 5 using neomycin reversed RNase 5-induced Akt activation. As a potential strategy for proliferation enhancement, RNase 5 increased the population of 5-bromo-2′-deoxyuridine (BrdU)-incorporated proliferating CECs with concomitant PI3-kinase/Akt activation, especially in CECs deprived of contact-inhibition. Specifically, RNase 5 suppressed p27 and up-regulated cyclin D1, D3, and E by activating PI3-kinase/Akt in CECs to initiate cell cycle progression. Together, our data indicate that RNase 5 facilitates corneal endothelial wound healing, and identify RNase 5 as a novel target for therapeutic exploitation. PMID:27526633

  5. Molecular Characterization of Synovial Sarcoma in Children and Adolescents: Evidence of Akt Activation1

    PubMed Central

    Bozzi, Fabio; Ferrari, Andrea; Negri, Tiziana; Conca, Elena; Luca, Da Riva; Losa, Marco; Casieri, Paola; Orsenigo, Marta; Lampis, Andrea; Meazza, Cristina; Casanova, Michela; Pierotti, Marco A; Tamborini, Elena; Pilotti, Silvana

    2008-01-01

    Synovial sarcoma (SS) is the most frequent nonrhabdomyosarcomatous soft tissue sarcoma encountered in adolescents and young adults, and despite advances in the treatment of local disease, metastases remain the main cause of death. The aim of this study was to characterize a single-center series of pediatric SS molecularly to seek any biomarkers or pathways that might make suitable targets for new agents. Seventeen cases of pediatric SS showing the SYT-SSX fusion transcript were screened immunohistochemically, biochemically, molecularly, and cytogenetically (depending on the available material) to investigate any expression/activation of epidermal growth factor receptor, platelet-derived growth factor receptor alpha (PDGFRα), PDGFRβ, Akt, and deregulated Wnt pathway. The most relevant outcome was the finding of activated epidermal growth factor receptor, PDGFRα, and PDGFRβ, which activated Akt in both the monophasic and biphasic histologic subtypes. Consistently, Akt activation was completely abolished in an SS cell line assay when stimulated by PDGF-AA and treated with the phosphatidylinositol 3-kinase inhibitor LY294002. Our results also showed the nuclear localization of β-catenin and cyclin D1 gene products in monophasic SS and the movement of β-catenin into the cytoplasm in the glandular component of the biphasic subtype. Although they need to be confirmed in larger series, these preliminary data suggest that therapeutic strategies including specific inhibitors of the phosphatidylinositol 3-kinase/Akt pathway might be exploited in SS. PMID:18633459

  6. Activation of PI3K-Akt-GSK3{beta} pathway mediates hepatocyte growth factor inhibition of RANTES expression in renal tubular epithelial cells

    SciTech Connect

    Gong Rujun . E-mail: rgong@Brown.edu; Rifai, Abdalla; Dworkin, Lance D.

    2005-04-29

    Hepatocyte growth factor (HGF) was recently reported to ameliorate renal inflammation in a rat model of chronic renal failure. HGF exerted its action through suppression of RANTES expression in renal tubules. In the present study, we utilized an in vitro model of human kidney proximal tubule epithelial cells (HKC) to elucidate the mechanisms of RANTES suppression by HGF. HGF significantly suppressed basal and TNF-{alpha}-induced mRNA and protein expression of RANTES in a time and dose dependent fashion. HGF elicited PI3K-Akt activation and inhibited GSK3, a downstream transducer of PI3K-Akt, by inhibitory phosphorylation at Ser-9. When the PI3K-Akt pathway was blocked by wortmannin, HGF inhibition of RANTES was abrogated, demonstrating that the PI3K-Akt pathway is necessary for HGF action. In addition, specific inhibition of GSK3 activity by lithium ion suppressed basal and TNF-{alpha}-induced RANTES expression, reminiscent of the action of HGF. To further investigate the role of GSK3 in modulating RANTES expression, we examined the effect of forced expression of wild type GSK3{beta} or an uninhibitable mutant GSK3{beta}, in which the regulatory Ser-9 residue is changed to alanine (S9A-GSK3{beta}) in HKC. Overexpression of wild type GSK3{beta} did not alter the inhibitory action of HGF on RANTES. In contrast, expression of S9A-GSK3{beta} abolished HGF inhibition of basal and TNF-{alpha} stimulated RANTES expression. These findings suggest that PI3K-Akt activation and subsequent inhibitory phosphorylation of GSK3{beta} are required for HGF-induced suppression of RANTES in HKC.

  7. Pancreatic fibroblasts smoothen their activities via AKT-GLI2-TGFα.

    PubMed

    Rustgi, Anil K

    2016-09-01

    Pancreatic stromal fibroblasts provide structural support. Activated fibroblasts are critical in the tumor microenvironment. In this issue of Genes & Development, Liu and colleagues (pp. 1943-1955) unravel the finding that depletion of Smoothened (Smo) in pancreatic stromal fibroblasts results in AKT activation and noncanonical GLI2 activation with subsequent TGFα secretion, activation of EGFR in pancreatic epithelial cells, and augmentation of acinar-ductal metaplasia. Additionally, Smo-mediated signaling has proproliferative effects on pancreatic tumor cells. PMID:27664234

  8. PI3K/Akt activity has variable cell-specific effects on expression of HIF target genes, CA9 and VEGF, in human cancer cell lines.

    PubMed

    Shafee, Norazizah; Kaluz, Stefan; Ru, Ning; Stanbridge, Eric J

    2009-09-01

    The phosphatidylinositol 3-kinase/Akt (PI3K) pathway regulates hypoxia-inducible factor (HIF) activity. Higher expression of HIF-1alpha and carbonic anhydrase IX (CAIX), a hypoxia-inducible gene, in HT10806TG fibrosarcoma cells (mutant N-ras allele), compared to derivative MCH603 cells (deleted mutant N-ras allele), correlated with increased PI3K activity. Constitutive activation of the PI3K pathway in MCH603/PI3K(act) cells increased HIF-1alpha but, surprisingly, decreased CAIX levels. The cell-type specific inhibitory effect on CAIX was confirmed at the transcriptional level whereas epigenetic modifications of CA9 were ruled out. In summary, our data do not substantiate the generalization that PI3K upregulation leads to increased HIF activity. PMID:19342157

  9. PERK Utilizes Intrinsic Lipid Kinase Activity To Generate Phosphatidic Acid, Mediate Akt Activation, and Promote Adipocyte Differentiation

    PubMed Central

    Bobrovnikova-Marjon, Ekaterina; Pytel, Dariusz; Riese, Matthew J.; Vaites, Laura Pontano; Singh, Nickpreet; Koretzky, Gary A.; Witze, Eric S.

    2012-01-01

    The endoplasmic reticulum (ER) resident PKR-like kinase (PERK) is necessary for Akt activation in response to ER stress. We demonstrate that PERK harbors intrinsic lipid kinase, favoring diacylglycerol (DAG) as a substrate and generating phosphatidic acid (PA). This activity of PERK correlates with activation of mTOR and phosphorylation of Akt on Ser473. PERK lipid kinase activity is regulated in a phosphatidylinositol 3-kinase (PI3K) p85α-dependent manner. Moreover, PERK activity is essential during adipocyte differentiation. Because PA and Akt regulate many cellular functions, including cellular survival, proliferation, migratory responses, and metabolic adaptation, our findings suggest that PERK has a more extensive role in insulin signaling, insulin resistance, obesity, and tumorigenesis than previously thought. PMID:22493067

  10. PERK utilizes intrinsic lipid kinase activity to generate phosphatidic acid, mediate Akt activation, and promote adipocyte differentiation.

    PubMed

    Bobrovnikova-Marjon, Ekaterina; Pytel, Dariusz; Riese, Matthew J; Vaites, Laura Pontano; Singh, Nickpreet; Koretzky, Gary A; Witze, Eric S; Diehl, J Alan

    2012-06-01

    The endoplasmic reticulum (ER) resident PKR-like kinase (PERK) is necessary for Akt activation in response to ER stress. We demonstrate that PERK harbors intrinsic lipid kinase, favoring diacylglycerol (DAG) as a substrate and generating phosphatidic acid (PA). This activity of PERK correlates with activation of mTOR and phosphorylation of Akt on Ser473. PERK lipid kinase activity is regulated in a phosphatidylinositol 3-kinase (PI3K) p85α-dependent manner. Moreover, PERK activity is essential during adipocyte differentiation. Because PA and Akt regulate many cellular functions, including cellular survival, proliferation, migratory responses, and metabolic adaptation, our findings suggest that PERK has a more extensive role in insulin signaling, insulin resistance, obesity, and tumorigenesis than previously thought.

  11. Copper ions strongly activate the phosphoinositide-3-kinase/Akt pathway independent of the generation of reactive oxygen species.

    PubMed

    Ostrakhovitch, Elena A; Lordnejad, Mohammad Reza; Schliess, Freimut; Sies, Helmut; Klotz, Lars-Oliver

    2002-01-15

    Copper is implicated in metabolic disorders, such as Wilson's disease or Alzheimer's disease. Analysis of signaling pathways regulating cellular survival and function in response to a copper stress is crucial for understanding the pathogenesis of such diseases. Exposure of human skin fibroblasts or HeLa cells to Cu(2+) resulted in a dose- and time-dependent activation of the antiapoptotic kinase Akt/protein kinase B, starting at concentrations as low as 3 microM. Only Cu(II), but not Cu(I), had this effect. Activation of Akt was accompanied by phosphorylation of a downstream target of Akt, glycogen synthase kinase-3. Inhibitors of phosphoinositide-3-kinase (PI3K) completely blocked activation of Akt by Cu(2+), indicating a requirement of PI3K for Cu(2+)-induced activation of Akt. Indeed, cellular PI3K activity was strongly enhanced after exposure to Cu(2+). Copper ions may lead to the formation of reactive oxygen species, such as hydrogen peroxide. Activation of Akt by hydrogen peroxide or growth factors is known to proceed via the activation growth factor receptors. In line with this, pretreatment with inhibitors of growth factor receptor tyrosine kinases blocked activation of Akt by hydrogen peroxide and growth factors, as did a src-family tyrosine kinase inhibitor or the broad-spectrum tyrosine kinase inhibitor genistein. Activation of Akt by Cu(2+), however, remained unimpaired, implying (i) that tyrosine kinase activation is not involved in Cu(2+) activation of Akt and (ii) that activation of the PI3K/Akt pathway by Cu(2+) is initiated independently of that induced by reactive oxygen species. Comparison of the time course of the oxidation of 2',7'-dichlorodihydrofluorescein in copper-treated cells with that of Akt activation led to the conclusion that production of hydroperoxides cannot be an upstream event in copper-induced Akt activation. Rather, both activation of Akt and generation of ROS are proposed to occur in parallel, regulating cell survival after a

  12. Activation of Erk1/2 and Akt following unilateral ureteral obstruction.

    PubMed

    Rodríguez-Peña, Ana B; Grande, Maria T; Eleno, Nélida; Arévalo, Miguel; Guerrero, Carmen; Santos, Eugerio; López-Novoa, José M

    2008-07-01

    Chronic unilateral ureteral obstruction is a well characterized model of renal injury leading to tubulointerstitial fibrosis and distinct patterns of cell proliferation and apoptosis in the obstructed kidney. In this study we assessed the contribution of the mitogen activated protein kinase (MAPK)-ERK1/2 and the phosphatidylinositol 3 kinase (PI3K)-Akt pathways to early renal changes following unilateral obstruction. Increased activation of small Ras GTPase and its downstream effectors ERK1/2 and Akt was detected in ligated kidneys. The use of specific pharmacological inhibitors to either ERK1/2 or Akt activation led to decreased levels of fibroblast-myofibroblast markers in the interstitium while inhibition of PI3K reduced the number of proliferating cells and the amount of interstitial extracellular matrix deposition. Treatment with an ERK1/2 inhibitor diminished the number of apoptotic tubule and interstitial cells. Our results suggest a role for the MAPK-ERK1/2 and PI3K-Akt systems in early changes induced by ureteral obstruction and that inhibition of these signaling pathways may provide a novel approach to prevent progression of renal fibrosis.

  13. [Pigment composition and photosynthetic activity of pea chlorophyll mutants].

    PubMed

    Ladygin, V G

    2003-01-01

    Pea chlorophyll mutants chlorotica 2004 and 2014 have been studied. The mutants differ from the initial form (pea cultivar Torsdag) in stem and leaf color (light green in the mutant 2004 and yellow-green in the mutant 2014), relative chlorophyll content (approximately 80 and 50%, respectively), and the composition of carotenoids: the mutant 2004 contains a significantly smaller amount of carotene but accumulates more lutein and violaxanthine; in the mutant 2014, the contents of all carotenoids are decreased proportionally to the decrease in chlorophyll content. It is shown that the rates of CO2 assimilation and oxygen production in the mutant chlorotica 2004 and 2014 plants are reduced. The quantum efficiency of photosynthesis in the mutants is 29-30% lower than in the control plants; in their hybrids, however, it is 1.5-2 higher. It is proposed that both the greater role of dark respiration in gas exchange and the reduced photosynthetic activity in chlorotica mutants are responsible for the decreased phytomass increment in these plants. On the basis of these results, the conclusion is drawn that the mutations chlorotica 2004 and 2014 affect the genes controlling the formation and functioning of various components of the photosynthetic apparatus.

  14. Differential Phosphatidylinositol-3-Kinase-Akt-mTOR Activation by Semliki Forest and Chikungunya Viruses Is Dependent on nsP3 and Connected to Replication Complex Internalization

    PubMed Central

    Biasiotto, Roberta; Eng, Kai; Neuvonen, Maarit; Götte, Benjamin; Rheinemann, Lara; Mutso, Margit; Utt, Age; Varghese, Finny; Balistreri, Giuseppe; Merits, Andres; Ahola, Tero; McInerney, Gerald M.

    2015-01-01

    ABSTRACT Many viruses affect or exploit the phosphatidylinositol-3-kinase (PI3K)-Akt-mammalian target of rapamycin (mTOR) pathway, a crucial prosurvival signaling cascade. We report that this pathway was strongly activated in cells upon infection with the Old World alphavirus Semliki Forest virus (SFV), even under conditions of complete nutrient starvation. We mapped this activation to the hyperphosphorylated/acidic domain in the C-terminal tail of SFV nonstructural protein nsP3. Viruses with a deletion of this domain (SFV-Δ50) but not of other regions in nsP3 displayed a clearly delayed and reduced capacity of Akt stimulation. Ectopic expression of the nsP3 of SFV wild type (nsP3-wt), but not nsP3-Δ50, equipped with a membrane anchor was sufficient to activate Akt. We linked PI3K-Akt-mTOR stimulation to the intracellular dynamics of viral replication complexes, which are formed at the plasma membrane and subsequently internalized in a process blocked by the PI3K inhibitor wortmannin. Replication complex internalization was observed upon infection of cells with SFV-wt and SFV mutants with deletions in nsP3 but not with SFV-Δ50, where replication complexes were typically accumulated at the cell periphery. In cells infected with the closely related chikungunya virus (CHIKV), the PI3K-Akt-mTOR pathway was only moderately activated. Replication complexes of CHIKV were predominantly located at the cell periphery. Exchanging the hypervariable C-terminal tail of nsP3 between SFV and CHIKV induced the phenotype of strong PI3K-Akt-mTOR activation and replication complex internalization in CHIKV. In conclusion, infection with SFV but not CHIKV boosts PI3K-Akt-mTOR through the hyperphosphorylated/acidic domain of nsP3 to drive replication complex internalization. IMPORTANCE SFV and CHIKV are very similar in terms of molecular and cell biology, e.g., regarding replication and molecular interactions, but are strikingly different regarding pathology: CHIKV is a relevant human

  15. Analysis of p53 mutants for transcriptional activity.

    PubMed Central

    Raycroft, L; Schmidt, J R; Yoas, K; Hao, M M; Lozano, G

    1991-01-01

    The wild-type p53 protein functions to suppress transformation, but numerous mutant p53 proteins are transformation competent. To examine the role of p53 as a transcription factor, we made fusion proteins containing human or mouse p53 sequences fused to the DNA binding domain of a known transcription factor, GAL4. Human and mouse wild-type p53/GAL4 specifically transactivated expression of a chloramphenicol acetyltransferase reporter in HeLa, CHO, and NIH 3T3 cells. Several mutant p53 proteins, including a mouse p53 mutant which is temperature sensitive for suppression, were also analyzed. A p53/GAL4 fusion protein with this mutation was also transcriptionally active only at the permissive temperature. Another mutant p53/GAL4 fusion protein analyzed mimics the mutation inherited in Li-Fraumeni patients. This fusion protein was as active as wild-type p53/GAL4 in our assay. Two human p53 mutants that arose from alterations of the p53 gene in colorectal carcinomas were 30- to 40-fold less effective at activating transcription than wild-type p53/GAL4 fusion proteins. Thus, functional wild-type p53/GAL4 fusion proteins activate transcription, while several transformation competent mutants do so poorly or not at all. Only one mutant p53/GAL4 fusion protein remained transcriptionally active. Images PMID:1944276

  16. p62 modulates Akt activity via association with PKC{zeta} in neuronal survival and differentiation

    SciTech Connect

    Joung, Insil . E-mail: ijoung@hanseo.ac.kr; Kim, Hak Jae; Kwon, Yunhee Kim . E-mail: kimyh@khu.ac.kr

    2005-08-26

    p62 is a ubiquitously expressed phosphoprotein that interacts with a number of signaling molecules and a major component of neurofibrillary tangles in the brain of Alzheimer's disease patients. It has been implicated in important cellular functions such as cell proliferation and anti-apoptotic pathways. In this study, we have addressed the potential role of p62 during neuronal differentiation and survival using HiB5, a rat neuronal progenitor cell. We generated a recombinant adenovirus encoding T7-epitope tagged p62 to reliably transfer p62 cDNA into the neuronal cells. The results show that an overexpression of p62 led not only to neuronal differentiation, but also to decreased cell death induced by serum withdrawal in HiB5 cells. In this process p62-dependent Akt phosphorylation occurred via the release of Akt from PKC{zeta} by association of p62 and PKC{zeta}, which is known as a negative regulator of Akt activation. These findings indicate that p62 facilitates cell survival through novel signaling cascades that result in Akt activation. Furthermore, we found that p62 expression was induced during neuronal differentiation. Taken together, the data suggest p62 is a regulator of neuronal cell survival and differentiation.

  17. Black raspberry extracts inhibit benzo(a)pyrene diol-epoxide-induced activator protein 1 activation and VEGF transcription by targeting the phosphotidylinositol 3-kinase/Akt pathway.

    PubMed

    Huang, Chuanshu; Li, Jingxia; Song, Lun; Zhang, Dongyun; Tong, Qiangsong; Ding, Min; Bowman, Linda; Aziz, Robeena; Stoner, Gary D

    2006-01-01

    Previous studies have shown that freeze-dried black raspberry extract fractions inhibit benzo(a)pyrene [B(a)P]-induced transformation of Syrian hamster embryo cells and benzo(a)pyrene diol-epoxide [B(a)PDE]-induced activator protein-1 (AP-1) activity in mouse epidermal Cl 41 cells. The phosphotidylinositol 3-kinase (PI-3K)/Akt pathway is critical for B(a)PDE-induced AP-1 activation in mouse epidermal Cl 41 cells. In the present study, we determined the potential involvement of PI-3K and its downstream kinases on the inhibition of AP-1 activation by black raspberry fractions, RO-FOO3, RO-FOO4, RO-ME, and RO-DM. In addition, we investigated the effects of these fractions on the expression of the AP-1 target genes, vascular endothelial growth factor (VEGF) and inducible nitric oxide synthase (iNOS). Pretreatment of Cl 41 cells with fractions RO-F003 and RO-ME reduced activation of AP-1 and the expression of VEGF, but not iNOS. In contrast, fractions RO-F004 and RO-DM had no effect on AP-1 activation or the expression of either VEGF or iNOS. Consistent with inhibition of AP-1 activation, the RO-ME fraction markedly inhibited activation of PI-3K, Akt, and p70 S6 kinase (p70(S6k)). In addition, overexpression of the dominant negative PI-3K mutant delta p85 reduced the induction of VEGF by B(a)PDE. It is likely that the inhibitory effects of fractions RO-FOO3 and RO-ME on B(a)PDE-induced AP-1 activation and VEGF expression are mediated by inhibition of the PI-3K/Akt pathway. In view of the important roles of AP-1 and VEGF in tumor development, one mechanism for the chemopreventive activity of black raspberries may be inhibition of the PI-3K/Akt/AP-1/VEGF pathway.

  18. Black raspberry extracts inhibit benzo(a)pyrene diol-epoxide-induced activator protein 1 activation and VEGF transcription by targeting the phosphotidylinositol 3-kinase/Akt pathway.

    PubMed

    Huang, Chuanshu; Li, Jingxia; Song, Lun; Zhang, Dongyun; Tong, Qiangsong; Ding, Min; Bowman, Linda; Aziz, Robeena; Stoner, Gary D

    2006-01-01

    Previous studies have shown that freeze-dried black raspberry extract fractions inhibit benzo(a)pyrene [B(a)P]-induced transformation of Syrian hamster embryo cells and benzo(a)pyrene diol-epoxide [B(a)PDE]-induced activator protein-1 (AP-1) activity in mouse epidermal Cl 41 cells. The phosphotidylinositol 3-kinase (PI-3K)/Akt pathway is critical for B(a)PDE-induced AP-1 activation in mouse epidermal Cl 41 cells. In the present study, we determined the potential involvement of PI-3K and its downstream kinases on the inhibition of AP-1 activation by black raspberry fractions, RO-FOO3, RO-FOO4, RO-ME, and RO-DM. In addition, we investigated the effects of these fractions on the expression of the AP-1 target genes, vascular endothelial growth factor (VEGF) and inducible nitric oxide synthase (iNOS). Pretreatment of Cl 41 cells with fractions RO-F003 and RO-ME reduced activation of AP-1 and the expression of VEGF, but not iNOS. In contrast, fractions RO-F004 and RO-DM had no effect on AP-1 activation or the expression of either VEGF or iNOS. Consistent with inhibition of AP-1 activation, the RO-ME fraction markedly inhibited activation of PI-3K, Akt, and p70 S6 kinase (p70(S6k)). In addition, overexpression of the dominant negative PI-3K mutant delta p85 reduced the induction of VEGF by B(a)PDE. It is likely that the inhibitory effects of fractions RO-FOO3 and RO-ME on B(a)PDE-induced AP-1 activation and VEGF expression are mediated by inhibition of the PI-3K/Akt pathway. In view of the important roles of AP-1 and VEGF in tumor development, one mechanism for the chemopreventive activity of black raspberries may be inhibition of the PI-3K/Akt/AP-1/VEGF pathway. PMID:16397275

  19. The physical association of the P2Y12 receptor with PAR4 regulates arrestin-mediated Akt activation.

    PubMed

    Khan, Aasma; Li, Dongjun; Ibrahim, Salam; Smyth, Emer; Woulfe, Donna S

    2014-07-01

    It is now well accepted that protease activated receptor (PAR) 1 and PAR4 have differential roles in platelet activation. PAR4, a low-affinity thrombin receptor in human platelets, participates in sustained platelet activation in a P2Y12-dependent manner; however, the mechanisms are not defined. Our previous studies demonstrated that thrombin induces the association of PAR4 with P2Y12, together with arrestin recruitment to the complex. Here we show that PAR4 and P2Y12 directly interact to coregulate Akt signaling after PAR4 activation. We observed direct and specific interaction of P2Y12 with PAR4 but not PAR1 by bioluminescent resonance energy transfer when the receptors were coexpressed in human embryonic kidney 293T cells. PAR4-P2Y12 dimerization was promoted by PAR4-AP and inhibited by P2Y12 antagonist. By using sequence comparison of the transmembrane domains of PAR1 and PAR4, we designed a mutant form of PAR4, "PAR4SFT," by replacing LGL194-196 at the base of transmembrane domain 4 with the corresponding aligned PAR1 residues SFT 220-222. PAR4SFT supported only 8.74% of PAR4-P2Y12 interaction, abolishing P2Y12-dependent arrestin recruitment to PAR4 and Akt activation. Nonetheless, PAR4SFT still supported homodimerization with PAR4. PAR4SFT failed to induce a calcium flux when expressed independently; however, coexpression of increasing concentrations of PAR4SFT, together with PAR4 potentiated PAR4-mediated calcium flux, suggested that PAR4 act as homodimers to signal to Gq-coupled calcium responses. In conclusion, PAR4 LGL (194-196) governs agonist-dependent association of PAR4 with P2Y12 and contributes to Gq-coupled calcium responses. PAR4-P2Y12 association supports arrestin-mediated sustained signaling to Akt. Hence, PAR4-P2Y12 dimerization is likely to be important for the PAR4-P2Y12 dependent stabilization of platelet thrombi.

  20. The physical association of the P2Y12 receptor with PAR4 regulates arrestin-mediated Akt activation.

    PubMed

    Khan, Aasma; Li, Dongjun; Ibrahim, Salam; Smyth, Emer; Woulfe, Donna S

    2014-07-01

    It is now well accepted that protease activated receptor (PAR) 1 and PAR4 have differential roles in platelet activation. PAR4, a low-affinity thrombin receptor in human platelets, participates in sustained platelet activation in a P2Y12-dependent manner; however, the mechanisms are not defined. Our previous studies demonstrated that thrombin induces the association of PAR4 with P2Y12, together with arrestin recruitment to the complex. Here we show that PAR4 and P2Y12 directly interact to coregulate Akt signaling after PAR4 activation. We observed direct and specific interaction of P2Y12 with PAR4 but not PAR1 by bioluminescent resonance energy transfer when the receptors were coexpressed in human embryonic kidney 293T cells. PAR4-P2Y12 dimerization was promoted by PAR4-AP and inhibited by P2Y12 antagonist. By using sequence comparison of the transmembrane domains of PAR1 and PAR4, we designed a mutant form of PAR4, "PAR4SFT," by replacing LGL194-196 at the base of transmembrane domain 4 with the corresponding aligned PAR1 residues SFT 220-222. PAR4SFT supported only 8.74% of PAR4-P2Y12 interaction, abolishing P2Y12-dependent arrestin recruitment to PAR4 and Akt activation. Nonetheless, PAR4SFT still supported homodimerization with PAR4. PAR4SFT failed to induce a calcium flux when expressed independently; however, coexpression of increasing concentrations of PAR4SFT, together with PAR4 potentiated PAR4-mediated calcium flux, suggested that PAR4 act as homodimers to signal to Gq-coupled calcium responses. In conclusion, PAR4 LGL (194-196) governs agonist-dependent association of PAR4 with P2Y12 and contributes to Gq-coupled calcium responses. PAR4-P2Y12 association supports arrestin-mediated sustained signaling to Akt. Hence, PAR4-P2Y12 dimerization is likely to be important for the PAR4-P2Y12 dependent stabilization of platelet thrombi. PMID:24723492

  1. MDM2 restrains estrogen-mediated AKT activation by promoting TBK1-dependent HPIP degradation.

    PubMed

    Shostak, K; Patrascu, F; Göktuna, S I; Close, P; Borgs, L; Nguyen, L; Olivier, F; Rammal, A; Brinkhaus, H; Bentires-Alj, M; Marine, J-C; Chariot, A

    2014-05-01

    Restoration of p53 tumor suppressor function through inhibition of its interaction and/or enzymatic activity of its E3 ligase, MDM2, is a promising therapeutic approach to treat cancer. However, because the MDM2 targetome extends beyond p53, MDM2 inhibition may also cause unwanted activation of oncogenic pathways. Accordingly, we identified the microtubule-associated HPIP, a positive regulator of oncogenic AKT signaling, as a novel MDM2 substrate. MDM2-dependent HPIP degradation occurs in breast cancer cells on its phosphorylation by the estrogen-activated kinase TBK1. Importantly, decreasing Mdm2 gene dosage in mouse mammary epithelial cells potentiates estrogen-dependent AKT activation owing to HPIP stabilization. In addition, we identified HPIP as a novel p53 transcriptional target, and pharmacological inhibition of MDM2 causes p53-dependent increase in HPIP transcription and also prevents HPIP degradation by turning off TBK1 activity. Our data indicate that p53 reactivation through MDM2 inhibition may result in ectopic AKT oncogenic activity by maintaining HPIP protein levels. PMID:24488098

  2. NF-κB is activated in response to temozolomide in an AKT-dependent manner and confers protection against the growth suppressive effect of the drug

    PubMed Central

    2012-01-01

    Background Most DNA-damaging chemotherapeutic agents activate the transcription factor nuclear factor κB (NF-κB). However, NF-κB activation can either protect from or contribute to the growth suppressive effects of the agent. We previously showed that the DNA-methylating drug temozolomide (TMZ) activates AKT, a positive modulator of NF-κB, in a mismatch repair (MMR) system-dependent manner. Here we investigated whether NF-κB is activated by TMZ and whether AKT is involved in this molecular event. We also evaluated the functional consequence of inhibiting NF-κB on tumor cell response to TMZ. Methods AKT phosphorylation, NF-κB transcriptional activity, IκB-α degradation, NF-κB2/p52 generation, and RelA and NF-κB2/p52 nuclear translocation were investigated in TMZ-treated MMR-deficient (HCT116, 293TLα-) and/or MMR-proficient (HCT116/3-6, 293TLα+, M10) cells. AKT involvement in TMZ-induced activation of NF-κB was addressed in HCT116/3-6 and M10 cells transiently transfected with AKT1-targeting siRNA or using the isogenic MMR-proficient cell lines pUSE2 and KD12, expressing wild type or kinase-dead mutant AKT1. The effects of inhibiting NF-κB on sensitivity to TMZ were investigated in HCT116/3-6 and M10 cells using the NF-κB inhibitor NEMO-binding domain (NBD) peptide or an anti-RelA siRNA. Results TMZ enhanced NF-κB transcriptional activity, activated AKT, induced IκB-α degradation and RelA nuclear translocation in HCT116/3-6 and M10 but not in HCT116 cells. In M10 cells, TMZ promoted NF-κB2/p52 generation and nuclear translocation and enhanced the secretion of IL-8 and MCP-1. TMZ induced RelA nuclear translocation also in 293TLα+ but not in 293TLα- cells. AKT1 silencing inhibited TMZ-induced IκB-α degradation and NF-κB2/p52 generation. Up-regulation of NF-κB transcriptional activity and nuclear translocation of RelA and NF-κB2/p52 in response to TMZ were impaired in KD12 cells. RelA silencing in HCT116/3-6 and M10 cells increased TMZ

  3. Activation of PI3K/Akt pathway limits JNK-mediated apoptosis during EV71 infection.

    PubMed

    Zhang, Hua; Li, Fengqi; Pan, Ziye; Wu, Zhijun; Wang, Yanhong; Cui, Yudong

    2014-11-01

    Apoptosis is frequently induced to inhibit virus replication during infection of Enterovirus 71 (EV71). On the contrary, anti-apoptotic pathway, such as PI3K/Akt pathway, is simultaneously exploited by EV71 to accomplish the viral life cycle. The relationship by which EV71-induced apoptosis and PI3K/Akt signaling pathway remains to be elucidated. In this study, we demonstrated that EV71 infection altered Bax conformation and triggered its redistribution from the cytosol to mitochondria in RD cells. Subsequently, cytochrome c was released from mitochondria to cytosol. We also found that c-Jun NH2-terminal kinase (JNK) was activated during EV71 infection. The JNK specific inhibitor significantly inhibited Bax activation and cytochrome c release, suggesting that EV71-induced apoptosis was involved into a JNK-dependent manner. Meanwhile, EV71-induced Akt phosphorylation involved a PI3K-dependent mechanism. Inhibition of the PI3K/Akt pathway enhanced JNK phosphorylation and the JNK-mediated apoptosis upon EV71 infection. Moreover, PI3K/Akt pathway phosphorylated apoptosis signal-regulating kinase 1 (ASK1) and negatively regulated the ASK1 activity. Knockdown of ASK1 significantly decreased JNK phosphorylation, which implied that ASK1 phosphorylation by Akt inhibited ASK1-mediated JNK activation. Collectively, these data reveal that activation of the PI3K/Akt pathway limits JNK-mediated apoptosis by phosphorylating and inactivating ASK1 during EV71 infection.

  4. Campylobacter jejuni induces an anti-inflammatory response in human intestinal epithelial cells through activation of phosphatidylinositol 3-kinase/Akt pathway.

    PubMed

    Li, Yi-Ping; Vegge, Christina S; Brøndsted, Lone; Madsen, Mogens; Ingmer, Hanne; Bang, Dang Duong

    2011-02-24

    Campylobacter jejuni (C. jejuni) is the most common cause of human acute bacterial gastroenteritis. Poultry is a major reservoir of C. jejuni and considered an important source of human infections, thus, it is important to understand the host response to C. jejuni from chicken origin. In this study, we demonstrated firstly that a chicken isolate SC11 colonized chicks faster than clinical isolate NCTC11168. Using the SC11, we further studied the host responds to C. jejuni in terms of inflammatory response and involvement of cellular signaling pathways. Infection of C. jejuni SC11 was able to activate phosphatidylinositol 3-kinase (PI3K)/Akt pathway and induce pro-inflammatory interleukin-8 (IL-8) as well as anti-inflammatory cytokine IL-10 in human intestinal epithelial cell line Colo 205. The signalling pathways PI3K/Akt and mitogen-activated protein (MAP) kinases ERK and p38 were involved in C. jejuni-induced IL-8 and IL-10 expression. Inhibition of PI3K resulted in augmentation of C. jejuni-induced IL-8 production, concomitant with down-regulation of IL-10 mRNA, indicating an anti-inflammatory response was activated and associated with the activation of P13K/Akt. Similar effect was observed for cytolethal distending toxin (CDT) deficient mutants. Moreover, we demonstrated that heat-killed bacteria were able to induce IL-8 and IL-10 expression to a lower level than live bacteria. We therefore conclude that C. jejuni activate a PI3K/Akt-dependent anti-inflammatory pathway in human intestinal epithelial cells which may benefit the intracellular survival of C. jejuni during infection.

  5. Green Tea Polyphenol Epigallocatechin Gallate Reduces Endothelin-1 Expression and Secretion in Vascular Endothelial Cells: Roles for AMP-Activated Protein Kinase, Akt, and FOXO1

    PubMed Central

    Reiter, Chad E. N.; Kim, Jeong-a; Quon, Michael J.

    2010-01-01

    Epigallocatechin gallate (EGCG), a green tea polyphenol, promotes vasodilation by phosphatidylinositol 3-kinase-dependent activation of Akt and endothelial nitric oxide synthase to stimulate production of nitric oxide. Reduction in endothelin-1 (ET-1) synthesis may also increase bioavailability of nitric oxide. We hypothesized that the phosphatidylinositol 3-kinase-dependent transcription factor FOXO1 may mediate effects of EGCG to regulate expression of ET-1 in endothelial cells. EGCG treatment (10 μm, 8 h) of human aortic endothelial cells reduced expression of ET-1 mRNA, protein, and ET-1 secretion. We identified a putative FOXO binding domain in the human ET-1 promoter 51 bp upstream from the transcription start site. Trans-activation of a human ET-1 (hET-1) promoter luciferase reporter was enhanced by coexpression of a constitutively nuclear FOXO1 mutant, whereas expression of a mutant FOXO1 with disrupted DNA binding domain did not trans-activate the hET-1 promoter. Disrupting the hET-1 putative FOXO binding domain by site-directed mutagenesis ablated promoter activity in response to overexpression of wild-type FOXO1. EGCG stimulated time-dependent phosphorylation of Akt (S473), FOXO1 (at Akt phosphorylation site T24), and AMP-activated protein kinase α (AMPKα) (T172). EGCG-induced nuclear exclusion of FOXO1, FOXO1 binding to the hET-1 promoter, and reduction of ET-1 expression was partially inhibited by the AMPK inhibitor Compound C. Basal ET-1 protein expression was enhanced by short interfering RNA knock-down of Akt and reduced by short interfering RNA knock-down of FOXO1 or adenovirus-mediated expression of dominant-negative Foxo1. We conclude that EGCG decreases ET-1 expression and secretion from endothelial cells, in part, via Akt- and AMPK-stimulated FOXO1 regulation of the ET-1 promoter. These findings may be relevant to beneficial cardiovascular actions of green tea. PMID:19887561

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

    PubMed Central

    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. PMID:25105067

  7. Histone deacetylase inhibitor AR42 regulates telomerase activity in human glioma cells via an Akt-dependent mechanism.

    PubMed

    Yang, Ya-Luen; Huang, Po-Hsien; Chiu, Hao-Chieh; Kulp, Samuel K; Chen, Ching-Shih; Kuo, Cheng-Ju; Chen, Huan-Da; Chen, Chang-Shi

    2013-05-24

    Epigenetic regulation via abnormal activation of histone deacetylases (HDACs) is a mechanism that leads to cancer initiation and promotion. Activation of HDACs results in transcriptional upregulation of human telomerase reverse transcriptase (hTERT) and increases telomerase activity during cellular immortalization and tumorigenesis. However, the effects of HDAC inhibitors on the transcription of hTERT vary in different cancer cells. Here, we studied the effects of a novel HDAC inhibitor, AR42, on telomerase activity in a PTEN-null U87MG glioma cell line. AR42 increased hTERT mRNA in U87MG glioma cells, but suppressed total telomerase activity in a dose-dependent manner. Further analyses suggested that AR42 decreases the phosphorylation of hTERT via an Akt-dependent mechanism. Suppression of Akt phosphorylation and telomerase activity was also observed with PI3K inhibitor LY294002 further supporting the hypothesis that Akt signaling is involved in suppression of AR42-induced inhibition of telomerase activity. Finally, ectopic expression of a constitutive active form of Akt restored telomerase activity in AR42-treated cells. Taken together, our results demonstrate that the novel HDAC inhibitor AR42 can suppress telomerase activity by inhibiting Akt-mediated hTERT phosphorylation, indicating that the PI3K/Akt pathway plays an important role in the regulation of telomerase activity in response to this HDAC inhibitor.

  8. Labdane diterpenes protect against anoxia/reperfusion injury in cardiomyocytes: involvement of AKT activation

    PubMed Central

    Cuadrado, I; Fernández-Velasco, M; Boscá, L; de las Heras, B

    2011-01-01

    Several labdane diterpenes exert anti-inflammatory and cytoprotective actions; therefore, we have investigated whether these molecules protect cardiomyocytes in an anoxia/reperfusion (A/R) model, establishing the molecular mechanisms involved in the process. The cardioprotective activity of three diterpenes (T1, T2 and T3) was studied in the H9c2 cell line and in isolated rat cardiomyocyte subjected to A/R injury. In both cases, treatment with diterpenes T1 and T2 protected from A/R-induced apoptosis, as deduced by a decrease in the percentage of apoptotic and caspase-3 active positive cells, a decrease in the Bcl-2/Bax ratio and an increase in the expression of antiapoptotic proteins. Analysis of cell survival signaling pathways showed that diterpenes T1 and T2 added after A/R increased phospho-AKT and phospho-ERK 1/2 levels. These cardioprotective effects were lost when AKT activity was pharmacologically inhibited. Moreover, the labdane-induced cardioprotection involves activation of AMPK, suggesting a role for energy homeostasis in their mechanism of action. Labdane diterpenes (T1 and T2) also exerted cardioprotective effects against A/R-induced injury in isolated cardiomyocytes and the mechanisms involved activation of specific survival signals (PI3K/AKT pathways, ERK1/2 and AMPK) and inhibition of apoptosis. PMID:22071634

  9. Axl is essential for VEGF-A-dependent activation of PI3K/Akt

    PubMed Central

    Ruan, Guo-Xiang; Kazlauskas, Andrius

    2012-01-01

    Herein, we report that vascular endothelial growth factor A (VEGF-A) engages the PI3K/Akt pathway by a previously unknown mechanism that involves three tyrosine kinases. Upon VEGF-A-dependent activation of VEGF receptor-2 (VEGFR-2), and subsequent TSAd-mediated activation of Src family kinases (SFKs), SFKs engage the receptor tyrosine kinase Axl via its juxtamembrane domain to trigger ligand-independent autophosphorylation at a pair of YXXM motifs that promotes association with PI3K and activation of Akt. Other VEGF-A-mediated signalling pathways are independent of Axl. Interfering with Axl expression or function impairs VEGF-A- but not bFGF-dependent migration of endothelial cells. Similarly, Axl null mice respond poorly to VEGF-A-induced vascular permeability or angiogenesis, whereas other agonists induce a normal response. These results elucidate the mechanism by which VEGF-A activates PI3K/Akt, and identify previously unappreciated potential therapeutic targets of VEGF-A-driven processes. PMID:22327215

  10. AKT activation promotes PTEN hamartoma tumor syndrome-associated cataract development.

    PubMed

    Sellitto, Caterina; Li, Leping; Gao, Junyuan; Robinson, Michael L; Lin, Richard Z; Mathias, Richard T; White, Thomas W

    2013-12-01

    Mutations in the human phosphatase and tensin homolog (PTEN) gene cause PTEN hamartoma tumor syndrome (PHTS), which includes cataract development among its diverse clinical pathologies. Currently, it is not known whether cataract formation in PHTS patients is secondary to other systemic problems, or the result of the loss of a critical function of PTEN within the lens. We generated a mouse line with a lens-specific deletion of Pten (PTEN KO) and identified a regulatory function for PTEN in lens ion transport. Specific loss of PTEN in the lens resulted in cataract. PTEN KO lenses exhibited a progressive age-related increase in intracellular hydrostatic pressure, along with, increased intracellular sodium concentrations, and reduced Na+/K+-ATPase activity. Collectively, these defects lead to lens swelling, opacities and ultimately organ rupture. Activation of AKT was highly elevated in PTEN KO lenses compared to WT mice. Additionally, pharmacological inhibition of AKT restored normal Na+/K+-ATPase activity in primary cultured lens cells and reduced lens pressure in intact lenses from PTEN KO animals. These findings identify a direct role for PTEN in the regulation of lens ion transport through an AKT-dependent modulation of Na+/K+-ATPase activity, and provide a new animal model to investigate cataract development in PHTS patients. PMID:24270425

  11. Activation of Akt by lithium: pro-survival pathways in aging.

    PubMed

    Tajes, Marta; Yeste-Velasco, Marc; Zhu, Xiongwei; Chou, Steven P; Smith, Mark A; Pallàs, Mercè; Camins, Antoni; Casadesús, Gemma

    2009-04-01

    The effects of lithium on senescence were investigated using the senescence-accelerated mouse prone 8 (SAMP8) mice and cultures of aging cerebellar granule cells. Our in vitro findings, using cerebellar granule neurons, demonstrate that lithium (1-10mM) exerts neuroprotective effects in young cultures (7-8 days) against LY294002-induced Akt inhibition. Furthermore, lithium (10mM) inhibits GSK-3beta activity by upregulating p-GSK-3beta (ser-9) and increases p-FOXO1 (Ser256) suggesting an effective anti-apoptotic effect. Our data also showed that lithium in aged cultures exerts anti-apoptotic effects via Akt activation and consequent inhibition of downstream targets regulated by this enzyme. Finally, the administration of lithium to senescence-accelerated mice (SAMP8) and senescence-accelerated resistant mice (SAMR1) at 3 months of age also caused increased Akt activity and p-FoxO-1. These results demonstrate the effectiveness of lithium in preventing age-related neural loss and the potential therapeutic applications of lithium in treatment/prevention of neurological disease.

  12. Akt isoforms in vascular disease.

    PubMed

    Yu, Haixiang; Littlewood, Trevor; Bennett, Martin

    2015-08-01

    The mammalian serine/threonine Akt kinases comprise three closely related isoforms: Akt1, Akt2 and Akt3. Akt activation has been implicated in both normal and disease processes, including in development and metabolism, as well as cancer and cardiovascular disease. Although Akt signalling has been identified as a promising therapeutic target in cancer, its role in cardiovascular disease is less clear. Importantly, accumulating evidence suggests that the three Akt isoforms exhibit distinct tissue expression profiles, localise to different subcellular compartments, and have unique modes of activation. Consistent with in vitro findings, genetic studies in mice show distinct effects of individual Akt isoforms on the pathophysiology of cardiovascular disease. This review summarises recent studies of individual Akt isoforms in atherosclerosis, vascular remodelling and aneurysm formation, to provide a comprehensive overview of Akt function in vascular disease.

  13. Akt isoforms in vascular disease

    PubMed Central

    Yu, Haixiang; Littlewood, Trevor; Bennett, Martin

    2015-01-01

    The mammalian serine/threonine Akt kinases comprise three closely related isoforms: Akt1, Akt2 and Akt3. Akt activation has been implicated in both normal and disease processes, including in development and metabolism, as well as cancer and cardiovascular disease. Although Akt signalling has been identified as a promising therapeutic target in cancer, its role in cardiovascular disease is less clear. Importantly, accumulating evidence suggests that the three Akt isoforms exhibit distinct tissue expression profiles, localise to different subcellular compartments, and have unique modes of activation. Consistent with in vitro findings, genetic studies in mice show distinct effects of individual Akt isoforms on the pathophysiology of cardiovascular disease. This review summarises recent studies of individual Akt isoforms in atherosclerosis, vascular remodelling and aneurysm formation, to provide a comprehensive overview of Akt function in vascular disease. PMID:25929188

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

    PubMed Central

    Tsai, Jia-Shiuan; Chao, Cheng-Han; Lin, Lih-Yuan

    2016-01-01

    Cadmium is a known environmental carcinogen. Exposure of Cd leads to the activation of several proto-oncogenes in cells. We investigated here the mechanism of c-Myc expression in hepatic cells under Cd treatment. The c-Myc protein and mRNA levels increased in dose- and time-dependent manners in HepG2 cells with Cd treatment. This increase was due to an increase in c-Myc mRNA stability. To explore the mechanism involved in enhancing the mRNA stability, several cellular signaling factors that evoked by Cd treatment were analyzed. PI3K, p38, ERK and JNK were activated by Cd. However, ERK did not participate in the Cd-induced c-Myc expression. Further analysis revealed that mTORC2 was a downstream factor of p38. PI3K, JNK and mTORC2 coordinately activated Akt. Akt was phosphorylated at Thr450 in the untreated cells. Cd treatment led to additional phosphorylation at Thr308 and Ser473. Blocking any of the three signaling factors resulted in the reduction of phosphorylation level at all three Akt sites. The activated Akt phosphorylated Foxo1 and allowed the modified protein to translocate into the cytoplasm. We conclude that Cd-induced accumulation of c-Myc requires the activation of several signaling pathways. The signals act coordinately for Akt activation and drive the Foxo1 from the nucleus to the cytoplasm. Reduction of Foxo1 in the nucleus reduces the transcription of its target genes that may affect c-Myc mRNA stability, resulting in a higher accumulation of the c-Myc proteins. PMID:26751215

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

    SciTech Connect

    Yamano, Noriko; Kimura, Tohru; Watanabe-Kushima, Shoko; Shinohara, Takashi; Nakano, Toru

    2010-02-12

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

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

    PubMed

    Shen, Shang-Hang; Yu, Ning; Liu, Xi-Yao; Tan, Guo-Wei; Wang, Zhan-Xiang

    2016-03-18

    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 of 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. PMID:26828272

  17. Regulation of protein kinase B/Akt activity and Ser473 phosphorylation by protein kinase Calpha in endothelial cells.

    PubMed

    Partovian, Chohreh; Simons, Michael

    2004-08-01

    Protein kinase Balpha (PKBalpha/Akt-1) is a key mediator of multiple signaling pathways involved in angiogenesis, cell proliferation and apoptosis among others. The unphosphorylated form of Akt-1 is virtually inactive and its full activation requires two phosphatidylinositol-3,4,5-triphosphate-dependent phosphorylation events, Thr308 by 3-phosphoinositide-dependent kinase-1 (PDK1) and Ser473 by an undefined kinase that has been termed PDK2. Recent studies have suggested that the Ser473 kinase is a plasma membrane raft-associated kinase. In this study we show that protein kinase Calpha (PKCalpha) translocates to the membrane rafts in response to insulin growth factor-1 (IGF-1) stimulation. Overexpression of PKCalpha increases Ser473 phosphorylation and Akt-1 activity, while inhibition of its activity or expression decreases IGF-1-dependent activation of Akt-1. Furthermore, in vitro, in the presence of phospholipids and calcium, PKCalpha directly phosphorylates Akt-1 at the Ser473 site. We conclude, therefore, that PKCalpha regulates Akt-1 activity via Ser473 phosphorylation and may function as PDK2 in endothelial cells. PMID:15157674

  18. Synthesis and SAR study of modulators inhibiting tRXRα-dependent AKT activation

    PubMed Central

    Wang, Zhi-Gang; Chen, Liqun; Chen, Jiebo; Zheng, Jian-Feng; Gao, Weiwei; Zeng, Zhiping; Zhou, Hu; Zhang, Xiao-kun; Huang, Pei-Qiang; Su, Ying

    2013-01-01

    RXRα represents an intriguing and unique target for pharmacologic interventions. We recently showed that Sulindac and a designed analog could bind to RXRα and modulate its biological activity, including inhibition of the interaction of an N-terminally truncated RXRα (tRXRα) with the p85α regulatory subunit of phosphatidylinositol-3-OH kinase (PI3K). Here we report the synthesis, testing and SAR of a series of novel analogs of Sulindac as potential modulators for inhibiting tRXRα-dependent AKT activation. A new compound 30 was identified to have improved biological activity. PMID:23434637

  19. Over-expression of astrocytic ET-1 attenuates neuropathic pain by inhibition of ERK1/2 and Akt(s) via activation of ETA receptor.

    PubMed

    Hung, Victor K L; Tai, Lydia W; Qiu, Qiu; Luo, Xin; Wong, K L; Chung, Sookja K; Cheung, C W

    2014-05-01

    A differential role of endothelin-1 (ET-1) in pain processing has recently been suggested. However, the function of central ET-1 in neuropathic pain (NP) has not been fully elucidated to date. We report here the action of endogenous central ET-1 in sciatic nerve ligation-induced NP (SNL-NP) in a transgenic animal model that over-expresses ET-1 in the astrocytes (GET-1 mice). We hypothesized that the over-expression of astrocytic ET-1 would exert anti-allodynic and anti-hyperalgesic effects in NP, as demonstrated by mechanical threshold and plantar withdrawal latency using the von Frey filament and heat stimuli. In our animal model, GET-1 mice showed an increase in the withdrawal threshold and latency in response to the mechanical and thermal stimuli, respectively, in pain behavior tests after SNL. ET-1 and endothelin type A receptor (ETA-R) levels were increased significantly in L4-L6 segments of the spinal cord (ipsilateral to SNL) of GET-1 mice at 7 and 21days after surgery. Moreover, intrathecal administration of a specific ETA-R antagonist, BQ-123, attenuated the anti-allodynic and anti-hyperalgesic phenotype in GET-1 mice. The effects of BQ-123 on the mRNA expression of extracellular signal-regulated protein kinase 1/2 (ERK1/2) and protein kinase B/serine protein kinase (Akt(s)) were assessed in the ipsilateral L4-L6 segments harvested 30min after BQ-123 administration on day 7 after surgery. Phosphorylation of ERK1/2 and Akt(s) in the ipsilateral spinal cord of GET-1 mice was reduced following SNL, whereas no reduction was observed after intrathecal injection of BQ-123. In conclusion, our results showed that the xover-expression of astrocytic ET-1 reduced SNL-induced allodynia and hyperalgesia by inhibiting the activation of ERK1/2 and Akt(s) via the ETA-R-mediated pathway.

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

    PubMed

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

    2011-01-01

    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-trisphosphate (PIP(3)) 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 PIP(3) 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 the 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.

  1. Co-activation of AKT and c-Met triggers rapid hepatocellular carcinoma development via the mTORC1/FASN pathway in mice

    PubMed Central

    Hu, Junjie; Che, Li; Li, Lei; Pilo, Maria G.; Cigliano, Antonio; Ribback, Silvia; Li, Xiaolei; Latte, Gavinella; Mela, Marta; Evert, Matthias; Dombrowski, Frank; Zheng, Guohua; Chen, Xin; Calvisi, Diego F.

    2016-01-01

    Activation of the AKT/mTOR cascade and overexpression of c-Met have been implicated in the development of human hepatocellular carcinoma (HCC). To elucidate the functional crosstalk between the two pathways, we generated a model characterized by the combined expression of activated AKT and c-Met in the mouse liver. Co-expression of AKT and c-Met triggered rapid liver tumor development and mice required to be euthanized within 8 weeks after hydrodynamic injection. At the molecular level, liver tumors induced by AKT/c-Met display activation of AKT/mTOR and Ras/MAPK cascades as well as increased lipogenesis and glycolysis. Since a remarkable lipogenic phenotype characterizes liver lesions from AKT/c-Met mice, we determined the requirement of lipogenesis in AKT/c-Met driven hepatocarcinogenesis using conditional Fatty Acid Synthase (FASN) knockout mice. Of note, hepatocarcinogenesis induced by AKT/c-Met was fully inhibited by FASN ablation. In human HCC samples, coordinated expression of FASN, activated AKT, and c-Met proteins was detected in a subgroup of biologically aggressive tumors. Altogether, our study demonstrates that co-activation of AKT and c-Met induces HCC development that depends on the mTORC1/FASN pathway. Suppression of mTORC1 and/or FASN might be highly detrimental for the growth of human HCC subsets characterized by concomitant induction of the AKT and c-Met cascades. PMID:26857837

  2. Biochemical and Cellular Evidence Demonstrating AKT-1 as a Binding Partner for Resveratrol Targeting Protein NQO2

    PubMed Central

    Hsieh, Tze-chen; Lin, Chia-Yi; Bennett, Dylan John; Wu, Erxi; Wu, Joseph M.

    2014-01-01

    Background AKT plays an important role in the control of cell proliferation and survival. Aberrant activation of AKT frequently occurs in human cancers making it an attractive drug targets and leading to the synthesis of numerous AKT inhibitors as therapeutic candidates. Less is known regarding proteins that control AKT. We recently reported that quinone reductase 2 (NQO2) inhibited AKT activity, by unknown mechanisms. Methodology/Principal Findings In this study, molecular modeling was used to query interaction between NQO2 and AKT. We found that pleckstrin homology (PH) and kinase domains of AKT bind to chains A and B of NQO2. Pull-down and deletion assays revealed that PH domain of AKT is essential for interaction with NQO2. Modeling analysis further revealed that kinase domain of AKT binds NQO2 in the vicinity of asparagine 161 located in the resveratrol-binding domain of NQO2. In studies to test whether exposure to resveratrol potentiates or diminishes AKT binding to NQO2, we showed that pre-binding by resveratrol in wild type but not histidine-161 (N161H) mutant NQO2 significantly affected this interaction. To obtain information on interplay between resveratrol and AKT, resveratrol affinity chromatography was performed. AKT binds with high affinity to the column suggesting that it is a target of resveratrol. The half-life of AKT mRNA decreased from ∼4 h in control cells to ∼1 h in NQO2-knockdown cells. The inhibition of AKT by resveratrol was attenuated in NQO2-expressing relative to NQO2-knockdown cells. Conclusion/Significance Both NQO2 and AKT are targets of resveratrol; NQO2:AKT interaction is a novel physiological regulator of AKT activation/function. PMID:24968355

  3. The Timing of Raf/ERK and AKT Activation in Protecting PC12 Cells against Oxidative Stress.

    PubMed

    Ong, Qunxiang; Guo, Shunling; Duan, Liting; Zhang, Kai; Collier, Eleanor Ann; Cui, Bianxiao

    2016-01-01

    Acute brain injuries such as ischemic stroke or traumatic brain injury often cause massive neural death and irreversible brain damage with grave consequences. Previous studies have established that a key participant in the events leading to neural death is the excessive production of reactive oxygen species. Protecting neuronal cells by activating their endogenous defense mechanisms is an attractive treatment strategy for acute brain injuries. In this work, we investigate how the precise timing of the Raf/ERK and the AKT pathway activation affects their protective effects against oxidative stress. For this purpose, we employed optogenetic systems that use light to precisely and reversibly activate either the Raf/ERK or the AKT pathway. We find that preconditioning activation of the Raf/ERK or the AKT pathway immediately before oxidant exposure provides significant protection to cells. Notably, a 15-minute transient activation of the Raf/ERK pathway is able to protect PC12 cells against oxidant strike that is applied 12 hours later, while the transient activation of the AKT pathway fails to protect PC12 cells in such a scenario. On the other hand, if the pathways are activated after the oxidative insult, i.e. postconditioning, the AKT pathway conveys greater protective effect than the Raf/ERK pathway. We find that postconditioning AKT activation has an optimal delay period of 2 hours. When the AKT pathway is activated 30min after the oxidative insult, it exhibits very little protective effect. Therefore, the precise timing of the pathway activation is crucial in determining its protective effect against oxidative injury. The optogenetic platform, with its precise temporal control and its ability to activate specific pathways, is ideal for the mechanistic dissection of intracellular pathways in protection against oxidative stress. PMID:27082641

  4. The Timing of Raf/ERK and AKT Activation in Protecting PC12 Cells against Oxidative Stress

    PubMed Central

    Ong, Qunxiang; Guo, Shunling; Duan, Liting; Zhang, Kai; Collier, Eleanor Ann; Cui, Bianxiao

    2016-01-01

    Acute brain injuries such as ischemic stroke or traumatic brain injury often cause massive neural death and irreversible brain damage with grave consequences. Previous studies have established that a key participant in the events leading to neural death is the excessive production of reactive oxygen species. Protecting neuronal cells by activating their endogenous defense mechanisms is an attractive treatment strategy for acute brain injuries. In this work, we investigate how the precise timing of the Raf/ERK and the AKT pathway activation affects their protective effects against oxidative stress. For this purpose, we employed optogenetic systems that use light to precisely and reversibly activate either the Raf/ERK or the AKT pathway. We find that preconditioning activation of the Raf/ERK or the AKT pathway immediately before oxidant exposure provides significant protection to cells. Notably, a 15-minute transient activation of the Raf/ERK pathway is able to protect PC12 cells against oxidant strike that is applied 12 hours later, while the transient activation of the AKT pathway fails to protect PC12 cells in such a scenario. On the other hand, if the pathways are activated after the oxidative insult, i.e. postconditioning, the AKT pathway conveys greater protective effect than the Raf/ERK pathway. We find that postconditioning AKT activation has an optimal delay period of 2 hours. When the AKT pathway is activated 30min after the oxidative insult, it exhibits very little protective effect. Therefore, the precise timing of the pathway activation is crucial in determining its protective effect against oxidative injury. The optogenetic platform, with its precise temporal control and its ability to activate specific pathways, is ideal for the mechanistic dissection of intracellular pathways in protection against oxidative stress. PMID:27082641

  5. Restoring Akt1 activity in outgrowth endothelial cells from South Asian men rescues vascular reparative potential.

    PubMed

    Cubbon, Richard M; Yuldasheva, Nadira Y; Viswambharan, Hema; Mercer, Ben N; Baliga, Vivek; Stephen, Sam L; Askham, Jonathan; Sukumar, Piruthivi; Skromna, Anna; Mughal, Romana S; Walker, Andrew M N; Bruns, Alexander; Bailey, Marc A; Galloway, Stacey; Imrie, Helen; Gage, Matthew C; Rakobowchuk, Mark; Li, Jing; Porter, Karen E; Ponnambalam, Sreenivasan; Wheatcroft, Stephen B; Beech, David J; Kearney, Mark T

    2014-10-01

    Recent data suggest reduced indices of vascular repair in South Asian men, a group at increased risk of cardiovascular events. Outgrowth endothelial cells (OEC) represent an attractive tool to study vascular repair in humans and may offer potential in cell-based repair therapies. We aimed to define and manipulate potential mechanisms of impaired vascular repair in South Asian (SA) men. In vitro and in vivo assays of vascular repair and angiogenesis were performed using OEC derived from SA men and matched European controls, prior defining potentially causal molecular mechanisms. SA OEC exhibited impaired colony formation, migration, and in vitro angiogenesis, associated with decreased expression of the proangiogenic molecules Akt1 and endothelial nitric oxide synthase (eNOS). Transfusion of European OEC into immunodeficient mice after wire-induced femoral artery injury augmented re-endothelialization, in contrast with SA OEC and vehicle; SA OEC also failed to promote angiogenesis after induction of hind limb ischemia. Expression of constitutively active Akt1 (E17KAkt), but not green fluorescent protein control, in SA OEC increased in vitro angiogenesis, which was abrogated by a NOS antagonist. Moreover, E17KAkt expressing SA OEC promoted re-endothelialization of wire-injured femoral arteries, and perfusion recovery of ischemic limbs, to a magnitude comparable with nonmanipulated European OEC. Silencing Akt1 in European OEC recapitulated the functional deficits noted in SA OEC. Reduced signaling via the Akt/eNOS axis is causally linked with impaired OEC-mediated vascular repair in South Asian men. These data prove the principle of rescuing marked reparative dysfunction in OEC derived from these men.

  6. Semaphorin 4A enhances lung fibrosis through activation of Akt via PlexinD1 receptor.

    PubMed

    Peng, Hai-Ying; Gao, Wei; Chong, Fa-Rong; Liu, Hong-Yan; Zhang, J I

    2015-12-01

    Semaphorin 4A plays a regulatory role in immune function and angiogenesis. However, its specific involvement in controlling lung fibrosis, a process that is closely related to angiogenesis and inflammation is still poorly understood. In the present study, we show that treatment of Sema4A on normal lung fibroblasts induces expression of proteins that contribute to a contractile phenotype, including alpha-smooth muscle actin (alpha-SMA), ezrin, moesin, and paxillin. We confirm that Sema4A enhances the ability of lung fibroblasts to contract collagen gel. Sema4A treatment led to resistance to apoptosis in normal lung fibroblasts. Relative to normal lung fibroblasts, fibroblasts cultured from scars of patients with the fibrotic disease Systemic Sclerosis (SSc) showed elevated Sema4A secretion, enhanced alpha-SMA, ezrin, moesin, and paxillin expression, and high ability to induce collagen gel contraction. Using neutralizing antibody against Sema4A receptor, PlexinD1, we found that endogenous Sema4A signalling in SSc fibroblast was through PlexinD1 receptor. We then identified the signalling mechanism through which Sema4A-PlexinD1 promotes the ability of normal fibroblasts to contract a collagen gel matrix. Western blot analysis showed that Sema4A activated the Akt pathway in lung fibroblasts, and the specific inhibitor of Akt pathway, Akt inhibitor III, blocked the ability of Sema4A to promote the ability of lung fibroblasts to contract a collagen gel matrix. Thus, blocking Sema4APlexinD1- Akt cascades might be beneficial in reducing pulmonary fibrosis.

  7. NTRK2 activation cooperates with PTEN deficiency in T-ALL through activation of both the PI3K–AKT and JAK–STAT3 pathways

    PubMed Central

    Yuzugullu, Haluk; Von, Thanh; Thorpe, Lauren M; Walker, Sarah R; Roberts, Thomas M; Frank, David A; Zhao, Jean J

    2016-01-01

    Loss of PTEN, a negative regulator of the phosphoinositide 3-kinase signaling pathway, is a frequent event in T-cell acute lymphoblastic leukemia, suggesting the importance of phosphoinositide 3-kinase activity in this disease. Indeed, hyperactivation of the phosphoinositide 3-kinase pathway is associated with the disease aggressiveness, poor prognosis and resistance to current therapies. To identify a molecular pathway capable of cooperating with PTEN deficiency to drive oncogenic transformation of leukocytes, we performed an unbiased transformation screen with a library of tyrosine kinases. We found that activation of NTRK2 is able to confer a full growth phenotype of Ba/F3 cells in an IL3-independent manner in the PTEN-null setting. NTRK2 activation cooperates with PTEN deficiency through engaging both phosphoinositide3-kinase/AKT and JAK/STAT3 pathway activation in leukocytes. Notably, pharmacological inhibition demonstrated that p110α and p110δ are the major isoforms mediating the phosphoinositide 3-kinase/AKT signaling driven by NTRK2 activation in PTEN-deficient leukemia cells. Furthermore, combined inhibition of phosphoinositide 3-kinase and STAT3 significantly suppressed proliferation of PTEN-mutant T-cell acute lymphoblastic leukemia both in culture and in mouse xenografts. Together, our data suggest that a unique conjunction of PTEN deficiency and NTRK2 activation in T-cell acute lymphoblastic leukemia, and combined pharmacologic inhibition of phosphoinositide 3-kinase and STAT3 signaling may serve as an effective and durable therapeutic strategy for T-cell acute lymphoblastic leukemia. PMID:27672444

  8. NTRK2 activation cooperates with PTEN deficiency in T-ALL through activation of both the PI3K-AKT and JAK-STAT3 pathways.

    PubMed

    Yuzugullu, Haluk; Von, Thanh; Thorpe, Lauren M; Walker, Sarah R; Roberts, Thomas M; Frank, David A; Zhao, Jean J

    2016-01-01

    Loss of PTEN, a negative regulator of the phosphoinositide 3-kinase signaling pathway, is a frequent event in T-cell acute lymphoblastic leukemia, suggesting the importance of phosphoinositide 3-kinase activity in this disease. Indeed, hyperactivation of the phosphoinositide 3-kinase pathway is associated with the disease aggressiveness, poor prognosis and resistance to current therapies. To identify a molecular pathway capable of cooperating with PTEN deficiency to drive oncogenic transformation of leukocytes, we performed an unbiased transformation screen with a library of tyrosine kinases. We found that activation of NTRK2 is able to confer a full growth phenotype of Ba/F3 cells in an IL3-independent manner in the PTEN-null setting. NTRK2 activation cooperates with PTEN deficiency through engaging both phosphoinositide3-kinase/AKT and JAK/STAT3 pathway activation in leukocytes. Notably, pharmacological inhibition demonstrated that p110α and p110δ are the major isoforms mediating the phosphoinositide 3-kinase/AKT signaling driven by NTRK2 activation in PTEN-deficient leukemia cells. Furthermore, combined inhibition of phosphoinositide 3-kinase and STAT3 significantly suppressed proliferation of PTEN-mutant T-cell acute lymphoblastic leukemia both in culture and in mouse xenografts. Together, our data suggest that a unique conjunction of PTEN deficiency and NTRK2 activation in T-cell acute lymphoblastic leukemia, and combined pharmacologic inhibition of phosphoinositide 3-kinase and STAT3 signaling may serve as an effective and durable therapeutic strategy for T-cell acute lymphoblastic leukemia. PMID:27672444

  9. NTRK2 activation cooperates with PTEN deficiency in T-ALL through activation of both the PI3K–AKT and JAK–STAT3 pathways

    PubMed Central

    Yuzugullu, Haluk; Von, Thanh; Thorpe, Lauren M; Walker, Sarah R; Roberts, Thomas M; Frank, David A; Zhao, Jean J

    2016-01-01

    Loss of PTEN, a negative regulator of the phosphoinositide 3-kinase signaling pathway, is a frequent event in T-cell acute lymphoblastic leukemia, suggesting the importance of phosphoinositide 3-kinase activity in this disease. Indeed, hyperactivation of the phosphoinositide 3-kinase pathway is associated with the disease aggressiveness, poor prognosis and resistance to current therapies. To identify a molecular pathway capable of cooperating with PTEN deficiency to drive oncogenic transformation of leukocytes, we performed an unbiased transformation screen with a library of tyrosine kinases. We found that activation of NTRK2 is able to confer a full growth phenotype of Ba/F3 cells in an IL3-independent manner in the PTEN-null setting. NTRK2 activation cooperates with PTEN deficiency through engaging both phosphoinositide3-kinase/AKT and JAK/STAT3 pathway activation in leukocytes. Notably, pharmacological inhibition demonstrated that p110α and p110δ are the major isoforms mediating the phosphoinositide 3-kinase/AKT signaling driven by NTRK2 activation in PTEN-deficient leukemia cells. Furthermore, combined inhibition of phosphoinositide 3-kinase and STAT3 significantly suppressed proliferation of PTEN-mutant T-cell acute lymphoblastic leukemia both in culture and in mouse xenografts. Together, our data suggest that a unique conjunction of PTEN deficiency and NTRK2 activation in T-cell acute lymphoblastic leukemia, and combined pharmacologic inhibition of phosphoinositide 3-kinase and STAT3 signaling may serve as an effective and durable therapeutic strategy for T-cell acute lymphoblastic leukemia.

  10. CSK negatively regulates nerve growth factor induced neural differentiation and augments AKT kinase activity

    SciTech Connect

    Dey, Nandini . E-mail: Don_Durden@oz.ped.emory.edu

    2005-07-01

    Src family kinases are involved in transducing growth factor signals for cellular differentiation and proliferation in a variety of cell types. The activity of all Src family kinases (SFKs) is controlled by phosphorylation at their C-terminal 527-tyrosine residue by C-terminal SRC kinase, CSK. There is a paucity of information regarding the role of CSK and/or specific Src family kinases in neuronal differentiation. Pretreatment of PC12 cells with the Src family kinase inhibitor, PP1, blocked NGF-induced activation of SFKs and obliterated neurite outgrowth. To confirm a role for CSK and specific isoforms of SFKs in neuronal differentiation, we overexpressed active and catalytically dead CSK in the rat pheochromocytoma cell line, PC12. CSK overexpression caused a profound inhibition of NGF-induced activation of FYN, YES, RAS, and ERK and inhibited neurite outgrowth, NGF-stimulated integrin-directed migration and blocked the NGF-induced conversion of GDP-RAC to its GTP-bound active state. CSK overexpression markedly augmented the activation state of AKT following NGF stimulation. In contrast, kinase-dead CSK augmented the activation of FYN, RAS, and ERK and increased neurite outgrowth. These data suggest a distinct requirement for CSK in the regulation of NGF/TrkA activation of RAS, RAC, ERK, and AKT via the differential control of SFKs in the orchestration of neuronal differentiation.

  11. A2B adenosine receptor contributes to penile erection via PI3K/AKT signaling cascade-mediated eNOS activation

    PubMed Central

    Wen, Jiaming; Grenz, Almut; Zhang, Yujin; Dai, Yingbo; Kellems, Rodney E.; Blackburn, Michael R.; Eltzschig, Holger K.; Xia, Yang

    2011-01-01

    Normal penile erection is under the control of multiple factors and signaling pathways. Although adenosine signaling is implicated in normal and abnormal penile erection, the exact role and the underlying mechanism for adenosine signaling in penile physiology remain elusive. Here we report that shear stress leads to increased adenosine release from endothelial cells. Subsequently, we determined that ecto-5′-nucleotidase (CD73) is a key enzyme required for the production of elevated adenosine from ATP released by shear-stressed endothelial cells. Mechanistically, we demonstrate that shear stress-mediated elevated adenosine functions through the adenosine A2B receptor (A2BR) to activate the PI3K/AKT signaling cascade and subsequent increased endothelial nitric oxide synthase (eNOS) phosphorylation. These in vitro studies led us to discover further that adenosine was induced during sustained penile erection and contributes to PI3K/AKT activation and subsequent eNOS phosphorylation via A2BR signaling in intact animal. Finally, we demonstrate that lowering adenosine in wild-type mice or genetic deletion of A2BR in mutant mice significantly attenuated PI3K/AKT activation, eNOS phosphorylation, and subsequent impaired penile erection featured with the reduction of ratio of maximal intracavernosal pressure to systemic arterial pressure from 0.49 ± 0.03 to 0.41 ± 0.05 and 0.38 ± 0.04, respectively (both P<0.05). Overall, using biochemical, cellular, genetic, and physiological approaches, our findings reveal that adenosine is a novel molecule signaling via A2BR activation, contributing to penile erection via PI3K/AKT-dependent eNOS activation. These studies suggest that this signaling pathway may be a novel therapeutic target for erectile disorders.—Wen, J., Grenz, A., Zhang, Y., Dai, Y., Kellems, R. E., Blackburn, M. R., Eltzschig, H. K., Xia, Y. A2B adenosine receptor contributes to penile erection via PI3K/AKT signaling cascade-mediated eNOS activation. PMID

  12. Integrin β1 mediates vaccinia virus entry through activation of PI3K/Akt signaling.

    PubMed

    Izmailyan, Roza; Hsao, Jye-Chian; Chung, Che-Sheng; Chen, Chein-Hung; Hsu, Paul Wei-Che; Liao, Chung-Lin; Chang, Wen

    2012-06-01

    Vaccinia virus has a broad range of infectivity in many cell lines and animals. Although it is known that the vaccinia mature virus binds to cell surface glycosaminoglycans and extracellular matrix proteins, whether additional cellular receptors are required for virus entry remains unclear. Our previous studies showed that the vaccinia mature virus enters through lipid rafts, suggesting the involvement of raft-associated cellular proteins. Here we demonstrate that one lipid raft-associated protein, integrin β1, is important for vaccinia mature virus entry into HeLa cells. Vaccinia virus associates with integrin β1 in lipid rafts on the cell surface, and the knockdown of integrin β1 in HeLa cells reduces vaccinia mature virus entry. Additionally, vaccinia mature virus infection is reduced in a mouse cell line, GD25, that is deficient in integrin β1 expression. Vaccinia mature virus infection triggers the activation of phosphatidylinositol 3-kinase (PI3K)/Akt signaling, and the treatment of cells with inhibitors to block P13K activation reduces virus entry in an integrin β1-dependent manner, suggesting that integrin β1-mediates PI3K/Akt activation induced by vaccinia virus and that this signaling pathway is essential for virus endocytosis. The inhibition of integrin β1-mediated cell adhesion results in a reduction of vaccinia virus entry and the disruption of focal adhesion and PI3K/Akt activation. In summary, our results show that the binding of vaccinia mature virus to cells mimics the outside-in activation process of integrin functions to facilitate vaccinia virus entry into HeLa cells.

  13. Withaferin A inhibits matrix metalloproteinase-9 activity by suppressing the Akt signaling pathway.

    PubMed

    Lee, Dae Hyung; Lim, In-Hye; Sung, Eon-Gi; Kim, Joo-Young; Song, In-Hwan; Park, Yoon Ki; Lee, Tae-Jin

    2013-08-01

    Withaferin A (Wit A), a steroidal lactone isolated from Withania somnifera, exhibits anti-inflammatory, immuno-modulatory and anti-angiogenic properties and antitumor activities. In the present study, we investigated the effects of Wit A on protease-mediated invasiveness of the human metastatic cancer cell lines Caski and SK-Hep1. We found that treatment with Wit A resulted in marked inhibition of the TGF‑β‑induced increase in expression and activity of matrix metalloproteinase (MMP)‑9 in Caski cell line. These effects of Wit A were dose-dependent and showed a correlation with suppression of MMP‑9 mRNA expression levels. Treatment with Wit A resulted in an ~1.6-fold induction of MMP-9 promoter activity, which was also suppressed by treatment with Wit A in Caski cells. We found that treatment with Wit A resulted in inhibition of TGF‑β‑induced phosphorylation of Akt, which was involved in the downregulation of expression of MMP-9 at the protein level. Introduction with constitutively active (CA)‑Akt resulted in a partial increase in the secretion of TGF-β-induced MMP-9 blocked by treatment with Wit A in Caski cells. According to these results, Wit A may inhibit the invasive and migratory abilities of Caski cells through a reduction in MMP-9 expression through suppression of the pAkt signaling pathway. These findings indicate that use of Wit A may be an effective strategy for control of metastasis and invasiveness of tumors.

  14. Lovastatin Inhibits VEGFR and AKT Activation: Synergistic Cytotoxicity in Combination with VEGFR Inhibitors

    PubMed Central

    Addison, Christina L.; Dimitroulakos, Jim

    2010-01-01

    Background In a recent study, we demonstrated the ability of lovastatin, a potent inhibitor of mevalonate synthesis, to inhibit the function of the epidermal growth factor receptor (EGFR). Lovastatin attenuated ligand-induced receptor activation and downstream signaling through the PI3K/AKT pathway. Combining lovastatin with gefitinib, a potent EGFR inhibitor, induced synergistic cytotoxicity in a variety of tumor derived cell lines. The vascular endothelial growth factor receptor (VEGFR) and EGFR share similar activation, internalization and downstream signaling characteristics. Methodology/Principal Findings The VEGFRs, particularly VEGFR-2 (KDR, Flt-1), play important roles in regulating tumor angiogenesis by promoting endothelial cell proliferation, survival and migration. Certain tumors, such as malignant mesothelioma (MM), also express both the VEGF ligand and VEGFRs that act in an autocrine loop to directly stimulate tumor cell growth and survival. In this study, we have shown that lovastatin inhibits ligand-induced VEGFR-2 activation through inhibition of receptor internalization and also inhibits VEGF activation of AKT in human umbilical vein endothelial cells (HUVEC) and H28 MM cells employing immunofluorescence and Western blotting. Combinations of lovastatin and a VEGFR-2 inhibitor showed more robust AKT inhibition than either agent alone in the H28 MM cell line. Furthermore, combining 5 µM lovastatin treatment, a therapeutically relevant dose, with two different VEGFR-2 inhibitors in HUVEC and the H28 and H2052 mesothelioma derived cell lines demonstrated synergistic cytotoxicity as demonstrated by MTT cell viability and flow cytometric analyses. Conclusions/Significance These results highlight a novel mechanism by which lovastatin can regulate VEGFR-2 function and a potential therapeutic approach for MM through combining statins with VEGFR-2 inhibitors. PMID:20838437

  15. Full activation of PKB/Akt in response to insulin or ionizing radiation is mediated through ATM.

    PubMed

    Viniegra, Juan Guinea; Martínez, Natalia; Modirassari, Pegah; Hernández Losa, Javier; Parada Cobo, Carlos; Sánchez-Arévalo Lobo, Víctor Javier; Aceves Luquero, Clara Isabel; Alvarez-Vallina, Luis; Ramón y Cajal, Santiago; Rojas, José María; Sánchez-Prieto, Ricardo

    2005-02-11

    The gene mutated in ataxia telangiectasia, ATM, has been implicated in several cell functions such as cell cycle control and response to DNA damage and insulin. PKB/Akt has also been implicated in the cellular response to insulin, gamma-radiation, and cell cycle control. Interestingly, lack of PKB/Akt function in vivo is able to mimic some phenotypic abnormalities associated with ataxia telangiectasia (AT). Here we show that ATM is a major determinant of full PKB/Akt activation in response to insulin or gamma-radiation. This effect is mediated through the phosphatidylinositol 3-kinase domain of ATM that specifically affects Akt serine 473 phosphorylation. This conclusion was inferred from the results obtained in transient transfection assays using exogenous PKB/Akt and ATM in Cos cells. Moreover, the use of ATM inhibitors or small interfering RNA confirmed our observation. Further supporting these results, we also observed that biological responses tightly regulated by Akt, such as transcription factor of the forkhead family activity after insulin treatment or gamma-radiation response, were altered in cell lines derived from AT patients and knockout mice for ATM in which phosphorylation in serine 473 was almost abolished. This study proposes new clues in the search of the unknown PDK2 and new explanations for the radiosensitivity or insulin intolerance described more than 30 years ago in AT patients. PMID:15546863

  16. A Hot-spot of In-frame Duplications Activates the Oncoprotein AKT1 in Juvenile Granulosa Cell Tumors

    PubMed Central

    Bessière, Laurianne; Todeschini, Anne-Laure; Auguste, Aurélie; Sarnacki, Sabine; Flatters, Delphine; Legois, Bérangère; Sultan, Charles; Kalfa, Nicolas; Galmiche, Louise; Veitia, Reiner A.

    2015-01-01

    Background Ovarian granulosa cell tumors are the most common sex-cord stromal tumors and have juvenile (JGCTs) and adult forms. In a previous study we reported the occurrence of activating somatic mutations of Gαs, which transduces mitogenic signals, in 30% of the analyzed JGCTs. Methods We have searched for alterations in other proteins involved in ovarian mitogenic signaling. We focused on the PI3K–AKT axis. As we found mutations in AKT1, we analyzed the subcellular localization of the mutated proteins and performed functional explorations using Western-blot and luciferase assays. Findings We detected in-frame duplications affecting the pleckstrin-homology domain of AKT1 in more than 60% of the tumors occurring in girls under 15 years of age. The somatic status of the mutations was confirmed when peritumoral DNA was available. The JGCTs without duplications carried point mutations affecting highly conserved residues. Several of these substitutions were somatic lesions. The mutated proteins carrying the duplications had a non-wild-type subcellular distribution, with a marked enrichment at the plasma membrane. This led to a striking degree of AKT1 activation demonstrated by a strong phosphorylation level and by reporter assays. Interpretation Our study incriminates somatic mutations of AKT1 as a major event in the pathogenesis of JGCTs. The existence of AKT inhibitors currently tested in clinical trials opens new perspectives for targeted therapies for these tumors, which are currently treated with standard non-specific chemotherapy protocols. PMID:26137586

  17. Holo-APP and G-protein-mediated signaling are required for sAPPα-induced activation of the Akt survival pathway

    PubMed Central

    Milosch, N; Tanriöver, G; Kundu, A; Rami, A; François, J-C; Baumkötter, F; Weyer, S W; Samanta, A; Jäschke, A; Brod, F; Buchholz, C J; Kins, S; Behl, C; Müller, U C; Kögel, D

    2014-01-01

    Accumulating evidence indicates that loss of physiologic amyloid precursor protein (APP) function leads to reduced neuronal plasticity, diminished synaptic signaling and enhanced susceptibility of neurons to cellular stress during brain aging. Here we investigated the neuroprotective function of the soluble APP ectodomain sAPPα (soluble APPα), which is generated by cleavage of APP by α-secretase along the non-amyloidogenic pathway. Recombinant sAPPα protected primary hippocampal neurons and SH-SY5Y neuroblastoma cells from cell death induced by trophic factor deprivation. We show that this protective effect is abrogated in neurons from APP-knockout animals and APP-depleted SH-SY5Y cells, but not in APP-like protein 1- and 2- (APLP1 and APLP2) depleted cells, indicating that expression of membrane-bound holo-APP is required for sAPPα-dependent neuroprotection. Trophic factor deprivation diminished the activity of the Akt survival pathway. Strikingly, both recombinant sAPPα and the APP-E1 domain were able to stimulate Akt activity in wild-type (wt) fibroblasts, SH-SY5Y cells and neurons, but failed to rescue in APP-deficient neurons or fibroblasts. The ADAM10 (a disintegrin and metalloproteinase domain-containing protein 10) inhibitor GI254023X exacerbated neuron death in organotypic (hippocampal) slice cultures of wt mice subjected to trophic factor and glucose deprivation. This cell death-enhancing effect of GI254023X could be completely rescued by applying exogenous sAPPα. Interestingly, sAPPα-dependent Akt induction was unaffected in neurons of APP-ΔCT15 mice that lack the C-terminal YENPTY motif of the APP intracellular region. In contrast, sAPPα-dependent rescue of Akt activation was completely abolished in APP mutant cells lacking the G-protein interaction motif located in the APP C-terminus and by blocking G-protein-dependent signaling with pertussis toxin. Collectively, our data provide new mechanistic insights into the physiologic role of APP in

  18. Holo-APP and G-protein-mediated signaling are required for sAPPα-induced activation of the Akt survival pathway.

    PubMed

    Milosch, N; Tanriöver, G; Kundu, A; Rami, A; François, J-C; Baumkötter, F; Weyer, S W; Samanta, A; Jäschke, A; Brod, F; Buchholz, C J; Kins, S; Behl, C; Müller, U C; Kögel, D

    2014-01-01

    Accumulating evidence indicates that loss of physiologic amyloid precursor protein (APP) function leads to reduced neuronal plasticity, diminished synaptic signaling and enhanced susceptibility of neurons to cellular stress during brain aging. Here we investigated the neuroprotective function of the soluble APP ectodomain sAPPα (soluble APPα), which is generated by cleavage of APP by α-secretase along the non-amyloidogenic pathway. Recombinant sAPPα protected primary hippocampal neurons and SH-SY5Y neuroblastoma cells from cell death induced by trophic factor deprivation. We show that this protective effect is abrogated in neurons from APP-knockout animals and APP-depleted SH-SY5Y cells, but not in APP-like protein 1- and 2- (APLP1 and APLP2) depleted cells, indicating that expression of membrane-bound holo-APP is required for sAPPα-dependent neuroprotection. Trophic factor deprivation diminished the activity of the Akt survival pathway. Strikingly, both recombinant sAPPα and the APP-E1 domain were able to stimulate Akt activity in wild-type (wt) fibroblasts, SH-SY5Y cells and neurons, but failed to rescue in APP-deficient neurons or fibroblasts. The ADAM10 (a disintegrin and metalloproteinase domain-containing protein 10) inhibitor GI254023X exacerbated neuron death in organotypic (hippocampal) slice cultures of wt mice subjected to trophic factor and glucose deprivation. This cell death-enhancing effect of GI254023X could be completely rescued by applying exogenous sAPPα. Interestingly, sAPPα-dependent Akt induction was unaffected in neurons of APP-ΔCT15 mice that lack the C-terminal YENPTY motif of the APP intracellular region. In contrast, sAPPα-dependent rescue of Akt activation was completely abolished in APP mutant cells lacking the G-protein interaction motif located in the APP C-terminus and by blocking G-protein-dependent signaling with pertussis toxin. Collectively, our data provide new mechanistic insights into the physiologic role of APP in

  19. Activation of Akt is essential for the propagation of mitochondrial respiratory stress signaling and activation of the transcriptional coactivator heterogeneous ribonucleoprotein A2.

    PubMed

    Guha, Manti; Fang, Ji-Kang; Monks, Robert; Birnbaum, Morris J; Avadhani, Narayan G

    2010-10-15

    Mitochondrial respiratory stress (also called mitochondrial retrograde signaling) activates a Ca(2+)/calcineurin-mediated signal that culminates in transcription activation/repression of a large number of nuclear genes. This signal is propagated through activation of the regulatory proteins NFκB c-Rel/p50, C/EBPδ, CREB, and NFAT. Additionally, the heterogeneous ribonucleoprotein A2 (hnRNPA2) functions as a coactivator in up-regulating the transcription of Cathepsin L, RyR1, and Glut-4, the target genes of stress signaling. Activation of IGF1R, which causes a metabolic switch to glycolysis, cell invasiveness, and resistance to apoptosis, is a phenotypic hallmark of C2C12 myoblasts subjected to mitochondrial stress. In this study, we report that mitochondrial stress leads to increased expression, activation, and nuclear localization of Akt1. Mitochondrial respiratory stress also activates Akt1-gene expression, which involves hnRNPA2 as a coactivator, indicating a complex interdependency of these two factors. Using Akt1(-/-) mouse embryonic fibroblasts and Akt1 mRNA-silenced C2C12 cells, we show that Akt1-mediated phosphorylation is crucial for the activation and recruitment of hnRNPA2 to the enhanceosome complex. Akt1 mRNA silencing in mtDNA-depleted cells resulted in reversal of the invasive phenotype, accompanied by sensitivity to apoptotic stimuli. These results show that Akt1 is an important regulator of the nuclear transcriptional response to mitochondrial stress.

  20. Sequential Activities of Phosphoinositide 3-Kinase, PKB/Akt, and Rab7 during Macropinosome Formation in Dictyostelium

    PubMed Central

    Rupper, Adam; Lee, Kyung; Knecht, David; Cardelli, James

    2001-01-01

    Macropinocytosis plays an important role in the internalization of antigens by dendritic cells and is the route of entry for many bacterial pathogens; however, little is known about the molecular mechanisms that regulate the formation or maturation of macropinosomes. Like dendritic cells, Dictyostelium amoebae are active in macropinocytosis, and various proteins have been identified that contribute to this process. As described here, microscopic analysis of null mutants have revealed that the class I phosphoinositide 3-kinases, PIK1 and PIK2, and the downstream effector protein kinase B (PKB/Akt) are important in regulating completion of macropinocytosis. Although actin-rich membrane protrusions form in these cell lines, they recede without forming macropinosomes. Imaging of cells expressing green fluorescent protein (GFP) fused to the pleckstrin homology domain (PH) of PKB (GFP-PHPKB) indicates that D3 phosphoinositides are enriched in the forming macropinocytic cup and remain associated with newly formed macropinosomes for <1 minute. A fusion protein, consisting of GFP fused to an F-actin binding domain, overlaps with GFP-PHPKB in the timing of association with forming macropinosomes. Although macropinocytosis is reduced in cells expressing dominant negative Rab7, microscopic imaging studies reveal that GFP-Rab7 associates only with formed macropinosomes at approximately the time that F-actin and D3 phosphoinositide levels decrease. These results support a model in which F-actin modulating proteins and vesicle trafficking proteins coordinately regulate the formation and maturation of macropinosomes. PMID:11553719

  1. Doxorubicin and cyclophosphamide induce cognitive dysfunction and activate the ERK and AKT signaling pathways.

    PubMed

    Salas-Ramirez, Kaliris Y; Bagnall, Ciara; Frias, Leslie; Abdali, Syed A; Ahles, Tim A; Hubbard, Karen

    2015-10-01

    Chemotherapy is associated with long-term cognitive deficits in breast cancer survivors. Studies suggest that these impairments result in the loss of cognitive reserve and/or induce a premature aging of the brain. This study has been aimed to determine the potential underlying mechanisms that induce cognitive impairments by chemotherapeutic agents commonly used in breast cancer. Intact and ovariectomized (OVX) female rats were treated intravenously with either saline or a combination of cyclophosphamide (40 mg/kg) and doxorubicin (4 mg/kg). All subjects were tested for anxiety, locomotor activity, working, visual and spatial memory consecutively. Although anxiety and visual memory were not affected, chemotherapy significantly decreased locomotor activity and impaired working and spatial memory in female rats, independent of their hormonal status. The cognitive deficits observed are hippocampal dependent. Therefore, as a first step to identity the potential signaling pathways involved in this cognitive dysfunction, the protein levels of extracellular signal-regulated kinase 1/2 (Erk1/2), Akt (neuroprotectant) BDNF and (structural protein) PSD95 in hippocampal lysates were measured. Erk1/2 and Akt pathways are known to modulate synaptic plasticity, neuronal survival, aging and cancer. We found an increased activation of Erk1/2 and Akt as well as an increase in the protein levels of PSD95 in OVX female rodents. However, OVX females had a higher overall BDNF level, independent of chemotherapy. These studies provide additional evidence that commonly used chemotherapeutic agents affect cognitive function and impact synaptic plasticity/aging molecules which may be part of the underlying biology explaining cognitive change and can be potential therapeutic targets.

  2. Inflammasome-independent role of AIM2 in suppressing colon tumorigenesis by interfering with DNA-PK–dependent Akt activation

    PubMed Central

    Wilson, Justin E; Petrucelli, Alex S; Chen, Liang; Koblansky, A Alicia; Truax, Agnieszka D; Oyama, Yoshitaka; Rogers, Arlin B; Brickey, W June; Wang, Yuli; Schneider, Monika; Mühlbauer, Marcus; Chou, Wei-Chun; Barker, Brianne R; Jobin, Christian; Allbritton, Nancy L; Ramsden, Dale A; Davis, Beckley K; Ting, Jenny P Y

    2015-01-01

    The inflammasome activates caspase-1 and the release of interleukin-1β (IL-1β) and IL-18, and several inflammasomes protect against intestinal inflammation and colitis-associated colon cancer (CAC) in animal models. The absent in melanoma 2 (AIM2) inflammasome is activated by double-stranded DNA, and AIM2 expression is reduced in several types of cancer, but the mechanism by which AIM2 restricts tumor growth remains unclear. We found that Aim2-deficient mice had greater tumor load than Asc-deficient mice in the azoxymethane/dextran sodium sulfate (AOM/DSS) model of colorectal cancer. Tumor burden was also higher in Aim2−/−/ApcMin/+ than in APCMin/+ mice. The effects of AIM2 on CAC were independent of inflammasome activation and IL-1β and were primarily mediated by a non–bone marrow source of AIM2. In resting cells, AIM2 physically interacted with and limited activation of DNA-dependent protein kinase (DNA-PK), a PI3K-related family member that promotes Akt phosphorylation, whereas loss of AIM2 promoted DNA-PK–mediated Akt activation. AIM2 reduced Akt activation and tumor burden in colorectal cancer models, while an Akt inhibitor reduced tumor load in Aim2−/− mice. These findings suggest that Akt inhibitors could be used to treat AIM2-deficient human cancers. PMID:26107252

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

    PubMed

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

    2014-01-01

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

  4. The novel AKT inhibitor afuresertib shows favorable safety, pharmacokinetics, and clinical activity in multiple myeloma

    PubMed Central

    Spencer, Andrew; Yoon, Sung-Soo; Harrison, Simon J.; Morris, Shannon R.; Smith, Deborah A.; Brigandi, Richard A.; Gauvin, Jennifer; Kumar, Rakesh; Opalinska, Joanna B.

    2014-01-01

    The PI3K/AKT pathway is constitutively active in hematologic malignancies, providing proliferative and antiapoptotic signals and possibly contributing to drug resistance. We conducted an open-label phase 1 study to evaluate the maximum tolerated dose (MTD), safety, pharmacokinetics, and clinical activity of afuresertib—an oral AKT inhibitor—in patients with advanced hematologic malignancies. Seventy-three patients were treated at doses ranging from 25 to 150 mg per day. The MTD was established at 125 mg per day because of 2 dose-limiting toxicities in the 150-mg cohort (liver function test abnormalities). The most frequent adverse events were nausea (35.6%), diarrhea (32.9%), and dyspepsia (24.7%). Maximum plasma concentrations and area under the plasma concentration-time curves from time 0 to 24 hours were generally dose proportional at >75-mg doses; the median time to peak plasma concentrations was 1.5 to 2.5 hours post dose, with a half-life of approximately 1.7 days. Three multiple myeloma patients attained partial responses; an additional 3 attained minimal responses. Clinical activity was also observed in non-Hodgkin lymphoma, Langerhan's cell histiocytosis, and Hodgkin disease. Single-agent afuresertib showed a favorable safety profile and demonstrated clinical activity against hematologic malignancies, including multiple myeloma. This trial was registered at www.clinicaltrials.gov as #NCT00881946. PMID:25075128

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

    SciTech Connect

    Montes, Daniela K.; Brenet, Marianne; Muñoz, Vanessa C.; Burgos, Patricia V.; Villanueva, Carolina I.; Figueroa, Carlos D.; González, Carlos B.

    2013-11-29

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

  6. Akt-mTORC1 signaling regulates Acly to integrate metabolic input to control of macrophage activation

    PubMed Central

    Covarrubias, Anthony J; Aksoylar, Halil Ibrahim; Yu, Jiujiu; Snyder, Nathaniel W; Worth, Andrew J; Iyer, Shankar S; Wang, Jiawei; Ben-Sahra, Issam; Byles, Vanessa; Polynne-Stapornkul, Tiffany; Espinosa, Erika C; Lamming, Dudley; Manning, Brendan D; Zhang, Yijing; Blair, Ian A; Horng, Tiffany

    2016-01-01

    Macrophage activation/polarization to distinct functional states is critically supported by metabolic shifts. How polarizing signals coordinate metabolic and functional reprogramming, and the potential implications for control of macrophage activation, remains poorly understood. Here we show that IL-4 signaling co-opts the Akt-mTORC1 pathway to regulate Acly, a key enzyme in Ac-CoA synthesis, leading to increased histone acetylation and M2 gene induction. Only a subset of M2 genes is controlled in this way, including those regulating cellular proliferation and chemokine production. Moreover, metabolic signals impinge on the Akt-mTORC1 axis for such control of M2 activation. We propose that Akt-mTORC1 signaling calibrates metabolic state to energetically demanding aspects of M2 activation, which may define a new role for metabolism in supporting macrophage activation. DOI: http://dx.doi.org/10.7554/eLife.11612.001 PMID:26894960

  7. TGF-{beta}2 inhibits AKT activation and FGF-2-induced corneal endothelial cell proliferation

    SciTech Connect

    Lu Jiawei; Lu Zhenyu; Reinach, Peter

    2006-11-01

    The corneal endothelial cells form a boundary layer between anterior chamber and cornea. This single cell layer is important to maintain cornea transparency by eliciting net fluid transport into the anterior chamber. Injuries of the corneal endothelial layer in humans lead to corneal swelling and translucence. This hindrance is thought to be due to limited proliferative capacity of the endothelial layer. Fibroblast growth factor 2 (FGF-2) and transforming growth factor-beta 2 (TGF-{beta}2) are both found in aqueous humor, and these two cytokines promote and inhibit cell growth, respectively. The intracellular signaling mechanisms by which TGF-{beta}2 suppresses the mitogenic response to FGF-2, however, remain unclear. We have addressed this question by investigating potential crosstalk between FGF-2-induced and TGF-{beta}2-regulated intracellular signaling events in cultured bovine corneal endothelial (BCE) cells. We found that TGF-{beta}2 and FGF-2 oppositely affect BCE cell proliferation and TGF-{beta}2 can override the stimulating effects of FGF-2 by increasing COX-2 expression in these cells. Consistent with these findings, overexpression of COX-2 significantly reduced FGF-2-induced cell proliferation whereas a COX-2 specific inhibitor NS398 reversed the effect of TGF-{beta}2 on FGF-2-induced cell proliferation. The COX-2 product prostaglandin E2 (PGE-2) blocks FGF-2-induced cell proliferation. Whereas FGF-2 stimulates cell proliferation by activating the AKT pathway, TGF-{beta}2 and PGE-2 both inhibit this pathway. In accordance with the effect of PGE-2, cAMP also inhibits FGF-2-induced AKT activation. These findings suggest that the mitogenic response to FGF-2 in vivo in the corneal endothelial layer may be inhibited by TGF-{beta}2-induced suppression of the PI3-kinase/AKT signaling pathway.

  8. Bufalin Reverses Resistance to Sorafenib by Inhibiting Akt Activation in Hepatocellular Carcinoma: The Role of Endoplasmic Reticulum Stress

    PubMed Central

    Zhai, Bo; Hu, Fengli; Yan, Haijiang; Zhao, Dali; Jin, Xin; Fang, Taishi; Pan, Shangha; Sun, Xueying; Xu, Lishan

    2015-01-01

    Sorafenib is the standard first-line therapeutic treatment for patients with advanced hepatocellular carcinoma (HCC), but its use is hampered by the development of drug resistance. The activation of Akt by sorafenib is thought to be responsible for this resistance. Bufalin is the major active ingredient of the traditional Chinese medicine Chan su, which inhibits Akt activation; therefore, Chan su is currently used in the clinic to treat cancer. The present study aimed to investigate the ability of bufalin to reverse both inherent and acquired resistance to sorafenib. Bufalin synergized with sorafenib to inhibit tumor cell proliferation and induce apoptosis. This effect was at least partially due to the ability of bufalin to inhibit Akt activation by sorafenib. Moreover, the ability of bufalin to inactivate Akt depended on endoplasmic reticulum (ER) stress mediated by inositol-requiring enzyme 1 (IRE1). Silencing IRE1 with siRNA blocked the bufalin-induced Akt inactivation, but silencing eukaryotic initiation factor 2 (eIF2) or C/EBP-homologous protein (CHOP) did not have the same effect. Additionally, silencing Akt did not influence IRE1, CHOP or phosphorylated eIF2α expression. Two sorafenib-resistant HCC cell lines, which were established from human HCC HepG2 and Huh7 cells, were refractory to sorafenib-induced growth inhibition but were sensitive to bufalin. Thus, Bufalin reversed acquired resistance to sorafenib by downregulating phosphorylated Akt in an ER-stress-dependent manner via the IRE1 pathway. These findings warrant further studies to examine the utility of bufalin alone or in combination with sorafenib as a first- or second-line treatment after sorafenib failure for advanced HCC. PMID:26381511

  9. Bufalin Reverses Resistance to Sorafenib by Inhibiting Akt Activation in Hepatocellular Carcinoma: The Role of Endoplasmic Reticulum Stress.

    PubMed

    Zhai, Bo; Hu, Fengli; Yan, Haijiang; Zhao, Dali; Jin, Xin; Fang, Taishi; Pan, Shangha; Sun, Xueying; Xu, Lishan

    2015-01-01

    Sorafenib is the standard first-line therapeutic treatment for patients with advanced hepatocellular carcinoma (HCC), but its use is hampered by the development of drug resistance. The activation of Akt by sorafenib is thought to be responsible for this resistance. Bufalin is the major active ingredient of the traditional Chinese medicine Chan su, which inhibits Akt activation; therefore, Chan su is currently used in the clinic to treat cancer. The present study aimed to investigate the ability of bufalin to reverse both inherent and acquired resistance to sorafenib. Bufalin synergized with sorafenib to inhibit tumor cell proliferation and induce apoptosis. This effect was at least partially due to the ability of bufalin to inhibit Akt activation by sorafenib. Moreover, the ability of bufalin to inactivate Akt depended on endoplasmic reticulum (ER) stress mediated by inositol-requiring enzyme 1 (IRE1). Silencing IRE1 with siRNA blocked the bufalin-induced Akt inactivation, but silencing eukaryotic initiation factor 2 (eIF2) or C/EBP-homologous protein (CHOP) did not have the same effect. Additionally, silencing Akt did not influence IRE1, CHOP or phosphorylated eIF2α expression. Two sorafenib-resistant HCC cell lines, which were established from human HCC HepG2 and Huh7 cells, were refractory to sorafenib-induced growth inhibition but were sensitive to bufalin. Thus, Bufalin reversed acquired resistance to sorafenib by downregulating phosphorylated Akt in an ER-stress-dependent manner via the IRE1 pathway. These findings warrant further studies to examine the utility of bufalin alone or in combination with sorafenib as a first- or second-line treatment after sorafenib failure for advanced HCC. PMID:26381511

  10. AKT and AMPK activation after high-fat and high-glucose in vitro treatment of prostate epithelial cells.

    PubMed

    Ribeiro, D L; Góes, R M; Pinto-Fochi, M E; Taboga, S R; Abrahamsson, P-A; Dizeyi, N

    2014-06-01

    Considering the increasing consumption of saturated fat and glucose in diets worldwide and its possible association to carcinogenesis, this investigation analysed the proliferation profile of nonmalignant human prostate epithelial cells after exposure to elevated levels of fat and glucose. PNT1A cells were cultured with palmitate (100 or 200 μM) and/or glucose (450 mg/dl) for 24 or 48 h. Treated cells were evaluated for viability test and cell proliferation (MTS assay). AKT and AMPK phosphorylation status were analysed by Western blotting. After 24 h of high-fat alone or associated with high-glucose treatment, there was an increase in AMPK and AKT activation associated to unchanged MTS-cell proliferation. Following 48 h of high-fat but not high-glucose alone, cells decreased AMPK activation and maintained elevated AKT levels. These data were associated to increased cell proliferation after further high-fat treatment. After longer high-fat exposure, MTS revealed that cells remained proliferating. High-glucose alone or associated to high-fat treatment was not able to increase cell proliferation and AKT activation. A high-fat medium containing 100 μM of palmitate stimulates proliferation in PNT1A cells by decreasing the activation of AMPK and increasing activation of AKT after longer exposure time. These findings improve the knowledge about the negative effect of high levels of this saturated fatty acid on proliferative disorders of prostate.

  11. Regulation of Bax/mitochondria interaction by AKT.

    PubMed

    Simonyan, Lilit; Renault, Thibaud T; Novais, Maria João da Costa; Sousa, Maria João; Côrte-Real, Manuela; Camougrand, Nadine; Gonzalez, Cécile; Manon, Stéphen

    2016-01-01

    Bax-dependent mitochondrial permeabilization during apoptosis is controlled by multiple factors, including the phosphorylation by the protein kinase AKT. We used the heterologous co-expression of human Bax and AKT1 in yeast to investigate how the kinase modulates the different steps underlying Bax activation. We found that AKT activated Bax and increased its cellular content. Both effects were dependent on Ser184, but a phosphorylation of this residue did not fully explain the effects of AKT. Additional experiments with mutants substituted on Ser184 suggested that the regulation of Bax dynamic equilibrium between the cytosol and mitochondria might be more tightly regulated by Bcl-xL when Bax is phosphorylated. PMID:26763134

  12. AKT activation drives the nuclear localization of CSE1L and a pro-oncogenic transcriptional activation in ovarian cancer cells

    SciTech Connect

    Lorenzato, Annalisa; Biolatti, Marta; Delogu, Giuseppe; Capobianco, Giampiero; Farace, Cristiano; Dessole, Salvatore; Cossu, Antonio; Tanda, Francesco; Madeddu, Roberto; Olivero, Martina; Di Renzo, Maria Flavia

    2013-10-15

    The human homolog of the yeast cse1 gene (CSE1L) is over-expressed in ovarian cancer. CSE1L forms complex with Ran and importin-α and has roles in nucleocytoplasmic traffic and gene expression. CSE1L accumulated in the nucleus of ovarian cancer cell lines, while it was localized also in the cytoplasm of other cancer cell lines. Nuclear localization depended on AKT, which was constitutively active in ovarian cancer cells, as the CSE1L protein translocated to the cytoplasm when AKT was inactivated. Moreover, the expression of a constitutively active AKT forced the translocation of CSE1L from the cytoplasm to the nucleus in other cancer cells. Nuclear accrual of CSE1L was associated to the nuclear accumulation of the phosphorylated Ran Binding protein 3 (RanBP3), which depended on AKT as well. Also in samples of human ovarian cancer, AKT activation was associated to nuclear accumulation of CSE1L and phosphorylation of RanBP3. Expression profiling of ovarian cancer cells after CSE1L silencing showed that CSE1L was required for the expression of genes promoting invasion and metastasis. In agreement, CSE1L silencing impaired motility and invasiveness of ovarian cancer cells. Altogether these data show that in ovarian cancer cells activated AKT by affecting RanBP3 phosphorylation determines the nuclear accumulation of CSE1L and likely the nuclear concentration of transcription factors conveying pro-oncogenic signals. - highlights: • CSE1L is a key player in nucleocytoplasmic traffic by forming complex with Ran. • AKT phosphorylates RanBP3 that regulates the nucleocytoplasmic gradient of Ran. • The activated oncogenic AKT drives the nuclear accumulation of CSE1L. • CSE1L in the nucleus up-regulates genes conveying pro-oncogenic signals. • CSE1L might contribute to tumor progression driven by the activated oncogenic AKT.

  13. Phosphorylation and activation of ubiquitin-specific protease-14 by Akt regulates the ubiquitin-proteasome system

    PubMed Central

    Xu, Daichao; Shan, Bing; Lee, Byung-Hoon; Zhu, Kezhou; Zhang, Tao; Sun, Huawang; Liu, Min; Shi, Linyu; Liang, Wei; Qian, Lihui; Xiao, Juan; Wang, Lili; Pan, Lifeng; Finley, Daniel; Yuan, Junying

    2015-01-01

    Regulation of ubiquitin-proteasome system (UPS), which controls the turnover of short-lived proteins in eukaryotic cells, is critical in maintaining cellular proteostasis. Here we show that USP14, a major deubiquitinating enzyme that regulates the UPS, is a substrate of Akt, a serine/threonine-specific protein kinase critical in mediating intracellular signaling transducer for growth factors. We report that Akt-mediated phosphorylation of USP14 at Ser432, which normally blocks its catalytic site in the inactive conformation, activates its deubiquitinating activity in vitro and in cells. We also demonstrate that phosphorylation of USP14 is critical for Akt to regulate proteasome activity and consequently global protein degradation. Since Akt can be activated by a wide range of growth factors and is under negative control by phosphoinosotide phosphatase PTEN, we suggest that regulation of UPS by Akt-mediated phosphorylation of USP14 may provide a common mechanism for growth factors to control global proteostasis and for promoting tumorigenesis in PTEN-negative cancer cells. DOI: http://dx.doi.org/10.7554/eLife.10510.001 PMID:26523394

  14. In vitro and In vivo Activity of Novel Small-Molecule Inhibitors Targeting the Pleckstrin Homology Domain of Protein Kinase B/AKT

    PubMed Central

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

    2010-01-01

    The phosphatidylinositol 3-kinase/AKT signaling pathway plays a critical role in activating survival and antiapoptotic pathways within cancer cells. Several studies have shown that this pathway is constitutively activated in many different cancer types. The goal of this study was to discover novel compounds that bind to the pleckstrin homology (PH) domain of AKT, thereby inhibiting AKT activation. Using proprietary docking software, 22 potential PH domain inhibitors were identified. Surface plasmon resonance spectroscopy was used to measure the binding of the compounds to the expressed PH domain of AKT followed by an in vitro activity screen in Panc-1 and MiaPaCa-2 pancreatic cancer cell lines. We identified a novel chemical scaffold in several of the compounds that binds selectively to the PH domain of AKT, inducing a decrease in AKT activation and causing apoptosis at low micromolar concentrations. Structural modifications of the scaffold led to compounds with enhanced inhibitory activity in cells. One compound, 4-dodecyl-N-(1,3,4-thiadiazol-2-yl) benzenesulfonamide, inhibited AKT and its downstream targets in cells as well as in pancreatic cancer cell xenografts in immunocompromised mice; it also exhibited good antitumor activity. In summary, a pharmacophore for PH domain inhibitors targeting AKT function was developed. Computer-aided modeling, synthesis, and testing produced novel AKT PH domain inhibitors that exhibit promising preclinical properties. PMID:19491272

  15. Elevated autocrine chemokine ligand 18 expression promotes oral cancer cell growth and invasion via Akt activation

    PubMed Central

    Hong, Yun; Wu, Tong; Chen, Xiaobing; Xia, Juan; Cheng, Bin

    2016-01-01

    Chemokine (C-C motif) ligand 18 (CCL18) has been implicated in the pathogenesis and progression of various cancers; however, in oral squamous cell carcinoma (OSCC), the role of CCL18 is unknown. In this study, we found that CCL18 was overexpressed in primary OSCC tissues and was associated with an advanced clinical stage. CCL18 was found in both the cytoplasm and cell membrane of OSCC cells and was predominantly produced by cancer epithelial cells, as opposed to tumor-infiltrating macrophages. In vitro studies indicated that the effects of endogenous CCL18 on OSCC cell growth, migration, and invasion could be blocked by treatment with a neutralizing anti-CCL18 antibody or CCL18 knockdown, while exogenous recombinant CCL18 (rCCL18) rescued those effects. Akt was activated in rCCL18-treated OSCC cells, while LY294002, a pan-PI3K inhibitor, abolished both endogenous and exogenous CCL18-induced OSCC cell invasion. In vivo, LY294002 treatment attenuated rCCL18-induced OSCC cell growth. Our results indicate that CCL18 acts in an autocrine manner via Akt activation to stimulate OSCC cell growth and invasion during OSCC progression. They also provide a potential therapeutic target for the treatment of oral cancer. PMID:26919103

  16. Acinus integrates AKT1 and subapoptotic caspase activities to regulate basal autophagy

    PubMed Central

    Nandi, Nilay; Tyra, Lauren K.; Stenesen, Drew

    2014-01-01

    How cellular stresses up-regulate autophagy is not fully understood. One potential regulator is the Drosophila melanogaster protein Acinus (Acn), which is necessary for autophagy induction and triggers excess autophagy when overexpressed. We show that cell type–specific regulation of Acn depends on proteolysis by the caspase Dcp-1. Basal Dcp-1 activity in developing photoreceptors is sufficient for this cleavage without a need for apoptosis to elevate caspase activity. On the other hand, Acn was stabilized by loss of Dcp-1 function or by the presence of a mutation in Acn that eliminates its conserved caspase cleavage site. Acn stability also was regulated by AKT1-mediated phosphorylation. Flies that expressed stabilized forms of Acn, either the phosphomimetic AcnS641,731D or the caspase-resistant AcnD527A, exhibited enhanced basal autophagy. Physiologically, these flies showed improvements in processes known to be autophagy dependent, including increased starvation resistance, reduced Huntingtin-induced neurodegeneration, and prolonged life span. These data indicate that AKT1 and caspase-dependent regulation of Acn stability adjusts basal autophagy levels. PMID:25332163

  17. B7-H4 Treatment of T Cells Inhibits ERK, JNK, p38, and AKT Activation

    PubMed Central

    Wang, Xiaojie; Hao, Jianqiang; Metzger, Daniel L.; Ao, Ziliang; Chen, Lieping; Ou, Dawei; Verchere, C. Bruce; Mui, Alice; Warnock, Garth L.

    2012-01-01

    B7-H4 is a newly identified B7 homolog that plays an important role in maintaining T-cell homeostasis by inhibiting T-cell proliferation and lymphokine-secretion. In this study, we investigated the signal transduction pathways inhibited by B7-H4 engagement in mouse T cells. We found that treatment of CD3+ T cells with a B7-H4.Ig fusion protein inhibits anti-CD3 elicited T-cell receptor (TCR)/CD28 signaling events, including phosphorylation of the MAP kinases, ERK, p38, and JNK. B7-H4.Ig treatment also inhibited the phosphorylation of AKT kinase and impaired its kinase activity as assessed by the phosphorylation of its endogenous substrate GSK-3. Expression of IL-2 is also reduced by B7-H4. In contrast, the phosphorylation state of the TCR proximal tyrosine kinases ZAP70 and lymphocyte-specific protein tyrosine kinase (LCK) are not affected by B7-H4 ligation. These results indicate that B7-H4 inhibits T-cell proliferation and IL-2 production through interfering with activation of ERK, JNK, and AKT, but not of ZAP70 or LCK. PMID:22238573

  18. Insulator dysfunction and oncogene activation in IDH mutant gliomas

    PubMed Central

    Flavahan, William A.; Drier, Yotam; Liau, Brian B.; Gillespie, Shawn M.; Venteicher, Andrew S.; Stemmer-Rachamimov, Anat O.; Suvà, Mario L.; Bernstein, Bradley E.

    2015-01-01

    Gain-of-function IDH mutations are initiating events that define major clinical and prognostic classes of gliomas1,2. Mutant IDH protein produces a novel onco-metabolite, 2-hydroxyglutarate (2-HG), that interferes with iron-dependent hydroxylases, including the TET family of 5′-methylcytosine hydroxylases3–7. TET enzymes catalyze a key step in the removal of DNA methylation8,9. IDH mutant gliomas thus manifest a CpG island methylator phenotype (G-CIMP)10,11, though the functional significance of this altered epigenetic state remains unclear. Here we show that IDH mutant gliomas exhibit hyper-methylation at CTCF binding sites, compromising binding of this methylation-sensitive insulator protein. Reduced CTCF binding is associated with loss of insulation between topological domains and aberrant gene activation. We specifically demonstrate that loss of CTCF at a domain boundary permits a constitutive enhancer to aberrantly interact with the receptor tyrosine kinase gene PDGFRA, a prominent glioma oncogene. Treatment of IDH mutant gliomaspheres with demethylating agent partially restores insulator function and down-regulates PDGFRA. Conversely, CRISPR-mediated disruption of the CTCF motif in IDH wildtype gliomaspheres up-regulates PDGFRA and increases proliferation. Our study suggests that IDH mutations promote gliomagenesis by disrupting chromosomal topology and allowing aberrant regulatory interactions that induce oncogene expression. PMID:26700815

  19. Abnormal grooming activity in Dab1(scm) (scrambler) mutant mice.

    PubMed

    Strazielle, C; Lefevre, A; Jacquelin, C; Lalonde, R

    2012-07-15

    Dab1(scm) mutant mice, characterized by cell ectopias and degeneration in cerebellum, hippocampus, and neocortex, were compared to non-ataxic controls for different facets of grooming caused by brief water immersions, as well as some non-grooming behaviors. Dab1(scm) mutants were strongly affected in their quantitative functional parameters, exhibiting higher starting latencies before grooming relative to non-ataxic littermates of the A/A strain, fewer grooming bouts, and grooming components of shorter duration, with an unequal regional distribution targeting almost totally the rostral part (head washing and forelimb licking) of the animal. Only bouts of a single grooming element were preserved. The cephalocaudal order of grooming elements appeared less disorganized, mutant and control mice initiating the grooming with head washing and forelimb licking prior to licking posterior parts. However, mutants differed from controls in that all their bouts were incomplete but uninterrupted, although intergroup difference for percentage of the incorrect transitions was not significant. In contrast to grooming, Dab1(scm) mice ambulated for a longer time. During walking episodes, they exhibited more body scratching than controls, possibly to compensate for the lack of licking different body parts. In conjunction with studies with other ataxic mice, these results indicate that the cerebellar cortex affects grooming activity and is consequently involved in executing various components, but not in its sequential organization, which requires other brain regions such as cerebral cortices or basal ganglia.

  20. N-methylhemeanthidine chloride, a novel Amaryllidaceae alkaloid, inhibits pancreatic cancer cell proliferation via down-regulating AKT activation

    SciTech Connect

    Guo, Guoli; Yao, Guangmin; Zhan, Guanqun; Hu, Yufeng; Yue, Ming; Cheng, Ling; Liu, Yaping; Ye, Qi; Qing, Guoliang; Zhang, Yonghui; Liu, Hudan

    2014-11-01

    We previously reported the isolation of a novel Amaryllidaceae alkaloid, N-methylhemeanthidine chloride (NMHC), from Zephyranthes candida, which exhibits potent cytotoxicity in a spectrum of tumor cells. However, the mechanism of action remains unclear. Using multiple cell lines derived from human pancreatic cancer, one of the most mortal and refractory human malignancies, we further studied the NMHC-mediated cytotoxicity and found that it induced drastic cytotoxicity in pancreatic cancer cells whereas an insignificant effect on a noncancerous cell line. The NMHC-mediated growth inhibition was more severe than the first-line chemotherapeutic agent gemcitabine, leading to cell cycle arrest, apoptotic death and decreased glycolysis. NMHC exerted its function through down-regulating AKT activation, and the ectopic expression of activated AKT rescued the growth inhibition. Consistently, NMHC injections in a pancreatic cancer xenograft model manifested the anti-tumor effect in vivo. Engrafted tumor cells underwent AKT attenuation and apoptotic death upon treatments. As such, we here demonstrate the AKT inhibition may be one of the mechanisms by which NMHC decreases tumor cell survival rate in vitro and in vivo. Our data thereby suggest that NMHC holds great promise as a potent chemotherapeutic agent against pancreatic cancer and sheds new light on obtaining such agents from natural products toward therapeutic purposes. - Highlights: • N-methylhemeanthidine chloride (NMHC) is a novel Amaryllidaceae alkaloid. • NMHC exhibits potent anti-neoplastic activity. • NMHC leads to cell cycle arrest, apoptotic death and decreased metabolism. • NMHC down-regulates the AKT signaling pathway.

  1. 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.

  2. Curcumin Attenuated Bupivacaine-Induced Neurotoxicity in SH-SY5Y Cells Via Activation of the Akt Signaling Pathway.

    PubMed

    Fan, You-Ling; Li, Heng-Chang; Zhao, Wei; Peng, Hui-Hua; Huang, Fang; Jiang, Wei-Hang; Xu, Shi-Yuan

    2016-09-01

    Bupivacaine is widely used for regional anesthesia, spinal anesthesia, and pain management. However, bupivacaine could cause neuronal injury. Curcumin, a low molecular weight polyphenol, has a variety of bioactivities and may exert neuroprotective effects against damage induced by some stimuli. In the present study, we tested whether curcumin could attenuate bupivacaine-induced neurotoxicity in SH-SY5Y cells. Cell injury was evaluated by examining cell viability, mitochondrial damage and apoptosis. We also investigated the levels of activation of the Akt signaling pathway and the effect of Akt inhibition by triciribine on cell injury following bupivacaine and curcumin treatment. Our findings showed that the bupivacaine treatment could induce neurotoxicity. Pretreatment of the SH-SY5Y cells with curcumin significantly attenuated bupivacaine-induced neurotoxicity. Interestingly, the curcumin treatment increased the levels of Akt phosphorylation. More significantly, the pharmacological inhibition of Akt abolished the cytoprotective effect of curcumin against bupivacaine-induced cell injury. Our data suggest that pretreating SH-SY5Y cells with curcumin provides a protective effect on bupivacaine-induced neuronal injury via activation of the Akt signaling pathway.

  3. Pyrrolidinium fullerene induces apoptosis by activation of procaspase-9 via suppression of Akt in primary effusion lymphoma

    SciTech Connect

    Watanabe, Tadashi; Nakamura, Shigeo; Ono, Toshiya; Ui, Sadaharu; Yagi, Syota; Kagawa, Hiroki; Watanabe, Hisami; Ohe, Tomoyuki; Mashino, Tadahiko; Fujimuro, Masahiro

    2014-08-15

    Highlights: • Seven fullerenes were evaluated in terms of their cytotoxic effects on B-lymphomas. • Pyrrolidinium fullerene induced apoptosis of KSHV-infected B-lymphoma PEL cells. • The activation of Akt is essential for PEL cell survival. • Pyrrolidinium fullerene activated caspase-9 by inactivating Akt in PEL cells. • Pyrrolidinium fullerene have potential as novel drugs for the treatment of PEL. - Abstract: Primary effusion lymphoma (PEL) is a subtype of non-Hodgkin’s B-cell lymphoma and is an aggressive neoplasm caused by Kaposi’s sarcoma-associated herpesvirus (KSHV) in immunosuppressed patients. In general, PEL cells are derived from post-germinal center B-cells and are infected with KSHV. To evaluate potential novel anti-tumor compounds against KSHV-associated PEL, seven water-soluble fullerene derivatives were evaluated as potential drug candidates for the treatment of PEL. Herein, we discovered a pyrrolidinium fullerene derivative, 1,1,1′,1′-tetramethyl [60]fullerenodipyrrolidinium diiodide, which induced apoptosis of PEL cells via a novel mechanism, the caspase-9 activation by suppressing the caspase-9 phosphorylation, causing caspase-9 inactivation. Pyrrolidinium fullerene treatment reduced significantly the viability of PEL cells compared with KSHV-uninfected lymphoma cells, and induced the apoptosis of PEL cells by activating caspase-9 via procaspase-9 cleavage. Pyrrolidinium fullerene additionally reduced the Ser473 phosphorylation of Akt and Ser196 of procaspase-9. Ser473-phosphorylated Akt (i.e., activated Akt) phosphorylates Ser196 in procaspase-9, causing inactivation of procaspase-9. We also demonstrated that Akt inhibitors suppressed the proliferation of PEL cells compared with KSHV-uninfected cells. Our data therefore suggest that Akt activation is essential for cell survival in PEL and a pyrrolidinium fullerene derivative induced apoptosis by activating caspase-9 via suppression of Akt in PEL cells. In addition, we evaluated

  4. Modification of Caffeic Acid with Pyrrolidine Enhances Antioxidant Ability by Activating AKT/HO-1 Pathway in Heart

    PubMed Central

    Ku, Hui-Chun; Lee, Shih-Yi; Yang, Kai-Chien; Kuo, Yueh-Hsiung; Su, Ming-Jai

    2016-01-01

    Overproduction of free radicals during ischemia/reperfusion (I/R) injury leads to an interest in using antioxidant therapy. Activating an endogenous antioxidant signaling pathway is more important due to the fact that the free radical scavenging behavior in vitro does not always correlate with a cytoprotection effect in vivo. Caffeic acid (CA), an antioxidant, is a major phenolic constituent in nature. Pyrrolidinyl caffeamide (PLCA), a derivative of CA, was compared with CA for their antioxidant and cytoprotective effects. Our results indicate that CA and PLCA exert the same ability to scavenge DPPH in vitro. In response to myocardial I/R stress, PLCA was shown to attenuate lipid peroxydation and troponin release more than CA. These responses were accompanied with a prominent elevation in AKT and HO-1 expression and a preservation of mnSOD expression and catalase activity. PLCA also improved cell viability and alleviated the intracellular ROS level more than CA in cardiomyocytes exposed to H2O2. When inhibiting the AKT or HO-1 pathways, PLCA lost its ability to recover mnSOD expression and catalase activity to counteract with oxidative stress, suggesting AKT/HO-1 pathway activation by PLCA plays an important role. In addition, inhibition of AKT signaling further abolished HO-1 activity, while inhibition of HO-1 signaling attenuated AKT expression, indicating cross-talk between the AKT and HO-1 pathways. These protective effects may contribute to the cardiac function improvement by PLCA. These findings provide new insight into therapeutic approaches using a modified natural compound against oxidative stress from myocardial injuries. PMID:26845693

  5. Modification of Caffeic Acid with Pyrrolidine Enhances Antioxidant Ability by Activating AKT/HO-1 Pathway in Heart.

    PubMed

    Ku, Hui-Chun; Lee, Shih-Yi; Yang, Kai-Chien; Kuo, Yueh-Hsiung; Su, Ming-Jai

    2016-01-01

    Overproduction of free radicals during ischemia/reperfusion (I/R) injury leads to an interest in using antioxidant therapy. Activating an endogenous antioxidant signaling pathway is more important due to the fact that the free radical scavenging behavior in vitro does not always correlate with a cytoprotection effect in vivo. Caffeic acid (CA), an antioxidant, is a major phenolic constituent in nature. Pyrrolidinyl caffeamide (PLCA), a derivative of CA, was compared with CA for their antioxidant and cytoprotective effects. Our results indicate that CA and PLCA exert the same ability to scavenge DPPH in vitro. In response to myocardial I/R stress, PLCA was shown to attenuate lipid peroxydation and troponin release more than CA. These responses were accompanied with a prominent elevation in AKT and HO-1 expression and a preservation of mnSOD expression and catalase activity. PLCA also improved cell viability and alleviated the intracellular ROS level more than CA in cardiomyocytes exposed to H2O2. When inhibiting the AKT or HO-1 pathways, PLCA lost its ability to recover mnSOD expression and catalase activity to counteract with oxidative stress, suggesting AKT/HO-1 pathway activation by PLCA plays an important role. In addition, inhibition of AKT signaling further abolished HO-1 activity, while inhibition of HO-1 signaling attenuated AKT expression, indicating cross-talk between the AKT and HO-1 pathways. These protective effects may contribute to the cardiac function improvement by PLCA. These findings provide new insight into therapeutic approaches using a modified natural compound against oxidative stress from myocardial injuries. PMID:26845693

  6. Treadmill exercise reduces spinal cord injury-induced apoptosis by activating the PI3K/Akt pathway in rats.

    PubMed

    Jung, Sun-Young; Kim, Dae-Young; Yune, Tae Young; Shin, Dong-Hoon; Baek, Sang-Bin; Kim, Chang-Ju

    2014-03-01

    Apoptosis occurring secondary to spinal cord injury (SCI) causes further neural damage and functional loss. In this study, a rat model was used to investigate the effect of treadmill exercise on SCI-induced apoptosis and expression of neurotrophic factors. To produce SCI, a contusion injury (10 g × 25 mm) was applied subsequent to laminectomy at the T9-T10 level. Following SCI, treadmill exercise was performed for six weeks. Hindlimb motor function was evaluated with a grid-walking test. The expression of neurotrophic factors and the level of apoptosis at the site of SCI were determined by western blotting. SCI reduced hindlimb motor function and suppressed expression of neurotrophin (NT)-3 and insulin-like growth factor (IGF)-1. Expression of phosphatidylinositol 3-kinase (PI3K), the ratio of phosphorylated Akt to Akt (pAkt/Akt) and the ratio of B-cell lymphoma 2 (Bcl-2) to Bax (Bcl-2/Bax) were decreased, and cleaved caspase-3 expression was increased by SCI. Treadmill exercise enhanced hindlimb motor function and increased expression of nerve growth factor (NGF), NT-3 and IGF-1 in the SCI rats. Treadmill exercise increased PI3K expression, the pAkt/Akt and the Bcl-2/Bax ratios, and suppressed cleaved caspase-3 expression in the injured spinal cord. This study demonstrated that treadmill exercise promotes the recovery of motor function by suppressing apoptosis in the injured spinal cord. The beneficial effect of exercise may be attributed to the increase in expression of neurotrophic factors via activation of the PI3K/Akt pathway.

  7. Fluorescence resonance energy transfer microscopy as demonstrated by measuring the activation of the serine/threonine kinase Akt

    PubMed Central

    Broussard, Joshua A; Rappaz, Benjamin; Webb, Donna J; Brown, Claire M

    2013-01-01

    This protocol describes procedures for performing fluorescence resonance energy transfer (FRET) microscopy analysis by three different methods: acceptor photobleaching, sensitized emission and spectral imaging. We also discuss anisotropy and fluorescence lifetime imaging microscopy–based FRET techniques. By using the specific example of the FRET probe Akind (Akt indicator), which is a version of Akt modified such that FRET occurs when the probe is activated by phosphorylation, indicating Akt activation. The protocol provides a detailed step-by-step description of sample preparation, image acquisition and analysis, including control samples, image corrections and the generation of quantitative FRET/CFP ratio images for both sensitized emission and spectral imaging. The sample preparation takes 2 d, equipment setup takes 2–3 h and image acquisition and analysis take 6–8 h. PMID:23306460

  8. TBP mutants defective in activated transcription in vivo.

    PubMed Central

    Arndt, K M; Ricupero-Hovasse, S; Winston, F

    1995-01-01

    The TATA box binding protein (TBP) plays a central and essential role in transcription initiation. At TATA box-containing genes transcribed by RNA polymerase II, TBP binds to the promoter and initiates the assembly of a multiprotein preinitiation complex. Several studies have suggested that binding of TBP to the TATA box is an important regulatory step in transcription initiation in vitro. To determine whether TBP is a target of regulatory factors in vivo, we performed a genetic screen in yeast for TBP mutants defective in activated transcription. One class of TBP mutants identified in this screen comprises inositol auxotrophs that are also defective in using galactose as a carbon source. These phenotypes are due to promoter-specific defects in transcription initiation that are governed by the upstream activating sequence (UAS) and apparently not by the sequence of the TATA element. The finding that these TBP mutants are severely impaired in DNA binding in vitro suggests that transcription initiation at certain genes is regulated at the level of TATA box binding by TBP in vivo. Images PMID:7729424

  9. Transgene expression study of CXCR4 active mutants

    PubMed Central

    Sharma, Menka; Afrin, Farhat; Tripathi, Rajendra P; Gangenahalli, Gurudutta

    2014-01-01

    Homing and engraftment, a determining factor in hematopoietic stem cell transplantation success is defined as a process through which hematopoietic stem/progenitor cells (HSPCs) lodge recipient bone marrow. SDF-1/CXCR4 axis acts as a principle regulator in homing and engraftment, however, CXCR4 signaling is dependent upon expression of CXCR4 and its ligand SDF-1, which is highly dynamic. Hence, present investigation was aimed to explore the potential of CXCR4 constitutive active mutants (CXCR4-CAMs) in overcoming the limitation of CXCR4 signaling and up-modulate its efficiency in homing and engraftment. Regulated transgene expression study of these mutants revealed their significantly enhanced cell adhesion efficiency to endothelium and extracellular matrix protein. This altogether indicates promising prospects of CXCR4-CAMs in research aimed to improve HSPCs engraftment efficiency. PMID:25482641

  10. Knockdown of Pokemon protein expression inhibits hepatocellular carcinoma cell proliferation by suppression of AKT activity.

    PubMed

    Zhu, Xiaosan; Dai, Yichen; Chen, Zhangxin; Xie, Junpei; Zeng, Wei; Lin, Yuanyuan

    2013-01-01

    Overexpression of Pokemon, which is an erythroid myeloid ontogenic factor protein, occurs in different cancers, including hepatocellular carcinoma (HCC). Pokemon is also reported to have an oncogenic activity in various human cancers. This study investigated the effect of Pokemon knockdown on the regulation of HCC growth. POK shRNA suppressed the expression of Pokemon protein in HepG2 cells compared to the negative control vector-transfected HCC cells. Pokemon knockdown also reduced HCC cell viability and enhanced cisplatin-induced apoptosis in HCC cells. AKT activation and the expression of various cell cycle-related genes were inhibited following Pokemon knockdown. These data demonstrate that Pokemon may play a role in HCC progression, suggesting that inhibition of Pokemon expression using Pokemon shRNA should be further evaluated as a novel target for the control of HCC. PMID:23924858

  11. Knockdown of Pokemon protein expression inhibits hepatocellular carcinoma cell proliferation by suppression of AKT activity.

    PubMed

    Zhu, Xiaosan; Dai, Yichen; Chen, Zhangxin; Xie, Junpei; Zeng, Wei; Lin, Yuanyuan

    2013-01-01

    Overexpression of Pokemon, which is an erythroid myeloid ontogenic factor protein, occurs in different cancers, including hepatocellular carcinoma (HCC). Pokemon is also reported to have an oncogenic activity in various human cancers. This study investigated the effect of Pokemon knockdown on the regulation of HCC growth. POK shRNA suppressed the expression of Pokemon protein in HepG2 cells compared to the negative control vector-transfected HCC cells. Pokemon knockdown also reduced HCC cell viability and enhanced cisplatin-induced apoptosis in HCC cells. AKT activation and the expression of various cell cycle-related genes were inhibited following Pokemon knockdown. These data demonstrate that Pokemon may play a role in HCC progression, suggesting that inhibition of Pokemon expression using Pokemon shRNA should be further evaluated as a novel target for the control of HCC.

  12. EGFR inhibition evokes innate drug resistance in lung cancer cells by preventing Akt activity and thus inactivating Ets-1 function.

    PubMed

    Phuchareon, Janyaporn; McCormick, Frank; Eisele, David W; Tetsu, Osamu

    2015-07-21

    Nonsmall cell lung cancer (NSCLC) is the leading cause of cancer death worldwide. About 14% of NSCLCs harbor mutations in epidermal growth factor receptor (EGFR). Despite remarkable progress in treatment with tyrosine kinase inhibitors (TKIs), only 5% of patients achieve tumor reduction >90%. The limited primary responses are attributed partly to drug resistance inherent in the tumor cells before therapy begins. Recent reports showed that activation of receptor tyrosine kinases (RTKs) is an important determinant of this innate drug resistance. In contrast, we demonstrate that EGFR inhibition promotes innate drug resistance despite blockade of RTK activity in NSCLC cells. EGFR TKIs decrease both the mitogen-activated protein kinase (MAPK) and Akt protein kinase pathways for a short time, after which the Ras/MAPK pathway becomes reactivated. Akt inhibition selectively blocks the transcriptional activation of Ets-1, which inhibits its target gene, dual specificity phosphatase 6 (DUSP6), a negative regulator specific for ERK1/2. As a result, ERK1/2 is activated. Furthermore, elevated c-Src stimulates Ras GTP-loading and activates Raf and MEK kinases. These observations suggest that not only ERK1/2 but also Akt activity is essential to maintain Ets-1 in an active state. Therefore, despite high levels of ERK1/2, Ets-1 target genes including DUSP6 and cyclins D1, D3, and E2 remain suppressed by Akt inhibition. Reduction of DUSP6 in combination with elevated c-Src renews activation of the Ras/MAPK pathway, which enhances cell survival by accelerating Bim protein turnover. Thus, EGFR TKIs evoke innate drug resistance by preventing Akt activity and inactivating Ets-1 function in NSCLC cells.

  13. Expression of constitutively activated Akt in the mammary gland leads to excess lipid synthesis during pregnancy and lactation.

    PubMed

    Schwertfeger, Kathryn L; McManaman, James L; Palmer, Carol A; Neville, Margaret C; Anderson, Steven M

    2003-06-01

    Expression of constitutively activated Akt in the mammary glands of transgenic mice results in a delay in post-lactational involution. We now report precocious lipid accumulation in the alveolar epithelium of mouse mammary tumor virus-myr-Akt transgenic mice accompanied by a lactation defect that results in a 50% decrease in litter weight over the first 9 days of lactation. Although ductal structures and alveolar units develop normally during pregnancy, cytoplasmic lipid droplets appeared precociously in mammary epithelial cells in early pregnancy and were accompanied by increased expression of adipophilin, which is associated with lipid droplets. By late pregnancy the lipid droplets had become significantly larger than in nontransgenic mice, and they persisted into lactation. The fat content of milk from lactating myr-Akt transgenic mice was 65-70% by volume compared to 25-30% in wild-type mice. The diminished growth of pups nursed by transgenic mothers could result from the high viscosity of the milk and the inability of the pups to remove sufficient quantities of milk by suckling. Transduction of the CIT3 mammary epithelial cell line with a recombinant human adenovirus encoding myr-Akt resulted in an increase in glucose transport and lipid biosynthesis, suggesting that Akt plays an important role in regulation of lipid metabolism. PMID:12700340

  14. Lysophosphatidic acid induces cell migration through the selective activation of Akt1

    PubMed Central

    Kim, Eun Kyoung; Yun, Sung Ji; Do, Kee Hun; Kim, Min Sung; Cho, Mong; Suh, Dong-Soo; Kim, Chi Dae; Kim, Jae Ho; Birnbaum, Morris J.

    2008-01-01

    Akt plays pivotal roles in many physiological responses including growth, proliferation, survival, metabolism, and migration. In the current studies, we have evaluated the isoform-specific role of akt in lysophosphatidic acid (LPA)-induced cell migration. Ascites from ovarian cancer patients (AOCP) induced mouse embryo fibroblast (MEF) cell migration in a dose-dependent manner. On the other hand, ascites from liver cirrhosis patients (ALCP) did not induce MEF cell migration. AOCP-induced MEF cell migration was completely blocked by pre-treatment of cells with LPA receptor antagonist, Ki16425. Both LPA- and AOCP-induced MEF cell migration was completely attenuated by PI3K inhibitor, LY294002. Furthermore, cells lacking Akt1 displayed defect in LPA-induced cell migration. Re-expression of Akt1 in DKO (Akt1-/-Akt2-/-) cells restored LPA-induced cell migration, whereas re-expression of Akt2 in DKO cells could not restore the LPA-induced cell migration. Finally, Akt1 was selectively phosphorylated by LPA and AOCP stimulation. These results suggest that LPA is a major factor responsible for AOCP-induced cell migration and signaling specificity of Akt1 may dictate LPA-induced cell migration. PMID:18779657

  15. AKT activation drives the nuclear localization of CSE1L and a pro-oncogenic transcriptional activation in ovarian cancer cells.

    PubMed

    Lorenzato, Annalisa; Biolatti, Marta; Delogu, Giuseppe; Capobianco, Giampiero; Farace, Cristiano; Dessole, Salvatore; Cossu, Antonio; Tanda, Francesco; Madeddu, Roberto; Olivero, Martina; Di Renzo, Maria Flavia

    2013-10-15

    The human homolog of the yeast cse1 gene (CSE1L) is over-expressed in ovarian cancer. CSE1L forms complex with Ran and importin-α and has roles in nucleocytoplasmic traffic and gene expression. CSE1L accumulated in the nucleus of ovarian cancer cell lines, while it was localized also in the cytoplasm of other cancer cell lines. Nuclear localization depended on AKT, which was constitutively active in ovarian cancer cells, as the CSE1L protein translocated to the cytoplasm when AKT was inactivated. Moreover, the expression of a constitutively active AKT forced the translocation of CSE1L from the cytoplasm to the nucleus in other cancer cells. Nuclear accrual of CSE1L was associated to the nuclear accumulation of the phosphorylated Ran Binding protein 3 (RanBP3), which depended on AKT as well. Also in samples of human ovarian cancer, AKT activation was associated to nuclear accumulation of CSE1L and phosphorylation of RanBP3. Expression profiling of ovarian cancer cells after CSE1L silencing showed that CSE1L was required for the expression of genes promoting invasion and metastasis. In agreement, CSE1L silencing impaired motility and invasiveness of ovarian cancer cells. Altogether these data show that in ovarian cancer cells activated AKT by affecting RanBP3 phosphorylation determines the nuclear accumulation of CSE1L and likely the nuclear concentration of transcription factors conveying pro-oncogenic signals. PMID:23948303

  16. SG2NA enhances cancer cell survival by stabilizing DJ-1 and thus activating Akt

    SciTech Connect

    Tanti, Goutam Kumar Pandey, Shweta; Goswami, Shyamal K.

    2015-08-07

    SG2NA in association with striatin and zinedin forms a striatin family of WD-40 repeat proteins. This family of proteins functions as scaffold in different signal transduction pathways. They also act as a regulatory subunit of protein phosphatase 2A. We have shown that SG2NA which evolved first in the metazoan evolution among the striatin family members expresses different isoforms generated out of alternative splicing. We have also shown that SG2NA protects cells from oxidative stress by recruiting DJ-1 and Akt to mitochondria and membrane in the post-mitotic neuronal cells. DJ-1 is both cancer and Parkinson's disease related protein. In the present study we have shown that SG2NA protects DJ-1 from proteasomal degradation in cancer cells. Hence, downregulation of SG2NA reduces DJ-1/Akt colocalization in cancer cells resulting in the reduction of anchorage dependent and independent growth. Thus SG2NA enhances cancer cell survival. Reactive oxygen species enhances SG2NA, DJ-1 and Akt trimerization. Removal of the reactive oxygen species by N-acetyl-cysteine thus reduces cancer cell growth. - Highlights: • Reactive oxygen species (ROS) play potential role in cancer cell proliferation. • It enhances the association between DJ-1 and Akt mediated by SG2NA. • In cancer cells SG2NA stabilizes DJ-1 by inhibiting it from proteosomal degradation. • DJ-1 then activates Akt and cancer cells get their property of enhanced proliferation by sustained activation of Akt. • Further study on this field could lead to new target for cancer therapy.

  17. CHIP promotes thyroid cancer proliferation via activation of the MAPK and AKT pathways.

    PubMed

    Zhang, Li; Liu, Lianyong; He, Xiaohua; Shen, Yunling; Liu, Xuerong; Wei, Jing; Yu, Fang; Tian, Jianqing

    2016-08-26

    The carboxyl terminus of Hsp70-interacting protein (CHIP) is a U box-type ubiquitin ligase that plays crucial roles in various biological processes, including tumor progression. To date, the functional mechanism of CHIP in thyroid cancer remains unknown. Here, we obtained evidence of upregulation of CHIP in thyroid cancer tissues and cell lines. CHIP overexpression markedly enhanced thyroid cancer cell viability and colony formation in vitro and accelerated tumor growth in vivo. Conversely, CHIP knockdown impaired cell proliferation and tumor growth. Notably, CHIP promoted cell growth through activation of MAPK and AKT pathways, subsequently decreasing p27 and increasing cyclin D1 and p-FOXO3a expression. Our findings collectively indicate that CHIP functions as an oncogene in thyroid cancer, and is therefore a potential therapeutic target for this disease.

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

    SciTech Connect

    Wang Lei; Sasai, Ken Akagi, Tsuyoshi; Tanaka, Shinya

    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 by 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.

  19. 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.

  20. The Akt1/IL-6/STAT3 pathway regulates growth of lung tumor initiating cells

    PubMed Central

    Malanga, Donatella; De Marco, Carmela; Guerriero, Ilaria; Colelli, Fabiana; Rinaldo, Nicola; Scrima, Marianna; Mirante, Teresa; De Vitis, Claudia; Zoppoli, Pietro; Ceccarelli, Michele; Riccardi, Miriam; Ravo, Maria; Weisz, Alessandro; Federico, Antonella; Franco, Renato; Rocco, Gaetano; Mancini, Rita; Rizzuto, Antonia; Gulletta, Elio; Ciliberto, Gennaro; Viglietto, Giuseppe

    2015-01-01

    Here we report that the PI3K/Akt1/IL-6/STAT3 signalling pathway regulates generation and stem cell-like properties of Non-Small Cell Lung Cancer (NSCLC) tumor initiating cells (TICs). Mutant Akt1, mutant PIK3CA or PTEN loss enhances formation of lung cancer spheroids (LCS), self-renewal, expression of stemness markers and tumorigenic potential of human immortalized bronchial cells (BEAS-2B) whereas Akt inhibition suppresses these activities in established (NCI-H460) and primary NSCLC cells. Matched microarray analysis of Akt1-interfered cells and LCSs identified IL-6 as a critical target of Akt signalling in NSCLC TICs. Accordingly, suppression of Akt in NSCLC cells decreases IL-6 levels, phosphorylation of IkK and IkB, NF-kB transcriptional activity, phosphorylation and transcriptional activity of STAT3 whereas active Akt1 up-regulates them. Exposure of LCSs isolated from NSCLC cells to blocking anti-IL-6 mAbs, shRNA to IL-6 receptor or to STAT3 markedly reduces the capability to generate LCSs, to self-renew and to form tumors, whereas administration of IL-6 to Akt-interfered cells restores the capability to generate LCSs. Finally, immunohistochemical studies in NSCLC patients demonstrated a positive correlative trend between activated Akt, IL-6 expression and STAT3 phosphorylation (n = 94; p < 0.05). In conclusion, our data indicate that aberrant Akt signalling contributes to maintaining stemness in lung cancer TICs through a NF-kB/IL-6/STAT3 pathway and provide novel potential therapeutic targets for eliminating these malignant cells in NSCLC. PMID:26486080

  1. Bacillus pumilus Cyanide Dihydratase Mutants with Higher Catalytic Activity

    PubMed Central

    Crum, Mary A.; Sewell, B. Trevor; Benedik, Michael J.

    2016-01-01

    Cyanide degrading nitrilases are noted for their potential to detoxify industrial wastewater contaminated with cyanide. However, such application would benefit from an improvement to characteristics such as their catalytic activity and stability. Following error-prone PCR for random mutagenesis, several cyanide dihydratase mutants from Bacillus pumilus were isolated based on improved catalysis. Four point mutations, K93R, D172N, A202T, and E327K were characterized and their effects on kinetics, thermostability and pH tolerance were studied. K93R and D172N increased the enzyme’s thermostability whereas E327K mutation had a less pronounced effect on stability. The D172N mutation also increased the affinity of the enzyme for its substrate at pH 7.7 but lowered its kcat. However, the A202T mutation, located in the dimerization or the A surface, destabilized the protein and abolished its activity. No significant effect on activity at alkaline pH was observed for any of the purified mutants. These mutations help confirm the model of CynD and are discussed in the context of the protein–protein interfaces leading to the protein quaternary structure. PMID:27570524

  2. Bacillus pumilus Cyanide Dihydratase Mutants with Higher Catalytic Activity.

    PubMed

    Crum, Mary A; Sewell, B Trevor; Benedik, Michael J

    2016-01-01

    Cyanide degrading nitrilases are noted for their potential to detoxify industrial wastewater contaminated with cyanide. However, such application would benefit from an improvement to characteristics such as their catalytic activity and stability. Following error-prone PCR for random mutagenesis, several cyanide dihydratase mutants from Bacillus pumilus were isolated based on improved catalysis. Four point mutations, K93R, D172N, A202T, and E327K were characterized and their effects on kinetics, thermostability and pH tolerance were studied. K93R and D172N increased the enzyme's thermostability whereas E327K mutation had a less pronounced effect on stability. The D172N mutation also increased the affinity of the enzyme for its substrate at pH 7.7 but lowered its k cat. However, the A202T mutation, located in the dimerization or the A surface, destabilized the protein and abolished its activity. No significant effect on activity at alkaline pH was observed for any of the purified mutants. These mutations help confirm the model of CynD and are discussed in the context of the protein-protein interfaces leading to the protein quaternary structure. PMID:27570524

  3. AKT/SGK-sensitive phosphorylation of GSK3 in the regulation of L-selectin and perforin expression as well as activation induced cell death of T-lymphocytes

    SciTech Connect

    Bhavsar, Shefalee K.; Merches, Katja; Bobbala, Diwakar; Lang, Florian

    2012-08-17

    Highlights: Black-Right-Pointing-Pointer Akt/SGK dependent phosphorylation of GSK3{alpha},{beta} regulates T lymphocytes. Black-Right-Pointing-Pointer T cells from mice expressing Akt/SGK insensitive GSK3{alpha},{beta} (gsk3{sup KI}) release less IL-2. Black-Right-Pointing-Pointer CD4{sup +} cells from gsk3{sup KI} mice express less CD62L. Black-Right-Pointing-Pointer CD8{sup +} cells from gsk3{sup KI} mice are relatively resistant to activation induced cell death. Black-Right-Pointing-Pointer Perforin expression is enhanced in gsk3{sup KI} T cells. -- Abstract: Survival and function of T-lymphocytes critically depends on phosphoinositide (PI) 3 kinase. PI3 kinase signaling includes the PKB/Akt and SGK dependent phosphorylation and thus inhibition of glycogen synthase kinase GSK3{alpha},{beta}. Lithium, a known unspecific GSK3 inhibitor protects against experimental autoimmune encephalomyelitis. The present study explored, whether Akt/SGK-dependent regulation of GSK3 activity is a determinant of T cell survival and function. Experiments were performed in mutant mice in which Akt/SGK-dependent GSK3{alpha},{beta} inhibition was disrupted by replacement of the serine residue in the respective SGK/Akt-phosphorylation consensus sequence by alanine (gsk3{sup KI}). T cells from gsk3{sup KI} mice were compared to T cells from corresponding wild type mice (gsk3{sup WT}). As a result, in gsk3{sup KI} CD4{sup +} cells surface CD62L (L-selectin) was significantly less abundant than in gsk3{sup WT} CD4{sup +} cells. Upon activation in vitro T cells from gsk3{sup KI} mice reacted with enhanced perforin production and reduced activation induced cell death. Cytokine production was rather reduced in gsk3{sup KI} T cells, suggesting that GSK3 induces effector function in CD8{sup +} T cells. In conclusion, PKB/Akt and SGK sensitive phosphorylation of GSK3{alpha},{beta} is a potent regulator of perforin expression and activation induced cell death in T lymphocytes.

  4. A Common Variant at the 14q32 Endometrial Cancer Risk Locus Activates AKT1 through YY1 Binding.

    PubMed

    Painter, Jodie N; Kaufmann, Susanne; O'Mara, Tracy A; Hillman, Kristine M; Sivakumaran, Haran; Darabi, Hatef; Cheng, Timothy H T; Pearson, John; Kazakoff, Stephen; Waddell, Nicola; Hoivik, Erling A; Goode, Ellen L; Scott, Rodney J; Tomlinson, Ian; Dunning, Alison M; Easton, Douglas F; French, Juliet D; Salvesen, Helga B; Pollock, Pamela M; Thompson, Deborah J; Spurdle, Amanda B; Edwards, Stacey L

    2016-06-01

    A recent meta-analysis of multiple genome-wide association and follow-up endometrial cancer case-control datasets identified a novel genetic risk locus for this disease at chromosome 14q32.33. To prioritize the functional SNP(s) and target gene(s) at this locus, we employed an in silico fine-mapping approach using genotyped and imputed SNP data for 6,608 endometrial cancer cases and 37,925 controls of European ancestry. Association and functional analyses provide evidence that the best candidate causal SNP is rs2494737. Multiple experimental analyses show that SNP rs2494737 maps to a silencer element located within AKT1, a member of the PI3K/AKT/MTOR intracellular signaling pathway activated in endometrial tumors. The rs2494737 risk A allele creates a YY1 transcription factor-binding site and abrogates the silencer activity in luciferase assays, an effect mimicked by transfection of YY1 siRNA. Our findings suggest YY1 is a positive regulator of AKT1, mediating the stimulatory effects of rs2494737 increasing endometrial cancer risk. Identification of an endometrial cancer risk allele within a member of the PI3K/AKT signaling pathway, more commonly activated in tumors by somatic alterations, raises the possibility that well tolerated inhibitors targeting this pathway could be candidates for evaluation as chemopreventive agents in individuals at high risk of developing endometrial cancer.

  5. Urocortin 3 activates AMPK and AKT pathways and enhances glucose disposal in rat skeletal muscle

    PubMed Central

    Roustit, Manon M; Vaughan, Joan M; Jamieson, Pauline M; Cleasby, Mark E

    2014-01-01

    Insulin resistance (IR) in skeletal muscle is an important component of both type 2 diabetes and the syndrome of sarcopaenic obesity, for which there are no effective therapies. Urocortins (UCNs) are not only well established as neuropeptides but also have their roles in metabolism in peripheral tissues. We have shown recently that global overexpression of UCN3 resulted in muscular hypertrophy and resistance to the adverse metabolic effects of a high-fat diet. Herein, we aimed to establish whether short-term local UCN3 expression could enhance glucose disposal and insulin signalling in skeletal muscle. UCN3 was found to be expressed in right tibialis cranialis and extensor digitorum longus muscles of rats by in vivo electrotransfer and the effects studied vs the contralateral muscles after 1 week. No increase in muscle mass was detected, but test muscles showed 19% larger muscle fibre diameter (P=0.030), associated with increased IGF1 and IGF1 receptor mRNA and increased SER256 phosphorylation of forkhead transcription factor. Glucose clearance into the test muscles after an intraperitoneal glucose load was increased by 23% (P=0.018) per unit mass, associated with increased GLUT1 (34% increase; P=0.026) and GLUT4 (48% increase; P=0.0009) proteins, and significantly increased phosphorylation of insulin receptor substrate-1, AKT, AKT substrate of 160 kDa, glycogen synthase kinase-3β, AMP-activated protein kinase and its substrate acetyl coA carboxylase. Thus, UCN3 expression enhances glucose disposal and signalling in muscle by an autocrine/paracrine mechanism that is separate from its pro-hypertrophic effects, implying that such a manipulation may have promised for the treatment of IR syndromes including sarcopaenic obesity. PMID:25122003

  6. Dopamine D2 receptor-mediated Akt/PKB signalling: initiation by the D2S receptor and role in quinpirole-induced behavioural activation.

    PubMed

    Chen, Han-Ting; Ruan, Nan-Yu; Chen, Jin-Chung; Lin, Tzu-Yung

    2012-09-24

    The short and long isoforms of the dopamine D2 receptor (D2S and D2L respectively) are highly expressed in the striatum. Functional D2 receptors activate an intracellular signalling pathway that includes a cAMP-independent route involving Akt/GSK3 (glycogen synthase kinase 3). To investigate the Akt/GSK3 response to the seldom-studied D2S receptor, we established a rat D2S receptor-expressing cell line [HEK (human embryonic kidney)-293/rD2S]. We found that in HEK-293/rD2S cells, the D2/D3 agonists bromocriptine and quinpirole significantly induced Akt and GSK3 phosphorylation, as well as ERK1/2 (extracellular-signal-regulated kinase 1/2) activation. The D2S receptor-induced Akt signals were profoundly inhibited by the internalization blockers monodansyl cadaverine and concanavalin A. Activation of the D2S receptor in HEK-293/rD2S cells appeared to trigger Akt/phospho-Akt translocation to the cell membrane. In addition to our cell culture experiments, we studied D2 receptor-dependent Akt in vivo by systemic administration of the D2/D3 agonist quinpirole. The results show that quinpirole evoked Akt-Ser473 phosphorylation in the ventral striatum. Furthermore, intra-accumbens administration of wortmannin, a PI3K (phosphoinositide 3-kinase) inhibitor, significantly suppressed the quinpirole-evoked behavioural activation. Overall, we demonstrate that activation of the dopamine D2S receptor stimulates Akt/GSK3 signalling. In addition, in vivo Akt activity in the ventral striatum appears to play an important role in systemic D2/D3 agonist-induced behavioural activation.

  7. Dopamine D2 receptor-mediated Akt/PKB signalling: initiation by the D2S receptor and role in quinpirole-induced behavioural activation

    PubMed Central

    Chen, Han-Ting; Ruan, Nan-Yu; Chen, Jin-Chung; Lin, Tzu-Yung

    2012-01-01

    The short and long isoforms of the dopamine D2 receptor (D2S and D2L respectively) are highly expressed in the striatum. Functional D2 receptors activate an intracellular signalling pathway that includes a cAMP-independent route involving Akt/GSK3 (glycogen synthase kinase 3). To investigate the Akt/GSK3 response to the seldom-studied D2S receptor, we established a rat D2S receptor-expressing cell line [HEK (human embryonic kidney)-293/rD2S]. We found that in HEK-293/rD2S cells, the D2/D3 agonists bromocriptine and quinpirole significantly induced Akt and GSK3 phosphorylation, as well as ERK1/2 (extracellular-signal-regulated kinase 1/2) activation. The D2S receptor-induced Akt signals were profoundly inhibited by the internalization blockers monodansyl cadaverine and concanavalin A. Activation of the D2S receptor in HEK-293/rD2S cells appeared to trigger Akt/phospho-Akt translocation to the cell membrane. In addition to our cell culture experiments, we studied D2 receptor-dependent Akt in vivo by systemic administration of the D2/D3 agonist quinpirole. The results show that quinpirole evoked Akt-Ser473 phosphorylation in the ventral striatum. Furthermore, intra-accumbens administration of wortmannin, a PI3K (phosphoinositide 3-kinase) inhibitor, significantly suppressed the quinpirole-evoked behavioural activation. Overall, we demonstrate that activation of the dopamine D2S receptor stimulates Akt/GSK3 signalling. In addition, in vivo Akt activity in the ventral striatum appears to play an important role in systemic D2/D3 agonist-induced behavioural activation. PMID:22909302

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

    PubMed

    Cheng, Yuan; Kim, Jaehong; Li, Xiao Xiao; Hsueh, Aaron J

    2015-01-01

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

  9. Up-regulation of Thrombospondin-2 in Akt1-null Mice Contributes to Compromised Tissue Repair Due to Abnormalities in Fibroblast Function*

    PubMed Central

    Bancroft, Tara; Bouaouina, Mohamed; Roberts, Sophia; Lee, Monica; Calderwood, David A.; Schwartz, Martin; Simons, Michael; Sessa, William C.; Kyriakides, Themis R.

    2015-01-01

    Vascular remodeling is essential for tissue repair and is regulated by multiple factors, including thrombospondin-2 (TSP2) and hypoxia/VEGF-induced activation of Akt. In contrast to TSP2 knock-out (KO) mice, Akt1 KO mice have elevated TSP2 expression and delayed tissue repair. To investigate the contribution of increased TSP2 to Akt1 KO mice phenotypes, we generated Akt1/TSP2 double KO (DKO) mice. Full-thickness excisional wounds in DKO mice healed at an accelerated rate when compared with Akt1 KO mice. Isolated dermal Akt1 KO fibroblasts expressed increased TSP2 and displayed altered morphology and defects in migration and adhesion. These defects were rescued in DKO fibroblasts or after TSP2 knockdown. Conversely, the addition of exogenous TSP2 to WT cells induced cell morphology and migration rates that were similar to those of Akt1 KO cells. Akt1 KO fibroblasts displayed reduced adhesion to fibronectin with manganese stimulation when compared with WT and DKO cells, revealing an Akt1-dependent role for TSP2 in regulating integrin-mediated adhesions; however, this effect was not due to changes in β1 integrin surface expression or activation. Consistent with these results, Akt1 KO fibroblasts displayed reduced Rac1 activation that was dependent upon expression of TSP2 and could be rescued by a constitutively active Rac mutant. Our observations show that repression of TSP2 expression is a critical aspect of Akt1 function in tissue repair. PMID:25389299

  10. Erbin loss promotes cancer cell proliferation through feedback activation of Akt-Skp2-p27 signaling

    SciTech Connect

    Huang, Hao; Song, Yuhua; Wu, Yan; Guo, Ning; Ma, Yuanfang; Qian, Lu

    2015-07-31

    Erbin localizes at the basolateral membrane to regulate cell junctions and polarity in epithelial cells. Dysregulation of Erbin has been implicated in tumorigenesis, and yet it is still unclear if and how disrupted Erbin regulates the biological behavior of cancer cells. We report here that depletion of Erbin leads to cancer cell excessive proliferation in vitro and in vivo. Erbin deficiency accelerates S-phase entry by down-regulating CDK inhibitors p21 and p27 via two independent mechanisms. Mechanistically, Erbin loss promotes p27 degradation by enhancing E3 ligase Skp2 activity though augmenting Akt signaling. Interestingly, we also show that Erbin is an unstable protein when the Akt-Skp2 signaling is aberrantly activated, which can be specifically destructed by SCF-Skp2 ligase. Erbin loss facilitates cell proliferation and migration in Skp2-dependent manner. Thus, our finding illustrates a novel negative feedback loop between Erbin and Akt-Skp2 signaling. It suggests disrupted Erbin links polarity loss, hyperproliferation and tumorigenesis. - Highlights: • Erbin loss leads to cancer cell excessive proliferation in vitro and in vivo. • Erbin loss accelerates cell cycle though down-regulating p21 and p27 expression. • Erbin is a novel negative modulator of Akt1-Skp2-p27 signaling pathway. • Our study suggests that Erbin loss contributes to Skp2 oncogenic function.

  11. Angiotensin II-induced Akt activation through the epidermal growth factor receptor in vascular smooth muscle cells is mediated by phospholipid metabolites derived by activation of phospholipase D.

    PubMed

    Li, Fang; Malik, Kafait U

    2005-03-01

    Angiotensin II (Ang II) activates cytosolic Ca(2+)-dependent phospholipase A(2) (cPLA(2)), phospholipase D (PLD), p38 mitogen-activated protein kinase (MAPK), epidermal growth factor receptor (EGFR) and Akt in vascular smooth muscle cells (VSMC). This study was conducted to investigate the relationship between Akt activation by Ang II and other signaling molecules in rat VSMC. Ang II-induced Akt phosphorylation was significantly reduced by the PLD inhibitor 1-butanol, but not by its inactive analog 2-butanol, and by brefeldin A, an inhibitor of the PLD cofactor ADP-ribosylation factor, and in cells infected with retrovirus containing PLD(2) siRNA or transfected with PLD(2) antisense but not control LacZ or sense oligonucleotide. Diacylglycerol kinase inhibitor II diminished Ang II-induced and diC8-phosphatidic acid (PA)-increased Akt phosphorylation, suggesting that PLD-dependent Akt activation is mediated by PA. Ang II-induced EGFR phosphorylation was inhibited by 1-butanol and PLD(2) siRNA and also by cPLA(2) siRNA. In addition, the inhibitor of arachidonic acid (AA) metabolism 5,8,11,14-eicosatetraynoic acid (ETYA) reduced both Ang II- and AA-induced EGFR transactivation. Furthermore, ETYA, cPLA(2) antisense, and cPLA(2) siRNA attenuated Ang II-elicited PLD activation. p38 MAPK inhibitor SB202190 [4-(4-flurophenyl)-2-(4-hydroxyphenyl)-5-(4-pyridyl)1H-imidazole] reduced PLD activity and EGFR and Akt phosphorylation elicited by Ang II. Pyrrolidine-1, a cPLA(2) inhibitor, and cPLA(2) siRNA decreased p38 MAPK activity. These data indicate that Ang II-stimulated Akt activity is mediated by cPLA(2)-dependent, p38 MAPK regulated PLD(2) activation and EGFR transactivation. We propose the following scheme of the sequence of events leading to activation of Akt in VSMC by Ang II: Ang II-->cPLA(2)-->AA-->p38 MAPK-->PLD(2)-->PA-->EGFR-->Akt. PMID:15525798

  12. Angiotensin II-induced Akt activation through the epidermal growth factor receptor in vascular smooth muscle cells is mediated by phospholipid metabolites derived by activation of phospholipase D.

    PubMed

    Li, Fang; Malik, Kafait U

    2005-03-01

    Angiotensin II (Ang II) activates cytosolic Ca(2+)-dependent phospholipase A(2) (cPLA(2)), phospholipase D (PLD), p38 mitogen-activated protein kinase (MAPK), epidermal growth factor receptor (EGFR) and Akt in vascular smooth muscle cells (VSMC). This study was conducted to investigate the relationship between Akt activation by Ang II and other signaling molecules in rat VSMC. Ang II-induced Akt phosphorylation was significantly reduced by the PLD inhibitor 1-butanol, but not by its inactive analog 2-butanol, and by brefeldin A, an inhibitor of the PLD cofactor ADP-ribosylation factor, and in cells infected with retrovirus containing PLD(2) siRNA or transfected with PLD(2) antisense but not control LacZ or sense oligonucleotide. Diacylglycerol kinase inhibitor II diminished Ang II-induced and diC8-phosphatidic acid (PA)-increased Akt phosphorylation, suggesting that PLD-dependent Akt activation is mediated by PA. Ang II-induced EGFR phosphorylation was inhibited by 1-butanol and PLD(2) siRNA and also by cPLA(2) siRNA. In addition, the inhibitor of arachidonic acid (AA) metabolism 5,8,11,14-eicosatetraynoic acid (ETYA) reduced both Ang II- and AA-induced EGFR transactivation. Furthermore, ETYA, cPLA(2) antisense, and cPLA(2) siRNA attenuated Ang II-elicited PLD activation. p38 MAPK inhibitor SB202190 [4-(4-flurophenyl)-2-(4-hydroxyphenyl)-5-(4-pyridyl)1H-imidazole] reduced PLD activity and EGFR and Akt phosphorylation elicited by Ang II. Pyrrolidine-1, a cPLA(2) inhibitor, and cPLA(2) siRNA decreased p38 MAPK activity. These data indicate that Ang II-stimulated Akt activity is mediated by cPLA(2)-dependent, p38 MAPK regulated PLD(2) activation and EGFR transactivation. We propose the following scheme of the sequence of events leading to activation of Akt in VSMC by Ang II: Ang II-->cPLA(2)-->AA-->p38 MAPK-->PLD(2)-->PA-->EGFR-->Akt.

  13. Krüppel-like factor 14 increases insulin sensitivity through activation of PI3K/Akt signal pathway.

    PubMed

    Yang, Min; Ren, Yan; Lin, Zhimin; Tang, Chenchen; Jia, Yanjun; Lai, Yerui; Zhou, Tingting; Wu, Shaobo; Liu, Hua; Yang, Gangyi; Li, Ling

    2015-11-01

    Genome-wide association studies (GWAS) have shown that Krüppel-like factor 14 (KLF14) is associated with type 2 diabetes mellitus (T2DM). However, no report has demonstrated a relationship between KLF14 and glucose metabolism. The aim of this study was to determine whether KLF14 is associated with glucose metabolism and insulin signaling in vitro. The mRNA and protein expressions of KLF14 were determined by Real-time PCR and Western blotting. Glucose uptake was assessed by 2-[(3)H]-deoxyglucose (2-DG) uptake. Western blotting was used to identify the activation of insulin signaling proteins. KLF14 mRNA and protein in fat and muscle were significantly decreased in HFD-fed mice, db/db mice and T2DM patients. Overexpression of KLF14 enhanced insulin-stimulated glucose uptake and the activation of Akt kinase in Hepa1-6 cells. The phosphorylation of insulin receptor (InsR), insulin receptor substrate-1(IRS-1), glycogen synthase kinase-3β (GSK-3β) and Akt also elevated significantly by up-regulation of KLF14. KLF14 overexpression in Hepa1-6 cells prevented the inhibition of glucose uptake and Akt phosphorylation induced by high glucose and/or high insulin, or T2DM serum. However, KLF14's ability to increase glucose uptake and Akt activation was significantly attenuated by LY294002, a PI3-kinase inhibitor. These data suggested that KLF14 could increase insulin sensitivity probably through the PI3K/Akt pathway. PMID:26226221

  14. Fucoidan/FGF-2 induces angiogenesis through JNK- and p38-mediated activation of AKT/MMP-2 signalling

    SciTech Connect

    Kim, Beom Su; Park, Ji-Yun; Kang, Hyo-Jin; Kim, Hyung-Jin; Lee, Jun

    2014-08-08

    Graphical abstract: Schematic diagram of the angiogenic activity mechanism by FGF-2/fucoidan treatment in HUVECs. Fucoidan enhances the FGF-2-induced phosphorylation of p38, JNK, and ERK MAPKs. However, p38 and JNK were involved in AKT phosphorylation and MMP-2 activation and resulted in enhanced angiogenic activity, such as tube formation and migration, in HUVECs. - Highlights: • The angiogenic activity of fucoidan in HUVECs was explored. • Fucoidan enhanced HUVEC proliferation, migration, and tube formation. • Fucoidan enhanced angiogenesis through p38 and JNK but not ERK in HUVECs. • Fucoidan targeted angiogenesis-mediated AKT/MMP-2 signalling in HUVECs. - Abstract: Angiogenesis is an important biological process in tissue development and repair. Fucoidan has previously been shown to potentiate in vitro tube formation in the presence of basic fibroblast growth factor (FGF-2). However, the underlying molecular mechanism remains largely unknown. This study was designed to investigate the action of fucoidan in angiogenesis in human umbilical vein endothelial cells (HUVECs) and to explore fucoidan-signalling pathways. First, we evaluated the effect of fucoidan on cell proliferation. Matrigel-based tube formation and wound healing assays were performed to investigate angiogenesis. Matrix metalloproteinase-2 (MMP-2) mRNA expression and activity levels were analysed by reverse transcription polymerase chain reaction (RT-PCR) and zymography, respectively. Additionally, phosphorylation of mitogen-activated protein kinases (MAPKs) and protein kinase B (AKT) was detected by Western blot. The results indicate that fucoidan treatment significantly increased cell proliferation in the presence of FGF-2. Moreover, compared to the effect of FGF-2 alone, fucoidan and FGF-2 had a greater effect on tube formation and cell migration, and this effect was found to be synergistic. Furthermore, fucoidan enhanced the phosphorylation of extracellular signal-regulated kinase (ERK

  15. Zinc promotes proliferation and activation of myogenic cells via the PI3K/Akt and ERK signaling cascade

    SciTech Connect

    Ohashi, Kazuya; Nagata, Yosuke; Wada, Eiji; Zammit, Peter S.; Shiozuka, Masataka; Matsuda, Ryoichi

    2015-05-01

    Skeletal muscle stem cells named muscle satellite cells are normally quiescent but are activated in response to various stimuli, such as injury and overload. Activated satellite cells enter the cell cycle and proliferate to produce a large number of myogenic progenitor cells, and these cells then differentiate and fuse to form myofibers. Zinc is one of the essential elements in the human body, and has multiple roles, including cell growth and DNA synthesis. However, the role of zinc in myogenic cells is not well understood, and is the focus of this study. We first examined the effects of zinc on differentiation of murine C2C12 myoblasts and found that zinc promoted proliferation, with an increased number of cells incorporating EdU, but inhibited differentiation with reduced myogenin expression and myotube formation. Furthermore, we used the C2C12 reserve cell model of myogenic quiescence to investigate the role of zinc on activation of myogenic cells. The number of reserve cells incorporating BrdU was increased by zinc in a dose dependent manner, with the number dramatically further increased using a combination of zinc and insulin. Akt and extracellular signal-regulated kinase (ERK) are downstream of insulin signaling, and both were phosphorylated after zinc treatment. The zinc/insulin combination-induced activation involved the phosphoinositide 3-kinase (PI3K)/Akt and ERK cascade. We conclude that zinc promotes activation and proliferation of myogenic cells, and this activation requires phosphorylation of PI3K/Akt and ERK as part of the signaling cascade. - Highlights: • Zinc has roles for promoting proliferation and inhibition differentiation of C2C12. • Zinc promotes activation of reserve cells. • Insulin and zinc synergize activation of reserve cells. • PI3K/Akt and ERK cascade affect zinc/insulin-mediated activation of reserve cells.

  16. Galectin-1 induces hepatocellular carcinoma EMT and sorafenib resistance by activating FAK/PI3K/AKT signaling

    PubMed Central

    Zhang, P-F; Li, K-S; Shen, Y-h; Gao, P-T; Dong, Z-R; Cai, J-B; Zhang, C; Huang, X-Y; Tian, M-X; Hu, Z-Q; Gao, D-M; Fan, J; Ke, A-W; Shi, G-M

    2016-01-01

    Galectin-1 (Gal-1) is involved in several pathological activities associated with tumor progression and chemoresistance, however, the role and molecular mechanism of Gal-1 activity in hepatocellular carcinoma (HCC) epithelial–mesenchymal transition (EMT) and sorafenib resistance remain enigmatic. In the present study, forced Gal-1 expression promoted HCC progression and sorafenib resistance. Gal-1 elevated αvβ3-integrin expression, leading to AKT activation. Moreover, Gal-1 overexpression induced HCC cell EMT via PI3K/AKT cascade activation. Clinically, our data revealed that Gal-1 overexpression is correlated with poor HCC survival outcomes and sorafenib response. These data suggest that Gal-1 may be a potential therapeutic target for HCC and a biomarker for predicting response to sorafenib treatment. PMID:27100895

  17. Galectin-1 induces hepatocellular carcinoma EMT and sorafenib resistance by activating FAK/PI3K/AKT signaling.

    PubMed

    Zhang, P-F; Li, K-S; Shen, Y-H; Gao, P-T; Dong, Z-R; Cai, J-B; Zhang, C; Huang, X-Y; Tian, M-X; Hu, Z-Q; Gao, D-M; Fan, J; Ke, A-W; Shi, G-M

    2016-01-01

    Galectin-1 (Gal-1) is involved in several pathological activities associated with tumor progression and chemoresistance, however, the role and molecular mechanism of Gal-1 activity in hepatocellular carcinoma (HCC) epithelial-mesenchymal transition (EMT) and sorafenib resistance remain enigmatic. In the present study, forced Gal-1 expression promoted HCC progression and sorafenib resistance. Gal-1 elevated αvβ3-integrin expression, leading to AKT activation. Moreover, Gal-1 overexpression induced HCC cell EMT via PI3K/AKT cascade activation. Clinically, our data revealed that Gal-1 overexpression is correlated with poor HCC survival outcomes and sorafenib response. These data suggest that Gal-1 may be a potential therapeutic target for HCC and a biomarker for predicting response to sorafenib treatment. PMID:27100895

  18. Imp2 regulates GBM progression by activating IGF2/PI3K/Akt pathway

    PubMed Central

    Mu, Qingchun; Wang, Lijun; Yu, Fengbo; Gao, Haijun; Lei, Ting; Li, Peiwen; Liu, Pengfei; Zheng, Xu; Hu, Xitong; Chen, Yong; Jiang, Zhenfeng; Sayari, Arash J; Shen, Jia; Huang, Haiyan

    2015-01-01

    Glioblastomas multiforme (GBM) are the most frequently occurring malignant brain cancers. Treatment for GBM consists of surgical resection and subsequent adjuvant radiation therapy and chemotherapy. Despite this, GBM patient survival is limited to 12–15 months, and researchers are continually trying to develop improved therapy options. Insulin-like growth factor 2 mRNA-binding protein 2 (Imp2) is known to be upregulated in many cancers and is known to regulate the signaling activity of insulin-like growth factor 2 (IGF2). However, relatively little is known about its role in malignant development of GBM. In this study, we first found Imp2 is upregulated in GBM tissues by using clinical samples and public database search. Studies with loss and gain of Imp2 expression in in vitro GBM cell culture system demonstrated the role of Imp2 in promoting GBM cell proliferation, migration, invasion and epithelial-to-mesenchymal transition (EMT). Additionally, our results show that Imp2 regulates the activity of IGF2, which further activates PI3K/Akt signaling, thereby to promote GBM malignancy. Inhibition of Imp2 was also found to sensitize GBM to temozolomide treatment. These observations add to the current knowledge of GBM biology, and may prove useful in development of more effective GBM therapy. PMID:25719943

  19. A novel form of constitutively active farnesylated Akt1 prevents mammary epithelial cells from anoikis and suppresses chemotherapy-induced apoptosis.

    PubMed

    Schmidt, M; Hövelmann, S; Beckers, T L

    2002-10-01

    Protein kinase B/Akt has been described as a central mediator of anti-apoptotic signals transduced by the PI3 kinase. Although the role of Akt in the suppression of apoptosis is well elucidated, a potential function of Akt in tumorigenesis and chemoresistance is less intensively documented. In this study, we describe the construction of a novel form of constitutively active Akt1, which relies on the deletion of its pleckstrin homology domain and the insertion of a C-terminal farnesylation sequence. Stable cell lines were generated with MCF10A mammary epithelial cells and A549 human NSCLC cells expressing constitutively active Akt1. Enigneered MCF10A cells were rendered resistant towards apoptosis resulting from loss of cellular substrate attachment (anoikis). We investigated the chemosensitivity of A549 cells expressing farnesylated Akt vs control cells. A profoundly decreased sensitivity towards Mitoxantrone and cisplatin was observed in cells expressing farnesylated Akt. No significant difference in sensitivity however was observed upon treatment with cell cycle specific chemotherapeutic agents like paclitaxel. Our data suggest, that Akt is a central mediator in the suppression of anoikis and modulation of chemotherapy-induced apoptosis. Therefore it represents a promising target for small molecule inhibitors to shift the apoptotic threshold in cancer cells after treatment with standard chemotherapy.

  20. 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

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

    PubMed

    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

  2. CYP2J2 and its metabolites (epoxyeicosatrienoic acids) attenuate cardiac hypertrophy by activating AMPKα2 and enhancing nuclear translocation of Akt1.

    PubMed

    Wang, Bei; Zeng, Hesong; Wen, Zheng; Chen, Chen; Wang, Dao Wen

    2016-10-01

    Cytochrome P450 epoyxgenase 2J2 and epoxyeicosatrienoic acids (EETs) are known to protect against cardiac hypertrophy and heart failure, which involve the activation of 5'-AMP-activated protein kinase (AMPK) and Akt. Although the functional roles of AMPK and Akt are well established, the significance of cross talk between them in the development of cardiac hypertrophy and antihypertrophy of CYP2J2 and EETs remains unclear. We investigated whether CYP2J2 and its metabolites EETs protected against cardiac hypertrophy by activating AMPKα2 and Akt1. Moreover, we tested whether EETs enhanced cross talk between AMPKα2 and phosphorylated Akt1 (p-Akt1), and stimulated nuclear translocation of p-Akt1, to exert their antihypertrophic effects. AMPKα2(-/-) mice that overexpressed CYP2J2 in heart were treated with Ang II for 2 weeks. Interestingly, overexpression of CYP2J2 suppressed cardiac hypertrophy and increased levels of atrial natriuretic peptide (ANP) in the heart tissue and plasma of wild-type mice but not AMPKα2(-/-) mice. The CYP2J2 metabolites, 11,12-EET, activated AMPKα2 to induce nuclear translocation of p-Akt1 selectively, which increased the production of ANP and therefore inhibited the development of cardiac hypertrophy. Furthermore, by co-immunoprecipitation analysis, we found that AMPKα2β2γ1 and p-Akt1 interact through the direct binding of the AMPKγ1 subunit to the Akt1 protein kinase domain. This interaction was enhanced by 11,12-EET. Our studies reveal a novel mechanism in which CYP2J2 and EETs enhanced Akt1 nuclear translocation through interaction with AMPKα2β2γ1 and protect against cardiac hypertrophy and suggest that overexpression of CYP2J2 might have clinical potential to suppress cardiac hypertrophy and heart failure.

  3. Protein kinase B (AKT) regulates SYK activity and shuttling through 14-3-3 and importin 7.

    PubMed

    Mohammad, Dara K; Nore, Beston F; Gustafsson, Manuela O; Mohamed, Abdalla J; Smith, C I Edvard

    2016-09-01

    The Protein kinase B (AKT) regulates a plethora of intracellular signaling proteins to fine-tune signaling of multiple pathways. Here, we found that following B-cell receptor (BCR)-induced tyrosine phosphorylation of the cytoplasmic tyrosine kinase SYK and the adaptor BLNK, the AKT/PKB enzyme strongly induced BLNK (>100-fold) and SYK (>100-fold) serine/threonine phosphorylation (pS/pT). Increased phosphorylation promoted 14-3-3 binding to BLNK (37-fold) and SYK (2.5-fold) in a pS/pT-concentration dependent manner. We also demonstrated that the AKT inhibitor MK2206 reduced pS/pT of both BLNK (3-fold) and SYK (2.5-fold). Notably, the AKT phosphatase, PHLPP2 maintained the activating phosphorylation of BLNK at Y84 and increased protein stability (8.5-fold). In addition, 14-3-3 was required for the regulation SYK's interaction with BLNK and attenuated SYK binding to Importin 7 (5-fold), thereby perturbing shuttling to the nucleus. Moreover, 14-3-3 proteins also sustained tyrosine phosphorylation of SYK and BLNK. Furthermore, substitution of S295 or S297 for alanine abrogated SYK's binding to Importin 7. SYK with S295A or S297A replacements showed intense pY525/526 phosphorylation, and BLNK pY84 phosphorylation correlated with the SYK pY525/526 phosphorylation level. Conversely, the corresponding mutations to aspartic acid in SYK reduced pY525/526 phosphorylation. Collectively, these and previous results suggest that AKT and 14-3-3 proteins down-regulate the activity of several BCR-associated components, including BTK, BLNK and SYK and also inhibit SYK's interaction with Importin 7. PMID:27381982

  4. AG and UAG induce β-casein expression via activation of ERK1/2 and AKT pathways

    PubMed Central

    Li, Sunan; Liu, Juxiong; Lv, Qingkang; Zhang, Chuan; Xu, Shiyao; Yang, Dongxue; Huang, Bingxu; Zeng, Yalong; Gao, Yingjie

    2016-01-01

    Abstract The ghrelin peptides were found to circulate in two major forms: acylated ghrelin (AG) and unacylated ghrelin (UAG). Previous studies showed that AG regulates β-casein (CSN2) expression in mammary epithelial cells. However, little is known about the mechanisms by which AG regulates CSN2 gene and protein expression. Evidence suggests that UAG has biological activity through GHSR1a-independent mechanisms. Here, we investigated the possible GHSR1a-mediated effect of UAG on the expression of CSN2 in primary bovine mammary epithelial cells (pbMECs) isolated from lactating cow. We found that both AG and UAG increase the expression of CSN2 in a dose-dependent manner in pbMECs in comparison with the control group. Increased expression of CSN2 was blocked by [D-Lys3]-GHRP-6 (an antagonist of the GHSR1a) and NF449 (a Gs-α subunit inhibitor) in pbMECs. In addition, both AG and UAG activated AKT/protein kinase B (AKT) and extracellular signal-regulated kinase 1/2 (ERK1/2) pathways, whereas [D-Lys3]-GHRP-6 and NF449 inhibited the phosphorylation of AKT and ERK1/2 in pbMECs respectively. Blockade of ERK1/2 and AKT signaling pathways prevented the expression of CSN2 induced by AG or UAG. Finally, we found that both AG and UAG cause cell proliferation through identical signaling pathways. Taken together, these results demonstrate that both AG and UAG act on ERK1/2 and AKT signaling pathways to facilitate the expression of CSN2 in a GHSR1a-dependent manner. PMID:26873999

  5. TIMP-2 Interaction with MT1-MMP Activates the AKT Pathway and Protects Tumor Cells from Apoptosis

    PubMed Central

    Valacca, Cristina; Tassone, Evelyne; Mignatti, Paolo

    2015-01-01

    Membrane-type 1 matrix metalloproteinase (MT1-MMP), a transmembrane proteinase with an extracellular catalytic domain and a short cytoplasmic tail, degrades a variety of extracellular matrix (ECM) components. In addition, MT1-MMP activates intracellular signaling through proteolysis-dependent and independent mechanisms. We have previously shown that binding of tissue inhibitor of metalloproteinases-2 (TIMP-2) to MT1-MMP controls cell proliferation and migration, as well as tumor growth in vivo by activating the Ras—extracellular signal regulated kinase-1 and -2 (ERK1/2) pathway through a mechanism that requires the cytoplasmic but not the proteolytic domain of MT1-MMP. Here we show that in MT1-MMP expressing cells TIMP-2 also induces rapid and sustained activation of AKT in a dose- and time-dependent manner and by a mechanism independent of the proteolytic activity of MT1-MMP. Fibroblast growth factor receptor-1 mediates TIMP-2 induction of ERK1/2 but not of AKT activation; however, Ras activation is necessary to transduce the TIMP-2-activated signal to both the ERK1/2 and AKT pathways. ERK1/2 and AKT activation by TIMP-2 binding to MT1-MMP protects tumor cells from apoptosis induced by serum starvation. Conversely, TIMP-2 upregulates apoptosis induced by three-dimensional type I collagen in epithelial cancer cells. Thus, TIMP-2 interaction with MT1-MMP provides tumor cells with either pro- or anti-apoptotic signaling depending on the extracellular environment and apoptotic stimulus. PMID:26331622

  6. Resistance exercise training increase activation of AKT-eNOS and Ref-1 expression by FOXO-1 activation in aorta of F344 rats

    PubMed Central

    Li, Meng; Li, Wei; Yoon, Jin-Hwan; Jeon, Byeong Hwa; Lee, Sang Ki

    2015-01-01

    Purpose This study investigated the effects of resistance exercise on the Akt-eNOS, the activation of antioxidant protein and FOXO1 in the aorta of F344 rats. Methods Male 7 week-old F344 rats were randomly divided into 2 groups: a climbing group (n = 6) and a sedentary group (n = 6). H&E staining and western blotting were used to analyze the rat aortas and target proteins. Results Resistance exercise training did not significantly affect aortic structure. Phosphorylation of AKT and eNOS and expression of MnSOD and Ref-1 were significantly increased while FOXO1 phosphorylation was significantly decreased in the resistance exercise group compared with the sedentary group. Conclusion We demonstrate that resistance exercise activates the Akt-eNOS and Ref-1 protein without changes to aortic thickness via FOXO-1 activation in the aorta of F344 rats. PMID:26526775

  7. 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

    2009-01-01

    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 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. PMID:19654415

  8. 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.

  9. Xenon-delayed postconditioning attenuates spinal cord ischemia/reperfusion injury through activation AKT and ERK signaling pathways in rats.

    PubMed

    Liu, Shiyao; Yang, Yanwei; Jin, Mu; Hou, Siyu; Dong, Xiuhua; Lu, Jiakai; Cheng, Weiping

    2016-09-15

    Previous studies have shown that xenon-delayed postconditioning for up to 2h after reperfusion provides protection against spinal cord ischemia/reperfusion (I/R) injury in rats. This study was designed to determine the roles of phosphatidylinositol 3-kinase (PI3K)-Akt and extracellular signal-regulated kinase (ERK) in this neuroprotection. The rats were randomly assigned to the following nine groups (n=16∗9): 1) I/R+N2 group, 2) I/R+Xe group, 3) I/R+PD98059+N2 group (ERK blocking agent), 4) I/R+wortmannin+N2 group (PI3K-Akt blocking agent), 5) I/R+PD98059+Xe group, 6) I/R+wortmannin+Xe group, 7) I/R+DMSO+Xe group (dimethyl sulfoxide, vehicle control), 8) I/R+DMSO+N2 group, and 9) sham group (no spinal cord ischemia and no xenon). Spinal cord ischemia was induced for 25min in male Sprague-Dawley rats. Neurological function was assessed using the Basso, Beattie, and Bresnahan (BBB) open-field locomotor scale at 6, 12, 24 and 48h after reperfusion. Histological examination of the lumbar spinal cord was performed using Nissl staining and TUNEL staining at 4 (n=8) and 48 (n=8)h after reperfusion. Western blotting was performed to evaluate p-Akt and p-ERK expression in the spinal cord at 4 (n=8) and 48 (n=8) h after reperfusion. Compared with the sham group, all rats in the I/R groups had lower BBB scores, fewer normal motor neurons, more apoptotic neurons and lower p-Akt and p-ERK levels at each time point (P<0.05). Compared with the I/R group, rats in the I/R+Xe group had higher neurological scores, more normal motor neurons, fewer apoptotic neurons and significantly higher levels of p-Akt and p-ERK at each time point (P<0.05). Compared with the I/R+Xe group, the I/R+PD98059+Xe and I/R+wortmannin+Xe groups showed worse neurological outcomes and less p-Akt and p-ERK at each time point (P<0.05). These results suggest that xenon-delayed postconditioning improves neurological outcomes to spinal cord I/R injury in rats through the activation of the AKT and ERK signaling

  10. Xenon-delayed postconditioning attenuates spinal cord ischemia/reperfusion injury through activation AKT and ERK signaling pathways in rats.

    PubMed

    Liu, Shiyao; Yang, Yanwei; Jin, Mu; Hou, Siyu; Dong, Xiuhua; Lu, Jiakai; Cheng, Weiping

    2016-09-15

    Previous studies have shown that xenon-delayed postconditioning for up to 2h after reperfusion provides protection against spinal cord ischemia/reperfusion (I/R) injury in rats. This study was designed to determine the roles of phosphatidylinositol 3-kinase (PI3K)-Akt and extracellular signal-regulated kinase (ERK) in this neuroprotection. The rats were randomly assigned to the following nine groups (n=16∗9): 1) I/R+N2 group, 2) I/R+Xe group, 3) I/R+PD98059+N2 group (ERK blocking agent), 4) I/R+wortmannin+N2 group (PI3K-Akt blocking agent), 5) I/R+PD98059+Xe group, 6) I/R+wortmannin+Xe group, 7) I/R+DMSO+Xe group (dimethyl sulfoxide, vehicle control), 8) I/R+DMSO+N2 group, and 9) sham group (no spinal cord ischemia and no xenon). Spinal cord ischemia was induced for 25min in male Sprague-Dawley rats. Neurological function was assessed using the Basso, Beattie, and Bresnahan (BBB) open-field locomotor scale at 6, 12, 24 and 48h after reperfusion. Histological examination of the lumbar spinal cord was performed using Nissl staining and TUNEL staining at 4 (n=8) and 48 (n=8)h after reperfusion. Western blotting was performed to evaluate p-Akt and p-ERK expression in the spinal cord at 4 (n=8) and 48 (n=8) h after reperfusion. Compared with the sham group, all rats in the I/R groups had lower BBB scores, fewer normal motor neurons, more apoptotic neurons and lower p-Akt and p-ERK levels at each time point (P<0.05). Compared with the I/R group, rats in the I/R+Xe group had higher neurological scores, more normal motor neurons, fewer apoptotic neurons and significantly higher levels of p-Akt and p-ERK at each time point (P<0.05). Compared with the I/R+Xe group, the I/R+PD98059+Xe and I/R+wortmannin+Xe groups showed worse neurological outcomes and less p-Akt and p-ERK at each time point (P<0.05). These results suggest that xenon-delayed postconditioning improves neurological outcomes to spinal cord I/R injury in rats through the activation of the AKT and ERK signaling

  11. Intestinal trefoil factor activates the PI3K/Akt signaling pathway to protect gastric mucosal epithelium from damage.

    PubMed

    Sun, Zhaorui; Liu, Hongmei; Yang, Zhizhou; Shao, Danbing; Zhang, Wei; Ren, Yi; Sun, Baodi; Lin, Jinfeng; Xu, Min; Nie, Shinan

    2014-09-01

    Intestinal trefoil factor (ITF, also named as trefoil factor 3, TFF3) is a member of the TFF-domain peptide family, which plays an essential role in the regulation of cell survival, cell migration and maintains mucosal epithelial integrity in the gastrointestinal tract. However, the underlying mechanisms and associated molecules remain unclear. The aim of this study was to explore the protective effects of ITF on gastric mucosal epithelium injury and its possible molecular mechanisms of action. In the present study, we show that ITF was able to promote the proliferation and migration of GES-1 cells via a mechanism that involves the PI3K/Akt signaling pathway. Western blot results indicated that ITF induced a dose- and time-dependent increase in the Akt signaling pathway. ITF also plays an essential role in the restitution of GES-1 cell damage induced by lipopolysaccharide (LPS). LPS induced the apoptosis of GES-1 cells, decreased cell viability significantly (P<0.01) and led to epithelial tight junction damage, which is attenuated via ITF treatment. The protective effect of ITF on the integrity of GES-1 was abrogated by inhibition of the PI3K/Akt pathway. Taken together, our results demonstrate that ITF promotes the proliferation and migration of gastric mucosal epithelial cells and preserves gastric mucosal epithelial integrity after damage is mediated by activation of the PI3K/Akt signaling pathway. This study suggested that the PI3K/Akt pathway could act as a key intracellular pathway in the gastric mucosal epithelium that may serve as a therapeutic target to preserve epithelial integrity during injury.

  12. Matrix adhesion and Ras transformation both activate a phosphoinositide 3-OH kinase and protein kinase B/Akt cellular survival pathway.

    PubMed Central

    Khwaja, A; Rodriguez-Viciana, P; Wennström, S; Warne, P H; Downward, J

    1997-01-01

    Upon detachment from the extracellular matrix, epithelial cells enter into programmed cell death, a phenomenon known as anoikis, ensuring that they are unable to survive in an inappropriate location. Activated ras oncogenes protect cells from this form of apoptosis. The nature of the survival signals activated by integrin engagement and usurped by oncogenic Ras are unknown: here we show that in both cases phosphoinositide 3-OH kinase (PI 3-kinase), but not Raf, mediates this protection, acting through protein kinase B/Akt (PKB/Akt). Constitutively activated PI 3-kinase or PKB/Akt block anoikis, while inhibition of PI 3-kinase abrogates protection by Ras, but not PKB/Akt. Inhibition of either PI 3-kinase or PKB/Akt induces apoptosis in adherent epithelial cells. Attachment of cells to matrix leads to rapid elevation of the levels of PI 3-kinase lipid products and PKB/Akt activity, both of which remain high in Ras-transformed cells even in suspension. PI 3-kinase acting through PKB/Akt is therefore implicated as a key mediator of the aberrant survival of Ras-transformed epithelial cells in the absence of attachment, and mediates matrix-induced survival of normal epithelial cells. PMID:9184223

  13. Fucoidan/FGF-2 induces angiogenesis through JNK- and p38-mediated activation of AKT/MMP-2 signalling.

    PubMed

    Kim, Beom Su; Park, Ji-Yun; Kang, Hyo-Jin; Kim, Hyung-Jin; Lee, Jun

    2014-08-01

    Angiogenesis is an important biological process in tissue development and repair. Fucoidan has previously been shown to potentiate in vitro tube formation in the presence of basic fibroblast growth factor (FGF-2). However, the underlying molecular mechanism remains largely unknown. This study was designed to investigate the action of fucoidan in angiogenesis in human umbilical vein endothelial cells (HUVECs) and to explore fucoidan-signalling pathways. First, we evaluated the effect of fucoidan on cell proliferation. Matrigel-based tube formation and wound healing assays were performed to investigate angiogenesis. Matrix metalloproteinase-2 (MMP-2) mRNA expression and activity levels were analysed by reverse transcription polymerase chain reaction (RT-PCR) and zymography, respectively. Additionally, phosphorylation of mitogen-activated protein kinases (MAPKs) and protein kinase B (AKT) was detected by Western blot. The results indicate that fucoidan treatment significantly increased cell proliferation in the presence of FGF-2. Moreover, compared to the effect of FGF-2 alone, fucoidan and FGF-2 had a greater effect on tube formation and cell migration, and this effect was found to be synergistic. Furthermore, fucoidan enhanced the phosphorylation of extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), p38, and AKT. MMP-2 activation was also significantly increased. Specific inhibitors of p38 (SB203580) and JNK (SP600125) inhibited tube formation and wound healing, while an ERK inhibitor (PD98059) did not. MMP-2 activation and AKT phosphorylation were also attenuated and associated with the suppression of p38 and JNK phosphorylation, but not with that of ERK. These results indicate that fucoidan, in the presence of FGF-2, induces angiogenesis through AKT/MMP-2 signalling by activating p38 and JNK. These findings provide basic molecular information on the effect of fucoidan on angiogenesis in the presence of FGF-2.

  14. 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

  15. Genistein activates endothelial nitric oxide synthase in broiler pulmonary arterial endothelial cells by an Akt-dependent mechanism

    PubMed Central

    Yang, Ying; Nie, Wei; Yuan, Jianmin; Zhang, Bingkun; Wang, Zhong

    2010-01-01

    Deregulation of endothelial nitric oxide synthase (eNOS) plays an important role in the development of multiple cardiovascular diseases. Our recent study demonstrated that genistein supplementation attenuates pulmonary arterial hypertension in broilers by restoration of endothelial function. In this study, we investigated the molecular mechanism by using broiler pulmonary arterial endothelial cells (PAECs). Our results showed that genistein stimulated a rapid phosphorylation of eNOS at Ser1179 which was associated with activation of eNOS/NO axis. Further study indicated that the activation of eNOS was not mediated through estrogen receptors or tyrosine kinase inhibition, but via a phosphatidylinositol 3-kinase (PI3K)/Akt-dependent signaling pathway, as the eNOS activity and related NO release were largely abolished by pharmacological inhibitors of PI3K or Akt. Thus, our findings revealed a critical function of Akt in mediating genistein-stimulated eNOS activity in PAECs, partially accounting for the beneficial effects of genistein on the development of cardiovascular diseases observed in animal models. PMID:20926919

  16. Knockdown of Slit2 promotes growth and motility in gastric cancer cells via activation of AKT/β-catenin.

    PubMed

    Shi, Rongliang; Yang, Zhen; Liu, Weiyan; Liu, Bingya; Xu, Ziping; Zhang, Ziping

    2014-02-01

    We previously showed that Slit2 was highly expressed in gastric cancer tissues that exhibit less advanced clinicopathological features, suggesting a tumor suppressor role for Slit2. In the present study, we investigated the effects of Slit2 knockdown on gastric cancer cells. Slit2-specific shRNAs were used to generate Slit2-knockdown SGC-7901 gastric cancer cells. Cell proliferation assay, Annexin V/PI double staining and cell cycle analysis were used to investigate the role of Slit2 knockdown in cell growth. Wound-healing and in vitro migration/invasion assays were performed. Subcutaneous tumor formation and peritoneal spreading in nude mice were employed to examine the in vivo effects of Slit2 knockdown. Cell signaling changes induced by Slit2 knockdown were analyzed by immunoblotting. Slit2 knockdown increased gastric cancer cell growth in monolayer and soft agar/Matrigel 3D culture. Slit2 knockdown inhibited apoptosis but did not alter cell cycle progression. Slit2-knockdown cells formed larger tumors and produced more peritoneal metastatic nodules in nude mice. Slit2 knockdown increased AKT phosphorylation, activated anti-apoptotic signaling, suppressed GSK3β activity and induced β-catenin activation. Blocking the effects of PI3K/AKT using pharmacological inhibitors abolished the ability of Slit2 knockdown to induce apoptosis resistance and cell migration/invasion. These results indicate that Slit2 knockdown promotes gastric cancer growth and metastasis through activation of the AKT/β‑catenin-mediated signaling pathway.

  17. Gecko proteins induce the apoptosis of bladder cancer 5637 cells by inhibiting Akt and activating the intrinsic caspase cascade

    PubMed Central

    Kim, Geun-Young; Park, Soon Yong; Jo, Ara; Kim, Mira; Leem, Sun-Hee; Jun, Woo-Jin; Shim, Sang In; Lee, Sang Chul; Chung, Jin Woong

    2015-01-01

    Gecko proteins have long been used as anti-tumor agents in oriental medicine, without any scientific background. Although anti-tumor effects of Gecko proteins on several cancers were recently reported, their effect on bladder cancer has not been investigated. Thus, we explored the anti-tumor effect of Gecko proteins and its cellular mechanisms in human bladder cancer 5637 cells. Gecko proteins significantly reduced the viability of 5637 cells without any cytotoxic effect on normal cells. These proteins increased the Annexin-V staining and the amount of condensed chromatin, demonstrating that the Gecko proteinsinduced cell death was caused by apoptosis. Gecko proteins suppressed Akt activation, and the overexpression of constitutively active form of myristoylated Akt prevented Gecko proteins-induced death of 5637 cells. Furthermore, Gecko proteins activated caspase 9 and caspase 3/7. Taken together, our data demonstrated that Gecko proteins suppressed the Akt pathway and activated the intrinsic caspase pathway, leading to the apoptosis of bladder cancer cells. [BMB Reports 2015; 48(9): 531-536] PMID:26246284

  18. PPAR-γ agonist stabilizes KLF4 protein via activating Akt signaling and reducing KLF4 ubiquitination

    SciTech Connect

    Sun, Yan; Zheng, Bin; Zhang, Xin-hua; He, Ming; Guo, Zong-wei; Wen, Jin-kun

    2014-01-10

    Highlights: •PPAR-γ increases KLF4 protein level but does not influence KLF4 gene transcription. •The increase of KLF4 protein levels induced by pioglitazone is PPAR-γ-dependent. •Pioglitazone stabilizes KLF4 protein via activating Akt signaling and reducing KLF4 ubiquitination. -- Abstract: Peroxisome proliferator activated receptor γ (PPAR-γ) plays important roles in cell cycle regulation, differentiation and apoptosis. Krüppel-like factor 4 (KLF4) modulates vascular smooth muscle cell (VSMC) phenotype. Both KLF4 and PPAR-γ are involved in VSMC proliferation and differentiation. However, the actual relationship between KLF4 and PPAR-γ in VSMCs is not clear. In this study, we found that PPAR-γ agonist pioglitazone increases KLF4 protein levels but does not influence KLF4 gene transcription. PPAR-γ overexpression increases, while PPAR-γ knockdown reduces KLF4 expression, suggesting that the increase in KLF4 protein levels induced by pioglitazone is PPAR-γ-dependent. Further study showed that pioglitazone enhances KLF4 protein stability through reducing KLF4 ubiquitination. Furthermore, we demonstrated that stabilization of KLF4 by pioglitazone was related to the activation of Akt signaling pathway. Taken together, we revealed that PPAR-γ agonist pioglitazone stabilizes KLF4 protein via activating Akt signaling and reducing KLF4 ubiquitination, providing further insights into PPAR-γ and KLF4 in regulating each other’s expression in VSMCs.

  19. Gecko proteins induce the apoptosis of bladder cancer 5637 cells by inhibiting Akt and activating the intrinsic caspase cascade.

    PubMed

    Kim, Geun-Young; Park, Soon Yong; Jo, Ara; Kim, Mira; Leem, Sun-Hee; Jun, Woo-Jin; Shim, Sang In; Lee, Sang Chul; Chung, Jin Woong

    2015-09-01

    Gecko proteins have long been used as anti-tumor agents in oriental medicine, without any scientific background. Although anti-tumor effects of Gecko proteins on several cancers were recently reported, their effect on bladder cancer has not been investigated. Thus, we explored the anti-tumor effect of Gecko proteins and its cellular mechanisms in human bladder cancer 5637 cells. Gecko proteins significantly reduced the viability of 5637 cells without any cytotoxic effect on normal cells. These proteins increased the Annexin-V staining and the amount of condensed chromatin, demonstrating that the Gecko proteinsinduced cell death was caused by apoptosis. Gecko proteins suppressed Akt activation, and the overexpression of constitutively active form of myristoylated Akt prevented Gecko proteins-induced death of 5637 cells. Furthermore, Gecko proteins activated caspase 9 and caspase 3/7. Taken together, our data demonstrated that Gecko proteins suppressed the Akt pathway and activated the intrinsic caspase pathway, leading to the apoptosis of bladder cancer cells. [BMB Reports 2015; 48(9): 531-536].

  20. Comparative analysis of MAPK and PI3K/AKT pathway activation and inhibition in human and canine melanoma.

    PubMed

    Fowles, J S; Denton, C L; Gustafson, D L

    2015-09-01

    The lack of advanced animal models of human cancers is considered a barrier to developing effective therapeutics. Canine and human melanomas are histologically disparate but show similar disease progression and response to therapies. The purpose of these studies was to compare human and canine melanoma tumours and cell lines regarding MAPK and PI3K/AKT signalling dysregulation, and response to select molecularly targeted agents. Pathway activation was investigated via microarray and mutational analysis. Growth inhibition and cell cycle effects were assessed for pathway inhibitors AZD6244 (MAPK) and rapamycin (PI3K/AKT) in human and canine melanoma cells. Human and canine melanoma share similar differential gene expression patterns within the MAPK and PI3K/AKT pathways. Constitutive pathway activation and similar sensitivity to AZD6244 and rapamycin was observed in human and canine cells. These results show that human and canine melanoma share activation and sensitivity to inhibition of cancer-related signalling pathways despite differences in activating mutations. PMID:23745794

  1. Loss of NDRG2 expression activates PI3K-AKT signalling via PTEN phosphorylation in ATLL and other cancers

    PubMed Central

    Nakahata, Shingo; Ichikawa, Tomonaga; Maneesaay, Phudit; Saito, Yusuke; Nagai, Kentaro; Tamura, Tomohiro; Manachai, Nawin; Yamakawa, Norio; Hamasaki, Makoto; Kitabayashi, Issay; Arai, Yasuhito; Kanai, Yae; Taki, Tomohiko; Abe, Takaya; Kiyonari, Hiroshi; Shimoda, Kazuya; Ohshima, Koichi; Horii, Akira; Shima, Hiroshi; Taniwaki, Masafumi; Yamaguchi, Ryoji; Morishita, Kazuhiro

    2014-01-01

    Constitutive phosphatidylinositol 3-kinase (PI3K)-AKT activation has a causal role in adult T-cell leukaemia-lymphoma (ATLL) and other cancers. ATLL cells do not harbour genetic alterations in PTEN and PI3KCA but express high levels of PTEN that is highly phosphorylated at its C-terminal tail. Here we report a mechanism for the N-myc downstream-regulated gene 2 (NDRG2)-dependent regulation of PTEN phosphatase activity via the dephosphorylation of PTEN at the Ser380, Thr382 and Thr383 cluster within the C-terminal tail. We show that NDRG2 is a PTEN-binding protein that recruits protein phosphatase 2A (PP2A) to PTEN. The expression of NDRG2 is frequently downregulated in ATLL, resulting in enhanced phosphorylation of PTEN at the Ser380/Thr382/Thr383 cluster and enhanced activation of the PI3K-AKT pathway. Given the high incidence of T-cell lymphoma and other cancers in NDRG2-deficient mice, PI3K-AKT activation via enhanced PTEN phosphorylation may be critical for the development of cancer. PMID:24569712

  2. Akt2 negatively regulates assembly of the POSH-MLK-JNK signaling complex.

    PubMed

    Figueroa, Claudia; Tarras, Samantha; Taylor, Jennifer; Vojtek, Anne B

    2003-11-28

    We demonstrate that POSH, a scaffold for the JNK signaling pathway, binds to Akt2. A POSH mutant that is unable to bind Akt2 (POSH W489A) exhibits enhanced-binding to MLK3, and this increase in binding is accompanied by increased activation of the JNK signaling pathway. In addition, we show that the association of MLK3 with POSH is increased upon inhibition of the endogenous phosphatidylinositol 3-kinase/Akt signaling pathway. Thus, the assembly of an active JNK signaling complex by POSH is negatively regulated by Akt2. Further, the level of Akt-phosphorylated MLK3 is reduced in cells expressing the Akt2 binding domain of POSH, which acts as a dominant interfering protein. Taken together, our results support a model in which Akt2 binds to a POSH-MLK-MKK-JNK complex and phosphorylates MLK3; phosphorylation of MLK3 by Akt2 results in the disassembly of the JNK complex bound to POSH and down-regulation of the JNK signaling pathway.

  3. Anthocyanins protected hearts against ischemic injury by reducing MMP-2 activity via Akt/P38 pathways

    PubMed Central

    Hao, Jie; Du, Hong; Li, Weiwei; Liu, Fan; Lu, Jingchao; Yang, Xiuchun; Cui, Wei

    2016-01-01

    Growing evidences suggest that there are close associations between anthocyanins and cardiac protection. However, little is known about the detailed roles of anthocyanins in regulating extracellular matrix (ECM) remodeling. Incubation of primary cultured fibroblasts with anthocyanins reduced both intracellular collagen expression and extracellular collagen secretion. Down-regulation of collagen production was also shown in infarcted cardiac tissues after permanent coronary artery ligation in mice treated with anthocyanins. The phosphorylation levels of Akt and/or P-38 were significantly increased by anthocyanins supplementation in primary cultured fibroblasts. Gelatin zymography analysis of matrix metalloproteinase-2 (MMP-2) activity in conditioned medium collected from fibroblasts demonstrated that anthocyanins treatment significantly reduced MMP-2 activity. These results demonstrated that anthocyanins play a role in mediating myocardial ECM remodeling and that the Akt/P-38 pathways mediate these protective effects on hearts. PMID:27158396

  4. Akt Activation Correlates with Snail Expression and Potentially Determines the Recurrence of Prostate Cancer in Patients at Stage T2 after a Radical Prostatectomy

    PubMed Central

    Chen, Wei-Yu; Hua, Kuo-Tai; Lee, Wei-Jiunn; Lin, Yung-Wei; Liu, Yen-Nien; Chen, Chi-Long; Wen, Yu-Ching; Chien, Ming-Hsien

    2016-01-01

    Our previous work demonstrated the epithelial-mesenchymal transition factor, Snail, is a potential marker for predicting the recurrence of localized prostate cancer (PCa). Akt activation is important for Snail stabilization and transcription in PCa. The purpose of this study was to retrospectively investigate the relationship between the phosphorylated level of Akt (p-Akt) in radical prostatectomy (RP) specimens and cancer biochemical recurrence (BCR). Using a tissue microarray and immunohistochemistry, the expression of p-Akt was measured in benign and neoplastic tissues from RP specimens in 53 patients whose cancer was pathologically defined as T2 without positive margins. Herein, we observed that the p-Akt level was higher in PCa than in benign tissues and was significantly associated with the Snail level. A high p-Akt image score (≥8) was significantly correlated with a higher histological Gleason sum, Snail image score, and preoperative prostate-specific antigen (PSA) value. Moreover, the high p-Akt image score and Gleason score sum (≥7) showed similar discriminatory abilities for BCR according to a receiver-operator characteristic curve analysis and were correlated with worse recurrence-free survival according to a log-rank test (p < 0.05). To further determine whether a high p-Akt image score could predict the risk of BCR, a Cox proportional hazard model showed that only a high p-Akt image score (hazard ratio (HR): 3.12, p = 0.05) and a high Gleason score sum (≥7) (HR: 1.18, p = 0.05) but not a high preoperative PSA value (HR: 0.62, p = 0.57) were significantly associated with a higher risk of developing BCR. Our data indicate that, for localized PCa patients after an RP, p-Akt can serve as a potential prognostic marker that improves predictions of BCR-free survival. PMID:27455254

  5. Myricitrin Attenuates High Glucose-Induced Apoptosis through Activating Akt-Nrf2 Signaling in H9c2 Cardiomyocytes.

    PubMed

    Zhang, Bin; Chen, Yaping; Shen, Qiang; Liu, Guiyan; Ye, Jingxue; Sun, Guibo; Sun, Xiaobo

    2016-01-01

    Hyperglycemia, as well as diabetes mellitus, has been shown to trigger cardiac cell apoptosis. We have previously demonstrated that myricitrin prevents endothelial cell apoptosis. However, whether myricitrin can attenuate H9c2 cell apoptosis remains unknown. In this study, we established an experiment model in H9c2 cells exposed to high glucose. We tested the hypothesis that myricitrin may inhibit high glucose (HG)-induced cardiac cell apoptosis as determined by TUNEL staining. Furthermore, myricitrin promoted antioxidative enzyme production, suppressed high glucose-induced reactive oxygen species (ROS) production and decreased mitochondrial membrane potential (MMP) in H9c2 cells. This agent significantly inhibited apoptotic protein expression, activated Akt and facilitated the transcription of NF-E2-related factor 2 (Nrf2)-mediated protein (heme oxygenase-1 (HO-1) and quinone oxidoreductase 1 (NQO-1) expression as determined by Western blotting. Significantly, an Akt inhibitor (LY294002) or HO-1 inhibitor (ZnPP) not only inhibited myricitrin-induced HO-1/NQO-1 upregulation but also alleviated its anti-apoptotic effects. In summary, these observations demonstrate that myricitrin activates Nrf2-mediated anti-oxidant signaling and attenuates H9c2 cell apoptosis induced by high glucose via activation of Akt signaling. PMID:27399653

  6. Arsenic Trioxide Overcomes Rapamycin-Induced Feedback Activation of AKT and ERK Signaling to Enhance the Anti-Tumor Effects in Breast Cancer

    PubMed Central

    Guilbert, Cynthia; Annis, Matthew G.; Dong, Zhifeng; Siegel, Peter M.; Miller, Wilson H.; Mann, Koren K.

    2013-01-01

    Inhibitors of the mammalian target of rapamycin (mTORi) have clinical activity; however, the benefits of mTOR inhibition by rapamycin and rapamycin-derivatives (rapalogs) may be limited by a feedback mechanism that results in AKT activation. Increased AKT activity resulting from mTOR inhibition can be a result of increased signaling via the mTOR complex, TORC2. Previously, we published that arsenic trioxide (ATO) inhibits AKT activity and in some cases, decreases AKT protein expression. Therefore, we propose that combining ATO and rapamycin may circumvent the AKT feedback loop and increase the anti-tumor effects. Using a panel of breast cancer cell lines, we find that ATO, at clinically-achievable doses, can enhance the inhibitory activity of the mTORi temsirolimus. In all cell lines, temsirolimus treatment resulted in AKT activation, which was decreased by concomitant ATO treatment only in those cell lines where ATO enhanced growth inhibition. Treatment with rapalog also results in activated ERK signaling, which is decreased with ATO co-treatment in all cell lines tested. We next tested the toxicity and efficacy of rapamycin plus ATO combination therapy in a MDA-MB-468 breast cancer xenograft model. The drug combination was well-tolerated, and rapamycin did not increase ATO-induced liver enzyme levels. In addition, combination of these drugs was significantly more effective at inhibiting tumor growth compared to individual drug treatments, which corresponded with diminished phospho-Akt and phospho-ERK levels when compared with rapamycin-treated tumors. Therefore, we propose that combining ATO and mTORi may overcome the feedback loop by decreasing activation of the MAPK and AKT signaling pathways. PMID:24392034

  7. {delta}-Opioid receptor-stimulated Akt signaling in neuroblastoma x glioma (NG108-15) hybrid cells involves receptor tyrosine kinase-mediated PI3K activation

    SciTech Connect

    Heiss, Anika; Ammer, Hermann; Eisinger, Daniela A.

    2009-07-15

    {delta}-Opioid receptor (DOR) agonists possess cytoprotective properties, an effect associated with activation of the 'pro-survival' kinase Akt. Here we delineate the signal transduction pathway by which opioids induce Akt activation in neuroblastoma x glioma (NG108-15) hybrid cells. Exposure of the cells to both [D-Pen{sup 2,5}]enkephalin and etorphine resulted in a time- and dose-dependent increase in Akt activity, as measured by means of an activation-specific antibody recognizing phosphoserine-473. DOR-mediated Akt signaling is blocked by the opioid antagonist naloxone and involves inhibitory G{sub i/o} proteins, because pre-treatment with pertussis toxin, but not over-expression of the G{sub q/11} scavengers EBP50 and GRK2-K220R, prevented this effect. Further studies with Wortmannin and LY294002 revealed that phophoinositol-3-kinase (PI3K) plays a central role in opioid-induced Akt activation. Opioids stimulate Akt activity through transactivation of receptor tyrosine kinases (RTK), because pre-treatment of the cells with inhibitors for neurotrophin receptor tyrosine kinases (AG879) and the insulin-like growth factor receptor IGF-1 (AG1024), but not over-expression of the G{beta}{gamma} scavenger phosducin, abolished this effect. Activated Akt translocates to the nuclear membrane, where it promotes GSK3 phosphorylation and prevents caspase-3 cleavage, two key events mediating inhibition of cell apoptosis and enhancement of cell survival. Taken together, these results demonstrate that in NG108-15 hybrid cells DOR agonists possess cytoprotective properties mediated by activation of the RTK/PI3K/Akt signaling pathway.

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

    PubMed Central

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

    2016-01-01

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

  9. Akt2 deficiency protects from acute lung injury via alternative macrophage activation and miR-146a induction in mice.

    PubMed

    Vergadi, Eleni; Vaporidi, Katerina; Theodorakis, Emmanuel E; Doxaki, Christina; Lagoudaki, Eleni; Ieronymaki, Eleftheria; Alexaki, Vassilia I; Helms, Mike; Kondili, Eumorfia; Soennichsen, Birte; Stathopoulos, Efstathios N; Margioris, Andrew N; Georgopoulos, Dimitrios; Tsatsanis, Christos

    2014-01-01

    Acute respiratory distress syndrome (ARDS) is a major cause of respiratory failure, with limited effective treatments available. Alveolar macrophages participate in the pathogenesis of ARDS. To investigate the role of macrophage activation in aseptic lung injury and identify molecular mediators with therapeutic potential, lung injury was induced in wild-type (WT) and Akt2(-/-) mice by hydrochloric acid aspiration. Acid-induced lung injury in WT mice was characterized by decreased lung compliance and increased protein and cytokine concentration in bronchoalveolar lavage fluid. Alveolar macrophages acquired a classical activation (M1) phenotype. Acid-induced lung injury was less severe in Akt2(-/-) mice compared with WT mice. Alveolar macrophages from acid-injured Akt2(-/-) mice demonstrated the alternative activation phenotype (M2). Although M2 polarization suppressed aseptic lung injury, it resulted in increased lung bacterial load when Akt2(-/-) mice were infected with Pseudomonas aeruginosa. miR-146a, an anti-inflammatory microRNA targeting TLR4 signaling, was induced during the late phase of lung injury in WT mice, whereas it was increased early in Akt2(-/-) mice. Indeed, miR-146a overexpression in WT macrophages suppressed LPS-induced inducible NO synthase (iNOS) and promoted M2 polarization, whereas miR-146a inhibition in Akt2(-/-) macrophages restored iNOS expression. Furthermore, miR-146a delivery or Akt2 silencing in WT mice exposed to acid resulted in suppression of iNOS in alveolar macrophages. In conclusion, Akt2 suppression and miR-146a induction promote the M2 macrophage phenotype, resulting in amelioration of acid-induced lung injury. In vivo modulation of macrophage phenotype through Akt2 or miR-146a could provide a potential therapeutic approach for aseptic ARDS; however, it may be deleterious in septic ARDS because of impaired bacterial clearance.

  10. Activation of the PI3K/AKT pathway correlates with prognosis in stage II colon cancer

    PubMed Central

    Malinowsky, K; Nitsche, U; Janssen, K-P; Bader, F G; Späth, C; Drecoll, E; Keller, G; Höfler, H; Slotta-Huspenina, J; Becker, K-F

    2014-01-01

    Background: Patients with UICC/AJCC stage II colon cancer have a high 5-year overall survival rate after surgery. Nevertheless, a significant subgroup of patients develops tumour recurrence. Currently, there are no clinically established biomarkers available to identify this patient group. We applied reverse-phase protein arrays (RPPA) for phosphatidylinositide-3-kinase pathway activation mapping to stratify patients according to their risk of tumour recurrence after surgery. Methods: Full-length proteins were extracted from formalin-fixed, paraffin-embedded tissue samples of 118 patients who underwent curative resection. RPPA technology was used to analyse expression and/or phosphorylation levels of six major factors of the phosphatidylinositide-3-kinase pathway. Oncogenic mutations of KRAS and BRAF, and DNA microsatellite status, currently discussed as prognostic markers, were analysed in parallel. Results: Expression of phospho-AKT (HR=3.52; P=0.032), S6RP (HR=6.3; P=0.044), and phospho-4E-BP1 (HR=4.12; P=0.011) were prognostic factors for disease-free survival. None of the molecular genetic alterations were significantly associated with prognosis. Conclusions: Our data indicate that activation of the PI3K/AKT pathway evidenced on the protein level might be a valuable prognostic marker to stratify patients for their risk of tumour recurrence. Beside adjuvant chemotherapy targeting of upregulated PI3K/AKT signalling may be an attractive strategy for treatment of high-risk patients. PMID:24619078

  11. Functional characterization of GhAKT1, a novel Shaker-like K⁺ channel gene involved in K⁺ uptake from cotton (Gossypium hirsutum).

    PubMed

    Xu, Juan; Tian, Xiaoli; Egrinya Eneji, A; Li, Zhaohu

    2014-07-15

    Shaker-like potassium (K(+)) channels in plants play an important role in K(+) absorption and transport. In this study, we characterized a Shaker-like K(+) channel gene GhAKT1 from the roots of Gossypium hirsutum cv. Liaomian17. Phylogenetic analysis showed that the GhAKT1 belongs to the AKT1-subfamily in the Shaker-like K(+) channel family. Confocal imaging of a GhAKT1-green fluorescent fusion protein (GFP) in transgenic Arabidopsis plants indicated that GhAKT1 is localized in the plasma membrane. Transcript analysis located GhAKT1 predominantly in cotton leaves with low abundance in roots, stem and shoot apex. Similarly, β-glucuronidase (GUS) activity was detected in both leaves and roots of PGhAKT1::GUS transgenic Arabidopsis plants. In roots, the GUS signals appeared in the epidermis, cortex and endodermis and root hairs, suggesting the contribution of GhAKT1 to K(+) uptake. In leaves, GhAKT1 was expressed in differentiated leaf primordial as well as mesophyll cells and veins of expanded leaves, pointing to its involvement in cell elongation and K(+) transport and distribution in leaves. Severe K(+) deficiency did not affect the expression of GhAKT1 gene. GhAKT1-overexpression in either the Arabidopsis wild-type or akt1 mutant enhanced the growth of transgenic seedlings under low K(+) deficiency and raised the net K(+) influx in roots at 100μM external K(+) concentration, within the range of operation of the high-affinity K(+) uptake system. The application of 2mM BaCl2 resulted in net K(+) efflux in roots, and eliminated the differences between GhAKT1-overexpression lines and their acceptors indicating that the K(+) uptake mediated by GhAKT1 is also as Ba(2+)-sensitive as AtAKT1.

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

    PubMed

    Hamilton, Garth; Abraham, Aswin G; Morton, Jennifer; Sampson, Oliver; Pefani, Dafni E; Khoronenkova, Svetlana; Grawenda, Anna; Papaspyropoulos, Angelos; Jamieson, Nigel; McKay, Colin; Sansom, Owen; Dianov, Grigory L; O'Neill, Eric

    2014-08-15

    Nucleophosmin (NPM) is known to regulate ARF subcellular localization and MDM2 activity in response to oncogenic stress, though the precise mechanism has remained elusive. Here we describe how NPM and ARF associate in the nucleoplasm to form a MDM2 inhibitory complex. We find that oligomerization of NPM drives nucleolar accumulation of ARF. Moreover, the formation of NPM and ARF oligomers antagonizes MDM2 association with the inhibitory complex, leading to activation of MDM2 E3-ligase activity and targeting of p53. We find that AKT phosphorylation of NPM-Ser48 prevents oligomerization that results in nucleoplasmic localization of ARF, constitutive MDM2 inhibition and stabilization of p53. We also show that ARF promotes p53 mutant stability in tumors and suppresses p73 mediated p21 expression and senescence. We demonstrate that AKT and PI3K inhibitors may be effective in treatment of therapeutically resistant tumors with elevated AKT and carrying gain of function mutations in p53. Our results show that the clinical candidate AKT inhibitor MK-2206 promotes ARF nucleolar localization, reduced p53(mut) stability and increased sensitivity to ionizing radiation in a xenograft model of pancreatic cancer. Analysis of human tumors indicates that phospho-S48-NPM may be a useful biomarker for monitoring AKT activity and in vivo efficacy of AKT inhibitor treatment. Critically, we propose that combination therapy involving PI3K-AKT inhibitors would benefit from a patient stratification rationale based on ARF and p53(mut) status.

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

    PubMed Central

    Morton, Jennifer; Sampson, Oliver; Pefani, Dafni E.; Khoronenkova, Svetlana; Grawenda, Anna; Papaspyropoulos, Angelos; Jamieson, Nigel; McKay, Colin; Sansom, Owen; Dianov, Grigory L.; O'Neill, Eric

    2014-01-01

    Nucleophosmin (NPM) is known to regulate ARF subcellular localization and MDM2 activity in response to oncogenic stress, though the precise mechanism has remained elusive. Here we describe how NPM and ARF associate in the nucleoplasm to form a MDM2 inhibitory complex. We find that oligomerization of NPM drives nucleolar accumulation of ARF. Moreover, the formation of NPM and ARF oligomers antagonizes MDM2 association with the inhibitory complex, leading to activation of MDM2 E3-ligase activity and targeting of p53. We find that AKT phosphorylation of NPM-Ser48 prevents oligomerization that results in nucleoplasmic localization of ARF, constitutive MDM2 inhibition and stabilization of p53. We also show that ARF promotes p53 mutant stability in tumors and suppresses p73 mediated p21 expression and senescence. We demonstrate that AKT and PI3K inhibitors may be effective in treatment of therapeutically resistant tumors with elevated AKT and carrying gain of function mutations in p53. Our results show that the clinical candidate AKT inhibitor MK-2206 promotes ARF nucleolar localization, reduced p53mut stability and increased sensitivity to ionizing radiation in a xenograft model of pancreatic cancer. Analysis of human tumors indicates that phospho-S48-NPM may be a useful biomarker for monitoring AKT activity and in vivo efficacy of AKT inhibitor treatment. Critically, we propose that combination therapy involving PI3K-AKT inhibitors would benefit from a patient stratification rationale based on ARF and p53mut status. PMID:25071014

  14. Angiogenin-induced protein kinase B/Akt activation is necessary for angiogenesis but is independent of nuclear translocation of angiogenin in HUVE cells

    SciTech Connect

    Kim, Hye-Mi; Kang, Dong-Ku; Kim, Hak Yong; Kang, Sang Sun; Chang, Soo-Ik . E-mail: sichang@cbnu.ac.kr

    2007-01-12

    Angiogenin, a potent angiogenic factor, binds to endothelial cells and is endocytosed and rapidly translocated to and concentrated in the nucleolus where it binds to DNA. In this study, we report that angiogenin induces transient phosphorylation of protein kinase B/Akt in cultured human umbilical vein endothelial (HUVE) cells. LY294002 inhibits the angiogenin-induced protein kinase B/Akt activation and also angiogenin-induced cell migration in vitro as well as angiogenesis in chick embryo chorioallantoic membrane in vivo without affecting nuclear translocation of angiogenin in HUVE cells. These results suggest that cross-talk between angiogenin and protein kinase B/Akt signaling pathways is essential for angiogenin-induced angiogenesis in vitro and in vivo, and that angiogenin-induced PKB/Akt activation is independent of nuclear translocation of angiogenin in HUVE cells.

  15. miR-190-mediated downregulation of PHLPP contributes to arsenic-induced Akt activation and carcinogenesis.

    PubMed

    Beezhold, Kevin; Liu, Jia; Kan, Hong; Meighan, Terry; Castranova, Vince; Shi, Xianglin; Chen, Fei

    2011-10-01

    The role of trivalent arsenic (As(3+)) on the regulation of the recently identified noncoding small RNAs, mainly microRNAs, has not been explored so far. In the present study, we provide evidence showing that As(3+) is a potent inducer for the expression of miR-190 in human bronchial epithelial cells. The induction of miR-190 by As(3+) is concentration dependent and associated with the expression of the host gene of miR-190, talin 2, a gene encoding a high-molecular-weight cytoskeletal protein. The elevated level of miR-190 induced by As(3+) is capable of downregulating the translation of the PH domain leucine-rich repeat protein phosphatase (PHLPP), a negative regulator of Akt signaling. Such a downregulation is occurred through direct interaction of the miR-190 with the 3'-UTR region of the PHLPP mRNA, leading to a diminished PHLPP protein expression and consequently, an enhanced Akt activation and expression of vascular endothelial growth factor, an Akt-regulated protein. Overexpression of miR-190 itself is able to enhance proliferation and malignant transformation of the cells as determined by anchorage-independent growth of the cells in soft agar. Accordingly, the data presented suggest that induction of miR-190 is one of the key mechanisms in As(3+)-induced carcinogenesis.

  16. Lowered Expression of Tumor Suppressor Candidate MYO1C Stimulates Cell Proliferation, Suppresses Cell Adhesion and Activates AKT

    PubMed Central

    Visuttijai, Kittichate; Pettersson, Jennifer; Mehrbani Azar, Yashar; van den Bout, Iman; Örndal, Charlotte; Marcickiewicz, Janusz; Nilsson, Staffan; Hörnquist, Michael; Olsson, Björn; Ejeskär, Katarina

    2016-01-01

    Myosin-1C (MYO1C) is a tumor suppressor candidate located in a region of recurrent losses distal to TP53. Myo1c can tightly and specifically bind to PIP2, the substrate of Phosphoinositide 3-kinase (PI3K), and to Rictor, suggesting a role for MYO1C in the PI3K pathway. This study was designed to examine MYO1C expression status in a panel of well-stratified endometrial carcinomas as well as to assess the biological significance of MYO1C as a tumor suppressor in vitro. We found a significant correlation between the tumor stage and lowered expression of MYO1C in endometrial carcinoma samples. In cell transfection experiments, we found a negative correlation between MYO1C expression and cell proliferation, and MYO1C silencing resulted in diminished cell migration and adhesion. Cells expressing excess of MYO1C had low basal level of phosphorylated protein kinase B (PKB, a.k.a. AKT) and cells with knocked down MYO1C expression showed a quicker phosphorylated AKT (pAKT) response in reaction to serum stimulation. Taken together the present study gives further evidence for tumor suppressor activity of MYO1C and suggests MYO1C mediates its tumor suppressor function through inhibition of PI3K pathway and its involvement in loss of contact inhibition. PMID:27716847

  17. Short-term psychosocial stress protects photoreceptors from damage via corticosterone-mediated activation of the AKT pathway.

    PubMed

    Forkwa, Tembei K; Neumann, Inga D; Tamm, Ernst R; Ohlmann, Andreas; Reber, Stefan O

    2014-02-01

    Apoptotic death of photoreceptors in hereditary retinal degenerations can be prevented by neuroprotective molecules. Here, we report that adrenal glucocorticoids (GC) released during psychosocial stress protect photoreceptors from apoptosis after light damage. Psychosocial stress is known to be the main type of stressor humans are exposed to and was induced here in mice by 10h of chronic subordinate colony housing (CSC). Photoreceptor damage was generated by subsequent exposure to white light. Short-term psychosocial stress prior to illumination significantly reduced the number of apoptotic photoreceptors, an effect that was absent in adrenalectomized (ADX) mice. The neuroprotective effect was completely restored in ADX mice substituted with GC. Moreover, phosphorylation of retinal AKT increased following CSC or exogenous GC treatment, an effect that was again absent in ADX mice exposed to CSC. Finally, inhibition of AKT signaling with triciribine blocked the stress- and GC-mediated neuroprotective effects on photoreceptors. In summary, we provide evidence that 1) short-term psychosocial stress protects photoreceptors from light-induced damage and 2) the protective effect is most likely mediated by GC-induced activation of the AKT signaling pathway.

  18. PI3K-C2γ is a Rab5 effector selectively controlling endosomal Akt2 activation downstream of insulin signalling

    PubMed Central

    Braccini, Laura; Ciraolo, Elisa; Campa, Carlo C.; Perino, Alessia; Longo, Dario L.; Tibolla, Gianpaolo; Pregnolato, Marco; Cao, Yanyan; Tassone, Beatrice; Damilano, Federico; Laffargue, Muriel; Calautti, Enzo; Falasca, Marco; Norata, Giuseppe D.; Backer, Jonathan M.; Hirsch, Emilio

    2015-01-01

    In the liver, insulin-mediated activation of the phosphatidylinositol 3-kinase (PI3K)/Akt pathway is at the core of metabolic control. Multiple PI3K and Akt isoenzymes are found in hepatocytes and whether isoform-selective interplays exist is currently unclear. Here we report that insulin signalling triggers the association of the liver-specific class II PI3K isoform γ (PI3K-C2γ) with Rab5-GTP, and its recruitment to Rab5-positive early endosomes. In these vesicles, PI3K-C2γ produces a phosphatidylinositol-3,4-bisphosphate pool specifically required for delayed and sustained endosomal Akt2 stimulation. Accordingly, loss of PI3K-C2γ does not affect insulin-dependent Akt1 activation as well as S6K and FoxO1-3 phosphorylation, but selectively reduces Akt2 activation, which specifically inhibits glycogen synthase activity. As a consequence, PI3K-C2γ-deficient mice display severely reduced liver accumulation of glycogen and develop hyperlipidemia, adiposity as well as insulin resistance with age or after consumption of a high-fat diet. Our data indicate PI3K-C2γ supports an isoenzyme-specific forking of insulin-mediated signal transduction to an endosomal pool of Akt2, required for glucose homeostasis. PMID:26100075

  19. Gardenamide A Protects RGC-5 Cells from H₂O₂-Induced Oxidative Stress Insults by Activating PI3K/Akt/eNOS Signaling Pathway.

    PubMed

    Wang, Rikang; Peng, Lizhi; Zhao, Jiaqiang; Zhang, Laitao; Guo, Cuiping; Zheng, Wenhua; Chen, Heru

    2015-01-01

    Gardenamide A (GA) protects the rat retinal ganglion (RGC-5) cells against cell apoptosis induced by H₂O₂. The protective effect of GA was completely abrogated by the specific phosphoinositide 3-kinase (PI3K) inhibitor LY294002, and the specific protein kinase B (Akt) inhibitor Akt VIII respectively, indicating that the protective mechanism of GA is mediated by the PI3K/Akt signaling pathway. The specific extracellular signal-regulated kinase (ERK1/2) inhibitor PD98059 could not block the neuroprotection of GA. GA attenuated the levels of reactive oxygen species (ROS) and malondialdehyde (MDA) induced by H₂O₂. Western blotting showed that GA promoted the phosphorylation of ERK1/2, Akt and endothelial nitric oxide synthase (eNOS), respectively, and effectively reversed the H₂O₂-inhibited phosphorylation of these three proteins. LY294002 completely inhibited the GA-activated phosphorylation of Akt, while only partially inhibiting eNOS. This evidence implies that eNOS may be activated directly by GA. PD98059 attenuated only partially the GA-induced phosphorylation of ERK1/2 with/without the presence of H₂O₂, indicating that GA may activate ERK1/2 directly. All these results put together confirm that GA protects RGC-5 cells from H₂O₂ insults via the activation of PI3K/Akt/eNOS signaling pathway. Whether the ERK1/2 signaling pathway is involved requires further investigations.

  20. Cell cycle regulation of breast cancer cells through estrogen-induced activities of ERK and Akt protein kinases.

    PubMed

    Geffroy, Nancy; Guédin, Aurore; Dacquet, Catherine; Lefebvre, Philippe

    2005-06-15

    The proliferative effect of estrogens on breast cancer cell (BCC) is mainly mediated through estrogen receptors (ER). Non-transcriptional effects of estrogens, exerted through activation of several protein kinases, may also contribute to BCC proliferation. However, the relative contribution of these two responses to BCC proliferation is not known. We characterized a novel estrogenic receptor ligand which possess Akt and ERK activating properties distinct from that of 17beta-estradiol. Early and delayed waves of activation of these kinases were detected upon estrogenic challenge of BCC, but only molecules able to promote a significant, delayed activation of ERK-induced BCC proliferation. Estrogen-induced cell cycle progression was not sensitive to the inhibition of ERK-regulating kinases MEK1 and 2. ERalpha was found to be necessary, but not sufficient for kinases activation. Thus, estrogens elicit a distinct pattern of early and delayed activation of ERK and Akt, and early protein kinase activation is probably not involved in BCC proliferation. Structural variations in the estrogen molecule may confer novel biological properties unrelated to estrogen-dependent transcriptional activation.

  1. Tanshinone IIA Prevents Leu27IGF-II-Induced Cardiomyocyte Hypertrophy Mediated by Estrogen Receptor and Subsequent Akt Activation.

    PubMed

    Weng, Yueh-Shan; Wang, Hsueh-Fang; Pai, Pei-Ying; Jong, Gwo-Ping; Lai, Chao-Hung; Chung, Li-Chin; Hsieh, Dennis Jine-Yuan; HsuanDay, Cecilia; Kuo, Wei-Wen; Huang, Chih-Yang

    2015-01-01

    IGF-IIR plays important roles as a key regulator in myocardial pathological hypertrophy and apoptosis, which subsequently lead to heart failure. Salvia miltiorrhiza Bunge (Danshen) is a traditional Chinese medicinal herb used to treat cardiovascular diseases. Tanshinone IIA is an active compound in Danshen and is structurally similar to 17[Formula: see text]-estradiol (E[Formula: see text]. However, whether tanshinone IIA improves cardiomyocyte survival in pathological hypertrophy through estrogen receptor (ER) regulation remains unclear. This study investigates the role of ER signaling in mediating the protective effects of tanshinone IIA on IGF-IIR-induced myocardial hypertrophy. Leu27IGF-II (IGF-II analog) was shown in this study to specifically activate IGF-IIR expression and ICI 182,780 (ICI), an ER antagonist used to investigate tanshinone IIA estrogenic activity. We demonstrated that tanshinone IIA significantly enhanced Akt phosphorylation through ER activation to inhibit Leu27IGF-II-induced calcineurin expression and subsequent NFATc3 nuclear translocation to suppress myocardial hypertrophy. Tanshinone IIA reduced the cell size and suppressed ANP and BNP, inhibiting antihypertrophic effects induced by Leu27IGF-II. The cardioprotective properties of tanshinone IIA that inhibit Leu27IGF-II-induced cell hypertrophy and promote cell survival were reversed by ICI. Furthermore, ICI significantly reduced phospho-Akt, Ly294002 (PI3K inhibitor), and PI3K siRNA significantly reduced the tanshinone IIA-induced protective effect. The above results suggest that tanshinone IIA inhibited cardiomyocyte hypertrophy, which was mediated through ER, by activating the PI3K/Akt pathway and inhibiting Leu27IGF-II-induced calcineurin and NFATC3. Tanshinone IIA exerted strong estrogenic activity and therefore represented a novel selective ER modulator that inhibits IGF-IIR signaling to block cardiac hypertrophy. PMID:26621443

  2. Niacin activates the PI3K/Akt cascade via PKC- and EGFR-transactivation-dependent pathways through hydroxyl-carboxylic acid receptor 2.

    PubMed

    Sun, Huawang; Li, Guo; Zhang, Wenjuan; Zhou, Qi; Yu, Yena; Shi, Ying; Offermanns, Stefan; Lu, Jianxin; Zhou, Naiming

    2014-01-01

    Niacin has been demonstrated to activate a PI3K/Akt signaling cascade to prevent brain damage after stroke and UV-induced skin damage; however, the underlying molecular mechanisms for HCA2-induced Akt activation remain to be elucidated. Using CHO-K1 cells stably expressing HCA2 and A431 cells, a human epidermoid cell line with high levels of endogenous expression of functional HCA2 receptors, we first demonstrated that niacin induced a robust Akt phosphorylation at both Thr308 and Ser473 in a time-dependent fashion, with a maximal activation at 5 min and a subsequent reduction to baseline by 30 min through HCA2, and that the activation was significantly blocked by pertussis toxin. The HCA2-mediated activation of Akt was also significantly inhibited by the PKC inhibitors GF109203x and Go6983 in both cell lines, by the PDGFR-selective inhibitor tyrphostin A9 in CHO-HCA2 cells and by the MMP inhibitor GM6001 and EGFR-specific inhibitor AG1478 in A431 cells. These results suggest that the PKC pathway and PDGFR/EGFR transactivation pathway play important roles in HCA2-mediated Akt activation. Further investigation indicated that PI3K and the Gβγ subunit were likely to play an essential role in HCA2-induced Akt activation. Moreover, Immunobloting analyses using an antibody that recognizes p70S6K1 phosphorylated at Thr389 showed that niacin evoked p70S6K1 activation via the PI3K/Akt pathway. The results of our study provide new insight into the signaling pathways involved in HCA2 activation.

  3. Opisthorchis viverrini infection activates the PI3K/ AKT/PTEN and Wnt/β-catenin signaling pathways in a Cholangiocarcinogenesis model.

    PubMed

    Yothaisong, Supak; Thanee, Malinee; Namwat, Nisana; Yongvanit, Puangrat; Boonmars, Thidarut; Puapairoj, Anucha; Loilome, Watcharin

    2014-01-01

    Opisthorchis viverrini (Ov) infection is the major etiological factor for cholangiocarcinoma (CCA), especially in northeast Thailand. We have previously reported significant involvement of PI3K/AKT/PTEN and Wnt/β- catenin in human CCA tissues. The present study, therefore, examined the expression and activation of PI3K/ AKT/PTEN and Wnt/β-catenin signaling components during Ov-induced cholangiocarcinogenesis in a hamster animal model. Hamsters were divided into two groups; non-treated and Ov plus NDMA treated. The results of immunohistochemical staining showed an upregulation of PI3K/AKT signaling as determined by elevated expression of the p85α-regulatory and p110α-catalytic subunits of PI3K as well as increased expression and activation of AKT during cholangiocarcinogenesis. Interestingly, the staining intensity of activated AKT (p-AKT) increased in the apical regions of the bile ducts and strong staining was detected where the liver fluke resides. Moreover, PTEN, a negative regulator of PI3K/AKT, was suppressed by decreased expression and increased phosphorylation during cholangiocarcinogenesis. We also detected upregulation of Wnt/β-catenin signaling as determined by increased positive staining of Wnt3, Wnt3a, Wnt5a, Wnt7b and β-catenin, corresponded with the period of cholangiocarcinogenesis. Furthermore, nuclear staining of β-catenin was observed in CCA tissues. Our results suggest the liver fluke infection causes chronic inflammatory conditions which lead to upregulation of the PI3K/AKT and Wnt/β-catenin signaling pathways which may drive CCA carcinogenesis. These results provide useful information for drug development, prevention and treatment of CCA. PMID:25556493

  4. The Novel Small Molecule Inhibitor, OSU-T315, Suppresses Vestibular Schwannoma and Meningioma Growth by Inhibiting PDK2 Function in the AKT Pathway Activation

    PubMed Central

    Mercado-Pimentel, ME; Igarashi, S; Dunn, AM; Behbahani, M; Miller, C; Read, CM; Jacob, A

    2016-01-01

    Activation of PKB/AKT signaling, which requires PDK1 and PDK2 function, drives Vestibular Schwannoma (VS) and meningioma growth. PDK2 function is defined as a molecule that phosphorylates AKT-Ser473. Integrin-Linked Kinase (ILK) functions as PDK2 in PKB/AKT activation in many cancers; therefore, we hypothesized that OSU-T315, a small molecule ILK inhibitor, will inhibit the ILK-PDK2 function in PKB/AKT signaling activation in VS and meningioma cell growth. OSU-T315 decreased cell viability at IC50 < 2μM in VS (HEI193) and meningioma (Ben-Men-1) cell lines, in primary cells at < 3.5μM, while in normal primary Schwann cells at 7.1μM. OSU-T315 inhibits AKT signaling by decreasing phosphorylation at AKT-Ser473, AKT-Thr308, ILK-Ser246 and ILK-Thr173. In addition, OSU-T315 affected the phosphorylation or expression levels of AKT downstream proliferation effectors as well as autophagy markers. Flow cytometry shows that OSU-T315 increased the percentage of cells arrested at G2/M for both, HEI193 (39.99%) and Ben-Men-1 (26.96%) cells, compared to controls (21.54%, 8.47%). Two hours of OSU-T315 treatment increased cell death in both cell lines (34.3%, 9.1%) versus untreated (12.1%, 8.1%). Though longer exposure increased cell death in Ben-Men-1, TUNEL assays showed that OSU-T315 does not induce apoptosis. OSU-T315 was primarily cytotoxic for HEI193 and Ben-Men-1 inducing a dysregulated autophagy. Our studies suggest that OSU-T315 has translational potential as a chemotherapeutic agent against VS and meningioma.

  5. Silymarin prevents palmitate-induced lipotoxicity in HepG2 cells: involvement of maintenance of Akt kinase activation.

    PubMed

    Song, Zhenyuan; Song, Ming; Lee, David Y W; Liu, Yanze; Deaciuc, Ion V; McClain, Craig J

    2007-10-01

    Whereas adipocytes have a unique capacity to store excess free fatty acids in the form of triglyceride in lipid droplets, non-adipose tissues, such as liver, have a limited capacity for storage of lipids. Saturated long-chain fatty acids, such as palmitate, are the major contributors to lipotoxicity. Silymarin is a mixture of flavonolignans, extracted from the milk thistle (Silibum marianum). Its hepatoprotective properties have been studied both in vitro and in vivo; however, its effect on palmitate-induced lipotoxicity has not been investigated. The objective of this study was to investigate (i) whether silymarin could protect HepG2 cells from palmitate-induced cell death in an in vitro model, and (ii) possible mechanisms involved in this hepatoprotective role of silymarin. HepG2 cells were treated with palmitate in the absence or presence of silymarin and supernatants or cell lysates were collected at varying time-points. Cell death was assayed by measuring DNA fragmentation, caspase-3 activity and lactate dehydrogenase release. Lipid peroxidation was assessed by measuring malondialdehyde and 4-hydroxyalkenals. Akt kinase activity was also measured. Incubation with palmitate caused significant death in HepG2 cells. Palmitate incubation did not cause significant changes in reactive oxygen species production or intracellular glutathione content, but markedly inhibited Akt kinase activity. Pre-treatment of HepG2 cells with silymarin prevented palmitate-induced inhibition of Akt kinase activity and attenuated cell death. Our results suggest that silymarin may be an effective agent in protecting hepatocytes from saturated fatty acids-induced cell death. These data also provide a further rationale for exploration of the use of silymarin in the treatment of non-alcoholic steatohepatitis.

  6. Methylmercury, an environmental electrophile capable of activation and disruption of the Akt/CREB/Bcl-2 signal transduction pathway in SH-SY5Y cells

    PubMed Central

    Unoki, Takamitsu; Abiko, Yumi; Toyama, Takashi; Uehara, Takashi; Tsuboi, Koji; Nishida, Motohiro; Kaji, Toshiyuki; Kumagai, Yoshito

    2016-01-01

    Methylmercury (MeHg) modifies cellular proteins via their thiol groups in a process referred to as “S-mercuration”, potentially resulting in modulation of the cellular signal transduction pathway. We examined whether low-dose MeHg could affect Akt signaling involved in cell survival. Exposure of human neuroblastoma SH-SY5Y cells of up to 2 μM MeHg phosphorylated Akt and its downstream signal molecule CREB, presumably due to inactivation of PTEN through S-mercuration. As a result, the anti-apoptotic protein Bcl-2 was up-regulated by MeHg. The activation of Akt/CREB/Bcl-2 signaling mediated by MeHg was, at least in part, linked to cellular defence because either pretreatment with wortmannin to block PI3K/Akt signaling or knockdown of Bcl-2 enhanced MeHg-mediated cytotoxicity. In contrast, increasing concentrations of MeHg disrupted Akt/CREB/Bcl-2 signaling. This phenomenon was attributed to S-mercuration of CREB through Cys286 rather than Akt. These results suggest that although MeHg is an apoptosis-inducing toxicant, this environmental electrophile is able to activate the cell survival signal transduction pathway at lower concentrations prior to apoptotic cell death. PMID:27357941

  7. Activation of PI3K/Akt/mTOR signaling in the tumor stroma drives endocrine therapy-dependent breast tumor regression

    PubMed Central

    Polo, María Laura; Riggio, Marina; May, María; Rodríguez, María Jimena; Perrone, María Cecilia; Stallings-Mann, Melody; Kaen, Diego; Frost, Marlene; Goetz, Matthew; Boughey, Judy; Lanari, Claudia; Radisky, Derek; Novaro, Virginia

    2015-01-01

    Improved efficacy of neoadjuvant endocrine-targeting therapies in luminal breast carcinomas could be achieved with optimal use of pathway targeting agents. In a mouse model of ductal breast carcinoma we identify a tumor regressive stromal reaction that is induced by neoadjuvant endocrine therapy. This reparative reaction is characterized by tumor neovascularization accompanied by infiltration of immune cells and carcinoma-associated fibroblasts that stain for phosphorylated ribosomal protein S6 (pS6), downstream the PI3K/Akt/mTOR pathway. While tumor variants with higher PI3K/Akt/mTOR activity respond well to a combination of endocrine and PI3K/Akt/mTOR inhibitors, tumor variants with lower PI3K/Akt/mTOR activity respond more poorly to the combination therapy than to the endocrine therapy alone, associated with inhibition of stromal pS6 and the reparative reaction. In human breast cancer xenografts we confirm that such differential sensitivity to therapy is primarily determined by the level of PI3K/Akt/mTOR in tumor cells. We further show that the clinical response of breast cancer patients undergoing neoadjuvant endocrine therapy is associated with the reparative stromal reaction. We conclude that tumor level and localization of pS6 are associated with therapeutic response in breast cancer and represent biomarkers to distinguish which tumors will benefit from the incorporation of PI3K/Akt/mTOR inhibitors with neoadjuvant endocrine therapy. PMID:26098779

  8. AKT and N-Ras co-activation in the mouse liver promotes rapid carcinogenesis via mTORC1, FOXM1/SKP2, and c-Myc pathways

    PubMed Central

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

    2011-01-01

    Activation of v-akt murine thymoma viral oncogene homolog (AKT) and Ras pathways is often implicated in carcinogenesis. However, the oncogenic cooperation between these two cascades in relationship to hepatocellular carcinoma (HCC) development remains undetermined. To investigate this issue, we generated a mouse model characterized by combined overexpression of activated forms of AKT and neuroblastoma Ras viral oncogene homolog (N-Ras) protooncogenes in the liver via hydrodynamic gene transfer. The molecular mechanisms underlying crosstalk between AKT and N-Ras were assessed in the mouse model and further evaluated in human and murine HCC cell lines. We found that co-expression of AKT and N-Ras resulted in a dramatic acceleration of liver tumor development when compared with mice overexpressing AKT alone, whereas N-Ras alone did not lead to tumor formation. At the cellular level, concomitant upregulation of AKT and N-Ras resulted in increased proliferation and microvascularization when compared with AKT injected mice. Mechanistic studies suggested that accelerated hepatocarcinogenesis driven by AKT and N-Ras resulted from a strong activation of mammalian target of rapamycin complex 1 (mTORC1). Furthermore, elevated expression of FOXM1/SKP2 and c-Myc also contributed to rapid tumor growth in AKT/Ras mice, yet via mTORC1-independent mechanisms. The biological effects of co-activation of AKT and N-Ras were then recapitulated in vitro using HCC cell lines, which supports the functional significance of mTORC1, FOXM1/SKP2 and c-Myc signaling cascades in mediating AKT and N-Ras induced liver tumor development. Conclusion Our data demonstrate the in vivo crosstalk between the AKT and Ras pathways in promoting liver tumor development, and the pivotal role of mTORC1-dependent and independent pathways in mediating AKT and Ras induced hepatocarcinogenesis. PMID:21993994

  9. Mutant strains of Tetrahymena thermophila defective in thymidine kinase activity: Biochemical and genetic characterization

    SciTech Connect

    Cornish, K.V.; Pearlman, R.E.

    1982-08-01

    Three mutant strains, one conditional, of Tetrahymena thermophila were defective in thymidine phosphorylating activity in vivo and in thymidine kinase activity in vitro. Nucleoside phosphotransferase activity in mutant cell extracts approached wild-type levels, suggesting that thymidine kinase is responsible for most, if not all, thymidine phosphorylation in vivo. Thymidine kinase activity in extracts of the conditional mutant strain was deficient when the cells were grown or assayed or both at the permissive temperature, implying a structural enzyme defect. Analysis of the reaction products from in vitro assays with partially purified enzymes showed that phosphorylation by thymidine kinase and nucleoside phosphotransferase occurred at the 5' position. Genetic analyses showed that the mutant phenotype was recessive and that mutations in each of the three mutant strains did not complement, suggesting allelism.

  10. GGAP2/PIKE-A directly activates both the Akt and NF-κB pathways and promotes prostate cancer progression

    PubMed Central

    Cai, Yi; Wang, Jianghua; Li, Rile; Ayala, Gustavo; Ittmann, Michael; Liu, Mingyao

    2009-01-01

    GGAP2/PIKE-A is a GTP-binding protein which can enhance Akt activity. Increased activation of the AKT and NF-κB pathways have been identified as critical steps in cancer initiation and progression in a variety of human cancers. We have found significantly increased expression GGAP2 in the majority of human prostate cancers and GGAP2 expression increases Akt activation in prostate cancer cells. Thus increased GGAP2 expression is a common mechanism for enhancing the activity of the Akt pathway in prostate cancers. In addition, we have found that activated Akt can bind and phosphorylate GGAP2 at serine 629, which enhances GTP binding by GGAP2. Phosphorylated GGAP2 can bind the p50 subunit of NF-κB and enhances NF-κB transcriptional activity. When expressed in prostate cancer cells, GGAP2 enhances proliferation, foci formation and tumor progression in vivo. Thus increased GGAP2 expression, which is present in three quarters of human prostate cancers, can activate two critical pathways that have been linked to prostate cancer initiation and progression. PMID:19176382

  11. Pathological Signaling via Platelet-Derived Growth Factor Receptor α Involves Chronic Activation of Akt and Suppression of p53 ▿

    PubMed Central

    Lei, Hetian; Velez, Gisela; Kazlauskas, Andrius

    2011-01-01

    In contrast to direct activation of platelet-derived growth factor (PDGF) receptor α (PDGFRα) via PDGF, indirect activation via growth factors outside the PDGF family failed to induce dimerization, internalization, and degradation of PDGFRα. Chronically activated, monomeric PDGFRα induced prolonged activation of Akt and suppressed the level of p53. These events were sufficient to promote both cellular responses (proliferation, survival, and contraction) that are intrinsic to proliferative vitreoretinopathy (PVR) and induce the disease itself. This signature signaling pathway appeared to extend beyond PVR since deregulating PDGFRα in ways that promote solid tumors also resulted in chronic activation of Akt and a decline in the level of p53. PMID:21357737

  12. Lithium-induced activation of Akt and CaM kinase II contributes to its neuroprotective action in a rat microsphere embolism model.

    PubMed

    Sasaki, Takuya; Han, Feng; Shioda, Norifumi; Moriguchi, Shigeki; Kasahara, Jiro; Ishiguro, Koichi; Fukunaga, Kohji

    2006-09-01

    Lithium used in bipolar mood disorder therapy protects neurons from brain ischemic cell death. Here, we documented that lithium administration under microsphere-embolism (ME)-induced brain ischemia restored decreased protein kinase B (Akt) and Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) activities 24 h after ischemia in rat brain. Akt activation was associated with increased phosphorylation of its potential targets forkhead transcription factor (FKHR) and glycogen synthase kinase-3beta (GSK-3beta). In parallel with decreased CaMKII autophosphorylation, we also found marked dephosphorylation of tau proteins 24-72 h after ME. Increased protein phosphatase 2A (PP2A) activity was found 24 h after ME. Inhibition of increased PP2A activity by lithium treatment apparently mediated restored tau phosphorylation. Taken together, activation of Akt and CaMKII by lithium was associated with neuroprotective activity in ME-induced neuronal injury.

  13. Activation of AMPK inhibits cervical cancer cell growth through AKT/FOXO3a/FOXM1 signaling cascade

    PubMed Central

    2013-01-01

    Background Although advanced-stage cervical cancer can benefit from current treatments, approximately 30% patients may fail after definitive treatment eventually. Therefore, exploring alternative molecular therapeutic approaches is imperatively needed for this disease. We have recently shown that activation of AMP-activated protein kinase (AMPK), a metabolic sensor, hampers cervical cancer cell growth through blocking the Wnt/β-catenin signaling activity. Here, we report that activated AMPK (p-AMPK) also inhibits cervical cancer cell growth by counteracting FOXM1 function. Methods Effect of the activation of AMPK on FOXM1 expression was examined by hypoxia and glucose deprivation, as well as pharmacological AMPK activators such as A23187, AICAR and metformin. RT Q-PCR and Western blot analysis were employed to investigate the activities of AMPK, FOXM1 and AKT/FOXO3a signaling. Results Consistent with our previous findings, the activation of AMPK by either AMPK activators such as AICAR, A23187, metformin, glucose deprivation or hypoxia significantly inhibited the cervical cancer cell growth. Importantly, we found that activated AMPK activity was concomitantly associated with the reduction of both the mRNA and protein levels of FOXM1. Mechanistically, we showed that activated AMPK was able to reduce AKT mediated phosphorylation of p-FOXO3a (Ser253). Interestingly, activated AMPK could not cause any significant changes in FOXM1 in cervical cancer cells in which endogenous FOXO3a levels were knocked down using siRNAs, suggesting that FOXO3a is involved in the suppression of FOXM1. Conclusion Taken together, our results suggest the activated AMPK impedes cervical cancer cell growth through reducing the expression of FOXM1. PMID:23819460

  14. Neuroprotective effects of preconditioning ischaemia on ischaemic brain injury through inhibition of mixed-lineage kinase 3 via NMDA receptor-mediated Akt1 activation.

    PubMed

    Yin, Xiao-Hui; Zhang, Quan-Guang; Miao, Bei; Zhang, Guang-Yi

    2005-05-01

    A number of works show that the mitogen-activated protein kinase (MAPK) signalling pathway responds actively in cerebral ischaemia and reperfusion. We undertook our present studies to clarify the role of mixed-lineage kinase 3 (MLK3), a MAPK kinase kinase (MAPKKK) in MAPK cascades, in global ischaemia and ischaemic tolerance. The mechanism concerning NMDA receptor-mediated Akt1 activation underlying ischaemic tolerance, was also investigated. Sprague-Dawley rats were subjected to 6 min of ischaemia and differing times of reperfusion. Our results showed MLK3 was activated in the hippocampal CA1 region with two peaks occurring at 30 min and 6 h, respectively. This activation returned to base level 3 days later. Both preconditioning with 3 min of sublethal ischaemia and NMDA pretreatment inhibited the 6-h peak of activation. However, pretreatment of ketamine before preconditioning reversed the inhibiting effect of preconditioning on MLK3 activation at 6 h of reperfusion. In the case of Akt1, however, preconditioning and NMDA pretreatment enhanced Akt1 activation at 10 min of reperfusion. Furthermore, ketamine pretreatment reversed preconditioning-induced increase of Akt1 activation. We also noted that pretreatment of LY294002 before preconditioning reversed both the inhibition of MLK3 activation at 6 h of reperfusion and the increase in Akt1 activation at 10 min of reperfusion. The above-mentioned results lead us to conclude that, in the hippocampal CA1 region, preconditioning inhibits MLK3 activation after lethal ischaemia and reperfusion and, furthermore, this effect is mediated by Akt1 activation through NMDA receptor stimulation.

  15. 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

  16. YB-1 overexpression promotes a TGF-β1-induced epithelial–mesenchymal transition via Akt activation

    SciTech Connect

    Ha, Bin; Lee, Eun Byul; Cui, Jun; Kim, Yosup; Jang, Ho Hee

    2015-03-06

    The Y-box binding protein-1 (YB-1) is a transcription/translation regulatory protein, and the expression thereof is associated with cancer aggressiveness. In the present study, we explored the regulatory effects of YB-1 during the transforming growth factor-β1 (TGF-β1)-induced epithelial-to-mesenchymal transition (EMT) in lung adenocarcinoma cells. Downregulation of YB-1 increased E-cadherin promoter activity, and upregulation of YB-1 decreased promoter activity, suggesting that the YB-1 level may be correlated with the EMT. TGF-β1 induced YB-1 expression, and TGF-β1 translocated cytosolic YB-1 into the nucleus. YB-1 overexpression promoted TGF-β1-induced downregulation of epithelial markers, upregulation of mesenchymal markers, and cell migration. Moreover, YB-1 overexpression enhanced the expression of E-cadherin transcriptional repressors via TGF-β1-induced Akt activation. Our findings afford new insights into the role played by YB-1 in the TGF-β1 signaling pathway. - Highlights: • YB-1 regulates E-cadherin expression in A549 cells. • TGF-β1 induces upregulating and nuclear localization of YB-1. • YB-1 overexpression accelerates TGF-β1-induced EMT and cell migration. • YB-1 regulates Snail and Slug expression via Akt activation.

  17. Kazinol B from Broussonetia kazinoki improves insulin sensitivity via Akt and AMPK activation in 3T3-L1 adipocytes.

    PubMed

    Lee, Hyejin; Li, Hua; Jeong, Ji Hye; Noh, Minsoo; Ryu, Jae-Ha

    2016-07-01

    In this study, we evaluated the insulin-sensitizing effect of flavans purified from Broussonetia kazinoki Siebold (BK) on 3T3-L1 adipocytes. Among the tested compounds, kazinol B enhanced intracellular lipid accumulation, gene expression of proliferator-activated receptorγ (PPARγ) and CCAAT/enhancer binding protein-alpha (C/EBPα), and consistently induced PPARγ transcriptional activation. To further investigate the insulin-sensitizing effect of kazinol B, we measured glucose analogue uptake by fully differentiated adipocytes and myotubes. Kazinol B increased 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxy-d-glucose (2-NBDG) uptake by cells by upregulating the gene expression and translocation of glucose transporter 4 (GLUT-4) into the plasma membrane in adipocytes. Kazinol B stimulated the gene expression and secretion of adiponectin, which is associated with a low risk of types 1 and 2 diabetes mellitus. We also suggested the mechanism of the antidiabetic effect of kazinol B by assaying Akt and AMP-activated protein kinase (AMPK) phosphorylation. In conclusion, kazinol B isolated from BK improved insulin sensitivity by enhancing glucose uptake via the insulin-Akt signaling pathway and AMPK activation. These results suggest that kazinol B might be a therapeutic candidate for diabetes mellitus. PMID:27223849

  18. Isorhamnetin Attenuates Atherosclerosis by Inhibiting Macrophage Apoptosis via PI3K/AKT Activation and HO-1 Induction

    PubMed Central

    Luo, Yun; Sun, Guibo; Dong, Xi; Wang, Min; Qin, Meng; Yu, Yingli; Sun, Xiaobo

    2015-01-01

    Background and Purpose Isorhamnetin (Iso) is a flavonoid compound extracted from the Chinese herb Hippophae rhamnoides L. Previous studies have revealed its anti-cancer, anti-inflammatory, and anti-oxidant activities. This study investigated the ability of Iso to inhibit oxidized low-density lipoprotein (ox-LDL)-induced cell apoptosis in THP-1-derived macrophages. The effects of Iso on atherosclerosis in vivo were also evaluated in apolipoprotein E knockout (ApoE-/-) mice fed a high fat diet. Methods and Results Iso showed significant inhibitory effects on ox-LDL-induced THP-1-derived macrophage injuries via decreasing reactive oxygen species levels, lipid deposition, and caspase-3 activation, restoring mitochondrial membrane potential, reducing the number of terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL)-positive cells, and regulating apoptosis-related proteins. We also determined the protective effects of Iso by PI3K/AKT activation and HO-1 induction. Iso reduced the atherosclerotic plaque size in vivo in ApoE-/- mice as assessed by oil red O, Sudan IV staining, and CD68-positive cells, and reduced macrophage apoptosis as assessed by caspase-3 and TUNEL assays in lesions. Conclusion In conclusion, our results show that Iso inhibited atherosclerotic plaque development in ApoE-/- mice by PI3K/AKT activation and HO-1 induction. PMID:25799286

  19. Endothelium-independent hypoxic contraction of porcine coronary arteries may be mediated by activation of phosphoinositide 3-kinase/Akt pathway.

    PubMed

    Liu, Huixia; Chen, Zhengju; Liu, Juan; Liu, Limei; Gao, Yuansheng; Dou, Dou

    2014-01-01

    Phosphoinositide 3-kinase (PI3K)/Akt signaling pathway plays an essential role in the regulation of vascular tone. The present study aimed to determine its role in hypoxic coronary vasoconstriction. Isometric tension of isolated porcine coronary arteries was measured with organ chamber technique; the protein levels of phosphorylated and total MLC were examined by Western blotting; the activities of PI3K and Rho kinase were determined by the phosphorylation of their respective target protein Akt and MTPT1. Acute hypoxia induced a rapid contraction followed by a short-term relaxation and then a sustained contraction in porcine coronary arteries. The rapid but not the sustained contraction was abolished by endothelium removal. The sustained contraction was attenuated by inhibitors of PI3K (LY294002) and Akt (Akt-I). The attenuation effect caused by LY294002 was not affected by nifedipine, but was abolished by Y27632, an inhibitor of Rho kinase. The sustained hypoxic contraction was associated with altered phosphorylation of MLC and Akt, which was inhibited by LY294002. The sustained hypoxic contraction was also accompanied with increased phosphorylation of MYPT1, which was inhibited by LY294002 and Y27632. This study demonstrates that sustained hypoxia causes porcine coronary artery to contract in an endothelium-independent manner. An increased PI3K/Akt/Rho kinase signaling may be involved. PMID:24685819

  20. Fear-induced suppression of nociceptive behaviour and activation of Akt signalling in the rat periaqueductal grey: role of fatty acid amide hydrolase.

    PubMed

    Butler, Ryan K; Ford, Gemma K; Hogan, Michelle; Roche, Michelle; Doyle, Karen M; Kelly, John P; Kendall, David A; Chapman, Victoria; Finn, David P

    2012-01-01

    The endocannabinoid system regulates nociception and aversion and mediates fear-conditioned analgesia (FCA). We investigated the effects of the fatty acid amide hydrolase (FAAH) inhibitor URB597, which inhibits the catabolism of the endocannabinoid anandamide and related N-acylethanolamines, on expression of FCA and fear and pain related behaviour per se in rats. We also examined associated alterations in the expression of the signal transduction molecule phospho-Akt in the periaqueductal grey (PAG) by immunoblotting. FCA was modelled by assessing formalin-evoked nociceptive behaviour in an arena previously paired with footshock. URB597 (0.3 mg/kg, i.p.) enhanced FCA and increased fear-related behaviour in formalin-treated rats. Conditioned fear per se in non-formalin-treated rats was associated with increased expression of phospho-Akt in the PAG. URB597 reduced the expression of fear-related behaviour in the early part of the trial, an effect that was accompanied by attenuation of the fear-induced increase in phospho-Akt expression in the PAG. Intra-plantar injection of formalin also reduced the fear-induced increase in phospho-Akt expression. These data provide evidence for a role of FAAH in FCA, fear responding in the presence or absence of nociceptive tone, and fear-evoked increases in PAG phospho-Akt expression. In addition, the results suggest that fear-evoked activation of Akt signalling in the PAG is abolished in the presence of nociceptive tone.

  1. Increased riboflavin production from activated bleaching earth by a mutant strain of Ashbya gossypii.

    PubMed

    Tajima, Satoshi; Itoh, Yoko; Sugimoto, Takashi; Kato, Tatsuya; Park, Enoch Y

    2009-10-01

    The production of riboflavin from vegetable oil was increased using a mutant strain of Ashbya gossypii. This mutant was generated by treating the wild-type strain with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). Riboflavin production was 10-fold higher in the mutant compared to the wild-type strain. The specific intracellular catalase activity after 3 d of culture was 6-fold higher in the mutant than in the wild-type strain. For the mutant, riboflavin production in the presence of 40 mM hydrogen peroxide was 16% less than that in the absence of hydrogen peroxide, whereas it was 56% less for the wild-type strain. The isocitrate lyase (ICL) activity of the mutant was 0.26 mU/mg of protein during the active riboflavin production phase, which was 2.6-fold higher than the wild-type strain. These data indicate that the mutant utilizes the carbon flux from the TCA cycle to the glyoxylate cycle more efficiently than the wild-type strain, resulting in enhanced riboflavin production. This novel mutant has the potential to be of use for industrial-scale riboflavin production from waste-activated bleaching earth (ABE), thereby transforming a useless material into a valuable bioproduct. PMID:19716523

  2. Seabuckthorn Pulp Oil Protects against Myocardial Ischemia-Reperfusion Injury in Rats through Activation of Akt/eNOS.

    PubMed

    Suchal, Kapil; Bhatia, Jagriti; Malik, Salma; Malhotra, Rajiv Kumar; Gamad, Nanda; Goyal, Sameer; Nag, Tapas C; Arya, Dharamvir S; Ojha, Shreesh

    2016-01-01

    Seabuckthorn (SBT) pulp oil obtained from the fruits of seabuckthorn [Hippophae rhamnoides L. (Elaeagnaceae)] has been used traditionally for its medicinal and nutritional properties. However, its role in ischemia-reperfusion (IR) injury of myocardium in rats has not been elucidated so far. The present study reports the cardioprotective effect of SBT pulp oil in IR-induced model of myocardial infarction in rats and underlying mechanism mediating activation of Akt/eNOS signaling pathway. Male albino Wistar rats were orally administered SBT pulp oil (5, 10, and 20 ml/kg/day) or saline for 30 days. On the day 31, ischemia was induced by one-stage ligation of left anterior descending coronary artery for 45 min followed by reperfusion for 60 min. SBT pulp oil pretreatment at the dose of 20 ml/kg observed to stabilize cardiac function and myocardial antioxidants such as glutathione, superoxide dismutase, catalase, and inhibited lipid peroxidation evidenced by reduced malondialdehyde levels as compared to IR-control group. SBT pulp oil also improved hemodynamic and contractile function and decreased tumor necrosis factor and activities of myocyte injury marker enzymes; lactate dehydrogenase and creatine kinase-MB. Additionally, a remarkable rise in expression of pAkt-eNOS, Bcl-2 and decline in expression of IKKβ/NF-κB and Bax was observed in the myocardium. The histopathological and ultrastructural salvage of cardiomyocytes further supports the cardioprotective effect of SBT pulp oil. Based on findings, it can be concluded that SBT pulp oil protects against myocardial IR injury mediating favorable modulation of Akt-eNOS and IKKβ/NF-κB expression. PMID:27445803

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

    SciTech Connect

    Xu, Yong; Fang, Shi-ji; Zhu, Li-juan; Zhu, Lun-qing; Zhou, Xiao-zhong

    2014-10-24

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

  4. A novel 7-bromoindirubin with potent anticancer activity suppresses survival of human melanoma cells associated with inhibition of STAT3 and Akt signaling.

    PubMed

    Liu, Lucy; Kritsanida, Marina; Magiatis, Prokopios; Gaboriaud, Nicolas; Wang, Yan; Wu, Jun; Buettner, Ralf; Yang, Fan; Nam, Sangkil; Skaltsounis, Leandros; Jove, Richard

    2012-11-01

    STAT3 and Akt signaling have been validated as potential molecular targets for treatment of cancers including melanoma. These small molecule inhibitors of STAT3 or Akt signaling are promising for developing anti-melanoma therapeutic agents. MLS-2438, a novel 7-bromoindirubin, a derivative of the natural product indirubin, was synthesized with a bromo-group at the 7-position on one indole ring and a hydrophilic group at the 3'-position on the other indole ring. We tested the anticancer activity of MLS-2438 and investigated its mechanism of action in human melanoma cell lines. Here, we show that MLS-2438 inhibits viability and induces apoptosis of human melanoma cells associated with inhibition of STAT3 and Akt signaling. Several pro-apoptotic Bcl-2 family proteins are involved in the MLS-2438 mediated apoptosis. MLS-2438 inhibits Src kinase activity in vitro and phosphorylation of JAK2, Src, STAT3 and Akt in cultured cancer cells. In contrast to the decreased phosphorylation levels of JAK2, Src, STAT3 and Akt, phosphorylation levels of the MAPK (Erk1/2) signaling protein were not reduced in cells treated with MLS-2438. These results demonstrate that MLS-2438, a novel natural product derivative, is a Src inhibitor and potentially regulates kinase activity of JAK2 and Akt in cancer cells. Importantly, MLS-2438 suppressed tumor growth with low toxicity in a mouse xenograft model of human melanoma. Our findings support further development of MLS-2438 as a potential small-molecule therapeutic agent that targets both STAT3 and Akt signaling in human melanoma cells.

  5. Pseudolaric acid B exerts antitumor activity via suppression of the Akt signaling pathway in HeLa cervical cancer cells.

    PubMed

    Li, Mingqun; Hong, Li

    2015-08-01

    Pseudolaric acid B (PAB) is a diterpene acid isolated from the bark of the root and trunk of Pseudolarix kaempferi Gordon (Pinaceae), which has demonstrated cytotoxic effects against various types of cancer. However, the mechanisms underlying the anticancer effects of PAB have remained to be elucidated. In the present study, the effects of PAB on the viability and apoptosis of HeLa cells were investigated by MTT assay, flow cytometric analysis of Annexin V-fluorescein isothiocyanate/propidium iodide staining, Rhodamine 123 staining and western blot analysis. The results demonstrated that PAB had antiproliferative and apoptosis-inducing effects on HeLa cells. PAB markedly inhibited HeLa cell viability in a time- and concentration-dependent manner. Flow cytometric analysis indicated that PAB induced apoptosis in HeLa cells in a dose-dependent manner. Treatment with PAB suppressed the expression of anti-apoptotic factor B cell lymphoma-2, and promoted the expression of pro-apoptotic factor Bcl-2-associated X protein. In addition, PAB induced an increase in Caspase-3 activity and loss of mitochondrial membrane potential, suggesting that this apoptosis may be mediated by mitochondrial pathways. Furthermore, the results of western blot analysis indicated that PAB was able to reduce Akt phosphorylation, thereby inhibiting the Akt pathway. These results suggested that PAB inhibited cell proliferation and induced apoptosis in HeLa cells, and that the anti-tumor effects of PAB were associated with inhibition of the Akt pathway. In conclusion, the results of the present study suggested that PAB may represent a novel therapeutic strategy for the treatment of human cervical cancer. However, additional studies are required to investigate the underlying apoptotic mechanisms.

  6. Apelin-36, a potent peptide, protects against ischemic brain injury by activating the PI3K/Akt pathway.

    PubMed

    Gu, Qin; Zhai, Lijing; Feng, Xing; Chen, Jing; Miao, Zhigang; Ren, Liyan; Qian, Xuanchen; Yu, Jian; Li, Yan; Xu, Xingshun; Liu, Chun-Feng

    2013-11-01

    Apelin is an endogenous ligand of G protein-coupled receptor-apelin and angiotensin-1-like receptor (APJ). The biological effects of apelin-APJ system are reported in multiple systems including cardiovascular, endocrinal, and gastrointestinal system. Previous studies had shown that apelin-13 is a potential protective agent on cardiac ischemia; however, the role of apelin in the central nervous system remained unknown. In this study, we investigated therapeutic effects of apelin-36, a long form of apelin, in ischemic brain injury models. We found that apelin-36 reduced cerebral infarct volume in the middle cerebral artery occlusion (MCAO) model and the neonatal hypoxic/ischemic (H/I) injury model. Apelin-36 improved neurological deficits in the MCAO model and promoted long-term functional recovery after H/I brain injury. We further explored the protective mechanisms of apelin-36 on H/I brain injury. We clearly demonstrated that apelin-36 significantly reduced the levels of cleaved caspase-3 and Bax, two well-established apoptotic markers after H/I injury, indicating the anti-apoptotic activity of apelin-36 in ischemic injury. Since apelin-36 increased the level of phosphorylated Akt after H/I injury, we treated neonates with a specific PI3K inhibitor LY294002. We found that LY294002 decreased the phosphorylated Akt level and attenuated protective effects of apelin-36 on apoptosis. These suggested that the PI3K/Akt pathway was at least in part involved in the anti-apoptotic mechanisms of apelin-36. Our findings demonstrated that apelin-36 was a promising therapeutic agent on the treatment of ischemic brain injury.

  7. The mRNA-edited form of GABRA3 suppresses GABRA3-mediated Akt activation and breast cancer metastasis

    PubMed Central

    Gumireddy, Kiranmai; Li, Anping; Kossenkov, Andrew V.; Sakurai, Masayuki; Yan, Jinchun; Li, Yan; Xu, Hua; Wang, Jian; Zhang, Paul J.; Zhang, Lin; Showe, Louise C.; Nishikura, Kazuko; Huang, Qihong

    2016-01-01

    Metastasis is a critical event affecting breast cancer patient survival. To identify molecules contributing to the metastatic process, we analysed The Cancer Genome Atlas (TCGA) breast cancer data and identified 41 genes whose expression is inversely correlated with survival. Here we show that GABAA receptor alpha3 (Gabra3), normally exclusively expressed in adult brain, is also expressed in breast cancer, with high expression of Gabra3 being inversely correlated with breast cancer survival. We demonstrate that Gabra3 activates the AKT pathway to promote breast cancer cell migration, invasion and metastasis. Importantly, we find an A-to-I RNA-edited form of Gabra3 only in non-invasive breast cancers and show that edited Gabra3 suppresses breast cancer cell invasion and metastasis. A-to-I-edited Gabra3 has reduced cell surface expression and suppresses the activation of AKT required for cell migration and invasion. Our study demonstrates a significant role for mRNA-edited Gabra3 in breast cancer metastasis. PMID:26869349

  8. Foxc2 enhances proliferation and inhibits apoptosis through activating Akt/mTORC1 signaling pathway in mouse preadipocytes

    PubMed Central

    Gan, Lu; Liu, Zhenjiang; Jin, Wei; Zhou, Zhongjie; Sun, Chao

    2015-01-01

    Forkhead box C2 (Foxc2) protein is a transcription factor in regulation of development, metabolism, and immunology. However, the regulatory mechanisms of Foxc2 on proliferation and apoptosis of preadipocytes are unclear. In this study, we found that high-fat-diet-induced obesity elevated the expression of Foxc2 and cyclin E after 6 weeks. Additionally, Foxc2 suppressed preadipocyte differentiation, increased cell counts and augmented G1-S transition of preadipocytes, along with the elevation of cyclin E expression and the reduction levels of p27 and p53. Furthermore, Foxc2 knockdown reduced early apoptotic cells with accompanying reduction of mitochondrial membrane potential and increased fragmentation of genomic DNA. We show that Foxc2 reduces the expression of Bax, caspase-9, and caspase-3 in both serum-starved and palmitic acid-induced cell apoptotic models, which confirms the anti-apoptotic role of Foxc2. Moreover, the protein kinase B (Akt)/mammalian target of rapamycin (mTOR)C1 signaling pathway and the ERK/mTORC1 signaling pathway were activated along with preadipocyte proliferation in response to Foxc2 overexpression, whereas apoptosis marker genes were downregulated during this process. Those effects were blocked by the interference of Foxc2 or signal pathways specific inhibitors. These data collectively reveal that Foxc2 enhances proliferation of preadipocytes and inhibits apoptosis of preadipocytes by activating the Akt/mTORC1 and ERK/mTORC1 signaling pathways. PMID:26113535

  9. Diacylglycerol kinase θ couples farnesoid X receptor-dependent bile acid signalling to Akt activation and glucose homoeostasis in hepatocytes.

    PubMed

    Cai, Kai; Sewer, Marion B

    2013-09-01

    DGKs (diacylglycerol kinases) catalyse the conversion of diacylglycerol into PA (phosphatidic acid), a positive modulator of mTOR (mammalian target of rapamycin). We have found that chenodeoxycholic acid and the synthetic FXR (farnesoid X receptor) ligand GW4064 induce the mRNA and protein expression of DGKθ in the HepG2 cell line and in primary human hepatocytes. Reporter gene studies using 1.5 kB of the DGKθ promoter fused to the luciferase gene revealed that bile acids increase DGKθ transcriptional activity. Mutation of putative FXR-binding sites attenuated the ability of GW4046 to increase DGKθ luciferase activity. Consistent with this finding, ChIP (chromatin immunoprecipitation) assays demonstrated that bile acid signalling increased the recruitment of FXR to the DGKθ promoter. Furthermore, GW4064 evoked a time-dependent increase in the cellular concentration of PA. We also found that GW4064 and PA promote the phosphorylation of mTOR, Akt and FoxO1 (forkhead box O1), and that silencing DGKθ expression significantly abrogated the ability of GW4046 to promote the phosphorylation of these PA-regulated targets. DGKθ was also required for bile-acid-dependent decreased glucose production. Taken together, our results establish DGKθ as a key mediator of bile-acid-stimulated modulation of mTORC2 (mTOR complex 2), the Akt pathway and glucose homoeostasis.

  10. A novel adriamycin analogue derived from marine microbes induces apoptosis by blocking Akt activation in human breast cancer cells.

    PubMed

    Yuan, Jie; He, Zhenjian; Wu, Jueheng; Lin, Yongcheng; Zhu, Xun

    2011-01-01

    1403P-3 is a novel anthracenedione derivative isolated from the secondary metabolites of endophytic fungus from the South China Sea. In previous studies, 1403P-3 was found to exhibit potent cytotoxicity against human cancer cells, but its molecular target and the mechanisms mediating its cytotoxicity remain unknown. In this study, we showed that 1403P-3 markedly inhibited the survival of the human breast cancer cells MCF-7 and MDA-MB-435 in a dose-dependent manner, with an IC₅₀ of 9.5 and 7.6 µM, respectively. Apoptosis induced by 1403P-3 was detected, as indicated by Annexin V-FITC/PI staining, elevated activated caspase-8 and -9, and cleavaged PARP determined by Western blot analysis. It is of note that the phosphorylation level of Akt was significantly reduced in 1403P-3-treated cells in a dose- and time-dependent manner. Taken together, our data demonstrated that 1403P-3 induced breast cancer cell apoptosis by blocking Akt activation, suggesting that 1403P-3 may be a promising candidate compound for anti-tumor drug development.

  11. 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

  12. Regulation of Matrix Metalloproteinase-2 Activity by COX-2-PGE2-pAKT Axis Promotes Angiogenesis in Endometriosis

    PubMed Central

    Ray, Amlan K.; DasMahapatra, Pramathes; Swarnakar, Snehasikta

    2016-01-01

    Endometriosis is characterized by the ectopic development of the endometrium which relies on angiogenesis. Although studies have identified the involvement of different matrix metalloproteinases (MMPs) in endometriosis, no study has yet investigated the role of MMP-2 in endometriosis-associated angiogenesis. The present study aims to understand the regulation of MMP-2 activity in endothelial cells and on angiogenesis during progression of ovarian endometriosis. Histological and biochemical data showed increased expressions of vascular endothelial growth factor (VEGF), VEGF receptor-2, cycloxygenase (COX)-2, von Willebrand factor along with angiogenesis during endometriosis progression. Women with endometriosis showed decreased MMP-2 activity in eutopic endometrium as compared to women without endometriosis. However, ectopic ovarian endometrioma showed significantly elevated MMP-2 activity with disease severity. In addition, increased MT1MMP and decreased tissue inhibitors of metalloproteinases (TIMP)-2 expressions were found in the late stages of endometriosis indicating more MMP-2 activation with disease progression. In vitro study using human endothelial cells showed that prostaglandin E2 (PGE2) significantly increased MMP-2 activity as well as tube formation. Inhibition of COX-2 and/or phosphorylated AKT suppressed MMP-2 activity and endothelial tube formation suggesting involvement of PGE2 in regulation of MMP-2 activity during angiogenesis. Moreover, specific inhibition of MMP-2 by chemical inhibitor significantly reduced cellular migration, invasion and tube formation. In ovo assay showed decreased angiogenic branching upon MMP-2 inhibition. Furthermore, a significant reduction of lesion numbers was observed upon inhibition of MMP-2 and COX-2 in mouse model of endometriosis. In conclusion, our study establishes the involvement of MMP-2 activity via COX-2-PGE2-pAKT axis in promoting angiogenesis during endometriosis progression. PMID:27695098

  13. Phosphorylation of GSK3α/β correlates with activation of AKT and is prognostic for poor overall survival in acute myeloid leukemia patients

    PubMed Central

    Ruvolo, Peter P.; Qiu, YiHua; Coombes, Kevin R.; Zhang, Nianxiang; Neeley, E. Shannon; Ruvolo, Vivian R.; Hail, Numsen; Borthakur, Gautam; Konopleva, Marina; Andreeff, Michael; Kornblau, Steven M.

    2015-01-01

    Background Acute myeloid leukemia (AML) patients with highly active AKT tend to do poorly. Cell cycle arrest and apoptosis are tightly regulated by AKT via phosphorylation of GSK3α and β isoforms which inactivates these kinases. In the current study we examine the prognostic role of AKT mediated GSK3 phosphorylation in AML. Methods We analyzed GSK3α/β phosphorylation by reverse phase protein analysis (RPPA) in a cohort of 511 acute myeloid leukemia (AML) patients. Levels of phosphorylated GSK3 were correlated with patient characteristics including survival and with expression of other proteins important in AML cell survival. Results High levels of p-GSK3α/β correlated with adverse overall survival and a lower incidence of complete remission duration in patients with intermediate cytogenetics, but not in those with unfavorable cytogenetics. Intermediate cytogenetic patients with FLT3 mutation also fared better respectively when p-GSK3α/β levels were lower. Phosphorylated GSK3α/β expression was compared and contrasted with that of 229 related cell cycle arrest and/or apoptosis proteins. Consistent with p-GSK3α/β as an indicator of AKT activation, RPPA revealed that p-GSK3α/β positively correlated with phosphorylation of AKT, BAD, and P70S6K, and negatively correlated with β-catenin and FOXO3A. PKCδ also positively correlated with p-GSK3α/β expression, suggesting crosstalk between the AKT and PKC signaling pathways in AML cells. Conclusions These findings suggest that AKT-mediated phosphorylation of GSK3α/β may be beneficial to AML cell survival, and hence detrimental to the overall survival of AML patients. Intrinsically, p-GSK3α/β may serve as an important adverse prognostic factor for a subset of AML patients. PMID:26674329

  14. Schwann cells expressing dismutase active mutant SOD1 unexpectedly slow disease progression in ALS mice

    PubMed Central

    Lobsiger, Christian S.; Boillee, Severine; McAlonis-Downes, Melissa; Khan, Amir M.; Feltri, M. Laura; Yamanaka, Koji; Cleveland, Don W.

    2009-01-01

    Neurodegeneration in an inherited form of ALS is non-cell-autonomous, with ALS-causing mutant SOD1 damage developed within multiple cell types. Selective inactivation within motor neurons of an ubiquitously expressed mutant SOD1 gene has demonstrated that mutant damage within motor neurons is a determinant of disease initiation, whereas mutant synthesis within neighboring astrocytes or microglia accelerates disease progression. We now report the surprising finding that diminished synthesis (by 70%) within Schwann cells of a fully dismutase active ALS-linked mutant (SOD1G37R) significantly accelerates disease progression, accompanied by reduction of insulin-like growth factor 1 (IGF-1) in nerves. Coupled with shorter disease duration in mouse models caused by dismutase inactive versus dismutase active SOD1 mutants, our findings implicate an oxidative cascade during disease progression that is triggered within axon ensheathing Schwann cells and that can be ameliorated by elevated dismutase activity. Thus, therapeutic down-regulation of dismutase active mutant SOD1 in familial forms of ALS should be targeted away from Schwann cells. PMID:19251638

  15. GM1 ganglioside activates ERK1/2 and Akt downstream of Trk tyrosine kinase and protects PC12 cells against hydrogen peroxide toxicity.

    PubMed

    Zakharova, Irina O; Sokolova, Tatyana V; Vlasova, Yulia A; Furaev, Victor V; Rychkova, Maria P; Avrova, Natalia F

    2014-11-01

    Ganglioside GM1 at micro- and nanomolar concentrations was shown to increase the viability of pheochromocytoma PC12 cells exposed to hydrogen peroxide and diminish the accumulation of reactive oxygen species and oxidative inactivation of Na(+),K(+)-ATPase, the effects of micromolar GM1 being more pronounced than those of nanomolar GM1. These effects of GM1 were abolished by Trk receptor tyrosine kinase inhibitor and diminished by MEK1/2, phosphoinositide 3-kinase and protein kinase C inhibitors. Hydrogen peroxide activates Trk tyrosine kinase; Akt and ERK1/2 are activated downstream of this protein kinase. GM1 was found to activate Trk receptor tyrosine kinase in PC12 cells. GM1 (100 nM and 10 µM) increased the basal activity of Akt, but did not change Akt activity in cells exposed to hydrogen peroxide. Basal ERK1/2 activity in PC12 cells was increased by GM1 at a concentration of 10 µM, but not at nanomolar concentrations. Activation of ERK1/2 by hydrogen peroxide was enhanced by GM1 at a concentration of 10 µM and to a lesser extent at a concentration of 100 nM. Thus, the protective and metabolic effects of GM1 ganglioside on PC12 cells exposed to hydrogen peroxide appear to depend on the activation of Trk receptor tyrosine kinase and downstream activation of Akt and ERK1/2.

  16. Isorhamnetin inhibits cell proliferation and induces apoptosis in breast cancer via Akt and mitogen-activated protein kinase kinase signaling pathways

    PubMed Central

    HU, SHAN; HUANG, LIMING; MENG, LIWEI; SUN, HE; ZHANG, WEI; XU, YINGCHUN

    2015-01-01

    Breast cancer is the most common cause of female cancer-associated mortality. Although treatment options, including chemotherapy, radiotherapy and surgery have led to a decline in the mortality rates associated with breast cancer, drug resistance remains one of the predominant causes for poor prognosis and high recurrence rates. The present study investigated the potential effects of the natural product, isorhamnetin on breast cancer, and examined the effects of isorhamnetin on the Akt/mammalian target of rapamycin (mTOR) and the mitogen-activated protein kinase (MAPK)/MAPK kinase (MEK) signaling cascades, which are two important signaling pathways for endocrine therapy resistance in breast cancer. The results of the present study indicate that isorhamnetin inhibits cell proliferation and induces cell apoptosis. In addition, isorhamnetin was observed to inhibit the Akt/mTOR and the MEK/extracellular signal-regulated kinase phosphorylation cascades. The inhibition of these two signaling pathways was attenuated by the two Akt and MEK1 inhibitors, but not by the nuclear factor-κB inhibitor. Furthermore, epidermal growth factor inhibited the effects of isorhamnetin via activation of the Akt and MEK signaling pathways. These results indicate that isorhamnetin exhibits antitumor effects in breast cancer, which are mediated by the Akt and MEK signaling pathways. PMID:26502751

  17. Isorhamnetin inhibits cell proliferation and induces apoptosis in breast cancer via Akt and mitogen‑activated protein kinase kinase signaling pathways.

    PubMed

    Hu, Shan; Huang, Liming; Meng, Liwei; Sun, He; Zhang, Wei; Xu, Yingchun

    2015-11-01

    Breast cancer is the most common cause of female cancer-associated mortality. Although treatment options, including chemotherapy, radiotherapy and surgery have led to a decline in the mortality rates associated with breast cancer, drug resistance remains one of the predominant causes for poor prognosis and high recurrence rates. The present study investigated the potential effects of the natural product, isorhamnetin on breast cancer, and examined the effects of isorhamnetin on the Akt/mammalian target of rapamycin (mTOR) and the mitogen-activated protein kinase (MAPK)/MAPK kinase (MEK) signaling cascades, which are two important signaling pathways for endocrine therapy resistance in breast cancer. The results of the present study indicate that isorhamnetin inhibits cell proliferation and induces cell apoptosis. In addition, isorhamnetin was observed to inhibit the Akt/mTOR and the MEK/extracellular signal-regulated kinase phosphorylation cascades. The inhibition of these two signaling pathways was attenuated by the two Akt and MEK1 inhibitors, but not by the nuclear factor-κB inhibitor. Furthermore, epidermal growth factor inhibited the effects of isorhamnetin via activation of the Akt and MEK signaling pathways. These results indicate that isorhamnetin exhibits antitumor effects in breast cancer, which are mediated by the Akt and MEK signaling pathways. PMID:26502751

  18. MiRNA‑542‑3p downregulation promotes trastuzumab resistance in breast cancer cells via AKT activation.

    PubMed

    Ma, Tao; Yang, Lu; Zhang, Jin

    2015-03-01

    Trastuzumab (Herceptin) has been widely used in breast cancer treatment. However, the majority of cancers that initially respond to trastuzumab begin to progress again within 1 year. Despite the high resistance rate, the molecular mechanisms underlying this desease are not well understood. In the present study, microRNA (miRNA‑542‑3p modulated trastuzumab resistance in SKBR3 and MCF7/Her2 breast cancer cell lines. Trastuzumab induced miRNA‑542‑3p expression in SKBR3 and MCF7/Her2 cells. Furthermore, knockdown of miRNA‑542‑3p in the two cell lines resulted in decreased drug sensitivity to trastuzumab and cell apoptosis. The blockage of G1/S checkpoint by trastuzumab was rescued as well. miRNA‑542‑3p knockdown also activated the phosphatidylinositol 3‑kinase (PI3K)‑Akt pathway, while LY294002 reversed the effect of miRNA‑542‑3p knockdown. In summary, the results suggested that miRNA‑542‑3p downregulation may contribute to the trastuzumab resistance in breast cancer via, at least in part, the PI3K‑akt pathway. Our findings provide new molecular mechanisms in trastuzumab resistance. PMID:25586125

  19. IL-8 induces the epithelial-mesenchymal transition of renal cell carcinoma cells through the activation of AKT signaling

    PubMed Central

    Zhou, Nan; Lu, Fuding; Liu, Cheng; Xu, Kewei; Huang, Jian; Yu, Dexin; Bi, Liangkuan

    2016-01-01

    The epithelial-mesenchymal transition (EMT) process has increasingly been examined due to its role in the progression of human tumors. Renal cell carcinoma (RCC) is one of the most common urological tumors that results in patient mortality. Previous studies have demonstrated that the EMT process is closely associated with the metastasis of RCC; however, the underlying molecular mechanism has not been determined yet. The present study revealed that interleukin (IL)-8 was highly expressed in metastatic RCC. IL-8 could induce the EMT of an RCC cell line by enhancing N-cadherin expression and decreasing E-cadherin expression. Furthermore, IL-8 could induce AKT phosphorylation, and the phosphatidylinositol-4,5-bisphosphate 3-kinase inhibitor LY294002 could inhibit the EMT of RCC cells that was induced by IL-8. Therefore, these results suggest that IL-8 is able to promote the EMT of RCC through the activation of the AKT signal transduction pathway, and this may provide a possible molecular mechanism for RCC metastasis. PMID:27588140

  20. Sulforaphane induces apoptosis in adipocytes via Akt/p70s6k1/Bad inhibition and ERK activation.

    PubMed

    Yao, Anjun; Shen, Yingzhuo; Wang, Anshi; Chen, Shiyong; Zhang, Huiqin; Chen, Fen; Chen, Zhongming; Wei, Hua; Zou, Zuquan; Shan, Yujuan; Zhang, Xiaohong

    2015-10-01

    Sulforaphane (SFN), an isothiocyanate isolated from cruciferous vegetables, possesses anti-oxidant and anti-cancer bioactivities. Moreover, SFN exerts its pro-apoptotic effects in some cancer lines. However, the effects and mechanisms of SFN on the regulation of apoptosis of adipocytes are still unknown. In this study, we found that SFN induced significant apoptosis in 3T3-L1 adipocytes and markedly decreased the cellular lipid content. Western blot demonstrated that SFN-induced apoptosis was mediated via the mitochondrial apoptosis pathway based on increased cleavage of poly-ADP-ribose-polymerase (PARP), release of cytochrome c into the cytoplasm, and activation of caspase-3, as well as decreased Bcl-2/Bax ratio. In addition, SFN markedly decreased phosphorylation of Akt and downstream proteins, p70s6k1 and Bad, and increased phosphorylation of ERK. Therefore, our findings clarified that SFN could induce 3T3-L1 adipocyte apoptosis via down-regulation of the Akt/p70s6k1/Bad pathway and up-regulation of the ERK pathway, suggesting SFN may be a promising agent for the treatment or prevention of obesity.

  1. Complement membrane attack complexes activate noncanonical NF-κB by forming an Akt+NIK+ signalosome on Rab5+ endosomes

    PubMed Central

    Jane-wit, Dan; Surovtseva, Yulia V.; Qin, Lingfeng; Li, Guangxin; Liu, Rebecca; Clark, Pamela; Manes, Thomas D.; Wang, Chen; Kashgarian, Michael; Kirkiles-Smith, Nancy C.; Tellides, George; Pober, Jordan S.

    2015-01-01

    Complement membrane attack complexes (MACs) promote inflammatory functions in endothelial cells (ECs) by stabilizing NF-κB–inducing kinase (NIK) and activating noncanonical NF-κB signaling. Here we report a novel endosome-based signaling complex induced by MACs to stabilize NIK. We found that, in contrast to cytokine-mediated activation, NIK stabilization by MACs did not involve cIAP2 or TRAF3. Informed by a genome-wide siRNA screen, instead this response required internalization of MACs in a clathrin-, AP2-, and dynamin-dependent manner into Rab5+endosomes, which recruited activated Akt, stabilized NIK, and led to phosphorylation of IκB kinase (IKK)-α. Active Rab5 was required for recruitment of activated Akt to MAC+ endosomes, but not for MAC internalization or for Akt activation. Consistent with these in vitro observations, MAC internalization occurred in human coronary ECs in vivo and was similarly required for NIK stabilization and EC activation. We conclude that MACs activate noncanonical NF-κB by forming a novel Akt+NIK+ signalosome on Rab5+ endosomes. PMID:26195760

  2. Characterization and RNA-seq analysis of underperformer, an activation-tagged potato mutant.

    PubMed

    Aulakh, Sukhwinder S; Veilleux, Richard E; Dickerman, Allan W; Tang, Guozhu; Flinn, Barry S

    2014-04-01

    The potato cv. Bintje and a Bintje activation-tagged mutant, underperformer (up) were compared. Mutant up plants grown in vitro were dwarf, with abundant axillary shoot growth, greater tuber yield, altered tuber traits and early senescence compared to wild type. Under in vivo conditions, the dwarf and early senescence phenotypes of the mutant remained, but the up plants exhibited a lower tuber yield and fewer axillary shoots compared to wild type. Southern blot analyses indicated a single T-DNA insertion in the mutant, located on chromosome 10. Initial PCR-based gene expression studies indicated transcriptional activation/repression of several genes in the mutant flanking the insertion. The gene immediately flanking the right border of the T-DNA insertion, which encoded an uncharacterized Broad complex, Tramtrac, Bric-a-brac; also known as Pox virus and Zinc finger (BTB/POZ) domain-containing protein (StBTB/POZ1) containing an Armadillo repeat region, was up-regulated in the mutant. Global gene expression comparisons between Bintje and up using RNA-seq on leaves from 60 day-old plants revealed a dataset of over 1,600 differentially expressed genes. Gene expression analyses suggested a variety of biological processes and pathways were modified in the mutant, including carbohydrate and lipid metabolism, cell division and cell cycle activity, biotic and abiotic stress responses, and proteolysis.

  3. Analysis of crystal structure of Arabidopsis MPK6 and generation of its mutants with higher activity

    PubMed Central

    Wang, Bo; Qin, Xinghua; Wu, Juan; Deng, Hongying; Li, Yuan; Yang, Hailian; Chen, Zhongzhou; Liu, Guoqin; Ren, Dongtao

    2016-01-01

    Mitogen-activated protein kinase (MAPK) cascades, which are the highly conserved signalling modules in eukaryotic organisms, have been shown to play important roles in regulating growth, development, and stress responses. The structures of various MAPKs from yeast and animal have been solved, and structure-based mutants were generated for their function analyses, however, the structures of plant MAPKs remain unsolved. Here, we report the crystal structure of Arabidopsis MPK6 at a 3.0 Å resolution. Although MPK6 is topologically similar to ERK2 and p38, the structures of the glycine-rich loop, MAPK insert, substrate binding sites, and L16 loop in MPK6 show notable differences from those of ERK2 and p38. Based on the structural comparison, we constructed MPK6 mutants and analyzed their kinase activity both in vitro and in planta. MPK6F364L and MPK6F368L mutants, in which Phe364 and Phe368 in the L16 loop were changed to Leu, respectively, acquired higher intrinsic kinase activity and retained the normal MAPKK activation property. The expression of MPK6 mutants with basal activity is sufficient to induce camalexin biosynthesis; however, to induce ethylene and leaf senescence, the expression of MPK6 mutants with higher activity is required. The results suggest that these mutants can be used to analyze the specific biological functions of MPK6. PMID:27160427

  4. The Akt switch model: Is location sufficient?

    PubMed

    Gray, Catheryn W; Coster, Adelle C F

    2016-06-01

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

  5. Glutathione biosynthesis is a metabolic vulnerability in PI3K/Akt-driven breast cancer

    PubMed Central

    Lien, Evan C.; Lyssiotis, Costas A.; Juvekar, Ashish; Hu, Hai; Asara, John M.; Cantley, Lewis C.; Toker, Alex

    2016-01-01

    Cancer cells often select for mutations that enhance signaling through pathways that promote anabolic metabolism1. Although the PI3K/Akt signaling pathway, which is frequently dysregulated in breast cancer2, is a well-established regulator of central glucose metabolism and aerobic glycolysis3,4, its regulation of other metabolic processes required for tumor growth is not well defined. Here we report that in mammary epithelial cells, oncogenic PI3K/Akt stimulates glutathione (GSH) biosynthesis by stabilizing and activating Nrf2 to up-regulate the GSH biosynthetic genes. Increased Nrf2 stability is dependent on the Akt-mediated accumulation of p21Cip1/WAF1 and GSK-3 inhibition. Consistently, in human breast tumors, up-regulation of Nrf2 targets is associated with PI3K pathway mutation status and oncogenic Akt activation. Elevated GSH biosynthesis is required for PI3K/Akt-driven resistance to oxidative stress, initiation of tumor spheroids, and anchorage-independent growth. Furthermore, inhibition of GSH biosynthesis with buthionine sulfoximine (BSO) synergizes with cisplatin (CDDP) to selectively induce tumor regression in PI3K pathway mutant breast cancer cells, both in vitro and in vivo. Our findings provide insight into GSH biosynthesis as a metabolic vulnerability associated with PI3K pathway mutant breast cancers. PMID:27088857

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    2015-01-01

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

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

    PubMed

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

    2015-01-01

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

  9. Coactivation of the PI3K/Akt and ERK signaling pathways in PCB153-induced NF-κB activation and caspase inhibition

    SciTech Connect

    Liu, Changjiang; Yang, Jixin; Fu, Wenjuan; Qi, Suqin; Wang, Chenmin; Quan, Chao; Yang, Kedi

    2014-06-15

    Polychlorinated biphenyls (PCBs) are a group of persistent and widely distributed environmental pollutants that have various deleterious effects, e.g., neurotoxicity, endocrine disruption and reproductive abnormalities. In order to verify the hypothesis that the PI3K/Akt and MAPK pathways play important roles in hepatotoxicity induced by PCBs, Sprague–Dawley (SD) rats were dosed with PCB153 intraperitoneally at 0, 4, 16 and 32 mg/kg for five consecutive days; BRL cells (rat liver cell line) were treated with PCB153 (0, 1, 5, and 10 μM) for 24 h. Results indicated that the PI3K/Akt and ERK pathways were activated in vivo and in vitro after exposure to PCB153, and protein levels of phospho-Akt and phospho-ERK were significantly increased. Nuclear factor-κB (NF-κB) activation and caspase-3, -8 and -9 inhibition caused by PCB153 were also observed. Inhibiting the ERK pathway significantly attenuated PCB153-induced NF-κB activation, whereas inhibiting the PI3K/Akt pathway hardly influenced phospho-NF-κB level. However, inhibiting the PI3K/Akt pathway significantly elevated caspase-3, -8 and -9 activities, while the ERK pathway only synergistically regulated caspase-9. Proliferating cell nuclear antigen (PCNA), a reliable indicator of cell proliferation, was also induced. Moreover, PCB153 led to hepatocellular hypertrophy and elevated liver weight. Taken together, PCB153 leads to aberrant proliferation and apoptosis of hepatocytes through NF-κB activation and caspase inhibition, and coactivated PI3K/Akt and ERK pathways play critical roles in PCB153-induced hepatotoxicity. - Highlights: • PCB153 led to hepatotoxicity through NF-κB activation and caspase inhibition. • The PI3K/Akt and ERK pathways were coactivated in vivo and in vitro by PCB153. • The ERK pathway regulated levels of phospho-NF-κB and caspase-9. • The PI3K/Akt pathway regulated levels of caspase-3, -8 and -9.

  10. High-affinity K(+) transport in Arabidopsis: AtHAK5 and AKT1 are vital for seedling establishment and postgermination growth under low-potassium conditions.

    PubMed

    Pyo, Young Jae; Gierth, Markus; Schroeder, Julian I; Cho, Myeon Haeng

    2010-06-01

    Potassium (K(+)) is a major plant nutrient required for growth and development. It is generally accepted that plant roots absorb K(+) through uptake systems operating at low concentrations (high-affinity transport) and/or high external concentrations (low-affinity transport). To understand the molecular basis of high-affinity K(+) uptake in Arabidopsis (Arabidopsis thaliana), we analyzed loss-of-function mutants in AtHAK5 and AKT1, two transmembrane proteins active in roots. Compared with the wild type under NH(4)(+)-free growth conditions, athak5 mutant plants exhibited growth defects at 10 mum K(+), but at K(+) concentrations of 20 mum and above, athak5 mutants were visibly indistinguishable from the wild type. While germination, scored as radicle emergence, was only slightly decreased in athak5 akt1 double mutants on low-K(+) medium, double mutants failed to grow on medium containing up to 100 mum K(+) and growth was impaired at concentrations up to 450 mum K(+). Moreover, transfer of 3-d-old plants from high to low K(+) concentrations led to growth defects and leaf chlorosis at 10 mum K(+) in athak5 akt1 double mutant plants. Determination of Rb(+)(K(+)) uptake kinetics in wild-type and mutant roots using rubidium ((86)Rb(+)) as a tracer for K(+) revealed that high-affinity Rb(+)(K(+)) uptake into roots is almost completely abolished in double mutants and impaired in single mutants. These results strongly indicate that AtHAK5 and AKT1 are the two major, physiologically relevant molecular entities mediating high-affinity K(+) uptake into roots during seedling establishment and postgermination growth and that residual Rb(+)(K(+)) uptake measured in athak5 akt1 double mutant roots is insufficient to enable plant growth. PMID:20413648

  11. 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

  12. Compensatory activation of Akt in response to mTOR and Raf inhibitors - a rationale for dual-targeted therapy approaches in neuroendocrine tumor disease.

    PubMed

    Zitzmann, Kathrin; Rüden, Janina von; Brand, Stephan; Göke, Burkhard; Lichtl, Jennifer; Spöttl, Gerald; Auernhammer, Christoph J

    2010-09-01

    Several studies have established a link between aberrant PI(3)K-Akt-mTOR- and Ras-Raf-MEK-Erk1/2 signaling and neuroendocrine tumor disease. In this study, we comparatively investigate the antitumor potential of novel small-molecule inhibitors targeting mTOR (RAD001), mTOR/PI(3)K (NVP-BEZ235) and Raf (Raf265) on human NET cell lines of heterogeneous origin. All inhibitors induced potent antitumor effects which involved the induction of apoptosis and G0/G1 arrest. However, the dual mTOR/PI(3)K inhibitor NVP-BEZ235 was more efficient compared to the single mTOR inhibitor RAD001. Consistently, NVP-BEZ235 prevented the negative feedback activation of Akt as observed after treatment with RAD001. Raf265 inhibited Erk1/2 phosphorylation but strongly induced Akt phosphorylation and VEGF secretion, suggesting the existence of a compensatory feedback loop on PI3K-Akt signaling. Finally, combined treatment with RAD001 or NVP-BEZ235 and Raf265 was more efficient than single treatment with either kinase inhibitor. Together, our data provide a rationale for dual targeting of PI(3)K-Akt-mTOR- and Ras-Raf-MEK-Erk1/2 signaling in NET disease.

  13. Estrogen receptor alpha transcriptionally activates casein kinase 2 alpha: A pivotal regulator of promyelocytic leukaemia protein (PML) and AKT in oncogenesis.

    PubMed

    Das, Nilanjana; Datta, Neerajana; Chatterjee, Uttara; Ghosh, Mrinal Kanti

    2016-06-01

    Protein kinase CK2α is frequently upregulated in different cancers. Alteration of CK2α expression and its activity is sufficient to induce dramatic changes in cell fate. It has been established that CK2α induces oncogenesis through modulation of both AKT and PML. CK2α has been found to be overexpressed in breast cancer. In contrary, statistical reports have shown low level of PML. However, the regulation of CK2α gene expression is not fully understood. In the current study, we found that CK2α and activated AKT positively correlate with ERα, whereas PML follows an inverse correlation in human breast cancer tissues. Modulation of ERα signalling leads to recruitment of activated ERα on the ERE sites of CK2α promoter, resulting in CK2α transactivation. Furthermore, the DMBA induced tumours in rat showed elevated level of active CK2α. Consequently it mediates enhancement of AKT activity and PML degradation, resulting in increased cellular proliferation, migration and metastasis. Syngeneic ERα overexpressing stable mouse 4T1 cells produce larger primary tumours and metastatic lung nodules in mice, corroborating our in vitro findings. Hence, our study provides a novel route of ERα dependent CK2α mediated oncogenesis that causes upregulation and consequent AKT activation along with degradation of tumour suppressor PML. PMID:27012497

  14. Crude Preparations of Helicobacter pylori Outer Membrane Vesicles Induce Upregulation of Heme Oxygenase-1 via Activating Akt-Nrf2 and mTOR–IκB Kinase–NF-κB Pathways in Dendritic Cells

    PubMed Central

    Ko, Su Hyuk; Rho, Da Jeong; Jeon, Jong Ik; Kim, Young-Jeon; Woo, Hyun Ae; Kim, Nayoung

    2016-01-01

    Helicobacter pylori sheds outer membrane vesicles (OMVs) that contain many surface elements of bacteria. Dendritic cells (DCs) play a major role in directing the nature of adaptive immune responses against H. pylori, and heme oxygenase-1 (HO-1) has been implicated in regulating function of DCs. In addition, HO-1 is important for adaptive immunity and the stress response. Although H. pylori-derived OMVs may contribute to the pathogenesis of H. pylori infection, responses of DCs to OMVs have not been elucidated. In the present study, we investigated the role of H. pylori-derived crude OMVs in modulating the expression of HO-1 in DCs. Exposure of DCs to crude H. pylori OMVs upregulated HO-1 expression. Crude OMVs obtained from a cagA-negative isogenic mutant strain induced less HO-1 expression than OMVs obtained from a wild-type strain. Crude H. pylori OMVs activated signals of transcription factors such as NF-κB, AP-1, and Nrf2. Suppression of NF-κB or Nrf2 resulted in significant attenuation of crude OMV-induced HO-1 expression. Crude OMVs increased the phosphorylation of Akt and downstream target molecules of mammalian target of rapamycin (mTOR), such as S6 kinase 1 (S6K1). Suppression of Akt resulted in inhibition of crude OMV-induced Nrf2-dependent HO-1 expression. Furthermore, suppression of mTOR was associated with inhibition of IκB kinase (IKK), NF-κB, and HO-1 expression in crude OMV-exposed DCs. These results suggest that H. pylori-derived OMVs regulate HO-1 expression through two different pathways in DCs, Akt-Nrf2 and mTOR–IKK–NF-κB signaling. Following this induction, increased HO-1 expression in DCs may modulate inflammatory responses in H. pylori infection. PMID:27185786

  15. Loss of Mel-18 induces tumor angiogenesis through enhancing the activity and expression of HIF-1α mediated by the PTEN/PI3K/Akt pathway.

    PubMed

    Park, J H; Lee, J Y; Shin, D H; Jang, K S; Kim, H J; Kong, Gu

    2011-11-10

    Mel-18 has been implicated in several processes in tumor progression, in which the Akt pathway is involved as an important key molecular event. However, the function of Mel-18 in human cancers has not been fully established yet. Here, we examined the effect of Mel-18 on tumor angiogenesis in human breast cancer, and found that Mel-18 was a novel regulator of HIF-1α. Mel-18 negatively regulated the HIF-1α expression and its target gene VEGF transcription during both normoxia and hypoxia. We demonstrated that Mel-18 regulated the HIF-1α expression and activity via the PI3K/Akt pathway. Loss of Mel-18 downregulated Phosphatase and tensin homolog deleted on chromosome 10 (PTEN) expression, consequently activating the PI3K/Akt/MDM2 pathway, and leading to an increase of HIF-1α protein level. Mel-18 modulated the HIF-1α transcriptional activity via regulating the cytoplasmic retention of FOXO3a, a downstream effector of Akt, and recruitment of HIF-1α/CBP complex to the VEGF promoter. Furthermore, our data shows that Mel-18 blocked tumor angiogenesis both in vitro and in vivo. Mel-18 overexpression inhibited in vitro tube formation in human umbilical endothelial cells (HUVECs). Xenografts in NOD/SCID mice derived from stably Mel-18 knocked down MCF7 human breast cancer cells showed increased tumor volume, microvessel density, and phospho-Akt and HIF-1α expression levels. In conclusion, our findings provide that Mel-18 is a novel regulator of tumor angiogenesis through regulating HIF-1α and its target VEGF expressions mediated by the PTEN/PI3K/Akt pathway, suggesting a new tumor-suppressive role of Mel-18 in human breast cancer.

  16. Propofol protects hippocampal neurons from apoptosis in ischemic brain injury by increasing GLT-1 expression and inhibiting the activation of NMDAR via the JNK/Akt signaling pathway.

    PubMed

    Gong, Hong-Yan; Zheng, Fang; Zhang, Chao; Chen, Xi-Yan; Liu, Jing-Jing; Yue, Xiu-Qin

    2016-09-01

    Ischemic brain injury (IBI) can cause nerve injury and is a leading cause of morbidity and mortality worldwide. The neuroprotective effects of propofol against IBI have been previously demonstrated. However, the neuroprotective effects of propofol on hippocampal neurons are not yet entirely clear. In the present study, models of IBI were established in hypoxia-exposed hippocampal neuronal cells. Cell viability assay and apoptosis assay were performed to examine the neuroprotective effects of propofol on hippocampal neurons in IBI. A significant decrease in cell viability and a significant increase in cell apoptosis were observed in the IBI group compared with the control group, accompanied by a decrease in glial glutamate transporter-1 (GLT‑1) expression as determined by RT-qPCR and western blot analysis. The effects of IBI were reversed by propofol treatment. The siRNA-mediated knockdown of GLT‑1 in the hypoxia-exposed hippocampal neuronal cells led to an increase in cell apoptosis, Jun N-terminal kinase (JNK) activation and N-methyl-D‑aspartate (NMDA) receptor (NR1 and NR2B) activation, as well as to a decrease in cell viability and a decrease in Akt activation. The effects of RNA interference-mediated GLT‑1 gene silencing on cell viability, JNK activation, NMDAR activation, cell apoptosis and Akt activation in the hippocampal neuronal cells were slightly reversed by propofol treatment. The JNK agonist, anisomycin, and the Akt inhibitor, LY294002, both significantly blocked the effects of propofol on hippocampal neuronal cell viability and apoptosis in IBI. The decrease in JNK activation and the increase in Akt activation caused by GLT‑1 overexpression were reversed by NMDA. Collectively, our findings suggest that propofol treatment protects hippocampal neurons against IBI by enhancing GLT‑1 expression and inhibiting the activation of NMDAR via the JNK/Akt signaling pathway. PMID:27430327

  17. Basic fibroblast growth factor promotes melanocyte migration via activating PI3K/Akt-Rac1-FAK-JNK and ERK signaling pathways.

    PubMed

    Shi, Hongxue; Lin, Beibei; Huang, Yan; Wu, Jiang; Zhang, Hongyu; Lin, Cai; Wang, Zhouguang; Zhu, Jingjing; Zhao, Yingzhen; Fu, Xiaobing; Lou, Zhencai; Li, Xiaokun; Xiao, Jian

    2016-09-01

    Vitiligo is a depigmentation disorder characterized by loss of functional melanocytes of the skin epidermis. The pathogenesis of vitiligo remains elusive. The purpose of this study is to investigate the effects of basic fibroblast growth factor (bFGF) on melanocyte migration, including its biochemical mechanism using transwell assay in vitro. We found that melanocyte treated with bFGF showed a significant increase in migration and cytoskeletal rearrangement. These changes were associated with increased activation of PI3K/Akt, Rac1, FAK, JNK, and ERK. Likewise, reduction of PI3K/Akt, Rac1, FAK, JNK, and ERK activity using selective inhibitors or siRNA was associated with impediment of bFGF-induced melanocyte migration. In addition, activity of Rac1, FAK, and JNK was reduced in cells in which PI3K/Akt was inhibited, activity of FAK and JNK was reduced in cells in which the Rac1 was inhibited, and activity of JNK was reduced in cells in which the FAK was inhibited. Collectively, these data demonstrate that bFGF facilitated melanocyte migration via PI3K/Akt-Rac1-FAK-JNK and ERK signaling pathways. © 2016 IUBMB Life, 68(9):735-747, 2016. PMID:27350596

  18. Biomarkers of Response to Akt Inhibitor MK-2206 in Breast Cancer

    PubMed Central

    Sangai, Takafumi; Akcakanat, Argun; Chen, Huiqin; Tarco, Emily; Wu, Yun; Do, Kim-Anh; Miller, Todd W.; Arteaga, Carlos L.; Mills, Gordon B.; Gonzalez-Angulo, Ana Maria; Meric-Bernstam, Funda

    2013-01-01

    Purpose We tested the hypothesis that allosteric Akt inhibitor MK-2206 inhibits tumor growth, and that PTEN/PIK3CA mutations confer MK-2206 sensitivity. Experimental Design MK-2206 effects on cell signaling were assessed in vitro and in vivo. Its antitumor efficacy was assessed in vitro in a panel of cancer cell lines with differing PIK3CA and PTEN status. Its in vivo efficacy was tested as a single agent and in combination with paclitaxel. Results MK-2206 inhibited Akt signaling and cell-cycle progression, and increased apoptosis in a dose-dependent manner in breast cancer cell lines. Cell lines with PTEN or PIK3CA mutations were significantly more sensitive to MK-2206; however, several lines with PTEN/PIK3CA mutations were MK-2206 resistant. siRNA knockdown of PTEN in breast cancer cells increased Akt phosphorylation concordant with increased MK-2206 sensitivity. Stable transfection of PIK3CA E545K or H1047R mutant plasmids into normal-like MCF10A breast cells enhanced MK-2206 sensitivity. Cell lines that were less sensitive to MK-2206 had lower ratios of Akt1/Akt2 and had less growth inhibition with Akt siRNA knockdown. In PTEN-mutant ZR75-1 breast cancer xenografts, MK-2206 treatment inhibited Akt signaling, cell proliferation, and tumor growth. In vitro, MK-2206 showed a synergistic interaction with paclitaxel in MK-2206–sensitive cell lines, and this combination had significantly greater antitumor efficacy than either agent alone in vivo. Conclusions MK-2206 has antitumor activity alone and in combination with chemotherapy. This activity may be greater in tumors with PTEN loss or PIK3CA mutation, providing a strategy for patient enrichment in clinical trials. PMID:22932669

  19. Similar PDK1-AKT-mTOR pathway activation in balloon cells and dysmorphic neurons of type II focal cortical dysplasia with refractory epilepsy.

    PubMed

    Lin, Yuan-xiang; Lin, Kun; Kang, De-zhi; Liu, Xin-xiu; Wang, Xing-fu; Zheng, Shu-fa; Yu, Liang-hong; Lin, Zhang-ya

    2015-05-01

    Dysmorphic neurons and balloon cells constitute the neuropathological hallmarks of type II focal cortical dysplasias (FCDs) with refractory epilepsy. The genesis of these cells may be critical to the histological findings in type II FCD. Recent work has shown enhanced activation of the mTOR cascade in both balloon cells and dysmorphic neurons, suggesting a common pathogenesis for these two neuropathological hallmarks. A direct comparative analysis of balloon cells and dysmorphic neurons might identify a molecular link between balloon cells and dysmorphic neurons. Here, we addressed whether PDK1-AKT-mTOR activation differentiates balloon cells from dysmorphic neurons. We used immunohistochemistry with antibodies against phosphorylated (p)-PDK1 (Ser241), p-AKT (Thr308), p-AKT (Ser473), p-mTOR (Ser2448), p-P70S6K (Thr229), and p-p70S6 kinase (Thr389) in balloon cells compared with dysmorphic neurons. Strong or moderate staining for components of the PDK1-AKT-mTOR signaling pathway was observed in both balloon cells and dysmorphic neurons. However, only a few pyramidal neurons displayed weak staining in control group (perilesional neocortex and histologically normal neocortex). Additionally, p-PDK1 (Ser241) and p-AKT (Thr308) staining in balloon cells were stronger than in dysmorphic neurons, whereas p-P70S6K (Thr229) and p-p70S6 kinase (Thr389) staining in balloon cells was weaker than in dysmorphic neurons. In balloon cells, p-AKT (Ser473) and p-mTOR (Ser2448) staining was comparable with the staining in dysmorphic neurons. Our data support the previously suggested pathogenic relationship between balloon cells and dysmorphic neurons concerning activation of the PDK1-AKT-mTOR, which may play important roles in the pathogenesis of type II FCD. Differential expression of some components of the PDK1-AKT-mTOR pathway between balloon cells and dysmorphic neurons may result from cell-specific gene expression.

  20. Expansion and long-term culture of human spermatogonial stem cells via the activation of SMAD3 and AKT pathways

    PubMed Central

    Guo, Ying; Liu, Linhong; Sun, Min; Hai, Yanan; Li, Zheng

    2015-01-01

    Spermatogonial stem cells (SSCs) can differentiate into spermatids, reflecting that they could be used in reproductive medicine for treating male infertility. SSCs are able to become embryonic stem-like cells with the potentials of differentiating into numerous cell types of the three germ layers and they can transdifferentiate to mature and functional cells of other lineages, highlighting significant applications of human SSCs for treating human diseases. However, human SSCs are very rare and a long-term culture system of human SSCs has not yet established. This aim of study was to isolate, identify and culture human SSCs for a long period. We isolated GPR125-positive spermatogonia with high purity and viability from adult human testicular tissues utilizing the two-step enzymatic digestion and magnetic-activated cell sorting with antibody against GPR125. These freshly isolated cells expressed a number of markers for SSCs, including GPR125, PLZF, GFRA1, RET, THY1, UCHL1 and MAGEA4, but not the hallmarks for spermatocytes and spermatozoa, e.g. SYCP1, SYCP3, PRM1, and TNP1. The isolated human SSCs could be cultured for two months with a significant increase of cell number with the defined medium containing growth factors and hydrogel. Notably, the expression of numerous SSC markers was maintained during the cultivation of human SSCs. Furthermore, SMAD3 and AKT phosphorylation was enhanced during the culture of human SSCs. Collectively, these results suggest that human SSCs can be cultivated for a long period and expanded whilst retaining an undifferentiated status via the activation of SMAD3 and AKT pathways. This study could provide sufficient cells of SSCs for their basic research and clinic applications in reproductive and regenerative medicine. PMID:26088866

  1. Seabuckthorn Pulp Oil Protects against Myocardial Ischemia–Reperfusion Injury in Rats through Activation of Akt/eNOS

    PubMed Central

    Suchal, Kapil; Bhatia, Jagriti; Malik, Salma; Malhotra, Rajiv Kumar; Gamad, Nanda; Goyal, Sameer; Nag, Tapas C.; Arya, Dharamvir S.; Ojha, Shreesh

    2016-01-01

    Seabuckthorn (SBT) pulp oil obtained from the fruits of seabuckthorn [Hippophae rhamnoides L. (Elaeagnaceae)] has been used traditionally for its medicinal and nutritional properties. However, its role in ischemia–reperfusion (IR) injury of myocardium in rats has not been elucidated so far. The present study reports the cardioprotective effect of SBT pulp oil in IR-induced model of myocardial infarction in rats and underlying mechanism mediating activation of Akt/eNOS signaling pathway. Male albino Wistar rats were orally administered SBT pulp oil (5, 10, and 20 ml/kg/day) or saline for 30 days. On the day 31, ischemia was induced by one-stage ligation of left anterior descending coronary artery for 45 min followed by reperfusion for 60 min. SBT pulp oil pretreatment at the dose of 20 ml/kg observed to stabilize cardiac function and myocardial antioxidants such as glutathione, superoxide dismutase, catalase, and inhibited lipid peroxidation evidenced by reduced malondialdehyde levels as compared to IR-control group. SBT pulp oil also improved hemodynamic and contractile function and decreased tumor necrosis factor and activities of myocyte injury marker enzymes; lactate dehydrogenase and creatine kinase-MB. Additionally, a remarkable rise in expression of pAkt–eNOS, Bcl-2 and decline in expression of IKKβ/NF-κB and Bax was observed in the myocardium. The histopathological and ultrastructural salvage of cardiomyocytes further supports the cardioprotective effect of SBT pulp oil. Based on findings, it can be concluded that SBT pulp oil protects against myocardial IR injury mediating favorable modulation of Akt-eNOS and IKKβ/NF-κB expression. PMID:27445803

  2. Transglutaminase 2 Promotes PDGF-Mediated Activation of PDGFR/Akt1 and β-catenin Signaling in Vascular Smooth Muscle Cells and Supports Neointima Formation

    PubMed Central

    Nurminskaya, Maria; Beazley, Kelly E.; Smith, Elizabeth P.; Belkin, Alexey M.

    2015-01-01

    Background Phenotypic switch of vascular smooth muscle cells (VSMCs) accompanies neointima formation and associates with vascular diseases. Platelet derived growth factor (PDGF)-induced activation of PDGFR/Akt1 and β-catenin signaling pathways in VSMCs has been implicated in vessel occlusion. Transglutaminase 2 (TG2) regulates these pathways and its levels are increased in the neointima. Objective To evaluate the role of TG2 in PDGF/β-catenin signaling cross-talk and assess its contribution to neointima. Methods Aortic VSMCs from wild-type and TG2 knockout mice were tested in vitro for levels of VSMC markers, proliferation, migration, and PDGF-induced activation of PDGFR/Akt1 and β-catenin pathways. Neointima in these mice was studied ex vivo in coronary vessels using heart slice model and in vivo using carotid artery ligation (CAL) model. Results Genetic deletion of TG2 attenuated the PDGF-induced phenotypic switch of aortic VSMCs, reduced their proliferation and migration rates, and inhibited PDGF-induced activation of PDGFR/Akt1 and β-catenin pathways in both ex vivo and in vivo neointima models. Importantly, genetic deletion of TG2 also markedly attenuated vessel occlusion. Conclusions TG2 promotes neointima formation by mediating the PDGF-induced activation of the PDGFR/Akt1 and β-catenin pathways in VSMCs. This study identifies TG2 as a potential therapeutic target for blocking neointima in blood vessels. PMID:25612735

  3. Cisplatin triggers atrophy of skeletal C2C12 myotubes via impairment of Akt signalling pathway and subsequent increment activity of proteasome and autophagy systems

    SciTech Connect

    Fanzani, Alessandro Zanola, Alessandra; Rovetta, Francesca; Rossi, Stefania; Aleo, Maria Francesca

    2011-02-01

    Cisplatin (cisPt) is an antineoplastic drug which causes an array of adverse effects on different organs and tissues, including skeletal muscle. In this work we show that cisPt behaves as a potent trigger to activate protein hypercatabolism in skeletal C2C12 myotubes. Within 24 h of 50 {mu}M cisPt administration, C2C12 myotubes displayed unchanged cell viability but showed a subset of hallmark signs typically recognized during atrophy, including severe reduction in body size, repression of Akt phosphorylation, transcriptional up-regulation of atrophy-related genes, such as atrogin-1, gabarap, beclin-1 and bnip-3, and loss of myogenic markers. As a consequence, proteasomal activity and formation of autophagosomes were remarkably increased in cisPt-treated myotubes, but forced stimulation of Akt pathway, as obtained through insulin administration or delivery of a constitutively activated Akt form, was sufficient to counter the cisPt-induced protein breakdown, leading to rescue of atrophic size. Overall, these results indicate that cisPt induces atrophy of C2C12 myotubes via activation of proteasome and autophagy systems, suggesting that the Akt pathway represents one sensitive target of cisPt molecular action in skeletal muscle.

  4. Fas apoptosis inhibitory molecule regulates T cell receptor-mediated apoptosis of thymocytes by modulating Akt activation and Nur77 expression.

    PubMed

    Huo, Jianxin; Xu, Shengli; Lam, Kong-Peng

    2010-04-16

    Fas apoptosis inhibitory molecule (FAIM) has been demonstrated to confer resistance to Fas-induced apoptosis of lymphocytes and hepatocytes in vitro and in vivo. Here, we show that FAIM is up-regulated in thymocytes upon T cell receptor (TCR) engagement and that faim(-/-) thymocytes are highly susceptible to TCR-mediated apoptosis with increased activation of caspase-8 and -9. Furthermore, injection of anti-CD3 antibodies leads to augmented depletion of CD4(+)CD8(+) T cells in the thymus of faim(-/-) mice compared with wild-type control, suggesting that FAIM plays a role in thymocyte apoptosis. Cross-linking of the TCR on faim(-/-) thymocytes leads to an elevated protein level of the orphan nuclear receptor Nur77, which plays a role in thymocyte apoptosis. Interestingly, in the absence of FAIM, there are reduced ubiquitination and degradation of the Nur77 protein. Faim(-/-) thymocytes also exhibit a defective TCR-induced activation of Akt whose activity we now show is required for Nur77 ubiquitination. Further analyses utilizing FAIM-deficient primary thymocytes and FAIM-overexpressing DO-11.10 T cells indicate that FAIM acts upstream of Akt during TCR signaling and influences the localization of Akt to lipid rafts, hence affecting its activation. Taken together, our study defined a TCR-induced FAIM/Akt/Nur77 signaling axis that is critical for modulating the apoptosis of developing thymocytes.

  5. TNF-{alpha} promotes human retinal pigment epithelial (RPE) cell migration by inducing matrix metallopeptidase 9 (MMP-9) expression through activation of Akt/mTORC1 signaling

    SciTech Connect

    Wang, Cheng-hu; Cao, Guo-Fan; Jiang, Qin; Yao, Jin

    2012-08-17

    Highlights: Black-Right-Pointing-Pointer TNF-{alpha} induces MMP-9 expression and secretion to promote RPE cell migration. Black-Right-Pointing-Pointer MAPK activation is not critical for TNF-{alpha}-induced MMP-9 expression. Black-Right-Pointing-Pointer Akt and mTORC1 signaling mediate TNF-{alpha}-induced MMP-9 expression. Black-Right-Pointing-Pointer SIN1 knockdown showed no significant effect on MMP-9 expression by TNF-{alpha}. -- Abstract: Tumor necrosis factor-alpha (TNF-{alpha}) promotes in vitro retinal pigment epithelial (RPE) cell migration to initiate proliferative vitreoretinopathy (PVR). Here we report that TNF-{alpha} promotes human RPE cell migration by inducing matrix metallopeptidase 9 (MMP-9) expression. Inhibition of MMP-9 by its inhibitor or its neutralizing antibody inhibited TNF-{alpha}-induced in vitro RPE cell migration. Reversely, exogenously-added active MMP-9 promoted RPE cell migration. Suppression Akt/mTOR complex 1(mTORC1) activation by LY 294002 and rapamycin inhibited TNF-{alpha}-mediated MMP-9 expression. To introduce a constitutively active Akt (CA-Akt) in cultured RPE cells increased MMP-9 expression, and to block mTORC1 activation by rapamycin inhibited its effect. RNA interference (RNAi)-mediated silencing of SIN1, a key component of mTOR complex 2 (mTORC2), had no effect on MMP-9 expression or secretion. In conclusion, this study suggest that TNF-{alpha} promotes RPE cell migration by inducing MMP-9 expression through activation of Akt/ mTORC1, but not mTORC2 signaling.

  6. Mutants with enhanced nitrogenase activity in hydroponic Azospirillum brasilense-wheat associations.

    PubMed

    Pereg Gerk, L; Gilchrist, K; Kennedy, I R

    2000-05-01

    The effect of a mutation affecting flocculation, differentiation into cyst-like forms, and root colonization on nitrogenase expression by Azospirillum brasilense is described. The gene flcA of strain Sp7 restored these phenotypes in spontaneous mutants of both strains Sp7 and Sp245. Employing both constitutive pLA-lacZ and nifH-lacZ reporter fusions expressed in situ, the colony morphology, colonization pattern, and potential for nitrogenase activity of spontaneous mutants and flcA Tn5-induced mutants were established. The results of this study show that the ability of Sp7 and Sp245 mutant strains to remain in a vegetative form improved their ability to express nitrogenase activity in association with wheat in a hydroponic system. Restoring the cyst formation and colonization pattern to the spontaneous mutant Sp7-S reduced nitrogenase activity rates in association with plants to that of the wild-type Sp7. Although Tn5-induced flcA mutants showed higher potentials for nitrogenase expression than Sp7, their potentials were lower than that of Sp7-S, indicating that other factors in this strain contribute to its exceptional nitrogenase activity rates on plants. The lack of lateral flagella is not one of these factors, as Sp7-PM23, a spontaneous mutant impaired in swarming and lateral-flagellum production but not in flocculation, showed wild-type nitrogenase activity and expression. The results also suggest factors of importance in evolving an effective symbiosis between Azospirillum and wheat, such as increasing the availability of microaerobic niches along the root, increased supply of carbon sources by the plant, and the retention of the bacterial cells in vegetative form for faster metabolism.

  7. Mutants with Enhanced Nitrogenase Activity in Hydroponic Azospirillum brasilense-Wheat Associations

    PubMed Central

    Pereg Gerk, Lily; Gilchrist, Kate; Kennedy, Ivan R.

    2000-01-01

    The effect of a mutation affecting flocculation, differentiation into cyst-like forms, and root colonization on nitrogenase expression by Azospirillum brasilense is described. The gene flcA of strain Sp7 restored these phenotypes in spontaneous mutants of both strains Sp7 and Sp245. Employing both constitutive pLA-lacZ and nifH-lacZ reporter fusions expressed in situ, the colony morphology, colonization pattern, and potential for nitrogenase activity of spontaneous mutants and flcA Tn5-induced mutants were established. The results of this study show that the ability of Sp7 and Sp245 mutant strains to remain in a vegetative form improved their ability to express nitrogenase activity in association with wheat in a hydroponic system. Restoring the cyst formation and colonization pattern to the spontaneous mutant Sp7-S reduced nitrogenase activity rates in association with plants to that of the wild-type Sp7. Although Tn5-induced flcA mutants showed higher potentials for nitrogenase expression than Sp7, their potentials were lower than that of Sp7-S, indicating that other factors in this strain contribute to its exceptional nitrogenase activity rates on plants. The lack of lateral flagella is not one of these factors, as Sp7-PM23, a spontaneous mutant impaired in swarming and lateral-flagellum production but not in flocculation, showed wild-type nitrogenase activity and expression. The results also suggest factors of importance in evolving an effective symbiosis between Azospirillum and wheat, such as increasing the availability of microaerobic niches along the root, increased supply of carbon sources by the plant, and the retention of the bacterial cells in vegetative form for faster metabolism. PMID:10788397

  8. Unglycosylated recombinant human glutathione peroxidase 3 mutant from Escherichia coli is active as a monomer.

    PubMed

    Song, Jian; Yu, Yang; Xing, Ruiqing; Guo, Xiao; Liu, Dali; Wei, Jingyan; Song, Hongwei

    2014-01-01

    Glutathione peroxidase 3 (GPx3) is a glycosylated member of GPx family and can catalyze the reaction of different types of peroxides with GSH to form their corresponding alcohols in vitro. The active center of GPx3 is selenocysteine (Sec), which is incorporated into proteins by a specific mechanism. In this study, we prepared a recombinant human GPx3 (rhGPx3) mutant with all Cys changed to Ser from a Cys auxotrophic strain of E. coli, BL21(DE3)cys. Although lacking post-translational modification, rhGPx3 mutant still retained the ability to reduce H2O2 and PLPC-OOH. Study on the quaternary structure suggested that rhGPx3 mutant existed as a monomer in solution, which is different from native tetrameric GPx3. Loss of the catalytic activity was considered to be attributed to both the absence of glycosylation and the failure of the tetramer. Further analysis was performed to compare the structures of rhGPx3 and GPx4 mutant, which were quite similar except for oligomerization loop. The differences of amino acid composition and electrostatic potentials on the oligomerization loop may affect the binding of large substrates to rhGPx3 mutant. This research provides an important foundation for biosynthesis of functionally selenium-containing GPx3 mutant in E.coli. PMID:25331785

  9. Enhanced dimerization drives ligand-independent activity of mutant epidermal growth factor receptor in lung cancer

    PubMed Central

    Valley, Christopher C.; Arndt-Jovin, Donna J.; Karedla, Narain; Steinkamp, Mara P.; Chizhik, Alexey I.; Hlavacek, William S.; Wilson, Bridget S.; Lidke, Keith A.; Lidke, Diane S.

    2015-01-01

    Mutations within the epidermal growth factor receptor (EGFR/erbB1/Her1) are often associated with tumorigenesis. In particular, a number of EGFR mutants that demonstrate ligand-independent signaling are common in non–small cell lung cancer (NSCLC), including kinase domain mutations L858R (also called L834R) and exon 19 deletions (e.g., ΔL747-P753insS), which collectively make up nearly 90% of mutations in NSCLC. The molecular mechanisms by which these mutations confer constitutive activity remain unresolved. Using multiple subdiffraction-limit imaging modalities, we reveal the altered receptor structure and interaction kinetics of NSCLC-associated EGFR mutants. We applied two-color single quantum dot tracking to quantify receptor dimerization kinetics on living cells and show that, in contrast to wild-type EGFR, mutants are capable of forming stable, ligand-independent dimers. Two-color superresolution localization microscopy confirmed ligand-independent aggregation of EGFR mutants. Live-cell Förster resonance energy transfer measurements revealed that the L858R kinase mutation alters ectodomain structure such that unliganded mutant EGFR adopts an extended, dimerization-competent conformation. Finally, mutation of the putative dimerization arm confirmed a critical role for ectodomain engagement in ligand-independent signaling. These data support a model in which dysregulated activity of NSCLC-associated kinase mutants is driven by coordinated interactions involving both the kinase and extracellular domains that lead to enhanced dimerization. PMID:26337388

  10. Berberine Induced Apoptosis of Human Osteosarcoma Cells by Inhibiting Phosphoinositide 3 Kinase/Protein Kinase B (PI3K/Akt) Signal Pathway Activation

    PubMed Central

    2016-01-01

    Background: Osteosarcoma is a malignant tumor with high mortality but effective therapy has not yet been developed. Berberine, an isoquinoline alkaloid component in several Chinese herbs including Huanglian, has been shown to induce growth inhibition and the apoptosis of certain cancer cells. The aim of this study was to determine the role of berberine on human osteosarcoma cell lines U2OS and its potential mechanism. Methods: The proliferation effect of U20S was exanimed by 3-(4,5)-dimethylthiahiazo(-z-y1)-3,5-di- phenytetrazoliumromide (MTT) and the percentage of apoptotic cells were determined by flow cytometric analysis. The expression of PI3K, p-Akt, Bax, Bcl-2, cleavage-PARP and Caspase3 were detected by Western blott. Results: Berberine treatment caused dose-dependent inhibiting proliferation and inducing apoptosis of U20S cell. Mechanistically, berberine inhibits PI3K/AKT activation that, in turn, results in up-regulating the expression of Bax, and PARP and down-regulating the expression of Bcl-2 and caspase3. In all, berberine can suppress the proliferation and induce the apoptosis of U2OS cell through inhibiting the PI3K/Akt signaling pathway activation. Conclusion: Berberine can suppress the proliferation and induce the apoptosis of U2OS cell through inhibiting the PI3K/Akt signaling pathway activation. PMID:27398330

  11. Phosphorylation of Akt Mediates Anti-Inflammatory Activity of 1-p-Coumaroyl β-D-Glucoside Against Lipopolysaccharide-Induced Inflammation in RAW264.7 Cells

    PubMed Central

    Vo, Van Anh; Lee, Jae-Won; Kim, Ji-Young; Park, Jun-Ho; Lee, Hee Jae; Kim, Sung-Soo; Kwon, Yong-Soo

    2014-01-01

    Hydroxycinnamic acids have been reported to possess numerous pharmacological activities such as antioxidant, anti-inflammatory, and anti-tumor properties. However, the biological activity of 1-p-coumaroyl β-D-glucoside (CG), a glucose ester derivative of p-coumaric acid, has not been clearly examined. The objective of this study is to elucidate the anti-inflammatory action of CG in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophage cells. In the present study, CG significantly suppressed LPS-induced excessive production of pro-inflammatory mediators such as nitric oxide (NO) and PGE2 and the protein expression of iNOS and COX-2. CG also inhibited LPS-induced secretion of pro-inflammatory cytokines, IL-1β and TNF-α. In addition, CG significantly suppressed LPS-induced degradation of IκB. To elucidate the underlying mechanism by which CG exerts its anti-inflammatory action, involvement of various signaling pathways were examined. CG exhibited significantly increased Akt phosphorylation in a concentration-dependent manner, although MAPKs such as Erk, JNK, and p38 appeared not to be involved. Furthermore, inhibition of Akt/PI3K signaling pathway with wortmannin significantly, albeit not completely, abolished CG-induced Akt phosphorylation and anti-inflammatory actions. Taken together, the present study demonstrates that Akt signaling pathway might play a major role in CG-mediated anti-inflammatory activity in LPS-stimulated RAW264.7 macrophage cells. PMID:24634601

  12. Myrcia bella Leaf Extract Presents Hypoglycemic Activity via PI3k/Akt Insulin Signaling Pathway

    PubMed Central

    Vareda, Priscilla Maria Ponce; Saldanha, Luiz Leonardo; Camaforte, Nathalia Aparecida de Paula; Violato, Natalia Moretti; Dokkedal, Anne Lígia; Bosqueiro, José Roberto

    2014-01-01

    Species of Myrcia are used by indigenous people and in traditional communities in Brazil for the treatment of Diabetes mellitus. We investigated the hypoglycemic effect of the extract of leaves of Myrcia bella in diabetic mice. The chemical fingerprinting of the 70% EtOH extract characterized as main constituents flavonoid aglycones, flavonoid-O-glycosides, and acylated flavonoid-O-glycosides derivatives of quercetin and myricetin. Mice were treated with saline or extract of M. bella (300 or 600 mg/Kg b.w.) for 14 days. Body weight and water and food intake were measured every day. Fasting blood glucose was measured weekly. At the end of the treatment, blood insulin, triglycerides, total cholesterol, and protein were measured. Glycogen content and expression of proteins of the insulin signaling pathway were measured in liver. The treatment with 600 mg/Kg reduced the fasting blood glucose in diabetic mice of the 7th day as water and food intake and increased hepatic glycogen. Total cholesterol and triglycerides were reduced in diabetic treated mice. The treatment increased the expression of IRS-1, PI3-K, and AKT in the livers of diabetic treated mice. The results indicate that the extract of the leaves of Myrcia bella has hypoglycemic properties and possibly acts to regulate glucose uptake by the liver. PMID:24872834

  13. Engineered epidermal growth factor mutants with faster binding on-rates correlate with enhanced receptor activation

    PubMed Central

    Lahti, Jennifer L.; Lui, Bertrand H.; Beck, Stayce E.; Lee, Stephen S.; Ly, Daphne P.; Longaker, Michael T.; Yang, George P.; Cochran, Jennifer R.

    2011-01-01

    Receptor tyrosine kinases (RTKs) regulate critical cell signaling pathways, yet the properties of their cognate ligands that influence receptor activation are not fully understood. There is great interest in parsing these complex ligand-receptor relationships using engineered proteins with altered binding properties. Here we focus on the interaction between two engineered epidermal growth factor (EGF) mutants and the EGF receptor (EGFR), a model member of the RTK superfamily. We found that EGF mutants with faster kinetic on-rates stimulate increased EGFR activation compared to wild-type EGF. These findings support previous predictions that faster association rates correlate with enhanced receptor activity. PMID:21439278

  14. The neuroprotective action of pyrroloquinoline quinone against glutamate-induced apoptosis in hippocampal neurons is mediated through the activation of PI3K/Akt pathway

    SciTech Connect

    Zhang Qi; Shen Mi; Ding Mei; Shen Dingding; Ding Fei

    2011-04-01

    Pyrroloquinoline quinone (PQQ), a cofactor in several enzyme-catalyzed redox reactions, possesses a potential capability of scavenging reactive oxygen species (ROS) and inhibiting cell apoptosis. In this study, we investigated the effects of PQQ on glutamate-induced cell death in primary cultured hippocampal neurons and the possible underlying mechanisms. We found that glutamate-induced apoptosis in cultured hippocampal neurons was significantly attenuated by the ensuing PQQ treatment, which also inhibited the glutamate-induced increase in Ca2+ influx, caspase-3 activity, and ROS production, and reversed the glutamate-induced decrease in Bcl-2/Bax ratio. The examination of signaling pathways revealed that PQQ treatment activated the phosphorylation of Akt and suppressed the glutamate-induced phosphorylation of c-Jun N-terminal protein kinase (JNK). And inhibition of phosphatidylinositol-3-kinase (PI3K)/Akt cascade by LY294002 and wortmannin significantly blocked the protective effects of PQQ, and alleviated the increase in Bcl-2/Bax ratio. Taken together, our results indicated that PQQ could protect primary cultured hippocampal neurons against glutamate-induced cell damage by scavenging ROS, reducing Ca2+ influx, and caspase-3 activity, and suggested that PQQ-activated PI3K/Akt signaling might be responsible for its neuroprotective action through modulation of glutamate-induced imbalance between Bcl-2 and Bax. - Research Highlights: >PQQ attenuated glutamate-induced cell apoptosis of cultured hippocampal neurons. >PQQ inhibited glutamate-induced Ca{sup 2+} influx and caspase-3 activity. >PQQ reduced glutamate-induced increase in ROS production. >PQQ affected phosphorylation of Akt and JNK signalings after glutamate injury. >PI3K/Akt was required for neuroprotection of PQQ by modulating Bcl-2/Bax ratio.

  15. Ochratoxin A activates opposing c-MET/PI3K/Akt and MAPK/ERK 1-2 pathways in human proximal tubule HK-2 cells.

    PubMed

    Özcan, Zeynep; Gül, Gizem; Yaman, Ibrahim

    2015-08-01

    Ochratoxin A (OTA) is a mycotoxin produced as a secondary metabolite by filamentous fungi, such as Aspergillus and Penicillium. Because OTA is a common contaminant of food and feeds, humans and animals are frequently exposed to OTA in daily life. It has been classified as a carcinogen in rodents and a possible carcinogen in humans. OTA has been shown to deregulate a variety of different signal transduction pathways in a cell type- and dosage-depending manner resulting in contrasting physiological effects, such as survival or cell death. While the ERK1-2 and JNK/SAPK MAPK pathways are major targets, knowledge about their role in OTA-mediated cell survival and death is fragmented. Similarly, the contribution of the PI3K/Akt pathway to the carcinogenic effect of OTA in proximal tubule cells has not been elucidated in detail. In this study, we demonstrated that OTA induced sustained activation of the PI3K/Akt and MEK/ERK1-2 signaling pathways in a dose- and time-dependent manner in HK-2 cells. Chemical inhibition of ERK1-2 activation or overexpression of dominant-negative and kinase-dead MEK1 leads to increased cell viability and decreased apoptosis in OTA-treated cells. Blockage of PI3K/Akt with Wortmannin aggravated the negative effect of OTA on cell viability and increased the levels of apoptosis. Moreover, we identified the c-MET proto-oncogene as an upstream receptor tyrosine kinase responsible for OTA-induced activation of PI3K/Akt signaling in HK-2 cells. Our data suggest that OTA may potentiate carcinogenesis by sustained activation of c-MET/PI3K/Akt signaling through suppression of apoptosis induced by MEK/ERK1-2 activation in damaged renal proximal tubule epithelial cells. PMID:25002221

  16. PTH-related protein upregulates integrin {alpha}6{beta}4 expression and activates Akt in breast cancer cells

    SciTech Connect

    Shen Xiaoli; Falzon, Miriam . E-mail: mfalzon@utmb.edu

    2006-11-15

    Breast cancer is the most common carcinoma that metastasizes to bone. Tumor-produced parathyroid hormone-related protein (PTHrP), a known stimulator of osteoclastic bone resorption, is a major mediator of the osteolytic process in breast cancer. We have previously shown that PTHrP increases breast cancer cell proliferation, survival, migration, and pro-invasive integrin {alpha}6{beta}4 expression. To determine the role of integrin {alpha}6{beta}4 in these PTHrP-mediated effects, we utilized two strategies to modulate expression of the {alpha}6 and {beta}4 subunits in parental and PTHrP-overexpressing MDA-MB-231 and MCF-7 cells: overexpression of {alpha}6{beta}4 by transfection with constructs encoding the {alpha}6 and {beta}4 subunits, and suppression of endogenous {alpha}6{beta}4 expression by transfection with siRNAs targeting these subunits. We now show that the effects of PTHrP are mediated via upregulation of integrin {alpha}6{beta}4 expression. We also show that integrin {alpha}6{beta}4 expression is modulated at the mRNA level, indicating a transcriptional and/or post-transcriptional mechanism of action for PTHrP. PTHrP expression also increased the levels of phosphorylated Akt, with a consequent increase in the levels of phosphorylated (inactive) glycogen synthase kinase-3 (GSK-3). The role of PTHrP in breast cancer growth and metastasis may thus be mediated via upregulation of integrin {alpha}6{beta}4 expression and Akt activation, with consequent inactivation of GSK-3.

  17. Angiotensin-converting enzyme 2/angiotensin-(1-7)/Mas axis activates Akt signaling to ameliorate hepatic steatosis.

    PubMed

    Cao, Xi; Yang, Fangyuan; Shi, Tingting; Yuan, Mingxia; Xin, Zhong; Xie, Rongrong; Li, Sen; Li, Hongbing; Yang, Jin-Kui

    2016-01-01

    The classical axis of renin-angiotensin system (RAS), angiotensin (Ang)-converting enzyme (ACE)/Ang II/AT1, contributes to the development of non-alcoholic fatty liver disease (NAFLD). However, the role of bypass axis of RAS (Angiotensin-converting enzyme 2 (ACE2)/Ang-(1-7)/Mas) in hepatic steatosis is still unclear. Here we showed that deletion of ACE2 aggravates liver steatosis, which is correlated with the increased expression of hepatic lipogenic genes and the decreased expression of fatty acid oxidation-related genes in the liver of ACE2 knockout (ACE2(-/y)) mice. Meanwhile, oxidative stress and inflammation were also aggravated in ACE2(-/y) mice. On the contrary, overexpression of ACE2 improved fatty liver in db/db mice, and the mRNA levels of fatty acid oxidation-related genes were up-regulated. In vitro, Ang-(1-7)/ACE2 ameliorated hepatic steatosis, oxidative stress and inflammation in free fatty acid (FFA)-induced HepG2 cells, and what's more, Akt inhibitors reduced ACE2-mediated lipid metabolism. Furthermore, ACE2-mediated Akt activation could be attenuated by blockade of ATP/P2 receptor/Calmodulin (CaM) pathway. These results indicated that Ang-(1-7)/ACE2/Mas axis may reduce liver lipid accumulation partly by regulating lipid-metabolizing genes through ATP/P2 receptor/CaM signaling pathway. Our findings support the potential role of ACE2/Ang-(1-7)/Mas axis in prevention and treatment of hepatic lipid metabolism. PMID:26883384

  18. Isolation and characterization of pediocin AcH chimeric protein mutants with altered bactericidal activity.

    PubMed

    Miller, K W; Schamber, R; Osmanagaoglu, O; Ray, B

    1998-06-01

    A collection of pediocin AcH amino acid substitution mutants was generated by PCR random mutagenesis of DNA encoding the bacteriocin. Mutants were isolated by cloning mutagenized DNA into an Escherichia coli malE plasmid that directs the secretion of maltose binding protein-pediocin AcH chimeric proteins and by screening transformant colonies for bactericidal activity against Lactobacillus plantarum NCDO955 (K. W. Miller, R. Schamber, Y. Chen, and B. Ray, 1998. Appl. Environ. Microbiol. 64:14-20, 1998). In all, 17 substitution mutants were isolated at 14 of the 44 amino acids of pediocin AcH. Seven mutants (N5K, C9R, C14S, C14Y, G37E, G37R, and C44W) were completely inactive against the pediocin AcH-sensitive strains L. plantarum NCDO955, Listeria innocua Lin11, Enterococcus faecalis M1, Pediococcus acidilactici LB42, and Leuconostoc mesenteroides Ly. A C24S substitution mutant constructed by other means also was inactive against these bacteria. Nine other mutants (K1N, W18R, I26T, M31T, A34D, N41K, H42L, K43N, and K43E) retained from <1% to approximately 60% of wild-type activity when assayed against L. innocua Lin11. One mutant, K11E, displayed approximately 2. 8-fold-higher activity against this indicator. About one half of the mutations mapped to amino acids that are conserved in the pediocin-like family of bacteriocins. All four cysteines were found to be required for activity, although only C9 and C14 are conserved among pediocin-like bacteriocins. Several basic amino acids as well as nonpolar amino acids located within the hydrophobic C-terminal region also were found to be important. The mutations are discussed in the context of structural models that have been proposed for the bacteriocin.

  19. Thrombin Receptor-Activating Protein (TRAP)-Activated Akt Is Involved in the Release of Phosphorylated-HSP27 (HSPB1) from Platelets in DM Patients

    PubMed Central

    Tokuda, Haruhiko; Kuroyanagi, Gen; Tsujimoto, Masanori; Matsushima-Nishiwaki, Rie; Akamatsu, Shigeru; Enomoto, Yukiko; Iida, Hiroki; Otsuka, Takanobu; Ogura, Shinji; Iwama, Toru; Kojima, Kumi; Kozawa, Osamu

    2016-01-01

    It is generally known that heat shock protein 27 (HSP27) is phosphorylated through p38 mitogen-activated protein (MAP) kinase. We have previously reported that HSP27 is released from human platelets associated with collagen-induced phosphorylation. In the present study, we conducted an investigation into the effect of thrombin receptor-activating protein (TRAP) on the release of HSP27 in platelets in type 2 diabetes mellitus (DM) patients. The phosphorylated-HSP27 levels induced by TRAP were directly proportional to the aggregation of platelets. The levels of phosphorylated-HSP27 (Ser-78) were correlated with the levels of phosphorylated-p38 MAP kinase and phosphorylated-Akt in the platelets stimulated by 10 µM TRAP but not with those of phosphorylated-p44/p42 MAP kinase. The levels of HSP27 released from the TRAP (10 µM)-stimulated platelets were correlated with the levels of phosphorylated-HSP27 in the platelets. The released platelet-derived growth factor-AB (PDGF-AB) levels were in parallel with the HSP27 levels released from the platelets stimulated by 10 µM TRAP. Although the area under the curve (AUC) of small aggregates (9–25 µm) induced by 10 µM TRAP showed no significant correlation with the released HSP27 levels, AUC of medium aggregates (25–50 µm), large aggregates (50–70 µm) and light transmittance were significantly correlated with the released HSP27 levels. TRAP-induced phosphorylation of HSP27 was truly suppressed by deguelin, an inhibitor of Akt, in the platelets from a healthy subject. These results strongly suggest that TRAP-induced activation of Akt in addition to p38 MAP kinase positively regulates the release of phosphorylated-HSP27 from human platelets, which is closely related to the platelet hyper-aggregation in type 2 DM patients. PMID:27187380

  20. Mechanical Stimulation and IGF-1 Enhance mRNA Translation Rate in Osteoblasts Via Activation of the AKT-mTOR Pathway.

    PubMed

    Bakker, Astrid D; Gakes, Tom; Hogervorst, Jolanda M A; de Wit, Gerard M J; Klein-Nulend, Jenneke; Jaspers, Richard T

    2016-06-01

    Insulin-like growth factor-1 (IGF-1) is anabolic for muscle by enhancing the rate of mRNA translation via activation of AKT and subsequent activation of the mammalian target of rapamycin complex 1 (mTOR), thereby increasing cellular protein production. IGF-1 is also anabolic for bone, but whether the mTOR pathway plays a role in the rate of bone matrix protein production by osteoblasts is unknown. We hypothesized that anabolic stimuli such as mechanical loading and IGF-1 stimulate protein synthesis in osteoblasts via activation of the AKT-mTOR pathway. MC3T3-E1 osteoblasts were either or not subjected for 1 h to mechanical loading by pulsating fluid flow (PFF) or treated with or without human recombinant IGF-1 (1-100 ng/ml) for 0.5-6 h, to determine phosphorylation of AKT and p70S6K (downstream of mTOR) by Western blot. After 4 days of culture with or without the mTOR inhibitor rapamycin, total protein, DNA, and gene expression were quantified. IGF-1 (100 ng/ml) reduced IGF-1 gene expression, although PFF enhanced IGF-1 expression. IGF-1 did not affect collagen-I gene expression. IGF-1 dose-dependently enhanced AKT and p70S6K phosphorylation at 2 and 6 h. PFF enhanced phosphorylation of AKT and p70S6K already within 1 h. Both IGF-1 and PFF enhanced total protein per cell by ∼30%, but not in the presence of rapamycin. Our results show that IGF-1 and PFF activate mTOR, thereby stimulating the rate of mRNA translation in osteoblasts. The known anabolic effect of mechanical loading and IGF-1 on bone may thus be partly explained by mTOR-mediated enhanced protein synthesis in osteoblasts.

  1. In Vivo Regulation of Brain-Derived Neurotrophic Factor in Dorsal Root Ganglia Is Mediated by Nerve Growth Factor-Triggered Akt Activation during Cystitis

    PubMed Central

    Qiao, Li-Ya; Yu, Sharon J.; Kay, Jarren C.; Xia, Chun-Mei

    2013-01-01

    The role of brain-derived neurotrophic factor (BDNF) in sensory hypersensitivity has been suggested; however the molecular mechanisms and signal transduction that regulate BDNF expression in primary afferent neurons during visceral inflammation are not clear. Here we used a rat model of cystitis and found that the mRNA and protein levels of BDNF were increased in the L6 dorsal root ganglia (DRG) in response to bladder inflammation. BDNF up-regulation in the L6 DRG was triggered by endogenous nerve growth factor (NGF) because neutralization of NGF with a specific NGF antibody reduced BDNF levels during cystitis. The neutralizing NGF antibody also subsequently reduced cystitis-induced up-regulation of the serine/threonine kinase Akt activity in L6 DRG. To examine whether the NGF-induced Akt activation led to BDNF up-regulation in DRG in cystitis, we found that in cystitis the phospho-Akt immunoreactivity was co-localized with BDNF in L6 DRG, and prevention of the endogenous Akt activity in the L6 DRG by inhibition of phosphoinositide 3-kinase (PI3K) with a potent inhibitor LY294002 reversed cystitis-induced BDNF up-regulation. Further study showed that application of NGF to the nerve terminals of the ganglion-nerve two-compartmented preparation enhanced BDNF expression in the DRG neuronal soma; which was reduced by pre-treatment of the ganglia with the PI3K inhibitor LY294002 and wortmannin. These in vivo and in vitro experiments indicated that NGF played an important role in the activation of Akt and subsequent up-regulation of BDNF in the sensory neurons in visceral inflammation such as cystitis. PMID:24303055

  2. Activation of PI3K/Akt signaling in rostral ventrolateral medulla impairs brain stem cardiovascular regulation that underpins circulatory depression during mevinphos intoxication.

    PubMed

    Tsai, Ching-Yi; Chang, Alice Y W; Chan, Julie Y H; Chan, Samuel H H

    2014-03-01

    As the most widely used pesticides in the globe, the organophosphate compounds are understandably linked with the highest incidence of suicidal poisoning. Whereas the elicited toxicity is often associated with circulatory depression, the underlying mechanisms require further delineation. Employing the pesticide mevinphos as our experimental tool, we evaluated the hypothesis that transcriptional upregulation of nitric oxide synthase II (NOS II) by NF-κB on activation of the PI3K/Akt cascade in the rostral ventrolateral medulla (RVLM), the brain stem site that maintains blood pressure and sympathetic vasomotor tone, underpins the circulatory depressive effects of organophosphate poisons. Microinjection of mevinphos (10 nmol) bilaterally into the RVLM of anesthetized Sprague-Dawley rats induced a progressive hypotension that was accompanied sequentially by an increase (Phase I) and a decrease (Phase II) of an experimental index for the baroreflex-mediated sympathetic vasomotor tone. There were also progressive augmentations in PI3K or Akt enzyme activity and phosphorylation of p85 or Akt(Thr308) subunit in the RVLM that were causally related to an increase in NF-κB transcription activity and elevation in NOS II or peroxynitrite expression. Loss-of-function manipulations of PI3K or Akt in the RVLM significantly antagonized the reduced baroreflex-mediated sympathetic vasomotor tone and hypotension during Phase II mevinphos intoxication, and blunted the increase in NF-κB/NOS II/peroxynitrite signaling. We conclude that activation of the PI3K/Akt cascade, leading to upregulation of NF-κB/NOS II/peroxynitrite signaling in the RVLM, elicits impairment of brain stem cardiovascular regulation that underpins circulatory depression during mevinphos intoxication.

  3. pGlcNAc Nanofiber Treatment of Cutaneous Wounds Stimulate Increased Tensile Strength and Reduced Scarring via Activation of Akt1

    PubMed Central

    Lindner, Haley Buff; Felmly, Lloyd McPherson; Demcheva, Marina; Seth, Arun; Norris, Russell; Bradshaw, Amy D.; Vournakis, John; Muise-Helmericks, Robin C.

    2015-01-01

    Treatment of cutaneous wounds with poly-N-acetyl-glucosamine containing nanofibers (pGlcNAc), a novel polysaccharide material derived from a marine diatom, results in increased wound closure, antibacterial activities and innate immune responses. We have shown that Akt1 plays a central role in the regulation of these activities. Here, we show that pGlcNAc treatment of cutaneous wounds results in a smaller scar that has increased tensile strength and elasticity. pGlcNAc treated wounds exhibit decreased collagen content, increased collagen organization and decreased myofibroblast content. A fibrin gel assay was used to assess the regulation of fibroblast alignment in vitro. In this assay, fibrin lattice is formed with two pins that provide focal points upon which the gel can exert force as the cells align from pole to pole. pGlcNAc stimulation of embedded fibroblasts results in cellular alignment as compared to untreated controls, by a process that is Akt1 dependent. We show that Akt1 is required in vivo for the pGlcNAc-induced increased tensile strength and elasticity. Taken together, our findings suggest that pGlcNAc nanofibers stimulate an Akt1 dependent pathway that results in the proper alignment of fibroblasts, decreased scarring, and increased tensile strength during cutaneous wound healing. PMID:25955155

  4. The cleaved FAS ligand activates the Na+/H+ exchanger NHE1 through Akt/ROCK1 to stimulate cell motility

    PubMed Central

    Monet, Michael; Poët, Mallorie; Tauzin, Sébastien; Fouqué, Amélie; Cophignon, Auréa; Lagadic-Gossmann, Dominique; Vacher, Pierre; Legembre, Patrick; Counillon, Laurent

    2016-01-01

    Transmembrane CD95L (Fas ligand) can be cleaved to release a promigratory soluble ligand, cl-CD95L, which can contribute to chronic inflammation and cancer cell dissemination. The motility signaling pathway elicited by cl-CD95L remains poorly defined. Here, we show that in the presence of cl-CD95L, CD95 activates the Akt and RhoA signaling pathways, which together orchestrate an allosteric activation of the Na+/H+ exchanger NHE1. Pharmacologic inhibition of Akt or ROCK1 independently blocks the cl-CD95L-induced migration. Confirming these pharmacologic data, disruption of the Akt and ROCK1 phosphorylation sites on NHE1 decreases cell migration in cells exposed to cl-CD95L. Together, these findings demonstrate that NHE1 is a novel molecular actor in the CD95 signaling pathway that drives the cl-CD95L-induced cell migration through both the Akt and RhoA signaling pathways. PMID:27302366

  5. Renal cortical hexokinase and pentose phosphate pathway activation through the EGFR/Akt signaling pathway in endotoxin-induced acute kidney injury.

    PubMed

    Smith, Joshua A; Stallons, L Jay; Schnellmann, Rick G

    2014-08-15

    While disruption of energy production is an important contributor to renal injury, metabolic alterations in sepsis-induced AKI remain understudied. We assessed changes in renal cortical glycolytic metabolism in a mouse model of sepsis-induced AKI. A specific and rapid increase in hexokinase (HK) activity (∼2-fold) was observed 3 h after LPS exposure and maintained up to 18 h, in association with a decline in renal function as measured by blood urea nitrogen (BUN). LPS-induced HK activation occurred independently of HK isoform expression or mitochondrial localization. No other changes in glycolytic enzymes were observed. LPS-mediated HK activation was not sufficient to increase glycolytic flux as indicated by reduced or unchanged pyruvate and lactate levels in the renal cortex. LPS-induced HK activation was associated with increased glucose-6-phosphate dehydrogenase activity but not glycogen production. Mechanistically, LPS-induced HK activation was attenuated by pharmacological inhibitors of the EGF receptor (EGFR) and Akt, indicating that EGFR/phosphatidylinositol 3-kinase/Akt signaling is responsible. Our findings reveal LPS rapidly increases renal cortical HK activity in an EGFR- and Akt-dependent manner and that HK activation is linked to increased pentose phosphate pathway activity.

  6. Impact of the PI3-kinase/Akt pathway on ITAM and hemITAM receptors: haemostasis, platelet activation and antithrombotic therapy.

    PubMed

    Moroi, Alyssa J; Watson, Steve P

    2015-04-01

    Phosphoinositide 3-kinases (PI3Ks) are a family of lipid kinases that are activated in response to various stimulants, and they regulate many processes including inflammation; the stress response; gene transcription; and cell proliferation, differentiation, and death. Increasing reports have shown that the PI3Ks and their downstream effector Akt are activated by several platelet receptors that regulate platelet activation and haemostasis. Platelets express two immunoreceptor tyrosine based activation motif (ITAM) receptors, collagen receptor glycoprotein VI (GPVI) and Fcγ receptor IIA (FcγRIIA), which are characterized by two YxxL sequences separated by 6-12 amino acids. Activation of an ITAM receptor initiates a reaction cascade via its YxxL sequence in which signaling molecules such as spleen tyrosine kinase (Syk), linker for activation of T cells (LAT) and phospholipase C γ2 (PLCγ2) become activated, leading to platelet activation. Platelets also express another receptor, C-type lectin 2 (CLEC-2), which has a single YxxL sequence, so it is appropriately called a hemITAM receptor. ITAM receptors and the hemITAM receptor share many signaling features. Here we will summarize our current knowledge about how the PI3K/Akt pathway regulates (hem)ITAM receptor-mediated platelet activation and haemostasis and discuss the possible benefits of targeting PI3K/Akt as an antithrombotic therapy.

  7. Acetaldehyde Induces Cytotoxicity of SH-SY5Y Cells via Inhibition of Akt Activation and Induction of Oxidative Stress

    PubMed Central

    Yan, Tingting; Zhao, Yan; Zhang, Xia

    2016-01-01

    Excessive alcohol consumption can lead to brain tissue damage and cognitive dysfunction. It has been shown that heavy drinking is associated with an earlier onset of neurodegenerative diseases such as Alzheimer's disease. Acetaldehyde, the most toxic metabolite of ethanol, is speculated to mediate the brain tissue damage and cognitive dysfunction induced by the chronic excessive consumption of alcohol. However, the exact mechanisms by which acetaldehyde induces neurotoxicity are not totally understood. In this study, we investigated the cytotoxic effects of acetaldehyde in SH-SY5Y cells and found that acetaldehyde induced apoptosis of SH-SY5Y cells by downregulating the expression of antiapoptotic Bcl-2 and Bcl-xL and upregulating the expression of proapoptotic Bax. Acetaldehyde treatment led to a significant decrease in the levels of activated Akt and cyclic AMP-responsive element binding protein (CREB). In addition, acetaldehyde induced the activation of p38 mitogen-activated protein kinase (MAPK) while inhibiting the activation of extracellular signal-regulated kinases (ERKs, p44/p42MAPK). Meanwhile, acetaldehyde treatment caused an increase in the production of reactive oxygen species and elevated the oxidative stress in SH-SY5Y cells. Therefore, acetaldehyde induces cytotoxicity of SH-SY5Y cells via promotion of apoptotic signaling, inhibition of cell survival pathway, and induction of oxidative stress. PMID:26649137

  8. Acetaldehyde Induces Cytotoxicity of SH-SY5Y Cells via Inhibition of Akt Activation and Induction of Oxidative Stress.

    PubMed

    Yan, Tingting; Zhao, Yan; Zhang, Xia

    2016-01-01

    Excessive alcohol consumption can lead to brain tissue damage and cognitive dysfunction. It has been shown that heavy drinking is associated with an earlier onset of neurodegenerative diseases such as Alzheimer's disease. Acetaldehyde, the most toxic metabolite of ethanol, is speculated to mediate the brain tissue damage and cognitive dysfunction induced by the chronic excessive consumption of alcohol. However, the exact mechanisms by which acetaldehyde induces neurotoxicity are not totally understood. In this study, we investigated the cytotoxic effects of acetaldehyde in SH-SY5Y cells and found that acetaldehyde induced apoptosis of SH-SY5Y cells by downregulating the expression of antiapoptotic Bcl-2 and Bcl-xL and upregulating the expression of proapoptotic Bax. Acetaldehyde treatment led to a significant decrease in the levels of activated Akt and cyclic AMP-responsive element binding protein (CREB). In addition, acetaldehyde induced the activation of p38 mitogen-activated protein kinase (MAPK) while inhibiting the activation of extracellular signal-regulated kinases (ERKs, p44/p42MAPK). Meanwhile, acetaldehyde treatment caused an increase in the production of reactive oxygen species and elevated the oxidative stress in SH-SY5Y cells. Therefore, acetaldehyde induces cytotoxicity of SH-SY5Y cells via promotion of apoptotic signaling, inhibition of cell survival pathway, and induction of oxidative stress.

  9. Reduced enzyme activity and starch level in an induced mutant of chloroplast phosphoglucose isomerase

    SciTech Connect

    Jones, T.W.; Gottlieb, L.D.; Pichersky, E.

    1986-06-01

    Ethyl methane sulfonate treatment was used to induce a mutation in the nuclear gene encoding the chloroplast isozyme of phosphoglucose isomerase in Clarkia xantiana. The mutation, which proved allelic to wild type activity, was backcrossed to wild type for five generations so that the two could be compared in a near isogenic background. An immunological analysis showed that the mutant, when homozygous, reduced the activity of the isozyme by about 50%. In contrast to wild type, the mutant showed little change in leaf starch level over a diurnal period or following a 72-hour continuous light treatment. By the end of the diurnal light period, the mutant accumulated only about 60% as much starch as wild type. However, mutant leaves had an increased sucrose level presumably because photosynthate was directly exported from the chloroplasts. The mutant also exhibited reduced leaf weight. These changes in metabolism and growth suggest that the wild type level of plastid phosphoglucose isomerase activity is necessary to achieve wild type carbohydrate status.

  10. Isolation and characterization of a starchless mutant of Arabidopsis thaliana (L. ) Heynh lacking ADPglucose pyrophosphorylase activity

    SciTech Connect

    Lin, Tsanpiao; Caspar, T.; Somerville, C.; Preiss, J. )

    1988-04-01

    A mutant of Arabidopsis thaliana lacking ADPglucose pyrophosphorylase activity (EC 2.7.7.27) was isolated (from a mutagenized population of plants) by screening for the absence of leaf starch. The mutant grows as vigorously as the wild type in continuous light but more slowly than the wild type in a 12 hours light/12 hours dark photoperiod. Genetic analysis showed that the deficiency of both starch and ADPglucose pyrophosphorylase activity were attributable to a single, nuclear, recessive mutation at a locus designated adg1. The absence of starch in the mutant demonstrates that starch synthesis in the chloroplast is entirely dependent on a pathway involving ADPglucose pyrophosphorylase. Analysis of leaf extracts by two-dimensional polyacrylamide gel electrophoresis followed by Western blotting experiments using antibodies specific for spinach ADPglucose pyrophosphorylase showed that two proteins, present in the wild type, were absent from the mutant. The heterozygous F{sub 1} progeny of a cross between the mutant and wild type had a specific activity of ADP-glucose pyrophosphorylase indistinguishable from the wild type. These observations suggest that the mutation in the adg1 gene in TL25 might affect a regulatory locus.

  11. Role of PECAM-1 in the shear-stress-induced activation of Akt and the endothelial nitric oxide synthase (eNOS) in endothelial cells.

    PubMed

    Fleming, Ingrid; Fisslthaler, Beate; Dixit, Madhulika; Busse, Rudi

    2005-09-15

    The application of fluid shear stress to endothelial cells elicits the formation of nitric oxide (NO) and phosphorylation of the endothelial NO synthase (eNOS). Shear stress also elicits the enhanced tyrosine phosphorylation of endothelial proteins, especially of those situated in the vicinity of cell-cell contacts. Since a major constituent of these endothelial cell-cell contacts is the platelet endothelial cell adhesion molecule-1 (PECAM-1) we assessed the role of PECAM-1 in the activation of eNOS. In human endothelial cells, shear stress induced the tyrosine phosphorylation of PECAM-1 and enhanced the association of PECAM-1 with eNOS. Endothelial cell stimulation with shear stress elicited the phosphorylation of Akt and eNOS as well as of the AMP-activated protein kinase (AMPK). While the shear-stress-induced tyrosine phosphorylation of PECAM-1 as well as the serine phosphorylation of Akt and eNOS were abolished by the pre-treatment of cells with the tyrosine kinase inhibitor PP1 the phosphorylation of AMPK was unaffected. Down-regulation of PECAM-1 using a siRNA approach attenuated the shear-stress-induced phosphorylation of Akt and eNOS, as well as the shear-stress-induced accumulation of cyclic GMP levels while the shear-stress-induced phosphorylation of AMPK remained intact. A comparable attenuation of Akt and eNOS (but not AMPK) phosphorylation and NO production was also observed in endothelial cells generated from PECAM-1-deficient mice. These data indicate that the shear-stress-induced activation of Akt and eNOS in endothelial cells is modulated by the tyrosine phosphorylation of PECAM-1 whereas the shear-stress-induced phosphorylation of AMPK is controlled by an alternative signaling pathway. PMID:16118242

  12. MiR-20a Induces Cell Radioresistance by Activating the PTEN/PI3K/Akt Signaling Pathway in Hepatocellular Carcinoma

    SciTech Connect

    Zhang, Yuqin; Zheng, Lin; Ding, Yi; Li, Qi; Wang, Rong; Liu, Tongxin; Sun, Quanquan; Yang, Hua; Peng, Shunli; Wang, Wei; Chen, Longhua

    2015-08-01

    Purpose: To investigate the role of miR-20a in hepatocellular carcinoma (HCC) cell radioresistance, which may reveal potential strategies to improve treatment. Methods and Materials: The expression of miR-20a and PTEN were detected in HCC cell lines and paired primary tissues by quantitative real-time polymerase chain reaction. Cell radiation combined with colony formation assays was administrated to discover the effect of miR-20a on radiosensitivity. Bioinformatics prediction and luciferase assay were used to identify the target of miR-20a. The phosphatidylinositol 3-kinase inhibitor LY294002 was used to inhibit phosphorylation of Akt, to verify whether miR-20a affects HCC cell radioresistance through activating the PTEN/PI3K/Akt pathway. Results: MiR-20a levels were increased in HCC cell lines and tissues, whereas PTEN was inversely correlated with it. Overexpression of miR-20a in Bel-7402 and SMMC-7721 cells enhances their resistance to the effect of ionizing radiation, and the inhibition of miR-20a in HCCLM3 and QGY-7701 cells sensitizes them to it. PTEN was identified as a direct functional target of miR-20a for the induction of radioresistance. Overexpression of miR-20a activated the PTEN/PI3K/Akt signaling pathway. Additionally, the kinase inhibitor LY294002 could reverse the effect of miR-20a–induced radioresistance. Conclusion: MiR-20a induces HCC cell radioresistance by activating the PTEN/PI3K/Akt pathway, which suggests that miR-20a/PTEN/PI3K/Akt might represent a target of investigation for developing effective therapeutic strategies against HCC.

  13. Runx2 activates PI3K/Akt signaling via mTORC2 regulation in invasive breast cancer cells

    PubMed Central

    2014-01-01

    Introduction The Runt-related transcription factor Runx2 is critical for skeletal development but is also aberrantly expressed in breast cancers, and promotes cell growth and invasion. A de-regulated serine/threonine kinase Akt signaling pathway is implicated in mammary carcinogenesis and cell survival; however, the mechanisms underlying Runx2 role in survival of invasive breast cancer cells are still unclear. Methods The phenotypic analysis of Runx2 function in cell survival was performed by gene silencing and flow cytometric analysis in highly invasive MDA-MB-231 and SUM-159-PT mammary epithelial cell lines. The expression analysis of Runx2 and pAkt (serine 473) proteins in metastatic breast cancer specimens was performed by immunohistochemistry. The mRNA and protein levels of kinases and phosphatases functional in Akt signaling were determined by real-time PCR and Western blotting, while DNA-protein interaction was studied by chromatin immunoprecipitation assays. Results The high Runx2 levels in invasive mammary epithelial cell lines promoted cell survival in Akt phosphorylation (pAkt-serine 473) dependent manner. The analysis of kinases and phosphatases associated with pAkt regulation revealed that Runx2 promotes pAkt levels via mammalian target of rapamycin complex-2 (mTORC2). The recruitment of Runx2 on mTOR promoter coupled with Runx2-dependent expression of mTORC2 component Rictor defined Runx2 function in pAkt-mediated survival of invasive breast cancer cells. Conclusions Our results identified a novel mechanism of Runx2 regulatory crosstalk in Akt signaling that could have important consequences in targeting invasive breast cancer-associated cell survival. PMID:24479521

  14. Ribosome display for selection of active dihydrofolate reductase mutants using immobilized methotrexate on agarose beads.

    PubMed

    Takahashi, Fumio; Ebihara, Takashi; Mie, Masayasu; Yanagida, Yasuko; Endo, Yaeta; Kobatake, Eiry; Aizawa, Masuo

    2002-03-01

    Ribosome display was applied to the selection of an enzyme. As a model, we selected and amplified the dihydrofolate reductase (DHFR) gene by ribosome display utilizing a wheat germ cell-free protein synthesis system based on binding affinity to its substrate analog, methotrexate, immobilized on agarose beads. After three rounds of selection, the DHFR gene could be effectively selected and preferentially amplified from a small proportion in a mixture also containing competitive genes. Active enzymes were expressed and amplified and by sequence analysis, four mutants of DHFR were identified. These mutants showed as much activity as the wild-type enzyme.

  15. Activation of protein kinase B (PKB/Akt) and risk of lung cancer among rural women in India who cook with biomass fuel

    SciTech Connect

    Roychoudhury, Sanghita; Mondal, Nandan Kumar; Mukherjee, Sayali; Dutta, Anindita; Siddique, Shabana; Ray, Manas Ranjan

    2012-02-15

    The impact of indoor air pollution (IAP) from biomass fuel burning on the risk of carcinogenesis in the airways has been investigated in 187 pre-menopausal women (median age 34 years) from eastern India who cooked exclusively with biomass and 155 age-matched control women from same locality who cooked with cleaner fuel liquefied petroleum gas. Compared with control, Papanicolau-stained sputum samples showed 3-times higher prevalence of metaplasia and 7-times higher prevalence of dysplasia in airway epithelial cell (AEC) of biomass users. Immunocytochemistry showed up-regulation of phosphorylated Akt (p-Akt{sup ser473} and p-Akt{sup thr308}) proteins in AEC of biomass users, especially in metaplastic and dysplastic cells. Compared with LPG users, biomass-using women showed marked rise in reactive oxygen species (ROS) generation and depletion of antioxidant enzyme, superoxide dismutase (SOD) indicating oxidative stress. There were 2–5 times more particulate pollutants (PM{sub 10} and PM{sub 2.5}), 72% more nitrogen dioxide and 4-times more particulate-laden benzo(a)pyrene, but no change in sulfur dioxide in indoor air of biomass-using households, and high performance liquid chromatography estimated 6-fold rise in the concentration of benzene metabolite trans,trans-muconic acid (t,t-MA) in urine of biomass users. Metaplasia and dysplasia, p-Akt expression and ROS generation were positively associated with PM and t,t-MA levels. It appears that cumulative exposure to biomass smoke increases the risk of lung carcinogenesis via oxidative stress-mediated activation of Akt signal transduction pathway. -- Highlights: ► Carcinogenesis in airway cells was examined in biomass and LPG using women. ► Metaplasia and dysplasia of epithelial cells were more prevalent in biomass users. ► Change in airway cytology was associated with oxidative stress and Akt activation. ► Biomass users had greater exposure to respirable PM, B(a)P and benzene. ► Cooking with biomass

  16. Motexafin gadolinium modulates levels of phosphorylated Akt and synergizes with inhibitors of Akt phosphorylation.

    PubMed

    Ramos, Jason; Sirisawad, Mint; Miller, Richard; Naumovski, Louie

    2006-05-01

    Motexafin gadolinium (MGd, Xcytrin) is a tumor-selective expanded porphyrin that targets oxidative stress-related proteins. MGd treatment of the follicular lymphoma-derived cell line HF-1 resulted in growth suppression and apoptosis whereas MGd treatment of the Burkitt's lymphoma-derived cell line Ramos resulted in growth suppression but not apoptosis. Because phosphorylation status of Akt/protein kinase B is regulated by oxidative stress, we monitored total and phosphorylated Akt (pAkt) in MGd-treated HF-1 and Ramos cells. Levels of pAkt increased within 30 minutes after MGd treatment of HF-1 but after 4 hours began to show a progressive decline to below baseline levels before cells underwent apoptosis. In MGd-treated Ramos cells, pAkt increased approximately 2-fold within 4 hours and remained persistently elevated. Because pAkt activates survival pathways, we determined if MGd-induced cell death could be enhanced by inhibiting phosphorylation of Akt. The addition of specific inhibitors of Akt phosphorylation (Akt inhibitor 1 or SH-5) reduced pAkt levels in MGd-treated HF-1 and Ramos cells and synergistically enhanced MGd-induced cell death. MGd was also evaluated in combination with celecoxib, an inhibitor of Akt phosphorylation, or docetaxel, a microtubule inhibitor that can decrease Akt phosphorylation. The combination of MGd/celecoxib or MGd/docetaxel resulted in decreased Akt phosphorylation and in synergistic cytotoxicity compared with either agent alone. These data point to a potential protective role for pAkt in MGd-induced apoptosis and suggest that MGd activity may be enhanced by combining it with agents that inhibit Akt phosphorylation.

  17. The Phosphoinositide-3-Kinase–Akt Signaling Pathway Is Important for Staphylococcus aureus Internalization by Endothelial Cells ▿

    PubMed Central

    Oviedo-Boyso, Javier; Cortés-Vieyra, Ricarda; Huante-Mendoza, Alejandro; Yu, Hong B.; Valdez-Alarcón, Juan J.; Bravo-Patiño, Alejandro; Cajero-Juárez, Marcos; Finlay, B. Brett; Baizabal-Aguirre, Víctor M.

    2011-01-01

    Internalization of Staphylococcus aureus in bovine endothelial cells (BEC) is increased by tumor necrosis factor alpha stimulation and NF-κB activation. Because the phosphoinositide-3-kinase (PI3K)–Akt signaling pathway also modulates NF-κB activity, we considered whether the internalization of S. aureus by BEC is associated with the activity of PI3K and Akt. We found a time- and multiplicity of infection-dependent phosphorylation of Akt on Ser473 in BEC infected with S. aureus. This phosphorylation was inhibited by LY294002 (LY), indicating the participation of PI3K. Inhibition of either PI3K with LY or wortmannin, or Akt with SH-5, strongly reduced the internalization of S. aureus. Transfection of BEC with a dominant-negative form of the Akt gene significantly decreased S. aureus internalization, whereas transfection with the constitutively active mutant increased the number of internalized bacterium. Inhibition of PDK1 activity with OSU-03012 did not affect the level of S. aureus internalization, demonstrating that phosphorylation of Akt on Thr308 is not important for this process. Compared to the untreated control, the adherence of S. aureus to the surface of BEC was unaltered when cells were transfected or incubated with the pharmacological inhibitors. Furthermore, Akt activation by internalized S. aureus triggered a time-dependent phosphorylation of glycogen synthase kinase-3α (GSK-3α) on Ser21 and GSK-3β on Ser9 that was partially inhibited with SH-5. Finally, treatment of BEC with LY prior to S. aureus infection inhibited the NF-κB p65 subunit phosphorylation on Ser536, indicating the involvement of PI3K. These results suggest that PI3K-Akt activity is important for the internalization of S. aureus and phosphorylation of GSK-3α, GSK-3β, and NF-κB. PMID:21844240

  18. Bis(hinokitiolato)zinc complex ([Zn(hkt)2]) activates Akt/protein kinase B independent of insulin signal transduction.

    PubMed

    Naito, Yuki; Yoshikawa, Yutaka; Masuda, Kazufumi; Yasui, Hiroyuki

    2016-07-01

    Since many Zn complexes have been developed to enhance the insulin-like activity and increase the exposure and residence of Zn in the animal body, these complexes are recognized as one of the new candidates with action mechanism different from existing anti-diabetic drugs. However, the molecular mechanism by which Zn complexes exert an anti-DM effect is unknown. Therefore, we evaluated the activity of Zn complexes, especially related to the phosphorylation of insulin signaling pathway components. We focused on the insulin-like effects of the bis(hinokitiolato)zinc complex, [Zn(hkt)2], using 3T3-L1 adipocytes. [Zn(hkt)2] was taken up by cells and induced Akt phosphorylation in a time-dependent manner. Additionally, it showed inhibitory activity against PTP1B and PTEN, which are major negative regulators of insulin signaling. It did not promote the phosphorylation of IR (insulin receptor)-β or IRS (insulin receptor substrate)-1 by itself, but in combination with insulin, it enhanced the phosphorylation of IRβ. We conclude that [Zn(hkt)2] has effects on the proteins of insulin signaling pathway without insulin receptor mediation, and [Zn(hkt)2] promotes insulin function and shows the anti-DM effects. Thus, [Zn(hkt)2] may be the basis for improved DM treatments.

  19. Bis(hinokitiolato)zinc complex ([Zn(hkt)2]) activates Akt/protein kinase B independent of insulin signal transduction.

    PubMed

    Naito, Yuki; Yoshikawa, Yutaka; Masuda, Kazufumi; Yasui, Hiroyuki

    2016-07-01

    Since many Zn complexes have been developed to enhance the insulin-like activity and increase the exposure and residence of Zn in the animal body, these complexes are recognized as one of the new candidates with action mechanism different from existing anti-diabetic drugs. However, the molecular mechanism by which Zn complexes exert an anti-DM effect is unknown. Therefore, we evaluated the activity of Zn complexes, especially related to the phosphorylation of insulin signaling pathway components. We focused on the insulin-like effects of the bis(hinokitiolato)zinc complex, [Zn(hkt)2], using 3T3-L1 adipocytes. [Zn(hkt)2] was taken up by cells and induced Akt phosphorylation in a time-dependent manner. Additionally, it showed inhibitory activity against PTP1B and PTEN, which are major negative regulators of insulin signaling. It did not promote the phosphorylation of IR (insulin receptor)-β or IRS (insulin receptor substrate)-1 by itself, but in combination with insulin, it enhanced the phosphorylation of IRβ. We conclude that [Zn(hkt)2] has effects on the proteins of insulin signaling pathway without insulin receptor mediation, and [Zn(hkt)2] promotes insulin function and shows the anti-DM effects. Thus, [Zn(hkt)2] may be the basis for improved DM treatments. PMID:27251140

  20. SKF-96365 activates cytoprotective autophagy to delay apoptosis in colorectal cancer cells through inhibition of the calcium/CaMKIIγ/AKT-mediated pathway.

    PubMed

    Jing, Zhao; Sui, Xinbing; Yao, Junlin; Xie, Jiansheng; Jiang, Liming; Zhou, Yubin; Pan, Hongming; Han, Weidong

    2016-03-28

    Store-operated Ca(2+) entry (SOCE) inhibitors are emerging as an attractive new generation of anti-cancer drugs. Here, we report that SKF-96365, an SOCE inhibitor, exhibits potent anti-neoplastic activity by inducing cell-cycle arrest and apoptosis in colorectal cancer cells. In the meantime, SKF-96365 also induces cytoprotective autophagy to delay apoptosis by preventing the release of cytochrome c (cyt c) from the mitochondria into the cytoplasm. Mechanistically, SKF-96365 treatment inhibited the calcium/calmodulin-dependent protein kinase IIγ (CaMKIIγ)/AKT signaling cascade in vitro and in vivo. Overexpression of CaMKIIγ or AKT abolished the effects of SKF-96365 on cancer cells, suggesting a critical role of the CaMKIIγ/AKT signaling pathway in SFK-96365-induced biological effects. Moreover, Hydroxychloroquine (HCQ), an FDA-approved drug used to inhibit autophagy, could significantly augment the anti-cancer effect of SFK-96365 in a mouse xenograft model. To our best knowledge, this is the first report to demonstrate that calcium/CaMKIIγ/AKT signaling can regulate apoptosis and autophagy simultaneously in cancer cells, and the combination of the SOCE inhibitor SKF-96365 with autophagy inhibitors represents a promising strategy for treating patients with colorectal cancer. PMID:26803057

  1. SKF-96365 activates cytoprotective autophagy to delay apoptosis in colorectal cancer cells through inhibition of the calcium/CaMKIIγ/AKT-mediated pathway.

    PubMed

    Jing, Zhao; Sui, Xinbing; Yao, Junlin; Xie, Jiansheng; Jiang, Liming; Zhou, Yubin; Pan, Hongming; Han, Weidong

    2016-03-28

    Store-operated Ca(2+) entry (SOCE) inhibitors are emerging as an attractive new generation of anti-cancer drugs. Here, we report that SKF-96365, an SOCE inhibitor, exhibits potent anti-neoplastic activity by inducing cell-cycle arrest and apoptosis in colorectal cancer cells. In the meantime, SKF-96365 also induces cytoprotective autophagy to delay apoptosis by preventing the release of cytochrome c (cyt c) from the mitochondria into the cytoplasm. Mechanistically, SKF-96365 treatment inhibited the calcium/calmodulin-dependent protein kinase IIγ (CaMKIIγ)/AKT signaling cascade in vitro and in vivo. Overexpression of CaMKIIγ or AKT abolished the effects of SKF-96365 on cancer cells, suggesting a critical role of the CaMKIIγ/AKT signaling pathway in SFK-96365-induced biological effects. Moreover, Hydroxychloroquine (HCQ), an FDA-approved drug used to inhibit autophagy, could significantly augment the anti-cancer effect of SFK-96365 in a mouse xenograft model. To our best knowledge, this is the first report to demonstrate that calcium/CaMKIIγ/AKT signaling can regulate apoptosis and autophagy simultaneously in cancer cells, and the combination of the SOCE inhibitor SKF-96365 with autophagy inhibitors represents a promising strategy for treating patients with colorectal cancer.

  2. The nuclear protein Sam68 is redistributed to the cytoplasm and is involved in PI3K/Akt activation during EV71 infection.

    PubMed

    Zhang, Hua; Cong, Haolong; Song, Lei; Tien, Po

    2014-02-13

    Nuclear proteins can be triggered to be redistributed to the cytoplasm to assist with EV71 virus replication. This process is frequently involved in cellular signal transduction upon virus infection. In this study, we have demonstrated that a new nuclear protein, 68-kDa Src-associated in mitosis protein (Sam68), was translocated to the cytoplasm and was co-localized with EV71 during virus infection. Confocal microscopy and subcellular fractionation assay confirmed that virus 3C protease triggered the redistribution of Sam68 to the cytoplasm. Knockdown of Sam68 expression using ShRNA significantly inhibited virus replication, suggesting that Sam68 may be a host factor involved in EV71 life cycle. In addition, EV71-induced Akt phosphorylation involved a PI3K-dependent mechanism. Sam68 is known to be an upstream regulator of PI3K and our immunoprecipitation studies confirmed that Sam68 interacted directly with the p85 regulatory subunit of PI3K and mediated PI3K/Akt activation during EV71 infection. On the contrary, silencing of Sam68 dramatically abrogated Akt phosphorylation. These data, plus the fact that Sam68 is known to be a signaling adaptor protein, indicated that Sam68 is a signal molecule with a functional role in the PI3K/Akt signal pathway during EV71 infection.

  3. Effects of different intensities of physical exercise on insulin sensitivity and protein kinase B/Akt activity in skeletal muscle of obese mice

    PubMed Central

    Marinho, Rodolfo; de Moura, Leandro Pereira; Rodrigues, Bárbara de Almeida; Pauli, Luciana Santos Souza; da Silva, Adelino Sanchez Ramos; Ropelle, Eloize Cristina Chiarreotto; de Souza, Claudio Teodoro; Cintra, Dennys Esper Corrêa; Ropelle, Eduardo Rochete; Pauli, José Rodrigo

    2014-01-01

    ABSTRACT Objective: To investigate the effects of different intensities of acute exercise on insulin sensitivity and protein kinase B/Akt activity in skeletal muscle of obese mice. Methods: Swiss mice were randomly divided into four groups, and fed either a standard diet (control group) or high fat diet (obese sedentary group and obese exercise group 1 and 2) for 12 weeks. Two different exercise protocols were used: swimming for 1 hour with or without an overload of 5% body weight. The insulin tolerance test was performed to estimate whole-body sensitivity. Western blot technique was used to determine protein levels of protein kinase B/Akt and phosphorylation by protein Kinase B/Akt in mice skeletal muscle. Results: A single bout of exercise inhibited the high fat diet-induced insulin resistance. There was increase in phosphorylation by protein kinase B/Akt serine, improve in insulin signaling and reduce of fasting glucose in mice that swam for 1 hour without overload and mice that swan for 1 hour with overload of 5%. However, no significant differences were seen between exercised groups. Conclusion: Regardless of intensity, aerobic exercise was able to improve insulin sensitivity and phosphorylation by protein kinase B/Ak, and proved to be a good form of treatment and prevention of type 2 diabetes. PMID:24728251

  4. Neuroprotective effect of allicin against traumatic brain injury via Akt/endothelial nitric oxide synthase pathway-mediated anti-inflammatory and anti-oxidative activities.

    PubMed

    Chen, Wei; Qi, Jun; Feng, Feng; Wang, Mao-de; Bao, Gang; Wang, Tuo; Xiang, Mu; Xie, Wan-Fu

    2014-03-01

    Allicin, one of the main biologically active compounds derived from garlic, has been shown to exert various anti-oxidative and anti-inflammatory activities in in vitro and in vivo studies. Here, we sought to investigate the potential neuroprotective effects of allicin against traumatic brain injury (TBI) in rats. We found that allicin treatment (10 and 50mg/kg, not 1mg/kg) significantly reduced brain edema and motor functional deficits, as well as apoptotic neuronal cell death in injured cortex. These protective effects could be observed even if the administration was delayed to 4h after injury. Moreover, allicin treatment decreased the expression levels of MDA and protein carbonyl, preserved the endogenous antioxidant enzyme activities, and suppressed the expression of inflammatory cytokines. The results of Western blot analysis showed that allicin increased the phosphorylation of Akt and endothelial nitric oxide synthase (eNOS). Blocking Akt/eNOS pathway activation by specific inhibitor LY294002 (10μL, 10mmol/L) or L-NIO (0.5mg/kg) partly reversed the protective effects of allicin and its anti-inflammatory activities. The allicin induced anti-oxidative activity was partly prevented by LY294002, but not L-NIO. In summary, our data strongly suggested that allicin treatment at an appropriate dose can exert protective effect against TBI through Akt/eNOS pathway-mediated anti-inflammatory and anti-oxidative activities.

  5. Activation of the PI3K/mTOR/AKT Pathway and Survival in Solid Tumors: Systematic Review and Meta-Analysis

    PubMed Central

    Ocana, Alberto; Vera-Badillo, Francisco; Al-Mubarak, Mustafa; Templeton, Arnoud J.; Corrales-Sanchez, Verónica; Diez-Gonzalez, Laura; Cuenca-Lopez, María D.; Seruga, Bostjan; Pandiella, Atanasio; Amir, Eitan

    2014-01-01

    Background Aberrations in the phosphatidylinositol 3-kinase (PI3K)/mammalian target of rapamycin (mTOR)/AKT pathway are common in solid tumors. Numerous drugs have been developed to target different components of this pathway. However the prognostic value of these aberrations is unclear. Methods PubMed was searched for studies evaluating the association between activation of the PI3K/mTOR/AKT pathway (defined as PI3K mutation [PIK3CA], lack of phosphatase and tensin homolog [PTEN] expression by immunohistochemistry or western-blot or increased expression/activation of downstream components of the pathway by immunohistochemistry) with overall survival (OS) in solid tumors. Published data were extracted and computed into odds ratios (OR) for death at 5 years. Data were pooled using the Mantel-Haenszel random-effect model. Results Analysis included 17 studies. Activation of the PI3K/mTOR/AKT pathway was associated with significantly worse 5-year survival (OR:2.12, 95% confidence intervals 1.42–3.16, p<0.001). Loss of PTEN expression and increased expression/activation of downstream components were associated with worse survival. No association between PIK3CA mutations and survival was observed. Differences between methods for assessing activation of the PI3K/mTOR/AKT pathway were statistically significant (p = 0.04). There was no difference in the effect of up-regulation of the pathway on survival between different cancer sites (p = 0.13). Conclusion Activation of the PI3K/AKT/mTOR pathway, especially if measured by loss of PTEN expression or increased expression/activation of downstream components is associated with poor survival. PIK3CA mutational status is not associated with adverse outcome, challenging its value as a biomarker of patient outcome or as a stratification factor for patients treated with agents acting on the PI3K/AKT/mTOR pathway. PMID:24777052

  6. Phosphatidylinositol 3-kinase is an upstream regulator of the phosphodiesterase 3B pathway of leptin signalling that may not involve activation of Akt in the rat hypothalamus.

    PubMed

    Sahu, A; Koshinaka, K; Sahu, M

    2013-02-01

    Leptin, the product of the obese gene, regulates energy homeostasis by acting primarily at the level of the hypothalamus. Leptin action through its receptor involves various pathways, including the signal transducer and activator of transcription (STAT)3, phosphatidylinositol 3-kinase (PI3K), and phosphodiesterase 3B (PDE3B)-cAMP signalling in the central nervous system and peripheral tissues. In the hypothalamus, leptin stimulates STAT3 activation, and induces PI3K and PDE3B activities, among others. We have previously demonstrated that PDE3B activation in the hypothalamus is critical for transducing the anorectic and body weight reducing effects of leptin. Similarly, PI3K has been implicated to play a critical role in leptin signalling in the hypothalamus. Although, in the insulin signalling pathway, PI3K is known to be an upstream regulator of PDE3B in non-neuronal tissues, it is still unknown whether this is also the case for leptin signalling in the hypothalamus. To address this possibility, the effect of wortmannin, a specific PI3K inhibitor, was examined on leptin-induced PDE3B activity in the hypothalamus of male rats. Intracerebroventricular injection of leptin (4 μg) significantly increased PDE3B activity by two-fold in the hypothalamus as expected. However, previous administration of wortmannin completely reversed the stimulatory effect of leptin on PDE3B activity in the hypothalamus. To investigate whether leptin stimulates phospho (p)-Akt levels and that there might be a possible upstream regulator of PDE3B, we examined the effects of i.c.v. leptin on p-Akt levels in the hypothalamus and compared them with the known stimulatory effect of insulin on p-Akt. We observed that insulin increased p-Akt levels but leptin failed to do so, although it increased p-STAT3 levels, in the rat hypothalamus. Immunocytochemistry confirmed the biochemical findings in that leptin failed but insulin increased the number of p-Akt positive cells in various hypothalamic

  7. Tetramethylpyrazine induces SH-SY5Y cell differentiation toward the neuronal phenotype through activation of the PI3K/Akt/Sp1/TopoIIβ pathway.

    PubMed

    Yan, Yong-Xin; Zhao, Jun-Xia; Han, Shuo; Zhou, Na-Jing; Jia, Zhi-Qiang; Yao, Sheng-Jie; Cao, Cui-Li; Wang, Yan-Ling; Xu, Yan-Nan; Zhao, Juan; Yan, Yun-Li; Cui, Hui-Xian

    2015-12-01

    Tetramethylpyrazine (TMP) is an active compound extracted from the traditional Chinese medicinal herb Chuanxiong. Previously, we have shown that TMP induces human SH-SY5Y neuroblastoma cell differentiation toward the neuronal phenotype by targeting topoisomeraseIIβ (TopoIIβ), a protein implicated in neural development. In the present study, we aimed to elucidate whether the transcriptional factors specificity protein 1 (Sp1) and nuclear factor Y (NF-Y), in addition to the upstream signaling pathways ERK1/2 and PI3K/Akt, are involved in modulating TopoIIβ expression in the neuronal differentiation process. We demonstrated that SH-SY5Y cells treated with TMP (80μM) terminally differentiated into neurons, characterized by increased neuronal markers, tubulin βIII and microtubule associated protein 2 (MAP2), and increased neurite outgrowth, with no negative effect on cell survival. TMP also increased the expression of TopoIIβ, which was accompanied by increased expression of Sp1 in the differentiated neuron-like cells, whereas NF-Y protein levels remained unchanged following the differentiation progression. We also found that the phosphorylation level of Akt, but not ERK1/2, was significantly increased as a result of TMP stimulation. Furthermore, as established by chromatin immunoprecipitation (ChIP) assay, activation of the PI3K/Akt pathway increased Sp1 binding to the promoter of the TopoIIβ gene. Blockage of PI3K/Akt was shown to lead to subsequent inhibition of TopoIIβ expression and neuronal differentiation. Collectively, the results indicate that the PI3K/Akt/Sp1/TopoIIβ signaling pathway is necessary for TMP-induced neuronal differentiation. Our findings offer mechanistic insights into understanding the upstream regulation of TopoIIβ in neuronal differentiation, and suggest potential applications of TMP both in neuroscience research and clinical practice to treat relevant diseases of the nervous system. PMID:26518113

  8. Exogenous hydrogen sulfide exerts proliferation, anti-apoptosis, migration effects and accelerates cell cycle progression in multiple myeloma cells via activating the Akt pathway.

    PubMed

    Zheng, Dong; Chen, Ziang; Chen, Jingfu; Zhuang, Xiaomin; Feng, Jianqiang; Li, Juan

    2016-10-01

    Hydrogen sulfide (H2S), regarded as the third gaseous transmitter, mediates and induces various biological effects. The present study investigated the effects of H2S on multiple myeloma cell progression via amplifying the activation of Akt pathway in multiple myeloma cells. The level of H2S produced in multiple myeloma (MM) patients and healthy subjects was measured using enzyme-linked immunosorbent assay (ELISA). MM cells were treated with 500 µmol/l NaHS (a donor of H2S) for 24 h. The expression levels of phosphorylated-Akt (p-Akt), Bcl-2 and caspase-3 were measured by western blot assay. Cell viability was detected by Cell Counting Kit 8 (CCK-8). The cell cycle was analyzed by flow cytometry. Our results show that the concentration of H2S was higher in MM patients and that it increased in parallel with disease progression. Treating MM cells with 500 µmol/l NaHS for 24 h markedly increased the expression level of Bcl-2 and the activation of p-Akt, however, the expression level of caspase-3 was decreased, cell viability was increased, and cell cycle progression was accelerated in MM cells. NaHS also induced migration in MM cells in transwell migration assay. Furthermore, co-treatment of MM cells with 500 µmol/l NaHS and 50 µmol/l LY294002 for 24 h significantly overset these effects. In conclusion, our findings demonstrate that the Akt pathway contributes to NaHS-induced cell proliferation, migration and acceleration of cell cycle progression in MM cells. PMID:27513630

  9. Osseointegration of chitosan coated porous titanium alloy implant by reactive oxygen species-mediated activation of the PI3K/AKT pathway under diabetic conditions.

    PubMed

    Li, Xiang; Ma, Xiang-Yu; Feng, Ya-Fei; Ma, Zhen-Sheng; Wang, Jian; Ma, Tian-Cheng; Qi, Wei; Lei, Wei; Wang, Lin

    2015-01-01

    Chitosan coated porous titanium alloy implant (CTI) is demonstrated a promising approach to improve osseointegration capacity of pure porous titanium alloy implant (TI). Since chitosan has been demonstrated to exhibit antioxidant activity, we propose CTI may ameliorate the ROS overproduction, thus reverse the poor osseointegration under diabetic conditions, and investigate the underlying mechanisms. Primary rat osteoblasts incubated on the TI and the CTI were subjected to normal serum (NS), diabetic serum (DS), DS + NAC (a potent ROS inhibitor) and DS + LY294002 (a PI3K/AKT-specific inhibitor). In vivo study was performed on diabetic sheep implanted with TI or CTI into the bone defects on crista iliaca. Results showed that diabetes-induced ROS overproduction led to osteoblast dysfunction and apoptosis, concomitant with the inhibition of AKT in osteoblasts on the TI substrate. While CTI stimulated AKT phosphorylation through ROS attenuation, thus reversed osteoblast dysfunction evidenced by improved osteoblast adhesion, increased proliferation and ALP activity, and decreased cytotoxicity and apoptotic rate, which exerted same effect to NAC treatment on the TI. These effects were further confirmed by the improved osseointegration within the CTI in vivo evidenced by Micro-CT and histological examinations. In addition, the aforementioned promotive effects afforded by CTI were abolished by blocking PI3K/AKT pathway with addition of LY294002. These results demonstrate that the chitosan coating markedly ameliorates diabetes-induced impaired bio-performance of TI via ROS-mediated reactivation of PI3K/AKT pathway, which elicits a new surface functionalization strategy for better clinical performance of titanium implant in diabetic patients.

  10. Photoproduction of Hydrogen by Sulfur-Deprived Chlamydomonas reinhardtii Mutants with Impaired Photosystem II Photochemical Activity

    SciTech Connect

    Makarova, V. V.; Kosourov, S.; Krendeleva, T. E.; Semin, B. K.; Kukarskikh, G. P.; Rubin, A. B.; Sayre, R. T.; Ghirardi, M. L.; Seibert, M.

    2007-01-01

    Photoproduction of H2 was examined in a series of sulfur-deprived Chlamydomonas reinhardtii D1-R323 mutants with progressively impaired PSII photochemical activity. In the R323H, R323D, and R323E D1 mutants, replacement of arginine affects photosystem II (PSII) function, as demonstrated by progressive decreases in O2-evolving activity and loss of PSII photochemical activity. Significant changes in PSII activity were found when the arginine residue was replaced by negatively charged amino acid residues (R323D and R323E). However, the R323H (positively charged or neutral, depending on the ambient pH) mutant had minimal changes in PSII activity. The R323H, R323D, and R323E mutants and the pseudo-wild-type (pWt) with restored PSII function were used to study the effects of sulfur deprivation on H2-production activity. All of these mutants exhibited significant changes in the normal parameters associated with the H2-photoproduction process, such as a shorter aerobic phase, lower accumulation of starch, a prolonged anaerobic phase observed before the onset of H2-production, a shorter duration of H2-production, lower H2 yields compared to the pWt control, and slightly higher production of dark fermentation products such as acetate and formate. The more compromised the PSII photochemical activity, the more dramatic was the effect of sulfur deprivation on the H2-production process, which depends both on the presence of residual PSII activity and the amount of stored starch.

  11. Photoproduction of hydrogen by sulfur-deprived C. reinhardtii mutants with impaired photosystem II photochemical activity.

    PubMed

    Makarova, Valeria V; Kosourov, Sergey; Krendeleva, Tatiana E; Semin, Boris K; Kukarskikh, Galina P; Rubin, Andrei B; Sayre, Richard T; Ghirardi, Maria L; Seibert, Michael

    2007-10-01

    Photoproduction of H2 was examined in a series of sulfur-deprived Chlamydomonas reinhardtii D1-R323 mutants with progressively impaired PSII photochemical activity. In the R323H, R323D, and R323E D1 mutants, replacement of arginine affects photosystem II (PSII) function, as demonstrated by progressive decreases in O2-evolving activity and loss of PSII photochemical activity. Significant changes in PSII activity were found when the arginine residue was replaced by negatively charged amino acid residues (R323D and R323E). However, the R323H (positively charged or neutral, depending on the ambient pH) mutant had minimal changes in PSII activity. The R323H, R323D, and R323E mutants and the pseudo-wild-type (pWt) with restored PSII function were used to study the effects of sulfur deprivation on H2-production activity. All of these mutants exhibited significant changes in the normal parameters associated with the H2-photoproduction process, such as a shorter aerobic phase, lower accumulation of starch, a prolonged anaerobic phase observed before the onset of H2-production, a shorter duration of H2-production, lower H2 yields compared to the pWt control, and slightly higher production of dark fermentation products such as acetate and formate. The more compromised the PSII photochemical activity, the more dramatic was the effect of sulfur deprivation on the H2-production process, which depends both on the presence of residual PSII activity and the amount of stored starch. PMID:17701084

  12. Temperature-sensitive retinoid isomerase activity of RPE65 mutants associated with Leber Congenital Amaurosis

    PubMed Central

    Li, Songhua; Hu, Jane; Jin, Robin J.; Aiyar, Ashok; Jacobson, Samuel G.; Bok, Dean; Jin, Minghao

    2015-01-01

    RPE65 is a membrane-associated retinoid isomerase involved in the visual cycle responsible for sustaining vision. Many mutations in the human RPE65 gene are associated with distinct forms of retinal degenerative diseases. The pathogenic mechanisms for most of these mutations remain poorly understood. Here, we show that three Leber congenital amaurosis -associated RPE65 mutants (R91W, Y249C and R515W) undergo rapid proteasomal degradation mediated by the 26 S proteasome non-ATPase regulatory subunit 13 (PSMD13) in cultured human retinal pigment epithelium (RPE) cells. These mutant proteins formed cytosolic inclusion bodies or high molecular weight complexes via disulfide bonds. The mutations are mapped on non-active sites but severely reduced isomerase activity of RPE65. At 30°C, however, the enzymatic function and membrane-association of the mutant RPE65s are significantly rescued possibly due to proper folding. In addition, PSMD13 displayed a drastically decreased effect on degradation of the mutant proteins in the cells grown at 30°C. These results suggest that PSMD13 plays a critical role in regulating pathogenicity of the mutations and the molecular basis for the PSMD13-mediated rapid degradation and loss of function of the mutants is misfolding of RPE65. PMID:25752820

  13. Atorvastatin overcomes gefitinib resistance in KRAS mutant human non-small cell lung carcinoma cells.

    PubMed

    Chen, J; Bi, H; Hou, J; Zhang, X; Zhang, C; Yue, L; Wen, X; Liu, D; Shi, H; Yuan, J; Liu, J; Liu, B

    2013-09-26

    The exact influence of statins on gefitinib resistance in human non-small cell lung cancer (NSCLC) cells with KRAS mutation alone or KRAS/PIK3CA and KRAS/PTEN comutations remains unclear. This work found that transfection of mutant KRAS plasmids significantly suppressed the gefitinib cytotoxicity in Calu3 cells (wild-type KRAS). Gefitinib disrupted the Kras/PI3K and Kras/Raf complexes in Calu3 cells, whereas not in Calu3 KRAS mutant cells. These trends were corresponding to the expression of pAKT and pERK in gefitinib treatment. Atorvastatin (1 μM) plus gefitinib treatment inhibited proliferation, promoted cell apoptosis, and reduced the AKT activity in KRAS mutant NSCLC cells compared with gefitinib alone. Atorvastatin (5 μM) further enhanced the gefitinib cytotoxicity through concomitant inhibition of AKT and ERK activity. Atorvastatin could interrupt Kras/PI3K and Kras/Raf complexes, leading to suppression of AKT and ERK activity. Similar results were also obtained in comutant KRAS/PTEN or KRAS/PIK3CA NSCLC cells. Furthermore, mevalonate administration reversed the effects of atorvastatin on the Kras/Raf and Kras/PI3K complexes, as well as AKT and ERK activity in both A549 and Calu1 cells. The in vivo results were similar to those obtained in vitro. Therefore, mutant KRAS-mediated gefitinib insensitivity is mainly derived from failure to disrupt the Kras/Raf and Kras/PI3K complexes in KRAS mutant NSCLC cells. Atorvastatin overcomes gefitinib resistance in KRAS mutant NSCLC cells irrespective of PIK3CA and PTEN statuses through inhibition of HMG-CoA reductase-dependent disruption of the Kras/Raf and Kras/PI3K complexes.

  14. Constitutive asymmetric dimerization drives oncogenic activation of epidermal growth factor receptor carboxyl-terminal deletion mutants

    PubMed Central

    Park, Angela K.J.; Francis, Joshua M.; Park, Woong-Yang; Park, Joon-Oh; Cho, Jeonghee

    2015-01-01

    Genomic alterations targeting the Epidermal Growth Factor Receptor (EGFR) gene have been strongly associated with cancer pathogenesis. The clinical effectiveness of EGFR targeted therapies, including small molecules directed against the kinase domain such as gefitinib, erlotinib and afatinib, have been proven successful in treating non-small cell lung cancer patients with tumors harboring EGFR kinase domain mutations. Recent large-scale genomic studies in glioblastoma and lung cancer have identified an additional class of oncogenic mutations caused by the intragenic deletion of carboxy-terminal coding regions. Here, we report that combinations of exonic deletions of exon 25 to 28 lead to the oncogenic activation of EGF receptor in the absence of ligand and consequent cellular transformation, indicating a significant role of C-terminal domain in modulating EGFR activation. Furthermore, we show that the oncogenic activity of the resulting C-terminal deletion mutants are efficiently inhibited by EGFR-targeted drugs including erlotinib, afatinib, dacomitinib as well as cetuximab, expanding the therapeutic rationale of cancer genome-based EGFR targeted approaches. Finally, in vivo and in vitro preclinical studies demonstrate that constitutive asymmetric dimerization in mutant EGFR is a key mechanism for oncogenic activation and tumorigenesis by C-terminal deletion mutants. Therefore, our data provide compelling evidence for oncogenic activation of C-terminal deletion mutants at the molecular level and we propose that C-terminal deletion status of EGFR can be considered as a potential genomic marker for EGFR-targeted therapy. PMID:25826094

  15. Induction of metastatic potential by TrkB via activation of IL6/JAK2/STAT3 and PI3K/AKT signaling in breast cancer.

    PubMed

    Kim, Min Soo; Lee, Won Sung; Jeong, Joon; Kim, Seong-Jin; Jin, Wook

    2015-11-24

    In metastatic breast cancers, the acquisition of metastatic ability, which leads to clinically incurable disease and poor survival, has been associated with acquisition of epithelial-mesenchymal transition (EMT) program and self-renewing trait (CSCs) via activation of PI3K/AKT and IL6/JAK2/STAT3 signaling pathways. We found that TrkB is a key regulator of PI3K/AKT and JAK/STAT signal pathway-mediated tumor metastasis and EMT program. Here, we demonstrated that TrkB activates AKT by directly binding to c-Src, leading to increased proliferation. Also, TrkB increases Twist-1 and Twist-2 expression through activation of JAK2/STAT3 by inducing c-Src-JAK2 complex formation. Furthermore, TrkB in the absence of c-Src binds directly to JAK2 and inhibits SOCS3-mediated JAK2 degradation, resulting in increased total JAK2 and STAT3 levels, which subsequently leads to JAK2/STAT3 activation and Twist-1 upregulation. Additionally, activation of the JAK2/STAT3 pathway via induction of IL-6 secretion by TrkB enables induction of activation of the EMT program via induction of STAT3 nuclear translocation. These observations suggest that TrkB is a promising target for future intervention strategies to prevent tumor metastasis, EMT program and self-renewing trait in breast cancer. PMID:26515594

  16. Investigation of gene effects and epistatic interactions between Akt1 and neuregulin 1 in the regulation of behavioral phenotypes and social functions in genetic mouse models of schizophrenia

    PubMed Central

    Huang, Ching-Hsun; Pei, Ju-Chun; Luo, Da-Zhong; Chen, Ching; Chen, Yi-Wen; Lai, Wen-Sung

    2015-01-01

    Accumulating evidence from human genetic studies has suggested several functional candidate genes that might contribute to susceptibility to schizophrenia, including AKT1 and neuregulin 1 (NRG1). Recent findings also revealed that NRG1 stimulates the PI3-kinase/AKT signaling pathway, which might be involved in the functional outcomes of some schizophrenic patients. The aim of this study was to evaluate the effect of Akt1-deficiency and Nrg1-deficiency alone or in combination in the regulation of behavioral phenotypes, cognition, and social functions using genetically modified mice as a model. Male Akt1+/−, Nrg1+/−, and double mutant mice were bred and compared with their wild-type (WT) littermate controls. In Experiment 1, general physical examination revealed that all mutant mice displayed a normal profile of body weight during development and a normal brain activity with microPET scan. In Experiment 2, no significant genotypic differences were found in our basic behavioral phenotyping, including locomotion, anxiety-like behavior, and sensorimotor gating function. However, both Nrg1+/− and double mutant mice exhibited impaired episodic-like memory. Double mutant mice also had impaired sociability. In Experiment 3, a synergistic epistasis between Akt1 and Nrg1 was further confirmed in double mutant mice in that they had impaired social interaction compared to the other 3 groups, especially encountering with a novel male or an ovariectomized female. Double mutant and Nrg1+/− mice also emitted fewer female urine-induced ultrasonic vocalization calls. Collectively, our results indicate that double deficiency of Akt1 and Nrg1 can result in the impairment of social cognitive functions, which might be pertinent to the pathogenesis of schizophrenia-related social cognition. PMID:25688191

  17. Dyrk1A, a serine/threonine kinase, is involved in ERK and Akt activation in the brain of hyperhomocysteinemic mice.

    PubMed

    Abekhoukh, Sabiha; Planque, Chris; Ripoll, Clémentine; Urbaniak, Paulina; Paul, Jean-Louis; Delabar, Jean-Maurice; Janel, Nathalie

    2013-02-01

    Hyperhomocysteinemia due to cystathionine beta synthase (CBS) deficiency is associated with diverse brain disease. Whereas the biological actions linking hyperhomocysteinemia to the cognitive dysfunction are not well understood, we tried to establish relationships between hyperhomocysteinemia and alterations of signaling pathways. In the brain of CBS-deficient mice, a murine model of hyperhomocysteinemia, we previously found an activation of extracellular signal-regulated kinase (ERK) pathway and an increase of Dyrk1A, a serine/threonine kinase involved in diverse functions ranging from development and growth to apoptosis. We then investigated the relationship between Dyrk1A and the signaling pathways initiated by receptor tyrosine kinases (RTK), the ERK and PI3K/Akt pathways. We found a significant increase of phospho-ERK, phospho-MEK, and phospho-Akt in the brain of CBS-deficient and Dyrk1a-overexpressing mice. This increase was abolished when CBS-deficient and Dyrk1A-transgenic mice were treated with harmine, an inhibitor of Dyrk1A kinase activity, which emphasizes the role of Dyrk1A activity on ERK and Akt activation. Sprouty 2 protein level, a negative feedback loop modulator that limits the intensity and duration of RTK activation, is decreased in the brain of CBS-deficient mice, but not in the brain of Dyrk1A transgenic mice. Furthermore, a reduced Dyrk1A and Grb2 binding on sprouty 2 and an increased interaction of Dyrk1A with Grb2 were found in the brain of Dyrk1A transgenic mice. The consequence of Dyrk1A overexpression on RTK activation seems to be a decreased interaction of sprouty 2/Grb2. These observations demonstrate ERK and Akt activation induced by Dyrk1A in the brain of hyperhomocysteinemic mice and open new perspectives to understand the basis of the cognitive defects in hyperhomocysteinemia. PMID:22923366

  18. Ciliary transport regulates PDGF-AA/αα signaling via elevated mammalian target of rapamycin signaling and diminished PP2A activity.

    PubMed

    Umberger, Nicole L; Caspary, Tamara

    2015-01-15

    Primary cilia are built and maintained by intraflagellar transport (IFT), whereby the two IFT complexes, IFTA and IFTB, carry cargo via kinesin and dynein motors for anterograde and retrograde transport, respectively. Many signaling pathways, including platelet- derived growth factor (PDGF)-AA/αα, are linked to primary cilia. Active PDGF-AA/αα signaling results in phosphorylation of Akt at two residues: P-Akt(T308) and P-Akt(S473), and previous work showed decreased P-Akt(S473) in response to PDGF-AA upon anterograde transport disruption. In this study, we investigated PDGF-AA/αα signaling via P-Akt(T308) and P-Akt(S473) in distinct ciliary transport mutants. We found increased Akt phosphorylation in the absence of PDGF-AA stimulation, which we show is due to impaired dephosphorylation resulting from diminished PP2A activity toward P-Akt(T308). Anterograde transport mutants display low platelet-derived growth factor receptor (PDGFR)α levels, whereas retrograde mutants exhibit normal PDGFRα levels. Despite this, neither shows an increase in P-Akt(S473) or P-Akt(T308) upon PDGF-AA stimulation. Because mammalian target of rapamycin complex 1 (mTORC1) signaling is increased in ciliary transport mutant cells and mTOR signaling inhibits PDGFRα levels, we demonstrate that inhibition of mTORC1 rescues PDGFRα levels as well as PDGF-AA-dependent phosphorylation of Akt(S473) and Akt(T308) in ciliary transport mutant MEFs. Taken together, our data indicate that the regulation of mTORC1 signaling and PP2A activity by ciliary transport plays key roles in PDGF-AA/αα signaling.

  19. Decreased phospho-Akt signaling in a mouse model of total parenteral nutrition: a potential mechanism for the development of intestinal mucosal atrophy

    PubMed Central

    Feng, Yongjia; McDunn, Jonathan E.

    2010-01-01

    Total parenteral nutrition (TPN) leads to a decline in phosphatidylinositol 3-kinase (PI3K)/phospho-Akt (p-Akt) activity, affecting downstream signaling, reducing epithelial cell (EC) proliferation, and contributing to intestinal mucosal atrophy. We hypothesized that promoting Akt activity would prevent these changes. We used a novel Akt-activating peptide, TCL1 (a head-to-tail dimer of the Akt-binding domain of T-cell lymphoma-1), or an inactive mutant sequence TCL1G conjugated to a transactivator of transcription peptide sequence to promote intracellular uptake. Four groups of mice were studied, enteral nutrition group (control), control mice given a functioning TCL1 (control + TCL1), TPN mice given TCL1G (control peptide, TPN + TCL1G); and TPN mice given TCL1. TPN mice given TCL1G showed a significant decrease in jejunal EC p-Akt (Ser473 and Thr308) abundance, whereas TPN + TCL1 mice showed increased p-Akt (Ser473) abundance. Phosphorylation of β-catenin and glycogen synthase kinase-3β (downstream targets of Akt signaling) were also decreased in the TPN + TCL1G group and completely prevented in the TPN + TCL1 group. Use of TCL1 nearly completely prevented the decline in EC proliferation seen in the TPN + TCL1G group, as well as partly returned EC apoptosis levels close to controls. The mammalian target of rapamycin pathway demonstrated a similar reduction in activity in the TPN + TCL1G group that was significantly prevented in the TPN + TCL1 group. These results support a significant loss of PI3K/p-Akt signaling upon replacing enteral nutrition with TPN, and prevention of this loss demonstrates the key importance of PI3K/p-Akt signaling in maintaining gut integrity including EC proliferation and reduction in apoptosis. PMID:20299605

  20. Receptor Interacting Protein 3 Suppresses Vascular Smooth Muscle Cell Growth by Inhibition of the Phosphoinositide 3-Kinase-Akt Axis*

    PubMed Central

    Li, Qian; Li, Geng; Lan, Xiaomei; Zheng, Ming; Chen, Kuang-Hueih; Cao, Chun-Mei; Xiao, Rui-Ping

    2010-01-01

    Proliferation of vascular smooth muscle cells (VSMCs) is a primary mechanism underlying cardiovascular proliferative disorders. Phosphoinositide 3-kinase (PI3K)-Akt (or protein kinase B) axis has been assigned at the center of pathways that regulate cell proliferation. Here we demonstrate that enhanced PI3K-Akt signaling by mitogenic stimulation or arterial injury profoundly elevates expression of receptor interacting protein 3 (RIP3) in primary cultured rat VSMCs and in vivo and that the up-regulation of RIP3 leads to VSMC growth arrest and apoptosis via inhibiting the PI3K-Akt signaling pathway, thereby alleviating balloon injury-induced neointimal formation. Specifically, mitogenic stimulation with platelet-derived growth factor-BB or angiotensin II leads to a profound increase in RIP3 expression, which is abolished by inhibition of PI3K or Akt, and increased PI3K-Akt signaling by expression of a constitutively active PI3K mutant also elevates RIP3 expression. Importantly, adenoviral overexpression of RIP3 not only triggers apoptosis but also causes cell cycle arrest at G1/G0 phases that is associated with suppressed Akt activation. In sharp contrast, RIP3 gene silencing enhances serum- and platelet-derived growth factor-induced cell proliferation and Akt activation. In vivo adenoviral gene delivery of rat RIP3 (rRIP3) increased apoptosis and reduced VSMC proliferation, thus, effectively alleviating balloon injury-induced neointimal formation. The growth-suppressive and pro-apoptotic effects are independent of rRIP3 Ser/Thr kinase activity, because overexpression of a kinase-inactive mutant of rRIP3, similar to its wild type, is sufficient to induce growth arrest and apoptosis. These findings reveal a novel growth-suppressive action of RIP3, marking RIP3 as an important factor to prevent excessive mitogenic stimulation- or injury-induced vascular smooth muscle cells hyperplasia. PMID:20042608

  1. Blunted Akt/FOXO signalling and activation of genes controlling atrophy and fuel use in statin myopathy.

    PubMed

    Mallinson, Joanne E; Constantin-Teodosiu, Dumitru; Sidaway, James; Westwood, F Russell; Greenhaff, Paul L

    2009-01-15

    Statins are used clinically for cholesterol reduction, but statin therapy is associated with myopathic changes through a poorly defined mechanism. We used an in vivo model of statin myopathy to determine whether statins up-regulate genes associated with proteasomal- and lysosomal-mediated proteolysis and whether PDK gene expression is simultaneously up-regulated leading to the impairment of muscle carbohydrate oxidation. Animals were dosed daily with 80 mg kg(-1) day(-1) simvastatin for 4 (n = 6) and 12 days (n = 5), 88 mg kg(-1) day(-1) simvastatin for 12 days (n = 4), or vehicle (0.5% w/v hydroxypropyl-methylcellulose and 0.1% w/v polysorbate 80; Control, n = 6) for 12 days by oral gavage. We found, in biceps femoris muscle, decreased Akt(Ser473), FOXO1(Ser253) and FOXO3a(Ser253) phosphorylation in the cytosol (P < 0.05, P < 0.05, P < 0.001, respectively) and decreased phosphorylation of FOXO1 in the nucleus after 12 days simvastatin when compared to Control (P < 0.05). This was paralleled by a marked increase in the transcription of downstream targets of FOXO, i.e. MAFbx (P < 0.001), MuRF-1 (P < 0.001), cathepsin-L (P < 0.05), PDK2 (P < 0.05) and PDK4 (P < 0.05). These changes were accompanied by increased PPARalpha (P < 0.05), TNFalpha (P < 0.01), IL6 (P < 0.01), Mt1A (P < 0.01) mRNA and increased muscle glycogen (P < 0.05) compared to Control. RhoA activity decreased after 4 days simvastatin (P < 0.05); however, activity was no different from Control after 12 days. Simvastatin down-regulated PI3k/Akt signalling, independently of RhoA, and up-regulated FOXO transcription factors and downstream gene targets known to be implicated in proteasomal- and lysosomal-mediated muscle proteolysis, carbohydrate oxidation, oxidative stress and inflammation in an in vivo model of statin-induced myopathy. These changes occurred in the main before evidence of extensive myopathy or a decline in the muscle protein to DNA ratio. PMID:19001041

  2. Protective effect of rutin on LPS-induced acute lung injury via down-regulation of MIP-2 expression and MMP-9 activation through inhibition of Akt phosphorylation.

    PubMed

    Chen, Wen-Ying; Huang, Yi-Chun; Yang, Ming-Ling; Lee, Chien-Ying; Chen, Chun-Jung; Yeh, Chung-Hsin; Pan, Pin-Ho; Horng, Chi-Ting; Kuo, Wu-Hsien; Kuan, Yu-Hsiang

    2014-10-01

    Lipopolysaccharide (LPS), also called endotoxin, is the important pathogen of acute lung injury (ALI), which is a clinical syndrome that still lacks effective therapeutic medicine. Rutin belongs to vitamin P and possesses various beneficial effects. In this study, we investigate the potential protective effects and the mechanisms of rutin on LPS-induced ALI. Pre-administration with rutin inhibited LPS-induced arterial blood gas exchange and neutrophils infiltration in the lungs. LPS-induced expression of macrophage inflammatory protein (MIP)-2 and activation of matrix metalloproteinase (MMP)-9 were suppressed by rutin. In addition, the inhibitory concentration of rutin on phosphorylation of Akt was similar as MIP-2 expression and MMP-9 activation. In conclusion, rutin is a potential protective agent for ALI via suppressing the blood gas exchange and neutrophil infiltration. The mechanism of rutin is down-regulation of MIP-2 expression and MMP-9 activation through inhibition of Akt phosphorylation.

  3. WISP-1 attenuates p53-mediated apoptosis in response to DNA damage through activation of the Akt kinase.

    PubMed

    Su, Fei; Overholtzer, Michael; Besser, Daniel; Levine, Arnold J

    2002-01-01

    WISP-1 (Wnt-1-induced secreted protein) was identified as an oncogene regulated by the Wnt-1-beta-catenin pathway. WISP-1 belongs to the CCN family of growth factors, which are cysteine-rich, heparin-binding, secreted proteins associated with the extracellular matrix, and can interact with cellular integrins. Expression of WISP-1 in some cells results in transformation and tumorigenesis. Here it is shown that WISP-1 can activate the antiapoptotic Akt/PKB signaling pathway. It also is demonstrated that WISP-1 can prevent cells from undergoing apoptosis following DNA damage through inhibition of the mitochondrial release of cytochrome c and up-regulation of antiapoptotic Bcl-X(L). Furthermore, the results show that WISP-1 protects cells from p53-dependent cell death, but not Fas-ligand activated cell death, suggesting that there may be cross talk between the tumor suppressor protein p53 and WISP-1 signaling pathways. WISP-1 acts to block cell death at a late stage in the p53-mediated apoptosis pathway.

  4. A natural diarylheptanoid promotes neuronal differentiation via activating ERK and PI3K-Akt dependent pathways.

    PubMed

    Tang, G; Dong, X; Huang, X; Huang, X-J; Liu, H; Wang, Y; Ye, W-C; Shi, L

    2015-09-10

    Neuronal differentiation is a critical developmental process that determines accurate synaptic connection and circuit wiring. A wide variety of naturally occurring compounds have been shown as promising drug leads for the generation and differentiation of neurons. Here we report that a diarylheptanoid from the plant Alpinia officinarum, 7-(4-hydroxyphenyl)-1-phenyl-4E-hepten-3-one (Cpd 1), exhibited potent activities in neuronal differentiation and neurite outgrowth. Cpd 1 induced differentiation of neuroblastoma Neuro-2a cells into a neuron-like morphology, and accelerated the establishment of axon-dendrite polarization of cultured hippocampal neurons. Moreover, Cpd 1 promoted neurite extension in both Neuro-2a cells and neurons. We showed that the effects of Cpd 1 on neuronal differentiation and neurite growth were specifically dependent on the activation of extracellular signal-regulated kinases (ERKs) and phosphoinositide 3-kinase (PI3K)-Akt signaling pathways. Importantly, intraperitoneal administration of Cpd 1 promoted the differentiation of new-born progenitor cells into mature neurons in the adult hippocampal dentate gyrus. Collectively, this study identifies a naturally occurring diarylheptanoid with beneficial effects on neuronal differentiation and neurite outgrowth in vitro and in vivo.

  5. Heterogeneity between triple negative breast cancer cells due to differential activation of Wnt and PI3K/AKT pathways.

    PubMed

    Martínez-Revollar, Gabriela; Garay, Erika; Martin-Tapia, Dolores; Nava, Porfirio; Huerta, Miriam; Lopez-Bayghen, Esther; Meraz-Cruz, Noemí; Segovia, José; González-Mariscal, Lorenza

    2015-11-15

    The lack of a successful treatment for triple-negative breast cancer demands the study of the heterogeneity of cells that constitute these tumors. With this aim, two clones from triple negative breast MDA-MB-231 cancer cells were isolated: One with fibroblast-like appearance (F) and another with semi-epithelial (SE) morphology. Cells of the F clone have a higher migration and tumorigenesis capacity than SE cells, suggesting that these cells are in a more advanced stage of epithelial to mesenchymal transformation. In agreement, F cells have a diminished expression of the tight junction proteins claudins 1 and 4, and an increased content of β-catenin. The latter is due to an augmented activity of the canonical Wnt route and of the EGFR/PI3K/mTORC2/AKT pathway favoring the cytoplasmic accumulation of β-catenin and its transcriptional activity. In addition, F cells display increased phosphorylation of β-catenin at Tyr654 by Src. These changes favor in F cells, the over-expression of Snail that promotes EMT. Finally, we observe that both F and SE cells display markers of cancer stem cells, which are more abundant in the F clone.

  6. Honokiol and magnolol stimulate glucose uptake by activating PI3K-dependent Akt in L6 myotubes.

    PubMed

    Choi, Sun-Sil; Cha, Byung-Yoon; Lee, Young-Sil; Yonezawa, Takayuki; Teruya, Toshiaki; Nagai, Kazuo; Woo, Je-Tae

    2012-01-01

    Honokiol and magnolol, ingredients of Magnolia officinalis, which is used in traditional Chinese and Japanese medicines, have been reported to have antioxidant, anticancer, and antiangiogenic effects. Effects of these compounds on glucose metabolism in adipocytes have also been reported. However, their effects on skeletal muscle glucose uptake and the underlying molecular mechanisms are still unknown. Here, we investigated the direct effects and signaling pathways activated by honokiol and magnolol in skeletal muscle cells using L6 myotubes. We found that honokiol and magnolol dose-dependently acutely stimulated glucose uptake without synergistic effects of combined administration in L6 myotubes. Treatment with honokiol and magnolol also stimulated glucose transporter-4 translocation to the cell surface. Honokiol- and magnolol-stimulated glucose uptake was blocked by the phosphatidylinositol-3 kinase inhibitor, wortmannin. Both honokiol and magnolol stimulated Akt phosphorylation, a key element in the insulin signaling pathway, which was completely inhibited by wortmannin. These results suggest that honokiol and magnolol might have beneficial effects on glucose metabolism by activating the insulin signaling pathway. PMID:22674833

  7. Dentin phosphophoryn in the matrix activates AKT and mTOR signaling pathway to promote preodontoblast survival and differentiation

    PubMed Central

    Eapen, Asha; George, Anne

    2015-01-01

    Dentin phosphophoryn (DPP) is an extracellular matrix protein synthesized by odontoblasts. It is highly acidic and the phosphorylated protein possesses a strong affinity for calcium ions. Therefore, DPP in the extracellular matrix can promote hydroxyapatite nucleation and can regulate the size of the growing crystal. Besides its calcium binding property, DPP can initiate signaling functions from the ECM (Extracellular matrix). The signals that promote the cytodifferentiation of preodontoblasts to fully functional odontoblasts are not known. In this study, we demonstrate that preodontoblasts on a DPP matrix, generates mechanical and biochemical signals. This is initiated by the ligation of the integrins with the RGD containing DPP. The downstream biochemical response observed is the activation of the AKT(protein kinase B) and mTOR (mammalian target of rapamycin) signaling pathways leading to the activation of the transcription factor NF-κB (Nuclear factor κB). Terminal differentiation of the preodontoblasts was assessed by identifying phosphate and calcium deposits in the matrix using von Kossa and Alizarin red staining respectively. Identifying the signaling pathways initiated by DPP in the dentin matrix would help in devising strategies for dentin tissue engineering. PMID:26300786

  8. A senescence secretory switch mediated by PI3K/AKT/mTOR activation controls chemoprotective endothelial secretory responses.

    PubMed

    Bent, Eric H; Gilbert, Luke A; Hemann, Michael T

    2016-08-15

    Cancer therapy targets malignant cells that are surrounded by a diverse complement of nonmalignant stromal cells. Therapy-induced damage of normal cells can alter the tumor microenvironment, causing cellular senescence and activating cancer-promoting inflammation. However, how these damage responses are regulated (both induced and resolved) to preserve tissue homeostasis and prevent chronic inflammation is poorly understood. Here, we detail an acute chemotherapy-induced secretory response that is self-limiting in vitro and in vivo despite the induction of cellular senescence. We used tissue-specific knockout mice to demonstrate that endothelial production of the proinflammatory cytokine IL-6 promotes chemoresistance and show that the chemotherapeutic doxorubicin induces acute IL-6 release through reactive oxygen species-mediated p38 activation in vitro. Doxorubicin causes endothelial senescence but, surprisingly, without a typical senescence secretory response. We found that endothelial cells repress senescence-associated inflammation through the down-regulation of PI3K/AKT/mTOR signaling and that reactivation of this pathway restores senescence-associated inflammation. Thus, we describe a mechanism by which damage-associated paracrine secretory responses are restrained to preserve tissue homeostasis and prevent chronic inflammation. PMID:27566778

  9. Olesoxime suppresses calpain activation and mutant huntingtin fragmentation in the BACHD rat.

    PubMed

    Clemens, Laura E; Weber, Jonasz J; Wlodkowski, Tanja T; Yu-Taeger, Libo; Michaud, Magali; Calaminus, Carsten; Eckert, Schamim H; Gaca, Janett; Weiss, Andreas; Magg, Janine C D; Jansson, Erik K H; Eckert, Gunter P; Pichler, Bernd J; Bordet, Thierry; Pruss, Rebecca M; Riess, Olaf; Nguyen, Huu P

    2015-12-01

    Huntington's disease is a fatal human neurodegenerative disorder caused by a CAG repeat expansion in the HTT gene, which translates into a mutant huntingtin protein. A key event in the molecular pathogenesis of Huntington's disease is the proteolytic cleavage of mutant huntingtin, leading to the accumulation of toxic protein fragments. Mutant huntingtin cleavage has been linked to the overactivation of proteases due to mitochondrial dysfunction and calcium derangements. Here, we investigated the therapeutic potential of olesoxime, a mitochondria-targeting, neuroprotective compound, in the BACHD rat model of Huntington's disease. BACHD rats were treated with olesoxime via the food for 12 months. In vivo analysis covered motor impairments, cognitive deficits, mood disturbances and brain atrophy. Ex vivo analyses addressed olesoxime's effect on mutant huntingtin aggregation and cleavage, as well as brain mitochondria function. Olesoxime improved cognitive and psychiatric phenotypes, and ameliorated cortical thinning in the BACHD rat. The treatment reduced cerebral mutant huntingtin aggregates and nuclear accumulation. Further analysis revealed a cortex-specific overactivation of calpain in untreated BACHD rats. Treated BACHD rats instead showed significantly reduced levels of mutant huntingtin fragments due to the suppression of calpain-mediated cleavage. In addition, olesoxime reduced the amount of mutant huntingtin fragments associated with mitochondria, restored a respiration deficit, and enhanced the expression of fusion and outer-membrane transport proteins. In conclusion, we discovered the calpain proteolytic system, a key player in Huntington's disease and other neurodegenerative disorders, as a target of olesoxime. Our findings suggest that olesoxime exerts its beneficial effects by improving mitochondrial function, which results in reduced calpain activation. The observed alleviation of behavioural and neuropathological phenotypes encourages further

  10. Mechanism of the Anticoagulant Activity of Thrombin Mutant W215A/E217A

    SciTech Connect

    Gandhi, Prafull S.; Page, Michael J.; Chen, Zhiwei; Bush-Pelc, Leslie; Di Cera, Enrico

    2009-09-15

    The thrombin mutant W215A/E217A (WE) is a potent anticoagulant both in vitro and in vivo. Previous x-ray structural studies have shown that WE assumes a partially collapsed conformation that is similar to the inactive E* form, which explains its drastically reduced activity toward substrate. Whether this collapsed conformation is genuine, rather than the result of crystal packing or the mutation introduced in the critical 215-217 {beta}-strand, and whether binding of thrombomodulin to exosite I can allosterically shift the E* form to the active E form to restore activity toward protein C are issues of considerable mechanistic importance to improve the design of an anticoagulant thrombin mutant for therapeutic applications. Here we present four crystal structures of WE in the human and murine forms that confirm the collapsed conformation reported previously under different experimental conditions and crystal packing. We also present structures of human and murine WE bound to exosite I with a fragment of the platelet receptor PAR1, which is unable to shift WE to the E form. These structural findings, along with kinetic and calorimetry data, indicate that WE is strongly stabilized in the E* form and explain why binding of ligands to exosite I has only a modest effect on the E*-E equilibrium for this mutant. The E* {yields} E transition requires the combined binding of thrombomodulin and protein C and restores activity of the mutant WE in the anticoagulant pathway.

  11. A cytochrome c mutant with high electron transfer and antioxidant activities but devoid of apoptogenic effect.

    PubMed Central

    Abdullaev, Ziedulla Kh; Bodrova, Marina E; Chernyak, Boris V; Dolgikh, Dmitry A; Kluck, Ruth M; Pereverzev, Mikhail O; Arseniev, Alexander S; Efremov, Roman G; Kirpichnikov, Mikhail P; Mokhova, Elena N; Newmeyer, Donald D; Roder, Heinrich; Skulachev, Vladimir P

    2002-01-01

    A cytochrome c mutant lacking apoptogenic function but competent in electron transfer and antioxidant activities has been constructed. To this end, mutant species of horse and yeast cytochromes c with substitutions in the N-terminal alpha-helix or position 72 were obtained. It was found that yeast cytochrome c was much less effective than the horse protein in activating respiration of rat liver mitoplasts deficient in endogenous cytochrome c as well as in inhibition of H(2)O(2) production by the initial segment of the respiratory chain of intact rat heart mitochondria. The major role in the difference between the horse and yeast proteins was shown to be played by the amino acid residue in position 4 (glutamate in horse, and lysine in yeast; horse protein numbering). A mutant of the yeast cytochrome c containing K4E and some other "horse" modifications in the N-terminal alpha-helix, proved to be (i) much more active in electron transfer and antioxidant activity than the wild-type yeast cytochrome c and (ii), like the yeast cytochrome c, inactive in caspase stimulation, even if added in 400-fold excess compared with the horse protein. Thus this mutant seems to be a good candidate for knock-in studies of the role of cytochrome c-mediated apoptosis, in contrast with the horse K72R, K72G, K72L and K72A mutant cytochromes that at low concentrations were less active in apoptosis than the wild-type, but were quite active when the concentrations were increased by a factor of 2-12. PMID:11879204

  12. Activation of protein kinase B (PKB/Akt) and risk of lung cancer among rural women in India who cook with biomass fuel.

    PubMed

    Roychoudhury, Sanghita; Mondal, Nandan Kumar; Mukherjee, Sayali; Dutta, Anindita; Siddique, Shabana; Ray, Manas Ranjan

    2012-02-15

    The impact of indoor air pollution (IAP) from biomass fuel burning on the risk of carcinogenesis in the airways has been investigated in 187 pre-menopausal women (median age 34years) from eastern India who cooked exclusively with biomass and 155 age-matched control women from same locality who cooked with cleaner fuel liquefied petroleum gas. Compared with control, Papanicolau-stained sputum samples showed 3-times higher prevalence of metaplasia and 7-times higher prevalence of dysplasia in airway epithelial cell (AEC) of biomass users. Immunocytochemistry showed up-regulation of phosphorylated Akt (p-Akt(ser473) and p-Akt(thr308)) proteins in AEC of biomass users, especially in metaplastic and dysplastic cells. Compared with LPG users, biomass-using women showed marked rise in reactive oxygen species (ROS) generation and depletion of antioxidant enzyme, superoxide dismutase (SOD) indicating oxidative stress. There were 2-5 times more particulate pollutants (PM(10) and PM(2.5)), 72% more nitrogen dioxide and 4-times more particulate-laden benzo(a)pyrene, but no change in sulfur dioxide in indoor air of biomass-using households, and high performance liquid chromatography estimated 6-fold rise in the concentration of benzene metabolite trans,trans-muconic acid (t,t-MA) in urine of biomass users. Metaplasia and dysplasia, p-Akt expression and ROS generation were positively associated with PM and t,t-MA levels. It appears that cumulative exposure to biomass smoke increases the risk of lung carcinogenesis via oxidative stress-mediated activation of Akt signal transduction pathway.

  13. Novel pathway in Bcr-Abl signal transduction involves Akt-independent, PLC-gamma1-driven activation of mTOR/p70S6-kinase pathway.

    PubMed

    Markova, B; Albers, C; Breitenbuecher, F; Melo, J V; Brümmendorf, T H; Heidel, F; Lipka, D; Duyster, J; Huber, C; Fischer, T

    2010-02-01

    In chronic myeloid leukemia, activation of the phosphoinositide 3-kinase (PI3K)/Akt pathway is crucial for survival and proliferation of leukemic cells. Essential downstream molecules involve mammalian target of rapamycin (mTOR) and S6-kinase. Here, we present a comprehensive analysis of the molecular events involved in activation of these key signaling pathways. We provide evidence for a previously unrecognized phospholipase C-gamma1 (PLC-gamma1)-controlled mechanism of mTOR/p70S6-kinase activation, which operates in parallel to the classical Akt-dependent machinery. Short-term imatinib treatment of Bcr-Abl-positive cells caused dephosphorylation of p70S6-K and S6-protein without inactivation of Akt. Suppression of Akt activity alone did not affect phosphorylation of p70-S6K and S6. These results suggested the existence of an alternative mechanism for mTOR/p70S6-K activation. In Bcr-Abl-expressing cells, we detected strong PLC-gamma1 activation, which was suppressed by imatinib. Pharmacological inhibition and siRNA knockdown of PLC-gamma1 blocked p70S6-K and S6 phosphorylation. By inhibiting the Ca-signaling, CaMK and PKCs we demonstrated participation of these molecules in the pathway. Suppression of PLC-gamma1 led to inhibition of cell proliferation and enhanced apoptosis. The novel pathway proved to be essential for survival and proliferation of leukemic cells and almost complete cell death was observed upon combined PLC-gamma1 and Bcr-Abl inhibition. The pivotal role of PLC-gamma1 was further confirmed in a mouse leukemogenesis model.

  14. Filamin A Expression Negatively Regulates Sphingosine-1-Phosphate-Induced NF-κB Activation in Melanoma Cells by Inhibition of Akt Signaling.

    PubMed

    Campos, Ludmila S; Rodriguez, Yamila I; Leopoldino, Andreia M; Hait, Nitai C; Lopez Bergami, Pablo; Castro, Melina G; Sanchez, Emilse S; Maceyka, Michael; Spiegel, Sarah; Alvarez, Sergio E

    2015-01-01

    Sphingosine-1-phosphate (S1P) is a bioactive lipid mediator that regulates many processes in inflammation and cancer. S1P is a ligand for five G-protein-coupled receptors, S1PR1 to -5, and also has important intracellular actions. Previously, we showed that intracellular S1P is involved in tumor necrosis factor alpha (TNF)-induced NF-κB activation in melanoma cell lines that express filamin A (FLNA). Here, we show that extracellular S1P activates NF-κB only in melanoma cells that lack FLNA. In these cells, S1P, but not TNF, promotes IκB kinase (IKK) and p65 phosphorylation, IκBα degradation, p65 nuclear translocation, and NF-κB reporter activity. NF-κB activation induced by S1P was mediated via S1PR1 and S1PR2. Exogenous S1P enhanced the phosphorylation of protein kinase Cδ (PKCδ), and its downregulation reduced S1P-induced the phosphorylation of IKK and p65. In addition, silencing of Bcl10 also inhibited S1P-induced IKK phosphorylation. Surprisingly, S1P reduced Akt activation in melanoma cells that express FLNA, whereas in the absence of FLNA, high phosphorylation levels of Akt were maintained, enabling S1P-mediated NF-κB signaling. In accord, inhibition of Akt suppressed S1P-mediated IKK and p65 phosphorylation and degradation of IκBα. Hence, these results support a negative role of FLNA in S1P-mediated NF-κB activation in melanoma cells through modulation of Akt. PMID:26552704

  15. Filamin A Expression Negatively Regulates Sphingosine-1-Phosphate-Induced NF-κB Activation in Melanoma Cells by Inhibition of Akt Signaling

    PubMed Central

    Campos, Ludmila S.; Rodriguez, Yamila I.; Leopoldino, Andreia M.; Hait, Nitai C.; Lopez Bergami, Pablo; Castro, Melina G.; Sanchez, Emilse S.; Maceyka, Michael

    2015-01-01

    Sphingosine-1-phosphate (S1P) is a bioactive lipid mediator that regulates many processes in inflammation and cancer. S1P is a ligand for five G-protein-coupled receptors, S1PR1 to -5, and also has important intracellular actions. Previously, we showed that intracellular S1P is involved in tumor necrosis factor alpha (TNF)-induced NF-κB activation in melanoma cell lines that express filamin A (FLNA). Here, we show that extracellular S1P activates NF-κB only in melanoma cells that lack FLNA. In these cells, S1P, but not TNF, promotes IκB kinase (IKK) and p65 phosphorylation, IκBα degradation, p65 nuclear translocation, and NF-κB reporter activity. NF-κB activation induced by S1P was mediated via S1PR1 and S1PR2. Exogenous S1P enhanced the phosphorylation of protein kinase Cδ (PKCδ), and its downregulation reduced S1P-induced the phosphorylation of IKK and p65. In addition, silencing of Bcl10 also inhibited S1P-induced IKK phosphorylation. Surprisingly, S1P reduced Akt activation in melanoma cells that express FLNA, whereas in the absence of FLNA, high phosphorylation levels of Akt were maintained, enabling S1P-mediated NF-κB signaling. In accord, inhibition of Akt suppressed S1P-mediated IKK and p65 phosphorylation and degradation of IκBα. Hence, these results support a negative role of FLNA in S1P-mediated NF-κB activation in melanoma cells through modulation of Akt. PMID:26552704

  16. Autolysis of Microbial Cells: Salt Activation of Autolytic Enzymes in a Mutant of Staphylococcus aureus

    PubMed Central

    Gilpin, Richard W.; Chatterjee, Anadi N.; Young, Frank E.

    1972-01-01

    The effect of various salts on the autolysis of cell wall of a ribitol teichoic acid-deficient mutant of Staphylococcus aureus H (strain 52A5 carrying tar-1) was compared with the parent strain. In the presence of high concentrations of certain salts such as 1.0 m NaCl, the mutant undergoes autolysis with the release of osmotically sensitive spheroplasts. The parent strain is not affected by these conditions. The stimulation of lysis is related to an activation of N-acylmuramyl-l-alanine amidase. Images PMID:4591480

  17. Understanding protein lids: kinetic analysis of active hinge mutants in triosephosphate isomerase.

    PubMed

    Sun, J; Sampson, N S

    1999-08-31

    In previous work we tested what three amino acid sequences could serve as a protein hinge in triosephosphate isomerase [Sun, J., and Sampson, N. S. (1998) Protein Sci. 7, 1495-1505]. We generated a genetic library encoding all 8000 possible 3 amino acid combinations at the C-terminal hinge and selected for those combinations of amino acids that formed active mutants. These mutants were classified into six phylogenetic families. Two families resembled wild-type hinges, and four families represented new types of hinges. In this work, the kinetic characteristics and thermal stabilities of mutants representing each of these families were determined in order to understand what properties make an efficient protein hinge, and why all of the families are not observed in nature. From a steady-state kinetic analysis of our mutants, it is clear that the partitioning between protonation of intermediate to form product and intermediate release from the enzyme surface to form methylglyoxal (a decomposition product) is not affected. The two most impaired mutants undergo a change in rate-limiting step from enediol formation to dihydroxyacetone phosphate binding. Thus, it appears that k(cat)/K(m)'s are reduced relative to wild type as a result of slower Michaelis complex formation and dissociation, rather than increased loop opening speed.

  18. Reactive oxygen species mediate Cr(VI)-induced carcinogenesis through PI3K/AKT-dependent activation of GSK-3β/β-catenin signaling

    SciTech Connect

    Son, Young-Ok; Pratheeshkumar, Poyil; Wang, Lei; Wang, Xin; Fan, Jia; Kim, Dong-Hern; Lee, Ju-Yeon; Zhang, Zhuo; Lee, Jeong-Chae; Shi, Xianglin

    2013-09-01

    Cr(VI) compounds are known human carcinogens that primarily target the lungs. Cr(VI) produces reactive oxygen species (ROS), but the exact effects of ROS on the signaling molecules involved in Cr(VI)-induced carcinogenesis have not been extensively studied. Chronic exposure of human bronchial epithelial cells to Cr(VI) at nanomolar concentrations (10–100 nM) for 3 months not only induced cell transformation, but also increased the potential of these cells to invade and migrate. Injection of Cr(VI)-stimulated cells into nude mice resulted in the formation of tumors. Chronic exposure to Cr(VI) increased levels of intracellular ROS and antiapoptotic proteins. Transfection with catalase or superoxide dismutase (SOD) prevented Cr(VI)-mediated increases in colony formation, cell invasion, migration, and xenograft tumors. While chronic Cr(VI) exposure led to activation of signaling cascades involving PI3K/AKT/GSK-3β/β-catenin and PI3K/AKT/mTOR, transfection with catalase or SOD markedly inhibited Cr(VI)-mediated activation of these signaling proteins. Inhibitors specific for AKT or β-catenin almost completely suppressed the Cr(VI)-mediated increase in total and active β-catenin proteins and colony formation. In particular, Cr(VI) suppressed autophagy of epithelial cells under nutrition deprivation. Furthermore, there was a marked induction of AKT, GSK-3β, β-catenin, mTOR, and carcinogenic markers in tumor tissues formed in mice after injection with Cr(VI)-stimulated cells. Collectively, our findings suggest that ROS is a key mediator of Cr(VI)-induced carcinogenesis through the activation of PI3K/AKT-dependent GSK-3β/β-catenin signaling and the promotion of cell survival mechanisms via the inhibition of apoptosis and autophagy. - Highlights: • Chronic exposure to Cr(VI) induces carcinogenic properties in BEAS-2B cells. • ROS play an important role in Cr(VI)-induced tumorigenicity of BEAS-2B cells. • PI3K/AKT/GSK-3β/β-catenin signaling involved in Cr

  19. Nobiletin protects against cerebral ischemia via activating the p-Akt, p-CREB, BDNF and Bcl-2 pathway and ameliorating BBB permeability in rat.

    PubMed

    Zhang, Lan; Zhao, Huiying; Zhang, Xiangjian; Chen, Linyu; Zhao, Xumeng; Bai, Xue; Zhang, Jian

    2013-07-01

    There is cumulative evidence that the serine-threonine kinase Akt and its downstream nuclear transcription factor CREB are involved in neuronal survival and protection. The Akt activates and phosphorylates CREB at Ser133, resulting in the up-regulation of pro-survival CREB target genes such as BDNF and Bcl-2. Thus, Akt/CREB signaling pathway may be one propitious target for treatment of ischemic cerebral injury. Nobiletin (NOB) exhibits a wide spectrum of beneficial biological properties including anti-inflammatory, antioxidant, anti-carcinogenic actions and contributes to reverse learning impairment in Alzheimer's disease rat. However, little is currently known regarding the exact role of NOB in ischemic stroke. Here, we designed to evaluate its possible therapeutic effect on cerebral ischemia. Adult male Sprague-Dawley rats were subjected to permanent middle cerebral artery occlusion (pMCAO) and randomly divided into five groups: Sham (sham-operated+0.05% Tween-80), MCAO (pMCAO+0.9% saline), Vehicle group (pMCAO+0.05% Tween-80), NOB-L (pMCAO+NOB 10 mg/kg) and NOB-H (pMCAO+NOB 25 mg/kg) groups. Rats were pre-administered intraperitoneally once daily for 3 days before surgery and then received once again immediately after surgery. Neurological deficit scores, brain water content and infarct volume were evaluated at 24 h after stroke. Additionally, the activities of Akt, CREB, BDNF, Bcl-2 and claudin-5 in ischemic brain cortex were analyzed by the methods of immunohistochemistry, western blot and RT-qPCR. Compared with Vehicle group, neurological deficits and brain edema were relieved in NOB-H group (P<0.05), infarct volume was lessened in both NOB-L and NOB-H groups (P<0.05) at 24 h after stroke. Immunohistochemistry, western blot and RT-qPCR analysis indicated that NOB dramatically promoted the activities of Akt, CREB, BDNF and Bcl-2 (P<0.05). Meanwhile, claudin-5 expression was also enhanced. On the basis of these findings, we concluded that NOB protected the

  20. Flexibility in Anaerobic Metabolism as Revealed in a Mutant of Chlamydomonas reinhardtii Lacking Hydrogenase Activity

    SciTech Connect

    Dubini, A.; Mus, F.; Seibert, M.; Grossman, A. R.; Posewitz, M. C.

    2009-03-13

    The green alga Chlamydomonas reinhardtii has a network of fermentation pathways that become active when cells acclimate to anoxia. Hydrogenase activity is an important component of this metabolism, and we have compared metabolic and regulatory responses that accompany anaerobiosis in wild-type C. reinhardtii cells and a null mutant strain for the HYDEF gene (hydEF-1 mutant), which encodes an [FeFe] hydrogenase maturation protein. This mutant has no hydrogenase activity and exhibits elevated accumulation of succinate and diminished production of CO2 relative to the parental strain during dark, anaerobic metabolism. In the absence of hydrogenase activity, increased succinate accumulation suggests that the cells activate alternative pathways for pyruvate metabolism, which contribute to NAD(P)H reoxidation, and continued glycolysis and fermentation in the absence of O2. Fermentative succinate production potentially proceeds via the formation of malate, and increases in the abundance of mRNAs encoding two malateforming enzymes, pyruvate carboxylase and malic enzyme, are observed in the mutant relative to the parental strain following transfer of cells from oxic to anoxic conditions. Although C. reinhardtii has a single gene encoding pyruvate carboxylase, it has six genes encoding putative malic enzymes. Only one of the malic enzyme genes, MME4, shows a dramatic increase in expression (mRNA abundance) in the hydEF-1 mutant during anaerobiosis. Furthermore, there are marked increases in transcripts encoding fumarase and fumarate reductase, enzymes putatively required to convert malate to succinate. These results illustrate the marked metabolic flexibility of C. reinhardtii and contribute to the development of an informed model of anaerobic metabolism in this and potentially other algae.

  1. Characterization of Bacillus licheniformis 6346 Mutants Which Have Altered Lytic Enzyme Activities

    PubMed Central

    Forsberg, C. W.; Rogers, H. J.

    1974-01-01

    Two groups of mutants altered in lytic enzyme activities have been isolated from Bacillus licheniformis 6346 MH-1 by screening clones for halo production in agar plates containing cell wall conjugated with Procion brilliant red. In the first group which produced halos during colony formation, two were shown to contain three- and eightfold more muramyl-l-alanine amidase than the parent. These strains liberated amidase and intracellular α-glucosidase into the culture medium during exponential growth in liquid medium. Isolated walls had a normal qualitative composition and in autolysing liberated N-terminal amino acids and reducing groups. Wall preparations from the second group of mutants which did not produce halos lysed very poorly at pH 9.5, the optimal pH for amidase activity, and poorly at pH 5.5 even though they had similar endo-N-acetylglucosaminidase activities to the parent. Two of these strains that were also deficient in phosphoglucomutase had only 3 to 5% of the membrane-bound amidase activity compared with that in the parent. Cell walls of the phosphoglucomutase-deficient mutants treated with sodium dodecyl sulfate to inactivate endogenous lytic enzymes were dissolved at 10% of the rate of those from the parent by added amidase, but their sensitivities to lysozyme were similar. Those from one mutant had 10 to 20% of the amidase-binding capacity of parent walls, whereas its isolated mucopeptide was essentially inactive in this respect. The failure of these phosphoglucomutase-deficient mutants to autolyse is likely to be due to the combined effects of both low amidase activity and resistant walls. As a result, daughter cells are unable to separate and long chains are formed during exponential growth. PMID:4828303

  2. Combination therapy for KIT-mutant mast cells: targeting constitutive NFAT and KIT activity.

    PubMed

    Macleod, Alison C; Klug, Lillian R; Patterson, Janice; Griffith, Diana J; Beadling, Carol; Town, Ajia; Heinrich, Michael C

    2014-12-01

    Resistant KIT mutations have hindered the development of KIT kinase inhibitors for treatment of patients with systemic mastocytosis. The goal of this research was to characterize the synergistic effects of a novel combination therapy involving inhibition of KIT and calcineurin phosphatase, a nuclear factor of activated T cells (NFAT) regulator, using a panel of KIT-mutant mast cell lines. The effects of monotherapy or combination therapy on the cellular viability/survival of KIT-mutant mast cells were evaluated. In addition, NFAT-dependent transcriptional activity was monitored in a representative cell line to evaluate the mechanisms responsible for the efficacy of combination therapy. Finally, shRNA was used to stably knockdown calcineurin expression to confirm the role of calcineurin in the observed synergy. The combination of a KIT inhibitor and a calcineurin phosphatase inhibitor (CNPI) synergized to reduce cell viability and induce apoptosis in six distinct KIT-mutant mast cell lines. Both KIT inhibitors and CNPIs were found to decrease NFAT-dependent transcriptional activity. NFAT-specific inhibitors induced similar synergistic apoptosis induction as CNPIs when combined with a KIT inhibitor. Notably, NFAT was constitutively active in each KIT-mutant cell line tested. Knockdown of calcineurin subunit PPP3R1 sensitized cells to KIT inhibition and increased NFAT phosphorylation and cytoplasmic localization. Constitutive activation of NFAT appears to represent a novel and targetable characteristic of KIT-mutant mast cell disease. Our studies suggest that combining KIT inhibition with NFAT inhibition might represent a new treatment strategy for mast cell disease.

  3. Bacteriorhodopsin mutants containing single substitutions of serine or threonine residues are all active in proton translocation

    SciTech Connect

    Marti, T.; Otto, H.; Mogi, T.; Roesselet, S.J.H.; Heyn, M.P.; Khorana, H.G. )

    1991-04-15

    To study their role in proton translocation by bacteriorhodopsin, 22 serine and threonine residues presumed to be located within and near the border of the transmembrane segments have been individually replaced by alanine or valine, respectively. Thr-89 was substituted by alanine, valine, and aspartic acid, and Ser-141 by alanine and cysteine. Most of the mutants showed essentially wild-type phenotype with regard to chromophore regeneration and absorption spectrum. However, replacement of Thr-89 by Val and of Ser-141 by Cys caused striking blue shifts of the chromophore by 100 and 80 nm, respectively. All substitutions of Thr-89 regenerated the chromophore at least 10-fold faster with 13-cis retinal than with all-trans retinal. The substitutions at positions 89, 90, and 141 also showed abnormal dark-light adaptation, suggesting interactions between these residues and the retinylidene chromophore. Proton pumping measurements revealed 60-75% activity for mutants of Thr-46, -89, -90, -205, and Ser-226, and about 20% for Ser-141----Cys, whereas the remaining mutants showed normal pumping. Kinetic studies of the photocycle and of proton release and uptake for mutants in which proton pumping was reduced revealed generally little alterations. The reduced activity in several of these mutants is most likely due to a lower percentage of all-trans retinal in the light-adapted state. In the mutants Thr-46----Val and Ser-226----Ala the decay of the photointer-mediate M was significantly accelerated, indicating an interaction between these residues and Asp-96 which reprotonates the Schiff base. Our results show that no single serine or threonine residue is obligatory for proton pumping.

  4. Inhibition of akt phosphorylation diminishes mitochondrial biogenesis regulators, tricarboxylic acid cycle activity and exacerbates recognition memory deficit in rat model of Alzheimer's disease.

    PubMed

    Shaerzadeh, Fatemeh; Motamedi, Fereshteh; Khodagholi, Fariba

    2014-11-01

    3-Methyladenine (3-MA), as a PI3K inhibitor, is widely used for inhibition of autophagy. Inhibition of PI3K class I leads to inhibition of Akt phosphorylation, a central molecule involved in diverse arrays of intracellular cascades in nervous system. Accordingly, in the present study, we aimed to determine the alterations of specific mitochondrial biogenesis markers and mitochondrial function in 3-MA-injected rats following amyloid beta (Aβ) insult. Our data revealed that inhibition of Akt phosphorylation downregulates master regulator of mitochondrial biogenesis, peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α). Our data also showed that decrease in PGC-1α level presumably is due to decrease in the phosphorylation of cAMP-response element binding and AMP-activated kinase, two upstream activators of PGC-1α. As a consequence, the level of some mitochondrial biogenesis factors including nuclear respiratory factor-1, mitochondrial transcription factor A, and Cytochrome c decreased significantly. Also, activities of tricarboxylic acid cycle (TCA) enzymes such as Aconitase, a-ketoglutarate dehydrogenase, and malate dehydrogenase reduced in the presence of 3-MA with or without Aβ insult. Decrease in mitochondrial biogenesis factors and TCA enzyme activity in the rats receiving 3-MA and Aβ were more compared to the rats that received either alone; indicating the additive destructive effects of these two agents. In agreement with our molecular results, data obtained from behavioral test (using novel objective recognition test) indicated that inhibition of Akt phosphorylation with or without Aβ injection impaired novel recognition (non-spatial) memory. Our results suggest that 3-MA amplified deleterious effects of Aβ by targeting central molecule Akt.

  5. Free energy simulations of active-site mutants of dihydrofolate reductase.

    PubMed

    Doron, Dvir; Stojković, Vanja; Gakhar, Lokesh; Vardi-Kilshtain, Alexandra; Kohen, Amnon; Major, Dan Thomas

    2015-01-22

    This study employs hybrid quantum mechanics-molecular mechanics (QM/MM) simulations to investigate the effect of mutations of the active-site residue I14 of E. coli dihydrofolate reductase (DHFR) on the hydride transfer. Recent kinetic measurements of the I14X mutants (X = V, A, and G) indicated slower hydride transfer rates and increasingly temperature-dependent kinetic isotope effects (KIEs) with systematic reduction of the I14 side chain. The QM/MM simulations show that when the original isoleucine residue is substituted in silico by valine, alanine, or glycine (I14V, I14A, and I14G DHFR, respectively), the free energy barrier height of the hydride transfer reaction increases relative to the wild-type enzyme. These trends are in line with the single-turnover rate measurements reported for these systems. In addition, extended dynamics simulations of the reactive Michaelis complex reveal enhanced flexibility in the mutants, and in particular for the I14G mutant, including considerable fluctuations of the donor-acceptor distance (DAD) and the active-site hydrogen bonding network compared with those detected in the native enzyme. These observations suggest that the perturbations induced by the mutations partly impair the active-site environment in the reactant state. On the other hand, the average DADs at the transition state of all DHFR variants are similar. Crystal structures of I14 mutants (V, A, and G) confirmed the trend of increased flexibility of the M20 and other loops. PMID:25382260

  6. Melatonin prevents cisplatin-induced primordial follicle loss via suppression of PTEN/AKT/FOXO3a pathway activation in the mouse ovary.

    PubMed

    Jang, Hoon; Lee, Ok-Hee; Lee, Youngeun; Yoon, Hyemin; Chang, Eun Mi; Park, Miseon; Lee, Jeong-Woong; Hong, Kwonho; Kim, Jung Oh; Kim, Nam Keun; Ko, Jung Jae; Lee, Dong Ryul; Yoon, Tae Ki; Lee, Woo Sik; Choi, Youngsok

    2016-04-01

    Premature ovarian failure (POF) is a major side effect of chemotherapy in young cancer patients. To develop pharmaceutical agents for preserving fertility, it is necessary to understand the mechanisms responsible for chemotherapy-induced follicle loss. Here, we show that treatment with cisplatin, a widely used anticancer drug, depleted the dormant follicle pool in mouse ovaries by excessive activation of the primordial follicles, without inducing follicular apoptosis. Moreover, we show that co-treatment with the antioxidant melatonin prevented cisplatin-induced disruption of the follicle reserve. We quantified the various stages of growing follicles, including primordial, primary, secondary, and antral, to demonstrate that cisplatin treatment alone significantly decreased, whereas melatonin co-treatment preserved, the number of primordial follicles in the ovary. Importantly, analysis of the PTEN/AKT/FOXO3a pathway demonstrated that melatonin significantly decreased the cisplatin-mediated inhibitory phosphorylation of PTEN, a key negative regulator of dormant follicle activation. Moreover, melatonin prevented the cisplatin-induced activating phosphorylation of AKT, GSK3β, and FOXO3a, all of which trigger follicle activation. Additionally, we show that melatonin inhibited the cisplatin-induced inhibitory phosphorylation and nuclear export of FOXO3a, which is required in the nucleus to maintain dormancy of the primordial follicles. These findings demonstrate that melatonin attenuates cisplatin-induced follicle loss by preventing the phosphorylation of PTEN/AKT/FOXO3a pathway members; thus, melatonin is a potential therapeutic agent for ovarian protection and fertility preservation during chemotherapy in female cancer patients.

  7. Clonal evolution enhances leukemia propagating cell frequency in T-cell acute lymphoblastic leukemia through Akt/mTORC1 pathway activation

    PubMed Central

    Blackburn, Jessica S.; Liu, Sali; Wilder, Jayme L.; Dobrinski, Kimberly P.; Lobbardi, Riadh; Moore, Finola E.; Martinez, Sarah A.; Chen, Eleanor Y.; Lee, Charles; Langenau, David M.

    2014-01-01

    SUMMARY Clonal evolution and intratumoral heterogeneity drive cancer progression through unknown molecular mechanisms. To address this issue, functional differences between single T-cell acute lymphoblastic leukemia (T-ALL) clones were assessed using a zebrafish transgenic model. Functional variation was observed within individual clones, with a minority of clones enhancing growth rate and leukemia propagating potential with time. Akt pathway activation was acquired in a subset of these evolved clones, which increased the number of leukemia propagating cells through activating mTORC1, elevated growth rate likely by stabilizing the Myc protein, and rendered cells resistant to dexamethasone, which was reversed by combined treatment with an Akt inhibitor. Thus, T-ALL clones spontaneously and continuously evolve to drive leukemia progression even in the absence of therapy-induced selection. PMID:24613413

  8. Methylglyoxal induces platelet hyperaggregation and reduces thrombus stability by activating PKC and inhibiting PI3K/Akt pathway.

    PubMed

    Hadas, Karin; Randriamboavonjy, Voahanginirina; Elgheznawy, Amro; Mann, Alexander; Fleming, Ingrid

    2013-01-01

    Diabetes is characterized by a dysregulation of glucose homeostasis and platelets from patients with diabetes are known to be hyper-reactive and contribute to the accelerated development of vascular diseases. Since many of the deleterious effects of glucose have been attributed to its metabolite methylgyloxal (MG) rather than to hyperglycemia itself, the aim of the present study was to characterize the effects of MG on platelet function. Washed human platelets were pre-incubated for 15 min with MG and platelet aggregation, adhesion on matrix-coated slides and signaling (Western blot) were assessed ex vivo. In vivo, the effect of MG on thrombus formation was determined using the FeCl3-induced carotid artery injury model. MG potentiated thrombin-induced platelet aggregation and dense granule release, but inhibited platelet spreading on fibronectin and collagen. In vivo, MG accelerated thrombus formation but decreased thrombus stability. At the molecular level, MG increased intracellular Ca(2+) and activated classical PKCs at the same time as inhibiting PI3K/Akt and the β3-integrin outside-in signaling. In conclusion, these findings indicate that the enhanced MG concentration measured in diabetic patients can directly contribute to the platelet dysfunction associated with diabetes characterized by hyperaggregability and reduced thrombus stability.

  9. EGF‑stimulated AKT activation is mediated by EGFR recycling via an early endocytic pathway in a gefitinib‑resistant human lung cancer cell line.

    PubMed

    Nishimura, Yukio; Takiguchi, Soichi; Ito, Shigeru; Itoh, Kazuyuki

    2015-04-01

    The receptor tyrosine kinase epidermal growth factor receptor (EGFR) and its ligand epidermal growth factor (EGF) are known to play important roles in malignant tumor cells, and the EGFR signaling pathway is one of the most important targets in various tumors, including non-small cell lung cancer (NSCLC). We reported recently that an aberration in certain steps of EGF-stimulated phosphorylated epidermal growth factor receptor (pEGFR) endocytic trafficking from the early endosomes to the late endosomes occurs in the gefitinib-resistant NSCLC cells, in which large amounts of sorting nexin 1 (SNX1) are colocalized with EGFR in the aggregated early endosomes where the internalized pEGFR is also accumulated of these cells. To further investigate the role of SNX1 in EGF‑stimulated pEGFR endocytosis, followed by downstream signaling leading to the activation of phosphatidylinositol 3-kinase (PI3K)--the serine/threonine kinase AKT pathway, we examined the effect of depletion of SNX1 knock-down expression by siRNA and an inhibition of targeting membrane recycling using monensin. Using immunofluorescence, we observed an efficient endocytic transport of pEGFR from early endosomes to late endosomes/lysosomes after EGF-stimulation in the cells transfected with siRNA‑SNX1, whereas the delayed endocytic delivery of pEGFR was evident in the siRNA-control-transfected cells. Furthermore, a large amount of endocytosed pEGFR was accumulated in the presence of monensin in the early endosomes of the SNX1 knock-down cells. In western blot analysis, EGF stimulation of both control and cells transfected with siRNA-SNX1 resulted in rapid phosphorylation of EGFR and enhanced AKT phosphorylation. Monensin-dependent inhibition of AKT phosphorylation was stronger in SNX1 knock-down cells than in controls. In contrast, however, monensin had no effect on AKT phosphorylation triggered by activation of the MET receptor tyrosine kinase. Collectively, we suggest that EGF-stimulated recycling of

  10. Haloperidol induces the nuclear translocation of phosphatidylinositol 3′-kinase to disrupt Akt phosphorylation in PC12 cells

    PubMed Central

    Dai, Yunxiu; Wei, Zelan; Sephton, Chantelle F.; Zhang, Di; Anderson, Deborah H.; Mousseau, Darrell D.

    2007-01-01

    Objective The antipsychotic drug haloperidol (HAL) has been linked to apoptosis and to inhibition of prosurvival Akt signalling in pheochromocytoma (PC12) and neuronal cell cultures. However, the mechanism involved is unclear. Methods We used HAL to induce cytotoxicity in preneuronal PC12 cells. The expression and the subcellular localization of selected components of the PI3K–Akt survival cascade were monitored with standard biochemical approaches, such as subcellular fractionation, western blot analysis, gene transfer and fluorescence microscopy. Results PC12 cell stimulation with the epidermal growth factor (used as a control) results in normal processing of phosphatidylinositol 3'-kinase (PI3K)–Akt signalling (e.g., localization of PI3K to the plasma membrane and phosphorylation of Akt (Ser473). Surprisingly, HAL induces PI3K-generated phosphoinositol [phosphatidylinositol-3,4,5-triphosphate (PIP3), which conflicts with its ability to inhibit Akt. In fact, the production of PIP3s is nuclear, as assessed by the localized concentration of a fluorophore-tagged PIP3-targeting pleckstrin homology protein and a fluorophore-tagged substrate-trapping mutant of the phosphoinositide phosphatase, phosphatase and tensin homologue deleted on chromosome 10 (PTEN). However, phosphoinositide-dependent protein kinase 1 (PDK1, the activating kinase of Akt) does not colocalize to the nucleus with the PI3K complex. This effectively inactivates both cytoplasmic and nuclear pools of Akt. Conclusion The differential compartmentalization of effectors of the PI3K–PDK1–Akt pathway is a unique means by which HAL disrupts Akt functioning in PC12 cells. PMID:17823648

  11. Subtoxic N-methyl-D-aspartate delayed neuronal death in ischemic brain injury through TrkB receptor- and calmodulin-mediated PI-3K/Akt pathway activation.

    PubMed

    Xu, Jing; Zhang, Quan-Guang; Li, Chong; Zhang, Guang-Yi

    2007-01-01

    Previous studies have shown that subtoxic NMDA moderated the neuronal survival in vitro and vivo. We performed this experiment to clarify the precise mechanism underlie subtoxic NMDA delayed neuronal death in ischemic brain injury. We found that pretreatment of NMDA (100 mg/kg) increased the number of the surviving CA1 pyramidal cells of hippocampus at 5 days of reperfusion. This dose of NMDA could also enhance Akt activation after ischemia/reperfusion (I/R). Here, we examined the possible mechanism that NMDA induced Akt activation. On the one hand, we found NMDA receptor-mediated Akt activation was associated with increased expression of BDNF (brain-derived neurotrophic factor) and activation of its high-affinity receptor TrkB after I/R in the hippocampus CA1 region, which could be held down by TrkB receptor antagonist K252a. On the other hand, we found that NMDA enhanced the binding of Ca2+-dependent calmodulin (CaM) to p85 (the regulation subunit of PI-3K), which led to the activation of Akt. W-13, an active CaM inhibitor, prevented the combination of CaM and p85 and subsequent Akt activation. Furthermore, NMDA receptor-mediated Akt activation was reversed by combined treatment with LY294002, the specific blockade of PI-3K. Taken together, our results suggested that subtoxic NMDA exerts the neuroprotective effect via activation of prosurvival PI-3K/Akt pathway against ischemic brain injury, and BDNF-TrkB signaling and Ca2+-dependent CaM cascade might contribute to NMDA induced activation of PI-3K/Akt pathway.

  12. Auranofin-mediated inhibition of PI3K/AKT/mTOR axis and anticancer activity in non-small cell lung cancer cells

    PubMed Central

    Li, Hongyu; Hu, Jing; Wu, Shuhong; Wang, Li; Cao, Xiaobo; Zhang, Xiaoshan; Dai, Bingbing; Cao, Mengru; Shao, Ruping; Zhang, Ran; Majidi, Mourad; Ji, Lin; Heymach, John V.; Wang, Michael; Pan, Shiyang; Minna, John; Mehran, Reza J.; Swisher, Stephen G.; Roth, Jack A.; Fang, Bingliang

    2016-01-01

    Auranofin, a gold complex that has been used to treat rheumatoid arthritis in clinics and has documented pharmacokinetic and safety profiles in humans, has recently been investigated for its anticancer activity in leukemia and some solid cancers. However, auranofin's single agent activity in lung cancer is not well characterized. To determine whether auranofin has single agent activity in lung cancer, we evaluated auranofin's activity in a panel of 10 non-small cell lung cancer (NSCLC) cell lines. Cell viability analysis revealed that auranofin induced growth inhibition in a subset of NSCLC cell lines with a half maximal inhibitory concentration (IC50) below 1.0 μM. Treatment with auranofin elicited apoptosis and necroptosis in auranofin-sensitive cell lines. Moreover, the susceptibility of NSCLC cells to auranofin was inversely correlated with TXNRD1 expression in the cells. Transient transfection of the TXNRD1-expressing plasmid in auranofin-sensitive Calu3 cells resulted in partial resistance, indicating that high TXNRD level is one of causal factors for resistance to auranofin. Further mechanistic characterization with proteomic analysis revealed that auranofin inhibits expression and/or phosphorylation of multiple key nodes in the PI3K/AKT/mTOR pathway, including S6, 4EBP1, Rictor, p70S6K, mTOR, TSC2, AKT and GSK3. Ectopic expression of TXNRD1 partially reversed auranofin-mediated PI3K/AKT/mTOR inhibition, suggesting that TXNRD1 may participate in the regulation of PI3K/AKT/mTOR pathway. Administration of auranofin to mice with xenograft tumors derived from NSCLC cells significantly suppressed tumor growth without inducing obvious toxic effects. Our results demonstrated feasibility of repurposing auranofin for treatment of lung cancer. PMID:26657290

  13. Fermented milk containing Lactobacillus GG alleviated DSS-induced colitis in mice and activated epidermal growth factor receptor and Akt signaling in intestinal epithelial cells.

    PubMed

    Yoda, Kazutoyo; He, Fang; Miyazawa, Kenji; Hiramatsu, Masaru; Yan, Fang

    2012-01-01

    Lactobacillus rhamnosus GG was assessed for its ability to alleviate DSS-induced colitis in mice and activate epidermal growth factor receptor and Akt signaling in intestinal epithelial cells. In this study mice were treated with DSS to induce colitis and they were given Lactobacillus GG fermented milk to assess the effect of probiotic on colitis. Lactobacillus GG fermented milk significantly reduced the colitis associated changes suggesting a protective effect against DSS induced colitis.

  14. Auranofin-mediated inhibition of PI3K/AKT/mTOR axis and anticancer activity in non-small cell lung cancer cells.

    PubMed

    Li, Hongyu; Hu, Jing; Wu, Shuhong; Wang, Li; Cao, Xiaobo; Zhang, Xiaoshan; Dai, Bingbing; Cao, Mengru; Shao, Ruping; Zhang, Ran; Majidi, Mourad; Ji, Lin; Heymach, John V; Wang, Michael; Pan, Shiyang; Minna, John; Mehran, Reza J; Swisher, Stephen G; Roth, Jack A; Fang, Bingliang

    2016-01-19

    Auranofin, a gold complex that has been used to treat rheumatoid arthritis in clinics and has documented pharmacokinetic and safety profiles in humans, has recently been investigated for its anticancer activity in leukemia and some solid cancers. However, auranofin's single agent activity in lung cancer is not well characterized. To determine whether auranofin has single agent activity in lung cancer, we evaluated auranofin's activity in a panel of 10 non-small cell lung cancer (NSCLC) cell lines. Cell viability analysis revealed that auranofin induced growth inhibition in a subset of NSCLC cell lines with a half maximal inhibitory concentration (IC50) below 1.0 μM. Treatment with auranofin elicited apoptosis and necroptosis in auranofin-sensitive cell lines. Moreover, the susceptibility of NSCLC cells to auranofin was inversely correlated with TXNRD1 expression in the cells. Transient transfection of the TXNRD1-expressing plasmid in auranofin-sensitive Calu3 cells resulted in partial resistance, indicating that high TXNRD level is one of causal factors for resistance to auranofin. Further mechanistic characterization with proteomic analysis revealed that auranofin inhibits expression and/or phosphorylation of multiple key nodes in the PI3K/AKT/mTOR pathway, including S6, 4EBP1, Rictor, p70S6K, mTOR, TSC2, AKT and GSK3. Ectopic expression of TXNRD1 partially reversed auranofin-mediated PI3K/AKT/mTOR inhibition, suggesting that TXNRD1 may participate in the regulation of PI3K/AKT/mTOR pathway. Administration of auranofin to mice with xenograft tumors derived from NSCLC cells significantly suppressed tumor growth without inducing obvious toxic effects. Our results demonstrated feasibility of repurposing auranofin for treatment of lung cancer. PMID:26657290

  15. Anti-Fibrotic Actions of Interleukin-10 against Hypertrophic Scarring by Activation of PI3K/AKT and STAT3 Signaling Pathways in Scar-Forming Fibroblasts

    PubMed Central

    Cai, Weixia; Bai, Xiaozhi; Fang, Xiaobing; Hu, Xiaolong; Wang, Yaojun; Wang, Hongtao; Zheng, Zhao; Su, Linlin; Hu, Dahai; Zhu, Xiongxiang

    2014-01-01

    Background The hypertrophic scar (HS) is a serious fibrotic skin condition and a major clinical problem. Interleukin-10 (IL-10) has been identified as a prospective scar-improving compound based on preclinical trials. Our previous work showed that IL-10 has anti-fibrotic effects in transforming growth factor (TGF)-β1-stimulated fibroblasts, as well as potential therapeutic benefits for the prevention and reduction of scar formation. However, relatively little is known about the mechanisms underlying IL-10-mediated anti-fibrotic and scar-improvement actions. Objective To explore the expression of the IL-10 receptor in human HS tissue and primary HS fibroblasts (HSFs), and the molecular mechanisms contributing to the anti-fibrotic and scar-improvement capabilities of IL-10. Methods Expression of the IL-10 receptor was assessed in HS tissue and HSFs by immunohistochemistry, immunofluorescence microscopy, and polymerase chain reaction analysis. Primary HSFs were treated with IL-10, a specific phosphatidylinositol 3 kinase (PI3K) inhibitor (LY294002) or a function-blocking antibody against the IL-10 receptor (IL-10RB). Next, Western blot analysis was used to evaluate changes in the phosphorylation status of AKT and signal transducers and activators of transcription (STAT) 3, as well as the expression levels of fibrosis-related proteins. Results HS tissue and primary HSFs were characterized by expression of the IL-10 receptor and by high expression of fibrotic markers relative to normal controls. Primary HSFs expressed the IL-10 receptor, while IL-10 induced AKT and STAT3 phosphorylation in these cells. In addition, LY294002 blocked AKT and STAT phosphorylation, and also up-regulated expression levels of type I and type III collagen (Col 1 and Col 3) and alpha-smooth muscle actin (α-SMA) in IL-10-treated cells. Similarly, IL-10RB reduced STAT3/AKT phosphorylation and blocked the IL-10-mediated mitigation of fibrosis in HSFs. Conclusion IL-10 apparently inhibits

  16. Growth-stimulatory activity of TIMP-2 is mediated through c-Src activation followed by activation of FAK, PI3-kinase/AKT, and ERK1/2 independent of MMP inhibition in lung adenocarcinoma cells.

    PubMed

    Kim, Han Ie; Lee, Hyun-Sung; Kim, Tae Hyun; Lee, Ju-Seog; Lee, Seung-Taek; Lee, Seo-Jin

    2015-12-15

    Tissue inhibitors of metalloproteinases (TIMPs) control extracellular matrix (ECM) homeostasis by inhibiting the activity of matrix metalloproteinases (MMPs), which are associated with ECM turnover. Recent studies have revealed that TIMPs are implicated in tumorigenesis in both MMP-dependent and MMP-independent manners. We examined a mechanism by which TIMP-2 stimulated lung adenocarcinoma cell proliferation, independent of MMP inhibition. The stimulation of growth by TIMP-2 in A549 cells required c-Src kinase activation. c-Src kinase activity, induced by TIMP-2, concomitantly increased FAK, phosphoinositide 3-kinase (PI3-kinase)/AKT, and ERK1/2 activation. Selective knockdown of integrin α3β1, known as a TIMP-2 receptor, did not significantly change TIMP-2 growth promoting activity. Furthermore, we showed that high TIMP-2 expression in lung adenocarcinomas is associated with a worse prognosis from multiple cohorts, especially for stage I lung adenocarcinoma. Through integrated analysis of The Cancer Genome Atlas data, TIMP-2 expression was significantly associated with the alteration of driving genes, c-Src activation, and PI3-kinase/AKT pathway activation. Taken together, our results demonstrate that TIMP-2 stimulates lung adenocarcinoma cell proliferation through c-Src, FAK, PI3-kinase/AKT, and ERK1/2 pathway activation in an MMP-independent manner.

  17. [Studies on construction of artificial mutants of Cucumber mosaic virus satellite RNA and their biological activity].

    PubMed

    Jin, Bo; Chen, Ji-Shuang; Zhang, Hua-Rong

    2005-08-01

    Based on the full length cDNA clone of a Cucumber mosaic virus satellite RNA, which was 369nt in size, artificial mutants were developed by the method of error-prone PCR and DNA shuffling. The new satellite cDNAs were transcribed in vitro into ssRNA and pseudo-recombined with a helper Cucumber mosaic virus, which contains no satellite RNA. Sequence analysis showed that A to T/G or G to A replacement all the four mutants, named MS1, MS5, MS6 and MS11 respectively, and there is no C to G or G to C replacement, but amongst, only the mutants MS11 could replicated when recombined with the helper virus strain. No satellite RNA could be detected by RT-PCR amplification and double-stranded RNA analysis for those pseudo-recombination constitution of Cucumber mosaic virus strain with mutants MS1, MS5 and MS6.Sequence homological comparison showed that the single replacement of mutants MS1, MS5 and MS6 occurred in the highly conservative regions and the T to A replacement of mutant MS11 was located in the normal-variation region. This is the first artificial mutation of satellite RNA of plant RNA viruses. The results indicated that single base in the region of satellite RNA maybe important to maintaining the biological activity of satellite RNA for its replication and stability. The variation and evolution of satellite RNA could be hopefully studied through combination directed evolution by DNA shuffling with pseudo-recombination in vitro.

  18. P-REX1 creates a positive feedback loop to activate growth factor receptor, PI3K/AKT, and MEK/ERK signaling in breast cancer

    PubMed Central

    Dillon, Lloye M.; Bean, Jennifer R.; Yang, Wei; Shee, Kevin; Symonds, Lynn K.; Balko, Justin M.; McDonald, W. Hayes; Liu, Shuying; Gonzalez-Angulo, Ana M.; Mills, Gordon B.; Arteaga, Carlos L.; Miller, Todd W.

    2014-01-01

    Phosphatidylinositol 3-kinase (PI3K) promotes cancer cell survival, migration, growth, and proliferation by generating phosphatidylinositol 3,4,5-trisphosphate (PIP3) in the inner leaflet of the plasma membrane. PIP3 recruits pleckstrin homology (PH) domain-containing proteins to the membrane to activate oncogenic signaling cascades. Anti-cancer therapeutics targeting the PI3K/AKT/mTOR pathway are in clinical development. In a mass spectrometric screen to identify PIP3-regulated proteins in breast cancer cells, levels of the Rac activator PIP3-dependent Rac exchange factor 1 (P-REX1) increased in response to PI3K inhibition, and decreased upon loss of the PI3K antagonist PTEN. P-REX1 mRNA and protein levels were positively correlated with ER expression, and inversely correlated with PI3K pathway activation in breast tumors as assessed by gene expression and phosphoproteomic analyses. P-REX1 increased activation of Rac1, PI3K/AKT, and MEK/ERK signaling in a PTEN-independent manner, and promoted cell and tumor viability. Loss of P-REX1 or inhibition of Rac suppressed PI3K/AKT and MEK/ERK, and decreased viability. P-REX1 also promoted insulin-like growth factor-1 receptor (IGF-1R) activation, suggesting that P-REX1 provides positive feedback to activators upstream of PI3K. In support of a model where PIP3-driven P-REX1 promotes both PI3K/AKT and MEK/ERK signaling, high levels of P-REX1 mRNA (but not phospho-AKT or a transcriptomic signature of PI3K activation) were predictive of sensitivity to PI3K inhibitors among breast cancer cell lines. P-REX1 expression was highest in ER+ breast tumors compared to many other cancer subtypes, suggesting that neutralizing the P-REX1/Rac axis may provide a novel therapeutic approach to selectively abrogate oncogenic signaling in breast cancer cells. PMID:25284585

  19. Anastrozole and everolimus in advanced gynecologic and breast malignancies: activity and molecular alterations in the PI3K/AKT/mTOR pathway

    PubMed Central

    Wheler, Jennifer J.; Moulder, Stacy L.; Naing, Aung; Janku, Filip; Piha-Paul, Sarina A.; Falchook, Gerald S.; Zinner, Ralph; Tsimberidou, Apostolia M.; Fu, Siqing; Hong, David S.; Atkins, Johnique T.; Yelensky, Roman; Stephens, Philip J.; Kurzrock, Razelle

    2014-01-01

    Background: Since PI3K/AKT/mTOR pathway activation diminishes the effects of hormone therapy, combining aromatase inhibitors (anatrozole) with mTOR inhibitors (everolimus) was investigated. Patients and Methods: We evaluated anastrozole and everolimus in 55 patients with metastatic estrogen (ER) and/or progesterone receptor (PR)-positive breast and gynecologic tumors. Endpoints were safety, antitumor activity and molecular correlates. Results: Full doses of anastrozole (1 mg PO daily) and everolimus (10 mg PO daily) were well tolerated. Twelve of 50 evaluable patients (24%) (median = 3 prior therapies) achieved stable disease (SD) ≥ 6 months/partial response (PR)/complete response (CR) (n = 5 (10%) with PR/CR): 9 of 32 (28%) with breast cancer (n=5 (16%) with PR/CR); 2 of 10 (20%), ovarian cancer; and 1 of 6 (17%), endometrial cancer. Six of 22 patients (27%) with molecular alterations in the PI3K/AKT/mTOR pathway achieved SD ≥ 6 months/PR/CR. Six of 8 patients (75%) with SD ≥ 6 months/PR/CR with molecular testing demonstrated at least one alteration in the PI3K/AKT/mTOR pathway: mutations in PIK3CA (n=3) and AKT1 (n=1) or PTEN loss (n=3). All three responders (CR (n = 1); PR (n=2)) who had next generation sequencing demonstrated additional alterations: amplifications in CCNE1, IRS2, MCL1, CCND1, FGFR1 and MYC and a rearrangement in PRKDC. Conclusions: Combination anastrozole and everolimus is well tolerated at full approved doses, and is active in heavily-pretreated patients with ER and/or PR-positive breast, ovarian and endometrial cancers. Responses were observed in patients with multiple molecular aberrations. Clinical Trails Included: NCT01197170 PMID:24912489

  20. Enoxaparin sensitizes human non-small-cell lung carcinomas to gefitinib by inhibiting DOCK1 expression, vimentin phosphorylation, and Akt activation.

    PubMed

    Pan, Yan; Li, Xin; Duan, Jianhui; Yuan, Lan; Fan, Shengjun; Fan, Jingpu; Xiaokaiti, Yilixiati; Yang, Haopeng; Wang, Yefan; Li, Xuejun

    2015-01-01

    Gefitinib is widely used for the treatment of lung cancer in patients with sensitizing epidermal growth factor receptor mutations, but patients tend to develop resistance after an average of 10 months. Low molecular weight heparins, such as enoxaparin, potently inhibit experimental metastasis. This study aimed to determine the potential of combined enoxaparin and gefitinib (enoxaparin + gefitinib) treatment to inhibit tumor resistance to gefitinib both in vitro and in vivo. A549 and H1975 cell migration was analyzed in wound closure and Transwell assays. Akt and extracellular signal-related kinase 1/2 signaling pathways were identified, and a proteomics analysis was conducted using SDS-PAGE/liquid chromatography-tandem mass spectrometry analysis. Molecular interaction networks were visualized using the Cytoscape bioinformatics platform. Protein expression of dedicator of cytokinesis 1 (DOCK1) and cytoskeleton intermediate filament vimentin were identified using an enzyme-linked immunosorbent assay, Western blot, and small interfering RNA transfection of A549 cells. In xenograft A549-luc-C8 tumors in nude mice, enoxaparin + gefitinib inhibited tumor growth and reduced lung colony formation compared with gefitinib alone. Furthermore, the combination had stronger inhibitory effects on cell migration than either agent used individually. Additional enoxaparin administration resulted in better effective inhibition of Akt activity compared with gefitinib alone. Proteomics and network analysis implicated DOCK1 as the key node molecule. Western blot verified the effective inhibition of the expression of DOCK1 and vimentin phosphorylation by enoxaparin + gefitinib compared with gefitinib alone. DOCK1 knockdown confirmed its role in cell migration, Akt expression, and vimentin phosphorylation. Our data indicate that enoxaparin sensitizes gefitinib antitumor and antimigration activity in lung cancer by suppressing DOCK1 expression, Akt activity, and vimentin phosphorylation

  1. The modulation of vascular ATP-sensitive K+ channel function via the phosphatidylinositol 3-kinase-Akt pathway activated by phenylephrine.

    PubMed

    Haba, Masanori; Hatakeyama, Noboru; Kinoshita, Hiroyuki; Teramae, Hiroki; Azma, Toshiharu; Hatano, Yoshio; Matsuda, Naoyuki

    2010-08-01

    The present study examined the modulator role of the phosphatidylinositol 3-kinase (PI3K)-Akt pathway activated by the alpha-1 adrenoceptor agonist phenylephrine in ATP-sensitive K(+) channel function in intact vascular smooth muscle. We evaluated the ATP-sensitive K(+) channel function and the activity of the PI3K-Akt pathway in the rat thoracic aorta without endothelium. The PI3K inhibitor 2-(4-morpholinyl)-8-phenyl-1(4H)-benzopyran-4-one hydrochloride (LY294002) (10(-5) M) augmented relaxation in response to the ATP-sensitive K(+) channel opener levcromakalim (10(-8) to 3 x 10(-6) M) in aortic rings contracted with phenylephrine (3 x 10(-7) M) but not with 9,11-dideoxy-11alpha,9alpha-epoxy-methanoprostaglandin F(2alpha) (U46619; 3 x 10(-8) M), although those agents induced similar contraction. ATP-sensitive K(+) channel currents induced by levcromakalim (10(-6) M) in the presence of phenylephrine (3 x 10(-7) M) were enhanced by the nonselective alpha-adrenoceptor antagonist phentolamine (10(-7) M) and LY294002 (10(-5) M). Levels of the regulatory subunits of PI3K p85-alpha and p55-gamma increased in the membrane fraction from aortas without endothelium treated with phenylephrine (3 x 10(-7) M) but not with U46619 (3 x 10(-8) M). Phenylephrine simultaneously augmented Akt phosphorylation at Ser473 and Thr308. Therefore, activation of the PI3K-Akt pathway seems to play a role in the impairment of ATP-sensitive K(+) channel function in vascular smooth muscle exposed to alpha-1 adrenergic stimuli.

  2. Downregulation of PI3-K/Akt/PTEN pathway and activation of mitochondrial intrinsic apoptosis by Diclofenac and Curcumin in colon cancer.

    PubMed

    Rana, Chandan; Piplani, Honit; Vaish, Vivek; Nehru, Bimla; Sanyal, S N

    2015-04-01

    Phosphatidylinositol 3-kinase (PI3-K)/PTEN/Akt signaling is over activated in various tumors including colon cancer. Activation of this pathway regulates multiple biological processes such as apoptosis, metabolism, cell proliferation, and cell growth that underlie the biology of a cancer cell. In the present study, the chemopreventive effects have been observed of Diclofenac, a preferential COX-2 inhibitory non-steroidal anti-inflammatory drugs, and Curcumin, a natural anti-inflammatory agent, in the early stage of colorectal carcinogenesis induced by 1,2-dimethylhydrazine dihydrochloride in rats. The tumor-promoting role of PI3-K/Akt/PTEN signal transduction pathway and its association with anti-apoptotic family of proteins are also observed. Both Diclofenac and Curcumin downregulated the PI3-K and Akt expression while promoting the apoptotic mechanism. Diclofenac and Curcumin administration significantly increased the expression of pro-apoptotic Bcl-2 family members (Bad and Bax) while decreasing the anti-apoptotic Bcl-2 protein. An up-regulation of cysteine protease family apoptosis executioner, such as caspase-3 and -9, is seen. Diclofenac and Curcumin inhibited the Bcl-2 protein by directly interacting at the active site by multiple hydrogen bonding, as also evident by negative glide score of Bcl-2. These drugs stimulated apoptosis by increasing reactive oxygen species (ROS) generation and simultaneously decreasing the mitochondrial membrane potential (ΔΨ M). Diclofenac and Curcumin showed anti-neoplastic effects by downregulating PI3-K/Akt/PTEN pathway, inducing apoptosis, increasing ROS generation, and decreasing ΔΨ M. The anti-neoplastic and apoptotic effects were found enhanced when both Diclofenac and Curcumin were administered together, rather than individually.

  3. IL-7 splicing variant IL-7{delta}5 induces human breast cancer cell proliferation via activation of PI3K/Akt pathway

    SciTech Connect

    Pan, Deshun; Liu, Bing; Jin, Xiaobao; Zhu, Jiayong

    2012-06-15

    Highlights: Black-Right-Pointing-Pointer This study confirms the role of IL-7{delta}5 in breast cancer cell proliferation. Black-Right-Pointing-Pointer IL-7{delta}5 promotes breast cancer cell proliferation and cell cycle progression. Black-Right-Pointing-Pointer IL-7{delta}5 promotes cell proliferation via activation of PI3K/Akt pathway. -- Abstract: Various tumor cells express interleukin 7 (IL-7) and IL-7 variants. IL-7 has been confirmed to stimulate solid tumor cell proliferation. However, the effect of IL-7 variants on tumor cell proliferation remains unclear. In this study, we evaluated the role of IL-7{delta}5 (an IL-7 variant lacking exon 5) on proliferation and cell cycle progression of human MDA-MB-231 and MCF-7 breast cancer cells. The results showed that IL-7{delta}5 promoted cell proliferation and cell cycle progression from G1 phase to G2/M phase, associated with upregulation of cyclin D1 expression and the downregulation of p27{sup kip1} expression. Mechanistically, we found that IL-7{delta}5 induced the activation of Akt. Inhibition of PI3K/Akt pathway by LY294002 reversed the proliferation and cell cycle progression of MDA-MB-231 and MCF-7 cells induced by IL-7{delta}5. In conclusion, our findings demonstrate that IL-7{delta}5 variant induces human breast cancer cell proliferation and cell cycle progression via activation of PI3K/Akt pathway. Thus, IL-7{delta}5 may be a potential target for human breast cancer therapeutics intervention.

  4. Aminofurazans as potent inhibitors of AKT kinase

    SciTech Connect

    Rouse, Meagan B.; Seefeld, Mark A.; Leber, Jack D.; McNulty, Kenneth C.; Sun, Lihui; Miller, William H.; Zhang, ShuYun; Minthorn, Elisabeth A.; Concha, Nestor O.; Choudhry, Anthony E.; Schaber, Michael D.; Heerding, Dirk A.

    2009-06-24

    AKT inhibitors containing an imidazopyridine aminofurazan scaffold have been optimized. We have previously disclosed identification of the AKT inhibitor GSK690693, which has been evaluated in clinical trials in cancer patients. Herein we describe recent efforts focusing on investigating a distinct region of this scaffold that have afforded compounds (30 and 32) with comparable activity profiles to that of GSK690693.

  5. Accelerated Tumor Growth Mediated by Sub-lytic Levels of Antibody-Induced Complement Activation is Associated with Activation of the PI3K/AKT Survival Pathway

    PubMed Central

    Wu, Xiaohong; Ragupathi, Govind; Panageas, Katherine; Hong, Feng; Livingston, Philip O.

    2013-01-01

    Purpose We addressed the possibility that low levels of tumor cell bound antibodies targeting gangliosides might accelerate tumor growth. Experimental Design To test this hypothesis, we treated mice with a range of mAb doses against GM2, GD2, GD3 and CD20 after challenge with tumors expressing these antigens and tested the activity of the same mAbs in-vitro. We also explored the mechanisms behind the complement-mediated tumor growth acceleration that we observed and an approach to overcome it. Results Serologically detectable levels of IgM-mAb against GM2 are able to delay or prevent tumor growth of high GM2-expressing cell lines both in-vitro and in a SCID mouse model, while very low levels of this mAb resulted in slight but consistent acceleration of tumor growth in both settings. Surprisingly, this is not restricted to IgM antibodies targeting GM2 but consistent against IgG-mAb targeting GD3 as well. These findings were mirrored by in-vitro studies with antibodies against these antigens as well as GD2 and CD20 (with Rituxan), and shown to be complement-dependent in all cases. Complement-mediated accelerated growth of cultured tumor cell lines initiated by low mAb levels was associated with activation of the PI3K/AKT survival pathway and significantly elevated levels of both p-AKT and p-PRAS40. This complement-mediated PI3K-activation and accelerated tumor growth in-vitro and in-vivo are eliminated by PI3K-inhibitors NVP-BEZ235 and Wortmannin. These PI3K-inhibitors also significantly increased efficacy of high doses of these 4 mAbs. Conclusion Our findings suggest that manipulation of the PI3K/AKT pathway and its signaling network can significantly increase the potency of passively administered mAbs and vaccine-induced-antibodies targeting a variety of tumor-cell-surface-antigens. PMID:23833306

  6. AtKC1 and CIPK23 Synergistically Modulate AKT1-Mediated Low-Potassium Stress Responses in Arabidopsis1[OPEN

    PubMed Central

    Chen, Li-Mei; Liu, Wen-Xin; Shen, Li-Ke; Wang, Feng-Liu; Zhou, Yuan

    2016-01-01

    In Arabidopsis (Arabidopsis thaliana), the Shaker K+ channel AKT1 conducts K+ uptake in root cells, and its activity is regulated by CBL1/9-CIPK23 complexes as well as by the AtKC1 channel subunit. CIPK23 and AtKC1 are both involved in the AKT1-mediated low-K+ (LK) response; however, the relationship between them remains unclear. In this study, we screened suppressors of low-K+ sensitive [lks1 (cipk23)] and isolated the suppressor of lks1 (sls1) mutant, which suppressed the leaf chlorosis phenotype of lks1 under LK conditions. Map-based cloning revealed a point mutation in AtKC1 of sls1 that led to an amino acid substitution (G322D) in the S6 region of AtKC1. The G322D substitution generated a gain-of-function mutation, AtKC1D, that enhanced K+ uptake capacity and LK tolerance in Arabidopsis. Structural prediction suggested that glycine-322 is highly conserved in K+ channels and may function as the gating hinge of plant Shaker K+ channels. Electrophysiological analyses revealed that, compared with wild-type AtKC1, AtKC1D showed enhanced inhibition of AKT1 activity and strongly reduced K+ leakage through AKT1 under LK conditions. In addition, phenotype analysis revealed distinct phenotypes of lks1 and atkc1 mutants in different LK assays, but the lks1 atkc1 double mutant always showed a LK-sensitive phenotype similar to that of akt1. This study revealed a link between CIPK-mediated activation and AtKC1-mediated modification in AKT1 regulation. CIPK23 and AtKC1 exhibit distinct effects; however, they act synergistically and balance K+ uptake/leakage to modulate AKT1-mediated LK responses in Arabidopsis. PMID:26829980

  7. The role of CAPE in PI3K/AKT/mTOR activation and oxidative stress on testis torsion.

    PubMed

    Dilber, Yagmur; Inan, Sevinc; Ercan, Gulinnaz Alper; Sencan, Aydin

    2016-01-01

    Ischemia reperfusion injury arises from testicular torsion resulting in a loss of spermatogenesis and significant germ cell apoptosis. This study evaluates the prooxidant/antioxidant effects of caffeic acid phenethyl ester (CAPE) through PI3K/AKT/mTOR signal pathways on testis torsion. A total of (28) male Wistar rats were divided randomly into 4 groups (n=7 for each group):group A (sham) group,group B torsion/detorsion group, group C (saturation group, during four days of CAPE, one dose (10 μmol/kg, i.p)) and group D (a single dose of CAPE 2h after torsion and before detorsion). At the end of the study, unilateral orchiectomies were performed for measurements of MDA and 8OHdG levels, histopathologic and immunohistochemical and TUNEL apoptotic cell examination. Testicular torsion-detorsion led to a significant decrease in the mean values of the Johnsen's scores and a significant increase in the apoptotic cell values of group B. There were no significant differences between group D and group A. In addition, the MDA and 8OHdG levels increased significantly in group B. The MDA and 8OHdG values were lower in group D. However, the 8OHdG levels were higher in group C than the groups A and D. On the other hand, CAPE suppresses mTOR activation and reduces the apoptosis on ischemia/reperfusion damage in rat testis. These results demonstrate that CAPE suppresses mTOR activation and reduces the apoptosis on ischemia/reperfusion damage in rat testis. PMID:26651953

  8. Systemic Bisperoxovanadium Activates Akt/mTOR, Reduces Autophagy, and Enhances Recovery following Cervical Spinal Cord Injury

    PubMed Central

    Walker, Chandler L.; Walker, Melissa J.; Liu, Nai-Kui; Risberg, Emelie C.; Gao, Xiang; Chen, Jinhui; Xu, Xiao-Ming

    2012-01-01

    Secondary damage following primary spinal cord injury extends pathology beyond the site of initial trauma, and effective management is imperative for maximizing anatomical and functional recovery. Bisperoxovanadium compounds have proven neuroprotective effects in several central nervous system injury/disease models, however, no mechanism has been linked to such neuroprotection from bisperoxovanadium treatment following spinal trauma. The goal of this study was to assess acute bisperoxovanadium treatment effects on neuroprotection and functional recovery following cervical unilateral contusive spinal cord injury, and investigate a potential mechanism of the compound's action. Two experimental groups of rats were established to 1) assess twice-daily 7 day treatment of the compound, potassium bisperoxo (picolinato) vanadium, on long-term recovery of skilled forelimb activity using a novel food manipulation test, and neuroprotection 6 weeks following injury and 2) elucidate an acute mechanistic link for the action of the drug post-injury. Immunofluorescence and Western blotting were performed to assess cellular signaling 1 day following SCI, and histochemistry and forelimb functional analysis were utilized to assess neuroprotection and recovery 6 weeks after injury. Bisperoxovanadium promoted significant neuroprotection through reduced motorneuron death, increased tissue sparing, and minimized cavity formation in rats. Enhanced forelimb functional ability during a treat-eating assessment was also observed. Additionally, bisperoxovanadium significantly enhanced downstream Akt and mammalian target of rapamycin signaling and reduced autophagic activity, suggesting inhibition of the phosphatase and tensin homologue deleted on chromosome ten as a potential mechanism of bisperoxovanadium action following traumatic spinal cord injury. Overall, this study demonstrates the efficacy of a clinically applicable pharmacological therapy for rapid initiation of neuroprotection post

  9. Septin4_i1 Regulates Apoptosis in Hepatic Stellate Cells through Peroxisome Proliferator-Activated Receptor-γ/Akt/B-Cell Lymphoma 2 Pathway

    PubMed Central

    Zhu, Dandan; Wang, Jianxin; Sun, Xiaolei; Chen, Jinling; Pan, Jing; Xu, Tianhua; Qin, Yongwei; He, Xingxin; Huang, Caiqun

    2015-01-01

    Apoptosis of activated hepatic stellate cells (HSCs) has been verified as a potential mechanism to aid in hepatic fibrosis remission. Earlier research suggests that Septin4_i1 may sensitize hepatocellular carcinoma cells to serum starvation-induced apoptosis. Here, we aimed to investigate the effect of Septin4_i1 on HSC apoptosis and explore the associated signaling pathways. We found that Septin4_i1 can induce apoptosis in LX-2 cells and that this is accompanied by an up-regulation in cleaved-caspase-3 and peroxisome proliferator-activated receptor-γ (PPAR-γ) expression and a down-regulation in α-SMA expression. Over-expression of Septin4_i1 reduced phosphorylated Akt and B-cell lymphoma 2 (Bcl-2) expression but had no effect on the expression of p53 and death receptor (DR)-5. The decreased expression of Bcl-2 and the increased expression of cleaved-caspase-3 induced by Sept4_i1 could be reversed by GW501516, a PPAR-β/δ agonist that has been reported by others to enhance Akt signaling. In addition, GW9662, an antagonist of PPAR-γ, could also inhibit apoptosis in LX-2 cells induced by Sept4_i1. In conclusion, our data suggest that Sept4_i1 induces HSC apoptosis by inhibiting Akt and Bcl-2 expression and up-regulating PPAR-γ expression. PMID:25527525

  10. Septin4_i1 regulates apoptosis in hepatic stellate cells through peroxisome proliferator-activated receptor-γ/Akt/B-cell lymphoma 2 pathway.

    PubMed

    Zhu, Dandan; Wang, Jianxin; Sun, Xiaolei; Chen, Jinling; Duan, Yinong; Pan, Jing; Xu, Tianhua; Qin, Yongwei; He, Xingxin; Huang, Caiqun

    2015-03-01

    Apoptosis of activated hepatic stellate cells (HSCs) has been verified as a potential mechanism to aid in hepatic fibrosis remission. Earlier research suggests that Septin4_i1 may sensitize hepatocellular carcinoma cells to serum starvation-induced apoptosis. Here, we aimed to investigate the effect of Septin4_i1 on HSC apoptosis and explore the associated signaling pathways. We found that Septin4_i1 can induce apoptosis in LX-2 cells and that this is accompanied by an up-regulation in cleaved-caspase-3 and peroxisome proliferator-activated receptor-γ (PPAR-γ) expression and a down-regulation in α-SMA expression. Over-expression of Septin4_i1 reduced phosphorylated Akt and B-cell lymphoma 2 (Bcl-2) expression but had no effect on the expression of p53 and death receptor (DR)-5. The decreased expression of Bcl-2 and the increased expression of cleaved-caspase-3 induced by Sept4_i1 could be reversed by GW501516, a PPAR-β/δ agonist that has been reported by others to enhance Akt signaling. In addition, GW9662, an antagonist of PPAR-γ, could also inhibit apoptosis in LX-2 cells induced by Sept4_i1. In conclusion, our data suggest that Sept4_i1 induces HSC apoptosis by inhibiting Akt and Bcl-2 expression and up-regulating PPAR-γ expression. PMID:25527525

  11. Effect of Thyrotropin on Osteopontin, Integrin αvβ3, and VCAM-1 in the Endothelium via Activation of Akt

    PubMed Central

    Yan, Yumeng; Jiang, Fengwei; Lai, Yaxin; Wang, Haoyu; Liu, Aihua; Wang, Chuyuan; Zhang, Yuanyuan; Teng, Weiping; Shan, Zhongyan

    2016-01-01

    Numerous epidemiological studies have shown that subclinical hypothyroidism (SCH) can impair endothelial function and cause dyslipidemia. Studies have evaluated the effects of thyroid stimulating hormone (TSH) on endothelial cells, but the mechanism underlying the proatherosclerotic effect of increased TSH levels remains unclear. In the present study, SCH rat models were established in thyroidectomized Wistar rats that were given l-T4 daily. The results showed that in vivo, the expression of osteopontin (OPN) vascular cell adhesion molecule (VCAM-1), and levels of integrin αvβ3 in the aortic tissue in SCH and Hypothyroidism (CH) groups was higher than in the control group. However, the effect in the SCH group was higher than in the CH group. In vitro, results showed that different concentration and time gradients of TSH stimulation could increase the expression of OPN, VCAM-1, and integrin αvβ3, and this was accompanied by extracellular signal regulated kinase 1/2 (Erk1/2) and Akt activation in human umbilical vein endothelial cells (HUVECs). TSH induced elevation of these proatherosclerotic factors was partially suppressed by a specific Akt inhibitor but not by a specific Erk inhibitor. Findings suggested that the endothelial dysfunction caused by SCH was related to increased proatherosclerotic factors induced by TSH via Akt activation. PMID:27657042

  12. Fas apoptosis inhibitory molecule is upregulated by IGF-1 signaling and modulates Akt activation and IRF4 expression in multiple myeloma.

    PubMed

    Huo, J; Xu, S; Lin, B; Chng, W-J; Lam, K-P

    2013-04-01

    Multiple myeloma (MM) is an incurable malignancy of terminally differentiated B-lymphoid cells. Here, we investigate the role of Fas apoptosis inhibitory molecule (FAIM) in MM. We demonstrate that insulin-like growth factor 1 (IGF-1) treatment upregulated FAIM expression in MM cells in a dose-dependent manner. Silencing of FAIM expression attenuates Akt signaling downstream of IGF-1 and compromises the viability of MM cells. We further showed that IGF-1 stimulation of MM cells leads to enhanced expression of IRF4, a known 'addictive' factor for MM. This upregulation of IRF4 expression by IGF-1 treatment of MM cells is abrogated when FAIM expression is silenced or Akt activation is inhibited. Thus, FAIM modulates IGF-1-induced Akt activation and IRF4 expression and has a role in MM cell survival. Consistent with these findings, FAIM expression is shown to be higher in plasma cells of symptomatic MM patients compared with normal individuals or patients with premalignant conditions. Moreover, a higher level of FAIM expression is shown to correlate with poorer survival outcomes of newly diagnosed MM patients treated with stem cell transplantation or relapsed MM patients treated in clinical trials with Bortezomib. Thus taken together, our study reveals a novel, as well as clinically relevant role for FAIM in MM.

  13. Effects of RAF inhibitors on PI3K/AKT signalling depend on mutational status of the RAS/RAF signalling axis

    PubMed Central

    Fritsche-Guenther, Raphaela; Witzel, Franziska; Kempa, Stefan; Brummer, Tilman; Sers, Christine; Blüthgen, Nils

    2016-01-01

    Targeted therapies within the RAS/RAF/MEK/ERK signalling axis become increasingly popular, yet cross-talk and feedbacks in the signalling network lead to unexpected effects. Here we look systematically into how inhibiting RAF and MEK with clinically relevant inhibitors result in changes in PI3K/AKT activation. We measure the signalling response using a bead-based ELISA, and use a panel of three cell lines, and isogenic cell lines that express mutant forms of the oncogenes KRAS and BRAF to interrogate the effects of the MEK and RAF inhibitors on signalling. We find that treatment with the RAF inhibitors have opposing effects on AKT phosphorylation depending on the mutational status of two important oncogenes, KRAS and BRAF. If these two genes are in wildtype configuration, RAF inhibitors reduce AKT phosphorylation. In contrast, if BRAF or KRAS are mutant, RAF inhibitors will leave AKT phosphorylation unaffected or lead to an increase of AKT phosphorylation. Down-regulation of phospho-AKT by RAF inhibitors also extends to downstream transcription factors, and correlates with apoptosis induction. Our results show that oncogenes rewire signalling such that targeted therapies can have opposing effects on parallel pathways, which depend on the mutational status of the cell. PMID:26799289

  14. Lactose metabolism in Streptococcus lactis: studies with a mutant lacking glucokinase and mannose-phosphotransferase activities

    SciTech Connect

    Thompson, J.; Chassy, B.M.; Egan, W.

    1985-04-01

    A mutant of Streptococcus lactis 133 has been isolated that lacks both glucokinase and phosphoenolpyruvate-dependent mannose- phosphotransferase (mannose-PTS) activities. The double mutant S. lactis 133 mannose-PTSd GK- is unable to utilize either exogenously supplied or intracellularly generated glucose for growth. Fluorographic analyses of metabolites formed during the metabolism of (/sup 14/C)lactose labeled specifically in the glucose or galactosyl moiety established that the cells were unable to phosphorylate intracellular glucose. However, cells of S. lactis 133 mannose-PTSd GK- readily metabolized intracellular glucose 6-phosphate, and the growth rates and cell yield of the mutant and parental strains on sucrose were the same. During growth on lactose, S. lactis 133 mannose-PTSd GK- fermented only the galactose moiety of the disaccharide, and 1 mol of glucose was generated per mol of lactose consumed. For an equivalent concentration of lactose, the cell yield of the mutant was 50% that of the wild type. The specific rate of lactose utilization by growing cells of S. lactis 133 mannose-PTSd GK- was ca. 50% greater than that of the wild type, but the cell doubling times were 70 and 47 min, respectively. High-resolution /sup 31/P nuclear magnetic resonance studies of lactose transport by starved cells of S. lactis 133 and S. lactis 133 mannose-PTSd GK- showed that the latter cells contained elev