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Sample records for active akt mutant

  1. Lycium barbarum polysaccharide attenuates the cytotoxicity of mutant huntingtin and increases the activity of AKT.

    PubMed

    Fang, Fang; Peng, Ting; Yang, Shiming; Wang, Weixi; Zhang, Yinong; Li, He

    2016-08-01

    Huntington's disease (HD) is an inherited neurodegenerative disease that is caused by the abnormal expansion of CAG repeats in the gene encoding huntingtin (Htt). Reduced AKT phosphorylation and inhibited AKT activity have been shown to be involved in mutant Htt (mHtt)-induced cell death. Lycium barbarum polysaccharide (LBP), the main bioactive component of Lycium barbarum, reportedly has neuroprotective roles in neural injuries, including neurodegenerative diseases. Here, we report that treatment with LBP can increased the viability of HEK293 cells that stably expressed mHtt containing 160 glutamine repeats and significantly improved motor behavior and life span in HD-transgenic mice. Furthermore, we found that in LBP-treated HEK293 cells expressing mHtt, mHtt levels were reduced and the phosphorylation of AKT at Ser473 (p-AKT-Ser473) was significantly increased. We also found that treatment with LBP increased p-AKT-Ser473 and decreased mHtt in the cortex, hippocampus and striatum in HD-transgenic mice. The level of phosphorylation of p-GSK3β-Ser9 remained unchanged in both cultured cells and HD-transgenic mice. Our findings suggest that LBP alleviates the cytotoxicity of mHtt by activating AKT and reducing mHtt levels, indicating that LBP may be potentially useful for treating HD. PMID:27196502

  2. Basal and treatment-induced activation of AKT mediates resistance to cell death by AZD6244 (ARRY-142886) in Braf-mutant human cutaneous melanoma cells

    PubMed Central

    Gopal, Y.N. Vashisht; Deng, Wanleng; Woodman, Scott E.; Komurov, Kakajan; Ram, Prahlad; Smith, Paul D.; Davies, Michael A.

    2014-01-01

    The majority of melanomas demonstrate constitutive activation of the RAS-RAF-MEK-MAPK pathway. AZD6244 is a selective MEK1/2 inhibitor which markedly reduces tumor P-MAPK levels, but it produced few clinical responses in melanoma patients. An improved understanding of the determinants of resistance to AZD6244 may lead to improved patient selection and effective combinatorial approaches. The effects of AZD6244 on cell growth and survival were tested in a total of 14 Braf-mutant and 3 wild-type human cutaneous melanoma cell lines. Quantitative assessment of phospho-protein levels in the Braf-mutant cell lines by reverse phase protein array (RPPA) analysis showed no significant association between P-MEK or P-MAPK levels and AZD6244 sensitivity, but activation-specific markers in the PI3K-AKT pathway correlated with resistance. We also identified resistant cell lines without basal activation of the PI3K-AKT pathway. RPPA characterization of the time-dependent changes in signaling pathways revealed that AZD6244 produced durable and potent inhibition of P-MAPK in sensitive and resistant Braf-mutant cell lines, but several resistant lines demonstrated AZD6244-induced activation of AKT. In contrast, sensitive cell lines demonstrated AZD6244 treatment-induced upregulation of PTEN protein and mRNA expression. Inhibition of AKT, TORC1/2, or IGF1R blocked AZD6244-induced activation of AKT and resulted in synergistic cell killing with AZD6244. These findings identify basal and treatment-induced regulation of the PI3K-AKT pathway as a critical regulator of AZD6244 sensitivity in Braf-mutant cutaneous melanoma cells, the novel regulation of PTEN expression by AZD6244 in sensitive cells, and suggest new combinatorial approaches for patients. PMID:20959481

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

    PubMed

    Iskandar, Kartini; Rezlan, Majidah; Pervaiz, Shazib

    2014-10-01

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

  4. Long non-coding RNA UCA1 induces non-T790M acquired resistance to EGFR-TKIs by activating the AKT/mTOR pathway in EGFR-mutant non-small cell lung cancer

    PubMed Central

    Ren, Shengxiang; Li, Xuefei; Wang, Qi; Pan, Hui; Zhao, Mingchuan; Li, Jiayu; Zhang, Yishi; Zhao, Chao; Chen, Xiaoxia; Fei, Ke; Zhou, Caicun; Hirsch, Fred R.

    2015-01-01

    The aim of this study was to explore the role of long non-coding RNA UCA1 (urothelial cancer-associated 1) in acquired resistance to epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) in EGFR-mutant non-small cell lung cancer (NSCLC). In our study, UCA1 expression was significantly increased in lung cancer cells and patients with acquired resistance to EGFR-TKIs. Over-expression of UCA1 was significantly associated with a shorter progression-free survival (PFS) [13.0 vs. 8.5 months, P < 0.01] in tumors with respond to EGFR-TKIs. The significant relationship was not observed in patients with T790M mutation (10.5 vs. 12.0 months, P = 0.778), but in patients with non-T790M (19.0 vs. 9.0 months, P = 0.023). UCA1 knockdown restored gefitinib sensitivity in acquired resistant cells with non-T790M and inhibited the activation of the AKT/mTOR pathway and epithelial-mesenchymal transition (EMT). The mTOR inhibitor was effective in UCA1-expressing cell PC9/R. Inhibiting mTOR could change the expression of UCA1, although there was no significant difference. In conclusion, the influence of over-expression of UCA1 on PFS for patients with acquired resistance to EGFR-TKIs was from the subgroup with non-T790M mutation. UCA1 may induce non-T790M acquired resistance to EGFR-TKIs by activating the AKT/mTOR pathway and EMT. PMID:26160838

  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 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

  7. Protein kinase Cδ regulates endothelial nitric oxide synthase expression via Akt activation and nitric oxide generation

    PubMed Central

    Sud, Neetu; Wedgwood, Stephen; Black, Stephen M.

    2008-01-01

    In this study, we explore the roles of the delta isoform of PKC (PKCδ) in the regulation of endothelial nitric oxide synthase (eNOS) activity in pulmonary arterial endothelial cells isolated from fetal lambs (FPAECs). Pharmacological inhibition of PKCδ with either rottlerin or with the peptide, δV1-1, acutely attenuated NO production, and this was associated with a decrease in phosphorylation of eNOS at Ser1177 (S1177). The chronic effects of PKCδ inhibition using either rottlerin or the overexpression of a dominant negative PKCδ mutant included the downregulation of eNOS gene expression that was manifested by a decrease in both eNOS promoter activity and protein expression after 24 h of treatment. We also found that PKCδ inhibition blunted Akt activation as observed by a reduction in phosphorylated Akt at position Ser473. Thus, we conclude that PKCδ is actively involved in the activation of Akt. To determine the effect of Akt on eNOS signaling, we overexpressed a dominant negative mutant of Akt and determined its effect of NO generation, eNOS expression, and phosphorylation of eNOS at S1177. Our results demonstrated that Akt inhibition was associated with decreased NO production that correlated with reduced phosphorylation of eNOS at S1177, and decreased eNOS promoter activity. We next evaluated the effect of endogenously produced NO on eNOS expression by incubating FPAECs with the eNOS inhibitor 2-ethyl-2-thiopseudourea (ETU). ETU significantly inhibited NO production, eNOS promoter activity, and eNOS protein levels. Together, our data indicate involvement of PKCδ-mediated Akt activation and NO generation in maintaining eNOS expression. PMID:18192589

  8. 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.

  9. 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

  10. 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

  11. v-Crk activates the phosphoinositide 3-kinase/AKT pathway in transformation

    PubMed Central

    Akagi, Tsuyoshi; Shishido, Tomoyuki; Murata, Kazutaka; Hanafusa, Hidesaburo

    2000-01-01

    v-Crk induces cellular tyrosine phosphorylation and transformation of chicken embryo fibroblasts (CEF). We studied the molecular mechanism of the v-Crk-induced transformation. Experiments with Src homology (SH)2 and SH3 domain mutants revealed that the induction of tyrosine phosphorylation of cellular proteins requires only the SH2 domain, but both the SH2 and SH3 domains are required for complete transformation. Analysis of three well defined signaling pathways, the mitogen-activated protein kinase (MAPK) pathway, the Jun N-terminal kinase (JNK) pathway, and the phosphoinositide 3-kinase (PI3K)/AKT pathway, demonstrated that only the PI3K/AKT pathway is constitutively activated in v-Crk-transformed CEF. Both the SH2 and SH3 domains are required for this activation of the PI3K/AKT pathway in CEF. We also found that the colony formation of CEF is strongly induced by a constitutively active PI3K mutant, and that a PI3K inhibitor, LY294002, suppresses the v-Crk-induced transformation. These results strongly suggest that constitutive activation of the PI3K/AKT pathway plays an essential role in v-Crk-induced transformation of CEF. PMID:10852971

  12. Subcutaneous Adipocytes Promote Melanoma Cell Growth by Activating the Akt Signaling Pathway

    PubMed Central

    Kwan, Hiu Yee; Fu, Xiuqiong; Liu, Bin; Chao, Xiaojuan; Chan, Chi Leung; Cao, Huihui; Su, Tao; Tse, Anfernee Kai Wing; Fong, Wang Fun; Yu, Zhi-Ling

    2014-01-01

    Tumorigenesis involves constant communication between tumor cells and neighboring normal cells such as adipocytes. The canonical function of adipocytes is to store triglyceride and release fatty acids for other tissues. This study was aimed to find out if adipocytes promoted melanoma cell growth and to investigate the underlying mechanism. Here we isolated adipocytes from inguinal adipose tissue in mice and co-cultured with melanoma cells. We found that the co-cultured melanoma had higher lipid accumulation compared with mono-cultured melanoma. In addition, fluorescently labeled fatty acid BODIPY® FLC16 signal was detected in melanoma co-cultured with the adipocytes that had been loaded with the fluorescent dye, suggesting that the adipocytes provide fatty acids to melanoma cells. Compared with mono-cultured melanoma, co-cultured melanoma cells had a higher proliferation and phospho-Akt (Ser-473 and Thr-450) expression. Overexpression of Akt mutants in melanoma cells reduced the co-culture-enhanced proliferation. A lipidomic study showed that the co-cultured melanoma had an elevated palmitic acid level. Interestingly, we found that palmitic acid stimulated melanoma cell proliferation, changed the cell cycle distribution, and increased phospho-Akt (Ser-473 and Thr-450) and PI3K but not phospho-PTEN (phosphophosphatase and tensin homolog) expressions. More importantly, the palmitic acid-stimulated proliferation was further enhanced in the Akt-overexpressed melanoma cells and was reduced by LY294002 or knockdown of endogenous Akt or overexpression of Akt mutants. We also found that palmitic acid-pretreated B16F10 cells were grown to a significantly larger tumor in mice compared with control cells. Taken together, we suggest that adipocytes may serve as an exogenous source of palmitic acid that promotes melanoma cell growth by activating Akt. PMID:25228694

  13. Somatic Activation of AKT3 Causes Hemispheric Developmental Brain Malformations

    PubMed Central

    Poduri, Annapurna; Evrony, Gilad D.; Cai, Xuyu; Elhosary, Princess Christina; Beroukhim, Rameen; Lehtinen, Maria K.; Hills, L. Benjamin; Heinzen, Erin L.; Hill, Anthony; Hill, R. Sean; Barry, Brenda J.; Bourgeois, Blaise F.D.; Riviello, James J.; Barkovich, A. James; Black, Peter M.; Ligon, Keith L.; Walsh, Christopher A.

    2012-01-01

    Summary Hemimegalencephaly (HMG) is a developmental brain disorder characterized by an enlarged, malformed cerebral hemisphere, typically causing epilepsy that requires surgical resection. We studied resected HMG tissue to test whether the condition might reflect somatic mutations affecting genes critical to brain development. We found that 2/8 HMG samples showed trisomy of chromosome 1q, encompassing many genes, including AKT3, which is known to regulate brain size. A third case showed a known activating mutation in AKT3 (c.49G→A, creating p.E17K) that was not present in the patient’s blood cells. Remarkably, the E17K mutation in AKT3 is exactly paralogous to E17K mutations in AKT1 and AKT2 recently discovered in somatic overgrowth syndromes. We show that AKT3 is the most abundant AKT paralogue in brain during neurogenesis and that phosphorylated AKT is abundant in cortical progenitor cells. Our data suggest that somatic mutations limited to brain could represent an important cause of complex neurogenetic disease. PMID:22500628

  14. 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.

  15. Early Endosomal Antigen 1 (EEA1) Is an Obligate Scaffold for Angiotensin II-induced, PKC-α-dependent Akt Activation in Endosomes*

    PubMed Central

    Nazarewicz, Rafal Robert; Salazar, Gloria; Patrushev, Nikolay; Martin, Alejandra San; Hilenski, Lula; Xiong, Shiqin; Alexander, R. Wayne

    2011-01-01

    Akt/protein kinase B (PKB) activation/phosphorylation by angiotensin II (Ang II) is a critical signaling event in hypertrophy of vascular smooth muscle cells (VSMCs). Conventional wisdom asserts that Akt activation occurs mainly in plasma membrane domains. Recent evidence that Akt activation may take place within intracellular compartments challenges this dogma. The spatial identity and mechanistic features of these putative signaling domains have not been defined. Using cell fractionation and fluorescence methods, we demonstrate that the early endosomal antigen-1 (EEA1)-positive endosomes are a major site of Ang II-induced Akt activation. Akt moves to and is activated in EEA1 endosomes. The expression of EEA1 is required for phosphorylation of Akt at both Thr-308 and Ser-473 as well as for phosphorylation of its downstream targets mTOR and S6 kinase, but not for Erk1/2 activation. Both Akt and phosphorylated Akt (p-Akt) interact with EEA1. We also found that PKC-α is required for organizing Ang II-induced, EEA1-dependent Akt phosphorylation in VSMC early endosomes. EEA1 expression enables PKC-α phosphorylation, which in turn regulates Akt upstream signaling kinases, PDK1 and p38 MAPK. Our results indicate that PKC-α is a necessary regulator of EEA1-dependent Akt signaling in early endosomes. Finally, EEA1 down-regulation or expression of a dominant negative mutant of PKC-α blunts Ang II-induced leucine incorporation in VSMCs. Thus, EEA1 serves a novel function as an obligate scaffold for Ang II-induced Akt activation in early endosomes. PMID:21097843

  16. Regulation of neutrophil apoptosis by modulation of PKB/Akt activation.

    PubMed

    Rane, Madhavi J; Klein, Jon B

    2009-01-01

    The serine/threonine kinase, Akt, also known as PKB (Protein Kinase B) is one important signal transduction pathway that mediates the delay of neutrophil apoptosis caused by inflammatory mediators. Proteins controlled by the PKB/Akt pathway have been reported to prevent or reverse apoptotic-signaling pathways and regulate cell survival. In this review we discuss the role of PKB/Akt activation in the regulation of neutrophil activation during inflammation, and the importance of resolving the inflammatory response by inhibiting PKB/Akt activation and neutrophil survival. Furthermore, we introduce the concept of a dynamic Akt signal complex that is altered when an extracellular signal is initiated such that changes in protein-protein interactions within the Akt signal complex regulates Akt activity and cell survival. Various substrates of PKB/Akt as well as positive and negative regulators of PKB/Akt activation are discussed which in turn inhibit or enhance cellular survival. PMID:19273208

  17. Active Akt and functional p53 modulate apoptosis in Abelson virus-transformed pre-B cells.

    PubMed

    Gong, Li; Unnikrishnan, Indira; Raghavan, Anuradha; Parmar, Kalindi; Rosenberg, Naomi

    2004-02-01

    Suppression of apoptosis is an important feature of the Abelson murine leukemia virus (Ab-MLV) transformation process. During multistep transformation, Ab-MLV-infected pre-B cells undergo p53-dependent apoptosis during the crisis phase of transformation. Even once cells are fully transformed, an active v-Abl protein tyrosine kinase is required to suppress apoptosis because cells transformed by temperature-sensitive (ts) kinase mutants undergo rapid apoptosis after a shift to the nonpermissive temperature. However, inactivation of the v-Abl protein by a temperature shift interrupts signals transmitted via multiple pathways, making it difficult to identify those that are critically important for the suppression of apoptosis. To begin to dissect these pathways, we tested the ability of an SH2 domain Ab-MLV mutant, P120/R273K, to rescue aspects of the ts phenotype of pre-B cells transformed by the conditional kinase domain mutant. The P120/R273K mutant suppressed apoptosis at the nonpermissive temperature, a phenotype correlated with its ability to activate Akt. Apoptosis also was suppressed at the nonpermissive temperature by constitutively active Akt and in p53-null pre-B cells transformed with the ts kinase domain mutant. These data indicate that an intact Src homology 2 (SH2) domain is not critical for apoptosis suppression and suggest that signals transmitted through Akt and p53 play an important role in the response. PMID:14747529

  18. 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

  19. 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

  20. Radiation-induced Akt activation modulates radioresistance in human glioblastoma cells

    PubMed Central

    Li, Hui-Fang; Kim, Jung-Sik; Waldman, Todd

    2009-01-01

    Background Ionizing radiation (IR) therapy is a primary treatment for glioblastoma multiforme (GBM), a common and devastating brain tumor in humans. IR has been shown to induce PI3K-Akt activation in many cell types, and activation of the PI3K-Akt signaling pathway has been correlated with radioresistance. Methods Initially, the effects of IR on Akt activation were assessed in multiple human GBM cell lines. Next, to evaluate a potential causative role of IR-induced Akt activation on radiosensitivity, Akt activation was inhibited during IR with several complementary genetic and pharmacological approaches, and radiosensitivity measured using clonogenic survival assays. Results Three of the eight cell lines tested demonstrated IR-induced Akt activation. Further studies revealed that IR-induced Akt activation was dependent upon the presence of a serum factor, and could be inhibited by the EGFR inhibitor AG1478. Inhibition of PI3K activation with LY294002, or with inducible wild-type PTEN, inhibition of EGFR, as well as direct inhibition of Akt with two Akt inhibitors during irradiation increased the radiosensitivity of U87MG cells. Conclusion These results suggest that Akt may be a central player in a feedback loop whereby activation of Akt induced by IR increases radioresistance of GBM cells. Targeting the Akt signaling pathway may have important therapeutic implications when used in combination with IR in the treatment of a subset of brain tumor patients. PMID:19828040

  1. 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.

  2. 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.

  3. Activated AKT/PKB signaling in C. elegans uncouples temporally distinct outputs of DAF-2/insulin-like signaling

    PubMed Central

    Gami, Minaxi S; Iser, Wendy B; Hanselman, Keaton B; Wolkow, Catherine A

    2006-01-01

    screen for suppressors of PI3K mutant phenotypes identified activating mutations in two known pathway components, providing insights into their regulation. In particular, the interdependence of akt-1 and pdk-1, even in activated forms, supports the existence of AGE-1-independent pathways for these phospholipid-dependent kinases. Phenotypic analysis of these alleles shows that the larval and adult outputs of AGE-1/PI3K are fully separable in these mutants. PMID:17020605

  4. 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

  5. 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.

  6. 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

  7. Interdomain conformational changes in Akt activation revealed by chemical cross-linking and tandem mass spectrometry.

    PubMed

    Huang, Bill X; Kim, Hee-Yong

    2006-06-01

    Akt, a serine/threonine kinase, plays a critical role in cell survival. Upon growth factor receptor stimulation, cytosolic Akt is recruited to the plasma membrane by phospholipid binding and activated through phosphorylation at Thr(308) and Ser(473). Although crystal structures for the parts of Akt have been reported, neither the three-dimensional structure of the whole molecule nor sequential conformational changes during activation have been demonstrated. In this study, we demonstrated that Akt undergoes dramatic interdomain conformational changes during activation processes by probing the three-dimensional structure of full-length Akt in solution using chemical cross-linking and tandem mass spectrometry. The cross-linking results not only provided new structural information but also revealed distinctive spatial arrangements of individual domains in the Akt molecule in resting, membrane-interacted, phosphorylated, and substrate-bound states. Our data allowed a new model for stepwise interdomain conformational changes in Akt activation sequence, setting a stage for the further investigation on Akt-membrane, Akt-protein, and/or Akt-drug interactions in solution to understand molecular mechanisms involved in physiological and pathophysiological processes of cell survival. PMID:16531397

  8. 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

  9. 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.

  10. 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

  11. Site Specific Activation of AKT Protects Cells from Death Induced by Glucose Deprivation

    PubMed Central

    Gao, Meng; Liang, Jiyong; Lu, Yiling; Guo, Huifang; German, Peter; Bai, Shanshan; Jonasch, Eric; Yang, Xingsheng; Mills, Gordon B.; Ding, Zhiyong

    2013-01-01

    The serine/threonine kinase AKT is a key mediator of cancer cell survival. We demonstrate that transient glucose deprivation modestly induces AKT phosphorylation at both Thr308 and Ser473. In contrast, prolonged glucose deprivation induces selective AKTThr308 phosphorylation and phosphorylation of a distinct subset of AKT downstream targets leading to cell survival under metabolic stress. Glucose deprivation-induced AKTThr308 phosphorylation is dependent on PDK1 and PI3K but not EGFR or IGF1R. Prolonged glucose deprivation induces the formation of a complex of AKT, PDK1, and the GRP78 chaperone protein, directing phosphorylation of AKTThr308 but AKTSer473. Our results reveal a novel mechanism of AKT activation under prolonged glucose deprivation that protects cells from metabolic stress. The selective activation of AKTThr308 phosphorylation that occurs during prolonged nutrient deprivation may provide an unexpected opportunity for the development and implementation of drugs targeting cell metabolism and aberrant AKT signaling. PMID:23396361

  12. 17β-estradiol activates mTOR in chondrocytes by AKT-dependent and AKT-independent signaling pathways

    PubMed Central

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

    2015-01-01

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

  13. Gastrointestinal growth factors and hormones have divergent effects on Akt activation

    PubMed Central

    Berna, Marc J.; Tapia, Jose A.; Sancho, Veronica; Thill, Michelle; Pace, Andrea; Hoffmann, K. Martin; Gonzalez-Fernandez, Lauro; Jensen, Robert T.

    2009-01-01

    Akt is a central regulator of apoptosis, cell growth and survival. Growth factors and some G-protein-coupled receptors (GPCR) regulate Akt. Whereas growth-factor activation of Akt has been extensively studied, the regulation of Akt by GPCR's, especially gastrointestinal hormones/neurotransmitters, remains unclear. To address this area, in this study the effects of GI growth factors and hormones/neurotransmitters were investigate in rat pancreatic acinar cells which are high responsive to these agents. Pancreatic acini expressed Akt and 5 of 7 known pancreatic growth-factors stimulate Akt phosphorylation (T308, S473) and translocation. These effects are mediated by p85 phosphorylation and activation of PI3K. GI hormones increasing intracellular cAMP had similar effects. However, GI-hormones/neurotransmitters[CCK, bombesin,carbachol] activating phospholipase C (PLC) inhibited basal and growth-factor-stimulated Akt activation. Detailed studies with CCK, which has both physiological and pathophysiological effects on pancreatic acinar cells at different concentrations, demonstrated CCK has a biphasic effect: at low concentrations(pM) stimulating Akt by a Src-dependent mechanism and at higher concentrations(nM) inhibited basal and stimulated Akt translocation, phosphorylation and activation, by de-phosphorylating p85 resulting in decreasing PI3K activity. This effect required activation of both limbs of the PLC-pathway and a protein tyrosine phosphatase, but was not mediated by p44/42 MAPK, Src or activation of a serine phosphatase. Akt inhibition by CCK was also found in vivo and in Panc-1 cancer cells where it inhibited serum-mediated rescue from apoptosis. These results demonstrate that GI growth factors as well as gastrointestinal hormones/neurotransmitters with different cellular basis of action can all regulate Akt phosphorylation in pancreatic acinar cells. This regulation is complex with phospholipase C agents such as CCK, because both stimulatory and inhibitory

  14. 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

  15. 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.

  16. Akt is activated in chronic lymphocytic leukemia cells and delivers a pro-survival signal: the therapeutic potential of Akt inhibition

    PubMed Central

    Zhuang, Jianguo; Hawkins, Stephen F.; Glenn, Mark A.; Lin, Ke; Johnson, Gillian G.; Carter, Anthony; Cawley, John C.; Pettitt, Andrew R.

    2010-01-01

    Background The aims of the present study were to ascertain the activation status of Akt in the primary cells of chronic lymphocytic leukemia and to investigate the effects of specific Akt inhibition on chronic lymphocytic leukemia-cell survival. Design and Methods Anti-phospho-Akt (Ser473 or Thr308) antibodies and western blotting were used to establish the activation status of Akt. The effects of two different, specific small-molecule inhibitors (A-443654 or Akti-1/2) or small interfering RNA on cell survival and downstream targets of Akt were assessed. Apoptosis was determined by fluorescence-activated cell sorting analysis of phosphatidylserine exposure and by measurement of PARP cleavage. The phosphorylation status of GSK-3 and MDM2, two immediate downstream substrates of Akt, levels of the anti-apoptotic proteins BCL2 and MCL1, and expression of p53 and p21 were all measured by western blotting. Results Fully activated Akt was demonstrable in all chronic lymphocytic leukemia clones examined (n=26). These results were validated with extensive controls and it was shown that a harsh method of cell extraction is needed for detection of the active enzyme. Specific inhibition of Akt induced extensive apoptosis of chronic lymphocytic leukemia cells, which was associated with both a rapid loss of MCL1 through proteasomal degradation and increased expression of p53. Moreover, the Akt inhibitors, at concentrations that induced extensive apoptosis in chronic lymphocytic leukemia cells, had little or no effect on normal peripheral blood mononuclear cells. Conclusions Chronic lymphocytic leukemia clones consistently contain activated Akt which plays a pivotal role in maintaining cell survival. Inhibition of the Akt pathway may be of potential value as a novel therapeutic strategy in chronic lymphocytic leukemia. PMID:19713228

  17. Prediabetes Linked to Excess Glucagon in Transgenic Mice with Pancreatic Active AKT1

    PubMed Central

    Albury-Warren, Toya M.; Pandey, Veethika; Spinel, Lina P.; Masternak, Michal M.; Altomare, Deborah A

    2015-01-01

    Protein Kinase B/AKT, has three isoforms (AKT1-3) and is renowned for its central role in the regulation of cell growth and proliferation, due to its constitutive activation in various cancers. AKT2, which is highly expressed in insulin responsive tissues, has been identified as a primary regulator of glucose metabolism as Akt2 knockout mice (Akt2−/−) are glucose intolerant and insulin resistant. However, the role of AKT1 in glucose metabolism is not as clearly defined. We previously showed that mice with myristoylated Akt1 (AKT1Myr) expressed through a bicistronic Pdx1-TetA and TetO-MyrAkt1 system were susceptible to islet cell carcinomas, and in this study we characterized an early onset, prediabetic phenotype. Beginning at weaning (3 weeks of age), the glucose intolerant AKT1Myr mice exhibited non-fasted hyperglycemia, which progressed to fasted hyperglycemia by 5 months of age. The glucose intolerance was attributed to a fasted hyperglucagonemia, and hepatic insulin resistance detectable by reduced phosphorylation of the insulin receptor following insulin injection into the inferior vena cava. In contrast, treatment with doxycycline diet to turn-off the transgene, caused attenuation of the non-fasted and fasted hyperglycemia, thus affirming AKT1 hyperactivation as the trigger. Collectively, this model highlights a novel glucagon-mediated mechanism by which AKT1 hyperactivation affects glucose homeostasis, and provides an avenue to better delineate the molecular mechanisms responsible for diabetes mellitus and the potential association with pancreatic cancer. PMID:26487674

  18. Prediabetes linked to excess glucagon in transgenic mice with pancreatic active AKT1.

    PubMed

    Albury-Warren, Toya M; Pandey, Veethika; Spinel, Lina P; Masternak, Michal M; Altomare, Deborah A

    2016-01-01

    Protein kinase B/AKT has three isoforms (AKT1-3) and is renowned for its central role in the regulation of cell growth and proliferation, due to its constitutive activation in various cancers. AKT2, which is highly expressed in insulin-responsive tissues, has been identified as a primary regulator of glucose metabolism as Akt2 knockout mice (Akt2(-/-)) are glucose-intolerant and insulin-resistant. However, the role of AKT1 in glucose metabolism is not as clearly defined. We previously showed that mice with myristoylated Akt1 (AKT1(Myr)) expressed through a bicistronic Pdx1-TetA and TetO-MyrAkt1 system were susceptible to islet cell carcinomas, and in this study we characterized an early onset, prediabetic phenotype. Beginning at weaning (3 weeks of age), the glucose-intolerant AKT1(Myr) mice exhibited non-fasted hyperglycemia, which progressed to fasted hyperglycemia by 5 months of age. The glucose intolerance was attributed to a fasted hyperglucagonemia, and hepatic insulin resistance detectable by reduced phosphorylation of the insulin receptor following insulin injection into the inferior vena cava. In contrast, treatment with doxycycline diet to turn off the transgene caused attenuation of the non-fasted and fasted hyperglycemia, thus affirming AKT1 hyperactivation as the trigger. Collectively, this model highlights a novel glucagon-mediated mechanism by which AKT1 hyperactivation affects glucose homeostasis and provides an avenue to better delineate the molecular mechanisms responsible for diabetes mellitus and the potential association with pancreatic cancer. PMID:26487674

  19. Activating Akt and the brain's resources to drive cellular survival and prevent inflammatory injury

    PubMed Central

    Chong, Z.Z.; Li, F.; Maiese, K.

    2008-01-01

    Summary Protein kinase B, also known as Akt, is a serine/threonine kinase and plays a critical role in the modulation of cell development, growth, and survival. Interestingly, Akt is ubiquitously expressed throughout the body, but its expression in the nervous system is substantially up-regulated during cellular stress, suggesting a more expansive role for Akt in the nervous system that may involve cellular protection. In this regard, a body of recent work has identified a robust capacity for Akt and its downstream substrates to foster both neuronal and vascular survival during apoptotic injury. Cell survival by Akt is driven by the modulation of both intrinsic cellular pathways that oversee genomic DNA integrity and extrinsic mechanisms that control inflammatory microglial activation. A series of distinct pathways are regulated by Akt that include the Forkhead family of transcription factors, GSK-3ß, ß-catenin, c-Jun, CREB, Bad, IKK, and p53. Culminating below these substrates of Akt are the control of caspase mediated pathways that promote genomic integrity as well as prevent inflammatory cell demise. With further levels of progress in defining the cellular role of Akt, the attractiveness of Akt as a vital and broad cytoprotectant for both neuronal and vascular cell populations should continue to escalate. PMID:15578447

  20. 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

  1. 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

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

    PubMed Central

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

    2008-01-01

    The PI3K/PTEN/Akt/mTOR pathway plays critical roles in the regulation of cell growth. The effects of this pathway on drug resistance and cellular senescence of breast cancer cells has been a focus of our laboratory. Introduction of activated Akt or mutant PTEN constructs which lack lipid phosphatase [PTEN(G129E)] or lipid and protein phosphatase [PTEN(C124S)] activity increased the resistance of the cells to the chemotherapeutic drug doxorubicin, and the hormonal drug tamoxifen. Activated Akt and PTEN genes also inhibited the induction of senescence after doxorubicin treatment; a phenomenon associated with unrestrained proliferation and tumorigenesis. Interference with the lipid phosphatase domain of PTEN was sufficient to activate Akt/mTOR/p70S6K as MCF-7 cells transfected with the mutant PTEN gene lacking the lipid phosphatase activity [PTEN(G129E)] displayed elevated levels of activated Akt and p70S6K compared to empty vector transfected cells. Cells transfected with mutant PTEN or Akt constructs were hypersensitive to mTOR inhibitors when compared with the parental or empty vector transfected cells. Akt-transfected cells were cultured for over two months in tamoxifen from which tamoxifen and doxorubicin resistant cells were isolated that were >10-fold more resistant to tamoxifen and doxorubicin than the original Akt-transfected cells. These cells had a decreased induction of both activated p53 and total p21Cip1 upon doxorubicin treatment. Furthermore, these cells had an increased inactivation of GSK-3β and decreased expression of the estrogen receptor-α. In these drug resistant cells, there was an increased activation of ERK which is associated with proliferation. These drug resistant cells were hypersensitive to mTOR inhibitors and also sensitive to MEK inhibitors, indicating that the enhanced p70S6K and ERK expression was relevant to their drug and hormonal resistance. Given that Akt is overexpressed in greater than 50% of breast cancers, our results point

  3. 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.

  4. Activation of the PI3K/AKT Pathway in Merkel Cell Carcinoma

    PubMed Central

    Baeurle, Anne; Ritter, Cathrin; Schrama, David; Landthaler, Michael; Becker, Juergen C.

    2012-01-01

    Merkel cell carcinoma (MCC) is a highly aggressive skin cancer with an increasing incidence. The understanding of the molecular carcinogenesis of MCC is limited. Here, we scrutinized the PI3K/AKT pathway, one of the major pathways activated in human cancer, in MCC. Immunohistochemical analysis of 41 tumor tissues and 9 MCC cell lines revealed high levels of AKT phosphorylation at threonine 308 in 88% of samples. Notably, the AKT phosphorylation was not correlated with the presence or absence of the Merkel cell polyoma virus (MCV). Accordingly, knock-down of the large and small T antigen by shRNA in MCV positive MCC cells did not affect phosphorylation of AKT. We also analyzed 46 MCC samples for activating PIK3CA and AKT1 mutations. Oncogenic PIK3CA mutations were found in 2/46 (4%) MCCs whereas mutations in exon 4 of AKT1 were absent. MCC cell lines demonstrated a high sensitivity towards the PI3K inhibitor LY-294002. This finding together with our observation that the PI3K/AKT pathway is activated in the majority of human MCCs identifies PI3K/AKT as a potential new therapeutic target for MCC patients. PMID:22363598

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

    PubMed Central

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

    2006-01-01

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

  6. Rapid accumulation of Akt in mitochondria following phosphatidylinositol 3-kinase activation.

    PubMed

    Bijur, Gautam N; Jope, Richard S

    2003-12-01

    We describe here a new component of the phosphatidylinositol 3-kinase/Akt signaling pathway that directly impacts mitochondria. Akt (protein kinase B) was shown for the first time to be localized in mitochondria, where it was found to reside in the matrix and the inner and outer membranes, and the level of mitochondrial Akt was very dynamically regulated. Stimulation of a variety of cell types with insulin-like growth factor-1, insulin, or stress (induced by heat shock), induced translocation of Akt to the mitochondria within only several minutes of stimulation, causing increases of nearly eight- to 12-fold, and the mitochondrial Akt was in its phosphorylated, active state. Two mitochondrial proteins were identified to be phosphorylated following stimulation of mitochondrial Akt, the beta-subunit of ATP synthase and glycogen synthase kinase-3beta. The finding that mitochondrial glycogen synthase kinase-3beta was rapidly and substantially modified by Ser9 phosphorylation, which inhibits its activity, following translocation of Akt to the mitochondria is the first evidence for a regulatory mechanism affecting mitochondrial glycogen synthase kinase-3beta. These results demonstrate that signals emanating from plasma membrane receptors or generated by stress rapidly modulate Akt and glycogen synthase kinase-3beta in mitochondria. PMID:14713298

  7. 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

  8. DNA-PK mediates AKT activation and apoptosis inhibition in clinically acquired platinum resistance.

    PubMed

    Stronach, Euan A; Chen, Michelle; Maginn, Elaina N; Agarwal, Roshan; Mills, Gordon B; Wasan, Harpreet; Gabra, Hani

    2011-11-01

    Clinical resistance to chemotherapy is a frequent event in cancer treatment and is closely linked to poor outcome. High-grade serous (HGS) ovarian cancer is characterized by p53 mutation and high levels of genomic instability. Treatment includes platinum-based chemotherapy and initial response rates are high; however, resistance is frequently acquired, at which point treatment options are largely palliative. Recent data indicate that platinum-resistant clones exist within the sensitive primary tumor at presentation, implying resistant cell selection after treatment with platinum chemotherapy. The AKT pathway is central to cell survival and has been implicated in platinum resistance. Here, we show that platinum exposure induces an AKT-dependent, prosurvival, DNA damage response in clinically platinum-resistant but not platinum-sensitive cells. AKT relocates to the nucleus of resistant cells where it is phosphorylated specifically on S473 by DNA-dependent protein kinase (DNA-PK), and this activation inhibits cisplatin-mediated apoptosis. Inhibition of DNA-PK or AKT, but not mTORC2, restores platinum sensitivity in a panel of clinically resistant HGS ovarian cancer cell lines: we also demonstrate these effects in other tumor types. Resensitization is associated with prevention of AKT-mediated BAD phosphorylation. Strikingly, in patient-matched sensitive cells, we do not see enhanced apoptosis on combining cisplatin with AKT or DNA-PK inhibition. Insulin-mediated activation of AKT is unaffected by DNA-PK inhibitor treatment, suggesting that this effect is restricted to DNA damage-mediated activation of AKT and that, clinically, DNA-PK inhibition might prevent platinum-induced AKT activation without interfering with normal glucose homeostasis, an unwanted toxicity of direct AKT inhibitors. PMID:22131882

  9. DNA-PK Mediates AKT Activation and Apoptosis Inhibition in Clinically Acquired Platinum Resistance12

    PubMed Central

    Stronach, Euan A; Chen, Michelle; Maginn, Elaina N; Agarwal, Roshan; Mills, Gordon B; Wasan, Harpreet; Gabra, Hani

    2011-01-01

    Clinical resistance to chemotherapy is a frequent event in cancer treatment and is closely linked to poor outcome. High-grade serous (HGS) ovarian cancer is characterized by p53 mutation and high levels of genomic instability. Treatment includes platinum-based chemotherapy and initial response rates are high; however, resistance is frequently acquired, at which point treatment options are largely palliative. Recent data indicate that platinum-resistant clones exist within the sensitive primary tumor at presentation, implying resistant cell selection after treatment with platinum chemotherapy. The AKT pathway is central to cell survival and has been implicated in platinum resistance. Here, we show that platinum exposure induces an AKT-dependent, prosurvival, DNA damage response in clinically platinum-resistant but not platinum-sensitive cells. AKT relocates to the nucleus of resistant cells where it is phosphorylated specifically on S473 by DNA-dependent protein kinase (DNA-PK), and this activation inhibits cisplatin-mediated apoptosis. Inhibition of DNA-PK or AKT, but not mTORC2, restores platinum sensitivity in a panel of clinically resistant HGS ovarian cancer cell lines: we also demonstrate these effects in other tumor types. Resensitization is associated with prevention of AKT-mediated BAD phosphorylation. Strikingly, in patient-matched sensitive cells, we do not see enhanced apoptosis on combining cisplatin with AKT or DNA-PK inhibition. Insulin-mediated activation of AKT is unaffected by DNA-PK inhibitor treatment, suggesting that this effect is restricted to DNA damage-mediated activation of AKT and that, clinically, DNA-PK inhibition might prevent platinum-induced AKT activation without interfering with normal glucose homeostasis, an unwanted toxicity of direct AKT inhibitors. PMID:22131882

  10. Optogenetic activation reveals distinct roles of PIP3 and Akt in adipocyte insulin action.

    PubMed

    Xu, Yingke; Nan, Di; Fan, Jiannan; Bogan, Jonathan S; Toomre, Derek

    2016-05-15

    Glucose transporter 4 (GLUT4; also known as SLC2A4) resides on intracellular vesicles in muscle and adipose cells, and translocates to the plasma membrane in response to insulin. The phosphoinositide 3-kinase (PI3K)-Akt signaling pathway plays a major role in GLUT4 translocation; however, a challenge has been to unravel the potentially distinct contributions of PI3K and Akt (of which there are three isoforms, Akt1-Akt3) to overall insulin action. Here, we describe new optogenetic tools based on CRY2 and the N-terminus of CIB1 (CIBN). We used these 'Opto' modules to activate PI3K and Akt selectively in time and space in 3T3-L1 adipocytes. We validated these tools using biochemical assays and performed live-cell kinetic analyses of IRAP-pHluorin translocation (IRAP is also known as LNPEP and acts as a surrogate marker for GLUT4 here). Strikingly, Opto-PIP3 largely mimicked the maximal effects of insulin stimulation, whereas Opto-Akt only partially triggered translocation. Conversely, drug-mediated inhibition of Akt only partially dampened the translocation response of Opto-PIP3 In spatial optogenetic studies, focal targeting of Akt to a region of the cell marked the sites where IRAP-pHluorin vesicles fused, supporting the idea that local Akt-mediated signaling regulates exocytosis. Taken together, these results indicate that PI3K and Akt play distinct roles, and that PI3K stimulates Akt-independent pathways that are important for GLUT4 translocation. PMID:27076519

  11. 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

  12. 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. PMID:25770423

  13. AKT activation controls cell survival in response to HDAC6 inhibition.

    PubMed

    Kaliszczak, M; Trousil, S; Ali, T; Aboagye, E O

    2016-01-01

    HDAC6 is emerging as an important therapeutic target for cancer. We investigated mechanisms responsible for survival of tumor cells treated with a HDAC6 inhibitor. Expression of the 20 000 genes examined did not change following HDAC6 treatment in vivo. We found that HDAC6 inhibition led to an increase of AKT activation (P-AKT) in vitro, and genetic knockdown of HDAC6 phenocopied drug-induced AKT activation. The activation of AKT was not observed in PTEN null cells; otherwise, PTEN/PIK3CA expression per se did not predict HDAC6 inhibitor sensitivity. Interestingly, HDAC6 inhibitor treatment led to inactivating phosphorylation of PTEN (P-PTEN Ser380), which likely led to the increased P-AKT in cells that express PTEN. Synergy was observed with phosphatidylinositol 3'-kinases (PI3K) inhibitor treatment in vitro, accompanied by increased caspase 3/7 activity. Furthermore, combination of HDAC6 inhibitor with a PI3K inhibitor caused substantial tumor growth inhibition in vivo compared with either treatment alone, also detectable by Ki-67 immunostaining and (18)F-FLT positron emission tomography (PET). In aggregate AKT activation appears to be a key survival mechanism for HDAC6 inhibitor treatment. Our findings indicate that dual inhibition of HDAC6 and P-AKT may be necessary to substantially inhibit growth of solid tumors. PMID:27362804

  14. Recurrent AKT mutations in human cancers: functional consequences and effects on drug sensitivity

    PubMed Central

    Yi, Kyung H.; Lauring, Josh

    2016-01-01

    Precision oncology trials based on tumor gene sequencing depend on robust knowledge about the phenotypic consequences of the genetic variants identified in patients' tumors. Mutations in AKT1-3 occur in 3-5% of human cancers. Although a single hotspot mutation, E17K, is the most common, well characterized activating mutations account for a minority of Akt variants that have been identified in large tumor sequencing studies to date. In order to determine the potential clinical relevance of both common and rare Akt mutations, we expressed a set of over twenty recurrent Akt mutants in three different cell lines and evaluated activation of Akt pathway signaling and effects on growth. We determined their relative sensitivity to allosteric and ATP-competitive Akt inhibitors in clinical development. Most Akt mutants did not activate pathway signaling compared to wild type Akt and did not affect growth properties. In addition, the most common activating Akt mutations, including Akt1 E17K, L52R, and Q79K conferred neither sensitivity nor resistance to Akt inhibitors. Equivocal evidence was found that Akt1 D323H and Akt2 W80C mutants are relatively resistant to the allosteric Akt inhibitor MK-2206, but not an ATP-competitive inhibitor. Our results suggest that the vast majority of rare Akt variants are passenger mutations with no effect on drug sensitivity. The hypothesis that activating Akt mutations predict for Akt inhibitor sensitivity remains to be tested clinically, but is not yet supported by our preclinical data. PMID:26701849

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

    PubMed

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

    2014-07-18

    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 Li2CO3 significantly inhibited phosphoinositide 3-kinase (PI3K)-mediated phosphorylation of Akt1 at Ser473, but Li2CO3 did not affect PI3K-mediated PI(3,4,5)P3 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 Li2CO3 on Akt1-induced phosphorylation of GSK-3β at Ser9, but otherwise Li2CO3 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 Li2CO3 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. PMID:24950409

  16. Activation of Akt by the Bacterial Inositol Phosphatase, SopB, is Wortmannin Insensitive

    PubMed Central

    Cooper, Kendal G.; Winfree, Seth; Malik-Kale, Preeti; Jolly, Carrie; Ireland, Robin; Knodler, Leigh A.; Steele-Mortimer, Olivia

    2011-01-01

    Salmonella enterica uses effector proteins translocated by a Type III Secretion System to invade epithelial cells. One of the invasion-associated effectors, SopB, is an inositol phosphatase that mediates sustained activation of the pro-survival kinase Akt in infected cells. Canonical activation of Akt involves membrane translocation and phosphorylation and is dependent on phosphatidyl inositide 3 kinase (PI3K). Here we have investigated these two distinct processes in Salmonella infected HeLa cells. Firstly, we found that SopB-dependent membrane translocation and phosphorylation of Akt are insensitive to the PI3K inhibitor wortmannin. Similarly, depletion of the PI3K regulatory subunits p85α and p85ß by RNAi had no inhibitory effect on SopB-dependent Akt phosphorylation. Nevertheless, SopB-dependent phosphorylation does depend on the Akt kinases, PDK1 and rictor-mTOR. Membrane translocation assays revealed a dependence on SopB for Akt recruitment to Salmonella ruffles and suggest that this is mediated by phosphoinositide (3,4) P2 rather than phosphoinositide (3,4,5) P3. Altogether these data demonstrate that Salmonella activates Akt via a wortmannin insensitive mechanism that is likely a class I PI3K-independent process that incorporates some essential elements of the canonical pathway. PMID:21779406

  17. Microcystin-LR promotes proliferation by activating Akt/S6K1 pathway and disordering apoptosis and cell cycle associated proteins phosphorylation in HL7702 cells.

    PubMed

    Liu, Jinghui; Wang, Hao; Wang, Beilei; Chen, Tao; Wang, Xiaofeng; Huang, Pu; Xu, Lihong; Guo, Zonglou

    2016-01-01

    Our previous studies had shown that MC-LR inhibited PP2A activity and hyperphosphorylated PP2A substrates at 24 h exposure in HL7702 cells. Although the cytoskeleton was rearranged, the cellular effects were not observed. The purpose of the present study with HL7702 cell exposed to MC-LR for 1-72 h was to further uncover the adverse effects of MC-LR comprehensively. The results showed that there were no obvious difference in apoptosis rate and cell-cycle distribution but the cell proliferation was changed since 36 h exposure while the uptake of MC-LR and its binding to PP2A/C kept unchanged since 1h exposure. PP2A activity had not manifested continued decline compare to 24h exposure and PP2A regulator α4 was found to release its associated PP2A/C since 1h exposure. The increasing of p-Akt-T308, p-Akt-S473, p-S6K1, p-S6, and p-4E-BP1 since 1h MC-LR exposure indicated that Akt/S6K1 cascade had been activated as early as 1h MC-LR treatment. And, PI3K/Akt inhibitor (LY294002) blocked MC-LR-induced Akt/S6K1 activation and proliferation. Besides, MC-LR also led to hyperphosphorylation of c-Myc, c-Jun, Bcl-2 and Bad and activation of Cdk1. Our study indicated that MC-LR exposure promoted HL7702 cell proliferation and the main mechanism was the activation of Akt/S6K1 cascade. Meanwhile, hyperphosphorylation of Bcl-2, Bad, c-Myc and c-Jun might also be involved. And, the inhibition of PP2A was the major reason for these molecular changes. PMID:26506538

  18. 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

  19. TRAIL-induced caspase/p38 activation is responsible for the increased catalytic and invasive activities of Akt

    PubMed Central

    SUN, BO K.; KIM, JOO-HANG; NGUYEN, HOAN N.; KIM, SO Y.; OH, SEEUN; LEE, YONG J.; SONG, JAE J.

    2010-01-01

    We previously observed that TRAIL induces acquired TRAIL resistance coinciding with increased Akt phosphorylation brought about by the Src-PI3K-Akt signaling pathways and mediated by c-Cbl. c-Cbl, a ubiquitously expressed cytoplasmic adaptor protein, is simultaneously involved in the rapid degradation of TRAIL receptors and Akt phosphorylation during TRAIL treatment. Here, we show that Akt phosphorylation is not exclusively responsible for acquired TRAIL resistance. Akt catalytic activation is known to increase during metabolic oxidative stress, but we show that TRAIL also dramatically induces the catalytic activation of Akt in TRAIL-sensitive cells, but not in TRAIL-resistant cells. This suggests that Akt catalytic activation during TRAIL-induced apoptosis is likely to play a compensatory role in the maintenance of cell homeostasis. In addition, activated p38 and phosphorylated HSP27 were found to act as downstream effector molecules of p38 during TRAIL treatment and were shown to be responsible for increased Akt catalytic and invasive activities. PMID:21109947

  20. 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.

  1. Induced Dwarf Mutant in Catharanthus roseus with Enhanced Antibacterial Activity

    PubMed Central

    Verma, A. K.; Singh, R. R.

    2010-01-01

    Evaluation of an ethyl methane sulphonate-induced dwarf mutant of Catharanthus roseus (L.) G. Don revealed that the mutant exhibited marked variation in morphometric parameters. The in vitro antibacterial activity of the aqueous and alcoholic leaf extracts of the mutant and control plants was investigated against medically important bacteria. The mutant leaf extracts showed enhanced antibacterial activity against all the tested bacteria except Bacillus subtilis. PMID:21695004

  2. 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.

  3. The majority of early primordial germ cells acquire pluripotency by AKT activation.

    PubMed

    Matsui, Yasuhisa; Takehara, Asuka; Tokitake, Yuko; Ikeda, Makiko; Obara, Yuka; Morita-Fujimura, Yuiko; Kimura, Tohru; Nakano, Toru

    2014-12-01

    Primordial germ cells (PGCs) are undifferentiated germ cells in embryos, the fate of which is to become gametes; however, mouse PGCs can easily be reprogrammed into pluripotent embryonic germ cells (EGCs) in culture in the presence of particular extracellular factors, such as combinations of Steel factor (KITL), LIF and bFGF (FGF2). Early PGCs form EGCs more readily than do later PGCs, and PGCs lose the ability to form EGCs by embryonic day (E) 15.5. Here, we examined the effects of activation of the serine/threonine kinase AKT in PGCs during EGC formation; notably, AKT activation, in combination with LIF and bFGF, enhanced EGC formation and caused ∼60% of E10.5 PGCs to become EGCs. The results indicate that the majority of PGCs at E10.5 could acquire pluripotency with an activated AKT signaling pathway. Importantly, AKT activation did not fully substitute for bFGF and LIF, and AKT activation without both LIF and bFGF did not result in EGC formation. These findings indicate that AKT signal enhances and/or collaborates with signaling pathways of bFGF and of LIF in PGCs for the acquisition of pluripotency. PMID:25359722

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

    PubMed Central

    Nguyen, Le Xuan Truong; Mitchell, Beverly S.

    2013-01-01

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

  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. PMID:19056378

  6. Akt-dependent NF-{kappa}B activation is required for bile acids to rescue colon cancer cells from stress-induced apoptosis

    SciTech Connect

    Shant, Jasleen; Cheng, Kunrong; Marasa, Bernard S.; Wang Jianying; 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-{kappa}B. Deoxycholyltaurine (DCT) treatment attenuated TNF-{alpha}-induced colon cancer cell apoptosis, and stimulated rapid and sustained NF-{kappa}B nuclear translocation and transcriptional activity (detected by NF-{kappa}B binding to an oligonucleotide consensus sequence and by activation of luciferase reporter gene constructs). Both DCT-induced NF-{kappa}B nuclear translocation and attenuation of TNF-{alpha}-stimulated apoptosis were dependent on EGFR activation. Inhibitors of nuclear translocation, proteosome activity, and I{kappa}B{alpha} kinase attenuated NF-{kappa}B transcriptional activity. Cell transfection with adenoviral vectors encoding a non-degradable I{kappa}B{alpha} 'super-repressor' blocked the actions of DCT on both NF-{kappa}B 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-{kappa}B transcriptional activity and TNF-{alpha}-induced apoptosis. Chemical inhibitors of Akt and NF-{kappa}B 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-{kappa}B activation. These findings provide a mechanism whereby bile acids increase resistance of colon cancer to chemotherapy and radiation.

  7. Reduced RKIP enhances nasopharyngeal carcinoma radioresistance by increasing ERK and AKT activity

    PubMed Central

    Yuan, Li; Yi, Hong-Mei; Yi, Hong; Qu, Jia-Quan; Zhu, Jin-Feng; Li, Li-Na; Xiao, Ta; Zheng, Zhen; Lu, Shan-Shan; Xiao, Zhi-Qiang

    2016-01-01

    Raf kinase inhibitory protein (RKIP) functions as a chemo-immunotherapeutic sensitizer of cancers, but regulation of RKIP on tumor radiosensitivity remains largely unexplored. In this study, we investigate the role and mechanism of RKIP in nasopharyngeal carcinoma (NPC) radioresistance. The results showed that RKIP was frequently downregulated in the radioresistant NPC tissues compared with radiosensitive NPC tissues, and its reduction correlated with NPC radioresistance and poor patient survival, and was an independent prognostic factor. In vitro radioresponse assay showed that RKIP overexpression decreased while RKIP knockdown increased NPC cell radioresistance. In the NPC xenografts, RKIP overexpression decreased while RKIP knockdown increased tumor radioresistance. Mechanistically, RKIP reduction promoted NPC cell radioresistance by increasing ERK and AKT activity, and AKT may be a downstream transducer of ERK signaling. Moreover, the levels of phospho-ERK−1/2 and phospho-AKT were increased in the radioresistant NPC tissues compared with radiosensitive ones, and negatively associated with RKIP expression, indicating that RKIP-regulated NPC radioresponse is mediated by ERK and AKT signaling in the clinical samples. Our data demonstrate that RKIP is a critical determinant of NPC radioresponse, and its reduction enhances NPC radioresistance through increasing ERK and AKT signaling activity, highlighting the therapeutic potential of RKIP-ERK-AKT signaling axis in NPC radiosensitization. PMID:26862850

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

    PubMed Central

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

    2008-01-01

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

  9. 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. PMID:26198194

  10. Pyruvate Kinase M2 Activates mTORC1 by Phosphorylating AKT1S1.

    PubMed

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

    2016-01-01

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

  11. Pyruvate Kinase M2 Activates mTORC1 by Phosphorylating AKT1S1

    PubMed Central

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

    2016-01-01

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

  12. 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

  13. 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

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

    PubMed Central

    Rauch, Jens; Kolch, Walter; Mahmoudi, Morteza

    2012-01-01

    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. PMID:23162692

  15. 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.

  16. Swimming activity in dystonia musculorum mutant mice.

    PubMed

    Lalonde, R; Joyal, C C; Cote, C

    1993-07-01

    Dystonia musculorum (dt) mutant mice, characterized by degeneration of spinocerebellar fibers, were evaluated in a visible platform swim test. It was found that dt mutants were slower to reach the platform than normal mice. However, the number of quadrants traversed was not higher in dt mutants. It is concluded that spinocerebellar fibers to the vermis are important in limb control during swimming but not in visuo-motor guidance (navigational skills) of the animal towards a visible goal, at least in regard to the quadrant measure. It is not excluded that a measure tracing their path may find a mild deviation from the goal. PMID:8327590

  17. Rapid activation of ERK1/2 and AKT in human breast cancer cells by cadmium

    SciTech Connect

    Liu Zhiwei; Yu Xinyuan; Shaikh, Zahir A.

    2008-05-01

    Cadmium (Cd), an endocrine disruptor, can induce a variety of signaling events including the activation of ERK1/2 and AKT. In this study, the involvement of estrogen receptors (ER) in these events was evaluated in three human breast caner cell lines, MCF-7, MDA-MB-231, and SK-BR-3. The Cd-induced signal activation patterns in the three cell lines mimicked those exhibited in response to 17{beta}-estradiol. Specifically, treatment of MCF-7 cells, that express ER{alpha}, ER{beta} and GPR30, to 0.5-10 {mu}M Cd for only 2.5 min resulted in transient phosphorylation of ERK1/2. Cd also triggered a gradual increase and sustained activation of AKT during the 60 min treatment period. In SK-BR-3 cells, that express only GPR30, Cd also caused a transient activation of ERK1/2, but not of AKT. In contrast, in MDA-MB-231 cells, that express only ER{beta}, Cd was unable to cause rapid activation of either ERK1/2 or AKT. A transient phosphorylation of ER{alpha} was also observed within 2.5 min of Cd exposure in the MCF-7 cells. While the estrogen receptor antagonist, ICI 182,780, did not prevent the effect of Cd on these signals, specific siRNA against hER{alpha} significantly reduced Cd-induced activation of ERK1/2 and completely blocked the activation of AKT. It is concluded that Cd, like estradiol, can cause rapid activation of ERK1/2 and AKT and that these signaling events are mediated by possible interaction with membrane ER{alpha} and GPR30, but not ER{beta}.

  18. 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

  19. 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.

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

    PubMed Central

    Hinton, Cimona V.; Fitzgerald, Latricia D.; Thompson, Marilyn E.

    2007-01-01

    Signaling pathways involved in regulating nuclear-cytoplasmic distribution of BRCA1 have not been previously reported. Here, we provide evidence that heregulin β1-induced activation of the Akt pathway increases the nuclear content of BRCA1. First, treatment of T47D breast cancer cells with heregulin β1 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 β1. 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. PMID:17428466

  1. Activation of the thrombopoietin receptor by mutant calreticulin in CALR-mutant myeloproliferative neoplasms.

    PubMed

    Araki, Marito; Yang, Yinjie; Masubuchi, Nami; Hironaka, Yumi; Takei, Hiraku; Morishita, Soji; Mizukami, Yoshihisa; Kan, Shin; Shirane, Shuichi; Edahiro, Yoko; Sunami, Yoshitaka; Ohsaka, Akimichi; Komatsu, Norio

    2016-03-10

    Recurrent somatic mutations of calreticulin (CALR) have been identified in patients harboring myeloproliferative neoplasms; however, their role in tumorigenesis remains elusive. Here, we found that the expression of mutant but not wild-type CALR induces the thrombopoietin (TPO)-independent growth of UT-7/TPO cells. We demonstrated that c-MPL, the TPO receptor, is required for this cytokine-independent growth of UT-7/TPO cells. Mutant CALR preferentially associates with c-MPL that is bound to Janus kinase 2 (JAK2) over the wild-type protein. Furthermore, we demonstrated that the mutant-specific carboxyl terminus portion of CALR interferes with the P-domain of CALR to allow the N-domain to interact with c-MPL, providing an explanation for the gain-of-function property of mutant CALR. We showed that mutant CALR induces the phosphorylation of JAK2 and its downstream signaling molecules in UT-7/TPO cells and that this induction was blocked by JAK2 inhibitor treatment. Finally, we demonstrated that c-MPL is required for TPO-independent megakaryopoiesis in induced pluripotent stem cell-derived hematopoietic stem cells harboring the CALR mutation. These findings imply that mutant CALR activates the JAK2 downstream pathway via its association with c-MPL. Considering these results, we propose that mutant CALR promotes myeloproliferative neoplasm development by activating c-MPL and its downstream pathway. PMID:26817954

  2. 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

  3. 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

  4. Acid sphingomyelinase regulates glucose and lipid metabolism in hepatocytes through AKT activation and AMP-activated protein kinase suppression

    PubMed Central

    Osawa, Yosuke; Seki, Ekihiro; Kodama, Yuzo; Suetsugu, Atsushi; Miura, Kouichi; Adachi, Masayuki; Ito, Hiroyasu; Shiratori, Yoshimune; Banno, Yoshiko; Olefsky, Jerrold M.; Nagaki, Masahito; Moriwaki, Hisataka; Brenner, David A.; Seishima, Mitsuru

    2011-01-01

    Acid sphingomyelinase (ASM) regulates the homeostasis of sphingolipids, including ceramides and sphingosine-1-phosphate (S1P). Because sphingolipids regulate AKT activation, we investigated the role of ASM in hepatic glucose and lipid metabolism. Initially, we overexpressed ASM in the livers of wild-type and diabetic db/db mice by adenovirus vector (Ad5ASM). In these mice, glucose tolerance was improved, and glycogen and lipid accumulation in the liver were increased. Using primary cultured hepatocytes, we confirmed that ASM increased glucose uptake, glycogen deposition, and lipid accumulation through activation of AKT and glycogen synthase kinase-3β. In addition, ASM induced up-regulation of glucose transporter 2 accompanied by suppression of AMP-activated protein kinase (AMPK) phosphorylation. Loss of sphingosine kinase-1 (SphK1) diminished ASM-mediated AKT phosphorylation, but exogenous S1P induced AKT activation in hepatocytes. In contrast, SphK1 deficiency did not affect AMPK activation. These results suggest that the SphK/S1P pathway is required for ASM-mediated AKT activation but not for AMPK inactivation. Finally, we found that treatment with high-dose glucose increased glycogen deposition and lipid accumulation in wild-type hepatocytes but not in ASM−/− cells. This result is consistent with glucose intolerance in ASM−/− mice. In conclusion, ASM modulates AKT activation and AMPK inactivation, thus regulating glucose and lipid metabolism in the liver.—Osawa, Y., Seki, E., Kodama, Y., Suetsugu, A., Miura, K., Adachi, M., Ito, H., Shiratori, Y., Banno, Y., Olefsky, J. M., Nagaki, M., Moriwaki, H., Brenner, D. A., Seishima, M. Acid sphingomyelinase regulates glucose and lipid metabolism in hepatocytes through AKT activation and AMP-activated protein kinase suppression. PMID:21163859

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

    PubMed

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

    2015-11-01

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

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

    PubMed

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

    2013-09-12

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

  7. Mutant p53: Multiple Mechanisms Define Biologic Activity in Cancer

    PubMed Central

    Kim, Michael Paul; Zhang, Yun; Lozano, Guillermina

    2015-01-01

    The functional importance of p53 as a tumor suppressor gene is evident through its pervasiveness in cancer biology. The p53 gene is the most commonly altered gene in human cancer; however, not all genetic alterations are biologically equivalent. The majority of alterations involve p53 missense mutations that result in the production of mutant p53 proteins. Such mutant p53 proteins lack normal p53 function and may concomitantly gain novel functions, often with deleterious effects. Here, we review characterized mechanisms of mutant p53 gain of function in various model systems. In addition, we review mutant p53 addiction as emerging evidence suggests that tumors may depend on sustained mutant p53 activity for continued growth. We also discuss the role of p53 in stromal elements and their contribution to tumor initiation and progression. Lastly, current genetic mouse models of mutant p53 in various organ systems are reviewed and their limitations discussed. PMID:26618142

  8. 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.

  9. Knockin of mutant PIK3CA activates multiple oncogenic pathways

    PubMed Central

    Gustin, John P.; Karakas, Bedri; Weiss, Michele B.; Abukhdeir, Abde M.; Lauring, Josh; Garay, Joseph P.; Cosgrove, David; Tamaki, Akina; Konishi, Hiroyuki; Konishi, Yuko; Mohseni, Morassa; Wang, Grace; Rosen, D. Marc; Denmeade, Samuel R.; Higgins, Michaela J.; Vitolo, Michele I.; Bachman, Kurtis E.; Park, Ben Ho

    2009-01-01

    The phosphatidylinositol 3-kinase subunit PIK3CA is frequently mutated in human cancers. Here we used gene targeting to “knock in” PIK3CA mutations into human breast epithelial cells to identify new therapeutic targets associated with oncogenic PIK3CA. Mutant PIK3CA knockin cells were capable of epidermal growth factor and mTOR-independent cell proliferation that was associated with AKT, ERK, and GSK3β phosphorylation. Paradoxically, the GSK3β inhibitors lithium chloride and SB216763 selectively decreased the proliferation of human breast and colorectal cancer cell lines with oncogenic PIK3CA mutations and led to a decrease in the GSK3β target gene CYCLIN D1. Oral treatment with lithium preferentially inhibited the growth of nude mouse xenografts of HCT-116 colon cancer cells with mutant PIK3CA compared with isogenic HCT-116 knockout cells containing only wild-type PIK3CA. Our findings suggest GSK3β is an important effector of mutant PIK3CA, and that lithium, an FDA-approved therapy for bipolar disorders, has selective antineoplastic properties against cancers that harbor these mutations. PMID:19196980

  10. 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

  11. 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

  12. 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.

  13. Mutant Erythropoietin without Erythropoietic Activity is Neuroprotective against Ischemic Brain Injury

    PubMed Central

    Gan, Yu; Xing, Juan; Jing, Zheng; Stetler, R. Anne; Zhang, Feng; Luo, Yumin; Ji, Xunmin; Gao, Yanqin; Cao, Guodong

    2012-01-01

    Background and Purpose Erythropoietin (EPO) confers potent neuroprotection against ischemic injury. However, treatment for stroke requires high doses and multiple administrations of EPO, which may cause deleterious side effects due to its erythropoietic activity. This study identifies a novel non-erythropoietic mutant EPO (MEPO) and investigates its potential neuroprotective effects and underlying mechanism in animal model of cerebral ischemia. Methods We constructed a series of MEPOs, each containing a single amino acid mutation within the erythropoietic motif, and tested their erythropoietic activity. Using cortical neuronal cultures exposed to NMDA neurotoxicity and a murine model of transient middle cerebral artery occlusion (MCAO), neuroprotection and neurofunctional outcomes were assessed as well as activation of intracellular signaling pathways. Results The serine to isoleucine mutation at position 104 (S104I-EPO) completely abolished the erythropoietic and platelet-stimulating activity of EPO. Administration of S104I-EPO significantly inhibited NMDA-induced neuronal death in primary cultures, and protected against cerebral infarction and neurological deficits with an efficacy similar to that of wild-type EPO. Both S104I-EPO and wild-type EPO activated similar pro-survival signaling pathways, such as PI3K/AKT, MAPK/ERK1/2 and STAT5. Inhibition of PI3K/AKT or MAPK/ERK1/2 signaling pathways significantly attenuated the neuroprotective effects of S104I-EPO, indicating that activation of these pathways underlies the neuroprotective mechanism of MEPO against cerebral ischemia. Conclusions S104I-EPO confers neuroprotective effects comparable to those of wild-type EPO against ischemic brain injury, with the added benefit of lacking erythropoietic and platelet-stimulating side effects. Our novel findings suggest that the non-erythropoietic mutant EPO is a legitimate candidate for ischemic stroke intervention. PMID:22984011

  14. 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

  15. 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

  16. [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. PMID:12942751

  17. 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

  18. Polydatin improves glucose and lipid metabolism in experimental diabetes through activating the Akt signaling pathway.

    PubMed

    Hao, Jie; Chen, Cheng; Huang, Kaipeng; Huang, Junying; Li, Jie; Liu, Peiqing; Huang, Heqing

    2014-12-15

    Recently, the effect of polydatin on lipid regulation has gained considerable attention. And previous study has demonstrated that polydatin has hypoglycemic effect on experimental diabetic rats. Repressed Akt pathway contributes to glucose and lipid disorders in diabetes. Thus, whether polydatin regulates glucose and lipid metabolism in experimental diabetic models through the Akt pathway arouses interest. The purpose was to explore the regulatory mechanism of polydain on glucose and lipid through Akt pathway. We used a diabetic rat model induced by high-fat and -sugar diet with low-dose of streptozocin and an insulin resistant HepG2 cell model induced by palmitic acid to clarify the role of polydatin on glucose and lipid metabolism. Here, we found that polydatin significantly attenuated fasting blood–glucose, glycosylated hemoglobin, glycosylated serum protein, total cholesterol, triglyceride, and low-density lipoprotein cholesterol in diabetic rats. Furthermore, polydatin significantly increased glucose uptake and consumption and decreased lipid accumulation in insulin resistant HepG2 cells. Polydatin markedly increased serum insulin levels in diabetic rats, and obviously activated the Akt signaling pathway in diabetic rat livers and insulin resistant HepG2 cells. Polydatin markedly increased phosphorylated GSK-3β, decreased the protein levels of G6Pase and SREBP-1c, and increased protein levels of GCK, LDLR, and phosphorylated IRS in livers and HepG2 cells. Overall, the results indicate that polydatin regulates glucose and lipid metabolism in experimental diabetic models, the underlying mechanism is probably associated with regulating the Akt pathway. The effect of polydatin on increased Akt phosphorylation is independent of prompting insulin secretion, but dependent of increasing IRS phosphorylation. PMID:25310908

  19. Activation of Akt pathway by transcription-independent mechanisms of retinoic acid promotes survival and invasion in lung cancer cells

    PubMed Central

    2013-01-01

    Background All-trans retinoic acid (ATRA) is currently being used in clinical trials for cancer treatment. The use of ATRA is limited because some cancers, such as lung cancer, show resistance to treatment. However, little is known about the molecular mechanisms that regulate resistance to ATRA treatment. Akt is a kinase that plays a key role in cell survival and cell invasion. Akt is often activated in lung cancer, suggesting its participation in resistance to chemotherapy. In this study, we explored the hypothesis that activation of the Akt pathway promotes resistance to ATRA treatment at the inhibition of cell survival and invasion in lung cancer. We aimed to provide guidelines for the proper use of ATRA in clinical trials and to elucidate basic biological mechanisms of resistance. Results We performed experiments using the A549 human lung adenocarcinoma cell line. We found that ATRA treatment promotes PI3k-Akt pathway activation through transcription-independent mechanisms. Interestingly, ATRA treatment induces the translocation of RARα to the plasma membrane, where it colocalizes with Akt. Immunoprecipitation assays showed that ATRA promotes Akt activation mediated by RARα-Akt interaction. Activation of the PI3k-Akt pathway by ATRA promotes invasion through Rac-GTPase, whereas pretreatment with 15e (PI3k inhibitor) or over-expression of the inactive form of Akt blocks ATRA-induced invasion. We also found that treatment with ATRA induces cell survival, which is inhibited by 15e or over-expression of an inactive form of Akt, through a subsequent increase in the levels of the active form of caspase-3. Finally, we showed that over-expression of the active form of Akt significantly decreases expression levels of the tumor suppressors RARβ2 and p53. In contrast, over-expression of the inactive form of Akt restores RARβ2 expression in cells treated with ATRA, indicating that activation of the PI3k-Akt pathway inhibits the expression of ATRA target genes

  20. 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

  1. Akt-mediated regulation of NFκB and the essentialness of NFκB for the oncogenicity of PI3K and Akt

    PubMed Central

    Bai, Dong; Ueno, Lynn; Vogt, Peter K.

    2009-01-01

    The serine/threonine kinase Akt (cellular homolog of murine thymoma virus akt8 oncogene), also known as PKB (protein kinase B), is activated by lipid products of phosphatidylinositol 3-kinase (PI3K). Akt phosphorylates numerous protein targets that control cell survival, proliferation and motility. Previous studies suggest that Akt regulates transcriptional activity of the nuclear factor-κB (NFκB) by inducing phosphorylation and subsequent degradation of inhibitor of κB (IκB). We show here that NFκB-driven transcription increases in chicken embryonic fibroblasts (CEF) transformed by myristylated Akt (myrAkt). Accordingly, both a dominant negative mutant of Akt and Akt inhibitors repress NFκB-dependent transcription. The degradation of the IκB protein is strongly enhanced in Akt-transformed cells, and the loss of NFκB activity by introduction of a super-repressor of NFκB, IκBSR, interferes with PI3K- and Akt-induced oncogenic transformation of CEF. The phosphorylation of the p65 subunit of NFκB at serine 534 is also upregulated in Akt-transformed cells. Our data suggest that the stimulation of NFκB by Akt is dependent on the phosphorylation of p65 at S534, mediated by IKK (IκB kinase) α and β. Akt phosphorylates IKKα on T23, and this phosphorylation event is a prerequisite for the phosphorylation of p65 at S534 by IKKα and β. Our results demonstrate two separate functions of the IKK complex in NFκB activation in cells with constitutive Akt activity: the phosphorylation and consequent degradation of IκB and the phosphorylation of p65. The data further support the conclusion that NFκB activity is essential for PI3K- and Akt-induced oncogenic transformation. PMID:19609947

  2. 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.

  3. miR-302 Attenuates Amyloid-β-Induced Neurotoxicity through Activation of Akt Signaling.

    PubMed

    Li, Hsin-Hua; Lin, Shi-Lung; Huang, Chien-Ning; Lu, Fung-Jou; Chiu, Pai-Yi; Huang, Wen-Nung; Lai, Te-Jen; Lin, Chih-Li

    2016-01-01

    Deficiency of insulin signaling has been linked to diabetes and ageing-related neurodegenerative diseases such as Alzheimer's disease (AD). In this regard, brains exhibit defective insulin receptor substrate-1 (IRS-1) and hence result in alteration of insulin signaling in progression of AD, the most common cause of dementia. Consequently, dysregulation of insulin signaling plays an important role in amyloid-β (Aβ)-induced neurotoxicity. As the derivation of induced pluripotent stem cells (iPSC) involves cell reprogramming, it may provide a means for regaining the control of ageing-associated dysfunction and neurodegeneration via affecting insulin-related signaling. To this, we found that an embryonic stem cell (ESC)-specific microRNA, miR-302, silences phosphatase and tensin homolog (PTEN) to activate Akt signaling, which subsequently stimulates nuclear factor erythroid 2-related factor 2 (Nrf2)/heme oxygenase-1 (HO-1) elevation and hence inhibits Aβ-induced neurotoxicity. miR-302 is predominantly expressed in iPSCs and is known to regulate several important biological processes of anti-oxidative stress, anti-apoptosis, and anti-aging through activating Akt signaling. In addition, we also found that miR-302-mediated Akt signaling further stimulates Nanog expression to suppress Aβ-induced p-Ser307 IRS-1 expression and thus enhances tyrosine phosphorylation and p-Ser 473-Akt/p-Ser 9-GSK3β formation. Furthermore, our in vivo studies revealed that the mRNA expression levels of both Nanog and miR-302-encoding LARP7 genes were significantly reduced in AD patients' blood cells, providing a novel diagnosis marker for AD. Taken together, our findings demonstrated that miR-302 is able to inhibit Aβ-induced cytotoxicity via activating Akt signaling to upregulate Nrf2 and Nanog expressions, leading to a marked restoration of insulin signaling in AD neurons. PMID:26890744

  4. The unfolded protein response selectively targets active smoothened mutants.

    PubMed

    Marada, Suresh; Stewart, Daniel P; Bodeen, William J; Han, Young-Goo; Ogden, Stacey K

    2013-06-01

    The Hedgehog signaling pathway, an essential regulator of developmental patterning, has been implicated in playing causative and survival roles in a range of human cancers. The signal-transducing component of the pathway, Smoothened, has revealed itself to be an efficacious therapeutic target in combating oncogenic signaling. However, therapeutic challenges remain in cases where tumors acquire resistance to Smoothened antagonists, and also in cases where signaling is driven by active Smoothened mutants that exhibit reduced sensitivity to these compounds. We previously demonstrated that active Smoothened mutants are subjected to prolonged endoplasmic reticulum (ER) retention, likely due to their mutations triggering conformation shifts that are detected by ER quality control. We attempted to exploit this biology and demonstrate that deregulated Hedgehog signaling driven by active Smoothened mutants is specifically attenuated by ER stressors that induce the unfolded protein response (UPR). Upon UPR induction, active Smoothened mutants are targeted by ER-associated degradation, resulting in attenuation of inappropriate pathway activity. Accordingly, we found that the UPR agonist thapsigargin attenuated mutant Smoothened-induced phenotypes in vivo in Drosophila melanogaster. Wild-type Smoothened and physiological Hedgehog patterning were not affected, suggesting that UPR modulation may provide a novel therapeutic window to be evaluated for targeting active Smoothened mutants in disease. PMID:23572559

  5. Denbinobin induces apoptosis in human lung adenocarcinoma cells via Akt inactivation, Bad activation, and mitochondrial dysfunction.

    PubMed

    Kuo, Chen-Tzu; Hsu, Ming-Jen; Chen, Bing-Chang; Chen, Chien-Chih; Teng, Che-Ming; Pan, Shiow-Lin; Lin, Chien-Huang

    2008-02-28

    Increasing evidence demonstrated that denbinobin, isolated from Ephemerantha lonchophylla, exert cytotoxic effects in cancer cells. The purpose of this study was to investigate whether denbinobin induces apoptosis and the apoptotic mechanism of denbinobin in human lung adenocarcinoma cells (A549). Denbinobin (1-20microM) caused cell death in a concentration-dependent manner. Flow cytometric analysis and annexin V labeling demonstrated that denbinobin increased the percentage of apoptotic cells. A549 cells treated with denbinobin showed typical characteristics of apoptosis including morphological changes and DNA fragmentation. Denbinobin induced caspase 3 activation, and N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (zVAD-fmk), a broad-spectrum caspase inhibitor, prevented denbinobin-induced cell death. Denbinobin induced the loss of the mitochondrial membrane potential and the release of mitochondrial apoptotic proteins including cytochrome c, second mitochondria derived activator of caspase (Smac), and apoptosis-inducing factor (AIF). In addition, denbinobin-induced Bad activation was accompanied by the dissociation of Bad with 14-3-3 and the association of Bad with Bcl-xL. Furthermore, denbinobin induced Akt inactivation in a time-dependent manner. Transfection of A549 cells with both wild-type and constitutively active Akt significantly suppressed denbinobin-induced Bad activation and cell apoptosis. These results suggest that Akt inactivation, followed by Bad activation, mitochondrial dysfunction, caspase 3 activation, and AIF release, contributes to denbinobin-induced cell apoptosis. PMID:18262737

  6. Antibiotic drug tigecycline reduces neuroblastoma cells proliferation by inhibiting Akt activation in vitro and in vivo.

    PubMed

    Zhong, Xiaoxia; Zhao, Erhu; Tang, Chunling; Zhang, Weibo; Tan, Juan; Dong, Zhen; Ding, Han-Fei; Cui, Hongjuan

    2016-06-01

    As the first member of glycylcycline bacteriostatic agents, tigecycline is approved as a novel expanded-spectrum antibiotic, which is clinically available. However, accumulating evidence indicated that tigecycline was provided with the potential application in cancer therapy. In this paper, tigecycline was shown to exert an anti-proliferative effect on neuroblastoma cell lines. Furthermore, it was found that tigecycline induced G1-phase cell cycle arrest instead of apoptosis by means of Akt pathway inhibition. In neuroblastoma cell lines, the Akt activator insulin-like growth factor-1 (hereafter referred to as IGF-1) reversed tigecycline-induced cell cycle arrest. Besides, tigecycline inhibited colony formation and suppressed neuroblastoma cells xenograft formation and growth. After tigecycline treatment in vivo, the Akt pathway inhibition was confirmed as well. Collectively, our data provided strong evidences that tigecycline inhibited neuroblastoma cells growth and proliferation through the Akt pathway inhibition in vitro and in vivo. In addition, these results were supported by previous studies concerning the application of tigecycline in human tumors treatment, suggesting that tigecycline might act as a potential candidate agent for neuroblastoma treatment. PMID:26687647

  7. CD133 promotes gallbladder carcinoma cell migration through activating Akt phosphorylation

    PubMed Central

    Zhen, Jiaojiao; Ai, Zhilong

    2016-01-01

    Gallbladder carcinoma (GBC) is the fifth most common malignancy of gastrointestinal tract. The prognosis of gallbladder carcinoma is extremely terrible partially due to metastasis. However, the mechanisms underlying gallbladder carcinoma metastasis remain largely unknown. CD133 is a widely used cancer stem cell marker including in gallbladder carcinoma. Here, we found that CD133 was highly expressed in gallbladder carcinoma as compared to normal tissues. CD133 was located in the invasive areas in gallbladder carcinoma. Down-regulation expression of CD133 inhibited migration and invasion of gallbladder carcinoma cell without obviously reducing cell proliferation. Mechanism analysis revealed that down-regulation expression of CD133 inhibited Akt phosphorylation and increased PTEN protein level. The inhibitory effect of CD133 down-regulation on gallbladder carcinoma cell migration could be rescued by Akt activation. Consistent with this, addition of Akt inhibitor Wortmannin markedly inhibited the migration ability of CD133-overexpressing cells. Thus, down-regulation of CD133 inhibits migration of gallbladder carcinoma cells through reducing Akt phosphorylation. These findings explore the fundamental biological aspect of CD133 in gallbladder carcinoma progression, providing insights into gallbladder carcinoma cell migration. PMID:26910892

  8. 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

  9. Activation of Akt Signaling in Prostate Induces a TGFβ Mediated Restraint on Cancer Progression and Metastasis

    PubMed Central

    Bjerke, Glen A.; Yang, Chun-Song; Frierson, Henry F.; Paschal, Bryce M.; Wotton, David

    2014-01-01

    Mutations in the PTEN tumor suppressor gene are found in a high proportion of human prostate cancers, and in mice, Pten deletion induces high-grade prostate intra-epithelial neoplasia (HGPIN). However, progression from HGPIN to invasive cancer occurs slowly, suggesting that tumorigenesis is subject to restraint. We show that Pten deletion, or constitutive activation of the downstream kinase AKT, activates the transforming growth factor (TGF) β pathway in prostate epithelial cells. TGFβ signaling is known to play a tumor suppressive role in many cancer types, and reduced expression of TGFβ receptors correlates with advanced human prostate cancer. We demonstrate that in combination either with loss of Pten, or expression of constitutively active AKT1, inactivation of TGFβ signaling by deletion of the TGFβ type II receptor gene relieves a restraint on tumorigenesis. This results in rapid progession to lethal prostate cancer, including metastasis to lymph node and lung. In prostate epithelium, inactivation of TGFβ signaling alone is insufficient to initiate tumorigenesis, but greatly accelerates cancer progression. The activation of TGFβ signaling by Pten loss or AKT activation suggests that the same signaling events that play key roles in tumor initiation also induce the activity of a pathway that restrains disease progression. PMID:23995785

  10. Hypoxia and classical activation limits Mycobacterium tuberculosis survival by Akt-dependent glycolytic shift in macrophages

    PubMed Central

    Matta, S K; Kumar, D

    2016-01-01

    Cellular reactive oxygen species (ROS) is a major antibacterial defense mechanism used by macrophages upon activation. Exposure of Mycobacterium tuberculosis (Mtb)-infected macrophages to hypoxia is known to compromise the survival of the pathogen. Here we report that the hypoxia-induced control of intracellular Mtb load in RAW 264.7 macrophages was mediated by regulating the cellular ROS levels. We show that similar to classical activation, hypoxia incubation of macrophages resulted in decreased mitochondrial outer membrane potential (MOMP) and a concomitant increase in the cellular ROS levels. Mitochondrial depolarization and consequently higher ROS could be blocked by knocking down Akt using siRNAs, which acted by inhibiting the switch to glycolytic mode of metabolism, an essential adaptive response upon classical activation or hypoxic incubation of macrophages. Moreover, in the classically activated macrophages or in the macrophages under hypoxia incubation, supplementation with additional glucose had similar effects as Akt knockdown. Interestingly, in both the cases, the reversal of phenotype was linked with the ability of the mitochondrial F0–F1 ATP synthase activity to maintain the MOMP in the absence of oxidative phosphorylation. Both Akt knockdown and glucose supplementation were also able to rescue Mtb survival in these macrophages upon classical activation or hypoxia incubation. These results provide a framework for better understanding of how the interplay between oxygen supply, which is limiting in the human tubercular granulomas, and nutrient availability could together direct the outcome of infections in vivo. PMID:27551515

  11. Deoxycholyltaurine rescues human colon cancer cells from apoptosis by activating EGFR-dependent PI3K/Akt signaling.

    PubMed

    Raufman, Jean-Pierre; Shant, Jasleen; Guo, Chang Yue; Roy, Sanjit; Cheng, Kunrong

    2008-05-01

    Recent studies indicate that secondary bile acids promote colon cancer cell proliferation but their role in maintaining cell survival has not been explored. We found that deoxycholyltaurine (DCT) markedly attenuated both unstimulated and TNF-alpha-stimulated programmed cell death in colon cancer cells by a phosphatidylinositol 3-kinase (PI3K)-dependent mechanism. To examine the role of bile acids and PI3K signaling in maintaining colon cancer cell survival, we explored the role of signaling downstream of bile acid-induced activation of the epidermal growth factor receptor (EGFR) in regulating both apoptosis and proliferation of HT-29 and H508 human colon cancer cells. DCT caused dose- and time-dependent Akt (Ser(473)) phosphorylation, a commonly used marker of activated PI3K/Akt signaling. Both EGFR kinase and PI3K inhibitors attenuated DCT-induced Akt phosphorylation and Akt activation, as demonstrated by reduced phosphorylation of a GSK-3-paramyosin substrate. Transfection of HT-29 cells with kinase-dead EGFR (K721M) reduced DCT-induced Akt phosphorylation. In HT-29 cells, EGFR and PI3K inhibitors as well as transfection with dominant negative AKT attenuated DCT-induced cell proliferation. DCT-induced PI3K/Akt activation resulted in downstream phosphorylation of GSK-3 (Ser(21/9)) and BAD (Ser(136)), and nuclear translocation (activation) of NF-kappaB, thereby confirming that DCT-induced activation of PI3K/Akt signaling regulates both proproliferative and prosurvival signals. Collectively, these results indicate that DCT-induced activation of post-EGFR PI3K/Akt signaling stimulates both colon cancer cell survival and proliferation. PMID:18064605

  12. RUNX2 and the PI3K/AKT axis reciprocal activation as a driving force for tumor progression.

    PubMed

    Cohen-Solal, Karine A; Boregowda, Rajeev K; Lasfar, Ahmed

    2015-01-01

    From the first reported role of the transcription factor RUNX2 in osteoblast and chondrocyte differentiation and migration to its involvement in promigratory/proinvasive behavior of breast, prostate, and thyroid cancer cells, osteosarcoma, or melanoma cells, RUNX2 currently emerges as a key player in metastasis. In this review, we address the interaction of RUNX2 with the PI3K/AKT signaling pathway, one of the critical axes controlling cancer growth and metastasis. AKT, either by directly phosphorylating/activating RUNX2 or phosphorylating/inactivating regulators of RUNX2 stability or activity, contributes to RUNX2 transcriptional activity. Reciprocally, the activation of the PI3K/AKT pathway by RUNX2 regulation of its different components has been described in non-transformed and transformed cells. This mutual activation in the context of cancer cells exhibiting constitutive AKT activation and high levels of RUNX2 might constitute a major driving force in tumor progression and aggressiveness. PMID:26204939

  13. AKT-independent signaling downstream of oncogenic PIK3CA mutations in human cancer.

    PubMed

    Vasudevan, Krishna M; Barbie, David A; Davies, Michael A; Rabinovsky, Rosalia; McNear, Chontelle J; Kim, Jessica J; Hennessy, Bryan T; Tseng, Hsiuyi; Pochanard, Panisa; Kim, So Young; Dunn, Ian F; Schinzel, Anna C; Sandy, Peter; Hoersch, Sebastian; Sheng, Qing; Gupta, Piyush B; Boehm, Jesse S; Reiling, Jan H; Silver, Serena; Lu, Yiling; Stemke-Hale, Katherine; Dutta, Bhaskar; Joy, Corwin; Sahin, Aysegul A; Gonzalez-Angulo, Ana Maria; Lluch, Ana; Rameh, Lucia E; Jacks, Tyler; Root, David E; Lander, Eric S; Mills, Gordon B; Hahn, William C; Sellers, William R; Garraway, Levi A

    2009-07-01

    Dysregulation of the phosphatidylinositol 3-kinase (PI3K) signaling pathway occurs frequently in human cancer. PTEN tumor suppressor or PIK3CA oncogene mutations both direct PI3K-dependent tumorigenesis largely through activation of the AKT/PKB kinase. However, here we show through phosphoprotein profiling and functional genomic studies that many PIK3CA mutant cancer cell lines and human breast tumors exhibit only minimal AKT activation and a diminished reliance on AKT for anchorage-independent growth. Instead, these cells retain robust PDK1 activation and membrane localization and exhibit dependency on the PDK1 substrate SGK3. SGK3 undergoes PI3K- and PDK1-dependent activation in PIK3CA mutant cancer cells. Thus, PI3K may promote cancer through both AKT-dependent and AKT-independent mechanisms. Knowledge of differential PI3K/PDK1 signaling could inform rational therapeutics in cancers harboring PIK3CA mutations. PMID:19573809

  14. AMIGO2, a novel membrane anchor of PDK1, controls cell survival and angiogenesis via Akt activation

    PubMed Central

    Park, Hyojin; Lee, Sungwoon; Shrestha, Pravesh; Kim, Jihye; Park, Jeong Ae; Ko, Yeongrim; Ban, Young Ho; Park, Dae-Young; Ha, Sang-Jun; Koh, Gou Young; Hong, Victor Sukbong; Mochizuki, Naoki; Kim, Young-Myeong; Lee, Weontae

    2015-01-01

    The phosphoinositide 3-kinase–Akt signaling pathway is essential to many biological processes, including cell proliferation, survival, metabolism, and angiogenesis, under pathophysiological conditions. Although 3-phosphoinositide–dependent kinase 1 (PDK1) is a primary activator of Akt at the plasma membrane, the optimal activation mechanism remains unclear. We report that adhesion molecule with IgG-like domain 2 (AMIGO2) is a novel scaffold protein that regulates PDK1 membrane localization and Akt activation. Loss of AMIGO2 in endothelial cells (ECs) led to apoptosis and inhibition of angiogenesis with Akt inactivation. Amino acid residues 465–474 in AMIGO2 directly bind to the PDK1 pleckstrin homology domain. A synthetic peptide containing the AMIGO2 465–474 residues abrogated the AMIGO2–PDK1 interaction and Akt activation. Moreover, it effectively suppressed pathological angiogenesis in murine tumor and oxygen-induced retinopathy models. These results demonstrate that AMIGO2 is an important regulator of the PDK1–Akt pathway in ECs and suggest that interference of the PDK1–AMIGO2 interaction might be a novel pharmaceutical target for designing an Akt pathway inhibitor. PMID:26553931

  15. Livers with Constitutive mTORC1 Activity Resist Steatosis Independent of Feedback Suppression of Akt

    PubMed Central

    Kenerson, Heidi L.; Subramanian, Savitha; McIntyre, Rebecca; Kazami, Machiko; Yeung, Raymond S.

    2015-01-01

    Insulin resistance is an important contributing factor in non-alcoholic fatty liver disease. AKT and mTORC1 are key components of the insulin pathway, and play a role in promoting de novo lipogenesis. However, mTORC1 hyperactivity per se does not induce steatosis in mouse livers, but instead, protects against high-fat diet induced steatosis. Here, we investigate the in vivo mechanism of steatosis-resistance secondary to mTORC1 activation, with emphasis on the role of S6K1-mediated feedback inhibition of AKT. Mice with single or double deletion of Tsc1 and/or S6k1 in a liver-specific or whole-body manner were generated to study glucose and hepatic lipid metabolism between the ages of 6–14 weeks. Following 8 weeks of high-fat diet, the Tsc1-/-;S6k1-/- mice had lower body weights but higher liver TG levels compared to that of the Tsc1-/- mice. However, the loss of S6k1 did not relieve feedback inhibition of Akt activity in the Tsc1-/- livers. To overcome Akt suppression, Pten was deleted in Tsc1-/- livers, and the resultant mice showed improved glucose tolerance compared with the Tsc1-/- mice. However, liver TG levels were significantly reduced in the Tsc1-/-;Pten-/- mice compared to the Pten-/- mice, which was restored with rapamycin. We found no correlation between liver TG and serum NEFA levels. Expression of lipogenic genes (Srebp1c, Fasn) were elevated in the Tsc1-/-;Pten-/- livers, but this was counter-balanced by an up-regulation of Cpt1a involved in fatty acid oxidation and the anti-oxidant protein, Nrf2. In summary, our in vivo models showed that mTORC1-induced resistance to steatosis was dependent on S6K1 activity, but not secondary to AKT suppression. These findings confirm that AKT and mTORC1 have opposing effects on hepatic lipid metabolism in vivo. PMID:25646773

  16. Akt2 influences glycogen synthase activity in human skeletal muscle through regulation of NH2-terminal (sites 2 + 2a) phosphorylation

    PubMed Central

    Birk, Jesper B.; Richter, Erik A.; Ribel-Madsen, Rasmus; Pehmøller, Christian; Hansen, Bo Falck; Beck-Nielsen, Henning; Hirshman, Michael F.; Goodyear, Laurie J.; Vaag, Allan; Poulsen, Pernille; Wojtaszewski, Jørgen F. P.

    2013-01-01

    Type 2 diabetes is characterized by reduced muscle glycogen synthesis. The key enzyme in this process, glycogen synthase (GS), is activated via proximal insulin signaling, but the exact molecular events remain unknown. Previously, we demonstrated that phosphorylation of Thr308 on Akt (p-Akt-Thr308), Akt2 activity, and GS activity in muscle were positively associated with insulin sensitivity. Here, in the same study population, we determined the influence of several upstream elements in the canonical PI3K signaling on muscle GS activation. One-hundred eighty-one nondiabetic twins were examined with the euglycemic hyperinsulinemic clamp combined with excision of muscle biopsies. Insulin signaling was evaluated at the levels of the insulin receptor, IRS-1-associated PI3K (IRS-1-PI3K), Akt, and GS employing activity assays and phosphospecific Western blotting. The insulin-stimulated GS activity was positively associated with p-Akt-Thr308 (P = 0.01) and Akt2 activity (P = 0.04) but not p-Akt-Ser473 or IRS-1-PI3K activity. Furthermore, p-Akt-Thr308 and Akt2 activity were negatively associated with NH2-terminal GS phosphorylation (P = 0.001 for both), which in turn was negatively associated with insulin-stimulated GS activity (P < 0.001). We found no association between COOH-terminal GS phosphorylation and Akt or GS activity. Employing whole body Akt2-knockout mice, we validated the necessity for Akt2 in insulin-mediated GS activation. However, since insulin did not affect NH2-terminal phosphorylation in mice, we could not use this model to validate the observed association between GS NH2-terminal phosphorylation and Akt activity in humans. In conclusion, our study suggests that although COOH-terminal dephosphorylation is likely necessary for GS activation, Akt2-dependent NH2-terminal dephosphorylation may be the site for “fine-tuning” insulin-mediated GS activation in humans. PMID:23321478

  17. Akt1 signaling coordinates BMP signaling and β-catenin activity to regulate second heart field progenitor development.

    PubMed

    Luo, Wen; Zhao, Xia; Jin, Hengwei; Tao, Lichan; Zhu, Jingai; Wang, Huijuan; Hemmings, Brian A; Yang, Zhongzhou

    2015-02-15

    Second heart field (SHF) progenitors exhibit continued proliferation and delayed differentiation, which are modulated by FGF4/8/10, BMP and canonical Wnt/β-catenin signaling. PTEN-Akt signaling regulates the stem cell/progenitor cell homeostasis in several systems, such as hematopoietic stem cells, intestinal stem cells and neural progenitor cells. To address whether PTEN-Akt signaling is involved in regulating cardiac progenitors, we deleted Pten in SHF progenitors. Deletion of Pten caused SHF expansion and increased the size of the SHF derivatives, the right ventricle and the outflow tract. Cell proliferation of cardiac progenitors was enhanced, whereas cardiac differentiation was unaffected by Pten deletion. Removal of Akt1 rescued the phenotype and early lethality of Pten deletion mice, suggesting that Akt1 was the key downstream target that was negatively regulated by PTEN in cardiac progenitors. Furthermore, we found that inhibition of FOXO by Akt1 suppressed the expression of the gene encoding the BMP ligand (BMP7), leading to dampened BMP signaling in the hearts of Pten deletion mice. Cardiac activation of Akt also increased the Ser552 phosphorylation of β-catenin, thus enhancing its activity. Reducing β-catenin levels could partially rescue heart defects of Pten deletion mice. We conclude that Akt signaling regulates the cell proliferation of SHF progenitors through coordination of BMP signaling and β-catenin activity. PMID:25670795

  18. Avian reovirus σA and σNS proteins activate the phosphatidylinositol 3-kinase-dependent Akt signalling pathway.

    PubMed

    Xie, Liji; Xie, Zhixun; Huang, Li; Fan, Qing; Luo, Sisi; Huang, Jiaoling; Deng, Xianwen; Xie, Zhiqin; Zeng, Tingting; Zhang, Yanfang; Wang, Sheng

    2016-08-01

    The present study was conducted to identify avian reovirus (ARV) proteins that can activate the phosphatidylinositol 3-kinase (PI3K)-dependent Akt pathway. Based on ARV protein amino acid sequence analysis, σA, σNS, μA, μB and μNS were identified as putative proteins capable of mediating PI3K/Akt pathway activation. The recombinant plasmids σA-pcAGEN, σNS-pcAGEN, μA-pcAGEN, μB-pcAGEN and μNS-pcAGEN were constructed and used to transfect Vero cells, and the expression levels of the corresponding genes were quantified by immunofluorescence and Western blot analysis. Phosphorylated Akt (P-Akt) levels in the transfected cells were measured by flow cytometry and Western blot analysis. The results showed that the σA, σNS, μA, μB and μNS genes were expressed in Vero cells. σA-expressing and σNS-expressing cells had higher P-Akt levels than negative control cells, pcAGEN-expressing cells and cells designed to express other proteins (i.e., μA, μB and μNS). Pre-treatment with the PI3K inhibitor LY294002 inhibited Akt phosphorylation in σA- and σNS-expressing cells. These results indicate that the σA and σNS proteins can activate the PI3K/Akt pathway. PMID:27233800

  19. Calorie restriction leads to greater Akt2 activity and glucose uptake by insulin-stimulated skeletal muscle from old rats.

    PubMed

    Wang, Haiyan; Arias, Edward B; Cartee, Gregory D

    2016-03-01

    Skeletal muscle insulin resistance is associated with many common age-related diseases, but moderate calorie restriction (CR) can substantially elevate glucose uptake by insulin-stimulated skeletal muscle from both young and old rats. The current study evaluated the isolated epitrochlearis muscle from ∼24.5-mo-old rats that were either fed ad libitum (AL) or subjected to CR (consuming ∼65% of ad libitum, AL, intake beginning at ∼22.5 mo old). Some muscles were also incubated with MK-2206, a potent and selective Akt inhibitor. The most important results were that in isolated muscles, CR vs. AL resulted in 1) greater insulin-stimulated glucose uptake 2) that was accompanied by significantly increased insulin-mediated activation of Akt2, as indicated by greater phosphorylation on both Thr(309) and Ser(474) along with greater Akt2 activity, 3) concomitant with enhanced phosphorylation of several Akt substrates, including an Akt substrate of 160 kDa on Thr(642) and Ser(588), filamin C on Ser(2213) and proline-rich Akt substrate of 40 kDa on Thr(246), but not TBC1D1 on Thr(596); and 4) each of the CR effects was eliminated by MK-2206. These data provide compelling new evidence linking greater Akt2 activation to the CR-induced elevation of insulin-stimulated glucose uptake by muscle from old animals. PMID:26739650

  20. FoxO1-Mediated Activation of Akt Plays a Critical Role in Vascular Homeostasis

    PubMed Central

    Yuan, Lei; Dupuis, Dylan; Beeler, David; Spokes, Katherine C.; Janes, Lauren; Sciuto, Tracey; Kang, Peter M.; Jaminet, Shou-Ching S.; Dvorak, Ann; Grant, Marianne A.; Regan, Erzsébet Ravasz; Aird, William C.

    2016-01-01

    Rationale Forkhead box-O transcription factors (FoxOs) transduce a wide range of extracellular signals, resulting in changes in cell survival, cell cycle progression, and a number of cell type-specific responses. FoxO1 is expressed in many cell types, including endothelial cells. Previous studies have shown that FoxO1 knockout in mice results in embryonic lethality at E11 due to impaired vascular development. In contrast, somatic deletion of FoxO1 is associated with hyperproliferation of endothelial cells. Thus, the precise role of FoxO1 in the endothelium remains enigmatic. Objective To determine the effect of endothelial-specific knockout and overexpression of FoxO1 on vascular homeostasis. Methods and Results We show that endothelial cell (EC)-specific disruption of FoxO1 in mice phenocopies the full knockout. While endothelial expression of FoxO1 rescued otherwise FoxO-null animals, overexpression of constitutively active FoxO1 resulted in increased EC size, occlusion of capillaries, elevated peripheral resistance, heart failure and death. Knockdown of FoxO1 in ECs resulted in marked inhibition of basal and VEGF-induced Akt-mTOR1 signaling. Conclusions Our findings suggest that in mice endothelial expression of FoxO1 is both necessary and sufficient for embryonic development. Moreover, FoxO1-mediated feedback activation of Akt maintains growth factor-responsive Akt/mTORC1 activity within a homeostatic range. PMID:24874427

  1. Dual PI3K/AKT/mTOR inhibitor BEZ235 synergistically enhances the activity of JAK2 inhibitor against cultured and primary human myeloproliferative neoplasm cells.

    PubMed

    Fiskus, Warren; Verstovsek, Srdan; Manshouri, Taghi; Smith, Jacqueline E; Peth, Karissa; Abhyankar, Sunil; McGuirk, Joseph; Bhalla, Kapil N

    2013-05-01

    Hemopoietic progenitor cells (HPC) from myeloproliferative neoplasms (MPN) such as myelofibrosis commonly express mutant JAK2-V617F or other mutations that are associated with increased activities of JAK-STAT5/3, RAS/RAF/MAPK, and PI3K/AKT/mTOR pathways. This confers proliferative and survival advantage on the MPN HPCs. Treatment with JAK tyrosine kinase inhibitor (TKI), for example, TG101209, TG101348 (SAR302503), or INCB018424 (ruxolitinib), inhibits mutant JAK2-mediated signaling. Although effective in reducing constitutional symptoms and splenomegaly, treatment with JAK-TKI does not ameliorate myelofibrosis or significantly improve survival of patients with advanced myelofibrosis. Here, we show that treatment with the dual phosphoinositide-3-kinase (PI3K)/AKT and mTOR inhibitor BEZ235 attenuated PI3K/AKT and mTOR signaling, as well as induced cell-cycle growth arrest and apoptosis of the cultured human JAK2-V617F-expressing HEL92.1.7 (HEL), UKE1 cells, and primary CD34+ myelofibrosis (MF)-MPN cells. Treatment with BEZ235 also induced significant apoptosis of the JAK2-TKI resistant HEL/TGR cells that were selected for resistance against JAK-TKI. Cotreatment with BEZ235 and JAK2-TKI (TG101209 and SAR302503) synergistically induced lethal activity against the cultured and primary CD34+ MPN cells while relatively sparing the normal CD34+ HPCs. These findings create a compelling rationale to determine the in vivo activity of dual PI3K/mTOR inhibitors in combination with JAK inhibitors against myelofibrosis HPCs. PMID:23445613

  2. 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

  3. 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.

  4. Induction of Autophagic Death in Cancer Cells by Agonizing TR3 and Attenuating Akt2 Activity.

    PubMed

    Wang, Wei-jia; Wang, Yuan; Hou, Pei-pei; Li, Feng-wei; Zhou, Bo; Chen, Hang-zi; Bian, Xue-li; Cai, Qi-xu; Xing, Yong-zhen; He, Jian-ping; Zhang, Hongkui; Huang, Pei-qiang; Lin, Tianwei; Wu, Qiao

    2015-08-20

    Apoptotic resistance is becoming a significant obstacle for cancer therapy as the majority of treatment takes the route of apoptotic induction. It is of great importance to develop an alternative strategy to induce cancer cell death. We previously reported that autophagic cell death mediated by nuclear receptor TR3 and driven by a chemical agonist, 1-(3,4,5-trihydroxyphenyl)nonan-1-one (THPN), is highly effective in the therapy of melanoma but not any other cancer types. Here, we discovered that the insensitivity of cancer cells to THPN originated from a high cellular Akt2 activity. Akt2 phosphorylation interferes with TR3 export to cytoplasm and targeting to mitochondria, which lead to the autophagic induction. Therefore, the TR3-mediated autophagy could be effectively induced in the otherwise insensitive cells by downregulating Akt2 activity. Highly effective antineoplastic compounds are developed through optimizing the structure of THPN. This study implicates a general strategy for cancer therapy by the induction of autophagic cell death. PMID:26235054

  5. Dormancy of Cancer Cells with Suppression of AKT Activity Contributes to Survival in Chronic Hypoxia

    PubMed Central

    Endo, Hiroko; Okuyama, Hiroaki; Ohue, Masayuki; Inoue, Masahiro

    2014-01-01

    A hypoxic microenvironment in tumors has been recognized as a cause of malignancy or resistance to various cancer therapies. In contrast to recent progress in understanding the acute response of cancer cells to hypoxia, the characteristics of tumor cells in chronic hypoxia remain elusive. We have identified a pancreatic cancer cell line, AsPC-1, that is exceptionally able to survive for weeks under 1% oxygen conditions while most tested cancer cell lines die after only some days under these conditions. In chronic hypoxia, AsPC-1 cells entered a state of dormancy characterized by no proliferation, no death, and metabolic suppression. They reversibly switched to active status after being placed again in optimal culture conditions. ATP turnover, an indicator of energy demand, was markedly decreased and accompanied by reduced AKT phosphorylation. Forced activation of AKT resulted in increased ATP turnover and massive cell death in vitro and a decreased number of dormant cells in vivo. In contrast to most cancer cell lines, primary-cultured colorectal cancer cells easily entered the dormant status with AKT suppression under hypoxia combined with growth factor–depleted conditions. Primary colorectal cancer cells in dormancy were resistant to chemotherapy. Thus, the ability to survive in a deteriorated microenvironment by entering into dormancy under chronic hypoxia might be a common property among cancer cells. Targeting the regulatory mechanism inducing this dormant status could provide a new strategy for treating cancer. PMID:24905002

  6. Antiplatelet activity of loureirin A by attenuating Akt phosphorylation: In vitro studies.

    PubMed

    Hao, Hong-Zhen; He, Ao-Di; Wang, Dao-Chun; Yin, Zhao; Zhou, Ya-Jun; Liu, Gang; Liang, Ming-Lu; Da, Xing-Wen; Yao, Guang-Qiang; Xie, Wen; Xiang, Ji-Zhou; Ming, Zhang-Yin

    2015-01-01

    Loureirin A is a flavonoid extracted from Dragon׳s Blood that has been used to promote blood circulation and remove stasis in Chinese traditional medicine. However, the mechanisms of these effects are not fully understood. We explored the anti-platelet activity and underlying mechanism of loureirin A in vitro. Our results indicated that loureirin A negatively affected agonist-induced platelet aggregation such as collagen, collagen-related peptide (CRP), ADP and thrombin. Loureirin A inhibited collagen-induced platelet ATP secretion and thrombin-stimulated P-selectin expression in a dose-dependent manner. Platelet spreading on immobilized fibrinogen was significantly impaired in the presence of loureirin A. Immunoblotting analysis indicated that 100μM of loureirin A almost completely eliminated collagen-induced Akt phosphorylation at Ser473. Interestingly, a submaximal dose (50μM) of loureirin A had an additive inhibitory effect with the phosphoinositide 3-kinase (PI3K) inhibitor Ly294002 on collage-induced Akt phosphorylation in platelets. Taken together, loureirin A had an inhibitory effect on platelet activation, perhaps through an impairment of PI3K/Akt signaling. PMID:25445049

  7. Akt Specific Activator SC79 Protects against Early Brain Injury following Subarachnoid Hemorrhage.

    PubMed

    Zhang, Dingding; Zhang, Huasheng; Hao, Shuangying; Yan, Huiying; Zhang, Zihuan; Hu, Yangchun; Zhuang, Zong; Li, Wei; Zhou, Mengliang; Li, Kuanyu; Hang, Chunhua

    2016-06-15

    A growing body of evidence demonstrates that Akt may serve as a therapeutic target for treatment of early brain injury following subarachnoid hemorrhage (SAH). The purpose of the current study was to evaluate the neuroprotective effect of Akt specific activator SC79 in an experimental rat model of SAH. SAH was induced by injecting 300 μL of blood into the prechiasmatic cistern. Intracerebroventricular (ICV) injection of SC79 (30 min post-SAH) induced the p-Akt (Ser473) expression in a dose-dependent manner. A single ICV dose treatment of SC79 (100 μg/rat) significantly increased the expression of Bcl-2 and p-GSK-3β (Ser9), decreased the protein levels of Bax, cytoplasm cytochrome c, and cleaved caspase-3, indicating the antiapoptotic effect of SC79. As a result, the number of apoptotic cells was reduced 24 h post SAH. Moreover, SC79 treatment alleviated SAH-induced oxidative stress, restored mitochondrial morphology, and improved neurological deficits. Strikingly, treatment of SC79 provided a beneficial outcome against neurologic deficit with a therapeutic window of at least 4 h post SAH by ICV injection and 30 min post SAH by intraperitoneal injection. Collectively, SC79 exerts its neuroprotective effect likely through the dual activities of antioxidation and antiapoptosis. These data provide a basic platform to consider SC79 as a novel therapeutic agent for treatment of SAH. PMID:26983552

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

    SciTech Connect

    Kim, Sang-pil; Ha, Jung Min; Yun, Sung Ji; Kim, Eun Kyoung; Chung, Sung Woon; Hong, Ki Whan; Kim, Chi Dae; Bae, Sun Sik

    2010-08-13

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

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

    PubMed Central

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

    2015-01-01

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

  10. Protein Kinase B (AKT) Mediates Phospholipase D Activation via ERK1/2 and Promotes Respiratory Burst Parameters in Formylpeptide-stimulated Neutrophil-like HL-60 Cells*

    PubMed Central

    Patel, Satyananda; Djerdjouri, Bahia; Raoul-Des-Essarts, Yannick; Dang, Pham My-Chan; El-Benna, Jamel; Périanin, Axel

    2010-01-01

    Phospholipase D (PLD), a major source of lipid second messengers (phosphatidic acid, diglycerides) in many cell types, is tightly regulated by protein kinases, but only a few of them have been identified. We show here that protein kinase B (AKT) is a novel major signaling effector of PLD activity induced by the formylpeptide f-Met-Leu-Phe (fMLP) in human neutrophil-like HL-60 cells (dHL-60 cells). AKT inhibition with the selective antagonist AKTib1/2 almost completely prevented fMLP-mediated activity of PLD, its upstream effector ERK1/2, but not p38 MAPK. Immunoprecipitation studies show that phosphorylated AKT, ERK, and PLD2 form a complex induced by fMLP, which can be prevented by AKTib1/2. In cell-free systems, AKT1 stimulated PLD activity via activation of ERK. AKT1 actually phosphorylated ERK2 as a substrate (Km 1 μm). Blocking AKT activation with AKTib1/2 also prevented fMLP- but not phorbol 12-myristate 13-acetate-mediated NADPH oxidase activation (respiratory burst, RB) of dHL-60 cells. Impaired RB was associated with defective membrane translocation of NADPH oxidase components p67phox and p47phox, ERK, AKT1, AKT2, but not AKT3. Depletion of AKT1 or AKT2 with antisense oligonucleotides further indicates a partial contribution of both isoforms in fMLP-induced activation of ERK, PLD, and RB, with a predominant role of AKT1. Thus, formylpeptides induce sequential activation of AKT, ERK1/2, and PLD, which represents a novel signaling pathway. A major primarily role of this AKT signaling pathway also emerges in membrane recruitment of NOX2 components p47phox, p67phox, and ERK, which may contribute to assembly and activation of the RB motor system, NADPH oxidase. PMID:20693286

  11. 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

  12. Modulation in Activation and Expression of PTEN, Akt1, and PDK1: Further Evidence Demonstrating Altered Phosphoinositide 3-kinase Signaling in Postmortem Brain of Suicide Subjects

    PubMed Central

    Dwivedi, Yogesh; Rizavi, Hooriyah S.; Zhang, Hui; Roberts, Rosalinda C.; Conley, Robert R.; Pandey, Ghanshyam N.

    2010-01-01

    Background Phosphoinositide 3-kinase (PI 3-K) signaling plays a crucial role in neuronal growth and plasticity. Recently, we demonstrated that suicide brain is associated with decreased activation and expression of selective catalytic and regulatory subunits of PI 3-K. The present investigation examined the regulation and functional significance of compromised PI 3-K in suicide brain at the level of upstream phosphatase and tensin homolog on chromosome ten (PTEN) and downstream substrates 3-phosphoinositide-dependent kinase 1 (PDK1) and Akt. Method mRNA expression of Akt1, Akt3, PTEN, and PDK1 by competitive RT-PCR; protein expression of Akt1, Akt3, PTEN, PDK1, phosphorylated-Akt1 (Ser473), phosphorylated-Akt1(Thr308), phosphorylated-PDK1, and phosphorylated-PTEN by Western blot; and catalytic activities of Akt1, Akt3, and PDK1 by enzymatic assays were determined in prefrontal cortex (PFC) and hippocampus obtained from suicide subjects and nonpsychiatric controls. Results No significant changes in the expression of Akt1 or Akt3 were observed; however, catalytic activity of Akt1, but not of Akt3, was decreased in PFC and hippocampus of suicide subjects, which was associated with decreased phosphorylation of Akt1 at Ser473 and Thr308. The catalytic activity of PDK1 and the level of phosphorylated-PDK1 were also decreased in both brain areas without any change in expression levels of PDK1. On the other hand, mRNA and protein expression of PTEN was increased, whereas the level of phosphorylated-PTEN was decreased. Conclusion Our study demonstrates abnormalities in PI 3-K signaling at several levels in brain of suicide subjects and suggests the possible involvement of aberrant PI 3-K/Akt signaling in the pathogenic mechanisms of suicide. PMID:20163786

  13. Role of Akt and c-Jun N-terminal Kinase 2 in Apoptosis Induced by Interleukin-4 Deprivation

    PubMed Central

    Cerezo, Ana; Martínez-A, Carlos; Lanzarot, Diego; Fischer, Siegmund; Franke, Thomas F.; Rebollo, Angelita

    1998-01-01

    We have shown previously that interleukin-4 (IL-4) protects TS1αβ cells from apoptosis, but very little is known about the mechanism by which IL-4 exerts this effect. We found that Akt activity, which is dependent on phosphatidylinositol 3 kinase, is reduced in IL-4-deprived TS1αβ cells. Overexpression of wild-type Akt or a constitutively active Akt mutant protects cells from IL-4 deprivation-induced apoptosis. Readdition of IL-4 before the commitment point is able to restore Akt activity. We also show expression and c-Jun N-terminal kinase 2 activation after IL-4 deprivation. Overexpression of the constitutively activated Akt mutant in IL-4-deprived cells correlates with inhibition of c-Jun N-terminal kinase 2 activity. Finally, TS1αβ survival is independent of Bcl-2, Bcl-x, or Bax. PMID:9802900

  14. 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

  15. Selective and potent Akt inhibition triggers anti-myeloma activities and enhances fatal endoplasmic reticulum stress induced by proteasome inhibition

    PubMed Central

    Mimura, Naoya; Hideshima, Teru; Shimomura, Toshiyasu; Suzuki, Rikio; Ohguchi, Hiroto; Rizq, Ola; Kikuchi, Shohei; Yoshida, Yasuhiro; Cottini, Francesca; Jakubikova, Jana; Cirstea, Diana; Gorgun, Gullu; Minami, Jiro; Tai, Yu-Tzu; Richardson, Paul G.; Utsugi, Teruhiro; Iwama, Atsushi; Anderson, Kenneth C.

    2014-01-01

    The PI3K/Akt pathway plays a crucial role in the pathogenesis of multiple myeloma (MM) in the bone marrow (BM) milieu. However, efficacy of selective and potent Akt inhibition has not yet been fully elucidated. In this study, we therefore examined the biologic impact of selective and potent Akt inhibition by a novel allosteric inhibitor TAS-117. TAS-117 induced significant growth inhibition, associated with downregulation of phosphorylated Akt (p-Akt), selectively in MM cell lines with high baseline p-Akt. Cytotoxicity of TAS-117 was also observed in patients MM cells, but not in normal peripheral blood mononuclear cells. Importantly, TAS-117 induced significant cytotoxicity in MM cells even in the presence of BM stromal cells, associated with inhibition of IL-6 secretion. Oral administration of TAS-117 significantly inhibited human MM cell growth in murine xenograft models. TAS-117 triggered apoptosis and autophagy, as well as induction of endoplasmic reticulum (ER) stress response with minimal expression of CHOP, a fatal ER-stress marker. Importantly, TAS-117 enhanced bortezomib-induced cytotoxicity, associated with increased CHOP and PARP cleavage and blockade of bortezomib-induced p-Akt, suggesting that TAS-117 augments bortezomib-induced ER stress and apoptotic signaling. Carfilzomib-induced cytotoxicity was similarly enhanced by TAS-117. Importantly, TAS-117 enhanced bortezomib-induced cytotoxicity in vivo, associated with prolonged host survival. Our results show that selective and potent Akt inhibition by TAS-117 triggers anti-MM activities in vitro and in vivo, as well as enhances cytotoxicity of proteasome inhibition, providing the preclinical framework for clinical evaluation of selective Akt inhibitors, alone and in combination with proteasome inhibitors in MM. PMID:24934808

  16. Selective and potent Akt inhibition triggers anti-myeloma activities and enhances fatal endoplasmic reticulum stress induced by proteasome inhibition.

    PubMed

    Mimura, Naoya; Hideshima, Teru; Shimomura, Toshiyasu; Suzuki, Rikio; Ohguchi, Hiroto; Rizq, Ola; Kikuchi, Shohei; Yoshida, Yasuhiro; Cottini, Francesca; Jakubikova, Jana; Cirstea, Diana; Gorgun, Gullu; Minami, Jiro; Tai, Yu-Tzu; Richardson, Paul G; Utsugi, Teruhiro; Iwama, Atsushi; Anderson, Kenneth C

    2014-08-15

    The PI3K/Akt pathway plays a crucial role in the pathogenesis of multiple myeloma (MM) in the bone marrow (BM) milieu. However, efficacy of selective and potent Akt inhibition has not yet been fully elucidated. In this study, we, therefore, examined the biologic impact of selective and potent Akt inhibition by a novel allosteric inhibitor TAS-117. TAS-117 induced significant growth inhibition, associated with downregulation of phosphorylated Akt (p-Akt), selectively in MM cell lines with high baseline p-Akt. Cytotoxicity of TAS-117 was also observed in patient MM cells, but not in normal peripheral blood mononuclear cells. Importantly, TAS-117 induced significant cytotoxicity in MM cells even in the presence of BM stromal cells, associated with inhibition of IL6 secretion. Oral administration of TAS-117 significantly inhibited human MM cell growth in murine xenograft models. TAS-117 triggered apoptosis and autophagy, as well as induction of endoplasmic reticulum (ER) stress response with minimal expression of C/EBP homologous protein (CHOP), a fatal ER stress marker. Importantly, TAS-117 enhanced bortezomib-induced cytotoxicity, associated with increased CHOP and PARP cleavage and blockade of bortezomib-induced p-Akt, suggesting that TAS-117 augments bortezomib-induced ER stress and apoptotic signaling. Carfilzomib-induced cytotoxicity was similarly enhanced by TAS-117. Importantly, TAS-117 enhanced bortezomib-induced cytotoxicity in vivo, associated with prolonged host survival. Our results show that selective and potent Akt inhibition by TAS-117 triggers anti-MM activities in vitro and in vivo, as well as enhances cytotoxicity of proteasome inhibition, providing the preclinical framework for clinical evaluation of selective Akt inhibitors, alone and in combination with proteasome inhibitors in MM. PMID:24934808

  17. Comparison of activity indexes for recognizing enzyme mutants of higher activity with uricase as model

    PubMed Central

    2013-01-01

    Background For screening a library of enzyme mutants, an efficient and cost-effective method for reliable assay of enzyme activity and a decision method for safe recognition of mutants of higher activity are needed. The comparison of activity concentrations of mutants in lysates of transformed Escherichia coli cells against a threshold is unsafe to recognize mutants of higher activity due to variations of both expression levels of mutant proteins and lysis efficiency of transformed cells. Hence, by a spectrophotometric method after verification to measure uricase activity, specific activity calculated from the level of total proteins in a lysate was tested for recognizing a mutant of higher activity. Results During uricase reaction, the intermediate 5-hydroxyisourate interferes with the assay of uric acid absorbance, but the measurement of absorbance at 293 nm in alkaline borate buffer was reliable for measuring uricase initial rates within a reasonable range. The level of total proteins in a lysate was determined by the Bradford assay. Polyacrylamide gel electrophoresis analysis supported different relative abundance of uricase mutant proteins in their lysates; activity concentrations of uricase in such lysates positively correlated with levels of total proteins. Receiver-operation-curve analysis of activity concentration or specific activity yielded area-under-the-curve close to 1.00 for recognizing a mutant with > 200% improvement of activity. For a mutant with just about 80% improvement of activity, receiver-operation-curve analysis of specific activity gave area-under-the-curve close to 1.00 while the analysis of activity concentration gave smaller area-under-the-curve. With the mean plus 1.4-fold of the standard deviation of specific activity of a starting material as the threshold, uricase mutants whose activities were improved by more than 80% were recognized with higher sensitivity and specificity. Conclusion Specific activity calculated from the level of

  18. AKT upregulates B-Raf Ser445 phosphorylation and ERK1/2 activation in prostate cancer cells in response to androgen depletion

    PubMed Central

    Hong, Seung-Keun; Jeong, Joseph H.; Chan, Andrew M.; Park, Jong-In

    2013-01-01

    Upregulated ERK1/2 activity is often correlated with AKT activation during prostate cancer (PCa) progression, yet their functional relation needs elucidation. Using androgen-deprived LNCaP cells, in which ERK1/2 activation occurs in strong correlation with AKT activation, we found that AKT-mediated B-Raf regulation is necessary for ERK1/2 activation. Specifically, in response to androgen deprivation, AKT upregulated B-Raf phosphorylation at Ser445 without affecting A-Raf or C-Raf-1. This effect of AKT was abolished by Arg25 to Ala mutation or truncating (Δ4-129) the pleckstrin homology domain of AKT, indicating that the canonical AKT regulation is important for this signaling. Intriguingly, although a constitutively active AKT containing N-terminal myristoylation signal could sufficiently upregulate B-Raf phosphorylation at Ser445 in LNCaP cells, subsequent MEK/ERK activation still required hormone deprivation. In contrast, AKT activity was sufficient to induce not only B-Raf phosphorylation but also MEK/ERK activation in the hormone refractory LNCaP variant, C4-2. These data indicate that androgen depletion may induce MEK/ERK activation through a synergy between AKT-dependent and -independent mechanisms and that the latter may become deregulated in association with castration resistance. In support, consistent AKT-mediated BRaf regulation was also detected in a panel of PCa lines derived from the cPten-/- L mice before and after castration. Our results also demonstrate that AKT regulates androgen receptor levels partly via the Raf/MEK/ERK pathway. This study reveals a novel crosstalk between ERK1/2 and AKT in PCa cells. PMID:23701950

  19. BCL-3 expression promotes colorectal tumorigenesis through activation of AKT signalling

    PubMed Central

    Urban, Bettina C; Collard, Tracey J; Eagle, Catherine J; Southern, Samantha L; Greenhough, Alexander; Hamdollah-Zadeh, Maryam; Ghosh, Anil; Paraskeva, Christos; Silver, Andrew; Williams, Ann C

    2016-01-01

    Objective Colorectal cancer remains the fourth most common cause of cancer-related mortality worldwide. Here we investigate the role of nuclear factor-κB (NF-κB) co-factor B-cell CLL/lymphoma 3 (BCL-3) in promoting colorectal tumour cell survival. Design Immunohistochemistry was carried out on 47 tumour samples and normal tissue from resection margins. The role of BCL-3/NF-κB complexes on cell growth was studied in vivo and in vitro using an siRNA approach and exogenous BCL-3 expression in colorectal adenoma and carcinoma cells. The question whether BCL-3 activated the AKT/protein kinase B (PKB) pathway in colorectal tumour cells was addressed by western blotting and confocal microscopy, and the ability of 5-aminosalicylic acid (5-ASA) to suppress BCL-3 expression was also investigated. Results We report increased BCL-3 expression in human colorectal cancers and demonstrate that BCL-3 expression promotes tumour cell survival in vitro and tumour growth in mouse xenografts in vivo, dependent on interaction with NF-κB p50 or p52 homodimers. We show that BCL-3 promotes cell survival under conditions relevant to the tumour microenvironment, protecting both colorectal adenoma and carcinoma cells from apoptosis via activation of the AKT survival pathway: AKT activation is mediated via both PI3K and mammalian target of rapamycin (mTOR) pathways, leading to phosphorylation of downstream targets GSK-3β and FoxO1/3a. Treatment with 5-ASA suppressed BCL-3 expression in colorectal cancer cells. Conclusions Our study helps to unravel the mechanism by which BCL-3 is linked to poor prognosis in colorectal cancer; we suggest that targeting BCL-3 activity represents an exciting therapeutic opportunity potentially increasing the sensitivity of tumour cells to conventional therapy. PMID:26033966

  20. 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

  1. 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

  2. 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

  3. Activation of AKT negatively regulates the pro-apoptotic function of death-associated protein kinase 3 (DAPK3) in prostate cancer.

    PubMed

    Das, Trinath P; Suman, Suman; Papu John, A M Sashi; Pal, Deeksha; Edwards, Angelena; Alatassi, Houda; Ankem, Murali K; Damodaran, Chendil

    2016-07-28

    The activation of AKT governs many signaling pathways and promotes cell growth and inhibits apoptosis in human malignancies including prostate cancer (CaP). Here, we investigated the molecular association between AKT activation and the function of death-associated protein kinase 3 (DAPK3) in CaP. An inverse correlation of pAKT and DAPK3 expression was seen in a panel of CaP cell lines. Inhibition of AKT by wortmannin/LY294002 or overexpression of DAPK3 reverts the proliferative function of AKT in CaP cells. On the other hand, ectopic expression of AKT inhibited DAPK3 function and induced proliferation of CaP cells. In addition, AKT over-expressed tumors exhibit aggressive growth when compared to control vector in xenograft models. The immunohistochemistry results revealed a down-regulation of DAPK3 expression in AKT over-expressed tumors as compared to control tumors. Finally, we examined the expression pattern of AKT and DAPK3 in human CaP specimens - the expected gradual increase and nuclear localization of pAKT was seen in higher Gleason score samples versus benign hyperplasia (BPH). On the contrary, reduced expression of DAPK3 was seen in higher Gleason stages versus BPH. This suggests that inhibition of DAPK3 may be a contributing factor to the carcinogenesis of the prostate. Understanding the mechanism by which AKT negatively regulates DAPK3 function may suggest whether DAPK3 can be a therapeutic target for CaP. PMID:27126362

  4. 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

  5. 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

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

    SciTech Connect

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

    2013-11-29

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

  7. 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

  8. Measurement of constitutive MAPK and PI3K/AKT signaling activity in human cancer cell lines

    PubMed Central

    Paraiso, Kim H.T.; Van Der Kooi, Kaisa; Messina, Jane L.; Smalley, Keiran S. M.

    2014-01-01

    The growth and survival of cancer cells is often driven by constitutive activity in the mitogen activated protein kinase (MAPK) and phospho-inositide 3-kinase (PI3K)/AKT signaling pathways. Activity in these signal transduction cascades is known to contribute to the uncontrolled growth and resistance to apoptosis that characterizes tumor progression. There is now a great deal of interest in therapeutically targeting these pathways in cancer using small molecule inhibitors. In this chapter we describe methods to measure constitutive MAPK and AKT activity in melanoma cell lines, with a focus upon Western blotting, phospho-flow cytometry and immunofluorescence staining techniques. PMID:21036250

  9. Phosphatidylinositol 3-kinase is required for integrin-stimulated AKT and Raf-1/mitogen-activated protein kinase pathway activation.

    PubMed Central

    King, W G; Mattaliano, M D; Chan, T O; Tsichlis, P N; Brugge, J S

    1997-01-01

    Cell attachment to fibronectin stimulates the integrin-dependent interaction of p85-associated phosphatidylinositol (PI) 3-kinase with integrin-dependent focal adhesion kinase (FAK) as well as activation of the Ras/mitogen-activated protein (MAP) kinase pathway. However, it is not known if this PI 3-kinase-FAK interaction increases the synthesis of the 3-phosphorylated phosphoinositides (3-PPIs) or what role, if any, is played by activated PI 3-kinase in integrin signaling. We demonstrate here the integrin-dependent accumulation of the PI 3-kinase products, PI 3,4-bisphosphate [PI(3,4)P2] and PI(3,4,5)P3, as well as activation of AKT kinase, a serine/threonine kinase that can be stimulated by binding of PI(3,4)P2. The PI 3-kinase inhibitors wortmannin and LY294002 significantly decreased the integrin-induced accumulation of the 3-PPIs and activation of AKT kinase, without having significant effects on the levels of PI(4,5)P2 or tyrosine phosphorylation of paxillin. These inhibitors also reduced cell adhesion/spreading onto fibronectin but had no effect on attachment to polylysine. Interestingly, integrin-mediated Erk-2, Mek-1, and Raf-1 activation, but not Ras-GTP loading, was inhibited at least 80% by wortmannin and LY294002. In support of the pharmacologic results, fibronectin activation of Erk-2 and AKT kinases was completely inhibited by overexpression of a dominant interfering p85 subunit of PI 3-kinase. We conclude that integrin-mediated adhesion to fibronectin results in the accumulation of the PI 3-kinase products PI(3,4)P2 and PI(3,4,5)P3 as well as the PI 3-kinase-dependent activation of the kinases Raf-1, Mek-1, Erk-2, and AKT and that PI 3-kinase may function upstream of Raf-1 but downstream of Ras in integrin activation of Erk-2 MAP and AKT kinases. PMID:9234699

  10. 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.

  11. 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

  12. 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

  13. Akt activation is a common event in pediatric malignant gliomas and a potential adverse prognostic marker: a report from the children’s oncology group

    PubMed Central

    Hamilton, Ronald L.; Murdoch, Geoffrey H.; Burger, Peter C.; Brat, Daniel J.; Rosenblum, Marc K.; Nikiforova, Marina N.; Holmes, Emiko J.; Zhou, Tianni; Cohen, Kenneth J.; Jakacki, Regina I.

    2010-01-01

    Aberrant activation of Akt is a common finding in adult malignant gliomas, resulting in most cases from mutations or deletions involving PTEN, which allows constitutive Akt phosphorylation. In contrast, we have previously reported that pediatric malignant gliomas, which are morphologically similar to lesions arising in adults, have a substantially lower incidence of genomic alterations of PTEN. The objective of this study was to determine whether Akt activation was also an uncommon finding in childhood malignant gliomas and whether this feature was associated with survival. To address this issue, we examined the frequency of Akt activation, determined by overexpression of the activated phosphorylated form of Akt (Se473) on immunohistochemical analysis, in a series of 53 childhood malignant gliomas obtained from newly diagnosed patients treated on the Children’s Oncology Group ACNS0126 and 0423 studies. The relationship between Akt activation and p53 over-expression, MIB1 labeling, and tumor histology was evaluated. The association between Akt activation and survival was also assessed. Overexpression of activated Akt was observed in 42 of 53 tumors, far in excess of the frequency of PTEN mutations we have previously observed. There was no association between Akt activation and either histology, p53 overexpression, or MIB1 proliferation indices. Although tumors that lacked Akt overexpression had a trend toward more favorable event-free survival and overall survival (p = 0.06), this association reflected that non-overexpressing tumors were significantly more likely to have undergone extensive tumor removal, which was independently associated with outcome. Activation of Akt is a common finding in pediatric malignant gliomas, although it remains uncertain whether this is an independent adverse prognostic factor. In view of the frequency of Akt activation, the evaluation of molecularly targeted therapies that inhibit this pathway warrants consideration for these tumors

  14. Coordinate direct input of both KRAS and IGF1 receptor to activation of PI 3-kinase in KRAS mutant lung cancer

    PubMed Central

    Molina-Arcas, Miriam; Hancock, David C.; Sheridan, Clare; Kumar, Madhu S.; Downward, Julian

    2013-01-01

    SUMMARY Using a panel of non-small cell lung cancer (NSCLC) lines, we show here that MEK and RAF inhibitors are selectively toxic for the KRAS mutant genotype, while PI 3-kinase (PI3K), AKT and mTOR inhibitors are not. IGF1 receptor (IGF1R) tyrosine kinase inhibitors also show selectivity for KRAS mutant lung cancer lines. Combinations of IGF1R and MEK inhibitors resulted in strengthened inhibition of KRAS mutant lines and also showed improved effectiveness in autochthonous mouse models of Kras induced NSCLC. PI3K pathway activity is dependent on basal IGF1R activity in KRAS mutant, but not wild-type, lung cancer cell lines. KRAS is needed for both MEK and PI3K pathway activity in KRAS mutant, but not wild-type, lung cancer cells, while acute activation of KRAS causes stimulation of PI3K dependent upon IGF1R kinase activity. Coordinate direct input of both KRAS and IGF1R is thus required to activate PI3K in KRAS mutant lung cancer cells. PMID:23454899

  15. 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

  16. Insulator dysfunction and oncogene activation in IDH mutant gliomas.

    PubMed

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

    2016-01-01

    Gain-of-function IDH mutations are initiating events that define major clinical and prognostic classes of gliomas. Mutant IDH protein produces a new onco-metabolite, 2-hydroxyglutarate, which interferes with iron-dependent hydroxylases, including the TET family of 5'-methylcytosine hydroxylases. TET enzymes catalyse a key step in the removal of DNA methylation. IDH mutant gliomas thus manifest a CpG island methylator phenotype (G-CIMP), although the functional importance of this altered epigenetic state remains unclear. Here we show that human IDH mutant gliomas exhibit hypermethylation at cohesin and CCCTC-binding factor (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 interact aberrantly with the receptor tyrosine kinase gene PDGFRA, a prominent glioma oncogene. Treatment of IDH mutant gliomaspheres with a demethylating agent partially restores insulator function and downregulates PDGFRA. Conversely, CRISPR-mediated disruption of the CTCF motif in IDH wild-type gliomaspheres upregulates 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

  17. 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

  18. 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.

  19. miR-93 Promotes Cell Proliferation in Gliomas through Activation of PI3K/Akt Signaling Pathway

    PubMed Central

    Jiang, Lili; Wang, Chanjuan; Lei, Fangyong; Zhang, Longjuan; Zhang, Xin; Liu, Aibin; Wu, Geyan; Zhu, Jinrong; Song, Libing

    2015-01-01

    The PI3K/Akt signaling pathway is frequently activated in various human cancer types and plays essential roles in development and progression of cancers. Multiple regulators, such as phosphatase and tensin homolog (PTEN) and PH domain leucine rich repeat protein phosphatases (PHLPP), have also found to be involved in suppression of the PI3K/Akt signaling pathway. However, how suppressive effects mediated by these regulators are concomitantly disrupted in cancers, which display constitutively activated PI3K/Akt signaling, remains puzzling. In the present study, we reported that the expression of miR-93 was markedly upregulated in glioma cell lines and clinical glioma tissues. Statistical analysis revealed that miR-93 levels significantly correlated with clinicopathologic grade and overall survival in gliomas. Furthermore, we found that overexpressing miR-93 promoted, but inhibition of miR-93 reduced, glioma cell proliferation and cell-cycle progression. We demonstrated that miR-93 activated PI3K/Akt signaling through directly suppressing PTEN, PHLPP2 and FOXO3 expression via targeting their 3′UTRs. Therefore, our results suggest that miR-93 might play an important role in glioma progression and uncover a novel mechanism for constitutive PI3K/Akt activation in gliomas. PMID:25823655

  20. 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

  1. Non-canonical Activation of Akt in Serum-Stimulated Fibroblasts, Revealed by Comparative Modeling of Pathway Dynamics

    PubMed Central

    Nim, Tri Hieu; Luo, Le; White, Jacob K.; Clément, Marie-Véronique; Tucker-Kellogg, Lisa

    2015-01-01

    The dynamic behaviors of signaling pathways can provide clues to pathway mechanisms. In cancer cells, excessive phosphorylation and activation of the Akt pathway is responsible for cell survival advantages. In normal cells, serum stimulation causes brief peaks of extremely high Akt phosphorylation before reaching a moderate steady-state. Previous modeling assumed this peak and decline behavior (i.e., “overshoot”) was due to receptor internalization. In this work, we modeled the dynamics of the overshoot as a tool for gaining insight into Akt pathway function. We built an ordinary differential equation (ODE) model describing pathway activation immediately upstream of Akt phosphorylation at Thr308 (Aktp308). The model was fit to experimental measurements of Aktp308, total Akt, and phosphatidylinositol (3,4,5)-trisphosphate (PIP3), from mouse embryonic fibroblasts with serum stimulation. The canonical Akt activation model (the null hypothesis) was unable to recapitulate the observed delay between the peak of PIP3 (at 2 minutes), and the peak of Aktp308 (at 30–60 minutes). From this we conclude that the peak and decline behavior of Aktp308 is not caused by PIP3 dynamics. Models for alternative hypotheses were constructed by allowing an arbitrary dynamic curve to perturb each of 5 steps of the pathway. All 5 of the alternative models could reproduce the observed delay. To distinguish among the alternatives, simulations suggested which species and timepoints would show strong differences. Time-series experiments with membrane fractionation and PI3K inhibition were performed, and incompatible hypotheses were excluded. We conclude that the peak and decline behavior of Aktp308 is caused by a non-canonical effect that retains Akt at the membrane, and not by receptor internalization. Furthermore, we provide a novel spline-based method for simulating the network implications of an unknown effect, and we demonstrate a process of hypothesis management for guiding efficient

  2. Activation of phosphatidylinositol 3-kinase/Akt signaling mediates sorafenib-induced invasion and metastasis in hepatocellular carcinoma.

    PubMed

    Wang, Haiyong; Xu, Litao; Zhu, Xiaoyan; Wang, Peng; Chi, Huiying; Meng, Zhiqiang

    2014-10-01

    Sorafenib, an antiangiogenic agent, can promote tumor invasion and metastasis. The phosphatidylinositol 3-kinase (PI3K)/Akt/Snail-dependent pathway plays an important role in tumor invasion and metastasis. Yet, little is known concerning the role of the PI3K/Akt/Snail-dependent pathway in sorafenib‑induced invasion and metastasis of hepatic carcinoma (HCC). A human HCC orthotopic xenograft model was established, and sorafenib (30 mg/kg/day) was administered orally. Tumor growth and intrahepatic metastasis were assessed, and immunohistochemistry was applied to analyze the activation of the PI3K/Akt/Snail-dependent pathway. HCC cell lines were treated with sorafenib (1, 5 and 10 µM), and proliferation, migration and invasion were assessed. Western blotting and real-time polymerase chain reaction (RT-PCR) were used to examine the related gene expression of epithelial-mesenchymal transition (EMT) markers and the PI3K/Akt/Snail-dependent pathway. Sorafenib inhibited tumor growth and promoted intrahepatic invasion and metastasis of the orthotopic tumors grown from SMMC7721-GFP cells in vivo. Additionally, sorafenib promoted EMT and invasion and metastasis of HCC cells in vitro. Importantly, sorafenib enhanced PI3K and Akt activation and upregulation of the expression of transcription factor Snail, a critical EMT mediator. The upregulation of transcription factor Snail expression by sorafenib may be related to activation of the PI3K/AKT signaling pathway. The PI3K/Akt/Snail-dependent pathway may mediate the pro-invasive and pro-metastatic effects of sorafenib on HCC by inducing EMT. PMID:25070581

  3. Non-canonical Activation of Akt in Serum-Stimulated Fibroblasts, Revealed by Comparative Modeling of Pathway Dynamics.

    PubMed

    Nim, Tri Hieu; Luo, Le; White, Jacob K; Clément, Marie-Véronique; Tucker-Kellogg, Lisa

    2015-11-01

    The dynamic behaviors of signaling pathways can provide clues to pathway mechanisms. In cancer cells, excessive phosphorylation and activation of the Akt pathway is responsible for cell survival advantages. In normal cells, serum stimulation causes brief peaks of extremely high Akt phosphorylation before reaching a moderate steady-state. Previous modeling assumed this peak and decline behavior (i.e., "overshoot") was due to receptor internalization. In this work, we modeled the dynamics of the overshoot as a tool for gaining insight into Akt pathway function. We built an ordinary differential equation (ODE) model describing pathway activation immediately upstream of Akt phosphorylation at Thr308 (Aktp308). The model was fit to experimental measurements of Aktp308, total Akt, and phosphatidylinositol (3,4,5)-trisphosphate (PIP3), from mouse embryonic fibroblasts with serum stimulation. The canonical Akt activation model (the null hypothesis) was unable to recapitulate the observed delay between the peak of PIP3 (at 2 minutes), and the peak of Aktp308 (at 30-60 minutes). From this we conclude that the peak and decline behavior of Aktp308 is not caused by PIP3 dynamics. Models for alternative hypotheses were constructed by allowing an arbitrary dynamic curve to perturb each of 5 steps of the pathway. All 5 of the alternative models could reproduce the observed delay. To distinguish among the alternatives, simulations suggested which species and timepoints would show strong differences. Time-series experiments with membrane fractionation and PI3K inhibition were performed, and incompatible hypotheses were excluded. We conclude that the peak and decline behavior of Aktp308 is caused by a non-canonical effect that retains Akt at the membrane, and not by receptor internalization. Furthermore, we provide a novel spline-based method for simulating the network implications of an unknown effect, and we demonstrate a process of hypothesis management for guiding efficient

  4. 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

  5. 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

  6. Shikonin Derivative DMAKO-05 Inhibits Akt Signal Activation and Melanoma Proliferation.

    PubMed

    Yang, Yao-Yao; He, Hui-Qiong; Cui, Jia-Hua; Nie, Yun-Juan; Wu, Ya-Xian; Wang, Rui; Wang, Gang; Zheng, Jun-Nian; Ye, Richard D; Wu, Qiong; Li, Shao-Shun; Qian, Feng

    2016-06-01

    DMAKO-05((S)-1-((5E,8E)-5,8-bis(hydroxyimino)-1,4-dimethoxy-5,8-dihydronaphthalen-2-yl)-4-methylpent-3-enyl 3-methylbutanoate) is a novel oxime derivative of shikonin, the major component extracted from Chinese herb Lithospermun erythrorhizon. Here, we report that DMAKO-05 had an antitumor activity against mouse melanoma cell line B16F0. Our studies indicated that DMAKO-05 not only inhibited B16F0 proliferation and migration but also led to cell cycle arrest at G1 phase and cell apoptosis, in which DMAKO-05 triggered mitochondrial-mediated apoptosis signal including caspase-9/3 and PARP. In response to DMAKO-05 treatment, the Akt-mediated survival signals were remarkably attenuated in B16F0 cells. Collectively, DMAKO-05 has a strong cytotoxicity in B16F0 cells via inhibiting Akt activation, inducing G1 arrest, and promoting B16F0 cell apoptosis. DMAKO-05 might serve as a potential candidate lead compound for melanoma. PMID:26804061

  7. Defects in Hemopoietic Stem Cell Activity in Ikaros Mutant Mice

    PubMed Central

    Nichogiannopoulou, Aliki; Trevisan, Maryanne; Neben, Steve; Friedrich, Christoph; Georgopoulos, Katia

    1999-01-01

    Here we provide evidence that the Ikaros family of DNA binding factors is critical for the activity of hemopoietic stem cells (HSCs) in the mouse. Mice homozygous for an Ikaros null mutation display a >30-fold reduction in long-term repopulation units, whereas mice homozygous for an Ikaros dominant negative mutation have no measurable activity. The defect in HSC activity is also illustrated by the ability of wild-type marrow to repopulate unconditioned Ikaros mutants. A progressive reduction in multipotent CFU-S14 (colony-forming unit-spleen) progenitors and the earliest erythroid-restricted precursors (BFU-E [burst-forming unit-erythroid]) is also detected in the Ikaros mutant strains consistent with the reduction in HSCs. Nonetheless, the more mature clonogenic erythroid and myeloid precursors are less affected, indicating either the action of a compensatory mechanism to provide more progeny or a negative role of Ikaros at later stages of erythromyeloid differentiation. In Ikaros mutant mice, a decrease in expression of the tyrosine kinase receptors flk-2 and c-kit is observed in the lineage-depleted c-kit+Sca-1+ population that is normally enriched for HSCs and may in part contribute to the early hemopoietic phenotypes manifested in the absence of Ikaros. PMID:10544193

  8. Antitumor activity of BRAF inhibitor vemurafenib in preclinical models of BRAF-mutant colorectal cancer.

    PubMed

    Yang, Hong; Higgins, Brian; Kolinsky, Kenneth; Packman, Kathryn; Bradley, William D; Lee, Richard J; Schostack, Kathleen; Simcox, Mary Ellen; Kopetz, Scott; Heimbrook, David; Lestini, Brian; Bollag, Gideon; Su, Fei

    2012-02-01

    The protein kinase BRAF is a key component of the RAS-RAF signaling pathway which plays an important role in regulating cell proliferation, differentiation, and survival. Mutations in BRAF at codon 600 promote catalytic activity and are associated with 8% of all human (solid) tumors, including 8% to 10% of colorectal cancers (CRC). Here, we report the preclinical characterization of vemurafenib (RG7204; PLX4032; RO5185426), a first-in-class, specific small molecule inhibitor of BRAF(V600E) in BRAF-mutated CRC cell lines and tumor xenograft models. As a single agent, vemurafenib shows dose-dependent inhibition of ERK and MEK phosphorylation, thereby arresting cell proliferation in BRAF(V600)-expressing cell lines and inhibiting tumor growth in BRAF(V600E) bearing xenograft models. Because vemurafenib has shown limited single-agent clinical activity in BRAF(V600E)-mutant metastatic CRC, we therefore explored a range of combination therapies, with both standard agents and targeted inhibitors in preclinical xenograft models. In a BRAF-mutant CRC xenograft model with de novo resistance to vemurafenib (RKO), tumor growth inhibition by vemurafenib was enhanced by combining with an AKT inhibitor (MK-2206). The addition of vemurafenib to capecitabine and/or bevacizumab, cetuximab and/or irinotecan, or erlotinib resulted in increased antitumor activity and improved survival in xenograft models. Together, our findings suggest that the administration of vemurafenib in combination with standard-of-care or novel targeted therapies may lead to enhanced and sustained clinical antitumor efficacy in CRCs harboring the BRAF(V600E) mutation. PMID:22180495

  9. 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.

  10. Dissecting signalling by individual Akt/PKB isoforms, three steps at once.

    PubMed

    Osorio-Fuentealba, Cesar; Klip, Amira

    2015-09-01

    The serine/threonine kinase Akt/PKB (protein kinase B) is key for mammalian cell growth, survival, metabolism and oncogenic transformation. The diverse level and tissue expression of its three isoforms, Akt1/PKBα, Akt2/PKBβ and Akt3/PKBγ, make it daunting to identify isoform-specific actions in vivo and even in isolated tissues/cells. To date, isoform-specific knockout and knockdown have been the best strategies to dissect their individual overall functions. In a recent article in the Biochemical Journal, Kajno et al. reported a new strategy to study isoform selectivity in cell lines. Individual Akt/PKB isoforms in 3T3-L1 pre-adipocytes are first silenced via shRNA and stable cellular clones lacking one or the other isoform are selected. The stably silenced isoform is then replaced by a mutant engineered to be refractory to inhibition by MK-2206 (Akt1(W80A) or Akt2(W80A)). Akt1(W80A) or Akt2(W80A) are functional and effectively recruited to the plasma membrane in response to insulin. The system affords the opportunity to acutely control the activity of the endogenous non-silenced isoform through timely addition of MK-2206. Using this approach, it is confirmed that Akt1/PKBα is the preferred isoform sustaining adipocyte differentiation, but both Akt1/PKBα and Akt2/PKBβ can indistinctly support insulin-dependent FoxO1 (forkhead box O1) nuclear exclusion. Surprisingly, either isoform can also support insulin-dependent glucose transporter (GLUT) 4 translocation to the membrane, in contrast with the preferential role of Akt2/PKBβ assessed by knockdown studies. The new strategy should allow analysis of the plurality of Akt/PKB functions in other cells and in response to other stimuli. It should also be amenable to high-throughput studies to speed up advances in signal transmission by this pivotal kinase. PMID:26348913

  11. 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

  12. 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. PMID:27233246

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

    PubMed

    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

    2015-08-01

    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

  14. 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

  15. 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

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

    PubMed Central

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

    2014-01-01

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

  17. 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

  18. Control of macrophage metabolism and activation by mTOR and Akt signaling

    PubMed Central

    Covarrubias, Anthony J.; Aksoylar, H. Ibrahim; Horng, Tiffany

    2015-01-01

    Macrophages are pleiotropic cells that assume a variety of functions depending on their tissue of residence and tissue state. They maintain homeostasis as well as coordinate responses to stresses such as infection and metabolic challenge. The ability of macrophages to acquire diverse, context-dependent activities requires their activation (or polarization) to distinct functional states. While macrophage activation is well understood at the level of signal transduction and transcriptional regulation, the metabolic underpinnings are poorly understood. Importantly, emerging studies indicate that metabolic shifts play a pivotal role in control of macrophage activation and acquisition of context-dependent effector activities. The signals that drive macrophage activation impinge on metabolic pathways, allowing for coordinate control of macrophage activation and metabolism. Here we discuss how mTOR and Akt, major metabolic regulators and targets of such activation signals, control macrophage metabolism and activation. Dysregulated macrophage activities contribute to many diseases, including infectious, inflammatory, and metabolic diseases and cancer, thus a better understanding of metabolic control of macrophage activation could pave the way to the development of new therapeutic strategies. PMID:26360589

  19. 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

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

    PubMed

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

    2015-10-01

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

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

    PubMed Central

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

    2015-01-01

    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. PMID:26150526

  2. 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. PMID:26150526

  3. The hepatocyte growth factor isoform NK2 activates motogenesis and survival but not proliferation due to lack of Akt activation.

    PubMed

    Mungunsukh, Ognoon; Lee, Young H; Bottaro, Donald P; Day, Regina M

    2016-08-01

    Hepatocyte growth factor (HGF) is a pleiotrophic factor involved in cellular proliferation, migration and morphogenesis. HGF is required for normal tissue and organ development during embryogenesis, but in the adult HGF has been demonstrated to drive normal tissue repair and inhibit fibrotic remodeling. HGF has two naturally occurring human isoforms as a result of alternative splicing, NK1 and NK2. While NK1 has been defined as an agonist for HGF receptor, Met, NK2 is defined as a partial Met antagonist. Furthermore, under conditions of fibrotic remodeling, NK2 is still expressed while full length HGF is suppressed. Furthermore, the mechanism by which NK2 partially signals through Met is not completely understood. Here, we investigated the mitogenic, motogenic, and anti-apoptotic activities of NK2 compared with full length HGF in primary human bronchial epithelial cells (BEpC) and bovine pulmonary artery endothelial cells (PAEC). In human BEpC, NK2 partial activated Met, inducing Met phosphorylation at Y1234/1235 in the tyrosine-kinase domain but not at Y1349 site in the multifunctional docking domain. Partial phosphorylation of Met by NK2 resulted in activation of MAPK and STAT3, but not AKT. This correlated with motogenesis and survival in a MAPK-dependent manner, but not cell proliferation. Overexpression of a constitutively active AKT complemented NK2 signaling, allowing NK2 to induce cell proliferation. These data indicate that NK2 and HGF drive motogenic and anti-apoptotic signaling but only HGF drives cell proliferation by activating AKT-pathway signaling. These results have implications for the biological consequences of differential regulation of the two isoforms under pro-fibrotic conditions. PMID:27224506

  4. A Ral GAP complex links PI 3-kinase/Akt signaling to RalA activation in insulin action

    PubMed Central

    Chen, Xiao-Wei; Leto, Dara; Xiong, Tingting; Yu, Genggeng; Cheng, Alan; Decker, Stuart; Saltiel, Alan R.

    2011-01-01

    Insulin stimulates glucose transport in muscle  and adipose tissue by translocation of glucose transporter 4 (GLUT4) to the plasma membrane. We previously reported that activation of the small GTPase RalA downstream of PI 3-kinase plays a critical role in this process by mobilizing the exocyst complex for GLUT4 vesicle targeting in adipocytes. Here we report the identification and characterization of a Ral GAP complex (RGC) that mediates the activation of RalA downstream of the PI 3-kinase/Akt pathway. The complex is composed of an RGC1 regulatory subunit and an RGC2 catalytic subunit (previously identified as AS250) that directly stimulates the guanosine triphosphate hydrolysis of RalA. Knockdown of RGC proteins leads to increased RalA activity and glucose uptake in adipocytes. Insulin inhibits the GAP complex through Akt2-catalyzed phosphorylation of RGC2 in vitro and in vivo, while activated Akt relieves the inhibitory effect of RGC proteins on RalA activity. The RGC complex thus connects PI 3-kinase/Akt activity to the transport machineries responsible for GLUT4 translocation. PMID:21148297

  5. 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

  6. 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

  7. The basal level of intracellular calcium gates the activation of phosphoinositide 3-kinase - Akt signaling by brain-derived neurotrophic factor in cortical neurons

    PubMed Central

    Zheng, Fei; Soellner, Deborah; Nunez, Joseph; Wang, Hongbing

    2008-01-01

    Brain derived neurotrophic factor (BDNF) mediates survival and neuroplasticity through the activation of phosphoinositide 3-kinase (PI3K)-Akt pathway. Although previous studies suggested the roles of MAPK, PLC-γ-mediated intra-cellular calcium ([Ca2+]i) increase, and extra-cellular calcium influx in regulating Akt activation, the cellular mechanisms are largely unknown. We demonstrated that sub-nanomolar BDNF significantly induced Akt activation in developing cortical neurons. The TrkB-dependent Akt phosphorylation at S473 and T308 required only PI3K, but not PLC and MAPK activity. Blocking NMDA receptors, L-type voltage-gated calcium channels, and chelating extra-cellular calcium by EGTA failed to block BDNF-induced Akt phosphorylation. In contrast, chelating [Ca2+]i by BAPTA-AM abolished Akt phosphorylation. Interestingly, sub-nanomolar BDNF did not stimulate [Ca2+]i increase under our culture conditions. Together with that NMDA- and membrane depolarization-induced [Ca2+]i increase did not activate Akt, we conclude that the basal level of [Ca2+]i gates BDNF function. Furthermore, inhibiting calmodulin by W13 suppressed Akt phosphorylation. On the other hand, inhibition of protein phosphatase 1 by okadaic acid and tautomycin rescued Akt phosphorylation in BAPTA- and W13-treated neurons. We further demonstrated that the phosphorylation of PDK1 did not correlate with Akt phosphorylation at T308. Our results suggested novel roles of basal [Ca2+]i, rather than activity-induced calcium elevation, in BDNF-Akt signaling. PMID:18485103

  8. Growth Hormone-Releaser Diet Attenuates Cognitive Dysfunction in Klotho Mutant Mice via Insulin-Like Growth Factor-1 Receptor Activation in a Genetic Aging Model

    PubMed Central

    Park, Seok Joo; Chung, Yoon Hee; Lee, Jeong Hyun; Dang, Duy-Khanh; Nam, Yunsung; Jeong, Ji Hoon; Kim, Yong Sun; Nabeshima, Toshitaka

    2014-01-01

    Background It has been recognized that a defect in klotho gene expression accelerates the degeneration of multiple age-sensitive traits. Accumulating evidence indicates that aging is associated with declines in cognitive function and the activity of growth hormone (GH)/insulin-like growth factor-1 (IGF-1). Methods In this study, we examined whether a GH-releaser diet could be effective in protecting against cognitive impairment in klotho mutant mice. Results The GH-releaser diet significantly induced the expression of IGF-1 and IGF-1 receptors in the hippocampus of klotho mutant mice. Klotho mutant mice showed significant memory impairments as compared with wild-type mice. In addition, the klotho mutation significantly decreased the expression of cell survival/antiapoptotic factors, including phospho-Akt (p-Akt)/phospho-glycogen synthase kinase3β (p-GSK3β), phospho-extracellular signal-related kinase (p-ERK), and Bcl-2, but significantly increased those of cell death/proapoptotic factors, such as phospho-c-jun N-terminal kinase (p-JNK), Bax, and cleaved caspase-3 in the hippocampus. Treatment with GH-releaser diet significantly attenuated both decreases in the expression of cell survival/antiapoptotic factors and increases in the expression of cell death/proapoptotic factors in the hippocampus of klotho mutant mice. In addition, klotho mutation-induced oxidative stress was significantly attenuated by the GH-releaser diet. Consequently, a GH-releaser diet significantly improved memory function in the klotho mutant mice. GH-releaser diet-mediated actions were significantly reversed by JB-1, an IGF-1 receptor antagonist. Conclusion The results suggest that a GH-releaser diet attenuates oxidative stress, proapoptotic changes and consequent dysfunction in klotho mutant mice by promoting IGF-1 expression and IGF-1 receptor activation. PMID:25309793

  9. 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.

  10. Activation of PI3K/Akt and ERK signaling pathways antagonized sinomenine-induced lung cancer cell apoptosis.

    PubMed

    Zhou, Liping; Luan, Hong; Liu, Qingpeng; Jiang, Tingshu; Liang, Hongyuan; Dong, Xihua; Shang, Hong

    2012-05-01

    Sinomenine (SIN) is a bioactive component derived from a Chinese medicinal plant. Our previous studies demonstrated that SIN has cytotoxic effects on human lung cancer cells. However, the antitumor molecular mechanisms of SIN have yet to be elucidated in detail. In the present study, we further explored the effects of SIN on NCI-H460 human lung cancer cell viability and apoptosis and investigated the regulation and function of PI3K/Akt and ERK signaling pathways during SIN-induced apoptosis in various lung cancer cell lines. NCI-H460 cells were incubated with 200 µg/ml SIN for the indicated times (0, 24, 48 or 72 h). Cell viability was assessed by MTT assay. Akt, p-Akt, ERK1/2 and p-ERK1/2 protein levels were detected by western blotting, respectively. Two different selective inhibitors (LY294002 for the PI3K pathway; PD98059 for the MEK/ERK pathway) were used to characterize the relative roles of PI3K/Akt and ERK in SIN-induced apoptosis. Apoptosis was determined by flow cytometry. SIN inhibited the proliferation of NCI-H460 cells in a time-dependent manner, which was accompanied with significant activation of pAkt and pERK. LY294002 and PD98059 both significantly increased SIN-induced apoptosis in NCI-H460, NCI-H226 and NCI-H522 cells. Our findings suggest that the activation of the PI3K/Akt and ERK signaling pathways antagonize SIN-induced lung cancer cell apoptosis and molecules that inhibit these pathways should potentiate the effects of SIN. This study represents a significant step forward in our understanding of the signal transduction pathways associated with the apoptosis elicited by SIN. PMID:22367396

  11. 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. PMID:27342662

  12. 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

  13. 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

  14. Clinical prognostic significance and pro-metastatic activity of RANK/RANKL via the AKT pathway in endometrial cancer.

    PubMed

    Wang, Jing; Liu, Yao; Wang, Lihua; Sun, Xiao; Wang, Yudong

    2016-02-01

    RANK/RANKL plays a key role in metastasis of certain malignant tumors, which makes it a promising target for developing novel therapeutic strategies for cancer. However, the prognostic value and pro-metastatic activity of RANK in endometrial cancer (EC) remain to be determined. Thus, the present study investigated the effect of RANK on the prognosis of EC patients, as well as the pro-metastatic activity of EC cells. The results indicated that those with high expression of RANK showed decreased overall survival and progression-free survival. Statistical analysis revealed the positive correlations between RANK/RANKL expression and metastasis-related factors. Additionally, RANK/RANKL significantly promoted cell migration/invasion via activating AKT/β-catenin/Snail pathway in vitro. However, RANK/RANKL-induced AKT activation could be suppressed after osteoprotegerin (OPG) treatment. Furthermore, the combination of medroxyprogesterone acetate (MPA) and RANKL could in turn attenuate the effect of RANKL alone. Similarly, MPA could partially inhibit the RANK-induced metastasis in an orthotopic mouse model via suppressing AKT/β-catenin/Snail pathway. Therefore, therapeutic inhibition of MPA in RANK/RANKL-induced metastasis was mediated by AKT/β-catenin/Snail pathway both in vitro and in vivo, suggesting a potential target of RANK for gene-based therapy for EC. PMID:26734994

  15. Clinical prognostic significance and pro-metastatic activity of RANK/RANKL via the AKT pathway in endometrial cancer

    PubMed Central

    Wang, Jing; Liu, Yao; Wang, Lihua; Sun, Xiao; Wang, Yudong

    2016-01-01

    RANK/RANKL plays a key role in metastasis of certain malignant tumors, which makes it a promising target for developing novel therapeutic strategies for cancer. However, the prognostic value and pro-metastatic activity of RANK in endometrial cancer (EC) remain to be determined. Thus, the present study investigated the effect of RANK on the prognosis of EC patients, as well as the pro-metastatic activity of EC cells. The results indicated that those with high expression of RANK showed decreased overall survival and progression-free survival. Statistical analysis revealed the positive correlations between RANK/RANKL expression and metastasis-related factors. Additionally, RANK/RANKL significantly promoted cell migration/invasion via activating AKT/β-catenin/Snail pathway in vitro. However, RANK/RANKL-induced AKT activation could be suppressed after osteoprotegerin (OPG) treatment. Furthermore, the combination of medroxyprogesterone acetate (MPA) and RANKL could in turn attenuate the effect of RANKL alone. Similarly, MPA could partially inhibit the RANK-induced metastasis in an orthotopic mouse model via suppressing AKT/β-catenin/Snail pathway. Therefore, therapeutic inhibition of MPA in RANK/RANKL-induced metastasis was mediated by AKT/β-catenin/Snail pathway both in vitro and in vivo, suggesting a potential target of RANK for gene-based therapy for EC. PMID:26734994

  16. 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. PMID:26486080

  17. 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

  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. 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.

  20. Rapamycin protects livers from ischemia and reperfusion Injury via both autophagy induction and mTORC2-Akt activation

    PubMed Central

    Zhu, Jianjun; Lu, Tianfei; Yue, Shi; Shen, Xiuda; Gao, Feng; Busuttil, Ronald W.; Kupiec-Weglinski, Jerzy W.; Xia, Qiang; Zhai, Yuan

    2014-01-01

    Background Although Rapamycin (RPM) have been studied extensively in ischemia models, its functional mechanisms remains to be defined. Methods We determined how RPM impacted the pathogenesis of ischemia reperfusion injury (IRI) in a murine liver partial warm ischemia model, with emphasis on its regulation of hepatocyte death. Results RPM protected livers from IRI in the presence of fully developed liver inflammatory immune response. RPM enhanced liver autophagy induction at the reperfusion stage. Dual mTOR1/2 inhibitor Torin 1, despite its ability to induced autophagy, failed to protect livers from IRI. The treatment with RPM, but not Torin 1, resulted in the enhanced activation of the mTORC2-Akt signaling pathway activation in livers post reperfusion. Inactivation of Akt by Triciribine abolished liver protective effect of RPM. The differential cytoprotective effect of RPM and Torin 1 was confirmed in vitro in hepatocyte cultures. RPM, but not Trin 1, protected hepatocytes from stress and TNF-α induced cell death; and inhibition of either autophagy by chloroquine or Akt by Triciribine abolished RPM-mediated cytoprotection. Conclusion RPM protected livers from IRI via both autophagy and mTORC2-Akt activation mechanisms. PMID:25340604

  1. 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. PMID:27259051

  2. Caveolin-1 is critical in the proliferative effect of leptin on osteoblasts through the activation of Akt.

    PubMed

    Zou, Lin; Zhang, Guichun; Liu, Lifeng; Chen, Chen; Cao, Xuecheng; Cai, Jinfang

    2016-09-01

    Osteoblasts are critical in bone remodeling and the repair of bone fractures. Leptin is involved in bone metabolism and osteoblast survival through the downstream signaling pathway, however, the exact mechanism of the effect of leptin on osteoblasts remains to be fully elucidated. In the present study, hFOB 1.19 cells were used to observe the effects of leptin on cell proliferation and apoptosis, and to investigate the underlying mechanism. The results confirmed that treatment of hFOB 1.19 cells with leptin significantly induced cell proliferation. Western blot analysis showed that the expression of caveolin‑1 and the activation of Akt in the cells treated with leptin were significantly increased, compared with the control cells. Additionally, inhibiting Akt activation eliminated the effects on cell proliferation induced by leptin. The rates of cell apoptosis and cell cycle distribution were examined using flow cytometry, which revealed a decrease in the apoptotic rate and an increase in the proportion of cells in the S phase. This indicated that leptin was capable of inducing cell proliferation by inhibiting apoptosis and stimulating cell progression to the S phase. Transfection of the cells with caveolin‑1 small interfering RNA showed that the activation of Akt induced by leptin was significantly inhibited. Furthermore, caveolin‑1 knockdown and inhibiting Akt activation eliminated the increased proliferation, increased proportion of cells in the S phase and increased anti‑apoptotic effects induced by leptin. Taken together, the data obtained in the present study demonstrated that caveolin‑1 was critical in the proliferative effect of leptin on osteoblasts via the activation of Akt. PMID:27430651

  3. 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

  4. 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

  5. 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

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

    PubMed

    MacDonald, Elizabeth M; Andres-Mateos, Eva; Mejias, Rebeca; Simmers, Jessica L; Mi, Ruifa; Park, Jae-Sung; Ying, Stephanie; Hoke, Ahmet; Lee, Se-Jin; Cohn, Ronald D

    2014-04-01

    The purpose of our study was to compare two acquired muscle atrophies and the use of myostatin inhibition for their treatment. Myostatin naturally inhibits skeletal muscle growth by binding to ActRIIB, a receptor on the cell surface of myofibers. Because blocking myostatin in an adult wild-type mouse induces profound muscle hypertrophy, we applied a soluble ActRIIB receptor to models of disuse (limb immobilization) and denervation (sciatic nerve resection) atrophy. We found that treatment of immobilized mice with ActRIIB prevented the loss of muscle mass observed in placebo-treated mice. Our results suggest that this protection from disuse atrophy is regulated by serum and glucocorticoid-induced kinase (SGK) rather than by Akt. Denervation atrophy, however, was not protected by ActRIIB treatment, yet resulted in an upregulation of the pro-growth factors Akt, SGK and components of the mTOR pathway. We then treated the denervated mice with the mTOR inhibitor rapamycin and found that, despite a reduction in mTOR activation, there is no alteration of the atrophy phenotype. Additionally, rapamycin prevented the denervation-induced upregulation of the mTORC2 substrates Akt and SGK. Thus, our studies show that denervation atrophy is not only independent from Akt, SGK and mTOR activation but also has a different underlying pathophysiological mechanism than disuse atrophy. PMID:24504412

  7. 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.

  8. CX3CR1-Mediated Akt1 Activation Contributes to the Paclitaxel-Induced Painful Peripheral Neuropathy in Rats.

    PubMed

    Li, Dai; Chen, Hui; Luo, Xiao-Huan; Sun, Yang; Xia, Wei; Xiong, Yuan-Chang

    2016-06-01

    Painful peripheral neuropathy is a serious dose-limiting side effect of paclitaxel therapy, which unfortunately often happens during the optimal clinical management of chemotherapy in cancer patients. Currently the underlying mechanisms of the painful peripheral neuropathy remain largely unknown. Here, we found that paclitaxel treatment (3 × 8 mg/kg, cumulative dose 24 mg/kg) upregulated the expression of CX3CR1 and phosphorylated Akt1 in DRG and spinal dorsal horn. Blocking of Akt1 pathway activation with different inhibitor (MK-2206 or LY294002) significantly attenuated mechanical allodynia and thermal hyperalgesia induced by paclitaxel. Furthermore, inhibition of CX3CR1 by using neutralizing antibody not only prevented Akt1 activation in DRG and spinal dorsal horn but also alleviated pain-related behavior induced by paclitaxel treatment. This study suggested that CX3CR1/Akt1 signaling pathway may be a potential target for prevention and reversion of the painful peripheral neuropathy induced by paclitaxel. PMID:26961886

  9. Sphingosine-1-Phosphate Protects Intestinal Epithelial Cells from Apoptosis Through the Akt Signaling Pathway

    PubMed Central

    Greenspon, Jose; Li, Ruiyun; Xiao, Lan; Rao, Jaladanki N.; Marasa, Bernard S.; Strauch, Eric D.; Wang, Jian-Ying; Turner, Douglas J.

    2009-01-01

    Objective The regulation of apoptosis of intestinal mucosal cells is important in maintenance of normal intestinal physiology. Summary Sphingosine-1-phosphate (S1P) has been shown to play a critical role in cellular protection to otherwise lethal stimuli in several nonintestinal tissues. Methods The current study determines whether S1P protected normal intestinal epithelial cells (IECs) from apoptosis and whether Akt activation was the central pathway for this effect. Results S1P demonstrated significantly reduced levels of apoptosis induced by tumor necrosis factor-alpha (TNF-α)/cycloheximide (CHX). S1P induced increased levels of phosphorylated Akt and increased Akt activity, but did not affect total amounts of Akt. This activation of Akt was associated with decreased levels of both caspase-3 protein levels and of caspase-3 activity. Inactivation of Akt by treatment with the PI3K chemical inhibitor LY294002 or by overexpression of the dominant negative mutant of Akt (DNMAkt) prevented the protective effect of S1P on apoptosis. Additionally, silencing of the S1P-1 receptor by specific siRNA demonstrated a lesser decrease in apoptosis to S1P exposure. Conclusion These results indicate that S1P protects intestinal epithelial cells from apoptosis via an Akt-dependent pathway. PMID:18654850

  10. 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

  11. MAT2B promotes adipogenesis by modulating SAMe levels and activating AKT/ERK pathway during porcine intramuscular preadipocyte differentiation.

    PubMed

    Zhao, Cunzhen; Chen, Xiaochang; Wu, Wenjing; Wang, Wusu; Pang, Weijun; Yang, Gongshe

    2016-05-15

    Intramuscular fat (IMF) has been demonstrated as one of the crucial factors of livestock meat quality. The MAT2B protein with MAT2α catalyzes the formation of methyl donor S- adenosylmethionine (SAMe) to mediate cell metabolism including proliferation and apoptosis. However, the regulatory effect of MAT2B on IMF deposition is still unclear. In this study, the effect of MAT2B on adipogenesis and its potential mechanism during porcine intramuscular preadipocyte differentiation was studied. The results showed that overexpression of MAT2B promoted adipogenesis and significantly up-regulated the mRNA and protein levels of adipogenic marker genes including FASN, PPARγ and aP2, consistently, knockdown of MAT2B inhibited lipid accumulation and down-regulated the mRNA and protein levels of the above genes. Furthermore, flow cytometry and EdU-labeling assay indicated that MAT2B regulate adipogenesis was partly due to influence intracellular SAMe levels and further affect cell clonal expansion. Also, increased expression of MAT2B activated the phosphorylations of AKT and ERK1/2, whereas knockdown of MAT2B blocked AKT signaling and repressed the phosphorylation of ERK1/2. Moreover, the inhibitory effect of LY294002 (a specific PI3K inhibitor) on the activities of AKT and ERK1/2 was partially recovered by overexpression of MAT2B in porcine intramuscular adipocytes. Finally, Co-IP experiments showed that MAT2B can directly interact with AKT. Taken together, our findings suggested that MAT2B acted as a positive regulator through modifying SAMe levels as well as activating AKT/ERK signaling pathway to promote porcine intramuscular adipocyte differentiation. PMID:26940012

  12. 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

  13. 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.

  14. 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

  15. 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

  16. Asymmetric Dimethylarginine Stimulates Akt1 Phosphorylation via Heat Shock Protein 70-Facilitated Carboxyl-Terminal Modulator Protein Degradation in Pulmonary Arterial Endothelial Cells.

    PubMed

    Sun, Xutong; Kellner, Manuela; Desai, Ankit A; Wang, Ting; Lu, Qing; Kangath, Archana; Qu, Ning; Klinger, Christina; Fratz, Sohrab; Yuan, Jason X-J; Jacobson, Jeffrey R; Garcia, Joe G N; Rafikov, Ruslan; Fineman, Jeffrey R; Black, Stephen M

    2016-08-01

    Asymmetric dimethylarginine (ADMA) induces the mitochondrial translocation of endothelial nitric oxide synthase (eNOS) through the nitration-mediated activation of Akt1. However, it is recognized that the activation of Akt1 requires phosphorylation events at threonine (T) 308 and serine (S) 473. Thus, the current study was performed to elucidate the potential effect of ADMA on Akt1 phosphorylation and the mechanisms that are involved. Exposure of pulmonary arterial endothelial cells to ADMA enhanced Akt1 phosphorylation at both threonine 308 and Ser473 without altering Akt1 protein levels, phosphatase and tensin homolog activity, or membrane Akt1 levels. Heat shock protein (Hsp) 90 plays a pivotal role in maintaining Akt1 activity, and our results demonstrate that ADMA decreased Hsp90-Akt1 interactions, but, surprisingly, overexpression of a dominant-negative Hsp90 mutant increased Akt1 phosphorylation. ADMA exposure or overexpression of dominant-negative Hsp90 increased Hsp70 levels, and depletion of Hsp70 abolished ADMA-induced Akt1 phosphorylation. ADMA decreased the interaction of Akt1 with its endogenous inhibitor, carboxyl-terminal modulator protein (CTMP). This was mediated by the proteasomal-dependent degradation of CTMP. The overexpression of CTMP attenuated ADMA-induced Akt1 phosphorylation at Ser473, eNOS phosphorylation at Ser617, and eNOS mitochondrial translocation. Finally, we found that the mitochondrial translocation of eNOS in our lamb model of pulmonary hypertension is associated with increased Akt1 and eNOS phosphorylation and reduced Akt1-CTMP protein interactions. In conclusion, our data suggest that CTMP is directly involved in ADMA-induced Akt1 phosphorylation in vitro and in vivo, and that increasing CTMP levels may be an avenue to treat pulmonary hypertension. PMID:26959555

  17. Totarol prevents neuronal injury in vitro and ameliorates brain ischemic stroke: Potential roles of Akt activation and HO-1 induction.

    PubMed

    Gao, Yuanxue; Xu, Xiaojun; Chang, Sai; Wang, Yunjie; Xu, Yazhou; Ran, Siqi; Huang, Zhangjian; Li, Ping; Li, Jia; Zhang, Luyong; Saavedra, Juan M; Liao, Hong; Pang, Tao

    2015-12-01

    The natural product totarol, a phenolic diterpenoid and a major constituent isolated from the sap of Podocarpus totara, has been reported to have a potent antimicrobial activity. In this study, we determined whether totarol possessed an additional neuroprotective activity in vitro and in vivo. We found that totarol prevented glutamate- and oxygen and glucose deprivation-induced neuronal death in primary rat cerebellar granule neuronal cells and cerebral cortical neurons. Totarol increased Akt and GSK-3β phosphorylation, Nrf2 and heme oxygenase-1 (HO-1) protein expressions and suppressed oxidative stress by increasing GSH and SOD activities. The PI3K/Akt inhibitor LY294002 prevented totarol neuroprotective effect by suppressing the totarol-induced changes in HO-1 expression and the activities of GSH and SOD. The HO-1 inhibitor ZnPPIX also prevented totarol-increased GSH and SOD activities. In a model of acute cerebral ischemic injury in Sprague-Dawley rats, produced by occlusion of the middle cerebral artery for 2h followed by 22 h or 46 h of reperfusion, totarol significantly reduced infarct volume and improved the neurological deficit. In this model, totarol increased HO-1 expression and the activities of GSH and SOD. These observations suggest that totarol may be a novel activator of the Akt/HO-1 pathway protecting against ischemic stroke through reduction of oxidative stress. PMID:26440581

  18. 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

  19. 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

  20. 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

  1. Angiogenesis effect of therapeutic ultrasound on HUVECs through activation of the PI3K-Akt-eNOS signal pathway.

    PubMed

    Huang, Jing-Juan; Shi, Yi-Qin; Li, Rui-Lin; Hu, An; Lu, Zhao-Yang; Weng, Liang; Wang, Shen-Qi; Han, Yi-Peng; Zhang, Lan; Li, Bao; Hao, Chang-Ning; Duan, Jun-Li

    2015-01-01

    Therapeutic angiogenic effects of low-intensity ultrasound have been reported in endothelial cells and animal models of hind limb ischemia. It has been shown that the proliferation, migration, and tube formation of endothelial cells play critical roles in angiogenesis. The purpose of this study was to determine the underlying mechanism of low-intensity continuous therapeutic ultrasound on angiogenesis in endothelial cells. In the present study, human umbilical vein endothelial cells (HUVECs) were simulated of low-intensity therapeutic ultrasound (TUS, 1 MHz, 0.3 W/cm(2), 9 minute per day) for 3 days, and we observed migration, tube formation, and expression of endothelial nitric oxide synthase (eNOS) and serine/threonine kinase (Akt) in HUVECs. Specific inhibitors of eNOS and phosphoinositide 3-kinase (PI3K) were added to the culture medium and TUS-induced changes in the pathways that mediate angiogenesis were investigated. After exposure to TUS, HUVECs tube formation and migration were significantly promoted, which was blocked by the eNOS inhibitor Immunofluorescence assay and Western blotting analysis demonstrated that eNOS expression in the HUVECs was significantly increased after TUS exhibition. Proteins of phosphorylated eNOS and Akt were both up-regulated after TUS stimulation. However, the specific inhibitor of PI3K not only significantly decreased the expression of p-Akt, but also down-regulated the p-eNOS. This suggested that the PI3K/Akt signal pathway might participate in modulating the activity of eNOS. In short, TUS therapy promotes angiogenesis through activation of the PI3K-Akt-eNOS signal cascade in HUVECs. PMID:26279754

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

    PubMed

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

    2016-04-01

    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

  3. FoxM1 promotes breast tumorigenesis by activating PDGF-A and forming a positive feedback loop with the PDGF/AKT signaling pathway.

    PubMed

    Yu, Guanzhen; Zhou, Aidong; Xue, Jianfei; Huang, Chen; Zhang, Xia; Kang, Shin-Hyuk; Chiu, Wen-Tai; Tan, Christina; Xie, Keping; Wang, Jiejun; Huang, Suyun

    2015-05-10

    The autocrine platelet-derived growth factor (PDGF)/PDGF receptor (PDGFR) signaling pathway promotes breast cancer tumorigenesis, but the mechanisms for its dysregulation in breast cancer are largely unknown. In the study, we identified PDGF-A as a novel transcriptional target of FoxM1. FoxM1 directly binds to two sites in the promoter of PDGF-A and activates its transcription. Mutation of these FoxM1-binding sites diminished PDGF-A promoter activity. Increased FoxM1 resulted in the upregulation of PDGF-A, which led to activation of the AKT pathway and increased breast cancer cell proliferation and tumorigenesis, whereas knockdown of FoxM1 does the opposite. Blocking AKT activation with a phosphoinositide 3-kinase/AKT inhibitor decreased FoxM1-induced cell proliferation. Moreover, PDGF/AKT pathway upregulates the expression of FoxM1 in breast cancer cells. Knockdown of PDGF-A or blockade of AKT activation inhibited the expression of FoxM1 in breast cancer cells. Furthermore, expression of FoxM1 significantly correlated with the expression of PDGF-A and the activated AKT signaling pathway in human breast cancer specimens. Our study demonstrates a novel positive regulatory feedback loop between FoxM1 and the PDGF/AKT signaling pathway; this loop contributes to breast cancer cell growth and tumorigenesis. PMID:25869208

  4. 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

  5. Epilepsy-Related Slack Channel Mutants Lead to Channel Over-Activity by Two Different Mechanisms.

    PubMed

    Tang, Qiong-Yao; Zhang, Fei-Fei; Xu, Jie; Wang, Ran; Chen, Jian; Logothetis, Diomedes E; Zhang, Zhe

    2016-01-01

    Twelve sodium-activated potassium channel (KCNT1, Slack) genetic mutants have been identified from severe early-onset epilepsy patients. The changes in biophysical properties of these mutants and the underlying mechanisms causing disease remain elusive. Here, we report that seven of the 12 mutations increase, whereas one mutation decreases, the channel's sodium sensitivity. Two of the mutants exhibit channel over-activity only when the intracellular Na(+) ([Na(+)]i) concentration is ∼80 mM. In contrast, single-channel data reveal that all 12 mutants increase the maximal open probability (Po). We conclude that these mutant channels lead to channel over-activity predominantly by increasing the ability of sodium binding to activate the channel, which is indicated by its maximal Po. The sodium sensitivity of these epilepsy causing mutants probably determines the [Na(+)]i concentration at which these mutants exert their pathological effects. PMID:26725113

  6. Sustained Akt Activity Is Required to Maintain Cell Viability in Seborrheic Keratosis, a Benign Epithelial Tumor.

    PubMed

    Neel, Victor A; Todorova, Kristina; Wang, Jun; Kwon, Eunjeong; Kang, Minjeong; Liu, Qingsong; Gray, Nathanael; Lee, Sam W; Mandinova, Anna

    2016-03-01

    Seborrheic keratoses (SKs) are common benign skin tumors that share many morphological features with their malignant counterpart, squamous cell carcinoma. SKs frequently have acquired oncogenic mutations in the receptor tyrosine kinase/phosphatidylinositol 3-kinase/Akt signaling cascade. We developed a reliable culture system to study SKs in vitro and screened these cells using a library of selective kinase inhibitors to evaluate effects on cell survival. These benign tumors are sensitive to inhibition by ATP-competitive Akt inhibitors, including A-443654 and GSK690693. RNA interference-mediated Akt suppression mimicked the effects of enzyme inhibition in cultured cells. Akt inhibition suppressed phosphorylation of downstream targets of Akt kinase that are critical for cell survival, including MDM2 and FOXO3a, and induced apoptosis. Cell death was also dependent on p53, mutations in which, although common in cutaneous squamous cell carcinoma, have not been identified in SKs. Intact explants of SKs were also sensitive to Akt inhibition. In addition to the obvious therapeutic implications of these findings, identifying the signaling characteristics that differentiate benign and malignant tumors may inform our understanding of the malignant state. PMID:26739095

  7. Age-related decrease in constructive activation of Akt/PKB in SAMP10 hippocampus.

    PubMed

    Nie, Kun; Yu, Jian-Chun; Fu, Yu; Cheng, Hai-Yan; Chen, Fu-Yan; Qu, You; Han, Jing-Xian

    2009-01-01

    Aging is the greatest risk factor for neurodegenerative diseases such as Alzheimer's disease (AD). Age-dependent alterations of cell signaling play an important role in the onset of AD. The serine/threonine kinase Akt is a critical cell signaling to neuronal survival. Using the senescence-accelerated mouse SAMP10, we investigated the effect of aging on AKT signaling in hippocampus tissue. During aging, the expression of Akt mRNA and protein remained stable. However, the constructive phosphorylation of Akt(Ser473) displayed a continuous decrease after 6 months in SAMP10. When compared with the control SAMR1, aged SAMP10 mice showed significant reduced phosphorylation of Akt(Ser473). SAMP10 at the age of 6 months showed obvious deterioration in performance of learning and memory tasks. Thus, the data reported here suggested a potential link between the age-related alteration of Akt(Ser473) and the deterioration in performance of learning and memory tasks in SAMP10 mouse. PMID:19013131

  8. 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

  9. 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

  10. 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]. PMID:26246284

  11. 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

  12. 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.

  13. 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

  14. 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

  15. Morphine peripheral analgesia depends on activation of the PI3Kγ/AKT/nNOS/NO/KATP signaling pathway

    PubMed Central

    Cunha, Thiago M.; Roman-Campos, Danilo; Lotufo, Celina M.; Duarte, Hugo L.; Souza, Guilherme R.; Verri, Waldiceu A.; Funez, Mani I.; Dias, Quintino M.; Schivo, Ieda R.; Domingues, Andressa C.; Sachs, Daniela; Chiavegatto, Silvana; Teixeira, Mauro M.; Hothersall, John S.; Cruz, Jader S.; Cunha, Fernando Q.; Ferreira, Sergio H.

    2010-01-01

    Morphine is one of the most prescribed and effective drugs used for the treatment of acute and chronic pain conditions. In addition to its central effects, morphine can also produce peripheral analgesia. However, the mechanisms underlying this peripheral action of morphine have not yet been fully elucidated. Here, we show that the peripheral antinociceptive effect of morphine is lost in neuronal nitric-oxide synthase null mice and that morphine induces the production of nitric oxide in primary nociceptive neurons. The activation of the nitric-oxide pathway by morphine was dependent on an initial stimulation of PI3Kγ/AKT protein kinase B (AKT) and culminated in increased activation of KATP channels. In the latter, this intracellular signaling pathway might cause a hyperpolarization of nociceptive neurons, and it is fundamental for the direct blockade of inflammatory pain by morphine. This understanding offers new targets for analgesic drug development. PMID:20147620

  16. Chronic resistance training activates autophagy and reduces apoptosis of muscle cells by modulating IGF-1 and its receptors, Akt/mTOR and Akt/FOXO3a signaling in aged rats.

    PubMed

    Luo, Li; Lu, A-Ming; Wang, Yan; Hong, An; Chen, Yulan; Hu, Juan; Li, Xiaoning; Qin, Zheng-Hong

    2013-04-01

    Resistance exercise training (RET) remains the most effective treatment for the loss of muscle mass and strength in elderly people. However, the underlying cellular and molecular mechanisms are not well understood. Recent evidence suggests that autophagic signaling is altered in aged skeletal muscles. This study aimed to investigate if RET affects IGF-1 and its receptors, the Akt/mTOR, and Akt/FOXO3a signaling pathways and regulates autophagy and apoptosis in the gastrocnemius muscles of 18-20 month old rats. The results showed that 9 weeks of RET prevented the loss of muscle mass and improved muscle strength, accompanied by reduced LC3-II/LC3-I ratio, reduced p62 protein levels, and increased levels of autophagy regulatory proteins, including Beclin 1, Atg5/12, Atg7, and the lysosomal enzyme cathepsin L. RET also reduced cytochrome c level in the cytosol but increased its level in mitochondrial fraction, and inhibited cleaved caspase 3 production and apoptosis. Furthermore, RET upregulated the expression of IGF-1 and its receptors but downregulated the phosphorylation of Akt and mTOR. In addition, RET upregulated the expression of total AMPK, phosphorylated AMPK, and FOXO3a. Taken together, these results suggest that the benefits of RET are associated with increased autophagy activity and reduced apoptosis of muscle cells by modulating IGF-1 and its receptors, the Akt/mTOR and Akt/FOXO3a signaling pathways in aged skeletal muscles. PMID:23419688

  17. Human umbilical cord blood mononuclear cells activate the survival protein Akt in cardiac myocytes and endothelial cells that limits apoptosis and necrosis during hypoxia.

    PubMed

    Henning, Robert J; Dennis, Steve; Sawmiller, Darrell; Hunter, Lorynn; Sanberg, Paul; Miller, Leslie

    2012-06-01

    We have previously reported that human umbilical cord blood mononuclear cells (HUCBC), which contain hematopoietic, mesenchymal, and endothelial stem cells, can significantly reduce acute myocardial infarction size. To determine the mechanism whereby HUCBC increase myocyte and vascular endothelial cell survival, we treated cardiac myocytes and coronary artery endothelial cells in separate experiments with HUCBC plus culture media or culture media alone and subjected the cells to 24 h of hypoxia or normoxia. We then determined in myocytes and endothelial cells activation of the cell survival protein Akt by Western blots. We also determined in these cells apoptosis by annexin V staining and necrosis by propidium iodide staining. Thereafter, we inhibited with API, a specific and sensitive Akt inhibitor, Akt activation in myocytes and endothelial cells cultured with HUCBC during hypoxia and determined cell apoptosis and necrosis. In cells cultured without HUCBC, hypoxia only slightly activated Akt. Moreover, hypoxia increased myocyte apoptosis by ≥ 226% and necrosis by 58% in comparison with myocytes in normoxia. Hypoxic treatment of endothelial cells without HUCBC increased apoptosis by 94% and necrosis by 59%. In contrast, hypoxia did not significantly affect HUCBC. Moreover, in myocyte + HUCBC cultures in hypoxia, HUCBC induced a ≥ 135% increase in myocyte phospho-Akt. Akt activation decreased myocyte apoptosis by 76% and necrosis by 35%. In endothelial cells, HUCBC increased phospho-Akt by 116%. HUCBC also decreased endothelial cell apoptosis by 58% and necrosis by 42%. Inhibition of Akt with API in myocytes and endothelial cells cultured with HUCBC during hypoxia nearly totally prevented the HUCBC-induced decrease in apoptosis and necrosis. We conclude that HUCBC can significantly decrease hypoxia-induced myocyte and endothelial cell apoptosis and necrosis by activating Akt in these cells and in this manner HUCBC can limit myocardial ischemia and injury. PMID

  18. p51/p63 Inhibits ultraviolet B-induced apoptosis via Akt activation.

    PubMed

    Ogawa, E; Okuyama, R; Ikawa, S; Nagoshi, H; Egawa, T; Kurihara, A; Yabuki, M; Tagami, H; Obinata, M; Aiba, S

    2008-01-31

    The epidermis must be protected against excess apoptotic cell death in response to ultraviolet-B (UV-B) irradiation. p53 is known to be critical for this protection. Although the p53 family member DeltaNp51B/DeltaNp63alpha (an N terminal-deleted form of p51/p63) is abundantly expressed in keratinocytes, its contribution to UV-B-dependent apoptosis is largely unknown. We found that, after a transient increase, DeltaNp51B is downregulated in UV-B-irradiated keratinocytes undergoing apoptosis, whereas p53 is upregulated with delayed kinetics. Furthermore, the reduction of DeltaNp51B by small interfering RNAs augmented UV-B-dependent apoptosis in keratinocytes, indicating that DeltaNp51B blocks keratinocyte apoptosis. Although the exogenous expression of DeltaNp51B in keratinocytes did not further block the UV-B-dependent apoptosis, to our surprise the expression of TAp51B (an isoform with a full NH(2)-terminal transactivation domain that is structurally and functionally similar to p53) decreased apoptosis significantly. The blockade of keratinocyte apoptosis by the p51 was dependent on the phosphorylation of Akt, resulting in the activation of a survival pathway. Thus, in addition to its indispensable roles in epithelial development, p51 acts in adult cells to protect the epidermis against UV-B irradiation by preventing excess depletion of keratinocytes. PMID:17653081

  19. Bacillus sphaericus asporogenous mutants: morphology, protein pattern and larvicidal activity.

    PubMed

    Charles, J F; Kalfon, A; Bourgouin, C; de Barjac, H

    1988-01-01

    Asporogenous mutants from Bacillus sphaericus strains 2297 and 1593-4, blocked at different stages of the sporulation process, were isolated. Two mutants (2297 Aspo30A and 2297 Aspo34) which were blocked early in sporulation did not possess any crystalline inclusions and were poorly toxic to Culex pipiens mosquito larvae. Other mutants (2297 Aspo115, 2297 Aspo24 and 1593-4 Aspo12) which were blocked at later stages synthesized crystal-like inclusions next to the forespores, and were highly toxic to mosquito larvae. Electrophoretic protein analysis of alkali extracts from mutants and wild type strains confirmed the absence of toxic crystal-related proteins in early-blocked mutants and their presence in later ones. Western blots with antisera directed against the crystal proteins confirmed those observations. PMID:3408593

  20. Synapsis and catalysis by activated Tn3 resolvase mutants

    PubMed Central

    Olorunniji, Femi J.; He, Jiuya; Wenwieser, Sandra V.C.T.; Boocock, Martin R.; Stark, W. Marshall

    2008-01-01

    The serine recombinase Tn3 resolvase catalyses recombination between two 114 bp res sites, each of which contains binding sites for three resolvase dimers. We have analysed the in vitro properties of resolvase variants with ‘activating’ mutations, which can catalyse recombination at binding site I of res when the rest of res is absent. Site I × site I recombination promoted by these variants can be as fast as res × res recombination promoted by wild-type resolvase. Activated variants have reduced topological selectivity and no longer require the 2–3′ interface between subunits that is essential for wild-type resolvase-mediated recombination. They also promote formation of a stable synapse comprising a resolvase tetramer and two copies of site I. Cleavage of the DNA strands by the activated mutants is slow relative to the rate of synapsis. Stable resolvase tetramers were not detected in the absence of DNA or bound to a single site I. Our results lead us to conclude that the synapse is assembled by sequential binding of resolvase monomers to site I followed by interaction of two site I-dimer complexes. We discuss the implications of our results for the mechanisms of synapsis and regulation in recombination by wild-type resolvase. PMID:19015124

  1. 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

  2. Role of mechanical strain-activated PI3K/Akt signaling pathway in pelvic organ prolapse

    PubMed Central

    LI, BING-SHU; GUO, WEN-JUN; HONG, LI; LIU, YAO-DAN; LIU, CHENG; HONG, SHA-SHA; WU, DE-BIN; MIN, JIE

    2016-01-01

    Mechanical loading on pelvic supports contributes to pelvic organ prolapse (POP). However, the underlying mechanisms remain to be elucidated. Our previous study identified that mechanical strain induced oxidative stress (OS) and promoted apoptosis and senescence in pelvic support fibroblasts. The aim of the present study is to investigate the molecular signaling pathway linking mechanical force with POP. Using a four-point bending device, human uterosacral ligament fibroblasts (hUSLF) were exposed to mechanical tensile strain at a frequency of 0.3 Hz and intensity of 5333 µε, in the presence or absence of LY294002. The applied mechanical strain on hUSLF resulted in apoptosis and senescence, and decreased expression of procollagen type I α1. Mechanical strain activated phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K)/Akt signaling and resulted in downregulated expression of glutathione peroxidase 1 and Mn-superoxide dismutase, and accumulation of intracellular reactive oxygen species. These effects were blocked by administration of LY294002. Furthermore, it was demonstrated that PI3K/Akt was activated in the uterosacral ligaments of POP patients, and that OS was increased and collagen type I production reduced. The results from the present study suggest that mechanical strain promotes apoptosis and senescence, and reduces collagen type I production via activation of PI3K/Akt-mediated OS signaling pathway in hUSLF. This process may be involved in the pathogenesis of POP as it results in relaxation and dysfunction of pelvic supports. PMID:27176043

  3. Neutrophil elastase enhances the proliferation and decreases apoptosis of leukemia cells via activation of PI3K/Akt signaling

    PubMed Central

    YANG, RONG; ZHONG, LIANG; YANG, XIAO-QUN; JIANG, KAI-LING; LI, LIU; SONG, HAO; LIU, BEI-ZHONG

    2016-01-01

    Neutrophil elastase (NE) is a neutrophil-derived serine proteinase with specificity for a broad range of substrates. NE has been reported to be associated with the pathogenesis of several conditions, particularly that of pulmonary diseases. Previous studies have shown that NE can cleave the pro-myelocyte - retinoic acid receptor-alpha chimeric protein and is important for the development of acute pro-myelocytic leukemia. To further elucidate the role of NE in acute pro-myelocytic leukemia, the present study successfully constructed a lentiviral vector containing the NE gene (LV5-NE), which was transfected into NB4 acute pro-myelocytic leukemia cells. The effects of NE overexpression in NB4 cells were detected using a Cell-Counting Kit-8 assay, flow cytometry and western blot analysis. The results showed that NE significantly promoted the proliferation of NB4 cells, inhibited cell apoptosis and apoptotic signaling, and led the activation of Akt. In an additional experiment, a vector expressing small hairpin RNA targeting NE was constructed to assess the effects of NE knockdown in U937 cells. Western blot analysis revealed that apoptotic signaling was increased, while Akt activation was decreased following silencing of NE. The results of the present study may indicate that NE activates the phosphoinositide-3 kinase/Akt signaling pathway in leukemia cells to inhibit apoptosis and enhance cell proliferation, and may therefore represent a molecular target for the treatment of pro-myelocytic leukemia. PMID:27035679

  4. 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

  5. Single Cell Chemical Cytometry of Akt Activity in Rheumatoid Arthritis and Normal Fibroblast-like Synoviocytes in Response to Tumor Necrosis Factor α.

    PubMed

    Mainz, Emilie R; Serafin, D Stephen; Nguyen, Tuong T; Tarrant, Teresa K; Sims, Christopher E; Allbritton, Nancy L

    2016-08-01

    The etiology of rheumatoid arthritis (RA) is poorly understood, and 30% of patients are unresponsive to established treatments targeting tumor necrosis factor α (TNFα). Akt kinase is implicated in TNFα signaling and may act as a barometer of patient responses to biologic therapies. Fluorescent peptide sensors and chemical cytometry were employed to directly measure Akt activity as well as proteolytic activity in individual fibroblast-like synoviocytes (FLS) from RA and normal subjects. The specificity of the peptide reporter was evaluated and shown to be a valid measure of Akt activity in single cells. The effect of TNFα treatment on Akt activity was highly heterogeneous between normal and RA subjects, which was not observable in bulk analyses. In 2 RA subjects, a bimodal distribution of Akt activity was observed, primarily due to a subpopulation (21.7%: RA Subject 5; 23.8%: RA Subject 6) of cells in which >60% of the reporter was phosphorylated. These subjects also possessed statistically elevated proteolytic cleavage of the reporter relative to normal subjects, suggesting heterogeneity in Akt and protease activity that may play a role in the RA-affected joint. We expect that chemical cytometry studies pairing peptide reporters with capillary electrophoresis will provide valuable data regarding aberrant kinase activity from small samples of clinical interest. PMID:27391352

  6. {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.

  7. Immunohistochemical Analysis of the Activation Status of the Akt/mTOR/pS6 Signaling Pathway in Oral Lichen Planus

    PubMed Central

    Prodromidis, Georgios; Nikitakis, Nikolaos G.; Sklavounou, Alexandra

    2013-01-01

    Introduction. Aberrations of the Akt/mTOR/pS6 pathway have been linked to various types of human cancer, including oral squamous cell carcinoma (OSCC). The purpose of this study was to evaluate the activation status of Akt, mTOR, and pS6 in oral lichen planus (OLP) in comparison with oral premalignant and malignant lesions and normal oral mucosa (NM). Materials and Methods. Immunohistochemistry for p-Akt, p-mTOR, and phospho-pS6 was performed in 40 OLP, 20 oral leukoplakias (OL), 10 OSCC, and 10 control samples of NM. Results. Nuclear p-Akt expression was detected in the vast majority of cases in all categories, being significantly higher in OL. Cytoplasmic p-Akt and p-mTOR staining was present only in a minority of OLP cases, being significantly lower compared to OL and OSCC. Phospho-pS6 showed cytoplasmic positivity in most OLP cases, which however was significantly lower compared to OL and OSCC. Conclusions. Overall, cytoplasmic p-Akt, p-mTOR, and phospho-pS6 levels appear to be significantly lower in OLP compared to OL and OSCC. However, the expression of these molecules in a subset of OLP cases suggests that activation of Akt/mTOR/pS6 may occur in the context of OLP, possibly contributing to the premalignant potential of individual cases. PMID:24228033

  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. Loss of desmocollin-2 confers a tumorigenic phenotype to colonic epithelial cells through activation of Akt/β-catenin signaling

    PubMed Central

    Kolegraff, Keli; Nava, Porfirio; Helms, My N.; Parkos, Charles A.; Nusrat, Asma

    2011-01-01

    Desmocollin-2 (Dsc2) and desmoglein-2 (Dsg2) are transmembrane cell adhesion proteins of desmosomes. Reduced expression of Dsc2 has been reported in colorectal carcinomas, suggesting that Dsc2 may play a role in the development and/or progression of colorectal cancer. However, no studies have examined the mechanistic contribution of Dsc2 deficiency to tumorigenesis. Here we report that loss of Dsc2 promotes cell proliferation and enables tumor growth in vivo through the activation of Akt/β-catenin signaling. Inhibition of Akt prevented the increase in β-catenin–dependent transcription and proliferation following Dsc2 knockdown and attenuated the in vivo growth of Dsc2-deficient cells. Taken together, our results provide evidence that loss of Dsc2 contributes to the growth of colorectal cancer cells and highlight a novel mechanism by which the desmosomal cadherins regulate β-catenin signaling. PMID:21325624

  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. Squamosamide derivative FLZ protects retinal pigment epithelium cells from oxidative stress through activation of epidermal growth factor receptor (EGFR)-AKT signaling.

    PubMed

    Cheng, Li-Bo; Chen, Chun-Ming; Zhong, Hong; Zhu, Li-Juan

    2014-01-01

    Reactive oxygen species (ROS)-mediated retinal pigment epithelium (RPE) cell apoptosis is attributed to age-related macular degeneration (AMD) pathogenesis. FLZ, a novel synthetic squamosamide derivative from a Chinese herb, Annona glabra, has displayed significant cyto-protective activity. In the current study, we explored the pro-survival effect of FLZ in oxidative stressed-RPE cells and studied the underlying signaling mechanisms. Our results showed that FLZ attenuated hydrogen peroxide (H2O2)-induced viability decrease and apoptosis in the RPE cell line (ARPE-19 cells) and in primary mouse RPE cells. Western blotting results showed that FLZ activated AKT signaling in RPE cells. The AKT-specific inhibitor, MK-2206, the phosphoinositide 3-kinase (PI3K)/AKT pan inhibitor, wortmannin, and AKT1-shRNA (short hairpin RNA) depletion almost abolished FLZ-mediated pro-survival/anti-apoptosis activity. We discovered that epidermal growth factor receptor (EGFR) trans-activation mediated FLZ-induced AKT activation and the pro-survival effect in RPE cells, and the anti-apoptosis effect of FLZ against H2O2 was inhibited by the EGFR inhibitor, PD153035, or by EGFR shRNA-knockdown. In conclusion, FLZ protects RPE cells from oxidative stress through activation of EGFR-AKT signaling, and our results suggest that FLZ might have therapeutic values for AMD. PMID:25329617

  12. Receptor for activated C kinase 1 (RACK1) promotes the progression of OSCC via the AKT/mTOR pathway.

    PubMed

    Zhang, Xuefeng; Liu, Na; Ma, Danhua; Liu, Ling; Jiang, Lu; Zhou, Yu; Zeng, Xin; Li, Jing; Chen, Qianming

    2016-08-01

    Our previous study suggested that receptor for activated C kinase 1 (RACK1) contribute to the progression of oral squamous cell carcinoma (OSCC). The aim of this study is to elucidate the mechanism by which RACK1 regulates cell growth in OSCC using in vitro and in vivo models. The effects of RACK1 knockdown with lentivirus based shRNA in stable cell lines were evaluated by Q-PCR and western blot analysis. RACK1 silencing effects on the cell cycle in OSCC cells were detected by flow cytometry and western blot analysis. The effect of RACK1 silencing on inhibiting the progression of OSCC was illustrated using a xenografted mouse model. RACK1 and relevant signaling pathways were investigated in tissues and cells using immunohistochemistry and/or western blot analysis. Stable silencing of the RACK1 gene resulted in a distinct G1 and G2 phase arrest by downregulating Cyclin B1 and Cyclin D1. Depleted RACK1 led to markedly decreased tumor volume and the expression of Ki67, CD34, and VEGF in vivo. The expression of RACK1 and p-AKT has a parallel pattern in different stages of oral carcinogenesis tissues. In addition, the protein level of RACK1 was positively correlated with p-AKT in OSCC tissue samples and cell lines. We found specific transient knockdown of RACK1 could downregulate the protein levels of p-AKT, p-mTOR, and p-S6 in a dose-dependent manner. This study demonstrates that RACK1-dependent OSCC growth and survival may be related to the increased activation of the AKT/mTOR/S6 pathway. PMID:27279145

  13. ADAM17 promotes breast cancer cell malignant phenotype through EGFR-PI3K-AKT activation

    PubMed Central

    Zheng, Xuguang; Jiang, Feng; Katakowski, Mark; Zhang, Zheng Gang; Lu, Qing-e; Chopp, Michael

    2009-01-01

    A disintegrin and metalloproteinase-17 (ADAM17) is involved in proteolytic ectodomain shedding of several membrane-bound growth factors and cytokines. The expression and activity of ADAM17 increase under some pathological conditions such as stroke and glioma. ADAM17 promotes neural progenitor cell migration and contributes to stroke-induced neurogenesis after stroke and brain tumor growth and invasion. In the present study, we sought to elucidate whether ADAM17 contributes to breast cancer progression and its mechanisms. To this end, we examined the role of ADAM17 in the proliferation, invasion, and tube formation of MDA-MB-231 breast cancer cells in vitro. Stable transfection of the MDA-MB-231 cell line with either a plasmid for over-expression of human ADAM17, or a siRNA to ADAM17 was employed in this study to establish high or low ADAM17 expression in breast cancer cells, respectively. For study of mechanism, the ADAM17 inhibitor TAPI-2 and the PI3K-AKT inhibitor LY294002 were used to counteract high ADAM17 expression or the activated PI3K-AKT pathway. Proliferation of MDA-MB-231 breast cancer cells were tested by MTT, Bromodeoxyuridine incorporation assay, growth curve, and sulforhodamine B assay. Matrigel invasion assays were used to assess the ability of MDA-MB-231 cells to penetrate the Extra Cellular Matrix. A Matrigel tube formation assay was performed to test capillary tube formation ability. EGFR-PI3K-Akt pathway activation in MDA-MB-231 cells under different ADAM17 expression levels were tested by Western blot and ELISA. Our data show that ADAM17 promotes the MDA-MB-231 malignant phenotype by increased proliferation, invasion and angiogenesis. TGF-α, VEGF secretion and VEGF expression was increasing by ADAM17 and counteracted by ADAM17 siRNA, TAPI-2, and LY294002 in MDA-MB-231 cells. ADAM17 activated, whereas ADAM17 siRNA, TAPI-2, and LY294002 deactivated the EGFR-PI3K-AKT signal pathway, which correlated with MDA-MB-231 cell malignant phenotype

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

    PubMed

    Lapierre, Jean-Marc; Eathiraj, Sudharshan; Vensel, David; Liu, Yanbin; Bull, Cathy O; Cornell-Kennon, Susan; Iimura, Shin; Kelleher, Eugene W; Kizer, Darin E; Koerner, Steffi; Makhija, Sapna; Matsuda, Akihisa; Moussa, Magdi; Namdev, Nivedita; Savage, Ronald E; Szwaya, Jeff; Volckova, Erika; Westlund, Neil; Wu, Hui; Schwartz, Brian

    2016-07-14

    The work in this paper describes the optimization of the 3-(3-phenyl-3H-imidazo[4,5-b]pyridin-2-yl)pyridin-2-amine chemical series as potent, selective allosteric inhibitors of AKT kinases, leading to the discovery of ARQ 092 (21a). The cocrystal structure of compound 21a bound to full-length AKT1 confirmed the allosteric mode of inhibition of this chemical class and the role of the cyclobutylamine moiety. Compound 21a demonstrated high enzymatic potency against AKT1, AKT2, and AKT3, as well as potent cellular inhibition of AKT activation and the phosphorylation of the downstream target PRAS40. Compound 21a also served as a potent inhibitor of the AKT1-E17K mutant protein and inhibited tumor growth in a human xenograft mouse model of endometrial adenocarcinoma. PMID:27305487

  15. 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.

  16. Platelets promote osteosarcoma cell growth through activation of the platelet-derived growth factor receptor-Akt signaling axis

    PubMed Central

    Takagi, Satoshi; Takemoto, Ai; Takami, Miho; Oh-hara, Tomoko; Fujita, Naoya

    2014-01-01

    The interactions of tumor cells with platelets contribute to the progression of tumor malignancy, and the expression levels of platelet aggregation-inducing factors positively correlate with the metastatic potential of osteosarcoma cells. However, it is unclear how tumor-platelet interaction contributes to the proliferation of osteosarcomas. We report here that osteosarcoma-platelet interactions induce the release of platelet-derived growth factor (PDGF) from platelets, which promotes the proliferation of osteosarcomas. Co-culture of platelets with MG63 or HOS osteosarcoma cells, which could induce platelet aggregation, enhanced the proliferation of each cell line in vitro. Analysis of phospho-antibody arrays revealed that co-culture of MG63 cells with platelets induced the phosphorylation of platelet derived growth factor receptor (PDGFR) and Akt. The addition of supernatants of osteosarcoma-platelet reactants also increased the growth of MG63 and HOS cells as well as the level of phosphorylated-PDGFR and -Akt. Sunitinib or LY294002, but not erlotinib, significantly inhibited the platelet-induced proliferation of osteosarcoma cells, indicating that PDGF released from platelets plays an important role in the proliferation of osteosarcomas by activating the PDGFR and then Akt. Our results suggest that inhibitors that specifically target osteosarcoma-platelet interactions may eradicate osteosarcomas. PMID:24974736

  17. 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

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

    PubMed

    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. EGFR Activation Leads to Cell Death Independent of PI3K/AKT/mTOR in an AD293 Cell Line.

    PubMed

    Treda, Cezary; Popeda, Marta; Ksiazkiewicz, Magdalena; Grzela, Dawid P; Walczak, Maciej P; Banaszczyk, Mateusz; Peciak, Joanna; Stoczynska-Fidelus, Ewelina; Rieske, Piotr

    2016-01-01

    The Epidermal Growth Factor Receptor (EGFR) and its mutations contribute in various ways to tumorigenesis and biology of human cancers. They are associated with tumor proliferation, progression, drug resistance and the process of apoptosis. There are also reports that overexpression and activation of wild-type EGFR may lead to cell apoptosis. To study this phenomenon, we overexpressed in an AD293 cell line two most frequently observed forms of the EGFR receptor: wild-type and the constitutively active mutant-EGFR variant III (EGFRvIII). Then, we compared the effect of EGF stimulation on cell viability and downstream EGFR signaling. AD293 cells overexpressing wild-type EGFR, despite a significant proliferation increase in serum supplemented medium, underwent apoptosis after EGF stimulation in serum free conditions. EGFRvIII expressing cells, however, were unaffected by either serum starvation or EGF treatment. The effect of EGF was completely neutralized by tyrosine kinase inhibitors (TKIs), indicating the specificity of this observation. Moreover, apoptosis was not prevented by inhibiting EGFR downstream proteins (PI3K, AKT and mTOR). Here we showed another EGFR function, dependent on environmental factors, which could be employed in therapy and drug design. We also proposed a new tool for EGFR inhibitor analysis. PMID:27153109

  20. 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

  1. Benomyl-resistant mutant strain of Trichoderma sp. with increased mycoparasitic activity.

    PubMed

    Olejníková, P; Ondrusová, Z; Krystofová, S; Hudecová, D

    2010-01-01

    Application of UV radiation to the strain Trichoderma sp. T-bt (isolated from lignite) resulted in the T-brm mutant which was resistant to the systemic fungicide benomyl. The tub2 gene sequence in the T-brm mutant differed from the parent as well as the collection strain (replacing tyrosine with histidine in the TUB2 protein). Under in vitro conditions this mutant exhibited a higher mycoparasitic activity toward phytopathogenic fungi. PMID:20336512

  2. Dual inhibition of Akt/mTOR pathway by nab-rapamycin and perifosine induces anti-tumor activity in multiple myeloma

    PubMed Central

    Cirstea, Diana; Hideshima, Teru; Rodig, Scott; Santo, Loredana; Pozzi, Samantha; Vallet, Sonia; Ikeda, Hiroshi; Perrone, Giulia; Patel, Kishan; Desai, Neil; Sportelli, Peter; Kapoor, Shweta; Vali, Shireen; Mukherjee, Siddhartha; Munshi, Nikhil C.; Anderson, Kenneth C.; Raje, Noopur

    2011-01-01

    The PI3K/Akt/mTOR pathway mediates multiple myeloma (MM) cell proliferation, survival, and development of drug resistance, underscoring the role of mTOR inhibitors such as rapamycin with potential anti-MM activity. However, recent data demonstrate a positive feedback loop from mTOR/S6K1 to Akt, whereby Akt activation confers resistance to mTOR inhibitors. We confirmed that suppression of mTOR signaling in MM cells by rapamycin was associated with upregulation of Akt phosphorylation. We hypothesized that inhibiting this positive feedback by a potent Akt inhibitor perifosine would augment rapamycin-induced cytotoxicity in MM cells. Perifosine inhibited rapamycin-induced p-Akt, resulting in enhanced cytotoxicity in MM.1S cells even in the presence of IL-6, IGF-1 or bone marrow stromal cells. Moreover, rapamycin induced autophagy in MM.1S MM cells as evidenced by electron microscopy and immunocytochemistry, was augmented by perifosine. Combination therapy increased apoptosis detected by Annexin/PI analysis and caspase/PARP cleavage. Importantly, in vivo antitumor activity and prolongation of survival in a MM mouse xenograft model after treatment was enhanced with combination of nab-rapamycin and perifosine. Utilizing the in silico predictive analysis we confirmed our experimental findings of this drug combination on PI3K, Akt, mTOR kinases, and the caspases. Our data suggests that mutual suppression of the PI3K/Akt/mTOR pathway by rapamycin and perifosine combination induces synergistic MM cell cytotoxicity, providing the rationale for clinical trials in patients with relapsed / refractory MM. PMID:20371718

  3. 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.

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

    PubMed Central

    Kumar, Ambuj; Purohit, Rituraj

    2013-01-01

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

  5. 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.

  6. Akt phosphorylates myc-associated zinc finger protein (MAZ), releases P-MAZ from the p53 promoter, and activates p53 transcription.

    PubMed

    Lee, Wei-Ping; Lan, Keng-Hsin; Li, Chung-Pin; Chao, Yee; Lin, Han-Chieh; Lee, Shou-Dong

    2016-05-28

    The p53 protein is a cell cycle regulator. When the cell cycle progresses, p53 plays an important role in putting a brake on the G1 phase to prevent unwanted errors during cell division. Akt is a downstream kinase of receptor tyrosine kinase. Upon activation, Akt phorphorylates IKK that then phosphorylates IκB and releases NF-κB, leading to transcriptional activation of Dmp1. Dmp1 is a transcriptional activator of Arf. It has been known that oncogene activation stabilizes p53 through transcriptional activation of Arf, which then binds and inhibits Mdm2. In the current study, we show that myc-associated zinc finger protein (MAZ) is a transcriptional repressor of the p53 promoter. Akt phosphorylates MAZ at Thr385, and the phosphorylated MAZ is released from the p53 promoter, leading to transcriptional activation of p53, a new mechanism that contributes to increased p53 protein pool during oncogene activation. PMID:26902421

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

    PubMed Central

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

    2010-01-01

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

  8. 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. PMID:17845508

  9. Nitric oxide activates intradomain disulfide bond formation in the kinase loop of Akt1/PKBα after burn injury.

    PubMed

    Lu, X-M; Tompkins, R G; Fischman, A J

    2013-03-01

    Severe burn injury is an acute inflammatory state with massive alterations in gene expression and levels of growth factors, cytokines and free radicals. During the catabolic processes, changes in insulin sensitivity and skeletal muscle wasting (unintended loss of 5-15% of lean body mass) are observed clinically. Here, we reveal a novel molecular mechanism of Akt1/protein kinase Bα (Akt1/PKBα) regulated via cross-talking between dephosphorylation of Thr308 and S-nitrosylation of Cys296 post severe burn injury, which were characterized using nano-LC interfaced with tandem quadrupole time-of-fight mass spectrometry (Q-TOF)micro tandem mass spectrometry in both in vitro and in vivo studies. For the in vitro studies, Akt1/PKBα was S-nitrosylated with S-nitrosoglutathione and derivatized by three methods. The derivatives were isolated by SDS-PAGE, trypsinized and analyzed by the tandem MS. For the in vivo studies, Akt1/PKBα in muscle lysates from burned rats was immunoprecipitated, derivatized with HPDP-Biotin and analyzed as above. The studies demonstrated that the NO free radical reacts with the free thiol of Cys296 to produce a Cys296-SNO intermediate which accelerates interaction with Cys310 to form Cys296-Cys310 in the kinase loop. MS/MS sequence analysis indicated that the dipeptide, linked via Cys296-Cys310, underwent dephosphorylation at Thr308. These effects were not observed in lysates from sham animals. As a result of this dual effect of burn injury, the loose conformation that is slightly stabilized by the Lys297-Thr308 salt bridge may be replaced by a more rigid structure which may block substrate access. Together with the findings of our previous report concerning mild IRS-1 integrity changes post burn, it is reasonable to conclude that the impaired Akt1/PKBα has a major impact on FOXO3 subcellular distribution and activities. PMID:23314241

  10. 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

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

    PubMed

    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

  12. Hydrogen sulfide promotes cell proliferation of oral cancer through activation of the COX2/AKT/ERK1/2 axis.

    PubMed

    Zhang, Shuai; Bian, Huan; Li, Xiaoxu; Wu, Huanhuan; Bi, Qingwei; Yan, Yingbin; Wang, Yixiang

    2016-05-01

    Hydrogen sulfide, the third gaseous transmitter, is one of the main causes of halitosis in the oral cavity. It is generally considered as playing a deleterious role in many oral diseases including oral cancer. However, the regulatory mechanisms involved in the effects of hydrogen sulfide on oral cancer growth remain largely unknown. In the present study, we investigated the underlying mechanisms through CCK-8 assay, EdU incorporation, real-time PCR, western blot and pathway blockade assays. Our results showed that hydrogen sulfide promoted oral cancer cell proliferation through activation of the COX2, AKT and ERK1/2 pathways in a dose-dependent manner. Blocking any of the three above pathways inhibited hydrogen sulfide-induced oral cancer cell proliferation. Meanwhile, blockade of COX2 by niflumic acid downregulated NaHS-induced p-ERK and p-AKT expression. Inactivation of the AKT pathway by GSK690693 significantly decreased NaHS‑induced p-ERK1/2 expression, and inhibition of the ERK1/2 pathway by U0126 markedly increased NaHS-induced p-AKT expression. Either the AKT or ERK1/2 inhibitor did not significantly alter the COX2 expression level. Our data revealed, for the first time, that hydrogen sulfide promotes oral cancer cell proliferation through activation of the COX2/AKT/ERK1/2 axis, suggesting new potential targets to eliminate the effect of hydrogen sulfide on the development of oral cancer. PMID:26987083

  13. 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

  14. 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

  15. Downregulation of Rab25 activates Akt1 in head and neck squamous cell carcinoma

    PubMed Central

    SEVEN, DIDEM; DOGAN, SOYDAN; KILIÇ, ERKAN; KARAMAN, EMIN; KOSEOGLU, HIKMET; BUYRU, NUR

    2015-01-01

    Several studies have suggested that Ras-associated binding 25 protein (Rab25) is involved in the pathogenesis of human cancer. Although it has been demonstrated that the development of head and neck squamous cell carcinoma (HNSCC) is the result of an accumulation of multiple sequential genetic and epigenetic alterations in key genes with important functions in cell growth and the cell cycle, recent studies have indicated that HNSCC is a complex and heterogenous disease. To the best of our knowledge, there is no data regarding the regulation of the Rab25 gene at the mRNA or protein level in HNSCC. Furthermore, available data on Rab25 expression in other types of cancer are conflicting. The aim of the present study was to investigate whether Rab25 is involved in the development and/or progression of HNSCC, and to analyze the mechanisms underlying its effects in this type of cancer. The expression of Rab25 mRNA in HNSCC tissues and adjacent non-tumor tissue samples was measured using reverse transcription-quantitative polymerase chain reaction, while the level of the Rab25, Akt1 and phosphorylated-Akt1 proteins was measured using western blotting. Expression of Rab25 mRNA and protein was downregulated in 69.1% and 56.1% of tumor tissue samples, respectively. This downregulation was associated with an increase in p-Akt1 expression, in the absence of a change in total Akt1 protein levels, in tumor tissues compared with normal tissues. The current findings suggest that Rab25 acts as a tumor suppressor in HNSCC. PMID:26622777

  16. Role of mechanical strain-activated PI3K/Akt signaling pathway in pelvic organ prolapse.

    PubMed

    Li, Bing-Shu; Guo, Wen-Jun; Hong, Li; Liu, Yao-Dan; Liu, Cheng; Hong, Sha-Sha; Wu, De-Bin; Min, Jie

    2016-07-01

    Mechanical loading on pelvic supports contributes to pelvic organ prolapse (POP). However, the underlying mechanisms remain to be elucidated. Our previous study identified that mechanical strain induced oxidative stress (OS) and promoted apoptosis and senescence in pelvic support fibroblasts. The aim of the present study is to investigate the molecular signaling pathway linking mechanical force with POP. Using a four‑point bending device, human uterosacral ligament fibroblasts (hUSLF) were exposed to mechanical tensile strain at a frequency of 0.3 Hz and intensity of 5333 µε, in the presence or absence of LY294002. The applied mechanical strain on hUSLF resulted in apoptosis and senescence, and decreased expression of procollagen type I α1. Mechanical strain activated phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K)/Akt signaling and resulted in downregulated expression of glutathione peroxidase 1 and Mn‑superoxide dismutase, and accumulation of intracellular reactive oxygen species. These effects were blocked by administration of LY294002. Furthermore, it was demonstrated that PI3K/Akt was activated in the uterosacral ligaments of POP patients, and that OS was increased and collagen type I production reduced. The results from the present study suggest that mechanical strain promotes apoptosis and senescence, and reduces collagen type I production via activation of PI3K/Akt-mediated OS signaling pathway in hUSLF. This process may be involved in the pathogenesis of POP as it results in relaxation and dysfunction of pelvic supports. PMID:27176043

  17. CpG-ODN Attenuates Pathological Cardiac Hypertrophy and Heart Failure by Activation of PI3Kα-Akt Signaling

    PubMed Central

    Yang, Liang; Cai, Xiangyu; Liu, Jie; Jia, Zhe; Jiao, Jinjin; Zhang, Jincai; Li, Changlin; Li, Jing; Tang, Xiang D.

    2013-01-01

    Phosphoinositide-3-kinase α (PI3Kα) represents a potential novel drug target for pathological cardiac hypertrophy (PCH) and heart failure. Oligodeoxynucleotides containing CpG motifs (CpG-ODN) are classic agonists of Toll-like receptor 9 (TLR9), which typically activates PI3K-Akt signaling in immune cells; however, the role of the nucleotide TLR9 agonists in cardiac myocytes is largely unknown. Here we report that CpG-ODN C274 could both attenuate PCH and improve cardiac dysfunction by activating PI3Kα-Akt signaling cascade. In vitro studies indicated that C274 could blunt reactivation of fetal cardiac genes and cell enlargement induced by a hypertrophic agent, isoproterenol. The anti-hypertrophic effect of C274 was suppressed by a pan-PI3K inhibitor, LY294002, or a small interfering RNA targeting PI3Kα. In vivo studies demonstrated that PCH, as marked by increased heart weight (HW) and cardiac ANF mRNA, was normalized by pre-administration with C274. In addition, Doppler echocardiography detected cardiac ventricular dilation, and contractile dysfunction in isoproterenol-treated animals, consistent with massive replacement fibrosis, reflecting cardiac cell death. As expected, pre-treatment of mice with C274 could prevent cardiac dysfunction associated with diminished cardiac cell death and fibrosis. In conclusion, CpG-ODNs are novel cardioprotective agents possessing antihypertrophic and anti-cell death activity afforded by engagement of the PI3Kα-Akt signaling. CpG-ODNs may have clinical use curbing the progression of PCH and preventing heart failure. PMID:23638055

  18. Erythropoietin pretreatment suppresses inflammation by activating the PI3K/Akt signaling pathway in myocardial ischemia-reperfusion injury

    PubMed Central

    RONG, REN; XIJUN, XIAO

    2015-01-01

    Erythropoietin (EPO), a glycoprotein originally known for its important role in the stimulation of erythropoiesis, has recently been shown to have significant protective effects in animal models of kidney and intestinal ischemia-reperfusion injury (IRI). However, the mechanism underlying these protective effects remains unclear. The aim of the current study was to evaluate the effects of EPO on myocardial IRI and to investigate the mechanism underlying these effects. A total of 18 male Sprague Dawley rats were randomly divided into three groups, namely the sham, IRI-saline and IRI-EPO groups. Rats in the IRI-EPO group were administered 5,000 U/kg EPO intraperitoneally 24 h prior to the induction of IRI. IRI was induced by ligating the left descending coronary artery for 30 min, followed by reperfusion for 3 h. Pathological changes in the myocardial tissue were observed and scored. The levels of the proinflammatory cytokines, interleukin (IL)-6, IL-1β and tumor necrosis factor (TNF)-α, were evaluated in the serum and myocardial tissue. Furthermore, the effects of EPO on phosphoinositide 3-kinase/protein kinase B (PI3K/Akt) signaling and EPO receptor (EPOR) phosphorylation were measured. Pathological changes in the myocardial tissue, increased expression levels of TNF-α, IL-6 and IL-1β in the myocardium, and increased serum levels of these mediators, as a result of IRI, were significantly decreased by EPO pretreatment. The effects of EPO were found to be associated with the activation of PI3K/Akt signaling, which suppressed the inflammatory responses, following the initiation of EPOR activation by EPO. Therefore, EPO pretreatment was demonstrated to decrease myocardial IRI, which was associated with activation of EPOR, subsequently increasing PI3K/Akt signaling to inhibit the production and release of inflammatory mediators. Thus, the results of the present study indicated that EPO may be useful for preventing myocardial IRI. PMID:26622330

  19. Brain-derived Neurotrophic Factor Prevents Phencyclidine-induced Apoptosis in Developing Brain by Parallel Activation of both the ERK and PI-3K/Akt Pathways

    PubMed Central

    Xia, Yan; Wang, Cheng Z.; Liu, Jie; Anastasio, Noelle C.; Johnson, Kenneth M.

    2009-01-01

    Summary Phencyclidine is an N-methyl D-aspartate receptor (NMDAR) blocker that has been reported to induce neuronal apoptosis during development and schizophrenia-like behaviors in rats later in life. Brain derived neurotrophic factor (BDNF) has been shown to prevent neuronal death caused by NMDAR blockade, but the precise mechanism is unknown. This study examined the role of the phosphatidylinositol-3 kinase (PI3K)/Akt and extracellular signal-regulated kinase (ERK) pathways in BDNF protection of PCP-induced apoptosis in corticostriatal organotypic cultures. It was observed that BDNF inhibited PCP-induced apoptosis in a concentration dependent fashion. BDNF effectively prevented PCP-induced inhibition of the ERK and PI-3K/Akt pathways and suppressed GSK-3β activation. Blockade of either PI-3K/Akt or ERK activation abolished BDNF protection. Western blot analysis revealed that the PI-3K inhibitor LY294002 prevented the stimulating effect of BDNF on the PI-3K/Akt pathway, but had no effect on the ERK pathway. Similarly, the ERK inhibitor PD98059 prevented the stimulating effect of BDNF on the ERK pathway, but not the PI-3K/Akt pathway. Co-application of LY294002 and PD98059 had no additional effect on BDNF-evoked activation of Akt or ERK. However, concurrent exposure to PD98059 and LY294002 caused much greater inhibition of BDNF-evoked phosphorylation of GSK-3β at serine 9 than did LY294002 alone. Finally, either BDNF or GSK-3β inhibition prevented PCP-induced suppression of cyclic-AMP response element binding protein (CREB) phosphorylation. These data demonstrate that the protective effect of BDNF against PCP-induced apoptosis is mediated by parallel activation of the PI-3K/Akt and ERK pathways, most likely involves inhibition of GSK-3β and activation of CREB. PMID:19887077

  20. BAI, a novel cyclin-dependent kinase inhibitor induces apoptosis in A549 cells through activation of caspases and inactivation of Akt.

    PubMed

    Kim, Shin; Lee, Jinho; Jang, Byeong-Churl; Kwon, Taeg Kyu; Park, Jong-Wook

    2013-02-01

    Previously, we have synthesized a novel cyclin-dependent kinase (CDK) inhibitor, 2-[1,1'biphenyl]-4-yl-N-[5-(1,1-dioxo-1λ(6) -isothiazolidin-2-yl)-1H-indazol-3-yl]acetamide (BAI) and reported its anti-cancer activity in head and neck cancer cells. In this study, we further evaluated the effect of BAI on growth of various human cancer cell lines, including A549 (nonsmall cell lung cancer), HCT116 (colon), and Caki (kidney). Profoundly, results of XTT and clonogenic assays demonstrated that BAI at nanomolar concentrations (20-60 nM) inhibited growth of A549, HCT116, and Caki cells, suggesting the anti-cancer potency. We show that BAI induced a dose-dependent apoptotic cell death in these human cancer cells, as measured by fluorescence-activated cell sorting (FACS). Interestingly, further biochemical analysis showed that treatment with BAI at 20 nM induced apoptosis in A549 cells in association with activation of caspases, cleavage of phospholipase C-γ1 (PLC-γ1), and inhibition of Akt in A549 cells. Importantly, pharmacological inhibition study revealed that pretreatment with z-VAD-fmk, a pan caspase inhibitor strongly blocked the BAI-induced apoptosis in A549 cells. Transfection analysis with Akt cDNA encoding constitutively active Akt further addressed the significance of Akt inhibition in the BAI-induced apoptosis in A549 cells. Notably, disruption of the PI3K/Akt pathway by LY294002, a PI3K/Akt inhibitor potentiated apoptosis in A549 cells by BAI at a subcytotoxic concentration. These findings collectively suggest that BAI potently inhibits growth of A549, HCT116, and Caki cells, and that the BAI-induced apoptosis in A549 cells is associated with activation of caspases, and inhibition of Akt. PMID:22887215

  1. Excess iodide downregulates Na(+)/I(-) symporter gene transcription through activation of PI3K/Akt pathway.

    PubMed

    Serrano-Nascimento, Caroline; Nicola, Juan Pablo; Teixeira, Silvania da Silva; Poyares, Leonice Lourenço; Lellis-Santos, Camilo; Bordin, Silvana; Masini-Repiso, Ana Maria; Nunes, Maria Tereza

    2016-05-01

    Transcriptional mechanisms associated with iodide-induced downregulation of NIS expression remain uncertain. Here, we further analyzed the transcriptional regulation of NIS gene expression by excess iodide using PCCl3 cells. NIS promoter activity was reduced in cells treated for 12-24 h with 10(-5) to 10(-3) M NaI. Site-directed mutagenesis of Pax8 and NF-κB cis-acting elements abrogated the iodide-induced NIS transcription repression. Indeed, excess iodide (10(-3) M) excluded Pax8 from the nucleus, decreased p65 total expression and reduced their transcriptional activity. Importantly, p65-Pax8 physical interaction and binding to NIS upstream enhancer were reduced upon iodide treatment. PI3K/Akt pathway activation by iodide-induced ROS production is involved in the transcriptional repression of NIS expression. In conclusion, the results indicated that excess iodide transcriptionally represses NIS gene expression through the impairment of Pax8 and p65 transcriptional activity. Furthermore, the data presented herein described novel roles for PI3K/Akt signaling pathway and oxidative status in the thyroid autoregulatory phenomenon. PMID:26872612

  2. Baf60c drives glycolytic metabolism in the muscle and improves systemic glucose homeostasis through Deptor-mediated Akt activation.

    PubMed

    Meng, Zhuo-Xian; Li, Siming; Wang, Lin; Ko, Hwi Jin; Lee, Yongjin; Jung, Dae Young; Okutsu, Mitsuharu; Yan, Zhen; Kim, Jason K; Lin, Jiandie D

    2013-05-01

    A shift from oxidative to glycolytic metabolism has been associated with skeletal muscle insulin resistance in type 2 diabetes. However, whether this metabolic switch is deleterious or adaptive remains under debate, in part because of a limited understanding of the regulatory network that directs the metabolic and contractile specification of fast-twitch glycolytic muscle. Here we show that Baf60c (also called Smarcd3), a transcriptional cofactor enriched in fast-twitch muscle, promotes a switch from oxidative to glycolytic myofiber type through DEP domain-containing mTOR-interacting protein (Deptor)-mediated Akt activation. Muscle-specific transgenic expression of Baf60c activates a program of molecular, metabolic and contractile changes characteristic of glycolytic muscle. In addition, Baf60c is required for maintaining glycolytic capacity in adult skeletal muscle in vivo. Baf60c expression is significantly lower in skeletal muscle from obese mice compared to that from lean mice. Activation of the glycolytic muscle program by transgenic expression of Baf60c protects mice from diet-induced insulin resistance and glucose intolerance. Further mechanistic studies revealed that Deptor is induced by the Baf60c-Six4 transcriptional complex and mediates activation of Akt and glycolytic metabolism by Baf60c in a cell-autonomous manner. This work defines a fundamental mechanism underlying the specification of fast-twitch glycolytic muscle and illustrates that the oxidative-to-glycolytic metabolic shift in skeletal muscle is potentially adaptive and beneficial in the diabetic state. PMID:23563706

  3. 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

  4. 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.

  5. Hyaluronan Activates Cell Motility of v-Src-transformed Cells via Ras-Mitogen–activated Protein Kinase and Phosphoinositide 3-Kinase-Akt in a Tumor-specific Manner

    PubMed Central

    Sohara, Yasuyoshi; Ishiguro, Naoki; Machida, Kazuya; Kurata, Hisashi; Thant, Aye Aye; Senga, Takeshi; Matsuda, Satoru; Kimata, Koji; Iwata, Hisashi; Hamaguchi, Michinari

    2001-01-01

    We investigated the production of hyaluronan (HA) and its effect on cell motility in cells expressing the v-src mutants. Transformation of 3Y1 by v-src virtually activated HA secretion, whereas G2A v-src, a nonmyristoylated form of v-src defective in cell transformation, had no effect. In cells expressing the temperature-sensitive mutant of v-Src, HA secretion was temperature dependent. In addition, HA as small as 1 nM, on the other side, activated cell motility in a tumor-specific manner. HA treatment strongly activated the motility of v-Src–transformed 3Y1, whereas it showed no effect on 3Y1- and 3Y1-expressing G2A v-src. HA-dependent cell locomotion was strongly blocked by either expression of dominant-negative Ras or treatment with a Ras farnesyltransferase inhibitor. Similarly, both the MEK1 inhibitor and the kinase inhibitor clearly inhibited HA-dependent cell locomotion. In contrast, cells transformed with an active MEK1 did not respond to the HA. Finally, an anti-CD44–neutralizing antibody could block the activation of cell motility by HA as well as the HA-dependent phosphorylation of mitogen-activated protein kinase and Akt. Taken together, these results suggest that simultaneous activation of the Ras-mitogen-activated protein kinase pathway and the phosphoinositide 3-kinase pathway by the HA-CD44 interaction is required for the activation of HA-dependent cell locomotion in v-Src–transformed cells. PMID:11408591

  6. Dynamic interplay between autophagic flux and Akt during melanoma progression in vitro.

    PubMed

    Maes, Hannelore; Martin, Shaun; Verfaillie, Tom; Agostinis, Patrizia

    2014-02-01

    Despite advances in cancer diagnosis and therapy, metastatic melanoma remains untreatable, due to its notorious resistance to apoptosis, deeming traditional therapies obsolete. Deregulated PI3K/Akt signalling is a common oncogenic event enabling melanocyte transformation and represents a significant and 'druggable' pathway in melanoma. Emerging data show that the ability of cancer cells to survive is also facilitated by alteration of vital homoeostatic mechanisms, such as autophagy. Although the role of autophagy in melanoma is still controversial, recent studies suggest that basal autophagy is down-modulated in primary melanomas. However, the dynamic connection between pro-tumorigenic PI3K/Akt and autophagy during melanoma progression has not been systematically studied. By using human primary melanocytes, incipient melanoma and metastatic melanoma cell lines, we show that early in melanomagenesis, increased Akt activity is associated with a low baseline autophagic flux. However, during melanoma progression, metastatic melanoma cells regain the ability to stimulate autophagic flux, supporting survival. Heightened autophagy is associated with an attenuated Akt activation status and can be suppressed by overexpressing a constitutive active mutant of Akt. On the other hand, blocking the higher Akt activity of primary melanoma is sufficient to incite autophagy. Interestingly, we found that although Akt supports survival of melanocytes and all melanoma cell lines, autophagy inhibition specifically targeted the metastatic melanoma cells, thus indicating a stage-specific requirement for Akt and autophagic flux, throughout melanoma progression. Therefore, this study highlights a dynamic interplay between Akt signalling and autophagic rescue in melanoma, which should be considered in the design of therapeutic strategies targeting these pathways. PMID:24313465

  7. Activated α2-macroglobulin binding to cell surface GRP78 induces T-loop phosphorylation of Akt1 by PDK1 in association with Raptor.

    PubMed

    Misra, Uma Kant; Pizzo, Salvatore Vincent

    2014-01-01

    PDK1 phosphorylates multiple substrates including Akt by PIP3-dependent mechanisms. In this report we provide evidence that in prostate cancer cells stimulated with activated α2-macroglobulin (α2M*) PDK1 phosphorylates Akt in the T-loop at Thr(308) by using Raptor in the mTORC1 complex as a scaffold protein. First we demonstrate that PDK1, Raptor, and mTOR co-immunoprecipitate. Silencing the expression, not only of PDK1, but also Raptor by RNAi nearly abolished Akt phosphorylation at Akt(Thr308) in Raptor-immunoprecipitates of α2M*-stimulated prostate cancer cells. Immunodepleting Raptor or PDK from cell lysates of cells treated with α2M* drastically reduced Akt phosphorylation at Thr(308), which was recovered by adding the supernatant of Raptor- or PDK1-depleted cell lysates, respectively. Studies of insulin binding to its receptor on prostate cancer cells yielded similar results. We thus demonstrate that phosphorylating the T-loop Akt residue Thr(308) by PDK1 requires Raptor of the mTORC1 complex as a platform or scaffold protein. PMID:24516643

  8. Distinct Time Course of the Decrease in Hepatic AMP-Activated Protein Kinase and Akt Phosphorylation in Mice Fed a High Fat Diet

    PubMed Central

    Shiwa, Mami; Yoneda, Masayasu; Okubo, Hirofumi; Ohno, Haruya; Kobuke, Kazuhiro; Monzen, Yuko; Kishimoto, Rui; Nakatsu, Yusuke; Asano, Tomoichiro; Kohno, Nobuoki

    2015-01-01

    AMP-activated protein kinase (AMPK) plays an important role in insulin resistance, which is characterized by the impairment of the insulin-Akt signaling pathway. However, the time course of the decrease in AMPK and Akt phosphorylation in the liver during the development of obesity and insulin resistance caused by feeding a high fat diet (HFD) remains controversial. Moreover, it is unclear whether the impairment of AMPK and Akt signaling pathways is reversible when changing from a HFD to a standard diet (SD). Male ddY mice were fed the SD or HFD for 3 to 28 days, or fed the HFD for 14 days, followed by the SD for 14 days. We examined the time course of the expression and phosphorylation levels of AMPK and Akt in the liver by immunoblotting. After 3 days of feeding on the HFD, mice gained body weight, resulting in an increased oil red O staining, indicative of hepatic lipid accumulation, and significantly decreased AMPK phosphorylation, in comparison with mice fed the SD. After 14 days on the HFD, systemic insulin resistance occurred and Akt phosphorylation significantly decreased. Subsequently, a change from the HFD to SD for 3 days, after 14 days on the HFD, ameliorated the impairment of AMPK and Akt phosphorylation and systemic insulin resistance. Our findings indicate that AMPK phosphorylation decreases early upon feeding a HFD and emphasizes the importance of prompt lifestyle modification for decreasing the risk of developing diabetes. PMID:26266809

  9. 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

  10. Fractionated Ionizing Radiation Promotes Epithelial-Mesenchymal Transition in Human Esophageal Cancer Cells through PTEN Deficiency-Mediated Akt Activation

    PubMed Central

    He, Enhui; Pan, Fei; Li, Guangchao; Li, Jingjing

    2015-01-01

    In some esophageal cancer patients, radiotherapy may not prevent distant metastasis thus resulting in poor survival. The underlying mechanism of metastasis in these patients is not well established. In this study, we have demonstrated that ionizing radiation may induce epithelial-mesenchymal transition (EMT) accompanied with increased cell migration and invasion, through downregulation of phosphatase and tensin homolog (PTEN), and activation of Akt/GSK-3β/Snail signaling. We developed a radioresistant (RR) esophageal squamous cancer cell line, KYSE-150/RR, by fractionated ionizing radiation (IR) treatment, and confirmed its radioresistance using a clonogenic survival assay. We found that the KYSE-150/RR cell line displayed typical morphological and molecular characteristics of EMT. In comparison to the parental cells, KYSE-150/RR cells showed an increase in post-IR colony survival, migration, and invasiveness. Furthermore, a decrease in PTEN in KYSE-150/RR cells was observed. We postulated that over-expression of PTEN may induce mesenchymal-epithelial transition in KYSE-150/RR cells and restore IR-induced increase of cell migration. Mechanistically, fractionated IR inhibits expression of PTEN, which leads to activation of Akt/GSK-3β signaling and is associated with the elevated levels of Snail protein, a transcription factor involved in EMT. Correspondingly, treatment with LY294002, a phosphatidylinositol-3-kinase inhibitor, mimicked PTEN overexpression effect in KYSE-150/RR cells, further suggesting a role for the Akt/GSK-3β/Snail signaling in effects mediated through PTEN. Together, these results strongly suggest that fractionated IR-mediated EMT in KYSE-150/RR cells is through PTEN-dependent pathways, highlighting a direct proinvasive effect of radiation treatment on tumor cells. PMID:26000878

  11. 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.

  12. Sestrin 3 Protein Enhances Hepatic Insulin Sensitivity by Direct Activation of the mTORC2-Akt Signaling

    PubMed Central

    Tao, Rongya; Xiong, Xiwen; Liangpunsakul, Suthat

    2015-01-01

    Sestrin proteins have been implicated in multiple biological processes including resistance to oxidative and genotoxic stresses, protection against aging-related pathologies, and promotion of metabolic homeostasis; however, the underlying mechanisms are incompletely understood. Some evidence suggests that sestrins may inhibit mTORC1 (mechanistic target of rapamycin complex 1) through inhibition of RagA/B GTPases or activation of AMPK; however, whether sestrins are also involved in mTORC2 regulation and function is unclear. To investigate the functions and mechanisms of Sestrin 3 (Sesn3), we generated Sesn3 liver-specific transgenic and knockout mice. Our data show that Sesn3 liver-specific knockout mice exhibit insulin resistance and glucose intolerance, and Sesn3 transgenic mice were protected against insulin resistance induced by a high-fat diet. Using AMPK liver-specific knockout mice, we demonstrate that the Sesn3 insulin-sensitizing effect is largely independent of AMPK. Biochemical analysis reveals that Sesn3 interacts with and activates mTORC2 and subsequently stimulates Akt phosphorylation at Ser473. These findings suggest that Sesn3 can activate Akt via mTORC2 to regulate hepatic insulin sensitivity and glucose metabolism. PMID:25377878

  13. The small GTPase Rap1b negatively regulates neutrophil chemotaxis and transcellular diapedesis by inhibiting Akt activation

    PubMed Central

    Kumar, Sachin; Xu, Juying; Kumar, Rupali Sani; Lakshmikanthan, Sribalaji; Kapur, Reuben; Kofron, Matthew; Chrzanowska-Wodnicka, Magdalena

    2014-01-01

    Neutrophils are the first line of cellular defense in response to infections and inflammatory injuries. However, neutrophil activation and accumulation into tissues trigger tissue damage due to release of a plethora of toxic oxidants and proteases, a cause of acute lung injury (ALI). Despite its clinical importance, the molecular regulation of neutrophil migration is poorly understood. The small GTPase Rap1b is generally viewed as a positive regulator of immune cell functions by controlling bidirectional integrin signaling. However, we found that Rap1b-deficient mice exhibited enhanced neutrophil recruitment to inflamed lungs and enhanced susceptibility to endotoxin shock. Unexpectedly, Rap1b deficiency promoted the transcellular route of diapedesis through endothelial cell. Increased transcellular migration of Rap1b-deficient neutrophils in vitro was selectively mediated by enhanced PI3K-Akt activation and invadopodia-like protrusions. Akt inhibition in vivo suppressed excessive Rap1b-deficient neutrophil migration and associated endotoxin shock. The inhibitory action of Rap1b on PI3K signaling may be mediated by activation of phosphatase SHP-1. Thus, this study reveals an unexpected role for Rap1b as a key suppressor of neutrophil migration and lung inflammation. PMID:25092872

  14. Development of a new model system to dissect isoform specific Akt signalling in adipocytes.

    PubMed

    Kajno, Esi; McGraw, Timothy E; Gonzalez, Eva

    2015-06-15

    Protein kinase B (Akt) kinases are critical signal transducers mediating insulin action. Genetic studies revealed that Akt1 and Akt2 signalling differentially contribute to sustain lipid and glucose homoeostasis; however Akt isoform-specific effectors remain elusive due to the lack of a suitable model system to mechanistically interrogate Akt isoform-specific signalling. To overcome those technical limitations we developed a novel model system that provides acute and specific control of signalling by Akt isoforms. We generated mutants of Akt1 and Akt2 resistant to the allosteric Akt inhibitor MK-2206. We then developed adipocyte cell lines, in which endogenous Akt1 or Akt2 has been replaced by their corresponding drug-resistant Akt mutant. Treatment of those cells with MK-2206 allowed for acute and specific control of either Akt1 or Akt2 function. Our data showed that Akt1(W80A) and Akt2(W80A) mutants are resistant to MK-2206, dynamically regulated by insulin and able to signal to Akt downstream effectors. Analyses of insulin action in this cellular system showed that Akt1 and Akt2 are both able to mediate insulin regulation of the transcription factor forkhead box O1 (FoxO1) and the glucose transporter 4 (GLUT4), revealing a redundant role for these Akt kinases in the control of glucose transport into fat cells. In contrast, Akt1 signalling is uniquely required for adipogenesis, by controlling the mitotic clonal expansion (MCE) of pre-adipocytes that precedes white adipose cell differentiation. Our data provide new insights into the role of Akt kinases in glucose transport and adipogenesis and support our model system as a valuable tool for the biochemical characterization of signalling by specific Akt isoforms. PMID:25856301

  15. Potential role of AKT/mTOR signalling proteins in hairy cell leukaemia: association with BRAF/ERK activation and clinical outcome

    PubMed Central

    Lakiotaki, Eleftheria; Levidou, Georgia; Angelopoulou, Maria K.; Adamopoulos, Christos; Pangalis, Gerassimos; Rassidakis, George; Vassilakopoulos, Theodoros; Gainaru, Gabriella; Flevari, Pagona; Sachanas, Sotirios; Saetta, Angelica A.; Sepsa, Athanasia; Moschogiannis, Maria; Kalpadakis, Christina; Tsesmetzis, Nikolaos; Milionis, Vassilios; Chatziandreou, Ilenia; Thymara, Irene; Panayiotidis, Panayiotis; Dimopoulou, Maria; Plata, Eleni; Konstantopoulos, Konstantinos; Patsouris, Efstratios; Piperi, Christina; Korkolopoulou, Penelope

    2016-01-01

    The potential role of AKT/mTOR signalling proteins and its association with the Raf-MEK-ERK pathway was investigated in hairy cell leukaemia (HCL). BRAFV600E expression and activated forms of AKT, mTOR, ERK1/2, p70S6k and 4E-BP1 were immunohistochemically assessed in 77 BM biopsies of HCL patients and correlated with clinicopathological and BM microvascular characteristics, as well as with c-Caspase-3 levels in hairy cells. Additionally, we tested rapamycin treatment response of BONNA-12 wild-type cells or transfected with BRAFV600E. Most HCL cases expressed p-p70S6K and p-4E-BP1 but not p-mTOR, being accompanied by p-ERK1/2 and p-AKT. AKT/mTOR activation was evident in BONNA-12 cells irrespective of the presence of BRAFV600E mutation and was implicated in cell proliferation enhancement. In multivariate analysis p-AKT/p-mTOR/p-4E-BP1 overexpression was an adverse prognostic factor for time to next treatment conferring earlier relapse. When p-AKT, p-mTOR and p-4E-BP1 were examined separately only p-4E-BP1 remained significant. Our findings indicate that in HCL, critical proteins up- and downstream of mTOR are activated. Moreover, the strong associations with Raf-MEK-ERK signalling imply a possible biologic interaction between these pathways. Most importantly, expression of p-4E-BP1 alone or combined with p-AKT and p-mTOR is of prognostic value in patients with HCL. PMID:26893254

  16. Notoginsenoside R1-mediated neuroprotection involves estrogen receptor-dependent crosstalk between Akt and ERK1/2 pathways: a novel mechanism of Nrf2/ARE signaling activation.

    PubMed

    Meng, X; Sun, G; Ye, J; Xu, H; Wang, H; Sun, X

    2014-04-01

    Notoginsenoside R1 (NGR1), a novel phytoestrogen isolated from Panax notoginseng, has antioxidant and anti-apoptotic properties. Oxidative stress plays a pivotal role in neurodegenerative diseases. To mimic oxidative stress in neurons and explore the neuroprotection of NGR1, H₂O₂-induced neurotoxicity in NGF-induced differentiation of PC12 cells was used. In this study, NGR1 preconditioning provided neuroprotective effects via suppressing H₂O₂-induced the intracellular ROS accumulation, the increase in the product of lipid peroxidation (MDA), protein oxidation (protein carbonyl), and DNA fragmentation (8-OHdG), and mitochondrial membrane depolarization as well as caspase-3 activation. Moreover, NGR1 treatment alone potently increased the nuclear translocation of Nrf2, augmented ARE enhancer activity, and upregulated the expression and activity of phase II antioxidant enzymes including HO-1, NQO-1, and γ-GCSc. NGR1 could also increase the ERE activity and activate Akt and ERK1/2 pathways. NGR1-mediated activation of Nrf2/ARE signaling and neuroprotection were abolished by genetic silencing of Nrf2 using siRNA or the pharmacological blockade of estrogen receptors using ICI-182780, and partially inhibited by Akt siRNA or ERK siRNA transfection. In addition, the phosphorylation of ERK1/2 mediated by NGR1 was markedly inhibited in PC12 cells transfected with Akt siRNA. On the contrary, ERK1/2 siRNA transfection hardly had any effect on the phosphorylation of Akt mediated by NGR1. NGR1-mediated activation of Akt and ERK1/2 pathways was blocked by ICI-182780. In conclusion, NGR1 provided neuroprotection via inducing an estrogen receptor-dependent crosstalk between Akt and ERK1/2 pathways, subsequently activating Nrf2/ARE signaling and thereby up-regulating phase II antioxidant enzymes. PMID:24437944

  17. Cell surface protein C23 affects EGF-EGFR induced activation of ERK and PI3K-AKT pathways.

    PubMed

    Lv, Shunzeng; Dai, Congxin; Liu, Yuting; Sun, Bowen; Shi, Ranran; Han, Mingzhi; Bian, Ruixiang; Wang, Renzhi

    2015-02-01

    The epidermal growth factor (EGF) pathway has been reported as canonical causes in cancer development. Meanwhile, the involvement of C23 in multiple signaling pathways has been also investigated (Lv et al., 2014). However, the effect of C23 on EGF pathway in glioblastoma is not fully characterized. In the present study, C23 and the epidermal growth factor receptor (EGFR) of U251 cell line were inhibited by C23 and EGFR antibodies, respectively; and then C23 and EGFR siRNAs were used to knock down endogenous C23 and EGFR, respectively. In addition, soft-agar and MTT assay were also introduced. Compared with control, either C23 or EGFR antibodies efficiently repressed the phosphorylation levels of ERK1/2 (p<0.000) and AKT (p<0.000). Similarly, either C23 or EGFR siRNAs indeed resulted in C23 and EGFR knockdown, and further suppressed the expression of p-ERK1/2 and p-AKT. Most importantly, immunoprecipitation revealed C23 interacted with EGFR once U251 was exposed to EGF treatment. In addition, the MTT and soft-agar assay also identified that C23 or EGFR siRNAs could obviously affected cell growth (p=0.004) and invasiveness, as cell viability and colony formation decreased markedly. Our results suggest that C23 plays a crucial role in activation of EGF-induced ERK and PI3K-AKT pathways via interacting with EGFR; furthermore, C23 could be indicative of an important factor in glioblastoma development and a useful target for glioblastoma treatment. PMID:25015231

  18. 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

  19. Naringenin induces apoptosis through downregulation of Akt and caspase-3 activation in human leukemia THP-1 cells.

    PubMed

    Park, Joon Hee; Jin, Cheng-Yun; Lee, Bok Kyu; Kim, Gi-Young; Choi, Yung Hyun; Jeong, Yong Kee

    2008-12-01

    Naringenin (NGEN), one of the most abundant flavonoids in citrus fruits, has been shown to inhibit in vitro growth of in human cancer cells, although the mechanism of action is poorly understood. Herein, we investigated NEGN's pro-apoptotic effect on human leukemia THP-1 cells. NGEN treatment inhibited THP-1 cells' growth a concentration-dependent manner by inducing apoptosis, as evidenced by the formation of apoptotic bodies and the accumulation of cells in the sub-G1 phase. NGEN-induced apoptosis was accompanied by increased hyperpolarization of the mitochondrial membrane potential, downregulation of Bcl-2, upregulation of Bax, activation of caspases and subsequent poly(ADP-ribose)polymerase (PARP) cleavages. z-DEVD-fmk, a caspase-3 inhibitor, significantly inhibited both the cytotoxic effect and apoptotic characteristics induced by NGEN treatment demonstrating caspase-3's important role in the observed cytotoxic effect. The induction of apoptosis was also associated with the inactivation of phosphatidylinositol 3-kinase (PI3K)/Akt, and PI3K inhibitor LY29004 significantly increases NGEN-induced cell death. These findings provide evidence that NEGN's pro-apoptotic effect is mediated by the activation of caspases and mitochondria dysfunctions that correlate with the inactivation of the PI3K/Akt pathway in THP-1 cells. Therefore, NGEN has a strong potential as a therapeutic agent for preventing cancers such as leukemia. PMID:18930780

  20. 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

  1. The sonic hedgehog signaling pathway stimulates anaplastic thyroid cancer cell motility and invasiveness by activating Akt and c-Met.

    PubMed

    Williamson, Ashley J; Doscas, Michelle E; Ye, Jin; Heiden, Katherine B; Xing, Mingzhao; Li, Yi; Prinz, Richard A; Xu, Xiulong

    2016-03-01

    The sonic hedgehog (Shh) pathway is highly activated in thyroid neoplasms and promotes thyroid cancer stem-like cell phenotype, but whether the Shh pathway regulates thyroid tumor cell motility and invasiveness remains unknown. Here, we report that the motility and invasiveness of two anaplastic thyroid tumor cell lines, KAT-18 and SW1736, were inhibited by two inhibitors of the Shh pathway (cyclopamine and GANT61). Consistently, the cell motility and invasiveness was decreased by Shh and Gli1 knockdown, and was increased by Gli1 overexpression in KAT-18 cells. Mechanistic studies revealed that Akt and c-Met phosphorylation was decreased by a Gli1 inhibitor and by Shh and Gli1 knockdown, but was increased by Gli1 overexpression. LY294002, a PI-3 kinase inhibitor, and a c-Met inhibitor inhibited the motility and invasiveness of Gli1-transfected KAT-18 cells more effectively than the vector-transfected cells. Knockdown of Snail, a transcription factor regulated by the Shh pathway, led to decreased cell motility and invasiveness in KAT-18 and SW1736 cells. However, key epithelial-to-mesenchymal transition (EMT) markers including E-cadherin and vimentin as well as Slug were not affected by cyclopamine and GANT61 in either SW1736 or WRO82, a well differentiated follicular thyroid carcinoma cell line. Our data suggest that the Shh pathway-stimulated thyroid tumor cell motility and invasiveness is largely mediated by AKT and c-Met activation with little involvement of EMT. PMID:26859575

  2. TCDD promotes lung tumors via attenuation of apoptosis through activation of the Akt and ERK1/2 signaling pathways.

    PubMed

    Chen, Rong-Jane; Siao, Shih-He; Hsu, Chung-Huei; Chang, Chu-Yung; Chang, Louis W; Wu, Chih-Hsiung; Lin, Pinpin; Wang, Ying-Jan

    2014-01-01

    2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a multiple-site, multiple-species carcinogen that induces cancer in multiple organs. The molecular mechanisms underlying TCDD-induced lung tumorigenesis remain unclear. In the present study, a two-stage lung tumorigenesis model was established by administrating a single low dose of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) combined with TCDD to female A/J mice. The results indicated that TCDD combined with low-dose NNK has a significant tumor-promoting effect compared with TCDD or low-dose NNK alone. Resistance to apoptosis is a hallmark of cancer and is thought to be one of the tumor-promoting mechanisms regulated by TCDD. We performed an additional series of experiments in the normal human bronchial epithelial cell line Beas2B cells, in which TCDD was combined with the apoptosis inducer staurosporine. Our in vitro results confirmed that TCDD could rescue cells from apoptosis induced by staurosporine. The inhibition of apoptosis is likely mediated by the activation of the Akt and ERK1/2 pathways, as determined by the effectiveness of pathway-specific inhibitors in abrogating the anti-apoptotic activity of TCDD. In conclusion, we demonstrated that TCDD promoted NNK-induced lung tumorigenesis and revealed that TCDD inhibits staurosporine-induced apoptosis, at least in part, through the Akt and ERK1/2 signaling pathways. PMID:24927102

  3. Inhibition of LN-308 glioma cell proliferation and migration by retinoic acid amide through activation of Akt pathway

    PubMed Central

    Zhu, Jun; Lu, Xiang-Dong; Si, Feng; Song, Chun-Yu; Meng, Qing-Hai

    2015-01-01

    The present study was performed to investigate the effect of retinoic acid amide (RAA) on the expression of integrin α3β1, rate of cell proliferation and migration in p53-deficient glioma cell line, LN-308. The results revealed promotion of integrin α3 expression, reduction in proliferation and migration in RAA treated cells compared to the control LN-308 glioma cells. Promotion of RAA induced integrin α3β1 expression led to the enhancement in cyclin-dependent kinase nuclear localization and activation of Akt pathway. In addition, RAA treatment inhibited the expression of nuclear factor-κB, Bcl-2 and epidermal growth factor receptor (EGFR). These factors are responsible for promoting the rate of cell proliferation and survival in the carcinoma cells. Thus RAA treatment inhibits rate of LN-308 glioma cell proliferation and migration through increase in integrin α3β1 expression and activation of Akt pathway. Therefore, RAA can be of therapeutic importance for the treatment of glioma. PMID:26823704

  4. The sonic hedgehog signaling pathway stimulates anaplastic thyroid cancer cell motility and invasiveness by activating Akt and c-Met

    PubMed Central

    Williamson, Ashley J.; Doscas, Michelle E.; Ye, Jin; Heiden, Katherine B.; Xing, Mingzhao; Li, Yi; Prinz, Richard A.; Xu, Xiulong

    2016-01-01

    The sonic hedgehog (Shh) pathway is highly activated in thyroid neoplasms and promotes thyroid cancer stem-like cell phenotype, but whether the Shh pathway regulates thyroid tumor cell motility and invasiveness remains unknown. Here, we report that the motility and invasiveness of two anaplastic thyroid tumor cell lines, KAT-18 and SW1736, were inhibited by two inhibitors of the Shh pathway (cyclopamine and GANT61). Consistently, the cell motility and invasiveness was decreased by Shh and Gli1 knockdown, and was increased by Gli1 overexpression in KAT-18 cells. Mechanistic studies revealed that Akt and c-Met phosphorylation was decreased by a Gli1 inhibitor and by Shh and Gli1 knockdown, but was increased by Gli1 overexpression. LY294002, a PI-3 kinase inhibitor, and a c-Met inhibitor inhibited the motility and invasiveness of Gli1-transfected KAT-18 cells more effectively than the vector-transfected cells. Knockdown of Snail, a transcription factor regulated by the Shh pathway, led to decreased cell motility and invasiveness in KAT-18 and SW1736 cells. However, key epithelial-to-mesenchymal transition (EMT) markers including E-cadherin and vimentin as well as Slug were not affected by cyclopamine and GANT61 in either SW1736 or WRO82, a well differentiated follicular thyroid carcinoma cell line. Our data suggest that the Shh pathway-stimulated thyroid tumor cell motility and invasiveness is largely mediated by AKT and c-Met activation with little involvement of EMT. PMID:26859575

  5. TCDD Promotes Lung Tumors via Attenuation of Apoptosis through Activation of the Akt and ERK1/2 Signaling Pathways

    PubMed Central

    Siao, Shih-He; Hsu, Chung-Huei; Chang, Chu-Yung; Chang, Louis W.; Lin, Pinpin; Wang, Ying-Jan

    2014-01-01

    2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a multiple-site, multiple-species carcinogen that induces cancer in multiple organs. The molecular mechanisms underlying TCDD-induced lung tumorigenesis remain unclear. In the present study, a two-stage lung tumorigenesis model was established by administrating a single low dose of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) combined with TCDD to female A/J mice. The results indicated that TCDD combined with low-dose NNK has a significant tumor-promoting effect compared with TCDD or low-dose NNK alone. Resistance to apoptosis is a hallmark of cancer and is thought to be one of the tumor-promoting mechanisms regulated by TCDD. We performed an additional series of experiments in the normal human bronchial epithelial cell line Beas2B cells, in which TCDD was combined with the apoptosis inducer staurosporine. Our in vitro results confirmed that TCDD could rescue cells from apoptosis induced by staurosporine. The inhibition of apoptosis is likely mediated by the activation of the Akt and ERK1/2 pathways, as determined by the effectiveness of pathway-specific inhibitors in abrogating the anti-apoptotic activity of TCDD. In conclusion, we demonstrated that TCDD promoted NNK-induced lung tumorigenesis and revealed that TCDD inhibits staurosporine-induced apoptosis, at least in part, through the Akt and ERK1/2 signaling pathways. PMID:24927102

  6. Myogenic signaling of phosphatidylinositol 3-kinase requires the serine-threonine kinase Akt/protein kinase B

    PubMed Central

    Jiang, Bing-Hua; Aoki, Masahiro; Zheng, Jenny Z.; Li, Jian; Vogt, Peter K.

    1999-01-01

    The oncogene p3k, coding for a constitutively active form of phosphatidylinositol 3-kinase (PI 3-kinase), strongly activates myogenic differentiation. Inhibition of endogenous PI 3-kinase activity with the specific inhibitor LY294002, or with dominant-negative mutants of PI 3-kinase, interferes with myotube formation and with the expression of muscle-specific proteins. Here we demonstrate that a downstream target of PI 3-kinase, serine-threonine kinase Akt, plays an important role in myogenic differentiation. Expression of constitutively active forms of Akt dramatically enhances myotube formation and expression of the muscle-specific proteins MyoD, creatine kinase, myosin heavy chain, and desmin. Transdominant negative forms of Akt inhibit myotube formation and the expression of muscle-specific proteins. The inhibition of myotube formation and the reduced expression of muscle-specific proteins caused by the PI 3-kinase inhibitor LY294002 are completely reversed by constitutively active forms of Akt. Wild-type cellular Akt effects a partial reversal of LY294002-induced inhibition of myogenic differentiation. This result suggests that Akt can substitute for PI 3-kinase in the stimulation of myogenesis; Akt may be an essential downstream component of PI 3-kinase-induced muscle differentiation. PMID:10051597

  7. A computational model on the modulation of mitogen-activated protein kinase (MAPK) and Akt pathways in heregulin-induced ErbB signalling.

    PubMed Central

    Hatakeyama, Mariko; Kimura, Shuhei; Naka, Takashi; Kawasaki, Takuji; Yumoto, Noriko; Ichikawa, Mio; Kim, Jae-Hoon; Saito, Kazuki; Saeki, Mihoro; Shirouzu, Mikako; Yokoyama, Shigeyuki; Konagaya, Akihiko

    2003-01-01

    ErbB tyrosine kinase receptors mediate mitogenic signal cascade by binding a variety of ligands and recruiting the different cassettes of adaptor proteins. In the present study, we examined heregulin (HRG)-induced signal transduction of ErbB4 receptor and found that the phosphatidylinositol 3'-kinase (PI3K)-Akt pathway negatively regulated the extracellular signal-regulated kinase (ERK) cascade by phosphorylating Raf-1 on Ser(259). As the time-course kinetics of Akt and ERK activities seemed to be transient and complex, we constructed a mathematical simulation model for HRG-induced ErbB4 receptor signalling to explain the dynamics of the regulation mechanism in this signal transduction cascade. The model reflected well the experimental results observed in HRG-induced ErbB4 cells and in other modes of growth hormone-induced cell signalling that involve Raf-Akt cross-talk. The model suggested that HRG signalling is regulated by protein phosphatase 2A as well as Raf-Akt cross-talk, and protein phosphatase 2A modulates the kinase activity in both the PI3K-Akt and MAPK (mitogen-activated protein kinase) pathways. PMID:12691603

  8. 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. PMID:24306493

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

    PubMed

    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. MPK6(F364L) and MPK6(F368L) 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

  10. 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

  11. MiRNA-486 regulates angiogenic activity and survival of mesenchymal stem cells under hypoxia through modulating Akt signal.

    PubMed

    Shi, Xue-Feng; Wang, Hua; Xiao, Feng-Jun; Yin, Yue; Xu, Qin-Qin; Ge, Ri-Li; Wang, Li-Sheng

    2016-02-12

    MicroRNA-486 (miR-486) was first identified from human fetal liver cDNA library and validated as a regulator of hematopoiesis. Its roles in regulating the biological function of bone marrow-derived mesnechymal stem cells (BM-MSCs) under hypoxia have not been explored yet. In this study, we demonstrated that exposure to hypoxia upregulates miR-486 expression in BM-MSCs. Lentivirus-mediated overexpression of miR-486 resulted in increase of hepatocyte growth factor (HGF) and vascular endothelial growth factor(VEGF) in both mRNA and protein levels. MiR-486 expression also promotes proliferation and reduces apoptosis of BM-MSCs. Whereas MiR-486 knockdown downregulated the secretion of HGF and VEGF and induced apoptosis of BM-MSCs. Furthermore, PTEN-PI3K/AKT signaling was validated to be involved in changes of BM-MSC biological functions regulated by miR-486. These results suggested that MiR-486 mediated the hypoxia-induced angiogenic activity and promoted the proliferation and survival of BM-MSCs through regulating PTEN-PI3K/AKT signaling. These findings might provide a novel understanding of effective therapeutic strategy for hypoxic-ischemic diseases. PMID:26801559

  12. 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

  13. 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

  14. Acidic Fibroblast Growth Factor Promotes Endothelial Progenitor Cells Function via Akt/FOXO3a Pathway

    PubMed Central

    Wang, Yuqiang; Cao, Qing; Sang, Tiantian; Liu, Fang; Chen, Shuyan

    2015-01-01

    Acidic fibroblast growth factor (FGF1) has been suggested to enhance the functional activities of endothelial progenitor cells (EPCs). The Forkhead homeobox type O transcription factors (FOXOs), a key substrate of the survival kinase Akt, play important roles in regulation of various cellular processes. We previously have shown that FOXO3a is the main subtype of FOXOs expressed in EPCs. Here, we aim to determine whether FGF1 promotes EPC function through Akt/FOXO3a pathway. Human peripheral blood derived EPCs were transduced with adenoviral vectors either expressing a non-phosphorylable, constitutively active triple mutant of FOXO3a (Ad-TM-FOXO3a) or a GFP control (Ad-GFP). FGF1 treatment improved functional activities of Ad-GFP transduced EPCs, including cell viability, proliferation, antiapoptosis, migration and tube formation, whereas these beneficial effects disappeared by Akt inhibitor pretreatment. Moreover, EPC function was declined by Ad-TM-FOXO3a transduction and failed to be attenuated even with FGF1 treatment. FGF1 upregulated phosphorylation levels of Akt and FOXO3a in Ad-GFP transduced EPCs, which were repressed by Akt inhibitor pretreatment. However, FGF1 failed to recover Ad-TM-FOXO3a transduced EPCs from dysfunction. These data indicate that FGF1 promoting EPC function is at least in part mediated through Akt/FOXO3a pathway. Our study may provide novel ideas for enhancing EPC angiogenic ability and optimizing EPC transplantation therapy in the future. PMID:26061278

  15. 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

  16. Modified Active Site Coordination in a Clinical Mutant of Sulfite Oxidase

    SciTech Connect

    Doonan, C.J.; Wilson, H.L.; Rajagopalan, K.V.; Garrett, R.M.; Bennett, B.; Prince, R.C.; George, G.N.

    2009-06-02

    The molybdenum site of the Arginine 160 {yields} Glutamine clinical mutant of the physiologically vital enzyme sulfite oxidase has been investigated by a combination of X-ray absorption spectroscopy and density functional theory calculations. We conclude that the mutant enzyme has a six-coordinate pseudo-octahedral active site with coordination of Glutamine O{sup {epsilon}} to molybdenum. This contrasts with the wild-type enzyme which is five-coordinate with approximately square-based pyramidal geometry. This difference in the structure of the molybdenum site explains many of the properties of the mutant enzyme which have previously been reported.

  17. ERK2-dependent reactivation of Akt mediates the limited response of tumor cells with constitutive K-RAS activity to PI3K inhibition

    PubMed Central

    Toulany, Mahmoud; Minjgee, Minjmaa; Saki, Mohammad; Holler, Marina; Meier, Friedegund; Eicheler, Wolfgang; Rodemann, H Peter

    2014-01-01

    K-RAS mutated (K-RASmut) non-small cell lung cancer (NSCLC) cells are resistant to EGFR targeting strategies. We investigated the impact of K-RAS activity irrespective of mutational status in the EGFR-independent increase in clonogenic cell survival. An analysis of the K-RAS activity status revealed a constitutively high K-RAS activity in K-RASmut NSCLC cells and also in head and neck squamous cell carcinoma (HNSCC) cells overexpressing wild-type K-RAS (K-RASwt). Similar to K-RAS-mutated cells, increased K-RAS activity in HNSCC cells overexpressing K-RASwt was associated with the stimulated production of the EGFR ligand amphiregulin and resistance to EGFR tyrosine kinase (EGFR-TK) inhibitors such as erlotinib. Expression of mutated K-RAS stimulated Akt phosphorylation and increased plating efficiency. Conversely, knockdown of K-RAS in K-RASmut NSCLC cells and in HNSCC cells presenting overexpression of K-RASwt resulted in sensitization to the anti-clonogenic activity of erlotinib. K-RAS activity results in EGFR-dependent and EGFR-independent Akt activity. The short-term treatment (2 h) of cells with EGFR-TK or PI3K inhibitors (erlotinib and PI-103) resulted in the repression of Akt activation, whereas long-term treatment (24 h) with inhibitors led to the reactivation of Akt and improved clonogenicity. The Akt re-activation was MAPK-ERK2-dependent and associated with a lack of complete response to anti-clonogenic activity of PI-103. A complete response was observed when PI-103 was combined with MEK inhibitor PD98059. Together, clonogenicity inhibition in tumor cells presenting constitutive K-RAS activity independent of K-RAS mutational status can be achieved by targeting of EGFR downstream pathways, i.e., PI3K alone or the combination of PI3K and MAPK inhibitors. PMID:24351425

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

    PubMed Central

    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. PMID:26389892

  19. 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

  20. 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

  1. 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.

  2. S9, a Novel Anticancer Agent, Exerts Its Anti-Proliferative Activity by Interfering with Both PI3K-Akt-mTOR Signaling and Microtubule Cytoskeleton

    PubMed Central

    Yang, Chun-hao; Ding, Hua-sheng; Luo, Cheng; Zhang, Yu; Wu, Mao-jiang; Zhang, Xiong-wen; Shen, Xu; Jiang, Hua-liang; Meng, Ling-hua; Ding, Jian

    2009-01-01

    Background Deregulation of the phosphatidylinositol 3-kinases (PI3K)/Akt/mammalian target of rapamycin (mTOR) pathway plays a central role in tumor formation and progression, providing validated targets for cancer therapy. S9, a hybrid of α-methylene-γ-lactone and 2-phenyl indole compound, possessed potent activity against this pathway. Methodology/Principal Findings Effects of S9 on PI3K-Akt-mTOR pathway were determined by Western blot, immunofluorescence staining and in vitro kinas assay. The interactions between tubulin and S9 were investigated by polymerization assay, CD, and SPR assay. The potential binding modes between S9 and PI3K, mTOR or tubulin were analyzed by molecular modeling. Anti-tumor activity of S9 was evaluated in tumor cells and in nude mice bearing human cancer xenografts. S9 abrogated EGF-activated PI3K-Akt-mTOR signaling cascade and Akt translocation to cellular membrane in human tumor cells. S9 possessed inhibitory activity against both PI3K and mTOR with little effect on other tested 30 kinases. S9 also completely impeded hyper-phosphorylation of Akt as a feedback of inhibition of mTOR by rapamycin. S9 unexpectedly arrested cells in M phase other than G1 phase, which was distinct from compounds targeting PI3K-Akt-mTOR pathway. Further study revealed that S9 inhibited tubulin polymerization via binding to colchicine-binding site of tubulin and resulted in microtubule disturbance. Molecular modeling indicated that S9 could potentially bind to the kinase domains of PI3K p110α subunit and mTOR, and shared similar hydrophobic interactions with colchicines in the complex with tubulin. Moreover, S9 induced rapid apoptosis in tumor cell, which might reflect a synergistic cooperation between blockade of both PI3-Akt-mTOR signaling and tubulin cytoskeleton. Finally, S9 displayed potent antiproliferative activity in a panel of tumor cells originated from different tissue types including drug-resistant cells and in nude mice bearing human tumor

  3. 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

  4. 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

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

    2016-08-01

    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

  5. 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

  6. 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

  7. β-Adrenergic Receptor-Mediated Transactivation Of Epidermal Growth Factor Receptor Decreases Cardiomyocyte Apoptosis Through Differential Subcellular Activation of ERK1/2 and Akt

    PubMed Central

    Grisanti, Laurel A.; Talarico, Jennifer A.; Carter, Rhonda L.; Yu, Justine E.; Repas, Ashley A.; Radcliffe, Scott W.; Tang, Hoang-ai; Makarewich, Catherine A.; Houser, Steven R.; Tilley, Douglas G.

    2014-01-01

    Rationale β-adrenergic receptor (βAR)-mediated transactivation of epidermal growth factor receptor (EGFR) has been shown to relay pro-survival effects via unknown mechanisms. Objective We hypothesized that acute βAR-mediated EGFR transactivation in the heart promotes differential subcellular activation of ERK1/2 and Akt, promoting cell survival through modulation of apoptosis. Methods and Results C57BL/6 mice underwent acute i.p. injection with isoproterenol (ISO) ± AG 1478 (EGFR antagonist) to assess the impact of βAR-mediated EGFR transactivation on phosphorylation of ERK1/2 (P-ERK1/2) and Akt (P-Akt) in distinct cardiac subcellular fractions. Increased P-ERK1/2 and P-Akt were observed in cytosolic, plasma membrane and nuclear fractions following ISO stimulation. Whereas the P-ERK1/2 response was EGFR-sensitive in all fractions, the P-Akt response was EGFR-sensitive only in the plasma membrane and nucleus, results confirmed in primary rat neonatal cardiomyocytes (RNCM). βAR-mediated EGFR-transactivation also decreased apoptosis in serum-depleted RNCM, as measured via TUNEL as well as caspase 3 activity/cleavage, which were sensitive to inhibition of either ERK1/2 (PD184352) or Akt (LY-294002) signaling. Caspase 3 activity/cleavage was also sensitive to inhibition of transcription, which, with an increase in nuclear P-ERK1/2 and P-Akt in response to ISO, suggested that βAR-mediated EGFR transactivation may regulate apoptotic gene transcription. An Apoptosis PCR Array identified tnfsf10 (TRAIL) to be altered by ISO in an EGFR-sensitive manner, results confirmed via RT-PCR and ELISA measurement of both membrane-bound and soluble cardiomyocyte TRAIL levels. Conclusions βAR-mediated EGFR transactivation induces differential subcellular activation of ERK1/2 and Akt leading to increased cell survival through the modulation of caspase 3 activity and apoptotic gene expression in cardiomyocytes. PMID:24566221

  8. 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

  9. Icariin stimulates angiogenesis by activating the MEK/ERK- and PI3K/Akt/eNOS-dependent signal pathways in human endothelial cells

    SciTech Connect

    Chung, Byung-Hee; Kim, Jong-Dai; Kim, Chun-Ki; Kim, Jung Huan; Won, Moo-Ho; Lee, Han-Soo; Dong, Mi-Sook; Ha, Kwon-Soo; Kwon, Young-Geun; Kim, Young-Myeong

    2008-11-14

    We investigated the molecular effect and signal pathway of icariin, a major flavonoid of Epimedium koreanum Nakai, on angiogenesis. Icariin stimulated in vitro endothelial cell proliferation, migration, and tubulogenesis, which are typical phenomena of angiogenesis, as well as increased in vivo angiogenesis. Icariin activated the angiogenic signal modulators, ERK, phosphatidylinositol 3-kinase (PI3K), Akt, and endothelial nitric oxide synthase (eNOS), and increased NO production, without affecting VEGF expression, indicating that icariin may directly stimulate angiogenesis. Icariin-induced ERK activation and angiogenic events were significantly inhibited by the MEK inhibitor PD98059, without affecting Akt and eNOS phosphorylation. The PI3K inhibitor Wortmannin suppressed icariin-mediated angiogenesis and Akt and eNOS activation without affecting ERK phosphorylation. Moreover, the NOS inhibitor NMA partially reduced the angiogenic activity of icariin. These results suggest that icariin stimulated angiogenesis by activating the MEK/ERK- and PI3K/Akt/eNOS-dependent signal pathways and may be a useful drug for angiogenic therapy.

  10. Metformin inhibits growth of eutopic stromal cells from adenomyotic endometrium via AMPK activation and subsequent inhibition of AKT phosphorylation: a possible role in the treatment of adenomyosis.

    PubMed

    Xue, Jing; Zhang, Hui; Liu, Wei; Liu, Ming; Shi, Min; Wen, Zeqing; Li, Changzhong

    2013-10-01

    Adenomyosis is a finding that is associated with dysmenorrhea and heavy menstrual bleeding, associated with PI3K/AKT signaling overactivity. To investigate the effect of metformin on the growth of eutopic endometrial stromal cells (ESCs) from patients with adenomyosis and to explore the involvement of AMP-activated protein kinase (AMPK) and PI3K/AKT pathways. Primary cultures of human ESCs were derived from normal endometrium (normal endometrial stromal cells (N-ESCs)) and adenomyotic eutopic endometrium (adenomyotic endometrial stroma cells (A-ESCs)). Expression of AMPK was determined using immunocytochemistry and western blot analysis. 3-(4, 5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide assays were used to determine the effects of metformin and compound C on ESCs and also to detect growth and proliferation of ESCs. AMPK and PI3K/AKT signaling was determined by western blotting. A-ECSs exhibited greater AMPK expression than N-ESCs. Metformin inhibited proliferation of ESCs in a concentration-dependent manner. The IC50 was 2.45 mmol/l for A-ESCs and 7.87 mmol/l for N-ESCs. Metformin increased AMPK activation levels (p-AMPK/AMPK) by 2.0±0.3-fold in A-ESCs, 2.3-fold in A-ESCs from the secretory phase, and 1.6-fold in the proliferation phase. The average reduction ratio of 17β-estradiol on A-ESCs was 2.1±0.8-fold in proliferative phase and 2.5±0.5-fold in secretory phase relative to the equivalent groups not treated with 17β-estradiol. The inhibitory effects of metformin on AKT activation (p-AKT/AKT) were more pronounced in A-ESCs from the secretory phase (3.2-fold inhibition vs control) than in those from the proliferation phase (2.3-fold inhibition vs control). Compound C, a selective AMPK inhibitor, abolished the effects of metformin on cell growth and PI3K/AKT signaling. Metformin inhibits cell growth via AMPK activation and subsequent inhibition of PI3K/AKT signaling in A-ESCs, particularly during the secretory phase, suggesting a greater

  11. Neuroprotective effects of salidroside through PI3K/Akt pathway activation in Alzheimer’s disease models

    PubMed Central

    Zhang, Bei; Wang, Ying; Li, Hui; Xiong, Ran; Zhao, Zongbo; Chu, Xingkun; Li, Qiongqiong; Sun, Suya; Chen, Shengdi

    2016-01-01

    Alzheimer’s disease (AD) is a devastating neurodegenerative disorder characterized by deposits of aggregated amyloid-β (Aβ) peptide and neurofibrillary tangles in the brain parenchyma. Despite considerable research to elucidate the pathological mechanisms and identify therapeutic strategies for AD, effective treatments are still lacking. In the present study, we found that salidroside (Sal), a phenylpropanoid glycoside isolated from Rhodiola rosea L., can protect against Aβ-induced neurotoxicity in four transgenic Drosophila AD models. Both longevity and locomotor activity were improved in Sal-fed Drosophila. Sal also decreased Aβ levels and Aβ deposition in brain and ameliorated toxicity in Aβ-treated primary neuronal culture. The neuroprotective effect of Sal was associated with upregulated phosphatidylinositide 3-kinase (PI3K)/Akt signaling. Our findings identify a compound that may possess potential therapeutic benefits for AD and other forms of neurodegeneration. PMID:27103787

  12. AtKC1 and CIPK23 Synergistically Modulate AKT1-Mediated Low-Potassium Stress Responses in Arabidopsis.

    PubMed

    Wang, Xue-Ping; Chen, Li-Mei; Liu, Wen-Xin; Shen, Li-Ke; Wang, Feng-Liu; Zhou, Yuan; Zhang, Ziding; Wu, Wei-Hua; Wang, Yi

    2016-04-01

    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, AtKC1(D), 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, AtKC1(D) 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

  13. 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. PMID:10788397

  14. EB-virus latent membrane protein 1 potentiates the stemness of nasopharyngeal carcinoma via preferential activation of PI3K/AKT pathway by a positive feedback loop.

    PubMed

    Yang, C-F; Yang, G-D; Huang, T-J; Li, R; Chu, Q-Q; Xu, L; Wang, M-S; Cai, M-D; Zhong, L; Wei, H-J; Huang, H-B; Huang, J-L; Qian, C-N; Huang, B-J

    2016-06-30

    Our previous study reported that Epstein-Barr virus(EBV)-encoded latent membrane protein 1 (LMP1) could induce development of CD44(+/High) stem-like cells in nasopharyngeal carcinoma (NPC). However, the molecular mechanisms that underlie modulation of cancer stem cells (CSCs) in NPC remain unclear. Here, we show that LMP1 induced CSC-like properties through promotion of the expression of epithelial-mesenchymal transition-like cellular markers and through alterations in differentiation markers. Furthermore, LMP1 activated and triggered phosphoinositide 3-kinase/protein kinase B (PI3K/AKT) pathway, which subsequently stimulated expression of CSC markers, development of side population and tumor sphere formation. This suggests that PI3K/AKT pathway has an important role in the induction and maintenance of CSC properties in NPC. Similarly, PI3K/AKT pathway was also activated by phosphorylase in LMP1-induced CD44(+/High) cells. In addition, LMP1 greatly increased expression of miR-21 and downregulated expression of the miR-21 target, PTEN. Overexpression of miR-21 by transfection of miR-21 mimics into LMP1-transformed cells led to phosphorylase-mediated activation of the PI3K/AKT pathway and induction of CSCs. On the contrary, phosphorylation of the PI3K/AKT pathway and the expression of CSC were reversed by an miR-21 inhibitor. The specific inhibitor (Ly294002) of PI3K/AKT pathway significantly decreased expression of miR-21 and CSC markers and upregulated the expression of PTEN, which indicates that miR-21 and PTEN are the downstream effectors of PI3K/AKT and that expression of these two effectors are related to the development of NPC CSCs. Taken together, our novel findings indicate that LMP1, PI3K/AKT, miR-21 and PTEN constitute a positive feedback loop and have a key role in LMP1-induced CSCs in NPC. PMID:26568302

  15. Bacillus subtilis ccpA gene mutants specifically defective in activation of acetoin biosynthesis.

    PubMed

    Turinsky, A J; Moir-Blais, T R; Grundy, F J; Henkin, T M

    2000-10-01

    A large number of carbon source utilization pathways are repressed in Bacillus subtilis by the global regulator CcpA, which also acts as an activator of carbon excretion pathways during growth in media containing glucose. In this study, CcpA mutants defective in transcriptional activation of the alsSD operon, which is involved in acetoin biosynthesis, were identified. These mutants retained normal glucose repression of amyE, encoding alpha-amylase, and acsA, encoding acetyl-coenzyme A synthetase, and normal activation of ackA, which is involved in acetate excretion; in these ccpA mutants the CcpA functions of activation of the acetate and acetoin excretion pathways appear to be separated. PMID:10986270

  16. 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

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

    PubMed

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

    2015-08-01

    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

  18. Downregulation of Foxc2 enhances apoptosis induced by 5-fluorouracil through activation of MAPK and AKT pathways in colorectal cancer

    PubMed Central

    YANG, CHAO; CUI, XIAOXIAN; DAI, XIAOQIN; LIAO, WENTING

    2016-01-01

    The chemotherapy drug 5-fluorouracil (5-FU) is fundamental for the treatment of colorectal cancer (CRC); however, drug resistance to 5-FU may occasionally occur. Abnormal expression of Forkhead box C2 gene (Foxc2) has been identified in several human cancers, but the role of Foxc2 in the progression of CRC remains unclear. The present study established a stable Foxc2-short hairpin (sh)RNA cell line, which was confirmed by western blot analysis and quantitative polymerase chain reaction. The Foxc2-shRNA cells were treated with 5-FU and the cell viability was determined by an MTT assay. Western blot analysis was performed to investigate the signaling pathway involved in 5-FU treatment. The present study identified that 5-FU increased the percentage of apoptotic CRC cells among the Foxc2/RNA interference-transfected cells compared with cells transfected with an empty vector. Therefore, the downregulation of Foxc2, induced by 5-FU, may enhance apoptosis by the downregulation of apoptotic factors, including B cell lymphoma-2 and pro-caspase-3, in Foxc2-shRNA CRC cells. Furthermore, the mitogen-activated protein kinase (MAPK) and phosphatidylinositide 3-kinases/protein kinase B (PI3K/AKT) pathways were essential for the sensitization effect of Foxc2 to 5-FU treatment. Overall, these findings reveal the mechanisms behind Foxc2 depletion and 5-FU treatment of CRC and suggest that Foxc2 enhances resistance to apoptosis, induced by 5-FU, through the activation of MAPK and P13K/AKT pathways, and may serve as a valuable clinical prognostic marker for CRC. PMID:26893778

  19. 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

  20. Weight Loss Upregulates the Small GTPase DIRAS3 in Human White Adipose Progenitor Cells, Which Negatively Regulates Adipogenesis and Activates Autophagy via Akt-mTOR Inhibition.

    PubMed

    Ejaz, Asim; Mitterberger, Maria C; Lu, Zhen; Mattesich, Monika; Zwierzina, Marit E; Hörl, Susanne; Kaiser, Andreas; Viertler, Hans-Peter; Rostek, Ursula; Meryk, Andreas; Khalid, Sana; Pierer, Gerhard; Bast, Robert C; Zwerschke, Werner

    2016-04-01

    Long-term weight-loss (WL) interventions reduce insulin serum levels, protect from obesity, and postpone age-associated diseases. The impact of long-term WL on adipose-derived stromal/progenitor cells (ASCs) is unknown. We identified DIRAS3 and IGF-1 as long-term WL target genes up-regulated in ASCs in subcutaneous white adipose tissue of formerly obese donors (WLDs). We show that DIRAS3 negatively regulates Akt, mTOR and ERK1/2 signaling in ASCs undergoing adipogenesis and acts as a negative regulator of this pathway and an activator of autophagy. Studying the IGF-1-DIRAS3 interaction in ASCs of WLDs, we demonstrate that IGF-1, although strongly up-regulated in these cells, hardly activates Akt, while ERK1/2 and S6K1 phosphorylation is activated by IGF-1. Overexpression of DIRAS3 in WLD ASCs completely inhibits Akt phosphorylation also in the presence of IGF-1. Phosphorylation of ERK1/2 and S6K1 is lesser reduced under these conditions. In conclusion, our key findings are that DIRAS3 down-regulates Akt-mTOR signaling in ASCs of WLDs. Moreover, DIRAS3 inhibits adipogenesis and activates autophagy in these cells. PMID:27211557

  1. 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.

  2. Inositol hexaphosphate represses telomerase activity and translocates TERT from the nucleus in mouse and human prostate cancer cells via the deactivation of Akt and PKC{alpha}

    SciTech Connect

    Jagadeesh, Shankar; Banerjee, Partha P. . E-mail: ppb@georgetown.edu

    2006-11-03

    Inositol hexaphosphate (IP6) has anti-proliferative effects on a variety of cancer cells, including prostate cancer. However, the molecular mechanism of anti-proliferative effects of IP6 is not entirely understood. Since the activation of telomerase is crucial for cells to gain immortality and proliferation ability, we examined the role of IP6 in the regulation of telomerase activity in prostate cancer cells. Here, we show that IP6 represses telomerase activity in mouse and human prostate cancer cells dose-dependently. In addition, IP6 prevents the translocation of TERT to the nucleus. Since phosphorylation of TERT by Akt and/or PKC{alpha} is necessary for nuclear translocation, we examined phosphorylation of Akt and PKC{alpha} after IP6 treatments. Our results show that IP6 inhibits phosphorylation of Akt and PKC{alpha}. These results show for the first time that IP6 represses telomerase activity in prostate cancer cells by posttranslational modification of TERT via the deactivation of Akt and PKC{alpha}.

  3. 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

  4. 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

  5. Hepatitis B virus: DNA polymerase activity of deletion mutants.

    PubMed

    Kim, Y; Hong, Y B; Jung, G

    1999-02-01

    The hepadnavirus P gene product is a multifunctional protein with priming, DNA- and RNA-dependent DNA polymerase, and RNase H activities. Nested N- or C-terminal deletion mutations and deletions of domain(s) in human HBV polymerase have been made. Wild-type and deletion forms of MBP-fused HBV polymerase were expressed in E. coli, purified by amylose column chromatography, and the DNA-dependent DNA polymerase activities of the purified proteins were compared. Deletion of the terminal protein or spacer regions reduced enzyme activity to 70%, respectively. However, deletion of the RNase H domain affected polymerase activity more than that of the terminal protein or spacer region. The polymerase domain alone or the N-terminal deletion of the polymerase domain still exhibited enzymatic activity. In this report, it is demonstrated that the minimal domain for the polymerizing activity of the HBV polymerase is smaller than the polymerase domain. PMID:10205676

  6. Insulin sensitizes FGF21 in glucose and lipid metabolisms via activating common AKT pathway.

    PubMed

    Yu, Dan; Ye, Xianlong; Wu, Qiang; Li, Shujie; Yang, Yongbi; He, Jinjiao; Liu, Yunye; Zhang, Xiaoyu; Yuan, Qingyan; Liu, Mingyao; Li, Deshan; Ren, Guiping

    2016-06-01

    Previous studies reveal that fibroblast growth factor 21 (FGF21) sensitizes insulin to achieve a synergy in regulating glucose metabolism. Here, we report that insulin sensitizes FGF21 in regulating both glucose and lipid metabolisms. db/db diabetic mice were subcutaneously administrated once a day for 6 weeks. Effective dose of insulin (1 U) could control blood glucose level of the db/db mice for maximum of 2 h, increased the body weight of the db/db mice and did not improve serum lipid parameters. In contrast, effective dose of FGF21 (0.5 mg/kg) could maintain blood glucose of the db/db mice at normal level for at least 24 h, repressed the weight gain of the mice and significantly improved lipid parameters. Ineffective doses of FGF21 (0.125 mg/kg) and insulin had no effect on blood glucose level of the db/db mice after 24 h administration, body weight or lipid parameters. However, combination of the two ineffective doses could maintain blood glucose level of the db/db mice for at least 24 h, suppressed weight gain and significantly improved lipid parameters. These results suggest that insulin sensitizes FGF21 in regulating both glucose and lipid metabolism. The results aimed to study the molecular basis of FGF21 sensitization indicates that combination of the two ineffective doses increased the mRNA expression of glut1, glut4, β-Klotho, sirt1, pgc-1α, ucp-1 and AKT phosphorylation, decreased fasn. The results demonstrate that insulin sensitizes FGF21 through elevating the phosphorylation of common gene Akt and amplifying FGF21 downstream signaling, including increasing expression of glut1 sirt1, pgc-1α, ucp-1, and decreasing fasn expression. In summary, we reports herein for the first time that insulin sensitizes FGF21 to achieve a synergy in regulating glucose and lipid metabolism. Along with previous studies, we conclude that the synergistic effect between FGF21 and insulin is realized through mutual sensitization. PMID:26607153

  7. 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.

  8. Pelota Regulates Epidermal Differentiation by Modulating BMP and PI3K/AKT Signaling Pathways.

    PubMed

    Elkenani, Manar; Nyamsuren, Gunsmaa; Raju, Priyadharsini; Liakath-Ali, Kifayathullah; Hamdaoui, Aicha; Kata, Aleksandra; Dressel, Ralf; Klonisch, Thomas; Watt, Fiona M; Engel, Wolfgang; Thliveris, James A; Krishna Pantakani, D V; Adham, Ibrahim M

    2016-08-01

    The depletion of evolutionarily conserved pelota protein causes impaired differentiation of embryonic and spermatogonial stem cells. In this study, we show that temporal deletion of pelota protein before epidermal barrier acquisition leads to neonatal lethality due to perturbations in permeability barrier formation. Further analysis indicated that this phenotype is a result of failed processing of profilaggrin into filaggrin monomers, which promotes the formation of a protective epidermal layer. Molecular analyses showed that pelota protein negatively regulates the activities of bone morphogenetic protein and phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT) signaling pathways in the epidermis. To address whether elevated activities of bone morphogenetic protein and PI3K/AKT signaling pathways were the cause for the perturbed epidermal barrier in Pelo-deficient mice, we made use of organotypic cultures of skin explants from control and mutant embryos at embryonic day 15.5. Inhibition of PI3K/AKT signaling did not significantly affect the bone morphogenetic protein activity. However, inhibition of bone morphogenetic protein signaling caused a significant attenuation of PI3K/AKT activity in mutant skin and, more interestingly, the restoration of profilaggrin processing and normal epidermal barrier function. Therefore, increased activity of the PI3K/AKT signaling pathway in Pelo-deficient skin might conflict with the dephosphorylation of profilaggrin and thereby affect its proper processing into filaggrin monomers and ultimately the epidermal differentiation. PMID:27164299

  9. 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

  10. PI3k/Akt signalling pathway plays a crucial role in the anti-inflammatory effects of curcumin in LPS-activated microglia.

    PubMed

    Cianciulli, Antonia; Calvello, Rosa; Porro, Chiara; Trotta, Teresa; Salvatore, Rosaria; Panaro, Maria Antonietta

    2016-07-01

    Microglia are resident macrophages in the central nervous system (CNS) deputed to defend against pathogens. Persistent or acute inflammation of microglia leads to CNS disorders, so regulation of pro-inflammatory responses of microglial cells is thought to be a promising therapeutic strategy to attenuate abnormal inflammatory responses observed in neurodegenerative disease. We hypothesized that curcumin supplementation could reduce the inflammatory responses of activated microglial cells modulating PI3K/Akt pathway. Different curcumin concentrations were administered as BV-2 microglia pre-treatment 1h prior to LPS stimulation. Nitric oxide (NO) and inducible nitric oxide synthase (iNOS) expression were determined by Griess reagent and western blotting, respectively. Inflammatory cytokines release was evaluated by ELISA and qRT-PCR. PI3K/Akt expression was analyzed by western blotting analysis. Curcumin significantly attenuated, in a dose-dependent manner, LPS-induced release of NO and pro-inflammatory cytokines, as well as iNOS expression. Interestingly, curcumin was able to reduce, again in a dose-dependent manner, PI3K/Akt phosphorylation as well as NF-κB activation in LPS-activated microglial cells. Overall these results suggest that curcumin plays an important role in the attenuation of LPS-induced inflammatory responses in microglial cells and that the mechanisms involve down-regulation of the PI3K/Akt signalling. PMID:27208432

  11. 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

  12. The Masticatory Contractile Load Induced Expression and Activation of Akt1/PKBα in Muscle Fibers at the Myotendinous Junction within Muscle-Tendon-Bone Unit

    PubMed Central

    Korkmaz, Yüksel; Klinz, Franz J.; Moghbeli, Mehrnoush; Addicks, Klaus; Raab, Wolfgang H. -M.; Bloch, Wilhelm

    2010-01-01

    The cell specific detection of enzyme activation in response to the physiological contractile load within muscle-tendon-bone unit is essential for understanding of the mechanical forces transmission from muscle cells via tendon to the bone. The hypothesis that the physiological mechanical loading regulates activation of Akt1/PKBα at Thr308 and at Ser473 in muscle fibers within muscle-tendon-bone unit was tested using quantitative immunohistochemistry, confocal double fluorescence analysis, and immunoblot analysis. In comparison to the staining intensities in peripheral regions of the muscle fibers, Akt1/PKBα was detected with a higher staining intensity in muscle fibers at the myotendinous junction (MTJ) areas. In muscle fibers at the MTJ areas, Akt1/PKBα is dually phosphorylated at Thr308 and Ser473. The immunohistochemical results were confirmed by immunoblot analysis. We conclude that contractile load generated by masticatory muscles induces local domain-dependent expression of Akt1/PKBα as well as activation by dually phosphorylation at Thr308 and Ser473 in muscle fibers at the MTJ areas within muscle-tendon-bone unit. PMID:20454577

  13. New insights on PI3K/AKT pathway alterations and clinical outcomes in breast cancer.

    PubMed

    Yang, Sherry X; Polley, Eric; Lipkowitz, Stanley

    2016-04-01

    PI3K/AKT signaling pathway plays an important role in tumorigenesis and regulates critical cellular functions including survival, proliferation and metabolism. PIK3CA mutations and AKT activation by phosphorylation (pAKT) are often detected in many cancers and especially at high frequencies in breast cancer. Mounting data suggest that PIK3CA mutations or pAKT are mostly associated with better or insignificant outcomes in estrogen receptor-positive (ER+) early stage breast cancer and tend to be with worse prognosis in ER- disease. pAKT expression has been identified to predict paclitaxel chemotherapy benefit in node-positive breast cancer. Preclinical and neoadjuvant trial data suggest that PIK3CA alterations confer resistance to HER2-targeted therapy and are associated with lower pathological complete response (pCR) rate in HER2-positive breast cancer. However, recent results from randomized clinical trials of adjuvant and metastatic settings show that patients with mutant and wildtype PIK3CA tumors derived similar benefit from anti-HER2 therapy. This article, with our new insights, aims to decipher the mixed data and discusses the influence of the potential confounding factors in the assessments. We also share our views for validation of PI3K/AKT alterations in relation to clinical outcome in the context of specific breast cancer subtypes and treatment modalities towards further advance of the precision medicine for breast cancer treatment. PMID:26995633

  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. Differential crosstalk between the AMPK and PI3K/Akt pathways in breast cancer cells of differing genotypes: Leptin inhibits the effectiveness of AMPK activation.

    PubMed

    El-Masry, Omar S; Al-Sakkaf, Kaltoom; Brown, Barry L; Dobson, Pauline R M

    2015-10-01

    AMP-activated protein kinase (AMPK), a sensor of cellular energy, is widely reported as a potential therapeutic target in treatment of breast and other cancers. The activated enzyme has been shown to be a promising anti-proliferative agent in breast cancer cell lines. However, little data exist on crosstalk between AMPK and the cellular survival axis of PI3K/Akt/mTOR pathway and the impact of microenvironment on cellular responses to AMPK activation. We present results which show differential crosstalk between AMPK and Akt, dependent on the cellular genetics of each breast cancer cell type. We also show that leptin blocks activation of AMPK and partially or completely attenuates the anti-proliferative effect of AMPK activation depending on the cell type. This suggests that leptin within the local environment might impose limitations on therapeutic usage of AMPK activators in cancer, thereby attenuating their effective use in many obese subjects. PMID:26260992

  17. Angiotensin-converting enzyme 2/angiotensin-(1–7)/Mas axis activates Akt signaling to ameliorate hepatic steatosis

    PubMed Central

    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. 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

  19. 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.

  20. Impaired exercise training-induced muscle fiber hypertrophy and Akt/mTOR pathway activation in hypoxemic patients with COPD.

    PubMed

    Costes, Frédéric; Gosker, Harry; Feasson, Léonard; Desgeorges, Marine; Kelders, Marco; Castells, Josiane; Schols, Annemie; Freyssenet, Damien

    2015-04-15

    Exercise training (ExTr) is largely used to improve functional capacity in patients with chronic obstructive pulmonary disease (COPD). However, ExTr only partially restores muscle function in patients with COPD, suggesting that confounding factors may limit the efficiency of ExTr. In the present study, we hypothesized that skeletal muscle adaptations triggered by ExTr could be compromised in hypoxemic patients with COPD. Vastus lateralis muscle biopsies were obtained from patients with COPD who were either normoxemic (n = 15, resting arterial Po2 = 68.5 ± 1.5 mmHg) or hypoxemic (n = 8, resting arterial Po2 = 57.0 ± 1.0 mmHg) before and after a 2-mo ExTr program. ExTr induced a significant increase in exercise capacity both in normoxemic and hypoxemic patients with COPD. However, ExTr increased citrate synthase and lactate dehydrogenase enzyme activities only in skeletal muscle of normoxemic patients. Similarly, muscle fiber cross-sectional area and capillary-to-fiber ratio were increased only in patients who were normoxemic. Expression of atrogenes (MuRF1, MAFbx/Atrogin-1) and autophagy-related genes (Beclin, LC3, Bnip, Gabarapl) remained unchanged in both groups. Phosphorylation of Akt (Ser473), GSK-3β (Ser9), and p70S6k (Thr389) was nonsignificantly increased in normoxemic patients in response to ExTr, but it was significantly decreased in hypoxemic patients. We further showed on C2C12 myotubes that hypoxia completely prevented insulin-like growth factor-1-induced phosphorylation of Akt, GSK-3β, and p70S6K. Together, our observations suggest a role for hypoxemia in the adaptive response of skeletal muscle of patients with COPD in an ExTr program. PMID:25701004

  1. Curcumin activates autophagy and attenuates oxidative damage in EA.hy926 cells via the Akt/mTOR pathway.

    PubMed

    Guo, Shouyu; Long, Mingzhi; Li, Xiuzhen; Zhu, Shushu; Zhang, Min; Yang, Zhijian

    2016-03-01

    Curcumin, which is the effective component of turmeric (Curcuma longa), has previously been shown to exert potent antioxidant, antitumor and anti‑inflammatory activities in vitro and in vivo. However, the mechanism underlying the protective effects of curcumin against oxidative damage in endothelial cells remains unclear. The present study aimed to examine the effects of curcumin on hydrogen peroxide (H2O2)‑induced apoptosis and autophagy in EA.hy926 cells, and to determine the underlying molecular mechanism. Cultured EA.hy926 cells were treated with curcumin (5‑20 µmol/l) 4 h prior to and for 4 h during exposure to H2O2 (200 µmol/l). Oxidative stress resulted in a significant increase in the rate of cell apoptosis, which was accompanied by an increase in the expression levels of caspase‑3 and B‑cell lymphoma 2 (Bcl‑2)‑associated X protein (Bax), and a decrease in the expression levels of Bcl‑2. Treatment with curcumin (5 or 20 µmol/l) significantly inhibited apoptosis, and reversed the alterations in caspase‑3, Bcl‑2 and Bax expression. Furthermore, curcumin induced autophagy and microtubule‑associated protein 1A/1B‑light chain 3‑Ⅱ expression, and suppressed the phosphorylation of Akt and mammalian target of rapamycin (mTOR). These results indicated that curcumin may protect cells against oxidative stress‑induced damage through inhibiting apoptosis and inducing autophagy via the Akt/mTOR pathway. PMID:26781771

  2. Characterization of antimicrobial activity against Listeria and cytotoxicity of native melittin and its mutant variants.

    PubMed

    Wu, Xi; Singh, Atul K; Wu, Xiaoyu; Lyu, Yuan; Bhunia, Arun K; Narsimhan, Ganesan

    2016-07-01

    Antimicrobial peptides (AMPs) are relatively short peptides that have the ability to penetrate the cell membrane, form pores leading to cell death. This study compares both antimicrobial activity and cytotoxicity of native melittin and its two mutants, namely, melittin I17K (GIGAVLKVLTTGLPALKSWIKRKRQQ) with a higher charge and lower hydrophobicity and mutant G1I (IIGAVLKVLTTGLPALISWIKRKRQQ) of higher hydrophobicity. The antimicrobial activity against different strains of Listeria was investigated by bioassay, viability studies, fluorescence and transmission electron microscopy. Cytotoxicity was examined by lactate dehydrogenase (LDH) assay on mammalian Caco-2 cells. The minimum inhibitory concentration of native, mutant I17K, mutant G1I against Listeria monocytogenes F4244 was 0.315±0.008, 0.814±0.006 and 0.494±0.037μg/ml respectively, whereas the minimum bactericidal concentration values were 3.263±0.0034, 7.412±0.017 and 5.366±0.019μg/ml respectively. Lag time for inactivation of L. monocytogenes F4244 was observed at concentrations below 0.20 and 0.78μg/ml for native and mutant melittin I17K respectively. The antimicrobial activity against L. monocytogenes F4244 was in the order native>G1I>I17K. Native melittin was cytotoxic to mammalian Caco-2 cells above concentration of 2μg/ml, whereas the two mutants exhibited negligible cytotoxicity up to a concentration of 8μg/ml. Pore formation in cell wall/membrane was observed by transmission electron microscopy. Molecular dynamics (MD) simulation of native and its mutants indicated that (i) surface native melittin and G1I exhibited higher tendency to penetrate a mimic of bacterial cell membrane and (ii) transmembrane native and I17K formed water channel in mimics of bacterial and mammalian cell membranes. PMID:27011349

  3. 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

  4. Thrombin Receptor-Activating Protein (TRAP)-Activated Akt Is Involved in the Release of Phosphorylated-HSP27 (HSPB1) from Platelets in DM Patients.

    PubMed

    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

  5. Mechanical Stimulation and IGF-1 Enhance mRNA Translation Rate in Osteoblasts Via Activation of the AKT-mTOR Pathway.

    PubMed

    Bakker, Astrid D; Gakes, Tom; Hogervorst, Jolanda M A; de Wit, Gerard M J; Klein-Nulend, Jenneke; Jaspers, Richard T

    2016-06-01

    Insulin-like growth factor-1 (IGF-1) is anabolic for muscle by enhancing the rate of mRNA translation via activation of AKT and subsequent activation of the mammalian target of rapamycin complex 1 (mTOR), thereby increasing cellular protein production. IGF-1 is also anabolic for bone, but whether the mTOR pathway plays a role in the rate of bone matrix protein production by osteoblasts is unknown. We hypothesized that anabolic stimuli such as mechanical loading and IGF-1 stimulate protein synthesis in osteoblasts via activation of the AKT-mTOR pathway. MC3T3-E1 osteoblasts were either or not subjected for 1 h to mechanical loading by pulsating fluid flow (PFF) or treated with or without human recombinant IGF-1 (1-100 ng/ml) for 0.5-6 h, to determine phosphorylation of AKT and p70S6K (downstream of mTOR) by Western blot. After 4 days of culture with or without the mTOR inhibitor rapamycin, total protein, DNA, and gene expression were quantified. IGF-1 (100 ng/ml) reduced IGF-1 gene expression, although PFF enhanced IGF-1 expression. IGF-1 did not affect collagen-I gene expression. IGF-1 dose-dependently enhanced AKT and p70S6K phosphorylation at 2 and 6 h. PFF enhanced phosphorylation of AKT and p70S6K already within 1 h. Both IGF-1 and PFF enhanced total protein per cell by ∼30%, but not in the presence of rapamycin. Our results show that IGF-1 and PFF activate mTOR, thereby stimulating the rate of mRNA translation in osteoblasts. The known anabolic effect of mechanical loading and IGF-1 on bone may thus be partly explained by mTOR-mediated enhanced protein synthesis in osteoblasts. PMID:26505782

  6. 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

  7. 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

  8. An integrin beta4-EGFR unit promotes hepatocellular carcinoma lung metastases by enhancing anchorage independence through activation of FAK-AKT pathway.

    PubMed

    Leng, Chao; Zhang, Zhan-Guo; Chen, Wei-Xun; Luo, Hong-Ping; Song, Jia; Dong, Wei; Zhu, Xuan-Ru; Chen, Xiao-Ping; Liang, Hui-Fang; Zhang, Bi-Xiang

    2016-06-28

    Anoikis, a form of programmed cell death, occurs when the cells are detached from the appropriate extracellular matrix. Anoikis resistance or anchorage independence is necessary for distant metastases of cancer. The mechanisms by which hepatocellular carcinoma (HCC) cells become resistant to anoikis are not fully understood. Integrin beta4 (ITGB4, also known as CD104) is associated with progression of many human cancers. In this study, we demonstrate that ITGB4 is over-expressed in HCC tissues and aggressive HCC cell lines. To explore the role of ITGB4 in HCC, we inhibited its expression using small interfering RNA in two HCC cell lines: HCCLM3 and HLF. We show that knockdown of ITGB4 significantly enhanced susceptibility to anoikis through inhibition of AKT/PKB signaling. Moreover, ITGB4 interacts with epidermal growth factor receptor (EGFR) in a ligand independent manner. Inactivation of EGFR inhibits the anchorage independence and AKT pathway promoted by ITGB4. Further investigation proved that the ITGB4-EGFR unit triggers the focal adhesion kinase (FAK) to activate the AKT signaling pathway. Finally, we demonstrate that over-expression of ITGB4 is positively associated with tumor growth and lung metastases of HCC in vivo. Collectively, we demonstrate for the first time that ITGB4 is overexpressed in HCC tissues and promotes metastases of HCC by conferring anchorage independence through EGFR-dependent FAK-AKT activation. PMID:26996299

  9. pGlcNAc Nanofiber Treatment of Cutaneous Wounds Stimulate Increased Tensile Strength and Reduced Scarring via Activation of Akt1.

    PubMed

    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

  10. Substance P Inhibits Hyperosmotic Stress-Induced Apoptosis in Corneal Epithelial Cells through the Mechanism of Akt Activation and Reactive Oxygen Species Scavenging via the Neurokinin-1 Receptor

    PubMed Central

    Yang, Lingling; Sui, Wenjie; Li, Yunqiu; Qi, Xia; Wang, Yao; Zhou, Qingjun; Gao, Hua

    2016-01-01

    Hyperosmolarity has been recognized as an important pathological factor in dry eye leading to ocular discomfort and damage. As one of the major neuropeptides of corneal innervation, substance P (SP) has been shown to possess anti-apoptotic effects in various cells. The aim of this study was to determine the capacity and mechanism of SP against hyperosmotic stress-induced apoptosis in cultured corneal epithelial cells. The cells were exposed to hyperosmotic stress by the addition of high glucose in the presence or absence of SP. The results showed that SP inhibited hyperosmotic stress-induced apoptosis of mouse corneal epithelial cells. Moreover, SP promoted the recovery of phosphorylated Akt level, mitochondrial membrane potential, Ca2+ contents, intracellular reactive oxygen species (ROS) and glutathione levels that impaired by hyperosmotic stress. However, the antiapoptotic capacity of SP was partially suppressed by Akt inhibitor or glutathione depleting agent, while the neurokinin-1 (NK-1) receptor antagonist impaired Akt activation and ROS scavenging that promoted by SP addition. In conclusion, SP protects corneal epithelial cells from hyperosmotic stress-induced apoptosis through the mechanism of Akt activation and ROS scavenging via the NK-1 receptor. PMID:26901348

  11. Akt signaling in platelets and thrombosis

    PubMed Central

    Woulfe, Donna S

    2010-01-01

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

  12. Renal cortical hexokinase and pentose phosphate pathway activation through the EGFR/Akt signaling pathway in endotoxin-induced acute kidney injury

    PubMed Central

    Smith, Joshua A.; Stallons, L. Jay

    2014-01-01

    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. PMID:24990892

  13. 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

  14. 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.

  15. 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

  16. l-carnitine protects human hepatocytes from oxidative stress-induced toxicity through Akt-mediated activation of Nrf2 signaling pathway.

    PubMed

    Li, Jinlian; Zhang, Yanli; Luan, Haiyun; Chen, Xuehong; Han, Yantao; Wang, Chunbo

    2016-05-01

    In our previous study, l-carnitine was shown to have cytoprotective effect against hydrogen peroxide (H2O2)-induced injury in human normal HL7702 hepatocytes. The aim of this study was to investigate whether the protective effect of l-carnitine was associated with the nuclear factor erythroid 2 (NFE2)-related factor 2 (Nrf2) pathway. Our results showed that pretreatment with l-carnitine augmented Nrf2 nuclear translocation, DNA binding activity and heme oxygenase-1 (HO-1) expression in H2O2-treated HL7702 cells, although l-carnitine treatment alone had no effect on them. Analysis using Nrf2 siRNA demonstrated that Nrf2 activation was involved in l-carnitine-induced HO-1 expression. In addition, l-carnitine-mediated protection against H2O2 toxicity was abrogated by Nrf2 siRNA, indicating the important role of Nrf2 in l-carnitine-induced cytoprotection. Further experiments revealed that l-carnitine pretreatment enhanced the phosphorylation of Akt in H2O2-treated cells. Blocking Akt pathway with inhibitor partly abrogated the protective effect of l-carnitine. Moreover, our finding demonstrated that the induction of Nrf2 translocation and HO-1 expression by l-carnitine directly correlated with the Akt pathway because Akt inhibitor showed inhibitory effects on the Nrf2 translocation and HO-1 expression. Altogether, these results demonstrate that l-carnitine protects HL7702 cells against H2O2-induced cell damage through Akt-mediated activation of Nrf2 signaling pathway. PMID:26889770

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

    PubMed

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

    2016-07-01

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

  18. 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.

  19. 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

  20. 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

  1. 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

  2. The Prolyl Peptidases PRCP/PREP Regulate IRS-1 Stability Critical for Rapamycin-induced Feedback Activation of PI3K and AKT*

    PubMed Central

    Duan, Lei; Ying, Guoguang; Danzer, Brian; Perez, Ricardo E.; Shariat-Madar, Zia; Levenson, Victor V.; Maki, Carl G.

    2014-01-01

    The phosphatidylinositol 3-kinase (PI3K)/protein kinase B (PKB/AKT)/mammalian target of rapamycin (mTOR) pathway conveys signals from receptor tyrosine kinases (RTKs) to regulate cell metabolism, proliferation, survival, and motility. Previously we found that prolylcarboxypeptidase (PRCP) regulate proliferation and survival in breast cancer cells. In this study, we found that PRCP and the related family member prolylendopeptidase (PREP) are essential for proliferation and survival of pancreatic cancer cells. Depletion/inhibition of PRCP and PREP-induced serine phosphorylation and degradation of IRS-1, leading to inactivation of the cellular PI3K and AKT. Notably, depletion/inhibition of PRCP/PREP destabilized IRS-1 in the cells treated with rapamycin, blocking the feedback activation PI3K/AKT. Consequently, inhibition of PRCP/PREP enhanced rapamycin-induced cytotoxicity. Thus, we have identified PRCP and PREP as a stabilizer of IRS-1 which is critical for PI3K/AKT/mTOR signaling in pancreatic cancer cells. PMID:24936056

  3. 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

  4. 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

  5. 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

  6. 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

  7. Isolation of a Fusarium solani mutant reduced in cutinase activity and virulence.

    PubMed Central

    Dantzig, A H; Zuckerman, S H; Andonov-Roland, M M

    1986-01-01

    Fusarium solani isolate T-8 produces an extracellular enzyme, cutinase, which catalyzes the degradation of cutin in the plant cuticle. Cutinase activity can be measured by the hydrolysis of either the artifical substrate, p-nitrophenylbutyrate (PNB), or radioactive cutin containing [14C]palmitic acid. In the present study, the culture filtrate contained basal levels of cutinase when T-8 was grown on acetate as a sole source of carbon. After mutagenesis, a cutinase-defective mutant (PNB-1) was identified by screening acetate-grown colonies for a loss of PNBase activity. The mutant possessed an 80 to 90% reduction in cutinase activity when grown for 3 to 5 days on acetate- or cutin-containing medium. Induction of cutinase by cutin or hydrolyzed cutin after growth on glucose medium was similarly reduced. Kinetic analysis indicated that cutinase from the mutant possessed a near normal Km for PNB and a 92% reduction in Vmax. Fluorography and Western blotting of 15% sodium dodecyl sulfate-polyacrylamide gels of separated 35S-labeled proteins from cutin induction medium revealed that in the mutant the 22,000-molecular-weight band corresponding to cutinase was reduced approximately 85%. The virulence of the mutant in a pea stem bioassay was decreased by 55% and was restored to nearly the parental level by the addition of purified cutinase. The data suggest that the mutant synthesizes reduced quantities of a functional and immunoreactive cutinase enzyme and that cutinase plays a critical role in infection. The PNB1 mutation may be within a regulatory gene or a promoter for cutinase. Images PMID:3782031

  8. FLT3-ITD confers resistance to the PI3K/Akt pathway inhibitors by protecting the mTOR/4EBP1/Mcl-1 pathway through STAT5 activation in acute myeloid leukemia.

    PubMed

    Nogami, Ayako; Oshikawa, Gaku; Okada, Keigo; Fukutake, Shusaku; Umezawa, Yoshihiro; Nagao, Toshikage; Kurosu, Tetsuya; Miura, Osamu

    2015-04-20

    FLT3-ITD and FLT3-TKD are the most frequent tyrosine kinase mutations in acute myeloid leukemia (AML), with the former associated with poor prognosis. Here, we show that the PI3K inhibitor GDC-0941 or the Akt inhibitor MK-2206 induced apoptosis through the mitochondria-mediated intrinsic pathway more efficiently in hematopoietic 32D cells driven by FLT3-TKD (32D/TKD) than FLT3-ITD (32D/ITD), which robustly activated STAT5. The resistance to GDC-0941 and MK-2206 was gained by expression of the constitutively activated STAT5 mutant STAT5A1*6 in 32D/TKD cells, while it was abrogated by the STAT5 inhibitor pimozide in 32D/ITD cells or FLT3-ITD-expressing human leukemic MV4-11 cells. GDC-0941 or MK-2206 induced dephosphorylation of 4EBP1 more conspicuously in 32D/TKD than in 32D/ITD, which was prevented or augmented by STAT5A1*6 or pimozide, respectively, and correlated with downregulation of the eIF4E/eIF4G complex formation and Mcl-1 expression. Furthermore, exogenous expression of Mcl-1 endowed resistance to GDC-0941 and MK-2206 on 32D/TKD cells. Finally, it was confirmed in primary AML cells with FLT3-ITD that pimozide enhanced 4EBP1 dephosphorylation and Mcl-1 downregulation to augment cytotoxicity of GDC-0941. These data suggest that the robust STAT5 activation by FLT3-ITD protects cells treated with the PI3K/Akt pathway inhibitors from apoptosis by maintaining Mcl-1 expression through the mTORC1/4EBP1/eIF4E pathway. PMID:25826077

  9. 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.

  10. DNA damage processing by human 8-oxoguanine-DNA glycosylase mutants with the occluded active site.

    PubMed

    Lukina, Maria V; Popov, Alexander V; Koval, Vladimir V; Vorobjev, Yuri N; Fedorova, Olga S; Zharkov, Dmitry O

    2013-10-01

    8-Oxoguanine-DNA glycosylase (OGG1) removes premutagenic lesion 8-oxoguanine (8-oxo-G) from DNA and then nicks the nascent abasic (apurinic/apyrimidinic) site by β-elimination. Although the structure of OGG1 bound to damaged DNA is known, the dynamic aspects of 8-oxo-G recognition are not well understood. To comprehend the mechanisms of substrate recognition and processing, we have constructed OGG1 mutants with the active site occluded by replacement of Cys-253, which forms a wall of the base-binding pocket, with bulky leucine or isoleucine. The conformational dynamics of OGG1 mutants were characterized by single-turnover kinetics and stopped-flow kinetics with fluorescent detection. Additionally, the conformational mobility of wild type and the mutant OGG1 substrate complex was assessed using molecular dynamics simulations. Although pocket occlusion distorted the active site and greatly decreased the catalytic activity of OGG1, it did not fully prevent processing of 8-oxo-G and apurinic/apyrimidinic sites. Both mutants were notably stimulated in the presence of free 8-bromoguanine, indicating that this base can bind to the distorted OGG1 and facilitate β-elimination. The results agree with the concept of enzyme plasticity, suggesting that the active site of OGG1 is flexible enough to compensate partially for distortions caused by mutation. PMID:23955443

  11. Akt kinase-mediated checkpoint of cGAS DNA sensing pathway

    PubMed Central

    Seo, Gil Ju; Yang, Aerin; Tan, Brandon; Kim, Sungyoon; Liang, Qiming; Choi, Younho; Yuan, Weiming; Feng, Pinghui; Park, Hee-Sung; Jung, Jae U.

    2015-01-01

    SUMMARY Upon DNA stimulation, cyclic GMP-AMP synthetase (cGAS) synthesizes the second messenger cyclic GMP-AMP (cGAMP) that binds to the STING, triggering antiviral interferon-β (IFN-β) production. However, it has remained undetermined how hosts regulate cGAS enzymatic activity after the resolution of DNA immunogen. Here, we show that Akt kinase plays a negative role in cGAS-mediated anti-viral immune response. Akt phosphorylated the S291 or S305 residue of the enzymatic domain of mouse or human cGAS, respectively, and this phosphorylation robustly suppressed its enzymatic activity. Consequently, expression of activated Akt led to the reduction of cGAMP and IFN-β production and the increase of herpes simplex virus 1 replication, whereas treatment with Akt inhibitor augmented cGAS-mediated IFN-β production. Furthermore, expression of the phosphorylation-resistant cGAS S291A mutant enhanced IFN-β production upon DNA stimulation, HSV-1 infection, and vaccinia virus infection. Our study identifies an Akt kinase-mediated checkpoint to fine-tune hosts’ immune responses to DNA stimulation. PMID:26440888

  12. Restoring assembly and activity of cystathionine β-synthase mutants by ligands and chemical chaperones

    PubMed Central

    Kopecká, Jana; Krijt, Jakub; Raková, Kateřina

    2010-01-01

    Misfolding and aggregation of mutant enzymes have been proposed to play role in the pathogenesis of homocystinuria due to cystathionine β-synthase (CBS) deficiency. Chemical chaperones have been recently shown to facilitate proper assembly of several CBS mutants. To asses the number of patients that may respond to chaperone therapy, we examined the effect of selected CBS ligands and osmolytes on assembly and activity of 27 CBS mutants that represent 70% of known CBS alleles. The mutant enzymes were expressed in a bacterial system, and their properties were assessed by native Western blotting and sensitive liquid chromatography tandem mass spectrometry (LC-MS/MS) assay, respectively. We studied the chaperoning activity of δ-aminolevulinic acid (δ-ALA)—a heme precursor—and of three osmolytes betaine, 2-aminoethanesulfonic acid (taurine), and glycerol. Fourteen mutants responded by at least 30% increase in the amount of correctly assembled tetramers and enzymatic activity to the coexpressional presence of either 0.5 mM δ-ALA, 100 mM betaine, and/or 750 mM glycerol. Eight of these mutants (p.R266K, p.P49L, p.R125Q, p.K102N, p.R369C, p.V180A, p.P78R, p.S466L) were rescuable by all of these three substances. Four mutants showed increased formation of tetramers that was not accompanied by changes in activity. Topology of mutations appeared to determine the chaperone responsiveness, as 11 of 14 solvent-exposed mutations were substantially more responsive than three of 13 buried mutations. This study identified chaperone-responsive mutants that represent 56 of 713 known patient-derived CBS alleles and may serve as a basis for exploring pharmacological approaches aimed at correcting misfolding in homocystinuria. Electronic supplementary material The online version of this article (doi:10.1007/s10545-010-9087-5) contains supplementary material, which is available to authorized users. PMID:20490928

  13. Dichloromethane fraction of Cimicifuga heracleifolia decreases the level of melanin synthesis by activating the ERK or AKT signaling pathway in B16F10 cells.

    PubMed

    Jang, Ji Yeon; Lee, Jun Hyuk; Kang, Byoung Won; Chung, Kyung Tae; Choi, Yung Hyun; Choi, Byung Tae

    2009-03-01

    Cimicifuga rhizoma has long been used in traditional Korean medicine. In particular, a Cimicifuga heracleifolia extract (CHE) was reported to inhibit the formation of glutamate and the glutamate dehydrogenase activity in cultured rat islet. Glutamate activates melanogenesis by activating tyrosinase. Accordingly, it was hypothesized that a CHE might inhibit the melanogenesis-related signal pathways including the inhibition of microphthalmia-associated transcription factor (MITF)-tyrosinase signaling and/or the activation of extracellular signal-regulated kinase (ERK)-Akt signaling. The results showed that CHE inhibits the cellular melanin contents, tyrosinase activity and expression of melanogenesis-related proteins including MITF, tyrosinase and tyrosinase-related protein (TRP)s in alpha-melanocyte-stimulating hormone-stimulated B16 cells. Moreover, CHE phosphorylates MEK, ERK1/2 and Akt, which are melanogenesis inhibitory proteins. The data suggest that CHE inhibits melanogenesis signaling by both inhibiting the tyrosinase directly and activating the MEK-ERK or Akt signal pathways-mediated suppression of MITF and its downstream signal pathway, including tyrosinase and TRPs. Therefore, C. heracleifolia would be a useful therapeutic agent for treating hyperpigmentation and an effective component in whitening and/or lightening cosmetics. PMID:18803655

  14. Hypoxanthine-Guanine Phosphoribosyltransferase Deficiency: Activity in Normal, Mutant, and Heterozygote-Cultured Human Skin Fibroblasts

    PubMed Central

    Fujimoto, Wilfred Y.; Seegmiller, J. Edwin

    1970-01-01

    Cultured skin fibroblasts from patients deficient for the enzyme hypoxanthine-guanine phosphoribosyltransferase (PRT) activity show very low but nevertheless significant levels of apparent PRT enzyme despite absence of detectable activity (<0.004% of normal) in erythrocytes of the same patients. In fibroblasts this mutant enzyme is more heat labile than the normal enzyme. These findings indicate that PRT deficiency in this disorder is not due to a deletion mutation of the PRT locus. Individual cultured skin fibroblasts from heterozygote females for PRT deficiency show normal, intermediate, or very low levels of PRT activity. The mosaicism demonstrated in the heterozygotes for this X-linked disorder accounts for the cells with normal and very low activities of PRT. Intermediate activity can best be explained by the phenomenon of metabolic cooperation presumably from the transfer of either PRT enzyme or messenger RNA, from normal to mutant cells. Images PMID:5267139

  15. Essential Opposite Roles of ERK and Akt Signaling in Cardiac Steroid-Induced Increase in Heart Contractility.

    PubMed

    Buzaglo, Nahum; Rosen, Haim; Ben Ami, Hagit Cohen; Inbal, Adi; Lichtstein, David

    2016-05-01

    Interaction of cardiac steroids (CS) with the Na(+), K(+)-ATPase elicits, in addition to inhibition of the enzyme's activity, the activation of intracellular signaling such as extracellular signal-regulated (ERK) and protein kinase B (Akt). We hypothesized that the activities of these pathways are involved in CS-induced increase in heart contractility. This hypothesis was tested using in vivo and ex vivo wild type (WT) and sarcoplasmic reticulum Ca(2+) atpase1a-deficient zebrafish (accordion, acc mutant) experimental model. Heart contractility was measured in vivo and in primary cardiomyocytes in WT zebrafish larvae and acc mutant. Ca(2+) transients were determined ex vivo in adult zebrafish hearts. CS dose dependently augmented the force of contraction of larvae heart muscle and cardiomyocytes and increased Ca(2+) transients in WT but not in acc mutant. CS in vivo increased the phosphorylation rate of ERK and Akt in the adult zebrafish heart of the two strains. Pretreatment of WT zebrafish larvae or cardiomyocytes with specific MAPK inhibitors completely abolished the CS-induced increase in contractility. On the contrary, pretreatment with Akt inhibitor significantly enhanced the CS-induced increase in heart contractility both in vivo and ex vivo without affecting CS-induced Ca(2+) transients. Furthermore, pretreatment of the acc mutant larvae or cardiomyocytes with Akt inhibitor restored the CS-induced increase in heart contractility also without affecting Ca(2+) transients. These results support the notion that the activity of MAPK pathway is obligatory for CS-induced increases in heart muscle contractility. Akt activity, on the other hand, plays a negative role, via Ca(2+) independent mechanisms, in CS action. These findings point to novel potential pharmacological intervention to increase CS efficacy. PMID:26941172

  16. ADAM17-siRNA inhibits MCF-7 breast cancer through EGFR-PI3K-AKT activation.

    PubMed

    Meng, Xiangchao; Hu, Baoshan; Hossain, Mohammad Monir; Chen, Guofu; Sun, Ying; Zhang, Xuepeng

    2016-08-01

    A disintegrin and metalloproteinase-17 (ADAM17) can cut and release a wide variety of epidermal growth factor receptor (EGFR) ligands to promote survival, invasion and proliferation of cancer cell, and therefore, is considered to be a potential therapeutic target for cancer. The main goal of the present study was to observe the effects of ADAM17 small interfering RNA (ADAM17-siRNA) on human MCF-7 breast cancer and investigate its activation pathway. In vitro, MCF-7 cells were divided into ADAM17-siRNA groups, nonsense siRNA groups, AG1478 (selective EGFR blocker) groups, LY294002 [phosphatidylinositol 3-kinase (PI3K) phosphorylation inhibitor] groups, PD0325901 [mitogen extracellular kinase (MEK) inhibitor] groups and control groups. In vivo, MCF-7 cells were implanted subcutaneously into nude mice and then these mice were randomly divided into ADAM17-siRNA groups, vector groups and control groups. Our data showed that compared with the control groups, ADAM17-siRNA, AG1478 and LY294002 could inhibit the migration and proliferation of MCF-7 cells, but PD0325901 and nonsense siRNA did not show this effect. Except that specific ADAM17-siRNA could inhibit the expression of ADAM17 mRNA, others did not change it. Western blot analysis further confirmed that EGFR-PI3K-AKT signaling pathway is involved in ADAM17-siRNA inhibiting migration and proliferation of MCF-7 cells. Similarly to the former, the growth of MCF-7 breast cancer in nude mice was significantly inhibited by ADAM17-siRNA. Compared with the control group and the vector group, the tumor volume was smaller in the ADAM17-siRNA group, the tissues developed large areas of necrosis, immunohistochemistry showed low expressions of ADAM17 and Ki-67 and western blot analysis proved that the expression of ADAM17 protein in the tissue was also reduced. The present study suggests that ADAM17-siRNA inhibits MCF-7 breast cancer and is activated through the EGFR-PI3K-AKT signaling pathway. PMID:27221510

  17. 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. PMID:25091621

  18. Apocynin and Diphenyleneiodonium Induce Oxidative Stress and Modulate PI3K/Akt and MAPK/Erk Activity in Mouse Embryonic Stem Cells

    PubMed Central

    Kučera, Jan; Binó, Lucia; Štefková, Kateřina; Jaroš, Josef; Vašíček, Ondřej; Večeřa, Josef; Kubala, Lukáš; Pacherník, Jiří

    2016-01-01

    Reactive oxygen species (ROS) are important regulators of cellular functions. In embryonic stem cells, ROS are suggested to influence differentiation status. Regulated ROS formation is catalyzed primarily by NADPH-dependent oxidases (NOXs). Apocynin and diphenyleneiodonium are frequently used inhibitors of NOXs; however, both exhibit uncharacterized effects not related to NOXs inhibition. Interestingly, in our model of mouse embryonic stem cells we demonstrate low expression of NOXs. Therefore we aimed to clarify potential side effects of these drugs. Both apocynin and diphenyleneiodonium impaired proliferation of cells. Surprisingly, we observed prooxidant activity of these drugs determined by hydroethidine. Further, we revealed that apocynin inhibits PI3K/Akt pathway with its downstream transcriptional factor Nanog. Opposite to this, apocynin augmented activity of canonical Wnt signaling. On the contrary, diphenyleneiodonium activated both PI3K/Akt and Erk signaling pathways without affecting Wnt. Our data indicates limits and possible unexpected interactions of NOXs inhibitors with intracellular signaling pathways. PMID:26788250

  19. 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

  20. PHLPP negatively regulates cell motility through inhibition of Akt activity and integrin expression in pancreatic cancer cells

    PubMed Central

    Smith, Alena J.; Wen, Yang-An; Stevens, Payton D.; Liu, Jingpeng; Wang, Chi; Gao, Tianyan

    2016-01-01

    Pancreatic adenocarcinoma is currently the fourth leading cause for cancer-related mortality. Malignant progression of pancreatic cancer depends not only on rapid proliferation of tumor cells but also on increased cell motility. In this study, we showed that increased PHLPP expression significantly reduced the rate of migration in pancreatic ductal adenocarcinoma (PDAC) cells whereas knockdown of PHLPP had the opposite effect. In addition, cell motility at the individual cell level was negatively regulated by PHLPP as determined using time-lapse imaging. Interestingly, the expression of β1 and β4 integrin proteins were decreased in PHLPP overexpressing cells and increased in PHLPP knockdown cells whereas the mRNA levels of integrin were not altered by changes in PHLPP expression. In determining the molecular mechanism underlying PHLPP-mediated regulation of integrin expression, we found that inhibition of lysosome activity rescued integrin expression in PHLPP overexpressing cells, thus suggesting that PHLPP negatively controls cell motility by inhibiting Akt activity to promote lysosome-dependent degradation of integrins. Functionally, the increased cell migration observed in PHLPP knockdown cells was effectively blocked by the neutralizing antibodies against β1 or β4 integrin. Taken together, our study identified a tumor suppressor role of PHLPP in suppressing cell motility by negatively regulating integrin expression in pancreatic cancer cells. PMID:26760962

  1. 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. PMID:26183020

  2. A caveolin-dependent and PI3K/AKT-independent role of PTEN in β-catenin transcriptional activity.

    PubMed

    Conde-Perez, Alejandro; Gros, Gwendoline; Longvert, Christine; Pedersen, Malin; Petit, Valérie; Aktary, Zackie; Viros, Amaya; Gesbert, Franck; Delmas, Véronique; Rambow, Florian; Bastian, Boris C; Campbell, Andrew D; Colombo, Sophie; Puig, Isabel; Bellacosa, Alfonso; Sansom, Owen; Marais, Richard; Van Kempen, Leon C L T; Larue, Lionel

    2015-01-01

    Loss of the tumour suppressor PTEN is frequent in human melanoma, results in MAPK activation, suppresses senescence and mediates metastatic behaviour. How PTEN loss mediates these effects is unknown. Here we show that loss of PTEN in epithelial and melanocytic cell lines induces the nuclear localization and transcriptional activation of β-catenin independent of the PI3K-AKT-GSK3β axis. The absence of PTEN leads to caveolin-1 (CAV1)-dependent β-catenin transcriptional modulation in vitro, cooperates with NRAS(Q61K) to initiate melanomagenesis in vivo and induces efficient metastasis formation associated with E-cadherin internalization. The CAV1-β-catenin axis is mediated by a feedback loop in which β-catenin represses transcription of miR-199a-5p and miR-203, which suppress the levels of CAV1 mRNA in melanoma cells. These data reveal a mechanism by which loss of PTEN increases CAV1-mediated dissociation of β-catenin from membranous E-cadherin, which may promote senescence bypass and metastasis. PMID:26307673

  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. High-Affinity K+ Transport in Arabidopsis: AtHAK5 and AKT1 Are Vital for Seedling Establishment and Postgermination Growth under Low-Potassium Conditions1[C][W][OA

    PubMed Central

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

    2010-01-01

    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 NH4+-free growth conditions, athak5 mutant plants exhibited growth defects at 10 μm K+, but at K+ concentrations of 20 μm 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 μm K+ and growth was impaired at concentrations up to 450 μm K+. Moreover, transfer of 3-d-old plants from high to low K+ concentrations led to growth defects and leaf chlorosis at 10 μm K+ in athak5 akt1 double mutant plants. Determination of Rb+(K+) uptake kinetics in wild-type and mutant roots using rubidium (86Rb+) 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

  5. Extension of polyphenolics by CWPO-C peroxidase mutant containing radical-robust surface active site.

    PubMed

    Pham, L T Mai; Kim, S Jin; Ahn, U Suk; Choi, J Weon; Song, B Keun; Kim, Y Hwan

    2014-01-01

    Expressed as insoluble forms in Escherichia coli, native cationic cell wall peroxidase (CWPO-C) from the poplar tree and mutant variants were successfully reactivated via refolding experiments and used to elucidate the previously presumed existence of an electron transfer (ET) pathway in the CWPO-C structure. Their catalytic properties were fully characterized through various analyses including steady-state kinetic, direct oxidation of lignin macromolecules and their respective stabilities during the polymerization reactions. The analysis results proved that the 74th residue on the CWPO-C surface plays an important role in catalyzing the macromolecules via supposed ET mechanism. By comparing the residual activities of wild-type CWPO-C and mutant 74W CWPO-C after 3 min, mutation of tyrosine 74 residue to tryptophan increased the radical resistance of peroxidase up to ten times dramatically while maintaining its capability to oxidize lignin macromolecules. Furthermore, extension of poly(catechin) as well as lignin macromolecules with CWPO-C Y74W mutant clearly showed that this radical-resistant peroxidase mutant can increase the molecular weight of various kinds of polyphenolics by using surface-located active site. The anti-oxidation activity of the synthesized poly(catechin) was confirmed by xanthine oxidase assay. The elucidation of a uniquely catalytic mechanism in CWPO-C may improve the applicability of the peroxidase/H2O2 catalyst to green polymer chemistry. PMID:24122664

  6. 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

  7. Crenolanib is a potent inhibitor of FLT3 with activity against resistance-conferring point mutants

    PubMed Central

    Galanis, Allison; Ma, Hayley; Rajkhowa, Trivikram; Ramachandran, Abhijit; Small, Donald; Cortes, Jorge

    2014-01-01

    Mutations of the type III receptor tyrosine kinase FLT3 occur in approximately 30% of acute myeloid leukemia patients and lead to constitutive activation. This has made FLT3-activating mutations an attractive drug target because they are probable driver mutations of this disease. As more potent FLT3 inhibitors are developed, a predictable development of resistance-conferring point mutations, commonly at residue D835, has been observed. Crenolanib is a highly selective and potent FLT3 tyrosine kinase inhibitor (TKI) with activity against the internal tandem duplication (FLT3/ITD) mutants and the FLT3/D835 point mutants. We tested crenolanib against a panel of D835 mutant cell lines and primary patient blasts and observed superior cytotoxic effects when compared with other available FLT3 TKIs such as quizartinib and sorafenib. Another potential advantage of crenolanib is its reduced inhibition of c-Kit compared with quizartinib. In progenitor cell assays, crenolanib was less disruptive of erythroid colony growth, which may result in relatively less myelosuppression than quizartinib. Finally, correlative data from an ongoing clinical trial demonstrate that acute myeloid leukemia patients can achieve sufficient levels of crenolanib to inhibit both FLT3/ITD and resistance-conferring FLT3/D835 mutants in vivo. Crenolanib is thus an important next-generation FLT3 TKI. This study is registered at clinicaltrials.gov (ID: NCT01657682). PMID:24227820

  8. Stimulation of peripheral Kappa opioid receptors inhibits inflammatory hyperalgesia via activation of the PI3Kγ/AKT/nNOS/NO signaling pathway

    PubMed Central

    2012-01-01

    Background In addition to their central effects, opioids cause peripheral analgesia. There is evidence showing that peripheral activation of kappa opioid receptors (KORs) inhibits inflammatory pain. Moreover, peripheral μ-opioid receptor (MOR) activation are able to direct block PGE2-induced ongoing hyperalgesia However, this effect was not tested for KOR selective activation. In the present study, the effect of the peripheral activation of KORs on PGE2-induced ongoing hyperalgesia was investigated. The mechanisms involved were also evaluated. Results Local (paw) administration of U50488 (a selective KOR agonist) directly blocked, PGE2-induced mechanical hyperalgesia in both rats and mice. This effect was reversed by treating animals with L-NMMA or N-propyl-L-arginine (a selective inhibitor of neuronal nitric oxide synthase, nNOS), suggesting involvement of the nNOS/NO pathway. U50488 peripheral effect was also dependent on stimulation of PI3Kγ/AKT because inhibitors of these kinases also reduced peripheral antinociception induced by U50488. Furthermore, U50488 lost its peripheral analgesic effect in PI3Kγ null mice. Observations made in vivo were confirmed after incubation of dorsal root ganglion cultured neurons with U50488 produced an increase in the activation of AKT as evaluated by western blot analyses of its phosphorylated form. Finally, immunofluorescence of DRG neurons revealed that KOR-expressing neurons also express PI3Kγ (≅ 43%). Conclusions The present study indicates that activation of peripheral KORs directly blocks inflammatory hyperalgesia through stimulation of the nNOS/NO signaling pathway which is probably stimulated by PI3Kγ/AKT signaling. This study extends a previously study of our group suggesting that PI3Kγ/AKT/nNOS/NO is an important analgesic pathway in primary nociceptive neurons. PMID:22316281

  9. 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

  10. 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

  11. Induction of apoptosis by aqueous extract of Cordyceps militaris through activation of caspases and inactivation of Akt in human breast cancer MDA-MB-231 Cells.

    PubMed

    Jin, Cheng-Yun; Kim, Gi-Young; Choi, Yung Hyun

    2008-12-01

    Cordyceps militaris is well known as a traditional medicinal mushroom and has been shown to exhibit immunostimulatory and anticancer activities. In this study, we investigated the apoptosis induced by an aqueous extract of C. militaris (AECM) via the activation of caspases and altered mitochondrial membrane permeability in human breast cancer MDA-MB-231 cells. Exposure to AECM induced apoptosis, as demonstrated by a quantitative analysis of nuclear morphological change and a flow cytometric analysis. AECM increased hyperpolarization of mitochondrial membrane potential and promoted the activation of caspases. Both the cytotoxic effect and apoptotic characteristics induced by AECM treatment were significantly inhibited by z-DEVD-fmk, a caspase-3 inhibitor, which demonstrates the important role of caspase-3 in the observed cytotoxic effect. AECM-induced apoptosis was associated with the inhibition of Akt activation in a time-dependent manner, and pretreatment with LY294002, a PI3K/Akt inhibitor, significantly increased AECM-induced apoptosis. The results indicated that AECM-induced apoptosis may relate to the activation of caspase-3 and mitochondria dysfunctions that correlate with the inactivation of Akt. PMID:19131705

  12. Helicobacter pylori CagA Suppresses Apoptosis through Activation of AKT in a Nontransformed Epithelial Cell Model of Glandular Acini Formation

    PubMed Central

    Vallejo-Flores, Gabriela; Torres, Javier; Sandoval-Montes, Claudia; Arévalo-Romero, Haruki; Meza, Isaura; Camorlinga-Ponce, Margarita; Torres-Morales, Julián; Chávez-Rueda, Adriana Karina; Legorreta-Haquet, María Victoria; Fuentes-Pananá, Ezequiel M.

    2015-01-01

    H. pylori infection is the most important environmental risk to develop gastric cancer, mainly through its virulence factor CagA. In vitro models of CagA function have demonstrated a phosphoprotein activity targeting multiple cellular signaling pathways, while cagA transgenic mice develop carcinomas of the gastrointestinal tract, supporting oncogenic functions. However, it is still not completely clear how CagA alters cellular processes associated with carcinogenic events. In this study, we evaluated the capacity of H. pylori CagA positive and negative strains to alter nontransformed MCF-10A glandular acini formation. We found that CagA positive strains inhibited lumen formation arguing for an evasion of apoptosis activity of central acini cells. In agreement, CagA positive strains induced a cell survival activity that correlated with phosphorylation of AKT and of proapoptotic proteins BIM and BAD. Anoikis is a specific type of apoptosis characterized by AKT and BIM activation and it is the mechanism responsible for lumen formation of MCF-10A acini in vitro and mammary glands in vivo. Anoikis resistance is also a common mechanism of invading tumor cells. Our data support that CagA positive strains signaling function targets the AKT and BIM signaling pathway and this could contribute to its oncogenic activity through anoikis evasion. PMID:26557697

  13. Tyrosine 1101 of Tie2 Is the Major Site of Association of p85 and Is Required for Activation of Phosphatidylinositol 3-Kinase and Akt

    PubMed Central

    Kontos, Christopher D.; Stauffer, Thomas P.; Yang, Wen-Pin; York, John D.; Huang, Liwen; Blanar, Michael A.; Meyer, Tobias; Peters, Kevin G.

    1998-01-01

    Tie2 is an endothelium-specific receptor tyrosine kinase that is required for both normal embryonic vascular development and tumor angiogenesis and is thought to play a role in vascular maintenance. However, the signaling pathways responsible for the function of Tie2 remain unknown. In this report, we demonstrate that the p85 subunit of phosphatidylinositol 3-kinase (PI3-kinase) associates with Tie2 and that this association confers functional lipid kinase activity. Mutation of tyrosine 1101 of Tie2 abrogated p85 association both in vitro and in vivo in yeast. Tie2 was found to activate PI3-kinase in vivo as demonstrated by direct measurement of increases in cellular phosphatidylinositol 3-phosphate and phosphatidylinositol 3,4-bisphosphate, by plasma membrane translocation of a green fluorescent protein-Akt pleckstrin homology domain fusion protein, and by downstream activation of the Akt kinase. Activation of PI3-kinase was abrogated in these assays by mutation of Y1101 to phenylalanine, consistent with a requirement for this residue for p85 association with Tie2. These results suggest that activation of PI3-kinase and Akt may in part account for Tie2’s role in both embryonic vascular development and pathologic angiogenesis, and they are consistent with a role for Tie2 in endothelial cell survival. PMID:9632797

  14. Quercetin protects oligodendrocyte precursor cells from oxygen/glucose deprivation injury in vitro via the activation of the PI3K/Akt signaling pathway.

    PubMed

    Wang, X-Q; Yao, R-Q; Liu, X; Huang, J-J; Qi, D-S; Yang, L-H

    2011-10-10

    The aim of this study was to investigate the protection of quercetin (QUE) on oligodendrocyte precursor cells (OPCs) from oxygen/glucose deprivation (OGD)-induced injury in vitro and explore whether the PI3K/Akt signaling pathway contributed to the protection provided by quercetin. The OGD condition was induced by including 2mM sodium dithionite (Na(2)S(2)O(4)) in glucose-free DMEM medium. The concentration of QUE in this study ranged from 3μM to 81μM. OPCs were identified by immunocytochemical staining. Cell viability was analyzed using the water soluble tetrazolium salt-8 (WST-8) and lactate dehydrogenase assay (LDH). The morphological changes of the nucleus were measured using Hoechst 33258 nuclear staining, and the ratio of apoptotic cells was determined by FITC annexin V- and propidium iodide (PI) flow cytometry assay kit. In addition, the levels of pro-apoptotic proteins such as cleaved-caspase-3 and Bax and the anti-apoptotic proteins p-Akt and Bcl-2 were quantified using western blotting. The results showed that the OPC cell survival rate was significantly increased by incubation in conditioned medium supplemented with QUE as measured by the WST-8 assay, while the LDH release rate was significantly decreased as analyzed by the LDH assay. Furthermore, apoptosis assay showed that the apoptosis ratio of OPCs was also dramatically reduced by QUE. Western blotting showed that the expression levels of Bax and cleaved-caspase-3 proteins were down-regulated, while Bcl-2 and p-Akt were up-regulated. Further study showed that the increase in p-Akt by QUE was reduced by the PI3K inhibitor LY294002. These results indicated that QUE effectively protected OPCs from OGD-induced injury and that the mechanism might be related to the activation of the PI3K/Akt signaling pathway. PMID:21803128

  15. 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

  16. Paeonia lactiflora Pall. protects against ANIT-induced cholestasis by activating Nrf2 via PI3K/Akt signaling pathway

    PubMed Central

    Ma, Xiao; Zhao, Yan-ling; Zhu, Yun; Chen, Zhe; Wang, Jia-bo; Li, Rui-yu; Chen, Chang; Wei, Shi-zhang; Li, Jian-yu; Liu, Bing; Wang, Rui-lin; Li, Yong-gang; Wang, Li-fu; Xiao, Xiao-he

    2015-01-01

    Background Paeonia lactiflora Pall. (PLP), a traditional Chinese herbal medicine, has been used for hepatic disease treatment over thousands of years. In our previous study, PLP was shown to demonstrate therapeutic effect on hepatitis with severe cholestasis. The aim of this study was to evaluate the antioxidative effect of PLP on alpha-naphthylisothiocyanate (ANIT)-induced cholestasis by activating NF-E2-related factor 2 (Nrf2) via phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway. Materials and m