Science.gov

Sample records for akt signaling activates

  1. The Akt signaling pathway

    PubMed Central

    Madhunapantula, SubbaRao V; Mosca, Paul J

    2011-01-01

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

  2. 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.

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

    PubMed

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

    2011-02-01

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

  4. An active IGF-1R-AKT signaling imparts functional heterogeneity in ovarian CSC population.

    PubMed

    Singh, Ram K; Dhadve, Ajit; Sakpal, Asmita; De, Abhijit; Ray, Pritha

    2016-11-07

    Deregulated IGF-1R-AKT signaling influences multiple nodes of cancer cell physiology and assists in migration, metastasis and acquirement of radio/chemoresistance. Enrichment of cancer stem cells (CSC) positively correlates with radio/chemoresistance development in various malignancies. It is unclear though, how IGF-1R-AKT signalling shapes CSC functionality especially in ovarian cancer. Previously we showed that upregulated IGF-1R expression is essential to initiate platinum-taxol resistance at early stage which declines with elevated levels of activated AKT at late resistant stage in ovarian cancer cells. Here, we investigated the effect of this oscillatory IGF-1R-AKT signalling upon CSC functionality during generation of chemoresistance. While gradual increase in CSC properties from early (ER) to late (LR) resistant stages was observed in three different (cisplatin/paclitaxel/cisplatin-paclitaxel) cellular models created in two ovarian cancer cell lines, the stemness gene expressions (oct4/sox2/nanog) reached a plateau at early resistant stages. Inhibition of IGF-1R only at ER and AKT inhibition only at LR stages significantly abrogated the CSC phenotype. Interestingly, real time bioluminescence imaging showed CSCs of ER stages possessed faster tumorigenic potential than CSCs belonging to LR stages. Together, our data suggest that IGF-1R-AKT signalling imparts functional heterogeneity in CSCs during acquirement of chemoresistance in ovarian carcinoma.

  5. 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. Copyright © 2015 Elsevier Inc. All rights reserved.

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

    PubMed

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

    2015-05-01

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

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

    SciTech Connect

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

    2016-03-18

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

  8. Akt1 Signaling Regulates Integrin Activation, Matrix Recognition, and Fibronectin Assembly*S

    PubMed Central

    Somanath, Payaningal R.; Kandel, Eugene S.; Hay, Nissim; Byzova, Tatiana V.

    2009-01-01

    Akt, a serine-threonine kinase, regulates multiple cellular processes in vascular cells. We have previously documented that Akt activates integrins and Akt1 deficiency results in matrix abnormalities in skin and blood vessels in vivo. Based on these observations, we hypothesized that Akt1 is necessary for integrin activation and matrix assembly by fibroblasts. In this study, using various cell systems, we show that Akt1 is essential for the inside-out activation of integrins in endothelial cells and fibroblasts, which in turn, mediates matrix assembly. Fibronectin is a major extracellular matrix component of the skin and the vascular basement membrane, which possesses binding sites for many integrins and extracellular matrix proteins. Akt1−/− fibroblasts and NIH fibroblasts expressing dominant negative Akt1 (K179M-Akt1) showed impaired fibronectin assembly compared with control fibroblasts. In contrast, expression of constitutively active Akt1 (myrAkt1) resulted in enhanced fibronectin assembly. Although increased fibronectin assembly by myrAkt1-expressing human foreskin fibroblasts was abolished by treatment with anti-integrin β1 blocking antibodies, treatment with β1-stimulating antibodies rescued the impaired fibronectin assembly that was due to lack of Akt activity. Finally, expression of myrAkt1 corrected the phenotype of Akt1−/− fibroblasts thus showing that Akt1 regulates fibronectin assembly through activation of integrin α5β1. PMID:17562714

  9. 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.

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

    SciTech Connect

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

  11. 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

  12. Subcutaneous adipocytes promote melanoma cell growth by activating the Akt signaling pathway: role of palmitic acid.

    PubMed

    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

    2014-10-31

    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.

  13. Myostatin signaling regulates Akt activity via the regulation of miR-486 expression.

    PubMed

    Hitachi, Keisuke; Nakatani, Masashi; Tsuchida, Kunihiro

    2014-02-01

    Myostatin, also known as growth and differentiation factor-8, is a pivotal negative regulator of skeletal muscle mass and reduces muscle protein synthesis by inhibiting the insulin-like growth factor-1 (IGF-1)/Akt/mammalian target of rapamycin (mTOR) pathway. However, the precise mechanism by which myostatin inhibits the IGF-1/Akt/mTOR pathway remains unclear. In this study, we investigated the global microRNA expression profile in myostatin knockout mice and identified miR-486, a positive regulator of the IGF-1/Akt pathway, as a novel target of myostatin signaling. In myostatin knockout mice, the expression level of miR-486 in skeletal muscle was significantly increased. In addition, we observed increased expression of the primary transcript of miR-486 (pri-miR-486) and Ankyrin 1.5 (Ank1.5), the host gene of miR-486, in myostatin knockout mice. In C2C12 cells, myostatin negatively regulated the expression of Ank1.5. Moreover, canonical myostatin signaling repressed the skeletal muscle-specific promoter activity of miR-486/Ank1.5. This repression was partially mediated by the E-box elements in the proximal region of the promoter. We also show that overexpression of miR-486 induced myotube hypertrophy in vitro and that miR-486 was essential to maintain skeletal muscle size both in vitro and in vivo. In addition, inhibition of miR-486 led to a decrease in Akt activity in C2C12 myotubes. Our findings indicate that miR-486 is one of the intermediary molecules connecting myostatin signaling and the IGF-1/Akt/mTOR pathway in the regulation of skeletal muscle size.

  14. 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.

  15. AKT/PKB Signaling: Navigating Downstream

    PubMed Central

    Manning, Brendan D.; Cantley, Lewis C.

    2009-01-01

    The serine/threonine kinase Akt, also known as protein kinase B (PKB), is a central node in cell signaling downstream of growth factors, cytokines, and other cellular stimuli. Aberrant loss or gain of Akt activation underlies the pathophysiological properties of a variety of complex diseases, including type-2 diabetes and cancer. Here, we review the molecular properties of Akt and the approaches used to characterize its true cellular targets. In addition, we discuss those Akt substrates that are most likely to contribute to the diverse cellular roles of Akt, which include cell survival, growth, proliferation, angiogenesis, metabolism, and migration. PMID:17604717

  16. Deoxycholyltaurine Rescues Human Colon Cancer Cells From Apoptosis by Activating EGFR-Dependent PI3K/Akt Signaling

    PubMed Central

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

    2010-01-01

    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-α-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 (Ser473) 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 (Ser21/9) and BAD (Ser136), and nuclear translocation (activation) of NF-κB, 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

  17. Zinc induces cell death in immortalized embryonic hippocampal cells via activation of Akt-GSK-3beta signaling.

    PubMed

    Min, Young Kyu; Lee, Jong Eun; Chung, Kwang Chul

    2007-01-15

    Zinc is an essential catalytic and structural element of many proteins and a signaling messenger that is released by neuronal activity at many central excitatory synapses. Excessive synaptic release of zinc followed by entry into vulnerable neurons contributes severe neuronal cell death. We have previously observed that zinc-induced neuronal cell death is accompanied by Akt activation in embryonic hippocampal progenitor (H19-7) cells. In the present study, we examined the role of Akt activation and its downstream signaling events during extracellular zinc-induced neuronal cell death. Treatment of H19-7 cells with 10 microM of zinc plus zinc ionophore, pyrithione, led to increased phosphorylation of Akt at Ser-473/Thr-308 and increased Akt kinase activity. Zinc-induced Akt activation was accompanied by increased Tyr-phosphorylated GSK-3beta as well as increased GSK-3beta kinase activity. Transient overexpression of a kinase-deficient Akt mutant remarkably suppressed GSK-3beta activation and cell death. Furthermore, tau phosphorylation, but not the degradation of beta-catenin, was dependent upon zinc-induced GSK-3beta activation and contributed to cell death. The current data suggest that, following exposure to zinc, the sequential activation of Akt and GSK-3beta plays an important role directing hippocampal neural precursor cell death.

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

    PubMed

    Gong, Yi-Qing; Huang, Wei; Li, Ke-Ran; Liu, Yuan-Yuan; Cao, Guo-Fan; Cao, Cong; Jiang, Qin

    2016-09-13

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

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

    PubMed Central

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

    2016-01-01

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

  20. 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

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

    PubMed

    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

    2016-02-19

    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.

  2. Activation of the EGFR/Akt/NF-κB/cyclinD1 survival signaling pathway in human cholesteatoma epithelium.

    PubMed

    Liu, Wei; Yin, Tuanfang; Ren, Jihao; Li, Lihua; Xiao, Zian; Chen, Xing; Xie, Dinghua

    2014-02-01

    Cholesteatoma is a benign keratinizing squamous epithelial lesion characterized by the hyper-proliferation of keratinocytes with abundant production of keratin debris in the middle ear. The epidermal growth factor receptor (EGFR)/Akt/nuclear factor-kappa B (NF-κB)/cyclinD1 signaling pathway is one of the most important pathways in regulating cell survival and proliferation. We hypothesized that the EGFR/Akt/NF-κB/cyclinD1 signaling pathway may be activated and involved in the cellular hyperplasia mechanism in acquired cholesteatoma epithelium. Immunohistochemical staining of phosphorylated EGFR (p-EGFR), phosphorylated Akt (p-Akt), activated NF-κB and cyclinD1 protein was performed in 40 cholesteatoma samples and 20 samples of normal external auditory canal (EAC) epithelium. Protein expression of p-EGFR, p-Akt, activated NF-κB and cyclinD1 in cholesteatoma epithelium was significantly increased when compared with normal EAC epithelium (p < 0.01). In cholesteatoma epithelium, a significant positive association was observed between p-EGFR and p-Akt expression and between the expressions of p-Akt and NF-κB, NF-κB and cyclinD1, respectively (p < 0.01). No significant relationships were observed between the levels of investigated proteins and the degree of bone destruction (p > 0.05). The increased protein expression of p-EGFR, p-Akt, NF-κB and cyclinD1 and their associations in cholesteatoma epithelium suggest that the EGFR/Akt/NF-κB/cyclinD1 survival signaling pathway is active and may be involved in the regulatory mechanisms of cellular hyperplasia in cholesteatoma epithelium.

  3. Effects of AFP-activated PI3K/Akt signaling pathway on cell proliferation of liver cancer.

    PubMed

    Zheng, Lu; Gong, Wei; Liang, Ping; Huang, XiaoBing; You, Nan; Han, Ke Qiang; Li, Yu Ming; Li, Jing

    2014-05-01

    This study aims to investigate effects of alpha-fetoprotein (AFP)-activated phosphatidylinositol 3-kinase/protein kinase B (PI3K/Akt) signaling pathway on hepatocellular carcinoma cell proliferation. Active cirrhosis patients after hepatitis B infection (n = 20) and viral hepatitis patients with hepatocellular carcinoma (HCC) (n = 20) were selected as the subjects of the present study. Another 20 healthy subjects were selected as the control group. The serum AFP expression and liver tissue PI3K and Akt gene mRNA expression were detected. The hepatoma cell model HepG2 which had a stable expression of AFP gene was used. Real-time quantitative PCR and Western blot and other methods were used to analyze the intracellular PI3K and Akt protein levels. Compared with control group and cirrhosis group, the serum AFP levels in HCC group significantly increased, and the tissue PI3K and Akt mRNA expression also significantly increased. HepG2 cells were intervened using AFP, in which the PIK and Akt protein expression significantly increased. After intervention by use of AFP monoclonal antibodies or LY294002 inhibitor, the PIK and Akt protein expression in HepG2 cell was significantly decreased (P < 0.05). AFP can promote the proliferation of hepatoma cells via activation of PI3K/Akt signaling pathway.

  4. MYO6 Regulates Spatial Organization of Signaling Endosomes Driving AKT Activation and Actin Dynamics.

    PubMed

    Masters, Thomas A; Tumbarello, David A; Chibalina, Margarita V; Buss, Folma

    2017-06-06

    APPL1- and RAB5-positive signaling endosomes play a crucial role in the activation of AKT in response to extracellular stimuli. Myosin VI (MYO6) and two of its cargo adaptor proteins, GIPC and TOM1/TOM1L2, localize to these peripheral endosomes and mediate endosome association with cortical actin filaments. Loss of MYO6 leads to the displacement of these endosomes from the cell cortex and accumulation in the perinuclear space. Depletion of this myosin not only affects endosome positioning, but also induces actin and lipid remodeling consistent with endosome maturation, including accumulation of F-actin and the endosomal lipid PI(3)P. These processes acutely perturb endosome function, as both AKT phosphorylation and RAC-dependent membrane ruffling were markedly reduced by depletion of either APPL1 or MYO6. These results place MYO6 and its binding partners at a central nexus in cellular signaling linking actin dynamics at the cell surface and endosomal signaling in the cell cortex. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

  5. Epigenetic silencing mediated through activated PI3K/AKT signaling in breast cancer.

    PubMed

    Zuo, Tao; Liu, Ta-Ming; Lan, Xun; Weng, Yu-I; Shen, Rulong; Gu, Fei; Huang, Yi-Wen; Liyanarachchi, Sandya; Deatherage, Daniel E; Hsu, Pei-Yin; Taslim, Cenny; Ramaswamy, Bhuvaneswari; Shapiro, Charles L; Lin, Huey-Jen L; Cheng, Alfred S L; Jin, Victor X; Huang, Tim H-M

    2011-03-01

    Trimethylation of histone 3 lysine 27 (H3K27me3) is a critical epigenetic mark for the maintenance of gene silencing. Additional accumulation of DNA methylation in target loci is thought to cooperatively support this epigenetic silencing during tumorigenesis. However, molecular mechanisms underlying the complex interplay between the two marks remain to be explored. Here we show that activation of PI3K/AKT signaling can be a trigger of this epigenetic processing at many downstream target genes. We also find that DNA methylation can be acquired at the same loci in cancer cells, thereby reinforcing permanent repression in those losing the H3K27me3 mark. Because of a link between PI3K/AKT signaling and epigenetic alterations, we conducted epigenetic therapies in conjunction with the signaling-targeted treatment. These combined treatments synergistically relieve gene silencing and suppress cancer cell growth in vitro and in xenografts. The new finding has important implications for improving targeted cancer therapies in the future.

  6. Epigenetic Silencing Mediated Through Activated PI3K/AKT Signaling in Breast Cancer

    PubMed Central

    Zuo, Tao; Liu, Ta-Ming; Lan, Xun; Weng, Yu-I; Shen, Rulong; Gu, Fei; Huang, Yi-Wen; Liyanarachchi, Sandya; Deatherage, Daniel E.; Hsu, Pei-Yin; Taslim, Cenny; Ramaswamy, Bhuvaneswari; Shapiro, Charles L.; Lin, Huey-Jen L.; Cheng, Alfred S.L.; Jin, Victor X.; Huang, Tim H.-M.

    2011-01-01

    Trimethylation of histone 3 lysine 27 (H3K27me3) is a critical epigenetic mark for the maintenance of gene silencing. Additional accumulation of DNA methylation in target loci is thought to cooperatively support this epigenetic silencing during tumorigenesis. However, molecular mechanisms underlying the complex interplay between the two marks remain to be explored. Here we demonstrate that activation of PI3K/AKT signaling can be a trigger of this epigenetic processing at many downstream target genes. We also find that DNA methylation can be acquired at the same loci in cancer cells, thereby reinforcing permanent repression in those losing the H3K27me3 mark. Because of a link between PI3K/AKT signaling and epigenetic alterations, we conducted epigenetic therapies in conjunction with the signaling-targeted treatment. These combined treatments synergistically relieve gene silencing and suppress cancer cell growth in vitro and in xenografts. The new finding has important implications for improving targeted cancer therapies in the future. PMID:21216892

  7. Berberine ameliorates hyperglycemia in alloxan-induced diabetic C57BL/6 mice through activation of Akt signaling pathway.

    PubMed

    Xie, Xi; Li, Wenyuan; Lan, Tian; Liu, Weihua; Peng, Jing; Huang, Kaipeng; Huang, Juan; Shen, Xiaoyan; Liu, Peiqing; Huang, Heqing

    2011-01-01

    Recently, it is implicated that the abnormality of Akt signaling pathway is involved in the diabetic pathology. Previous studies have demonstrated that berberine could decrease blood glucose by elevating liver glycogen synthesis. However, the underlying mechanism is still unclear. In the present study, we investigated the effects of berberine on fasting blood glucose, liver glycogen, Akt, Glycogen synthase kinase-3, glucokinase and insulin receptor substrate (IRS) in alloxan-induced diabetic mice, exploring its possible hypoglycemic mechanism. We found that in alloxan-induced diabetic mice, the high blood glucose was significantly lowered by berberine treatment. Liver glycogen content, the expression and activity of glucokinase and the phosphorylated Akt and IRS were all significantly reduced in diabetic mice whereas berberine blocked these changes. Berberine also depressed the increasing of phosphorylated GSK-3β in diabetic mice. Collectively, Berberine upregulates the activity of Akt possibly via insulin signaling pathway, eventually lowering high blood glucose in alloxan-induced diabetic mice.

  8. 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

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

    PubMed

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

    2015-08-01

    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.

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

    PubMed

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

    2013-08-01

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

  11. Akt signaling leads to stem cell activation and promotes tumor development in epidermis.

    PubMed

    Segrelles, Carmen; García-Escudero, Ramón; Garín, Maria I; Aranda, Juan F; Hernández, Pilar; Ariza, José M; Santos, Mirentxu; Paramio, Jesús M; Lorz, Corina

    2014-07-01

    Hair follicle stem cells (HF-SCs) alternate between periods of quiescence and proliferation, to finally differentiate into all the cell types that constitute the hair follicle. Also, they have been recently identified as cells of origin in skin cancer. HF-SCs localize in a precise region of the hair follicle, the bulge, and molecular markers for this population have been established. Thus, HF-SCs are good model to study the potential role of oncogenic activations on SC physiology. Expression of a permanently active form of Akt (myrAkt) in basal cells leads to Akt hyperactivation specifically in the CD34(+)Itga6(H) population. This activation causes bulge stem cells to exit from quiescence increasing their response to proliferative stimuli and affecting some functions such as cell migration. HF-SC identity upon Akt activation is preserved; in this sense, increased proliferation does not result in stem cell exhaustion with age suggesting that Akt activation does not affect self-renewal an important aspect for normal tissue maintenance and cancer development. Genome-wide transcriptome analysis of HF-SC isolated from myrAkt and wild-type epidermis underscores changes in metabolic pathways characteristic of cancer cells. These differences manifest during a two-step carcinogenesis protocol in which Akt activation in HF-SCs results in increased tumor development and malignant transformation.

  12. Bamboo leaf extract ameliorates diabetic nephropathy through activating the AKT signaling pathway in rats.

    PubMed

    Ying, Changjiang; Mao, Yizhen; Chen, Lei; Wang, Shanshan; Ling, Hongwei; Li, Wei; Zhou, Xiaoyan

    2017-03-27

    Cleaved Caspase-3 levels in STZ-diabetic rats. In conclusion, our study suggested that bamboo leaf extract ameliorated DN in diabetic rats, and this protective effect is possibly related to suppressing oxidative stress through activating AKT signaling pathway. Bamboo leaf extract treatment may be a potential promising therapy for DN.

  13. Oxidative stress induces proliferation of colorectal cancer cells by inhibiting RUNX3 and activating the Akt signaling pathway.

    PubMed

    Kang, Kyoung Ah; Kim, Ki Cheon; Bae, Suk Chul; Hyun, Jin Won

    2013-11-01

    We recently reported that the tumor suppressor Runt-related transcription factor 3 (RUNX3) is silenced in colorectal cancer cells via oxidative stress-induced hypermethylation of its promoter. The resulting downregulation of RUNX3 expression influences cell proliferation. Activation of the Akt signaling pathway is also associated with cell survival and proliferation; however, the effects of oxidative stress on the relationship between RUNX3 and Akt signaling are largely unknown. Therefore, this study investigated the mechanisms involved in cell proliferation caused by oxidative stress-induced silencing of RUNX3. The levels of RUNX3 mRNA and protein were downregulated in response to treatment of the human colorectal cancer cell line SNU-407 with H2O2. Treatment of the cells with H2O2 also upregulated Akt mRNA and protein expression, and inhibited the binding of RUNX3 to the Akt promoter. The inverse correlation between the expression levels of RUNX3 and Akt in H2O2-treated cells was also associated with nuclear translocation of β-catenin and upregulation of cyclin D1 expression, which induced cell proliferation. H2O2 treatment also increased the binding of β-catenin to the cyclin D1 promoter. The results presented here demonstrate that reactive oxygen species silence the tumor suppressor RUNX3, enhance the Akt-mediated signaling pathway, and promote the proliferation of colorectal cancer cells.

  14. Activation of Akt signaling in rat brain by intracerebroventricular injection of ouabain: a rat model for mania.

    PubMed

    Yu, Hyun-Sook; Kim, Se Hyun; Park, Hong Geun; Kim, Yong Sik; Ahn, Yong Min

    2010-08-16

    Intracerebroventricular (ICV) injection of ouabain, a specific Na-K ATPase inhibitor, induces behavioral changes in rats resembling the manic phenotypes of bipolar disorder. The binding of ouabain to the Na-K ATPase affects signal events in vitro including Akt, a possible molecular target of mood disorders. However, the effects of ouabain on Akt in the brain need further clarification. In this study, we investigated changes in the phosphorylation state of Akt in the rat brain after ICV injection of ouabain. Consistent with our previous report, the locomotor activity of rats within 30 min after ouabain ICV injection changed according to the dose with higher doses of ouabain, 0.5 and 1 mM, inducing significant hyperactivity. In addition, ouabain administration induced a dose-dependent increase in the immunoreactivity of p-Akt (Ser473) in the frontal cortex, striatum, and hippocampus after 30 min, and reached statistical significance with 1mM of ouabain. Phosphorylation of GSK-3beta (Ser9), FOXO1 (Ser256), and eNOS (Ser1177), which are downstream molecules of Akt, was also increased in a dose-dependent manner within the same brain regions. Moreover, hyperactivity was seen for 8h after a single 1mM injection of ouabain and increased phosphorylation of Akt (Ser473), GSK-3beta (Ser9), FOXO1 (Ser256), and eNOS (Ser1177) was also observed in the cortex, striatum, and hippocampus. Thus, intrabrain injection of ouabain induces activation of Akt signaling accompanied by hyperactivity, suggesting the possible role of Akt in ouabain rat model of mania. Copyright (c) 2010 Elsevier Inc. All rights reserved.

  15. Insulin-mediated signaling promotes proliferation and survival of glioblastoma through Akt activation

    PubMed Central

    Gong, Yuanying; Ma, Yufang; Sinyuk, Maksim; Loganathan, Sudan; Thompson, Reid C.; Sarkaria, Jann N.; Chen, Wenbiao; Lathia, Justin D.; Mobley, Bret C.; Clark, Stephen W.; Wang, Jialiang

    2016-01-01

    Background Metabolic complications such as obesity, hyperglycemia, and type 2 diabetes are associated with poor outcomes in patients with glioblastoma. To control peritumoral edema, use of chronic high-dose steroids in glioblastoma patients is common, which can result in de novo diabetic symptoms. These metabolic complications may affect tumors via profound mechanisms, including activation of insulin receptor (InsR) and the related insulin-like growth factor 1 receptor (IGF1R) in malignant cells. Methods In the present study, we assessed expression of InsR in glioblastoma surgical specimens and glioblastoma response to insulin at physiologically relevant concentrations. We further determined whether genetic or pharmacological targeting of InsR affected oncogenic functions of glioblastoma in vitro and in vivo. Results We showed that InsR was commonly expressed in glioblastoma surgical specimens and xenograft tumor lines, with mitogenic isoform-A predominating. Insulin at physiologically relevant concentrations promoted glioblastoma cell growth and survival, potentially via Akt activation. Depletion of InsR impaired cellular functions and repressed orthotopic tumor growth. The absence of InsR compromised downstream Akt activity, but yet stimulated IGF1R expression. Targeting both InsR and IGF1R with dual kinase inhibitors resulted in effective blockade of downstream signaling, loss of cell viability, and repression of xenograft tumor growth. Conclusions Taken together, our work suggests that glioblastoma is sensitive to the mitogenic functions of insulin, thus significant insulin exposure imposes risks to glioblastoma patients. Additionally, dual inhibition of InsR and IGF1R exhibits promise for treating glioblastoma. PMID:26136493

  16. Intracellular calcium promotes radioresistance of non-small cell lung cancer A549 cells through activating Akt signaling.

    PubMed

    Wang, Yiling; He, Jiantao; Zhang, Shenghui; Yang, Qingbo

    2017-03-01

    Radiotherapy is a major therapeutic approach in non-small cell lung cancer but is restricted by radioresistance. Although Akt signaling promotes radioresistance in non-small cell lung cancer, it is not well understood how Akt signaling is activated. Since intracellular calcium (Ca(2+)) could activate Akt in A549 cells, we investigated the relationship between intracellular calcium (Ca(2+)) and Akt signaling in radioresistant A549 cells by establishing radioresistant non-small cell lung cancer A549 cells. The radioresistant cell line A549 was generated by dose-gradient irradiation of the parental A549 cells. The cell viability, proliferation, and apoptosis were, respectively, assessed using the cell counting kit-8, EdU labeling, and flow cytometry analysis. The phosphorylation of Akt was evaluated by Western blotting, and the intracellular Ca(2+) concentration was assessed by Fluo 4-AM. The radioresistant A549 cells displayed mesenchymal morphology. After additional irradiation, the radioresistant A549 cells showed decreased cell viability and proliferation but increased apoptosis. Moreover, the intracellular Ca(2+) concentration and the phosphorylation level on the Akt473 site in radioresistant A549 cells were higher than those in original cells, whereas the percentage of apoptosis in radioresistant A549 cells was less. All these results could be reversed by verapamil. In conclusion, our study found that intracellular Ca(2+) could promote radioresistance of non-small cell lung cancer cells through phosphorylating of Akt on the 473 site, which contributes to a better understanding on the non-small cell lung cancer radioresistance, and may provide a new target for radioresistance management.

  17. 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.

  18. 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

  19. Dysfunctional MnSOD leads to redox dysregulation and activation of prosurvival AKT signaling in uterine leiomyomas

    PubMed Central

    Vidimar, Vania; Gius, David; Chakravarti, Debabrata; Bulun, Serdar E.; Wei, Jian-Jun; Kim, J. Julie

    2016-01-01

    AKT signaling promotes cell growth and survival and is often dysregulated via multiple mechanisms in different types of cancer, including uterine leiomyomas (ULMs). ULMs are highly prevalent fibrotic tumors that arise from the smooth muscular layer of the uterus, the myometrium (MM). ULMs pose a major public health issue because they can cause severe morbidity and poor pregnancy outcomes. ‬We investigate the mechanisms driving ULM growth and survival via aberrant activation of AKT. We demonstrate that an acetylation-mediated impairment of the manganese superoxide dismutase (MnSOD) activity is prevalent in ULM cells compared to the normal-matched MM from the same patients. This impairment increases the levels of superoxide and oxidative stress, which activate AKT via oxidative inactivation of the phosphatase and tensin homolog deleted on chromosome 10 (PTEN). Redox activation of AKT promotes ULM cell survival under conditions of moderate but persistent oxidative stress that are compatible with ULM’s prooxidative microenvironment. Moreover, because of impaired MnSOD activity, ULM cells are sensitive to high levels of reactive oxygen species (ROS) and superoxide-generating compounds, resulting in decreased ULM cell viability. On the contrary, MM cells with functional MnSOD are more resistant to high levels of oxidants. This study demonstrates a causative role of acetylation-mediated MnSOD dysfunction in activating prosurvival AKT signaling in ULMs. The specific AKT and redox states of ULM cells provide a potential novel therapeutic rationale to selectively target ULM cells because of their defective ROS-scavenging system.‬‬‬‬‬‬‬‬ PMID:27847869

  20. 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.

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

    PubMed Central

    2016-01-01

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

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

    PubMed

    Hou, Ya-Qin; Yao, Yao; Bao, Yong-Li; Song, Zhen-Bo; Yang, Cheng; Gao, Xiu-Li; Zhang, Wen-Jing; Sun, Lu-Guo; Yu, Chun-Lei; Huang, Yan-Xin; Wang, Guan-Nan; Li, Yu-Xin

    2016-01-01

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

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

    SciTech Connect

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

    2014-12-12

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

  4. Metastasis and AKT activation.

    PubMed

    Qiao, Meng; Sheng, Shijie; Pardee, Arthur B

    2008-10-01

    Metastasis is responsible for 90% of cancer patient deaths. More information is needed about the molecular basis for its potential detection and treatment. The activated AKT kinase is necessary for many events of the metastatic pathway including escape of cells from the tumor's environment, into and then out of the circulation, activation of proliferation, blockage of apoptosis, and activation of angiogenesis. A series of steps leading to metastatic properties can be initiated upon activation of AKT by phosphorylation on Ser-473. These findings lead to the question of how this activation is connected to metastasis. Activated AKT phosphorylates GSK-3beta causing its proteolytic removal. This increases stability of the negative transcription factor SNAIL, thereby decreasing transcription of the transmembrane protein E-cadherin that forms adhesions between adjacent cells, thereby permitting their detachment. How is AKT hyperactivated in metastatic cells? Increased PI3K or TORC2 kinase activity- or decreased PHLPP phosphatase could be responsible. Furthermore, a positive feedback mechanism is that the decrease of E-cadherin lowers PTEN and thereby increases PIP3, further activating AKT and metastasis.

  5. 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

  6. FAM83B-mediated activation of PI3K/AKT and MAPK signaling cooperates to promote epithelial cell transformation and resistance to targeted therapies

    PubMed Central

    Cipriano, Rocky; Miskimen, Kristy L.S.; Bryson, Benjamin L.; Foy, Chase R.; Bartel, Courtney A.; Jackson, Mark W.

    2013-01-01

    Therapies targeting MAPK and AKT/mTOR signaling are currently being evaluated in clinical trials for several tumor types. However, recent studies suggest that these therapies may be limited due to acquired cancer cell resistance and a small therapeutic index between normal and cancer cells. The identification of novel proteins that are involved in MAPK or AKT/mTOR signaling and differentially expressed between normal and cancer cells will provide mechanistically distinct therapeutic targets with the potential to inhibit these key cancer-associated pathways. We recently identified FAM83B as a novel, previously uncharacterized oncogene capable of hyperactivating MAPK and mTOR signaling and driving the tumorigenicity of immortalized human mammary epithelial cells (HMEC). We show here that elevated FAM83B expression also activates the PI3K/AKT signaling pathway and confers a decreased sensitivity to PI3K, AKT, and mTOR inhibitors. FAM83B co-precipitated with the p85α and p110α subunits of PI3K, as well as AKT, and increased p110α and AKT membrane localization, consistent with elevated PI3K/AKT signaling. In tumor-derived cells harboring elevated FAM83B expression, ablation of FAM83B decreased p110α and AKT membrane localization, suppressed AKT phosphorylation, and diminished proliferation, AIG, and tumorigenicity in vivo. We propose that the level of FAM83B expression may be an important factor to consider when combined therapies targeting MAPK and AKT/mTOR signaling are used. Moreover, the identification of FAM83B as a novel oncogene and its integral involvement in activating PI3K/AKT and MAPK provides a foundation for future therapies aimed at targeting FAM83B in order to suppress the growth of PI3K/AKT- and MAPK-driven cancers. PMID:23676467

  7. 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.

  8. Increase in reactive oxygen species and activation of Akt signaling pathway in neuropathic pain.

    PubMed

    Guedes, Renata P; Araújo, Alex S R; Janner, Daiane; Belló-Klein, Adriane; Ribeiro, Maria Flávia M; Partata, Wania A

    2008-12-01

    Neuropathic pain occurs as a result of peripheral or central nervous system injury. Its pathophysiology involves mainly a central sensitization mechanism that may be correlated to many molecules acting in regions involved in pain processing, such as the spinal cord. It has been demonstrated that reactive oxygen species (ROS) and signaling molecules, such as the serine/threonine protein kinase Akt, are involved in neuropathic pain mechanisms. Thus, the aim of this study was to provide evidence of this relationship. Sciatic nerve transection (SNT) was used to induce neuropathic pain in rats. Western blot analysis of Akt and 4-hydroxy-2-nonenal (HNE)-Michael adducts, and measurement of hydrogen peroxide (H(2)O(2)) in the lumbosacral spinal cord were performed. The main findings were found seven days after SNT, when there was an increase in HNE-Michael adducts formation, total and p-Akt expression, and H(2)O(2) concentration. However, one and 15 days after SNT, H(2)O(2) concentration was raised in both sham (animals that were submitted to surgery without nerve injury) and SNT groups, showing the high sensibility of this ROS to nociceptive afferent stimuli, not only to neuropathic pain. p-Akt also increased in sham and SNT groups one day post injury, but at 3 and 7 days the increase occurred exclusively in SNT animals. Thus, there is crosstalk between intracellular signaling pathways and ROS, and these molecules can act as protective agents in acute pain situations or play a role in the development of chronic pain states.

  9. 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.

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

    PubMed

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

    2014-12-12

    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 (H₂O₂). SCF activated its receptor c-Kit in osteoblasts, which was required for its cyto-protective effects against H₂O₂. 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 H₂O₂ was mediated via activation of c-Kit-dependent Akt pathway. Inhibition of Akt activation, through pharmacological or genetic means, suppressed SCF-mediated anti-H₂O₂ activity in osteoblasts. In summary, we have identified a new SCF-c-Kit-Akt physiologic pathway that protects osteoblasts from H₂O₂-induced damages, and might minimize the risk of osteonecrosis caused by oxidative stress.

  11. Dystrophin Glycoprotein Complex-associated Gβγ Subunits Activate Phosphatidyl Inositol-3-Kinase/Akt signaling in Skeletal Muscle in a Laminin-dependent Manner

    PubMed Central

    Xiong, Yongmin; Zhou, Yanwen; Jarrett, Harry W.

    2010-01-01

    Previously, we showed that laminin-binding to the dystrophin glycoprotein complex (DGC) of skeletal muscle causes a heterotrimeric G-protein, (Gαβγ) to bind, changing the activation state of the Gsα subunit. Others have shown that laminin-binding to the DGC also leads to Akt activation. Gβγ, released when Gsα is activated, is known to bind phosphatidylinositol 3-kinase (PI3K), which activates Akt in other cells. Here, we investigate whether muscle Akt activation results from Gβγ, using immunoprecipitation and immunoblotting, and purified Gβγ. In the presence of laminin, PI3K-binding to the DGC increases and Akt becomes phosphorylated and activated (pAkt), and glycogen synthase kinase is phosphorylated. Antibodies, which specifically block laminin-binding to α-dystroglycan, prevent PI3K-binding to the DGC. Purified bovine brain Gβγ also caused PI3K and Akt activation. These results show that DGC-Gβγ is binding PI3K and activating pAkt in a laminin-dependent manner. Mdx mice, which have greatly diminished amounts of DGC proteins, display elevated pAkt signaling and increased expression of integrin β1 compared to normal muscle. This integrin binds laminin, Gβγ, and PI3K. Collectively, these suggest that PI3K is an important target for the Gβγ, which normally binds to DGC syntrophin, and activates PI3K/Akt signaling. Disruption of the DGC in mdx mouse is causing dis-regulation of the laminin-DGC-Gβγ-PI3K-Akt signaling and is likely to be important to the pathogenesis of muscular dystrophy. Up-regulating integrin β1 expression and activating the PI3K/Akt pathway in muscular dystrophy may partially compensate for the loss of the DGC. The results suggest new therapeutic approaches to muscle disease. PMID:19117013

  12. Dystrophin glycoprotein complex-associated Gbetagamma subunits activate phosphatidylinositol-3-kinase/Akt signaling in skeletal muscle in a laminin-dependent manner.

    PubMed

    Xiong, Yongmin; Zhou, Yanwen; Jarrett, Harry W

    2009-05-01

    Previously, we showed that laminin-binding to the dystrophin glycoprotein complex (DGC) of skeletal muscle causes a heterotrimeric G-protein (Galphabetagamma) to bind, changing the activation state of the Gsalpha subunit. Others have shown that laminin-binding to the DGC also leads to Akt activation. Gbetagamma, released when Gsalpha is activated, is known to bind phosphatidylinositol-3-kinase (PI3K), which activates Akt in other cells. Here, we investigate whether muscle Akt activation results from Gbetagamma, using immunoprecipitation and immunoblotting, and purified Gbetagamma. In the presence of laminin, PI3K-binding to the DGC increases and Akt becomes phosphorylated and activated (pAkt), and glycogen synthase kinase is phosphorylated. Antibodies, which specifically block laminin-binding to alpha-dystroglycan, prevent PI3K-binding to the DGC. Purified bovine brain Gbetagamma also caused PI3K and Akt activation. These results show that DGC-Gbetagamma is binding PI3K and activating pAkt in a laminin-dependent manner. Mdx mice, which have greatly diminished amounts of DGC proteins, display elevated pAkt signaling and increased expression of integrin beta1 compared to normal muscle. This integrin binds laminin, Gbetagamma, and PI3K. Collectively, these suggest that PI3K is an important target for the Gbetagamma, which normally binds to DGC syntrophin, and activates PI3K/Akt signaling. Disruption of the DGC in mdx mouse is causing dis-regulation of the laminin-DGC-Gbetagamma-PI3K-Akt signaling and is likely to be important to the pathogenesis of muscular dystrophy. Upregulating integrin beta1 expression and activating the PI3K/Akt pathway in muscular dystrophy may partially compensate for the loss of the DGC. The results suggest new therapeutic approaches to muscle disease.

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

    PubMed

    Yang, Rong; Zhong, Liang; Yang, Xiao-Qun; Jiang, Kai-Ling; Li, Liu; Song, Hao; Liu, Bei-Zhong

    2016-05-01

    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.

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

    PubMed

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

    2014-08-08

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

  15. Alpha-fetoprotein activates AKT/mTOR signaling to promote CXCR4 expression and migration of hepatoma cells.

    PubMed

    Zhu, Mingyue; Guo, Junli; Xia, Hua; Li, Wei; Lu, Yan; Dong, Xu; Chen, Yi; Xie, Xieju; Fu, Shigan; Li, Mengsen

    2015-01-01

    CXCR4, stromal cell-derived factor-1α(SDF 1α) receptor, stimulates growth and metastasis of hepatocellular carcinoma (HCC). Alpha-fetoprotein(AFP) governs the expression of some metastasis-related genes. Here we report that AFP and CXCR4 levels correlated in HCC tissues. AFP-expressing vectors induced CXCR4. In agreement, AFP depletion by siRNA decreased CXCR4. AFP co-localized and interacted with PTEN, thus inducing CXCR4 by activating AKT(Ser473) phosphorylation. In turn, phospho-mTOR(Ser2448) entered the nucleus and bound the CXCR4 gene promoter. Thus, AFP promoted migration of HCC cells. In concusion, AFP induced CXCR4 by activating the AKT/mTOR signal pathway.

  16. Alpha-fetoprotein activates AKT/mTOR signaling to promote CXCR4 expression and migration of hepatoma cells

    PubMed Central

    Li, Wei; Lu, Yan; Dong, Xu; Chen, Yi; Xie, Xieju; Fu, Shigan; Li, Mengsen

    2015-01-01

    CXCR4, stromal cell-derived factor-1α(SDF 1α) receptor, stimulates growth and metastasis of hepatocellular carcinoma (HCC). Alpha-fetoprotein(AFP) governs the expression of some metastasis-related genes. Here we report that AFP and CXCR4 levels correlated in HCC tissues. AFP-expressing vectors induced CXCR4. In agreement, AFP depletion by siRNA decreased CXCR4. AFP co-localized and interacted with PTEN, thus inducing CXCR4 by activating AKT(Ser473) phosphorylation. In turn, phospho-mTOR(Ser2448) entered the nucleus and bound the CXCR4 gene promoter. Thus, AFP promoted migration of HCC cells. In concusion, AFP induced CXCR4 by activating the AKT/mTOR signal pathway. PMID:25815363

  17. Early activation of mTORC1 signalling in response to mechanical overload is independent of phosphoinositide 3-kinase/Akt signalling

    PubMed Central

    Miyazaki, Mitsunori; McCarthy, John J; Fedele, Mark J; Esser, Karyn A

    2011-01-01

    Abstract The mammalian target of rapamycin complex 1 (mTORC1) functions as a central integrator of a wide range of signals that modulate protein metabolism and cell growth. However, the contributions of individual pathways regulating mTORC1 activity in skeletal muscle are poorly defined. The purpose of this study was to determine the regulatory mechanisms that contribute to mTORC1 activation during mechanical overload-induced skeletal muscle hypertrophy. Consistent with previous studies, mechanical overload induced progressive hypertrophy of the plantaris muscle which was associated with significant increases in total RNA content and protein metabolism. mTORC1 was activated after a single day of overload as indicated by a significant increase in S6K1 phosphorylation at T389 and T421/S424. In contrast, Akt activity, as assessed by Akt phosphorylation status (T308 and S473), phosphorylation of direct downstream targets (glycogen synthase kinase 3 β, proline-rich Akt substrate 40 kDa and tuberous sclerosis 2 (TSC2)) and a kinase assay, was not significantly increased until 2–3 days of overload. Inhibition of phosphoinositide 3-kinase (PI3K) activity by wortmannin was sufficient to block insulin-dependent signalling but did not prevent the early activation of mTORC1 in response to overload. We identified that the mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK)-dependent pathway was activated at day 1 after overload. In addition, a target of MEK/ERK signalling, phosphorylation of TSC2 at S664, was also increased at this early time point. These observations demonstrate that in vivo, mTORC1 activation at the early phase of mechanical overload in skeletal muscle occurs independently of PI3K/Akt signalling and provide evidence that the MEK/ERK pathway may contribute to mTORC1 activation through phosphorylation of TSC2. PMID:21300751

  18. 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

  19. DYRK1B blocks canonical and promotes non-canonical Hedgehog signaling through activation of the mTOR/AKT pathway

    PubMed Central

    Singh, Rajeev; Dhanyamraju, Pavan Kumar; Lauth, Matthias

    2017-01-01

    Hedgehog (Hh) signaling plays important roles in embryonic development and in tumor formation. Apart from the well-established stimulation of the GLI family of transcription factors, Hh ligands promote the phosphorylation and activation of mTOR and AKT kinases, yet the molecular mechanism underlying these processes are unknown. Here, we identify the DYRK1B kinase as a mediator between Hh signaling and mTOR/AKT activation. In fibroblasts, Hh signaling induces DYRK1B protein expression, resulting in activation of the mTOR/AKT kinase signaling arm. Furthermore, DYRK1B exerts positive and negative feedback regulation on the Hh pathway itself: It negatively interferes with SMO-elicited canonical Hh signaling, while at the same time it provides positive feed-forward functions by promoting AKT-mediated GLI stability. Due to the fact that the mTOR/AKT pathway is itself subject to strong negative feedback regulation, pharmacological inhibition of DYRK1B results in initial upregulation followed by downregulation of AKT phosphorylation and GLI stabilization. Addressing this issue therapeutically, we show that a pharmacological approach combining a DYRK1B antagonist with an mTOR/AKT inhibitor results in strong GLI1 targeting and in pronounced cytotoxicity in human pancreatic and ovarian cancer cells. PMID:27903983

  20. 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.

  1. Membrane type 1-matrix metalloproteinase/Akt signaling axis modulates TNF-α-induced procoagulant activity and apoptosis in endothelial cells.

    PubMed

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

    2014-01-01

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

  2. Signal propagation from membrane messengers to nuclear effectors revealed by reporters of phosphoinositide dynamics and Akt activity

    PubMed Central

    Ananthanarayanan, Bharath; Ni, Qiang; Zhang, Jin

    2005-01-01

    Among various second messengers, phosphatidylinositol 3,4,5-triphosphate (PIP3) and phosphatidylinositol 3,4-bisphosphate [PI(3,4)P2] regulate a variety of cellular processes, such as cell survival, polarization, and proliferation. Many of these functions are achieved via activation of serine/threonine kinase Akt. To investigate the spatiotemporal regulation of these lipids, we constructed a genetically targetable phosphoinositide (PI) indicator by sandwiching pleckstrin homology (PH) domain of Akt and a “pseudoligand” containing acidic amino acid residues, between cyan and yellow mutants of GFP. In living cells, elevations in PIP3 and PI(3,4)P2 by growth factor-induced activation of phosphatidylinositol 3-kinase (PI3K) resulted in a change in fluorescence resonance energy transfer (FRET) between the fluorescent proteins, increasing yellow to cyan emission ratios by 10–30%. This response can be reversed by inhibiting PI3K and abolished by mutating the critical residues responsible for PI binding. Differential dynamics of PIs were observed at plasma membrane of NIH 3T3 cells, stimulated by various growth factors. On the other hand, the nuclear targeted indicator showed no response within an hour after platelet-derived growth factor stimulation, suggesting that no appreciable amounts of accessible PIP3 and PI(3,4)P2 were produced in the nucleus. Furthermore, simultaneous imaging of a plasma membrane-targeted PI indicator and a nuclear-targeted Akt activity reporter revealed a gradual and sustained accumulation of Akt activity in the nucleus after rapid and transient production of PIP3 and PI(3,4)P2 at plasma membrane in the same cell. Thus, signal propagation from the lipid messengers at plasma membrane to the effectors in the nucleus is precisely controlled by kinases as well as lipid and protein phosphatases. PMID:16214892

  3. Signal propagation from membrane messengers to nuclear effectors revealed by reporters of phosphoinositide dynamics and Akt activity.

    PubMed

    Ananthanarayanan, Bharath; Ni, Qiang; Zhang, Jin

    2005-10-18

    Among various second messengers, phosphatidylinositol 3,4,5-triphosphate (PIP3) and phosphatidylinositol 3,4-bisphosphate [PI(3,4)P2] regulate a variety of cellular processes, such as cell survival, polarization, and proliferation. Many of these functions are achieved via activation of serine/threonine kinase Akt. To investigate the spatiotemporal regulation of these lipids, we constructed a genetically targetable phosphoinositide (PI) indicator by sandwiching pleckstrin homology (PH) domain of Akt and a "pseudoligand" containing acidic amino acid residues, between cyan and yellow mutants of GFP. In living cells, elevations in PIP3 and PI(3,4)P2 by growth factor-induced activation of phosphatidylinositol 3-kinase (PI3K) resulted in a change in fluorescence resonance energy transfer (FRET) between the fluorescent proteins, increasing yellow to cyan emission ratios by 10-30%. This response can be reversed by inhibiting PI3K and abolished by mutating the critical residues responsible for PI binding. Differential dynamics of PIs were observed at plasma membrane of NIH 3T3 cells, stimulated by various growth factors. On the other hand, the nuclear targeted indicator showed no response within an hour after platelet-derived growth factor stimulation, suggesting that no appreciable amounts of accessible PIP3 and PI(3,4)P2 were produced in the nucleus. Furthermore, simultaneous imaging of a plasma membrane-targeted PI indicator and a nuclear-targeted Akt activity reporter revealed a gradual and sustained accumulation of Akt activity in the nucleus after rapid and transient production of PIP3 and PI(3,4)P2 at plasma membrane in the same cell. Thus, signal propagation from the lipid messengers at plasma membrane to the effectors in the nucleus is precisely controlled by kinases as well as lipid and protein phosphatases.

  4. 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

  5. β-Caryophyllene Pretreatment Alleviates Focal Cerebral Ischemia-Reperfusion Injury by Activating PI3K/Akt Signaling Pathway.

    PubMed

    Zhang, Qian; An, Ruidi; Tian, Xiaocui; Yang, Mei; Li, Minghang; Lou, Jie; Xu, Lu; Dong, Zhi

    2017-02-24

    β-Caryophyllene (BCP) has been reported to be protective against focal cerebral ischemia-reperfusion (I/R) injury by its anti-oxidative and anti-inflammatory features. Recent study demonstrates that the BCP exhibits potential neuroprotection against I/R injury induced apoptosis, however, the mechanism remains unknown. Therefore, we investigate the underlying anti-apoptotic mechanism of BCP pretreatment in I/R injury. Sprague-Dawley rats (pretreated with BCP suspensions or solvent orally for 7 days) were subjected to transient Middle Cerebral Artery Occlusion (MCAO) for 90 min, followed by 24 h reperfusion. Results showed that BCP pretreatment improved the neurologic deficit score, lowered the infarct volume and decreased number of apoptotic cells in the hippocampus. Moreover, in western blot and RT-qPCR detections, BCP pretreatment down-regulated the expressions of Bax and p53, up-regulated the expression of Bcl-2, and enhanced the phosphorylation of Akt on Ser473. Blockage of PI3K activity by wortmannin not only abolished the BCP-induced decreases in infarct volume and neurologic deficit score, but also dramatically abrogated the enhancement of AKt phosphorylation. Our results suggested that BCP pre-treatment protects against I/R injury partly by suppressing apoptosis via PI3K/AKt signaling pathway activation.

  6. Acadesine Inhibits Tissue Factor Induction and Thrombus Formation by Activating the Phosphoinositide 3-Kinase/Akt Signaling Pathway

    PubMed Central

    Zhang, Weiyu; Wang, Jianguo; Wang, Huan; Tang, Rong; Belcher, John D.; Viollet, Benoit; Geng, Jian-Guo; Zhang, Chunxiang; Wu, Chaodong; Slungaard, Arne; Zhu, Chuhong; Huo, Yuqing

    2013-01-01

    Objective Acadesine, an adenosine-regulating agent and activator of AMP-activated protein kinase, has been shown to possess antiinflammatory activity. This study investigated whether and how acadesine inhibits tissue factor (TF) expression and thrombus formation. Methods and Results Human umbilical vein endothelial cells and human peripheral blood monocytes were stimulated with lipopolysaccharide to induce TF expression. Pretreatment with acadesine dramatically suppressed the clotting activity and expression of TF (protein and mRNA). These inhibitory effects of acadesine were unchanged for endothelial cells treated with ZM241385 (a specific adenosine A2A receptor antagonist) or AMP-activated protein kinase inhibitor compound C, and in macrophages lacking adenosine A2A receptor or α1–AMP-activated protein kinase. In endothelial cells and macrophages, acadesine activated the phosphoinositide 3-kinase/Akt signaling pathway, reduced the activity of mitogen-activated protein kinases, and consequently suppressed TF expression by inhibiting the activator protein-1 and NF-κB pathways. In mice, acadesine suppressed lipopolysaccharide-mediated increases in blood coagulation, decreased TF expression in atherosclerotic lesions, and reduced deep vein thrombus formation. Conclusion Acadesine inhibits TF expression and thrombus formation by activating the phosphoinositide 3-kinase/Akt pathway. This novel finding implicates acadesine as a potentially useful treatment for many disorders associated with thrombotic pathology, such as angina pain, deep vein thrombosis, and sepsis. PMID:20185792

  7. 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

  8. The mitochondrial-derived peptide humanin activates the ERK1/2, AKT, and STAT3 signaling pathways and has age-dependent signaling differences in the hippocampus

    PubMed Central

    Kim, Su-Jeong; Guerrero, Noel; Wassef, Gabriella; Xiao, Jialin; Mehta, Hemal H.; Cohen, Pinchas; Yen, Kelvin

    2016-01-01

    Humanin is a small secreted peptide that is encoded in the mitochondrial genome. Humanin and its analogues have a protective role in multiple age-related diseases including type 2 diabetes and Alzheimer's disease, through cytoprotective and neuroprotective effects both in vitro and in vivo. However, the humanin-mediated signaling pathways are not well understood. In this paper, we demonstrate that humanin acts through the GP130/IL6ST receptor complex to activate AKT, ERK1/2, and STAT3 signaling pathways. Humanin treatment increases phosphorylation in AKT, ERK 1/2, and STAT3 where PI3K, MEK, and JAK are involved in the activation of those three signaling pathways, respectively. Furthermore, old mice, but not young mice, injected with humanin showed an increase in phosphorylation in AKT and ERK1/2 in the hippocampus. These findings uncover a key signaling pathway of humanin that is important for humanin's function and also demonstrates an age-specific in vivo effect in a region of the brain that is critical for memory formation in an age-dependent manner. PMID:27384491

  9. The mitochondrial-derived peptide humanin activates the ERK1/2, AKT, and STAT3 signaling pathways and has age-dependent signaling differences in the hippocampus.

    PubMed

    Kim, Su-Jeong; Guerrero, Noel; Wassef, Gabriella; Xiao, Jialin; Mehta, Hemal H; Cohen, Pinchas; Yen, Kelvin

    2016-07-26

    Humanin is a small secreted peptide that is encoded in the mitochondrial genome. Humanin and its analogues have a protective role in multiple age-related diseases including type 2 diabetes and Alzheimer's disease, through cytoprotective and neuroprotective effects both in vitro and in vivo. However, the humanin-mediated signaling pathways are not well understood. In this paper, we demonstrate that humanin acts through the GP130/IL6ST receptor complex to activate AKT, ERK1/2, and STAT3 signaling pathways. Humanin treatment increases phosphorylation in AKT, ERK 1/2, and STAT3 where PI3K, MEK, and JAK are involved in the activation of those three signaling pathways, respectively. Furthermore, old mice, but not young mice, injected with humanin showed an increase in phosphorylation in AKT and ERK1/2 in the hippocampus. These findings uncover a key signaling pathway of humanin that is important for humanin's function and also demonstrates an age-specific in vivo effect in a region of the brain that is critical for memory formation in an age-dependent manner.

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

    SciTech Connect

    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. - Highlights: • miR-486 is a hypoxia-induced miRNA. • miR-486 regulates the secretion of HGF and VEGF, promotes proliferation, and inhibits apoptosis of BM-MSCs. • miR-486 enhances PI3K/AKT activity signaling by targeting PTEN molecule.

  11. Cruciferous vegetable phytochemical sulforaphane affects phase II enzyme expression and activity in rat cardiomyocytes through modulation of Akt signaling pathway.

    PubMed

    Leoncini, Emanuela; Malaguti, Marco; Angeloni, Cristina; Motori, Elisa; Fabbri, Daniele; Hrelia, Silvana

    2011-09-01

    The isothiocyanate sulforaphane (SF), abundant in Cruciferous vegetables, is known to induce antioxidant/detoxification enzymes in many cancer cell lines, but studies focused on its cytoprotective action in nontransformed cells are just at the beginning. Since we previously demonstrated that SF elicits cardioprotection through an indirect antioxidative mechanism, the aim of this study was to analyze the signaling pathways through which SF exerts its protective effects. Using cultured rat cardiomyocytes, we investigated the ability of SF to activate Akt/protein kinase B (PKB) and extracellular signal-regulated kinase 1 and 2 (ERK1/2) signaling pathways, which are implicated in cardiac cell survival, and to increase the phosphorylation of Nuclear factor E2-related factor 2 (Nrf2) and its binding to the antioxidant response element. By means of specific inhibitors, we demonstrated that the Phosphatidylinositol 3-kinase (PI3K)/Akt pathway represents a mechanism through which SF influences both expression and activity of glutathione reductase, glutathione-S-transferase, thioredoxin reductase, and NAD(P)H:quinone oxidoreductase-1, analyzed by western immunoblotting and spectrophotometric assay, respectively, and modulates Nrf2 binding and phosphorylation resulting in a cytoprotective action against oxidative damage. Results of this study confirm the importance of phase II enzymes modulation as cytoprotective mechanism and support the nutritional assumption of Cruciferous vegetables as source of nutraceutical cardioprotective agents. © 2011 Institute of Food Technologists®

  12. {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.

  13. Akt signalling in health and disease.

    PubMed

    Hers, Ingeborg; Vincent, Emma E; Tavaré, Jeremy M

    2011-10-01

    Akt (also known as protein kinase B or PKB) comprises three closely related isoforms Akt1, Akt2 and Akt3 (or PKBα/β/γ respectively). We have a very good understanding of the mechanisms by which Akt isoforms are activated by growth factors and other extracellular stimuli as well as by oncogenic mutations in key upstream regulatory proteins including Ras, PI3-kinase subunits and PTEN. There are also an ever increasing number of Akt substrates being identified that play a role in the regulation of the diverse array of biological effects of activated Akt; this includes the regulation of cell proliferation, survival and metabolism. Dysregulation of Akt leads to diseases of major unmet medical need such as cancer, diabetes, cardiovascular and neurological diseases. As a result there has been substantial investment in the development of small molecular Akt inhibitors that act competitively with ATP or phospholipid binding, or allosterically. In this review we will briefly discuss our current understanding of how Akt isoforms are regulated, the substrate proteins they phosphorylate and how this integrates with the role of Akt in disease. We will furthermore discuss the types of Akt inhibitors that have been developed and are in clinical trials for human cancer, as well as speculate on potential on-target toxicities, such as disturbances of heart and vascular function, metabolism, memory and mood, which should be monitored very carefully during clinical trial.

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

    PubMed Central

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

    2013-01-01

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

  15. Runx2 activates PI3K/Akt signaling via mTORC2 regulation in invasive breast cancer cells

    PubMed Central

    2014-01-01

    Introduction The Runt-related transcription factor Runx2 is critical for skeletal development but is also aberrantly expressed in breast cancers, and promotes cell growth and invasion. A de-regulated serine/threonine kinase Akt signaling pathway is implicated in mammary carcinogenesis and cell survival; however, the mechanisms underlying Runx2 role in survival of invasive breast cancer cells are still unclear. Methods The phenotypic analysis of Runx2 function in cell survival was performed by gene silencing and flow cytometric analysis in highly invasive MDA-MB-231 and SUM-159-PT mammary epithelial cell lines. The expression analysis of Runx2 and pAkt (serine 473) proteins in metastatic breast cancer specimens was performed by immunohistochemistry. The mRNA and protein levels of kinases and phosphatases functional in Akt signaling were determined by real-time PCR and Western blotting, while DNA-protein interaction was studied by chromatin immunoprecipitation assays. Results The high Runx2 levels in invasive mammary epithelial cell lines promoted cell survival in Akt phosphorylation (pAkt-serine 473) dependent manner. The analysis of kinases and phosphatases associated with pAkt regulation revealed that Runx2 promotes pAkt levels via mammalian target of rapamycin complex-2 (mTORC2). The recruitment of Runx2 on mTOR promoter coupled with Runx2-dependent expression of mTORC2 component Rictor defined Runx2 function in pAkt-mediated survival of invasive breast cancer cells. Conclusions Our results identified a novel mechanism of Runx2 regulatory crosstalk in Akt signaling that could have important consequences in targeting invasive breast cancer-associated cell survival. PMID:24479521

  16. Expression of constitutively active Akt/protein kinase B signals GLUT4 translocation in the absence of an intact actin cytoskeleton.

    PubMed

    Eyster, Craig A; Duggins, Quwanza S; Olson, Ann Louise

    2005-05-06

    The actin cytoskeleton has been shown to be required for insulin-dependent GLUT4 translocation; however, the role that the actin network plays is unknown. Actin may play a role in formation of an active signaling complex, or actin may be required for movement of vesicles to the plasma membrane surface. To distinguish between these possibilities, we examined the ability of myr-Akt, a constitutively active form of Akt that signals GLUT4 translocation to the plasma membrane in the absence of insulin, to signal translocation of an HA-GLUT4-GFP reporter protein in the presence or absence of an intact cytoskeleton in 3T3-L1 adipocytes. Expression of myr-Akt signaled the redistribution of the GLUT4 reporter protein to the cell surface in the absence or presence of 10 microm latrunculin B, a concentration sufficient to completely inhibit insulin-dependent redistribution of the GLUT4 reporter to the cell surface. These data suggest that the actin network plays a primary role in organization of the insulin-signaling complex. To further support this conclusion, we measured the activation of known signaling proteins using a saturating concentration of insulin in cells pretreated without or with 10 microm latrunculin B. We found that latrunculin treatment did not affect insulin-dependent tyrosine phosphorylation of the insulin receptor beta-subunit and IRS-1 but completely inhibited activation of Akt/PKB enzymatic activity. Phosphorylation of Akt/PKB at Ser-473 and Thr-308 was inhibited by latrunculin B treatment, indicating that the defect in signaling lies prior to Akt/PKB activation. In summary, our data support the hypothesis that the actin network plays a role in organization of the insulin-signaling complex but is not required for vesicle trafficking and/or fusion.

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

    PubMed

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

    2015-11-01

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

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

    PubMed Central

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

    2015-01-01

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

  19. Regulator of G protein signaling 1 suppresses CXCL12-mediated migration and AKT activation in RPMI 8226 human plasmacytoma cells and plasmablasts.

    PubMed

    Pak, Hyo-Kyung; Gil, Minchan; Lee, Yoonkyung; Lee, Hyunji; Lee, A-Neum; Roh, Jin; Park, Chan-Sik

    2015-01-01

    Migration of plasma cells to the bone marrow is critical factor to humoral immunity and controlled by chemokines. Regulator of G protein signaling 1 (RGS1) is a GTPase-activating protein that controls various crucial functions such as migration. Here, we show that RGS1 controls the chemotactic migration of RPMI 8226 human plasmacytoma cells and human plasmablasts. LPS strongly increased RGS1 expression and retarded the migration of RPMI 8226 cells by suppressing CXCL12-mediated AKT activation. RGS1 knockdown by siRNA abolished the retardation of migration and AKT suppression by LPS. RGS1-dependent regulation of migration via AKT is also observed in cultured plasmablasts. We propose novel functions of RGS1 that suppress AKT activation and the migration of RPMI 8226 cells and plasmablasts in CXCL12-mediated chemotaxis.

  20. 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

  1. AKT/GSK3 signaling pathway and schizophrenia

    PubMed Central

    Emamian, Effat S.

    2012-01-01

    transcription factors related to schizophrenia will be reviewed to see how hypo-activity of AKT signaling pathway may impact such transcriptional mechanisms. PMID:22435049

  2. Betulinic acid protects against cerebral ischemia/reperfusion injury by activating the PI3K/Akt signaling pathway.

    PubMed

    Jiao, Shujie; Zhu, Hongcan; He, Ping; Teng, Junfang

    2016-12-01

    Betulinic acid (BA), a naturally occurring pentacyclic lupane group triterpenoid, has been demonstrated to protect against ischemia/reperfusion-induced renal damage. However, the effects of BA on cerebral ischemia/reperfusion (I/R) injury remain unclear. Hence, this study was to investigate the effects of BA on oxygen and glucose deprivation/reperfusion (OGD/R) induced neuronal injury in rat hippocampal neurons. Our results showed that BA pretreatment greatly attenuated OGD/R-induced neuronal injury. BA also inhibited OGD/R-induced intracellular ROS production and MDA level in rat hippocampal neurons. Furthermore, the down-regulation of Bcl-2, up-regulation of Bax and the consequent activation of caspase-3 induced by OGD/R were reversed by BA pretreatment. Mechanistic studies demonstrated that BA pretreatment up-regulated the expression levels of p-PI3K and p-Akt in hippocampal neurons induced by OGD/R. Taken together, these data suggested that BA inhibits OGD/R-induced neuronal injury in rat hippocampal neurons through the activation of PI3K/Akt signaling pathway.

  3. 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

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

    PubMed

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

    2012-11-01

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

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

    PubMed Central

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

    2012-01-01

    STAT3 and Akt signaling have been validated as potential molecular targets for treatment of cancers including melanoma. These small molecule inhibitors of STAT3 or Akt signaling are promising for developing anti-melanoma therapeutic agents. MLS-2438, a novel 7-bromoindirubin, a derivative of the natural product indirubin, was synthesized with a bromo-group at the 7-position on one indole ring and a hydrophilic group at the 3′-position on the other indole ring. We tested the anticancer activity of MLS-2438 and investigated its mechanism of action in human melanoma cell lines. Here, we show that MLS-2438 inhibits viability and induces apoptosis of human melanoma cells associated with inhibition of STAT3 and Akt signaling. Several pro-apoptotic Bcl-2 family proteins are involved in the MLS-2438 mediated apoptosis. MLS-2438 inhibits Src kinase activity in vitro and phosphorylation of JAK2, Src, STAT3 and Akt in cultured cancer cells. In contrast to the decreased phosphorylation levels of JAK2, Src, STAT3 and Akt, phosphorylation levels of the MAPK (Erk1/2) signaling protein were not reduced in cells treated with MLS-2438. These results demonstrate that MLS-2438, a novel natural product derivative, is a Src inhibitor and potentially regulates kinase activity of JAK2 and Akt in cancer cells. Importantly, MLS-2438 suppressed tumor growth with low toxicity in a mouse xenograft model of human melanoma. Our findings support further development of MLS-2438 as a potential small-molecule therapeutic agent that targets both STAT3 and Akt signaling in human melanoma cells. PMID:22895078

  6. Repression of AKT signaling by ARQ 092 in cells and tissues from patients with Proteus syndrome

    PubMed Central

    Lindhurst, Marjorie J.; Yourick, Miranda R.; Yu, Yi; Savage, Ronald E.; Ferrari, Dora; Biesecker, Leslie G.

    2015-01-01

    A somatic activating mutation in AKT1, c.49G>A, pGlu17Lys, that results in elevated AKT signaling in mutation-positive cells, is responsible for the mosaic overgrowth condition, Proteus syndrome. ARQ 092 is an allosteric pan-AKT inhibitor under development for treatment in cancer. We tested the efficacy of this drug for suppressing AKT signaling in cells and tissues from patients with Proteus syndrome. ARQ 092 reduced phosphorylation of AKT and downstream targets of AKT in a concentration-dependent manner in as little as two hours. While AKT signaling was suppressed with ARQ 092 treatment, cells retained their ability to respond to growth factor stimulation by increasing pAKT levels proportionally to untreated cells. At concentrations sufficient to decrease AKT signaling, little reduction in cell viability was seen. These results indicate that ARQ 092 can suppress AKT signaling and warrants further development as a therapeutic option for patients with Proteus syndrome. PMID:26657992

  7. Repression of AKT signaling by ARQ 092 in cells and tissues from patients with Proteus syndrome.

    PubMed

    Lindhurst, Marjorie J; Yourick, Miranda R; Yu, Yi; Savage, Ronald E; Ferrari, Dora; Biesecker, Leslie G

    2015-12-11

    A somatic activating mutation in AKT1, c.49G>A, pGlu17Lys, that results in elevated AKT signaling in mutation-positive cells, is responsible for the mosaic overgrowth condition, Proteus syndrome. ARQ 092 is an allosteric pan-AKT inhibitor under development for treatment in cancer. We tested the efficacy of this drug for suppressing AKT signaling in cells and tissues from patients with Proteus syndrome. ARQ 092 reduced phosphorylation of AKT and downstream targets of AKT in a concentration-dependent manner in as little as two hours. While AKT signaling was suppressed with ARQ 092 treatment, cells retained their ability to respond to growth factor stimulation by increasing pAKT levels proportionally to untreated cells. At concentrations sufficient to decrease AKT signaling, little reduction in cell viability was seen. These results indicate that ARQ 092 can suppress AKT signaling and warrants further development as a therapeutic option for patients with Proteus syndrome.

  8. FOXO3 induces FOXO1-dependent autophagy by activating the AKT1 signaling pathway

    PubMed Central

    Zhou, Jingyi; Liao, Wenjuan; Yang, Jing; Ma, Ke; Li, Xue; Wang, Yachen; Wang, Donglai; Wang, Lina; Zhang, Yu; Yin, Yuxin; Zhao, Ying; Zhu, Wei-Guo

    2012-01-01

    Forkhead box O (FOXO) transcriptional protein family members, including FOXO1 and FOXO3, are involved in the modulation of autophagy. However, whether there is redundancy between FOXO1 and FOXO3 in the ability to induce autophagy remains unclear. In this study, we showed that FOXO3 induced a transcription-dependent autophagy, and FOXO1 was required for this process. Overexpression of wild-type FOXO3 (WT) or FOXO3 (3A), which harbors alanine mutations at residues Thr32, Ser253 and Ser315, but not transcription-inactive FOXO3 (∆DB3A), significantly induced autophagy in the human embryonic kidney cell line HEK293T and mouse embryonic fibroblast (MEF) cell lines. Interestingly, depletion of FOXO1 by siRNA attenuated FOXO3-induced autophagy. Our data also showed that FOXO3 overexpression did not increase the expression of FOXO1 at the protein level, although FOXO3 was capable of binding the promoter region of FOXO1 and inducing an increase in the transcription of FOXO1 mRNA. Furthermore, our results showed that FOXO3 promoted the translocation of FOXO1 from the nucleus to the cytoplasm, resulting in an increase in FOXO1-induced autophagy. Moreover, our results supported a mechanism whereby FOXO3 dramatically increased the expression of the class I PtdIns3K catalytic subunit PIK3CA, leading to an increase in AKT1 activity, which resulted in the phosphorylation and nuclear export of FOXO1. To the best of our knowledge, our data are the first to suggest that FOXO1 plays a central role in FOXO3-induced autophagy. PMID:22931788

  9. Dodeca-2(E),4(E)-dienoic acid isobutylamide enhances glucose uptake in 3T3-L1 cells via activation of Akt signaling.

    PubMed

    Choi, Kyeong-Mi; Kim, Wonkyun; Hong, Jin Tae; Yoo, Hwan-Soo

    2017-02-01

    Dodeca-2(E),4(E)-dienoic acid isobutylamide (DDI), an alkamide derived from the plant Echinacea purpurea, promotes adipocyte differentiation and activates peroxisome proliferator-activated receptor γ, which is associated with enhanced insulin sensitivity. In the present study, we investigated whether DDI may increase glucose uptake through activation of the insulin signaling pathway in 3T3-L1 adipocytes. DDI increased insulin-stimulated glucose uptake, and expression and translocation of glucose transporter 4 in adipocytes treated with sub-optimal levels of insulin. Additionally, DDI enhanced Akt phosphorylation, whereas phosphoinositide 3-kinase/Akt inhibitors suppressed DDI-induced glucose uptake. These results suggest that DDI may improve insulin sensitivity through the activation of Akt signaling, which leads to enhanced glucose uptake.

  10. Liraglutide reduces lipogenetic signals in visceral adipose of db/db mice with AMPK activation and Akt suppression.

    PubMed

    Shao, Yimin; Yuan, Geheng; Zhang, Junqing; Guo, Xiaohui

    2015-01-01

    Liraglutide, a glucagon-like peptide-1 analog, has been proved to reduce body weight and visceral adipose tissue (VAT) in human studies. In this study, we aimed at examining lipogenetic signal changes in VAT after weight-loss with liraglutide in db/db mice. The mice were divided into two groups: liraglutide-treated group (n=14, 8-week-old, fasting glucose. >10 mmol/L, liraglutide 300 μg/kg twice a day for 4 weeks) and control group (n=14, saline). We found body weight gain and food intake were reduced after liraglutide treatment (P<0.05). Compared to the control group, the VAT weights were significantly lower in the treated group (2.32±0.37 g versus 3.20±0.30 g, P<0.01) than that in control group. In VAT, compared with control group, the lipogenetic transcription factors PPARγ and C/EBPα expressions were both reduced with pAMPK and pACC increased 3.5-fold and 2.31-fold respectively, while pAkt and pP38MAPK were reduced 0.38-fold and 0.62-fold respectively (P<0.01). In conclusion, VAT was reduced after weight loss with AMPK activation and Akt suppression with liraglutide treatment, which was associated with reduction of lipogenetic process in VAT.

  11. 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

  12. Metastasis and AKT activation.

    PubMed

    Sheng, Shijie; Qiao, Meng; Pardee, Arthur B

    2009-03-01

    Metastasis, responsible for 90% of cancer patient deaths, is an inefficient process because many tumor cells die. The survival of metastatic tumor cells should be considered as a critical therapeutic target. This review provides a new perspective regarding the role of AKT in tumor survival, and the rationale to target AKT in anti-metastasis therapies.

  13. Calcium Signaling Involvement in Cadmium-Induced Astrocyte Cytotoxicity and Cell Death Through Activation of MAPK and PI3K/Akt Signaling Pathways.

    PubMed

    Jiang, Jiao Hua; Ge, Guo; Gao, Kai; Pang, Ying; Chai, Rui Chao; Jia, Xi Hua; Kong, Jin Ge; Yu, Albert Cheung-Hoi

    2015-09-01

    Cadmium (Cd), a highly ubiquitous toxic heavy metal, can contaminate the environment, including agricultural soil, water and air, via industrial runoff and other sources of pollution. Cd accumulated in the body via direct exposure or through the food chain results in neurodegeneration and many other diseases. Previous studies on its toxicity in the central nervous system (CNS) focused mainly on neurons. To obtain a more comprehensive understanding of Cd toxicity for the CNS, we investigated how astrocytes respond to acute and chronic Cd exposure and its toxic molecular mechanisms. When primary cultures of cerebral cortical astrocytes incubated with 1-300 μM CdCl2, morphological changes, LDH release and cell death were observed in a time and dose-dependent manner. Further studies demonstrated that acute and chronic Cd treatment phosphorylated JNK, p38 and Akt to different degrees, while ERK1/2 was only phosphorylated under low doses of Cd (10 μM) exposure. Inhibition of JNK and PI3K/Akt, but not of p38, could partially protect astrocyte from cytotoxicity in chronic and acute Cd exposure. Moreover, Cd also induced a strong calcium signal, while BAPTA, a specific intracellular calcium (Ca(2+)) chelator, prevented Cd-induced intracellular increase of calcium levels in astrocytes; inhibited the Cd-induced activation of ERK1/2, JNK, p38 and Akt; and also significantly reduced astrocyte cell death. All of these results suggested that the Cd-Ca(2+)-MAPK and PI3K/Akt signaling pathways were involved in Cd-induced toxicity in astrocytes. This toxicity involvement indicates that these pathways may be exploited as a target for the prevention of Cd-induced neurodegenerative diseases.

  14. 5-LOX Inhibitor Zileuton Reduces Inflammatory Reaction and Ischemic Brain Damage Through the Activation of PI3K/Akt Signaling Pathway.

    PubMed

    Tu, Xian-Kun; Zhang, Hua-Bin; Shi, Song-Sheng; Liang, Ri-Sheng; Wang, Chun-Hua; Chen, Chun-Mei; Yang, Wei-Zhong

    2016-10-01

    Previous studies from our laboratories showed that an anti-inflammatory drug, 5-lipoxygenase inhibitor zileuton, attenuates ischemic brain damage via inhibiting inflammatory reaction. However, it was elusive whether zileuton attenuates inflammatory reaction and ischemic brain damage through the modulation of PI3K/Akt signaling pathway. In the present study, we, for the first time, investigated whether PI3K/Akt pathway was involved in zileuton's anti-inflammatory and neuroprotective properties against brain damage following experimental ischemic stroke. Adult male Sprague-Dawley rats underwent middle cerebral artery occlusion (MCAO), then received treatment with zileuton or vehicle after the onset of ischemia. LY294002 was injected intracerebroventricularly to inhibit the activation of PI3K/Akt signaling pathway selectively. Neurological deficit scores, cerebral infarct volume, morphological characteristic and cerebral water content were assessed 24 h after cerebral ischemia. The enzymatic activity of myeloperoxidase (MPO) was measured 24 h after cerebral ischemia. Expression of p-Akt, t-Akt and COX-2 in ischemic brain were determined by western blot. NF-κB p65 immuno-positive cells in ischemic brain were detected 24 h after cerebral ischemia. The content of TNF-α in blood was examined by ELISA. 5-LOX inhibitor zileuton significantly reduces neurological deficit scores, cerebral infarct volume, cerebral water content, ischemic neuronal injury and the enzymatic activity of MPO, all of which were abolished by LY294002 administration. Zileuton significantly up-regulates the expression of p-Akt, which was inhibited by LY294002 administration. Zileuton significantly down-regulates the over-expression of NF-κB p65 and COX-2, and attenuates the release of TNF-α, all of which were disminished by LY294002 administration. These results suggest that zileuton attenuates ischemic brain damage by inhibiting inflammatory reaction through the activation of PI3K/Akt signaling

  15. 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.

  16. Nontranscriptional activation of PI3K/Akt signaling mediates hypotensive effect following activation of estrogen receptor β in the rostral ventrolateral medulla of rats

    PubMed Central

    2012-01-01

    Background Estrogen acts on the rostral ventrolateral medulla (RVLM), where sympathetic premotor neurons are located, to elicit vasodepressor effects via an estrogen receptor (ER)β-dependent mechanism. We investigated in the present study nontranscriptional mechanism on cardiovascular effects following activation of ERβ in the RVLM, and delineated the involvement of phosphatidylinositol 3-kinase (PI3K)/serine/threonine kinase (Akt) signaling pathway in the effects. Methods In male Sprague–Dawley rats maintained under propofol anesthesia, changes in arterial pressure, heart rate and sympathetic neurogenic vasomotor tone were examined after microinjection bilaterally into RVLM of 17β-estradiol (E2β) or a selective ERα or ERβ agonist. Involvement of ER subtypes and PI3K/Akt signaling pathway in the induced cardiovascular effects were studied using pharmacological tools of antagonists or inhibitors, gene manipulation with antisense oligonucleotide (ASON) or adenovirus-mediated gene transfection. Results Similar to E2β (1 pmol), microinjection of ERβ agonist, diarylpropionitrile (DPN, 1, 2 or 5 pmol), into bilateral RVLM evoked dose-dependent hypotension and reduction in sympathetic neurogenic vasomotor tone. These vasodepressive effects of DPN (2 pmol) were inhibited by ERβ antagonist, R,R-tetrahydrochrysene (50 pmol), ASON against ERβ mRNA (250 pmol), PI3K inhibitor LY294002 (5 pmol), or Akt inhibitor (250 pmol), but not by ERα inhibitor, methyl-piperidino-pyrazole (1 nmol), or transcription inhibitor, actinomycin D (5 or 10 nmol). Gene transfer by microinjection into bilateral RVLM of adenovirus encoding phosphatase and tensin homologues deleted on chromosome 10 (5 × 108 pfu) reversed the vasodepressive effects of DPN. Conclusions Our results indicate that vasodepressive effects following activation of ERβ in RVLM are mediated by nongenomic activation of PI3K/Akt signaling pathway. This study provides new insight in the intracellular signaling

  17. Akt is required for Stat5 activation and mammary differentiation

    PubMed Central

    2010-01-01

    Introduction The Akt pathway plays a central role in regulating cell survival, proliferation and metabolism, and is one of the most commonly activated pathways in human cancer. A role for Akt in epithelial differentiation, however, has not been established. We previously reported that mice lacking Akt1, but not Akt2, exhibit a pronounced metabolic defect during late pregnancy and lactation that results from a failure to upregulate Glut1 as well as several lipid synthetic enzymes. Despite this metabolic defect, however, both Akt1-deficient and Akt2-deficient mice exhibit normal mammary epithelial differentiation and Stat5 activation. Methods In light of the overlapping functions of Akt family members, we considered the possibility that Akt may play an essential role in regulating mammary epithelial development that is not evident in Akt1-deficient mice due to compensation by other Akt isoforms. To address this possibility, we interbred mice bearing targeted deletions in Akt1 and Akt2 and determined the effect on mammary differentiation during pregnancy and lactation. Results Deletion of one allele of Akt2 in Akt1-deficient mice resulted in a severe defect in Stat5 activation during late pregnancy that was accompanied by a global failure of terminal mammary epithelial cell differentiation, as manifested by the near-complete loss in production of the three principal components of milk: lactose, lipid, and milk proteins. This defect was due, in part, to a failure of pregnant Akt1-/-;Akt2+/- mice to upregulate the positive regulator of Prlr-Jak-Stat5 signaling, Id2, or to downregulate the negative regulators of Prlr-Jak-Stat5 signaling, caveolin-1 and Socs2. Conclusions Our findings demonstrate an unexpected requirement for Akt in Prlr-Jak-Stat5 signaling and establish Akt as an essential central regulator of mammary epithelial differentiation and lactation. PMID:20849614

  18. Statins upregulate cystathionine γ-lyase transcription and H2S generation via activating Akt signaling in macrophage.

    PubMed

    Xu, Yuan; Du, Hua-Ping; Li, Jiaojiao; Xu, Ran; Wang, Ya-Li; You, Shou-Jiang; Liu, Huihui; Wang, Fen; Cao, Yong-Jun; Liu, Chun-Feng; Hu, Li-Fang

    2014-09-01

    Hydrogen sulfide (H2S), the third gaseous transmitter, is implicated in various pathophysiologic processes. In the cardiovascular system, H2S exerts effects of cardioprotection, vascular tone regulation, and atherogenesis inhibition. Recent studies demonstrated that atorvastatin, the inhibitor of 3-hydroxyl-3-methyl coenzyme A reductase, affected H2S formation in kidney and other organs. However, the underlying mechanisms are not fully understood. In this study, we examined the effects of three different statins (fluvastatin, atorvastatin and pravastatin) on H2S formation in raw264.7 macrophages. There was a remarkable rise in H2S level in fluvastatin- and atorvastatin-stimulated macrophages, while pravastatin failed to show any significant effect on it. Moreover, fluvastatin and atorvastatin enhanced the mRNA and protein expression of cystathionine γ-lyase (CSE) in dose- and time-dependent manners. Fluvastatin also markedly enhanced the CSE activity. However, fluvastatin did not alter the mRNA or protein expression of another H2S-producing enzyme 3-mercaptopyruvate sulfurtransferase. Blockade of CSE with its inhibitor dl-propargylglycine (PAG) or siRNA markedly reduced the H2S level in fluvastatin-stimulated macrophages. In addition, fluvastatin elevated Akt phosphorylation, which occurred as early as 15 min after treatment, peaked at 1h, and lasted at least 3h. Both PI3K inhibitor LY294002 (10 μM) and Akt inhibitor perifosine (10μM) were able to reverse the increases of CSE mRNA and H2S production in fluvastatin-stimulated macrophages. Last, we showed that fluvastatin reduced the mRNA levels of pro-inflammatory molecules such as IL-1β and MCP-1 in LPS-treated macrophages, which were completely reversed by CSE inhibitor PAG. Taken together, the findings demonstrate that statins may up-regulate CSE expression/activity and subsequently elevate H2S generation by activating Akt signaling pathway and also imply that CSE-H2S pathway plays a critical role in the anti

  19. Akt phosphorylates and regulates the osteogenic activity of Osterix.

    PubMed

    Choi, You Hee; Jeong, Hyung Min; Jin, Yun-Hye; Li, Hongyan; Yeo, Chang-Yeol; Lee, Kwang-Youl

    2011-08-05

    Osterix (Osx), a zinc-finger transcription factor is required for osteoblast differentiation and new bone formation during embryonic development. Akt is a member of the serine/threonine-specific protein kinase and plays important roles in osteoblast differentiation. The function of Osterix can be also modulated by post-translational modification. But, the precise molecular signaling mechanisms between Osterix and Akt are not known. In this study, we investigated the potential regulation of Osterix function by Akt in osteoblast differentiation. We found that Akt phosphorylates Osterix and that Akt activation increases protein stability, osteogenic activity and transcriptional activity of Osterix. We also found that BMP-2 increases the protein level of Osterix in an Akt activity-dependent manner. These results suggest that Akt activity enhances the osteogenic function of Osterix, at least in part, through protein stabilization and that BMP-2 regulates the osteogenic function of Osterix, at least in part, through Akt.

  20. Sericin can reduce hippocampal neuronal apoptosis by activating the Akt signal transduction pathway in a rat model of diabetes mellitus☆

    PubMed Central

    Chen, Zhihong; He, Yaqiang; Song, Chengjun; Dong, Zhijun; Su, Zhejun; Xue, Jingfeng

    2012-01-01

    In the present study, a rat model of type 2 diabetes mellitus was established by continuous peritoneal injection of streptozotocin. Following intragastric perfusion of sericin for 35 days, blood glucose levels significantly reduced, neuronal apoptosis in the hippocampal CA1 region decreased, hippocampal phosphorylated Akt and nuclear factor kappa B expression were enhanced, but Bcl-xL/Bcl-2 associated death promoter expression decreased. Results demonstrated that sericin can reduce hippocampal neuronal apoptosis in a rat model of diabetes mellitus by regulating abnormal changes in the Akt signal transduction pathway. PMID:25767499

  1. GDNF-independent ureteric budding: role of PI3K-independent activation of AKT and FOSB/JUN/AP-1 signaling

    PubMed Central

    Tee, James B.; Choi, Yohan; Dnyanmote, Ankur; Decambre, Marvalyn; Ito, Chiharu; Bush, Kevin T.; Nigam, Sanjay K.

    2013-01-01

    Summary A significant fraction of mice deficient in either glial cell-derived neurotrophic factor (GDNF) or its co-receptors (Gfrα1, Ret), undergoes ureteric bud (UB) outgrowth leading to the formation of a rudimentary kidney. Previous studies using the isolated Wolffian duct (WD) culture indicate that activation of fibroblast growth factor (FGF) receptor signaling, together with suppression of BMP/Activin signaling, is critical for GDNF-independent WD budding (Maeshima et al., 2007). By expression analysis of embryonic kidney from Ret(−/−) mice, we found the upregulation of several FGFs, including FGF7. To examine the intracellular pathways, we then analyzed GDNF-dependent and GDNF-independent budding in the isolated WD culture. In both conditions, Akt activation was found to be important; however, whereas this occurred through PI3-kinase in GDNF-dependent budding, in the case of GDNF-independent budding, Akt activation was apparently via a PI3-kinase independent mechanism. Jnk signaling and the AP-1 transcription factor complex were also implicated in GDNF-independent budding. FosB, a binding partner of c-Jun in the formation of AP-1, was the most highly upregulated gene in the ret knockout kidney (in which budding had still occurred), and we found that its siRNA-mediated knockdown in isolated WDs also blocked GDNF-independent budding. Taken together with the finding that inhibition of Jnk signaling does not block Akt activation/phosphorylation in GDNF-independent budding, the data support necessary roles for both FosB/Jun/AP-1 signaling and PI3-kinase-independent activation of Akt in GDNF-independent budding. A model is proposed for signaling events that involve Akt and JNK working to regulate GDNF-independent WD budding. PMID:24143282

  2. GDNF-independent ureteric budding: role of PI3K-independent activation of AKT and FOSB/JUN/AP-1 signaling.

    PubMed

    Tee, James B; Choi, Yohan; Dnyanmote, Ankur; Decambre, Marvalyn; Ito, Chiharu; Bush, Kevin T; Nigam, Sanjay K

    2013-01-01

    A significant fraction of mice deficient in either glial cell-derived neurotrophic factor (GDNF) or its co-receptors (Gfrα1, Ret), undergoes ureteric bud (UB) outgrowth leading to the formation of a rudimentary kidney. Previous studies using the isolated Wolffian duct (WD) culture indicate that activation of fibroblast growth factor (FGF) receptor signaling, together with suppression of BMP/Activin signaling, is critical for GDNF-independent WD budding (Maeshima et al., 2007). By expression analysis of embryonic kidney from Ret((-/-)) mice, we found the upregulation of several FGFs, including FGF7. To examine the intracellular pathways, we then analyzed GDNF-dependent and GDNF-independent budding in the isolated WD culture. In both conditions, Akt activation was found to be important; however, whereas this occurred through PI3-kinase in GDNF-dependent budding, in the case of GDNF-independent budding, Akt activation was apparently via a PI3-kinase independent mechanism. Jnk signaling and the AP-1 transcription factor complex were also implicated in GDNF-independent budding. FosB, a binding partner of c-Jun in the formation of AP-1, was the most highly upregulated gene in the ret knockout kidney (in which budding had still occurred), and we found that its siRNA-mediated knockdown in isolated WDs also blocked GDNF-independent budding. Taken together with the finding that inhibition of Jnk signaling does not block Akt activation/phosphorylation in GDNF-independent budding, the data support necessary roles for both FosB/Jun/AP-1 signaling and PI3-kinase-independent activation of Akt in GDNF-independent budding. A model is proposed for signaling events that involve Akt and JNK working to regulate GDNF-independent WD budding.

  3. 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

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

    PubMed

    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

    2016-02-19

    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.

  5. Mefloquine exerts anticancer activity in prostate cancer cells via ROS-mediated modulation of Akt, ERK, JNK and AMPK signaling

    PubMed Central

    YAN, KUN-HUANG; YAO, CHIH-JUNG; HSIAO, CHI-HAO; LIN, KE-HSUN; LIN, YUNG-WEI; WEN, YU-CHING; LIU, CHUNG-CHI; YAN, MING-DE; CHUANG, SHUANG-EN; LAI, GI-MING; LEE, LIANG-MING

    2013-01-01

    Mefloquine (MQ) is a prophylactic anti-malarial drug. Previous studies have shown that MQ induces oxidative stress in vitro. Evidence indicates that reactive oxygen species (ROS) may be used as a therapeutic modality to kill cancer cells. This study investigated whether MQ also inhibits prostate cancer (PCa) cell growth. We used sulforhodamine B (SRB) staining to determine cell viability. MQ has a highly selective cytotoxicity that inhibits PCa cell growth. The antitumor effect was most significant when examined using a colony formation assay. MQ also induces hyperpolarization of the mitochondrial membrane potential (MMP), as well as ROS generation. The blockade of MQ-induced anticancer effects by N-acetyl cysteine (NAC) pre-treatment confirmed the role of ROS. This indicates that the MQ-induced anticancer effects are caused primarily by increased ROS generation. Moreover, we observed that MQ-mediated ROS simultaneously downregulated Akt phosphorylation and activated extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK) and adenosine monophosphate-activated protein kinase (AMPK) signaling in PC3 cells. These findings provide insights for further anticancer therapeutic options. PMID:23760395

  6. 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.

  7. 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

  8. Calcium oxalate crystals induces tight junction disruption in distal renal tubular epithelial cells by activating ROS/Akt/p38 MAPK signaling pathway.

    PubMed

    Yu, Lei; Gan, Xiuguo; Liu, Xukun; An, Ruihua

    2017-11-01

    Tight junction plays important roles in regulating paracellular transports and maintaining cell polarity. Calcium oxalate monohydrate (COM) crystals, the major crystalline composition of kidney stones, have been demonstrated to be able to cause tight junction disruption to accelerate renal cell injury. However, the cellular signaling involved in COM crystal-induced tight junction disruption remains largely to be investigated. In the present study, we proved that COM crystals induced tight junction disruption by activating ROS/Akt/p38 MAPK pathway. Treating Madin-Darby canine kidney (MDCK) cells with COM crystals induced a substantial increasing of ROS generation and activation of Akt that triggered subsequential activation of ASK1 and p38 mitogen-activated protein kinase (MAPK). Western blot revealed a significantly decreased expression of ZO-1 and occludin, two important structural proteins of tight junction. Besides, redistribution and dissociation of ZO-1 were observed by COM crystals treatment. Inhibition of ROS by N-acetyl-l-cysteine (NAC) attenuated the activation of Akt, ASK1, p38 MAPK, and down-regulation of ZO-1 and occludin. The redistribution and dissociation of ZO-1 were also alleviated by NAC treatment. These results indicated that ROS were involved in the regulation of tight junction disruption induced by COM crystals. In addition, the down-regulation of ZO-1 and occludin, the phosphorylation of ASK1 and p38 MAPK were also attenuated by MK-2206, an inhibitor of Akt kinase, implying Akt was involved in the disruption of tight junction upstream of p38 MAPK. Thus, these results suggested that ROS-Akt-p38 MAPK signaling pathway was activated in COM crystal-induced disruption of tight junction in MDCK cells.

  9. Pressure activates Src-dependent FAK-Akt and ERK1/2 signaling pathways in rat hepatic stellate cells.

    PubMed

    Wu, Han-Jun; Zhang, Zong-Qi; Yu, Bang; Liu, Su; Qin, Kai-Rong; Zhu, Liang

    2010-01-01

    Hepatic fibrosis is associated with elevated sinusoidal pressure, which can be transmitted to the hepatic stellate cells (HSCs) in the perisinusoidal space of Disse. Here, we sought to determine the effects of pressure on cellular growth and Src-dependent signaling pathways in the rat HSCs. Cultured rat HSCs were exposed to pressures (0 to 80 mmHg) by using a pressure-inducing apparatus. The proliferation of the cells was determined by a 5-bromo-2'-deoxy-uridine (BrdU) incorporation assay. Reverse transcription-PCR and Western-blot analysis were used to examine the mRNA and protein levels of representative molecules in Src-dependent signaling pathways. Pressure at 10 to 20 mmHg applied to the HSCs over 1 h upregulated Brdu incorporation and expression of proliferating cell nuclear antigen (PCNA) and type I collagen, while a higher pressure of 40-80 mmHg did not have noticeable effect. The mRNA level of beta (3) integrin was increased by 1-h application of 5 to 20 mmHg. Immunoblot with phospho-specific antibodies demonstrated the phosphorylation of Src (Tyr418), focal adhesion kinase (FAK) (Tyr397), Akt (Ser473) and extracellular signal-regulated kinases 1 and 2 (ERK1/2) (Thr421/Ser424) was increased in response to 10-mmHg pressure. Herbimycin A, an inhibitor of Src phosphorylation, attenuated the pressure-induced HSC proliferation and phosphorylation of above-mentioned signaling molecules. Our data demonstrated that pressure alone induced HSC proliferation involving the activation of Src-dependent signaling pathways. Copyright 2010 S. Karger AG, Basel.

  10. Isoflurane Promotes Non-Small Cell Lung Cancer Malignancy by Activating the Akt-Mammalian Target of Rapamycin (mTOR) Signaling Pathway

    PubMed Central

    Zhang, Wenhua; Shao, Xueqian

    2016-01-01

    Background Lung cancer is one of the leading causes of cancer mortalities worldwide, and non-small cell lung cancer (NSCLC) accounts for the majority of all lung cancer cases. Surgery remains one of the front-line treatment options for NSCLC, but events within the perioperative period were found to affect cancer prognosis, such as anesthesia procedures. Isoflurane, a commonly used volatile anesthetic, enhances the malignant potential of renal, prostate, and ovarian cancer cells, but its effects on NSCLC development have not been previously reported. Material/Methods CCK-8 and MTT cell proliferation assays were used to analyze NSCLC cell proliferation. Metastatic ability was examined by wound healing and transwell assays. We used Western blot analysis to study the mechanism of effect of Isoflurane in NSCLC development. Results We demonstrated that isoflurane promotes proliferation, migration and invasiveness of NSCLC cells, as well as upregulation of the Akt-mTOR signaling pathway in NSCLC cells. Pharmacological inhibition of Akt-mTOR signaling abolished the ability of isoflurane to promote proliferation, migration, and invasion of NSCLC cells, indicating that isoflurane promotes NSCLC cell malignancy by activating the Akt-mTOR signaling pathway. Conclusions Isoflurane promotes NSCLC proliferation, migration and invasion by activating the Akt-mTOR signaling pathway. PMID:27897153

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

    PubMed

    Um, Moonkyoung; Lodish, Harvey F

    2006-03-03

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

  12. Phosphatidylserine is a critical modulator for Akt activation

    PubMed Central

    Huang, Bill X.; Akbar, Mohammed; Kevala, Karl

    2011-01-01

    Akt activation relies on the binding of Akt to phosphatidylinositol-3,4,5-trisphosphate (PIP3) in the membrane. Here, we demonstrate that Akt activation requires not only PIP3 but also membrane phosphatidylserine (PS). The extent of insulin-like growth factor–induced Akt activation and downstream signaling as well as cell survival under serum starvation conditions positively correlates with plasma membrane PS levels in living cells. PS promotes Akt-PIP3 binding, participates in PIP3-induced Akt interdomain conformational changes for T308 phosphorylation, and causes an open conformation that allows for S473 phosphorylation by mTORC2. PS interacts with specific residues in the pleckstrin homology (PH) and regulatory (RD) domains of Akt. Disruption of PS–Akt interaction by mutation impairs Akt signaling and increases susceptibility to cell death. These data identify a critical function of PS for Akt activation and cell survival, particularly in conditions with limited PIP3 availability. The novel molecular interaction mechanism for Akt activation suggests potential new targets for controlling Akt-dependent cell survival and proliferation. PMID:21402788

  13. Signaling Networks Associated with AKT Activation in Non-Small Cell Lung Cancer (NSCLC): New Insights on the Role of Phosphatydil-Inositol-3 kinase

    PubMed Central

    Scrima, Marianna; De Marco, Carmela; Fabiani, Fernanda; Franco, Renato; Pirozzi, Giuseppe; Rocco, Gaetano; Ravo, Maria; Weisz, Alessandro; Zoppoli, Pietro; Ceccarelli, Michele; Botti, Gerardo; Malanga, Donatella; Viglietto, Giuseppe

    2012-01-01

    Aberrant activation of PI3K/AKT signalling represents one of the most common molecular alterations in lung cancer, though the relative contribution of the single components of the cascade to the NSCLC development is still poorly defined. In this manuscript we have investigated the relationship between expression and genetic alterations of the components of the PI3K/AKT pathway [KRAS, the catalytic subunit of PI3K (p110α), PTEN, AKT1 and AKT2] and the activation of AKT in 107 surgically resected NSCLCs and have analyzed the existing relationships with clinico-pathologic features. Expression analysis was performed by immunohistochemistry on Tissue Micro Arrays (TMA); mutation analysis was performed by DNA sequencing; copy number variation was determined by FISH. We report that activation of PI3K/AKT pathway in Italian NSCLC patients is associated with high grade (G3–G4 compared with G1–G2; n = 83; p<0.05) and more advanced disease (TNM stage III vs. stages I and II; n = 26; p<0.05). In addition, we found that PTEN loss (41/104, 39%) and the overexpression of p110α (27/92, 29%) represent the most frequent aberration observed in NSCLCs. Less frequent molecular lesions comprised the overexpression of AKT2 (18/83, 22%) or AKT1 (17/96, 18%), and KRAS mutation (7/63, 11%). Our results indicate that, among all genes, only p110α overexpression was significantly associated to AKT activation in NSCLCs (p = 0.02). Manipulation of p110α expression in lung cancer cells carrying an active PI3K allele (NCI-H460) efficiently reduced proliferation of NSCLC cells in vitro and tumour growth in vivo. Finally, RNA profiling of lung epithelial cells (BEAS-2B) expressing a mutant allele of PIK3 (E545K) identified a network of transcription factors such as MYC, FOS and HMGA1, not previously recognised to be associated with aberrant PI3K signalling in lung cancer. PMID:22363436

  14. Sevoflurane Postconditioning Protects Rat Hearts against Ischemia-Reperfusion Injury via the Activation of PI3K/AKT/mTOR Signaling

    PubMed Central

    Zhang, Jing; Wang, Chen; Yu, Shuchun; Luo, Zhenzhong; Chen, Yong; Liu, Qin; Hua, Fuzhou; Xu, Guohai; Yu, Peng

    2014-01-01

    Phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) pathway plays a key role in myocardial ischemia-reperfusion (I/R) injury. Mammalian target of rapamycin (mTOR), a downstream target of PI3K/AKT signaling, is necessary and sufficient to protect the heart from I/R injury. Inhaled anesthetic sevoflurane is widely used in cardiac surgeries because its induction and recovery are faster and smoother than other inhaled anesthetics. Sevoflurane proved capable of inducing postconditioning effects in the myocardium. However, the underlying molecular mechanisms for sevoflurane-induced postconditioning (SPC) were largely unclear. In the present study, we demonstrated that SPC protects myocardium from I/R injury with narrowed cardiac infarct focus, increased ATP content, and decreased cardiomyocyte apoptosis, which are mainly due to the activation of PI3K/AKT/mTOR signaling and the protection of mitochondrial energy metabolism. Application of dactolisib (BEZ235), a PI3K/mTOR dual inhibitor, abolishes the up-regulation of pho-AKT, pho-GSK, pho-mTOR, and pho-p70s6k induced by SPC, hence abrogating the anti-apoptotic effect of sevoflurane and reducing SPC-mediated protection of heart from I/R injury. As such, this study proved that PI3K/AKT/mTOR pathway plays an important role in SPC induced cardiac protection against I/R injury. PMID:25471136

  15. TC21 mediates transformation and cell survival via activation of phosphatidylinositol 3-kinase/Akt and NF-kappaB signaling pathway.

    PubMed

    Rong, Rong; He, Qin; Liu, Yusen; Sheikh, M Saeed; Huang, Ying

    2002-02-07

    The signaling pathways of TC21-mediated transformation and cell survival are not well-established. In this study, we have investigated the role of PI3-K/Akt signaling pathway in oncogenic-TC21-mediated transformation and cell survival. We found that oncogenic-TC21 stimulated the PI3-K activity. This was associated with the activation of Akt, a key component of PI3-K signaling pathway. We also found that TC21 interacted and formed complex with PI3-K. Mutations in the GTP-binding region of TC21, which enhanced GTP-binding potential of this protein, also stimulated its association with PI3-K, suggesting that PI3-K may preferentially interact with the GTP-bound form. Suppression of PI3-K and Akt by specific inhibitors LY294002 and Wortmannin reversed TC21-induced transformation. Likewise, inhibition of PI3-K activity by the PI3-K phosphotase PTEN reduced TC21-mediated focus formation in NIH3T3 cells. Investigation of TC21's effect on cell survival revealed that mutant-TC21 expressing cells were more resistant to etoposide- and cisplatin-induced cell death, and this was associated with the activation of anti-apoptotic protein NF-kappaB, a downstream target of Akt. Treatment of PI3-K inhibitor LY294002 significantly suppressed TC21-mediated NF-kappaB activation. In conclusion, we have identified PI3-K as an effector of TC21 and demonstrated that the PI3-K/Akt signaling pathway plays important roles in TC21-mediated transformation and cell survival.

  16. Galangin Activates the ERK/AKT-Driven Nrf2 Signaling Pathway to Increase the Level of Reduced Glutathione in Human Keratinocytes.

    PubMed

    Madduma Hewage, Susara Ruwan Kumara; Piao, Mei Jing; Kang, Kyoung Ah; Ryu, Yea Seong; Fernando, Pattage Madushan Dilhara Jayatissa; Oh, Min Chang; Park, Jeong Eon; Shilnikova, Kristina; Moon, Yu Jin; Shin, Dae O; Hyun, Jin Won

    2016-11-08

    Previously, we demonstrated that galangin (3,5,7-trihydroxyflavone) protects human keratinocytes against ultraviolet B (UVB)-induced oxidative damage. In this study, we investigated the effect of galangin on induction of antioxidant enzymes involved in synthesis of reduced glutathione (GSH), and investigated the associated upstream signaling cascades. By activating nuclear factor-erythroid 2-related factor (Nrf2), galangin treatment significantly increased expression of glutamate-cysteine ligase catalytic subunit (GCLC) and glutathione synthetase (GSS). This activation of Nrf2 depended on extracellular signal-regulated kinases (ERKs) and protein kinase B (AKT) signaling. Inhibition of GSH in galangin-treated cells attenuated the protective effect of galangin against the deleterious effects of UVB. Our results reveal that galangin protects human keratinocytes by activating ERK/AKT-Nrf2, leading to elevated expression of GSH-synthesizing enzymes.

  17. SMND-309 promotes neuron survival through the activation of the PI3K/Akt/CREB-signalling pathway.

    PubMed

    Wang, Youlei; Zhang, Jinjin; Han, Meng; Liu, Bo; Gao, Yulin; Ma, Peng; Zhang, Songzi; Zheng, Qingyin; Song, Xiaodong

    2016-10-01

    Context In clinical practice, the promotion of neuron survival is necessary to recover neurological functions after the onset of stroke. Objective This study aimed to investigate the post-ischaemic neuroprotective effect of SMND-309, a novel metabolite of salvianolic acid, on differentiated SH-SY5Y cells. Materials and methods SH-SY5Y cells were differentiated by pre-treating with 5 μM all-trans-retinoic acid for 6 d. The differentiated SH-SY5Y cells were exposed to oxygen-glucose deprivation (OGD) for 2 h and reperfusion (R) for 24 h to induce OGD/R injury. After OGD injury, differentiated SH-SY5Y cells were treated with or without SMND-309 (5, 10, 20 μM) for another 24 h. Cell viability was detected through Cell counting kit-8 assay and lactate dehydrogenase leakage assay. Apoptosis was evaluated through flow cytometry, caspase-3 activity assay. Changes in protein levels were assessed through Western blot. Results SMND-309 ameliorated the degree of injury in the differentiated SH-SY5Y cells by increasing cell viabilities (5 μM, 65.4% ± 4.1%; 10 μM, 69.8% ± 3.7%; 20 μM, 75.3% ± 5.1%) and by reducing LDH activity (20 μM, 2.5 fold) upon OGD/R stimulation. Annexin V-fluorescein isothiocyanate/propidium iodide staining results suggested that apoptotic rate of differentiated SH-SY5Y cells decreased from 43.8% induced by OGD/R injury to 19.2% when the cells were treated with 20 μM SMND-309. SMND-309 significantly increased the Bcl-2 level of the injured differentiated SH-SY5Y cells but decreased the caspase-3 activity of these cells by 1.6-fold. In contrast, SMND-309 did not affect the Bax level of these cells. SMND-309 evidently increased the protein expression of BDNF when Akt and CREB were activated. This function was antagonized by the addition of LY294002. Conclusion SMND-309 can prevent neuronal cell death in vitro. This process may be related to the activation of the PI3K/Akt/CREB-signalling pathway.

  18. NEDD4 ubiquitin ligase is a putative oncogene in endometrial cancer that activates IGF-1R/PI3K/Akt signaling.

    PubMed

    Zhang, Yuping; Goodfellow, Renee; Li, Yujun; Yang, Shujie; Winters, Christopher J; Thiel, Kristina W; Leslie, Kimberly K; Yang, Baoli

    2015-10-01

    The PI3K/Akt pathway is frequently dysregulated in endometrial cancer, the most common gynecologic malignancy. Emerging evidence identifies the ubiquitin ligase NEDD4 as a key regulator of the PI3K/Akt pathway via activation of insulin-like growth factor-1 receptor (IGF-1R). Our objective was to understand the role of NEDD4 in endometrial cancer. NEDD4 expression was assessed by immunohistochemistry in a tissue microarray with 77 endometrial lesions ranging from normal benign endometrium to tumor specimens of varying stage and grade. Studies were extended to a panel of eight endometrial cancer cell lines phenotypically representing the most common endometrial patient tumors. Immunohistochemistry demonstrated robust staining of NEDD4 in endometrial tumor specimens, with greater NEDD4 expression in the most aggressive tumors. Expression of NEDD4 was detected in a majority of endometrial cancer cell lines surveyed. Exogenous overexpression of murine Nedd4 in endometrial cancer cell lines with modest endogenous NEDD4 expression resulted in a significant increase in the rate of proliferation. Nedd4 overexpression also promoted an increase in cell surface localization of IGF-1R and activation of Akt. Inhibition of PI3K/Akt signaling reversed the enhanced cell growth in Nedd4-overexpressing endometrial cancer cells. In addition, the expression of NEDD4 in endometrial tumors positively correlated with the Akt downstream effector FoxM1. This study identifies NEDD4 as a putative oncogene in endometrial cancer that may augment activation of the IGF-1R/PI3K/Akt signaling pathway. Copyright © 2015 Elsevier Inc. All rights reserved.

  19. Asiatic acid enhances Nrf2 signaling to protect HepG2 cells from oxidative damage through Akt and ERK activation.

    PubMed

    Qi, Zhimin; Ci, Xinxin; Huang, Jingbo; Liu, Qinmei; Yu, Qinlei; Zhou, Junfeng; Deng, Xuming

    2017-04-01

    Asiatic acid (AA), a natural triterpene isolated from the plant Centella asiatica, have antioxidative potential, but the molecular mechanism of AA against oxidative stress remains unclear. Our study was performed to investigate the antioxidative effect of AA against oxidative stress and the antioxidative mechanism in tert-butyl hydroperoxide (t-BHP) -stimulated the HepG2 cells. The results showed that AA suppressed t-BHP-induced cytotoxicity, apoptosis, and reactive oxygen species (ROS) generation. Additionally, AA activated the nuclear factor erythroid 2-related factor 2 (Nrf2) signal, which was closely related to induction Nrf2 nuclear translocation, reduction the expression of Keap1 and up-regulation the activity of the antioxidant response element (ARE). Meanwhile, activation of Nrf2 signal upregulated the protein expressions of antioxidant genes, including heme oxygenase-1 (HO-1), NAD(P)H: quinone oxidase (NQO-1), and glutamyl cysteine ligase catalytic subunit (GCLC). Excitingly, Knockout of Nrf2 almost abolished AA-mediated antioxidant activity and cytoprotection against t-BHP. Further studies showed the mechanism underlying that AA induced Nrf2 activation in HepG2 cells via Akt and ERK signal activation. We found Akt and ERK inhibitors treatment attenuated AA-mediated Nrf2 nuclear translocation. Furthermore, treatment with either Akt or ERK inhibitor also decreased AA-mediated cytoprotection against t-BHP-induced cellular damage. Collectively, these results presented in this study indicate that AA has the protective effect against t-BHP-induced cellular damage and oxidative stress by modulating Nrf2 signaling through activating the signals of Akt and ERK.

  20. PI3K is an upstream regulator of the PDE3B pathway of leptin signaling that may not involve activation of Akt in the rat hypothalamus

    PubMed Central

    Sahu, Abhiram; Koshinaka, Keiichi; Sahu, Maitrayee

    2012-01-01

    Leptin, the product of the obese gene, regulates energy homeostasis by acting primarily at the level of the hypothalamus. Leptin action through its receptor involves various pathways including the signal transducer and activator of transcription (STAT3), phosphatidylinositol 3-kinase (PI3K), and phosphodiesterase 3B (PDE3B)-cAMP signaling in the CNS and peripheral tissues. In the hypothalamus, leptin stimulates STAT3 activation, and induces PI3K and PDE3B activities, among others. We have previously demonstrated that PDE3B activation in the hypothalamus is critical for transducing anorectic and body weight reducing effects of leptin. Similarly, PI3K has been implicated toplay a critical role in leptin signaling in the hypothalamus. Whereas in insulin signaling pathway, PI3K is known to be an upstream regulator of PDE3B in non-neuronal tissues, it is still unknown whether this is also the case for leptin signaling in the hypothalamus. To address this possibility, the effect of wortmannin, a specific PI3K inhibitor, was examined on the leptin-induced PDE3B activity in the hypothalamus of male rats. Intracerebroventricular (icv) injection of leptin (4 μg) significantly increased PDE3B activity by 2-fold in the hypothalamus as expected. However, prior administration of wortmannin completely reversed the stimulatory effect of leptin on PDE3B activity in the hypothalamus. To demonstrate whether leptin stimulates p-Akt levels and there by a possible upstream regulator of PDE3B, we examined the effects of icv leptin on p-Akt levels in the hypothalamus and compared that with the known stimulatory effect of insulin on p-Akt. We observed that insulin increased p-Akt levels but leptin failed to do so although it increased p-STAT3 levels in the rat hypothalamus. Immunocytochemistry confirmed the biochemical finding in that leptin failed but insulin increased the number of p-Akt positive cells in various hypothalamic nuclei. Altogether these results implicate PI3K but not Akt

  1. Berberine Induced Apoptosis of Human Osteosarcoma Cells by Inhibiting Phosphoinositide 3 Kinase/Protein Kinase B (PI3K/Akt) Signal Pathway Activation.

    PubMed

    Chen, Zhi-Ze

    2016-05-01

    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. 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. 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. Berberine can suppress the proliferation and induce the apoptosis of U2OS cell through inhibiting the PI3K/Akt signaling pathway activation.

  2. 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

  3. 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.

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

    PubMed

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

    2016-09-01

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

  5. 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

  6. AKT Regulates BRCA1 Stability in Response to Hormone Signaling

    PubMed Central

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

    2015-01-01

    BRCA1, with its binding partner BARD1, regulates the cellular response to DNA damage in multiple tissues, yet inherited mutations within BRCA1 result specifically in breast and ovarian cancers. This observation, along with several other lines of evidence, suggests a functional relationship may exist between hormone signaling and BRCA1 function. Our data demonstrates that AKT activation promotes the expression of BRCA1 in response to estrogen and IGF-1 receptor signaling. Further, we have identified a novel AKT phosphorylation site in BRCA1 at S694 which is responsive to activation of these signaling pathways. This rapid increase in BRCA1 protein levels appears to occur independently of new protein synthesis and treatment with the clinically utilized proteasome inhibitor bortezomib similarly leads to a rapid increase in BRCA1 protein levels. Together, these data suggest that AKT phosphorylation of BRCA1 increases total protein expression by preventing proteasomal degradation. AKT activation also appears to support nuclear localization of BRCA1, and co-expression of activated AKT with BRCA1 decreases radiation sensitivity, suggesting this interaction has functional consequences for BRCA1's role in DNA repair. We conclude that AKT regulates BRCA1 protein stability and function through direct phosphorylation of BRCA1. Further, the responsiveness of the AKT-BRCA1 regulatory pathway to hormone signaling may, in part, underlie the tissue specificity of BRCA1 mutant cancers. Pharmacological targets within this pathway could provide strategies for modulation of BRCA1 protein, which may prove therapeutically beneficial for the treatment of breast and ovarian cancers. PMID:20085797

  7. 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

  8. Activation of PI3K/Akt signaling in rostral ventrolateral medulla impairs brain stem cardiovascular regulation that underpins circulatory depression during mevinphos intoxication.

    PubMed

    Tsai, Ching-Yi; Chang, Alice Y W; Chan, Julie Y H; Chan, Samuel H H

    2014-03-01

    As the most widely used pesticides in the globe, the organophosphate compounds are understandably linked with the highest incidence of suicidal poisoning. Whereas the elicited toxicity is often associated with circulatory depression, the underlying mechanisms require further delineation. Employing the pesticide mevinphos as our experimental tool, we evaluated the hypothesis that transcriptional upregulation of nitric oxide synthase II (NOS II) by NF-κB on activation of the PI3K/Akt cascade in the rostral ventrolateral medulla (RVLM), the brain stem site that maintains blood pressure and sympathetic vasomotor tone, underpins the circulatory depressive effects of organophosphate poisons. Microinjection of mevinphos (10 nmol) bilaterally into the RVLM of anesthetized Sprague-Dawley rats induced a progressive hypotension that was accompanied sequentially by an increase (Phase I) and a decrease (Phase II) of an experimental index for the baroreflex-mediated sympathetic vasomotor tone. There were also progressive augmentations in PI3K or Akt enzyme activity and phosphorylation of p85 or Akt(Thr308) subunit in the RVLM that were causally related to an increase in NF-κB transcription activity and elevation in NOS II or peroxynitrite expression. Loss-of-function manipulations of PI3K or Akt in the RVLM significantly antagonized the reduced baroreflex-mediated sympathetic vasomotor tone and hypotension during Phase II mevinphos intoxication, and blunted the increase in NF-κB/NOS II/peroxynitrite signaling. We conclude that activation of the PI3K/Akt cascade, leading to upregulation of NF-κB/NOS II/peroxynitrite signaling in the RVLM, elicits impairment of brain stem cardiovascular regulation that underpins circulatory depression during mevinphos intoxication.

  9. 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

  10. Compensation of the AKT signaling by ERK signaling in transgenic mice hearts overexpressing TRIM72

    SciTech Connect

    Ham, Young-Mi; Mahoney, Sarah Jane

    2013-06-10

    The AKT and ERK signaling pathways are known to be involved in cell hypertrophy, proliferation, survival and differentiation. Although there is evidence for crosstalk between these two signaling pathways in cellulo, there is less evidence for cross talk in vivo. Here, we show that crosstalk between AKT and ERK signaling in the hearts of TRIM72-overexpressing transgenic mice (TRIM72-Tg) with alpha-MHC promoter regulates and maintains their heart size. TRIM72, a heart- and skeletal muscle-specific protein, downregulates AKT-mTOR signaling via IRS-1 degradation and reduces the size of rat cardiomyocytes and the size of postnatal TRIM72-Tg hearts. TRIM72 expression was upregulated by hypertrophic inducers in cardiomyocytes, while IRS-1 was downregulated by IGF-1. TRIM72 specifically regulated IGF-1-dependent AKT-mTOR signaling, resulting in a reduction of the size of cardiomyocytes. Postnatal TRIM72-Tg hearts were smaller than control-treated hearts with inhibition of AKT-mTOR signaling. However, adult TRIM72-Tg hearts were larger than of control despite the suppression of AKT-mTOR signaling. Activation of ERK, PKC-α, and JNK were observed to be elevated in adult TRIM72-Tg, and these signals were mediated by ET-1 via the ET receptors A and B. Altogether, these results suggest that AKT signaling regulates cardiac hypertrophy in physiological conditions, and ERK signaling compensates for the absence of AKT signaling during TRIM72 overexpression, leading to pathological hypertrophy. -- Highlights: • TRIM72 inhibits AKT signaling through ubiquitination of IRS-1 in cardiac cells. • TRIM72 regulates the size of cardiac cells. • TRIM72 regulates size of postnatal TRIM72-overexpressing transgenic mice hearts. • Adult TRIM72-overexpressing transgenic mice hearts showed cardiac dysfunction. • Adult TRIM72 transgenic mice hearts showed higher expression of endothelin receptors.

  11. 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.

  12. Simvastatin inhibits glucose uptake activity and GLUT4 translocation through suppression of the IR/IRS-1/Akt signaling in C2C12 myotubes.

    PubMed

    Li, Weihua; Liang, Xiaojing; Zeng, Zhipeng; Yu, Kaizhen; Zhan, Shaopeng; Su, Qiang; Yan, Yinzhi; Mansai, Huseen; Qiao, Weitong; Yang, Qi; Qi, Zhongquan; Huang, Zhengrong

    2016-10-01

    Simvastatin,a 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase inhibitor, is clinically used in the prevention and treatment of cardiovascular diseases. Numerous studies demonstrate that statins increase the risk of new-onset diabetes in long-term therapy, but mechanisms underpinning this effect are still unclear. Here, we investigated whether simvastatin inhibited the glucose uptake activity and the underlying mechanisms in C2C12 myotubes. Our studies showed that simvastatin significantly inhibited glucose uptake activity and GLUT4 translocation, whereas the effect was reversible with mevalonolactone (ML), which acts as an intermediate of cholesterol synthesis pathway. Mechanistically, the inhibition of glucose uptake and GLUT4 translocation elicited by simvastatin were associated with the suppression of the insulin receptor (IR)/IR substrate (IRS)/Akt signaling cascade. Simvastatin suppressed the phosphorylation of IR, IRS-1 and Akt, and total expression of IR or IRS-1, but did not affect Akt. Furthermore, simvastatin decreased Rac1 GTP binding. In conclusion, our findings indicate that simvastatin suppresses glucose uptake activity and GLUT4 translocation via IR-dependent IRS-1/PI3K/Akt pathway. These results provide an important new insight into the mechanism of statins on insulin sensitivity which may be associated with new-onset diabetes.

  13. Protection afforded by quercetin against H2O2-induced apoptosis on PC12 cells via activating PI3K/Akt signal pathway.

    PubMed

    Chen, Liang; Sun, Lejin; Liu, Zhene; Wang, Hongxia; Xu, Cunli

    2016-01-01

    Cell damage and apoptosis induced by oxidative stress have been involved in various neurodegenerative diseases. This study aims to explore the neuro-protective effects of quercetin on PC12 cells apoptosis induced by hydrogen peroxide (H(2)O(2)) and the underlying mechanisms. The cell viability was detected, as well as the production of reactive oxygen species (ROS), lactate dehydrogenase (LDH) leakage, and the activities of glutathione peroxidase (GSH-Px), superoxide dismutase (SOD) and malondialdehyde (MDA) of the cells in control, H(2)O(2) and quercetin groups. It finally turned out that quercetin might protect PC12 cells against the negative effect of H(2)O(2) by decreasing of LDH release, ROS concentration and MDA level and regaining the GSH-Px and SOD activities. To investigate the mechanism, LY294002 was introduced, the phosphatidylinositol-3-kinase (PI3K) inhibitor. Bax/Bcl-2 ratio and Akt phosphorylation (p-Akt) were examined by Western blot analysis. The data showed that LY294002 almost had the same effects with H(2)O(2), which was also significantly reversed by quercetin could enhance Bax/Bcl-2 ratio and adjust the p-Akt expression, which indicated quercetin might protect PC12 cells against the negative effect of H(2)O(2) via activating the PI3K/Akt signal pathway.

  14. PI3K/AKT Signaling Regulates Bioenergetics in Immortalized Hepatocytes

    PubMed Central

    Li, Chen; Li, Yang; He, Lina; Agarwal, Amit R.; Zeng, Ni; Cadenas, Enrique; Stiles, Bangyan L.

    2013-01-01

    Regulation of cellular bioenergetics by PI3K/AKT signaling was examined in isogenic hepatocyte cell lines lacking the major inhibitor of PI3K/AKT signaling, PTEN (phosphatase and tensin homolog deleted on Chromosome 10). PI3K/AKT signaling was manipulated using the activator (IGF-1) and the inhibitor (LY 294002) of the PI3K/AKT pathway. Activation of PI3K/AKT signaling resulted in an enhanced anaerobic glycolysis and mitochondrial respiration. AKT, when phosphorylated and activated, translocated to mitochondria and localized within the membrane structure of mitochondria, where it phosphorylated a number of mitochondrial residence proteins including the subunits α and β of ATP synthase. Inhibition of GSK3β by either phosphorylation by AKT or lithium chloride resulted in activation of pyruvate dehydrogenase, i.e., decrease of its phosphorylated form. AKT-dependent phosphorylation of ATP synthase subunits α and β resulted in an increased complex activity. AKT translocation to mitochondria was associated with an increased expression and activity of complex I. These data suggest that the mitochondrial signaling pathway AKT-GSK3β-PDH, AKT-dependent phosphorylation of ATP synthase, and upregulation of mitochondrial complex I expression and activity are involved in the control of mitochondrial bioenergetics by increasing substrate availability and regulating the mitochondrial catalytic/energy-transducing capacity. PMID:23376468

  15. Fucoidan Induces ROS-Dependent Apoptosis in 5637 Human Bladder Cancer Cells by Downregulating Telomerase Activity via Inactivation of the PI3K/Akt Signaling Pathway.

    PubMed

    Han, Min Ho; Lee, Dae-Sung; Jeong, Jin-Woo; Hong, Su-Hyun; Choi, Il-Whan; Cha, Hee-Jae; Kim, Suhkmann; Kim, Heui-Soo; Park, Cheol; Kim, Gi-Young; Moon, Sung-Kwon; Kim, Wun-Jae; Hyun Choi, Yung

    2017-02-01

    Preclinical Research Fucoidan, a sulfated polysaccharide, is a compound found in various species of seaweed that has anti-viral, anti-bacterial, anti-oxidant, anti-inflammatory, and immunomodulatory activities; however, the underlying relationship between apoptosis and anti-telomerase activity has not been investigated. Here, we report that fucoidan-induced apoptosis in 5637 human bladder cancer cells was associated with an increase in the Bax/Bcl-2 ratio, the dissipation of the mitochondrial membrane potential (MMP, Δψm), and cytosolic release of cytochrome c from the mitochondria. Under the same experimental conditions, fucoidan-treatment decreased hTERT (human telomerase reverse transcriptase) expression and the transcription factors, c-myc and Sp1. This was accompanied by decreased telomerase activity. Fucoidan-treatment also suppressed activation of the PI3K/Akt signaling pathway. Inhibition of PI3K/Akt signaling enhanced fucoidan-induced apoptosis and anti-telomerase activity. Meanwhile, fucoidan treatment increased the generation of intracellular ROS, whereas the over-elimination of ROS by N-acetylcysteine, an anti-oxidant, attenuated fucoidan-induced apoptosis, inhibition of hTERT, c-myc, and Sp1 expression, and reversed fucoidan-induced inactivation of the PI3K/Akt signaling pathway. Collectively, these data indicate that the induction of apoptosis and the inhibition of telomerase activity by fucoidan are mediated via ROS-dependent inactivation of the PI3K/Akt pathway. Drug Dev Res 78 : 37-48, 2017.   © 2016 Wiley Periodicals, Inc.

  16. Induction of metastatic potential by TrkB via activation of IL6/JAK2/STAT3 and PI3K/AKT signaling in breast cancer

    PubMed Central

    Kim, Min Soo; Lee, Won Sung; Jeong, Joon; Kim, Seong-Jin; Jin, Wook

    2015-01-01

    In metastatic breast cancers, the acquisition of metastatic ability, which leads to clinically incurable disease and poor survival, has been associated with acquisition of epithelial-mesenchymal transition (EMT) program and self-renewing trait (CSCs) via activation of PI3K/AKT and IL6/JAK2/STAT3 signaling pathways. We found that TrkB is a key regulator of PI3K/AKT and JAK/STAT signal pathway-mediated tumor metastasis and EMT program. Here, we demonstrated that TrkB activates AKT by directly binding to c-Src, leading to increased proliferation. Also, TrkB increases Twist-1 and Twist-2 expression through activation of JAK2/STAT3 by inducing c-Src-JAK2 complex formation. Furthermore, TrkB in the absence of c-Src binds directly to JAK2 and inhibits SOCS3-mediated JAK2 degradation, resulting in increased total JAK2 and STAT3 levels, which subsequently leads to JAK2/STAT3 activation and Twist-1 upregulation. Additionally, activation of the JAK2/STAT3 pathway via induction of IL-6 secretion by TrkB enables induction of activation of the EMT program via induction of STAT3 nuclear translocation. These observations suggest that TrkB is a promising target for future intervention strategies to prevent tumor metastasis, EMT program and self-renewing trait in breast cancer. PMID:26515594

  17. Cadmium induces carcinogenesis in BEAS-2B cells through ROS-dependent activation of PI3K/AKT/GSK-3β/β-catenin signaling

    PubMed Central

    Son, Young-Ok; Wang, Lei; Poyil, Pratheeshkumar; Budhraja, Amit; Hitron, J. Andrew; Zhang, Zhuo; Lee, Jeong-Chae; Shi, Xianglin

    2012-01-01

    Cadmium has been widely used in industry and is known to be carcinogenic to humans. Although it is widely accepted that chronic exposure to cadmium increases the incidence of cancer, the mechanisms underlying cadmium-induced carcinogenesis are unclear. The main aim of this study was to investigate the role of reactive oxygen species (ROS) in cadmium-induced carcinogenesis and the signal transduction pathways involved. Chronic exposure of human bronchial epithelial BEAS-2B cells to cadmium induced cell transformation, as evidenced by anchorage-independent growth in soft agar and clonogenic assays. Chronic cadmium treatment also increased the potential of these cells to invade and migrate. Injection of cadmium-stimulated cells into nude mice resulted in the formation of tumors. In contrast, the cadmium-mediated increases in colony formation, cell invasion and migration were prevented by transfection with catalase, superoxide dismutase-1 (SOD1), or SOD2. In particular, chronic cadmium exposure led to activation of signaling cascades involving PI3K, AKT, GSK-3β, and β-catenin and transfection with each of the above antioxidant enzymes markedly inhibited cadmium-mediated activation of these signaling proteins. Inhibitors specific for AKT or β-catenin almost completely suppressed the cadmium-mediated increase in total and active β-catenin proteins and colony formation. Moreover, there was a marked induction of AKT, GSK-3β, β-catenin, and carcinogenic markers in tumor tissues formed in mice after injection with cadmium-stimulated cells. Collectively, our findings suggest a direct involvement of ROS in cadmium-induced carcinogenesis and implicate a role of AKT/GSK-3β/β-catenin signaling in this process. PMID:22884995

  18. PI3K/Akt Signaling Pathway Activates the WNK-OSR1/SPAK-NCC Phosphorylation Cascade in Hyperinsulinemic db/db Mice

    PubMed Central

    Nishida, Hidenori; Sohara, Eisei; Nomura, Naohiro; Chiga, Motoko; Alessi, Dario R; Rai, Tatemitsu; Sasaki, Sei; Uchida, Shinichi

    2013-01-01

    Metabolic syndrome patients have insulin resistance, which causes hyperinsulinemia, which in turn causes aberrant increased renal sodium reabsorption. The precise mechanisms underlying this greater salt-sensitivity of hyperinsulinemic patients remain unclear. Abnormal activation of the recently-identified WNK kinase-OSR1/SPAK kinases-NCC transporter phosphorylation cascade results in the salt-sensitive hypertension of pseudohypoaldosteronism type II. Here, we report a study of renal WNK-OSR1/SPAK-NCC cascade activation in the db/db mouse model of hyperinsulinemic metabolic syndrome. Thiazide sensitivity was increased, suggesting greater activity of NCC in db/db mice. In fact, increased phosphorylation of OSR1/SPAK and NCC was observed. In both SpakT243A/+ and Osr1T185A/+ knock-in db/db mice, which carry mutations that disrupt the signal from WNK kinases, increased phosphorylation of NCC and elevated blood pressure were completely corrected, indicating that phosphorylation of SPAK and OSR1 by WNK kinases is required for the increased activation and phosphorylation of NCC in this model. Renal phosphorylated Akt was increased in db/db mice, suggesting that increased NCC phosphorylation is regulated by the PI3K/Akt signaling cascade in the kidney in response to hyperinsulinemia. A PI3K inhibitor (NVP-BEZ235) corrected the increased OSR1/SPAK-NCC phosphorylation. Another more specific PI3K inhibitor (GDC-0941) and an Akt inhibitor (MK-2206) also inhibited increased NCC phosphorylation. These results indicate that the PI3K/Akt signaling pathway activates the WNK-OSR1/SPAK-NCC phosphorylation cascade in db/db mice. This mechanism may play a role in the pathogenesis of salt-sensitive hypertension in human hyperinsulinemic conditions such as the metabolic syndrome. PMID:22949526

  19. Phosphatidylinositol 3-kinase/Akt signaling pathway activates the WNK-OSR1/SPAK-NCC phosphorylation cascade in hyperinsulinemic db/db mice.

    PubMed

    Nishida, Hidenori; Sohara, Eisei; Nomura, Naohiro; Chiga, Motoko; Alessi, Dario R; Rai, Tatemitsu; Sasaki, Sei; Uchida, Shinichi

    2012-10-01

    Metabolic syndrome patients have insulin resistance, which causes hyperinsulinemia, which in turn causes aberrant increased renal sodium reabsorption. The precise mechanisms underlying this greater salt sensitivity of hyperinsulinemic patients remain unclear. Abnormal activation of the recently identified with-no-lysine kinase (WNK)-oxidative stress-responsive kinase 1 (OSR1)/STE20/SPS1-related proline/alanine-rich kinase (SPAK)-NaCl cotransporter (NCC) phosphorylation cascade results in the salt-sensitive hypertension of pseudohypoaldosteronism type II. Here, we report a study of renal WNK-OSR1/SPAK-NCC cascade activation in the db/db mouse model of hyperinsulinemic metabolic syndrome. Thiazide sensitivity was increased, suggesting greater activity of NCC in db/db mice. In fact, increased phosphorylation of OSR1/SPAK and NCC was observed. In both SpakT243A/+ and Osr1T185A/+ knock-in db/db mice, which carry mutations that disrupt the signal from WNK kinases, increased phosphorylation of NCC and elevated blood pressure were completely corrected, indicating that phosphorylation of SPAK and OSR1 by WNK kinases is required for the increased activation and phosphorylation of NCC in this model. Renal phosphorylated Akt was increased in db/db mice, suggesting that increased NCC phosphorylation is regulated by the phosphatidylinositol 3-kinase/Akt signaling cascade in the kidney in response to hyperinsulinemia. A phosphatidylinositol 3-kinase inhibitor (NVP-BEZ235) corrected the increased OSR1/SPAK-NCC phosphorylation. Another more specific phosphatidylinositol 3-kinase inhibitor (GDC-0941) and an Akt inhibitor (MK-2206) also inhibited increased NCC phosphorylation. These results indicate that the phosphatidylinositol 3-kinase/Akt signaling pathway activates the WNK-OSR1/SPAK-NCC phosphorylation cascade in db/db mice. This mechanism may play a role in the pathogenesis of salt-sensitive hypertension in human hyperinsulinemic conditions, such as the metabolic syndrome.

  20. Lung injury after simulated cardiopulmonary bypass in an isolated perfused rat lung preparation: Role of mitogen-activated protein kinase/Akt signaling and the effects of theophylline

    PubMed Central

    Markou, Thomais; Chambers, David J.

    2014-01-01

    Objectives Lung deflation and inflation during cardiac surgery with cardiopulmonary bypass contributes to pulmonary dysfunction postoperatively. Theophylline treatment for lung diseases has traditionally been thought to act by phosphodiesterase inhibition; however, increasing evidence has suggested other plausible mechanisms. We investigated the effects of deflation and reinflation on signaling pathways (p38-mitogen-activated protein kinase [MAPK], extracellular signal-regulated kinase 1 and 2 [ERK1/2], and Akt) and whether theophylline influences the deflation-induced lung injury and associated signaling. Methods Isolated rat lungs were perfused (15 mL/min) with deoxygenated rat blood in bicarbonate buffer and ventilated. After 20 minutes' equilibration, the lungs were deflated (60 minutes, aerobic perfusion 1.5 mL/min), followed by reinflation (60 minutes, anaerobic reperfusion 15 mL/min). Compliance, vascular resistance, and kinase phosphorylation were assessed during deflation and reinflation. The effects of SB203580 (50 μM), a p38-MAPK inhibitor, and theophylline (0.083 mM [therapeutic] or 3 mM [supratherapeutic]) on physiology and signaling were studied. Results Deflation reduced compliance by 44% compared with continuously ventilated lungs. p38-MAPK and Akt phosphorylation increased (three to fivefold) during deflation and reinflation, and ERK1/2 phosphorylation increased (approximately twofold) during reinflation. SB203580 had no effect on lung physiology or ERK1/2 and Akt activation. Both theophylline doses increased cyclic adenosine monophosphate, but only 3 mM theophylline improved compliance. p38-MAPK phosphorylation was not affected by theophylline; 0.083 mM theophylline inhibited reinflation-induced ERK1/2 phosphorylation (72% ± 3%); and 3 mM theophylline inhibited Akt phosphorylation during deflation (75% ± 5%) and reinflation (87% ± 4%). Conclusions Lung deflation and reinflation stimulates differential p38-MAPK, ERK1/2, and Akt activation

  1. Lung injury after simulated cardiopulmonary bypass in an isolated perfused rat lung preparation: Role of mitogen-activated protein kinase/Akt signaling and the effects of theophylline.

    PubMed

    Markou, Thomais; Chambers, David J

    2014-11-01

    Lung deflation and inflation during cardiac surgery with cardiopulmonary bypass contributes to pulmonary dysfunction postoperatively. Theophylline treatment for lung diseases has traditionally been thought to act by phosphodiesterase inhibition; however, increasing evidence has suggested other plausible mechanisms. We investigated the effects of deflation and reinflation on signaling pathways (p38-mitogen-activated protein kinase [MAPK], extracellular signal-regulated kinase 1 and 2 [ERK1/2], and Akt) and whether theophylline influences the deflation-induced lung injury and associated signaling. Isolated rat lungs were perfused (15 mL/min) with deoxygenated rat blood in bicarbonate buffer and ventilated. After 20 minutes' equilibration, the lungs were deflated (60 minutes, aerobic perfusion 1.5 mL/min), followed by reinflation (60 minutes, anaerobic reperfusion 15 mL/min). Compliance, vascular resistance, and kinase phosphorylation were assessed during deflation and reinflation. The effects of SB203580 (50 μM), a p38-MAPK inhibitor, and theophylline (0.083 mM [therapeutic] or 3 mM [supratherapeutic]) on physiology and signaling were studied. Deflation reduced compliance by 44% compared with continuously ventilated lungs. p38-MAPK and Akt phosphorylation increased (three to fivefold) during deflation and reinflation, and ERK1/2 phosphorylation increased (approximately twofold) during reinflation. SB203580 had no effect on lung physiology or ERK1/2 and Akt activation. Both theophylline doses increased cyclic adenosine monophosphate, but only 3 mM theophylline improved compliance. p38-MAPK phosphorylation was not affected by theophylline; 0.083 mM theophylline inhibited reinflation-induced ERK1/2 phosphorylation (72%±3%); and 3 mM theophylline inhibited Akt phosphorylation during deflation (75%±5%) and reinflation (87%±4%). Lung deflation and reinflation stimulates differential p38-MAPK, ERK1/2, and Akt activation, suggesting a role in lung injury during

  2. Cadmium induces carcinogenesis in BEAS-2B cells through ROS-dependent activation of PI3K/AKT/GSK-3β/β-catenin signaling

    SciTech Connect

    Son, Young-Ok; Wang, Lei; Poyil, Pratheeshkumar; Budhraja, Amit; Hitron, J. Andrew; Zhang, Zhuo; Lee, Jeong-Chae; Shi, Xianglin

    2012-10-15

    Cadmium has been widely used in industry and is known to be carcinogenic to humans. Although it is widely accepted that chronic exposure to cadmium increases the incidence of cancer, the mechanisms underlying cadmium-induced carcinogenesis are unclear. The main aim of this study was to investigate the role of reactive oxygen species (ROS) in cadmium-induced carcinogenesis and the signal transduction pathways involved. Chronic exposure of human bronchial epithelial BEAS-2B cells to cadmium induced cell transformation, as evidenced by anchorage-independent growth in soft agar and clonogenic assays. Chronic cadmium treatment also increased the potential of these cells to invade and migrate. Injection of cadmium-stimulated cells into nude mice resulted in the formation of tumors. In contrast, the cadmium-mediated increases in colony formation, cell invasion and migration were prevented by transfection with catalase, superoxide dismutase-1 (SOD1), or SOD2. In particular, chronic cadmium exposure led to activation of signaling cascades involving PI3K, AKT, GSK-3β, and β-catenin and transfection with each of the above antioxidant enzymes markedly inhibited cadmium-mediated activation of these signaling proteins. Inhibitors specific for AKT or β-catenin almost completely suppressed the cadmium-mediated increase in total and active β-catenin proteins and colony formation. Moreover, there was a marked induction of AKT, GSK-3β, β-catenin, and carcinogenic markers in tumor tissues formed in mice after injection with cadmium-stimulated cells. Collectively, our findings suggest a direct involvement of ROS in cadmium-induced carcinogenesis and implicate a role of AKT/GSK-3β/β-catenin signaling in this process. -- Highlights: ► Chronic exposure to cadmium induces carcinogenic properties in BEAS-2B cells. ► ROS involved in cadmium-induced tumorigenicity of BEAS-2B cells. ► Cadmium activates ROS-dependent AKT/GSK-3β/β-catenin-mediated signaling. ► ROS

  3. Carbohydrate-binding motif in Chitinase 3-like 1 (CHI3L1/YKL-40) specifically activates Akt signaling pathway in colonic epithelial cells

    PubMed Central

    Chen, Chun-Chuan; Llado, Victoria; Eurich, Katrin; Tran, Hoa T.; Mizoguchi, Emiko

    2011-01-01

    Host-microbial interactions play a key role during the development of colitis. We have previously shown that chinase 3-like 1 (CHI3L1) is an inducible molecule overexpressed in colonic epithelial cells (CECs) under inflammatory conditions. In this study, we found that chitin-binding motif (CBM) of CHI3L1 is specifically associated with the CHI3L1-mediated activation of the Akt-signaling in CEC by transfecting the CBM-mutant CHI3L1 vectors in SW480 CECs. Downstream, CHI3L1 enhanced the secretion of IL-8 and TNFα in a dose-dependent manner. We previously show that 325 through 339 amino-acids in CBM are crucial for the biological function of CHI3L1. Here we demonstrated that 325th–339th residues of CBM in CHI3L1 is a critical region for the activation of Akt, IL-8 production, and for a specific cellular localization of CHI3L1. In conclusion, CBM region of CHI3L1 is critical in activating Akt signaling in CECs, and the activation may be associated with the development of chronic colitis. PMID:21546314

  4. Squamosamide derivative FLZ protected dopaminergic neuron by activating Akt signaling pathway in 6-OHDA-induced in vivo and in vitro Parkinson's disease models.

    PubMed

    Bao, Xiu-Qi; Kong, Xiang-Chen; Kong, Li-Bing; Wu, Liang-Yu; Sun, Hua; Zhang, Dan

    2014-02-14

    Parkinson's disease (PD) is a neurodegenerative disease affecting up to 80% of dopaminergic neurons in the nigrostriatal pathway. FLZ, a novel synthetic squamosamide derivative from a Chinese herb, has been shown to have neuroprotective effects in experimental PD models. In this study, we carried out a set of in vitro and in vivo experiments to address the neuroprotective effect of FLZ and related mechanism. The results showed that FLZ significantly improved motor dysfunction and dopaminergic neuronal loss of rats injured by 6-hydroxydopamine (6-OHDA). The beneficial effects of FLZ attributed to the elevation of dopaminergic neuron number, dopamine level and tyrosine hydroxylase (TH) activity. Mechanistic study showed that FLZ protected TH activity and dopaminergic neurons through decreasing α-synuclein (α-Syn) expression and the interaction between α-Syn and TH. Further studies indicated the involvement of phosphoinositide 3-kinases (PI3K)/Akt signaling pathway in the protective effect of FLZ since it showed that blocking PI3K/Akt signaling pathway prevented the expression of α-Syn and attenuated the neuroprotection of FLZ. In addition, FLZ treatment reduced the expression of RTP801, an important protein involved in the pathogenesis of PD. Taken together, these results revealed that FLZ suppressed α-Syn expression and elevated TH activity in dopaminergic neuron through activating Akt survival pathway in 6-OHDA-induced PD models. The data also provided evidence that FLZ had potent neuroprotecive effects and might become a new promising agent for PD treatment.

  5. Genistein attenuates monocrotaline-induced pulmonary arterial hypertension in rats by activating PI3K/Akt/eNOS signaling.

    PubMed

    Zheng, Zeqi; Yu, Songping; Zhang, Wan; Peng, Yongchao; Pu, Mingyu; Kang, Ting; Zeng, Junyi; Yu, Yuefei; Li, Guorong

    2017-01-01

    pulmonary artery pressure, improve right heart function and ameliorate survival rate in the rat model of PAH. Our study suggested that its mechanism was related with PI3K/Akt/eNOS signal pathway. Phytoestrogen genistein may become a new and effective drug for patients with PAH.

  6. Novel pathway in Bcr-Abl signal transduction involves Akt-independent, PLC-gamma1-driven activation of mTOR/p70S6-kinase pathway.

    PubMed

    Markova, B; Albers, C; Breitenbuecher, F; Melo, J V; Brümmendorf, T H; Heidel, F; Lipka, D; Duyster, J; Huber, C; Fischer, T

    2010-02-04

    In chronic myeloid leukemia, activation of the phosphoinositide 3-kinase (PI3K)/Akt pathway is crucial for survival and proliferation of leukemic cells. Essential downstream molecules involve mammalian target of rapamycin (mTOR) and S6-kinase. Here, we present a comprehensive analysis of the molecular events involved in activation of these key signaling pathways. We provide evidence for a previously unrecognized phospholipase C-gamma1 (PLC-gamma1)-controlled mechanism of mTOR/p70S6-kinase activation, which operates in parallel to the classical Akt-dependent machinery. Short-term imatinib treatment of Bcr-Abl-positive cells caused dephosphorylation of p70S6-K and S6-protein without inactivation of Akt. Suppression of Akt activity alone did not affect phosphorylation of p70-S6K and S6. These results suggested the existence of an alternative mechanism for mTOR/p70S6-K activation. In Bcr-Abl-expressing cells, we detected strong PLC-gamma1 activation, which was suppressed by imatinib. Pharmacological inhibition and siRNA knockdown of PLC-gamma1 blocked p70S6-K and S6 phosphorylation. By inhibiting the Ca-signaling, CaMK and PKCs we demonstrated participation of these molecules in the pathway. Suppression of PLC-gamma1 led to inhibition of cell proliferation and enhanced apoptosis. The novel pathway proved to be essential for survival and proliferation of leukemic cells and almost complete cell death was observed upon combined PLC-gamma1 and Bcr-Abl inhibition. The pivotal role of PLC-gamma1 was further confirmed in a mouse leukemogenesis model.

  7. Quercetin attenuates cell apoptosis in focal cerebral ischemia rat brain via activation of BDNF-TrkB-PI3K/Akt signaling pathway.

    PubMed

    Yao, Rui-Qin; Qi, Da-Shi; Yu, Hong-Li; Liu, Jing; Yang, Li-Hua; Wu, Xiu-Xiang

    2012-12-01

    Many studies have demonstrated that apoptosis play an important role in cerebral ischemic pathogenesis and may represent a target for treatment. Neuroprotective effect of quercetin has been shown in a variety of brain injury models including ischemia/reperfusion. It is not clear whether BDNF-TrkB-PI3K/Akt signaling pathway mediates the neuroprotection of quercetin, though there has been some reports on the quercetin increased brain-derived neurotrophic factor (BDNF) level in brain injury models. We therefore first examined the neurological function, infarct volume and cell apoptosis in quercetin treated middle cerebral artery occlusion (MCAO) rats. Then the protein expression of BDNF, cleaved caspase-3 and p-Akt were evaluated in either the absence or presence of PI3K inhibitor (LY294002) or tropomyosin receptor kinase B (TrkB) receptor antagonist (K252a) by immunohistochemistry staining and western blotting. Quercetin significantly improved neurological function, while it decreased the infarct volume and the number of TdT mediated dUTP nick end labeling positive cells in MCAO rats. The protein expression of BDNF, TrkB and p-Akt also increased in the quercetin treated rats. However, treatment with LY294002 or K252a reversed the quercetin-induced increase of BDNF and p-Akt proteins and decrease of cleaved caspase-3 protein in focal cerebral ischemia rats. These results demonstrate that quercetin can decrease cell apoptosis in the focal cerebral ischemia rat brain and the mechanism may be related to the activation of BDNF-TrkB-PI3K/Akt signaling pathway.

  8. P-REX1 creates a positive feedback loop to activate growth factor receptor, PI3K/AKT and MEK/ERK signaling in breast cancer.

    PubMed

    Dillon, L M; Bean, J R; Yang, W; Shee, K; Symonds, L K; Balko, J M; McDonald, W H; Liu, S; Gonzalez-Angulo, A M; Mills, G B; Arteaga, C L; Miller, T W

    2015-07-23

    Phosphatidylinositol 3-kinase (PI3K) promotes cancer cell survival, migration, growth and proliferation by generating phosphatidylinositol 3,4,5-trisphosphate (PIP3) in the inner leaflet of the plasma membrane. PIP3 recruits pleckstrin homology domain-containing proteins to the membrane to activate oncogenic signaling cascades. Anticancer therapeutics targeting the PI3K/AKT/mTOR (mammalian target of rapamycin) pathway are in clinical development. In a mass spectrometric screen to identify PIP3-regulated proteins in breast cancer cells, levels of the Rac activator PIP3-dependent Rac exchange factor-1 (P-REX1) increased in response to PI3K inhibition, and decreased upon loss of the PI3K antagonist phosphatase and tensin homolog (PTEN). P-REX1 mRNA and protein levels were positively correlated with ER expression, and inversely correlated with PI3K pathway activation in breast tumors as assessed by gene expression and phosphoproteomic analyses. P-REX1 increased activation of Rac1, PI3K/AKT and MEK/ERK signaling in a PTEN-independent manner, and promoted cell and tumor viability. Loss of P-REX1 or inhibition of Rac suppressed PI3K/AKT and MEK/ERK, and decreased viability. P-REX1 also promoted insulin-like growth factor-1 receptor activation, suggesting that P-REX1 provides positive feedback to activators upstream of PI3K. In support of a model where PIP3-driven P-REX1 promotes both PI3K/AKT and MEK/ERK signaling, high levels of P-REX1 mRNA (but not phospho-AKT or a transcriptomic signature of PI3K activation) were predictive of sensitivity to PI3K inhibitors among breast cancer cell lines. P-REX1 expression was highest in estrogen receptor-positive breast tumors compared with many other cancer subtypes, suggesting that neutralizing the P-REX1/Rac axis may provide a novel therapeutic approach to selectively abrogate oncogenic signaling in breast cancer cells.

  9. 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.

  10. 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.

  11. Relaxin attenuates aristolochic acid induced human tubular epithelial cell apoptosis in vitro by activation of the PI3K/Akt signaling pathway.

    PubMed

    Xie, Xiang-Cheng; Zhao, Ning; Xu, Qun-Hong; Yang, Xiu; Xia, Wen-Kai; Chen, Qi; Wang, Ming; Fei, Xiao

    2017-04-06

    Aristolochic acid nephropathy remains a leading cause of chronic kidney disease (CKD), however few treatment strategies exist. Emerging evidence has shown that H2 relaxin (RLX) possesses powerful antifibrosis and anti-apoptotic properties, therefore we aimed to investigate whether H2 relaxin can be employed to reduce AA-induced cell apoptosis. Human proximal tubular epithelial (HK-2) cells exposed to AA-I were treated with or without administration of H2 RLX. Cell viability was examined using the WST-8 assay. Apoptotic morphologic alterations were observed using the Hoechst 33342 staining method. Apoptosis was detected using flow cytometry. The expression of caspase 3, caspase 8, caspase 9, ERK1/2, Bax, Bcl-2, and Akt proteins was determined by Western blot. Co-treatment with RLX reversed the increased apoptosis observed in the AA-I only treated group. RLX restored expression of phosphorylated Akt which found to be decreased in the AA-I only treated cells. RLX co-treatment led to a decrease in the Bax/Bcl-2 ratio as well as the cleaved form of caspase-3 compared to the AA-I only treated cells. This anti-apoptotic effect of RLX was attenuated by co-administration of the Akt inhibitor LY294002. The present study demonstrated H2 RLX can decrease AA-I induced apoptosis through activation of the PI3K/Akt signaling pathway.

  12. Redox-Sensitive Oxidation and Phosphorylation of PTEN Contribute to Enhanced Activation of PI3K/Akt Signaling in Rostral Ventrolateral Medulla and Neurogenic Hypertension in Spontaneously Hypertensive Rats

    PubMed Central

    Wu, Kay L.H.; Wu, Chiung-Ai; Wu, Chih-Wei; Chan, Samuel H.H.; Chang, Alice Y.W.

    2013-01-01

    Abstract Aims: The activity of phosphoinositide 3-kinase (PI3K)/serine/threonine protein kinase (Akt) is enhanced under hypertension. The phosphatase and tensin homolog deleted on chromosome 10 (PTEN) is a negative regulator of PI3K signaling, and its activity is redox-sensitive. In the rostral ventrolateral medulla (RVLM), which is responsible for the maintenance of blood pressure, oxidative stress plays a pivotal role in neurogenic hypertension. The present study evaluated the hypothesis that redox-sensitive inactivation of PTEN results in enhanced PI3K/Akt signaling in RVLM, leading to neurogenic hypertension. Results: Compared to age-matched normotensive Wistar-Kyoto (WKY) rats, PTEN inactivation in the form of oxidation and phosphorylation were greater in RVLM of spontaneously hypertensive rats (SHR). PTEN inactivation was accompanied by augmented PI3K activity and PI3K/Akt signaling, as reflected by the increase in phosphorylation of Akt and mammalian target of rapamycin. Intracisternal infusion of tempol or microinjection into the bilateral RVLM of adenovirus encoding superoxide dismutase significantly antagonized the PTEN inactivation and blunted the enhanced PI3K/Akt signaling in SHR. Gene transfer of PTEN to RVLM in SHR also abrogated the enhanced Akt activation and promoted antihypertension. Silencing PTEN expression in RVLM with small-interfering RNA, on the other hand, augmented PI3K/Akt signaling and promoted long-term pressor response in normotensive WKY rats. Innovation: The present study demonstrated for the first time that the redox-sensitive check-and-balance process between PTEN and PI3K/Akt signaling is engaged in the pathogenesis of hypertension. Conclusion: We conclude that an aberrant interplay between the redox-sensitive PTEN and PI3k/Akt signaling in RVLM underpins neural mechanism of hypertension. Antioxid. Redox Signal. 18, 36–50. PMID:22746319

  13. Salidroside inhibits oxygen glucose deprivation (OGD)/re-oxygenation-induced H9c2 cell necrosis through activating of Akt-Nrf2 signaling.

    PubMed

    Zheng, Koulong; Sheng, Zhenqiang; Li, Yefei; Lu, Huihe

    2014-08-15

    Oxygen glucose deprivation (OGD)/re-oxygenation has been applied to cultured cardiomyocytes to create a cellular model of ischemic heart damage. In the current study, we explored the potential role of salidroside against OGD/re-oxygenation-induced damage in H9c2 cardiomyocytes, and studied the underlying mechanisms. We found that OGD/re-oxygenation primarily induced necrosis in H9c2 cells, which was inhibited by salidroside. Salidroside suppressed OGD/re-oxygenation-induced reactive oxygen species (ROS) production, p53 mitochondrial translocation and cyclophilin D (Cyp-D) association as well as mitochondrial membrane potential (MMP) decrease in H9c2 cells. Meanwhile, salidroside activated Akt and promoted transcription of NF-E2-related factor 2 (Nrf2)-regulated genes (heme oxygenase-1 (HO-1) and quinone oxidoreductase 1 (NQO-1)). Significantly, Nrf2 shRNA knockdown or Akt inhibitors (LY 294002 and wortmannin) not only prevented salidroside-induced HO-1/NQO-1 transcription, but also alleviated salidroside-mediated cytoprotective effect against OGD/re-oxygenation in H9c2 cells. These observations suggest that salidroside activates Nrf2-regulated anti-oxidant signaling, and protects against OGD/re-oxygenation-induced H9c2 cell necrosis via activation of Akt signaling. Copyright © 2014 Elsevier Inc. All rights reserved.

  14. Oncogenic S1P signalling in EBV-associated nasopharyngeal carcinoma activates AKT and promotes cell migration through S1P receptor 3.

    PubMed

    Lee, Hui Min; Lo, Kwok-Wai; Wei, Wenbin; Tsao, Sai Wah; Chung, Grace Tin Yun; Ibrahim, Maha Hafez; Dawson, Christopher W; Murray, Paul G; Paterson, Ian C; Yap, Lee Fah

    2017-02-27

    Undifferentiated nasopharyngeal carcinoma (NPC) is a cancer with high metastatic potential that is consistently associated with Epstein-Barr virus (EBV) infection. In this study, we have investigated the functional contribution of sphingosine-1-phosphate (S1P) signalling to the pathogenesis of NPC. We show that EBV infection or ectopic expression of the EBV-encoded latent genes (EBNA1, LMP1 and LMP2A) can up-regulate sphingosine kinase 1 (SPHK1), the key enzyme that produces S1P, in NPC cell lines. Exogenous addition of S1P promotes the migration of NPC cells through the activation of AKT; shRNA knockdown of SPHK1 resulted in a reduction in the levels of activated AKT and inhibition of cell migration. We also show that S1P receptor 3 (S1PR3) mRNA is over-expressed in EBV-positive NPC patient-derived xenografts and a subset of primary NPC tissues, and that knockdown of S1PR3 suppressed the activation of AKT and the S1P-induced migration of NPC cells. Taken together, our data point to a central role for EBV in mediating the oncogenic effects of S1P in NPC and identify S1P signalling as a potential therapeutic target in this disease.

  15. Gremlin promotes retinal pigmentation epithelial (RPE) cell proliferation, migration and VEGF production via activating VEGFR2-Akt-mTORC2 signaling

    PubMed Central

    Liu, Yuan; Chen, Zhijun; Cheng, Haixia; Chen, Juan; Qian, Jing

    2017-01-01

    Retinopathy of prematurity (ROP) is characterized by late-phase pathologic retinal vasoproliferation. Gremlin is a novel vascular endothelial growth factors (VEGF) receptor 2 (VEGFR2) agonist and promotes angiogenic response. We demonstrated that gremlin expression was significantly increased in retinas of ROP model mice, which was correlated with VEGF upregulation. In retinal pigmentation epithelial (RPE) cells, gremlin activated VEGFR2-Akt-mTORC2 (mammalian target of rapamycin complex 2) signaling, and promoted cell proliferation, migration and VEGF production. VEGFR inhibition (by SU5416) or shRNA knockdown almost abolished gremlin-mediated pleiotropic functions in RPE cells. Further, pharmacological inhibition of Akt-mTOR, or shRNA knockdown of key mTORC2 component (Rictor or Sin1) also attenuated gremlin-exerted activities in RPE cells. We conclude that gremlin promotes RPE cell proliferation, migration and VEGF production possibly via activating VEGFR2-Akt-mTORC2 signaling. Gremlin could be a novel therapeutic target of ROP or other retinal vasoproliferation diseases. PMID:27894090

  16. Gremlin promotes retinal pigmentation epithelial (RPE) cell proliferation, migration and VEGF production via activating VEGFR2-Akt-mTORC2 signaling.

    PubMed

    Liu, Yuan; Chen, Zhijun; Cheng, Haixia; Chen, Juan; Qian, Jing

    2017-01-03

    Retinopathy of prematurity (ROP) is characterized by late-phase pathologic retinal vasoproliferation. Gremlin is a novel vascular endothelial growth factors (VEGF) receptor 2 (VEGFR2) agonist and promotes angiogenic response. We demonstrated that gremlin expression was significantly increased in retinas of ROP model mice, which was correlated with VEGF upregulation. In retinal pigmentation epithelial (RPE) cells, gremlin activated VEGFR2-Akt-mTORC2 (mammalian target of rapamycin complex 2) signaling, and promoted cell proliferation, migration and VEGF production. VEGFR inhibition (by SU5416) or shRNA knockdown almost abolished gremlin-mediated pleiotropic functions in RPE cells. Further, pharmacological inhibition of Akt-mTOR, or shRNA knockdown of key mTORC2 component (Rictor or Sin1) also attenuated gremlin-exerted activities in RPE cells. We conclude that gremlin promotes RPE cell proliferation, migration and VEGF production possibly via activating VEGFR2-Akt-mTORC2 signaling. Gremlin could be a novel therapeutic target of ROP or other retinal vasoproliferation diseases.

  17. PDGF-D/PDGFRβ promotes tongue squamous carcinoma cell (TSCC) progression via activating p38/AKT/ERK/EMT signal pathway.

    PubMed

    Zhang, Hong; Sun, Jia-Dong; Yan, Ling-Jian; Zhao, Xiao-Peng

    2016-09-16

    Platelet-derived growth factor D (PDGF-D) signaling plays significant roles during the development and progression of human malignancies via interacting with the receptor of PDGF-D (PDGFR). Meanwhile, the majority of human tumor metastasis is closely associated with epithelial-mesenchymal transition (EMT). However, the underlying mechanism between PDGF-D/PDGFR signaling and EMT which involved in tumor metastasis remain dismal. This study aimed to investigate the role of PDGF-D signaling during EMT process of tongue squamous cell carcinoma (TSCC). In our study, the expression of PDGF-D and PDGFR were examined in primary TSCC samples and the expression of PDGF-D was also determined in TSCC cell lines. In addition, the correlation between PDGF-D expression and TSCC aggressive histopathological features was analyzed. Our results implied that upregulation of PDGFRβ in UM1 cells induced with exogenous PDGF-D can remarkably promote tumor cells invasiveness; conversely, when using small interfering RNA (siRNA), the invasiveness can be severely prohibited. Furthermore, PDGF-D downstream signal molecules p38, AKT, ERK and EMT biomarkers (E-cadherin, N-cadherin, Vimentin and snail) were measured using Western blot. Our results showed that PDGF-D can induce p38, AKT and ERK phosphorylation; downregulate epithelial markers and upregulate mesenchymal markers. On the contrary, PDGFRβ siRNA significantly prohibited p38, AKT and ERK phosphorylation; inhibited EMT process. Function analysis revealed that PDGFRβ siRNA obviously interfered with UM1 cell migration and invasion, according to transwell and wound healing assay. In conclusion, this study suggested that EMT process can be triggered by the PDGF-D/PDGFRβ axis in TSCC, and then involved in the tumor cell invasion via activation of p38/AKT/ERK/EMT pathway.

  18. SYUNZ-16, a newly synthesized alkannin derivative, induces tumor cells apoptosis and suppresses tumor growth through inhibition of PKB/AKT kinase activity and blockade of AKT/FOXO signal pathway.

    PubMed

    Deng, Rong; Tang, Jun; Xie, Bing-Fen; Feng, Gong-Kan; Huang, Yu-Han; Liu, Zong-Chao; Zhu, Xiao-Feng

    2010-07-01

    Alkannin is the major bioactive compound of Arnebia euchroma roots, which is used in many therapeutic remedies in Chinese traditional medicine. SYUNZ-16 is a new derivative of alkannin. In this study, anticancer effects of SYUNZ-16 on human lung adenocarcinoma cell line GLC-82 and human hepatocarcinoma cell line Hep3B were tested in vitro. The results showed SYUNZ-16 could obviously inhibit the proliferation of these cancer cell lines via induction of apoptosis, with the evidence of increasing AnnexinV-positive cells and cleaved caspase-3 and PARP fragments. More importantly, we found that SYUNZ-16 could inhibit AKT activity in cell-free system. Treatment of cancer cells with SYUNZ-16 decreased the phosphorylation of AKT. Additionally, SYUNZ-16 partially attenuated the phosphorylation levels of FKHR and FKHRL1 in a dose-dependent and time-dependent fashion, and led to an increase in the nuclear accumulation of exogenous FKHR, and upregulated the mRNA expression of Bim and TRADD in cancer cells. Further study showed that constitutively activated AKT1 transfection could reduce apoptosis induction mediated by SYUNZ-16. The in vivo experiments showed that SYUNZ-16 had inhibitory effects on S-180 sarcoma implanted to mice. And in GLC-82 xenograft models, SYUNZ-16 at 20 mg/kg/qod remarkably inhibited the tumor growth with the T/C value of 45.3%. Taken together, SYUNZ-16 might be a potent inhibitor of AKT signaling pathway in tumor cells. These data provide evidence for the development of SYUNZ-16 as a potential antitumor drug candidate for further research and development.

  19. Crop milk protein is synthesised following activation of the IRS1/Akt/TOR signalling pathway in the domestic pigeon (Columba livia).

    PubMed

    Hu, X-C; Gao, C-Q; Wang, X-H; Yan, H-C; Chen, Z-S; Wang, X-Q

    2016-12-01

    The experiment was conducted to study whether insulin receptor substance 1 (IRS1) / Protein kinase B (Akt)/target of the rapamycin (TOR) signalling pathway activation stimulates crop milk protein synthesis in the domestic pigeon (Columba livia). Crop milk was collected from ten 1-d-old squabs and analysed for nutrient content. During the non-breeding period and the first day of lactation, blood samples were collected from 5 pairs of breeding pigeons and the levels of prolactin and insulin were determined. Crop samples were collected from 5 pairs of breeders at d 14 and 16 of the incubation period and d 1, 3 and 7 of the lactation period. Crop samples were evaluated for changes in crop weight and thickness and changes in the expression patterns of IRS1/Akt/TOR signalling pathway-related proteins. The results demonstrated that prolactin induces a gradual increase in the relative weight and thickness of the crop, with crops reaching a maximum size at the third day of lactation. Pigeon crop milk contains 64.1% crude protein and 29.7% crude fat based on dry weight. Serum prolactin and insulin levels in the lactation period were significantly higher than those in the non-breeding period. Compared with non-breeding pigeons, the expression of the phosphorylated IRS1 phosphorylated Akt, phosphorylated TOR, phosphorylated ribosomal protein S6 kinase, phosphorylated S6, phosphorylated eukaryotic initiation factor 4E binding protein 1 and eukaryotic initiation factor 4E were significantly up-regulated in the crop of pigeons in the lactation period. In conclusion, prolactin might induce changes in crop tissue and form the physiological structure for crop milk synthesis. Furthermore, the synthesis of crop milk protein is regulated by activation of the IRS1/Akt/TOR signalling pathway.

  20. 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 methods Liquid chromatography-mass spectrometry (LC-MS) was performed to identify the main compounds present in PLP. The mechanism of action of PLP and its therapeutic effect on cholestasis, induced by ANIT, were further investigated. Serum indices such as total bilirubin (TBIL), direct bilirubin (DBIL), aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), γ-glutamyl transpeptidase (γ-GT), and total bile acid (TBA) were measured, and histopathology of liver was also performed to determine the efficacy of treatment with PLP. Moreover, in order to illustrate the underlying signaling pathway, liver glutathione (GSH) content and mRNA or protein levels of glutamate-cysteine ligase catalytic subunit (GCLc), glutamate-cysteine ligase modulatory subunit (GCLm), Akt, heme oxygenase-1 (HO-1), NAD(P)H/quinone oxidoreductase 1 (Nqo1), and Nrf2 were further analyzed. In addition, validation of PLP putative target network was also performed in silico. Results Four major compounds including paeoniflorin, albiflorin, oxypaeoniflorin, and benzoylpaeoniflorin were identified by LC-MS analysis in water extract of PLP. Moreover, PLP could remarkably downregulate serum levels of TBIL, DBIL, AST, ALT, ALP, γ-GT, and TBA, and alleviate the histological damage of liver tissue caused by ANIT. It enhanced antioxidative system by activating PI3K/Akt/Nrf2 pathway through increasing Akt, Nrf2, HO-1, Nqo1, GCLc, and GCLm expression. The putative

  1. CB2 cannabinoid receptor activation promotes colon cancer progression via AKT/GSK3β signaling pathway

    PubMed Central

    Martínez-Martínez, Esther; Martín-Ruiz, Asunción; Martín, Paloma; Calvo, Virginia; Provencio, Mariano; García, José M.

    2016-01-01

    The pharmacological activation of the cannabinoid receptor type 2, CB2, has been shown to elicit anti-tumoral mechanisms in different cancer types. However, little is known about its endogenous role in tumor pathophysiology, and different studies have attributed pro-tumorigenic properties to this receptor. In a previous work, we showed that CB2 expression is a poor prognostic factor in colon cancer patients. Here we report that activation of CB2 with low doses of specific agonists induce cell proliferation and favor the acquisition of aggressive molecular features in colon cancer cells. We show that sub-micromolar concentrations of CB2-specific agonists, JWH-133 and HU-308, promote an increase in cell proliferation rate through the activation of AKT/PKB pathway in colon cancer in vitro and in vivo. AKT activation promotes GSK3β inhibition and thus, a more aggressive cell phenotype with the subsequent elevation of SNAIL levels, E-cadherin degradation and β-catenin delocalization from cell membrane. Taken together, our data show that CB2 activation with sub-micromolar doses of agonists, which could be more similar to endogenous levels of cannabinoids, promote colon cancer progression, implicating that CB2 could have a pro-tumorigenic endogenous role in colon cancer. PMID:27634891

  2. Andrographolide Inhibits Nuclear Factor-κB Activation through JNK-Akt-p65 Signaling Cascade in Tumor Necrosis Factor-α-Stimulated Vascular Smooth Muscle Cells

    PubMed Central

    Chen, Yu-Ying; Hsieh, Cheng-Ying; Lee, Lin-Wen; Sheu, Joen-Rong

    2014-01-01

    Critical vascular inflammation leads to vascular dysfunction and cardiovascular diseases, including abdominal aortic aneurysms, hypertension, and atherosclerosis. Andrographolide is the most active and critical constituent isolated from the leaves of Andrographis paniculata, a herbal medicine widely used for treating anti-inflammation in Asia. In this study, we investigated the mechanisms of the inhibitory effects of andrographolide in vascular smooth muscle cells (VSMCs) exposed to a proinflammatory stimulus, tumor necrosis factor-α (TNF-α). Treating TNF-α-stimulated VSMCs with andrographolide suppressed the expression of inducible nitric oxide synthase in a concentration-dependent manner. A reduction in TNF-α-induced c-Jun N-terminal kinase (JNK), Akt, and p65 phosphorylation was observed in andrographolide-treated VSMCs. However, andrographolide affected neither IκBα degradation nor p38 mitogen-activated protein kinase or extracellular signal-regulated kinase 1/2 phosphorylation under these conditions. Both treatment with LY294002, a phosphatidylinositol 3-kinase/Akt inhibitor, and treatment with SP600125, a JNK inhibitor, markedly reversed the andrographolide-mediated inhibition of p65 phosphorylation. In addition, LY294002 and SP600125 both diminished Akt phosphorylation, whereas LY294002 had no effects on JNK phosphorylation. These results collectively suggest that therapeutic interventions using andrographolide can benefit the treatment of vascular inflammatory diseases, and andrographolide-mediated inhibition of NF-κB activity in TNF-α-stimulated VSMCs occurs through the JNK-Akt-p65 signaling cascade, an IκBα-independent mechanism. PMID:25114952

  3. Membrane Heterogeneity in Akt Activation in Prostate Cancer

    DTIC Science & Technology

    2009-11-01

    construct from the top, Fig. 1), proved to be incompatible with Akt1 activity. The prenylation signal derived from K- Ras rendered Akt1 catalytically...with either cyclo- dextrin (CD) or water-soluble cholesterol ( Chol ) or with CD followed by cholesterol treatment (CD Chol ). Cells incubated in serum

  4. Membrane Heterogeneity in Akt Activation in Prostate Cancer

    DTIC Science & Technology

    2009-07-01

    variant (third construct from the top, Fig. 1), proved to be incompatible with Akt1 activity. The prenylation signal derived from K- Ras rendered Akt1...water-soluble cholesterol ( Chol ) or with CD followed by cholesterol treatment (CD Chol ). Cells incubated in serum-free medium served as controls

  5. Desmocollin 3 mediates follicle stimulating hormone-induced ovarian epithelial cancer cell proliferation by activating the EGFR/Akt signaling pathway.

    PubMed

    Yang, Xiao; Wang, Jing; Li, Wen-Ping; Jin, Zhi-Jun; Liu, Xiao-Jun

    2015-01-01

    Follicle-stimulating hormone (FSH) is associated with the pathogenesis of ovarian cancer. We sought to explore whether desmocollin 3 (Dsc3) mediates FSH-induced ovarian epithelial cancer cell proliferation and whether the EGFR/Akt signaling pathway may be involved in this process. Dsc3 positivity in ovarian tissue specimens from 72 patients was assessed by immunohistochemistry. The positive expression rates of Dsc3 were similar in ovarian cancer tissues (24/31:77.4%) and borderline ovarian tumor tissues (18/22:81.8%) (P>0.05), but were significantly higher in these cancerous tissues than in benign ovarian cyst tissues (3/19:15.8%) (P<0.05). Consistently, the expression of Dsc3 in four out of five ovarian cancer cells (HO8910, Skov3ip, Skov and Hey cells, but not ES-2 and in borderline ovarian MCV152 tumor cells was higher than in the immortalized ovarian epithelial cell line, Moody. FSH up-regulated the expression of Dsc3 and EGFR in a dose- and time-dependent manner. Furthermore, a converse relationship between the expression of Dsc3, EFGR and PI3K/Akt signaling was elucidated using RNA interference and PI3K/Akt inhibitor in the absence and presence of FSH. A role for these proteins in FSH-induced cell proliferation was verified, highlighting their interdependence in mediating ovarian cancer cell function. These results suggest that Dsc3 can mediate FSH-induced ovarian cancer cell proliferation by activating the EGFR/Akt signaling pathway.

  6. Akt and MAPK signaling mediate pregnancy-induced cardiac adaptation.

    PubMed

    Chung, Eunhee; Yeung, Fan; Leinwand, Leslie A

    2012-05-01

    Although the signaling pathways underlying exercise-induced cardiac adaptation have been extensively studied, little is known about the molecular mechanisms that result in the response of the heart to pregnancy. The objective of this study was to define the morphological, functional, and gene expression patterns that define the hearts of pregnant mice, and to identify the signaling pathways that mediate this response. Mice were divided into three groups: nonpregnant diestrus control, midpregnancy, and late pregnancy. Both time points of pregnancy were associated with significant cardiac hypertrophy. The prosurvival signaling cascades of Akt and ERK1/2 were activated in the hearts of pregnant mice, while the stress kinase, p38, was decreased. Given the activation of Akt in pregnancy and its known role in cardiac hypertrophy, the hypertrophic response to pregnancy was tested in mice expressing a cardiac-specific activated (myristoylated) form of Akt (myrAkt) or a cardiac-specific constitutively active (antipathologic hypertrophic) form of its downstream target, glycogen synthase kinase 3β (caGSK3β). The pregnancy-induced hypertrophic responses of hearts from these mice were significantly attenuated. Finally, we tested whether pregnancy-associated sex hormones could induce hypertrophy and alter signaling pathways in isolated neonatal rat ventricular myocytes (NRVMs). In fact, progesterone, but not estradiol treatment increased NRVM cell size via phosphorylation of ERK1/2. Inhibition of MEK1 effectively blocked progesterone-induced cellular hypertrophy. Taken together, our study demonstrates that pregnancy-induced cardiac hypertrophy is mediated by activation of Akt and ERK1/2 pathways.

  7. Akt and MAPK signaling mediate pregnancy-induced cardiac adaptation

    PubMed Central

    Chung, Eunhee; Yeung, Fan

    2012-01-01

    Although the signaling pathways underlying exercise-induced cardiac adaptation have been extensively studied, little is known about the molecular mechanisms that result in the response of the heart to pregnancy. The objective of this study was to define the morphological, functional, and gene expression patterns that define the hearts of pregnant mice, and to identify the signaling pathways that mediate this response. Mice were divided into three groups: nonpregnant diestrus control, midpregnancy, and late pregnancy. Both time points of pregnancy were associated with significant cardiac hypertrophy. The prosurvival signaling cascades of Akt and ERK1/2 were activated in the hearts of pregnant mice, while the stress kinase, p38, was decreased. Given the activation of Akt in pregnancy and its known role in cardiac hypertrophy, the hypertrophic response to pregnancy was tested in mice expressing a cardiac-specific activated (myristoylated) form of Akt (myrAkt) or a cardiac-specific constitutively active (antipathologic hypertrophic) form of its downstream target, glycogen synthase kinase 3β (caGSK3β). The pregnancy-induced hypertrophic responses of hearts from these mice were significantly attenuated. Finally, we tested whether pregnancy-associated sex hormones could induce hypertrophy and alter signaling pathways in isolated neonatal rat ventricular myocytes (NRVMs). In fact, progesterone, but not estradiol treatment increased NRVM cell size via phosphorylation of ERK1/2. Inhibition of MEK1 effectively blocked progesterone-induced cellular hypertrophy. Taken together, our study demonstrates that pregnancy-induced cardiac hypertrophy is mediated by activation of Akt and ERK1/2 pathways. PMID:22345431

  8. Reversal of muscle atrophy by Zhimu and Huangbai herb pair via activation of IGF-1/Akt and autophagy signal in cancer cachexia.

    PubMed

    Zhuang, Pengwei; Zhang, Jinbao; Wang, Yan; Zhang, Mixia; Song, Lili; Lu, Zhiqiang; Zhang, Lu; Zhang, Fengqi; Wang, Jing; Zhang, Yanjun; Wei, Hongjun; Li, Hongyan

    2016-03-01

    Muscle atrophy is the prominent clinical feature of cancer-induced cachexia. Zhimu and Huangbai herb pair (ZBHP) has been used since ancient China times and have been phytochemically investigated for constituents that might cause anti-cancer, diabetes, and their complication. In this study, the effects and mechanisms of ZBHP on reversal of muscle atrophy were explored. C57BL/6 mice implanted with colon-26 adenocarcinoma were chosen to develop cancer cachexia for evaluating the effects of ZBHP on reversal of muscle atrophy. The body weight, survival time, inflammatory cytokines, and pathological changes of muscle were monitored. In addition, IGF-1/Akt and autophagy pathway members were analyzed to interpret the mechanism of drug response. The function and morphology of skeletal muscle in cachexia model were significantly disturbed, and the survival time was shortened. Consistently, inflammatory cytokines and muscle atrophy-related atrogin-1, MuRF1, and FOXO3 were significantly increased, and IGF-1/Akt and autophagy signal pathways were depressed. Treatment with ZBHP significantly alleviated tumor-free body weight reduction and cachexia-induced changes in cytokines and prolonged survival. ZBHP treatment not only inhibited the muscle atrophy-related genes but also activated the IGF-1/Akt and autophagy signal pathways to facilitate the protein synthesis. The results revealed that ZBHP treatment could inhibit the muscle atrophy induced by cancer cachexia and prolong the survival time, and ZBHP may be of value as a pharmacological alternative in treatment of cancer cachexia.

  9. Jaceosidin, a natural flavone, promotes angiogenesis via activation of VEGFR2/FAK/PI3K/AKT/NF-κB signaling pathways in endothelial cells.

    PubMed

    Lee, Tae Hoon; Jung, Hana; Park, Keun Hyung; Bang, Myun Ho; Baek, Nam-In; Kim, Jiyoung

    2014-10-01

    Angiogenesis, the growth of new blood vessels from pre-existing vasculature, plays an important role in physiological and pathological processes such as embryonic development wound healing and revascularization of tissues after exposure to ischemia. We investigated the effects of jaceosidin, a main constituent of medicinal herbs of the genus Artemisia, on angiogenesis and signaling pathways in endothelial cells. Jaceosidin stimulated proliferation, migration and tubulogenesis of ECs as well as ex vivo sprouting from aorta rings, which are phenomena typical of angiogenesis. Jaceosidin activated vascular endothelial growth factor receptor 2 (VEGFR2, FLk-1/KDR) and angiogenic signaling molecules such as focal adhesion kinase, phosphatidylinositol 3-kinase, and its downstream target, the serine-threonine kinase AKTWe also demonstrated that jaceosidin activated the NF-κB-driven expression of a luciferase reporter gene and NF-κB binding to DNA. Jaceosidin-induced proliferation and migration of human umbilical vascular endothelial cells were strongly inhibited by the phosphatidylinositol 3-kinase inhibitor LY294002 and NF-κB inhibitor BAY11-7082, indicating that the PI3K/AKT/NF-κB signaling pathway is involved in jaceosidin-induced angiogenesis. Our results suggest that jaceosidin stimulates angiogenesis by activating the VEGFR2/FAK/PI3K/AKT/NF-κB signaling pathway and that it may be useful in developing angiogenic agents to promote the growth of collateral blood vessels in ischemic tissues.

  10. Negative Immune Regulator TIPE2 Promotes M2 Macrophage Differentiation through the Activation of PI3K-AKT Signaling Pathway

    PubMed Central

    Geng, Wenwen; Chen, Youhai H.; Zhang, Cui

    2017-01-01

    Macrophages play important roles in the regulation of the innate and adaptive immune responses. Classically activated macrophages and alternatively activated macrophages are the two major forms of macrophages and have opposing functionalities. Tumor necrosis factor-α-induced protein 8–2 is expressed primarily by immune cells and negatively regulates type 1 innate and adaptive immune responses to maintain immune tolerance. While previous studies indicate that TIPE2 promotes M2 but inhibits M1 macrophage differentiation, the underlying molecular mechanism by which TIPE2 promotes M2 macrophage differentiation remains unclear. Our current study shows that TIPE2-deficient bone-marrow cells are defective in IL-4 induced M2 macrophage differentiation in vitro. Mechanistic studies revealed that TIPE2 promotes phosphoinositide metabolism and the activation of the down-stream AKT signaling pathway, which in turn leads to the expression of markers specific for M2 macrophages. In addition, our results showed that Tipe2-deficiency does not affect the activation of the JAK-STAT6 signaling pathway that also plays an important role during M2 macrophage differentiation. Taken together, these results indicate that TIPE2 promotes M2 macrophage differentiation through the activation of PI3K-AKT signaling pathway, and may play an important role during the resolution of inflammation, parasite control, as well as tissue repair. PMID:28122045

  11. Morphine mediates a proinflammatory phenotype via μ-opioid receptor-PKCɛ-Akt-ERK1/2 signaling pathway in activated microglial cells.

    PubMed

    Merighi, Stefania; Gessi, Stefania; Varani, Katia; Fazzi, Debora; Stefanelli, Angela; Borea, Pier Andrea

    2013-08-15

    Anti-nociceptive tolerance to opioids severely limits their clinical efficacy for the treatment of chronic pain syndromes. Glia has a central role in the development of morphine tolerance. Here, we characterized the receptor-proximal signaling events that link μ-opioid receptors to activation of Akt and ERKs in lipopolysaccharide (LPS)-stimulated murine microglial cells with the aim to define the molecular mechanism contributing to the ability of morphine to increase inflammatory mediators such as nitric oxide (NO), tumor necrosis factor (TNF)-α, interleukin (IL)-1β and IL-6 in activated microglial cells. In particular, the role of PKCɛ isoform in μ-opioid-induced inflammatory response in microglia was investigated. The results indicate that morphine increases the LPS-induced expression and activation of PKCɛ and stimulates Akt pathway upstream of ERK1/2 and iNOS. Furthermore, we found that morphine enhanced the release of IL-1β, TNF-α, IL-6, and of NO via μ-opioid receptor-PKCɛ signaling pathway in activated microglial cells, mediating a proinflammatory phenotype in mouse microglial cells. Together, these data suggest that the modulation of μ-opioid receptor signaling on microglia through PKCɛ selective inhibition may provide a means to attenuate glial activation and, as a consequence, to treat opioid development of tolerance and dependence.

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

    PubMed

    Li, Mingqun; Hong, Li

    2015-08-01

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

  13. Rumex acetosa L. induces vasorelaxation in rat aorta via activation of PI3-kinase/Akt- AND Ca(2+)-eNOS-NO signaling in endothelial cells.

    PubMed

    Sun, Y Y; Su, X H; Jin, J Y; Zhou, Z Q; Sun, S S; Wen, J F; Kang, D G; Lee, H S; Cho, K W; Jin, S N

    2015-12-01

    Rumex acetosa L. (RA) (Polygonaceae) is an important traditional Chinese medicine (TCM) commonly used in clinic for a long history in China and the aerial parts of RA has a wide variety of pharmacological actions such as diuretic, anti-hypertensive, anti-oxidative, and anti-cancer effects. However, the mechanisms involved are to be defined. The purpose of the present study was to evaluate the vasorelaxant effect and define the mechanism of action of the ethanol extract of Rumex acetosa L. (ERA) in rat aorta. ERA was examined for its vascular relaxant effect in isolated phenylephrine-precontracted rat thoracic aorta and its acute effects on arterial blood pressure. In addition, the roles of the nitric oxide synthase (NOS)-nitric oxide (NO) signaling in the ERA-induced effects were tested in human umbilical vein endothelial cells (HUVECs). The phosphorylation levels of Akt and eNOS were assessed by Western blot analysis in the cultured HUVECs. ERA induced endothelium-dependent vasorelaxation. The ERA-induced vasorelaxation was abolished by L-NAME (an NOS inhibitor) or ODQ (a sGC inhibitor), but not by indomethacin. Inhibition of PI3-kinase/Akt signaling pathway markedly reduced the ERA-induced vasorelaxation. In HUVECs, ERA increased NO formation in a dose-dependent manner, which was inhibited by L-NAME and by removing extracellular Ca(2+). In addition, ERA promoted phosphorylation of Akt and eNOS, which was prevented by wortmannin and LY294002, indicating that ERA induces eNOS phosphorylation through the PI3-kinase/Akt pathway. Further, in anesthetized rats, intravenously administered ERA decreased arterial blood pressure in a dose-dependent manner through an activation of the NOS-NO system. In summary, the ERA- induced vasorelaxation was dependent on endothelial integrity and NO production, and was mediated by activation of both the endothelial PI3-kinase/Akt- and Ca(2+)-eNOS-NO signaling and muscular NO-sGC-cGMP signaling.

  14. 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.

  15. 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

  16. Notch-1 signaling activates NF-κB in human breast carcinoma MDA-MB-231 cells via PP2A-dependent AKT pathway.

    PubMed

    Li, Li; Zhang, Jing; Xiong, Niya; Li, Shun; Chen, Yu; Yang, Hong; Wu, Chunhui; Zeng, Hongjuan; Liu, Yiyao

    2016-04-01

    Breast cancer has a high incidence in the world and is becoming a leading cause of death in female patients due to its high metastatic ability. High expression of Notch-1 and its ligand Jagged-1 correlates with poor prognosis in breast cancer. Our previous work has shown that Notch-1 signaling pathway upregulates NF-κB transcriptional activity and induces the adhesion, migration and invasion of human breast cancer cell line MDA-MB-231. However, the role of Notch-1 in NF-κB activation is still poorly understood. Here, we aim to understand the exact mechanism that Notch-1 regulates NF-κB activity. In MDA-MB-231 cells where Notch-1 is constitutively activated, the phosphorylation of p85 and AKT (Tyr308/Ser473) is upregulated, indicating PI3K/AKT pathway is activated. Notch-1 activation caused the increase of PP2A phosphorylation at Tyr307, indicating Notch-1 inhibits PP2A activity. NF-κB transcriptional activity was evaluated by dual-luciferase reporter assay, and the results showed that, while silencing of Notch-1, PP2A activity was upregulated and NF-κB activity was downregulated, whereas PP2A inhibitor okadaic acid (OA) restored NF-κB activity. Immunofluorescence and Western blots showed that OA treatment antagonized the decrease of p65 nuclear translocation caused by Notch-1 silencing. Moreover, OA treatment also upregulated MMP-2, MMP-9 and VEGF mRNA expression levels, indicating OA rescues Notch-1 silencing that caused low cell invasion. Taken together, our results suggest that Notch-1-activating PI3K/AKT/NF-κB pathway is PP2A dependent; PP2A may be a potential therapeutic target in breast cancer.

  17. Id-1 promotes tumorigenicity and metastasis of human esophageal cancer cells through activation of PI3K/AKT signaling pathway.

    PubMed

    Li, Bin; Tsao, Sai Wah; Li, Yuk Yin; Wang, Xianghong; Ling, Ming Tat; Wong, Yong Chuan; He, Qing Yu; Cheung, Annie L M

    2009-12-01

    Id-1 (inhibitor of differentiation or DNA binding) is a helix-loop-helix protein that is overexpressed in many types of cancer including esophageal squamous cell carcinoma (ESCC). We previously reported that ectopic Id-1 expression activates the phosphatidylinositol-3-kinase (PI3K)/protein kinase B (AKT) signaling pathway in human esophageal cancer cells. In this study, we confirmed a positive correlation between Id-1 and phospho-AKT (Ser473) expressions in ESCC cell lines, as well as in ESCC on a tissue microarray. To investigate the significance of Id-1 in esophageal cancer progression, ESCC cells with stable ectopic Id-1 expression were inoculated subcutaneously into the flank of nude mice and were found to form larger tumors that showed elevated Ki-67 proliferation index and increased angiogenesis, as well as reduced apoptosis, compared with control cells expressing the empty vector.The Id-1-overexpressing cells also exhibited enhanced metastatic potential in the experimental metastasis assay. Treatment with the PI3K inhibitor LY294002 attenuated the tumor promotion effects of Id-1, indicating that the effects were mediated by the PI3K/AKT signaling pathway. In addition, our in vitro experiments showed that ectopic Id-1 expression altered the expression levels of markers associated with epithelial-mesenchymal transition and enhanced the migration ability of esophageal cancer cells. The Id-1-overexpressing ESCC cells also exhibited increased invasive potential, which was in part due to PI3K/AKT-dependent modulation of matrix metalloproteinase-9 expression. In conclusion, our results provide the first evidence that Id-1 promotes tumorigenicity and metastasis of human esophageal cancer in vivo and that the PI3K inhibitor LY294002 can attenuate these effects.

  18. HO-1 attenuates hippocampal neurons injury via the activation of BDNF-TrkB-PI3K/Akt signaling pathway in stroke.

    PubMed

    Qi, Dashi; Ouyang, Changjie; Wang, Yulan; Zhang, Shichun; Ma, Xijuan; Song, YuanJian; Yu, HongLi; Tang, Jiali; Fu, Wei; Sheng, Lei; Yang, Lihua; Wang, Mei; Zhang, Weihao; Miao, Lei; Li, Tengteng; Huang, Xiaojing; Dong, Hongyan

    2014-08-19

    Although recent studies have found that HO-1 plays an important role in neuronal survival, little is known about the precise mechanisms occurring during cerebral ischemia/reperfusion (I/R). Therefore, the aim of this study was to investigate the neuroprotective mechanisms of HO-1 against ischemic brain injury induced by cerebral I/R and to explore whether the BDNF-TrkB-PI3K/Akt signaling pathway contributed to the protection provided by HO-1. Over-expressed HO-1 plasmids were employed to induce the overexpression of HO-1 through hippocampi CA1 injection 5 days before the cerebral I/R animal model was induced by four-vessel occlusion for 15 min transient ischemia and followed by reperfusion in Sprague-Dawley rats. Immunoblotting was carried out to examine the expression of the related proteins, and HE-staining was used to detect the percentage of living neurons in the hippocampal CA1 region. The results showed that over-expressed HO-1 could significantly protect neurons against cerebral I/R. Furthermore, the protein expression of BDNF, TrkB and p-Akt also increased in the rats treated with over-expressed HO-1 plasmids. However, treatment with tropomyosin receptor kinase B (TrkB) receptor antagonist (K252a) reversed the HO-1-induced increase in BDNF and p-Akt protein levels and decreased the level of cleaved caspase-3 protein in I/R rats. In summary, our results imply that HO-1 can decrease cell apoptosis in the I/R rat brain and that the mechanism may be related to the activation of the BDNF-TrkB-PI3K/Akt signaling pathway.

  19. Protein O-fucosyltransferase 1 promotes trophoblast cell proliferation through activation of MAPK and PI3K/Akt signaling pathways.

    PubMed

    Liu, Chang; Liang, Xiaohua; Wang, Jiao; Zheng, Qin; Zhao, Yue; Khan, Muhammad Noman; Liu, Shuai; Yan, Qiu

    2017-04-01

    Protein O-fucosylation is an important glycosylation modification and plays an important role in embryonic development. Protein O-fucosyltransferase 1 (poFUT1) is an essential enzyme that catalyzes the synthesis of protein O-fucosylation. Our previous studies showed that poFUT1 promoted trophoblast cell migration and invasion at the fetal-maternal interface, but the role of poFUT1 in trophoblast cells proliferation remains unclear. Here, immunohistochemistry data showed that poFUT1 and PCNA levels were decreased in abortion patient's trophoblasts compared with women with normal pregnancies. Our results also showed that poFUT1 promoted trophoblast cell proliferation by CCK-8 assay and cell cycle analysis. PoFUT1 increased the phosphorylation of ERK1/2, p38 MAPK, and PI3K/Akt, while inhibitors of ERK1/2(PD98059), p38 MAPK(SB203580), and PI3K (LY294002) prevented ERK1/2, p38 MAPK, and Akt phosphorylation. Moreover, poFUT1 stimulation of trophoblast cells proliferation correlated with increased cell cycle progression by promoting cells into S-phase. The underlying mechanism involved increased cyclin D1, cyclin E, CDK 2, CDK 4, and pRb expression and decreased levels of the cyclin-dependent kinase inhibitors p21 and p27, which were blocked by inhibitors of the upstream signaling molecules MAPK and PI3K/Akt. In conclusion, poFUT1 promotes trophoblast cell proliferation by activating MAPK and PI3K/Akt signaling pathways.

  20. ERK and Akt signaling pathways function through parallel mechanisms to promote mTORC1 signaling

    PubMed Central

    Winter, Jeremiah N.; Jefferson, Leonard S.

    2011-01-01

    The mammalian target of rapamycin (mTOR) is a protein kinase that, when present in a complex referred to as mTOR complex 1 (mTORC1), acts as an important regulator of growth and metabolism. The activity of the complex is regulated through multiple upstream signaling pathways, including those involving Akt and the extracellular-regulated kinase (ERK). Previous studies have shown that, in part, Akt and ERK promote mTORC1 signaling through phosphorylation of a GTPase activator protein (GAP), referred to as tuberous sclerosis complex 2 (TSC2), that acts as an upstream inhibitor of mTORC1. In the present study we extend the earlier studies to show that activation of the Akt and ERK pathways acts in a synergistic manner to promote mTORC1 signaling. Moreover, we provide evidence that the Akt and ERK signaling pathways converge on TSC2, and that Akt phosphorylates residues on TSC2 distinct from those phosphorylated by ERK. The results also suggest that leucine-induced stimulation of mTORC1 signaling occurs through a mechanism distinct from TSC2 and the Akt and ERK signaling pathways. Overall, the results are consistent with a model in which Akt and ERK phosphorylate distinct sites on TSC2, leading to greater repression of its GAP activity, and consequently a magnified stimulation of mTORC1 signaling, when compared with either input alone. The results further suggest that leucine acts through a mechanism distinct from TSC2 to stimulate mTORC1 signaling. PMID:21289294

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

    PubMed Central

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

    2014-01-01

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

  2. 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

    2014-08-28

    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

  3. Fenofibrate pre-treatment suppressed inflammation by activating phosphoinositide 3 kinase/protein kinase B (PI3K/Akt) signaling in renal ischemia-reperfusion injury.

    PubMed

    Yang, Feng-jie; He, Yong-hua; Zhou, Jian-hua

    2015-02-01

    The aim of this study was to investigate the possible beneficial effects of Fenofibrate on renal ischemia-reperfusion injury (IRI) in mice and its potential mechanism. IRI was induced by bilateral renal ischemia for 60 min followed by reperfusion for 24 h. Eighteen male C57BL/6 mice were randomly divided into three groups: sham-operated group (sham), IRI+saline group (IRI group), IRI+Fenofibrate (FEN) group. Normal saline or Fenofibrate (3 mg/kg) was intravenously injected 60 min before renal ischemia in IRI group and FEN group, respectively. Blood samples and renal tissues were collected at the end of reperfusion. The renal function, histopathologic changes, and the expression levels of pro-inflammatory cytokines [interleukin-8 (IL-8), tumor necrosis factor alpha (TNF-α) and IL-6] in serum and renal tissue homogenate were assessed. Moreover, the effects of Fenofibrate on activating phosphoinositide 3 kinase/protein kinase B (PI3K/Akt) signaling and peroxisome proliferator-activated receptor-α (PPAR-α) were also measured in renal IRI. The results showed that plasma levels of blood urea nitrogen and creatinine, histopathologic scores and the expression levels of TNF-α, IL-8 and IL-6 were significantly lower in FEN group than in IRI group. Moreover, Fenofibrate pretreatment could further induce PI3K/Akt signal pathway and PPAR-α activation following renal IRI. These findings indicated PPAR-α activation by Fenofibrate exerts protective effects on renal IRI in mice by suppressing inflammation via PI3K/Akt activation. Thus, Fenofibrate could be a novel therapeutic alternative in renal IRI.

  4. G protein-coupled receptors (GPCRs) That Signal via Protein Kinase A (PKA) Cross-talk at Insulin Receptor Substrate 1 (IRS1) to Activate the phosphatidylinositol 3-kinase (PI3K)/AKT Pathway.

    PubMed

    Law, Nathan C; White, Morris F; Hunzicker-Dunn, Mary E

    2016-12-30

    G protein-coupled receptors (GPCRs) activate PI3K/v-AKT thymoma viral oncoprotein (AKT) to regulate many cellular functions that promote cell survival, proliferation, and growth. However, the mechanism by which GPCRs activate PI3K/AKT remains poorly understood. We used ovarian preantral granulosa cells (GCs) to elucidate the mechanism by which the GPCR agonist FSH via PKA activates the PI3K/AKT cascade. Insulin-like growth factor 1 (IGF1) is secreted in an autocrine/paracrine manner by GCs and activates the IGF1 receptor (IGF1R) but, in the absence of FSH, fails to stimulate YXXM phosphorylation of IRS1 (insulin receptor substrate 1) required for PI3K/AKT activation. We show that PKA directly phosphorylates the protein phosphatase 1 (PP1) regulatory subunit myosin phosphatase targeting subunit 1 (MYPT1) to activate PP1 associated with the IGF1R-IRS1 complex. Activated PP1 is sufficient to dephosphorylate at least four IRS1 Ser residues, Ser(318), Ser(346), Ser(612), and Ser(789), and promotes IRS1 YXXM phosphorylation by the IGF1R to activate the PI3K/AKT cascade. Additional experiments indicate that this mechanism also occurs in breast cancer, thyroid, and preovulatory granulosa cells, suggesting that the PKA-dependent dephosphorylation of IRS1 Ser/Thr residues is a conserved mechanism by which GPCRs signal to activate the PI3K/AKT pathway downstream of the IGF1R.

  5. Jujuboside A Protects H9C2 Cells from Isoproterenol-Induced Injury via Activating PI3K/Akt/mTOR Signaling Pathway

    PubMed Central

    Han, Dandan; Wan, Changrong; Liu, Fenghua; Xu, Xiaolong; Jiang, Linshu; Xu, Jianqin

    2016-01-01

    Jujuboside A is a kind of the saponins isolated from the seeds of Ziziphus jujuba, which possesses multiple biological effects, such as antianxiety, antioxidant, and anti-inflammatory effects; however, its mediatory effect on isoproterenol-stimulated cardiomyocytes has not been investigated yet. In this study, we tried to detect the protective effect and potential mechanism of JUA on ISO-induced cardiomyocytes injury. H9C2 cells were treated with ISO to induce cell damage. Cells were pretreated with JUA to investigate the effects on the cell viability, morphological changes, light chain 3 conversion, and the activation of PI3K/Akt/mTOR signaling pathway. Results showed that ISO significantly inhibited the cell viability in a time- and dose-dependent manner. JUA pretreatment could reverse the reduction of cell viability and better the injury of H9C2 cells induced by ISO. Western blot analysis showed that JUA could accelerate the phosphorylation of PI3K, Akt, and mTOR. Results also indicated that JUA could significantly decrease the ratio of microtubule-associated protein LC3-II/I in H9C2 cells. Taken together, our research showed that JUA could notably reduce the damage cause by ISO via promoting the phosphorylation of PI3K, Akt, and mTOR and inhibiting LC3 conversion, which may be a potential choice for the treatment of heart diseases. PMID:27293469

  6. PTEN mutations and activation of the PI3K/Akt/mTOR signaling pathway in papillary tumors of the pineal region.

    PubMed

    Goschzik, Tobias; Gessi, Marco; Denkhaus, Dorota; Pietsch, Torsten

    2014-08-01

    Papillary tumors of the pineal region (PTPR) are recognized as a distinct entity in the World Health Organization classification of CNS tumors. Papillary tumors of the pineal region frequently show loss of chromosome 10, but no studies have investigated possible target genes on this chromosome. Chromosome 10 harbors the PTEN (phosphatase and tensin homolog) gene, the inactivation of which, by mutation or epigenetic silencing, has been observed in different brain tumors, including high-grade gliomas. In this study, we investigated copy number changes by molecular inversion probe (MIP) analysis and the mutational status of PTEN in 13 PTPR by direct sequencing. MIP analysis of 5 PTPR showed chromosome 10 loss in all cases. In addition, there were losses of chromosomes 3, 14, 22, and X, and gains of whole chromosomes 8, 9, and 12 in more than 1 case. One case had a homozygous PTEN deletion; and 2 point mutations in exon 7 of PTEN (G251D and Q261stop) were found. Immunohistochemistry revealed decrease or loss of the PTEN protein and increased expression of p-Akt and p-S6. These results indicated that PTEN mutations and activation of the PI3K/Akt/mTOR signaling pathway may play a role in the biology of PTPR. This evidence may lead to the possible use of PI3K/Akt/mTOR inhibitors in therapy for patients with PTPR.

  7. Fisetin inhibits UVB-induced cutaneous inflammation and activation of PI3K/AKT/NFκB signaling pathways in SKH-1 hairless mice.

    PubMed

    Pal, Harish Chandra; Athar, Mohammad; Elmets, Craig A; Afaq, Farrukh

    2015-01-01

    Solar ultraviolet B (UVB) radiation has been shown to induce inflammation, DNA damage, p53 mutations and alterations in signaling pathways eventually leading to skin cancer. In this study, we investigated whether fisetin reduces inflammatory responses and modulates PI3K/AKT/NFκB cell survival signaling pathways in UVB-exposed SKH-1 hairless mouse skin. Mice were exposed to 180 mJ cm(-2) of UVB radiation on alternate days for a total of seven exposures, and fisetin (250 and 500 nmol) was applied topically after 15 min of each UVB exposure. Fisetin treatment to UVB-exposed mice resulted in decreased hyperplasia and reduced infiltration of inflammatory cells. Fisetin treatment also reduced inflammatory mediators such as COX-2, PGE2 as well as its receptors (EP1-EP4) and MPO activity. Furthermore, fisetin reduced the level of inflammatory cytokines TNFα, IL-1β and IL-6 in UVB-exposed skin. Fisetin treatment also reduced cell proliferation markers as well as DNA damage as evidenced by increased expression of p53 and p21 proteins. Further studies revealed that fisetin inhibited UVB-induced expression of PI3K, phosphorylation of AKT and activation of the NFκB signaling pathway in mouse skin. Overall, these data suggest that fisetin may be useful against UVB-induced cutaneous inflammation and DNA damage. © 2014 The American Society of Photobiology.

  8. Fisetin inhibits UVB-induced cutaneous inflammation and activation of PI3K/AKT/NFκB signaling pathways in SKH-1 hairless mice†

    PubMed Central

    Pal, Harish Chandra; Athar, Mohammad; Elmets, Craig A.; Afaq, Farrukh

    2014-01-01

    Solar ultraviolet B (UVB) radiation has been shown to induce inflammation, DNA damage, p53 mutations, and alterations in signaling pathways eventually leading to skin cancer. In the present study, we investigated whether fisetin reduces inflammatory responses and modulates PI3K/AKT/NFκB cell survival signaling pathways in UVB exposed SKH-1 hairless mouse skin. Mice were exposed to 180 mJ/cm2 of UVB radiation on alternate days for a total of seven exposures, and fisetin (250 and 500 nmol) was applied topically after 15 min of each UVB exposure. Fisetin treatment to UVB exposed mice resulted in decreased hyperplasia and reduced infiltration of inflammatory cells. Fisetin treatment also reduced inflammatory mediators such as COX-2, PGE2 as well as its receptors (EP1- EP4), and MPO activity. Furthermore, fisetin reduced the level of inflammatory cytokines TNFα, IL-1β and IL-6 in UVB exposed skin. Fisetin treatment also reduced cell proliferation markers as well as DNA damage as evidenced by increased expression of p53 and p21 proteins. Further studies revealed that fisetin inhibited UVB-induced expression of PI3K, phosphorylation of AKT, and activation of the NFκB signaling pathway in mouse skin. Overall, these data suggest that fisetin may be useful against UVB-induced cutaneous inflammation and DNA damage. PMID:25169110

  9. Activation of focal adhesion kinase by Salmonella suppresses autophagy via an Akt/mTOR signaling pathway and promotes bacterial survival in macrophages.

    PubMed

    Owen, Katherine A; Meyer, Corey B; Bouton, Amy H; Casanova, James E

    2014-06-01

    Autophagy has emerged as an important antimicrobial host defense mechanism that not only orchestrates the systemic immune response, but also functions in a cell autonomous manner to directly eliminate invading pathogens. Pathogenic bacteria such as Salmonella have evolved adaptations to protect themselves from autophagic elimination. Here we show that signaling through the non-receptor tyrosine kinase focal adhesion kinase (FAK) is actively manipulated by the Salmonella SPI-2 system in macrophages to promote intracellular survival. In wild-type macrophages, FAK is recruited to the surface of the Salmonella-containing vacuole (SCV), leading to amplified signaling through the Akt-mTOR axis and inhibition of the autophagic response. In FAK-deficient macrophages, Akt/mTOR signaling is attenuated and autophagic capture of intracellular bacteria is enhanced, resulting in reduced bacterial survival. We further demonstrate that enhanced autophagy in FAK(-/-) macrophages requires the activity of Atg5 and ULK1 in a process that is distinct from LC3-assisted phagocytosis (LAP). In vivo, selective knockout of FAK in macrophages resulted in more rapid clearance of bacteria from tissues after oral infection with S. typhimurium. Clearance was correlated with reduced infiltration of inflammatory cell types into infected tissues and reduced tissue damage. Together, these data demonstrate that FAK is specifically targeted by S. typhimurium as a novel means of suppressing autophagy in macrophages, thereby enhancing their intracellular survival.

  10. Rapamycin Prevents cyclophosphamide-induced Over-activation of Primordial Follicle pool through PI3K/Akt/mTOR Signaling Pathway in vivo.

    PubMed

    Zhou, Linyan; Xie, Yanqiu; Li, Song; Liang, Yihua; Qiu, Qi; Lin, Haiyan; Zhang, Qingxue

    2017-08-16

    Primordial follicular depletion has thought to be a common adverse effect of chemotherapy especially for female of reproductive age. The study aimed to evaluate the protective effect of rapamycin on the primordial follicles and its potential mechanism for patients receiving chemotherapy. 8-week old BALB/c female mice were randomly assigned into four groups (control; rapamycin; cyclophosphamide; and rapamycin combined with cyclophosphamide). Hematoxylin staining, immunohistochemical, TUNEL, western blotting and ELISA were employed to assess inter-group differences using Student's t-test and Mann-Whitney test. Cyclophosphamide depleted the follicular reserve and induced the phosphorylation of the key proteins of PI3K/Akt/mTOR pathway in mice in a dose-dependent manner. Co-treatment with rapamycin significantly reduced primordial follicle loss at all cyclophosphamide dose groups and prevent the follicle growth wave caused by cyclophosphamide treatment (P < 0.05). TUNEL staining showed that no apoptosis occured in the primordial follicles in all groups and fewer apoptosis in large growing follicles were observed in ovaries from rapamycin + cyclophosphamide group compared to that received cyclophosphamide alone. Serum anti-Müllerian hormone (AMH) was significantly reduced in cyclophosphamide alone group, in contrast to the normal level in rapamycin + cyclophosphamide group. Compared to p-Akt/Akt and p-mtor/mtor, p-rps6/rps6 was significantly decreased in rapamycin + cyclophosphamide group (P < 0.05), indicating that rapamycin attenuated the increased level of phosphorylation of rpS6 after cyclophosphamide treatment. Rapamycin can prevent the primordial follicle activation induced by cyclophosphamide through PI3K/Akt/mTOR signaling pathway and thus plays a role in preserving the follicle pool. These results suggest that rapamycin may be an effective protection for ovarian function during chemotherapy, which means a new nonsurgical application for protection of

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

    PubMed Central

    Hellesøy, Monica; Lorens, James B.

    2015-01-01

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

  12. Anti-Osteoclastogenic Activity of Praeruptorin A via Inhibition of p38/Akt-c-Fos-NFATc1 Signaling and PLCγ-Independent Ca2+ Oscillation

    PubMed Central

    Choi, Sik-Won; Moon, Seong-Hee; Park, Young Sik; Ryu, Byung Jun; Oh, Jaemin; Kim, Min Seuk; Erkhembaatar, Munkhsoyol; Son, Young-Jin; Kim, Seong Hwan

    2014-01-01

    Background A decrease of bone mass is a major risk factor for fracture. Several natural products have traditionally been used as herbal medicines to prevent and/or treat bone disorders including osteoporosis. Praeruptorin A is isolated from the dry root extract of Peucedanum praeruptorum Dunn and has several biological activities, but its anti-osteoporotic activity has not been studied yet. Materials and Methods The effect of praeruptorin A on the differentiation of bone marrow–derived macrophages into osteoclasts was examined by phenotype assay and confirmed by real-time PCR and immunoblotting. The involvement of NFATc1 in the anti-osteoclastogenic action of praeruptorin A was evaluated by its lentiviral ectopic expression. Intracellular Ca2+ levels were also measured. Results Praeruptorin A inhibited the RANKL-stimulated osteoclast differentiation accompanied by inhibition of p38 and Akt signaling, which could be the reason for praeruptorin A-downregulated expression levels of c-Fos and NFATc1, transcription factors that regulate osteoclast-specific genes, as well as osteoclast fusion-related molecules. The anti-osteoclastogenic effect of praeruptorin A was rescued by overexpression of NFATc1. Praeruptorin A strongly prevented the RANKL-induced Ca2+ oscillation without any changes in the phosphorylation of PLCγ. Conclusion Praeruptorin A could exhibit its anti-osteoclastogenic activity by inhibiting p38/Akt-c-Fos-NFATc1 signaling and PLCγ-independent Ca2+ oscillation. PMID:24586466

  13. Inhibition of Phosphatidylinositol 3-Kinase/Akt Signaling Suppresses Tumor Cell Proliferation and Neuroendocrine Marker Expression in GI Carcinoid Tumors

    PubMed Central

    Pitt, Susan C.; Chen, Herbert; Kunnimalaiyaan, Muthusamy

    2010-01-01

    Background Over-activation of PI3K/Akt signaling facilitates tumor proliferation in several cancers. We have shown that various signal transduction pathways promote tumorigenesis in carcinoid tumors, which exhibit endogenously high levels of active, phosphorylated Akt. Therefore, we hypothesized that inhibition of the PI3K/Akt pathway would suppress carcinoid tumor cell growth and neuroendocrine (NE) marker production. Methods Human carcinoid BON cells were treated in vitro with LY294002, a PI3 kinase inhibitor, or transfected with Akt1 siRNA. Tumor cell proliferation was measured by MTT for six days. The effect of LY294002 or Akt1 siRNA treatment was assessed by western analysis. We examined the levels of phosphorylated Akt, total Akt, Akt1, and the NE markers human achaete-scute homolog1 (ASCL1) and chromogranin A (CgA). Results Treatment of BON cells with LY294002 reduced tumor cell proliferation (76%) in a dose-dependent manner. Growth also decreased in Akt1 siRNA transfected cells (29%). Levels of active, phosphorylated Akt and the NE tumor markers, ASCL1 and CgA, were diminished with both LY294002 and Akt1 siRNA treatments proportional to the degree of Akt inhibition. Total Akt, Akt2, and Akt3 levels were unaffected by these experiments. Conclusions These data indicate that PI3K/Akt signaling performs a critical role in human carcinoid tumor cell survival and NE hormone generation. Furthermore, the development of novel therapeutics targeting Akt1 or components of the PI3K/Akt pathway may enhance the management of carcinoid disease. Synopsis Carcinoid tumor cells were treated with a PI3K inhibitor, LY294002, and Akt1 siRNA to delineate the role of PI3K/Akt signaling in carcinoids. The effects of treatment on cellular proliferation and neuroendocrine marker expression were observed. PMID:19588205

  14. Inhibition of c-Met activation sensitizes osteosarcoma cells to cisplatin via suppression of the PI3K-Akt signaling.

    PubMed

    Wang, Kelai; Zhuang, Yan; Liu, Chunlan; Li, Yang

    2012-10-01

    Osteosarcoma is a common malignant bone tumor. Cisplatin (CDDP) achieves a high response rate in osteosarcoma. However, osteosarcoma usually exhibits cisplatin resistance. Many members of receptor tyrosine kinases (RTKs)(1) have been demonstrated to be overexpressed and constitutively activated in various tumors including osteosarcoma, resulting in malignant progression and insensitivity to chemotherapy. Hepatocyte growth factor receptor (HGFR/c-Met) also appears overexpressed and activated in osteosarcoma cells. Nevertheless, which role of c-Met activation in cisplatin efficacy against osteosarcoma cells remains still elusive. This study found that inhibition of c-Met activity by PHA-665752 or blockade of the interaction of autocrined HGF with c-Met with neutralizing anti-HGF antibody promoted cisplatin efficacy in osteosarcoma cells, while addition of recombinant human HGF (rh-HGF) counteracts cisplatin cytotoxicity. Specifically, we demonstrated that inhibition of c-Met activity led to suppression of the PI3K-Akt pathway, thus enhancing cisplatin chemosensitivity. Our study clearly suggests that inhibition of c-Met activity can effectively sensitize osteosarcoma cells to cisplatin via suppression of the PI3K-Akt signaling.

  15. Akt phosphorylates Tal1 oncoprotein and inhibits its repressor activity.

    PubMed

    Palamarchuk, Alexey; Efanov, Alexey; Maximov, Vadim; Aqeilan, Rami I; Croce, Carlo M; Pekarsky, Yuri

    2005-06-01

    The helix-loop-helix transcription factor Tal1 is required for blood cell development and its activation is a frequent event in T-cell acute lymphoblastic leukemia. The Akt (protein kinase B) kinase is a key player in transduction of antiapoptotic and proliferative signals in T cells. Because Tal1 has a putative Akt phosphorylation site at Thr90, we investigated whether Akt regulates Tal1. Our results show that Akt specifically phosphorylates Thr90 of the Tal1 protein within its transactivation domain in vitro and in vivo. Coimmunoprecipitation experiments showed the presence of Tal1 in Akt immune complexes, suggesting that Tal1 and Akt physically interact. We further showed that phosphorylation of Tal1 by Akt causes redistribution of Tal1 within the nucleus. Using luciferase assay, we showed that phosphorylation of Tal1 by Akt decreased repressor activity of Tal1 on EpB42 (P4.2) promoter. Thus, these data indicate that Akt interacts with Tal1 and regulates Tal1 by phosphorylation at Thr90 in a phosphatidylinositol 3-kinase-dependent manner.

  16. Lucidone Promotes the Cutaneous Wound Healing Process via Activation of the PI3K/AKT, Wnt/β-catenin and NF-κB Signaling Pathways.

    PubMed

    Yang, Hsin-Ling; Tsai, Yu-Cheng; Korivi, Mallikarjuna; Chang, Chia-Ting; Hseu, You-Cheng

    2017-01-01

    Lucidone, which comprises a naturally occurring cyclopentenedione, has been investigated for its in vitro and in vivo wound healing properties, and the underlying molecular signaling cascades in the wound healing mechanism have been elucidated. We demonstrated the cell-/dose-specific responses of lucidone (0.5-8μM) on proliferation and migration/invasion of keratinocyte HaCaT and fibroblast Hs68 cells. In keratinocytes, lucidone-induced nuclear translocation of β-catenin was accompanied by increased transcriptional target genes, including c-Myc and cyclin-D1, through GSK3β-dependent pathway. Correspondingly, lucidone promoted the cell-cycle by increasing PCNA/CDK4 and decreasing p21/p27 expressions. Lucidone induced EMT through the downregulation of epithelial (E-cadherin/occludin) and upregulation of mesenchymal (vimentin/Twist/Snail) marker proteins. Activated MMP-9/-2 and uPA/uPAR as well as suppressed TIMP-1/-2 and PAI-1 expressions by lucidone may promote the migration/invasion of keratinocytes. Notably, lucidone activated NF-κB signaling via IKK-mediated-IκB degradation, and its inhibition abolished MMP-9 activation and keratinocyte migration. Inhibition of PI3K/AKT signaling impaired the lucidone-induced proliferation/migration with corresponding suppression of β-catenin/c-Myc/cyclin-D1 and NF-κB/MMP-9 expressions. Results indicate that lucidone-induced PI3K/AKT signaling anchored the β-catenin/NF-κB-mediated healing mechanism. β-catenin knockdown substantially diminished lucidone-induced keratinocyte migration. Furthermore, lucidone increased endothelial cell proliferation/migration and triggered angiogenesis (MMP-9/uPA/ICAM-1). In macrophages, lucidone-activated NF-κB-mediated inflammation (COX-2/iNOS/NO) and VEGF, which may contribute to the growth of keratinocytes/fibroblasts and endothelial cells. Punched wounds on mice were rapidly healed with the topical application of lucidone (5mM) compared with control ointment-treated mice. Taken

  17. Flotillin-2 promotes metastasis of nasopharyngeal carcinoma by activating NF-κB and PI3K/Akt3 signaling pathways

    PubMed Central

    Liu, Jie; Huang, Wei; Ren, Caiping; Wen, Qiuyuan; Liu, Weidong; Yang, Xuyu; Wang, Lei; Zhu, Bin; Zeng, Liang; Feng, Xiangling; Zhang, Chang; Chen, Huan; Jia, Wei; Zhang, Lihua; Xia, Xiaomeng; Chen, Yuxiang

    2015-01-01

    Lipid raft proteins have been confirmed to be important in cell signal transduction. Some reports have shown that the aberrant expression of lipid raft proteins is associated with malignant phenotypes in some cancers. However, the role of the lipid raft protein flotillin-2 (Flot-2) in nasopharyngeal carcinoma (NPC) remains to be comprehensively characterized. Here, overexpression of Flot-2 in NPC tissues and cell lines was detected by immunostaining, and Flot-2 expression was found to be positively associated with NPC metastasis. Furthermore, inhibiting Flot-2 expression impaired the malignancy of the highly metastatic NPC cell line 5-8F by constraining its growth and proliferation, mobility and migration, and decreasing the capacity of 5-8F cells to metastasize in nude mice. In contrast, forced overexpression of Flot-2 increased the malignancy of 6-10B, a non-metastatic NPC cell line that weakly expresses Flot-2. Moreover, in 5-8F-shFlot-2 cells, which have inhibited Flot-2 expression, the NF-κB and PI3K/Akt3 pathways were inactivated. Subsequently, MMPs expression were decreased, and Foxo1 activity was increased. In addition, enhanced NF-κB and PI3K/Akt3 activities were observed in Flot-2 overexpressing 6-10B cells. Thus, Flot-2 exerts a pro-neoplastic role in NPC and is involved in tumor progression and metastasis. Moreover, Flot-2 exerts its role through NF-κB and PI3K/Akt3 signaling. PMID:26206082

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

    PubMed

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

    2013-06-01

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

  19. Raloxifene activates G protein-coupled estrogen receptor 1/Akt signaling to protect dopamine neurons in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine mice.

    PubMed

    Bourque, Mélanie; Morissette, Marc; Di Paolo, Thérèse

    2014-10-01

    Raloxifene, used in the clinic, is reported to protect brain dopaminergic neurons in mice. Raloxifene was shown to mediate an effect through the G protein-coupled estrogen receptor 1 (GPER1). We investigated if raloxifene neuroprotective effect in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated male mice is mediated through GPER1 by using its antagonist G15. Striatal concentrations of dopamine, 3,4-dihydroxyphenylacetic acid, homovanillic acid to dopamine ratio as well as dopamine transporter and vesicular monoamine transporter 2 showed that raloxifene neuroprotection of dopaminergic neurons was blocked by G15. Protection by raloxifene was accompanied by activation of striatal Akt signaling (but not ERK1/2 signaling) and increased Bcl-2 and brain-derived neurotrophic factor levels; these effects were abolished by coadministration with G15. The effect of raloxifene was not mediated through increased levels of 17β-estradiol. MPTP mice had decreased plasma testosterone, dihydrotestosterone, and 3β-diol levels; this was prevented in raloxifene-treated MPTP mice. Our results suggest that raloxifene acted through GPER1 to mediate Akt activation, increase Bcl-2 and brain-derived neurotrophic factor levels, and protection of dopaminergic neurons and plasma androgens.

  20. Ankyrin repeat and suppressor of cytokine signaling (SOCS) box-containing protein (ASB) 15 alters differentiation of mouse C2C12 myoblasts and phosphorylation of mitogen-activated protein kinase and Akt.

    PubMed

    McDaneld, T G; Spurlock, D M

    2008-11-01

    Ankyrin repeat and suppressor of cytokine signaling box-containing protein (ASB) 15 is a novel ASB gene family member predominantly expressed in skeletal muscle. We have previously reported that overexpression of ASB15 delays differentiation and alters protein turnover in mouse C(2)C(12) myoblasts. However, the extent of ASB15 regulation of differentiation and molecular pathways underlying this activity are unknown. The extracellular signal-regulated kinase (Erk) 1/2 and phosphatidylinositol-3 kinase-Akt (PI3K/Akt; Akt is also known as protein kinase B) signaling pathways have a role in skeletal muscle growth. Activation (phosphorylation) of the Erk1/2 signaling pathway promotes proliferation, whereas activation of the PI3K/Akt signaling pathway promotes myoblast differentiation. Accordingly, we tested the hypothesis that ASB15 controls myoblast differentiation through its regulation of these kinases. Stably transfected myoblasts overexpressing ASB15 (ASB15+) demonstrated decreased differentiation, whereas attenuation of ASB15 expression (ASB15-) increased differentiation. However, ASB15+ cells had less abundance of the phosphorylated mitogen-activated protein kinase (active) form, despite decreased differentiation relative to control myoblasts (ASB15Con). The mitogen-activated protein kinase kinase inhibitor, U0126, effectively decreased mitogen-activated protein kinase phosphorylation and stimulated differentiation in ASB15- and ASB15Con cells. However, inhibition of the Erk1/2 pathway was unable to overcome the inhibitory effect of overexpressing ASB15 on differentiation (ASB15+), suggesting that the Erk1/2 pathway is likely not the predominant mediator of ASB15 activity on differentiation. Expression of ASB15 also altered phosphorylation of the PI3K/Akt pathway, as ASB15+ and ASB15- cells had decreased and increased Akt phosphorylation, respectively. These data were consistent with observed differences in differentiation. Administration of IGF-I, a PI3K/Akt

  1. Berberine reduces ischemia/reperfusion-induced myocardial apoptosis via activating AMPK and PI3K-Akt signaling in diabetic rats.

    PubMed

    Chen, Keke; Li, Guohua; Geng, Fenghao; Zhang, Zhao; Li, Jiani; Yang, Min; Dong, Ling; Gao, Feng

    2014-06-01

    Diabetes increases the risk of cardiovascular diseases. Berberine (BBR), an isoquinoline alkaloid used in Chinese medicine, exerts anti-diabetic effect by lowering blood glucose and regulating lipid metabolism. It has been reported that BBR decreases mortality in patients with chronic congestive heart failure. However, the molecular mechanisms of these beneficial effects are incompletely understood. In the present study, we sought to determine whether BBR exerts cardioprotective effect against ischemia/reperfusion (I/R) injury in diabetic rats and the underlying mechanisms. Male Sprague-Dawley rats were injected with low dose streptozotocin and fed with a high-fat diet for 12 weeks to induce diabetes. The diabetic rats were intragastrically administered with saline or BBR (100, 200 and 400 mg/kg/d) starting from week 9 to 12. At the end of week 12, all rats were subjected to 30 min of myocardial ischemia and 3 h of reperfusion. BBR significantly improved the recovery of cardiac systolic/diastolic function and reduced myocardial apoptosis in diabetic rats subjected to myocardial I/R. Furthermore, in cultured neonatal rat cardiomyocytes, BBR (50 μmol/L) reduced hypoxia/reoxygenation-induced myocardial apoptosis, increased Bcl-2/Bax ratio and decreased caspase-3 expression, together with enhanced activation of PI3K-Akt and increased adenosine monophosphate-activated protein kinase (AMPK) and eNOS phosphorylation. Pretreatment with either PI3K/Akt inhibitor wortmannin or AMPK inhibitor Compound C blunted the anti-apoptotic effect of BBR. Our findings demonstrate that BBR exerts anti-apoptotic effect and improves cardiac functional recovery following myocardial I/R via activating AMPK and PI3K-Akt-eNOS signaling in diabetic rats.

  2. Notoginsenoside R1 ameliorates podocyte injury in rats with diabetic nephropathy by activating the PI3K/Akt signaling pathway

    PubMed Central

    Huang, Guodong; Lv, Jianzhen; Li, Tongyu; Huai, Guoli; Li, Xiang; Xiang, Shaowei; Wang, Longlong; Qin, Zhenlin; Pang, Jianli; Zou, Bingyu; Wang, Yi

    2016-01-01

    The present study was designed to examine the protective effect of notoginsenoside R1 (NR1) on podocytes in a rat model of streptozotocin (STZ)-induced diabetic nephropathy (DN), and to explore the mechanism responsible for NR1-induced renal protection. Diabetes was induced by a single injection of STZ, and NR1 was administered daily at a dose of 5 mg/kg (low dose), 10 mg/kg (medium) and 20 mg/kg (high) for 16 weeks in Sprague-Dawley rats. Blood glucose levels, body weight and proteinuria were measured every 4 weeks, starting on the day that the rats received NR1. Furthermore, on the day of sacrifice, blood, urine and kidneys were collected in order to assess renal function according to general parameters. Pathological staining was performed to evaluate the renal protective effect of NR1, and the expression of the key slit diaphragm proteins, namely neprhin, podocin and desmin, were evaluated. In addition, the serum levels of inflammatory cytokines [tumor necrosis factor-α (TNF-α), tumor growth factor-β1 (TGF-β1), interleukin (IL)-1 and IL-6] as well as an anti-inflammatory cytokine (IL-10) were assessed, and the apoptosis of podocytes was quantified. Finally, the phosphoinositide 3-kinase (PI3K)/Akt signaling pathway and the involvement of nuclear factor-κB (NF-κB) inactivation was further analyzed. In this study, NR1 improved renal function by ameliorating histological alterations, increasing the expression of nephrin and podocin, decreasing the expression of desmin, and inhibiting both the inflammatory response as well as the apoptosis of podocytes. Furthermore, NR1 treatment increased the phosphorylation of both PI3K (p85) and Akt, indicating that activation of the PI3K/Akt signaling pathway was involved. Moreover, NR1 treatment decreased the phosphorylation of NF-κB (p65), suggesting the downregulation of NF-κB. This is the first study to the best of our knowledge, to clearly demonstrate that NR1 treatment ameliorates podocyte injury by inhibiting both

  3. EPO-dependent activation of PI3K/Akt/FoxO3a signalling mediates neuroprotection in in vitro and in vivo models of Parkinson's disease.

    PubMed

    Jia, Yu; Mo, Shi-Jing; Feng, Qi-Qi; Zhan, Ma-Li; OuYang, Li-Si; Chen, Jia-Chang; Ma, Yu-Xin; Wu, Jia-Jia; Lei, Wan-Long

    2014-05-01

    Erythropoietin (EPO) may become a potential therapeutic candidate for the treatment of the neurodegenerative disorder -- Parkinson's disease (PD), since EPO has been found to prevent neuron apoptosis through the activation of cell survival signalling. However, the underlying mechanisms of how EPO exerts its neuroprotective effect are not fully elucidated. Here we investigated the mechanism by which EPO suppressed 6-hydroxydopamine (6-OHDA)-induced neuron death in in vitro and in vivo models of PD. EPO knockdown conferred 6-OHDA-induced cytotoxicity. This effect was reversed by EPO administration. Treatment of PC12 cells with EPO greatly diminished the toxicity induced by 6-OHDA in a dose- and time-dependent manner. EPO effectively reduced apoptosis of striatal neurons and induced a significant improvement on the neurological function score in the rat models of PD. Furthermore, EPO increased the expression of phosphorylated Akt and phosphorylated FoxO3a, and abrogated the 6-OHDA-induced dysregulation of Bcl-2, Bax and Caspase-3 in PC12 cells and in striatal neurons. Meanwhile, the EPO-dependent neuroprotection was notably reversed by pretreatment with LY294002, a specific inhibitor of phosphatidylinositol 3-kinase (PI3K). Our data suggest that PI3K/Akt/FoxO3a signalling pathway may be a possible mechanism involved in the neuroprotective effect of EPO in PD.

  4. Ergosterol peroxide activates Foxo3-mediated cell death signaling by inhibiting AKT and c-Myc in human hepatocellular carcinoma cells

    PubMed Central

    Hu, Liming; Du, William W.; Jiao, Chunwei; Pan, Honghui; Sdiri, Mouna; Wu, Nan; Xie, Yizhen; Yang, Burton B.

    2016-01-01

    Sterols are the important active ingredients of fungal secondary metabolites to induce death of tumor cells. In our previous study, we found that ergosterol peroxide (5α, 8α-epidioxiergosta-6, 22-dien-3β-ol), purified from Ganoderma lucidum, induced human cancer cell death. Since the amount of purified ergosterol peroxide is not sufficient to perform in vivo experiments or apply clinically, we developed an approach to synthesize ergosterol peroxide chemically. After confirming the production of ergosterol peroxide, we examined the biological functions of the synthetic ergosterol peroxide. The results showed that ergosterol peroxide induced cell death and inhibited cell migration, cell cycle progression, and colony growth of human hepatocellular carcinoma cells. We further examined the mechanism associated with this effect and found that treatment with ergosterol peroxide increased the expression of Foxo3 mRNA and protein in HepG2 cells. The upstream signal proteins pAKT and c-Myc, which can inhibit Foxo3 functions, were clearly decreased in HepG2 cells treated with ergosterol peroxide. The levels of Puma and Bax, pro-apoptotic proteins, were effectively enhanced. Our results suggest that ergosterol peroxide stimulated Foxo3 activity by inhibiting pAKT and c-Myc and activating pro-apoptotic protein Puma and Bax to induce cancer cell death. PMID:27058618

  5. PDGFRα signaling in the primary cilium regulates NHE1-dependent fibroblast migration via coordinated differential activity of MEK1/2–ERK1/2–p90RSK and AKT signaling pathways

    PubMed Central

    Clement, Ditte L.; Mally, Sabine; Stock, Christian; Lethan, Mette; Satir, Peter; Schwab, Albrecht; Pedersen, Stine F.; Christensen, Søren T.

    2013-01-01

    Summary In fibroblasts, platelet-derived growth factor receptor alpha (PDGFRα) is upregulated during growth arrest and compartmentalized to the primary cilium. PDGF-AA mediated activation of the dimerized ciliary receptor produces a phosphorylation cascade through the PI3K–AKT and MEK1/2–ERK1/2 pathways leading to the activation of the Na+/H+ exchanger, NHE1, cytoplasmic alkalinization and actin nucleation at the lamellipodium that supports directional cell migration. We here show that AKT and MEK1/2–ERK1/2–p90RSK inhibition reduced PDGF-AA-induced cell migration by distinct mechanisms: AKT inhibition reduced NHE1 activity by blocking the translocation of NHE1 to the cell membrane. MEK1/2 inhibition did not affect NHE1 activity but influenced NHE1 localization, causing NHE1 to localize discontinuously in patches along the plasma membrane, rather than preferentially at the lamellipodium. We also provide direct evidence of NHE1 translocation through the cytoplasm to the leading edge. In conclusion, signals initiated at the primary cilium through the PDGFRαα cascade reorganize the cytoskeleton to regulate cell migration differentially through the AKT and the MEK1/2–ERK1/2–p90RSK pathways. The AKT pathway is necessary for initiation of NHE1 translocation, presumably in vesicles, to the leading edge and for its activation. In contrast, the MEK1/2–ERK1/2–p90RSK pathway controls the spatial organization of NHE1 translocation and incorporation, and therefore specifies the direction of the leading edge formation. PMID:23264740

  6. PDGFRα signaling in the primary cilium regulates NHE1-dependent fibroblast migration via coordinated differential activity of MEK1/2-ERK1/2-p90RSK and AKT signaling pathways.

    PubMed

    Clement, Ditte L; Mally, Sabine; Stock, Christian; Lethan, Mette; Satir, Peter; Schwab, Albrecht; Pedersen, Stine F; Christensen, Søren T

    2013-02-15

    In fibroblasts, platelet-derived growth factor receptor alpha (PDGFRα) is upregulated during growth arrest and compartmentalized to the primary cilium. PDGF-AA mediated activation of the dimerized ciliary receptor produces a phosphorylation cascade through the PI3K-AKT and MEK1/2-ERK1/2 pathways leading to the activation of the Na(+)/H(+) exchanger, NHE1, cytoplasmic alkalinization and actin nucleation at the lamellipodium that supports directional cell migration. We here show that AKT and MEK1/2-ERK1/2-p90(RSK) inhibition reduced PDGF-AA-induced cell migration by distinct mechanisms: AKT inhibition reduced NHE1 activity by blocking the translocation of NHE1 to the cell membrane. MEK1/2 inhibition did not affect NHE1 activity but influenced NHE1 localization, causing NHE1 to localize discontinuously in patches along the plasma membrane, rather than preferentially at the lamellipodium. We also provide direct evidence of NHE1 translocation through the cytoplasm to the leading edge. In conclusion, signals initiated at the primary cilium through the PDGFRαα cascade reorganize the cytoskeleton to regulate cell migration differentially through the AKT and the MEK1/2-ERK1/2-p90(RSK) pathways. The AKT pathway is necessary for initiation of NHE1 translocation, presumably in vesicles, to the leading edge and for its activation. In contrast, the MEK1/2-ERK1/2-p90(RSK) pathway controls the spatial organization of NHE1 translocation and incorporation, and therefore specifies the direction of the leading edge formation.

  7. The Nuclear Zinc Finger Protein Zfat Maintains FoxO1 Protein Levels in Peripheral T Cells by Regulating the Activities of Autophagy and the Akt Signaling Pathway*

    PubMed Central

    Ishikura, Shuhei; Iwaihara, Yuri; Tanaka, Yoko; Luo, Hao; Nishi, Kensuke; Doi, Keiko; Koyanagi, Midori; Okamura, Tadashi; Tsunoda, Toshiyuki; Shirasawa, Senji

    2016-01-01

    Forkhead box O1 (FoxO1) is a key molecule for the development and functions of peripheral T cells. However, the precise mechanisms regulating FoxO1 expression in peripheral T cells remain elusive. We previously reported that Zfatf/f-CD4Cre mice showed a marked decline in FoxO1 protein levels in peripheral T cells, partially through proteasomal degradation. Here we have identified the precise mechanisms, apart from proteasome-mediated degradation, of the decreased FoxO1 levels in Zfat-deficient T cells. First, we confirmed that tamoxifen-inducible deletion of Zfat in Zfatf/f-CreERT2 mice coincidently decreases FoxO1 protein levels in peripheral T cells, indicating that Zfat is essential for maintaining FoxO1 levels in these cells. Although the proteasome-specific inhibitors lactacystin and epoxomicin only moderately increase FoxO1 protein levels, the inhibitors of lysosomal proteolysis bafilomycin A1 and chloroquine restore the decreased FoxO1 levels in Zfat-deficient T cells to levels comparable with those in control cells. Furthermore, Zfat-deficient T cells show increased numbers of autophagosomes and decreased levels of p62 protein, together indicating that Zfat deficiency promotes lysosomal FoxO1 degradation through autophagy. In addition, Zfat deficiency increases the phosphorylation levels of Thr-308 and Ser-473 of Akt and the relative amounts of cytoplasmic to nuclear FoxO1 protein levels, indicating that Zfat deficiency causes Akt activation, leading to nuclear exclusion of FoxO1. Our findings have demonstrated a novel role of Zfat in maintaining FoxO1 protein levels in peripheral T cells by regulating the activities of autophagy and the Akt signaling pathway. PMID:27226588

  8. The PI3K/Akt signal hyperactivates Eya1 via the SUMOylation pathway

    PubMed Central

    Sun, Ye; Kaneko, Satoshi; Li, Xiaokun; Li, Xue

    2014-01-01

    Eya1 is a conserved critical regulator of organ-specific stem cells. Ectopic Eya1 activities, however, promote transformation of mammary epithelial cells. Signals that instigate Eya1 oncogenic activities remain to be determined. Here, we show that Akt1 kinase physically interacts with Eya1 and phosphorylates a conserved consensus site of the Akt kinase. PI3K/Akt signaling enhances Eya1 transcription activity, which largely attributes to the phosphorylation-induced reduction of Eya1 SUMOylation. Indeed, SUMOylation inhibits Eya1 transcription activity; and pharmacologic and genetic activation of PI3K/Akt robustly reduces Eya1 SUMOylation. Wild type but not Akt phosphorylation site mutant Eya1 variant rescues the cell migratory phenotype of EYA1-silenced breast cancer cells, highlighting the importance of Eya1 phosphorylation. Furthermore, knockdown EYA1 sensitizes breast cancer cells to the PI3K/Akt1 inhibitor and irradiation treatments. Thus, the PI3K/Akt signal pathway activates Eya1. These findings further suggest that regulation of SUMOylation by PI3K/Akt signaling is likely an important aspect of tumorigenesis. PMID:24954506

  9. K6PC-5, a novel sphingosine kinase 1 (SphK1) activator, alleviates dexamethasone-induced damages to osteoblasts through activating SphK1-Akt signaling.

    PubMed

    Ji, Feng; Mao, Li; Liu, Yuanyuan; Cao, Xiaojian; Xie, Yue; Wang, Shouguo; Fei, Haodong

    2015-03-13

    Long-term glucocorticoid usage is a common cause of non-traumatic femoral head osteonecrosis. Glucocorticoids (i.e. dexamethasone (Dex)) could directly induce damages to osteoblasts. In the current study, we investigated the potential activity of K6PC-5 [N-(1,3-dihydroxyisopropyl)-2-hexyl-3-oxo-decanamide], a novel sphingosine kinase 1 (SphK1) activator, against this process. Our data revealed that both osteoblastic-like MC3T3-E1 cells and primary murine osteoblasts were responsible to K6PC-5. K6PC-5 activated SphK1, increased sphingosine-1-phosphate (S1P) production and induced Akt phosphorylation in cultured osteoblasts. Functionally, K6PC-5 protected osteoblasts from Dex-induced apoptosis and necrosis. Such signaling and functional effects by K6PC-5 were prevented by the SphK1 inhibitor N,N-dimethylsphingosine (DMS), and by SphK1-siRNAs. On the other hand, exogenously-added S1P activated Akt and reduced Dex-induced osteoblast damages. LY294002 and MK-2206, two established Akt inhibitors, alleviated K6PC-5- or S1P-mediated osteoblast protection against Dex. Together, our results suggest that K6PC-5 alleviates Dex-induced osteoblast injuries through activating SphK1-Akt signaling. K6PC-5 might be further investigated in animal or clinical studies for its anti-glucocorticoids-associated osteonecrosis potential.

  10. Roles of oxidative stress and Akt signaling in doxorubicin cardiotoxicity

    SciTech Connect

    Ichihara, Sahoko . E-mail: saho@gene.mie-u.ac.jp; Yamada, Yoshiji; Kawai, Yoshichika; Osawa, Toshihiko; Furuhashi, Koichi; Duan Zhiwen; Ichihara, Gaku

    2007-07-20

    Cardiotoxicity is a treatment-limiting side effect of the anticancer drug doxorubicin (DOX). We have now investigated the roles of oxidative stress and signaling by the protein kinase Akt in DOX-induced cardiotoxicity as well as the effects on such toxicity both of fenofibrate, an agonist of peroxisome proliferator-activated receptor-{alpha}, and of polyethylene glycol-conjugated superoxide dismutase (PEG-SOD), an antioxidant. Mice injected intraperitoneally with DOX were treated for 4 days with fenofibrate or PEG-SOD. Fenofibrate and PEG-SOD each prevented the induction of cardiac dysfunction by DOX. Both drugs also inhibited the activation of the transcription factor NF-{kappa}B and increase in lipid peroxidation in the left ventricle induced by DOX, whereas only PEG-SOD inhibited the DOX-induced activation of Akt and Akt-regulated gene expression. These results suggest that fenofibrate and PEG-SOD prevented cardiac dysfunction induced by DOX through normalization of oxidative stress and redox-regulated NF-{kappa}B signaling.

  11. Activation of Akt predicts poor outcome in neuroblastoma.

    PubMed

    Opel, Daniela; Poremba, Christopher; Simon, Thorsten; Debatin, Klaus-Michael; Fulda, Simone

    2007-01-15

    Whereas aberrant activation of the phosphatidylinositol 3'-kinase (PI3K)/Akt pathway, a key survival cascade, has previously been linked to poor prognosis in several human malignancies, its prognostic effect in neuroblastoma has not yet been explored. We therefore investigated the phosphorylation status of Akt, S6 ribosomal protein as target of mammalian target of rapamycin, and extracellular signal-regulated kinase (ERK) in 116 primary neuroblastoma samples by tissue microarray and its correlation with established prognostic markers and survival outcome. Here, we provide for the first time evidence that phosphorylation of Akt at serine 473 (S473) and/or threonine 308 (T308), S6 ribosomal protein, and ERK frequently occurs in primary neuroblastoma. Importantly, we identified Akt activation as a novel prognostic indicator of decreased event-free or overall survival in neuroblastoma, whereas phosphorylation of S6 ribosomal protein or ERK had no prognostic effect. In addition, Akt activation correlated with variables of aggressive disease, including MYCN amplification, 1p36 aberrations, advanced disease stage, age at diagnosis, and unfavorable histology. Monitoring Akt at T308 or both phosphorylation sites improved the prognostic significance of Akt activation in neuroblastoma specimens compared with S473 phosphorylation. Parallel experiments in neuroblastoma cell lines revealed that activation of Akt by insulin-like growth factor (IGF)-I significantly inhibited tumor necrosis factor-related apoptosis-inducing ligand- or chemotherapy-induced apoptosis in a PI3K-dependent manner because the PI3K inhibitor LY294002 completely reversed the IGF-I-mediated protection of neuroblastoma cells from apoptosis. By showing that activation of Akt correlates with poor prognosis in primary neuroblastoma in vivo and with apoptosis resistance in vitro, our findings indicate that Akt presents a clinically relevant target in neuroblastoma that warrants further investigation.

  12. Serotonin, but not N-Methyltryptamines, activates the Serotonin 2A Receptor via a βarrestin2/Src/Akt signaling complex in vivo

    PubMed Central

    Schmid, Cullen L.; Bohn, Laura M.

    2010-01-01

    Hallucinogens mediate many of their psychoactive effects by activating serotonin 2A receptors (5-HT2AR). While serotonin is the cognate endogenous neurotransmitter and is not considered hallucinogenic, metabolites of serotonin also have high affinity at 5-HT2AR and can induce hallucinations in humans. Here we report that serotonin differs from the psychoactive N-methyltryptamines by its ability to engage a βarrestin2-mediated signaling cascade in the frontal cortex. Serotonin and 5-hydroxy-L-tryptophan (5-HTP) induce a head twitch response in wild-type (WT) mice which is a behavioral proxy for 5-HT2AR activation. The response in βarrestin2 knockout (βarr2-KO) mice is greatly attenuated until the doses are elevated, at which point, βarr2-KO mice display a head twitch response that can exceed that of WT mice. Direct administration of N-methyltryptamines also produces a greater response in βarr2-KO mice. Moreover, the inhibition of N-methyltransferase blocks 5-HTP-induced head twitches in βarr2-KO mice indicating that N-methyltrypatmines, rather than serotonin, primarily mediate this response. Biochemical studies demonstrate that serotonin stimulates Akt phosphorylation in the frontal cortex and in primary cortical neurons through the activation of a βarrestin2/PI3-K/Src/Akt cascade, while N-methyltryptamines do not. Further, disruption of any of the components of this cascade prevents 5-HTP-, but not N-methyltryptamine-induced, head twitches. We propose that there is a bifurcation of 5-HT2AR signaling that is neurotransmitter- and βarrestin2-dependent. This demonstration of agonist-directed 5-HT2AR signaling in vivo, may significantly impact drug discovery efforts for the treatment of disorders wherein hallucinations are part of the etiology, such as schizophrenia, or manifest as side effects of treatment, such as depression. PMID:20926677

  13. mTOR Activation by PI3K/Akt and ERK Signaling in Short ELF-EMF Exposed Human Keratinocytes

    PubMed Central

    Patruno, Antonia; Pesce, Mirko; Grilli, Alfredo; Speranza, Lorenza; Franceschelli, Sara; De Lutiis, Maria Anna; Vianale, Giovina; Costantini, Erica; Amerio, Paolo; Muraro, Raffaella; Felaco, Mario; Reale, Marcella

    2015-01-01

    Several reports suggest that ELF-EMF exposures interact with biological processes including promotion of cell proliferation. However, the molecular mechanisms by which ELF-EMF controls cell growth are not completely understood. The present study aimed to investigate the effect of ELF-EMF on keratinocytes proliferation and molecular mechanisms involved. Effect of ELF-EMF (50 Hz, 1 mT) on HaCaT cell cycle and cells growth and viability was monitored by FACS analysis and BrdU assay. Gene expression profile by microarray and qRT-PCR validation was performed in HaCaT cells exposed or not to ELF-EMF. mTOR, Akt and MAPKs expressions were evaluated by Western blot analysis. In HaCaT cells, short ELF-EMF exposure modulates distinct patterns of gene expression involved in cell proliferation and in the cell cycle. mTOR activation resulted the main molecular target of ELF-EMF on HaCaT cells. Our data showed the increase of the canonical pathway of mTOR regulation (PI3K/Akt) and activation of ERK signaling pathways. Our results indicate that ELF-EMF selectively modulated the expression of multiple genes related to pivotal biological processes and functions that play a key role in physio-pathological mechanisms such as wound healing. PMID:26431550

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

    SciTech Connect

    Lee, Seung Jin; Lee, Yi Sle; Seo, Kyo Won; Bae, Jin Ung; Kim, Gyu Hee; Park, So Youn; Kim, Chi Dae

    2012-04-01

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

  15. Effects of SIRT1 gene knock-out via activation of SREBP2 protein-mediated PI3K/AKT signaling on osteoarthritis in mice.

    PubMed

    Yu, Fei; Zeng, Hui; Lei, Ming; Xiao, De-Ming; Li, Wei; Yuan, Hao; Lin, Jian-Jing

    2016-10-01

    This study investigated the effects of SIRT1 gene knock-out on osteoarthritis in mice, and the possible roles of SREBP2 protein and the PI3K/AKT signaling pathway in the effects. Mice were randomly divided into a normal group and a SIRT1 gene knock-out group (6 mice in each group). In these groups, one side of the knee anterior cruciate ligament was traversed, and the ipsilateral medial meniscus was cut to establish an osteoarthritis model of knee joint. The countralateral synovial bursa was cut out, serving as controls. The knee joint specimens were then divided into four groups: SIRT1(+/+) control group (group A, n=6); SIRT1(+/+) osteoarthritis group (group B, n=6); SIRT1(-/-) control group (group C, n=6); SIRT1(-/-) osteoarthritis group (group D, n=6). HE staining, Masson staining, Safranin O-Fast Green staining and Van Gieson staining were used to observe the morphological changes in the articular cartilage of the knee. Immunohistochemical staining was employed to detect the expression of SIRT1, SREBP2, VEGF, AKT, HMGCR and type II collagen proteins. SA-β-gal staining was utilized to evaluate chondrocyte aging. The results showed clear knee joint cartilage destruction and degeneration in the SIRT1(-/-) osteoarthritis group. The tidal line was twisted and displaced anteriorly. Type II collagen was destroyed and distributed unevenly. Compared with the SIRT1(+/+) osteoarthritis group and SIRT1(-/-) control group, SIRT1 protein expression was not obviously changed in the SIRT1(-/-) osteoarthritis group (P>0.05), while the expression levels of the SREBP2, VEGF and HMGCR proteins were significantly increased (P<0.05) and the levels of AKT and type II collagen proteins were significantly decreased (P<0.05). SIRT1 gene knock-out may aggravate cartilage degeneration in osteoarthritis by activating the SREBP2 protein-mediated PI3K/AKT signalling pathway, suggesting that SIRT1 gene may play a protective role against osteoarthritis.

  16. Ganglioside GM3 inhibits hepatoma cell motility via down-regulating activity of EGFR and PI3K/AKT signaling pathway.

    PubMed

    Huang, X; Li, Y; Zhang, J; Xu, Y; Tian, Y; Ma, K

    2013-07-01

    Two related sublines derived from murine ascites hepatoma cell lines Hca-F25, which were selected for their markedly different metastatic potential to lymph nodes, were found to be distinct in their ganglioside patterns. The low metastatic cell line (HcaP) contained a major ganglioside GM3, whereas the high metastatic cell line (HcaF) contained a major ganglioside GM2. Suppression of GM3 by P4 enhanced the mobility and migration of the low metastatic HcaP cells in vitro. Increase in GM3 content in high metastatic HcaF cells by addition of exogenous GM3 inhibited the mobility and migration. These results suggested that the differences in lymphatic metastasis potential between these two cell lines could be attributed to the differences in their ganglioside compositions, and GM3 could suppress the motility and migration of these cells. Further, we investigated the mechanism by which GM3 suppressed the cell mobility and migration. The results showed that suppression of GM3 synthesis by P4 in low metastatic HcaP cells promoted PKB/Akt phosphorylation at Ser473 and Thr308, and phosphorylation of EGFR at the Tyr1173. In contrast, increase in GM3 content in high metastatic HcaF cells by addition of exogenous GM3 into the culture medium suppressed phosphorylation of PKB/Akt and EGFR at the same residues. Taken together, these results suggested that the mechanism of GM3-suppressed cell motility and migration may involve the inhibition of phosphorylation of EGFR and the activity of PI3K/AKT signaling pathway. Copyright © 2013 Wiley Periodicals, Inc.

  17. TRPM4 regulates Akt/GSK3-β activity and enhances β-catenin signaling and cell proliferation in prostate cancer cells.

    PubMed

    Sagredo, Alfredo I; Sagredo, Eduardo A; Cappelli, Claudio; Báez, Pablo; Rodrigo, Andaur M; Blanco, Constanza; Tapia, Julio C; Echeverría, César; Cerda, Oscar; Stutzin, Andrés; Simon, Felipe; Marcelain, Katherine; Armisén, Ricardo

    2017-06-14

    Increased expression of the TRPM4 channel has been reported to be associated with the progression of prostate cancer. However, the molecular mechanism underlying its effect remains unknown. This work found that decreasing TRPM4 levels leads to the reduced proliferation of PC3 cells. This effect was associated with a decrease in total β-catenin protein levels and its nuclear localization, and a significant reduction in Tcf/Lef transcriptional activity. Moreover, TRPM4 silencing increases the Ser33/Ser37/Thr41 β-catenin phosphorylated population and reduces the phosphorylation of GSK-3β at Ser9, suggesting an increase in β-catenin degradation as the underlying mechanism. Conversely, TRPM4 overexpresssion in LNCaP cells increases the Ser9 inhibitory phosphorylation of GSK-3β and the total levels of β-catenin and its non-phosphorylated form. Finally, PC3 cells with reduced levels of TRPM4 showed a decrease of basal and stimulated phospho-activation of Akt1, which is likely responsible for the decrease in GSK-3β activity in these cells. Our results also suggest that the effect of TRPM4 on Akt1 is probably mediated by an alteration in the calcium/calmodulin-EGFR axis, linking TRPM4 activity with the observed effects in β-catenin related signaling pathways. These results suggest a role for TRPM4 channels in β-catenin oncogene signaling and underlying mechanisms, highlighting this ion channel as a new potential target for future therapies in prostate cancer. Molecular Oncology (2017) © 2017 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.

  18. Alpha1-ACT Functions as a Tumour Suppressor in Hepatocellular Carcinoma by Inhibiting the PI3K/AKT/mTOR Signalling Pathway via Activation of PTEN.

    PubMed

    Zhu, Hanzhang; Liu, Qiaoyu; Tang, Junwei; Xie, Yu; Xu, Xiaoliang; Huang, Ruyi; Zhang, Yuanguangyan; Jin, Kangpeng; Sun, Beicheng

    2017-01-01

    To investigate the expression and prognostic value of α1-ACT (Alpha1-antichymotrypsin) in patients with HCC (hepatocellular carcinoma) and identify the mechanism by which α1-ACT inhibits proliferation and promotes apoptosis of HCC. We first measured α1-ACT expression levels and determined their relationship with the clinicopathological characteristics and prognosis of patients with HCC.We then established stable HCC cell lines with both α1-ACT overexpression and knockdown and performed a functional analysis in vitro.We first examined the relationship between α1-ACT and the PTEN/PI3K/AKT/mTOR pathway using Western blotting. Then, we determined whether α1-ACT can directly bind to PTEN using co-immunoprecipitation. Finally, we measured α1-ACT expression to evaluate its correlation with the PI3K/AKT/mTOR pathway-related apoptosis proteins in a xenograft tumour mouse model using immunohistochemistry. The α1-ACT expression level was significantly lower in the HCC tissues than in the paratumour tissues and was negatively positively correlated with the level of Ki67, AFP, the AJCC stage, tumour size and tumour invasion. The overexpression of α1-ACT can inhibit cell proliferation and increase cell apoptosis by activating PI3K/AKT/mTOR-mediated apoptosis via binding to PTEN and activating it in vitro. Additionally, the overexpression of α1-ACT can also increase the proportion of cells in the G0/G1 stage by increasing cyclin p21 expression and inhibiting the migration and invasion abilities of HCC cells by regulating MMP2 and MMP9. The xenotransplantation studies with nude mice also showed that overexpression of α1-ACT inhibited tumourigenesis and knockdown of α1-ACT had the opposite effect. Our study demonstrates that α1-ACT suppresses liver cancer development and metastasis via targeting the PTEN/PI3K/AKT/mTOR signalling pathway, which may be a potential target for therapeutic intervention in HCC. © 2017 The Author(s). Published by S. Karger AG, Basel.

  19. 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

  20. Impaired Striatal Akt Signaling Disrupts Dopamine Homeostasis and Increases Feeding

    PubMed Central

    Davis, Adeola R.; Owens, W. Anthony; Matthies, Heinrich J. G.; Saadat, Sanaz; Kennedy, Jack P.; Vaughan, Roxanne A.; Neve, Rachael L.; Lindsley, Craig W.; Russo, Scott J.; Daws, Lynette C.; Niswender1, Kevin D.; Galli, Aurelio

    2011-01-01

    Background The prevalence of obesity has increased dramatically worldwide. The obesity epidemic begs for novel concepts and therapeutic targets that cohesively address “food-abuse” disorders. We demonstrate a molecular link between impairment of a central kinase (Akt) involved in insulin signaling induced by exposure to a high-fat (HF) diet and dysregulation of higher order circuitry involved in feeding. Dopamine (DA) rich brain structures, such as striatum, provide motivation stimuli for feeding. In these central circuitries, DA dysfunction is posited to contribute to obesity pathogenesis. We identified a mechanistic link between metabolic dysregulation and the maladaptive behaviors that potentiate weight gain. Insulin, a hormone in the periphery, also acts centrally to regulate both homeostatic and reward-based HF feeding. It regulates DA homeostasis, in part, by controlling a key element in DA clearance, the DA transporter (DAT). Upon HF feeding, nigro-striatal neurons rapidly develop insulin signaling deficiencies, causing increased HF calorie intake. Methodology/Principal Findings We show that consumption of fat-rich food impairs striatal activation of the insulin-activated signaling kinase, Akt. HF-induced Akt impairment, in turn, reduces DAT cell surface expression and function, thereby decreasing DA homeostasis and amphetamine (AMPH)-induced DA efflux. In addition, HF-mediated dysregulation of Akt signaling impairs DA-related behaviors such as (AMPH)-induced locomotion and increased caloric intake. We restored nigro-striatal Akt phosphorylation using recombinant viral vector expression technology. We observed a rescue of DAT expression in HF fed rats, which was associated with a return of locomotor responses to AMPH and normalization of HF diet-induced hyperphagia. Conclusions/Significance Acquired disruption of brain insulin action may confer risk for and/or underlie “food-abuse” disorders and the recalcitrance of obesity. This molecular model

  1. HPV16 E6-E7 induces cancer stem-like cells phenotypes in esophageal squamous cell carcinoma through the activation of PI3K/Akt signaling pathway in vitro and in vivo.

    PubMed

    Xi, Ruxing; Pan, Shupei; Chen, Xin; Hui, Beina; Zhang, Li; Fu, Shenbo; Li, Xiaolong; Zhang, Xuanwei; Gong, Tuotuo; Guo, Jia; Zhang, Xiaozhi; Che, Shaomin

    2016-08-30

    High-risk human papillomavirus (HPV), especially HPV16, correlates with cancerogenesis of human esophageal squamous cell carcinoma (ESCC) and we have reported that HPV16 related with a poor prognosis of ESCC patients in China. We aim to investigate the potential role and mechanism of HPV16 in ESCC development and progress. Our following researches demonstrated that ESCC cells which were stably transfected by HPV16 E6-E7 lentiviral vector showed a remarkable cancer stem-like cells (CSCs) phenotype, such as: migration, invasion, spherogenesis, high expression of CSCs marker in ESCC---p75NTR, chemoresistance, radioresistance, anti-apoptosis ability in vitro and cancerogenesis in vivo. HPV16 E6-E7 induced PI3K/Akt signaling pathway activation and this affect could be effectively inhibited by LY294002, a specific PI3K inhibitor. It was also indicated that the inhibition of PI3K/Akt signaling pathway by PI3K and Akt siRNA reverse the effect which induced by HPV16 E6-E7 in ESCC cells. Taken together, the present study demonstrates that HPV16 E6-E7 promotes CSCs phenotype in ESCC cells through the activation of PI3K/Akt signaling pathway. Targeting the PI3K/Akt signaling pathway in HPV16 positive tissues is an available therapeutic for ESCC patients.

  2. HPV16 E6-E7 induces cancer stem-like cells phenotypes in esophageal squamous cell carcinoma through the activation of PI3K/Akt signaling pathway in vitro and in vivo

    PubMed Central

    Xi, Ruxing; Pan, Shupei; Chen, Xin; Hui, Beina; Zhang, Li; Fu, Shenbo; Li, Xiaolong; Zhang, Xuanwei; Gong, Tuotuo; Guo, Jia; Zhang, Xiaozhi; Che, Shaomin

    2016-01-01

    High-risk human papillomavirus (HPV), especially HPV16, correlates with cancerogenesis of human esophageal squamous cell carcinoma (ESCC) and we have reported that HPV16 related with a poor prognosis of ESCC patients in China. We aim to investigate the potential role and mechanism of HPV16 in ESCC development and progress. Our following researches demonstrated that ESCC cells which were stably transfected by HPV16 E6-E7 lentiviral vector showed a remarkable cancer stem-like cells (CSCs) phenotype, such as: migration, invasion, spherogenesis, high expression of CSCs marker in ESCC---p75NTR, chemoresistance, radioresistance, anti-apoptosis ability in vitro and cancerogenesis in vivo. HPV16 E6-E7 induced PI3K/Akt signaling pathway activation and this affect could be effectively inhibited by LY294002, a specific PI3K inhibitor. It was also indicated that the inhibition of PI3K/Akt signaling pathway by PI3K and Akt siRNA reverse the effect which induced by HPV16 E6-E7 in ESCC cells. Taken together, the present study demonstrates that HPV16 E6-E7 promotes CSCs phenotype in ESCC cells through the activation of PI3K/Akt signaling pathway. Targeting the PI3K/Akt signaling pathway in HPV16 positive tissues is an available therapeutic for ESCC patients. PMID:27489353

  3. Rutin attenuates H2O2-induced oxidation damage and apoptosis in Leydig cells by activating PI3K/Akt signal pathways.

    PubMed

    Sun, Jianhua; Wang, Heng; Liu, Bei; Shi, Wenhao; Shi, Juanzi; Zhang, Zhou; Xing, Junping

    2017-04-01

    Oxidative stress is a primary factor in the pathology of male infertility. The strong antioxidative capacity of rutin has been proven by numerous studies, but a protective role in the context of male reproduction remains to be elucidated. To explore the biological role of rutin in protecting male reproductive function and the potential underlying mechanism, H2O2-induced Leydig cells were used as a cell model of oxidation damage. Our findings showed that rutin at concentrations of 10, 20, and 40μmol/L remarkably increased cell survival rate of H2O2-induced Leydig cells to 70.1%, 86.8%, and 80.3% respectively. Next, rutin with concentrations of 10, 20, and 40μmol/L decreased reactive oxygen species (ROS) and malondialdehyde (MDA) levels but increased the levels of glutathione (GSH) and testosterone in H2O2-induced Leydig cells. The activities of superoxide dismutase (SOD), catalase (CAT) and peroxidase (POD) were remarkably increased by rutin treatment with concentrations of 20 and 40μmol/L, but glutathione peroxidase (GSH-Px) activity was notably decreased. Moreover, rutin with concentrations of 10, 20, and 40μmol/L increased Bcl-2 protein levels but decreased protein levels of Bax and caspase-3. Furthermore, 20μmol/L rutin significantly abrogated the decrease in levels of phosphoinositide 3-kinase (PI3K) and phosphorylated serine/threonine kinase (p-AKT) induced by H2O2. Pretreatment with LY294002, a PI3K inhibitor, antagonized protective action of 20μmol/L rutin against H2O2-induced cell activities, intracellular oxidant, testosterone, antioxidant enzyme activities, and the apoptosis related protein expression. Taken together, these results suggest that rutin attenuates H2O2-induced oxidation damage and apoptosis in Leydig cells by activating PI3K/Akt signal pathways, providing a promising strategy to decrease oxidative stress associated with male infertility.

  4. (+)-Catechin Attenuates NF-κB Activation Through Regulation of Akt, MAPK, and AMPK Signaling Pathways in LPS-Induced BV-2 Microglial Cells.

    PubMed

    Syed Hussein, Sharifah Salwa; Kamarudin, Muhamad Noor Alfarizal; Kadir, Habsah Abdul

    2015-01-01

    (+)-Catechin is a flavanol that possesses various health and medicinal values, which include neuroprotection, anti-oxidation, antitumor and antihepatitis activities. This study investigated the modulatory effects of (+)-catechin on the lipopolysaccharides (LPS)-stimulated BV-2 cells. (+)-catechin attenuated LPS-induced inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) and inhibited microglial NO and ROS production. Additionally, (+)-catechin suppressed the production of tumor necrosis factor-α (TNF-α) and interleukin (IL)-6, while augmenting IL-4. (+)-catechin attenuated LPS-induced nuclear factor-κB (NF-κB) p65 nuclear translocation via the inhibition of IκB-α phosphorylation. Moreover, (+)-catechin blocked the activation of Akt and its inhibition was shown to play a crucial role in LPS-induced inflammation in BV-2 microglial cells. (+)-catechin also attenuated the LPS-induced phosphorylation of extracellular signal-regulated kinase (ERK1/2), and p-38 mitogen activated protein kinases (p38 MAPK) and specific inhibitors of ERK1/2 (UO126) and p38 MAPK (SB202190) subsequently down-regulated the expression of the proinflammatory mediators iNOS and COX-2. Further mechanistic study revealed that (+)-catechin acted through the amelioration of the LPS-induced suppression of adenosine monophosphate-activated protein kinase (AMPK) activity. Taken together, our data indicate that (+)-catechin exhibits anti-inflammatory effects in BV-2 cells by suppressing the production of proinflammatory mediators and mitigation of NF-κB through Akt, ERK, p38 MAPK, and AMPK pathways.

  5. Targeting protein kinase-b3 (akt3) signaling in melanoma.

    PubMed

    Madhunapantula, SubbaRao V; Robertson, Gavin P

    2017-03-01

    Deregulated Akt activity leading to apoptosis inhibition, enhanced proliferation and drug resistance has been shown to be responsible for 35-70% of advanced metastatic melanomas. Of the three isoforms, the majority of melanomas have elevated Akt3 expression and activity. Hence, potent inhibitors targeting Akt are urgently required, which is possible only if (a) the factors responsible for the failure of Akt inhibitors in clinical trials is known; and (b) the information pertaining to synergistically acting targeted therapeutics is available. Areas covered: This review provides a brief introduction of the PI3K-Akt signaling pathway and its role in melanoma development. In addition, the functional role of key Akt pathway members such as PRAS40, GSK3 kinases, WEE1 kinase in melanoma development are discussed together with strategies to modulate these targets. Efficacy and safety of Akt inhibitors is also discussed. Finally, the mechanism(s) through which Akt leads to drug resistance is discussed in this expert opinion review. Expert opinion: Even though Akt play key roles in melanoma tumor progression, cell survival and drug resistance, many gaps still exist that require further understanding of Akt functions, especially in the (a) metastatic spread; (b) circulating melanoma cells survival; and (c) melanoma stem cells growth.

  6. Antimitogenic and proapoptotic activities of methylseleninic acid in vascular endothelial cells and associated effects on PI3K-AKT, ERK, JNK and p38 MAPK signaling.

    PubMed

    Wang, Z; Jiang, C; Ganther, H; Lü, J

    2001-10-01

    Inhibiting the mitogenic response of vascular endothelial cells may in part mediate the antiangiogenic and anticancer activity of supranutritional selenium supplements. Our previous work had shown that methylseleninic acid (MSeA), a precursor of the critical anticancer methylselenol metabolite pool, was a potent inhibitor of the growth and survival of human umbilical vein endothelial cells (HUVECs). Here we investigated the effects of MSeA on selected protein kinase signaling transduction pathways to characterize their role in methylselenium induction of HUVEC cell cycle arrest and apoptosis. Exposure of asynchronous HUVECs for 30 h to 3-5 microM MSeA led to a profound G(1) arrest, and exposure to higher levels of MSeA not only led to G(1) arrest but also to DNA fragmentation and caspase-mediated cleavage of poly(ADP-ribose)polymerase, both biochemical hallmarks of apoptosis. Immunoblot analyses indicated that G(1) arrest induced by the sublethal doses of MSeA was associated with dose-dependent reductions of the levels of phospho-protein kinase B (also known as AKT or PKB), phospho-extracellular signal regulated kinase (ERK) 1/2, and phospho-Jun NH(2)-terminal kinases 1/2 in the absence of any change in p38 mitogen-activated protein kinase (MAPK) phosphorylation. Apoptosis induced by MSeA was associated with an increased phosphorylation of p38 MAPK in addition to the dephosphorylation of the above kinases. In HUVECs deprived of endothelial cell growth supplement (ECGS) for 48 h, resumption of ECGS stimulation resulted in an approximately 10-fold increase in mitogenic response, as indicated by [(3)H]thymidine incorporation into DNA. The ECGS-stimulated mitogenic response was inhibited in a dose-dependent manner by MSeA exposure with a IC(50) approximately 1 microM and a complete blockage at 3 microM. Wortmannin, an inhibitor of phosphatidylinositol 3-kinase (PI3K) upstream of AKT, potently inhibited the ECGS-stimulated DNA synthesis (IC(50), approximately 40 n

  7. 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.

  8. 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

  9. ON 01910.Na (rigosertib) inhibits PI3K/Akt pathway and activates oxidative stress signals in head and neck cancer cell lines

    PubMed Central

    Prasad, Anil; Khudaynazar, Nagina; Tantravahi, Ramana V.; Gillum, Amanda M.; Hoffman, Benjamin S.

    2016-01-01

    Squamous cell carcinoma of the head and neck (HNSCC) is characterized by high morbidity and mortality. Treatment failure, drug resistance and chemoradiation toxicity have necessitated the development of alternative treatment strategies. Styryl benzyl sulfones, a family of novel small molecule inhibitors, are being evaluated as anti-neoplastic agents in multiple clinical trials. The activity of these compounds has been well characterized in several preclinical tumor studies, but their activity has yet to be fully examined in HNSCC. We tested ON 01910.Na (rigosertib), a styryl benzyl sulfone in late-stage development, in HNSCC preclinical models. Rigosertib induced cytotoxicity in both HPV(+) and HPV(−) HNSCC cells in a dose-dependent manner. Characterization of the underlying molecular mechanism indicated that rigosertib induced inhibition of the PI3K/Akt/mTOR pathway, induced oxidative stress resulting in increased generation of reactive oxygen species (ROS), and activated extracellular signal-regulated kinases (ERK1/2) and c-Jun NH2-terminal kinase (JNK). Increased phosphorylation and cytoplasmic translocation of ATF-2 were also observed following rigosertib treatment. These changes in cell signaling led us to consider combining rigosertib with HNSCC standard-of-care therapies, such as cisplatin and radiation. Our study highlights the promising preclinical activity of rigosertib in HNSCC irrespective of HPV status and provides a molecular basis for rigosertib in combination with standard of care agents for HNSCC. PMID:27764820

  10. Protease-activated receptor-2 promotes kidney tubular epithelial inflammation by inhibiting autophagy via the PI3K/Akt/mTOR signalling pathway.

    PubMed

    Du, Chunyang; Zhang, Tao; Xiao, Xia; Shi, Yonghong; Duan, Huijun; Ren, Yunzhuo

    2017-08-02

    Protease-activated receptor-2 (PAR2), which belongs to a specific class of the G-protein-coupled receptors, is central to several inflammation processes. However, the precise molecular mechanism involved remains undefined. Autophagy has been previously shown to affect inflammation. In the present study, we examine the effect of PAR2 on kidney tubular epithelial autophagy and on autophagy-related inflammation and reveal the underlying mechanism involved. Autophagic activity and levels of autophagic marker LC3 were examined in human kidney tubular epithelial cells with PAR2 knockdown or overexpression. We administered the mammalian target of rapamycin (mTOR) inhibitor (rapamycin) or activator (MHY1485) to investigate the function of the phosphoinositide 3-kinase (PI3K)/Akt/mTOR pathway. We also used transforming growth factor-β1 (TGF-β1)-induced HK-2 cell inflammation models to investigate the role of PAR2-associated autophagy in kidney tubular epithelial inflammation. PAR2 antagonist and rapamycin were administered to mice after unilateral ureteral obstruction to detect the correlations between PAR2, autophagy, and inflammation. Our results show that PAR2 overexpression in HK-2 cells led to a greater reduction in autophagy via the PI3K/Akt/mTOR pathway activation and induces autophagy-related inflammation. Meanwhile, a knockdown of PAR2 via PAR2 RNAi transfection greatly increased autophagy and alleviated autophagy-associated inflammation. In unilateral ureteral obstruction (UUO) kidneys, PAR2 antagonist treatment greatly attenuated renal inflammation and interstitial injury by enhancing autophagy. Moreover, inhibition of mTOR, rapa, markedly increased autophagy and inhibited the UUO-induced inflammation. We conclude that PAR2 induces kidney tubular epithelial inflammation by inhibiting autophagy via the PI3K/Akt/mTOR signalling pathway. Our results are suggestive that PAR2 inhibition may play a role in the treatment of diseases with increased inflammatory

  11. Activation of GRs-Akt-nNOs-NR2B signaling pathway by second dose GR agonist contributes to exacerbated hyperalgesia in a rat model of radicular pain.

    PubMed

    Zhang, Jing; Zhang, Wei; Sun, Yu'e; Liu, Yue; Song, Lihua; Ma, Zhengliang; Gu, Xiaoping

    2014-06-01

    Central Akt, neuronal nitric oxide synthase (nNOS) and N-methyl-D-aspartate receptor subunit 2B (NR2B) play key roles in the development of neuropathic pain. Here we investigate the effects of glucocorticoid receptors (GRs) on the expression and activation of spinal Akt, nNOS and NR2B after chronic compression of dorsal root ganglia (CCD). Thermal hyperalgesia test and mechanical allodynia test were used to measure rats after intrathecal injection of GR antagonist mifepristone or GR agonist dexamethasone for 21 days postoperatively. Expression of spinal Akt, nNOS, NR2B and their phosphorylation state after CCD was examined by western blot. The effects of intrathecal treatment with dexamethasone or mifepristone on nociceptive behaviors and the corresponding expression of Akt, nNOS and NR2B in spinal cord were also investigated. Intrathecal injection of mifepristone or dexamethasone inhibited PWMT and PWTL in CCD rats. However, hyperalgesia was induced by intrathecal injection of dexamethasone on days 12 to 14 after surgery. Treatment of dexamethasone increased the expression and phosphorylation levels of spinal Akt, nNOS, GR and NR2B time dependently, whereas administration of mifepristone downregulated the expression of these proteins significantly. GRs activated spinal Akt-nNOS/NR2B pathway play important roles in the development of neuropathic pain in a time-dependent manner.

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

    PubMed Central

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

    2015-01-01

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

  13. Phosphodiesterase 5a Inhibition with Adenoviral Short Hairpin RNA Benefits Infarcted Heart Partially through Activation of Akt Signaling Pathway and Reduction of Inflammatory Cytokines

    PubMed Central

    Jin, Zhe; Zhang, Jian; Paul, Christian; Wang, Yigang

    2015-01-01

    Introduction Treatment with short hairpin RNA (shRNA) interference therapy targeting phosphodiesterase 5a after myocardial infarction (MI) has been shown to mitigate post-MI heart failure. We investigated the mechanisms that underpin the beneficial effects of PDE5a inhibition through shRNA on post-MI heart failure. Methods An adenoviral vector with an shRNA sequence inserted was adopted for the inhibition of phosphodiesterase 5a (Ad-shPDE5a) in vivo and in vitro. Myocardial infarction (MI) was induced in male C57BL/6J mice by left coronary artery ligation, and immediately after that, the Ad-shPDE5a was injected intramyocardially around the MI region and border areas. Results Four weeks post-MI, the Ad-shPDE5a-treated mice showed significant mitigation of the left ventricular (LV) dilatation and dysfunction compared to control mice. Infarction size and fibrosis were also significantly reduced in Ad-shPDE5a-treated mice. Additionally, Ad-shPDE5a treatment decreased the MI-induced inflammatory cytokines interleukin (IL)-1β, IL-6, tumor necrosis factor-α, and transforming growth factor-β1, which was confirmed in vitro in Ad-shPDE5a transfected myofibroblasts cultured under oxygen glucose deprivation. Finally, Ad-shPDE5a treatment was found to activate the myocardial Akt signaling pathway in both in vivo and in vitro experiments. Conclusion These findings indicate that PDE5a inhibition by Ad-shPDE5a via the Akt signal pathway could be of significant value in the design of future therapeutics for post-MI heart failure. PMID:26709517

  14. Hydrogen Sulfide Preconditioning Protects Rat Liver against Ischemia/Reperfusion Injury by Activating Akt-GSK-3β Signaling and Inhibiting Mitochondrial Permeability Transition

    PubMed Central

    Zhang, Hao; Xu, Fengying; Zou, Zui; Liu, Meng; Wang, Quanxing; Miao, Mingyong; Shi, Xueyin

    2013-01-01

    Hydrogen sulfide (H2S) is the third most common endogenously produced gaseous signaling molecule, but its impact on hepatic ischemia/reperfusion (I/R) injury, especially on mitochondrial function, remains unclear. In this study, rats were randomized into Sham, I/R, ischemia preconditioning (IPC) or sodium hydrosulfide (NaHS, an H2S donor) preconditioning groups. To establish a model of segmental (70%) warm hepatic ischemia, the hepatic artery, left portal vein and median liver lobes were occluded for 60 min and then unclamped to allow reperfusion. Preconditioning with 12.5, 25 or 50 μmol/kg NaHS prior to the I/R insult significantly increased serum H2S levels, and, similar to IPC, NaHS preconditioning decreased alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels in the plasma and prevented hepatocytes from undergoing I/R-induced necrosis. Moreover, a sub-toxic dose of NaHS (25 μmol/kg) did not disrupt the systemic hemodynamics but dramatically inhibited mitochondrial permeability transition pore (MPTP) opening and thus prevented mitochondrial-related cell death and apoptosis. Mechanistic studies revealed that NaHS preconditioning markedly increased the expression of phosphorylated protein kinase B (p-Akt), phosphorylated glycogen synthase kinase-3 beta (p-GSK-3β) and B-cell lymphoma-2 (Bcl-2) and decreased the release of mitochondrial cytochrome c and cleaved caspase-3/9 levels. Therefore, NaHS administration prior to hepatic I/R ameliorates mitochondrial and hepatocellular damage through the inhibition of MPTP opening and the activation of Akt-GSK-3β signaling. Furthermore, this study provides experimental evidence for the clinical use of H2S to reduce liver damage after perioperative I/R injury. PMID:24058562

  15. FTY720 prevents ischemia/reperfusion injury-associated arrhythmias in an ex vivo rat heart model via activation of Pak1/Akt signaling.

    PubMed

    Egom, E Eroume A; Ke, Yunbo; Musa, Hanny; Mohamed, Tamer M A; Wang, Tao; Cartwright, Elizabeth; Solaro, R John; Lei, Ming

    2010-02-01

    Recent studies demonstrated a role of sphingosine-1-phosphate (S1P) in the protection against the stress of ischemia/reperfusion (I/R) injury. In experiments reported here, we have investigated the signaling through the S1P cascade by FTY720, a sphingolipid drug candidate displaying structural similarity to S1P, underlying the S1P cardioprotective effect. In ex vivo rat heart and isolated sinoatrial node models, FTY720 significantly prevented arrhythmic events associated with I/R injury including premature ventricular beats, VT, and sinus bradycardia as well as A-V conduction block. Real-time PCR and Western blot analysis demonstrated the expression of the S1P receptor transcript pools and corresponding proteins including S1P1, S1P2, and S1P3 in tissues dissected from sinoatrial node, atrium and ventricle. FTY720 (25 nM) significantly blunted the depression of the levels of phospho-Pak1 and phospho-Akt with ischemia and with reperfusion. There was a significant increase in phospho-Pak1 levels by 35%, 199%, and 205% after 5, 10, and 15 min of treatment with 25 nM FTY720 compared with control nontreated myocytes. However, there was no significant difference in the levels of total Pak1 expression between nontreated and FTY720 treated. Phospho-Akt levels were increased by 44%, 63%, and 61% after 5, 10, and 15 min of treatment with 25 nM FTY720, respectively. Our data provide the first evidence that FTY720 prevents I/R injury-associated arrhythmias and indicate its potential significance as an important and new agent protecting against I/R injury. Our data also indicate, for the first time, that the cardioprotective effect of FTY720 is likely to involve activation of signaling through the Pak1.

  16. Multiple roles and therapeutic implications of Akt signaling in cancer

    PubMed Central

    Calvo, Emiliano; Bolós, Victoria; Grande, Enrique

    2009-01-01

    The prominence of the PI3K-Akt signaling pathway in several tumors indicates a relationship with tumor grade and proliferation. Critical cellular processes are driven through this pathway. More detailed knowledge of the pathogenesis of tumors would enable us to design targeted drugs to block both membrane tyrosine kinase receptors and the intracellular kinases involved in the transmission of the signal. The newly approved molecular inhibitors sunitinib (an inhibitor of vascular endothelial growth factor receptor, platelet-derived growth factor receptor, and other tyrosine kinase receptors), sorafenib (a serine–threonine kinase inhibitor that acts against B-Raf) and temsirolimus (an mTOR inhibitor) shown clinical activity in advanced kidney cancer. Chronic myeloid leukemia has changed its natural history thanks to imatinib and dasatinib, both of which inhibit the intracellular bcr/abl protein derived from the alteration in the Philadelphia chromosome. Intracellular pathways are still important in cancer development and their blockade directly affects outcome. Cross-talk has been observed but is not well understood. Vertical and horizontal pathway blockade are promising anticancer strategies. Indeed, preclinical and early clinical data suggest that combining superficial and intracellular blocking agents can synergize and leverage single-agent activity. The implication of the Akt signaling pathway in cancer is well established and has led to the development of new anticancer agents that block its activation. PMID:20616901

  17. Crocin Inhibits Oxidative Stress and Pro-inflammatory Response of Microglial Cells Associated with Diabetic Retinopathy Through the Activation of PI3K/Akt Signaling Pathway.

    PubMed

    Yang, Xinguang; Huo, Fuquan; Liu, Bei; Liu, Jing; Chen, Tao; Li, Junping; Zhu, Zhongqiao; Lv, Bochang

    2017-02-25

    Diabetic retinopathy (DR) is a serious microvascular complication of diabetes mellitus that is closely associated with the degeneration and loss of retinal ganglion cells (RGCs) caused by diabetic microangiopathy and subsequent oxidative stress and an inflammatory response. Microglial cells are classed as neurogliocytes and play a significant role in neurodegenerative diseases. Over-activated microglial cells may cause neurotoxicity and induce the death and apoptosis of RGCs. Crocin is one of the two most pharmacologically bioactive constituents in saffron. In the present study, we focused on the role of microglial cells in DR, suggesting that DR may cause the over-activation of microglial cells and induce oxidative stress and the release of pro-inflammatory factors. Microglial cells BV-2 and N9 were cultured, and high-glucose (HG) and free fatty acid (FFA) were used to simulate diabetes. The results showed that HG-FFA co-treatment caused the up-regulated expression of CD11b and Iba-1, indicating that BV-2 and N9 cells were over-activated. Moreover, oxidative stress markers and pro-inflammatory factors were significantly enhanced by HG-FFA treatment. We found that crocin prevented the oxidative stress and pro-inflammatory response induced by HG-FFA co-treatment. Moreover, using the PI3K/Akt inhibitor LY294002, we revealed that PI3K/Akt signaling plays a significant role in blocking oxidative stress, suppressing the pro-inflammatory response, and maintaining the neuroprotective effects of crocin. In total, these results provide a new insight into DR and DR-induced oxidative stress and the inflammatory response, which provide a potential therapeutic target for neuronal damage, vision loss, and other DR-induced complications.

  18. Activation of phosphatidylinositol 3-kinase/Akt-mammalian target of Rapamycin signaling pathway in the hippocampus is essential for the acquisition of morphine-induced place preference in rats.

    PubMed

    Cui, Yue; Zhang, X Q; Cui, Y; Xin, W J; Jing, J; Liu, X G

    2010-11-24

    Hippocampus is a critical structure for the acquisition of morphine-induced conditioned place preference (CPP), which is a usual learning paradigm for assessing drug reward. However, the precise mechanisms remain largely unknown. Phosphatidylinositol 3-kinase (PI3K) and its downstream targets, including Akt, mammalian target of Rapamycin (mTOR) and 70-kDa ribosomal S6 kinase (p70S6K), are critical molecules implicated in learning and memory. Here, we tested the role of PI3K/Akt-mTOR-p70S6K signaling pathway in morphine-induced CPP in the hippocampus. Our results showed that the acquisition of morphine CPP increased phosphorylation of Akt in the hippocampal CA3, but not in the nucleus accumbens (NAc), the ventral tegmental area (VTA) or the CA1. Moreover, the phosphorylated Akt exclusively expressed in the CA3 neurons. Likewise, levels of phosphorylated mTOR and p70S6K were significantly enhanced in the CA3 following morphine CPP. The alterations of these phosphorylated proteins are positively correlated with the acquisition of morphine CPP. More importantly, microinjection of PI3K inhibitor (LY294002) or mTOR inhibitor (Rapamycin) into the CA3 prevented the acquisition of CPP and inhibited the activation of PI3K-Akt signaling pathway. In addition, pre-infusion of β-FNA (β-funaltrexamine hydrochloride), a selective irreversible μ opioid receptor antagonist, into CA3 significantly prevented the acquisition of CPP and impaired Akt phosphorylation. All these results strongly implied that the PI3K-Akt signaling pathway activated by μ opioid receptor in hippocampal CA3 plays an important role in acquisition of morphine-induced CPP.

  19. Integrin β1, myosin light chain kinase and myosin IIA are required for activation of PI3K-AKT signaling following MEK inhibition in metastatic triple negative breast cancer

    PubMed Central

    Choi, Cheolwon; Kwon, Junyeob; Lim, Sunyoung; Helfman, David M.

    2016-01-01

    The effectiveness of targeted therapies against the Ras-ERK signaling pathway are limited due to adaptive resistance of tumor cells. Inhibition of the Ras-ERK pathway can result in activation of the PI3K-AKT pathway, thereby diminishing the therapeutic effects of targeting ERK signaling. Here we investigated the crosstalk between the Ras-ERK and PI3K-AKT pathways in MDA-MB-231 breast cancer cell lines that have a preference to metastasize to lung (LM2), brain (BrM2) or bone (BoM2). Inhibition of the Ras-ERK pathway reduced motility in both parental and BoM2 cells. In contrast, inhibition of the Ras-ERK pathway in BrM2 and LM2 cells resulted in activation of PI3K-AKT signaling that was responsible for continued cell motility. Analysis of the cross talk between Ras-ERK and PI3K-AKT signaling pathways revealed integrin β1, myosin light chain kinase (MLCK) and myosin IIA are required for the activation of PI3K-AKT following inhibition of the Ras-ERK pathway. Furthermore, feedback activation of the PI3K-AKT pathway following MEK suppression was independent of the epidermal growth factor receptor. Thus, integrin β1, MLCK, and myosin IIA are factors in the development of resistance to MEK inhibitors. These proteins could provide an opportunity to develop markers and therapeutic targets in a subgroup of triple negative breast cancer (TNBC) that exhibit resistance against MEK inhibition. PMID:27563827

  20. AKT/GSK3β signaling pathway is critically involved in human pluripotent stem cell survival

    PubMed Central

    Romorini, Leonardo; Garate, Ximena; Neiman, Gabriel; Luzzani, Carlos; Furmento, Verónica Alejandra; Guberman, Alejandra Sonia; Sevlever, Gustavo Emilio; Scassa, María Elida; Miriuka, Santiago Gabriel

    2016-01-01

    Human embryonic and induced pluripotent stem cells are self-renewing pluripotent stem cells (PSC) that can differentiate into a wide range of specialized cells. Basic fibroblast growth factor is essential for PSC survival, stemness and self-renewal. PI3K/AKT pathway regulates cell viability and apoptosis in many cell types. Although it has been demonstrated that PI3K/AKT activation by bFGF is relevant for PSC stemness maintenance its role on PSC survival remains elusive. In this study we explored the molecular mechanisms involved in the regulation of PSC survival by AKT. We found that inhibition of AKT with three non-structurally related inhibitors (GSK690693, AKT inhibitor VIII and AKT inhibitor IV) decreased cell viability and induced apoptosis. We observed a rapid increase in phosphatidylserine translocation and in the extent of DNA fragmentation after inhibitors addition. Moreover, abrogation of AKT activity led to Caspase-9, Caspase-3, and PARP cleavage. Importantly, we demonstrated by pharmacological inhibition and siRNA knockdown that GSK3β signaling is responsible, at least in part, of the apoptosis triggered by AKT inhibition. Moreover, GSK3β inhibition decreases basal apoptosis rate and promotes PSC proliferation. In conclusion, we demonstrated that AKT activation prevents apoptosis, partly through inhibition of GSK3β, and thus results relevant for PSC survival. PMID:27762303

  1. Lentiviral shRNA against KCa3.1 inhibits allergic response in allergic rhinitis and suppresses mast cell activity via PI3K/AKT signaling pathway

    PubMed Central

    Lin, Hai; Zheng, Chunquan; Li, Jing; Yang, Chen; Hu, Li

    2015-01-01

    Calcium-activated potassium ion channel-3.1 (KCa3.1) plays a pivotal role in the potassium-calcium exchange involved in atopy. This study aimed to explore the impact of lentiviral-mediated shRNA silencing KCa3.1 on allergic response in a murine allergic rhinitis (AR) model. The BALB/c mice were divided into four groups: untreated AR group, negative control AR group, lentiviral KCa3.1-shRNA treated AR group and normal control group. Concentrations of ovalbumin (OVA)-specific IgE, histamine and leukotrienes C4 (LTC4) in serum, and IL-4, IL-9 and IL-17 in nasal lavage fluid (NLF) were analyzed. Goblet cells and mast cells were counted. KCa3.1 positive cells were counted after immunolabelling by immunofluorescence method. KCa3.1, Mucin 5AC (MUC5AC), and tryptase mRNA levels were determined using real-time polymerase chain reaction. Furthermore, P815 cell line was used to explore the role and mechanism of lentiviral KCa3.1-shRNA on mast cells. The results showed that LV-KCa3.1-shRNA intervention effectively attenuated allergic responses in LV-KCa3.1-shRNA treated mice. LV-KCa3.1-shRNA intervention effectively suppressed KCa3.1 levels and phosphorylation of AKT in P815 cells, leading to the downregulation of tryptase, IL-6 and IL-8 levels. LV-KCa3.1-shRNA intervention effectively attenuated the allergic responses in AR and suppressed mast cell activity by inhibiting PI3K/AKT signaling pathway. PMID:26272420

  2. Epoxyeicosatrienoic acid agonist regulates human mesenchymal stem cell-derived adipocytes through activation of HO-1-pAKT signaling and a decrease in PPARγ.

    PubMed

    Kim, Dong Hyun; Vanella, Luca; Inoue, Kazuyoshi; Burgess, Angela; Gotlinger, Katherine; Manthati, Vijaya Lingam; Koduru, Sreenivasulu Reddy; Zeldin, Darryl C; Falck, John R; Schwartzman, Michal L; Abraham, Nader G

    2010-12-01

    Human mesenchymal stem cells (MSCs) expressed substantial levels of CYP2J2, a major CYP450 involved in epoxyeicosatrienoic acid (EET) formation. MSCs synthesized significant levels of EETs (65.8 ± 5.8 pg/mg protein) and dihydroxyeicosatrienoic acids (DHETs) (15.83 ± 1.62 pg/mg protein), suggesting the presence of soluble epoxide hydrolase (sEH). The addition of an sEH inhibitor to MSC culture decreased adipogenesis. EETs decreased MSC-derived adipocytes in a concentration-dependent manner, 8,9- and 14,15-EET having the maximum reductive effect on adipogenesis. We examined the effect of 12-(3-hexylureido)dodec-8(Z)-enoic acid, an EET agonist, on MSC-derived adipocytes and demonstrated an increased number of healthy small adipocytes, attenuated fatty acid synthase (FAS) levels (P < 0.01), and reduced PPARγ, C/EBPα, FAS, and lipid accumulation (P < 0.05). These effects were accompanied by increased levels of heme oxygenase (HO)-1 and adiponectin (P < 0.05), and increased glucose uptake (P < 0.05). Inhibition of HO activity or AKT by tin mesoporphyrin (SnMP) and LY2940002, respectively, reversed EET-induced inhibition of adipogenesis, suggesting that activation of the HO-1-adiponectin axis underlies EET effect in MSCs. These findings indicate that EETs decrease MSC-derived adipocyte stem cell differentiation by upregulation of HO-1-adiponectin-AKT signaling and play essential roles in the regulation of adipocyte differentiation by inhibiting PPARγ, C/EBPα, and FAS and in stem cell development. These novel observations highlight the seminal role of arachidonic acid metabolism in MSCs and suggest that an EET agonist may have potential therapeutic use in the treatment of dyslipidemia, diabetes, and the metabolic syndrome.

  3. Lipid raft-mediated Akt signaling as a therapeutic target in mantle cell lymphoma

    PubMed Central

    Reis-Sobreiro, M; Roué, G; Moros, A; Gajate, C; de la Iglesia-Vicente, J; Colomer, D; Mollinedo, F

    2013-01-01

    Recent evidence shows that lipid raft membrane domains modulate both cell survival and death. Here, we have found that the phosphatidylinositol-3-kinase (PI3K)/Akt signaling pathway is present in the lipid rafts of mantle cell lymphoma (MCL) cells, and this location seems to be critical for full activation and MCL cell survival. The antitumor lipids (ATLs) edelfosine and perifosine target rafts, and we found that ATLs exerted in vitro and in vivo antitumor activity against MCL cells by displacing Akt as well as key regulatory kinases p-PDK1 (phosphatidylinositol-dependent protein kinase 1), PI3K and mTOR (mammalian TOR) from lipid rafts. This raft reorganization led to Akt dephosphorylation, while proapoptotic Fas/CD95 death receptor was recruited into rafts. Raft integrity was critical for Ser473 Akt phosphorylation. ATL-induced apoptosis appeared to correlate with the basal Akt phosphorylation status in MCL cell lines and primary cultures, and could be potentiated by the PI3K inhibitor wortmannin, or inhibited by the Akt activator pervanadate. Classical Akt inhibitors induced apoptosis in MCL cells. Microenvironmental stimuli, such as CD40 ligation or stromal cell contact, did not prevent ATL-induced apoptosis in MCL cell lines and patient-derived cells. These results highlight the role of raft-mediated PI3K/Akt signaling in MCL cell survival and chemotherapy, thus becoming a new target for MCL treatment. PMID:23727661

  4. Blockade of the phosphatidylinositol-3-kinase-Akt signaling pathway enhances the induction of apoptosis by microtubule-destabilizing agents in tumor cells in which the pathway is constitutively activated.

    PubMed

    Fujiwara, Yusuke; Hosokawa, Yoshihisa; Watanabe, Kazushi; Tanimura, Susumu; Ozaki, Kei-ichi; Kohno, Michiaki

    2007-03-01

    Constitutive activation of the phosphatidylinositol-3-kinase (PI3K)-Akt signaling pathway is associated with the neoplastic phenotype in many human tumor cell types. Given the antiapoptotic role of this pathway, we examined whether its specific blockade might sensitize human tumor cells to the induction of apoptosis by various anticancer drugs. Although specific blockade of the PI3K-Akt pathway alone with inhibitors such as LY294002 did not induce cell death, it resulted in marked and selective enhancement of the induction of apoptosis by microtubule-destabilizing agents such as vincristine. This effect was apparent only in tumor cells in which the PI3K-Akt pathway is constitutively activated. Blockade of the PI3K-Akt pathway induced the activation of glycogen synthase kinase-3beta, which phosphorylates microtubule-associated proteins such as tau and thereby reduces their ability to bind and stabilize microtubules. The consequent destabilization of microtubules induced by the inhibition of PI3K-Akt signaling appeared to increase their sensitivity to low concentrations of microtubule-destabilizing agents that alone do not lead to the disruption of cytoplasmic microtubules in tumor cells. Such a synergistic effect on microtubule integrity was not apparent for stable microtubules in the neurites of neuronal cells. These results suggest that the administration of a combination of a PI3K-Akt pathway inhibitor and a microtubule-destabilizing agent is a potential chemotherapeutic strategy for the treatment of tumor cells in which this signaling pathway is constitutively activated.

  5. Curcumin Inhibits Heat-Induced Apoptosis by Suppressing NADPH Oxidase 2 and Activating the Akt/mTOR Signaling Pathway in Bronchial Epithelial Cells.

    PubMed

    Peng, Yuan; Pu, Junliang; Tang, Chengyong; Wu, Zhongjun

    2017-01-01

    underlying curcumin inhibiting heat-induced apoptosis is through suppressing NADPH Oxidase 2 and activating the Akt/mTOR signaling pathway in bronchial epithelial cells. © 2017 The Author(s). Published by S. Karger AG, Basel.

  6. High expression of G-protein signaling modulator 2 in hepatocellular carcinoma facilitates tumor growth and metastasis by activating the PI3K/AKT signaling pathway.

    PubMed

    He, Xiao-Qin; Zhang, Yue-Feng; Yu, Jia-Jun; Gan, Yuan-Yuan; Han, Na-Na; Zhang, Mei-Xia; Ge, Wei; Deng, Jun-Jian; Zheng, Yong-Fa; Xu, Xi-Ming

    2017-03-01

    The aim of this study was to investigate the role of G-protein signaling modulator 2 in the carcinogenesis and progression of hepatocellular carcinoma. We previously showed that G-protein signaling modulator 2 was upregulated in hepatitis B virus-related hepatocellular carcinoma tissues through a hierarchical clustering analysis. With this study, we first assessed the expression pattern of G-protein signaling modulator 2 in hepatocellular carcinoma specimens and adjacent noncancerous tissues; clinical data were analyzed, along survival times, utilizing the Kaplan-Meier method. Moreover, the functions of G-protein signaling modulator 2 were examined using small-interfering RNAs in vitro. The results showed that G-protein signaling modulator 2 was clearly overexpressed in hepatocellular carcinoma tissues and cell lines and that the G-protein signaling modulator 2 expression level was related to tumor size and hepatitis B virus infection. Furthermore, G-protein signaling modulator 2 knockdown studies suggested that G-protein signaling modulator 2 accelerates cell growth, cell cycle, migration, and invasion and inhibits apoptosis, acting as an oncogene in hepatocellular carcinoma. Western blotting indicated that silencing of G-protein signaling modulator 2 in HepG2 and SMMC-7721 cells increased the expression levels of Bax, caspase-3, and E-cadherin, while notably suppressing the cyclin-dependent kinase 4, cyclin-dependent kinase 6, CyclinD1, Snail1, Vimentin, and matrix metallopeptidase 9 expression levels, compared with that in the control groups. In addition, we found that G-protein signaling modulator 2 can affect the expression of key proteins involved in protein kinase B activation. In conclusion, high expression of G-protein signaling modulator 2 was involved in the pathological processes of hepatocellular carcinoma through activation of the phosphatidylinositol 3-kinase/protein kinase B signaling pathway, which may provide an attractive potential diagnostic

  7. Hesperidin attenuates learning and memory deficits in APP/PS1 mice through activation of Akt/Nrf2 signaling and inhibition of RAGE/NF-κB signaling.

    PubMed

    Hong, Yan; An, Zhongping

    2015-09-21

    Numerous studies have demonstrated that oxidative stress and inflammation play a pivotal role in the pathophysiology of Alzheimer disease (AD). Hesperidin (HP) has various pharmacological effects including anti-oxidative, anti-inflammatory and neuroprotective properties. In this study, APP/PS1 mice were used to evaluate the neuroprotective effects of HP. We reported that intragastric administration of HP (40 mg/kg) for 90 days significantly attenuated cognitive impairment in APP/PS1 mice. HP treatment suppressed oxidative stress by reducing the levels of ROS, LPO, protein carbonyl and 8-OHdG and increasing the activity of HO-1, SOD, catalase, and GSH-Px. HP treatment also inhibited inflammation by decreasing the levels of TNF-α, C-reactive protein and MCP-1 and reducing the activity of NF-κB. Moreover, HP could reverse the decreased phosphorylation of Akt, the decreased phosphorylation of GSK-3β, the lessened Nrf2 and the reduced expression of HO-1. HP could also inhibit the increased the RAGE expression, the enhanced phosphorylation of IκBα, and the augmented nuclear translocation of NF-κB/p65 in cortex of APP/PS1 mice. Taken together, HP suppresses oxidative stress and inflammation via activation of Akt/Nrf2 signaling and inhibition of RAGE/NF-κB signaling and further confers neuroprotection.

  8. 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.

  9. 4-Ketopinoresinol, a novel naturally occurring ARE activator, induces the Nrf2/HO-1 axis and protects against oxidative stress-induced cell injury via activation of PI3K/AKT signaling.

    PubMed

    Chen, Huang-Hui; Chen, Yu-Tsen; Huang, Yen-Wen; Tsai, Hui-Ju; Kuo, Ching-Chuan

    2012-03-15

    The Nrf2/ARE pathway plays an important role in inducing phase II detoxifying enzymes and antioxidant proteins and has been considered a potential target for cancer chemoprevention because it eliminates harmful reactive oxygen species or reactive intermediates generated from carcinogens. The objectives of this study were to identify novel Nrf2/ARE activators and to investigate the mechanistic signaling pathway involved in the activation of Nrf2-mediated cytoprotective effects against oxidative-induced cell injury. A stable ARE-driven luciferase reporter cell line was established to screen a potentially cytoprotective compound. 4-Ketopinoresinol (4-KPR), the (α-γ) double-cyclized type of lignan obtained from adlay (Coix lachryma-jobi L. var. ma-yuen Stapf), activates ARE-driven luciferase activity more effectively than the classical ARE activator tert-butylhydroquinone. 4-KPR treatment resulted in a transient increase in AKT phosphorylation and subsequent phosphorylation and nuclear translocation of Nrf2, along with increased expression of ARE-dependent cytoprotective genes, such as heme oxygenase-1 (HO-1), aldo-keto reductases, and glutathione synthetic enzyme. 4-KPR suppresses oxidative stress-induced DNA damage and cell death via upregulation of HO-1. Inhibition of PI3K/AKT signaling by chemical inhibitors or RNA interference not only suppressed 4-KPR-induced Nrf2/HO-1 activation, but also eliminated the cytoprotective effect against oxidative damage. These observations in an ARE-regulated gene system suggest that 4-KPR is a novel Nrf2/ARE-mediated transcription activator, activates the Nrf2/HO-1 axis, and protects against oxidative stress-induced cell injury via activation of PI3K/AKT signaling.

  10. Phospholipase D2 mediates survival signaling through direct regulation of Akt in glioblastoma cells.

    PubMed

    Bruntz, Ronald C; Taylor, Harry E; Lindsley, Craig W; Brown, H Alex

    2014-01-10

    The lack of innovative drug targets for glioblastoma multiforme (GBM) limits patient survival to approximately 1 year following diagnosis. The pro-survival kinase Akt provides an ideal target for the treatment of GBM as Akt signaling is frequently activated in this cancer type. However, the central role of Akt in physiological processes limits its potential as a therapeutic target. In this report, we show that the lipid-metabolizing enzyme phospholipaseD(PLD) is a novel regulator of Akt inGBM.Studies using a combination of small molecule PLD inhibitors and siRNA knockdowns establish phosphatidic acid, the product of the PLD reaction, as an essential component for the membrane recruitment and activation of Akt. Inhibition of PLD enzymatic activity and subsequent Akt activation decreases GBM cell viability by specifically inhibiting autophagic flux. We propose a mechanism whereby phosphorylation of beclin1 by Akt prevents binding of Rubicon (RUN domain cysteine-rich domain containing beclin1-interacting protein), an interaction known to inhibit autophagic flux. These findings provide a novel framework through which Akt inhibition can be achieved without directly targeting the kinase.

  11. Phospholipase D2 Mediates Survival Signaling through Direct Regulation of Akt in Glioblastoma Cells*♦

    PubMed Central

    Bruntz, Ronald C.; Taylor, Harry E.; Lindsley, Craig W.; Brown, H. Alex

    2014-01-01

    The lack of innovative drug targets for glioblastoma multiforme (GBM) limits patient survival to approximately 1 year following diagnosis. The pro-survival kinase Akt provides an ideal target for the treatment of GBM as Akt signaling is frequently activated in this cancer type. However, the central role of Akt in physiological processes limits its potential as a therapeutic target. In this report, we show that the lipid-metabolizing enzyme phospholipase D (PLD) is a novel regulator of Akt in GBM. Studies using a combination of small molecule PLD inhibitors and siRNA knockdowns establish phosphatidic acid, the product of the PLD reaction, as an essential component for the membrane recruitment and activation of Akt. Inhibition of PLD enzymatic activity and subsequent Akt activation decreases GBM cell viability by specifically inhibiting autophagic flux. We propose a mechanism whereby phosphorylation of beclin1 by Akt prevents binding of Rubicon (RUN domain cysteine-rich domain containing beclin1-interacting protein), an interaction known to inhibit autophagic flux. These findings provide a novel framework through which Akt inhibition can be achieved without directly targeting the kinase. PMID:24257753

  12. Angiotensin II Signaling in Human Preadipose Cells: Participation of ERK1,2-Dependent Modulation of Akt

    PubMed Central

    Dünner, Natalia; Quezada, Carolina; Berndt, F. Andrés; Cánovas, José; Rojas, Cecilia V.

    2013-01-01

    The renin-angiotensin system expressed in adipose tissue has been implicated in the modulation of adipocyte formation, glucose metabolism, triglyceride accumulation, lipolysis, and the onset of the adverse metabolic consequences of obesity. As we investigated angiotensin II signal transduction mechanisms in human preadipose cells, an interplay of extracellular-signal-regulated kinases 1 and 2 (ERK1,2) and Akt/PKB became evident. Angiotensin II caused attenuation of phosphorylated Akt (p-Akt), at serine 473; the p-Akt/Akt ratio decreased to 0.5±0.2-fold the control value without angiotensin II (p<0.001). Here we report that the reduction of phosphorylated Akt associates with ERK1,2 activities. In the absence of angiotensin II, inhibition of ERK1,2 activation with U0126 or PD98059 resulted in a 2.1±0.5 (p<0.001) and 1.4±0.2-fold (p<0.05) increase in the p-Akt/Akt ratio, respectively. In addition, partial knockdown of ERK1 protein expression by the short hairpin RNA technique also raised phosphorylated Akt in these cells (the p-Akt/Akt ratio was 1.5±0.1-fold the corresponding control; p<0.05). Furthermore, inhibition of ERK1,2 activation with U0126 prevented the reduction of p-Akt/Akt by angiotensin II. An analogous effect was found on the phosphorylation status of Akt downstream effectors, the forkhead box (Fox) proteins O1 and O4. Altogether, these results indicate that angiotensin II signaling in human preadipose cells involves an ERK1,2-dependent attenuation of Akt activity, whose impact on the biological functions under its regulation is not fully understood. PMID:24098385

  13. Infectious bursal disease virus activates the phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway by interaction of VP5 protein with the p85{alpha} subunit of PI3K

    SciTech Connect

    Wei Li; Hou Lei; Zhu Shanshan; Wang Jing; Zhou Jiao; Liu Jue

    2011-08-15

    Phosphatidylinositol 3-kinase (PI3K)/Akt signaling is commonly activated upon virus infection and has been implicated in the regulation of diverse cellular functions such as proliferation and apoptosis. The present study demonstrated for the first time that infectious bursal disease virus (IBDV), the causative agent of a highly contagious disease in chickens, can induce Akt phosphorylation in cultured cells, by a mechanism that is dependent on PI3K. Inhibition of PI3K activation greatly enhanced virus-induced cytopathic effect and apoptotic cell death as evidenced by cleavage of poly-ADP ribose polymerase and activation of caspase-3. Investigations into the mechanism of PI3K/Akt activation revealed that IBDV activates PI3K/Akt signaling through binding of the non-structural protein VP5 to regulatory subunit p85{alpha} of PI3K resulting in the suppression of premature apoptosis and improved virus growth after infection. The results presented here provide a basis for understanding molecular mechanism of IBDV infection.

  14. Positive feedback loop of IL-1β/Akt/RARα/Akt signaling mediates oncogenic property of RARα in gastric carcinoma

    PubMed Central

    Huang, Gui-Li; Liu, Yu; Shen, Jin-Xing; Zhou, Pan; Liu, Wen-Ming; Shen, Dong-Yan

    2017-01-01

    Abnormal expression and function of retinoic acid receptor α (RARα) have been reported to be associated with various cancers including acute promyelocytic leukemia and hepatocellular carcinoma. However, the role and the mechanism of RARα in gastric carcinoma (GC) were unknown. Here, the expression of RARα was frequently elevated in human GC tissues and cell lines, and its overexpression was closely correlated with tumor size, lymph node metastasis and clinical stages in GC patients. Moreover, RARα overexpression was related with pathological differentiation. Functionally, RARα knockdown inhibited the proliferation and metastasis of GC cells, as well as enhanced drug susceptibility both in vitro and in vivo. Additionally, RARα knockdown suppressed GC progression through regulating the expression of cell proliferation, cell cycle, invasion and drug resistance associated proteins, such as PCNA, CyclinB1, CyclinD2, CyclinE, p21, MMP9 and MDR1. Mechanistically, the above oncogenic properties of RARα in GC were closely associated with Akt signaling activation. Moreover, overexpression of RARα was induced by IL-1β/Akt signaling activation, which suggested a positive feedback loop of IL-1β/Akt/RARα/Akt signaling in GC. Taken together, we demonstrated that RARα was frequently elevated in GC and exerted oncogenic properties. It might be a potential molecular target for GC treatment. PMID:28035062

  15. Survival signalling by Akt and eIF4E in oncogenesis and cancer therapy.

    PubMed

    Wendel, Hans-Guido; De Stanchina, Elisa; Fridman, Jordan S; Malina, Abba; Ray, Sagarika; Kogan, Scott; Cordon-Cardo, Carlos; Pelletier, Jerry; Lowe, Scott W

    2004-03-18

    Evading apoptosis is considered to be a hallmark of cancer, because mutations in apoptotic regulators invariably accompany tumorigenesis. Many chemotherapeutic agents induce apoptosis, and so disruption of apoptosis during tumour evolution can promote drug resistance. For example, Akt is an apoptotic regulator that is activated in many cancers and may promote drug resistance in vitro. Nevertheless, how Akt disables apoptosis and its contribution to clinical drug resistance are unclear. Using a murine lymphoma model, we show that Akt promotes tumorigenesis and drug resistance by disrupting apoptosis, and that disruption of Akt signalling using the mTOR inhibitor rapamycin reverses chemoresistance in lymphomas expressing Akt, but not in those with other apoptotic defects. eIF4E, a translational regulator that acts downstream of Akt and mTOR, recapitulates Akt's action in tumorigenesis and drug resistance, but is unable to confer sensitivity to rapamycin and chemotherapy. These results establish Akt signalling through mTOR and eIF4E as an important mechanism of oncogenesis and drug resistance in vivo, and reveal how targeting apoptotic programmes can restore drug sensitivity in a genotype-dependent manner.

  16. PTEN differentially regulates expressions of ICAM-1 and VCAM-1 through PI3K/Akt/GSK-3β/GATA-6 signaling pathways in TNF-α-activated human endothelial cells.

    PubMed

    Tsoyi, Konstantin; Jang, Hwa Jin; Nizamutdinova, Irina Tsoy; Park, Kyungok; Kim, Young Min; Kim, Hye Jung; Seo, Han Geuk; Lee, Jae Heun; Chang, Ki Churl

    2010-11-01

    Phosphotase and tensin homolog deleted on chromosome 10 (PTEN) is a potent negative regulator of PI3K/Akt pathway. Here, we tried to elucidate the role of PTEN in the regulation of endothelial adhesion molecules, vascular cell adhesion molecule (VCAM)-1 and intracellular adhesion molecule (ICAM)-1, induced by TNF-α in human endothelial cells (ECs). Transfection with PTEN overexpressing vector resulted in the significant decrease in phosphorylation of Akt in TNF-α-treated ECs. PTEN strongly inhibited VCAM-1 but not ICAM-1, however this inhibitory effect was reversed by co-transfection with constitutively active-Akt (CA-Akt-HA) in TNF-α-stimulated ECs. Additionally, silencing of PTEN with specific siRNA showed significant increase of phosphor-Akt compared with TNF-α alone treated ECs. siPTEN significantly upregulated VCAM-1 but was indifferent to ICAM-1 in TNF-α-treated cells. Further, chromatin immunoprecipitation (ChIP) assay showed that PTEN targets GATA-6 but not IRF-1 binding to VCAM-1 promoter. In addition, GATA-6 is associated with glycogen synthesis kinase-3beta (GSK-3β) which is in turn regulated by PTEN-dependent Akt activity. Finally, PTEN significantly prevented monocyte adhesion to TNF-α-induced ECs probably through VCAM-1 regulation. It is concluded that PTEN selectively inhibits expression of VCAM-1 but not ICAM-1 through modulation of PI3K/Akt/GSK-3β/GATA-6 signaling cascade in TNF-α-treated ECs.

  17. 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.

  18. Kallikrein/kinin protects against myocardial apoptosis after ischemia/reperfusion via Akt-glycogen synthase kinase-3 and Akt-Bad.14-3-3 signaling pathways.

    PubMed

    Yin, Hang; Chao, Lee; Chao, Julie

    2005-03-04

    Our previous study has shown that human tissue kallikrein protected against ischemia/reperfusion-induced myocardial injury. In the present study, we investigated the protective role of local kallikrein gene delivery in ischemia/reperfusion-induced cardiomyocyte apoptosis and its signaling mechanisms in promoting cardiomyocyte survival. Adenovirus carrying the human tissue kallikrein gene was delivered locally into the heart using a catheter-based technique. Expression and localization of recombinant human kallikrein in rat myocardium after gene transfer were determined immunohistochemically. Kallikrein gene delivery markedly reduced reperfusion-induced cardiomyocyte apoptosis identified by both in situ nick end-labeling and DNA fragmentation. Delivery of the kallikrein gene increased phosphorylation of Src, Akt, glycogen synthase kinase (GSK)-3beta, and Bad(Ser-136) but reduced caspase-3 activation in rat myocardium after reperfusion. The protective effect of kallikrein on apoptosis and its signaling mediators was blocked by icatibant and dominant-negative Akt, indicating a kinin B2 receptor-Akt-mediated event. Similarly, kinin or transduction of kallikrein in cultured cardiomyocytes promoted cell viability and attenuated apoptosis induced by hypoxia/reoxygenation. The effect of kallikrein on cardiomyocyte survival was blocked by dominant-negative Akt and a constitutively active mutant of GSK-3beta, but it was facilitated by constitutively active Akt, catalytically inactive GSK-3beta, lithium, and caspase-3 inhibitor. Moreover, kallikrein promoted Bad.14-3-3 complex formation and inhibited Akt-GSK-3beta-dependent activation of caspase-3, whereas caspase-3 administration caused reduction of the Bad.14-3-3 complex, indicating an interaction between Akt-GSK-caspase-3 and Akt-Bad.14-3-3 signaling pathways. In conclusion, kallikrein/kinin protects against cardiomyocyte apoptosis in vivo and in vitro via Akt-Bad.14-3-3 and Akt-GSK-3beta-caspase-3 signaling pathways.

  19. HER2-induced metastasis is mediated by AKT/JNK/EMT signaling pathway in gastric cancer

    PubMed Central

    Choi, Yiseul; Ko, Young San; Park, Jinju; Choi, Youngsun; Kim, Younghoon; Pyo, Jung-Soo; Jang, Bo Gun; Hwang, Douk Ho; Kim, Woo Ho; Lee, Byung Lan

    2016-01-01

    AIM To investigated the relationships between HER2, c-Jun N-terminal kinase (JNK) and protein kinase B (AKT) with respect to metastatic potential of HER2-positive gastric cancer (GC) cells. METHODS Immunohistochemistry was performed on tissue array slides containing 423 human GC specimens. Using HER2-positve GC cell lines SNU-216 and NCI-N87, HER2 expression was silenced by RNA interference, and the activations of JNK and AKT were suppressed by SP600125 and LY294002, respectively. Transwell assay, Western blot, semi-quantitative reverse transcription-polymerase chain reaction and immunofluorescence staining were used in cell culture experiments. RESULTS In GC specimens, HER2, JNK, and AKT activations were positively correlated with each other. In vitro analysis revealed a positive regulatory feedback loop between HER2 and JNK in GC cell lines and the role of JNK as a downstream effector of AKT in the HER2/AKT signaling pathway. JNK inhibition suppressed migratory capacity through reversing EMT and dual inhibition of JNK and AKT induced a more profound effect on cancer cell motility. CONCLUSION HER2, JNK and AKT in human GC specimens are positively associated with each other. JNK and AKT, downstream effectors of HER2, co-operatively contribute to the metastatic potential of HER2-positive GC cells. Thus, targeting of these two molecules in combination with HER2 downregulation may be a good approach to combat HER2-positive GC. PMID:27895401

  20. Constitutive activation of Akt contributes to the pathogenesis and survival of mantle cell lymphoma

    PubMed Central

    Rudelius, Martina; Pittaluga, Stefania; Nishizuka, Satoshi; Pham, Trinh H.-T.; Fend, Falko; Jaffe, Elaine S.; Quintanilla-Martinez, Leticia; Raffeld, Mark

    2006-01-01

    To determine whether the PI3K/Akt signaling pathway is involved in the pathogenesis of mantle cell lymphoma (MCL), we investigated the phosphorylation status of Akt and multiple downstream targets in primary MCL cases and cell lines. Akt was phosphorylated in 12 of 12 aggressive blastoid MCL variants and in 4 of 4 MCL cell lines. In contrast, phosphorylated Akt was present in only 5 of 16 typical MCL, 3 at comparable levels to the blastoid cases, and 2 at low levels. The presence of p-Akt was accompanied by the phosphorylation of p27kip1, FRKHL-1, MDM2, Bad, mTOR, and p70S6K. Inhibition of the PI3K/Akt pathway in the MCL cell lines abrogated or reduced the phosphorylation of Akt, p27kip1, FRKHL-1, MDM2, Bad, mTOR, GSK-3β, IκB, and led to cell-cycle arrest and apoptosis. Six MCL cases (5 with activated Akt and 1 with inactive Akt) and 3 of 4 cell lines showed loss of PTEN expression. PIK3CA mutations were not detected. We conclude that constitutive activation of the PI3K/Akt pathway contributes to the pathogenesis of MCL and preferentially occurs in blastoid variants. One possible mechanism of activation is loss of PTEN expression. These data suggest that PI3K/Akt inhibitors may be effective in the treatment of Akt-activated MCL. PMID:16645163

  1. Gli1 promotes colorectal cancer metastasis in a Foxm1-dependent manner by activating EMT and PI3K-AKT signaling

    PubMed Central

    Zhang, Yue; Ji, Bing; Wang, Sen; Sun, Ye; Zhu, Chunyan; Zhang, Dongsheng; Sun, Yueming

    2016-01-01

    Colorectal cancer(CRC) is one of the most commonly diagnosed cancers in human beings and metastasis is the main death reason. Recently, Gli1 has been reported to be a key regulator of various cancer biologies and genes expressions. However, the detailed molecular mechanism of Gli1 in CRC metastasis remains largely unknown. In this study, we aimed to investigate the role of Gli1 in CRC metastasis. We used qRT-PCR, Immunohistochemistry and Western blot to test the expression levels of Gli1, Foxm1 and other target genes in the tissues and cells; Lentivirus stable transfection to change the expression levels of Gli1 and Foxm1; Wound-healing, cell invasion, migration assays and tail vein metastatic assay to test the role of Gli1 in CRC metastasis in vitro and vivo. We demonstrated that Gli1 was significantly overexpressed in colorectal cancer tissues and cells. Foxm1 level had a positive correlation with Gli1. Furthermore, we found that Gli1 promotes colorectal cancer cells metastasis in a Foxm1-dependent manner by activating EMT and PI3K-AKT signaling. Thus, we proved that Gli1 plays important role in CRC metastasis and provided a new visual field on the therapy of CRC metastasis. PMID:27863385

  2. Induction of CCL8/MCP-2 by Mycobacteria through the Activation of TLR2/PI3K/Akt Signaling Pathway

    PubMed Central

    Liu, Haipeng; Liu, Zhonghua; Chen, Jianxia; Chen, Ling; He, Xin; Zheng, Ruijuan; Yang, Hong; Song, Peng; Weng, Dong; Hu, Haili; Fan, Lin; Xiao, Heping; Kaufmann, Stefan H. E.; Ernst, Joel; Ge, Baoxue

    2013-01-01

    Pleural tuberculosis (TB), together with lymphatic TB, constitutes more than half of all extrapulmonary cases. Pleural effusions (PEs) in TB are representative of lymphocytic PEs which are dominated by T cells. However, the mechanism underlying T lymphocytes homing and accumulation in PEs is still incompletely understood. Here we performed a comparative analysis of cytokine abundance in PEs from TB patients and non-TB patients by protein array analysis and observed that MCP-2/CCL8 is highly expressed in the TB-PEs as compared to peripheral blood. Meanwhile, we observed that CCR5, the primary receptor used by MCP-2/CCL8, is mostly expressed on pleural CD4+ T lymphocytes. Furthermore, we found that infection with either Mycobacterium bovis Bacillus Calmette-Guérin (BCG) or Mycobacterium tuberculosis H37Rv induced production of MCP-2/CCL8 at both transcriptional and protein level in Raw264.7 and THP-1 macrophage cells, mouse peritoneal macrophages as well as human PBMC monocyte-derived macrophages (MDMs). The induction of MCP-2/CCL8 by mycobacteria is dependent on the activation of TLR2/PI3K/Akt and p38 signaling pathway. We conclude that accumulation of MCP-2/CCL8 in TB-PEs may function as a biomarker for TB diagnosis. PMID:23418602

  3. Apamin inhibits PDGF-BB-induced vascular smooth muscle cell proliferation and migration through suppressions of activated Akt and Erk signaling pathway.

    PubMed

    Kim, Jung-Yeon; Kim, Kyung-Hyun; Lee, Woo-Ram; An, Hyun-Jin; Lee, Sun-Jae; Han, Sang-Mi; Lee, Kwang-Gill; Park, Yoon-Yub; Kim, Kee-Sik; Lee, Young-Soo; Park, Kwan-Kyu

    2015-07-01

    The increased proliferation and migration of vascular smooth muscle cells (VSMC) are key process in the development of atherosclerosis lesions. Platelet-derived growth factor (PDGF) initiates a multitude of biological effects that contribute to VSMC proliferation and migration. Apamin, a component of bee venom, has been known to block the Ca(2+)-activated K(+) channels. However, the effects of apamin in the regulation PDGF-BB-induced VSMC proliferation and migration has not been identified. In this study, we investigate the inhibitory effect of apamin on PDGF-BB-induced VSMC proliferation and migration. Apamin suppressed the PDGF-BB-induced VSMC proliferation and migration with no apparent cytotoxic effect. In accordance with these findings, apamin induced the arrest of cell cycle progression at G0/G1 phase. Apamin also decreased the expressions of G0/G1 specific regulatory proteins including proliferating cell nuclear antigen (PCNA), cyclin D1, cyclin-dependent kinases (CDK) 4, cyclin E and CDK2, as well as increased the expression of p21(Cip1) in PDGF-BB-induced VSMC. Moreover, apamin inhibited PDGF-BB-induced phosphorylation of Akt and Erk1/2. These results suggest that apamin plays an important role in prevention of vascular proliferation and migration through the G0/G1 cell cycle arrest by PDGF signaling pathway. Thus, apamin may be a promising candidate for the therapy of atherosclerosis.

  4. Activated AKT regulates NF-kappaB activation, p53 inhibition and cell survival in HTLV-1-transformed cells.

    PubMed

    Jeong, Soo-Jin; Pise-Masison, Cynthia A; Radonovich, Michael F; Park, Hyeon Ung; Brady, John N

    2005-10-06

    AKT activation enhances resistance to apoptosis and induces cell survival signaling through multiple downstream pathways. We now present evidence that AKT is activated in HTLV-1-transformed cells and that Tax activation of AKT is linked to NF-kappaB activation, p53 inhibition and cell survival. Overexpression of AKT wild type (WT), but not a kinase dead (KD) mutant, resulted in increased Tax-mediated NF-kappaB activation. Blocking AKT with the PI3K/AKT inhibitor LY294002 or AKT SiRNA prevented NF-kappaB activation and inhibition of p53. Treatment of C81 cells with LY294002 resulted in an increase in the p53-responsive gene MDM2, suggesting a role for AKT in the Tax-mediated regulation of p53 transcriptional activity. Further, we show that LY294002 treatment of C81 cells abrogates in vitro IKKbeta phosphorylation of p65 and causes a reduction of p65 Ser-536 phosphorylation in vivo, steps critical to p53 inhibition. Interestingly, blockage of AKT function did not affect IKKbeta phosphorylation of IkappaBalpha in vitro suggesting selective activity of AKT on the IKKbeta complex. Finally, AKT prosurvival function in HTLV-1-transformed cells is linked to expression of Bcl-xL. We suggest that AKT plays a role in the activation of prosurvival pathways in HTLV-1-transformed cells, possibly through NF-kappaB activation and inhibition of p53 transcription activity.

  5. TMEFF2 modulates the AKT and ERK signaling pathways

    PubMed Central

    Chen, Xiaofei; Ruiz-Echevarría, Maria J

    2013-01-01

    The transmembrane protein with epidermal growth factor (EGF) and two follistatin (FS) motifs 2 (TMEFF2) has a limited tissue distribution with strong expression only in brain and prostate. While TMEFF2 is overexpressed in prostate cancer indicating an oncogenic role, several studies indicate a tumor suppressor role for this protein. This dual mode of action is, at least in part, the result of metalloproteinase-dependent shedding that generates a soluble TMEFF2 ectodomain with a growth promoting function. While recent studies have shed some light on the biology of different forms of TMEFF2, little is known about the molecular mechanisms that influence its oncogenic/tumor suppressive function. In several non-prostate cell lines, it has been shown that a recombinant form of the TMEFF2 ectodomain can interact with platelet derived growth factor (PDGF)-AA to suppress PDGF receptor signaling and can promote ErbB4 and ERK1/2 phosphorylation. However, the role of the full length TMEFF2 in these pathways has not been examined. Using prostate cell lines, here we examine the role of TMEFF2 in ERK and Akt activation, two pathways implicated in prostate cancer progression and that have been shown to cross talk in several cancers. Our results show that different forms of TMEFF2 distinctly affect Akt and ERK activation and this may contribute to a different cellular response of either proliferation or tumor suppression. PMID:23936739

  6. Essential Role of the Zinc Transporter ZIP9/SLC39A9 in Regulating the Activations of Akt and Erk in B-Cell Receptor Signaling Pathway in DT40 Cells

    PubMed Central

    Taniguchi, Masanari; Fukunaka, Ayako; Hagihara, Mitsue; Watanabe, Keiko; Kamino, Shinichiro; Kambe, Taiho; Enomoto, Shuichi; Hiromura, Makoto

    2013-01-01

    The essential trace element zinc is important for all living organisms. Zinc functions not only as a nutritional factor, but also as a second messenger. However, the effects of intracellular zinc on the B cell-receptor (BCR) signaling pathway remain poorly understood. Here, we present data indicating that the increase in intracellular zinc level induced by ZIP9/SLC39A9 (a ZIP Zrt-/Irt-like protein) plays an important role in the activation of Akt and Erk in response to BCR activation. In DT40 cells, the enhancement of Akt and Erk phosphorylation following BCR activation requires intracellular zinc. To clarify this event, we used chicken ZnT5/6/7-gene-triple-knockout DT40 (TKO) cells and chicken Zip9-knockout DT40 (cZip9KO) cells. The levels of Akt and ERK phosphorylation significantly decreased in cZip9KO cells. In addition, the enzymatic activity of protein tyrosine phosphatase (PTPase) increased in cZip9KO cells. These biochemical events were restored by overexpressing the human Zip9 (hZip9) gene. Moreover, we found that the increase in intracellular zinc level depends on the expression of ZIP9. This observation is in agreement with the increased levels of Akt and Erk phosphorylation and the inhibition of total PTPase activity. We concluded that ZIP9 regulates cytosolic zinc level, resulting in the enhancement of Akt and Erk phosphorylation. Our observations provide new mechanistic insights into the BCR signaling pathway underlying the regulation of intracellular zinc level by ZIP9 in response to the BCR activation. PMID:23505453

  7. Notoginsenoside R1 attenuates glucose-induced podocyte injury via the inhibition of apoptosis and the activation of autophagy through the PI3K/Akt/mTOR signaling pathway

    PubMed Central

    Huang, Guodong; Zou, Bingyu; Lv, Jianzhen; Li, Tongyu; Huai, Guoli; Xiang, Shaowei; Lu, Shilong; Luo, Huan; Zhang, Yaping; Jin, Yi; Wang, Yi

    2017-01-01

    Injury to terminally differentiated podocytes contributes ignificantly to proteinuria and glomerulosclerosis. The aim of this study was to examine the protective effects of notoginsenoside R1 (NR1) on the maintenance of podocyte number and foot process architecture via the inhibition of apoptosis, the induction of autophagy and the maintenance pf podocyte biology in target cells. The effects of NR1 on conditionally immortalized human podocytes under high glucose conditions were evaluated by determining the percentage apoptosis, the percentage autophagy and the expression levels of slit diaphragm proteins. Our results revealed that NR1 protected the podocytes against high glucose-induced injury by decreasing apoptosis, increasing autophagy and by promoting cytoskeletal recovery. The phosphoinositide 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) signaling pathway was further investigated in order to elucidate the mechanisms responsible for the protective effects of NR1 on podocytes. Our data indicated that treatment with NR increased the phosphorylation levels of PI3K, Akt and mTOR, leading to the activation of the PI3K/Akt/mTOR signaling pathway in podocytes. To the best of our knowledge, this is the first in vitro study to demonstrate that NR1 protects podocytes by activating the PI3K/Akt/mTOR pathway. PMID:28112381

  8. Acemannan accelerates cell proliferation and skin wound healing through AKT/mTOR signaling pathway.

    PubMed

    Xing, Wei; Guo, Wei; Zou, Cun-Hua; Fu, Ting-Ting; Li, Xiang-Yun; Zhu, Ming; Qi, Jun-Hua; Song, Jiao; Dong, Chen-Hui; Li, Zhuang; Xiao, Yong; Yuan, Pei-Song; Huang, Hong; Xu, Xiang

    2015-08-01

    Acemannan is a bioactive polysaccharides promoting tissue repair. However, the roles of acemannan in skin wound healing and the underlying molecular mechanisms are largely unclear. The goal of this study is to investigate the positive role of acemannan in cutaneous wound healing and its mechanism. Mouse skin wound model and skin primary fibroblasts were used to demonstrate the positive effect of acemannan on cutaneous wound healing. The expressions of cell proliferation nuclear antigen ki-67, cyclin D1 and activity of AKT/mTOR signaling were analyzed in acemannan-treated fibroblasts and mice. Rapamycin and AKT inhibitor VIII were used to determine the key role of AKT/mTOR signaling in acemannan-promoting cutaneous wound healing. We found that acemannan significantly accelerated skin wound closure and cell proliferation. Acemannan promoted the expression of cyclin D1 in cultured fibroblasts, which was mediated by AKT/mTOR signal pathway leading to enhanced activity of the eukaryotic translation initiation factor-4F (eIF4F) and increased translation of cyclin D1. In contrast, pharmaceutical blockade of AKT/mTOR signaling by mTOR inhibitor rapamycin or AKT inhibitor VIII abolished acemannan-induced cyclin D1 translation and cell proliferation. In vivo studies confirmed that the activation of AKT/mTOR by acemannan played a key role in wound healing, which could be reversed by rapamycin. Acemannan promoted skin wound healing partly through activating AKT/mTOR-mediated protein translation mechanism, which may represent an alternative therapy approach for cutaneous wound. Copyright © 2015. Published by Elsevier Ireland Ltd.

  9. α-Lipoic acid interaction with dopamine D2 receptor-dependent activation of the Akt/GSK-3β signaling pathway induced by antipsychotics: potential relevance for the treatment of schizophrenia.

    PubMed

    Deslauriers, Jessica; Desmarais, Christian; Sarret, Philippe; Grignon, Sylvain

    2013-05-01

    Chronic administration of antipsychotics has been associated with dopamine D2 receptor (D2R) upregulation and tardive dyskinesia. We have previously shown that haloperidol, a first-generation antipsychotic (FGA), exerted an increase in D2R expression and oxidative stress and that (±)-α-lipoic acid reversed its effect. Previous studies have implicated the Akt/glycogen synthase kinase-3β (GSK-3β) signaling pathway in antipsychotic action. These findings led us to examine whether the Akt/GSK-3β pathway was involved in D2R upregulation and oxidative stress elicited by antipsychotics and, in (±)-α-lipoic acid-induced reversal of these phenomena, in SH-SY5Y cells. Antipsychotics increased phosphorylation of Akt and GSK-3β, and additive effects were observed with (±)-α-lipoic acid. GSK-3β inhibitors reversed haloperidol-induced overexpression of D2R mRNA levels but did not affect haloperidol-induced oxidative stress. Sustained antipsychotic treatment increased β-arrestin-2 and D2R receptor interaction. Regarding Akt/GSK-3β downstream targets, antipsychotics increased β-catenin levels, whereas (±)-α-lipoic acid induced an elevation of mTOR activation. These results suggest (1) that the effect of antipsychotics on the Akt/GSK-3β pathway in SH-SY5Y cells is reminiscent of their in vivo action, (2) that (±)-α-lipoic acid partially synergizes with antipsychotic drugs (APDs) on the same pathway, and (3) that the Akt/GSK-3β signaling cascade is not involved in the preventive effect of (±)-α-lipoic acid on antipsychotics-induced D2R upregulation.

  10. PI3K/Akt/mTOR signaling pathway in cancer stem cells: from basic research to clinical application

    PubMed Central

    Xia, Pu; Xu, Xiao-Yan

    2015-01-01

    Cancer stem cells (CSCs) are a subpopulation of tumor cells that possess unique self-renewal activity and mediate tumor initiation and propagation. The PI3K/Akt/mTOR signaling pathway can be considered as a master regulator for cancer. More and more recent studies have shown the links between PI3K/Akt/mTOR signaling pathway and CSC biology. Herein, we provide a comprehensive review on the role of signaling components upstream and downstream of PI3K/Akt/mTOR signaling in CSC. In addition, we also summarize various classes of small molecule inhibitors of PI3K/Akt/mTOR signaling pathway and their clinical potential in CSC. Overall, the current available data suggest that the PI3K/Akt/mTOR signaling pathway could be a promising target for development of CSC-target drugs. PMID:26175931

  11. 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. © 2015 Authors.

  12. FAM3A enhances adipogenesis of 3T3-L1 preadipocytes via activation of ATP-P2 receptor-Akt signaling pathway.

    PubMed

    Chi, Yujing; Li, Jing; Li, Na; Chen, Zhenzhen; Ma, Liping; Peng, Weikang; Pan, Xiuying; Li, Mei; Yu, Weidong; He, Xiangjun; Geng, Bin; Cui, Qinghua; Liu, Yulan; Yang, Jichun

    2017-07-11

    FAM3A plays important roles in regulating hepatic glucose/lipid metabolism and the proliferation of VSMCs. This study determined the role and mechanism of FAM3A in the adipogenesis of 3T3-L1 preadipocytes. During the adipogenesis of 3T3-L1 preadipocytes, FAM3A expression was significantly increased. FAM3A overexpression enhanced 3T3-L1 preadipocyte adipogenesis with increased phosphorylated Akt (pAkt) level, whereas FAM3A silencing inhibited 3T3-L1 preadipocyte adipogenesis with reduced pAkt level. Moreover, FAM3A silencing reduced the expression and secretion of adipokines in 3T3-L1 cells. FAM3A protein is mainly located in mitochondrial fraction of 3T3-L1 cells and mouse adipose tissue. FAM3A overexpression increased, whereas FAM3A silencing decreased ATP production in 3T3-L1 preadipocytes. FAM3A-induced adipogenesis of 3T3-L1 preadipocytes was blunted by inhibitor of P2 receptor. In white adipose tissues of db/db and HFD-fed obese mice, FAM3A expression was reduced. One-month rosiglitazone administration upregulated FAM3A expression, and increased cellular ATP content and pAkt level in white adipose tissues of normal and obese mice. In conclusion, FAM3A enhances the adipogenesis of preadipocytes by activating ATP-P2 receptor-Akt pathway. Under obese condition, a decrease in FAM3A expression in adipose tissues plays important roles in the development of adipose dysfunction and type 2 diabetes.

  13. Canstatin inhibits hypoxia-induced apoptosis through activation of integrin/focal adhesion kinase/Akt signaling pathway in H9c2 cardiomyoblasts

    PubMed Central

    Yamawaki, Hideyuki

    2017-01-01

    A hypoxic stress which causes apoptosis of cardiomyocytes is the main problem in the ischemic heart disease. Canstatin, a non-collagenous fragment of type IV collagen α2 chain, is an endogenous anti-angiogenic factor. We have previously reported that canstatin has a cytoprotective effect on cardiomyoblasts. In the present study, we examined the effects of canstatin on hypoxia-induced apoptosis in H9c2 cardiomyoblasts. Cell counting assay was performed to determine a cell viability. Western blotting was performed to detect expression of cleaved casepase-3 and phosphorylation of focal adhesion kinase (FAK) and Akt. Immunocytochemical staining was performed to observe a distribution of αv integrin. Hypoxia (1% O2, 48 h) significantly decreased cell viability and increased cleaved caspase-3 expression. Canstatin (10–250 ng/ml) significantly inhibited these changes in a concentration-dependent manner. Cilengitide (1 μM), an αvβ3 and αvβ5 integrin inhibitor, significantly prevented the protective effects of canstatin on cell viability. Canstatin significantly increased phosphorylation of FAK and Akt under hypoxic condition, which were inhibited by cilengitide. LY294002, an inhibitor of phosphatidylinositol-3 kinase/Akt pathway, suppressed the canstatin-induced Akt phosphorylation and reversed the protective effects of canstatin. It was observed that hypoxia caused a localization of αv integrin to focal adhesion. In summary, we for the first time clarified that canstatin inhibits hypoxia-induced apoptosis via FAK and Akt pathways through activating integrins in H9c2 cardiomyoblasts. PMID:28235037

  14. FAM3A enhances adipogenesis of 3T3-L1 preadipocytes via activation of ATP-P2 receptor-Akt signaling pathway

    PubMed Central

    Li, Na; Chen, Zhenzhen; Ma, Liping; Peng, Weikang; Pan, Xiuying; Li, Mei; Yu, Weidong; He, Xiangjun; Geng, Bin; Cui, Qinghua; Liu, Yulan; Yang, Jichun

    2017-01-01

    FAM3A plays important roles in regulating hepatic glucose/lipid metabolism and the proliferation of VSMCs. This study determined the role and mechanism of FAM3A in the adipogenesis of 3T3-L1 preadipocytes. During the adipogenesis of 3T3-L1 preadipocytes, FAM3A expression was significantly increased. FAM3A overexpression enhanced 3T3-L1 preadipocyte adipogenesis with increased phosphorylated Akt (pAkt) level, whereas FAM3A silencing inhibited 3T3-L1 preadipocyte adipogenesis with reduced pAkt level. Moreover, FAM3A silencing reduced the expression and secretion of adipokines in 3T3-L1 cells. FAM3A protein is mainly located in mitochondrial fraction of 3T3-L1 cells and mouse adipose tissue. FAM3A overexpression increased, whereas FAM3A silencing decreased ATP production in 3T3-L1 preadipocytes. FAM3A-induced adipogenesis of 3T3-L1 preadipocytes was blunted by inhibitor of P2 receptor. In white adipose tissues of db/db and HFD-fed obese mice, FAM3A expression was reduced. One-month rosiglitazone administration upregulated FAM3A expression, and increased cellular ATP content and pAkt level in white adipose tissues of normal and obese mice. In conclusion, FAM3A enhances the adipogenesis of preadipocytes by activating ATP-P2 receptor-Akt pathway. Under obese condition, a decrease in FAM3A expression in adipose tissues plays important roles in the development of adipose dysfunction and type 2 diabetes. PMID:28515350

  15. Salidroside attenuates myocardial ischemia-reperfusion injury via PI3K/Akt signaling pathway.

    PubMed

    Xu, Mao-Chun; Shi, Hai-Ming; Gao, Xiu-Fang; Wang, Hao

    2013-01-01

    To investigate the cardioprotective effects of salidroside on myocardial ischemia-reperfusion injury (IRI) in rabbits and the underlying action mechanisms in PI3K/Akt signaling pathway, a rabbit ischemia/reperfusion model was created by ligating the left anterior descending coronary arterial branch for 30 min and by releasing the ligature to allow reperfusion for 120 min. Salidroside or salidroside+PI3K inhibitor (LY294002) was administered via intracoronary injections at the onset of reperfusion. Apoptosis of cardiomyocytes was assessed by terminal dUTP nick-end labeling assay, and the expression of apoptosis-related proteins was observed by immunohistochemistry. The expressions of total Akt and phosphorylated Akt (p-Akt) were detected by western blot analysis. The results showed that intracoronary injection of salidroside at the onset of reperfusion markedly reduced the apoptosis of cardiomyocytes, significantly increasing Bcl-2 and p-Akt proteins expressions and decreasing Bax and caspase-3 expressions in the hearts subjected to ischemia followed by 120-min reperfusion. However, the anti-apoptotic effect induced by salidroside was inhibited by LY294002, which blocked the activation of Akt. These results suggested that intracoronary administration of salidroside at the onset of reperfusion could significantly reduce the IRI-induced apoptosis of cardiomyocytes, and this protective mechanism seemed to be mediated by the PI3K-Akt signaling pathway.

  16. Synergistic inhibition of cell migration by tetraspanin CD82 and gangliosides occurs via the EGFR or cMet-activated Pl3K/Akt signalling pathway.

    PubMed

    Li, Ying; Huang, Xiaohua; Zhang, Jianing; Li, Yuzhong; Ma, Keli

    2013-11-01

    The metastasis suppressor CD82/KAI-1, which is a member of the tetraspanin superfamily, has been proposed to exert its activity together with glycosphingolipids. However, the mechanism of CD82 inhibition has not been fully elucidated. The present study aimed to investigate the synergistic inhibition of cell migration by the tetraspanin CD82 and gangliosides and to correlate this inhibition with activation of epidermal growth factor receptor (EGFR) and hepatocyte growth factor receptor (HGFR/cMet) in Hepa1-6 cell lines, whose motility and migration is stimulated by epidermal growth factor (EGF) and hepatocyte growth factor (HGF) in vitro. We found that Hepa1-6 cells transfected with the CD82 gene exhibited decreased migration in response to EGF and HGF. EGF-stimulated phosphorylation of EGFR at Tyr1173 was inhibited in these cells, which contributed to the attenuation of EGFR. Ectopic expression of CD82 in Hepa1-6 cells inhibited HGF-stimulated tyrosine phosphorylation of cMet at Tyr1313 and Tyr1365 without affecting the expression of cMet. These inhibitory effects were enhanced when CD82 was introduced with Ganglioside GM3 alone or GM2/GM3. Reduction of CD82 expression by RNA interference together with depletion of glycosphingolipids with P4 significantly enhanced cell motility and increased the expression of EGFR and its phosphorylation at Tyr1173 in response to EGF. Increased cell motility and HGF-dependent activation of cMet at Tyr1313 and Tyr1365 resulted from decreased CD82 levels and increased GM3. Furthermore, CD82 expression selectively attenuated EGFR and cMet signalling via phosphatidylinositol 3-kinase/Akt but had no affect on the activity of the MAPK signalling pathway. These results suggest that the synergistic effects of CD82 and GM3 or GM2/GM3 on EGFR expression and phosphorylation and cMet activation are responsible for CD82 inhibition of EGF- and HGF-dependent cell motility and migration of Hepa1-6 cells. Copyright © 2013 Elsevier Ltd. All rights

  17. Myricitrin attenuates endothelial cell apoptosis to prevent atherosclerosis: An insight into PI3K/Akt activation and STAT3 signaling pathways.

    PubMed

    Qin, Meng; Luo, Yun; Meng, Xiang-bao; Wang, Min; Wang, Hong-wei; Song, Shi-yu; Ye, Jing-xue; Pan, Rui-le; Yao, Fan; Wu, Ping; Sun, Gui-bo; Sun, Xiao-bo

    2015-07-01

    Blood vessel endothelial dysfunction induced by oxidized low-density lipoprotein (ox-LDL) has been implicated in the pathogenesis of atherosclerosis and vasculopathy. The ox-LDL-elicited reactive oxygen species (ROS) release has been assumed to serve a critical function in endothelial damage. Myricitrin (from Myrica cerifera) is a natural antioxidant that has strong anti-oxidative, anti-inflammatory, and anti-nociceptive activities. However, the protective effect of myricitrin on ROS-induced endothelial cell injury and its related molecular mechanisms have never been investigated. This study demonstrates that myricitrin can inhibit ox-LDL-induced endothelial apoptosis and prevent plaque formation at an early stage in an atherosclerotic mouse model. The administration of myricitrin in vivo decreases the thickness of the vascular wall in the aortic arch of ApoE-/- mice. In vitro study shows that ox-LDL-induced human umbilical vein endothelial cell apoptosis can be reduced upon receiving myricitrin pre-treatment. Treatment with myricitrin significantly attenuated ox-LDL-induced endothelial cell apoptosis by inhibiting LOX-1 expression and by increasing the activation of the STAT3 and PI3K/Akt/eNOS signaling pathways. At the same time, our result demonstrates that myricitrin treatment optimizes the balance of pro/anti-apoptosis proteins, including Bax, Bad, XIAP, cIAP-2, and survivin. Our study suggests that myricitrin treatment can effectively protect cells from ox-LDL-induced endothelial cell apoptosis, which results in reduced atherosclerotic plaque formation. This result indicates that myricitrin can be used as a drug candidate for the treatment of cardiovascular diseases. Copyright © 2015. Published by Elsevier Inc.

  18. LTβR signalling preferentially accelerates oncogenic AKT-initiated liver tumours.

    PubMed

    Scarzello, Anthony J; Jiang, Qun; Back, Timothy; Dang, Hien; Hodge, Deborah; Hanson, Charlotte; Subleski, Jeffrey; Weiss, Jonathan M; Stauffer, Jimmy K; Chaisaingmongkol, Jitti; Rabibhadana, Siritida; Ruchirawat, Mathuros; Ortaldo, John; Wang, Xin Wei; Norris, Paula S; Ware, Carl F; Wiltrout, Robert H

    2016-10-01

    The relative contributions of inflammatory signalling and sequential oncogenic dysregulation driving liver cancer pathogenesis remain incompletely understood. Lymphotoxin-β receptor (LTβR) signalling is critically involved in hepatitis and liver tumorigenesis. Therefore, we explored the interdependence of inflammatory lymphotoxin signalling and specific oncogenic pathways in the progression of hepatic cancer. Pathologically distinct liver tumours were initiated by hydrodynamic transfection of oncogenic V-Akt Murine Thymoma Viral Oncogene Homolog 1 (AKT)/β-catenin or AKT/Notch expressing plasmids. To investigate the relationship of LTβR signalling and specific oncogenic pathways, LTβR antagonist (LTβR-Fc) or agonist (anti-LTβR) were administered post oncogene transfection. Initiated livers/tumours were investigated for changes in oncogene expression, tumour proliferation, progression, latency and pathology. Moreover, specific LTβR-mediated molecular events were investigated in human liver cancer cell lines and through transcriptional analyses of samples from patients with intrahepatic cholangiocarcinoma (ICC). AKT/β-catenin-transfected livers displayed increased expression of LTβ and LTβR, with antagonism of LTβR signalling reducing tumour progression and enhancing survival. Conversely, enforced LTβR-activation of AKT/β-catenin-initiated tumours induced robust increases in proliferation and progression of hepatic tumour phenotypes in an AKT-dependent manner. LTβR-activation also rapidly accelerated ICC progression initiated by AKT/Notch, but not Notch alone. Moreover, LTβR-accelerated development coincides with increases of Notch, Hes1, c-MYC, pAKT and β-catenin. We further demonstrate LTβR signalling in human liver cancer cell lines to be a regulator of Notch, pAKTser473 and β-catenin. Transcriptome analysis of samples from patients with ICC links increased LTβR network expression with poor patient survival, increased Notch1 expression and Notch

  19. Restoration of SHIP activity in a human leukemia cell line downregulates constitutively activated phosphatidylinositol 3-kinase/Akt/GSK-3beta signaling and leads to an increased transit time through the G1 phase of the cell cycle.

    PubMed

    Horn, S; Endl, E; Fehse, B; Weck, M M; Mayr, G W; Jücker, M

    2004-11-01

    The inositol 5-phosphatase SHIP (SHIP-1) is a negative regulator of signal transduction in hematopoietic cells and targeted disruption of SHIP in mice leads to a myeloproliferative disorder. We analyzed the effects of SHIP on the human leukemia cell line Jurkat in which expression of endogenous SHIP protein is not detectable. Restoration of SHIP expression in Jurkat cells with an inducible expression system caused a 69% reduction of phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P(3)) and a 65% reduction of Akt kinase activity, which was associated with reduced phosphorylation of glycogen synthase kinase 3beta (GSK-3beta) (Ser-9) without changing the phosphorylation of Bad (Ser-136), FKHR (Ser-256) or MAPK (Thr-202/Tyr-204). SHIP-expressing Jurkat cells showed an increased transit time through the G1 phase of the cell cycle, but SHIP did not cause a complete cell cycle arrest or apoptosis. Extension of the G1 phase was associated with an increased stability of the cell cycle inhibitor p27(Kip1) and reduced phosphorylation of the retinoblastoma protein Rb at serine residue 780. Our data indicate that restoration of SHIP activity in a human leukemia cell line, which has lost expression of endogenous SHIP, downregulates constitutively activated phosphatidylinositol 3-kinase/Akt/GSK-3beta signaling and leads to an increased transit time through the G1 phase of the cell cycle.

  20. Activation of sonic hedgehog signaling enhances cell migration and invasion by induction of matrix metalloproteinase-2 and -9 via the phosphoinositide-3 kinase/AKT signaling pathway in glioblastoma.

    PubMed

    Chang, Liang; Zhao, Dan; Liu, Hui-Bin; Wang, Qiu-Shi; Zhang, Ping; Li, Chen-Long; Du, Wen-Zhong; Wang, Hong-Jun; Liu, Xing; Zhang, Zhi-Ren; Jiang, Chuan-Lu

    2015-11-01

    Aberrant hedgehog signaling contributes to the development of various malignancies, including glioblastoma (GBM). However, the potential mechanism of hedgehog signaling in GBM migration and invasion has remained to be elucidated. The present study showed that enhanced hedgehog signaling by recombinant human sonic hedgehog N‑terminal peptide (rhSHH) promoted the adhesion, invasion and migration of GBM cells, accompanied by increases in mRNA and protein levels of matrix metalloproteinase‑2 (MMP‑2) and MMP‑9. However, inhibition of hedgehog signaling with cyclopamine suppressed the adhesion, invasion and migration of GBM cells, accompanied by decreases in mRNA and protein levels of MMP‑2 and ‑9. Furthermore, it was found that MMP‑2- and MMP‑9-neutralizing antibodies or GAM6001 reversed the inductive effects of rhSHH on cell migration and invasion. In addition, enhanced hedgehog signaling by rhSHH increased AKT phosphorylation, whereas blockade of hedgehog signaling decreased AKT phosphorylations. Further experiments showed that LY294002, an inhibitor of phosphoinositide-3 kinase (PI3K), decreased rhSHH‑induced upregulation of MMP‑2 and ‑9. Finally, the protein expression of glioblastoma-associated oncogene 1 was positively correlated with levels of phosphorylated AKT as well as protein expressions of MMP‑2 and ‑9 in GBM tissue samples. In conclusion, the present study indicated that the hedgehog pathway regulates GBM-cell migration and invasion by increasing MMP-2 and MMP-9 production via the PI3K/AKT pathway.

  1. The protective effects of Chinese herb-Taikong Yangxin Prescription on the atrophic remodeling of cardiac muscle in rats induced by hindlimb unloading through activating Akt/GSK-3beta signaling pathway

    NASA Astrophysics Data System (ADS)

    Ming, Yuan; Min, Yuan; Jianfeng, Zhang; Zhili, Li; Huijuan, Wang; Desheng, Wang; Yinghui, Li; Yongzhi, Li; Shizhong, Jiang

    Objective To test the hypothesis that traditional Chinese herb-TaiKong Yangxin Prescrip-tion can activate the Akt/GSK-3β signaling pathway and alleviate the atrophic remodeling of cardiac muscle in rats induced by hindlimb unloading. Methods The physiological effects of simulated microgravity was induced by 7d hindlimb unloading in rats. TaiKong Yangxin Pre-scription was given daily by gastric irrigation as countermeasure against effects of simulated microgravity. The frozen sections of left ventricular cardiac muscles were stained by FITC la-beled lectin and visualized by laser scanning confocal microscopy, the cross section areas(CSA) of cardiomyocytes were calculated by IPP6.0 Image software. The protein expression of TnI, phosphorylation level of Akt and GSK-3β were measured by Western blot. Results Simulated microgravity decreased the CSA of cardiomyocytes and protein expression of TnI in left ven-tricular cardiac muscles, inhibited the phosphorylation level of Akt at serine 473 and GSK-3β at serine 9. The traditional Chinese herb-TaiKong Yangxin Prescription alleviated the atrophic remodeling of cardiac muscles, reversed the declined protein expression of TnI and phosphoryla-tion levels of Akt at serine 473 and GSK-3β at serine 9 in hindlimb-unloading rats. Conclusion The traditional Chinese herb-TaiKong Yangxin Prescription has significant countermeasure effects on the atrophic remodeling of cardiac muscle induced by hindlimb unloading in rats, in which activating Akt/GSK-3β signaling pathway plays an important role.(Funded by Advanced space medico-engineering research project of China, grant NO. 2005SY5206005 and SJ200801)

  2. Vascular neuroprotection via TrkB- and Akt-dependent cell survival signaling.

    PubMed

    Guo, Shuzhen; Som, Angel T; Waeber, Christian; Lo, Eng H

    2012-11-01

    The cerebral endothelium can be a vital source of signaling factors such as brain-derived neurotrophic factor that defends the neuronal parenchyma against stress and injury. But the underlying mechanisms remain to be fully defined. Here, we use cell models to ask how vascular neuroprotection is sustained. Human brain endothelial cells were grown in culture, and conditioned media were transferred to primary rat cortical neurons. Brain endothelial cell-conditioned media activated neuronal Akt signaling and protected neurons against hypoxia and oxygen-glucose deprivation. Blockade of Akt phosphorylation with the PI3-kinase inhibitor LY294002 negated this vascular neuroprotective effect. Upstream of Akt signaling, the brain-derived neurotrophic factor receptor TrkB (neurotrophic tyrosine kinase receptor, type 2) was involved because depletion with TrkB/Fc eliminated the ability of endothelial-conditioned media to protect neurons against hypoxia. Downstream of Akt signaling, activation of GSK-3β (glycogen synthase kinase 3 beta), caspase 9, caspase 3 and Bad pathways were detected. Taken together, these findings suggest that the molecular basis for vascular neuroprotection involves TrkB-Akt signaling that ameliorates neuronal apoptosis. Further investigation of these mechanisms may reveal new approaches for augmenting endogenous vascular neuroprotection in stroke, brain injury, and neurodegeneration.

  3. PDGF inactivates forkhead family transcription factor by activation of Akt in glomerular mesangial cells.

    PubMed

    Ghosh Choudhury, Goutam; Lenin, Mahimainathan; Calhaun, Cheresa; Zhang, Jian-Hua; Abboud, Hanna E

    2003-02-01

    Regulation of the forkhead domain transcription factors by PDGF has not been studied. In this report, we investigated the role of PDGF-induced Akt in regulating forkhead domain protein FKHRL1 in glomerular mesangial cells. PDGF increased phosphorylation of FKHRL1 in a time- and PI 3 kinase-dependent manner. Expression of dominant negative Akt by adenovirus-mediated gene transfer blocked PDGF-induced FKHRL1 phosphorylation. PDGF inhibited transcription of a forkhead DNA binding element-driven reporter gene. This inhibition was mimicked by constitutively active myristoylated Akt. Moreover, FKHR1-mediated transcription of the reporter gene was completely attenuated by both PDGF and Myr-Akt. One of the targets of forkhead transcription factors is the proapoptotic Fas ligand (FasL) gene. PDGF, as well as Myr-Akt, inhibited transcription of FasL. In contrast, inhibition of PI 3 kinase and dominant negative Akt increased FasL gene transcription, suggesting that suppression of PI 3 kinase/Akt signalling may induce apoptosis in mesangial cells via upregulation of FasL expression. However, expression of dominant negative Akt by adenovirus did not induce apoptosis in mesangial cells, suggesting that Akt-independent antiapoptotic mechanisms also exist. Together, our data demonstrate for the first time that PDGF inactivates forkhead domain transcription factor by Akt-dependent phosphorylation and that suppression of Akt signalling is not sufficient to induce apoptosis in mesangial cells.

  4. Insulin Regulates Adipocyte Lipolysis via an Akt-Independent Signaling Pathway ▿

    PubMed Central

    Choi, Sarah M.; Tucker, David F.; Gross, Danielle N.; Easton, Rachael M.; DiPilato, Lisa M.; Dean, Abigail S.; Monks, Bob R.; Birnbaum, Morris J.

    2010-01-01

    After a meal, insulin suppresses lipolysis through the activation of its downstream kinase, Akt, resulting in the inhibition of protein kinase A (PKA), the main positive effector of lipolysis. During insulin resistance, this process is ineffective, leading to a characteristic dyslipidemia and the worsening of impaired insulin action and obesity. Here, we describe a noncanonical Akt-independent, phosphoinositide-3 kinase (PI3K)-dependent pathway that regulates adipocyte lipolysis using restricted subcellular signaling. This pathway selectively alters the PKA phosphorylation of its major lipid droplet-associated substrate, perilipin. In contrast, the phosphorylation of another PKA substrate, hormone-sensitive lipase (HSL), remains Akt dependent. Furthermore, insulin regulates total PKA activity in an Akt-dependent manner. These findings indicate that localized changes in insulin action are responsible for the differential phosphorylation of PKA substrates. Thus, we identify a pathway by which insulin regulates lipolysis through the spatially compartmentalized modulation of PKA. PMID:20733001

  5. Insulin regulates adipocyte lipolysis via an Akt-independent signaling pathway.

    PubMed

    Choi, Sarah M; Tucker, David F; Gross, Danielle N; Easton, Rachael M; DiPilato, Lisa M; Dean, Abigail S; Monks, Bob R; Birnbaum, Morris J

    2010-11-01

    After a meal, insulin suppresses lipolysis through the activation of its downstream kinase, Akt, resulting in the inhibition of protein kinase A (PKA), the main positive effector of lipolysis. During insulin resistance, this process is ineffective, leading to a characteristic dyslipidemia and the worsening of impaired insulin action and obesity. Here, we describe a noncanonical Akt-independent, phosphoinositide-3 kinase (PI3K)-dependent pathway that regulates adipocyte lipolysis using restricted subcellular signaling. This pathway selectively alters the PKA phosphorylation of its major lipid droplet-associated substrate, perilipin. In contrast, the phosphorylation of another PKA substrate, hormone-sensitive lipase (HSL), remains Akt dependent. Furthermore, insulin regulates total PKA activity in an Akt-dependent manner. These findings indicate that localized changes in insulin action are responsible for the differential phosphorylation of PKA substrates. Thus, we identify a pathway by which insulin regulates lipolysis through the spatially compartmentalized modulation of PKA.

  6. Mutual inhibition of insulin signaling and PHLPP-1 determines cardioprotective efficiency of Akt in aged heart

    PubMed Central

    Xing, Yuan; Sun, Wanqing; Wang, Yishi; Gao, Feng; Ma, Heng

    2016-01-01

    Insulin protects cardiomyocytes from myocardial ischemia/reperfusion (MI/R) injury through activating Akt. However, phosphatase PHLPP-1 (PH domain leucine-rich repeat protein phosphatase-1) dephosphorylates and inactivates Akt. The balanced competitive interaction of insulin and PHLPP-1 has not been directly examined. In this study, we have identified the effect of mutual inhibition of insulin signaling and PHLPP-1 on the cardioprotective efficiency of Akt in aged heart. Young (3 mon) and aged (20 mon) Sprague Dawley (SD) rats were subjected to MI/R in vivo. The PHLPP-1 level was higher in aged vs. young hearts at base. But, insulin treatment failed to decrease PHLPP-1 level during reperfusion in the aged hearts. Consequently, the cardioprotection of insulin-induced Akt activation was impaired in aged hearts, resulting in more susceptible to MI/R injury. In cultured rat ventricular myocytes, PHLPP-1 knockdown significantly enhanced insulin-induced Akt phosphorylation and reduced simulated hypoxia/reoxygenation-induced apoptosis. Contrary, PHLPP-1 overexpression terminated Akt phosphorylation and deteriorated myocytes apoptosis. Using in vivo aged animal models, we confirmed that cardiac PHLPP-1 knockdown or enhanced insulin sensitivity by exercise training dramatically increased insulin-induced Akt phosphorylation. Specifically, MI/R-induced cardiomyocyte apoptosis and infarct size were decreased and cardiac function was increased. More importantly, we found that insulin regulated the degradation of PHLPP-1 and insulin treatment could enhance the binding between PHLPP-1 and β-transducin repeat-containing protein (β-TrCP) to target for ubiquitin-dependent degradation. Altogether, we have identified a new mechanism by which insulin suppresses PHLPP-1 to enhance Akt activation. But, aged heart possesses lower insulin effectiveness and fails to decrease PHLPP-1 during MI/R, which subsequently limited Akt activity and cardioprotection. PHLPP-1 could be a promising

  7. Interleukin-11 promotes epithelial-mesenchymal transition in anaplastic thyroid carcinoma cells through PI3K/Akt/GSK3β signaling pathway activation

    PubMed Central

    Jiang, Yue; Sun, Ruimei; Chen, Xue; Chu, Hongying; Zeng, Musheng; Sun, Chuanzheng

    2016-01-01

    Metastasis is the major cause of treatment failure in anaplastic thyroid carcinoma (ATC) patients. In the preliminary study, we demonstrated that interleukin (IL)-11 expression is positively correlated with distant metastasis in ATC. However, the mechanisms underlying remain largely unknown. Here, we found that cobalt chloride (a hypoxia mimetic) promoted IL-11 expression via HIF-1α activation. Furthermore, the resultant increase in IL-11 expression significantly induced epithelial-mesenchymal transition (EMT) in ATC cells, accompanied by Akt/GSK3β pathway activation and increased invasive and migratory abilities. Conversely, HIF-1α or IL-11 knockdown, or treating cells with a neutralizing antibody against IL-11, a PI3K inhibitor, or Akt inhibitor V, significantly suppressed the induction of EMT and counteracted the enhancements in invasive and migratory abilities. These results indicate that hypoxia increases IL-11 secretion in ATC cells via HIF-1α induction and that IL-11 then induces EMT in these cells via the PI3K/Akt/GSK3β pathway, ultimately improving their invasive and migratory potential. This study elucidates the prometastatic role played by IL-11 in ATC metastasis and indicates it as a potential target for the treatment of cancer metastasis. However, many questions remain to be explored. PMID:27487122

  8. The neuroprotective effect of a novel agent N2 on rat cerebral ischemia associated with the activation of PI3K/Akt signaling pathway.

    PubMed

    Huang, Jinru; Kodithuwakku, Nandani Darshika; He, Wei; Zhou, Yi; Fan, Wenxiang; Fang, Weirong; He, Guangwei; Wu, Qiang; Chu, Shaoxing; Li, Yunman

    2015-08-01

    Ischemic stroke is the third leading cause of death and the main reason for severe disabilities in the world today. N2, 4 - (2 - (1H - imidazol - 1 - yl) ethoxy) - 3 - methoxybenzoic acid is considered as a novel potent agent for cerebral ischemia due to its effect in preventing neuronal cell death after ischemic stroke. In the present study, we investigated the post-ischemic neuroprotective effect of N2 and its underlying mechanisms. Using a MCAO rat model, we found that N2 reversed brain infarct size, reduced cerebral edema and decreased the neurological deficit score significantly. Moreover, N2 diminished TUNEL positive cells, down-regulated bax expression and up-regulated bcl-2 expression notably. In addition, we evaluated the oxygen glucose deprivation/reoxygenation (OGD/R) injury induced neuron cell death in rat primary cortical neuron and assessed the neuroprotective effect of our drug. N2 increased cell viability, ameliorated neuron cell injury by decreasing LDH activity, and inhibited cell apoptotic rate while suppressed apoptotic signaling via inhibiting the bax expression, and elevating the bcl-2 expression. Furthermore, the neuroprotective effect of N2 was associated with the PI3K/Akt pathway which was proved by the use of PI3K inhibitor LY294002. The combination of our findings disclosed that N2 can be used as an effective neuroprotective agent for ischemic stroke due to its significant effect on preventing neuronal cell death after cerebral ischemia both in vivo and in vitro and the effectiveness was dose dependent.

  9. Cobalt chloride stimulates phosphoinositide 3-kinase/Akt signaling through the epidermal growth factor receptor in oral squamous cell carcinoma.

    PubMed

    Ryu, Mi Heon; Park, Jeong Hee; Park, Ji Eun; Chung, Jin; Lee, Chang Hun; Park, Hae Ryoun

    2010-04-01

    Tumor cells are often found under hypoxic conditions due to the rapid outgrowth of their vascular supply, and, in order to survive hypoxia, these cells induce numerous signaling factors. Akt is an important kinase in cell survival, and its activity is regulated by the upstream phosphoinositide 3-kinase (PI3K) and receptor tyrosine kinases (RTKs). In this study, we examined Akt activation and RTKs/PI3K/Akt signaling using the hypoxia-mimetic cobalt chloride in oral squamous carcinoma cells. Cobalt chloride increases Akt phosphorylation in both a dose- and time-dependent manner. Blocking the activation of the PI3K/Akt pathway using LY294002 abolished Akt activation in response to cobalt chloride, suggesting that Akt phosphorylation by cobalt chloride is dependent on PI3K. In addition, activation of the PI3K/Akt pathway seems to rely on the epidermal growth factor receptor (EGFR), since the inhibition of EGFR attenuated cobalt chloride-induced Akt activation. The results in this study also demonstrate that cobalt chloride increases EGFR protein levels and induces oral squamous cell carcinoma cells to enter S phase.

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

    PubMed Central

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

    2012-01-01

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

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

    PubMed

    Matta, Sumit Kumar; Kumar, Dhiraj

    2015-09-01

    Autophagy is considered as an innate defense mechanism primarily due to its role in the targeting of intracellular pathogens for lysosomal degradation. Here we report inhibition of autophagy as an adaptive response in classically activated macrophages that helps achieve high cellular ROS production and cell death-another hallmark of innate mechanisms. We show prolonged classical activation of Raw 264.7 macrophages by treating them with IFN-γ and LPS inhibited autophagy. The inhibition of autophagy was dependent on nitric oxide (NO) production which activated the AKT-mTOR signaling, the known negative regulators of autophagy. Autophagy inhibition in these cells was accompanied with a shift to aerobic glycolysis along with a decline in the mitochondrial membrane potential (MOMP). The decline in MOMP coupled with autophagy inhibition led to increased mitochondrial content and considerably elevated cellular ROS, eventually causing cell death. Next, using specific siRNA mediated knockdowns we show AKT was responsible for the glycolytic shift and autophagy inhibition in activated macrophages. Surprisingly, AKT knockdown in activated macrophages also rescued them from cell death. Finally we show that AKT mediated autophagy inhibition in the activated macrophages correlated with the depletion of glucose from the extracellular medium, and glucose supplementation not only rescued autophagy levels and reversed other phenotypes of activated macrophages, but also inhibited cell death. Thus we report here a novel link between AKT mediated glycolytic metabolism and autophagy in the activated macrophages, and provide a possible mechanism for sustained macrophage activation in vivo. Copyright © 2015 Elsevier Ltd. All rights reserved.

  12. Low expression of phosphatase and tensin homolog in clear-cell renal cell carcinoma contributes to chemoresistance through activating the Akt/HDM2 signaling pathway

    PubMed Central

    CHEN, JUN; ZHU, HE; ZHANG, YAN; CUI, MAN-HUA; HAN, LI-YING; JIA, ZHAN-HUI; WANG, LING; TENG, HONG; MIAO, LI-NING

    2015-01-01

    Clear-cell renal cell carcinoma (CCRCC) is the most frequent primary malignancy in the adult kidney. Most patients with advanced CCRCC have poor prognosis as CCRCC remains resistant to chemotherapy. The present study explored the possible mechanism underlying CCRCC resistance to chemotherapy and found that loss of PTEN in CCRCC may be involved. Knockdown of PTEN in the CCRCC cell line ACHN blocked etoposide-induced apoptosis and etoposide-impaired cell proliferation was also inhibited. It has been demonstrated that most chemotherapy drugs exert their anti-cancer effects via p53-mediated apoptosis, and in accordance, with this, the present study showed that treatment with etoposide significantly increased p53 levels. Silencing of PTEN in ACHN inhibited the Akt/HDM2 signaling cascade and depressed p53 expression, and the interaction between HDM2 and p53 was also enhanced. This was further verified in CCRCC tissue specimens from patients The results of the present study suggested that loss of PTEN, which deactivated Akt/HDM2 signaling followed by degradation of p53, may contribute to the development of etoposide resistance in CCRCC. PMID:25954860

  13. 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

  14. Cytoplasmic localization of wild-type survivin is associated with constitutive activation of the PI3K/Akt signaling pathway and represents a favorable prognostic factor in patients with acute myeloid leukemia

    PubMed Central

    Serrano-López, Juana; Serrano, Josefina; Figueroa, Vianihuini; Torres-Gomez, Antonio; Tabares, Salvador; Casaño, Javier; Fernandez-Escalada, Noemi; Sánchez-Garcia, Joaquín

    2013-01-01

    Survivin is over-expressed in most hematologic malignancies but the prognostic significance of the subcompartmental distribution of wild-type or splicing variants in acute myeloid leukemia has not been addressed yet. Using western blotting, we assessed the expression of wild-type survivin and survivin splice variants 2B and Delta-Ex3 in nuclear and cytoplasmic protein extracts in samples taken from 105 patients at the time of their diagnosis of acute myeloid leukemia. Given that survivin is a downstream effector of the PI3K/Akt signaling pathway, survivin expression was also correlated with pSer473-Akt. Wild-type survivin and the 2B splice variant were positive in 76.3% and 78.0% of samples in the nucleus, cytoplasm or both, whereas the Delta-Ex3 isoform was only positive in the nucleus in 37.7% of samples. Cytoplasmic localization of wild-type survivin was significantly associated with the presence of high levels of pSer473-Akt (P<0.001). Inhibition of the PI3K/Akt pathway with wortmannin and Ly294002 caused a significant reduction in the expression of cytoplasmic wild-type survivin. The presence of cytoplasmic wild-type survivin and pSer473-Akt was associated with a lower fraction of quiescent leukemia stem cells (P=0.02). The presence of cytoplasmic wild-type survivin and pSer473-Akt were favorable independent prognostic factors. Moreover, the activation of the PI3K/Akt pathway with expression of cytoplasmic wild-type survivin identified a subgroup of acute myeloid leukemia patients with an excellent outcome (overall survival rate of 60.0±21.9% and relapse-free survival of 63.0±13.5%). Our findings suggest that cytoplasmic wild-type survivin is a critical downstream effector of the PI3K/Akt pathway leading to more chemosensitive cells and a more favorable outcome in acute myeloid leukemia. PMID:23812937

  15. Inhibitors beta-amyloid-induced toxicity by modulating the Akt signaling pathway.

    PubMed

    Nakagami, Yasuhiro

    2004-12-01

    The Akt signaling pathway plays a crucial role in neuronal survival, leading to inhibition of apoptosis. Many stimulants including neurotrophins are reported to activate this pathway in preclinical studies; however, there are no drugs for neurodegenerative diseases adopting such a concept on the market so far. Among neurodegenerative diseases, Alzheimer's disease is the most common and characterized by senile plaques and neurofibrillary tangles, which consist of beta-amyloid and hyperphosphorylated tau, respectively. Recent studies suggest that activation of Akt inhibits toxicity of beta-amyloid and formation of neurofibrillary tangles, leading to protection of neurons against apoptosis. This review discusses the possibility of treatment of Alzheimer's disease by activating the Akt signaling pathway.

  16. Exposure to Ionizing Radiation Causes Long-Term Increase in Serum Estradiol and Activation of PI3K-Akt Signaling Pathway in Mouse Mammary Gland

    SciTech Connect

    Suman, Shubhankar; Johnson, Michael D.; Fornace, Albert J.; Datta, Kamal

    2012-10-01

    Purpose: Exposure to ionizing radiation is an established risk factor for breast cancer. Radiation exposure during infancy, childhood, and adolescence confers the highest risk. Although radiation is a proven mammary carcinogen, it remains unclear where it acts in the complex multistage process of breast cancer development. In this study, we investigated the long-term pathophysiologic effects of ionizing radiation at a dose (2 Gy) relevant to fractionated radiotherapy. Methods and Materials: Adolescent (6-8 weeks old; n = 10) female C57BL/6J mice were exposed to 2 Gy total body {gamma}-radiation, the mammary glands were surgically removed, and serum and urine samples were collected 2 and 12 months after exposure. Molecular pathways involving estrogen receptor-{alpha} (ER{alpha}) and phosphatidylinositol-3-OH kinase (PI3K)-Akt signaling were investigated by immunohistochemistry and Western blot. Results: Serum estrogen and urinary levels of the oncogenic estrogen metabolite (16{alpha}OHE1) were significantly increased in irradiated animals. Immunostaining for the cellular proliferative marker Ki-67 and cyclin-D1 showed increased nuclear accumulation in sections of mammary glands from irradiated vs. control mice. Marked increase in p85{alpha}, a regulatory sub-unit of the PI3K was associated with increase in Akt, phospho-Akt, phospho-BAD, phospho-mTOR, and c-Myc in irradiated samples. Persistent increase in nuclear ER{alpha} in mammary tissues 2 and 12 months after radiation exposure was also observed. Conclusions: Taken together, our data not only support epidemiologic observations associating radiation and breast cancer but also, specify molecular events that could be involved in radiation-induced breast cancer.

  17. Membrane Heterogeneity in Akt Activation in Prostate Cancer

    DTIC Science & Technology

    2008-03-01

    ELEMENT NUMBER 6. AUTHOR(S) 5d. PROJECT NUMBER Martin H. Hager 5e. TASK NUMBER Email: Martin.Hager@childrens.harvard.edu 5f. WORK UNIT...SUPPLEMENTARY NOTES 14. ABSTRACT This project focuses on the novel finding from our group that the serine-threonine kinase Akt1 partitions into...signaling protein that is frequently activated in prostate cancer. I have hypothesized in this project that cholesterol accumulation in prostate

  18. Resveratrol Regulates Activated Hepatic Stellate Cells by Modulating NF-κB and the PI3K/Akt Signaling Pathway.

    PubMed

    Zhang, De-Quan; Sun, Peng; Jin, Quan; Li, Xia; Zhang, Yu; Zhang, Yu-Jing; Wu, Yan-Ling; Nan, Ji-Xing; Lian, Li-Hua

    2016-01-01

    In the present study, we investigated whether resveratrol could suppress the hepatic fibrogenesis in activated hepatic stellate cells. The immortalized rat hepatic stellate cells, t-HSC/Cl-6, were treated with resveratrol 1 h prior to lipopolysaccharide (LPS, 1 μg/mL). Resveratrol decreased t-HSC/Cl-6 cell viability at much lower concentrations within 24 h. Resveratrol pretreatment also decreased the LPS-induced protein expression of α-SMA and collagen I. In addition, resveratrol significantly reduced the protein expression of Toll-like receptor 4 (TLR4) and myeloid differentiation primary response gene 88 (MyD88), and the expression of phosphorylated phosphatidylinositol 3-kinase (PI3K) and phosphorylated serine/threonine kinase B (Akt). Moreover, resveratrol markedly blocked the translocation of nuclear factor (NF)-κB in LPS-activated HSCs. Furthermore, resveratrol inhibited HSCs activation through stimulating LXRβ, but did not influence LXRα. Overall, we conclude that the antifibrotic effect of resveratrol is the result of blocking NF-κB activation and PI3K/Akt phosphorylation, which inhibits HSC activation to obstruct liver fibrosis. Thus, resveratrol may be a natural agent for preventing hepatic fibrosis.

  19. Functional proteomic analysis of a three-tier PKCepsilon-Akt-eNOS signaling module in cardiac protection.

    PubMed

    Zhang, Jun; Baines, Christopher P; Zong, Chenggong; Cardwell, Ernest M; Wang, Guangwu; Vondriska, Thomas M; Ping, Peipei

    2005-02-01

    Cardiac protective signaling networks have been shown to involve PKCepsilon. However, the molecular mechanisms by which PKCepsilon interacts with other members of these networks to form task-specific modules remain unknown. Among 93 different PKCepsilon-associated proteins that have been identified, Akt and endothelial nitric oxide (NO) synthase (eNOS) are of importance because of their independent abilities to promote cell survival and prevent cell death. The simultaneous association of PKCepsilon, Akt, and eNOS has not been examined, and, in particular, the formation of a module containing these three proteins and the role of such a module in the regulation of NO production and cardiac protection are unknown. The present study was undertaken to determine whether these molecules form a signaling module and, thereby, play a collective role in cardiac signaling. Using recombinant proteins in vitro and PKCepsilon transgenic mouse hearts, we demonstrate the following: 1) PKCepsilon, Akt, and eNOS interact and form signaling modules in vitro and in the mouse heart. Activation of either PKCepsilon or Akt enhances the formation of PKCepsilon-Akt-eNOS signaling modules. 2) PKCepsilon directly phosphorylates and enhances activation of Akt in vitro, and PKCepsilon activation increases phosphorylation and activation of Akt in PKCepsilon transgenic mouse hearts. 3) PKCepsilon directly phosphorylates eNOS in vitro, and this phosphorylation enhances eNOS activity. Activation of PKCepsilon in vivo increased phosphorylation of eNOS at Ser(1177), indicating eNOS activation. This study characterizes, for the first time, the physical, as well as functional, coupling of PKCepsilon, Akt, and eNOS in the heart and implicates these PKCepsilon-Akt-eNOS signaling modules as critical signaling elements during PKCepsilon-induced cardiac protection.

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

    PubMed Central

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

    2015-01-01

    Apoptosis plays an important role in neural development and neurological disorders. In this study, we found that O-GlcNAcylation, a unique protein posttranslational modification with O-linked β-N-acetylglucosamine (GlcNAc), promoted apoptosis through attenuating phosphorylation/activation of AKT and Bad. By using co-immunoprecipitation and mutagenesis techniques, we identified O-GlcNAc modification at both Thr308 and Ser473 of AKT. O-GlcNAcylation-induced apoptosis was attenuated by over-expression of AKT. We also found a dynamic elevation of protein O-GlcNAcylation during the first four hours of cerebral ischemia, followed by continuous decline after middle cerebral artery occlusion (MCAO) in the mouse brain. The elevation of O-GlcNAcylation coincided with activation of cell apoptosis. Finally, we found a negative correlation between AKT phosphorylation and O-GlcNAcylation in ischemic brain tissue. These results indicate that cerebral ischemia induces a rapid increase of O-GlcNAcylation that promotes apoptosis through down-regulation of AKT activity. These findings provide a novel mechanism through which O-GlcNAcylation regulates ischemia-induced neuronal apoptosis through AKT signaling. PMID:26412745

  1. Aberrant CD8+ T-Cell Responses and Memory Differentiation upon Viral Infection of an Ataxia-Telangiectasia Mouse Model Driven by Hyper-Activated Akt and mTORC1 Signaling

    PubMed Central

    D'Souza, Anthony D.; Parish, Ian A.; McKay, Sharen E.; Kaech, Susan M.; Shadel, Gerald S.

    2011-01-01

    Immune system-related pathology is common in ataxia-telangiectasia (A-T) patients and mice that lack the protein kinase, A-T mutated (ATM). However, it has not been studied how ATM influences immune responses to a viral infection. Using the lymphocytic choriomeningitis virus (LCMV) infection model, we show that ATM−/− mice, despite having fewer naïve CD8+ T cells, effectively clear the virus. However, aberrant CD8+ T-cell responses are observed, including defective expansion and contraction, effector-to-memory differentiation, and a switch in viral-epitope immunodominance. T-cell receptor-activated, but not naïve, ATM−/− splenic CD8+ T cells have increased ribosomal protein S6 and Akt phosphorylation and do not proliferate well in response to IL-15, a cytokine important for memory T-cell development. Accordingly, pharmacological Akt or mammalian target of rapamycin complex 1 (mTORC1) inhibition during T-cell receptor activation alone rescues the IL-15 proliferation defect. Finally, rapamycin treatment during LCMV infection in vivo increases the number of memory T cells in ATM−/− mice. Altogether, these results show that CD8+ T cells lacking ATM have hyperactive Akt and mTORC1 signaling in response to T-cell receptor activation, which results in aberrant cytokine responses and memory T-cell development. We speculate that similar signaling defects contribute to the immune system pathology of A-T, and that inhibition of Akt and/or mTORC1 may be of therapeutic value. PMID:21641396

  2. Inhibition of fatty acid synthase suppresses U-2 OS cell invasion and migration via downregulating the activity of HER2/PI3K/AKT signaling pathway in vitro

    SciTech Connect

    Wang, Tao Fang; Wang, Heng; Peng, Ai Fen; Luo, Qing Feng; Liu, Zhi Li; Zhou, Rong Ping; Gao, Song; Zhou, Yang; Chen, Wen Zhao

    2013-10-18

    Highlights: •We investigate the relationship between FASN and HER2 or p-HER2 by IHC in OS tissues. •We construct FASN-specific RNAi plasmid. •Inhibiting FASN down-regulates HER2/PI3K/AKT cell signaling in U-2 OS. •Inhibiting FASN blocks U-2 OS cell invasion and migration. -- Abstract: FASN plays an important role in the malignant phenotype of various tumors. Our previous studies show that inhibition FASN could induce apoptosis and inhibit proliferation in human osteosarcoma (OS) cell in vivo and vitro. The aim in this study was to investigate the effect of inhibition FASN on the activity of HER2/PI3K/AKT axis and invasion and migration of OS cell. The expression of FASN, HER2 and p-HER2(Y1248) proteins was detected by immunohistochemistry in OS tissues from 24 patients with pulmonary metastatic disease, and the relationship between FASN and p-HER2 as well as HER2 was investigated. The results showed that there was a positive correlation between FASN and HER2 as well as p-HER2 protein expression. The U-2 OS cells were transfected with either the FASN specific RNAi plasmid or the negative control RNAi plasmid. FASN mRNA was measured by RT-PCR. Western blot assays was performed to examine the protein expression of FASN, HER2, p-HER2(Y1248), PI3K, Akt and p-Akt (Ser473). Migration and invasion of cells were investigated by wound healing and transwell invasion assays. The results showed that the activity of HER2/PI3K/AKT signaling pathway was suppressed by inhibiting FASN. Meanwhile, the U-2OS cells migration and invasion were also impaired by inhibiting the activity of FASN/HER2/PI3K/AKT. Our results indicated that inhibition of FASN suppresses OS cell invasion and migration via down-regulation of the “HER2/PI3K/AKT” axis in vitro. FASN blocker may be a new therapeutic strategy in OS management.

  3. Non-immunosuppressive triazole-based small molecule induces anticancer activity against human hormone-refractory prostate cancers: the role in inhibition of PI3K/AKT/mTOR and c-Myc signaling pathways

    PubMed Central

    Chan, She-Hung; Hsu, Jui-Ling; Liu, Shih-Ping; Chan, Mei-Ling; Yu, Chia-Chun; Hsu, Lih-Ching; Chou, Yen-Lin; Chang, Wei-Ling; Hou, Duen-Ren; Guh, Jih-Hwa

    2016-01-01

    A series of triazole-based small molecules that mimic FTY720-mediated anticancer activity but minimize its immunosuppressive effect have been produced. SPS-7 is the most effective derivative displaying higher activity than FTY720 in anti-proliferation against human hormone-refractory prostate cancer (HRPC). It induced G1 arrest of cell cycle and subsequent apoptosis in thymidine block-mediated synchronization model. The data were supported by a decrease of cyclin D1 expression, a dramatic increase of p21 expression and an associated decrease in RB phosphorylation. c-Myc overexpression replenished protein levels of cyclin D1 indicating that c-Myc was responsible for cell cycle regulation. PI3K/Akt/mTOR signaling pathways through p70S6K- and 4EBP1-mediated translational regulation are critical to cell proliferation and survival. SPS-7 significantly inhibited this translational pathway. Overexpression of Myr-Akt (constitutively active Akt) completely abolished SPS-7-induced inhibitory effect on mTOR/p70S6K/4EBP1 signaling and c-Myc protein expression, suggesting that PI3K/Akt serves as a key upstream regulator. SPS-7 also demonstrated substantial anti-tumor efficacy in an in vivo xenograft study using PC-3 mouse model. Notably, FTY720 but not SPS-7 induced a significant immunosuppressive effect as evidenced by depletion of marginal zone B cells, down-regulation of sphingosine-1-phosphate receptors and a decrease in peripheral blood lymphocytes. In conclusion, the data suggest that SPS-7 is not an immunosuppressant while induces anticancer effect against HRPC through inhibition of Akt/mTOR/p70S6K pathwaysthat down-regulate protein levels of both c-Myc and cyclin D1, leading to G1 arrest of cell cycle and subsequent apoptosis. The data also indicate the potential of SPS-7 since PI3K/Akt signalingis responsive for the genomic alterations in prostate cancer. PMID:27769069

  4. Akt signaling is critical for memory CD8(+) T-cell development and tumor immune surveillance.

    PubMed

    Rogel, Anne; Willoughby, Jane E; Buchan, Sarah L; Leonard, Henry J; Thirdborough, Stephen M; Al-Shamkhani, Aymen

    2017-02-14

    Memory CD8(+) T cells confer long-term immunity against tumors, and anticancer vaccines therefore should maximize their generation. Multiple memory CD8(+) T-cell subsets with distinct functional and homing characteristics exist, but the signaling pathways that regulate their development are ill defined. Here we examined the role of the serine/threonine kinase Akt in the generation of protective immunity by CD8(+) T cells. Akt is known to be activated by the T-cell antigen receptor and the cytokine IL-2, but its role in T-cell immunity in vivo has not been explored. Using CD8(+) T cells from pdk1(K465E/K465E) knockin mice, we found that decreased Akt activity inhibited the survival of T cells during the effector-to-memory cell transition and abolished their differentiation into C-X-C chemokine receptor 3 (CXCR3)(lo)CD43(lo) effector-like memory cells. Consequently, antitumor immunity by CD8(+) T cells that display defective Akt signaling was substantially diminished during the memory phase. Reduced memory T-cell survival and altered memory cell differentiation were associated with up-regulation of the proapoptotic protein Bim and the T-box transcription factor eomesodermin, respectively. These findings suggest an important role for effector-like memory CD8(+) T cells in tumor immune surveillance and identify Akt as a key signaling node in the development of protective memory CD8(+) T-cell responses.

  5. Akt signaling is critical for memory CD8+ T-cell development and tumor immune surveillance

    PubMed Central

    Rogel, Anne; Willoughby, Jane E.; Buchan, Sarah L.; Leonard, Henry J.; Thirdborough, Stephen M.; Al-Shamkhani, Aymen

    2017-01-01

    Memory CD8+ T cells confer long-term immunity against tumors, and anticancer vaccines therefore should maximize their generation. Multiple memory CD8+ T-cell subsets with distinct functional and homing characteristics exist, but the signaling pathways that regulate their development are ill defined. Here we examined the role of the serine/threonine kinase Akt in the generation of protective immunity by CD8+ T cells. Akt is known to be activated by the T-cell antigen receptor and the cytokine IL-2, but its role in T-cell immunity in vivo has not been explored. Using CD8+ T cells from pdk1K465E/K465E knockin mice, we found that decreased Akt activity inhibited the survival of T cells during the effector-to-memory cell transition and abolished their differentiation into C-X-C chemokine receptor 3 (CXCR3)loCD43lo effector-like memory cells. Consequently, antitumor immunity by CD8+ T cells that display defective Akt signaling was substantially diminished during the memory phase. Reduced memory T-cell survival and altered memory cell differentiation were associated with up-regulation of the proapoptotic protein Bim and the T-box transcription factor eomesodermin, respectively. These findings suggest an important role for effector-like memory CD8+ T cells in tumor immune surveillance and identify Akt as a key signaling node in the development of protective memory CD8+ T-cell responses. PMID:28137869

  6. Inositol pyrophosphates inhibit Akt signaling, regulate insulin sensitivity and weight gain

    PubMed Central

    Chakraborty, Anutosh; Koldobskiy, Michael A.; Bello, Nicholas T.; Maxwell, Micah; Potter, James J.; Juluri, Krishna R.; Maag, David; Kim, Seyun; Huang, Alex S.; Dailey, Megan J.; Saleh, Masoumeh; Snowman, Adele M.; Moran, Timothy H.; Mezey, Esteban; Snyder, Solomon H.

    2011-01-01

    Summary The inositol pyrophosphate IP7 (5-diphosphoinositolpentakisphosphate), formed by a family of three inositol hexakisphosphate kinases (IP6Ks), modulates diverse cellular activities. We now report that IP7 is a physiologic inhibitor of Akt, a serine/threonine kinase which regulates glucose homeostasis and protein translation respectively via the GSK3β and mTOR pathways. Thus Akt, mTOR and GSK3β signaling are dramatically augmented in skeletal muscle, white adipose tissue, and liver of mice with targeted deletion of IP6K1. IP7 impacts this pathway by potently inhibiting the PDK1 phosphorylation of Akt, preventing its activation and thereby impacting insulin signaling. IP6K1 knockout mice manifest insulin sensitivity and are resistant to obesity elicited by high fat diet or aging. Inhibition of IP6K1 may afford a therapeutic approach to obesity and diabetes. PMID:21145457

  7. Activation of endoplasmic reticulum stress promotes autophagy and apoptosis and reverses chemoresistance of human small cell lung cancer cells by inhibiting the PI3K/AKT/mTOR signaling pathway

    PubMed Central

    Yu, Xin-Shuang; Du, Juan; Fan, Yu-Jun; Liu, Feng-Jun; Cao, Li-Li; Liang, Ning; Xu, De-Guo; Zhang, Jian-Dong

    2016-01-01

    Objective This study aims to investigate the effects of endoplasmic reticulum stress (ERS) on autophagy, apoptosis and chemoresistance of human small cell lung cancer (SCLC) cells via the PI3K/AKT/mTOR signaling pathway. Results The expressions of ERS-related proteins (PEAK, eIF2α and CHOP) up-regulated, autophagy-related proteins (LC3, LC3-II and Beclin1) and apoptosis-related proteins (Bax and procaspase-3) down-regulated in NCI-H446 and H69 cells after tunicamycin treatment for 24 h. Compared with the blank group, the tunicamycin, BEZ235 and tunicamycin + BEZ235 groups exhibited decreased expressions of p-PI3K, p-AKT and p-mTOR, and increased expressions of autophagy-related proteins (LC3, LC3-II and Beclin1) and apoptosis proteins (Bax and procaspase-3), and the most obvious changes were observed in the tunicamycin + BEZ235 group. Materials and Methods CCK-8 assay was applied to select the best cell line from five SCLC cell lines (NCI-H446, H69, H526, H146 and H209). Finally, NCI-H446 and H69 cells were selected for further experiments. NCI-H446/CDDP and H69/CDDP were selected and divided into the blank group, tunicamycin (an ESR inducer) group, BEZ235 (inhibitors of PI3K/AKT/mTOR pathway) group and tunicamycin + BEZ235 group. Cell apoptosis was detected by flow cytometry. Autophagy was observed by fluorescence microscopy and flow cytometry. Western blotting was used to detect the expressions of ERS-related proteins, autophagy-related proteins, apoptosis-related proteins and PI3K/AKT/mTOR pathway-related proteins. Conclusions Our findings provide evidence that the activation of ERS could promote autophagy and apoptosis and reverse chemoresistance of human SCLC cells by inhibiting the PI3K/AKT/mTOR pathway. PMID:27765907

  8. 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

  9. Focal adhesion kinase mediates atrial fibrosis via the AKT/S6K signaling pathway in chronic atrial fibrillation patients with rheumatic mitral valve disease.

    PubMed

    Zhang, Peide; Wang, Wei; Wang, Xin; Wang, Xu; Song, Yunhu; Zhang, Jing; Zhao, Hui

    2013-10-09

    Atrial fibrosis, as a hallmark of atrial structural remodeling, plays a critical role in the maintenance of chronic atrial fibrillation (AF), but the mechanisms responsible for atrial fibrosis are still uncertain. Fibrogenesis represents a complex process in which focal adhesion kinase (FAK) plays an important role. Therefore, we investigated the role of FAK-mediated signaling in atrial fibrosis in patients with chronic AF related to rheumatic mitral valve disease (RMVD). Atrial appendages were excised from 45 patients with RMVD and either chronic AF (n=25, AF >6 months) or sinus rhythm (n=20). Fibrosis was assessed by histology, and FAK and its two downstream pathways (AKT/S6K and ERK1/2) were evaluated by western blotting. We further evaluated the role of FAK in fibrogenesis by culturing neonatal rat cardiac fibroblasts to determine the importance of FAK-regulated signaling in cardiac myofibroblast differentiation induced by transforming growth factor-β1 (TGFβ1). Our study revealed that FAK can regulate its downstream signaling to cause fibrosis in atrial tissue and activate isolated fibroblasts. Histology revealed a significant increase in atrial fibrosis in AF patients. The phosphorylation of FAK and its downstream AKT/S6K signaling was increased secondary to TGFβ1-induced high expression of α-SMA, a marker of myofibroblast activity. FAK and AKT inhibitors suppressed α-SMA expression in TGFβ1-induced fibroblasts. However, ERK1/2 signaling seemed to be unrelated to the fibrotic process in AF patients. The FAK-mediated AKT/S6K signaling pathway participated in atrial fibrogenesis and this finding may contribute to the prevention of atrial fibrosis associated with chronic AF in patients with underlying cardiac disease. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

  10. AKT Signaling Pathway in the Nucleus Accumbens Mediates Excessive Alcohol Drinking Behaviors

    PubMed Central

    Neasta, Jérémie; Ben Hamida, Sami; Yowell, Quinn V.; Carnicella, Sebastien; Ron, Dorit

    2011-01-01

    Background Neuroadaptations within the nucleus accumbens (NAc) have been implicated in molecular mechanisms underlying the development and/or maintenance of alcohol abuse disorders. We recently reported that the activation of mammalian target of rapamycin complex 1 (mTORC1) signaling pathway in the NAc of rodents, following exposure to alcohol, contributes to alcohol drinking behaviors. The kinase AKT, is the main upstream activator of the mTORC1 pathway. We therefore hypothesized that the activation of AKT in the NAc in response to alcohol exposure plays an important role in mechanisms that underlie excessive alcohol consumption. Methods Western blot analysis was used to assess the phosphorylation levels of enzymes. Acute exposure of mice to alcohol was achieved by the administration of 2 g/kg alcohol intraperitoneally, (i.p.). Two-bottle choice and operant self-administration procedures were used to assess drinking behaviors in rats. Results We found that acute systemic administration of alcohol and recurring cycles of excessive voluntary consumption of alcohol and withdrawal result in the activation of AKT signaling in the NAc of rodents. Importantly, we show that inhibition of AKT, or its upstream activator, phosphatidylinositol-3-kinase (PI3K), within the NAc of rats attenuates binge drinking as well as alcohol but not sucrose operant self-administration. Conclusions Our results suggest that the activation of the AKT pathway in the NAc in response to alcohol exposure is an important contributor to the molecular mechanisms underlying alcohol-drinking behaviors. AKT signaling pathway inhibitors are therefore potential candidates for drug development for the treatment of alcohol use and abuse disorders. PMID:21549353

  11. AKT signaling pathway in the nucleus accumbens mediates excessive alcohol drinking behaviors.

    PubMed

    Neasta, Jérémie; Ben Hamida, Sami; Yowell, Quinn V; Carnicella, Sebastien; Ron, Dorit

    2011-09-15

    Neuroadaptations within the nucleus accumbens (NAc) have been implicated in molecular mechanisms underlying the development and/or maintenance of alcohol abuse disorders. We recently reported that the activation of mammalian target of rapamycin complex 1 (mTORC1) signaling pathway in the NAc of rodents, after exposure to alcohol, contributes to alcohol drinking behaviors. The kinase AKT is a main upstream activator of the mTORC1 pathway. We therefore hypothesized that the activation of AKT in the NAc in response to alcohol exposure plays an important role in mechanisms that underlie excessive alcohol consumption. Western blot analysis was used to assess the phosphorylation levels of enzymes. Acute exposure of mice to alcohol was achieved by the administration of 2 g/kg alcohol intraperitoneally (i.p.). Two-bottle choice and operant self-administration procedures were used to assess drinking behaviors in rats. We found that acute systemic administration of alcohol and recurring cycles of excessive voluntary consumption of alcohol and withdrawal result in the activation of AKT signaling in the NAc of rodents. We show that inhibition of AKT or its upstream activator, phosphatidylinositol-3-kinase (PI3K), within the NAc of rats attenuates binge drinking as well as alcohol but not sucrose operant self-administration. Our results suggest that the activation of the AKT pathway in the NAc in response to alcohol exposure is an important contributor to the molecular mechanisms underlying alcohol-drinking behaviors. AKT signaling pathway inhibitors are therefore potential candidates for drug development for the treatment of alcohol use and abuse disorders. Copyright © 2011 Society of Biological Psychiatry. Published by Elsevier Inc. All rights reserved.

  12. The Asian-American variant of human papillomavirus type 16 exhibits higher activation of MAPK and PI3K/AKT signaling pathways, transformation, migration and invasion of primary human keratinocytes.

    PubMed

    Hochmann, Jimena; Sobrinho, João S; Villa, Luisa L; Sichero, Laura

    2016-05-01

    Asian-American (AA) HPV-16 variants are associated with higher risk of cancer. Abnormal activation of intracellular signaling play a critical role in cancer development and progression. Our aim was to elucidate mechanisms underlying the higher oncogenic potential attributed to AA variant. We evaluated activation of MAPK and PI3K/AKT pathways in primary human keratinocytes (PHKs) transduced with E6/E7 of three HPV-16 variants: E-P, AA, E-350G. Phenotypes examined included migration, anchorage independent growth and invasion. AA PHKs presented the highest levels of active proteins involved in all cascades analyzed: MAPK-ERK, MAPK-p38 and PI3K-AKT. AA PHKs were more efficient in promoting anchorage independent growth, and in stimulating cell migration and invasion. MEK1 inhibition decreased migration. The mesenchymal phenotype marker vimentin was increased in AA PHKs. Our results suggest that MEK1, ERK2, AKT2 hyperactivation influence cellular behavior by means of GSK-3b inactivation and EMT induction prompting AA immortalized PHKs to more efficiently surpass carcinogenesis steps.

  13. 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

  14. Pseudoginsenoside-F11 (PF11) exerts anti-neuroinflammatory effects on LPS-activated microglial cells by inhibiting TLR4-mediated TAK1/IKK/NF-κB, MAPKs and Akt signaling pathways.

    PubMed

    Wang, Xiaoxiao; Wang, Chunming; Wang, Jiming; Zhao, Siqi; Zhang, Kuo; Wang, Jingmin; Zhang, Wei; Wu, Chunfu; Yang, Jingyu

    2014-04-01

    Pseudoginsenoside-F11 (PF11), an ocotillol-type ginsenoside, has been shown to possess significant neuroprotective activity. Since microglia-mediated inflammation is critical for induction of neurodegeneration, this study was designed to investigate the effect of PF11 on activated microglia. PF11 significantly suppressed the release of ROS and proinflammatory mediators induced by LPS in a microglial cell line N9 including NO, PGE2, IL-1β, IL-6 and TNF-α. Moreover, PF11 inhibited interaction and expression of TLR4 and MyD88 in LPS-activated N9 cells, resulting in an inhibition of the TAK1/IKK/NF-κB signaling pathway. PF11 also inhibited the phosphorylation of Akt and MAPKs induced by LPS in N9 cells. Importantly, PF11 significantly alleviated the death of SH-SY5Y neuroblastoma cells and primary cortical neurons induced by the conditioned-medium from activated microglia. At last, the effect of PF11 on neuroinflammation was confirmed in vivo: PF11 mitigated the microglial activation and proinflammatory factors expression obviously in both cortex and hippocampus in mice injected intrahippocampally with LPS. These findings indicate that PF11 exerts anti-neuroinflammatory effects on LPS-activated microglial cells by inhibiting TLR4-mediated TAK1/IKK/NF-κB, MAPKs and Akt signaling pathways, suggesting its therapeutic implication for neurodegenerative disease associated with neuroinflammation.

  15. Cross Talk between the Akt and p38α Pathways in Macrophages Downstream of Toll-Like Receptor Signaling

    PubMed Central

    McGuire, Victoria A.; Gray, Alexander; Monk, Claire E.; Santos, Susana G.; Lee, Keunwook; Aubareda, Anna; Crowe, Jonathan; Ronkina, Natalia; Schwermann, Jessica; Batty, Ian H.; Leslie, Nick R.; Dean, Jonathan L. E.; O'Keefe, Stephen J.; Boothby, Mark; Gaestel, Matthias

    2013-01-01

    The stimulation of Toll-like receptors (TLRs) on macrophages by pathogen-associated molecular patterns (PAMPs) results in the activation of intracellular signaling pathways that are required for initiating a host immune response. Both phosphatidylinositol 3-kinase (PI3K)–Akt and p38 mitogen-activated protein kinase (MAPK) signaling pathways are activated rapidly in response to TLR activation and are required to coordinate effective host responses to pathogen invasion. In this study, we analyzed the role of the p38-dependent kinases MK2/3 in the activation of Akt and show that lipopolysaccharide (LPS)-induced phosphorylation of Akt on Thr308 and Ser473 requires p38α and MK2/3. In cells treated with p38 inhibitors or an MK2/3 inhibitor, phosphorylation of Akt on Ser473 and Thr308 is reduced and Akt activity is inhibited. Furthermore, BMDMs deficient in MK2/3 display greatly reduced phosphorylation of Ser473 and Thr308 following TLR stimulation. However, MK2/3 do not directly phosphorylate Akt in macrophages but act upstream of PDK1 and mTORC2 to regulate Akt phosphorylation. Akt is recruited to phosphatidylinositol 3,4,5-trisphosphate (PIP3) in the membrane, where it is activated by PDK1 and mTORC2. Analysis of lipid levels in MK2/3-deficient bone marrow-derived macrophages (BMDMs) revealed a role for MK2/3 in regulating Akt activity by affecting availability of PIP3 at the membrane. These data describe a novel role for p38α-MK2/3 in regulating TLR-induced Akt activation in macrophages. PMID:23979601

  16. K-RAS(V12) Induces Autocrine Production of EGFR Ligands and Mediates Radioresistance Through EGFR-Dependent Akt Signaling and Activation of DNA-PKcs

    SciTech Connect

    Minjgee, Minjmaa; Toulany, Mahmoud; Kehlbach, Rainer; Giehl, Klaudia; Rodemann, H. Peter

    2011-12-01

    Purpose: It is known that postirradiation survival of tumor cells presenting mutated K-RAS is mediated through autocrine activation of epidermal growth factor receptor (EGFR). In this study the molecular mechanism of radioresistance of cells overexpressing mutated K-RAS(V12) was investigated. Methods and Materials: Head-and-neck cancer cells (FaDu) presenting wild-type K-RAS were transfected with empty vector or vector expressing mutated K-RAS(V12). The effect of K-RAS(V12) on autocrine production of EGFR ligands, activation of EGFR downstream pathways, DNA damage repair, and postirradiation survival was analyzed. Results: Conditioned medium collected from K-RAS(V12)-transfected cells enhanced activation of the phosphatidylinositol-3-kinase-Akt pathway and increased postirradiation survival of wild-type K-RAS parental cells when compared with controls. These effects were reversed by amphiregulin (AREG)-neutralizing antibody. In addition, secretion of the EGFR ligands AREG and transforming growth factor {alpha} was significantly increased upon overexpression of K-RAS(V12). Expression of mutated K-RAS(V12) resulted in an increase in radiation-induced DNA-dependent protein kinase catalytic subunit (DNA-PKcs) phosphorylation at S2056. This increase was accompanied by increased repair of DNA double-strand breaks. Abrogation of DNA-PKcs phosphorylation by serum depletion or AREG-neutralizing antibody underscored the role of autocrine production of EGFR ligands, namely, AREG, in regulating DNA-PKcs activation in K-RAS mutated cells. Conclusions: These data indicate that radioresistance of K-RAS mutated tumor cells is at least in part due to constitutive production of EGFR ligands, which mediate enhanced repair of DNA double-strand breaks through the EGFR-phosphatidylinositol-3-kinase-Akt cascade.

  17. Cyclophilin A as a downstream effector of PI3K/Akt signalling pathway in multiple myeloma cells.

    PubMed

    Lin, Zuo-Lin; Wu, Hsin-Jou; Chen, Jin-An; Lin, Kuo-Chih; Hsu, Jung-Hsin

    2015-12-01

    Cyclophilin A (Cyp A), a member of the peptidyl-prolyl isomerase (PPI) family, may function as a molecular signalling switch. Comparative proteomic studies have identified Cyp A as a potential downstream target of protein kinase B (Akt). This study confirmed that Cyp A is a downstream effector of the phosphatidylinositide 3-kinase (PI3K)/Akt signalling pathway. Cyp A was highly phosphorylated in response to interleukin-6 treatment, which was consistent with the accumulation of phosphorylated Akt, suggesting that Cyp A is a phosphorylation target of Akt and downstream effector of the PI3K/Akt pathway. Cyclosporine A (CsA), a PPI inhibitor, inhibited the growth of multiple myeloma (MM) U266 cells. Moreover, CsA treatment inhibited the activation of the signal transducer and activator of transcription 3 (STAT3) in MM U266 cells. Several Cyp A mutants were generated. Mutants with mutated AKT phosphorylation sites increased the G1 phase arrest in MM U266 cells. The other mutants that mimicked the phosphorylated state of Cyp A decreased the percentage of G1 phase. These results demonstrated that the states of phosphorylation of Cyp A by Akt can influence the progress of the cell cycle in MM U266 cells and that this effect is probably mediated through the Janus-activated kinase 2/STAT3 signalling pathway.

  18. Antitumor Activity of a Novel Antisense Oligonucleotide Against Akt1

    PubMed Central

    Yoon, Heejeong; Kim, Deog Joong; Ahn, Eun Hyun; Gellert, Ginelle C.; Shay, Jerry W.; Ahn, Chang-Ho; Lee, Young Bok

    2010-01-01

    The AKT pathway is an important therapeutic target for cancer drug discovery as it functions as a main point for transducing extracellular and intracellular oncogenic signals. Moreover, alternations of the AKT pathway have been found in a wide range of cancers. In the present study, we found that an Akt1 antisense oligonucleotide (Akt1 AO) significantly downregulated the expression of AKT1 at both the mRNA and protein levels and inhibited cellular growth at nanomolar concentrations in various types of human cancer cells. Combined treatment of Akt1 AO with several cytotoxic drugs resulted in an additive growth inhibition of Caki-1 cells. The in vivo effectiveness of Akt1 AO was determined using two different xenograft nude mouse models. Akt1 AO (30 mg/kg, i.v. every 48 h) significantly inhibited the tumor growth of nude mouse subcutaneously implanted with U251 human glioblastoma cells after 27 days treatment. Akt1 AO (30 mg/kg, i.p continuously via osmotic pump) also significantly inhibited the tumor formation in nude mice implanted with luciferase-expressing MIA human pancreatic cancer cells (MIA-Luc) after 14 days of treatment. The luciferase signals from MIA-Luc cells were reduced or completely abolished after 2 weeks of treatment and the implanted tumors were barely detectable. Our findings suggest that Akt1 AO alone or in combination with other clinically approved anticancer agents should be further explored and progressed into clinical studies as a potential novel therapeutic agent. PMID:19693774

  19. Gallic acid inhibits gastric cancer cells metastasis and invasive growth via increased expression of RhoB, downregulation of AKT/small GTPase signals and inhibition of NF-κB activity

    SciTech Connect

    Ho, Hsieh-Hsun; Chang, Chi-Sen; Ho, Wei-Chi; Liao, Sheng-You; Lin, Wea-Lung; Wang, Chau-Jong

    2013-01-01

    Our previous study demonstrated the therapeutic potential of gallic acid (GA) for controlling tumor metastasis through its inhibitory effect on the motility of AGS cells. A noteworthy finding in our previous experiment was increased RhoB expression in GA-treated cells. The aim of this study was to evaluate the role of RhoB expression on the inhibitory effects of GA on AGS cells. By applying the transfection of RhoB siRNA into AGS cells and an animal model, we tested the effect of GA on inhibition of tumor growth and RhoB expression. The results confirmed that RhoB-siRNA transfection induced GA to inhibit AGS cells’ invasive growth involving blocking the AKT/small GTPase signals pathway and inhibition of NF-κB activity. Finally, we evaluated the effect of GA on AGS cell metastasis by colonization of tumor cells in nude mice. It showed GA inhibited tumor cells growth via the expression of RhoB. These data support the inhibitory effect of GA which was shown to inhibit gastric cancer cell metastasis and invasive growth via increased expression of RhoB, downregulation of AKT/small GTPase signals and inhibition of NF-κB activity. Thus, GA might be a potential agent in treating gastric cancer. Highlights: ► GA could downregulate AKT signal via increased expression of RhoB. ► GA inhibits metastasis in vitro in gastric carcinoma. ► GA inhibits tumor growth in nude mice model.

  20. L-3-n-Butylphthalide Activates Akt/mTOR Signaling, Inhibits Neuronal Apoptosis and Autophagy and Improves Cognitive Impairment in Mice with Repeated Cerebral Ischemia-Reperfusion Injury.

    PubMed

    Xu, Jing; Huai, Yaping; Meng, Nan; Dong, Yanhong; Liu, Zhijuan; Qi, Qianqian; Hu, Ming; Fan, Mingyue; Jin, Wei; Lv, Peiyuan

    2017-06-15

    L-3-n-Butylphthalide (L-NBP) exerts neuroprotective effects in animal models of cerebral ischemia, but its potential benefits in repeated cerebral ischemia-reperfusion (RCIR) injury remain unknown. We investigated the effect of L-NBP on cognitive impairment induced by RCIR in mice. Male C57Bl/6 mice received sham surgery or bilateral common carotid artery occlusion (3 times, 20 min each) and were orally administered preoperative L-NBP (30 mg/kg/day, 7 days), postoperative L-NBP (30 or 60 mg/kg/day, 28 days) or postoperative vehicle (28 days). Learning and memory were assessed by the Morris water maze task and step-down passive avoidance test. Nissl staining was used to identify pathologic changes in the hippocampal CA1 region. The expressions of proteins associated with signaling, apoptosis and autophagy were assessed by quantitative PCR and western blot. RCIR induced deficits in learning and memory that were alleviated by preoperative or postoperative L-NBP administration. Pathologic lesions in the hippocampal CA1 region induced by RCIR were less severe in mice treated with L-NBP. Preoperative or postoperative L-NBP administration in mice receiving RCIR promoted hippocampal expression of phospho-Akt and phospho-mTOR (suggesting activation of Akt/mTOR signaling), increased the Bcl-2/Bax ratio (indicating suppression of apoptosis) and reduced the LC3-II/LC3-I ratio (implying inhibition of autophagy). Preoperative or postoperative L-NBP administration also depressed hippocampal levels of beclin-1 mRNA (indicating suppression of autophagy). These findings suggest that the effect of L-NBP to alleviate learning and memory deficits in mice following RCIR may involve activation of Akt/mTOR signaling and regulation of the expressions of proteins related to apoptosis and autophagy.

  1. Hispolon suppresses SK-Hep1 human hepatoma cell metastasis by inhibiting matrix metalloproteinase-2/9 and urokinase-plasminogen activator through the PI3K/Akt and ERK signaling pathways.

    PubMed

    Huang, Guan-Jhong; Yang, Chih-Min; Chang, Yuan-Shiun; Amagaya, Sakae; Wang, Hsiao-Chieh; Hou, Wen-Chi; Huang, Shyh-Shyun; Hu, Miao-Lin

    2010-09-08

    Cancer metastasis is a primary cause of cancer death. Hispolon is an active phenolic compound of Phellinus linteus, a mushroom that has recently been shown to have antioxidant and anticancer activities. In this study, we first observed that hispolon exerted a dose-dependent inhibitory effect on invasion and motility, but not on adhesion, of the highly metastatic SK-Hep1 cells in the absence of cytotoxicity. Mechanistically, hispolon decreased the expression of matrix metalloproteinase-2 (MMP-2), matrix metalloproteinase-9 (MMP-9), and urokinase-plasminogen activator (uPA) in a concentration-dependent manner. Hispolon also inhibited phosphorylation of extracellular signaling-regulating kinase1/2 (ERK1/2), phosphatidylinositol-3-kinase/serine/threonine protein kinase (or protein kinase B (PI3K/Akt), and focal adhesion kinase (FAK). Furthermore, treatment of SK-Hep1 cells with an inhibitor specific for ERK1/2 (PD98256) decreased the expression of MMP-2, and MMP-9. These results demonstrate that hispolon can inhibit the metastasis of SK-Hep1 cells by reduced expression of MMP-2, MMP-9, and uPA through the suppression of the FAK signaling pathway and of the activity of PI3K/Akt and Ras homologue gene family, member A (RhoA). These findings suggest that hispolon may be used as an antimetastatic agent.

  2. Interference with Akt signaling protects against myocardial infarction and death by limiting the consequences of oxidative stress

    PubMed Central

    Kerr, Bethany A.; Ma, Lining; West, Xiaoxia Z.; Ding, Liang; Malinin, Nikolay L.; Weber, Malory E.; Tischenko, Mira; Goc, Anna; Somanath, Payaningal R.; Penn, Marc S.; Podrez, Eugene A.; Byzova, Tatiana V.

    2014-01-01

    The intricacy of multiple feedback loops in the pathways downstream of Akt allows Akt to control multiple cellular processes in the cardiovascular system and precludes inferring consequences of its activation in specific pathological conditions. Akt1, the major Akt isoform in heart and vasculature, has a protective role in the endothelium during atherosclerosis. However, Akt1 activation has been proposed to have detrimental consequences in the cardiovascular system. Mice lacking the apolipoprotein E (ApoE), which promotes clearance of remnant lipoproteins, and the high-density lipoprotein receptor SR-BI are a model of spontaneous myocardial infarction and severe dyslipidemia. Akt1 was activated in these mice, and this activation correlated with cardiac dysfunction, hypertrophy, and fibrosis; increased infarct area; macrophage cholesterol accumulation and atherosclerosis; and reduced lifespan. Akt1 activation was associated with inflammation, oxidative stress, accumulation of oxidized lipids and increased abundance of CD36, a major sensor of oxidative stress, which created a positive feedback loop that exacerbated the consequences of oxidative stress. Thus, interference with Akt1 signaling in vivo could be protective and improve survival in dyslipidemia in the absence of SR-BI by reducing oxidative stress and responses to oxidized lipids. PMID:23921086

  3. Melatonin inhibits AP-2β/hTERT, NF-κB/COX-2 and Akt/ERK and activates caspase/Cyto C signaling to enhance the antitumor activity of berberine in lung cancer cells.

    PubMed

    Lu, Jian-Jun; Fu, Lingyi; Tang, Zhipeng; Zhang, Changlin; Qin, Lijun; Wang, Jingshu; Yu, Zhenlong; Shi, Dingbo; Xiao, Xiangsheng; Xie, Fangyun; Huang, Wenlin; Deng, Wuguo

    2016-01-19

    Melatonin, a molecule produced throughout the animal and plant kingdoms, and berberine, a plant derived agent, both exhibit antitumor and multiple biological and pharmacological effects, but they have never been combined altogether for the inhibition of human lung cancers. In this study, we investigated the role and underlying mechanisms of melatonin in the regulation of antitumor activity of berberine in lung cancer cells. Treatment with melatonin effectively increased the berberine-mediated inhibitions of cell proliferation, colony formation and cell migration, thereby enhancing the sensitivities of lung cancer cells to berberine. Melatonin also markedly increased apoptosis induced by berberine. Further mechanism study showed that melatonin promoted the cleavage of caspse-9 and PARP, enhanced the inhibition of Bcl2, and triggered the releasing of cytochrome C (Cyto C), thereby increasing the berberine-induced apoptosis. Melatonin also enhanced the berberine-mediated inhibition of telomerase reverses transcriptase (hTERT) by down-regulating the expression of AP-2β and its binding on hTERT promoter. Moreover, melatonin enhanced the berberine-mediated inhibition of cyclooxygenase 2 (COX-2) by inhibiting the nuclear translocation of NF-κB and its binding on COX-2 promoter. Melatonin also increased the berberine-mediated inhibition of the phosphorylated Akt and ERK. Collectively, our results demonstrated that melatonin enhanced the antitumor activity of berberine by activating caspase/Cyto C and inhibiting AP-2β/hTERT, NF-κB/COX-2 and Akt/ERK signaling pathways. Our findings provide new insights in exploring the potential therapeutic strategies and novel targets for lung cancer treatment.

  4. Melatonin inhibits AP-2β/hTERT, NF-κB/COX-2 and Akt/ERK and activates caspase/Cyto C signaling to enhance the antitumor activity of berberine in lung cancer cells

    PubMed Central

    Zhang, Changlin; Qin, Lijun; Wang, Jingshu; Yu, Zhenlong; Shi, Dingbo; Xiao, Xiangsheng; Xie, Fangyun; Huang, Wenlin; Deng, Wuguo

    2016-01-01

    Melatonin, a molecule produced throughout the animal and plant kingdoms, and berberine, a plant derived agent, both exhibit antitumor and multiple biological and pharmacological effects, but they have never been combined altogether for the inhibition of human lung cancers. In this study, we investigated the role and underlying mechanisms of melatonin in the regulation of antitumor activity of berberine in lung cancer cells. Treatment with melatonin effectively increased the berberine-mediated inhibitions of cell proliferation, colony formation and cell migration, thereby enhancing the sensitivities of lung cancer cells to berberine. Melatonin also markedly increased apoptosis induced by berberine. Further mechanism study showed that melatonin promoted the cleavage of caspse-9 and PARP, enhanced the inhibition of Bcl2, and triggered the releasing of cytochrome C (Cyto C), thereby increasing the berberine-induced apoptosis. Melatonin also enhanced the berberine-mediated inhibition of telomerase reverses transcriptase (hTERT) by down-regulating the expression of AP-2β and its binding on hTERT promoter. Moreover, melatonin enhanced the berberine-mediated inhibition of cyclooxygenase 2 (COX-2) by inhibiting the nuclear translocation of NF-κB and its binding on COX-2 promoter. Melatonin also increased the berberine-mediated inhibition of the phosphorylated Akt and ERK. Collectively, our results demonstrated that melatonin enhanced the antitumor activity of berberine by activating caspase/Cyto C and inhibiting AP-2β/hTERT, NF-κB/COX-2 and Akt/ERK signaling pathways. Our findings provide new insights in exploring the potential therapeutic strategies and novel targets for lung cancer treatment. PMID:26672764

  5. ROS-mediated Activation of AKT Induces Apoptosis Via pVHL in Prostate Cancer Cells

    PubMed Central

    Chetram, Mahandranauth A.; Bethea, Danaya A.; Jones, Kia J.; Don-Salu-Hewage, Ayesha S.; Odero-Marah, Valerie A.; Hinton, Cimona V.

    2013-01-01

    Reactive oxygen species (ROS) play a central role in oxidative stress, which leads to the onset of diseases, such as cancer. Furthermore, ROS contributes to the delicate balance between tumor cell survival and death. However, the mechanisms by which tumor cells decide to elicit survival or death signals during oxidative stress are not completely understood. We have previously reported that ROS enhanced tumorigenic functions in prostate cancer cells, such as transendothelial migration and invasion, which depended on CXCR4 and AKT signaling. Here, we report a novel mechanism by which ROS facilitated cell death through activation of AKT. We initially observed that ROS increased expression of phosphorylated AKT (p-AKT) in 22Rv1 human prostate cancer cells. The tumor suppressor PTEN, a negative regulator of AKT signaling, was rendered catalytically inactive through oxidation by ROS, although the expression levels remained consistent. Despite these events, cells still underwent apoptosis. Further investigation into apoptosis revealed that expression of the tumor suppressor pVHL increased, and contains a target site for p-AKT phosphorylation. pVHL and p-AKT associated in vitro, and knockdown of pVHL rescued HIF1α expression and the cells from apoptosis. Collectively, our study suggests that in the context of oxidative stress, p-AKT facilitated apoptosis by inducing pVHL function. PMID:23315288

  6. Leukemia-associated mutations in SHIP1 inhibit its enzymatic activity, interaction with the GM-CSF receptor and Grb2, and its ability to inactivate PI3K/AKT signaling.

    PubMed

    Brauer, Helena; Strauss, Julia; Wegner, Wiebke; Müller-Tidow, Carsten; Horstmann, Martin; Jücker, Manfred

    2012-11-01

    The inositol 5-phosphatase SHIP1 is a negative regulator of the PI3K/AKT pathway, which is constitutively activated in 50-70% of acute myeloid leukemias (AML). Ten different missense mutations in SHIP1 have been described in 3% of AML patients suggesting a functional role of SHIP1 in AML. Here, we report the identification of two new SHIP1 mutations T162P and R225W that were detected in 2 and 1 out of 96 AML patients, respectively. The functional analysis of all 12 AML-associated SHIP1 mutations, one ALL-associated SHIP1 mutation (Q1076X) and a missense SNP (H1168Y) revealed that two mutations i.e. Y643H and P1039S abrogated the ability of SHIP1 to reduce constitutive PI3K/AKT signaling in Jurkat cells. The loss of function of SHIP1 mutant Y643H which is localized in the inositol phosphatase domain was due to a reduction of the specific activity by 84%. Because all other SHIP1 mutants had a normal enzymatic activity, we assumed that these SHIP1 mutants may be functionally impaired due to a loss of interaction with plasma membrane receptors or adapter proteins. In agreement with this model, we found that the SHIP1 mutant F28L located in the FLVR motif of the SH2 domain was incapable of binding tyrosine-phosphorylated proteins including the GM-CSF receptor and that the SHIP1 mutant Q1076X lost its ability to bind to the C-terminal SH3 domain of the adapter protein Grb2. In addition, SHIP1 mutant P1039S which does not reduce PI3K/AKT signaling anymore is located in a PXXP SH3 domain consensus binding motif suggesting that mutation of the conserved proline residue interferes with binding of SHIP1 to a so far unidentified SH3 domain containing protein. In summary, our data indicate that SHIP1 mutations detected in human leukemia patients impair the negative regulatory function of SHIP1 on PI3K/AKT signaling in leukemia cells either directly by reduced enzymatic activity or indirectly by disturbed protein interaction with tyrosine-phosphorylated membrane receptors or

  7. Activation of p53/p21/PUMA alliance and disruption of PI-3/Akt in multimodal targeting of apoptotic signaling cascades in cervical cancer cells by a pentacyclic triterpenediol from Boswellia serrata.

    PubMed

    Bhushan, Shashi; Malik, Fayaz; Kumar, Ajay; Isher, Harpreet Kaur; Kaur, Indu Pal; Taneja, Subhash Chandra; Singh, Jaswant

    2009-12-01

    Cervical carcinoma is a growing menace to women health worldwide. This study reports the apoptotic cell death in human cervical cancer HeLa and SiHa cells by a pentacyclic triterpenediol (TPD) from Boswellia serrata by a mechanism different from reported in HL-60 cells. It caused oxidative stress by early generation of nitric oxide and reactive oxygen species that robustly up regulated time-dependent expression of p53/p21/PUMA while conversely abrogating phosphatidylinositol-3-kinase (PI3K)/Akt pathways in parallel. TPD also decreased the expression of PI3K/pAkt, ERK1/2, NF-kappaB/Akt signaling cascades which coordinately contribute to cancer cell survival through these distinct pathways. The tumor suppressor p53 pathway predominantly activated by TPD further up-regulated PUMA, which concomitantly decreased the Bcl-2 level, caused mitochondrial membrane potential loss with attendant translocation of Bax and drp1 to mitochondria and release of pro-apoptotic factors such as cytochrome c and Smac/Diablo to cytosol leading to caspases-3 and -9 activation. In addition both the phospho-p53 and p21 were found to accumulate heavily in the nuclear fraction with attendant decrease in topoisomarase II and survivin levels. On the contrary, TPD did not affect the extrinsic signaling transduction pathway effectively through apical death receptors. Interestingly, N-acetyl cysteine, ascorbate and s-methylisothiourea (sMIT) rescued cells significantly from TPD induced DNA damage and caspases activation. TPD may thus find usefulness in managing and treating cervical cancer.

  8. Deuterohemin-AlaHisLys mitigates the symptoms of rats with non-insulin dependent diabetes mellitus by scavenging reactive oxygen species and activating the PI3-K/AKT signal transduction pathway.

    PubMed

    Lei, Liyan; Zhang, Guangji; Li, Pengfei; Zhang, Yuan; Guo, Youming; Zhang, Wenqi; Zhang, Wenbo; Hu, Bing; Wang, Liping

    2014-09-05

    Damage to pancreatic β-cells plays an important role in the development of type 2 diabetes, and oxidative stress is a likely contributor. In the present study, we investigated the effect of deuterohemin-AlaHisLys (DhHP-3), a microperoxidase-11 mimic, on rats with non-insulin dependent diabetes mellitus and examined the action mechanisms of DhHP-3. The induced hyperglycemia, glucose intolerance, and insulin resistance in diabetic rats were associated with increased oxidative stress and damage to pancreatic islets. DhHP-3 (3 mg/kg) ameliorated hyperglycemia and insulin resistance, protected pancreas islet, decreased the content of malondialdehyde, and increased the activity of superoxide dismutase in plasma and pancreatic tissue by reducing ROS levels. Furthermore, DhHP-3 stimulated the proliferation of INS-1 cells and inhibited apoptosis by activating the phosphatidylinositol 3-kinase/protein kinase B (PI3-K/AKT) signaling pathway. Our results demonstrated for the first time that DhHP-3 decreased blood glucose level in rats with non-insulin dependent diabetes mellitus, scavenged reactive oxygen species, activated the PI3-K/AKT signaling pathway, and protected pancreatic β-cells against apoptosis.

  9. Erythropoietin alleviates hepatic insulin resistance via PPARγ-dependent AKT activation

    PubMed Central

    Ge, Zhijuan; Zhang, Pengzi; Hong, Ting; Tang, Sunyinyan; Meng, Ran; Bi, Yan; Zhu, Dalong

    2015-01-01

    Erythropoietin (EPO) has beneficial effects on glucose metabolism and insulin resistance. However, the mechanism underlying these effects has not yet been elucidated. This study aimed to investigate how EPO affects hepatic glucose metabolism. Here, we report that EPO administration promoted phosphatidylinositol 3-kinase (PI3K)/AKT pathway activation in palmitic acid (PA)-treated HepG2 cells and in the liver of high-fat diet (HFD)-fed mice, whereas adenovirus-mediated silencing of the erythropoietin receptor (EPOR) blocked EPO-induced AKT signalling in HepG2 cells. Importantly, a peroxisome proliferator-activated receptor γ (PPARγ) antagonist and PPARγ small interfering RNA (siRNA) abrogated the EPO-induced increase in p-AKT in HepG2 cells. Lentiviral vector-mediated hepatic PPARγ silencing in HFD-fed C57BL/6 mice impaired EPO-mediated increases in glucose tolerance, insulin sensitivity and hepatic AKT activation. Furthermore, EPO activated the AMP-activated protein kinase (AMPK)/sirtuin 1 (SIRT1) signalling pathway, and AMPKα and SIRT1 knockdown each attenuated the EPO-induced PPARγ expression and deacetylation and PPARγ-dependent AKT activation in HepG2 cells. In summary, these findings suggest that PPARγ is involved in EPO/EPOR-induced AKT activation, and targeting the PPARγ/AKT pathway via EPO may have therapeutic implications for hepatic insulin resistance and type 2 diabetes. PMID:26643367

  10. IDH1 R132H Mutation Enhances Cell Migration by Activating AKT-mTOR Signaling Pathway, but Sensitizes Cells to 5-FU Treatment as NADPH and GSH Are Reduced

    PubMed Central

    Qiu, Jiangdong; Huang, Keting; Wu, Mindan; Xia, Chunlin

    2017-01-01

    Aim of study Mutations of isocitrate dehydrogenase 1 and 2 (IDH1 and IDH2) gene were recently discovered in vast majority of World Health Organization (WHO) grade II/III gliomas. This study is to understand the effects of IDH1 R132H mutation in gliomagenesis and to develop new strategies to treat glioma with IDH1 R132H mutation. Materials and methods Over expression of IDH1 R132H in U87MG cells was done by transfecting cells with IDH1 R132H plasmid. MTT assay, scratch repair assay and western blot were performed to study effects of IDH1 R132H mutation on cell proliferation, migration, regulating AKT-mTOR signaling pathway and cell death respectively. NADP+/NADPH and GSH quantification assays were performed to evaluate effects of IDH1 R132H mutation on the production of antioxidant NADPH and GSH. Results We found that over expression of IDH1 R132H mutation decreased cell proliferation consistent with previous reports; however, it increased cell migration and enhanced AKT-mTOR signaling pathway activation. Mutations in isocitrate dehydrogenase (IDH) 1 also change the function of the enzymes and cause them to produce 2-hydroxyglutarate and not produce NADPH. We tested the level of NADPH and GSH and demonstrated that IDH1 R132H mutant stable cells had significantly low NADPH and GSH level compared to control or IDH1 wild type stable cells. The reduced antioxidants (NADPH and GSH) sensitized U87MG cells with IDH R132H mutant to 5-FU treatment. Conclusion Our study highlights the important role of IHD1 R132H mutant in up- regulating AKT-mTOR signaling pathway and enhancing cell migration. Furthermore, we demonstrate that IDH1 R132H mutation affects cellular redox status and sensitizes gliomas cells with IDH1 R132H mutation to 5FU treatment. PMID:28052098

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

    PubMed Central

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

    2012-01-01

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

  12. Crosstalk between Akt/GSK3β signaling and dynamin-1 regulates clathrin-mediated endocytosis

    PubMed Central

    Reis, Carlos R; Chen, Ping-Hung; Srinivasan, Saipraveen; Aguet, François; Mettlen, Marcel; Schmid, Sandra L

    2015-01-01

    Clathrin-mediated endocytosis (CME) regulates signaling from the plasma membrane. Analysis of clathrin-coated pit (CCP) dynamics led us to propose the existence of a rate-limiting, regulatory step(s) that monitor the fidelity of early stages in CCP maturation. Here we show that nascent endocytic vesicles formed in mutant cells displaying rapid, dysregulated CME are defective in early endosomal trafficking, maturation and acidification, confirming the importance of this “checkpoint.” Dysregulated CME also alters EGF receptor signaling and leads to constitutive activation of the protein kinase Akt. Dynamin-1, which was thought to be neuron specific, is activated by the Akt/GSK3β signaling cascade in non-neuronal cells to trigger rapid, dysregulated CME. Acute activation of dynamin-1 in RPE cells by inhibition of GSK3β accelerates CME, alters CCP dynamics and, unexpectedly, increases the rate of CCP initiation. CRISPR-Cas9n-mediated knockout and reconstitution studies establish that dynamin-1 is activated by Akt/GSK3β signaling in H1299 non-small lung cancer cells. These findings provide direct evidence for an isoform-specific role for dynamin in regulating CME and reveal a feed-forward pathway that could link signaling from cell surface receptors to the regulation of CME. PMID:26139537

  13. G-Protein-Coupled Lysophosphatidic Acid Receptors and Their Regulation of AKT Signaling

    PubMed Central

    Riaz, Anjum; Huang, Ying; Johansson, Staffan

    2016-01-01

    A hallmark of G-protein-coupled receptors (GPCRs) is their ability to recognize and respond to chemically diverse ligands. Lysophospholipids constitute a relatively recent addition to these ligands and carry out their biological functions by activating G-proteins coupled to a large family of cell-surface receptors. This review aims to highlight salient features of cell signaling by one class of these receptors, known as lysophosphatidic acid (LPA) receptors, in the context of phosphatidylinositol 3-kinase (PI3K)–AKT pathway activation. LPA moieties efficiently activate AKT phosphorylation and activation in a multitude of cell types. The interplay between LPA, its receptors, the associated Gαi/o subunits, PI3K and AKT contributes to the regulation of cell survival, migration, proliferation and confers chemotherapy-resistance in certain cancers. However, detailed information on the regulation of PI3K–AKT signals induced by LPA receptors is missing from the literature. Here, some urgent issues for investigation are highlighted. PMID:26861299

  14. G-Protein-Coupled Lysophosphatidic Acid Receptors and Their Regulation of AKT Signaling.

    PubMed

    Riaz, Anjum; Huang, Ying; Johansson, Staffan

    2016-02-05

    A hallmark of G-protein-coupled receptors (GPCRs) is their ability to recognize and respond to chemically diverse ligands. Lysophospholipids constitute a relatively recent addition to these ligands and carry out their biological functions by activating G-proteins coupled to a large family of cell-surface receptors. This review aims to highlight salient features of cell signaling by one class of these receptors, known as lysophosphatidic acid (LPA) receptors, in the context of phosphatidylinositol 3-kinase (PI3K)-AKT pathway activation. LPA moieties efficiently activate AKT phosphorylation and activation in a multitude of cell types. The interplay between LPA, its receptors, the associated Gαi/o subunits, PI3K and AKT contributes to the regulation of cell survival, migration, proliferation and confers chemotherapy-resistance in certain cancers. However, detailed information on the regulation of PI3K-AKT signals induced by LPA receptors is missing from the literature. Here, some urgent issues for investigation are highlighted.

  15. Exercise Reduces Lung Fibrosis Involving Serotonin/Akt Signaling.

    PubMed

    Pereira, Paulo Rogerio; Oliveira-Junior, Manoel Carneiro; Mackenzie, Breanne; Chiovatto, Jaime Eduardo Davino; Matos, Yves; Greiffo, Flavia Regina; Rigonato-Oliveira, Nicole Cristine; Brugemman, Thayse Regina; Delle, Humberto; Idzko, Marco; Albertini, Regiane; Ligeiro Oliveira, Ana Paula; Damaceno-Rodrigues, Nilsa Regina; Caldini, Elia Garcia; Fernandez, Isis Ensil; Castro-Faria-Neto, Hugo Caire; Dolhnikoff, Marisa; Eickelberg, Oliver; Vieira, Rodolfo Paula

    2016-07-01

    Idiopathic pulmonary fibrosis (IPF) is a chronic fibrosing interstitial pneumonia, which involves aberrant serotonin (5-hydroxytryptamine [5-HT]) and Akt signaling. As protective effects of chronic aerobic training (AT) have been demonstrated in the context of lung injury, this study investigated whether AT attenuates bleomycin-induced lung fibrosis partly via a reduction of 5-HT and AKT signaling. Seventy-two C57BL/6 male mice were distributed in Control (Co), Exercise (Ex), Fibrosis (Fi), and Fibrosis + Exercise (Fi + Ex) groups. Bleomycin (1.5 UI·kg) was administered on day 1 and treadmill AT began on day 15 and continued for 60 min·d, 5 d·wk for 4 wk. We evaluated total and differential cell counts in bronchoalveolar lavage (BAL), interleukin (IL)-1β, IL-6, CXCL1/KC, IL-10, tumor necrosis factor α, and transforming growth factor β levels in BAL, collagen content in lung parenchyma, 5-HT levels in BAL fluid and in serum, the expression of 5-HT2B receptor, and Akt phosphorylation in lung tissue. AT reduced bleomycin-increased number of total cells (P < 0.001), neutrophils (P < 0.01), macrophages (P < 0.01), and lymphocytes (P < 0.05) in BAL. It also reduced the levels of IL-1β (P < 0.01), IL-6 (P < 0.05), CXCL1/KC (P < 0.001), tumor necrosis factor α (P < 0.001), and transforming growth factor β (P < 0.001). It increased expression of ant-inflammatory cytokine IL-10 (P < 0.001). It reduced bleomycin-increased 5-HT levels in BAL (P < 0.001) and in serum (P < 0.05). Reductions in collagen fiber deposition (P < 0.01), 5-HT2B receptor expression (P < 0.01), and Akt phosphorylation in lung tissue were observed. AT accelerates the resolution of lung inflammation and fibrosis in a model of bleomycin-induced lung fibrosis partly via attenuation of 5-HT/Akt signaling.

  16. Optogenetic clustering of CNK1 reveals mechanistic insights in RAF and AKT signalling controlling cell fate decisions

    PubMed Central

    Fischer, Adrian; Warscheid, Bettina; Weber, Wilfried; Radziwill, Gerald

    2016-01-01

    Scaffold proteins such as the multidomain protein CNK1 orchestrate the signalling network by integrating and controlling the underlying pathways. Using an optogenetic approach to stimulate CNK1 uncoupled from upstream effectors, we identified selective clusters of CNK1 that either stimulate RAF-MEK-ERK or AKT signalling depending on the light intensity applied. OptoCNK1 implemented in MCF7 cells induces differentiation at low light intensity stimulating ERK activity whereas stimulation of AKT signalling by higher light intensity promotes cell proliferation. CNK1 clustering in response to increasing EGF concentrations revealed that CNK1 binds to RAF correlating with ERK activation at low EGF dose. At higher EGF dose active AKT binds to CNK1 and phosphorylates and inhibits RAF. Knockdown of CNK1 protects CNK1 from this AKT/RAF crosstalk. In C2 skeletal muscle cells CNK1 expression is induced with the onset of differentiation. Hence, AKT-bound CNK1 counteracts ERK stimulation in differentiated but not in proliferating cells. Ectopically expressed CNK1 facilitates C2 cell differentiation and knockdown of CNK1 impaired the transcriptional network underlying C2 cell differentiation. Thus, CNK1 expression, CNK1 clustering and the thereto related differential signalling processes decide on proliferation and differentiation in a cell type- and cell stage-dependent manner by orchestrating AKT and RAF signalling. PMID:27901111

  17. Pharmacological manipulation of the akt signaling pathway regulates myxoma virus replication and tropism in human cancer cells.

    PubMed

    Werden, Steven J; McFadden, Grant

    2010-04-01

    Viruses have evolved an assortment of mechanisms for regulating the Akt signaling pathway to establish a cellular environment more favorable for viral replication. Myxoma virus (MYXV) is a rabbit-specific poxvirus that encodes many immunomodulatory factors, including an ankyrin repeat-containing host range protein termed M-T5 that functions to regulate tropism of MYXV for rabbit lymphocytes and certain human cancer cells. MYXV permissiveness in these human cancer cells is dependent upon the direct interaction between M-T5 and Akt, which has been shown to induce the kinase activity of Akt. In this study, an array of compounds that selectively manipulate Akt signaling was screened and we show that only a subset of Akt inhibitors significantly decreased the ability of MYXV to replicate in previously permissive human cancer cells. Furthermore, reduced viral replication efficiency was correlated with lower levels of phosphorylated Akt. In contrast, the PP2A-specific phosphatase inhibitor okadaic acid promoted increased Akt kinase activation and rescued MYXV replication in human cancer cells that did not previously support viral replication. Finally, phosphorylation of Akt at residue Thr308 was shown to dictate the physical interaction between Akt and M-T5, which then leads to phosphorylation of Ser473 and permits productive MYXV replication in these human cancer cells. The results of this study further characterize the mechanism by which M-T5 exploits the Akt signaling cascade and affirms this interaction as a major tropism determinant that regulates the replication efficiency of MYXV in human cancer cells.

  18. c-Src activation promotes nasopharyngeal carcinoma metastasis by inducing the epithelial-mesenchymal transition via PI3K/Akt signaling pathway: a new and promising target for NPC

    PubMed Central

    Lu, Jinping; Xia, Weixiong; Yu, Yahui; Peng, Yongjian; Wang, Li; Wang, Gang; Ye, Yanfang; Yang, Jing; Liang, Hu; Kang, Tiebang; Lv, Xing

    2016-01-01

    Aberrant activation of cellular Src (c-Src), a non-receptor tyrosine kinase, could promote cancer progression through activating its downstream signaling pathways. However, the roles of c-Src and phosphorylated-Src (p-Src) in nasopharyngeal carcinoma (NPC) progression are rarely investigated. Herein, we have identified high c-Src concentrations in the serum of NPC patients with distant metastasis using high-throughput protein microarrays. Levels of c-Src in serum and p-Src in human primary NPC samples were unfavorable independent prognostic factors for cancer-specific survival, disease-free survival, and distant metastasis-free survival. Depletion or inactivation of c-Src in NPC cells using sgRNA with CRISPR/Cas9 system or PP2 decreased cell viability, colony formation, migration and invasion in vitro and metastasis in vivo. In contrast, these malignancies could be up-regulated by overexpressed c-Src in a NPC cell line with low-metastasis potential. Furthermore, p-Src was involved in promoting NPC cell metastasis by inducing the epithelial-mesenchymal transition (EMT) process via activating the PI3K/Akt pathway and cytoskeleton remodeling. The p-Src-induced EMT process could be retarded by PP2, which mediated by down-regulating the PI3K/Akt pathway. In conclusion, elevated levels of c-Src in serum and p-Src in primary NPC tissue correlated with poor outcomes of NPC patients. And aberrant activation of c-Src facilitated NPC cells with malignant potential, especially metastasis ability, which mediated by the PI3K/Akt pathway activation and sequentially induced the EMT process. These findings unveiled a promising approach for targeted therapy of advanced NPC. PMID:27078847

  19. Insulin sensitization via partial agonism of PPARγ and glucose uptake through translocation and activation of GLUT4 in PI3K/p-Akt signaling pathway by embelin in type 2 diabetic rats.

    PubMed

    Gandhi, Gopalsamy Rajiv; Stalin, Antony; Balakrishna, Kedike; Ignacimuthu, Savarimuthu; Paulraj, Michael Gabriel; Vishal, Rajagopal

    2013-01-01

    The present study was aimed at isolating an antidiabetic molecule from a herbal source and assessing its mechanism of action. Embelin, isolated from Embelia ribes Burm. (Myrsinaceae) fruit, was evaluated for its potential to regulate insulin resistance, alter β-cell dysfunction and modulate key markers involved in insulin sensitivity and glucose transport using high-fat diet (HFD) fed-streptozotocin (STZ) (40mg/kg)-induced type 2 diabetic rats. Molecular-dockings were performed to investigate the binding modes of embelin into PPARγ, PI3K, p-Akt and GLUT4 active sites. Embelin (50mg/kg b wt.) reduced body weight gain, blood glucose and plasma insulin in treated diabetic rats. It further modulated the altered lipid profiles and antioxidant enzymes with cytoprotective action on β-cell. Embelin significantly increased the PPARγ expression in epididymal adipose tissue compared to diabetic control group; it also inhibited adipogenic activity; it mildly activated PPARγ levels in the liver and skeletal muscle. It also regulated insulin mediated glucose uptake in epididymal adipose tissue through translocation and activation of GLUT4 in PI3K/p-Akt signaling cascade. Embelin bound to PPARγ; it disclosed stable binding affinities to the active sites of PI3K, p-Akt and GLUT4. These findings show that embelin could improve adipose tissue insulin sensitivity without increasing weight gain, enhance glycemic control, protect β-cell from damage and maintain glucose homeostasis in adipose tissue. Embelin can be used in the prevention and treatment of type 2 diabetes mellitus caused due to obesity. Copyright © 2012 Elsevier B.V. All rights reserved.

  20. MicroRNA-130a alleviates human coronary artery endothelial cell injury and inflammatory responses by targeting PTEN via activating PI3K/Akt/eNOS signaling pathway

    PubMed Central

    Song, Chun-Li; Liu, Bin; Shi, Yong-Feng; Liu, Ning; Yan, You-You; Zhang, Ji-Chang; Xue, Xin; Wang, Jin-Peng; Zhao, Zhuo; Liu, Jian-Gen; Li, Yang-Xue; Zhang, Xiao-Hao; Wu, Jun-Duo

    2016-01-01

    Our study aims to investigate the roles of microRNA-130a (miR-130a) in human coronary artery endothelial cells (HCAECs) injury and inflammatory responses by targeting PTEN through the PI3K/Akt/eNOS signaling pathway. HCAECs were treated with 1.0 mmol/L homocysteine (HCY) and assigned into eight groups: the blank group, the negative control (NC) group, the miR-130a mimics group, the miR-130a inhibitors group, the si-PTEN group, the Wortmannin group, the miR-130a inhibitors + si-PTEN group and the miR-130a mimics + Wortmannin group. Luciferase reporter gene assay was used to validate the relationship between miR-130a and PTEN. The expressions of miR-130a, PTEN and PI3K/Akt/eNOS signaling pathway-related proteins were detected by qRT-PCR assay and Western blotting. MTT assay and Hoechst 33258 staining were adopted to testify cell growth and apoptosis. The NO kit assay was used to detect the NO release. ELISA was conducted to measure serum cytokine levels. Luciferase reporter gene assay confirmed the target relationship between miR-130a and PTEN. Compared with the blank and NC groups, the miR-130a mimics and si-PTEN groups showed significant increases in the expressions of PI3K/Akt/eNOS signaling pathway-related proteins, cell viability and the NO release, while serum cytokine levels and cell apoptosis were decreased; by contrast, an opposite trend was observed in miR-130a inhibitors and Wortmannin groups. However, no significant difference was found in the miR-130a inhibitors + si-PTEN and miR-130a mimics + Wortmannin groups when compared with the blank group. These results indicate that miR-130a could alleviate HCAECs injury and inflammatory responses by down-regulating PTEN and activating PI3K/Akt/eNOS signaling pathway. PMID:27713121

  1. GIV/girdin links vascular endothelial growth factor signaling to Akt survival signaling in podocytes independent of nephrin.

    PubMed

    Wang, Honghui; Misaki, Taro; Taupin, Vanessa; Eguchi, Akiko; Ghosh, Pradipta; Farquhar, Marilyn G

    2015-02-01

    Podocytes are critically involved in the maintenance of the glomerular filtration barrier and are key targets of injury in many glomerular diseases. Chronic injury leads to progressive loss of podocytes, glomerulosclerosis, and renal failure. Thus, it is essential to maintain podocyte survival and avoid apoptosis after acute glomerular injury. In normal glomeruli, podocyte survival is mediated via nephrin-dependent Akt signaling. In several glomerular diseases, nephrin expression decreases and podocyte survival correlates with increased vascular endothelial growth factor (VEGF) signaling. How VEGF signaling contributes to podocyte survival and prevents apoptosis remains unknown. We show here that Gα-interacting, vesicle-associated protein (GIV)/girdin mediates VEGF receptor 2 (VEGFR2) signaling and compensates for nephrin loss. In puromycin aminonucleoside nephrosis (PAN), GIV expression increased, GIV was phosphorylated by VEGFR2, and p-GIV bound and activated Gαi3 and enhanced downstream Akt2, mammalian target of rapamycin complex 1 (mTORC1), and mammalian target of rapamycin complex-2 (mTORC2) signaling. In GIV-depleted podocytes, VEGF-induced Akt activation was abolished, apoptosis was triggered, and cell migration was impaired. These effects were reversed by introducing GIV but not a GIV mutant that cannot activate Gαi3. Our data indicate that after PAN injury, VEGF promotes podocyte survival by triggering assembly of an activated VEGFR2/GIV/Gαi3 signaling complex and enhancing downstream PI3K/Akt survival signaling. Because of its important role in promoting podocyte survival, GIV may represent a novel target for therapeutic intervention in the nephrotic syndrome and other proteinuric diseases.

  2. Akt and mTOR in B Cell Activation and Differentiation.

    PubMed

    Limon, Jose J; Fruman, David A

    2012-01-01

    Activation of phosphoinositide 3-kinase (PI3K) is required for B cell proliferation and survival. PI3K signaling also controls key aspects of B cell differentiation. Upon engagement of the B cell receptor (BCR), PI3K activation promotes Ca(2+) mobilization and activation of NFκB-dependent transcription, events which are essential for B cell proliferation. PI3K also initiates a distinct signaling pathway involving the Akt and mTOR serine/threonine kinases. It has been generally assumed that activation of Akt and mTOR downstream of PI3K is essential for B cell function. However, Akt and mTOR have complex roles in B cell fate decisions and suppression of this pathway can enhance certain B cell responses while repressing others. In this review we will discuss genetic and pharmacological studies of Akt and mTOR function in normal B cells, and in malignancies of B cell origin.

  3. Orexins protect neuronal cell cultures against hypoxic stress: an involvement of Akt signaling.

    PubMed

    Sokołowska, Paulina; Urbańska, Anna; Biegańska, Kaja; Wagner, Waldemar; Ciszewski, Wojciech; Namiecińska, Magdalena; Zawilska, Jolanta B

    2014-01-01

    Orexins A and B are peptides produced mainly by hypothalamic neurons that project to numerous brain structures. We have previously demonstrated that rat cortical neurons express both types of orexin receptors, and their activation by orexins initiates different intracellular signals. The present study aimed to determine the effect of orexins on the Akt kinase activation in the rat neuronal cultures and the significance of that response in neurons subjected to hypoxic stress. We report the first evidence that orexins A and B stimulated Akt in cortical neurons in a concentration- and time-dependent manner. Orexin B more potently than orexin A increased Akt phosphorylation, but the maximal effect of both peptides on the kinase activation was very similar. Next, cultured cortical neurons were challenged with cobalt chloride, an inducer of reactive oxygen species and hypoxia-mediated signaling pathways. Under conditions of chemical hypoxia, orexins potently increased neuronal viability and protected cortical neurons against oxidative stress. Our results also indicate that Akt kinase plays an important role in the pro-survival effects of orexins in neurons, which implies a possible mechanism of the orexin-induced neuroprotection.

  4. Clusterin facilitates metastasis by EIF3I/Akt/MMP13 signaling in hepatocellular carcinoma

    PubMed Central

    Wang, Ning; Luo, Qin; Zhang, Yurong; Gao, Dongmei; Jiang, Kai; Gu, Dishui; Shen, Qiujing; Huo, Xisong; Hu, Fangyuan; Ge, Tianxiang; Zhao, Fangyu; Chu, Wei; Shu, Huiqun; Yao, Ming; Cong, Wenming; Qin, Wenxin

    2015-01-01

    Clusterin (CLU) is a stress-induced chaperone that confers proliferative and survival advantages to cancer cells. However, effects and molecular mechanisms of CLU in hepatocellular carcinoma (HCC) metastasis are still unknown. In this study, HCC tissue array (n = 198) was utilized to investigate correlation between CLU expression and clinicopathological features. Overexpression of CLU in HCC tissues was correlated with shorter overall survival and higher tumor recurrence. In vitro and in vivo assays demonstrated that silencing CLU attenuated the invasion and metastasis of HCC cells, whereas ectopic overexpression of CLU resulted in the forced metastasis of HCC cells. We also revealed that CLU activated Akt signaling through complexing with eukaryotic translation initiation factor 3 subunit I (EIF3I), which in turn promoted matrix metalloproteinase 13 (MMP13) expression and HCC metastasis. Positive correlations between CLU and MMP13, p-Akt, or EIF3I were found in HCC tissues. We further observed that CLU knockdown using the CLU inhibitor OGX-011 significantly suppressed HCC metastasis in two metastatic models through inhibiting EIF3I/Akt/MMP13 signaling. These findings indicate that CLU is an independent predictive factor for prognosis of HCC and it facilitates metastasis through EIF3I/Akt/MMP13 signaling. CLU suppression using OGX-011 may represent a promising therapeutic option for suppressing HCC metastasis. PMID:25609201

  5. Cyproheptadine-induced myeloma cell apoptosis is associated with inhibition of the PI3K/AKT signaling.

    PubMed

    Li, Jie; Cao, Biyin; Zhou, Shunye; Zhu, Jingyu; Zhang, Zubin; Hou, Tingjun; Mao, Xinliang

    2013-12-01

    Recent studies revealed that the anti-allergic cyproheptadine displays anti-blood cancer activity. However, its mechanism is still elusive. In this study, cyproheptadine was found to decrease the expression of anti-apoptotic proteins, including Bcl-2, Mcl-1, and XIAP. More importantly, cyproheptadine-induced apoptosis was accompanied by suppressing AKT activation in myeloma cells. In the subsequent study, cyproheptadine was found to inhibit insulin-like growth factor 1-triggered AKT activation in a time- and concentration-dependent manner. Specifically, cyproheptadine blocked AKT translocation from nuclei for phosphorylation. This inhibition led to suppressed activation of p70S6K and 4EBP1, two key downstream signaling proteins in the PI3K/AKT pathway. However, cyproheptadine did not display inhibition on activation of IGF-1R or STAT3, possible upstream signals of AKT activation. These results further demonstrated that cyproheptadine suppresses the PI3K/AKT signaling pathway, which is probably critical for cyproheptadine-induced MM cell apoptosis.

  6. Role of Akt signaling in resistance to DNA-targeted therapy

    PubMed Central

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

    2016-01-01

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

  7. mTOR Promotes Survival and Astrocytic Characteristics Induced by Pten/Akt Signaling in Glioblastoma1

    PubMed Central

    Hu, Xiaoyi; Pandolfi, Pier Paolo; Li, Yi; Koutcher, Jason A; Rosenblum, Marc; Holland, Eric C

    2005-01-01

    Abstract Combined activation of Ras and Akt leads to the formation of astrocytic glioblastoma multiforme (GBM) in mice. In human GBMs, AKT is not mutated but is activated in approximately 70% of these tumors, in association with loss of PTEN and/or activation of receptor tyrosine kinases. Mechanistic justification for the therapeutic blockade of targets downstream of AKT, such as mTOR, in these cancers requires demonstration that the oncogenic effect of PTEN loss is through elevated AKT activity. We demonstrate here that loss of Pten is similar to Akt activation in the context of glioma formation in mice. We further delineate the role of mTOR activity downstream of Akt in the maintenance of Akt+KRas-induced GBMs. Blockade of mTOR results in regional apoptosis in these tumors and conversion in the character of surviving tumor cells from astrocytoma to oligodendroglioma. These data suggest that mTOR activity is required for the survival of some cells within these GBMs, and mTOR appears required for the maintenance of astrocytic character in the surviving cells. Furthermore, our study provides the first example of conversion between two distinct tumor types usually thought of as belonging to specific lineages, and provides evidence for signal transduction-mediated transdifferentiation between glioma subtypes. PMID:15967113

  8. 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.

  9. Connective tissue growth factor induces cardiac hypertrophy through Akt signaling

    SciTech Connect

    Hayata, Nozomi; Fujio, Yasushi; Yamamoto, Yasuhiro; Iwakura, Tomohiko; Obana, Masanori; Takai, Mika; Mohri, Tomomi; Nonen, Shinpei; Maeda, Makiko; Azuma, Junichi

    2008-05-30

    In the process of cardiac remodeling, connective tissue growth factor (CTGF/CCN2) is secreted from cardiac myocytes. Though CTGF is well known to promote fibroblast proliferation, its pathophysiological effects in cardiac myocytes remain to be elucidated. In this study, we examined the biological effects of CTGF in rat neonatal cardiomyocytes. Cardiac myocytes stimulated with full length CTGF and its C-terminal region peptide showed the increase in cell surface area. Similar to hypertrophic ligands for G-protein coupled receptors, such as endothelin-1, CTGF activated amino acid uptake; however, CTGF-induced hypertrophy is not associated with the increased expression of skeletal actin or BNP, analyzed by Northern-blotting. CTGF treatment activated ERK1/2, p38 MAPK, JNK and Akt. The inhibition of Akt by transducing dominant-negative Akt abrogated CTGF-mediated increase in cell size, while the inhibition of MAP kinases did not affect the cardiac hypertrophy. These findings indicate that CTGF is a novel hypertrophic factor in cardiac myocytes.

  10. Crosstalking between Androgen and PI3K/AKT Signaling Pathways in Prostate Cancer Cells*

    PubMed Central

    Lee, Suk Hyung; Johnson, Daniel; Luong, Richard; Sun, Zijie

    2015-01-01

    Both androgen action and PI3K medicated signaling pathways have been implicated in prostate tumorigenesis. Our androgen receptor (AR) conditional transgenic mice developed murine prostatic intraepithelial neoplasia (mPIN) and prostatic adenocarcinoma lesions recapitulating human prostate cancer development and progression. Role of transgenic AR contributing to malignancy was demonstrated by high degree of transgenic AR expression in atypical and tumor cells in mPIN as well as prostatic adenocarcinoma lesions of the transgenic mice, but not in adjacent normal tissue. Interestingly, reduced PI3K/Akt activation also appeared in these mouse atypical and tumor cells, suggesting an interaction between androgen and PI3K/AKT pathways. In this study, we further investigated this interaction. We showed that the androgen depletion or knockdown of AR expression results in elevated levels of active phosphorylated AKT in prostate cancer cells. Castration of conditional Pten knock-out mice showed increased Akt, phosphorylated Akt, and pS6 expression in the mouse prostate. Using a series of newly generated Ar reporter and Pten knock-out compound mice, we showed that Pten loss directly represses endogenous Ar expression in prostatic epithelial cells. Moreover, Pten loss and PI3K/Akt activation reduced Ar-mediated transcription in purified Pten-null cells. This study provides novel evidence demonstrating interplay between androgen and PI3K pathways, as well as introduces unique and relevant mouse models for further studies of PI3K and AR pathways in the context of prostate tumorigenesis. PMID:25527506

  11. Palmitic acid stimulates energy metabolism and inhibits insulin/PI3K/AKT signaling in differentiated human neuroblastoma cells: The role of mTOR activation and mitochondrial ROS production.

    PubMed

    Calvo-Ochoa, Erika; Sánchez-Alegría, Karina; Gómez-Inclán, Cecilia; Ferrera, Patricia; Arias, Clorinda

    2017-09-15

    The high consumption of saturated lipids has been largely associated with the increasing prevalence of metabolic diseases. In particular, saturated fatty acids such as palmitic acid (PA) have been implicated in the development of insulin resistance in peripheral tissues. However, how neurons develop insulin resistance in response to lipid overload is not fully understood. Here, we used cultured rat cortical neurons and differentiated human neuroblastoma cells to demonstrate that PA blocks insulin-induced metabolic activation, inhibits the activation of the insulin/PI3K/Akt pathway and activates mTOR kinase downstream of Akt. Despite the fact that fatty acids are not normally used as a significant source of fuel by neural cells, we also found that short-term neuronal exposure to PA reduces the NAD(+)/NADH ratio, indicating that PA modifies the neuronal energy balance. Finally, inhibiting mitochondrial ROS production with mitoTEMPO prevented the deleterious effect of PA on insulin signaling. This work provides novel evidence of the mechanisms behind saturated fatty acid-induced insulin resistance and its metabolic consequences on neuronal cells. Copyright © 2017 Elsevier Ltd. All rights reserved.

  12. Immunohistochemical Evaluation of AKT Protein Activation in Canine Mast Cell Tumours

    PubMed Central

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

    2011-01-01

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

  13. 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.

  14. Tissue Kallikrein Reverses Insulin Resistance and Attenuates Nephropathy in Diabetic Rats by Activation of PI3 kinase/Akt and AMPK Signaling Pathways

    PubMed Central

    Yuan, Gang; Deng, Juanjuan; Wang, Tao; Zhao, Chunxia; Xu, Xizheng; Wang, Peihua; Voltz, James W.; Edin, Matthew L.; Xiao, Xiao; Chao, Lee; Chao, Julie; Zhang, Xin A.; Zeldin, Darryl C.; Wang, Dao Wen

    2007-01-01

    We previously reported that intravenous delivery of the human tissue kallikrein (HK) gene reduced blood pressure and plasma insulin levels in fructose-induced hypertensive rats with insulin resistance. In the current study, we evaluated the potential of a recombinant adeno-associated viral vector expressing the HK cDNA (rAAV·HK) as a sole, long term therapy to correct insulin resistance and prevent renal damage in streptozotocin-induced type-2 diabetic rats. Administration of streptozotocin in conjunction with a high fat diet induced systemic hypertension, diabetes and renal damage in rats. Delivery of rAAV·HK resulted in a long-term reduction in blood pressure, and fasting plasma insulin was significantly lower in the rAAV·HK group than in the control group. The expression of PI3-kinase p110 catalytic subunit, and the levels of phosphorylation at residue Thr-308 of Akt, insulin receptor B and AMP-activated protein kinases (AMPK) were significantly decreased in organs from diabetic animals. These changes were significantly attenuated following rAAV-mediated HK gene therapy. Moreover, rAAV·HK significantly decreased urinary microalbumin excretion, improved creatinine clearance and increased urinary osmolarity. HK gene therapy also attenuated diabetic renal damage as assessed by histology. Together, these findings demonstrate that rAAV·HK delivery can efficiently attenuate hypertension, insulin resistance and diabetic nephropathy in streptozotocin-induced diabetic rats. PMID:17272402

  15. FANCI is a negative regulator of Akt activation.

    PubMed

    Zhang, Xiaoshan; Lu, Xiaoyan; Akhter, Shamima; Georgescu, Maria-Magdalena; Legerski, Randy J

    2016-01-01

    Akt is a critical mediator of the oncogenic PI3K pathway, and its activation is regulated by kinases and phosphatases acting in opposition. We report here the existence of a novel protein complex that is composed minimally of Akt, PHLPP1, PHLPP2, FANCI, FANCD2, USP1 and UAF1. Our studies show that depletion of FANCI, but not FANCD2 or USP1, results in increased phosphorylation and activation of Akt. This activation is due to a reduction in the interaction between PHLPP1 and Akt in the absence of FANCI. In response to DNA damage or growth factor treatment, the interactions between Akt, PHLPP1 and FANCI are reduced consistent with the known phosphorylation of Akt in response to these stimuli. Furthermore, depletion of FANCI results in reduced apoptosis after DNA damage in accord with its role as a negative regular of Akt. Our findings describe an unexpected function for FANCI in the regulation of Akt and define a previously unrecognized intersection between the PI3K-Akt and FA pathways.

  16. Activation of PI3Kγ/Akt pathway increases cardiomyocyte HMGB1 expression in diabetic environment

    PubMed Central

    Song, Jia; Liu, Qian; Tang, Han; Tao, Aibin; Wang, Hao; Kao, Raymond; Rui, Tao

    2016-01-01

    Background The high mobility group box 1 (HMGB1) protein mediates the cardiomyocyte–cardiac fibroblast interaction that contributes to induction of myocardial fibrosis in diabetes mellitus (DM). In the present study, we aim to investigate the intracellular signaling pathway that leads to cardiomyocyte HMGB1 expression under a diabetic environment. Results HMGB1 expression is increased in high concentration of glucose (HG)-conditioned cardiomyocytes. Challenging cardiomyocytes with HG also increased PI3Kγ and Akt phosphorylation. Inhibition of PI3Kγ (CRISPR/Cas9 knockout plasmid or AS605240) prevented HG-induced Akt phosphorylation and HMGB1 expression by the cardiomyocytes. In addition, inhibition of Akt (Akt1/2/3 siRNA or A6730) attenuated HG-induced HMGB1 production. Finally, challenging cardiomyocytes with HG resulted in increased reactive oxygen species (ROS) production. Treatment of cardiomyocytes with an antioxidant (Mitotempo) abolished HG-induced PI3Kγ and Akt activation, as well as HMGB1 production. Materials and Methods Isolated rat cardiomyocytes were cultured with a high concentration of glucose. Cardiomyocyte phosphatidylinositol 3-kinase gamma (PI3Kγ) and Akt activation were determined by Western blot. Cardiomyocyte HMGB1 production was evaluated with Western blot and enzyme-linked immunosorbent assay (ELISA), while cardiomyocyte oxidative stress was determined with a DCFDA fluorescence probe. Conclusions Our results suggest that the cardiomyocytes incur an oxidative stress under diabetic condition, which subsequently activates the PI3Kγ/Akt cell-signaling pathway and further increases HMGB1 expression. PMID:27821807

  17. Centipede grass exerts anti-adipogenic activity through inhibition of C/EBPβ, C/EBPα, and PPARγ expression and the AKT signaling pathway in 3T3-L1 adipocytes

    PubMed Central

    2012-01-01

    Background Centipede grass (CG) originates from China and South America and is reported to contain several C-glycosyl flavones and phenolic constituents, including maysin and luteolin derivatives. This study aimed to investigate, for the first time, the antiobesity activity of CG and its potential molecular mechanism in 3T3-L1 cells. Methods To study the effect of CG on adipogenesis, differentiating 3T3-L1 cells were treated every day with CG at various concentrations (0–100 μg/ml) for six days. Oil-red O staining and triglyceride content assay were performed to determine the lipid accumulation in 3T3-L1 cells. The expression of mRNAs or proteins associated with adipogenesis was measured using RT-PCR and Western blotting analysis. We examined the effect of CG on level of phosphorylated Akt in 3T3-L1 cells treated with CG at various concentration s during adipocyte differentiation. Results Differentiation was investigated with an Oil-red O staining assay using CG-treated 3T3-L1 adipocytes. We found that CG suppressed lipid droplet formation and adipocyte differentiation in 3T3-L1 cells in a dose-dependent manner. Treatment of the 3T3-L1 adipocytes with CG resulted in an attenuation of the expression of adipogenesis-related factors and lipid metabolic genes. The expression of C/EBPα and PPARγ, the central transcriptional regulators of adipogenesis, was decreased by the treatment with CG. The expression of genes involved in lipid metabolism, aP2 were significantly inhibited following the CG treatment. Moreover, the CG treatment down-regulated the phosphorylation levels of Akt and GSK3β. Conclusions Taken collectively, these data indicated that CG exerts antiadipogenic activity by inhibiting the expression of C/EBPβ, C/EBPα, and PPARγ and the Akt signaling pathway in 3T3-L1 adipocytes. PMID:23181522

  18. 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. © 2014. Published by The Company of Biologists Ltd.

  19. Exercise regulates Akt and glycogen synthase kinase-3 activities in human skeletal muscle.

    PubMed

    Sakamoto, Kei; Arnolds, David E W; Ekberg, Ingvar; Thorell, Anders; Goodyear, Laurie J

    2004-06-25

    Activation of Akt and deactivation of GSK3 are critical signals regulating a number of cellular processes in multiple systems. Whether physical exercise alters Akt and GSK3 activity in human skeletal muscle is controversial. beta-Catenin, a GSK3 substrate and important Wnt signaling protein that alters gene transcription, has not been investigated in human skeletal muscle. In the present study, eight healthy human subjects performed 30min of cycling exercise at 75% of maximum workload (submaximal) followed by 6 bouts of 60s at 125% maximum workload (maximal). Biopsies of vastus lateralis muscle were taken at rest (basal), and within 15s following cessation of the submaximal and maximal exercise bouts. Exercise at both submaximal and maximal intensities significantly increased Akt activity (40% and 110%, respectively). Increases in Akt activity were accompanied by increases in Akt Thr(308) and Ser(473) phosphorylation, decreased GSK3alpha activity ( approximately 30% at both intensities), and increased phosphorylation of GSK3alpha Ser(21). Exercise at both intensities also decreased beta-catenin Ser(33/37)Thr(41) phosphorylation (50-60% at both intensities). These results demonstrate that Akt, GSK3, and beta-catenin signaling are regulated by exercise in human skeletal muscle, and as such identify them as possible molecular mediators of exercise's effect on metabolic and transcriptional processes in skeletal muscle.

  20. Active form of AKT controls cell proliferation and response to apoptosis in hepatocellular carcinoma

    PubMed Central

    KUNTER, IMGE; ERDAL, ESRA; NART, DENIZ; YILMAZ, FUNDA; KARADEMIR, SEDAT; SAGOL, OZGUL; ATABEY, NESE

    2014-01-01

    Hepatocellular carcinoma (HCC) is the third most common cause of cancer-related mortality worldwide. Deregulation of the AKT signaling pathway has been found in HCC. However, the effect of AKT activation on the proliferation and apoptosis in HCC is not clear. Herein, expression of phosphorylated form of AKT (Ser 473) was investigated in HCC tumor (n=73), cirrhosis (n=17), normal liver (n=22) samples and in HCC cell lines (n=8). The results showed that expression of p-AKT was higher in tumor (53%) than in cirrhotic tissues (12%) while it was absent in normal liver (p<0.0001). p-AKT expression was also associated with number of tumor nodules and differentiation status (p<0.05). LY294002 induced cell cycle arrest at G0/G1 in SNU-449 and Mahlavu cells by decreasing expression of CDK2, CDK4, CycD1, CycD3, CycE, CycA and increasing expression of p21 and p27 as well; it also caused a decrease in the E2F1 transcriptional activity through declining phosphorylated Rb. LY294002 did not affect the basal level of apoptosis; however, it amplified cisplatin-induced apoptosis in SNU-449 cells. When the p-AKT level was decreased specifically after transfection with the DN-AKT plasmid, SNU-449 cells became more sensitive to cisplatin-induced apoptosis. HuH-7 cells with no basal p-AKT, were markedly affected by the treatment of doxorubicin. Thus, Akt signaling controls growth and chemical-induced apoptosis in HCC and p-AKT may be a potential target for therapeutic interventions in HCC patients. PMID:24337632

  1. Glycitin regulates osteoblasts through TGF-β or AKT signaling pathways in bone marrow stem cells

    PubMed Central

    Zhang, Liyan; Chen, Jiying; Chai, Wei; Ni, Min; Sun, Xin; Tian, Dan

    2016-01-01

    The aim of the present study was to examine the effect of glycitin on the regulation of osteoblasts from bone marrow stem cells (BMSCs) through transforming growth factor (TGF)-β or protein kinase B (AKT) signaling pathways. BMSCs were extracted from New Zealand white rabbits and used to analyze the effect of glycitin on BMSCs. BMSCs were cleared using xylene and observed via light microscopy. BMSCs were subsequently induced with glycitin (0.01, 0.5, 1, 5 and 10 µM) for 7 days, and stained with Oil Red O. The mechanism of action of glycitin on BMSCs was investigated, in which contact with collagen type I (Col I), alkaline phosphatase (ALP), TGF-β and AKT was studied. Firstly, BMSCs appeared homogeneously mazarine blue, and which showed that BMSCs were successful extracted. Administration of glycitin increased cell proliferation and promoted osteoblast formation from BMSCs. Furthermore, glycitin activated the gene expression of Col I and ALP in BMSCs. Notably, glycitin suppressed protein expression of TGF-β and AKT in BMSCs. These results indicated that glycitin may regulate osteoblasts through TGF-β or AKT signaling pathways in BMSCs. PMID:27882117

  2. The Histamine H3 Receptor Differentially Modulates Mitogen-activated Protein Kinase (MAPK) and Akt Signaling in Striatonigral and Striatopallidal Neurons.

    PubMed

    Rapanelli, Maximiliano; Frick, Luciana R; Horn, Kyla D; Schwarcz, Rivka C; Pogorelov, Vladimir; Nairn, Angus C; Pittenger, Christopher

    2016-09-30

    The basal ganglia have a central role in motor patterning, habits, motivated behaviors, and cognition as well as in numerous neuropsychiatric disorders. Receptors for histamine, especially the H3 receptor (H3R), are highly expressed in the striatum, the primary input nucleus of the basal ganglia, but their effects on this circuitry have been little explored. H3R interacts with dopamine (DA) receptors ex vivo; the nature and functional importance of these interactions in vivo remain obscure. We found H3R activation with the agonist R-(-)-α-methylhistamine to produce a unique time- and cell type-dependent profile of molecular signaling events in the striatum. H3 agonist treatment did not detectably alter extracellular DA levels or signaling through the cAMP/DARPP-32 signaling pathway in either D1- or D2-expressing striatal medium spiny neurons (MSNs). In D1-MSNs, H3 agonist treatment transiently activated MAPK signaling and phosphorylation of rpS6 and led to phosphorylation of GSK3β-Ser(9), a novel effect. Consequences of H3 activation in D2-MSNs were completely different. MAPK signaling was unchanged, and GSK3β-Ser(9) phosphorylation was reduced. At the behavioral level, two H3 agonists had no significant effect on locomotion or stereotypy, but they dramatically attenuated the locomotor activation produced by the D1 agonist SKF82958. H3 agonist co-administration blocked the activation of MAPK signaling and the phosphorylation of rpS6 produced by D1 activation in D1-MSNs, paralleling behavioral effects. In contrast, GSK3β-Ser(9) phosphorylation was seen only after H3 agonist treatment, with no interactive effects. H3R signaling has been neglected in models of basal ganglia function and has implications for a range of pathophysiologies.

  3. Myogenic Akt signaling attenuates muscular degeneration, promotes myofiber regeneration and improves muscle function in dystrophin-deficient mdx mice

    PubMed Central

    Kim, Michelle H.; Kay, Danielle I.; Rudra, Renuka T.; Chen, Bo Ming; Hsu, Nigel; Izumiya, Yasuhiro; Martinez, Leonel; Spencer, Melissa J.; Walsh, Kenneth; Grinnell, Alan D.; Crosbie, Rachelle H.

    2011-01-01

    Duchenne muscular dystrophy, the most common form of childhood muscular dystrophy, is caused by X-linked inherited mutations in the dystrophin gene. Dystrophin deficiencies result in the loss of the dystrophin–glycoprotein complex at the plasma membrane, which leads to structural instability and muscle degeneration. Previously, we induced muscle-specific overexpression of Akt, a regulator of cellular metabolism and survival, in mdx mice at pre-necrotic (<3.5 weeks) ages and demonstrated upregulation of the utrophin–glycoprotein complex and protection against contractile-induced stress. Here, we found that delaying exogenous Akt treatment of mdx mice after the onset of peak pathology (>6 weeks) similarly increased the abundance of compensatory adhesion complexes at the extrasynaptic sarcolemma. Akt introduction after onset of pathology reverses the mdx histopathological measures, including decreases in blood serum albumin infiltration. Akt also improves muscle function in mdx mice as demonstrated through in vivo grip strength tests and in vitro contraction measurements of the extensor digitorum longus muscle. To further explore the significance of Akt in myofiber regeneration, we injured wild-type muscle with cardiotoxin and found that Akt induced a faster regenerative response relative to controls at equivalent time points. We demonstrate that Akt signaling pathways counteract mdx pathogenesis by enhancing endogenous compensatory mechanisms. These findings provide a rationale for investigating the therapeutic activation of the Akt pathway to counteract muscle wasting. PMID:21245083

  4. Tetramethylpyrazine Analogue CXC195 Protects Against Dopaminergic Neuronal Apoptosis via Activation of PI3K/Akt/GSK3β Signaling Pathway in 6-OHDA-Induced Parkinson's Disease Mice.

    PubMed

    Chen, Lin; Cheng, Li; Wei, Xinbing; Yuan, Zheng; Wu, Yanmei; Wang, Shuaishuai; Ren, Zhiping; Liu, Xinyong; Liu, Huiqing

    2016-12-22

    Parkinson's disease (PD) is a progressive neurodegenerative disorder and characterized by motor system disorders resulting in loss of dopaminergic (DA) neurons. CXC195, a novel tetramethylpyrazine derivative, has been shown strongest neuroprotective effects due to its anti-apoptotic activity. However, whether CXC195 protects against DA neuronal damage in PD and the mechanisms underlying its beneficial effects are unknown. The purpose of our study was to investigate the potential neuroprotective role of CXC195 and to elucidate its mechanism of action against 6-hydroxydopamine (6-OHDA)-induced mouse model of PD. CXC195 administration improved DA neurodegeneration in PD mice induced by 6-OHDA. Our further findings confirmed treatment of CXC195 at the dose of 10 mg/kg significantly inhibited the apoptosis by decreasing the level of cleaved caspase-3 and Bax, and increasing the level of Bcl-2 in 6-OHDA-lesioned mice. Meanwhile, 6-OHDA also decreased the amount of phosphorylated Akt while increased GSK-3β activity (the amount of phosphorylated GSK-3β at Ser9 was decreased) which was prevented by CXC195. Wortmannin, a specific PI3K inhibitor, dramatically abolished the changes induced by CXC195. Our study firstly demonstrated that CXC195 protected against DA neurodegeneration in 6-OHDA-induced PD model by its anti-apoptotic properties and PI3K/Akt/GSK3β signaling pathway was involved in it.

  5. Computational Modeling of PI3K/AKT and MAPK Signaling Pathways in Melanoma Cancer.

    PubMed

    Pappalardo, Francesco; Russo, Giulia; Candido, Saverio; Pennisi, Marzio; Cavalieri, Salvatore; Motta, Santo; McCubrey, James A; Nicoletti, Ferdinando; Libra, Massimo

    2016-01-01

    Malignant melanoma is an aggressive tumor of the skin and seems to be resistant to current therapeutic approaches. Melanocytic transformation is thought to occur by sequential accumulation of genetic and molecular alterations able to activate the Ras/Raf/MEK/ERK (MAPK) and/or the PI3K/AKT (AKT) signalling pathways. Specifically, mutations of B-RAF activate MAPK pathway resulting in cell cycle progression and apoptosis prevention. According to these findings, MAPK and AKT pathways may represent promising therapeutic targets for an otherwise devastating disease. Here we show a computational model able to simulate the main biochemical and metabolic interactions in the PI3K/AKT and MAPK pathways potentially involved in melanoma development. Overall, this computational approach may accelerate the drug discovery process and encourages the identification of novel pathway activators with consequent development of novel antioncogenic compounds to overcome tumor cell resistance to conventional therapeutic agents. The source code of the various versions of the model are available as S1 Archive.

  6. Computational Modeling of PI3K/AKT and MAPK Signaling Pathways in Melanoma Cancer

    PubMed Central

    Pappalardo, Francesco; Russo, Giulia; Candido, Saverio; Pennisi, Marzio; Cavalieri, Salvatore; Motta, Santo; McCubrey, James A.; Nicoletti, Ferdinando; Libra, Massimo

    2016-01-01

    Background Malignant melanoma is an aggressive tumor of the skin and seems to be resistant to current therapeutic approaches. Melanocytic transformation is thought to occur by sequential accumulation of genetic and molecular alterations able to activate the Ras/Raf/MEK/ERK (MAPK) and/or the PI3K/AKT (AKT) signalling pathways. Specifically, mutations of B-RAF activate MAPK pathway resulting in cell cycle progression and apoptosis prevention. According to these findings, MAPK and AKT pathways may represent promising therapeutic targets for an otherwise devastating disease. Result Here we show a computational model able to simulate the main biochemical and metabolic interactions in the PI3K/AKT and MAPK pathways potentially involved in melanoma development. Overall, this computational approach may accelerate the drug discovery process and encourages the identification of novel pathway activators with consequent development of novel antioncogenic compounds to overcome tumor cell resistance to conventional therapeutic agents. The source code of the various versions of the model are available as S1 Archive. PMID:27015094

  7. PI3K/AKT Signaling Pathway Is Essential for Survival of Induced Pluripotent Stem Cells

    PubMed Central

    Hossini, Amir M.; Quast, Annika S.; Plötz, Michael; Grauel, Katharina; Exner, Tarik; Küchler, Judit; Stachelscheid, Harald; Eberle, Jürgen; Rabien, Anja

    2016-01-01

    Apoptosis is a highly conserved biochemical mechanism which is tightly controlled in cells. It contributes to maintenance of tissue homeostasis and normally eliminates highly proliferative cells with malignant properties. Induced pluripotent stem cells (iPSCs) have recently been described with significant functional and morphological similarities to embryonic stem cells. Human iPSCs are of great hope for regenerative medicine due to their broad potential to differentiate into specialized cell types in culture. They may be useful for exploring disease mechanisms and may provide the basis for future cell-based replacement therapies. However, there is only poor insight into iPSCs cell signaling as the regulation of apoptosis. In this study, we focused our attention on the apoptotic response of Alzheimer fibroblast-derived iPSCs and two other Alzheimer free iPSCs to five biologically relevant kinase inhibitors as well as to the death ligand TRAIL. To our knowledge, we are the first to report that the relatively high basal apoptotic rate of iPSCs is strongly suppressed by the pancaspase inhibitor QVD-Oph, thus underlining the dependency on proapoptotic caspase cascades. Furthermore, wortmannin, an inhibitor of phosphoinositid-3 kinase / Akt signaling (PI3K-AKT), dramatically and rapidly induced apoptosis in iPSCs. In contrast, parental fibroblasts as well as iPSC-derived neuronal cells were not responsive. The resulting condensation and fragmentation of DNA and decrease of the membrane potential are typical features of apoptosis. Comparable effects were observed with an AKT inhibitor (MK-2206). Wortmannin resulted in disappearance of phosphorylated AKT and activation of the main effector caspase-3 in iPSCs. These results clearly demonstrate for the first time that PI3K-AKT represents a highly essential survival signaling pathway in iPSCs. The findings provide improved understanding on the underlying mechanisms of apoptosis regulation in iPSCs. PMID:27138223

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

    PubMed

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

    2014-05-09

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

  9. Disruption of Akt kinase activation is important for immunosuppression induced by measles virus.

    PubMed

    Avota, E; Avots, A; Niewiesk, S; Kane, L P; Bommhardt, U; ter Meulen, V; Schneider-Schaulies, S

    2001-06-01

    Surface-contact-mediated signaling induced by the measles virus (MV) fusion and hemagglutinin glycoproteins is necessary and sufficient to induce T-cell unresponsiveness in vitro and in vivo. To define the intracellular pathways involved, we analyzed interleukin (IL)-2R signaling in primary human T cells and in Kit-225 cells. Unlike IL-2-dependent activation of JAK/STAT pathways, activation of Akt kinase was impaired after MV contact both in vitro and in vivo. MV interference with Akt activation was important for immunosuppression, as expression of a catalytically active Akt prevented negative signaling by the MV glycoproteins. Thus, we show here that MV exploits a novel strategy to interfere with T-cell activation during immunosuppression.

  10. Low-intensity pulsed ultrasound activates ERK1/2 and PI3K-Akt signalling pathways and promotes the proliferation of human amnion-derived mesenchymal stem cells.

    PubMed

    Ling, Li; Wei, Tianqin; He, Lianli; Wang, Yaping; Wang, Yan; Feng, Xiushan; Zhang, Wenqian; Xiong, Zhengai

    2017-09-22

    This study was to investigate the effect and mechanism of low-intensity pulsed ultrasound (LIPUS) on the proliferation of human amnion-derived mesenchymal stem cells (hAD-MSCs). Human amnion-derived mesenchymal stem cells were isolated from the amnion of term placentas and identified by flow cytometry and differentiation culture. Proliferation of hAD-MSCs was investigated by Cell Counting Kit-8, cell cycle and EdU assays. Western blotting was used to determine the protein expression levels. Human amnion-derived mesenchymal stem cells were successfully isolated from the amnion and identified as multipotent mesenchymal stem cells. Low-intensity pulsed ultrasound promoted the proliferation of hAD-MSCs. Cell cycle analysis showed that LIPUS promoted cells to enter S and G2/M phases from G0/G1 phase. Western blot results showed that LIPUS promoted the phosphorylation and activation of ERK1/2 and Akt and significantly upregulated expression of cyclin D1, cyclin E1, cyclin A2 and cyclin B1. ERK1/2 inhibitor (U0126) and PI3K inhibitor (LY294002) significantly reduced LIPUS-induced phosphorylation of ERK1/2 and Akt, respectively, which in turn reduced the LIPUS-induced proliferation of hAD-MSCs. Low-intensity pulsed ultrasound can promote the proliferation of hAD-MSCs, and ERK1/2 and PI3K-Akt signalling pathways may play important roles in this process. © 2017 John Wiley & Sons Ltd.

  11. The phosphatidylinositol 3-kinase/Akt/mTOR signaling network as a therapeutic target in acute myelogenous leukemia patients

    PubMed Central

    Martelli, Alberto M.; Evangelisti, Camilla; Chiarini, Francesca; McCubrey, James A.

    2010-01-01

    The phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) signaling axis plays a central role in cell proliferation, growth, and survival under physiological conditions. However, aberrant PI3K/Akt/mTOR signaling has been implicated in many human cancers, including acute myelogenous leukemia (AML). Therefore, the PI3K/Akt/mTOR network is considered as a validated target for innovative cancer therapy. The limit of acceptable toxicity for standard polychemotherapy has been reached in AML. Novel therapeutic strategies are therefore needed. This review highlights how the PI3K/Akt/mTOR signaling axis is constitutively active in AML patients, where it affects survival, proliferation, and drug-resistance of leukemic cells including leukemic stem cells. Effective targeting of this pathway with small molecule kinase inhibitors, employed alone or in combination with other drugs, could result in the suppression of leukemic cell growth. Furthermore, targeting the PI3K/Akt/mTOR signaling network with small pharmacological inhibitors, employed either alone or in combinations with other drugs, may result in less toxic and more efficacious treatment of AML patients. Efforts to exploit pharmacological inhibitors of the PI3K/Akt/mTOR cascade which show efficacy and safety in the clinical setting are now underway. PMID:20671809

  12. Upregulation of Akt/NF-κB-regulated inflammation and Akt/Bad-related apoptosis signaling pathway involved in hepatic carcinoma process: suppression by carnosic acid nanoparticle

    PubMed Central

    Tang, Bo; Tang, Fang; Wang, Zhenran; Qi, Guangying; Liang, Xingsi; Li, Bo; Yuan, Shengguang; Liu, Jie; Yu, Shuiping; He, Songqing

    2016-01-01

    Primary liver cancer is globally the sixth most frequent cancer, and the second leading cause of cancer death and its incidence is increasing in many countries, becoming a serious threat to human health. Many researches focused on the treatment and prevention of liver cancer. However, due to the underlying molecular mechanism of liver cancer still not fully understood, the studies and development of treatments were forced to be delayed. Akt has been suggested to play an essential role in the progression of inflammation response and apoptosis. Hence, in this study, Akt-knockout mice and cells of liver cancer were used as a model to investigate the molecular mechanism of Akt-associated inflammatory and apoptotic signaling pathway linked with NF-κB and Bcl-2-associated death promoter (Bad) for the progression of liver cancer. Carnosic acid (CA), as a phenolic diterpene with anticancer, antibacterial, antidiabetic, as well as neuroprotective properties, is produced by many species from Lamiaceae family. Administration of CA nanoparticles was sufficient to lead to considerable inhibition of liver cancer progression. The results indicated that, compared to the normal liver cells, the expression of Akt was significantly higher in liver cancer cell lines. Also, we found that Akt-knockout cancer cell lines modulated inflammation response and apoptosis via inhibiting NF-κB activation and inducing apoptotic reaction. Our results indicated that the downstream signals, including cytokines regulated by NF-κB and caspase-3-activated apoptosis affected by Bad, were re-modulated for knockout of Akt. And CA nanoparticles, acting as Akt-knockout, could inhibit inflammation and accelerate apoptosis in liver cancer by altering NF-κB activation and activating caspase-3 through Bad pathway. These findings demonstrated that the nanoparticulate drug CA performed its effective role owing to its ability to reduce inflammatory action and enhance apoptosis for the overexpression of NF

  13. Upregulation of Akt/NF-κB-regulated inflammation and Akt/Bad-related apoptosis signaling pathway involved in hepatic carcinoma process: suppression by carnosic acid nanoparticle.

    PubMed

    Tang, Bo; Tang, Fang; Wang, Zhenran; Qi, Guangying; Liang, Xingsi; Li, Bo; Yuan, Shengguang; Liu, Jie; Yu, Shuiping; He, Songqing

    Primary liver cancer is globally the sixth most frequent cancer, and the second leading cause of cancer death and its incidence is increasing in many countries, becoming a serious threat to human health. Many researches focused on the treatment and prevention of liver cancer. However, due to the underlying molecular mechanism of liver cancer still not fully understood, the studies and development of treatments were forced to be delayed. Akt has been suggested to play an essential role in the progression of inflammation response and apoptosis. Hence, in this study, Akt-knockout mice and cells of liver cancer were used as a model to investigate the molecular mechanism of Akt-associated inflammatory and apoptotic signaling pathway linked with NF-κB and Bcl-2-associated death promoter (Bad) for the progression of liver cancer. Carnosic acid (CA), as a phenolic diterpene with anticancer, antibacterial, antidiabetic, as well as neuroprotective properties, is produced by many species from Lamiaceae family. Administration of CA nanoparticles was sufficient to lead to considerable inhibition of liver cancer progression. The results indicated that, compared to the normal liver cells, the expression of Akt was significantly higher in liver cancer cell lines. Also, we found that Akt-knockout cancer cell lines modulated inflammation response and apoptosis via inhibiting NF-κB activation and inducing apoptotic reaction. Our results indicated that the downstream signals, including cytokines regulated by NF-κB and caspase-3-activated apoptosis affected by Bad, were re-modulated for knockout of Akt. And CA nanoparticles, acting as Akt-knockout, could inhibit inflammation and accelerate apoptosis in liver cancer by altering NF-κB activation and activating caspase-3 through Bad pathway. These findings demonstrated that the nanoparticulate drug CA performed its effective role owing to its ability to reduce inflammatory action and enhance apoptosis for the overexpression of NF

  14. Embryonic liver fordin is involved in glucose glycolysis of hepatic stellate cell by regulating PI3K/Akt signaling.

    PubMed

    Tu, Wei; Ye, Jin; Wang, Zhi-Jun

    2016-10-14

    To investigate the role of embryonic liver fordin (ELF) in liver fibrosis by regulating hepatic stellate cells (HSCs) glucose glycolysis. The expression of ELF and the glucose glycolysis-related proteins were evaluated in activated HSCs. siRNA was used to silence ELF expression in activated HSCs in vitro and the subsequent changes in PI3K/Akt signaling and glucose glycolysis-related proteins were observed. The expression of ELF increased remarkably in HSCs of the fibrosis mouse model and HSCs that were cultured for 3 wk in vitro. Glucose glycolysis-related proteins showed an obvious increase in the activated HSCs, such as phosphofructokinase, platelet and glucose transporter 1. ELF-siRNA, which perfectly silenced the expression of ELF in activated HSCs, led to the induction of glucose glycolysis-related proteins and extracellular matrix (ECM) components. Moreover, pAkt, which is an important downstream factor in PI3K/Akt signaling, showed a significant change in response to the ELF silencing. The expression of glucose glycolysis-related proteins and ECM components decreased remarkably when the PI3K/Akt signaling was blocked by Ly294002 in the activated HSCs. ELF is involved in HSC glucose glycolysis by regulating PI3K/Akt signaling.

  15. Embryonic liver fordin is involved in glucose glycolysis of hepatic stellate cell by regulating PI3K/Akt signaling

    PubMed Central

    Tu, Wei; Ye, Jin; Wang, Zhi-Jun

    2016-01-01

    AIM To investigate the role of embryonic liver fordin (ELF) in liver fibrosis by regulating hepatic stellate cells (HSCs) glucose glycolysis. METHODS The expression of ELF and the glucose glycolysis-related proteins were evaluated in activated HSCs. siRNA was used to silence ELF expression in activated HSCs in vitro and the subsequent changes in PI3K/Akt signaling and glucose glycolysis-related proteins were observed. RESULTS The expression of ELF increased remarkably in HSCs of the fibrosis mouse model and HSCs that were cultured for 3 wk in vitro. Glucose glycolysis-related proteins showed an obvious increase in the activated HSCs, such as phosphofructokinase, platelet and glucose transporter 1. ELF-siRNA, which perfectly silenced the expression of ELF in activated HSCs, led to the induction of glucose glycolysis-related proteins and extracellular matrix (ECM) components. Moreover, pAkt, which is an important downstream factor in PI3K/Akt signaling, showed a significant change in response to the ELF silencing. The expression of glucose glycolysis-related proteins and ECM components decreased remarkably when the PI3K/Akt signaling was blocked by Ly294002 in the activated HSCs. CONCLUSION ELF is involved in HSC glucose glycolysis by regulating PI3K/Akt signaling. PMID:27784964

  16. Cyclic Compressive Stress Regulates Apoptosis in Rat Osteoblasts: Involvement of PI3K/Akt and JNK MAPK Signaling Pathways

    PubMed Central

    Jiang, Dawei; Wang, Tianchen; Zhang, Yinquan; Ma, Hui

    2016-01-01

    It is widely accepted that physiological mechanical stimulation suppresses apoptosis and induces synthesis of extracellular matrix by osteoblasts; however, the effect of stress overloading on osteoblasts has not been fully illustrated. In the present study, we investigated the effect of cyclic compressive stress on rat osteoblasts apoptosis, using a novel liquid drop method to generate mechanical stress on osteoblast monolayers. After treatment with different levels of mechanical stress, apoptosis of osteoblasts and activations of mitogen-activated protein kinases (MAPKs) and PI3-kinase (PI3K)/Akt signaling pathways were investigated. Osteoblasts apoptosis was observed after treated with specific inhibitors prior to mechanical stimulation. Protein levels of Bax/Bcl-2/caspase-3 signaling were determined using western blot with or without inhibitors of PI3K/Akt and phosphorylation of c-jun N-terminal kinase (JNK) MAPK. Results showed that mechanical stimulation led to osteoblasts apoptosis in a dose-dependent manner and a remarkable activation of MAPKs and PI3K/Akt signaling pathways. Activation of PI3K/Akt protected against apoptosis, whereas JNK MAPK increased apoptosis via regulation of Bax/Bcl-2/caspase-3 activation. In summary, the PI3K/Akt and JNK MAPK signaling pathways played opposing roles in osteoblasts apoptosis, resulting in inhibition of apoptosis upon small-magnitude stress and increased apoptosis upon large-magnitude stress. PMID:27806136

  17. Phosphoinositide 3-kinase signaling to Akt promotes keratinocyte differentiation versus death.

    PubMed

    Calautti, Enzo; Li, Jian; Saoncella, Stefania; Brissette, Janice L; Goetinck, Paul F

    2005-09-23

    Signaling pathways regulating the differentiation program of epidermal cells overlap widely with those activated during apoptosis. How differentiating cells remain protected from premature death, however, is still poorly defined. We show here that the phosphoinositide 3-kinase (PI3K)/Akt pathway is activated at early stages of mouse keratinocyte differentiation both in culture and in the intact epidermis in vivo. Expression of active Akt in keratinocytes promotes growth arrest and differentiation, whereas pharmacological blockade of PI3K inhibits the expression of "late" differentiation markers and leads to death of cells that would otherwise differentiate. Mechanistically, the activation of the PI3K/Akt pathway in keratinocyte differentiation depends on the activity of the epidermal growth factor receptor and Src families of tyrosine kinases and the engagement of E-cadherin-mediated adhesion. During this process, PI3K associates increasingly with cadherin-catenin protein complexes bearing tyrosine phosphorylated YXXM motifs. Thus, the PI3K signaling pathway regulates the choice between epidermal cell differentiation and death at the cross-talk between tyrosine kinases and cadherin-associated catenins.

  18. IFNγ-induced suppression of β-catenin signaling: evidence for roles of Akt and 14.3.3ζ

    PubMed Central

    Nava, Porfirio; Kamekura, Ryuta; Quirós, Miguel; Medina-Contreras, Oscar; Hamilton, Ross W.; Kolegraff, Keli N.; Koch, Stefan; Candelario, Aurora; Romo-Parra, Hector; Laur, Oskar; Hilgarth, Roland S.; Denning, Timothy L.; Parkos, Charles A.; Nusrat, Asma

    2014-01-01

    The proinflammatory cytokine interferon γ (IFNγ ) influences intestinal epithelial cell (IEC) homeostasis in a biphasic manner by acutely stimulating proliferation that is followed by sustained inhibition of proliferation despite continued mucosal injury. β-Catenin activation has been classically associated with increased IEC proliferation. However, we observed that IFNγ inhibits IEC proliferation despite sustained activation of Akt/β-catenin signaling. Here we show that inhibition of Akt/β-catenin–mediated cell proliferation by IFNγ is associated with the formation of a protein complex containing phosphorylated β-catenin 552 (pβ-cat552) and 14.3.3ζ. Akt1 served as a bimodal switch that promotes or inhibits β-catenin transactivation in response to IFNγ stimulation. IFNγ initially promotes β-catenin transactivation through Akt-dependent C-terminal phosphorylation of β-catenin to promote its association with 14.3.3ζ. Augmented β-catenin transactivation leads to increased Akt1 protein levels, and active Akt1 accumulates in the nucleus, where it phosphorylates 14.3.3ζ to translocate 14.3.3ζ/β-catenin from the nucleus, thereby inhibiting β-catenin transactivation and IEC proliferation. These results outline a dual function of Akt1 that suppresses IEC proliferation during intestinal inflammation. PMID:25079689

  19. Eupafolin suppresses prostate cancer by targeting phosphatidylinositol 3-kinase-mediated Akt signaling.

    PubMed

    Liu, Kangdong; Park, Chanmi; Chen, Hanyong; Hwang, Joonsung; Thimmegowda, N R; Bae, Eun Young; Lee, Ki Won; Kim, Hong-Gyum; Liu, Haidan; Soung, Nak Kyun; Peng, Cong; Jang, Jae Hyuk; Kim, Kyoon Eon; Ahn, Jong Seog; Bode, Ann M; Dong, Ziming; Kim, Bo Yeon; Dong, Zigang

    2015-09-01

    Phosphatase and tensin homolog (PTEN) loss or mutation consistently activates the phosphatidylinositol 3-kinase (PI3-K)/Akt signaling pathway, which contributes to the progression and invasiveness of prostate cancer. Furthermore, the PTEN/PI3-K/Akt and Ras/MAPK pathways cooperate to promote the epithelial-mesenchymal transition (EMT) and metastasis initiated from prostate stem/progenitor cells. For these reasons, the PTEN/PI3-K/Akt pathway is considered as an attractive target for both chemoprevention and chemotherapy. Herein we report that eupafolin, a natural compound found in common sage, inhibited proliferation of prostate cancer cells. Protein content analysis indicated that phosphorylation of Akt and its downstream kinases was inhibited by eupafolin treatment. Pull-down assay and in vitro kinase assay results indicated that eupafolin could bind with PI3-K and attenuate its kinase activity. Eupafolin also exhibited tumor suppressive effects in vivo in an athymic nude mouse model. Overall, these results suggested that eupafolin exerts antitumor effects by targeting PI3-K. © 2014 Wiley Periodicals, Inc.

  20. Eupafolin suppresses prostate cancer by targeting phosphatidylinositol 3-kinase-mediated Akt signaling

    PubMed Central

    Liu, Kangdong; Park, Chanmi; Chen, Hanyong; Hwang, Joonsung; Thimmegowda, N.R.; Bae, Eun Young; Lee, Ki Won; Kim, Hong-Gyum; Liu, Haidan; Soung, Nak Kyun; Peng, Cong; Jang, Jae Hyuk; Kim, Kyoon Eon; Ahn, Jong Seog; Bode, Ann M.; Dong, Ziming; Kim, Bo Yeon; Dong, Zigang

    2014-01-01

    Phosphatase and tensin homolog (PTEN) loss or mutation consistently activates the phosphatidylinositol 3-kinase (PI3-K)/Akt signaling pathway, which contributes to the progression and invasiveness of prostate cancer. Furthermore, the PTEN/PI3-K/Akt and Ras/MAPK pathways cooperate to promote the epithelial-mesenchymal transition (EMT) and metastasis initiated from prostate stem/progenitor cells. For these reasons, the PTEN/PI3-K/Akt pathway is considered as an attractive target for both chemoprevention and chemotherapy. Herein we report that eupafolin, a natural compound found in common sage, inhibited proliferation of prostate cancer cells. Protein content analysis indicated that phosphorylation of Akt and its downstream kinases was inhibited by eupafolin treatment. Pull-down assay and in vitro kinase assay results indicated that eupafolin could bind with PI3-K and attenuate its kinase activity. Eupafolin also exhibited tumor suppressive effects in vivo in an athymic nude mouse model. Overall, these results suggested that eupafolin exerts antitumor effects by targeting PI3-K. PMID:24700667

  1. Therapeutic Implications of Targeting AKT Signaling in Melanoma

    PubMed Central

    Madhunapantula, SubbaRao V.; Robertson, Gavin P.

    2011-01-01

    Identification of key enzymes regulating melanoma progression and drug resistance has the potential to lead to the development of novel, more effective targeted agents for inhibiting this deadly form of skin cancer. The Akt3, also known as protein kinase B gamma, pathway enzymes regulate diverse cellular processes including proliferation, survival, and invasion thereby promoting the development of melanoma. Accumulating preclinical evidence demonstrates that therapeutic agents targeting these kinases alone or in combination with other pathway members could be effective for the long-term treatment of advanced-stage disease. However, currently, no selective and effective therapeutic agent targeting these kinases has been identified for clinical use. This paper provides an overview of the key enzymes of the PI3K pathway with emphasis placed on Akt3 and the negative regulator of this kinase called PTEN (phosphatase and tensin homolog deleted on chromosome 10). Mechanisms regulating these enzymes, their substrates and therapeutic implications of targeting these proteins to treat melanoma are also discussed. Finally, key issues that remain to be answered and future directions for interested researchers pertaining to this signaling cascade are highlighted. PMID:21461351

  2. Protective Role of PI3-kinase/Akt/eNOS Signaling in Mechanical Stress Through Inhibition of p38 Mitogen-Activated Protein Kinase in Mouse Lung

    DTIC Science & Technology

    2010-01-01

    81. 6 Iwakiri Y. Tsai MH. McCabe TJ. Gratton JP. Fulton D. Groszmann RJ. et al. Phosphorylation of eNOS initiates excessive NO production in early...Crit care Med 2008; 177(Abstracts issue): A760. 21 Gratton JP. Morales-Ruiz M. Kureishi Y. Fulton D. Walsh K. Sessa WC. Akt down-regulation of p38

  3. Akt Regulates TNFα Synthesis Downstream of RIP1 Kinase Activation during Necroptosis

    PubMed Central

    McNamara, Colleen R.; Ahuja, Ruchita; Osafo-Addo, Awo D.; Barrows, Douglas; Kettenbach, Arminja; Skidan, Igor; Teng, Xin; Cuny, Gregory D.; Gerber, Scott; Degterev, Alexei

    2013-01-01

    Necroptosis is a regulated form of necrotic cell death that has been implicated in the pathogenesis of various diseases including intestinal inflammation and systemic inflammatory response syndrome (SIRS). In this work, we investigated the signaling mechanisms controlled by the necroptosis mediator receptor interacting protein-1 (RIP1) kinase. We show that Akt kinase activity is critical for necroptosis in L929 cells and plays a key role in TNFα production. During necroptosis, Akt is activated in a RIP1 dependent fashion through its phosphorylation on Thr308. In L929 cells, this activation requires independent signaling inputs from both growth factors and RIP1. Akt controls necroptosis through downstream targeting of mammalian Target of Rapamycin complex 1 (mTORC1). Akt activity, mediated in part through mTORC1, links RIP1 to JNK activation and autocrine production of TNFα. In other cell types, such as mouse lung fibroblasts and macrophages, Akt exhibited control over necroptosis-associated TNFα production without contributing to cell death. Overall, our results provide new insights into the mechanism of necroptosis and the role of Akt kinase in both cell death and inflammatory regulation. PMID:23469174

  4. Akt Regulates TNFα synthesis downstream of RIP1 kinase activation during necroptosis.

    PubMed

    McNamara, Colleen R; Ahuja, Ruchita; Osafo-Addo, Awo D; Barrows, Douglas; Kettenbach, Arminja; Skidan, Igor; Teng, Xin; Cuny, Gregory D; Gerber, Scott; Degterev, Alexei

    2013-01-01

    Necroptosis is a regulated form of necrotic cell death that has been implicated in the pathogenesis of various diseases including intestinal inflammation and systemic inflammatory response syndrome (SIRS). In this work, we investigated the signaling mechanisms controlled by the necroptosis mediator receptor interacting protein-1 (RIP1) kinase. We show that Akt kinase activity is critical for necroptosis in L929 cells and plays a key role in TNFα production. During necroptosis, Akt is activated in a RIP1 dependent fashion through its phosphorylation on Thr308. In L929 cells, this activation requires independent signaling inputs from both growth factors and RIP1. Akt controls necroptosis through downstream targeting of mammalian Target of Rapamycin complex 1 (mTORC1). Akt activity, mediated in part through mTORC1, links RIP1 to JNK activation and autocrine production of TNFα. In other cell types, such as mouse lung fibroblasts and macrophages, Akt exhibited control over necroptosis-associated TNFα production without contributing to cell death. Overall, our results provide new insights into the mechanism of necroptosis and the role of Akt kinase in both cell death and inflammatory regulation.

  5. Dioscin inhibits osteoclast differentiation and bone resorption though down-regulating the Akt signaling cascades

    SciTech Connect

    Qu, Xinhua; Zhai, Zanjing; Liu, Xuqiang; Li, Haowei; Ouyang, Zhengxiao; Wu, Chuanlong; Liu, Guangwang; Fan, Qiming; Tang, Tingting; Qin, An; Dai, Kerong

    2014-01-10

    Highlights: •A natural-derived compound, dioscin, suppresses osteoclast formation and bone resorption. •Dioscin inhibits osteolytic bone loss in vivo. •Dioscin impairs the Akt signaling cascades pathways during osteoclastogenesis. •Dioscin have therapeutic value in treating osteoclast-related diseases. -- Abstract: Bone resorption is the unique function of osteoclasts (OCs) and is critical for both bone homeostasis and pathologic bone diseases including osteoporosis, rheumatoid arthritis and tumor bone metastasis. Thus, searching for natural compounds that may suppress osteoclast formation and/or function is promising for the treatment of osteoclast-related diseases. In this study, we for the first time demonstrated that dioscin suppressed RANKL-mediated osteoclast differentiation and bone resorption in vitro in a dose-dependent manner. The suppressive effect of dioscin is supported by the reduced expression of osteoclast-specific markers. Further molecular analysis revealed that dioscin abrogated AKT phosphorylation, which subsequently impaired RANKL-induced nuclear factor-kappaB (NF-κB) signaling pathway and inhibited NFATc1 transcriptional activity. Moreover, in vivo studies further verified the bone protection activity of dioscin in osteolytic animal model. Together our data demonstrate that dioscin suppressed RANKL-induced osteoclast formation and function through Akt signaling cascades. Therefore, dioscin is a potential natural agent for the treatment of osteoclast-related diseases.

  6. Loss of Akt activity increases circulating soluble endoglin release in preeclampsia: identification of inter-dependency between Akt-1 and heme oxygenase-1

    PubMed Central

    Cudmore, Melissa J.; Ahmad, Shakil; Sissaoui, Samir; Ramma, Wenda; Ma, Bin; Fujisawa, Takeshi; Al-Ani, Bahjat; Wang, Keqing; Cai, Meng; Crispi, Fatima; Hewett, Peter W.; Gratacós, Eduard; Egginton, Stuart; Ahmed, Asif

    2012-01-01

    Aims Endothelial dysfunction is a hallmark of preeclampsia. Desensitization of the phosphoinositide 3-kinase (PI3K)/Akt pathway underlies endothelial dysfunction and haeme oxygenase-1 (HO-1) is decreased in preeclampsia. To identify therapeutic targets, we sought to assess whether these two regulators act to suppress soluble endoglin (sEng), an antagonist of transforming growth factor-β (TGF-β) signalling, which is known to be elevated in preeclampsia. Methods and results Vascular endothelial growth factor-A (VEGF-A), fibroblast growth factor (FGF-2), angiopoietin-1 (Ang-1), and insulin, which all activate the PI3K/Akt pathway, inhibited the release of sEng from endothelial cells. Inhibition of the PI3K/Akt pathway, by overexpression of phosphatase and tensin homolog (PTEN) or a dominant-negative isoform of Akt (Aktdn) induced sEng release from endothelial cells and prevented the inhibitory effect of VEGF-A. Conversely, overexpression of a constitutively active Akt (Aktmyr) inhibited PTEN and cytokine-induced sEng release. Systemic delivery of Aktmyr to mice significantly reduced circulating sEng, whereas Aktdn promoted sEng release. Phosphorylation of Akt was reduced in preeclamptic placenta and this correlated with the elevated level of circulating sEng. Knock-down of Akt using siRNA prevented HO-1-mediated inhibition of sEng release and reduced HO-1 expression. Furthermore, HO-1 null mice have reduced phosphorylated Akt in their organs and overexpression of Aktmyr failed to suppress the elevated levels of sEng detected in HO-1 null mice, indicating that HO-1 is required for the Akt-mediated inhibition of sEng. Conclusion The loss of PI3K/Akt and/or HO-1 activity promotes sEng release and positive manipulation of these pathways offers a strategy to circumvent endothelial dysfunction. PMID:21411816

  7. Snail promotes cell migration through PI3K/AKT-dependent Rac1 activation as well as PI3K/AKT-independent pathways during prostate cancer progression

    PubMed Central

    Henderson, Veronica; Smith, Basil; Burton, Liza J; Randle, Diandra; Morris, Marisha; Odero-Marah, Valerie A

    2015-01-01

    Snail, a zinc-finger transcription factor, induces epithelial-mesenchymal transition (EMT), which is associated with increased cell migration and metastasis in cancer cells. Rac1 is a small G-protein which upon activation results in formation of lamellipodia, the first protrusions formed by migrating cells. We have previously shown that Snail promotes cell migration through down-regulation of maspin tumor suppressor. We hypothesized that Snail's regulation of cell migration may also involve Rac1 signaling regulated by PI3K/AKT and/or MAPK pathways. We found that Snail overexpression in LNCaP and 22Rv1 prostate cancer cells increased Rac1 activity associated with increased cell migration, and the Rac1 inhibitor, NSC23766, could inhibit Snail-mediated cell migration. Conversely, Snail downregulation using shRNA in the aggressive C4–2 prostate cancer cells decreased Rac1 activity and cell migration. Moreover, Snail overexpression increased ERK and PI3K/AKT activity in 22Rv1 prostate cancer cells. Treatment of Snail-overexpressing 22Rv1 cells with LY294002, PI3K/AKT inhibitor or U0126, MEK inhibitor, decreased cell migration significantly, but only LY294002 significantly reduced Rac1 activity, suggesting that Snail promotes Rac1 activation via the PI3K/AKT pathway. Furthermore, 22Rv1 cells overexpressing Snail displayed decreased maspin levels, while inhibition of maspin expression in 22Rv1 cells with siRNA, led to increased PI3K/AKT, Rac1 activity and cell migration, without affecting ERK activity, suggesting that maspin is upstream of PI3K/AKT. Overall, we have dissected signaling pathways by which Snail may promote cell migration through MAPK signaling or alternatively through PI3K/AKT-Rac1 signaling that involves Snail inhibition of maspin tumor suppressor. This may contribute to prostate cancer progression. PMID:26207671

  8. Photoreceptor Neuroprotection: Regulation of Akt Activation Through Serine/Threonine Phosphatases, PHLPP and PHLPPL.

    PubMed

    Rajala, Raju V S; Kanan, Yogita; Anderson, Robert E

    2016-01-01

    Serine/threonine kinase Akt is a downstream effector of insulin receptor/PI3K pathway that is involved in many processes, including providing neuroprotection to stressed rod photoreceptor cells. Akt signaling is known to be regulated by the serine/threonine phosphatases, PHLPP (PH domain and leucine rich repeat protein phosphatase) and PHLPPL (PH domain and leucine rich repeat protein phosphatase-like). We previously reported that both phosphatases are expressed in the retina, as well as in photoreceptor cells. In this study, we examined the PHLPP and PHLPPL phosphatase activities towards non-physiological and physiological substrates. Our results suggest that PHLPP was more active than PHLPPL towards non-physiological substrates, whereas both PHLPP and PHLPP dephosphorylated the physiological substrates of Akt1 and Akt3 with similar efficiencies. Our results also suggest that knockdown of PHLPPL alone does not increase Akt phosphorylation, due to a compensatory increase of PHLPP, which results in the dephosphorylation of Akt. Therefore, PHLPP and PHLPPL regulate Akt activation together when both phosphatases are expressed.

  9. Effects of RAF inhibitors on PI3K/AKT signalling depend on mutational status of the RAS/RAF signalling axis

    PubMed Central

    Fritsche-Guenther, Raphaela; Witzel, Franziska; Kempa, Stefan; Brummer, Tilman; Sers, Christine; Blüthgen, Nils

    2016-01-01

    Targeted therapies within the RAS/RAF/MEK/ERK signalling axis become increasingly popular, yet cross-talk and feedbacks in the signalling network lead to unexpected effects. Here we look systematically into how inhibiting RAF and MEK with clinically relevant inhibitors result in changes in PI3K/AKT activation. We measure the signalling response using a bead-based ELISA, and use a panel of three cell lines, and isogenic cell lines that express mutant forms of the oncogenes KRAS and BRAF to interrogate the effects of the MEK and RAF inhibitors on signalling. We find that treatment with the RAF inhibitors have opposing effects on AKT phosphorylation depending on the mutational status of two important oncogenes, KRAS and BRAF. If these two genes are in wildtype configuration, RAF inhibitors reduce AKT phosphorylation. In contrast, if BRAF or KRAS are mutant, RAF inhibitors will leave AKT phosphorylation unaffected or lead to an increase of AKT phosphorylation. Down-regulation of phospho-AKT by RAF inhibitors also extends to downstream transcription factors, and correlates with apoptosis induction. Our results show that oncogenes rewire signalling such that targeted therapies can have opposing effects on parallel pathways, which depend on the mutational status of the cell. PMID:26799289

  10. Differential regulation of mTOR signaling determines sensitivity to AKT inhibition in diffuse large B cell lymphoma

    PubMed Central

    Ezell, Scott A.; Wang, Suping; Bihani, Teeru; Lai, Zhongwu; Grosskurth, Shaun E.; Tepsuporn, Suprawee; Davies, Barry R.; Huszar, Dennis; Byth, Kate F.

    2016-01-01

    Agents that target components of the PI3K/AKT/mTOR pathway are under investigation for the treatment of diffuse large B cell lymphoma (DLBCL). Given the highly heterogeneous nature of DLBCL, it is not clear whether all subtypes of DLBCL will be susceptible to PI3K pathway inhibition, or which kinase within this pathway is the most favorable target. Pharmacological profiling of a panel of DLBCL cell lines revealed a subset of DLBCL that was resistant to AKT inhibition. Strikingly, sensitivity to AKT inhibitors correlated with the ability of these inhibitors to block phosphorylation of S6K1 and ribosomal protein S6. Cell lines resistant to AKT inhibition activated S6K1 independent of AKT either through upregulation of PIM2 or through activation by B cell receptor (BCR) signaling components. Finally, combined inhibition of AKT and BTK, PIM2, or S6K1 proved to be an effective strategy to overcome resistance to AKT inhibition in DLBCL. PMID:26824321

  11. Anti-proliferative effect of RCE-4 from Reineckia carnea on human cervical cancer HeLa cells by inhibiting the PI3K/Akt/mTOR signaling pathway and NF-κB activation.

    PubMed

    Bai, Caihong; Yang, Xiaojiao; Zou, Kun; He, Haibo; Wang, Junzhi; Qin, Huilin; Yu, Xiaoqin; Liu, Chengxiong; Zheng, Juyan; Cheng, Fan; Chen, Jianfeng

    2016-06-01

    Cervical cancer is the second leading cause of cancer deaths in women worldwide. In recent years, the studies find that inflammation is a critical component of tumor progression, and the ideal therapeutic methods should be aimed at the inflammation reaction triggers. (1β,3β,5β,25S)-spirostan-1,3-diol1-[α-L-rhamnopyranosyl-(1 → 2)-β-D-xylopyranoside] (RCE-4) was the main active composition of Reineckia carnea (Andr.) Kunth. It significantly induced apoptosis in cervical cancer Caski cells through the mitochondrial pathway in our previous studies; however, its underlying mechanism remains poorly understood. This study aimed to further evaluate the effect of RCE-4 on human cervical cancer HeLa cells. Based on this observation, we investigated the anti-cervical cancer effect of RCE-4 by modulating phosphatidylinositol 3-kinase/protein kinase-B/mammalian target of rapamycin (PI3K/Akt/mTOR) signaling pathway, nuclear factor-kappa B (NF-κB) activation, and inflammation-related key factors in HeLa cells. The results indicated that the HeLa cell was the most sensitive with an IC50 of 7.01 μM; RCE-4 significantly promoted the release of cellular lactate dehydrogenase (LDH); increased DNA fragmentation and apoptosis; reduced PI3K, Akt, mTOR, and NF-κBp65 phosphorylation levels; increased the Bax and cleaved poly (ADP-ribose) polymerase (PARP) protein levels; suppressed Bcl-2 protein expression; elevated the Bax/Bcl-2 expression ratio; and decreased the interleukin-1 beta (IL-1β) and interleukin-6 (IL-6) mRNA expressions in HeLa cells in a concentration-dependent manner. These findings suggest that RCE-4 exerted beneficially anti-cervical cancer effect on HeLa cells, mainly inhibiting PI3K/Akt/mTOR signaling pathway phosphorylation and NF-κB activation, promoting HeLa cell apoptosis. Graphical abstract Anti-tumor effect of RCE-4 on HeLa cells.

  12. IL-29 and IFN-α regulate the expression of MxA, 2',5'-OAS and PKR genes in association with the activation of Raf-MEK-ERK and PI3K-AKT signal pathways in HepG2.2.15 cells.

    PubMed

    Chai, Yu; Huang, Hai-Liang; Hu, Dao-Jun; Luo, Xin; Tao, Qian-Shan; Zhang, Xiao-Ling; Zhang, Sheng-Quan

    2011-01-01

    Interferons (IFNs) can activate the PI3K-AKT and Raf-MEK-ERK signal pathways and induce antiviral proteins (MxA, 2',5'-OAS and PKR) expression in specific cell lines. However, the relationship between those antiviral proteins expression and signal pathways remains unknown at present. Thus our experiments were designed to determine the exact relationship in HepG2.2.15 cell line. The results demonstrated that IFN-α and IL-29 were both able to activate PI3K-AKT and Raf-MEK-ERK signal pathways, and IFN-α up-regulated the expression of MxA, 2',5'-OAS and PKR whereas IL-29 increased mRNA expression of MxA and 2',5'-OAS and had no influence on PKR. Furthermore, MxA, 2',5'-OAS and PKR expression were down-regulated while PI3K-AKT signal pathway was blocked by LY294002. And MxA was up-regulated after Raf-MEK-ERK signal pathway being blocked by PD98059. These findings indicate that the expression of MxA, 2',5'-OAS and PKR are up-regulate by PI3K-AKT signal pathway, and Raf-MEK-ERK signal pathway has a negative regulatory effect on the expression of MxA and no significant effect on 2',5'-OAS and PKR.

  13. Hydrogen peroxide inhibits transforming growth factor-β1-induced cell cycle arrest by promoting Smad3 linker phosphorylation through activation of Akt-ERK1/2-linked signaling pathway

    SciTech Connect

    Choi, Jiyeon; Park, Seong Ji; Jo, Eun Ji; Lee, Hui-Young; Hong, Suntaek; Kim, Seong-Jin; Kim, Byung-Chul

    2013-06-14

    Highlights: •H{sub 2}O{sub 2} inhibits TGF-β1-induced cell cycle arrest. •H{sub 2}O{sub 2} induces Smad3 linker phosphorylation through Akt-ERK1/2 pathway. •H{sub 2}O{sub 2}-mediated suppression of TGF-β signal requires Smad3 linker phosphorylation. •This is a first report about interplay between H{sub 2}O{sub 2} and growth inhibition pathway. -- Abstract: Hydrogen peroxide (H{sub 2}O{sub 2}) functions as a second messenger in growth factor receptor-mediated intracellular signaling cascade and is tumorigenic by virtue of its ability to promote cell proliferation; however, the mechanisms underlying the growth stimulatory action of H{sub 2}O{sub 2} are less understood. Here we report an important mechanism for antagonistic effects of H{sub 2}O{sub 2} on growth inhibitory response to transforming growth factor-β1 (TGF-β1). In Mv1Lu and HepG2 cells, pretreatment of H{sub 2}O{sub 2} (0.05–0.2 mM) completely blocked TGF-β1-mediated induction of p15{sup INK4B} expression and increase of its promoter activity. Interestingly, H{sub 2}O{sub 2} selectively suppressed the transcriptional activation potential of Smad3, not Smad2, in the absence of effects on TGF-β1-induced phosphorylation of the COOH-tail SSXS motif of Smad3 and its nuclear translocation. Mechanism studies showed that H{sub 2}O{sub 2} increases the phosphorylation of Smad3 at the middle linker region in a concentration- and time-dependent manner and this effect is mediated by activation of extracellular signal-activated kinase 1/2 through Akt. Furthermore, expression of a mutant Smad3 in which linker phosphorylation sites were ablated significantly abrogated the inhibitory effects of H{sub 2}O{sub 2} on TGF-β1-induced increase of p15{sup INK4B}-Luc reporter activity and blockade of cell cycle progression from G1 to S phase. These findings for the first time define H{sub 2}O{sub 2} as a signaling molecule that modulate Smad3 linker phosphorylation and its transcriptional activity, thus providing

  14. The Hepatitis B Virus (HBV) HBx Protein Activates AKT To Simultaneously Regulate HBV Replication and Hepatocyte Survival

    PubMed Central

    Rawat, Siddhartha

    2014-01-01

    ABSTRACT Chronic infection with hepatitis B virus (HBV) is a risk factor for developing liver diseases such as hepatocellular carcinoma (HCC). HBx is a multifunctional protein encoded by the HBV genome; HBx stimulates HBV replication and is thought to play an important role in the development of HBV-associated HCC. HBx can activate the phosphatidylinositol 3-kinase (PI3K)/AKT signaling pathway in some cell lines; however, whether HBx regulates PI3K/AKT signaling in normal hepatocytes has not been evaluated. In studies described here, we assessed HBx activation of PI3K/AKT signaling in an ex vivo model of cultured primary hepatocytes and determined how this HBx activity affects HBV replication. We report that HBx activates AKT in primary hepatocytes and that the activation of AKT decreases HBV replication and HBV mRNA and core protein levels. We show that the transcription factor hepatocyte nuclear factor 4α (HNF4α) is a target of HBx-regulated AKT, and we link HNF4α to HBx-regulated AKT modulation of HBV transcription and replication. Although we and others have shown that HBx stimulates and is likely required for HBV replication, we now report that HBx also activates signals that can diminish the overall level of HBV replication. While this may seem counterintuitive, we show that an important effect of HBx activation of AKT is inhibition of apoptosis. Consequently, our studies suggest that HBx balances HBV replication and cell survival by stimulating signaling pathways that enhance hepatocyte survival at the expense of higher levels of HBV replication. IMPORTANCE Chronic hepatitis B virus (HBV) infection is a common cause of the development of liver cancer. Regulation of cell signaling pathways by the HBV HBx protein is thought to influence the development of HBV-associated liver cancer. HBx stimulates, and may be essential for, HBV replication. We show that HBx activates AKT in hepatocytes to reduce HBV replication. While this seems contradictory to an

  15. Psoralidin, An Herbal Molecule Inhibits PI3K Mediated Akt Signaling In Androgen Independent Prostate Cancer (AIPC) Cells

    PubMed Central

    Kumar, Raj; Srinivasan, Sowmyalakshmi; Koduru, Srinivas; Pahari, Pallab; Roh